opce-str.vp a word from the new editor-in-chief dear authors, readers, and friends of acta botanica croatica first of all, i want to express my thanks to the editorial board of acta botanica croatica for its vote of confidence by appointing me the new editor-in-chief of the journal. i feel privileged to assume this important role in our botanical journal with a long and distinguished tradition dating back to 1925. acta botanica croatica has been fortunate to have had an outstanding editor over the last fifteen years, and i gratefully acknowledge my immediate predecessor, professor damir vili~i}, for having established and maintained a particularly high standard of editorship. thanks to his dedicated work and enthusiasm, significant progress in the quality and management of the journal was achieved between 1998 and 2013. since 1998, abc has been published regularly twice a year in english, and an international editorial board was formed. since 2001, open access to abc was established and in 2010 its impact factor was rated at 0.386, with a tendency to increase. today abc is listed in the scopus database, at the thomson reuters master journal list and the web of science database. since 2011 abc has been published in a partnership between the faculty of science, university of zagreb and a foreign co-publisher versita-walter de gruyter. i hope this good practice will increase the reputation of the journal in the future as well. professor vili~i} has a watching brief as a new member of the editorial advisory board. i am grateful to current and new members of the editorial board; the subject editors, the technical editor-in-chief and other technical editors, the managing editor, language editor and the editorial advisory board for their willingness to continue their collaboration and support. i hope and believe that with the excellent collaboration of all members of the editorial board we will continue the tradition and contribute to the further improvement of the quality of the journal. best regards editor’s word.ps u:\acta botanica\acta-botan 1-14\editor’s word.vp 19. o ujak 2014 16:37:26 color profile: generic cmyk printer profile composite default screen 102 acta bot. croat. 77 (1), 2018 acta bot. croat. 77 (1), 102–104, 2018 coden: abcra 25 doi: 10.1515/botcro-2017-0020 issn 0365-0588 eissn 1847-8476 short communication erigeron acris l. subsp. angulosus (gaudin) vacc. (asteraceae), a new taxon in the flora of poland artur pliszko* department of taxonomy, phytogeography and paleobotany, institute of botany, jagiellonian university, kopernika 31, 31-501 kraków, poland abstract – the paper reports for the first time the occurrence of erigeron acris subsp. angulosus in poland. this rare european temperate plant was found in august 2014 in a former sand and gravel quarry, close to the sobolewo reservoir in the town of suwałki, north-eastern poland. species composition of the habitat is characterized by a phytosociological relevé based on the braun-blanquet method, diagnostic characters in comparison to the morphologically similar e. acris subsp. droebachiensis are presented using scanning electron microscopy imaging, and an identification key for e. acris s. l. in poland is given. keywords: distribution, erigeron, indumentum, poland, scanning electron microscopy * corresponding author, e-mail: artur.pliszko@uj.edu.pl introduction erigeron acris l. subsp. angulosus (gaudin) vacc. (asteraceae), a european temperate representative of e. acris l. s. l. (greuter 2003, 2006–2016), is confined mainly to the alpine zone of the alps (šída 1998, 2000, 2004). it has been reported from france, italy, switzerland, liechtenstein, austria, slovenia, germany, the czech republic, slovakia, hungary, romania and estonia (greuter 2006–2016). this biennial or perennial herb usually occurs in scree and gravel river bank plant communities of the class thlaspietea rotundifolii (mucina 1997). one of the most reliable diagnostic characters that allows e. acris subsp. angulosus to be distinguished from most of the other subspecies of e. acris is the absence of villous indumentum on stems, leaves, and involucral bracts, with the exception of the short-stipitate glandular trichomes on involucral bracts (šída 1998). in poland, a recent study based on herbarium materials of e. acris s. l. deposited in the polish herbaria (pliszko 2015) revealed the occurrence of three subspecies, namely e. acris subsp. acris, e. acris subsp. droebachiensis (o. f. müll.) arcang., and e. acris subsp. serotinus (weihe) greuter. the present paper provides the first record of e. acris subsp. angulosus in poland and is aimed at showing the morphological characteristics of the taxon as well as its distribution and habitat. materials and methods erigeron acris subsp. angulosus was identified based on morphological features given by šída (1998, 2000, 2004) and its voucher specimens are deposited in the herbarium of the institute of botany of the jagiellonian university in kraków (kra). a phytosociological relevé followed the braun-blanquet method (braun-blanquet 1964). names of syntaxa and diagnostic species followed mucina (1997). associated plants were identified after wójciak (2003) and rutkowski (2004). names of taxa followed mirek et al. (2002) and ochyra et al. (2003). in morphometric and sem studies e. acris subsp. angulosus was compared to the morphologically similar e. acris subsp. droebachiensis. measurements were taken on herbarium specimens. indumentum terminology followed werker (2000). specimens of e. acris subsp. droebachiensis used in the morphometric study were collected in brzozowiec (south-eastern poland) in july 2013 and 2014 (gps coordinates: 49°25'54,36"n/22°11'57,36"e), whereas those used in the sem study were collected in toruń (north-central poland) in july 2013 (gps coordinates: 53°1'21,06"n/18°33'22,68"e). samples of dry cauline leaves, peduncles, and capitula were mounted on aluminum stubs coated with double-sided conductive carbon tabs. next, the samples were coated with gold in a sputter-coater and examined with a hitachi s-4700 scanning electron microscope at erigeron acris subsp. angulosus in poland acta bot. croat. 77 (1), 2018 103 an accelerating voltage of 10 kv, part of the standard procedure for viewing biological specimens in the sem method (bozzola and russell 1999). results and discussion erigeron acris subsp. angulosus was discovered in august 2014 on the south-eastern outskirts of the town of suwałki, north-eastern poland (gps coordinates: 54°4'18,06"n/22°57'48,84"e; altitude: 157 m.a.s.l.). according to zając (1978), the locality is situated within the fb18 square unit of the atpol cartogram grid. a small population consisting of 25 individuals was found in a former sand and gravel quarry, close to the sobolewo reservoir (fig. 1). vegetation in this anthropogenic habitat is represented by populations of grassland and ruderal plant species. the species composition in the site occupied by e. acris subsp. angulosus is characterized by the following phytosociological relevé: size of relevé: 25 m²; cover of herb layer: 20%; cover of moss layer: 40%; ch.cl. artemisietea vulgaris: poa compressa 2, melilotus albus +; ch.cl. koelerio-corynephoretea: helichrysum arenarium 1, arenaria serpyllifolia +, herniaria glabra +, ceratodon purpureus (d) 3; ch.cl. festucobrometea: artemisia campestris 1, centaurea stoebe +; others: erigeron acris subsp. angulosus 1, hieracium pilosella 1, oenothera sp. 1, echium vulgare +, erigeron acris subsp. acris +, pinus sylvestris +. taking into consideration the selected quantitative features (tab. 1), e. acris subsp. angulosus clearly shows shorter stems and synflorescences, and a lower number of capitula than e. acris subsp. droebachiensis. the number of cauline leaves is similar in both subspecies; however, the lower cauline leaves of e. acris subsp. angulosus are much narrower than those of e. acris subsp. droebachiensis. regarding the sem micrographs (on-line suppl. figs. 1–3), in contrast to e. acris subsp. droebachiensis, e. acris subsp. angulosus has no unbranched multicellular uniseriate non-glandular trichomes on involucral bracts, peduncles, and cauline leaves. on the other hand, the unbranched short-stipitate multicellular biseriate glandular trichomes are densely distributed on involucral bracts and sparsely on peduncles in both subspecies. moreover, involucral bracts in e. acris subsp. angulosus are serrated relatively more deeply at the apex than those in e. acris subsp. droebachiensis (online suppl. fig. 2). the occurrence of e. acris subsp. angulosus in north-eastern poland suggests that there may be a continuity of its geographical distribution between central europe and the baltic states, since the taxon is present in estonia (greuter 2006– 2016). furthermore, it is worth mentioning that most of the specimens collected in suwałki have relatively more cauline leaves than those characterized by šída (1998, 2000, 2004). this can be explained by the fact that they were found flowering in late summer. erigeron acris subsp. angulosus typically flowers in june, and at higher altitudes in july (šída 2000, 2004). for example, summer specimens of e. acris subsp. acris often produce more cauline leaves than typical late spring specimens, especially after mowing or grazing (pliszko 2015). in this connection, it should also be mentioned that similar specimens with numerous glabrous cauline leaves and a low number of capitula were collected between miedzierza and sielpia wielka (south-central poland) by walas in september 1929 (kra 0124518). these specimens were mistakenly identified as e. acris subsp. droebachiensis (pliszko 2015). however, it is hard to decide if they belong to e. acris subsp. angulosus because their atypical features seem to be a result of mowing or grazing. the locality between miedzierza and sielpia wielka needs to be re-examined and the identity of the plant should be confirmed in the flowering stage in june or july. e. acris subsp. angulosus is the rarest subspecies of e. acris in poland. although the habitat in which the plant was discovered shows an anthropogenic character, it should be viewed as a native taxon. its presence was confirmed in july 2016 with one flowering specimen. the expansion of trees and shrubs during secondary succession and recreational use of motorcycles are the main factors that can pose a threat to the e. acris subsp. angulosus population in suwałki. fig. 1. erigeron acris subsp. angulosus in suwałki, north-eastern poland: a – upper part of the plant; b – habitat (photos by a. pliszko). tab. 1. morphological comparison of the selected quantitative characters of erigeron acris subsp. angulosus and e. acris subsp. droebachien n – number of examined individuals. numbers indicate mean value (out of brackets), minimum and maximum values (in brackets). character erigeron acris subsp. angulosus (n=15) erigeron acris subsp. droebachiensis (n=15) height of stem (cm) 31.9 (22.0–45.0) 46.6 (28.5–62) number of cauline leaves 17.4 (12–24) 19.2 (10–25) size of lower cauline leaf (cm) 3.8 × 0.4 (2.5–6.5 × 0.3–0.6) 7.6 × 1.0 (4.5–12.0 × 0.7–1.4) number of capitula 6.2 (3–9) 21.3 (6–46) lenght of synflorescence (cm) 6.6 (4.5–10.0) 14.1 (7–24) pliszko a. 104 acta bot. croat. 77 (1), 2018 acknowledgements i would like to thank m.sc. eng. anna łatkiewicz (department of mineralogy, petrology and geochemistry of the institute of geological sciences of the jagiellonian university in kraków) for her help in taking sem images. i also thank identification key for erigeron acris s. l. in poland 1. stems and leaves densely villous, involucral bracts abaxially densely villous and sparsely short-stipitate glandular ..............................................................................2 stems and leaves glabrous or sparsely villous at the base, involucral bracts abaxially densely short-stipitate glandular, glabrous or sparsely villous ............................3 2. cauline leaves 4–16, flat, usually erect; internodes 1.0–8.0 cm; capitula in paniculiform synflorescence ..........................erigeron acris subsp. acris the anonymous reviewers for their suggestions and comments. this work was financially supported by the jagiellonian university in kraków (grants for research projects ds/mnd/ wbinoz/ib/4/2013 and ds/mnd/wbinoz/ib/2/2014). references bozzola, j. j., russell, l.d., 1999: electron microscopy. principles and techniques for biologists. 2nd ed. jones and bartlett publishers, boston, toronto, london, singapoure. braun-blanquet, j., 1964: pflanzensoziologiae, grundzüge der vegetationskunde. 3rd ed. springer-verlag, wien-new york. greuter, w., 2003: the euro+med treatment of astereae (compositae) – generic concepts and required new names. willdenowia 33, 45–47. greuter, w., 2006–2016: compositae (pro parte majore). in: greuter, w., raab-straube, e. von (eds.), compositae. euro+med plantbase – the information resource for euromediterranean plant diversity. retrieved january 27, 2016 from http://ww2.bgbm.org mirek, z., piękoś-mirkowa, h., zając, a., zając, m., 2002: flowering plants and pteridophytes of poland, a checklist. w. szafer institute of botany, polish academy of sciences, kraków. mucina, l., 1997: conspectus of classes of european vegetation. folia geobotanica et phytotaxonomica 32, 117–172. ochyra, r., żarnowiec, j., bednarek-ochyra, h., 2003: census catalogue of polish moses. w. szafer institute of botany, polish academy of sciences, kraków. pliszko, a., 2015: taxonomic revision and distribution of erigeron acris s. l. (asteraceae) in poland. phytotaxa 208, 021–033. rutkowski, l., 2004: a key for identification of vascular plants of lowland poland (in polish). wydawnictwo naukowe pwn, warszawa. šída, o., 1998: taxonomic problems in erigeron sect. trimorpha (compositae) in eurasia. preslia 70, 259–269. šída, o., 2000: erigeron acris agg. in the czech republic and slovakia. zprávy české botanické společnosti 35, 1–33 (in czech). šída, o., 2004: erigeron l. in: slavík, b., štěpánková, j. (eds.), flora of the czech republic vol. 7, 140–153. academia, praha (in czech). werker, e., 2000: trichome diversity and development. in: hallahan, d. l., gray, j. c. (eds.), advances in botanical research. incorporating advances in plant pathology. plant trichomes, vol. 31, 1–35. academic press, london. wójciak, h., 2003: lichens, bryophytes, pteridophytes. flora of poland. multico oficyna wydawnicza, warszawa (in polish). zając, a., 1978: atlas of distribution of vascular plants in poland (atpol). taxon 27, 481–484. cauline leaves 12–36, broadly undulate, recurved; internodes 0.2–3.8 cm; capitula in racemiform or paniculiform synflorescence ............... erigeron acris subsp. serotinus 3. basal leaves with acute apex; cauline leaves narrowly lanceolate to linear; peduncles sparsely short-stipitate glandular or glabrous; capitula in racemiform synflorescence .................erigeron acris subsp. angulosus basal leaves with obtuse apex; cauline leaves lanceolate; peduncles densely to sparsely villous and short-stipitate glandular; capitula in paniculiform synflorescence .........erigeron acris subsp. droebachiensis acta bot. croat. 77 (2), 2018 189 acta bot. croat. 77 (2), 189–192, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0015 issn 0365-0588 eissn 1847-8476 occurrence of the sexual morph of erysiphe macleayae on chelidonium majus in romania vasilică-claudiu chinan* alexandru ioan cuza university, faculty of biology, bd. carol i, no. 20a, 700505, iaşi, romania abstract – the sexual morph (chasmothecium) of erysiphe macleayae on chelidonium majus has been found in romania for the first time. although the asexual morph of this powdery mildew on c. majus is known from many countries, the occurrence of chasmothecia on this host is rather rare. a description of morphological characters and an analysis of the phylogenetic relationship of its sequences of the romanian specimens are presented. key words: greater celandine, powdery mildew, chasmothecia, its * corresponding author, e-mail: vasilechinan@yahoo.com introduction thecia) were also found in germany in 2014 (braun 2014) and slovakia in 2014 and 2015 (pastirčáková et al. 2016). recently, the anamorph of e. macleayae on c. majus was also reported from ukraine (heluta and kravchuk 2015). based on a collection from moldavia (romania), the anamorph of this powdery mildew on c. majus was described by iacob and drobotă (2008) as a new species, oidium chelidonii iacob. because of the absence of both a latin diagnosis and a holotype, this name is invalid (pastirčáková et al. 2016). the aim of this paper is to report the new occurrence of the teleomorph of e. macleayae on c. majus, which is new for romania. furthermore, a description of the romanian specimens and a phylogeny inferred including the newly obtained its sequences are provided. materials and methods leaves of chelidonium majus infected with powdery mildew were collected from the urban area of iași, romania. for light microscopic examination, fresh samples were mounted in 2% koh. the sexual and asexual features (chasmothecia, asci, ascospores, conidia, and conidiophores) were described, measured, and photographed. the given size ranges of all structures are based on 30 measurements. statistical data (length and width) are given as arithmetic mean ± standard deviation (sd). for the examination of the conidial germination, the moist chamber method with saturated aqueous solution of potassium sulphate as described in braun and erysiphe macleayae was originally described from china as a parasite of macleaya cordata (willd.) r. br. and papaver nudicaule l. (zheng and chen 1981). besides these hosts, it was also reported as causal agent of powdery mildew on macleaya microcarpa (maxim.) fedde (park et al. 2012), meconopsis cambrica (l.) vig. (schmidt and scholler 2011, braun and cook 2012), and chelidonium majus l. (jiang et al. 2015), all of them plant species of the family papaveraceae. outside this family, it was recently found on torenia fournieri linden ex e. fourn. (men et al. 2014), a species of linderniaceae. in the last decade, the powdery mildew of chelidonium majus (greater celandine) has been a disputed topic in the context of the extension of its range of distribution and the absence, until recently, of the sexual morph. the first report of this disease based on dna analysis of the anamorph was published by jankovics (2007) from hungary, who identified the powdery mildew on c. majus as oidium neolycopersici. in a study of the powdery mildews of papaveraceae species in germany, schmidt and scholler (2011) mentioned that the powdery mildew of c. majus may be related to or even conspecific with e. macleayae. later, pastirčáková and pastirčák (2013) confirmed on the basis of morphological characteristics that the anamorph of the c. majus powdery mildew in the czech republic and slovakia is caused by a pseudoidium species. finally, jiang et al. (2015) published the first collection of the chasmothecia on c. majus in china and identified the causal agent as erysiphe macleayae. fruiting bodies (chasmoshort communication chinan v. c. 190 acta bot. croat. 77 (2), 2018 cook (2012) was used. the analyzed specimens are deposited in the herbarium of alexandru ioan cuza university of iasi, romania (index herbariorum abbreviation: i). for the molecular characterization, dna was extracted from the chasmothecia (voucher specimen i 183221) and the anamorph (voucher specimen i 183222). the nuclear ribosomal internal transcribed spacer (its) region was amplified using primers its1f 5’-cttggtcatttagaggaagtaa-3’ (gardes and bruns 1993) and its4, 5′-tcctccgcttattgatatgc-3′ (white et al. 1990) and sequenced at alvalab (santander, spain). the newly obtained sequences were deposited in genbank (accession numbers ku756266 and ku756267). for the phylogenetic analysis, sequences of the powdery mildew specimens isolated from chelidonium majus (jankovics 2007, jiang et al. 2015, kovács et al. 2011, pastirčáková et al. 2016, takamatsu et al. 2015), macleaya cordata (jiang et al. 2015, pastirčáková et al. 2016, takamatsu et al. 2015), macleaya microcarpa (park et al. 2012), and torenia fournieri (men et al. 2014) were downloaded from genbank and aligned with the clusatlw algorithm (larkin et al. 2007). sequences of erysiphe aquilegiae dc., e. magnoliae (sawada) u. braun & s. takam. from takamatsu et al. (2015), and e. deutziae (bunkina) u. braun & s. takam. from denton and henricot (2007) were used as outgroup taxa as these species were recovered as basal to the e. aquilegiae clade in the comprehensive phylogenetic analysis of takamatsu et al. (2015). a list of all the 35 sequences used in the analysis is included in the on-line suppl. tab. 1. maximum likelihood (ml) analysis was used as a tree building method. the k2 (kimura 2-parameter) model of nucleotide substitution was selected using bic (bayesian information criterion). the reliability of phylogenetic relationships was evaluated using a non-parametric bootstrap analysis with 1000 replicates and ml bootstrap values (bp) are presented at branch nodes. the bootstrap values exceeding 75 were considered well supported. all computations were performed using mega v6.0.5 (tamura et al. 2013). results and discussion in the autumn of 2014, several specimens of c. majus with powdery mildew were collected in iasi, romania, including a single leaf with a few young chasmothecia. a year later, alongside a building wall near the headquarters of the alexandru ioan cuza university of iași, a powdery mildew specimen on c. majus with mature chasmothecia on basal leaves was found. of the 12 leaves of the plant, nine showed disease symptoms. besides this, although other specimens of greater celandine with powdery mildew were investigated, only a single additional leaf with chasmothecia on another specimen was found. the collected specimens belong to e. macleayae. the occurrence of the teleomorph of this powdery mildew is new for romania. erysiphe macleayae r.y. zheng & g.q. chen, sydowia 34: 290 (1981) mycelium white, amphigenous, but mostly epiphyllous, covering the entire surface of the older basal leaves, or forming patches mainly on the upper lobes of the leaf (fig. 1a). hyphae 4–6 µm wide, with lobed or nipple-shaped appressoria. conidiophores straight or slightly curved, hyaline, 70–135 (mean 99.8 ± 14.7) × 8–12 µm (mean 10.0 ± 0.9), foot-cells cylindrical, followed by 1–2 shorter cells. conidia formed singly, ellipsoid, doliiform-cylindrical, hyaline, 25– 50 (mean 38.7 ± 5.4) × 10–20 µm (mean 14.6 ± 2.7). germ tubes formed subapically (pseudoidium type) (fig. 1d). chasmothecia scattered to gregarious, subglobose, 75–116 µm (mean 92.3 ± 10.2) in diameter, initially light brown, then dark brown. appendages 15–25 per chasmothecium, simple, often tortuous-sinuous, up to 700 µm long, light brown (fig. 1b). asci 3–6 per chasmothecium, 45–70 (mean 57.1 ± 7.6) × 30–55 µm (mean 37.4 ± 7.9), obovoid-saccate, short-stalked, 3–6-spored. ascospores 16–26 (mean 21.6 ± 2.7) × 10–13 µm (mean 10.7 ± 0.8), ellipsoid-ovoid (fig. 1c). specimens examined: on living leaves of chelidonium majus, romania, iasi, anastasie fătu botanic garden, dumbrava roşie street, 7 oct. 2014, leg. et det. v. chinan, voucher specimen i 183224 (anamorph and immature chasmothecia); iasi, pinului street, near the alexandru ioan cuza university, 10 oct. 2015, leg. et det. v. chinan, voucher specimens: i 183221(teleomorph, genbank accession number ku756266), i 183222 (anamorph, genbank accession number ku756267); 20 oct. 2015, leg. et det. v. chinan, voucher specimen i 183223 (teleomorph). phylogenetic analysis: the sequences obtained from the chasmothecia (ku756266) and the anamorph (ku756267) of the romanian specimens are identical, and positioned well with the other sequences of erysiphe macleayae parasitizing c. majus, m. cordata, m microcarpa and t. fournieri fig. 1. erysiphe macleayae: a – symptoms on chelidonium majus, b – chasmothecium, conidia and mycelium on the leaf surface, c – crushed mature chasmothecium, asci with ascospores, d – germinated conidium. erysiphe macleayae in romania acta bot. croat. 77 (2), 2018 191 (bp = 96) (fig. 2). these results confirm the species identity and the conspecificity of the asexual and sexual morphs found on c. majus in romania. the intraspecific molecular variability of e. macleayae is very low: specimens of diverse geographical origins differ at most in two substitutions (0– 0.4% uncorrected pairwise distance). both the sexual and the asexual morph of the romanian collections agree well morphologically with the descriptions of e. macleayae from m. cordata (zheng and chen 1981) and c. majus (jiang et al. 2015, pastirčáková et al. 2016). it is noteworthy that in europe chasmothecia of e. macleayae on c. majus were found in autumn 2014, simultaneously in germany (braun 2014), slovakia (pastirčáková et al. 2016) and romania (in this study). in these countries, the anamorph on this host was first found in 2003 (jage et al. 2010), 2006 (pastirčáková and pastirčák 2013), and 2007 (iacob and drobotă 2008) respectively. this confirms that mafig. 2. rooted maximum likelihood tree based on its sequences. the sequences of erysiphe macleayae from romania obtained in this work are indicated by a star. bootstrap support values from 1000 pseudoreplicates are reported above nodes. ny powdery mildew fungi do not produce chasmothecia for several years after extending their geographical areas or host ranges (kiss 2002, kiss et al. 2002). the annual monitoring of this powdery mildew made in slovakia by pastirčáková et al. (2016), in the period 2006–2015 fully supports this observation. furthermore, homoor heterothallism of the species concerned may play an important role. heterothallic species often need more time to find compatible thalli to be able to reproduce sexually. the individual life cycle of e. macleayae is still unknown, but heterothallism appears to prevail among powdery mildews (wolfenbarger et al. 2015). acknowledgments i would like to thank lucian fusu for all of the help with the phylogenetic analysis and the anonymous reviewers, whose comments and suggestions helped improve this manuscript. chinan v. c. 192 acta bot. croat. 77 (2), 2018 references braun, u., 2014: fungi selecti exsiccati ex herbario universitatis halensis – nos. 211–220. schlechtendalia 28, 35–37. braun, u., cook r. t. a., 2012: taxonomic manual of the erysiphales (powdery mildews). cbs biodiversity series 11. cbsknaw fungal biodiversity centre, utrecht, the netherlands. denton, g., henricot, b., 2007: first report of powdery mildew on deutzia spp. in the uk. plant pathology 56, 353–353. gardes, m., bruns. t. d., 1993: its primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. molecular ecology 2, 113–118. heluta, v. p., kravchuk, h. a., 2015: first records of a new invasive fungus, erysiphe macleayae (erysiphales), in ukraine. ukrainian botanical journal 72, 39–45. iacob, v., drobotă, i., 2008: new parasitic and saprophytic micromycetes on cultivated horticultural plants from moldavia. lucrări științifice, seria horticultură 51, 1049–1054. jage, h., klenke, f., kummer, v., 2010: new records and remarkable confirmations of phytoparasitic microfungi in germany – erysiphales (powdery mildews). schlechtendalia 21, 1–140. jankovics, t., 2007: first report of powdery mildew (oidium sp.) on greater celandine (chelidonium majus). plant pathology 56, 353–353. jiang, w., liu, s., an, b., wanga, l., li, y., takamatsu, s., braun, u., 2015: chasmothecia of erysiphe macleayae on chelidonium majus confirm species identification. mycoscience 56, 132–135. kiss, l., 2002: advances in the identification of emerging powdery mildew fungi using morphological and molecular data. acta microbiologica et immunologica hungarica 49, 245–248. kiss, l., bolay, a., takamatsu, s., cook, r. t. a., limkaisang, s., ale-agha, n., szentivanyi, o., boal, r. j., jeffries, p., 2002: spread of the north american snowberry powdery mildew fungus, erysiphe symphoricarpi (syn. microsphaera symphoricarpi), to europe. mycological research 106, 1086–1092. kovács, g. m., jankovics t., kiss l., 2011: variation in the nrdna its sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information? european journal of plant pathology 131, 135–141. larkin, m. a., blackshields, g., brown, n. p., chenna r., mcgettigan, p. a., mcwilliam, h., valentin, f., wallace, i. m., wilm, a., lopez, r., thompson, j. d., gibson, t. j., higgins, d. g., 2007: clustal w and clustal x version 2.0. bioinformatics 23, 2947–2948. men, x.-y., liu, s.-y., jiang, w.-t., li, y., 2014: first report of powdery mildew caused by erysiphe macleayae on torenia fournieri in china. plant disease 98, 1277–1277. pastirčáková k., pastirčák, m., 2013: a powdery mildew (pseudoidium sp.) found on chelidonium majus in the czech republic and slovakia. czech mycology 65, 125–132. pastirčáková, k., jankovics, t., komáromi, j., pintye, a., pastirčák, m., 2016: genetic diversity and host range of powdery mildews on papaveraceae. mycological progress 15: 36. pаrk, m. j., cho, s. e., piątek, m., shin, h. d., 2012: first report of powdery mildew cаused by erysiphe mаcleаyаe on mаcleаyа microcаrpа in polаnd. plant disease 96, 1376–1376. schmidt, a., scholler, m., 2011: studies in erysiphales anamorphs (4): species on hydrangeaceae and papaveraceae. mycotaxon 115, 287–301. takamatsu, s., ito arakawa, h., shiroya, y., kiss, l., heluta, v., 2015. first comprehensive phylogenetic analysis of the genus erysiphe (erysiphales, erysiphaceae) i. the microsphaera lineage. mycologia 107, 475–489. tamura, k., stecher, g., peterson, d., filipski, a., kumar, s., 2013: mega6: molecular evolutionary genetics analysis version 6.0. molecular biology and evolution 30, 2725–2729. white, t. j., bruns, t., lee, s., taylor, j. w., 1990: amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis, m. a. et al. (eds.), pcr protocols: a guide to methods and applications, 315–322. academic press inc., new york. wolfenbarger, s. n., twomey, m. c., gadoury, d. m., knaus, b. j., grünwald, n.j., gent, d. h., 2015: identification and distribution of mating‐type idiomorphs in populations of podosphaera macularis and development of chasmothecia of the fungus. plant pathology, 64, 1094–1102. zheng, r. y., chen, g. q., 1981: the genus erysiphe in china. sydowia 34, 214–327. acta bot. croat. 77 (1), 2018 105 acta bot. croat. 77 (1), 105–108, 2018 coden: abcra 25 doi: 10.1515/botcro-2017-0021 issn 0365-0588 eissn 1847-8476 short communication rare plants of threatened habitats – the croatian case of corrigiola litoralis l. (caryophyllaceae) nina vuković1, vedran šegota2*, antun alegro1, zorana sedlar3 1 university of zagreb, faculty of science, department of biology, division of botany, marulićev trg 20/ii, hr-10000 zagreb 2 university of zagreb, faculty of science, department of biology, division of botany, herbarium croaticum (za), marulićev trg 20/ii, hr-10000 zagreb 3 croatian natural history museum, department of botany, demetrova 1, hr-10000 zagreb abstract – corrigiola litoralis is the only member of the genus in the croatian flora. most available data are more than 50 years old, and include only four localities in croatia, exclusively in the islands of the mediterranean region. during a floristic survey of the island of molat (northern dalmatia) we recorded a small population in zapuntelsko polje, in a damp, shallow depression, seasonally occurring as a temporary pond. comparison with the existing literature shows that c. litoralis often occurs in mediterranean temporary ponds and similar globally important and threatened habitats. we strongly believe that careful studies of populations of c. litoralis and other species with similar ecologies are necessary in order to preserve these habitats and therefore propose actions to achieve this goal. keywords: endangered habitats, iucn, mediterranean temporary ponds, molat * corresponding author, e-mail: vedran.segota@biol.pmf.hr introduction according to flora europaea, the genus corrigiola is represented in europe by two species: c. litoralis l. (with two subspecies, c. litoralis l. ssp. litoralis and c. litoralis l. ssp. telephiifolia (pourr.) briq.), and c. imbricata lapeyr (walters and akeroyd 1993). according to the croatian checklist, c. litoralis, and a typical subspecies (c. litoralis l. ssp. litoralis) have so far been registered in croatia (nikolić 2016). c. litoralis is a short, prostrate annual, with slender stem, very small flowers (2 mm diameter when expanded), and alternate leaves (unlike most caryophyllaceae) (coker 1962, walters and akeroyd 1993). it grows on periodically wet, sandy or gravel ground, in damp, open environments; often in sites along rivers, lakes and ponds subject to fluctuating water levels and with seasonally exposed shores (walters and akeroyd 1993, mchugh 2007, hamston 2010). native distribution of c. litoralis is mediterranean-atlantic, with occurrences in north, east and south africa (coker 1962, rankou et al. 2015). the introduced range includes australia (rankou et al. 2015) and probably america (coker 1962, rankou et al. 2015). in the mediterranean parts of south and west europe, the species is very widespread and common (rankou et al. 2015). according to the iucn, corrigiola litoralis is globally considered a species of least concern (lc), for the overall trend of the population appears stable, although it is very rare and threatened locally, for example in england, at the northern fringe of its distribution (hamston 2010). in croatia, only four localities were known prior to this study, and moreover, only one historical record was confirmed during the 21st century (visiani 1852, lusina 1938, trinajstić 1979, škunca et al. 2008) (fig. 1). the first known records date from the middle of the 19th century, when visiani (1852) noted this species for the islands krk (northern adriatic) and korčula (southern adriatic). the record from krk was confirmed later by schlosser (1856) (za collection), tommasini (1875) and trinajstić (1965), while a finding from korčula was cited once without confirmation (trinajstić 1985), and never mentioned in the literature since. as much as 86 years after the first records, a new record from lusina (1938) was published, of a finding of c. litoralis on the island of unije (northern adriatic). this record was confirmed by the same author almost two decades later (lusina 1956), and cited in recent times but without confirmation (wallnöfer 2008). finally, the species was found by trinajstić (1979) on the island of lastovo (southern adriatic), and confirmed in recent times during a floristic study of the lastovsko otočje nature park (škunca et al. vuković n., šegota v., alegro a., sedlar z. 106 acta bot. croat. 77 (1), 2018 2008). the data on c. litoralis on krk and unije published by rottensteiner (2014), refer to previously mentioned historical data, and were not confirmed in recent times. literature data show that corrigiola litoralis often occurs in croatia on sandy soil, regularly around ponds or lakes, exclusively in the mediterranean part of the country (visiani 1852, hayek 1924–1927, lusina 1938, 1956, trinajstić 1965, topić and vukelić 2009, rottensteiner 2014). trinajstić (1965, 2008) recorded it in ponikve (krk) along the shores of a temporary pond (dry at the time) within the association plantagini intermediae-crypsidetum schoenoidis trinajstić 1965, a community associated with mediterranean temporary ponds (topić and vukelić 2009). although c. litoralis is extremely rare in croatia, it was never estimated according to the iucn criteria. materials and methods in october 2015, several fieldtrips were undertaken within a floristic study of the island of molat in the zadar archipelago (northern dalmatia). the north-western part of the island, in the area of zapuntelsko polje, was researched on 25th october. gps coordinates (according to wgs84) were recorded and lists of vascular plants were prepared for four selected sites in that area, while some plant taxa were photographed. specimens of corrigiola litoralis were collected and stored in herbarium croaticum (za). shortly after the fieldtrip, the flora croatica database (nikolić 2016) was searched for c. litoralis, and all the related literature was closely examined. both herbarium croaticum (za) and herbarium ivo and marija horvat (zaho) were searched for c. litoralis (appendix 1). results and discussion corrigiola litoralis was found in the zapuntelsko polje (fig. 2), at coordinates 44°14'59"n, 14°48'28"e. it was recorded in a shallow depression in the north-western part of the field, dry at the time of our visit, but occasionally existing as a pond (fig. 3). the water regime varies from year to year, depending on the rainfall, but the pond seasonally dries out, and the drought period usually extends from late spring, till late autumn. the pond is not particularly maintained by the local inhabitants; nevertheless the nearby area is occasionally used for pasturing (mainly by sheep). although the area was dry at the time, the presence of some plants commonly found in moist habitats, such as agrostis stolonifera l., poa trivialis l. ssp. sylvicola (guss.) h. lindb., ranunculus sardous crantz, oenanthe pimpinelloides l., carex divulsa stokes and others, clearly indicates the importance of water in this particular site. only a few plants of corrigiola litoralis were found, growing on patches of bare soil, in periodically flooded parts of the field (fig. 3). all individuals were vigorous and generally in a good state, showing no signs of herbivory or pathogens, and flowering at the time (fig. 4). the species was recorded in a typical microhabitat; it prefers open sites, with sparse vegetation preferably not higher than 2–4 cm, in areas where occasional flooding and moderate trampling reduce the competition from more robust plants (coker 1962). in addition, the same microhabitat in zapuntelsko polje was occupied by some peculiar and rare ephemeral liverworts (fossombronia echinata macvicar, fossombronia caespitiformis de not. ex rabenh. ssp. multispira (schiffn.) j. r. bray & d. c. cargill, riccia sorocarpa bisch., riccia bifurca hoffm. and riccia subbifurca warnst. ex croz.), typically growing on patches of bare ground, usually in damp micro-depressions subject to trampling. notably, the invasive grass paspalum paspaloides (michx.) scribn was recorded on the same localfig. 1. all known localities of corrigiola litoralis in croatia (from the top downwards: krk, unije, molat, korčula, lastovo); our record is denoted with a red dot. the years of the last known field confirmations are shown. fig. 2. aerial photographs of molat, showing the exact location of corrigiola litoralis in zapuntelsko polje. corrigiola litoralis in croatia acta bot. croat. 77 (1), 2018 107 ity, aggressively occupying the area around the pond, as well as other moist micro-depressions in near vicinity. another invasive species, aster squamatus (spreng.) hieron, was also recorded in the area. due to the extremely low number of findings in croatia, and the affiliation with specific microhabitats, c. litoralis is presumably threatened with extinction, and should be evaluated according to the iucn criteria. at the best, even without insights into the current state of the population or its habitats, assuming that the species is still present in all historically known sites, it could qualify as vulnerable (vu) according to criterion d (iucn 2013). in order to obtain more comprehensive data on the croatian distribution of c. litoralis, historical localities should be searched for the presence of its populations, since only records from molat and lastovo have been confirmed within the last 50 years. in particular, other temporary ponds along the eastern adriatic coast, including the islands, should be carefully studied. mediterranean temporary ponds are, beyond any doubt, globally significant in the context of the preservation of rare and endangered species (zacharias et al. 2007). unfortunately, these habitats are simultaneously facing a rapid decline worldwide (rhazi et al. 2012), accordingly being listed as a priority habitat in the habitats directive (natura 2000 priority habitat type 3170, anonymous 2007). other rare/endangered plants with similar ecologies, such as pilularia minuta durie ex. a. braun, cicendia filiformis (l.) delarbre, damasonium polyspermum cosson, crypsis aculeata (l.) aiton, crypsis schoenoides (l.) lam., fimbristylis bisumbellata (forssk.) bubani, lythrum tribracteatum salzm. ex spreng. etc., are typically recorded in these and similar habitats (nikolić and topić 2005, anonymous 2007, topić and vukelić 2009, boršić and posavec vukelić 2012, vuković and jelaska 2015), within ephemeral communities or their fragments. due to their specific ecological requirements, the survival of these species and communities is not supported beyond the very specific conditions of the corresponding habitats. keeping this in mind, it is strongly advisable to study these species, including corrigiola litoralis, more closely. for the future, we propose careful studies of mediterranean temporary ponds through fieldtrips during the droughty period when ephemeral plants and mosses can be detected and recorded, their population size estimated, and threats identified. to monitor their population trends and quantify the level of habitat loss, we advise these activifig. 3. a temporary pond in zapuntelsko polje (molat) where corrigiola litoralis occurs, photographed in 2015. (a) 28th february, (b) 09th april, (c) 08th june, (d) 25th october (time of our visit). photos by z. sedlar. fig. 4. corrigiola litoralis in bloom, on 25th october 2015. photo by a. alegro. vuković n., šegota v., alegro a., sedlar z. 108 acta bot. croat. 77 (1), 2018 ties be performed periodically (preferably yearly). finally, we would suggest re-introducing pond maintenance (mowing, grazing) as a way to prevent further loss of this rare and endangered habitat and species. acknowledgements the fieldwork was undertaken within the frames of the “molat – zeleni otok” project, funded by the city of zadar. references park lastovsko otočje. in: prvan, m., čavrak, v. v. 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hydrobiologia 689, 23–36. rottensteiner, w.k., 2014: excursionflora für istrien. verlag des naturwissenschaftlichen vereins für kärnten, klagenfurt. škunca, l., magajne, m., šegota, v., kirin, t., hruševar, d., dobrijević, t., 2008: inventarization of flora of the nature appendix 1. specimina visa schlosser c. j.: island of krk (in arenosis insulae veglia), 1856 (za), šegota, v., vuković, n., alegro, a., sedlar, z.: island of molat, zapuntelsko polje, 2015 (za). opce-str.vp acta bot. croat. 68 (2), 231–237, 2009 coden: abcra 25 issn 0365–0588 a new species of sellaphora (sellaphoraceae) from hannaberry lake, arkansas, u.s.a. mihaela d. enache*, marina potapova the academy of natural sciences, 1900 benjamin franklin parkway, philadelphia, pa, 19103, usa a new small-size species of sellaphora was found in sediments from hannaberry lake, arkansas, during the national lakes assessment project conducted by the united states environmental protection agency. the species was studied with light microscopy and scanning electron microscopy. it differs from previously reported sellaphora species by its small and delicate frustule with striation irresolvable in light microscopy. here we present details on its morphology and size variation and report the characteristics of the lake where the species was found. key words: diatom, sellaphora, morphology, ultrastructure, size, lake hannaberry, arkansas, usa abbreviations: nla – national lakes assessment, epa – environmental protection agency, lm – light microscopy(e), sem – scanning electron microscopy(e) introduction several new species of sellaphora were reported in recent studies of freshwater bodies from north america (antoniades et al. 2007, potapova and ponader 2008). the north american findings came to complement the abundant recent literature describing new species or combinations since the resurrection of the genus sellaphora by mann (1989) who showed that this genus is different from navicula bory s. str. in many characteristics of both protoplast and frustule. in addition to the more than 30 new species described from south america (kusber and jahn 2003, metzeltin et al. 2005, metzeltin and langebertalot 2007), 6 species were separated within the sellaphora pupula complex (mann et al. 2004) and another 6 species were described from the european lakes prespa and ohrid (levkov et al. 2003, 2007). here we present details of frustule morphology and habitat characteristics of a small species that we place within sellaphora. acta bot. croat. 68 (2), 2009 231 * corresponding author: enache@ansp.org u:\acta botanica\acta-botan 2-09\enache.vp 5. listopad 2009 15:13:16 color profile: disabled composite 150 lpi at 45 degrees materials and methods sediment cores were collected from freshwater bodies within the national lakes assessment (nla) across all states of the u.s.a. during the summer-fall season of 2007. the survey included natural and man-made freshwater lakes, ponds, and reservoirs greater than 10 acres (4 hectares) in the conterminous u.s., excluding the great lakes. more details on sampling design can be found at the epa web page http://www.epa.gov/owow/lakes/ lakessurvey/siteselect_factsheet. html. sediment cores were retrieved from the deepest basins of the lakes using a glew corer (internal diameter 7.62 cm) that preserves an undisturbed sediment-water interface (glew et al. 2001). sediment samples from the top (0–1 cm, representing sediments recently accumulated or modern samples) and from the bottom of each core (> 35 cm, representing pre-industrial sediments) were analyzed by different laboratories to allow comparisons between modern and pre-industrial diatom species composition. within this project, more than 500 sediment samples were analyzed at the academy of natural sciences of philadelphia (ansp). sediment sample digestion for diatom analysis followed the ansp protocols (charles et al. 2002). diatom permanent slides were prepared using naphraxr mounting medium. for light microscopy (lm) we used a zeiss axio imager microscope equipped with axiocam mrm digital camera. for scanning electron microscopy (sem), cleaned material was dried on aluminum stubs; stubs were sputter-coated with pt-pd and observed under a zeiss supra 50 vp fe sem (carl zeiss, jena, germany) at an accelerating voltage of 10 kv. terminology used to describe valve structure follows anonymous (1975), ross et al. (1979), and mann et al. (2004). the water chemistry characteristics for hannaberry lake, the site where the new sellaphora species was found, were obtained from the epa. results sellaphora pulchra enache et potapova sp. nov. (figs. 1–13) descriptio: valvae ellipticae-lanceolatae apicibus subcapitatis vel capitatis, 7.6–13.3 mm longae, 3.3–4.0 mm latae. area axialis angusta, recta. area centralis transverse rectangulata versus marginem dilatata. striae transapicales uniseriatae radiantes in media parte, ad apices parallelae vel paulo convergentes, 30–40 in 10 mm ad area centralis, 35–45 in 10 mm in media parte et ad apicem. areolae parvae circulares, 60–100 in 10 mm. striae non aspectabiles microscopio photonico. raphe recta, filiformis, poris centralibus simples paulo flexis, fissuris terminalibus curvatis ad latus secundum valvae. plica ad partem conjunctem frontis valvae et limbi praesens. areae terminales non vel leviter distinguibiles. description: valves elliptic-lanceolate with sub-capitate to capitate ends, 7.6–13.3 mm long and 3.3–4.0 mm wide. axial area narrow and straight. central area bow-tie-shaped with alternating short and long striae. transapical striae uniseriate and strongly radiate in mid-valve, becoming parallel or slightly convergent at valve ends, 30–40 in 10 mm around central area, 35–45 in 10 mm from mid-valve to apices. areolae small, rounded, 60–100 in 10 mm transapically. striae irresolvable under lm. raphe straight, filiform, with simple 232 acta bot. croat. 68 (2), 2009 enache m. d., potapova m. u:\acta botanica\acta-botan 2-09\enache.vp 9. listopad 2009 13:08:24 color profile: disabled composite 150 lpi at 45 degrees central endings, only slightly expanded, and terminal fissures curved towards secondary side of the valve. a fold can be observed at the junction of the valve face and the mantle. polar bars indistinct or very slightly developed. holotype: slide ansp gc64854, diatom herbarium, academy of natural sciences of philadelphia (ansp). isotype: slide ansp gc64855, diatom herbarium, academy of natural sciences of philadelphia (ansp). type locality: hannaberry lake, jefferson county, arkansas, mississippi alluvial plane, coordinates: 34.1177 n; 91.5542 w, lake surface sediment. etymology: specific epithet refers to the beauty of the species’ delicate and elegantly shaped frustule. sellaphora pulchra is distinguished from other naviculoid diatoms by its delicate frustule, striae irresolvable under lm (pl. 1, figs. 1–6), small size and characteristic shape. the raphe of sellaphora pulchra is difficult to observe in lm, which may lead to its misidentification as a monoraphid diatom. sem observations show that the raphe is filiform, straight, with simple, slightly expanded (figs. 8–10) or drop-like in larger specimens (figs. 7, 12) external proximal endings. the terminal raphe fissures are curved and extend to valve mantle (pl. 2, figs. 7–13). the areolae are small, poroid, arranged in uniseriate striae. at the valve apices the interstriae are occasionally wide, thus forming structures that may be interpreted as 'polar bars' (fig. 13). discussion several genera have been recently established to accommodate small naviculoid diatoms with uniseriate striae previously placed within navicula sensu lato. these genera are often based on a few morphological characters, such as the shape of the valve, raphe structure, and position of the sieve membranes in relation to the valve external or internal surface. adlafia and mayamaea, for instance, have sieve membranes positioned closer to the internal valve surface within areolae, while eolimna, fistulifera, as well as sellaphora have membranes near external valve surface (moser et al. 1998). the species discovered in acta bot. croat. 68 (2), 2009 233 a new species of sellaphora pl. 1. sellaphora pulchra sp. nov., lm. type material, hannaberry lake, arkansas. fig. 1 – holotype, ansp gc64854; figs. 2–5 – variation in size and shape. fig. 6 – girdle view. scale bar = 5 mm. u:\acta botanica\acta-botan 2-09\enache.vp 5. listopad 2009 15:13:17 color profile: disabled composite 150 lpi at 45 degrees hannaberry lake obviously does not have areolae occluded externally, and therefore cannot be placed in adlafia or mayamaea. it also cannot be placed in fistulifera, which does not possess terminal raphe fissures. the difference between eolimna and sellaphora is currently not clear. the diatom known as eolimna minima (grunow) lange-bertalot, for example, has been shown to be a sister taxon to sellaphora in a molecular phylogenetic study (evans et al. 2008). eolimna has fimbriae in the valvocopula and raphe on a raised sternum (moser et al. 1998). these features were not present in our specimens, and, therefore, we placed the new species in sellaphora. 234 acta bot. croat. 68 (2), 2009 enache m. d., potapova m. pl. 2. sellaphora pulchra sp. nov., type material, sem. figs. 7–9 – valve face in external view showing uniseriate striae, bow-tie-shaped central area, filiform raphe with simple only slightly expanded central endings, and terminal fissures curved towards secondary side of the valve. figs. 10–11 – the marginal fold is visible at the junction between valve face and mantle, arrowed. fig. 12 – external valve view and partial internal view of the opposite valve. fig. 13 – detail of valve apex with indistinct polar bar. scale bars = 2 mm. u:\acta botanica\acta-botan 2-09\enache.vp 5. listopad 2009 15:13:22 color profile: disabled composite 150 lpi at 45 degrees morphologically, the species most similar to sellaphora pulchra is in our opinion naviculadicta rionautensis rumrich et lange-bertalot (rumrich et al. 2000, pl. 70, figs. 14–18), from which s. pulchra is distinguished particularly by its much finer striae and shape. sellaphora species with fine striation, such as sellaphora wallacei (reimer) potapova et ponader (potapova and ponader 2008: 172, fig. 1) and s. rioplatensis metzeltin, lange-bertalot et garcia-rodríguez (metzeltin et al. 2005: 212, pl. 67, figs. 13–27) have already been reported from the americas. s. pulchra differs from these two species by the valve shape, bow-tie-shaped central area, and the absence of distinct polar bars. navicula impexa hustedt (hustedt 1961: 151, fig. 1282; simonsen 1987: 475, pl. 728, figs. 15–18), another small-size species with irresolvable striae, differs from s. pulchra by the absence of a bow-tie-shaped central area and by the valve shape. sellaphora pulchra displays some similarities to other small-size species of navicula sensu lato, such as n. medioconvexa hustedt (hustedt 1961: 151, fig. 1283), n. digna hustedt (simonsen 1987: 440, pl. 657, figs. 10–12), n. schmassmannii hustedt (simonsen 1987: 475, pl. 122, figs. 11–12; 174, pl. 276, figs. 22–30), n. mediopunctata hustedt (simonsen 1987: 174, pl. 276, figs. 17–21), and sellaphora hustedtii (krasske) lange-bertalot (lange-bertalot et al. 1996: 116, pl. 18, figs. 4–5). however, it can be easily distinguished from these species by its morphological features. n. medioconvexa has coarser striae (~ 30 striae/10 mm), different valve shape, broader ends, and slightly larger size (12–16 mm long and 4–4.5 mm wide). navicula digna, a species with morphological features very similar to n. medioconvexa but with finer striae (36/10 mm) differs from our new species by the shape of the valve and smaller central area. n. schmassmannii differs from s. pulchra by the lack of bow-tie-shaped central area and smaller size (6–10 mm long and 2.5–3 mm wide). s. hustedtii is larger than s. pulchra and displays coarser striae. n. mediopunctata has similar valve outline with s. pulchra, but the bow-tie-shaped central area is absent and transapical striae are parallel. sellaphora pulchra was found only in two top sediment samples from hannaberry lake (ansp slides nls00252 and nls00254). presently, hannaberry lake is a freshwater eutrophic lake, with ca concentration (1559 meq l–1) higher than the average for lakes sampled within the nla project (median = 1053 meq l–1), and with average levels of silica (tab. 1). the high productivity of the lake is indicated by the high nutrient (tab. 1) and chlorophyll a (1360 mg l–1) concentrations. acta bot. croat. 68 (2), 2009 235 a new species of sellaphora tab. 1. limnological characteristics of hannaberry lake parameter hannaberry lake ph 8.3 secchi (m) 0.5 conductivity (ms cm–1, 25 °c) 410 turbidity (ntu) 9.4 color (pcu) 10 dissoled organic carbon (mg l–1) 5.4 total phosphorus (mg l–1) 100 total nitrogen (mg l–1) 956 sio2 (mg l –1) 6.8 u:\acta botanica\acta-botan 2-09\enache.vp 5. listopad 2009 15:13:22 color profile: disabled composite 150 lpi at 45 degrees acknowledgements m. enache’s work has been supported by the epa funding for the nla project and by the phycology section at ansp directed by d. f. charles. we acknowledge the use of the centralized research facilities in the college of engineering at drexel university, philadelphia. references anonymous, 1975: proposals for a standardization of diatom terminology and diagnoses. nova hedwigia 53, 323–354. antoniades, d., hamilton p. b., douglas m. s. v., smol, j. p., 2007: diatoms of north america: the freshwater floras of prince patrick, ellef ringnes and northern ellesmere islands from the canadian arctic archipelago. iconographia diatomologica 17. koeltz scientific books. koenigstein. charles, d. f., knowles c., davis, r. s., 2002: protocols for the analysis of algal samples collected as part of the u.s. geological survey national water-quality assessment program. the academy of natural sciences, philadelphia, report no. 02–06. evans, k. m., wortley, a. h., simpson, g. e., chepurnov, v. a., mann, d. g., 2008: a molecular systematic approach to explore diversity within the sellaphora pupula species complex (bacillariophyta). journal of phycology 44, 215–231. glew, j. r., smol, j. p. last, w. m., 2001: sediment core collection and extrusion. in: last, w. m., smol, j. p. (eds), tracking environmental change using lake sediments, 1: basin analysis, coring, and chronological techniques, 73–105. kluwer academic publishers, dordrecht. hustedt, f., 1961: kryptogamen-flora von deutschland, osterreich und der schweiz 7. kieselalgen 3, 1. akademische verlagsgesellschaft. leipzig. kusber, w.-h., jahn, r., 2003: annotated list of diatom names by horst lange-bertalot and co-workers. version 3.0; http://www.algaterra.org/names_version3_0.pdf lange-bertalot, h., külbst, k., lauser, t., nörpel-schempp, m., willmann, m., 1996: diatom taxa introduced by georg krasske documentation and revision. iconographia diatomologica 3. koeltz scientific books. koenigstein. levkov, z., krstic, s., metzeltin, d., nakov, t., 2007: diatoms of lakes prespa and ochrid. about 500 taxa from ancient lake system. iconographia diatomologica 16. koeltz scientific books. koenigstein. levkov, z., krstic, s., nakov, t., 2003: diversity of diatom microflora of lake prespa-preliminary results. in melovski lj. (ed.), proceedings 2 congress of ecologists of republic of macedonia, ohrid, 443–449. mann, d. g., 1989: the diatom genus sellaphora: separation from navicula. british phycological journal 24, 1–20. mann, d. g., mcdonald, s. m., bayer, m. m., droop, s. m., chepurnov, v. a., loke, r. e., ciobanu, a., du buf, j. m. h., 2004: the sellaphora pupula species complex (bacillariophyceae): morphometric analysis, ultrastructure and mating data provide evidence for five new species. phycologia 43, 459–482. 236 acta bot. croat. 68 (2), 2009 enache m. d., potapova m. u:\acta botanica\acta-botan 2-09\enache.vp 5. listopad 2009 15:13:22 color profile: disabled composite 150 lpi at 45 degrees metzeltin, d., lange-bertalot, h., 2007: tropical diatoms of south america ii. special remarks on biogeographic disjunction. iconographia diatomologica 18. koeltz scientific books. koenigstein. metzeltin, d., lange-bertalot, h., garcía-rodríguez, f., 2005: diatoms of uruguay. compared with other taxa from south america and elsewhere. iconographia diatomologica 15. koeltz scientific books. koenigstein. moser, g., lange-bertalot, h., metzeltin, d., 1998: insel der endemiten. geobotanisches phanomen neukaledonien. bibliotheca diatomologica 38. j. cramer, berlinstuttgart. potapova m. g., ponader, k. c., 2008: new species and combinations in the diatom genus sellaphora (sellaphoraceae) from southeastern united states. harvard papers in botany 13, 171–181. ross, r., cox, e. j., karayeva, n. i., mann, d. g., paddock, t. b. b., simonsen, r., sims, p. a., 1979: an amended terminology for the siliceous components of the diatom cell. nova hedwigia 64, 513–533. rumrich, u., lange-bertalot, h., rumrich, m., 2000: diatomeen der anden. iconographia diatomologica 9. koeltz scientific books. koenigstein. simonsen, r., 1987: atlas and catalogue of the diatom types of friedrich hustedt j. cramer, berlin. acta bot. croat. 68 (2), 2009 237 a new species of sellaphora u:\acta botanica\acta-botan 2-09\enache.vp 5. listopad 2009 15:13:22 color profile: disabled composite 150 lpi at 45 degrees acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 173 acta bot. croat. 74 (1), 173-179, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication re-evaluation of the panicum capillare complex (poaceae) in croatia gergely király1*, antun alegro2 1 university of west hungary, institute of silviculture and forest protection, ady e. u. 5, h-9400 sopron, hungary 2 university of zagreb, faculty of science, department of botany and botanical garden, marulićev trg 9a, hr-10000 zagreb, croatia abstract – the panicum capillare complex includes several taxa, among them p. capillare l., which is usually considered to be an established alien throughout europe, whereas other species are recorded only as casuals. a new representative of the complex, p. riparium h. scholz was described from germany in 2002, and shortly after its description was recorded in several countries on the continent. in the course of herbarium revisions and recent fi eld studies the authors documented several localities of the species in croatia as well. the paper presents a new key for the determination of croatian species of the complex and anticipates the invasion of p. riparium in the sub-mediterranean regions of the balkan peninsula. keywords: balkan peninsula, determination key, invasive alien, neophyte, panicum capillare agg. introduction the genus panicum l. is one of the largest genera of grasses with about 300 species worldwide. the majority of species are of tropical or subtropical origin (zuloaga and soderstrom 1985, freckmann and lelong 2003, 2007, shouliang and renvoize 2006). recently, the number of species in the genus has been considerably reduced through segregation of many species into smaller genera (aliscioni et al. 2003). several species play an important role as feed and forage in different ecosystems of the world, and, under anthropogenic infl uences many species have signifi cantly expanded their distribution as agricultural weeds or ruderals (ryves et al. 1996, van de wouw et al. 2008, moravcová et al. 2010). the majority of panicum species recognized in central and southern europe belong to sect. * corresponding author, e-mail: kbgergely@gmail.com copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. király g., alegro a. 174 acta bot. croat. 74 (1), 2015 panicum l. (leaf sheaths rounded and hairy, lower glumes acute to attenuate) and sect. dichotomifl ora (hitch.) honda (leaf sheaths compressed, glabrous, lower glumes truncate to subacute) (conert 1979, clayton 1980, freckmann and lelong 2003). the panicum capillare complex (sect. panicum, »witchgrasses« native to north america) includes taxa, which were taxonomically evaluated in different ways. employing a typical central european »disaggregate approach«, scholz (2002) distinguished several taxa at species rank, whereas anglo-saxon taxonomists (»aggregate approach«) tend to reduce the number of species through assessment of taxa at the rank of subspecies or variety (clements el al. 2004, freckmann and lelong 2003, 2007). these contractions do not signify that these taxa are conspecifi c, but some authors consider the differences to be too small and the degree of variety too great to accept them as »good species«. it should be noted that the american determination keys to the discussed complex usually focus only on the length of mature spikelets and ignore other features (e.g. form of lemmas, form of panicle). among the representatives of the complex, panicum capillare l. is described as a naturalized alien throughout europe (clayton et al. 1980), whereas other species were recorded in certain countries (e.g. p. gattingeri nash and p. hillmannii chase in austria and slovenia; jogan 2007, fischer 2008) as rare casuals only. from croatia only p. capillare has been reported (domac 1994, euro+med 2006, nikolić 2014). panicum riparium h. scholz (2002: 275) was recently described as a new species from the elbe valley in germany. whereas scholz (2002) emphasized that p. riparium and p. barbipulvinatum nash are not conspecifi c, the exhaustive study of amarell (2013a, b) showed that the names are probably synonymous. although some authors (hohla 2013, verloove 2014) have already adopted the name p. barbipulvinatum for some newer records, widespread use of this name in the case of central european plants should be discouraged until type material of all related north american taxa has been investigated. herbarium revisions of the p. capillare complex have shown that p. riparium is not a new invader but an overlooked, long-established taxon in central europe, and, in some parts of the synanthropic european range, is more abundant than p. capillare s. str. (wilhalm 2011, nagy et al. 2012, amarell 2013b). according to these fi ndings, its occurrence in croatia was to be expected, which has necessitated a re-evaluation of the complex in the area. material and methods specimens in the following herbaria (acronyms given according to thiers 2014) were searched for records of the panicum capillare complex: bp, bpu, de, gjo, jpu, lju, pecs, w, za, zaho, and the private herbarium of the fi rst author. not only were the historical collections revised, but a new voucher specimen of p. riparium was placed in za. characterization of p. capillare s. str. and p. riparium was based on the revision of 20 specimens for each as a reference derived from the herbaria listed above, the keys of scholz (2002), fischer et al. (2008), király et al. (2009) and amarell (2013b) were also applied. field studies in northern croatia were conducted in 2012 and 2013. for the new locality of panicum riparium, geo-coordinates were determined using a trimble nomad gps handheld device in wgs 84 projection. quadrant numbers (»mtb«) of the central-european flora mapping system are presented after nikolić et al. (1998). the panicum capillare complex in croatia acta bot. croat. 74 (1), 2015 175 results and discussion panicum riparium is readily distinguished from p. capillare s. str. using macroscopic characters by the structure of the panicle and the form of spikelets. for further confi rmation of the determination some measurements of spikelets and fruits are useful (see the key below and fig. 1). determination key for croatian species of panicum capillare complex: 1a. pedicel of subterminal spikelet longer than 5 mm, patent (angle between subterminal and terminal pedicels 20–60°). mature spikelet 2.2–2.7× as long as broad, acute, short pointed; lemma (7–)9 veined. caryopsis 1.5–1.7× as long as broad . . . . . p. capillare s. str. 1b. pedicel of subterminal spikelet shorter than 3 mm, appressed to the branch (angle between subterminal and terminal pedicels < 10°). mature spikelet 2.7–3.4× as long as broad, acuminate with a long tapering apex; lemma 5(–7) veined. caryopsis 1.8–2.2× as long as broad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. riparium in the course of systematic revision of herbarium material of the p. capillare complex, several overlooked specimens of p. riparium, a new species for the croatian fl ora, were found in bp and za. the earliest collection is dated to the second half of the 1800s, however, its exact location and date are not given. the fi rst precisely dated specimen was collected in the early 1900s in the present-day rijeka. from the 1950s p. riparium was repeatedly collected in the vicinity of zagreb, and further fi ndings are known from the fig. 1. panicum capillare l.: (a) panicle; (b) detail of panicle; (c) mature spikelets; panicum riparium h. scholz: (d) panicle; (e) detail of panicle; (f) mature spikelets. del. j. táborská. király g., alegro a. 176 acta bot. croat. 74 (1), 2015 sub-continental (slavonski brod), sub-mediterranean (knin, rijeka) and mediterranean (vodice) parts of croatia. in the course of the fl oristic investigation of the ivanščica mountain, a recent occurrence of p. riparium was documented in 2013 (see appendix 1 and fig. 2). during the revision we recorded only fi ve croatian collections of p. capillare s. str. (see appendix 2), thus we consider that it is distinctly rare in the country. shortly after its description in germany (scholz 2002), p. riparium was reported from several countries in central europe: austria (hohla 2006), hungary (király et al. 2009), germany (amarell 2010), switzerland (ciardo et al. 2011), italy (wilhalm 2011), france and great britain (amarell 2013b), and belgium (verloove 2014), due to actual fl oristic research in the fi eld and herbarium revisions. accompanying p. capillare s. str., p. riparium was often recorded as well, thus showing that both species coexist on the continent. the relative abundance of these taxa is not yet known in the individual countries. nevertheless, p. riparium seems to have a slight sub-atlantic character with the (hitherto known) european distribution centre north of the alps. representatives of the p. capillare complex grow usually in ruderal habitats (roadsides, building sites) in europe; their occurrences in agricultural habitats are much less frequent, whereas they are serious weeds in north america, infesting predominantly corn, soybean and winter wheat cultures (darbyshire and cayouette 1995, clements et al. 2004). p. riparium was reported from hungary exceptionally as an abundant agricultural weed on acidic sandy soils (nagy et al. 2012). fig. 2. distribution of panicum riparium h. scholz in croatia. the panicum capillare complex in croatia acta bot. croat. 74 (1), 2015 177 in croatia p. capillare has been described as an invasive alien (boršić et al. 2008), and as a particularly noxious agricultural weed (hulina 2010). however, several former reports without voucher may refer to p. riparium, newly published records of which are a clear indication of a presumable invasion in the sub-mediterranean and mediterranean regions. the croatian records from knin and vodice represent the southernmost reported localities of the latter species in europe. exhaustive fi eld studies and herbarium revisions are needed to assess the actual distribution and role of p. riparium in the northern part of the balkan peninsula because of the prospect of rapid invasion in secondary habitats. acknowledgements thanks are due to uwe amarell (offenburg, germany) for discussions on the origin and distribution of panicum riparium in europe. richard lansdown (stroud, great britain) kindly improved our english. the study of gergely király was supported by project agrárklíma.2 vksz-12-1-2013-0034. references aliscioni, s., guissani, l., zuloaga, f., kellogg, e., 2003: a molecular phylogeny of panicum (poaceae: paniceae): tests of monophyly and phylogenetic placement within the panicoideae. american journal of botany 90, 796–821. amarell, u., 2010: bemerkenswerte neophytenfunde aus baden-württemberg und nachbargebieten (2004–2008). berichte der botanischen arbeitsgemeinschaft südwestdeutschlands 6, 3–21. amarell, u., 2013a: panicum barbipulvinatum (= panicum riparium) in baden-württemberg. berichte der botanischen arbeitsgemeinschaft südwestdeutschlands 7, 3–10. amarell, u., 2013b: panicum riparium h. scholz – eine neoindigene art europas? kochia 6, 1–24. boršić, i., milović, m., dujmović, i., bogdanović, s., cigić, p., rešetnik, i., nikolić, t., mitić, b., 2008: preliminary check-list of invasive alien plant species (ias) in croatia. natura croatica 17, 55–71. ciardo, f., jutzeler, s., hoffer-massard, f., bornand, c. (eds.), 2011: notes fl oristiqes vaudoises 2011. bulletin du cercle vaudois de botanique (lausanne) 40, 117–147. clayton, w. d., 1980: panicum l. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a., chater, a. o., richardson, i. b. k. (eds.), flora europaea 5, 261. cambridge university press, cambridge. clements, d. r., ditommaso, d., darbyshire, s. j., cavers, p. b., sartonov, a. d., 2004: the biology of canadian weeds. 127. panicum capillare l. canadian journal of plant science 84, 327–341. conert, h., 1979: panicum. in: conert, h., jäger, e., kadereit, j., schultze-motel, w., wagenitz, g., weber, h. (eds.), gustav hegi, illustrierte flora von mitteleuropa 1/3, 37–45. parey, berlin. darbyshire, s., cayouette, j., 1995: identifi cation of the species in the panicum capillare complex (poaceae) from eastern canada and adjacent new york state. canadian journal of botany 73, 333–348. király g., alegro a. 178 acta bot. croat. 74 (1), 2015 domac, r., 1994: small fl ora of croatia (in croatian). školska knjiga, zagreb. euro+med, 2006: euro+med plantbase – the information resource for euro-mediterranean plant diversity. retrieved january 21 2014 from http://ww2.bgbm.org/europlusmed/ fischer, m., oswald, k., adler, w., 2008: exkursionsfl ora für österreich, liechtenstein und südtirol. land oberösterreich, biologiezentrum der oberösterreichischen landesmuseen, linz. freckmann, r. w., lelong, m. g., 2003: panicum l. in: barkworth, m. e., capels, k. m., long, s., piep, m. b. (eds.), flora of north america, 25, 450–488. oxford university press, new york. freckmann, r. w., lelong, m. g., 2007: panicum l. in: barkworth, m. e., anderton, l. a., capels, k. m., long, s., piep, m. b. (eds.), manual of grasses for north america, 289–296. intermountain herbarium & utah state university press, logan, utah. hohla, m., 2006: panicum riparium (poaceae) – neu für österreich – und weitere beiträge zur kenntnis der adventivfl ora oberösterreichs. neilreichia 4, 9–44. hohla, m., 2013: eragrostis amurensis, euphorbia serpens und lepidium latifolium – neu für oberösterreich sowie weitere beiträge zur flora österreichs. stapfi a 99, 35–51. hulina, n., 2010: »planta hortifuga« in fl ora of the continental part of croatia. agriculturae conspectus scientifi cus 75, 57–65. jogan, n., 2007: poaceae. in: martinčič, a., wraber, t., ravnik, v., jogan, n., podobnik, a., turk, b., vreš, b. (eds.), small fl ora of slovenia (in slovenian), 826–932. tehniška založba slovenije, ljubljana. király, g., baranyai-nagy, a., kerekes, sz., király, a., korda, m., 2009: additions to the knowledge of the alien fl ora of hungary (in hungarian). flora pannonica 7, 3–31. moravcová, l., pyšek, p., jarošík, v., havlíčková, v., zákravský, p. 2010: reproductive characteristics of neophytes in the czech republic: traits of invasive and non-invasive species. preslia 82, 365–390. nagy, m., király, g., magyar, l., nagy, l. simon, z., 2012: distribution and threats of panicum riparium in hungary (in hungarian). agrofórum 23(5), 10–18. nikolić, t. (ed.), 2014: flora croatica database. retrieved january 21, 2014 from http:// hirc.botanic.hr/fcd nikolić, t., bukovec, d., šopf, j., jelaska, s. d., 1998: mapping the fl ora of croatia: possibilities and standards (in croatian). natura croatica 7 (suppl. 1), 1–62. ryves, t. b., clement, e. j., foster, m. c., 1996: alien grasses of the british isles. bsbi, london. scholz, h., 2002: panicum riparium h. scholz – eine neue indigene art der flora mitteleuropas. feddes repertorium 113, 273–280. shouliang, c., renvoize, s. a., 2006: panicum linnaeus. in: w. zhengyi, w., raven, p. h., deyuan, h. (eds.), flora of china 22, 504–510. science press & missouri botanical garden, beijing & st. louis. thiers, b., 2014: index herbariorum: a global directory of public herbaria and associated staff. retrieved february 10, 2014 from http://sweetgum.nybg.org/ih/ the panicum capillare complex in croatia acta bot. croat. 74 (1), 2015 179 van de wouw, m., jorge, m. a., bierwirth, j. hanson, j., 2008: characterization of a collection of perennial panicum species. tropical grasslands 42, 40–53. verloove, f., 2014: manual of the alien plants of belgium. panicum capillare. retrieved january 21, 2014 from http://alienplantsbelgium.be wilhalm, t., 2011: ergänzungen und korrekturen zum katalog der gefäßpfl anzen (4). gredleriana 11, 71–82. zuloaga, f., soderstrom, t. r., 1985: classifi cation of the outlying species of new world panicum (poaceae: paniceae). smithsonian contributions to botany 59, 1–63. király g., alegro a. 180 acta bot. croat. 74 (1), 2015 appendix 1. recorded localities of panicum riparium h. scholz in croatia. »in agris«; c. j. schlosser (as »p. capillare«), n. d., za16220; fiume (today rijeka), »in ruderatis viae ferrae« (probably mtb 0652.4), g. lengyel (as »p. capillare«), viii. 1908, bp356966; kamena gorica, n of the settlement, n46.162662°; e16.256507° (mtb 9863.2), g. király & d. schmidt, 07. ix. 2013, za; knin, railway station (mtb 1963.3), m. milović (as »p. capillare«), 12. xi. 2000, za16208; slavonski brod, poloj, bank of drava river (mtb 0874.3), l. marković (as »p. capillare«), 08. ix. 1969, za16214; stubičke toplice (mtb 0061.2), n. fiket (as »p. capillare«), 29. viii. 1955, za16218; vodice (mtb 2260.2), m. milović (as »p. dichotomifl orum«), 10. viii. 1999, za16210; zagreb, »garden« (mtb?), i. horvat (as »p. capillare«), 18. ix. 1948, zaho; zagreb, borovje (mtb 0161.4), l. marković, l. gospodarić (as »p. capillare«), 09. ix. 1954, za16211; zagreb, lanište (mtb 0261.2), a. kumbarić (as »p. capillare«), 25. viii. 1992, za16216; zagreb, savski most (mtb 0261.2), l. marković (as »p. capillare«), 26. viii. 1959, za16217; zagreb, w of »savski most« (mtb 0261.2), l. marković (as »p. capillare«), 03. x. 1969, za16215; zagreb, sesvete (mtb 0162.4), b. hundozi (as »p. capillare«), 04. ix. 1964, za16214. appendix 2. herbarium specimens of panicum capillare l. found in croatian herbaria. gunja, near the railway bride on sava (mtb 1179.1), l. marković, 10. ix. 1969, za16213; medvednica, adolfovac, forest cutting (mtb 0161.2), l. marković, 08. ix. 1974, za11846; negoslavci, on row crops (probably mtb 0779.2), m. jakovlević, 15. viii. 1956, za16219; ozalj, on row crops sw of railway station (mtb 0358.4), l. ilijanić (as »p. dichotomifl orum« but fragments of the collection belong to p. capillare), 12. xi. 1986, za; trakošćan, on row crops (mtb 9761.2), s. kečkeš (as »p. miliaceum«), 13. viii. 1956, za16207. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 245 acta bot. croat. 74 (2), 245–252, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0020 a new european record of diadesmis fukushimae and its transference to humidophila genus (bacillariophyta) csilla kövér1, jános korponai1, sándor harangi2, krisztina buczkó3* 1 university of west hungary, faculty of natural science, károlyi gáspár tér 4. h-9700 szombathely, hungary (kover.csilla@ttk.nyme.hu) 2 university of debrecen, faculty of natural science, egyetem tér 1. h-4032 debrecen, hungary 3 department of botany, hungarian natural history museum, könyves kálmán krt. 40. h-1476 budapest, hungary abstract – diadesmis fukushimae, a rare oligotraphenic diatom, was found in some high mountain lakes of romania. its occurrence in the parâng and retezat mountains is the second european record of the species. to date d. fukushimae has been known only from the type locality (shenandoah national park, virginia, usa) and from a spring (grotta guernica, dolomiti bellunesi national park (south-eastern alps, italy). investigation by scanning electron microscopy showed that this species should be transferred to the recently established genus humidophila. a new combination is proposed, humidophila fukushimae. the morphological details of the european population are also presented. keywords: diadesmis, diatoms, humidophila, mountain lakes, oligotrophy, taxonomy introduction during studies on diatom assemblages in high mountain lakes a characteristic species was recently recorded. the heavily silicifi ed diatom at fi rst glance resembles representatives of the genus diadesmis (grunow 1860: 552), but was not identical to the known and illustrated european taxa (e.g., krammer and lange-bertalot 1991, lange-bertalot and metzeltin 1996). after detailed investigation we identifi ed it as diadesmis fukushimae langebertalot, m. werum & broszinski (werum and lange-bertalot 2004). the only previous european record of this diatom has been from the grotta guernica spring (dolomiti bellunesi national park, south-eastern alps, italy) as reported by cantonati and spitale (2009). * corresponding author, e-mail: krisztina@buczko.eu kövér c., korponai j., harangi s., buczkó k. 246 acta bot. croat. 74 (2), 2015 recently, lowe et al. (2014) published a study on the taxa and populations of the genus diadesmis, subgenus paradiadesmis, from the hawaiian islands. on the basis of the structure of valves, they concluded that most of the known diadesmis taxa considerably differ from diadesmis confervacea kutzing (1844: 109), which is the type species of the genus diadesmis. the establishment of a new genus, humidophila lowe, kociolek, johansen, van de vijver, lange-bertalot & kopalová 2014, was necessary and was accomplished by splitting off the genus humidophila from the genus diadesmis (kützing 1844) by lowe et al. (2014). a total of 47 former diadesmis species were transferred to humidophila, but diadesmis fukushimae was not mentioned in this compilation. a species of genus diadesmis is fairly easily distinguished from other naviculoid diatoms by the combination of striae and raphe structure, and valve shape (round et al. 1990). according to the defi nition of the genus humidophila (lowe et al. 2014), the species belonging to the new genus are linear, linear-elliptical to elliptical. the valve face is fl at; a narrow ridge can be seen between the valve face and mantle. striae on the valve face are composed of one elliptical to ovoid areola. on the valve mantle, one single, elongated areola opening is in line with each stria. the raphe is fi liform, straight with variable external proximal raphe endings but never defl ected, bent or hooked. distal external raphe endings never extend onto the mantle. internally, areolae openings are covered by a porous hymen. the central nodule is small and rounded. proximal raphe endings are straight or slightly anchor-shaped; distal raphe endings terminate in small helictoglossae. in algaebase there are 65 records of diadesmis taxa listed (guiry and guiry 2014), and 75 species names are found in the california academy of sciences catalogue of diatom names (fourtanier and kociolek 2014), which speaks to the richness of this group. material and methods a total of 40 high mountain lakes, situated in three characteristic mountain regions of romania: the făgăraş, parâng and retezat mountains were sampled in 2012, 2013 and 2014. the results of the sampling are under preparation and investigation. the diatom assemblage analysis is complete but the results have not been published. for analyses of the siliceous algae, samples were prepared using standard digestion procedures (battarbee 1986). aliquot-evaporated suspensions were embedded in zrax and pleurax. approximately 400 valves were counted from each sample using a light microscope (leica dm lb2 with 100 hcx plan apo objective and vsi–3.om(h) digital camera). for scanning electron microscope analysis (sem), cleaned samples were air-dried on an aluminium stub. coating with gold-palladium was accomplished using an xc7620 mini sputter coater for 120 s at 16 ma. a hitachi s–2600n scanning electron microscope operated at 20 kv and 5–8 mm distance was used. morphological terminology follows barber and haworth (1981) and round et al. (1990). valve measurements were made from digital images using the camera software. the following samples were used for documentation in this paper: – bp 2013/152; lake peleaga, sample from the deepest part of the lake, at 4.1 m water depth, collected on 21st july 2013. – bp 2013/160; lake negru, the deepest part of the lake, at 27.2 m water depth, collected on 25th july 2013. humidophila fukushimae in europe acta bot. croat. 74 (2), 2015 247 results diadesmis fukushimae was found in three lakes in the romanian carpathians, one in the parâng mountains and two in the retezat mountains. the localities and some chemical parameters of the lakes where d. fukushimae was detected are shown in tab. 1. the relative abundance of it was low in all the samples. it was most numerous in lake negru, although its relative abundance remained below 3% in this lake. on the basis of the description of humidophila we should transfer diadesmis fukushimae into this newly defi ned genus. observation humidophila fukushimae (lange-bertalot, m. werum et broszinski) buczkó and kövér comb. nov. figs. 1a–n, figs. 2a–d, figs. 3a–d. tab. 1. locations and some chemical parameters of the lakes where humidophila fukushimae was found. lake mountain region altitude (m) a.s.l. n e ph water depth (m) conductivity (μs cm–1) rosiile parâng 1978 45°20’41,3” 23°33’15,8” 8.54 17.2 12.5 negru retezat 2036 45°21’33,2” 22°49’36,6” 7.71 27.7 15.0 peleaga retezat 2122 45°21’51,9” 22°54’07,6” 7.51 4.1 13.0 fig. 1. humidophila fukushimae: lm micrographs of population on lake negru (a–m), valve view (a–l); scanning electron micrograph (sem), entire frustule in girdle view in oblique position (n). scale bar represents 10 μm except for n where scale bar = 5 μm. kövér c., korponai j., harangi s., buczkó k. 248 acta bot. croat. 74 (2), 2015 basionym. diadesmis fukushimae lange-bert, m. werum et broszinski 2001 diatom 17, pp 6–9, pl. 1, figs. 33–38, pl. 2, figs. 47–53, pl. 3. 54–57. morphological observations light microscopy (figs. 1a–m) valves are linear-elliptical to elliptical-lanceolate, ends not protracted (figs.1a–l). frustules in girdle view are broadly rectangular (fig. 1m). valve dimensions (n = 20): length 14–21.7 μm, (x = 17.4 ± 2.2), width 4.5–6 μm (x = 5 ± 0.4), frustule’s width more than 4 μm (fig. 1m). raphe is fi liform, straight, with distinct central pores. axial area is broad, but strongly narrowed and lanceolate to the ends. transapical striae are very short, and diffi cult to resolve in lm. fig. 2. humidophila fukushimae: scanning electron micrograph (sem), external valve view; valve face and open bands (a); details of the central area with central pores that are entirely fl anked by depressions (b), note relief–like patches in the area between raphe and areolae; details of apices with the simple raphe ends (c), note ridge of silica at the junction of valve face and mantle; entire frustule (d). scale bars represent 5 μm for a and d, and for b and c scale bars = 2 μm. humidophila fukushimae in europe acta bot. croat. 74 (2), 2015 249 scanning electron microscopy (fig. 1n, figs. 2a–d, figs. 3a–d) valves are fl at (fig. 1n, figs. 2a–d). junction of the valve face and mantle bears a ridge of silica (fig. 1n, figs. 2b–c). striae are uniseriate, containing round poroids (fig. 1n, fig. 2a–d), the number of areolae is 38–40 in 10 μm. the central pores are fl anked by short but comparatively deep depressions (figs. 2a,b,d). the hymens lie on the inner surface of the transapical ribs making the valve interior appear somewhat featureless (figs. 3a,b; round at al. 1990). sometimes there are polar excavations on the sternum in the inside view (fig. 3b). axial area is broad (fig. 1n, figs. 2a–d). distal raphe endings are fig. 3. humidophila fukushimae (a–d) and humidophila perpusilla (e–g): scanning electron micrograph (sem), internal valve view (a–d); internal overview of an entire valve (a–b), note the not quite t–shaped central raphe endings; the hymens lie on the inner surface of the transapical ribs, partly corroded (b), note the polar excavations (marked by arrow); details of apices with simply raphe endings and intercalated shorter striae (c); details of central part (d); external girdle view (e); valves of an entire frustulum with inside and outside view (f), note the mantle areolae located on the valve face (lowe et al. 2014); external view of a valve (g). scale bars represent 5 μm except for d where scale bar = 2 μm. kövér c., korponai j., harangi s., buczkó k. 250 acta bot. croat. 74 (2), 2015 simple (fig. 3c), proximal raphe endings not quite t-shaped (fig. 3d). the cingulum consists of a few to many open bands (two bands on fig. 2d, four bands on fig. 1n), with one transverse row of round poroids (fig. 1n, figs. 2a–d). irregular relief-like patches in the area between raphe and areolae can be observed (fig. 1n, figs. 2b–d) but no such structures are seen on fig. 2a. intercalated shorter striae are in the apices (fig. 3c). taxonomical remarks: the valves of the carpathian population are markedly smaller (14–21 μm) than is indicated in the description (18–25 μm). the width of the valves – a more stable feature of a species – is 4.5–6 μm (in the type population the width is 5–6 μm). the number of striae is higher (38–40/10 μm) than the 34–36 /10 μm in the holotype population (werum and lange-bertalot 2000). another difference, of deep polar excavations on either side of the sternum in the inside view, as documented by lange-bertalot and werum (2001) and werum and lange-bertalot (2004), can be observed only on a few valves; the majority of our population is without such a structure. humidophila fukushimae is similar in outlines to the most common h. perpusilla (grunow 1860: 552) (lowe et al. 2014). however h. perpusilla (figs. 3e–g) is signifi cantly smaller. humidophila perpusilla co-occurred with h. fukushimae in lake negru. distribution: humidophila fukushimae was originally described in shenandoah national park near »hogback overlook«, virginia, usa. it was collected from a freshwater spring trickling over rocks (leg. anja broszinski, november 1999), and was dedicated to hiroshi fukushima on the occasion of his 77th birthday (lange-bertalot and werum 2001) in the journal diatom. three years later the description with the same photographic documentation was published (werum and lange-bertalot 2004) on page 136, plate 62 figs. 33–38, plate 63 figs. 1–7, and plate 64 figs. 1–4. recently cantonati and spitale (2009) published a record away from its type locality, in europe. they reported h. fukushimae from spring grotta guernica, where this diatom also had very low relative abundance (0.2%), as in our samples. discussion it is noteworthy that the representatives of genus humidophila are known from aerial/ subaerial habitats and most of the species were collected from springs and/or wet surfaces. we have found h. fukushimae in mountain lakes, furthermore from the deepest part of the lakes (water depth varied between 4.1 and 27.7 meter). it was the most frequent in a deep lake, lake negru. on the other hand, the sample from the lake bottom was rich in silt, perhaps as a result of erosion from the lakeshore, which could be an explanation for the unusual habitat. the obtained results confi rm the presence of a typical and highly specifi c limnoterrestrial diatom fl ora in the high mountain lakes of the carpathians. the taxonomic revision of the diatom assemblages of poorly studied regions, such as antarctica (e.g. van de vijver et al. 2011) or lake baikal (kulikovskiy et al. 2012), revealed a biogeographic pattern of several diatoms; they are not »cosmopolitan« with worldwide distribution, as has been generally accepted for a long time. our fi nding somewhat contradicts the previous idea, as we found a species whose distribution to date was believed to be restricted to the american continent. humidophila fukushimae appears to be a peculiar species with regard to its valve outline and dimensions, and its valve face structure, which enabled us to identify it easily. it humidophila fukushimae in europe acta bot. croat. 74 (2), 2015 251 can be considered a rare diatom species, and its discovery in the carpathians draws attention to how restricted our knowledge of diatom distribution is, even in a well-investigated area like europe. this new record also highlights the diatom species richness of high mountain regions and their importance for diatom biodiversity conservation. acknowledgements we are very grateful to rex lowe for sharing his personal/unpublished data on diadesmis/humidophila taxa. we thank the hungarian scientifi c fund for fi nancial support (otka 83999). references barber, h. g., haworth, e. y., 1981: a guide to the morphology of the diatom frustule with a key to the british freshwater genera. freshwater biological association scientific publication 44, 1–112. battarbee, r. w., 1986: diatom analysis. in: berglund, b. e. (ed.), handbook of holocene palaeoecology and palaeohydrology. john wiley & sons, chichester, new york, brisbane, toronto, singapore, 527–570. cantonati, m., spitale, d., 2009: the role of environmental variables in structuring epiphytic and epilithic diatom assemblages in springs and streams of the dolomiti bellunesi national park (south-eastern alps). fundamental and applied limnology 174, 117–133. fourtanier, e., kociolek, j. p., 2014: catalogue of diatom names, california academy of sciences. retrieved september 15, 2014 from http://research.calacademy.org/research/ diatoms/names/index.asp grunow, a., 1860: über neue oder ungenügend gekannte algen. erste folge, diatomeen, familie naviculaceen. verhandlungen der kaiserlich-königlichen zoologisch-botanischen gesellschaft in wein 10: 503–582, pls iii–vii. guiry, m. d., guiry, g. m., 2014: algaebase. world-wide electronic publication, national university of ireland, galway. retrieved march 21, 2014 from http://www.algaebase. org. krammer, k., lange-bertalot, h., 1986: bacillariophyceae. 1. teil: naviculaceae. in: ettl, h., gärtner, g., gerloff, j ., heynig, h., mollenhauer, d. (eds.), süsswasserfl ora von mitteleuropa, bd 2/1, 1–876. gustav fischer verlag: stuttgart, jena. kulikovskiy, m. s., lange-bertalot, h., metzeltin, d., witkowski, a., 2012: lake baikal: hotspot of endemic diatoms i. in: lange-bertalot, h. (ed.), iconographia diatomologica 23, 7–607. koeltz scientifi c books. koenigstein. kützing, f. t., 1844: die kieselschaligen. bacillarien oder diatomeen. nordhausen. annals and magazine of natural history 15, 1–152. lange-bertalot, h., metzeltin, d., 1996: oligotrophie-indikatoren. 800 taxa in drei ökologisch diversen seen-typen. in: lange-bertalot, h. (ed.), iconographia diatomologica. annotated diatom micrographs 2, 1–390. koeltz scientifi c books, koenigstein. kövér c., korponai j., harangi s., buczkó k. 252 acta bot. croat. 74 (2), 2015 lange-bertalot, h., werum, m., 2001: diadesmis fukushimae sp. nov. and some new or rarely observed taxa of the subgenus paradiadesmis lange-bertalot and le cohu. diatom 17, 3–19. lowe, r. l., kociolek, p., johansen, j. r., van de vijver, b., lange-bertalot, h., kopalová, k., 2014: humidophila gen. nov., a new genus for a group of diatoms (bacillariophyta) formerly within the genus diadesmis: species from hawai’i, including one new species. diatom research 29, 351–360. round, f. e., crawford, r. m., mann, d. g., 1990: the diatoms: biology and morphology of the genera. cambridge university press, cambridge. van de vijver, b., zidarova, r., sterken, m., verleyen, e., de haan, m., vyverman, w., hinz, f., sabbe, k., 2011: revision of the genus navicula s.s. (bacillariophyceae) in inland water of the sub-antarctic and antarctic with the description of 5 new species. phycologia 50, 281–297. werum, m., lange-bertalot, h., 2004: diatoms in springs from central europe and elsewhere under the infl uence of hydrologeology and anthropogenic impacts. in: langebertalot h. (ed.), iconographia diatomologica. annotated diatom micrographs 13, 1–417. a. r. g. gantner verlag k. g., ruggell. acta bot. croat. 77 (1), 2018 97 acta bot. croat. 77 (1), 97–101, 2018 coden: abcra 25 doi: 10.1515/botcro-2017-0022 issn 0365-0588 eissn 1847-8476 short communication identification and expression profiling of flax (linum usitatissimum l.) polyamine oxidase genes in response to stimuli seung hee eom1, jae kook lee1, dong-ho kim2, heekyu kim3, keum-il jang2, hojin ryu4, tae kyung hyun1 1 department of industrial plant science and technology, college of agricultural, life and environmental sciences, chungbuk national university, cheongju 28644, republic of korea 2 department of food science and biotechnology, college of agricultural, life and environmental sciences, chungbuk national university, cheongju 28644, republic of korea 3 nature environment research park of gangwon province, hongcheon 250-884, republic of korea 4 department of biology, college of natural science, chungbuk national university, cheongju 28644, republic of korea abstract – polyamine oxidases (paos) are known to be involved in either the terminal catabolism or the back conv– ersion of polyamines, which affect a range of physiological processes, including growth, development, and stress responses. in this study, based on genome-wide analysis, we identified five putative pao genes (lupao1 to lupao5) in flax (linum usitatissimum l.) that contain the amino-oxidase domain and fad-binding-domain. the expression analysis using quantitative real-time pcr revealed spatial variations in the expression of lupaos in different organs. in addition, the expression level of lupaos in the flax cell suspension culture was increased by treatment with methyl-jasmonate (meja) or pectin, but not with salicylic acid or chitosan. this indicates that lupaos might be involved in the meja-mediated biological activities. taken together, our genome-wide analysis of pao genes and expression profiling of these genes provide the first step toward the functional dissection of lupaos. keywords: cell suspension culture, flax, methyl-jasmonate, pectin, polyamine oxidase * corresponding author, e-mail: taekyung7708@chungbuk.ac.kr introduction polyamines (pas), including spermine (spm), spermidine (spd), and putrescine (put), are low-molecular-mass aliphatic polycations that are ubiquitously distributed in organisms. due to the cationic nature of pas, they bind to macromolecules, such as dna, rna, and proteins, through electrostatic linkages that can cause either stabilization or destabilization (kusano et al. 2008). thus, they have been implicated in a range of fundamental cellular processes, including the regulation of gene expression, translation, cell proliferation, cell growth, differentiation, modulation of cell signaling, membrane stabilization, and modulation of ion-channel function and stability (kusano et al. 2008, jiménez-bremont et al. 2014, minocha et al. 2014, tiburcio et al. 2014). endogenous pa contents depend upon the regulation of biosynthesis, transport, and catabolism in both prokaryotes and eukaryotes, including plants (kusano et al. 2008, takahashi et al. 2010). pas are oxidatively deaminated by two types of amine oxidases: copper-containing amine oxidases (cuaos, ec 1.4.3.6) and fad-dependent polyamine oxidases (paos, ec 1.5.3.6) (cona et al. 2006). the extracellular paos, such as the paos from monocotyledonous plants oxidize the carbon on the endo-side of the n4-nitrogen of spd and spm to produce 4-aminobutanal and n-(3-aminopropyl)-4-aminobutanal, respectively, along with 1,3-diaminopropane and h2o2, and are thus considered involved in the terminal catabolism of pas. differently, intracellular (cytosolic and peroxisomal) paos oxidize the carbon at the exo-side of the n4-nitrogen of spd and spm with the production of spd from spm and put from spd, 3-aminopropanal, and h2o2, and are considered involved in a polyamine back-conversion pathway (planas-portell et al. 2013, ahou et al. 2014). although it has been explained that paos in monocotyledonous plants are involved in the terminal catabolism of pas, four rice paos were found to mailto:taekyung7708@chungbuk.ac.kr eom s. h., lee j. k., kim d.-h., kim h., jang k.-i., ryu h., hyun t. k. 98 acta bot. croat. 77 (1), 2018 be involved in the pa back-conversion pathway (ono et al. 2012). this suggests that the pa back-conversion pathway also exists in monocotyledonous plants. plant paos have been suggested to play an important role in physiological processes, including growth, development, and responses to abiotic and biotic stresses (angelini et al. 2010). the physiological role of pao proteins is mediated by the regulation of cellular pa levels, but also by h2o2 (an important signaling molecule in the promotion of plant cell death and biotic or abiotic stress response) synthesis via the terminal catabolism and back conversion of pas (minocha et al. 2014, tiburcio et al. 2014). although accumulating evidence has shown that paos play roles in modulating a range of physiological processes, most pao family members in higher plants, except rice and arabidopsis paos, are poorly understood. therefore, in this study, we identified genes potentially encoding paos in flax (linum usitatissimum l.), which is a medicinally important oil seed crop. based on in-silico analysis, gene structures, sequence homology, intron phase, and cis-elements in the promoter regions of five pao genes were investigated. in addition, the expression patterns of flax paos in elicitor-treated flax cell suspensions were examined. our systematic analysis provides new insights into the understanding of the potential roles of flax paos in response to stimuli. materials and methods identification and sequence analysis of lupao genes and promoters protein sequences of arabidopsis and rice paos were used as queries in a search against the flax genome sequence (phytozome v9.1; http://www.phytozome.net /search. php?method=org_lusitatissimum). the information on lupao gene features, including introns and exons, was obtained from phytozome v9.1. the intron phases of different introns were analyzed using wise 2.0 (http://www.ebi.ac.uk/ tools/wise2). in addition, the molecular weight (mw) and the theoretical isoelectric point (pi) were calculated using the compute pi/mw tool available on the expert protein analysis system site (http://web.expasy.org/compute_pi/), and the amino acid sequences of putative lupao were analyzed to predict subcellular localization using hybridgoloc web services (http://bioinfo.eie.polyu.edu.hk/hybridgoserver/) and wolf psort (https://wolfpsort.hgc. jp/). the program meme (http://meme.sdsc.edu/meme4_6_1/ cgi-bin/meme.cgi) was used for the recognition of motifs in lupaos. the phylogenetic analysis was performed with the use of the phylogeny.fr server (http://www.phylogeny.fr) in the “one-click” mode, as described by hyun et al. (2014). for the cis-element analysis, all 1000-bp upstream sequences of lupao genes, except lupao1 (437-bp upstream), were compared with known cis-regulatory elements in the collection of the place database (http://www.dna.affrc.go.jp/place/). plant growth flax seeds (golden variety) were obtained from danong co. ltd in south korea. the seeds were germinated and grown in soil at 22 °c±2 °c /16±2 °c, at a light intensity of 180 μmol m–2 s–1 and a 16-h-light/8-h-dark cycle. the seeds, cotyledons and young leaves were harvested for tissue specific pao gene expression analysis. cell culture treatment the cell suspension culture of flax (golden variety), described previously by hano et al. (2006), was used as the experimental system. for the stress treatment, cell suspension cultures were sub-cultured every two weeks and incubated on a rotary shaker set to 120 rpm in darkness at 25 °c. for elicitor treatment, suspension-cultured cells were treated with 50 µm methyl-jasmonate (meja), 1 mm salicylic acid (sa), 50 mg l–1 chitosan, or 50 mg l–1 pectin. the cells were harvested at different time points (5 h, 24 h, and 48 h after treatment) by centrifugation and stored at –80 °c until analysis. quantitative real-time pcr analysis total rna was extracted using the favorprep plant total rna purification mini kit (favorgen, ping-tung, taiwan) according to the manufacturer’s instructions and was reverse-transcribed into cdna using the quantitect® reverse transcription kit (qiagen) in accordance with the manufacturer’s recommendations. quantitative real-time pcr (qrt-pcr) was performed using the ampigene qpcr green mix (enzo life sciences inc., lausen, switzerland) in the ecotm real-time pcr system (illumina) with default parameters. the expression levels of different genes were normalized to the constitutive expression level of flax actin. specific primer pairs are listed in on-line suppl. tab. 1. tab. 1. gene catalog and nomenclature of polyamine oxidases (paos) in linum usitatissimum. the subcellular locations of polyamine oxidases were predicted by hybridgo-loc web services (a) and wolf psort (b). name gene id location cds (bp) aa intron nr. pi kda subcellular localization lupao1 lus10020726 scaffold 303:384767 – 390953 1473 490 9 5.43 54.27 peroxisome a lupao2 lus10005021 scaffold 637:175302 – 177892 1395 464 9 5.67 50.82 peroxisome a lupao3 lus10039599 scaffold 15:686508 – 691160 1491 496 9 4.95 55.52 plastid a lupao4 lus10029495 scaffold 55:372940 – 377831 1419 472 9 4.75 52.29 extracellular b lupao5 lus10019725 scaffold 420:540284 – 543964 1446 481 6 6.78 53.69 extracellular b http://www.phytozome.net https://wolfpsort.hgc polyamine oxidase genes in flax acta bot. croat. 77 (1), 2018 99 determination of pao activity pao activity was determined according to han et al. (2014) with slight modifications. soluble proteins were extracted by grinding cultured cells in 0.1 m sodium phosphate buffer (ph 6.5). after centrifugation (10 min, 10,000 g) at 4°c, the supernatant was used in the assays. reaction solutions (1.5 ml) contained 0.9 ml of 0.1 m sodium phosphate buffer (ph 6.5), 0.45 ml of crude enzyme extracts, 0.05 ml of peroxidase (200 u ml–1), 0.1 ml of 4-aminoantipyrine and n, n’-dimethylaniline solution. the reaction was initiated by the addition of 7.5 μl spd (20 mm) for the determination of pao activity. the reaction mixture was incubated at 25 °c for 20 min, and then terminated with the addition of 0.25 ml of 10% trichloroacetic acid. a 0.001 change in the absorbance value at 550 nm was regarded as one enzyme activity unit. protein concentration was determined according to the method described by bradford (1976) with bovine serum albumin as the standard. statistical analysis statistical differences were analyzed using anova based on duncan's multiple range tests (p < 0.05). all experiments were repeated at least three times, and all data were expressed as means ± standard error. results and discussion the availability of the flax genome sequence (phytozome v9.1) has made it possible to identify the putative pao family in this plant species for the first time. in order to identify pao genes, sequences of paos from arabidopsis and rice were analyzed using blastp against all scaffold sequences of flax. the redundant sequences were removed according to the self-blast of sequences, resulting in a total of five putative pao genes from flax (tab. 1). then, to further support the hypothesis that the five computationally predicted lupao proteins belong to the pao family, the presence of the amino-oxidase domain (pf01593) and fad-bindingdomain, which are conserved in paos (sebela et al. 2001, gaweska and fitzpatrick 2011), was analyzed using smart (http://smart.embl-heidelberg.de/) and pfam. based on the phylogenetic analysis of the pao proteins from different plants, the paos were classified into four major classes (i, ii, iii, and iv). class i contained lupao1 and lupao2, whereas lupao3 and lupao4 were clustered into class iii. in addition, lupao5 belonged to class iv (online suppl. fig. 1).it was not clear whether flax lacked class ii of the pao family or whether class ii of lupaos might be not sequenced. multiple-sequence alignments of putative lupaos showed that two paos (lupao 1 and lupao2) contained a putative peroxisomal targeting signal (on-line suppl. fig. 2), which was defined as a tripeptide of the c-terminus ([sa] [rk][lm]) (reumann 2004). in addition, these lupao proteins were predicted to be peroxisomal proteins, whereas lupao 4 and 5 localize to the extracellular (table 1). arabidopsis (atpao2, atpao3 and atpao4) and rice (ospao3, ospao4, and ospao5) polyamine oxidases, clustered into class i (on-line suppl. fig. 1), are known as peroxisomal proteins like lupao1 and lupao2 (ono et al. 2012; planasportell et al. 2013). this indicates that class i paos are peroxisomal proteins (on-line suppl. fig. 1) and are involved in a pa back-conversion pathway. furthermore, lupao3 was predicted as a plastid-associated pao (tab. 1). the occurrence of pas at all stage of plastid development suggested that pas serve as a nitrogen source for proteins and chlorophyll synthesis, which play a role in plastid differentiation (sobieszczuk-nowicka and legocka 2014). pa content depends not only on biosynthesis, but also on the catabolism (sobieszczuk-nowicka and legocka 2014), suggesting that plastid-associated paos including lupao3 should be involved in the plastid differentiation via controlling pa catabolism. conserved gene structures, including the same number of nucleotides in the exons and the conserved intron phases, indicate the similarities between the studied genes (von schantz et al. 2006). as shown in fig. 1, lupao1 and 2 shared eight exons, with the same number of nucleotides and the same intron phase, whereas lupao3 and 4 shared six exons. in addition, we used the meme program to identify the conserved motifs in lupaos. as shown in on-line suppl. fig. 3, we found a total of three conserved motifs with low e values. three motifs were shared by lupao3, lupao4, and lupao5 proteins, and motif 3 was not found in lupao1 and lupao2. these differences represent the evolutionary and functional relationship between lupaos. to investigate the spatial organization of transcripts for lupaos, the expression patterns of lupao genes in differfig. 1. phylogenetic analysis and intron-exon structures of pao gene family in flax. default values were used except for 100 bootstraps. numbers in boxes are nucleotide length of each exon, and the connecting thin boxes indicate the positions of the introns. the numbers above the introns indicate the phase of the intron. eom s. h., lee j. k., kim d.-h., kim h., jang k.-i., ryu h., hyun t. k. 100 acta bot. croat. 77 (1), 2018 ent tissues and cultured cells were analyzed by qrt-pcr. as shown in fig. 2, the transcription levels of all lupaos, except lupao3, were detected in all the tested tissues with high expression level compared to the cultured cells. plant paos have been reported to be involved in plant responses to abiotic and biotic stresses (angelini et al. 2010). therefore, we analyzed the expression patterns of lupaos in response to external stimuli by subjecting suspension flax cell cultures to different treatments, including meja, sa, chitosan, and pectin. when flax-cultured cells were treated with meja or pectin, increased expression levels of all lupaos were observed, whereas the expression of no lupaos was significantly affected by sa and chitosan treatments (fig. 3a). in addition, lupaos exhibited different expression patterns during response to meja or pectin, indicating the divergent functions of lupaos in response to stimuli. although copper amine oxidases are also able to oxidize put and spd, with the subsequent release of h2o2 (planas-portell et al. 2013), the increased transcription level of lupaos by meja or pectin treatment resulted in the induction of enzymatic activity for oxidizing spd (fig. 3b). in addition, the enzymatic activity was not changed by treatment with sa or chitosan (fig. 3b). however, the cis-elements like t/gboxatpin2 for jasmonate signaling were not found in lupaos (on-line suppl. fig. 4), indicating the presence of a novel jasmonate-responsive element in the lupao promoters. pa accumulation depends on de novo synthesis and catabolism under stress conditions (kusano et al. 2008, takahashi et al. 2010), suggesting that the expression of paos under stress conditions is required for the induction of a pa-mediated response. in fact, stress-induced pao expressions have been observed in higher plants (planas-portell et al. 2013, wang and liu 2015). in addition, several stress-responsive elements were found in the lupao gene promoters, including the w box (wboxntchn48), elrecorepcrp1 motif (elicitor responsive element), myb1at (dehydration-responsive), gt1consensus (consensus gt-1 binding site in many light-regulated genes), and bihd1os (bell homeodomain transcription factor in disease resistance responses) (on-line suppl. fig. 4). the presence of the aforementioned putative cis-elements in lupao promoters indicates the contribution of pao to stress defense responses. in conclusion, based on genome-wide analysis, we identified five flax pao genes, which belong to three groups. plant paos are known to be responsible for either the terminal catabolism or the back conversion of pas. therefore, a further motivating challenge would be to investigate the fig. 2. tissue-specific expression of linum usitatissimum polyamine oxidase (lupao) genes. the expression levels for each gene in different tissue samples were calculated relative to its expression in the cultured cells. the y-axis represents the normalized relative expression values (log2). data represent the means ± se of three independent experiments. values with different superscript letters are significantly different (p < 0.05). n.d = not detected. fig. 3. effects of elicitation on the expression of linum usitatissimum polyamine oxidase (lupao) genes and enzymatic activity in suspension-cultured cells. (a) the expression analysis of lupao genes. transcript levels of lupao1-5 were normalized to the constitutive expression level of flax actin, and were expressed relative to the values at 0 hour. the y-axis represents the normalized relative expression values (log2). 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altabella, t., bitrián, m., alcázar, r., 2014: the roles of polyamines during the lifespan of plants: from development to stress. planta 240, 1–18. von schantz, m., jenkins, a., archer, s. n., 2006: evolutionary history of the vertebrate period genes. journal of molecular evolution 62, 701–707. wang, w., liu, j. h., 2015: genome-wide identification and expression analysis of the polyamine oxidase gene family in sweet orange (citrus sinensis). gene 555, 421–429. specific roles of each lupao in metabolism. an in-depth analysis of lupao gene expression patterns under different stress conditions suggested that lupao should be involved in the meja-mediated biological activities. taken together, our genome-wide analysis and expression analysis provide a solid foundation for developing further understanding of the potential function of paos. acknowledgements this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education (nrf2015r1a4a1041869) untitled 144 acta bot. croat. 75 (1), 2016 acta bot. croat. 75 (1), 144–148, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0019 issn 0365-0588 eissn 1847-8476 a comparison of the infl uences of fl otation and wet sieving on certain carbonized legume and cereal remains sara mareković*, renata šoštarić department of biology, faculty of science, university of zagreb, marulićev trg 20/2, hr-10000 zagreb, croatia abstract – in order to determine the infl uence of recovery techniques with water (fl otation and wet sieving) on carbonized plant remains, a certain amount of wheat, barley, millet, horsebean and lentil macrofossils from archaeological sites was taken and treated with water. moist recovery was also applied to in-laboratory, artifi cially, charred barley, millet and lentil samples. after the treatments, the investigated remains were re-counted and the percentages of still recognizable remains for every plant species and for each method were recorded. comparisons were made of the sensitivities of the investigated species and of the differences in the degree of macrofossil breakup depending on the method of recovery. our investigation proved that fl otation is a less aggressive method than wet sieving and that barley, horsebean and wheat carbonized macrofossils are resistant to moist treatments, while the breakup percentage of lentil and millet (from archaeological sites) is higher than 30%, which should be taken into account when deciding on the (non)use of water recovery in the investigations. key words: carbonized plant remains, cereals, fl otation, legumes, wet sieving * corresponding author, e-mail: sara.marekovic@biol.pmf.hr introduction at archaeological investigation sites, botanical (as well as zoological and archaeological) material is often collected together with large amounts of soil and before analysis of that material, it is necessary to conduct a recovery process in order to remove the soil and to make sample analysis easier and faster. the most common recovery methods use water: fl otation or wet-sieving (water screening). in water screening, soil is placed on a screen and washed with a water jet. all the particles smaller than the screen mesh are washed through the screen and the larger materials (like botanical fi ndings) are recovered and later analyzed. flotation is based on the difference in density between organic and inorganic material. in the fl otation system, soil is poured into a body of water. agitation breaks up the soil, allowing light materials (seeds, charcoal) to fl oat and heavier materials (pebbles, potsherds) to sink. archaeobotanists analyse the organic material collected from sieves with different mesh sizes, to which the water with fl oating material is directed (jacomet and kreuz 1999, pearsall 2000). wagner (1982) in his paper introduces the poppy seed recovery test for determining the effectiveness and consistency of any particular botanical fl otation system. this is important because it helps investigators to be aware of the recovery rate of found remains in their fl otation devices. the aim of this study is to examine whether the wet sieving destroys carbonized plant remains more than the fl otation method and besides that, to investigate whether there is a difference in sensitivity among the species horsebean, lentil, wheat, barley and millet. in our previous studies, we accidentally noticed that the lentil has greater sensitivity to water treatments than wheat and barley grains and therefore our goal was to verify whether this difference is constant and to what extent the difference between the species exists. tanno and willcox (2006) hypothesised that the rarity of faba bean fi nds in their investigation might have been due to their fragility (and devastation during fl otation and transportation). because of the limited access to real archaeological material not previously treated with water, in this study we could not include all the legumes and cereals which could theoretically be found on archaeological sites. but the species that were analyzed in this paper, are certainly some of the most common species found in samples, so we believe that this analysis will give some important answers to archaeobotanists, when the need to make a decision about the right water recovery method for their sample or want to discuss the ratio of different carbonized fi ndings previously water recovered. a comparison of the influences of flotation and wet sieving acta bot. croat. 75 (1), 2016 145 wright (2005) concluded that the results of archaeobotanical analysis depend on fl otation sample size, how the sample is measured and processed and how well the plant material within the sample withstands the rigors of fl otation. some other previous studies have also shown that wet sieving affects plant residues of different species differently, so hosch and zibulski (2003) demonstrated in their paper that the number of fragile remains (e. g. cereal chaff remains and malus sylvestris mill. pericarp fragments) were reduced by increasing wet-sieving intensity, but more or less round and robust remains were scarcely affected. they suggested that a less destructive technique for fragile plant macroremains should be used, although the researchers will need more time for the recovery of the sample. tolar et al (2010) did an experiment with macroremains found in the neolithic pile dwelling site of stare gmajne, ljubljansko barje (slovenia). they compared three subsamples which were differently rough-handled during the wet sieving and their study showed that some sensitive species were almost completely destroyed and no longer recognizable. this study clearly shows that the proportion of species found in the sample can largely depend on the method of recovery of the sample and that this should be considered when selecting methods and in the interpretation of results. we are aware that the above mentioned studies deal with waterlogged sediments, which are certainly not the same as mineral soils, but there are also some similarities that can help us draw conclusions about recovery techniques. it should be taken into account that the plant remains can be damaged not only by the mechanical force of water rinsing, but also because of the contact of the carbonized material with the water and during the drying processes, which affects the mechanical structure and cracking of the fragile carbonized structures. pearsall (2000) describes the work of jarman et al. (1972), which analyses the percentage of destroyed carbonized macroremains after repetition of the fl otation and drying. their experiment showed that after the fi rst fl otation and drying, only 4% of the sample was destroyed. however after the second repetition of the procedure, an additional 56% of the sample was destroyed and the third fl otation with drying destroyed the remaining carbonized macrofossils. badham and jones (1985) also confi rmed that carbonized are far more sensitive than mineralized remains and therefore water screening should be avoided for the sake of fi nding such remains. materials and methods species that we have studied are: horsebean (vicia faba l.), lentil (lens culinaris medik.), wheat (mix: triticum turgidum ssp. dicoccon (schrank) thell.+ t. aestivum ssp. aestivum l. + t. aestivum ssp. spelta (l.) thell. + triticum turgidum ssp. dicoccon/t. aestivum ssp. spelta), barley (hordeum vulgare l.) and millet (panicum miliaceum l.). for all these species we took 300 well-preserved whole grains from the late bronze age sites kalnik-igrišče (mareković et al. 2015), for both pretreatments that we explored. additionally, we have treated 180 carbonized lentil seeds from the roman site poreč (which was explored in 2008) and 100 horsebean seeds from bronze age settlements nova bukovica (šoštarić 2001). as we had just one additional (beside those from kalnik-igriše site) archaeological carbonized sample, for horsebean and lentil species only, in the laboratory we made 6 extra samples with 30 grains for millet, barley and lentil species. millet and lentils were chosen because they in previous research they showed high rates of destruction during moist treatments, and barley was chosen as the control species, because it had shown great resistance to the treatments. the material was charred in a muffl e furnace. the barley and lentil species were heated up to 500 °c, for 3 or 10 minutes. millet was heated up to 300 °c, for approximately 10 minutes. in our experiment we found the best heating temperature and duration by consulting literature (wright 2003, babrauskas 2013) and by using the method of trial and error for each plant species. for each plant we took 3 samples for fl otation and another three for wet sieving. in the fl otation treatment we put the macroremains in a container, which was supplied with water via a rubber tube placed in lower part of the container. a steady supply of water caused the carbonized plant remains to move around the container and fi nally overfl ow the container on which they were collected in a sieve. the entire procedure lasted between 5 and 10 minutes for each sample. in the second treatment we put the macroremains on metal sieve, and rinsed it with the water jet from above. the strength of the water stream was regulated to medium strength, and the process lasted for ca 5 to 10 minutes. because different soils interact with macrofossils in different ways (some are much more viscous than others, for example), we deliberately did fl otation and wet sieving only with carbonized remains, in order to stay focused just on the fragility of the plant remains of every species, regardless of the infl uence of the soil type. after rinsing, the macrofossils were left to dry in the open air. subsequently they were all examined under the microscope, and we counted a number of carbonized remains still recognizable after the wet treatments. we calculated the percentage of recognizable, slightly damaged plant remains for every species and compared for any signifi cant difference between our two treatments and among the different species investigated. photographs of lentil and barley demonstrate the differences in plant remains before and after treatment, and also show grains or seeds that are either recognisable or unrecognisable (figs. 1, 2) the experiment with the plant material carbonized in the laboratory was, because of absence of decomposition after the fi rst moistening, repeated 5 and 10 times to check whether repeated water treatments and drying lead to greater destruction of the material. we wanted to see if the artifi cially charred remains can show us fragility differences among species similar to those found in genuinely archaeological remains, however much they are more resistant due to their date of origin as well as the absence of pedological and elemental infl uences that cannot easily be simulated in laboratory conditions. mareković s., šoštarić r. 146 acta bot. croat. 75 (1), 2016 statistical analysis of the obtained data was performed with the software package statistica (ver 8), manufacturers statsoft inc., usa. the aim was to check statistically whether there is a difference in the proportion (percentage, labelled p1) of recognizable seed after the fl otation and the proportion (percentage, labelled p2) of recognizable seed after the wet sieving treatment. beside that we wanted to see whether there are signifi cant differences among species in the same pretreatment. for this purpose, we set the null hypothesis h0: p1 = p2 and the alternative hypothesis h1: p1 ≠ p2. we tagged with n1 the total number of seeds before the fl otation and with n2 the total number of seeds before the wet sieving treatment. the value of the test statistics is calculated with the following formula: where beside the value of the test statistic z we needed also the corresponding p-value which is calculated as follows: – p = p{z ≥ z} if alternative hypothesis is in this form h1 : p1 − p2 > 0, – p = p{z ≤ z} if alternative hypothesis is in this form: p1 − p2 < 0, where z is a random variable with a standard normal distribution. p-values were counted with the probability calculator in the software package statistica. calculated p-values are compared with the level of signifi cance α = 0.05 = 5% (α is the maximum probability that we will make mistakes, if we reject the null hypothesis, i.e., maximum probability that we will reject the null hypothesis if it is correct – so called error of the fi rst kind) and one of the following two conclusions is made: – if p < α we reject the null hypothesis and at the level of signifi cance α we accept the alternative hypothesis h1, – if p > α we conclude that we do not have enough arguments to support the decision to reject the null hypothesis. results the results of treatment comparisons made on macrofossils collected from archaeological sites and afterwards on those carbonized in the laboratory are shown in figs. 3–5. horsebean collected from the kalnik-igrišče site showed the least sensitivity to treatments and as many as 91% of specimens were recognizable after both pretreatments. horsebean is followed by barley grains, preserved 85% after fl otation and 82.3% after wet sieving. wheat also proved to be a resistant macrofossil, because both treatments preserved almost three quarters of the grains in a recognizable form (75.3% after fl otation and 74.7% after wet sieving) (fig. 3). during fl otation as well as during wet sieving the remains most destroyed were the carbonized remains of the lentil, as only 68.7% of these remains (kalnik-igrišče) and 35% (poreč) were recognizable after fl otation, and, that percentage decreased after wet sieving to 52% (kalnikigrišče) and 22.2% (poreč) (figs. 4, 5).   p1 p2 1 1 1 n1 n2            z p' p' n1 p1 n2 p2 n1 n2      p' 0 10 20 30 40 50 60 70 80 90 100 vicia faba hordeum vulgare triticum mix. panicum miliaceum lens culinaris fig. 1. the lentil seeds from the site kalnik-igrišče: a) before the treatment, b) upon wet sieving: the recognizable lentil remains are on the top and unrecognizable ones on the bottom of fi gure. fig. 2. the barley grains from the site kalnik-igrišče: a) before the treatment, b) upon wet sieving: the recognizable barley remains are on the top and unrecognizable ones on the bottom of fi gure. fig. 3. comparison of percentage of recognizable macrofossils after fl otation (black bars) and wet sieving (grey bars) on the site kalnik-igrišče. a comparison of the influences of flotation and wet sieving acta bot. croat. 75 (1), 2016 147 the results show that in all cases, except for the horsebean sample from the site kalnik-igrišče, carbonized material is destroyed more by the wet sieving method. the biggest difference in destruction beyond recognition, is evident on the lentil (16.7% and 12.8%) and millet (8.3%) sample. the results show that for the horsebean sample from both sites, we cannot claim there is a signifi cant difference (if we use the level of signifi cance α = 0.05) between the fl otation and wet sieving treatments. for wheat and barley, there are no very strong arguments for stating that the proportions (percentages) of recognizable seeds differ according to the treatment. in both lentil samples it has been proven that (by the level of signifi cance α = 0.05) wet sieving damages the carbonized remains more than fl otation. at the level of signifi cance 0.05, we accepted the hypothesis that the proportion of recognizable millet seeds is greater after fl otation than after wet sieving. the samples of millet, lentil and barley experimentally carbonised in a furnace are much more resistant and less prone to destruction real archaeological material. the experiments with the laboratory-carbonized, material showed that the disintegration of the plant remains, with both recovery methods and regardless of the number of repetitions, was the biggest in millet samples. barley samples were the most resistant and barley grains remained undamaged during all fl otation and wet sieving procedures (tab. 1). the number of repetitions of the procedures done on the millet and lentil samples, increased the decomposition of the grains, and the samples were more damaged after wet sieving, than after fl otation. as for the fl otation process and the wet sieving of laboratory-carbonised material (lentils, barley, millet), we cannot (at the level of signifi cance α = 0.05) prove a statistically signifi cant difference in the destruction of the material beyond recognition, regardless of the number of repetitions of procedures (1, 5 and 10). however, when the sensitivity of millet is compared with that of barley, it can be proven (at the level of signifi cance α = 0.05) that sensitivity is signifi cantly bigger when fl otation is repeated 10 times, and with wet sieving after the fi rst repetition. therefore it became apparent that wet sieving is a more aggressive procedure and affects the carbonized remains of millet more than fl otation. when we compared the sensitivity of the lentil in relation to barley, we came to the conclusion that their sensitivities are not statistically signifi cantly different (at the level of signifi cance α = 0.05) after fl otation or wet sieving, even after ten repetitions. the sensitivity of millet compared to that of lentil is statistically signifi cantly different (at α = 0.05) only after the tenth repetition of wet sieving. discussion laboratory samples of carbonized lentil, millet and barley clearly showed that recent material, carbonized in a furnace, is more resilient and less prone to disintegration than real archaeological material. in previous investigations it has been already noted (pearsall 1980, king 1987, goette et al. 1994, king 1994,) that it is not easy to get lab samples 0 10 20 30 40 50 60 70 80 90 100 vicia faba hordeum vulgare triticum mix. lens culinaris panicum miliaceum fig. 4. the percentage of recognizable carbonized remains after fl otation. black bars represent the results from the site kalnikigrišče, grey bar shows the results from nova bukovica and the white one from poreč site. 0 10 20 30 40 50 60 70 80 90 100 vicia faba hordeum vulgare triticum mix. panicum miliaceum lens culinaris fig. 5. the percentage of recognizable carbonized remains after wet sieving. black bars represent results from the site kalnikigrišče, the white bar result from nova bukovica and the grey bar the result of the lentil from poreč site. tab. 1. the comparison of the preservation percentage of carbonized macrofossils obtained by methods of repeated fl otation and wet sieving. no. of repetitions lentil barley millet flotation wet sieving flotation wet sieving flotation wet sieving 1 x 100 % 100 % 100 % 100 % 97.77 % 96.67 % 5 x 100 % 99.44 % 100 % 100 % 96.67 % 93.33 % 10 x 98.89 % 98.33 % 100 % 100 % 94.47 % 86.67 % mareković s., šoštarić r. 148 acta bot. croat. 75 (1), 2016 (corn) the structure and properties of which identical or at least satisfactorily similar to those found in real archaeological samples. brady (1989) investigated the impact of fl otation on the preservation of laboratory samples of carbonized wood and concluded that there is no signifi cant difference in the conservation of mass during fl otation, regardless of the differences in the density of the wood in different plant species, which can also be result of greater resistance of wood in the case of laboratory-carbonized material. therefore we presume that although it is impossible to get accurate numerical data on the rate of destruction of some plant species in wet recovery treatments on the basis of studies done on laboratory carbonized material, it is possible to conclude which species are more sensitive and which method is more aggressive. this study showed that the carbonized grains of lentils and millet are sensitive and therefore samples that contain these grains should preferably be spared wet sieving. carbonized remains of beans, wheat and barley after both treatments keep a preservation percentage greater than 70% and therefore we believe that they are suffi ciently robust and their presence should not be a reason for avoiding wet sieving. therefore, we propose that, before the water recovery, a preliminary insight into the diversity of plant remains from each site, should be acquired. if the sample contains carbonized lentils and millet, and it is possible to conduct sieving without water, wet recovery techniques should defi nitely be avoided. however if the soil around the sample is so compact that it demands the use of water, we recommend avoiding wet sieving, because it will do more damage to sensitive carbonized material than fl otation. if wet sieving has already been conducted or must be used to accelerate the recovery process, we certainly suggest researchers should consider the fact that the decomposition of lentils may be greater than 45% and the decomposition of millet greater than 40%. that fact should be noted in order to obtain a more accurate proportion ratio of found taxa. in conclusion, our study is in agreement with previous reports of badham and jones (1985) that the wet sieving is a more aggressive method than fl otation and that it should be used only when the sample does not contain many fragile remains. references babrauskas, v. 2013: temperatures in fl ames and fi res. retrieved february 10, 2013 from http://www.doctorfi re.com/fl ametmp. html badham, k., jones, g., 1985: an experiment in manual processing of soil samples for plant remains. circaea 3, 15–26. brady, j. t., 1989: the infl uence of fl otation on the rate of recovery of wood charcoal from archaeological sites. journal of ethnobiology 9, 207–227. goette, s., williams, m., johannessen, s., hastorf, c. a., 1994: toward reconstructing ancient maize: experiments in processing and charring. journal of ethnobiology 14, 1–21. hosch, s., zibulski p., 2003: the infl uence of inconsistent wetsieving procedures on the macroremain concentration in waterlogged sediments. journal of archaeological science 30, 849–857. jacomet s., kreuz s., 1999: archäobotanik – aufgaben, methoden und ergebnisse vegetations-und agrargeschichtlicher forschung. eugen ulmer, stuttgart. jarman, h. n., legge, a. j., charles, j. a., 1972: retrieval of plant remains from archaeological sites by froth fl otation. in: higgs, e. (ed.) papers in economic prehistory, 39–48. cambridge university press. king, f. b., 1987: prehistoric maize in eastern north america: an evolutionary evaluation. dissertation, university of illinois. king, f. b., 1994: variability in cob and kernel characteristics of north american maize cultivars. in: johannessen, s., hastorf, c. a. (eds.) corn and culture in prehistoric new world, 35– 54. westview press, boulder, colorado. mareković, s., karavanić, s., kudelić, a., šoštarić, r., 2015: the botanical macroremains from the prehistoric settlement kalnik-igrišče (nw croatia) in the context of current knowledge about cultivation and plant consumption in croatia and neighboring countries during the bronze age. acta societatis botanicorum poloniae 84, 227–235. pearsall, d. m., 2000: paleoethnobotany, a handbook of procedures. academic press, san diego. pearsall, d. m., 1980: analysis of archaeological maize kernel cache from manabi province, ecuador. economic botany 34, 344–351. šoštarić, r., 2001: carbonized plant remains of the prehistoric locality in nova bukovica on the site sjenjak. prilozi instituta za arheologiju u zagrebu. 18, 79–82. tanno, k., willcox, g., 2006: the origins of cultivation of cicer arietinum l. and vicia faba l.: early fi nds form tell el-kerkh, north-west syria, late 10th millennium b.p. vegetation history and archaeobotany 15, 197–204. tolar, t., jacomet, s., velušček, a., čufar, k., 2010: recovery techniques for waterlogged archaeological sediments: a comparison of different treatment methods for samples from neolithic lake shore settlements. vegetation history and archaeobotany 19, 53–67. wagner, g., 1982: testing fl otation recovery rates. american antiquity 47, 127–132. wright, p. j., 2003: preservation or destruction of plant remains by car bonization?. journal of archaeological science 30, 577– 583. wright, p. j., 2005: flotation samples and some paleoethnobotanical implications. journal of archaeological science 32, 19–26. untitled acta bot. croat. 75 (1), 2016 39 acta bot. croat. 75 (1), 39–44, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0014 issn 0365-0588 eissn 1847-8476 embryological features, pollen and seed viability of arnica montana (asteraceae) – a threatened endemic species in europe elina yankova-tsvetkova*, petka yurukova-grancharova, georgi baldjiev, antonina vitkova department of plant and fungal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, 1113 sofi a, gagarin 2 street, bulgaria abstract – the embryological features, mode of reproduction and reproductive capacity (pollen and seed viability) on two naturalized populations of arnica montana in bulgaria were studied. the embryological study shows that a. montana is a facultative apomictic species in which sexual reproduction predominates. in this species, it was established that there is a comparatively high viability of the mature pollen and embryos, which enables the successful realization of its reproductive capacity. the results of the study on a. montana reveal that both sexual and asexual vegetative reproduction with rhizomes undoubtedly play more important roles than the apomixis (namely diplospory) for support and preservation of the populations. keywords: apomixis, arnica montana, embryology, male and female gametophyte, pollen and seed viability, sexual reproduction * corresponding author, e-mail: e_jankova@abv.bg; e_jankova@mail.bg introduction arnica montana l. is a rosette-forming perennial plant of the family asteraceae dum., subfamily asteroideae, tribe madieae (noyes 2007). it is a diploid species with 2n = 38 (ekenäs 2008), highly self-incompatible with entomophily (luijten et al. 1996, 2000). arnica montana has been used for medical purposes since the 1500s because of the contents of various active compounds with antiseptic, antifungal, antimicrobial and antibiotic activities. it occurs from the lowlands to the alpine belt. in the mountains, arnica montana is a characteristic species of nardus stricta grasslands and hay meadows (oberdorfer 1994). the excessive exploitation of this medicinal plant has affected the state of its natural reserves and reproductive capacity. at present a. montana is considered a rare and endemic species in europe (maguire 1943, ferguson 1976, ekenäs 2008), and is included in many european countries in the category “endangered” and listed in annex v of the euffh-directive (directive 92/43/eec 1992). the species is regarded as: “critically endangered” in belgium, bosnia, croatia and luxembourg; “endangered” in belarus and the netherlands; “vulnerable” in estonia, germany, latvia, lithuania, portugal and romania; and “near threatened” in denmark and norway. in bulgaria, a. montana was found more than 100 years ago in the rila mts (herbarium specimen so 86331 deposited, exists in the herbarium of biological faculty of the sofi a university “st. kliment ohridski”) but up to now its occurrence in the country has not been confi rmed. hitherto, this species has been primarily an object of chorological, karyological and phytochemical studies but scanty and fragmentary data exist on its embryology in the accessible literature. the aim of the present study is to reveal some of the main characteristics of the reproductive biology of arnica montana: peculiarities of the male and female gametophyte; embryoand endospermogenesis; pollen and seed viability, in connection with the realization of its reproductive capacity that infl uence the shape and size of the populations. material and methods the material of two naturalized populations (two-years old) of a. montana were studied, as follows: beli rid experimental station – on mt vitosha (a population from the carpathian mountains, ukraine) and the experimental station in the village of beglica – rhodopi mts (western) – a population from the botanical garden, chemnitz, germany. the mt vitosha population consists of 167 individuals and yankova-tsvetkova e., yurukova-grancharova p., baldjiev g., vitkova a. 40 acta bot. croat. 75 (1), 2016 the rhodopi mts population contains 146 individuals. voucher specimens were deposited in the herbarium of institute of biodiversity and ecosystem research, bulgarian academy of sciences (som 168516, 168517 – from mt vitosha and som 169535 – from the rhodopi mts). embryological study for embryological study, 50 fl ower buds and capitula at different stages of development were collected from 20 individuals of each of the two naturalized populations (above mentioned) and fi xed in a mixture of faa (formalin: glacial acetic acid: 70% ethanol in correlation 5:5:90 parts). consecutively, the plant material was treated according to the classical paraffi n methods (sundara 2000), embedded in paraffi n and cut into 10–25 μm sections with a rotary microtome. the sections were stained with heidenhain’s haematoxylin and embedded in enthelan in order to get permanent slides. pollen and seed viability study mature pollen grains were isolated from the two studied populations and their viability was estimated using the acetocarmine test (singh 2003). for this purpose, anthers from 50 open fl owers from 20 plants of each population studied were collected, placed in 1% acetocarmine solution, dispersed on the slides and the stained (viable) and unstained (unviable) pollen grains were counted in 30 anthers from each population using a light microscope (visual fi eld, enlargement 100×). for the study on seed viability, 400 mature seeds were collected from the two populations. to estimate the seed (embryo) viability, a quick tetrazolium test was applied (peters 2000). the embryos were isolated with a dissection needle, incubated in water at 30–35 °c and subsequently in a diluted 1% solution of 2,3,5-triphenyltetrazolium chloride for 24 hours. initially, the tetrazolium solution is colourless, but it changes to red when it comes to contact with hydrogen (a reduction process), deriving from enzymes of the respiration process of the seeds. the observations during the present study were carried out using a stereomicroscope “leica ez4”, lm “olympus” cx2. the microphotographs were made with digital camera 1.4 mpx. results embryological features anther and development of the male gametophyte the anthers are tetrasporangiate. the anther wall formation follows the dicotyledonous type and consists of four layers: an epidermis, an endothecium, one middle layer and a tapetum (fig. 1a). the epidermis comprises one row of almost rectangular uninucleate cells that vastly enlarge during the anther ontogenesis. the middle layer is ephemeral and degenerates up to end of the meiosis in microspore mother cells (mmcs). the endothecium develops not clearly expressed fi brous thickenings of its consisting cells. this layer becomes completely disorganized at the stage of the mature three-celled pollen. initially, the tapetum is glandular, consisting of one row of uninucleate cells (fig. 1a). during the meiosis in mmcs, a rapid lengthening and multiplication of the nuclei of tapetum cells (as result of consecutive mitoses) is observed (fig. 1c) and they become fourto eight-nucleate at the stage of microspore tetrads. after the formation of uninucleate pollen grains, the tapetum transforms from glandular to ameboid (fig. 1e). at the time of anther dehiscence, the anther wall comprises only epidermis the sporogenous tissue is one-, two-rowed (fig. 1a). the meiosis in mmcs passes with some deviations, such as: individual lagging chromosomes (fig. 1c) and chromosome out of the division spindle, especially during the fi rst (heterotypic) division of the meiosis. after simultaneous cytokinesis, the resultant microspore tetrads (fig. 1b) were classifi ed on the basis of 50 enumerated tetrads, according to schmid (1982), as: “usually” tetrahedral (71%), “occasionally” isobilateral (22%), “quite occasionally” t-shaped (5%) and “rarely” linear (2%). sporadically monads and dyads were also observed. in a number of fl orets of some capitula degenerating microspore tetrads presented (fig. 1d).at the time of shedding, the pollen grains are usually morphologically uniform, three-celled, tricolporate with fig. 1. anther and development of the male gametophyte: a) anther wall and two-rowed sporogenous tissue, b) lagging chromosomes in the metaphase i of the meiosis in microspore mother cells, c) different type of microspore tetrads and one-rowed glandular tapetum with multinucleate cells, d) degenerating microspore tetrads in the anther locules, e) one-nucleate pollen and ameboid tapetum, f) viable and sterile pollen grains in an anther locule. ep -epidermis, en – endothecium, ml – middle layer, tp – tapetum, st – sporogenous tissue, mt – microspore tetrad, pg – pollen grain, atp – amoeboid tapetum, lch – lagging chromosome. scale bars = 20 μm. embryological features of arnica montana acta bot. croat. 75 (1), 2016 41 echinate exine. often, besides the normal viable pollen grains, small-sizedand dark-stained sterile grains were observed in the anthers of some fl orets in one and the same capitulum (fig. 1f). ovule and development of the female gametophyte the gynoecium is syncarpous, inferior with a unilocular ovary in which only one ovule forms. the well-developed ovule is anatropous, tenuinucellate and unitegmic. in it unicellular archesporium forms hypodermally. the archesporium cell functions directly as a megaspore mother cell (fig. 2a), which later undergoes meiosis to produce a linear megaspore tetrad (fig. 2b). the embryo sac (es) development runs according to the polygonum (monosporic)-type from the chalazal megaspore of tetrad that functions as an embryo sac mother cell. after three mitoses, successively two-, fourand eight-nucleate es forms. the mature es consists of a three-celled egg apparatus (a usually pearshaped egg cell and two synergids), two polar nuclei (after their fusion a secondary nucleus forms) and a three-celled antipodal apparatus in the chalazal part of es (fig. 2c). the synergids degenerate after fertilization. the antipodals are ephemeral and begin to degenerate after the formation of a secondary nucleus. the endothelium differentiates from the innermost layer of the single integument after onenucleate es and the cells of which it consists progressively lengthen in a radial direction during the es development. it is important to notice that in the megaspore mother cells of some ovules in the fl orets of the capitula a restitution nucleus forms after inhibited meiosis that leads to a dyad of megaspores, instead of a tetrad (fig. 2d). later on, in these cases, from the chalazal megaspore a meiotic diplosporous es of taraxacum-type develops. usually, the embryo and endosperm form after double fertilization accompanied with a destruction of one synergid from the pollen tube penetrating it through the micropyle of ovule. the fi rst division of the zygote is transverse and usually runs before the endospermogenesis (fig. 2e). the embryogenesis follows the asterad type. in the mature seed, the embryo is nearly straight with two equal cotyledons. in fi ve ovules (18% from 28 observed in total with globular embryo), the presence of a globular embryo together with two undamaged, long living synergids (in contrast to the stage of legitimate globular embryo in which the synergids are missing) provides reason to suppose that in these cases the embryo forms parthenogenetically from the unfertilized egg cell in es that develops after a meiotic diplosporous taraxacum-type, mentioned above (fig. 2f). at the beginning, the endosperm passes from a free nucleate to a cellular stage, when a globular embryo forms. the mature seed completely lacks endosperm (fig. 4d). pollen and seed viability after acetocarmine staining, the cytoplasm and nuclei of viable pollen grains were stained in red while unviable, empty and shrunken pollen grains remain unstained (figs. 3a and d). the results of the study show the high viability of the mature pollen (over 80%) in the two studied naturalized populations of a. montana (tab. 1). according to the intensity of staining with tetrazolium solution, the viable embryos are stained in red, while embryos partially stained fig. 2. ovule and development of the female gametophyte: a) megaspore mother cell in the ovule, b) lineare megaspore tetrad in the ovule, c) three-celled egg apparatus (egg cell and two synergides) and secondary nucleus in the embryo sac, d) megaspore dyad (initiation of the diplosporous taraxacum-type development of embryo sac from the chalazal megaspore), e) two-celled proembryo and developing endosperm, f) parthenogenetic globular embryo together with preserved two synergids and nuclear endosperm. mmc – megaspore mother cell, mgt – megaspore tetrad, sg – synergid, egc – egg cell, cc – central cell, md – megaspore dyad, em – embryo, sp – sperm, pem – parthenogenetic embryo. scale bars = 20 μm (a–e) and 50 μm (f). fig. 3. pollen of arnica montana analyzed by acetocarmine test: a) mature pollen grains without acetocarmine staining, b) viable pollen grains, stained in red, c) tricolporate viable pollen grain, stained in red, d) unviable, unstained pollen grain. scale bars = 100 μm (a), and 20 μm (b–d). yankova-tsvetkova e., yurukova-grancharova p., baldjiev g., vitkova a. 42 acta bot. croat. 75 (1), 2016 or unstained are unviable (figs 4a and 4d). on basis of tetrazolium testing, the seeds (embryos) were differentiated in three classes (fig. 5): class i – viable embryos (whole embryo stained in red); class ii – unviable embryos (only the root of embryo stained in red); class iii – unviable embryos (unstained). it was established that the majority of embryos are viable (68.97% 63.46% for the mt vitosha and rhodopi mts populations, respectively (fig. 5). discussion embryological features pullaiah (1983) studied the embryology of the tribe senecioneae, including the genus arnica. the results of his investigation revealed only sexual reproduction in the studied representatives of this tribe. in the majority of the species in the genus arnica, which are largely polyploids, game tophytic apomixis has found – diplospory and parthenogenetic development of the embryo (afzelius 1936, flovik 1940, engell 1970). for that reason, nordenstam (1977) excluded the genus arnica from the tribe senecioneae. in the new systematical classifi cations, this genus is included in the tribe madieae (baldwin et al. 2002, noyes 2007). the tapetum of the four-layered anther wall in a. montana is glandular and one-layered, consisting of multinucleate cells. after the meiosis in the mmcs it becomes ameboid (partial spreading of the cell walls occurs), as in most representatives of the family asteraceae (solntseva 1987). the formation of microspore dyads, polyads and monads in the anthers, as result of deviations during the meiosis, was often reported for apomictic representatives of the family asteraceae (some species of the genera crepis l., taraxacum cass., rudbeckia l., arnica l., hieracium l. (solntseva 1987). in contrast with the apomictic species, in which a high percentage of sterile pollen grains is observed (afzelius 1936, gustafsson 1937, babcock and stebbins 1938, fagerlind 1939, battaglia 1947), in arnica montana we found the formation of morphologically uniform and highly fertile pollen in the majority of the anthers. the development of the female gametophyte in a. montana follows exclusively the polygonum (monosporic)-type that begins from the chalazal megaspore of tetrad observed in the most asteraceae (solntseva 1987, poddubnaya arnoldi 1982), namely: chrysanthemum multicaule,(deng at al. 2010);, tugarinovia mongolica, (ma and wang 2000), calendula offi cinalis, (ao 2007), chrysanthemum grandifl orum, (deng et al. 2010). the polygonum -type is not the only type for es development in the asteraceae family in which are observed, but more rarely, the adoxa-type (liu, 2001a), drusa-type (liu 2001b), oenothera-type (teng et al. 2008, li et al. 2009) and pyrethrum cinerariaefolium type (hu 2005). the formation of a restitution nucleus in some ovules, a dyad of megaspores instead of tetrads as well as some other embryological peculiarities established during our study suggest that the female gametophyte development most likely follows the taraxacum-type of meiotic diplospory (gametophytic apomixis). consequently, we found indications that the embryo develops parthenogenetically and the proof of that is the preservation of the two synergids together with a multicellular globular embryo in the es cavity. the same phenomenon we have observed in the species erigeron annuus of the asteraceae family (yurukova-grancharova at al. 2012) in conclusion, on the grounds that during this study in a. montana both sexual and apomictic reproduction were established, we assume that this diploid species with 2n = тab. 1. pollen viability in studied populations, analyzed by acetocarmine staining. no. – number. populations no. of pollen grains viability (% ± sd) germany (rhodopi mts) 2897 79.99 ± 4.22 ukraine (vitosha mt) 2810 83.77 ± 7.12 fig. 4. seed (embryo) viability examined by tetrazolium test: a) seed (achene) on the right side and an isolated embryo (on the left), b) isolated viable embryo (only the root stained in red), c) unviable unstained embryo (on the left side) and viable embryo, stained in dark red (on the right), d) unviable, unstained embryo (on the left) and two viable embryos, stained in red. scale bars = 20 μm. fig. 5. frequency of seeds (embryos) viability (%) assessed by tetrazolium test: 1 – class i, viable embryos (stained in red); 2 – class ii, unviable embryos (only the root of embryo stained); 3 – class iii, unviable embryos (unstained). embryological features of arnica montana acta bot. croat. 75 (1), 2016 43 2x = 38 probably occupies an isolated position within the genus arnica, in which polyploids with gametophytic apomixis (diplospory) predominate (kao 2007, 2008; noyes 2007). the endosperm in the family asteraceae was shown to be usually ab initio cellular by davis (1966). solntseva (1987) and poddubnaya-arnoldi (1982) described two types of endosperm (nuclear and cellular) in this family. it was established that in arnica montana the endosperm is not ab initio cellular, but passes a free nuclear stage. pollen and seed viability the estimated comparatively high viability of pollen (over 80%) and seeds (68.97% 63.46% for the mt vitosha and rhodopi mts populations, respectively) of a. montana in our study provides for the successful realization of its reproductive capacity, an important condition for the preservation of the size of its population. pollen viability (potential fertility), seed production, viability and germination ability are closely connected to one another. in arnica montana in particular, strykstra et al. (1998) show that lighter seeds (achenes) are better dispersed by the wind but have a lower germination ability. conclusion in the present study the main embryological features, mode of reproduction and reproductive capacity (pollen and seed viability) in arnica montana, a threatened endemic species in europe, were established. the study found a high plasticity of the female generative sphere compensating for the large number of sterile ovules and degenerating es in the fl orets of capitula observed. besides, this high plasticity has a signifi cant importance for the better adaptation of the studied species. according to the results obtained, a. montana may be considered a facultative apomictic species in which sexual reproduction predominates (only 18% of apomictic embryos were observed). thus, it may be concluded that sexual reproduction combined with vegetative reproduction determine the state of its populations rather than the diplospory (gametophytic apomixis), established during the study. acknowledgements the authors are grateful to national science fund of the ministry of education, youth and science in bulgaria for the fi nancial support of the study under contract dtk 02/38. references afzelius, k., 1936: apomixis in der gattung arnica. svensk botanisk tidskrift 30, 572–579. ao, c. q. 2007: comparative anatomy of bisexual and female fl orets, embryology in calendula offi cinalis (asteraceae), a natural horticultural plant. scientia horticulturae, 144, 214–219. babcock, e. b., stebbins, g. l., 1938: the american species of crepis: their interrelationships and distribution as affected by polyploidy. carnegie institution publication, 504. carnegie institution, washington. baldwin, b. g., wessa, b. l., panero, j., 2002: nuclear rdna evidence for major lineages of helenioid heliantheae (compositae). systematic botany 27, 161–198. battaglia, e., 1947: ricerche cariologische ed embriologische sul genere rudbeckia. 9. le anomalie del gametofi to femminile cellularizzatio di r. laciniata. nuovo giornale botanico. italiano 54, 377–405. davis, g, 1966: systematic embryology of the angiosperms. john wiley, new york. deng, y., chen, s, teng, n., chen, f., li, f., song, a., guan, z., 2010: flower morphologic anatomy and embryological characteristics in chrysanthemum multicaule (asteraceae). acta horticulturae 124 500–505. hu, s. y., 2005: reproductive biology of angiosperm. higher education press, beijing directive 92/43/eec, 1992: council directive 92/43/eec of 21 may 1992 on the conservation of natural habitats and of wild fauna and flora, pp. 7–50. oj l 206, 22.07.1992. ekenäs, c., 2008: phylogenies and secondary chemistry in arnica (asteraceae). abstract of dissertation, 58 p., acta universitat upsalensis, uppsala. digital comprehensive summaries of uppsala dissertations from the faculty of science and technology 392. retrieved from http://uu.diva portal.org/smash/ search.jsf. engell, k., 1970: embryological investigations in arnica alpina from greenland. svensk botanisk tidsskrift 65, 225–44. fagerlind, f., 1939: kritische und revidierende untersuchungen über das vorkommen des adoxa (lilium)-typus. acta horticulturae berg. 13, 1–49. ferguson, i. k., 1976: arnica l. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. 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(in russian). strykstra, r. j., pegtel, d. m., bergsma, a., 1998: dispersal distance and achene quality of the rare anemochorous species arnica montana l.: implications for conservation. acta botanica neerlandica 47, 45–56. sundara, r. s., 2000: practical manual of plant anatomy and embryology. anmol publ. pvt ltd, new delhi. teng, n. j., chen, f. d., ma, x., hou, x. l., 2008: investigation on megasporogenesis, megagametogenesis and embryogenesis in dendranthema nankingense. journal of nanjing agricultural university 31, 47–51. yurukova-grancharova, p., yankova-tsvetkova, e., baldjiev, g., vladimirov, v., 2012: on the reproductive biology of the invasive alien species erigeron annuus (asteraceae) in the bulgarian fl ora. – comptes rendus de l’academie bulgaredes sciences, 65, 933–938. 88 acta bot. croat. 77 (1), 2018 acta bot. croat. 77 (1), 88–92, 2018 coden: abcra 25 doi: 110.1515/botcro-2017-0019 issn 0365-0588 eissn 1847-8476 short communication assessment of the genetic relationship of turkish olives (olea europaea subsp. europaea) cultivars based on cpdna trnl-f regions ergun kaya1, recep vatansever2, ertugrul filiz3* 1 department of molecular biology and genetics, mugla sitki kocman university, 48000, kotekli, mugla, turkey 2 department of biology, faculty of science and arts, marmara university, 34722, istanbul, turkey 3 department of crop and animal production, cilimli vocational school, duzce university, 81750, duzce, turkey abstract – the olive tree (olea europaea l.) is one of the major cultivated species in the world, and mediterranean countries produce about 90% of world cultivated olives. in this study, the genetic relationship of seven turkish olive cultivars was investigated using non-coding trnl-f regions in chloroplastic genome. cultivars demonstrated a similar sequence length of 330-340 bp with an average 35.26% g+c content. variable (polymorphic/segregating), parsimony informative and total numbers of the insertion or the deletion of bases in the dna (indel sites) were 4, 3, and 28, respectively. nucleotide diversities π and θ were found as 0.00631 and 0.00644 respectively, while tajima’s d was –0.786. cpdna trnl-f regions of sequenced turkish olive cultivars had a low level of genetic variations, and these non-coding regions were strictly conserved in all analyzed cultivars. geographically distant shared more sequence similarities than relatively close cultivars. the phylogenetic analyses indicated that the biogeographic distribution of cultivars does not demonstrate any association inferring cultivar source. these results indicate the possibility of germplasm exchanges among countries or that some indel mutations contribute to variations of the turkish olive gene pool. thus, the authorities should develop the necessary programs to preserve the purity of native germplasms. keywords: germplasm, intergenic, lineage, non-coding trnl-f, pairwise, plastome * corresponding author, e-mail: ertugrulfiliz@gmail.com introduction the olive tree (olea europaea l.) is one of the major cultivated species in the mediterranean basin. o. europaea, which belongs to section olea, demonstrates a wide spectrum of distribution with six natural subspecies, including o. europaea subsp. europaea (mediterranean basin), o. europaea subsp. cuspidata (from south africa throughout east africa and arabia to south west china), o. europaea subsp. guanchica (canaries), o. europaea subsp. cerasiformis (madeira), o. europaea subsp. maroccana (morocco), and o. europaea subsp. laperrinei (algeria, sudan and niger). the mediterranean form, o. europaea subsp. europaea contains two varieties, cultivated (var. europaea) and wild (var. sylvestris) (green 2002). in o. europaea members, seven main cpdna lineages were reported such as e1 (mediterranean area and saharan mountains), e2 and e3 (western mediterranean area), m (macaronesia), c1 and c2 (from southern asia to eastern africa), and a (tropical african olives) (green 2002). in turkey, the olive grows in the aegean, marmara, mediterranean and southeastern anatolia regions. turkey hosts a large number of cultivated and wild cultivars/germplasms, including very old cultivars such as “gemlik”, “ayvalık” amd “uslu”(mendilcioglu 1999, ercisli 2004). in addition, turkey is one of the most important olive producers in the world, along with spain, italy and greece. olea species have been used in various molecular studies, including plastome sequencing (besnard et al. 2011), restriction endonuclease studies (amane et al. 2000), ssr (hannachi et al. 2010), rapd (hess et al. 2000) and issr marker analyses (kaya 2015). besides, chloroplast dna (cpdna) sequences have been used as genomic resources in plant phylogenetic mailto:ertugrulfiliz@gmail.com http://en.wikipedia.org/wiki/morocco genetic relationship of turkish olives based on trnl-f acta bot. croat. 77 (1), 2018 89 studies. the cpdna trnl–trnf intergenic spacer region has been used for intraand interspecific levels in plants (mes et al. 2000). for example, four plastid sequences such as trnltrnf, trnt-trnl, trns-trng, and matk were studied in 71 olea samples, resulting in 261 variable sites and 121 potentially parsimony-informative characters (besnard et al. 2009). in a different study, two non-coding chloroplast loci, rps16 intron and trnl-f were used in the phylogenetic analysis of 76 species of the oleaceae family; rps16 and trnl-f datasets contained 265 and 240 informative characters, respectively (wallander and albert 2000). in addition, besnard et al. (2011) reported that nucleotide divergence between olive cpdna lineages was low (<0.07%) in eight complete cpdna genomes of olea. moreover, chloroplast genome organization and gene order of o. europaea, subsp. europaea var. europaea was reported to be conserved among numerous angiosperm species, indicating the lack of gene inversions, duplications, insertions, inverted repeat expansions and intron losses (mariotti et al. 2010). recent studies have reported genetic analyses of some turkish olive cultivars using dna-based molecular markers (ipek et al. 2012, çelikkol et al. 2014, kaya 2015). however, no studies are available regarding the usage of trnl-f intergenic spacer regions for genetic assessment in turkish olive cultivars. therefore, in this study, we have analyzed the seven turkish olive cultivars using trnl-f intergenic spacer regions to investigate the genetic relationships among turkish olive cultivars. materials and methods plant materials and genomic dna isolation seven cultivated turkish olive cultivars (o. europaea subsp. europaea var. europaea) were obtained from six different locations in turkey (tab. 1 and on-line suppl. fig. 1). total genomic dna was isolated from 0.5 g of powdered fresh leaves with the use of the ctab method (doyle and doyle 1987). the dna concentration of each sample was measured with the use of biospec-nano (shimadzu, japan) and then elutions were diluted with distilled water for a final concentration of 50 ng μl–1. trnl-f amplification and sequencing the cpdna trnl-f regions in seven turkish olive cultivars were amplifiedwith the use of pcr with trnl-f primers. for pcr amplification, reaction mixture was prepared for 25 µl total volume, containing 2.0 mm mgcl2, nucleotides datp, dttp, dctp, and dgtp (200 µm each), 0.2 µm primers, 50 ng template dna, and 0.5 units (u) of taq dna polymerase (thermo sci, usa). thermal cycling conditions were chosen as: 3 min at 94 °c; 36 cycles of 45 s at 94 °c, 1 min at annealing temperature of each primer pair, and 1 min at 72 °c, and a final extension step of 5 min at 72 °c. pcr products were analyzed on 1% agarose gel in 1tbe buffer, stained with safeview dna stain (nbs scientific, uk), and visualized with quantum st5 imaging system. then, pcr products were purified using a genejet gel extraction kit (thermo scientific, usa), and sequenced by iontek sequencing service. data analysis to supplement the seven turkish olive cultivars, 25 additional trnl-f sequences from various olea species were obtained from the ncbi database (www.ncbi.nlm.nih.gov). thus, a total of 32 trnl-f sequences were used in analyses. additional species include o. borneensis, o. capensis subsp. capensis, o. capensis subsp. enervis, o. capensis subsp. macrocarpa, o. chimanimani, o. dioica, o. europaea subsp. cerasiformis, o. europaea subsp. cuspidate, o. europaea subsp. guanchica, o. europaea subsp. laperrinei, o. javanica, o. lancea, o. paniculata, o. neriifolia, o. rosea, o. salicifolia, o. schliebenii, o. tsoongii, o. welwitschii and o. europaea subsp. europaea. the ncbi accession numbers of additional sequences were indicated on phylogenetic trees. the sequence alignment was done by clustalw (thompson et al. 2002) and a phylogenetic tree was constructed with mega v. 6.0 using the maximum likelihood (ml) method for 1000 bootstrap values (tamura et al. 2013). bootstrap values lower than 70% were not shown on the branch. the dnasp v 5.10 was used to calculate the nucleotide diversity with π (nei 1987) and θ (watterson 1975), segregating/polymorphic sites (s), and tajima’s d (tajima 1989, librado and rozas 2009). genotype/cultivar comparisons were performed by arlequin 3.5.2 software with the use of the population comparison tool with the following settings; pairwise differences (π) with tajima and nei’s method for 100 permutations and 0.05 significance level (excoffier and lischer 2010). tab. 1. geographic location and some gene features of seven turkish olive cultivars (olea europaea subsp. europaea). ncbi access. genotype name locality geographical regions gps coordinates trnl-f gene (bp) g+c content (%) kj670690 yaglık izmir aegean 38°27’53.7”n, 27°14’22.9”e 337 35.01 kj670691 mugla mugla aegean 37°07’43.1”n, 28°27’05.0”e 330 35.45 kj670688 gemlik bursa marmara 40°31’08.7”n, 29°06’05.0”e 339 35.69 kj670692 hatay hatay mediterranean 36°46’59.4”n, 36°15’47.7”e 339 35.10 kj670693 samsun samsun black sea 41°38’42.3”n, 35°26’07.9”e 337 35.01 kj670694 tekir izmir aegean 38°27’53.7”n, 27°14’22.9”e 340 35.59 kj670689 burhaniye izmir aegean 38°30’28.5”n, 27°01’58.9”e 340 35.00 kaya e., vatansever r., filiz e. 90 acta bot. croat. 77 (1), 2018 results and discussion sequence analysis of trnl-f genes the cpdna trnl-f regions of the seven sequenced turkish olive cultivars demonstrated similar sequence lengths, ranging from 330 bp (mugla cultivar) to 340 bp (tekir and burhaniye cultivars), with an average of 35.26% g+c content (table 1). similar results were also indicated in a previous study, in which the length of trnl-f sequences in olea species was reported to range between 327-343 bp, and the number of variable/potentially informative characters and mean g+c content were calculated as 23/6, and 35%, respectively (besnard et al. 2009). to analyze the conservancy or divergency degrees in turkish olive cultivars, sequences were multiply aligned (fig. 1). the alignment analysis demonstrated that non-coding trnl-f sequences are strictly conserved in all turkish olive cultivars. despite the high degree of similarities between sequences, we also searched for the presence of any informative variable site/s. the variable (polymorphic/segregating), parsimony informative sites and total numbers of indel sites were found to be 4, 3, and 28, respectively. in addition, nucleotide diversity π and θ values were 0.00631 and 0.00644 respectively, while tajima’s d was -0.786. the nucleotide diversity and parsimony informative sites in turkish olive cultivars were found to be low. in the olea subgenus, the cpdna substitution rate was predicted to be between 1.2 × 10–10 and 2 × 10–10. these values were ten times lower than plastid mutation rates previously reported in other plant species. this slower molecular evolution might be related with the long generation time of olive trees (besnard et al. 2009). the plastome sequence comparisons also demonstrated that cpdna of olive cultivars represent low level genetic variations (mariotti et al. 2009). in a study from moroccan olives, low cpdna variation was reported using various restriction endonuclease enzymes (amane et al. 2000). therefore, the low level of genetic variations in the cpdna trnl-f regions of the seven turkish olive cultivars sequenced in this study complies with the reports of previous studies. furthermore, negative and positive tajimas’s d values were reported to show an excess of low-frequency and intermediate polymorphisms (luo et al. 2012). in this study, tajimas’s d was found to be negative (–0.786), suggesting a possibility of selection at trnl-f loci in analyzed olea members. pairwise-comparison of turkish olive cultivars in order to have insights about the genetic relationships of turkish olives, pairwise similarity and difference comparisons of trnl-f sequences were conducted. the similarity matrix (on-line suppl. tab. 1) ranged between lowest 0.927 (yaglık-mugla cultivars) and highest 0.988 (samsun-hatay cultivars) values. however, it was interesting that different regional cultivars such as those from the samsun, hatay, black sea and mediterranean regions had the highest similarity value, while the same regional cultivars such as those from the mugla, yaglık and aegean region had the lowest value. besides, the difference matrix (fig. 2) demonstrated that hatay and samsun have lowest (0.00) pairwise difference, therefore the highest similarity, while mugla and gemlik have highest (4.052) pairwise difference, and therefore the lowest similarity in analyzed samples. accordingly, it seems that geographically distant cultivars share more sequence similarities than relatively close varieties. previous studies have also demonstrated similar results. for example, in ssr analysis, the turkish olive cultivars analyzed were not grouped according to their their locations of cultivation; southeastern anatolia and aegean region cultivars were grouped together (ipek et al. 2012). in a different study, sarri et al. (2006) reported that 118 olive cultivars from different regions were genetically identical in the mediterranean basin. in another study, all gemlik (turkish cultivar) individuals collected from the same region indicated 100% identity (çelikkol et al. 2014). the low level of genetic variations in cpdna trnl-f regions in turkish olive cultivars analyzed could have arisen from indel events. in this study, a total of 28 indel events were found in cultivars. fig. 1. the alignment of trnl-f regions in seven turkish olive cultivars. the sequences were aligned by clustalw, and identical and similar nucleotide residues were shaded black and grey, respectively, with 100% threshold value. the sequences were annotated with ncbi accession numbers and cultivar names. genetic relationship of turkish olives based on trnl-f acta bot. croat. 77 (1), 2018 91 fig. 2. the pairwise difference comparisons of seven turkish olive cultivars. the blue, orange and green colors represent nei’s distance, within population and between population variations, respectively. europaea species, a phylogenetic tree was constructed using 13 trnl-f sequences (fig. 3). phylogeny showed two major groups, a and b. the group a was then subdivided into two subgroups based on tree topology, named a1 and a2. all o. europaea subsp. europaea individuals from various countries such as australia, morocco, algeria, egypt, italy and syria were clustered in subgroup a1, while five turkish cultivars, including gemlik, yaglık, burhaniye, hatay and samsun were in subgroup a2, with two of the turkish cultivars, mugla and tekir, being separated from others and clustered in group b. the close phylogenetic relationship of five turkish cultivars (gemlik, yaglık, burhaniye, hatay and samsun) with various o. europaea subsp. europaea members from different countries indicated their common ancestral origin. however, two turkish aegean cultivars, mugla and tekir demonstrated a clear divergence from all other olea individuals, indicating that either these two cultivars are native to turkey or they share a totally different origin. overall, germplasm exchanges between countries or some mutations may have been a major contributing factor in variations of turkish olive gene pool. thus, the relevant authorities should develop the necessary breeding programs in order to preserve the purity of of germplasms. moreover, we also constructed an olea genus-level phylogenetic tree using a total of 32 trnl-f sequences (on-line suppl. fig. 3). the phylogeny included two major groups, a and b. the group a was then subdivided into three subgroups a1, a2 and a3, based on the tree topology. subgroup a1 included cultivars from various geographic locations such as algeria, italy, egypt, pakistan, morocco, portugal, spain, australia and syria, subgroup a2 had cultivars from neighboring countries such as tanzania, kenya, zimbabwe, south africa and madagascar, subgroup a3 only contained turkish varieties, and group b had cultivars from countries relatively close to each other, such as laos, philippines, thailand, indonesia and china. the phylogenetic analysis indicated that germplasms could be relatively more conserved in group a2, a3 and b countries. however, clustering of different cultivars in the same group could indicate the possibility of germplasm exchanges befig. 3. phylogenetic tree of various o. europaea subsp. europaea taxa, including turkish olive cultivars. the tree was constructed using trnl-f sequences with maximum likelihood method for 1000 bootstraps. turkish olive cultivars are indicated with a diamond symbol, “ ”. phylogenetic analysis of trnl-f genes in addition to seven turkish cultivars, 25 additional trnl-f sequences from various olea species were obtained from ncbi. thus, a total of 32 trnl-f sequences were comparatively analyzed in three separate phylogenetic trees. the phylogenetic tree of turkish olive cultivars (on-line suppl. fig. 2) demonstrated two main groups, a and b. group a was then subdivided into two subgroups based on the tree topology, a1 and a2. the subgroup a1 included mugla and tekir cultivars, subgroup a2 contained hatay and samsun cultivars, and group b had yaglık, gemlik and burhaniye cultivars. thus, phylogenetic distribution of cultivars did not demonstrate any association inferring the regional localization in terms of cpdna trnl-f regions. to further understand the phylogenetic distribution of turkish olive cultivars along with various o. europaea subsp. kaya e., vatansever r., filiz e. 92 acta bot. croat. 77 (1), 2018 tween countries. previous studies have also reported similar results. besnard et al. (2001) showed that olive genotypes from different countries grouped together without respect to geographical origins. slovenian and croatian olive cultivars were clustered together based on ssr markers (poljuha et al. 2008). a phylogenetic analysis of approximately 40 olea taxa revealed that different country cultivars were combined in a phylogenetic tree based on four plastid regions (besnard et al. 2009). hess et al. (2000) reported that different country cultivars were grouped in a phylogenetic tree in terms of its1 sequences. all these studies indicate that geographical origin was not so effective for olive cultivars as to allow any inference for phylogenetic relationships to be made. overall, the analyzed turkish olive cultivars demonstrated a low level of genetic variation in terms of cpdna trnl-f regions. this could indicate the genetic stability of plastid genomes. in addition, no particular relationship was observed between biogeographic distribution and cultivar localization. what is more, since turkey has 88 different local olive cultivars (iooc 2011), further studies with more cultivars are required for a better understanding of the phylogenetic relationships of turkish olive cultivars with the use of chloroplast genomes. references amane, m., ouazzani, n., lumaret, l., debain, c., 2000: chloroplast-dna variation in the wild and cultivated olives (olea europaea l.) of morocco. euphytica 116, 59–64. besnard, g., breton, c., baradat, p., khadari, b., berville, a., 2001: cultivar identification in olive based on rapd markers. journal of the american society for horticultural science 126, 668–675. besnard, g., casas, r. r., christin, p. a., vargas, p., 2009: phylogenetics of olea (oleaceae) based on plastid and nuclear ribosomal dna sequences: tertiary climatic shifts and lineage differentiation times. annals of botany 104, 143–160. besnard, g., hernández, p., khadari, b., dorado, g., savolainen, v., 2011: genomic profiling of plastid dna variation in the mediterranean olive tree. bmc plant biology 11, 80. çelikkol, a., özkan, u., şan, g., dolgun, b., dağdelen, o., bozdoğan a., konuşkan, d., 2014: genetic stability in a predominating turkish olive cultivar, gemlik, assessed by rapd, microsatellite, and aflp marker systems. turkish journal of botany 38, 430–438. doyle, j. j., doyle, j. l., 1987: a rapid dna isolation procedure from small quantities of fresh leaf tissue. phytochemical bulletin 19, 11–15. ercisli, s., 2004: a short review of the fruit germplasm resources of turkey. genetic resources and crop evolution 51, 419–435. excoffier, l., lischer, h. e., 2010: arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under linux and windows. molecular ecology resources 10, 564–567. green, p. s., 2002: a revision of olea l. kew bulletin 57, 91–140. hannachi, h., breton, c., msallem, m., hadj, s. b., gazzah, m., bervillé, a., 2010: genetic relationships between cultivated and wild olive trees (olea europaea l. var. europaea and var. sylvestris) based on nuclear and chloroplast ssr markers. natural resources 1, 95–103. hess, j., kadereit, j. w., vargas, p., 2000: the colonization history of olea europaea l. in macaronesia based on internal transcribed spacer 1 (its-1) sequences, randomly amplified polymorphic dnas (rapd), and intersimple sequence repeats (issr). molecular ecology 9, 857–868. iooc, 2011: international olive council. retrieved from http:// www.internationaloliveoil.org/. ipek, a., barut, e., gulen, h., ipek, m., 2012: assessment of interand intra-cultivar variations in olive using ssr markers. scientia agricola 69, 327–335. kaya, e., 2015: issr analysis for determination of genetic diversity and relationship in eight turkish olive (olea europaea l.) cultivars. notulae botanicae horti agrobotanici cluj-napoca 43, 96–99. librado, p., rozas, j., 2009: dnasp v5: a software for comprehensive analysis of dna polymorphism data. bioinformatics 25, 1451–1452. luo, n., yu, x., liu, j., jiang, y., 2012: nucleotide diversity and linkage disequilibrium in antioxidant genes of brachypodium distachyon. plant science 197, 122–129. mariotti, r., cultrera, n.g.m., muñoz díez, c., baldoni, l., rubini, a., 2010: identification of new polymorphic regions and differentiation of cultivated olives (olea europaea l.) through plastome sequence comparison. bmc plant biology 10, 211. mendilcioglu, k., 1999: subtropical fruit species (olive). ege university agricultural faculty 12, 43. mes, t. h., kuperus, p., kirschner, j., stepanek, j., oosterveld, p., storchova, h., den nijs, j. c., 2000: hairpins involving both inverted and direct repeats are associated with homoplasious indels in non-coding chloroplast dna of taraxacum (lactuceae: asteraceae). genome 43, 634–641. nei, m., 1987: molecular evolutionary genetics. columbia university, new york. poljuha, d., sladonja, b., setic, e., milotic, a., bandelj, d., jakse, j., javornik, b., 2008: dna fingerprinting of olive varieties in istria (croatia) by microsatellite markers. scientific horticulture 115, 223–230. sarri, v., baldon, l., porceddu, a., cultrera, n. g. m., contento, a., frediani, m., belaj, a., trujillo, i., cionini, p. g., 2006: microsatellite markers are powerful tools for discriminating among olive cultivars and assigning them to geographically defined populations. genome 49, 1606–1615. tajima, f., 1989: statistical method for testing the neutral mutation hypothesis by dna polymorphism. genetics 123, 585–595. tamura, k., stecher, g., peterson, d., filipski, a., kumar, s., 2013: mega6: molecular evolutionary genetics analysis version 6.0. molecular biology and evolution 30, 2725–2729. thompson, j. d., gibson, t., higgins, d. g., 2002: multiple sequence alignment using clustalw and clustalx. current protocols in bioinformatics, 2–3. wallander, e., albert, v. a., 2000: phylogeny and classification of oleaceae based on rps16 and trnl-f sequence data. american journal of botany 87, 1827–1841. watterson, g. a., 1975: on the number of segregating sites in genetical models without recombination. theoretical population biology 7, 188–193. opce-str.vp acta bot. croat. 68 (1), 117–126, 2009 coden: abcra 25 issn 0365–0588 nutlet size, shape and surface ornamentation in 14 onosma species (boraginaceae) riza binzet1*, öznur e. akçin2 1 adiyaman university, vocational school of kahta, 02400 adiyaman, turkey 2 ordu university, faculty of art and science, department of biology, 52750 ordu, turkey nutlets of 14 species of onosma l. in turkey were examined with scanning electron microscope. nutlet (mericarp) morphology of the examined specimens exhibits some variation in shape and size. nutlets of species vary in the range 2.5–6 ´ 2–4.5 mm. the studied nutlet surfaces show some variations. according to surface ornamentations 5 types were defined and illustrated: type 1 (ruminate type), type 2, type 3 (rugose-type), type 4(reticulate-type) and type 5 (pusticulate-type). the reticulate is the most common type in the examined species. six of the species observed belong to the reticulate type, i.e. o. papillosum, o. lycaonicum, o. caerulescens, o. rascheyanum, o. mersinana and o. riedliana. in addition, the nutlet features of o. papillosum h. riedl and o. lycaonicum hub.-mor., previously unpublished, are given for the first time here in detail. it is clear that external nutlet characteristics could especially help in the classification of the species of the complex genus onosma in the future. keywords: onosma, boraginaceae, nutlet, mericarp, morphology, ornamentation, sem introduction the family boraginaceae comprises about 131 genera and 2500 species, mainly annual, bi-annual or perennial herbs and shrubs, some trees and a few lianes, distributed throughout the temperate and subtropical regions of the world. the family is mainly distributed in dry, cliffy and sunny habitats of eurasia, the mediterranean region and western north america (retief and van wyk 1997), with a maximum concentration in iran (willis 1973). this family has about 34 genera and 350 taxa in turkey (yildirimli 2000). the genus onosma l. is the largest genus with about 97 species in turkey and the rate of endemism among the native species is about 50 % (riedl 1978, davis et al. 1988, yildirimli 2000, riedl et al. 2005, binzet and orcan 2007). riedl (1978) reported that the onosma genus has been divided into three sections: protonosma, podonosma and onosma. protonosma and podonosma sections are represented by 1 species, the other onosma species belong to onosma sections. this section is separated into two subsections according to indumentum type: asterotricha (boiss.) gürke and haplotricha (boiss.) gürke. riedl (1978) reported that the acta bot. croat. 68 (1), 2009 117 * corresponding author, e-mail: rbinzet@gmail.com u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:01 color profile: disabled composite 150 lpi at 45 degrees classification of onosma species appears to be partly artificial and in need of re-investigation and that new data may provide useful reference points in a future classification. there have been karyological studies (teppner 1980, 1981, 1988, 1991, 1996a, 1996b); anatomical studies (akçin and engin 2001, binzet and orcan 2003, akçin 2004, akçin and engin 2005, 2007a); palynological studies (binzet and orcan 2003); chemical studies (mellidis and papageorgiou 1987, khajuria & jain 1993, el-shazly et al. 2003, özgen et al. 2004) of onosma species. the first study of the ultramorphology of boraginaceae including onosma was done by khatoon et al. (1994). the first study on nutlet micromorphology of turkish onosma has confirmed micromorphology as a useful tool in the onosma taxonomy (akçin 2007b). material and methods nutlets of 14 species from two subsections of onosma were collected in 1998–2006. o. sintenisii hasskn. ex bornm, o. papillosum h. riedl and o. bulbotrichum dc belong to haplotricha subsect. and o. lycaonicum hub.-mor., o. intertextum hub.-mor., o. briquetii 118 acta bot. croat. 68 (1), 2009 binzet r., akçin ö. e. tab. 1. the localities of the examined species. taxa locality o. sintenisii hasskn. ex bornm sivas, divrigi-kemaliye 44 km, slopes, 26.v.2006, 1060 m, binzet 99. o. papillosum h. riedl adana: yesilkent-tufanbeyli 7 km, steppe, 1560 m, 16.06.2005, binzet 22. o. bulbotrichum dc adiyaman, karahöyük village, slopes, 24.v.2006, 650 m, binzet 85. o. lycaonicum hub.-mor. mersin: mut-karaman, sertavul around, steppe, 1670 m, 19.06.2004, binzet 89. o. intertextum hub.-mor. gümüshane, around karaca cave, 1300m, akçin 1049. o. briquetii czecz. amasya, akdag, çalardi plateau, 28.v. 2006, binzet 128. o. bourgaei boiss. çankiri: ilgaz, roadside, 2000 m, akçin 996. o. rascheyanum boiss. kahramanmaras: çaglayancerit-pazarcik 15 km, stony and rocky slopes, 970 m, 26.05.2004, binzet 87. o. caerulescens boiss. kahramanmaras: pazarcik-gölbasi 20 km, roadside, 900 m, 27.05.2004, binzet 2. o. angustissimum hasskn. et bornm. adana: gülek, gülekkale around, stony and rocky slopes, 1550 m, 26.06.2004, binzet 61. o. ambigens lacaita çorum: iskilip to tosya, 450 m, ergen 1008. o. giganteum lam. osmaniye: yarpuz-hasanbeyli 15 km, roadside, 750 m, 30.06.2004, binzet 52 o. mersinana riedl, binzet et orcan mersin: müglü deresi village, rocky slopes, 1100 m, 07.05.2003, binzet 18. o. riedliana binzet et orcan mersin: gülnar-ermenek 42 km, roadside open field, 1300 m, 05.06.2004, binzet 24. u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:01 color profile: disabled composite 150 lpi at 45 degrees czecz., o. bourgaei boiss., o. rascheyanum boiss., o. caerulescens boiss., o. angustissimum hasskn. et bornm., o. ambigens lacaita, o. giganteum lam., o. mersinana riedl, binzet et orcan and o. riedliana binzet et orcan belong to asterotricha subsect. sample specimens are stored at the herbarium of the department of biology at the university of mersin and university of ordu. collection data of the studied specimens are given in table 1. in order to determine the average nutlet sizes, 15 nutlets from each species were measured. for scanning electron microscopy, dried mature nutlets were mounted on stubs using double-sided adhesive tape. samples were coated with 12.5–15 nm of gold. coated nutlets were examined and photographed with jms-6400 scanning electron microscope. observations were made on the surface patterns of nutlets (stearn 1973). results nutlet size studied nutlet size shows some variations. nutlets of studied onosma species vary in the range 2.5–6 ´2–4.5 mm. o. riedliana has the smallest nutlets (2.5 ´2 mm) and o. bulbotrichum has the largest (6 ´4.5 mm). nutlet colour colour of studied nutlets shows some variations. the colour of the nutlets is brown in o. sintenisii, o. intertextum and o. bourgaei; gray-pale brown in o. papillosum, o. mersinana, o. briquetii and o. riedliana; pale brownish in o. ambigens and o. rascheyanum; pale gray in o. bulbotrichum; gray in o. lycaonicum; reddish brown in o. caerulescens; gray brown in o. angustissimum and o. giganteum. nutlet shape nutlets of 14 taxa were examined in detail in this study. nutlet shape shows some variations. nutlet shape is oblong-ovoid in o. sintenisii; ovoid in o. bulbotrichum, o. lycaonicum, o. bourgaei, o. rascheyanum, o. caerulescens, o. angustissimum, o. giganteum; ovoid-globose in o. intertextum; oblong in o. briquetii and o. mersinana; oblong globose in o. riedliana. in the nutlets of o. bulbotrichum and o. caerulescens in particular sharp ventral and dorsal keels were seen. nutlet ornamentation some variations in nutlet surfaces were determined. five main types in onosma species can be distinguished according to surface ornamentation: type 1 (ruminate type), type 2, type 3 (rugose type), type 4 (reticulate type) and type 5 (pusticulate type); within these some types, variants can be recognised. type 1 (figs. 1–3) is characterised by a surface pushed into a series of large ridges superimposed on the pattern formed by the cell walls. two species observed belong to this type, i.e. o. ambigens and o. bourgaei. type 2 (figs. 4–6) has epidermal cells of nutlet surface formed in an elongated type with varied sizes and shapes. three species observed belong to the elongated type, i.e. o. acta bot. croat. 68 (1), 2009 119 nutlet morphologies of onosma species u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:01 color profile: disabled composite 150 lpi at 45 degrees 120 acta bot. croat. 68 (1), 2009 binzet r., akçin ö. e. plate 1. sem micrographs of nutlet surfaces. type 1: 1 – o. ambigens, 2,3 – o. bourgaei; type 2: 4 – o. briquetii, 5 – o. intertextum, 6 – o. sintenisii; type 3: 7 – o. angustisimum, 8 – o. giganteum; u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:04 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 121 nutlet morphologies of onosma species plate 1. – continued. type 4: 9 – o. papillosum, 10 – o. lycaonicum, 11 – o. caerulescens, 12 – o. rascheyanum, 13 – o. mersinana, 14 – o. riedliana; type 5: 15 – o. bulbotrichum. u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:06 color profile: disabled composite 150 lpi at 45 degrees 122 acta bot. croat. 68 (1), 2009 binzet r., akçin ö. e. t a b . 2 . a c o m p a ri so n o f c h a ra c te rs st u d ie d fo r o n o sm a ta x a n u tl e ts . t a x a n u tl e t si z e s h a p e c o lo u r n u tl e t su rf a c e s u b se c ti o n p h y to g e o g ra p h ic re g io n o . si n te n is ii h a ss k n . e x b o rn m 3 .5 ´ 3 m m o b lo n g -o v o id , a c u te , w it h v e n tr a l a n d d o rs a l k e e l, sh o rt b e a k e d b ro w n t y p e ii h a p lo tr ic h a ir .t u r. e le m e n t o . p a p il lo su m h . r ie d l 4 ´ 2 .5 m m o b lo n g -o v o id , a c u m in a te , w it h v e n tr a l a n d d o rs a l k e e l, sh o rt b e a k e d g ra y -p a le b ro w n t y p e iv h a p lo tr ic h a ir .t u r. e le m e n t o . b u lb o tr ic h u m d c 6 ´ 4 .5 m m o v o id , o b tu se , w it h sh a rp v e n tr a l a n d in d is ti n c t d o rs a l k e e l, b e a k st ro n g ly in c u rv e d p a le g ra y t y p e v h a p lo tr ic h a ir .t u r. e le m e n t o . ly c a o n ic u m h u b .m o r. 3 ´ 2 m m o v o id , a c u te -a c u m in a te , w it h v e n tr a l a n d d o rs a l k e e l, sh o rt b e a k e d g ra y t y p e iv a st e ro tr ic h a ir .t u r. e le m e n t o . in te rt e x tu m h u b .m o r. 4 ´ 3 m m o v o id -g lo b o se , a c u m in a te b ro w n t y p e ii a st e ro tr ic h a ir .t u r. e le m e n t? o . b ri q u e ti i c z e c z . 3 ´ 2 .5 o b lo n g -o v o id , a c u m in a te g ra y -p a le b ro w n t y p e ii a st e ro tr ic h a ir .t u r. e le m e n t o . b o u rg a e i b o is s. 3 ´ 2 .5 m m o v o id , a c u te b e a k , in d is ti n c t d o rs a l k e e l b ro w n t y p e i a st e ro tr ic h a ir .t u r. e le m e n t o . ra sc h e y a n u m b o is s. 5 ´ 3 m m o v o id , b e a k a c u te , in c u rv e d , w it h p ro m in e n t v e n tr a l k e e l a n d la te ra l ri d g e s p a le b ro w n is h t y p e iv a st e ro tr ic h a ir .t u r. e le m e n t o . c a e ru le sc e n s b o is s 4 ´ 3 m m o v o id , a c u m in a te , w it h sh a rp v e n tr a l a n d le ss p ro m in e n t d o rs a l k e e l, sh o rt b e a k e d r e d is h -b ro w n t y p e iv a st e ro tr ic h a ir .t u r. e le m e n t o . a n g u st is si m u m h a ss k n . e t b o rn m . 3 .5 ´ 3 m m o v o id , a c u m in a te -a c u te , w it h v e n tr a l a n d d o rs a l k e e l, sh o rt b e a k e d g ra y -b ro w n t y p e ii i a st e ro tr ic h a ir .t u r. e le m e n t o . a m b ig e n s l a c a it a 3 ´ 2 .5 d o rs a ll y c o m p re ss e d , b e a k a c u te p a le b ro w n is h t y p e i a st e ro tr ic h a ir .t u r. e le m e n t o . g ig a n te u m l a m . 3 .5 ´ 2 m m o v o id , a c u te , w it h v e n tr a l a n d d o rs a l k e e l, v e ry sh o rt b e a k e d g ra y -b ro w n t y p e ii i a st e ro tr ic h a m e d it . e le m e n t st ri g h t o . m e rs in a n a r ie d l, b in z e t e t o rc a n 3 ´ 2 m m o b lo n g , o b tu se -a c u m in a te w it h d o rs a l k e e l g ra y -p a le b ro w n t y p e iv a st e ro tr ic h a m e d it . e le m e n t o . ri e d li a n a b in z e e t o rc a n 2 .5 ´ 2 m m o b lo n g -g lo b o se , a c u m in a te , w it h d o rs a l k e e l a n d sh o rt b e a k g ra y -p a le b ro w n t y p e iv a st e ro tr ic h a m e d it . e le m e n t u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:06 color profile: disabled composite 150 lpi at 45 degrees briquetii, o. intertextum and o. sintenisii. in o. briquetii, the surface epidermis cells are elongated rectangular and narrow-large. cells walls are clear and wavy. o. intertextum has elongated rectangular and large epidermis cells. cell walls are clear and straight. in the nutlet of o. sintenisii, the surface epidermis cells are elongated-rectangular and large-narrow. cell walls are clear and straight. type 3 (figs. 7–8) is the rugose type, characterised by the epidermal cells of the nutlet surface having small or fine wrinkles. this type is recognised on the nutlet of two studied species, i.e. o. angustisimum and o. giganteum. type 4 (figs. 9–14) is the reticulate surface type. the epidermal cells of the nutlet surface are formed in a reticulate ornamentation with varied sizes and shapes of mesh. most species of onosma have the type 4 nutlet surface, such as o. papillosum, o. lycaonicum, o. caerulescens, o. rascheyanum, o. mersinana and o. riedliana. in these species, o. mersinana is especially distinct from the others. in o. mersinana, the cells are clear. boundaries of cells are elevated and thick. o. caerulescens and o. riedliana are similar. in these species, the cells are small and isodiametric. cell walls are ±clear. the other similarity is seen in o. papillosum and o. rascheyanum. the surface type is reticulate. cells are ±clear. boundaries of cells are elevated. in o. lycaonicum, the surface type is reticulate. cells are ±clear, cell walls are elevated. type 5 (fig. 15) is characterised by the pusticulate pattern and by the epidermal cells of the nutlet surface having compressed tubercules. boundaries and cells are not clear. this type is recognised on the nutlet of o. bulbotrichum. discussion the examined 14 species belong to the section onosma. three species, o. sintenisii, o. papillosum and o. bulbotrichum belong to the subsection haplotricha. the other species belong to the subsection asterotricha. nine species are endemic for turkey. riedl (1978) needs to include new characteristics that may provide useful reference points in a future classification. the species of onosma members are very similar and this similarity often causes identification problems. boraginaceae fruits are characterized by one-seeded nutlets with a sclerified exocarp protecting the seeds (diana et al. 2002). in the present study, the nutlet colour of the studied taxa appeared to be brown or gray; however it is difficult to distinguish taxa on the basis of fruit colour. therefore the colour of nutlet was not used as diagnostic character to distinguish onosma species in this study. there is an opinion that external nutlet characters, size, shape, colour and ornamentation are of limited taxonomic value (riedl 1978). however, the sculpturing of the nutlet surface patterns, as seen by scanning electron microscope, shows specific variations. nutlet size and shape show specific characteristics. onosma riedliana has the smallest nutlets (2.5´2 mm), whereas o. bulbotrichum has the largest nutlets (6´4.5 mm). nutlet shape in o. bulbotrichum and o. caerulescens is characterized by sharp ventral and dorsal keels. in this study, five types of fruit surface were examined. most species of onosma have nutlet coat type 4, such as o. papillosum, o. lycaonicum, o. caerulescens, o. rascheyanum, o. mersinana and o. riedliana. the other species determined belong to types 1, 2, 3 acta bot. croat. 68 (1), 2009 123 nutlet morphologies of onosma species u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:06 color profile: disabled composite 150 lpi at 45 degrees and 5. o. ambigens and o. bourgaei are morphologically similar and have type 1, and this type is very distinct. o. briquetii, o. intertextum and o. sintenisii are type 2. o. angustisimum and o. giganteum have type 3 nutlet coat sculpturing. o. bulbotrichum has type 5. o.sintenisii, o. papillosum and o. bulbotrichum belong to the subsection haplotricha. these species have a different surface type. except for o. bulbotrichum, the same surface type was seen in both species belonging to the subsection asterotricha and species belonging to he subsection haplotricha. accordingly it is difficult to use nutlet surface features at subsection level. in this study, the nutlet features of onosma papillosum h. riedl and o. lycaonicum hub.-mor. are given for the first time, and i the morphology is described as a useful tool for species identification, as indicated previously (akçin 2007b). mericarp characters are useful for the identification of anchusa l. species (aytasz akçin and ulu 2008). fruit surface and seed coat morphologies are considered useful characters for species identification of cynoglossum (akçin 2008). in these studies, the micromorphology of several plant seeds and fruits has been examined with sem and their importance in plant taxonomy has been emphasized (olgun 1997, çoszkunçelebi et al. 2000, khalik et al 2008). the external nutlet characters, especially surface ornamentation, could help in classification species of the complex genus onosma in the future. acknowledgements the authors would like to thank personnel of the electron microscopy laboratory of karadeniz technical university. references akçin, o. e., 2004: endemik onosma bornmuelleri hausskn.’nin morfolojisi anatomisi ve ekolojisi üzerine bir arasztirma (an investigation on the morphology, anatomy and ecology of endemic onosma bornmuelleri hausskn.). ecology 13, 13–19. akçin, o. e., 2007a: the morphological and anatomical properties of endemic onosma armenum dc. (boraginaceae) species. international journal of natural and engineering sciences 1, 37–43. akçin, o. e., 2007b: nutlets micromorphology of some onosma l. (boraginaceae) species from turkey. biologia, bratislava 62, 684–689. akçin, o. e., 2008: seed coat and fruit surface micromorphology of some cynoglossum l. (boraginaceae) species. bangladesh journal of botany 37, 115–119. akçin, o. e., engin, a., 2001: comparative morphological and anatomical study on species of onosma isauricum boiss. et heldr. and o. stenolobum hausskn. ex h. riedl the herb journal of systematic botany 8, 75–95. akçin, o. e., engin, a., 2005: the morphological and anatomical and ecological properties of endemic onosma bracteosum hausskn. & bornm. (boraginaceae) species. turkish journal of botany 29, 317–325. 124 acta bot. croat. 68 (1), 2009 binzet r., akçin ö. e. u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:06 color profile: disabled composite 150 lpi at 45 degrees aytasz akçin, t., ulu, sz., 2008: micromorphological characters of fruit of some anchusa l. (boraginaceae) species from turkey. international journal of natural and engineering sciences 2, 63–67. binzet, r. orcan, n., 2003: morphological and palynological studies on onosma roussaei dc. and onosma giganteum lam. (boraginaceae). the herb journal of systematic botany 10, 57–76. binzet, r., orcan, n., 2007: a new species of onosma l. (boraginaceae) from southern turkey. a journal for botanical nomenclature 17, 8–10. çoszkunçelebi, k., kandemir, a., beyazogh lu, o., 2000: scanning electron microscopic examination of the seeds of ornithogalum (liliaceae) species distributed in black sea region of turkey. biologia, bratislava 55(4), 397–401. davis, p. h., mill, r. r., tan, k., 1988: flora of turkey and the east aegean islands, 10, edinburgh university press, edinburgh. diana, n., förther, h., hilger, h. h., 2002: a systematic analysis of heliotropium, tourneforti, and allied taxa of the heliotropiaceae(boraginales) based on itsi sequences and morphological data. american journal of botany 89, 287–295. el-shazly, a., abdel-ghani, a., wink, m., 2003: pyrrolizidine alkaloids from onosma arenaria (boraginaceae). biochemical systematics and ecology 31, 477–485. khalik, k. a., el-ghani, m. a., el-kordy, a., 2008: fruit and seed morphology in galium l. (rubiaceae) and its importance for taxonomic identification. acta botanica croatica 67, 1–20. khajuria, r. k., jain, s. m., 1993: two new naphthoquinones from the roots of onosma hispidum. indian journal of chemistry 32, 390–391. khatoon, s., mehrotra, s., bajbai, v. k., mehrotra, b. n., 1994: ultramorphology of some boraginaceous taxa used as ratanjot. fedes repertorium 105, 61–712. mellidis, a. s., papageorgiou, v. p., 1987: lipids from roots of onosma heterophylla. phytochemistry 26, 451–454. olgun, a., beyazogh lu, o. 1997. micromorphological studies of carex section mitratae (cyperaceae) in turkey. rhodora 99, 368–375. özgen, u., maksut, c., kazaz, c., secen, h., 2004: naphthoquinones from the roots of onosma argentatum hub.-mor. (boraginaceae). turkish journal of chemistry 28, 451–454. retief, e., van wyk, a. e., 1997: palynology of southern african boraginaceae: the genera lobostemon, echiostachys and echium. grana 36, 271–278. riedl, h., 1978: boraginaceae, in: davis, p. h. (ed.), flora of turkey and the east aegean islands 6, 237–437. edinburgh university press, edinburgh. riedl, h., binzet, r., orcan, n., 2005: a new species of onosma (boraginaceae-lithospermeae) from southern turkey. edinburgh journal of botany 61, 127–130. stearn, w. t., 1973: botanical latin. david and charles newton, london. teppner, h., 1980: die onosma alboroseum-gruppe (boraginaceae). phyton (austria) 20, 135–157. acta bot. croat. 68 (1), 2009 125 nutlet morphologies of onosma species u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:06 color profile: disabled composite 150 lpi at 45 degrees teppner, h. 1981: karyosystematick von onosma stellulatum, o. pygmaeum und o. leptanthum (boraginaceae). botanische jahrbücher für systematik 102, 297–306. teppner, h., 1988: onosma kaheirei sp. nova und o. erectum (boraginaceae) aus griechenland. phyton (austria) 28, 115–131. teppner, h., 1991: karyology of some greek onosma species (boraginaceae). botanica chronica 10, 271–292. teppner, h., 1996a: remarks to the onosma species o. bourgaei, o. spruneri and o. stellulata (boraginaceae) offered. samentauschverzeichnis, botanical garden of the institute of botany, graz, 33–39. teppner, h., 1996b: die onosma-arten (boraginaceae-lithospermeae) rumaniens. stapfia 45, 47–54. willis, j. c., 1973: a dictionary of the flowering plants and ferns. university press, cambridge. yildirimli, sz., 2000: the chorology of the turkish species of boraginaceae family. the herb journal of systematic botany 7, 257–272. 126 acta bot. croat. 68 (1), 2009 binzet r., akçin ö. e. u:\acta botanica\acta-botan 1-09\binzet.vp 16. travanj 2009 11:52:06 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 437 acta bot. croat. 73 (2), 437–446, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 distribution and morphological variations of invasive macrophytes elodea nuttallii (planch.) h. st. john and elodea canadensis michx in croatia aleksandra kočić1, janja horvatić1, sven d. jelaska2 1 josip juraj strossmayer university of osijek, department of biology, cara hadrijana 8a, hr-31000 osijek, croatia 2 university of zagreb, faculty of science, division of biology, marulićev trg 20/2, hr-10000 zagreb, croatia abstract – the invasive species elodea nuttallii was recorded for the fi rst time in the croatian fl ora in 2006, in the drainage channels of kopački rit (baranja). after its establishment, e. nuttallii begins to spread to the eastern and northern part of the drainage channel network from 2006–2009. high water levels are responsible for the linear spreading direction of e. nuttallii, e. nuttallii and e. canadensis show a wide range of morphological variation, which is characteristic of successful invaders. to show morphological variations of two elodea species, the most impor tant characters indicated in the literature were measured on 24 fresh collected samples from the seven sites in croatia. in spite of some overlap in leaf length and width between the two elodea species, the differences of all morphological traits except internode length are statistically signifi cant. in e. nuttallii leaf width, length and internode length show a higher morphological variability as a result of the higher adaptive strategy to environmental parameters. the most reliable morphological characters distinguishing e. nuttallii and e. canadensis are leaf width 0.5 mm below the tip and the angle at the apex. e. nuttallii can be expected to spread to other areas of croatia. keywords: distribution, elodea canadensis, elodea nuttallii, invasive species, morphological variation introduction the genus elodea michx. (hydrocharitaceae) consists of rooted, submerged, perennial, freshwater macrophytes, native to north america. since the 19th century, several elodea species have become adventive in europe. the fi rst record was elodea canadensis michx. * corresponding author, e-mail: akocic@biologija.unios.hr copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. kočić a., horvatić j., jelaska s. d. 438 acta bot. croat. 73 (2), 2014 from the fi rst fi nding reported in 1836 in a pond in ireland (simpson 1984), it became widespread in north and central european countries. by the middle of the last century it seems to have reached the maximum extent of its distribution (simpson 1986, 1990, rodwell 1995). in croatia it was recorded for the fi rst time in 1894 in ješkovo pond in gola, podravina (nikolić 2009). the second species, elodea nuttallii, has been known in europe since 1939. the plant was fi rst recorded in belgium (wolff 1980, simpson 1984) and it is spreading rapidly. it has displaced e. canadensis and has become as frequent in northern europe as e. canadensis was before. so far, e. nuttallii has colonised most of croatia’s neighbouring countries: hungary (király et al. 2007b), slovenia (király et al. 2007a, mazej grudnik and savinek 2010, mazej grudnik and germ 2013), serbia (vukov et al. 2006, ljevnaić-mašić 2010), as well as all national stretches of the danube, upstream (germany, austria, slovakia) and downstream (serbia, bulgaria, romania) from croatia. it is only in croatia in the danube river basin that it has not been recorded (janauer et al. 2007). since only female plants of the two elodea species were introduced in europe, both propagate mainly by vegetative reproduction (simpson 1986, james et al. 1999). the rapid growth, the rapid spread provided by vegetative reproduction, and the phenotypic plasticity are all characteristics of successful invaders. due to low genetic variability of invasive species in the introduced range (riis et al. 2010) phenotypic plasticity is an essential of elodea invasiveness in aquatic environments (hérault et al. 2008). by producing a better phenotype-environment match (agrawal 2001) it maximizes fi tness in variable conditions. in e. nuttallii, the extremes of the morphological variation are such that they appear to be distinct taxa in europe, one with long, fl at leaves, and the other with shorter, broader, strongly refl exed leaves (simpson 1988, thiébaut and di nino 2009). simpson (1988) described e. nuttallii as the most variable of the species in the british isles. however, as a result of its wide range of morphological variations there are some differences among authors about the main distinguishing characters of elodea species. these are linked to leaf length and width and the curvature of the plants. because of this variability, we measured the most important characters indicated in the literature on fresh material of elodea species collected in the fi eld surveys to identify morphological features that are the most important for quick determination in fi eld surveys. thus, the fi rst aim of our study was to show the distribution of e. nuttallii as a new species and the new sites of e. canadensis, which is well established in croatia. the second aim was to indicate the main differences between two elodea species in croatia and give an identifi cation key with the most important morphological characters. materials and methods study area one of the largest remaining fl oodplain areas in central europe stretches on territory belonging to hungary, croatia and serbia and lies where the danube and drava rivers meet. in the croatian part, the croatian baranja region (fig. 1), the fl oodplain area lies within the triangle between the danube and drava rivers and is protected as the kopački rit nature park. in the past, the area was covered by alluvial forests and marshes and was under the constant impact of fl ood water. the marshy and fl ooded area was considerably reduced durdistribution and variations of two invasive elodea species in croatia acta bot. croat. 73 (2), 2014 439 ing the 19th century by reclamation activities. after the drainage network system was constructed, parts of the former fl oodplain were turned into large agricultural fi elds. the water in the drainage channel system is controlled by the danube and drava rivers. high water levels in the channel system are most frequent in may and june and low water levels from august to october. occasionally, water level can also be high in autumn, due to the high amount of precipitation. the drainage network system of baranja mainly drains the arable land. resulting fertilizer run-off causes eutrophication, leads to channels being overgrown with vegetation and consequently, due to mud sedimentation, the channels become shallower. thanks to fi nanfig. 1. a) the spread of e. nuttallii in baranja (croatia) in the investigated period 2006–2009. corine land cover map with classes: agriculture, forest, urban and wetland is used as background of baranja. the height of the bar represents the adjusted braun-blanquet scale of abundance. b) distribution map of e. nuttallii and e. canadensis in croatia. distribution map of e. canadensis represents the new sites in the lonja river basin (▲) and known distribution according to flora croatica database – fcd (▲). kočić a., horvatić j., jelaska s. d. 440 acta bot. croat. 73 (2), 2014 cial investment in the waterways of baranja during the last few years, the drainage channels are subject to management with regular harvesting and maintaining. methods field work was carried out at 75 sites that were monitored during 2006, 2007, 2008 and 2009 in the summer (july–august). e. canadensis and e. nuttallii were found at seven sites. the percentage cover occupied by the elodea species was determined visually from the 50 m reference channel length (haury et al. 1996) and converted to a braun-blanquet scale (braun-blanquet 1964) adjusted as follows; 1= a few scattered specimens, 2 ≤ 5%, 3 = 5–25%, 4 = 25–50%, 5 = 50–75%, 6 > 75%. the elodea species were identifi ed according to casper and krausch (1980) and simpson (1986). during the investigated period, e. canadensis was found at three sites and seven samples were collected at different times, while e. nuttallii was found at four sites and 17 samples were collected at different times. voucher specimens were deposited in a private herbarium. a few plants of elodea species were randomly collected to investigate the morphological variations of each elodea sample. the average values of collected plants for each sample were used in further analysis for each measured morphological trait. all collected samples were analysed morphometrically in the laboratory. samples of the fresh plants were captured by digital imaging equipment. scion image beta 4.0.3 software (scion corporation 1997–2005) was used to measure the morphological traits of each elodea plant: leaf length, leaf width at the mid-point of the fully developed upper leaves, leaf width 0.5 mm below the tip, the angle at the apex between leaf margin and midrib, as well as the internode length, obtained as the length of ten internodes below the three-cm long apex. also, the width/ length ratio was further calculated and the curvature of the leaves was visually determined. the t-test was used to compare the differences of the morphological traits between the two species. results during the period of investigation, e. nuttallii and e. canadensis had geographically separated distributions in two different river basins (fig. 1b): e. canadensis appears in the lonjsko polje fl oodplain (sava river basin) and e. nuttallii in the kopački rit fl oodplain (the mouth of the river drava into the danube). e. nuttallii was found at four sites in the drainage channel system of baranja (fig. 1b). after it was established in the period from 2006–2008 in kopački rit fl oodplain, linear spreading along the drainage network channels was noted in 2009 (fig. 1a). e. canadensis was found at three new sites in lonja river basin in the central part of croatia during the investigation period. at all investigated sites, both elodea species for the most part grew in relatively rich communities and never as the dominant species. the most signifi cant taxonomical character of the two elodea species is connected to the male fl owers (dandy 1980) and the leaf curvature (bowmer et al. 1995, sell and murrell 2007). with an evident lack of petals, male fl ower of e. nuttallii breaks off and fl oats on the surface in anthesis. however, only female plants have been found in europe (simpson 1988). furthermore, most of the samples collected in the research area of e. nuttallii had only straight and no recurved leaves (fig. 2a). in both elodea species the leaves are usually in whorls of three (rarely more, sometimes two). in e. nuttallii leaves were longer (7.62–14.9 mm) and distribution and variations of two invasive elodea species in croatia acta bot. croat. 73 (2), 2014 441 narrower at the mid-point (0.63–1.64 mm) and at 0.5 mm below the tip (0.33–0.72 mm). in e. canadensis leaves are shorter (5.62–9.61 mm) and wider at the mid-point (1.83–2.38 mm) and at 0.5 mm below the tip (1.03–1.51 mm). internode length is similar in both elodea species. the angle at the leaf apex in e. nuttallii did not exceed 34º, while in e. canadensis it is in a range between 50º and 60º. the width/length ratio is also higher in e. canadensis (0.24– 0.40) than in e. nuttallii (0.09–0.17). the most reliable characters for distinguishing e. canadensis from e. nuttallii were the width of a leaf measured at a point approximately 0.5 mm below the apex, the angle at the apex and the width/length ratio (tab. 1). they were greater in e. canadensis and show the shape of the leaf apex, which is the most important taxonomical feature indicated in identifi cation keys (simpson 1984, dandy 1980). in e. nuttallii leaf apices are acuminate or narrowly acute, while in e. canadensis they are obtuse or broadly acute (fig. 2). tab. 1. morphological differences between species elodea canadensis and e. nuttallii. the mean ± standard deviation and total range of parameter values (in parentheses) were given with sample sizes for e. canadenis (n = 7) and for e. nuttallii (n = 17). the morphological characters were compared by t-tests and the signifi cance levels (p) are shown. e. canadensis e. nuttallii statistics leaf width at the mid-point (mm) 2.05±0.22 (1.83–2.38) 1.14±0.27 (0.63–1.64) t = 6.602 p < 0.001 leaf length (mm) 7.22±1.38 (5.64–9.61) 10.45±2.11 (7.62–14.01) t = –4.449 p < 0.001 width/length ratio 0.29±0.06 (0.24–0.40) 0.11±0.03 (0.09–0.17) t = 7.039 p < 0.001 leaf width 0.5 mm below the tip (mm) 1.22±0.17 (1.03–1.51) 0.50±0.09 (0.33–0.72) t = 8.152 p < 0.001 angle at the leaf apex (°) 54.34±3.49 (50.89–60.35) 28.31±3.55 (19.92–33.97) t = 10.814 p < 0.001 internode length (mm) 58.65±8.11 (38.35–81.78) 52.64±18.64 (29.52–87.43) t = 0.405 p = 0.692 our investigation showed that leaf length and width can be used to separate species, although these characteristics exhibited some overlap between e. canadensis and e. nuttallii (tab. 1). the variability of leaf length, width and internode length and some overlap between these characteristics in the investigated elodea species show that they are more dependent on external environmental parameters. leaf width, length and internode length (tab. 1) of e. nuttallii show a higher morphological variability in relation to e. canadensis. in our investigation the invasive species e. nuttallii was found for the fi rst time in croatia. we propose an identifi cation key for two elodea species that exist at the moment in croatia according to literature (dandy 1980, simpson 1986, bowmer et al. 1995, erhard 2005) and our results: e. canadensis: leaves linear-oblong or ovate. most of leaves are 1.75 mm wide or more. leaf apices are broadly acute or obtuse. angle at the leaf apex is greater than 45º. towards the stem apex leaves usually overlapping in regular rows and lying along the stem, often oblong or ovate. kočić a., horvatić j., jelaska s. d. 442 acta bot. croat. 73 (2), 2014 e. nuttallii: leaves linear or linear-lanceolate. most of leaves are less than 1.75 mm wide. leaf apices are narrowly acute or acuminate. at least some leaves are strongly recurved. leaf lamina is often strongly twisted. angle at the leaf apex is less than 45º. leaves are usually folded along the midrib, somewhat recurved, with undulate margins, rarely more than 10 mm long. discussion the main direction of the spread of e. nuttallii in the danube river basin is from western europe down the river into the delta area (janauer et al. 2008), so the introduction pathway of the species in croatia was probably from hungary. in fact, e. nuttallii was noticed in the southern part of hungary, along the danubian river basin near the croatianhungarian border (király et al. 2007b). fig. 2. (a–c) elodea nuttallii: a) part of the plant, b) whorl with the leaves, c) female fl ower. (d–e) elodea canadensis: d) part of the plant, e) whorl with the leaves. photo by a. kočić. distribution and variations of two invasive elodea species in croatia acta bot. croat. 73 (2), 2014 443 since the fi rst fi nding of e. nuttallii in 2006 and its establishment in baranja (croatia), it has spread to the eastern and northern part of the drainage channel network within the investigated period (fig. 1a). the spread and direction of the e. nuttallii pathways in croatia are probably caused by high water levels, which are a key factor in the fl ooded research area that links the water of the main river with the drainage network system (fig. 1a) and interconnects the stagnant waters in the channels. our results show that the direction in which e. nuttallii spreads is along the channels. the linear spread of e. nuttallii was also recorded in some neighbouring countries: in hungary along the danube river (király et al. 2007b), in serbia along the banatska palankanovi bečej canal, which is directly connected with the danube river (ljevnaić-mašić 2010). linear spread was recorded in some other parts of europe, for example in poland along the vistula river (kamiński et al. 2010). e. nuttallii was recently also found in the rivers drava and lendava in slovenia (király et al. 2007a, mazej grudnik and savinek 2010, mazej grudnik and germ 2013). thus, the second possible direction of introduction that we can expect in croatia is from slovenia along the drava river. however, since e. nuttallii is easily mistaken for e. canadensis, its present distribution in croatia might be more extensive than has been actually established. the cause of the linear spread of e. nuttallii in the baranja drainage system could be the mechanical control conducted for several years. this method did not reduce the population of e. nuttallii, but, on the contrary, probably had an important role in increasing the rate of spread due to transportation of cut-off fragments in the water, as found by barrat-segretain (2005). moreover, regular cleaning reduces the biological resistance and favours the establishment of alien species. thus, in slovenia the spread of e. canadensis is described as the consequence of habitat change and destruction (kuhar et al. 2010). our investigation showed the spread of e. canadensis at some new sites in the lonja river basin. it was recorded along the sava river in croatia (nikolić 2009) and it is well established in croatia (fig. 1b). during the period of investigation it grew in relatively rich communities without any remarkable increases in the cover at researched sites. simpson (1984) describes increases in the cover of e. canadensis as rare events. a few months after elodea has invaded habitats, its cover increases for a brief period and then declines (barratsegretain and cellot 2007). stands of e. canadensis were noted in croatia after establishment in a hydro-power plant reservoir (lodeta et al. 2009). the measured morphological characters of two elodea species in general confi rm that e. canadensis is a species with broader and shorter leaves and e. nuttallii with longer and narrower leaves (simpson 1986, 1988, dandy 1980). however, results of morphometric comparison shows that in both species the leaves are smaller than stated by the above mentioned authors, and for e. nuttallii they are similar to the fi ndings of erhard (2005) and thiébaut and di nino (2009). as regarding leaf width, dandy (1980) and simpson (1986, 1988) take 2 mm as the limiting value (e. canadensis have leaves broader then 2 mm, and e. nuttallii have narrower leaves). our research shows that some specimens of e. canadensis have narrower leaves (below 2 mm), which is in accordance with research of bowmer et al. (1995) who indicate that if the majority of leaves are 1.75 mm wide or more the species is e. canadensis and if the majority of leaves are < 1.75 mm wide then it is e. nuttallii. in relation to identifi cation keys in both species the leaves were smaller in size. the morphological differences can be explained by ecological parameters and shallow water of fl oodplains (down to 2 m) in our research. as thiébaut and di nino (2009) explained for e. nuttallii, the kočić a., horvatić j., jelaska s. d. 444 acta bot. croat. 73 (2), 2014 shorter and broader-leaved phenotype typically occurs in shallow streams, whereas the longer and narrower-leaved phenotype occurs in deeper waters. high morphological variations in leaf length and its dependency to environmental parameters exclude it as a relevant distinguishing character of elodea species. we found that the angle at the leaf apex proved to be a good character for distinguishing e. canadensis and e. nuttallii with a limit around 45°, which is similar to the fi nding of erhard (2005). the width of a leaf measured at a point approximately 0.5 mm below the apex, the angle at the apex and the width/length ratio were greater in e. canadensis, and show the shape of the leaf apex that is the most important taxonomical feature indicated in identifi cation keys (dandy 1980, simpson 1984). simpson (1986) and sell and murrell (2007) describe the leaf apices as important distinguishing leaf characters. in e. canadensis they are obtuse or broadly acute and in e. nuttallii acuminate to narrowly acute. moreover, according to erhard (2005) in e. nuttallii the lateral leaf margins are more or less parallel, and in e. canadensis are convex. leaf size characters and internode length showed high morphological variability especially in e. nuttallii, which simpson (1988) describes as the most variable of the species in the british isles. the higher morphological variability of e. nuttallii is explained by trémolières et al. (2007) as a higher adaptation strategy in relation to e. canadensis. the impact of e. nuttallii at the established sites in the channel drainage network of baranja is currently negligible; however, because of the interactions between species and new habitats a spread of the taxon is expected (larson and willén 2007). in many countries, the introduction of e. nuttallii has resulted in displacement of e. canadensis from many waters (simpson 1990). barrat-segretain (2005) explains that after e. nuttallii fragments colonize the area with e. canadensis, the two elodea species will probably coexist at least for a time, but displacement often occurs over a relatively short time, in one or two years (james et al. 1999). in croatia, there are no overlaps at the moment between the distributions of the two species. considering that e. nuttallii is in its expansionary phase in europe and that it spreads much more rapidly than e. canadensis, we can expect the spread of e. nuttallii to the new areas. as a new invasive species in croatia, its current invasions and spreading need to be carefully monitored. the fi rst record in croatia of the invasive species e. nuttallii was in 2006 in the drainage network channels of kopački rit (baranja). high water levels are responsible for e. nuttallii spreading in a linear direction in the investigated period in the fl ooded area. as regards the species e. canadensis already established in croatia, we have located some new sites.a higher morphological variability of leaf width, length and internode length in e. nuttallii in relation to e. canadensis is a result of higher adaptivity to environmental parameters. to distinguish e. nuttallii and e. canadensis the most reliable parameters are leaf width 0.5 mm below the tip and the angle at the apex. because of the rapid spread of e. nuttallii in europe we can expect its spread to new areas in croatia. acknowledgements this research was partly sponsored by croatian ministry of science, education and sports (project number 285-0000000-3175). we are grateful to the two anonymous reviewers for the constructive comments that signifi cantly improve the paper. distribution and variations of two invasive elodea species in croatia acta bot. croat. 73 (2), 2014 445 references agrawal, a. a., 2001: phenotypic plasticity in the interactions and evolution of species. science 294, 321–326. barrat-segretain, m.-h., 2005: competition between invasive and indigenous species: impact of spatial pattern and developmental stage. plant ecology 180, 153–160. barrat-segretain, m.-h., cellot, b., 2007: response of invasive macrophyte species to drawdown: the case of elodea sp. aquatic botany 87, 255–261. bowmer, k. h., jacobs, s. w. l., sainty, g. r., 1995: identifi cation, biology and management of elodea canadensis, hydrocharitaceae. journal of aquatic plant management 33, 13–19. braun-blanquet, j., 1964: pfl anzensoziologie. springer verlag, wien, new york. casper, s. j., krausch h. d., 1980: süsswasserfl ora von mitteleuropa. band 23: pteridophyta und anthophyta. veb gustav fischer verlag, jena. dandy, j. e., 1980: elodea. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. 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serbia. proceedings 36th international conference of iad. austrian committee danube research / iad, vienna, 127–131. wolff, p., 1980: die hydrilleae (hydrocharitaceae) in europa. göttinger floristische rundbriefe 14(2), 33–56. untitled acta bot. croat. 75 (1), 2016 11 acta bot. croat. 75 (1), 11–16, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0015 issn 0365-0588 eissn 1847-8476 selenium induced selenocysteine methyltransferase gene expression and antioxidant enzyme activities in astragalus chrysochlorus özgür çakir1*, neslihan turgut-kara1, şule ari1,2 1 department of molecular biology and genetics, faculty of science, istanbul university, 34134 vezneciler istanbul, turkey 2 research and application center for biotechnology and genetic engineering, istanbul university, 34134 vezneciler, istanbul, turkey abstract – astragalus sp. are used in folk medicine because of their biological activities and are known for the ability to accumulate high levels of selenium (se). the purpose of this study was to explore gene expression of selenocysteine methyltransferase (smt), responsible for forming mesecys, and activities of ascorbate peroxidase (apx), peroxidase (pox), catalase (cat) and glutathione reductase (gr) enzymes in callus tissues of astragalus chrysochlorus growing in different se-containing media. quantitative real-time polymerase chain reaction assay was done for quantifi cation of smt gene transcript and it was normalized to actin gene. it was found that transcript level of callus tissues grown at 5.2 μm and 26.4 μm se-enriched media was lower than that of the control callus. in contrast, a high level of se (132.3 μm) in the medium caused an approximately 4.26 times higher level of smt transcript in callus than the control. apx, pox, cat and gr enzymes were all effected by different se concentrations. while pox and apx activities were higher then control, cat and gr activities decreased. these results show that an increase of smt gene expression led to a rise in apx and pox, but a suppression of cat and gr enzymes activities in astragalus chrysochlorus. this suggests that se could be involved in the antioxidant metabolism in astragalus chrysochlorus. keywords: antioxidant enzymes, callus, real time pcr, selenium, selenocysteine methyltransferase * corresponding author, e-mail: ozgurckr@istanbul.edu.tr introduction antioxidant selenoproteins contain selenium (se) which is a very important element for animals, microorganisms and some other eukaryotes (birringer et al. 2002, berken et al. 2002). these organisms contain the selenocysteine (secys) that may be incorporated in selenoproteins. in addition to nutritional needs for se, the element also has health effi cacy. it is also effective in reducing the incidence of some debilitating disorders (mckenzie et al. 2001, foresta et al. 2002, beck et al. 2003, soriano-garcia 2004) and there is some evidence that se plays a role as a cancer preventive agent when given in certain amounts (ip 1998, combs and gray 1998, fleming et al. 2001, whanger 2004). selenium is metabolized through the sulfur (s) assimilation pathway. selenocysteine methyltransferase (smt) is the key enzyme in se metabolism and it was isolated from the se hyperaccumulator astragalus bisulcatus for the fi rst time (neuhierl et al. 1999). the smt enzyme couples the methyl group with selenocysteine (secys) to produce semethylselenocysteine. a. bisulcatus smt gene was overexpressed in arabidopsis thaliana and brassica juncea and it was shown that selenium accumulation and tolerance were enhanced in smt transgenics (ellis et al. 2004, leduc et al. 2004). in our previous work, smt cdna sequences were isolated from astragalus chrysochlorus (çakir and ari 2013). smt was expressed continuously in the tissues of the se hyperaccumulator astragalus bisulcatus (pickering et al. 2003). while most plants cannot survive with high concentrations of se, some of them can tolerate high concentrations and accumulate se, like a. bisulcatus. thus, the amount of se is of vital importance for plants because it could be toxic at high concentrations. se can change a plant’s antioxidant capacity. this can be achieved on two ways; fi rst, by changing the antioxidant activity of selenocompounds, and second, by se inducing a plant’s antioxidants (hartikainen 2005). enzymes of antioxidant metabolism such as ascorbate peroxidase (apx), peroxidase (pox), glutathione reductase (gr) and catalase (cat) play an important role against reactive oxygen species (apel and çakir ö., turgut-kara n., ari ş. 12 acta bot. croat. 75 (1), 2016 hirt 2004, habibi and hajiboland 2012). because of the biological activities like hepatoprotective, antioxidative, immunostimulative and antiviral properties, astragalus species have been used in traditional medicine (ionkova et al. 1997). the chemical contents of them have been broadly investigated because of their economic and medicinal importance (calis et al. 1997, bedir et al. 1998, yahara et al. 2000). one of the turkish endemics, a. chrysochlorus, has been determined as a secondary se accumulator and a cdna sequence of the smt gene (accession number: gq844862) named achsmt was isolated (ari et al. 2010). the achsmt cdna has an open reading frame (orf) of 1020 bp and this orf encodes 339 amino acid residues with a 36.94 kd molecular mass (çakir and ari 2013). the aim of this work was to investigate the expression pattern of the smt gene and determine the alteration of activities of enzymes related to the antioxidant metabolism of a. chrysochlorus treated with se. here we report the activities of apx, pox, cat and gr enzymes in callus tissues of a. chrysochlorus grown in different concentrations of se and the expression of a crucial gene related to se metabolism by real-time quantitative pcr. materials and methods plant material plants and seeds were collected from sertavul, karaman, turkey and were identifi ed by prof. dr. tuna ekim (istanbul university, faculty of science, department of botany, istf no: 40006, istanbul university, faculty of science herbarium). germination of seeds and callus induction seeds were surface-sterilized in 70% alcohol for 1 min, then in 5% commercial bleach for 15 min, followed by three rinses for 15 min with sterile distilled water. seeds were germinated aseptically in petri dishes containing 25 ml of growth-regulator-free ms (murashige and skoog 1962) medium supplemented with 3% (w/v) sucrose and solidifi ed with 0.8% agar (w/v). the ph of the ms medium was adjusted to 5.8 before sterilization by autoclaving at 121 °c at 105 kpa for 20 min. the ph of the ms medium was confi rmed after sterilization. for callus induction, 30-day-old hypocotyl parts of seedlings were used. callus proliferation was achieved with 0.5 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-d) and was subcultured every three weeks. the calli used in this study were grown in ms medium supplemented with 5.2 μm, 26.4 μm and 132.3 μm sodium selenate (sigma, s8295). seed germination, callus induction and subculture were carried out in a growth chamber illuminated with fl uorescent light (ca. 1400 μmol m–2 s–1) over a 16/8 day and night at 25 ± 2 ºc. calli were treated with se for three weeks, and after treatment, the calli were harvested, and their fresh weights were determined and samples were taken for various analyses. enzyme extractions and assays catalase (cat, ec 1.11.1.6) activity was done according to aebi (1984). callus samples (0.5 g) were homogenized in 50 mm sodium phosphate buffer (ph 7.0) with prechilled mortar and pestle. the extract was centrifuged at 15000 g for 15 min at 4 °c, and the supernatant was used as enzyme extract. the reaction mixture contained 3% h2o2 and 0.1 mm edta in 50 mm sodium phosphate buffer. to measure the activity, absorbance was read at 240 nm, and it was calculated as μmol per minute. peroxidase (pox, ec 1.11.1.7) activity was determined according to putter (1974), using the guaiacol oxidation method in a 3 ml reaction mixture containing 50 mm sodium phosphate buffer (ph 7.0), 20 mm guaiacol and 20 mm h2o2. the increase in absorbance was recorded at 470 nm within 20 s through 2 min after h2o2 was added. a unit of peroxidase activity was expressed as μmol h2o2 decomposed per minute. ascorbate peroxidase (apx, ec 1.11.1.11) activity was determined according to the method of nakano and asada (1981). 50 mm sodium phosphate buffer (ph 7.0), 0.5 mm ascorbate, 0.1 mm h2o2, and 0.1 ml enzyme extract in a fi nal assay volume of 1 ml were the components of the reaction. ascorbate oxidation was measured by reading absorbance at 290 nm. to calculate the oxidized ascorbate concentration, the extinction coeffi cient (ε = 2.8 mm−1 cm−1) was used. 1 μmol ascorbate oxidized per minute was described as one unit apx. glutathione reductase (gr, e.c. 1.6.4.2) activity was determined according to the method of foyer and halliwell (1976). gr activity was determined spectrophotometrically at 340 nm in a reaction mixture containing 50 mm tris-hcl buffer (ph 7.6), 1 mm gssg, and 10 mm nadph. 1 μmol nadph oxidized per minute was used as one unit gr. protein concentration was determined according to bradford (1976), using bovine serum albumin as a standard. the specifi c enzyme activity for all enzymes was expressed as units (u) per mg of proteins. rna isolation and cdna synthesis for rna isolation 5.2 μm, 26.4 μm and 132.3 μm sodium selenate-treated calli and control without sodium selenate were used. at the end of third week of treatment, total rna was extracted (trizol reagent, invitrogen, carlsbad, ca) and reverse-transcribed (superscript first-strand synthesis system, invitrogen, 11904-018) from a. chrysochlorus. rna isolation and fi rst strand cdna synthesis were done according to the manufacturers instructions. real-time pcr conditions and analysis quantitative real time pcr (qpcr) was performed with the dynamo™ hs sybr® green qpcr kit (thermo scientifi c) following the manufacturer’s instructions. primers are given in table 1. the qpcr conditions were set as follows: 2 min at 50 °c (uracil-dna glycosylase incubation), 5 min at 94 °c (pre-incubation), followed by 40 cycles of 30 s at 94 °c, 30 s at 64 °c and 30 s at 72 °c. to obtain melting curve data collection, a gradual temperature increase from 55 °c to 95 °c at the rate of 1 °c/10 s was added as a fi nal step. a non-template control was run alongside this, and serial dilutions (1, 1:5, 1:25 and 1:125) of the reference (actin) and smt gene were included with every assay. selenium induced antioxidative metabolism changes in astragalus acta bot. croat. 75 (1), 2016 13 amplifi cation specifi city of each reaction was verifi ed by melting curve analysis. actin was used to normalize expression levels. mx3000 stratagene software (agilent technologies, inc., santa clara, usa) was used to determine relative gene expression. all qpcr experiments were done in triplicate experiments for each sample. statistical analysis all tests were done in triplicates. analysis of variance was performed using graphpad prism ver. 5.0 trial version. the data were presented as the means for each treatment. data were compared using the one-way anova test with post tukey test at the 5% probability level. results establishment of callus cultures the hypocotyl parts of 30 day old seedlings produced friable and green callus on ms medium supplemented with 0.5 mg l–1 2,4-d after a 15-day culture period (fig. 1a). after induction of callus, 2,4-d was used for proliferation purpose and a. chrysochlorus callus was maintained for over ten years by subculture at three week intervals on ms medium supplemented with 0.5 mg l–1 2,4-d. the calli were then taken and grown in media containing 5.2 μm, 26.4 μm and 132.3 μm sodium selenate for 21 days (fig. 1b, c, d). the color red was observed when the concentration of selenium was 132.3 μm (fig. 1d). effect of selenium treatments on antioxidant enzyme activities the activity of cat decreased by 13%, 22% and 27% in the 5.2 μm, 26.4 μm and 132.3 μm se-treated calli of a. chrysochlorus, respectively (fig. 2a). in all calli, the activity of this enzyme was not modifi ed and the decrease was not signifi cant statistically according to treatment with se. the pox activity increased 4, 3 and 2 fold when compared to the control calli, respectively, after treatment with se (fig. 2b). lower doses of se (5.2 μm and 26.4 μm) have increased the pox activity more than 132.3 μm se. the increase in the pox activity was statistically signifi cant in 5.2 μm and 26.4 μm se-treated calli. the apx activity was signifi cantly increased in 5.2 μm and 26.4 μm se-treated calli (fig. 2c). the activity of this enzyme was higher in 132.3 μm se treated callus tissue than in control tissues but the increase was not signifi cant. treatment with se resulted in an increase of gr activity in 5.2 μm-treated callus tissues and a decrease in 26.4 μm and 132.3 μm-treated calli tissues, respectively (fig. 2d). however, in the 5.2 μm setreated callus tissues, this enzyme activity did not signifi cantly change. isolation and quality detection of total rna electrophoresis of isolated rna on 1% agarose gel stained with etbr showed distinct 28s and 18s rrna bands, indicating a good quality of total rna (on-line suppl. fig. 1). the a260/a230 absorbance ratio is usually calculated to check polysaccharide or polyphenolic contamination, and the a260/a280 ratio for protein contamination. we determined that a260/a230 ratios were greater than 2, and the a260/a280 ratios between 1.9 and 2.1. this result showed that the isolated rnas were intact and not contaminated. expression analysis of smt gene to evaluate the expression of the transcript, the reversetranscribed cdnas of samples (control, and treated tissues with 5.2 μm, 26.4 μm and 132.3 μm of se) were used as templates. smt was amplifi ed by the method for an sybr green qpcr assay. we can conclude that pcr amplifi cation curves show that amplifi cation has good reproducibility in each sample. the se metabolism pathway was studied extensively, and the conservation level of this pathway is high in plants. the expression of smt were observed by qpcr in all samples of selenium-treated astragalus plant (fig. 3). from the results obtained, we conclude that smt transcript levels for 5.2 μm and 26.4 μm selenium-treated callus were lower than for the control callus tissues. in contrast, the smt expression level of 132.3 μm in selenium-treated callus was 4.26 times higher than in the control callus. discussion selenium hyperaccumulation is involved with smt enzymatic activity and accumulation of s-methylcysteine (mecys) and mesecys (sors et al. 2005). in this study, our tab. 1. primers used in quantitative real time pcr reactions. smt – selenocysteine methyltransferase. target sequences (5′-3′) smt forward revers acggagcttgaacgtcatggtg atcaagatgcacctggcgaatgag actin forward revers tcaagacgaaggatg ttggattctggtgat fig. 1. astragalus chrysochlorus callus: untreated control (a), and selenium-treated tissue: 5.2 μm (b), 26.4 μm (c), and 132.3 μm (d). çakir ö., turgut-kara n., ari ş. 14 acta bot. croat. 75 (1), 2016 main object was to investigate the expression profi le of the smt gene (achsmt, accession number:gq844862) involved in selenium metabolism in a. chrysochlorus callus under increasing concentrations of selenium. we used qpcr to measure smt gene expression. as a result, smt expressions in 5.2 μm and 26.4 μm selenium-treated callus were slightly lower than in the control callus. in contrast, the smt expression level of 132.3 μm selenium-treated callus was found to be higher and the fold change was 4.26. in our previous work (çakir and ari 2013), a. chrysochlorus plantlets were treated with 0, 5.2 μm, 26.4 μm and 132.3 μm sodium selenate and analysed with semi-quantitative reverse transcription pcr. according to the results, differentially, smt expression sodium selenate treated plantlets were constitutively expressed like absmt from hyperaccumulator a. bisulcatus in all selenate treatments. smt gene has been isolated from a se hyperaccumulator a. bisulcatus (neuhierl et al. 1999, neuhierl and bock 1996). it was thought that smt is the key player in the accumulation of selenium and protection of the plants from the toxic effects (neuhierl et al. 1999). pickering et al. (2003) concluded that smt gene expression was continuous in tissues of a. bisulcatus. in contrast, lyi et al. (2005) reported that broccoli smt (bosmt) expression is up-regulated by selenate treatment. its expression in leaf has been determined to be involved in development. tao et al. (2012) reported that in young leaves of selenium-treated tea plant, the expression level of smt gene was similar to mature leaves, and it was the highest in roots. these fi ndings suggested that selenite is immediately converted to organic se and remains in the root tissues. in the present study, we determined that smt expression level was differentially affected upon treatements with increasing concentrations of se. in low concentrations (5.2 μm and 26.4 μm) smt expression was lower than in control tissues (0.397 and 0.597 times, respectively). our results revealed that the transcriptional levels of smt gene fluctuated after se treatment. the changes or variations in expression levels of smt gene after treatment with different concentrations of se may be due to the delayed response of se metabolism. se assimilation and hyperaccumulation pathway are a very complicated metabolic process. one of the important approaches is to investigate smt gene expression. also further research into smt at genetic translation level and the content of related amino acids in different parts of the a. chrysochlorus plant is needed to validate smt status in the process of selenium metabolism. selenium is an important micronutrient for many organisms. a. chrysochlorus has been found to be secondary se fig. 2. antioxidant enzyme activities in callus tissue of astragalus chrysochlorus treated with different concentrations of selenium: a) catalase (cat), b) peroxidase c), ascorbate peroxidase (apx), d) glutathione reductase (gr). asterisk (*) means statistically signifi cant (p < 0.05); bars indicate ± standard deviation. fig. 3. relative expression levels of selenocysteine methyltransferase gene in callus tissue of astragalus chrysochlorus treated with different concentrations of selenium in comparison to untreated control. the ratio of the relative quantity of the target gene and the relative quantities of reference genes (actin) of each treatment was normalized against the untreated control (value = 1) and plotted using arbitrary units. each data point represents the mean ± se, n = 3. selenium induced antioxidative metabolism changes in astragalus acta bot. croat. 75 (1), 2016 15 accumulator in our previous study (ari et al. 2010). se is very important for antioxidant metabolism and response. in this study the activities of apx, pox, cat and gr enzymes were also investigated. antioxidant responses of a. chrysochlorus callus tissues varied in a concentration-dependent manner. cat levels decreased gradually in increasing concentrations of se, nevertheless this decrease was not signifi cant when compared to control. pox and apx showed similar activity patterns. at 5.2 μm level, there was a signifi cant increase, on the other hand at 26.4 μm and 132.3 μm levels the activities were decreased compared to the 5.2 μm level. gr activity was increased at 5.2 μm and decreased signifi cantly at higher concentrations. under the concentration of 132.3 μm, se stimulated antioxidant metabolism. as shown in fi gure 2, despite the decrease in cat and gr activity, pox and apx activities were increased signifi cantly (p < 0.05). it is thought that the activities of pox and apx are more convenient to reduce the oxidative stress caused by se. the results indicate that the increase in pox and apx activities were suffi cient to protect callus cells. at the higher concentration of 132.3 μm, growth rate of callus cultures was decreased and pigmentation started. it is thought that this pigmentation is related to biosynthesis of secondary metabolites and also it is an environmental stress response of a. chrysochlorus. however, pox activity was still higher than in control callus cultures. in our previous studies, crude ethanol and ethyl acetate extracts of this plant exhibited cytotoxic activity and apoptosis-inducing properties (karagoz et al. 2007) and also antioxidant effects (hasancebi, unpublished data). besides the biotechnological importance of astragalus species, in callus tissues of the secondary se accumulator a. chrysochlorus it was found that antioxidant enzymes and smt gene expression were affected by se treatment in callus tissues. therefore, it is thought that it could be related and important for accumulation and tolerance to se of these species. in conclusion, se triggered significant transcriptional responses in a. chrysochlorus. the smt gene was repressed by low concentrations of se. results of a cross comparison showed that the smt gene was up-regulated by 132.3 μm se in a. chrysochlorus. the results obtained provide novel information for understanding the effects of different se concentrations on plant gene expression and will help to reveal the se metabolism of plants. acknowledgements this work was supported by the research fund of istanbul university. project no: 26494. references aebi, h., 1984: catalase in vitro. methods in enzymology 105, 121–126. apel, k., hirt, h., 2004: reactive oxygen species: metabolism, 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(ed.), methods of enzymatic analysis, 685–690. academic press, new york. soriano-garcia, m., 2004 organoselenium compounds as potential therapeutic and chemopreventive agents: a review. current medicinal chemistry 11, 1657–69. sors, t. g., ellis, d. r., na, g. n., lahner, b., lee, s., leustek, t., pickering, i. j., salt, d. e., 2005: analysis of sulfur and selenium assimilation in astragalus plants with varying capacities to accumulate selenium. plant journal 42, 785–97. tao, s., li, j., gu, x., wang, y., xia, q., qin, b., zhu, l., 2012: quantitative analysis of atp sulfurylase and selenocysteine methyltransferase gene expression in different organs of tea plant (camellia sinensis). american journal of plant sciences 3 51–59. whanger, p. d., 2004: selenium and its relationship to cancer: an update. british journal of nutrition 91, 11–28. yahara, s., kohjyouma, m., kohoda, h., 2000: flavonoid glycosides and saponins from astragalus shikokianus. phytochemistry 53, 469–71. selenium induced antioxidative metabolism changes in astragalus acta bot. croat. 75 (1), 2016 1 on-line suppl. fig. 1. representative rna isolated from astragalus chrysochlorus callus and analyzed by gel electrophoresis: a) untreated control, and selenium-treated tissue in the following concentrations: b) 5.2 μm, c) 26.4 μm, d) 132.3 μm. untitled 60 acta bot. croat. 75 (1), 2016 acta bot. croat. 75 (1), 60–66, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0001 issn 0365-0588 eissn 1847-8476 resurrection of ornithogalum brevipedicellatum (asparagaceae) with morphological and molecular data candan aykurt*1, i̇smail g. deniz2, duygu sari3, mecit vural4, hüseyin sümbül1 1 department of biology, faculty of science, akdeniz university, antalya, turkey 2 department of biology education, faculty of education, akdeniz university, antalya, turkey 3 department of field crops, faculty of agriculture, akdeniz university, antalya, turkey 4 department of biology, faculty of science, gazi university, ankara, turkey abstract – this study evaluates ornithogalum brevipedicellatum, which was previously accepted as a synonym of o. oligophyllum, as a separate distinct species and discusses the similarities and differences between o. brevipedicellatum and its related species (o. oligophyllum and o. pamphylicum). similarities and differences among these species were identifi ed by morphological and molecular studies. the leaf morphology and infl orescence of o. brevipedicellatum and o. pamphylicum are similar to each other, and in terms of these features, they show differences from o. oligophyllum. some diagnostic characteristics are quite different in o. brevipedicellatum and o. pamphylicum, such as the size of tepals, length of fruiting pedicels and style. morphological data were supported by the results obtained from molecular studies. according to a dendrogram obtained by molecular studies, o. brevipedicellatum and o. pamphylicum are similar. o. oligophyllum is more closely related to o. pyrenaicum used as an out-group. additionally, the seeds of o. brevipedicellatum were examined with the use of scanning electron microscopy. key words: asparagaceae, endemic, molecular, morphology, ornithogalum, scanning electron microscopy, synonym, turkey. * corresponding author, e-mail: candan@akdeniz.edu.tr introduction asparagaceae juss. (1789) (including agavaceae dumort., aphyllanthaceae burnett, hesperocallidaceae traub, hyacinthaceae batsch ex borkh., laxmanniaceae bubani, ruscaceae m.roem., themidaceae salisb.) include 143 plant genera (apg iii 2009). hyacinthaceae can be treated as subfamily scilloideae of asparagaceae, and the subfamilies hyacinthoideae, ornithogaloideae, urgineoideae and oziroëoideae of hyacinthaceae are then treated as tribes hyacintheae, ornithogaleae, oziroëeae and urgineeae (e.g. apg iii 2009). ornithogaloideae treated as one of the subfamilies in hyacinthaceae show a distribution through europe, south-west asia and africa, and include about 280 species (speta 1998). nowadays, as a result of molecular phylogenetic studies on this subfamily, 19 monophyletic genera are accepted within ornithogaloideae: albuca, avonsera, battandiera, cathissa, coilonox, dipcadi, eliokarmos, elsiea, ethesia, galtonia, honorius, loncomelos, me lomphis, neopatersonia, nicipe, ornithogalum, pseudo gal tonia, stellarioides and trimelopter (martínez-azorín et al. 2011). recently, molecular tools have gained importance in identifying taxonomic relations. in ornithogaloideae, matk, trnl intron, trnl-f spacer, and rbcl plastid dna sequences have been used for phylogenetic analysis (manning et al. 2009, martínez-azorín et al. 2011) because plastid sequences comprise an important source for phylogenetic reconstruction, mostly at interspecifi c or higher taxonomic levels (clegg and zurawski 1992, cameron 2004, kress and erikson 2007). in flora of turkey (cullen 1984), the species of the genus ornithogalum l. were evaluated under 4 subgenera, subg. beryllis (salisb.) baker, subg. ornithogalum, subg. myogalum (link) baker and subg. caruelia (parl.) baker. the recent arrangements recombined beryllis subg. as lancomelos raf., myogalum subg. as honorius gray and caruelia subg. as melomphis raf. (martínez-azorín et al. 2011). anatolia is an important area for the distribution of the genus ornithogalum in asia (uysal et al. 2005). since the last revision by cullen (1984) for the flora of turkey, nutaxonomic notes on ornithogalum brevipedicellatum acta bot. croat. 75 (1), 2016 61 merous new taxa, records and combinations of ornithogalum have been introduced from turkey (e.g. davis et al. 1988, özhatay 2000, düşen and sümbül 2002, 2003, düşen and deniz 2005, uysal et al. 2005, özhatay and kültür 2006, bağcı et al. 2009, 2011, özhatay et al. 2009, yıl dırımlı 2009, koca and yıldırımlı 2010, özhatay et al. 2011, mutlu and karakuş 2012). a total of 61 species of the genus ornithogalum are distributed in turkey, 31 of them endemic to the country (uysal 2012). the fi rst specimens of ornithogalum brevipedicellatum in turkey were collected by bourgeau in 1860 in the higher mountain steppes of the elmalı (antalya) district, the region known as ‘lycia’ in antiquity. these specimens were evaluated under the name of “o. brevipedicellatum” by boissier, and subsequently were introduced to science in 1873 by baker (boissier 1884, cullen 1984). o. brevipedicellatum was reported as the synonym of the o. oligophyllum species in the flora of turkey (cullen 1984), the plant list of turkey (uysal 2012) and in “the plant list” database. o. oligophyllum has a substantially wide area of occupancy in turkey. it was previously reported in the flora of turkey that the specimens of the species o. oligophyllum, which were collected in antalya, isparta and konya, would be classifi ed as o. brevipedicellatum provided that the distinguishing determinant characteristics were supported by a suffi cient number of samples (cullen 1984). materials and methods plant samples and morphological studies the plant specimens of the genus ornithogalum were collected by fi eld studies between 2012 and 2014 in the muğla and antalya provinces. during the fi eld studies, gps locations of o. brevipedicellatum were taken, and the individuals of its populations were numbered. this data was used to determine the threat category of o. brevipedicellatum according to the categories and criteria of iucn (version 11, 2014). the extent of occurrence (eoo) and area of occupancy (aoo) values were calculated. in the present study, ornithogalum brevipedicellatum known as a synonym of o. oligophyllum is morphologically described in detail and compared with its related species, o. oligophyllum and o. pamphylicum o.d.düşen & sümbül, with the data obtained from morphological and molecular studies. the individuals of these species were observed during fi eld studies and the morphological evaluations were done both in the fi eld and in laboratory. specimens collected and used in molecular studies within the scope of this study, are shown in tab. 1. the digital isotype photographs obtained from mnhn (museum national d’histoire naturelle) of o. brevipedicellatum were also examined. additionally, a number of herbarium specimens of o. oligophyllum collected from different parts of turkey in the iste (istanbul university, herbarium of the faculty of pharmacy) and gazi (gazi university herbarium) were morphologically examined (see appendix). seed micromorphology of ornithogalum brevipedicellatum was investigated using scanning electron microscopy (sem) techniques. for sem study, the seeds were treated with gold conjugate on stub. the microphotographs were taken with a zeiss leo-1430 scanning electron microscope. molecular study: dna isolation, pcr amplifi cation and sequencing complete dna of ornithogalum brevipedicellatum and o. pamphylicum was extracted from the leaves of herbarium specimens using the cetyl trimethyl ammonium bromide (ctab) method of doyle and doyle (1990). dna concentration and quality were tested with 1% agarose gel against a dna standard. two different plastid regions were used for molecular analysis to identify the phylogenetic similarities of the species. amplifi cation of these regions was conducted with the universal primers used in previous tab. 1. locality data of collected specimens of ornithogalum for the current study. species locality collection data o. brevipedicellatum c2 muğla, fethiye, seki, 6 km from seki to yuva, ak mountain, steppe, 1980 m, 27.iv.2012. c. aykurt 3071(akdu) muğla, fethiye, seki, 4–6 km from seki to yuva, ak mountain, 1900–1980 m, steppe, 4.v.2012. c. aykurt 3084 (akdu) muğla: fethiye, seki, 4–6 km from seki to yuva, ak mountain, 1900–1980 m, steppe, 22.v.2012. c. aykurt 3137* (akdu) antalya: kaş, gömbe, between gömbe-i̇kizgöller, subaşı environs, plateau, 2080 m, 25.05.2012. c. aykurt 3156 (akdu) muğla, fethiye, seki, 6 km from seki to yuva, ak mountain, steppe, 1975 m, 30.04.2013. c. aykurt 3741* (akdu) muğla, fethiye, seki, 6 km from seki to yuva, ak mountain, steppe, 1827 m, 30.04.2013. c. aykurt 3742* (akdu) muğla, fethiye, seki, 6 km from seki to yuva, ak mountain, steppe, 1825 m, 23.05.2013. c. aykurt 3868 (akdu) muğla, fethiye, seki, 6 km from seki to yuva, ak mountain, steppe, 1975 m, 23.05.2013. c. aykurt 3869 (akdu) o. pamphylicum antalya, above fesleğen plateau, sakarpınar, 1850–1900 m, calcerous slopes, 4.v.2012. c. aykurt 3087 (akdu) antalya, elmalı, i̇mecik plateau, bey mountains, 1800 m, 29.04.2012. c. aykurt 3089* (akdu) antalya, elmalı, kızlarsivrisi mountain, karakuyu district, 1900 m, openings in cedrus libani forest, calcareous slopes, 09.v.2012. i̇.g. deniz 4528 (akdu) antalya: çalbalı mountain, 1700 m, calcareous slopes, 15.v.2014. i̇.g. deniz 5548 (akdu) o. oligophyllum c3 antalya: sarıçınar, 36 s 274566, 4096808, 1380 m, 14.iv.2014. c. aykurt 3882 (akdu) c2 antalya: finike, west side of finike, under cedrus libani, 1200 m, 24.iv.2014. c. aykurt 3911 (akdu) aykurt c., deniz i. g., sari d., vural m., sümbül h. 62 acta bot. croat. 75 (1), 2016 studies. the trnl intron and trnl spacers were amplifi ed with c (5’-cgaaatcggtagacgctacg) and f (5’-at ttgaactg-gtgacacgag) primers described in ta ber let et al. (1991). amplifi cation of the rbcl gene was performed using a forward primer (427f), 5’-gcttattcaaaaac t ttccaaggcccgcc, and a reverse primer (724r), 5’-tcgcatgtacctgcagttgc (lledó et al. 1998). polymerase chain reaction (pcr) for both trnl and rbcl regions contained 2 mm mgcl2, 0.2 mm of each dntp, 10 pm μl–1 of each primer, 1 u of taq dna polymerase (fermentas life sciences, burlington, canada) with supplied reaction buffer at 10× concentration, and 40 ng of template dna. the amplifi cations were performed on a programmable thermocycler (bioneer, mygenie™) with the following program: one cycle of 4 min at 94 °c, 28 cycles of 1 min at 94 ºc, 30 s at 48 °c for rbcl, or 1 min at 50 °c for trnl, 1 min at 72 °c, and for fi nal extension one cycle of 7 min at 72 °c. pcr products were cleaned up using the genejet gel extraction kit (thermo scientifi c fermentas, vilnius, lithuania). sequencing process was carried out at iontek laboratory in istanbul, turkey as direct sequencing from pcr products. the sequences of other species (o. oligophyllum and o. pyrenaicum) were retrieved from genbank databases at the national center for biotechnology information (ncbi) and compared with o. brevipedicellatum trnl and rbcl sequences. a phylogenetic tree was constructed using the software mega 5. bootstrap values are displayed at tree nodes (tamura et al. 2011). results morphological studies ornithogalum brevipedicellatum boiss. ex baker, j. linn. soc., bot. 13: 263 (1873) (figs. 1–3) geophyte, 5–10(–15) cm high; bulb 1.3–1.8(–2) × 1.3– 1.8(–2) cm, ovoid-spherical, without bulblets; outer tunics cream to pale-brown. leaf synanthous, 4–6(–7), 9–37 × 2.3–0.5 cm, linear-lanceolate, canaliculate, with a whitish median line, gradually narrowing towards the base, longer than scape, margins entire, glabrous. scape below the ground level, 3–12 cm, slender. infl orescence congested racemose, racem borne at ground level, dense, 2–6 × 3–4.5 cm, erect, with (1–)5–15(–18) fl owers. bracts lanceolate, long acuminate, (12–)15–23 × 3–5 mm, membranous, longer than pedicels. flowers sessile or subpedicellate up to 3 mm at anthesis and fruiting period. tepals eliptic to ovateeliptic, obtuse to acute at apex; outers 12–18 × 4–5 mm; inners 13–18 × 4.5–5 mm, white inside, green with narrowly white margins outside. filaments white, conspicuously tapering towards the apex, 4–6 × 1–2 mm; anthers medifi xed, milky white, 2–2.2 mm length. ovary ovoid, 3.5–5 × 2–3.5 mm, pale green, longer than style; style 1.5–2 mm length; stigma capitate. capsule 10–15 × 10–15 mm, ovoid, erect, distinctly winged, pale brown. seeds numerous, 1.8–2 × 1.2–1.8 mm, elliptic-subglobose to globose, black, strongly apiculate; testa reticulate, with reticules formed by prominent crests, testa cells irregular. hitherto ornithogalum brevipedicellatum was evaluated as the synonym of o. oligophyllum; however the species is morphologically more related to o. pamphylicum, described in 2002. the infl orescence type of o. brevipedicellatum is similar to o. pamphylicum. the raceme of o. brevipedicellatum is very condensed in fl owering and fruiting period and borne at ground level. its fl owers are sessile or the fl oral or fruiting pedicels are up to 3 mm long. the infl orescence of o. pamphylicum resembles o. brevipedicellatum, but the internodes are more elongated and the pedicels are fl accid and longer at fruiting time. on the other hand, the lower pedicels of o. oligophyllum are longer then the fl owers; fruiting pedicels are 10–30 mm long. therefore, the infl orescence of o. oligophyllum can be evaluated as corymbose or pseudocorymbose. moreover, the infl orescence of o. brevipedicellatum is different due to its sessile or subpedicellate fl owers. fig. 1. ornithogalum brevipedicellatum (from c. aykurt 3071): a) habit, b): fl ower, c) stamen, d) pistil, e) capsule. scale bars: 1 cm (a,b,e), and 1 mm (c,d). fig. 2. sem photographs of the seeds of ornithogalum brevipedicellatum (from c. aykurt 3137): a) seed, b) seed surface. taxonomic notes on ornithogalum brevipedicellatum acta bot. croat. 75 (1), 2016 63 the other morphological differences of o. brevipedicellatum, o. oligophyllum and o. pamphylicum are shown in tab. 2. habitat, distribution and conservation status ornithogalum brevipedicellatum shares its habitats with several herbaceous plants such as cerastium macranthum boiss. ceratocephala falcata (l.) pers., corydalis erdelii zucc., crocus bifl orus mill. subsp. isauricus (siehe ex bowles) b. mathew, fumaria asepala boiss., gagea villosa (m. bieb.) sweet subsp. hermonis dafni & heyn, scilla bifolia l., tussilago farfara l., viola heldreichiana boiss. an old specimen collection record was documented in flora orientalis indicating that specimens of the ornithogalum brevipedicellatum species were collected on mount trodos in southern cyprus (boissier 1884). however, recent data supporting these dubious records was not documented and for that reason, the species o. brevipedicellatum was evaluated as endemic to turkey. the conservation status of ornithogalum brevipedicellatum is described below according to the rules of citation. considering the iucn (2014) categories and criteria (version 11), the species should be placed in the en (endangered) category, according to the criteria for critically endangered, endangered and vulnerable. the subpopulations of the species were identifi ed in seki (muğla) and gömbe (kas, antalya) (fig. 4). no artifi cial factors endangering the development of the species in its area of occupancy were present. therefore, it was projected that the population of the species in terms of percentage did not decrease through time as a result of anthropological effects. item a cannot therefore be evaluated. the distance between the two locations of the species is 25 km. the eoo value of the species was determined as 130 km2 (en b1b(iii)) taking both locations of occupancy and the area contained within the shortest continuous imaginary boundary. the aoo value in this area, where the species was identifi ed was calculated as 13 km2 (en b2b(ii)). the number of individuals identifi ed in the subpopulations of the species was determined as 400 at the seki location and 200 at the gömbe location (en c). molecular studies individuals of ornithogalum brevipedicellatum and o. pamphylicum were collected from different localities for molecular analysis (tab. 1). the sequences of these specimens were compared and evaluated with o. oligoliphylum fig. 3. ornithogalum brevipedicellatum on ak mountain (elmalı, antalya). tab. 2. comparison of the morphological characters of ornithogalum brevipedicellatum, o. oligophyllum and o. pamphylicum. features o. brevipedicellatum o. oligophyllum o. pamphylicum scape length (cm) 3–12 (borne at ground level) 4–15 3–15 number of leaves 4–6(–7) 2–3 (rarely 4) (3–)4–11(–13) leaves with a whitish median line without a whitish median line with a whitish median line leaves width 2–5 mm 5–20 mm 1–4 mm infl orescence congested racemose corymbose or pseudocorymbose laxly racemose (internodes more elongated) number of fl owers (1–)5–15(–18) 3–10(–20) 3–25 fruiting pedicels capsules sessile or subpedicellate (up to 3 mm long), erect 10–30 mm, become fl accid at base capsules pedicettale (6–12 mm), erect flowers odour odourless odourless tepals (mm) 12–18 × 4–5 – 20–30 × 5–9 style length (mm) 1.5–2 2–3.5 4–5 fig. 4. distribution areas of ornithogalum brevipedicellatum (), ornithogalum oligophyllum (●) and ornithogalum pamphylicum (◼) in turkey. aykurt c., deniz i. g., sari d., vural m., sümbül h. 64 acta bot. croat. 75 (1), 2016 and o. pyrenaicum retrieved from the genbank database. to amplify the trnl and rbcl region of o. brevipedicellatum and o. pamphylicum species, c/f and 427f/724r primer pairs were used, respectively, as described in methodology section. about 1200 bp amplicons for trnl and 300 bp amplicons for rbcl were yielded in pcr assays. these amplicons were sequenced and compared with those of o. oligophyllum species (fig. 5) available in the genbank using the blast similarity search tool. in the trnl tree, two groups were identifi ed. all of the individuals of o. brevipedicellatum were aligned in the fi rst group. o. pamphylicum was the closest species to o. brevipedicellatum while o. oligophyllum and o. pyrenaicum were more distant from this group. similarly, three individuals of o. brevipedicellatum were located together in the rbcl tree. although o. pamphylicum was placed in a different branch, it was the closest species to o. brevipedicellatum while o. oligophyllum and o. pyrenaicum species were positioned in another group. discussion the ornithogalum genus, previously classifi ed into numerous subcategories by several different researchers, was rearranged recently in the light of the novel molecular phylogenetic studies conducted on the ornithogaloideae subfamily (martínez-azorín et al. 2011). moret and galland (1992) indicated that many taxa have been described in subg. ornithogalum which is a taxonomically problematic subgenus on the basis of subtle morphological variations, usually with little biological signifi cance. the complex taxonomy of subg. ornithogalum may be due to intraspecifi c variations, the plasticity of individuals, their habits being strongly dependent on environmental factors and poor conservation of the types (moret and galland 1992). martínez-azorín et al. (2010) reported that some authors have used the infl orescence structure and/or the length of the fl oral bracts relative to their pedicels as diagnostic characteristics. when ornithogalum brevipedicellatum, o. pamphylicum and o. oligohyllum species are evaluated with respect to the infl orescence type and length of the pedicels, it is seen that the specimens of o. brevipedicellatum are distinct; distinguished from the other species by the lack of pedicels, or the presence of very short pedicelseither during fl owering or fruiting periods. the presence of very short internodes between the fl owers in the subpedicellate or the sessile fl owers allowed the infl orescence of the species to be evaluated as congested racemose, rather than corymb or pseudocorymb. furthermore, the habitus of these three species show differences, while the fi rst fl owers of o. brevipedicellatum and o. pamphylicum are at ground level but the infl orescence of o. oligophyllum has generally a scape above ground level. the pedicels of o. oligophyllum are distinctly longer than the pedicels of o. brevipedicellatum and o. pamphylicum in fl owering and fruiting periods. ornithogalum brevipedicellatum and o. pamphylicum are similar to each other with respect to their leaf morphology and infl orescence in fl owering time. they have narrower leaves from o. oligophyllum. we observed the white line on the leaf surface of o. pamphylicum and the ovary of the species is distinctly winged, contrary to the description of the species by düşen and sümbül (2002). therefore, o. pamphylicum was included in subg. ornithogalum in the present study but, on the contrary, in subg. myogalum by düşen and sümbül (2002). the closer morphologic proximity between o. brevipedicellatum and o. pamphylicum as opposed to o. oligophyllum was supported by the data obtained from molecular studies. considering to the phylogenetic tree obtained from trnl and rbcl sequences, the individuals of o. brevipedicellatum were separated from o. oligophyllum in two different dendograms. o. brevipedicellatum are closer to o. pamphylicum compared with o. oligophyllum. the most remarkable morphological differences between o.brevipedicellatum and o. pamphylicum can be defi ned as follows: (1) the capsules of o. brevipedicellatum are generally sessile, sometimes subpedicellate but they are pedicellate and pendant in o. pamphylicum; (2) lengths of fig. 5. phylogenetic trees of trnl (a) and rbcl (b) sequences from six ornithogalum specimens. locations (*) and genbank accession numbers (**) are given next to the ornithogalum name. taxonomic notes on ornithogalum brevipedicellatum acta bot. croat. 75 (1), 2016 65 the style are quite different; (3) o. brevipedicellatum has more congested infl orescence while the internodes are elongated in o. pamphylicum; (4) the tepals are more narrow in o. brevipedicellatum (tab. 2). the seed size, shape, color, surface ornamentation and shape of the cells, raphe, micropylar and chalazal poles are useful diagnostic characteristics for seed micro-morphological studies conducted on the genus ornithogalum (bednorz and czarna 2008, çitak et al. 2015). moret et al. (1990) and martínez-azorín et al. (2010) reported that subg. ornithogalum shows globose and apiculate seeds with reticulate testa. similarly, the seeds of o. brevipedicellatum are elliptic-subglobose to globose, distinctly apiculate with reticulate testa. the results obtained from the present study clearly indicate that o. brevipedicallatum is a distinct species and cannot be claimed to be a synonym of o. oligophyllum. acknowledgements the authors are indebted to akdeniz university scientifi c research projects unit (project number: 2012.01. 0115.004) for fi nancial support to fi eld studies and thanks to the curators of akdu, iste and gazi. appendix additional specimens examined o. oligophyllum: a1(e) kırklareli: demirköy, mahyadağ, mareşal road, fagus gap, 860 m, 22.5.1974, a. baytop et e. tuzlacı (28271 iste). kırklareli: demirköy, mahyadağ, 1030 m, 22.5.1974, a. baytop et e. tuzlacı (28304 iste). kırklareli: merkez, between dereköy and boundary, 1.5.1973, g. ertem (24281 iste). kırklareli: centre, woodlands in dereköy, kocakaynak environs, 26.4.1974, n. özhatay, e. özhatay (27642 iste), kırklareli: demirköy, between yeniceköy and velika, 1 km to haydut mountain, 24.4.1988, g. dalgıç, n. başak (59757 iste). kırklareli: centre, dereköy, boundary, under fagus, 23.4.1988, g. dalgıç, n. başak (59758 iste). kırklareli: demirköy, velika, balaban village picnic areas, under woodlands, 24.4.1988, g. dalgıç, n. başak (59766 iste). a3 bolu: between bolu and mudurnu, under quercus, 1250–1350 m, 03.05.1993, z. aytaç 5789 et al. (gazi). bolu: kale, tenekeci river, streamside, woodlands, 800 m, i̇. kılınç 1029 (gazi). a4 ankara: kızılcahamam, soğuksu national park, çakmaklı environs, 1450–1500 m, 12.05.1990, ö. eyüboğlu (gazi). ankara: çubuk, between ovacık and saraycık villages, borucağın hill, shrubs, 1250–1380 m, 20.5.1992, e. dündar 1193 (gazi). çankırı: atkaracalar, dumanlı mountain, atkaracalar plateau, abaza river environs, steppe, 1300–1450, 09.05.1992, a. duran 1516 (gazi). ankara: ayaş, ayaşbeli, çal hill, steppe, 1300– 1380 m, 13.04.1986, m. vural 4029 (gazi). kırıkkale: koçubaba town, 1200 m, 31.03.1990, a.a. dönmez 1696 (gazi). a9 kars: arpaçay, karakale village, under rumex shrubs, 2250 m, 16.06.1984, h. ocakverdi 1714 (gazi). ardahan: posof, baykent village, birch woodlands, 1900 m, 25.6.2007, sezgin esen (87083 iste). ardahan: çataldere village birch woodlands, 9.6.2008, sezgin esen (87275 iste). b4 kırşehir: çiçek mountain, halaçlı village, almanderebaşı environs, quercus shrubs, slopes, 1550 m, 09.05.1993, f.a. karavelioğulları 1026 et al. (gazi). b5 nevşehir: between uçhisar and göreme, erosional dry slopes, 1250 m, 19.04.1989, m. vural 4497 et al. (gazi). b6 kayseri: sarız, yalak environs, binboğa mountain, steppe, 1800–2200 m, 07.05.1991, z. aytaç 3709 et al. (gazi). kahramanmaraş: binboğa mountain, west of kaşvut village, alpine steppe, under cedrus libani, cliffs, 1750–2250 m, 08.05.1991, z. aytaç 3733 et al. (gazi). kayseri: sarız, binboğa mountain, doğankonak, karagöz, 1450–1900 m, 21.04.1992, z. aytaç 4486 et al. (gazi). kayseri: pınarbaşı, south of aşağıbeyçayırı village, eroded west slopes, 29.4.2004, b. yıldız, t. arabacı (89931 iste). b7 malatya: kubbe mountain, kubbe plateau, mountain steppe, 1700 m, 1.6.1992, b.yıldız 9378 (iste). b8 erzurum: between aşkale and tercan, kükürtlü environs, slopes and shrubs, 1950 m, 23.5.2004, m. koyuncu, m. fırat, m. armağan (88864 iste). b9 bitlis: adilcevaz, süphan mountain hillside, süte plateau, meadows, 1900 m, 04.06.1993, y. altan 4796 (gazi). van: gevaş, west slopes of çadır mountain, steppe, 2300 m, 06.06.1993, y. altan 5077 (gazi). c3 isparta: barla, gelincik mountain, tahtacıçukuru hills environs, pinus nigra, 1800–1900 m, 09.05.2009, h. duman 9921 (gazi). c6 kahramanmaraş: engizek mountain, küçükcerit village, under quercus, 1400–1500 m, 21.04.1987, h. duman 2486 (gazi). kahramanmaraş: engizek mountain, south of şaç rocks, wetlands, 2400–2500 m, 20.05.1987, h. duman 2680 (gazi). kahramanmaraş: erince mountain, under quercus, 1300–1400 m, 22.04.1987, h. duman 2507 (gazi). kahramanmaraş: between göksun and geben, kayranlı mountain, east slopes, 30.4.2014, b. yıldız, t. arabacı (89959 iste). c7 şanşıurfa: karacadağ, 2 km from karabahça to dağ, reme river environs, stony areas, 1400 m, i̇. eker 181-b (gazi). references apg iii., angiosperm phylogeny group, 2009: an update of the angiosperm phylogeny group classifi cation for the orders and families of fl owering plants: apg iii. botanical journal of the linnean society 161, 105–121. bağcı, y., savran, a., başköse, i̇., 2009: ornithogalum nurdaniae (liliaceae), a new species from northwest anatolia, turkey. turkish journal of botany 33, 163–167. bağcı, y., savran, a., düşen, o. d., tutar, l., 2011: ornithogalum beyazoglui (hyacinthaceae), a new species from west anatolia, turkey. bangladesh journal of plant taxonomy 18, 51–55. bednorz, l., czarna, a., 2008: sem and stereoscope microscope observations on the seeds of some ornithogalum (hyacinthaceae) species. biologia 63, 642–646. boissier, e., 1884: flora orientalis 5. geneva & basle. cameron, k. m., 2004: utility of plastid psab gene sequences for investigating intrafamilial relationships within orchidaceae. molecular phylogenetics and evolution 31, 1157–1180. clegg, m. t., zurawski, g., 1992: chloroplast dna and the study of plant phylogeny: present status and future prospects. in: soltis, p. s., soltis, d. e., doyle, j. j. 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(liliaceae) from sw anatolia, turkey. the herb journal of systematic botany 16, 1–8. acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 49 acta bot. croat. 76 (1), 49–54, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0038 issn 0365-0588 eissn 1847-8476 response of dihaploid tobacco roots to salt stress tihana marček1*, željka vidaković-cifrek2, mirta tkalec2, marin ježić2, mirna ćurković-perica2 1 subdepartment of biology and microbiology, faculty of food technology osijek, josip juraj strossmayer university of osijek, franje kuhača 20, hr-31000 osijek, croatia 2 department of biology, faculty of science, university of zagreb, rooseveltov trg 6, hr-10000 zagreb, croatia abstract – salinity is a common abiotic factor that affects plant growth and development. seedlings of tobacco (nicotiana tabacum l.) f1 hybrid dh10 and three dihaploid lines (207b, 238c and 239k) obtained by diploidization of anther-derived haploids of hybrid dh10 were subjected to 0, 100 and 200 mm nacl in in vitro conditions for 33 days and the effect on roots was evaluated. in all lines and in the hybrid dh10 exposed to 200 mm nacl evident root growth inhibition and increased proline content were noticed. however, in some cases lines differed in the activity of antioxidative enzymes, which could account for differences in their salinity tolerance. increased activity of catalase and peroxidase in roots of line 239k could contribute to the more pronounced salinity tolerance previously reported for shoots of this line. keywords: antioxidative enzymes, nicotiana tabacum l., proline, root, salinity, tobacco dihaploid lines * corresponding author, e-mail: tihana.marcek@ptfos.hr introduction salinity is one of the most adverse environmental factors that reduce the growth and productivity of agricultural crops (horie et al. 2012). in nature, it often comes together with drought and heat and all these stress factors may cause changes in plant water status, decreased activity and denaturation of structural proteins and leakiness of membranes caused by phospholipid bilayer disruption (wang et al. 2003, mahajan and tuteja 2005). generally, the detrimental effects of salinity in plants are characterized by a decrease in growth rate, a change in root/shoot ratio and development of chlorosis (parida and das 2005). reduced growth mainly occurs due to low water potential in the soil. in such conditions the possibility of cell dehydration is increased due to diffi culty in the acquisition of water by plant roots. in addition, increased concentration of na+ and cl– in plant organs disturbs the intake of essential microelements such as k+, leading to altered k+/ na+ ratios and disruption of cellular homeostasis (apse and blumwald 2007, munns and tester 2008). in order to adjust osmotic potential, plants accumulate compatible osmolytes. the most common among them is the amino acid proline, which is broadly present in many organisms. besides being an osmolyte, proline acts as a molecular chaperone: it enhances the activities of the enzymes, controls plant development and acts as a signal molecule. proline also shows antioxidant property through reactive oxygen species (ros) scavenging activity (szèkely et al. 2008, szábados and savourè 2009). under the conditions of increased salinity, the levels of ros can dramatically increase. in order to deal with these highly reactive species, antioxidative enzymes such as catalase (cat), non-specifi c peroxidase (pod), superoxide dismutase (sod) and ascorbate peroxidase (apx) cooperate together to mitigate cellular damage such as unspecifi c oxidation of proteins, membrane lipids and nucleic acids. sod converts superoxide radical (o2•–) into oxygen and hydrogen peroxide while pod and cat are involved in h2o2 detoxifi cation (bandeoğlu et al. 2004, parvanova et al. 2004). numerous studies emphasize that the antioxidative response strongly correlates with susceptibility and resistance to increased salinity (acar et al. 2001, tűrkan et al. 2005). in previous work we investigated the response of shoots of f1 hybrid dh10 and four dihaploid tobacco lines derived from that hybrid (207b, 238c, 239k, 244b) to 100 and 200 mm nacl. f1 hybrid and dihaploid lines were previously proved to be tolerant to potato virus y (pvy) (šmalcelj and ćurković-perica 2000). salt stress caused growth retardation and induced proline and sodium accumulation in shoots of all dihaploids and hybrid dh10. in line 239k, salt induced higher activities of sod, cat and pod. our results revealed that lines 238c and 244b were more susceptible than lines 207b and 239k to increased salinity, suggesting that tolerance to the virus is not associated with salinity tolerance (marček et al. 2014). although the roots are the fi rst to encounter excess salinity and are potentially the fi rst sites of damage or “line of defence” (rewald et al. 2013), most studies of plant tolerance to high salinity marček t., vidaković-cifrek ž., tkalec m., ježić m., ćurković-perica m. 50 acta bot. croat. 76 (1), 2017 focus on traits in the aboveground tissues; data on the phenotypical and physiological plasticity of root systems under salt stress are rare. except having a role in the uptake of water and nutrients, roots also act as a sensory system, integrating changes in nutrient availability, water content and salinity in order to adjust root morphology for better exploitation of available resources (gruber et al. 2013). therefore, the purpose of this study was to expand our previous research by investigating the effect of nacl on roots of dihaploid tobacco lines and hybrid dh10 in order to reveal if root responses to salinity may contribute to the salt tolerance of investigated tobacco lines. materials and methods plant material and treatments tobacco (nicotiana tabacum l.) f1 hybrid dh10 (hybrid of cv. virginia d and line gv3) and four dihaploid lines (207b, 238c, 239k, 244b) obtained by diploidization of anther-derived haploids of the hybrid dh10 (šmalcelj and ćurković-perica 2000) were tested for salinity tolerance. seeds, provided by the duhanski institut zagreb (croatia), were grown in pots containing a mixture of peat-sand (2:1). ten weeks after planting, the seedlings were surface sterilised in 70% ethanol for 10–15 s and then for 15 min in 1% (w/v) sodium hypochlorite (naocl) supplemented with 0.05% (v/v) tween. after extensive rinses in sterile distilled water, seedlings were transferred to solid murashige and skoog (ms) medium (murashige and skoog 1962) containing 3% (w/v) sucrose and 0.8% agar (w/v) without growth regulators (ph 5.7). plants were grown in a climate chamber at 24±2 °c under 16 h light/8 h dark photoperiod and artifi cial fl uorescent lamp (90 μmol s–1 m–2) and multiplied by subcultivation every 4 to 6 weeks using nodal segments. four-week-old plants were exposed to salt stress by supplementing ms medium with 100 or 200 mm nacl. ms medium without added nacl was used as control. after 33 days of treatment, roots were gently separated from shoots and carefully washed to remove medium residues between root branches. root tissue was stored at –80 °c until analyses. due to severe growth retardation in plants exposed to the higher concentration of nacl (200 mm), roots from several plant samples had to be pooled together. line 244b did not develop a root system at any salt treatment hence this line was omitted from further analyses. proline content free proline content was determined according to bates et al. (1973). 10–25 mg of fresh plant tissue was homogenized in 1.5 ml of 3% (w/v) sulphosalicylic acid and the residue was removed by centrifugation at 15 000 g for 15 min. supernatant, ninhydrin and glacial acetic acid were heated at 100 °c for 1 h, and the reaction was stopped in an ice bath. the absorbance of the free proline fraction with toluene aspirated from the liquid phase was read spectrophotometrically at 520 nm. proline concentration was determined using a calibration curve obtained with l-proline solutions ranging from 10 to 160 μm and expressed as micromols per gram of fresh weight [μmol g–1 fw]. enzyme extraction root tissue (50 mg) was homogenized in 1 ml ice cold 50 mm potassium phosphate buffer (ph 7.0) containing 0.1 mm ethylenediaminetetraacetic acid (edta), 5 mm ascorbic acid and polyvinylpolypyrrolidone (pvpp). homogenate was centrifuged at 25 000 g for 30 min at 4 °c. the content of soluble proteins in the supernatant was determined according to bradford (1976) using bovine serum albumin as a standard. obtained supernatants were used for assays of antioxidative enzyme activities. assays of antioxidant enzyme activities pod (ec 1.11.1.7) activity was analysed by measuring the oxidation of guaiacol in the presence of hydrogen peroxide at 470 nm according to chance and maehly (1955). cat (ec 1.11.1.6) activity was estimated according to aebi (1984) by monitoring the decline in absorbance as a consequence of hydrogen peroxide consumption at 240 nm. the activity of sod (ec 1.15.1.1) was determined by measuring the inhibition of the photochemical reduction of nitro blue tetrazolium (nbt) at 560 nm as described by beauchamp and fridovich (1971). one unit of sod activity was defi ned as the amount of enzyme that caused 50% inhibition of nbt reduction. the enzyme activities were expressed as units (u) of enzyme activity per milligram of protein [μmol min–1 mg–1proteins]. statistical analysis all results were expressed as means of three replicates with the corresponding standard errors, except for lines 238c and 239k at the highest salt concentration (200 mm nacl) where only one replicate was obtained. data were subjected to the analysis of variance (anova) with a 3×4 factorial approach replicated three times with missing values. differences between means were compared at p≤0.05 of signifi cance using fisher’s least signifi cant difference test. principal component analysis (pca) was performed to evaluate and discriminate the roots responses of different tobacco lines exposed to nacl. the data set used for pca comprised 5 variables (protein concentration, proline content, activity of pod, cat and sod). data were analysed with statistica 10.0 (stat soft inc., usa) software package. results after 33 days of treatment all control plants of tested lines (207b, 239k and 238c) and hybrid dh10 as well as those treated with 100 mm nacl developed roots. in all tobacco lines roots of control plants were fi brous and thick, 3.5–5 cm long, with lateral branches, while plants exposed to 100 mm nacl had shorter roots (1–2.5 cm). the most pronounced effect of salt stress was noticed at 200 mm nacl where growth of roots was severely inhibited and roots were only 0.5–1 cm long (fig. 1) or even not developed, as in line 244b which was therefore omitted from further analysis. dihaploid tobacco roots under salinity acta bot. croat. 76 (1), 2017 51 considering total protein concentration, results revealed that in line 207b protein concentration was higher in roots of plants exposed to 200 mm nacl, especially compared to untreated (control) plants (30% above the control value in 207b). among different tobacco lines control plants of line 239k had the lowest protein content (fig. 2a). salt stress caused remarkable proline accumulation in the roots of all tobacco lines treated with 200 mm nacl in comparison to their respective controls (from 2-fold above the control level in hybrid dh10 to above 8-fold in 207b). the proline accumulation was lowest in roots of 239k line. treatment with 100 mm nacl caused signifi cant proline accumulation in all dihaploid lines (5-fold in 207b and 4-fold in 239k and 238c) but not in hybrid dh10 (fig. 2b). when the proline content in the control plants of all experimental groups was compared, a signifi cantly higher level was measured in hybrid dh10. salt stress mostly did not cause any considerable differences in activities of antioxidative enzymes in the roots of all the tobacco lines tested. however, statistical analysis revealed a higher cat activity in roots of line 239k exposed to 100 mm nacl than in the roots of line 207b exposed to the same salt concentration (fig. 3a). in a comparison of pod activity in control plants of different tobacco lines, line 239k had signifi cantly higher pod activity than line fig. 1. roots of line 207b after 33 days of treatment with 0, 100 and 200 mm nacl. bc bc ab d bcd bc bcd cdcd a ab bcd 0 0.04 0.08 0.12 dh10 207b 238c 239k p ro te in c o n te n t (m g g -1 f w ) tobacco lines 0 mm 100 mm 200 mm a c d d d c bc c c a a ab bc 0 2 4 6 dh10 207b 238c 239k p ro li n e c o n te n t (m m o l g -1 f w ) tobacco lines 0 mm 100 mm 200 mm b abc c bc abc abc bc abc a abc bc abc ab 0 0.03 0.06 0.09 0.12 0.15 dh10 207b 238c 239k c a t a c ti v it y (u m g -1 p ro te in s ) tobacco lines 0 mm 100 mm 200 mm bc c bc ab bc abc bc a ab abc bc ab 0 50 100 150 dh10 207b 238c 239k p o d a c ti v it y (u m g -1 p ro te in s ) tobacco lines 0 mm 100 mm 200 mm b a bcd bc bc cd ab bcd bcd d ab bc bcd 0 1000 2000 3000 4000 dh10 207b 238c 239k s o d a c ti v it y (u m g -1 p ro te in s ) tobacco lines 0 mm 100 mm 200 mm a c fig. 2. protein concentration (a) and proline content (b) in roots of tobacco f1 hybrid dh10 and dihaploid lines 207b, 238c and 239k exposed to 0, 100 and 200 mm nacl for 33 days. values are means±se (n=3). different letters indicate signifi cantly different values (p<0.05) between means. fig. 3. activity of catalase (a), peroxidase (b) and superoxide dismutase (c) in roots of tobacco f1 hybrid dh10 and dihaploid lines 207b, 238c and 239k exposed to 0, 100 and 200 mm nacl for 33 days. values are means±se (n=3). different letters indicate signifi cantly different values (p<0.05) between means. marček t., vidaković-cifrek ž., tkalec m., ježić m., ćurković-perica m. 52 acta bot. croat. 76 (1), 2017 207b and increased enzyme activity at 100 mm nacl in comparison to 238c and dh10 (fig. 3b). the activity of sod signifi cantly decreased in hybrid dh10 in nacl supplemented medium as compared to its respective control. among different tobacco lines the highest sod activity was observed in control plants of hybrid dh10 while salttreated roots of line 207b had signifi cantly higher sod activity than hybrid dh10 under the same treatment (fig. 3c). pca yielded two signifi cant components explaining 70% of data variance. the fi rst component (pc1) was largely determined by pod and cat activity as well as protein content and sod activity and the second component (pc2) by protein and proline contents (fig. 4a). the corresponding scores plot (fig. 4b) showed that unstressed plants of lines 207b, 238c and hybrid dh10 grouped together in cluster i while unstressed plants of line 239k separated due to lower protein content. due to higher cat activity and higher sod activity, plants of lines 239k and 207b separated from other lines treated with 100 mm nacl (dh10 and 238c, that form cluster ii). plants of all lines exposed to 200 mm nacl were differentiated from others due to their high proline content but they further separated into two clusters, cluster iii with plants from lines 207b and 238c characterized with higher protein content, and cluster iv with plants from lines 239k and hybrid dh10 characterized with increased pod activity. discussion root growth reduction is one of the most common effects of increased salinity (ceccoli et al. 2011). in our experiment, salt stress caused prominent growth inhibition of the roots of all tobacco lines and hybrid dh10. it even completely inhibited root development, especially in line 244b which was therefore omitted from further analysis. low water potential of nutrient medium, osmotic and toxic effect of nacl and disturbed uptake of essential minerals can all account for the observed effect (haq et al. 2009). previously obtained results revealed that in tobacco shoots salt stress also reduced growth but not very severely, especially in lines 207b and 239k (marček et al. 2014). this might indicate that shoots at least partially avoided toxic effect of nacl, retaining it by apoplastic barriers in root or by disposal into parenchyma cells of the root cortex (munns and tester 2008). the same effect was observed in wheat (triticum sp.) (datta et al. 2009) and persian clover (trifolium resupinatum l.) (ates and tekeli 2007). with the application of higher nacl concentration (200 mm) a signifi cant increase in root protein content in line 207b was observed. this effect was determined earlier in roots of wheat cultivars (shaddad et al. 2013). increased protein content can be an indicator of salinity tolerance if it is caused by synthesis of stress proteins or proteins contributing to antioxidative defence (mohammadkhani and heidari 2008, manaa et al. 2011). however, a degradation process caused by growth retardation can also increase protein level (parvaiz and satyavati 2008, goudarzi and pakniyat 2009, keshtehgar et al. 2013, gupta and huang 2014). con trary to the results obtained in this study, protein content in shoots, investigated previously, remained unchanged within lines 207b and 238c exposed to salinity (marček et al. 2014). it has been reported that salinity can cause different changes in the protein content in various parts of the plant (elsamad and shaddad 1997). in all dihaploid lines and hybrid dh10, salt stress caused signifi cant increase in proline production in roots, implying that proline contributes to osmotic adjustment. furthermore, remarkable proline production in 207b, 238c and 239k at both salt treatments suggests that in these genotypes proline could be connected with salt resistance (ahmed et al. 2008). interestingly, despite higher basal proline content in control plants, roots of hybrid dh10 did not show any more prominent tolerance to 100 mm nacl. according to tammam (2003), sodium accumulation in root causes increased proline production thus protecting root cells against dehydration. increased proline content was noticed in the root of a cat pod -1.0 -0.5 0.0 0.5 1.0 factor 1 : 45.34% -1.0 -0.5 0.0 0.5 1.0 f a c to r 2 : 2 6 .1 5 % sod pro proteins dh10-200 207-0 207-100 207-200 239-0 239-100 239-200 238-0 238-200 -4 -3 -2 -1 0 1 2 3 4 factor 1: 45.34% -4 -3 -2 -1 0 1 2 3 4 f a c to r 2 : 2 6 .1 5 % dh10-0 dh10-100 238-100 a b fig. 4. principal component analysis of combined tobacco lines data sets. loadings (a) and scores (b) of the fi rst two factors. protein concentration (proteins), proline content (pro) and activity of sod, pod and cat represent variables. numbers 0, 100 and 200 following names of tobacco lines (dh10, 207b, 238c and 239k) represent concentrations of nacl – 0, 100 and 200 mm, respectively. dihaploid tobacco roots under salinity acta bot. croat. 76 (1), 2017 53 tobacco cultivar (n. tabacum l. wisconsin) exposed to 300 mm nacl (razavizadeh et al. 2009). when proline production in shoots of tobacco dihaploid lines and hybrid dh10 exposed to nacl was considered, signifi cant proline production in plants at 200 mm nacl was recorded (marček et al. 2014). much higher proline content in shoots than in the roots of these lines suggests that tobacco, like most glycophytes, could have a poor ability to exclude salt, so na+ is concentrated in the cell vacuoles of transpiring leaves. in such conditions, proline should accumulate in the cytoplasm and organelles to balance the osmotic pressure of the ions in the vacuole (munns 2002). similar results have already been reported for barley (hordeum vulgare l.) (ueda et al. 2007) and maize (zea mays l.) (versules and sharp 1999). cat, pod and sod are considered major scavenging enzymes involved in removal of ros produced under various stress conditions, including salinity (parvanova et al. 2004). among investigated tobacco lines, roots of line 239k treated with 100 mm nacl exhibited higher cat activity than line 207b and increased pod activity compared to line 238c and hybrid dh10; those increased activities of both enzymes in line 239k might ensure better scavenging of ros under 100 mm nacl. on the other hand, roots of lines 207b and 238c treated with 200 mm nacl showed remarkable induction of sod activity compared to hybrid dh10 which implies that in the roots of these tobacco lines sod might be more involved in defence response. in our previous study (marček et al. 2014), shoots of line 239k also had pronounced sod, cat and pod activity under salt levels so it seems that line 239k generally had more effective antioxidative defence mechanism and is thus less susceptible to salt stress than other dihaploids and hybrid dh10. untreated roots of hybrid dh10 had higher sod activity than other tobacco lines indicating that its expression is constitutive under physiological level (rangani et al. 2016). moreover, roots of hybrid dh10 exposed to salt treatments showed the same inhibition of sod activity as shoots (marček et al. 2014) which could be connected with the lack of zinc (tavallali et al. 2010), an enzyme cofactor, since salinity disturbs plant nutrient uptake and ion distribution (fernández-garcía et al. 2004, bameri et al. 2012). the decline in sod activity in the salt-exposed roots was also demonstrated in maize salt-sensitive cultivar (sk) (keyster et al. 2013). observed differences in enzyme activity between lines included in this experiment might be the result of their genetic properties. compared to shoots (marček et al. 2014), the roots of all dihaploids and hybrid dh10 exhibited high cat, pod and sod activity as a response to increased salinity suggesting that roots of tobacco dihaploid lines have a stronger antioxidative defence. similarly, more prominent antioxidative response of root than of shoot has been reported for the lentil (lens culinaris m.) 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(ranunculaceae), a protected species in poland, we found that the seed set is impaired. the fl ower is considered an adaptation that has coevolved to achieve effective pollination and successful fertilization. therefore we have focused on the morphological and anatomical characteristics of the fl owers of a. sylvestris l. as a prelude to the study of the species’ pollination biology and plant breeding system. the large size of the fl ower (50.6 ± 16.4 mm in dimensions) and its bowl shape fulfi l both the biotic pollination syndrome and the aerodynamic requirements for pollen dispersal and capture. the opening and closing of the perianth provide a shelter for beetles. the odourless perianth, absence of nectar, scarcity of pollen (approximately 200 000 pollen grains per fl ower) and its traits – small size (axis p = 18.52 ± 1.0 μm; e = 16.59 ± 0.9 μm), lack of balsam on the exine surface, starch accumulation in more than 95% of pollen grains correspond to the specialization in anemophily. the stigma is papillous, the dense hairs are situated between single carpels indicating adaptation to capturing dry pollen and specialization in the wind pollination syndrome. the fl ower of a. sylvestris is an example for an intermediate form between entomophily and anemophily, i.e. a secondary and more advanced feature among ranunculaceae. keywords: androecium, anemone sylvestris, anther, gynoecium, papille, pollen, ranunculaceae, trichomes * corresponding author, e-mail: bozena.denisow@up.lublin.pl introduction different interdependent factors may infl uence the effi ciency of pollen dispersal between individuals, i.e. density of population or pollen vectors (nishikawa and kudo 1995, aizen and harder 2007). the most signifi cant adaptation that co-evolved in order to maximize pollination is that based on fl oral morphology (faegri and van der pijl 1979, konarska 2014, sulborska et al. 2014). typically, colour, shape, scent, display size, and symmetry start the reaction chain of direct or indirect action that leads to pollination (willmer 2011). however, some fl oral structures, e.g. nectaries, characteristics of the style and stamens, or pollen traits have been recognized as fundamental traits of specialization in the pollination syndrome (e.g. anemophily vs. entomophily vs. self-pollination), although numerous exceptions from typical adaptation exist (friedman and barrett 2009). the ranunculaceae is a family with 59 genera and is considered the most primitive of angiosperms (tamura 1995), as well as one of the basic groups of eudicots in apg iii (chase and reveal 2009). within this botanical family, diverse evolutionary tendencies in the fl ower morphology and pollination syndrome occur (endress 1995, tamura 1995, willmer 2011). some genera, i.e. ranunculus, hepatica, adonis, or ficaria possess fl owers with morphological traits fulfi lling the requirements of generalist insect pollinators (denisow et al. 2014). the species that develop spurs or pockets from the perianth and share nectaries contribute to the specialized insect pollination syndrome, e.g. aconitum or aquilegia (endress 1995, denisow and antoń 2012, antoń and denisow 2014), whereas others are adapted to wind pollination, e.g. species from the genus thalictrum (friedman and barrett 2009). a combination of insect and wind pollination has been reported for some species from the genus clematis (tamura 1995). the genus anemone l. s. str. (ranunculaceae) is closely related to hepatica, clematis, anemonella, and ranunculus (chase and reveal 2009). the anemone species are distributed primarily in the northern temperate zones (hegi 1974, tamura 1995). in europe, approximately 17 species from four different subgenera may be found in natural populations (müller 2002, ziman et al. 2011). four species are native to polish fl ora; they occur in different habitats, i.e. deflower morphology of anemone sylvestris acta bot. croat. 75 (1), 2016 75 ciduous forests, xerothermic grasslands, or in mountain fl ora (zając and zając 2009). flowers from the genus anemone were recognized as nectarless and exclusively polleniferous (horovitz 1991, nishikawa and kudo 1995, szklanowska 1995, denisow and bożek 2006); however, nectar production has been documented in anemone nemorosa (erbar and leins 2013). generally, fl owers devoid of nectaries are considered less specialized with regard to the pollination system, than fl owers that do develop nectaries (erbar et al. 1999). anemone sylvestris l. is a perennial herb native to eurasia and distributed from central europe through the caucasus, south siberia to the northwest regions of china (hegi 1974). it is a common species in the balkans (albania, bosnia and herzegovina, bulgaria, croatia, greece, macedonia, montenegro, serbia, and slovenia), occurring in semi-shady steppe-forest communities (zimman et al. 2011). in northern europe, a. sylvestris is found in xerothermic grasslands of the festuco-brometea class and is a characteristic species for the geranio-anemonetum sylvestris association or grows in thermophilous thickets of corylus avellana (piękoś-mirkowa and mirek 2006, maciejewska-rutkowska and antkowiak 2013). in poland, it is an endangered plant, protected by law (piękoś-mirkowa and mirek 2006, kwiatkowska-falińska and faliński 2007). recovery programs for a. sylvestris primarily aim at conservation and management of the habitat (wrzesień and denisow 2006, chmura et al. 2013). it is a long-lived, rhizomatous clonal species. individuals may produce several fl owers each year. the species also has a capability of sexual reproduction (ehrendorfer et al. 2009). a number of studies have pointed out that sexual reproduction is a prerequisite for maintenance and development of sustainable population, even for clonal species, since it counteracts the inbreeding depression (müller 2002). the monitoring of three wild populations of a. sylvestris located in se poland conducted in shortand long-term perspective revealed their decrease in size (denisow and wrzesień 2015, in press). vegetation changes, e.g. shrub encroachment, are possible explanations (kwiatkowskafalińska and faliński 2007). in addition, there are problems with seed set, as we observed only 9–12% of seeds developed in relation to one-ovuled pistils. in the present paper, we focused on the morphological characteristics of the fl owers of a. sylvestris l. as a prelude to the study of the species’ pollination biology and breeding system. in particular, we focused on (i) primary and secondary fl oral attractants, (ii) pollen production and pollen traits. materials and methods flowering of anemone sylvestris l. (ranunculaceae) was observed in 2013 and 2014 in a population located in stawska góra (51°13’n, 23°25’e; 224.8 m a.s.l.) in the lublin upland, se poland. the a. sylvestris was a component of a loose grassland patch of brachypodio-teucrietum from the festuco-brometea class. additionally, for microscopic examinations performed in 2014, we used fl owers from individuals cultivated in the botanical garden of maria curie-skłodowska university, lublin, se poland (51°15’ 44’’n, 22°30’48’’e). the collection was established on the basis of individuals derived from the stawska góra population. flower biology the duration of the fl ower life span was recorded in 2013 (n = 11 fl owers) and in 2014 (n = 15 fl owers). we defi ned the total fl ower lifetime as the period from fl ower opening to corolla shedding. to determine the temporal separation of stigma receptivity and anther dehiscence, the fl ower development was monitored from the bud stage until the end of pollen presentation. the number of anthers and the number of pistils per fl ower (n = 23 in 2013 and n = 27 in 2014) were established. every day we recorded the degree to which stigmas were exerted as well as the number of dehiscing anthers. stigma receptivity the timing of stigma receptivity was determined; 30% hydrogen peroxide was applied for detection of peroxidase activity (spa) (dafni 1992). the entire gynoecium from the fl owers was extracted and was subdivided for three parts (low, medial, apical). each part was placed on a glass slide separately and coated with a drop of h2o2. stigmas that produced bubbles within 2–3 min were considered receptive. the number of receptive stigmas was counted for each individual fl ower (n = 9 fl owers per consecutive day of anthesis) under a binocular microscope (nikon smz-2b). flower size and micromorphology the fl ower size was established by means of fl ower diameter measurements (n = 20 fl owers) at the full fl owering stage. the length between the external points of the petaloid sepals was measured. these measurements were performed using a digital calliper with an accuracy of 0.02 mm. the morphological and anatomical details were examined by means of light microscopy (lm) and scanning electron microscopy (sem). the photographic documentation was made on freshly cut material using an olympus szx12 stereomicroscope equipped with a canon eos 550d digital camera. the material used for sem was fi xed in 2.5% glutaraldehyde in phosphate buffer (ph 7.4; 0.1 m) at a temperature of 4 °c for 12 hours. next, the material was washed in phosphate buffer and dehydrated in graded acetone series, respectively. afterwards, the plant material was critical-point dried using liquid co2, sputter coated with gold, and examined at an accelerating voltage of 30 kv with a tescan/vega lmu scanning electron microscope. pollen production and pollen characteristics the number of pollen grains per anther and fl ower and pollen grain size were determined. the anthers (n = 16 in 2013 and n = 20 in 2014) were dissected from closed fl owdenisow b., antoń s., wrzesień m. 76 acta bot. croat. 75 (1), 2016 ers (n = 11 in 2013 and n = 14 in 2014). next, the anthers were placed on a microscopic glass, the pollen sacs were squashed, and the anther walls were carefully removed. afterwards, we put on a drop of aniline blue with glycerine and the number of pollen grains was counted. pollen grain dimensions were determined in glycerol-gelatine slides (erdtman 1954). the lengths of the polar axis (p) and the equatorial axis (e) were determined (n = 4 × 50 per year). these observations were conducted using a nikon eclipse 200 light microscope. the protein content was detected in dry samples collected during the study period. the kjeldahl method was used for nitrogen content determination and crude protein was estimated using factor 6.25 (roulston and cane 2000). starch accumulation was detected with the lugol’s iodine solution in 200 pollen grains per year. data analysis standard anova was applied to assess inter-year differences in the mean values of the analyzed criteria. in order to detect differences between the means, post hoc comparison was made by means of the tukey test. data are presented as mean values ± standard deviation (sd). the level of statistical signifi cance required to measure differences between the means for all the analyses was p = 0.05. all data analyses were performed using statistica 10.0 (statsoft inc.) software. results the fl owering of a. sylvestris lasted 4–6 weeks, with peak fl owering periods differing up to 4 weeks between the years (tab. 1). the peak of the fl owering time of the species was recorded in april (2013) or in may (2014). the reproductive shoot is unbranched, 10–15 cm high. the fl ower is perfect, actinomorphic, and odour-less. the pentamerous sepals are white and obovate; the fl ower lacks petals (figs. 1a–c). the mean dimension of the fl ower is 50.6 ± 16.4 mm (n = 20). anthesis of a single fl ower typically lasted 4–6 days (n = 11–15). for the fi rst 2–3 days, the fl ower opened between 9.00 and 11.00 and closed at ca. 17.00. a temperature decrease (< 10 °c) kept the fl owers closed, or their closure was observed before 17.00. beetles, e.g. mordellistena sp. were observed in the fl owers. during the course of fl owering, sepal turgor gradually decreased, and from the third day of anthesis, the sepals in many fl owers were not able to open fully. when the fl ower opened the fi rst time, multiple stigmas were inserted over the top of the immature anthers. the fi rst anthers dehisced on day 3–4 after the fi rst fl ower opening, and the remaining anthers dehisced progressively during the successive days. the fl ower development of a. sylvestris allowed us to distinguish 5 relatively distinct stages: stage 1 – tight whitegreenish bud, no stigma receptivity and no pollen release; stage 2 – petals closed, beginning of stigma receptivity; stage 3 – petals opened, stigma receptivity in > 30% of pistils, stage 4 – intermezzo – early male stage, dehiscence of the fi rst anthers, stigma receptivity; stage 5 – > 30% of anthers had dehisced, aborted ovules, or developed achenes. androecium and gynoecium characteristics the fl oral organs are free and arranged in a spiral sequence on a convex receptacle (figs. 1c–d). nectariferous tissue is absent. the androecium consists of numerous stamens, ranging from 57 to 146 per fl ower (tab. 1). the number of stamens per fl ower varied signifi cantly from year to year (f1,11 = 19.7, p = 0.044). the anthers are ovoid-elongated. the anthers within the androecium dehisce gradually, starting at the middle. opening of the ripe anther is longitudinal. three-fold disparities in the number of pollen grains produced per anther were found to occur between the study seasons (f1,16 = 9.6, p = 0.034; tab. 2). the gynoecium possesses numerous apocarpic carpels (figs. 1c–d), whose number ranged from 94 to 184 per fl ower. the number of pistils per fl ower varied signifi cantly from year to year (f1,11 = 12.1, p = 0.032). flower micromorphology the adaxial epidermis of the petaloid sepals is smooth (fig. 2a), whereas the abaxial epidermis bears numerous, tab. 1. phenology and fl oral characteristics (mean ± standard deviation, with range in parentheses) of anemone sylvestris from natural population in stawska góra, se poland, during a 2 year study. means with the same small letter do not differ signifi cantly between years at p < 0.05, based on hsd tukey test. feature 2013 2014 flowering period 10 april – 10 may 26 april – 5 june duration of fl owering (days) 31 55 no. of anthers per fl ower 103.5a ± 36.2 (87 – 131) (n = 23) 87.9b ± 23.1 (57 – 146) (n = 27) no. of pistils per fl ower 112.3a ± 26.8 (94 – 126) (n = 23) 157.6b ± 53.1 (108 – 184) (n = 27) fig. 1. macrophotographs of the of anemone sylvestris: a) plants in the experimental population (bar = 10 cm); b) solitary, white fl owers (bar = 5 cm); c) spirally arranged, multi-staminate androecium (bar = 1 cm); d) longitudinal section through gynoecium showing numerous carpels (bar = 0.5 cm). flower morphology of anemone sylvestris acta bot. croat. 75 (1), 2016 77 long, unicellular hairs with the greatest density observed at the base (figs. 2b, e). in cross sections, the petaloid sepals are relatively thin with 4–6 mesophyll layers and have reduced vascular bundles (fig. 2c). numerous hairs between distinct apocarpic carpels are present (fig. 1d; figs. 2d–h). each carpel contains a single ovule, which develops into a one-seeded achene. the receptacle enlarges after anthesis and continues to enlarge during achene formation in a fruit cluster of fruitlets. stigmatic tissue is visible at the apex of the style (figs. 2f–h). the stigmatic area is easily distinguishable from the style, bearing a number of unicellular and conical papillae, whereas the surface of the style is covered by smooth epidermal cells (figs. 2h–i). the presented stigma is dry. the anther walls consist of the epidermis, endothecium, middle layer, and tapetum (figs. 3a–d). the endothecium has thickenings that are differentiated in the lateral walls of the cells (figs. 3c–d). the pollen grains are dry, tricolpate with a reticulate surface (fig. 3e), and prolato-spheroid (shape index 1.09–1.13). the diameter of the polar axis (p) fig. 3. scanning electron (a–e) and light (f) micrographs of the anther and pollen grains of a. sylvestris: a) general aspect of the stamen (bar = 500 μm); b) longitudinal section through anther with visible connective (c) (bar = 200 μm); c–d) section through anther showing tapetum (arrow), endothecium cells with wall thicknesses (arrowhead) and numerous pollen grains (pg) (bars = 20 μm); e) tricolpate pollen grains (bar = 20 μm); f) pollen grains stained with lugol’s iodine (bar = 10 μm). fig. 2. scanning electron micrographs of the fl oral organs of a. sylvestris: a) smooth adaxial surface of the petaloid-sepal (bar = 500 μm); b) abaxial surface of the petaloid-sepal with numerous hairs (bar = 500 μm);c) longitudinal section through petaloid-sepal, note reduced vascular bundle (bar = 20 μm); d) section through gynoecium showing numerous pistils (bar = 500 μm); e) longitudinal section through fl ower, from the external to internal part: petaloid-sepals, stamens, pistil (bar = 200 μm); f, g) general aspect of the multiple gynoecium with distinct apocarpic carpels (bars = 200 μm and 500 μm, respectively); h) details of a single carpel with easily distinguishable stigmatic area (bar = 100 μm); i) stigmatic area of pistils with a number of unicellular and conical papillae (bar = 20 μm). tab. 2. pollen production and pollen grain characteristics (mean ± standard deviation, with range in parentheses) of anemone sylvestris from natural population in stawska góra, se poland, during a 2 year study. means with the same small letter do not differ signifi cantly between years at p < 0.05, based on tukey test. year no. of pollen grains length of axis (μm) shape index (p/e)per anther per fl ower polar (n =200) equatorial (n = 200) 2013 930.2a ± 484.0 (110 – 1790) (n = 16) 96275 (80927 – 121856) 18.31a ± 1.02 (16.56 – 19.68) 16.71a ± 0.61 (14.02 – 17.23) 1.09 2014 3361.0b ±825.3 (70 – 4480) (n = 20) 295432 (191577 – 490706) 18.73a ± 0.91 (17.34 – 20.88) 16.47a ± 0.85 (14.67 – 17.96) 1.13 mean for years 2144.7 195853 18.52 ± 1.01 16.59 ± 0.92 1.11 denisow b., antoń s., wrzesień m. 78 acta bot. croat. 75 (1), 2016 ranges from 16.56 to 20.88 μm and the equatorial axis (e) is in the range of 14.02–17.96 μm. no year effect on the mean values of the diameter of pollen axis was found (f1,6 = 2.3, p = 0.124 for polar and f1,6 = 1.2, p = 0.085 for equatorial). starch was present in more than 95% of pollen grains (fig. 3f). the mean protein content in the pollen is 24.6% of dry matter ± 0.75. discussion the morphological architecture and extrafl oral attributes of a. sylvestris fl owers imply that two pollination modes may interact to affect pollen dispersal. secondary attractants, sensu faegri and van der pijl (1979), i.e. visual and/or temperature attractants, indicate that the fl owers of a. sylvestris may lure insects as potential pollen vectors. on the other hand, the specifi city of fl oral reward, the traits of pollen grains, as well as some gynoecium traits indicate involvement of wind in the dispersal of pollen. a. sylvestris fl owers are large and bowl-shaped, according to the classifi cation of faegri and van der pijl (1979). in general, this fl ower type seems to function as an advertising organ for luring insect visitors (willmer 2011); it also fulfi ls the aerodynamic requirements for pollen dispersal and capture (friedman and barrett 2009). the perianth is scentless and the feature corresponds to that in anemophilous taxa whose perianth is still visually attractive (willmer 2011). from the third day of anthesis, the turgor of the sepals decreased, which apparently impaired sepal opening. we also observed closing of the fl ower for the night and during the day when temperature dropped below 10 °c. closed fl owers prevent the wind from serving as a pollen dispersal agent. however, the anthers of a. sylvestris inserted on thin fi laments are likely to contact stigmas when the fl ower is blown by the wind; therefore, self-pollination is highly possible. closed fl owers are also temporarily unavailable for insects. we found beetles both in closed fl owers (presumably warming up or avoiding the wind) and in opened fl owers on sunny days (presumably maximizing exposure to solar radiation in order to build up heat before take-off). the syndrome of the opening and closure of the fl ower, which provides a shelter for ectotherms, is referred to as a temperature attractant (faegri and van der pijl 1979) and has already been documented for several species, e.g. adonis vernalis (denisow et al. 2014), anemone patens (ordway 1986), a. canadensis (douglas and cruden 1994), or a. nemorosa (van doorn and van meeteren 2003). however, the signifi cance of insects that take shelter inside a fl ower is not obvious, and requires further experimental investigations to establish their effi ciency in the pollination process, e.g. the number of pollen grains they actually transfer and deposit on the stigmas. in most species, perianth movements of a thermonastic type are associated with a difference in the growth rate of mesophyll cells (van doorn and van meeteren 2003). presumably, the relatively thin mesophyll layer of the petaloid sepals revealed in a. sylvestris optimizes the movement at a minimal metabolic cost. among the noteworthy results of the current study is the fi nding that a. sylvestris fl owers lack nectar-secreting structures. the attribute can be considered as promotion of anemophily (friedman and barrett 2009) and is interpreted as a co-evolutionary adaptation to attraction of specifi c type of insects (waldbauer and friedman 1991). absence of nectar was confi rmed for other anemone species, i.e. a. coronaria, a. canadensis, a. fl accida, or a. japonica, the fl owers of which were visited mainly by beetles; however, fl ies or primitive apoidea (anthophoridae, halictidae) were also observed (horovitz 1975, horowitz 1991, douglas and cruden 1994, nishikawa and kudo 1995, denisow and bożek 2006). conversely, nectar-producing fl owers of a. nemorosa were reported to be visited by bombylius spp., honeybees, or bumblebees (szklanowska 1995, erbar and leins 2013). probably, the nectar defi ciency in the fl owers of a. sylvestris restricts the range of the insect spectrum, resulting in the scarcity of dipterans and bee visitors in our experimental population (manuscript in preparation). not only was nectar absent, but a relatively small amount of pollen was produced in a single fl ower of a. sylvestris. altogether, the primary attractant characteristics possibly explain the low interest of insect visitors. although the fl owers are multi-staminate, they released only approx. 200 000 pollen grains per fl ower. the value is ten-fold lower than that established for anemone coronaria (approx. two million). generally, taxa from the family ranunculaceae are reported to produce copious amounts of pollen (szklanowska 1995, denisow and bożek 2006, denisow and antoń 2012). the low pollen production in a. sylvestris may be related to the genetic potential of the species; pollen production is reported to be highly genetically dependent (szklanowska 1995, denisow 2011). however, the year-toyear disparities in the number of pollen grains produced indicate that pollen production in a. sylvestris is sensitive to external factors. for example, weather conditions (shortage of precipitation or air temperature anomalies) are known to limit pollen production in a number of taxa (denisow and bożek 2006, aizen and harder 2007, denisow et al. 2014). the pollen grains of a. sylvestris are small, within a range of 10–25 μm (erdtman 1954). the protein content of pollen is approximately 25% of the dry mass. the protein concentration in pollen grains is a highly conserved trait within plant genera and families; however, the correlation between the protein content and the type of pollination is not always obvious (roulston and cane 2000, denisow 2011). nonetheless, small pollen grains, lack of balsam on the exine surface, and starch accumulation correspond well to specialization in anemophily (friedman and barrett 2009). similar pollen traits were described for anemone coronaria by horowitz (1991), who proved that pollen was dispersed by air currents up to the distance of < 1.5 m from the fl ower. we found papillous stigma and dense hairs situated between single carpels. it is possible that these properties enhance the capture of dry pollen and may indicate specialization in the wind pollination syndrome, as suggested by horowitz (1991). however, the pistils placed on the convex receptacle are well exposed and provide an unobstructed flower morphology of anemone sylvestris acta bot. croat. 75 (1), 2016 79 path for pollen transported by both abiotic and biotic agents (friedman and barrett 2009). the other ecologically significant adjustment of these dense hairs seems to be protection of pistils and ovules from overheating and/or heat loss. low temperatures are particularly frequent during early spring, and they are often hazardous for carpels, which are considered the most sensitive plant structures (hedhly 2011). the anthers of a. sylvestris dehisce longitudinally. we found lignifi ed wall thickenings in the endothecium cells. many investigation results are consistent in showing that cell wall thickenings control the dynamics of anther opening by regulating the dehydration and shrinkage of cells, which may indirectly support pollen dispersal (keijzer 1987). the abaxial surface of the petaloid sepals is velvety. it is associated with the presence of numerous hairs on the abaxial epidermis. the same characteristic of abaxial epidermis was evidenced in the fl owers of adonis vernalis (gostin 2009). epidermal hairs are reported to protect epidermal and mesophyllous cells against excessive heating or water loss, which is especially important in xerothermic habitats (karabourniotis et al. 1992). the adaptive signifi cance of epidermal hairs in the defence against phytophagous insects has also been confi rmed (hanley et al. 2007). a. sylvestris fl owers are protogynous, which has been confi rmed by the fi ndings for other anemone species (horowitz 1991, denisow and bożek 2006). protogyny is commonly described in cantharophilous fl owers (faegri and van der pijl 1979) as well as in anemophilous ones (friedman and barrett 2009). in the protogynous fl owers of a. sylvestris, the absence of nectar may affect the function of the female phase by decreasing the attractiveness of the phase to potential pollinators and infl uence the mating opportunity of the fl ower. presumably, the fl ower of a. sylvestris is an example of an intermediate form between entomophily and anemophily, i.e. a secondary and more advanced feature among ranunculaceae (endress 1995). the fl oral morphology and the type of the fl oral attractant indicate the existence of alternative modes of pollination (partial wind, self-pollination, or biotic pollination). acknowledgements the material from protected species was collected in compliance with polish law under permit from the regional nature conservator in lublin. the research was supported fi nancially by the ministry of science and higher education of poland as a part of the statutory activities of the department of botany, university of life sciences in lublin (project: okb/ds/2) and the department of geobotany, institute of biology and biochemistry, maria curieskłodowska university. references aizen, m. a., harder, l. d., 2007: expanding the limits of the pollen limitation concept: effects of pollen quantity and quality. ecology 99, 271–281. antoń, 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(ed.), natürliche pfl anzenfamilien, 223–519. second ed., duncker & humblot, berlin, germany. waldbauer, g. p., friedman, s., 1991: self-selection of optimal diets by insects. annual review of entomology 36, 43–63. willmer, p., 2011: pollination and fl oral ecology. princeton univ press, princeton. wrzesień, m., denisow, b., 2006: the share of nectariferous and polleniferous taxons in chosen patches of thermophilous grasslands of the lublin upland. acta agrobotanica 59, 213– 221. van doorn, w.g., van meeteren, u., 2003: flower opening and closure: a review. journal of experimental botany 54, 1801–1812. zając, m., zając, a., 2009: the geographical elements of native fl ora of poland. laboratory of computer chorology, institute of botany, jagiellonian university, kraków. ziman, s., bulakh, e., tsarenko, o., 2011: anemone l. (ranunculaceae): comparative morphology and taxonomy of the species from the balkan fl ora. botanica serbica 35, 87–97. opce-str.vp acta bot. croat. 68 (2), 301–312, 2009 coden: abcra 25 issn 0365–0588 distribution of periphytic diatoms in the rivers of the lake ladoga basin (northwestern russia) alexander g. rusanov1*, elena v. stanislavskaya1, eva acs2 1 institute of limnology of the russian academy of sciences, sevastyanova 9, 196 105, st. petersburg, russia. 2 institute of ecology and botany of the hungarian academy of sciences, hungarian danube research station, jávorka s. u. 14, 2131 göd, hungary. relationships between distribution of periphytic diatoms and environmental variables in 19 rivers of the lake ladoga basin (northwestern russia) were examined using gradient analysis. on the basis of geology and river water chemistry, the lake ladoga basin could be separated into two main parts, the northern and the southern sub-basin. the rivers in the northern sub-basin are slightly acidic and low in conductivity (mean value 53 ms cm–1); the rivers in the southern sub-basin have neutral to slightly alkaline waters with higher conductivities (mean value 168 ms cm–1). a detrended correspondence analysis (dca) defined two groups of rivers generally corresponding to the two main parts of the lake ladoga basin. fragilaria capucina var. rumpens, frustulia saxonica and tabellaria flocculosa were the typical species for the northern sub-basin, whereas cocconeis placentula var. euglypta, ulnaria ulna and gomphonema parvulum were characteristic species for the southern sub-basin. a canonical correspondence analysis (cca) identified conductivity, ph, bicarbonate, total phosphorus and water colour as the most important environmental variables related to changes in assemblage structure. both dca and cca ordination showed that conductivity related to geology was the most important variable, while concentration of total phosphorus was the second most important variable. weighted averaging was used to infer total phosphorus from relative biomass of diatoms. the predictive ability of the inference model was sufficiently strong with r2 = 0.71 and rmsep = 1.9 mg l–1. these results strongly support the use of a diatom-based inference phosphorus model for indicating eutrophication in the rivers of the lake ladoga basin. keywords: diatom, periphyton, distribution, gradient, eutrophication, lake ladoga, russia introduction periphytic diatoms are excellent indicators of ecological condition of rivers and streams, because of their ability to respond rapidly to changes in nutrient concentrations. the sensitivity of diatoms to eutrophication has led to development of monitoring methods acta bot. croat. 68 (2), 2009 301 * corresponding author: a_rusanov@yahoo.com u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:08 color profile: disabled composite 150 lpi at 45 degrees and indices to assess water quality of rivers. several diatom-based indices are being used to estimate trophic status of european rivers (e.g. kelly and whitton 1995, rott et al. 2003). however, the applicability of these indices may be limited to specific geographic regions, because autecological metrics of diatoms can vary across different geographic areas (potapova and charles 2007). species response to nutrient enrichment may also depend on other environmental factors, which are related to geology of the underlying bedrocks, land use and other landscape characteristics. therefore, in order to improve diatom-based water-quality assessment, autecological metrics of indicator species should be developed by quantifying species distribution along environmental gradients within a particular region. in this study, distributional patterns of periphytic diatoms in relation to environmental variables in 18 inflow rivers of the lake ladoga and the outflowing neva river were investigated using multivariate ordination methods. in addition, this study assessed the potential use of periphytic diatoms as indicators of eutrophication in the rivers of the lake ladoga basin by developing a diatom-based inference model for total phosphorus. characteristics of the water bodies the lake ladoga basin (republic of karelia and leningrad oblast, northwestern russia) has a length of more than 1,000 km (north-south) and a catchment area of 260,000 km2. it can be divided into two main parts, the northern and the southern sub-basin, which differ in geomorphology and geology. the northern part is located on the baltic shield comprised of acid crystalline and metamorphic rocks (granite in majority and gneiss). the southern part is dominated by terrigenous and carbonaceous sedimentary rocks (sandstone, limestone and dolomite) of the russian platform. the rivers flowing into lake ladoga on the northern and eastern coasts (burnaya, khiitolan, iijoki, mijnola, yanis, uksun, tulema, vidlitsa, tuloksa, olonka, svir) belong to the northern sub-basin, while rivers of the western, southern and southeastern coasts (avloga, morje, lava, volkhov, syas, pasha, oyat,) belong to the southern sub-basin (fig. 1). the division of the lake ladoga basin into two areas is reflected in differences in the water chemistry of their rivers (solovieva 1967). the rivers of the northern sub-basin generally have slightly acidic waters with low conductivities, whereas the rivers of the southern sub-basin have neutral to slightly alkaline waters with high conductivities (up to 350 ms cm–1) (tab. 1). the studied rivers range widely in water colour, which reflects differences in the percentage of lakes/wetlands in the river catchments. the pasha, tulema, iijoki, avloga, olonka, tuloksa and morje rivers with wetland-dominated catchments are characterized by darker water colour with values ranging from 150 to 275 pt-co units (tab. 1). in contrast, the rivers khiitolan, svir and burnaya with a high percentages of lakes in the catchments (14–20%) have less coloured water (<50 pt-co units). due to anthropogenic eutrophication in the 1970s and 1980s, the amount of nutrients transported by the rivers increased considerably (raspletina 1982). the highest concentrations of total phosphorus were recorded where the drainage area was subjected to the greatest anthropogenic impact, mainly in the southern rivers volkhov and lava, rivers of the western coast morje and avloga, some rivers of the eastern coast olonka and tuloksa, and in the northern river iijoki (tab. 1). only in the rivers of the northern and northeastern coast, less subject to anthropogenic influence (burnaya, yanis, uksun, tulema and svir), did the concentration of nutrients remain at the level of the 1960s (total phosphorus: 15–24 mg l–1). the hydrochemistry of the neva river source is largely determined by 302 acta bot. croat. 68 (2), 2009 rusanov a. g., stanislavskaya e. v., acs e. u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:08 color profile: disabled composite 150 lpi at 45 degrees hydrochemical regime of lake ladoga and to a lesser extent by the water mass supplied by the major inflows of lake ladoga, the volkhov and burnaya rivers (raspletina et al. 2006). materials and methods periphytic diatom samples were collected from stones and macrophytes in the mouths of 18 tributaries of lake ladoga and in the source of the neva river in may, july and september 2000–2001. in all, 135 diatom samples were collected and analyzed. water samples were taken simultaneously with diatom samples. they were analyzed for electric conductivity, bicarbonate, ph, soluble reactive phosphorus, total phosphorus and water colour. after acid cleaning and mounting diatoms on slides, diatom valves were identified and enumerated using a light microscope (1000× magnification), by scanning transects until acta bot. croat. 68 (2), 2009 303 periphytic diatoms in the lake ladoga basin fig. 1. location of the sampling sites in the rivers of the lake ladoga basin. open (group a) and filled (group b) circles are rivers which were included in groups defined by dca ordination of diatom composition data. u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:10 color profile: disabled composite 150 lpi at 45 degrees 300 valves were counted. diatoms were identified to species level using krammer and lange-bertalot (1986–1991). for common taxa the dimensions of 20–25 cells (fewer cells measured on rare taxa) were measured with an ocular micrometer and were used to calculate biovolume. biovolume for each diatom taxon was estimated with the geometrical equations proposed by hillebrand et al. (1999). the 61 most abundant diatom taxa contributing on average 96.8% (min 62.1%, max 99.7%) to the total diatom biovolume in periphyton samples were included in the statistical analyses. ordination analyses of periphytic diatom assemblages were performed using canoco (ter braak and smilauer 1998). detrended correspondence analysis (dca) was used to elucidate main patterns in diatom assemblage and to assess similarity among rivers in terms of assemblage composition. canonical correspondence analysis (cca) was subsequently used to investigate species-environment relationships. the variance explained in the dca and cca ordinations were 3–4% more using counts based upon percent biovolume than percent cell abundance of taxa; therefore diatom assemblage structure was expressed as percent biovolume. weighted-averaging (wa) regression-calibration models were developed for electric conductivity, ph and total phosphorus (tp) using the computer program c2 (juggins 2003). the strength of the inference models was assessed by the determination coefficient (r2) between the observed and inferred values, and the root mean squared errors of prediction (rmsep). prior to the analyses, all environmental variables were log-transformed and percent biovolumes of diatom taxa were arcsine square root transformed to approximate a normal distribution. 304 acta bot. croat. 68 (2), 2009 rusanov a. g., stanislavskaya e. v., acs e. tab. 1. hydrological and average hydrochemical characteristics for the 19 study rivers. river discharge (m3 sec–1) tp (mg l–1) srp (mg l–1) conductivity (ms cm–1) hco3 (mg l–1) ph color (pt-co units) neva 2400.0 28 5 89 34 7.5 29 svir 661.0 24 6 55 22 7.1 49 burnaya 613.0 16 5 65 15 7.1 30 volkhov 535.0 68 30 197 76 7.5 108 syas 63.8 34 14 193 129 7.8 131 pasha 73.7 34 9 115 59 7.3 149 ojat 58.6 30 14 86 47 7.3 129 yanis 41.7 18 5 34 8 6.8 69 olonka 35.2 88 50 61 21 6.9 187 tulema 21.8 20 8 33 11 6.8 149 khiitolan 14.7 35 10 92 25 7.1 38 vidlitsa 18.5 46 8 44 19 6.9 102 uksun 15.0 17 3 26 6 6.6 128 tuloksa 8.6 70 35 41 16 6.6 218 mijnola 5.2 32 15 58 19 6.8 130 lava 4.2 102 67 347 196 8.1 123 morje 4.5 149 80 61 16 6.6 275 avloga 1.8 395 360 258 121 7.3 155 iijoki 1.5 190 161 61 18 7.1 179 u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:10 color profile: disabled composite 150 lpi at 45 degrees two trophic diatom indices tdi and tid were calculated following kelly and whitton (1995) and rott et al. (2003) using the omnidia software (lecointe et al. 1993). the relationships between the diatom indices and tp were assessed using simple linear regression. results diatom-environmental relationships the eigenvalues of the first two dca axes accounted for 17% of the variance in the data set. the first axis was significantly correlated with electric conductivity, bicarbonate and ph (correlation coefficient r = –0.67, –0.65 and –0.63, respectively). the second axis was most correlated with tp and srp (correlation coefficient r = 0.51, 0.47, respectively). the dca diagram shows centroids of seasonal samples of the investigated rivers in the ordination space of the first and second axes (fig. 2). the dca diagram separates all rivers along the first axis into two groups based on diatom species composition. group a in the left side of the dca diagram consists of rivers from the southern part of the lake ladoga basin (including the neva river), except for one northern river, the khiitolan (figs. 1, 2). group b in the right side of the dca diagram mainly consists of rivers from the northern part of the lake ladoga basin with one exception, the southern river morje (figs. 1, 2). conductivity, bicarbonate and ph in group a were significantly higher than in group b (tab. 2). it is noticeable that tp and srp were not significantly different between the two river groups. species replacement from group a to group b was evident in diatom assemblages (tab. 2). diatom assemblages in group a were dominated by cocconeis placentula var. euglypta, ulnaria ulna and gomphonema parvulum. the dominance of these three species was replaced by that of tabellaria flocculosa and fragilaria capucina var. rumpens in group b. comparisons between groups a and b for relative biomasses of these species showed staacta bot. croat. 68 (2), 2009 305 periphytic diatoms in the lake ladoga basin 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 lava volkhov khiitolan avloga syas neva ojat pasha morje iijoki mijnola vidlitsa olonka burnaya svir tuloksa uksun tulema yanis a b dca axis 1 d c a a x is 2 fig. 2. dca diagram showing centroids of seasonal periphyton samples for each river. group a mostly consists of rivers from the southern part of the lake ladoga basin, whereas group b mainly contains rivers from the northern part of the lake ladoga basin (explanation in text). u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:10 color profile: disabled composite 150 lpi at 45 degrees tistical differences (tab. 2). other common species, which had a significantly higher relative biomass in group b than group a, included frustulia saxonica, eunotia bilunaris, e. incisa and e. praerupta. the eigenvalues of the first two cca axes were both significant (p < 0.01; monte carlo permutation test), and they explained 14% of the variance in the species data. the diatom-environment correlations for cca axis 1 (0.86) and 2 (0.83) were high, indicating a strong relation between diatoms and the measured environmental variables. conductivity, bicarbonate and ph were the most significant factors contributing to the first axis, whereas tp, srp and colour were the most important variables along the second axis (fig. 3). cca separated rivers primarily according to their conductivity, ph and bicarbonate concentration, related to geology, and secondarily according to their trophic status related to phosphorus concentration. the first axis mainly separated rivers with high conductivity and neutral to slightly alkaline waters in the southern sub-basin from rivers with low conductivity and slightly acidic waters in the northern sub-basin. on the right side of the first axis alkaliphilous taxa (according to van dam et al. 1994) like amphora ovalis, cocconeis placentula var. euglypta, diatoma vulgaris, frustulia vulgaris, gomphonema olivaceum, navicula tripunctata, planothidium lanceolatum, rhoicosphenia abbreviata, and ulnaria ulna were located, while on the left side of the first axis acidophilous species (according to 306 acta bot. croat. 68 (2), 2009 rusanov a. g., stanislavskaya e. v., acs e. tab. 2. differences in mean values (and range) of environmental variables and relative biomass of dominant diatom taxa between the two river groups defined by dca. p-values are the results of mann-whitney u test. variables river group p a (n = 57) b (n = 78) environmental variables conductivity (ms cm–1) 161 (43–363) 48 (21–90) < 0.001 ph 7.5 (6.8–8.3) 6.8 (5.9–7.6) < 0.001 hco3 (mg l –1) 81 (19–201) 16 (3–36) < 0.001 tp (mg l–1) 79 (14–450) 54 (10–335) ns srp (mg l–1) 53 (1–430) 28 (1–300) ns color (pt-co units) 110 (25–242) 136 (25–360) ns diatoms (% biovolume) cocconeis placentula var. euglypta 19.1 (0–97.4) 4.6 (0–68.6) < 0.001 ulnaria ulna 14.5 (0–63.7) 8.5 (0–56.6) < 0.05 gomphonema parvulum 11.3 (0–81.9) 5.8 (0–79.5) < 0.01 tabellaria flocculosa 2.5 (0–12.3) 21.2 (0–83.6) < 0.001 fragilaria capucina var. rumpens 3.2 (0–14.8) 12.5 (0–66.5) < 0.001 frustulia saxonica 0.5 (0–17.2) 3.2 (0–48.9) < 0.05 eunotia bilunaris 0.3 (0–4.7) 4.1 (0–64.1) < 0.05 eunotia incisa 0.4 (0–19.2) 3.3 (0–46.9) < 0.001 eunotia praerupta 0.1 (0–2.3) 2.3 (0–31.9) < 0.05 melosira varians 12.6 (0–65.1) 16.1 (0–89.4) ns ns – differences are not significant (p > 0.05). u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:10 color profile: disabled composite 150 lpi at 45 degrees van dam et al. 1994) such as tabellaria flocculosa, eunotia bilunaris, e. incisa, e. pectinalis var. undulata, e. praerupta, e. tenella, e. veneris, fragilariforma virescens, and frustulia saxonica were positioned (fig. 3). the second axis primarily separated phosphorus-enriched and humic rivers, such as the morje, avloga, iijoki and tuloksa, from oligo-mesotrophic, clear-water rivers, including the rivers burnaya, svir and yanis. species indicative of high phosphorus concentration such as navicula capitatoradiata, placoneis elginensis, sellaphora pupula, frustulia vulgaris and pinnularia gibba were positioned on the positive side of the second axis, whereas species indicative of low nutrient status such as eunotia tenella, e. pectinalis var. undulata, achnanthidium minutissimum and fragilaria capucina var. rumpens were on the negative side of the second axis (fig. 3). such ordination of these species corresponded to their trophic preferences known from the literature (e.g. van dam et al. 1994). some species such as frustulia saxonica and eunotia praerupta, often found at low nutrient concentrations (van dam et al. 1994), grouped with eutrophic taxa (fig. 3). these species reached the highest relative abundances (48.9% and 31.9%, respectively) in eutrophic humic rivers such as the morje and the iijoki. weighted-averaging models a series of weighted-averaging (wa) models were developed to test for their suitability to infer water quality variables. environmental variables should be considered only if the ratio of the first eigenvalue (l1) to the second eigenvalue (l2) is large in a cca constrained for an environmental variable (hall and smol 1992). analyses constrained by conductivacta bot. croat. 68 (2), 2009 307 periphytic diatoms in the lake ladoga basin -0.6 1.0 -0 .6 1 .0 pbi ami ptl aov enm ensctu cpe cci dmo dte dvu fcr fcv spi uul fua ffv fsa fvu gac gan gol gpa gtr gya ebi eve ein epu ete eex epr mva mun mci hca nra ncp ncr nme nrh nvi nvr ntp spu pel ndi npa nli nan rab pgi pin pvi psc pma san sbi tfe tfl tp srp color temp ph hco3 ec cca axis 1 c c a a x is 2 fig. 3. cca diagram showing environmental variables and diatom species. codes for environmental variables: ec – electric conductivity, hco3 – bicarbonate, ph – water ph, tp – total phosphorus, srp – soluble reactive phosphorus, color – water color, and temp – water temperature. species codes can be found in table 3. u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:10 color profile: disabled composite 150 lpi at 45 degrees ity (l1/l2 = 0.61), ph (l1/l2 = 0.57), and tp (l1/l2 = 0.54) yielded ratios that suggested the potential for developing a model. thus, diatom inference models were constructed for each of these three variables using weighted-averaging (wa). considering both rmsep and coefficients of determination (r2) between measured and inferred values of the environmental variables, wa with tolerance down-weighting and classical de-shrinking procedure performed best in the construction of conductivity, ph and tp models. the inference models for conductivity, ph and tp had rmsep/r2 values of 0.199/0.80, 0.021/0.74 0.291/0.71, respectively. the wa estimated optima of conductivity, ph and tp for the 61 most common diatom taxa are listed in table 3 together with the number of occurrences. diatom indices the tdi values for the lake ladoga tributaries data set ranged from 16.3 to 89.4 on a scale of 1 (oligotrophic) to 100 (eutrophic). the index values were not significantly related to tp (r = 0.16, p > 0.05). the tid values ranged from 3.2 to 15.5 on a scale of 1 (eutrophic) to 20 (oligotrophic). values for the tid were significantly related to tp (r = –0.47, p < 0.001), but the coefficient of determination value (r2 = 0.22) indicated that only a relatively small amount of the variance in the relationship was explained. 308 acta bot. croat. 68 (2), 2009 rusanov a. g., stanislavskaya e. v., acs e. tab. 3. code, the number of occurrences (n) (of 135 samples) and optima values for electric conductivity (ec, ms cm–1), ph, and total phosphorus (tp, mg l–1) of 61 most common diatom taxa in the lake ladoga rivers. code taxon n ec ph tp adm achnanthidium minutissimum (kütz.) czarn. 80 72 7.1 35 aov amphora ovalis (kütz.) kütz. 10 242 7.9 46 cpe cocconeis placentula var. euglypta (ehrenb.) grun. 72 124 7.5 52 cci cymbella cistula (ehrenb.) kirchn. 4 64 7.1 71 ctu cymbella tumida (breb. ex kütz.) v. h. 11 58 6.9 60 dmo diatoma moniliformis kütz. 8 83 7.0 30 dte diatoma tenuis ag. 14 123 7.1 37 dvu diatoma vulgaris bory 21 107 7.4 35 enm encyonema minutum (hilse) d.g. mann 69 74 7.2 14 ens encyonema silesiacum (bleisch) d.g. mann 25 100 7.3 72 ebi eunotia bilunaris (ehrenb.) mills 35 51 6.9 74 eex eunotia exigua (breb. ex kütz.) rabenh. 14 48 6.8 104 ein eunotia incisa w. smith ex greg. 32 47 6.7 52 epu eunotia pectinalis var. undulata (ralfs) rabenh. 14 53 6.9 24 epr eunotia praerupta ehrenb. 12 63 6.8 159 ete eunotia tenella (grun.) cleve 30 50 6.9 20 eve eunotia veneris (kütz.) de toni 7 69 6.8 28 fcr fragilaria capucina var. rumpens (kütz.) lange-b. 100 57 6.9 30 fcv fragilaria capucina var. vaucheriae (kütz.) lange-b. 25 81 7.1 48 fua fragilaria ulna (nitz.) lange-b. var. acus (kütz.) lange-b. 6 72 7.2 23 u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:10 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 309 periphytic diatoms in the lake ladoga basin code taxon n ec ph tp ffv fragilariforma virescens (ralfs) williams et round 6 34 6.6 28 fsa frustulia saxonica rabenh. 20 64 6.9 105 fvu frustulia vulgaris (thwaites) de toni 17 119 7.3 122 gac gomphonema acuminatum ehrenb. 21 50 6.8 36 gan gomphonema angustatum (kütz.) rabenh. 15 117 7.2 71 gol gomphonema olivaceum (hornemann) breb. 22 101 7.2 49 gpa gomphonema parvulum (kütz.) kütz. 110 70 7.0 67 gtr gomphonema truncatum ehrenb. 29 94 7.4 33 gya gyrosigma acuminatum (kütz.) rabenh. 13 84 7.2 79 hca hippodonta capitata (ehr.) lange-b., metzeltin et witkowski 13 127 7.4 43 mun melosira undulata (ehrenb.) kütz. 5 158 7.7 34 mva melosira varians ag. 62 77 7.1 68 mci meridion circulare (grev.) ag. 35 48 6.8 51 ncp navicula capitatoradiata germain 13 203 7.3 189 ncr navicula cryptocephala kütz. 103 72 7.1 50 nme navicula menisculus schum. 7 93 7.4 16 nra navicula radiosa kütz. 33 76 7.1 41 nrh navicula rhynchocephala kütz. 55 67 7.1 42 ntp navicula tripunctata (o.f. müll.) bory 11 196 7.6 71 nvi navicula viridula (kütz.) ehrenb. 14 92 7.2 47 nvr navicula viridula var. rostellata (kütz.) cleve 11 82 7.0 81 nan nitzschia angustata (w. smith) grun. 3 362 8.1 102 ndi nitzschia dissipata (kütz.) grun. 20 87 7.2 34 nli nitzschia linearis (ag.) w. smith 10 116 7.3 52 npa nitzschia palea (kütz.) w. smith 40 75 7.0 71 pgi pinnularia gibba (ehrenb.) ehrenb. 15 91 7.0 91 pin pinnularia interrupta w. smith 11 35 6.7 20 pma pinnularia major (kütz.) rabenh. 5 59 7.1 112 psc pinnularia subcapitata greg. 14 103 7.0 162 pvi pinnularia viridis (nitz.) ehrenb. 4 124 7.2 41 pel placoneis elginensis (greg.) e.j. cox 11 103 7.4 115 ptl planothidium lanceolatum (breb. ex kütz.) lange-b. 47 106 7.2 64 pbi psammothidium bioretii (germain) bukhtiyarova et round 7 57 7.1 28 rab rhoicosphenia abbreviata (ag.) lange-b. 12 261 7.8 237 spu sellaphora pupula (kütz.) meresck. 12 150 7.1 168 spi staurosirella pinnata (ehrenb.) williams et round 27 137 7.4 32 san surirella angusta kütz. 15 106 7.2 55 sbi surirella biseriata breb. et godey 7 66 7.0 88 tfe tabellaria fenestrata (lyngb.) kütz. 41 60 7.0 70 tfl tabellaria flocculosa (roth) kütz. 56 37 6.7 32 uul ulnaria ulna (nitz.) compère 86 82 7.2 51 tab. 3. – continued u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:11 color profile: disabled composite 150 lpi at 45 degrees discussion in our study, two main groups of rivers were identified. they reflected the two geomorphological regions of the lake ladoga basin, which are characterized by granitoid crystalline rocks in the northern sub-basin and carbonaceous sedimentary rocks in the southern sub-basin and corresponding differences in hydrochemistry. in the northern sub-basin, crystalline rocks with low buffering capacity led to slightly acidic rivers, whereas in the southern sub-basin, carbonaceous sedimentary rocks led to neutral to slightly alkaline waters. this was reflected by differences in diatom assemblages with acidophilus taxa occurring in the north and alkaliphilous taxa in the south. misclassified rivers such as the morje and khiitolan indicate that local features of river catchments can influence hydrochemistry and distribution pattern of the diatom assemblages. for example, the southern river morje had a high percentage of wetlands in its catchment and a significantly lower ph than the mean ph of the southern sub-basin rivers. it was also characterized by acidophilous diatom assemblages similar to those in the northern sub-basin. ordination showed that electric conductivity was the most important factor for diatom assemblages of rivers in the lake ladoga basin, similar to findings in other studies on periphytic diatom assemblages (soininen 2007, soininen et al. 2004). potapova and charles (2003) showed that conductivity and major ions (including bicarbonate) explained a significant amount of the variation in assemblage composition of benthic diatoms in us rivers. conductivity reflects watershed processes such as bedrock weathering and consequent dissolution of chemical constituents as well as nutrient enrichment due to agricultural land use (biggs 1995). phosphorus was the second most important factor. if there are large regional differences between watersheds in dominant rock type, environmental variables reflecting geology such as conductivity, alkalinity and ph may be more important determinants of periphytic diatom assemblages than variables reflecting trophic status such as nutrient concentrations (leland 1995, rimet et al. 2004). furthermore, at regional spatial scales changes in conductivity may also be related to trophic status, reflecting changes in land use across different regions (denicola et al. 2004). both geology and land use were important in our study. the southern sub-basin is characterized by more intensive agricultural land use than the northern sub-basin (raspletina 1982). this was evident from a marked shift in dominant diatom taxa between the sub-basins. diatom assemblages of the southern sub-basin were dominated by taxa typical of meso-eutrophic and eutrophic conditions such as cocconeis placentula var. euglypta, ulnaria ulna and gomphonema parvulum (according to van dam et al. 1994). in contrast, diatom assemblages of the northern sub-basin were dominated by tabellaria flocculosa and fragilaria capucina var. rumpens, indicating oligo-mesotrophic waters (van dam et al. 1994). the wa model for tp provided good estimates of measured tp, accurate within ±1.9 mg l–1, with a predictive ability (apparent r2 = 0.71) similar to other studies (c. 0.6–0.8, leland and porter 2000, winter and duthie 2000, denicola et al. 2004). optima for tp ranged from 14 mg l–1 for encyonema minutum to 237 mg l–1 for rhoicosphenia abbreviata. taxa indicative of eutrophic conditions including gomphonema parvulum, navicula capitatoradiata, sellaphora pupula, placoneis elginensis, and nitzschia palea and those typical of low tp concentration such as eunotia tenella, e. pectinalis var. undulata, fragilaria capucina var. rumpens, and tabellaria flocculosa corresponded to classifica310 acta bot. croat. 68 (2), 2009 rusanov a. g., stanislavskaya e. v., acs e. u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:11 color profile: disabled composite 150 lpi at 45 degrees tions by van dam et al. (1994). the calculated optima for several species were different from data suggested in the literature. for example, frustulia saxonica and eunotia praerupta listed as oligotrophic species in van dam et al. (1994) appeared in rivers with high total phosphorus concentrations. these rivers were also rich in humic substances. it is possible that phosphorus was bound by humic acids and therefore not freely available for algae (jones et al. 1988, meili 1992). values for two diatom indices tdi and tid calculated in this study were poor indicators of trophic state. weak relationships between diatom index values and phosphorus concentrations in the studied rivers as well as the observed differences in species responses to nutrient enrichment show the need to develop a regional method for the assessment of river trophic status. the autecological data provided by this study can be used for the development of such a biomonitoring tool for the rivers in the lake ladoga basin. acknowledgement we would like to thank the referees for their useful comments on the manuscript. this work was supported by the russian foundation for fundamental research – grant no. 08-04-01544. references biggs, b. j. f., 1995: the contribution of flood disturbance, catchment geology and land use to the habitat template of periphyton in stream ecosystems. freshwater biology 33, 419–438. denicola, d. m., de eyto, e., wemaere, a., irvine, k., 2004: using epilithic algal communities to assess trophic status in irish lakes. journal of phycology 40, 481–495. hall, r. i., smol, j. p., 1992: a weighted-averaging regression and calibration model for inferring total phosphorus concentration from diatoms in british columbia (canada) lakes. freshwater biology 27, 417–434. hillebrand, h., durselen, c. d., kirschtel, d. b., pollingher, u., zohary, t., 1999: biovolume calculation for pelagic and benthic microalgae. journal of phycology 35, 403–424. jones, r. t., salonen, k., de hann, h., 1988: phosphorus transformations in the epilimnion of humic lakes: abiotic interactions between dissolved humic materials and phosphate. freshwater biology 19, 357–369. juggins, s., 2003: c2 user guide: software for ecological and paleoecological data analysis and visualization. university of newcastle. kelly, m. g., whitton, b. a., 1995: the trophic diatom index: a new index for monitoring eutrophication in rivers. journal of applied phycology 7, 433–444. krammer, k., lange-bertalot, h., 1986–1991: bacillariophyceae. in: ettl, h., gerloff, j., heynig, h., mollenhauer, d. (eds.), süsswasserflora von mitteleuropa, 2, 1–4. g. fischer verlag, jena. lecointe, c., coste, m., prygiel, j., 1993: »omnidia«: a software for taxonomy, calculation of diatom indices and inventories management. hydrobiologia 269/270, 509–513. acta bot. croat. 68 (2), 2009 311 periphytic diatoms in the lake ladoga basin u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:11 color profile: disabled composite 150 lpi at 45 degrees leland, h. v., 1995: distribution of phytobenthos in the yakima river basin, washington, in relation to geology, land use, and other environmental factors. canadian journal of fisheries and aquatic sciences 52, 1108–1129. leland, h. v., porter, s. d., 2000: distribution of benthic algae in the upper illinois river basin in relation to geology and land use. freshwater biology 44, 279–301. meili, m., 1992: sources, concentrations and characteristics of organic matter in softwater lakes and streams of the swedish forest region. hydrobiologia 229, 23–41. potapova, m., charles, d. f., 2002: benthic diatoms in usa rivers: distributions along spatial and environmental gradients. journal of biogeography 29, 167–187. potapova, m., charles, d. f., 2007: diatom metrics for monitoring eutrophication in the rivers of the united states. ecological indicators 7, 48–70. raspletina, g. f., 1982: changes in hydrochemical regime of tributaries due to economic activity on the watershed. in: petrova, t.n. (ed.), anthropogenic eutrophication of lake ladoga (in russian), 42–50. nauka, leningrad. raspletina, g. f., kulish, t. p., petrova, t. n., 2006: hydrochemical characteristics of the lake ladoga tributaries and the neva river. in: trifonova, i. s. (ed.), assessment of ecological condition in rivers of the lake ladoga basin using hydrochemical characteristics and structure of hydrobiocenosis (in russian), 11–35. lema, st. petersburg. rimet, f., ector, l., cauchie, h. m., hoffmann, l., 2004: regional distribution of diatom assemblages in the headwater streams of luxembourg. hydrobiologia 520, 105–117. rott, e., pipp, e., pfister, p., 2003: diatom methods developed for river quality assessment in austria and a cross-check against numerical trophic indication methods used in europe. algological studies 110, 91–115. soininen, j., 2007: environmental and spatial control of freshwater diatoms – a review. diatom research 22, 473–490. soininen, j., paavola, r., muotka, t., 2004: benthic diatom communities in boreal streams: community structure in relation to environmental and spatial gradients. ecography 27, 330–342. solovieva, n. f., 1967: hydrochemistry of the lake ladoga tributaries and the neva river. in: alekin, o. a. (ed.), hydrochemistry and hydrooptics of the lake ladoga (in russian), 5–59. nauka, leningrad. ter braak, c. j. f, smilauer, p., 1998: canoco reference manual and users guide to canoco for windows: software for community ordination (version 4). microcomputer power. ithaca, ny. van dam, h., mertens, a., sinkeldam, j., 1994: a coded checklist and ecological indicator values of freshwater diatoms from the netherlands. netherlands journal of aquatic ecology 28, 117–133. winter, j. g., duthie, h. c., 2000: epilithic diatoms as indicators of stream total n and total p concentration. journal of the north american benthological society 19, 32–49. 312 acta bot. croat. 68 (2), 2009 rusanov a. g., stanislavskaya e. v., acs e. u:\acta botanica\acta-botan 2-09\rusanov.vp 6. listopad 2009 10:22:11 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2016 za web.indd acta bot. croat. 75 (2), 2016 179 acta bot. croat. 75 (2), 179–185, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0036 issn 0365-0588 eissn 1847-8476 photochemical effi ciency, content of photosynthetic pigments and phenolic compounds in different pitcher parts of sarracenia hybrids martina tušek1, marcela curman2, marija babić3, mirta tkalec3* 1 galovec začretski 43b, hr-49223 sveti križ začretje, croatia 2 mokrice 72, hr-49243 oroslavje, croatia 3 sveučilište u zagrebu, prirodoslovno-matematički fakultet, biološki odsjek, rooseveltov trg 6, hr-10000 zagreb, croatia abstract – sarracenia is a genus of carnivorous plants characterised by leaves modifi ed into pitchers which lure, trap and digest insects. the aim of this study was to analyse the photochemical effi ciency and contents of photosynthetic pigments and phenolic compounds in different pitcher parts – operculum, wing, pitchertube upper part and pitcher-tube lower part of three morphologically different sarracenia hybrids. the photochemical effi ciency of the operculum and the pitcher-tube upper part was lower than that of the pitcher-tube lower part and wing, especially in hybrid b. in all hybrids, the wing had higher amount of chlorophyll a than other pitcher parts. in contrast, a higher amount of phenolic compounds, in particular anthocyanins, was measured in the operculum and the pitcher-tube upper part, parts which are red-coloured and participate in luring and trapping insects. although there were some differences among the hybrids, the results show that amount of phenolic compounds and photosynthetic pigments as well as photochemical effi ciency are related to the function of the pitcher part analysed. keywords: anthocyanins, carnivorous plants, carotenoids, chlorophyll, fl avonoids, photosynthesis, sarracenia * corresponding author, e-mail: mtkalec@zg.biol.pmf.hr m. tušek and m. curman equally contributed to the work introduction sarracenia is a genus comprising 11 carnivorous species. those rosette-forming perennials have modifi ed their leaves into pitchers, which perform a dual role – photosynthesis and the capture of insects (ellison and gotelli 2002, pavlovič et al. 2007). sarracenia plants attract their insect prey with extrafl oral nectaries and the pitchers’ colourful patterns (rodenas 2012). sarracenia species, as well as their numerous hybrids that occur in the wild and cultivation, greatly differ in morphology and the colouration of pitchers (rice 2006). the pitchers may be erect or decumbent, yellow-green to red and pitcher-tube upper part and hood can have red veins and/or white translucent areolation (schnell 2002). there are three types of pigments involved in the sarracenia pitcher colouration: chlorophylls, carotenoids and fl avonoids. the red of pitchers can be attributed to the presence of fl avonoids, in particular anthocyanins (sheridan and griesbach 2001, rodenas 2012). besides attracting the insects, fl avonoids have a signifi cant role in protection from high intensities of visible and uv radiation, as well as being antioxidants (close and mcarthur 2002). the main pitcher parts (fig. 1) in sarracenia species are the pitcher tube, pitcher mouth, nectar roll (peristome), lid (operculum) and wing (ala) (rice 2006). the pitcher mouth is surrounded by a peristome covered with nectar-secreting glands that attract insects into the pitcher tube. above the pitcher mouth, there is a pitcher operculum which is often coloured red and covered with glands that also secrete nectar and attract prey. besides preventing rain from fi lling the pitcher tube, the pitcher lid also serves as a landing platform for fl ying insects (d`amato 2013). the wing has nectar trails that lead insects to the pitcher mouth. the pitcher tube can be divided into upper and lower part; the upper part secretes nectar and smooth waxy material, therefore taking a role in attracting insects and preventing their escape (rice 2006). in lower part of the pitcher there are glands that secrete digestive enzymes enabling digestion of prey and absorption of minerals (glenn and bodri 2012). since the fi rst darwin’s experiments, it has been assumed that carnivorous plants compensate for the lack of minerals in their natural habitat by capturing and digesting the insect prey. recent studies have proven the benefi t of tušek m., curman m., babić m., tkalec m. 180 acta bot. croat. 75 (2), 2016 carnivory through increased rate of photosynthesis, biomass and fertility (givnish et al. 1984, farnsworth and ellison 2008, pavlovič et al. 2014). however, carnivory entails also some costs due to energy investment in attracting and trapping the prey (givnish et al. 1984, ellison and farnsworth 2005). namely, to catch and digest their prey, carnivorous plants have transformed some of their photosynthetic leaves into traps with metabolically active glands, resulting in an overall decrease of photochemical effi ciency (adamec 2010). the recent studies have shown that some sarracenia species have different metabolic activities in wing and pitcher tube, which has more glandular tissue, suggesting that there might be also a difference in photosynthetic activity between different pitcher parts (adamec 2010). in the genus darlingtonia, which is similar to the genus sarracenia, the pitcher tube contained a higher amount of chlorophyll in regard to the hood-shaped operculum, which is more reddish and has many translucent areoles. also, pitcher parts with extrafl oral nectaries have a lower amount of photosynthetic pigments (ellison and farnsworth 2005). the aim of this study was to analyse content of photosynthetic pigments and phenolic compounds, as well as photochemical effi ciency of different pitcher parts in morphologically different hybrids of the genus sarracenia. materials and methods morphological characteristics of hybrids the research was carried out on three morphologically different hybrids of the genus sarracenia l., named hybrid a, hybrid b and hybrid c (fig. 1). the plants were grown in plastic pots containing peat, and watered daily with distilled water. there was always 2–3 cm of water in the plates beneath the pots. during spring and summer, the plants were grown outdoors where they were exposed to photosynthetically active radiation and uv irradiance of maximum daily levels ca. 2000 μmol photons m−2 s−1 (quantum sensor hansatech quantitherm, uk) and 30 w m−2 (uv meter yx-35uv, taiwan, china), respectively. the average day/night temperature was 26/12 ± 3 °c. in all hybrids, operculum and the upper part of the pitcher tube were red-coloured with numerous red veins, while wing and the pitcher-tube lower part were green. there were some differences between hybrids – in pitcher size, operculum shape as well as in the colouration of operculum and pitcher-tube upper part. pitchers of hybrid a (ca. 28 cm high) had a reduced wing. the back side of the hoodshaped operculum and upper part of tube had translucent areoles with a network of red veins. the mouth of the pitcher was smaller than in the other two hybrids. hybrid b (ca. 35 cm high) had undulating operculum and red veins spread all over operculum and pitcher-tube upper part. the space between the veins was more greenish, not white as in hybrid a. the wing was larger than in hybrids a and c. hybrid c (ca. 23 cm high) had a more reddish operculum than the other two hybrids. the operculum was less undulating than in hybrid b. although hybrids used in this study are of unknown origin, according to morphological features one of the parents of hybrid a is s. psittacina while one of the parents of hybrid b and c is s. purpurea. for each hybrid, three representative and morphologically similar pitchers were used. four different pitcher parts: pitcher-tube upper part, pitcher-tube lower part, wing and operculum were analysed in the experiment (fig. 1). photochemical effi ciency of psii fluorescence of chlorophyll a in vivo was measured using fl uorometer (qubit systems inc., canada), with the method described by lichtenthaler and babani (2004) and lichtenthaler et al. (2005). before measurement, the pitcher samples were kept on well-watered fi lter paper in the dark for 20 minutes. the dark-adapted pitcher sample was placed on the fl uorometer stand, on wet fi lter paper, and exposed to modulated red light of low intensity (1 μmol photons m−2 s−1) to obtain the minimal fl uorescence (f0). the sample was then exposed to continuous actinic light of high intensity (1500 μmol photons m−2 s−1), which resulted in transient increase of the fl uorescence signal from f0 to maximum fl uorescence (fm). during fi ve minutes of light exposure, the fl uorescence signal decreased, reaching steady -state fl uorescence condition (fs). results were recorded by software logger pro 3.2. recorded data were used for calculation of maximum quantum yield (fv/fm), fv/f0 and fl uorescence decrease ratio (rfd) according to lichtenthaler et al. (2005). photosynthetic pigments for determination of photosynthetic pigments, 50 mg of samples were homogenised with mortar and pestle in 1.5 ml of cold 80% (v/v) acetone with the addition of calcium carbonate. homogenates were centrifuged for 10 minutes at 5000 g and 4 °c. the absorbance of supernatants was measured at 470 nm, 646 nm and 663 nm using a uv/vis specfig. 1. pitchers of three different sarracenia hybrids (a, b and c) and their main parts used in the study. photosynthetic efficiency, pigments and phenolic compounds in sarracenia acta bot. croat. 75 (2), 2016 181 trophotometer specord (analytik jena). the content of chlorophyll a, chlorophyll b and total carotenoids was de termined according to lichtenthaler (1987) and wellburn (1994). total phenolics, fl avonoids and anthocyanins pitcher samples (50 mg) were homogenised with mortar and pestle in 1.5 ml of cold 50% (v/v) ethanol. after incubation at 60 °c for 30 minutes, the homogenates were centrifuged at 12000 g for 10 minutes. the supernatants were used to measure total phenolics, fl avonoids and anthocyanins. for total phenolics content, a mixture of 1580 μl of distilled water, 20 μl of plant extract, 100 μl of folin–ciocalteu reagent and 300 μl of 1.88 m sodium carbonate was prepared. after incubation at 45 °c for 60 minutes, the absorbance at 765 nm was measured using a uv/vis spectrophotometer (singleton et al. 1999). the results were expressed as mg of gallic acid equivalents (gae) per g of fresh weight. for total fl avonoids content, an aliquot of the plant extract (100 μl) was mixed with 20 μl of 10% (w/v) alcl3, 500 μl of 1 m potassium acetate and 380 μl of distilled water. the mixture was incubated at 24 °c for 30 minutes and absorbance at 420 nm was measured using a uv/vis spectrophotometer (pourmorad et al. 2006). the results were expressed as mg of quercetin equivalents (qe) per g of fresh weight. for total anthocyanins content, 500 μl of plant extract was mixed with 500 μl of 50% (v/v) ethanol and 84 μl of 37% hcl. after incubation at 60 °c for 30 minutes, the absorbance was measured at 530 nm using a uv/vis spectrophotometer (paiva et al. 2003). total anthocyanin content was determined using a molar extinction coeffi cient of 34300 m–1 cm–1 and a molecular weight of 449.2 g mol–1, and expressed as mg of cyanidin-3-glucoside equivalents (c3ge) per g of fresh weight. statistical analysis results were shown as means ± standard errors. determination of phenolic compounds and photosynthetic pigments content was performed in six replicates. determination of photochemical effi ciency was performed in triplicate. for processing data microsoft excel 2007 and statistica 10 (statsoft inc., sad) were used. the results obtained for different pitcher parts within each hybrid were compared by analysis of variance (anova) and post hoc tukey’s test. differences between means were considered statistically signifi cant at p ≤ 0.05. results photochemical effi ciency of psii all pitcher parts of all hybrids had a similar maximum quantum yield of psii (fv/fm) with values ranging from 0.72 to 0.78 (tab. 1). the exceptions were much lower fv/ fm values noticed in the pitcher-tube upper part and operculum of hybrid b, the operculum showing an extremely low value (0.37). the highest value of fv/f0 was detected in the wing of all hybrids investigated, while the lowest value was found in the operculum. analysis of pitcher parts of hybrid a showed a signifi cant decrease of fv/f0 in the pitcher-tube upper part and operculum compared to the wing. amongst hybrids, hybrid b stands out with the lowest fv/f0 values measured in the operculum and the pitcher-tube upper part. in hybrid c, there was no signifi cant difference in fv/f0 between pitcher parts, though the highest value was observed in the wing (tab. 1). in hybrid a, there was no signifi cant difference in rfd values among different pitcher parts although a slight decrease was determined in the pitcher-tube lower part. in hybrid b, the highest and the lowest rfd values were measured in the wing and operculum, respectively. the operculum of hybrid b had the lowest rfd value. there was no signifi cant difference in rfd values among the different pitcher parts in hybrid c (tab. 1). content of photosynthetic pigments in all three hybrids, the chlorophyll a content was signifi cantly higher in the wing than in the other parts of pitchers. the wing of hybrid b had the highest amount of chlorophyll a. a high chlorophyll a content was also detected in the pitcher-tube lower part of hybrid b. in hybrids a and c, chlorophyll a contents in the pitcher-tube upper and lower part as well as operculum did not differ from each other (fig. 2a). in hybrids b and c, the highest amounts of chlorophyll b and total carotenoids were detected in the wing while in hybrid a the highest amount was detected in the operculum. tab 1. chlorophyll a fl uorescence parameters: maximum quantum yield of psii (fv/fm), ratio between variable and minimum fl uorescence (fv/f0) and chlorophyll fl uorescence decrease ratio (rfd), in three different sarracenia hybrids (a, b and c). four pitcher parts: pitcher-tube upper part (pu), pitcher-tube lower part (pl), wing (w) and operculum (o) were analysed. the results were expressed as the average of 3 replicates ± standard error. different letters in each column denote signifi cantly different results (p ≤ 0.05) among different pitcher parts within each hybrid, ns – not signifi cant. sarracenia hybrids pitcher part fluorescence parameters fv/fm fv/f0 rfd a pu 0.74±0.01ab 2.86±0.12a 2.83±0.09ns pl 0.77±0.01ab 3.30±0.14ab 2.15±0.22ns w 0.78±0.003b 3.62±0.06b 2.66±0.33ns o 0.72±0.02a 2.63±0.25a 2.72±0.22ns b pu 0.57±0.07b 1.41±0.34a 1.88±0.44ab pl 0.77±0.003c 3.36±0.05b 2.43±0.40ab w 0.78±0.001c 3.59±0.02b 2.94±0.09b o 0.37±0.04a 0.61±0.11a 1.18±0.19a c pu 0.75±0.01ns 3.02±0.20ns 3.21±0.07ns pl 0.73±0.02ns 2.77±0.25ns 2.83±0.33ns w 0.77±0.01ns 3.37±0.13ns 3.06±0.21ns o 0.74±0.02ns 2.81±0.24ns 3.76±0.32ns tušek m., curman m., babić m., tkalec m. 182 acta bot. croat. 75 (2), 2016 the wing of hybrid b had the highest amount of chlorophyll b and carotenoids. the lowest contents of chlorophyll b and carotenoids were observed in the pitcher-tube lower part of hybrid a, as well as in the pitcher-tube lower and upper part of hybrid c when compared to the wing and operculum (figs. 2b and c). in hybrids a and b the chlorophyll a/b ratio and the chlorophylls/carotenoids ratio were higher in the wing and pitcher-tube lower part than in the other two pitcher parts. in hybrid c the highest values of both ratios were observed in the wing, while the operculum had the lowest chlorophyll a/b ratio (tab. 2). content of phenolic compounds among different pitcher parts, the highest amount of total phenolics was detected in the operculum, especially in hybrids a and c. in hybrids a and b, the pitcher-tube lower part and the wing had the lowest amount of total phenolics, while in hybrid c the lowest amount was measured in the pitcher-tube upper part (fig. 3a). in all analysed hybrids, the operculum showed the highest and the pitcher-tube lower part the lowest content of fl avonoids. in hybrid c, the wing also contained a higher amount of fl avonoids than the pitcher-tube upper and lower part (fig. 3b). the operculum was the pitcher part with the highest amount of anthocyanins, especially in hybrid a. in hybrids b and c, the pitcher-tube upper part also contained a high amount of anthocyanins. in all hybrids, the pitcher-tube lower part and the wing had lower anthocyanin content than the other pitcher parts (fig. 3c). discussion the optimal effi ciency of psii (fv/fm) is an important indicator of photochemical effi ciency in plants (maxwell and johnson 2000). in all the hybrids investigated, the measured fv/fm values (tab. 1) were in accordance with theoretical values (0.74 to 0.85) for non-carnivorous plants (maxwell and johnson 2000, lichtenthaler et al. 2005). however, fv/f0, a more sensitive fl uorescence parameter (lichtenthaler et al. 2005), showed values below the critical value of 3.8, indicating a decrease of photochemical effi ciency (chatzistathis et al. 2011) in all pitchers parts (tab. 1). a lower photochemical effi ciency has been often found in carnivorous plants (bruzzese et al. 2010) and it has been correlated with the costs of carnivory (adamec 2010) as well as with low foliar nitrogen content and slow growth of carnivorous plants (ellison and adamec 2011, pavlovič and saganová 2015). the lowest values of both parameters, fv/fm and fv/f0 were found mostly in the operculum and upper part of the pitcher tube, which are predominantly red-coloured areas with numerous nectar-secreting glands. moreover, in these pitcher parts a lower amount of chlorophyll a and higher tab 2. chlorophyll a/ b ratio (chl a/b) and chlorophylls/carotenoids ratio (chl/car) in three different sarracenia hybrids (a, b and c). four pitcher parts: pitcher-tube upper part (pu), pitchertube lower part (pl), wing (w) and operculum (o) were analysed. the results were expressed as the average of 6 replicates ± standard error. different letters in each column denote signifi cantly different results (p ≤ 0.05) among different pitcher parts within each hybrid. sarracenia hybrids pitcher part chl a/b chl/car a pu 2.12±0.38ab 2.11±0.12b pl 2.75±0.06b 2.47±0.06bc w 2.98±0.08b 2.54±0.04c o 1.75±0.28a 1.74±0.12a b pu 1.91±0.17a 1.83±0.12a pl 2.44±0.06b 2.33±0.12b w 2.37±0.1b 2.9±0.13c o 1.86±0.08a 1.49±0.11a c pu 2.35±0.11b 2.11±0.12ab pl 2.28±0.16ab 1.82±0.06a w 2.58±0.15b 2.45±0.09b o 1.8±0.17a 1.89±0.1a a b c a a b a ab b c a a a b a 0 0.1 0.2 0.3 0.4 0.5 0.6 pu pl w o pu pl w o pu pl w o hybrid a hybrid b hybrid c c on te nt o f c hl or op hy ll a (m g g f w ) ab a ab b a a b a a a b b 0 0.05 0.1 0.15 0.2 0.25 0.3 pu pl w o pu pl w o pu pl w o hybrid a hybrid b hybrid c c on te nt o f c hl or op hy ll b (m g g f w ) ab a bc c a a b a a a b b 0 0.05 0.1 0.15 0.2 0.25 0.3 pu pl w o pu pl w o pu pl w o hybrid a hybrid b hybrid c c on te nt o f t ot al c ar ot en oi ds (m g g f w ) fig. 2. content of chlorophyll a (a), chlorophyll b (b) and total carotenoids (c) in three different sarracenia hybrids. four pitcher parts: pitcher-tube upper part (pu) and lower part (pl), wing (w) and operculum (o) were analyzed. the results were expressed as the average of 6 replicates ± standard error. different letters above the bars denote signifi cantly different results (p ≤ 0.05) among different pitcher parts within each hybrid. fw – fresh weight. photosynthetic efficiency, pigments and phenolic compounds in sarracenia acta bot. croat. 75 (2), 2016 183 amount of phenolic compounds were found (figs. 2 and 3), which is in accordance with higher anthocyanin and lower chlorophyll content found in the traps of nepenthes (pavlovič and saganová 2015). on the other hand, the highest photochemical effi ciency and chlorophyll content were recorded in the green-coloured wing of sarracenia, a pitcher part that has a role in guiding insects to the pitcher mouth (d`amato 2013). however, the lower part of the pitcher tube, although green-coloured, had lower photochemical effi ciency, probably due to the presence of digestive glands needed for the digestion and absorption of minerals (d`amato 2013). the lower fv/fm values and photosynthesis rate in the trapping organs of carnivorous plants other than sarracenia have already been described (pav lovič et al. 2009). we also measured the fl uorescence decrease ratio (rfd), a parameter directly related to rate of photosynthesis (lichtenthaler and babani 2004). the measured rfd values (tab. 1) were mostly between those of sun-exposed leaves (3 to 5) and those of shade-exposed leaves (1 to 2.5). higher rfd values in sun-exposed leaves refl ect their higher photosynthetic capacity and co2 fi xation rate (lichtenthaler et al. 2005). carnivorous plants grow mostly in sunny habitats (zamora et al. 1998) suggesting that rfd values should be in the range for sun-exposed plants. however, as already mentioned, carnivorous plants mostly have a reduced rate of photosynthesis due to low foliar nitrogen content and nitrogen incorporation into other than photosynthesis-related molecules (chlorophylls, proteins), such as those involved in adjustments for trapping and digesting insects (ellison and adamec 2011, pavlovič and saganová 2015). unexpectedly, in hybrid a and c high rfd values were observed not only for the green-coloured wings but also for red-coloured pitcher-tube upper part and operculum. the wing of all hybrids investigated had signifi cantly higher content of chlorophyll a, than the operculum and pitcher tube (fig. 2a). in the genus sarracenia, red-coloured veins with nectar-producing glands (newell and nastase 1998) are mostly distributed in the area of operculum, pitcher upper part and peristome (płachno 2007). replacement of cells that contain chlorophyll with glands that have an important role in the attraction and digestion of insects is considered to be one of the costs of carnivory (hájek and adamec 2010). in the genus darlingtonia, it has been found that pitcher parts that contain more extrafl oral nectaries have a lower amount of photosynthetic pigments (ellison and farnsworth 2005). unlike chlorophyll a, high values of chlorophyll b and carotenoids were found also in the operculum, especially in hybrids a and c (fig. 2b) which coincided with the high phenolic content (especially anthocyanins, figs. 3a and c). as anthocyanins could absorb some part of photosynthetically active radiation (gould et al. 2010), we hypothesize that increased content of chlorophyll b and carotenoids could be involved in spectral broadening of light absorbed by the operculum. the relatively high photosynthetic rates observed in hybrids a and c support the idea. additionally, carotenoids could also have a role in protection from high light stress (ramel et al. 2012) as the operculum is the pitcher part most exposed to the sunlight. the lower chlorophyll a/b ratio found in the operculum may be considered an enlargement of the antenna system of photosystem ii while a lower chlorophylls/carotenoids ratio is a typical characteristic of sun-exposed leaves (lichtenthaler and buschmann 2001). similar results were reported by tkalec et al. (2015) who observed a higher content of anthocyanins as well as a lower chlorophylls/carotenoids ratio in sun-exposed drosera rotundifolia and a lower chlorophyll a/b ratio in plants growing in low-light conditions. the highest content of total phenolic compounds and fl avonoids found in the operculum of sarracenia hybrids is probably related to the presence of red-coloured phenolic compounds, like anthocyanins (sheridan and griesbach 2001). the operculum is variegated with a lot of red veins that have an important role in luring insects (newell and nastase 1998). furthermore, phenolic compounds with their antioxidant potential can protect the operculum, the pitcher part most exposed to sunlight, from light-induced formation of free radicals (close and mcarthur 2002). interestingly, in all hybrids the lower part of pitcher tube had a b c a a a b bc a a c b a a c 0 0.3 0.6 0.9 1.2 1.5 pu pl w o pu pl w o pu pl w o hybrid a hybrid b hybrid c c on te nt o f a nt ho cy an in s ( m g e -c 3g g f w ) a a a b b a b c a a b b 0 3 6 9 12 pu pl w o pu pl w o pu pl w o hybrid a hybrid b hybrid c c on te nt o f f la vo no id s (m g e -k g f w ) b a ab c ab a a b a ab ab b 0 5 10 15 20 25 pu pl w o pu pl w o pu pl w o hybrid a hybrid b hybrid c c on te nt o f t ot al p he no lic s (m g e -g a g f w ) fig. 3. content of total phenolics (a), fl avonoids (b) and anthocyanins (c) in three different sarracenia hybrids. four pitcher parts: pitcher-tube upper part (pu) and lower part (pl), wing (w) and operculum (o) were analyzed. the results were expressed as the average of 6 replicates ± standard error. different letters above the bars denote signifi cantly different results (p ≤ 0.05) among different pitcher parts within each hybrid. fw – fresh weight. tušek m., curman m., babić m., tkalec m. 184 acta bot. croat. 75 (2), 2016 the lowest content of fl avonoids, probably because its function is not luring the prey, but digestion of pray trapped in the tube (glenn and bodri 2012). by contrast, the content of fl avonoids was relatively high in the wing, the green-coloured pitcher part with the highest content of chlorophyll a and fv/f0. it is possible that fl avonoids protect photosynthetically active tissue from oxidative stress, due to their antioxidant potential and free radical quenching ability (banasiuk et al. 2012). as already mentioned, anthocyanins are known to be responsible for the red colouration of sarracenia pitchers, so the highest content could be expected to be found in the operculum (fig. 3c), the most red-coloured part of the pitcher. besides the operculum, the upper pitcher part had also high content of anthocyanins, pointing to its role in the attraction of insects (rice 2006). it has been found that some species of the genera drosera and dionaea exposed to mineral defi ciency conditions, especially nitrogen, can increase the intensity of red colour to attract more prey (ischiishi et al. 1999, gao et al. 2015). the lowest anthocyanin content was observed in the operculum of hybrid b which had a more greenish tissue between the veins. interestingly, the operculum of hybrid a which had translucent aureoles showed the highest content of anthocyanins (figs. 1 and 3c). it is possible that leucoanthocyanins, which are not coloured, contribute to the high content of anthocyanins, but it is yet to be determined. in conclusion, our results show that variances in photochemical effi ciency, content of photosynthetic pigments and phenolic compounds correlate with the function of pitcher part analysed. in all sarracenia hybrids investigated, the green-coloured wing had the highest content of chlorophyll a and photochemical effi ciency which is connected with its role in photosynthesis. the red-coloured operculum and upper pitcher part, whose role is luring and catching prey, contained more phenolic compounds, especially anthocyanins, which help to attract insects. references adamec, l., 2010: dark respiration of leaves and traps of 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zamora, r., gómez, j. m., hódar, j. a., 1998: fitness responses of a carnivorous plant in contrasting ecological scenarios. ecology 79, 1630–1644. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 363 acta bot. croat. 74 (2), 363–376, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0021 similar small-scale variation of diatom assemblages on different substrates in a mesotrophic stream ma ria kahlert*, ivana savatijević rašić swedish university of agricultural sciences, department of aquatic sciences and assessment, box 7050, 750 07 uppsala, sweden abstract – the aim of the present study was to analyze if small-scale spatial variation of benthic diatom assemblages has consequences for biomonitoring. benthic diatom samples were collected at one sampling site in a mesotrophic stream in middle-sweden from stone and plant (macrophytes and mosses) substrate. our results showed that spatial variation of both the diatom species composition and the calculated bioindices were similar on both small (distance of centimeter) and medium (distance of decimeters) scales. spatial variation was also similar on both studied substrates. this implies that it does not matter if a small or a larger area is sampled for biomonitoring as long as no major environmental factors impact certain sites systematically. diatom assemblages and indices were signifi cantly different between substrates. spatial variation did not contribute much to this variation, and variation on a slide was unimportant. these results confi rm earlier fi ndings that smallscale spatial variation is not a problem when using diatoms to detect anthropogenic impacts to a stream or lake. keywords: bioindices, diatoms, diversity, epilithon, epiphyton, freshwater, monitoring, sampling introduction diatoms are frequently used for monitoring water quality status in streams and also recently in lakes (sis 2014). many indices are in use to refl ect eutrophication or pollution (birk et al. 2012). usually diatoms refl ect water chemistry well (rimet 2012). the european standard for diatom sampling in freshwater for biomonitoring (sis 2014) allows sampling from different substrates. still, diatom assemblages can differ between substrates, e.g. stones and macrophytes. it is not well studied how these differences impact diatom indices and recommendations about which substrate to sample are contradictory. the european sampling standard (sis 2014) recommends sampling from stones in opposite to besse-lototskaya et al. (2006) recommending macrophytes. kröpfl et al. (2006) even * corresponding author, e-mail: maria.kahlert@slu.se kahlert m., savatijević rašić i. 364 acta bot. croat. 74 (2), 2015 found differences of diatom indices between different artifi cial substrates. winter and duthie (2000) showed that differences between substrates have not been consistent over time. they conclude that more studies are needed to evaluate detailed information about the effect of substrate on freshwater diatom assemblages from different environments. it is important to keep a comparable sampling strategy when studying the effect of substrate on the attached algal assemblage to avoid differences that may occur due to it being a different sampling area. differences in index values might otherwise been related to the fact that a diatom assemblage sampled from a restricted area represents local factors whereas a sample pooled from many stones across the stream rather represent a streams’ water quality. a diatom sample taken according to eu standard is pooled from at least fi ve subsamples. macrophytes often grow in relatively restricted places, whereas stones mostly can be sampled across the whole river or lake section. a pooled sample taken from macrophytes would therefore represent the local environment around a spot, whereas a pooled sample taken from stones would refl ect the environmental conditions of the watercourse. in other words, observed differences could be due to the fact that the pooled sample from macrophytes has a low spatial variation and might deviate from a whole-stream average value just because it is taken from a relatively restricted area. the small-scale variation of benthic diatom assemblages on different substrates is not very well studied in freshwater and it is not known what impact it has on the bioindices derived by a different sampling strategy when a substrate is restricted to a small area. many studies have focused on spatial variation at larger scales (soininen 2007). a general view has been established that history is important but local factors are steering diatom communities (soininen 2007, vyverman et al. 2007, mann and vanormelingen 2013). confi rming this, most studies on space have proved that diatom indices are suffi ciently robust to refl ect an anthropogenic impact despite spatial or temporal variation of the species composition (rothfritz et al. 1997, kelly 2002, lavoie et al. 2005, king et al. 2006). the studied »small« spatial scale is usually represented by a distance of meters or rarely decimeters (rothfritz et al. 1997, prygiel et al. 2002, lavoie et al. 2005, o’driscoll et al. 2014, svoboda et al. 2014), the scale usually sampled in a standard investigation (sis 2014). variation at smaller spatial scales is rarely studied. a rare exception is machova-cerna and neustupa (2009) who investigated small-scale variation in a peat-bog in comparison to larger-scale variation and found signifi cant differences in species composition even at the smallest sampled scale. more information is available about small-scale variation from marine and brackish water studies on soft bottom algal assemblages (e.g. saburova et al. 1995, coleman 2002). these studies found that spatial variation increases with scale, but not linearly. variation at the laboratory scale has been found to usually be of minor importance compared with variation at spatial and other scales, both regarding slide replicates (e.g. lavoie et al. 2005, besse-lototskaya et al. 2006) and replicate counts on one slide (prygiel et al. 2002). still, it is necessary to have an idea about the laboratory variation to assess the variability of e.g. spatial scales. the present study analyzes and compares the size of small-scale variation of diatom assemblages in a mesotrophic stream on different substrates. we calculate if spatial variation is smaller on a restricted area than across an entire stream section and assess if eventual differences are refl ected in diatom bioindices with consequences for monitoring. our hypothesis are 1) small-scale variation of diatom assemblage is smaller than medium-scale variation, 2) spatial variation of diatom assemblages is similar on stones and plants (macrophytes and mosses) if sampled at the same scale, and 3) diatom indices pooled small-scale variation of diatoms on different substrates acta bot. croat. 74 (2), 2015 365 according to the eu standard can be different between stones and plants if plants are sampled on a restricted spot and stones at a larger spatial scale. materials and methods sampling benthic diatom samples were collected at one sampling site in broströmmen, a mesotrophic stream in middle-sweden (598280,7+0187355,3/ iso 6709, average water chemistry: 44 μg total phosphorus l–1, ph 7.1), august 2010. sampling followed eu standard (sis 2003). samples were taken with a syringe brush sampler (opening diameter of 1 cm) at random in a nested design (figs. 1a–b) to cover the variance at small (cm) and medium fig. 1. a) nested sampling design used in studied stream. five subsamples were taken with a distance of some centimeters (cm-scale). this cm-scale sampling was performed in two different randomly chosen areas. additionally, fi ve single samples were taken from one substrate with a distance of 20–100 cm (dm-scale); b) nested sampling design for both substrates, schematic. larger circles with solid line represent sub-area for epilithon samples, while those with dashed line represent sub-area for epiphyton samples. the fi ve subsamples in cm-scale are shown as shaded grey small circles, the two sub-areas are marked with cm 1 and 2 for each substrate. the fi ve subsamples in dm-scale are shown as fi lled small circles for each substrate. note that samples were taken at random, so this schematic picture is not refl ecting a map. a) b) kahlert m., savatijević rašić i. 366 acta bot. croat. 74 (2), 2015 scale (dm). two different diatom assemblages were sampled in the same area using the same design: epilithon from cobble stones and epiphyton from a mixture of submerged macrophytes and mosses. five subsamples were taken with a distance of some centimeters (cm-scale). this cm-scale sampling was performed in two different randomly chosen areas. additionally, fi ve single samples were taken from one substrate with a distance of 20–100 cm (dm-scale). all samples were analyzed following standard procedures (sis 2005), identifi cation followed swedish standards (dyntaxa 2013). to get an assessment of the variation at laboratory scale, two stone samples were counted in four laboratory replicates. to meet the demands of the eu standard for diatom sampling, we also pooled a subsample of the replicates taken at the two restricted areas, and of the replicates taken at medium scale. in that way it was possible to compare the effect of sampling on the pooled samples directly. diatom species composition for a comparison of diatom species composition non-metric multidimensional scaling (nmds) (kruskal 1964) was used to display dissimilarities (metric: relative sorensen) between samples and between substrates. unidentifi ed taxa, taxa identifi ed to genus, and species which were only observed once were removed from the analysis. diatom relative abundance values were arcsine square root transformed prior to the analyses. non-parametric multivariate analysis of variance (npmanova) (anderson 2001) was used pooling all samples to test if species composition differed in general signifi cantly between substrates. simper analysis (similarity percentage, (clarke 1993), bray-curtis metric as default for dissimilarity) was used to identify the species which were typical for either substrate. nmds and npmanova were performed with the software pcord 6.15 (mccune and mefford 2011), simper with past 2.17 (hammer et al. 2001). additionally, we calculated the number of diatom taxa and diversity (shannon 1948) for each level as general metrics for species composition. diatom indices to analyze the impact of community differences on diatom indices, we calculated the indices used for swedish monitoring, i.e. indice de polluo-sensibilité spécifi que (ips) (cemagref 1982), trophic diatom index (tdi) and proportion of pollution tolerant (% pt) valves (kelly and whitton 1995, kelly 1998). to test if the indices varied depending on substrate, we calculated t-tests with the fi ve dm-scale samples as replicates. spatial scale variation the impact of the fi xed factor substrate and the random factors dmand cmscale on the variation of the diatom indices was assessed by calculating coeffi cients of variance (cv%) for each sampled scale (cm-scale n = 5, dm-scale n = 5) for the diatom indices and for the number of taxa and the diversity of both substrates. the variation of the spatial scales was compared with the variation between substrates, for which the pooled standard samples for each substrate were averaged (n = 2). for stones, also the cv% of the laboratory scale (n = 4) was assessed. boxplots were created to visualize the results. small-scale variation of diatoms on different substrates acta bot. croat. 74 (2), 2015 367 results overall, we found that the variation was similar at the smallest sampled scale of centimeters and at the medium-scale of decimeters in the studied stream. the variation was about the same for both studied substrates and contributed only negligible to the large differences between substrates. diatom species composition 161 diatom taxa were found in broströmmen, 105 of them were used for the analyses of differences in species composition. the fi ve most common taxa were cyclotella meneghiniana kützing (average relative abundance 16%), amphora pediculus (kützing) grunow (12%), cocconeis placentula incl. varieties ehrenberg (9%), planothidium frequentissimum lange-bertalot (7%) and navicula capitatoradiata germain (6%). small-scale variation of the diatom assemblage was on the plant substrate smaller than in dm-scale. however, for stones, one of the small areas had a lower and the other one a higher variation than the larger area, which is illustrated by the nmds analyses (figs. 2–3). after the removal of singletons, 70 taxa were included in the nmds analysis for stones and 68 taxa for plants. three major gradients captured most of the variance in the epilithon communities, with a fi nal stress of 9.8. for epiphyton, two major gradients captured most of the variance, giving a fi nal stress of 10. so, small-scale variation of the diatom assemblage was not always smaller than medium-scale variation, thereby falsifying our fi rst hypothesis. spatial variation of assemblage species composition was about the same for stones and plants, thereby verifying our second hypothesis. this similar size of variation is illustrated by the nmdsanalysis (fig. 4) where two major gradients captured most of the variance when analyzing both communities together, giving a fi nal stress of 10.1. the diatom fl ora was signifi cantly different between stones and plants (bray-curtis dissimilarity 57.7%, npmanova, p < fig. 2. differences in diatom assemblages scraped from stones with replicates at different spatial scales. δ, ○ – replicates in cm-scale (2 different areas),▼ – replicates on dm-scale. nmds – non-metric multidimensional scaling (kruskal 1964) on 70 diatom taxa, fi nal stress for 3-dimensional solution = 9.8. kahlert m., savatijević rašić i. 368 acta bot. croat. 74 (2), 2015 fig. 3. differences in diatom assemblages scraped from plants (submerged macrophytes and mosses) with replicates at different spatial scales. δ, ○ – replicates in cm-scale (2 different areas),▼ – replicates on dm-scale. nmds – non-metric multidimensional scaling (kruskal 1964) on 68 diatom taxa, fi nal stress for 2-dimensional solution = 10. fig. 4. differences in diatom assemblages scraped from stones (left) and plants (right, submerged macrophytes and mosses) with replicates at different spatial scales. stones: δ, ○ – replicates in cm-scale (2 different areas),▼ – replicates on dm-scale. plants: ◊,  – cm-scale, ■ – dmscale. nmds – non-metric multidimensional scaling (kruskal 1964) on 100 diatom taxa, fi nal stress for 2-dimensional solution = 10.1. small-scale variation of diatoms on different substrates acta bot. croat. 74 (2), 2015 369 0.001). differences in the relative abundance of only 16 taxa explained 50% of the difference in taxon assemblages between stones and plants (simper similarity percentage analysis, tab. 1). the number of taxa and the diversity was higher on stones than on plants (tab. 2). consequences for diatom indices the main index ips was signifi cant higher on stones than on plants (t-test, p < 0.01, tab. 2, fig. 5). the results of the comparison of the samples pooled according to the eu standard confi rmed that ips still was very different between substrates, independently of sampling scale (tab. 2, fig. 5). the same results were found for the supporting index tdi (t-test, p < 0.01, tab. 2, fig. 6). following this, our third hypothesis was rejected, at least in the investigated stream. the variation of ips and tdi at the spatial scales refl ected the variation of the diatom assemblages, with somewhat lower variation at cm-scale than dmscale for the plant substrate, and about similar variation at both scales for the stone subtab. 1. taxa contributing most to the signifi cant difference of diatom assemblages on stones and plants, respectively, in broströmmen (simper analysis, npmanova, p < 0.001. braycurtis dissimilarity 57.7). for each taxon, the sensitivity values for the diatom indices ips -indice de polluo-sensibilité spécifi que (cemagref 1982) and tdi – trophic diatom index (kelly and whitton 1995, kelly 1998) are given. stone substrate plant substrate code taxon ipss tdis code taxon ipss tdis aped amphora pediculus (kützing) grunow 4 5 cmen cyclotella meneghiniana kützing 2 0 plfr planothidium frequentissimum lange-bertalot 3.4 5 ncpr navicula capitatoradiata germain 3 3 kala karayevia laterostrata (hustedt) bukhtiyarova 4.5 4 cpla cocconeis placentula incl. varieties ehrenberg 4 3 kasu karayevia suchlandtii (hustedt) bukhtiyarova 4.5 4 scon staurosira construens var. construens ehrenberg 4 4 cbac caloneis bacillum (grunow) cleve 4 3 ssve staurosira venter (ehrenberg) cleve & moeller 4 4 prst planothidium rostratum lange-bertalot 4.4 5 nzsu nitzschia supralitorea lange-bertalot 1.5 4 ptla planothidium lanceolatum lange-bertalot 4.6 5 nsem navicula seminulum grunow 1.5 5 eomi eolimna minima (grunow) lange-bertalot 2.2 5 ptco platessa conspicua lange-bertalot 4 5 kahlert m., savatijević rašić i. 370 acta bot. croat. 74 (2), 2015 strate (tab. 2, figs. 5–6). the laboratory replicates of ips and tdi varied less than the replicates taken in the fi eld (tab. 2, figs. 5–6). % pt did not differ between substrates (t-test, p > 0.05, tab. 2, fig. 7). discussion we found that already at very small spatial scales, with a distance of a few centimeters, diatom species composition can vary substantially, similar to the differences at medium distance of several decimeters. as expected, laboratory variation contributes only a minor tab. 2. means and coeffi cients of variance (cv%) for ips20 – indice de polluo-sensibilité spécifi que (cemagref 1982), tdi – trophic diatom index, % pt – proportion of pollution tolerant valves (kelly and whitton 1995, kelly 1998) and number of taxa (nr taxa), and diversity (shannon) at different scales and substrates in broströmmen. substrate scale n ips20 cv% tdi100 cv% % pt cv% nr taxa cv% diversity both stream reach 2 11.7 18.1 70.9 17.1 6.9 20.5 43.0 52.6 4.0 stone dm 5 13.8 6.9 79.6 4.0 9.6 50.0 39.0 11.2 4.6 stone cm 1 5 13.4 3.1 86.9 4.0 14.0 27.1 37.4 13.4 4.0 stone cm 2 5 13.8 6.5 83.5 2.8 10.7 35.1 39.2 14.8 4.3 stone laboratory 1 4 14.3 1.8 83.3 1.2 5.5 66.8 36.5 3.5 4.0 stone laboratory 2 4 13.6 2.7 83.5 2.7 10.6 20.4 38.5 4.5 4.1 plant dm 5 10.8 10.8 62.7 13.7 12.5 35.8 31.4 10.0 3.7 plant cm 1 5 10.9 2.8 56.1 4.2 8.8 25.1 37.2 11.0 3.7 plant cm 2 5 11.3 5.6 62.5 5.3 8.8 20.8 37.6 7.7 3.8 lab 1 lab 2 cm 1 cm 2 dm cm 1 cm 2 dm 8 9 10 11 12 13 14 15 16 ip s 20 plantsstones x x fig. 5. variation of diatom index ips – indice de polluo-sensibilité spécifi que (cemagref 1982) at different scales and substrates (stones and plants) in broströmmen. median, interquartile range and total range are shown in the boxplots for scales at laboratory (n = 4), samples take with cm distance (n = 5) and dm distance (n = 5). × represents the pooled sample taken according to eu standard procedures (sis 2014). small-scale variation of diatoms on different substrates acta bot. croat. 74 (2), 2015 371 part to that variation. the spatial variation was similar on stones and on plants. however, the spatial variation was not contributing signifi cantly to the much larger variation of the diatom assemblages between stones and plants. this substrate variation in the studied mesotrophic stream was the same even if stones from a large area were compared with plants from a small area, a sampling strategy that might be applied when plants only are available from a restricted area. factors impacting at spatial scale earlier studies on the diatom fl ora of intertidal sandfl ats indicate that patchiness at small scales is random up to a scale of several decimeters (saburova et al. 1995). similar results were found for algae on hard substrate in marine and freshwater environments (rindi and cinelli 2000, soininen 2003). these large scale patterns have been attributed to environlab 1 lab 2 cm 1 cm 2 dm cm 1 cm 2 dm 50 55 60 65 70 75 80 85 90 95 100 t d i1 00 plantsstones x x lab 1 lab 2 cm 1 cm 2 dm cm 1 cm 2 dm 0 2 4 6 8 10 12 14 16 18 20 22 % p t stones plants x x fig. 6. variation of diatom index tdi – trophic diatom index (kelly and whitton 1995, kelly 1998) at different scales and substrates (stones and plants) in broströmmen. see fig. 5 for more information. fig. 7. variation of diatom index % pt – proportion of pollution tolerant valves (kelly and whitton 1995, kelly 1998) at different scales and substrates (stones and plants) in broströmmen. see fig. 5 for more information. kahlert m., savatijević rašić i. 372 acta bot. croat. 74 (2), 2015 mental factors such as water chemistry and tidal effects in the example of sandfl ats. the patterns at smaller scales have been attributed to biotic species interactions and as saburova et al. (1995) point it out »a complex of abiotic conditions in the sediments«. other studies highlight the impact of dispersal. still, both machova-cerna and neustupa (2009) and our study indicate that variation at the smallest scale with a distance of centimeters can be substantial. machova-cerna and neustupa (2009) discussed dispersal limitation and niche adaptation as explanations. according to our study, we can assume that similar factors impact the spatial distribution of diatom species on the two substrates because spatial variation is similar on both studied scales when the substrate is sampled from the same spot in the stream. certainly spatial variation depends on the sampled environment. we suggest that in a medium-order stream that it is mainly velocity that is shaping the diatom community at the decimeter scale, as shown by passy (2001). at smaller scales, grazing could have an important impact on algal communities (gothe et al. 2013, o’driscoll et al. 2014), probably together with historical events following dispersal (müller-haeckel 1976) and also the named »complex interaction of abiotic conditions« (e.g. kemp and dodds 2001). the small scale variation can be of similar size as the medium scale variation, which shows that factors acting on very small scales can cause similar large variations in diatom diversity as shown for velocity (passy 2001). however, to analyze the importance of these factors, it is necessary to include them directly in an analysis along with the temporal aspect as earlier events are shaping the community of later stages. bioindices regarding the impact of diatom fl ora variation on the bioindices used for monitoring, we found the difference on substrates to be most important. the diatom index ips on stones indicated a moderate ecological status class with a tendency to good ecological status according to the swedish classifi cation (naturvårdsverket 2007). on plants, ips also indicated a moderate ecological status class which shows the robustness of the method to assess ecological status of a water body. however, ips had a clear tendency to poor ecological status class on plants. contrary to this result, the tdi was higher on stones than on plants, indicating a higher nutrient level on stones (kelly and whitton 1995). diatom taxa on stones had on average higher sensitivity values for both ips and tdi than the taxa on plants, explaining the somewhat unexpected differences of ips and tdi. ips was constructed to refl ect a general pollution, integrating both aspects of eutrophication and organic pollution (cemagref 1982), whereas tdi was constructed to solely refl ect nutrient conditions (kelly and whitton 1995). a streams’ diatom fl ora is expected to refl ect mainly a streams’ integrated water chemistry, the factor diatom indices were developed and are meant to use for. however, the species composition certainly always refl ects other aspects of the environment than just water chemistry. in our study, the taxa on plants being mainly responsible for the difference to the stone diatom fl ora can be divided into three subgroups, probably representing three types of diatom groups. first, cyclotella meneghiniana kützing, staurosira construens var. construens ehrenberg and s. venter (ehrenberg) cleve & moeller are tychoplanktonic taxa. they are probably entangled in the plant substrate and would be swept away on smooth stones. second, cocconeis placentula ehrenberg is known as epiphyte, adapted to a life on plant, moss or macroalgal substrate. third, nitzschia supralitorea lange-bertalot belongs to the genus nitzschia where many, maybe all, species have the ability to grow heterotrophic on small-scale variation of diatoms on different substrates acta bot. croat. 74 (2), 2015 373 organic substrates in complete darkness (tuchman 1996). probably not only tychoplanktonic diatoms, but also detritus gets entangled within the plant substrate, offering the opportunity for the diatoms to use included organic substances. the combination of the factors restricted current, availability of organic substances and adaptation to plant substrate then leads to a diatom assemblage which has a on average low ips value. at the same time, the assemblage on plants has a relatively low average tdi value. a possible explanation could be that the above mentioned factors are more important on the plant substrate than the nutrient level from the overlying water. c. meneghiniana, comprising on average 30% of the plant assemblage, has no appointed tdi value as the tdi excludes planktonic taxa. similar results for the difference in ips and tdi have been shown in a large study covering streams across europe (besse-lototskaya et al. 2006), which indicates that the discussed mechanisms could be valid for other streams than only our study site. these differences between substrates should be kept in mind when sampling from plant substrate. better information about the effect of substrate type on diatom assemblages is yet missing, and the existing studies are contradicting. differences are obviously not consistent but varying at least with time, with diatom assemblages getting more dissimilar between substrates during succession (winter and duthie 2000, machova-cerna and neustupa 2009). so depending on time, diatom samples could be quite similar even on different substrates. there is not a simple typical plant or stone diatom fl ora, different taxa have been reported to dominate different substrates in different studies (reavie and smol 1997, winter and duthie 2000). still, besse-lototskaya et al. (2006) large european study showed that differences in diatom indices was larger between streams with a different impact than between substrates sampled within each stream, so indices are robust enough to refl ect an anthropogenic impact, a result confi rmed by other studies showing that between stream variation is larger than within stream variation on different substrates (juttner et al. 1996, rothfritz et al. 1997). we want to point out that our study only refl ects the conditions of a single stream and a single occasion. still, not much has been done in freshwater to unravel the impact of very small scales on diatom species composition, and other conditions might give different results especially with respect to diatom bioindices. in conclusion, our results show that spatial variation was similar on both small and medium scales, and on both studied substrates, in the studied stream. this implies that it does not matter if a small or a larger area is sampled for biomonitoring as long as no major environmental factor impacts certain sites systematically. this high patchiness at both studied scales implies that by chance, one can get a quite homogenous pooled sample by taking fi ve subsamples, but also high variation in other cases. only frequent sampling can minimize the risk of getting outliers into the analysis. in comparison to the studied environmental factor substrate, spatial variation was relatively small and variation on a slide was unimportant, results that confi rm earlier fi ndings that small-scale spatial variation is not a problem when using diatoms to detect deteriorate impacts to a stream or lake (lavoie et al. 2005, machova-cerna and neustupa 2009). acknowledgements this project was funded by the swedish agency for marine and water management (swam) and the sepa research department (waterbody assessment tools for ecological reference conditions and status in sweden (waters)). we also thank claire irving and two anonymous referees for their 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(eds.), algal ecology: freshwater benthic ecosystems, 299–320. academic press, san diego. winter, j., duthie, h., 2000: stream epilithic, epipelic and epiphytic diatoms: habitat fi delity and use in biomonitoring. aquatic ecology 34, 345–353. vyverman, w., verleyen, e., sabbe, k., vanhou tte, k., sterken, m., hodgson, d. a., mann, d. g., juggins, s., vijver, b. v. d., jones, v., flower, r., roberts, d., chepurnov, v. a., kilroy, c., vanormelingen, p., wever, a. d., 2007: historical processes constrain patterns in global diatom diversity. ecology 88, 1924–1931. acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 109 acta bot. croat. 74 (1), 109–121, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 heat tolerance indicators in pakistani wheat (triticum aestivum l.) genotypes sami u. khan1, jalal u. din2, abdul qayyum1*, noor e. jan2, matthew a. jenks3 1 department of agriculture sciences, university of haripur, 22620, pakistan. 2 plant physiology program, crop sciences institute, national agricultural research centre, islamabad, 45500, pakistan. 3 division of plant and soil sciences, west virginia university, morgantown, wv 26506-6108, usa. abstract – the effect of high temperature stress on six wheat cultivars exposed to 35–40 °c for 3 h each day for fi ve consecutive days was examined. high temperature signifi cantly affected total proline, soluble protein content, membrane stability index (msi), yield, and various yield components, and had a direct effect on growth and other physiological attributes of wheat at anthesis and the milky seed stages. the wheat cultivar as2002 achieved better osmotic adjustment by accumulating more leaf proline. higher msi was also observed in as-2002, as well as inqalab-91. the anthesis growth stage was found to be more sensitive to heat stress than seed development at the milky stage. overall heat stress reduced yield 75% at anthesis and 40% at the milky stage. as-2002 performed better on the basis of yield and yield components. seed weight per spike was highest in as2002, and lowest in sh-2002. the cumulative response of as-2002 was better on the basis of physiological and yield attributes. in addition to yield, plant breeders should also include proline and msi as selection parameter in the breeding program for development of heat tolerant wheat cultivars. most of the evaluated wheat cultivars/lines were developed for cultivation in the rainfed areas of pakistan. keywords: anthesis, grain yield, heat stress, membrane stability index, milky seed stage, proline, soluble proteins, triticum aestivum l., wheat introduction high temperature is a major problem in fi eld cropping systems world-wide, with unexpected spatial and temporal variations causing reduced plant growth and productivity (parent et al. 2010). it has been estimated that a rise in temperature of just 1 °c in wheat during the growing season reduces wheat yields by about 3–10% (you et al. 2009). wheat is the * corresponding author, e-mail: aqayyum@uoh.edu.pk copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. khan s. u., din j. u., qayyum a., jan n. e., jenks m. a. 110 acta bot. croat. 74 (1), 2015 major staple food crop of pakistan, where the estimated per capita consumption is about 124 kg year–1, among highest in the world. in order to meet the demand for food in pakistan, an increase in wheat production of at least 4% is required to keep up with population growth. in pakistan, wheat yields are especially sensitive to heat stress during the fl owering to seed maturing stages. during this period, heat stress shortens the growth cycle and forces premature ripening, reduces the number of grains per spike, lowers grain weight, and ultimately results in grain yield and quality deterioration (khan et al. 2007, wahid et al. 2007, din et al. 2010). therefore, an urgent need exists to develop wheat cultivars which are better able to withstand heat stress during later growth stages, or else mature earlier to escape the heat stress typically occurring later in the growing season. understanding the means by which crop plants like wheat can tolerate heat would lay the foundation for work to develop plants with improved heat tolerance. one of the most common responses of crop plants to high temperature stress is an increase in proline accumulation (ahmed and hasan 2011). under high temperature, free proline is involved in osmotic adjustment to protect pollen and plant enzymes from heat injury, and also provides a source of nitrogen and other metabolites (verslues and sharma 2010). accumulation of proline has been shown to occur under heat stress in arabidopsis (wei-tao et al. 2011), cotton (ronde et al. 2001) and wheat (hasan et al. 2007), and genotypic variation in proline accumulation have been reported for these species. under high temperature certain heat shock genes are triggered, resulting in the synthesis of heat shock proteins, whereas other soluble and insoluble proteins have also been shown to exhibit changes in abundance under high temperature stress (simmonds 1995, he et al. 2005). high temperature can cause a loss of membrane integrity, damage to primary photosynthetic processes, and changes in lipid composition, and protein denaturation (wahid et al. 2007). membrane thermal stability due to heat stress, typically measured as ion leakage from the cell, has been used for screening wheat germplasm for thermal tolerance (yildirim et al. 2009). blum et al. (2001) showed a higher yield in spring wheat lines having greater membrane-thermostability in fl ag leaves at anthesis. the study presented here was conducted to explore the physiological basis for heat stress tolerance during later growth stages in wheat, and recommend reliable screening strategies for heat tolerance that can be utilized in wheat breeding programs for pakistan and elsewhere. investigated genotypes were developed for cultivation in rainfed areas of pakistan so they could be further used as parent material for development of heat tolerant wheat cultivars in the domestic wheat breeding program. materials and methods six wheat genotypes as-2002, inqalab-91, punjab-96, nr-234, wafaq-2001 and sh2002 obtained from wheat program, crop sciences institute, national agricultural research centre (narc), pakistan, were used in the study. wheat plants were grown in pots (30 × 40 cm size) containing 10 kg sandy loam soil in a greenhouse under natural daylight at narc, islamabad (latitude 33.38°n, longitude 73.00°e) during the winter/spring with average day/night temperature 30 ± 8 °c and 13 ± 5 °c, respectively. a recommended dose of npk (120-100-60 kg ha–1) was applied as urea, diammonium phosphate and potassium sulphate. the pots were arranged in factorial, randomized, complete block design. plants heat tolerance indicators in wheat acta bot. croat. 74 (1), 2015 111 were subjected to specifi c heat stress treatments immediately at anthesis (80 days after sowing) and at milky growth stages (120 days after sowing). at anthesis when the fi rst anther extrusion occurred, pots (three replications) each containing three plants were moved to a greenhouse where temperature was maintained at 35–40 °c and 14/10 h day/night, 50–70% relative humidity, and illumination of 335 μmol m–2 s–2. after high temperature treatment for 3 h daily for fi ve consecutive days, pots were moved back to normal temperature (average day/night temperature 30 ± 8 °c and 13 ± 5 °c) conditions in open greenhouse atmosphere. these heat treatments were also applied to the second set of potted plants during the milky growth stage. after daily 3-h heat stress treatments on fi ve consecutive days, fl ag leaf from the control and stressed plants was sampled for analysis of proline, soluble protein, and membrane stability index (msi). total proline was determined using the method of bates et al. (1973). fresh plant tissue was extracted with 3% aqueous 5-sulfosalicylic acid and the fi ltrate was reacted with glacial acetic acid and ninhydrin solution at 100 °c for 1 h. the reaction mixture was extracted with toluene and the absorbance of the chromophore contain toluene was read at 520 nm. the leaf msi was determined according to sairam et al. (2002). leaf strips (0.2 g) of uniform size were taken in a test tube containing 10 ml double distilled water in two sets. test tubes in one set were kept at 40 °c in a water bath for 30 min and electrical conductivity of the water containing the sample was measured (c1) using a conductivity bridge. test tubes of the other set were incubated at 100 °c in boiling water for 15 min and their electrical conductivity was measured as above (c2). msi was calculated using formula as below: msi = [1 – (c1 / c2)] ×100 soluble protein content was determined according to bradford (1976). fresh plant tissue was extracted with 0.15 m nacl and the fi ltrate was reacted with bradford reagent (bio-rad protein assay dye reagent). protein concentration of the sample was calculated using the calibration curve of bovine serum albumin and expressed on a fresh weight basis. at physiological maturity, grain yield per plant, fl orets per spike, number of seeds, seed weight per spike, and number of sterile fl orets per spike of both the heat stressed and the control plants were recorded. the analysis of variance of the data for each attribute was carried out using minitab version 13.1. the mean values were compared with duncan’s multiple range (dmr) test at signifi cant differences (anova) (p < 0.05) following snedecor and cochran 1980. results impact of heat on proline, total protein, and the membrane stability index (msi) heat stress imposed at anthesis and milky growth stages signifi cantly increased proline concentration in leaves of all the wheat genotypes in comparison to their control values. at anthesis, the highest increase was recorded in as-2002 (89%) and lowest in punjab-96 (76%) (fig. 1a). at the milky stage, the trend of relative increase in proline concentration was lower in as-2002 (76%) followed by inqalab-91 (77%) and even higher in the rest of the genotypes (fig. 1b). khan s. u., din j. u., qayyum a., jan n. e., jenks m. a. 112 acta bot. croat. 74 (1), 2015 under heat stress, increases in soluble protein content were recorded at both anthesis and milky growth stages (figs. 2a, b). the increase in soluble protein concentration was comparable in all the genotypes, and there was only slight, non-signifi cant change in leaf protein content. high temperature decreased the msi at both anthesis and milky growth stages in all wheat genotypes tested. at anthesis, the decrease in msi was greater in sh-2002 (23%) and least in as-2002 (11%) and inqalab-91 (15%). a similar trend toward decreased msi was exhibited by all the genotypes during the milky growth stage (fig. 3). impact of heat on grain yield, and the number of fl orets and seeds per spike the heat treatment signifi cantly decreased the grain yield per plant in all the tested wheat genotypes, at both anthesis and milky growth stages, and there was signifi cant variation in grain yield within the genotypes, both at anthesis and milky growth stages (tab. 1). anthesis growth was more sensitive to heat stress, as heat treatments during this stage reduced yield by 75%, as compared to milky stage treatments that decreased yield by only 0 250 500 750 1000 as-2002 inqalab-91 punjab-96 nr-234 wafaq-2001 sh-2002 p ro li n e c o n te n t (μ g g -1 f w ) wheat genotypes control stresseda 0 250 500 750 1000 as-2002 inqalab-91 punjab-96 nr-234 wafaq-2001 sh-2002 p ro li n e c o n te n t (μ g g -1 f w ) wheat genotypes control stressedb fig. 1. effect of heat stress on the leaf proline concentration (μg g–1 fresh weight): (a) at anthesis, and (b) milky growth stages of wheat genotypes. the vertical bars indicate standard error (± se) of mean (n = 3). all means are signifi cantly different at p < 0.05. heat tolerance indicators in wheat acta bot. croat. 74 (1), 2015 113 40%, relative to non-treated controls. at anthesis, the magnitude of decrease in grain yield was least in as-2002 (62.9%), whereas the decrease in grain yield in other genotypes was almost equal. at the milky stage, the smallest decrease in grain yield occurred for as-2002 (16.2%), whereas the greatest decrease occurred for sh-2002 (57.5%). similarly, heat treatment signifi cantly decreased seed weight per spike at anthesis growth stage, in all the tested wheat genotypes except cv. as-2002 (tab. 2). the highest seed weight per spike after stress occurred with inqalab-91 (1.06 g), whereas the lowest seed weight after stress treatment was found in nr-234 (0.54 g). at milky stage, seed weight decreased for all the genotypes, and their responses to the heat treatments were similar. however this effect was not statistically signifi cant among the tested wheat genotypes. the number of fl orets per spike decreased as a result of heat treatment for all the tested genotypes; however the effect was not statistically signifi cant. the decrease in number of fl orets was greater at anthesis and least at milky stage (tab. 3). heat stress signifi cantly increased the number of sterile fl orets per spike in all the tested wheat genotypes at the anthesis growth stage but not at the milky stage (tab. 4). the highest and signifi cant number of 0 100 200 300 400 as-2002 inqalab-91 punjab-96 nr-234 wafaq-2001 sh-2002 s o lu b le p ro te in c o n te n t (μ g g 1 f w ) wheat genotypes control stressed b 0 200 400 600 as-2002 inqalab-91 punjab-96 nr-234 wafaq-2001 sh-2002s o lu b le p ro te in c o n te n t (μ g g -1 f w ) wheat genotypes control stressed a fig. 2. effect of heat stress on the soluble leaf protein concentration (μg g–1 fresh weight): (a) at anthesis, and (b) milky growth stages of wheat genotypes. the vertical bars indicate standard error (± se) of mean (n = 3). all means are signifi cantly different at p < 0.05. khan s. u., din j. u., qayyum a., jan n. e., jenks m. a. 114 acta bot. croat. 74 (1), 2015 40 50 60 70 80 90 100 as-2002 inqalab-91 punjab-96 nr-234 wafaq-2001 sh-2002 m e m b ra n e s ta b il it y i n d e x ( % ) wheat genotypes control stressed a 40 50 60 70 80 as-2002 inqalab-91 punjab-96 nr-234 wafaq-2001 sh-2002 m e m b ra n e s ta b il it y i n d e x ( % ) wheat genotypes control stressed b fig. 3. effect of heat stress on the leaf membrane stability index (%): (a) at anthesis, and (b) milky growth stages of wheat genotypes. the vertical bars indicate standard error (± se) of mean (n = 3). all means are signifi cantly different at p < 0.05. tab. 1. effect of heat stress applied at anthesis and milky growth stages on the grain yield per plant (g plant–1) of wheat genotypes. values in columns having the same letter are not signifi cantly different at p > 0.05, duncan’s multiple range test. least signifi cant difference (lsd) value (0.05) for variety × treatment interaction (v × t) = 1.535 at anthesis, lsd value (0.05) for variety × treatment interaction (v × t) = 1.674 at milky stage. the data in parentheses indicate percent decrease in grain yield per plant (g plant–1) of wheat genotypes at anthesis and milky growth stages in comparison to their control values. genotypes anthesis stage milky stage control heat stress control heat stress as-2002 19.36±1.13 b 7.18±0.24 d (62.9) 19.36±1.13 b 16.23± 0.58 d (16.2) inqalab-91 16.40±0.55 c 5.00±0.05 e (69.5) 16.40±0.55 cd 10.56± 0.33 f (35.9) punjab-96 21.13±0.84 a 4.59±0.05 e (78.3) 21.13±0.84 a 14.26± 0.23 e (32.5) nr-234 22.20±0.86 a 4.42±0.01 e (80.0) 22.20±0.86 a 13.73± 0.26 e (38.2) wafaq-2001 22.73±0.47 a 4.43±0.02 e (80.5) 22.73±0.47 a 9.83±0.15 f (56.8) sh-2002 18.00±0.15 b 4.62±0.04 e (74.3) 18.00±0.15 bc 7.66±0.33 g (57.4) means 19.97 5.04 (75%) 19.97 12.05 (40%) heat tolerance indicators in wheat acta bot. croat. 74 (1), 2015 115 tab. 2. effect of heat stress applied at anthesis and milky growth stages on the seed weight per spike (g spike–1) of wheat genotypes. values in columns having the same letter are not signifi cantly different at p > 0.05, duncan’s multiple range test. there were non-signifi cant statistical differences for milky stage. least signifi cant difference (lsd) value (0.05) for variety × treatment interaction (v × t) = 0.316 at anthesis, lsd value (0.05) for variety × treatment interaction (v × t) = 0.801 at milky stage. genotypes anthesis stage milky stage control heat stress control heat stress as-2002 1.99±0.15 d 1.02±0.10 d 1.99±0.15 1.77±0.10 inqalab-91 2.09±0.20 b 1.06±0.04 d 2.09±0.20 1.41±0.28 punjab-96 2.46±0.11 a 0.83±0.18 de 2.46±0.11 1.12±0.04 nr-234 2.09±0.07 b 0.54±0.009 e 2.09±0.07 1.28±0.15 wafaq-2001 1.93±0.07 bc 0.75±0.11 de 1.93±0.07 1.25±0.29 sh-2002 1.70±0.14 c 0.63±0.05 e 1.70±0.14 1.22±0.15 tab. 3. effect of heat stress applied at anthesis and milky growth stages on the number of fl orets per spike of wheat genotypes. there were non-signifi cant statistical differences for anthesis and milky stage. least signifi cant difference (lsd) value (0.05) for variety × treatment interaction (v × t) = 10.28 at anthesis, lsd value (0.05) for variety × treatment interaction (v × t) = 8.312 at milky stage. genotypes anthesis stage milky stage control heat stress control heat stress as-2002 54.66±0.75 49.33±0.88 54.66±0.75 46.00±1.17 inqalab-91 64.66±1.53 57.66±1.20 64.66±1.53 64.00±3.17 punjab-96 46.66±0.75 32.33±1.85 46.66±0.75 54.30±3.33 nr-234 57.66±1.53 50.00±1.45 57.66±1.53 57.00±2.45 wafaq-2001 58.66±0.75 44.00±1.60 58.66±0.75 49.00±1.05 sh-2002 55.66±0.96 42.00±2.08 55.66±0.96 46.00±1.97 tab. 4. effect of heat stress applied at anthesis and milky growth stages on the number of sterile fl orets per spike of wheat genotypes. values in columns having the same letter are not signifi cantly different at p > 0.05, duncan’s multiple range test. there were non-signifi cant statistical differences for milky stage. least signifi cant difference (lsd) value (0.05) for variety × treatment interaction (v × t) = 4.193 at anthesis, and lsd value (0.05) for variety × treatment interaction (v × t) = 6.649 at milky stage. genotypes anthesis stage milky stage control heat stress control heat stress as-2002 8.66±1.20 d 24.33±1.20 b 8.66±1.20 11.00±0.61 inqalab-91 8.66±0.66 d 33.33±1.20 a 8.66±0.66 13.30±0.55 punjab-96 7.66±0.29 d 26.00±1.52 b 7.66±0.29 7.00±0.96 nr-234 4.33±0.24 d 15.33±1.85 c 4.33±0.24 8.00±0.22 wafaq-2001 4.66±0.41 d 23.66±1.45 b 4.66±0.41 9.23±0.90 sh-2002 6.00±1.00 d 17.33±2.72 c 6.00±1.00 11.20±0.77 means 6.67 23.32 6.67 9.95 khan s. u., din j. u., qayyum a., jan n. e., jenks m. a. 116 acta bot. croat. 74 (1), 2015 sterile fl orets was recorded in inqalab-91 (33.33) and punjab-96 (26.0), and least in nr-234 (15.33) at anthesis stage. at anthesis growth stage, the number of seeds per spike also decreased signifi cantly in all the tested wheat genotypes, except cv. punjab-96 (14.8) (tab. 5). signifi cant variations were observed among the genotypes. at anthesis stage, the quantity of decrease in number of seeds per spike was greater in nr-234 (41.7%) followed by sh2002 (38.8%). at milky stage all the tested genotypes exhibited similar trends of fl oret number and sterility as a result of applied heat stress. tab. 5. effect of heat stress applied at anthesis and milky growth stages on the number of seeds per spike of wheat genotypes. values in columns having the same letter are not signifi cantly different at p > 0.05, duncan’s multiple range test. there were non-signifi cant statistical differences for milky stage. least signifi cant difference (lsd) value (0.05) for variety × treatment interaction (v × t) = 7.432 at anthesis, lsd value (0.05) for variety × treatment interaction (v × t) = 5.715 at milky stage. the data in parentheses indicate percent decrease in number of seeds spike–1 of wheat genotypes at anthesis and milky growth stages in comparison to their control values. genotypes anthesis stage milky stage control heat stress control heat stress as-2002 47.5±2.2 bcd 40.3±1.2 de (15.2) 47.5±2.2 44.7±0.9 (5.8) inqalab-91 64.0±1.2 a0 51.7±0.6 bc (19.2) 64.0±1.2 59.3±3.1 (7.3) punjab-96 51.3±0.9 bc 43.7±0.3 cd (14.8) 51.3±0.9 50.7±2.7 (1.1) nr-234 52.7±1.6 b0 30.7±0.6 f (41.7)0 52.7±1.6 48.7±2.3 (7.6) wafaq-2001 50.7±0.4 bc 33.0±0.7 ef (34.9) 50.7±0.4 47.4±1.1 (6.5) sh-2002 49.0±1.2 bc 30.0±0.7 f (38.8)0 49.0±1.2 45.7±1.3 (6.7) means 52.53 38.23 (27%) 52.53 49.42 (6%) discussion in this study, heat stress imposed after anthesis and the milky stages resulted in changes in physiological attributes such as proline content, total soluble protein, msi, yield parameters. susceptibility to high temperatures may vary with the stage of plant development, but all vegetative and reproductive stages are affected by heat stress to some extent (wahid et al. 2007). heat stress induced modifi cations in plants may be observed as changes in specifi c physiological processes, or in varying effects on development, and these responses may vary from one growth stage to another. in the present study, heat stress application during reproductive stages, at either anthesis or the milky seed development stage, increased signifi cantly the proline concentration in fl ag leaves of all the wheat genotypes examined. at anthesis stage, the increase was greater in as-2002 and lowest in sh-2002, whereas at the milky stage, the increase was lower in as-2002 and inqalab-2001, and higher in rest of the genotypes. one of the most common responses of many plant species exposed to abiotic stresses is the accumulation of compatible organic solutes such as proline. proline has been suggested to play a protective role in plants acting as a cellular osmotic regulator between cytoplasm and vacuole, and by its ability to detoxify reactive oxygen species (ros) and thereby protecting membrane integrity and stabilizing antioxidant enzymes (ashraf and foolad 2007). under stress conditions, accumulation of proline in plants results either heat tolerance indicators in wheat acta bot. croat. 74 (1), 2015 117 from increased expression of proline synthetic enzymes or due to repressed activity of proline degradation (hong et al. 2000). in our case, the increase in proline accumulation was greater in as-2002 and lowest in sh-2002. genotypic differences in proline accumulation under high temperatures were previously reported in 20 wheat genotypes (ahmed and hassan 2011). leaf proline level is thought to serve as an effective index to screen wheat genotypes for relative differences in heat tolerance. in the present study, increase in total leaf protein under heat stress was observed at both reproductive growth stages. it seems likely that this increase in total soluble proteins under heat stress is due to the induction of stress proteins, as such stress induced protein expression has been shown to be an important adaptive strategy of crop plants. further, the majority of stress induced proteins is soluble in water and therefore contributes to stress tolerance presumably via hydration of cellular structures (wahid and close 2007). in our study, increased protein concentration resulting from heat treatments was similar in all the genotypes. likewise, din et al. (2011) observed no differences in leaf soluble protein concentration in various canola cultivars under water stress. further studies are needed to determine if other wheat genotypes show variation for total leaf protein content as a response to heat stress. increased solute leakage is an indication of decreased cell membrane thermostability, and has long been used as an indirect measure of heat-stress tolerance in diverse plant species, including wheat (blum et al. 2001, wahid et al. 2007). in the present investigation, high temperature decreased the msi at both growth stages in all the tested wheat genotypes. at anthesis, the decrease in msi was greater in sh-2002 (23%) and lowest in as-2002 (11%) and inqalab-91 (15%). a similar trend of decreased msi was exhibited by all the genotypes at milky growth stage. yildirim et al. (2009) and dhanda and munjal (2006) concluded from their fi ndings that membrane thermal stability was a useful selection criterion for heat stress tolerance in wheat, reporting differences in msi among different wheat cultivars at various growth stages. as in that study, they also showed that the msi of genotypes decreased towards later developmental stages, i.e. the milky stage. membrane instability at the milky growth stage might be associated with the beginning of senescence. sikder et al. (2001) found signifi cant correlation between membrane stability of fl ag leaf and grain yield, and suggested that membrane thermostability can be used to determine the heat tolerance of wheat varieties under heat stress conditions. heat stress is a common constraint during anthesis and grain fi lling stages in many cereal crops in both arid and temperate regions. at the reproductive phases, fertilization has been shown to be highly sensitive to high temperatures in various plants, whereas heat stress during wheat grain fi lling is known to reduce kernel growth and cause a reduction in kernel density and weight (foolad 2005, laghari et al. 2012) in the present study, heat treatment signifi cantly increased the number of sterile fl orets per plant in all the tested wheat genotypes at anthesis growth stages. at anthesis, the highest number of sterile fl orets per spike after heat treatment occurred in inqalab-91 (33.33), and the lowest in nr-234 (15.33) (tab. 4). these results indicate that the genotypes examined here utilize unique responses for heat tolerance, as the lines showing the best performance as measured in proline, total protein, and msi response show different heat responses regarding sterility and other heat associated traits. heat treatment decreased the grain yield per plant in all the tested wheat genotypes at both reproductive growth stages; however the most signifi cant decrease was observed by khan s. u., din j. u., qayyum a., jan n. e., jenks m. a. 118 acta bot. croat. 74 (1), 2015 heat treatments during anthesis. there were signifi cant variations in grain yield per plant within the genotypes both at anthesis and milky growth stages. at anthesis, the highest decease in grain yield was observed in wafaq-2001 (80.5%), nr-234 (80.0%) and punjab-96 (78.3%), and the least effect was observed for as-2002 (62.9%). khan and hussain (2006) tested as-2002 for heat tolerance in fi eld studies, and found it had the best performance with respect to grain yield, followed by inqalab-91. at the milky stage, the same cultivar also exhibited the least effect of heat stress on grain yield. comparable studies show similar genotypic effects of heat on grain yield for wheat and other crop species (khan et al. 2007, nahar et al. 2010, balouchi 2011). during the onset of meiosis in the male generative tissues until completion of anthesis, wheat grain setting is reduced by rises in temperature above optimum (ferris et al. 1998). our results are consistent with these previous reports, showing the heat effects on yield for new genotypes in wheat. this study also shows that heat stress signifi cantly decreases seed number and weight per spike in these wheat genotypes, more during the anthesis growth stage than the milky stage. for heat stress applied during anthesis, the relative seed number reduction was lowest in punjab-96 followed by as-2002 and inqalab-91, and highest in nr-234 followed closely by sh-2002 and wafaq-2001. similarly, for anthesis applied heat stress, the seed weight per spike was greater in inqalab-91 and as-2002, and lowest in nr-234. for heat stress applied during the milky stage, the seed number and weight per spike was reduced, but the effect was small and not signifi cant. in the present study, high temperature treatment at anthesis stage increased the number of sterile fl orets in all the tested genotypes, and most notably in the inqalab-91, consequently reducing the number of grains per plant and the yield. genotypic variation in grain yield for heat effects on the number of sterile fl orets was also observed. among the reproductive stages, fertilization (1–3 days after anthesis) is one of the most sensitive to high temperatures (foolad 2005). pollen viability, patterns of assimilates partitioning, and growth and development of seed/grain, ear or spike are likewise highly affected by heat. cereal crops can tolerate only narrow temperature ranges, and if these are exceeded during the fl owering stages they can damage fertilization and seed set, resulting in yield reduction (porter 2005). it has been shown for example that supra-optimal temperatures during grain fi lling decreased wheat yield by reducing kernel weight (gibson and paulsen 1999). in the current project, the decline in grain yield due to heat treatment at the milky stage is due to reduced seed weight per spike. in wheat, genotypic variation with respect to translocation of assimilates from source to sink under high temperature has already been reported (mohammadi et al. 2009). the high grain yield and seed weight of the as-2002 and inqalab-91 cultivars may be due to their greater effi ciency in mobilizing reserves from leaves, stem or other plant parts towards sink (gupta et al. 2011). furthermore, high temperatures can reduce the number of grains per spike, by causing either fl ower sterility or seed abortion, which reduces grain yield and ultimately harvest index causing smaller grain yield. wardlaw (2002) found reductions in grain weight due to high temperature during both anthesis and milky growth stages under fi eld conditions. conclusion tolerance to heat stress is a complex phenomenon and is controlled by multiple genes imparting a number of physiological and biochemical changes. no single trait fully explains why some wheat varieties are able to generate better yield under heat stress. wide heat tolerance indicators in wheat acta bot. croat. 74 (1), 2015 119 variation in tolerance to heat stress existed in the tested wheat genotypes. thus, there is a dire need for the development of heat tolerant wheat genotypes through a breeding program for cultivation under heat stress environments like those prevailing in pakistan. the cumulative response of as-2002 was better on the basis of physiological and yield attributes, indicating as-2002 may represent an excellent parental material for a breeding program. plant breeders should also use proline and msi as selection markers in the breeding 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wardlaw, i. f., 2002: interaction between drought and chronic high temperature during kernel fi lling in wheat in a controlled environment. annals of botany 90, 469–476. wei-tao, l., lin, b., zhang, m., hua, x. j., 2011: proline accumulation is inhibitory to arabidopsis seedlings during heat stress. plant physiology 156, 1921–1933. yildirim, m., bahar, b., koc, m., barutcular, c., 2009: membrane thermal stability at different developmental stages of spring wheat genotypes and their diallel cross populations. tarim bilimleri dergisi 15, 293–300. you, l., rosegrant, m. w., wood, s., sun, d., 2009: impact of growing season temperature on wheat productivity in china. agricultural and forest meteorology 149, 1009–1014. acta botanica 2-2016 za web.indd acta bot. croat. 75 (2), 2016 199 acta bot. croat. 75 (2), 199–205, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0032 issn 0365-0588 eissn 1847-8476 the application of benthic diatoms in water quality assessment (mlava river, serbia) olga s. jakovljević1*, slađana s. popović1, danijela p. vidaković1, katarina z. stojanović2, jelena ž. krizmanić1 1 university of belgrade, faculty of biology, institute of botany and botanical garden »jevremovac«, takovska 43, 11000 belgrade, serbia 2 university of belgrade, faculty of biology, institute of zoology, student square 16, 11 000 belgrade, serbia abstract–the main objective of this study was to assess the ecological status of the mlava river based on epilithic diatoms and to test the use of diatom indices as a tool for estimating the quality of fl owing waters in serbia. quantitative analysis showed that in april achnanthidium minutissimum was dominant at each site, except at the fi fth site, where amphora pediculus was dominant. in july and september, achnanthidium minutissimum, achnanthidium pyrenaicum, amphora pediculus, denticula tenuis, diatoma vulgaris, gomphonema elegantissimum, cocconeis pseudolineata and cocconeis placentula var. lineata dominated. detrended correspondence analysis (dca) was used to detect the major patterns of variation in species composition. the fi rst dca axis summarizes the distribution of the diatom community, mainly through temperature, conductivity, oxygen and water hardness gradient. the second dca axis was weakly correlated with few variables. based on the average values of the pollution sensitivity index (ips), commission for economical community metric (cee) and biological diatom index (ibd), the water of the mlava river belonged to water class i during all three seasons. values of the diatom-based eutrophication/pollution index (epi-d) indicated class ii water quality. according to calculated trophic diatom index (tdi) values, water of the mlava river was characterized by intermediate nutrient concentrations during three seasons. principal components analysis was used to represent the correlation between diatom indices, and the highest correlation among the selected diatom indices is seen between epi-d, ips and ibd. key words: biomonitoring, diatoms, indices, mlava river * corresponding author, e-mail: olga.jakovljevic@bio.bg.ac.rs introduction the best way to evaluate the ecological status of waters is the simultaneous use of physico-chemical and biological analysis. use of only physico-chemical methods gives unreliable results, because water quality may change over a short period of time. the really current state of the ecosystem can only be obtained by using biological methods. according to the water framework directive (wfd 2000/60/ ec), biological indicators play a key role in the assessment of ecological status. biological assessment is expressed according to a numerical scale between zero and one – the ‘ecological quality ratio’ (eqr). values of this scale ranged from zero to one, with high status represented by values close to one and bad status by values close to zero. ‘macrophytes and phytobenthos‘ are together one of the biological quality elements (bqes) for assessment of the ecological status of european rivers and lakes. in total, 66 macrophyte and phytobenthos assessment systems (32 for macrophytes, 30 for phytobenthos and 4 combined) have been developed and intercalibrated (poikane et al. 2016). when it comes to phytobenthos, benthic diatoms are a valuable tool in water quality assessment and monitoring because they occur in most aquatic ecosystems, at any time, are easy to collect and sensitive to physical, chemical and biological changes in the water (vasiljević et al. 2014). however, there are some diffi culties in the use of diatoms in water quality assessment. one disadvantage is the large number of taxa involved, which is partly resolved by using an index based on genera (rumeau and coste 1988). also, considerable taxonomic expertise is required (ács et al. 2004). in addition, ph, conductivity, total phosphorus, temperature, alkalinity, altitude, nitrates, calcium, biological oxygen demand (bod), chlorophyll a and substrate type are major environmental factors that affect diatom distribution in streams, in addition to light availability, total phosphorus and grazing pressure (toman et al. 2014). jakovljević o. s., popović s. s., vidaković d. p., stojanović k. z., krizmanić j. ž. 200 acta bot. croat. 75 (2), 2016 diatoms have been used for water evaluation purposes for more than a decade in many countries of europe (austria, switzerland, germany, belgium, france, poland, finland, luxemburg, united kingdom, spain, portugal, italy). according with the requirements of the wfd, the epilithic diatoms were investigated for ecological status assessment of the aksu and isparta streams, as well as the porsuk and karasu rivers in turkey (solak 2011). ecological status assessment, based on diatom indices, is still a new topic in turkey and the number of studies on this topic is constantly on the increase (solak and àcs 2011). diatom indices have been widely used for polish rivers and streams. algological investigation of 38 polish rivers was conducted and it was found that generic diatom index and pollution sensitivity index (ips) indices are the most appropriate for polish conditions. many rivers were characterized by high and good ecological status (solak 2011). diatom studies have been sporadic and focused on large rivers in hungary. ács et al. (2009) investigated 398 streams and found that more than half had good ecological status based on the values of ips, austrian saprobic index and austrian trophic index. the implementation of eu standards has been started and these countries possess information on ecological status of their most important rivers (solak 2011). the offi cial gazette of the republic of serbia (»sl. glasnik rs«, no. 74/2011) prescribes the use of two diatom indices: ips (coste in cemagref 1982) and commission for economical community metric (cee) (descy and coste 1991). the wfd has not been suffi ciently integrated in serbia. examination of diatom indices applicability in the assessment of the ecological status of fl owing waters in serbia started during 2012 (andrejić 2012). in that study, the trophic diatom index (tdi) was used in assessment of the water quality of the nišava river and its tributaries the jerma and temska. after that, the ecological status of the djetinja river was determined using the diatom pollution index (daipo) (krizmanić et al. 2013). in all, 17 diatom indices were calculated with the help of a software package omnidia (lecointe et al. 1993) based on which the quality of the raška river was estimated (vidaković 2013). water quality assessment of the study area of the danubetisza-danube (dtd) hydrosystem has been evaluated by four diatom indices (watanabe’s index-daipo, biological diatom index-ibd, tdi and ips) (jakovljević et al. 2014). serbia is in the early stage of preparation in the fi eld of environment and climate changes (denić et al. 2015). it is necessary to audit the »ordinance on the parameters of the ecological and chemical status of surface waters and the parameters of the chemical and quantitative status of groundwater«, (»sl. glasnik rs«, br. 74/2011) in the part of the parameters list and class boundaries for individual parameters of the biological quality elements. based on the category of ecological status expressed through ecological quality ratios (eqr) values (wfd cis guidance no. 7. 2003), excellent ecological status is achieved when the eqr value is greater than 0.80. in all, 6 samples over 3 years are needed for reliable ecological status classifi cation (wfd-uktag 2014). the aim of this study was to assess the ecological status of the mlava river based on epilithic diatoms and to test the use of diatom indices as tool for estimating the quality of fl owing waters in serbia. materials and methods the mlava river is located in the northwestern part of eastern serbia with a course of 120 km, covering a drainage area of 1830 km2, and fl ows into the danube. it is one of the longest rivers in eastern serbia. the mlava river is particularly specifi c in physical-geographical characteristics (geological structure of the terrain and water level of the basin) (manojlović et al. 2012). the upper part of the river fl ows through a gorge and the downstream part through a wide valley (babić mladenović 2009). a trout fi sh pond was created in the upper part of the investigated section of the mlava river, between the fi rst and second sampling site. some basic data about the sites are given in tab. 1. the sampling was conducted during three seasons (april, july and september 2011) from 5 localities (ml1ml5), along the mlava river. benthic diatoms were collected from the stones by scraping with a toothbrush. the material was preserved in 4% formalin. water temperature, conductivity, ph, ammonium ions, nitrates, oxygen, biochemical oxygen demand, alkalinity, total phosphorus, orthophosphates and water hardness were also determined in the water samples from each sampling site. diatom frustules were cleaned using the standard method with concentrated sulfuric acid (h2so4), potassium permanganate (kmno4) and oxalic acid to remove the organic content (krammer and lange-bertalot 1986). when ph was approximately 7, the material was mounted in naphrax® mounting medium and permanent slides were made. microscopic analysis of the permanent slides was performed using a zeiss axio-imager m1 microscope with a digital camera axiocam mrc5 and axiovision 4.8 software. tab. 1. characteristics of the 5 investigated sites (ml1-ml5), along the mlava river (serbia). site ml1 ml2 ml3 ml4 ml5 altitude [m] 314 311 310 297 296 water depth [m] 0.29 0.17 0.2 0.16 0.23 flow width [m] 12 7.27 9.63 6.81 7.16 flow velocity [m s–1] 0.1 0.44 0.39 0.65 0.37 watercourse bottom gravel: 40% stones: 75% stones: 75% stones: 70% stones: 75% coordinates 44°11.493n 021°47.012e 44°11.842n 021°46.516e 44°11.781n 021°46.158e 44°12.133n 021°45.131e 44°13.488n 021°44.635e water quality monitoring using benthic diatoms acta bot. croat. 75 (2), 2016 201 relative abundance of each diatom taxa was determined by counting 400 valves in each slide. seventeen diatom indices are calculated based on the indicator values of identifi ed taxa using the omnidia software (lecointe et al. 1993), and fi ve were taken into consideration for the water quality assessment. these were: ips (coste in cemagref 1982), eutrophication and/or pollution index – diatom based – epi-d (dell’uomo 2004), cee (descy and coste 1991), ibd (coste et al. 2009) and tdi (kelly and whitton 1995). ips, epi-d, cee and ibd are scaled from 1 to 20, while tdi is scaled from 1 to 100. higher values of the fi rst four indices indicate water quality improvement while higher values of tdi indicate bigger trophy of water (tab. 2) (lecointe et al. 1993). statistical analyses, detrended correspondence analysis and principal component analysis, were done using canoco for windows version 5 (ter braak and šmilauer 2012). results qualitative and quantitative analysis of diatoms an investigation of the benthic diatoms from the mlava river during three seasons resulted in the description of 86 diatom taxa, belonging to 27 genera. the greatest taxa richness was recorded within the following genera: navicula bory sp. (12), gomphonema ehrenberg sp. (11), and nitzschia hassall sp. (8). samples from the third sampling site (ml3) had the highest diversity of benthic diatoms in all seasons. forty-fi ve taxa were recorded in april, 39 taxa in july and 30 in september. the lowest number of diatom taxa was recorded at the site ml1. in april, 16 taxa were recorded, 25 taxa in july and 17 in september. according to the frequency of the taxa, amphora pediculus (kützing) grunow, denticula tenuis kützing, cocconeis placentula var. lineata (ehrenberg) van heurck, planothidium frequentissimum lange-bertalot, reimeria sinuata (gregory) kociolek & stoermer, rhoicosphenia abbreviata (c.agardh) lange-bertalot and encyonema minutum (hilse) d.g.mann were the taxa that occurred at the most investigated sites. cymbella excisa kützing, surirella brebissonii var. kuetzingii krammer and lange-bertalot, nitzschia pusilla grunow, cymbella excisiformis krammer and fragilaria parasitica var. parasitica (w. smith) grunow were documented at only one site (fig. 1). quantitative analysis showed that in april achnanthidium minutissimum (kützing) czarnecki was dominant at each locality, except at the fi fth site, where amphora pediculus was dominant. also, planothidium frequentissimum, gomphonema micropus kützing, achnanthidium pyrenaicum (hustedt) h.kobayasi, amphora pediculus and reimeria sinuata (gregory) kociolek & stoermer were dominant taxa during april. a. minutissimum, a. pyrenaicum (hustedt) h.kobayasi, a. pediculus, denticula tenuis, diatoma vulgaris bory, gomphonema elegantissimum e.reichardt & h.lange-bertalot, cocconeis pseudolineata (geitler) lange bertalot and cocconeis placentula var. lineata dominated in july and september. the percentage participation of dominant taxa was 10% or more at least in one site. tab. 2. class boundary limits for diatom indices. ps – pollution sensitivity index, epi-d –diatom-based eutrophication/pollution index, cee – commission for economical community metric, ibd – biological diatom index, tdi – trophic diatom index. water quality class ecological status ips, epi-d, cee, ibd tdi trophic status average % of taxa used for the index calculation i high 17–20 <20 oligotrophic 100 (ips) ii good 13–16 20–40 oligo-mesotrophic 73.91 (epi-d) iii moderate 9–12 40–60 mesotrophic 74.23 (cee) iv poor 5–8 60–80 eutrophic 93.61 (ibd) v bad 1–4 80–100 hypertrophic 82.49 (tdi) fig. 1. light micrographs of diatom species that occurred at most investigated sites (1–7); species that occurred at only one site (8– 13). 1 – cocconeis placentula var. lineata; 2 – rhoicosphenia abbreviata; 3 – encyonema minutum; 4 – planothidium frequentissimum; 5 – reimeria sinuata; 6 – denticula tenuis; 7 – amphora pediculus; 8 – cymbella excisa; 9 – surirella brebissonii var. kuetzingii; 10 – cymbella excisiformis; 11 – fragilaria parasitica var. parasitica; 12 – nitzschia pusilla. scale bar = 10 μm. jakovljević o. s., popović s. s., vidaković d. p., stojanović k. z., krizmanić j. ž. 202 acta bot. croat. 75 (2), 2016 diatom-environmental relationships the values of physico-chemical parameters of the mlava river are presented in tab. 3. detrended correspondence analysis (dca) was used to detect the major patterns of variation in species composition (fig. 2). the eigenvalues of the fi rst and second axis were 0.7398 and 0.3210 respectively. the high eigenvalue of the fi rst axis indicates its good explanatory power. the fi rst dca axis summarizes the distribution of the diatom community, mainly through temperature, conductivity, oxygen and water hardness gradient. sites, sampling time and taxa from the left side of the ordination diagram have a tendency toward higher oxygen and water hardness, but lower temperature and conductivity level, while sites, sampling time and taxa from the right side of the diagram show the opposite. as for the second axis, it is more diffi cult to interpret, since there are several variables weakly correlated with it, for example nitrates and orthophosphates. higher levels of orthophosphates correspond to the lower part of the ordination diagram, and higher levels of nitrates to the upper part of the diagram. correlation coeffi cients of the selected variables and the two dca axes are given in online suppl. tab. 1. it is also worth saying that the sampling site ml1 had the lowest values of ammonia and total phosphorous and highest values of oxygen and water hardness. the species response curves that explain difference for the selected species distribution along the fi rst dca axis using generalized linear model are shown in on-line suppl. fig. 1. since the fi rst dca axis shows the highest negative correlation with oxygen and highest positive correlation with temperature, species response curves of selected taxa show that achnanthidium minutissimum, planothidium frequentissimum and gomphonema micropus are more abundant at higher oxygen levels, while cocconeis placentula var. lineata and cocconeis pseudolineata are more to be found at higher temperatures. diatom indices and water quality the values of the fi ve diatom indices used indicated a similar ecological status of the water at the studied sites. pca was used to represent the correlation between diatom indices, where the highest correlation is seen between epi, ips and ibd. also, cee is correlated with them. these three indices had the highest values at sampling site ml2 during september. cee had the highest value at sampling site ml1 during september and tdi at sampling site ml5 during april (fig. 3). based on the average values of the ips, cee and ibd diatom index, water of the mlava river belong to water class i during all three seasons, which corresponded to high ecological status. values of the epi-d index indicated class ii water quality (good ecological status), although these values were on the border with values indicating class i water quality. according to calculated tdi values, water of the mlava river is mesotrophic with moderate nutrient concentrations and corresponds to water class iii during all seafig. 2. detrended correspondence analysis (dca) of dominant diatom taxa (adpy – achnanthidium pyrenaicum, gmic – gomphonema micropus, plfr – planothidium frequentissimum, amin – achnanthidium minutissimum, rsin – reimeria sinuata, apen – amphora pediculus, dten – denticula tenuis, gelg – gomphonema elegantissimum, dvul – diatoma vulgaris, cpli – cocconeis placentula var. lineata, copl – cocconeis pseudolineata) in the mlava river (serbia) in the ordination space of fi rst and second axis with locality (squares; ml1–ml5), sampling season (circles; apr, jul, sep) and physico-chemical characteristics (oxygen level-o2, water hardness-wh, biochemical oxygen demand-bod, orthophosphates-op, ammonia-amm, total phosphorous-tp, ph, temperature-t, nitrates-no3, conductivity-cond and alkalinity-alc) as a supplementary variable. tab. 3. chemical water parameters of the mlava river at the studied sites (ml1–ml5). bod – biological oxygen demand. parameter ml1 ml2 ml3 ml4 ml5 temperature [°c] 10.4–11.4 10.5–15.2 12.8–16.8 10.6–14.6 10.8–15 conductivity [μs cm–1] 378–435 374–490 355–506 340–490 351–475 ph 7.14–7.38 7.53–7.8 7.62–7.95 7.94–8.12 7.97–8.04 ammonia [mg l–1] <0.0189–0.0471 0.143–0.4556 0.246–0.379 0.1648–0.3396 0.0505–0.176 nitrates [mg l–1] 6.85–7.5 6.1–8 6.8–8.2 6.8–8.8 3.5–8.8 oxygen [mg l–1] 11.1–12.2 7.7–10.8 9.4–11.2 10.6–10.9 9.1–10.8 bod [mg l–1] 1.65–5.1 1.4–5.7 1.55–5.25 2–5.4 2.25–5.15 alkalinity [mmol l–1] 3.6–4 3.6–4.1 1.29–4 3.4–4.2 3.5–4.2 total phosphates [mg p l–1] 0.0335–0.052 0.0544–0.0817 0.0389–0.0942 0.0252–0.1128 0.0282–0.1151 orthophosphates [mg p l–1] 0.0192–0.036 0.033–0.0344 0.0275–0.039 0.0134–0.0468 0.0171–0.0392 water hardness [ºdh] 11.4–12.06 1.27–12.16 1.29–12.06 1.3–12.27 1.3–12.15 water quality monitoring using benthic diatoms acta bot. croat. 75 (2), 2016 203 sons. tdi values during the fi rst (april) and second season (july) were on the border with values indicating the high nutrient concentrations. values of all fi ve indices increase in the second and third season (september) (on-line suppl. tab. 2). discussion some of the most signifi cant factors affecting the distribution of benthic diatoms in the rivers are the physicochemical characteristics of water, type of substrate, water velocity, amount of light and presence of predators (stevenson et al. 1996). the infl uence of the physico-chemical characteristics of water has been widely studied by many authors. patrick (1973) recorded that acidic waters do not support an abundance of bacillariophyceae, but in alkaline waters with ph above 8.0, their density is much higher. some studies indicate that ph in the neutral range (7.0–8.0), like the values recorded in our study (7.14–8.12), supports a good population of diatoms (kumar and oommen 2011). optimal temperatures for benthic diatoms are in the range from 25 to 30 °c with diversity declining at the temperatures above 30 °c. according to rusanov et al. (2009), conductivity related to geology, as well as total phosphorus, were the most important variables related to changes in the diatom assemblage structure. phosphates, nitrates and silica are generally considered the most important nutrients in primary production, and it is documented that diatoms reach their maximum growth rate at a concentration of total phosphorus by 0.5 mg l–1 (chételator et al. 1999). the dca performed showed that the highest correlations with the fi rst dca axis were with temperature and oxygen. even though the temperature range in our study was quite low (10.4–16.8), dca showed that some species of the genus cocconeis (cocconeis placentula var. lineata and cocconeis pseudolineata) have a tendency toward higher temperatures (they are placed at the right side of the ordination diagram). andrejić (2012) reported that optimal temperatures for cocconeis genus are > 25 °c. it is known that cocconeis placentula var. lineata has a wide ecological range, especially with regard to the electrolyte content and the trophic situation, even found in poorer electrolyte, silicate-dominated streams (hofmann et al. 2013). one of the indicators of oxygen-rich waters, a. minutissimum, (noga et al. 2014) is one of the most frequent taxa in epilithic diatom communities in streams and rivers in general (virtanen and soininen 2011). it is a cosmopolitan species that has a wide ecological spectrum recorded in water bodies with the conditions from oligoto eutrophic with a wide range of ph (4.3–9.2). there are records of numerous populations that develop in high mountain streams (van dam et al. 1994, kawecka 2012). it is also one of the most common and most frequently noted diatoms in the podkarpacie region, poland, especially in the upper courses of rivers and streams (noga 2012, pajączek et al. 2012). this diatom requires a continuously high concentration of dissolved oxygen. other taxa requiring a fairly high oxygen level (above 75% saturation) (van dam et al. 1994) are planothidium frequentissimum and gomphonema micropus. dca showed good correlation between all these three taxa and higher oxygen level, but also with higher water hardness. it is worth mentioning that planothidium frequentissimum was an indicator of high levels of organic pollution and electrolyte contents in rivers in the northeast of spain and in france (tornés et al. 2007, rimet 2009) and appeared in samples with a phosphates concentration up to 5 mg l–1 and nitrates concentration up to 35 mg l–1. in our samples, orthophosphates concentration was much lower and nitrates concentration was similar and varied 3.5–8.8. amphora pediculus is an alkaliphilous species, often found in waters with moderate conductivity and tolerant to increased concentrations of organic nitrogen (van dam et al. 1994). it colonized oligosaprobic and mesosaprobic habitats (hofmann et al. 2013) corresponding to those of the studied sites of the mlava river. based on pca analysis conducted by studying diatoms of the nišava river, it was observed that a. pediculus is positively correlated with nutrients (andrejić 2012). it can be seen from our study that this species is correlated with nitrates but also with conductivity. achnanthidium pyrenaicum and reimeria sinuata are placed on the opposite sides of the dca second axis, but it is not sure which factors contribute the most to their position along the second axis. it is known that a. pyrenaicum is an alkaliphilous taxon mainly occurring at ph > 7 and a nitrogen-autotrophic taxon, tolerating elevated concentrations of organically bound nitrogen (van dam et al. 1994, noga et al. 2014). a. pyrenaicum had an optimum in oligoto mesotrophic calcium rich waters with medium to high concentration of electrolytes (krammer and lange-bertalot 1991). it was very abundant in the territory of the podkarpacie province, poland, in the upper course of the wisłok river (noga 2012). fig. 3. principal components analysis (pca) of selected diatom indices (epi-d – eutrophication and/or pollution index – diatom based, ips – specifi c pollution index, ibd-biological diatom index, cee – commission for economical community metric, tdi – trophic diatom index) with sampling time (april, july and september) and sampling site (ml1–ml5) as supplementary variables. jakovljević o. s., popović s. s., vidaković d. p., stojanović k. z., krizmanić j. ž. 204 acta bot. croat. 75 (2), 2016 many taxa already mentioned above occurred at most of the sampling sites and were the dominant taxa in the diatom community. achnanthidium minutissimum, achnanthidium pyrenaicum and amphora pediculus were dominant taxa in all three seasons. in general, in our study a great diversity of diatom species was documented and a similar diversity was found in an investigation of the porsuk river in turkey (solak 2011) and the raška river in serbia (vidaković 2013). navicula, gomphonema and nitzschia are usually the most numerous genera in rivers in europe, and in serbia as well (solak 2011, andrejić 2012, noga et al. 2013, vidaković 2013, vasiljević et al. 2014). as mentioned, the lowest number of diatom taxa was recorded at ml1. results showed that the sampling site ml1 had the lowest values of ammonia and total phosphorous and highest values of oxygen and water hardness (seen also in dca ordination diagram), as expected, due to the fi shpond located directly after the ml1. the higher number of diatom taxa from the second locality was caused by the higher level of nutrients, especially phosphates. the water quality studies of the mlava river during april, july and september 2011 indicated good and high water qualities. this was confi rmed by most of the measured physico-chemical characteristics which indicated class i water quality. diatom indices calculation showed that at the studied sites there are no major variations in water quality, regardless of the trout pond which is located between the fi rst and second locality. based on pca analysis, a correlation was noticed between the selected indices, indicating their applicability to the rivers in serbia. the epi-d index was used in monitoring seven rivers located in the central eastern apennine sector (italy) (torrisi and dell’ uomo 2006). as in our study, a high correlation was shown between this index and the ips and ibd indices. ips and cee are the most sensitive to eutrophication and organic pollution (descy and coste 1991). cee had the highest value at sampling site ml1, which can be explained by the position of the pond (between the fi rst and the second locality), although these values still are within the fi rst class quality. according to szabó et al. (2004), among ips, ibd, cee and epi-d diatom indices, which are good for hungarian rivers, ips is probably the best. the suitability of the index is dependent on the percentage of taxa used for the index calculation. in our study, the average percentage of taxa used for the ips calculation is 100, so ips is the most suitable index for this reason. tdi is not correlated with the other four indices in our study, which is expected, since it is the only index in which the values range from 1 to 100 while the values of all other indices range from 1 to 20. values of all fi ve indices increase in the second and third season which indicates the improvement of water quality. the data provided by this study can be used for the development of a biomonitoring tool for the rivers in serbia. acknowledgments financial support was provided by the ministry of education and science of the republic of serbia (project no. tr 037009). references ács, é., borics, g., fehér, g., kiss, k.t., reskóne, n.m., stengerkovács, cs., vábríró, g., 2009: implementation of the european water framework directive to assessment the water quality of hungarian running waters with diatoms. datomededelingen 33, 29–33. àcs, é., szabó, k., tóth, b., kiss, k.t., 2004: investigation of benthic algal communities, especially diatoms of some hungarian streams in connection with reference conditions of the water framework directives. acta botanica hungarica 46, 255–277. andrejić, j., 2012: floristic-ecological analysis of diatoms (bacillariophyta) from the nišava river and tributaries jerma and temska rivers. doctoral dissertation, university of belgrade, belgrade. babić mladenović m., 2009: sub-basin level fl ood action plan: velika morava river basin and right danube tributaries between the sava river mouth and rsbg border. ministry of agriculture, forestry and water management, republic directorate for water, in cooperation with institute for development of water resources »jaroslav černi«, belgrade, and republic hydrometeorological service of serbia, belgrade and ministry of environment and water, sofi a danube river basin directorate, sofi a. chételator, j., pick, j., morin, a., hamilton, g., 1999: periphyton biomass and community compoition in rivers of different nutrient status. canadian journal of fisheries and aquatic sciences 56, 560–569. coste in cemagref, 1982: etude des méthodes biologiques quantitative d’appréciation de la qualité des eaux. rapport division qualité des eaux lyon – agence fi nancière de bassin rhôneméditerranée-corse, pierre-bénite. coste, m., boutry, s., tison-rosebery, j., delmas, f., 2009: improvements of the biological diatom index (bdi): description and effi ciency of the new version (bdi-2006). ecological indicators 9, 621–650. dell’uomo, a., 2004: l’indice diatomico de eutrofi zzacione/ polluzione (epi-d) nel monitoraggio delle acque correnti. linee guida. apat: roma. denić, lj., čađo, s., đuraković, a., novaković, b., dopuđaglišić, t., veljković, n., stojanović, z., milovanović, j., domanović, m., 2015: status of surface waters of serbia: analysis and elements for monitoring design. agencija za zaštitu životne sredine, ministarstvo poljoprivrede i zaštite životne sredine, republika srbija (in serbian). descy, j. p., coste, m., 1991: a test of methods for assessing water quality based on diatoms. verhandlungen der internationalen vereinigung für theoretische und angewandte limnologie 24, 2112–2116. hofmann, g., werum, m., lange-bertalot, h., 2013: diatomeen im süßwasser – benthos von mitteleuropa. bestimmungsfl ora kieselalgen für die ökologische praxis. koeltz scientifi c books, königstein. jakovljević, o., krizmanić, j., cvijan, m., 2014: water quality assessment of the dtd hydrosystem by diatom indices. matica srpska journal for natural sciences 127, 22–33. water quality monitoring using benthic diatoms acta bot. croat. 75 (2), 2016 205 kawecka, b., 2012: diatom diversity in streams of the tatra national park (poland) as indicator of environmental conditions. w. szafer institute of botany, polish academy of sciences, kraków. kelly, m. g., whitton, b. a., 1995: the trophic diatom index: a new index for monitoring eutrophication in rivers. journal of applied phycology 7, 433–444. krammer, k., lange-bertalot, h., 1986: bacillariophyceae. 1. teil: naviculaceae. in: ettl, h., gerloff, j., heynig, h., mollenhauer, d. 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environment and space in shaping stream diatom communities. european journal of phycology 47, 160–168. wfd cis guidance document no. 7., 2003: monitoring under the wfd, produced by working group 2.7-monitoring, european communities. wfd, 2000: water framework directive – directive of european parliament and of the council 2000/60/ec – establishing a framework for community action in the field of water policy. european union, the european parliament and council, luxembourg. wfd-uktag, 2014: phytobenthos – diatoms for assessing river and lake ecological quality (river darleq2). water framework directive – united kingdom technical advisory group (wfd-uktag), scotland. acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 55 acta bot. croat. 76 (1), 55–63, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0042 issn 0365-0588 eissn 1847-8476 genetic analysis of microsatellite markers for salt stress in two contrasting maize parental lines and their ril population ayşen yumurtaci1*, hülya sipahi2, li zhao3 1 marmara university, faculty of science and letters, department of biology, 34722, istanbul, turkey 2 sinop university, faculty of the arts and sciences, department of biology, 57000, sinop, turkey 3 the state key laboratory of plant cell and chromosome engineering, institute of genetics and developmental biology, chinese academy of sciences, beijing, china abstract – salt stress considerably hinders the growth and productivity of maize (zea mays l.). identifi cation of salt tolerant genotypes and integration of alternative molecular markers have important roles in enhancing breeding processes. in this study, 3308 maize expressed sequence tags (ests) from salt stress-related libraries were assembled to mine repetitive sequences for development of applicable markers. in this core est data, 208 simple and 18 non-simple repetitive regions were detected in 312 contigs and 1121 singletons. the di-nucleotide repeats were the most abundant type and accounted for 79.3%, followed by tri (19.7%), and tetra-nucleotide (1%). among 59 est-simple sequence repeats (ssrs), a total of 55 were screened for polymorphism between f35 (salt sensitive) and f63 (salt tolerant) parents and 48 out of 55 were detected as monomorphic. signifi cantly, seven of them (12.7%) were found to be polymorphic and were used for genotyping of 158 f5 derived recombinant inbred maize lines, and four of them were located on chromosome 1 and 3. using in silico mapping, 44 out of 59 est-ssr markers were mapped on 10 maize chromosomes. analysis of sequence homology revealed different functional groups such as: membrane transport, cell defense, cell division, signaling components, photosynthesis and cell metabolism. these est-ssrs might be used as new functional molecular markers in the diversity analysis, identifi cation of quantitative trait loci (qtls) and comparative genomic studies in maize in the future. keywords: est, maize molecular markers, salinity, ssr * corresponding author, e-mail: yumurtaciay@yahoo.com introduction soil salinization is an important external stress factor for plants except halophytes, and has proved to be a serious drawback for plant growth from seed germination to adult plant stage (singh 2015). nearly 50% of the world’s territorial areas may be under pressure from salinity in the next forty years, due to the rapid shifts that are occurring in global weather conditions (bray et al. 2000). therefore, improvement of salt tolerance in plants has gained importance because of the need to provide suffi cient food for the world’s increasing population (bita and gerats 2013, yumurtaci 2015). maize (zea mays l.) is the third most cultivated crop after wheat and provides many commercial benefi ts for clean energy production, food and livestock feed (klopfenstein et al. 2013). since detrimental declines in yield and agro-morphological traits have seen under saline conditions, maize is accepted as a salt sensitive crop (schubert et al. 2001). in this perspective, investigation of the genetic control and identifi cation of genome regions associated with salt tolerance are of great signifi cance. implementation of various dna markers and quantitative trait loci (qtl) mapping techniques have contributed to an improved knowledge of the genetic bases of agriculturally significant traits and assisted the progress of plant breeding (lee 2007, xue et al. 2010). the development and mapping of dna markers are essential for using qtl mapping of salt tolerance and marker-assisted selection of this trait in maize genotypes. maize is one of the fi rst major crop species to have had maps of different molecular markers (davis et al. 1999, sharopova et al. 2002, sibov et al. 2003, lima et al. 2009, xu et al. 2013, zhou et al. 2011). recently, research has focused on genetic dissection in maize using qtl mapping of recombinant inbred lines yumurtaci a., si̇pahi̇ h., zhao l. 56 acta bot. croat. 76 (1), 2017 (rils) derived from a cross between two maize inbred lines with contrasting salinity tolerance. a high density genetic map based on single nucleotide polymorphism (snp) markers identifi ed 20 qtls on seven maize chromosomes (cui et al. 2015). signifi cantly, three qtls had only additive effects, while 12 had both additive and additive x treatment interaction effects. these qtls were mainly clustered on maize chromosomes 1, 3 and 5. the fi ve unconditional and three conditional qtls could individually explain more than 20% of the phenotypic variation. xiang et al. (2015) identifi ed six sequence-related amplified polymorphism (srap) markers linked to salt tolerance using bulk segregant analysis of dna pools from two salt-tolerant and saltsensitive maize genotypes. compared with other types of molecular markers, simple sequence repeats (ssrs) have many advantages because of their multi-allelic nature and co-dominant inheritance, as well as simple and inexpensive developmental methodology. expressed sequence tag ssrs (est-ssrs) are derived from expressed genome parts, which are more evolutionarily conserved than non-coding sequences and therefore have transferability. also, est-ssrs have close associations with gene/qtl regions that offer a practical strategy for molecular plant breeding. thus, est mining can be accepted as a reconstruction of genome-scale analyses. many maize ests contained simple sequence repeats and could be readily converted to functional markers (lee 2007). in recent years, a number of maize est-ssrs have been developed (xu et al. 2013) and applied to construct linkage maps (sharopova et al. 2002, zhou et al. 2011, orsini et al. 2012, sa et al. 2012), qtl mapping and markerassisted breeding (orsini et al. 2012, cui et al. 2015). in addition, banisetti et al. (2012) identifi ed candidate genebased ssr markers for lysine, tryptophan pathway and opaque2 modifi ers. a total of twenty-four ssr loci were developed and found to be useful markers for fi ne mapping and high-density mapping of opaque2 modifi ers. despite an increasing number of dna markers, the identifi cation of comprehensive markers for screening of salinity specifi c regions in maize is lagging behind and needs to be further developed. in the present study, motif structures and density estimations of ssr regions were identifi ed through in silico analysis of publicly available maize est libraries which were constructed under salt stress at seedling stage. a set of primer pairs fl anking repetitive regions was developed from these analyses and validated using two contrasting parental genotypes and their recombinant inbred lines (f35-salt sensitive and f63-salt tolerant) for the genetic basis of salt tolerance. also, relative in silico map positions were defi ned and functional annotations of these ssrs were carried out to detect the functional marker source for maize. materials and methods plant material maize parental lines f63 (salt tolerant) and f35 (salt sensitive) and their segregating population of 158 f5 rils were used in this study. these materials were previously developed by cui et al. (2015) in the state key laboratory of plant cell and chromosome engineering, institute of genetics and developmental biology, chinese academy of sciences, beijing, china. ssr detection two publicly available cdna libraries, constructed by wang/bohnert lab under salinity stress in maize b73 line, were used; library zm14 from root and library zm13 from shoot tissue. this study was performed at four main computational steps. at fi rst, a total of 3308 est sequences were downloaded in fasta format from ncbi genbank (http:// www.ncbı.nlm.nıh.gov). for scanning vector contaminated sites in ests, ncbi vecscreen module and dnadragon (http://www.sequentix.de/download/dnadragon.zip) were used. after trimming of contaminated sites, the dnadragon software “sequence assemble” module was used to cluster all ests into condensed non-redundant groups. nonclustered (singletons) and clustered ests (contigs) were analyzed on etra 1.0 software, developed by bilgen et al. (2004) and karaca et al. (2005), under the following parameters: up and down fi ltering was selected as 5%, minimum and maximum motif length was 2 and 10, respectively. repeat index and repeat percentages were automatically calculated on etra 1.0 software. pcr conditions genomic dna was extracted from young leaves of each ril using cetyltrimethyl ammonium bromide (ctab) method (doyle and doyle 1987). polymerase chain reaction (pcr) was conducted in 15 μl volume containing 5 ng of genomic dna template, fi nal concentrations of 0.25 pm of both forward and reverse primers, 1.5 mm of mgcl2, 0.2 mm of dntps, 1× pcr buffer, and 0.1 u of taq polymerase enzymes. the cycling conditions for pcr were used to an initial denaturation of 2 min at 95 °c, followed by 33 cycles of 94 °c for 30 sec, annealing temperatures at 59 °c (for zm13-ai977889, zm14-contig119), 56 °c (for the remaining primers) for 30 sec, and extension at 72 °c for 30 sec. this was followed by fi nal extension of 7 min at 72 °c. pcr products were separated electrophoretically on polyacrylamide gels (12%) for detailed fragment separation and results were scored manually. a total of 55 primers were generated and used in the validation studies. data analysis and linkage map construction salt tolerant (f63) and sensitive (f35) parents were screened with newly developed markers. subsequently, polymorphic ones were also tested in a segregating population of 158 f5 rils derived from a cross between f63 and f35. chi-square analysis was performed at a signifi cance threshold of 5% in order to detect deviations from the expected mendelian segregation ratio of 1:1. linkage analysis was performed using the program qtl icimapping 3.3 (li et al. 2007, 2008). the linkage groups were created with lod threshold of 3.0. map distances (centimorgan) were calculated according to the kosambi mapping function (kosambi 1943). genetic analysis of microsatellite markers in maize acta bot. croat. 76 (1), 2017 57 chromosomal localization and annotation studies of ssrs for nucleotide-protein similarities in contig and singleton groups, blastx (http://www.ncbi.nlm.nih.gov) search was retained for the best hit match score with the following criteria; e-value<10–5 and 90% accepted as for minimum identity. est-based three hundred and twenty-six ssr contained sequences were aligned with several linkage maps of maize genomes, using e-pcr module (rotmistrovsky et al. 2004) under default stringency parameters (http://www. ncbi.nlm.nih.gov/projects/e-pcr/). an in silico consensus map was drawn on mapchart 2.0 software according to voorrips (2002). results based on the in silico analysis, 3308 ests consisting of 477119 base pairs were analyzed to fi nd ssr loci. redundancy profi les revealed 312 contigs and 1121 singletons. average lengths of contig sequences were 714 bp and 632 bp for root and shoot tissues, respectively, but these values decreased to 483bp and 373bp for singleton sequences in root and shoot tissue. among contig sequences, repeat index was 0.314 and 0.266 for shoot and root tissue, respectively. additionally, singleton sequences had lower repeat indices for shoot (0.158) and root (0.156) tissues. overall statistics for all clustered sequences indicated a repeat index as low as 0.186. here, 226 repeats, 208 simple and 18 non-simple, were detected in a total of 1433 contigs and singletons. di-nucleotide repeats were the most abundant type and accounted for 79.3%, followed by tri(19.7%), and tetra-nucleotide (1%). the vast majority of class i type dinucleotide repeats were found in shoot tissue, in the percentage of 84.9% and root tissue specifi c class i type trinucleotide repeats (30.1%) marked as a higher level than shoot (13.5%) (tab. 1). among di-nucleotide motifs, aa (18.75%) and tt (13%) had the highest frequency, while gg had the lowest frequency, 7.2%. thirty different tri-nucleotide motif repeats were identifi ed. the most frequent tri-nucleotide motif was ccg (7.3%). some tri-nucleotide repeats (ctc, ctg, ctt, tta) that are responsible for leucine synthesis were found only in root tissue-specifi c sequences. only two tetra-nucleotide motifs (ataa, acca) specifi c to shoot tissue were identifi ed. a total of 55 primer pairs were designed from 59 est-ssr containing regions (tabs. 2, 3). of the 55 tested maize est-ssrs, 48 generated amplifi cation of monomorphic products and 7 others (12.7%) produced clear and consistent polymorphic banding patterns between the parental lines f35 and f63. these seven polymorphic markers (zm14-contig119, zm13-ai964577, zm13 -ai977889, zm13-contig37, zm13-contig83, zm14-ai 855336, zm13-ai966933) were also genotyped across the 158 individuals of the ril population; four of them were assigned to two maize chromosomes. the loci zm14contig119 and zm13ai964577 were located on the same tab. 1. distribution of root and shoot tissue specifi c expressed sequence tag (est) based simple sequence repeats (ssr) and sequence tagged site (sts) marker densities and number of classi and classii type repeats. est shoot tissue root tissue number of contigs number of singletons number of contigs number of singletons total 188 647 124 474 only ssr included 46 90 25 65 only sts included 40 66 32 69 both (ssr + sts) 14 18 9 18 classi (di/tri/tetra repeats) 11/2/2 4/2/– classii (di/tri/tetra repeats) 95/15/– 54/23/– tab. 2. detailed blastx similarity matches and motifs with repeat types of both simple sequence repeats (ssr) and sequence tagged site sts contained sequences in maize shoot tissues. “p” indicates perfect repeats, “ip” imperfect repeats and “*” shows unmapped markers. zm13 is the library name. sequence id motifs/repeat type organism protein e-value primer pairs (5’-3’) product size (bp) zm13-contig13 (ta)6/ p z. mays glyceraldehyde-3dehydrogenase 2e-91 cagattattcgacgaaagaga gcatggttgatgaaacaaata 148 zm13-contig19 (gat)6/p z. mays hypothetical protein 8e-36 caaccaagtcctaaattgtca ttaagcagagctcaaaaactg 153 zm13-contig33* (ca)5,(gt)5/p(gt)3,(ta)5ip z. mays jacalin like lectin protein 3e-100 aggttcgacgagagcttgac cgttttgttcgtgctgaaga 800 zm13-contig37 (tc) 10/p z. mays phospholipid transfer protein precursor 1e-38 aataaaccaaacagccaaaa aataaaatctctccgtgtgtg 163 zm13-contig 60* (ta)6/p z. mays unknown protein 1e-11 ggaggaacagaggtttgttat actttcaaggtggtggaag 335 yumurtaci a., si̇pahi̇ h., zhao l. 58 acta bot. croat. 76 (1), 2017 tab. 2. – continued sequence id motifs/repeat type organism protein e-value primer pairs (5’-3’) product size (bp) zm13-contig63 (gcg)6/p z. mays unknown protein 3e-43 tttgatacacggaggaagata aaccagagattcatcagagtg 427 zm13-contig65 (ga)6,(gcag)4/ip z. mays hypothetical protein 5e-96 gttccttcacacagacacagt cttttacatggtggacgac 508 zm13-contig72 (gc)6/p z. mays unknown protein 3e-19 aacaactggctgcatctataa acagaagcagaaccagca 504 zm13-contig83 (ta)5/ p z. mays nonspecifi c lipid transfer protein 8e-04 gcaggccacacatacataata gaccaagctaataagcctacc 169 zm13-contig99* (ta)6/ p z. mays unknown protein 2e-29 gccatagcaaaagctcaaatcc ctcccgatcagccgactctac 482 zm13-contig111* (gag)5,(acg)4/ip z. mays chlorophyll ab binding protein 1e-107 agtatcccctttttacactgg cgtcgtcgtcgttgtt 941 zm13-contig136 (aa)6/ p z. mays putative glutation peroxidase 2e-97 catacagaaagggcgaaaca acacgcttcaaggctgagta 501 zm13-contig148 (acca)5/ p z. mays putative protein kinase 1e-18 aaatatacggccccaagaaaa caagcaagatcggtggaaac 327 zm13-contig175 (cca)7/ p-(ta)5/ p z. mays unknown protein 5e-46 atgagtacagcgtcggagt atggatgggatacaaatacaa 900 zm13-ai649785* (ca)5/ p z. mays unknown protein 9e-33 gaaacaagaaagcaggaactc gcgctacaactccaacac 552 zm13-ai649789 (tt)10/ p z. mays penta tricopeptid repeat 1e-89 gccactctgtttggtggatag cggccttgttaattaccttga 537 zm13-ai649800* (ac)7,(ca)3/ p z. mays transcription factor 5e-32 ctaagcacaaattcaagaac tagatagcctcgatcactc 406 zm13-ai964452 (ct)8,(aa)5/ p z. mays hypothetical protein 2e-37 gaagttgcagttgttgttttt taggcaaggtgttattactgg 207 zm13-ai964488 (at)5,(ta)4/ip z. mays carbonic anhydrase 4e-38 ctcaagacctaccccttcgtc actcctcgcattcacatatcg 215 zm13-ai964426 (ct)5,(ca)5/ p z. mays phospholipide transfer protein 1e-13 taaataaagaccgcatgaact aagcacttgccatctacc 399 zm13-ai964559* (ac)3,(aa)5/ip z. mays nadh ubiquinon oxidoreductase 6e-59 gagaggggtatttgctgtatt cctactctgttgtacgtggat 397 zm13-ai964577 (ct)13/ p g. max anaphase entrance complex 0.44 ccacaaaggcatcaaaagaat attgtccgccttaaccagaag 356 zm13-ai977807* (cg)5/ p z. mays hypothetical protein 7e-108 ctcatcatctcgttgtcctc gacgcacctgtacctgaa 453 zm13-ai977889 (gagaagg)4, (gg)3,(ga)5/ip t. versicolor unknown protein 8.2 cgccaatggtgagtgaaagaa gcaaacgacacctccaaccac 281 zm13-ai977895 (ta)5/ p z. mays nucleotide sugar transporter 3e-32 gctgcacatggaaacacttca acgcaggtggcgttagaaagg 463 zm13-ai977907 (cgt)5/ p z. mays hypothetical protein 3e-09 atctcgatctgcttcatcat gtaacctcaacgtcagcttg 253 zm13-ai977935 (gct)5/ p no similarity – – caaacaaaagagacatcagga cccaagcattcaaaactacta 303 zm13-ai966834 (aa)5/ p z. mays 40s ribosomal protein 2e-48 tccattttgctctgtttctt agccttcttctgccacag 285 zm13-ai966924* (tt)8/ p z. mays phosphoglycomutase 1e-35 atccgatacctcttcggaaat caaaggctcaaacaaaaccag 505 zm13-ai966933 (aata)5/p-(ggt)5/ ip,(cgg)3/ip z. mays unknown protein 2e-21 atttgagaacggaagcaagta accaccaccaccgacaag 268 zm13-ai966957* (aa)5,(ag)5/ p z. mays unknown protein 5e-30 catccaccgtgttgtgtggaa gtggttatggcagtcggcaac 281 zm13-ai967009* (gc)5/ p z. mays alcohol dehydrogenase 1e-56 tcaactgcgtgtccccctatg tggaatgttcaatcatctgttgg 400 genetic analysis of microsatellite markers in maize acta bot. croat. 76 (1), 2017 59 tab. 3. detailed blastx similarity matches and motifs with repeat types of both simple sequence repeats (ssr) and sequence tagged site sts contained sequences in maize root tissues. “p” indicates perfect repeats, “ip” imperfect repeats and “*” shows unmapped markers. zm14 is the library name. sequence id motifs/repeat type organism protein e-value primer pairs (5’-3’) product size (bp) zm14-contig13 (gt)6/ p-(gcg)6, (cg)3/ ip z. mays putative ring zinc fi nger protein 7e-38 atacatttttacgtccac gtttcgtttggagggtgtctt 214 zm14-contig24* (ca)6/ p z. mays unknown protein 3e-26 tttgtccaatcaagcgagata ttgtgtccgtgcaaattggtg 488 zm14-contig27* (ta)9,(ac)3/ ip z. mays jacalin like lectin protein 3e-93 cggtagcgaaaatacagt tccatctccttcatctacatc 504 zm14-contig53 (ccg)6/ p z. mays unknown protein 3e-140 not suitable for primer design – zm14-contig68* (at)5/ p z. mays aquaporin plasma membran protein 1e-32 aaacagggagtcttcttcttaac aaacagggagtcttcttcttaac 382 zm14-contig79 (cg)5/ p z. mays unknown protein 3e-166 not suitable for primer design – zm14-contig106* (tt)6/ p z. mays hypothetical protein 1e-48 gcccatattacatacaaaag caacaagaaggtttattctg 708 zm14-contig114 (gg)5/ p z. mays unknown protein 5e-48 tggaccaaacatcggttgagc gaatggaaggaagaggggtggt 530 zm14-contig119 (aca)5,(cca)6/ p(ccg)5,(cca)3/ip z. mays hypothetical protein 6e-39 tggccccacctgatgaaataa ggagtggaggcggaggatct 615 zm14-ai649544 (ta)3,(tgga)5/ ip z. mays oxin transporter protein 1e-81 cgatgccgaaaacccattctt gcatctgctcgtggaggaaaa 303 zm14-ai649556 (ctg)5/ p z. mays lysin decarboxylase 3e-07 not suitable for primer design – zm14-ai855154 (gag)3,(ctg)5/ ip z. mays hypothetical protein 0.002 tggtcctgctgcttctcttgc caaacggtccacctccacctc 316 zm14-ai855158 (at)5/ p z. mays ribosome biogenesis protein 1e-94 not suitable for primer design – zm14-ai855164 (at)5/ p z. mays unknown protein 4e-08 gtgcagagacggacagcgagt ttttgagttttgccggagtgg 346 zm14-ai855182 (at)6/ p z. mays glyceraldehyde-3dehydrogenase 1e-47 cgactcccaacaggaaatgga gtcgcctggtacgacaacgag 349 zm14-ai855214 (ta)5/ p z. mays unknown protein 5e-61 tggatcgaagcaactcgcact caacagcgtcaagagcgtcaa 310 zm14-ai855258 (tc)13/ p(ta)3, (cacag)6/ ip z. mays unknown protein 3e-19 gagataggcgaggaaggtgag atcgtcatcattcgagcagag 180 zm14-ai855272 (agg)6/ p z. mays putative myb binding protein 2e-43 gacctcaacctcgacctctg ggcttcgttcacttcatcttg 275 zm14-ai855286 (ctg)9/ p z. mays unknown protein 3e-34 ccctctcttttctcagcccta atccagaggacgagggtttt 288 zm14-ai855336 (cgc)5/ p z. mays hypothetical protein 2e-28 gagaagccgagaacagtagca cacgaggcagagtcgtagttt 365 zm14-ai855352 (gc)5/ p(cgc)6, (cga)3/ ip z. mays hypothetical protein 8e-11 cgagagcgccattagaagtcg ttttgtggaacgaagcgatgg 401 zm14-ai855361 (acc)9,(ta)5/ p z. mays unknown protein 2e-47 gacctggagtggtggttc gatgggatacaaatacaatac 481 zm14-ai649556 (ctg)5/ p z. mays lysin decarboxylase 3e-07 ccgggttttggactttggaga tgttgtggctctttgcctgtg 301 zm14-ai861145 (tt)5,(gtc)5/ p z. mays unknown protein 4e-56 ttgtttttgcctttccttgaa tgctgtacccaaatccttctg 447 zm14-ai857227 (gcc)6/ p z. mays putative cytochrome p450 superfamily protein 5e-43 gccgtgccaacttttaatttc ctggaggtggaaggagagg 367 zm14-ai855418 (ag)5/ p z. mays putative phototrophic-responsive nph3 family 0.082 gcctgcctgtccatcaatcaa catccacctcccacccagaac 242 zm14ai857233 (atg)6/ p z. mays protein transport protein sec31-like atctgcacctcaacctgaatg attggatggttcttgtgttgg 236 yumurtaci a., si̇pahi̇ h., zhao l. 60 acta bot. croat. 76 (1), 2017 chromo some 1, but they were located far from each other (50.58 cm). on the other hand, the loci zm13-contig37 and zm13-contig83 were located together on the other chromosome 2, with 154.58 cm. the in silico mapping of 44 est-ssr markers in comparison to maize genome assembly is given in fig. 1. all primer pairs were assigned to ten maize chromosomes by in silico mapping while only 4 polymorphic primer pairs were assigned to two chromosomes by classical genetic mapping. the putative functions of ssr contained sequences were assigned in the genbank database using similarity search of blastx (tabs. 2, 3). the data indicated that 26 (44.06%) of 59 loci have shown similarity to known genes and have a range of functions, such as metabolic enzymes, structural proteins, disease signaling and transcription factors. more than half (52.7%) of the 55 primer pairs did not yield any signifi cant annotation. discussion dna sequences retrieved from maize cdna libraries in ncbi data-base were searched for repetitive regions. the repeat percentage was found to be 1.46% under salt stress in maize cdna libraries from ncbi genbank. this frequency is similar to that previously reported by kantety et al. (2002) (1.5% percentage of maize est-ssrs). the most abundant repeats were di-nucleotide (78.8%). this result is consistent with those obtained by wang et al. (1994) in temperate maize and sibov et al. (2003) in tropical maize. the most repeated di-nucleotide motifs were (tt)31, (ct)27, (ct)32 and (ga)26, (ga)34. jayashree et al. (2006) observed (ct) and (ga) repeats to be the most common di-nucleotide motifs in cereals and legume plants. four tri-nucleotide repeats (ctc, ctg, ctt, tta) responsible for leucine synthesis were found only in root tissue specifi c sequences. the triplet codons for proline and arginine were mostly detected in contigs and singletons from root and shoot tissue. proline containing repeats were detected in zm14contig119 and zm13-contig175 sequences for root and shoot tissue respectively, while zm14-ai855272, zm14ai855352 and zm14-ai855336 sequences contained triplets for arginine synthesis. proline is a well-known osmoprotectant molecule and responsible for balancing the cell water potential in plants (hu et al. 1992). in case of decreased photosynthesis rate, proline has also functions in protein deamination to provide extra energy from proteins (mattioli et al. 2009, hayat et al. 2012). thus, it has been pointed that these ests may especially have the potential to develop markers for salt stress. although many dna markers have been developed from maize, ssr markers have not been developed specifi cally from ests constructed under salt stress condition. in this research, 55 primer pairs were specifi cally developed by using salt stress-induced maize ests. fragments with expected sizes were clearly amplifi ed in all primer pairs. molecular markers can be mapped by using classical genetic mapping or in silico mapping. in this study, many markers have not been mapped classically due to the lack of polymorphism between the parents of the mapping population. on the other hand, according to in silico mapping, 44 markers were assigned to ten maize chromosomes (fig. 1). in silico mapping position of four markers (zm14-contig119, zm13-ai964577, zm13-contig37, zm13-contig83) were matched with the conventional linkage map, in terms of both chromosome arm location and order. fig. 1. in silico relative map locations of 44 expressed sequence tagged-simple sequence repeats (est-ssrs) on haploid maize chromosomes constructed on mapchart 2.0 software and mapping unit represented as centimorgan (cm). genetic analysis of microsatellite markers in maize acta bot. croat. 76 (1), 2017 61 in classical genetic mapping analysis, segregation deviation from expected mendelian segregation ratios was detected for two markers (zm14-contig119 and zm13-ai 964577). these markers mapped to chromosomes 1 were skewed towards the f63 (tolerant) parent. deviation from the expected mendelian ratios was previously reported in maize (pereira and lee 1995, sharopova et al. 2002). segregation distortions are often due to differential gametophytic selection (lu et al. 2002), chromosomal rearrangements (lu et al. 2002). sa et al. (2012) indicated that segregation distortions might be infl uenced by the mapping population of f2, a backcross or recombinant inbred line, and by sizes of the mapping population. expansion of the knowledge of the function of ests will increase the likelihood that est based markers for salttolerance in maize molecular breeding will be identifi ed. in this study, there are many ests exhibiting sequence homology in sequence databases (tabs. 2, 3). two contig sequences (contig33, contig27) relating to maize root and shoot tissues were matched with jacalin-like plant stress proteins. xiang et al. (2011) have proved that jacalin is a core compound for plant disease resistance mechanism. in addition, two contigs (zm13-contig37 and zm13-contig83) assigned to chromosome 3 showed homology to nonspecific lipid transfer proteins (nsltp). ltps in maize (zmltps) have critical roles in resistance to biotic and abiotic stress. they provide high salinity resistance with decreasing solute permeability of cell membrane (liu et al. 2015). 63 nsltp genes identifi ed and differentially expressed under drought, salt and cold stresses were unevenly assigned to ten maize chromosomes by in silico mapping (wei and zhong 2014). six zmltps (zmltpd6, zmltpd7, zmltpd8, zmltp1.1, zmltp1.2, zmltp1.3) were placed on chromosome 3 (wei and zhong 2014). sharapova et al. (2002) used an ibm population b73xmo17 and mapped the microsatellite marker p-umc1010, as an anchor marker for phospholipid transfer protein homolog2 (plt2), on maize chromosome 3. this marker region amplifi ed a (ga) motif with ten repeats. in our study, we have identifi ed two different alternative marker regions associated to phospholipid transfer protein in maize f63xf35 rils. these marker loci (zm13-contig37 and zm13-contig83) were placed on chromosome 3 and amplifi ed the repeat regions for (tc)10 and (ta)5. in addition, zm14-contig119, which covered three different types of triple motifs (aca, ccg and cca) and was located on maize chromosome 1, matched with hypothetical proteins. in sorghum, ngara et al. (2012) observed 22 hypothetical protein inductions after salt stress application in moderately salt tolerant plants. similarly, zahra et al. (2013) observed hypothetical protein induction after salt stress application. shinozaki et al. (2005) reported that hypothetical proteins with uncharacterized domains were relevant to salt tolerance. another est-ssr tagged as zm13ai964577 showed similarity to glycine max anaphase entrance complex. however, this sequence showed a polymorphic fragment pattern, and blastx e-value resulting was out of the accepted score limits. parental genotypes and their 158 f5 derived rils which were used in our study were previously tested with 3072 snp markers (cui et al. 2015) and 81 of these snp markers clustered on chromosomes 1, 3 and 5. results suggested that some qtls were related to the traits of shoot sodium and potassium concentration in maize. in our study, we have identifi ed four est-ssr markers that were annotated to two different maize chromosomes (1 and 3). signifi cantly, annotation analysis of these est-ssrs showed a close relatedness to some salt tolerance proteins. another matched est (ai964488) displayed homology with carbonic anhydrase protein. yu et al. (2007) demonstrated that carbonic anhydrase gene was expressed in oryza under environmental stress such as salt stress. further, the root specifi c contig68 showed a similarity with aquaporin plasma membrane protein. plasma membrane proteins control osmotic pressure of the cell and they are closely correlated to specifi c transport proteins such as syp121. this protein was identifi ed as vesicle transport protein and controls potassium traffi c in plant cells (besserer et al. 2012). potassium is an essential, and the most abundant, cation in plants and it has protective effects for plants under salinity (cakmak 2005). lastly, est (ai855272) derived from root tissue displayed high similarity with the myb binding protein. genes coding myb transcription factors were fi rst discovered from maize (paz-ares et al. 1987). these proteins have various regulatory functions in gene expression mechanism under salinity (hasegawa et al. 2000). for screening of high potential parts of plant genomes, there are different types of molecular marker techniques such as the pcr based; ssr, issr (inter simple sequence repeat) and hybridization based; dart (diversity array technology) and sequencing based markers such as snps (xie et al. 2006, kalia et al. 2010). ests are attractive tools for marker development since they represent coding regions of the genome. there are a number of advantages for markers developed from est-derived sequences (davis et al. 1999). when an est marker is found to be genetically associated with a trait of interest, the corresponding mapped gene can directly affect the trait. also, genetic mapping with ests would provide a more rapid transfer of linkage information between species (cato et al. 2001). ssr markers are abundantly distributed throughout the maize genome and they are cost effective markers for screening qtls (xu et al. 2013). in this study, both advantages of ests and ssrs were merged to characterize the new marker sources for maize breeding. in terms of overall evaluation of homology analysis, the new primers that were designed from computationally extracted est-ssrs and tested in tolerant and sensitive maize genotypes might be used for scanning other maize germplasm sources. furthermore, integration of molecular validation of these ssrs may serve cost effective molecular markers for improvement of salt tolerant maize. with the improvement of feasible molecular markers, these candidate est-ssrs might have the ability to broaden the genetic base of maize. yumurtaci a., si̇pahi̇ h., zhao l. 62 acta bot. croat. 76 (1), 2017 acknowledgements the authors would like to thank professor huabang chen, state key laboratory of plant cell and chromosome engineering, institute of genetics and developmental biology, chinese academy of sciences, for his great support during experiments. this work was fi nancially supported by a scientifi c research projects (bap project no. fef-1901.14-02) sponsored by the university of sinop, turkey. references banisetti, k., agrawal, p. k., gupta, h. s., kumar, a., bhatt, j. c., 2012: identifi cation of candidate gene based ssr markers for lysine and tryptophan metabolic pathways in maize. plant breeding 131, 20–27. besserer, a., burnotte, e., bienert, g. p., chevalier, a. s., errachid, a., grefen, c., blatt, m. r., 2012: selective regulation of maize plasma membrane aquaporin traffi cking and activity by the snare syp121. plant cell 24, 3463–3481. bilgen, m., karaca, m., onus, a. n., ince, a. g., 2004: a software program combining sequence motif searches with keywords for fi nding repeats containing dna sequences. bioinformatics 20, 3379–3386. bita, c. e., gerats, t., 2013: plant tolerance to high temperature in a changing environment: scientifi c fundamentals and production of heat stress-tolerant crops. frontiers in plant science 4, 273. bray, e. a., bailey-serres, j., weretilnyk, e., 2000: responses to abiotic stresses. chapter 22. in: gruissem w., buchannan b., jones r. 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surface that is immersed in seawater becomes rapidly covered with an unavoidable biofi lm. such biofi lm formation, also known as fouling, is a complex multistage process and not yet thoroughly investigated. in this study, the succession of diatoms and bacteria was investigated during a one month exposure on an artifi cial substrate of plexiglass (polymer of methyl methacrylate) mounted above the seafl oor at a depth of 5 m. for biofi lm analyses, the fouling was investigated using selective agar plates, epifl uorescence, light and electronic microscopy, as well as high performance liquid chromatography (hplc) pigment analysis. during biofi lm development, the abundance of all biofi lm components increased and reached maximum values after a one month exposure. in the bacterial community, heterotrophic marine bacteria were dominant and reached 1.96 ± 0.79 × 104 colony forming units (cfu) cm–2. despite the fact that faecal coliforms and intestinal enterococci were detected in the water column, faecal coliforms were not detected in the biofi lm and intestinal enterococci appeared after one month of exposure but in the negligible number of 60 ± 10 cfu cm–2. the phototrophic component of the biofi lm was dominated by diatoms and reached a concentration of 6.10 × 105 cells cm–2, which was supported by pigment analysis with fucoxanthin as dominant pigment in a concentration up to 110 ng cm–2. the diatom community was dominated by cylindrotheca closterium and other pennate benthic diatoms. a detailed taxonomic analysis by electronic microscopy revealed 30 different taxa of diatoms. the study confi rmed that a plexiglass surface in a marine environment is susceptible to biofouling within 30 days of contact. furthermore, the co lonization process sequence fi rstly involved bacteria and cyanobacteria, and secondly diatoms, which together formed a primary biofi lm in the sea. keywords: bacteria, biofi lm, biofouling, diatoms, succession * corresponding author, e-mail: mmejdandzic@gmail.com mejdandžić m., ivanković t., pfannkuchen m., godrijan j., marić pfannkuchen d., et al. 408 acta bot. croat. 74 (2), 2015 introduction a biofi lm is an assemblage of adhered cells and their products on a surface, a ‘coating’ or ‘covering’ composed of organisms like bacteria, protozoa, algae and invertebrate animals (o’toole et al., 2000, stoodley et al., 2002). as a general rule, biofi lm growth can be explained in several phases: (i) adsorption – binding of dissolved chemical compounds, macromolecules such as glycoproteins, polysaccharides and proteoglycans, in the fi rst moments of contact with any surface immersed in the seawater, (ii) immobilization – reversible binding of bacterial cells with weak links and interactions on the surface of the substrate, (iii) consolidation – irreversible binding of bacterial cells on the surface of the substrate in which bacterial cells begin to secrete extracellular polymeric substances (eps), which creates a permanent bond between cells and surfaces, (iv) settling – the fi nal stage in the development of biofi lms, when other micro-organisms such as unicellular algae inhabit bacterial colonies and biofi lm takes a three-dimensional structure. colonization of microorganisms on the solid surfaces is a worldwide problem: from offshore oil platforms and bridges that can collapse due to biofouling, through freighters, cruisers and naval vessels to fi shing facilities and a variety of fi shing gear. the biggest problem of fouling on ships is the possibility of corrosion of the stern, which increases ships fuel consumption up to 30% (de rincon et al. 2001). consequently, there is an increased release of greenhouse gases contributing to the major environmental problem of our time. because of fouling on aquaculture installations for fi sh, shellfi sh and other organisms their cultivation is faced with environmental problems such as anoxia, eutrophication, increased turbidity, and all of these can lead to plague organisms and major economic losses (lewis et al. 1997). settling allows for the colonization of multicellular organisms such as larvae, invertebrates, multicellular fi lamentous algae and other macro-invertebrates (lehaitre and compère 2007). periphyton includes all plant and animal organisms attached to various types of substrates that are submerged in water, and that do not penetrate the surface and is divided into ‘euperiphyon’ (basic part of the periphyton, formed by attached organisms adapted to a sessile lifestyle) and ‘pseudoperiphyton’ (part that is associated with the periphyton formed by communities of organisms that move freely among attached species, depending on them as a source of food and protection from predators and planktonic organisms caught and retained in a dense network of organic matter). investigations of biofi lm formation in the marine environment are scarce. it is known that colonization of artifi cial differs from that of natural substrates (hamilton and duthie 1984, sabater et al. 1998). periphyton communities in the northern adriatic consist mainly of diatoms with a distinct seasonal variation. the highest abundance and biomass were observed in the period between february and october (557.156 ± 82.602 cells cm–2), while in the period between january and february abundance and biomass was much lower (365 ± 407 cells cm–2) (totti et al. 2007). also, no signifi cant difference in the periphyton community structure and composition were observed in different artifi cial substrates. investigation of periphyton development on artifi cial substrates in the highly stratifi ed estuary of the karstic zrmanja river (middle adriatic) (caput et al. 2008), showed high diatom abundance after a two-week exposure (2.3 × 107 cells cm–2) with species richness 41, while after 4 weeks, abundance doubled and richness increased to 50. periphyton was composed mostly of amphora coffeaeformis and navicula veneta after 2 weeks; while after 4 weeks, melosira moniliformis was co-dominant. on the other hand, romagnoli et al. (2007) described the diatom communities associated with eudendrium racemosum as a natural substrate colonization of diatoms and bacteria in the ne adriatic acta bot. croat. 74 (2), 2015 409 throughout an annual cycle in the ligurian sea. diatom abundance values ranged from 46.752 ± 24.684 cells mm–2 in february 2003 to 917 ± 331 cells mm–2 in october 2003, while biomass ranged from 1.94 ± 1.94 to 0.013 ± 0.003 μg c mm–2 for the same periods. in contrast, fi lamentous cyanobacteria appeared with high densities between late spring and summer with maximum abundance of 29.872 ± 4.482 cells mm–2 and biomass of 0.32 ± 0.18 μg c mm–2. on average, motile diatoms represented the most abundant fraction of diatom communities (73%), followed by adnate (17%), erect (7%) and tube-dwelling growth forms (3%). considering biomass, motile diatoms represented 48% of the total biomass value, followed by erect (25%), adnate (18%) and tube-dwelling diatoms (9%) (romagnoli et al. 2007). the aim of this study was to investigate the succession and settling of benthic microalgae in the process of formation of primary biofi lm in the northeast adriatic sea. an additional importance of this research is the interdisciplinary approach in which various methods were combined to obtain a better insight into the biofi lm formation. thus, the objectives of this study were to (i) determine the quantitative and qualitative composition of diatoms and bacteria on artifi cial plexiglass plates, (ii) to demonstrate their distribution through the investigated time of 30 days and (iii) to provide insight in their probable mutual infl uence on the growth and development of the biofi lm. materials and methods sampling periphyton sampling was carried out in the bay val de lesso in the city of rovinj (45.060n, 13.3748e) in the period from september 9th to october 7th 2013. the bay is under moderate anthropogenic infl uence due to the surrounding inhabited area. it is shallow (maximum depth up to 10 m) and the seabed is covered by seagrass cymodocea nodosa. the late summer/early autumn was chosen for the exposure period according to hillebrand and sommer (1997) due to the appropriate temperature for the endorsement of speed of settlement on artifi cial substrates. continuous weather measurements were conducted at a meteorological station located at sv. ivan na pučini, rovinj, western istrian coast (45.04746n, 13.62167e). the data were supplied by the croatian meteorological and hydrological service (tab. 1). all data regarding abundance of bacteria and diatoms were statistically analyzed by software statistica 7.0 and 2007 microsoft offi ce suite service pack 3. plexiglass was used as a substrate for biofi lm formation following the recommendation of slàdečkovà (1962a) because of its convenience compared to a natural substrate. dimensions of plates were 70 × 20 × 2 mm (l/w/h) for bacteriological sampling and 100 × 70 × 2 mm (l/w/h) for algological sampling. the plates were set in a steel structure with plastic rails (fig. 1a, 1b). in an effort to refi ne the terminology slàdečkovà (1962a) discovered periphyton exclusive of epiphytic and epizoic forms. periphyton was subdivided into ‘euperiphyon’, immobile organisms attached to the substrate by means of rhizoides, gelatinous stalks, or other holdfast mechanisms, and ‘pseudoperiphyton’ forms that are free-living, mobile, or creeping among or within the euperiphyton. the plates were set vertically in order to reduce the accumulation of sludge and biomass of ‘pseudoperiphytic’ diatoms (slàdečkovà 1962a). the construction was set in the sea at a depth of 5 m in the meadow of seagrass cymodocea nodosa and raised 30 cm above the sediment. the depth of fi ve mejdandžić m., ivanković t., pfannkuchen m., godrijan j., marić pfannkuchen d., et al. 410 acta bot. croat. 74 (2), 2015 meters ensures enough light for biofi lm development, while benthic impacts of waves and tides do not interfere with the succession. two large and two small plexiglass plates were placed into the construction for each sample with a total of 24. plexiglas plates were previously disinfected with 70% ethanol and stored in sterile containers. the collected material for diatom analyses was preserved in 4% formaldehyde. bacteriological analysis for bacteriological analysis, plates were removed from the structure after 1 h, 12 h, 24 h, one week and one month of contact. plates were gently washed with 100 ml of sterile saline (0.8% nacl) and immersed in schott bottles containing 150 ml of sterile saline solution so that the entire surface of the plate was immersed in liquid. biofi lm was dispersed using an ultrasonic probe (40 w, 120 one second cycles). after treatment with ultrasound tab. 1. hydrographic parameters of sampling events at bay val de lesso, rovinj in the period from september 9th to october 7th 2013, avg. – average value, wind strength is shown in values according to beaufort scale (0–12). parameters / date 9-sept 10-sept 11-sept 13-sept 16-sept 7-oct avg. daily temperature (°c) 24.2 23.1 18.3 17.3 18.6 14.2 avg. sea temperature (°c) 22.20 22.00 22.00 22.20 22.00 19.90 daily pressure (hpa) 1014.4 1013.3 1011.3 1016.9 1003.7 1020.9 precipitation (mm) 0.3 0.0 0.2 0.5 48.9 20.8 wind direction sse se se ese ne ene wind strength 4 1 1 1 3 1 fig. 1. construction with plexiglass plates placed in meadow of cymodocea nodosa seagrass (a), plexiglass plates placed in steel structure with plastic rails (b). colonization of diatoms and bacteria in the ne adriatic acta bot. croat. 74 (2), 2015 411 supernatant, samples were collected and inoculated on the appropriate culture medium in duplicates. dispersed biofi lm for marine heterotrophic bacteria analysis was decimally diluted, while for coliform and enterococci analysis the samples were fi ltered through a nitrocellulose fi lter (0.2 μm) and then inoculated onto selective agar plates in duplicates. the number of heterotrophic marine bacteria was determined on marine agar, difco, usa, (25 °c, 72 h). the number of faecal coliforms was determined on m-fc agar, biolife, italy (44.5 °c, 24 h). the number of intestinal enterococci was determined on slanetz-bartley agar, biolife, italy (35 °c, 72 h). diatom and phytoplankton analysis for algological analysis, plates were sampled after 1 h, 12 h, 24 h, 48 h, 96 h, one week and one month and placed in sterile bags with care that the biofi lm was not damaged. fouling was scraped off the inner and outer side of the plates using a brush and dispersed in a known volume of 2% formaldehyde solution. cell counts were obtained by the inverted microscope zeiss axiovert 200 using a previously described method (utermöhl 1958). subsamples of 10 ml were analysed microscopically after sedimentation for 24 h. microplankton (micro) cells (longer than 20 μm) were counted under a magnifi cation of 400× (1–2 transects), as well as 200× and 100× (transects along the rest of the counting chamber base plate). the samples were acid cleaned (hcl/h2so4) prior to qualitative analysis using scanning electron microscope (sem) philips 515. for direct plate analyses plexiglass plates were pre-dried at room temperature. fouling on such prepared plates was observed by sem philips 515 and, after being dyed with dapi, by epifl uorescence microscopy (zeiss axio imager z1). to get insight into the natural periphytic community, leaves of seagrass cymodocea nodosa were sampled and phytoplankton samples also, to establish stabile surrounding phytoplankton assemblages. leaves of seagrass c. nodosa were sampled on the fi rst day of the experiment and the periphytic community was acid cleaned (hcl/h2so4) and examined by sem philips 515. for analysis of the surrounding phytoplankton community, samples were taken at a depth of 5 m by 5 l niskin bottle after 1 h, 24 h, 48 h, 96 h, one week and one month. samples of 50 ml were sedimented for 24 h and analysed following the utermöhl method (1958) with an inverted microscope (zeiss axiovert 200). pigment analysis in order to gather information about the chemotaxonomic composition of the periphytic community, pigments were analyzed using high performance liquid chromatography (hplc). re-suspended samples were fi ltered on 0.7 μm pore size whatman glass fibre filters (gf/f) and preserved in liquid nitrogen until the analysis. the extraction in 4 ml of cold 90% acetone was performed by sonication, and the extract was clarifi ed by centrifugation. pigments were separated by reversed phase hplc following the protocol of barlow et al. (1997). extracts were mixed 1:1 (v/v) with 1 m ammonium acetate and injected into an hplc system equipped with 3 mm thermo hypersil column mos2 (c-8, 120 a pore size, 150 × 4.6 mm) (thermo hypersil-keystone). pigments were separated at a fl ow rate of 1 ml min–1 using a linear gradient program with a duration of 40 min. solvent a consisted of 70:30 (v/v) methanol: 1 m ammonium acetate and solvent b was 100% methanol. chlorophyll and carotenoids were detected by absorbance at 440 nm (spectra system, model uv mejdandžić m., ivanković t., pfannkuchen m., godrijan j., marić pfannkuchen d., et al. 412 acta bot. croat. 74 (2), 2015 2000). qualitative and quantitative analyses of individual pigments were performed by external standard calibration using authentic pigment standards (vki, denmark). results during sampling, weather conditions varied from day to day, which was refl ected in the structure of the water column and consequently the structure of the periphytic community and biofi lm formation. after 30 days of exposure, a slimy mucous biofi lm was present on plexiglass plates. the main living constituents of the biofi lm were attached cells of bacteria, cyanobacteria, diatoms, dinofl agellates, green (ulvophyceae) and brown (phaeophyceae) algae. bacteria heterotrophic marine bacteria reached the number of 1.96 ± 0.79 × 104 cfu cm–2 during one month of exposure (fig. 2a). despite the fact that faecal coliforms and intestinal enterococci were present in the water column at the beginning of the experiment (faecal fig. 2. abundance (expressed as logarithmic value) of heterotrophic marine bacteria through exposure time from 1 h to one month (a); abundance (expressed as logarithmic value) of periphytic diatoms through exposition time from 12 h to one month (b); concentration of pigments fucoxanthin and chl a through exposure time from 24 h to one month (c). colonization of diatoms and bacteria in the ne adriatic acta bot. croat. 74 (2), 2015 413 coliforms 255 ± 78 cfu l–1, intestinal enterococci 180 ± 28 cfu l–1) no coliforms were detected in the biofi lm during the entire experiment. enterococci were present after one month of exposure in a negligible number (60 ± 10 cfu cm–2). diatoms periphytic diatoms also showed a steady increase in abundance during the month of exposure of the plexiglass plates (fig. 2b). the absolute maximum value of abundance of periphytic diatoms was 6.10 × 105 cells cm–2 at 30 days of exposure. the pioneer species cylindroteca closterium and nitzschia longissima were determined and they re-occurred with planktonic species dactyliosolen fragilissimus, diploneis bombus, proboscia alata and group pseudo-nitzschia pseudodelicatissima »sensu lato« (tab. 2). these were followed by thalassionema nitzschioides, leptocylindrus danicus, microtabella interrupta, pleurosigma angulatum, licmophora sp. and melosira nummuloides. the species with the greatest abundance was cylindroteca closterium (5.5 × 104 cells cm–2). a total of 30 diatom taxa were determined (tab. 3) in the periphyton assemblage from the plexiglass plates and the dominant taxa were: navicula sp. (71%), thalassiosira sp. (71%), amphora ovalis (71%), amphora sp. (43%), licmophora sp. (43%), mastogloia sp. (43%), proschkinia bulnheimii (43%), thalassionema nitzschioides (43%), cyclotella sp. (43%). the qualitative analysis of stable diatom community on cymodocea nodosa revealed a total of 10 species of which amphora ovalis, cyclophora sp., navicula sp. and proschkinia bulnheimii were most frequent. three taxa were recorded only by analyzing leaves of cymodocea nodosa: achnanthes sp., cyclophora sp. and microtabella interrupta. tab. 2. list of all diatom taxa present in biofi lm at different exposure times during research period as well as their abundance. taxa / exposure time abundance (cells cm–2) 1 h 12 h 24 h 48 h 96 h 1 week 1 month dactyliosolen fragilissimus (bergon) hasle 2 cylindroteca closterium (ehrenberg) reimann & j. c. lewin 16 61 114 151 7234 55764 diploneis bombus ehrenberg 2 leptocylindrus danicus cleve 26 licmophora sp. 1426 37176 melosira nummuloides c. agardh 703 microtabella interrupta (ehrenberg) round 201 88 nitzschia longissima (brébisson) ralfs 16 25 23 pleurosigma angulatum (quekett) w. smith 2 proboscia alata (brightwell) sundström 65 23 25 pseudo-nitzschia pseudodelicatissima »sensu lato« 194 163 137 182 thalassionema nitzschioides (grunow) mereschkowsky 25 82 524 780 251 unindentifi ed pennate diatoms 307 53 225 2234 4909 25722 515814 mejdandžić m., ivanković t., pfannkuchen m., godrijan j., marić pfannkuchen d., et al. 414 acta bot. croat. 74 (2), 2015 tab. 3. list of all diatom taxa analyzed with sem at different exposure times, c – diatom taxa found on leaves of seagrass cymodocea nodosa, fr. – occurrence frequency of taxa (diatom taxa found on c. nodosa were excluded), 0* – zero frequency for taxa found only on leaves of c. nodosa. taxa (30 in total) exposure time c fr. (%)1 h 12 h 24 h 48 h 96 h 1 week 1 month achnanthes sp. + 0* amphora coffeaeformis cleve + + + + + 58 amphora ovalis (kützing) kützing + + + + + + 71 amphora sp. + + + + 43 bacteriastrum sp. + 14 cocconeis sp. + + + 29 cyclophora sp. + 0* cyclophora tenuis castracane + 14 cyclotella sp. + + + 43 cylindrotheca closterium (ehrenberg) reimann & j. c. lewin + + 29 diploneis bombus ehrenberg + 14 diploneis sp. + + + 43 fallacia sp. + 14 gomphonema sp. + 14 grammatophora marina (lyngbye) kützing + 14 licmophora fl abellata (grev.) c. agardh + 14 licmophora sp. + + + 43 mastogloia sp. + + + + 43 mastogloia undulata grunow + 14 microtabella interrupta (ehrenberg) round + 0* navicula sp. + + + + + 71 nitzchia sp. + + + 29 paralia sulcata (ehrenberg) cleve + 14 pleurosigma angulatum (quekett) w. smith + 14 proschkinia bulnheimii (grunow) karayeva + + + + 43 psammodictyon mediterraneum (hustedt) d. g. mann + + + 29 rhizosolenia sp. + 14 thalassionema nitzschioides (grunow) mereschkowsky + + + 43 thalassiosira sp. + + + + + 71 triceratium sp. + 14 colonization of diatoms and bacteria in the ne adriatic acta bot. croat. 74 (2), 2015 415 the surrounding phytoplankton assemblage was composed of 24 dominant taxa of bacillariophyceae, six dominant taxa in the group dinophyceae and the groups cryptophyceae and chlorophyceae. the composition of phytoplankton throughout the exposure period was relatively consistent with the dominant group pseudo-nitzschia pseudodelicatissima »sensu lato« whose greatest abundance was 10.2 × 104 cells l–1. biofi lm pigments hplc pigment analysis revealed 10 different pigments belonging to different groups: peridinin, diadinoxanthin, butinin, chlorophyll a (chl a), chlorophyll c (chl c), fucoxanthin, hexanoiloxifucoxanthin, zeaxanthin, β-carotene and prasinoxanthin, fucoxanthin (concentration up to 110 ng cm–2) and chl a (concentration up to 135 ng cm–2) being the dominant pigments (fig. 2c). during this study chl c, prasinoxanthin and zeaxanthin were detected after 96 h of exposure. chl a, fucoxanthin and peridinin were dominant from beginning. also, the concentration of fucoxanthin was signifi cantly lower than the concentration of chl a in the fi rst four days of exposure, and then increasingly reached similar concentrations (fucoxanthin 108.272 ng cm–2, chl a 132.424 ng cm–2) (fig. 2c). direct biofi lm visualisation during the colonization of the plexiglass plates and biofi lm generation some parts of the surface remained uninhabited, while the others developed a comprehensive three-dimensional biofi lm (figs. 3, 4). biofi lm thickness was not homogeneous in all parts of the substrate and varied from a few μm to a few cm depending on the state of succession on the plates. discussion research of periphytic algae on natural and artifi cial substrates is faced with an array of challenges: quantifying the substrate surface as well as the diversity and inaccuracies of the techniques of removing fouling makes it diffi cult to compare published results (barbiero 2000). the best practice for studying processes of migration, colonization and growth is: (i) carefully to choose the artifi cial substrate, its material surface, texture and size (cattaneo and amireault 1992), (ii) to adjust for easy handling (weitzel et al. 1979), (iii) to determine the appropriate location and duration of exposure of the substrate (iv) and to adjust material for the selective attachment of benthic fl ora (snoeijs 1991). in our study plexiglass was selected as artifi cial substrate which facilitated removal and the measurement of surface fouling, allowed easier handling and reduced the possibility of settling of organisms that are not of interest for the development of primary biofi lms. moreover, we confi rmed that the plexiglass surface in a marine environment is susceptible to biofouling within 30 days of contact. however, the dominating factor involved in the initial attachment of a bacterial cell to a surface has remained elusive.today it is thought that a multitude of factors are involved in processes of settling, including surface conditioning, mass transport, surface charge, hydrophobicity, surface roughness and surface micro-topography (palmer et al. 2007). our fi nding that faecal coliforms and intestinal enterococci were detected in the water column, but not in a biofi lm may be explained with chemical composition, surface roughness and micro-topography. coliforms are known to inhabit mixed-population biomejdandžić m., ivanković t., pfannkuchen m., godrijan j., marić pfannkuchen d., et al. 416 acta bot. croat. 74 (2), 2015 fi lms in water distribution systems (camper et al. 1996). in laboratory conditions the persistence of coliforms in mixed biofi lms on polycarbonate surfaces was highly dependent on the growth rate of the inoculum and type of substratum. signifi cantly higher numbers of both heterotrophs and coliforms were found on steel (reactive surface) than on polycarbonate (inert surface) (camper et al. 1996). bacteria (including coliforms) that initially successfully colonized the surface can readily acclimatize to low-nutrient conditions (seawater) (novitsy and morita 1978, kurath and morita 1983, camper et al. 1996). thus, the absence of faecal coliforms and intestinal enterococci in the biofi lm in our experiment was likely due to the choice of substratum; the mentioned species were unable to initially attach to inert surface of plexiglass and continue their growth in the biofi lm. an important factor during the fi rst week of succession is the strength and direction of the wind because it contributes to the mixing of the water column. the occurrence of plankfig. 3. direct plate analysis under epifl uorescence microscope through the exposure time from 1 h to 1 month; b – bacterial cells; cb – cyanobacterial chains; cc – cells of cylindrotheca closterium; ma – macro-aggregate. bars = 10 μm. colonization of diatoms and bacteria in the ne adriatic acta bot. croat. 74 (2), 2015 417 fig. 4. direct plate analysis under scanning electron microscope through the exposure time from 1 h to 1 month; b – bacterial cells; bc – cells of bacteriastrum sp.; cc – cells of cylindrotheca closterium; eps – extracellular polymer substances produced by bacterial and diatom cells; pb – cells of proschkinia bulnheimii; pd – unidentifi ed pennate diatoms; pl – cells of pleurosigma sp. mejdandžić m., ivanković t., pfannkuchen m., godrijan j., marić pfannkuchen d., et al. 418 acta bot. croat. 74 (2), 2015 tonic diatoms among the pioneer species on the plates might be explained by these environmental conditions. in this study we recorded dactyliosolen fragilissimus, proboscia alata, thalassionema nitzschioides, and leptocylindrus danicus, all common planktonic diatoms of the north-eastern adriatic sea (godrijan et al. 2013). records of frustules of planktonic taxa found in a dense biofi lm can be explained by their natural settling and entanglement after death. frustules were caught and retained in the dense organic network of the biofi lm. the composition of phytoplankton throughout the exposure period was relatively consistent with the dominant group being pseudo-nitzschia pseudodelicatissima »sensu lato« whose greatest abundance was observed after a month of exposure (10.2 × 104 cells l–1). the group p. pseudodelicatissima »sensu lato« is positively correlated with temperature in the northern adriatic, as was the case in the western mediterranean, where higher abundances were recorded in the warmer part of the year, for example, in the late summer and autumn (ljubešić et al. 2011, marić et al. 2012). as succession progressed, planktonic species were replaced by benthic diatoms the abundance of which increased exponentially. the taxonomic composition and abundance of periphytic diatoms in this study confi rms previous research done in the northern adriatic (totti et al. 2007, caput et al. 2008). according to denicola and mcintyre (1990), diatoms of the genus licmophora, cocconeis and achnantes are among the most prominent colonizers after the fi rst week of exposure. they are accompanied by other motile pennate diatoms which are competitors for light and nutrients such as those from the genus amphora. as confi rmed by a quantitative analysis of the biofi lm, the qualitative analysis shows that in the initial formation of biofi lms, from the fi rst to the fourth day of immigration, the most important factor is planktonic species (stevenson 1986), while later (from the 7th to the 30th day) there is a signifi cant development of benthic diatoms, which are attached with the entire surface of the valve (e. g. genera cocconeis and amphora) and eps production in the form of stands (licmophora sp.), apical plate, mucilage plates and cell membranes (hoagland et al. 1993). additionally munda (2005) in her study near piran (gulf of trieste) describes primary colonizers as achnanthes and licmophora species which form dense epilithic populations due to their attachment by stalks. also noteworthy is the centric diatom paralia sulcata, which is regarded as an indicator species of coastal upwelling situations and is common in plankton and the benthos under low light conditions, which are an advantage to it (munda 2005). in our study p. sulcata was recorded at fi rst sampling event when the water column was mostly under wind pressure resulting in mixed conditions. our study correlates with previous fi ndings (caput et al. 2008) and the abundances of amphora coffeaeformis and navicula veneta that dominated (f ≥ 83%, abundance > 106 cells cm–2) in the zrmanja estuary. in our study, in addition to these two species, nitzschia longissima (f = 80%, abundance 1.5 × 106 cells cm–2) was co-dominant. the genera cyclophora, achnanthes and species microtabella interrupta were not recorded in the biofi lm on the plates at any sampling event. this could be explained by hypothesizing that they settle later during the biofi lm formation or rather that they prefer natural substrates such as seagrass. according to romagnoli et al. (2007) who compared microalgae grown on artifi cial substrata and those that settled on e. racemosum, on average diatom abundance values were signifi cantly lower on artifi cial substrata (2.521 ± 1.784 cells mm–2) than on the hydroid (4.737 ± 3.919 cells mm–2, p < 0.01). the same pattern was observed for diatom biomass, which showed signifi cantly lower values on mimic substrata colonization of diatoms and bacteria in the ne adriatic acta bot. croat. 74 (2), 2015 419 (0.033 ± 0.027 μg c mm–2) than on e. racemosum (0.093 ± 0.088 μg c mm–2, p < 0.005). many studies have shown that macrophytes generally serve as a source of nutrients for periphyton, which makes plexiglass a relatively poor substrate (cattaneo and kalff 1979, carignan and kalff 1982, burkholder and wetzel 1990, cattaneo and amireault 1992). the chemotaxonomic analysis of the biofi lm provided an excellent confi rmation method for our microscopic results. chl c, prasinoxanthin and zeaxanthin were detected after 96 h of exposition which nicely correlated with the appearance of cyanobacteria on the plates after the fi rst week. the presence of chl a and fucoxanthin from the beginning revealed diatoms as primary colonizers. descriptive methods of direct plate visualisation with epifl uorescence and electron microscopy in this study gave a better insight into the sequence of colonization on artifi cial substrates and into the three-dimensional structure of this early biofouling stages. so far, these methods have not been used in research and the data obtained in this study can serve as starting point for the development of such methods in the future. during the fi rst hours of contact with seawater we could observe the deposition of a thin fi lm on the substrate. subsequently, we confi rmed that the bacteria are the fi rst colonizers in the process of immobilization (reversible binding), where they fi rst examine the substrate and check for the availability of nutrients, which is followed by the process of consolidation (irreversible binding) when they begin to exude eps (lehaitre and compère 2007). on this sticky mixture of proteins, proteoglycans and carbohydrate diatoms start to attach, and they tend to form permanently adhesive complex structures such as pads, stalks, capsules and tubes. even though we observed a distinct succession pattern in the biofi lm formation, the rather two-dimensional structure of the observed biofi lm seems to indicate, that colonization is a rather individual process of direct interaction between the cells and the substrate. however the maturation of the supposedly organic but abiotic thin fi lm covering the substrate surface might explain the observed succession pattern. also, differences in a presumed time lag of growth induction after attachment to a surface might contribute to the succession of species observed during the biofi lm formation. in conclusion, this study confi rmed that plexiglass surfaces in a marine environment are susceptible to biofouling within 30 days of contact. abundance of bacterial cells and diatom cells increased through the investigated period and they interacted, creating an optimized micro-environment. with hplc analysis this study confi rmed a temporal distribution pattern during the process of biofi lm formation: fi rst colonizers are bacteria and cyanobacteria, and second are diatoms, which together form a primary biofi lm in the sea. in natural biofi lms, niche formation might, in addition to other factors, explain the success and distribution pattern of certain diatoms and associated bacteria. they can support each other by equilibrium of the cross-feeding, possibly optimized by exchange of chemical factors. these associations can be specifi c or random. it is likely that cross-feeding partners may change due to various factors such as light, temperature, water currents etc., or presence of other microorganisms and their secretions. further, these interactions appear to initialize the formation of diatom biofi lms and aggregates, as has been shown with marine microbial communities. however, we were able to demonstrate a certain spatial independence between bacterial and diatom early settlers, which indicates the importance of abiotic components in the early biofi lm formation. mejdandžić m., ivanković t., pfannkuchen m., godrijan j., marić pfannkuchen d., et al. 420 acta bot. croat. 74 (2), 2015 acknowledgments this work was in part supported by croatian science foundation under the project 6433 and under projects of ministry of science, education and sports of the republic of croatia no. 119-1191189-1228, 119-1191155-1203 and 098-0982705-2731. the authors are grateful to paolo paliaga for his help with epifl uorescence microscopy. references barbiero, r. p., 2000: a multi-lake comparison of epilithic diatom communities on natural and artifi cial substrates. hydrobiologia 438, 157–170. brotas, v., plante-cuny, m-r., 2003: the use of hplc pigment analysis to study 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(ed.), methods and measurements of periphyton communities: a review, american society for testing and materials. 138 acta bot. croat. 76 (2), 2017 acta bot. croat. 76 (2), 138–145, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0005 issn 0365-0588 eissn 1847-8476 anatomical and micromorphological properties of some tanacetum l. (asteraceae) taxa from turkey and their systematic implications saban dere1, tulay aytas akcin2* 1 ersan sancı anatolian high school, ladik, samsun, turkey 2 ondokuz mayıs university, faculty of arts and science, department of biology, samsun, turkey abstract – in this study, the anatomy and trichome micromorphology of tanacetum macrophyllum (waldst.& kit.) schultz, t. parthenium (l.) schultz, t. poteriifolium (ledeb.) grierson and t. vulgare l. were examined by light microscopy and scanning electron microscopy. some anatomical characters such as presence of secretory cavities and pith in root and mesophyll type in leaf provide information of taxonomical significance. in addition, the existence of a parenchymatic layer, which consists of elongated parenchymatic cells in the stem of t. macrophyllum, is a distinguishing character. the results obtained from scanning electron microscope studies showed that trichome micromorphology varies among examined taxa. in t. macrophyllum, the eglandular and glandular trichomes especially on disc florets, ligulate florets and cypselas are more sparse, whereas disc florets and cypselas of the other taxa are covered with abundant glandular trichomes. additionally, t. poteriifolium and t. parthenium has a distinct distribution of glandular trichomes forming in a row across the entire cypsela surface. keywords: asteraceae, anatomy, scanning electron microscopy, tanacetum, trichome micromorphology * corresponding author, e-mail: aytasakcin@hotmail.com introduction tanacetum l. is a large genus in the asteraceae (compositae) family containing 150–200 species (brown et al. 1999). in the flora of turkey, the genus tanacetum is represented by nearly 60 taxa (fahn 1979, güner et al. 2000). tanacetum species have been used in standard and traditional medicines since medieval times (holetz et al. 2002, stevović et al. 2010). the essential oils and extracts of members of the tanacetum genus exhibit anti-inflammatory (brown et al. 1997), antibacterial (stefanović et al. 1988), antifungal (hethelyi et al. 1991, neszmelyi et al. 1992), insecticidal (hough-golstein and hahn 1992), antioxidant (bandoniene et al. 2000, mantle et al. 2000), antimalarial (jansen 2006) and vasorelaxing effects (lahlou et al. 2008). it has also been reported that the essential oil in tansy (t. vulgare) helps as an indicator of plant adaptation to environmental stress conditions such as drought, high temperature, intense radiation and heavy metal content (abu-darwish and abu-dieyeh 2009, stevović et al. 2009). there are a number of biochemical studies on tanacetum (williams et al. 1999). it has been indicated that sesquiterpene lactones are a predominant compounds in this genus (marles et al. 1995). some morphological and anatomical studies of the asteraceae family have been carried out. metcalfe and chalk (1950) reported that due to the diversity of their habitats, asteraceae species show various anatomical differentiations, especially in the structures of leaves. the effects of environmental factors on morphological and anatomical structure of tansy (t. vulgare) have also been determined. fahn (1979) observed that secretory structures have an important diagnostic value in asteraceae. these secretory structures such as ducts, cavities, idioblasts, laticifers, hydathodes, extrafloral nectaries and trichomes presented diagnosis value at genus level (castro et al. 1997). it has been suggested that trichome micromorphology is useful in the systematics of asteraceae (ciccarelli et al. 2007). napp-zinn and eble (1978) carried out a detailed ultrastructure study of the glandular and non-glandular hairs of twenty genera of the anthemidae. glandular hairs in asteraceae may be widely distributed both on the vegetative and the floral parts (ciccarelli et al. 2007). in several species of the asteraceae, these trichomes have been used to anatomy and micromorphology of some tanacetum taxa acta bot. croat. 76 (2), 2017 139 elucidate the biosynthesis of terpenes (neszmelyi et al. 1992, maes et al. 2011, majdi et al. 2011, 2013). micromorphological characters of cypsela have been used as discriminative characters to delimit the various taxa in the family asteraceae (zhu et al. 2006, abid and qaiser 2008a, b, akcin and akcin 2014). cypselar external micromorphology and surface sculpturing in members of asteraceae may provide useful data for the delimitation of genera (garg and sharma 2007, pandey and kumari 2007). abid and qaiser (2009) found the surface pattern of cypsela of the genus tanacetum very useful for specific delimitation. regarding the medicinal and economic importance of tanacetum species, studies on anatomy and trichome micromorphology of turkish tanacetum species are rather limited. so far, there have been no detailed anatomical and micromorphological studies on tanacetum species naturally distributed in the northern anatolian region. therefore, in this research our objective is to determine the anatomical and micromorphological characters of t. macrophyllum, t. parthenium, t. poteriifolium and t. vulgare distributed in northern anatolia and their contribution to the taxonomy of tanacetum taxa. material and methods plant material belonging to the taxa involved was collected from north anatolia in the year 2011, during the flowering period. the taxonomical descriptions of the specimens were made according to grierson (1975). voucher samples were deposited in the herbarium of the department of biology of ondokuz mayıs university (omub), samsun, turkey. the collection data for the studied taxa are listed in tab. 1. samples for anatomical studies were fixed in 70% alcohol. anatomical studies were carried out on 30 samples. in these samples, cross sections of root, stem and leaves and surface sections of leaves were used. sections were stained with safranin-fast green and mounted in entellan (johansen 1944). photographs were taken with a nikon-coolpix p5100 digital camera. all anatomical measurements were made with the use of image j program on the figures. the mean and standard deviation (sd) of measurements were calculated with the use of the statistical package programme spss (version 10.0). the significance of these measurements was determined by duncan test with one-way analysis of variance (anova). micromorphological studies were carried out on stem, leaf, phyllaries, ligulate floret, disc floret and cypsela obtained from these samples. for scanning electron microscopic observations (sem), dried samples were mounted on stubs using double-sided adhesive tape, coated with gold, examined and photographed with a jeol-neoscope jcm-5000 scanning electron microscope. the terminology of the cypselar characters follows abid and qaiser (2009). results anatomical properties tanacetum macrophyllum root anatomy – in transverse section, the periderm layer on the outermost surface is thick and multilayered. secondary cortex is 8–9 layered and consists of parenchymatic cells. secretory canals in cortex are present. the mean diameter of these canals, which are elliptic in shape, is 49.26±8.87 µm (tab. 2). cambium cells are not distinguishable. phloem and xylem elements can be distinguished in the vascular tissue. the xylem consists of tracheary elements. in the center, there is pith composed of primary xylem elements. the mean diameter of vessel members is 19.75±6.60 µm (tab. 2). stem anatomy – epidermis consists of prismatic thickwalled cells with thick cuticle. under the epidermis, parenchyma cells, usually one-layered, are located. a multilayered lacunar collenchyma layer is more developed at the corners of the stem. the sclerenchyma tissues are located under the cortex and are 60.13±7.65 µm in thickness. cambium cells are distinguishable. xylem and phloem elements are clear. the trachea diameter is 18.60±7.67 µm. the pith is large and composed of parenchymatic cells with intercellular spaces in the center of stem (fig. 1c). leaf anatomy – the upper and the lower epidermis are covered with a thin cuticle layer (fig. 1f). both epidermises consist of uniseriate, oval or rectangular cells. the leaf is of the bifacial type. the mesophyll is composed of elongated rectangular palisade parenchyma cells and isodiametric spongy parenchyma cells. palisade parenchyma cells are 11.59±1.39 µm in width (tab. 2). vascular bundles are surrounded by a parenchymatic bundle sheath. the stomata are anomocytic and are found only in the lower epidermis. the dimensions of stomata are 29.76±2.65 × 23.13±1.92 µm (tab. 2). tab. 1. list of studied tanacetum l. taxa and their localities and collectors. taxa specimen number and collector locality t. macrophyllum ş.dereli 132 a5 amasya: merzifon, tavşan mountain, gelinsini village, valley side, 1500 m., 09.08.2011. t. parthenium ş.dereli 118 a5 amasya: merzifon, tavşan mountain, 10 km. from gelinsini village to hacıveli, field sides, 1250 m., 07.08.2011. t. poteriifolium ş.dereli 126 a5 samsun: bafra, yeraltı village, around ağcaalan stream, 150 m., 13.07.2011. a5 amasya: merzifon, tavşan mountain, gelinsini village, road sides, 1500 m., 20.07.2011. t. vulgare ş.dereli 143 a5 samsun: bafra, around dereler village, forest openings, 100 m., 13.07.2011. a5 samsun: bafra, yer altı village, near ağcaalan stream, meadows, 150 m., 16.07.2011. dere s., aytas akcin t. 140 acta bot. croat. 76 (2), 2017 ta b. 2 . a na to m ic al c ha ra ct er is tic s of f ou r ta na ce tu m l . t ax a. s d – s ta nd ar d de vi at io n, * d en ot es a bs en ce o f ch ar ac te r. m ea ns in th e co lu m n m ar ke d w ith th e sa m e le tte r ar e no t s ta tis tic al ly s ig ni fic an t (p <0 .0 5) . t. m ac ro ph yl lu m t. p ar th en iu m t. p ot er iif ol iu m t. v ul ga re w id th (µ m ) l en gt h (µ m ) w id th (µ m ) l en gt h (µ m ) w id th (µ m ) l en gt h (µ m ) w id th (µ m ) l en gt h (µ m ) m ea n± sd m ea n± sd m ea n± sd m ea n± sd r oo t pe ri de rm a ce ll 1 8. 78 ±3 .7 9b 44 .8 9± 9. 33 c 1 2. 09 ±3 .2 7a 33 .8 4± 7. 40 b 17 .4 7± 3. 23 b 24 .6 6± 4. 09 a 13 .7 8± 2. 94 a 27 .5 0± 5. 43 a d ia m et er o f c or te x 16 8. 46 ±8 .6 2b – 12 7. 47 ±5 .2 5d – 14 9. 80 ±1 1. 03 c – 21 2. 23 ±1 8. 87 a – t hi ck ne ss o f p hl oe m 5 8. 03 ±7 .0 9d – 18 0. 91 ±9 .3 9a – 14 8. 07 ±1 0. 43 b – 7 9. 05 ±2 4. 13 c – t hi ck ne ss o f x yl em 3 73 .8 0± 14 .2 1c – 5 77 .8 4± 23 .9 5b – 87 4. 29 ±2 9. 79 a – 12 8. 13 ±2 0. 54 d – d ia m et er o f v es se l 1 9. 75 ±6 .6 0b – 4 2. 11 ±1 3. 16 a – 12 .3 9± 3. 24 b – 14 .7 2± 5. 54 b – st em e pi de rm is c el l 1 2. 81 ±2 .0 9c 14 .6 5± 3. 57 d 3 .7 2± 0. 64 a 3 .9 0± 0. 75 a 10 .3 9± 1. 70 b 8 .1 5± 1. 45 b 11 .1 8± 2. 57 b 12 .7 9± 2. 85 c c ol le nc hy m a ce ll 9 .5 2± 1. 36 b 13 .9 9± 1. 66 b 3 .2 7± 0. 53 a 5 .0 9± 0. 46 a 8 .6 6± 2. 69 b 16 .8 0± 3. 58 c 15 .3 6± 2. 34 c 20 .9 6± 2. 33 d t hi ck ne ss o f c or te x 8 8. 84 ±1 0. 33 b – 5 6. 68 ±1 7. 95 c – 50 .6 8± 3. 92 c – 18 4. 83 ±7 2. 12 a – t hi ck ne ss o f s cl er en ch ym a 6 0. 13 ±7 .6 5b – 6 7. 18 ±1 6. 81 b – 71 .1 3± 5. 64 b – 9 5. 37 ±2 1. 41 a – t hi ck ne ss o f p hl oe m 6 1. 54 ±9 .6 3b – 6 5. 44 ±1 5. 89 b – 67 .6 4± 8. 08 b – 9 1. 18 ±2 5. 75 a – c am bi um 1 6. 33 ±2 .6 8c – 2 5. 91 ±8 .8 5b – 39 .7 4± 7. 68 a – 46 .7 2± 7. 14 a – t hi ck ne ss o f x yl em 1 35 .5 0± 17 .1 5c – 8 6. 30 ±1 8. 17 d – 17 3. 68 ±2 2. 91 b – 20 9. 66 ±2 1. 81 a – d ia m et er o f v es se l 1 8. 60 ±7 .6 7a – 1 7. 00 ±3 .6 9a – 18 .6 1± 7. 58 a – 15 .3 2± 3 07 a – pi th c el l 3 2. 99 ±7 .4 2c – 4 8. 45 ±1 4. 30 c – 7 5. 60 ±1 7. 42 a – 5 1. 63 ±1 1. 53 b – l ea f e pi de rm is c el l 1 9. 33 ±5 .0 6c 2 2. 05 ±4 .5 1a b 1 6. 77 ±2 .9 8a b 25 .8 8± 8. 39 b 1 8. 19 ±3 .9 4b c 23 .1 1± 6. 98 b 15 .4 5± 2. 47 a 18 .2 6± 5. 04 a pa lis ad e ce ll 1 1. 59 ±1 .3 9a 33 .5 5± 2. 63 a 1 6. 18 ±2 .0 3b 47 .4 5± 8. 09 b 14 .9 6± 2. 49 b 47 .6 1± 6. 82 b 11 .7 6± 2. 23 a 4 3. 97 ±1 0. 52 b sp on gy c el l 1 1. 34 ±1 .5 6a 19 .1 0± 4. 35 a 1 3. 57 ±2 .1 2b 19 .9 5± 2. 86 a 16 .3 9± 3. 25 c 22 .7 8± 5. 16 b 15 .5 4± 2. 62 c 30 .9 4± 6. 58 c t hi ck ne ss o f p hl oe m 3 8. 39 ±6 .3 6b – 3 5. 84 ±8 .8 4b – 43 .8 0± 5. 73 b – 6 1. 52 ±1 2. 65 a – t hi ck ne ss o f x yl em 8 1. 51 ±9 .0 0a – 8 9. 42 ±9 .9 1a – 61 .4 9± 7. 61 b – 63 .2 0± 9. 01 b – a da xi al s to m at a * * 2 2. 17 ±1 .4 8a 31 .0 8± 3. 92 a * * 25 .3 3± 2. 86 a 34 .8 9± 2. 44 a a ba xi al s to m at a 2 3. 13 ±1 .9 2b 29 .7 6± 2. 65 b 2 4. 35 ±1 .4 4a b 30 .3 0± 3. 17 b 25 .5 7± 2. 18 a 37 .1 7± 2. 01 a 25 .5 2± 1. 98 a 33 .8 7± 3. 40 a anatomy and micromorphology of some tanacetum taxa acta bot. croat. 76 (2), 2017 141 tanacetum parthenium root anatomy – periderm is 3–4 layered. there is a parenchymatic cortex under the periderm and the diameter of this layer is 127.47±5.25 µm (tab. 2). cambium cells are undistinguishable. the xylem consists of vessel members and tracheids. the trachea diameter is 42.11±13.16 µm (tab. 2). the pith consists of primary xylem elements with pith rays in 1–4 rows. stem anatomy – the stem is clearly quadrangular in transverse section. the epidermis consists of oval or rectangular cells and is covered by a thick cuticula. under the epidermis, a collenchyma layer is located at the corners of the stem. the cortex is composed of irregular oval or circular parenchymatic cells. 2–4 layered sclerenchymatic layer is present above the phloem. there is a distinguishable cambium between the phloem and the xylem. the mean trachea diameter is 17.00±3.69 µm (tab. 2). in the whole center of the stem, there is a pith which is composed of circular, oval and hexagonal large parenchymatic cells. leaf anatomy – there is a single layered epidermis on the upper and lower surface of the leaf. mesophyll consists of single layer of palisade parenchyma cells and 4–6 layers of spongy parenchyma cells with large intercellular spaces. spongy parenchyma cells are 19.95±2.86 µm in length (tab. 2). vascular bundles are surrounded by a parenchymatic bundle sheath. there is one large vascular bundle in the center. the leaf surface shows stomata of the anomocytic type. stomata occur on both epidermal surfaces and are more abundant on the lower epidermis. tanacetum poteriifolium root anatomy – periderm is multilayered. the cortex layer is parenchymatic and the mean diameter of cortex 149.80±11.03 µm (tab. 2). the cambium is inconspicuous. most of the root volume is occupied by secondary xylem. diameters of vessel members are 12.39±3.24 µm (tab. 2). the pith consists of large parenchymatic cells that are oval or rounded in shape (fig. 1a). stem anatomy – a transverse section taken from the stem showed that it was covered by a uniseriate epidermis with thick cuticle (fig. 1d). the epidermal cells of both surfaces are more or less rectangular to oval. seven-eight layers of lacunar collenchyma are located under the epidermis. cortex parenchyma cells are multilayered. the endodermis is located between the cortex and the vascular tissue. the cells of the endodermis layer are one-layered. the cambium is hardly visible and undistinguishable. the size of vascular bundles at the corners is larger than the bundles between corners. the mean diameter of vessel members is 18.61±7.58 µm. pith cells are large and parenchymatic (fig. 1d). leaf anatomy – the epidermis at both surfaces of the leaves is single-layered. a well developed cuticle on the surface of leaves was observed. upper and lower epidermises consist of a single layer of rectangular cells. the mesophyll is differentiated into a 1–2 layered palisade and a 3–4 layered spongy parenchyma. palisade parenchyma cells are elongated. spongy parenchyma cells are 22.78± 5.16 µm in length (tab. 2). vascular bundles are collateral and well developed. the leaves are hypostomatic. the dimensions of the anomocytic type stomata on the lower surface are 37.17±2.01 × 25.57±2.18 µm (tab. 2). tanacetum vulgare root anatomy – -in transverse section, the root is well advanced in a secondary structure formation (fig. 1b). under the multilayered periderm, there is a cortex layer consisting of oval cells. the cells of this layer are 13.78±2.94 µm in width and 27.50±5.43 µm in length (tab. 2). there are sclerenchymatic cells that form groups between the cortex and the phloem. the cambium is not distinguishable. the xylem consists of vessel members and tracheids. vessel members are circular or hexagonal. the xylem rays are composed of 2–4 rows of rectangular cells. the pith containing parenchymatic cells occupies the center of the root (fig. 1b). stem anatomy – a transverse section taken from the middle part of plants clearly showed a quandrangular shape fig. 1. cross-sections of roots, stems and leaves of investigated tanacetum taxa: (a) t. poteriifolium root, (b) t. vulgare root, (c) t. macrophyllum stem, (d) t. poteriifolium stem, (e) t. vulgare stem, (f) t. macrophyllum leaf, (g) t. vulgare leaf. c – cortex, cl – collenchyma, e – epidermis, p – periderm, ph – phloem, pr – parenchyma, pt – pith, pp – palisade parenchyma, s – sclerenchyma, sp – spongy parenchyma, x – xylem. dere s., aytas akcin t. 142 acta bot. croat. 76 (2), 2017 (fig. 1e). epidermal cells consist of a single layer and are orbicular or rectangular. underneath the epidermis, there is a lacunar collenchyma layer which is more developed at the corner of the stem. the length of collenchyma cells is 20.96±2.33µm (tab. 2).the cortex is composed of multilayered irregular oval or rectangular parenchymatic cells with intercellular spaces. the cambium between xylem and phloem is hardly visible. there are multilayered sclerenchymatic cells surrounding the phloem. these bundles are different sizes. the xylem and phloem elements are visible. the pith consists of large parenchymatic cells with intercellular spaces (fig. 1e). leaf anatomy – in transverse section, the upper and lower epidermises are composed of uniseriate rectangular cells (fig. 1g). epidermis cells are covered with a cuticula layer. the leaf is of the isobilateral type. the mesophyll consists of 2 or 3 layers of upper and lower palisade cells and isodiametric spongy parenchymatic cells with large intercellular spaces. spongy parenchyma cells are 3–4 layered and orbicular or oval in shape. the vascular bundles are collateral and surrounded by a parenchymatic bundle sheath. the stomata type is anomocytic and the stomata occur on the surfaces of both sides but are more abundant on the lower surface. the dimensions of stomata on the lower surface of leaf are 33.87±3.40 × 25.52±1.98 µm (tab. 2). micromorphology of the stem, leaf and phyllary the micromorphological characters and distribution of the trichomes on stem, leaf and phyllaries of the tanacetum taxa examined showed a considerable variation (tab. 3). in our study, two basic types of trichomes were observed on the vegetative organs: non-glandular and glandular trichomes. non-glandular trichomes are acicular or curved, simple and made up from one or more cells (figs. 2a–b), whereas glandular trichomes were peltate or capitate (figs. 2c–f). both these types of trichomes were common in the four species of the studied tanacetum genus (figs.2a–f). however, glandular trichomes are absent from the stem of all tanacetum taxa. in additional, these kinds of trichome are very rare on the phyllaries of t. vulgare (tab. 3), although they are present on the leaves and phyllaries of the other taxa. non-glandular trichomes especially are present in large numbers on both surfaces of leaves, phyllaries and stem in t. macrophyllum, t. parthenium and t. poteriifoli­ um (figs. 2a–b, tab. 3). micromorphology of flower and cypsela glandular trichomes are generally abundant on the disc florets and ray florets of tanacetum taxa (figs. 3a–d). these kinds of trichome are the most common type and are observed in all investigated tanacetum species. however, they are usually sparsely found on disc and ray florets of t. macrophyllum (tab. 3). sem showed that the disc florets have greater trichome density than the ray florets in tanace­ tab. 3. distribution of glandular (gland) and non-glandular (egland) trichomes on different parts of studied tanacetum l. species. (–) denotes absence of trichomes, (+) denotes a few trichomes, (++,+++) denote increasing presence of trichomes, (*) denotes absence of character. plant material t. macrophylum t. parthenium t. poteriifolium t. vulgare gland egland gland egland gland egland gland egland stem – ++ – + – + – + adaxial leaf surface ++ +++ +++ ++ ++ +++ +++ + abaxial leaf surface +++ +++ +++ ++ + +++ +++ + phyllaries ++ +++ ++ + ++ +++ – ++ disc floret + – + – ++ – ++ – ray floret + – ++ – ++ – * * cypsela + – +++ – +++ – +++ – fig. 2. scanning electron micrographs (sem) of trichomes of studied tanacetum l. taxa: (a) eglandular trichomes on leaf of t. macrophyllum, (b) eglandular trichomes on phyllaries of t. mac­ rophyllum, (c) glandular trichomes on the lower surface of leaf of t. macrophyllum, (d) glandular trichomes on phyllaries of t. parthenium, (e) glandular trichomes on the lower surface of leaf of t. poteriifolium, (f) glandular trichomes on the lower surface of leaf of t. vulgare. anatomy and micromorphology of some tanacetum taxa acta bot. croat. 76 (2), 2017 143 tum (figs. 3b–d).trichome density on disc florets is higher on the ovary than the corolla. the non-glandular trichomes are absent from the florets of the tanacetum taxa studied (tab. 3). surface characteristics and ornamentation features of the cypselas of 4 turkish tanacetum species are given in tab. 4. in cypselas of examined tanacetum species there are 4–8 ribs on all sides (figs.4a–d). as evidenced in tab. 4, in all species studied here, the cypselas have glandular surfaces. however, in t. poteriifolium and t. parthenium glandular trichomes on cypselas are mainly situated between the ribs especially forming in order over the cypsela surface (figs. 4a–d, tab.4). discussion the present study provides useful information on the anatomy and micromorphology of t. macrophyllum, t. parthenium,t. poteriifolium and t. vulgare. this is the first detailed anatomical and micromorphological report on the four examined taxa of tanacetum. all taxa investigated in the present study were found to have the same general characteristics as other members of the asteraceae. metcalfe and chalk (1979) gave some information about general anatomical characters of the family asteraceae. some studies on asteraceae reported the existence of secretory structures especially in the root (bremer 1994, heywood 1976). in the present study, we determined that t. macrophyllum has secretory canals within the root cortex. our findings are consistent with those of metcalfe and chalk (1979) and those of some other studies of asteraceae (castro et al. 1997, milan et al. 2006). the results in the present study indicated that there was a similarity in root anatomical structure among the taxa. however, in t. poteriifolium and t. vul­ gare, the pith was occupied with large parenchymatic cells, whereas it was filled with primary xylem elements in the other examined taxa. the transverse section of stem showed that the stem of investigated tanacetum taxa has a well-defined collenchyma layer in the corners. metcalfe and chalk (1979) reported that stems of many genera and species of the family asteraceae have distinct collenchyma. it was also determined that this layer is more distinguishable and thicker in t. vulgare. as well as cortex, sclerenchyma and vascular bundles are the thickest in t. vulgare. a distinguishable endodermis was seen between the cortex and vascular bundle. özörgücü et al. (1991) pointed out that endodermis is usually distinguishable in the stem of asteraceae. the parenchymatic layer, which consists of elongated cells, in the stem of t. macro­ phyllum is a useful character for distinguishing the examined species. sclerenchyma was observed in the transverse sections of the four tanacetum taxa as previously described by melo de pinna and menezes (2002) and stevović et al. (2010). in t. poteriifolium, pith in stem occupied a large place compared to the other species. the studied species showed some anatomical differences in the leaf. in this study, it was seen that all the examined species have bifacial leaf types except for t. vulgare. metcalfe and chalk (1950) pointed out that there was generally bifasial mesophyll in the family asteraceae. whereas in t. vulgare, the leaf is of the isobilateral type. stevović et al. (2010) also reported that the mesophyll in t. vulgare is of the isobilateral type. our findings are consistent with those of stevović et al. (2010). the mesophyll of the studied species varied according to the number of cell layers composfig. 3. scanning electron micrographs (sem) of glandular trichomes of studied tanacetum l. taxa: (a) on ray floret of t. parthenium, (b) on the disc floret of t. parthenium, (c) on the disc floret of t. poteriifolium, (d) on the disc floret of t. vulgare. fig. 4. scanning electron micrographs (sem) of cypselas of studied tanacetum l. taxa: (a) t. macrophyllum, (b) t. parthenium, (c) t. poteriifolium, (d) t. vulgare. tab.4. surface characteristics and ornamentation features of the cypselas of studied tanacetum l. taxa. taxa surface and ornamentation characteristics t. macrophylum 4–5 ribbed, sparsely glandular on the ribs t. parthenium 6–8 ribbed, densely glandular in between the ribs t. poteriifolium 6–8 ribbed, densely glandular in between the ribs t. vulgare 6–8 indistinct longitudinal ribbed, glandular on the ribs dere s., aytas akcin t. 144 acta bot. croat. 76 (2), 2017 ing the palisade and spongy parenchyma. in this research, it was found that all examined taxa had anomocytic stomata. in addition, unlike in t. macrophyllum and t. poteriifolium, which have hypostomatic leaves, the leaves are amphistomatic in t. parthenium and t. vulgare. metcalfe and chalk (1950) reported that there were both anomocytic and anisocytic stomata in the family asteraceae. these findings support those of metcalfe and chalk (1950). the value of trichomes in identification of some members of asteraceae has been previously reported by some workers (adedeji 2004, ciccarelli et al. 2007, majdi et al. 2011). according to observations in the current study all the species examined here have eglandular trichomes on the stem and leaves, and glandular trichomes are not present on the stem. the glandular trichomes are absent on the phyllaries of t. vulgare only. ciccarelli et al. (2007) reported that glandular hairs can be used as taxonomical characters in the asteraceae. the secretion of terpenoids by glandular hairs was shown by several studies of this family (pagni et al. 2003). it was suggested that some volatile terpenoids may attract pollinating insects to flowers, while other may protect the plant (kelsey et al. 1984). in the present study, the majority of the glandular hairs were found in disc and ray florets of the all taxa. however, this type of trichome is sparser in t. vulgare than in the other taxa. our results show that within the flowers of the examined tanacetum taxa, the disc florets contain higher glandular trichome density. majdi et al. (2011) reported that trichome densityis highest on the disc florets, followed by leaves, while stems contain a lower density of trichomes in t. parthenium. these variations in the distribution of trichome forms in tanacetum species can help to differentiate species. the importance of cypsela characters such as the presence of ribs and non-glandular and glandular hairs has been previously emphasized (bremer 1994, abid and qaiser 2009, akcin and akcin 2010, 2014). according to bremer (1994), cypselar morphology is very important as a distinguishing character in the classification of asteraceae mainly at the generic and species level. the sem investigation of the cypselas showed that the examined tanacetum taxa have a glandular surface. abid and qaiser (2009) determined that the surface of cypsela in the tanacetum species can be glabrous, papillate or glandular. the genus tanace­ tum is characterized by the presence of 4–8 ribs on all sided of the cypsela (abid and qaiser 2009). according to our studies, t. macrophyllum is characterized by 4–5 ribbed cypselas, while 6–8 ribbed cypselas are found in the other taxa examined. in addition, the cypselas of t. poteriifolium and t. parthenium could be separated from the remaining species by the distribution of the glandular trichomes on the ribs. these species have glandular trichomes, especially present between the ribs. the present results confirmed those of abid and qaiser (2009). in conclusion, some of the anatomical and micromorphological characters found in this study will provide useful characters for distinguishing species in the genus tana­ cetum. acknowledgements we wish to thank ondokuz mayıs university scientific projects research fund (project no: pyo.fen.1904.11. 028) for financial support and gazi university for sem micrographs. references abid, r., qaiser, m., 2008a: cypsela morphology of gnaphalium l. and its allied genera (gnaphalieae-asteraceae) from pakistan. pakistan journal of botany 40, 25–32. abid, r., qaiser, m., 2008b: cypsela morphology of some genera in the tribe gnaphalieae(asteraceae) from pakistan. pakistan journal of botany 40, 73–485. abid, r., qaiser, m., 2009: taxonomic significance of the cypsela morphology in the tribe anthemideae (asteraceae) from pakistan and kashmir. pakistan journal of botany 41, 555–579. abu-darwish, m. s., abu-dieyeh, z. h. m., 2009: essential oil content and heavy metals composition of thymus vulgaris cultivated in various climatic regions of jordan. international journal of agricultural biology 11, 59–63. adedeji, o., 2004:leaf epidermal studies of the species of emilia cass. 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[chrysanthemum (fam. compositae)]. bulletin academie serbe des sciences et des arts tome xcv, classe des sciences mathematiques et naturelles, sciences naturelles 28, 23–43. stevović s., surčinskimikovilović, v., ćalić-dragosavac, d., 2009: environmental adaptibility of tansy (tanacetum vulgare l.). african journal of biotechnology 8, 6290–6294. stevović, s., mikovilović, v., ćalić-dragosavac, d., 2010: environmental impact on morphological and anatomical structure of tansy. african journal of biotechnology 9, 2413–2421. williams, a. c., harborne, j. b.,geiger, h., holut, j. r. s., 1999: the flavonoids of tanacetum parthenium and tanacetum vul­ gare and their antiinflamatory properties. phytochemistry 51, 417–423. zhu, s. x., qin, h. n., shih, c., 2006:achene wall anatomy and surface sculpturing of lactuca l. and related genera (compositae: lactuceae) with notes on their systematic significance. journal of integrative plant biology 48, 390–399. acta botanica 2-2016 za web.indd 244 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 244–252, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0028 issn 0365-0588 eissn 1847-8476 micropropagation of the narrow endemic hladnikia pastinacifolia (apiaceae) jana ambrožič-dolinšek1, 2, 3*, terezija ciringer1, mitja kaligarič1, 3 1 university of maribor, faculty of natural sciences and mathematics, koroška 160, maribor, slovenia 2 university of maribor, faculty of education, koroška 160 maribor, slovenia 3 university of maribor, faculty of agriculture and life sciences, pivola 10, 2311 hoče, slovenia abstract – the monotypic hladnikia pastinacifolia rchb. is a narrow endemic species, with an extremely small distribution area in slovenia, prone to any kind of threat that could lead to species extinction. tissue culture techniques are proposed as a conservation measure for rapid propagation and ex-situ conservation. tissue culture was initiated from seeds and juvenile plants obtained from natural sites on a solid murashige and skoog (ms) medium, with and without growth regulators. we tested various combinations and concentrations of growth regulators, and the best proliferation of axillary shoots, on average 14, was obtained on ms medium with 5 μm bap and 3 μm iba and 3% sucrose. rooting was achieved after transferral of the shoots to an ms medium with 2 μm iba and 3% sucrose. the rooted plants were acclimatized on a mixture of limestone sand, potting soil and vermiculite in a ratio of 10:2:2, with ph in the range of 7.5–8.0. in vitro propagation methods provide an important opportunity for the propagation and preservation of h. pastinacifolia by rapidly increasing the number of plants, without disturbing the wild population. keywords: apiaceae, biotechnology, conservation, endemic, hladnikia pastinacifolia, micropropagation, tissue culture abbreviation: bap – 6-benzylaminopurine, iba – indol-3-butyric acid, 2ip – 2-isopentinyl adenine, k – kinetin, ms – murashige and skoog medium, naa – naphthalene acetic acid, tdz – thidiazuron, z – zeatin * corresponding author, e-mail: jana.ambrozic@um.si introduction in the distant past and during more recent geological periods, complex biogeographical and evolutionary processes resulted in an extraordinarily high number of endemic alpine taxa (berthouzoz et al. 2013). five hundred and one endemic taxa have been described in the alps, comprising ca. 9% of the 4,485 taxa inhabiting this mountain range (aeschimann et al. 2011). the most emblematic species are the narrow endemics. however, only a few of them have been objects of intensive ecological and/or genetic studies (berthouzoz et al. 2013). hladnikia pastinacifolia rchb. (apiaceae) is one of the four endemic monotypic genera of the alps – in addition to berardia, physoplexis and rhizobotrya (wraber 2009). it has an extremely narrow distribution area of 4 km2 within the high karst plateau of trnovski gozd in slovenia (aeschimann et al. 2004), which represents a bridge between the alps and the north-western dinaric alps. the species is also famous for its uniquely isolated phylogenetic position within the apiaceae family (šajna et al. 2012). it is not restricted to a specifi c niche; on the contrary, it thrives in a variety of the habitats (šajna et al. 2012, 2014) that are available within the entire area of the high plateaus of the dinaric alps (mayer 1960). it is still unknown why the distribution range is so narrow. thus, the species’ inability to populate favorable habitats in an area wider than a few isolated populations within a few square kilometers is the reason for concern regarding its persistence and has placed the plant at risk. rare narrow endemic species occurring in a few small populations have to cope with random genetic drift, inbreeding, a stronger founder effect, and a greater potential for demographic bottlenecks that result in low genetic variability (gaston and kunin 1997). small habitat populations decrease the chances of the plant outcrossing. the low potential for range expansion and recolonization was additionally explained by poor seed dispersal (šajna et al. 2012). thus, along with all mentioned restrictions for its dispersal across appropriate habitats and potential for further adaptation, its unique taxonomic position too makes h. pastinacifolia vulnerable and a potential candidate for extinction, especially on account micropropagation of endemic hladnikia pastinacifolia acta bot. croat. 75 (2), 2016 245 of the forecast global climatic changes (dawson et al. 2011). species that persist in small, isolated populations with low genetic diversity will have limited ability to adapt to new climate conditions (lavergne et al. 2010). hladnikia pastinacifolia has another peculiarity: the small distribution range is divided into two populations by a large dense forest, which is unsuitable as habitat, in between. it has been presumed that such isolated distribution would have caused genetic divergence after the last glaciation. for that reason, its genetic variability has been tested, but divergence was not confi rmed. a general loss of genetic diversity has been proved with molecular techniques using rapd markers (šajna et al. 2012). the authors concluded that the genetically homogeneous populations of hladnikia pastinacifolia are the result of severe bottlenecks, which dramatically reduced or eliminated some populations, regardless of the time of colonization (before or after glaciation); the extant populations were founded by a single lineage, starting from a founding population. the conservation status of hladinika pastinacifolia is satisfactory. it is protected by the decree on rare and threatened wild plant species (offi cial gazette of the republic of slovenia 2002, 2010), with a status 4, meaning that measures for favorable conservation status on its habitat should be implemented. furthermore, this species is listed as a “natura 2000 species” in annex ii of the habitat directive (council directive 1992), which also envisages the designation of a natura 2000 site on the basis of its occurrence. there have already been some attempts at ex situ conservation in the botanical garden of the university of ljubljana, where a seed collection and living exemplars are stored. both methods have disadvantages. one is connected with the short life cycle, since monocarpic h. pastincifolia (šajna et al. 2009, 2011, 2014) fl owers once in its lifetime, and dies after fl owering. the other is connected with problematic seed germination established in the apiaceae family (hendrawati et al. 2012, baskin and baskin 2014). for such exceptional botanical rarities, it is nowadays usual to apply, besides the conventional propagation methods, in vitro propagation methods, which provide an important opportunity for the propagation and preservation of endemic, rare and/or endangered plant species in general (fay 1992, rao 2004, pence 2010, 2011, cruz-cruz et al. 2013). only in cases where a previously optimized and tested propagation protocol exists would this kind of plant escape extinction from sudden changes in natural conditions or other factors that place the plant at risk of local extinction (which is here the defi nitive level). over-collecting and competition with other species, along with reduction and fragmentation or degradation of natural habitats could diminish the population substantially. micropropagation is a convenient measure suggested for preserving endangered species by rapidly increasing the number of plants in the case of natural population loss in a relatively short period of time and introducing them to their natural or to new environments (sarasan et al. 2006, kapai et al. 2010, cruz-cruz et al. 2013, gonzález-benito and martín 2011). beside this, tissue culture allows genotype conservation (tavares et al. 2010b), constitutes a kind of living ex situ collection and is a basic method for other biotechnological conservation measures like cryopreservation of shoots (cruz-cruz et al. 2013). several institutions, like the kew and missouri botanical gardens (sarasan et al. 2006), and the botanic garden of the university of coimbra for endemic apiaceae, are now using this strategy to propagate and maintain endangered and endemic species (tavares et al. 2009–2010, 2010b). the problems with such propagation include the potential for somaclonal variation, as a consequence of either genetic or epigenetic changes in the tissue culturepropagated material (bairu et al. 2011). to obtain plant material that is as genetically stable and uniform as possible, with a low probability of genetic or epigenetic variation, micropropagation must be carried out through shoot tip or axillary bud proliferation (pierik 1991). the aim of the present work was to establish and evaluate the in vitro propagation protocol for hladnikia pastinacifolia through axillary shoots in order to obtain enough plants that can be used for several purposes allowed because of legislative restrictions connected with sample quantities and for establishment of ex situ collections in botanical gardens, production of seeds for seed banking, reintroduction to the natural environment, for basic research on the ecology and biology of threatened plant species, and for long-term maintenance in a tissue culture collection – all, without disturbing the wild population. material and methods plant material, explant source and culture condition all plant material, seeds and juvenile plants were collected at natural secondary sites near the route to predmeja, slovenia (figs. 1a, b), with the permission of the responsible authority in 2004 and 2013. seeds and juvenile plants of hladnikia pastinacifolia were used as initial explants (figs. 1c, d). since the species is protected by law, we performed only one culture initiation by seed germination, and one initiation of the culture from the juvenile plants collected at the natural sites in 2004. tissue culture was initiated once more in 2013, with sterilized juvenile plants collected at the natural sites. each plant was the source of one tissue culture line for the propagation of plant material. seeds and all plant material were surface sterilized with 70% ethanol (sigma aldrich) for 30 seconds and then soaked in 1% or 2% commercial bleach 6.7% naocl (šampionka), with a drop of the detergent tween 80 (merck) for 15–20 min and rinsed three times with sterile deionized water . we tested several factors to increase the germination rate: cold treatment, scarifi cation and sterilization of seeds. sterilized and non-sterilized seeds germinated in sterile petri-dishes closed with parafi lm (bemis), on moistened paper bridges at 20 °c in the growth chamber or in the fridge at 4 °c in the dark. the cultures in the growth chamber were kept at 23 ± 2 °c, with a photoperiod of 16 h at 38–50 μmol m–2 s–1 (osram l 58w/77 – fluora) and 50% relative humidity. ambrožič-dolinšek j., ciringer t., kaligarič m. 246 acta bot. croat. 75 (2), 2016 culture medium for shoot proliferation and multiplication in 2004 and the next two years, tissue culture was initiated from 12 sterilized juvenile plants from the natural sites and 10 sprouts from seeds (figs. 1c, d), and the best concentrations were additionally tested on juvenile plants from the natural site in 2013. the sterilized juvenile plants and sprouts were placed on solid ms medium (murashige and skoog 1962). supplemented with 0.8% difco-bacto-agar (medias international), with 3% sucrose (duchefa biochemie), with and without growth regulators: 6-benzylaminopurine (0–20 μm bap, sigma aldrich), 2-isopentinyl adenine (0–20 μm 2ip, sigma aldrich), thidiazuron (0–10 μm tdz, sigma aldrich), kinetin (0–20 μm k, sigma aldrih), zeatin (0–20 μm z, sigma aldrich), naphthalene acetic acid (0–1 μm naa, sigma aldrich), indol-3-butyric acid (0–3μm iba, sigma aldrich) (tab. 1) adjusted to ph 5.7– 5.8, and later on autoclaved. root induction and acclimatization the regenerated shoots were rooted on ms media with different combinations and concentrations of the growth regulators bap (0–1 μm) and iba (2–10 μm). in the rooting experiment, the following combinations and concentrations of the growth regulators were tested: 2 μm iba and 1 μm bap; 5 μm iba and 1 μm bap; 10 μm iba and 1 μm bap; 5μm iba and 2 μm bap; 10 μm iba a nd 2 μm bap; 2 μm iba and 0 μm bap; 5 μm iba and 0 μm bap; 10 μm iba and 0 μm bap. rooted plants were transferred to the prepared substrate containing a mixture of limestone sand : potting soil : vermiculite (10:1:2) with a ph of 7.0–7.5, or in a mixture of limestone sand : potting soil : vermiculite (vermit group) (10:2:2) with a ph of 7.5–8.5, and transferred to in vivo greenhouse conditions. the plants were protected against excessive water loss by regular spraying with water, and by plastic covers with adjustable aeration openings. they were acclimatized with a progressive increase in aeration through the adjustable aeration openings for the fi rst two weeks, and with progressive opening of the whole cover over the next three weeks. statistical analysis the statistical package spss® 21.0 was used for data analysis. student’s t-test and the 2 x 2 chi-squared test (χ2) were used for evaluating levels of statistical signifi cance (p) between treatments. statistical signifi cance was shown between control on ms without and with growth regulators, unless otherwise denoted. the symbols used in the fi gures are as follows: ns denotes not signifi cant, symbol (*) denotes p < 0.05, symbol (**) denotes p < 0.01, symbol (***) denotes p < 0.001. all experiments were repeated twice with similar results. fig. 1. micropropagation of hladnikia pastinacifolia: a, b) plants from the natural habitat in predmeja (bar = 5 cm); c) sterilized seeds germinated into sprouts (bar = 1 cm); d) initiation of the culture with sprouts from natural sites (bar = 1 cm); e) the shoots developed on ms medium with 5 μm 6-benzylaminopurine (bap) and 3 μm – indol-3-butyric acid (iba); f) detached juvenile shoots are the most suitable for multiplication of h. pastinacifolia shoots; g) the effect of bap on the length of shoots (bar = 2.5 cm); h) fl ower shoots developed sporadically from vegetative shoots (bar = 2.5 cm); i) rooted h. pastinacifolia shoots after 4 weeks on ms medium with 2 μm bap (bar = 2.5 cm); j) rooted plants transferred to plastic pots with substrate for acclimatization (bar = 2.5 cm); k) acclimated plants after 6 months at outdoor conditions (bar = 1 cm). micropropagation of endemic hladnikia pastinacifolia acta bot. croat. 75 (2), 2016 247 tab. 1. effect of different cytokinins and auxins on shoot development of detached shoots of hladnikia pastinacifolia after two months of culture. bap – 6-benzylaminopurine, 2ip – 2-isopentinyl adenine, tdz – thidiazuron, k – kinetin, z – zeatin, iba – indol-3-butyric acid, 2,4-d – 2,4-dichlorophenoxyacetic acid, naa – naphthalene acetic acid, sd – standard deviation. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001. signifi cance was shown between shoots on ms without and with hormones, except if it is denoted different: awith 2 μm bap; bwith 5 μm bap; cwith 10 μm bap; dwith 0 μm tdz. hormones [μm] explants with new shoots [%] average number of shoots ± sdbap 2ip tdz kin z iba 2,4-d naa 0 0 0 0 0 0 0 0 9.3 3.7±0.4 2 0 0 0 0 0 0 0 27.3 6.4±1.5 * a 2 0 0 0 0 2 0 0 69.5 10.0±1.9 * a 2 0 0 0 0 3 0 0 71.0 9.1±2.5 * a 2 0 0 0 0 0 3 0 – callus 2 0 0 0 0 0 5 0 – callus 2 0 0 0 0 0 10 0 – callus 5 0 0 0 0 0 0 0.5 25.0 6.8±1.1 ** 5 0 0 0 0 0 0 1 10.3 6.1±0.9 * 5 0 0 0 0 0 0 0 38.5 6.6±0.7*** b 5 0 0 0 0 0.5 0 0 37.0 5.8±0.7 ** 5 0 0 0 0 1 0 0 39.7 5.2±0.2 ** 5 0 0 0 0 2 0 0 68.0 10.5±1. 6 ** b 5 0 0 0 0 3 0 0 90.2 14.0±2.7 ** b 5 0 0 0 0 0 10 0 – callus 10 0 0 0 0 0 0 0 23.3 7.7±1.1 ** c 10 0 0 0 0 0 0 0.5 57.0 6.5±1.0 * 10 0 0 0 0 0 0 1 43.5 6.3±0.4 *** 10 0 0 0 0 0.5 0 0 35.0 6.0±0.6 ** 10 0 0 0 0 1 0 0 55.3 7.3±1.4 *** 10 0 0 0 0 2 0 0 67.3 12.0±2.2 ** c 10 0 0 0 0 3 0 0 72.7 10.1±2.3 * c 20 0 0 0 0 0 0 0 17.0 5.6±0.3 *** 20 0 0 0 0 3 0 0 53.3 8.5±2.1 0 2 0 0 0 0 0 0 10.3 4.5±0.7 0 5 0 0 0 0 0 0 20.0 6.3±1.4 * 0 10 0 0 0 0 0 0 39.0 8.1±1.7 ** 0 10 0 0 0 0 0 0.5 40.0 6.8±1.6 * 0 10 0 0 0 0.5 0 0 27.3 5.3±1.0 0 20 0 0 0 0 0 0 50.3 8.6±2.0 ** 0 20 0 0 0 0 0 0.5 34.7 4.7±0.5 0 20 0 0 0 0.5 0 0 38.0 5.4±1.8 * 0 0 0.25 0 0 0 0 0 46.3 7.3±1.7 * 0 0 0.5 0 0 0 0 0 74.3 8.50±2.12 * 0 0 0.5 0 0 2 0 0 73.7 9.81±1.5 *** 0 0 1 0 0 0 0 0 34.7 5.1±1.1 0 0 2 0 0 0 0 0 39.0 6.2±1.0 * 0 0 5 0 0 0 0 0 26.3 5.2±0.8 0 0 10 0 0 0 0 0 27.0 4.3±0.7 0 0 0 1 0 0 0 0 6.3 3.6±0.3 0 0 0 2 0 0 0 0 14.0 4.1±0.6 0 0 0 5 0 0 0 0 14.0 4.0±0.3 0 0 0 10 0 0 0 0 11.3 3.8±0.4 0 0 0 20 0 0 0 0 13.3 4.2±0.7 0 0 0 0 0.5 0 0 0 8.5 3.6±0.3 0 0 0 0 2 0 0 0 21.0 5.5±1.2 0 0 0 0 3 0 0 0 21.0 5.2±1.4 0 0 0 0 5 0 0 0 28.0 4.5±0.6 0 0 0 0 10 0 0 0 52.5 4.6±2.2 * 0 0 0 0 20 0 0 0 34.0 4.1±0.1 0 0 0 0 0 0 1 0 callus 0 0 0 0 0 0 3 0 callus 0 0 0 0 0 0 5 0 callus 0 0 0 0 0 0 10 0 callus ambrožič-dolinšek j., ciringer t., kaligarič m. 248 acta bot. croat. 75 (2), 2016 results in vitro seed germination and initiation of the culture the seeds of h. pastinacifolia started to germinate after 4 months in the culture (fig. 1c) and achieved their highest germination rate after one year. we tried several factors which could infl uence the germination rate: room temperature and cold treatment, scarifi cation and sterilization of seeds. only those seeds under permanent cold treatment germinated but failed to germinate at all if they were kept in the growth chamber. scarifi cation did not promote germination. between 7% of the sterilized and 30% of the unsterilized seeds germinated after 4 months and between 45% of the sterilized and 60% of the unsterilized seeds germinated after one year of cold treatment. sterilization decreased and slowed the rate of early germination. this was signifi cantly evident (χ2 = 10.960; df = 1; p = 0.001) after four months of germination but no longer signifi cantly evident after one year of germination. tissue culture was initiated from shoots excised from juvenile sprouts (fig. 1c) germinated from sterilized seeds, from sterilized juvenile sprouts germinated from the unsterilized seeds and from juvenile plants from the natural sites (fig. 1d). shoot proliferation and multiplication permission for the application, which allowed only a small number of explants, limited our research. that is why we began our experiments with a small number of initial explants. in these early experiments we fi rst cultivated a small number of explants on ms medium with and without 2–20 μm bap (data not shown); this was later repeated several times on a higher numbers of explants (fig. 2a). shoot multiplication was observed on all ms media, with or without growth regulators. shoot proliferation was optimal when 5 and 10 μm bap were used, and the best growth medium produced on average, 7.3 shoots per explant (fig. 2a). this plant material was used for further experiments, where several concentrations and combinations of cytokinins and auxins were tested (tab. 1). in further experiments, the development of new shoots was observed on almost all media tested (tab. 1). there was a signifi cant difference within the various treatments using different combinations and concentrations of growth regulators. the addition of auxins to cytokinins additionally improved shoot development (tab. 1). new shoots developed on almost all the explants. the best proliferation of shoots, on average 14 (fig. 1e, f, g), was obtained on ms medium with 5 μm bap and 3 μm iba (fig. 2b, tab. 1). shoots developed on 90% of explants. slightly fewer shoots, on average 12, were obtained on ms medium with 10 μm bap and 2 μm iba (tab. 1). shoots developed on 67% of these explants. the new shoots began to proliferate after 10 to 14 days. over the next 15 to 20 days, the shoots elongated to a length appropriate for detachment and rooting. these detached shoots exhibited both organized and unorganized growth (tab. 1). unorganized growth was observed only on the ms medium with growth regulator 2,4-dichlorophenoxyacetic acid (2,4-d) (tab. 1). flowers developed sporadically from the new shoots. well-developed fl ower shoots formed several infl orescences at the shoot tip, which developed into a considerable number of fl owers (fig. 1h). growth regulators did infl uence the shoot length. with an increased concentration of bap, smaller shoots developed, if bap alone was added to the media. this was signifi cantly evident when concentrations exceeded 5 μm bap (fig. 1g). root development and acclimatization roots did not develop on those media without growth regulators. to obtain roots, we tested several combinations and concentrations of growth regulators. we started by decreasing the amounts of cytokinin bap and increasing the auxin iba and fi nished by increasing only the auxin iba. fig. 2. effect of plant growth regulators on the shoot and root development in the micropropagation of hladnikia pastinacifolia: a) the effect of 6-benzylaminopurine (bap) on the number of shoots (n = 28) after 35 days of culture; b) the effect of bap and indol-3butyric acid (iba) on the number of shoots (n = 28) after 35 days of culture; c) root development (n = 25) on ms media supplemented by different concentrations of iba after 4 weeks of culture. statistically signifi cant differences (t-test) are shown between the control without hormones (a, b) or ms medium with 2 μm iba (c) and different treatments. sd -standard deviation, * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001. micropropagation of endemic hladnikia pastinacifolia acta bot. croat. 75 (2), 2016 249 the best rooting was achieved on the ms medium with iba alone (fig.. 1i, 2c). roots started to develop after 2 weeks. optimal rooting was obtained on the ms medium with 2 μm or 5 μm iba after 4 weeks of culture. the greatest number, 44% of shoots with roots, was obtained on the ms medium with 2 μm bap and the smallest, 28% of shoots with roots, was obtained on the ms medium with 5μm iba. shoots with roots developed an average 10.8 of roots on medium with 2 μm iba. a lower average of 6.9 roots developed on the medium with 5 μm iba, and the signifi cantly lower average of 2.6 roots appeared on the ms medium with 10 μm iba. the higher concentration, 10 μm iba, induced both roots and callus to develop. roots developed on the detached side of the shoots. two growth substrates with different ph were assessed for effi ciency in supporting ex vitro growth. both mixtures are attempts to imitate conditions in the natural habitat. they were prepared with limestone sand, which increases the ph of the substrate, potting soil and vermiculite. after 5 months, 66% of the plants survived in the substrate with a higher ph between 7.5 and 8.5 (fig. 1j) and only 22% in substrate with a lower ph between 7 and 7.5. plants of h. pastiancifolia obtained by this procedure are transferred to outside conditions (fig. 1k). discussion like many other alpine plant endemics (aeschimann et al. 2011), hladnikia pastinacifolia, has an extremely restricted distribution. due to its inability to colonize appropriate habitats in a wider range due to several reasons, due to genetic depauperation, which make its capacity of adaptation to climate change even weaker, hladnikia pastinacifolia is a strictly protected species (šajna et al. 2012). this is the reason for the restrictions on the collecting of seeds for seed banks and research activities. micropropagation of h. pastinacifolia provides the possibility for conservation in tissue culture collection, without disturbing the wild population and allows rapid propagation of h. pastinacifolia for research efforts. seed-banking is the primary method for ex-situ conservation. we had shown that h. pastinacifolia had problematic seed germination. seeds from cold wet environments have been shown to be relatively short lived in storage, and successful long-term seed conservation for alpine plants may be diffi cult (mondoni et al. 2011). therefore h. pastinacifolia has to be conserved either in living collections or through micropropagation and/or cryogenic storage, and this article presents the latter method. in vitro seed germination and initiation of the culture micropropagation was started with the introduction of plant material in aseptic conditions. we started with limited sample quantities and with different sources of plant material. seeds, juvenile sprouts from seeds, and juvenile plants of h. pastinacifolia were used as initial explants for initiation of the culture. the starting material had a range of disadvantages connected with germination and/or the establishment of aseptic culture conditions. h. pastinacifolia has problematic seed germination. these seeds did not germinate in conditions that usually promote germination. when they did germinate, they did so neither in high percentages nor immediately and simultaneously. for that reason, the initiation of culture from seeds was time consuming, uncertain in outcome and in need of further consideration. the seeds began to germinate between four and twelve months after initiation of the culture. the seeds in our experimental system germinated only after cold treatment and did not germinate at all in growth chamber conditions. the reason for this late and low germination could be dormancy, which is not unusual for seeds of the apiaceae family (hendrawati et al. 2012, baskin and baskin 2014), or possible loss of viability (makunga et al. 2003, mondoni et al. 2011). sterilization only slowed the rate of early germination and slightly infl uences the fi nal germination of cold treated seeds. that is why the initiation of the culture through seeds or juvenile sprouts from seeds could be problematic, since this lengthens the initial stage of micropropagation by several months. the introduction of unsterilized plant material in aseptic conditions was also problematic, because of damage during sterilization. when we started the culture with sterilized seeds, they germinated into viable, juvenile sprouts. when we started the culture with unsterilized seeds that germinated into unsterilized juvenile sprouts, there were losses due to contamination and/ or mechanical damage during their transfer to aseptic conditions by sterilization. problems with the establishment of an aseptic culture make seeds more appropriate than juvenile sprouts from seeds. when tissue culture was initiated from sterilized juvenile plants from the natural sites, the initiation of the culture was problematic because sterilization did not always remove contamination. these sources of contamination include surface sterilization resistant plant endophytic microorganisms and common environmental microorganisms, both of which may become pathogenic in culture (cassells 2012). contamination can resist sterilization and sooner or later damage plant material. cover contamination makes juvenile plants taken from natural sites the least appropriate for tissue culture initiation. shoot multiplication the development of new shoots was observed on almost all media tested, even on the ms medium free of growth regulators. growth regulators on optimal media more than tripled the number of shoots. the best proliferation of shoots was obtained on the ms medium with 5 μm bap and 3 μm, and slightly worse on the ms medium with 10 μm bap and 2 μm iba. a combination of bap and iba was used for shoot proliferation of apiaceae anethum graveolens (sharma et al. 2004) and centella asiatica (banerjee et al. 1999), fi rst in a combination of 2.2 μm bap and 0.5 μm iba, and second in a combination of 8.9 μm bap and 0.5 μm iba (banerjee et al. 1999, sharma et al. 2004). other researchers (hosain et al. 2000, nath and buragohain 2003, sharma and wakhlu 2003, karuppusamy et al. 2007, das et al. 2008, jaheduzzaman et al. 2012) used a combination of bap and naa or a combination of bap ambrožič-dolinšek j., ciringer t., kaligarič m. 250 acta bot. croat. 75 (2), 2016 and iaa (gaddaguti et al. 2013) for proliferation of shoots in apiaceaes and an optimal concentration ranging from 2 to 17.5 μm of bap and from 0.5 to 2.7 μm of naa. the combination of bap with naa in our case was not as effective in shoot regeneration. growth regulators infl uence the shoot length of h. pastinacifolia. with an increased concentration of bap, when bap alone was added to the media, smaller shoots developed. however, the trend toward shoot lowering was not evident when bap was combined with different concentrations of auxin iba (data not shown). the opposite trend was observed when 2ip alone was added to the media, where with the increased concentration of 2ip, taller shoots developed (data not shown). sporadically observed fl ower development, instead of vegetative shoots, indicates the end of the life cycle for these plants. since it occurred infrequently, it did not disturb the micropropagation procedure. flower shoots with infl orescences were previously observed in apiaceae tissue culture (tavares et al. 2010a). root initiation rooting of shoots during propagation is genotype-dependent, and shoots of some species which root naturally without a rooting stage need a special rooting medium, with or without growth regulators. hladnikia pastinatifolia shoots developed roots only on the ms medium with growth regulators. optimal rooting was obtained on ms medium with 2 μm iba. concentrations higher than 5 μm iba induced both roots and the development of callus. root development with an intermediary callus indicated that at that roots and shoots were not well connected with vascular tissue and that plants are insuffi ciently supplied with nutrients (george 1993). successful rooting in other members of the apiaceae family was also achieved with 1–5 μm iba (tavares et al. 2010a, das et al. 2008, jana and shekhawat 2011, coste et al. 2012, jaheduzzaman et al. 2012), and with higher concentrations of iba (hossain et al. 2000, sharma and wakhlu 2001, banerjee et al. 1999, tavares et al. 2009–2010), with naa or iaa (karuppusamy et al. 2007, jaheduzzaman et al. 2012) and only rarely without growth regulators (makunga et al. 2003). acclimatization and transfer to in vivo conditions acclimatization of h. pastinacifolia required a specially prepared substrate. the substrates usually used for acclimatization with other members of that family (sharma and wakhlu 2001, nath and buragohain 2003, tavares et al. 2010a, coste et al. 2012, hendrawati et al. 2012, jaheduzzaman et al. 2012) were far different from the substrate which promoted acclimatization of our plant species. h. pastinacifolia did not acclimatize at all if the substrates were rich in organic matter (data not shown). that is why we tested two artifi cial substrates, which imitated the crumbling granulated texture and the alkaline nature of the substrate from the natural sites (čušin 2004). we used two mixtures composed of limestone sand, potting soil and vermiculite in different ratios. our plants survived better in the substrate with a more alkaline reaction, with a ph between 7.5 and 8.5. nevertheless, less than half the shoots were successfully acclimatized. plants of h. pastiancifolia obtained by this procedure are now growing in outside conditions, showing the potential of this method to increase the number of plants available for conservation purposes. do we need alternative ex situ conservation programs for alpine plants, especially endemic species like h. pastinacifolia? alpine plants and their habitats are extremely fragile, easily disturbed and prone to any kind of climate change effects. during the past few decades, human activity has increased in the alpine environment, and disturbance is probably the greatest threat to alpine plants. protected areas with specifi c conservation measures are an effi cient way to protect alpine biodiversity, but uncertainty still persists, owing to a range of different climate-change driven effects. more than half the species growing within the european natura 2000 network might lose suitable habitats in these areas. hence, it will be necessary to prepare alternative ex situ measures (vittoz et al. 2013), of which the micropropagation presented in this paper is claimed to be the most effi cient. conclusion as an ancient paleoendemic and one of the most remarkable among the pleistocene survivors, h. pastinacifolia is of great importance for understanding the effects of changing environment and deserves further research efforts in the direction of biodiversity studies and conservation programs (šajna 2012). in our study we established a protocol for in vitro propagation of hladnikia pastinacifolia, from preparation of plant material, establishment of aseptic culture, multiplication, in vitro rooting, transplanting and acclimatization of plants. furthermore, tissue culture collection has been successfully propagated continuously since 2006. we have maintained it for 10 years without loss of either juvenile character or shoot and root proliferation capacity, the only exception being the sporadically observed fl ower development, which indicates the end of the life cycle for these plants. since the rest of the material retained its juvenility, we can confi rm that we did achieve the goal of having a long-term collection of h. pastinacifolia in tissue culture. in 2013, we additionally started a tissue culture of h. pastinacifolia and obtained several new lines. with these lines, we additionally tested the pre-designed micropropagation protocol, with repeated results. the tissue culture of h. pastinacifolia represents a living collection or back-up gene pool of this species. new plants can be used for several purposes: reintroduction to nature, assisted migration, strengthening the natural population, for ex situ collections in botanical or rock gardens, for studies on the ecology and biology of species, for its nutritional value, or simply for educational and ornamental purposes. further studies on this species will focus on other specifi c biotechnological tools successfully used in several programs of plant conservation, like assessment of the degree of genetic variability of plants in culture and introduction of techniques for long-term storage at low temperatures. micropropagation of endemic hladnikia pastinacifolia acta bot. croat. 75 (2), 2016 251 acknowledgements we would like to thank nina šajna for collecting plant material at the natural sites. this research was supported by the slovene ministry of higher education, 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[texts], lenarčič, m. [photos]: the alps: a bird’s-eye view, 479–481. panalp, nazarje. untitled acta bot. croat. 75 (1), 2016 17 acta bot. croat. 75 (1), 17–24, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0016 issn 0365-0588 eissn 1847-8476 selenium uptake and se compounds in se-treated buckwheat aleksandra golob1, mateja germ1, ivan kreft2, igor zelnik1, urška kristan3, vekoslava stibilj3* 1 university of ljubljana, biotechnical faculty, jamnikarjeva 101, si-1000 ljubljana, slovenia 2 slovenian forestry institute, večna pot 2, si-1000 ljubljana, slovenia 3 jožef stefan institute, jamova 39, si-1000 ljubljana, slovenia abstract – in fi eld experiments, tartary buckwheat and hybrid buckwheat were foliarly sprayed with an aqueous solution of sodium selenate (20 mg se l–1). in treated plants, the selenium content was signifi cantly higher than in controls, irrespective of the plant part and taxon of buckwheat. the highest average se concentrations in hybrid and tartary buckwheat were found in seeds. the main se species found in seeds was semethionine. selenium-sprayed plants had higher photochemical effi ciency of photosystem ii in both taxa and higher electron transport system activity in hybrid buckwheat, suggesting a positive effect of se on physiological characteristics. because of the concentration of se in both buckwheat taxa and selenomethionine as the dominant species of se, se-enriched buckwheat is a potential source of dietary se for animals and humans. keywords: hybrid buckwheat, selenite, semet, speciation, tartary buckwheat abbreviations: ets – electron transport system, fv, fm, f0 – variable, maximal and minimal chlorophyll fl uorescence level from dark-adapted leaves, psii – photosystem ii, se – selenium, semet – se-methionine * corresponding author, e-mail: vekoslava.stibilj@ijs.si introduction buckwheat is becoming a very important alternative crop in europe because it is well suited to ecological cultivation, and its use in the diet has many positive effects on the health and nutritional status of consumers (bonafaccia et al. 2003b). it has favourable medicinal properties, because of its antioxidative (holasova et al. 2002), anti-infl ammatory and anti-carcinogenic effects and because it can increase the strength of blood vessels. buckwheat also has signifi cant health benefi ts for individuals with diabetes, obesity and constipation (li and zhang 2001). the buckwheat herb, especially tartary buckwheat (fagopyrum tataricum gaertn.), known to be a rich source of fl avonoids, is used in herbal medicinal products, for green buckwheat tea, for producing buckwheat green leaf fl our as an additive to some food products. in addition the fresh green plant parts are used as a vegetable (fabjan et al. 2003, kreft et al. 2006). tartary buckwheat seeds contain proteins with a high biological value and a well-balanced amino acid composition, relatively high crude fi ber content and vitamins b1, b2 and b6 (bonafaccia et al. 2003b). its seeds are also an important dietary source of zn, cu and mn (bonafaccia et al. 2003a, pongrac et al. 2011) and of phenolic compounds with antioxidant properties, such as rutin, quercetin and quercitrin (fabjan et al. 2003). tartary buckwheat is grown and used in mountainous regions of south-west china (sichuan), in northern india, in bhutan and nepal (bonafaccia et al. 2003b). in europe, tartary buckwheat is currently grown as a crop only in a small area of north-west europe (bonafaccia et al. 2003a), and recently in slovenia and sweden. under the name fagopyrum hybridum the most productive progenies f10 and later generations of hybrids f. tataricum (4x = 32) × f. giganteum are united. the taste and other characteristics of hybrid buckwheat grain are mostly similar to those of tetraploid bitter buckwheat. it possesses a rough grain of intermediate type with a high fraction of hulls (fesenko and fesenko 2010). since hybrid buckwheat is a new buckwheat taxon, recently obtained in orel, russia, by interspecifi c crossing (fesenko and fesenko 2010), very little is known about its characteristics and properties. selenium (se) is an element essential in humans and animals for normal functioning of a number of se-dependent antioxidant enzymes, such as glutathione peroxidase and thioredoxin reductase (brown and arthur 2001). it also exerts benefi cial effects in cancer prevention and can posigolob a., germ m., kreft i., zelnik i., kristan u., stibilj v. 18 acta bot. croat. 75 (1), 2016 tively infl uence many functions by reducing infl ammation, and heart disease and regulating blood pressure (brozmanová et al. 2010). this element, however, can also be toxic in higher amounts, its toxicity depending on its chemical form, concentration and other environmentally regulating variables. worldwide, defi ciency of se in human diet is common and natural supplements have been recommended to increase daily se intake (pyrzynska 2009). defi ciency of se is associated with many diseases and with deterioration in the health status of animals and humans. the se supply in almost all european countries is below the recommended daily intake, which is 30–70 μg se per day for adult europeans (german nutrition society 2002). in two separate locations around the rivers drava and sava in eastern croatia, the mean content of selenium in foods was measured and the authors concluded that the dietary se intake in both areas is likely to be between suboptimal and adequate (klapec et al. 2004). in another study in croatia, matek et al. (2000) produced a report on the estimated dietary se intake, which showed that se intake for the observed group of females from the zagreb area is lower than in the majority of european countries, and lower than the value recommended by the world health organisation. the authors claimed however that the risk for the inhabitants of the zone of developing se defi ciency is low. se bioavailability strongly depends on the chemical form of se found in the diet. organic se compounds are more bioavailable than inorganic forms. se-enriched plants seem an ideal source of se for animals and humans, because they contain many different chemical forms of se, many of which are highly bioavailable (navarro-alarcon and cabrera-vique 2008). the se content in vegetables from many areas of the world refl ects low levels in soil, and hence crops and food enriched with se during cultivation could be an effective means of producing se-rich foodstuffs (pyrzynska 2009). foliar spraying of buckwheat plants with a solution of sodium selenate at the beginning of fl owering has a benefi cial effect on the se content in all above-ground plant organs, such as seeds, leaves, and stems (smrkolj et al. 2006b, vogrinčič et al. 2009). as plants are the main source of dietary se, metabolism of se in plants is important for the se nutrition of humans and animals. knowledge of locations of se accumulation and its speciation is important because the anti-carcinogenic effects of se depend on the species (carey et al. 2012). the essential nature of se to plants remains unclear. even though there is some evidence for its positive effect at low concentrations on many plant processes (germ et al. 2007a), at higher concentrations it can induce negative effects on plant physiology (hartikainen et al. 2000). some research work has proposed that se at low concentrations can increase the tolerance of plants exposed to various abiotic stressors (germ et al. 2007a, djanaguiraman et al. 2010). thus the purpose of the current study was to determine the effect of foliar spraying with se-containing compounds on the physiological responses of plants determined by measurements of primary and terminal fl ow of electrons, on the content and the species of se in the above-ground parts of the new and promising hybrid buckwheat and to compare the results with those in tartary buckwheat. because of the potential toxicity of se we will examine the plant vitality and the end of vegetative phenological phase by measuring electron transport activity. we hypothesized that se treatment would signifi cantly benefi t physiological plant responses, including photochemical effi ciency of photosystem ii (psii) and respiratory potential, in tartary as well as in hybrid buckwheat. we also hypothesized that se treatment would increase the content of bioavailable se in seeds of both buckwheat taxa. material and methods plant material and growth conditions tartary (f. tataricum) and hybrid (f. hybridum) buckwheat seeds were sown on a 10 m2 experimental plot of the biotechnical faculty of the university of ljubljana, ljubljana. the experiment lasted from late june to late september, during which time the mean temperature was 21.1 °c and the mean humidity 67% (http://meteo.arso.gov.si/ met/sl/ archive/). the control and treated groups of both buckwheat taxa were grown under the same conditions. at the beginning of fl owering, half of the plants, the treated groups, were sprayed with a sodium selenate solution containing 20 mg l–1 of se and a detergent (0.2 ml l–1 triton x-100, sigma), while control groups were sprayed only with detergent. two weeks after se spraying, chlorophyll fl uorescence and terminal electron transport system (ets) activity were measured. from the date of spraying with se to the date of measurement of chlorophyll fl uorescence and ets activity the mean temperature was 24.5 °c and the mean humidity 62.3%. in that time there was a total of 181 hours of sunshine (http://meteo.arso.gov.si/met/sl/ archive/). thirteen weeks after sowing, when the majority of grains reached their ripe phase, samples of both taxa and both groups were collected. for each taxon and treatment one sample of plants (10–15 plants) was taken for determination of se content and se speciation. plants were air-dried and separated into individual parts: stems, leaves, seeds and husks and lyophilized using a christ alpha freeze dryer, then homogenized in an agate planar micro mill at 2600 rpm (fritsch, pulverisette 7, idar-oberstein, germany). determination of physiological parameters fluorescence measurement, a non-destructive method, allows the rapid assessment of the quantum yield of electron fl ow through the psii. this method has been widely used for detection of various stresses in plants. murchie and lawson (2013) reported that chlorophyll fl uorescence can be used to determine the primary signals by active and passive methods. chlorophyll fl uorescence was measured in situ with a fl uorometer (pam 2500 portable chlorophyll fluorometer, walz) in 6 vital plants (on the fi rst fully developed leaf) from each treatment group and from both taxa of buckwheat. measurements of minimal (f0) and maximal (fm) chlorophyll fl uorescence were made after 15 min of darkness, provided by dark-adaptation clips. fluorescence selenium compounds in buckwheat acta bot. croat. 75 (1), 2016 19 was excited with a saturating beam of “white light” (ppfd = 8000 μmol m–2 s–1, 0.8 s). the difference between fm and f0 is termed the variable fl uorescence (fv). the potential photochemical effi ciency of psii was evaluated in terms of the ratio fv/fm. the effective photochemical effi ciency was determined after saturation with a 0.8 s pulse of white light (ppfd = 9000 μmol m–2 s–1). the effective photochemical effi ciency was calculated as (fm’ – f)/fm’ = ∆f/fm’, where fm’ is the maximum fl uorescence signal of an illuminated leaf after a pulse of saturating light and f is the steady state fl uorescence (schreiber et al. 1996). the respiratory potential of mitochondria was measured in the plants by terminal electron transport system (ets) activity as described by packard (1971) and modifi ed by kenner and ahmed (1975). fresh leaves of plants (0.006 – 0.012 g) were homogenized in a fi nal volume of 4 ml of ice-cold homogenization buffer (ph = 8.4), followed by ultrasonic homogenization (4710, cole-parmer, vernon hills, il, usa) for 20 sec at 40 w. the homogenates were centrifuged for 4 min at 0 °c at 10000 rpm (sigma 2–16 pk, germany). within 10 min, 0.5 ml of supernatant (in triplicate) was added to a mixture of 1.5 ml substrate solution containing nadh, nadph, triton x-100, and 0.5 ml 2-(piodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (int) reagent solution and incubated for 40 min at 20 °c. formazan production was determined spectrophotometrically (lambda 12, perkin-elmer, norwalk, ct, usa) from the absorbance of the sample at 490 nm against the blank within 10 min of stopping the reaction with a 1:1 mixture of formaldehyde and phosphoric acid. ets activity per dry weight (dw) was calculated according to kenner and ahmed (1975). ets activity was measured on 5 plants from each treatment group and from both taxa of buckwheat. determination of total se content the total se content was determined using hydride generation atomic fl uorescence spectrometry (hg-afs) in all plant parts of se-treated and control buckwheat. we weighed 0.2 g of a lyophilized sample into a tefl on tube and se content was determined in three aliquots. digestion of samples was carried out in the closed tubes with a mixture of h2so4, hno3, h2o2 and v2o5. hf was added only to samples containing fi bers. subsequently, the reduction of se(vi) to se(iv) was carried out by adding concentrated hcl and heating at 90 °c for 10 min. after digestion and reduction of samples, they were diluted with milli-q water and the se content was determined by hg-afs. details of the method of digestion and optimal measurement conditions have been described in detail by smrkolj and stibilj (2004). the accuracy of the method was checked with the certifi ed reference material spinach leaves (national institute of standards and technology – nist 1570a) and good agreement was found between the values obtained (115 ± 2 ng se g–1) and the certifi ed value (117 ± 9 ng se g–1). se in residues from the enzymatic extraction process was determined by the same procedure mentioned above. for total se determination in supernatants from the enzymatic extraction process, 1 ml of concentrated hno3 was added to 0.5 g of supernatant. the mixture was heated for 30 min at 80 °c and then for 15 min at 160 °c in a closed tefl on tube. after cooling, 0.5 ml h2o2 was added followed by heating of the mixture for 10 min at 120 °c, and this procedure was repeated twice more. afterwards the reduction of se(vi) to se(iv) was carried out by the addition of concentrated hcl and heating at 90 °c (pograjc et al. 2012). selenium species determination soluble se species were extracted from samples by enzymatic hydrolysis. the sample (0.6 g) and a solution (8 g) containing 60 mg of the enzyme protease xiv were placed in a centrifuge tube and the mixture was shaken for 24 h in a water bath at 37 °c at 200 rpm. after extraction the samples were centrifuged at 11000 rpm at 4 °c for 60 min (5804r eppendorf). the supernatants were separated from the residue and fi ltered successively through 0.45 μm and 0.22 μm millex gv fi lters (millipore corporation). supernatants and residues for total se determination were stored at –20 °c prior to analysis. working standard solutions in milli-q water of 100 ng g–1 of se species containing selenite (se(iv)), selenate (se(vi)), se-methionine (semet), se-cystine (secys2) and se-methylselenocysteine (semesecys) were prepared daily by dilution of a stock solution (10 μg g–1). the concentrations of extracted se species were determined using an ion-exchange high-performance liquid chromatography system coupled to an inductively coupled plasma mass spectrometry (hplc-icp-ms) apparatus (agilent 7500ce, tokyo, japan). the se species were separated on a hamilton prp-x 100 anion exchange column and a zorbax 300-scx cation exchange column. we used 1 mm and 10 mm citrate buffers in 2% methanol as the successive mobile phases for anion-exchange chromatography, and 3 mm pyridine buffer in 2% methanol as mobile phase for cation-exchange chromatography. the method and optimal operating conditions have been described in detail elsewhere (cuderman et al. 2008). the accuracy and precision of the results obtained for se species determination were tested using the reference material wheat gluten (nist 8418), for which semet data can be found in the literature. we found good agreement between our values (1.39 ± 0.08 μg g–1) and the published values (1.53 ± 0.03 μg g–1) obtained by wolf and goldschmidt (2007). statistical analysis for statistical analysis spss statistic software, version 20, (ibm) was used. the normal distribution of the data was tested with shapiro-wilk tests. differences in ets activity and in chlorophyll fl uorescence between control and treated plants were evaluated by one-way anova followed by tukey’s post-hoc multiple comparison tests. differences at p < 0.05 were considered to be statistically signifi cant. for se concentration, only one sample was made for each treatment and each taxon, analysed in three aliquots, making it impossible to perform statistical analysis of the data. results were given as means of three aliquots and standard deviation. golob a., germ m., kreft i., zelnik i., kristan u., stibilj v. 20 acta bot. croat. 75 (1), 2016 results plant vitality after se treatment hybrid buckwheat had a signifi cantly higher fv/fm than tartary buckwheat in the control as well as in the se-treated groups. addition of se signifi cantly increased the potential photochemical effi ciency in both taxa of buckwheat (fig. 1). in addition to the potential photochemical effi ciency, we also monitored the effective photochemical effi ciency of psii. however, there were no statistically signifi cant differences in effective photochemical effi ciency between control and treated groups of both taxa (data not shown). hybrid buckwheat had a higher ets activity than tartary buckwheat in both (control and treated) groups of plants (fig. 2). addition of se signifi cantly increased ets activity in hybrid buckwheat but not in tartary buckwheat. concentration of se in different plant parts the se content in the control groups of both buckwheat taxa was low (< 0.05 μg se g–1) in all plant parts. the measured values were near or below the detection limit, which was between 0.01 and 0.02 μg se g–1 dry weight. the content of se was about 30% lower in all plant parts of hybrid buckwheat in comparison to tartary buckwheat in se-treated plants (fig. 3). foliar fertilization with sodium selenate increased the content of se in all plant parts, especially in leaves and seeds where values in the treated groups were 186-fold (leaves) and 64-fold (seeds) higher than in the control groups for hybrid buckwheat and 83-fold (leaves) and 276-fold (seeds) higher for tartary buckwheat (fig. 3). in se-treated buckwheat the leaves and seeds within each taxon contained similar concentrations of se, while in stems and husks lower concentrations of se than in seeds and leaves were found (fig. 3). a similar trend in the distribution of se was also observed in both control groups of buckwheat (tab. 1). selenium speciation in se-enriched buckwheat seeds, approximately 47% (hybrid buckwheat) and 44% (tartary buckwheat) of the total se found in seeds was soluble (tab. 2). to determine if losses of se occur during enzymatic extraction, a mass balance was drawn up between the total se in the lyophilized sample and the content of se in the supernatant and residue. the mass balance for seeds and leaves of both taxa was approximately 100%, indicating that there was no loss of se during extraction. the only soluble se species identifi ed in seeds by hplc-icp-ms was semet. semet in supernatant was confi rmed by the standard addition method (fig. 4). semet represented only 37% of the total se content in seeds of hybrid buckwheat and 32% of the total se content in 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 se treated control p o te n ti a l p h o to c h e m ic a l e ff ic ie n c y o f p s ii f. hybridum f. tataricum a b b c f. hybridum f. tataricum steams leaves seeds husks s e c o n te n t (µ g s e g d w ) – 1 0 0.5 1 1.5 2 2.5 3 3.5 4 se treated control f. hybridum f. tataricum 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 e t s (µ l o m g h ) 2 – 1 – 1 d w a c b c fig. 1. potential photochemical effi ciency of photosystem ii (psii) in selenium-treated hybrid (fagopyrum hybridum) and tartary (f. tataricum) buckwheat. columns not sharing the same letter are signifi cantly different (p < 0.05), (n = 6). fig. 3. distribution of selenium in plant parts of selenium-treated hybrid (fagopyrum hybridum) and tartary (f. tataricum) buckwheat. data represents means ± standard deviations of three aliquots of composite sample. dw – dry weight. fig. 2. terminal electron transport system (ets) activity in selenium-treated hybrid (fagopyrum hybridum) and tartary (f. tataricum) buckwheat. columns not sharing the same letter are signifi cantly different (p < 0.05), (n = 5). dw – dry weight. tab. 1. concentration of selenium (μg g–1 dw) in controls and in selenium-enriched samples of hybrid and tartary buckwheat. results are expressed as the average of three measurements of one sample ± standard deviation. dw – dry weight, se – selenium. plant part hybrid buckwheat tartary buckwheat control se-enriched control se-enriched stems 0.011±0.007 0.40±0.03 0.019±0.0007 0.95±0.18 leaves 0.005±0.001 0.93±0.05 0.041±0.002 3.39±0.16 seeds 0.019±0.004 1.22±0.06 0.013±0.006 3.59±0.52 husks 0.005±0.004 0.31±0.04 0.048±0.009 1.08±0.30 selenium compounds in buckwheat acta bot. croat. 75 (1), 2016 21 seeds of tartary buckwheat (tab. 2). semet in the extract was stable, since the peaks of semet in extracts from both taxa two and four days after extraction were comparable. after enzymatic hydrolysis of se-enriched buckwheat leaves, only 6.6% of the se was present in a soluble form in hybrid buckwheat and 3.7% in tartary buckwheat, but using hplc-icp-ms under the described conditions, we were unable to identify any of the se species in the supernatants. discussion plant vitality after se treatment the control and treated groups of hybrid and tartary buckwheat had a lower potential photochemical effi ciency (fv/fm) than the theoretical maximum value (fig. 1), which ranges from 0.80 to 0.83 for a variety of unstressed dark adapted plants (schreiber et al. 1996). the values of fv/fm refl ect the maximum effi ciency at which light absorbed by light harvesting antennae of psii is converted to chemical energy (baker and rosenqvist 2004). the fv/fm ratio refl ects photooxidative damage of psii occurring during stress (critchley 1998). higher values of fv/fm in hybrid buckwheat in comparison to tartary buckwheat in the control as well as in the se-treated groups could mean that the hybrid buckwheat has a better ability than tartary buckwheat to suppress photoinhibition reactions in the case of stressful environmental conditions, which would represent an advantage of this taxon in increasingly stressful environmental conditions. the addition of se signifi cantly increased the potential photochemical effi ciency in both taxa of buckwheat (fig. 1), suggesting a positive effect of se in reducing the photoinhibitory effects of environmental stressors. potential photochemical effi ciency was also higher in the foliarly setreated progeny of tartary buckwheat plants sprayed with 10 mg se(vi) l–1 than the controls in the study from kreft et al. (2013). a positive effect of se on fv/fm was demonstrated in the cultivation of strawberries in soil enriched with se (0.1 mg se kg–1soil and 1 mg se kg–1 soil in the form of h2seo4), but the same treatment had no positive effect on barley (valkama et al. 2003). zhang et al. (2014) found out that soil application of selenite (50 g se h–1) enhanced the activity of the photosynthetic system by increasing fv/fm. in the study by diao et al. (2014) addition of 0.05 mm se (na2seo3) in hydroponic experiments caused an increase in the maximum quantum yield of psii (fv/fm) in two cultivars of tomato. further, foliar spraying with naselenate solution did not infl uence the potential photochemical effi ciency in pumpkins (1.5 mg se l–1) (germ 2005), in chicory (1 mg se l–1) (germ et al. 2007b), in common buckwheat (1 mg se l–1) (breznik et al. 2005, tadina et al. 2007), red cabbage (soil fertilized 30 times with se (vi) 2 μg l–1, or twice with 0.5 mg l–1 se) (mechora et al. 2011), or in cucumber plants (2–80 μm of se) under hydroponic conditions (hawrylak-nowak et al. 2015). the general metabolic activity of different organisms may be assessed by measurement of the ets activity in mitochondria (packard 1971). the addition of se enhanced ets activity in hybrid buckwheat but not in tartary buckwheat. tadina et al. (2007) studied the effect of se addition on two cultivars of common buckwheat, pyra and siva. foliar spraying with selenate (1 mg l–1) signifi cantly increased ets activity in the cultivar siva and decreased it in the cultivar pyra. however, breznik et al. (2005) did not notice signifi cant differences in ets activity between control and se treated (foliar application of na-selenate solution at a concentration 1 mg l–1) groups of tartary buckwheat or of common buckwheat. foliar treatment with selenate (1.5 mg l–1) had no effect on ets activity in pumpkin plants (germ 2005). in accordance with our results, the addition of se increased ets activity in chicory (foliarly sprayed with na-selenate solution with concentra0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 0 1000 retention time (s) sample sample + 100 ng/g semet sample + 200 ng/g semet in te n s it y 7 8 s e fig. 4. chromatogram of selenium (se) species in enzyme hydrolysis extracts of the seeds of foliarly treated tartary buckwheat (semet, se-methionine) on the anion column (prp-x 100), with addition of standard solution of semet. tab. 2. selenium (se) speciation and selenium content (μg g–1 dw) in supernatants and residues of se-enriched hybrid (fagopyrum hybridum) and tartary (f. tataricum) buckwheat seeds and leaves. results are expressed as the average of three measurements of one sample ± standard deviation; nd – not detected; * according to total se content. dw – dry weight, semet – se-methionine. plant part se content (μg g–1) % of soluble se semet residue supernatant (μg g–1) (%)* seeds f. hybridum 0.63±0.048 0.57±0.012 47.5 0.45±0.074 37.5 f. tataricum 2.10±0.044 1.70±0.080 44.7 1.22±0.018 32.1 leaves f. hybridum 0.99±0.048 0.07±0.006 6.6 nd nd f. tataricum 3.6±0.19 0.14±0.012 3.7 nd nd golob a., germ m., kreft i., zelnik i., kristan u., stibilj v. 22 acta bot. croat. 75 (1), 2016 tion 1 mg l–1) (germ et al. 2007b), in foliarly sprayed young pea plants (10 mg l–1) (smrkolj et al. 2006a) and three weeks after germination in the se-treated progeny of tartary buckwheat plants (kreft et al. 2013). a possible explanation for the increased ets activity in se-treated buckwheat plants could be increased glutathione peroxidase (gpx) activity in mitochondria. hartikainen et al. (2000) observed that se exposure induced gpx activity in ryegrass and hawrylak-nowak et al. (2015) investigated the effect of selenite or selenate with different concentrations (2–80 μm) on cucumber plants. the root respiratory activity considerably increased with increasing selenite concentrations, suggesting the upregulation of mitochondrial dehydrogenases activity. increased ets activity in hybrid buckwheat by the addition of se is in line with fi ndings that the addition of se to vigna radiata enhanced respiratory as well as succinate dehydrogenase activity and the involvement of se in mitochondrial membrane functions (easwari and lalitha 1995). germ et al. (2009) also observed that se is involved in activation of energy resources in green alga zygnema sp. concentration of se in different plant parts the se content in plants is largely dependent on the se content in soil. se concentrations in cultivated plants are usually low (< 0.1 μg se g–1) (ihnat 1989). our results revealed that hybrid and tartary buckwheat were moderate sources of se (tab. 1). for comparison, the se content in seafood ranges from 0.4 to 1.5, in meat from 0.1 to 0.4 and in fruits and vegetables less than 0.1 μg se g–1, all on a dry weight basis (navarro-alarcon and cabrera-vique 2008). all plant parts in both buckwheat taxa treated with se showed the ability of buckwheat to accumulate high concentrations of se. the highest concentration of se in se enriched plants was found in seeds and leaves while considerably lower concentrations were found in stems and husks in both taxa studied. similar distribution patterns of se in common buckwheat plants were observed by vogrinčič et al. (2009), who used a lower concentration of se for foliar spraying. higher contents of se in seeds and leaves than in stems was also observed by ožbolt et al. (2008) in an experiment in which buckwheat was grown from seeds previously soaked in solutions of sodium selenate and sodium selenite. the results indicated that selenate applied by foliar spraying was effi ciently transported from leaves to seeds. similar conclusions were obtained for tartary buckwheat in the study performed by golob et al. (2015) who added se to soil in concentrations of 0.002, 0.5 and 10 mg l–1 or sprayed plants with se at concentrations of 0.5 mg l–1. successful transport of se from leaves to seeds of a foliarly sprayed barley crop was also observed by macleod et al. (1998). it was found that hybrid buckwheat had signifi cantly lower concentrations of se in stems, leaves, seeds and husks than tartary buckwheat. hybrid buckwheat displayed a higher photochemical effi ciency of psii (fig. 1) and a higher respiratory potential (fig. 2) than tartary buckwheat in both control and treated groups. these results indicated that hybrid buckwheat was less sensitive to selenium spraying and was more metabolically active than tartary buckwheat. selenium speciation in se-enriched buckwheat seeds around 47% (hybrid buckwheat) and 44% (tartary buckwheat) of the total se found in seeds was soluble (tab. 2). for comparison, smrkolj et al. (2006b) reported that the great majority of se(vi) added to tartary buckwheat by foliar application was transformed to semet, 99% of the total se in seeds. vogrinčič et al. (2009) also observed a higher percentage of semet, 59% of the total se content in common buckwheat seeds, compared to the present results. the lower percentage of soluble se in seeds in the present study could be due to the use of other plant taxa, a different concentration of se in the foliar solution or different growing conditions. the experiments of smrkolj et al. (2006b) were carried out under controlled conditions in the laboratory, whereas our experiment took place outdoors in the fi eld, where plants were exposed to varying weather and radiation conditions, and to other factors that can infl uence plant metabolism. good conversion of selenate to selenomethionine in seeds was also demonstrated in cereals. stadlober et al. (2001) reported that semet is the predominant form of se found in seeds of cereals (wheat, barley and rye) grown in soil to which fertilizer supplemented with selenate had been added. the results showed that the majority of se in leaves was insoluble, and as such could not be harmful to the plants, although in tartary buckwheat the concentration of se was high, > 3 μg g–1. these results are consistent with those of smrkolj et al. (2006b), who found about 7% of soluble se in buckwheat leaves which had been foliarly sprayed, (15 mg se l–1), of which only 3% was identifi ed as se(vi). vogrinčič et al. (2009) also obtained similar results for the solubility of se in se-enriched common buckwheat leaves (foliar spraying, 10 mg se l–1). after enzymatic hydrolysis with protease xiv they found around 14% of soluble se. because of the concentration of se in both buckwheat taxa and semet as the dominant species of se, se-enriched buckwheat is a potential source of dietary se for animals and humans. hybrid buckwheat responded to enhanced levels of se by higher photochemical effi ciency of psii and by enhanced potential respiratory activity, but tartary buckwheat only by higher photochemical effi ciency of psii. this revealed that se at this concentration did not have negative effects on the physiological characteristics of buckwheat plants. on the contrary, additional se increased the vitality of the plants. a large proportion of the soluble se in se-enriched buckwheat seeds was in the form of semet, which is known to be the major nutritional source of se for animals and humans. all this indicates that se-enriched buckwheat could form a potential source of supplementary dietary se for animals and humans. in all plant parts of hybrid buckwheat we found about one third lower se concentrations than in tartary buckwheat. the data presented in this study constitute the fi rst report on potential and effective photochemical effi ciency of psii, on terminal electron transport system (ets) activity in mitochondria and on se uptake and distribution as well as se speciation in plant parts for hybrid buckwheat. selenium compounds in buckwheat acta bot. croat. 75 (1), 2016 23 acknowledgments this research was fi nanced by the ministry of education, science and sport, republic of slovenia, through the programmes “biology of plants” (p1-0212), “young researchers” (34326), “nutrition and public health” (p30395), and projects j4-4224 and j4-5524 and supported by the european project euforinno (regpot no. 315982) of the fp7 infrastructures program. the authors are grateful to anthony r. byrne and bill milne for revising the english text. references baker, n. r., rosenqvist, e., 2004: applications of chlorophyll fl uorescence can improve crop production strategies: an examination of future possibilities. journal of experimental botany 55, 1607–1621. bonafaccia, g., gambelli, l., fabjan, n., kreft, i., 2003a: trace elements in fl our and bran from common and tartary buckwheat. food chemistry 83, 1–5. bonafaccia, g., marocchini, m., kreft, i., 2003b: composition and technological properties of the fl our and bran from common and tartary buckwheat. food chemistry 80, 9–15. breznik, b., germ, m., gaberščik, a., kreft, i., 2005: combined effects of elevated uv-b radiation and the addition of selenium on common (fagopyrum esculentum moench) and tartary (fagopyrum tataricum (l.) gaertn.) buckwheat. photosynthetica 43, 583–589. brown, k. m., arthur, j. r., 2001: selenium, selenoproteins and human health: a review. public health nutrition 4, 593–599. brozmanová, j., mániková, d., vlčková, v., chovanec, m., 2010: selenium: a double-edged sword for defense and offence in cancer. archives of toxicology 84, 919–938. carey, a. m., scheckel, k. g., lombi, e., newville, m., choi, y., norton, g. j., price, a. h., meharg, a. a., 2012: grain accumulation of selenium species in rice (oryza sativa l.). environmental science and technology 46, 5557–5564. critchley, c., 1998: photoinhibition. in: raghevendra, a. s. 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bioanalytical chemistry 387, 2449– 2452. acta botanica 1-2017 za web.indd 80 acta bot. croat. 76 (1), 2017 acta bot. croat. 76 (1), 80–90, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0051 issn 0365-0588 eissn 1847-8476 do benthic diatom assemblages refl ect abiotic typology: a case study of croatian streams and rivers koraljka kralj borojević1, marija gligora udovič1*, petar žutinić1, gábor várbíró2, anđelka plenković-moraj1 1 university of zagreb, faculty of science, department of biology, rooseveltov trg 6, hr-10000 zagreb, croatia 2 mta centre for ecological research, danube research institute, department of tisza river research, bem sqr. 18/c, debrecen, h-4026, hungary abstract – benthic diatoms are widely used in europe and worldwide to access ecological status of running waters. one of key goals of water framework directive is to classify rivers and streams using biological quality elements and type specifi c reference conditions. according to system b which incorporates additional abiotic descriptors, there are 24 water types in croatia. for biological analyses 92 rivers and streams with 140 sampling points were chosen and sampled for benthic diatoms and water chemistry simultaneously. self organizing map (som) analysis was used to defi ne biotypes from species composition and abundance of benthic diatoms. grouping of samples in som resulted in 10 distinctive groups. based on their geographical position and site characteristics, groups represent sites with similar properties (as waterbed, catchment size, altitude, size of stream) belonging to different ecoregions in croatia. analysis of variance revealed statistically signifi cant differences (p<0.05) among som groups concerning ammonia, nitrates and total phosphorus. indicator species analysis (indval) singled out species that were signifi cantly characteristic (p<0.05) for som and abiotic types. compared to abiotic groups, in which 7 out of 24 have no indicator species, all som groups have one or several characteristic diatom species, thus indicating diatom assemblages as valuable site descriptors. canonical analysis of principal coordinates analysis also indicated that som grouping of samples is statistically reliable. grouping of similar sites, although placed into different abiotic types, makes som groups with its corresponding representative species an easy tool for water quality assessment and description of reference assemblage. keywords: benthic diatoms, self organizing map, water framework directive, water typology. abbreviations: bmu – best matching unit, som – self organizing map, * corresponding author, e-mail: marija.gligora.udovic@biol.pmf.hr introduction according to water framework directive (wfd), european union member states have to achieve a good ecological status for all streams whose catchment area exceeds 10 km2. since the assessment is to be performed by observing deviations from the reference conditions, one of the key goals of the water framework directive was to classify rivers and streams using biological quality parameters and to describe hydromorphological, physicochemical and biological type specifi c reference conditions (ec 2000). that should be determined using fi ve biological quality elements: phytoplankton, phytobenthos, macrophytes, benthic invertebrates and fi sh. as commonly dominant phytobenthic representatives, benthic diatoms are used worldwide to assess the ecological status of running waters (reid et al. 1995, ács et al. 2005, kireta et al. 2012, martin and reyes fernandez 2012, kahlert et al. 2016). they are widespread and can be found in almost any type of running water which, combined with short generation time and ability to clearly defi ne nutrient status of their habitat, makes them great indicators of water quality. also, they are relatively easy to sample and their ecological valences and habitats have been known for more than 100 years (kolkwitz and marsson 1908). during the past decades numerous indices, mostly based on trophic and saprobic status, have been used specifi cally for that purpose (sládeček 1986, kelly and whitton 1995). unlike the previously set index class boundaries, water quality assessment according to wfd is performed by comparison to reference conditions. as different habitats do benthic diatom assemblages reflect abiotic typology acta bot. croat. 76 (1), 2017 81 in their undisturbed stages support different assemblages, it is essential to describe reference condition for every type of habitat (bennion et al. 2014). as a starting point, all water bodies should be classifi ed according to wfd hierarchical water body typology. it is organized by fi rstly placing the surface water bodies into the broad categories such as rivers, lakes, transitional/coastal bodies, etc. two typologies were acknowledged within those categories: “system a” fi xed typology, and “system b” alternative typology, which comprises a mixture of obligatory and optional factors (like catchment size, elevation, fl ow velocity, granulometric properties of the waterbed, tufa forming conditions, permanence of fl ow, etc.). abiotic typology is then used as a base for describing type specifi c reference conditions. eventually, water quality is determined by a comparison of the actual conditions to the ones that have been referenced. using water type system b, 24 different water types were discerned in croatia (mihaljević et al. 2011). types were fi rst separated by region (continental, pannonian and coastal), then by catchment size, type of waterbed, altitude of stream fl ow and stream fl ow discharge which is determined from the size, altitude and slope (tab. 1). such a high number of types is a refl ection of geographical position and shape of the country of which two thirds belong to karst infl uenced by high mountains separating the coastal area from the continent, along with a rather fl at pannonian area which is extensively used for agriculture. croatia has scarce and non-continuous data on attached diatoms (plenković-moraj 1995) with parts of the country, especially poorly populated areas, that have never been explored. this study yielded the fi rst comprehensive diatom dataset on most waterbodies in croatia. the aim of this paper is to investigate relationship between previously determined 24 types of rivers and streams in croatia and potential biotypes that are derived from benthic diatom assemblage data. considering the number of types in neighbouring countries and previous data, it is expected that diatom assemblages will show regional differences along with some differences from other factors, but will altogether group certain abiotic types and thus reduce them. materials and methods sampling design data on diatom species composition, in the form of proportion of total diatom valves in each sample, has been gathered within the two projects for nation-wide survey covering all relevant important types of running waters along with concurrent and relevant environmental variables. the fi nal goal of those projects was to describe water quality elements for determination of water class category actab. 1. abiotic water types (codes from ab1-ab24) in croatia according to system b with factor values. abiotic type altitude / m catchment / km2 discharge (q / m3 s–1) region waterbed ab1 >600 10–100 q<2 pannonian silicate ab2 200–600 10–100 q<2 pannonian carbonate-silicate ab3 <200 10–100 q<2 pannonian silicate ab4 <200 100–1000 2<q<20 pannonian silicate ab5 <200 1000–10000 q>20 pannonian silicate ab6 <200 1000–10000 q>20 pannonian silicate ab7 <200 1000–10000 q>20 pannonian carbonate ab8 <200 1000–10000 q>20 pannonian silicate ab9 >600 10–100 q<2 continental carbonate ab10 >600 10–100 q<2 continental carbonate ab11 200–600 10–100 q<2 continental carbonate ab12 200–600 100–1000 2<q<20 continental carbonate ab13 200–600 1000–10000 2<q<20 continental carbonate ab14 <200 100–1000 2<q<20 continental carbonate ab15 <200 1000–10000 q>20 continental carbonate-silicate ab16 200–600 10–100 q<2 coastal carbonate ab17 <200 10–100 q<2 coastal carbonate ab18 200–600 10–100 2<q<20 coastal carbonate ab19 <200 100–1000 2<q<20 coastal carbonate ab20 200–600 1000–10000 q>20 coastal carbonate ab21 <200 1000–10000 q>20 coastal carbonate-silicate ab22 200–600 100–1000 2<q<20 coastal carbonate ab23 200–600 10–100 q<2 coastal carbonate-silicate ab24 <200 100–1000 2<q<20 coastal carbonate kralj borojević k., gligora udovič m., žutinić p., várbíró g., plenković-moraj a. 82 acta bot. croat. 76 (1), 2017 cording to the water framework directive. investigations were performed during spring and summer of 2006, 2007 and 2009. altogether, samples were collected on 140 sampling points from 92 different rivers and streams in croatia. diatom sampling, preparation of slides and counting followed cen standards (european committee for standardization 2003, 2004). at least 5 stones were randomly chosen at each site, where diatoms were scraped from a surface area of approximately 10 cm2 using a toothbrush or a scalpel (depending on substrate type). diatoms were acid cleaned (sulphuric acid and sodium nitrate) and mounted in naphrax (brunel microscopes, uk). on each slide at least 400 valves were counted using random transects under light microscopy at a magnifi cation of 1000x. identifi cation of diatoms was performed using krammer and lange-bertalot (1991a, b, 1997a, b) and lange-bertalot (2001). as supportive data, a dataset with physical and chemical variables of the investigated sites was also used. values of water temperature, ph, conductivity, oxygen concentration, and oxygen saturation were measured in situ using a wtw multi 340i handheld multimeter. concentrations of nitrate, nitrite, ammonia, orthophosphate and total phosphorus were determined from the water samples that were simultaneously collected and measured in the laboratory using standard methods (apha 1995). self organizing map in order to analyze the pattern of diatom assemblages self organizing map (som) module was applied according to kohonen (2001). the som is a neural network analysis tool which converts complex statistical relationships between high-dimensional data into a simple, low-dimension by compressing information while preserving the important topological and metric relationships in the original data (kohonen 2001). the som analytical method has been used in diatom ecology describing benthic algal assemblages in france, luxembourg and hungary (rimet et al. 2004, gosselain et al. 2005, varbiro et al. 2007). the data matrix consisted of 141 samples and 182 variables as species, with additional 4 environmental variables. environmental variables used for describing abiotic types were used as nominal variables, and coded as follows: region categories – continental, pannonian and coastal regions as 1, 2 and 3 respectively; catchment size of 10–100 km2 – creeks and small streams (1), 100–1000 km2 – medium sized rivers (2), 1000–10000 km2 – large rivers (3) and >10000 km2 – very large rivers (4); type of waterbed with silicates (1), carbonates (2), organic (3) and mixed (4); altitude of stream’s fl ow – lowland streams <200 m (1), submountain streams 200– 600 m (2) and mountain streams >600 m (3); stream fl ow discharge (determined from the size, altitude and slope) as 3 groups: (1) low (q<2 m3 s–1), (2) medium (2 m3 s–1<q<20 m3 s–1) and high (q>20 m3 s–1). the assemblage data for each taxon were normalized before the rescaling process by logarithmic transformation [log (x=1)]. the som consists of two layers: an input layer containing the samples and its variables, and an output layer containing so-called virtual units (vu). a vu can be considered a virtual sample unit with its virtual set of variables. in selection phase, weights of output layer were randomly assigned. then a sample unit was chosen randomly and a best matching unit (bmu) was selected by calculating the euclidean distance between the weights of the input layer and the weights of the output layer. it is important to note that the selection of the bmus was based exclusively on diatom species abundances and environmental variables were masked out. this was done by applying a mask function that assigns a weight of 1 to the biological variables used for the selection of the soms vu and a weight of 0 to environmental variables. in the learning phase, the bmu weights (variables) in the output layer defi ned in the selection phase were updated with the weights of the input unit. the update process included both biological and environmental weights. the bmu was not the only grid unit updated: a neighborhood was defi ned around the bmu and all units within this neighborhood were also updated. in this way the values of environmental variables can also be visualized on an som that was previously trained, but only with the biological variables (céréghino and park 2009). the resulting hexagon map with its weights was visualized using the som toolbox as component planes. each component plane represents variables that the som algorithm has learned. the som toolbox (http://www.cis.hut.fi /projects/somtoolbox) was used to implement the som in a matlab™ environment. the detailed algorithm of the som can be found in kohonen (2001) and lek and guégan (1999) for ecological applications. to divide the output som into different diatoms groups according to their similarity, a hierarchical cluster analysis of the ward linkage method with euclidean distances was used (park et al. 2004, 2006). after clustering the som the bmus of the original data matrix were calculated, thus we could obtain in which cluster a given sampling event would occur. species which were important and stable for a given cluster were obtained using the indicator value index (indval) according to dufrene and legendre (1997) based on the original data matrix. statistical analysis to test the differences among assigned groups (both biotic and abiotic), one-way analyses of variance (anova) with abiotic and som groups as grouping factors, followed by post-hoc tukey honest signifi cant difference (hsd) tests were employed with a statistically signifi cant value of p<0.05. anovas were performed using statistical program statistica 9.0. primer 6 (clarke and warwick 2001) was used to allocate physical and chemical parameters that had the greatest impact on the assemblage. firstly, the bio-env procedure which allocates the combination of environmental variables explaining the highest proportion of variance within the assemblage was used. for the analysis both bray-curtis similarity matrix of biotic data and of normalized environmental data were used. afterwards, canonical analysis of principal coordinates (cap) was performed. its purpose was to fi nd axes through multivariate cloud of points that either: (i) are the best at discriminating among a priori groups or (ii) have the strongest correlation with some other set of variables. discriminant analysis of cap do benthic diatom assemblages reflect abiotic typology acta bot. croat. 76 (1), 2017 83 procedure was used to identify proportion of the samples that were correctly allocated to both som and abiotic types used as nominal factors of initial matrix. the other way of using cap is to analyze how well multivariate data predict positions of samples along a continuous or quantitative gradient (anderson et al. 2008). for discriminant analysis only bray-curtis similarity matrix of biotic data was used, and for canonical analysis, a matrix with normalized environmental data was used additionally. results typology grouping of samples in som (online suppl. fig. 1) resulted in 10 distinctive biotypes. based on their geographical position and site characteristics, groups represent sites with similar properties (such as waterbed, catchment size, altitude, size of stream) belonging to different ecoregions of croatia (fig. 1). most of the samples of group d1 are small to medium sized streams on higher altitudes on carbonate waterbed in continental region. groups d2, d3 and d4 combine small to medium streams on lower (group d3) and higher altitudes (groups d2 and d4) on silicate (group d2) or carbonate waterbed (groups d3 and d4) mostly in coastal region. groups d5, d6, d8 and d10 combine streams on silica waterbed in continental and pannonian region (group d5, d6, d8, d10) – they differ in size of stream and its altitude. sites from groups d7 and d9 belong to coastal region and combine medium to large lowland rivers (group d7) or small streams in higher altitudes (group d9) on karstic carbonate waterbed (online suppl. tab. 1). environmental variables one-way anova revealed that som groups statistically signifi cantly differ (f=2.16, p<0.001) concerning combination of all environmental variables. to single out variables that contributed mostly towards statistical signifi cance, post-hoc tukey hsd test was performed. it revealed that group 6 is statistically different (p<0.05) from all other groups concerning concentrations of ammonia, nitrates, orthophosphates and total phosphorus. infl uence of nominal variables that were the base for abiotic typology can be seen in component planes of the som for the typological attribute categories (fig. 2). fig. 1. map of croatian investigation sites divided into diatom-based self organizing map (som) groups. som cluster groups are assigned with codes from d1–d10. lines denote different ecoregions of croatia. kralj borojević k., gligora udovič m., žutinić p., várbíró g., plenković-moraj a. 84 acta bot. croat. 76 (1), 2017 among abiotic groups anova also revealed statistically signifi cant differences (f=1.59, p<0.001), but post-hoc tukey hsd test did not single out any particular parameter for any of the groups to be statistically signifi cant (threshold: p<0.05). biotic data one-way anova on diatom abundance data set revealed that for both typologies their corresponding groups statistically signifi cantly differ one from another (f=138.08, p<0.001 and f=2.4, p<0.001, for som and abiotic groups, respectively). indval analysis was employed to discover which of the variables species have signifi cant indicator value for each of the groups. the analysis confi rmed species with highest abundance and/or frequency within each som group as signifi cant representatives of each group (tab. 2), which can also be seen from som component planes (fig. 3). unlike som groups, some of the abiotic groups (groups ab4, ab8, ab9, ab12, ab15, ab18 and ab19) were not represented by any of the species (tab. 3). tab. 2. self organizing map (som) groups with indicator species according to indicator species analysis (indval). abundance is represented as average percentage (%) of total number of valves of particular species for each site belonging to that group ± standard deviation. cluster code species name abundance indval p-value d1 adbi achnanthidium biasolettianum (grunow) bukhtiyarova 74.42±4.09 0.64 <0.001 d2 acaf achnanthidium affi ne (grunow) czarnecki 7.36±3.18 0.41 <0.001 cpla cocconeis placentula ehrenberg 23.51±5.37 0.34 <0.001 d3 caff cymbella affi nis kützing 3.55±0.99 0.23 0.01 cals caloneis sp. 0.09±0.06 0.13 0.05 cyms cymbella sp. 1.32±0.65 0.27 0.01 npal nitzschia palea (kützing) w.smith 2.45±0.71 0.23 0.02 nrhy navicula rhynchocephala kützing 0.22±0.12 0.19 0.01 spho stauroneis phoenicenteron (nitzsch) ehrenberg 0.04±0.03 0.13 0.05 srma staurosira martyi (heribaud) lange-bertalot 0.56±0.31 0.25 0.01 ssmu staurosira mutabilis (w.smith) pfi tzer 1.67±1.02 0.25 0.01 d4 encm encyonopsis microcephala (grunow) krammer 6.8±1.93 0.42 <0.001 d5 cmts cymatopleura sp. 0.04±0.03 0.20 <0.001 gyac gyrosigma acuminatum (kützing) rabenhorst 1.54±0.78 0.23 <0.001 gyrs gyrosigma sp. 0.17±0.11 0.19 <0.001 namp nitzschia amphibia grunow f. amphibia grunow 0.36±0.27 0.19 <0.001 oroe orthoseira roeseana (rabenhorst) o’meara 2.19±1.99 0.20 <0.001 ptds planothidium sp. 0.76±0.46 0.20 <0.001 d6 augr aulacoseira granulata (ehrenberg) simonsen 2.75±1.19 0.30 <0.001 cyls cyclotella sp. 7.68±4.58 0.51 <0.001 fpyg fallacia pygmaea (kützing) stickle & mann ssp. pygmaea lange-bertalot 7.04±2.69 0.56 <0.001 gyat gyrosigma attenuatum (kützing) rabenhorst 1.02±0.61 0.40 <0.001 hhun hippodonta hungarica (grunow) lange-bertalot, metzeltin & witkowski 0.56±0.39 0.34 <0.001 naci nitzschia acicularis (kützing) w.smith 15.23±4.60 0.53 <0.001 nvlc nitzschia valdecostata lange-bertalot et simonsen 2.14±1.05 0.40 <0.001 shan stephanodiscus hantzschii grunow 10.7±6.59 0.62 <0.001 d7 admi achnanthidium minutissimum (kütz.) czarnecki 59.95±7.32 0.31 <0.001 ecae encyonema caespitosum kützing 4.82±2.57 0.53 <0.001 esle encyonema silesiacum (bleisch) d.g.mann 2.55±1.58 0.30 <0.001 fig. 2. component planes of the self organizing map (som) for the typological attribute categories. darker cells mean higher values of the given attributes. do benthic diatom assemblages reflect abiotic typology acta bot. croat. 76 (1), 2017 85 cluster code species name abundance indval p-value d7 fras fragilaria sp. 0.29±0.15 0.13 0.05 fsap fistulifera saprophila (lange-bertalot & bonik) lange-bertalot 0.37±0.17 0.43 <0.001 gtru gomphonema truncatum ehrenberg 1.13±0.96 0.37 <0.001 d8 afor asterionella formosa hassall 14.19±4.61 0.69 <0.001 apel amphipleura pelucida kützing 1.06±0.32 0.29 <0.001 atri achnanthes trinodis (w.smith) grunow 1.68±0.74 0.32 <0.001 ccmp cymbella compacta østrup 0.82±0.44 0.27 <0.001 clbe cymbella lange-bertalotii krammer 3.05±1.15 0.38 <0.001 dobl diploneis oblongella (nägeli ex kützing) cleve-euler 7.74±2.53 0.65 <0.001 dvca diatoma vulgaris var. capitulatum grunow 14.07±3.91 0.63 <0.001 epro encyonema prostratum (berkeley) kützing 5±1.55 0.53 <0.001 etur epithemia turgida kützing 0.68±0.31 0.21 <0.001 farc fragilaria arcus (ehrenberg) cleve 2.23±1.81 0.34 <0.001 gdec geissleria decussis (østrup) lange-bert. & metzeltin 1.49±0.57 0.43 <0.001 sspe staurosira sp. 0.7±0.27 0.35 <0.001 d9 cped cocconeis pediculus ehrenberg 6.41±1.82 0.21 0.04 enve encyonema ventricosum (c.agardh) grunow 1.45±0.42 0.19 0.03 d10 adsu achnanthidium subatomus (hustedt) lange-bertalot 11.01±5.38 0.19 0.01 crbu craticula buderi (hustedt) lange-bertalot 3.88±2.30 0.22 0.03 fvul frustulia vulgaris (thwaites) de toni 0.12±0.09 0.15 0.03 nmen navicula menisculus schumann var. menisculus 5.98±2.52 0.25 <0.001 numb nitzschia umbonata (ehrenberg) lange-bertalot 1.27±0.45 0.38 <0.001 sbre surirella brebissonii krammer & lange-bertalot var. brebissonii 1.81±0.96 0.20 0.02 stan stauroneis anceps ehrenberg 0.05±0.04 0.15 0.03 tab. 2. – continued tab. 3. abiotic groups with indicator species according to indicator species analysis (indval). average represents percentage (%) of total number of valves of particular species for each site belonging to that group. std – standard deviation. abiotic group species average std p-value ab1 achnanthidium affi ne (grunow) czarnecki 8.4 0.101 <0.01 cocconeis placentula ehrenberg 30.0 0.202 <0.05 craticula cuspidata (kützing) mann 5.8 0.115 <0.05 cymbella tumida (brébisson) van heurck 3.6 0.072 <0.05 gomphonema gracile ehrenberg 3.0 0.060 <0.05 ab2 diatoma tenuis agardh 0.5 0.012 <0.05 navicula trophicatrix lange-bertalot <0.1 0.001 <0.05 sellaphora pupula (kützing) mereschkowksy 4.8 0.081 <0.05 ab3 achnanthidium subatomus (hustedt) lange-bertalot 18.5 0.227 <0.05 ab5 aulacoseira granulata (ehrenberg) simonsen 2.3 0.033 <0.05 gyrosigma sp. 0.2 0.003 <0.05 ab6 asterionella formosa hassall 35.3 0.483 <0.05 cymbella stuxbergii (cleve) cleve 1.2 0.017 <0.01 fallacia pygmaea (kützing) stickle & mann 12.3 0.173 <0.05 ab7 asterionella formosa hassall 32.7 0.463 <0.01 ab10 encyonema ventricosum (agardh) grunow 5.9 <0.05 nitzschia dissipata (kützing) grunow 4.8 <0.05 nitzschia sp. 1.2 <0.01 ab11 achnanthidium biasolettianum (grunow) bukhtiyarova 62.2 0.400 <0.05 aulacoseira ambigua (grunow) simonsen 0.2 0.004 <0.05 cymbella subhelvetica krammer 0.1 0.003 <0.05 nitzschia linearis w.smith 0.2 0.004 <0.05 kralj borojević k., gligora udovič m., žutinić p., várbíró g., plenković-moraj a. 86 acta bot. croat. 76 (1), 2017 bio-env procedure showed that the main environmental variables describing the data set were nitrogen fractions nh4+ and no3– and total p (rho=0.235; signifi cance level of sample statistic=0.1%, number of permutations performed 999). cap procedure in primer that corresponds to canonical analysis of principal coordinates (to test correlation to environmental variables) did not completely confi rm those results (fig. 4). the results show strong and signifi cant correlations between the diatom abundance and the en vi ronmental variables (p=0.001). first canonical correlation was 0.74 and the second one was 0.67. cap axis 1 was correlated with oxygen saturation (r=–0.924) and cap axis 2 with total p (r=0.571) and no3– (r=0.490). discriminant cap analysis showed that 10 som groups are indeed distinguishable one from another. first two canonical correlations are quite high (δ1=0.90 and δ2=0.76). diagnostic showed that the choice of 3 pca axes includes 34.29% of total variation, i.e. that many samples were correctly classifi ed, with values of q0m’hq0m=3.17 (p=0.001) and δ12=0.81 (p=0.001). discriminant cap on abiotic groups showed that abiotic typology is also relevant – with fi rst two canonical correlations being 0.91 and 0.74 (δ1 and δ2, respectively). although permutation test revealed high statistical signifi cance (q0m’hq0m=4.85 (p=0.001) and δ12=0.84 (p=0.001), abiotic grouping correctly allocated only 19.29% of samples. tab. 3. – continued abiotic group species average std p-value ab13 didymosphenia geminata (lyngbye) mart.schmidt 0.1 0.002 <0.05 encyonema ventricosum (agardh) grunow 2.7 0.028 <0.05 gomphonema olivaceum (hornemann) brébisson 3.9 0.059 <0.05 navicula slesvicensis grunow 0.5 0.010 <0.05 nitzschia palea (kützing) w.smith 7.5 0.110 <0.05 surirella ovalis brébisson 0.1 0.001 <0.05 ab14 aulacoseira italica (ehrenberg) simonsen 7.4 0.127 <0.01 campylodiscus noricus ehrenberg 0.1 0.003 <0.01 fragilaria crotonensis kitton 6.1 0.106 <0.01 ulnaria ulna var. acus (kützing) lange-bertalot 7.9 0.137 <0.01 ab16 gomphonema acuminatum ehrenberg 0.2 0.005 <0.05 ab17 cymbella compacta østrup 2.6 0.046 <0.01 denticula tenuis kützing 8.4 0.135 <0.05 epithemia argus (ehrenberg) kützing 0.1 0.001 <0.01 gomphonema pala reichardt 0.2 0.003 <0.01 gomphonema subclavatum grunow 0.2 0.004 <0.05 navicula hofmanniae lange-bertalot 6.0 0.056 <0.05 ab20 cyclotella ocellata pantocsek 5.6 0.083 <0.05 navicula cryptotenelloides lange-bertalot 2.9 0.051 <0.01 ab21 cyclotella ocellata pantocsek 4.0 0.080 <0.05 encyonema caespitosum kützing 5.9 0.119 <0.05 melosira arenaria moore ex ralfs 12.5 0.250 <0.05 urosolenia eriensis (h.l.smith) round & r.m.crawford 0.2 0.004 <0.05 ab22 encyonopsis microcephala (grunow) krammer 27.8 <0.01 sellaphora minima (grunow) mann 1.2 <0.01 ab23 epithemia sp. 0.6 0.012 <0.05 frustulia creuzburgensis (krasske) hustedt 1.7 0.034 <0.05 nitzschia palea (kützing) w.smith 5.4 0.063 <0.05 stauroneis smithii grunow 0.3 0.007 <0.05 ab24 navicula antonii lange-bertalot 2.4 0.041 <0.01 navicula weinzierlii schimanski 1.0 0.018 <0.05 reimeria sinuata (gregory) kociolek & stoermer 0.4 0.008 <0.05 surirella angusta kützing 0.3 0.005 <0.05 surirella linearis w.smith 0.1 0.001 <0.05 surirella ovalis brébisson 0.1 0.001 <0.05 surirella sp. 4.1 0.071 <0.05 do benthic diatom assemblages reflect abiotic typology acta bot. croat. 76 (1), 2017 87 finally, all investigated sites plotted according to environmental variables and biotic data were effi ciently associated with both biotic (som) and abiotic groups (tab. 4). discussion determination of water quality according to wfd greatly depends on reliable typifi cation of waterbodies, which includes entire range of hydrological, geographical, physical, chemical and biological characteristics of sampling sites. since the status itself is derived by comparison to reference conditions, it is essential to have all sites and their corresponding reference assemblages described and grouped into usable groups. as abiotic typology in croatia resulted in 24 different types, which in relation to surface area seem rather high, especially in comparison to germany (20 types, schmedtje et al. 2001), hungary (26 types, van dam et al. 2007), great britain (14 types, davy-bowker et al. 2006) or sweden (11 types, davy-bowker et al. 2006), it was expected for some types to group together. som analysis revealed 10 groups according to diatom assemblages, which is a number of types comparable to other countries. as shown in som component planes, type of waterbed, altitude, size of catchment area and type of stream (its size) were variables infl uencing the development of diatom assemblage, as also shown in the other studies (biggs 1995, pan et al. 2000, potapova and charles 2003). all of those variables are in a direct relation to measured physical and chemical parameters of an investigated site, although there tab. 4. relationship among benthic diatom-based som (self organizing map) groups and abiotic typology for croatian rivers and streams. sum represents a number of sites belonging to the group/site. croatian abiotic type sumab 1 ab 2 ab 3 ab 4 ab 5 ab 6 ab 7 ab 8 ab 9 ab 10 ab 11 ab 12 ab 13 ab 14 ab 15 ab 16 ab 17 ab 18 ab 19 ab 20 ab 21 ab 22 ab 23 ab 24 so m ty pe d1 1 1 1 1 2 3 1 1 1 1 13 d2 3 1 2 1 2 9 d3 1 1 1 1 1 4 1 2 3 1 16 d4 1 1 2 2 2 2 1 5 1 2 1 1 21 d5 2 1 2 1 1 1 1 1 10 d6 1 1 3 1 3 9 d7 1 1 1 1 1 1 1 1 1 9 d8 3 2 1 1 9 1 1 1 1 20 d9 1 1 1 1 1 2 1 1 1 2 1 2 1 1 2 1 20 d10 2 6 2 1 1 1 13 sum 4 6 12 10 11 2 2 17 10 1 5 3 4 3 11 4 3 11 6 3 4 1 4 3 140 fig. 3. component planes of the self organizing map (som) for characteristic diatom species. som cluster groups are assigned with codes from d1–d10. darker cells mean relative higher abundance of the given diatom species. abbreviations of diatom names are the same as in tables 2 and 3. fig. 4. canonical analysis of principal coordinates (cap) ordination plot relating benthic diatom assemblages to environmental variables. kralj borojević k., gligora udovič m., žutinić p., várbíró g., plenković-moraj a. 88 acta bot. croat. 76 (1), 2017 are other variables, such as type of vegetation, size of substrate or exposure, that also greatly infl uence the nutrient availability. altitude is usually related to fl ow velocity which also greatly infl uences the development of the assemblage (primc-habdija et al. 2001). size of the catchment area and size of the stream, in a way, refl ect the number of tributaries which bring additional nutrients to the stream. subsequently, it also extends the possible presence of settlements such as industrial zones and agricultural estates which greatly infl uence nutrient infl ow. increasing phosphorus levels can be mainly related to those variables and not exclusively to the natural sources phosphorus levels. as published in many other studies (bothwell 1989, 1988, stanley et al. 1990) phosphorus, both as orthophosphate and total phosphorus, was shown to be an important factor in the development of benthic diatom assemblage. phosphorus and nitrogen fractions have been generally considered to be most critical variables (hutchinson 1957), as shown in bio-env and cap procedures. composition of diatom assemblages and the characteristic (indicator) species as achnanthidium, cymbella, encyonopsis or nitzschia species, from this set of croatian samples closely corresponded to those observed in other geographical areas (tison et al. 2005, park et al. 2006, tornés et al. 2007, dohet et al. 2008, tornés et al. 2012). diatom assemblages are infl uenced by many variables including those that are site specifi c at various temporal and special scales (denicola et al. 2004, pan et al. 2004), as well as those that refl ect human interventions in the environment. statistically, both types of typology proved to be signifi cant, but abiotic typology resulted in several groups being without one or several discriminant species. on the other hand, som groups had several characteristic species for each of the groups. typical species for som group d1, small to medium, mountain, continental streams on carbonate waterbed, achnanthidium biasolettianum (grunow) bukhtiyarova is a species reported from upstream, low human impact sites at siliceous streams (soininen et al. 2004). presence of a. biasolettianum in carbonate streams can be explained by the fact that all streams from group d1 belong to continental part of croatia that is not karstic and carbonates are prevailing but are not exclusive in the waterbed. similar sized streams, but on carbonate mostly karst waterbed, belong to groups d3 and d4 whose indicative species cymbella affi nis kützing and encyonopsis microcephala (grunow) krammer were reported from mediterranean mineralized headwaters in spain (tornés et al. 2007) and pyrenean calcareous springs (sabater and roca 1992). also, cymbella species seem to have high tendency to inhabit such calcium-rich waters, as reported from numerous streams in usa by potapova and charles (2003). species typical for group d2 (mountain, siliceous headwater streams) are achnanthidium affi ne (grunow) czarnecki and cocconeis placentula ehrenberg. those two species are most common in the streams with higher velocities, a. affi ne as a primary colonizer and c. placentula as a tightly attached, fl ow resistant species (hoagland et al. 1982, plenković-moraj and jasprica 2000, kralj et al. 2006). groups d5, d6 and d10 represent mostly lowland, larger or smaller silicate rivers and streams impacted by agriculture (predominantly in pannonian region) or urban settlements as shown by typical planktonic species like aulacoseira granulata (ehrenberg) simonsen and nitzschia acicularis (kützing) w.smith with a benthic species navicula menisculus schumann, all favoring greater nutrient load (ács and kiss 1991, ács et al. 2003). group d8 showed statistically proven differences in many aspects from all other groups. as a group it is specifi ed by the lack of a common descriptive species and presence of species like achnanthes trinodis (ralfs) grunow in van heurck, cymbella lange-bertalotii krammer, encyonema prostratum (berkeley) kützing or planktonic asterionella formosa hassall. group d8 proved to be specifi c because it encompasses sites in lower reaches of large rivers that can be considered almost as lentic sites. achnanthidium minutissimum (kützing) czarnecki as a characteristic species in group d7, (small, mountain, karstic creeks) refl ects more the nature of sites than their nutrient load. the species is known as an early colonizer that favors high fl ow velocities (primc-habdija et al. 2001, kelly 2002, kralj et al. 2006, stenger-kovács et al. 2013, b-béres et al. 2016) and is commonly reported as a dominant species of similar habitats. group d9 collects sites from istra, the largest croatian peninsula that differs from the rest of croatian coast (prelogović et al. 1995), consisting of upper reaches of small karstic streams and a few continental streams. although differing signifi cantly, they share a species nitzschia palea (kützing) w.smith, commonly reported from sites affected by agriculture and industry (john 2002, soininen 2002). therefore, group d9 encompasses streams affected by high artifi cial nutrient load. since complete data set included mostly undisturbed and slightly disturbed sites, due to war in croatia at the beginning of 1990s and consequential deterioration of entire industry, all of the data, except from extremely disturbed sites were used. that approach ignores the differences in disturbance to similar sites, but general trends which can help in further research and monitoring were observed. benthic diatom assemblages do refl ect abiotic typology by grouping similar sites and therefore reducing the number of types. that reduction implies that diatom comminutes simplify abiotic groups, as also shown in hungary (van dam et al. 2007). also, the fact that this study described typical assemblage for each som group, in comparison to many undefi ned abiotic groups, as well as practicality of considering only 10 in comparison to 24 groups, makes this approach valuable contribution towards description of biotypes and easier determination of water quality according to wfd. conclusions as particular species have different ecological preferences, some with narrow and some with wide ecological valences, diatom assemblage itself does not seem to show clear preference to particular type of stream. diatom assemblage clearly groups similar types of rivers and streams, indicating that for estimation of water quality purposes there do benthic diatom assemblages reflect abiotic typology acta bot. croat. 76 (1), 2017 89 references ács, é., kiss, k. t., 1991: 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science, university of zagreb 125th anniversary celebration (1889–2014) the oldest university botanical garden in croatia, the botanical garden of the faculty of science, university of zagreb, this year, through a variety of events, celebrates 125 years of continuous work. throughout its existence, the garden has operated within the university, at fi rst as a part of the faculty of philosophy, while today it belongs to the faculty of science. it has always had an important role in university teaching, scientifi c research and professional work in the fi eld of botany, as well as in educating the public about the importance of joint actions with the aim of protecting and preserving the wealth of the national fl ora. the idea that it was necessary to establish a botanical garden within the botany and physiology department of the faculty of philosophy originated with dr bohuslav jiruš (1841–1901), the fi rst professor of botany in the university of zagreb. he was fully supported by the then rector of the university, dr stjepan spevec (1839–1905). at the university’s initiative, in 1884 the royal land government decided to constitute a botanical garden for the royal university. the idea was put into action in 1889 by the university professor of botany, dr antun heinz (1861–1919). 120 years ago, the location of the garden was on the outskirts of a city with a population of only 38,000. the largest part of the garden was set in the english landscape style with winding paths and freely growing groups of trees and shrubs. only the fl ower parterre in the southern side of the greenhouses was constructed in the french style of strict order and symmetry. today, the botanical garden is an integral part of a sequence of handsome old zagreb squares and parks known by the name of lenuci’s or the green horseshoe. because of its great educational, cultural, historical and touristic values and its overall signifi cance for zagreb and croatia, since 1971 the garden has been statutorily protected as a monument of park architecture. copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. fig. 1. the main entrance to the royal university botanical garden, 1900. s2 acta bot. croat. 73 (2), 2014 only a decade after its foundation, the garden already had a diverse plant collection, which today numbers more than 5,000 plant taxa. winter resistant woody plants (shrubs and trees) are grown in the arboretum, while sensitive species from warm climates overwinter in the greenhouses. decorative perennials are cultivated in the fl ower parterre, around the small lakes and in the fl owerbeds of the fl ower rainbow. particularly valuable parts of the collection are indigenous plants, belonging to the rich and diverse native fl ora, grown in phytogeographical groups – rock gardens. according to new legislation, growing in the botanical garden at this moment are 109 statutorily strictly protected vascular plant species of croatian fl ora (13.6% of all), 46 taxa from the red book of vascular flora of croatia (19.5% of categories re, cr, en and vu), and seven natura-2000-species of interest to the european union (38.9% of all). the two most recognized croatian endemics, the velebit degenia (degenia velebitica) and dubrovnik cornfl ower (centaurea ragusina), are periodically available for purchase. tropical and subtropical plants are cultivated in the greenhouses, and are displayed in the open during summer. marsh plants grow in the small lakes, the ponds and in the little domed greenhouse in the fl ower parterre. an important part of the collection is also situated in the systematic fi eld, intended for university teaching in botany. recent activities of the botanical garden fulfi l all the conditions from the widely accepted defi nition of botanical gardens and their difference from other public gardens and parks. accordingly, a botanical garden is an institution holding documented collections of living plants for the purposes of scientifi c research, conservation, display and education. hence, our plant collection is properly documented and has been used for more than a century in university research and the teaching of botany. the garden has also been open to the public without an entrance fee from its beginning. a variety of educational and popularization activities organized by the garden curators attract the attention of many visitors, which is evident from the fact that the garden is the third most visited tourist site in zagreb. the garden is a member of the botanical gardens conservation international (bgci), an assembly of nearly 1,000 botanical gardens in the world, as well as the international plant exchange network (ipen). these memberships greatly facilitate our efforts to develop different activities, within our fi nancial constraints, by sharing the experiences with fig. 2. flower parterre with a view of greenhouses, around 1920. acta bot. croat. 73 (2), 2014 s3 other botanical gardens around the globe. the activities of botanical gardens in the 21st century are largely devoted to research, conservation of wild plants and public education. botanical gardens around the world, like ours, contribute to the accomplishment of a number of adopted international conventions and agreements, with the common goal of preserving and protecting rare and endangered plant species. with the restoration of the exhibition pavilion in 2007, the garden acquired a representative venue for exhibition and education, intended for various workshops, lectures and exhibitions, related to the marvellous world of plants. those events are exceptionally well attended, e.g. the fantastic forest exhibition, organized on the occasion of the international year of forests in 2011, was visited by more than 11,000 people in only fi ve months, along with more than 4,000 foreign tourists. every year the exhibition pavilion hosts several dozen workshops for children and adults, dedicated to plants in the broadest sense. since most fig. 3. fountain and ponds with water lilies, 1924. fig. 4. fountain and ponds, 2014. s4 acta bot. croat. 73 (2), 2014 children who live in cities have few opportunities to grow plants, a small children’s fl ower and vegetable garden was also set up in our botanical garden. with the help of their teachers and garden staff, children from the nearby school and kindergarten sow and plant their own fl owers and vegetables, and nurture them during the season until the autumn harvest. during the entire 2014 season different events are going to take place in the garden to mark the 125th anniversary. the program is beginning in april with opening of the botanical garden to visitors and awarding prizes to junior winners of the quiz held during the 2013 exhibition on croatia’s contribution to eu-wide natura 2000 ecological network. the 4th week of croatian botanical gardens and arboreta opening ceremony will be held in our garden on may 12, for the 4th year in a row organized by the section of croatian botanical gardens and arboreta within the croatian botanical society. the members of the european botanic gardens consortium (ebgc), national representatives of botanical gardens from eu countries, switzerland and norway, meet in our garden for the fi rst time in june. their regular meeting is going to be held prior to the celebration ceremony at the invitation of the garden’s manager and croatian representative to ebgc. the central ceremony marking the 125th anniversary of the garden, attended by the representatives of the croatian government, the city of zagreb and the university of zagreb will be organized in the hall of the university. national representatives of the ebgc will join the anniversary celebration ceremony. in september, the garden is hosting the annual garden party of the university of zagreb, to mark the beginning of the 346th academic year. biserka juretić, manager of the botanical garden and croatian representative to ebgc botanical garden of the faculty of science, university of zagreb, marulićev trg 9a, hr-10000 zagreb, croatia fig. 5. workshop botanical magic: let’s play at making a herbarium! held during the 3rd week of croatian botanical gardens and arboreta, 2013. acta botanica 2-2016 za web.indd 210 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 210–212, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0024 issn 0365-0588 eissn 1847-8476 short communication myosotis refracta boiss. (boraginaceae), an unexpected forget-me-not in the slovene fl ora simona strgulc krajšek1*, marko accetto2, nejc jogan1 1 university of ljubljana, biotechnical faculty, department of biology, večna pot 111, si-1000 ljubljana, slovenia 2 trnovski pristan 18, si-1000 ljubljana, slovenia abstract – myosotis refracta boiss. is reported as a new and unexpected fi nding for the slovene fl ora. the species was found in three collections stored in the herbarium lju from south-east slovenia, in the kolpa river valley bordering croatia. all plants thrived under overhanging dolomite rocks. on account of an earlier misidentifi cation, the respective plant community had been described as the association arabido alpinaemyosotidetum strictae accetto 2008, which is here corrected to arabido alpinae-myosotidetum refractae accetto 2008 corr. strgulc krajšek, accetto & jogan 2016. myosotis refracta has a disjunct mediterraneansouthwest asian distribution. the reported new localities extend its known range more than 500 km in north-west direction, from its nearest known occurrences on the southern balkan peninsula. key words – fl ora, forget-me-not, kolpa valley, myosotis refracta, phytocenology, slovenia * corresponding author, e-mail: simona.strgulc@bf.uni-lj.si introduction myosotis l., forget-me-not (boraginaceae) has about 41 species reported from europe (grau and merxmüler 1972). the genus is considered taxonomically diffi cult, due to the many morphologically similar species that are for practical reasons grouped in several aggregates, such as m. alpestris agg., m. sylvatica agg., and m. scorpioides agg. (greuter et al. 1984). eleven species of myosotis were recorded from slovenia (strgulc krajšek 2007, király et al. 2007) before the revision of herbarium material in herbarium lju in 2015, carried out by strgulc krajšek. during the revision, an unexpected species, m. refracta boiss., was identifi ed. m. refracta is morphologically distinct, but easily confused with other small myosotis species. the plants are annual, branching from base, with few-leaved, delicate, stiff branches terminating in long, many-fl owered partial infl orescences. the leaves are lanceolate, to 4 cm long. the fl owers are small, corolla to 1.5 mm in diameter and pale blue. the calyx is densely covered with patent hooked hairs. hooked hairs on the base of calyx are strongly defl exed towards the very short pedicel. during ripening, the calyces elongate to ca 4 mm and their pedicels become defl exed, giving the plant a very characteristic appearance in that state. the nutlets are narrow, almost spindle-shaped (grau and merxmüler 1972). phylogenetically (based on its), m. refracta has a basal position relative to a clade containing most of the eurasian myosotis species (winkworth et al. 2002), but poor resolution and plastid marker incongruencies would need more sampling. the known distribution of m. refracta is apparently linked to the southern edge of the eurasian mountains formed during the tertiary alpine orogeny of the tethyan (palaeo-mediterranian) region, stretching from spain to nw india. it has an interesting distribution range from the iberian peninsula in the west to the western outskirts of himalaya in the east with at least two disjunctions separated by 1000 or more km and possibly another disjunction in the eastern part of the range, but the data quality for western asia is less reliable so it could have been expected also in the mountain ranges around the black and the caspian sea. in the western mediterranean its records are limited to the south of spain. the eastern mediterranean disjunction ranges from albania and greece to syria, and the eastern disjunction spans the area from pakistan to himalaya (chowdhery and wadhwa 1984). in spain it is reported for sierra nevada where its locus classicus is (boissier 1841). in the former ussr it is reported only for eastern crimea (fedorov 1981), from albania several localities were reported just recently (barina et al. 2009) on limestone areas, with only one older record known from the vicinity of borsh. in macedonia bornmüller 1928 reports it only for drenovo, but recently matevski 2010 added several localities scattered in the mountains. in greece several localities are reported from various mountain ranges (strid 1991) and crete (jahn and schönfelder 1995). meikle 1985 reports it myosotis refracta in slovenia acta bot. croat. 75 (2), 2016 211 from 3 localities in the mountains of western cyprus. heller & heyn 1986 reported m. refracta for cyprus, a major part of asian turkey (similarly also grau 1978), lebanon, syria and israel (especially mountains near mediterranean coast), ne iraq and w iran. to date, four subspecies of m. refracta were described (grau and merxmüler 1972, greuter et al. 1984, kazmi 1971): – m. refracta subsp. refracta: 2n = 44, distribution range: spain, e mediterranean region and krym. – m. refracta subsp. paucipilosa grau: 2n = 20, distribution range: s greece, kriti. turkey. this subspecies has been recognised as a separate species due to karyological, morphological and distributional differences: myosotis paucipilosa (grau) ristow & hand (hand 2015). – m. refracta subsp. aegagrophylla greuter & grau: chromosome number unknown, distribution range: kriti (greuter and grau 1970). this taxon is not generally accepted and has been synonymized with nominal subspecies (hand 2015). – m. refracta subsp. chitralica kazmi: w and c asia (afghanistan, pakistan, kashmir, nw india, turki stan, pamir alaj, tien shan.) (kazmi 1971, nasir 2015). according to hand 2015, this taxon may need critical reconsideration, but see dickoré 2016. the differences between subspecies are in the shape of leaves, distribution of hooked hairs on calyx, the extent of calyx defl exion in the fruiting state and in the shape of nutlets (grau and merxmüler 1972), but, on the other hand, all these characters are reported as highly variable across the distribution range (riedl 1967). the author of m. refracta (boissier 1841) reported its rocky habitats in the subalpine to alpine belt, and obviously the species is a high mountain dweller in the southern parts of its distribution. in cooler areas, as in macedonia it was found to range from the lowlands to the montane belt, 200– 1000 m a.s.l. (matevski 2010), and in crimea is found in shaded rocky places in the lowlands (fedorov 1981). all authors reported rocky habitats and pioneer plant communities. more specifi c ecological conditions are reported for macedonia, where the species can also be found along paths (matevski 2010). meikle 1985 reports it from diabase and serpentine gravel on slopes, and, most precisely its ecology is described in mountain fl ora of greece as including »rocky pastures, often in somewhat damp, semi-shaded, nutrient rich places« (strid 1991). material and methods a revision of all available material of the genus myosotis stored in the herbarium lju (university of ljubljana, biotechnical faculty, department of biology) was carried out in 2015. identifi cations were checked using the keys in flora europaea (grau and merxmüler 1972), and characters given in other publications including original descriptions: boissier 1841, greuter and grau 1970, hand 2015. results and discussion three specimens of myosotis refracta boiss. from the kolpa valley area in southeast slovenia were identifi ed in the herbarium lju. all had been collected by m. accetto between the years 1999 and 2007, and previously misidentifi ed as m. stricta link ex roemer & schultes: 1) 0454/3 slovenia: se slovenia, valley of river kolpa, osilnica, above ribjek by kolpa, foothill of overhanging rock wall, e from mož, 200 m a.s.l., leg. m. accetto, 2. 5. 2007. lju10133797. 2) 0454/4 slovenia: se slovenia, valley of river kolpa, osilnica, rock wall e of fistov rep, 940 m a.s.l., leg. m. accetto, 6. 6. 1999. lju10119844. 3) 0454/3 slovenia: se slovenia, valley of river kolpa, osilnica, rock wall fistov rep, 710 m a.s.l., leg. m. accetto, 30. 5. 1999. lju10119845. the slovene plants can clearly be referred to the type subspecies, myosotis refracta subsp. refracta (fig. 1) and with habitus and morphology very similar to the illustration of the type specimen from spain (boissier 1841). the fl owering time of the species is early spring. specimens from slovenia, sampled in may, were already in fruiting state. the ecological conditions in all three localities in slovenia, where herbarium specimens of m. refracta were collected, were very similar: narrow rocky shelves below overhanging dolomite rocks (slightly sheltered from rain), in open habitats on fi ne dolomite debris, slopes facing north-west or east. myosotis refracta was growing together with: arabis alpina subsp. alpina, poa bulbosa, sesleria juncifolia subsp. kalnikensis, cardaminopis arenosa, taraxacum laevigatum agg., anisantha tectorum, hornungia petraea and few other species. due the specifi c habitat and species composition, a new association from the alysso-sedion alliance, »arabido alpinae-myosotidetum strictae« had been described (accetto 2008). myosotis refracta (at fig. 1. the herbarium specimen of myosotis refracta, collected on 2nd of may 2007 in slovenia in valley of river kolpa by m. accetto (lju10133797), photo by: a. kladnik. strgulc krajšek s., accetto m., jogan n. 212 acta bot. croat. 75 (2), 2016 that time erroneously determined as m. stricta) actually comprises the edifi cator species of the new association, selected on account of its relatively high mean cover value. in accordance with international code of phytosociological nomenclature (weber et al. 2000) the name of association has now to be changed to arabido alpinae-myosotidetum refractae accetto 2008 corr. strgulc krajšek, accetto & jogan 2016, nom. corr. hoc loco. the gap between the known distribution range (grau and merxmüler 1972) and the newly discovered localities in slovenia is more than 500 km. due to early fl owering and extreme ecological conditions of m. refracta habitats it is highly possible that the species could have been overlooked in similar habitats along the dinarides. myosotis refracta is not the only representative of boraginaceae with such extreme ecology; a comparable one is asperugo procumbens l. (martinčič 2007). with their obvious adaptation to epizoochorous fruit dispersal we can think of some animal species adapted to rocky cliffs as potential vectors. wild goats have already been mentioned in literature as sharing habitats with m. refracta, but as the diasporae are really tiny, also some bird of the rocky habitats as tichodroma muraria (l., 1766) (wallcreeper) might have been a vector. that would be especially important for longdistance dispersal. according to the previously known distribution and its recent discovery occurrence in slovenia, it seems possible that myosotis refracta may be found in other localities along the dinaric arc, such as in croatia and montenegro. acknowledgements this work was supported by slovenian research agency grant no. p1-0212. the authors thank tinka bačič for valuable discussions, aleš kladnik for photo of herbarium specimen, and reviewers for comments and additions to the manuscript. references accetto, m., 2008: floristic and vegetation curiosities at the foot of the overhanging rock face with the rock shelter above ribjek upon the kolpa river area (s slovenia). hladnikia 21, 3–17. (in slovene). barina, z., pifkó, d., mesterházy, a., 2009: contributions to the fl ora of albania. willdenowia 39, 293–299. boissier, e., 1841: voyage botanique dans le midi de l’espagne 1, tab. 125a, 2, p. 433. boissier, e., 1879: flora orientalis 4. lugduni, genevae & basileae. bornmüller, j., 1928: beiträge zur flora mazedoniens ii–iii. verlag von max weg, leipzig. chowdhery, h. j., wadhwa, b. m., 1984: flora of himachal pradesh analysis. botanical survey of india, dept. of environment. dickoré, b. the himalayan uplands plant database (hup version 1). global mountain biodiversity assessment gmba. retrieved januar 19, 2016 from: http://www.gbif.org/occurrence/615358638. fedorov, a. (ed.), 1981: flora of the european part of ussr, vol 5. (in russian). nauka, leningrad. grau, j., 1978: myosotis l. in: davis, p. d. (ed.), flora of turkey, vol. 6, 264–280. edinburgh university press. grau, j., merxmüller, h., 1972: 29. myosotis l. in: tutin, t. g, heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.): flora europaea, vol. 3, 111–117. university press, cambridge. greuter, w., burdet, h. m., long, g., 1984: med-checklist, vol. 1. conservatoire et jardin botanique ville de genève. greuter, w., grau, j., 1970: zum vorkommen von drei unterarten der myosotis refracta boiss. auf kreta. candollea 25, 7–10. hand, r., 2015: supplementary notes to the fl ora of cyprus viii. willdenovia 45, 245–259. heller, d., heyn, c. c., 1986: conspectus florae orientalis. the israel academy of sciences and humanities, yerusalem. jahn, r., schonfelder, p., 1995: ekskursionsfl ora für kreta. ulmer verlag, stuttgart. kazmi, s. m. a., 1971: a revision of the boraginaceae of west pakistan and kashmir. journal of the arnold arboretum 52, 666–690. király, g., mesterházy, a., bakan, b., 2007: elodea nuttalii (planch.) h. st. john, myosotis laxa lehm. and pyrus austriaca kern., new for slovenia, as well as other fl oristic records. hladnikia 20, 11–15. martinčič, a., 2007: 122. family: boraginaceae. in: martinčič, a. (ed.), little fl ora of slovenia (in slovene), 530–545. tehniška založba slovenije, ljubljana. matevski, v., 2010: flora of the republic of macedonia (macedonian academy of sciences and arts, skopje (in macedonian). meikle, r. d., 1985: flora of cyprus, vol. 2. rbg, kew. nasir, y. j., 2015: flora of pakistan. tropicos.org. missouri botanical garden. retrieved october 22, 2015 from: http://www. tropicos.org/name/50320245?projectid=32. riedl, h., 1967: boraginaceae, flora iranica 48. akademische druck und verlagsanstalt, graz. strgulc krajšek, s., 2007: myosotis l. in: martinčič, a. (ed.), little fl ora of slovenia (in slovene), 541–543. tehniška založba slovenije, ljubljana. strid, a., 1991: myosotis l. in: strid, a., tan, k. (eds.), mountain fl ora of greece 2, 47–57. university press, edinburgh. weber, h. e., moravec, j., theurillat, j.-p., 2000: international code of phytosociological nomenclature. 3rd edition. journal of vegetation science 11, 739–768. winkworth, r. c., grau, j., robertson, a. w., lockhart, p. j., 2002: the origins and evolution of the genus myosotis l. (boraginaceae). molecular phylogenetics and evolution 24, 180–193. acta botanica 2-2015.indd a word from the guest editor dear readers it is my great pleasure to introduce this volume of acta botanica croatica dedicated to the 8th central european diatom meeting (8th cedm). the 8th cedm was organized by croatian botanical society and held from april 10 to april 13, 2014, at the university of zagreb. as guest editor of this volume, i would like to express my gratitude to the authors who supported the volume with their contributions, as well as to the referees who applied their professional expertise to review those contributions. the results of this excellent and stimulating collaboration are presented in this volume. it was at the 6th central european diatom meeting in innsbruck, 2012 that professor eugen rott suggested to dr nenad jasprica that the algology section of croatian botanical society should host a symposium. i would like to thank dr nenad jasprica for accepting this invitation and suggesting to the algology group, faculty of science, university of zagreb and the croatian botanical society that it should host the symposium. we were honoured to obtain this opportunity. the organization of an international symposium is extremely challenging, and i would like especially to thank dr nenad jasprica and dr regine jahn for sharing their experience and expertise in leading the organization committee, as well for being there through all the challenges. without their help the symposium would not have been so successful. i would also like to thank the group from the ruđer bošković institute, laboratory for processes in the marine ecosystem, rovinj for accepting the invitation to join the organizing committee and for their devotion to the process. together we created a great team and without the help of each one of us, it would not have been possible. thank you all for this adventure through which all of us have grown as scientists, group members and leaders. the 8th cedm was organized at the university of zagreb, to which we are grateful for support with all the facilities and for being such great hosts. at the opening ceremony the participants were welcomed with opening remarks by dean amir hamzić, faculty of science, university of zagreb, rector aleksa bjeliš, university of zagreb, staša skenzić, head of the offi ce for international cooperation at ministry of science, education and sports of the republic of croatia and the mayor of zagreb milan bandić. i had, as symposium chair, the honour to declare the symposium open. a total of 72 participants representing 17 countries, mostly european, but also from the usa, japan and mexico attended the 8th cedm and contributed with 4 plenary lectures, 17 oral and 38 poster presentations. presentations were assigned to two categories – the morphology, taxonomy and phylogeny of diatoms and the ecology, paleoecology and use of diatoms in monitoring. during the symposium, a round table entitled »a cellular model of ecosystem functioning (probing aquatic ecosystems)« was moderated by dr martin pfannkuchen from the ruđer bošković institute, cim, rovinj. added value was imparted to the symposium by the microscope and book exhibitions of the sponsors. on the last day of the symposium an excursion to the plitvice lakes national park was organized; the participants had the pleasure not just to enjoy the amazing beauty of nature, but also the chance to investigate the diatoms of the national park. i would like to express my sincere thanks to the sponsors of the symposium: the croatian ministry of science and sports; the zagreb tourist board; the croatian environmental protection and energy effi ciency fund; np plitvice lakes; the university of zagreb, faculty of science, department of biology; koeltz scientifi c books, koenigstein, germany; zeiss; inel; algoritam; ets medical and microlux. special thanks are due to mirela gašpar and her team from pbz card travel agency, the offi cial partner of the symposium, for their excellent and highly professional support. thanks to renata horvat for setting up and maintaining the offi cial web page, as well as for the technical support that she provided. it was highly appreciated. many thanks to adam maguire for designed the symposium logo. a warm thank you goes to all the members of organization committee and symposium support group, colleagues from the algology group from the university of zagreb, faculty of science, ruđer bošković institute, laboratory for processes in the marine ecosystem, cim, rovinj and the institute for marine and coastal research in dubrovnik, as well as to the students from university of zagreb, faculty of science for their support, team work and devotion to the main goal. the 8th cedm organization was a joint project and because of the great team work and devotion the symposium was a big success. i do hope that all the participants enjoyed not only the scientifi c part of the symposium, but also all the warm welcome to zagreb through social events. the 8th central european diatom meeting included 3 very intensive days, with excellent lectures and poster presentations as well as discussions and a less formal day at the plitvice lakes national park. new collaboration was established and previous relationships extended. i hope that this volume of acta botanica croatica will not only have scientifi c value but be a fi tting and pleasant reminder of the 8th cedm. zrinka ljubešić chair of the 8th central european diatom meeting guest editor of acta botanica croatica 74(2)2015 zagreb, august 2015 acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 27 acta bot. croat. 76 (1), 27–31, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0052 issn 0365-0588 eissn 1847-8476 a morphological study on the dioecious endemic erodium somanum h. peşmen (geraniaceae), critically endangered in turkey dilek oskay* celal bayar university, faculty of science and arts, department of biology, manisa, turkey abstract – morphological features of the endemic erodium somanum are investigated based on the specimens collected from natural populations from soma in manisa. almost all morphological characteristics are expanded and some morphological characteristics are fi rstly determined in this study. e. somanum is a dioecious species and in this study drawings of male and female individuals are given for the fi rst time. an umbel is 3–5 fl owered in female plants and 6–11 fl owered in male plants, pedicels accrescent to 20–25 mm in fruit. flower morphology was identifi ed in detail and drawings are given for the fi rst time. stigma color ranges from yellow to red in populations. the fruit is long-beaked 4.8–8 cm, stout and adpressed pilose, glandular below. mericarp morphology investigated for fruit characters has a special diagnostic value for systematic studies. the mericarp micromorphology and seed micromorphology were determined for the fi rst time. mericarp size 9–12 mm, mericarps have two apical shallow pits (foveoles) without furrow beneath. mericarp surface ornamentation is foveate with crowded bristles of dissimilar size, some longer and others shorter. mericarp pit is crowded with eglandular hairs and some sparse, long-stalked glands which are also at the start of the awn. seed size is 4.5–6×1.5–2 mm, seed type is narrowly ovate, seed surface is ruminate. keywords: dioecious plant, endangered endemic plant, erodium somanum, fl ower morphology, micromorphology. * corresponding author, e-mail: dilek.oskay@cbu.edu.tr introduction geraniaceae have fi ve genera characterized by their fl ower features, erodium, geranium, monsonia, sarcocaulon, pelargonium (takhtajan 1997). a sixth genus, california, has been segregated from erodium primarily because of the absence of staminodes (aldasoro et al. 2002). the genus erodium is distributed across all continents. a major center of diversity is in the mediterranean region (63 species), whereas in other regions only a few native species have been observed; north america (1), south america (1), australia (5), and asia (4) (fiz et al. 2006). the fi rst revision of erodium species in turkey and the east aegean islands was made by davis who recognized 27 taxa (davis 1967). since then, fi ve new species have been described (davis et al. 1988, güner et al. 2000, yıldırımlı and doğrukoca 2004) and the total has now reached 32 taxa. sixteen of these erodium taxa are endemic in turkey. erodium somanum h. peşmen is an endemic species known from soma in manisa, west of turkey (davis et al. 1988). it was reported that e. somanum, in terms of general appearance is similar to erodium sibthorpianum boiss. subsp. sibthorpianum and erodium absinthoides willd. subsp. absinthoides. but e. somanum is clearly different from e. sibthorpianum subsp. sibthorpianum in that it has less canescent leaves, lilac petals and a shorter (3.5–4.5 cm) beak to the fruit; also e. somanum is clearly different from e. absinthoides subsp. absinthoides in that it has lax rosettes, white petals and a longer (2–2.8 mm) sepal mucro (davis et al. 1988). then, it was seen that a new combination had been made as erodium sibthorpianum boiss. var. somanum (peşmen) el-oqlah (el-oqlah 1989). moreover, it was seen that a new name erodium chrysanthum subsp. somanum had been given but the new name of the author was not provided (fiz et al. 2006). as shown there are contradictions in the nomenclature of this species. to overcome confusion in this regard, it seems that a detailed study about the species mentioned is needed. in this study, e. somanum was used because of the fi rst name given to the type specimen. this species was fi rst classifi ed into the endangered (en) category based on iucn criteria (ekim et al. 2000) and later classifi ed as critically endangered (cr) according to data obtained from population studies and based on iucn criteria (oskay and altan 2015). oskay d. 28 acta bot. croat. 76 (1), 2017 e. somanum naturally grows in rocky habitats, above 800 m, after the tree line. total distribution area of populations is approximately 5 km2, in the surroundings of the east-west extension of güllük mountains in soma. distribution soils are slightly alkaline, without salt and a generally limey structure, adequately ferrous but poor in phosphor. climate type is semi-arid upper mediterranean where the winters in particular are cool (oskay and altan 2015). studies on its morphology are limited. pollen features and chromosome numbers of e. somanum have been investigated (oskay et al. 2011). the present study is aimed at expanding morphological descriptions of this endemic species, including its detailed fl oral features. mericarp surface is a very important diagnostic character for erodium species; mericarp and seed surface have been investigated and scanning electron microscopy (sem) photos are given fi rst time in this study. material and methods e. somanum was collected from natural populations during a project supported by scientifi c research projects from celal bayar university. the specimens are dried according to standard herbarium techniques and stored at celal bayar university science and education faculty. the taxonomical description of the plants was made according to e. somanum in “flora of turkey vol. 10” by davis et al. (1988). for the morphological studies, dried samples were used. specifi c characters were measured at least twenty times. photographs and drawings with general views of the male and female individuals, fl ower parts and fruit have been added to the study because it is a dioecious species. all measurements with fruit and seed were made at least thirty times. fruit and seed were also directly placed on prepared stubs and covered with gold for micromorphological features by sem. photographs are taken with a jeol jsm 6060. for fruit terminology the following literature was consulted (davis 1967, fiz et al. 2006). seed terminology from the following literature was consulted (stearn 1996). results morphology of vegetative parts e. somanum is a dioecious perennial plant (fig. 1, online suppl. fig. 1, on-line suppl. fig. 2). plant populations consist of female and male individuals. plants are very wide hard cushions up to 60 cm diameter. rootstock is vertical, woody and black colored. basal leaves are bipinnatisect, oblong elliptic, 6–16×15–37 mm with 4–20 mm petiole. leaves are 3–5 segments, linear, acute, adpressed eglandular pubescent with stipules. stems are erect, 1.3–19.5 cm (excluding peduncules) and simple or scarcely branched, covered with folded mixed villi, bearing 2–4 peduncules. peduncules are 12–70 mm eglandular hirsute and have stipitate glands. the umbel infl orescences have 3–5 fl owers in female plants and 6–11 fl owers in male plants. umbels are multibracteate and bracts are pale grey. pedicels are very slender, 8–20 mm (accrescent to 20–25 mm in fruit) and glandular pubescent. morphology of reproductive parts there are two types of fl owers in e. somanum. female plants have only female fl owers while male plants have only male fl owers. the fl owers are medium size, actinomorphic corolla and borne in bracteate umbels. e. somanum have pentamerous fl owers consisting of fi ve sepals, fi ve petals, fi ve antipetalous nectaries and fi ve staminodes. five fertile stamens and fi ve staminodes enclose the vestigial gynoecium in male fl owers while fi ve vestigial stamens and fi ve staminodes enclose a gynoecium with fi ve carpels in female fl owers (on-line suppl. fig. 3). petals are pale sulphur yellow in colour, broadly obovate, 3–8×6.5–12 mm, pilose towards base. petal veins are usually darker than the rest of the petal (fig. 2). sepals appearance is transparent, sepal veins are usually green and darker than the rest of the sepal (fig. 2). sepal shape is oblong-elliptic, 2–5×4–8 mm (accrescent to 4–8×10–14 mm in fruit), obtuse with 0.5–1.5 mm mucro (on-line suppl. fig. 4). dorsal part of sepal is covered with dense stipitate glands and eglandular villose. the sepals are not deciduous after fruit formation. the synfig. 1. general appearance of erodium somanum in nature. fig. 2. reproductive parts of erodium somanum: (a, b) female fl owers; (c) nectaria and pistil in female fl owers; (d, e) male fl owers; (f) nectaria and stamens in male fl owers. a morphological study on erodium somanum acta bot. croat. 76 (1), 2017 29 carpous ovary has fi ve carpels, with each chamber having a single ovule with basal placentation. the long style is terminated by fi ve furcated stigmas. stigma color ranges from yellow to red in populations. the stamens are antisepalous, extrorse and versatile. anthers are two-celled and opened longitudinally. fruit type is a schizocarp and it is divided into fi ve mericarps (fig. 3, on-line suppl. fig. 4). fruit is longbeaked with beak 4.8–8 cm long, stout and adpressed pilose, glandular below. the outer part of the style separates into fi ve long awns that usually remain attached to the mericarps. awns are spirally twisted below and rarely deciduous. mericarp is sized 9–12 mm and is light brown with whitish bristles (fig. 3). mericarps have two apical shallow pits (foveoles) without a furrow beneath. mericarp awn is not plumose. mericarp surface ornamentation is foveate with crowded bristles of dissimilar size, some longer and others shorter (fig. 4). mericarp pit is crowded with eglandular hairs and some sparse, long-stalked glands which are also at the start of the awn (fig. 4). seed is glabrous, light brown-dark green brown (fig. 5a). seed is 4.5–6 mm long and 1.5–2 mm wide, seed shape is oblanceolate, almost terete, dorsal section of the seed is plain, ventral section of the seed is slightly swollen. apex of the seed is rounded. hilum not recessed and is clearly evident. hilum is linear and sub basal, in other words extending upwards from the base of the seed. when sem photographs of seed surface are examined, it is determined to be ruminate (figs. 5b, c) according to the following literature (stearn 1996). discussion e. somanum, a critically endangered endemic species in turkey, was investigated morphologically in the present study in order to contribute to the description and to elucidate some morphological details. the taxonomical description was carried out according to e. somanum in “flora of turkey” by davis et al. (1988). almost all morphological characteristics are expanded and some morphological characteristics, such as mericarp micromorphology, seed micromorphology and fl ower morphology are determined for the fi rst time. mericarp morphology investigated for fruit characters has a particular diagnostic value for systematic studies. some differences were found in the morphological characteristic from the description of the taxon in “flora of turkey” by davis et al. (1988). these differences are shown in table 1. the differences in this study occur because of the goal behind the assessment, the number of samples investigated, the greater time devoted to the task and spent on the related fi eld work. in addition, climatic conditions vary from year to year and may affect the adaptation of plants. this plant is an example of dioecious species, so female and male fl owers morphology were identifi ed in detail and drawings are given for the fi rst time. some differences were found (tab. 1) in the fl ower characters from the description of the taxon (davis et al. 1988). some characteristics are new fi ndings: there are fi ve fertile stamens and fi ve staminodes that enclose the vestigial gynoecium in male fl owers and fi ve vestigial stamens and fi ve staminodes that enclose a gynoecium with fi ve carpels in female fl owers; the petal veins are usually darker than the rest of the petal; sepals are transparent; sepal veins are usually green and darker than the rest of the sepal; dorsal part of sepals covered with dense stipitate glands and are eglandular villous; the sepals fig. 3. fruit (a) and mericarp (b) of erodium somanum. fig. 4. scanning electron microscopy photographs of erodium somanum mericarps: (a) mericarp surface; (b) long and short bristles; (c) glandular hairs; (d) foveoate surface ornamentation. fig. 5. seeds of erodium somanum: (a) general view; (b, c) scan ning electron microscopy photographs of seed surface at different magnifi cation. oskay d. 30 acta bot. croat. 76 (1), 2017 are not deciduous after the fruit formation; stigma color ranges from yellow to red in populations. the phylogenetic relationships and evolution in erodium have been described according to gene sequences by fiz et al. (2006). this work is very important in the sense that 74 erodium taxa have been collected from all over the world. the morphological characters used in the phylogenetic analyses of authors are noteworthy because about half of these characters are mericarp features. we have benefi ted from this work and from the “flora of turkey” when evaluating the characters of the mericarp. it was reported that mericarps were hispid, not foveolate, in the “flora of turkey” (davis et al. 1988). in contrast, the results of this study show that mericarps are hispid but there are two quite shallow apical pits (foveoles) and these pits are densely hispid (tab. 1). approximately 32 erodium taxa are distributed in turkey; however the micromorphology of most species has not been investigated. micromorphological investigations on the mericarp of some annual taxa of erodium were conducted by oskay and eş (2015) who ascertained the mericarp micromorphology of e. hoefftianum c.a. meyer, e. botrys (cav.) bertol, e. malacoides (l.) l’hérit, e. moschatum (l.) l’hérit, e. cicutarium (l.) l’hérit, sub sp. cicutarium, e. ciconium (l.) l’hérit taxa. authors investigated several fruit characters of erodium species, such as fruit type, length of fruit, length of mericarp, form of mericarp apex (presenting pits, ridges or furrows), and glandulose structures (glands, glandular hairs and papilles) and found signifi cant differences between species. in a study by coşkunçelebi et al. (2012) e. hendrikii, a closely related taxon to e. malacoides, was compared with respect to characters of basal leaves, stem indumentum, trichome type on mericarp surface, petal colour, sepal length, beak of fruit and mericarp. and so these two closely related taxa were separated thanks to the new fi ndings. there is no detailed information about seed microsculpturing in erodium species. seed macro and micro morphological characters of 19 taxa belonging to the family geraniaceae were investigated by ather et al. (2012). they found data that could be useful in providing additional information for taxonomic delimitation at various levels. also they stated that the morphological and phylogenetic relationship of the taxa within the family geraniaceae correlates well with seed morphological data. the fi ndings of seed characteristics in this study are compared with the fi ndings of seed characteristics by ather et al. (2012), and it is seen that e. somanum has its own distinct characters. in conclusion, in this study we aimed to introduce morphological details of the endemic e. somanum, a perennial dioecious plant species. the morphological features of the investigated taxon were quite similar to those reported by davis et al. (1988) in “flora of turkey”. however, morphological fi ndings concerning the reproductive parts of e. somanum are, with some exceptions, presented for the fi rst time. acknowledgements i would like to thank scientifi c research projects from celal bayar university (project number: fef–2007–12) for the fi nancial support. i also would like to thank dr. cem azeri for illustrations. tab. 1. comparison of plant characters of erodium somanum. plant characters unit “flora of turkey” (davis et al. 1988) in this study hard cushion s cm up to 40 diameter up to 60 diameter basal leaves mm 10–15 × 25–30 6–16 × 15–37 petiole mm 15–20 4–20 stems cm 7–18 1.3–19.5 peduncules mm 15–45 12–70 pedicels mm 10–15 8–20 sepals mm 2–2.5 × 6–6.5 2–5 × 4–8 sepals in fruit mm 4–5 × 10–12 4–8× 10–14 sepal mucro mm 0.5–1 0.5–1.5 petals mm 6.5 × 6.5–7 3–8 × 6.5–12 fruit cm 5–5.5 4.8–8 mericarp mm 8 9–12 mericarps – not foveolate with 2 shallow apical pits (foveoles) references aldasoro, j. j., navarro, c., vargas, p., sa’ez, l. l., aedo, c., 2002: california, a new genus of geraniaceae endemic to the southwest of north america. anales del jardín botánico de madrid 59, 209–216. ather, a., abid, r., qaiser, m., 2012: the seed atlas of pakistanvii. geraniaceae. pakistan journal of botany 44, 1059–1064. coşkunçelebi, k., terzioğlu, s., karaköse, m., güzel, m. e., 2012: contributions to the description and molecular properties of erodium hendrikii alpınar (geraniaceae), endemic to turkey. turkish journal of botany 36, 455–461. davis, p. h., 1967: erodium l’hérit. in: davis p. h, (ed.), flora of turkey and the east aegean islands, vol. 2, 475–487. edinburgh university press, edinburgh, uk. davis, p. h., mill, r. r., tan, k., 1988: erodium l’hérit. in: davis, p. h., mill, r. r., tan, k., (eds.), flora of turkey and the east aegean islands, vol. 10 (suppl. i), 105–106. edinburgh university press, edinburgh, uk. ekim, t., koyuncu, m., vural, m., duman, h., aytaç, z., adıgüzel, n., 2000: red data book of turkish plants, pteridophyta and spermatophyta, ankara (in turkish). el-oqlah, a. a., 1989: a revision of the genus erodium l’he´ritier in the middle east. feddes repertorium 100, 3–4. fiz, o., vargas, p., alarcon, m. l., aldasoro, j. j., 2006: phylogenetic relationships and evolution in erodium (geraniaceae) based on trnl-trnf sequences. systematic botany 31, 739– 763. güner, a., özhatay, n., ekim, t., başer, k. h. c., 2000: erodium l’hérit. in: güner, a., özhatay, n., ekim, t., başer, k. h. a morphological study on erodium somanum acta bot. croat. 76 (1), 2017 31 c., (eds.), flora of turkey and east eagean islands, vol 11 (suppl. ii), 74–75. edinburgh university press, edinburgh, uk. oskay, d., altan, y., kesercioğlu, t., 2011: investigation of pollen features and chromosome numbers of erodium somanum, biological diversity and conservation 4, 175–179. oskay, d., altan, y., 2015: an investigation on habitat and population properties of local endemic erodium somanum. ekoloji 24, 32–39. oskay, d., eş, ö., 2015: micromorphological investigations on mericarp of some annual taxa of erodium. c.b.u. journal of science 11, 59–68. takhtajan, a., 1997: diversity and classifi cation of fl owering plants. columbia university press, new york, usa. stearn, w.t., 1996: botanical latin, 4th edition. publisher david & charles. yıldırımlı, ş., doğru-koca, a., 2004: a new species from turkey, erodium aytacii yıldırımlı & a. doğru-koca (geraniaceae). the herb journal of systematic botany 11, 1–6. opce-str.vp acta bot. croat. 69 (1), 83–92, 2010 coden: abcra 25 issn 0365–0588 pollen morphology of the genus lathyrus (fabaceae) section cicercula in thrace (european turkey) fatma gunes1*, ali cirpici2 1 kafkas university, faculty of arts and sciences, department of biology, kars, turkey 2 marmara university, faculty of arts and sciences, department of biology, göztepe, istanbul, turkey we examined the pollen morphology of four taxa from the cicercula section of lathyrus, grown in the thrace region (european turkey), including l. annuus l., l. gorgoni parl. var. pilosus c. c. townsend, l. cicera l. and l. hirsutus l. the pollen grains are 3-zonocolporate of subprolate and prolate types (p/e=1.2378-1.4491), medium to large sized, elliptical or rectangular-obtuse-convex (equatorial view) and circular to slightly triangular-obtuse-convex (polar view). the ornamentation is reticulate. keywords: pollen, morphology, lathyrus, cicercula, turkey introduction pollen morphology is not affected by environmental conditions; therefore it has been accepted as a reliable taxonomic marker in the identification of higher plants (aytug 1959). the genus lathyrus l. (fabaceae), comprising nearly 200 species with annual and perennial plants, is centered in mediterranean countries and mainly distributed in the northern hemisphere and the highlands of tropical africa (seen 1938; davis 1970, 1988; heywood 1978; kupicha 1983). the flora of turkey contains 64 species belonging to the sections orobus, platystylis, pratensis, orobastrum, orobon, lathyrus, cicercula, aphaca, nissolia and clymenum; a total of 24 of these are endemic (davis 1970, 1988; guner et al. 2000). the pollen morphology have been already investigated in lathyrus digitatus (aytug 1967, aytug et al. 1971), l. undulatus, l. sylvestris and l. ochrus (gunes and cirpici 1998). l. sylvestris, l. pratensis, l. maritimus, l. nissolia and l. montanus (moore et al. 1991), l. grandiflorus, l. latifolius, l. sylvestris, l. tuberosus, l. alpestris, l. aureus, l. linifolius, l. niger, l. palustris, l. transsilvanicus, l. venetus and l. vernus (tosheva et al. 2004, tosheva and tonkov 2005), l. emodii, l. cicera, l. humulis and l. pratensis (perveen and qaiser 1998). the authors indicated that the pollen grains are mainly tricolporatae. in addition to pollen type, a tectate type of exine structure and a reticulate acta bot. croat. 69 (1), 2010 83 * corresponding author, e-mail: drgunes@gmail.com u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:17 color profile: disabled composite 150 lpi at 45 degrees type of sculpture were also reported in all lathyrus species. furthermore, moore et al. (1991) also noted that the endexine thickens 2–3 times around the colpus and the porus and form a large costae formation. material and methods four taxa of lathyrus were collected from their natural habitats in the thrace region of turkey between 1996 and 1998. all collected specimen were identified and deposited at the herbarium of the biology department, faculty of arts and sciences of the marmara university (mufe). pollen grains for lm examination were prepared following the standard procedure of wodehouse (1959) and the acetolysis method (erdtman 1960). fifteen pollen characters were investigated by light microscope (lm). p (polar diameter), e (equatorial diameter) were measured at a magnification of 400 times and clg (colpus length), clt (colpus width), plg (porus lenght regarding the poles), plt (porus width regarding the equatorial diameter), t (one edge of polar triangle), exine thickness, intine thickness and costa were also measured under a magnification of 1000´. structure (exine), sculpture (exine ornamentation) were also shown. for sem examination, pollen grains were coated with gold. the microphotographs were obtained using jeol-jsm-5200 scanning electron microscope (sem) at a magnification of 2000–5000´. the arithmetic means, standard deviation and variations were calculated for each characteristic. standard deviations are not given for characteristics having no requirement for multiple measurements. in table 1, fresh pollen measurements are given in light and the fossilized pollen measurements are given in bold. to determine the structure and sculpture characteristics, the specimens specifically prepared with the acetolysis method were analyzed under lm microscopy. reticulate type of sculpture was shown by photographs taken in sem (fig. 2). the pollen morphological descriptions follow the terminology of iversen and troels-smith (1950), reitsma (1970), moore et al. (1991) and punt et al. (1994). results the following shapes of pollen were found in the four lathyrus species: 3-zonocolporate type, a tectate type of exine structure and a reticulate type of sculpture, subprolate and prolate. colpus width in fossilized grains appears to have decreased based on the values determined as described above. as the pollens fossilize they increase in the direction of the poles, that is they increase in length. the colpus measurements were done at the regions close to the pores. although the colpus above the pores in l. gorgoni enlarged as much as the pore itself, the colpus above the pore narrowed to a line in l. cicera. lathyrus annuus l. (tab. 1, figs. 1, 2) a1(e). edirne: saçlimüsellim-sigh ircik village road, 1 km from rice-field edges, 30 m, 25.05.1997, f. gunes, mufe 5392. pollen class: 3-zonocolporate. pollen group: subprolate [p/e=1.112 (wodehouse) and p/e=1.114 (acetolysis)] dimensions: medium size [p ´ e = 36.679 ´ 33.085 (wodehouse), 39.676 ´ 35.620 (acetolysis)]. 84 acta bot. croat. 69 (1), 2010 gunes f., cirpici a. u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:17 color profile: disabled composite 150 lpi at 45 degrees apertures: ectoapertures – colpi: short, narrow, deep, with acute ends, thick costae along the margins of the colpi, colpus membrane covered by clearly visible granules; clg: 20.300 (wodehouse), 32.5 (acetolysis) mm, clt:1.8 (wodehouse), 2.3 (acetolysis) mm. endoapertures – pori : lalongate (wodehouse), lolongate (acetolysis) methods., plg: 7.0 (wodehouse), 12.6 (acetolysis) mm, plt: 9.0 (wodehouse), 9.9 (acetolysis) mm and plg/plt= 0.8 (wodehouse), 1.3 (acetolysis). outlines: equatorial view – elliptic to rectangular-obtuse-convex; polar view circular ornamentation: reticulate; reticules distinct, big, irregular. heads of single columellae are visible inside the lumina. colpus area, porus and apocolpium are slightly reticulate. ex/int (wodehouse): 1/1 exine (acetolysis): 2 mm thick, tectate – infrastructure, nexine/sexine = 1/1 lathyrus gorgoni parl. var. pilosus (tab. 1; fig. 1–4). a1(e) i · stanbul: büyükçekmece-highway way out, through road, meadow, 30 m, 11.05.1996, f. gunes, mufe 5007. pollen class: 3-zonocolporate. pollen group: subprolate [p/e=1.1426 (wodehouse)] – prolate [p/e=1.3944 (acetolysis)]. dimensions: medium size [p ´ e = 41.228 ´ 36.081 (wodehouse), 56.992 ´ 40.872 (acetolysis)]. apertures: ectoapertures – colpi: long, narrow, deep, nearly reaching the poles, with acute ends, thick costae along the margins of the colpi, colpus membrane covered by clearly visible granules; clg: 34.266 (wodehouse), 55.240 (acetolysis) mm, clt: 2.737 (wodehouse), 3.383 (acetolysis) mm. endoapertures – pori: lalongate to slightly lalongate, plg: 9.790 (wodehouse), 13.440 (acetolysis) mm, plt: 11.437 (wodehouse), 14.252 (acetolysis) mm and plg/plt= 0.856 (wodehouse), 0.943 (acetolysis). outlines: equatorial view – elliptic to rectangular-obtuse-convex; polar view circular ornamentation: reticulate; reticules distinct, big and irregular. heads of single columellae are visible inside the lumina. colpus area, porus and apocolpium are slightly reticulate. ex/int (wodehouse): 1/2 exine (acetolysis): 3 mm thick, tectate – infrastructure, nexine/sexine = 1/1 lathyrus cicera l. (tab. 1; figs. 1–6). a1(e) çanakkale: gelibolu-eceabat road, 20 km. 0–10 m, 24.04.1998, f. gunes and a. cirpici, mufe 5646. pollen class: 3-zonocolporate. pollen group: subprolate [p/e=1.321 (wodehouse)) – prolate (p/e=1.574 (acetolysis)]. dimensions: medium size [p ´ e = 41.707 ´ 31.581 (wodehouse), 60.320 ´ 38.324 (acetolysis)]. apertures: ectoapertures – colpi: long, slightly wide, nearly reaching the poles, with acute ends, thick costae along the margins of the colpi, colpus membrane covered by small granules; clg: 31.390 (wodehouse), 58.804 (acetolysis) mm, clt: 4.031 (wodehouse), 3.864 (acetolysis) mm. endoapertures – pori: lalongate, plg: 4.379 (wodehouse), 10.366 (acetolysis) mm, plt: 8.120 (wodehouse), 14.868 (acetolysis) mm and plg/plt= 0.539 (wodehouse), 0.6972 (acetolysis). outlines: equatorial view – elliptic to rectangular-obtuse-convex; polar view circular to slightly triangular acta bot. croat. 69 (1), 2010 85 pollen morphology in lathyrus u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:17 color profile: disabled composite 150 lpi at 45 degrees 86 a c t a b o t .c r o a t .69 (1),2010 g u n e s f .,c ir p ic i a . tab. 1. mean pollen characteritics (m), structure and sculpture (ornamentation). s – standard deviation, var. – variations of line numbers of polen lamela, w – wodehouse-metod, a – asetolysis-metod, p – polar diameter, e – equatorial diameter, p/e – pollen shape, ex – exine thickness, ex/int – the ratio of exine to the intine, clg – colpus length, clt – colpus width, plg – porus length regarding the poles, plt – porus width regarding the equatorial diameter, plg/plt – porus shape, t – one edge of polar triangle. lathyrus annuus lathyrus gorgoni var. pilosus lathyrus cicera lathyrus hirsutus m s var. m s var. m s var. m s var. p w 36.679 ±1.617 11–13 41.228 ±1.597 13–16 41.707 ±1.354 13–15 38.976 ±2.504 13–14.5 a 39.676 ±2.590 12–17 56.992 ±2.257 20–24 60.320 ±3.247 20–25 52.832 ±2.463 18–23 e 33.085 ±1.462 10–13 36.081 ±1.476 11–14 31.581 ±0.974 10–11.5 32.799 ±1.227 10–11.5 35.620 ±2.031 12–16 40.872 ±2.146 14–17 38.324 ±2.108 13–16 30.784 ±1.198 10–14 p/e 1.112 subprolate 1.143 subprolate 1.321 subprolate 1.188 subprolate 1.114 subprolate 1.394 prolate 1.574 prolate 1.716 prolate ex not measured not measured i · nce not measured not measured not measured not measured not measured 2 3 3 2 ect/end 1/1 1/1 1/1 1/1 ex/int 1/1 not measured ½ not measured 2/1 not measured 1/1 not measured clg 20.300 ±1.477 16–21 34.266 ±1.317 27–32 31.390 ±1.631 24–30 27.882 ±1.211 22–2 6 32.523 ±2.343 20–26 55.240 ±3.553 35–44 58.804 ±3.493 39–47 47.796 ±2.761 29–38 clt 1.856 ±0.568 1–2 2.7377 ±0.950 1–5 4.031 ±0.733 2–5 3.712 ±0.843 2–4 2.275 ±0.500 1–2 3.383 ±1.063 1–4 3.864 ±1.548 1–5 1.960 ±0.686 1–2 plg 7.018 ±0.498 5–7 9.790 ±0.903 7–10 4.379 ±0.710 3–5 8.294 ±0.611 6–8 12.593 ±1.565 7–11 13.440 ±1.313 7–12 10.366 ±1.249 5–9 10.556 ±0.978 6–9 u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 0 \ g u n e s . v p 9 . t r a v a n j 2 0 1 0 1 3 : 0 7 : 1 8 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s a c t a b o t .c r o a t .69 (1),2010 87 p o l l e n m o r p h o l o g y in l a t h y r u s tab. 1. – continued lathyrus annuus lathyrus gorgoni var. pilosus lathyrus cicera lathyrus hirsutus m s var. m s var. m s var. m s var. plt 9.048 ±0.696 7–9 11.438 ±0.957 8–12 8.120 ±0.935 5–9 10.701 ±0.661 8–10 9.870 ±1.744 5–10 14.252 ±1.810 8–13 14.868 ±1.768 8–13 10.136 ±0.856 6–8 plg/plt 0.776 not measured not measured 0.856 not measured 0.539 not measured not measured 0.775 not measured not measured 1.276 not measured not measured 0.943 not measured 0.697 not measured not measured 1.041 not measured not measured t not measured not measured not measured 14.500 ±1.249 10–15 17.110 ±1.264 13–16 not measured not measured not measured structure tectateinfrastructure tectateinfrastructure tectateinfrastructure tectateinfrastructure sculpture reticulate, distinct, big, irregular reticulate, distinct, big and irregular reticulate, distinct, big and irregular reticulate, sligylt, distinct, regular and small u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 0 \ g u n e s . v p 9 . t r a v a n j 2 0 1 0 1 3 : 0 7 : 1 8 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s ornamentation: reticulate; reticules slightly distinct, big and irregular. colpus area, porus and apocolpium are slightly reticulate. ex/int (wodehouse): 2/1 exine (acetolysis): 3 mm thick, tectate – infrastructure, nexine/sexine = 1/1 lathyrus hirsutus l. (tab. 1, figs. 1 – 8). a1(e) edirne: edirne-doyran village entrance, through water channels, 60 m, 31.05.1998, f. gunes and a. cirpici, mufe 5739. pollen class: 3-zonocolporate. 88 acta bot. croat. 69 (1), 2010 gunes f., cirpici a. fig. 1. pollen grains of lathyrus annuus (1, 2), l. gorgoni (3, 4), l. cicera (5, 6) and l. hirsutus (7, 8). 1, 3, 5, 7 – equatorial view; 2, 4, 6, 8 – polar view (lm). bar denotes 15 mm. u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:28 color profile: disabled composite 150 lpi at 45 degrees pollen group: subprolate [p/e = 1.188 (wodehouse)) – prolate (p/e=1.716 (acetolysis)]. dimensions: medium size [p ´ e = 38.976 ´ 32.799 (wodehouse), 52.832 ´ 30.784 (acetolysis)]. apertures: ectoapertures – colpi: long, slightly wide (wodehouse), narrow (acetolysis), nearly reaching the poles (wodehouse), with acute ends, thick costae along the margins of the colpi, colpus membrane covered by small granules; clg: 27.882 (wodehouse), 47.796 acta bot. croat. 69 (1), 2010 89 pollen morphology in lathyrus fig. 2. ornamentation in pollen grains of lathyrus annuus (1, 2); l. gorgoni (3, 4); l. cicera (5, 6); l. hirsutus (7, 8) (sem). 1, 3, 5, 7 – equatorial view; 2, 4, 6, 8 – polar view. u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:30 color profile: disabled composite 150 lpi at 45 degrees (acetolysis) mm, clt: 3.712 (wodehouse), 1.960 (acetolysis) mm. endoapertures – pori: lalongate to circular, plg: 8.294 (wodehouse), 10.556 (acetolysis) mm, plt: 10.701 (wodehouse), 10.136 (acetolysis) mm and plg/plt= 0.775 (wodehouse), 1.041 (acetolysis). outlines: equatorial view – eliptic to rectangular-obtuse-convex; polar view circular ornamentation: reticulate; reticules sligylt, distinct, regular and small. colpus area is psilate. porus and apocolpium are slightly reticulate. ex/int (wodehouse): 1/1 exine (acetolysis): 2 mm thick, tectate – infrastructure, nexine/sexine = 1/1 discussion pollen dimensions varied in three of the taxa; the exception is l. annuus. the subprolate type of pollen shape (p/e) was found in fresh pollens of l. gorgoni, l. cicera and l. hirsutus, but the prolate shape was found in fossilized pollens of those three species. in contrast to l. gorgoni, l. cicera, l. hirsutus, subprolate shape (p/e) was observed in both fresh and fossilized pollens of l. annuus (tab. 1). pollen dimensions and shape differed in fresh and fossilized pollens (tab. 1). the ratio of plg to plt in fresh pollens was 0.775 in l. annuus, 0.856 in l. gorgoni, 0.539 in l. cicera, and 0.775 in l. hirsutus. the values found in fossilized pollens were 1.276 in l. annuus, 0.943 in l. gorgoni, 0.697 in l. cicera, and 1.041 in l. hirsutus. looking at the pore length, the ratio of plg to plt in fossilized pollens revealed an increase in the four species. although a tectate-infrastructure (exine) and a reticulate ornamentation were seen in all four taxa (iversen and troels-smith 1950, aytug et al. 1971, moore et al. 1991, tosheva and tonkov 2005), our study showed that the reticulate size and its arrangement were distinct from each other (tab. 1, fig. 2) and that l. annuus possessed a distinct, big, and evenly shaped reticulation. a very distinct and unevenly shaped reticulation was also seen in l. gorgoni. similar to these results in l. gorgoni, a very big distinct and uneven shaped reticulation was observed in l. cicera. a slightly distinct, evenly shaped and small sized reticulation was found in l. hirsutus. although a reticulate pattern in all lathyrus species is clearly distinct in the center of its pollens, a distinguishable reticulate characteristic appeared to be diminished in the split and holes of pollens. reticulation was clearly distinguishable in the polar regions of l. annuus, l. gorgoni and l. cicera, whereas l. hirsutus (fig. 2). operculum above pores and colpus had a reticulated appearance. the contribution of this study to previously reported data on the study of pollen morphology can be summarized as follows: 1. the pollen grains are 3-zonocolporate of subprolate – prolate type (p/e = 1.112 – 1.188 (wodehouse), 1.114 – 1.716 mm (acetolysis)), medium to large in size. the smallest pollen grains belong to l. annuus (p´e = 36.679 ´ 33.085 mm in wodehouse and asetolysis) and the biggest to l. cicera (p/e = 41.707 ´ 31.581 [wodehouse) – (p/e = 60.320 ´ 38.324 mm (acetolysis)]. equatorial view is elliptic to rectangular-obtuse-convex. the polar view in all pollen grains is circular. 2. the aperture system consists of ectoapertures – colpi and endoapertures – pori. the colpi are generally straight, narrow, with acute ends and thick costae along the colpus regions. there are small granules in colpus membrane. the pori are large, circular to lalongate. 90 acta bot. croat. 69 (1), 2010 gunes f., cirpici a. u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:30 color profile: disabled composite 150 lpi at 45 degrees 3. the thickness of the exine is 1 mm in wodehouse and 2–3 mm in acetolyse, the nexine as thick as the sexine. 4. the ornamentation is reticulate in all four taxa, but the size and the arrangement of the lumina are different from each other. the reticulate ornementation is clear in mesocolpium, while the apocolpium, the colpus area and the porus are psilate or small. although pollens in observed taxa seemed to be of a type 3-zonocolporate characteristic, the shapes and largeness of apertures, the largeness of the polar triangle, the clearness of the aperture borders, the shape of the pollens (p/e), the thickness of the exine and the intine, played an important key for the diagnostic of the differentiation of the taxa aytug (1959). in conclusion, it can be stated that the four taxa discussed, although belonging to the same section, can clearly be differentiated based on the palynological findings as described. acknowledgements this work is a part of the project supported by the institution of science at marmara university (1996 fen-16). references aytug, b., 1959: role of palinology in taxonomy and classification (in turkish). journal of forestry faculty of istambul university, serial b, 9, 118–125. aytug, b., 1967: pallinologic researches in polen morphology and important gymnosperms of turkey (in turkish). istanbul university press, istanbul. aytug, b., aykut, s., merev, n., edis, g., 1971: pollen atlas of plants from environs of istanbul (in turkish). yayin 1654, 174. davis, p. h.,1970: lathyrus l. in: davis, p. h. (ed.), flora of turkey, 3. university press, edinburg. davis, p. h., 1988: flora of turkey, 10. university press, edinburg. erdtman, g., 1960: the acetolysis method, a revised description. upsala, svensk botanic tidskrift 54, 561–564. guner, a., ozhatay, n., ekim, t., baser, k. h. c. (eds.), 2000: flora of turkey and the east aegean islands, 11 (supplement 2). university press, edinburg. gunes, f., cirpici, a., 1998: pollen morphology of some lathyrus species (l. undulatus boiss., l. sylvestris l., l. ochrus (l.) dc.) of istanbul area (in tukish). proceedings of the symposium on quercus vulcanica and flora of turkey, çantay kitapevi, laleli-istanbul, 431–440. heywood, vh., 1978: flowering plants of the world. oxford univ.press, oxford. iversen j., og j. troels-smith., 1950: pollen morphological definitions and types. denmark geological research, kobenhaven. kupicha, fk., 1983: the infrageneric structure of lathyrus. notes from the royal botanic garden edinburg 41, 209–244. acta bot. croat. 69 (1), 2010 91 pollen morphology in lathyrus u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:30 color profile: disabled composite 150 lpi at 45 degrees moore, pd., webb, ja., collinson, m., 1991: pollen analysis. blackwell sci. publication. london. perveen, a., qaiser, m., 1998: pollen flora of pakistan, 8. leguninosae (subfamly: papilionoideae), turkish journal of botany 22, 73–91. punt, w., blackmore, s., nilsson l. e., thomas, a., 1994: glossary of pollen and spores terminology. laboratory for paleobotany and palynology, utrecht. reitsma, t., 1970: suggestions towards unification of descriptive terminology of angiospermae pollen grains. 10: 39–60. seen, ha., 1938: experimental data for the revision of the genus lathyrus. american journal of botany 25, 67–78. tosheva, a., tonkov., s. dimitrov, n., 2004: pollen morphology of bulgarian species from the section lathyrus (lathyrus, fabaceae). phytologia balcanica 9, 529–536. tosheva, a., tonkov, s., 2005: pollen morphology of bulgarian species from the section orobus (l.) gren. et godr. (genus lathyrus, fabaceae). acta botanica croatica 64, 275–287. wodehouse, r. p., 1959: pollen graine. mcgraw-hill, new york. 92 acta bot. croat. 69 (1), 2010 gunes f., cirpici a. u:\acta botanica\acta-botan 1-10\gunes.vp 9. travanj 2010 13:07:30 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 76 (2), 2017 191 acta bot. croat. 76 (2), 191–195, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0003 issn 0365-0588 eissn 1847-8476 short communication bouché’s star of bethlehem, ornithogalum boucheanum (kunth) asch. (hyacinthaceae), a new species in flora of croatia dragica purger1*, sanja kovačić2, jános csiky1 1 department of ecology, institute of biology, faculty of sciences, university of pécs, ifjúság útja 6, h-7624 pécs, hungary. 2 botanical garden, department of biology, faculty of science, university of zagreb, trg marka marulića 9a, 10000, zagreb, croatia. abstract – populations of bouché’s star of bethlehem (ornithogalum boucheanum (kunth) asch., fam. hyacinthaceae) were recorded on bansko hill (baranja, croatia) in 2007. since this species has not been previously confirmed in croatia, it should be treated as a new taxon in the country and included in the flora croatica database. in this paper we present a short morphological description of o. boucheanum and diagnostic morphological characters for differentiation from the related o. nutans l. we suggested o. boucheanum be evaluated as a critically endangered (cr) species of the croatian flora, considering the small number of individuals and the small extension of its population. the recording of its populations on the edge of the loess cliff in bansko hill a part of which belongs to the important plant area and natura 2000, confirms the significance of this unique habitat in preserving rare and endangered plants. key words: bansko hill, baranja, loess, myogalum boucheanum, ornithogalum nutans, threatened plants * corresponding author, e-mail: dragica@gamma.ttk.pte.hu introduction bouché’s star of bethlehem (ornithogalum boucheanum (kunth) asch.; österr. bot. zeitschr. (1866) 16: 192), (syn.: myogalum boucheanum kunth, honorius boucheanus (kunth) holub, ornithogalum nutans l. subsp. boucheanum (asch.) hayek; liliales, hyacinthaceae), is a lowland-colline plant, an eastern-sub-mediterranean-pannonic (pontic) floristic element (soó 1973). the species is distributed in central and southeastern europe, where it is considered native, while in north and western europe it is treated as adventive (zahariadi 1980). the species is distributed in the caucasus, around the black sea, through anatolia and the balkans to the eastern part of the danube valley and the carpathian basin (soó 1973). according to jávorka (1925) it does not occur in croatia. during a survey of the loess flora and vegetation on bansko hill in baranja (ne croatia) we found two small populations of ornithogalum boucheanum. this species is not included in the recent croatian botanical literature (cf. domac 2002; nikolić 2000; nikolić and topić 2005) or in the national database of the croatian flora (nikolić 2016a,b). there are no data about this species in the papers dealing with the flora of bansko hill (sturc 1988) and baranja (panjković 1990, zahirović 2000). the occurrence of a related species, the nodding star of bethlehem (ornithogalum nutans l.) is reported from the northern part of croatia (rauš and šegulja 1983) and several adriatic islands (nikolić 2016a). the native range of this species is smaller than that of the related o. boucheanum and it comprises anatolian and balkan peninsulas. in most of europe it is considered to be an alien species, or else it is of unknown status (euro+med 2006–). ornithogalum boucheanum and o. nutans are two species of the subgenus myogalum (link) baker in europe spe cimens of which are difficult to separate on the basis of mor phological characters, especially after they have bloomed (zahariadi 1980). since some authors of the last century (e.g. hayek 1933) considered o. boucheanum a subspecies of o. nutans (o. nutans l. subsp. boucheanum (asch.) hayek), there is a possibility, that these two taxa were not properly distinguished when collected in the last century. therefore our aim was to check the specimens in herbarium croaticum (za) and herbarium in budapest (bp) and to clarify the status of these two related taxa in croatia. in this paper we present a short morphological description of o. boucheanum and diagnostic morphological characters for differentiation from the related o. nutans. purger d., kovačić s., csiky j. 192 acta bot. croat. 76 (2), 2017 material and methods for determination of plant species we used the recent croatian (domac 2002) and hungarian (király 2009) handbooks. the description of the species is given based on characteristics of the collected specimens and according to related literature (e.g. speta 2008, meriç et al. 2011). the distribution map of o. boucheanum in croatia (fig. 1) was compiled according to the central european flora mapping system (nikolić et al. 1998). geocoding of site was made by a gps device. for classification of o. boucheanum into iucn red list categories we used criteria after nikolić (2005) and iucn (2012) amended for croatia in 2014. examples of plants have been collected (on april 16, 2008) and a voucher specimen was deposited in herbarium croaticum (za). study area bansko hill (fig. 1.) is a 21 km long elevation (243 m a.s.l.) extending in a ne–sw direction, and consists of loess sediments. its southeastern part is a sheer, 25–58 m high loess cliff, facing the floodplain of the river danube (purger and csiky 2008). results and discussion during a survey of loess flora and vegetation on bansko hill (ne croatia) from zmajevac to batina (0178/4 central european flora mapping quadrate) two small populations of o. boucheanum were recorded (fig. 1.), at the very border of the natura 2000 site hr2001309 “dunav north of kopački rit” (anonymous 2013a). wgs 84 coordinates, altitude, population size and brief habitat descriptions: 1. n 45°49’11.10”, e 18°49’40.14”, 100 m a.s.l., small population of fewer than 10 individuals, on the edge of the plateau, in a secondary xerophilous forest with fraxinus ornus, acer campestre, robinia pseudacacia in the tree layer, crataegus monogyna, prunus spinosa, viburnum lantana in the shrub layer and anthriscus cerefolium, ballota nigra, bromus sterilis, chelidonium majus, erodium ciconium, ga lium aparine, geranium rotundifolium, stellaria media and veronica hederifolia in the herb layer (leg./det.: j. csiky, d. purger, 26.04.2007, zmajevac); 2. n 45°49’13.99”, e 18°49’42.39”, 100 m a.s.l., small popu lation of fewer than 100 individuals, in a secondary xerophilous forest with robinia pseudacacia, fraxinus ornus in the tree layer, crataegus monogyna, prunus spinosa, euonymus europaeus, ulmus minor in the shrub layer, anthriscus cerefolium, artemisia campestris, arum orientale, ballota nigra, bromus sterilis, carduus acanthoides, chelidonium majus, securigera varia, erodium ciconium, euphorbia cyparissias, e. helioscopia, fumaria schleicheri, galium aparine, g. mollugo, geranium pusillum, geum urbanum, lamium purpureum, melandrium album, muscari neglectum, onopordon acanthium, sisymbrium orientale, stellaria media, veronica hederifolia, vicia narbonensis subsp. serratifolia, v. pannonica, viola cf. odorata in the herb layer (leg./det.: j. csiky, d. purger, 16.04.2008., zmajevac [za]). the populations on bansko hill occurred in the shrubs and transitional zone of grassland and forest in the very border of the loess plateau. these are the only known and proved localities of o. boucheanum in croatia. since this species has not been previously identified in the country, it should be treated as a new taxon of the vascular flora of croatia and included in the flora croatica database (nikolić 2016a). we also checked data of similar, related species o. nutans to clarify its status in the country. in the old botanical literature occurrence of this species (“between vukovar and sotin”) is mentioned (strein ap. schlosser and vukotinović fig. 1. distribution of ornithogalum boucheanum (*) on bansko hill (croatia). a grid square measures approximately 6500 (latitudinal) ×5500 (longitudinal) metres. abbreviations: ba – batina; bm – beli manastir; br – branjina; bv – branjin vrh; dr – draž; ga – gajić; ka – kamenac; kr – karanac; ko – kotlina; kv – kneževi vinogradi; pd – podolje; pp – popovac; su – suza; zm – zmajevac (drawn by j. csiky and t. nikolić). ornithogalum boucheanum the new species in flora of croatia acta bot. croat. 76 (2), 2017 193 fig. 2. ornithogalum boucheanum near zmajevac on bansko hill: a) flowers and b) fruits (photo: j. csiky and d. purger). 1869). the specimen of star of bethlehem collected by pavich in the 19th century near sava river (slavonia), identified as o. nutans is deposited in the herbarium croaticum (za). it was the only record of this species reported from the continental part of croatia (nikolić 2016a). after checking this specimen in za, we found that it was ornithogalum umbellatum s.l. misidentified as o nutans. probably upon these old data the nodding star of bethlehem (o. nutans) is listed in the flora of slavonia and baranja (rauš and šegulja 1983), though without any localities or recent findings. accordingly, we can conclude, that the distribution of o. nutans in croatia is restricted to the mediterranean part of the country (nikolić 2016a). there are three specimens in the herbarium in budapest which are supposed to be o. boucheanum and are said to originate from croatia. on these sheets data about locality, habitat and date of collecting are missing or ambiguous, therefore upon that material we could not confirm the occurrence of o. boucheanum in the mediterranean part of croatia. according to the data of its distribution in hungary, it seems that this species becomes rare towards the south-west (towards croatia) and it is already rare in baranya county (bartha and király 2015). as the handbook for the identification of plants in croatia (domac 2002) does not contain o. boucheanum, here we provide a description of this species and additional information for its determination. according to our own knowledge and literature data (zahariadi 1980, hrouda 2002, mesterházy and király 2009, meriç et al. 2011), we summarised the morphological and anatomical features of o. boucheanum and the closely related o. nutans, which are very similar, but differ in several characters (tab. 1). ornithogalum boucheanum is a 20–60 cm high plant. bulb ovoid-globose, 30×40 mm, tunica light brown. scape erect, cylindrical, light green. leaves (3–6) bright green, as long as the scape or shorter, 1.0–1.5 cm wide, 20–40 cm long, margin entire, edges are parallel and gradually tapering to acute apex, at flowering time withered. the adaxial face of the leaf has white median line. raceme corymbose, dense, 10–20 flowered. flowers scentless. bract 22–25 mm long, lanceolate-acuminate, light brown and membrane-like structured. pedicel 5–10 mm, 10–13 mm in fruit. perianth segments 18–25 (–30) mm long, 2–3.5 mm wide, lanceolate, milky-white with green fascia inside and outside (fig. 2a). anthers 2–3 (–3.5) mm, white, light yellow, filament 4–5 (–6) mm, lanceolate, wide and winged, ending with two lateral teeth. stigma spatulate. ovary ovoid-elliptic (green), as long as style (white). capsule ovoid (fig. 2b) 18×12 mm, erect. seeds numerous, black, subglobose to globose, black and reticulate with wrinkled, undulate walls. according to the results of bednorz and czarna (2008) seeds of o. boucheanum and o. nutans, two closely related and morphologically very similar species, are practically undistinguishable. although the species are similar in regard to the scape anatomy, their leaf anatomy is different: mesophyll in o. boucheanum is thicker, differentiates as palisade parenchyma which is present on both the adaxial and abaxial side and spongy parenchyma, which contains large lacuna (meriç et al. 2011). ornithogalum is a taxonomically difficult genus; its morphology is poorly correlated with the variation in chromosome number and karyotype. the chromosome numbers of o. boucheanum have been determined as 2n=56 (basic chromosome number x= 8), while the chromosome numbers of o. nutans are determined as 2n=14 and 2n=35 (basic chromosome number x= 7) (dalgıc and ozhatay 1997, dalgıc et al. 2009). o. boucheanum rarely occurs in southern transdanubia (hungary), in grasslands (purger 2008) and spring weed vegetation in vineyards (pál 2007); in this part of the countab. 1. diagnostic characters of ornithogalum boucheanum and o. nutans. characters o. boucheanum o. nutans raceme (5)10–15(20) flowered (3)6–10(12) flowered perianth segments (length) 20–35 mm 15–20 mm perianth segments (colour) green and white inner side, dark green stripe on outer side white inner side, greenish stripe on outer side ovary (length) 4–7 mm 3–5 mm style (length) 5–7 mm <5 mm toothed filaments 6 3 seed 2–2.5 mm, broadly ovoid-orbicular 1.5–2 mm, ovoid-orbicular leaf anatomy mesophyll equifacial (1000 μm) mesophyll unifacial (700 μm) pollen grains (p – polar axis, e – equatorial axis) p = 70.84±0.35 μm e = 50.27±1.22 μm p = 64.02±0.15 μm e = 42.01±0.20 μm purger d., kovačić s., csiky j. 194 acta bot. croat. 76 (2), 2017 try it is considered to be a strongly endangered plant (pál 2005). in hungary it grows in clay (loamy) soils on loess and in humic sandy soils and can be found in different habitats, mostly in forest clearings, sparse oak forests, on open slopes, in valleys along streams, in lowland-colline areas with altitudinal range up to 500 m (soó 1973). according to borhidi (1995) it is a natural weed, a plant of disturbed habitats which belongs to the vegetation of class artemisietea. in serbia it occurs only in the pannonian part (vojvodina province): the deliblato sandy area, the vicinity of kikinda (diklić 1975), riđica, sombor, bački monoštor (obradović et al. 1981); fruška gora mountains: irig (butorac 1992). focus on the carpathian basin: o. boucheanum is a sparse but not endangered species in hungary (király 2007), rare and critically endangered in austria (speta 2008). in slovakia this species is endangered (en) according to feráková et al. (2001), however recently eliáš et al. (2015) considered it as least concern (lc). in serbia it is a statutorily protected but not threatened species (stevanović 1999). so far, ornithogalum boucheanum is known in croatia from only the two (very close) localities presented here, its estimated ‘area of occupancy’ being less than 10 km2 (criterion b1a and b2a) and population number of the species is estimated to fewer than 1000 mature individuals (criterion c), therefore it could be evaluated as a critically endangered (cr) species of the croatian flora (nikolić 2005; iucn 2012). accordingly, we also propose that this species should be included among the statutorily strictly protected species of croatian flora (acc. to anonymous 2013b). since loess cliff habitats are very rare and sporadically distributed in ne croatia, a part of this area is considered an ipa (important plant area) site and belongs to the natura 2000 network. this habitat is endangered by the invasion of the black-locust tree (robinia pseudacacia) and tree of heaven (ailanthus altissima) (purger and csiky 2008). the recording of o. boucheanum on the edge of a loess cliff in bansko hill confirms the extreme importance of this habitat for preserving this rare and endangered plant species. acknowledgement we thank vedran šegota and lajos somlyay for helping us to access the sheets of za and bp. this survey was carried out with permission given by the croatian ministry of culture (klasa: up/i-612-07/07-33/739, urbroj: 53208-01-01/3-07-02, zagreb, may 2007) and it was supported by interreg iii a, slo-hu-cro 2006/01/167/hu project. references anonymous, 2013a: regulation of ecological network natura 2000 croatia. official gazette (og) 124/2013 (in croatian). anonymous, 2013b: ordinance of strictly protected species. offi -: ordinance of strictly protected species. offi -ordinance of strictly protected species. official gazette (og) 144/2013 (in croatian). bartha, d., király, g. 2015: distribution atlas of vascular plants of hungary. university of west hungary press, sopron. bednorz, l., czarna, a., 2008: sem and stereoscope microscope observations on the seeds of some ornithogalum (hyacinthaceae) species. biologia 63, 642–646. borhidi, a., 1995: social behaviour types of the hungarian flora, its naturalness and relative ecological indicator values. acta botanica hungarica 39, 91–181. butorac, b., 1992: vegetation of loess plateau of the fruška gora mt. matica srpska, novi sad (in serbian). dalgıc, g., ozhatay, n., 1997: the genus ornithogalum (liliaceae) and its karyotype variation in european turkey. bocconea 5, 743–747. dalgıc, g., dane, f., aksoy, o., 2009: a new record for the flora of turkey: ornithogalum boucheanum (hyacinthaceae). in: iva-ivanova, d. 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(eds.), exkursionsflora von österreich, liechtenstein und südtirol. 3. auflage, 1069–1077. biologiecentrum der oberösterreichischen landesmuseen, linz. stevanović, v. (ed.), 1999: red data book of serbian flora 1. extinct and critically endangered taxa. ministry of environministry of environment of the republic of serbia, faculty of biology, university of belgrade, institution for protection of nature of the republic of serbia, belgrade (in serbian). sturc, b., 1988: floristic and geobotanical data from loess hills in baranya. pécsi műszaki szemle 33, 19–24 (in hungarian). zahariadi, c., 1980: ornithogalum l. in: tutin, t. g., heywood, v. h. burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea. volume 5, 35–40. cambridge university press, cambridge. zahirović, ž., 2000: rare and endangered plants of north-eastern part of croatia. msc thesis, faculty of sciences, university of zagreb, zagreb (in croatian). 120 acta bot. croat. 76 (2), 2017 acta bot. croat. 76 (2), 120–131, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0013 issn 0365-0588 eissn 1847-8476 new xenophytes from the canary islands (gran canaria and tenerife; spain) filip verloove botanic garden of meise, nieuwelaan 38, b-1860 meise, belgium abstract. – recent fieldwork in gran canaria and tenerife (canary islands, spain), mostly between 2012 and 2016, yielded new chorological data for several non-native vascular plant species. the following are considered naturalized and/or potentially invasive: callistemon viminalis, casuarina glauca, chloris barbata, cyperus difformis, eucalyptus gomphocephala, e. sideroxylon, nephrolepis cordifolia, rumex palustris, senna × artemisioides (s.l.) and s. × floribunda and are reported for the first time from the canary islands. other first records include: cascabela thevetia (tenerife), cyclospermum leptophyllum (gran canaria), digitaria radicosa (gran canaria, tenerife), dysphania anthelmintica (tenerife), erythrostemon gilliesii (tenerife), heliotropium supinum (tenerife), limoniastrum monopetalum (tenerife), nerium oleander (tenerife), pascalia glauca (tenerife), phytolacca americana (tenerife), podranea ricasoliana (gran canaria), psidium guajava (gran canaria), rumex cristatus (tenerife), schinus terebinthifolia (tenerife), solandra maxima (tenerife), tipuana tipu (tenerife) and youngia japonica (gran canaria). more than 20 additional taxa also represent chorological novelties but are considered ephemerals. finally, miscellaneous notes are added for diplachne fusca subsp. uninervia, eclipta prostrata, pluchea carolinensis, prosopis juliflora and sida rhombifolia from gran canaria. keywords: canary islands, chorology, invasion, naturalization, new records, vascular plants, xenophytes * corresponding author, e-mail: filip.verloove@botanicgardenmeise.be introduction despite being well-documented (hohenester and welss 1993, acebes ginovés et al. 2009), the flora of the canary islands (spain) requires permanent updates. an increase of taxonomic knowledge allows the recognition of poorly known or neglected native species or even the description of new taxa. also, in our globalized world, geographical boundaries have become obsolete, which has enhanced the almost unlimited exchange of goods and their stowaways (diaspores, etc.). in addition to these inadvertently introduced alien species, humans are also responsible for the introduction of economically or otherwise important species to new areas, for instance garden ornamentals. in the past centuries the canary islands, with their subtropical climate, became home for hundreds of ornamental, subtropical plant species. an increasing number of these are not only welladapted to the local climate but started to reproduce locally, in some cases eventually naturalizing, spreading or even becoming invasive. this is a well-known phenomenon that has been widely studied in recent times (foxcroft et al. 2008). moreover, islands are particularly vulnerable, invasive alien species being among the primary threats to biodiversity on islands (vitousek 2002). the early taxonomic recognition of newly detected aliens is of the utmost importance, enabling, if appropriate, the immediate implementation of management practices. even the first, seemingly anecdotic, reproduction of an ornamental is worth reporting, since it may constitute the first (but essential) step in a future naturalization process (böcker et al. 1995). in the past years, mainly between 2012 and 2016, the author documented the presence of numerous non-native species that had not been recorded before from the canary islands or that appear to be acquisitions for the islands of gran canaria and/or tenerife (see also verloove and reyesbetancort 2011 and verloove 2013 for previous reports). some of these probably were introduced unintentionally (as weeds) although most are obviously escapes from cultivation. their degree of naturalization varies from ephemerals to (locally or potentially) invasive aliens. in this paper proportionately more attention is paid to the latter group, which should be included in future editions of the ‘lista de especies silvestres de canarias’ (acebes ginovés et al. 2009). new xenophytes from canary islands acta bot. croat. 76 (2), 2017 121 materials and methods voucher specimens of all taxa are preserved in the public herbaria of the jardín de aclimatación de la orotava, tenerife (ort), the jardín botánico canario viera y clavijo, gran canaria (lpa), the botanic garden of meise, belgium (br) and/or the united states national herbarium, washington, u.s.a. (us). the presence or absence on the islands of gran canaria or tenerife of the non-native taxa here presented was each time compared with data provided by acebes ginovés et al. (2009). for some recently introduced species several additional papers were checked as well. the present paper is divided into three main parts: the first part deals with apparently naturalized and/or (potentially) invasive species, the second one with (presumably) ephemeral aliens and in a third part some miscellaneous notes are presented for diplachne fusca subsp. uninervia, eclipta prostrata, pluchea carolinensis, prosopis juliflora and sida rhombifolia, five taxa of interest from gran canaria. in each part taxa are presented in alphabetical order. for naturalized taxa, each entry includes the scientific name of the taxon, the family to which the taxon belongs (see below), the kind of chorological novelty and the estimated degree of naturalization, enumeration of herbarium collections, origin of the taxon and details about its secondary distribution. for ephemeral records in the second part, in most cases only herbarium data are referred to. the degree of naturalization for each taxon was assessed according to richardson et al. (2000). in some cases, however, the research period was too short for an accurate estimation of invasion status. this particularly holds true for trees and shrubs with long life cycles. in such cases, the degree of naturalization should be considered indicative rather than decisive and takes into account behavior of the species in similar conditions outside of its native range. familial and generic circumscriptions are in accordance with apg iii (2009). authorities of plant names follow tropicos (www.tropicos.org). results naturalized and/or (potentially) invasive species callistemon viminalis (sol. ex gaertn.) g. don (myrtaceae) (pl. 1a) new to the flora of the canary islands. tenerife: los realejos, between calle puerto franco and barranco de palo blanco, foot of wall, 5–10 self-sown individuals, young bushes (under plantation), 12.11.2014, f. verloove 11186 (br); gran canaria: ayagaures, barranco de ayagaures, gc-504 (km 6–7), dry riverbed, few individuals, up to 4 m tall, abundantly seeding, 27.11.2015, f. verloove 12013 (br, lpa). a native of eastern australia, c. viminalis is widely cultivated as an ornamental in warm-temperate and subtropical regions of the world (cullen and knees 2011), also in the canary islands. it is here reported for the first time as an escape. although at present it cannot be considered genuinely naturalized, future naturalization is likely, especially in ayagaures in gran canaria where mature, abundantly seeding specimens were found growing spontaneously in a barranco, in a habitat similar to that in its area of origin. it is considered an invasive species in areas where it was initially introduced as an ornamental, for instance in the southern united states (fleppc 2015). cascabela thevetia (l.) lippold (apocynaceae) new to the flora of tenerife. tenerife: armeñime, barranco de las salinas at tf-47, gravelly riverbed, a single shrub (self-sown), 31.10.2014, f. verloove 11183 (br). this species is originally native in tropical america but it is widely cultivated as a garden ornamental in the (sub-) tropical regions of the world (cullen and knees 2011), also in the canary islands. since 2012 it has been recorded as an escape from cultivation in gran canaria and la palma (verloove 2013, otto and verloove 2016), initially as a mere ephemeral species. however, in the past years it is increasingly seen, also in tenerife, from where it is here reported for the first time. in addition to the locality cited above, it was also observed in several other localities since 2014: santa cruz de tenerife (barranco ifara), playa paraiso (barranco el pinque; several individuals), puerto de la cruz (barranco de martiánez), la estrella (las galletas), palm-mar, etc. also in gran canaria it has been recorded on several instances since its discovery in 2012, e.g. in el pedrazo (barranco del negro) and in arguineguín. in some of these localities this species seems to be established in small populations and a future naturalization is very likely. similar behavior has been observed in, for instance, the pacific islands, australia, south africa, etc. (starr et al. 2002, http://www.invasives.org.za/). in some areas it is considered a high risk invasive weed. casuarina glauca sieber ex spreng. (casuarinaceae) (pl. 1b) new to the flora of the canary islands. gran canaria: san agustín, barranco los guinchos, close to the sea, dry riverbed, very invasive, 17.11.2015, f. verloove 11979 (br, lpa). originally native in a narrow belt along the southeastern coast of australia, c. glauca is more or less widely cultivated elsewhere in warm-temperate and subtropical regions of the world (cullen and knees 2011), either as an ornamental tree or for timber, windbreaks, etc. outside of its area of origin, it probably is the most widespread species of the genus, along with c. cunninghamiana and c. equisetifolia, both already known as escapes from the canary islands (acebes ginovés et al. 2009). all these species are very similar in general appearance and may have been confused so far. c. glauca is readily distinguished from both other species by the higher number of leaf teeth (at least 10, usually 12–14; vs. less than 10). it regenerates copiously through vigorous root suckers and often forms dense stands. in areas where it was once introduced vegetative reproducverloove f. 122 acta bot. croat. 76 (2), 2017 tion is much more important than sexual reproduction. in fact, the species being dioecious, cones are rarely produced outside of australia and most reports of seed-producing c. glauca in the southern u.s.a. probably refer to hybrids (woodall and geary 1985; see also gaskin et al. 2009). however, in a single barranco in san agustín in gran canaria (barranco los guinchos), where apparently both sexes have been planted nearby in the past, plants of this species produce high amounts of samaras and also regenerate vegetatively. the species competes with other invasive escapes such as arundo donax, eucalyptus camaldulensis, schinus terebinthifolia and washingtonia robusta. similar occurrences are known from e.g. hawaii, new zealand and south africa (wilmot-dear 2000, starr et al. 2002, etc.). chloris barbata swartz (poaceae) (pl. 1c) new to the flora of the canary islands. tenerife: callao salvaje, playa ajabo, beach and adjacent rough ground, very common, 24.06.2014, f. verloove 10838 (br, ort); callao salvaje, barranco de las barandas, gravelly riverbed, wasteland, etc., very common, 24.06.2014, f. verloove 10842 (br); callao salvaje, barranco de las barandas, gravelly area, close to the sea, locally very common, 31.10.2014, f. verloove 11195 (br). chloris barbata is widespread in warm temperate, subtropical and tropical regions of the world (anderson 1974) and occurs in a wide variety of habitats: waste areas, cultivated fields, along beaches, etc. in the mediterranean area it is known, as a native species, from algeria, israel and morocco (euro+med plantbase 2006+). in 2014 c. barbata was discovered in callao salvaje in southern tenerife where it was confirmed subsequently. it is very common and obviously established since some time close to the beach and in the dry, gravelly riverbed (barranco), as well as in adjacent waste land. it can be considered invasive and a future expansion to nearby suitable habitats seems likely, the species being reputed to be a noxious weed in many areas worldwide (holm et al. 1979). four species of chloris are known to occur in the canary islands (in addition to c. barbata: c. gayana, c. pycnothryx and c. truncata). for convenience, they are distinguished in the following key: 1. sterile florets 2–4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 sterile floret 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2. all sterile florets awned. the uppermost sterile floret strongly inflated. annual . . . . . . . . . . . . . . . . . . . . . . . . . . chloris barbata only the lowermost sterile floret awned, the others awnless. sterile florets not inflated. perennial . . . . . . . .c. gayana 3. sterile floret 0.5–0.9 mm wide. spikelets black at maturity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .c. truncata sterile floret at most 0.2 mm wide. spikelets not black at maturity . . . . . . . . . . . . . . . . . . . . . . . . .c. pycnothrix cyclospermum leptophyllum (pers.) sprague ex britton (apiaceae) new to the flora of gran canaria. gran canaria: san agustín, gc-500, lawn, locally, 24.03.2013, f. verloove 10037 (br, lpa). a weed from south america (ronse et al. 2010) but naturalized in many warm-temperate and (sub-) tropical regions of the world, this species was known so far in the canary islands from la palma, tenerife and fuerteventura (acebes ginovés et al. 2009). in san agustín in gran canaria it was observed as a weed in irrigated lawns, along with other lawn weeds such as youngia japonica (see there). cyperus difformis l. (cyperaceae) new to the flora of the canary islands. gran canaria: fataga, n of the village, water reservoir, scattered specimens, 14.09.2013, f. verloove 10559 (br, lpa). cyperus difformis is a widespread weed in the (sub-) tropical and warm-temperate regions of the world, its exact area of origin being unclear (verloove 2014). although relatively frequent in the mediterranean area and also known to occur in madeira and the azores, it has not been recorded before in the canary islands. digitaria radicosa (j. presl) miq. (poaceae) new to the flora of gran canaria and tenerife. gran canaria: agaete, botanic garden in city center, weed in irrigated lawn, 13.09.2013, f. verloove 10598 (br); taurito, palmeral near entrance of tf-1 motorway, plantation weed, 24.11.2015, f. verloove 11996 (br, lpa); tenerife: puerto de la cruz, calle de la sala, flower bed, 04.11.2014, f. verloove 11243 (br). digitaria radicosa is a weed from tropical asia and the pacific islands (veldkamp 1973), locally naturalized elsewhere. it is reminiscent of d. ciliaris and may have been overlooked so far in the canary islands. characteristic features are its weak, trailing habit, few digitate racemes (usually 2–4 in number) and smooth-edged rachis (otto and verloove 2016). in europe it has been recorded from corsica (verloove 2008), probably as an ephemeral introduction. more stable populations were recently detected in the canary islands, more precisely in la palma (otto and scholz 2009), where it has been confirmed subsequently (comm. r. otto). in 2013 it was recorded in agaete in gran canaria where it grows as a weed in an irrigated lawn in the small botanic garden ‘huerto de las flores’. in similar circumstances it was also discovered in 2014 in puerto de la cruz in tenerife and in 2015, again in gran canaria, in taurito. dysphania anthelmintica (l.) mosyakin & clemants (amaranthaceae) new to the flora of tenerife. tenerife: bufadero (n of santa cruz de tenerife), barranco del bufadero, dry gravelly riverbed, very common, 29.06.2014, f. verloove 10854 (br, ort). a native from the americas (clemants and mosyakin 2003), this is the widely naturalized species that was previously known as ‘chenopodium ambrosioides’ in the canary islands. it is a common species in gran canaria and la palma (verloove 2013; otto and verloove 2016) and is here new xenophytes from canary islands acta bot. croat. 76 (2), 2017 123 plate 1. naturalized species from the canary islands. a) callistemon viminalis, ayagaures, barranco de ayagaures, november 2015; b) casuarina glauca, san agustín, barranco los guinchos, november 2015; c) chloris barbata. callao salvaje, playa ajabo, october 2014; d) senna × artemisioides subsp. filifolia, arguineguin, november 2015; e) limoniastrum monopetalum, poris de abona, june 2014; f) senna × artemisioides subsp. coriacea, playa paraiso, barranco de las galgas, june 2014 (photo f. verloove). verloove f. 124 acta bot. croat. 76 (2), 2017 formally reported for the first time from tenerife. contrary to dysphania ambrosioides it has an inflorescence that is ebracteate or with bracts inconspicuous and shorter than the glomerules. it seems to be a much more thermophytic species, totally absent north of the mediterranean area. erythrostemon gilliesii (wall. ex hook.) link (leguminosae s.l.) new to the flora of tenerife. tenerife: callao salvaje, barranco de las barandas, rocks, a single shrub (subspontaneous), 24.06.2014, f. verloove 10830 (ort); adeje, av. francisco ucelay sabina, rough ground close to barranco, several tens (well-established), 31.10.2014, f. verloove 11170 (br). erythrostemon gilliesii, is a native of south america (argentina and possibly uruguay; cullen and knees 2011) but widely introduced as an ornamental shrub in tropical, subtropical and warm-temperate countries. in the canary islands it was known so far – as an invasive species – from la gomera, gran canaria, fuerteventura and lanzarote (acebes ginovés et al. 2009). it is here reported for the first time from tenerife where it was observed on several occasions since 2014. in addition to the localities cited above, e. gilliesii was also seen, for instance, in el fraile where it is found in abundance in natural vegetation dominated by species such as cenchrus ciliaris, launaea arborescens, plocama pendula, schizogyne sericea, etc. the latter observation seems to confirm the species’ invasive behavior in the canary islands. eucalyptus gomphocephala dc. (myrtaceae) new to the flora of the canary islands. tenerife: las galletas, ten-bel, calle diana, rough ground, several young self-sown individuals (also planted nearby), 08.06.2015, f. verloove 12030 (br, ort). originally confined to a narrow coastal corridor in the extreme southwest of australia (chippendale 1988), this species is sometimes used as a windbreak in coastal areas, in forestation for wood production, as an ornamental or for stabilization of sands near the sea. it is very characteristic for its distinctly flattened, thick pedicels and mushroomshaped flower buds. in las galletas this species is planted on the verge of a holiday resort and prolifically self-seeds in the adjacent rough ground. in the mediterranean area it is known from italy, morocco, spain, malta and sicily (euro+med plantbase 2006+). at least in malta it is considered an invasive species (http://www.maltawildplants.com), while in the iberian peninsula it is confined to the southernmost regions (paiva 2000). eucalyptus sideroxylon a. cunn. ex woolls (myrtaceae) new to the flora of the canary islands. gran canaria: tauro, barranco playa del cura, calle vaticano, dry riverbed, scattered individuals, with e. camaldulensis, 24.11.2015, f. verloove 12006 (br). eucalyptus sideroxylon is originally native to southeastern australia (chippendale 1988) but is widely introduced elsewhere, e.g. in the iberian peninsula where it is rather frequent (paiva 2000). it is here reported for the first time from the canary islands, although it may have been overlooked so far, as a result of confusion with e. camaldulensis. several self-sown individuals of both these species grow in a barranco near tauro in the south of gran canaria. it is sometimes considered an invasive species outside its area of origin, e.g. in south africa (henderson 2001). heliotropium supinum l. (boraginaceae) new to the flora of tenerife. tenerife: la estrella (las galettas), water reservoir, exposed bank, ca. 10 individuals, 31.10.2014, f. verloove 11173 (br, ort); guargacho, water reservoir e of calle olimpia, exposed bank, scattered individuals, 01.11.2014, f. verloove 11178 (br); palm-mar, on the verge of rasca mountain, temporarily moist, sandy area, close to the sea, rather frequent but only locally, 20.02.2015, f. verloove 11262 (br); la estrella (costa del silencio), water reservoir, exposed pond, common, 07.06.2015, f. verloove 11552 (br); palm-mar, on the verge of rasca reserve, temporary wet open habitat, close to the sea, very numerous, 09.06.2015, f. verloove 11445 (br). a native from the mediterranean area, southwestern europe and southwestern asia (brummitt 1972), heliotropium supinum was known so far from fuerteventura in the canary islands, as a possibly native species (acebes ginovés et al. 2009). since 2014 it has been discovered repeatedly on exposed pond margins and similar habitats in several localities in southern tenerife. in most localities it was accompanied by verbena supina. its residence status in the canary islands remains uncertain. however, since this species has not been recorded before in tenerife, in habitats that are frequently visited by botanists, it most likely is a recent introduction. limoniastrum monopetalum (l.) boiss. (plumbaginaceae) (pl. 1e) new to the flora of tenerife. tenerife: poris de abona, rough ground, close to the sea cliffs, ca. 20 bushes, naturalized, 17.03.2014, f. verloove 10639 (br); poris de abona, sea cliffs and adjacent rough ground, established population, 25.06.2014, f. verloove 10840 (br, ort). limoniastrum monopetalum is originally native in the western mediterranean area (pignatti 1972). it is sometimes grown as an ornamental and easily escapes. as such, it was known to occur in the canary islands in gran canaria and fuerteventura (barone et al. 1995; acebes ginovés et al. 2009) and is here reported for the first time from tenerife. in poris de abona l. monopetalum is locally naturalized on sea cliffs, in a habitat that closely matches the species’ natural biotope. it was considered to be a species with a high invasive potential in the canary islands by barone et al. (1995). nephrolepis cordifolia (l.) c. presl (davalliaceae) (online suppl. pl. 1a) new to the flora of the canary islands. tenerife: puerto de la cruz, barranco tafuriaste, damp shady rocks, +/– 6 plants, 26.06.2014, f. verloove 10847 (br). new xenophytes from canary islands acta bot. croat. 76 (2), 2017 125 the congeneric nephrolepis exaltata is commonly cultivated as an ornamental fern in the canary islands and it easily escapes, especially in the damper, shady places in the northern parts of the islands (gran canaria, la palma and tenerife, according to acebes ginovés et al. 2009) where it is often seen as an epiphyte on palm trunks. n. cordifolia apparently is less frequently cultivated and has not been recorded so far in the wild although it may have been overlooked. a small population was discovered on damp rocks of a barranco in puerto de la cruz in tenerife in 2014. it is quite similar to n. exaltata but its rachis scales are distinctly bicolored (with a dark point of attachment) and its pinnae are more densely arranged (points of pinnae attachment 5–12 mm apart vs. 7–21 mm apart). also, it often (but not always) produces tubers while these are always absent in n. exaltata (nauman 1993). both species are a known threat to native plant species (riefner and smith 2015). nerium oleander l. (apocynaceae) new to the flora of tenerife. tenerife: golf del sur, barranco del saltadero, barranco, one young bush (not planted), 20.03.2014, f. verloove 10640 (br); golf del sur, barranco del saltadero, dry gravelly riverbed, a single individual, 02.11.2014, f. verloove 11161 (ort). nerium oleander is native in the mediterranean area but widely cultivated elsewhere as an ornamental shrub (cullen and knees 2011), also in the canary islands. it is surprisingly rare as an escape from cultivation, probably because fruit is rarely produced (herrera 1991). since 2013 spontaneous occurrences are known from la palma (otto and verloove 2016) and in 2014 and 2015 it was seen several times in tenerife as well (e.g. güímar, playa paraiso, playa de las américas, puerto de la cruz). pascalia glauca ortega (asteraceae) new to the flora of tenerife. tenerife: las galletas, ten-bel, weed in abandoned resort, locally (several tens), 02.03.2015, f. verloove 11273 (br, ort). this poisonous weed species is originally native in south america (strother 1991) and well-naturalized in warm-temperate regions elsewhere in the world, also in spain (carretero 1988, robledo et al. 1996). in las galletas a small population was discovered in an abandoned resort in 2015 and subsequently confirmed. up to the present it has only been recorded before in the canary islands in la palma (otto and verloove 2016). phytolacca americana l. (phytolaccaceae) new to the flora of tenerife. tenerife: la orotava, barranco de la arena, gravelly riverbed, shady, several tens (naturalized), 02.07.2014, f. verloove 10898 (br). phytolacca americana has been claimed before from tenerife (acebes ginovés et al. 2009) but these claims were probably erroneous and referable to the much more widely cultivated (and morphologically very different) p. dioica (verloove and reyes-betancort 2011). however, its genuine presence in tenerife could be demonstrated in 2014 when it was found plentifully in a shady barranco near to la orotava. it was recently also detected in similar habitats in gran canaria (verloove 2013). podranea ricasoliana (tanfani) sprague (bignoniaceae) new to the flora of gran canaria and tenerife. gran canaria: puerto rico (motor grande), barranco de puerto rico, riverbed, 20.11.2015, f. verloove 12008 (br). a native of south africa (cullen and knees 2011) this species is widely cultivated as an ornamental vine in the canary islands. like solandra maxima it is very vigorous, covering large surfaces and taking root wherever branches touch the soil; accordingly, planted and escaped plants are often hard to distinguish. voucher specimens were collected in 2015 in puerto rico in gran canaria but it has also been recorded in other localities in gran canaria (e.g. arucas 2015; teror, 2015; etc.) and tenerife (e.g. el socorro, barranco del mulato, 2014; golf del sur, barranco del saltadero, 2014; güímar, barranco badajoz, 2014; la orotava, barranco de la arena, 2014; playa de las américas, 2015; puerto de la cruz, barranco tafuriaste, 2014; idem, malpais, calle chinyero, 2014; santa cruz, barranco valle seco, 2014; tegueste, barranco agua de dios, 2014; etc.). it was previously reported from la palma in the canary islands (otto and verloove 2016). psidium guajava l. (myrtaceae) (on-line suppl. pl. 1b) new to the flora of gran canaria. gran canaria: carrizal, barranco de aromeros between gc-191 and gc-1 motorway, dry riverbed, near sewer, a single individual, ca. 250 cm tall, 26.11.2015, f. verloove 11995 (br, lpa); ayagaures, barranco de ayagaures, gc-504 (km 7–8), dry riverbed, scattered individuals but only locally, 27.11.2015, f. verloove 12012 (br). an evergreen shrub or small tree native to the caribbean, central america and south america, p. guajava is widely cultivated in tropical and subtropical regions of the world for its edible fruit (guava) or as an ornamental (cullen and knees 2011), also in the canary islands. it was recently reported for the first time in the wild from the island of la palma (santos guerra and reyes-betancort 2014). in 2015 it was also observed in two barrancos in gran canaria. although it is probably not yet genuinely naturalized (except perhaps in ayagaures), future naturalization and subsequent invasive behavior can be expected. p. guajava is often considered one of the most significant invasive species in areas where it was initially introduced (sheil 1994, meyer 2000, henderson 2007). rumex cristatus dc. (polygonaceae) new to the flora of tenerife. tenerife: tejina, barranco de los aguas de dios, damp area, near artificial pond, five individuals, 27.06.2014, f. verloove 10856 (br, ort); la cuesta (la laguna), barranco de santos, dry gravelly riverbed, 28.06.2014, f. verloove 10899 (br); la laguna, barranco de santos e of tf13, gravelly riverbed, frequent, 03.07.2014, f. verloove verloove f. 126 acta bot. croat. 76 (2), 2017 10870 (br); la laguna (bronco), camino la rúa, roadside, very common, 06.11.2014, f. verloove 11189 (ort). rumex cristatus, originally restricted to a relatively small area in southcentral europe (rechinger and akeroyd 1993), is a fast spreading alien in parts of the mediterranean area (quesada et al. 2007). until recently, it had not been recorded from the canary islands (acebes ginovés et al. 2009) but since 2012 it is known from a single locality in la palma (otto and verloove 2016). in 2014 it was recorded on several occasions in tenerife. it is in fact relatively widely distributed between tejina and santa cruz de tenerife, especially in barranco de santos. it occurs in temporarily wet, ruderalized habitats such as (artificial) pond margins, ditches, gravelly riverbeds, roadsides, etc. given its actual abundance and distribution r. cristatus either is a fast spreading recent introduction (probably easily dispersed as a result of regular flooding in the barranco) or a previously neglected or overlooked species (the latter option being less likely since confusion with other species of rumex is unlikely in tenerife). r. cristatus is a fairly variable species and easily hybridizes with other species (jauzein 1990). plants currently found in the canary islands, as well as in large parts of western europe, are somewhat atypical in having fruiting valves that are slightly smaller than in its area of origin. rumex palustris sm. (polygonaceae) new to the flora of the canary islands. tenerife: armeñime, water reservoir alongside tf-47, muddy bank, in monospecific stands, several 100s, 31.10.2014, f. verloove 11163 (ort); playa paraiso, barranco de las galgas, dried out water reservoir, very common, 29.11.2016, f. verloove 12693 (br). r. palustris is native in large parts of europe and western asia (rechinger and akeroyd 1993). it is here reported for the first time from the canary islands. in the locality cited above it is a very common and dominant species. it looks firmly established and plants were also seen in the adjacent barranco de las salinas, as well as in water reservoirs in playa paraiso. this species now occurs in many areas outside of its original distribution range but it is rarely (if ever) considered an invasive species. schinus terebinthifolia raddi (anacardiaceae) new to the flora of tenerife. tenerife: golf del sur, barranco del saltadero, barranco, at least 10 bushes, old and young (naturalized), 20.03.2014, f. verloove 10641 (br); guarguacho, barranco, dry riverbed, near sewage drain, 27.02.2015, f. verloove 11276 (br). originally native to south america, s. terebinthifolia is commonly cultivated as an ornamental shrub or tree in the warm-temperate and (sub-) tropical areas of the world (cullen and knees 2011). in recent years it was reported for the first time as an escape from the canary islands, at first from gran canaria and soon afterwards also from fuerteventura (verloove 2013, verloove and guiggi 2013). although much less frequent in tenerife, it was recorded in several different localities since 2014. in addition to those localities listed above, it was also seen in playa paraiso (el pinque), callao salvaje (playa ajabo), santa cruz de tenerife (barranco tahodio), las caletillas, guargacho, puerto de la cruz (parque taoro), etc. birds that feed on the fruits of s. terebinthifolia seem to play an important role in the processes of naturalization of this species in the canary islands (see also panetta and mckee 2006; williams et al. 2007; mink et al. 2015). a future wider naturalization and subsequent invasive behavior in the canary islands is predictable. senna × artemisioides (gaudich. ex dc.) randell (leguminosae s.l.) cf. subsp. coriacea (benth.) randell (pl. 1d) tenerife: playa paraiso, barranco de las galgas, dry gravelly riverbed, a single shrub (subspontaneous), 23.06.2014, f. verloove 10837 (br, ort); playa paraiso, barranco de las galgas, rough ground, foot of bridge, a single shrub (an additional one elsewhere in the same barranco), 31.10.2014, f. verloove 11171 (br); palm-mar, abandoned resort close to the sea, rough ground, several young, self-sown individuals, 20.02.2015, f. verloove 11263 (br); palm-mar, center of village, rough ground (vacant lot in residential area), a single young shrub, 28.02.2015, f. verloove 11275 (br). subsp. filifolia randell, j. adelaide bot. gard. 12(2): 227– 229. 1989. = cassia eremophila auct. hort. non cunn. ex j. vogel (sánchez de lorenzo cáceres 2005) (pl. 1f) gran canaria: arguineguín, barranco av. mencey, bare, stony ground, several tens, self-sown (not seen planted nearby), 20.11.2015, f. verloove 12032 (br, lpa). endemic to much of mainland arid australia, senna × artemisioides is widely grown as landscape plant in desert areas across the world (cullen and knees 2011). several (notho-) subspecies with quite variable foliage are considered to be a group of complex hybrids. single populations often contain three or more of these taxa and are interpreted as hybrid swarms (randell 1989). in the canary islands two very distinct representatives of this complex were recently recorded as escapes from cultivation, subsp. × filifolia in gran canaria and cf. subsp. × coriacea in tenerife (see above). the former has filiform, terete leaflets (usually two pairs) and is slightly glaucous-greyish in appearance; the latter, in contrast, has usually more numerous and wider, elliptic to obovate leaflets and a dull greenish foliage. both abundantly reproduce from seed and will probably naturalize in suitable habitats in the southernmost parts of these islands. while subsp. × coriacea was only seen in the vicinity of plantations, × filifolia was found growing in relative abundance in the absence of parent plants. senna × artemisioides apparently is a recent escape from cultivation in europe. it was probably first recorded in cyprus, subsequently also in spain (laguna et al. 2010). in other climatologically favorable areas, e.g. the southern united states (ditomaso and healy 2007), it is sometimes considered a troublesome invasive species. senna × floribunda (cav.) h.s. irwin & barneby. (leguminosae s.l.) (on-line suppl. pl. 1c) new xenophytes from canary islands acta bot. croat. 76 (2), 2017 127 new to the flora of the canary islands. tenerife: puerto de la cruz, barranco de martíanez, dry, gravelly riverbed, rocks, several tens, saplings as well as flowering and fruiting individuals, 25.06.2014, f. verloove 10868 (br, ort). senna × floribunda, of putative s. multiglandulosa × s. septemtrionalis parentage, is an artificial cross that is widely grown as a garden ornamental (cullen and knees 2011). like other species of senna (e.g. s. bicapsularis and s. corymbosa) it easily escapes, despite being only partly fertile. in the barranco de martíanez in puerto de la cruz it was locally seen with several tens of individuals in various stages of development (saplings as well as fruiting individuals). it seems more or less established there. senna × floribunda belongs to sect. chamaefistula ser. coluteoideae. it is highly reminiscent of s. septemtrionalis and both have been confused, for instance in australia and new zealand (webb et al. 1988). both have leaflets that are acute to acuminate at apex, unlike the other species of senna known at present from the canary islands (the annual and probably ephemeral s. occidentalis). senna × floribunda is distinguished from s. septemtrionalis by leaflets (at least when young), pedicels and base of ovary that are finely pilosulous with weak incurved or subappressed hairs up to 0.2–0.7 mm (an influence from s. multiglandulosa, while s. septemtrionalis is completely glabrous) and its inflorescence is often corymbose-paniculate and ± exserted from foliage (irwin and barneby 1982). also, it is partly sterile and its leaflets seem thinner in texture (often more or less leathery in s. septemtrionalis). senna × floribunda is considered an invasive species in australia (numerous references on the internet). solandra maxima (sessé & moc.) p.s. green (solanaceae) new to the flora of tenerife. tenerife: san lorenzo, barranco de chija at tf-28, foot of bridge, escaped from cultivation, at least two individuals, 22.06.2014, f. verloove 10876 (br). s. maxima is native to mexico, central america and northern south america but widely cultivated as an attractive liana in (sub-) tropical regions of the world (cullen and knees 2011). it is a very vigorous species that was recently reported for the first time in the wild from the canary islands (la palma; otto and verloove 2016). in 2014 it was recorded on several occasions in tenerife as well, escaping from nearby gardens (often in barrancos). however, in some instances it was obviously no longer directly associated with gardens, apparently growing from washed-up rhizomes or perhaps even from seed. tipuana tipu (benth.) kuntze (leguminosae s.l.) new to the flora of tenerife. tenerife: santa cruz de tenerife, ermita n.s. de la candelaria, barranco de santos, riverbed, in shallow depressions, numerous young, self-sown individuals (planted nearby), 28.06.2014, f. verloove 10862 (br); santa cruz de tenerife, barranco tahodio, dry, gravelly riverbed, ca. 25 young shrubs (self-sown), some fruiting, 29.06.2014, f. verloove 10855 (br, ort); güímar, barranco badajoz, former gravel pit/dump, one shrub, 01.07.2014, f. verloove 10885 (br); los christianos, n of montaña chayofita, abandoned plantation, two young individuals, self-sown, 10.06.2015, f. verloove (br). native to south america, t. tipu is widely planted as an ornamental tree in the (sub-) tropics (cullen and knees 2011). it was recently recorded for the first time in the wild in the canary islands (in gran canaria; verloove 2013). since 2014 it was observed, obviously self-sown, in several places in tenerife as well (usually in barrancos). mostly, only saplings or single individuals were recorded. however, in the barranco tahodio in santa cruz de tenerife t. tipu looks more or less established, several of the young, selfsown individuals bearing fruit and thus representing a selfsustaining population. t. tipu reproduces abundantly and fruits are easily wind-dispersed. a future, wider naturalization is predictable. the species occurs on the ‘alien invasive plants list’ for south africa (http://www.invasives. org.za/). youngia japonica (l.) dc. (asteraceae) new to the flora of gran canaria. gran canaria: san agustín, gc-500, lawn, locally, 24.03.2013, f. verloove 10037 (br, lpa). originally native in southeast asia, this weed now occurs in many warm-temperate and (sub-) tropical areas of the world (spurr 2006). in the canary islands it was recorded for the first time in 2010 in tenerife (lawn weed in puerto de la cruz; siverio núñez et al. 2013). it was regularly confirmed subsequently and seems perfectly naturalized now. in 2013 y. japonica was observed for the first time in gran canaria: it grows in disturbed irrigated lawns in san agustín and puerto de mogan (see above; also http:// invasionesbiologicas.blogspot.be/2013/09/dos-nuevas-especies-introducidas-en.html). y. japonica is a rather variable species. molecular data shed new light on its taxonomy. plants from the canary islands have brownish achenes ca. 2 mm long and belong to subsp. japonica, the widespread weedy taxon (nakamura et al. 2013). ephemeral species new to the flora of the canary islands commelina erecta l. (commelinaceae) (on-line suppl. pl. 1d) gran canaria: telde, lomo cementerio, gc-100 near barranco la rocha, roadside, crack in concrete, few plants, 18.11.2015, f. verloove 12045 (br, us). cucumis metuliferus naudin (cucurbitaceae) tenerife: la cuesta (la laguna), barranco de santos, dry, gravelly riverbed, one individual (flowering and fruiting), 28.06.2014, f. verloove 10866 (br). cyperus longus l. (s.str.; excl. c. badius desf.) (cyperaceae) tenerife: playa de las américas, tf-1 motorway in front of siam park, weed in flowerbed, three individuals, 23.06.2014, f. verloove (br). verloove f. 128 acta bot. croat. 76 (2), 2017 cyperus papyrus l. (cyperaceae) tenerife: la quinta, pool, 01.01.1971, j.e. de langhe 92 (nam). hypoestes phyllostachya baker (acanthaceae) tenerife: tejina, barranco cuevas (centro ciudad), dry riverbed, one individual, 05.11.2014, f. verloove 11236 (br). lycium barbarum l. (solanaceae) (on-line suppl. pl. 1e) gran canaria: jinámar, barranco de telde w of gc-1 motorway, on the verge of dry riverbed, scattered shrubs in two populations, 05.11.2012, f. verloove 11978 (br, lpa); idem, 15.11.2015, f. verloove 12050 (br). lycium ferocissimum miers (solanaceae) tenerife: santa cruz de tenerife, taco, av. san matias, rough ground, a single shrub, 03.11.2014, f. verloove 11208 (br). macfadyena unguis-cati (l.) gentry (bignoniaceae) gran canaria: fataga, gc-60 s of the village, climbing in palm tree, 18.03.2013, f. verloove 10042 (lpa). mangifera indica l. (anacardiaceae) tenerife: igueste de san andres, barranco de igueste, dry, gravelly riverbed, one specimen (not planted), 16.03.2014, f. verloove 10631(br). salvia hispanica l. (lamiaceae) tenerife: valle tabares (la laguna), barranco de santos, dry gravelly riverbed, a single individual, 28.06.2014, f. verloove 10937 (br). also observed in various other points of barranco de santos in la laguna and santa cruz de tenerife, usually in small number. schefflera arboricola (hayata) merr. (araliaceae) tenerife: buenavista del norte, golf court (w-side), epiphyte on phoenix, 30.06.2014, f. verloove 10884 (br); torviscas, close to barranco colon, epiphyte on phoenix, 31.10.2014, f. verloove s.c.; las galletas, ten-bel, epiphyte on phoenix, 11.06.2015, f. verloove s.c.; playa de las américas, fañabe, hotel riu, epiphyte on phoenix, 15.06.2015, f. verloove s.c. sphagneticola trilobata (l.) pruski (asteraceae) (on-line suppl. pl. 1f) gran canaria: maspalomas, meloneras golf (e-side), small barranco, escaping from nearby plantation, far creeping, 18.09.2013, f. verloove 10560 (br, lpa). new to the flora of gran canaria and/or tenerife cucumis melo l. (cucurbitaceae) gran canaria: playa del inglés, barranco de los vicentes, from sewage sludge (with citrullus lanatus, cucurbita moschata), 17.11.2015, f. verloove 12000 (br). cucurbita moschata duchesne (cucurbitaceae) tenerife: güímar, barranco fregenal at tf-28, dry, gravelly riverbed, 10.11.2014, f. verloove 11198 (ort). also seen in numerous other localities since 2014 in tenerife and gran canaria. this species is by far the most frequent cucurbita species in the canary islands (see also otto and verloove 2016). diplotaxis tenuifolia (l.) dc. (brassicaceae) tenerife: tabaiba alta, barranco de las higueras at tf-28, riverbed, a single plant, 01.07.2014, f. verloove 10873 (ort); puerto de la cruz, ctra. las tapias, foot of tree, 04.11.2014, f. verloove 11169 (ort). ficus microcarpa l. (moraceae) gran canaria: arguineguín, barranco in city center, close to the sea, crack in wall, young tree, self-sown, 18.09.2013, f. verloove 10579 (lpa). this ornamental is an increasing epiphyte on palm trees (mainly phoenix) and is also found on old masonry and concrete structures in the canary islands, germinating from seed dispersed in figs eaten by birds or small mammals. the seedlings subsequently establish and grow without human intervention or intentional summer watering. a future naturalization and even invasive behavior is possible (riefner 2016). gossypium barbadense l. (malvaceae) tenerife: palm-mar, rough ground, close to the sea, 21.03.2014, f. verloove 10643 (br). this species has been repeatedly recorded in the past years in gran canaria and tenerife, usually single individuals. at least in palm-mar, however, where it can be found in several places, an incipient naturalization process is discernable. grevillea robusta cunn. ex r. br. (proteaceae) tenerife: santa maría del mar, calle atamán, water reservoir, crack in concrete, a single individual, self-sown, 08.11.2014, f. verloove 11176 (br). paspalum notatum flüggé var. saurae parodi (poaceae) gran canaria: maspalomas, centro commercial yumbo, ruderalized lawn, locally, 23.03.2013, f. verloove 10040 (br, lpa). passiflora edulis sims (passifloraceae) tenerife: playa de las américas, fañabe, calle helsinki, barranco de fañabe, 14.06.2015, f. verloove 11529 (br); santa cruz de tenerife, barranco santos at calle de diego crosa, dry riverbed, a single individual, 13.11.2016, f. verloove 12691 (br). pteris cretica l. (pteridaceae) tenerife: puerto de la cruz, barranco de martíanez, damp shady rock, 25.06.2014, f. verloove 10850 (br). tecoma stans (l.) kunth (bignoniaceae) tenerife: bufadero (ne of santa cruz de tenerife), barranco del bufadero, dry gravelly riverbed, a single shrub, selfsown, ca. 200 cm tall, 29.06.2014, f. verloove 10875 (br). also observed in several other localities in tenerife since 2014, mostly in small number: torviscas, barranco new xenophytes from canary islands acta bot. croat. 76 (2), 2017 129 colon (old wall), puerto de la cruz (la paz), san andres (barranco cercades de san andres), santa cruz de tenerife (port area), etc. miscellaneous notes diplachne fusca (l.) p. beauv. ex roem. & schult. subsp. uninervia (j. presl) p.m. peterson & n. snow (poaceae) gran canaria: maspalomas, palmeral close to lagoon, pond margins and ditches, very common, 17.11.2015, f. verloove 12016 (br, lpa). this weed is known from a garden center in montaña los vélez since 2011 (verloove 2013). it is still present there but does not seem to spread. the very similar diplachne malabarica was claimed from the la charca lagoon in maspalomas, also in gran canaria (scholz and böcker 1996). the latter is best distinguished by its much longer anthers (1.3–2.7 mm long vs. 0.2–0.6 mm long) and paler spikelets (greenish rather than lead-colored). in 2015 its presence was confirmed in that area: it is a prolific weed of ditches and pond margins and obviously well-naturalized. however, it corresponds in all characters with d. fusca subsp. uninervia, not with d. malabarica. the latter name needs to be omitted from acebes ginovés et al. (2009). d. fusca subsp. uninervia has also been recorded on several occasions in fuerteventura since 2008, where it has been associated with grass seed for lawns (scholz et al. 2013; sub leptochloa uninervia). eclipta prostrata (l.) l. (asteraceae) gran canaria: maspalomas, close to faro (lighthouse), irrigated lawn, a troublesome weed but only very locally, 18.09.2013, f. verloove 10517 (br). this pantropical weed was recently observed for the first time in the canary islands. it was found in two localities in the northern part of gran canaria (verloove 2013) where its presence was confirmed in 2015. an additional record in 2013, in irrigated lawns in maspalomas in the southern part of the island, seems to suggest a recent naturalization in gran canaria. pluchea carolinensis (jacq.) g. don (asteraceae) gran canaria: arguineguín, barranco de arguineguín, close to the sea, dry, gravelly riverbed (shady, under eucalyptus), 18.09.2013, f. verloove 10581 (br, lpa); idem, 20.11.2015, f. verloove 11980 (br); ayagaures, barranco de ayagaures, gc-504 (km 3), dry riverbed, two individuals (flowering and fruiting), 27.11.2015, f. verloove 12014 (br). this highly invasive xenophyte was recently reported as new for the canary islands. a single shrub was found growing in a barranco near arguineguín in gran canaria in 2011 (verloove 2013). it was expected that this species would be present elsewhere in this area. indeed, in september 2013 several additional individuals were discovered in the same barranco, under eucalyptus canopy, close to its estuary in arguineguín. in 2015 some plants of p. carolinensis were also recorded in a barranco in ayagaures. this species looks well-established in southern gran canaria and a future, wider naturalization is predictable, similar to that of the congeneric, highly invasive p. ovalis in tenerife (see padrón-mederos et al. 2009, verloove and reyes-betancort 2011). prosopis juliflora (sw.) dc. (leguminosae) (on-line suppl. pl. 1g) gran canaria: arinaga, barranco del polvo, estuary, scattered mature individuals (flowering and fruiting) and numerous saplings, naturalizing, 16.11.2015, f. verloove 11998 (br); pedrazo, barranco del negro, dry riverbed, a single individual (flowering and fruiting), 17.11.2015, f. verloove 12005 (br); pozo izquierdo, llanos de tenefé, barranco de tirajana, gravelly, dry riverbed, close to the sea, ca. 5 individuals (not planted), 22.11.2015, f. verloove 12023 (br). this species (mesquite) has been known since 2011 as an escape from cultivation in the drier, southernmost parts of gran canaria and a future naturalization was predicted (verloove 2013). in 2015 it was recorded in several additional localities, all in barrancos. in one of these, in the estuary of barranco del polvo in arinaga, it is present in relative abundance and in various stages of development, in a natural coastal vegetation. at least in this locality it can be considered naturalized. scattered individuals of a similar species, prosopis glandulosa (honey mesquite), grow on the verge of the tirajana barranco near el doctoral [el doctoral, barranco de tirajana e of tf 1 motorway, on the verge of dry riverbed, three individuals, 28.11.2015, f. verloove 12004 (br, lpa)]. its leaflets are more widely spaced and are at least 5x as long as wide. the origin of these trees is uncertain (originally planted?) but, at least at present, it does not seem to reproduce. like p. juliflora, however, it has the potential to naturalize. sida rhombifolia l. (malvaceae) gran canaria: el hormiguero (e of galdar), dry roadside in the village, scattered individuals, 06.11.2012, f. verloove 10619 (br, lpa). claims of this species from gran canaria often have been considered erroneous, being referable to the similar but much more widespread malvastrum coromandelianum (comm. a. reyes-betancort). its genuine presence in gran canaria, however, was confirmed in 2012. discussion in the present paper eleven non-native taxa are reported for the first time from the canary islands that can be considered either (locally) naturalized and/or potentially or genuinely invasive. several additional species, most of them also behaving as invasive species, are recorded for the first time from the islands of gran canaria and/or tenerife. the paper further includes records of alien species that, at least at present, have not yet naturalized; some, however, probably will do so in the near future. as has been shown before on numerous occasions (dehnen-schmutz et al. 2007), ornamental horticulture is the most important pathway for plant invasion world-wide. among the taxa reported as naturalized or invasive in this verloove f. 130 acta bot. croat. 76 (2), 2017 paper the proportion of species initially introduced for utilitarian or ornamental purposes is high. all species that have been included in the ‘black list’ of invasive plants on the island of tenerife are escaped ornamentals and the same applies to almost the entire ‘grey list’ (machado carillo 2000). despite this, the influx of new ornamentals in the canary islands seems unstoppable and additional legislative measurements are urgently needed to prevent further plant invasions resulting from ornamental horticulture. finally, this paper also stresses the need for a sufficient taxonomic expertise in understanding and managing plant invasions. taxonomy plays a critical role and is essential for the effective management of invasive plants since incorrect identifications can impede ecological studies (pyšek et al. 2013). several of the species reported in this paper may have been overlooked up to present, as a result of confusion with similar or related species. acknowledgements dr. robert faden (washington, u.s.a.) kindly confirmed the identity of commelina erecta. references acebes ginovés, j. r., león arencibia, m. c., rodríguez navarro, m. l., et al. 2009: pteridophyta, spermatophyta. in: arechavaleta, m., rodríguez, s., zurita, n., garcía, a. 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asheville, n.c., u.s. dept. of agriculture, forest service, southeastern forest experiment station. acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 31 acta bot. croat. 74 (1), 31–41, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 morphological and anatomical features of seeds of turkish romulea taxa (iridaceae) and their taxonomic signifi cance mehmet cengi̇z karai̇smai̇loğlu istanbul university, faculty of science, department of biology, 034116 istanbul, turkey abstract – this paper reports on the assessment of morphological (macro and micro) and anatomical characters of seeds of romulea taxa distributed in turkey with the use of oneway analysis of variance, cluster analysis and principal component analysis. morphological characteristics such as size, shape, color and surface of seeds were examined with the use of light and scanning electron microscopes. thicknesses of testa and phytomelan layer, sizes of embryo in seeds were studied anatomically. the outcomes revealed that taxa were similar in some aspects such as color and shape of seeds. however, seed size, thickness of testa and phytomelan layer, shape of the epidermal cells in testa and sizes of embryo were different among taxa, and have taxonomic value in the distinction of these taxa from each other. in addition, the seed surfaces were more or less different for the examined taxa on an interspecifi c level. consequently, seed morphology and anatomy with a few exceptions demonstrated diversity and they had taxonomic importance in terms of distinguishing among species. keywords: anatomy, morphology, phytomelan, romulea, scanning electron microscopy, seed, turkey introduction romulea maratti belongs to the subfamily crocoideae (ixioideae) in the family iridaceae. the ixioideae is the biggest subfamily of iridaceae, with more than 800 species of 30 genera (isik and donmez 2007). the ixoideae is a consistent subfamily in terms of morphologic and anatomic features (rudall and goldblatt 1991, ozdemir et al. 2011). the romulea genus originated from south africa, and spreads over a wide area, including the mediterranean zone, has more than 90 species in a range of distribution areas. the genus romulea is represented by 7 taxa in turkey, all of which are listed in table 1 (marais 1984, erol and kucuker 2003). * corresponding author, e-mail: biology_61@hotmail.com copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. karai̇smai̇loğlu m. c. 32 acta bot. croat. 74 (1), 2015 in the classifi cation of romulea species sexual parts such as anther, fi lament and stigma are often used to separate the taxa from each other. however, classifi cation of romulea taxa in turkey as well as in sicily and south-west europe is diffi cult because of the frequent emergence of gynodioecism, which is the presence of only hermaphrodite or female fl owers in a population. namely, if there are only female fl owers on the plants, romulea taxa look morphologically similar (davis 1984, moret et al. 1992, moret et al. 1993). this situation leads to some problems in their classifi cation from time to time, thus additional characters that will support the existing identifi cation are needed in the classifi cation of turkish romulea taxa. seeds and small fruits have typical morphological features, such as shape, colour, dimension, microstructure (including ultrastructure) and they may provide valuable contributions to plant taxonomy. in a practical sense, many signifi cant data gathered from seed or fruit epidermal surface analyses are acquired with scanning electron microscope (sem). these surface features include micromorphological characteristics, such as shape of the cells, seed surface ornamentation and epicuticular protrusions. these features demonstrate a large diversity from subspecies to family level and they are not readily changed under the infl uence of habitat conditions (barthlott 1981). however, anatomical features are usually as helpful as morphological features for plant diagnostics, and they often are useful in the separation of closely related taxa (karamian et al. 2012, akalin-urusak and kizilarslan 2013). the signifi cance of morphology and anatomy in the classifi cation of taxa in the iridaceae has been revealed by various studies (marais 1984, erol and kucuker 2003, isik and donmez 2007). accordingly, the main objective of this study is to investigate morphological and anatomical features of the seeds of the romulea species native to turkey with light microscope (lm) and sem in order to discover differences and specifi cities of the seed morphology and anatomy of examined taxa with the use of statistical analysis. exhaustive description of seed structures of the examined species are offered in this paper for the fi rst time. materials and methods plant material seeds of 7 taxa, which had been gathered from native populations in turkey and cultivated at the alfred heilbronn botanical garden in istanbul university, were utilized for tab. 1. examined taxa and their origin. no taxa locality (origin) r1 r. bulbocodium (l.) seb. & mauri var. bulbocodium izmir, çeşmealti r2 r. bulbocodium (l.) seb. & mauri var. crocea (boiss. & heldr.) baker antalya, manavgat r3 r. bulbocodium (l.) seb. & mauri var. leichtliniana (heldr. ex hai.) bég. hatay, samandag r4 r. tempskyana freyn antalya, falezler r5 r. ramifl ora ten. subsp. ramifl ora balikesir, patria r6 r. linaresii pari. subsp. graeca bég. istanbul, kurtköy r7 r. columnae seb. & mauri subsp. columnae izmir, yamanlar morphological and anatomical studies of romula taxa seeds acta bot. croat. 74 (1), 2015 33 morphological and anatomical observations. investigations were performed with 10 ripe seeds for each taxon. the taxa examined and their origins are given in table 1. morphological and anatomical analyses macromorphological features such as the shape, size, raphe presence or absence and colour of the seeds were examined using an olympus zs51 stereomicroscope and kameram imaging software. for micromorphological observations of seed surface ornamentation, specimens were prepared for electron microscopy by mounting with silver adhesive on the stub, coated with gold, and analysed with a jeol neoscope-5000 scanning electron microscope. for anatomical features such as testa thickness, phytomelan layer thickness, shape of epidermal cells and embryo size, cross sections were taken with a fully automatic microtome (thermo shonda met finesse) from the middle of the seeds. afterwards, they were passed through a variety of alcohol and xylene series, and stained with haematoxylin (harris-rrsp67-e) in a staining device (asc 720 medite), and were covered with entellan for anatomical structures to be examined (inceer et al. 2010). anatomical characters were observed using an olympus cx21fs1 microscope and kameram imaging software. the terminology of morphological and anatomical characters was carried out in accordance with stearn (1985). statistical analyses data of studied parameters were performed with the spss computer program. the duncan multiple-range test was utilized to determine the statistical signifi cance of differences among the values obtained for different taxa. grouping of taxa was carried out utilizing the clustering analysis method (upgma) in accordance with quantitative characters in tab. 2. in addition, principal component analysis (pca) ordination and similarity matrix were performed based on some morphological and anatomical characters of seeds such as seed dimensions, testa thickness, phytomelan layer thickness and embryo sizes. results seed size, raphe and shape characters of taxa were evaluated macromorphologically and differences among the studied taxa were observed (tab. 2). in seed dimensions, r6 and r7 had the greatest differences among the examined species. their values range from 2.04 mm to 4.54 mm for length and from 2.88 mm to 3.67 mm for width, respectively. colour of seeds in all cases is orange, but colour tones are different in r1 (darker). however, colour of seeds is determined to be unimportant in the distinction of the taxa of romulea examined. the shape of seeds in these taxa indicates major differences. likewise, the ratio values of the seed sizes show variations, which vary between 0.60 and 1.28. seeds are ovate in r1 (0.60), r2 (1.28), r4 (0.72), r5 (1.10), r6 (0.70), and elliptical in r3 (1.25), r7 (1.23). however, the raphes of the taxa also differ. the raphe is very prominent in r4 and r7, compared to other species (fig. 1). seed surfaces of the studied taxa exhibited diversity of surface ornamentation (fig. 2 and tab. 2). the seed surface ornamentation, which is a useful character in the separation of karai̇smai̇loğlu m. c. 34 acta bot. croat. 74 (1), 2015 ta b. 2 . m or ph ol og ic al a nd a na to m ic al fe at ur es in s ee ds o f t he t ur ki sh r om ul ea ta xa . r es ul ts re pr es en t m ea n va lu es ± s ta nd ar d de vi at io n; m ea ns w ith d if fe re nt le tte rs a re s ig ni fi c an t a t p = 0 .0 5 le ve l ( d un ca n’ s m ul tip le -r an ge te st ), fo r t ax a ab br ev ia tio ns s ee t ab . 1 ; l – le ng th , w – w id th . ta xa c ol ou r sh ap e se ed s iz e r at io (l /w ) se ed su rf ac e or na m en ta tio n te st a th ic kn es s (μ m ) ph yt om el an la ye r th ic kn es s (μ m ) te st a ep id er m al ce lls e m br yo s iz es l (m m ) w (m m ) l (μ m ) w (μ m ) r 1 d ar k or an ge o va te tr an sv er se 2. 14 ± 0. 15 de 3. 53 ± 0. 11 c 0. 60 r et ic ul at e 21 8. 84 ± 10 .7 0a b 54 .2 0± 5. 81 d u nd ul at ed 29 8. 38 ± 11 .3 2d 22 4. 47 ± 15 .3 5c d r 2 o ra ng e o va te l at e 3. 87 ± 0. 12 a 3. 02 ± 0. 21 de 1. 28 r et ic ul at efo ve at e 21 3. 05 ± 8. 64 b 86 .0 1± 6. 37 b c ru sh ed 31 7. 91 ± 7. 55 c 28 9. 25 ± 11 .6 5b r 3 o ra ng e e lli pt ic al l at e 3. 04 ± 0. 28 c 2. 43 ± 0. 08 e 1. 25 r et ic ul at ea re ol at e 81 .9 3± 10 .5 6e 54 .1 1± 10 .6 9c d c ru sh ed 17 5. 98 ± 35 .9 9f 15 6. 82 ± 21 .2 4e r 4 o ra ng e o va te l at is si m e 2. 51 ± 0. 17 d 3. 48 ± 0. 04 b 0. 72 r et ic ul at e 23 8. 31 ± 5. 58 a 77 .0 3± 11 .3 8b c c ru sh ed 24 6. 04 ± 34 .0 8e 19 6. 41 ± 18 .6 3d r 5 o ra ng e o va te l at e 3. 56 ± 0. 08 b 3. 21 ± 0. 12 d 1. 10 r et ic ul at ea re ol at e 18 3. 21 ± 14 .6 3c 56 .0 1± 9. 41 cd c ru sh ed 38 8. 80 ± 11 .7 9a 32 8. 60 ± 12 .4 2a r 6 o ra ng e o va te d ep re ss e 2. 04 ± 0. 16 e 2. 88 ± 0. 19 de 0. 70 r et ic ul at e 13 5. 79 ± 19 .2 5d 99 .3 2± 3. 54 a c ru sh ed 35 1. 44 ± 17 .4 3b 28 1. 12 ± 33 .4 9b c r 7 o ra ng e e lli pt ic al 4. 54 ± 0. 49 a 3. 67 ± 0. 38 a 1. 23 a lv eo la te 15 6. 53 ± 20 .9 1c d 63 .6 3± 2. 93 c fl at 29 6. 63 ± 12 .6 8d 24 5. 33 ± 10 .6 6c morphological and anatomical studies of romula taxa seeds acta bot. croat. 74 (1), 2015 35 taxa, varies from slightly (r3) or intensively (r2) reticulate to alveolate (r7). however, seeds of r5 have a vaguely reticulateareolate sculpture, while there are elevated protrusions on the seed surface of r1 which is distinguished by these protrusions from r4 and r6 (fig. 2). on the other hand, the seed surface patterns of r4 and r6 are not signifi cant in terms of the separation of taxa (tab. 2). the anatomical characteristics of the seeds of turkish romulea are given in table 2. accordingly, the mean values of testa thickness vary from 81.93 μm to 238.31 μm, and this character varied signifi cantly among the examined taxa. a thick testa is noted in r4, whereas the testa in r3 is thin (tab. 2). in other respects, shapes of epidermal cells of testa differ in the studied taxa (tab. 2), which have elevated-fl at, crushed and undulating cells in unequal or equal forms, and thick or thin walls in cross sections (fig. 3). in addition to these fi ndings, embryo sizes of the seeds have been shown to range from 175.98–388.80 μm to 156.82– 328.60 μm, and embryos in seeds formed oval (r4 and r5), globular (r2 and r3) and prolonged (r1, r6 and r7) in shapes (fig. 3). fig. 1. turkish romulea seeds; a: r1, b: r2, c: r3, d: r4, e: r5, f: r6, g: r7; for taxa abbreviations see tab. 1; scale bars = 1 mm. fig. 2. seed surfaces of romulea taxa; a: r1; reticulate, b: r2; reticulate-foveate, c: r3; reticulate -areolate, d: r5; slightly reticulate-areolate, e: r7; alveolate; for taxa abbreviations see tab. 1. the arrow shows protrusion on the seed surface. karai̇smai̇loğlu m. c. 36 acta bot. croat. 74 (1), 2015 an unweighted pair group method with arithmetic mean (upgma) dissimilarity clustering dendrogram for turkish romulea taxa according to the examined quantitative characters in tab. 2 is given in fig. 4. accordingly, branches in the dendrogram are divided into two main clads and sub-clads. r7 composes the fi rst main branch; other taxa make up the second main branch. also, r7 differs clearly from other taxa in terms of characters deterfig. 3. cross section structures of turkish romulea seeds; 1: overall appearance; anticlinal cell types: 2: fl at cell (r7), 3: crushed cell (r2, r3, r4, r5 and r6), 4: undulated cell (r1), types of embryo, 5: oval (r4 and r5), 6: prolonged (r1, r6 and r7), 7: globular (r2 and r3); for taxa abbreviations see tab. 1; e – embryo, en – endosperm, sr – storage reserve, t – testa, ec – epidermal cells of testa, fc – fl at cell, ph – phytomelan layer, cc – crushed cell, uc – undulated cell; white scale bars = 100 μm, black scale bars = 50 μm. fig. 4. upgma clustering of romulea taxa in turkey based on morphological and anatomical characters of the seeds; for taxa abbreviations see tab. 1. morphological and anatomical studies of romula taxa seeds acta bot. croat. 74 (1), 2015 37 mined in tab. 2. furthermore, pca ordination and similarity matrix based on some morphological and anatomical characters of seeds such as seed dimensions, testa thickness, phytomelan layer thickness and embryo sizes are presented in fig. 5 and tab. 3. among the studied taxa, the closest and the most distant species are determined. according to these results, r6 and r2 are the most closely related species (percentage similarity: 0.789), r6 and r7 are the most distantly related species (percentage similarity: –0.169) (tab. 3 and fig. 5). in addition, the cumulative variance value of principal components achieved 63.14 % (axis 1: 32.29%, axis 2: 30.85%). fig. 5. principal component analysis of romulea taxa in turkey based on morphological and anatomical characters of the seeds; for taxa abbreviations see tab. 1. tab. 3. similarity matrix among the turkish romulea taxa based on some morphological and anatomical characters of seeds; for taxa abbreviations see tab. 1. taxa r1 r2 r3 r4 r5 r6 r7 r1 1 – – – – – – r2 0.310 1 – – – – – r3 0.070 0.514 1 – – – – r4 –0.0440 0.383 0.633 1 – – – r5 0.070 0.514 0.514 0.383 1 – – r6 0.292 0.789 0.310 0.450 0.310 1 – r7 0.070 0.029 0.271 –0.1180 0.029 –0.1690 1 karai̇smai̇loğlu m. c. 38 acta bot. croat. 74 (1), 2015 discussion the morphological (macro and micro) and anatomical characters of seeds frequently indicate major differences among taxa of the same genus or family, and these differences provide a signifi cant contribution to delimiting various taxonomic levels of taxa and to the establishment of taxonomic relationships. although reliability and sustainability of seed properties in classifi cation are known (dahlgren and clifford 1982, grilli caiola et al. 2010), no researches appeared to have been performed so far concerning seed morphology and anatomy of romulea taxa in turkey. in this investigation, macromorphological features such as size, shape and raphe characters of seeds of romulea taxa in turkey have indicated the presence of variations among the examined taxa. although it is reported as being only globular in the flora of turkey and aegean islands by marais (1984), seed shape in almost all of the examined romulea taxa varied (excluding seed shapes of r2 and r5) (tab. 2). the seed sizes of romulea taxa are quite variable. the broadest seeds are found in r7, and the smallest seeds in r6. both of these measurements are considerably different from those obtained from other examined taxa. these variations in the seed shape are compatible with the diagnostic characters in flora of turkey (davis 1984) for the romulea species. in contrast to the fi ndings of marais (1984), the seed colour of turkish romulea taxa has been found to be orange and its tones rather than brown, and the colour of seed has no use taxonomically. one of the remarkable characters is also the presence of raphes on seeds, and their size. the raphes of r4 and r7 are more pronounced than on other examined taxa, and there is no prominent raphe in r1 (fig. 1). at the same time, the sizes of seeds having a raphe are generally smaller than those do not have a marked raphe. this result is attributable to the principle that wind-dispersed seeds should be in small size and should bear raphe-like structures (tebbitt 2005). the seed morphology has not been suffi ciently utilized in the taxonomy of iridaceae family (erol et al. 2006). results obtained from this research indicate that some macromorphological features, particularly the shapes and sizes of seeds of romulea taxa reveal as distinctive characters in terms of classifi cation, because of their diversity among taxa, so they may be used as supporting characters in taxonomic studies. seed surface structures have been utilized for some purposes such as the solution of systematic problems, interpretation of evolutionary interactions, and illumination of the adaptive features of the seed surface (heywood 1971, sulaiman 1995). in this investigation, the surface patterns range from slightly (r3) or intensively (r2) reticulate to alveolate (r7) in the examined taxa. r5 has very weak reticulate-areolate surface and the seed surface of r1 has visible raised protrusions (fig. 2). and so, the surface patterns of seed coats are found helpful in taxonomy for some romulea. nonetheless, variations in the obtained results indicate that the seed surface characters support the frequently used characters in the systematics of the genus (according to davis 1984) (except for r4 and r6), and can contribute to classifi cation of turkish romulea species. the testa thicknesses and epidermal cell types of the seeds can provide precious data about phylogenetic classifi cation of fl owering plants (corner 1976). in this investigation, the differences in anatomical features such as testa thickness, phytomelan thickness, shape of epidermal cells of testa and embryo size of the seeds of turkish romulea have been identifi ed. the most distant species are r3 (81.93 μm) and r4 (238.31 μm), while the other species examined are located between 81.93 μm and 238.31 μm. likewise, the epidermal morphological and anatomical studies of romula taxa seeds acta bot. croat. 74 (1), 2015 39 cells of testa indicate difference in turkish romulea species. they form different shapes such as undulating (r1), polygonal crushed (r2–r6) and fl at (r7) in the cross sections of the seeds of the examined taxa (tab. 2). accordingly, epidermal cells can be used in the separation of r1 and r7 from other taxa, and can be of some use in the taxonomy of examined taxa. in this regard, the results obtained are consistent with a previous similar study in iridaceae (grilli caiola et al. 2010). the taxonomic signifi cance of phytomelan-covered seeds was explained by huber (1969), and the presence-absence and thickness (if any) of phytomelan layer are of notable taxonomic signifi cance in the distinction of taxa in the iridaceae family (dahlgren and clifford 1982). the thickness of the phytomelan layer in turkish romulea species ranged from 54.11 μm to 99.32 μm and changed signifi cantly in examined romulea species (tab. 2). this fi nding attributed as the thickness of phytomelan layer can be useful in the classifi cation of turkish romulea species because of differentiated thickness in almost all of the examined taxa in this investigation (tab. 2). the embryos of the turkish romulea taxa exhibit a wide variety in terms of shapes and sizes (tab. 2). while the embryo in r2 and r3 is usually swollen and well arranged, the embryo in other examined taxa is more or less elongated (as if not well developed).the anatomical fi ndings of this investigation are compatible with those of grilli cailo et al. (2010), who have made a study with similar variables including anatomical features on some crocus seeds. one of the most important characters which distinguish r1, r4 and r6 from each other is the colour and length of fi laments (davis 1984). however, it is impossible to separate the taxa from each other in the case of gynodioecism in one of the populations of these taxa since there will be only female fl owers. in this investigation, seed shape, size and presence of raphe on seeds morphologically; thickness of phytomelan layer, embryo size anatomically are applicable characters in distinguishing these taxa. upgma of cluster analysis was used to evaluate the morphological and anatomical features of the seeds among the analyzed taxa. in this dendrogram, branches that are markedly dissimilar to other taxa were created for r1 and r7. this fi nding can be attributed to r1 and r7 being different from the other analyzed taxa in terms of the studied characters. generally, the branches contained nearly related taxa are compatible with the traditional taxonomic rank of turkey’s romulea species. in other words, the morphological and anatomical features of the seeds supported the characters used in the distribution of romulea species in the fl ora of turkey (according to davis 1984). principal component analysis can be helpful in giving data about the variability of quantitative characters. the obtained cumulative variance values of principal components show that the studied characters in turkish romulea taxa can be of use in explaining the differences among the taxa because of high variance value. in addition, the morphological and anatomical features of the seeds chosen for pca to evaluate the characteristics that are signifi cant in description change among the analyzed specimens (tab. 3). likelihood ratios among the taxa were determined. the closest relationship was seen between r6 and r2, additionally the most distant relationship was found between r6 and r7. these results indicate that a similarity matrix can give information about the classifi cation of turkish romulea taxa if there is gynodioecism in a population. karai̇smai̇loğlu m. c. 40 acta bot. croat. 74 (1), 2015 in conclusion, this investigation supports the use of seed surface, shape, size and the cross section of seed such as testa and phytomelan layer thickness, shape of testa epidermal cells and embryo sizes as the distinctive characters in the classifi cation within the romulea taxa in turkey. acknowledgements the author thanks dr. osman erol for supplying seeds of the studied taxa and veysel süzerer and almıla çiftçi for technical assistance. references akalin-urusak, e., kizilarslan, c., 2013: fruit anatomy of some ferulago (apiaceae) species in turkey. turkish journal of botany 37, 434–445. barthlott, w., 1981: epidermal and seed surface characters of plants: systematic applicability and some evolutionary aspects. nordic journal of botany 1, 345–355. corner, e. j., 1976: the seeds of the dicotyledons. vol. 2. cambridge university press, cambridge. dahlgren, r. f., clifford, h. t., 1982: the monocotyledons: a comparative study, academic press, london. davis, p. h., 1984: flora of turkey and the east aegean islands, vol. 8. edinburgh university press, edinburgh. erol, o., kucuker, o., 2003: morpho-anatomical observations on three romulea (iridaceae) taxa of turkey. bocconea 16, 607–613. erol, o., uzen, e., kucuker, o., 2006: preliminary sem observations on the seed testa structure of gladiolus l. species from turkey. international journal of botany 2, 125– 127. grilli caiola, m., leonardi, d., canini, a., 2010: seed structure in crocus sativus l. x, c. cartwrightianus herb., c. thomasii ten., and c. hadriaticus herb. at sem. plant systematics and evolution 285, 111–120. heywood, w. h., 1971: scanning elentron microscopy. academic press, london. huber, h., 1969: die samenmerkmale und verwandtschafts-verhaltnisse der liliifl orae. mitteilungen der botanischen staatssammlung münchen 8, 219–538. inceer, h., bal, m., ceter, t., pinar, n. m., 2010: fruit structure of 12 turkish endemic tripleurospermum sch. bip. (asteraceae) taxa and its taxonomic implications. plant systematics and evolution 298, 845–855 isik, s., donmez, e. o., 2007: pollen morphology of the turkish romulea maratti (iridaceae). turkish journal of botany 31, 171–182. karamian, r., behjou, a. m., ranjbar, m., 2012: anatomical fi ndings of onobrychis sect. heliobrychis (fabaceae) in iran and their taxonomic implications. turkish journal of botany 36, 27–37. marais, w., 1984: romulea maratti in: davis ph (ed.), flora of turkey and the east aegean islands, vol. 8, pp. 438–441. edinburgh university press, edinburgh. morphological and anatomical studies of romula taxa seeds acta bot. croat. 74 (1), 2015 41 moret, j., bari, a., le thomas, a., goldblatt, p., 1992: gynodioecy, herkogamy and sexratio in romulea bulbocodium var. dioica (iridaceae). evolutionary trends in plants 6, 99–109. moret, j., bari, a., le thomas, a., 1993: evolution of herkogamy and gynodioecy in moroccan species of romulea (iridaceae). plant systematics and evolution 184, 241–257. ozdemir, c., bozdag, b., akyol, y., sen, u., sepet, h., yetisen, k., 2011: morphological and anatomical investigations of romulea bulbocodium var. bulbocodium and romulea bulbocodium var. leichtliniana (iridaceae). thaiszia journal of botany 21, 65–72. rudall, f. l. s., goldblatt, p., 1991: leaf anatomy and phylogeny of ixioideae (iridaceae). botanical journal of the linnean society 106, 329–345. sulaiman, i. m., 1995: scanning electron microscopic studies seed coat patterns of fi ve endangered himalayan species of meconopsis (papaveraceae). annals of botany 76, 323– 376. stearn, w. t., 1985: botanical latin: history, grammar syntax, terminology and vocabulary. david & charles, london. tebbitt, m. c., 2005: begonias, cultivation, identifi cation and natural history. timber press, portland. opce-str.vp acta bot. croat. 68 (2), 455–463, 2009 coden: abcra 25 issn 0365–0588 'araphid' diatom classification and the 'absolute standard' david m. williams department of botany, the natural history museum, cromwell road, london sw7 5bd, united kingdom 'araphid' diatom classification is discussed from the point of view of an 'absolute standard' for taxonomic rank. the 'absolute standard' is the phylogenetic tree, its nodes, the included monophyletic groups and sub-groups. to illustrate this point a few species from the genus licmophora are re-analysed and the resulting phylogenetic tree is discussed in terms of a possible classification, the groups and sub-groups and their ranks. keywords: diatom, araphid, classification, phylogeny introduction at the 18th international diatom symposium i discussed diatom phylogenetic trees and their interpretation. some of my presentation appeared in a recent book on the classification of 'algae' (williams 2007) and in a shorter review paper (williams and kociolek 2007). since that time a few more papers have appeared on 'araphid' diatom phylogeny, particularly from a molecular perspective (sato et al. 2008a, 2008b, medlin et al. 2008a, 2008b). a contribution of some significance is medlin et al. (2008b), as it presents a large quantity of data and proposes one of the first trees of relationships to include a good number of the diverse 'araphid' diatoms. in this short note i want to add a few more comments on the relationship between phylogenies (branching diagrams) and classification with respect to 'araphid' diatoms. the purpose of this paper is not to examine in detail the phylogenetic trees presented in medlin et al. (2008b) but to offer some commentary on a few general points. in particular i will focus on an idea previously articulated in mann (1997) but recently stated more succinctly, concerning the apparent elusive 'absolute standard' of classification (sato et al. 2008a: 386). i begin with a short discussion on the parameters of classification. acta bot. croat. 68 (2), 2009 455 * corresponding author: dmw@nhm.ac.uk u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:42 color profile: disabled composite 150 lpi at 45 degrees genera, taxa, monophyly and the 'absolute standard' over a decade ago, the japanese journal of diatomology (diatom 13, 1997) presented a series of essays in honour of the late professor hiromu kobayasi (1926–1996). the topic was the species concept in diatoms but, inevitably, discussion spilled over into concerns with other taxa at all ranks, partly because from the late 1980s onwards a veritable flood of new genera had been described (e.g. round et al. 1990; see kociolek 1997, 1998; lange-bertalot 1997; round 1997a, b). reading the series of papers in diatom today leaves one with the feeling that the core problem remains unresolved. a general question might be framed from those contributions: how might one find the right 'measure' for any particular taxon, the genus, for example? according to sato et al. (2008a: 386), there is no absolute standard for the amount of sequence difference that justifies generic status. particular questions, such as 'what is a genus?' and 'what is a species?' might benefit from re-structuring. one might more profitably view the problem from the perspective of artificial and natural classifications rather than a quest for some measure. these two approaches – artificial and natural classification – might differ, inasmuch as the first is concerned with naming organisms, the second with naming schemes of relationships. examples of artificial diatom classifications (smith 1853, hustedt 1930) clearly considered identification to be of great importance and the primary purpose of a classification. examples of natural classifications are harder to come across, as by their very definition they are temporary, subject to change. some examples of persons clearly searching for such a system are agardh (1823–1828) and merezhkowsky (1902) (see williams 2007 for a fuller review). that the two – a set of names for identification and a classification of relationships – might differ has been accepted for quite some time (williams and kociolek 2007), although that may not be an altogether happy circumstance (williams and ebach 2007, 2009). it seems fair to suggest that, over time, the goals of natural classification have been lost, or at least disappeared, even considered of little significance. this might possibly be because there are a vast number of general biologists who simply require names for their organisms, rather than a classification that reflects relationships. medlin et al. (2008b) did not concern themselves with classification beyond the generic level, as their intention was to test existing genera and to test some relatively new proposals. happily, there is less literature concerning the genus concept than there is concerning the species concept. that does not necessarily make it easier to digest. i will not attempt to do so (for those interested, these more general references provide an easily accessible sample from the last 60 years: bartlett 1940, anderson 1940, greenman 1940, sherff 1940, legendre 1971, clayton, 1972, 1983, stevens 1983). here i simply quote from the conclusion of peter stevens’s paper: »if a decision is made that the classification is not to be at least congruent with a phylogeny, and no attempt is made to produce such a phylogeny, then the work we produce will be of limited relevance to biologists« (stevens 1983: 463). phylogeny might be directly equated with natural classification. there are now many attempts at producing phylogenies, the data source usually being dna sequences but that need not be the case, as morphology is equally as useful (williams and reid 2006). so the issue might be seen as relating the one – phylogenetic trees – to the other – classification. the issue of an 'absolute standard', regardless of whether data are molecular or morphological, is explored in the rest of this paper, with an example derived from a re-analysis of the 'araphid' phylogenetic tree. 456 acta bot. croat. 68 (2), 2009 williams d. m. u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:42 color profile: disabled composite 150 lpi at 45 degrees re-analysis of the 18s rdna 'araphid' diatom data results from the most comprehensive 18s rdna sequence analyses of 'araphid' diatoms were summarised in a tree with a total of 45 nodes (medlin et al. 2008b: figs. 1, 2). the groups so formed were recognised, informally, as five clades, labelled 1–5; clade 5 is subdivided into four sub-clades, 5a–5d, clade 2 is further subdivided into two sub-clades, 2a and 2b (medlin et al. 2008b: figs. 1, 2). none of the clades or sub-clades was named. clade 3 has 5 specimens related as in figure 1 (redrawn from medlin et al. 2008b: fig. 2). clade 3 is interesting for a number of reasons: (1) all the specimens belong to different genera, therefore the sample is insufficient to determine any generic relationships; (2) clade 3 includes striatella, a genus usually most closely related to all pennate diatoms but »…18s rdna analyses undertaken so far have placed s.[triatella] unipunctata in various phylogenetic positions«, thus its position in this tree is also somewhat anomalous (sato et al. 2008a: 386); (3) a larger number of species from the genus licmophora are now available in genbank, hence a more detailed analysis can be undertaken. to reinvestigate 'araphid' relationships, 164 18s rdna sequences were aligned using the program bioedit (1997–2004) and clustalw (larkin et al. 2007), the latter from within bioedit's accessory applications (clustalw was implemented using bioedit's default options). of the 164 sequences, 7 (arcocellulus mammifer, cymatosira belgica, extubocellulus spinifer, minutocellus polymorphus, minutocellus sp. ccmp1701?, papiliocellulus elegans, and talaroneis posidoniae) are included as outgroup taxa, all from cymatosiraceae, with the exception of talaroneis posidoniae, which currently resides in plagiogrammaceae (sato et al. 2008b). a problem with both this re-analysis and that of medlin et al. (2008b) is the lack of any raphid, pennate diatoms, which should be included as it is now accepted that 'araphid' diatoms are paraphyletic, not a natural group. the reason for their omission in this study was to make the analysis of 'araphid' data and the recognition of their sub-groups more tractable, given the size a matrix would need to be if relevant raphid diatoms were included. in any case, the focus of attention is the relationships of the genus licmophora. the complete alignment, including all introduced gaps, is 2830 bases long. actual taxon sequence length ranges from 981 (synedropsis sp. ccmp2747) to 2350 (hyalosira sp. ccmp469); average sequence length ranges between 1750–1800 (a few relevant taxa with sequences in genbank were omitted as they had too few bases to offer anything of significance). acta bot. croat. 68 (2), 2009 457 'araphid' diatoms and the absolute standard fig. 1. part of the phylogenetic tree from medlin et al. 2008b, figure 2, illustrating members and their relationships of their clade 3. u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:42 color profile: disabled composite 150 lpi at 45 degrees using the program winclada (nixon 1999–2002), uninformative characters (those that do not specify any relationships, those not shared by more than two taxa) can be removed, which discards a total of 2210, leaving 620 (c. 28%); if uninformative characters are removed using the option available in the parsimony program nona (goloboff 1999), a total of 2245 are discarded, leaving 585 (c. 21%). analysis of the informative character matrix (585) with nona yields in excess of 10,000 trees (the maximum tree number to be retained was set to 10,000; the implication being that there are more trees to be found should the search be prolonged and the number of trees retained increased); trees are of length 3079, ci=51, ri=83; using the parsimony rachet (nixon 1999, goloboff 1999), a subset of 67 trees are found, also of length 3079, ci=51, ri=83 (the strict consensus tree has a length of 3148, ci=50, ri=83). naming the tree (fig. 2) the analysis undertaken here includes a group of 13 specimens, of which 11 are named as species in the genus licmophora (distributed among 7 species); the remaining two specimens are named as cyclophora tenuis and protoraphis atlantica. the relevant part of the complete tree is fully resolved with 11 nodes (fig. 2, taken from the consensus tree). the 11 specimens recognised as belonging to licmophora include a node relating cyclophora tenuis and protoraphis atlantica most closely to licmophora flabellata and l. communis (fig. 2, node 4) rendering licmophora non-monophyletic (paraphyletic), if the genus name is applied to the root of the tree (fig. 2, node 0). that is, all species of licmophora relate to more than one node (fig. 2, nodes 6, 7, and 9). one option would be to transfer both cyclophora tenuis and protoraphis atlantica to licmophora creating a highly variable, but monophyletic, genus at node 0 (fig. 2). however, several branches in the tree have monophyletic groups that include some species of licmophora and could be named if one so desired: nodes 3, 6, 7, and 9, of which only one can be licmophora s.s., the group that includes its type, licmophora argentescens c.a. agardh. (vanlandingham understands licmophora argentescens to be synonymous with l. flabellata. if this is so, species from node 9 will be the genus licmophora s.s. vanlandingham 1971, see round et al. 1990.) 458 acta bot. croat. 68 (2), 2009 williams d. m. fig. 2. part of phylogenetic tree derived from a new analysis of 164 18s rdna sequences, which includes a group of 13 specimens, 11 from the genus licmophora, one each from cyclophora tenuis and protoraphis atlantica. u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:43 color profile: disabled composite 150 lpi at 45 degrees the branch leading to licmophora juergensii (figure 2, node 7) relates two specimens of the same species, basal to node 2. in this case, the only reasonable option is to place l. juergensii in a separate genus even though it is, at this time, monotypic. cyclophora and protoraphis are represented by one species apiece; other species in each genus are said to exist but have yet to be investigated relative to rdna data (possibly one more for cyclophora; possibly two more for protoraphis). thus, at this time, it would be wise to retain their generic names representing (probable) monophyletic groups. in total, then, for the species in figure 2, it is possible to name six genera: three monophyletic genera (nodes 3, 6, and 9) and one monospecific genus (node 7) for the species of licmophora, plus cyclophora and protoraphis (fig. 2, terminals 12 and 13). while the taxonomic level of genus is arbitrary, it is the fact that the groups recognised are monophyletic that is of significance. higher-level classification is more problematic, with a variety of ways of solving the problem. here i offer one. the tree divides into two groups at nodes 1 and 2. node 1 contains genus 1, from node 3 (node 5 is here ignored) and genus 2, from node 6 (fig. 2). node 2 subdivides into node 4 and genus 3, from node 7 (fig. 2). node 4 further subdivides into nodes 8 and 9, where node 8 relates cyclophora and protoraphis and node 9 relates most closely the two species in genus 4 (fig. 2). tables 1–3 illustrate all possible nodes and their inclusive taxa (tab. 1), related to groups and sub-groups (tab. 2) and to possible ranks (tab. 3). not all nodes need naming; alternatives schemes are possible – but once again the 'absolute standard' is monophyly. node 7, the monotypic genus for licmophora juergensii, remains a curiosity. if we assume an appropriate rank for the entire group (node 0) is an order, the next sub-division is into two families (nodes 1 and 2) (tab. 3). family 1 is composed of two genera. family 2 is composed of further sub-divisions, the first being licmophora juergensii plus all taxa from node 4. as node 4 has itself two further sub-divisions, licmophora juergensii and all species included in node 4 could be sub-families. thus, licmophora juergensii will be in its own sub-family as well as its own genus. the meaning of monotypic taxa is that the relationships of the included single species is unknown, relative to other closely related species, beyond their shared more basal node. in the case of licmophora juergensii, its relationships are unknown other than it is being basal acta bot. croat. 68 (2), 2009 459 'araphid' diatoms and the absolute standard tab. 1. nodes and inclusive taxa, derived from the tree in figure 2. nodes taxa 0 1 3 licmophora abbreviata 0 1 3 licmophora gracilis 0 1 6 licmophora grandis 0 1 6 licmophora reichardtii 0 2 7 licmophora juergensii 0 2 4 8 cyclophora tenuis 0 2 4 8 protoraphis atlantica 0 2 4 9 licmophora flabellata 0 2 4 9 licmophora communis u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:44 color profile: disabled composite 150 lpi at 45 degrees to all species from node 2. a monotypic taxon – a single species – is neither mono-, paranor polyphyletic; it is, strictly speaking, only most closely related to itself. monotypy in this sense, serves a proper function, indicating the extent of our knowledge (or, rather, ignorance) (fig. 2). this use is in contrast to that of cyclophorales, for example, which was placed in its own family, cyclophoraceae, with the intention of indicating its uniqueness in some unspecified way (round et al. 1990). thus, cyclophorales and cyclophoraceae are uninformative expressions of a certain amount of ignorance rather than acknowledgement of some unquantifiable uniqueness (patterson 1982: 32). discussion above i presented part of a phylogenetic tree for species of licmophora derived from a parsimony analysis of 18s rdna sequence data. i also presented a classification derived from that tree. the tree allows precision in establishing the systematic position of each 460 acta bot. croat. 68 (2), 2009 williams d. m. tab. 3. possible ranks for groups and sub-groups, derived from the tree in figure 2. ranks taxon order 1 (0) family 1 (1) genus 1 (3) licmophora abbreviata genus 1 (3) licmophora gracilis genus 2 (6) licmophora grandis genus 2 (6) licmophora reichardtii family 2 (2) sub-family 1 (7) genus 3 (7) licmophora juergensii sub-family 2 (4) tribe 1 (8) genus 4 (12) cyclophora tenuis genus 5 (13) protoraphis atlantica tribe 2 (9) genus 6 (10) licmophora flabellata genus 6 (14) licmophora communis tab. 2. groups, sub-groups and genera, derived from the tree in figure 2. groups and sub-groups taxon group 1 sub-group 1 (1) genus 1 (3) licmophora abbreviata genus 1 (3) licmophora gracilis genus 2 (6) licmophora grandis genus 2 (6) licmophora reichardtii sub-group 2 (2) sub-sub-group 1 (7) genus 3 (7) licmophora juergensii sub-sub-sub-group 1 (8) genus 4 (12) cyclophora tenuis genus 5 (13) protoraphis atlantica sub-sub-group 2 (4) sub-sub-sub-group 2 (9) genus 6 (10) licmophora flabellata genus 6 (14) licmophora communis u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:44 color profile: disabled composite 150 lpi at 45 degrees taxon. regardless of rank, each taxon relates to a particular node. other classifications are possible but the tree – the nodes, the monophyletic groups – is the 'absolute standard'. of further significance, the tree allows precision when conducting further studies. for example, should another species of licmophora be studied it will relate to some part of the tree. should it relate directly to either node 3, 6 or 9, for example, it is part of that particular genus (fig. 2). should it relate to node 7 instead then support is gained for the genus based on licmophora juergensii, providing it with a second species and demonstrating its monophyly (fig. 2). in short, when further species are investigated their position and taxonomic status is determined in an absolute sense by the tree. this can be contrasted with the viewpoint recently expressed and noted above. defending the naming of the new monotypic genus pseudostriatella, sato, mann and medlin sato et al. offered the following argument: »there are many morphological and ecological similarities between p. oceanica and s. [striatella] unipunctata…[its most closest relative] [but] there are also many differences, which we regard as sufficient to differentiate these taxa at the rank of genus« (sato et al. 2008a: 383). of course, the notion of 'sufficient' remains unexplored and unquantified. further, they state, that genetic distance may also be considered: »18s rdna phylogeny places p. oceanica among the pennate diatoms and supports a close relationship between p. oceanica and s. unipunctata, but the genetic distance between them, coupled with the morphological differences, justifies separation at genus level« (sato et al. 2008a: 383). thus, 'many' differences linked to an unspecified genetic distance become 'sufficient' for 'justifying' a particular rank. but oddly, »…there is no absolute standard for the amount of sequence difference that justifies generic status« (sato et al. 2008a: 386). one must assume sato et al. guessed. i have tried to show above that there is an 'absolute standard', which resides in the nodes of the phylogenetic tree, with the congruence of classification and phylogeny; in short, monophyly (williams and kociolek 2007). i have also tried to show that classifications derived in this way are predictive, in the sense that any further information will relate – or meaningfully test – the relationships proposed; this is the essence of congruence – of which morphology provides data in abundance – and the essence of scientific investigation in general. acknowledgements i wish to thank mark carine, malte ebach, tony gill and pat kociolek for their comments on various drafts of this paper and two anonymous referees for their comments. references agardh, c. a. 1823–1828: systema algarum. litteris berlingianis, lundae. anderson, e. 1940: the concept of the genus: ii. a survey of modern opinion. bulletin of the torrey botanical club 67, 363–369. bartlett, h. h. 1940: the concept of the genus: i. history of the generic concept in botany. bulletin of the torrey botanical club 67, 349–362. clayton, w. d. 1972: some aspects of the genus concept. kew bulletin 27, 281–287. acta bot. croat. 68 (2), 2009 461 'araphid' diatoms and the absolute standard u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:44 color profile: disabled composite 150 lpi at 45 degrees clayton, w. d. 1983: the genus concept in practice. kew bulletin 38, 149–153. greenman, j. m. 1940: the concept of the genus: iii. genera from the standpoint of morphology. bulletin of the torrey botanical club 67, 371–374. goloboff, p. 1999: nona (no name) ver. 2. published by the author, tucumán, argentina. hustedt, f. 1930. bacillariophyta (diatomeae). in: pascher, a. (ed), die susswasserflora mitteleuropas, 10 verlag von gustav fischer, germany. kociolek, j. p. 1997: historical constraints, species concepts and the search for a natural classification of diatoms. diatom 13, 3–8. kociolek, j. p. 1998: does each genus of diatoms have at least one unique feature? – a reply to round. diatom research 13, 177–179. lange-bertalot, h. 1997: as a practical diatomist, how does one deal with the flood of new names? diatom 13, 9–12. larkin, m. a., blackshields, g., brown, n. p., chenna, r., mcgettigan, p. a., mcwilliam, h., valentin, f., wallace, i. m., wilm, a., lopez, r., thompson, j. d., gibson, t. j., higgins, d. g. 2007: clustal w and clustal x version 2.0. bioinformatics 23:2947– 2948; doi:10.1093/bioinformatics/btm404 legendre, p. 1971: circumscribing the concept of the genus. taxon 20, 137–139. mann, d. g. 1997: shifting sands: the use of lower taxonomic ranks in diatoms. diatom 13, 13–17. medlin l. k., sato s., mann d. g., kooistra w. h. c. f. 2008a: molecular evidence confirms sister relationship of ardissonea, climacosphenia and toxarium within the bipolar centric diatoms. journal of phycology 44, 1340–1348. medlin l. k., jung, i., bahulikar, r., mendgen, k., kroth, p., kooistra, w. h. c. f. 2008b: evolution of the diatoms. vi. assessment of the new genera in the araphids using molecular data. nova hedwigia 133, 81–100. merezhkowsky, c. 1902: on the classification of diatoms. annals and magazine of natural history, 9, 65–68. nixon, k. c. 1999: the parsimony ratchet, a new method for rapid parsimony analysis. cladistics 15, 407–414. nixon, k. c. 1999–2002: winclada ver. 1.0000. published by the author, ithaca, ny, usa. patterson, c. 1982: morphological characters and homology. in: joysey, k. a., friday, a. e.(eds), problems of phylogenetic reconstruction, 21–74. academic press, london. round, f. e. 1997a: genera, species and varieties – are problems real or imagined? diatom 13, 25–29. round, f. e. 1997b: does each diatom genus have a unique feature? diatom research 12, 341–345. round, f. e., crawford, r. m., mann, d. g. 1990: the diatoms – biology and morphology of the genera. cambridge university press, cambridge. sato, s., mann, d. g., matsumoto, s., medlin, l. k. 2008a: pseudostriatella (bacillariophyta): a description of a new araphid diatom genus based on observations of frustule and auxospore structure and 18s rdna phylogeny. phycologia 47, 371–391. 462 acta bot. croat. 68 (2), 2009 williams d. m. u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:44 color profile: disabled composite 150 lpi at 45 degrees sato, s., kooistra, w. h. c. f., watanabe, t., matsumoto, s., medlin, l. k. 2008b: a new araphid diatom genus psammoneis gen. nov. (plagiogrammaceae, bacillariophyta) with three new species based on ssu and lsu rdna sequence data and morphology. phycologia 47, 510–528. sherff, e. e. 1940: the concept of the genus: iv. the delimitations of genera from the conservative point of view. bulletin of the torrey botanical club 67, 375–380. smith, w. 1853: a synopsis of the british diatomaceae, 1. smith and beck, london. stevens, p. f. 1983: the genus concept in practice – but for what practice? kew bulletin 40, 457–465. vanlandingham, s. l. 1971: catalogue of the fossil and recent genera and species of diatoms and their synonyms. part iv. fragilaria through naunema. j. cramer, lehre. williams, d. m. 2007: classification and diatom systematics: the past, the present and the future. in: brodie, j., lewis, j. (eds), unravelling the algae, 57–91. crc press, florida. williams, d. m, ebach, m. c. 2007: the foundations of systematics and biogeography. springer, new york. williams, d. m, ebach, m. c. 2009: what, exactly, is cladistics? re-writing the history of systematics and biogeography. acta biotheoretica 57, 249–268. williams, d. m, kociolek, j. p. 2007: pursuit of a natural classification of diatoms: history, monophyly and the rejection of paraphyletic taxa. european journal of phycology 42, 313–319. williams, d. m., reid, g. 2006: amphorotia nov. gen., a new genus in the family eunotiaceae (bacillariophyceae), based on eunotia clevei grunow in cleve et grunow. diatom monographs 6, 1–153. acta bot. croat. 68 (2), 2009 463 'araphid' diatoms and the absolute standard u:\acta botanica\acta-botan 2-09\williams.vp 6. listopad 2009 13:43:44 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2016 za web.indd 266 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 266–271, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0026 issn 0365-0588 eissn 1847-8476 short communication infl uence of soil traits on polyphenols level in moltkia petraea (tratt.) griseb. (boraginaceae) dario kremer1, renata jurišić grubešić1, dalibor ballian2, danijela stešević3, ivan kosalec1, jadranka vuković rodríguez1, marija vukobratović4, siniša srečec4* 1 faculty of pharmacy and biochemistry, university of zagreb, a. kovačića 1, zagreb hr-10000, croatia 2 faculty of forestry, university of sarajevo, zagrebačka 20, bih-71000, bosnia and herzegovina 3 faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81000 podgorica, montenegro 4 križevci college of agriculture, m. demerca 1, hr-45260 križevci, croatia abstract – the illyric–balkan endemic species moltkia petraea (tratt.) griseb. is very interesting as a potential horticultural and medicinal plant. the aim of this study was to investigate soil conditions of m. petraea habitats, the phenolic content in plant parts, and the infl uence of soil properties on the phenolic contents. the results were evaluated using spearman rank order correlations. analyzed soil samples contained very low to intermediate levels of physiologically active phosphorus, but were very rich in potash. organic matter content of soil was high. phenolic compound content was higher in leaves than in fl owers or stems. the analyses showed that m. petraea possesses considerable quantities of phenolic compounds and has no specifi c demands for particular soil conditions. a negative correlation was found between soil phosphorus content and total phenols content in leaves and stems, and with the total phenolic acids content in fl owers. organic matter in soil also found to have a negative infl uence on total tannins content in stems. among the tested geographical locations, the mljet population showed a higher degree of separation from the remaining locations. keywords: endemic, moltkia petraea, phenolic compounds, plant habitats, soil traits * corresponding author, e-mail: ssrecec@vguk.hr introduction the north-western balkan peninsula, particularly the area of dinaric alps, though poorly investigated, is known to be very rich in endemic plant species. from the biogeographical perspective, the dinaric alps mountain complex towards the north and the mediterranean region towards the south are hotspots of endemicity. this is the meeting point of two large phytogeographic regions: the euro siberian – north american and the alpine – high nordic regions (ređić et al. 2011). moltkia petraea (tratt.) griseb. is a typical representative of an endemic plant with horticultural and medicinal potential. it is an endemic, lithophytic, xerothermic illyric– balkan species distributed along the adriatic coast in croatia, bosnia and herzegovina, montenegro, albania, and greece, mostly in mediterranean and sub-mediterranean regions, at altitudes between near sea level to 2000 m. it is a dense, dwarf shrub that grows up to 40 cm and blooms from may to july with very decorative, deep violet-blue, tubular fl owers (šilić 2005). m. petraea is a strictly protected and threatened species in croatia (anonymous 2013). among biologically active compounds, phenolic compounds have attracted a great deal of public and scientifi c interest due to their health-promoting effects as antioxidants. the content of biologically active compounds varies greatly among species, including closely related species, making them useful chemotaxonomic markers. the differences between populations of a species could very often be signifi cant (dunkić et al. 2012). these differences could be associated with the region of origin, growth phase, habitat condition, and seasonal environmental variability, which encompass biotic and abiotic factors (buchwald et al. 2015, ramegowda and senthil-kumar 2015). according to dapkevicius et al. (2002), it is possible to increase the content of pharmacologically desirable compounds through agriculture techniques, such as irrigation or using photo bioreactor systems. on the other hand, buchwald et al. (2015) did not observe a statistically signifi cant infl uence of mineral fertilization on the level of main active compounds. to date, chemical compound contents in m. petraea have only been investigated by zovko končić et al. (2010). the aim of this study was to investigate the soil traits of m. petraea habitats and to evaluate the infl uence of soil phenolic substances in moltkia petraea acta bot. croat. 75 (2), 2016 267 properties on phenolic compound accumulation. the evaluation of soil traits in native populations of m. petraea is the fi rst step towards the determination of m. petraea as a plant with possible horticultural signifi cance. materials and methods plant material samples of moltkia petraea (tratt.) griseb. were collected during the blooming period in june and july of 2011 at ten locations along croatian adriatic coast, and in the dinarides and durmitor mountain ranges of montenegro and bosnia and herzegovina (on-line suppl. fig. 1). altitude and latitude of each habitat locality were determined by a gps locator (tab. 1). voucher specimens of herbal material were deposited in the fran kušan herbarium of the department of pharmaceutical botany with fran kušan pharmaceutical botanical garden at the faculty of pharmacy and biochemistry, university of zagreb, croatia. above-ground parts of several dozen randomly selected plants were harvested from mature plants on a dry day and mixed to obtain the randomly selected sample. samples were air-dried for three weeks in a well-ventilated room at 60% relative humidity and room temperature (22 °c), single-layered and protected from direct sunlight. air-dried samples were placed in double paper bags labelled with the sample number, and stored in a dry place at room temperature (22 °c; 60% of humidity) protected from light for fi ve months until analysis. soil sampling simultaneously with the sampling of plant material, soil was also collected in situ from all localities. soil sampling was provided with a soil probe, from depths of 0–15 cm, due to the very shallow depth of the undeveloped karst soil. soil samples were prepared for chemical analyses according to the iso 11464:1994 method (pernar et al. 2013). soil analyses the ph of soil in h2o and 1 m solution of kcl were analyzed according to the iso 10390:1994 method (pernar et al. 2013), while organic matter content (%) was analyzed according to tjurin’s method (lal et al. 2002). total nitrogen (%) was analyzed according to the iso 11261:1995 method (pernar et al. 2013), total content of caco3 was analyzed according to the scheibler method (tatzber et al. 2007), and contents of physiological active phosphorus and potash, calculated in mg of p2o5 and k2o per 100 grams of soil, were analyzed according to the egner-riehm-domingo’s ammonium lactate method (page 1982). all soil analyses were performed in triplicate. phenolic compounds analyses total polyphenols and tannins contents were determined according to schneider (1976). this procedure is based on a reaction with folin-ciocalteu’s phenol reagent (fcr) and spectrophotometric determination of total polyphenols and tannins (indirectly, after precipitation with casein) at 720 nm. tannin was used as the standard substance. tab. 1. habitats of moltkia petraea and collection data of researched plant and soil samples. states and locality of sampling voucher no. latitude; longitude altitude (m) abbreviation croatia omiš hfk-hr-113-2011 42°26’17’’ n; 16°42’01’’ e 30 om vošac hfk-hr-117-2011 43°18’46’’ n; 17°03’07’’ e 1295 vo mljet hfk-hr-122-2011 42°42’35’’ n; 17°40’36’’ e 225 ml sniježnica hfk-hr-133-2011 42°34’08’’ n; 18°21’28’’ e 1152 sn bosnia and herzegovina diva grabovica hfk-hr-124-2011 43°35’59’’ n; 17°41’04’’ e 251 dg drežnica hfk-hr-125-2011 43°31’48’’ n; 17°42’22’’ e 208 dr rujišta hfk-hr-123-2011 43°27’30’’ n; 17°57’02’’ e 964 ru rakitnica hfk-hr-127-2011 43°34’39’’ n; 18°05’20’’ e 768 ra montenegro orjen hfk-hr-142-2011 42°33’45’’ n; 18°47’47’’ e 760 or lovćen hfk-hr-146-2011 42°23’44’’ n; 18°48’28’’ e 1365 lo kremer d., jurišić grubešić r., ballian d., stešević d., kosalec i., et al. 268 acta bot. croat. 75 (2), 2016 the total fl avonoid content (quercetin type) was determined using the method according to christ and müller (1960). this procedure includes hydrolysis of glycosides, extraction of total fl avonoid aglycones with ethyl acetate and complex formation with alcl3 at 425 nm. the yield was calculated as quercetin according to the following expression: total fl avonoids (%) = a × 0.772 / b a = absorbance; 0.772 = conversion factor related to specifi c absorbance of quercetin at 425 nm; b = mass of dry herbal material (g) total phenolic acids content was determined according to the monograph of rosmarini folium in european pharmacopoeia (2007). phenolic acids in the extracts were measured spectrophotometrically at 505 nm (three independent analyses), using the nitrite-molybdate reagent of arnow, in a sodium hydroxide medium, and the percent of their content, expressed as rosmarinic acid, was calculated from the expression: total phenolic acids (%) = a × 2.5 / m a = absorbance; 2.5 = conversion factor related to specifi c absorbance of rosmarinic acid at 505 nm; m = mass of the substance to be examined (g), taking the specifi c absorbance of rosmarinic acid to be 400. the contents of total polyphenols, tannins, total fl avonoids, and total phenolic acids were evaluated from three independent analyses and were expressed as the percentages of dry mass of herbal material. a uv/vis spectrophotometer agilent 8453 (agilent, germany) with pc-hp 845x uv-visible system (agilent, germany) and 1 cm quartz cells was used for all absorbance measurements. statistical analysis statistical comparisons of phenolic compound contents among investigated populations and between plant organs were conducted using one-way anova followed by scheffe’s post-hoc test at the p ≤ 0.05 level. prior to anova, data was transformed using angular (i.e. arcsin) transformation. the results were evaluated using multivariate analysis. principal component analysis (pca) calculation was based on the correlation matrix between the values of the characteristics, meaning that the contribution of each variable was independent of the range of its values. to confi rm the results of the pca, the unweighted pair-group method with arithmetic mean (upgma) with euclidean distance (de) was conducted. upgma generally yields results that are the most accurate for classifi cation purposes. interactions between soil traits and the content of different biologically active compounds were analyzed using the spearman rank order correlation matrices. prior to analysis, data were transformed using angular transformation. statistical analyses were performed using the statistica 7 software package (statsoft inc., tulsa, ok, usa). results and discussion soil properties chemical properties of soil where native populations of m. petraea grow are presented in tab. 2. soil samples measured in a 1 m solution of kcl showed a neutral to slightly alkaline reaction. soils from all sites contained a very low to intermediate amount of physiologically active phosphorus (p2o5), but were very rich in potash (k2o). soil organic matter content was very high at all localities. due to the very shallow depth of the undeveloped karst soils, the high organic matter content was related to mulch not humus (tab. 2). according to descriptive statistics, very high variability of caco3, organic matter and potash contents was obtained in soil samples from different sites, indicating that tab. 2. variability of chemical properties of soil in different habitats of moltkia petraea. al – ammonium-lactate, blq – below limit of quantifi cation. habitat (locality abbreviation) ph caco3 (%) organic matter (%) nitrogen (%) al-method (mg/100 g) h2o 1 m kcl p2o5 k2o omiš 7.78 7.33 29.93 5.04 0.41 8.50 26.61 vošac 7.70 7.32 39.84 7.89 0.64 3.25 68.14 mljet 7.68 7.25 27.10 12.85 0.48 2.79 61.36 sniježnica 7.45 6.96 2.63 19.89 0.57 3.25 45.42 diva grabovica 7.33 6.89 blq 35.69 0.68 4.68 46.10 drežnica 7.66 7.31 50.59 8.35 0.68 2.43 38.72 rujišta 7.76 7.21 34.10 4.19 0.33 1.22 26.88 rakitnica 7.04 6.59 blq 36.26 0.70 5.29 36.45 orjen 7.24 6.97 6.45 13.76 0.41 11.09 125.00 lovćen 7.71 7.17 17.97 9.67 0.40 1.55 27.81 mean 7.54 7.10 26.08 15.36 0.53 4.41 50.25 stand. dev. 0.26 0.24 16.35 11.78 0.14 3.16 29.82 var. 0.07 0.06 267.32 138.68 0.02 9.98 889.28 coef. of var. 3.39 3.40 62.70 76.67 26.37 71.70 59.35 stand. error 0.08 0.08 5.78 3.72 0.04 0.10 9.43 phenolic substances in moltkia petraea acta bot. croat. 75 (2), 2016 269 m. petraea has no specifi c demands regarding particular soil conditions. phenolic compounds the contents of total polyphenols (tp), tannins (t), total fl avonoids (tf), and total phenolic acids (tpa) in leaves, fl owers, and stems of the investigated m. petraea populations are presented in tab. 3. tp, tf and tpa contents were highest in leaves, while t content was highest in fl owers in most populations. in general, the concentrations of the analyzed bioactive compounds were lowest in stems. the smallest differences between populations were observed in tf content. it can be concluded that tf content in m. petraea is under a lesser infl uence of habitat conditions than the contents of tp, t and tpa. the results confi rmed that the content of biological active compounds in analyzed plants varied between populations. according to dunkić et al. (2012), the contents of tp, t and tf in aerial parts of the investigated populations of satureja montana l. and s. subspicata vis. (lamiaceae) revealed a statistically signifi cant within-species difference, depending on the locality and plant organ used for determination. variations between locations could be ascribed to biotic (vermin, alleopathy, diseases) and abiotic (climate, soil, fertilization) factors (young et al. 2005). the content of biologically active compounds also depends on plant age and harvesting time (kołodziej and sugier 2013). with regard to the analyzed phenolic compounds, the pca and upgma separated the investigated m. petraea populations as presented in fig. 1. the fi rst principal component (pc 1) explained 46.1% of the total variance, the second 25.9%, and the third component 13.9%. thus, the fi rst three components accounted for 85.9% of the variance, emphasizing the usefulness of the pca. the most similar populations were vo and lo, om and dg, and sn and or, respectively (fig. 1a). the population ml showed a higher tab. 3. content of total polyphenols, tannins, total fl avonoids and total phenolic acids in leaves, fl owers, and stems of moltkia petraea expressed as mean ± standard deviation of the three independent analyses. capital letters and symbols in superscript denote difference between populations for leaves (a, b, d, e, f, g, h, i, j), fl owers (k, l, m, n, o, p, r, s, t, u) and stems (v, w, x, y, z, “, γ, λ, π, σ) related to certain investigated trait. sample plant part total polyphenols (%) tannins (%) total fl avonoids (%) total phenolic acids (%) omiš leaves 4.29±0.10a 1.04±0.05a 0.42±0.00a 2.06±0.06a omiš fl owers 3.91±0.02k 1.06±0.02k 0.19±0.01k 1.72±0.02k omiš stems 3.33±0.09v 1.01±0.05v 0.04±0.01v 1.76±0.03v vošac leaves 5.78±0.09ab 1.95±0.01ab 0.44±0.00ab 3.43±0.02ab vošac fl owers 6.07±0.07kl 2.45±0.05kl 0.28±0.00kl 3.12±0.08kl vošac stems 4.44±0.03vw 1.53±0.07vw 0.15±0.00vw 2.20±0.02vw mljet leaves 5.88±0.11ad 0.39±0.09abd 0.37±0.01abd 3.00±0.02abd mljet fl owers 5.53±0.07kln 1.17±0.02lmn 0.21±0.00lmn 2.58±0.04klmn mljet stems 5.53±0.06vwxy 0.16±0.02vwxy 0.12±0.00vwxy 2.92±0.04vwxy sniježnica leaves 4.62±0.02bde 1.07±0.02bde 0.39±0.00abde 1.89±0.06abde sniježnica fl owers 4.64±0.07kmno 1.20±0.02lmo 0.18±0.00lmo 1.71±0.06lmno sniježnica stems 3.26±0.03wxyz 0.53±0.04vxyz 0.31±0.00vwxyz 1.56±0.02vwxyz diva grabovica leaves 3.97±0.02bdf 0.14±0.03abef 0.84±0.01abdef 2.01±0.05bdf diva grabovica fl owers 4.46±0.02klmnp 0.81±0.09lmnop 0.43±0.02klmnop 2.20±0.02klmnop diva grabovica stems 3.37±0.05wx” 0.88±0.05wxyz” 0.09±0.02vwxyz” 1.70±0.06wy” drežnica leaves 6.01±0.05aefg 1.93±0.03adefg 0.81±0.01abdefg 2.59±0.02abdefg drežnica fl owers 5.19±0.03klmopr 1.48±0.02klpr 0.45±0.01lmnor 2.49±0.03klmopr drežnica stems 5.26±0.00vwxz”γ 1.83±0.00vxyz”γ 0.16±0.01vyz”γ 2.15±0.09vxyz”γ rujišta leaves 6.28±0.45aefgh 3.00±0.43abdefgh 0.91±0.00abdefgh 1.98±0.06bdgh rujišta fl owers 4.30±0.01lmnrs 1.48±0.06klps 0.52±0.01klmnoprs 2.12±0.03klmnors rujišta stems 5.09±0.22vwxz”λ 2.55±0.20vwyz” γλ 0.18±0.01vyz” 1.72±0.03wyzγλ rakitnica leaves 5.31±0.28afghi 1.61±0.26dfh 1.13±0.01abdefghi 2.57±0.05abefhi rakitnica fl owers 4.65±0.21klmnrt 1.11±0.12lmprst 0.48±0.01klmnoprst 1.92±0.05klmnoprst rakitnica stems 3.87±0.15vwyz”γλπ 1.00±0.11wxyzγλπ 0.19±0.01vwyz”γ 2.20±0.05vxyz”λπ orjen leaves 5.42±0.10afgj 1.24±0.08bdfgh 0.50±0.00abdefghij 1.58±0.03abdefghij orjen fl owers 5.55±0.04klmopstu 1.51±0.02klpt 0.27±0.00kmnoprstu 2.00±0.05klmnopru orjen stems 3.88±0.04vwxz”γλς 0.30±0.02vwxz”γλπς 0.20±0.00vwyz”γ 1.50±0.02vwxy”γλπς lovćen leaves 6.61±0.04aefgij 1.71±0.02adfh 0.37±0.00abdefghij 2.53±0.03abdefhj lovćen fl owers 5.90±0.06klmoprst 1.77±0.01klnopt 0.21±0.00lmoprstu 3.26±0.03kmnoprstu lovćen stems 5.32±0.07vwxyz”πς 1.19±0.04xyzγλπς 0.19±0.00vwyz”γ 2.44±0.04vwxyz”γλπς kremer d., jurišić grubešić r., ballian d., stešević d., kosalec i., et al. 270 acta bot. croat. 75 (2), 2016 degree of separation. this population is situated on an island in the adriatic sea and it is under the stronger infl uence of the mediterranean climate than the remaining populations studied. the populations vo and lo are situated at similar altitudes on mt biokovo and mt lovćen, respectively. both mountains are near the adriatic sea and under similar climatic conditions. consequently, the geographical position of the populations vo and lo could explain the similarities obtained in the multivariate analysis. the populations sn and or are geographically close and their similarities in the multivariate analysis were expected. the similarity between the populations om and dg is more diffi cult to explain. both populations are found in canyons, om in the cetina river valley, and dg in the neretva river valley. it is possible that this environmental factor also played a signifi cant role in the accumulation of phenolic compounds. although some studies of plant species such as tanacetum cinerariifolium (trevir.) sch. bip. (grdiša et al. 2014), campanula pyramidalis l. (lakušić et al. 2013), edraianthus tenuifolius (waldst. et kit.) a. dc. (surina et al. 2011), and cardamine maritima dc. (kučera et al. 2008) showed a greater or lesser phylogeographical or taxonomical split in the area of the neretva river valley, such a split was not confi rmed here. similar results to the pca were obtained using upgma, which separated three groups of populations (fig. 1b). similar populations were vo (vošac), lo (lovćen) and ml (mljet) which formed one large group. the remaining populations formed the second group containing two subgroups. the most similar populations were sn and or, which formed a single cluster connected at a euclidean distance of 2.64. correlation between phenolic substances and chemical properties of soil spearman rank order correlations between soil reaction (ph in h2o and in 1 m kcl), nitrogen content, and potash content (expressed as content of k2o in mg per 100 grams of soil) in soils of all habitats (as independent variables) and biologically active substances (i.e. total polyphenols, tannins, total fl avonoids, and total phenolic acids in leaves, fl owers and stems as dependent variables) in all plant samples of m. petraea were not signifi cant (on-line suppl. tab. 1). however, correlations between the content of phosphorus and content of total polyphenols in leaves and stems, as well as the content of total phenolic acids in fl owers of m. petraea were strongly negative. spearman rank order correlations between the content of caco3 in soil of m. petraea habitats and the content of biologically active substances were calculated only for eight (of ten) habitats of m. petraea, due to the very low caco3 content (below minimal quantities) in soil samples from the two remaining locations. however, only one comparison of caco3 content in the soil and tannin content in the stem of m. petraea indicated a strong positive correlation. comparison of the organic matter content in soil of habitats (independent variable) and the content of biologically active substances (dependent variable) showed a moderately negative correlation in comparison with the total tannin content in the stems of m. petraea. no other comparisons gave signifi cant correlations (on-line suppl. tab. 1). the results of this study showed negative correlations between the phosphorus content in soil and tp content in leaves and stems. also, a negative correlation was found between the phosphorus content in soil and tpa content in fl owers. these results are in line with results of gerschenzon (1983) who found that soil defi ciencies in phosphorus, sulphur, iron, calcium, and magnesium stimulates the production of phenolic compounds in plant tissues. higher phenolic contents have also been reported as a response to phosphate starvation in phaseolus vulgaris l. (juszczuk et al. 2004). according to tavarini et al. (2015) nitrogen fertilization of soil in the amount of 150 kg per ha will optimize the content of total phenols and fl avonoids in leaves of stevia rebaudiana bertoloni (asteraceae). buchwald et al. (2015) showed that mineral fertilization with nitrogen, phosphorus and potassium did not substantially affect the level of phenolic acids in the raw material of rhodiola rosea l. it was also found that using bio-fertilizer signifi cantly increased total fl avonoid contents in anethum graveolens l. (apiaceae) (said-al ahl et al. 2015). accordingly, it can be concluded that m. petraea has no specifi c demands regarding particular soil conditions. two locations (sn and or) showing similar phenolic compounds levels, had quite different soil chemical properties. this fig. 1. principal component analysis (a) and the unweighted pairgroup method with arithmetic mean (upgma) (b) of the phenolic compounds content in moltkia petraea populations. om – omiš, vo – vošac, ml – mljet, sn – sniježnica, dg – diva grabovica, dr – drežnica, ru – rujišta, ra – rakitnica, or – orjen, lo – lovćen. phenolic substances in moltkia petraea acta bot. croat. 75 (2), 2016 271 suggests that biosynthesis of phenolic compounds in m. petraea is largely affected by other factors (genetic factors, topography, and exposition of plant site in relief, possible infl uence of aeolian deposits in site, or other soil properties) rather than the investigated soil properties. however, due to the very nice habitus (particularly because of the shape and colour of fl owers) and low soil and water demands, m. petraea may be considered a potentially valuable horticultural plant in landscape architecture. acknowledgements these researches were supported by the ministry of science, education and sports of the republic of croatia (project grant no. 006–0000000–3178). authors would like to thank to dr valentina papić bogadi and linda zanella, msc for helpful comments on the manuscript and for correcting the english style. references anonymous, 2013: ordinance of strictly protected species (in croatian). narodne novine 144, 7–84. buchwald, w., mordalski, r., kucharski, w. a., gryszczyńska, a., adamczak, a., 2015: effect of fertilization on roseroot (rhodiola rosea l.) yield and content of active compounds. acta scientiarum polonorum hortorum cultus 14, 109–121. christ, b., müller, k. h., 1960: zur serienmäßigen bestimmung des gehaltes an fl avanol-derivaten in drogen. archiv der pharmazie 293, 1033–1042. dapkevicius, a., van beek, t. a., lelyveld, g. p., van veldhuizen, a., de groot, a., linssen, j. p., venskutonis, r., 2002: isolation and structure elucidation of radical scavengers from thymus vulgaris leaves. journal of natural products 65, 892–896. dunkić, v., kremer, d., dragojević müller, i., stabentheiner, e., kuzmić, s., jurišić grubešić, r., vujić, l., kosalec, i., randić, m., srečec, s., bezić, n., 2012: chemotaxonomic and micromorphological traits of satureja montana l. and s. subspicata vis. 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acta bot. croat. 74 (1), 2015 71 acta bot. croat. 74 (1), 71–94, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 carbon gain optimization in fi ve broadleaf deciduous trees in response to light variation within the crown: correlations among morphological, anatomical and physiological leaf traits rosangela catoni1*, loretta gratani1, francesco sartori2, laura varone1, mirko u. granata2 1 sapienza university of rome, department of environmental biology, p. le a. moro, 5, 00185 rome, italy 2 university of pavia, department of earth and environmental sciences, via s. epifanio 14, 27100 pavia, italy abstract – leaf trait variations in fi ve deciduous species (quercus robur, corylus avellana, populus alba, acer campestre, robinia pseudoacacia) growing in an old broadleaf deciduous forest in response to light variation within the tree crown was analyzed. net photosynthetic rate (pn), leaf respiration rate (r) and the photosynthetic nitrogen use effi ciency were, on average, more than 100% higher in sun than in shade leaves. a. campestre and c. avellana sun leaves had the highest specifi c leaf area (sla, 156.0 ± 17.9 cm2 g–1) and the lowest total leaf thickness (l, 101.9 ± 8.8 μm) underlining their shade-tolerance. among the shade-intolerant species (q. robur, p. alba and r. pseudoacacia), q. robur had the lowest sla and the highest l in sun leaves (130.6 ± 10.0 cm2 g–1 and 160.8 ± 9.6 μm, respectively) since shade-intolerant species typically have thicker leaves. the higher pn decrease in respect to r decrease from sun to shade leaves attested the higher sensitivity of pn than r to light variations within the crown. this determined a 69% lower r/pn in sun than in shade leaves. this result is further attested by the signifi cant correlation between pn and the relative photosynthetic photon fl ux density. the shade-tolerant species have a 76% higher r/pn ratio than the shade-intolerant ones. the measured leaf phenotypic plasticity (pi = 0.35) was in the range of broadleaf deciduous species. plasticity is a key trait useful to quantify plant response to environmental stimuli. it is defined as the ability of a genotype to produce different phenotypes depending on the environment. among the considered species, q. robur showed the highest pi (0.39) and p. alba the lowest (0.29). knowledge on phenotypic plasticity is important in making hypotheses about the dynamics of the studied forest in consideration of environmental stress factors, including invasive species competition and global climate change. keywords: deciduous trees, forest, gas exchange, light gradient, shade tolerance, specifi c leaf area * corresponding author, e-mail: rosangela.catoni@uniroma1.it copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. catoni r., gratani l., sartori f., varone l., granata m. u. 72 acta bot. croat. 74 (1), 2015 abbreviations: pn – net photosynthesis; gs – stomatal diffusive conductance to water vapour; e – transpiration rates; r – leaf respiration rates; r/ pn – ratio of leaf respiration to photosynthesis; pnue – photosynthetic nitrogen use; na – leaf nitrogen content per unit of leaf area; chl a + b – chlorophyll a + b; chl/car – ratio of chlorophyll to carotenoid content; chl a/b – ratio of chlorophyll a to chlorophyll b content; chl/n – ratio of chlorophyll to nitrogen content; la – projected leaf surface area; dm – leaf dry mass; sla – specific leaf area. introduction in forest ecosystems, the heterogeneous light environment within a tree crown due to self-shading and shading by neighboring trees (sack et al. 2006, wyka et al. 2012) determines leaf trait variations (ellsworth and reich 1993, gratani 1997, gratani and foti 1998, gratani et al. 2006, yoshimura 2010). large trees support leaves acclimated to high light intensities (sun leaves) in the upper canopy and may produce leaves acclimated to low light intensities (shade leaves) in lower canopy layers (hölscher 2004). the range of variations refl ects the optimization of whole plant gas exchange and the resource investment strategy (gratani 1997, wyka et al. 2012). most studies of plant response to light have been aimed at underlining the ecological implications of the tolerance to extremes (i.e. tolerance to sun and shade), but noticeably less effort has been invested in the exploration of trends in the plastic response to light (valladares et al. 2000). sun leaves with respect to shade leaves generally exhibit a higher photosynthetic rate on a leaf area basis, a higher chl a to chl b ratio, a lower light-harvesting chl a/b protein (lhcp), a lower stacking degree of thylakoids (lichtenthaler et al. 1982), and a higher nitrogen (n) content per unit of leaf area (hikosaka 2005) since approximately half of n is invested in photosynthetic proteins (evans 1989). hirose and werger (1987) suggest that n varies with light availability in the plant crown in such a way as to optimize daily crown photosynthesis. differentiation in n distribution patterns in sun and shade leaves within the crown increases the crown photosynthetic nitrogen use effi ciency (pnue) (hikosaka 2003). pnue underlines the effi ciency with which species utilize n to grow (garnier et al. 1995). there is a strong relationship between photosynthesis and respiration (r) as respiration relies on photosynthetic substrates (atkin et al. 2007). plant respiration accounts for a large fraction of carbon cycling in forest ecosystems and may be of comparable importance to photosynthesis as a determinant of net primary productivity (ryan et al. 1997). in general, upper-canopy leaves had much higher leaf respiration than lower-canopy leaves, refl ecting a greater metabolic activity related to a higher light availability and thus, higher net photosynthetic rates (gunderson et al. 2002). at a morphological level, the specifi c leaf area (sla) varies vertically in the forest canopy (niinemets 1995) altering the amount of light that can be intercepted per unit of leaf dry mass (evans and poorter 2001). in general, plants growing in high light conditions have thicker leaves with a lower sla than plants growing in shade conditions (björkman 1981) partially due to longer or extra layers of palisade cells (hanson 1917). species can be classifi ed into obligate or facultative shade plants and obligate or facultative sun plants (damascos and rapoport 2002). however, comparative studies indicate that there are few species that are either extremely shade-tolerant or light-demanding, with most species having intermediate and thus, overlapping light preferences (wright et al. 2003). one of the components of shade tolerance resides in the ability of individuals to opcarbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 73 timize carbon gain under low light environments, by maximizing light interception and minimizing carbon loss by respiration (walters and reich 1999), according to the carbon gain hypothesis (valladares and niinemtes 2008). light competition and inter-specific differences in shade tolerance are frequently important determinants of forest structure and dynamics (gravel et al. 2010). nevertheless, the potential correlation between plasticity of morphological and physiological leaf traits and the tolerance to shade is still under discussion (robakowski et al. 2003, portsmuth and niinemets 2007). a frequent assumption in literature is that shade-intolerant species display a larger plasticity than shade-tolerant ones (strauss de benedetti and bazzaz 1991), although the reverse is sometimes found. other authors have observed a large variability in plasticity that depended on the studied traits with no real connection to shade tolerance (valladares et al. 2000). the main objective of this research was to analyze leaf trait variations in response to the light gradient within the crown of fi ve tree species growing in an old broadleaf deciduous forest characterized by different tolerance to shade. intra-canopy plasticity has important impacts on many aspects of tree biology, potentially contributing to whole-crown performance via effects on light penetration through the crown and on the energy, carbon, and water balance of individual leaves (hikosaka 2005). considering that information about the correlation between plasticity and shade tolerance is limited and the evidence is contrasting (portsmuth and niinemets 2007), we tested the relationship between leaf trait plasticity and shade tolerance of the selected trees, assuming that shade-tolerant tree species might have a lower plasticity index than the shade-intolerants. material and methods the study was carried out in the period may – july 2012 in an old broadleaf deciduous forest developing in the natural reserve siro negri (45°12'39"n; 09°03'26"e, 74 m a. s. l, italy) extending over 10 ha. the forest largely covered the fl uvial valleys along the ticino river from the 19th century, and no logging has been carried out since the establishment of the reserve in 1970 (sartori 1984, castagneri et. al. 2013). the reserve represents one of the best conserved relicts of the original alluvial forest which in the past largely covered the banks of the ticino river, and contains trees that are more than 100-years old (castagneri et al. 2013). the reserve has been reported as part of a sites of community importance (sic) it 2080014 »bosco siro negri e moriano«. the following broadleaf species were considered for measurements: acer campestre l., corylus avellana l., populus alba l., robinia pseudoacacia l. and quercus robur l. c. avellana is a shade-tolerant species (kull and niinemets 1993, gratani and foti 1998) growing in the understory of european deciduous forests (tutin 2001) and able to colonize large gaps (kull and niinemets 1993). a. campestre is a shade-tolerant species growing in the understory of deciduous mixed forests in europe (mills 1996). p. alba is a shade-intolerant species (cooper et al. 1999) with a great geographic distribution, including the centre and south of europe, north africa, western and central asia (fao 1980) where it grows on the banks of rivers (delledonne et al. 2001). r. pseudoacacia is a shade-intolerant species (motta et al. 2009) originating from the south-eastern united states (ferraris et al. 2000). the dispersion of the r. pseudoacacia into italian native forests started in catoni r., gratani l., sartori f., varone l., granata m. u. 74 acta bot. croat. 74 (1), 2015 the early 20th century (motta et al. 2009). it is mainly a threat for nutrient-poor sites based on its ability to fix nitrogen by symbiosis (roloff et al. 1994). this species can cause an unwanted and long-lasting shift in vegetation composition toward nitrogen rich and species-poor plant communities (kowarik 2010). q. robur is a predominant european oak species (scotti-saintagne et al. 2004) growing in more open habitats; it is a more light–demanding species (niinemets 1996, valladares et al. 2002). meteorological data the climate of the area was characterized by a mean annual rainfall of 654 mm, most of it falling in spring and autumn. the mean minimum air temperature (tmin) of the coldest month (january) was –0.2 ± 1.8 °c, the mean maximum air temperature (tmax) of the hottest month (july) was 30.1 ± 1.3 °c and the mean annual temperature (tm) was 13.7 ± 8.2 °c. in the period may – july, total rainfall was 136 mm, tm 22.2 ± 3.1 °c and tmax (july) 30.3 ± 1.2 °c (lombardia regional agency for environmental protection, meteorological station of pavia, ponte ticino ss35, data for the period 2002 to 2012). floods occurred sporadically every 5–10 years during the last 40 years, with water levels up to 1.50 m height in the forest during exceptional events (motta et al. 2009, castagneri et al. 2013). on average, groundwater level was around –4.50 m in winter reaching –3.50 m in summer due to irrigation in the surrounding areas (sartori, unpublished). forest measurements and microclimate measurements of forest structure were carried out in 10 representative sample areas (400 m2 each) randomly selected in the considered forest. plant traits included: plant height (h) and the diameter at breast height (d). total forest density and total tree basal area were calculated. leaf area index (lai) was estimated at the end of june by the »lai2000 plant canopy analyzer« (li-cor inc., lincoln, nebraska, usa). microclimate and leaf trait measurements were made by an aerial lift for the selected species (four representative mature plants per species) at the top (sun leaves) and at the bottom (shade leaves) of the crown of each considered tree, according to sack et al. (2006). the photosynthetic photon fl ux density (ppfd, μmol photons m–2 s–1) was measured at the top and at the bottom of the crown for each sampled tree by a quantum radiometer photometer (li-189 li-cor, usa) with the quantum sensor li-190sa. simultaneously, ppfd was determined in the open near the forest, in order to provide references irradiance level for calculation of relative irradiance (ppfd%), according to wyka et al. (2012). measurements were carried out in june and july on overcast days from 09.00 a. m. to 12.00 p. m to provide a reliable estimate of the average light conditions during the »in-leaf« growing season, according to tobin and reich (2009). air humidity (rh, %) and air temperature (ta, °c) were measured by thermo-hygrometers (hd8901, delta ohm, it). leaf morphology and anatomy fully expanded leaves (n = 20 per species and per crown position) were collected from the selected trees at the end of june, sealed in plastic bags and transported immediately to the laboratory for measurements. measurements included leaf surface area (la, cm2), obtained by the image analysis system (delta-t devices, uk), and leaf dry mass (dm, mg), carbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 75 determined by drying leaves at 80 °c to constant mass. specifi c leaf area (sla, cm2 g−1) was calculated by the ratio of la and dm. fresh leaf sections from fully expanded leaves (n = 10 per species and per crown position) were hand cut and analyzed by light microscopy using an image analysis system (arkon, a&p, i). the following parameters were measured: total leaf thickness (l, μm), palisade and spongy parenchyma thickness, adaxial and abaxial epidermis and cuticle thickness. all measurements were restricted to vein-free areas. gas exchange gas exchange measurements were carried out in june – july (fi ve leaves per species per crown position per each sampling occasion), from 09:00 a. m. to 12:00 p. m. to ensure that the maximum rates were measured (reich et al. 1995). leaves were retained in their natural position during measurements. net photosynthetic rate (pn, μmol co2 m−2 s−1), stomatal conductance to water vapor (gs, mol h2o m−2 s−1), leaf transpiration rate (e, mmol h2o m−2 s−1), photosynthetic photon fl ux density (ppfd, μmol photons m−2 s−1) and leaf temperature (tl, °c) were measured by an infrared gas analyzer (lc-pro+, adc, uk) equipped with a leaf chamber (plc, parkinson leaf chamber, uk). on each sampling occasion, respiration rates (r, μmol co2 m−2 s−1) were measured after pn ones (on the same leaves) as co2 effl ux, by darkening the leaf chamber with a black paper, according to cai et al. (2005) for 30 min prior to each measurement, to avoid the release of co2 transient post-irradiation bursts (atkin et al. 1998). the shown r and pn rates represented the mean values of three days of measurements per month characterized by the same weather conditions, under clear sky. the ratio between r and pn was calculated. leaf nitrogen and pigment content leaf samples were collected on the same occasions as those for gas exchange measurements. immediately after collection leaf samples were kept cool in the dark and transported immediately to the laboratory. leaf nitrogen content per leaf area (na, g m–2) was determined by drying leaf samples at 70 °c (6 leaf samples, 0.5 g of leaf dry mass each, per species and per crown position in each sampling occasion) and grinding them into a fi ne powder. the n content was measured by the kjeldahl method (mendes et al. 2001). photosynthetic nitrogen use effi ciency (pnue, μmol co2 g−1 na s−1) was calculated by the ratio between pn rates and na content. chlorophyll content (chl, mg g−1 fresh weight) and carotenoid content (car, mg g−1 fresh weight) were determined after grinding leaves in acetone (6 samples, 1.5 g of leaf fresh weight each, per species and per crown position in each sampling occasion). the homogenates were centrifuged in a refrigerated centrifuge (4237r. a. l. c., i). absorbance of the supernatants was measured by a jasco model 7800lcd (japan) spectrophotometer at the wavelengths of 645, 663, and 440 nm. chl content was calculated according to maclachlan and zalik (1963) and car content according to holm (1954). the chl a + b content, the ratio chl a/b, the ratio chl/car, and the ratio chl/n were calculated. catoni r., gratani l., sartori f., varone l., granata m. u. 76 acta bot. croat. 74 (1), 2015 plasticity index the plasticity index of morphological (pim), anatomical (pia) and physiological (pip) leaf traits for each of the species was calculated by the difference between the minimum and the maximum mean value between sun and shade leaves divided by the maximum mean value, according to valladares et al. (2000). the mean plasticity index (pi), which had a scale ranging from 0 to 1, was calculated by averaging pim, pia and pip per species, according to valladares et al. (2000). statistics all statistical tests were performed using a statistical software package (statistica, statsoft, usa). differences in the considered leaf traits were determined by the analysis of variance (anova) and the tukey test for multiple comparisons. kolmogorov–smirnov and levene tests were used to verify the assumption of normality and homogeneity of variances, respectively. regression analysis was carried out to examine the relationship among the considered leaf traits. principal component analysis (pca) was carried out in order to detect structure in the correlations between the considered leaf traits (pn, gs, e, r, r/pn, na, pnue, chl/n, chl a+b, chl a/b, chl /car, sla, leaf thickness, palisade parenchyma thickness, spongy parenchyma thickness, adaxial epidermis thickness, adaxial cuticle thickness, abaxial epidermis thickness, abaxial cuticle thickness). the analysis was performed on a standardized matrix. the matrix was subjected to a rotated principal component analysis with the objective of summarizing the main factors determining the variation of the analyzed leaf traits. in order to evaluate the similarity among the species in terms of phenotypic plasticity a correspondence analysis (ca) was carried out. the analysis was performed using the species as group variable and pi as category variable. in particular, three pi categories were considered: physiological, morphological and anatomical pi. results forest structure and microclimate the forest structure was characterized by a dominant tree layer consisting of p. alba, populus nigra l., q. robur and r. pseudoacacia, (h = 30 ± 3 m), a dominated tree layer of r. pseudoacacia, a. campestre and c. avellana (h = 20 ± 3 m), two shrub layers of sambucus nigra l., ulmus minor mill. and c. avellana (h = 6.0 ± 1.5 m and 1.5 ± 0.5 m, respectively) and a grass layer (tab. 1). total tree density was 237 ± 100 stems ha–1 and the total basal area 74.5 ± 24.6 m2 ha–1. lai was 4.5 ± 0.3. on average, ppfd% ranged from 6.1 ± 3.5% at the bottom to 97.4 ± 1.1% at the top of the crown. the ta and rh ranged from 28.1 ± 0.5 °c and 48.0 ± 0.9%, respectively, at the bottom, to 30.4 ± 0.6 °c and 35.1 ± 2. 1%, respectively, at the top of the crown of the trees considered. carbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 77 leaf morphology there were signifi cant differences between sun and shade leaves among the species (tab. 2). on average, sla was 81% higher in shade than in sun leaves. in particular, c. avellana had the highest sla both in sun and in shade leaves (168.8 ± 15.1 cm2 g–1 and 400.7 ± 60.0 cm2 g–1, respectively), q. robur the lowest sla in sun leaves (130.6 ± 10.0 cm2g–1) and p. alba the lowest sla in shade leaves (168.4 ± 16.8 cm2 g–1). leaf anatomy on average, l and palisade parenchyma thickness were 23% and 38% higher in sun than in shade leaves, respectively, and spongy parenchyma thickness was 17% higher in sun than in shade leaves (tab. 3). in particular, q. robur had the highest l thickness in sun leaves (160.8 ± 9.6 μm), p. alba in shade leaves (128.9 ± 4.7 μm) and c. avellana the lowest l thickness both in sun and in shade leaves (95.7 ± 10.1 μm and 85.3 ± 5.3 μm, respectively). the ratio between palisade parenchyma thickness and l in sun leaves was the highest in r. pseudoacacia (55%) and the lowest in c. avellana (33%), while in shade leaves it was the highest in p. alba (53%) and the lowest in c. avellana (30%). q. robur had the highest spongy parenchyma thickness, the highest adaxial and abaxial epidermis thickness and cuticle thickness both in sun and in shade leaves, and c. avellana the lowest palisade parenchyma thickness, adaxial and abaxial epidermis thickness and cuticle thickness both in sun and in shade leaves. tab. 1. structural traits of the considered species; h – plant height, d – diameter at breast height. species h (m) d (cm) acer campestre 15 ± 6 33.0 ± 7.9 corylus avellana 8 ± 2 12.3 ± 3.6 populus alba 28 ± 5 79.5 ± 26.8 robinia pseudoacacia 20 ± 6 27.2 ± 8.8 quercus robur 26 ± 5 75.3 ± 8.0 tab. 2. morphological leaf traits of sun and shade leaves of acer campestre, corylus avellana, populus alba, quercus robur and robinia pseudoacacia. mean values (± sd) are shown (n = 20). mean values with the same letters are not significantly different between sun and shade leaves (tukey test, p ≥ 0.05). la – projected leaf surface area; dm – leaf dry mass; sla – specific leaf area. a. campestre c. avellana p. alba q. robur r. pseudoacacia sun shade sun shade sun shade sun shade sun shade la (cm2) 20.2± 4.2a 57.7± 7.7b 31.0± 9.6a 88.5± 17.8b 14.8± 3.2a 26.8± 2.1b 19.8± 3.0a 50.6± 11.1b 130.1± 22.5a 470.4± 73.8b dm (mg) 157.4± 41.4a 198.4± 31.2b 190.8± 57.5a 227.2± 62.8b 109.6± 24.4a 161± 22.2b 150.9± 33.7a 263.4± 59.4b 932± 168a 1838± 376b sla (cm2 g–1) 143.2± 15.1a 305.1± 35.3b 168.8± 15.1a 400.7± 60.0b 135.6± 7.3a 168.4± 16.8b 130.6± 10.0a 192.7± 10.2b 139.9± 4.8a 258.3± 15.4b catoni r., gratani l., sartori f., varone l., granata m. u. 78 acta bot. croat. 74 (1), 2015 gas exchange on average, pn was more than twice as high in sun leaves than in shade leaves. pn ranged between 20.3 ± 0.3 μmol m–2 s–1 (p. alba) and 5.8 ± 0.1 μmol m–2 s–1 (c. avellana) in sun leaves, and between 3.6 ± 0.3 μmol m–2 s–1 (p. alba) and 1.8 ± 0.2 μmol m–2 s–1 (a. campestre) in shade leaves (tab. 4). the gs had the same pn trend with the highest rates in sun leaves (0.20 ± 0.07 mol m–2 s–1) decreasing, on average, by 49% in shade leaves. in particular, p. alba had the highest gs in sun leaves (0.27 ± 0.06 mol m–2 s–1) and c. avellana the lowest one (0.08 ± 0.01 mol m–2 s–1). r. pseudoacacia showed the highest gs in shade leaves (0.19 ± 0.02 mol m–2 s–1) and c. avellana the lowest (0.02 ± 0.01 mol m–2 s–1). there was a signifi cant (p < 0.01) positive correlation between pn and gs and between pn and palisade parenchyma thickness, showing that 46% and 63% of pn variations were explained by the two parameters, respectively. the highest r were monitored in sun leaves (1.10 ± 0.33 μmol m–2 s–1) decreasing by 36% in shade leaves (tab. 4). among the considered species, r. pseudacacia had the highest r both in sun and in shade leaves (1.33 ± 0.23 and 1.18 ± 0.06 μmol m–2 s–1, respectively), c. avellana had the lowest r in sun leaves (0.60 ± 0.07 μmol m–2 s–1) and q. robur in shade leaves (0.21 ± 0.06 μmol m–2 s–1). the ratio r/pn was the highest in shade leaves (more than 100%), a. campestre having the highest r/pn ratio both in sun and in shade leaves (0.17 ± 0.01 and 0.63 ± 0.18, respectively). tab. 3. anatomical leaf traits of sun and shade leaves of acer campestre, corylus avellana, populus alba, quercus robur and robinia pseudoacacia. mean values (± sd) are shown (n = 10). mean values with the same letters are not significantly different between sun and shade leaves (tukey test, p ≥ 0.05). leaf traits a. campestre c. avellana p. alba q. robur r. pseudoacacia sun shade sun shade sun shade sun shade sun shade leaf thickness (μm) 108.2± 6.7a 93.2± 1.4b 95.7± 10.1a 85.3± 5.3a 142.6± 8.3a 128.9± 4.7b 160.8± 9.6a 120.8± 5.9b 146.3± 12.0a 101.9± 2.5b palisade parenchyma thickness (μm) 42.0± 2.4a 39.6± 0.9a 31.9± 7.5a 25.6± 5.4a 75.3± 2.4a 68.4± 5.0b 60.7± 3.9a 37.3± 3.5b 80.2± 9.7a 43.4± 5.1b spongy parenchyma thickness (μm) 43.2± 5.5a 34.2± 2.5b 46.0± 8.5a 44.4± 4.6a 46.7± 5.3a 42.3± 6.7a 71.7± 4.9a 56.9± 5.0b 51.2± 11.2a 42.6± 4.6a adaxial epidermis thickness (μm) 12.4± 1.6a 10.5± 2.8a 9.2± 0.7a 7.6± 2.1a 11.2± 1.7a 10.7± 0.8a 15.2± 1.6a 14.5± 3.0a 8.2± 1.1a 7.9± 1.4a adaxial cuticle thickness (μm) 1.0± 0.2a 0.9± 0.1a 0.9± 0.2a 0.8± 0.1a 1.1± 0.3a 1.0± 0.3a 1.3± 0.4a 1.1± 0.4a 1.0± 0.3a 0.9± 0.2a abaxial epidermis thickness (μm) 8.7± 1.6a 7.4± 0.9a 7.0± 1.3a 5.7± 0.9a 8.0± 1.9a 5.7± 0.6a 10.9± 2.1a 10.3± 1.4a 7.0± 1.3a 6.2± 0.8a abaxial cuticle thickness (μm) 0.9± 0.1a 0.8± 0.1a 0.7± 0.1a 0.6± 0.1a 0.8± 0.2a 0.7± 0.1a 1.0± 0.4a 0.9± 0.2a 0.9± 0.1a 0.8± 0.1a carbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 79 leaf nitrogen content and pigment content the na was, on an average, 54% higher in sun than in shade leaves, r. pseudoacacia having the highest na in sun leaves (2.51 ± 0.08 g m–2) and a. campestre the lowest one (0.91 ± 0.11 g m–2). p. alba had the highest na in shade leaves (1.56 ± 0.04 g m–2). pnue was 8.4 ± 3.0 μmol g–1 s–1 in sun leaves, decreasing by 68% in shade leaves, p. alba having the highest value in sun leaves (12.7 ± 0.2 μmol g–1 s–1) and c. avellana the lowest one (4.2 ± 0.2 μmol g–1 s–1). the chl a + b was 82% higher in shade than in sun leaves. among the considered species, q. robur and p. alba had the highest chl a + b in sun leaves (1.61 ± 0.26 mg g–1) and c. avellana the lowest both in sun and in shade leaves (0.76 ± 0.10 and 1.14 ± 0.20 mg g–1, respectively). a. campestre had the highest chl a + b in shade leaves (3.61 ± 0.39 mg g–1). the ratio chl a/b was 8% higher in sun than in shade leaves, p. alba having the highest ratio both in sun and in shade leaves (2.80 ± 0.03 and 2.72 ± 0.27, respectively), r. pseudoacacia the lowest ratio in sun leaves (2.02 ± 0.03) and q. robur in shade leaves (1.85 ± 0.04) (fig. 1a). the ratios chl/car and chl/n were 32% and 44% higher in shade than in sun leaves, respectively. in particular, among the species considered, q. robur had the highest chl/car in sun leaves (7.84 ± 0.45) and a. campestre in shade leaves (9.84 ± 0.87). the ratio chl/n was the highest in a. campestre both in sun and in shade leaves (0.102 ± 0.010 and 0.174 ± 0.014, respectively) (figs. 1b and 1c). plasticity index the pi for the considered species was, on average, 0.35, q. robur having the highest pi (0.39), followed by r. pseudoacacia (0.38), a. campestre (0.34), c. avellana (0.33) and p. alba (0.29) (tab. 5). pim was, on average, 0.45, r. pseudoacacia showing the highest value (0.56) and p. alba the lowest (0.32). among the considered leaf morphological traits, la had the largest variation (pila = 0.62, mean value). the pia was, on average, 0.15, r. pseudoacacia showing the highest (0.18) and p. alba the lowest value (0.12). among the considered anatomical leaf traits, palisade parenchyma thickness had the largest variation (0.24, mean value). the pip was, on an average, 0.44, q. robur having the highest value (0.53). among the considered physiological traits, pn had the largest variation (0.79, mean value). tab. 4. values of photosynthesis and respiration rates of sun and shade leaves of acer campestre, corylus avellana, populus alba, quercus robur and robinia pseudoacacia. mean values (± sd) are shown (n = 15). mean values with the same letters are not significantly different between sun and shade leaves (tukey test, p ≥ 0.05). pn – net photosynthesis; r – leaf respiration rate. a. campestre c. avellana p. alba q. robur r. pseudoacacia sun shade sun shade sun shade sun shade sun shade pn (μmol m–2 s–1) 7.5± 0.2a 1.8± 0.5b 5.8± 0.1a 1.9± 0.6b 20.3± 0.3a 3.6± 0.3b 16.2± 0.3a 2.0± 0.1b 19.5± 1.5a 2.6± 0.2b r (μmol m–2 s–1) 1.30± 0.09a 1.12± 0.07b 0.60± 0.07a 0.53± 0.07b 1.32± 0.36a 0.48± 0.05b 0.92± 0.32a 0.21± 0.06b 1.33± 0.23a 1.18± 0.06a catoni r., gratani l., sartori f., varone l., granata m. u. 80 acta bot. croat. 74 (1), 2015 a a b a a a a b a a a. campestre c. avellana p. alba q. robur r. pseudoacacia 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 c h l a / b a) ab ab bc b a b a ab b a a. campestre c. avellana p. alba q. robur r. pseudoacacia 0 2 4 6 8 10 12 c h l / c a r b) c a b b a c a b b a a. campestre c. avellana p. alba q. robur r. pseudoacacia 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 c h l / n c) fig. 1. a) ratio of chlorophyll a to chlorophyll b (chl a/b), b) ratio of chlorophyll to carotenoid content (chl /car) and c) ratio of chlorophyll to nitrogen content (chl/n) of acer campestre, corylus avellana, populus alba, quercus robur and robinia pseudoacacia. lowercase and capital letters indicate the inter-specifi c differences in sun (white bars) and shade (grey bars) leaves, respectively. the means with the same letters are not signifi cantly different (anova, p ≥ 0.05). mean values (± sd) are shown (n = 36). carbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 81 regression and multivariate analysis there was a signifi cant (p < 0.01) positive correlation between pn and na and a signifi cant (p < 0.01) negative correlation between sla and l, 52% of pn variations being explained by na, and 51% of sla variation by l. the regression analysis between pn and ppfd% showed a high correlation coeffi cient between these two variables (r = 0.81). tab. 5. phenotypic plasticity index for physiological (pip), morphological (pim) and anatomical (pia) leaf traits of acer campestre, corylus avellana, populus alba, quercus robur and robinia pseudoacacia. pn – net photosynthesis, gs – stomatal diffusive conductance to water vapour, e – transpiration rates, r – leaf respiration rates, r/ pn – ratio of leaf respiration to photosynthesis, pnue – photosynthetic nitrogen use, na – leaf nitrogen content per unit of leaf area, chl a + b – chlorophyll a + b content, chl/car – ratio of chlorophyll to carotenoid content, chl a/b – ratio of chlorophyll a to chlorophyll b content, chl/n – ratio of chlorophyll to nitrogen content, la – projected leaf surface area, dm – leaf dry mass, sla – specific leaf area, pi – mean plasticity index. plasticity index a. campestre c. avellana p. alba q. robur r. pseudoacacia physiological traits pn 0.76 0.65 0.82 0.87 0.86 gs 0.29 0.75 0.63 0.51 0.09 e 0.11 0.73 0.74 0.65 0.60 r 0.14 0.12 0.64 0.77 0.11 r/pn 0.73 0.66 0.50 0.67 0.87 pnue 0.68 0.27 0.82 0.90 0.75 na 0.26 0.53 0.03 0.24 0.46 chl a+b 0.66 0.33 0.28 0.53 0.16 chl/car 0.47 0.08 0.19 0.09 0.24 chl a/b 0.02 0.03 0.03 0.21 0.07 chl/n 0.41 0.25 0.13 0.44 0.17 mean value pip 0.41 0.40 0.44 0.53 0.40 morphological traits la 0.65 0.65 0.45 0.61 0.72 dm 0.21 0.16 0.32 0.43 0.49 sla 0.53 0.58 0.19 0.32 0.46 mean value pim 0.46 0.46 0.32 0.45 0.56 anatomical traits leaf thickness 0.14 0.11 0.10 0.25 0.30 palisade parenchyma thickness 0.06 0.20 0.09 0.39 0.46 spongy parenchyma thickness 0.21 0.03 0.09 0.21 0.17 adaxial cuticle thickness 0.10 0.11 0.09 0.15 0.10 abaxial cuticle thickness 0.11 0.14 0.12 0.10 0.11 adaxial epidermis thickness 0.15 0.17 0.04 0.04 0.04 abaxial epidermis thickness 0.15 0.18 0.28 0.06 0.11 mean value pia 0.13 0.13 0.12 0.17 0.18 pi 0.34 0.33 0.29 0.39 0.38 catoni r., gratani l., sartori f., varone l., granata m. u. 82 acta bot. croat. 74 (1), 2015 pca extracted two factors which accounted for 56.5% of the total variance and of which 38.8% was due to factor 1 and 17.8% to factor 2 (fig. 2). the analysis showed a colinearity among the considered leaf traits. factor 1 was signifi cantly correlated to pn (r = 0.92), total leaf thickness (r = 0.88), e (r = 0.86), sla (r = –0.84), pnue (r = 0.82), na (r = 0.78), palisade parenchyma thickness (r = 0.77) and r/pn (r = – 0.71). factor 2 was signifi cantly correlated to chl a + b (r = 0.77), adaxial epidermis thickness (r = 0.73) and chl/na (r = 0.73). along the factor 1, which explained most of the total variance, sun leaves were completely separated from shade leaves. among the considered species, a. campestre and c. avellana sun leaves were closer to shade leaf behavior and p. alba shade leaves were closer to sun leaf behavior. the ca showed a greater similarity between p. alba and q. robur because of the similar pip while a. campestre and c. avellana were closer on the base of pim. r. pseudoacia was further from the other species (fig. 3). discussion light gradient within the crown in a forest ecosystem depends on the structure and stage of development of trees (larcher 2003). moreover, the distribution, size and orientation of leaves control such processes as leaf development, leaf water use, and photosynthesis (norman and campbell 1989). the relationship between canopy structure and the spatial distribution of light availability differs between primary and secondary growth forests (nicotra et al. 1999) and light availability in the understory is frequently associated with regeneration processes and the long-term survival of forest tree species (woods 2000). -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 -2 -1 0 1 2 fa ct or 2 factor 1 a. campestre sun a. campestre shade c. avellana sun c. avellana shade p. alba shade r. pseudoacacia sun r. pseudoacacia shade q. robur sun q. robur shade p. alba sun fig. 2. principal component analysis (pca) carried out using the physiological, morphological and anatomical leaf traits. factor 1 and factor 2 accounts for 38.8% and 17.8% of the total variance, respectively. open symbols for each species indicate sun leaves and dark symbol indicate shade leaves. carbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 83 light response of broadleaf deciduous species is of particular interest because regeneration, either natural or via silvicultural practices, is infl uenced by their shade tolerance (welander and ottosson 2000). the studied forest is characterized by a tree density of 237 ± 100 stem ha–1 and a lai of 4.5 ± 0.3 which is within the range of broadleaf deciduous forests in italy (schirone et al. 1985, piccoli and borelli 1988, gratani and foti 1998, gratani and crescente 2000). lai is an important variable for characterizing vegetation structure and functioning including estimation of plant productivity and canopy cover density (garrigues et al. 2008). lai drives both the withinand the below-canopy microclimate since lai and ppfd absorption are closely related parameters (kull et al. 1999; porte et al. 2000). any change in forest lai by frost, storm, defoliation, drought and management practices is accompanied by modifi cations of plant productivity (bréda 2003). our results show that the ppfd% through the considered tree crown decreases, on average, by 94% from the top to the bottom involving a large number of leaf trait variation as confi rmed by the pca that shows a complete separation between sun and shade leaves. in particular, sla is 81% higher in shade than in sun leaves (mean value of the considered species), according to the results of witkowski and lamont (1991), gratani (1997) and gratani et al. (2006). the higher sla of shade leaves is mostly the result of a decreased total leaf thickness, as attested by the signifi cant (p < 0.01) negative correlation between sla and l. in fact, the larger and thinner shade leaves are more advantageous for light capture in low light conditions and, in general, species which grow in shade conditions are characterized by a larger sla and a lower l than those growing in more open areas (gratani and foti 1998). -0.04 -0.02 0 0.02 0.04 0.06 0.08 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 a xi s 2 axis 1pip pim r. pseudoacacia q. robur p. alba a. campestre c. avellana pia fig. 3. correspondence analysis (ca) performed using the species as group variable and plasticity index as category variable, pim – morphological plasticity, pia – anatomical plasticity, pip – physiological plasticity. catoni r., gratani l., sartori f., varone l., granata m. u. 84 acta bot. croat. 74 (1), 2015 moreover, sla differs between shade-tolerant and intolerant species (abrams and kubiske 1990, kull and niinmets 1993), and is the trait most strongly correlated to shade tolerance (janse-ten klooster et al. 2007). the considered shade-tolerant species (a. campestre and c. avellana) have a 49% higher sla and a 28% lower l than the shade-intolerant species (p. alba, r. pseudoacacia and q. robur) as confi rmed by the pca. the analysis shows that sun leaves of a. campestre and c. avellana are closer in behavior to shade leaves, underlining their ability to tolerate shade in comparison to others species. among the shade-intolerant species, q. robur has the lowest sla (130.6 ± 10.0 cm2 g–1) and the highest l in sun leaves (160.8 ± 9.6 μm), since shade-intolerant species typically have thicker leaves (niinemets et al. 1998). leaf thickening under higher irradiance can be interpreted as a means for the plant to optimize light use through irradiance attenuation (jacquemoud and baret 1990, ustin et al. 2001). moreover, the structure of the mesophyll is associated with the photosynthetic performance of leaves via regulation of the internal light and carbon dioxide profiles (evans 1999). in particular, the palisade parenchyma reflects the leaf ability to capture solar energy accounting for the photosynthetic efficiency (kumar et al. 2012). this is attested by the signifi cant (p < 0.01) positive correlation between pn and palisade parenchyma thickness. on average, pn is more than 100% higher in sun than in shade leaves, associated with a 38% higher palisade parenchyma thickness, while shade leaves are characterized by a 3% higher spongy parenchyma thickness, that enhances light capture by scattering light (mendes et al. 2001, sack et al. 2006), in respect to palisade parenchyma. the higher pn rates in sun leaves than in shade leaves for the considered species are supported by a more than twice as high gs value indicating that the larger stomata opening in sun leaves (schulze et al. 1975, farquhar and sharkey 1982) determines a higher intercellular co2 concentration. this result is supported by the signifi cant (p < 0.01) correlation between pn and gs. the shade-intolerant species have a greater difference between pn in sun and shade leaves than shade-tolerant species, depending on the different level of pn light saturation (koike et al. 2001). moreover, shade-tolerant species have lower pn rates both in sun than in shade leaves (6.7 ± 1.2 and 1.9 ± 0.1 μmol m–2 s–1 respectively) than shade-intolerant species, according to the results of koike et al. (2001), valladares et al. (2002) and valladares and niinemets (2008). variations in light availability within the crown also induce changes in co2 release through r, this last refl ecting the acclimation response to the light variations within the crown (zha et al. 2002). high ppfd increases r as a result of higher maintenance costs caused by higher pn (amthor 1989, niinemets et al. 1998), higher protein turnover and increased need for secondary compounds such as fl avonoids or carotenoids (delagrange et al. 2004). the ratio r/pn can be considered a simple approach to leaf carbon balance because it indicates the percentage of photosynthates that are respired (loveys et al. 2002). the 69% lower r/pn ratio in sun than in shade leaves (mean value of the considered species) is mainly due to the higher pn decrease from sun to shade leaves, with respect to the r decrease, underlining the higher pn sensitivity to light variations within the crown. this result is further attested by the signifi cant correlation between pn and ppfd%. because they have the lowest pn rates, the shade-tolerant species have a 76% higher r/pn ratio than those that are shade-intolerant. the strong and direct effect of light on the photosynthetic capacity is mainly related to light-induced variations in the pool of proteins, pigments and enzymes that supports light carbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 85 capture and co2 fi xation by leaves (niinemets et al. 1998, le roux et al. 1999). thus, crown photosynthesis is a function of leaf na distribution within the crown profile (gratani et al. 2006) which is attested by the signifi cant correlation between these two variables, and may be explained by more than 70% leaf n being allocated to the photosynthetic apparatus (poorter and evans 1998). sun leaves have a 54% higher na than shade leaves which enhances their capacity to convert high ppfd to organic matter (mendes et al. 2001). moreover, the higher pnue (greater than 100%) in sun leaves underlines the effi ciency to allocate n to rubisco, according to poorter and evans (1998) and gratani et al. (2006). the shade-intolerant species have a 21% higher pnue than the shade-tolerant species, demonstrating different light demands (kull and niinemets 1993). the acclimation of the photosynthetic pigment apparatus to high irradiance in sun leaves determines a higher chl a/b ratio (8%, mean value) and a lower (22%) chl/car ratio than in shade leaves, according to the results of lichtenthaler et al. (2007). the higher chl b content in shade leaves is indicative of acclimation to low irradiance, because it is usually the main component of the lhcp (koike et al. 2001), while the lower chl/car ratio in sun leaves is primarily caused by the highest activity in the xanthophyll cycle (demmig-adams 1998). moreover, the ratio chl/n, which is an indicator of leaf n allocation to chlorophyll-protein complexes in the light-harvesting component (koike et al. 2001), is 44% higher in the shade than in the sun leaves of the species considered. the increase in the proportional investment of leaf n in thylakoids, which improves incident light-use effi ciency, is a major response of n partitioning within the leaf to decreased irradiance (evans 1989). among the considered species, a. campestre has the highest chl/n both in sun and shade leaves (0.102 ± 0.010 and 0.174 ± 0.014, respectively) with a large increase from sun to shade leaves, thus attesting its shadetolerance. in fact, there is a wide consensus that a higher increase of chl/n is even more signifi cant for species of greater shade tolerance (evans 1989). plasticity is a key trait useful to quantify plant response to environmental stimuli (nicotra et al. 2010) and phenotypic plasticity is defined as the ability of a genotype to produce different phenotypes depending on the environment (sultan 2000). the analysis of leaf phenotypic plasticity attests the considered species responsiveness to light variation. the pi for the considered species (0.35) is in the range of broadleaf deciduous species (valladares et al. 2002, wyka et al. 2007, 2012). nevertheless, there are signifi cant differences among the species. in particular, the shade-intolerant q. robur and r. pseudoacacia have the highest pi (0.39 ± 0.01, mean value) while p. alba has the lowest (0.29). this last can be explained by the preference of p. alba to grow along the edge of forests and on river banks (madejón et al. 2004) considering that forest edges have a signifi cant infl uence on the solar radiation pattern on nearby landscape units, in relation to the number and size of openings, the distance of openings from the edge, and the orientation of openings relative to the path of the sun (galo et al. 1992). the presence of p. alba along the edge of forests and its vertically extended crown structure (chiusoli 1991) are typical traits of shade-intolerant species (delagrange et al. 2004). moreover, p. alba has the lowest pim (0.32) and pia (0.12), which are mainly explained by the lowest sla and l variations between sun and shade leaves, due to the lack of deep shade in its crown. the shade-intolerant species have a higher pip (0.46 ± 0.07) than the shade-tolerant species, in agreement with other studies (strauss-debenedetti and bazzaz 1996, groninger et al. 1996, valladares et al. 2002, bloor 2003, delagrange et al. 2004, sánchez-gómez et al. 2006, portsmuth and niinemets 2007). this result is also attested by the results of the correspondence analysis (ca). pip is catoni r., gratani l., sartori f., varone l., granata m. u. 86 acta bot. croat. 74 (1), 2015 also linked to an enhanced capacity to colonize gaps and open areas (valladares et al. 2002, niinemets and valladares 2004) because it ensures adjustments of gas exchange in response to stress factors changes in the short term (zunzunegui et al. 2009). a high physiological plasticity allows species to achieve rapid growth increasing the capability to colonize early successional habitats (walters and reich 1999). in particular, among the considered physiological traits, the photosynthetic plasticity (pipn) is signifi cantly higher in shade-intolerant than in shade-tolerant species (ducrey 1994). several studied show that that shade-intolerant species have greater photosynthetic acclimation potential than shade-tolerant species (strauss-debenedetti and bazzaz. 1996). this higher photosynthetic plasticity determines a large photosynthetic capacity to use full sunlight, which results in a more effi cient avoidance of photo-inhibition (valladares et al. 2002). the capacity for photosynthetic acclimation to light could be considered speciesspecifi c (duan et al. 2005). q. robur has the highest pip (0.53) which is related to its shadeintolerant behavior, according to the results of valladeres et al. (2002). moreover, its highest pipn (0.87) allows a rapid acclimation of photosynthesis which leads to an increased carbon gain, thereby facilitating growth and competitive ability in high light conditions (valladares et al. 2002). the high r. pseudoacacia pi confi rms that phenotypic plasticity allows invasive species to occupy a wide range of new environments (callaway et al. 2003, pigliucci 2005, rejmanek et al. 2005) and the highest pim (0.56) and pia (0.18) of r. pseudoacaia contribute to its adaptability. the higher pim of a. campestre and c. avellana is linked to an enhanced capacity to survive and grow in the understory (valladares et al. 2002, niinemets and valladares 2004), because it has an important role in resource acquisition (navas and garnier 2002, yamashita et al. 2000). on the whole, our results support the overall trend that light-demanding species are more plastic than shade-tolerant species (strauss-debenedetti and bazzaz 1991, 1996, muth and bazzaz 2002, longuetaud et al. 2013). however, from this general scheme, p. alba, despite its shade intolerance, exhibits the lowest pi which can be related to its crown type and the environment where it grows. moreover, our results support the trend for which the early-successional species (r. pseudoacacia and q. robur) are more plastic than the late successional ones (strauss-debenedetti and bazzaz 1991, 1996). in this context, the presence of large openings could favor both q. robur and r. pseudoacacia regeneration. knowledge on phenotypic plasticity is important to make hypotheses about the dynamics of the studied forest in consideration of environmental stress factors, including invasive species competition and global climate change. these stress factors might alter the composition of the forest in the long term, infl uencing the competition of the early-successional species. in particular, the increase of air temperature and co2 concentration might act as potent agents of natural selection among species favoring the more phenotypically plastic species (chown et al. 2007). in particular, air temperature increase might allow the reproduction of phythophora cinammomi, a pathogen largely affecting q. robur in europe (la porta et al. 2008) while the co2 concentration increase might favor r. pseudoacacia because of its high growth rate (mohan et al. 2007). considerations can be made for the conservative management of the forest carried out since the establishment of the reserve, which has probably limited the presence of r. pseudoacacia until 1980 when it knowingly became established (motta et al. 2009). thus, it is important to maintain this type of management in the future since creating gaps could allow a greater seed regeneration of r. carbon gain optimization in response to light gradient within the tree crown acta bot. croat. 74 (1), 2015 87 pseudoacacia over q. robur because of its higher growth rate. consequently, land use is a fundamental determinant in shaping vegetation composition and structure with important implications for forest management. in particular, forests with old-growth characteristics (bauhus et al. 2009), like the investigated forest, are important reference sites for more natural management approaches involving a broad range of ecosystem functions and services (wirth et al. 2009). in recent years, interest in understanding the natural processes in forest ecosystems has increased because of a few primary forests remaining in many regions (e.g., europe, peterken 1996). knowledge on the studied species, their plant and leaf traits, shade-tolerance, plasticity and information on the forest history may be important in making hypotheses about the current dynamics of individual trees and of the whole forest considering the increase of environmental stress factors including global climate change. acknowledgements this work was supported by grants from the university of pavia, ‘functional and structural characterizations of tree species inside 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environmental and spatial variables on the distribution of surface sediment diatoms in an upland loch, scotland hong yang*, roger j. flower, richard w. battarbee environmental change research centre, university college london, pearson building, gower street, london, wc1e 6bt, uk the spatial distribution of surface sediment diatoms was analyzed using arcgis in the round loch of glenhead, an acid upland lake in south-west scotland. the assemblages were composed almost entirely of benthic species. tabellaria quadriseptata was fairly evenly distributed across the loch but some species (navicula madumensis, brachysira brebissonii, aulacoseira perglabra and eunotia vanheurckii var 1) showed rather patchy distributions. ordination analysis was performed to assess the influence of environmental and spatial variables on the diatom composition of the samples. loss of ignition was significantly negatively correlated with redundancy analysis species axis 1 (r = –0.77), indicating the influence of substrate on the diatom assemblages. the positive relationship between theoretical bottom shear stress resulting from wind stress and redundancy analysis (r = 0.31) suggests wind stress also influences the spatial distribution of diatoms within the loch. spatial variables [(principal coordinates of neighbour matrices (pcnm 1 and pcnm3) positively correlated with redundancy analysis axis 2], indicated that spatial variables, ignored in former studies, are a further influence on diatom distribution. unique environmental and spatial variables explained 27.3% and 8.6% of diatom variability respectively. environmental and spatial interactive variables combined explained 4.8% of variation. although the pure contribution of spatial variables was only 8.6%, the study highlighted the importance of differences in the spatial distribution of different benthic diatom species in this upland lake. keywords: diatom, distribution, composition, benthic, substrate, wind, lake, scotland abbreviations: dca – detrended correspondence analysis, gam – generalizes additive models, i – downward irradiance, kd(par) – diffuse attenuation coefficient for downward irradiance, loi – sediment organic matter content, par – photosyntheticall active radiation, rda – redundancy analysis, tbss – theoretical bottom shear stress acta bot. croat. 68 (2), 2009 367 * corresponding author: hongyanghy@gmail.com u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:57:58 color profile: disabled composite 150 lpi at 45 degrees introduction it is often assumed that sediment deposition below a certain water depth is conformable and representative. however, the complexity of diatom deposition in lake basins is receiving increasing attention as a result of multicore studies (anderson 1989, 1990a, b; anderson 1998; adler and hubener 2007). in these studies, research focused on the selection of a representative core site from multiple cores collected from a range of water depths and locations within a lake. studies of transects across lakes from littoral to pelagic zones also demonstrated the variability in surface sediment diatom composition (meriläinen 1971, bradbury and winter 1976, kingston et al. 1983, kauppila 1998). the majority of sites investigated spatially have been lowland lakes. in upland lakes, the spatial variability of living benthic diatom communities has been surveyed, but only from samples spatially limited to the littoral zone (jones and flower 1986) or, in the case of surface sediment diatom assemblages, from samples collected along transects (allott 1991). however, transect sampling is an imperfect method of capturing whole-lake distributions (watts and halliwell 1996). a stratified random sample (srs) method that ensures good area coverage and preserves the advantage of randomness is preferred (watts and halliwell 1996). diatom samples collected according to the srs method can be analysed using multivariate statistical techniques to identify the principal factors that explain the distribution. until now, only environmental variables have been included in such analyses (kauppila 1998). here we also consider the importance of spatial variables. spatial patterns in the abundance and distribution of organisms are inherent properties of ecological systems (fortin and dale 2005), and therefore need to be included (legendre and fortin 1989, borcard et al. 1992). consequently, we use principal coordinates of neighbour matrices (pcnm) a technique introduced by borcard and legendre (2002) and now used to construct spatial models in various fields of ecology (borcard et al. 2004, crist et al. 2006). the respective contributions of environmental and spatial variables to the variability of surface sediment diatom can then be calculated using the variation partitioning technique (borcard et al. 1992). here we use this approach to analyse the spatial patterns of benthic diatoms in a scottish upland lake, the round loch of glenhead. materials and methods study site the round loch of glenhead (british national grid reference nx 450 804, 55°5’ n, 4°25’ w) is a small lake located in the galloway hills of south-west scotland (fig. 1). the climate of galloway is mild oceanic with an annual average precipitation of ca. 2500 mm yr–1 and an average temperature 8.5 °c with a range from –6 °c to 27 °c (yang 2009). soils in the catchment are mainly deep peats and peaty podsols; skeletal soils and bare rock occur on the steepest slopes (jones et al. 1989). the lake itself is situated within a rugged moorland landscape at 295 m altitude and has an open water area of 12.5 ha with a mean water depth of 4.3 m (maximum depth is 14.0 m). the average ph of the lake (04/2005–03/2006) is 5.2 and dissolved organic carbon is 362.5 µmol l–1 (for more details of water chemistry, see shilland et al. 2006). 368 acta bot. croat. 68 (2), 2009 yang h., flower r. j., battarbee r. w. u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:57:58 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 369 spatial distribution of surface sediment diatoms fig. 1. location and map of the round loch of glenhead, south-west scotland, uk, showing lake bathymetry, the main distribution areas of the dominant aquatic macrophytes (isoetes lacustris, juncus bulbosus var. fluitans, lobelia dortmanna), loss on ignition (as histograms), the location of the weather station and the position of the ten stakes. note that , which occurs sporadically in the loch, is not shown because its main growth period was after the main diatom sampling in april 2007. u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:09 color profile: disabled composite 150 lpi at 45 degrees diatom habitats in the lake are confined to the littoral zone. they are dominated by the epilithon growing on stones and boulders that occur around the whole perimeter of the lake and the epiphyton growing principally on submerged plants. the dominants are juncus bulbosus var. fluitans, lobelia dortmanna, and isoetes lacustris (shilland et al. 2006). juncus bulbosus var. fluitans is abundant in sandy bays, especially in the eastern littoral areas, whereas lobelia dortmanna occurs around almost all the margins of the lake and isoetes lacustris grows on mud surfaces with water depths of up to 3 m (yang 2009). the photic depth is about 6 m deep and epipsammic and epipelic diatoms can be found down to 6 m (yang 2009). sampling the stratified random sampling (srs) method was used to collect the surface sediment samples in the loch from 22nd to 28th april 2007. the loch area was divided into 40 × 40 m grids (fig. 1) and gps positions (as osgb 36) of the centre points of each grid were set up on a garmin gps iii. the boat was navigated according to the waypoints and anchored at the centre of each grid square. when the boat stopped drifting, one surface sample was collected randomly within each grid using either an epipelic sampler (yang and flower 2009), when the water depth was less than 1.5 m, or a renberg corer (renberg 1991) when water depth was more than 2.0 m. the top 1 cm sediment was removed for surface sediment samples. shallow water samples (from ca. 0.5 m depth) were collected using the epipelic sampler at ten georeferenced points (fig. 1) around the lake shore. all samples were transported back to the laboratory and stored at 4 °c. diatom analysis each sample was treated in the same way with no attempt to separate live and dead cells. subsequently the samples were oxidised using hydrogen peroxide (h2o2) by heating in a water bath (renberg 1990). microspheres were added to calculate the diatom concentration (battarbee and kneen 1982). all samples were mounted on microscope slides using naphrax as a mountant. diatom valves were identified and counted using a leitz research microscope under oil immersion at 1000 times magnification and phase contrast. a minimum of 500 valves was identified and counted for each sample. the abundance contours of the dominant taxa were analysed using the idw method in arcgis 9.0. environmental data environmental variables used in the analysis included photosynthetically active radiation (par), wind speed and direction, bottom shear stress and sediment organic matter content (loi). par was measured using a licor li-250 underwater light meter at 0.5 m depth intervals between 10 am and 2 pm in the loch centre. diffuse attenuation coefficients for downward irradiance kd (par) were calculated from the slope of the linear regression of the natural logarithm of downward irradiance (i) versus depth (z), and values only from a fit r2 > 0.95 were accepted (kirk 1994): 370 acta bot. croat. 68 (2), 2009 yang h., flower r. j., battarbee r. w. u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:09 color profile: disabled composite 150 lpi at 45 degrees kd(par) = –1/z ln(iz/i0) where kd(par) is the diffuse attenuation coefficient for downward irradiance, iz is the par measurement at depth z, and i0 is the par measurement at the water surface. the kd(par) and surface par (i0) were used to infer the par at different water depths. the temperatures at different water depths (1–12 m), wind speed and direction data were monitored by the weather station installed on a platform floating in the lake centre in october 2005. data were provided by d. monteith. the theoretical bottom shear stress (tbss) t was calculated as the following formula (james et al. 2004): t = h r u p( (2 / ) ) 2sinh(2 ) 3 0.5 t kh     where t is the calculated bottom shear stress, h is the wave height, r is the density of water, t is the wave period, u is the kinematic viscosity, k is the wave number (2p/l where l = wavelength, cm), and h is the water depth. wave characteristics (h, t and l) were calculated from wind speed, water depth and fetch distance using wave models according to the coastal engineering research center (1984). wind speed was measured from weather station located near the loch centre and the one point measurement was cautiously extrapolated to the whole loch. for loi, the percentage dry weights (dw) of sediment samples were calculated after drying a known weight of sample overnight at 105 °c and then combusting in a muffle furnace at 550 °c for 2 hours. spatial data in the field, bng (british national grid) coordinates of each sampling site were recorded. the original coordinate values were z-score transformed and these standard coordinates were used to create the dataset of spatial variables derived from pcnm using the program spacemaker2 (borcard and legendre 2004, http://www.bio.umontreal.ca/legendre/). a matrix of euclidean distances between samples was created and subsequently truncated based on truncation distance, which was equal to or larger than the largest distance between neighbours. data analysis all environmental data were z-score transformed (legendre and gallagher 2001) if the normality criteria were not satisfied. species data were hellinger transformed, because this method minimises the effects of the large number of zeros common in species abundance data (legendre and gallagher 2001). species with percent abundance of less than 3% per single sample were considered as »rare« taxa and were excluded from further ordination analysis. preliminary detrended correspondence analysis (dca) was performed using the program canoco 4.5 to measure the patterns of compositional variation and the biological species turnover (the gradient length) (ter braak and prentice 1988). redundancy analysis or canonical correspondence analysis was selected based on gradient length to explore the relationships between diatom assemblages and environmental variables (ter acta bot. croat. 68 (2), 2009 371 spatial distribution of surface sediment diatoms u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 12:10:03 color profile: disabled composite 150 lpi at 45 degrees braak and prentice 1988). to reduce the possible effects of the difference in the number of variables included in each set of explanatory variables, we only used those environmental variables that were significant based on a forward selection (p £ 0.05 after 999 random permutations) (økland and eilertsen 1994). to explore the relationship between single species distribution and environmental variables, gam (hastie and tibshirani 1990) were performed using the r-language (r development core team 2006) mgcv package (wood 2008). with respect to the spatial variables, the coordinates were created using pcnm and those eigenvectors with positive eigenvalues were retained for inclusion in the subsequent ordination analysis. variation partitioning was used to quantify the proportion of the variation in diatom assemblage explained by variation in each of the combinations of environmental, and spatial variable sub-models (borcard et al. 1992). the unadjusted r2 value was biased (peres-neto et al. 2006) and therefore they were adjusted into r2a. based on the proportion of variation explained (r2a) in the analyses, the contributions of every component to the total variation in the diatom assemblages were calculated (borcard et al. 1992). the significances of fractions were tested by means of 999 permutations under a reduced model. all the analyses were performed using the r-language (r development core team 2006) function varpart in the vegan package (oksanen et al. 2008). results distribution of surface sediment diatoms a total of 18 genera and 94 species were collected during the study. the mean diatom abundance was 5.802 × 103 valves dw mg–1 with a sd of 4.766 × 103 valves dw mg–1. most species (80%) belonged to the genera eunotia, navicula, frustulia or tabellaria. the most abundant species were navicula leptostriata jørgensen, frustulia rhomboides var. saxonica (rabenhorst) de toni, eunotia incisa w. sm. ex greg., tabellaria quadriseptata knudson, brachysira brebissonii r. ross in hartley, aulacoseira perglabra østrup and eunotia vanheurckii var. 1 r.j.flower. the environmental variables are summarised in table 1. gam models that fitted the distribution of navicula leptostriata abundance included loi, par and tbss and they were able to account for 54.8% of the variance. the selected models for frustulia rhomboides var. saxonica and eunotia incisa both included loi and par, amounting to 35.9% and 24.9% of the total variance, respectively. although the models that fitted tabellaria quadriseptata and eunotia vanheurckii var 1 only included loi, 372 acta bot. croat. 68 (2), 2009 yang h., flower r. j., battarbee r. w. tab. 1. summary statistics for environmental variables. loi=loss of ignition, par = photosynthetically active radiation, and tbss= theoretical bottom shear stress. min max mean sd water depth 0.5 14.0 4.9 3.8 dry weight 0.07 79.14 15.21 21.89 loi 0.64 61.29 31.93 15.30 par 0.07 398.72 100.15 106.19 temperature 9.7 11.3 10.7 0.5 tbss 0 0.0221 0.0011 0.0041 u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:09 color profile: disabled composite 150 lpi at 45 degrees the models accounted for 40.7% and 32.6% of the total variance, respectively. the models that fitted brachysira brebissonii and aulacoseira perglabra included par and tbss and they amounted to 17.8 and 24.0% of the total variance, respectively. the spatial distributions of the dominant taxa are shown in figures 2 and 3. navicula leptostriata was common in the northern shallow water but was seldom found in the eastern and southern basin (fig. 2). frustulia rhomboides var saxonica and eunotia incisa occurred mostly in the middle and southern parts of the lake basin. navicula madumensis (=kobayasiella madumensis, lange-bertalot 1999) jørgensen (1948) occurred mostly acta bot. croat. 68 (2), 2009 373 spatial distribution of surface sediment diatoms fig. 2. spatial distribution of surface sediment diatom concentrations (103 valves dw mg–1) in the round loch of glenhead in april 2007. upper left, navicula leptostriata; upper right, frustulia rhomboides var. saxonica; bottom left, eunotia incisa; botttom right, navicula madumensis. u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:20 color profile: disabled composite 150 lpi at 45 degrees in the eastern region of the loch. tabellaria quadriseptata was widely distributed in the loch but with three small high abundance patches in the southern basin (fig. 3). brachysira brebissonii and aulacoseira perglabra were concentrated in the loch centre. eunotia vanheurckii var. 1 was mainly found in the northern and middle basin. ordination analysis the gradient length of the first axis explored by dca is 1.75 sd. this indicates that species turnover was at a range where linear species response models rda could be suitable. 374 acta bot. croat. 68 (2), 2009 yang h., flower r. j., battarbee r. w. fig. 3. spatial distribution of surface sediment diatom concentrations (103 valves dw mg–1) in the round loch of glenhead in april 2007. upper left, tabellaria quadriseptata; upper right, brachysira brebissonii; bottom left, aulacoseira perglabra; botttom right, eunotia vanheurckii var. 1 u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:36 color profile: disabled composite 150 lpi at 45 degrees the environmental variables included dry weight, loi, par, temperature and theoretical bottom shear stress (tbss). forward selection identified loi, par and tbss as significant environmental variables for the later ordination analysis and variation partition methods. spatial variables pcnm1, pcnm2, pcnm3 and pcnm4 were calculated and all four variables were included in the later analysis. rda captures the variance in the species-environment and space relationship quite well; 77.3% by the first two axes (tab. 2). the first ordination axis was negatively related to loi (r = – 0.772) and par (r = –0.437). par, tbss, pcnm1 and pcnm 3 were positively related to axis 2. variation partitioning the variation in surface sediment diatom assemblages was partitioned into four parts: environmental variation (e), spatial variation (s), spatially structured environmental variation (es) and unexplained variation (u). their respective percentages were 27.3%, 8.6%, 4.8% and 59.3%. discussion spatial distribution of surface sediment diatoms georeferencing and organized sampling techniques are of major benefit in assessing the spatial distribution of modern diatoms in a lake (watts and halliwell 1996). for the round loch of glenhead these techniques have demonstrated major differences in the disacta bot. croat. 68 (2), 2009 375 spatial distribution of surface sediment diatoms tab. 2. summary statistics for the first four axes of the redundancy analysis (rda) of diatom-environment and space with hellinger transformation of diatom species, z – standardization of environmental variables and down-weighting of rare species. only environmental variables selected in the forward selection procedure are presented in the ordination. tbss = theoretical bottom shear stress, loi = loss of ignition, par= photosynthetically active radiation, pcnm = principal coordinates of neighbour matrices. axes 1 2 3 4 eigenvalue: 0.184 0.094 0.036 0.018 species-environment and space correlations: 0.858 0.75 0.705 0.669 cumulative percentage variance of species data: 18.4 27.8 31.3 33.1 of species-environment and space relation: 51.2 77.3 87.2 92.2 weighted correlation with species axes tbss 0.005 0.311 0.610 –0.050 par –0.437 0.456 –0.097 0.061 loi –0.772 –0.203 –0.105 0.103 pcnm1 0.262 0.314 –0.390 0.406 pcnm2 0.196 0.019 0.312 0.327 pcnm3 –0.157 0.378 –0.181 –0.284 pcnm4 0.139 0.128 –0.188 –0.179 u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:36 color profile: disabled composite 150 lpi at 45 degrees tribution of benthic diatoms according to species and to spatial location. tabellaria quadriseptata occurred almost evenly everywhere in the loch. although it can be transported around the lake basin, its wide spatial distribution indicated that it could be a habitat generalist taxon. on the other hand, there were clearly patchy distributions of navicula madumensis, brachysira brebissonii, eunotia vanheurckii var. 1 and aulacoseira perglabra. the reasons for the different spatial distribution of surface sediment diatom assemblages are discussed below. in lake salkolanjärvi, results indicated that water depth was the main environmental variable controlling the distribution of surface sediment diatoms (kauppila 1998). arguably, water depth is an integrated variable, correlated directly or indirectly with light, temperature and other environmental variables. in this study, underwater light par and water temperature, rather than water depth, were considered. rda indicated that there was a significantly negative correlation between loi and rda axis 1 (r = –0.77), suggesting the influence of substrate on the diatom assemblages. eunotia vanheurckii var. 1 is a typical epipsammic diatom in upland lochs (allott 1991). one of its aggregation areas was a sandy area in the loch (fig. 3) and this patchy distribution probably supports the influence of substrate on the distribution of this diatom. par correlated to rda axis 1 and 2. par is a major regulator of photosynthesis and consequently plays an important role in driving algal productivity and determining species composition in lakes (hill 1996). tbss correlated to axis 2, indicating the influence of wind stress on the spatial distribution of diatoms in the loch. although the gam that fitted the distribution of tabellaria quadriseptata did not include tbss, wind and wave action may be responsible for the relatively even distribution of this species. aulacoseira perglabra, a planktonic diatom species, is not found living in the loch today (e.g. jones 1987, allott 1991, yang 2009), but was abundant in the early holocene (jones 1987). the current occurrence of these diatoms in the deeper water surface sediments are probably the result of sediment resuspension processes from the exposure of old sediment surfaces in this region of the loch. the gam models that fitted aulacoseira perglabra included tbss, supporting this interpretation. comparisons between the distribution of macrophytes and surface sediment diatoms show no clear relationship, probably because the epiphytic diatoms were removed and re-distributed effectively by water turbulence. the influence of spatial variables although the role of spatial variables is increasingly recognised in the ordination analysis of organism composition (e.g. wagner 2003, borcard et al. 2004, beisner et al. 2006), spatial variables have seldom been considered in the analysis of diatom distributions. spatial structures observed in floristic composition can arise from: (1) autogenous structure (s), independent of any environmental variation; and (2) exogenous structure (es), which results when species respond to environmental variables that are themselves spatially structured (fortin and dale 2005, jones et al. 2008). before the introduction of the variation partition technique (borcard et al. 1992), s and es were not distinguished and both considered together as spatial variables in most studies. in the present study, we can extract the variation explained commonly by environmental and spatial variables (es) by partial ordination analysis. the result indicates that 36% (=4.8/(4.8+8.6)) of the variation in the spatial sub-models was due to environmental interaction. the contribution of unique spa376 acta bot. croat. 68 (2), 2009 yang h., flower r. j., battarbee r. w. u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:36 color profile: disabled composite 150 lpi at 45 degrees tial variables to the total variability of the surface sediment diatom assemblage was only 8.6%. however, although this is a relatively small figure the result highlights the importance of considering spatial variables separately from environmental variables in accounting for variability in surface sediment diatom composition. the small value of total diatom variation explained by spatial structure alone (8.6%) suggests that no significant unique spatial-structuring of diatom variation was missed in the round loch of glenhead. the positive relationship between both pcnm1 and pcnm3 and rda species axis 2 (tab. 2) confirmed the influence of spatial variables on surface sediment diatom composition. the unique spatial pattern (s) is more important to species that are habitat generalists or that aggregate in habitat patches (crist et al. 2006). further research in this study, the combined contribution of environmental and spatial variables to the variation in diatom composition was only 40.7%. the high unexplained contribution may result from both insufficient measurement of variables and the limitations of using a single time period for sampling in a system that is quite dynamic (beisner et al. 2006). in addition the factors that control the distribution of dead cells from living ones will differ. in the case of dead cells it is important to understand the taphonomic processes that control the transport, deposition and resuspension of cells across the lake basin, and in the case of living cells diatom distributions may be controlled as much by seasonal variability in hydrochemistry and by grazer suppression as by water depth and habitat (koetsier 2005). identifying the factors that control diatom distributions across lake basins with respect to both living and dead cells remains an important area for research (yang et al, in preparation). this is relevant to attempts to improve the design of field sampling programmes, especially those that aim to characterise the floras of a whole lake rather than of a localised sampling site within a lake. conclusions study of the spatial distribution of surface sediment diatoms in the round loch of glenhead has indicated that many taxa (navicula madumensis, brachysira brebissonii, aulacoseira perglabra and eunotia vanheurckii var. 1, in particular) were unevenly distributed in the loch. the results of ordination analysis showed that loi and par were negatively correlated to rda diatom variation axis 1, while par, tbss and the spatial variables (pcnm 1 and pcnm3) were positively correlated with axis 2. these observations suggest that substrate, wind-induced water turbulence and the spatial configuration of the loch basin significantly influence benthic diatom distributions in the loch. we conclude that the distribution of diatom taxa is related to the habitats in the lake as well as to transport processes and sediment resuspension. in the case of aulacoseira perglabra, resuspension of diatom valves from exposed old sediment surfaces is suspected. unique spatial variables, not considered in former studies on diatom distribution, explained 8.6% of the total variability of the surface sediment diatom composition. although this value is not very high, the result confirmed the importance of spatial variables independent of environmental variables in explaining the variability in surface sediment diatom composition. acta bot. croat. 68 (2), 2009 377 spatial distribution of surface sediment diatoms u:\acta botanica\acta-botan 2-09\yang.vp 6. listopad 2009 11:58:36 color profile: disabled composite 150 lpi at 45 degrees acknowledgements we are grateful to scottish natural heritage and the eu euro-limpacs project (goce-ct-2003-505540) for funds to support this study and to the dorothy hodgkin’s postgraduate award scheme for supporting hong yang. fieldwork was greatly assisted by support from the forestry commission staff especially by neil grieve, sany white and geoffrey shaw, from the ecrc, ucl, especially james shilland, ewan shilland, simon turner, david hunt and yan zhao, from the geography department, ucl, especially julian thompson and raja singh, from tim allott, manchester university, and all the others who helped with fieldwork. 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0365-0588 eissn 1847-8476 short communication two moss species from mt durmitor new to the bryophyte flora of montenegro anja vulević1, snežana dragićević2, danka petrović1 1 faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81 000 podgorica, montenegro 2 natural history museum of montenegro, trg vojvode bećir-bega osmanagića 16, 81 000 podgorica, montenegro abstract the present paper reports the first records of two moss species, orthotrichum obtusifolium brid. and dicranoweisia cirrata (hedw.) lindb., in the flora of montenegro. the mosses were found in the tepački kraj area on mt durmitor. some morphological features, the ecological characteristics and the distribution of the species are given, along with photographs of diagnostic details and their threat status in the countries of se europe. keywords: bryophyta, dicranoweisia cirrata, orthotrichum obtusifolium, se europe * corresponding author, e-mail: sneza.dragicevic@t-com.me introduction knowledge on the diversity and distribution of mosses in montenegro has increased significantly in the last decade. and yet the bryophyte flora of a number of regions remains unexplored. the area of durmitor is one of the bryologically richest and best explored areas in montenegro (dragićević and veljić 2006 and references therein, papp and erzberger 2010, 2011). of the 694 bryophyte species (545 mosses and 149 hepatics) registered for montenegro, 360 taxa have been recorded for the national park of durmitor alone, which thus accounts for almost the half of the known number of bryophytes in montenegro (papp and erzberger 2010). nevertheless, there are many zones in the area of durmitor that remain underexplored. bryological studies of a section of tepačke forest, which included two management units (podgora and tmora) revealed that this area has a rich bryoflora. in total, 37 bryophyte species were recorded, of which 34 are mosses and 3 belong to liverwort species (vulević 2012). another study included 34 management units of tepačke forest and reported 132 bryophytes, with 104 mosses and 28 liverwort species (vulević 2015). according to hill et al. (2006), the genus orthotrichum hedw. in montenegro comprises 15 species and 2 varieties, whereas the genus dicranoweisia milde is represented by a single species, dicranoweisia crispula (hedw.) milde, with a single record (dragićević and veljić 2006). material and methods study area durmitor is a high mountainous massif in the northwest of montenegro, spreading over an area that is about 55 km long and 18–21 km wide (lješević and stijepović 1996). it belongs to one of the most attractive natural areas in montenegro, with rocky vertical peaks, numerous valleys between the peaks, and mountain lakes and unique landscapes (cerović 1986). limestone is the dominant substrate, and therefore, the hydrographic network is poorly developed. limestone and dolomite dark soil on carbonate substrates and various types of brown soils are present in this area (fuštić and đuretić 2000). the climate is alpine (long, cold winters and short summers), modified by continental and maritime influence, as the mountain range of durmitor, acting as a barrier, hampers the flow of warm air from the southern regions to the north and the stream of continental air from the north to the south. sub-zero temperatures are frequent – the town of žabljak has on average 164 frosty days per year-and they are not uncommon even in june and september. this territory has the greatest cloud cover in montenegro, and only july, august and september have average monthly cloud cover below 50%. the climate is humid, with the average yearly rainfall between 1100 and 1700 mm. most of the rainfall occurs during the autumn, and july and august are the driest months, although there is no clear period of drought (ivezić 1984). two new mosses for the bryoflora of montenegro acta bot. croat. 76 (2), 2017 197 a part of durmitor was declared a national park in 1978 and a unesco world natural heritage site in 1980 (minje vić 1996). the rich and diverse flora of durmitor includes 1771 taxa, a large number of which belong to the ancient tertiary flora (ludajić 2000). in line with the geographical position, altitude, and the significantly desegregated terrain and complex historical processes, the vegetation of the durmitor region is also very complex and diverse. tepačke forest belongs to the durmitor mountain range and covers a plateau northwest from the town of žabljak (fig. 1). the area is dominated by forest vegetation. the collection site for the two moss species described in this paper is located in the zone of coniferous forests from the association piceo abietis–pinetum sylvestris stefanović 1960. the floristic composition of the community is characterized by the following: edificators in the tree layer (picea abies (l.) h. karst. and pinus sylvestris l.) are equally represented, but other species are very rare. in the shrub layer, beside the edificators, dominant species are: lonicera xylosteum l., rosa pendulina l., rhamnus fallax boiss. and ru bus idaeus l. the forest floor is well developed and dominated by brachypodium sylvaticum (huds.) p. beauv., euphorbia amygdaloides l., carex humilis leyss. and vaccinium myrtillus l. during bryological field studies, material was collected from many different natural and anthropogenic micro-locations including tree bark and the decaying wood of a cottage. the results were compared with the bryophyte checklists of montenegro (dragićević and veljić 2006, sabov ljević et al. 2008, ros et al. 2013). voucher specimens are deposited in the natural history museum of montenegro in podgorica (montenegro). the nomenclature followed that of hill et al. (2006). results and discussion during field studies conducted in the area of tepačke forest in 2014, we registered two moss species, orthotrichum obtusifolium and dicranoweisia cirrata. this is the first record of these species in the bryophyte flora of montenegro. orthotrichum obtusifolium brid. (orthotrichaceae) (= nyholmiella obtusifolia (brid.) holmen & warncke, stroemia obtusifolia (brid.) i. hagen) o. obtusifolium is a circumpolar, tropical/montane-arctic subcontinental-suboceanic species, basiphyte, moderate xerophyte, photophyte (growing in well-lit sites or under full sun), preferably moderate thermophyte, nitrophyte (medium nitrogen content), a pioneer on exposed roots, trunks, and twigs of trees and shrubs (preferably on fraxinus, alnus, and populus), occasionally found on old fence rails, and in alpine and arctic areas on calcareous rocks and cliffs (dierβen 2001). site: mt durmitor, žabljak, podgora village, omar božovića, 43°12’ 07.51’ n, 19°08’ 39.86’ e, 1417 m a.s.l. o. obtusifolium was collected on the bark of an elm tree. the population was solitary, of about 30 small tufts, up to 0.5 cm high, and concentrated on a small part of the tree (fig. 2 a). on the same micro-location (tree bark and exposed roots) we found the mosses antitrichia curtipendula (hedw.) brid., leucodon sciuroides (hedw.) schwchw., pterigynandrum filiforme hedw., pseudoleskea incurvata (hedw.) loeske and syntrichia ruralis (hedw.) f.weber & d.mohr, the liverworts frullania dilatata (l.) dumort. and radula complanata (l.) dumort., as well as lichens of the genus cladonia. morphological characteristics of the collected specimens of o. obtusifolium fit the description of this moss species: leaves rounded with numerous green clavate gemmae, leaf margin flat, costa ending below the apex (fig. 2 b). this small population of o. obtusifolium was collected from a solitary elm tree growing in a wide meadow. it is not threatened by cutting, but future studies will confirm whether this species should be protected. the region of mt durmitor is abundant in environments with no industry, heavy traffic, and rapid urbanization. therefore, o. obtusifolium, which is susceptible to atmospheric pollution (smith 2004), is expected to be present in pristine areas. this record contributes to our knowledge of the distribution of o. obtusifolium in southeastern europe. the species is registered in all countries of southeastern europe, except european turkey (sabovljević et al. 2008). in serbia, it has the status of vulnerable species (sabovljević et al. 2004) and in hungary and great britain the status of near threatened species (hodgetts 2015). fig. 1. location of the investigated area in montenegro and its position in se europe. country abbreviations: at – austria; al – albania; ba – bosniaherzegovina; bg – bulgaria; hr – croatia; gr – greece; hu – hungary; it – italy; ko – kosovo; mk – republic of macedonia; me – montenegro; ro – romania; rs – serbia; tr – turkey. vulević a., dragićević s., petrović d. 198 acta bot. croat. 76 (2), 2017 dicranoweisia cirrata (hedw.) lindb. (rhabdoweisiaceae) (= mnium cirratum l, barbula cirrata brid., grimmia cirrata schrad., tortula cirrata clairv., blindia cirrata c.m., weissia cirrata hedw.) according to dierβen (2001) dicranoweisia cirrata is: a cosmopolitan, austral-tropical/montane-hemiboreal. subcontinental–suboceanic species, acidophyte (-subneutrophyte), mesophyte–xerophyte (moderately dry), photophyte (growing in well-lit sites or under full sun), mesotherm, nitrophyte (medium nitrogen content), usually on tree trunks or wood in early stages of decomposition, on rocks and rooftops and stone or concrete walls. site: mt durmitor, žabljak, podgora village, omar božovića, 43°12’ 04.66’ n, 19°09’ 43.37’ e, 1441 m a.s.l. the moss dicranoweisia cirrata was found only at one micro-location, on the decaying roof of a cottage. the cottage is situated near the elm tree that was the collection site of o. obtusifolium, on the edge of the piceo abietis–pinetum sylvestris forest. on the same substrate, we found the mosses dicranum tauricum sapjegin, herzogiella seligeri (brid.) z.iwats., hypnum cupressiforme hedw., the liverworts blepharostoma trichophyllum (l.) dumort., lophozia incisa (schrad.) dumort., and ptilidium ciliare (l.) hampe, lichens from the genus cladonia. this population of dicranoweisia cirrata was small, com posed of a few green tufts or cushions, up to 1 cm high. some individuals had sporophytes (fig. 3 a).the leaves were crisped and incurved when dry, recurved at the middle, with costa ending below the apex. in addition, the leaves had dark, cylindrical gemmae up to 150 μm long (fig. 3 b). dicranoweisia cirrata is a southeastern european species and in slovenia and bulgaria has been reported as a data deficient species (dd), whereas in romania, where it was recently recorded, it is listed as endangered (hodgetts 2015). in other european countries in which the species has been confirmed, it is placed on the endangered list only in latvia as data deficient but recently recorded (dd*) (hodgetts 2015). the montenegrin population may gain endangered status with the destruction or removal of the cottage and therefore should be protected. acknowledgements authors thank dr. beata papp (hungarian natural history museum, budapest) who revised the material. fig. 2. the moss orthotrichum obtusifolium: a) montenegrin population on the bark of an elm tree, b) general view and leaf with gemmae. fig. 3. the moss dicranoweisia cirrata: a) greenish cushions up to 3 cm tall, b) leaf and gemmae. two new mosses for the bryoflora of montenegro acta bot. croat. 76 (2), 2017 199 references cerović, b., 1986: durmitor and the tara canyon. durmitor national park, žabljak. dierβen, k., 2001: distribution, ecological amplitude and phytosociological characterization of european bryophytes. bryophytorum bibliotheca 56. j. cramer, berlin – stuttgart. dragićević, s., veljić, m., 2006: survey of bryophyta of montenegro. special edition 1. natural history museum of montenegro, podgorica (in montenegrian). fuštić, b., đuretić, g., 2000: soils of montenegro. university of montenegro. biotechnical faculty, podgorica (in montenegrian) hill, m. o., bell, n., bruggeman-nannenga, m. a., brugués, m., cano, m. j., enroth, j., flatberg, k. i., frahm, j. p., gallego, m. t., garilleti, r., guerra, j., hedenäs, l., holyoak, d. t., hy vönen, j., ignatov, m. s., lara, f., mazimpaka, v., munoz, j., söderström, l., 2006: bryological monograph an annotated checklist of the mosses of europe and macaronesia. journal of bryology 28, 198–267. hodgetts, n. g., 2015: checklist and country status of european bryophytes – towards a new red list for europe. irish wildlife manuals no. 84. national parks and wildlife services, department of arts, heritage and the gaeltacht. ireland. ludajić, k., 2000: material for the flora of durmitor (review of the published papers). thesis, university of belgrade, faculty of biology, belgrade (in serbian). lješević, m., stijepović, m., 1996: the nature of national park durmitor. in: lješević, m. (ed.), the nature of national park durmitor, special editions, volume 8, 1–6. faculty of geography university of belgrade, belgrade (in serbian). ivezić, d., 1984: the climate of the durmitor. in: nonveiller, g. (ed.), the fauna of durmitor, volume 1, special edition, book 18, department of natural science, book 11, 55–61. the montenegrin academy of sciences and arts, titograd (in montenegrian). minjević, d., 1996: the natural landscape of durmitor, in: lješević, m. (ed.), the nature of national park durmitor, special editions, volume 8, 285–299. faculty of geography university of belgrade, belgrade (in serbian). papp, b., erzeberger, p., 2010: contributions to the bryophyte flora of durmitor national park, montenegro. nova hedwigia 138, 147–163. papp, b., erzeberger, p., 2011: additions to the bryophyte flora of the tara river canyon and the durmitor area, montenegro. studia botanica hungarica 42, 31–39. ros, r.m.,, mazimpaka, v., abou-salama, u., aleffi, m., blockeel, t.l., brugués, m., cros, r.m., dia, m.g., dirkse, g.m., draper, i., el-saadawi, w., erdağ, a., ganeva, a., gabriel, r., gonzáles-mancebo, j. m., granger, c., herrnstadt, i., hugonnot, v., khalil, k., kürschner, h., losada-lima, a., luis, l., mifsud, s., privitera, m., puglisi, m., sabovljević, m., sérgio, c., shabbara, h. m., sim-sim, m., sotiaux, a., tacchi, r., van derpoorten, a., werner, o., 2013: mosses of the mediterranean, an annotated checklist. cryptogamie bryologie 34, 99–283. sabovljević, m., cvetić, t., stevanović, v., 2004: bryophyte red list of serbia and montenegro. biodiversity and conservation 13, 1781–1790. sabovljević, m., natcheva, r., tsakiri, e., dihoru, g., dragićević, s., erdağ, a., papp, b., 2008: check-list of the mosses of south -eastern europe. phytologia balcanica 14, 207–244. smith,a. j. e., 2004: the moss flora of britain and ireland. university press, cambridge. vulević, a., 2012: taxonomic and ecologycal analysis of the bryoflora in tepačke forest on the durmitor mountain. thesis, uni versity of montenegro, faculty of science, department of biology, podgorica (in montenegrian). vulević, a., 2015: bryological studies of the tepački kraj with review of the bryological habitats. master thesis, university of montenegro, faculty of science, department of biology, podgorica (in montenegrian). 10 acta bot. croat. 77 (1), 2018 acta bot. croat. 77 (1), 10–17, 2018 coden: abcra 25 doi: 10.1515/botcro-2017-0015 issn 0365-0588 eissn 1847-8476 species delimitation and relationship in crocus l. (iridaceae) masoud sheidai1, melica tabasi1, mohammad-reza mehrabian1, fahimeh koohdar1, somayeh ghasemzadeh-baraki1, zahra noormohammadi2* 1 faculty of biological sciences, shahid beheshti university, tehran, iran 2 department of biology, science and research branch, islamic azad university, tehran, iran abstract – the genus crocus l. (iridaceae) is monophyletic and contains about 100 species throughout the world. crocus species have horticultural, medicinal and pharmacological importance. saffron is the dried styles of c. sativus and is one of the world’s most expensive spices by weight. controversy exits about the taxonomy of the genus and the species relationship. exploring genetic diversity and inter-specific cross-ability are important tasks for conservation of wild taxa and for breeding of cultivated c. sativus. the present study was performed to study genetic variability and population structure in five crocus l. species including crocus almehensis brickell & mathew, c. caspius fischer & meyer, c. speciosus marschall von biberstein, c. haussknechtii boissier, and c. sativus l. by inter simple sequence repeat (issr) molecular markers. we also used published internal transcribed spacer (its) sequences to study species relationship and compare the results with issr data. the results revealed a high degree of genetic variability both within and among the studied species. neighbor joining (nj) tree and network analysis revealed that issr markers are useful in crocus species delimitation. population fragmentation occurred in c. caspius and c. sativus. both issr and sequenced based analyses separated c. sativus from the other studied species. close genetic affinity of c. sativus and c. pallisii and inter-specific gene flow was supported by both data sets. key words: crocus, gene flow, issr, its * corresponding author, e-mail: marjannm@yahoo.com, z-nouri@srbiau.ac.ir introduction pollinated by e.g. bees, bumble-bees or moths (jensen and jacobsen 2003). nine wild species of crocus l. have been described from persia and some adjacent areas (idem and mathew 1975, wendelbo 1977, matine 1978). crocus taxonomy is controversial and has been based primarily on morphology, as well as chromosome number. the genus crocus is divided into two subgenera: subgenus crociris containing c. banaticus and subgenus crocus comprising the remaining species. the subgenus crocus is further divided into two sections: section crocus and section nudiscapus. however, the lack of clear distinctive characters, the wide range of habitats and the heterogeneity of the morphological traits and cytological data make the taxonomy of crocus difficult (norbak 2002). the crocus species grow in various geographical regions and therefore face different environmental and ecological conditions. adaptation of plant taxa to such a dispersed distributhe genus crocus l. (iridaceae) is monophyletic and consists of about 100 recognized species (petersen et al. 2008). these taxa occur from western europe and northwestern africa to western china with the center of species diversity in asia minor and on the balkan peninsula (harpke et al. 2003). the genus is well characterized and morphologically distinct but karyologically very heterogeneous. the genus is of ecological, horticultural, culinary and pharmacological importance (sik et al. 2008). saffron is the dried styles of c. sativus and is one of the world’s most expensive spices by weight. moreover, the styles of c. sativus and some other crocus species contain carotenoids that inhibit cancer cell proliferation (chryssanthi et al. 2007). crocus l. is a genus of perennial geophytes. the floral and foliar organs are completely formed before the summer drought when they enter dormancy and wait for moisture from autumn rains and melting snow. the flowers are crossmailto:marjannm@yahoo.com mailto:z-nouri@srbiau.ac.ir molecular systematic of crocus l. acta bot. croat. 77 (1), 2018 11 tion usually is accompanied by extensive genetic variability that can be present either in the form of allelic variability or allelic uniqueness of some populations (petit et al. 1998). there have been several attempts to study crocus taxa by molecular markers revealing species genetic variability (see for example, caiola et al. 2004, alavi-kia et al. 2008, sik et al. 2008, beiki et al. 2013). however, none was aimed at studying population genetic structure and gene flow among the species and populations. exploring the genetic structure and genetic variability of the wild relatives of cultivated plants is important for breeding purpose. tracing the useful traits among wild plants can broaden the gene pool available for human use. these traits and genes can be incorporated in the crop plant through hybridization. the genus crocus contains out-crossing species and most of its species are cross-compatible even with c. sativus. thus, studying the genetic diversity and population fragmentation in the genus is an important task for future breeding of this important crop plant (larsen 2011). the objectives of the present study are: i) to explore genetic variability within and among the species, ii) to identify population fragmentation and gene flow among these species, and iii) to reveal the species relationship. neutral molecular markers have been used extensively for species delimitation and genetic diversity analyses (see for example, sheidai et al. 2013, 2014). these molecular markers are extensively used in population genetics analyses that can shed light on different levels of genetic variation and the partitioning of variability within/among populations. it can also identify inbreeding as well as selfing versus outcrossing, effective population size and population bottleneck. these analyses may be of help in planning effective management strategies for endangered and/or invasive species (chen 2000). at present no investigation into population genetic structure, gene flow or genetic fragmentation of crocus species in iran has been reported. we have carried out population genetic analysis of 14 populations of five crocus species for the first time in the country. we used inter-simple sequence repeat (issr) markers to study genetic diversity since this marker is reproducible, cheap and easy to work with (sheidai et al. 2013, 2014). moreover, recent studies have shown that arbitrary amplified dominant markers like issrs can solve phylogenetic relationships of closely related recently radiated taxa at low taxonomic levels (poczai 2011). we used its sequences of the ncbi to build up a phylogenetic tree of crocus species growing in iran and compare the result with the issr tree obtained. materials and methods plant material ninety plant specimens were collected from 14 populations of five crocus l. species. the species studied are: 1 crocus almehensis brickell & mathew, 2c. caspius fischer & meyer, 3c. speciosus marschall von biberstein, 4c. haussknechtii boissier, and 5c. sativusl. (= c. officinalis pers.). details of the studied populations are provided in table 1. plants of each species are marked with numbers: c. sativus = 1–26, c. almehensis = 27–33, c. haussknechtii = 34– 41, c. caspicus = 42–83, c. speciosus = 84–90. dna extraction and issr assay fresh leaves were collected randomly in each of the studied populations and dried in silica gel powder. genomic dna was extracted using ctab protocol with activated charcoal (krizman et al. 2006). the quality of extracted dna was examined by running it on 0.8% agarose gel. ten issr primers; (agc)5gt, (ca)7gt, (agc)5gg, ubc810, (ca)7at, (ga)9c, ubc807, ubc811, (ga)9a and (gt)7ca commercialized by ubc (the university of british columbia) were used. pcr reactions were performed in a 25 μl volume containing 10 mm tris-hcl buffer at ph 8, 50 mm kcl, 1.5 mm mgcl2, 0.2 mm of each dntp (bioron, germany), 0.2 μm of a single primer, 20 ng genomic dna and 1 u of taq dna polymerase (bioron, germany). the amplifications’ reactions were performed in technethermocycler (germany) with the following program: 5 min initial denaturation step at 94 °c, 30 s at 94 °c; 1 min at 50 °c and 1 min at 72 °c. the reaction was completed by a final extension step of 7 min at 72 °c. the amplification products were visualized by running them on 2% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). issr bands obtained were coded as binary characters (presence = 1, absence = 0). the following genetic diversity parameters were determined in each population: percentage of allelic polymorphism, allele diversity (weising 2005), nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (freeland et al. 2011). fig. 1. distribution map of the studied crocus populations. populations are marked with numbers from 1-14 according to the tab. 1. sheidai m., tabasi m., mehrabian m.-r., koohdar f., ghasemzadeh-baraki s., noormohammadi z. 12 acta bot. croat. 77 (1), 2018 its sequences its sequence data were obtained from ncbi for representative crocus species growing in iran. the species and their accession numbers are: crocus biflorus subsp. albocoronatus (lt222365), c. almehensis (he801162), c. cancellatus subsp. pamphylicus (lm993449), c. speciosus subsp. ilgazensis (he801120), c. caspius (lt222445), c. gilanicus (he801172), c. sativus (dq094185) and c. pallasii (he664002). data analysis genetic diversity and population structure issr bands obtained were scored as binary characters. genetic diversity parameters were determined in each population. these parameters were nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (weising 2005, freeland et al. 2011). nei’s genetic distance was determined among the studied populations and used for clustering (weising 2005, freeland et al. 2011). for grouping of the plant specimens, neighbor joining (nj) clustering and neighbornet methods of networking were performed after bootstrapping 100 times (freeland et al. 2011, huson and bryant 2006 ). the mantel test was performed to check the correlation between geographical distance and the genetic distance of the studied species (podani 2000). past ver. 2.17 (hamer et al. 2012), darwin ver. 5 (2012) and splitstree4 v4.13.1 (2013) programs were used for these analyses. significant genetic difference among the studied populations and provinces were determined by amova (analysis of molecular variance) test (with 1000 permutations) by using genalex 6.4 (peakall and smouse 2006), and nei,s gst analysis of genodive ver.2 (2013) (meirmans and van tienderen 2004). the population genetic differentiation was studied by gst_est (standardized measure of genetic differentiation) (hedrick 2005), and d_est (jost measure of differentiation (jost 2008). in order to overcome potential problems caused by the dominance of issr markers, a bayesian program, hickory (ver. 1.0) (holsinger et al. 2003) was used to estimate parameters related to genetic structure (theta b value). the genetic structure of populations was studied in two different approaches. firstly with the use of the bayesian based model structure analysis (pritchard et al. 2000), and secondly by the maximum likelihood-based method of k-means clustering. for structure analysis, data were scored as dominant markers (falush et al. 2007). the evanno test was performed on the structure result to find the proper number of k by using delta k value (evanno et al. 2005). we performed k-means clustering as done in genodive ver. 2. (2013). two summary statistics, pseudo-f, and bayesian information criterion (bic), provide the best fit for k-means clustering (meirmans 2012). gene flow gene flow was determined by two different approaches. first was the calculation of nm, an estimate of gene flow from gst by popgen ver. 1.32 (1997) as: nm = 0.5 (1 – gst)/ coefficient of gene differentiation (gst). this approach considers equal amounts of gene flow among all populations (which may not be correct for all situations), secondly, a population assignment test based on maximum likelihood (ml) as performed in genodive ver. 2. (2013). the latter approach does not consider equal amounts of gene flow among the studied populations. recently, frichot et al. (2013) introduced the statistical model called latent factor mixed models (lfmm), which tab. 1. crocus species and populations, their locality and voucher number. species province locality/population (no. sample) altitude (m) longitude latitude voucher no. c. sativus khorasan-razavi jovein/pop1 (1-7) 1100 36.42 57.25 2014600 c. sativus khorasan-razavi kashmar/pop2 (8-11) 1603 35.14 58.28 2014601 c. sativus khorasan-razavi torbate-heydariyyeh/pop3 (12-15) 1450 35.1 59 2014602 c. sativus khorasan-razavi bardaskan/pop4 (16-19) 1370 33.25 59.42 2014603 c. sativus khorasan-razavi tabas village/pop5 (20-26) 1500 36.24 51.42 2014604 c. almehenis golestan almeh mountain/pop6 (27-33) 2165 37.22 56.38 2014605 c. haussknechtti markazi shazand/pop7 (34-41) 1900 33.55 49.24 2014606 c. caspius gilan rudbar-reshtehrud/pop8 (42-48) 320 37 49.27 2014607 c. caspius gilan rezvanshahr/pop9 (49-55) 1200 37.32 48.49 2014608 c. caspius mazandaran sari, agriculture university/pop10 (56-62) 30 36.34 53.11 2014609 c. caspius mazandaran sari, jamkhanehvillage/pop11 (63-69) 64 36.35 53.14 20146010 c. caspius mazandaran ghaemshahr/pop12 (70-76) 55 36.27 52.51 20146011 c. caspius rostamabad gilan/pop13 (77-83) 2500 36.53 49.14 20146012 c. speciosus gilan rostam-abad/pop14 (84-90) 2500 36.53 49.14 20146013 molecular systematic of crocus l. acta bot. croat. 77 (1), 2018 13 tests correlations between environmental and genetic variations while estimating the effects of hidden factors that represent background residual levels of population structure. we used this method to check if issr markers showed correlation with the environmental features of the studied populations. the analysis was done with the lfmm program version: 1.2 (2013). results genetic diversity genetic diversity parameters were first determined for the studied populations and then for the five studied species. data with regard to genetic diversity in 14 studied populations are presented in table 2. the highest values for an effective number of alleles occurred in population pop13 (1.37), followed by pop9 (1.32). the highest value of genetic diversity due to population (hs) occurred in population pop13 (0.25), followed by pop10 (0.23). the genetic diversity parameters in the species studied are presented in table 3. the highest value of gene diversity (he) occurred in c. saspicus (0.23), while c. haussknechtii had the lowest value (0.10). the highest value of genetic polymorphism occurred in c. caspicus (92.45), while the lowest one occurred in c. speciosus (32.08). population genetic structure amova test produced significant genetic difference (phipt = 0.51, p = 0.010) among the studied populations. moreover, gst = 0.49 (p = 0.001), the hedrick standardized fixation index (g'st = 0.59, p = 0.001) and the jost differentiation index (d-est = 0.20, p = 0.001) revealed the genetic differentiation of the studied populations. pair-wise fst values determined among 14 studied populations revealed that all populations differed significantly due to their genetic differences. among species, amova analysis produced significant difference among the studied species (phipt = 0.31, p = 0.010). it also revealed that genetic variability is higher within species (69%) than among species (31%). the hickory test also produced a high theta b value (0.4), supporting amova. since neighbor joining (nj) tree and neighbor-net diagram of issr molecular markers produced similar results, only nj is presented and discussed (fig. 2). plant specimens of each species were placed in separate clusters. this reveals that issr molecular markers can delimit the studied species. the nj tree and neighbor-net diagram also revealed withinspecies genetic variability (among populations genetic difference), as some of the populations in one species were located far from the other members of the same species. for example, populations of s. caspius were distributed in two major clusters. the nj tree of issr data revealed close genetic affinity between c. speciosus, c. almehensis and c. caspicus. k-means clustering produced k = 2 according to pseudo-f value (13.94), which is in agreement with the number of major clusters produced by the nj tree, while it produced k = 10 according to the bic value (577.12). this is in agreement with the number of sub-clusters in the nj tree. moreover, the evanno test produced the best k = 9 based on delta k. therefore obtaining k = 9–10 indicates the population genetic fragmentation in crocus species studied. structure plot (fig. 3) based on k = 9, separated the five populations of c. sativus studied into two genetic groups. populations pop1 and pop2, which formed the first genetic group, differed in their allele content from populations pop3–5 of the second genetic group. all these populations are located in khorasan-razavi province. therefore, c. sativus showed population fragmentation in this province. c. caspius (pop8-13) also showed population fragmentation. these populations are located in the gilan and mazandaran provinces. populations pop8 and pop9 of gilan province showed genetic similarity, but differed in their allele composition from the other populations. the populations pop10-13 of mazandaran province showed great allele diversity and differed significantly from each other. the mantel test performed after 5000 permutations produced significant correlation (r = 0.41, p < 0.01) between getab. 2. genetic diversity parameters in 14 studied crocus populations. ae = effective number of alleles, hs = genetic diversity due to population, and ht = total genetic diversity. population sample no. ae hs hs/ht pop1 10 1.196 0.128 0.05 pop2 4 1.098 0.069 0.03 pop3 4 1.223 0.164 0.07 pop4 4 1.151 0.119 0.05 pop5 4 1.158 0.123 0.05 pop6 7 1.256 0.174 0.07 pop7 8 1.201 0.142 0.06 pop8 7 1.271 0.18 0.08 pop9 7 1.326 0.226 0.1 pop10 7 1.352 0.234 0.1 pop11 7 1.185 0.128 0.05 pop12 7 1.223 0.151 0.06 pop13 7 1.374 0.253 0.11 pop14 7 1.234 0.153 0.06 tab. 3. genetic diversity parameters in the studied crocus species. n= number of populations, na = number of alleles, ne = number of effective alleles, i = shanon information index, he = gene diversity, uhe = unbiassed gene diversity, %p = percentage of polymorphism. species n na ne i he uhe %p c. sativus 26 1.245 1.277 0.268 0.171 0.175 62.26 c. almehensis 7 0.849 1.189 0.186 0.120 0.129 39.62 c. haussknechtii 8 0.830 1.171 0.165 0.105 0.113 37.74 c. caspicus 42 1.849 1.355 0.373 0.233 0.235 92.45 c. speciosus 7 0.679 1.209 0.177 0.120 0.129 32.08 sheidai m., tabasi m., mehrabian m.-r., koohdar f., ghasemzadeh-baraki s., noormohammadi z. 14 acta bot. croat. 77 (1), 2018 netic distance and geographical distance of the studied populations. therefore, ibd (isolation by distance) occurred in crocus species. gene flow the mean nm = 1.03 was obtained for issr loci studied. this is a high value and indicates a high level of gene flow among the studied populations. moreover, the structure plot revealed some degree of genetic admixture in the studied species. for example, population pop6 (c. almehensis) had some alleles from population pop14 (c. speciosus). similarly, populations pop12 and pop13 as well as pop10 and pop13 of c. caspicus had some shared alleles (similarly colored segments). the reticulograms of the species based on the nj tree (fig. 2) also showed some degree of gene flow among the studied species. it particularly revealed gene flow among c. sativus and the other crocus species. lfmm analysis revealed that some of the issr loci had -log10 (p-value)>1.50 (p<0.05). these loci are adaptive loci in the studied populations, such as issr loci 1, 2, 10, 19, 24, 25, and 29. some of these loci are among highly shared loci (nm >1), while some had lower nm values. therefore, both shared alleles and those that were confined to some geographical populations had adaptive value. species relationship based on its sequences the model test analysis revealed that k2 + g (the kimura two parameter model + gamma) is the best model to fit its data in the studied species. the species relationship determined by ml, nj, and maximum parsimony produced simifig. 2. reticulogram of the crocus species studied based on a neighbor joining tree of inter simple sequence repeat (issr) data. populations are marked with numbers from 1–90 according to the tab. 1. fig. 3. structure plot of crocus species and populations based on inter simple sequence repeat (issr) data. populations are marked with numbers from 1–14 according to the tab. 1. molecular systematic of crocus l. acta bot. croat. 77 (1), 2018 15 fig. 4. maximum likelihood phylogeny tree of crocus species based on its sequences. fig. 5. reticulogram of the crocus species based on its sequences. lar results. therefore, the ml tree is presented and discussed here (fig. 4). the ml tree produced two major clades. the first major clade consists of 5 species, c. almahensis, c. biflorus, c. speciosus, c. cancellatus and c. caspius. c. almahensis and c. biflorus showed a higher degree of genetic affinity and were placed close to each other. similarly, c. speciosus and c. cancellatus were placed close to each other, while c. caspius joined the other species at a great distance. the second major cluster contained c. gilanicus, c. pallasii and c. sativus. the phylogenetic tree obtained on its was in agreement with the issr tree presented above. both molecular analyses revealed close genetic affinity among c. almahensis, c. speciosus, and c. caspius. similarly, both analyses revealed that c. sativus is genetically different from these three species and joins them at some distance. the reticulogram (fig. 5.) obtained based on its was in agreement with issr reticulograms and revealed the occurrence of gene flow and/or ancestral shared alleles among the studied species. discussion exploring the wild relatives of cultivated plants is important in order to have better picture on the genetic variability of related plant taxa, thereby broadening the available gene pool for human needs. usually, wild relatives of important crop plants may contain useful genes that can be introduced to the crop plant that has been under selection pressure for long time. extensive selection and uniform breeding practice can lead to genetic erosion and lower genetic variability of crop plants (poczai 2011). the present investigation revealed the presence of a high degree of genetic variability both within and among crocus species. due to the high crossability potential among crocus species, there are possibilities of transferring useful genes in these taxa. to recognize different gene pools in plant species, it is necessary to study the possible population genetic fragmentation due to habitat differences. the present study showed that some crocus species do show genetic fragmentation of populations. the population fragmentation is connected with their geographical location. this was particularly true for the cultivated species c. sativus and for c. caspius. genetic differences of geographical populations in c. sativus are particularly interesting as these plants can be selected for desirable agronomic traits and be used for further breeding purpose. larsen (2011) studied hybridization compatibility among species of subgenus crocus series crocus sativus by making 88 crosses. the results indicated that essentially all species of series crocus can be hybridized. this occurs due to cross-pollination and self-incompatibility suppressing selffertilization in these species (chichiricco 1990, 1996). it is suggested that the considerable genetic resources found in different crocus species could be used to genetically improve c. sativus. in fact, the hybrids obtained in the genus crocus show an increased frequency of unreduced gametes and it could be possible to use these hybrids in the production of future triploids (øragaard et al. 1995. habitat fragmentation along with extensive use of plants by humans and deforestation result in reduced rate of gene flow among populations (hou and lou 2011). structure analysis and k-means clustering revealed population fragmentation in the studied crocus species. however, these populations are not completely disconnected as the population assignment test, reticulation analysis and nm estimation showed some degree of gene flow among them. therefore, these populations obtain some degree of genetic variability. this situation is similar to a meta-population. a meta-population is an assemblage of local populations that usually are small and are linked by loose relationships, i.e., there is some gene flow among them. in such cases, gene flow among local populations could mitigate losses of genetic variation caused by genetic drift in local populations and thus save them from extinction via so-called ‘‘genetic rescue’’ (richards 2000). genetic diversity is an important aspect of species genetic continuity. it could be used for study of plant adaptation potential that copes with environmental changes during a plant’s life history (çalişkan 2012, sheidai et al. 2013, 2014). a high degree of within-population genetic diversity was observed in the studied populations (amova test revealed that 69% of total genetic variability is present within a population and 31% was present among populations). such a result could be related to the out-crossing nature of crocus taxa (larsen 2011). the mantel test revealed a pattern of isolation by distance across the distribution range of the studied crocus populations. therefore, gene flow is most likely to occur between neighboring populations and as a result, more closely located populations tend to be more genetically similar to one another (hutchison and templeton 1999). larsen (2011) reported between-population genetic differentiation in crocus hadrisheidai m., tabasi m., mehrabian m.-r., koohdar f., ghasemzadeh-baraki s., noormohammadi z. 16 acta bot. croat. 77 (1), 2018 aticus of mainland greece and peloponnese. he considered isolation by distance as the main reason for this genetic differentiation. he stated that individuals tended to cross with individuals from neighboring locations rather than with individuals from more distant sites. this results in adaption to local conditions. lfmm analysis of issr data in our study revealed that some of the genetic loci have an adaptive nature. therefore, the combination of genetic divergence, limited gene flow and local adaptation has played a role in the diversification of crocus. in conclusion the present study may provide some useful information about population genetic structure and genetic variability of crocus species that may be used in the conservation of these important species and also for hybridization with cultivated crocus sativus. c. sativus has 2n = 3x = 24 chromosomes and morphologically belongs to series crocus. it is believed that the ancestors of the species should be searched between one or two diploid species of series crocus with 2n = 16. six species fulfill these criteria: crocus cartwrightianus, c. thomasii, c. hadriaticus, c. oreocreticus, c. matheweii, and c. pallasii. the ancestor of c. sativus is still in question. for example, based on morphological, cytological and molecular analyses larsen (2011) considered c. cartwrightianus the most probable ancestor for c. sativus. zubor et al. (2004) found that c. sativus shares a high number of morphological similarities with c. cartwrightianus and c. thomasii, while frizzi et al. (2007) considered c. cartwrightianus the only ancestor of c. sativus. however, according to caiola et al. (2004), c. cartwrightianus, c. hadriaticus, c. asumaniae and c. pallasii share most similar genetic fragments with c. sativus. recently, alsayied et al. 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(2nd ed.). crc press, boca rayton, usa. wendelbo, p., 1977: tulips and irises of iran, and their relatives, botanical institute of iran, tehran. zubor, á. a., surányi, g., gyóri, z., borbély, g., prokisch, j., 2004: molecular biological approach of the systematics of crocus sativus l. and its allies. acta horticulturae 650, 85– 93. http://www.ncbi.nlm.nih.gov/pubmed/?term=richards cm%5bauthor%5d&cauthor=true&cauthor_uid=10718733 http://www.ncbi.nlm.nih.gov/pubmed/10718733 acta botanica 2-2016 za web.indd 206 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 206–209, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0022 issn 0365-0588 eissn 1847-8476 short communication phytolacca acinosa roxb. (phytolaccaceae), a new alien species in the croatian fl ora valentina borak martan1, renata šoštarić2* 1 kamaufova ulica 10, hr10000, zagreb, croatia 2 university of zagreb, faculty of science, department of botany with the botanical garden, marulićev trg 20/ii, hr10000, zagreb, croatia abstract: phytolacca acinosa roxb., an east asian plant species naturalised in many parts of the european continent, has been recorded for the fi rst time in croatia in two anthropogenic habitats in varaždin city (nw croatia). this study reports the newly discovered localities and presents the characteristics of the new alien species in the fl ora of croatia. a determination key is given for phytolacca taxa registered in croatia and neighbouring countries. keywords: alien plants, croatia¸ phytolacca acinosa, varaždin * corresponding author, e-mail: rsostar@biol.pmf.hr introduction phytolacca l. is the largest genus of the family phytolaccaceae, with a number of species, ranging from 25 (dequan and larsen 2003, king 2011) to over 35 (willis 1966). the genus is distributed worldwide, is nearly cosmopolitan and mostly native to south america, with a few species in africa and asia (dequan and larsen 2003). several species are found in cultivation and occasionally escape and become naturalised (king 2011). according to drake (2009), on the list of alien species in europe there are six phytolacca species: ph. acinosa roxb., ph. americana l. (syn. ph. decandra l., ph. vulgaris crantz), ph. dioica l. (syn. pircunia dioica (l.) moq.), ph. esculenta van houtte, ph. heterotepala h. walter and ph. polyandra batalin. in euro+med plantbase (2015), the occurrence of ph. pruinosa fenzl in cyprus, lebanon and syria is quoted. in europe, the most commonly recorded species from the genus is ph. americana, originating from north america and widely naturalised in southern europe, locally also in western and central europe (webb and akeroyd 1964). unlike ph. americana the species ph. acinosa is much less common in europe (wyrzykiewicz-raszewska 2009) and it is of east asian origin. ph. acinosa is native in china, eastern asia (japan, korea, taiwan), the indian subcontinent and indo-china (dequan and larsen 2003). it was brought to europe as a vegetable as well as an ornamental plant (wyrzykiewicz-raszewska 2009). according to available literature, the occurrence of ph. acinosa has been reported in several countries: belgium (alien plants of belgium 2015, daisie 2015, q-bank 2015), denmark (daisie 2015, q-bank 2015), bulgaria (q-bank 2015), slovenia (lešnik 2009), sweden, united kingdom, netherlands (q-bank 2015) and france (daisie 2015). considering the species ph. esculenta as a synonym of ph. acinosa, the species has also been recorded in austria (essl 1998, q-bank 2015), germany (jäger et al. 2013, floraweb 2015), switzerland (info flora 2015), in the czech republic (pyšek et al. 2012), romania (webb and akeroyd 1964, q-bank 2015) hungary (balogh 2005) and bulgaria (zieliń ski et al. 2012). taxonomical status of the species ph. acinosa and ph. esculenta has been the subject of several discussions. according to webb and akeroyd (1964) the name ph. acinosa is a synonym of the species ph. esculenta, while for clement (1982) there are the clearly separate species, ph. acinosa and ph. esculenta (tab. 1). recent taxonomists (see for instance dequan and larsen 2003) tend to include all eastern asian’related taxa that are cultivated for ornament (ph. esculenta and ph. latbenia buch.-ham. h. walter) in a broadly circumscribed ph. acinosa (alien plants of belgium 2015). in croatia, ph. americana is reported in cultivation and naturalised (nikolić 2014). this species is invasive in croatia (nikolić 2015) and in some other european countries like france (dumas 2011), italy (siniscalco et al. 2011), portugal (invasoras 2015), switzerland (wittenberg 2005), greece (arianoutsou et al. 2010), czech republic (pyšek et al. 2012) and bulgaria (petrova et al. 2013). so far, no other species of the genus have been recorded in the territory of the republic of croatia. phytolacca acinosa in croatia acta bot. croat. 75 (2), 2016 207 material and methods the city of varaždin is the county town of varaždin county and after zagreb it is the second biggest city in the north-west of croatia with 46,946 inhabitants (fig. 1). according to koppen’s climate classifi cation, the broad va ra ždin area has a temperate humid climate with warm summers (cfa) (šegota and filipčić 2003). the forest community of sessile oak and common hornbeam (querceto-carpinetum orientalis) (horvatić 1967) would be the zonal vegetation but recently has been transformed into various kinds of anthropogenic and semi-natural vegetation due long human infl uence. plant identifi cation was done according to webb and akeroyd (1964) and clement (1982). the plants were photographed with canon digital camera and geocoding of the locations was performed with use of a gps device. results and discussion during a fl oristic investigation of varaždin in 2014 ph. acinosa was found at two localities. at the fi rst location (gps coordinates: 5601192, 5131810) a single specimen was recorded on june 8th 2014 in a landfi ll for various types of waste (occasional fi re site). on august 11th 2014 two specimens were found at the second location (gps coordinates: 5603637, 5129550) in the green area around a building (under a fi r) (fig. 1). as the taxonomical status of the species ph. acinosa and ph. esculenta is still a matter of discussion (webb and akeroyd 1964, clement 1982, tab. 1), we consider both opinions in the analysis of specimens found in varaždin. the material had all characteristics of the species ph. acinosa s. str., except the anther colour. according to clement (1982) the anthers of ph. acinosa are white, while our specimens had pink anthers. something similar was noticed in belgium where collections of the ph. acinosa group are usually more or less intermediate between ph. acinosa and ph. esculenta: infl orescence axes are often scabrid-glandular (as in ph. acinosa) but the fl oral characters resemble ph. esculenta (alien plants of belgium 2015). whether this intermediate characteristic manifests possible hybridization is not quite clear and further investigation should give the answer. ph. acinosa s. str. (fig. 2) is a perennial, growing to a height of 1.5(–3) m (nienaber and thieret 2003). roots are thick, fl eshy. stems are erect, green or reddish purple, longitudinally grooved, fl eshy, branched (dequan and larsen 2003), naked, juicy, branching in the upper part (wyrzykiewicz-raszewska 2009). leaves are spirally arranged, leaf blade is elliptic or lanceolate-elliptic, 10–30 cm long, and 4.5–15 cm wide. leaf base is cuneate, apex acuminate or sharply pointed. petiole is 1.5–3 cm long (dequan and larsen 2003). numerous, densely clustered flowers form a cylindrical raceme of 15–20 cm in length, which grows sympodially and is erect not only during flowering, but also during fruiting. flowers are radial, bisexual, of approx. 8 mm in diameter, growing from axils on peduncles 6–10(13) mm long. the simple perianth is composed of five petaloid sepals, initially white in colour, later changing into green to become purple-red during fruit ripening. sepals are elliptical to ovoid, 3–4 mm in length and 2 mm in width. after pollination of flowers they do not drop, but tilt backwards. there are 8–10 stamens, equal in length to the perianth, fi laments are persistent, white, subulate, wider at the base with pinkish, elliptical anthers. the pistil of hypogynous fl ower is composed of 7–15 free carpels. the fruit (fig. 2), generally defined as a berry, is juicy and composed of 7–15, most often eight, adjacent single-seeded berries, forming a compound berry, approx. 7 mm in diameter. each berry has fig. 1. localities with coordinates of phytolacca acinosa roxb. in croatia. tab. 1. differences between phytolacca esculenta van houtte and phytolacca acinosa roxb. according to clement (1982). flowers characteristics ph. esculenta ph. acinosa pedicels and infl orescence axis almost glabrous scabrid-glandular perianth white greenish white to pinkish anthers pink white fig. 2. phytolacca acinosa roxb. in location number 2 (photo by v. borak martan). borak martan v., šoštarić r. 208 acta bot. croat. 75 (2), 2016 an excrescence on the top, a remnant of the style. at maturity fruits are shiny purple-black. seeds are kidney-shaped, smooth, slightly 3-angulate, approx. 3 mm in length (wyrzy kiewicz-raszewska 2009). all three specimens have been found in anthropogenic habitats, similar to cases in the neighbouring countries (essl 1998, lešnik 2009, zieliński et al. 2012). ph. acinosa is a gradually spreading, locally naturalised garden escaper. it most often occurs in gardens or parks (under trees or shrubs, foot of walls), in cemeteries or in urban wastelands. ph. acinosa often occurs in single specimens but bigger local populations are increasingly recorded (alien plants of belgium 2015). ph. americana survives in most environments, in woodlands, pastures, fi elds, forest margins and disturbed sites such as ornamental landscapes, urban waste areas (di tomaso et al. 2013) so it is possible to fi nd both species in the same type of habitat. according to the available literature ph. acinosa always is a much smaller plant (rarely exceeding 100 cm) with an erect infl orescence and broader leaves (wyrzykiewicz-raszewska 2009, alien plants of belgium 2015) and fruit composed of eight free segments (representing the eight carpels) and erect infl orescences, which remain erect even after the ripening of the fruits (petrova et al. 2013). the specimen found at locality number 1 was burnt down during a fi re in the habitat, whereas the other two specimens found at locality number 2 survived through the winter, showing that they are adjusted to a moderate continental climate. the same was also noticed in slovenia. according to lešnik (2009) ph. acinosa is a perennial and it recovers from the buds on a thickened root. since it is the fi rst fi nding of the species ph. acinosa in croatia, it is necessary to follow its spread so it can be classifi ed according to criteria applied for alien species, established by richardson et al. (2000) (casual plants, naturalised plants, invasive plants) and adjusted for croatia according to mitić et al. (2008). according to the above stated criteria, the mentioned species has been already naturalised in several countries of europe: switzerland (wittenberg 2005), hungary (balogh 2005), the czech republic (pyšek et al. 2012), belgium (alien plants of belgium 2015) and germany (floraweb 2015). in neighbouring hungary the occurrence of both species (ph. acinosa and ph. esculenta) has been recorded (balogh 2005). ph. esculenta is considered an invasive alien species in hungary (balogh 2005, tiborcz et al. 2012), so it is important to emphasise the possibility of the species spreading into the territory of the republic of croatia. determination key for phytolacca species in croatia and neighbouring countries: 1 carpels 7–15, usually 8, free; racemes erect in ripening; 8 single seed berries forming a compound berry; leaves broadly ovate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 carpels 10, united; nodding racemes; typical single berry (10-seeded); leaves ovate-lanceolate . . . ph. americana 2 flower pedicels and infl orescence axis almost glabrous; perianth white; anthers pink . . . . . . . . . . . ph. esculenta 2 flower pedicels and infl orescence axis scabrid-glandular; perianth greenish white to pinkish; anthers white . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ph. acinosa references alien plants of belgium, 2015: retrieved may 15, 2015 from: http://alienplantsbelgium.be/content/phytolacca-acinosa arianoutsou, m., bazos, i., delipetrou, p., kokkoris, y., 2010: the alien flora of greece: taxonomy, life traits and habitat preferences. biological invasions 12, 3525–3549. balogh, l., 2005: a phytolacca esculenta van houtte szelíd inváziója a magyarországi településfl órábon. flora pannonica 3, 135–161. clement, e. j., 1982: pokeweeds (phytolacca sp.) in britain. botanical society of britain and ireland news 32, 22–23. daisie, 2015: european invasive alien species gateway. phytolacca acinosa. retrieved may 15, 2015 from: http://www.europe-aliens.org/speciesfactsheet.do?speciesid=8652 dequan, l., larsen, k., 2003: phytolaccaceae. in: wu z., raven, p. h. (eds.), flora of china. vol 5., 435–436. science press, beijing, and missouri botanical garden press, st. louis. retrived from http://www.efl oras.org/fl orataxon.aspx?fl ora_id= 600&taxon_id=10687 di tomaso, j. m., kyeser, g. b., oneto, r. s., wilson, r. g., orloff, s. b., anderson, l. w., wright, s. d., roncoroni, j. a., miller, t. l., prather, t. s., ransom, c., beck, k. g., duncan, c., wilson, k. a., mann, j. j., 2013: weed control in natural areas in the western united states. weed research and information center, university of california, 544 pp. drake, j. a. (ed.), 2009: list of species alien in europe and to europe. in: daisie handbook of alien species in europe, 133– 265. springer, berlin. dumas, y., 2011: que savons-nous du raisin d’amérique (phytolacca americana), espèce exotique envahissante? rendezvous techniques 33–34, 48–57. euro+med (2006–): euro+med plantbase – the information resource for euro-mediterranean plant diversity. retrieved november 22, 2015 from: http://ww2.bgbm.org/europlusmed/ essl, f., 1998: floristische beobachtungen aus dem östlichen oberösterreichischen alpenvorland ii. beiträge zur naturkunde oberösterreichs 6, 107–126. floraweb, 2015: retrieved may 15, 2015 from: http://www.fl oraweb.de/pfl anzenarten/artenhome.xsql?suchnr=6502& horvatić, s., 1967: phytogeographical characteristics and division of yugoslavia (in croatian). in: horvatić, s. (ed.), analitička fl ora jugoslavije 1, 23–61. info flora, 2015: das nationale datenund informationszentrum der schweizer flora. retrieved may 15, 2015 from: https:// www.infoflora.ch/de/flora/5242-phytolacca-esculenta. html#map invasoras, 2015: invasive plants in portugal. phytolacca americana. retrieved november 23, 2015 from: http://invasoras.pt/ wp-content/uploads/2012/10/phytolacca-americana_en.pdf jäger, e. j., müller, f., ritz, c. m., welk, e., wesche, k., 2013: rothmaler exkursionsfl ora von deutschland, gefäβpfl anzen: atlasband, 12. aufl age, page 524. springer-verlag, berlin heidelberg. king, c. j., 2011: phytolacca linnaeus. in: cullen, j., knees s. g., cubey h. s. (eds.), the european garden fl ora. flowering plants: a manual for the identifi cation of plants cultivated in phytolacca acinosa in croatia acta bot. croat. 75 (2), 2016 209 europe, both of-doors and undress glass. vol 2., 2nd edition, 133–134. cambridge university press, new york. lešnik, m., 2009: new weed species in slovenia – estimation of dynamics of transition from ruderal to fi eld crop and perennial crop weed communities. proceedings 9th slovenian conference on plant protection. nova gorica, 299–308 (in slovenian). mitić, b., boršić, i., dujmović, i., bogdanović, s., milović, m., cigić, p., rešetnik, i., nikolić, t., 2008: alien fl ora of croatia: proposals for standards in terminology, criteria and related database. natura croatica 17, 73–90. nienaber, m. a, thieret, j., 2003: phytolacacceae. in: flora of north america editorial committee, eds. 1993+. flora of north america north of mexico. 18+ vols. new york and oxford. vol. 4, 3–12. retrieved from http://www.efl oras.org/ fl ora taxon.aspx?fl ora_id=1&taxon_id=200007011 nikolić, t., 2014: phytolacca americana l. in: nikolić, t., mitić, b., boršić, i., 2014: flora of croatia: invasive plants (in croatian), 242–245. alfa, zagreb. nikolić, t., (ed.), 2015: flora croatica database. retrived may 5, 2015 from: http://hirc.botanic.hr/fcd/invazivnevrste/detalji. aspx?idvrste=7450 petrova, a., vladimirov, v., georgiev, v., 2013: invasive alien species of vascular plants in bulgaria. institute of biodiversity and ecosystem research, bulgarian academy of sciences, sofi a. pyšek, p., danihelka, j., sádlo, j., chrtek, j., chytry, m., jarošík, v., kaplan, z., krahulec, f., moravcová, l., pergl, j., štajerová, k., tichý, l., 2012: catalogue of alien plants of the czech republic (2nd edition): checklist update, taxonomic diversity and invasion patterns. preslia 84, 155–255. q-bank, 2015: comprehensive databases of quarantine plant pests and diseases. retrieved may 15, 2015 from: http://www.qbank.eu/plants/biolomics.aspx?table=plants%20-%20 species&rec=1124&fields=all richardson, d. m., pyšek, p., rejmánek, m., barbour, m. g., panetta, f. d., west, c. j., 2000: naturalization and invasion of alien plants: concepts and defi nitions. diversity and distributions 6, 93–107. siniscalco, c., barni, e., bacaro, g., 2011: non-native species distribution along the elevation gradient in the western italian alps. plant biosystems 145, 1–15. šegota, t., filipčić, a., 2003: koppen’s classifi cation of climates and the problem of corresponding croatian terminology (in croatian). geoadria 8, 17–37. tiborcz, v., csiszár, á., korda m., schmidt, d., šporčić, d., teleki, b., zagyvai, g., bartha, d., 2012: distribution of some invasive alien plant species in hungary. international scientific conference on sustainable development and ecological footprint. sopron, 1–5. webb, d. a, akeroyd, j. r., 1964: phytolacca l. in: tutin t. g., burges n. a., chater a. o., edmondson, j. r., heywood, v. h., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea. vol 1., 2nd edition, 134. cambridge university press, cambridge. willis, j. c., 1966: a dictionary of fl owering plants and ferns. cambridge at the university press, london. wittenberg, r. (ed.) 2005: an inventory of alien species and their threat to biodiversity and economy in switzerland. cabi bioscience switzerland centre report to the swiss agency for environment, forests and landscape. the environment in practice no. 0629. federal offi ce for the environment, bern. wyrzykiewicz-raszewska, m., 2009: phytolacca acinosa roxb. – a new anthropophyte in the fl ora of poland. botanika – steciana 13, 3–7. zieliński, j., petrova, a., natcheva, r., 2012: new species for the bulgarian fl ora. phytologia balcanica 18, 197–204. untitled acta bot. croat. 75 (1), 2016 129 acta bot. croat. 75 (1), 129–143, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0008 issn 0365-0588 eissn 1847-8476 epilithic diatom assemblages and environmental quality of the su gologone karst spring (centraleastern sardinia, italy) giuseppina g. lai1*, bachisio m. padedda1, carlos e. wetzel2, antonella lugliè1, nicola sechi1, luc ector2 1 università degli studi di sassari, dipartimento di architettura, design e urbanistica (dadu), via piandanna 4, i-07100 sassari, italy 2 luxembourg institute of science and technology (list), environmental research and innovation department (erin), 41 rue du brill, l-4422 belvaux, luxembourg abstract – karst springs are considered among the most vulnerable groundwater-dependent ecosystems. despite their ecological value and importance as strategic water sources, mediterranean karst springs are still poorly investigated. the aim of this study was to analyse the epilithic diatom assemblages and to test their usefulness as indicators of environmental quality on the su gologone spring (central-eastern sardinia, italy), a biotope of great natural value and a precious source of drinking water. a total of 89 diatom taxa were found with 25 new records for sardinian running waters. species richness, shannon-wiener and pielou indices showed good biotic integrity. the dominant taxa were alkaliphilous, halophobous-oligohalobous exigent, xeno-oligosaprobic and characteristic of oligotrophic waters. the eutrophication/pollution index − diatom based (epi-d) and the navicula nitzschia surirella indices indicated respectively an excellent/good biological water quality and a low physical disturbance. however, the biological and chemical oxygen demand, and the microbiological variables (e. coli, fecal and total coliforms) revealed an organic contamination of the water, although moderate. the judgment provided by the epi-d should be verifi ed after updating of the index. in fact, 10 taxa found in this study are not currently considered by the epi-d method. keywords: bacillariophyta, biological quality, diatom indices, groundwater-dependent ecosystems, karst springs, mediterranean region, physical disturbance, sardinia * corresponding author, e-mail: lai.gg@tiscali.it introduction springs are aquatic habitats with unique characteristics and a high ecological value (odum 1971, cantonati 2003, cantonati et al. 2006). they belong to the group of groundwater-dependent ecosystems (gdes) (kløve et al. 2011) and provide contacts and connections between groundwater, surface water and terrestrial ecosystems (webb et al. 1998, scarsbrook et al. 2007, cantonati et al. 2012a, b). their nature of multiple ecotones creates a complex mosaic structure of different microhabitats (weigand 1998) that makes them important hotspots of biodiversity (cantonati et al. 2006, scarsbrook et al. 2007, ilmonen et al. 2012). springs are considered insular biotopes (mac arthur and wilson 1967, whittaker et al. 2001) or water islands (werum 2001) capable of hosting specifi c biocenoses because of their disjointed distribution within the landscape (cantonati et al. 2012b). in addition, they show a greater stability of physico-chemical parameters than other surface aquatic ecosystems (van der kamp 1995, glazier 1998). when pristine or still relatively sheltered from heavy human impacts, they are an important source of high quality water and can host endemic, rare, threatened and relict taxa (botosaneanu 1995, cantonati et al. 2006). springs are among the most interesting aquatic environments for the study of algal microfl ora, especially diatoms, since they are often the dominant algae and are considered useful indicators of environmental quality, because they can refl ect the ecological integrity of spring habitats (cantonati and lange-bertalot 2010, smol and stoermer 2010). the diatom fl ora of springs from south europe was investigated in spain (aboal et al. 1998, penalta-rodríguez and lópez-rodríguez 2007, delgado et. al. 2013), pyrenees (sabater and roca 1990, 1992), slovenia (menegalija and kosi 2008), bosnia and herzegovina (hafner 2008, kapetanović lai g. g., padedda b. m., wetzel c. e., luglič a., sechi n., ector l. 130 acta bot. croat. 75 (1), 2016 and hafner 2007), republic of macedonia (stavrevaveselinovska and todorovska 2010), greece (economouamilli and anagnostidis, 1981). studies on the diatom fl ora of italian springs have been carried out, especially in recent years, and in particular in the alpine region (dell’uomo 1975, cantonati 1998a, b, 1999, 2001, 2003, cantonati and ortler 1998, cantonati and pipp 2000, battegazzore et al. 2004, falasco et al. 2012, cantonati et al. 2006, 2012a, cantonati and spitale 2009, angeli et al. 2010, falasco and bona 2011, spitale and cantonati 2011, battegazzore 2012, battegazzore and morisi 2012, spitale et al. 2012a, b) and in the apennines (dell’uomo 1986, dell’uomo and torrisi 2000, 2001, torrisi and dell’uomo 2001, 2009). by contrast, very few studies have investigated springs of the mediterranean region and, in particular, of the main islands, such as sicily (mannino 2007) and sardinia (lange-bertalot et al. 2003). the diatom fl ora of karst springs, for example, has been explored only occasionally (dell’uomo 1990), despite these aquatic ecosystems are particularly important for the mediterranean area (civita 2008). furthermore, this geographic area is one of the major hotspot of plant biodiversity (myers et al. 2000, zachos and habel 2011). karst springs are considered among the most vulnerable gdes for both water quantity and quality (leibundgut 1998). they respond quickly to heavy rainfall and drought periods (meyer et al. 2003), which are typical of the mediterranean climate, with wide variations in discharge (white 1988). their load of suspended solids frequently varies with the discharge (herman et al. 2007) and can increase signifi cantly during the rainfall period, producing siltation events (weigand 1998). karst springs are also increasingly exposed to water abstractions and are particularly sensitive to pollution due to rapid infi ltration, thin or absent soil cover, high fl ow velocity of the water and poor self-purifi cation capacity of the karst aquifer (sasowsky and wicks 2000, daly et al. 2002). these factors can signifi cantly infl uence the composition and structure of their aquatic communities (smith et al. 2003, danehy and bilby 2009). because of the natural scarcity of permanent surface freshwater (fadda and pala 1992), in sardinia karst springs represent a precious and strategic source of drinking water. they are almost the exclusive source of drinking water for many urban centres (de waele and murgia 2001). some studies have emphasized the importance of gaining a greater knowledge and understanding of the biocenoses and ecological dynamics of karst springs in sardinia, also considering the signifi cant potential vulnerability of these ecosystems (de waele and murgia 2001, de waele 2003). diatoms, as bioindicators, can provide important information on the environmental integrity of these ecosystems. moreover, the geographic and ecological isolation of sardinia, located in the middle of the mediterranean sea, is recognized as an important prerequisite for the potential presence of endemic diatom species (lange-bertalot et al. 2003). this study focused on the su gologone spring, which is the most important spring in sardinia, whose algal fl ora was investigated at the end of the 80s (dell’uomo 1990). the main objectives were: a) to describe the current taxonomic composition and structure of the epilithic diatom assemblages of the spring and compare them with data from the previous study; b) to document the presence of interesting taxa by light and scanning electron microscopy; c) to assess for the fi rst time the environmental quality of the spring on the basis of physico-chemical and microbiological parameters and diatom indices. materials and methods study area the su gologone spring is the most important spring system of sardinia (bianco 1993). it is the main resurgence of the supramonte massif, a vast and complex karst system that extends in central-eastern sardinia (fig. 1) (de waele 2008). the hydrogeological basin that feeds the spring system covers a total surface area of about 160 km2 and is composed of middle jurassic-upper cretaceous dolostones and limestones covering a crystalline palaeozoic basement made out of granites and metamorphic rocks. it is an aquifer of regional importance with a hydrodynamic behaviour that is still in need of proper understanding (de waele 2008). the su gologone spring system is relatively isolated geographically due the surrounding impervious and largely inaccessible mountains. it is inside the gennargentu and the gulf of orosei national park (presidential decree 30/03/ 1998), which, however, has never been operative due to the opposition of the local communities. the su gologone is also a natural monument (regional decree 845/1998) and a respect zone (legislative decree 152/1999). the potential sources of disturbance in this territory are mainly local animal farming and hiking activities. the spring system (fig. 1) is located in the guthiddai valley at the foot of the north-eastern slope of mount uddè (806 m) and is composed of two points of water emergence, sa vena manna, a limno-rheocrene spring (104.5 m a.s.l.) and sa vena, a rheocrene spring (103.7 m a.s.l.). sa vena fig. 1. geographic location of the su gologone karst springs. diatoms and environmental quality of a karst spring of sardinia acta bot. croat. 75 (1), 2016 131 manna, is the largest point of water emergence (bianco 1993) and is a typical vauclusian spring (de waele 2008). the water gushes permanently from a fracture in the limestone rock and forms at the opening a narrow and deep pool, trapped between two high and almost vertical rock walls. the water fl ows slowly in the initial portion and faster in the following stretch. the discharge is high but very irregular, rarely less than 200 l s–1 in the drought period (bianco 1993) and up to 10 000 l s–1 during rainfalls (de waele 2008). sa vena is the smallest of the two water emergences of the su gologone karst system and has a much more modest water discharge. the water emerges from a small rock from which it is abstracted by a small aqueduct (bianco 1993) and immediately forms a small brook. water abstraction from sa vena supplies drinking water to the municipalities of oliena and dorgali (about 16 000 inhabitants). waters from both sa vena manna and sa vena fl ow, after a short distance, into the cedrino river and further downstream into lake cedrino, a eutrophic artifi cial lake, which supplies water to the downstream municipalities (about 20 000 inhabitants) (bianco 1993, padedda and sechi 2008). when fl oods of the cedrino river occur (on average twice a year), the spring system is submerged by the waters of the lake for a time ranging from a couple of hours to several days, making it impossible to supply drinking water (bianco 1993, de waele 2008). sampling the sampling was carried out at sa vena between december 2010 and june 2011. water samples for chemical and microbiological analyses were collected each month using 1 l polyethylene and glass bottles. the water samples were preserved in cold and dark conditions until the laboratory analyses were performed. epilithic diatoms were collected in december 2010 and june 2011 by scraping the upper surface of hard natural substrates (fi ve cobbles randomly selected in fl owing water for a total surface of at least 100 cm2) with a hard bristled toothbrush according to the methods of kelly et al. (1998) and ispra (2007). all diatom samples were preserved in 100 ml polyethylene bottles and fi xed in situ with a formaldehyde solution (4% v/v). measurements and analyses temperature, ph, conductivity and dissolved oxygen were measured in situ with a multi-parameter probe (ysi mps 556). alkalinity, chlorides (cl–), hardness, biological oxygen demand (bod), chemical oxygen demand (cod), soluble reactive phosphorus (p-po43–), total phosphorus (tp), ammonia nitrogen (n-nh4+), nitrites (n-no2–), nitrates (nno3–), total nitrogen (tn), reactive silica (rsi), total suspended solids (tss) and some ions (ca2+, mg2+, fe2+ and mn2+) were determined in the laboratory according to standard methods reported by cnr-irsa (1994) and apha (1998). escherichia coli, fecal and total coliforms were analysed using a membrane fi ltration method according to the cnr-irsa (1994). diatom subsamples (50 ml) were treated in the laboratory, after natural decantation for 48 h. the organic matter was eliminated by boiling the samples in hydrogen peroxide (30%). diluted hydrochloric acid (37%) was added to remove carbonates (ispra 2007). after being washed with distilled water, the cleaned frustules were mounted on permanent microscope slides using styrax® resin (refractive index = 1.59). diatoms were examined using a zeiss axiovert 10 light microscope (lm) at a 1000× magnifi cation. in the fi rst stage, the identifi cation of all species was done according to krammer and lange-bertalot (1986, 1988, 1991a, b, 2000), lange-bertalot et al. (2003), reichardt (2004), werum and lange-bertalot (2004), levkov (2009), lange-bertalot et al. (2011), żelazna-wieczorek (2011). light microscope images were taken with an axiocam zeiss digital camera mounted on the microscope and connected to a computer. afterwards, for each sample, at least 400 valves and/or frustules were counted. for the purpose of corroborating the lm study, several species were also examined using a zeiss evo ls10 environmental scanning electron microscope (sem). subsamples of the diatom suspension were air-dried on aluminium sheets and fi xed on aluminium stubs that were sputter-coated with gold (sputter coater edwards s-150a). the sem identifi cation of some diatom taxa was made after consultation of the current taxonomic literature available (e.g., idei and kobayasi 1986, reichardt 2009, van de vijver et al. 2011, jovanovska et al. 2013). data processing all species observed in the samples were used to draw up a complete fl oristic list which was compared with that obtained in a previous study performed on the algal microfl ora of the su gologone spring at the end of the 80s (dell’uomo 1990). the ecological preferences of all diatom taxa were investigated primarily by referring to dell’uomo (2004), torrisi and dell’uomo (2009) and van dam et al. (1994). in addition, the fi rst indications on the vulnerability degree of the observed taxa were taken from the german red list of threatened diatoms proposed by langebertalot and steindorf (1996). this red list, although compiled for the local diatom fl ora and not updated since 1996, is the only international reference currently available for the classifi cation of diatoms based on their vulnerability. the taxa present in the counts were used for the analysis of structure of the diatom assemblages and the evaluation of the environmental quality and physical disturbance of the spring. the abundance values of diatom taxa in each sample were transformed into relative abundance values which express the percentage contribution of each species compared to the total contribution of all species present in the count of each sample. the relative percentage abundance of each taxon was calculated by dividing the number of valves and/or frustules by the total number of valves and/ or frustules of all the taxa counted in each sample and multiplying this quotient by 100. the biotic integrity of the spring was estimated by calculating species richness, shannon-wiener diversity index, 2 based logarithm (h’), and evenness (j’) (shannon 1948, shannon and weaver 1949, pielou 1975). synthetic ecological spectra of ph, salinity, organic lai g. g., padedda b. m., wetzel c. e., luglič a., sechi n., ector l. 132 acta bot. croat. 75 (1), 2016 matter and nutrients were obtained considering both the presence and the relative abundances of the taxa. the taxa with a wider ecological range were placed between the respective autecological levels, dividing equally the values of their relative percentage abundance. biological water quality was evaluated using the eutrophication/pollution index − diatom based (epi-d) (dell’uomo 2004). the epi-d index was chosen because it is the only index developed in italy, after a long period of research conducted mainly on diatom communities of the central apennines, but also of the southern alps and apennines. moreover, this index has already been applied with good results to different italian springs, including karst springs such as the clitunno springs (torrisi and dell’uomo 2001). this index, based on the zelinka and marvan formula (zelinka and marvan 1961), considers the sensitivity (affi nity/tolerance) of diatoms to nutrients, organic matter and degree of water mineralization, providing an estimation of the general quality of the water body. the values of the epi-d were expressed in the original scale from 0 to 4, with values close to 0 indicating excellent quality and values close to 4 indicating very bad quality. the results around the threshold values were considered as transition classes (transition interval ± 0.05). the degree of physical disturbance (siltation) was inferred by applying the navicula nitzschia surirella indices (nns and nns’) (battegazzore et al. 2003, 2004, 2007). the nns and nns’, based on the work by hill et al. (2001) and bahls (1993), provide an estimate of the physical disturbance in an aquatic ecosystem due to natural and anthropogenic factors assuming that siltation events determine an increase in the proportion of motile taxa within the community, both in terms of number of taxa and of number of individuals. the nns (qualitative index) was calculated as a percentage ratio between the number of motile taxa belonging to the genera navicula, nitzschia and surirella and the total number of taxa recorded in each sample. the nns’ (quantitative index) was calculated as a percentage ratio between the number of individuals belonging to three genera navicula, nitzschia and surirella and the total number of individuals recorded in each sample. the values of both indices range from 0 to 100. values close to 0 represent a low tab. 1. monthly and average values of the physico-chemical and microbiological variables measured and analyzed in the karst spring su gologone (sa vena). d. l. – detection level; n/a – not available; bod – biological oxygen demand; cod – chemical oxygen demand; tp – total phosphorus; tn – total nitrogen; tss – total suspended solids; rsi – reactive silica; ufc – units forming colony. variables d. l. 07/12/10 12/01/11 10/02/2011 07/03/11 18/04/11 18/05/11 13/06/11 average temperature (°c) -5 °c 12.4 12.0 12.0 12.0 12.0 13.0 13.0 12.3 ph (units) 0 7.6 7.9 8.0 7.8 8.3 8.1 7.5 7.9 conductivity (μs cm–1) 0 356 254 353 336 338 322 344 329 alkalinity (meq l–1) n/a 2.7 2.7 2.9 2.7 2.6 2.8 2.6 2.7 dissolved oxygen (mg l–1) 0 9.2 10.6 11.6 7.7 11.7 12.2 10.7 10.5 oxygen saturation (%) 0 86 98 107 72 109 116 73 94 bod (mg l–1) n/a 3.6 1.6 3.3 0.9 2.9 4.6 2.9 2.8 cod (mg l–1) 5 – < d. l. < d. l. 18.1 < d. l. 9.2 23.7 17.0 cl– (mg l–1) 5 21.3 14.2 14.2 12.4 16.0 13.5 17.7 15.6 hardness (mg l–1) 5 120 105 143 133 145 143 155 135 p-po43– (μg l–1) 4 4.0 4.0 4.0 < d.l. 4.0 4.0 < d.l. 4.0 tp (μg l–1) 4 14.0 38.0 11.0 12.0 13.0 13.0 12.0 16.0 n-nh4+ (μg l–1) 5 14.0 10.0 15.0 10.0 13.0 13.0 33.0 15.0 n-no2– (μg l–1) 1 < d. l. < d. l. < d. l. 1.0 < d. l. < d. l. 1.0 1.0 n-no3– (μg l–1) 50 554.0 560.0 271.0 388.0 544.0 458.0 551.0 475.0 tn (μg l–1) 300 719.0 679.0 762.0 643.0 679.0 769.0 943.0 742.0 rsi (mg l–1) 0.05 1.8 2.1 1.8 1.5 2.0 2.0 2.0 1.9 ca2+ (mg l–1) n/a 40.1 40.1 88.0 52.0 44.0 46.0 49.0 51.0 fe2+ (mg l–1) 0.001 0.008 0.005 0.011 0.013 0.008 0.007 0.013 0.009 mg2+ (mg l–1) n/a 4.9 1.2 19.0 1.0 8.5 6.6 7.9 6.9 mn2+ (mg l–1) 0.01 < d. l. < d. l. 0.011 0.019 < d. l. 0.010 0.023 0.016 tss (mg l–1) n/a 3.0 – 1.0 3.0 1.0 13.0 0.4 3.6 escherichia coli (ufc 100 ml–1) 1 38 69 5 2 80 25 158 54 fecal coliforms (ufc 100 ml–1) 1 51 115 20 1 103 170 342 115 total coliforms (ufc 100 ml–1) 1 95 202 25 8 184 210 606 190 diatoms and environmental quality of a karst spring of sardinia acta bot. croat. 75 (1), 2016 133 level of physical disturbance, whereas values close to 100 indicate a high level of physical disturbance. all indices applied in this study, except the nns and nns’, were calculated using the software omnidia 7 v. 8.1 (lecointe et al. 1993). statistical analyses principal component analysis (pca) was performed using r 3.1.3 (venables et al. 2015) on environmental variables to detect temporal differences among samplings and to better characterize the diatom samplings made in december and june. for the ordination analysis data were normalized using a (x-mean)/standard deviation. in the pca analysis, we included temperature (temp), conductivity (cond), hardness (hardn), biological oxygen demand (bod), chlorides (cl–), nitrates (n-no3–), ammonia nitrogen (n-nh4+), soluble reactive phosphorus (p-po43–). differences in the specifi c composition of the two seasonal diatom assemblages were analysed with a t-test performed on the abundances of the taxa present in each diatom sample, using r 3.1.0 (r core team 2012). results environmental variables the monthly and average values of the physico-chemical and microbiological variables are reported in tab. 1. during our study, the water temperature varied from a minimum of 12 °c (january–april) to a maximum of 13 °c (may–june). waters had a slightly basic ph (7.5–8.3), with an intermediate level of hardness (105–155 mg l–1 caco3). ca2+ was the most abundant cation and ranged between 40.1 mg l–1 (december–january) and 88.0 mg l–1 (february). its highest concentration coincided with the maximum of mg2+ (19.0 mg l–1), which was always much lower in the other months (1.0–8.5 mg l–1). fe2+ and mn2+were present as trace elements in very small amounts. the conductivity ranged from 254 to 356 μs cm–1. the percent water oxygenation was generally > 75%. slight supersaturation was observed in february, april and may (107–116). p-po43– showed values of 4.0 μg l–1 in almost all months, while tp values ranged from 11.0 to 38.0 μg l–1. n-no3– was the most abundant inorganic nitrogen compound (271.0–560.0 μg l–1). tn concentrations were in the range of 643.0– 943.0 μg l–1. bod and cod values showed peaks in may (4.6 mg l–1) and june (23.7 mg l–1). the bacterial load varied widely with higher densities observed in the spring, from april to june. suspended solids had very low concentrations with the exception of an isolated highest value in may (13 mg l–1). the pca on environmental variables showed relationships among variables and between variables and samples (fig. 2). the fi rst axis explained 38.3% of the variance and the second 23.7%. n-nh4+ (0.48), water temperature (0.45) and hardness (0.44) were positively related to the fi rst axis, characterizing the diatom sampling in june. the second axis was associated with n-no3– (0.54), cl– (0.48) and p-po43– (0.44) at opposite positions, characterizing the diatom sampling in december. diatom assemblages the complete list of taxa and their ecological preferences are reported (tab. 2). the list is composed of a total of 89 diatom taxa, belonging to 36 genera. the genera with the highest number of species were navicula (10), nitzschia (9) and achnanthidium (7), followed by amphora, cocconeis, diploneis and gomphonema (6). according to van dam et al. (1994), the majority of the species observed mainly occur in water bodies. only 9 taxa (10% of the total) are not strictly linked to aquatic environments and belong to the categories “nearly exclusively occurring outside water bodies” and “mainly occurring on wet and moist or temporarily dry places”. of the total 89 taxa identifi ed in our study, 52 (58% of the total) are included in different categories of the german diatom red list (lange-bertalot and steindorf 1996). however, only 5 taxa are classifi ed as “in regression” and 2 taxa “extremely rare”. the diatom assemblages included 25 new records for the su gologone spring, and more for sardinian running waters in general. lm and sem images of some abundant, frequent, rare and occasional taxa, including some new records, were reported respectively in figs. 5 a–p and figs. 6 a–o. overall, there were 39 taxa present in the counts (44% of the total), belonging to 20 genera with 1 genus of centrales (ellerbeckia) and 19 genera of pennales (38 taxa). they included 6 abundant taxa (relative abundance more than 5%), 10 frequent (relative abundance between 1.5 and 5%) and 23 rare (relative abundance less than 1.5%). achnanthidium subatomus, amphora pediculus and achnanthidium minutissimum were the most abundant taxa in the fig. 2. principal component analysis (pca) biplot with the environmental variables. temp – water temperature; cond – conductivity; hardn – hardness; bod – biological oxygen demand; cl– – chlorides; n-no3– – nitrates; n-nh4+ – ammonia nitrogen; p-po43– – soluble reactive phosphorus. dec – december; feb – february; mar – march; apr – april; may – may; jun – june. lai g. g., padedda b. m., wetzel c. e., luglič a., sechi n., ector l. 134 acta bot. croat. 75 (1), 2016 tab. 2. list of the diatom taxa observed in the karst spring su gologone (sa vena) and their ecological preferences. all the acronyms used for ph, salinity, saprobity and trophic state are explained in the legend of the fi gure 3. ma – maximum relative abundance in at least one sample: r – rare: < 1.5%, f – frequent: 1.5 – 5%, a – abundant: > 5%; rl – germain red list of diatom taxa (lange-bertalot and steindorf 1996): * – currently not considered endangered, ** – surely not endangered, 2 – highly endangered, 3 – endangered, d – insuffi cient data, g – considered at risk, v – in regression, r – extremely rare; moisture: 1 – never, or only very rarely, occurring outside water bodies, 2 – mainly occurring in water bodies, sometimes in wet places, 3 – mainly occurring in water bodies, also rather regularly on wet and moist places, 4 – mainly occurring in wet and moist or temporarily dry places, 5 – nearly exclusively occurring outside water bodies. in bold: new diatom records for the su gologone karst spring. taxa highlighted in gray color are not currently included in the eutrophication/pollution index − diatom based method. taxa 19 85 –1 98 6 20 10 –2 01 1 m a r l ph sa lin ity sa pr ob ity tr op hi c st at e m oi st ur e achnanthes coarctata (brébisson) grunow χ x * * n hb-oe x-o hypo-oligo 5 achnanthidium acsiae wojtal, e. morales, van de vijver & ector x – – – – – – achnanthium affi ne (grunow) czarnecki x * achnanthidium bioretii (h. germain) o. monnier, lange-bertalot & ector x v n hb-oe x-o hypo-oligo 4 achnanthidium exiguum (grunow) czarnecki x * * ak oe-ot o-β oligo-meso 3 achnanthidium lineare w. smith x x – n – – – – achnanthidium minutissimum sensu lato (kützing) czarnecki x x a – n-ak oe o oligo 3 achnanthidium sp. x – – – – – – achnanthidium subatomus (hustedt) lange-bertalot x a * – – – – – amphipleura pellucida (kützing) kützing x r * ak oe o-β oligo-meso 2 amphora indistincta levkov x * – – – – – amphora meridionalis levkov x – – – – – – amphora ovalis (kützing) kützing x x r * * ak ot o-β oligo-meso 1 amphora pediculus (kützing) grunow ex a. schmidt x x a * * ak ot x-β oligo-meso 3 amphora vetula levkov x – – – – – – asterionella formosa hassall x * * ak oe-ot o-β oligo-meso 1 caloneis bacillum (grunow) cleve x * * caloneis fontinalis (grunow) cleve-euler x f – – – – – – caloneis lancettula (schulz) lange-bertalot & witkowski x – – – – – – campylodiscus hibernicus ehrenberg x * cocconeis euglypta ehrenberg x x r – ak ot-h o-α oligo-eu 2 cocconeis neothumensis krammer x v akb – o – – cocconeis placentula sensu lato ehrenberg x x f – ak ot x-β oligo-meso 2 cocconeis placentula var. lineata (ehrenberg) van heurck x x * * ak ot x-β oligo-meso 2 cocconeis placentula var. placentula sensu jahn et al. 2009 x * * – – – – – cocconeis pseudolineata (geitler) lange-bertalot x f d ak ot x-β oligo-meso – cyclotella ocellata pantocsek x * cymbella affi nis kützing x * ak hb-os o oligo 2 cymbella helvetica kützing x v cymbella lanceolata (ehrenberg) van heurck x v cymbopleura cuspidata (kützing) krammer x v denticula tenuis var. crassula (nägeli) hustedt x v diatoma hyemalis (roth) heiberg x * diatoma mesodon (ehrenberg) kützing x x r * n hb-oe o oligo 2 diploneis elliptica (kützing) cleve x * a-k oe x-o hypo-oligo 3 diploneis minuta j.b. petersen x r – – – – 5 diploneis cf. oculata x f – – – – – – diploneis oculata (brébisson) cleve x * diploneis separanda lange-bertalot x r – – – – – – diploneis sp. 1 x r – – – – – – diploneis sp. 2 x – – – – – – diatoms and environmental quality of a karst spring of sardinia acta bot. croat. 75 (1), 2016 135 tab. 2. – continued taxa 19 85 –1 98 6 20 10 –2 01 1 m a r l ph sa lin ity sa pr ob ity tr op hi c st at e m oi st ur e ellerbeckia arenaria (moore ex ralfs) r.m. crawford x x r * * a-k hb o hypo 4 encyonema elginense (krammer) d.g. mann x 2 encyonema silesiacum (bleisch) d.g. mann x * n ot β meso 1 encyonema ventricosum (c. agardh) grunow x x * n oe o oligo – encyonema vulgare krammer x v – – – – – eolimna minima (grunow) lange-bertalot x r * * ak h α eu 3 epithemia argus (ehrenberg) kützing x * epithemia sp. x – – – – – – eunotia mucophila (lange-bertalot, nörpel & alles) lange-bertalot x g eunotia pectinalis (kützing) rabenhorst x r v ac hb-oe x-o hypo-oligo 3 eunotia sp. x – – – – – eunotia valida hustedt x – fallacia mitis (hustedt) d.g. mann x r – ak oe x oligo – fallacia subhamulata (grunow) d.g. mann x f * n oe o-β meso 3 fragilaria capucina desmazières x r – n-ak oe o oligo – fragilaria mesolepta rabenhorst x * * fragilaria recapitellata lange-bertalot & metzeltin x – n oe o-β meso – frustulia vulgaris (thwaites) de toni x * * ak oe o-β meso 3 geissleria decussis (østrup) lange-bertalot & metzeltin x * * ak oe o oligo 3 gomphonema acuminatum ehrenberg x * * gomphonema angustius e. reichardt x – – – – – – gomphonema clavatum ehrenberg x * n-i hb x-o oligo 3 gomphonema elegantissimum e. reichardt & lange-bertalot x r – – – – – – gomphonema micropus kützing x * ak α-meso β eu – gomphonema productum (grunow) lange-bertalot & e. reichardt x d gomphonema pumilum (grunow) e. reichardt & lange-bertalot x * gomphonema pumilum var. rigidum e. reichardt & lange-bertalot x – – – – – – gomphonema truncatum ehrenberg x x * ak ot o-β oligo-meso – gomphosphenia grovei var. lingulata (hustedt) lange-bertalot x – – – – – – gyrosigma sciotense (sullivant & wormley) cleve x halamphora montana (krasske) levkov x * ak oe o-β meso 4 humidophila contenta r.l. lowe et al. x * * ak oe-ot o-β oligo-meso 4 karayevia clevei (grunow) bukhtiyarova x r * ak oe o-β oligo-meso 1 karayevia ploenensis var. gessneri (hustedt) bukhtiyarova x r – – – – – – luticola goeppertiana (bleisch) d.g. mann x r * * ak h-β α-p eu-hyper 3 melosira varians c. agardh x x * * ak ot x-α oligo-eu 2 meridion circulare (greville) c. agardh x x f * * ak hb-oe x-o oligo 1 navicula antonii lange-bertalot x r * * – – – – – navicula chiarae lange-bertalot & genkal x – – – – – – navicula cincta (ehrenberg) ralfs x * * ak h α eu 4 navicula cryptocephala kützing x * * navicula cryptotenella lange-bertalot x x f – ak oe o-β oligo-meso 2 navicula cryptotenelloides lange-bertalot x r * * ak – – – – navicula gregaria donkin x r * * ak h α meso 3 navicula menisculus schumann x v navicula reichardtiana lange-bertalot x – ak oe o-β oligo-meso – navicula tripunctata (o.f. müller) bory x x a * * ak hb-oe o oligo 3 lai g. g., padedda b. m., wetzel c. e., luglič a., sechi n., ector l. 136 acta bot. croat. 75 (1), 2016 winter sample (respectively, 28%, 23% and 14%). prevailing in the spring sample were a. pediculus, a. subatomus and planothidium frequentissimum (26%, 15% and 9% respectively %). the results of the biotic integrity indices are reported in tab. 3. species richness varied from 25 in winter to 31 in late spring and the diatom samples showed 20 taxa that were common to both seasons. the values of the shannonwiener diversity index and the pielou index (evenness) were respectively 3.27 and 0.70 in winter and 3.69 and 0.75 in late spring. the t-test, performed on the abundances of the taxa present in each sample, indicated signifi cant differences between the diatom assemblages in winter and late spring (p < 0.01). synthetic ecological spectra of ph, salinity, organic matter and nutrients are reported in figs. 3a–d. the ph spectrum (fig. 3a) showed a dominance of alkaliphilous species (69%), while circumneutral diatoms were present in insignifi cant amounts (4%). in the salinity spectrum (fig. 3b), the halophobous and oligohalobous exigent (38%) prevailed over the oligohalobous tolerant diatoms (32%) that tolerate moderate concentrations of dissolved salts and, in particular, of chlorides. the halophilous forms that prefer water with a higher salt content were present in a negligible quantity (2%). the saprobic spectrum (fig. 3c) revealed a greater presence of xenosaprobic and oligosaprobic taxa (43%) in respect to the β-mesosaprobic species (18%). the presence of the α-mesosaprobic (7%) and polysaprobic species (4%), which require a higher quantity of organic compounds, was negligible. with regard to preferences for the tab. 2. – continued taxa 19 85 –1 98 6 20 10 –2 01 1 m a r l ph sa lin ity sa pr ob ity tr op hi c st at e m oi st ur e navicula radiosa kützing x * * navicula veneta kützing x r * * ak h-β α-p eu-hyper 3 navicula vilaplanii (lange-bertalot & sabater) lange-bertalot & sabater x r – – – – – nitzschia commutata grunow x r * – – – – – nitzschia dissipata (kützing) grunow x x f – ak ot o-α meso 3 nitzschia fonticola (grunow) grunow x x f – ak oe o-β oligo-meso 1 nitzschia frustulum (kützing) grunow x – ak ot-h β-α meso-eu 3 nitzschia inconspicua grunow x r * * ak ot-h β-α meso-eu 3 nitzschia linearis (c. agardh) w. smith x x r – ak ot-h β-α meso-eu 3 nitzschia recta hantzsch ex rabenhorst x – ak ot β meso 1 nitzschia sigmoidea (nitzsch) w. smith x * * nitzschia sociabilis hustedt x f * * n ot-h β-α meso-eu 1 nitzschia solgensis cleve-euler x v ak oe-ot o-β oligo-meso 4 placoneis clementis (grunow) e.j. cox x * ak oe-ot o-β oligo-meso 3 planothidium delicatulum (kützing) round & bukhtiyarova x * planothidium ellipticum (cleve) m.b. edlund x – planothidium frequentissimum (lange-bertalot) lange-bertalot x a – ak hb-oe α-poli ind – planothidium lanceolatum (brébisson ex kützing) lange-bertalot x x a – ak hb-oe o oligo 3 platessa hustedtii (krasske) lange-bertalot x * ak hb-oe o oligo 4 pleurosigma elongatum w. smith x * reimeria sinuata (w. gregory) kociolek & stoermer x * * n oe o oligo 3 reimeria uniseriata s.e. sala, j.m. guerrero & ferrario x – – – – – – rhoicosphenia abbreviata (c. agardh) lange-bertalot x x r * * ak oe o-β oligo-meso 2 sellaphora sp. x – – – – – – simonsenia delognei (grunow) lange-bertalot x * * – ot α eu 3 staurosira construens ehrenberg x ak oe o oligo 1 ulnaria acus (kützing) aboal x * ulnaria ulna (nitzsch) compère x x * ak ot β meso 2 tab. 3. seasonal values of the biotic integrity indices applied to diatom assemblages of the karst spring su gologone (sa vena). species richness diversity (h’) evenness (j’) december 2010 25 3.27 0.70 june 2011 31 3.69 0.75 diatoms and environmental quality of a karst spring of sardinia acta bot. croat. 75 (1), 2016 137 trophic characteristics of the water bodies (fig. 3d), the species characteristic of oligotrophic waters (40%) were most abundant in respect to the species characteristic of mesotrophic waters (23%). the eutraphentic taxa, characteristic of more nutrient-rich water bodies, were insignifi cant (2%) as were indifferent taxa with a wide ecological range (7%). diatom indices the epi-d values in winter (0.99) and in late spring (1.01) revealed an excellent/good water quality (class i–ii) in both seasons (fig. 4). the nns and nns’ indices revealed a low physical disturbance both in winter and in late spring. in particular, the nns index showed a slightly greater number of mobile fig. 3. synthetic ecological spectra of the diatom taxa observed in the karst spring su gologone (sa vena): a) ph: ak – alkaliphilous, n – circumneutral; b) salinity: hb – halophobous, oe – oligohalobous exigent, ot – oligohalobous tolerant, h – halophilous; c) saprobity: x – xenosaprobic, o – oligosaprobic, β – β-mesosaprobic, α – α-mesosaprobic; p – polysaprobic; d) trophic state; taxa characteristic of environment: oligo – oligotrophic, meso – mesotrophic, eu – eutrophic; ind – indifferent taxa; for all spectra: n.d. – no data (ecological preferences poorly known). fig. 4. seasonal values of the indices epi-d (the eutrophication/ pollution index − diatom based, scale from 0 to 4), nns and nns’ (the navicula nitzschia surirella indices, scale from 0 to 100) used for the evaluation of the biological water quality and physical disturbance of the karst spring su gologone (sa vena). fig. 5. light microscopy. a) achnanthidium lineare w. smith; b-c) achnanthidium subatomus (hustedt) lange-bertalot; d) amphora indistincta levkov; e) caloneis fontinalis (grunow) cleve-euler; f) caloneis lancettula (schulz) lange-bertalot & witkowski; g) cocconeis neothumensis krammer; h) amphora meridionalis levkov; i) diatoma mesodon (ehrenberg) kützing; j) diploneis separanda lange-bertalot; k) diploneis sp. 1; l) diploneis sp. 2; m) diploneis minuta j.b. petersen; n) gomphonema angustius e. reichardt, initial valve; o) meridion circulare (greville) c. agardh; p) navicula vilaplanii (lange-bertalot & sabater) lange-bertalot & sabater in rumrich et al.. scale bar = 10 μm. fig. 6. scanning electron microscopy. a) achnanthidium lineare w. smith, internal view of raphe valve; b) achnanthidium subatomus (hustedt) lange-bertalot, external view of raphe valve; c) cocconeis neothumensis krammer, external view of raphe valve; d) amphora indistincta levkov, external view; e) amphora vetula levkov, external view; f) diatoma mesodon (ehrenberg) kützing, external view; g) caloneis fontinalis (grunow) cleve-euler, external view; h) caloneis lancettula (schulz) lange-bertalot & witkowski, external view; i) diploneis minuta j.b. petersen, external view; j) diploneis sp. 1, external view; k) diploneis separanda lange-bertalot, external view; l) diploneis sp. 2., external view; m) gomphonema angustius e. reichardt, internal view of initial valve; n) meridion circulare (greville) c. agardh, external view; o) navicula vilaplanii (lange-bertalot & sabater) lange-bertalot & sabater in rumrich et al., external view. scale bars = 5 μm. lai g. g., padedda b. m., wetzel c. e., luglič a., sechi n., ector l. 138 acta bot. croat. 75 (1), 2016 taxa in spring (41.9%) than in winter (40.0%). also the nns’ index showed a slightly higher number of individuals belonging to the mobile genera in late spring (23.4%) than in winter (16.7%). discussion despite its small dimensions, the su gologone spring at sa vena hosts a diatom fl ora with high total species richness, refl ecting a high diversity of microhabitats and a moderate level of anthropic disturbance. the moderate current, due to the mild slope of the terrain, may have contributed to the high number of species found. in fact, according to many authors, the fast current generally present in the rheocrene springs may favour the rheophilous taxa, determining a loss in species number and diversity (sabater and roca 1992, cantonati 1998a, 2001, cantonati et al. 2012a). moreover, rheocrene springs on carbonate substrate seem to be generally characterized by epilithic diatom communities with a lower diversity of species than rheocrene springs on siliceous substrate (e.g., cantonati 1998a, gesierich and kofl er 2010). the total species richness (89 taxa) was higher than that observed in the previous investigation carried out at the end of the 80s (49 taxa) (dell’uomo 1990), suggesting a possible role of the protective measures present since 1998 in the studied area. in fact, in other geographic areas, like for example the alpine region, the species richness found in protected areas is higher than in non-protected areas (e.g., falasco and bona 2011). the comparison between our list of species and that reported by dell’uomo (1990) indicated considerable differences in the su gologone spring after 25 years, with the presence of 21 taxa in common in both studies, 29 taxa detected only in the previous study and 68 taxa observed only in this study. the latter included 25 new records for the spring and more in general for running waters of sardinia. the differences found seem mostly due to advances of taxonomic knowledge because several taxa like navicula anto nii, navicula vilaplanii, diploneis separanda, gomphonema angustius, amphora indistincta, amphora meridionalis, amphora vetula, were described some years after the study carried out in 1985–1986. except for the temperature, previous environmental data of the spring are not available. however, differences found could be also related with environmental changes. in fact, species like campylodiscus hibernicus, cymbella helvetica, encyonema elginense, cyclotella ocellata, navicula radiosa, (xenosaprobic and characteristic of hypotrophic or ultraoligotrophic environments) were not detected during our investigation, and nor were planothidium ellipticum and diatoma hyemalis, the only two taxa belonging to the nordic-alpine fl ora (dell’uomo 1990). in the central apennines, d. hyemalis is reported as a species “at risk of extinction”. it seems to be increasingly rare and exclusively located at high altitudes, in cold well-oxygenated waters, and with a fast current (torrisi and dell’uomo 2009). a longer study could ascertain the real state of p. ellipticum and d. hyemalis in the su gologone spring, also taking into account a possible effect of global warming on these cold water stenotherm species. instead, the new records found are probably explicable by the lack of specifi c studies on communities of benthic diatoms in the running waters of sardinia. in accordance with the results of the previous study, the diatom species of the su gologone spring have mostly a cosmopolitan and boreal-mountain distribution. the diatom assemblages showed some common features with some alpine springs, like the presence of abundant and frequent taxa, such as achnanthidium minutissimum, meridion circulare and planothidium lanceolatum (cantonati 1998a, battegazzore et al. 2004, gesierich and kofl er 2010, falasco and bona 2011) and a high number of subdominant and rare taxa (e.g., cantonati 1998a, gesierich and kofl er 2010). however, a comparison with the fl oristic lists from other italian springs showed a higher number of species in common with the diatom communities found in different springs and headwaters of rivers with the calcareous substrate found in the central apennine region (e.g., torrisi and dell’uomo 2009). several taxa were also in common with diatom communities of springs in the castellón province (aboal et al. 1998), pyrenean springs (sabater and roca 1992) and spring-fed streams on majorca island (delgado et al. 2013). overall, the spring hosted a small group of taxa that are not closely linked to the aquatic environment according to van dam et al. (1994). for example, achnanthes coarctata and diploneis minuta were reported as species occurring nearly exclusively outside water bodies. although this group of species accounted for 10% of the total species observed, they are part of the biodiversity of the spring and underline its nature of ecotone. the vulnerable species according to the german red list were achnanthidium bioretii, cocconeis neothumensis, encyonema vulgare and nitzschia solgensis, considered species “in regression”, d. minuta and n. vilaplanii included as “extremely rare”. all these species were occasional members of the diatom communities of the spring. this information has only a purely indicative and preliminary value for sardinia. in fact, data on distribution and abundance of diatom taxa are recent and still scarce and prevent an extended comparison with the inland waters of the island. however, these fi rst indications could integrate those from other regions and provide a contribution to a fi rst possible diatom red list for the italian territory. the indices of biotic integrity revealed high species richness and diversity and a balanced distribution of the species in the current diatom assemblages. the species richness and diversity were higher in late spring, probably due to a greater degree of light irradiation. in permanent springs with a constant temperature, the light regime is an important factor for the seasonal changes in the algal communities (e.g., ward and dufford 1979). spring is also reported to be the season with the highest diatom diversity (e.g., cantonati 1998a). the t-test analysis indicated signifi cant seasonal changes in the diatom assemblages, unlike what was reported for example for the alpine springs on a carbonate substrate (cantonati 1998a). diatoms and environmental quality of a karst spring of sardinia acta bot. croat. 75 (1), 2016 139 the observed diatom assemblages were dominated by achnanthidium subatomus in winter and amphora pediculus in late spring. as to a. subatomus, no information is available about its preference for organic matter, while with respect to its trophic preferences krammer and lange-bertalot (1991b) indicated that it privileges oligo-mesotrophic waters. amphora pediculus is a xeno-β-mesosaprobic species and prefers oligo-mesotrophic environments. other dominant species were a. minutissimum, navicula tripunctata, planothidium frequentissimum, caloneis fontinalis, nitzschia dissipata and p. lanceolatum. among these species, n. dissipata is an oligo-α-mesosaprobic species typical of mesotrophic environments, whereas p. frequentissimum is an α-meso-polisaprobic species and colonizes environments with different trophic states (van dam et al. 1994). all other species are oligosaprobic and typical of oligotrophic waters, except for c. fontinalis, about which no autecological information is available. in contrast, fragilaria mesolepta, a species xeno-oligosaprobic and typical of hypo-oligotrophic waters, dominated the diatom assemblages in a previous study (dell’uomo 1990). the ecological preferences of the dominant species in the current assemblages suggest a possible slight deterioration of the water quality, especially of the organic type, over the years. in general, the synthetic ecological spectra highlighted the dominance of species that prefer alkaline waters and low to moderate concentrations of dissolved salts, organic matter and nutrients. these results were very consistent with those of the physico-chemical analyses and epi-d index. in fact, the water was characterized by a slightly basic ph and a medium hardness and degree of water mineralization. with respect to the nutrient concentrations, in particular of phosphorus, the su gologone spring may be considered an ecosystem with a low trophic level, which is comparable with that of the alpine springs (cantonati 1998a). however, their values and in particular nitrate values, do not refl ect the condition of pristine environments. moreover, bod and especially cod, and microbiological variables indicated a not negligible degree of organic contamination in certain months. it is potentially attributable to pasture activities carried out in the surrounding area, the pollutant load of the cedrino river and the periodic submersion of the spring by the cedrino lake. the highest densities of both escherichia coli and fecal and total coliforms were observed in the spring season, probably due to the reduction of the discharge. this kind of contamination, although moderate, is important for the water quality, especially in view of its use as drinking water. the epi-d index revealed an excellent/good biological water quality (class i–ii) in both seasons. however, a slight deterioration of the biological quality was observed in late spring, probably as a result of some peaks of bod and cod in the months before the diatom sampling (june). the judgment provided by the epi-d index seems fairly reliable and probably only slightly overestimates the quality of water. in our study eolimna minima and navicula gregaria, species strictly α-mesosaprobic and nitzschia inconspicua and nitzschia sociabilis, species β-α-mesosaprobic, showed very low relative abundances (0.1–1.6%). among α-polysaprobic species p. frequentissimum was the taxon with higher relative abundance (5.1% in winter and 9.1% in late spring). the epi-d index integrates the sensitivity of each species to salinity, organic matter and nutrients and it mediates the sensitivity of each species among different environmental variables. it provides a global judgment on the quality of the water body refl ecting the interactions of several variables (dell’uomo 2004), rather than just organic matter. the pca analysis performed on the environmental variables also indicated that the bod was less important than other variables in december and june and may have affected to a lesser extent the structure of the diatom assemblages. however, 10 taxa found in this study (26% of the total), including p. frequentissimum, currently are not considered by the epi-d method and their values of “i” and “r” are not present in the omnidia software. for this reason, our result should be checked after the update of the epi-d index in order to obtain a more accurate assessment. the nns and nns’ indices indicated a low degree of physical disturbance, in accordance with the low concentrations of suspended solids observed throughout the period of the study. both indices indicated a slight worsening in late spring, following an anomalous peak of the concentration of suspended solids in may. these results were also consistent with the low physical disturbance reported for the su gologone spring on the basis of the disturbance index for karst environments (kdi) (de waele 2009). this study has contributed to a greater understanding of the diatom fl ora of the su gologone spring, which is the most important of sardinia. it has led to an initial ecological characterization of the diatom assemblages, albeit limited to winter and late spring (wet period). moreover it is the fi rst work on the use of epilithic diatoms as indicators of biological quality and physical disturbance in the karst springs of sardinia. despite the presence of some potential impacts in the territory, the su gologone spring showed a high environmental quality, probably only slightly overestimated by the epi-d index, and a good level of biotic integrity, refl ecting the scarce human presence and the very high degree of naturalness of the hydrogeological basin. the high species richness and the presence of diatom taxa included in the german red list also underline the importance of protecting and preserving this important biotope. however, the periodic submersion of the spring due to fl oods of the cedrino river should be assessed more accurately in order to evaluate the role of fl oods as stressors for the ecosystem and their aquatic communities. in addition, the organic contamination detected in this study, albeit moderate, remains a problem that should no longer be neglected. the biocenoses and ecological dynamics of the sardinian karst springs are still largely unknown. yet these peculiar environments deserve greater consideration because of their high natural value and vulnerability and their strategic importance as sources of drinking water. further investigations will contribute to the gathering of useful information for their long-term monitoring and management. lai g. g., padedda b. m., wetzel c. e., luglič a., sechi n., ector l. 140 acta bot. croat. 75 (1), 2016 acknowledgements this study was funded by the province of nuoro, and in the framework of the taxodia project (list luxembourg institute of science and technology).. the authors thank bastianina manca and pasqualina farina for their precious help in the laboratory analysis and salvatore marceddu for the work done with the sem. the authors are also grateful to jo de waele and francesco murgia for 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molero and montserrat 2007) still accept the the genus lavatera for them. the annual mallows lavatera maroccana (batt. & trab.) maire, l. punctata all., and l. trimestris l. are traditionally placed in lavatera sect. lavatera (= stegia dc.) and are represented by plants with solitary flowers, long petioles, epicalyces with very broad and shallow lobes, and lavateroid-type mericarps (see e.g. ray 1995; fernandes 1993). the morphological similarity among these taxa is not, however, supported by the molecular analyses, and these affinities might be homoplasious (escobar et al. 2009). before a new extensive study on the phylogenetic relationships among the annual mallows is made, a nomenclatural investigation of the names related to this group is presented here as part of the revision of the malvaceae for the new edition of the flora d’italia, editor prof. s. pignatti (e.g. iamonico 2010, 2014a, 2014b, 2016; iamonico and peruzzi 2014; iamonico et al. 2015). materials and methods the paper is based on both examination of specimens preserved in the herbaria bm, linn, mpu, p, ro, and to (abbreviations according to thiers 2016) and analysis of relevant literature (protologues included). the articles cited through the text agree with the melbourne code (mcneill et al. 2012). the taxa are presented in alphabetical order. nomenclature on annual mallows acta bot. croat. 77 (1), 2018 29 results lavatera africana mill. miller (1768: lavatera no. 2) provided a short diagnosis (“lavatera (africana) foliis infimis cordato-angulatis, supernè sagittatis, pedunculis unifloris, caule herbaceo hirsuto”), cited one synonym from boerhaave (1727: 268, “lavatera africana, flore pulcherrimo”), and stated “the second sort [lavatera africana] grows naturally at the cape of good hope… is annual”. miller’s name makes lavatera africana cav. (cavanilles 1788: 282), which does not belong to the sect. lavatera, a later homonym and an illegitimate name according to art. 53.2. as a consequence, the combination malva africana (cav.) banfi, soldano, and galasso by banfi et al. (2005) was erroneous. the correct basionym was choosen by molero and monserrat (2005, 2006), and iamonico (2010: 312) who proposed the combinations m. subovata (dc.) molero & montserrat subsp. rupestris (pomel) molero & montserrat, and m. subovata subsp. bicolor (rouy) iamonico, respectively. a study on l. sect. axolopha dc. (iamonico 2016) revealed that miller’s lavatera africana includes plants whose features cannot be associated with perennial taxa belonging to this section; rather, it is related to l. sect. lavatera. i found a sheet at bm (code bm000603431; fig. 1) that bears a plant whose features of leaves and flowers match the diagnosis. this sheet bears three labels: the first one (on the top right of the sheet) represents an identification by g. krebs (15 december 1985, as l. trimestris), the second label (on the bottom right, “lavatera africanna miller 1768 gard. dict. ed 8. no. 2 | type specimen | journ. bot. 1913. 132”) might be steran’s or dandy’s script (j. hunnex pers. comm.), the third label (on the top left, “hort.”) looks as if it might be from j. banks (j. hunnex pers. comm.). since there are doubts about the collector of this exsiccata, and the collection date is lacking, i prefer to avoid the bm000603431 for lectotypification purposes. since there are no further specimens, which can be considered as part of the original material used to describe lavatera africana mill., i here designate bm000603431 as the neotype of lavatera africana mill. lavatera biennis m.bieb. bieberstein (1798: 116) described lavatera biennis from the caspian region through a short diagnosis and the habitat (“occurrit passim in pascuis, graminosis et ad agrorum versuras”); the species was placed into the group named “caule herbaceo”. some years later, bieberstein (1808: 143) again reported the original diagnosis, adding a description, the provenance (“habitat in caucasi orientalis…”), and providing a comparison with l. thuringiaca l. (“…affini l. thuringiaca… petalorum forma; calycis quoque interioris foliola in longius acumen sunt producta. arilli transuersim sutilissime rugosi”). there is one specimen at le (bieberstein’s collection, code 01009603) that bears a terminal part of one plant with some cauline leaves and flowers; the plant was collected in 1796 in “ex planitiebus sihirvanicis”, where shirvan is a province of azerbaijan (v. shvanova, pers. comm.). the plant mounted on the sheet shows features that match bieberstein’s diagnosis and the specimen le-01009603 is here designated as the lectotype of the name lavatera biennis. lavatera grandiflora moench moench (1794: 614) created the name lavatera grandiflora, citing as synonym the validly published l. trimestris l. as a consequence, moench’s name is a superfluous nomen novum for l. trimestris, and is therefore illegitimate according to art. 52.2 of the icn. lavatera moschata miergues and lavatera moschata moris lavatera moschata miergues is a later homonym of l. moschata moris, which was published 31 years earlier in the 2nd volume of stirpium sardoarum elenchus (moris 1827: 9). as a consequence miergues’ name is illegitimate under art. 53.1 of the icn. miergues (1858: 593) observed an algerian population (locality arba) of a lavatera species morphologically similar to l. trimestris, and recognized this population as a new species, named l. moschata. according to the protologue, this species would differ from l. trimestris in having a musky smell, higher branching, soft leaf blades, epicalyx with entire and glabrous margins, and nails of the petals not coloured. one specimen at p (code 04694665) bears a plant that is part of miegues’ collection and it was collected in the locality “l’arba…” in “may”; the morphology of the plant fig. 1. neotype of the name lavatera africana (bm-000603431!). iamonico d. 30 acta bot. croat. 77 (1), 2018 (two pieces) matches miergues’ diagnosis. since the date of collection is lacking, i cannot be sure that this specimen is an ante-1858 addition to the collection, and, consequently, i prefer to avoid it as a possible lectotype (arts. 9.2, and 9.3 of the icn). however, since no other unquestionably original material was found, i here below designate the specimen as neotype of lavatera moschata miergues. examination of the original material and other specimens that are part of moris’ collection (herbarium to) showed that l. moschata moris cannot be identified with any of the species belonging to l. sect. lavatera. on the basis of the morphology of these exsiccatae, l. moschata moris should be included in l. sect. glandulosae fernandes, which is characterized by mixed indumentum (simple, stellate, and glandular hairs) and flowers arranged in groups of about 4–6 (fernandes 1968a, 1968b, escobar et al. 2009). a taxonomic and nomenclatural study on the whole l. sect. glandulosae is in preparation (g. bacchetta and collaborators). lavatera punctata all. this species was first described by allioni (1789: 26) who provided a diagnosis, a detailed description, and the provenance “in agro nicaeensis frequens”; no synonyms, illustrations, or specimens were listed. at to (allioni’s collection), i found three specimens bearing plants whose features match the diagnosis and description of l. punctata; labels (one per specimen) also occur, and include in allioni’s script “lavatera punctata ex nicaea”. although the locality matches the provenance cited in the protologue, the date of collection is lacking, and it is not possible to know if the plants were collected anteor post-1789 (l. guglielmone, pers. comm.). the typification of allioni’s names is not indeed a simple issue, since allioni’s original material is often affected by various types of complication, mainly with respect to 1) the scarcity of information reported on the specimens’ labels and 2) the lack on the sheets of specific annotations which link the protologues to the specimens (see e.g., dal vesco et al. 1987–1988). as a consequence, we cannot be sure that these specimens are part of the original material (art. 9.3), and their use as lectotypes should be avoided (see art. 9.2). another possible repository of original material for allioni’s name is the bellardi collection (kept at to). c. a. l. bellardi was a student and close collaborator of c. allioni, who often examined bellardi’s material for his researches (l. guglielmone, pers. comm.). one specimen at to bears bellardi’s annotation “lavatera trimestris linn. punctata all.”, so bellardi originally identified the plant as l. trimestris, but later revised the exsiccatum as l. punctata. furthermore, there is a second label: “ex horto x. d. johannis. inveni abunde in vineis, et campis a monaco ad mentonum et non longe a nicaea” (the san giovanni botanical garden was part of the san giovanni hospital area, and it is part of the faculty of medicine). it is interesting to note that the code of l. punctata in allioni’s auctarium (“1424”) corresponds to that given to l. trimestris in flora pedemontana (allioni 1785: 42) where the author indicated “loc. olivetis villafrancae nicaeensis reperitur”, also associating to the species the illustration nos. 67 (volume 14), and 87 (volume 17) of the iconographia taurinensis (allioni 1765, 1771 – see forneris 2008). unfortunately, the date of collection is lacking in bellardi’s specimens, and i am not able to establish if the exsiccata is an ante-or post-1789 addition to the collection, so i prefer to avoid its use as lectotype. lacking other specimens that are unquestionably original material, i am forced to choose one of them as a neotype (art. 9.7). below i designate the specimen at to (no. 1) as the neotype of the name lavatera punctata since it bears a more complete plant, including lower, middle and cauline leaves, well preserved flowers, and fruits. lavatera punctata var. maroccana batt. & trab. battandier and trabut (1888: 14) published lavatera punctata var. maroccana through a detailed description; the provenance (“ravins de taourirt”), and the collector (“m. ducellier”) were also provided. there is one specimen at mpu (code 001641) that bears a piece of a branch (terminal part of the inflorescence) with one opened flower and a mature schizocarp, whose features match the diagnosis. the plant was collected by l. ducellier in “maroc oriental: taourirt, ravins” on may 7, 1916. the specimen is undoubtedly original material, and it is here designated as the lectotype of the name l. punctata var. maroccana. the taxon is currently accepted at species level as l. maroccana (batt. and trab.) verloove and lambinon [see e.g., fernandes 1968b: 234 sub l. maroccana (batt. and trab.) maire, and verloove and lambinon 2011: 40]. lavatera rosea medik. medikus (1787: 40) proposed the name lavatera rosea citing a synonym from jacquin (1770), “jacq. h. v. t. i, tab. 72”. the latter author published an illustration (image available at http://www.biodiversitylibrary.org/item/10249#page/3/ mode/1up) labelled as “lavatera trimestris” (see jacquin 1770: 29). as a consequence, medikus’ (1787) new name is superfluous and illegitimate since the linnaean name should have been adopted (arts. 52.1 and 52.2). lavatera trimestris var. brachypoda pérez lara near jerez de la frontera (localities cortijo del pino, and cortijo del alijar, province of cadíz, southwestern spain), pérez lara (1896: 329) found a population of l. trimestris characterized by having the indument composed of scattered, small and stellate hairs (l. trimentis s.str. has hairs that are simple or in fascicles, not stellate, long and densely arranged). the author described the cadíz population at varietal rank, as l. trimestris var. brachypoda. there is one specimen at maf (code 35596) bearing two pieces of one plant collected by pérez lara in the locality cortijo del pino on august 19, 1880; it appears to be the only extant exsiccata that is part of the original material, and it is here chosen as the lectotype of the name lavatera trimestris var. brachypoda. http://www.biodiversitylibrary.org/item/10249#page/3/mode/1up http://www.biodiversitylibrary.org/item/10249#page/3/mode/1up nomenclature on annual mallows acta bot. croat. 77 (1), 2018 31 lavatera trimestris var. trimestris f. colorata gatt. & maire lavatera trimestris f. colorata gatt. & maire was described by maire (1949: 130) to distinguish forms with purplish petals (“a typo non differ nisi floribus vivide purpureis (nec roseis)”). there is one specimen at mpu (code 004731) bearing a terminal flowered branch collected by j. gattefossé (no. 2058) in morocco (locality fedhala) on may 15, 1946. the date of collection is ante-1949, and the other label data completely match the protologue; moreover, the label was signed by r. maire. since no further specimens (possible syntypes) were found, and according to the wide discussion by mcneill (2014), i here designate the mpu sheet as the lectotype of maire’s name. olbia deflexa moench moench (1802: 200) published olbia deflexa citing as synonym lavatera punctata, wrongly attributed to linnaeus and referring to “sp. pl. iii. p. 797”. the latter quotation refers to a willdenow’s description in the 4th edition of species plantarum (willdenow 1800: 797) of l. punctata all. the moench name can be interpreted as a replacement name of l. punctata. since this latter name was validly and legitimately published, o. deflexa is a superfluous and illegitimate name according to arts. 51.1 and 52.2. stegia lavatera dc. the name stegia lavatera was published by candolle (1805: 856) citing as synonym lavatera trimestris l. candolle’s name is, therefore, a new, superfluous and illegitimate name according to arts. 52.1 and 52.2. discussion lavatera africana mill. the synonym cited by miller (1768: lavatera no. 2) from boerhaave (1727: 268), was reported by linnaeus (1738: 348, 1748: 203) as a synonym of, respectively, “lavatera foliis trilobis, lacinia media productiore, caule herbaceo”, and “lavatera foliis glabris, caule scabro herbaceo, pedunculis unifloris, fructibus orbiculo tectis”. in the 1st edition of species plantarum linnaeus (1753: 692) published the name l. trimestris using a diagnosis taken directly from the hortus upsaliensis (linnaeus 1748: 203); moreover, he cited the synonym from the hortus cliffortianus (linnaeus 1738: 348). however, i have not been able to trace boerhaave’s polynomial in any of linnaeus’ works published after 1753 (1st edition of species plantarum). there are three sheets at bm (codes 000646478, 000646479, 000646480; images available at http://www.nhm.ac.uk/resources/research-curation/projects/clifford-herbarium/lgimages/bm000646478. jpg, http://www.nhm.ac.uk/resources/research-curation / projects/clifford-herbarium/lgimages/bm000646479.jpg, and http://www.nhm.ac.uk/ resources/research-curation/ projects/clifford-herbarium/lgimages/bm000646480.jpg) bearing, respectively, the original annotations “lavatera folio et facie altheae”, “lavatera africana flore pulcherrimo”, and “malva vel lavatera folio et facie altheae” [“lavatera folio et facie altheae”, and “malva vel lavatera folio et facie altheae” are listed by boerhaave (1727: 268)]. all these sheets are original material for the name l. trimestris, and the pinned plants can be identified as l. trimestris according to the current concept of this species (see e.g. pignatti 1982; fernandes 1968b, 1993). furthermore, these plants show features that match miller’s description of his l. africana. also the chosen lectotype of lavatera africana mill. (see below) is identifiable as l. trimestris, showing the following characteristics: herbaceous plant with leaves glabrous, the upper suborbicular, 3-lobed with base rounded to cordate and margins crenate, the more distal leaves sagittate, 3-lobed with central lobe much longer then the lateral ones and margins dentate; flowers solitary, with epicalyx segments wide and acuminate, calyx longer than the epicalyx with lobes acute, and petal 3–4 times longer than the calyx. concerning the distribution area of lavatera africana, miller (1768) indicated that the species “…grows naturally at the cape of good hope”. this information apparently contrasts with the current known distribution of l. trimestris [western europe, north-western africa, and south-western asia (see valdés 2011)]. however, according to recent floristic data (see e.g., sanbi 2012a), l. trimestris also occurs in south africa where it is considered a naturalized alien. miller (1768) also reported “…cape of good hope, from whence the seed were brought to holland, and the plants were cultivated…”. on the basis of the j. banks annotation at the top left of the neotype preserved at bm, it is likely that p. miller examined specimens based on cultivated plants growing from seed collected in south africa. considering all the above, it is here proposed to treat lavatera africana mill. as a heterotypic synonym of l. trimestris, which holds nomenclatural priority (1768 vs. 1753). lavatera biennis m.bieb. on the basis of current knowledge (see e.g. pignatti 1982, fernandes 1968b, 1993), the comparison of types and diagnoses of l. biennis, l. trimestris, l. maroccana, and l. punctata, and the examination of further specimens, the name l. biennis can be synonymised with l. punctata on the basis of two main characters: 1) the carpophore not expanded over the mericarps, 2) upper leaves 3-lobed with the apical lobe clearly longer than the lateral ones. both l. trimestris and l. maroccana have the carpophore more or less expanded over the mericarps, and the upper leaves 3-5-lobed with the central lobe usually not longer than lateral ones. lavatera punctata has nomenclatural priority over l. biennis (1789 vs. 1798). lavatera moschata miergues and lavatera moschata moris based on the comparison between l. moschata miergues and l. trimestris s. str. (protologues, types and other specimens), and the examination of treatments in recent floras (see e.g., fernandes 1968b, 1993), it appears that the only reliable diagnostic character of l. moschata miergues is the http://www.nhm.ac.uk/resources/research-curation/projects/clifford-herbarium/lgimages/bm000646478.jpg http://www.nhm.ac.uk/resources/research-curation/projects/clifford-herbarium/lgimages/bm000646478.jpg http://www.nhm.ac.uk/resources/research-curation/projects/clifford-herbarium/lgimages/bm000646478.jpg http://www.nhm.ac.uk/resources/research-curation /projects/clifford-herbarium/lgimages/bm000646479.jpg http://www.nhm.ac.uk/resources/research-curation /projects/clifford-herbarium/lgimages/bm000646479.jpg http://www.nhm.ac.uk/ resources/research-curation/projects/clifford-herbarium/lgimages/bm000646480.jpg http://www.nhm.ac.uk/ resources/research-curation/projects/clifford-herbarium/lgimages/bm000646480.jpg iamonico d. 32 acta bot. croat. 77 (1), 2018 musky smell of the plants, while l. trimestris s. str. has a herbaceous smell. battandier and trabut (1888: 115) proposed the varietal rank for miergues’ species. although l. moschata miergues is illegitimate, the combination proposed by battandier and trabut (l.c.) is to be considered legitimate and should be cited as l. trimestris var. moschata batt. & trab. (art. 58.1 ex.2 of icn). considering all the above, i here propose to consider l. trimestris var. malvaeformis as a heterotypic synonym of l. trimestris var. trimestris, while l. trimestris var. moschata batt. & trab. is a pro parte synonym of both l. trimestris var. trimestris, and l. moschata by miergues. although further taxonomic investigations (e.g., molecular ones) are needed to definitively establish the taxonomic value of the miergues variety, on the basis of both the characteristics of this taxon (i.e. the musky smell), as well as its distribution (which seems to be a restricted area of n-algeria, in the blinda province), i here propose to accept the miergues lavatera, by maintaining the varietal rank. a new combination of l. trimestris var. moschata batt. & trab. is here proposed (see the paragraph “taxonomic treatment”). lavatera trimestris var. brachypoda pérez lara fernandes (1993: 234) stated that l. trimestris var. brachypoda seemed to be extinct in spain, since no subsequent population was found. however, ortiz and lópez (2012) recently rediscovered two populations in the province of cadíz, in the localities cañada de los marchantes, and peña arpada. the latter authors confirm that the indumentum represents the main diagnostic character separating l. trimetris var. brachypoda and l. trimetris var. trimestris, as previously reported by fernandes (1993: 234), sub stegia trimestris (l.) luque & devesa var. brachypoda (pérez lara) luque & devesa]. outside spain, l. trimetris var. brachypoda was recently recorded in belgium (verloove and lambinon 2011), where the taxon is considered alien. the taxonomic status of l. trimetris var. brachypoda needs further investigation (i.e. new field surveys), so i prefer to provisionally maintain it at the varietal rank, in agreement with the spanish authors (fernandes 1993). lavatera trimestris var. trimestris f. colorata gatt. & maire on the basis of the current concept, the colour of the petals in l. trimestris s. str. shows a wide range of variation, from white to deep-rose (see e.g., fernandes 1993). i consider the coloured form named by maire (1949) to be part of this variation. so, maire’s name is here treated as a heterotypic synonym of lavatera trimestris var. trimestris. taxonomic treatment lavatera punctata all. var. maroccana batt. & trab., bull. soc. hist. nat. afrique n. 9: 14. 1918 ≡ malva maroccana (batt. & trab.) verloove & lambinon, new j. bot. 1(1): 40. 2011 [31 august 2011] ≡ lavatera maroccana (batt. & trab.) maire, bull. soc. hist. nat. afrique n. 17: 107. 1926. lectotype (here designated): morocco, taourirt, ravines, 07 may 1916, ducellier s.n. (mpu-001641!). image of the lectotype available at http://www.herbier-mpu.org/ zoomify/zoomify.php?fichier=mpu001641 − malva maroccana (batt. & trab.) soldano, banfi and galasso, atti soc. ital. sci. nat. mus. civico storia nat. milano 152: 95. 2011, isonym [november 2011]. − malva maroccana (batt. & trab.) valdés, willdenowia 41(2): 319. 2011, isonym [20 december 2011]. lavatera punctata all., auct. fl. pedem.: 26. 1789 [january-march 1789] ≡ malva punctata (all.) alef., oesterr. bot. z. 12: 258 1862 ≡ olbia deflexa moench, suppl. meth. (moench): 200. 1802, nom. nov. pro lavatera punctata all., nom. superfl. et illeg. (art. 52.2 of icn) ≡ althaea punctata (all.) kuntze, revis. gen. pl. 1: 66. 1891. neotype (here designated by d. iamonico and l. guglielmone): italy, ex nicaea, s.d., s.coll. s.n. (to-1!) − fig. 2. = lavatera biennis m.bieb., tabl. prov. mer casp. 116. 1798 ≡ althaea biennis (m.bieb.) kuntze, revis. gen. pl. 1: 66. 1891. lectotype (here designated): azerbaidzhan, ex planitiebus sihirvanicis, 1796, bieberstein s.n. (le-01009603!) − fig. 3. − malva punctata (l.) g.h.loos, jahrb. bochum. bot. vereins. 1: 125. 2010, isonym [february 2010]. lavatera trimestris l., sp. pl. 2: 692. 1753 ≡ malva trimestris (l.) salisb. var. trimestris, prodr. stirp. chap. allerton: 381. 1796 ≡ lavatera rosea medik., malv.: 40. 1787, nom. nov. pro lavatera trimestris l., nom. superfl. et illeg. (arts. 52.1, fig. 2. neotype of the name lavatera punctata (to-1!). http://www.herbier-mpu.org/zoomify/zoomify.php?fichier=mpu001641 http://www.herbier-mpu.org/zoomify/zoomify.php?fichier=mpu001641 nomenclature on annual mallows acta bot. croat. 77 (1), 2018 33 and 52.2 of icn) ≡ lavatera grandiflora moench, meth.: 614. 1794, nom. nov. pro lavatera trimestris l., nom. superfl. et illeg. (arts. 52.1, and 52.2 of icn) ≡ stegia lavatera dc., fl. fr., ed. 5, 4(2): 856. 1805, nom. nov. pro lavatera trimestris l., nom. superfl. et illeg. (arts. 52.1, and 52.2 of icn) ≡ stegia trimestris (l.) risso, fl. nice: 96. 1844 ≡ althaea trimestris (l.) kuntze, revis. gen. pl. 1: 66. 1891. lectotype (designated by fernandes 1968a: 400): herb. linn., no. 871.11 (linn!). image of the lectotype available at http://linnean-online.org/8081/ = lavatera africana mill., gard. dict., ed. 8.: lavatera n. 2. 1768 [16 apr 1768]. neotype (here designated): england, chelsea physic garden from seed growing in the cape of good hope (south africa), s.d., s.n. (bm-000603431!) − fig. 1. = lavatera trimestris var. malvaeformis ball, spic. fl. maroc.: 376. 1877. type: not designated. = lavatera trimestris var. trimestris f. colorata gatt. & maire, bull soc. nat. af. n. 39(7–8): 130. 1949. lectotype (here designated): morocco, fedhala, gattefossé 2058 (mpu-004731!, image of the lectotype available at http://www.herbier-mpu.org/zoomify/zoomify.php?fichier =mpu004731). – lavatera trimestris var. moschata batt. and trab., fl. alg. 1: 115. 1888, p. p. − stegia trimestris (l.) t.luque & j.a.devesa, lagascalia 14(2): 237. 1986, isonym. − malva trimestris (l.) g.h.loos, jahrb. bochum. bot. vereins. 1: 125. 2010, isonym [febraury 2010]. lavatera trimestris var. brachypoda pérez lara, soc. esp. hist. nat. 24: 329. 1896 ≡ malva trimestris (l.) salisb. var. brachypoda (pérez lara) verloove & lambinon, new j. bot. 1(1): 40. 2011 [31 august 2011]. lectotype (here designated): spain, cadíz, cortijo del pino, 19 august 1880, pérez lara s.n. (maf-35596!) − fig. 4. malva trimestris (l.) salisb. var. moschata (batt. and trab.) iamonico, comb. nov. ≡ lavatera trimestris var. moschata batt. and trab., fl. alg. 1: 115. 1888 ≡ lavatera moschata miergues, bull. soc. bot. france 5: 593. 1858, nom. illeg. (art. 52.1), non lavatera moschata moris, stirp. sard. elench. 1: 9. 1827. neotype (here designated): algeria, l’arba, may, miergues s.n. (p-04994665!, image of the neotype available at http:// sonneratphoto.mnhn.fr/2012/09/27/10/p04694665.jpg). acknowledgements thanks are due to v. shvanova and i. tatanov (herbarium of vascular plants of the komarov botanical institute, st. petersburg) for permission to reproduce the image of the lectotype of lavatera biennis m. bieb., to j. hunnex (natural history museum, london) for information provided and help in the interpretation of the herbarium annotation on the type of lavatera africana, and to b. valdés (university of seville, seville) for the useful discussion about lavatera trimestris var. brachypoda. fig. 3. lectotype of the name lavatera biennis (le-01009603!). fig. 4. lectotype of the name lavatera trimestris var. brachypoda (maf-35596!). http://linnean-online.org/8081/ http://www.herbier-mpu.org/zoomify/zoomify.php?fichier =mpu004731 http://www.herbier-mpu.org/zoomify/zoomify.php?fichier =mpu004731 http://sonneratphoto.mnhn.fr/2012/09/27/10/p04694665.jpg http://sonneratphoto.mnhn.fr/2012/09/27/10/p04694665.jpg iamonico d. 34 acta bot. croat. 77 (1), 2018 references allioni, c., 1765: iconographia taurinensis 14. augustae taurinorum. allioni, c., 1771: iconographia taurinensis 17. augustae taurinorum. allioni, c., 1785: flora pedemontana, sive enumeratio methodica stirpium indigenarum pedemontii 2. ioannes michael briolus, augustae taurinorum. allioni, c., 1789: auctarium ad floram pedemontanam cum notis et emendationibus. j.m. briolus, augustae taurinorum. banfi, e., galasso, g., soldano, a., 2005: notes on systematics and taxonomy for the italian vascular flora 1. atti della società italiana di scienze naturali del museo civico di storia naturale di milano 146, 219–244. battandier, j. a., trabut, l. q1c., 1888: flore de l’algérie, ancienne flore d’alger transformée contenant la description de toutes les plantes signalées jusqu'à ce jour comme spontanées en algérie. 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(malvaceae). lagascalia 26, 153–155. molero, j., montserrat, j. m., 2007: a new species of lavatera sect. olbia (medik.) dc. (malvaceae) from north-east morocco. botanical journal of the linnaean society 153, 445–454. moris, j. h., 1827: stirpium sardoarum elenchus 2. ex typis regis, carali. ortiz, j. h., lópez, f. v., 2012: redescubrimiento de lavatera trimestris var. brachypoda. revista de la sociedad gaditana de historia natural 6, 23–24. pérez lara, j. m., 1896: florula gaditana seu recensio celer omnium plantarum in provincia gaditana hucusque notarum. pars quinta. anales de la sociedad española de historia natural, ser. 2, 24, 279–35. pignatti, s., 1982: flora d’italia 2. edagricole, bologna. ray, m. f., 1995: systematics of lavatera and malva (malvaceae, malveae): a new perspective. plant systematic and evolution 198, 29–53. nomenclature on annual mallows acta bot. croat. 77 (1), 2018 35 sanbi, 2012a: lavatera trimestris l. – red list of south african plants. retrieved from http://redlist.sanbi.org/species. php?species=2586-4. sanbi, 2012b: lavatera trimestris l. var. malvaeformis ball. – red list of south african plants. retrieved from http://www.ville-ge.ch/musinfo/bd/cjb/africa/details.php? langue=an&id=166564]. tate, j. a., fuertes aguilar, j., wagstaff, s. j., la duke, j. c., slotta, t. a. b., simpson, b. b., 2005: phylogenetic relationships within the tribe malveae (malvaceae, subfamily malvoideae) as inferred from its sequence data. american journal of botany 92, 584–602. thiers, b., 2016: index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden's virtual herbarium. valdés, b., 2011: malvaceae juss. in: greuter, w., raus, t. (eds.), med-cheklist notulae 30. willdenowia 41, 318–319. verloove, f., lambinon, j., 2011: the non-native vascular flora of belgium: new names and combinations. new journal of botany 1, 38–42. willdenow, c. l., 1800: species plantarum, 4th ed. tomus 3. impensis g. c. berlin ("berolini"), germany. http://redlist.sanbi.org/species.php?species=2586-4 http://redlist.sanbi.org/species.php?species=2586-4 http://www.ville-ge.ch/musinfo/bd/cjb/africa/details.php? langue=an&id=166564 http://www.ville-ge.ch/musinfo/bd/cjb/africa/details.php? langue=an&id=166564 acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 b1 book review endemi u hrvatskoj fl ori by toni nikolić, milenko milović, sandro bogdanović and nenad jasprica. 2015, 492 pp. isbn 978-953-297-763-9. publisher: alfa d.d., zagreb, print: grafi ka markulin it is believed that the fi rst who used the term endemism in botany was de candolle in 1820. although the term was already used in medicine, where endemic referred to a disease occurring with high frequency in a defi ned geographical area, but not necessarily limited to it (garbari 1990), de candolle (1820) gave it a slightly different meaning, i.e. to designate a species growing exclusively in a defi ned area (siljak-yakovlev and peruzzi 2012). de candolle (1875) recognized that endemic taxa are not just randomly distributed, while range restrictions indicating survival in refugia have been regarded as a driving force in the generation of distributional patterns of endemics. a relationship of areas of high endemism with presumed refugia has been well documented worldwide, especially for the mediterranean basin (médail and diadema 2009); in croatia alone, three glacial refugia have been registered: the istrian peninsula, the velebit and biokovo mountains. the present book has taken a step further, trying to thoroughly present and elucidate the phenomenon of adriatic endemism on a much larger geographical scale, dealing with taxa of limited distribution ranges occurring also or exclusively in croatia. the book has 492 pages divided into the following chapters: introduction, endemic taxa in croatia, two appendices and 13 boxes. the text is accompanied by no less than 615 numerated pictures and more than 800 quality photographs (contributed by the authors and colleagues) and 200 hand drawings (most frequently redrawn with permission from šilić’s excellent book endemične biljke (1984)) and an extensive bibliography for each taxon. the book is a joint effort of four authors: toni nikolić (tn), milenko milović (mm), sandro bogdanović (sb) and nenad jasprica (nj). in the introduction the author (tn) sets the scene by explaining some basic terms linked to the fl ora of croatia, the current state of knowledge on systematics and distribution of vascular plants in croatia, and phenomena of endemism on both a global and a local scale including defi nitions on the categorization of endemics with some justifi cations of the criteria applied. however, it is exactly the classifi cation of endemic taxa according to selected, well-accepted criteria (e.g. ecology, age, distribution range) that is one of the most controversial and intriguing issues in biology but the present book offers a purely technical and somewhat unusual proposal. for example: a taxon is termed “stenoendemic” if it is restricted to a narrow range completely within the borders of croatia occupying up to approximately 4000 km2 (e.g., in an alphabetical order: allium croaticum, a. telmateum, asplenium hybridum, campanula fenestrellata s.l. etc.). however, while in certain cases even more b2 acta bot. croat. 74 (2), 2015 narrowly distributed taxa that occur in croatia as well as in other areas (e.g. moehringia tommasinii, the distribution range of which spans italy, slovenia and croatia), are classifi ed as “endemic”. nevertheless, according to the given criteria and biogeographic analyses, authors thoroughly and in painstaking detail have described 155 (40%) endemic taxa out of 384 (7.6% of the total fl oristic inventory of the country) occurring in croatia, while 53 (14%) taxa are mentioned in less detail. due to the lack of information, 102 taxa were not taken into consideration. the extensive database, which was a basis for spatial analyses of endemism in croatia, enabled some interesting insights into the spatial distribution of endemism. for example: the areas with the highest number of stenoendemics per unit area in croatia are kvarner, velebit, the krka river estuary with the šibenik archipelago, adriatic islands in central and southern dalmatia, mt. biokovo and konavle, while for endemics the most important areas are gorski kotar, mts mosor, kozjak and učka, ćićarija, kapela, mt lička plješevica and plitvice lakes, mts dinara and omiška dinara, the žumberak and samoborsko gorje hills, mt medvednica, strahinščica, ivanščica and the hills near požega. the next 438 pages of the chapter endemi u hrvatskoj fl ori are pure gold. each genus with its subordinate taxa are thoroughly described, covering the name of a taxon, place of publication, and common names in six languages: croatian, english, italian, german, french and slovenian. then follow: species description, distribution, habitat preferences and ecology, threats, additional information and literature. in 13 boxes authors give additional information on important contributors to the knowledge of croatian fl ora, e.g. muzio tommasini, august von hayek, čedomil šilić, vinczé von borbas and paul kitaibel, to name just a few. although the selection of the presented taxa did follow a certain logic and defi ned criteria, it is, however, not clear why the authors did not present in detail some remarkable taxa such as: campanula austroadriatica, c. marchesettii, c. pyramidalis, carlina fi umensis, cerinthe glabra subsp. smithiae and subsp. velebitica, c. tristis, inula verbascifolia subsp. metanea, onosma visiani subsp. biokovoense, peucedanum crassifolium, satureja montana subsp. variegata, s. subspicata subsp. liburnica and subsp. subspicata, scabiosa silenifolia, saxifraga prenja, trinia carniolica (longipes), etc., instead of taxonomically (t) and/or chorologically (ch; not according to selection criteria) “dubious” anthyllis montana subsp. atropurpurea (t, ch), aquilegia dinarica (ch – its occurrence in croatia is doubtful), armeria canescens subsp. dalmatica (t), asperula wettsteinii (ch – its occurrence in croatia is doubtful), cardamine chelidonia (ch), trifolium dalmaticum (ch) etc. with the exception of puccinelllia teyberi, there are no other representatives of grasses (poaceae). an extensive literature list at the end of the description of each taxon is more than satisfactory, although in certain cases one gets the impression that the new insights into the systematics and taxonomics, although cited, are somewhat neglected or only partially accepted, e.g. in the case of brassica incana agg., onosma echioides agg., dianthus sylvestris agg., and the genera cerinthe and centaurea. at the very end of the book, in the fi rst appendix, authors give us a complete list of endemic taxa of vascular plants in croatia, while the second appendix in fact represents index of latin names of vascular plants. this book is supposed to be a textbook for students attending the course flora of croatia at the university of zagreb, but it will be valuable source of information to any nature explorer, and a must for any botanist interested in the biodiversity and biogeography of vascular plants of the balkans. it will also serve as a promoter and conservation source acta bot. croat. 74 (2), 2015 b3 of vascular plants, as well as a sound grounding for further scientifi c activities in the area. all in all, the book, which has signifi cantly raised the standard for future similar publications even on the international market, makes an impression on the reader and one must give all due credit to the authors for a great effort – an example to be followed. the book layout is fabulous and the print is spotless. well, well done. dr. sc. boštjan surina natural history museum rijeka, croatia references de candolle, a. p., 1820: essai e´lementaire de geographie botanique. strasbourg. de candolle, a. p., 1875: sur les causes de l’inégale distribution des plantes rares dans la chaîne des alpes. atti del congresso internazionale botanico tenuto in firenze 1874, 92–104. garbari f., 1990: l’endemismo vegetale: genesi, tipi e signifi cato biogeografi co. studi trentini sc nat 66, 113–120. médail, f. and diadema k., 2009: glacial refugia infl uence plant diversity patterns in the mediterranean basin. journal of biogeography 36, 1333-1345. siljak-yakovlev, s. and peruzzi l., 2012: cytogenetic characterization of endemics: past and future. plant biosystems 146, 694-702. šilić, č., 1984: endemic plants. sarajevo, svjetlost, oour zavod za udžbenike i nastavna sredstva. (in croatian) opce-str.vp acta bot. croat. 68 (2), 325–338, 2009 coden: abcra 25 issn 0365–0588 the diatom chaetoceros in ships’ ballast waters – survivorship of stowaways georgia klein1*, irena kaczmarska1, james m. ehrman2 1 department of biology, mount allison university, sackville, nb, e4l 1g7, canada 2 digital microscopy facility, mount allison university, sackville, nb, e4l 1g7, canada ship ballast water discharged by vessels into the receiving port is recognised today as an important vector for the spread of non-indigenous species and facilitates the introduction of potential invasive species. here, we report on 18 species (of about 30 identified), both vegetative cells and spores, of the diatom genus chaetoceros ehrenberg found in ballast water collected from ships arriving at canadian ports on the west coast (wc), east coast (ec) and the great lakes (gl). we found live, vegetative chaetoceros cells (one of the most abundant taxa) in 49% of the 57 ballast water samples. the highest density of viable spores enumerated in our counts was 414 cells l–1. in 62% of 52 samples examined using scanning electron microscopy (sem), chaetoceros spores were found, though fewer live, identifiable spores were found using light microscopy. three reportedly harmful species, c. convolutus, c. danicus, c. debilis were encountered in wc samples, and additionally, c. cf. hispidus, a species not yet reported from canada. c. ceratosporus and c. cf. subsecundus, to date reported only from the ec of the usa, now have been transported to the port of vancouver, british columbia. our findings contribute to the assessment of the effectiveness of ballast water treatment via water exchange, and serve to evaluate the diversity of diatom vegetative cells and spores transported in ballast water tanks. keywords: diatom, chaetoceros, phytoplankton, non-indigenous species, ballast water, resting stage, spore, ultrastructure introduction the rapid increase in large container shipping since the 1970s has facilitated rapid inter-connection between ports worldwide. this has provided marine biota with an unprecedented anthropogenic means of worldwide dispersal via ballast water (carlton 1996, ruiz et al. 2000). studies of invasive, non-indigenous aquatic macro-organisms in canada (e.g. breen and metaxas 2008, jokela and ricciardi 2008) also focused interest on ship ballast transport of microalgae, which, until recently, have received little systematic scientific attention. extensive literature shows ballast waters and sediments to be vectors for the introduction of dinoflagellate resting stages into recipient ports (e.g. hallegraeff et al. acta bot. croat. 68 (2), 2009 325 * corresponding author: gklein@mta.ca u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:28 color profile: disabled composite 150 lpi at 45 degrees 1990, zhang and dickman 1999, hamer et al. 2000, pertola et al. 2006), particularly of potentially toxic dinoflagellates (e.g. hallegraeff and bolch 1991, bolch and desalas 2007). however, diatoms, one of the most species rich and abundant ballast water micro-biota, require more attention than they have thus far received. the literature on diatom resting stages in ballast water is scarce and only a few publications identify diatom spores (e.g. hallegraeff and bolch 1992), despite their potential for being harmful (bates and strain 2006) and/or invasive, and their long-term tolerance to growth-adverse conditions (mcquoid and hobson 1995). depending on ship type and age, as well as weather conditions for ballast water exchange, 1–5% of the water plus sediment remains un-exchanged in the ballast tank due to limitation of pumping technology, but the sediments are likely to contain more species, some in resting stages, than the overlaying water. diatom and dinoflagellate resting stages (spores, cysts) may remain viable in growth-limiting environments for years (sicko-goad et al. 1989, hallegraeff and bolch 1992, mcquoid et al. 2002). there are both environmental and economical implications of the introduction of non-indigenous aquatic species. damage due to invasive harmful algae can be considerable and it is not only toxin producing diatoms that are harmful (e.g. skov et al. 1999). chaetoceros species can cause mechanical damage by piercing the gills of fish and mass die-off of high cell densities of blooming species can cause hypoxia in the water column (e.g. bates and strain 2006). furthermore, biodiversity issues may result from the introduction of invasive species. there is potential for the successful establishment of non-indigenous aquatic species that may displace and replace native species (edlund et al. 2000). local biodiversity may be compromised, leading to a cascade of changes with the potential for weakening or altering an ecosystem. the aim of our study within the canadian aquatic invasive species network (caisn) was to evaluate the effectiveness of open-ocean exchange of ballast water prior to arriving in canadian ports, as currently regulated by the imo (international maritime organisation). we present an initial assessment of diversity and abundance of cells and resting spores from the diatom genus chaetoceros arriving in ship ballasts in 2007. materials and methods ship sampling vegetative cells and spores were identified and enumerated in a total of 67 ballast water samples collected from the west coast (wc, 30 samples; vancouver harbour) from 28 april to 6 september 2007; east coast (ec, 24 samples; arrival ports sept-îles, baie-comeau and port-cartier) 29 april to 13 august 2007; great lakes (gl, 13 samples; arrival ports toledo, milwaukee, detroit (usa), sarnia and windsor (canada)) 30 july to 26 november 2007. the sampling protocol for water samples (developed by caisn) targeted tanks that were at least 50% full. bulk carriers were primarily sampled, but container ships and tankers were also included. with the use of a 5 l niskin-bottle, water was collected at 3 depths to represent the tank water column (subsurface, about middle depth – depending on water level in the tank, and ca 1–2 m above bottom), combined to a total of 15 l and subsampled for various projects. for diatoms, 3 l of each sample were transferred into plastic bottles, preserved with acidified lugol’s solution and stored in a cool and dark loca326 acta bot. croat. 68 (2), 2009 klein g., kaczmarska i., ehrman j.m. u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:28 color profile: disabled composite 150 lpi at 45 degrees tion until shipment to our laboratory. specifics regarding ship type (e.g. double-bottom, wing-tank, top-side), location (starboard, portside, aft, peak), filling level of the tank, ballast water volume discharged, and physicochemical properties of the tank waters (temperature, salinity and ph) were recorded. laboratory procedures samples were carefully decanted and contents concentrated to a final volume of 20 ml and stored at 4 °c until processed. diatom identification and enumeration were performed on a 10 ml sub-sample of the concentrate in a settling chamber (hydrobios, kiel, germany) and expressed as cells l–1 or cells per ballast volume discharged as required. autofluorescence of the chloroplast served as an indicator of cell viability at the time of fixation, and only fluorescing cells were counted. to reverse the quenching of chloroplast autofluorescence by lugol’s fixation, 4 drops of a saturated sodium thiosulfate solution in distilled water were added to the sub-sample prior to settling in the chamber. counts were carried out using a zeiss axiovert 200 with epifluorescence illumination (hbo 50/ac, mercury shortarc) and 20 times and 40 times objectives. using lm, fluorescing chaetoceros cells (vegetative cells and spores ³ 10 µm in shortest linear dimension) were identified to the lowest practical taxonomic level, most often to species. cell morphometrics and scanning electron microscopy (sem) were used to verify identification. resting stages were counted when detected in lm however, many spores were too small (< 10 µm in longest dimension) for confident identification or enumeration. therefore viable vegetative cells and larger spores were reliably identified and enumerated in lm, but much greater diversity and abundance of spores was found in sem samples prepared directly from water mounts, due to the presence of the very small spores. for sem examination, after lm counts were completed, the contents of the settling chamber were prepared by acid cleaning (modified from drebes 1974) and dispersed onto filters as described in kaczmarska et al. (2005). statistical analysis hierarchical cluster analysis using average linking and euclidean distance was performed on the entire data set using systat version 10 (systat software, inc., chicago, il). results ballast water we found members of the genus chaetoceros in 52 out of 67 ballast water (bw) samples. each bw source differed either spatially or temporally (coast of japan, europe, america, mid-pacific or mid-atlantic) depending on if and where exchange took place. during the 2007 sampling season, a variety of bw treatments were applied to ships arriving at canadian ports. following international maritime organization (imo) regulations, only ships that have undergone ballast water exchange may enter wc, ec or gl ports unless they commute from neighbouring waters (ports north of cape blanco, or, usa, on the pacific and north of cape cod, ma, usa, on the atlantic coast). ballast water exchange may occur anywhere on the route, adding to the variability of the biotic and acta bot. croat. 68 (2), 2009 327 chaetoceros in ballast waters u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:28 color profile: disabled composite 150 lpi at 45 degrees physicochemical properties of the tank waters. the application of cluster analysis to the entire sample set did not reveal any statistically significant patterns or similarities. this is not surprising considering the great variability of the ballast water sources, varying ballast water treatments and the varied abundance and species composition of very small spores (unidentifiable in light microscopy). therefore, we did not pursue any more complex statistical analyses. among the 180 viable diatom species encountered in lm/sem, we found the following resting stage or spore forming taxa: stephanopyxis (with spore in formation), leptocylindrus, thalassiosira and chaetoceros. here, we report on diversity and abundance of vegetative cells and spores belonging to the genus chaetoceros. the volume of ballast water discharged (regardless of vessel-type or ballast water treatment) into the port ranged between 197 m³ and 22.897 m³. depending on this discharged volume, we calculated between 2.2 x 105 (exchanged bw) and 3.6 x 108 (un-exchanged bw) chaetoceros resting spores per tank for those found to contain chaetoceros spores. in 30 of 52 samples containing chaetoceros (seen in lm) and prepared for sem we found spores of approximately 30 species and other diatom and non-diatom taxa (e.g. chrysophyceae). due to the fact that many spores were smaller than 10–15 µm and could not be confidently identified and counted in the lm settling chambers, the data set comprising samples with enumerated spore densities is very conservative (tab. 1). using lm data, we recorded approximately 5 times higher (168–414 spores l–1 larger than 10 µm) spore densities in un-exchanged ships than in exchanged ships (1–75 spores l–1; tab. 1). the diversity of (large) spores encountered in lm counts is lower and not directly comparable to that found using sem, which includes both large and small spores. vegetative cells of species from the genus chaetoceros were found in 49% of the samples. they were present in 18 wc, 13 ec and 2 gl samples. the most common and abundant chaetoceros species in our samples identified to date by lm and sem are listed below. most common species encountered the following list details the most common chaetoceros species found in the ballast water tanks (figs. 1–16) and includes taxonomic references used to identify particular species, an indication of whether vegetative cells and/or spores were encountered, illustration of a specimen, size range (apical axis) of the cells, indication of abundance, and report of occurrence on canadian or northern us-coasts, in that order. the abbreviation la indicates length of apical axis. 1. chaetoceros cf. affinis lauder: pitcher (1990), cupp (1943); spores (fig. 1); la: 7.7–12.9 µm; abundant in one gl ballast sample; reported from ec/wc. note: spores of c. affinis seem to bear more robust spines whereas ours feature rather delicate and thin spines. apart from this character, the spore is closest to the description of c. affinis given by cupp (1943). 2. chaetoceros ceratosporus ostenfeld: rines and hargraves (1988); vegetative cell and spore (fig. 2); la: 4.1–13.5 µm; found mainly in wc, rare in ec ballast; reported from wc (usa) and ec. note: the allocation of the spore to c. ceratosporus might be disputable, however, it meets hustedt’s (1962: p. 761) criteria describing it with the secondary valve smooth and the optional possession of spines: »sekundärschale in der mitte glatt, die langen fortsätze sind zuweilen vorhanden«. in some of our species 328 acta bot. croat. 68 (2), 2009 klein g., kaczmarska i., ehrman j.m. u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:28 color profile: disabled composite 150 lpi at 45 degrees spines were observed (as in rines and hargraves 1988), but these images are of low resolution and therefore not for publication. 3. chaetoceros cinctus gran: hasle and syvertsen (1997); spore with basal plate (fig. 3); la: 6.4–15 µm; found only in wc samples; reported from wc (usa) and ec. 4. chaetoceros compressus lauder: see pitcher (1990) for spores; spores; la: 5.5–9.8 µm; found in ec ballast only; reported from ec and wc. 5. chaetoceros cf. compressus var. hirtisetus rines et hargraves: rines and hargraves (1990); spore size ca. 7 µm (fig. 4); found in wc ballast; reported from ec. 6. chaetoceros constrictus gran: rines and hargraves (1988); vegetative cells and spores identified in lm; la: 21–25 µm; found in wc samples; reported from ec and wc (usa). 7. chaetoceros convolutus castracane: rines and hargraves (1988); vegetative cells; la: 9.1–17.4 µm; found only in wc ballast; reported from ec and wc. 8. chaetoceros debilis cleve: rines and hargraves (1988); vegetative cell and spore (fig. 5); la: 9.8–18.1 µm; found in ec and wc ballast; reported from ec and wc. 9. chaetoceros diadema (ehrenberg) gran: rines and hargraves (1988); spore (figs. 6,7) la: 7.4 – 31 µm; found mainly in ec, with a few in wc ballast; reported from ec and wc. 10. chaetoceros cf. diadema (ehrenberg) gran: hustedt (1930); spore (fig. 15); la: 12.1 µm; found in wc ballast; reported from ec and wc (usa). note: the closest match we could find in literature is the description by suto (2003) of monocladia humilis and its assigned synonym c. subsecundus (syn. c. diadema). in general the literature describes the number of processes on the spore valve of c. diadema with more than 2, never with one singular process. however, the depicted spore might also represent c. similis. 11. chaetoceros didymus ehrenberg: rines and hargraves (1988); vegetative cell and spore (fig. 8); la: 8.8–29.8 µm; present only in wc ballast; reported from ec and wc. 12. chaetoceros furcillatus bailey: peterson et al (1999); spore (fig. 9); size: 7.8 µm; found in one wc sample; reported from ec. 13. chaetoceros cf. hispidus (ehr.) brightw.: cleve-euler (1951); vegetative cell and spore (fig. 10); la: 11–27.4 µm; found only in wc ballast; no records on extant biogeography, poss. tertiary. 14. chaetoceros laciniosus schütt: rines and hargraves (1988); vegetative cell and spore; la: 4.9– 0.5 µm; found in wc, ec and gl ballast; reported from ec and wc. 15. chaetoceros lorenzianus grunow: rines and hargraves (1988); vegetative cell and spore (figs. 11, 12); la: 5.3–24.6 µm; mainly in ballast from wc, rare in ec arrivals; reported from ec and wc. note: although fig. 11 could also represent c. decipiens, rines and hargraves (1988) comment: »c. lorenzianus is extremely variable, and its varieties and forms are not well documented. intermediate forms between c. lorenzianus and c. decipiens exist, at times making it difficult to separate these closely related taxa«, and a range of variability is also present in our material. 16. chaetoceros cf. radicans schütt: rines and hargraves (1988); vegetative cell and probable spore; la: 5.7–16.2 µm; found mainly in wc, rare in ec ballast; reported from ec and wc. acta bot. croat. 68 (2), 2009 329 chaetoceros in ballast waters u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:28 color profile: disabled composite 150 lpi at 45 degrees 17. chaetoceros cf. similis cleve: rines and hargraves (1988); vegetative cell and spore (fig. 13); la: 8.2–10.6 µm; found in wc ballast only; reported from ec and wc. 18. chaetoceros cf. socialis var. radians (schütt) proschkina-lavrenko: proschkina-lavrenko (1953); vegetative cell and spore (fig. 14); la: 5.1–10.4 µm; mainly in wc, rarely in ec ballast; reported from ec and wc. 19. spore no.1 (fig. 16); identity could not be confirmed because we did not find valves of the vegetative stage attached. it is however similar to chaetoceros coronatus. discussion quantities of vegetative and resting chaetoceros propagules our results serve to estimate chaetoceros propagule pressure in canadian coastal waters. compared to pseudo-nitzschia species found in the same ballast tanks (16 species; up to 1.2 x 1010 cells l–1), chaetoceros species arrive at canadian ports in more moderate cell densities but higher species diversity (over 30) which we consider representative of ports receiving shipments from a wide variety of sources. vegetative cell densities of chaetoceros spp. ranged from 1–5.6 x 104 cells l–1, while large spores identifiable using light microscopy (apical axis > 10 µm) were less frequent; with 1–414 live spores l–1. we found ballast water cell densities comparable to that found in one sample from the receiving port of vancouver, where a bloom of one chaetoceros species produced 2.7 x 103 viable cells l–1 and 87 spores l–1. however, late spring blooms of canadian ec chaetoceros spp. often carry cell densities that are an order of magnitude higher (martin et al. 2001, 2006). the highest densities of spores carried to receiving ports were found in unexchanged ships traveling along the coasts of north america (414 spores l–1), while the vessels that underwent mid-ocean exchange brought in, on average, fewer than 10 (large) live spores l–1. exchanged ships carried up to 75 spores l–1 in their ballast. if the low densities of large spores are also representative of the scarcity of all chaetoceros spores in transoceanic vessels exchanging ballast in the open oceans, then the spore density and diversity originating from exotic coastal waters (across the ocean) were diminished due to ballast water exchange. this was particularly evident in ballast arriving in trans-atlantic vessels, where spores were detected in sem, but below countable quantities in lm. placed in the context of ballast tank volumes discharged, however, these moderate cell densities can become a sizable cell inoculate. we sampled tanks deballasting considerable inocula, up to 4.3 x 1010 live vegetative chaetoceros spp. cells l–1 and 3.6 x 108 live chaetoceros spores l–1 into the port waters. apart from these particular examples of quantities discharged, the annual volumes of ballast waters discharged are considerable as well. if the compiled data set for 2007 is a true representation of a typical year of ballasted ship traffic in canada, 22.7 x 106 t of ballast water were discharged in the atlantic region, 16.3 x 106 t in the pacific and 1.7 x 106 t in the great lakes/st. lawrence regions (lo pers. com.). these volumes combined (just one year in one country) equal the total of 40.7 x 106 t. divided by the population of canada (33 million) this results in about 1.25 t of ballast water per capita in the country. also, we found a higher than anticipated species diversity in our chaetoceros-containing samples. twenty nine species were encountered, with approximately half of these present as both vegetative cells and spores. within the genus chaetoceros, 73 spore forming species have been reported (stockwell and hargraves 1984). 330 acta bot. croat. 68 (2), 2009 klein g., kaczmarska i., ehrman j.m. u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:28 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 331 chaetoceros in ballast waters tab. 1. summary of densities of viable vegetative cells and spores in examined samples (lm) and presence/absence information for spores from sem. sample exchange type total spores (cells/l) total vegetative cells (cells/l) sample exchange type total spores (cells/l) total vegetative cells (cells/l) ec002 mo – – wc001 nx d 56,993 ec004 mo d 7,441 wc002 nx – – ec005 mo – 11 wc003 mo – 280 ec008 mo d – wc004 mo d 1,525 ec009 mo d 1,690 wc005 nx – – ec011 co d – wc006 mo d 18 ec013 mo d 604 wc007 mo – 11 ec016 co – – wc008 mo d 96 ec020 mo d – wc009 mo 1 – ec024 mo d 128 wc010 mo d 43 ec025 mo – 26 wc011 mo – – ec026 mo – – wc013 co 75 163 ec027 mo – 62 wc014 mo 5 – ec030 co – 1,176 wc015 co d – ec032 co d – wc016 nx 168 19 ec034 mo – – wc017 nx 240 540 ec036 mo – – wc018 co – – ec038 mo – 133 wc019 nx d 991 ec039 mo d 13 wc020 mo – 1 ec041 mo 3 337 wc021 mo d – ec044 mo d 33 wc022 co d – ec046 mo d – wc023 co d – ec049 mo – – wc024 mo – – ec050 mo d 18 wc026 nx 414 523 gl001 mo – – wc028 co d 955 gl002 mo – – wc029 mo d – gl003 mo – – wc030 mo d – gl004 mo 27 – wc032 mo – – gl005 co – – wc033 mo 67 5 gl006 mo – – wc034 co 5 18 gl007 co 9 57 gl008 nx – 22 gl009 mo 11 – gl010 mo – – gl011 co – – gl012 mo – 8 gl014 mo – – mo = mid-ocean exchange; co = coastal exchange; nx = no exchange; d = detected in sem; – = not detected in lm or sem u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:28 color profile: disabled composite 150 lpi at 45 degrees our study distinguished 31 spore types, 18 of which could be identified to species level, albeit with uncertainty in some cases. in addition, 12 species (including those that are not known to form spores) were present as vegetative cells. this number of chaetoceros species recovered from just 67 tanks arriving in canada within a four month time-period rivals the diversity reported from multiannual monitoring in the entire quoddy region of the bay of fundy (martin et al. 2001, 2006) and is comparable to the number of species reported from considerably larger water bodies such as the gulf of st. lawrence (bérard-therriault et al. 1999) or narragansett bay (rines and hargraves 1988). furthermore, residual ballast water and resuspended sediments increase the diversity we encountered, especially since the viability of spores found using only sem could not be determined. 332 acta bot. croat. 68 (2), 2009 klein g., kaczmarska i., ehrman j.m. figs. 1–8. chaetoceros spores and/or vegetative cells (sem). 1 – c. cf. affinis; 2 – c. ceratosporus; 3 – c. cinctus; 4 – c. cf. compressus var. hirtisetus; 5 – c. debilis; 6, 7 – c. diadema; 8 – c. didymus. u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:32 color profile: disabled composite 150 lpi at 45 degrees it is difficult to assess whether the observed diversity of spore bearing species is an unusual characteristic only of our samples (though this would be unexpected), because other ballast water diatom studies have rarely reported or identified chaetoceros spores. two major factors may have contributed to a high diversity of spores in our samples. first, most ballast examined was taken up during may–june, a time when a later spring diatom bloom becomes rich in chaetoceros species in boreal temperate waters. second, some spores we encountered may have been residual occupants of the tanks, that is they could have been re-suspended from the bottom of the tank sediment during ballast water exchange. deacta bot. croat. 68 (2), 2009 333 chaetoceros in ballast waters figs. 9–16. chaetoceros spores and/or vegetative cells (sem). 9 – c. furcillatus; 10 – c. cf. hispidus; 11– c. lorenzianus/c. decipiens?, 12 – c. lorenzianus; 13 – c. cf. similis; 14 – c. cf. socialis var. radians; 15 – c. cf. diadema (close to c. similis); 16 – spore no.1., similar to c. coronatus. u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:36 color profile: disabled composite 150 lpi at 45 degrees pending on ship type and atmospheric conditions during ballast exchange, up to 5% of the tank bottom waters and sediments are retained despite exchange. this would allow accumulation of sediments in ballast tanks and retention of associated biota, including resting spores (hallegraeff and bolch 1992). additional concern related to the discharge of non-indigenous organisms (or genotypes) is the presence of potentially harmful taxa. among the species found in our study are those listed by bates and strain (2006) as harmful: chaetoceros concavicornis, c. convolutus and c. debilis; implicated in fish mortality due to gill irritation on the canadian west coast and also reported from the east coast, albeit in low cell numbers to date. c. concavicornis, c. convolutus and possibly c. danicus are listed by horner et al. (1997) as harmful species on the u.s. west coast. of these, we found c. convolutus, c. danicus and c. debilis (with spores) in both ec and wc samples. in the natural environment, sporulation often occurs when growth conditions become suboptimal. light and nutrient depletion, and to a lesser degree oscillations of water temperature (hollibaugh et al. 1981) may trigger spore production (mcquoid and hobson 1995, 1996). the deprivation of nitrate in ambient waters, for example, has been repeatedly shown to induce spore formation (garrison 1981, kuwata and takahashi 1999). it is possible that the physicochemistry within a ballast water tank facilitates sporulation, as prolonged darkness, decline of oxygen concentration, biomass decay and oscillation of mineral nutrient concentration are expected in ship ballast tanks. several studies have shown the survival potential of diatom dormant stages (spores and resting cells), some germinating after years (e.g. anil et al. 2007), even after exposure to high temperatures (itakura et al. 1997). a period of dormancy of approximately two years seems to be common in many coastal species, with an upper limit of nine years for diatom spores (mcquoid et al. 2002) and decades for resting cells (sicko-goad et al. 1989). conditions within residual ballast tank sediments are not unlike those found in coastal sediments, including anoxia, darkness and changes in temperature. spore-producing coastal species, such as members of the genus chaetoceros, may be well adapted to man-made environments such as ballast tanks, an advantage that may further contribute to their cosmopolitan distribution. our study also revealed that not only a considerable number of species, some potentially harmful, but also species not yet recorded from at least some canadian coasts (such as c. cf. hispidus) are being discharged via ballast water. for example, abundant specimens of the cosmopolitan coastal marine species c. affinis were found in one ballast tank destined for the great lakes. in addition (including species found in bw samples but not represented in our list), c. cf. compressus var. hirtisetus, c. coronatus, c. densus, c. cf. subsecundus, and c. simplex var. calcitrans were found in ballast water samples arriving in vancouver, while our extensive literature search shows them reported thus far only from the ec. some species known from the canadian atlantic coast and more southern locations of the u.s. pacific (c. cinctus, c. ceratosporus, c. dichaeta) were found in ballast destined for vancouver bc, but not yet reported from western canadian coastal waters, probably due to the scarcity of floristic studies. the latter example underscores challenges facing microbial invasive biology. the »waxing-and-waning« of diatom populations, scarcity of long term and historical base-line research for large regions of the world (smayda 2007) and changing taxonomy (round et al. 1990) all add to impairment of our capacity to detect and recognise non-indigenous micro-biota. 334 acta bot. croat. 68 (2), 2009 klein g., kaczmarska i., ehrman j.m. u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:36 color profile: disabled composite 150 lpi at 45 degrees in summary, we found a considerable number of species from the genus chaetoceros arriving live at canadian ports in just 67 sampled ballast tanks. the highest densities of vegetative cells and spores were brought by vessels coming from the close southern neighbourhood, along the continental shelf from the same bioprovince. because the species composition of this source of ballast waters is expected to be similar to that from canada, such vessels do not undergo ballast water exchange. vessels undergoing ballast water exchange carry smaller diatom inoculums per cargo vessel. considering the shipping traffic magnitude and the volume of ballast waters globally translocated annually, the rate and consistency of the inocula and diversity of diatom species introduced to foreign ports should be of concern to canada and other countries. acknowledgements funding for this study was provided by nserc to the canadian aquatic invasive species network (caisn), project 1.3. »propagule pressure in relation to shipping mode and route«. the authors would like to thank the caisn sampling teams, especially beth piercey, andrea weise and colin van overdijk. we would like to thank the two anonymous reviewers for their helpful comments and discussion on spore taxonomy and nomenclature. references anil, a. c., mitbavkar, s., d’silva, m. s., hegde, s., d’costa, p. m., meher, s. s., banerjee, d., 2007: effect of aging on survival of benthic diatom propagules. journal of experimental marine biology and ecology 343, 37–43. bates, s. s., strain, p. m., 2006: nutrients and phytoplankton in prince edward island inlets during late summer to fall: 2001–2003. canadian technical report of fisheries and aquatic sciences 2668 xii, 1–136. bérard-therriault, l., poulin, m., bossé, 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diatoms and dinoflagellates. marine ecology progress series 176, 243–251. 338 acta bot. croat. 68 (2), 2009 klein g., kaczmarska i., ehrman j.m. u:\acta botanica\acta-botan 2-09\klein.vp 6. listopad 2009 11:07:36 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 317 acta bot. croat. 74 (2), 317–331, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0022 diatoms of the dojkinci river (stara planina nature park, serbia) jelena krizmanić1*, marija ilić2, danijela vidaković1, gordana subakov-simić1, jelena petrović1, katarina cvetanović3 1 university of belgrade, faculty of biology, institute of botany and botanical garden »jevremovac«, 43 takovska, 11000 belgrade, serbia 2 university of belgrade, institute for biological research »siniša stanković«, 142 bulevar despota stefana, 11060 belgrade, serbia 3 university of belgrade, faculty of chemistry, 12–16 studentski trg, 11000 belgrade, serbia abstract – diatom samples were collected during july 2010 at 15 localities from different types of substrate (stone surfaces, sand, mud, fi lamentous algae and submerged mosses) from the dojkinci river. during the research period, 124 taxa were determined within 43 genera. among numerous common diatoms we recorded three taxa for the fi rst time in serbia: brachysira intermedia (øst.) lange-bertalot, chamaepinnularia mediocris (krass.) lange-bertalot and navicula tridentula krass. also, we observed 21 taxa which are rarely recorded taxa for serbia. the most interesting was diatomella balfouriana grevill. that was previously known only from the river tisa near titel. in the studied material, it was identifi ed only in samples collected from the surface of boulders with mosses at the third locality. their morphology, distribution and ecology are presented in this paper. key words: brachysira intermedia, chamaepinnularia mediocris, diatomella balfouriana, diatoms, distribution, dojkinci river, navicula tridentula. introduction in lotic ecosystems, diatoms are considered to be the most diverse group of algae (round et al. 1990). regularly found diatom species in european freshwater habitats have well known ecological preferences (van dam et al. 1994, coste et al. 2009). however, data about the distribution and ecology of diatom species that are rarely observed may be incomplete (e.g. wojtal 2001, 2004, szabo et al. 2007, van de vijver and lange-bertalot 2009, kelly et al. 2009, andrejić et al. 2012). as diatoms are very important indicators of * corresponding author, e-mail: kjelena@bio.bg.ac.rs krizmanić j., ilić m., vidaković d., subakov-simić g., petrović j., cvetanović k. 318 acta bot. croat. 74 (2), 2015 environmental changes and for the biological monitoring of lotic ecosystems (stevenson and pan 1999, bere and tundisi 2010), it is of great signifi cance to describe their prevalence and ecology as well as possible. the mountain stara planina (balkan mountain) belongs to the system of balkan mountains which ranges from black sea in the east to vrška čuka in the west (marković 1980). it is one of the fl oristically and faunistically most diverse parts of serbia and the entire balkan peninsula. the overall richness and diversity of species, as well as the presence of numerous endemic and relic forms point to a specifi c genesis thаt infl uenced the remarkable diversity of this mountain, which makes it distinct from other parts of the balkans and europe from the biogeographical aspect. in 1997, at the suggestion of the institute for nature conservation of serbia, the government of the republic of serbia adopted the regulation on the protection of stara planina nature park which put stara planina under protection as a natural resource of great importance, and led to its being placed within the fi rst category of protection (mijović 2001). the total protected area of stara planina nature park is about 114.332 ha and it has a threelevel system of protection (the fi rst degree of protection is applied to an area of 3.680 ha, or 3.23%; the second degree of protection applies to an area 20.159 ha, or 17.63%, and the third degree of protection to an area 90.493 ha, or 79.14%) (official gazette rs, 2009). stara planina nature park is a protected natural resource nominated for the program »man and the biosphere« (unesco-mab) (ivančević et al. 2007). the dojkinci river is formed by the merging of two streams: belčin dol and tri kladenca. it is about 25 km long and ends in its confl uence with the jelovička river. this way the visočica river is formed which belongs to the black sea basin. the substrate through the longest part of the dojkinci river runs is red fi ne-grained sandstone, whose color comes from the large amount of fe2o3 and is extremely rich in quartz sand (andjelković 1958), as opposed to most of rivers in serbia, which have their beds made dominantly of carbonate (marković 1980). these siliceous, low-conductivity mountain habitats are currently being shown to host benthic diatom communities of high species richness (cantonati and langebertalot 2011, cantonati et al. 2011, liu et al. 2011). the morphology, distribution and ecology of algal fl ora of stara planina is poorly known (simić 1995, 1996, 2002), especially for diatoms from dojkinci river (obušković 1993, obušković et al. 1994). in this paper, we present the results of a fl oristic study of the diatoms of the dojkinci river (stara planina, serbia). among numerous common diatoms three new taxa for serbia were identifi ed, as well as 21 taxa that are rarely recorded in serbia. materials and methods during july of 2010 the material which is used in the present study was collected from 15 localities along the dojkinci river. from the gravel surface and boulders epilithic samples were scraped by scalpel blade and brush. epipsamic samples were collected from the surface of sand and mud substrates, while the epiphyton was taken from mosses plagiomnium sp. and philonotis sp. and liverwort marchantia polymorpha line subsp. polymorpha. samples were immediately fi xed with formaldehyde to a fi nal concentration of 4%. in order to remove organic matter, fi eld samples were treated with concentrated acid (h2so4) and diatoms of the dojkinci river in serbia acta bot. croat. 74 (2), 2015 319 kmno4, and then washed several times with distilled water in a laboratory. afterwards the material was airdried on cover glasses and mounted in naphrax®. permanent slides, prepared material and aliquots of the samples were deposited in the diatom collection of the university of belgrade, faculty of biology. light microscope observations and micrographs were made using a zeiss axioimagerm.1 microscope with dic optics and axiovision4.8 software. conductivity, oxygen, ph and water temperature were measured with a lovibond multimeter wtw 340i at each sampling site. ammonium ions, nitrates, phosphates, alkalinity and total hardness were measured using a lovibond multidirect photometer. terminology of valve morphology is based according barber and haworth (1981) and round et al. (1990). the abundance was estimated by counting 400 valves of each taxa present on a slide. sampling sites along with levels of protection in stara planina nature park are presented on the map (fig. 1). fig. 1. sampling sites along the dojkinci river with levels of protection in stara planina nature park. results a total of 125 diatom taxa (tab. 1) belonging to 43 genera were determined in material from the dojkinci river. the genus with the highest number of species was eunotia ehrenberg with 18 taxa. eunotia paludosa was the dominant taxon with an abundance of as much krizmanić j., ilić m., vidaković d., subakov-simić g., petrović j., cvetanović k. 320 acta bot. croat. 74 (2), 2015 tab. 1. diatom species list from dojkinci river. achnanthes coarctat a (brébisson) grunow frustulia saxonica rabenhorst achnanthidium exile (kützing) heiberg frustulia vulgaris (thwaites) de toni achnanthidium lineare w. smith geissleria decussis (østrup) lange-bertalot & metzeltin achnanthidium microcephalum kützing gomphonema acuminatum ehrenberg achnanthidium minutissimum (kützing) czarnecki gomphonema exilissimum (grunow) langebertalot & e. reichardt amphora sp. gomphonema gracile ehrenberg aulacoseira sp. gomphonema micropus kützing aulacoseira granulata (ehrenberg) simonsen gomphonema parvulius (l.-b. & reichardt) lange-bertalot & reichardt brachysira brebissonii r. ross gomphonema parvulum (kützing) kützing brachysira intermedia (østrup) lange-bertalot gomphonema pumilum (grunow) e. reichardt & lange-bertalot caloneis silicula (ehrenberg) cleve hantzschia amphioxys (ehrenberg) grunow chamaepinnularia mediocris (krasske) lange-bertalot & krammer humidophila contenta (grunow) lowe et al. cocconeis pediculus ehrenberg humidophila ingeae (van de vijver) lowe et al. cocconeis placentula ehrenberg humidophila perpusilla (grunow) lowe et al. cocconeis placentula var. lineata (ehrenberg) van heurck luticola mutica (kützing) d.g. mann cocconeis placentula var. pseudolineata geitler melosira varians c. agardh cymatopleura solea (brébisson) w. smith meridion circulare (greville) c. agardh cymbella compacta østrup navicula sp. cymbella parva (w. smith) kirchner navicula angusta grunow cymbella perparva krammer navicula cryptocephala kützing cymbopleura anglica (lagerstedt) krammer navicula cryptotenella lange-bertalot cymbopleura naviculiformis (auerswald ex heiberg) krammer navicula exilis kützing diatoma ehrenbergii kützing navicula gregaria donkin diatoma ehrenbergii f. capitulata (grunow) lange-bertalot navicula lanceolata ehrenberg diatoma hyemalis (roth) heiberg navicula recens (lange-bertalot) lange-bertalot diatoma mesodon (ehrenberg) kützing navicula reichardtiana lange-bertalot diatoma moniliformis (kützing) d.m. williams navicula tridentula krasske diatomella balfouriana greville navicula tripunctata (o. f. müller) bory de saint-vincent diploneis sp. neidium sp. encyonema lunatum (w. smith) van heurck neidium subampliatum (grunow) flower encyonema silesiacum (bleisch) d.g. mann nitzschia sp. encyonema ventricosum (c. agardh) grunow nitzschia acicularis (kützing) w. smith encyonopsis falaisensis (grunow) krammer nitzschia alpina hustedt diatoms of the dojkinci river in serbia acta bot. croat. 74 (2), 2015 321 encyonopsis microcephala (grunow) krammer nitzschia fonticola (grunow) grunow epithemia adnata (kützing) brébisson nitzschia linearis w. smith eunotia sp. nitzschia palea (kützing) w. smith eunotia bilunaris (ehrenberg) schaarschmidt nitzschia recta hantzsch ex rabenhorst eunotia boreoalpina lange-bertalot & nörpel-schempp orthoseira sp. eunotia circumborealis lange-bertalot & norpel pinnularia sp. eunotia diodon ehrenberg pinnularia acoricola hustedt eunotia exigua (brébisson ex kützing) rabenhorst pinnularia borealis ehrenberg eunotia fallax a. cleve pinnularia borealis var. rectangularis carlson eunotia fl exuosa (brébisson ex kützing) kützing pinnularia microstauron var. nonfasciata krammer eunotia groenlandica (grunow) nörpelschempp & lange-bertalot pinnularia subcapitata var. subrostrata krammer eunotia minor (kützing) grunow pinnularia viridiformis krammer eunotia nymanniana grunow placoneis sp. eunotia paludosa grunow planothidium sp. eunotia paratridentula lange-bertalot & kulikovskiy planothidium frequentissimum (lange-bertalot) lange-bertalot eunotia praerupta ehrenberg planothidium lanceolatum (brébisson ex kützing) bukhtiyarova eunotia rhomboidea hustedt psammothidium sp eunotia subherkiniensis lange-bertalot psammothidium subatomoides (hustedt) l. bukhtiyarova & round eunotia tetraodon ehrenberg reimeria sinuata (gregory) kociolek & stoermer eunotia trinacria krasske rhoicosphenia abbreviata (c. agardh) langebertalot eunotia valida hustedt stauroneis anceps ehrenberg fallacia subhamulata (grunow) d. g. mann stauroneis gracilis ehrenberg fragilaria arcus (ehrenberg) cleve stauroneis smithii grunow fragilaria biceps (kützing) lange-bertalot staurosirella pinnata (ehrenberg) d. m. williams & round fragilaria capucina desmazières surirella brebissonii var. kuetzingii krammer & lange-bertalot fragilaria capucina var. gracilis (oestrup) hustedt surirella linearis w. smith fragilaria ulna (nitzsch) lange-bertalot surirella minuta brébisson fragilaria vaucheriae (kützing) j. b. petersen tabellaria fl occulosa (roth) kützing fragilariforma virescens (ralfs) d. m. williams & round tetracyclus rupestris (braun) grunow frustulia crassinervia (brébisson) langebertalot & krammer krizmanić j., ilić m., vidaković d., subakov-simić g., petrović j., cvetanović k. 322 acta bot. croat. 74 (2), 2015 as 80% and was found on all types of substrates. subdominant taxon was achnanthidium minutissimum with the abundance of 9.28–42.13%. of these identifi ed taxa, three were new records to serbia: brachysira intermedia, chamaepinnularia mediocris and navicula tridentula. they were rarely distributed with very low abundance in samples. twenty one rarely observed taxa were: achnanthes coarctata (brébisson) grunow, achnanthidium exile (kützing) heiberg, brachysira brebissonii r. ross (pl. 2, figs. 13–18), humidophila ingeae (van de vijver) lowe et al. (pl. 1, figs. 18–24), h. perpusilla (grunow) lowe et al. (pl 1, plate 1. light microscope micrographs. eunotia tetraodon (figs. 1–3); e. fl exuosa (fig. 4); e. diodon (fig. 5); e. subherkiniensis (fig. 6); e. rhomboidea (fig. 7); e. exigua (figs. 8–13); psammothidium subatomoides (figs. 14–17); humidophila ingeae (figs. 18–24); h. perpusilla (figs. 25–26); tetracyclus rupestris (figs. 27–30). scale bar = 10 μm. diatoms of the dojkinci river in serbia acta bot. croat. 74 (2), 2015 323 figs. 25–26), diatomella balfouriana greville (pl. 2, figs. 21–30), eunotia diodon ehrenberg (pl. 1, fig. 5), e. exigua (brébisson ex kützing) rabenhorst (pl. 1, figs. 8–13), e. fl exuosa (brébisson ex kützing) kützing (pl. 1, fig. 4), e. praerupta ehrenberg, e. rhomboidea hustedt (pl. 1, fig. 7), e. subherkiniensis lange-bertalot (pl. 1, fig. 6), e. tetraodon ehrenberg (pl. 1, figs. 1–3), fallacia subhamulata (grunow) d.g. mann, frustulia crassinervia (brébisson) lange-bertalot & krammer (pl. 2, fig. 1–4), f. saxonica rabenhorst, navicula angusta grunow (pl. 2, fig. 5–6), nitzschia alpina hustedt (pl. 2, fig. 8), plate 2. light microscope micrographs. frustulia crassinervia (figs. 1–4); navicula angusta (figs. 5–6); navicula tridentula (fig. 7); nitzschia alpina (fig. 8); brachysira intermedia (figs. 9–12); b. brebissonii (figs. 13–18); chamaepinnularia mediocris (figs. 19–20); diatomella balfouriana (figs. 21–30). scale bar = 10 μm. krizmanić j., ilić m., vidaković d., subakov-simić g., petrović j., cvetanović k. 324 acta bot. croat. 74 (2), 2015 pinnularia acoricola hustedt, psammothidium subatomoides (hustedt) l. bukhtiyarova & round (pl. 1, figs. 14–17) and tetracyclus rupestris (braun) grunow (pl. 1, figs. 27–30). the most interesting was diatomella balfouriana. the paper presents the description and distribution of four species and their ecology. main valve measurements of the populations in the dojkinci river are shown in tab. 2, and chemical analyses of the water in tab. 3. description of the three new taxa for serbian diatom fl ora and one rarely observed taxon brachysira intermedia (østrup) lange-bertalot in lange-bertalot and moser (1994) (pl. 2, figs. 9–12) description: valves are lanceolate, gradually narrowed and at the ends almost pointed, length 22.13–27.3 μm, breadth 5.18–6.26 μm. the axial area is narrow linear near the apices, small and rhombic at the central area. the raphe is fi liform. the striae are parallel, at the ends sometimes parallel to slightly convergent, 26–27 in 10 μm. ecology: water temperature range from 6.4 °c to 12.5 °c, ph varied from 5.46 to 6.5 and concentrations of other nutrients were low. distribution (serbia): along the upper course of the dojkinci river (locality 1–5), on the surface of boulders, mud and on the mosses, with low abundance (0.23–0.61%), at the altitude from 1442.5 to 1723.5 m a.s.l. tab. 2. main valve measurements of the four species in the dojkinci river. taxon length (μm) width (μm) striae in 10 μm brachysira intermedia 22.13–27.3 5.18–6.26 26–27 chamaepinnularia mediocris 10.26–12.74 2.81–3.02 17–20 navicula tridentila 15 4.1 diatomella balfouriana 13.6–23.86 4.97–5.83 19–20 tab. 3. physico-chemical parameters of the water during sampling period. variable values water temperature (°с) 6.4–12.5 altitude (m) 924–1723.5 conductivity (μs cm–1) 213–302 ph 5.46–6.5 hardness (mg l–1) 25–67 total alkalinity (mg l–1) 0.1 alkalinity p (mg l–1) 0.1 no3-n (μg l–1) 0.635 po4-p (μg l–1) 1.45 diatoms of the dojkinci river in serbia acta bot. croat. 74 (2), 2015 325 chamaepinnularia mediocris (krasske) lange-bertalot and krammer in lange-bertalot and metzeltin (1996) (pl. 2, figs. 19–20) description: valves are linear to broadly oval, with rounded apices and a gibbous middle, length 10.26–12.74 μm, breath 2.81–3.02 μm. the axial area is narrow near the apices, becoming wider near the central area. the central area is a transverse fascia. the raphe is fi liform and slightly arched. the striae are parallel, 17–20 in 10 μm. ecology: water temperature 12.5 °c, ph 6.5 and concentrations of other nutrients were low. distribution (serbia): in the upper course of the dojkinci river at only one locality (4) with very low abundance (0.12%), on surface of mud, at the altitude 1432 m a.s.l. navicula tridentula krasske (1923) (pl. 2, fig. 7) description: valve is linear, triundulate, with capitate ends, length 15 μm, breath 4.1 μm. the axial area is very narrow, linear, central area is variables size, often diffi cult to see. the raphe is fi liform. ecology: water temperature 12.5 °c, ph 6.5 and concentrations of other nutrients were low. distribution (serbia): in the lower course of the dojkinci river at locality 11, only one specimen, on surface of sand at the altitude of 1015.5 m a.s.l. diatomella balfouriana greville (1855) (pl. 2, figs. 21–30) description: valves are linear with rounded apices, lenght 13.6–23.86 μm, breath 4.97– 5.83 μm.the axial area is wide. the raphe is fi liform, with dilated external proximal raphe ends that terminate relatively distant from one another. the striae are short and parallel to radiate, 19–20 in 10 μm. the general character of this species is a septum which is present on each valvocopula. the septum extends the entire length of the valve and has three large openings, the largest in the center and two smaller at the ends. ecology: water temperature 12.5 °c, ph 6.5 and concentrations of other nutrients were low. distribution (serbia): in the upper course of the dojkinci river at only one locality (3) with an abundance of 23.78%, on surface of boulders with mosses, at the altitude 1503.5 m a.s.l.; tisa river at titel (szabados 1966). discussion the main purpose of the study was to collect records on diatom assemblages from the dojkinci river, which has a bottom substrate (red sandstone) unusual when compared with the substrates of other rivers in serbia. in many mountainous areas of the word the highest elevations are reached on siliceous crystalline mountain ranges. these sites are often pristine, relatively remote, and included in nature preserves because of their value for biodiversity conservation, recreation, and as storages of good-quality water (cantonati and lange-bertalot 2011). stara planina nature park has similar characteristics and very rich valuable fl ora and fauna with an important role in biodiversity conservation in serbia, as well as, in the balkan peninsula. krizmanić j., ilić m., vidaković d., subakov-simić g., petrović j., cvetanović k. 326 acta bot. croat. 74 (2), 2015 the diatom fl ora of dojkinci river was mainly formed by acidophilic, limno-terrestial, moss-inhabiting, widely distributed species. the large development of acidophilic species is related to siliceous riverbed rocks of the dojkinci river. the dominant diatom genus in the dojkinci river was eunotia, with 18 taxa. according to lange-bertalot et al. (2011) the genus eunotia is mainly restricted to freshwater oligotrophic and oligosaprobic habitats, which enables it to play an important role as indicator in water monitoring (alles et al. 1991, kwandrans 2007). physico-chemical conditions of the dojkinci river are suitable for dominance of different eunotia species. according to pavlov and levkov (2013) and wojtal et al. (1999) e. paludosa was established as dominant taxon in the samples taken from the mud substrates and from mosses. achnanthidium minutissimum is one of the most frequently occurring diatoms in freshwater benthic samples globally (patrick and reimer 1966; krammer and lange-bertalot 1991; potapova and hamilton 2007; hofmann et al. 2013). this species has been reported from different type of waters, alkaline and acidic, oligotrophic and hypertrophic (van dam et al. 1994; potapova and hamilton 2007). a brachysira intermedia is widespread in waters of mountain regions on silica surfaces with low electrolyte content and in oligoto dystrophic waters. according to lange-bertalot and moser (1994) and hofmman et al. (2013), valve length is 25–33 μm, valve breadth 5–6.5 μm, striae 26–30 in 10 μm. valve measurements in our samples were similar to those in the literature. chamaepinnularia mediocris can be found in oligoto dystrophic freshwaters of mountain regions (springs, streams and lakes) on silica substrates. according to van dam et al. (1994), c. mediocris is an acidophilic species, mainly occurring at ph < 7. hofmman et al. (2013) state that this species prefers moderately acidic waters. our results showed that the water of the dojkinci river is moderately acidic (ph was 6.5) at the site at which this alga was recorded. c. mediocris mainly occurs on wet and moist or temporarily dry places. fránková et al. (2009) found c. mediocris in sphagnum fens, as did kapetanović et al. (2011). also, buczkó and wojtal (2005) and van de vijver and beyens (1997) found this species on different type of mosses. in our sample c. mediocris was found on a mud substrate, at an altitude of 1432 m a.s.l., which is higher than that shown by kapetanović et al. (2011) (at an altitude 930 m a.s.l.). researches of buczkó and wojtal (2005) noted c. mediocris along with eunotia exigua which is complementary with our fi ndings. morphological data show that the valve is 9–16 μm long, 2–3 μm wide and has 20–30 striae in 10 μm (hofmman et al. 2013). according to vesela and johansen (2009), valve length is 10–12 μm, valve breadth 2.7 μm, striae 18–21 in 10 μm. valve measurements in our samples were similar those in literatures. chamaepinnularia mediocris is known from: strømness bay area, south georgia, antarctica (vandevijver and beyens 1997); laurentian great lakes (stoermer et al. 1999); hungary (buczkó and wojtal 2005, buczkó 2006); great smoky mountains national park, usa (johansen et al. 2007); penza oblast, european russia (kulikovskii 2008); elbsandsteingebirge region, czech republic (vesela and johansen 2009); western carpathian spring fens, between the czech republic and slovakia (fránková et al. 2009); mt. zvijezda near sarajevo, bosnia and herzegovina (kapetanović et al. 2011); lakes in the azores archipelago, portugal (pereira et al. 2014). diatoms of the dojkinci river in serbia acta bot. croat. 74 (2), 2015 327 navicula tridentula has cosmopolitan widespread, sporadically at typical habitat, such as springhead of stream, peat bogs, swamp. krammer and lange-bertalot (1986) state that the species prefers fresh water with low concentrations of electrolytes. according to van dam et al. (1994), n. tridentula is an alcalophilic species, mainly occurring at ph < 7, in wet and moist or temporarily dry places. in the dojkinci river n. tridentula was found at one locality at ph 6.5, on a sandy surface. in thailand (leelahakriengkra 2013) the species was recorded at an altitude 478–489 m a.s.l., but in dojkinci river the same species was recorded on higher altitude (1015.5 m a.s.l.). valve measurements of the population from the dojkinci river had similar values, as well as, values according to krammer and lange-bertalot (1986): valve length 11–19 μm, breadth 3.5–4 μm, striae are not visible on lm. navicula tridentula is known from: north western united states (bahls 2009); western carpathian spring fens, between the czech republic and slovakia (fránková 2009); chiang dao district, thailand (leelahakriengkra 2013). new species for diatom fl ora of serbia recorded at localities of the dojkinci river were found with very low abundances, although they were on substratum typical for them. a cosmopolitan species, diatomella balfouriana is widespread from the arctic to antarctica. it is characteristic for oligoto dystrophic boreo-alpine mountain freshwaters, with low conductivity and nutrient poor (krammer and lange-bertalot 1986). however, d. balfouriana was recorded in temporary pools of brackish water, as well as in moist soil near the ocean round antarctica (van de vijver et al. 2012). until now, there is only one published record about d. balfouriana for serbia, in the tisa river (plain river in northern part of serbia, ph mainly occurring about 7) (szabados 1966). this can be considered poorly reliable information, because there are no microphotographs which can confi rm the fi nding. in addition, the type of substrate (mud at the bottom of the river) and ph are not typical of d. balfouriana. our fi ndings are complementary with literature data: oligoto dystrophic freshwater, low-conductivity and nutrient-poor, boreo-alpine mountain habitats. krammer and lange-bertalot (1986) states that the valve is 12–52 μm long, 6–8 μm wide and 18– 21 striae in 10 μm. according to kociolek (2011), valve length is 12–40 μm, breadth is 3.5–6 μm and there are 18–22 striae in 10 μm. valve measurements of the population from the dojkinci river had values similar to those given by kociolek (2011), but the breadths of the valves reported here are smaller than those given by krammer and lange-bertalot (1986). acknowledgments financial support was provided by the ministry of education and science of the republic of serbia (project no. tr 037009). references alles, e., nörpel-schempp, m., lange-bertalot, h., 1991: zur systematik und ökologie charakteristischer eunotia-arten (bacillariophyceae) in elektrolytarmen bachberläufen. nova hedwigia 53, 171–213. krizmanić j., ilić m., vidaković d., subakov-simić g., petrović j., cvetanović k. 328 acta bot. croat. 74 (2), 2015 andrejić, j., krizmanić, j., cvijan, m., 2012: three new records for diatoms from 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europe. algological studies supplement 112, 89–94. opce-str.vp acta bot. croat. 68 (2), 401–419, 2009 coden: abcra 25 issn 0365–0588 morphotype variations in subfossil diatom species of aulacoseira in 24 michigan lakes, usa kalina m. manoylov1*, nadja ognjanova-rumenova2, robert jan stevenson3 1 department of biological and environmental sciences, georgia college and state university, milledgeville, ga 31061 usa 2 institute of geology, bulgarian academy of sciences, acad. g. bonchev str. 24, 1113 sofia, bulgaria 3 department of zoology, michigan state university, east lansing, mi 48824 usa diatom assemblages preserved in lake sediment records can provide proxy data of past environmental changes in biological conditions. in order to investigate past changes in the environment of north-central michigan, diatom assemblages were studied in sediment cores retrieved from 24 lakes. diatoms were analyzed from the 'top' and 'bottom' of each core to reconstruct land-use changes in this area. aulacoseira taxa were identified and evaluated with light and scanning electron microscopy. results of these observations showed the presence of some variability of the morphological features within north american species populations. diatom species composition in surface sediments and differences between tops and bottoms corresponded to changes in land use surrounding the lakes, ranging from predominantly forest and rangeland to urban and agriculturally impacted. diatom-inferred past conditions revealed that the observed morphotypes probably represent taxa with different ecological preferences. the main factors influencing the variability of these morphotypes are changes in the trophic status of the lakes. key words: diatom, aulacoseira, ultrastructure, sediment, distribution, morphotype, michigan lakes introduction in the last few decades, lakes within large watersheds adjacent to the great lakes have been of considerable interest (wolin and stoermer 2005). information on changes due to human activities is imprinted in the lacustrine sediment composition (stoermer et al. 1985, fritz et al. 1993, bradbury et al. 2002) and inferences of past conditions are possible (stoermer and smol 1999). following changes in diatom assemblages allows the distinguishing of natural disturbances from those caused by the development of the many coastal areas of the great lakes (enache and prairie 2002). acta bot. croat. 68 (2), 2009 401 * corresponding author: kalina.manoylov@gcsu.edu u:\acta botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:41 color profile: disabled composite 150 lpi at 45 degrees species within aquatic ecosystems change with the surrounding environment. algae go through such changes faster than other organisms (barinova et al. 2008) and are considered sensitive indicators of past and current changes. studies of past environmental conditions are limited to inorganic biological remnants such as diatom frustules and other preserved proxies of past conditions. in sediments, diatoms are abundant and well preserved (stoermer and smol 1999), and diatom species respond sensitively to anthropogenic alterations in land use and cover and resulting changes in nutrient concentration and conductivity (dixit et al. 2001). many reconstructions of lake history based on sub-fossil diatoms have used changes in ratios of centric and pennate diatoms in sediments (e.g. wolin and duthie 1999, stone and fritz 2004). centric diatoms dominate lacustrine sediments (stoermer and smol 1999) and the genus aulacoseira has been reported in most lakes (zohary 2004, wolin and stoermer 2005). changes in aulacoseira species can bring region-specific understanding in freshwater habitats (usoltseva and likhoshway 2007). closely related species have been lumped in many studies because fine level distinctions in taxonomy are too challenging without extensive taxonomic experience. the goals of this study were first to trace coarse changes in centric and pennate diatom species composition between 'top' and 'bottom' sediments and second to compare the dominant aulacoseira species and diatoms were analyzed from the 'top' and 'bottom' of each core. the 'top/bottom' comparison approach has been used in palaeolimnological reconstruction of recent climate change and extensive catchment land-use changes in different locations (dixit et al. 1999, messerli et al. 2000, yang et al. 2002, 2008). materials and methods sediment cores were retrieved from 24 lakes in the muskegon river watershed, michigan (stevenson et al. 2007). the muskegon river watershed drains approximately 2,723 square miles of land and is located in north-central michigan. the river is approximately 219 miles long from its source at houghton and higgins lakes down to its mouth at muskegon lake and, eventually, lake michigan. the muskegon river watershed is one of the largest watersheds in the state of michigan (http://www.cevl.msu.edu/mrweap/mriver map.html). lakes were sampled at their deepest point, in the deepest basin (glew et al. 2001). glew corer was used for sediment retrieval. lake metrics were measured and summarized as part of the muskegon watershed project. in this study, tops were the surface sediment, bottoms were the deepest intact sediment retrieved by the corer. using a knife, tops and bottoms were sliced and represented 1cm of sediment (v. lougheed personal communication). preparation of diatom samples followed standard methods (battarbee 1986). diatom relative abundances were determined by subsampling and acid-cleaning a subsample of the sediment algal sample. subsamples of cleaned diatoms were mounted on microscope slides using naphrax® as mounting medium. a minimum of 600 diatom valves were identified and counted at 1000 times magnification using a leica dmlb microscope and differential interference contrast optics. samples were scanned for aulacoseira species after enumeration of 600 valves. sem observations were made with acid-cleaned, gold-coated specimens and a scanning electron microscope (sem, jeol, ja402 acta bot. croat. 68 (2), 2009 manoylov k. m., ognjanova-rumenova n., stevenson r. j. u:\acta botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:41 color profile: disabled composite 150 lpi at 45 degrees pan electron optics laboratories) 6400v with a lab6 emitter (noran eds) at the center for advanced microscopy at michigan state university. diatoms were identified primarily with the use of hustedt (1927–1966), krammer and lange-bertalot (1986, 1988, 1991a, b), patrick and reimer (1966, 1975), as well as more recent or more specific references such as the pirla diatom iconograph (camburn et al. 1986, krammer 1991a, b, siver and kling 1997, crawford and likhoshway 1998, likhoshway and crawford 2001, houk and klee 2007) and diatoms of north america (siver et al. 2005). aulacoseira species have been identified by the following characters: frustule dimensions (ranges in valve diameter and pervalvar axis length); areolar rows on the mantle in 10 µm; form and ultrastructure of the areola-type and vola-covered occlusions; form and structure of the linking spines; form and structure of the separation spines; structure of the ringleist – shallow or deep; thick, thin or hollow; number, position and form of the rimoportula; type of the external opening of the rimoportula; other species features – presence of spines; granulae, etc. the following community characteristics were calculated for each sample: richness (total number of taxa observed in a site); shannon-weaver diversity index h= –s(i–s)pilog(pi), where pi is the proportion of the total count arising from the i th species (shannon and weaver 1949); evenness (equitability)=h’/ln(richness) (pielou 1969) and simpson’s dominance index (simpson 1949), which measures the likelihood of two randomly chosen individuals in a sample being the same species. the correlation coefficients (r2) were used to identify relationships between physicochemical variables in top and bottom-reference sites. significant correlations of more than 50% were discussed. all statistical analyses were performed with systat ® 10 (wilkinson 1989). results lakes varied in depth from sapphire lake with 2.2 m to fremont and ryerson lakes with more than 20 m. total anthropogenic land use varied from less than 4% for 3 lakes to more than 80% in fremont lake. diatom species composition in surface sediments and differences between tops and bottoms corresponded to the current land use surrounding the lakes, which ranged from predominantly forest (goose and long lakes, missauke county) and rangeland to urban and agriculturally impacted (cadillac lake, wexford county). particular combinations of size, depth and human impact conditions allowed the classification of 13 lakes with good conditions, 8 lakes with fair and 3 lakes with poor conditions. for example, the shallow and eutrophic brooks lake with only 23% total land use had the highest cultural eutrophication index and was classified as poor (tab. 1). we identified 153 diatom species. centric diatoms were dominant in 55% of the 'top' sediment counts and in 92% of the 'bottom' sediment counts. aulacoseira species were present in all lakes and dominant in top and bottom counts. no community diversity attributes were significantly different in 'top' and 'bottom' counts. mean shannon diversity was close to 1 in both 'top' and 'bottom' counts. shannon diversity was highest in the 'top' sediment count for goose lake (a good quality lake) and the 'bottom' sediment count of round lake (a fair quality lake). in the three lakes with poor quality, diversity significantly deacta bot. croat. 68 (2), 2009 403 aulacoseira species in sediments u:\acta botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:42 color profile: disabled composite 150 lpi at 45 degrees 404 a c t a b o t .c r o a t .68 (2),2009 m a n o y l o v k .m .,o g n ja n o v a -r u m e n o v a n .,s t e v e n s o n r .j. tab. 1. lake identity and characteristics. number abbreviations correspond to numbers in map 1. t_land – total land use, where 0% land use means no human alteration or 100 % forest; depth in m; s_ctp – sum of total p in ppb; cei – cultural eutrophication; a_oligo – algae as percent oligotrophic taxa according to stevenson’s mid atlantic trophic index; lake_ibi – lake index of biological indicator and lake condition (muskegon watershed web). n lake county t_land depth s_ctp secchi cei a_oligo lake_ibi lake condition 1 baptist lake newaygo 0.53 14.33 14.31 5.77 –0.42 0.06 52.02 good 2 brooks lake newaygo 0.22 4.13 16.30 0.93 1.03 0.00 8.91 poor 3 cadillac lake wexford 0.76 3.75 24.60 1.58 0.71 0.01 14.68 poor 4 doc and tom lake clare 0.34 6.33 16.59 2.50 0.04 0.04 62.78 good 5 fremont lake newaygo 0.83 20.67 19.75 3.82 0.62 0.03 29.45 fair 6 goose lake missauke 0.03 3.50 13.44 1.91 0.06 0.13 72.83 good 7 haymarsh lake mecosta 0.07 8.67 16.33 3.08 0.05 0.02 36.99 fair 8 hess lake newaygo 0.42 3.80 13.70 0.60 1.42 0.00 8.47 poor 9 hillsview lake mecosta 0.13 10.30 14.40 2.00 0.77 0.12 38.01 fair 10 horsehead lake mecosta 0.34 13.33 14.20 3.50 –0.18 0.04 42.70 good 11 little whitefish lake newaygo 0.20 12.00 22.90 3.62 –0.30 0.01 47.24 good 12 long lake c clare 0.31 17.70 10.10 5.10 –0.50 – 49.35 good 13 long lake m missauke 0.04 5.00 12.89 2.83 –0.01 0.02 59.65 good 14 mccoy lake osceola 0.61 8.33 33.01 2.58 0.07 0.08 53.75 good 15 pickerel lake newaygo 0.34 19.25 21.10 4.13 –0.02 0.00 29.29 fair 16 rogers dam pond mecosta 0.48 4.40 34.36 1.53 0.64 0.06 33.38 fair 17 round lake mecosta 0.41 12.50 23.83 2.67 0.40 0.03 33.71 fair 18 ryerson lake newaygo 0.48 24.17 22.79 5.17 –0.16 0.03 40.80 good 19 sapphire lake missauke 0.21 2.20 15.00 1.50 –0.11 0.07 57.78 good 20 school section lake mecosta 0.40 7.00 20.94 3.57 –0.23 0.06 54.82 good 21 second lake newaygo 0.65 12.33 13.76 3.37 0.26 0.04 36.13 fair 22 silver lake clare 0.27 14.92 13.76 4.67 –0.34 0.33 64.77 good 23 townline lake mecosta 0.46 11.43 14.20 2.90 0.32 0.07 43.45 good 24 winfield lake newaygo 0.64 14.33 33.67 1.95 0.90 0.03 19.67 fair u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ m a n o y l o v . v p 6 . l i s t o p a d 2 0 0 9 1 2 : 3 8 : 4 2 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s creased with an increase in human influence 1.01 (± 0.00001 se 'bottom') to 0.89 (± 0.001 se 'top'). baptist lake had the lowest diversity in both 'top' and 'bottom' counts, so lake quality alone was not a good predictor of diatom sediment diversity. simpson’s index did not differ between top and bottom (mean 0.74 for both) even though the dominant taxa changed. in 'bottom' sediment samples, taxa observed in counts varied from 21 to 69 taxa (evenness 0.14 to 0.34), while in the 'top' 1 cm sediment species ranged from 17 to 63 diatom taxa (evenness 0.06 to 0.34, tab. 2). a fairly diverse aulacoseira bottom community reduced to a. subarctica and a. ambigua in the top samples. the main factors influencing the variability of these morphotypes were changes in the trophic status of the lakes (tab. 3). aulacoseira taxa from the 'bottom' sediment counts were negatively correlated with depth, cultural eutrophication, and increase in total phosphorus where pennate diatoms dominated the community. in the 'top' diatom community aulacoseira taxa were positively correlated with depth and oligotrophic conditions (tab. 4). acta bot. croat. 68 (2), 2009 405 aulacoseira species in sediments tab. 2. community attributes in top and bottom sediment sites. se – standard error; p – significance level. attributes bottom top p mean ±se min max mean ±se min max diversity 1.01 0.045 0.42 1.4 1 0.059 0.19 1.4 ns richness 46 2.55 21 69 43 2.36 17 63 ns evennes 0.26 0.009 0.14 0.337 0.26 0.013 0.07 0.341 ns simpson’s dominance 0.736 0.009 0.663 0.863 0.737 0.013 0.659 0.934 ns tab. 3. relative abundance (ra) of aulacoseira species in top and bottom sediment samples of the 24 michigan lakes. taxon ra top, mean (range) ra bottom, mean (range) aulacoseira ambigua (grunow) simonsen 0.22 (0.006–0.54) 0.11 (0.003–0.29) aulacoseira granulata (ehrenberg) simonsen 0.02 (0.002–0.13) 0.02 (0.001–0.08) aulacoseira granulata morphotype curvata 0,003 aulacoseira italica (ehrenberg) simonsen 0,003 0,003 aulacoseira nygaardii (camburn) camburn et charles 0,003 aulacoseira perglabra (østrup) haworth 0,08 aulacoseira pseudoamericana (camburn) siver et kling 0,08 aulacoseira subarctica (müller) haworth 0.19 (0.003–0.52) 0.11 (0.002–0.68) aulacoseira valida (grunow) krammer 0,003 u:\acta botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:42 color profile: disabled composite 150 lpi at 45 degrees 406 a c t a b o t .c r o a t .68 (2),2009 m a n o y l o v k .m .,o g n ja n o v a -r u m e n o v a n .,s t e v e n s o n r .j. tab. 4. correlations between algal groups and lake dimensions. percent aulacoseira in 'top' and 'bottom' samples, other centric and pennate diatoms and factors as abbreviated in table 1. depth s_ctp secchi cei a_oligo lake_ibi au_t centr-t pen_t au_b centr-b pen_b depth 1.00 sumctpppb (s_ctp) 0.23 1.00 secchi –0.48 0.51 1.00 culturaleut (cei) 0.11 0.99 0.65 1.00 (a_oligo) 0.91 –0.18 –0.67 –0.29 1.00 lake_ibi 0.57 –0.12 –0.91 –0.29 0.59 1.00 aulacoseira -top (au_t) 1.00 0.26 –0.47 0.14 0.90 0.58 1.00 other centrics-top (centr-t) 0.42 0.98 0.41 0.95 0.01 –0.04 0.44 1.00 pennate-top 0.50 –0.56 –0.38 –0.56 0.76 0.06 0.47 –0.39 1.00 aulacoseira -bottom (au_b) –0.83 –0.73 0.02 –0.64 –0.54 –0.30 –0.85 –0.85 –0.06 1.00 other centrics-bottom (centr-b) 0.33 0.24 0.45 0.35 –0.45 –0.01 0.35 0.04 0.19 0.19 1.00 pennate-bottom 0.73 0.68 0.24 0.65 0.48 –0.06 0.74 0.80 0.23 –0.92 –0.41 1.00 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ m a n o y l o v . v p 6 . l i s t o p a d 2 0 0 9 1 2 : 3 8 : 4 2 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s aulacoseira species descriptions aulacoseira ambigua (grunow) simonsen (simonsen 1979; pl. 1, figs. 1–10) basionym: melosira crenulata var. ambigua grunow (van heurck, h., 1880–1885) melosira ambigua (grunow) o. müller aulacoseira ambigua (grunow) simonsen (kobayasi and nozawa 1981: figs. 1–22) melosira ambigua (grunow) o. müller var. ambigua (camburn and kingston 1986: 20, pl. i, figs. 1–5) aulacoseira ambigua (grunow) simonsen (siver and kling 1997: 1808, figs. 1–12) aulacoseira ambigua (grunow) simonsen (camburn and charles 2000; 13, pl. 1, figs. 18–22) aulacoseira ambigua (grunow) simonsen; (siver et al. 2005: 33, pl. 1, figs. 22, 25–27, pl. 3, fig. 3) valve diameter ranges of 4–14 µm; the height of valve mantle 5.50–14 µm; the areolae on the mantle are in spiral rows 16–20 in 10 µm. the areolae have circular form with deep-seated vela. the linking spines are notched bifid, and terminate each mantle costa. the ringleist is hollow (pl. 1, fig. 10). the rimoportulae are two per valve, without stalk. they are located on the ringleist (likhoshway and crawford 2001). their external openings are large, visible in lm (pl. 2, fig.5). the separating spines are longer, pointed (le cohu 1991). ecology: known from mesotrophic to eutrophic environments, dominant in shallow lakes (van dam et al. 1994, trifonova and genkal 2001, barinova et al. 2008). this taxon disappeared from the top of cadillac lake (being with 29% ra) in the bottom sediment, and remained in low abundance in doc and tom lake (both lakes with poor conditions). a 15-fold increase from bottom to top was observed in fremont lake. this taxon was not observed in any lake classified as 'good' (tab. 1). aulacoseira granulata (ehrenberg) simonsen (simonsen 1979; pl. 2, figs. 1–6) basionym: gallionella granulata ehrenberg 1841 melosira granulata (ehrenberg.) ralfs (pritchard 1861: 820) melosira granulata (ehrenberg.) ralfs (stoermer et al. 1981: 348, pl. 2, figs. 21–33) aulacoseira granulata (ehrenberg) simonsen (siver and kling 1997: 1813, figs. 23–28) valve diameter ranges of 3–11.50 µm; the height of valve mantle 10–17 µm; the areolae on the mantle are in straight or spiral rows 10–15 in 10 µm. the areolae are coarse, round to square, covered with velar complex (crawford and likhoshway 1998). the velar plate occurs on the outside. the linking spines are notched triangular. the rimoportulae are found on the valve face/mantle junction, near the ringleist (pl. 1, fig. 5). the external opening is large, visible in lm (likhoshway and crawford 2001). internally, the rimoportula has tightly curved stalks lying close to the mantle surface (likhoshway and crawford 2001). the separating spines are conical with varying length, from short to almost the length of the valve mantle. ecology: known from mesotrophic to highly eutrophic environments, dominant in shallow lakes (van dam et al. 1994, siver and kling 1997, trifonova and genkal 2001, barinova et al. 2008). acta bot. croat. 68 (2), 2009 407 aulacoseira species in sediments u:\acta botanica\acta-botan 2-09\manoylov.vp 9. listopad 2009 13:41:30 color profile: disabled composite 150 lpi at 45 degrees 408 acta bot. croat. 68 (2), 2009 manoylov k. m., ognjanova-rumenova n., stevenson r. j. pl. 1. aulacoseira ambigua (grun.) simonsen fig. 1 – girdle view – lm, round lake, top sediments; figs. 2, 3 – girdle view with cross-section focus – lm, silver lake, bottom sediments; figs. 4 a, b – girdle view with cross-section focus – lm, cadillac lake, top sediments; fig. 5 – sibling valves, showing spiral rows of areolae with external openings of the rimoportulae, sem, round lake, top sediments; figs. 6, 7 – sibling valves, sem, cadillac lake, top sediments; fig. 8 – detail of linking spines – triangular with bicuspid ends, sem, cadillac lake, top sediments; fig. 9 – sibling valves with dissolution, see the prominent form of the linking spines – the ends are clavate, sem, mccoy lake, bottom sediments; fig. 10 – inner surface of the girdle, the ringleist is hollow, sem, cadillac lake, top sediments. u:\acta botanica\acta-botan 2-09\manoylov.vp 9. listopad 2009 13:41:32 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 409 aulacoseira species in sediments pl. 2. aulacoseira granulata (ehrenber) simonsen. fig. 1 – girdle view – lm, ryerson lake, top sediments; fig. 2 – girdle view – lm, second lake, top sediments; figs. 3a, b – girdle view with cross-section focus – lm, ryerson lake, bottom sediments; fig. 4 – girdle view – lm, haymarsh lake, bottom sediments; fig. 5 – aulacoseira ambigua (above) and a. granulata (below) with long pointed separating spines, sem, cadillac lake, top sediments; fig. 6 – a. granulata, external opening of the rimoportula arrowed, sem, cadillac lake, top sediments; fig. 7 – a. italica (ehrenberg) simonsen, lm, horsehead lake, top sediments; figs. 8 a, b – a. nygaardii (camburn) camburn and charles, girdle view with cross section focus, lm, mccoy lake, bottom sediments; fig. 9 – a. nygaardii (camburn) camburn and charles, sibling valves with t-shaped tips of the spines, sem, mccoy lake, bottom sediments. u:\acta botanica\acta-botan 2-09\manoylov.vp 9. listopad 2009 13:41:33 color profile: disabled composite 150 lpi at 45 degrees aulacoseira granulata mophotype curvata (hustedt) krammer basionym: melosira granulata var. curvata grunow (van heurck, h., 1880–1885) aulacoseira granulata mophotype curvata (hustedt) krammer (krammer 1991a: figs. 6–8, 19) valve diameter ranges 5–7.50 µm; the height of valve mantle 12.50–16.50 µm; the areolae on the mantle are in straight or spiral rows 12 in 10 µm. ecology: known from mesotrophic to eutrophic environments as a. granulata (van dam et al. 1994) typical of shallow lakes (trifonova and genkal 2001). in muskegon lakes we found it in townline lake, which has good conditions. aulacoseira italica (ehrenberg) simonsen (simonsen 1979; pl. 2, fig. 7) basionym: gallionella italica ehrenberg 1838 melosira italica (ehrenberg) kützing (kützing 1884: 55, pl. 2, fig. 4) aulacoseira italica (ehrenberg) simonsen (kobayasi and nozawa 1981: figs. 1–11) aulacoseira italica (ehrenberg) simonsen (kramer 1991b: fig. 44) aulacoseira italica (ehrenberg) simonsen emend. crawford, likhoshway et jahn (crawford et al. 2003: 17, figs. 2–14) valve diameter ranges of 8–11 µm; the height of valve mantle 9–14 µm; the areolae on the mantle are in straight or sinistrorse spiral rows 16–18 in 10 µm. the areolae are subcircular, or, more usually, elongated to fine slits (crawford et al. 2003). the velum, a spongiform plate, occurs on the inner aperture. the rimoportulae are usually two per valve and positioned 4–5 areolae distant from the ringleist. ringleist is solid, narrow and shallow (crawford et al. 2003). the linking spines are large and t-shaped. the separation spines are the same length as linking spines, but are pointed and are rarely found on each valve. ecology: known from mesotrophic to eutrophic environments, dominant in shallow lakes (van dam et al. 1994, trifonova and genkal 2001). aulacoseira nygaardii (camburn) camburn et charles (camburn and charles 2000; pl. 2, figs. 8–9) basionym: melosira nygaardii camburn 1986 aulacoseira nygaardii (camburn) camburn et charles (camburn and charles 2000: 14, pl. 3, figs. 9, 16–22) aulacoseira nygaardii (camburn) camburn et charles (siver and hamilton 2005: 258, figs. 1–16) aulacoseira nygaardii (camburn) camburn et charles (siver et al. 2005: 35, pl. 1, figs. 11–16, pl. 2, figs. 6–8) valve diameter 10 µm; the height of valve mantle 6 µm; the areolae on the mantle are in straight rows 22 in 10 µm. the areolae are circular to rectangular; the position of the velum (type 'rotae’) is attached down into the base of areola (siver and hamilton 2005). the ringleist is very shallow. the linking spines are short, with equal length and t-shaped endings. ecology: known from acidic, nutrient-poor waters (siver and hamilton 2005, siver et al. 2005) this taxon was rare in our study and appeared in the 'bottom' sediment of mccoy lake. 410 acta bot. croat. 68 (2), 2009 manoylov k. m., ognjanova-rumenova n., stevenson r. j. u:\acta botanica\acta-botan 2-09\manoylov.vp 9. listopad 2009 13:41:33 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 411 aulacoseira species in sediments pl. 3. aulacoseira perglabra (østrup) haworth. fig. 1 – valve view, lm, mccoy lake, bottom sediments; figs. 2, 3, 4, 5 – girdle view – lm, mccoy lake, bottom sediments; fig. 6 – girdle view, see the long and narrow linking spines, sem, mccoy lake, bottom sediments; fig. 7 – valve view with peripheral ring of elongated areolae, the second (inner) ring is partial with smaller and cruciate areolae with vela, sem, mccoy lake, bottom sediments; fig. 8 – valve view with more regular second peripheral areolar ring, sem, mccoy lake, bottom sediments; fig. 9 – the velum of the areola, sem, mccoy lake, bottom sediments. u:\acta botanica\acta-botan 2-09\manoylov.vp 9. listopad 2009 13:41:35 color profile: disabled composite 150 lpi at 45 degrees aulacoseira perglabra (østrup) haworth (haworth 1988; pl. 3, figs. 1–9) basionym: melosira perglabra østrup 1910 melosira perglabra østrup (camburn and kingston 1986: 28, pl. 4, figs. 59–65, pl. 5, figs. 77–78) aulacoseira perglabra (østrup) haworth (siver and kling 1997: 1818, figs. 48–53, 60–63) aulacoseira perglabra (østrup) haworth (camburn and charles 2000: 14, pl. 4, figs. 1–8) aulacoseira perglabra (østrup) haworth (siver et al. 2005: 1, figs. 7–10, 23–24: 2, figs. 3–5) valve diameter ranges 5.50–13.50 µm; the height of valve mantle 1.50–4.00 µm; the areolae on the mantle are in very short rows (12–21 in 10 µm) and are normally obscured by the overlapping spines of the sibling valve. the areolae are covered by vela. the rimoportula is single, small, and sits in a narrow band as in likhoshway and crawford (2001). there is a lack of any noticeable ringleist (siver and kling 1997). the linking spines are long and narrow, with flat ending as shown in crawford and likhoshway (1998, fig. 11). they are carried on buttresses spanning the margin of the face and mantle. on the valve face there is one peripheral ring of elongated areolae that extends onto the mantle, but sometimes smaller crucial areolae form second (inner) partial ring. ecology: known from oligotrophic conditions (siver and kling 1997, trifonova and genkal 2001) this taxon was not observed in 'top' sediment samples. we found it in 'bottom' sediments of mccoy lake. aulacoseira pseudoamericana (camburn) siver et kling (siver and kling 1997; pl. 4, figs. 1–9) basionym: melosira pseudoamericana camburn 1986 valve diameter ranges 8.50–13.00 µm; the height of valve mantle 3.00–6.00 µm; the mantle is unornamented. the valve face is ornamented with two, sometimes three, peripheral rings of small and irregularly arranged areolae. the single rimoportula is positioned near the junction of the valve face and mantle (siver and kling 1997). the ringleist is very shallow. the linking spines are spatulate with apiculate tips (pl. 4, fig. 5), or long and narrow – similar to aulacoseira perglabra (østrup) haworth (pl. 3, fig. 6). ecology: this taxon was rare and appeared in bottom sediments of mccoy lake, where trophic conditions are good. previously this taxon was reported together with a. perglabra in oligotrophic environments (siver and kling 1997). aulacoseira subarctica (o. müller) haworth (haworth 1988; pl. 5, figs. 1–11) basionym: melosira italica ssp. subarctica o. müller 1906 melosira italica ssp. subarctica o. müller (camburn and kingston 1986: 24, pl. 2, figs. 34–35, 37–40) aulacoseira subarctica (o. müller) haworth (siver and kling 1997: 1811, figs. 13–22) aulacoseira subarctica (o. müller) haworth (camburn and charles 2000: 15, pl. 4, figs. 23–31) 412 acta bot. croat. 68 (2), 2009 manoylov k. m., ognjanova-rumenova n., stevenson r. j. u:\acta botanica\acta-botan 2-09\manoylov.vp 9. listopad 2009 13:41:35 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 413 aulacoseira species in sediments pl. 4. aulacoseira pseudoamericana (camburn) siver et kling. figs. 1–4 – girdle view – lm, mccoy lake, bottom sediments; figs. 5, 6 – sibling valves, unornamented mantle and spatulate spines with apiculate tips, sem, mccoy lake, bottom sediments; fig. 7 – valve view with peripheral, irregularly arranged areolae, sem, mccoy lake, bottom sediments; fig. 8 – valve inside view with internal projections of the rimoportulae, very shallow ringleist, sem, mccoy lake, bottom sediments; fig. 9 – valve inside view, detail with the peripheral rings of areolae, sem, mccoy lake, bottom sediments. u:\acta botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:49 color profile: disabled composite 150 lpi at 45 degrees 414 acta bot. croat. 68 (2), 2009 manoylov k. m., ognjanova-rumenova n., stevenson r. j. pl. 5. aulacoseira subarctica (o. müller) haworth. fig. 1 – girdle view – lm, second lake, top sediments; figs. 2, 3, 4 – girdle view – lm, ryerson lake, top sediments; fig. 3 – girdle view – lm, ryerson lake, bottom sediments; fig. 5 – girdle view – lm, hillsview lake, bottom sediments; fig. 6 – valves of a. ambigua and a. subarctica – on the valve face of a. subarctica there are a few areolae, located close to the margin, sem, pickerel lake, bottom sediments; fig. 7 – sibling valves with clearly overlapping cingulae, sem, pickerel lake, bottom sediments; figs. 8, 9, 10 – girdle view, the isomorphic spines are of equal length; see, arrowed, the external opening of the rimoportula (with curved stalk), located near the well developed ringleist, sem, pickerel lake, bottom sediments; fig. 11 – girdle view – a long narrow valve, left – ligula shaped cingulum, sem, ryerson lake, top sediments. u:\acta botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:51 color profile: disabled composite 150 lpi at 45 degrees valve diameter ranges 4–22.5 µm; the height of valve mantle 6–15 µm; the areolae on the mantle (14–25 in 10 µm) are in spiral pervalvar rows. the areolae are small with deep-set vela (crawford and likhoshway 1998). the rimoportulae are two per valve, without stalk. they are found near the ringleist (fig. 9) and their external openings are large and visible in lm (likhoshway and crawford 2001). the ringleist is well developed. to our knowledge only isomorphic spines have been described for this taxon (gibson et al. 2003, wolfe and edlund 2005). all spines are pointed and of equal length. ecology: similarly to literature findings of abundance in oligotrophic conditions (van dam et al. 1994, siver and kling 1997, trifonova and genkal 2001, tuji and houki 2004, barinova et al. 2008), this taxon was abundant in both top and bottom sediment in lakes of good and fair trophic conditions, but appeared only in the bottom sediment of brooks and hess lakes, classified as lakes with poor conditions. aulacoseira valida (grunow) krammer 1991 basionym: melosira crenulata var. valida grunow (van heurck 1880–1885) melosira italica var. valida (grunow) hustedt 1927 melosira italica var. valida (grunow) hustedt (camburn and kingston 1986: 25, pl. 2, fig. 36) aulacoseira valida (grunow) krammer (krammer 1991b: figs. 23–29, 31, 36–39) aulacoseira valida (grunow) krammer specimens conspecific with aulacoseira italica (ehrenberg) simonsen (siver and kling 1997: 1815, figs. 42–45) aulacoseira valida (grunow) krammer (camburn and charles 2000: 15, pl. 5, figs. 1–4). aulacoseira valida (grunow) krammer (siver et al. 2005: 40, pl. 3, figs. 1–2). valve diameter 8 µm; the height of valve mantle 10 µm; the areolae on the mantle are 12 in 10 µm, formed dextrorse spirals. areolae become enlarged longitudinally towards the valve mantle (houk and klee 2007). the rimoportula is located in the inner side of the ringleist (likhoshway and crawford 2001). the linking spines are long, relatively thick, with t-shaped enlarged distal edge. ecology: this taxon, reported from neutral to mesotrophic conditions (siver et al. 2005) was rare and appeared in bottom sediments of mccoy lake. discussion within diatom communities from michigan lake sediment cores we saw a 44% decrease in aulacoseira species diversity from bottom to top sediment layers. no other diatom group, such as centric diatoms or pennate diatoms, experienced such a decrease. diatom species composition between tops and bottoms corresponded to the current land use surrounding the lakes, which ranged from predominantly forest and rangeland to urban and agriculturally impacted. inferred past conditions revealed that the observed morphotypes probably represent taxa with different ecological preferences. 'bottom' aulacoseiras like a. granulata and a. italica did not change with human influence, while a. subarctica and a. ambigua increased in abundance. aulacoseira granulata morphotype curvata, a. nygaardii, a. perglabra and a. pseudoamericana were absent in the 'top' sediment and were replaced with a. ambigua and a. subarctica. the main factors influencing the variability of these morphotypes were changes in the trophic status of the lakes. acta bot. croat. 68 (2), 2009 415 aulacoseira species in sediments u:\acta botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:51 color profile: disabled composite 150 lpi at 45 degrees diatom-inferred indices for changes in lake water levels and trophic status have been based on species presence or absence (wolin 1996). in this study we linked aulacoseira species with the trophic status of lakes in michigan, where good quality lakes are abundant. species such as a. nygaardii, a. perglabra and a. pseudoamericana that occurred frequently in bottom sediments were absent in the top sediment and were replaced predominantly with a. ambigua. in poor quality lakes, a. subarctica disappeared in the top sediment. we concentrated on centric diatoms and aulcoseira, but changes in pennate diatoms remain unclear. acknowledgement we are very grateful to the field crew led by v. lougheed for collecting the samples. the authors are grateful to two anonymous reviewers for constructive suggestions that improved the manuscript significantly. this research was supported by a grant to dr. r. j. stevenson from the great lakes fisheries trust. references barinova, s. s., medvedeva, l. a., anisimova, o. v., 2008: diversity of algal indicators in environmental assessment (in russian). pilies studio, jerusalem, israel. battarbee, r. w., 1986: diatom analysis. in: berglund, b. e. 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botanica\acta-botan 2-09\manoylov.vp 6. listopad 2009 12:38:52 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 233 acta bot. croat. 74 (2), 233–244, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0019 morphological study of chaetoceros wighamii brightwell (chaetocerotaceae, bacillariophyta) from lake vrana, croatia sunčica bosak1*, marija gligora udovič1, diana sarno2 1 university of zagreb, faculty of science, department of biology, rooseveltov trg 6, hr-10000 zagreb, croatia 2 stazione zoologica anton dohrn, villa comunale, 80121 napoli, italy abstract – chaetoceros wighamii brightwell is a planktonic diatom species originally described from brackish waters. since its original description, the species has been reported in a wide variety of habitats, ranging from freshwater to marine. varying descriptions exist in the taxonomic literature and several taxa have been considered as synonyms, including freshwater species chaetoceros amanita. in this study we provide morphological and ultrastructural information on a cultured strain isolated from freshwater sample collected in the lake vrana (vransko jezero) in croatia, in april 2011. the cells form short and robust chains with very narrow apertures, often partially occluded by silica membranes. other distinctive features observable in light microscopy are the shape and orientation of the setae which are very long, straight and robust, diverging in various directions from the chain axis and the single parietal chloroplast extending from valve to valve. distinct ultrastructural characteristics are the absence of processes either in intercalary or terminal valves and the ornamentation of the valve face with densely distributed ribs spreading from an irregular eccentric hyaline area without a clearly defi ned annulus. the outer surface of the terminal valve is ornamented with small spines and setae are composed of fl at longitudinal fi laments interconnected with short bars and ornamented with small spines tightly arranged around the setae. our description agrees well with that reported for the freshwater morphotypes of c. wighamii (syn. c. amanita) and contributes for a reliable distinction of this intriguing taxon from similar morphotypes. this fi nding supports the interpretation of chaetoceros wighamii as a freshwater/brackish species and represents the fi rst report of a chaetoceros species in lacustrine environment in croatia and possibly in any central european habitats. key words: chaetoceros, diatom taxonomy, lake vrana, morphology * corresponding author, e-mail: suncica.bosak@biol.pmf.hr bosak s., gligora udovič m., sarno d. 234 acta bot. croat. 74 (2), 2015 introduction chaetoceros ehrenberg is one of the most species-rich genera among planktonic diatoms, including approximately 170 species (vanlandingham 1968). this genus has been the subject of a number of taxonomical investigations due to its ecological relevance, extraordinary morphological diversity and complexity (hustedt 1930, rines and hargraves 1988, hernández-becerril 1996, jensen and moestrup 1998). although the species are primarily marine a few taxa occur at very low salinities in estuaries and freshwater lakes, e.g. the small, often solitary and lightly silicifi ed species chaetoceros muelleri lemmermann or the more robust c. elmorei boyer (rushforth and johansen 1986). the taxonomy and morphology of the most frequently occurring species chaetoceros muelleri and similar non-marine taxa has been the subject of a revision in which two distinct varieties were recognized (johansen and rusforth 1985): the nominate variety c. muelleri var. muelleri and c. muelleri var. subsalsum (lemmermann) johansen et rushforth. chaetoceros wighamii brightwell was originally described (brightwell 1856) shortly after the genus chaetoceros was established (ehrenberg 1844), with special attention to the distinctive spore morphology, and a quite vague illustration of the chain features. the description leaves no doubts on the continental nature of the type locality: chaetoceros wighamii »occurred in a gathering made from a dirty ditch of brackish water at the back of a small public-house, called the burney arms, which is marked on the ordnance maps« in a sample rich in brackish/freshwater diatom taxa such as campylodiscus clypeus and one or more mastogloia species (brightwell 1856, p. 108). the species chaetoceros amanita cleve euler (cleve-euler 1915) was established at a later date based on the description of fossil resting spores, characterized by a spiny domeshaped primary valve and a truncate secondary valve, which were similar to resting spores described by brightwell for c. wighamii. kaczmarska et al. (1985) provided the fi rst detailed description of the vegetative forms of c. amanita from the blue lake spring, utah, usa, and discussed the similarity and the possible conspecifi city of c. amanita and c. wighamii, that was already considered in previous studies (kolbe and krieger 1942, cleve-euler 1951). over the years, c. wighamii has been reported in a wide variety of habitats, ranging from freshwater (sanchez castillo et al. 1992) to marine (cupp 1943, snoeijs 1993, jensen and moestrup 1998, haecky et al. 1998, bérard-therriault et al. 1999) and was considered as the valid synonym of different marine species, including c. bottnicus cleve, c. biconcavum gran, c. caspicum ostenfeld (hustedt 1930, vanlandingham 1968, hasle and syvertsen 1997) and c. fallax proschkina lavrenko (jensen and moestrup 1998). sanchez castillo et al. (1992) discussed the identity of c. wighamii in relation with the taxa traditionally associated to it, i.e. c. amanita, c. bottnicus, c. biconcavum and c. capsicum. the authors compared the original descriptions and material from different areas, including the type locality of c. wighamii, and recognized the existence of two different species: 1) c. wighamii, which corresponded to specimens from brackish/freshwater environments and has as the only valid synonym c. amanita, and 2) c. bottnicus, originally described from the baltic sea (aurivillius 1896), which included most of the examined marine morphotypes. this view has been disregarded in some of the following studies (hasle and syvertsen 1997, jensen and moestrup 1998) or partially considered in other investigations where the name c. wighamii was retained but a possible misinterpretation was taken into account (snoeijs 1993, bérard-therriault et al. 1999). the morphology of chaetoceros wighamii from lake vrana acta bot. croat. 74 (2), 2015 235 as a result of this complicated taxonomic history, c. wighamii has been often perceived as a highly variable species (hustedt 1930, cupp 1943, rines and hargraves 1988, jensen and moestrup 1998), whereas there are indications (sanchez castillo et al. 1992) that some of the morphological descriptions, synonyms and reports from different habitats, refer indeed to morphologically similar but yet distinct species. possible misidentifi cations can be primarily attributed to the lack of strongly distinctive characters in the vegetative cell morphology reported by brightwell (1856) and to the fact that the presence of the very typical resting spores was rarely used to support a positive identifi cation of the species. in this study we investigated the morphology of a strain of c. wighamii (syn. c. amanita) isolated from the freshwater lake vrana in croatia using light (lm), scanning electron (sem), and transmission electron (tem) microscopy. the aim is to provide detailed information, especially on the ultrastructural features of the frustule, allowing for a better delineation of this intriguing taxon and its distinction from apparently similar morphotypes. material and methods study area situated in the central part of the eastern adriatic coast, lake vrana and its surroundings were declared a nature reserve on july 21, 1999. it is the largest croatian lake (length 13.6 km and width between 1.4 and 3.4 km). the lake area is between 2980 ha and 3020 ha and extends parallel to the coast in a northwest-southeast direction, separated from the sea by an 800–2500 m wide limestone ridge (up to 113 m above sea level). the basin is a karst drainage system in the area of ravni kotari with a surface area of 49400 ha. it is a polymictic karstic cryptodepression connected to the adriatic sea by a narrow channel at its southern side. in the northwestern section the lake is between 0.5 and 1 m deep, and 4–6 m in the southeast. the fl ow of water in the lake is infl uenced by conduction waves caused by the south-eastern and north-westerly winds. the channel prosika (length 800 m, width 4 m, depth 5–6 m) connects the lake with the adriatic sea. the channel is used for drainage and was constructed in 1895 but was subsequently expanded, deepened and regulated. the extensive fl oodplain around lake vrana is a natural marsh with high biodiversity. the physico-chemical parameters presented in table 1 were measured approximately bimonthly from february to october 2011 (n = 7) at a single station, situated in the central part of the lake (43°54’27’’n, 15°33’57’’e). the observed ranges of values agree well with the results of previous investigations (gligora et al. 2007), indicating considerable infl uence of the mediterranean climate conditions both on the nutrient levels and on temperature. during 2011 the salinity in the lake ranged between 2.4 and 6.5 psu, in april 2011, when the diatom strain was isolated in culture the salinity was 2.9 psu. strain isolation, culture condititons and morphological analyses the water sample was collected at the surface (0.5 m depth) of the lake on april 26, 2011 and examined fresh upon the arrival at the laboratory, in a plastic petri dish. the individual cell chains were isolated using a pasteur micropipette and an inverted olympus ckx41 light microscope (olympus, tokyo, japan) equipped with bright-fi eld optics and phase contrast. the chains were fi rst placed in sterile 35-mm falcon polystyrene tissue culture dish (model 353001 bd labware, le pont de claix, france) fi lled with ca. 2 ml f/2 bosak s., gligora udovič m., sarno d. 236 acta bot. croat. 74 (2), 2015 medium (guillard 1975) and observed each day during the fi rst week for growth and conditions of the cells. the culturing medium was prepared using guillard’s (f/2) marine water enrichment solution (sigma-aldrich) according to the manufacturer instructions. the water used for medium preparation was collected from the lake vrana and sterilized by fi ltration through 0.2-μm nucleopore fi lters. when the cell density was high enough the established clonal culture was transferred from a dish to 70 ml falcon polystyrene cell culture fl asks (model 353082; bd biosciences, le pont de claix, france) fi lled with 30 – 40 ml of f/2 medium. the strain designated pmf m1 was maintained in a growth culture chamber under cool white (40 w) fl uorescent light (30 μmol photons m−2 s−1) at a room temperature of 20 °c in a 16 h:8 h light:dark cycle and sub-cultured every 1–2 weeks. the material for morphological studies was obtained in the exponential growth phase within the fi rst month after establishment of the culture. for lm observations, a zeiss axiophot microscope (carl zeiss, oberkochen, germany), equipped with bright-fi eld, phase contrast and nomarski differential interference contrast (dic) optics was used. light micrographs were taken with a zeiss axiocam mrc digital camera and processed with an axiovision 4.8.2 digital image processing software. the culture material was subjected to cleaning treatment in order to remove the organic matter from diatom frustules. the sample was fi rst desalinized by rinsing with distilled water, treated with strong acids (1:1:4; sample:h2so4:hno3), boiled for a few seconds and then washed again with distilled water. for sem observations, the cleaned material was fi ltered and air dried on 3-μm nucleopore polycarbonate fi lter (nucleopore, pleasanton, tab. 1. physico-chemical variables in lake vrana from february to october 2011. min – minimum, max – maximum, avg – average, stdev – standard deviation. number of sampling = 7. the values measured in april 2011 when the strain of chaetoceros wighamii was isolated in culture are presented separately. variable min max avg stdv april 2011 temperature (°c) 2.8 25.6 17.6 8.1 13.9 ph 7.9 9.0 8.5 0.4 7.98 conductivity (μs cm–1) 3870 8760 5434 1797 4090 total suspended solids (mg l–1) 3.6 14 8.6 3.8 14 alkalinity m-value (mg caco3 l–1) 66 196 121 61 196 hardness (mg caco3 l–1) 422 1242 706 279 689 o2 (mg l–1) 7.8 12.9 9.5 1.8 9.1 saturation o2 (%) 83 119 97 13 88.3 nh4+ (mg l–1) <0.010 0.129 0.047 0.041 0.031 no2– (mg l–1) 0.002 0.016 0.007 0.005 0.016 no3– (mg l–1) 0.031 1.980 0.722 0.790 1.340 total inorganic n (mg l–1) 0.064 2.004 0.769 0.805 1.387 organic n (mg l–1) 0.215 0.328 0.274 0.035 0.268 po4 (mg l–1) 0.008 0.099 0.034 0.039 0.008 total p (mg l–1) 0.021 0.233 0.084 0.070 0.067 the morphology of chaetoceros wighamii from lake vrana acta bot. croat. 74 (2), 2015 237 ca), mounted on a stub, sputter coated with gold and examined in a jeol jsm-6500f sem (jeol-usa inc., peabody, ma, usa). tem observations were made by deposition of cleaned and rinsed material on formvar/carbon coated 200-mesh nickel grids and examined in a leo 912ab tem (leo, oberkochen, germany). preserved material and tem grids are available from the authors upon request. the general diatom terminology used for the morphological descriptions follows proposals recommended by anonymous (1975), ross et al. (1979), round et al. (1990), hasle and syvertsen (1997). the specifi c terminology for chaetoceros genus follows evensen and hasle (1975), rines and hargraves (1988) and kooistra et al. (2010). results chaetoceros wighamii brightwell 1856: 108, pl. 7: fi gs 19–36 described as chaetoceros amanita cleve euler in kaczmarska et al. (1985), rushforth and johansen (1986), rines and hargraves (1988) and as c. wighamii in sanchez castillo et al. (1992). light microscopy observations chains are straight, robust and usually short, composed of 4–7 cells. cells are elliptical in valve view and rectangular in girdle view with the pervalvar axis often shorter (8–15 μm) than the apical axis (14–26 μm). each cell contains a single large chloroplast in the shape of a parietal plate extending from valve to valve (figs. 2b–c). the valve face is fl at, sometimes with a central infl ation which is usually more visible on the terminal valves. no process was visible in either intercalary or terminal valves on > 30 observed chains. valve mantle is low with a very slight constriction near the abvalvar margin. the girdle is usually equi-dimensional in height with the mantle. valves of adjacent cells touch at the corners. apertures are slit-shaped and very narrow, partially occluded by silica membranes which are sometimes visible in lm (not shown). intercalary setae originate from the valve apices fig. 1. position of sampling site at lake vrana (croatia). bosak s., gligora udovič m., sarno d. 238 acta bot. croat. 74 (2), 2015 and cross immediately at chain margin, without basal part (figs. 2b–c). setae lack chloroplasts. intercalary setae are very long (up to 300 μm), straight, and stiff diverging in various directions, some perpendicular but more often they are oriented at a variable angle to the chain axis (fig. 2a). in most cases sibling setae diverge from each other at an angle of 90° belonging to brunel group iii (fig. 2d), but in the same chain they can also diverge equally from the apical plane at an angle of 20–30° conforming to brunel group ii. terminal setae have the same morphology as intercalary ones but they are oriented more parallel to the chain axis, diverging in a broad uor v-shaped curve towards the end of the chain (fig. 2a). electron microscopy observations the valve is ornamented with very densely distributed anastomosing ribs spreading from an irregularly shaped hyaline area positioned slightly eccentrically on the valve face (figs. 3b–c). the valve face is not perforated by poroids. the outer surface of the terminal valves is covered by minute spines (fig. 3f). the process was not observed on ten intercalary and terminal valves examined with em (figs. 3a–e). in all valves the marginal ridge between the valve face and the valve mantle is ornamented with a high hyaline rim (fig. 3e). in intercalary valves, the aperture between sibling cells is partially occluded by silica membranes appearing to project from the hyaline rim (fig. 3a). the setae are circular in fig. 2. light micrographs of chaetoceros wighamii, strain pmf m1. a) the complete chain showing orientation of the setae. b) cells with one parietal chloroplast per cell extending from valve to valve. c) four-celled chain. d) sibling valves in valve view with setae diverging from each other at an angle of 90°, belonging to brunel group iii. scale bars: a = 50 μm; b–d = 10 μm. the morphology of chaetoceros wighamii from lake vrana acta bot. croat. 74 (2), 2015 239 cross-section, 0.6–0.8 μm in diameter, composed of fl at, thick longitudinal fi laments arranged in spiral pattern around the setae length and interconnected by very short transverse bars. the space between bars corresponds to a spiral row of minute poroids. the fi laments are ornamented with spines (3 per 1 μm) arranged in a helicoidal pattern around the setae. (figs. 3g–h). discussion a combination of morphological and ultrastructural characters allows for the ascription of the morphotype from lake vrana to c. wighamii brightwell (syn. c. amanita) sensu sanchez castillo et al. (1992). the robust colonies have very long, stiff and straight intercalary setae projecting in various directions from the chain axis and terminal setae oriented more parallel to the chain axis, diverging in a broad u or vshaped curve towards the end of the chain. the apertures between sibling valves are partially occluded by a silica membrane which is sometimes visible in lm. the partial sealing of the apertures was considered peculiar for c. wighamii syn. c. amanita (kaczmarska et al. 1985, rushforth and johansen 1986), but was not described in c. wighamii from lake chica (sanchez castillo et al. 1992) where apparently only terminal valves were observed by electron microscopy. a silica membrane partially occluding the apertures is not unique for c. wighamii as a similar structure (silica wall) has been observed in other very different species such as c. decipiens (evensen and hasle 1975) and c. diversus (hernández-becerril 1996). fig. 3. sem (a, d, e, g) and tem (b, c, f, h) micrographs of chaetoceros wighamii, strain pmf m1. a) two sibling valves with aperture partially occluded by silica. b) two overlapped sibling intercalary valves showing the valve pattern with anastomosing ribs. c) detail of the valve with the irregularly shaped hyaline eccentric area. d) internal view of the terminal valve. e) terminal valve in girdle view showing the hyaline rim extending from the marginal ridge (arrow). f) detail of the terminal valve with minute spines and the structure probably corresponding to the girdle band. g) detail of a seta with spirally positioned spines. h) detail of a seta. scale bars: a, b, d, e = 5 μm; c, g, h = 1 μm; f = 0.5 μm. bosak s., gligora udovič m., sarno d. 240 acta bot. croat. 74 (2), 2015 a rather distinctive feature in our material is the absence of any processes and central annulus in either intercalary or terminal valves. a process is present on terminal valves in most of the species in the subgenus hyalochaete, and generally on every valve in the subgenus chaetoceros (evensen and hasle 1975, hernández-becerril 1996). the lack of processes was recognized as a common feature in several unicellular inland taxa which are nowadays considered as synonyms of c. muelleri var. subsalsum (lemmermann) johansen & rushforth (reinke 1984, johansen and rusforth 1985) and was already reported in specimens from freshwater fi eld material of c. wighamii (kaczmarska et al. 1985 as c. amanita, sanchez castillo et al. 1992). kaczmarska et al. (1985) were not able to confi rm the presence of any process after examination of 200 valves but suggested that a rimoportula may be produced under some circumstances. our observation in cultivated material, although based on a limited number of examined valves, constitutes a further evidence of the lack of the process in the freshwater specimens of c. wighamii. the presence of the central process bearing a long external tube was illustrated in cultures of c. wighamii from the baltic sea (syn. c. bottnicus, c. biconcavum, c. capsicum and c. fallax, jensen and moestrup 1998). the projection is clearly visible in lm illustrations and sometimes is present in sibling cells, probably as a result of impending chain division with the process-bearing intercalary valves becoming terminal valves. the baltic morphotype appears rather similar to the croatian morphotype as regarding the very variable orientation of the setae, but looks different for the more delicate aspect and the wider apertures of the chains. as noted by snoeijs (1993), detailed studies on the species from the baltic sea are required to reveal if it corresponds to c. wighamii or to c. bottnicus, which was indeed considered as a synonym of c. wighamii by jensen and moestrup (1998). the absence of a distinct annulus is not common among hyalochaete species that generally have a thickened annulus surrounding the hyaline area from which the dichotomously branching ribs extend towards the valve margin (evensen and hasle 1975, hernándezbecerril 1996). in c. wighamii, the valve face shows a peculiar ornamentation pattern with densely distributed ribs spreading from an irregularly shaped hyaline area. kaczmarska et al. (1985) described a similar pattern as »composed of anastomosing areas of light silifi cation« probably referring to the hyaline area between the ribs. the lack of a clearly defi ned annulus was also observed in the freshwater species chaetoceros muelleri var. subsalsum (johansen and rushforth 1985) but the ribs are thicker and do not diverge in such a dense branching pattern as in c. wighamii. the presence of small spines or protrusions on the outer valve surface in c. wighamii was already noticed in previous studies (kaczmarska et al. 1985 as c. amanita, sanchez castillo et al. 1992). the seta ultrastructure has been considered as useful taxonomic character used in species delineation in chaetoceros since the fi rst em studies were published (evensen and hasle 1975). this idea was recently developed further by lee et al. (2014) who proposed the use of several setae characters as the identifi cation criteria in the subgenus chaetoceros, which includes species with thick and chloroplasts containing setae. chaetoceros wighamii belongs to the subgenus hyalochaete which comprises species possessing relatively thin setae without chloroplasts. in c. wighamii, the setae are composed of long longitudinal fi laments, which are arranged in a spiral pattern around the seta axis and are connected by short transverse bars. the arrangement of longitudinal fi laments and transverse bars results in a reticulate pattern when setae are observed in tem (kaczmarska et al. 1985). the fi laments are adorned with somewhat strong small spines in a helicoidal arrangement. the the morphology of chaetoceros wighamii from lake vrana acta bot. croat. 74 (2), 2015 241 same pattern was previously illustrated in c. wighamii (kaczmarska et al. 1985 as c. amanita, sanchez castillo et al. 1992). generally, hyalochaete species possess setae with spines, with the exception of c. vixvisibilis (hernández-becerril et al. 2010), but their size and pattern shows differences among species (hernández-becerril 1996, kooistra et al. 2010). spines adorning setae in c. wighamii are tightly arranged around the axis and appear slightly longer and more silicifi ed compared to the spines present in other species, as, for example, chaetoceros curvisetus or c. lauderi (kooistra et al. 2010, evensen and hasle 1975).the details on chloroplast number, shape and position have not been known previously in chaetoceros wighamii syn c. amanita, as the previous observations were based mostly on cleaned material (kaczmarska et al. 1985, sanchez castillo et al. 1992). according to our observations on live material, chaetoceros wighamii has one chloroplast, which is plate-like or slightly lobed, situated parietally within the cell but extending from valve to valve, differently from, for example, c. affi nis which has the single parietal plastid positioned around the girdle (rines and hargraves 1988). the apical length size was considered by cleve-euler (1951) and later adopted by kaczmarska et al. (1985) as one of the characters considered on which their specimens were designated as c. amanita and not as c. wighamii. the authors compared their measurements with the data on apical length reported for marine morphotypes named c. wighamii by hustedt (1930) and cupp (1943) which range between 7 and 18 μm, these are considerably smaller than the c. wighamii syn c. amanita range. however, the morphotype illustrated and described by hustedt (1930) is very different from the original description of c. wighamii, with characters such as delicate chains, thin setae and cells with drawn up poles, which probably correspond to a different species with marine preferences. the cell apical length measured in our study ranged between 14 and 26 μm, which is comparable to the range reported in other studies of c. wighamii syn. c. amanita: 18–40 μm, 15–20 μm, 19–31 μm, (kaczmarska et al. 1985, rines and hargraves 1988, sanchez castillo et al. 1992), respectively. one of the most distinctive characters of c. wighamii is a resting spore with primary valve covered with spines and secondary valve shaped as a truncated cone (brightwell 1856). unfortunately, the resting spore has not been observed in this study. however, resting spores morphologically similar to those reported for c. wighamii by brightwell (1856) and sanchez castillo et al. (1992) as well as by kaczmarska et al. (1985) and rines and hargraves (1988) as c. amanita, have been frequently found in the sediments from the lake vrana (fig. 4, i. galović, personal communication). the distinct cone-shaped secondary fig. 4. the lm micrograph (photo by i. galović) of a resting spore in lake vrana, having the spinose secondary valve distinctively shaped as a truncated cone considerably narrower in diameter than the primary valve. scale bar = 10 μm. bosak s., gligora udovič m., sarno d. 242 acta bot. croat. 74 (2), 2015 valve is a feature observed in some other freshwater chaetoceros species, e.g. c. elmorei and c. muelleri, which however generally form smooth spores (rushforth and johansen 1986). the results of this study support the idea that the freshwater morphotypes of c. wighamii are distinct from the marine morphotypes. however, morphological and molecular studies on material from different geographical areas are required to defi nitely clarify if (1) c. wighamii is a very variable species which includes phenotypically different populations able to adapt to ecologically distinct habitats, or (2) different species have been identifi ed over time as c. wighamii. future studies should also investigate phylogenetic relationships among different freshwater chaetoceros species which share similar distinctive features such as absence of process and related spore morphology. conclusion our observations on morphology of chaetoceros wighamii isolated from lake vrana, croatia agree well with the description reported for the freshwater morphotypes of c. wighamii (syn. c. amanita) and contribute to a better distinction of this taxon from similar species. c. wighamii is a freshwater/brackish species whereas the taxonomical affi liation of the marine synonyms should be further investigated. additional species-specifi c ultrastructural features are described, such as the particular valve ornamentation pattern and the position and shape of the chloroplast. until now the morphotype corresponding to chaetoceros wighamii (syn. c. amanita) including observations of both vegetative cells and resting spores has been reported from inland waters in usa (kaczmarska et al. 1985), southern (sanchez castillo et al. 1992) and northern europe (brightwell 1856). this report represents the fi rst report of this species from croatia and from central european habitats. acknowledgements special thanks to franco iamunno (szn, naples, italy) on his valuable help with electron microscopy. the presented research was funded by the ministry of science, education and sport of the republic of croatia projects no. 119-1191189-1228 and 119-00000001229 and by the european community – research infrastructure action under the fp7 »capacities« specifi c programme (ref. assemble grant agreement no. 227799). we are thankful to dr. ines galović, croatian geological institute, zagreb, croatia for the image of c. wighamii resting spore. references anonymou s, 1975: proposals for a standardization of 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opulus press, uppsala. vanlandingham, s. l., 1968: catalogu e of the fossil and recent genera and species of diatoms and their synonyms. part ii. bacteriastrum through coscinodiscus. verlag von j. cramer, lehre. acta botanica 1-2017 za web.indd 64 acta bot. croat. 76 (1), 2017 acta bot. croat. 76 (1), 64–71, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0044 issn 0365-0588 eissn 1847-8476 antifungal potential of thyme essential oil as a preservative for storage of wheat seeds sabina anžlovar, matevž likar, jasna dolenc koce* department of biology, biotechnical faculty, university of ljubljana, večna pot 111, si-1000 ljubljana, slovenia abstract – plant essential oils are potential food preservatives due to their inhibitory effects on bacterial and fungal growth. antifungal activities of common thyme (thymus vulgaris) essential oil were tested against endophytic fungi grown from wheat (triticum aestivum) grain, molecularly identifi ed as alternaria alternata, alternaria infectoria, aspergillus fl avus, epicoccum nigrum and fusarium poae. their susceptibility to thyme essential oil was tested in vitro, and ranged from fungicidal to fungistatic. treatment combinations of prior grain surface sterilization with hypochlorite and direct/indirect treatment with the essential oil were used, which showed strong effects on infection incidence and germination. direct soaking of the wheat grain in the essential oil was particularly effective, but inhibited both fungal growth and seed germination. in contrast, indirect treatment of the grain with the essential oil (i.e., fumigation) inhibited fungal growth without negative effects on seed germination. in combination with grain surface sterilization with hypochlorite, indirect treatment with thyme essential oil reduced these fungal infections even more. since thyme essential oil is safe for plants and consumers, in the form of fumigation it could be used as a protectant of storage containers for wheat grain intended for sowing and for food production. keywords: antifungal activity, essential oil, seed spoilage, thymus vulgaris, wheat * corresponding author, e-mail: jasna.dolenc.koce@bf.uni-lj.si introduction wheat is an important cereal crop that can be attacked by a number of fungi. from seed germination to harvest, soil-borne and seed-borne diseases can reduce the vigour and yield of wheat plants (soliman and badeaa 2002). several wheat-associated fungi are carried from the fi eld with the harvested grain, and can spread further during post-harvest storage, and can reduce their later germination (özer 2005, perelló and larrán 2013, rajput et al. 2005). among these fungi, fusarium spp., aspergillus spp. and penicillium spp. are known to have negative impacts on grain during storage, which can result in serious risk for animal and human health through fungal production of a range of mycotoxins (d'mello et al. 1998). furthermore, infected grain and grain transmission represent a primary source of infection in the fi eld, from which these fungi can spread to broader areas, which can potentially lead to fungal epidemics (perelló and larrán 2013). treatment of cereal plants with synthetic fungicides in the fi eld before harvest has resulted in fungal resistance to antifungal agents, and it can often be problematic due to the high toxicity of fungicide residues to mammals (chen et al. 2008). post-harvest synthetic fungicide treatment of stored grain can infl uence the quality of cereals and can involve serious health hazards for consumers (osman and abdulrahman 2003). therefore considerable interest has developed in recent years in the preservation of grain using more consumerand nature-friendly protectants. plants contain a broad spectrum of antimicrobially active compounds that fi t into this category and show effective inhibition of fungal growth. essential oils are posed to become an important seed-decontamination alternative to synthetic seed preservatives, because in addition to strong fungicidal effects (krisch et al. 2011) they are biodegradable and show low toxicity to humans and animals (krisch et al. 2011, sivakumar and bautista-baños 2014) in recent years, essential oils from different plants have been used to prevent fungal growth and the consequent mycotoxin accumulation in cereals (batish et al. 2006, sumalan et al. 2013). in particular, oils from aromatic and spice plants have been applied, because of their safety and their common use in the food industry for centuries. many studies have documented the antimicrobial activities of thymus spp. essential oils and extracts (bouzidi et al. 2013, gonçalves et al. 2010, soković et al. 2009, solomakos et al. 2008). in a previous study, we reported that the antifungal potential of thyme essential oil acta bot. croat. 76 (1), 2017 65 essential oil from common thyme (thymus vulgaris) shows promising antibacterial and antifungal activities against foodborne isolates of a fusarium sp. and armillaria mellea, as model fungi from basidiomycetes and ascomycetes, respectively (anžlovar et al. 2014). the main aim of the present study was to determine the antifungal activities and potential use of common thyme essential oil as a disinfectant against fungi grown on wheat grain to be used for sowing or germination. we hypothesised that treatment with thyme essential oil will decrease the infection rate of the stored wheat grain, although high concentrations might also have negative impact on germination of the treated grain. to test our hypothesis, we: (i) isolated fungal endophytes from wheat grain and tested the effectiveness of thyme essential oil on their radial growth in vitro; (ii) evaluated the effectiveness of the thyme essential oil on fungal infection rates of wheat grain; and (iii) tested for potential negative impact of the essential oil treatments on germination of the treated wheat grain. different concentrations of thyme essential oil were tested, as well as in combination with initial surface sterilization of the grain with hypochlorite, to defi ne the best possible antifungal treatments for the wheat grain, for effective real-life treatments. materials and methods preparation of thyme essential oil seedlings of common thyme (thymus vulgaris l. cv. ‘deutcher winter’) were grown from seeds under greenhouse conditions and transplanted to the experimental fi eld of the biotechnical faculty (university of ljubljana, slovenia: 46°2’53.7’’n, 14°28’30.47’’e; altitude, 292 m a.s.l.) in march 2010. the plantation was regularly cultivated and was grown in the experimental fi eld until october 2010, when the non-fl owering shoots were harvested and processed to extract the essential oil. the thyme essential oil was isolated by water-steam distillation using a clevenger apparatus (anžlovar et al. 2014), following the procedures of the european pharmacopoeia monograph on thymi herba. isolation and molecular identifi cation of fungi from wheat grain wheat grain with developed fungal colonies on their surface were left in the dark at 20 °c until a single colony was large enough for transfer to fresh potato dextrose agar (pda) medium (biolife). the cultures are deposited in the fungal bank of the plant physiology laboratory at the department of biology (biotechnical faculty, university in ljubljana, slovenia), under accession numbers kp271953kp271958. the fungal mycelia were ground in liquid nitrogen and the dna was extracted using geneelute plant genomic dna miniprep kits (sigma), following the manufacturer instructions. all of the pcr reactions were carried out in a thermal cycler (mj research) using taq dna polymerase (promega). the 25-μl reaction mixture contained: 2.5 μl 10× pcr buffer, 2.5 mm mgcl2, 200 μm of each nucleotide, 500 nm of each primer, 0.75 u dna polymerase, and 12.5 μl 100-fold–diluted template. the pcr conditions for the its1f-its4 primer pairs (gardes and bruns, 1993, white et al., 1990) were: 1 min at 94 °c, followed by 35 cycles of 45 s denaturation at 94 °c, followed by 53 s annealing at 55 °c, and 30 s elongation at 72 °c. the elongation step was at 72 °c for 10 min. the pcr amplicons were cleaned and sequenced by macrogen (the netherlands), using the its1f and its4 primers. the nucleotide sequences obtained were subjected to blast analysis, to determine their homology with other sequences available in the genbank database. fungal growth inhibition assay the inhibitory effect of the thyme essential oil on radial growth of fungal mycelia was tested using the agar dilution method, as described by zabka et al. (2009). the concentrated essential oil was added to the autoclaved pda medium (medium temperature, ~50 °c) to prepare fi nal essential oil concentrations of 0.05%, 0.01%, 0.005% and 0.0025% (vessential oil/vmedium). fungal mycelia disks (diameter, 5 mm) were cut from the margin of the 7-day-old original cultures and aseptically inoculated by placing them in the centre of the medium with the essential oil in a petri dish (diameter, 90 mm). the control samples were prepared at the same time, but without the essential oil. the fungal colonies were incubated at 23 °c in the dark for 7 days, each experiment being carried out twice. inhibition of fungal radial growth was calculated according to the following formula: inhibition (%) = (dc – dt) / dc × 100 where dc is the diameter of the control colonies, and dt is the diameter of the treated colonies. to evaluate the responses of the fungal isolates to the thyme essential oil and to calculate the 50% effective concentration (ec50) and 90% effective concentration (ec90), the drc software package, version 2.3, was used (ritz and streibig 2005). to determine the nature of the essential oil toxicity, the inhibited fungal disks grown on the medium with 0.05% thyme essential oil were re-inoculated onto fresh medium without essential oil, and the revival of the fungal growth was assessed after 7 days. the essential oil toxicity was designated as fungistatic when the fungal colonies grew again, and fungicidal when there was no growth of the fungal colonies (kumar et al. 2008). essential oil treatment of wheat grain in the present study, two types of wheat (triticum aestivum l.) grain were used: conventionally grown grain obtained from the agricultural institute of slovenia (t. aestivum cv. ‘savinja’; designated as ‘conventional grain’ hereafter), and grain from the ecological farm vila natura (slovenia, prlekija, vučja vas; designated as ‘eco-grain’ hereafter). each of the grain types was divided in two groups: one group was left without any disinfection of the grain (nonsterile grain), while the other group was sterilized by soakanžlovar s., likar m., dolenc koce j. 66 acta bot. croat. 76 (1), 2017 ing in 3% hypochlorite for 3 min, to eliminate saprophytic microorganisms on the grain surface, followed by washing four times in sterilized distilled water (sterile grain). the non-sterile and sterile wheat grains (10 g) were treated with the thyme essential oil, with each treatment repeated twice. three concentrations of the thyme essential oil were prepared, as 2%, 0.2% and 0.05% by dilution of the essential oil in 10% dimethylsulphoxide (dmso) in distilled water. the grain was treated with the thyme essential oil according to two protocols: (i) direct treatment, where the grain was submerged and incubated in the essential oil; and (ii) indirect treatment, where sterile whatman fi lter paper was placed into the inner side of the top of the petri dishes and impregnated with 4 ml of essential oil suspension, according to concentration (i.e., exposure to the essential oil vapour only). the grains were distributed on the bottom of each petri dish, which were then sealed with parafi lm. for these essential oil treatments, the grains were incubated under shaking (50 rpm) for 24 h at 25 °c. the control grains were soaked in the vehicle, as 10% dmso, or exposed to the 10% dmso vapours, for direct and indirect exposure controls, respectively. germination and fungal contamination of wheat grain the assessments of the fungal infection and seed germination were performed by the direct plating method (sumalan et al. 2013). ten subsamples (where each subsample contained 10 wheat grains) from each previously described treatment were placed on 2% pda in petri dishes (diameter, 90 mm) and incubated at 25±2 °c in the dark. after 72 h, the development of fungal colonies on the surface of the wheat grain was determined, with the seed contamination index calculated according to doolotkeldieva (2010). at the same time, the germination of the wheat grain was determined by counting the number of germinated grains, expressed as a percentage of all grains used under each condition. statistical analysis all of the statistical analyses were performed with the software package r 3.1.2 (http://cran.r-project.org). the concentration/response analysis for growth of fungal isolates was performed using the drc package (v2.5-12) and a four-parameter log-logistic function. the effects of the seed type, prior sterilization, essential oil treatments, and essential oil concentrations on the fungal infection rates and the germination rates of the wheat grain were tested using multiway-anova, with the level of signifi cance set at p<0.05. all post-hoc comparisons were performed using holm-sidak post-hoc tests, with the level of signifi cance set at p<0.05. results fungal isolation and growth inhibition tests five fungal species were isolated from the wheat grain: alternaria alternata (2 strains), alternaria infectoria (=lewia infectoria), aspergillus fl avus, epicoccum nigrum, and fusarium poae (tab. 1). the majority of these fungal endophytes were saprophytes, although f. poae is considered a plant pathogen. all of these fungal species were isolated from conventional grain as well as eco-grain, although the level of fungal infection was higher for eco-grain. the potential of the thyme essential oil as an antifungal preservative was tested against all of the isolated fungal endophytes (fig. 1). the thyme essential oil showed in vivo fungitoxicity against all of the tested fungi, with a. infectoria, e. nigrum and f. poae showing the lowest ec50 and ec90 values. the highest tolerance to the thyme essential oil was for a. fl avus, with an ec50 of 0.010% and an ec90 of 0.018% (vessential oil/vmedium). after re-inoculation, the nature of the essential oil toxicity was determined as fungicidal for a. alternaria, e. nigrum and f. poae, and fungistatic for a. infectoria and a. fl avus (tab. 1). infection and germination of wheat grain after essential oil treatments the conventional grain and eco-grain were subjected to four treatment combinations, without and with surface sterilization with 3% hypochlorite, and direct and indirect exposure to the thyme essential oil, which yielded similar inhibitory effects on the fungal infection rates and different effects on the germination of the wheat grain (fig. 2, tab. 2). the thyme essential oil signifi cantly reduced the fungal infection of the wheat grain under all of the treatment combinations (fig. 2a). several factors affected the infection rates of the wheat grain (tab. 3), including the source of the grain (conventional grain vs. eco-grain: f=59288, p<0.0001). tab. 1. details of the fungal endophytes isolated from the wheat grain. e-value – expectation value. end – endophyte, sapro – saprophyte, path – pathogen. isolate no. accession no. nearest match e-value maximum identity (%) putative ecological niche essential oil toxicity 9c kp271953 kj739880 alternaria alternata 0.0 99 end/sapro fungicidal 11c kp271955 jf440581 alternaria alternata 0.0 100 end/sapro fungicidal 13c kp271957 gu584953 alternaria infectoria 0.0 99 end/sapro fungistatic 12c kp271956 jx164075 aspergillus fl avus 0.0 99 sapro/path fungistatic 10c kp271954 jq781728 epicoccum nigrum 0.0 100 sapro fungicidal 14c kp271958 jq912669 fusarium poae 0.0 99 path fungicidal antifungal potential of thyme essential oil acta bot. croat. 76 (1), 2017 67 fig. 1. fungal growth–response curves in the presence of thyme essential oil (eo) in the growth media. data are means±standard errors of two independent experiments (● experiment 1, ▲ experiment 2) with three individual measurements (n=6). a 95% confi dence interval is shown in grey. fig. 2. infection rates (a) and germination rates (b) of the wheat grain according to the concentrations and treatments with thyme essential oil (eo). data are means±standard errors (n=40). data with different letters are signifi cantly different between treatments (holmsidak post hoc tests, p<0.05). anžlovar s., likar m., dolenc koce j. 68 acta bot. croat. 76 (1), 2017 in addition, the surface sterilization of the seeds prior to essential oil exposure also reduced the infection rates of the seeds (non-sterile vs. sterile: f=111096, p<0.0001). the inhibition of fungal growth depended on the essential oil concentrations (0.05% vs. 0.2% vs. 2%; f=53974, p<0.0001) and the concentrations of the essential oil showed interactions with the treatment methods (direct vs. indirect: f= 102801, p<0.0001), with the direct essential oil treatment showing greater reduction of the infection rate than the indirect essential oil treatment (tab. 2). on the other hand, the germination rates of the grain showed different susceptibility to these essential oil treatments. direct treatment with the essential oil (i.e., submergence of grain in the essential oil) signifi cantly reduced the germination rate of the grain, while the indirect treatment did not affect the germination rate, which remained at the same level as for the control treatment (fig. 2b). when compared to the above-described fungal infection rates, more of the factors affected the germination rates of the seeds (tab. 4). in addition to the essential oil treatments (i.e., method and concentrations), the source of seeds was also an important factor (conventional grain vs. eco-grain; f=4212, p=0.041). as previously indicated, prior surface sterilization signifi cantly contributed to the reduction of the fungal infection rates (tab. 2). both the conventional grain and the ecograin were similarly contaminated with fungi when no sterilization was applied (99%, 100%, respectively) (tab. 2). the surface sterilization without the essential oil treatments signifi cantly reduced the fungal contamination (nonsterile vs. sterile; f=111096, p<0.0001), which on average dropped to 58% (tab. 2). the additional direct and indirect treatments with thyme essential oil further reduced the fungal contamination of the wheat grain (tab. 2). the direct treatment was generally more effective than the indirect tab 2. infection and germination rates of wheat grain after combinations of seed sources, surface sterilization and essential oil (eo) treatments. data are means±standard errors (n=10). g ra in s ou rc e pr io r g ra in st er ili za tio n e o tr ea tm en t eo concentration (%) infection rate (%) germination rate (%) c on ve nt io na l n on -s te ri le d ir ec t 0 100±0.00 97±1.53 0.05 27±4.73 52±6.63 0.2 15±4.53 0±0.00 in di re ct 0 99±1.00 100±0.00 0.2 38±7.57 99±1.00 2 31±5.26 98±1.33 st er ile d ir ec t 0 60±13.74 99±1.00 0.05 3±1.53 2±1.33 0.2 7±2.13 0±0.00 in di re ct 0 37±4.23 97±1.53 0.2 11±3.15 99±1.00 2 2±1.47 97±1.47 e co lo gi ca l n on -s te ri le d ir ec t 0 100±0.00 70±3.33 0.05 85±6.37 83±3.67 0.2 47±5.78 3±1.53 in di re ct 0 100±0.00 96±2.21 0.2 69±4.07 98±1.33 2 61±4.82 98±1.33 st er ile d ir ec t 0 72±8.00 86±3.71 0.05 57±13.75 65±9.69 0.2 11±5.04 0±0.00 in di re ct 0 63±6.33 97±1.53 0.2 32±2.91 99±1.00 2 23±3.67 95±3.07 tab. 3. anova for the differences in the infection rates among the grain sources, sterilization, and essential oil treatments and concentrations. df – degrees of freedom, ss – sum of squares, ms – mean square. factor df ss ms f-ratio p source 1.00 34031 34031 59288 <0.0001 sterilization 1.00 63769 63769 111096 <0.0001 treatment (essential oil) 1.00 108 108 0.19 0.666 concentration (essential oil) 1.00 30981 30981 53974 <0.0001 source: sterilization 1.00 77 77 0.14 0.714 source: treatment 1.00 368 368 0.64 0.424 sterilization: treatment 1.00 1835 1835 3197 0.075 source: concentration 1.00 307 307 0.53 0.466 sterilization: concentration 1.00 559 559 0.97 0.325 treatment: concentration 1.00 59008 59008 102801 <0.0001 source: sterilization: treatment 1.00 285 285 0.50 0.482 source: sterilization: concentration 1.00 830 830 1445 0.231 source: treatment: concentration 1.00 53 53 0.09 0.761 sterilization: treatment: concentration 1.00 490 490 0.85 0.357 source: sterilization: treatment: concentration 1.00 1752 1752 3053 0.082 residuals 223 128001 574 antifungal potential of thyme essential oil acta bot. croat. 76 (1), 2017 69 treatment, and the highest fungal inhibition was seen for 0.2% thyme essential oil directly applied to the wheat grain. further comparisons of these treatments showed that the indirect essential oil treatment had similar antifungal effects to the surface sterilization with hypochlorite (tab. 2). with the conventional grain, the fungal infection rate was 31% when the grain were fumigated (i.e., indirect treatment) with 2% essential oil, and 37% when only surface sterilization was used. for the eco-seeds, the corresponding values were 61% and 63% (tab. 2). also, direct essential oil treatments resulted in range of fungal inhibition comparable to indirect essential oil treatments but at ~one-tenth of the concentrations used (e.g., 7% infection with direct treatment of the grain with 0.2% essential oil, and 2% infection with indirect treatment of the grain with 2% essential oil). the combined effects of the surface sterilization with hypochlorite and the indirect treatment with the thyme essential oil had no effects on seed germination, but provided greatly decreased fungal infection, thus retaining the benefi ts of both of these treatments (fig. 3). tab. 4. anova for the evaluation of the differences in the germination rates according to the grain sources, sterilisation, and essential oil treatments and concentrations. df – degrees of freedom, ss – sum of squares, ms – mean square. factor df ss ms f-ratio p source 1 1123 1123 4212 0.041 sterilization 1 1507 1507 5655 0.018 treatment (essential oil) 1 157609 157609 591348 <0.0001 concentration (essential oil) 1 1703 1703 6390 0.012 source: sterilization 1 894 894 3353 0.068 source: treatment 1 1692 1692 6349 0.012 sterilization: treatment 1 959 959 3597 0.059 source: concentration 1 2 2 0.01 0.931 sterilization: concentration 1 14 14 0.05 0.820 treatment: concentration 1 145536 145536 546050 <0.0001 source: sterilization: treatment 1 687 687 2576 0.110 source: sterilization: concentration 1 93 93 0.35 0.555 source: treatment: concentration 1 35 35 0.13 0.719 sterilization: treatment: concentration 1 23 23 0.09 0.771 source: sterilization: treatment: concentration 1 727 727 2726 0.100 residuals 223 59435 267 fig. 3. correlations between infection and germination rates for the non-sterile (a) and sterile (b) wheat grain according to the concentrations and treatments with thyme essential oil (eo). the sizes of the symbols depict the essential oil concentrations. black squares denote direct essential oil treatment; white diamonds denote indirect essential oil treatment. anžlovar s., likar m., dolenc koce j. 70 acta bot. croat. 76 (1), 2017 discussion during plant growth as well as after harvest and during storage, wheat and other cereal grains are exposed to fungal colonisation. the use of such colonised seed for future sowing only exacerbates the problems of fungal pathogens and requires the application of fungicides. as essential oils from spice herbs are known to be safe for animal and human health, they represent a possible source for use in the decontamination and storage of grain that is intended not only for sowing, but also for food production (e.g., fl our and its products, sprouts, dietary supplements). in the present study, fi ve indigenous fungal species were isolated from wheat grain and identifi ed using molecular methods: a. alternata, a. infectoria, a. fl avus, e. nigrum and f. poae were isolated from the grain from wheat grown in conventionally managed fi elds and in fi elds with sustainable eco-management. with the exception of a. fl avus, these species appear to be common colonisers of wheat grain (nicolaisen et al. 2014). however, in contrast to nicolaisen et al. (2014), who identifi ed the genera phaeosphearia and microdochium as the core operational taxonomic units in 99% of wheat grain samples, we did not isolate any fungi from these genera. in addition to fusarium spp. and alternaria spp., which are both known as soil fungi, the so-called storage fungi of penicillium spp., aspergillus spp. and rhizopus spp. have also been reported for wheat grain (bensassi et al. 2011, bottalico and perrone 2002, gohari et al. 2007). in the present study, only a. fl avus was isolated, which suggests the potential contamination of the grain with afl atoxins, which would negatively impact on the usability of the grain for animal and human consumption. accordingly, the fungitoxic effects of thyme essential oil on the growth of a. fl avus might prove to be extremely benefi cial, as it would reduce the spoilage of food due to contamination with fungal toxins. as post-harvest synthetic fungicide treatments of stored products can infl uence the quality of the grain (osman and abdulrahman 2003), natural essential oils have advantages over synthetic agents due to their biodegradability and low toxicity. in the present study, in vitro thyme essential oil showed a broad spectrum of fungitoxicity against all of the fungi isolated from the wheat grain. the ec90 of the thyme essential oil was < 0.02% for all of these tested fungi, with a mainly fungicidal nature of the toxicity. the ec90 for thyme essential oil against the fi ve fungi in this study fi t well within ranges of values reported in other studies, where the fungitoxicity of thyme essential oil has been given as between 0.025% (soliman et al. 2002) and 0.07% (kumar et al. 2008), although zabka et al. (2009) reported a higher mic for a. fl avus (0.23%) and a fusarium sp. (~0.15%). the antifungal effects of thyme essential oil can be explained by modifi cations of fungal morphogenesis and growth through interference of the essential oil components with the fungal enzymes that are responsible for cell wall synthesis, which will lead to changes in hyphae integrity and to plasma membrane disruption and mitochondrial destruction (rassoli et al. 2006). the antifungal activities of essential oils are generally strongly associated with monoterpenic phenols, and especially thymol, carvacrol and eugenol (bluma et al. 2008). both thymol and carvacrol are also present in thyme essential oil and they are characterised by their high antimicrobial activity (bouzidi et al. 2013, and references therein, gonçalves et al. 2010, so kovič et al. 2009, šegvić klarić et al. 2007). chemical analysis of the thyme essential oil used in the present study has shown that the major constituent is indeed thymol (68.9%), whereas carvacrol constitutes only 1.6% (an žlo var et al. 2014). due to the possible negative effects of essential oils on seed germination, we examined the effects of this thyme essential oil treatment on seed germination. our results show that treatment with thyme essential oil is effective for the reduction of fungal contamination of wheat grain, although it can also affect the germination of the treated grain, which is an important aspect for the potential use of these treated grains. direct treatment with thyme essential oil signifi cantly inhibited seed germination, and so despite the good reduction in infection seen, this treatment is less appropriate as a prevention method for stored grain. in contrast, the indirect essential oil treatment had no effects on the germination of these wheat grains, but did successfully inhibit fungal growth, which was even more pronounced in combination with the prior surface hypochlorite sterilization that was also used here. our results are in agreement with observations from several studies that have reported negative effects of direct essential oil treatments on seed germination (kordali et al. 2008, kotan et al. 2013, paudel and gupta 2008). in contrast, kedia et al. (2014) demonstrated that wheat grain that has been indirectly fumigated with cumin seed essential oil retain viability even 12 months after storage. accordingly, in combination with other studies, our results indicate that indirect treatment (i.e., fumigation) with thyme essential oil has great potential as a bioprotection technique. we also show that the combination of surface sterilization and indirect thyme essential oil treatment further diminishes fungal infection without negative effects on the germination of the treated wheat grain. to conclude, fumigation with thyme essential oil shows a good in vivo effi cacy for the protection of wheat grain from fungal colonisers. indirect treatment with thyme essential oil in combination with prior surface sterilization of wheat grain provided an even more effi cient treatment that signifi cantly reduced fungal infection and spread on these grain and was accompanied by retention of high germination rates of the treated seeds. for the sake of safety for plants and consumers, thyme essential oil can be applied as a protection agent in storage containers for grain intended for sowing and food production. acknowledgments this study was fi nancially supported by the slovenian research agency, grant no. p1-0212. the authors are thankful to tjaša pršin for technical help and christopher berrie for language editing. antifungal potential of thyme essential oil acta bot. croat. 76 (1), 2017 71 references anžlovar, s., baričevič, d., ambrožič avguštin, j., dolenc koce, j., 2014: essential oil of common thyme as a natural antimicrobial food additive. food technology and biotechnology 52, 263–268. batish, d. r., singh, h. p., setia, n., kaur, s., kohli, r. k., 2006: chemical composition and phytotoxicity of volatile essential oil from intact and fallen leaves of eucalyptus citriodora. verlag der zeitschrift für naturforschung c 61, 465–471. bensassi, f., mahdi, c., bacha, h., hajlaoui, m. r., 2011: survey of the mycobiota of freshly harvested wheat grains in the main production areas of tunisia. african journal of food science 5, 292–298. bluma, r., amaiden, m. r., etcheverry, m., 2008: screening of argentine plant extracts: impact of growth parameters and aflatoxin b1 accumulation by aspergillus section flavi. international journal of food microbiology 122, 114–125. bottalico, a., perrone, g., 2002: toxigenic fusarium species and my cotoxins associated with head blight in small-grain cereals in europe. european journal of plant pathology 108, 611–624. bouzidi, l. e., jamali, c. a., bekkouche, k., hassani, l., wohlmuth, h., leach, d., abbad, a., 2013: chemical composition, antioxidant and antimicrobial activities of essential oils obtained from wild and cultivated moroccan thymus species. industiral crops and products 43, 450–456. chen, p. j., moore, t., nesnow, s., 2008: cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes. toxicology in vitro 22, 1476–1483. d’mello, j. p. f., macdonald, a. m. c., postel, d., dijksma, w. t. p., dujardin, a., placinta, c. m., 1998: pesticide use and mycotoxin production in fusarium and aspergillus phytopathogens. european journal of plant pathology 104, 741–751. doolotkeldieva, t. d., 2010: microbiological control of fl ourmanufacture: dissemination of mycotoxins producing fungi in cereal products. microbiology insights 3, 1–15. gardes, m., bruns, t. d., 1993: its primers with enhanced specifi city of basidiomycetes: application to the identifi cation of mycorrhizae and rusts. molecular ecology 2, 113–118. gohari, a. m., sedaghat, n., nikkhah, m. j., saber-riseh, r., 2007: mycofl ora of wheat grains in the main production area in kerman province. international journal of agriculture and biology 9, 635–637. gonçalves, m. j., cruz, m. t., cavaliero, c., lopes, m. c., salgueiro, l., 2010: chemical, antifungal and cytotoxic evaluation of the essential oil of thymus zygis subsp. sylvestris. industrial crops and products 32, 70–75 kedia, a., prakash, b., mishra, p. k., dubey, n. k., 2014: antifungal and antiafl atoxigenic properties of cuminum cyminum (l.) seed essential oil and its effi cacy as a preservative in stored commodities. international journal of food microbiology 168–169, 1–7. kordali, s., cakir, a., ozer, h., cakmakci, r., kesdek, m., mete, e., 2008: antifungal and insecticidal properties of essential oil isolated from turkish origanum acutidens and its three components, carvacrol, thymol and p-cymene. bioresource technology 99, 8788–8795. kotan, r., dadasoğlu, f., karagoz, k., cakir, a., ozer, h., kordali, s., cakmakci, r., dikbas, n., 2013: antibacterial activity of the essential oil and extracts of satureja hortensis against plant pathogenic bacteria and their potential use as seed disinfectants. scientia horticulturae 153, 34–41. krisch, j., tserennadmid, r., vagvölgyi, c., 2011: essential oils against yeasts and moulds spoilage. in: mendez-vilas, a. 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(eds.), pcr-protocols and applications-a laboratory manual, 315–322. academic, london. zabka, m., pavela, r., slezakova, l., 2009: antifungal effect of pimenta dioica essential oil against dangerous pathogenic and toxinogenic fungi. industrial crops and products 30, 250–253. opce-str.vp acta bot. croat. 68 (2), 183–197, 2009 coden: abcra 25 issn 0365–0588 historical abundance and morphology of didymosphenia species in naknek lake, alaska danielle p. pite1,2, kelly a. lane1, anna k. hermann1,3, sarah a. spaulding1,4*, bruce p. finney5 1 instaar campus box 450, university of colorado, 1560 30th street, boulder co 80309, usa 2 smith college, northampton ma 01063, usa 3 tulane university, new orleans la 70118, usa 4 u.s. geological survey, fort collins science center, fort collins, co 80526-8118, usa 5 idaho state university, department of biological sciences, pocatello id 83209, usa since the 1980s, nuisance blooms of didymosphenia geminata (lyngbye) m. schmidt have been documented in sites that are warmer and more mesotrophic than historical records indicate. while the invasion of d. geminata in new zealand is well documented, it is less clear whether nuisance blooms in north america are a new phenomenon. in order to test the hypothesis that d. geminata blooms have increased in recent years, we examined the historical record of this species in sediments of naknek lake, in katmai national park, alaska. chronological control was established by relating the presence of two ash layers to known volcanic eruptions. we identified two species of didymosphenia within the sediment record: d. geminata and d. clavaherculis (ehrenberg) metzeltin et lange-bertalot. this is the first published record of d. clavaherculis in north america. we found no statistically significant change in the numerical presence of d. geminata or d. clavaherculis, as a group, in naknek lake between the years 1218 and 2003. while there has been no sudden, or recent, increase in abundance of didymosphenia in naknek lake, morphological features of d. geminata populations in naknek lake are distinct compared to morphological features of d. geminata in streams containing nuisance blooms from sites in north america and new zealand. variance in the morphology of didymosphenia cells may help determine relationships between distinct sub-populations and establish the history of habitat invasion. key words: diatom, didymosphenia geminata, didymosphenia clavaherculis, morphology, counting, bloom, stream, lake, invasion, history, alaska introduction until recently, didymosphenia geminata (lyngbye) m. schmidt was thought to be limited to cold, low-nutrient, well-oxygenated waters in northern latitudes. nuisance blooms acta bot. croat. 68 (2), 2009 183 * corresponding author: e-mail: sarah.spaulding@usgs.gov u:\acta botanica\acta-botan 2-09\pite.vp 9. listopad 2009 13:01:38 color profile: disabled composite 150 lpi at 45 degrees of this diatom, however, have been documented in warmer and more mesotrophic sites since the 1980s (noga 2003, kawecka and sanecki 2003, subakov-simi] and cvijan 2004). didymosphenia geminata is of concern in stream ecosystems because of its capacity to form thick masses, impacting biological and physical stream conditions. further, this species is reported in streams and rivers across the united states (kumar et al. 2009). this diatom has demonstrated its invasive ability, evidenced by the presence of d. geminata in south island of new zealand in 2004 (kilroy et al. 2007). although some reports assert that the nuisance blooms of d. geminata in north america (kumar et al. 2009) and europe (kawecka and sanecki 2003) are of recent occurrence, there are few to no historical data to establish the historical abundance of d. geminata. the purpose of this study is to examine the historical record of didymosphenia in order to test the hypothesis that blooms have increased in recent years. this paper documents the record of didymosphenia over 800 years in the sediments of naknek lake, alaska. didymosphenia geminata was first recorded from the faroe islands in 1819 (lyngbye 1819). other records in the early literature also mention the presence of d. geminata, including a reference to large masses in the kanchou region of china (skvortzow 1935). it is possible that extensive blooms are a normal part of this diatom’s life history, but few data exist to quantify stream growth habits. recognition of the patterns of d. geminata growth is needed to understand the current blooms in north america (kumar et al. 2009) and europe (beltrami et al. 2008) and the expansion to new zealand. while the invasion of d. geminata in new zealand is well documented, it is less clear whether nuisance blooms in north america are a new phenomenon. although several species of didymosphenia are known from lakes (skvortzow and meyer 1928, kociolek et al. 2000), d. geminata is most often recorded in streams and rivers (sheath et al. 1986, lapierriere et al. 1989, miller et al. 1992, sheath et al. 1996, ellwood and whitton 2007) where it appears to reach its greatest biomass (kilroy et al. 2007). determining the history of diatoms in streams and rivers, however, is more problematic than in lakes, as streams are high flow systems that typically do not leave a continuous sedimentary record that can be interpreted (smol 2002). even so, reconstruction of environmental change in rivers by examining stream diatoms deposited in the sediments of lakes has been shown to be successful (smol 2002). in sites where streams or rivers flow into lakes, records of historical change in river systems may be archived in lake sediments. for example, the relative abundance of stream diatoms [hannaea arcus (ehrenberg) patrick and meridion circulare (greville) agardh] found in lake sediments was used to reconstruct historical river discharge in the high arctic (ludlam et al. 1996, antoniades and douglas 2002). because didymosphenia reaches its greatest abundance in streams and rivers, we propose that the concentration of cells in lake sediments is directly related to the concentration of cells in stream inflows. the presence of diatom cells preserved in lake sediments and the accurate dating of those sediments provide that opportunity to examine changes in cell abundance over time. differences in valve morphology of d. geminata cells from different regions of europe and asia have been noted (antoine and benson-evans 1983, stoermer et al. 1986, metzeltin and lange-bertalot 1995) and these morphological differences are thought to reflect distinct sub-populations. although differences in the valve margins of diatoms are easily recognized visually, until recently few tools have existed to evaluate statistically sig184 acta bot. croat. 68 (2), 2009 pite d. p., lane k. a., hermann a. k., spaulding s. a., finney b. p. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:37 color profile: disabled composite 150 lpi at 45 degrees nificant differences in shape (stoermer et al. 1986). diatoms, in particular, are a group of organisms whose species concepts and boundaries are based largely on differences in valve shape (stoermer et al. 1986). qualitative evaluation of valve morphology of cells of didymosphenia may provide insights into the nature of the relatedness of populations and the history of range expansion and invasion into new habitats. furthermore, preliminary evaluation of molecular markers in the internal transcribed spacer (its) regions of the 18s rrna gene shows a distinction between populations of d. geminata from different geographic regions (cary et al. 2008). these results not only indicate a marked separation between the european and north american populations of d. geminata, but the close affiliation of north american and new zealand populations. the second objective of this paper is to examine the morphology of d. geminata cells preserved in sediments dating to 800 years b.p. and compare valve shape to modern, bloom-forming populations of d. geminata from around the world. materials and methods naknek lake is located in katmai national park, alaska, near the base of the alaskan peninsula (latitude 58°40’n and longitude 156°12’w) (fig. 1). the lake is 64 km long and up to 13 km wide and has a maximum depth of 173 m (laperriere 1997). the lake is fed acta bot. croat. 68 (2), 2009 185 didymosphenia in naknek lake fig. 1. map showing alaska (inset) with the naknek lake watershed in katmai national park. bathymetric map of naknek and brooks lakes with contours at 10 m intervals. the site where the sediment core was taken is marked by an »x«. brooks lake flows into naknek lake via brooks river, providing much of the clear water inflow. the savanoski and utak rivers and margot creek are the major inflows to the southern arm of naknek lake. the outlet of naknek lake is the naknek river. map courtesy of the national park service, based on 1963 data. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:37 color profile: disabled composite 150 lpi at 45 degrees both by glacial meltwater and clearwater streams such as the major inflowing tributary, brooks river. naknek lake drains west into bristol bay through the naknek river. naknek lake is bounded by terminal moraines that were deposited by glaciers that flowed from east to west during the last glaciation. based on dating of the terminal moraines, naknek lake is estimated to have formed approximately 14,000 years bp. the lake today receives a large input of silt particles from active glaciers in its watershed. the sediments are grey in color and suspended silt particles prevent light penetration in much of the lake (laperriere and edmundson 2000). nutrient concentrations are low and the lakes are considered oligotrophic (goldman 1960, laperriere and jones 2002). cores were obtained from naknek lake in 2003 using both a hammer core and piston core. the coring location, at a water depth of 61 m, is shown in figure 1. the tops of the hammer cores were extruded at 1 cm intervals in the field in order to preserve the integrity of recent sediments. the top of the second hammer core, the middle of the first hammer core, and the bottom of the piston core were combined to make a total composite core of 452 cm in length. the cores were correlated at the two major tephras encountered with 1.3 m included from the first hammer core, 1.94 m from the second hammer core, and 1.28 m from the piston core. sediments were sectioned, freeze dried, and stored at the university of alaska fairbanks. the cores were dated based on the 1912 katmai ash layer and the »brown ash« layer. the age of the brown ash was determined by ams dating of terrestrial macrofossils from cores from nearby lakes, and determined to be 1570 +/– 30 yr cal bp. analyses for loss on ignition (loi) and percent biogenic silica (opal) were made at the university of alaska fairbanks. subsamples from 38 sections of the core were sent to institute of arctic and alpine research (instaar) for diatom preparation and analysis. dried sediment from each sample was weighed to the nearest milligram to obtain approximately 1000 mg of dry sediment. the samples were hydrated with 15 ml of deionized water for 12 hours in 50 ml centrifuge tubes. organic material was oxidized using 15 ml of 30% h2o2 in a digestion over 6 days (renberg 1990). following the digestion, deionized water was added to bring the total volume to 50 ml. the samples were allowed to settle for 8 hours, decanted, and rinsed with deionized water 6 times to remove h2o2. the cleaned sediments were well mixed by shaking and 0.500 ml was placed on glass cover slips. five replicate cover slips were made for each of the 38 samples. the cover slips were allowed to dry and were mounted on glass microslides using a high refractive mounting medium (zrax). permanent slides and cleaned material are archived in the university of colorado instaar diatom database (accession 10753–10790). ideally, a calibration between cell abundance in a river inflow and the sediment records should be able to be established. in this study, however, we were not able to obtain modern collections from the river inflow to determine the relation between river and sediment cell abundances. reconstruction of the river abundance based on the lake sediments is based on an assumption of constant sedimentation rate within naknek lake. although studies of streams mention the presence of d. geminata (patrick and freese 1961, foged 1981, laperriere et al. 1989, hein 1990) it is not noted to be of great abundance. other limnological surveys in the area do not mention the presence of cells (goldman 1960, oswood 1989, laperriere 1997, laperriere and edmundson 2000, laperriere and jones 2002) in lakes or streams. the inflow (brooks river) to naknek lake is one of the most frequent 186 acta bot. croat. 68 (2), 2009 pite d. p., lane k. a., hermann a. k., spaulding s. a., finney b. p. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:37 color profile: disabled composite 150 lpi at 45 degrees sites of visitors to the area (j. scherer, national park service, personal comm.). the core was collected in 2003, and four years later in 2007, reports of masses of didymosphenia were made on brooks falls (d. bogan, university of alaska, anchorage, personal comm.). however, we are not able to evaluate the correlation between river and sediment abundance. we systematically examined the entirety of each of 185 slides for didymosphenia cells using the light microscope (olympus vanox, zeiss universal, and nikon optiphot) at low magnification (40 times to 200 times). a distinction between d. geminata and d. clavaherculis (ehrenberg) metzeltin et lange-bertalot was not made for all specimens because fragments of cells were included in the counts. we were not able conclusively to determine species level determinations of didymosphenia on valve fragments. where whole valves were located, their images were recorded and several morphological measurements were made. for each slide, cymbella mexicana (ehrenberg) cleve cells were also counted. cymbella and didymosphenia are both members of the family cymbellaceae. they share many characteristics such as asymmetry about the apical axis and similar apical pore fields (kociolek and stoermer 1988). because of these shared traits, as well as the fact that they have been seen to grow in similar places and produce a profuse amount of stalk, it has been proposed that the number of c. mexicana present will co-vary with the number of didymosphenia cells. electronic images of cells were captured using an olympus vanox microscope equipped with a 63 times oil immersion lens (1.4 na) and a 3.3 m q imaging camera and software. we were able to differentiate between the various cymbella forms at low magnification because the striae density in c. mexicana have a unique light refraction that makes the cells appear light blue under low magnification. the calibration and measuring capabilities of the imaging software were used to measure six key dimensions of didymosphenia specimens from naknek lake: length, width, footpole, footpole-constriction, headpole, and headpole-constriction (figs. 2–9). images were obtained of d. geminata populations during blooms, from sites archived in the instaar diatom database including matapédia river, québec (10752), popo agie river, wyoming (10745), blue river, colorado (10610) and waiau river, new zealand (10580). measurements of the six morphological features were made for 15 individuals from each population. relationships between diatom morphology and sites were explored using principal components analysis (pca) and analysis of variance (anova). all ordination and statistical analyses were run in r software using default functions in the vegan package of r (r development core team 2006). results our analysis was based on enumeration of all didymosphenia valves and valve fragments observed within the five duplicate slides from selected strata in the sediment record. during our examination, however, we noted two species of didymosphenia were present in naknek lake (tab. 1). we identified d. geminata (figs. 2–5) and propose that the second species belongs to d. clavaherculis (ehrenberg) metzeltin et lange-bertalot (figs. 6–9), which most noticeably differs in the ratio of the width of the central valve to the headpole width. this is the first known record of the d. clavaherculis in north america. both d. acta bot. croat. 68 (2), 2009 187 didymosphenia in naknek lake u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:38 color profile: disabled composite 150 lpi at 45 degrees geminata and d. clavaherculis were found in the core, although only a few complete specimens of d. clavaherculis were present. we included fragments of cells in our analysis, which precluded identification to the species level. because of the small number of d. clavaherculis valves, we grouped both species together to examine abundance of didymosphenia in naknek lake over the past 800 years. the total abundance of d. geminata and d. clavaherculis in the sediment record that we analyzed ranged from 5–30 valves mg–1 sediment, while the total abundance of c. mexicana ranged from 5–300 valves mg–1 sediment (fig. 10). didymosphenia is present throughout the sediment record with no trends in the total abundance between the years 1218 and 2003, based on comparison of the sample means and standard deviations. the biogenic silica in the sediments, the source of which is considered to be the cell walls of diatoms, reached it greatest percent of the total near 1900. while the peak value in opal represents a peak in diatom concentration in the sediments, and therefore diatom biomass, there was no concurrent increase in didymosphenia cells. similarly, the minor changes measured in organic matter, measured as percent loss on ignition, does not appear to be related 188 acta bot. croat. 68 (2), 2009 pite d. p., lane k. a., hermann a. k., spaulding s. a., finney b. p. tab. 1. comparison of morphological features of d. clavaherculis, three morphotypes of d. geminata based on metzeltin and lange-bertalot (1995), and four complete d. clavaherculis specimens in naknek lake. note that d. geminata var. stricta m. schmidt is a later homonym of d. clavaherculis. didymosphenia clavaherculis was described from diatomites (»infusorial earths«) of ireland (ehrenberg 1842). feature comparison of features of d. clavaherculis to those of d. geminata d. clavaherculis from naknek lake. n = 4 1) general valve outline valve outline symmetrical, no cymbelloid type bend in the apical axis, as in d. geminata. slight asymmetry (fig. 9) 2) size generally larger size range (l = 90–180 mm, w = 34–45 mm) size range (l = 111–118, w = 22–30) 3) midvalve to footpole outline shape of outline from midvalve to footpole less concave. the footpole is relatively broader. ratio of the central valve to headpole is 1.1 – 1.3 (much lower than in d. geminata). shape of outline from midvalve to footpole less concave. the footpole is relatively broader. ratio of the central valve to headpole is 1.0 – 1.1 4) central area central area thin and narrowly elliptic to lanceolate not as described 5) striae striae number 7–9 in 10 mm, as compared to 8–10 in d. geminata. striae more strongly diverge toward the headpole and footpole than in other didymosphenia species. striae number 8–9 in 10 mm. striae not as described. 6) number of stigmata range 2–7 stigmata per valve. specimens from spitzbergen were found to possess 1–2 stigmata, while specimens from angara river (russia) and lake koko nor (tibet) consistently possessed 4–7 stigmata. (d. metzeltin pers. comm.). stigmata number 2–3 per valve. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:38 color profile: disabled composite 150 lpi at 45 degrees to a change in concentration of didymosphenia or c. mexicana, at least not within the measurements of those changes that we had the ability to obtain. we found no statistically significant relationship between didymosphenia and any of the sediment variables. on the other hand, we found a statistically significant relation between valve concentration of didymosphenia and c. mexicana (r2 = 0.09, p < 0.01) (fig. 11). therefore, cells of didymosphenia and c. mexicana responded in a similar manner to one another. we observed differences in shape between d. geminata in naknek lake in other regions of the world. these differences caused us to ask how the morphologically different forms in naknek lake compare to the variations seen in other geographical areas, namely the matapedia river, québec, popo agie river, wyoming, blue river, colorado and waiau river, new zealand where nuisance blooms of d. geminata occur (kilroy 2008, simard and simoneau 2008, spaulding et al. 2008). specifically, we were interested in determining if a »nuisance form« could be identified. acta bot. croat. 68 (2), 2009 189 didymosphenia in naknek lake figs. 2–9. light micrographs of didymosphenia. figs. 2–5: didymosphenia geminata. figs. 6–9: didymosphenia clavaherculis. scale bar in fig. 6 is equal to 10 mm and applies to all images. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:41 color profile: disabled composite 150 lpi at 45 degrees results of measurement of six morphological dimensions of d. geminata specimens from naknek lake including length, width, footpole, footpole-constriction, headpole, and headpole-constriction are shown in table 2. we excluded specimens of d. clavaherculis from the analysis. the mean valve length from the naknek lake population was less than the mean of all the other sites, except for blue river, colorado. in contrast, the standard deviation of valve length from naknek lake specimens was the greatest of all sites. we found more consistency among the morphological measurements of d. geminata from québec, wyoming, new zealand, and colorado compared with d. geminata in naknek lake as demonstrated by mean and standard deviation (tab. 2). a correlation matrix showed that valve length and headpole width were highly correlated with footpole width in all samples (tab. 3). therefore, footpole width was removed from further analysis. principal components analysis (pca) of the remaining variables (fig. 12) shows that the first two principal axes are significant, as indicated by a broken stick model (fig. 13), and account for 82% of the total variance. while cell width, length and headpole width 190 acta bot. croat. 68 (2), 2009 pite d. p., lane k. a., hermann a. k., spaulding s. a., finney b. p. fig. 10. graph showing cymbella mexicana abundance (valves mg–1 sediment), didymosphenia abundance (valves mg–1 sediment), percent biogenic silica and percent organic matter (g cc–1) measured as loss on ignition at 550 °c against calendar year from 1218 to 2003 a.d. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:42 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 191 didymosphenia in naknek lake fig. 11. graph showing relationship between didymosphenia and c. mexicana valves / mg sediment). the linear regression of valve concentration was statistically significant (r2 = 0.09, p < 0.01). tab. 2. mean, standard deviation and number of d. geminata valves measured for valve length, width, footpole width, footpole constriction, headpole width, and headpole constriction (all in mm) for naknek lake, matapédia river (québec), popo agie river (wyoming), blue river (colorado) and waiau river (new zealand). length width footpole footpole constriction headpole headpole constriction naknek, n = 14 mean std dev 123.3 10.1 37.6 4.2 19.8 1.9 16.8 1.3 27.2 3.5 22.3 2.4 matapédia, n = 15 mean std dev 126.6 4.5 40.5 1.4 22.5 0.9 16.9 1.1 30.0 1.0 21.4 0.9 popo agie, n = 15 mean std dev 146.7 4.1 44.6 1.0 24.3 0.9 18.2 1.2 31.6 1.1 22.4 1.8 blue, n = 15 mean std dev 118.9 6.1 42.0 1.8 20.6 1.0 15.8 1.6 28.7 1.1 20.7 2.0 waiau, n = 15 mean std dev 126.8 2.2 40.4 1.6 22.5 0.9 17.7 1.8 29.1 1.8 22.2 1.7 u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:42 color profile: disabled composite 150 lpi at 45 degrees co-vary with one another, the constriction of the footpole and headpole co-vary with one another. the naknek lake specimens have the greatest variation in morphology when compared to populations from other sites. the specimens from blue river are also variable, but not to the extent of the naknek lake population. specimens from popo agie, waiau and matapedia rivers are all more closely clustered to one another. results of an 192 acta bot. croat. 68 (2), 2009 pite d. p., lane k. a., hermann a. k., spaulding s. a., finney b. p. tab. 3. correlation matrix of measurements of morphological features. because length and footpole measures were highly correlated (0.8), the footpole measure was removed from the principal components analysis. length width footpole footpole constriction headpole headpole constriction length 1.0 width 0.6 1.0 footpole 0.8 0.7 1.0 footpole-constriction 0.5 0.4 0.6 1.0 headpole 0.7 0.7 0.8 0.5 1.0 headpole-constriction 0.2 0.1 0.4 0.7 0.4 1.0 fig. 12. principal components analysis (pca) of valve morphology of five populations of d. geminata. each point represents a single specimen with five measures (valve length, width, headpole, footpole constriction, and headpole constriction) in samples from naknek lake, wyoming, new zealand, colorado and québec. origin of the biplot represents the average value for each variable. variables increase in the direction of the arrow and the length of the arrows represent strength of that variable. the angles between arrows are approximate representation of correlation between variables. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:42 color profile: disabled composite 150 lpi at 45 degrees analysis of variance (anova) test show that the populations were significantly different in terms of valve length, width and headpole constriction (tab. 4). discussion we used the total abundance of d. geminata and d. clavaherculis valves in lake sediments to infer the historical pattern of valves in streams flowing into naknek lake. because we were not able to obtain modern collections, however, our interpretation is based on the assumption that the relationship between river abundance and lake sediment concentration is constant over time. ideally, a comparison of cell densities linked to a known acta bot. croat. 68 (2), 2009 193 didymosphenia in naknek lake fig. 13. plot of pca axes against eigenvalues for the calculated axes (solid line) and axes based on the broken stick model (dashed line). the values for axes 1 and 2 fall above the broken stick model and are therefore the only significant axes. axes 1 and 2 account for 82% of the total variance. tab. 4. results of analysis of variance test of morphological differences between populations from alaska, québec, wyoming, new zealand, and colorado. the populations were significantly different in terms of length, width and headpole constriction. measurement f value level of significance (p > f) length 18.492 0.0001 width 26.364 0.0001 footpole constriction 0.0143 ns headpole 3.2800 0.1 headpole constriction 19.855 0.0001 u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:42 color profile: disabled composite 150 lpi at 45 degrees bloom of d. geminata could be linked to historical cell densities in the lake sediments. further work to establish the relationship between river cell abundance and the sediment record is likely to be instructive. such a comparison is needed to resolve whether large masses occurred in brooks river prior to 2003, the time of coring. our results show that between ad 1218 and 2003, the concentrations of d. geminata and d. clavaherculis and c. mexicana remained relatively constant and responded in a similar manner. the abundance of these three diatom species did not appear to be correlated with percent loss on ignition or percent carbon/nitrogen ratio, d15n and d13c (finney, unpublished data). the d15n ratio in sockeye salmon nursery lakes such as naknek lake, has been shown to reflect abundance of sockeye spawning in and around the lake (finney et al. 2000). while recent work has suggested that the growth of d. geminata in rivers and streams is a new phenomenon in regions of north america (spaulding et al. 2008), we show that didymosphenia abundance has not changed over time in naknek lake, alaska. the presence of d. geminata in a high latitude site such as naknek lake is to be expected, based on historical records from canada and alaska (cleve 1894–1986, patrick and freese 1961, foged 1981, sheath et al. 1986, lapierriere et al. 1989, hein 1990, miller et al. 1992, sheath et al. 1996). it is interesting to note, however, that much of the work on diatoms from high latitudes has concerned analysis of sediment cores for paleolimnological reconstruction, and these studies do not report the presence of didymosphenia (ludlam et al. 1996, antoniades and douglas 2002). we believe the absence may be the result of using fixed counts of total diatom valves (usually 100–600 valves). our results demonstrate that a method of counting using low magnification objectives and examining entire slides for large diatoms results is useful for 1) detecting and documenting large, rare species and 2) obtaining an adequate number of total cells for a representative count. furthermore, this method may be more time-efficient than traditional microslide counts. in future work, inclusion of a greater number of specimens (microslides) identified and counted would allow for greater precision for each taxon to determine potential trends over time. our results indicate that this approach to determining the historical abundance of didymosphenia could be applied in lakes downstream of nuisance blooms. this approach could be repeated in regions in north america and europe to quantify how, and whether, d. geminata is changing abundance and expanding its geographic range. the variance in cell morphology within naknek lake is larger than in other didymosphenia geminata populations that we examined from québec, wyoming, new zealand, and colorado. to date, we are not aware of published records of d. clavaherculis in north america, although hein (1990: pl. 14, fig. 1) shows an image of a specimen that could belong to this species. we suggest that in the examination of specimens from areas with invasive or nuisance blooms, morphological analysis combined with molecular markers would be a promising approach to determining if such nuisance blooms represent a new strain of d. geminata. acknowledgements thank you to dr. paul strode for his support and encouragement. we also thank wendy freeman at instaar sediment laboratory and andrea krumhardt, university of alaska fairbanks for assistance with samples. we appreciate the contribution of ditmar metzeltin, 194 acta bot. croat. 68 (2), 2009 pite d. p., lane k. a., hermann a. k., spaulding s. a., finney b. p. u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:42 color profile: disabled composite 150 lpi at 45 degrees who brought the species d. clavaherculis to our attention. jeff scherer, national park service, provided maps, literature and background information. any use of trade, product or firm names is for descriptive purposes only and does not imply endorsement by the u.s. government. references antoine, s. e., benson-evans, k., 1983: polymorphism and size variation in didymosphenia geminata from great britain. british phycological journal 18, 199–200. 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characteristics. algological studies 114, 53–66. acta bot. croat. 68 (2), 2009 197 didymosphenia in naknek lake u:\acta botanica\acta-botan 2-09\pite.vp 5. listopad 2009 13:22:43 color profile: disabled composite 150 lpi at 45 degrees acta botanica 1-2017 za web.indd 98 acta bot. croat. 76 (1), 2017 acta bot. croat. 76 (1), 98–102, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0048 issn 0365-0588 eissn 1847-8476 short communication five new alien species in the fl ora of montenegro: coreopsis tinctoria nutt., ipomoea indica (burm.) merr., lupinus × regalis bergmans, physalis angulata l., and solidago canadensis l. and new possible threats to the biodiversity danijela stešević1*, nada bubanja2 1 faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81 000 podgorica, montenegro 2 natural history museum of montenegro, trg vojvode bećir-bega osmanagića 16, 81000 podgorica, montenegro abstract – the alien fl ora of montenegro is enlarged by 5 new species: coreopsis tinctoria, ipomoea indica, lupinus × regalis, physalis angulata, and solidago canadensis. all species are grown as ornamentals, and thus horticulture is considered the most possible pathway for their introduction. nevertheless, in the case of physalis angulata, the occurrence might have originated in the remains of picnic meals. in order to defi ne the alien status of these species, monitoring is needed, and thus we propose including the species on the national list of indicators in environmental protection. special attention should be paid to the species with invasive potentials: solidago canadensis, ipomoea indica and physalis angulata. keywords: alien plants, coreopsis, ipomoea, lupinus, montenegro, physalis, solidago. * corresponding author, e-mail: danijela.stesevic@ac.me introduction the interest in alien fl ora in montenegro has grown rapidly in the last decade, with many new alien species having been reported. still, information on alien plants is incomplete and frequently updated with new records. according to recent research, 51 alien species are considered invasive in montenegro (stešević and petrović 2010, stešević and caković 2013). taking into account the rather small size of the country (13,812 km2), this number can be considered as troublesome, while in croatia, with a state area of 57,000 km2, the number of invasive alien species (ias) is 74 (nikolić 2015), in serbia with a state area of 88 361 km2 the number of ias is 68 (lazarević et al. 2012), in vojvodina with an area of 21, 506 km2 the number of ias is 152 (anačkov et al. 2013), in slovenia with a state area of 20,151 km2 the number of ias is 32+71 naturalized and potentially invasive (jogan et al. 2012) etc. nevertheless, those numbers are not fully comparable because the lists of ias in different countries have been assembled in different ways. out of 51 invasive species in montenegro, only one, ambrosia artemisiifolia is monitored and partly managed (ste šević et al. 2014). nevertheless, improvements in this fi eld are expected soon, due to the recently adopted regulation on the national list of indicators in environmental protection. according to this document, alien (a) and invasive species (is) are considered an indicator (b05) of pressure in the driving forces – pressures – state – impact – response (dpsir) model and they will be monitored with a 10 year dynamic (offi cial gazette no 29/2013). this paper presents a supplement to the national list of alien plant species in montenegro as well as to the general distribution of the mentioned aliens in se europe. material and methods plant material of fi ve new alien species for the fl ora of montenegro was collected during fi eld investigations of the coastal and central part of the country, in the period from august 2013 to october 2014. site geocoding was done with a gps device, garmin e-trex vista c, while the specimens were identifi ed with following keys: ipomoea indica with backer and bakhuizen van den brink (1965), physalis angulata (hawkes 1972), coreopsis tinctoria, solidago canadensis (pignatti 1982), lupinus × regalis (webb 1988). voucher specimens were deposited in the herbarium collection at the university of montenegro (tgu), voucher numfive new alien species in the flora of montenegro acta bot. croat. 76 (1), 2017 99 bers 560413-56016 and in the herbarium collection of the natural history museum of montenegro, voucher numbers 5591-5592. species are presented in alphabetical order. results and discussion coreopsis tinctoria nutt. during the fl oristic survey conducted in june 2014 in the hinterland of long beach in ulcinj (n41°53’51”, e19°17’52”) we recorded a population of coreopsis tinctoria (fig 1a). there were ca. 20 individuals scattered over an area of 16 m2. besides the typical form with yellow ligules tinged reddish brown towards the center of the fl ower heads, also the form coreopsis atropurpurea coexisted (2 individuals). it is characterized by completely reddish brown heads (fig 1b) (smith and parker 1971). the population had grown on trampled vegetation along the road, accompanied by plantago major l., plantago lanceolata l., cichorium intybus l., crepis sancta (l.) bornm., knautia integrifolia (l.) bertol, poa annua l., lolium perenne l., vicia villosa roth. subsp. varia (host) corb, erigeron annuus (l.) desf. etc. since both forms of c. tinctoria are grown as ornamentals in the štoj area, we suppose that horticulture is the pathway of introduction. up to the date the only coreopsis species reported in the fl ora of montenegro was the alien north american species c. lanceolata l. (stešević et al. 2008). c. tinctoria is also a north american species introduced to europe in 1835, when it has become a common garden plant (gajić 1975). in the native range it inhabits low wet areas along the coast (smith and parker 1971), while in non-native areas it grows on disturbed places up to 1000m (strother 2006). nowadays it is spread over almost all of europe as largely cultivated in spain, portugal, poland, serbia; as casual, in austria, belgium (euro+med 2006), the british isles (stace 1997), bulgaria (v. vladimirov 2014, personal communication), the czech republic (pyšek et al. 2002), croatia (ni kolić 2015), denmark (alanen et al. 2004), france, germany, hungary, italy, norway (euro+med 2006), romania (anastasiu et al. 2011), ukraine (volutsa 2010); or with a unclear status: in belarus, finland and sweden (euro+med 2006). the oldest record of this species in europe dates from 1883 (czech republic, pyšek et al. 2002). voucher specimen: montenegro, long beach in ulcinj, n41°53’51”, e19°17’52”, 1 m.a.s.l., roadside vegetation, (n. bubanja, 19.07.2014., no. 5591). ipomoea indica (burm.) merr.,syn. convolvulus indicus burm., ipomoea congesta r. br., i. acuminata (vahl) roemer &schultes, i. learii paxton the coastal part of the country hosted another alien species: i. indica. a wild population was recorded at several sites between the settlements of meljine and igalo in the bay of boka kotorska (n42°26’59”, e18°32’59”). the plant mainly formed very dense stands on the walls of abandoned buildings, or on trees in abandoned yards, or spreading over the ground. at the locality presented in the fig. 1c, it covered an area of ca. 300 m2. in two sites, a few individuals were growing sparsely on boulders close to the sea (fig. 1d). up to this research the only ipomoea species reported in the fl ora of montenegro was the tropical american ipomoea purpurea (l.) roth (stešević et al. 2008). the origin of i. indica is unclear. according to one opinion it originates probably from south america (weber 2003), and to another from tropical asia (swarbrick and skarratt 1994). it was introduced to europe (maltese islands) in the last 500 years (mufsud 2002–2013), while the oldest record of escaped and established individuals dates from the beginning of the 1900s in spain. the minimum residence time is estimated as being 104 years (gassó et al. 2012). in spain (gassó et al. 2012), portugal (invasoras 2012), macronesia (silva et al. 2008), and italy (acta plantarum 2007) the species is considered invasive, and in greece, kriti (yannitsaros 1998), france (fried 2012) and maltese islands (mufsud 2002– 2013) is held to be naturalized. it usually inhabits coastal sites, moist forests and it is also known as an opportunistic colonizer of open, disturbed habitats. under favorable conditions it can grow very rapidly, smothering all other vegefig. 1. a) coreopsis tinctoria, b) coreopsis tinctoria f. atropurpurea, c) stand of ipomoea indica in meljine, d) single individual of i. indica in meljine, along the sea shore, e) lupinus × regalis in the old ruin in kolašin, f) physalis angulata, g) solidago canadensis habitus, h) s. canadensis stem, i) s. canadensis infl orescence (photo d. stešević). stešević d., bubanja n. 100 acta bot. croat. 76 (1), 2017 tation. its climbing habit makes it as a very successful competitor (muyt 2001). it reproduces primarily from broken fragments, hence, the most common mode of dispersal is believed to be unwanted vegetative material dumped by gardeners (csurhes 2008). we suppose that the same scenario occurred in montenegro. voucher specimen: montenegro, boka kotorska, meljine n42°26’59”, e18°32’59”, 1 m.a.s.l., wall of the abandoned yard (d. stešević, 19.10.2013., tgu-560413). lupinus × regalis bergmans = l. arboreus sims × l. polyphyllus lind i. several individuals of lupinus × regalis (fig. 1e) were recorded in the urban area of kolašin, in one old ruin frequently used for dumping unwanted vegetative material and waste (n42°49’24”, e19°31’40”). on the same site several ornamental plants grown in the city emerged: cosmos bipinnatus cav., reynoutria × bohemica chrtek & chrtková and symphoricarpos albus (l.) s. f. blake. thus, we consider horticulture the pathway of introduction. lupinus × regalis is a garden hybrid (or hybrid complex) with the parents being l. polyphyllus lindl. and l. arboreus sims. (both native to north america), and possibly involving some annual species (huxley 1999). it was introduced to the european market in 1935 as an ornamental (fremstad 2010) and is frequently cultivated in central and north europe; it often occurs as a casual and may be locally naturalized (franco and silva 1968). in the uk it has gradually started to replace l. polyphyllus (fremstad 2010). due to the similarity of these species it is probable that many modern records refer to lupinus × regalis, or backcrosses with l. arboreus (clement and foster 1994). the study conducted in norway (lid and lid 2005) showed that l. × regalis seems to be less hardy than l. polyphyllus and has not established itself outside gardens so far. in france the species is sporadically established in some northern and central part of the country, but still not in corsica (julve 2014). with respect to ecology, the species grows on rough ground, motorway verges, riverside shingle (preston et al. 2002), along railway lines (preston et al. 2002), meadows (groom 2007), forest edges (rich et al. 1996), urban habitats (lawley 2010). voucher specimen: montenegro, kolašin n42°49’24”, e19°31’40”, 960 m.a.s.l., old ruin (d. stešević, 08.07.2014., tgu-560414). physalis angulata l., syn. p. pendula rydb. the autumn fl oristic survey in the hinterland of long beach in ulcinj resulted in another new record for the alien fl ora of montenegro: physalis angulate (fig. 1f). it inhabited moist waysides in an open coastal pinus halepensis mill. forest on sandy dunes in the hinterland of long beach (n41°54’33”, e19°15’12”). population counted ca. 15 plants, surrounded by numerous individuals of ephedra campylopoda c. a. mey., digitaria ischaemum (scherb) muhl., solanum nigrum l., vicia villosa subsp. varia. in the area of štoj the plant is grown as an ornamental, and thus horticulture is considered to have been one introduction pathway. the species is not used as an edible plant in montenegro, but due to the fact that the long beach in ulcinj is a very attractive touristic destination for foreign visitors, there is possibility that its occurrence also originates in the remains of picnic meals, as is the case in greece (travlos 2012). up to this record physalis alkekengi l. was the only species out of the genus reported for our fl ora (rohlena 1942). it is native in europe, unlike p. angulata, which is considered to be native in tropical america (hawkes 1972). nowadays the species has a pantropical distribution as a weed of crops, gardens and plantations (mairura 2008). it was introduced into warm areas of the world in post-columbian times, with the voyages of exploration, discovery and commercial exploitation that began in the 16th century (sturtevant 1919). in many regions of the world it is naturalized, while in europe is cultivated locally for edible fruits (hawkes 1972). it has the status of casual species in austria (essl and rabitsch 2002), belgium (valdés 2012), the czech republic (pyšek et al. 2002), great britain (clement and foster, 1994), and slovenia (lešnik 2009); and of naturalized species in croatia (milović et al. 2010), while it is considered invasive in greece (travlos 2012). the species is also present in albania (tan and mullaj 2000) and denmark (nobanis 2005), but the status of naturalization is unclear. the means of introduction differs from country to country: escape from cultivation (albania, austria, slovenia), soya bean importation (belgium, verloove 2011, croatia, milović et al. 2010, slovenia, lešnik 2014, personal communication), cereals (croatia, milović et al. 2010), oil seed and wool (britain, clement and foster 1994), remains of picnic meals (greece, bőhling 2001). the oldest record of this species in europe dates from 1961 (belgium, verloove 2011).with respect to ecology the species grows best in moist, fertile soils, it is tolerant to partial shade and occurs widely as a weed of crops and pastures, and in waste areas. it can be found up to 3000 m altitude. it can tolerate light frost (mairura 2008). voucher specimen: montenegro, long beach in ulcinj n41°54’33”, e19°15’12”, 2 m.a.s.l., moist wayside in open pinus halepensis forest (d. stešević 06.10.2014., tgu560416; n. bubanja, no. 5591). solidago canadensis l., syn. s. altissima l., s. canadensis ssp. altissima (l.) bolos & vigo, s. canadensis var. scabra torr. & a. gray. along the roadside in the village vir, near the city of nikšić a population of solidago canadensis was recorded (n42°50’11”, e18°55’41”). it formed a dense patch that covered approximately 10 m2. due to strong anthropogenic pressure there were several alien and invasive species in the vicinity of the patch: artemisia verlotiorum lamotte and erigeron annuus, as well as typical ruderal species common in the vegetation along the roadsides: salvia verticillata l., cirsium arvense (l.) scop., clematis vitalba l., daucus carota l., diplotaxis tenuifolia (l.) dc., geum urbanum l., picris hieracioides l., arrhenatherum elatius (l.) p. beauv. ex j. presl & c. presl etc. since it is planted sporadically in the area of nikšić, we suppose that horticulture is the introduction pathway. five new alien species in the flora of montenegro acta bot. croat. 76 (1), 2017 101 the species (figs. 1g–i) usually forms extensive colonies with high shoot density covering large areas, eliminating almost all other species. individual clones expand rapidly by vegetative lateral growth. the annual stem (fig. 1h) dies in autumn and new shoots arise from the rhizome in spring. once established the plant remains dominant for a long period of time. seeds are abundantly produced and dispersed by wind (weber 2003). it is native to north america and was introduced to europe as an ornamental plant (weber 1998). its earliest record dates from 1645 in england (kowarik 2003). because the species is attractive and easy to grow, it was widely used by gardeners. the fi rst observations of wild populations in europe dates to around 1850 (wagenitz, 1964), and the plant spread rapidly until 1950 (weber 1998). the species soon extended its range in europe (weber 2000). nowadays it is present over most of europe with the status of invasive alien plant (parker 2008). in its native range the species is often a weedy component of vegetation in abandoned pastures and roadsides, in abandoned fi elds, grasslands, forest edges and human disturbed habitats in urban areas and settlements (walck et al. 1999). in its alien range it inhabits grasslands, forest edges, riparian habitats, disturbed sites (weber 2003). voucher specimen: montenegro, village vir, nikšić municipality n42°50’11”, e18°55’41”, 645 m a.s.l., roadside vegetation (d. stešević, 04.09.2014., tgu-560415). in order to defi ne the status of these fi ve new alien species in montenegro, monitoring is needed, land thus we propose the inclusion of the species on the national list of indicators in environmental protection (b05). particular attention should be paid to species with an invasive potential: solidago canadensis, ipomoea indica and physalis angulata. acknowledgements authors would like to thank f. essl, b. beckmann, j., jogan, m, lešnik, v. vladimirov, g. fried, f. verloove, m. rat, v. matevski for valuable help with the literature and information on alien plant species and snežana bulajić for improving our use of english. references acta plantarum, 2007, scheda ipfi, acta plantarum. retrieved december 26, 2014 from http://www.actaplantarum.org/fl ora/ fl ora_info.php?id=4188 alanen, a., bongard, t., einarsson, e., hansen, h., hedlund, l., jansson, k., josefsson, m., philipp, m., sandlund, o.t., svart, a.e., svart, h.e., weidema, i., 2004: introduced species in the nordic countries (denmark) under nordic council of ministers (nmr), subgroup naturog friluftslivs gruppen, copenhagen. anastasiu, p., negrean, g., samoilǎ, c., memedemin, d., cogalniceanu, da., 2011: a comparative analysis of alien plant species along the romanian black sea coastal area. the role of harbours. journal of coastal conservation 15, 595–606. anačkov, g. t., rat, m. m., radak, b. d., igić, r. s., vukov, d. m., rućando, m. m., krstivojević, m. m., radulović, s. b., cvijanović, d. l., milić, d. m., panjković, b. i., szabados, k. l., perić, d., kiš, a. m., stojšić, v. r., boža, p. p., 2013: alien invasive neophytes of the southeastern part of the pannonian plain. central european journal of biology 8, 1032–1043. backer, c. a., bakhuizen van den brink, r. c., 1965: flora of java (spermatophytes physalis angulata only). vol. ii. n. v. p. noordhoff, groningen. böhling, n., 2001: physalis angulata l., centaurea cyanus l., phagnalon saxatile (l.) cass., scorzonera judaica eig., juglans regia l. in: greuter, w., raus, t. 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(eds.), flora of new zealand, volume iv naturalised pteridophytes, gymnosperms, dicotyledons, 635–710. botany division, department of scientifi c and industrial research, christchurch. weber, e., 1998: the dynamic of plant invasion: a case of three exotic goldenrod species (solidago l.) in europe. journal of biogeography 25, 147–154. weber, e., 2000: biological fl ora of central europe: solidago altissima l. flora 195, 123–134. weber, e., 2003: invasive plant species of the world a reference guide to environmental weeds. cab international, oxford. yannitsaros, a., 1998: additions to the fl ora of kithira (greece) i. willdenowia 28, 77–94. acta botanica 1-2017 za web.indd 72 acta bot. croat. 76 (1), 2017 acta bot. croat. 76 (1), 72–79, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0053 issn 0365-0588 eissn 1847-8476 chemical composition and antifungal potential of medicinal plants against seedborne mycofl ora of eggplant (solanum melongena l.) bushra ashiq1, sobia chohan1*, rashida perveen1, muhammad abid1 and mirza abid mehmood2 1 department of plant pathology, faculty of agricultural sciences and technology, bahauddin zakariya university, multan, pakistan. 2 department of plant pathology, mian nawaz sharif university of agriculture, multan, pakistan abstract – antifungal activities of medicinal plants were observed against seedborne mycofl ora of eggplant (solanum melongena). the effect of ethanolic leaf extracts of mangifera indica, mentha spicata, citrus limon, eucalyptus camaldulensis against four isolated fungal species including fusarium oxysporum, aspergillus fl avus, rhizopus stolonifer and penicillium digitatum was evaluated at various concentrations, by using the poisoned food technique. the impact of the extracts on seed germination and growth of eggplant was assessed by seed treatment and growth in a greenhouse experiment. total fl avonoids of e. camaldulensis were analyzed through spectrophotometer, using quercetin as a standard. physico-chemical parameters were also determined. antifungal activity showed that maximum inhibition percentage of p. digitatum (67.78%) and f. oxysporum (64.44%) was observed at the highest concentration (80%) of c. limon and e. camaldulensis extracts, respectively, followed by m. spicata extract against a. fl avus (63.33%) and r. stolonifer (52.22%). least inhibition percentage of f. oxysporum, p. digitatum, r. stolonifer and a. fl avus was 6.67, 7.78, 14.44 and 16.67%, respectively, at the lowest (20%) concentration of m. spicata. the greenhouse experiment showed variations in seedling germination and post-germination growth. e. camaldulensis extract showed an increase in percent germination (78.98%) over untreated control (62.83%), root and shoot length and fresh and dry weight of root and shoot with the consequent reduction in disease symptoms. phytochemical analysis depicted the presence of alkaloids, fl avonoids, tannins, saponins in all extracts while steroids and glycosides were absent. a fair amount (10.38 mg qe g–1df) of fl avonoid was present in leaf extract of e. camaldulensis. physico-chemical analysis showed ph of 4.6, ash content of 0.41% and weight loss on drying of 8.14%. keywords: medicinal plants, physico-chemical parameters, secondary metabolites, seedborne mycofl ora, solanum melongena * corresponding author, e-mail:sobia_mustafa2006@hotmail.com introduction eggplant, also commonly known as brinjal or aubergine (solanum melongena l.), is an important solanaceous vegetable crop of the sub-tropics and tropics and is cultivated for its fl eshy fruits (pandey 2010). it was later domesticated in india and is grown widely in pakistan, india, bangladesh, china and the philippines. in pakistan it is grown for local consumption with an annual production of 88148 tones over an area of 8673 hectares (gop 2009). this important summer crop is prone to numerous diseases and pests during various stages of crop growth in all seasons in most of the tropical zones (ali et al. 2012). the severity in any serious disease depends on the season and the region in which the crop is grown (dhamdhere et al. 1995). seedborne diseases are considered important because they bring about a signifi cant reduction in yield. different microorganisms affect the quality of seed by reducing its vigor, ultimately weakening the plant at its initial growth stage causing seed rot, preand post-germination death of seedlings (ellis et al. 1975). pathogenic fungi are the largest group of plant pathogens. seed-borne diseases caused by fungi are relatively diffi cult to control. a primary inoculum can be avoided by using disease-free seed, choosing pathogen-free seedbed soil, treating infected seed and following a three year rotation. however, some pre-emptive measures in selection of seed such as color and texture can be helpful in avoiding future losses. healthy seed is always recommended for plantation, to save time and labor. however, if seed is not disease free, it can be treated with pesticides or with hot antifungal activity of medicinal plants acta bot. croat. 76 (1), 2017 73 water at 50 °c (panwar and chand 1970). al-kassimand monawar (2000) identifi ed more than 30 fungal species belonging to 19 genera on eggplant seeds. the most prevalent seed borne fungi of brinjal were aspergillus niger, a. fl avus, a. clavatus, penicilliumdigitatum, pythium sp., rhizoctonia sp., rhizopusarrhizus, r. stolonifer, alternaria alternate and fusarium oxysporum. large quantities of fungicides have been used all over the world by farmers to control fungal diseases in their fi elds, thus increasing production costs and contaminating food and the environment with toxic substances (bowers and locke 2000). consequently, indiscriminate use of fungicides poses a serious threat to the environment and health. use of bio pesticides gives a hope that synthetic pesticides can be replaced.. these pesticides are made up of natural products, and are easily biodegradable. a potentially useful substitute for expensive and possibly toxic fungicides could befound in plant extracts (joseph et al. 2008). the plant extracts of many higher plants have for centuries been known for their antimicrobial properties. medicinal plants are storehouses of secondary phytomedicines. these compounds have additive or synergistic effects on the physiological processes of single or a multiple targets. ethno-botany surveys all over the world support the medicinal use of these extracts against human and plant pests (shariff et al. 2006, gupta et al. 2008). recently, the antimicrobial activity of some of the higher plant products that are biodegradable and safe for human health has gained the attention of researchers working on plant defenses against microbial ingress (koirala et al. 2005). numerous chemical compounds are present in medicinal plants including saponins, tannins, fl avonoids, alkaloids, terpenoids, steroids, glycosides and proteins. these substances are potent bioactive compounds and can be used for remedial purposes (sofowora 1993). although use of plant extracts is less laborious, more economical and more eco-friendly than that of fungicides, it is still not popular among stakeholders. the aim of this research was to uncover the potentials of these natural occurring chemicals against existing mycofl ora associated with seeds. the present study was undertaken to exploit different plant species that have antifungal properties, primarily under in vitro and greenhouse conditions, to control the seedborne diseases of eggplant as an alternative to chemical fun gicide. materials and methods sample collection eggplant seeds were obtained from the vegetable section of ayub agriculture research institute, faisalabad and preserved in brown paper bags in the laboratory of plant pathology at bahauddin zakariya university (bzu) multan, pakistan, until further investigation. detection and isolation of fungi on seeds two hundred seeds were taken at random, surface sterilized with 1% (v/v) sodium hypochlorite solution and subjected to the standard blotter paper method for isolations of fungal mycofl ora (ista1993). ten seeds were placed on three-layered moistened blotter paper in 9cm diameter petri plates. after one week of incubation, fungal colonies emerging around the seeds were isolated by the single-hyphal isolation technique on potato dextrose agar (pda) medium and incubated under a 12 h photoperiod at 27 °c for seven days for further identifi cation and purifi cation. the fungi were identifi ed according to the morphological characters and relevant literature (barnet and hunter 1972, ellis et al. 1975, nelson et al. 1983). pathogenicity was confi rmed according to koch’s postulates. preparation of plant extracts a previously described method (neycee 2012) was used for extraction procedure. fresh healthy leaves of indigenous medicinal plants: mangifera indica, citrus limon, eucalyptus camaldulensis and mentha spicata were collected in the premises of bahauddin zakariya university, pakistan. samples were thoroughly washed with tap water, followed by distilled water and fi nally with 70% ethanol (merck) to eliminate any traces of contaminants. blot dried leaves of each sample were then dried in the oven at 50 °c for 2 h, later shade dried and homogenized to fi ne powder and stored in airtight bottles. 10 g of powdered material was extracted using soxhlet apparatus (j. p. selecta-spain) with 100 ml ethanol for 48 h. the extract solutions were centrifuged at 6000 rpm for 10 min, fi ltered with fi lter paper (whatman no.1) and concentrated over a water bath at 40 °c. after complete solvent evaporation extract residues were sealed in dark bottles at 4 °c for further use. fungal growth assay (in vitro) four out of eight isolated fungal strains including fusarium oxysporum, aspergillus fl avus, rhizopus stolonifer and penicillium digitatum were tested. the comparative toxicity of plant extracts on the growth of fungi was evaluated by the poisoned food technique (nene and thapilyal 2000). prerequisite amounts of different concentrations (20, 30, 40, 60 and 80%) of plant extracts were incorporated aseptically into potato dextrose agar medium for inoculation of test fungi in 9 cm sterilized petri plates. medium without any plant extract served as a control. a mycelial disk (0.6 cm) of test pathogens was placed at the center of each petri plate and incubated for 7 days at 27 °c under a 12 h photoperiod. the experiment was carried out in triplicate. mycelial growth (cm) of fungus was measured after 7 days of incubation. the inhibition percentage (p) was calculated according to the equation of singhand tripathi (1999) where dc denotes average increase in mycelial growth in control and dt denotes average increase in mycelial growth in treatment: p=(dc–dt)/dc×100 greenhouse experiment fresh plant extracts were prepared by the method described above with distilled water (50:50 w/v) and fi ltered ashiq b., chohan s., perveen r., abid m. and mehmood m. a. 74 acta bot. croat. 76 (1), 2017 through cheese cloth. further, these extracts were diluted with distilled water to obtain an 80% concentration. four hundred eggplant seeds per treatment were immersed in each extract solution for 30 min (ista 1996). then the excess extract was drained off and seeds were blot dried and kept in the open air. the pots were arranged in a randomized complete block design with four replications. the seed rate was 10 seeds/ pot in each replication. data regarding germination percentage, seedling height, shoot length, root length, fresh shoot weight, fresh root weight, dry root weight, dry shoot weight and average biomass parameters were recorded. germination percentage was observed at 18 days after sowing (das) while other growth characters were recorded at 35 das. phytochemical analysis plant extract were subjected to qualitative phytochemical screening using the methods of sofowora (1993) and harborne (1973). for analysis of alkaloids (wagner’s test), a 20 mg of ethanolic extract was warmed with 2% sulphuric acid (h2so4) for 1–2 min, fi ltered and treated with a few drops of wagner’s reagent. presence of reddish brown precipitation or turbidity indicated the presence of alkaloids. for tannins (ferric chloride test) a 20 mg plant extract was dissolved in ethanol, a few drops of 0.1% ferric chloride were added and the extract was observed for the formation of blue black coloration. for steroids, a 2 ml of acetic anhydride and concentrated h2so4 were added to 50 mg ethanolic plant extract. a blue green ring indicates the presence of steroids. for terpenoids (salkowski test), a 5 ml extract of each sample was mixed in chloroform (2 ml) and h2so4 (3 ml) was added carefully to form a layer. formation of reddish brown coloration at the interface indicates the presence of terpenoids. for analysis of saponins a 20 mg powdered sample was boiled in 5 ml distilled water and shaken vigorously for a stable persistent froth. three drops of olive oil were mixed vigorously with the frothing and observed for the formation of emulsion. for fl avonoids analysis, a powdered sample (20 mg) was heated with 10 ml of ethyl acetate for 3 min and fi ltered. the fi ltrate (4 ml) was mixed with 1 ml of dilute ammonia solution. a yellow coloration that disappears on addition of concentrated hcl indicated the presence of fl avonoids. estimation of total fl avonoid contents total fl avonoid contents were determined following the procedure of chang et al. (2002) with a slight modifi cation. quercetin was used as a standard to make the calibration curve. plant extract (0.5 ml) was mixed with 1.5 ml methanol, 10% (w/v) aluminum chloride (0.1 ml), 1 m potassium acetate (0.1 ml) and distilled water (2.8 ml). it was kept at room temperature for 30 min. absorbance was measured at 415 nm by spectrophotometer (uv-3000).the calibration curve was prepared using quercetin standard solutions of 25, 50 and 100 mg l–1 in ethanol. total fl avonoid values are expressed in terms of mg quercetin equivalent per g of dried fraction (mg qe g–1df). the experiment was run in triplicate. physico-chemical analysis total ash contents, ph values and weight loss on drying were determined for ethanolic extract of e. camaldulensis, following the methods of vaghasiya et al. (2008). statistical analysis the data obtained were subjected to analysis of variance (anova) and treatment means were compared with fisher’s least signifi cance difference (lsd) test at 5% level of signifi cance (steel and torrie 1980) by using the statistical software sigma plot 11. results eight fungal species belonging to 5 genera were isolated by the standard blotter method from seeds of eggplant. the highest percentage frequency was recorded by penicillium digitatum (49.67%) followed by aspergillus fl avus, fusarium oxysporum, rhizopus stolonifer,alternaria alternata, curvularia lunata, aspergillus niger and fusarium solani with 47.33, 38.00, 33.00, 15.00, 13.67, 8.33 and 5.67% recovery percentage, respectively (fig. 1). out of recovered fungi, c. lunata, a. alternata, a. niger and f. solani were found in minimum incidence percentages, hence only predominant fungi, viz. f. oxysporum, a. fl avus, p. digitatum and r. stolonifer were purifi ed and subjected to antifungal assay with four plant extracts at fi ve different concentrations (20, 30, 40, 60 and 80%). all plant extracts revealed varied inhibitory effects on the mycelial growth of test fungi, by affecting their normal growth. the data regarding mean mycelial growth inhibition of four fungal species and ethanolic leaf extract of four extracts with different concentrations was statistically signifi cant (fig. 2). extract of m. spicata at 20% concentration was found less effective against a. fl avus, r. stolonifer, p. digitatum and f. oxysporum with 7.5, 7.7, 8.3 and 8.4 cm growth, respectively. at 30% concentration, mycelial growth of p. digitatum (7.9 cm), f. oxysporum (7.7 cm), a. fl avus and r. stolonifer (6.8 cm) was observed. less mycefig.1. recovery percentage (%±sd) of fungi isolated from eggplant seeds by using the standard blotter method. antifungal activity of medicinal plants acta bot. croat. 76 (1), 2017 75 lial growth, of 5.8 and 5.9 cm, was found for a. fl avus and r. stolonifer, while more growth, of 7.2 and 7.5 cm, was observed for f. oxysporum and p. digitatum respectively at 40% concentration of m. spicata extract. maximum mycelial growth at 60% concentration of m. spicata extract was of f. oxysporum (6.7 cm) and p. digitatum (6.6 cm) and minimum growth was of r. stolonifer (5.1 cm) and a. fl avus (4.7 cm). at 80% concentration of m. spicata extract, the mycelial growth of a. fl avus was best inhibited with only 3.3 cm growth followed by r. stolonifer (4.3 cm), p. digitatum (6.1 cm) and f. oxysporum (6.4 cm) compared to control fungi with 9.0 cm growth (fig. 2a). at 20% concentration of c. limon, the diagonal growth of f. oxysporum (7.9 cm), a. fl avus (7.3 cm), p. digitatum (7.1 cm) and r. stolonifer (6.9 cm) was observed. r. stolonifer, p.digitatum, a. fl avus and f. oxysporum showed 6.2, 6.4, 6.6 and 7.5 cm mycelial growth respectively at 30% concentration of c. limon extract. the minimum growth (5.7 cm) was of p. digitatum after r. stolonifer (5.8 cm), a. fl avus (6.1 cm) and f. oxysporum (7.1 cm) at 40% concentration of c. limon extract. maximum mycelial growth (6.3 cm) was of f. oxysporum followed by a. fl avus (5.5 cm), r. stolonifer (4.9 cm) and p. digitatum (4.1 cm) at 60% concentration of c. limon extract. on the other hand, 80% concentration of c. limon extract was found to be the most effective and illustrated maximum growth inhibition of p. digitatum (2.9 cm) and minimum inhibition in r. stolonifer, a. fl avus, f. oxysporum, with 4.7, 5.2 and 5.9 cm growth respectively compared to control fungi with 9 cm growth (fig. 2b). the mycelial growth of p. digitatum, f. oxysporum, r. stolonifer and a. fl avus on agar plate containing different concentrations of ethanolic leaf extract of e. camaldulensis was highest with 7.9, 7.8, 7.7 and 7.4 cm at 20% concentration. at 30% concentration, the growth of p. digitatum and r. stolonifer was 7.3 and 7.4, whereas in f. oxysporum and a. fl avus it was 6.9 and 6.6 cm respectively. maximum mycelial growth (6.9 cm) was of r. stolonifer followed by p. digitatum (6.4 cm), a. fl avus (5.8 cm) and f. oxysporum (5.4 cm) at 40% concentration of e. camaldulensis extract. mycelial growth of f. oxysporum (4.3 cm), a. fl avus (4.9 cm), p. digitatum (5.1 cm) and r. stolonifer (5.8 cm) decreased at 60% concentration e. camaldulensis extract. minimum mycelial growth of 3.2, 3.6, 3.9 and 4.3 cm was observed in f. oxysporum, p. digitatum, a. fl avus and r. stolonifer respectively, at 80% concentration of e. camaldulensis extract compared to control fungi with 9 cm growth (fig. 2c). at 20% concentration of ethanolic leaf extract of m. indica, mycelial growth of f. oxysporum (8.3 cm) was greater than in p. digitatum (8.1 cm), r. stolonifer (7.8 cm) and a. fl avus (7.4 cm). at a 30% concentration, the mycelial growths of a. fl avus (6.9 cm), r. stolonifer (7.2 cm), p. digitatum (7.6 cm) and f. oxysporum (7.7 cm) were observed. growth of mycelia was 6.4, 6.7, 6.8 and 7 cm in a. fl avus, r. stolonifer, p. digitatum and f. oxysporum respectively at a 40% concentration of m. indica extract. at a 60% concentration, maximum growth (6.6 cm) was seen for f. oxysporum followed by r. stolonifer (6.3 cm), p. digitatum (6.2 cm) and a. fl avus (5.7 cm). minimum mycelial growth (4.8 fig. 2. mycelial growth (mean±se) of four tested fungi after using different concentrations of ethanolic leaf extract of four plants: a) mentha spicata, b) citrus limon, c) eucalyptus camaldulensis, d) mangifera indica. ashiq b., chohan s., perveen r., abid m. and mehmood m. a. 76 acta bot. croat. 76 (1), 2017 cm) was observed in a. fl avus after r. stolonifer (5.4 cm), p. digitatum (5.7 cm) and f. oxysporum (6.1 cm) at 80% concentration of m. indica extract, compared to control fungi with 9 cm growth (fig. 2d). m. indica at 20% concentration showed minimum mycelial growth inhibition (13.33%) while maximum inhibition (52.22%) was observed for m. spicata and e. camaldulensis extracts at 80% concentration against r. stolonifer. growth of a. fl avus was inhibited by 16.67% and 63.33% at 20% and 80% concentration of m. spicata extract, respectively. mean mycelial growth inhibition of f. oxysporum was lower (6.67%) at 20% concentration but maximum inhi bition of 64.44% was observed at 80% concentration of e. camaldulensis. a maximum inhibition percentage (67.78%) of p. digitatum was observed at 80% concentration of c. limon with, while a minimum inhibition (7.78%) was observed at a 20% concentration of m. spicata extract (fig 3a–d). results of variance analysis for the germination experiment showed that germination percentage and growth characters of eggplant were statistically signifi cant except for dry root weight. maximum germination was observed after treatment with e. camaldulensis extract (78.98%) followed by extracts of c. limon (73.05%), m. spicata (71.00%) and m. indica (64.10%), and control untreated plants (62.83%), respectively. maximum seedling height was observed in seeds treated with e. camaldulensis (18.57 cm) and c. limon extracts (18.27 cm) and a minimum in control plants (12.87 cm). shoot length was greater (15.25 cm) after treatment with e. camaldulensis extract than in control plants table 1. effect of different plant extracts on eggplant seed germination and growth parameters. means sharing different letters are statistically different at 5% level of signifi cance. values are means of four replications. t1 – eucalyptus camaldulensis, t2 – mangifera indica, t3 – citrus limon, t4 – mentha spicata, t5 – negative control; lsd – least signifi cant difference. treatments germination (%) seedling height (cm) shoot length (cm) root length (cm) fresh shoot weight (g) fresh root weight (g) dry shoot weight (g) dry root weight (g) biomass (g) t1 78.98 a 18.575 a 15.25 a 4.05 a 0.825 a 0.0723 a 0.0805 a 0.0080 a 1.015 a t2 64.10 c 14.175 c 11.95 bc 2.73 c 0.550 c 0.0463 d 0.0588 d 0.0053 a 0.685 cd t3 73.05 b 18.275 a 14.85 a 3.85 ab 0.900 ab 0.0660 b 0.0730 b 0.0078 a 0.898 b t4 71.00 b 16.125 b 12.90 b 3.40 b 0.750 abc 0.0593 c 0.0660 c 0.0065 a 0.738 c t5 62.83 c 12.875 d 11.53 c 2.80 c 0.625 bc 0.0478 d 0.0565 d 0.0070 a 0.648 d lsd p=0.05 2.129 1.113 1.323 0.514 0.205 0.005 0.006 0.0028 0.075 fig. 3. mycelial inhibition (mean±se) by four different plant extracts at different concentrations: a) mentha spicata, b) citrus limon, c) eucalyptus camaldulensis, d) mangifera indica. antifungal activity of medicinal plants acta bot. croat. 76 (1), 2017 77 (11.53 cm). minimum root length was observed in seeds treated with m. indica extract (2.73 cm) and maximum in seeds treated with e. camaldulensis extract (4.05 cm). fresh shoot weight was greater after seed treatment with c. limon extract (0.90 g) than after treatment with m. indica extract (0.55 g). highest fresh root weight was after seed treatment with e. camaldulensis extract (0.072 g) and the lowest after treatment with m. indica extract (0.046 g). greatest dry shoot weight and biomass was observed in seeds treated with e. camaldulensis (0.08 g and 1.015 g) and lowest in control (0.05 g and 0.64 g), respectively (tab. 1). ethanol leaf extract of e. camaldulensis was found rich in fl avonoids, alkaloids, tannins and saponins while steroids and glycosides were absent. quantitative analysis of ethanol extract resulted in 10.38 mg qe g–1 fl avonoid contents in eucalyptus leaves. the results of physico-chemical analysis revealed ph value of 4.6, ash content of 0.41% and weight loss on drying of 8.14%. discussion seedborne microorganisms play an important role in affecting the quality of seed, causing signifi cant crop losses. predominant fungi associated with seed samples of eggplant were identifi ed as fusarium oxysporum, aspergillus fl avus, penicillium digitatum and rhizopus stolonifer, causing quantitative and qualitative losses to seeds during storage (neergard 1977). opportunistic pathogens such as aspergillus sp. and penicillum sp. were found associated with seed infection and caused severe damage to both quality and quantity of seed production (kumar et al. 2005). traditionally, seeds have been treated with fungicides to kill microorganisms. however, the world health organization (who) banned many agriculturally important pesticides due to their toxicity against non-target organisms including humans, and because they are known to cause pollution problem (barnard et al. 1977). many plant extracts and oils, such as tea tree and clove had been used as contemporary antiseptics, or had been reported to have antimicrobial properties (hoffman 1987, rice 2012). extracts from different plants have been reported to have antifungal, antibacterial and antioxidant activities. the antifungal activity of diverse plants against different fungi has been reported (farrag and moharam 2012, vidua-martos et al. 2008). results in the present study showed that four fungal species were isolated from eggplant seeds. their growth was best during february, march and confi rms the results of pandey (2010) who detected fi ve fungi on brinjal viz. f. solani, helminthosporium spiciferum, phomopsis vexan, c. lunata, trichothecium roseum. the growth of these fungi was particularly luxurious from october to november, and february to april. different concentrations of different plant extracts exhibited different activities. in this study fi ve extract concentrations (20, 30, 40, 60 and 80%) were used against test fungi and illustrated varying results. some concentrations were weak while few were effective in controlling mycelial growth of test fungi. similarly, joseph et al. (2008) used different concentrations, i.e., 5, 10, 15 and 20% of plant extracts. among the different extracts 20% of azardiachta indica was found most effective followed by rheum emodi, eucalyptus globulus, artemessia annua and ocimum sanctum. in our study, in the case of r. stolonifer and a. fl avus the best inhibition was exhibited by e. camaldulensis and m. spicata extracts, respectively, at 80% concentration. similar results were also reported by different scientists at different times (satish et al. 2007). on the other hand, tzortzakis and economakis (2007) found lemongrass (cympopogon citratus) extract to be effective against r. stolonifer and a. niger. in our study, f. oxysporum was best inhibited by e. camaldulensis extract at 80% concentration. the results correlated with previous fi ndings that aqueous leaf extracts of e. citriodora, m. indica, accacia nilotica, a. indica and syzygium cumini signifi cantly reduced the incidence of the two most frequent seed-borne fungi, viz., f. solani, and a. alternata (shafi que et al. 2007, marzoug et al. 2011). p. digitatum was best inhibited at higher concentration by c. limon extract in our study. these fi ndings are in line with the results of kanan et al. (2008), who evaluated the effect of different plant extracts and liquid fractions against citrus post-harvest disease agent p. digitatum. similarly, ragab et al. (2012) found that mint, thyme, peppermint and clove had signifi cant inhibitory effects on fungal mycelia. fungal mycelial growth decreased signifi cantly as the concentrations of extracts increased. mango leaf extract moderately reduce the growth of pathogenic fungi. in the same way suvarna and patil (2009) showed that extract of m. indica had moderate activity against the human pathogenic fungi candida albicans. seed germination and other growth parameters of eggplant were greater in seeds treated with e. camaldulensis extract as compared to control treatment. conversely e. camaldulensis signifi cantly reduced seed germination of sorghum (mohamadi and rajaie 2009). m. indica extract was found least effective among all the extracts used. present results are in harmony with the fi ndings of kumar et al. (2005) and alberts et al. (2006). they reported that preand post-harvest bio-deterioration of crop seeds are mainly due to seed infestation by microorganisms and insects, causing up to 100% losses. contrary to our results, leaf extract of moringa oleifera increased seed germination of eggplant up to 92% over control treatment (kuri et al. 2011). e. camaldulensis extract was found most effective in reducing the fungal growth of seedborne mycofl ora, eventually increasing the germination of eggplant seed. similarly e. camaldulensis exhibited pronounced antifungal activity, among different species of eucalyptus (may and ash 1990, rukhsana. 2005, babayi et al. 2004, ghalem and mohamed 2008) and this might be due to the presence of secondary compounds. thus the phytochemical and physico-chemical parameters of e. camaldulensis were studied. phytochemical screening of leaf extract of e. camaldulensis extract revealed the presence of tannins, fl avonoid, alkaloids and saponins, while steroids and glycosides were absent, and these results are similar to earlier fi ndings (vaghasiya et al. 2008). ashiq b., chohan s., perveen r., abid m. and mehmood m. a. 78 acta bot. croat. 76 (1), 2017 references abd-alla, m. f., el-negoumy, s. i., el-lakany, m. h., saleh, n. a. m., 1980: flavonoid glycosides and the chemosystematics of eucalyptus camaldulensis. phytochemistry 19, 2629–2632. alberts, j. f., engelbrecht, y., steyn, p. s., holzapfel, w. h., vanzyl, w. h., 2006: biological degradation of afl atoxin b1 by rhodococcus erythropolis cultures. international journal of food microbiology 109, 121–126. ali, m., ashfaq, m., akram, w., sahi, s. t., ali, a., 2012: the physiomorphic characters of the brinjal (solanum melongena l.) plant and their relationship with the jassid (amrasca bigutulla) population fl uctuation. pakistan journal of agricultural science 49, 67–71. al-kassim, m. y., monawar, m. n., 2000: seed-borne fungi of some vegetable seeds in gazan province and their chemical control. saudi journal 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(m.p.). indian journal of entomological research 19, 71–77. el-ghorab, a. h., el-massry, k. f., marx, f., fadel, h. m., 2003: antioxidant activity of eucalyptus camaldulensis var. brevirotsris leaf extracts. food/nahrung 47, 41–45. ellis, m. a., ilyas, m. b., sincair, j. b., 1975: effect of three fungicides on internally seedbornefungi and germination of soybean seeds. phytopathology 65, 553–556. farrag, e. s., moharam, m. h., 2012: pathogenic fungi transmitted through cucumber seeds and safely elimination by application of peppermint extract and oil. notulae scientia biologicae 4, 83–91. ghalem, b. r., mohamed, b., 2008: antibacterial activity of leaf essential oils of eucalyptus globulus and eucalyptus camaldulensis. african journal of pharmacy and pharmacology 2, 211–215. gharekhani, m., ghorbani, m., rasoulnejad, n., 2012: microwave-assisted extraction of phenolic and fl avonoid compounds from eucalyptus camaldulensis dehn leaves as compared with ultrasound-assisted extraction. latin american applied research 42, 305–310. government of pakistan, gop, 2009: fruits vegetables and condiments statistics of pakistan. ministry of food and agriculture (economic wing) islamabad. pp. 11–12. gupta, c., garg, a.p., uniyal, r. c., kumari, a., 2008: antimicrobial activity of some herbal soils against common food borne pathogens. african journal of microbiology research 2, 254– 261. harborne, j. b., 1973: phytochemical methods: a guide to modern techniques of plant analysis. chapman and hall ltd, london. hoffman, d. l., 1987: the herb user’s guide. thorsons publishing group, wellingborough, uk. ista, 1993: international rules for seed treating. seed science and technology 21, 160–186. ista, 1996: international rules for seed testing. international seed testing association, switzerland. joseph, b., muzafar, a., vinod, k., 2008: bioeffi cacy of plant extracts to control fusarium solani f. sp. melongenae incitant of brinjal wilt. global journal of biotechnology and biochemistry 3, 56–59. kanan, g. j., rasha, a., al-najar., 2008: in vitro antifungal activities of various plant crude extracts and fractions against citrus post-harvest disease agent penicillium digitatum. jordan journal of biological science 22, 89–99. koirala, p., kumar, s., yadar, b. k., premarajan, k. c., 2005: occurrence of afl atoxin in some of the food and feed in nepal. indian journal of medical sciences 59, 331–336. kumar, r. s., sivakumar, t., sunderam, r. s., 2005: antioxidant and antimicrobial activity of bauhinia racemosa l. stem bark. brazilian journal of medical and biological research 38, 1015–1024. a good amount of fl avonoids were also detected in leaves of e. camaldulensis, confi rming their role in plant growth and defense against infections (abd-alla et al. 1980, larson 1998, pourmorad et al. 2006). the present results are in harmony with previous researchers who also extracted higher contents of fl avonoids from leaves of e. camaldulensis in ethanol solvent (el-ghorab et al. 2003, gharekhani et al. 2012). according to takahashi et al. (2004) three fl avonoids (2’,6’-dihydroxy-3’-methyl-4’-methoxy-dihydro chalcone,8-desmethyl-eucalyptin and eucalyptin) isolated from eucalyptus maculata leaf extracts commonly exhibited potent antimicrobial activities against seven micro-organisms causing food poisoning. the physico-chemical analysis showed that the compounds present in e. camaldulensis were acidic in nature. these results were correlated with previous results obtained by vaghasiya et al. (2008) when analyzing ph, ash values and loss on drying. conclusion it was presumed that different plant extracts performed differently at different concentrations and exhibited signifi cant antifungal activity against different pathogenic fungi. the antifungal effect exhibited by the selected plants might be credited to the existence of either single or synergistic consequences of more than one compound. the results of the present study can be further applied to the formulation of an integrated disease management program for eggplant seedborne mycofl ora. more investigations are needed to examine the separation and depiction of antifungal moieties and acclamation in fi eld applications. acknowledgments we are very thankful to seed certifi cation department, islamabad for providing seeds of eggplant. antifungal activity of medicinal plants acta bot. croat. 76 (1), 2017 79 kuri, s. k., islam, m. r., mondal, u., 2011: antifungal potentiality of some botanical extracts against important seedborne fungal pathogen associated with brinjal seeds, solanum melongena l. journal of agricultural technology 7, 1139–1153. larson, r. a., 1988: the antioxidants of higher plants. phytochemistry 27, 969–978. marzoug, h. n. b., romdhane, m., lebrihi, a., mathieu, f., couderc, f., abderraba, m., khouja, m.l., bouajila, j., 2011: eucalyptus oleosa essential oils: chemical composition and antimicrobial and antioxidant activities of the oils from different plant parts (stems, leaves, fl owers and fruits). molecules 16, 1695–1709. may, f. e., ash, j. e., 1990: an assessment of the allelopathic potential of eucalyptus. australian journal of botany 38, 245– 254. mohamadi, n., rajaie, p., 2009: effect of aqueous eucalyptus (e. camaldulensis labill) extracts on seed germination, seedling growth and physiological responses of phaseolus vulgaris and sorghum bicolor. research journal of biological sciences 4, 1291–1296. neergard, p., 1977: seed pathology. new york: john wiley. nelson, p. e., toussoun, t. a., marasas, w. f. o., 1983: fusarium species – an illustrated manual for fusarium research. the pennsylvania state university press, university park and london. nene, y., thapilyal, l., 2000: poisoned food technique of fungicides in plant disease control. 3rd edn, oxford and ibh publishing company, new delhi. neycee, m. a., nematzadeh, g. h. a., dehestani, a., alavi, m., 2012: assessment of antifungal effects of shoot extracts in chinaberry (melia azedarach) against 5 phytopathogenic fungi. international journal of agriculture and crop science 4, 474–477. pandey, a., 2010: studies on fungal diseases of eggplant in relation to statistical analysis and making of a disease calendar. recent research in science and technology 2, 1–3. panwar, n. s., chand, j. n.,1970: phomopsis fruit rot of brinjal (solanum melongena) in the punjab. viability of fungus and role of seeds in disease development. journal of researchpunjab agricultural university 7, 641–643. pourmorad, f., hosseinmehr, s. j., shanabimajd, n., 2006: antioxidant activity, phenol and fl avonoid contents of some selected iranian medicinal plants. african journal of biotechnology 5, 1142–1145. ragab, m. m., ashour, a. m. a., abdel-kader, m. m., el-mohamady, r., 2012: in vitro evaluation of some fungicides alternatives against fusarium oxysporum the causal of wilt disease of pepper (capsicum annum l.). international journal of agriculture forestry 2, 70–77. rice, e. l., 2012: allelopathy. academic press. rukhsana, b., 2005: antifungal activity of allelopathic plant extracts vi: in vitro control of fungal pathogen by aqueous leaf extracts of eucalyptus. mycopath 3, 7–12. satish, s., mohana, d.c., ranhavendra, m.p., raveesha, k.a., 2007: antifungal activity of some plant extracts against important seed borne pathogens of aspergillus sp. journal of agricultural technology 3, 109–119. shafi que, s., javaid, a., bajwa, r., 2007: effect of aqueous leaf extracts of allelopathic trees on germination and seed borne mycofl ora of wheat. pakistan journal of botany 39, 2619– 2624. shariff, n., sudarshana, m. s., umesha, s., hariprasad, p., 2006: antimicrobial activity of rauvolfi a etraphylla and physalis minima leaf and callus extracts. african journal of biotechnology 5, 946–950. singh, j., tripathi, n. n., 1999: inhibition of storage fungi of blackgram (vigna mungo) by some essential oils. flavor and fragrance journal 14, 1–4. sofowora, a., 1993: medicinal plants and traditional medicine in africa. chichester john, willey and sons, new york, p. 256. steel, r. g. d., torrie, j. h., 1980: principles and procedure of statistics approach. 2ndedn, mcgraw hill company, new york. suvarna, v., patil, s., 2009: antifungal activity of selected plant extracts against human fungal pathogens. journal of herbal medicine and toxicology 3, 151–153. takahashi, t., kokubo, r., sakaino, m., 2004: antimicrobial activities of eucalyptus leaf extracts and fl avonoids from eucalyptus maculata. letters in applied microbiology 39, 60–64. tzortzakis, n. g., economakis, c. d., 2007: antifungal activity of lemongrass (cymbopogon citratus l.) essential oil against key postharvest pathogens. innovative food science and emerging technologies 8, 253–258. vaghasiya, y., nair, r., chanda, s., 2008: antibacterial and preliminary phytochemical and physico-chemical analysis of eucalyptus citriodora hk leaf. natural product research 22, 754–762. viuda-martos, m., ruiz-navajas, y., fernandez-lopez, j., perezálvarez, j., 2008: antifungal activity of lemon (citrus lemon l.), mandarin (citrus reticulata l.), grapefruit (citrus paradisi l.) and orange (citrus sinensis l.) essential oils. food control 19, 1130–1138. opce-str.vp acta bot. croat. 69 (1), 1–6, 2010 coden: abcra 25 issn 0365–0588 orobanche pseudorosmarina a. pujadas et muñoz garm. sp. nov. (orobanchaceae) from the eastern mediterranean region antonio j. pujadas salvà1, jose félix muñoz garmendia2 1 departamento de ciencias y recursos agrícolas y forestales, campus de rabanales, universidad de córdoba, e-14071 córdoba, spain. 2 real jardín botánico de madrid, csic, plaza de murillo 2, e-28014 madrid, spain. the analysed plants from dalmatia (croatia) identified by beck as orobanche rosmarina beck do not fit the lectotype for orobanche rosmarina originally used by foley [bm 574992]. as a consequence, these orobanche specimens from the eastern mediterranean region remain unnamed, but are described here as a new species orobanche pseudorosmarina a. pujadas et muñoz garm. keywords: nomenclature, orobanche pseudorosmarina, typification, croatia, dalmatia, mediterranean. introduction the name orobanche rosmarina beck [subgen. phelipanche (pomel) tzvelev=sect. trionychon wallr.] was first proposed at the specific rank in a publication on the flora of south dalmatia (croatia) by ginzberger (1921: 243) where he wrote »orobanche rosmarina (welw.) beck« and, in the footnote, »=o. muteli schltz. var. stenosiphon beck«. also, in his preparatory account of the genus orobanche for flora iberica, foley (2001a: 231–232) lectotypified orobanche rosmarina beck based on a sheet of welwitsch’s exsiccata deposited in bm: »flora lusitanica, sect. ii (da). no. 779. trionychium rosmarinum [sic.] nov. sp. s. de arrabida. annis 1848–50 leg. dr. welwitsch« (bm 574992) (fig. 1). the problem arose when the characters of the croatian and portuguese specimens were found not to coincide, so the plants could not belong to the same taxon. thus, while examining the lectotype of foley (bm 574992) and the iberian specimens which should seemingly be given its name, we found their morphological features to depart from the description and illustration of beck (1890: 96, tab. 1, fig. 13) for his o. mutelii var. stenosiphon. also, beck’s description and illustration differ from those by foley (2001b: 40–41). thus, beck (1890: 96) described var. stenosiphon as possessing some differential characters: »spicis brevibus, densifloris, in apice rotundato-obtusis; floribus …valde pronus curvatis, subhorizontaliter patentibus,…«; also, in his iconography, beck (1890: acta bot. croat. 69 (1), 2010 1 * corresponding author: cr1pusaa@uco.es u:\acta botanica\acta-botan 1-10\pujadas salva.vp 9. travanj 2010 10:32:41 color profile: disabled composite 150 lpi at 45 degrees tab. 1, fig. 13) depicted a flower with linear lanceolate bracteoles, a calyx with long acuminate narrow triangular teeth and a densely pubescent patent corolla. in the lectotype (bm 574992), however, the spike was pauciflora and bore only three flowers rather than being densiflora; bracteoles were lanceolate rather than linear lanceolate; calyx teeth were triangular rather than narrow triangular long acuminate; corollas were erect to erecto–patent rather than subhorizontally patent or sharply inflected patent; and the corolla had scarce short glandular hairs not greater than 0.2 mm in size rather than being densely pubescent as in beck’s icon (beck 1890: pl. 1, fig. 13). the lectotype characters (bm 574992) are consistent with those originally reported by foley (2001a: 232; 2001b: 40). therefore, our morphological observations reveal that the lectotype proposed by foley differs markedly from the description provided by beck (1890: 96). although we must concede that, most probably, beck used plants from istria or dalmatia to describe the variety stenosiphon – he wrote »o. stenosiphon imprimis in istria et dalmatia observatur« – (beck 1890: 96), the welwitsch exsiccata specimen was the only one cited in the protologue and was thus the sole syntype. foley had therefore no choice under art. 9.10 (mcneill et al. 2006) but to select a specimen of this exsiccata number. results description of the new species the croatian plants that were previously identified as o. rosmarina beck belong to an unnamed taxon other than that of the iberian ones and are described as the following new species here: 2 acta bot. croat. 69 (1), 2010 pujadas salvà a. j., muñoz garmendia j. f. fig. 1. (bm 574992) lectotype for orobanche rosmarina beck u:\acta botanica\acta-botan 1-10\pujadas salva.vp 13. travanj 2010 9:33:13 color profile: disabled composite 150 lpi at 45 degrees orobanche pseudorosmarina a. pujadas et muñoz garm., sp. nov. = o. mutelii var. stenosiphon beck in biblioth. bot. 19: 96, taf. i, fig. 13(1) (1890) p.p. = o. rosmarina beck in ginzberger, oesterr. bot. z. 70 (9/12): 243 (1921) p.p. holotype –reise nach den dalmatinischen inseln. / 15. mai bis 15. juni 1911 / =o. mutelii var. stenosiphon beck [handwritten by beck] / orobanche rosmarina (welw.) g. beck. [handwritten by beck] / (auf rosmarinus officinalis) / insel lissa, umgebung von comisa: schiff am der nordseite der bricht. / 21. mai. / leg. a. ginzberger. a. teyber / (wu) other studied material dalmatia: dalmat: ins. brazza a species nova? (w), hb. portenschlag, [illegible]: 118 b. (wu) [sub o. caerulea, identified by beck as orobanche stenosiphon beck]. scoglie san andrea. […illegible.], 26-v-1876, g. c. spreitzenhofer (wu 7791) [sub phelipaea caerulea c. a. meyer; with a label handwritten by beck: »orobanche muteli schultz / var. o. stenosiphon mihi / beck«]. dalmatien, insel lissa, bei comisa, 23-v1901, a. ginzberger (wu) [identified by beck as orobanche mutelii var. stenosiphon beck]. dalmatien, comisa, insel lissa, 19-v-1905, e. kindt (wu) [identified by beck as orobanche mutelii var. stenosiphon beck]. insel busi, oberhalb porto basi, machie, 20 to 26-v-1911, a. ginzberger and a. teyber (wu) [identified by beck as orobanche rosmarina beck =o. mutelii var. stenosiphon beck]. insel san andrea westl. von lissa, oberhalb porto slatina, 6 to 9-vi-1911, a. ginzberger and a. teyber (wu) [identified by beck as orobanche rosmarina beck =o. mutelii var. stenosiphon beck]. insel san andrea westl. von lissa, […illegible], 6 to 9-vi-1911, a. ginzberger and a. teyber (wu) [identified by beck as orobanche rosmarina beck =o. mutelii var. stenosiphon beck]. we should note that the voucher housed as type material in beck’s herbarium at prc – »o. rosmarina gb [pencil handwriting] / =orob. muteli schultz var. stenosiphon m. [beck’s handwriting] / det. dr. g. beck (prc)« – also belongs to this taxon. however, it has no collection label, which was probably lost at some time. therefore, we believe it is not amenable to typification iconography: beck (1890: tab. 1, fig. 13); fig. 2. diagnosis planta humilis, 9–14 cm alta. caulis gracilis, debilis, 2.5–3.3 mm latus in medio. spica 3.5–5.5 ´ 2.2–2.7 cm, breviter cylindracea, rotundata in apice, subdensa. bracteae 4.5–5.5 mm longae, quam calyx breviores, ovatae, cum pilis glandulosis usque ad 0.4 mm longis. bracteolae 3.5–6 ´ 0.3–0.8 mm, linear–lanceolatae. calyx 6–9 mm longus, triangularibus longiacuminatis dentibus, perspicuis percursis nervis, interdum indumento occultis. corolla 13–16 mm longa, curvata, valde prona, patens ad erecto–patentem; conspique constricta supra insertionem staminum, basi alba, apice in siccitate atroviolaceo; labrorum lobulis obtusis, rotundis; labrorum marginibus breviglanduloso–pilosis, pilis usque ad 0.4 mm longis. filamenta glabra infra, supra pilis glanduliferis perpaucis obsita. antherae 0.9–1.1 mm, ciliis comosis basi, pilis 0.3–0.4 mm longis. germen parce glanduloso–pilosum, subglabrum interdum, pilis usque ad 0.2 mm. stylus parce glanduloso–pilosus, subglabrus interdum, pilis usque ad 0.1 mm longis. acta bot. croat. 69 (1), 2010 3 orobanche pseudorosmarina sp. nov. u:\acta botanica\acta-botan 1-10\pujadas salva.vp 9. travanj 2010 10:32:43 color profile: disabled composite 150 lpi at 45 degrees description plant 9–14 cm tall. stem 2.5–3.3 mm in diameter, thin, slightly swollen at the base, up to 8 mm in diameter. leaves 5–7(8) mm, ovate, spread. inflorescence 3.5–5.5 ´ 2.2–2.7 cm, short, cylindrical, with a round apex, subdense, densely pubescent glandular rachis, with hairs up to 0.4 mm long. sessile flowers. bracts 4.5–5.5 ´ 2.5–3 mm, smaller than the calyx, ovate, with dense glandular hairs up to 0.4 mm, usually blue to dark purple when dry. bracteoles 3.5–6 ´ 0.3–0.8 mm, linear-lanceolate. calyx 6–9 mm, with long acuminate tri4 acta bot. croat. 69 (1), 2010 pujadas salvà a. j., muñoz garmendia j. f. fig. 2. (wu) holotype for orobanche pseudorosmarina a. pujadas et muñoz garm. u:\acta botanica\acta-botan 1-10\pujadas salva.vp 13. travanj 2010 9:33:52 color profile: disabled composite 150 lpi at 45 degrees angular teeth, matching its tube, conspicuous nerves, occasionally concealed by an indument of glandular hairs 0.2–0.4 (0.6) mm long. corolla 13–16 mm, patent to erecto-patent, uniform and strongly curved, base slightly thickened and thinner above the insertion point of filaments and tubular, slightly infundibuliform at the apex, shortly pubescent with glandular and eglandular hairs up to 0.3(0.4) mm, hairs uniformly, densely distributed in the upper 2/3 of the corolla, glabrous or glabrescent at the base, with a white base and a purplish blue apex when dry. upper lip bilobate, round, emarginate, obtuse, the lower one longer than the upper one, having subequal, round, obtuse lobes with shortly hairy glandular margin, hairs up to 0.4 mm long. filaments obliquely inserted, adaxial filaments inserted 4.5–5.2 mm above the corolla base, abaxial filaments inserted at 4–5 mm, glabrous at the base and hairs nearly 0.1 mm in size, dispersed in the upper 2/3, occasionally subglabrous, scant subssesile glandular hairs (c. 0.05 mm) below the anthers; anthers 0.9–1.1 mm (apiculum 0.1–0.2 mm), ovate, apiculate, white when dry, hairy at the base, hairs 0.3–0.4 mm long. ovary with short glandular hairs up to 0.2 mm in size, in the upper half, occasionally subglabrous. style with hairs up to 0.1 mm long, disperse, occasionally subglabrous. stigma scarcely bilobate. geographical distribution and habitat orobanche pseudorosmarina occurs on the coastal islands of croatia (dalmatia), where it seems to be very scarce. we have found no herbarium material from other regions corresponding to this taxon. it often parasites on rosmarinus officinalis l. other studied material orobanche rosmarina beck portugal. estremadura: s. de arrábida, 1848–1850 [1852], dr. welwitsch, no 779 (bm 574992) [sub trionychium rosmarinum nov. sp.; foley’s lectotype for o. rosmarina]. serra da arrábida, …, in serra de montejunto, v–vi, welwitsch (lisu p34461) [sub phelipaea (trionychion) rosmarini welw.]. serra da arrábida, casal da pimenta, 15-iv-1903, a. guimarães (lisu p 34460) [sub orobanche muteli f. schultz a. (o.) stenosiphon beck]. serra da arrábida, casal do pimenta, sobre rosmarinus, -iv-1903, a. guimarães, n° 2270 (lisu p34459) [sub o. ramosa l. b. mutelii (f. schultz) var. stenosiphon beck (sic.)]. serra da arrábida, in collib. calcar. pr. freitas, 150 m, 20-v-1936, w. rothmaler, fl. lusit. 419 (je). serra da arrábida, in rupestribus inter portinho et torres, 21-v-1936, w. rothmaler, fl. lusit. 515 (je). discussion the new species can be easily distinguished from orobanche rosmarina (= o. mutelii var. stenosiphon beck), with which it has so far been confused, owing to its thin stem, 2.5–3.3 mm in diameter [(3)5–7 mm in diameter, relatively robust, in o. rosmarina]; inflorescence 3.5–5.5 ´ 2.2–2.7 cm, short [5–12 ´ (1.5)2–2.3 cm, longer in o. rosmarina]; bracts 4.5–5.5 ´ 2.5–3 mm (6–8 ´ 3–4.5 mm in o. rosmarina); bracts with dense glandular hairs up to 0.4 mm [with very short (0.1–0.2 mm), glandular hairs in o. rosmarina]; bracteoles 3.5–6 ´ 0.3–0.8 mm, linear–lanceolate (4–7 ´ 1–2 mm, lanceolate in o. rosmarina); acta bot. croat. 69 (1), 2010 5 orobanche pseudorosmarina sp. nov. u:\acta botanica\acta-botan 1-10\pujadas salva.vp 9. travanj 2010 10:32:47 color profile: disabled composite 150 lpi at 45 degrees calyx with triangular long acuminate teeth (with triangular teeth in o. rosmarina); corolla 13–16 mm, patent to erecto-patent, uniform and strongly curved (erecto–patent, occasionally erect, straight to uniform and gently curved in o. rosmarina); corolla with shortly hairy glandular margin, hairs up to 0.4 mm long (with a glabrous or glabrescent margin in o. rosmarina); staminal filaments glabrous at the base and hairs up to 0.1 mm long, disperse in the upper 2/3, occasionally subglabrous (shortly hairy in the lower half and glabrous in the upper 2/3 or with subsessile glandular hairs below the anthers in o. rosmarina); anthers 0.9–1.1 mm (1.2–1.4 mm in o. rosmarina); anthers hairy at the base with hairs 0.3-0.4 mm long (glabrous in o. rosmarina); ovary with short glandular hairs up to 0.2 mm in size in the upper half, occasionally subglabrous (glabrous in o. rosmarina); and style with hairs up to 0.1 mm, disperse, occasionally subglabrous (glabrous in o. rosmarina). acknowledgements we are deeply indebted to the keepers and staff of the herbaria bm, je, lisu, prc, w, wu for the loan of the studied specimens, santos cabello pérez for his latin diagnosis, and john mcneill for providing very helpful comments. references beck, g., 1890: monographie der gattung orobanche. bibliotheca botanica 19, 1–275. foley, m. j. y., 2001a: orobanchaceae in the »flora iberica« area: new taxa, excluded taxa, and typification. anales del jardín botánico de madrid 58, 223–233. foley, m. j. y., 2001b: orobanche l. in: paiva, j., sales, f., hedge, i. c., aedo, c., aldasoro, j. j., castroviejo, s., herrero, a., velayos, m. (eds.), flora iberica 14, 32–72. real jardín botánico, madrid. ginzberger, a., 1921: beitrag zur kenntnis der flora der scoglien und kleineren inseln süd-dalmatiens. österreichische botanische zeitschrift 70, 233–248. mcneill, j., barrie, f. r., burdet, h. m., demoulin, v., hawksworth, d. l., marhold, k., nicolson, d. h., prado, j., silva, p. c., skog, j. e., wiersema, j. h., turland, n. j. (eds.), 2006: international code of botanical nomenclature (vienna code). regnum vegetabile 146. gantner verlag, ruggell. 6 acta bot. croat. 69 (1), 2010 pujadas salvà a. j., muñoz garmendia j. f. u:\acta botanica\acta-botan 1-10\pujadas salva.vp 13. travanj 2010 9:34:16 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (2), 285–300, 2009 coden: abcra 25 issn 0365–0588 periphytic diatom community in a mediterranean salt wedge estuary: the ebro estuary (ne iberian peninsula) laia rovira*, rosa trobajo, carles ibáñez irta – aquatic ecosystems, carretera poble nou km 5.5, 43540, st. carles de la ràpita, catalonia, spain. the ebro river discharge is the main factor controlling hydrological dynamics in the ebro microtidal salt wedge estuary. the aim of this study was to describe the species composition of the periphytic diatom communities, and to elucidate the main environmental factors affecting them. samples of periphytic diatoms were collected at 8 sites along the estuary in october 2007 and january 2008. the diatom community was sampled both from natural and artificial substrata. water depth, velocity, ph, dissolved oxygen, temperature, conductivity, total chlorophyll and water chlorophyll a, total periphytic chlorophyll, dissolved nutrients (p-po4 3–, n-no3 –, n-no2 –, n-nh4 +, si-sio4 4–), total dissolved nitrogen and total dissolved phosphorous were determined in each campaign. altogether, more than 120 taxa of diatoms were identified. the most abundant genera were cocconeis, amphora, navicula and tabularia. the variability of the diatom community was analyzed with multivariate analysis methods. water stratification affected diatom community in both the horizontal and the vertical gradient, according mainly to salinity, dissolved oxygen and nutrient concentration differences. key words: diatom, estuary, salt wedge, periphyton, distribution, taxonomic composition, ebro, mediterranean, spain introduction diatoms are valuable indicators of ecological quality: they respond directly and sensitively to many physical, chemical and biological changes in aquatic environment. they are found in almost all aquatic habitats and their high contribution to primary production has been pointed out by several authors. they also have among the shortest generation times of all biological indicators, allowing them to respond rapidly to environmental changes and to provide early warning of potential changes in nutrient status for different water bodies (rott 1991, stevenson and pan 1999). acta bot. croat. 68 (2), 2009 285 * corresponding author: laia.rovira@irta.cat u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:12 color profile: disabled composite 150 lpi at 45 degrees despite the ecological importance of diatoms as a periphytic component, the knowledge of lentic communities is less than the knowledge of lotic periphytic communities. the scarcity of diatom studies in estuarine and transitional water bodies may be caused by the complexity of these systems (with high fluctuations among environmental parameters), but also due to the taxonomical difficulty of identifying estuarine diatom flora (trobajo et al. 2004). however, this fluctuating dynamics is interesting to study because it may help us to understand the responses of diatoms to environmental gradients. nowadays these studies are focused mainly in tidal estuaries and there is a lack of knowledge of periphytic communities in stratified estuaries (with small tidal effect). the ebro estuary is classified as a mediterranean salt wedge or highly stratified estuary (ibáñez et al. 1997), with two completely different water layers and low tidal range (around 20 cm). the ebro river discharge is the main factor that affects the dynamics of the salt wedge, but other factors like the topography of the estuarine bed must be also considered. the river discharge has been highly regulated by dams built since the 1960s, which decreased the annual ebro river discharge and, therefore, increased the presence of the salt wedge. this stratification in two water layers has an ecological significance since biological communities in the estuary will receive either freshwater or saline water, depending on the dynamics of the salt wedge. the ebro river has also great socioeconomic importance since it is the largest river in spain and its flow is the water source for irrigation in the ebro basin (ibáñez et al. 1996). although during the last 20 years a number or research projects concerned with the ebro river and its estuary have produced a large amount of ecological and hydrological data of these systems (muñoz and prat 1989, guillén and palanques 1992, ibáñez et al. 1999, sierra et al. 2002, 2004, falcó et al. 2006), there have been no studies on its periphytic diatom communities. the aim of this preliminary study was to carry out the first description of the periphytic diatom community in the ebro estuary and to explore the main factors that may affect their distribution. this is an initial analysis as a part of a broader study of diatom community of mediterranean estuarine systems. materials and methods study site the ebro river is the largest river in spain. its estuary covers an approximate area of 7 km2 and it is considered a »micro-tidal salt wedge estuary«. tides have little or no influence because of the low tidal range (around 20 cm), the ebro river discharge being the main factor controlling the hydrological dynamics of this transitional system. the ebro estuary is 30 km long with a mean depth of 6.8 m and a mean width of 237 m. the microtidal range (around 20 cm), favours the stratification of the water column and the existence of a salt wedge, with a maximum intrusion into the ebro river channel of 32 km. this salt wedge disappears when the river flow is above 400 m3s–1. when the river discharge is between 400 and 300 m3s–1, the salt wedge can occupy the last 5 km of the estuary, but with discharges lower than 300 m3s–1 the salt wedge advances quickly up to 18 km from the mouth. when the river discharge is less than 100 m3s–1 , the salt wedge reaches its maximum extent (i.e. 32 km from the mouth) (ibáñez et al. 1997). 286 acta bot. croat. 68 (2), 2009 rovira l., trobajo r., ibáñez c. u:\acta botanica\acta-botan 2-09\rovira.vp 12. listopad 2009 11:39:50 color profile: disabled composite 150 lpi at 45 degrees sampling eight sampling points were established along the estuary (fig. 1). the distance between sampling points was approximately 5 km. the first point was above the maximum extent of the salt wedge (e-3), thus it did not receive saline water, and the last point was located at the river mouth. every site was sampled in october 2007 and january 2008. water depth, temperature, electrical conductivity (ec25), dissolved oxygen (do2) and ph were measured in situ in all sampling sites with an ysi 556 multiprobe. flow direction and velocity were also measured using a braystoke bfm 001 current flow meter. irradiance was measured with a qsp-2100 submersible scalar par sensor. periphytic samples were collected from two different substrata: natural substratum (mainly macrophytes potamogeton pectinatus and ceratophyllum sp., but also wood debris where macrophytes were not available) and from artificial substrata (fired clay bricks, fig. 2), to avoid variability due to substratum. a known area of periphyton (4 cm2) was scraped with a brush from the artificial substrata in each replicate. three fragments from natural substrata were included in each replicate. in order to study the effects of the salt wedge on diatom communities, bricks were placed in the superficial water layer (0.5 m water depth) and in the deep-water layer (which ranged from 2–8 m). bricks were considered the most appropriate artificial substratum due to their resistance to high flows and to the sudden variations of flows that characterize the lower ebro river. unfortunately, due to this flow dynamics, in some sites, artificial substrata were not encountered. two replicates from superficial and deep water bricks (when they were available) were processed. two replicates from natural superficial substrata were also processed. acta bot. croat. 68 (2), 2009 287 periphytic diatoms in the ebro estuary fig. 1. ebro estuary map showing the sampling points. u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:12 color profile: disabled composite 150 lpi at 45 degrees diatom identification and valve counting the periphytic samples were cleaned of organic material using distilled water, h2so4 and kno3 (hustedt 1930). clean valves were permanently mounted in naphrax® (refractive index 1.74). the permanent slides were examined using a leica dmi 3000b light microscope equipped with differential interference contrast (dic) with a 100 times oil immersion objective (n.a.=1.40). for the scanning electron microscopy (sem) observations, the cleaned material was gold coated and studied under a jeol – 6400 sem. the relative abundance of species was determined by counting a minimum of 400 frustules in each substratum replicate. identification of diatoms to species level was based mainly on appropriate keys (krammer and lange-bertalot 1991a, b; 1997a, b; witkowski et al. 2000; lange-bertalot 2001). nutrient and chlorophyll analysis analysis of dissolved inorganic nutrients: silicate (si-sio4 4–), nitrate (n-no3 –), nitrite (n-no2 –), phosphate (p-po4 3–); total dissolved nitrogen (tdn) and total dissolved phosphorus (tdp) were measured following grasshoff et al. (1999), while ammonium (n-nh4 +) was measured following the method proposed by the equipment manufacturer, alliance instruments, sa. total periphytic chlorophyll and water chlorophyll were extracted using 90% acetone and measured with a spectrophotometer using jeffrey and humphrey expressions (rowan 1989). data analysis diatom community variation along the ebro estuary was analysed with a correspondence analysis (ca) using canoco 4.5 version with diatom relative abundance. to avoid the effect of rare species, only diatom species with a relative abundance (ra > 0.2%) and present in more than 10% of the samples (total number of samples = 32) were included in the analysis. relative abundance data was square-root transformed in order to reduce the effect of highly variable population densities on ordination scores. relationships between 288 acta bot. croat. 68 (2), 2009 rovira l., trobajo r., ibáñez c. fig. 2. artificial substrata (fired clay bricks) used for diatom colonization both in superficial and deep-water layers. u:\acta botanica\acta-botan 2-09\rovira.vp 9. listopad 2009 13:15:25 color profile: disabled composite 150 lpi at 45 degrees ca dimensions and environmental parameters were determined with pearson correlations using the spss 15.0 software for windows. only correlations with p < 0.05 were considered. environmental data (except ph, water depth and water velocity) were logarithmically transformed before analysis. results physical and chemical parameters the average values for the water physicochemical parameters measured in october 2007 and january 2008 at each sampling point are shown (tab. 1). mean monthly river discharge was 175.95 m3 s–1 in october and 110.41 m3 s–1 in january. in both sampling periods the salt wedge was detected up to the e-9 site (fig. 1). in october we found and sampled the limit of the salt wedge, whereas in january the salt wedge was detected further upstream (between e-7 and e-9 deep water layers). the superficial layer was in both sampling periods oligohaline; salinity ranged approximately from 0.70 to near 3.00, whereas in the deep-water layer salinity ranged from oligohaline (0.70–0.80) in e-3, e-5 and e-7 to euhaline (36.00–37.00) in e-9, e-11, e-13, e-14 and e-15. irradiance values were 37 me m–2 s–1 at 6 m depth and 1104 me m–2 s–1 at surface (october); in january values ranged from 44 me m–2 s–1 at 7 m depth to 1407 me m–2 s–1 at surface. dissolved oxygen concentration (do2) was lower in october. in both sampling periods, do2 concentration was lower in the salt wedge than in the superficial freshwater layer and e-9d showed the lowest do2 concentrations among all sampling points. nutrients were usually higher in the freshwater layer than in the salt wedge. it should be noted that for both sampling periods, e-9d presented the highest nutrient concentrations among all the salt wedge sites (except in the case of n-no3 – in october 2007) and the lowest values for ph and do2 among all sampling points (considering both freshwater layer and salt wedge). e-9d also showed the highest values of conductivity, p-po4 3–, tdp and n-nh4 + among all sampling points in october. chlorophyll the highest values of both water chlorophyll a and total water chlorophyll concentrations (tab. 2) were found in the sampling points where the salt wedge was present (e-9, e-11, e-13, e-14 and e-15). the average minimum water chlorophyll a values were 0.87 mg l–1 in the freshwater layer and 0.79 mg l–1 in the salt wedge, both in january. minimum water total chlorophyll values were 1.10 mg l–1 in the freshwater layer and 1.24 mg l–1 in the salt wedge, both in january. periphytic total chlorophyll (a + b + c) was always higher in superficial samples than in the deep ones, independently of the presence of the salt wedge (tab. 2). diatom community altogether, 122 diatom species were identified in the 32 analysed samples. the most abundant genera (considering all species) were cocconeis (24%), navicula (21%), nitzschia (17%) and tabularia (11%). navicula was the genus with the highest number of taxa acta bot. croat. 68 (2), 2009 289 periphytic diatoms in the ebro estuary u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:13 color profile: disabled composite 150 lpi at 45 degrees 290 a c t a b o t .c r o a t .68 (2),2009 r o v ir a l .,t r o b a jo r .,ib á ñ e z c . tab. 1. water physicochemical parameters measured in october 2007 and january 2008. the negatives values in water velocity mean that water flowed in the opposite direction to river flow. s – superficial water layer, d – deep water layer sampling point water depth (m) temp (°c) ph do2 (mg/l) salinity (psu) conductivity (ms/cm) p-po4 3– (mg/l) tdp (mg/l) n-nh4 (mg/l) n-no2 (mg/l) n-no3 – (mg/l) tdn (mg/l) si-sio4 4– (mg/l) water velocity (m/s) october 2007 e-3s 0.2 22.8 8.2 7.1 0.72 1375.0 0.028 0.067 0.086 0.014 1.79 2.46 0.85 0.34 e-3d 1.0 22.8 8.2 6.9 0.72 1374.4 0.029 0.070 0.028 0.014 1.63 2.40 0.65 0.16 e-5s 0.2 22.9 8.3 7.9 0.72 1373.7 0.041 0.077 0.070 0.014 1.75 2.35 0.76 0.17 e-5d 7.0 22.8 8.2 7.0 0.72 1367.9 0.028 0.066 0.075 0.013 1.73 2.50 0.79 0.12 e-7s 0.2 22.9 8.3 8.1 0.72 1373.8 0.021 0.052 0.127 0.011 1.68 2.38 1.31 0.19 e-7d 3.0 22.9 8.3 8.1 0.72 1372.6 0.024 0.058 0.061 0.011 1.70 2.42 1.04 0.17 e-9s 0.2 23.0 8.4 8.9 0.73 1408.0 0.022 0.050 0.075 0.012 1.75 2.34 1.24 0.28 e-9d 4.0 23.7 8.0 2.2 35.41 52248.4 0.049 0.095 0.295 0.014 0.04 0.40 0.91 –0.03 e-11s 0.2 23.2 8.4 8.9 1.09 2053.9 0.022 0.049 0.081 0.012 1.65 2.34 1.87 0.31 e-11d 6.0 21.5 8.3 5.5 35.80 50474.1 0.013 0.057 0.080 0.002 0.05 0.19 0.22 –0.07 e-13s 0.2 22.8 8.3 8.6 1.59 2917.8 0.017 0.044 0.031 0.013 1.77 2.26 2.61 0.39 e-13d 7.0 22.3 8.3 6.4 36.25 51887.9 0.006 0.042 0.017 0.000 0.03 0.16 0.08 –0.05 e-14s 0.2 22.5 8.4 9.1 1.98 3577.3 0.013 0.043 0.073 0.012 1.68 2.56 2.52 0.45 e-14d 6.8 22.1 8.4 7.8 36.42 51823.3 0.003 0.039 0.024 0.000 0.02 0.14 0.63 –0.14 e-15s 0.2 22.3 8.3 8.5 2.89 5074.8 0.016 0.047 0.047 0.012 1.56 2.08 2.08 0.47 e-15d 4.5 21.9 8.4 8.2 36.00 51139.7 0.003 0.037 0.022 0.001 0.06 0.12 0.52 –0.23 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ r o v i r a . v p 6 . l i s t o p a d 2 0 0 9 1 0 : 1 7 : 1 3 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s a c t a b o t .c r o a t .68 (2),2009 291 p e r ip h y t ic d ia t o m s in t h e e b r o e s t u a r y sampling point water depth (m) temp (°c) ph do2 (mg/l) salinity (psu) conductivity (ms/cm) p-po4 3– (mg/l) tdp (mg/l) n-nh4 (mg/l) n-no2 (mg/l) n-no3 – (mg/l) tdn (mg/l) si-sio4 4– (mg/l) water velocity (m/s) january 2008 e-3s 0.2 11.0 7.8 13.7 0.76 1105.9 0.050 0.064 0.024 0.019 3.49 3.77 0.99 0.27 e-3d 3.0 11.0 7.9 13.2 0.76 1105.6 0.038 0.061 0.024 0.018 3.51 3.75 1.06 0.18 e-5s 0.2 11.7 7.9 13.7 0.74 1093.9 0.029 0.055 0.104 0.018 3.54 4.07 0.87 0.12 e-5d 7.0 11.4 7.9 13.7 0.75 1098.5 0.030 0.046 0.045 0.017 3.50 3.87 0.80 0.10 e-7s 0.2 11.6 8.0 16.5 0.78 1142.9 0.021 0.036 0.022 0.017 3.34 3.70 0.92 0.15 e-7d 3.0 11.4 8.0 14.8 0.79 1156.4 0.015 0.038 0.019 0.017 3.46 3.74 1.10 0.11 e-9s 0.2 11.7 8.0 17.7 0.91 1335.4 0.027 0.045 0.007 0.021 3.53 3.93 1.34 0.27 e-9d 5.0 13.3 7.9 9.0 36.36 42631.2 0.029 0.054 0.089 0.015 0.13 0.27 0.45 0.00 e-11s 0.2 11.9 8.1 19.1 1.34 1932.9 0.020 0.045 0.027 0.022 3.69 3.86 1.05 0.29 e-11d 6.0 13.3 8.0 11.3 36.60 42892.7 0.009 0.020 0.042 0.008 0.09 0.14 0.16 0.00 e-13s 0.2 11.8 8.0 15.6 1.63 2322.8 0.026 0.050 0.031 0.023 3.45 3.81 0.77 0.35 e-13d 7.0 13.2 8.0 10.4 36.78 43053.6 0.004 0.015 0.038 0.006 0.09 0.13 0.10 –0.03 e-14s 0.2 11.8 8.1 20.5 1.84 2604.0 0.031 0.048 0.040 0.023 3.42 3.78 0.92 0.65 e-14d 8.0 13.3 8.0 10.6 37.34 43688.1 0.002 0.015 0.036 0.004 0.11 0.16 0.08 –0.06 e-15s 0.2 11.5 8.0 13.6 2.57 3547.3 0.037 0.051 0.049 0.023 3.24 3.70 0.66 0.55 e-15d 4.0 13.3 8.0 10.4 37.36 43776.1 0.001 0.012 0.029 0.003 0.08 0.14 0.05 –0.09 tab. 1. continued u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ r o v i r a . v p 6 . l i s t o p a d 2 0 0 9 1 0 : 1 7 : 1 3 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s 292 acta bot. croat. 68 (2), 2009 rovira l., trobajo r., ibáñez c. tab. 2. mean values of total periphytic chlorophyll, water chlorophyll a and total water chlorophyll measured in october 2007 and january 2008. the range values (minimum – maximum) for each sample are represented in brackets. chl – chlorophyll, s – superficial water layer, d – deep water layer sampling point water depth (m) total periphytic chl (mg/cm2) water chl a (mg/l) total water chl (mg/l) october 2007 e-3s 0.20 2.80 (0.88–4.72) 1.47 (1.26–1.90) 2.35 (1.31–2.45) e-3d 1.00 0.23 (0.20–0.26) 1.83 (1.48–2.31) 3.34 (1.75–4.49) e-5s 0.20 2.63 (1.89–3.39) 2.00 (1.70–2.32) 3.57 (2.18–4.15) e-5d 7.00 0.27 (0.22–0.32) 2.21 (2.14–2.24) 3.82 (2.82–3.21) e-7s 0.20 7.07 (4.88–9.25) 2.19 (2.12–2.26) 4.14 (0.00–3.16) e-7d 3.00 – – 2.25 (2.04–2.36) 3.79 (2.44–3.26) e-9s 0.20 0.40 (0.28–0.51) 3.19 (3.07–3.38) 5.12 (3.54–4.48) e-9d 4.00 0.28 (0.21–0.36) 2.69 (2.72–2.73) 4.19 (3.01–3.62) e-11s 0.20 7.88 (4.61–11.15) 3.42 (3.30–3.57) 5.23 (3.75–5.58) e-11d 6.00 – – 3.23 (3.19–3.31) 4.66 (3.83–4.07) e-13s 0.20 – – 2.65 (2.16–3.42) 4.99 (2.49–6.42) e-13d 7.00 1.95 (1.35–2.56) 5.13 (4.90–5.24) 7.65 (5.97–6.43) e-14s 0.20 – – 2.40 (1.92–2.67) 5.28 (2.39–5.42) e-14d 6.80 – – 5.41 (5.22–5.67) 8.32 (6.51–7.33) e-15s 0.20 – – 2.62 (2.46–2.79) 4.71 (3.35–4.46) e-15d 4.50 0.85 (0.77–0.93) 3.79 (3.65–4.07) 5.71 (4.39–5.53) january 2008 e-3s 0.20 5.87 (5.40–6.33) 1.09 (0.81–1.31) 1.69 (0.87–2.93) e-3d 3.00 0.24 – 1.37 (0.92–2.07) 1.43 (0.84–2.20) e-5s 0.20 – – 1.08 (0.68–1.55) 1.59 (0.74–2.80) e-5d 7.00 – – 1.10 (1.04–1.24) 1.24 (1.03–1.65) e-7s 0.20 – – 0.87 (0.70–1.00) 1.10 (0.70–1.77) e-7d 3.00 – – 0.79 (0.06–1.19) 2.13 (1.60–2.66) e-9s 0.20 – – 2.44 (2.16–2.69) 3.19 (2.49–3.64) e-9d 5.00 – – 1.17 (1.02–1.33) 1.91 (1.44–2.37) e-11s 0.20 3.72 (2.04–5.40) 6.21 (6.00–6.44) 7.87 (7.61–8.13) e-11d 6.00 0.55 (0.45–0.65) 1.43 (0.90–2.26) 1.84 (1.17–2.87) e-13s 0.20 8.71 – 4.32 (4.16–4.52) 5.48 (5.09–5.74) e-13d 7.00 0.68 (0.64–0.71) 1.78 (0.80–3.43) 2.75 (0.79–5.87) e-14s 0.20 2.16 – 4.34 (4.00–4.65) 5.11 (4.61–5.48) e-14d 8.00 – – 1.55 (1.02–2.09) 2.85 (1.44–4.27) e-15s 0.20 – – 3.40 (3.28–3.51) 4.14 (4.09–4.19) e-15d 4.00 – – 1.14 (0.70–1.37) 1.39 (0.70–1.44) u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:13 color profile: disabled composite 150 lpi at 45 degrees (20), followed by nitzschia (13). the 45 diatom taxa with a relative abundance > 0.2% and present in more than 10% of the samples are listed in table 3. species richness and shannon-wiener diversity index for natural and artificial samples are shown in table 4. no major differences were observed in diatom community diversity between substrata type. diatom community changed along the horizontal and vertical gradients (fig. 3). the diatom community of the natural substrata in october 2007 (fig. 3a) was dominated by cocconeis placentula ehrenberg (figs. 4a, b). bacillaria paradoxa gmelin (fig. 4h) and tabularia fasciculata (agardh) williams et round (figs. 4c, d) also appeared as abundant species in most sites (e-3 to e-13). there was a change in diatom community structure in e-14, navicula perminuta grunow (fig. 4n) being the dominant species and rhoicosphenia abbreviata (agardh) lange-bertalot (figs. 4f, g) abundant. another change occurred in e-15, where fallacia cf. clepsidroides witkowski (fig. 4j) and nitzschia cf. inconspicua grunow (fig. 4k) dominated the sample. in january 2008 (fig. 3b), the diaacta bot. croat. 68 (2), 2009 293 periphytic diatoms in the ebro estuary tab. 3. diatom taxa of all samples (ranged alphabetically) with relative abundance (ra) > 0.2% and present in more than 10% of the ebro estuary samples. diatom taxa % ra achnanthes amoena hustedt 1.29 achnanthes minutissima kützing 1.26 amphora aff. helenensis giffen 0.35 amphora inariensis krammer 0.99 amphora lybica ehrenberg 3.55 amphora ovalis (kützing) kützing 0.37 amphora pediculus (kützing) grunow 6.51 amphora sp.1 0.45 bacillaria paradoxa gmelin 3.75 cocconeis pediculus ehrenberg 0.47 cocconeis placentula ehrenberg 23.00 cyclotella meneghiniana kützing 0.48 fallacia sp.1 3.33 gomphonema cf. olivaceum (hornemann) brébisson 0.33 gomphonema clevei fricke 0.29 melosira varians agardh 0.32 navicula aff. mollis (w. smith) cleve 1.01 navicula antonii lange-bertalot 1.40 navicula capitatoradiata germain 0.25 navicula cryptotenella lange-bertalot 1.96 navicula aff. cryptotenelloides lange-bertalot 0.50 navicula gregaria donkin 0.83 navicula cf. margalithii (lange-bertalot) 0.91 navicula perminuta grunow 4.47 diatom taxa % ra navicula recens (lange-bertalot) lange-bertalot 2.76 navicula cf. subminuscula manguin 0.52 navicula sp.1 0.57 navicula tripunctata (o.f. müller) bory 0.36 nitzschia amphibia grunow 0.58 nitzschia constricta (kützing) ralfs 0.41 nitzschia dissipata (kützing) grunow 3.66 nitzschia filiformis (w. smith) van heurck 1.63 nitzschia cf. fonticola (grunow) grunow 0.33 nitzschia cf. frustulum (kützing) grunow 4.22 nitzschia cf. inconspicua grunow 2.89 nitzschia microcephala grunow 0.32 nitzschia palea (kützing) w. smith 1.26 nitzschia cf. palea (kützing) w. smith 1.36 nitzschia prolongata hustedt 0.67 nitzschia cf. sociabilis hustedt 0.26 pleurosira laevis (ehrenberg) compère 0.61 rhoicosphenia abbreviata (agardh) lange-bertalot 5.86 stephanodiscus aff. alpinus hustedt 0.21 synedra ulna ehrenberg 0.33 tabularia fasciculata (agardh) williams et round 6.94 u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:13 color profile: disabled composite 150 lpi at 45 degrees 294 acta bot. croat. 68 (2), 2009 rovira l., trobajo r., ibáñez c. tab. 4. species richness and shannon–wiener index for natural and artificial samples in october 2007 and january 2008. october 2007 species richness shannon–wiener index natural samples 42 3.03 artificial samples 34 3.22 january 2008 species richness shannon–wiener index natural samples 40 3.61 artificial samples 42 3.73 fig. 3. diatom community of the ebro estuary. a) natural substrata in october 2007, b) natural substrata in january 2008, c) artificial substrata in october 2007, d) artificial substrata in january 2008. natural substrata were only collected at superficial level. s – superficial water layer, and d – deep water layer. only diatom taxa with a relative abundance > 0.1 are shown u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:14 color profile: disabled composite 150 lpi at 45 degrees tom community was more diverse than in october. in this period a change in the diatom community along the estuary was also observed. in e-13, cocconeis placentula and nitzschia dissipata (kützing) grunow (fig. 4l) drastically decreased and other diatom acta bot. croat. 68 (2), 2009 295 periphytic diatoms in the ebro estuary fig. 4. representative diatom taxa from the ebro estuary. a, b: – cocconeis placentula ehrenberg; c, d – tabularia fasciculata (agardh) williams et round; e – amphora libyca ehrenberg; f, g – rhoicosphenia abbreviata (agardh) lange-bertalot; h – bacillaria paradoxa gmelin; i – amphora pediculus (kützing) grunow; j – fallacia cf. clepsidroides witkowski; k – nitzschia cf. inconspicua grunow; l – nitzschia dissipata (kützing) grunow; m – fallacia sp.1; n – navicula perminuta grunow; o – nitzschia cf. palea (kützing) w. smith; p – nitzschia cf. frustulum (kützing) grunow. scale bar denotes 10 mm. u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:16 color profile: disabled composite 150 lpi at 45 degrees taxa became representative, like navicula perminuta, nitzschia cf. palea (kützing) w. smith (fig. 4o) and nitzschia cf. frustulum (kützing) grunow (fig. 4p), among others. the diatom community of the artificial substrata in october (fig. 3c), showed a clear change in e-9d (the limit of the salt wedge), where cocconeis placentula, amphora libyca ehrenberg (fig. 4e) and amphora pediculus (kützing) grunow (fig. 4i) decreased; and fallacia sp.1 (fig. 4m) clearly dominated the sample. unfortunately, several artificial substrata could not be recovered in the january campaign and thus only few data were available (fig. 3d). factors affecting diatom distribution results of the correspondence analysis (ca) show that the first dimension of the ca (dim1) explained 24.6% of diatom community variability, and seems to differentiate the upstream points (with no salt wedge) from those closer to the sea (fig. 5). this dimension was significantly and negatively correlated with phosphorous (tdp) and positively with water velocity and salinity (tab. 5). the second ca dimension (dim2) explained 13.5% of variation and differentiated e-9o (deep layer, october) from the rest of samples. it was significantly and positively correlated with salinity and water temperature; and negatively with oxygen (do2), and nitrogen (n-no3 – and tdn). 296 acta bot. croat. 68 (2), 2009 rovira l., trobajo r., ibáñez c. fig. 5. sample ordination in the plane defined by the two first ca dimensions. o = october, j = january. only diatom taxa with a relative abundance (ra) > 0.2% and present in more than 10% of the samples are included in the analysis. as – artificial superficial samples, ad – artificial deep samples, n – natural samples (only collected at superficial level). u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:16 color profile: disabled composite 150 lpi at 45 degrees discussion water chlorophyll values found in this study were lower than those reported in previous ebro estuary papers. the minimum total chlorophyll (a + b + c) values in july 1991 were 21.5 mg l–1 in the freshwater layer and 3.2 mg l–1 in the salt wedge (ibáñez et al. 1995). from 1989 to 1992 in the freshwater layers the minimum chlorophyll a values were 20 mg l–1 and 7 mg l–1 in the salt wedge (casamayor et al. 2001); and in july 1999 the minimum chlorophyll a values were around 9 mg l–1 in the freshwater layer and 2 mg l–1 in the salt wedge (falcó et al. 2006). this decrease in phytoplankton has been attributed to a decrease in riverine phosphorous (ibáñez et al. 2008), which has allowed light to reach the salt wedge and probably has permitted periphytic communities to become established. in july 1989, the light intensity was practically zero at 4.8 m depth, below the interface (casamayor et al. 2001). in our study, the light reached the river bed (6 m depth in october and 7 m depth in january). in addition, light also reached the river bed in april 2008 (10 m) and july 2008 (7 m), thus the increase in water transparency was not a sampling period effect. however, due to the lower light intensity, the periphytic total chlorophyll concentrations in deep substrata were considerably lower than those found in superficial substrata. this study allowed identification of the importance in terms of abundance of some periphytic diatom species in the ebro estuary, like cocconeis placentula, and the need to study it at infraespecific level in further studies, because different varieties may have different ecological responses. some of the most representative diatom species found in the ebro estuary (cocconeis placentula in freshwater layers, navicula perminuta in saline layers, amphora pediculus or rhoicosphenia abbreviata) are not found in the same proportion and/or occurrence in other estuarine studies (mcintire and overton 1971, moore and mcintire 1977, mcintire 1978, underwood 1994, nayar 2005). the ebro estuary has specific characteristics (high stratification, irregular and sudden salt wedge intrusions) that are not met in other estuarine systems studied (e.g. atlantic estuaries, fiords), and these differences could explain differences in diatom flora. the diatom community structure changed along the ebro estuary. a change in diatom community structure was observed in points closer to the sea (from e-13 to e-15) with higher salinity. in these points the marine and riverine influences can be both strong and acta bot. croat. 68 (2), 2009 297 periphytic diatoms in the ebro estuary tab. 5. pearson correlation coefficients between ca dimensions and environmental parameters. only significant correlations at p < 0.05 and with a pearson correlation coefficient higher than 0.4 are listed. environmental parameters dim 1 dim 2 % explained variance 24.6 13.5 salinity (ppt) 0.431 0.539 tdp (mg l) –0.465 – do2 (mg l) – –0.601 n-no3 – (mg l) – –0.609 tdn (mg l) – –0.510 water velocity (m s–1) 0.477 – water temperature (°c) – 0.404 u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:16 color profile: disabled composite 150 lpi at 45 degrees they change depending on the river flow, sea storms and winds, resulting in a more dynamic situation at superficial layers. fluctuating conditions have been previously reported as an important factor affecting diatom community structure, diversity and composition of such environments (sullivan 1978, underwood 1994, trobajo et al. 2004). another important change in the diatom community was found in e-9d in october. this point was the limit of the salt wedge, the interface between fresh and saline water. in this zone the water could remain still at the bottom of the river for a long period. it may well be that the decomposition of organic matter promoted the oxygen depletion found in october. however, the observed oxygen depletion did not reach anoxic conditions that were previously recorded (ibáñez et al. 1995, casamayor et al. 2001, falcó et al. 2006). in january, due to the lower river flow, the limit of the salt wedge had to be further upstream (between e-7 and e-9), and unfortunately it was not sampled. artificial substrata should allow the comparison of diatom communities from superficial and deep layers, avoiding substratum variability. several investigations had used artificial substrata to study periphytic diatom communities in estuaries and other transitional systems (mcintire and overton 1971; mcintire 1973, 1978; moore and mcintire 1977; lai 2001; trobajo et al. 2004; nayar et al. 2005). the ca analysis did not clearly segregate the samples according to the type of substrata (natural vs. artificial). therefore, it seems that the periphytic diatom community in the ebro estuary is more affected by the environmental conditions (mainly salinity, dissolved oxygen, nutrient concentrations) than by the substrata type. similar results were found by snoeijs (1994) in the study of epiphytes from the baltic sea, indicating that epiphytic diatom community composition was more affected by environmental parameters than by macroalgal hosts. the preliminary hypothesis would indicate that diatom community in the ebro estuary is determined by salt wedge intrusions, which cause high and irregular fluctuations of salinity and nutrient concentrations. the diatom community could be also affected by the water residence period, which could cause the oxygen decrease observed from the river mouth to the limit of the salt wedge. these initial results suggest that the factors affecting the diatom community and its distribution in the ebro estuary are salinity, phosphorous, nitrogen, oxygen, water temperature and water velocity. the results show a longitudinal variation correlated with salinity and inversely with phosphorous (tdp), and as we expected it could be related to the presence of the salt wedge, which causes a system shift. the highest water residence period was reached in the limit of the salt wedge intrusion. the particular conditions in this site (high salinity, low oxygen and nitrogen depletion) also affected diatom community composition and structure. therefore, it seems that the salt wedge dynamics (not only vertical and longitudinal salinity gradients but also the magnitude and frequency of salinity oscillations) have an influence on the periphytic diatom composition of the ebro estuary and have to be taken into account in further studies. estuarine dynamics is different in more mixed estuaries where salinity gradients and salinity changes are more regular and predictable (due to tidal circulation). acknowledgements this research was supported by the ministerio de educación y ciencia of the spanish government (research project cgl 2006-01487) and by the agència catalana de l’aigua and departament d’innovació, universitats i empresa of the government of catalonia. 298 acta bot. croat. 68 (2), 2009 rovira l., trobajo r., ibáñez c. u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:16 color profile: disabled composite 150 lpi at 45 degrees references casamayor, e. o., garcía-cantizano, j., mas, j., pedrós-alió, c., 2001: primary production in estuarine oxic/anoxic interfaces: contribution of microbial dark co2 fixation in the ebro river salt wedge estuary. marine 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u:\acta botanica\acta-botan 2-09\rovira.vp 6. listopad 2009 10:17:16 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (2), 263–283, 2009 coden: abcra 25 issn 0365–0588 epilithic diatoms (bacillariophyta) from cloud forest and alpine streams in bolivia, south america 3: diatoms from sehuencas, carrasco national park, department of cochabamba eduardo a. morales1,2,3*, erika fernández4, patrick j. kociolek5 1 herbario criptogámico universidad católica boliviana, carrera de ingeniería ambiental, casilla de correos 5381, cochabamba, bolivia 2 patrick center for environmental research, the academy of natural sciences of philadelphia, pa 19103-1195, usa 3 unidad de limnología y recursos acuáticos, universidad mayor de san simón, casilla de correos 992, cochabamba, bolivia 4 centro de biodiversidad y genética, universidad mayor de san simón, casilla de correos 538, cochabamba, bolivia 5 museum of natural history, ucb 218, university of colorado, boulder, co 80309, usa studies on bolivian diatoms are scarce and they do not represent the great geographic variability of the country. one of the regions with the highest biological diversity in bolivia is the yungas (cloud forest), a 90.500 km2 strip located between the andean puna and the amazonian lowlands. the carrasco national park is the park with the largest extension of yungas within its boundaries. this park is located east from cochabamba, the third largest city in bolivia, and has an area of ca 6.226 km2, serving as a refuge to 5.000 recorded species of plants and more than 300 species of vertebrates. very little is known about the aquatic biota in the zone and there are no studies on diatoms. one of the preferred tourist spots within the park is sehuencas, located 17°31'42" s and 65°16'17" w and characterized by numerous lotic waterbodies. the present work was carried on 5 epilithic samples from which 118 species, varieties and forms were identified using light (lm) and scanning electron microscopy (sem). forty-two (36%) of these taxa were not found in the literature for south america or other regions of the world. this high percentage of unknown taxa suggests a high potential for the contribution of new organisms to science, many of which are possibly endemic to the region, thus justifying an additional effort to preserve the aquatic habitats in the park. two new species are described herein (fragilaria cochabambina morales sp. nov. and achnanthidium sehuencoensis morales acta bot. croat. 68 (2), 2009 263 * corresponding author, e-mail: edu.morales2006@gmail.com u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:41 color profile: disabled composite 150 lpi at 45 degrees sp. nov.) and comparisons with published morphologically similar taxa are presented. unknowns remain undescribed until larger populations that allow detailed lm and sem studies are found. key words: diatom, fragilaria cochabambina, achnanthidium sehuencoensis, ultrastructure, cloud forest, yungas, carrasco national park, bolivia introduction bolivian diatoms have been poorly studied and a review of the literature (morales et al. 2008) shows that from the 54 references published for specific algal groups, only 12 (22%) deal with diatoms. furthermore, this literature is largely restricted to the altiplano (high mountain plateau) and the lowlands of the amazon. the taxonomy presented in these publications is poor and names based on european floras have been force-fitted giving the impression that the flora is not too diverse and that it does not differ too much from other parts of the world. however, the recent works of lange-bertalot and collaborators (metzeltin and lange-bertalot 1998, rumrich et al. 2000, metzeltin and lange-bertalot 2007), among others, show that the diatom flora in south america, except for a few cosmopolitan elements, is indeed distinct. the south american cloud forest (or peruvian-bolivian yungas) is one of the most biologically diverse ecosystems in south america (ibish and merida 2003, navarro and maldonado 2004). it runs along the eastern cordillera of the andes, a branch of the mountainous chain that projects in a southeastern direction, between 13 °–17 °s and 69 ° –63 °w. the wide ranges in geographic and climatic characteristics favor the development of a diverse set of habitats populated by a highly diverse flora and fauna. the yungas is also characterized by hundreds of small and medium-sized rivers and streams with diverse physical and chemical characteristics (navarro and maldonado 2004), but the consequences of such a diversity on the aquatic biota are less known, and in the case of algae, completely neglected. two of the most recent surveys on soft-bodied algae and diatoms (mcclintic et al. 2003 and morales and vis 2007, respectively) show that the potential of the yungas for the contribution of new species to science, many of which could be endemic to the region, is very high. the conservation efforts directed to the yungas have been considerable, from both the bolivian government and many international organizations (parks watch 2005). at least 4 national parks contain portions of the yungas within its territories, carrasco national park in the department of cochabamba being the park with the highest proportion of yungas within its borders. however, all conservation practices have concentrated on terrestrial ecosystems and there is no clear intention (national or international) to preserve lotic and lentic systems in the area. the current paper presents a taxonomic assessment, using lm and sem, of the epilithic diatom flora in five courses of water located in sehuencas, a region within carrasco national park, which has received considerable attention from tourists and sport fishermen. the increased inflow of people has had a visually striking impact on the local aquatic systems, but no scientific assessments have been produced to measure such an impact. we propose that diatoms offer an opportunity to seriously consider conservation practices of aquatic systems within the park. 264 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:41 color profile: disabled composite 150 lpi at 45 degrees materials and methods epilithic material from two medium-sized rivers and 3 streams in sehuencas (about 150 km from the city of cochabamba) (tab. 1) were studied as part of a larger project directed to a preliminary assessment of the epilithic diatom diversity in the cloud forest of bolivia. acta bot. croat. 68 (2), 2009 265 bolivian cloud forest diatoms fig. 1. map of bolivia showing the national parks system. carrasco national park is located in the center of the country in the department of cochabamba (number 6). sehuencas is depicted by a white spot pointed by the black arrow. modified from www.sernap.gov.bo (visited october, 2008). u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:45 color profile: disabled composite 150 lpi at 45 degrees samples were collected in 2007, scraping with a stiff metal brush, five medium-sized rocks selected from each water course, rinsing scrapings with river water onto a plastic tray, and transferring the mixture into plastic bottles for transport to the laboratory. samples were fixed with about four drops of formalin per 200 ml of sample, sufficient to kill zooplankton and arrest algal growth. the preservation of samples was not sought since our interest was concentrated on inorganic diatom remains. location and physicochemical data were collected directly from the stream at the same time samples were collected (tab. 1). 266 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. tab. 1. information data for the five lotic systems considered in this work. descriptions of the sites were done directly in the field as samples were collected. river / stream geographic location and altitude water physicochemistry description río ibirizu 17° 29.8" s, 65° 16.4" w 2.200 m.a.s.l. t 12 °c; ph 6.8; cond. 31 ms/cm clear water. width of channel variable (about 50 m in some places). depth very variable, but no more than 50 cm. fast flowing current. bed formed by rocks of different sizes, even large boulders are present. water contains debris, mainly leaves from nearby trees; logs are also present. area affected by camping. stream 1 17° 31.6" s, 65° 16.2" w 2.400 m.a.s.l. t 10 °c; ph 6.9; cond. 31 ms/cm clear water. width of channel variable (about 2 m at sampling site). depth variable, but no more than 30 cm. stream cuts off secondary road, water flows fast, but sample was collected from a pool a few steps upstream. bed composed of fine pebbles, small stones and large boulders. area affected by road traffic (about one car every hour during the day). stream 2 not determined, 2.450 m.a.s.l. t 10 °c; ph 6.1; cond. 20 ms/cm clear water. width of channel variable (about 50 cm at sampling site). depth variable, but no more than 5 cm. thread of water coming downhill on the side of the secondary road. many wet rocks and pebbles, as well as silts characterize the area. moss and algal growth evident on wet rocks. coordinates were not determined due to heavy cloud cover, but site is about 400 m up the road from río sehuencas. río sehuencas 17° 32.6" s, 65° 16.12" w 2.600 m.a.s.l. t 10.2 °c; ph 6.7; cond. 23 ms/cm clear water. width of channel variable (about 20 m at sampling site). depth variable, but no more than 50 cm. fast flowing current. river cuts off road and there is no bridge to cross it. some debris present, as well as fine sandy deposits, especially along area where traffic crosses the river. smaller boulders and rocks are also seen in the river. stream 3 17° 33.2" s, 65° 16.2" w 2.650 m.a.s.l. t 9 °c; ph 6; cond. 21 ms/cm clear water. width of channel variable (about 2 m at sampling site). depth variable, but no more than 20 cm. stream cuts off secondary road, large rocks, pebbles and some isolated sandy deposits compose the bed. debris present in the form of leaves and twigs. green and brown algal growths evident on rocks. u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:45 color profile: disabled composite 150 lpi at 45 degrees for lm analysis, subsamples were digested with 70% nitric acid, 1:1 by volume with the sample, and boiling the solution for 20 min. repeated rinsing and decanting of samples with distilled water led to a neutral diatom suspension used for the preparation of permanent slides using cumar r-9 as mounting medium (holmes et al. 1981). slides were analyzed using a zeiss universal microscope equipped with dic and a spot insight 2 color digital camera model 18.2. images were directly captured using spotsoftware version 4.6 at magnifications ranging between 1250x and 2000x. measurements of at least 30 valves for the new taxa were taken using the same software and directly on a computer screen. two slides were analyzed per site to cover as much diatom diversity as possible. identifications were performed using local floras, frequently checking also floras for europe and other continents. this literature is cited in the discussion section below. in order to assess quickly which were the most common taxa in the samples, counts of 100 valves were performed along randomly placed transects (width determined by the field of view of the microscope) on one slide of each locality and at 1250x magnification. all identification and count data were stored in an excel spreadsheet, version 2003. sem work was performed using a leica® stereoscan 430i operated at 20 kv. aluminium stubs were prepared by filtering aliquots of clean sample through polycarbonate filters with a 30 mm mesh, which were then fixed to the stubs using double sided carbon tape. coating was accomplished using a bal-tec med 020 modular high vacuum coating system. images were directly captured and stored in the computer. all images were manipulated and plates made using adobe photoshop v. 7.0. morphological terminology follows anonymous (1975), ross et al. (1979) and round et al. (1990). results a total of 118 taxa at the species and variety levels distributed in 39 genera were encountered. a high percentage of these taxa (36%) could not be identified and were recorded as »sp.« or »cf.« (tab. 2). the taxa bearing published names were readily identified using south american floras such as metzeltin and lange-bertalot (1998), rumrich et al. (2000), metzeltin et al. (2005), metzeltin and lange-bertalot (2007). taxonomic articles by frenguelli (1939), manguin (1964), servant-vildary (1986), morales and vis (2007), morales et al. 2007 and the annotated checklist by hohn (1966), also proved useful. cosmopolitan taxa and taxa characteristic of eutrophic waters (diatoma mesodon kützing, encyonema silesiacum (bleisch) mann, melosira varians agardh, and others were identified easily using süsswasserflora von mitteleuropa series (krammer and lange-bertalot 1986, 1988, 1991a, 1991b, lange-bertalot 1993). references specialized in certain genera such as navicula bory (lange-bertalot 2001), pinnularia ehrenberg (krammer 2000), brachysira kützing (lange-bertalot and moser 1994) and cymbelloids (krammer 1997a, 1997b) were useful, as well. other books that are frequently used for identification of south american taxa were not useful or did not contain taxa found in the yungas region. the epilithic flora in sehuencas is richer in biraphid taxa (84 spp.), with monoraphids and araphids being represented by 16 and 13 species each. as expected, centric diatoms are acta bot. croat. 68 (2), 2009 267 bolivian cloud forest diatoms u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:45 color profile: disabled composite 150 lpi at 45 degrees 268 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. tab. 2. relative abundance of diatoms found in counts of 100 valves along random transects. diatoms marked with »+« were rare and did not appear during counts. 1: río ibirizu, 2: stream 1, 3: stream 2, 4: río sehuencas, 5: stream 4. taxon/sample 1 2 3 4 5 achnanthes inflata (kützing) grunow 1 + achnanthidium minutissimum (kützing) czarnecki 10 1 + 6 1 achnanthidium rivulare potapova et ponader + achnanthidium sp. 1 sehuencas 7 10 2 6 + achnanthidium sp. 2 sehuencas + + adlafia cf. muscora (kociolek et reviers) moser, lange-bertalot et metzeltin + + 6 + + adlafia minuscula (grunow) lange-bertalot + + 2 + brachysira minor (krasske) lange-bertalot 8 1 caloneis sp. 1 sehuencas + cocconeis placentula var. euglypta (ehrenberg) grunow + cymbella naviculiformis auerswald ex heiberg + diadesmis contenta (grunow ex van heurck) mann 4 + diadesmis gallica smith + diatoma mesodon kützing 1 44 51 4 64 encyonema minutum (hilse) mann + + + encyonema silesiacum (bleisch) mann 21 6 + 9 3 encyonema sp. 1 coroico + eolimna minima (grunow) lange-bertalot 6 + + eolimna subminuscula (manguin) moser, lange-bertalot et metzeltin + epithemia adnata (kützing) brébisson + + eunotia cf. soleirolii (kützing) rabenhorst + 2 2 1 1 eunotia cf. tropico-arcus metzeltin et lange-bertalot + + eunotia fallax var. groenlandica (grunow) lange-bertalot et nörpel + eunotia rabenhorstii cleve et grunow + eunotia sp. 1 coroico + eunotia sp. 1 sehuencas + eunotia sp. 2 sehuencas + eunotia sudetica müller + fistulifera pelliculosa (brébisson ex kützing) lange-bertalot + fragilaria capucina var. mesolepta rabenhorst + fragilaria capucina var. rumpens (kützing) lange-bertalot + fragilaria sp. 2 sorata 1 + + fragilaria vaucheriae (kützing) petersen 3 + frankophila similioides lange-bertalot et rumrich + frustulia crassinervia (brébisson) lange-bertalot et krammer + frustulia crassipunctata metzeltin et lange-bertalot + frustulia kosmolliana lange-bertalot et rumrich + + + gomphonema acuminatum ehrenberg 4 u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:45 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 269 bolivian cloud forest diatoms taxon/sample 1 2 3 4 5 gomphonema cf. angustatum (kützing) rabenhorst + 1 + 1 gomphonema cf. gracile ehrenberg + gomphonema cf. lagenula kützing 2 + + + gomphonema cf. parvulum (kützing) kützing + gomphonema coronatum ehrenberg + + + gomphonema parvulius (lange-bertalot et reichardt) lange-bertalot et reichardt + + gomphonema pumilum var. elegans reichardt et lange-bertalot + + 1 1 gomphonema pumilum var. rigidum reichardt et lange-bertalot + gomphonema punae lange-bertalot et rumrich + + + gomphonema sp. 1 sehuencas + + gomphonema sp. 2 coroico + 2 1 + gomphonema sp. 2 sehuencas + + + gomphonema sp. 3 sehuencas + gomphonema sp. 3 sorata + gomphonema sp. 4 sehuencas + + gomphonema sp. 5 sehuencas + gomphonema sp. 6 sehuencas + gomphonema sp. 7 sehuencas + gomphonema sp. 8 sehuencas + gomphonema subclavatum (grunow) grunow + 2 + + hannaea arcus (ehrenberg) patrick 30 1 + 7 + hantzschia abundans lange-bertalot + + luticola acidoclinata lange-bertalot 1 luticola goeppertiana (bleisch) mann + luticola sp. 1 sehuencas + melosira varians agardh + 7 navicula angusta grunow + navicula arvensis hustedt + + navicula cf. lundii reichardt 4 + navicula cryptocephala kützing + navicula gregaria donkin + + navicula notha wallace 2 2 1 navicula rhynchocephala kützing + navicula vaucheriae petersen + 2 nitzschia acidoclinata lange-bertalot + + + nitzschia amphibia grunow + nitzschia cf. tubicola grunow + nitzschia clandestina manguin 21 + 3 nitzschia dissipata (kützing) grunow + + 1 nitzschia inconspicua grunow + tab. 2. – continued u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:45 color profile: disabled composite 150 lpi at 45 degrees 270 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. taxon/sample 1 2 3 4 5 nitzschia intermedia hantzsch ex cleve et grunow + + + nitzschia palea (kützing) smith + nitzschia palea fo. major rabenhorst + nitzschia palea var. debilis (kützing) grunow + nitzschia recta hantzsch ex rabenhorst 1 1 nitzschia sp. 1 sehuencas 1 + 1 12 nitzschia sp. 4 sorata + + + nupela praecipua (reichardt) reichardt + + + + + orthoseira dendroteres (ehrenberg) round, crawford et mann + + + orthoseira roeseana (rabenhorst) o’meara + + orthoseira roeseana (rabenhorst) o’meara (morphotype spiralis) + orthoseira sp. 1 sehuencas + pinnularia biceps gregory + pinnularia cf. viridis (nitzsch) ehrenberg + pinnularia parvulissima krammer + + pinnularia subcapitata var. hilseana (janisch) müller + + + planothidium biporomum (hohn et hellermann) lange-bertalot + planothidium frequentissimum (lange-bertalot) lange-bertalot 6 + planothidium haynaldii (schaarschmidt) lange-bertalot 1 8 13 17 2 planothidium lanceolatum (brébisson ex kützing) lange-bertalot + + + + + planothidium salvadorianum (hustedt) lange-bertalot + planothidium sp. 1 coroico + 1 psammothidium subatomoides (hustedt) bukhtiyarova et round + + + pseudostaurosira brevistriata (grunow) williams et round + pseudostaurosira laucensis (lange-bertalot et rumrich) morales et vis 3 2 reimeria sinuata (gregory) kociolek et stoermer 4 + + 11 rhoicosphenia abbreviata (agardh) lange-bertalot + 4 6 2 10 sellaphora seminulum (grunow) mann + + + + + stauroneis kriegerii patrick + stauroneis sp. 1 sehuencas + stephanodiscus sp. 1 sehuencas + surirella angusta kützing + + surirella susanae metzeltin et lange-bertalot + synedra cf. ulna var. andina manguin + + synedra sp. 15 nawqa morales + + tabellaria flocculosa (roth) kützing + + 2 ulnaria sp. 1 sehuencas 1 + + + ulnaria sp. 2 sehuencas + + + 10 ulnaria sp. 3 sehuencas + ulnaria sp. 4 sehuencas + 100 100 100 100 100 tab. 2. – continued u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:46 color profile: disabled composite 150 lpi at 45 degrees less rich (6 spp.) and are mainly represented by orthoseira thwaites and melosira agardh, frequently found in benthic collections. taking compiled relative abundance into account (the sum of relative abundances for all five sites), araphids are by far the most abundant (45%) followed by biraphid diatoms (36%). monoraphids accounted for 19% and centric diatoms for only 1%. of the 39 genera recorded, gomphonema agardh, nitzschia hassall, eunotia ehrenberg and navicula accounted for the most species (23, 13, 8 and 8 spp., respectively) while the remaining genera are represented by 6 species or fewer. the high number of species within navicula is due to the inclusion of several small, unknown entities within this genus for lack of a better placement. the genera with the highest number of unknown taxa are gomphonema, eunotia and ulnaria (kützing) compère (14, 5 and 4, respectively). compiling the abundance found at all five sites (in order to estimate the overall abundance of each taxon in sehuencas), diatoma bory, planothidium bukhtiyarova et round, nitzschia, encyonema kützing and hannaea patrick are among the most abundant genera. at the species level, diatoma mesodon, planothidium haynaldii (schaarschmidt) lange-bertalot, encyonema silesiacum, hannaea arcus (ehrenberg) patrick, achnanthidium sehuencoensis sp. nov. are among the most abundant (tab. 3). from the list of 10 species with the highest relative abundance presented in table 3, two taxa were undetermined, namely achnanthidium sehuencoensis sp. nov. and nitzschia sp. 1 (from sehuencas). the achnanthidium representative was found in high proportions under sem and thus we describe it here as a new taxon. although less frequent (but easily found under the microscope due to its size), fragilaria cochabambina sp. nov. was also studied under sem. acta bot. croat. 68 (2), 2009 271 bolivian cloud forest diatoms tab. 3. relative abundance data compiled (summed) for the five sites included in this study. left two columns: 10 most abundant genera; right two columns: 10 most abundant species. relative abundance at each site was calculated counting 100 valves along a randomly placed transect. *small nitzschia morphologically related to n. acidoclinata lange-bertalot, but with no defined central area spacing between fibulae. most abundant genera relative abundance compiled for 5 sites most abundant species relative abundance compiled for 5 sites diatoma 164 diatoma mesodon kützing 164 planothidium 48 planothidium haynaldii (schaarschmidt) lange-bertalot 41 nitzschia 41 encyonema silesiacum (bleisch) mann 39 encyonema 39 hannaea arcus (ehrenberg) patrick 38 hannaea 38 achnanthidium sehuencoensis sehuencas 25 rhoicosphenia 22 nitzschia clandestina manguin 24 reimeria 15 rhoicosphenia abbreviata (agardh) lange-bertalot 22 gomphonema 15 achnanthidium minutissimum (kützing) czarnecki 18 navicula 11 reimeria sinuata (gregory) kociolek et stoermer 15 ulnaria 10 nitzschia sp. 1 sehuencas 14 u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:46 color profile: disabled composite 150 lpi at 45 degrees taxonomic section fragilaria cochabambina morales sp. nov. lm figs. 2–7; sem figs. 15–20 holotype: national botanical garden, meise, belgium br-4143 isotype: herbario criptogámico universidad católica boliviana, cochabamba h.c. ucb d-1 type locality: río ibirizu, sehuencas, provincia carrasco, cochabamba, bolivia. 17° 29.8" s, 65° 16.4" w. 272 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. pl. 1. lm images from type material of the two taxa described as new herein (figs. 2–14). fragilaria cochabambina sp. nov. (figs. 2–7). images 2–4 have been taken at higher magnification to highlight rimoportulae (2 per valve) and striae with a single row of transapically elongated areolae. notice formation of palisade colonies in figure 7. achnanthidium sehuencoensis sp. nov. (figs. 8–14). notice clear central area in raphe valve and the absence of such an area in the rapheless valve. figure 14 shows two contiguous frustules, probably the products of recent vegetative cell division. u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:48 color profile: disabled composite 150 lpi at 45 degrees etymology: epithet makes reference to the department of cochabamba, bolivia. descriptio: frustula longa rectangularia indentata ad apices in margines limbi et valvocopulae junctura. valvae longae subrostratis nonnunquam subcapitatis late rotundatis apicibus contracti non nisi ad polos, 88–296 mm longae, 8–9 mm latae, striae 8–10 in 10 mm. area axialis anguste linearis inter strias aequalia ubique valvam. striae alternae parallelae modice radiantes ad apices. striae continuae in medio limbis valvae sed interruptae in limbi et valvae junctura per spinas. costae elevatae aspectu externo. area centralis fasciis quadratis vel rectangularibus. aliquot phasma-striae in fasciarum valvaribus marginibus. spinae spathulatae solidae obfirmans finitimae valvae. areolae rimiformes apicaliter elongatae in striis plerumque uniseriatis. parviores rotundatae areolae et rimiformes alternantibus in striis versus polos nonnunquam. volae ramosae crescentes ex ambitu interno areolae. area apicalis pororum evoluta typus ocellulimbus constata ex seriebus rotundatis pororum. duae structurae cornuatae in extremitatibus arearum apicalis pororum. duae rimoportulae, unum in quoque apicibus valvarum. valvocopulae clausae cum singulari serie areolarum. valvocopulae margo ab valva modice curvatus itaque valvocopulae vadosior ad apices. copulae non visae. chromatophora incognita. description: frustules long and rectangular with indentations at the apex, where the valve mantle edges meet the valvocopulae. valves long with subrostrate (subcapitate in some cases), broadly rounded apices that taper only at the end of the valves. length: 88–296 mm, width: 8–9 mm, striae density 8–10 in 10 mm. axial area narrow and straight, delimited by striae of equal length throughout the valve. striae alternate and parallel becoming slightly radiate toward the apices. each stria continues onto the middle of the valve mantle, but is interrupted at the valve face-mantle junction by spines. costae are raised in outer view. central area with clear square or rectangle-shaped fascia. some ghost striae can be seen on the valve face edges of the fascia. spines are spatulate and solid, and effectively interlock neighboring valves. areolae are slit-like, apically elongated, and mostly forming a single row along each stria. sometimes striae toward the poles exhibit two smaller, round areolae alternating with the regular slit-like openings. volae are branched and grow from the internal contour of the areola. apical pore fields well-developed, of the ocellulimbus type, and composed of several rows of round pores. two terminal horn-like structures are located on top of each apical pore field. two rimoportulae are present, one on each valve apex. valvocopulae are closed and bear a single row of areolae of similar characteristics to those along striae. the abvalvar edge of the valvocopula is slightly curved and as consequence, the valvocopula is shallower at the apex. copulae not observed. plastids unknown. achnanthidium sehuencoensis morales sp. nov. lm figs. 8–14; sem figs. 21–26 holotype: national botanical garden, meise, belgium br-4144 isotype: herbario criptogámico universidad católica boliviana, cochabamba h.c. ucb d-2 type locality: unnamed stream (stream 1 in table 1), sehuencas, provincia carrasco, cochabamba, bolivia. 17° 31.6" s, 65° 16.2" w etymology: epithet makes reference to the name of the region where the type locality is found. descriptio: frustula rectangularia modice arcuatae itaque raphovalva concava. raphovalvae lanceolatae vel subellipticae apicibus modice angustatis late rotundatis. longiacta bot. croat. 68 (2), 2009 273 bolivian cloud forest diatoms u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:48 color profile: disabled composite 150 lpi at 45 degrees 274 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. pl. 2. sem images of the new taxa presented in this manuscript (figs. 15–26). fragilaria cochabambina sp. nov. (figs. 15–20) from type material. figures 15, 17 and 19, and 16, 18 and 20 are of the same valve. notice presence of two rimoportulae per valve, the clear central area extending to both sides, the spines located along the striae, and the transapically elongated areolae. figures 16 and 20 show side views of the apical pore fields bearing two short horn-like spines, and the ends of a closed valvocopula bearing a single row of pores. achnanthidium sehuencoensis sp. nov. (figs. 21–26) from type material. figures 21 and 22 show outer details of the raphe valves. figure 23 shows an inner view of a raphe valve. notice clear fusion of the helictoglossa with apical thickenings. figure 24 depicts an outer view of the rapheless valve. a single row of areolae on mantle can be seen, with more clarity toward valve ends. figures 25 and 26 show inner details of rapheless valves. u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:50 color profile: disabled composite 150 lpi at 45 degrees tudo: 12–22 mm, latitudo: 2–4 mm, striae 20–24 in 10 mm. sternum lanceolatum elevatum interne cum costis nonnunquam area axiali elongata rhombica. raphe filiformis. extrema centralia declinata unilateraliter extensa ultra strias in area hyalina affini valva. interne extrema terminalia helictoglossis elevatis connatis ad condensationem valvae apicis. externe extrema centralia modice expansis non nisi per microscopium photonicum. interne extrema centralia uncinata declinata invicem. striae parallelae sed fortiter radiantes densiores ad apices. stria curta unilateraliter vel bilateraliter in area centrali. striae modice separatior in area centrali. areolae rotundatae interne et externe similares. extremitas striae in valva procul valvae-limbi marginem pergens ab singulari areola in limbo etiam procul valvae-limbi marginem itaque area hyalina cingenti limbo formans. hymenes versus aperturam externam areolae. area centralis stauro conspicuo rectangulari praeditae extenso trans valvam nonnunquam sejunctis areolis. araphovalvae acutioribus apicibus striationibus similibus quam raphovalvae sed latioribus dispositionibus striarum ad apices stauro inconspicuo. striae 21–24 in 10 mm. singularis series pororum in limbo ad apices. hymenes non visae. chromatophora incognita. description: frustules rectangular and slightly arched, producing a concave raphe valve. raphe valves lanceolate to subelliptical with slightly tapering, broadly rounded apices. length: 12–22 mm, width: 2–4 mm, striae density 20–24 in 10 mm. sternum lanceolate or sometimes showing an elongated diamond-shaped axial area, and bearing a filiform raphe with terminal endings that are externally only slightly deflected in the same direction. these terminal raphe endings extend beyond the striae and onto a clear area that borders the entire valve face. internally, the terminal raphe endings are terminated in helictoglossa that are raised and fused to a thickening of the valve apex. proximal raphe fissures externally slightly expanded, feature visible only under lm. internally, proximal raphe endings are conspicuously deflected away from each other and are hooked. striae vary from parallel to strongly radiate toward the apices, where they also increase in density. areolae round and do not vary much in internal and external views. each stria ends on the valve face and at a distance from the valve face-mantle edge, then it is continued by a single round areola on the valve mantle, also situated at a distance from the valve mantle-valve face edge, forming as a consequence a clear area that surrounds the entire valve mantle. hymenes located toward the outer opening of the areolae (not shown here). central area with a clear, rectangular stauros extending across entire valve face. sometimes, isolated areolae can be present in the stauros. rapheless valves with more acute apices than raphe counterparts. pattern and characteristics of striation are very similar, except that the spacing between striae toward apices is wider and there is no clear stauros. sometimes a shorter stria can be present at one or both sides of the central area. striae in the central area are slightly more separated from each other. striae density 21–24 in 10 mm. sternum lanceolate and raised together with the costae in internal view. single row of pores on the mantle stops before the valve apices. hymenes not observed. girdle bands not observed. plastids unknown. discussion the high number of taxa found in only 5 epilithic gatherings from sites located relatively close to each other is remarkable. sehuencas is only a small part of the carrasco national park and therefore, the total number of taxa in the park could be much higher, espeacta bot. croat. 68 (2), 2009 275 bolivian cloud forest diatoms u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:50 color profile: disabled composite 150 lpi at 45 degrees cially if other compartments within rivers and lakes are included in future sampling efforts. taking into account that 36% of the recorded flora corresponds to unknown entities, it is possible that a large fraction of these diatoms is endemic to bolivia and to the region. conservation of aquatic systems could be easily justified if diatoms and other algae and organisms (for which there are currently no available data) are taken into account. traditionally, national parks have been established based on organisms that man has historically considered as »important«, that is, higher plants and vertebrates. however, the recognized value of algae, especially diatoms, as primary producers and in the maintenance of aquatic food webs has been neglected in conservation practices, with adverse affects on applied studies such as environmental impact and bioremediation. further studies are needed to assess the true biodiversity potential of bolivian parks and ecosystems. at present, bolivia is considered among the 20 countries with most biodiversity in the planet. if the algae were included, the country’s ranking could be increased considerably. to assure taxonomic precision and consistency, it is important to recognize the diversity of diatoms in bolivian water ecosystems by careful taxonomic identification using a combined approach between lm and sem. it is also of utmost importance that pertinent floras are used as source of names, because the use of floras from parts of the world that are ecologically different from the ecosystems found in tropical south america could lead to serious biodiversity estimation errors (mann and droop 1996, kociolek and spaulding 2000) and to biased assessments of biogeographic patterns (edlund and jahn 2000). currently, even if all the literature on bolivian diatoms is combined, an estimation of the species richness of this group would be difficult to determine, precisely because most of that literature is not specialized and because taxa have been force-fitted into european-based concepts (morales et al. 2008). the epilithic flora found in sehuencas is dominated by cosmopolitan taxa commonly found in waters with high nutrient content (van dam et al. 1994) (tab. 3). this is evidence of the degree of impact of uncontrolled tourism in the area. it is very common to find bottles, cans, plastic bags, and even piles of organic and other garbage items in the area contiguous to the sampling sites. an added effect comes from cattle and farming practices that would increase the nutrient content in the water favoring the development of taxa common world-wide. from the list of the most abundant species presented in table 3, eight have been characterized ecologically (van dam et al. 1994). based on these taxa, the 5 rivers sampled for this study can be determined to be meso to eutrophic, with circumneutral ph, probably with high concentrations of organically bound nitrogen, and oxygen levels above saturation. the two remaining species not found in van dam et al. (1994), namely nitzschia clandestina and nitzschia sp. 1 (from sehuencas) do not have published ecological information. nitzschia clandestina was originally described from río fortaleza near lima, peru at 1400 m.a.s.l., where it was found forming epilithic growths (manguin 1964). on the other hand, n. sp. 1 (from sehuencas) was found growing on rocks in río ibirizu, río sehuencas and stream 3, which share similar ecological conditions (cold, circumneutral, and low conductivity waters). two of the taxa that were found in sufficient abundance during sem analyses were described above in an initial effort to solve the taxonomy of those 36% of taxa that could not be identified during our study. the rest of the taxa will have to be described at a later date and when developed populations are found in additional field collections. 276 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:50 color profile: disabled composite 150 lpi at 45 degrees fragilaria cochabambina sp. nov. has features similar to other taxa currently included in fragilaria lyngbye. a recent review by tuji and williams (2006) showed that the presence of a rimoportula at each valve apex is not a restriction for a taxon to be placed in this genus (many fragilaria spp. have only one). fragilaria capucina desmazières has two rimoportulae per valve, spines interrupting the striae, valvocopulae that are shallower toward the apices and bear a single row of areolae, all features found in f. cochabambina. although the revision of the generitype of fragilaria (fragilaria pectinalis (müller) lyngbye) is pending, it is possible that its general structure does not differ too much from that of f. capucina and f. cochabambina. one feature that is different in f. cochabambina is the closed nature of the girdle bands (open in f. capucina and other fragilarioids). ulnaria has closed girdle bands, but consideration of only this feature is insufficient to place f. cochabambina in ulnaria. characters such as striae, areolae, spine structure and position, apical pore fields, and other features of the girdle bands such as arching and presence of a single row of areolae, are all congruent with other taxa in fragilaria. fragilaria cochabambina sp. nov. is unique among fragilarioid taxa due to its large size and robust architecture. it shares this characteristic with taxa in ulnaria, especially u. ungeriana (grunow) compère, which is very similar in gross morphological features. in u. ungeriana, however, the degree of tapering of the valve apices is not as pronounced as in f. cochabambina and its apices are also more broadly rounded (morales 2003, morales et al. 2007). the striae in ulnaria ungeriana vary from opposite to alternate on the same valve and are more radiate toward the apices. also, the areolae in u. ungeriana are less elongated and more broadly elliptical. the volae in u. ungeriana are conspicuously developed and are composed of a solid plate positioned in the middle of the foramina and attached by several siliceous bridges to the internal contour of the areola. this type of vola has not been observed in f. cochabambina, but rather branched volae are present. another difference lies in the valvocopulae, which are arched in f. cochabambina, but straight in u. ungeriana. one of the most conspicuous differences between these two taxa is the position of spines, which interrupt the striae in f. cochabambina, but are on the costae in u. ungeriana. achnanthidium sehuencoensis possesses all features currently considered within the genus achnanthidium kützing. the raphe valves of the new taxon are concave, as in all species currently placed in achnanthidium. the characteristics of the raphe, the progressively less coarse striae toward the apices and the characteristics of areolation are all congruent with other species within the genus. there are a number of taxa with striae interrupted at the central stauros as in a. sehuencoensis, but they differ from the latter in valve outline, characteristics of the raphe, striation pattern, and areolae shape and distribution on valve face and mantle of both raphe and rapheless valves (tab. 4). it is worth noting that the type of the genus, achnanthidium minutissimum (kützing) czarnecki also has the clear central stauros, but the valve outline and striation pattern are different from those of a. sehuencoensis (compare our sem material to that of the generitype presented in potapova and hamilton 2007). additionally, a. minutissimum of the type has slits on the valve mantle (and sometimes on the valve face toward the valve face-mantle junction), instead of the round areolae present in a. sehuencoensis. it is interesting that the raphe structure is very similar in these two taxa and that the degree of fusion of the helictoglossa with the thickened apex of the valve sets them apart from the rest of achnanthidium spp. for which pubacta bot. croat. 68 (2), 2009 277 bolivian cloud forest diatoms u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:51 color profile: disabled composite 150 lpi at 45 degrees lished sem information is available (see for example kobayasi 1997, potapova and ponader 2004, cantonati and lange-bertalot 2006, potapova 2006, ponader and potapova 2007, etc.). other taxa in achnanthidium have the helictoglossa fused to the apex, but the thickening of the latter is not as pronounced (e.g., a. atomoides monnier, lange-bertalot et ector, in monnier et al. 2004) (tab. 4). in other cases, there is a clear depression between the raised termination of the helictoglossa and the apex (as in a. rivulare potapova et ponader potapova and ponader 2004). the significance of the degree of fusion of the helictoglossa to the valve apex is not known, and further assessments are needed to explore its usefulness as a distinguishing character within achnanthidium. one of the distinguishing features of a. sehuencoensis is the considerable deviation of the proximal raphe endings seen in internal view. this feature is also present in achnan278 acta bot. croat. 68 (2), 2009 morales e. a., fernández e., kociolek p. j. tab. 4. comparison of relevant morphological features among taxa most similar to achnnthidium sehuencoensis and for which lm and sem of type material are available. taxon raphe central stauros/central area striae external view internal view a. minutissimum filamentous, with proximal raphe ends slightly enlarged and ending opposite to each other. distal ends continue onto valve mantle at apex and curve slightly to the same side. sometimes at least one of the terminal ends stops before the valve apex, while the other reaches the mantle. runs along raised sternum. branches slightly deviated from each other along apical axis so they end away from each other. proximal ends slighlty hooked and bent in opposite directions. helictoglossae raised and fused with the apex by a slight thickening. clear in raphe valve, sometimes with a few isolated areolae. no apparent stauros in rapheless valve although some shortened and more spaced striae are present in this area. raised sternum in internal view lacking. radiate throughout in both valves and increasing in density toward apices. a. sehuencoensis filamentous, with proximal raphe ends slightly enlarged and ending opposite to each other. distal ends touch only slightly the valve mantle at apex and curve slightly to the same side. runs along raised sternum. branches sometimes slightly deviated from each other along apical axis so they end away from each other. proximal ends slighlty hooked and bent in opposite directions. helictoglossae raised and fused with the apex by a clearly elevated thickening. clear in raphe valve, sometimes with a few isolated areolae. no apparent stauros in rapheless valve although some shortened and more spaced striae are present in this area. raised sternum in internal view lacking. parallel to radiate in raphe valves; slightly to strongly radiate in rapheless valves (sometimes a few striae are parallel at the center of the valve). increasing in density toward apices in both valves. u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:51 color profile: disabled composite 150 lpi at 45 degrees thidium affine (grunow) czarnecki (sensu lange-bertalot and krammer 1989, not the type). the rest of the features are different between a. sehuencoensis and a. affine. the shape of the valve is predominately lanceolate, the raised helictoglossa is separated from the valve apex, and the row of valve mantle areolae travel around the valve apex in both the raphe and rapheless valves in a. affine, which is not the case of a. sehuencoensis. in 1966, hohn described achnanthes kryophiloides from río rondos in the peruvian amazon, a taxon that is very similar to achnanthidium sehuencoensis. however, a. kryophiloides has slightly wider valves (4.2 mm) and higher average striae density (24 in 10 mm). a preliminary analysis of the type slide deposited by hohn in philadelphia (ansp. g.c. 25858a) yielded no specimens of achnanthes kryophiloides (m. potapova pers. comm.). examining the protologue of the species (hohn 1966) it appears that hohn based acta bot. croat. 68 (2), 2009 279 bolivian cloud forest diatoms taxon raphe central stauros/central area striae external view internal view a. pyrenaicum filamentous and running along slightly raised sternum. proximal raphe ends slightly enlarged and ending opposite to each other. distal ends never reach valve apex and deflect markedly in the same direction and stop at valve face clear perimeter, never reaching the mantle. runs along raised sternum. branches sometimes slightly deviated from each other along apical axis so they end away from each other. proximal ends not hooked, but deviate at an open angle and in opposite directions. helictoglossae raised and not fused with the apex, leaving a clear gap between it and the valve apex internal mantle face. sometimes clear in raphe valve, but often with shortened or even complete striae. no apparent stauros in rapheless valve although some shortened and more spaced striae are present in this area. raised sternum in internal view present. mostly parallel or slightly radiate, especially toward apex in both valves. increase in density toward apices is not conspicuous. a. atomoides filamentous and running along slightly raised sternum with conspicuously swollen central nodule. proximal raphe ends slightly enlarged and ending opposite to each other. distal ends never reach the valve apex, but not the mantle. deflection of the distal ends in the same direction is barely noticeable. runs along raised sternum with swollen central nodule. proximal ends not hooked, but deviate at an open angle and in opposite directions. helictoglossae slightly raised and with a shallow gap between it and the valve apex internal mantle face. clear in raphe valve. no apparent stauros in rapheless valve although some shortened and more spaced striae are present in this area. raised sternum in internal view lacking. strongly radiate in raphe valve and mostly parallel to slightly radiate toward apices in rapheless valve. increase in density toward apices is more marked in raphe valves. tab. 4. – continued u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:51 color profile: disabled composite 150 lpi at 45 degrees his description on a single specimen and that the range of striae measurements given by him (»22–26/10 mm«) might actually correspond to striae density on the raphe and rapheless valves, respectively. investigation of type material for a. kryophiloides is underway. another taxon that is morphologicaly similar to achnanthidium sehuencoensis is a. pyrenaicum (hustedt) kobayasi (tab. 3). both taxa produce rapheless valves that are similar to each other under lm, except that the striae are more dense (up to 38 in 10 mm following potapova and ponader 2004) and are mostly parallel becoming radiate only toward the apices in a. pyrenaicum. a. sehuencoensis has up to 24 striae in 10 mm, only a few are parallel toward the valve center and they quickly become radiate toward the apices. achnanthidium pyrenaicum has more fusiform valves, especially true for the raphe valves (see study of the type presented by potapova and ponader 2004); the shape of the valves in a. sehuencoensis is subelliptical with more broadly rounded apices. at the sem level, the differences between a. sehuencoensis and a. pyrenaicum are more conspicuous. the terminal ends of the raphe are markedly deflected in the same direction in a. pyrenaicum while in a. sehuencoensis the deflection is slight. the internal view of the raphe valves in a. pyrenaicum shows a slightly raised sternum that becomes even more raised toward the valve apices, ending in helictoglossae that are clearly separated from the valve apices (potapova and ponader 2004, fig.107). a. sehuencoensis does not have a clearly raised sternum and the helictoglossae are clearly fused with the valve apices. the two species described in this manuscript as new thrive in circumneutral ph and low conductivity waters (tab. 1). the temperature of the water is relatively low for both type localities, and both are affected by human-related disturbance, although the waters remain clear. nutrients were not measured during this investigation due to high costs. acknowledgements we thank the california academy of sciences and the museum of natural history, the university of colorado at boulder, usa for partial financial support of this project. eam thanks the academy of natural sciences of philadelphia (especially dr. d. charles) for travel support to visit the gabriel lippman research center in luxembourg, where the sem analysis was performed. l. ector and c. wetzel made this latter analysis possible, and eam is greatly indebted to them. dr. m. potapova, kindly provided some comments 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(bacillariophyceae) and the identity of the type of an alleged synonym, fragilaria capucina desm. taxon 55, 193–199. van dam, h., mertens, a., sinkeldam, j., 1994: a coded checklist and ecological indicator values of freshwater diatoms from the netherlands. netherlands journal of aquatic ecology 28, 117–133. acta bot. croat. 68 (2), 2009 283 bolivian cloud forest diatoms u:\acta botanica\acta-botan 2-09\morales.vp 5. listopad 2009 16:19:51 color profile: disabled composite 150 lpi at 45 degrees acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 107 acta bot. croat. 76 (1), 107–110, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0043 issn 0365-0588 eissn 1847-8476 short communication temperature-dependent chlorophyll accumulation and photosystem ii assembly during etioplast to chloroplast transition in sunfl ower cotyledons hrvoje lepeduš1*, mario jakopec2, jasenka antunović dunić3, goran krizmanić4, sanida osmanović5, vera cesar3 1 faculty of humanities and social sciences, josip juraj strossmayer university of osijek, lorenza jägera 9, 31000 osijek, croatia 2 vodovod osijek, poljski put 1, 31000 osijek, croatia 3 department of biology, josip juraj strossmayer university of osijek, ulica cara hadrijana 8/a, 31000 osijek, croatia 4 agricultural institute osijek, južno predgrađe 17, 31000, osijek, croatia 5 faculty of natural sciences, university of tuzla, univerzitetska 4, 75000 tuzla, bosnia and herzegovina abstract – despite numerous data dealing with the biogenesis of photosynthetic membranes connected with specifi c functional alterations in higher plants this is still an insuffi ciently understood topic and is one of the most promising areas of research in plant biochemistry. the main goal of our study was to detect the impact of different temperatures on chlorophyll biosynthesis and the maximum quantum yield of psii (fv/fm). therefore, we investigated the greening processes in etiolated sunfl ower cotyledons (helianthus annuus l.) grown at different temperatures (10, 20 and 30 °c) during 24 h. the dynamics of chlorophyll a and b (chl a and chl b) accumulation as well as photosystem ii (psii) effi ciency were observed. we also evaluated combined effects of different temperatures (20 and 30 °c) and short-term application of increased irradiation (800 μmol m–2 s–1) on effective quantum yield of psii (δf/f’m) and non photochemical quenching (npq) in cotyledons with fully developed psii. our results showed reduced chlorophyll accumulation and the arrest of psii assembly at 10 °c in comparison with 20 and 30 °c. further, the increased irradiance induced equal down regulation of effective quantum yield of psii at 20 and 30 °c, with signifi cantly higher capability of heat dissipation at 30 °c. keywords: chlorophyll, chloroplast biogenesis, photosynthesis, photosystem ii, thylakoid membranes * corresponding author, e-mail: hlepedus@yahoo.com introduction thylakoid membranes in chloroplasts of higher plants are arranged in a highly fl exible way so as to conduct effi cient photosynthesis (tikkanen and aro 2014). they comprise several multiprotein complexes responsible for key photochemical processes such as light harvesting, trapping of excitons and electron transport to its fi nal acceptor (nadp+). a lot of studies have been undertaken with the aim of elucidating the de novo development of thylakoid membranes with special emphasis on the precise order of multiprotein complex assembly as well as on their interplay with photosynthetic pigments and their precursors (rudowska et al. 2012). still, some key processes have remained insuffi ciently understood and thus became muchdiscussed topics in scientifi c debates (pudelski et al. 2009). however, it is well known that the key step in the transition of etioplast to chloroplast is regulated by catalytic activity of protochlorophyllide oxidoreductase a (por a). the enzyme was found in etioplast prolamellar bodies and was shown to catalyze the reduction of protochlorophyllide to chlorophyllide in angiosperm plants upon exposure to light (reinbothe et al. 2010). one of the key regulatory components of the thylakoid electron-transport chain is chloroplast water-plastoquinone oxidoreductase, known as photosystem ii (psii), which is divided into three functionally distinct parts: antennae, reaction centre (rc) and oxygen evolving complex (oec) (barber et al. 1997). the effi ciency of psii can be quantifi ed thanks to the fl uorescence feature of chlorophyll molecules. the methodology enables determination of the maximum quantum yield of psii (fv/ fm) refl ecting the light-harvesting effi ciency of psii and is widely used to evaluate its general functionality (schreiber et al. 1995). lepeduš h., jakopec m., antunović dunić j., krizmanić g., osmanović s., cesar v. 108 acta bot. croat. 76 (1), 2017 the aim of our study was to investigate the dynamics of chlorophyll a and b accumulation as well as psii effi ciency during greening process in etiolated sunfl ower cotyledons exposed to the different temperatures (10, 20, 30 °c). since it is generally well known that low temperatures inhibit enzymatic activity, we hypothesized that the lowest temperature would result in slower accumulation of chlorophylls in developing chloroplasts. however, we aimed to place particular emphasis on the way this would be refl ecteed in the fv/fm and chl a/b ratio, the important parameter that infl uences psii stability (sakuraba et al. 2010). also, we endeavored to evaluate the effects of greening temperature and short-term application of increased irradiation on the effective quantum yield of psii (δf/f’m) and its capacity for non photochemical quenching (npq) as very important protective process against photoinhibition. materials and methods sunfl ower (helianthus annuus l.) seedlings were germinated in the darkness at room temperature for 7 days. etiolated cotyledons were then removed from the darkgrown plants and subjected to greening by exposure to 100 μmol m–2 s–1 of white light (osram powerstar hqi bt 400 w/d e40), for 3, 6, 12 and 24 h. three separate experiments were done by changing the temperature during greening process: 10, 20 and 30 °c. chlorophylls a and b were quantifi ed spectrophotometrically, and concentrations were calculated according to lichtenthaler (1987). in vivo chlorophyll a fl uorescence measurement (mini-pam, waltz) was used to quantify several parameters of photosystem ii (psii) effi ciency: the maximum quantum yield of photosystem ii (fv/fm), the effective quantum yield of photosystem ii (δf/f’m) and non-photochemical quenching (npq) at 800 μmol m–2 s–1 (schreiber et al. 1995). a student t-test as well as factorial analysis of variance followed by least signifi cance difference (lsd) test was performed to analyze statistical signifi cance (statistica 7.1.). results and discussion chloroplast biogenesis, usually seen as greening (fig. 1), is characterized by coordinated biosynthesis of photosynthetic pigments and other molecules essential for the assembly of a fully functional photosynthetic apparatus. since functional chloroplasts contain up to 4 thousand different proteins, most of them encoded by nuclear genes, it is likely that different environmental signals (e.g. light or temperature) would have a great infl uence on signaling during chloroplast biogenesis (fey et al. 2005). in order to quantify green color appearance after a certain time of greening, changes in chl a and b concentrations were measured. our results (tab. 1) revealed a permanent increase of chl a during 24 h of greening at temperatures of 20 and 30 °c, as well as a constant increase of chl b at 30 °c. also, delay in chl a accumulation was observed at 10 °c, as well as in chl b accumulation at 10 and 20 °c during the fi rst 6 h of greening. further, chl b accumulation at 10 and 20 °c was slowed down after 12 h of greening. generally, inferior chlorophyll biosynthesis was revealed in greening cotyledons at lower temperatures, especially at 10 °c. this was also evident from the value of chl a/b ratio after 24 h of greening, which was lower in cotyledons that were greening at 10 °c in comparison to these that were greening at 20 and 30 °c (tab. 1). tewari and tripathy (1998) revealed 90% inhibition of chl biosynthesis during greening of etiolated cucumber seedlings at 7 °c caused by the inhibition of enzymes involved in protoporphyrin ix biosynthesis, which directly decreased the biosynthesis of protochlorophyllide. so, the lower chlorophyll concentrations in such cases would be the result of diminished biosynthesis. further, low temperatures diminish co2 fi xation and thus increase the chance for over-reduction of the electron-transport chain and consequently for photo-damage of psii due to inhibition of the repair cycle of d1 protein, the essential protein for optimal psii functioning (giardi et al. 1997). fig. 1. greening of etiolated sunfl ower cotyledons (0 h) exposed to different temperatures (10 °c, 20 °c, 30 °c) and the light intensity of 100 μmol m–2 s–1 for 24 h. color changes were recorded after 3, 6, 12 and 24 h. tab. 1. changes in concentrations of chlorophyll a (chl a), chlorophyll b (chl b) and chlorophyll a/b ratio (chl a/b ) in sunfl ower cotyledons, measured after 3, 6, 12 and 24 h of the greening period, at 10 °c, 20 °c and 30 °c. data represent arithmetic means±standard deviations of ten replications. factorial analysis of variance followed by least signifi cance difference test was done separately for each temperature. different lowercase letters indicate a signifi cant difference at p<0.05. fw – fresh weight. g re en in g te m pe ra tu re l ig ht ex po su re chl a (mg g–1 fw) chl b (mg g–1 fw) chl a/b 10 °c 3 h 0.034±0.012a 0.047±0.022a 0.828±0.372a 6 h 0.027±0.007a 0.039±0.015a 0.747±0.214a 12 h 0.053±0.013b 0.080±0.024b 0.691±0.172a 24 h 0.078±0.013c 0.084±0.025b 0.958±0.172a 20 °c 3 h 0.065±0.010a 0.057±0.019a 1.311±0.586a 6 h 0.122±0.033b 0.064±0.022a 2.018±0.526b 12 h 0.253±0.048c 0.097±0.034b 2.747±0.654c 24 h 0.336±0.079d 0.113±0.035b 3.048±0.374c 30 °c 3 h 0.082±0.011a 0.066±0.024a 1.352±0.383a 6 h 0.172±0.037b 0.097±0.042b 1.916±0.493b 12 h 0.372±0.084c 0.125±0.024c 2.967±0.264c 24 h 0.440±0.046d 0.166±0.025d 2.707±0.473c temperature dependent chloroplast biogenesis acta bot. croat. 76 (1), 2017 109 changes in maximum quantum yield of psii (fv/fm) are shown in fig. 2. continuous increase of fv/fm was observed during fi rst 12 h of greening at 10 °c (fig. 2a). however, the upcoming arrest of psii assembly that was observed after 24 h of greening at 10 °c (fig. 2) was most likely due to the inability of thylakoid membranes to accumulate de novo synthesized chloroplast encoded proteins (such as d1) relatively to nuclear encoded ones (allen and ort 2001). this would cause improper chlorophyll-protein complex assembly as well as the formation of fully competent grana (rudowska et al. 2012) required for fv/fm values usually observed in mature leaves. such a scenario consequently led to the irreversible photoinhibition that was revealed (fig. 2a). values of the fv/fm in fully competent and healthy photosynthetic leaves are usually reported to be about 0.83 (schreiber et al. 1995). bolhar-nordenkampf et al. (1989) reported the fv/fm of 0.75 as the lowest boundary value of psii functionality. judging from our results (fig. 2b, c), cotyledons that were greening at 20 and 30 °c developed fully functional psii. muraja ljubičić et al. (1998) reported parallel increase of light-harvesting proteins of psii (lhcii) with chlorophyll accumulation during greening of potato. at the temperature of 30 °c psii functionality was achieved after as little as 6 h of greening (fv/fm=0.77), while at the temperature of 20 °c it needed a longer period (24 h for fv/fm to be 0.76). increased accumulation of chl a and b, as well as increased chl a/b ratio in these cotyledons compared to those grown at 10 °c was required for the assembly of functional psii. since the measurement of fv/fm revealed functional psii in cotyledons subjected to greening at 20 and 30 °c, additional characterization of psii effi ciency was performed. after 24 h of greening cotyledons were exposed to short-term high irradiation (800 μmol m–2 s–1) and the effective quantum yield of psii (δf/f’m) as well as non-photochemical quenching (npq) were measured (fig. 3). although there were no differences in δf/f’m upon exposure to high irradiation between cotyledons that were greening at 20 and 30 °c, cotyledons that were greening at 30 °c revealed a better capacity for npq than cotyledons that were greening at 20 °c (fig. 3). npq represents the measure for dissipation of the excess excitation energy as heat relative to dark adapted state (maxwell and johnson 2000). precise mechanisms of npq are under constant debate and new evidence is being continuously provided (jurić et al. 2013). basically, suffi cient amounts of xanthophyll cycle carotenoids and structural rearrangement of psii antennae are essential for effi cient npq. bilger and björkman (1991) showed up-regulation of the rate of violaxanthin de-epoxidation by temperature increase. it seems that higher temperature during greening process (30 °c) of etiolated sunfl ower cotyledons favored zeaxanthin formation, thereby enabling more effi cient heat dissipation by npq. it can be concluded that low temperature (10 °c) decreased biosynthesis of chl a and chl b in comparison to two higher investigated temperatures, which was directly linked with impaired assembly of psii after a prolonged period of greening (24 h) at 10 °c. lebkuecher et al. (1999) reported that assembly of psii during the transition from etioplast to chloroplast in sunfl ower cotyledons at 25 °c was accompanied by small initial lhcii complexes and reaction centers (rcs) that are defi cient in qa to qb electron transport. further greening in their experiment (up to 12 h) enabled conversion of qb-nonreducing to qb-reducing rcs and effi cient electron transport. hence, it is most likely that the arrest of psii assembly after 24 h of greening at 10 °c that we have observed was due to over-reduction damage in the psii reaction center. also, fully developed psii at temfig. 2. changes in maximum quantum yield of psii (fv/fm) in sunfl ower cotyledons, measured after 3, 6, 12 and 24 h of the greening period, at 10 °c (a), 20 °c (b) and 30 °c (c). data represent arithmetic means±standard deviations of fi ve replications. differences between presented values were evaluated using least signifi cance difference test. different lowercase letters indicate a signifi cant difference at p<0.05. fig. 3. the effect of short-term high irradiation (800 μmol m–2 s–1) on effective quantum yield of psii (δf/f’m) and non-photochemical quenching (npq) of sunfl ower cotyledons, after 24 h of greening at different temperatures (20 °c and 30 °c). data represent arithmetic means±standard deviations of fi ve replications. differences between presented values were evaluated using student’s ttest. asterisk (*) indicate a signifi cant difference at p<0.05. lepeduš h., jakopec m., antunović dunić j., krizmanić g., osmanović s., cesar v. 110 acta bot. croat. 76 (1), 2017 peratures of 20 and 30 °c was further challenged by shortterm exposure to increased irradiance (800 μmol m–2 s–1). although the equal down regulation of psii effi ciency was revealed regardless of the greening temperature, a more effi cient capability of heat dissipation was achieved by greening at 30 °c. this might be attributed to higher effi ciency of the xanthophyll cycle as well as to the different structures and connectivity of lhcii proteins (bilger and björkman 1991, tikkanen and aro 2012) and requires further investigation. acknowledgements this research was supported by the croatian ministry of science, education and sport, as part of projects no. 0730731674-1673 and 073-0731674-0841. references allen, d. j., ort, d. r., 2001: impacts of chilling temperatures on photosynthesis in warm-climate plants. trends in plant science 6, 36–42. barber, j., nield, j., morris, e. p., zheleva, d., hankamer, b., 1997: the structure, function and dynamics of photosystem two. physiologia plantarum 100, 817–827. bilger, w., björkman, o., 1991: temperature dependence of violaxanthin de-epoxidation and non-photochemical fl uorescence quenching in intact leaves of gossypium hirsutum l. and malva parvifl ora l. planta 184, 226–234. bolhàr-nordenkampf, h. r., long, s. p., baker, n. r., öquist, g., schreiber, u., lechner, e. g., 1989: chlorophyll fl uorescence as a probe of the photosynthetic competence of leaves in the fi eld: a review of current instrumentation. functional ecology 3, 497–514. fey, v., wagner, r., bräutigam, k., pfannschmidt, t., 2005: photosynthetic redox control of nuclear gene expression. journal of experimental botany 56, 1491–1498. giardi, m. t., masojídek, j., g odde, d., 1997: effects of abiotic stresses on the turnover of the d1 reaction centre ii protein. physiologia plantarum 101, 635–642. jurić, s., vojta, l., fulgosi, h., 2013: e lectron transfer routes in oxygenic photosynthesis: regulatory mechanisms and new perspectives. in: dubinsky, z. (ed.), photosynthesis, 23–46. intech, rijeka. lebkuecher, j. g., haldeman, k. a., harris, c. e., holz, s. l., joudah, s. a., minton, d. a., 1999: development of photosystem-ii activity during irradiance of etiolated helianthus (asteraceae) seedlings. american journal of botany 86, 1087– 1092. lichtenthaler, h. k., 1987: chlorophylls and carotenoids: pigments of photosynthetic biomembranes. methods in enzymology 148, 350–382. maxwell, k., johnson, g. n., 2000: chlorophyll fl uorescence – a practical guide. journal of experimental botany 51, 659–668. muraja ljubičić, j., wrischer, m., ljubešić, n., 1998: formation of the photosynthetic apparatus in plastids during greening of potato microtubers. plant physiology and biochemistry 36, 747–752. pudelski, b., soll, j., philippar, k., 2009: a search for factors infl uencing etioplast-chloroplast transition. proceedings of the national academy of sciences of the united states of america 106, 12201–12206. reinbothe, c., bakkouri, m. e., buhr, f., muraki, n., nomata, j., kurisu, g., fujita, y., reinbothe, s., 2010: chlorophyll biosynthesis: spotlight on protochlorophyllide reduction. trends in plant science 15, 614–624. rudowska, l., gieczewska, k., mazur, r., garstka, m., mostowska a., 2012: chloroplast biogenesis – correlation between structure and function. biochimica et biophysica acta 1817, 1380–1387. sakuraba, y., yokono, m., akimoto, s., tanaka, r., tanaka, a., 2010: deregulated chlorophyll b synthesis reduces the energy transfer rate between photosynthetic pigments and induces photodamage in arabidopsis thaliana. plant and cell physiology 51, 1055–1065. schreiber, u., bilger, w., neubauer, c., 1995 : chlorophyll fl uorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. in: schulze, e. d., caldwell, m. m. (eds.), ecophysiology of photosynthesis: ecological studies, 49–70. springer verlag, berlin. tewari, a. k., tripathy, b. c., 1998: temperature-stress-induced impairment of chlorophyll biosynthetic reactions in cucumber and wheat. plant physiology 117, 851–858. tikkanen, m., aro, e. m., 2012: thylakoid protein phosphorylation in dynamic regulation of photosystem ii in higher plants. biochimica et biophysica acta – bioenergetics 1817, 232– 238. tikkanen, m., aro, e. m., 2014: integrative regulatory network of plant thylakoid energy transduction. trends in plant science 19, 10–17. opce-str.vp acta bot. croat. 68 (1), 147–151, 2009 coden: abcra 25 short communication issn 0365–0588 a new nothospecies in the genus potentilla l. (rosaceae) jeremi kolv odziejek* department of geobotany and plant ecology, university of lv ódzb, banacha 12/16, 90-237 lv ódzb, poland the morphological, ecological and chorological features of a new hybrid from poland are described and illustrated. the morphological characteristics of the new nothospecies potentilla ´gabarae kolodziejek are compared with those of related species. keywords: rosaceae, potentilla, hybrid, morphology, taxonomy introduction during an expedition to czezstochowska upland, poland, in june 2003, the author found five specimens (from the same plant) of potentilla that differ from congeners in poland and the neighbouring countries. material and methods after a morphological study, literature searches (wolf 1908, szafer and pawlv owski 1955, soják j. 1995, gerstberger 2002, kurtto et al. 2004) and examination of many species, the author concluded that it represents a new nothospecies, a hybrid of p. leucopolitana p. j. müll. and p. incana p. gertner, b. meyer et scherb. results and discussion potentilla ´gabarae kolodziejek, nothosp. nova = p. leucopolitana p. j. müll. ´ p. incana p. gertner, b. meyer et scherb (fig. 1). type: poland. czezstochowska upland: jaroszów village near zarki 50°39’21”n/ 19°21’32”e, thermophilous grassland, 21.vi.2003, j. kolv odziejek (holotype lod). habitu et indumento inter parentes media. p. leucopolitana foliolis supra plerumque dense sericeo-pilosa vel cano-tomentosa, subtus cano-tomentosa. p. incana foliolis densissime stellato-tomentosa et cinerascentia, supra parce pilosa vel subglabra et viridia. caules basi ascendentes vel suberecti (14.2–)15.4(–16.7) cm longi raro longiores, ramis paniculato-corymbosis multifloris. folia basalia et caulina inferna 5–7-nata, foliolis plerumque 1.5–2 cm longis, basi longe cuneatis, antice breviter late obovatis multiacta bot. croat. 68 (1), 2009 147 * corresponding author, e-mail: kolo@biol.uni.lodz.pl u:\acta botanica\acta-botan 1-09\kolodziejek1.vp 22. travanj 2009 12:40:22 color profile: disabled composite 150 lpi at 45 degrees dentata, dentibus utrinque 4–5 obtusis aequalibus. dens terminalis foliorum (1.8–)2(–2.3) mm longis non aut parum prominens. folia caulina media 5–nata; folia caulina supera 3-nata. pagina inferior foliolorum cana, plerumque parce pilosa, subtus dense tomentosa; pilis crassiusculis rectis et imperfecte stellatis composito obtecta. flores numerosi, cymosi, 5-partiti, 8–10 mm lati. petala luteola,(3.2–)3.6(–3.9) mm, obovata sepali externi (2.7–) 2.9(–3.2) mm longi, acuto-elliptici, pilis flexuosibus dense villosi. stamina 20 antheris parvis subrotundo-ovatis. carpella valde numerosa, stylus non typice coniformis basi papilloso-incrassatus. floret vi–viii. 148 acta bot. croat. 68 (1), 2009 kolv odziejek j. fig. 1. potentilla ´gabarae (from holotype). u:\acta botanica\acta-botan 1-09\kolodziejek1.vp 17. travanj 2009 11:10:41 color profile: disabled composite 150 lpi at 45 degrees description: flowering stems (14.2–)15.4(–16.7) cm, lateral; glabrous to white-tomentose. stock with a terminal rosette of leaves during flowering. leaves digitate; leaflets 5–7, (18–)20(–24.2) ´ (9.4–)9.9(–10.3) mm, oblong-obovate with 4–5 teeth; terminal tooth of leaflet (1.8–)2(–2.3) ´ (1.5–)1.8(–2.2) mm, smaller in size than adjacent lateral (2.8–)3 (–3.2) ´ (1.7–)1.8(–1.9) mm. cauline leaves with 5 leaflets (7.6–)9.1(–12.1) ´ (3.4–)4.5 (–5.3) mm with 2–3 teeth; terminal tooth of leaflet (1.2–)1.5(–1.9) ´ (0.8–)1.1–(–1.4) mm, smaller in size than lateral (1.7–)1.9 (–2.5) ´ (1.5–)1.7(–1.8) mm. leaflets sparsely sericeous hairy with numerous unicellular imperfect hairs, having a variable number of arms (3–7) radiating from a large basal cell. flowers numerous, (7.8–)8.5(–9.9) mm in diameter; sepals ovate (2.7–)2.9(–3.2) ´ (0.4)0.5(–0.6) mm; epicalyx segments linear-lanceolate (1.7–)2.1(–2.4) ´ (0.4–)0.5(–0.6) mm, shorter than sepals; petals (3.2–)3.6(–4.2) ´ (1.8–)2.3(–2.9) mm, only slightly longer than sepals. stamens (19–)20; carpel styles 30, longitudinally filiform-elongated, i.e. from the middle up to the stigma equally thick. achenes ovate, laterally flattened. apex obtuse, curved, base obtuse. surface dull, glabrous, strongly rugose-ribbed, between ribs reticulate. interspaces wider than ribs; ribs ca 10 mm wide and 10 mm high. the style scar is well above the middle of the achene (subterminal), notch prominent ca. 60 mm deep. attachment scar subbasal, without aril, 40 mm long and 30 mm wide. dorsal margin with a ridge 10 mm high. ventral suture 40 mm long and 10 mm wide. colour pale brown. size: 0.84–1.20 (0.97±0.13) mm long, 0.61–0.80 (0.64±0.09) mm wide, 0.59–0.87 (0.67±0.11) mm thick. flowering: v–vi, fruiting: vii– viii. acta bot. croat. 68 (1), 2009 149 a new nothospecies in potentilla a b c d e f g a b c d e f g a b c d e f g a b c d e f g fig. 2. distribution of potentilla ´gabarae – cartographic square in poland atpol (zajac 1978). u:\acta botanica\acta-botan 1-09\kolodziejek1.vp 17. travanj 2009 11:10:41 color profile: disabled composite 150 lpi at 45 degrees distribution: the rare species occurs in a restricted number of localities in poland, i.e. only on the czestochowska upland, in the vicinity of jaroszów and zaborze village (fig. 2). in zaborze, the nothospecies occurs in mixed populations with acinos arvensis, brachythecium albicans, carlina vulgaris, centaurea stoebe, cladonia sp., clinopodium vulgare, dianthus carthusianorum, euphorbia cyparissias, festuca ovina, lolium perenne, origanum vulgare, peucedanum oreoselinum, plantago media, poa compressa, potentilla incana, p. leucopolitana, p. tabernaemontani, scabiosa columbaria, sedum acre, stachys recta, thymus serpyllum, trifolium arvense. this type of vegetation can be classified as origano-brachypodietum medw.-korn. et kornasb 1963. 150 acta bot. croat. 68 (1), 2009 kolv odziejek j. fig. 3. a – leaf ultrastructure morphology (from holotype), upper leaf surface; b – imperfectly stellate hairs, lower leaf surface; c – lower leaf surface; d – achene of outline ovoid, laterally flattened; e – surface of achene strongly rugose-ribbed; f – close up of achene surface between ribs with thick-walled reticulate microsculpture. u:\acta botanica\acta-botan 1-09\kolodziejek1.vp 22. travanj 2009 12:40:26 color profile: disabled composite 150 lpi at 45 degrees etymology: the species epithet honours professor barbara gabara, lódz, for her contribution to the knowledge of the plant anatomy and embryology. further specimens examined: czezstochowska upland: zaborze near @arki 50°40’31”n/ 19°20’00”e, 333 m, thermophilous grassland, 3 june 2003, 17 july 2007, 14 june 2004, 30 june 2004 (j. kolv odziejek lod). affinities: p. ´gabarae resembles p. leucopolitana in having dense tomentum and leaves with 5–7 leaflets. however, there are many differences that render identification easy. the former has numerous incomplete stellate hairs on the upper side of leaves, terminal tooth of leaflet smaller in size than adjacent lateral and smaller leaves and flowers. p. ´ gabarae also resembles to some extent potentilla incana and p. ´ subarenaria borbás ex zimmeter by having stellate hairs. leaves of p. incana are covered with stellate hairs mixed with long hairs which make the plant greyish; p. ´ subarenaria, which is considered to be of hybrid origin (p. incana ´ tabernaemontani ascherson), has curved and stellate hairs on the surface. the stellate hairs of p. incana, which were described by wolf (1908) as »sternhaare«, may consist of up to 30 identically long rays, while those of p. ´ subarenaria have about 10 or less identically rays. leaflets of p. ´ gabarae are covered with straight and crispate hairs mixed with imperfectly stellate hairs which make the leaves sparsely sericeous hairy; the imperfectly stellate hairs (»zackenhaare«) consist of up to 7 not identically long arms (fig. 3 a, b). references gerstberger, p., 2002: potentilla l. in: conert, h. j., hamann, a., schultze-motel, w., wagenitz, g. (eds), g. hegi: illustrierte flora von mitteleuropa 4(2c), 109–205. blackwell, berlin. kurtto, a., lampinen, r., junikka, l., 2004: 13. rosaceae (spiraea to fragaria, excl. rubus). in: kurtto a., lampinen r., junikka l. (eds.), atlas florae europaeae. distribution of vascular plants in europe 13. rosaceae (spirea to fragaria, escl. rubus). the committee for mapping the flora of europe societas biologica fennica, vanamo, helsinki. soják, j., 1995: potentilla l. in: slavik, b. (ed.), flora of the czech republic 4, 283 – 314. academia, praha. szafer, w., pawlv owski, b., 1955: potentilla l. in: szafer, w., pawlv owski, b. (eds.), flora polska 7, 96–143. wydawnictwo naukowe pwn, warszawa-kraków. wolf, t., 1908: monographie der gattung potentilla l. bibliotheca botanica 71, 6 + [1] + 714 + 2 maps. zajac, a., 1978: atlas of distribution of vascular plants in poland. taxon 27, 481 – 484. acta bot. croat. 68 (1), 2009 151 a new nothospecies in potentilla u:\acta botanica\acta-botan 1-09\kolodziejek1.vp 22. travanj 2009 12:40:26 color profile: disabled composite 150 lpi at 45 degrees bogdanovic.pdf acta bot. croat. 74 (1), 2015 165 acta bot. croat. 74 (1), 165–172, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication aegilops uniaristata vis. (poaceae): typifi cation and occurrence in croatia sandro bogdanović1*, ivica ljubičić1, moreno clementi2 1 university of zagreb, faculty of agriculture, department of agricultural botany, svetošimunska 25, 10000 zagreb, croatia 2 university of padova, department of biology, via ugo bassi 58 b, 35131 padova, italy abstract – after 88 years, occurrence of aegilops uniaristata vis. (poaceae) in croatian fl ora was confi rmed and its distribution is supplemented by new localities. it has been confi rmed and its distribution supplemented by new localities. populations of a few specimens were found in southern istria, in the vicinity of the small town of bale, in the village of krnica and on the rt kamenjak promontory, growing within dry mediterranean grasslands. based on herbarium revision a lectotype from visiani’s collection in herbarium pad and an epitype from the herbarium w were designated. keywords: aegilops, epitype, herbarium pad, herbarium w, istria, lectotype, poaceae, rare species introduction the genus aegilops l. (poaceae) consists of more than 20 species and has been a widely studied genus among grasses since it was confi rmed to have the secondary gene pool for cultivated wheat (van slageren 1994, belkadi et al. 2003). furthermore, in some approaches the genus triticum l. is included in the genus aegilops. the species of this genus are adapted to diverse disturbed and ruderal environments, pastures, dry grasslands, edges of cultivated fi elds (belkadi et al. 2003), and are mainly distributed in southwest and central asia and throughout the mediterranean basin (cabi et al. 2010). in the fl ora of croatia the genus aegilops is represented by six species: a. cylindrica host, a. geniculata roth, a. lorentii hochst., a. neglecta req. ex bertol., a. triuncialis l. and a. uniaristata vis. (nikolić 2014). a. uniaristata was described from the zadar area as an annual species morphologically similar to a. cylindrica (visani 1852: 345). in croatia this species is protected (anonymus 2009) and it has the category of near threatened (nt) plant (nikolić and topić 2005). morphologically a. uniaristata is easy to distinguish from * corresponding author, e-mail: sbogdanovic@agr.hr copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. bogdanović s., ljubičić i., clementi m. 166 acta bot. croat. 74 (1), 2015 other aegilops species by the solitary awns on both glumes of its terminal spikelet (fig. 1). a. uniaristata can be diploid and tetraploid (2n = 14, 28) and with a. comosa sm. in sibth. et sm. it belongs to the section comopyrum (jaub. & spach) zhuk. (tutin and humphries 1980, witcombe 1983, cabi 2010). aegilops uniaristata is distributed mainly in the central and eastern mediterranean: croatia, bosnia and herzegovina, montenegro, italy, albania and greece (pignatti 1982, witcombe 1983, van slageren 1994, perrino 2011, perrino et al. 2014, nikolić 2014) while in turkey it is probably extinct (cabi 2010). it grows within mediterranean dry grasslands and sometimes on anthropogenic habitats especially at the border of olive groves (van slageren 1994, perrino 2011, perrino et al. 2014). in the croatian fl ora, according to literature data, a. uniaristata was previously known from zadar (visiani 1852), from the wider area of pula in southern istria (freyn 1878) and from the island of plavnik in the kvarner archipelago (horvatić 1927). several specimens deposited in herbarium za were collected in the period from 1872 to 1925 in poreč, rijeka, on the islands of lošinj and unije, from surroundings of pula, and along the kvarner littoral. since then, new localities have not been reported. material and methods based on fi eld surveys performed in istria in 2012 and 2013, and on herbarium revisions, new insight regarding the distribution and occurrence of a. uniaristata along the eastern adriatic coast was provided. herbarium specimens were studied in cnhm, nhmr, pad, za, zagr, zaho, w, and virtual herbaria database. abbreviations of herbaria follow thiers (2014). newly collected herbarium specimens of a. uniaristata from istria are deposited in zagr and are accessible through virtual zagr herbarium (bogdanović 2014). relevant taxonomic literature and determination keys were used for identifi cation according to witcombe (1983), van slageren (1994) and cabi (2010). a distribution map was generated using geocoded herbarium localities and literature data combined with the flora croatica database (nikolić 2014). typifi cation was performed in accordance with the icn code (mc neill et al. 2012). results and discussion aegilops uniaristata vis., fl. dalm. 3: 345 (1852). homotypic synonyms: chennapyrum uniaristatum (vis.) á. löve, triticum uniari sta tum (vis.) k. richter. fig. 1. infl orescence of aegilops uniaristata vis. (photo by s. bogdanović). aegilops uniaristata in croatia acta bot. croat. 74 (1), 2015 167 heterotypic synonyms: aegilops notarisii clementi, aegilops uniaristata steud. (nom. illeg.). species description: a. uniaristata is an annual species, with numerous stems, up to 40 cm height. leaves are glabrous or shortly hairy, with glabrous sheaths or the lower patenthairy. spike subovoid rarely moniliform, tapering towards apex, 1–3.5 cm long (excl. awns), with usually 2–3 fertile spikelets and 2(–3) rudimentary spikelets. each spikelet has one caryopsis and the whole spike breaking off as a unit at maturity. lower glume of lowest spikelets is 6–8 mm long and 4–6 mm wide, awned or toothed. glumes of apical spikelet with a fl at 3–5 cm long awn without lateral teeth or awns, glumes of lateral spikelets distinctly ventricose, bifi d into a sharp tooth and an increasing short awn towards apex. awns are variable in number and length, often patent at maturity (fig. 1). distribution in croatia: istria, kvarner archipelago and central dalmatia (town of zadar and the island of hvar) (fig. 2). our fi ndings in istria: the village of krnica near the small town of bale (vicinity of the town of rovinj) and rt kamenjak promontory provide the fi rst records of this species in the croatian fl ora after 88 years. the area of distribution of a. uniaristata has been extended from istria and kvarner archipelago to central dalmatia because of one herbarium specimen from the island of hvar found in w, while at the locus classicus in zadar the species was not confi rmed (fig. 2). a. uniaristata seems to be more widespread along the adriatic coasts of italy and croatia as concluded in perrino (2011) and perrino et al. (2014). further fi ndings of this rare species could be expected in similar habitats along croatian coast and islands. typifi cation research of original type material deposited in pad and w enabled designation of a lectotype and an epitype in accordance with art. 9.2 and art. 9.8 of the icn code (mc neill et al. 2012). fig. 2. present distribution of aegilops uniaristata vis. in croatia (▲ literature data, ● herbarium data). bogdanović s., ljubičić i., clementi m. 168 acta bot. croat. 74 (1), 2015 lectotype: croatia. circa zara [zadar], s. d., alsch. [a. alschinger] (lectotype here designated: pad-h0028902) (fig. 3, the right specimen). fig. 3. lectotype of aegilops uniaristata vis. deposited in pad, the right specimen (reproduced with permission). aegilops uniaristata in croatia acta bot. croat. 74 (1), 2015 169 epitype: croatia. circa zara [zadar], s. d., s. n. (epitype here designated: w-rchb. 1889-0251356) (fig. 4). fig. 4. epitype of aegilops uniaristata vis. deposited in w (© naturhistorisches museum wien, reproduced with permission). bogdanović s., ljubičić i., clementi m. 170 acta bot. croat. 74 (1), 2015 the sterile specimen that we have selected, from the general collection in pad (padh0028902) fully corresponds to the protologue (»in herbidis circa zara, unde communicavit prof. alschinger«). the label is in visiani’s handwriting. the lectotype is mounted on the same sheet as another, later specimen (pad-h0028901, fig. 3, left specimen). what appeared to be a fruiting duplicate is available from visiani’s main collection (herbarium dalmaticum in pad, pad-hd00406), but unfortunately the spikelets conserved in the envelope pinned to the herbarium sheet clearly do not belong to the genus aegilops and were certainly attached there by mistake. that specimen, therefore, does not correspond with the protologue and could not be selected as type. since the spike is fundamental for the morphological identifi cation of this species, we designate, as an epitype, a second specimen by visiani, in w (w-rchb. 1889-0251356), which was incorrectly recognised as an isotype by m. van slageren (wag), in 1989. this specimen was not suitable as lectotype since it cannot be shown to have been collected by alschinger as cited in the protologue. specimina visa in total forty two herbarium specimens were consulted and studied from cnhm, nhmr, pad, za, zagr, zaho and w: croatia: plavnik, 14.06.1925, s. horvatić (za14867); flora lussinensis, unija-polje, 22.05.1912, a. haračić (za14868); flora lussinensis, unie, polje, illeg., 22.05.1912, a. haračić (za14862); cigale, 24.05.1897, a. haračić (za14864); cigale, 24.05.1897, a. haračić (za14872); flora lussinensis, illeg. cigale, 12.06.1891, a. haračić (za14861); flora lussinensis, blatina, strada tur. che conduce in slatina, 15.06.1890, a. haračić (za14863); kod rieke, 06.1872, lj. rossi (za14871); istriae-australis circa parentians, m.g.s. tommasini (za14865); im val bandon b. pola, 28.08.1872, e. hackel (za14866); istria. austria, prope oppidium parentium, 06.1884, c. marchesetti (za14869); istria, is. fenera pr. promontore, 06.1893, c. marchesetti (za14870); istra, okolica mjesta bale, uz cestu, 30.05.2012, s. bogdanović & i. ljubičić (zagr33401); istra, premantura, rt kamenjak, 30.05.2013, s. bogdanović & i. ljubičić (zagr33402); hrvatska, istra, gornji kamenjak, rt bumbište-crvene stine, suhi travnjak, 30.05.2012, s. bogdanović & i. ljubičić (zagr31701); hrvatska, istra, krnica, 28.05.2014, i. vitasović kosić & m. britvec (zagr36459); isola di lesina, dalmazia, s.d., anonymous coll. (w1974-0008324); macchien-mte foibor, pola, 08.06.1901, k. untchj (w1949-0004005); auf grasplätzen um parengo und sonst hie und da in südistrien, 05.19##, m.g.s. de tommasini (w19400021473); istrien, straßenränder am monte grande bei pola, 08.06.1909, e. korb (w19510002571); austria, isria, in graminosis prope oppidum parentium, 06.1884, c. von marchesetti (w1887-0001937); auf wiesen um pola, rovigno, 05.1863, m.g.s. de tommasini (w-hackel 1916-0013598); in herbidis istria australis circa parenzo, 05.1863, m.g.s. de tommasini (w1912-0020319); istria. is. fenera pr. promontore, 1898, c. von marcchesetti (w1900-0003890); in herbidis circa zara, s.d., r. de visiani (w-rchb. 1889-0251356); val bandon b. pola, 20.05.1872, e. hackel (w-hackel 1916-0013599); auf einem grabplatz des prato grande bei pola, 08.06.1880, e. witting (w1887-0006684); flora lussinensis, blatine, 12.06.1891, a. haračić (w1927-0017653); istria. in pratis apricis ad mare prope parentium, s.d., c. von marchesetti (w 1887-0006685); ad parnzo, in graminosis, 05.1863, m.g.s. de tommasini (w0001020); istrien, an straßenrändern auf der insel brioni maggiore, 13.06.1909. e. korb (w1951-0002570); sull’isola di unie, quarnero, 23.06.1843, aegilops uniaristata in croatia acta bot. croat. 74 (1), 2015 171 m.g.s. de tommasini (pad-hd00405); circa zara, s.d., a. alschinger (pad-hd00405); zara, s.d., m.g.s. de tommasini (pad-hd00404); contorni di parenzo, frà la stanza artori e val del rio, 19.05.1843, m.g.s. de tommasini (pad-h0028901); circa zara, s.d., a. alschinger (pad-h0028902). greece: constatinople. lieux herbeux prés de zékériékeny, 02.06.21901, g.v. aznavour (w1974-0008547); flora de constantinople. lieux herbeux près de zékériékeny, 06.02.1901, g.v. aznavour (w1902-0010391); insel kérkira (nom. kerkíras). talsenke sündlich von der brücke der straße paleokastrítsa-kérkira, 22,-4 km s(-sse) skriperó, niederung unweit der straße, 09.05.2000, w. gutermann et al. 34636 (w2006-0015228); insel kérkira (nom. kerkíras). hügelland s giannádes: an der fahrpiste knapp 1 km in richtung ermónes, 15.05.2000, w. gutermann et al. 34942 (w2006-0015231); erimanthos, s.d., i. trinajstić (cnhm26920). italy: leucaspide prope tarentum in saxosis, 19.05.1881, h. groves (w-hackel 19160013597). acknowledgements the authors would like to thank curators of herbaria cnhm, nhmr, pad, za, zagr, zaho and w during investigation of herbarium specimens. this study was fi nanced by the project »vascular fl ora mapping of gornji kamenjak: pilot study 2«. references anonymous, 2009: ordinance on designating wild taxa protected and strictly protected (in croatian). narodne novine 99/09. belkadi, b., assali, n., benlhabibet, o., 2003: variation of specifi c morphological traits and ploidy level of fi ve aegilops l. species in morocco. acta botanica malacitana 28, 47–58. bogdanović, s., (ed.) 2014: virtual herbarium zagr. university of zagreb, faculty of agriculture. retrieved march 27, 2014 from http://herbarium.agr.hr. cabi, e., 2010: taxonomic revision of the tribe triticeae dumortier (poaceae) in turkey. phd thesis. faculty of arts and sciences, middle east technical university, department of biological sciences, ankara, 1–364. cabi, e., dogan, m., özler, h., akaydin, g., karagöz, a., 2010: taxonomy, morphology and palynology of aegilops vavilovii (zhuk.) chennav. 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(in croatian). ministarstvo kulture, državni zavod za zaštitu prirode, zagreb, 16–393. pignatti, s., 1982: flora d’italia, vol. 3. edagricole, bologna. perrino, e. v., 2011: new data on aegilops uniaristata vis. in italy. natura croatica 20, 117–123. perrino, e. v., wagensommer, r. p., medagli, p., 2014: the genus aegilops l. (poaceae) in italy: taxonomy, geographical distribution, ecology, vulnerability and conservation. systematics and biodiversity 12, 331–349. thiers, b., 2014: index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden’s virtual herbarium. retrieved march 27, 2014 from: http://sweetgum.nybg.org/ih/. tutin, t. g., humphries, c. j., 1980: aegilops l. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea vol. 5, 200–203, cambridge university press, cambridge. van slageren, m. w., 1994: wild wheats: a monograph of aegilops l. and amblyopyrum (jaub. & spach) eig. (poaceae). wageningen agriculture university, 1–513. virtual herbaria database. retrieved march 27, 2014 from http://herbarium.univie.ac.at/. visiani, r. de, 1852: flora dalmatica vol 3, 345, lipsiae. witcombe, j. r., 1983: a guide to the species of aegilops l. their taxonomy, morphology and distribution. international board for plant genetic resources – wheat programme. rome, 1–74. acta botanica 2-2016 za web.indd 260 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 260–265, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0025 issn 0365-0588 eissn 1847-8476 effects of biodynamic production on growth and essential oil content in basil slavica dudaš1, danijela poljuha2*, ivana šola3, sabina šegula4, sanja varga1, barbara sladonja2 1 polytechnic of rijeka, agricultural department, poreč, karla huguesa 6, poreč, croatia 2 institute of agriculture and tourism, karla huguesa 8, poreč, croatia 3 department of biology, faculty of science, university of zagreb, horvatovac 102a, zagreb, croatia 4 biotechnical centre naklo, strahinj 99, naklo, slovenia abstract – the effects of a biodynamic sowing calendar on the growth (plant height, fresh herb yield, nodes number) and quality (percentage of leaf mass, essential oil content) of three basil species, ocimum americanum l., ocimum × hybrida and ocimum basilicum l., represented by the cultivars ‘rosso’ and ‘eco genovese’, were tested. statistical analyses showed that the species had greater impact on the observed parameters than either the sowing date or the species and sowing date in combination. the species showed a signifi cant infl uence on all fi ve tested parameters, while sowing date and interaction of both factors signifi cantly infl uenced plant height, leaf percentage and essential oil content. the best yield per plant was obtained for o. × hybrida and o. basilicum ‘eco genovese’. the lowest species o. × hybrida produced the highest amount of essential oil. »nodes number« parameter most clearly separated the species, but not the cultivars. even though o. americanum gave the tallest plants, it did not yield either the highest amount of fresh herb or essential oil. this species’ height was most consistent, considering the signifi cant impact of biodynamic rhythm. sowing date was not crucial for basil fresh yield; however if there is a need for taller plants with a higher percentage of leaf mass and more essential oil, sowing date needs to be controlled. key words: biodynamic production, essential oil, ocimum americanum l., ocimum basilicum ‘eco genovese’, ocimum basilicum ‘rosso’, ocimum × hybrida, sowing calendar * corresponding author, e-mail: danijela@iptpo.hr introduction the genus ocimum belongs to the large botanical family lamiaceae which includes a high number of aromatic plants, well-known herbs and ornamental plants grown worldwide. sweet basil (ocimum basilicum l.) is the most widely grown ocimum species in the world for the fresh market, but also for essential oil production (zheljazkov et al. 2008). sweet basil essential oil is used in medicine, for aromatherapy, and the cosmetic and food industries (putievsky and galambosi 1999), and as an insecticide (umerie et al. 1998, keita et al. 2001, pascual-villalobos and ballesta-acosta 2003). due to its insecticide effects, sweet basil is also used in intercropping, for example with cotton (shader et al. 2005). after more than 200 analyses of essential oils isolated from o. basilicum l., lawrence (1988) classifi ed essential oils of sweet basil into four main chemotypes: methyl chavicol-rich, linalool-rich, methyleugenol-rich, methyl cinnamate-rich, and also found numerous subtypes. o. americanum is not often used as a culinary herb, unlike the related o. basilicum, but more often as a medicinal plant (vieira et al. 2003). the essential oils in this species have strong fungicidal activity (dubey 1991), and the leaves have been used as an insecticide against postharvest damage (weaver et al. 1991). on the global market of agricultural products, organic, safe, high quality agricultural products, including herbs and spices, achieve the best prices. organic agricultural production implies, in general, production without easily soluble fertilizers and chemical pesticides. according to ec reg. 834/2007 it combines best environmental practices, a high level of biodiversity, the preservation of natural resources and the application of high animal welfare standards. the objectives of organic plant production are to work within natural systems and cycles, to maintain soil fertility, use renewable resources and to produce safe, nutritious, wholesome food (mikkelsen 2008). the concept of biodynamic agricultural production, devised by rudolf steiner (1924), respects the principles of organic production, supplemented by the use of biodynamic effects of biodynamic production on basil acta bot. croat. 75 (2), 2016 261 preparations (demeter international production standards 2005) and implies the use of a strict biodynamic sowing and planting calendar (wistinghausen et al. 1998). namely, it has been proven that time of sowing has a signifi cant effect on plant growth or yield (ejimofor ogbonna et al. 2012, ehsanullah et al. 2014). biodynamic preparations include strictly prescribed herbal and mineral additives and extracts for plant conditioning and compost activation, biostimulants and organic fertilizers, manures and composts (wistinghausen et al. 1998). the use of biodynamic preparations in organic production is also allowed by eu regulation (council reg. 834/2007). organic production in croatia is a small, but growing, branch of food production; it occupies about 2.5% (ministry of agriculture, 2013) of all farmland and is subsidized by the ministry of agriculture. biodynamic production, however, is not yet recognized as very promising. the aim of this study was to test the effect of four sowing dates recommended in the biodynamic sowing and planting calendar (thun and thun 2011) on the growth and essential oil content of three basil species (including two cultivars of one species) grown in pots for fresh seasoning herb. for that purpose we screened: 1) plant height, 2) number of nodes, 3) yield of fresh herb, 4) percentage of leaves mass, and 5) essential oil content of o. basilicum ‘rosso’, o. basilicum ‘eco genovese’, o. × hybrida and o. americanum. material and methods the practical experiment was conducted in summer 2012 with different basil species/cultivars (x): two cultivars of sweet basil, o. basilicum ‘rosso’ and ‘eco genovese’, bushy basil o. × hybrida, and the citral type basil o. americanum l. the experiment was conducted in a plastic tunnel and on an open area of the institute of agriculture and tourism in poreč. the organic seeds used in the experiment were supplied for o. basilicum ‘rosso’ and ‘eco genovese’ by amarant (slovenia) and for o. × hybrida and o. americanum by reinsaat (austria). four species/cultivars were tested and each one was sown on four different sowing dates. sowing was done under the usual climatic conditions for this geographic area and period (monthly average temperatures were for june 23.1 °c, july 26.1 °c, august 25.1 °c; total monthly precipitation was for june 18.6 l m–2, july 2.2 l m–2, august 3.8 l m–2) (croatian meteorological and hydrological service, 2012). basil was sown in containers fi lled with standard substrate, klasmann 1 (producer: klasmann-deilmann, germany) on four different sowing dates (y) chosen according to the biodynamic rhythm sowing scheme (thun and thun 2011), recommended for leafy plants in the mediterranean climatic zone: (1) june 7th 2012 after 1.00 pm – suitable for root plants; (2) june 8th 2012 after 6.00 pm – suitable for sowing of fl owering plants; (3) june12th 2012 after 8.00 am – suitable for sowing of leafy plants; (4) june 14th 2012 after 5.00 pm – suitable for sowing of fruity plants. from the scientifi c viewpoint, these sowing periods will be evaluated as different sowing dates with minimal timings. at the 4 true leaf stage, basil plants were transplanted into pots (644 ml volume) in klasmann 2 (producer: klasmann-deilmann, germany) standard substrate. pot basil was placed in an open area. irrigation with tap water was done daily, fertilisation twice after transplantation with 2.0 dl per pot of 1.0% standard organic fl uid fertiliser bio plantella (producer: unichem agro ltd). every treatment was represented in three repetitions with 15 pots per repetition. four species/cultivars (x) were tested and each one was sown in four different sowing dates. species and cultivars with the same sowing date began to fl ower homogenously, with only 1–2 days difference. the duration of the cultivation was considered as a period in days from sowing to harvest, where the fi rst day was the sowing day and the last day was the day of harvest. it lasted on average between 44 and 54 days, depending on the sowing date (tab. 1). the shortest-lasting cultivation period was for the basil sown on june 7th and june 8th, and the longest for the basil sown on june 12th and 14th. the plant height, number of nodes per main stem, fresh yield per plant, leaf mass percentage and the essential oil content were measured. the harvest of basil was done at the beginning of the fl owering period, at the time of the appearance of the fi rst fl owers in 10% of plants. measurements of fresh herb were made just before harvesting. fresh herb was then air dried in a shady and airy place in standard room conditions. after drying, leaves were separated from the stems; the leaf-to-stem ratio presents the value obtained from the mass of dried leaves divided by the mass of dried stems. plant height was defi ned as the length of the main stem in cm and the number of nodes was determined by counting from the fi rst fully developed leaves in the basal part of the main stem until the last fully developed and visible nodes. leaf mass percentage was determined as the percentage of leaves in the whole mass of dried herb. essential oil content was expressed in ml per 100 g of leaf dried weight. the essential oil was distilled with the neo-clevenger apparatus, using the method described in the european pharmacopeia 5.0 (2005). each sample, represented by three repetitions, was distilled twice. experimental data were statistically analysed using factorial (two way) anova and post hoc tukey test for multiple comparisons between groups with p ≤ 0.05. tab. 1. biodynamic calendar of basil. sowing date germination (days after sowing) transplanting into pots (days after sowing) flowering beginning (days after sowing) harvesting date june 7th 4 22 44 july 23rd june 8th 4 23 45 july 23rd june 12th 4 20 52 august 8th june 14th 4 22 54 august 8th dudaš s., poljuha d., šola i., šegula s., varga s., sladonja b. 262 acta bot. croat. 75 (2), 2016 results statistical analysis of tested factors throughout all observed parameters showed that the largest source of variation in all parameters was the species, which is the factor with the largest impact (tab. 2). plant height, percentage of leaf mass and essential oil content were signifi cantly affected not only by the species/cultivar, but also by the sowing date (y) and by the interaction between the species/cultivar and the sowing date (x×y). shortly before fl owering, basil formed on average between 8.0 and 10.2 nodes per main stem, depending on the species and cultivar (tab. 3). the highest number of nodes was observed in o. × hybrida, which formed signifi cantly more nodes than other species/cultivars tested. the highest number of nodes and the lowest plant height were found in o. × hybrida, indicating its very short internodes i.e. its compact, bushy habitus. the lowest number of nodes was found in o. basilicum cultivars. the number of nodes was not infl uenced by sowing date, or by interaction of species/ cultivar and sowing date. the fresh yield of basil ranged between 12.5 and 17.2 g/ plant. o. × hybrida had a signifi cantly higher yield than o. americanum or o. basilicum ‘rosso’ (tab. 3). the second best cultivar in terms of fresh yield was o. basilicum ‘eco genovese’. the yield per plant was not infl uenced by sowing date or by interaction of species/cultivar and sowing date. in fig. 1, the parameters infl uenced by both species/cultivars and sowing date, as well as their interaction, are presented. the height of basil plants at the beginning of fl owering was between 18.1 and 38.3 cm (fig. 1). this large range is a result of the genetic potential of species/cultivars and the different sowing dates, but is also, as the statistical analysis showed, impacted by the interaction between these two factors (tab. 2). o. americanum dominated in growth. at the last sowing date, it was signifi cantly higher than all the other species and cultivars. additionally, at all sowing dates, o. americanum was signifi cantly taller than o. × hybrida. leaf proportion in dried herbs ranged between 45.6 and 73.1% (fig.1). signifi cantly the highest leaf mass proportion was found in o. basilicum ‘rosso’ sown on june 7th, and the lowest in ‘eco genovese’ sown on june 8th. the most pronounced differences among species/cultivars were found for essential oil content (fig. 1), which was predominantly infl uenced by the species (tab. 2), and to a lower extent by the sowing date, and by the interaction of both factors. the essential oil content of o. × hybrida was signifi cantly higher than that of any of the other species/ cultivars with the same sowing dates, while the lowest essential oil content was found in o. basilicum cultivar ‘rosso’. the o. × hybrida of the last sowing date reached maximum essential oil content. discussion conventional farming is being progressively replaced by more ecology-friendly and sustainable organic agricultural production. basil is grown worldwide for seasoning and for essential oil extraction. for culinary purposes, fresh basil leaves are used; harvesting takes place at the period with the best leaf/stem proportion (over 1.0), mostly reached shortly before or at the beginning of fl owering. for essential oil extraction, basil is harvested in full bloom, because then it has the highest tab. 2. impact of analysed factors (species/cultivar (x) and sowing date (y)) and their interaction (x×y) on quantitative and qualitative plant parameters (two-way anova). the numbers denote squared total value of the parameter variation caused by species/cultivar, sowing date or their interaction. *signifi cant with p ≤ 0.05, df – degrees of freedom. source of variation df mean square (s2) plant height (cm) nodes number yield (g per plant) percentage of leaves mass essential oil content (ml per 100 g dw) species/cultivar (x) 3 5320.2* 60.4* 245.1* 245.9* 2.3* sowing date (y) 3 1704.5* 1.3 22.6 197.3* 0.1* x×y 9 342.5* 3.2 26.0 120.0* 0.2* error 222 24.5 2.0 18.4 1.8 0.0 tab. 3. infl uence of analysed factors (species/cultivar (x) and sowing date (y)) and their interaction (x×y) on quantitative plant parameters. *signifi cant with p ≤ 0.05. different letters in the same column indicate signifi cant difference. sd – standard deviation. factor nodes number ± sd yield of fresh herb (g per plant) ± sd species/cultivar (x) o. basilicum ‘rosso’ 8.3±0.6 c 12.5±1.0 c o. basilicum ‘eco genovese’ 8.0±0.9 c 15.8±0.9 ab o. × hybrida 10.2±1.0 a 17.2±0.8 a o. americanum 9.5±0.7 b 14.6±0.6 b sowing date (y) june 7th 2012 after 3.00 pm 9.1±1.2 a 15.1±1.1 a june 8th 2012 after 8.00 pm 9.1±0.6 a 14.1±1.0 a june 12th 2012 after 9.00 pm 8.9±0.9 a 15.5±2.0 a june 14th 2012 after 5.00 pm 8.9±1.0 a 15.4±0.8 a anova, tukey test x * * y x×y effects of biodynamic production on basil acta bot. croat. 75 (2), 2016 263 essen tial oil content (sifola and barbieri 2006). sifola and barbieri (2006) investigated the cultivation of three basil cultivars, which lasted from the end of may to mid-june. they harvested basil 49 days after transplanting, in the full bloom stage. the full bloom stage, as mentioned above, is considered to be the most appropriate for commercial harvesting of basil for essential oil production. these two authors report the branching of basil plant, a parameter comparable to the number of nodes per main stem. higher bud and node numbers can be an indicator of branching formation potential and yield. in their experiment, basil formed between 9.3 and 9.8 branches. this is in conformity with our determined numbers of nodes of between 8.0 and 10.2 per main stem in pot basil (tab. 3). the range in numbers of nodes in pot basil is affected mostly by the characteristics of the species/cultivar, which is also in accordance with our result. additionally, we observed that node number signifi cantly depended on the tested species, but not on the cultivars. in the study of frąszczak et al. (2011) on two basil cultivars grown in small pots in different daily light regimes and different temperatures, a signifi cant infl uence of the cultivar, temperature and photoperiod on the monitored parameters was found. these results are in accordance with ours, where the species, i.e. genetic factor, was confi rmed as the main signifi cant factor that infl uenced the monitored parameters. in our experiment, the differences between species/cultivars of basil were much more expressed, thus clarifying the intensity of species/cultivar infl uence on the monitored parameters. strong cultivar infl uence on the yield of basil was also confi rmed by politycka and gołcz (2004), who confi rmed that the yield of dry leaves of green basil cultivars was up to 100% higher than that of cultivars with anthocyanin. we further detected that sowing date itself, as well as the interaction of species/cultivar and sowing date, signifi cantly affected the plant height, leaf share and the essential oil content of basil, but not the number of nodes and overall yield (tab. 2). the quality of herbs is, among other factors, infl uenced by the proportion of leaves and stems (werker et al. 1993, ioannidis et al. 2002), as well as by essential oil content (rey and saez 2002). a higher proportion of leaves than stems represents a higher quality, as the concentration of oil glands is the highest in mature leaves (werker et al. 1993, ioannidis et al. 2002) and in basil, leaves and calyxes have the highest concentration of oil glands (putievsky and galambosi 1999). the leaf proportion in our experiment is analogous to the leaf-to-stem ratio used by sifola and barbieri (2006). the leaf-to-stem ratio in their investigation varied between 0.7 and 1.2. all values over 1.0 mean that the amount of leaves is over 50%. the percentage of leaf mass in our investigation depended on the species/cultivars, but it was also infl uenced by the sowing date and the interaction of species/cultivar × sowing date (tab. 2). only in o. basilicum ‘eco genovese’, which was sown on june 8th (second sowing date), was the percentage of leaves under 50%, while all other combinations of species/cultivars and sowing dates had leaf percentages of over 50%. essential oil content, as the second quality aspect, in four basil species and cultivars, ranged between 0.4 and 1.8 ml per 100 g dried leaves, depending on the species/cultivar and sowing date (fig. 1). the species with the highest amount of essential oil in this study was o. × hybrida, while o. basilicum ‘rosso’, as expected, had the lowest amount. interestingly, even though o. × hybrida had the least plant height, it contained the highest amount of essential oil, indicating that internodes negligibly contribute to essential oil production. o. basilicum ‘rosso’ is a cultivar for decorative purposes; it is purple basil with high anthocyanidin content. compared to the essential oil content in o. basilicum cultivars from the experimental garden in prague (klimánková et al. 2008), our o. basilicum ‘rosso’ is most similar to the czech cultivar v, while o. basilicum ‘eco genovese’ is more similar to the czech cultivars i–iv. based on the obtained data we conclude the following: 1) species had greater impact on the observed parameters than sowing date or species and sowing date in combinafig. 1. plant parameters infl uenced by both species/cultivar and sowing date, as well as their interaction: a) height, b) leaf mass and c) essential oil. data are the means of three replicates ± sd (standard deviation). the means labelled by different letters are signifi cantly different (two way anova and tukey’s test), p ≤ 0.05. dudaš s., poljuha d., šola i., šegula s., varga s., sladonja b. 264 acta bot. croat. 75 (2), 2016 tion; 2) three parameters – plant height, percentage of leaf mass and essential oil content – were also signifi cantly affected by the sowing date and the interaction between species/cultivars and sowing date; 3) the best yield per plant was given by o. × hybrida and o. basilicum ‘eco genovese’; 4) the highest production of essential oil was in the lowest species o. × hybrida indicating that ocimum stem negligibly contributes to the essential oil production. the lowest essential oil content was confi rmed in the o. basilicum purple cultivar ‘rosso’; 5) the percentage of leaf mass was over 50.0% in all cases, except in o. basilicum ‘eco genovese’, sown on june 8th, with 45.6%; 6) the most clear species separation was according to the »number of nodes« parameter, while the cultivars did not differ; 7) even though o. americanum gave the tallest plants, it did not yield the highest amount of fresh herb or essential oil; 8) the species with the most consistent height, considering the signifi cant impact of biodynamic rhythm, was o. americanum; 9) fi nally, if we were interested in yield only, then the sowing date would not be crucial, however if we needed higher plants with a higher percentage of leaf mass and more essential oil (i.e. more leaves) then the sowing date should be controlled. acknowledgements the present research was not funded by any national or international institution but the authors are grateful to the polytechnic of rijeka, agricultural department in poreč, to the institute of agriculture and tourism, department of biology at the faculty of science in zagreb, and to the biotechnical centre naklo for materials and infrastructural support given during the research. references croatian meteorological and hydrological service. retrieved october 28, 2014 from http://www.dhmz.htnet.hr/. demeter association inc. biodynamic farm standard. philomath, usa. retrieved october 28, 2014 from: http://www.demeterusa.org/downloads/demeter-farm-standard.pdf. demeter international production standards. demeter international e.v. retrieved october 28, 2014 from: http://organicrules. org/567/1/demeter_international_production_standards_2005_june_en.pdf. dubey, r. c., 1991: fungicidal effect of essential oils of three higher plants on sclerotia of macrophomia phaseolina. indian phytopathology 44, 241–244. ehsanullah, s. a. a., ashraf, u., rafi q, h., tanveer, m., khan, i., 2014: effect of sowing dates and weed control methods on weed infestation, growth and yield of direct-seeded rice. the philippine agricultural scientist 97, 307–312. ejimofor ogbonna, p., gbacinku umar-shaba, y., 2012: time of sowing affects the growth and yield of sesame in a derived savanna agroecology of south-eastern nigeria. the philippine agricultural scientist 95, 153–159. european pharmacopeia 5.0. 2005: council of europe, strasbourg, 5th edition 2, 2667–2668. frąszczak, b., kałużewicz, a., krzesiński, v., lisiecka, j., spiżewski, t., 2011: effect of differential temperature and photoperiod on growth of ocimum basilicum. zemdirbysteagriculture 98, 375–382. hiltunen, r., holm, y., 1999: basil. industrial profi les. harwood academic publisher, new jersey. ioannidis, d., bonner, l., johnson, c. b., 2002: uv-b is required for normal development of oil glands in ocimum basilicum l. 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(lamiaceae). annals of botany 71, 43–50. wistinghausen, c. h. v., scheibe, w., wistinghausen, e. v., könig u. j., 1998: instructions for preparing the biodynamic fi eld sprays and fertilizer preparations. 3rd. expanded edition, stuttgart (in german). zheljazkov, v. d., cantrell, c. l., evans, v. b., ebelhar, w. b., coker, c. h., 2008: yield and composition of ocimum basilicum l. and ocimum sanctum l. grown at four locations. horticultural science 43, 737–741. 132 acta bot. croat. 76 (2), 2017 acta bot. croat. 76 (2), 132–137, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0010 issn 0365-0588 eissn 1847-8476 morphological, anatomical and karyological investigations of the turkish endemic species lathyrus woronowii bornm. (fabaceae) fatma güneş1, çiler meriç2 1department of pharmaceutical botany, faculty of pharmacy, trakya university, edirne 22100, turkey. 2department of biology, faculty of sciences, trakya university, edirne 22100, turkey abstract – lathyrus woronowii bornm., an endemic species of turkey, is threatened with extinction due to dam construction. it exists only in the çoruh valley, artvin. this annual species is in the critically endangered (cr) category according to the international union of conservation of nature (iucn) criteria. its morphology, anatomy and karyology are studied here for the first time. a detailed description is given and the general appearance of the species has been drawn; cross sections from the stem and leaf have been taken and examined; and the diploid chromosome number (2n = 14) has been reported and illustrated for the first time. key words: anatomy, endemic, karyology, lathyrus, morphology, turkey. * corresponding author, e-mail: drgunes@gmail.com introduction the genus lathyrus l. (fabaceae) is represented by 13 sections and about 187 subspecies worldwide (allkin et al. 1983), most of which are annual or perennial plants, predominantly centred in the mediterranean region (kupicha 1983). in turkey, lathyrus is divided into 10 sections with 76 species, 26 of which are endemic (davis 1970, davis et al. 1988, güneş and özhatay 2000, genç and şahin 2011, güneş and çırpıcı 2012, güneş 2014). lathyrus woronowii bornm. belongs to the section orobastrum, which contains eight species, two of which (l. woronowii and l. tauricola p. h. davis) are endemic to turkey. the main characteristics of this section are that they are annual; are procumbent to climbing; have angled or sometimes very narrowly winged stems; have tendrilous or aristate leaves with single to several-paired, linear to elliptic, parallel-veined leaflets; have peduncles that are single-flowered; and have legumes that are glabrous or pubescent, with the upper suture not winged. only l. saxatilis (vent.) vis., l. vinealis boiss. & noё and l. woronowii have more than one pair of 2 or 3 leaflets, whereas all the other species in the section have only one pair. leaves of l. saxatilis and l. woronowii are mucronate and shortly aristate, respectively, whereas all other species have leaves with simple tendrils, at least on the upper parts of the plant. the flower colour is creamy to yellow in l. saxatilis and l. woronowii; red in l. vinealis, l. sphaericus retz. and l. setifolius l.; and lavender in l. inconspicuus l. and l. tauricola. pods are hairy in l. inconspicuus and l. setifolius but glabrous in other lathyrus species (davis 1970). pollen morphology and seed morphology of the orobastrum section have been investigated by chernoff et al. (1992), pastor-cavada et al. (2011), güneş (2011a, 2012, 2013) and güneş and çırpıcı (2011). it was previously reported that the pollen type is 3-zonocolporate and pollen shape is prolate in l. woronowii and that the shape is subprolate or spheroidal in eight other studied taxa. the pollen structure of all studied taxa has a tectate infrastructure and reticulate ornamentation; the seed shape is spheroidal in l. woronowii and subprolate in l. vinealis and l. sphaericus; and the seed surface is mostly smooth. some karyology studies were previously conducted within this genus, including the studies of senn (1938), ress and hazarika (1969), fouzdar and tandon (1975), yamamoto and fujiwara (1984), kuriyan and narayan (1988), kumar and sinha (1989), kakoli and sumitra (1991), şahin and babaç (1995), şahin et al. (2000), klamt and schifino-wittmann (2000), seijo and fernandez (2003), ayaz and ertekin (2008), and güneş (2011b). these studies indicated that the genus lathyrus generally has a diploid chromosome number of 2n = 14, as reported in approximately 95 lathyrus species worldwide. tetraploid counts of 2n = 28 have been reported in l. pratensis l. (darlington and wylie 1955), l. venosus sweet. (başaran et al. 2007) and l. brachypterus (güneş 2011b). hexaploid counts of 2n = 42 have been reported in l. palustris l. subsp. palustris (ball 1968, güneş and çırpıcı 2008). several investigations of lathyrus woronowii in turkey acta bot. croat. 76 (2), 2017 133 studies were conducted on the species of the orobastrum section in the elâzığ area by şahin and altan (1990), in the black sea region by özcan et al. (2006) and in the european region of turkey by güneş and çırpıcı (2008). karyotype analysis is an important tool for distinguishing controversial species, especially when there are very similar morphological characters, such as those in l. spatulatus čel. and l. elongatus (bornm.) sirj. (güneş 2011b). there are several anatomical studies on lathyrus species. lathyrus latifolius l. was investigated for its morphological, anatomical and physiology characteristics by krstic et al. (2002). the study by çildir (2011) of the morphology, anatomy and systematics of the 25 taxa belonging to the genus lathyrus in central anatolia (turkey) has been presented. in addition, there are a few studies on the anatomy of turkish lathyrus (mantar et al. 2002, mantar et al. 2003, celep et al. 2011, kahraman et al. 2014). since there have been no previous detailed morphological, anatomical or karyological studies on the turkish endemic l. woronowii, the goal of this study is to determine the morphological, anatomical and karyological properties of l. woronowii, which is under threat of extinction due to several dam construction projects in the çoruh valley, and to use this information to aid in identification of the species and raise awareness about its threatened status. materials and methods in 2009, 72 specimens of lathyrus woronowii were collected from four different localities in artvin province of north-eastern turkey during their flowering time between april and may. the specimens were deposited in the herbarium of the faculty of pharmacy. morphological observations and biometric measurements were made on fresh material and herbarium specimens. they were examined and drawn using an olympus szx7 microscope. for anatomical studies, the material was stored in 70% alcohol. transverse sections of the stems and leaves and the surface sections of leaves from fixed samples were made with a razor blade. some sections were stained with alcian blue (sigma) for pectic substances and others with safranin (sigma) for lignin, using 20 μl alcian blue + 20 μl safranin in 10 ml of 25% glycerine for 4 hours. stained and unstained sections were mounted in glycerine–gelatin to preserve the preparations, according to the methods of jensen (1962). slides were examined using an olympus bh2 microscope fitted with a digital camera. images were taken with a progres c12 plus digital camera. for karyological studies, germinating root tips were collected and pre-treated in a saturated solution of α-bromonaphthalene for 24 hours at 4 °c and then fixed in carnoy’s solution. the root tips were hydrolysed in 1 n hcl for 10– 20 minutes at 60 °c and stained with feulgen solution. semi-permanent slides were examined with a light microscope. mitotic chromosomes were photographed on a nikon e600 trinocular photomicroscope using a 100 × magnification oil immersion objective. chromosome measurements were made using 4000 × magnified photographs of five well-spread metaphase plates. short arm lengths (s) and long arm lengths (l) were measured, and the total length of the chromosomes (c), arm ratio (r = l/s), centromeric index (i) and relative length (r) were calculated. the classification of chromosomes by centromeric indices followed the methods of levan et al. (1964). results the habitat (fig. 1) is given, while tab. 1 shows the morphological features of l. woronowii. previously, seed and pollen properties were studied by güneş (2012, 2013). the general appearance and flower parts have been drawn (fig. 2). three-paired leaflets were observed in some specimens of l. woronowii in this study. although peduncles were reported as being single-flowered in the flora of turkey (davis 1970), 2-flowered peduncles have also been found here. the standard colour of l. woronowii is yellow when it is fresh, turning violet when pressed in herbarium specimens. stems are covered with a single-layered epidermis, with a 5–6-layered parenchymatic cortex underneath. the endodermis is not distinct. the central cylinder contains four units of collateral vascular bundles. the bundles have a sclerenchyma cap above the phloem. the vascular cambium is not distinct between the phloem and xylem. the pith is filled with parenchymatous cells (fig. 3a). the leaves are covered with a single layer of epidermis. the mesophyll is bifacial and differentiated as palisade and spongy parenchyma. unfig. 1. habit and general view of lathyrus woronowii, (a) flower, and (b) pod (güneş 2190). güneş f., meriç ç 134 acta bot. croat. 76 (2), 2017 tab. 1. morphological features of lathyrus woronowii. p – polar axis, e – eguatorial diameter, m – median point, m – median region, sm – submedian region, st – subterminal region. features p. h. davis 1970 our results habit low, slender annual and glabrous low, slender annual and glabrous stem stems are ascending or procumbent stems are ascending or procumbent stem length (cm) 8–18 8–57; some stems drying blackish stem width (mm) not winged 0.5–1.5; angled leaves apex (mm) shortly aristate shortly aristate, 1–2 leaflets, length and width (mm) ovate-elliptic, the lowest often with 1-paired leaflets the remainder 2-paired, c. 12 × 5 ovate-elliptic, the lowest with 1-paired leaflets, the remainder 2 or 3-paired, 3-12 × 1–6 vein type unspecified 5–7 parallel-veined petiol length (mm) unspecified 3–15 stipules, length × width (mm) lanceolate-subulate, semi-sagittate lanceolate-subulate, semi-sagittate, 1–5 × 0.1 peduncles (mm) 1-flowered, shorter than leaf 1 (–2) flowered, 4–15 pedicel (mm) unspecified 3–4 calyx (mm) c. 5 mm, glabrous 5–6 (–7) glabrous calyx tube (mm) unspecified 2–2.5 (–3) calyx teeth (mm) subequal, lanceolate, a little longer than the tube subequal, lanceolate, upper teeth 3–4, the lowest 4–5 flowers length (mm) unspecified 8–14 standard colour and length (mm) standard violet standard yellow with red veins, drying violet or purple, 9–14 × 8–12 wings (mm) wings cream yellow, 11–13 × 3–5 keel (mm) unspecified cream, 11–14 × 4–6 style (mm) unspecified 5–6 × c.1 stigma (mm) unspecified hairy, enlarged legume, length × width (mm) 3.5 × 0.6 cm altan (2001) glabrous, reticulate nerved, upper sture not winged, 18–28 × (3–)4–4.5 (–5), with 6–10 seeds seeds, length × width [min. value (mean) max. value (mm)], color and surface structure unspecified spheroidal (p/e = 1.04), smooth, pressed, 1.71 (2.21) 2.66 × 1.69 (2.13) 2.58, yellow-green tones and speckled (güneş 2013) hilum length × width [min. value (mean) max. value (mm)] unspecified 0.32 (0.44) 0.55 × 0.18 (0.24) 0.33 (güneş 2013) pollen shape length × width (µm) unspecified prolate (p/e = 1.36), p × e = 35.78 x 26.31 (güneş 2012) chromosome number and type unspecified 2n = 14; m, m, sm-st, m, sm, m-sm, msat flowering time may april – may habitat schictone screes schictone screes tab. 2. chromosome characters of lathyrus woronowii. mc – mean chromosome, mr – mean ratio, m – median point, m – median region, sm – submedian region, st – subterminal region chromosome pairs total length (c, µm) long arm (l, µm) short arm (s, µm) arm ratio r=l/s centromeric index (i) relative length (r) chromosome type i 2.56 1.31 1.25 1.05 48.79 8.83 m ii 2.25 1.38 0.88 1.57 38.88 7.76 m iii 2.00 1.50 0.50 3.00 25.00 6.90 sm-st iv 2.00 1.00 1.00 1.00 50.00 6.90 m v 2.00 1.38 0.63 2.2 31.25 6.90 sm vi 2.00 1.25 0.75 1.67 37.50 6.90 m-sm vii 1.44 0.75 0.69 1.09 47.92 4.97 m average mc=2.04 mr=1.65 investigations of lathyrus woronowii in turkey acta bot. croat. 76 (2), 2017 135 der the upper epidermis, the mesophyll contains two layers of palisade cells, which are elongated cells. under the lower epidermis, spongy parenchyma is composed of two layers of oval-shaped cells with intercellular spaces. the vascular bundles of leaves are collateral (fig. 3b). the leaves are amphistomatic, and stomata are found along the upper and lower epidermis. the stomata index is 23.38 for the upper epidermis and 28.02 for the lower epidermis (figs. 3c, d). the chromosome number was determined to be 2n = 14, and the detailed characteristics of chromosomes are shown in tab. 2 and fig. 4. discussion lathyrus woronowii is a critically endangered endemic species [cr c2a(i)] (iucn 2001), restricted to the province of artvin (a8 çoruh valley) in north-eastern turkey (ekim et al. 2013). it is faced with the threat of flooding due to 15 dams having been planned along the çoruh river. its distribution area is approximately 30 km in diameter. specimens of living plants and seeds are preserved in the nezahat gökyiğit botanical garden (ngbb) in i̇stanbul. altan (2001) reported l. woronowii stem length (60 cm) and pod size (3.5 × 0.6 cm). our results supported the stem length (8–57 cm; tab. 1) but not the pod size (18–28 × (3–) 4–4.5 (–5) mm). the anatomical features of l. woronowii are reported for the first time in this study. metcalfe and chalk (1950) suggested that the presence or absence and structure of the fig. 2. lathyrus woronowii schematic view: a) general habit, b) flower, c) calyx, d) corolla (a – standard, b – wing, c – keel), e) androecium, f) gynoecium and g) fruit. author: f. güneş (güneş 2190, güneş 2244). fig. 3. anatomical sections of lathyrus woronowii. a) cross-section of stem; b) cross-section of leaf; c) upper surface section of leaf; d) lower surface section of leaf. (co – cortex, ep – epidermis, le – lower epidermis, ph – phloem, pi – pith, pp – palisade parenchyma, sc – sclerenchyma, sp – spongy parenchyma, st – stomata, ue – upper epidermis, xy – xylem. table 3. chromosome number and chromosome type of orobastrum. m – median point, m – median region, sm – submedian region, st – subterminal region taxa chromosome number (2n) chromosome types total length of largest and smallest chromosome (µm) reference l. saxatilis (vent.) vis. 14 sm,stsat,sm,sm,st,st,m 4.89 and 2.86 (şahin and altan 1990) l. vinealis boiss. & noe 14 msat,sm,sm,sm,sm,m,m 4.66 and 2.66 (şahin and altan 1990) l. sphaericus retz. 14 m,m,m,sm,m,m,m m,msat,sm,m,m,m,sm m,m,sm,m,sm,m,m 6.92 and 4.68 3.96 and 2.72 4.50 and 2.83 (genç and şahin 2001) (özcan et al. 2006) (güneş and çırpıcı 2008) l. inconspicuus l. 14 msat,sm,sm,m,sm,sm,m msat,m,sm,m,sm,m,sm 4.44 and 2.64 4.66 and 3.28 (şahin and altan 1990) (genç and şahin 2001) l. tauricola p. h. davis 14 msat, sm,m,sm,sm,m,m 6.06 and 3.98 (genç and şahin 2001) l. setifolius l. 14 msat,sm,st,sm,sm,m,m msat,sm,sm,sm,sm,m,m msat,sm,st,sm,sm,m,m 6.87 and 2.66 7.16 and 3.46 5.25 and 3.00 (şahin and altan 1990) (genç and şahin 2001) (güneş and çırpıcı 2008) güneş f., meriç ç 136 acta bot. croat. 76 (2), 2017 wings in the stem are taxonomically important in the distinction of lathyrus species. the stem of l. woronowii has no wings like those of l. cilicicus hayek & siehe (celep et al. 2011); l. inconspicuus l.; l. vinealis (mantar et al. 2002); and l. nissolia l. (kahraman et al. 2014). stems of l. maritimus bigel, l. pratensis, l. sylvestris l. (metcalfe and chalk 1950), l. latifolius l. (krstic et al. 2002), l. hirsutus l. and l. sativus l. (mantar et al. 2003) contain wings. çildir (2011) reported that l. incurvus (roth.) willd., l. cassius boiss., l. cicera l. and l. chloranthus boiss. have winged stems, while stems of l. aureus (steven) brandza, l. brachypterus, l. haussknechtii širj., l. armenus (boiss & huet) širj., l. digitatus (m. bieb.) fiori, l. tukhtensis czeczott, l. spathulatus čelak., l. pratensis, l. laxiflorus (desf.) o. kuntze subsp. laxiflorus, l. czeczottianus bässler, l. roseus steven subsp. roseus, l. tuberosus l., l. saxatilis, l. sphaericus, l. aphaca l. var. biflorus post and l. aphaca l. var. affinis (guss.) arc. contain no wings. the pith is composed of large parenchyma cells in l. woronowii., l. aureus, l. armenus, l. laxiflorus subsp. laxiflorus, l. roseus subsp. roseus, l. cilicicus, l. maritimus and l. pratensis, while l. latifolius stems are hollow at the centre. however, the stems of l. incurvus, l. brachypterus, l. haussknechtii, l. digitatus, l. tukhtensis, l. spathulatus, l. pratensis, l. czeczottianus, l. tuberosus, l. cassius, l. hirsutus, l. chloranthus, l. sativus, l. cicera, l. saxatilis, l. inconspicuus var. inconspicuus, l. sphaericus, l. nissolia, l. aphaca var. biflorus and l. aphaca var. affinis are hollow (metcalfe and chalk 1950, krstic et al. 2002, mantar et al. 2002, 2003, celep et al. 2011, çildir 2011, kahraman et al. 2014). l. woronowii has amphistomatic leaflets, like those of l. incurvus, l. pratensis, l. czeczottianus, l. sphaericus, l. cassius, l. cicera, l. latifolius and l. cilicicus, whereas l. roseus subsp. roseus and l. laxiflorus subsp. laxiflorus have hypostomatic leaves (krstic et al. 2002, celep et al. 2011, çildir 2011). the chromosome measurements of lathyrus species belonging to section orobastrum are shown in tab. 3. the largest and smallest chromosomes of l. woronowii were measured as 2.56 and 1.44 µm, respectively. the total lengths of the largest and smallest chromosomes of other species belonging to the orobastrum section were stated by the authors. in this section, l. woronowii has the smallest chromosome set. the largest set belongs to l. setifolius, and the chromosomal numbers of lathyrus species in the present study (tab. 3) agree with previous records (şahin and altan 1990, genç and şahin 2001, özcan et al. 2006, güneş and çırpıcı 2008). according to ress and hazarika (1969); yamamoto et al. (1984); and güneş and çırpıcı (2008), the perennial species of the genus lathyrus have larger chromosomes in comparison with those of the annual species. acknowledgement the authors thank the scientific and technological research council of turkey (tübi̇tak) for financial support (project number 107t127). fig. 4. mitotic metaphase chromosomes (a), karyogram (b), and idiogram (c) of lathyrus woronowii (güneş 2244). references allkin, r., macfarlane, t. d., white, r. j., bisby, f. a., adey, m. e., 1983: names and synonyms of species and subspecies in the vicieae. issue 2, vicieae database project publication no. 2, southampton, uk. altan, y., 2001: some interesting endemic plants collected from the east and the northeast of turkey and their threatened categories. pakistan journal of botany 33, 157–166. ayaz, e., ertekin, s. a., 2008: karyotype analysis of two species of genus lathyrus from southeastern anatolia turkey. international journal of agriculture and biology 10, 569–572. ball, p. w., 1968: lathyrus l., in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. 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0365-0588 eissn 1847-8476 short communication long time no see – rediscovery of peculiar ephemeral fern anogramma leptophylla (l.) link in croatia vedran šegota*, vladimir hršak, antun alegro university of zagreb, faculty of science, department of biology, division of botany, marulićev trg 20/ii, hr-10000 zagreb abstract – anogramma leptophylla is one of the rarest fern species in balkan peninsula. in croatia, several localities were noted prior to this study, when its presence was confi rmed with a discovery of a small population on the island of mljet (southern adriatic). this was, after almost 80 years, the fi rst reliable fi nding of this species along eastern adriatic. the establishment of a. leptophylla on the western part of the island of mljet may be attributable to certain favourable environmental conditions, but essentially to higher air and soil humidity. its unusual bryophyte-like life strategy, with short-living annual sporophytes, facilitates its survival under mediterranean climate, generally unfavourable for pteridophytes. keywords: balkan peninsula, distribution, island of mljet, pteridophytes abbreviations: cnhm – herbarium of croatian natural history museum, za – herbarium croaticum, zagr – herbarium facultatis agronomiae, zaho – herbarium of ivo and marija horvat * corresponding author, e-mail: vedran.segota@biol.pmf.hr introduction unlike the majority of fern species that exhibit perennial sporophytes and short-living gametophytes, a few peculiar ferns utilize bryophyte-like life strategy (proskauer 1964, pangua et al. 2011). with its ephemeral annual sporophyte and dormant perennial gametophyte, genus anogramma (adiantaceae) is one of the most well-known members of this group. the distribution centre of the genus is in the neotropics (tropical areas of mexico, central and south america), where all fi ve species except a. ascensionis (endemic from ascension island in south atlantic) can be found, including cosmopolitan a. leptophylla (l.) link (tyron et al. 1990, molnár et al. 2008). aside from neotropics, a. leptophylla grows in the mediterranean basin, macaronesia, atlantic europe, tropical and south africa, australia and new zealand (paugua et al. 2011). consequently, anogramma leptophylla is the only representative of the genus in european fl ora (tutin 1993), occurring along the atlantic coast, the mediterranean basin and in crimea (jalas and suominen 1972). a. leptophylla is a cosmopolitan species restricted to temperate and tropical zones (landolt 2010), with preference for humid regions, usually with alternating wet and dry seasons (nakazato and gastony 2003, studnička 2009). in european context, it is considered an oceanic-suboceanic (meusel et al. 1965, dostál 1984) or oceanic-submediterranean species (fischer et al. 2008). similarly, in the fl ora of croatia, the species was defi ned as an atlantic-mediterranean element (regula-bevilaqua and ilijanić 1984). a. leptophylla is recognized in all croatian checklists (mayer and horvatić 1967, hršak 1994, nikolić 2016). however, only several localities were noted in croatia prior to this study, all in the southern part of the country. first known record dates from mid19th century, when visiani (1852) noted the species in the town of dubrovnik, cited again later by schlosser and vukotinović (1869), mannagetta (1901) (including also the adjacent village zaton) and hirc (1905). the species was found again in dubrovnik (precisely on lapad peninsula) in 1868 by vodopić, being the only currently available specimen of this species deposited in croatian herbariums (za3132). this locality was cited later also by hirc (1905), and confi rmed by latzel (1914). the latter author found the species on one more locality – on the island of mljet, in the vicinity of the sea-port sobra (latzel 1914), which was cited later in the analysis of the fl ora of mljet performed by regula-bevilaqua and ilijanić (1984). finally, the fern was recorded on the island of hvar (village of vrisnik near jelsa) by rechinger (1934), and never reported in the southern adriatic since. recent šegota v., hršak v., alegro a. 92 acta bot. croat. 76 (1), 2017 notice on fi nding of a. leptophylla on the northern adriatic island of krk, between town of baška and halm hill in 2009 (rottensteiner 2014), with no herbarium specimen available, although unexpected, might be authentic; nevertheless, it should be carefully examined. historical and recent fi ndings of a. leptophylla along the southern adriatic coast appeared to be on the northernmost border of its areal on balkan peninsula, extending southward through montenegro and macedonia, down to the greek mainland and islands, including crete. within former yugoslavia, mayer and horvatić (1967) mention only southern dalmatia (croatia) as a locality for a. lep tophylla, however it was recorded in montenegro (boka kotorska: rose, sutorina, vrmac hill above place muo) by studniczka (1890), what is cited later by several authors (mannagetta 1901, hayek 1927, pulević 2005, stešević and caković 2013) and macedonia (markovi kuli near prilep and belasina mt.) (hayek 1927, micevski 1985). although a. leptophylla is obviously extremely rare in croatia, it was not estimated according to the iucn criteria. material and methods during an extensive fl oristic study of the national park mljet (southern adriatic), a number of fi eldtrips were undertaken in the period from 2008 to 2011. nearly all basic 1/64 mtb fi elds of the central european fl ora mapping grid (1.5×1.4 km) (nikolić et al. 1998) within the park were accessed at regular intervals throughout four growing seasons. collected specimen of a. leptophylla, was identifi ed in 2012 using several identifi cation keys and iconographies (pigniati 1982, tutin 1993, kopp and schneebeli-graf 1998). historical literature regarding distribution of a. leptophylla on eastern adriatic coast, and all available herbarium collections in croatia (za, zaho, cnhm and zagr) were checked. results and discussion a small population of a. leptophylla was discovered on 6th may 2010 in the vicinity of the footpath along the northern coastline of the mljet national park, opposite to the neighbouring islet of kobrava. the fi ndspot is located within 3270.213 1/64 mtb fi eld, between tatinica cove and rt križice (rt kula) promontory. between these two localities, there are two coves (pod veja gora and čartonica) and the rt travno kocje promontory, spreading from west to east. the same direction is followed by the hills above them (veja gora, 265 m a. s. l.; vriješće, 296 m a. s. l. and hripe, 183 m a. s. l.). some other fern species, such as polypodium cambricum l., asplenium ceterach l., a. onopteris l. and a. adianthum-nigrum l., were noted on the same site. at the time of our visit, hardly few plants of a. leptophylla were observed; all were in a good state, bearing matured sporangia. around 10 cm high stem did not bear leaf bases of previous years, clearly indicating that the aboveground sporophyte is, beyond any doubt, annual. one specimen was collected, with both aboveground green sporophyte and subterranean pea-like structure called tubercle, and deposited in za collection (fig 1). the population was not further monitored, due to the geographic distance of the island. an examination of major croatian herbarium collections revealed only one herbarium specimen of a. leptophylla, collected in 1898 on lapad peninsula (part of the town of dubrovnik), but with only aboveground sporophyte containing ripened sporangia as well (fig 1). within the national park mljet, the specimens of a. leptophylla were observed on bare wet soil in limestone rock crevices surrounded by green mats of mosses. the fi ndspot was in the close vicinity of the hiking footpath, which is approximately 5 to 10 m a. s. l. the similar habitat, on the rocks, was also briefl y described at an old fi ndspot on the same island (sea-port sobra) (latzel 1914). similarly, on iberian peninsula, the species grows in rocky habitats, mainly in wide cracks of siliceous rocks fi lled with soil (nogueira 1986); however, it spreads from the lowlands up to about 1000 m a. s. l., under climates varying from typical oceanic to mediterranean. a. leptophylla is considered to be, to some extent, a pioneer species occupying competitive-free habitats such as rock fi ssures with little soil, where, in mediterranean, it is often accompanied with selaginella denticulata (l.) spring (dostál 1984). similarly, we observed that these kinds of microhabitats on island mljet were often occupied by s. denticulata, and it appeared to be rather frequent along fig 1. anogramma leptophylla, scanned herbarium specimens from island of mljet, 6th may 2010 (right) and lapad peninsula (dubrovnik) 1868 (left), using a3+ scanner expression 11000xl. anogramma leptophylla in croatia acta bot. croat. 76 (1), 2017 93 the footpath where a. leptophylla was detected. in order to avoid its competitors, a. leptophylla fi nishes its short lifecycle at the end of spring when ephemeral mosses, liverworts and minute seed plants start to inhabit the unhostile surfaces. this way it uses its competitive advantage for fast development in favourable seasons. in some inland populations, such as the southern slopes of swiss and italian alps, the species occupies similar microhabitats, like mouths of caves, which provide a suitable microclimate (lauber and wagner 1998). in macedonia, as an extremely rare species, it occupies moist and shaded places beneath large granite rocks (micevski 1985). the most remote inland population, more than 1000 km from the closest populations in alps and southern adriatic, was recently discovered in carpathian basin (ne hungary). using precise fi eld measurements of the climatic conditions of the site, this remarkable discovery has been explained by the microclimate which provides above-freezing air temperature and high humidity throughout the year (molnár et al. 2008). to summarize, the species in europe undoubtedly prefers very specifi c microhabitats characterized by rocky fi ssures with small amount of soil sediment, away from full sun exposure, yet out of reach of frost, in positions at least partially sheltered and shaded by adjacent rocks and/or higher shrub and tree vegetation (dostál 1984). this is the case on island of mljet, where species was found in a site shaded by edges of very compact evergreen vegetation dominated by quercus ilex l., juniperus phoenicea l., phillyrea lattifolia l., pistacia lentiscus l., cistus creticus l. subsp. eriocephalus (viv) greuter et burdet, erica arborea l., arbutus unedo l., myrtus communis l., viburnum tinus l. and emerus majus mill. subsp. emeroides (boiss. et spruner) soldano et f. conti. this vegetation is practically impenetrable, owing to dense ruscus aculeatus l. in the understory and smilax aspera l. that densely climbs to the canopies. according to trinajstić (1986), island of mljet is situated in steno-mediterranean zone of the mediterranean region attributed to rather hot and arid conditions, which is unfavourable for a. leptophylla establishment. however, the mean annual precipitation on the western part of the island is surprisingly high (up to 1000 mm) (perčec tadić 2008), although unevenly distributed. the locality of a. leptophylla, situated on the exposed fl ank of the island and in close vicinity to the sea, enables additional water condensations over the cooler nights in the summer draught period. therefore, it is likely that soil retains somewhat more water under the partial shadow of higher and dense tree and shrub vegetation. this more humid microclimate of the site is also refl ected in the presence of additional submediterranean elements such as pistacia terebinthus l., fraxinus ornus l., laurus nobilis l., and particularly sesleria autumnalis (scop.) f.w.schultz. narrow habitat preferences of a. leptophilla might not be the only reason for its rarity in western balkan. considering the rather short life-cycle of the aboveground sporophyte, some populations have most likely not been observed by botanists. the sporophyte generation does not begin to develop from dormant embryos on perennial tubercles before december, reaching its maturity at the beginning of april. spore ripening takes place in april and may, followed by sporophyte withering at the beginning of june. thus, specimens are easily overlooked until next spring. however prothallia, newly produced from the spores, photosynthesize during june and july, and then, if no suitable growing conditions exist, they form a dormant tubercle (pangua et al. 2011). if in some years a very dry spring occurs, sporophyte might not appear at all within the same season, and tubercle remains in a resting state. according to molnár et al. (2008), dormant tubercles are able to survive for 2.5 years in stressful drought conditions. this, among ferns a rare aspect of the life-cycle, indicates its therophytic behaviour and misleads to concluding that a. leptophylla is an annual species. moreover, it resembles bryophyte-like life strategy, which gives this fern some advantages in colonisation and has probably contributed to it wide geographic range (pangua 2011). in contrast to other ferns among which environmental sex determination is a rule, in a. leptophylla sex is genetically controlled, as in the majority of land plants (pangua et al. 2011). our discovery of a. leptophylla on island of mljet in may confi rms that its life-cycle is in accordance with those reported for other european populations. a. leptophylla is a cosmopolitan species, and has not been reported as threatened so far. even though there is an extremely low number of recent fi ndings in croatia, and there is no evidence that it is still present at other historically known sites, its specifi c habitat is not endangered at all. therefore, according to iucn criteria, the species qualifi es as data defi cient (dd) and calls for larger fi eld surveys. considering its specifi c ecological requirements, climate change and global warming could present a major threat for a. leptophylla populations, especially in the mediterranean basin. anogramma leptophylla is one of the rarest fern species in croatia; however, other rare ferns such as asplenium cuneifolium viv., a. sagittatum (dc.) bange, hymenophyllum tunbrigense (l.) sm., notholaena marantae (l.) desv. and woodsia ilvensis (l.) r. br., each with barely few fi nding localities, also occur in the state territory. thus, targeted fi eld investigations of these species, including a. leptophylla, are of great importance for widening the knowledge on their distribution in the future. acknowledgements the fi eld research was undertaken within the frames of the “flora of mljet np” project, funded by the national park mljet. references dostál, j., 1984: famile gymnogramminaceae. 112–115. in: kramer, k. u. (ed.), illustrierte flora von mitteleuropa. dritte, völlige neubearbeitete aufl age. verlag paul parey, berlin. fischer, m. a., oswald, k., adler, w., 2008: exkursionsfl ora für österreich, lichtenstein und südtirol. biologiezentrum der oberösterreichischen landesmuseum, linz. hayek, a., 1927: prodromus fl orae peninsulae balcanicae. repertorium specierum novarum regni vegetabilis. band i. pteridošegota v., hršak v., alegro a. 94 acta bot. croat. 76 (1), 2017 phyta, gymnospermae, dicotyledone (apetalae et choripetalae) 19. dahlem. hirc, d., 1905: revision of croatian fl ora. rad jazu 161, 145– 239 (in croatian). hršak, v., 1994: anogramma leptophylla (l.) link. in: nikolić (ed.), flora croatica. index fl orae croaticae. pars 1. natura croatica 4, suppl. 2, 28. jalas, j., suominen, j. (eds.), 1972: atlas fl orae europaeae. distribution of vascular plants in europe. vol. 1. the comittee for mapping the fl ora of europe and societas biologica fennica vanamo, helsinki. kopp, e., schneebeli-graf, r, 1998: illustrierter leitfaden zum bestimmen der farne und farnverwandten pfl anzen der schweiz und angrenzender gebiete. schweizerische vereinigung der farnfreunde. switzerland. landolt, e., 2010: flora indicativa. ökologische zeigerwerte und biologische kennzeichen zur flora der schweiz und der alpen. 1. aufl age. haupt verlag. latzel, a., 1914: neuere ergebnisse der botanischen erfoschung dalmatiens und der herzegovina. gesellschaft deutscher naturforscher und ärzte. leipzig. lauber, k., wagner, g., 1998: flora helvetica. verlag paul haupt. bern-stuttgart-wien. mannagetta, b.g. von, 1901: die vegetationsverhältnisse der illyrischen länder. wilhelm engelmann, leipzig, 1–534. mayer, e., horvatić, s., 1967: anogramma leptophylla. in: horvatić, s. (ed.), analytical fl ora of yugoslavia 1/1. šumarski fakultet sveučilišta u zagrebu. sveučilišna naklada liber (in croatian). meusel, h, jäger, e., wienest, e., 1965: vergleichende chorologie der zentraleuropäischen flora. text. veb gustav fischer verlag, jena. micevski, k., 1985: flora of fr macedonia. 1/1, 52. makedonska akademija na naukite umetnostite, skopje (in macedonian). molnár, c., baros, z., pintér, i., türke, i. j., molnár, a., sramkó, g., 2008: remote, inland occurrence of the oceanic anogramma leptophylla (l.) link (pteridaceae: taenitidoideae) in hungary. american fern journal 98/3, 128–138. nakazato, t., gastony, g. j., 2003: molecular phylogenetocs of anogramma species and related genera (pteridaceae: taenitioideae). systematic botany 28, 490–502. nikolić, t. (ed.), 2016: anogramma leptophylla. in: flora croatica database. on-line. university of zagreb. faculty of science, division of botany, zagreb. retrieved february 30, 2016 from http://hirc.botanic.hr/fcd. nikolić, t., bukovec, d., šopf, s., jelaska, s. d., 1998: mapping of croatian fl ora – possibilities and standards. natura croatica, periodicum musei historiae naturalis croatici. 7, suppl. 1, 1–62. nogueira, i., 1986: anogramma link. in: castroviejo, s., laínz, m., lópez gonzaléz, g., montserrat, p., munoz garmendia, f., paiva, j., villar, l. (eds.), flora iberica 1, 63–65. real jardín botaníco, csic, madrid. pangua, e., pérey/ruyafa, i., pajarón, s., 2011: gametophyte features in peculiar annual fern, anogramma leptphylla. annales botanici fennici 48, 465–472. perčec tadić, m., 2008: mean annual precipitation (map). in: zaninović, k. (ed.), climatological atlas of croatia 1961– 1990. 1971–2009. dhmz, zagreb (in croatian). pignatti, s., 1982: flora d’italia vol. 1. edagricole, bologna. proskauer, j., 1964: riccia tuberosa taylor = anogramma leptophylla (l.) link, or on the importance of being bryophytic. journal of the indian botanical society 42, 185–188. pulević, v., 2005: material for vascular fl ora of montenegro. addition to „conspectus fl orae montenegrinae” of j. rohlena. republički zavod za zaštitu prirode crne gore. posebna izdanja, book 2, podgorica (in montenegrin). rechinger, k. h., 1934: zur kenntnis der flora der halbinsel pelješac (sabioncello) und einiger inseln des jugoslawischen adriagebietes (dalmatien). magyar botanikai lapok 33, 24– 42. regula-bevilacqua lj., ilijanić, lj., 1984: analyse der flora der insel mljet. acta botanica croatica 43, 119–142. rottensteiner, w. k., 2014: exkursionsfl ora für istrien. verlag des aturwissenschaftlichen vereins für kärnten. klagenfurt. schlosser, j. c. k., vukotinović, lj., 1869: flora croatica. sumptibus et auspiciis academiae scientiarum et articum slavorum meridionalium, zagreb, i–cxli, 1–1362. stešević, d., caković, d., 2013: catalogue of vascular fl ora of montenegro i. crnogorska akademija nauka i umetnosti, 33 (in montenegrin) studnička, m., 2009: ferns. atlas of autochthonous and exotic species. academia, praha (in czech). studniczka, c., 1890: beiträge zur flora von süddalmatien. verhandlungen der zoologisch-botanischen gesellschaft in wien 40, 55–84. trinajstić, i., 1986: phytogeographical division of the forest vegetation of the eastern adriatic mediterranean area – basic point in the organisation of management of the mediterranean forests. glasnik za šumske pokuse, special edition 2, 53–67 (in croatian). tutin, t. g., 1993: anogramma leptophylla. in: tutin, t. g., burges, n. a., chater, a. o., edmondson, j. r., heywood, v. h., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea vol. 1, 2. ed. psilotaceae to platanaceae, 13. cambridge university press, new york. tyron, r., tyron, f., kramer, k. u., 1990: pteridaceae. 230–256, in: kramer, u. k., green, p. s. (eds.), the families and genera of vascular plants, vol. 1. pterydophytes and gymnosperms. springer verlag, berlin. visiani, r., 1852: flora dalmatica, lipsiae. appendix specimina visa vodopić, mato: lapad (dubrovnik) (ad saxa viae in olivato opuić, lapad), 1868 (za): šegota, vedran and vladimir hršak: national park mljet, between tatinica cove and rt križice (rt kula) promontory, 2010 (za) literature data visiani: dubrovnik (ad muros et rupes agri ragusini, ubi legit fr. neumayer. fruct. a vere in aestatum), 1852; schlosser and vukotinović: dubrovnik, 1869; mannagetta, b. g. von: dubrovnik, village of zaton (reicher an besonderheiten ist die festlandküste um ragusa, d. h. von malfi bis ragusa vecchia.), 1901; hirz: dubrovnik, lapad, 1905; latzel: sobra (island of mljet), lapad peninsula (dubrovnik) (an den felsen in der nähe des porto sovra auf der insel meleda; auf lapad bei ragusa), 1914; hayek: da (dalmatia), 1927; rechinger: village of vrisnik (island of hvar) (h: grund v. wegmauern b. vrisnik, 150 m), 1934; regula-bevilaqua and ilijanić: mljet, 1984; rottensteiner: krk, baška – halm, 2014. opce-str.vp acta bot. croat. 68 (2), 351–365, 2009 coden: abcra 25 issn 0365–0588 seasonal distribution of plankton diatoms in lim bay, northeastern adriatic sea sun^ica bosak1*, zrinka buri]1, tamara djakovac2, damir vili^i]1 1 division of biology, faculty of science, university of zagreb, rooseveltov trg 6, hr-10000 zagreb, croatia 2 centre for marine research, ru|er bo{kovi} institute, g. paliaga 5, hr-52210 rovinj, croatia the seasonal distribution of planktonic diatoms is presented in relation to the hydrographic and chemical parameters in lim bay, a 10 km long fjord-like embayment situated in the north east adriatic sea. water samplings for physicochemical and biological variables were conducted from march 2002 to november 2007 at three stations along the bay. the phytoplankton was dominated by diatoms throughout the year, with the minimum identified and classified according to their seasonal occurrence in the plankton; some were consistently present throughout the whole year, but a large fraction showed a distinct seasonal cycle. the inner two stations differed significantly in terms of phytoplankton biomass and nutrient content from the outer station, indicating a higher nutrient input in the inner part. the majority of the dominant diatoms recorded in this study prefer nutrient-enriched conditions. due to the anticipated increase of human impact in the area, this study can serve as a base for future environmental studies in lim bay. keywords: diatom, phytoplankton, seasonality, lim bay, adriatic sea introduction the adriatic sea is the northernmost basin in the mediterranean, with a length of 800 km and a width of 200–250 km. it may be divided into three parts according to the bathymetry and latitude: the northern adriatic basin with an average depth of 40 m, the middle adriatic with depressions up to 280 m and the southern adriatic circular basin with depths of up to 1230 m. the circulation of the adriatic is cyclonic due to the considerable freshwater input from the northern adriatic rivers, which generates the south-easterly outflowing west adriatic current (orli] et al. 2007). thus the nutrient enriched waters reach the middle and south adriatic basin. the eastern adriatic region is exposed to the east adriatic current which brings highly saline and low-nutrient waters from the ionian and levantine seas (orli] et al. 2007). these hydrographical features combine to make acta bot. croat. 68 (2), 2009 351 * corresponding author: sbosak@biol.pmf.hr u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:21 color profile: disabled composite 150 lpi at 45 degrees species diversity in summer and the maximum in autumn. a total of 100 diatoms were the system quite heterogeneous with across-shelf and longitudinal trophic gradient resulting in the asymmetric distribution of the phytoplankton composition, abundance and biomass (polimene et al. 2007). the literature concerning phytoplankton distribution and dynamics of the northern (revelante and gilmartin 1976, 1980; revelante et al. 1984), middle (caroppo et al. 1999, totti et al. 2000) and southern (vili^i] et al. 1995) adriatic is wide. each of these studies stresses the influence of above-mentioned environmental forcings on the spatial and temporal high variability of phytoplankton biomass and qualitative distribution. the po river discharge and the meteorological forcing factors are the main components triggering the alternation of stratification and mixing of the water column and that strongly affect the phytoplankton annual dynamics in the northern adriatic (revelante et gilmartin 1976). diatoms dominate the phytoplankton assemblages (both microplankton and nanoplankton fractions) over most of the year while flagellates dominate only in oligotrophic conditions, in june–july (bernardi aubry et al. 2004). diatom dominance is often described in connection with the appearance of large mucilaginous aggregates that often occur during late spring/early summer in the northern basin (totti et al. 2005). the exact role of diatoms remains elusive, but some studies indicate the importance of the intensive production of extracellular polysaccharide by several diatom species (e.g. skeletonema marinoi, chaetoceros spp., ceratoneis closterium) commonly found during mucilage occurrences (totti et al. 2005). some toxin-producing diatom species of the genus pseudo-nitzschia have been described from different parts of the adriatic sea. diatoms belonging to the genus pseudo-nitzschia are present the entire year and are generally considered to be dominant in the phytoplankton of the adriatic sea (vili^i] et al. 1995, 2009), but the actual species composition and species succession remain to be elucidated. lundholm et al. (2003) reported the occurrence of p. calliantha in samples from the northern and middle part, buri] et al. (2008) described p. calliantha from the middle part and carropo et al. (2005) identified p. calliantha and p. delicatissima from the southern part of the basin. the first report on the taxonomic composition of phytoplankton from the north east adriatic sea (revelante 1985) has been recently supplemented with records from the middle and southern adriatic (vili^i] et al. 2002). a total of 829 phytoplankton taxa were recorded for the eastern adriatic sea; these taxa comprise 328 pennate diatoms and 179 centric diatoms, 220 dinoflagellates, 94 prymnesiophytes, 3 chrysophytes, 1 euglenophyte, 2 cryptophytes, 1 raphidophyte and 1 chlorophyte. diatoms represent the dominant taxonomic group of the northern adriatic phytoplankton assemblages throughout most of the year (vili^i] et al. 2009), and the aim of this study was to assess their seasonal distribution in lim bay, in the north east part of the adriatic sea. study area lim bay is a fjord-like embayment, 11 km long and 0.5 km wide, located on the west side of the istrian peninsula in the north east adriatic sea (fig. 1.). the maximum depth is about 33 m in the outer part while the inner part is about 17 m deep. many effluents charac352 acta bot. croat. 68 (2), 2009 bosak s., buri] z., djakovac t., vili^i] d. u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:21 color profile: disabled composite 150 lpi at 45 degrees teristic of the karstic region and a number of underwater freshwater springs, especially in the inner part, contribute to the productivity of lim bay. freshwater inflow becomes important during heavy rainfall periods, which are usually in the period from september to december and in april. (penzar et al. 2001). some shellfish and fish farming operations are to be found in the inner and middle parts of the bay (krajnovi]-ozreti] et al. 2001) which is also known for providing a good spawning ground as well as a hiding place for many commercial fish (huljev and strohal 1983). the climate is classified as moderate mediterranean, with an annual precipitation averaging 873 mm (gaji]-^apka et al. 2003). materials and methods sampling was carried out five times from march to august 2002 and seven times yearly from february 2003 to november 2007 at three stations located along lim bay (tab. 1). in all, 414 samples were collected and analyzed throughout the study period. water samples were collected with 5 l niskin bottles at stations lim1 and lim2 at five (surface, 5m, 10m, 20 m and 2 m above bottom) or at station lim3 at four depths (surface, 5 m, 10 m and 2 m above bottom). at each station, water temperature and salinity were measured using a ctd probe (seabird, usa). dissolved oxygen concentration was determined by the winkler titration method (parsons et al. 1984), while the saturation percent of dissolved oxygen in each water sample was calculated (from the quotient between the measured oxygen concentration and the oxygen solubility) following the benson and krause equation (unesco 1986). acta bot. croat. 68 (2), 2009 353 seasonality of diatoms in lim bay fig. 1. location of the sampling stations in lim bay, north east adriatic sea, visited from march 2002 to november 2007. u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:21 color profile: disabled composite 150 lpi at 45 degrees subsamples for the determination of nitrate (no3), phosphate (po4) and silicic acid (si(oh)4) were measured using a shimadzu uv-mini 1240 spectrophotometer (ivan^i] and degobbis 1984, parsons et al. 1984). subsamples for the determination of chlorophyll (chl) a were filtered onto whatman gf/c filters. following a 3 hour extraction in 90% acetone (at dark, with grinding), chl a concentrations were determined on a turner td-700 fluorometer (parsons et al. 1984). for the enumeration of phytoplankton cells, 150 ml samples were preserved with 2% (final concentration) disodium tetraborate (borax) buffered formaldehyde, as well as additional net samples for qualitative analyses. cells were identified and enumerated using an inverted microscope (zeiss axiovert 200) operating with phase contrast and brightfield optics (utermöhl 1958, lund et al. 1958, vili^i] et al. 2008). we used the following references for phytoplankton identification: hustedt 1959, ricard 1987, hasle and syversten 1997, round et al 1990, bérard-therriault et al. 1999, horner 2002, throndsen et al. 2007. the data set represents presence/absence observations combined from the net and bottle samples as well as abundance data from the bottle samples. our monthly observations of diatom flora were combined into seasonal categories as follows a (autumn): october, november w (winter): december, january w/s (winter/spring transition): february, march s (spring): april, may s/su (spring/summer transition): june su (summer): july, august su/a (summer/autumn transition): september the categories were defined by taking into consideration local meteorological variables (monitored by the croatian meteorological and hydrological service dhmz, available at http:// meteo.hr) two diversity indices were calculated using the statistical program primer v5. these were the margalef species richness index (d) (margalef 1968), which represents a measure of the number of different species (s) present in the sample, making some allowance for the number of individuals (n), which was calculated using the formula: d = (s-1)/log 354 acta bot. croat. 68 (2), 2009 bosak s., buri] z., djakovac t., vili^i] d. tab. 1. distribution of sampling during 2002–2007. the number of samples collected for each period is indicated. total number of samples is 414. month year jan feb mar apr may jun jul aug sep oct nov dec 2002 9 6 6 6 6 2003 12 12 12 11 10 12 10 2004 11 11 11 12 11 11 9 2005 11 12 11 8 3 10 11 2006 10 12 12 10 13 12 12 2007 13 13 7 11 10 12 13 u:\acta botanica\acta-botan 2-09\bosak.vp 9. listopad 2009 13:24:23 color profile: disabled composite 150 lpi at 45 degrees (n) and the shannon-wiener (h’) diversity index (shannon and weaver 1963) using the formula h’ = si p i ln (pi) where pi is the proportion of abundance of species. environmental and biological data were analysed by one-way anovas with post-hoc bonferroni tests using the program statistica, version 6.0. a logarithmic transformation (log (x+1)) was used on nutrient and diatom abundance data to obtain the normal distribution. results the range of variation in the environmental and biological variables measured in lim bay over the six-year period is listed in table 2. salinity and temperature profiles (fig. 2a, b) indicate a vertical instability of the water column most of the year, except in summer, which is characterized by a shallow thermocline in the 5–10 m layer. salinity was rather constant, but with some occasional low values (< 30) recorded in the surface layer (0–5 m) of the inner stations in the period february to may, due to winter and early spring precipitation events. mean phosphate concentrations were at least two times higher at the inner two stations than at the outer one and showed a slight increase from late spring to autumn (fig. 3a). mean nitrate and silicic acid concentrations were lower during the summer stratification period (fig. 3b, d). the seasonal distribution of diatom abundances and chlorophyll a concentrations showed a slight increase during the summer and autumn period in all three stations (fig. 4). one-way anova with post-hoc bonferroni test run on nutrient (phosphate, nitrate, silicid acid) and chlorophyll a data showed a statistically significant difference (p < 0.001) between the two inner (lim2, lim3) and the outer station (lim1). the same test run on diatom abundance data showed significant difference between the middle (lim2) and other two (lim1, lim3) stations (p < 0.001). acta bot. croat. 68 (2), 2009 355 seasonality of diatoms in lim bay tab. 2. descriptive statistics of physical, chemical and biological variables measured in the water column at three stations of lim bay between march 2002 and november 2007. n: number of observations. n.d.: not detected. min mean max sd n temperature (°c) 7.8 17.3 27.6 5.3 519 salinity (psu) 23.2 37.4 38.5 1.4 519 oxygen (%) 41 99 135 13 519 total phytoplankton (cells l–1) 160 9.38 x 104 245 x 104 20.5 x 104 414 diatoms (cells l–1) 40 8.44 x 104 243 x 104 20.2 x 104 414 chlorophyll a (µg l–1) 0.19 1.04 9.55 0.93 492 no3 (µmol l –1) 0.07 2.14 30.10 2.72 519 po4 (µmol l –1) n.d. 0.06 0.60 0.08 519 si(oh)4 (µmol l –1) 0.02 4.63 40.07 4.58 515 u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:21 color profile: disabled composite 150 lpi at 45 degrees a total of 100 diatom taxa were identified from lim bay (tab. 3) together with seasonal distribution and qualitative occurrence. although the majority of taxa are marine planktonic diatoms, there are several benthic species that occurred regularly or occasionally. eleven taxa are characterized as dominant with maximum abundance greater than 105 cells l–1 and occurrence greater than 20% in the samples: cerataulina pelagica, chaetoceros socialis, chaetoceros sp., dactyliosolen fragilissimus, guinardia striata, leptocylindrus danicus, nitzschia longissima, proboscia alata, pseudo-nitzschia spp., rhizosolenia imbricata and thalassionema nitzschioides. some diatoms showed no seasonal variation but were consistently present throughout the year: ceratoneis closterium, chaetoceros sp., coscinodiscus spp., dactyliosolen fragilissimus, diploneis bombus, guinardia flaccida, g. striata, hemiaulus hauckii, pleurosigma sp., pseudosolenia calcar-avis, rhizosolenia imbricata, rhizosolenia sp. and thalassiosira spp. a large fraction of the diatoms recorded showed a distinct seasonal cycle, defined by their presence in one season only or all year round but with a most frequent occurrence at a given season. the most frequent occurrence defined as more than three observations for a given seasonal category is sometimes, but not always, accompanied by the highest abundance (tab. 3). both the species richness (d) and biodiversity index (h’) showed high values in autumn as opposed to low summer values although phytoplankton cell abundances were similar (figs. 4a, 5). the high diversity recorded in autumn is mainly explained by the high number of different species of the planktonic diatoms chaetoceros and bacteriastrum. 356 acta bot. croat. 68 (2), 2009 bosak s., buri] z., djakovac t., vili^i] d. fig. 2. distribution of temperature (a) and salinity (b) at station lim3 in lim bay in the period march 2002 to november 2007. u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:22 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 357 seasonality of diatoms in lim bay fig. 3. annual distribution of nitrate (no3) (a), phosphate (po4) (b) and silicic acid (si(oh)4) (c), chlorophyll a (d) and diatom abundance (e) at three stations in lim bay from march 2002 to november 2007, at stations lim1 (�), lim2 (�) and lim3 (�). data are presented as the monthly average obtained from the entire data set, clustered for each month per each station. u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:22 color profile: disabled composite 150 lpi at 45 degrees 358 acta bot. croat. 68 (2), 2009 bosak s., buri] z., djakovac t., vili^i] d. fig. 4. annual distribution of species richness (d) (a) and diversity index (h’) (b) in lim bay from march 2002 to november 2007. u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:23 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 359 seasonality of diatoms in lim bay tab. 3. list of planktonic diatoms recorded at three stations in lim bay between march 2002 and november 2007. information on seasonality follows after each species name. w: winter; w/s: winter/spring; s: spring; s/su: spring/summer; su: summer; su/a: summer/autumn; a: autumn; bold character indicates the most frequent occurrence, sometimes accompanied by highest cell abundance. only name of the month indicates single occurrence, not in all months within defined category. actinocyclus spp. mar, nov actinoptychus splendens (shadbolt) ralfs nov amphora spp. aug, oct amphiprora spp. feb, nov asterionellopsis glacialis (castracane) round su/a, a, mar, dec asteromphalus flabellatus (brébisson) greville su/a a. heptactis (brébisson) ralfs s/su, aug bacillaria paxillifera (o.f. müller) hendey s/su, su/a, oct bacteriastrum delicatulum cleve all year, s, su/a, a b. elongatum cleve nov b. hyalinum lauder jan, mar, su/a, a b. mediterraneum pavillard w/s, su, a bacteriastrum spp. mar, su, su/a, a biddulphia titiana grunow nov cerataulina pelagica (cleve) hendey all year, s/su, su ceratoneis closterium ehrenberg all year chaetoceros affinis lauder w/s, su/a, a, may, dec c. anastomosans grunow su c. brevis schutt mar, su c. compressus lauder mar, may, jul, su/a, a c. convolutus castracane feb, aug, su/a, oct c. costatus pavillard w/s, aug, a, dec c. curvisetus cleve all year, mar, aug c. danicus cleve all year, su/a, a c. decipiens cleve all year, su/a, a c. densus (cleve) cleve mar, dec c. didymus ehrenberg feb, nov c. diversus cleve may, aug, su/a, nov c. eibenii (grunow) meunier jan, a c. lauderi ralfs su, nov c. lorenzianus grunow may, s/su, jul, su/a, a c. perpusillus cleve su/a c. peruvianus brightwell nov c. rostratus lauder jul, aug, a c. simplex ostenfeld may, s/su, nov u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:23 color profile: disabled composite 150 lpi at 45 degrees 360 acta bot. croat. 68 (2), 2009 bosak s., buri] z., djakovac t., vili^i] d. c. socialis lauder all year, su chaetoceros spp. all year c. tenuissimus meunier su/a, a c. throndsenii marino, montresor et zingone s/su c. tortissimus gran su/a c. vixvisibilis schiller may, s/su, su, oct cocconeis scutellum ehrenberg su, nov c. placentula ehrenberg s/su coscinodiscus granii gough nov c. perforatus ehrenberg nov coscinodiscus spp. all year cyclotella spp. all year, s/su, su dactyliosolen blavyanus (peragallo) hasle may, nov d. fragilissimus (bergon) hasle all year d. phuketensis (sundström) hasle jan, w/s detonula pumila (castracane) gran w, w/s, nov diploneis bombus ehrenberg all year d. crabro ehrenberg su, su/a, a ditylum brightwellii (west) grunow w/s, nov eucampia cornuta (cleve) grunow mar, aug, a e. zodiacus ehrenberg jan, feb grammatophora sp. oct guinardia delicatula (cleve) hasle su/a guinardia flaccida (castracane) peragallo all year g. striata (stolterfoth) hasle all year gyrosigma balticum (ehrenberg) rabenhorst feb, nov hemiaulus hauckii grunow all year h. sinensis greville w/s, s/su, su, a lithodesmium undulatum ehrenberg jan lauderia annulata cleve jan, feb, aug leptocylindrus adriaticus schroeder feb l. danicus cleve w/s, may, jun, aug l. mediterraneus (h. peragallo) hasle mar, aug, a licmophora spp. w/s, s lioloma pacificum (cupp) hasle jul, a, dec lyrella lyra(ehrenberg) karayeva aug neocalyptrella robusta (norman) hernández-becerril et meave w, w/s, may, nov navicula spp. su, a nitzschia incerta (grunow) m. peragallo aug tab. 3. continued u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:23 color profile: disabled composite 150 lpi at 45 degrees discussion the northern adriatic basin is a complex system in which the spatial distribution and seasonal variability of phytoplankton are mainly driven by the po river discharge, coupled with the stratification/mixing regime and circulation (revelante and gilmartin 1976). lim bay is a semi-enclosed area without any strong freshwater source, unlike the nearby gulf of trieste, which has a strong seasonal pattern related to riverine nutrient input (malej et al. 1995). the freshwater input from underwater springs was not very prominent, which can be seen on the salinity profile (fig. 2). the minimum salinity values were recorded in the surface layer at the inner stations due to the winter and early spring precipitation events. nutrients in lim bay are generally low, the results corroborating those obtained from the adjacent northern adriatic basin (degobbis et al. 2005, totti et al. 2005). no major unacta bot. croat. 68 (2), 2009 361 seasonality of diatoms in lim bay nitzschia longissima (brébisson) ralfs all year, s/su, su nitzschia spp. s/su, su/a, oct odontella mobiliensis (bailey) grunow nov opephora marina (gregory) petit mar, oct paralia sulcata (ehrenberg) cleve all year, su/a pleurosigma angulatum (quekett) smith su, su/a, a p. normanii ralfs aug pleurosigma spp. all year proboscia alata (brightwell) sundström all year, jun p. indica (h. peragallo) hernández-becerril may psammodictyon panduriforme (gregory) mann aug, a pseudo-nitzschia spp. all year, su/a, aug, oct pseudosolenia calcar-avis (schultze) sundström all year rhizosolenia hebetata bailey s/su, su r. imbricata brightwell all year rhizosolenia spp. all year skeletonema marinoi sarno et zingone w, w/s, aug, su/a, a striatella unipunctata (lyngbye) agardh mar, s, s/su, su synedra toxoneides castracane jul thalassionema nitzschioides (grunow) mereschkowsky all year, su/a, a t. frauenfeldii (grunow) hallegraeff dec, su, su/a, a thalassiosira eccentrica (ehrenberg) cleve feb t. rotula meunier feb thalassiosira spp. all year thalassiothrix longissima cleve et grunow oct toxarium undulatum bailey s/su tab. 3. continued u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:23 color profile: disabled composite 150 lpi at 45 degrees usual patterns in the seasonal distribution of nutrients were noticed during the seven years investigated. the only exception is the maximum phosphate concentration of 0.60 µm, recorded in the middle part of the bay (station lim2), and the generally higher concentrations of this nutrient during the summer and autumn months (> 0.3 µm) in the inner part, which could be possibly related to the influence of fish farms. the high concentrations of nitrate and silicic acid could be attributed to freshwater inputs during the precipitation season. the high availability of three major nutrients could contribute to high phytoplankton biomass values, especially in the period from july to november; this could be seen in the seasonal chlorophyll a distribution (fig. 4b). in summer, the nutrient input from the northern adriatic basin could also be possible after the formation of the istrian coastal countercurrent (iccc), the surface current that usually appears along the istrian coastal line and runs counter to the general adriatic-wide cyclonic flow (supi] et al. 2000). the seasonal distribution of chlorophyll a in the northeast adriatic sea in the last decade (1993–2000) shows a bimodal pattern with a more pronounced maximum in autumn than in spring, which may be attributed to hydrological changes in the po river (supi] et al. 2006, vili^i] et al. 2009). this trend was in accord with the results from this study. a detailed taxonomic analysis showed a more or less similar composition of plankton diatoms recorded in both lim bay and north east adriatic sea, with some differences. some benthic pennate diatoms, such as cocconeis spp., licmophora sp., lyrella lyra and psammodictyon panduriforme, were probably released from the bottom by turbulence in the water column and no apparent seasonal distributional pattern was detected. the majority of the dominant diatoms in lim bay i.e. cerataulina pelagica, chaetoceros socialis, dactyliosolen fragilissimus, guinardia striata, leptocylindrus danicus, nitzschia longissima, pseudo-nitzschia spp., rhizosolenia imbricata and thalassionema nitzschioides, showed a preference for nutrient-enriched conditions in the adriatic sea (revelante and gilmartin 1980, 1985, pucher-petkovi] and marasovi] 1980), which agrees well with the classification of species according to their eutrophication level preferences (yamada et al. 1980). comparison of lim bay with other, similar, systems in the eastrern-central adriatic sea, characterized as moderately eutrophic environments, novigrad sea and [ibenik harbour (buri] et al. 2005, cetini] et al. 2006) showed that the same plankton diatom taxa were frequent, but not equally abundant. high nutrient levels in the novigrad sea, the lower part of the oligotrophic zrmanja estuary, were of natural origin (buri] et al. 2005) while in [ibenik harbour, in the lower part of the krka estuary, a higher phosphate content indicated a more prominent anthropogenic influence (cetini] et al. 2006). the composition of dominant species during the summer-autumn period in lim bay was more similar to that of [ibenik harbour than that of novigrad sea. the eutrophic centric diatom skeletonema marinoi occurs most of the year in lim bay, being found in 18% of samples, with a maximum abundance of 1.45 x105 cells l–1 (recorded november 2005) but with decreasing abundance in the adjacent oligotrophic coastal northeast adriatic sea (vili^i] et al. 2009). in [ibenik harbour it was recorded as one of the dominant species, while in novigrad sea it was not detected at all. its presence in lim bay could be one of the indicators of the higher anthropogenic influence in this area. although our data set covers a relatively short time interval and it is more restricted than some other studies, it brings new information on the composition and average yearly fluctuation of the diatom flora. it can serve as a base for future studies in lim bay where an increase of the human impact can be anticipated. 362 acta bot. croat. 68 (2), 2009 bosak s., buri] z., djakovac t., vili^i] d. u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:23 color profile: disabled composite 150 lpi at 45 degrees acknowledgements the research was financially supported by the ministry of science, education and sports of the republic of croatia (croatian national monitoring programme – jadran, and project no. 119-1191189-1228). the authors are grateful to the crew of the rv vila velebita (centre for marine research, ru|er bo{kovi} institute, rovinj, croatia) and especially to jelena godrijan and daniela mari} for their assistance with sampling. we are thankful to the anonymous reviewers for constructive comments and helpful suggestions on the manuscript. references bérard-therriault, l., poulin, m., bossé, l., 1999: guide d’identification du phytoplancton marin de l’estuaire et du golfe du saint-laurent incluant également certains protozoaires. publication spéciale canadienne des sciences halieutiques et aquatiques 128, 1–387 bernardi aubry, f., berton, a., bastianini, m., socal, g., acri f., 2004: phytoplankton succession in a coastal area 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oluji], g., 2008: phytoplankton abundance and pigment biomarkers in the oligotrophic eastern adriatic estuary. environmental monitoring and assessment 142, 199–218. yamada, m., tsurita, a., yoshida, y., 1980: a list of phytoplankton as eutrophic level indicator. bulletin of the japanese society of scientific fisheries 46, 1435–1438. acta bot. croat. 68 (2), 2009 365 seasonality of diatoms in lim bay u:\acta botanica\acta-botan 2-09\bosak.vp 6. listopad 2009 11:51:23 color profile: disabled composite 150 lpi at 45 degrees acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 95 acta bot. croat. 76 (1), 95–97, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0047 issn 0365-0588 eissn 1847-8476 short communication hieracium pollinense (asteraceae), an endemic species to the pollino national park (southern italy) rediscovered günter gottschlich1, filippo scafi di2, emilio di gristina3,* 1 hermann-kurz-straße 35, 72074 tübingen, germany 2 department stebicef, section of botany and plant ecology, university of palermo, via archirafi 38, 90123 palermo, italy 3 botanical garden and herbarium mediterraneum panormitanum, university of palermo, via lincoln 2, 90133 palermo, italy abstract – the presence of hieracium pollinense zahn in italy is confi rmed here after 132 years since its fi rst description based on a single collection made in 1877 in the mt. pollino. it is a calcicolous species, so far represented by one population, belongs to the h. sect. villosa. in line with the iucn criteria its conservation status assessment is “endangered”. keywords: basilicata, distribution, hieracium, pollino massif, taxonomy, vascular fl ora * corresponding author, e-mail: emilio.digristina@unipa.it introduction the pollino national park is the largest protected area (196,000 ha, bernardo 1995) in italy (di sanzo et al. 2013). it is located in the southern italian apennines along the border between the calabria and basilicata regions (fig. 1). the pollino national park exhibits several very particular fl oristic features, due to a geographical location which allows different kind of biogeographical links to be established (puglisi et al. 2009). the biogeographical relation with the southern balkan peninsula is the most evident and it is testifi ed by the occurrence of a great number of amphiadriatic species (pinus heldreichii h. christ, festuca bosniaca kumm. & sendtn., carex kitaibeliana beck, edraianthus graminifolius a. dc., sesleria autumnalis (scop.) f. w. schultz, gentianella crispata (vis.) holub, cytisus spinescens c. presl, etc.) (puglisi et al. 2009). also the pollino massif is the southernmost limit for various boreal or arcticalpine species such as orthilia secunda (l.) house, pyrola minor l., chrysosplenium dubium ser., saxifraga aizoides l., carex pallescens l., c. vesicaria l., senecio alpinus (l.) scop., etc.), which covered southwards the italian peninsula during the ice ages and which remained isolated as relics in the postglacial period (puglisi et al. 2009). by virtue of its ecological and fl oristic diversity, the fl ora of the pollino national park is currently under revision. recent studies in fact resulted in the description of new taxa (conti et al. 2014, di gristina et al. 2014 and 2015) that add to the considerable number of about 1500 species (gargano et al. 2014) as already estimated. regarding hieracium l. (asteraceae) s. str., 12 taxa have been reported from this territory: h. hypochoeroides subsp. serinense (zahn) greuter, h. murorum s.l., h. pallescens subsp. tephrochlorum (zahn) gottschl., h. schmidtii tausch, h. pollinense zahn, h. portanum belli, h. racemosum subsp. crinitum (sm.) rouy, h. racemosum subsp. virgaurea (coss.) zahn, h. scorzonerifolium subsp. divaricatum nägeli & peter, h. terraccianoi di grist. et al. and h. valoddae subsp. austroitalicum (zahn) zahn (gavioli 1947, di gristina et al. 2014). fig. 1. the only known location of hieracium pollinense. gottschlich g., scafidi f., di gristina e. 96 acta bot. croat. 76 (1), 2017 hieracium pollinense zahn is a little-known endemic species of the pollino national park. more than hundred years ago, zahn (1901) described the plant, based on material collected in 1877 by huter, porta and rigo on mt pollino, and distributed in the exsiccata “huter, porta, rigo, ex itinere italico iii, nr. 661b” (fig. 2) (not 616 as errorneously indicated in the protologue by zahn). lectotype: boz [brix-2462] (gottschlich 2007), isolectotypes: bp-449969, fi s.n., w-1889-86148. in 1904 belli and arvet-touvet described a species named h. rigoanum (belli 1904), whose type was from the same locality as indicated for h. pollinense by zahn, but with the reference to an exsiccata specimen “huter, porta et rigo nr. 661”, not 661b, an orthographic mistake (fig. 2). therefore h. rigoanum must be regarded as superfl uous (illegitimate) name. beyond that it was a younger homonym to h. rigoanum zahn 1902. so far, the presence of h. pollinense in italy was known from the type specimens only. pignatti (1982) reports the plant but as doubtfully present (“to be searched”), whereas conti et al. (2005) do not account for this species in their checklist of the italian fl ora. during our recent fl oristic inventory of the pollino massif (lucanian side, potenza province) (fig. 1), a small population of hieracium clearly related to h. pollinense was found. the comparison with the plants collected by huter, porta and rigo, has allowed us to ascertain defi nitively the identiy of this population to h. pollinense and to confi rm its presence in the italian fl ora after more than a century. material and methods the relevant herbarium specimens were found during general revisions of hieracium specimens in boz, fi, bp, w (acronyms are according to thiers 2015). additional material was collected during our fi eld research in southern italy from 2014 to 2016. this material is deposited as pal108621 and hb. gottschlich-63953. results and discussion nomenclature: hieracium pollinense zahn, allg. bot. z. syst. 7: 145 (1901) ≡ h. leucophaeum subsp. pollinense (zahn) zahn in engler, pfl anzenreich [heft 75] iv 280: 116 (1921) ≡ h. rigoanum arv.-touv. & belli in fiori & paoletti, fl. italia 3: 481 (1904), nom. illeg (non zahn 1902) because the original diagnosis (zahn 1901) is very short we provide a detailed description of the taxon: perennial, phyllopodous, rosulate hemicryptophyte. rhi zome thick, horizontal to oblique. stem erect, solid, (20–) 25–30(–45) cm high, sparsely covered with 8–12 mm long sericeous white simple hairs, without glandular and stellate hairs. basal leaves (2–)3–5(–6), petiolate, petiole (1–)2–3(– 4) cm long, moderate covered with 8–10 mm long sericeus white simple hairs lamina of outer basal leaves ovate (1.5–2 ×3–4 cm), rounded or truncate at base, entire, other ovatelanceolate to lanceolate (5–7×1–2 cm), hastate at base, entire, denticulate or with one prominent tooth on each side, rarely whole margin dentate, glaucous; surface of leaves glabrous, margin and lower side with sericeous simple hairs up to 5 mm long. cauline leaves 1(–2), often reduced, entire to denticulate. synfl orescence usually furcate, branches (0)1(–3), erect, 3–8 cm long, capitula altogether (1–)2(–4); acladium 3–4 cm long. peduncles sparsely covered with simple and glandular hairs, stellate hairs rather dense. capitula 8–10(–11) mm long, subglobose. involucral bracts linear-lanceolate, acute, dark olive green, covered with moderate sericeus white simple hairs up to 5 mm long and sparse glandular hairs, 0.2–0.5 mm long, stellate hairs lacking or only at base along margins. ligules liguliform, yellow, glabrous at apex. styles dark. achenes 3–3.5 mm long, dark brown. taxonomic relationship. regarding to hieracium pollinense, zahn (1901) noted (in translation): “synfl orescence refers to h. scorzonerifolium grex schizocladum, the glandular hairs and the few stellate hairs point to h. humile, whose infl uence becomes evident also on the leaves. h. pollinense differs from h. bernense and its subspecies especially by the broad leaves”. however, after having examined further material from switzerland, he placed it together with other earlier described species (like h. bernense christener, h. leucophaeum gren. & godr., h. gremlii arv.-touv., h. asterinum arv.-touv. & briq., h. diabolinum nägeli & peter, h. godetii christener) and some subspecies described by himself, into the collective species h. leucophaeum, relegating h. pollinense and the other taxa to the rank of subspecies (zahn 1921–1923, 1922–1938).fig. 2. hieracium pollinense: lectotype (fi). hieracium pollinense in the pollino national park acta bot. croat. 76 (1), 2017 97 recently the taxon was recognized at specifi c rank (greuter 2008). the examination of recent collections showed that the plant deserves the rank of species and the interpretation about an introgression of h. humile jacq. seems to be not appropriate. in fact there can be found some very isolated glandular hairs on the margins of the leaves, but this and also the shape of leaves are over-diagnosed with regard to an introgression of h. humile. the coincidence with the western alpine complex of similar taxa may be an analogy. so we favour to take h. pollinense as a separate species endemic to southern italy. concerning the relationship to h. scorzonerifolium vill., the long sericeous simple hairs at the lower part of the stem, the petioles, the phyllaries and the somewhat glaucous leaves could indicate some plausibility for an introgression of a species from h. sect. villosa (griseb.) gremli. the second parent probably belongs to a species of h. sect. grovesiana gottschl. phenology. flowering time: july to the fi rst decade of august (fig. 3). fruiting time: july–august. distribution and ecology. within the pollino national park, h. pollinense is currently known only from the lucanian side of the pollino massif. it is a calcicolous taxon represented by a small population consisting about 100 individuals occuring on north-exposed rocks and stony slopes facing the bosco di chiaromonte (chiaromonte, potenza province), between 1700 and 1800 m of elevation. within this narrow mountain belt, it grows together with adenostyles australis (ten.) iamonico & pignatti, arabis collina ten., astragalus depressus l., hypochoeris laevigata (l.) ces. et al., sanicula europaea l., veronica montana l., physospermum verticillatum (waldst. & kit.) vis., etc. conservation status. only one site is currently known for hieracium pollinense within the pollino national park. despite its limited distribution (less than 1 km2) and the low number of plants – in the only know location 40–60 mature individuals were estimated – it does not seem to be currently subject to threats that may cause a decrease of the number of mature individuals. therefore, according to the iucn (2014) criteria for the conservation status assessment, h. pollinense should be classifi ed as “endangered” (en): d. acknowledgements financial support by the international foundation pro herbario mediterraneo and by the università degli studi di palermo (fondi di ateneo per la ricerca) are acknowledged. fig. 3. hieracium pollinense on the locus classicus. references belli, s., 1904: hieracium. in: fiori, a., paoletti, g. (eds.), flora ana lytica d’italia. vol. iii: 442–505. tipografi a del seminario, padua. bernardo, l., 1995: fiori e piante del parco del pollino. edizioni prometeo, castrovillari. conti, f., abbate, g., alessandrini, a., blasi, c., 2005: an annotated checklist of the italian vascular fl ora. palombi, roma. conti, f., bernardo, l., cusma velari, t., kosovel v., feoli chiapella l., 2014: morphometric and karyological study of genista sericea (cytiseae-fabaceae). phytotaxa 181, 61–78. di gristina, e., gottschlich, g., raimondo, f. m., 2014: hieracium terraccianoi (asteraceae), a new species endemic to the pollino national park (southern italy). phytotaxa 188, 55–60. di gristina, e., scafi di, f., domina, g., 2015: a new species of isatis (brassicaceae) from the pollino national park (basilicata, s italy). flora mediterranea 25 (special issue), 297–303. di sanzo, p., de martino, l., mancini, e., de feo, v., 2013: medicinal and useful plants in the tradition of rotonda, pollino national park, southern italy. journal of ethnobiology and ethnomedicine 9, 1–9. gargano, d., bonacci, a., de vivo, g., marchianò, v., schettino, a., bernardo, l., 2014: a permanent fi eld laboratory in the pollino national park: vegetation dynamics in mountain herbaceous communities. italian journal of forest and mountain environments 69, 125–133. gavioli, o., 1947: synopsis florae lucanae. giornale botanico italiano 54, 1–278. gottschlich, g., 2007: die gattung hieracium l. (compositae) im herbarium rupert huter (vinzentinum brixen, brix). kommentiertes verzeichnis mit taxonomischen und nomenklatorischen ergänzungen unter besonderer berücksichtigung der typus-belege. veröffentlichungen des tiroler landesmuseums ferdinandeum 86, 5–416. greuter, w., 2008: med-checklist 2. dicotyledones (compositae). optima secretariat, palermo, berlin. pignatti, s., 1982: flora d’italia vol. 3. edagricole, bologna. puglisi, m., privitera, m., di pietro, r., 2009: new interesting bryophyte records from the pollino national park (southern italy). flora mediterranea 19, 5–9. thiers, b., 2015: index herbariorum, a global directory of public herbaria and associated staff. new york botanical garden’s virtual herbarium. retrieved september 16, 2016 from http:// sweetgum.nybg.org/ih/. zahn, k. h., 1901: beitrag zur kenntnis südeuropäischer hieracien. allgemeine botanische zeitschrift für systematik, floristik, pfl anzengeographie etc. 7, 113–115, 145–147, 177–179. zahn, k. h., 1921–23: hieracium. in: engler, a., (ed.), das pfl anzenreich 75, 1–288, 76, 289–576, 77, 577–864 (1921); 79, 865– 1146 (1922); 82, 1147–1705 (1923). engelmann, leipzig. zahn, k. h., 1922–38: hieracium. in: ascherson, p. f. a., graebner, k. o. p. p. (ed.), synopsis der mitteleuropäischen flora 12(1), 1–80 (1922), 81–160 (1924), 161–400 (1929), 401–492 (1930); 12(2), 1–160 (1930), 161–480 (1931), 481–640 (1934), 641–790 (1935); 12(3), 1–320 (1936), 321–480 (1937), 481– 708 (1938). borntraeger, leipzig and berlin. acta botanica 2-2016 za web.indd 194 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 194–198, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0030 issn 0365-0588 eissn 1847-8476 an eco-physiological and biotechnological approach to conservation of the world-wide rare and endangered aquatic liverwort riella helicophylla (bory et mont.) mont. marko s. sabovljević1*, josé gabriel segarra-moragues2, felisa puche3, milorad vujičić1, annalena cogoni4, aneta sabovljević1 1 institute of botany and botanical garden, faculty of biology, university of belgrade, takovska 43, 11000 belgrade, serbia 2 departamento de biología vegetal, facultad de ciencias biológicas, universitat de valència. avda. dr. moliner, 50, e-46100, burjassot (valencia), spain 3 departamento de botánica, facultad de ciencias biológicas, universitat de valència. avda. dr. moliner, 50, e-46100, burjassot (valencia), spain 4 dipartimento di scienze della vita e dell’ambiente – sezione botanica ed orto botanico,università di cagliari, italy abstract – the rare aquatic liverwort riella helicophylla (bory et mont.) mont., inhabitant of temporary shallow ponds around the mediterranean basin, is considered threatened throughout its distribution range. in addition, little is known of its biology and ecology or of its role in such an important ecosystem where environmental conditions vary yearly in unpredictable ways. in these variable habitats, due to the seasonal fl uctuation of water levels, there is no guarantee of yearly spore input into the spore bank. spore germination rate and the effects of different culture media in an axenic culture establishment, as well as propagation procedures of r. helicophylla, were tested. new insights into the ecology and biology of r. helicophylla are given. spore dormancy is documented, and the protocols for the in vitro culture establishment, propagation and acclimatization of this liverwort are developed. dry storage at 20 ± 2 °c for about three months broke the dormancy of spores, which subsequently germinated in a high percentage (over 90%). a two phase (solid and liquid) culture media system was developed for the purpose of achieving fully developed gametophytes. the liquid phase contained electrolytes simulating brackish water. key words: conservation, ecology, propagation, riella, temporary ponds * corresponding author, e-mail: marko@bio.bg.ac.rs introduction temporary mediterranean ponds are threatened ha bitats of shallow water bodies that remain fl ooded for a suffi ciently long period of time during winter and spring to allow the development of (semi-) aquatic vegetation and animal communities. these ponds can be of various types, differing in size, shape, depth, altitude, substrates (soil and rock types and combination of these), sources of water, salt concentration in the water, and duration of water reservoir, thus infl uencing their biological diversity. mediterranean temporary ponds comprise a prio ri ty habitat according to the natura 2000 network of the european union (natura code 3170, habitats directive 92/43/ec) which are scattered primarily in peri-mediterranean countries with dry and semi-arid climates. conservation biologists are aware of the importance of these sites, as they play a signifi cant role in global biogeochemical cycles and biodiversity maintenance (miracle et al. 2010). threats to these habitats may vary on different spatial scales and ultimately depend on the conditions at a specifi c site. the main threats derive from inadequate manage ment of the ponds due to human activities that include agriculture, over-exploitation of aquifers, draining and dredging as well as silting, which, although it constitutes a process in the natural evolution of the ponds, can be accelerated by human activities (asem et al. 2014). despite their high biodiversity levels, not all the organisms in these habitats have received similar attention from a conservation perspective (della bella et al. 2005). conservation efforts have mainly focused on vascular plants, amphibians, crustaceans and other invertebrates such as rotipropagation of riella helicophylla acta bot. croat. 75 (2), 2016 195 fers (zacharias et al. 2007). bryophytes have been subject to very few conservation initiatives, most likely because they represent a very minor proportion of the species diversity in these aquatic habitats. among bryophytes, a small group of thallose liverworts in the sphaerocarpales includes the genus riella mont. this genus includes some 28 species (söderström et al. 2016), some of which have been described only recently, indicating how poor the knowledge of this genus is (segarra-moragues et al. 2012, 2014; cargill and milne 2013; segarra-moragues and puche 2014). species of riella grow commonly submerged in clean, shallow, fresh or brackish waters of seasonal ponds, more rarely in permanent waters of arid and semiarid regions and have disjunct distributions and scattered populations (ci rujano et al. 1988). species of riella are rare and/or under-recorded due to their ephemeral habit and fl uctuant popu lations (segarra-moragues et al. 2014). the delicate gametophyte is completely intolerant to desiccation and population persistence in drought periods is ensured through their spores which can remain viable through decades in the spore bank (proctor 1972). some species of riella, such as r. helicophylla (bory et mont.) mont., inhabit brackish water temporary ponds making them, from an ecological perspective, unique amongst bryophytes. in this study, we aimed at establishing the protocol for axenic micropropagation of r. helicophylla, an endangered species included in annex ii of the european union habitats directive 92/43/eec. we tested the effect of different culture media in the axenic establishment and propagation of r. helicophylla using samples originally collected from a spanish population, hereafter referred as spanish genotype, for the purpose of ex situ conservation and future research on its biology. materials and methods plant material the initial plant materials were collected in spring of 2010 at marjal del moro, sagunto, valencia province, spain by jgs-m and fp. plants with sporophytes were kept in cultures containing sediments from the original locality until ripe spores were observed. ripe unopened sporophytes were separated from the plants, air dried and kept in darkness, at room temperature. sporophytes were collected at different time intervals in order to cover a range of time from the onset of drought conditions (15, 30, 45, 60, 75 and 90 days). species of riella have been observed to present spore dormancy which prevents immediate germination of the spores (thompson 1941). they require a drought period, which in natural conditions may correspond to the summer months but that can be extended further in particularly dry years, to break up dormancy. thus, our sampling of sporophytes covered the minimum drought period necessary to restore the germination capability of the spores. sporophyte sterilization assays for the purpose of axenic culture establishment and to prevent undesired contamination of the cultures, surface ste rilization of sporophytes was required. sodium dichloroisocyanurate (nadcc) and commercial bleach (naocl, 8% of active chlorine) were tested for sporophyte surface sterilization. sporophytes were sterilized by soaking them for up to 10 minutes using different concentrations (1%, 3%, 5% and 7%) of nadcc or commercial bleach solutions. culture media assays in this study we used different culture media depending on the experiment. liquid and solid media were used to germinate the spores whereas plant growth was assessed on solid media or in bi-phasic cultures corresponding to a combination of liquid and solid media. as liquid media we used distilled water or a solution enriched in electrolytes. this electrolyte solution was composed of: ca(no3)2 × 4 h2o 2876 mg l–1, kh2po4 500 mg l–1, mgso4 × 7 h2o 1000 mg l–1, kno3 1000 mg l–1, kcl 1000 mg l–1, khco3 400 mg l–1, and fe-edta 130 mg l–1. three types of solid media, namely ms (murashige and skoog 1962), bcd (sabovljević et al. 2009) and knop (reski and abel, 1985), were used in plant growth experiments. ms medium contained: nh4no3 1650 mg l–1, kno3 1900 mg l–1, cacl2 × 2 h2o 440 mg l–1, mgso4 × 7 h2o 370 mg l–1, kh2po4 170 mg l–1, na2edta 37.3 mg l–1, feso4 × 7 h2o 27.8 mg l–1, h3bo3 6.2 mg l–1, mnso4 × 4 h2o 22.3 mg l–1, znso4 × 4 h2o 8.6 mg l–1, ki 0.83 mg l–1, na2moo4 × 2 h2o 0.25 mg l–1, cocl2 × 6 h2o 0.025 mg l–1, cuso4 × 5 h2o 0.025 mg l–1, agar 8000 mg l–1, and myo-inositole 100 mg l–1. bcd medium contained: mgso4 × 7 h2o 250 mg l–1, kh2po4 250 mg l–1, kno3 1010 mg l–1, feso4 × 7 h2o 12.5 mg l–1, and agar 8000 mg l–1. finally, knop medium contained: kh2po4 25000 mg l–1, kcl 25000 mg l–1, mgso4 25000 mg l–1, ca(no3)2 × 4 h2o 100000 mg l–1, feso4 × 7 h2o 250 mg l–1, and 12000 mg l–1 of agar. additionally, cultures on ms medium were assayed on half strength ms or supplemented with sucrose 15000 mg l–1. the ph of the media was adjusted to 5.8 before sterilization of the media at 121 °c for 25 min. for the axenic culture establishment experiments, spore germination was tested on the three aforementioned solid media: knop, ms (standard composition and half strength), and bcd. the different media were prepared following the protocol described above and replicates supplemented with sucrose or plant growth regulators were also tested. the unopened sporophytes were surface sterilized, and then opened in an air fl ow chamber to release the spores on sterile solidphase media described above and kept at 25 ± 2 °c under long day conditions (16 h light / 8 h darkness). spore germination assays in order to test whether spore germination was affected by the time from which the sporophytes were subjected to drought conditions, they were collected at the aforementioned six time intervals and then kept dried up to 10 days prior to experimentation. spore (total content of three spore capsules) germination was recorded seven days after incubation in tubes by quantifi cation of germlings using a hemocytometer slide. water or electrolyte solution containing gibberellins (0.03 mm, 0.1 mm, 1 mm and 10 mm) (ga3 sabovljević m. s., segarra-moragues j. g., puche f., vujičić m., cogoni a., sabovljević a. 196 acta bot. croat. 75 (2), 2016 and ga7) were tested in order to investigate the infl uence of these phytohormones on spore germination, since dormancy of spores has been reported in riella (thompson 1941). axenic culture establishment assays as plants of riella inhabit shallow water ponds and remain attached to sediments by their rhizoids, bi-phasic axenic culture media were established and tested. spores were fi rstly sown on knop, ms or bcd solid sterile medium under long day conditions. once the spore germinated, the green structure appeared in less than 10 days after sowing. thin solid basal media containing plants were subsequently covered with liquid solutions of sterilized distilled water or electrolytes (liquid phase). bi-phasic cultures were grown at 18 ± 2 °c under long day conditions, at 47 μmol m–2 s–1 photosynthetic photon fl ux density provided by cool-white fl uorescent tubes. cultures were grown for six weeks under these conditions. four replicates of each culture were established in order to account for the variation in total biomass. quantifi cation of vegetative growth it was not possible to estimate the vegetative growth of r. helicophylla through the multiplication index (that represents the number of newly grown shoots which derived from one starting shoot) because of its thallose habit. to overcome this, estimation of the vegetative growth was based on quantifi cation of total biomass of individual gametophores. for this purpose individual gametophores were kept fully hydrated, to avoid variations in water content and then weighed on a sartorius analytical balance (weight estimated to the nearest 0.0001 g). results spores collected in the fi eld and immediately spread on solid medium of any type did not germinate. the exposure to light or dark during the three weeks after spread on media did not affect germination. our results showed an effect of the sporophyte (i.e. spores) drought duration time on the germination ability of r. helicophylla spores. germination proportions were negligible for spores that had remained dry less than two months (fig. 1). spore germination increased after 75 days since drying out and reached maximum values at 90 days since drought (fig. 1). although germ ination percentages were higher for spores germinated on the electrolyte solution than on distilled water, the differences between the two treatments were not signifi cant (fig. 1). gibberellins, which are widely known to break seed dormancy, did not bring the expected results in breaking dormancy in spores that had been dried out for up to ten days (fig. 2). the treatments of spores with 0.03, 0.1, 1 and 10 mm ga3 and ga7 added to basal media did not break the germination inhibition either in dark or in light condition at 18 ºc. the imitation of the natural dry and hot period (fig. 1) breaks spore dormancy. the dormancy noted caused the problems with in vitro establishment of r. helicophylla. once, we have found the way to break dormancy in spores we precede to axenic in vitro culture establishment. the most effective way to get viable axenic spores was treatment with 3% nadcc for 7 minutes. the percentage of uncontaminated media was over 95%. the spores (released from dormancy) were germinated in a high percentage in all tested solid media under long day conditions. however, the gametophytes did not equally develop (fig. 3), and instead a callus-like tissue was formed that afterwards started developing some buds (fig. 4a). fig. 1. spore germination rate of riella helicophylla expressed in percentage (± standard deviations) after dry storage at room temperature (in days) prior to test for seven days in liquids. fig. 3. biomass production of riella helicophylla gametophytes on different solid media covered with water or electrolyte solution. ms – murashige and skoog medium; ms/2 – half strength murashige and skoog medium; ms/2 + s – half strength murashige and skoog medium enriched with 15 g l–1 of sucrose. fig. 2. test of breaking dormancy with different concentrations of selected gibberellins (ga) dissolved in water or electrolyte solution. the difference in control (0 mm ga) clearly indicates the presence of some variation within spores from the same liverwort population. propagation of riella helicophylla acta bot. croat. 75 (2), 2016 197 fully developed gametophytes were obtained from biphasic axenic in vitro cultures that imitated the shallow water ecological conditions in the wild. when all the tested solid media were covered with distilled water, fully developed gametophytes were formed (fig. 3). but when they were covered with the electrolyte solution, the total biomass production was signifi cantly increased on all the media tested. discussion the results clearly show the existence of spore dormancy in the tested riella helicophylla population. this is rather expected since this species inhabits temporary ponds which can be dried out after few months and stay without water for couple of years. dormancy is a response to the harsh environment during the drought period and in r. helicophylla is a prime example of genotype-environment interaction. by the dormancy present in r. helicophylla, it can be inferred that the drought period is anticipated genetically. it can, however, be broken by signals from the environment. as usually stated, dormancy is complex phenomenon, and induction, maintenance and breaking of dormancy in vascular plants is quite unknown and not consistent across all species. even less is known for non-tracheophytes, such as bryophytes. this evidence opens many other questions such as defi ning external signals in processes such as induction, establishment and breaking of spore dormancy in the interesting aquatic liverwort r. helicophylla. it was shown that photomorphogenesis in bryophytes is positively infl uenced by light, as one of the most important environmental factors that infl uences bryophyte development (nakazato et al. 1999). nevertheless, bi-phase culture with electrolytes infl uenced full development of the gametophytes. having in mind that these plants grow in brackish environments and that the uptake of minerals takes place on the whole through the thallose gametophyte, fully developed liverwort in such conditions is rather expected than in a bi-phase culture with water where ions are dissolved from the solid medium (figs. 4b, 4c). it has been previously reported that gibberellins have an infl uence on the process of morphogenesis of selected moss species (sabovljević et al. 2010). however, the exposures of spores to selected concentrations of two widely used gibberellins did not cause germination. therefore, we decided to test out simulation of natural conditions, with the aim of achieving the spore germination ability. we chose to keep the spores in dry conditions at 20 ºc, since in natural condition spores survive a long dry summer period. the germination dormancy was not broken after 15, 30, 45, 60 and 75 days. however, spores germinated after 90 days in dry and relatively hot conditions, up to 91%. thus, dryness and warmth, here combined, break the spore dormancy in the tested r. helicophylla population. the germinated spores showed slightly better growth in bcd medium. the other tested media did not show a statistically signifi cant difference in basal media preferential (anova p value 0.914). accordingly, the other basal media tested did not have any signifi cant effect on spore germination. solid, but soft, media were good for spore germination in contrast to the completely liquid media tested. two phase system (solid and liquid) culture media were developed for the purpose of achieving fully developed gametophytes. spores were able to germinate on a solid rather than in purely liquid medium, and they developed some kind of callus tissue that developed into green plants, gametophores, after being transferred to the two phase culture. two types of liquid phases: (1) distilled water and (2) water containing electrolytes simulating brackish water (electrolyte solution), were tested over bcd medium, in which callus mass had good growth. the best plant morphogenesis was achieved on bcd medium covered with electrolyteenriched liquid phase. experiments on sporophyte development induction (i.e. spore production) by growing female and male plants and acclimation to xenic condition are ongoing. the aim is to develop biotechnical model, which can be widely used for reintroduction and release of the riella plants to potential natural and semi-natural aquatic habitats. acknowledgements the serbian ministry of education, science and technological development is thanked for the support given by grants nos. 173024 and 173030. fig. 4. a) development of a callus-like tissue and buds from spores germinated on solid bcd medium, b) test of various solid media types covered with water on the development of riella helicophylla gametophytes, c) fully developed gametophytes in biphasic axenic cultures on solid bcd medium covered with electrolytes. sabovljević m. s., segarra-moragues j. g., puche f., vujičić m., cogoni a., sabovljević a. 198 acta bot. croat. 75 (2), 2016 references asem, a., eimanifar a., djamali, m., de los rios, p., wink, m., 2014: biodiversity of the hypersaline urmia lake national park (nw iran). diversity 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(eds.), protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants. methods in molecular biology. humana press, pp.117–128. segarra-moragues, j. g., sabovljević, m., puche, f., 2012: riella heliospora (riellaceae) a new monoicous species of riella subgenus trabutiella from california. systematic botany 37, 307–319. segarra-moragues, j. g., puche, f., 2014: advances in the knowledge of south african riella (sphaerocarpales) and a new endemic species, riella trigonospora. south african journal of botany 94, 166–176. segarra-moragues, j. g., puche, f., sabovljević, m., infante, m., heras, m., 2014: taxonomic revision of riella subgen. trabutiella (riellaceae, sphaerocarpales). phytotaxa 159, 131–174. söderström, l., hagborg, a., von konrat, m., bartholomew-began, s., bell, d., briscoe, l., brown, e., cargill, d. c., costa, d. p., crandall-stotler, b. j., cooper, e. d., dauphin, g., engel, j. j., feldberg, k., glenny, d., gradstein, s. r., he, x., ilkiu-borges, a. l., heinrichs, j., hentschel, j., katagiri, t., konstantinova, n. a., larraín, j., long, d. g., nebel, m., pócs, t., puche, f., reiner-drehwald, m. e., renner, m. a. m., sass-gyarmati, a., schäfer-verwimp, a., segarra-moragues, j. g., stotler, r. e., sukkharak, p., thiers, b. m., uribe, j., váňa, j., villarreal, j. c., wigginton, m., zhang, l., zhu, r.-l., 2016: world checklist of hornworts and liverworts. phytokeys, 59, 1–828. thompson, r. h., 1941: morphology of riella affi nis. i. germination of the spore and development of the thallus. american journal of botany 28, 845–855. zacharias, i., dimitrou, e., dekker, a., dorsman, e., 2007: overview of temporary ponds in the mediterranean region: threats, management and conservation issues. journal of environmental biology 28, 1–9. acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 15 acta bot. croat. 76 (1), 15–26, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0041 issn 0365-0588 eissn 1847-8476 ecological, fl oristic and functional analysis of zonal forest vegetation in bosnia and herzegovina vladimir stupar1*, andraž čarni2, 3 1 university of banjaluka, forestry faculty, department of forest ecology, s.stepanovića 75a, ba-78000 banjaluka, bosnia and herzegovina 2 institute of biology, research centre of the slovenian academy of sciences and arts, novi trg 2, si-1000 ljubljana, slovenia 3 university of nova gorica, vipavska 13, si-5000 nova gorica, slovenia abstract – zonal vegetation is a large-scale expression of macro-climate and, due to the climatic diversity of the country, there are seven traditionally recognized zonal forest plant communities in bosnia and herzegovina. using data from bosnia and herzegovina, this study aimed to reveal whether macro-climate is indeed the most important factor determining the existence of zonal forest plant communities (zfpc). detrended correspondence analysis of 398 relevés of seven zfpcs revealed that the species turnover along the fi rst axis is strongly related to the macro-climatic gradient (annual mean temperature, mean temperature of the coldest quarter and precipitation of the warmest quarter). no correlation was detected between this gradient and topographic factors (slope and aspect) and soil reaction. floristic analysis revealed clear separation of zfpcs in terms of diagnostic species. functional analysis of all layers showed that competitive ecological strategy has the highest proportion, while analysis of the herb layer alone expressed a shift of csr signatures towards the middle of the c–s axis. ruderality was overall poorly expressed. statistically signifi cant differences among communities were discovered along the c–s axis. in terms of life forms, statistically signifi cant differences in the proportions of phanerophytes, geophytes and hemicryptophytes among communities were discovered. our study confi rms that macro-climatic gradient is the most important determinant of the species turnover along zfpcs. csr signatures show that zonal forest vegetation is represented by productive communities in a terminal stage of succession. this does not refer to degraded quercus ilex stands (maquis), which are in the middle stage of secondary succession. keywords: balkans, climatic gradient, chorotypes, ecological strategies, life forms, ordination, plant functional types, zonal communities * corresponding author, e-mail: vladimir.stupar@sfbl.org introduction every plant community is a result of a complex interaction of various ecological factors in a given place and time. in terms of natural vegetation, there are three types of vegetation that generally develop in accordance with the biotic, climatic and soil conditions: zonal, extrazonal and azonal (dierschke 1994, ellenberg 2009, surina 2014). zonal vegetation is a large-scale expression of climate dominating a particular area, while it is not confi ned to specifi c soil conditions, i.e., it most precisely refl ects the macroclimatic conditions of particular regions (kovar-eder and kvaček 2007). zonal vegetation often does not represent a single homogeneous plant association but rather a number of similar communities, which can differ to some extent and, consequently, it is possible to talk of a ‘zonal vegetation group’ (ellenberg 2009). for example, when different bedrocks and soil series occur within the same climatic zone or there is a species turnover due to minor biogeographical differences, several different, yet similar associations make up the zonal vegetation group. furthermore, a mountainous relief of a particular climatic zone leads to vertical differentiation of the climatic factors and, accordingly, vertical differentiation of zonal vegetation communities, which are then often called ‘altitudinal belts’. although in the last several thousand years man has deforested great parts of europe (gunia et al. 2012), the potential natural vegetation and, consequently, the zonal vegetation of most of temperate europe is forest (bohn and neuhäusl 2004, ellenberg 2009). the same is valid for bosnia and herzegovina (b&h) (horvat et al. 1974, stefanović stupar v., čarni a. 16 acta bot. croat. 76 (1), 2017 et al. 1983). b&h is situated in the western part of the balkan peninsula in se europe (fig. 1). its climate is very diverse, since two major climatic zones overlap here: central european from the north and mediterranean from the south. the climate of the transitional zone is highly modifi ed by the infl uence of mountain massifs (dinaric alps), while in the eastern part of country the infl uence of the drier continental climate can be felt (milosavljević 1976). while vegetation studies of forests in the neighboring regions have produced synthetic overviews (borhidi et al. 2012, vukelić 2012, chytrý 2013, tomić and rakonjac 2013), despite the fairly long tradition of phytosociological studies in b&h (horvat 1933, 1941, horvat and pawlowski 1939, tregubov 1941) and the considerable number of relevés, the ecology, nomenclature and syntaxonomical position of the majority of b&h forests still remains unsettled (redžić 2007). with the exception of thermophilous deciduous forest communities (stupar et al. 2015), this also applies to zonal vegetation, which has only been the subject of a few studies in the past (horvat et al. 1974, stefanović et al. 1983, beus 1984). however, following the macro-climatic diversity, these authors generally agree that seven zonal forest plant communities (zfpcs) can be distinguished for the territory of b&h (tab. 1, fig. 1). four communities are represented by various oak forests, which occupy the lowlands and hilly region of b&h, while the other three are altitudinal belts (montane, altimontane and subalpine) above the oak forests, mainly built by various types of beech forests (pure and mixed). there are two different conceptual frameworks for the study of plant communities. the traditional approach to classifi cation is performed at the species level, while a more recent ‘functional’ approach is based on plant functional types (duckworth et al. 2000, shipley 2010, škornik et al. 2010). plant functional types are non-phylogenetic groupings of species that show close similarities in their response to environmental and biotic factors and are derived from plant traits based on species morphology, physiology and/or life history (pérez-harguindeguy et al. 2013). plant functional types can aid in the understanding of ecological processes, such as the assembly and stability of communities and succession, and facilitate the detection and prediction of response to environmental change on a range of scales (duckworth et al. 2000). with plant functional types, comparisons between communities of widely differing composition can be facilitated (diaz et al. 2004). there are two main approaches to the classifi cation of functional types fig. 1. location of the study area in bosnia and herzegovina. the potential zonal forest plant communities (zfpcs) are indicated (horvat et al. 1974, modifi ed after stefanović et al. 1983). numbers on the map correspond to community numbers in tab. 1. tab. 1. zonal forest plant communities in bosnia and herzegovina. community numbers correspond to those used in tabs. 3–4, and figs. 1–4. asterisk (*) denotes provisional invalid names still in use in bosnia and herzegovina. community no. forest type related syntaxa no. of relevés no. of resampled relevés 1 quercus ilex quercion ilicis br.-bl. 1931 (1936) 5 5 2 quercus pubescenscarpinus orientalis querco pubescenti-carpinetum orientalis horvatić 1939 (carpinion orientalis horvat 1958) 30 16 3 quercus frainetto quercetum frainetto-cerridis (rudski 1949) trinajstić et al. 1996 (quercion frainetto horvat 1954) 38 24 4 quercus petraeacarpinus betulus querco-carpinetum illyricum horvat et al. 1974* (erythronio-carpinion betuli (horvat 1938) marinček in wallnöfer et al. 1993) 43 26 5 pure fagus / mixed fagus-abies fagetum montanum illyricum fukarek et stefanović 1958*, abieti-fagetum dinaricum tregubov 1957* (aremonio-fagion török et al. ex marinček et al. 1993) 231 162 6 mixed piceaabies-fagus piceo-abieti-fagetum stefanović et al. 1983* (abieti-piceenion br.-bl. in br.-bl. et al. 1939) 191 123 7 subalpine fagus sylvatica fagetum subalpinum s. lato* (saxifrago rotundifoliae-fagenion marinček et al. 1993) 74 42 zonal forest vegetation of bosnia and herzegovina acta bot. croat. 76 (1), 2017 17 (shipley 2010). the fi rst is used especially by plant geographers, concentrating on the morphological properties of plants, e.g., raunkiaer’s life-forms (raunkiaer 1934) and how, on average, such morphologies change as a function of major climatic variables. a second approach is based on the notion of ecological ‘strategies’, for which grime’s model of csr triangle (grime 1974, 1977, 2001) is often used (pugnaire and valladares 2007). this model is a classifi cation based on how plants deal with two groups of external factors, i.e., stress and disturbance, which results in three primary plant strategies: competitors (c), stress-tolerators (s), and ruderals (r) and four secondary, intermediate ones. the position of each species, as well as each relevé, can be determined in a csr triangle. the whole community is thus given a functional signature, which can be very useful in comparative studies involving widely differing samples (hunt et al. 2004). while the use of ecological strategies is quite common in studies of herbaceous vegetation (hunt et al. 2004, zelnik and čarni 2008, škornik et al. 2010, pipenbaher et al. 2013), it has lately also been gaining ground in the study of forest and woodland communities (kilinç et al. 2010, paušič and čarni 2012, juvan et al. 2013, košir et al. 2013b, rozman et al. 2013). its use is most often related to studies of changes in communities’ composition as a response to disturbance, succession, environmental changes or in gradient analysis. the aim of this study was to test whether zonal vegetation is an expression of macro-climatic conditions or whether there are also other environmental factors involved. the underlying assumptions were (1) that the macro-climatic gradient is the most important for the species and structure turnover along the gradient of zfpcs in b&h; and (2) that zfpcs would demonstrate similarities in grime’s ecological strategies, due to the fact that the communities are in terminal stages of succession, with a low level of degradation, occupying habitats on moderately fertile soils. materials and methods data collection and preparation seven zfpcs were identifi ed for b&h (tab. 1, fig. 1, ste fanović et al. 1983, beus 1984): (1) eu-mediterranean ever green quercus ilex forests (a very small area of the warmest, southernmost part of b&h; represented by maquis); (2) sub-mediterranean thermophilous deciduous quercus pubescens-carpinus orientalis forests (a major part of the lowland and hilly region in southern b&h); (3) central balkans thermophilous quercus frainetto forests (relatively narrow lowland and hilly belt of eastern b&h); (4) illyrian mesophilous quercus petraea-carpinus betulus forests (major part of northern b&h and marginally in central b&h); (5) montane mesoneutrophilous pure fagus or mixed fagus-abies forests (altitudinal belt above oak forests in all b&h but mainly in the mountainous region of central b&h (dinaric alps); (6) alti-montane, colder and more acidophilous mixed picea-abies-fagus forests (altitudinal belt above community 5, mainly in the dinaric mountainous region); and (7) subalpine fagus sylvatica forests (uppermost forest altitudinal belt). relevés were extracted from the forest vegetation database of bosnia and herzegovina stored in the global index of vegetation-plot databases (dengler et al. 2011) with the id eu-ba-001. this database consists of 2810 published and available unpublished forest vegetation relevés in b&h. we compiled all relevés that were assigned to one of the seven zfpcs by their authors (tab. 1), with the exception of two zonal communities of thermophilous deciduous forests (communities 2 and 3), for which we used the results of formalized classifi cation (stupar et al. 2015). we did not consider relevés of stands with less than 70% of canopy cover, nor those with edifi er tree species cover value less than 3 (25%) on the braun-blanquet scale, considering them structurally degraded. only stands of high, productive forests were thus taken into account, except in the case of community 1 (quercus ilex stands) because wellestablished stands do not exist in b&h, and all relevés were made in structurally degraded maquis. all relevés were made using the standard central european phytosociological method (braun-blanquet 1964). only relevés that could be georeferenced relatively precisely and those that contained complete species records were taken into consideration. in all 612 relevés were compiled in the turboveg database (hennekens and schaminée 2001) and exported to juice 7 software (tichý 2002) for further analysis. mosses, as well as taxa determined only to the genus level, were removed from the data set prior to numerical analysis. all vegetation layers were merged into one layer. taxonomy and nomenclature followed flora europaea (tutin et al. 1968–1993) unless a more modern taxon concept or circumscription suggested otherwise. these taxa, as well as those from taxonomically critical groups that were combined into aggregates (agg.) or species that included several subspecies that were not always recorded or recognized by authors and were combined under the abbreviation ‘s.l.’ (sensu lato), were listed in on-line suppl. tab. 1. the dubious taxon quercus dalechampii was treated as part of quercus petraea agg., following di pietro et al. (2012). records of fagus moesiaca were treated as f. sylvatica (marinšek et al. 2013). to avoid geographic overrepresentation of some vegetation types due to oversampling of certain regions, we performed geographical stratifi cation and resampling of the initial data set (knollová et al. 2005), a frequent strategy in recent national and regional level vegetation studies (chytrý 2013, košir et al. 2013a, rodríguez-rojo et al. 2014). stratifi cation was performed in a geographical grid with 1 km2 size. if two or more relevés assigned to the same community fell in the same grid cell, only one of them was selected. stratifi cation was not performed on community 1 since it consisted of only fi ve relevés. the resulting stratifi ed data set contained 398 relevés and 669 species. data analysis the data set was then subjected to detrended correspondence analysis (dca) in r software, version 2.10.1 (r development core team 2009) using the vegan package (http:// cc.oulu.fi /~jarioksa/softhelp/vegan.html) on presence-absence data. to extract the main gradients in species composition, stupar v., čarni a. 18 acta bot. croat. 76 (1), 2017 398 relevés, together with the selected ecological variables were projected onto the two-dimensional ordination space of dca. unweighted average species ecological indicator values (eivs) for soil reaction (pignatti et al. 2005) and selected climatic variables available from the worldclim database (hijmans et al. 2005) were used as explanatory ecological variables. the signifi cance of eivs correlation with the dca relevé scores was tested using the modifi ed permutation test proposed by zelený and schaffers (2012). climate variables best explaining variation in species composition were selected through forward selection in canonical correspondence analysis (cca) in canoco 4.5 software (microcomputer power, ithaca, ny, us). other explanatory variables (altitude, inclination of slope, aspect, chorotypes, latitude and longitude, life forms and ecological strategies) were selected and projected based on the strength of correlation with the fi rst and second dca axis. the signifi cances of correlations between these explanatory variables and dca relevé scores were calculated using the kendall tau coeffi cient in statistica software (statsoft, inc.; v 7.0, http:// www.statsoft.com). chorological spectra of the zonal forest communities (gajić 1980, pignatti et al. 2005) were calculated for each relevé using presence-absence data. endemic illyrian species were separated from southeast european species in a broader sense. in order to reveal the fl oristic differences among the seven zfpcs, we calculated their diagnostic species in the resampled data set using phi coeffi cient in the juice 7 program (chytrý et al. 2002), after standardization to a relevé group size equal to a seventh of the total data set size (tichý and chytrý 2006). fisher’s exact test was calculated giving a zero fi delity value to species whose phi values were not statistically signifi cant (p>0.001). the threshold phi value for a species to be considered diagnostic was set at 0.25. the functional study of zonal forest communities was performed using data of plant functional traits, i.e., life forms (raunkiaer 1934) and ecological strategies (grime 1977). plants were classifi ed according to grime’s primary and secondary strategies into seven functional types using data from the biolflor database (klotz et al. 2002). we thus obtained data for about 80% of species. ecological strategies for the remaining species were calculated using the dichotomous key suggested by véla (2002). averages of each strategy type weighted by cover-percentage were calculated and standardized for each relevé as suggested by hunt et al. (2004). ecological strategy scores for each group of relevés were then calculated with the csr signature calculator 1.2 program (hunt et al. 2004) and represented on a csr ternary plot. we did a separate analysis for all layers and for the herb layer alone because herb functional signatures respond more quickly to environmental changes in the course of late succession (paušič and čarni 2012). plant life forms obtained from pignatti et al. (2005) and supplemented by our expert knowledge were used for the calculation of life forms spectra for each relevé using presence-absence data, and mean proportions were compared between zfpcs. the occurrence of an individual functional trait in each of the zfpcs was compared using the scheffé post hoc test for normal distributions and kruskal-wallis test by ranks and median for non-normal distributions using statistica software. a normality check was performed using lilliefors test. results gradient analysis within zfpcs in bosnia and herzegovina forward selection suggested that the climatic variables with the highest explanatory value of the variation in species composition are annual mean temperature (bio1), mean temperature of the coldest quarter (bio11) and precipitation of the warmest quarter (bio18). the fi rst dca axis represents the main gradient in the data set, and all three climatic variables are signifi cantly related to the fi rst dca axis at p<0.001 (tab. 2, fig. 2). it runs from the coldtab. 2. correlations (kendall-tau coeffi cient) between detrended correspondence analysis (dca) relevé scores and explanatory variables. bio1 – annual mean temperature; bio11 – mean temperature of the coldest quarter; bio18 – precipitation of the warmest quarter; eur – european and eurasian; seur – south and southeast european; eurimed – euri-mediterranean; stmed – steno-mediterranean; illyr – endemic illyrian; bor – boreal; seoro – south european orophytes; cosm – widespread species; p – phanerophytes; np – nanophanerophytes; ch – chamaephytes; h – hemicryptophytes; g – geophytes; t – terophytes; c – competitors; s – stress-tolerators; r – ruderals. asterisk (*) denotes signifi cant correlation at p<0.001. ecological factors geographical factors altitude aspect slope bio1 bio11 bio18 latitude longitude dca 1 –0.521* 0.046 0.050 0.569* 0.566* –0.451* –0.200* 0.367* dca 2 –0.151* –0.034 –0.103 0.118* 0.103 –0.011 0.028 –0.041 chorotypes eur seur eurimed stmed illyr bor seoro cosm dca 1 0.088 0.203* 0.445* 0.309* –0.172* –0.313* –0.510* –0.155* dca 2 0.077 –0.034 0.035 –0.004 –0.066 –0.009 –0.138* 0.077 life forms ecological strategies p np ch h g t c s r dca 1 0.182* 0.020 0.045 0.080 –0.317* 0.109 0.045 –0.048 0.014 dca 2 0.010 –0.059 0.110 0.057 –0.121* 0.126* –0.049 –0.067 0.109 zonal forest vegetation of bosnia and herzegovina acta bot. croat. 76 (1), 2017 19 est and most mesophilous subalpine beech forests (community 7) on the left side of the diagram to the most xerothermophilous quercus ilex maquis (community 1) on the far right side of diagram. it is positively correlated with annual mean temperature and mean temperature of the coldest quarter, and negatively with the precipitation of the warmest quarter (fig. 2, tab. 2). these are strong indicators that the fi rst dca axis represents a macro-climatic gradient that runs from wet summers, cold winters and lower annual temperatures to dry summers but warmer winters and higher overall annual temperatures. there is also a high negative correlation with altitude, which is again related to macro-climatic factors. the positive correlation with longitude and negative correlation with latitude refl ects the fact that south and east parts of b&h are warmer and dryer than the north and west. correlations between the fi rst axis and slope and aspect are not statistically signifi cant (tab. 2). a modifi ed permutation test also showed that the correlation between dca 1 and eivs for soil reaction was not statistically signifi cant (r2=0.125, p=0.195). in terms of chorotypes, dca axis 1 shows a positive correlation with the proportions of southand southeast european, euri-mediterranean and steno-mediterranean chorotypes, and a negative correlation with boreal and south european orophytic chorotypes, while the correlation with the most abundant european and eurasian chorotypes is not statistically signifi cant. correlations between the fi rst axis and life forms are signifi cant only for the proportions of phanerophytes (positive) and geophytes (negative). there is no signifi cant correlation between ecological strategies and dca axes (tab. 2). floristic differentiation analysis revealed differences in the fl oristic composition among the seven zfpcs. tab. 3 shows a simplifi ed frequency-fi delity synoptic table of the zfpcs of b&h based on a data set of 398 resampled relevés (full version is given in on-line suppl. tab. 2). fig. 2. detrended correspondence analysis (dca) spider plot of 398 relevés of zonal forest plant communities with climatic variables, ecological indicator values (eivs) for soil reaction and altitude passively projected. the surface variable is annual mean temperature. the length of the fi rst dca axis is 7.02 sd units, the length of the second axis is 2.85 sd units. centroids of clusters are indicated by numbers that refer to tab. 1. tab. 3. frequency-fi delity table of zonal forest plant communities (zfpcs) in bosnia and herzegovina. frequencies of species are presented as percentages with phi values multiplied by 100 shown in superscript. diagnostic species (phi values higher than 0.25) for each community are shaded (only ten species with the highest phi value for every community are presented). proportions of chorotypes and altitudinal ranges of each community are also shown. community numbers correspond to those used in tab. 1 and fig. 1. community number 1 2 3 4 5 6 7 no. of relevés 5 16 24 26 162 123 42 altitudinal range (m) 0–150 100–650 150–700 200–900 700–1400 800–1600 1400–1800 chorotype (% of all species in a community) widespread species 3 1 3 5 7 7 4 european and eurasian 13 29 56 67 59 55 49 south and southeast european 21 30 21 16 13 10 12 eurimediterranean 29 29 13 5 1 1 0 stenomediterranean 33 7 0 0 0 0 0 endemic illyrian 1 3 1 0 1 2 3 boreal 0 1 6 6 10 13 13 south european orophytes 0 0 0 1 9 12 19 zfpc 1 quercus ilex 100 96.5 6 – . . . . . arbutus unedo 80 88 . . . . . . teucrium polium ssp. capitatum 80 84.1 6 – . . . . . centaurium erythraea 80 83.2 . . 8 – . . . pistacia terebinthus 100 82.6 38 18.3 . . . . . juniperus phoenicea 60 75 . . . . . . pistacia lentiscus 60 75 . . . . . . juniperus oxycedrus ssp. macrocarpa 40 60.3 . . . . . . stupar v., čarni a. 20 acta bot. croat. 76 (1), 2017 community number 1 2 3 4 5 6 7 no. of relevés 5 16 24 26 162 123 42 altitudinal range (m) 0–150 100–650 150–700 200–900 700–1400 800–1600 1400–1800 crepis sancta 40 60.3 . . . . . . cistus salvifolius 40 60.3 . . . . . . zfpc 2 quercus pubescens 20 – 100 84.5 12 – . . . . cornus mas . 88 83.6 8 – 8 – . . . acer monspessulanum . 62 76.7 . . . . . sesleria autumnalis 20 – 81 76.5 . . 1 – 1 – . frangula rupestris . 56 72.4 . . . . . viola hirta . 75 69.4 17 – 12 – . . . rubus ulmifolius . 50 67.9 . . . . . carex halleriana 20 – 62 64.2 . . . . . brachypodium sylvaticum . 75 61.1 12 – 19 – 5 – 6 – 7 – juniperus oxycedrus 40 – 69 60 . . . . . zfpc 3 quercus frainetto . 19 – 100 90.3 . . . . chamaecytisus hirsutus agg. . . 58 70.4 4 – . 1 – . quercus cerris . 50 23 100 69.8 27 – 1 – 1 – . thymus pulegioides . . 54 65.7 . 4 – 1 – 2 – lychnis coronaria . . 46 64.8 . . . . lathyrus niger . 12 – 58 63 4 – . . . euphorbia cyparissias . 19 – 58 61.7 . . . . dianthus armeria . . 42 61.6 . . . . carex caryophyllea . 19 – 58 57.8 8 – . . . silene viridifl ora . 12 – 46 55.4 . . . . zfpc 4 carpinus betulus . . 42 19.6 100 77.3 11 – . . cruciata glabra . . 4 – 69 67.5 11 – 8 – . prunus avium . . 25 – 65 60.7 6 – 1 – . luzula luzuloides . . . 58 59.9 1 – 8 – 12 – pteridium aquilinum . . 38 – 77 57.7 12 – 15 – . stellaria holostea . . 8 – 50 56.6 1 – . 7 – melampyrum pratense . . 17 – 50 54.5 . 1 – 2 – erythronium dens-canis . . . 27 49 . . . crataegus monogyna . 44 – 29 – 69 45.3 15 – 2 – . hieracium racemosum . . . 23 45.2 . . . zfpc 5 galium odoratum . . . 12 – 76 46.5 59 – 36 – acer pseudoplatanus . . . 12 – 81 44.6 69 – 52 – cardamine bulbifera . . . 12 – 67 42.3 30 – 55 – lonicera xylosteum . . . . 31 38.9 15 – 2 – cardamine enneaphyllos . . . . 62 38.1 35 – 62 – daphne mezereum . . . . 51 35.1 37 – 38 – salvia glutinosa . . 4 – 4 – 31 34.9 14 – 2 – glechoma hirsuta . . 17 – 19 – 50 34.6 26 – 12 – rubus hirtus . . 12 – 42 – 57 33.1 36 – 14 – cardamine trifolia . . . . 28 32.9 20 – 2 – zfpc 6 picea abies . . . . 51 – 100 59.7 69 – tab. 3. – continued zonal forest vegetation of bosnia and herzegovina acta bot. croat. 76 (1), 2017 21 analysis of functional traits in the standard csr triangle plot, communities are positioned in the upper right part of the diagram (fig. 3a). this means that zonal forest plant communities are dominated by species with considerable competitive capacity. however, there is apparent divergence of communities 1, 2 and 4 from the main cluster along the c–s axis, whereby statistically signifi cant differences were discovered (tab. 4). the highest ratio of stress tolerant-species occurs in community 1, much less in community 2, while the most competitive ability is expressed by community 4. csr signatures of the herb layer are shifted to the middle of the c–s axis (fig. 3b). the most signifi cant differences in functional traits of a life form were detected for phanerophytes and geophytes between communities 1–4 and communities 5–7, and for hemicryptophytes between communities 1 and 5 and the rest of the data set (tab. 4). the highest ratios of phanerofig. 3. csr triangle of the ordination of zonal forest plant communities in bosnia and herzegovina according to ecological strategy scores (proportion of competitors – c, stress-tolerators – s and ruderals – r components in each community). community numbers correspond to those used in tab. 1 and fig. 1. (a) all layers together; (b) herb layer. community number 1 2 3 4 5 6 7 no. of relevés 5 16 24 26 162 123 42 altitudinal range (m) 0–150 100–650 150–700 200–900 700–1400 800–1600 1400–1800 abies alba . . . 8 – 73 – 98 52.5 76 – athyrium fi lix-femina . . . 8 – 38 – 63 49 14 – galium rotundifolium . . . . 9 – 41 46.5 12 – oxalis acetosella . . . . 64 – 83 46.1 69 – lonicera nigra . . . . 19 – 46 43.9 17 – sorbus aucuparia . . . . 33 – 63 42.1 50 – senecio nemorensis s.l. . . . . 38 – 59 40.8 38 – prenanthes purpurea . . . 4 – 46 – 68 38.9 67 – lamiastrum galeobdolon . . . 27 – 56 – 73 38.8 52 – zfpc 7 saxifraga rotundifolia . . . . 12 – 24 – 76 66.7 luzula sylvatica . . . . 3 – 20 – 67 65.6 adenostyles alliariae . . . . 5 – 23 – 64 61.8 valeriana montana . . . . 4 – 8 – 45 55.3 cicerbita alpina . . . . 1 – 10 – 43 53.9 veronica urticifolia . . . . 4 – 23 – 52 52.8 ranunculus platanifolius . . . . 2 – 5 – 38 52.4 astrantia major . . . . . 1 – 31 51.8 homogyne alpina . . . . . . 29 50.5 veratrum lobelianum . . . . . . 24 46 species diagnostic for more than one community phillyrea latifolia 100 78.2 50 28.4 . . . . . clematis fl ammula 80 64.5 50 33 . . . . . asparagus acutifolius 60 40.5 81 62.1 . . . . . carpinus orientalis . 88 68.3 54 34.5 . . . . fraxinus ornus 60 – 100 50.4 83 36.5 31 – 2 – 1 – . genista tinctoria . . 54 50.6 35 26.7 1 – . . quercus petraea . . 71 43.7 100 71.3 1 – 1 – . acer campestre . 12 – 42 27.5 58 45.1 4 – . . fagus sylvatica . . 12 – 46 – 100 39.8 100 39.8 100 – vaccinium myrtillus . . . 4 – 12 – 57 40.3 57 40.6 tab. 3. – continued stupar v., čarni a. 22 acta bot. croat. 76 (1), 2017 phytes and nanophanerophytes are found in the fi rst two communities, with a decline in phanerophytes towards community 7 (fig. 4). beech-dominated forests (communities 5–7) have twice the ratio of geophytes compared to oak-dominated forests (communities 1–4). there is the highest proportion of chamaephytes in community 1, while the biggest share of therophytes is found in communities 1 and 3. discussion our study strongly suggests macro-climatically based differentiation of zfpcs in b&h. while gradient analysis revealed a major infl uence of climatic factors on the species turnover, there is little or no impact of topographic (slope and aspect) or edaphic conditions (fig. 2, tab. 2), which is congruent with the traditional understanding of zonal vegetation (dierschke 1994, ellenberg 2009, surina 2014). macro-climatic gradient is supported by the signifi cant correlation with the geographic factors (altitude, longitude and latitude), which are all determinants of climate on a larger scale (ellenberg 2009, adams 2010). a recent study of zonal forests of korean peninsula similarly showed that the main factor discriminating individual forest types was temperature (annual mean temperature, as well as temperature extremes) followed by precipitation, altitude and aridity (černý et al. 2015). while chorotypes indicate units that describe distribution patterns shared by several species, explaining origins and history of development of particular fl oras (pignatti and pignatti 2014, passalacqua 2015), some studies suggest that there is a correlation between chorotypes and climatic conditions (ferrer-castán and vetaas 2003, abbate et al. 2012). in the case of our study, the proportion of ‘warmer’ (euri-mediterranean and steno-mediterranean) chorotypes increases in a direction of the main gradient, while the proportion of ‘colder’ (boreal and orophytic) chorotypes decreases (tab. 3), which again suggests the macro-climatic gradient. floristic analysis showed a clear separation of seven zfpcs in b&h. community 1 (quercus ilex maquis, tab. 3, col. 1) occupies only a small area (about 20 km2) in the extreme south of b&h. it is represented by the secondary succession stage of the eastern adriatic eu-mediterranean zonal association fraxino orni-quercetum ilicis (kutleša and lakušić 1964). mean annual temperatures are above 15 °c (fig. 2) and elevations 0–150 m. diagnostic species are mainly of steno-mediterranean, euri-mediterranean and south and southeast european chorotypes. diagnostic herb species are mainly indicators of rocky habitats due to structural degradation. although in the last 50 years this community has expanded its distribution area in b&h, due to the abandonment of coppicing, burning and goat breeding, there are still no high stands (drešković et al. 2011). the situation is similar, though a little better, in neighboring croatia (vukelić 2012). community 2 (quercus pubesfig. 4. proportions of life forms in zonal forest plant communities in bosnia and herzegovina. community numbers correspond to those used in tab. 1 and fig. 1. p – phanerophytes; np – nanophaneorphytes; ch – chamaephytes; h – hemicryptophytes; g – geophytes; t – therophytes. tab. 4. comparison of differences in functional traits occurrences between each pair of zonal forest plant communities (zfpcs). statistical signifi cance established using kruskal-wallis test by ranks and median, except for r, h and g, for which the scheffé post hoc test for a normal distribution was used. community numbers correspond to those used in tab. 1 and fig. 1. ft – functional type; c – competitors; s – stress tolerators; r – ruderals; p – phanerophytes; np – nanophanerophytes; ch – chamaephytes; h – hemicryptophytes; g – geophytes; t – therophytes. *** p<0.001; ** p<0.01; * p<0.05. ft compared pairs of zfpcs 1– 2 1– 3 1– 4 1– 5 1– 6 1– 7 2– 3 2– 4 2– 5 2– 6 2– 7 3– 4 3– 5 3– 6 3– 7 4– 5 4– 6 4– 7 5– 6 5– 7 6– 7 c *** * ** * ** *** * s ** *** ** * ** *** *** * *** ** *** r ** *** ** p * *** * ** *** *** ** *** *** *** *** np *** *** *** * *** ch ** * ** *** * * ** h *** *** ** ** *** ** *** * *** *** *** *** g *** ** *** *** *** *** *** *** *** *** *** *** *** * t * ** *** ** *** zonal forest vegetation of bosnia and herzegovina acta bot. croat. 76 (1), 2017 23 cens-carpinus orientalis forests, tab. 3, col. 2) is represented by high, not degraded stands of zonal thermophilous deci duous forest of the association querco pubescenti-carpinetum orientalis (stupar et al. 2015) found in the lowlands and hilly area of sub-mediterranean b&h. however, due to the negative human impact, they have mainly been replaced by the secondary scrub community rusco aculeati-carpinetum orientalis (muratspahić et al. 1991), while their present distribution area is restricted to small patches of mainly private old groves (stupar et al. 2015). mean annual temperatures are between 12 and 15 °c (fig. 2), occupying elevations between 100 and 650 m. diagnostic species are thermophilous and xerophilous plants of mainly s/se european or euri-mediterranean distribution, although some widespread nemoral herbs and shrubs appear with high frequency, e.g.: brachypodium sylvaticum, dactylis glomerata, veronica chamaedrys, crataegus monogyna, hedera helix etc., indicating more mesophilous microclimatic conditions under the closed canopy. community 3 (quercus frainetto forests, tab. 3, col. 3) is found in the eastern parts of b&h, where the infl uence of the continental drier climate increases. this is a zonal community of central balkans lowlands and hilly areas (horvat et al. 1974) and is represented by the fairly heterogeneous association quercetum frainetto-cerridis (tomić and rakonjac 2013, stupar et al. 2015). although we included in the analysis only stands with 70% and more canopy cover, these forests are mainly found in the proximity of human settlements, so they are frequently degraded by grazing and browsing, as well as occasional fi res. mean annual temperatures are between 10 and 12 °c (fig. 2), and elevations are between 150 and 700 m. they are dominated by a mixture of thermophilous, acid tolerant and widespread nemoral species, as well as some more light-demanding herbs, indicating wood pasture and cutting. community 4 is represented by sessile oak-common hornbeam forests, which are considered a zonal forest vegetation community for the area of nw balkans (quercus petraea-carpinus betulus forests, tab. 3, col. 4). in b&h they are found mainly in the hilly area of northern parts of the country but can also penetrate deeper into the south (e.g., central bosnian basin, fig. 1). habitats of these forests have been under intensive anthropogenic infl uence since the neolithic (horvat et al. 1974), so it is hard to fi nd stands of high, productive forests with quercus petraea cover value in the tree layer more than 3 on the braun-blanquet scale (25–50% of cover) and more than 70% of overall canopy cover. they are often, due to bad management, structurally degraded to carpinus betulus coppice. mean annual temperatures are between 9 and 10 °c (fig. 2) and they occupy elevations between 200 and 900 m. several types of these forests occur in b&h but neither the syntaxonomy nor nomenclature has been settled (lakušić et al. 1978, redžić 2007). in addition, there is disagreement about the syntaxonomical position of these forests on the regional level. while croatian and slovenian authors assign these forest to the illyrian alliance erythronio-carpinion betuli (vukelić 2012, košir et al. 2013a), some other authors question this view, arguing that the group is weakly characterized by diagnostic species (willner and grabherr 2007, borhidi et al. 2012). this is sustained by our results, in the sense that this zfpc is the only one in b&h that completely lacks endemic illyrian species (tab. 3). diagnostic species are mainly represented by mesophilous european and eurasian elements. community 5 (pure fagus / mixed fagus-abies forests, tab. 3, col. 5) represents the fi rst altitudinal belt above oak forests and is made up of mesoneutrophilous montane pure beech, as well as mixed fi r-beech forests. the syntaxonomy and nomenclature of these forests have not yet been the subject of thorough analysis but two main types, or widely understood associations, are included: fagetum montanum illyricum and abieti-fagetum dinaricum (beus 1984). they belong to the alliance aremonio-fagion (marinšek et al. 2013). mean annual temperatures are between 6 and 8 °c (fig. 2) and they are mainly found at elevations between 700 and 1400 m. floristically and ecologically similar to this type is community 6 (mixed picea-abies-fagus forests tab. 3, col. 6), which also has a considerable amount of boreal and south european orophytic elements, while annual mean temperatures are between 5 and 7 °c (fig. 2). in the view of beus (1984), which is supported by our results, the main difference between community 6 and the previous community is that community 6 has a higher proportion of spruce (100% frequency) and acidophilous vaccinio-piceetea species, while the proportion of mesoneutrophilous aremoniofagion species is remarkably lower. in addition, it comes as an altitudinal belt above community 5 (elevations between 800 and 1600 m) and can be found exclusively in the central range of dinaric alps, while community 5 extends more to the north and south (fig. 1). this is explained by the fact that the hot dry summers of the northern and southern chains of dinaric alps do not favor spruce (beus 1984). the syntaxonomy and nomenclature of community 6 have also not been settled, so in b&h this complex type is identifi ed under the invalid provisional name piceo-abieti-fagetum. community 7 is the highest altitudinal belt, represented by subalpine fagus sylvatica forests (tab. 3, col. 7). this is yet another group the syntaxonomy and nomenclature of which in b&h has not been analyzed and it is known by the generic name of fagetum subalpinum s. lato. these forests belong to the suballiance saxifrago rotundifoliaefagenion of the alliance aremonio-fagion (marinšek et al. 2013). these are mainly mesoneutrophilous pure beech forests found mainly on the mountains of the central and southern chains of the dinaric alps (fig.1). mean annual temperatures are between 5 and 6 °c (fig. 2), with elevations between 1400 and 1800 m. this community harbors the highest proportion (32%) of boreal and south european orophytic elements. functional analysis of ecological strategies showed small overall differences among zfpcs. based on the communities’ locations on the csr triangle, competitive strategy (c) is dominant in all seven communities, followed by csr and sc strategies (fig. 3). considering the course of succession (fig. 3a), site production is high (grime 1974), while the importance of ruderality is insignifi cant, which indicates late stages of succession, as pointed out by prévosto et al. (2011). the terminal stage of zonal communistupar v., čarni a. 24 acta bot. croat. 76 (1), 2017 ties is supported by the position of the communities on the csr plot when only the herb layer was analyzed (fig. 3b). in this plot, stress tolerance becomes more important since shading and lack of nutrients in the herb layer coincide with the development of large long-lived forest trees (grime 1977, 1988). only community 1 (quercus ilex maquis), which has the largest share of ruderals, expresses conditions of moderate productivity and middle to late stage of secondary succession (maquis). the increased proportion of stress-tolerators in community 1 and also, to a much lesser extent, in community 2 is related to drought stress during the summer season in the mediterranean limestone region of b&h. on the other hand, community 4 (quercus petraea-carpinus betulus forests) has an increased proportion of competitors, which indicates higher productive capacities. also, this community lies in the middle of the macro-climatic gradient (fig. 2), which altogether suggests the most favorable conditions for forest vegetation development in b&h. changes in the life form proportions of different communities are expressed in the greater share of woody species in the more thermophilous vegetation types, which corresponds with the statement that ‘the phanerophyte is the plant type that belongs to warm regions’ (raunkiaer 1934). similar results were obtained in the study of the gradient from warm to mesic temperate forests on the galičica mountain range in macedonia (čarni et al. 2016). the proportion of geophytes is higher in communities 5–7 (beech dominated forests), which have spring ephemerals (mainly bulbous geophytes), also abundant in oak communities (popović et al. 2016), in addition to harboring a considerable number of non-ephemeral rhizomatous ferns and species from the family liliaceae. this is congruent with the notion that rhizomatous geophytes are adapted to life in regions with a severe unfavorable period (e.g., hard winters), but have at the same time a long period of vegetation (raunkiaer 1934). the proportion of therophytes is insignifi cant except in communities 1 and 3, which can be explained by water shortage in the conditions of harsher mediterranean and continental climates (kavgaci et al. 2012, raju et al. 2014). the same can be said for the proportion of chamaephytes in community 1. on the other hand, according to bloch-petersen et al. (2006) plant life forms differentiate not only due to climatic variations, but seem also to relate to humane disturbance and management. prasad (1995) studied the effects of grazing on the plant species and life form composition and found that the percentage of therophytes was higher on grazed areas than on protected areas, which refl ects the disturbance through grazing in communities 1 and 3. to conclude, the results of our analysis support the hypothesis that zonal forest vegetation in b&h is an expression of macro-climatic conditions and that there are no remarkable differences in csr plant strategies among the zfpcs in b&h (excluding community 1). however, having in mind that the currently protected forest area in b&h is very small and covers only a few types of zfpcs (stupar 2011), in order to facilitate further research into the ecological, fl oristic and functional characteristics of zfpcs there is a need for establishment of a national network of protected areas which would cover all zfpc types, thus preserving the representative stands in as natural conditions as possible (milanović et al. 2015). particular emphasis should be given to thermophilous forests, especially to community 1 (stands of quercus ilex) which should be converted from maquis to high forests. acknowledgements the research was partly carried out by the bh-si bilateral project »vegetation of thermophilous oak forests of the pre-pannonian area of the western balkans« no. bi-ba/ 12-13-015. ač also acknowledges the fi nancial support of the slovenian research agency (p1-0236). we would like to express our gratitude to the institute of biology, research center of the slovenian academy of sciences and arts (zrc sazu) and forestry faculty, university of banja luka for logistic support. references abbate, g., iberite, m., bonacquisti, s., giovi, e., iamonico, d., scassellati, e., 2012: taxonomical and chorological diversity of native woody fl ora of italy at regional scale. bocconea 24, 169–175. adams, j., 2010: vegetation-climate interaction. springer berlin heidelberg, berlin, heidelberg. beus, v., 1984: vertical zonation of forests in the light of real and primary vegetation in yugoslavia. radovi anubih 76, odjeljenje prirodno-matematičkih nauka 23, 23–32 (in serbo-croatian). bloch-petersen, m., brandt, j., olsen, m., 2006: integration of european habitat monitoring based on plant life form composition as an indicator of environmental change and change in biodiversity. geografi sk tidsskrift-danish journal of geography 106, 61–74. bohn, u., neuhäusl, r. 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oxidative stress balance. the present study aimed to investigate the effects of osmotic stress on the croatian perennial species fibigia triquetra (dc.) boiss, adapted to a hot and dry habitat. plants grown in culture conditions were subjected to isoosmotic concentrations of mannitol and polyethylene glycol (peg) and certain physiological and oxidative stress parameters were analyzed during a period of 14 days. dry weight and proline content in fibigia triquetra shoots increased in response to osmotic stress while the relative water content decreased. after an initial rise, chlorophyll and carotenoid levels in treated plants dropped to untreated plant levels. oxidative damage to proteins and especially to lipids was evident upon peg-induced osmotic stress. superoxide dismutase and ascorbate peroxidase appear to play an essential protective role in stressed plants. regardless of the osmotic agent, accumulation of heatshock proteins of 70 kda was noticed under osmotic stress. the tolerance of the plant species to osmotic stress seems to be associated with increased capacity of the antioxidative system and effi cient photoprotective system. keywords: antioxidative enzymes, lipid peroxidation, mannitol, osmotic stress, oxidative damage, polyethylene glycol, proline, stress proteins abbreviations: apox – ascorbate peroxidase, cat – catalase, dw – dry weight, fw – fresh weight, mda – malondialdehyde, page – polyacrylamide gel electrophoresis, peg – polyethylene glycol, pox – pyrogallol peroxidase, rwc – relative water content, sod – superoxide dismutase introduction drought is one of the most common abiotic stressors limiting plant productivity. under such stress, water defi cit in plant tissue develops and the photosynthetic rate decreases due to stomatal closure or metabolic impairment (reddy et al. 2004). plants have evolved differ* corresponding author, e-mail: vvujcic@biol.pmf.hr copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. vujčić v., radić brkanac s. 348 acta bot. croat. 73 (2), 2014 ent mechanisms to limit water loss, including adaptations at morphological and physiological levels – thick cuticle, sunken stomata, reduced leaf surface area, shoot growth inhibition, enhanced root growth, changes in carbon metabolism and synthesis of compatible solutes such as proline, glycine betaine, various sugars and polyols (xiong and zhu 2002, chaves et al. 2009). accumulation of proline is a common metabolic response of plants to various abiotic stress conditions including water defi cit and salt stress (bartels and sunkar 2005, hayat et al. 2012). aside from acting as an osmoprotectant, proline has been reported to play an important role in the removal of reactive oxygen species (ros), maintaining protein stability and the regulation of ph in the cytoplasm (hayat et al. 2012). inhibition of co2 assimilation coupled with changes in photosystem activities and photosynthetic electron transport capacity results in increased generation of ros which may cause damage to membrane lipids and proteins and inactivate sh-containing enzymes (reddy et al. 2004). this enhanced ros production is however kept under tight control by a versatile antioxidant system that includes enzymes such as superoxide dismutase (sod), catalase (cat) and ascorbate peroxidase (apx) and low molecular weight antioxidants such as ascorbic acid, tocopherol, carotenoids and other (cruz de carvalho 2008). in addition to the activation of oxidative stress protection mechanisms, the combined effects of multiple stress factors (drought, extreme temperatures, increased salinity etc.) often induce synthesis of various stress proteins such as heat-shock proteins (hsps) involved in the folding of nascent proteins and the refolding of denatured proteins. among hsps, the stressinducible heat shock protein 70 kda (hsp70) has been proposed as a biomarker for monitoring environmental stressors (wang et al. 2004). fibigia triquetra (dc.) boiss. is a croatian rare stenoendemic plant species growing on sunny and dry habitats of calcareous rocks and thus often exposed to water defi cit (pevalekkozlina et al. 1997). because of the natural habitat of f. triquetra, we assume that the plant has developed tolerance to drought and high temperatures as well as to oxidative stress. osmotic stress in experimental conditions is often simulated by using polyethylene glycol (peg, non-ionic-impermeable osmoticum) and mannitol (non-ionic-permeable osmoticum). since mannitol partially enters the cell, the use of high molecular weight peg (6000 or 8000) is recommended under laboratory conditions (verslues et al. 2006). several studies carried out with different osmotic agents correlated effi cient antioxidant defense with tolerance mechanisms (radić et al. 2006, 2013). we hypothesized that f. trique tra tolerance to osmotic stress could be associated with induction of the antioxidative system and hsp70 stress protein as well as effective osmoregulation mechanisms. hence, this study reports on: 1) a comparative analysis of the changes in the activities of antioxidative enzymes, 2) analysis of proline (compatible solute and antioxidant), and 3) a possible accumulation of hsp70 upon pegand mannitol-induced osmotic stress. material and methods plant material and osmotic treatments f. triquetra seeds were collected from their natural habitat. the sterilized seeds were inoculated in test tubes fi lled with 15 ml of ms ½ medium containing 0.1 g l–1 myoinositol (sigma–aldrich), 0.1 mg l–1 thiamine × hcl (sigma–aldrich), 0.5 mg l–1 pyridoxine × hcl (sigma–aldrich), 0.5 mg l–1 nicotinic acid (sigma–aldrich), 2.9 μm gibberellic acid (ga3; responses of fibigia triquetra to osmotic stress acta bot. croat. 73 (2), 2014 349 sigma–aldrich), 0.5 μm 6-benzylaminopurine (ba; sigma–aldrich), 30 g l–1 sucrose (kemika) and 8 g l–1 agar (sigma–aldrich) (murashige and skoog 1962, pevalek-kozlina et al. 1997). the ph value of nutrient medium was adjusted to 7 with 0.1 m koh (kemika) and the medium was autoclaved at 118 kpa and 120 °c for 20 min. the cultures were grown under a 16 h photoperiod of fl uorescent light (80 me m–2 s–1) at 24±2 °c. four-week old plants were subcultured to liquid ms ½ medium and, following root initiation, were transferred to medium of the same composition supplemented with mannitol (sigma–aldrich) or peg (sigma–aldrich). except for the control medium, osmotica were added to the medium as 51 g l–1 mannitol or 168 g l–1 peg 6000 corresponding to −1.0 mpa in both cases. the water potentials of nutrient solutions were determined by a cryoscopic osmometer (knauer). plants were exposed to mannitoland peg-mediated osmotic stress for a period of 14 days. shoot samples for all analyses were collected after 1, 7 and 14 days of experiment. growth parameters dry weight (dw) was measured after oven drying of the samples at 70 °c for 48 h. relative water content (rwc) was calculated as: rwc (%) = (fw − dw)/fw × 100 where fw denotes fresh weight. prior to determination of fresh weight, shoots were washed with distilled water and dried with towels. proline content free proline content was measured by the method of bates et al. (1973). plant tissue was homogenized in 3% (w/v) sulphosalycylic acid (sigma–aldrich) and centrifuged at 700 × g for 3 min. after addition of ninhydrin reagent (riedel-dehaën), mixtures were heated at 100 °c for 1 h and cooled in an ice-bath. the chromophore obtained was extracted from liquid phase with toluene (kemika) and the absorbance of the organic layer was read at 520 nm. proline concentration was determined from the calibration curve using l-proline as standard and expressed as nmol g–1 fw. chlorophylls and carotenoids content chlorophyll a, b and total carotenoid contents were measured and calculated according to lichtenthaler (1987). in brief, fresh leaves were homogenized with 80% (v/v) cold acetone (kemika), centrifuged at 5,000 × g for 10 min. the absorbances of the supernatant were read at 663, 646 and 470 nm. mda content lipid peroxidation was determined by an estimation of the amount of malondialdehyde (mda) content with the use of the thiobarbituric acid method described by heath and packer (1968). the crude extracts were mixed with 0.25% (w/v) thiobarbituric acid (sigma–aldrich) solution containing 10% (w/v) trichloroacetic acid (sigma–aldrich), heated at 95 °c for 30 min and the reaction was stopped in an ice-bath. the cooled mixtures were centrifuged at 10,000 × g for 10 min and the mda content calculated from the absorbance at 532 nm (correction was done by subtracting the absorbance at 600 nm for non-specifi c turbidity) by using extinction coeffi cient of 155 mm–1 cm–1. vujčić v., radić brkanac s. 350 acta bot. croat. 73 (2), 2014 carbonyl groups content the amount of protein oxidation was estimated by the reaction of carbonyl groups (c=o) with 2, 4-dinitrophenylhydrazine (sigma–aldrich), as described in levine et al. (1990). after the 2,4-dinitrophenylhydrazine reaction, the c=o content was calculated by absorbance at 370 nm, using an extinction coeffi cient for aliphatic hydrazones of 22 mm–1 cm–1 and expressed as nmol mg–1 protein. enzyme determinations plant tissue was homogenized in 50 mm potassium phosphate (k2hpo4/kh2po4) buffer (ph 7.0) including 5 mm sodium ascorbate (sigma-aldrich), 1 mm ethylene diamine tetraacetic acid (sigma–aldrich) and polyvinylpolypyrrolidone (pvpp, sigma-aldrich). the homogenates were centrifuged (sigma 3k18 centrifuge, germany) at 22,000 × g for 20 min at 4 °c. supernatant was used for enzyme activity and protein content assays. total soluble protein contents of the enzyme extracts were esti mated according to bradford (1976) using bovine albumin serum (bsa, sigma) as standard. the activity of sod was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (sigma–aldrich) following the method of giannopolitis and ries (1977). one unit of sod was taken as the volume of the enzyme extract causing 50% inhibition of nitroblue tetrazolium reduction. cat activity was determined by the decomposition of h2o2 and was measured spectrophotometrically by following the decrease in absorbance at 240 nm (aebi 1984). activity was calculated using the extinction coeffi cient of 0.04 mm–1 cm–1 and mmol h2o2 g –1 fw min–1 was defi ned as unit of cat. apx activity was done according to nakano and asada (1981). the ascorbate oxidation was followed at 290 nm and its concentration calculated using the molar extinction coeffi cient (e = 2.8 mm–1 cm–1). corrections were done for low, non-enzymatic oxidation of ascorbate by h2o2. one enzyme unit was defi ned as mmol oxidized ascorbate g–1 fw min–1. the activity of pox was measured by monitoring the formation of purpurogallin at 430 nm (e = 2.47 mm–1 cm–1) according to chance and maehly (1955). the reaction mixture contained 50 mm potassium phosphate buffer (ph 7), 1 mm h2o2, 20 mm pyrogallol (sigma-aldrich) and enzyme extract. the specifi c enzyme activity for all enzymes was expressed as units per mg of protein. imunodetection of hsp70 to analyze hsp70, plant tissue were homogenized in tris-hcl extraction buffer ph 8.0 containing 17.1% (w/v) sucrose (kemika), 0.1% (w/v) ascorbic acid (sigma-aldrich), and 0.1% (w/v) cysteinehydrochloride (sigma-aldrich) with addition of pvpp and then centrifuged at 29,700 × g for 50 min. total protein concentration in the supernatant was determined according to bradford (1976). aliquots of each homogenate were mixed with corresponding volumes of denaturing 0.065 m tris-hcl buffer containing 6% (w/v) sodium dodecyl sulfate (sds, sigma-aldrich), 6% (v/v) b-mercaptoethanol (sigma-aldrich), 30% (v/v) glycerol, and 0.01% (w/v) of bromphenol blue (sigma-aldrich). the extracts were boiled for 2 min. constant protein weights 9 or 12 mg of total protein per lane were analyzed by sds-polyacrylamide gel electrophoresis (page) (bio-rad, hercules, ca, usa) and subsequent western blotting at 60 v (bio-rad). the resolving gel was made at 10% of polyacrylamide (w/v). standard proteins of known molecular weights (fermentas, glen burnie, md, usa) were run in the same gel. the membranes were blocked with 10% (w/v) nonfat powdered milk solution made in phosphate-buffered saline (58 mm na2hpo4, 17 responses of fibigia triquetra to osmotic stress acta bot. croat. 73 (2), 2014 351 mm nah2po4, 68 mm nacl) ph 7.4 containing 1% (v/v) of tween 20 (sigma-aldrich) and incubated with a rabbit monoclonal antibody raised against the pea hsp70 (diluted 1:1000) overnight at 4 °c. the secondary antibody was an alkaline phosphatase–anti-rabbit igg (sigmaaldrich) diluted 1:2000. the membranes were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (sigma-aldrich). statistical analysis data were analyzed by one-way analysis of variance (anova) using statistica 7.1. (statsoft, inc.) software package, and differences between corresponding controls and exposure treatment were considered as statistically signifi cant at p < 0.05. each data point is the average of six replicates (n = 6). results exposure of f. triquetra to peg-induced osmotic stress caused a signifi cant decrease in plant water status after only one day of exposure, while after 7and 14-day periods a signifi cant drop in water content (between 7 and 9% compared to control) was evident in response to both osmotica (fig. 1 a). mannitol induced a marked rise (a 32% rise compared fig. 1. parameters: (a) rwc, (b) dw, (c) proline, (d) chlorophyll a, (e) chlorophyll b and (f) carotenoid contents evaluated in f. triquetra plants under control conditions (c) and upon stresses caused by mannitol (m) and peg (p) after 14day growth period. values are mean ± sd based on six replicates. bars with different letters are signifi cantly different at p < 0.05. vujčić v., radić brkanac s. 352 acta bot. croat. 73 (2), 2014 to control) in shoot dw after 7and 14-day period while peg caused more conspicuous increase of dw (over 40% rise compared to control) which was noted even after the fi rst day of stress (fig. 1 b). osmotic stress had a signifi cant effect on proline contents following 7and 14-day periods (fig. 1 c) though peg produced a more conspicuous rise in the amino acid content (a 2.5-fold increase compared to control) than mannitol (35 and 73% increase compared to control following the 7and the 14-day period, respectively). content of chlorophyll a, chlorophyll b and carotenoids signifi cantly increased after 24 h of plant exposure to mannitol and peg as compared to control plants (figs. 1 d–f). after seven days of experiment, chlorophyll and carotenoid contents under mannitol treatment were not signifi cantly different from respective values in control plants while in plants exposed to peg the content of these pigments was still signifi cantly elevated. however, at the end of the experiment the contents of the studied photosynthetic pigments in osmoticstressed shoots were similar to those in control plants. the level of lipid peroxidation, expressed as mda content, in f. triquetra shoots sampled after 24 h period increased by 43% in response to mannitol (fig. 2 a). following longer exposure (a 7and a 14-day growth period), the mda content of mannitol-treated plants leveled with that of control. unlike mannitol, peg caused a signifi cant rise in the fig. 2. parameters: (a) mda, (b) c=o – carbonyl groups, (c) sod, (d) cat, (e) apox and (f) pox measured in f. triquetra plants under control conditions (c) and upon stresses caused by mannitol (m) and peg (p) after 14-day growth period. values are mean ± sd based on six replicates. bars with different letters are signifi cantly different at p < 0.05. responses of fibigia triquetra to osmotic stress acta bot. croat. 73 (2), 2014 353 fig. 3. patterns of heat-shock protein of 70 kda (hsp70) in f. triquetra plants under control conditions (c) and upon stresses caused by mannitol (m) and peg (p) after 1-, 7and 14-day growth period. extent of lipid peroxidation during the entire experimental period (increase by 40–60%, compared to control). regarding the level of carbonyl groups, peg induced a much stronger effect than the lower molecular weight osmoticum. namely, the carbonyl group content of peg-treated f. triquetra was markedly elevated compared to control plants after a 7-day period of exposure while mannitol did not show any signifi cant effect on the parameter over the entire experimental period (fig. 2 b). the activity of sod in the shoots of f. triquetra exposed to either mannitol or peg remained unchanged after 1and 7-day growth periods and signifi cantly increased only at the end of the experiment (fig. 2 c). cat activity of f. triquetra shoots was not affected by either osmotica during the entire experimental period (fig. 2 d). regardless of the substrate used, the activity of the peroxidases (apox and pox) of f. triquetra shoots was markedly induced by both mannitol and peg over the entire experimental period (figs. 2 e–f). however, after 1and 14-day growth periods, the apox activity of peg-treated plants was over 30% higher than the enzyme activity of mannitol-treated plants. under osmotic treatment, the increase of pox activity in f. triquetra shoots exceeded 50% in comparison to control after 1and 7-day growth period. the rise in the activity of pox caused by osmotic stress was even more expressed after a 14-day growth period – both mannitol and peg caused a 2.5-fold increase in the enzyme activity compared to control. proteins of plant species f. triquetra separated by page in the denatured conditions were transferred to nitrocellulose membrane followed by immunodetection with hsp70 antibodies. after a 1-day period of exposure, the expression of the hsp70 stress protein was already stronger in treated than in control plants (fig. 3). the protein accumulated even more in treated f. triquetra shoots following a longer period of exposure to mannitol and peg. vujčić v., radić brkanac s. 354 acta bot. croat. 73 (2), 2014 discussion the results presented here indicate that the use of peg as an osmotic agent led to more severe osmotic effects in f. triquetra shoots than when mannitol was used. although both agents produced similar effects on rwc following a longer growth period, peg caused signifi cant change in the parameter after only a 24 h period. the initially stronger effects of high-molecular-weight peg compared to low-molecular-weight mannitol on rwc could be related to the inhibitory action of peg on the capacity of roots to supply water to the leaves (chazen et al. 1995). stronger effects of peg on rwc, compared to mannitol, were observed in the halophytic species sesuvium portulacastrum exposed to osmotic stress for a 12-day period (slama et al. 2007). such effects of peg were previously linked to greater viscosity of peg solutions while mannitol was assumed to attenuate osmotic gradient between the medium and the cell due to uptake by cells (hohl and schopfer 1991, slama et al. 2007). many studies have shown that osmotic stress caused by either use of osmotica or by the withholding of water leads to a reduction i n plant rwc which is often accompanied with a decrease of leaf fw or dw (fu and huang 2001, egert and tevini 2002, slama et al. 2007). however, in our study, dw of f. triquetra shoots signifi cantly increased under both mannitol and peg treatment. similar results with respect to dw and rwc were obtained with drought-tolerant bean cultivar phaseolus acutifolius grown on the medium with peg for a 14-day period (türkan et al. 2005). the increase in dw of stressed plants noted in our study could, at least partly, be ascribed to increased accumulation of proline. however, regarding the level of proline accumulation in osmotically stressed f. triquetra shoots, especially under mannitol, the observed increase in dw might be the result of increased synthesis of other organic compounds which are used in osmotic adjustment (hare et al. 1998, slama et al. 2007, farooq et al. 2009) or different proteins such as hsp proteins and dehydrins engaged in the adaptation to abiotic stress (wang et al. 2004). a rise in proline noted in our study was too low to suggest that the amino acid acts as an osmolyte (slama et al. 2007). thus, it is likely that proline accumulation in f. triquetra shoots is related to some other proline roles such as ros detoxifi cation, protection of cellular macromolecules or maintenance of cellular ph. the greater accumulation of proline in response to peg than to mannitol, described here in response to osmotic stress, has also been observed in osmotically stressed rice (pandey et al. 2004). the tolerance to drought may be closely related with effi cient photoprotective system (farooq et al. 2009). in the present study, dynamics of changes in chlorophyll and carotenoid contents of stressed f. triquetra over time was similar – the pigments showed an initial rise, chlorophyll a and carotenoids were still elevated under peg-stress after 7 day-period while after 14 day-period the levels of those pigments also leveled with control ones. increased or unaffected levels of chlorophylls and carotenoids under osmotic stress imply a better photoprotection and capacity for light harvesting and are considered to be a defensive response which restricts the harmful effects of drought (farooq et al. 2009). similar results with respect to chlorophyll and carotenoid levels had been reported in drought-tolerant plant species exposed to drought stress conditions (fu and huang 2001, egert and tevini 2002, kalefetoglu macer and ekmekci 2009). the results presented here indicate that osmotic stress increased antioxidative defense in f. triquetra shoots, especially in the case of peg. however, regarding parameters of oxidative injury to vital biomolecules, f. triquetra proved to be less susceptible to mannitol responses of fibigia triquetra to osmotic stress acta bot. croat. 73 (2), 2014 355 than to peg-induced osmotic stress. the indicator of lipid peroxidation, mda, after initial increase under both mannitol and peg treatment, remained at high levels only in pegstressed plants. the high-molecular peg also caused transient increase in carbonyl groups content indicating oxidative damage to proteins. the results suggest far greater ros production under pegthan mannitol-stress despite the relatively higher induction of antioxidative defense in response to peg. concerning chlorophyll and carotenoid contents under peg-stress, the targets for harmful action of ros might not necessarily be thylakoid membranes but possibly other ros-generating sites like mitochondria, plasma membranes, cell wall etc. (moran et al. 1994). several plant species exposed to peg-induced osmotic stress showed the same pattern of change in mda, c=o groups and the activities of antioxidative enzymes described here i.e. the increase in the extent of lipid peroxidation or protein carbon ylation with simultaneous induction of antioxidative enzymes upon peg action (sivritepe et al. 2008, pyngrope et al. 2013). sod, an enzyme that breaks down superoxide radicals to hydrogen peroxide and oxygen, is often activated in response to osmotic stress especially in plants tolerant to the stress conditions (bor et al. 2003, türkan et al. 2005, huang et al. 2013, radić et al. 2013). sod activity in shoots of treated plants f. triquetra increased after 14 days of the experiment which indicates that this species is well adapted to the conditions of osmotic stress and can activate the mechanism of elimination of superoxide radicals. hydrogen peroxide requires further degradation enabled by the plant apox, non-specifi c peroxidase (pox) and cat. two enzymes engaged in the decomposition of hydrogen peroxide were induced in the f. triquetra shoots, though in some plant species, depending on the type and severity of stress, an increased activity of only one enzyme can be apparent. the main enzyme for degrading h2o2 in chives and centaurea ragusina leaves exposed to osmotic stress was apox (egert and tevini 2002, radić et al. 2013) while in pea plants exposed to drought it was nonspecifi c peroxidase (moran et al. 1994). in our study, the activities of peroxidases, rather than activity of cat, seem to have a major role in the degradation of hydrogen peroxide produced upon osmotic stress. on the other hand, osmotic stress in cherry rootstock caused activation of all three h2o2-degrading enzymes (sivritepe et al. 2008). a number of different proteins, including hsp proteins, are synthesized or accumulated in response to osmotic stress (rizhsky et al. 2002). studies on arabidopsis and spinach demonstrated that over-expression of hsp70 chaperones results in enhanced tolerance to salt, water and high-temperature stress in plants (wang et al. 2004). in our study, accumulation of hsp70 in f. triquetra exposed to mannitol and peg was evident after a period of only 24 h. similar results with respect to expression of hsp70 were obtained in fucus serratus and lemna minor upon osmotic stress conditions (ireland et al. 2004). in conclusion, both osmotica, especially peg, caused oxidative stress in f. triquetra, but also a rapid activation of antioxidant and protective defense mechanisms (enzymatic and non-enzymatic antioxidants, accumulation of hsp70). based on the results and previous fi ndings on the plant’s morphology, it can be concluded that f. triquetra is relatively tolerant to osmotic stress due to inducible defense systems. acknowledgements this study was supported by croatian ministry of science, education and sport, as part of project no. 119-1191196-1202. vujčić v., radić brkanac s. 356 acta bot. croat. 73 (2), 2014 references aebi, h., 1984: catalase in vitro. methods in enzymology 105, 121–126. bartels, d., sunkar, r., 2005: drought and salt tolerance 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agarwal, m., katiyar-agarwal, s., zhu j., zhu, j.-k., 2006: methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. the plant journal 45, 523–539. wang, w., vinocur, b., shoseyov, o., altman, a., 2004: role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. trends in plant science 9, 244– 252. xiong, l., zhu j. k., 2002: molecular and genetic aspects of plant responses to osmotic stress. plant, cell and environment 25, 131–139. untitled acta bot. croat. 75 (1), 2016 99 acta bot. croat. 75 (1), 99–108, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0004 issn 0365-0588 eissn 1847-8476 spring weed communities of rice agrocoenoses in central nepal arkadiusz nowak1,2*, sylwia nowak1, marcin nobis3 1 department of biosystematics, laboratory of geobotany & plant conservation, opole university, oleska st. 22, 45-052 opole, poland 2 department of biology and ecology, university of ostrava, 710 00 ostrava, czech republic 3 department of plant taxonomy, phytogeography and herbarium, institute of botany, jagiellonian university, kopernika st. 27, 31-501 kraków, poland abstract – rice fi eld weed communities occurring in central nepal are presented in this study. the research was focussed on the classifi cation of segetal plant communities occurring in paddy fi elds, which had been poorly investigated from a geobotanical standpoint. in all, 108 phytosociological relevés were sampled, using the braun-blanquet method. the analyses classifi ed the vegetation into 9 communities, including 7 associations and one subassociation. four new plant associations and one new subassociation were proposed: elatinetum triandro-ambiguae, mazo pumili-lindernietum ciliatae, mazo pumili-lindernietum ciliatae caesulietosum axillaris, rotaletum rotundifoliae and ammanietum pygmeae. due to species composition and habitat preferences all phytocoenoses were included into the oryzetea sativae class and the ludwigion hyssopifolio-octovalvis alliance. as in other rice fi eld phytocoenoses, the main discrimination factors for the plots are depth of water, soil trophy and species richness. the altitudinal distribution also has a signifi cant infl uence and separates the rotaletum rotundifoliae and elatinetum triandro-ambiguae associations. the study shows that anthropogenic rice fi elds can harbour relatively rich rush and water vegetation. more than 80 species were noted in the vegetation plots. several of them are considered to be extremely rare and have been recorded on the world red list. key words: anthropogenic habitats, oryzetea sativae, rice fi elds, segetal communities * corresponding author, e-mail: anowak@uni.opole.pl introduction rice production in nepal plays the most important role in the agricultural economy and in food supply for the society. a half of the total crop area is devoted to rice cultivation, giving ca 60% of total grain production. however, this fi gure has dropped considerably in the last decade (gumma et al. 2011). rice paddy fi elds are located from lowlands (e.g. tarai) to ca. 3,000 m in the western himalayas (jumla valley), which is the highest elevation of rice cultivation in the world (paudel 2011). more than 70% of the rice-growing area in nepal is under rainfed conditions. rice is usually grown only once a year, in the wet season; monsoon rain is the single source of water supply for rice cultivation. more than 60% of the rice production comes from lowland tarai from the plain valleys of gandaki, narayani, karnali. rainfed paddies in hills intermountain basins supply mainly local societies and are less effective. although nepal is the world’s second richest country in water resources, more than 18% of the fi elds are irrigated and some southern areas are threatened by draught (cbs 2007). rice is the most economically important food crop in nepal. its production employs two-thirds of the national labour and supplies more than 40% of calories for the people and is crucial for national food security (gumma et al. 2011). in most parts of nepal, rice is grown on small familybased subsistence farms with an average size varying from less than 0.1 to 1.0 ha. this fragmentation of holdings promotes extensifi cation of rice production and persistence of many weed species in rice segetal agroecosystems. weeds are considered one of the major problems in rice production in asia, including nepal (papademetriou et al. 2000, bhatt et al. 2009). losses due to weeds have been estimated at 12% of the crop yields (manandhar et al. 2007). considerable progress in weed control has been achieved with various measures such as ensuring the purity of rice seed, the proper selection of cultivar and seeding rate, the proper planting method, good land preparation and nowak a., nowak s., nobis m. 100 acta bot. croat. 75 (1), 2016 water management, hand weeding and chemical weed control, as well as crop rotation (de datta 1981), used together in a system of integrated weed management. the frequency and abundance of weeds varies with different methods of cultivation. in nepal, there are several types of rice cultivation, such as upland, dry-seeded, deepwater, transplanted, and wet-seeded or pre-germinated direct-seeded rice. in transplanted or deepwater plantings the weeds are limited by unsuitable conditions for establishment of plants. direct seeded rice crops are the most weed-infested with yearly yield losses of up to 75% (manandhar et al. 2007). rice fi elds are still recognized globally as well as in southern asia as important refuges for many wetland and water species. many of them are highlighted by the iucn as important in terms of biodiversity conservation and red-listed. among others, lindernia ciliata, ammannia multifl ora, ludwigia perennis, aeschynomene indica, cyperus michelianus, geissaspis cristata, limnophila erecta, marsilea minuta, hydrocera trifolia and monochoria vaginalis can be mentioned (iucn 2013). the increasing food demand will lead inevitably to intensifi cation of rice production and then impoverishment and degradation of rice fi eld phytoco enoses. perhaps this is the last chance to document and classify the vegetation types of rice paddies in south asia. as to maintain the withdrawing segetal weeds and to stop the impoverishment of agrocoenoses, many conservation actions and research works have been implemented in recent years (e.g. van elsen et al. 2006). recent studies were focused mainly on the effect of cultivation methods on the fl oristic composition and richness of weed communities (kent et al. 2001, hyvönen and salonen 2002, chauhan 2013). many studies have recorded basic fl oristic and vegetation data for rice crops, often focussing on their conservation value or ecological services (barrett and seaman 1980, camenisch and cook 1996, turki and sheded 2002, bambaradeniya et al. 2004). specifi c biological features which are supposed to be responsible for weed decline have received less attention (e.g. gibson et al. 2006). phytosociological studies of rice fi eld communities have been carried out for several years mainly in the countries of southern europe (bolòs and masclans 1955, piccoli and gerdol 1981, carretero 1989, parras and lorca 1993, garcia and benzal 2009), japan (miyawaki 1960), the korean peninsula (kolbek et al. 1996, kolbek and jarolímek 2013, shimoda and song 2014), tajikistan (nowak et al. 2013) and thailand (nowak et al. 2015a). to date, no detailed research into the plant communities of these specifi c ecosystems has been conducted in other important regions of rice cultivation, for example, in indonesia, india, madagascar or central asian countries. thus the main aim of the presented research is to reveal the diversity of weed associations in rice crops in central nepal and their syntaxonomical classifi cation within the oryzetea sativae class. material and methods study area nepal is located in southern asia between 80°03′ and 88°11′e, and 26°21′ and 30°26′n, situated at altitudes between 78 m and 8,848 m (sagarmatha peak; fig. 1). it shares its northern border with china and from the east, south and west it is surrounded by india. the country covers 147,181 km2 with a population of ca 29 million. the country is divided into fi ve main ecological zones: himalayas, high hills, mid hills, siwalik and lowland terai. the area of the country is drained by three main tributaries of the ganges: the kosi, gandaki and karnali rivers. because of the extreme variations in altitude, topography and physical condition, the agro-climatic conditions, the cultivation methods and types are very diverse in nepal. on account of the climate there is a considerable variability of rice landraces found throughout the country, adapted to specifi c environments. thus, nepal is considered a world biodiversity centre for oryza sativa (asian rice) diversity (van dusen et al. 2007). it is estimated that in nepal about 2000 different landraces of cultivated rice are in use and 4 taxa of wild rice still occurs (o. nivara, o. ruffi pogon, o. offi cinalis and o. granulata; gupta et al. 1996). the rice paddy fi elds are categorized according to the source of irrigation: seasonal streams (kholapani), rivers of snow-melt-water (gadkule) or waterlogged marshes (sim; bajracharya et al. 2006). nepal has tremendously varied climate conditions, from tropical in the south to temperate in the north with alpine and arctic conditions in the highest elevations of the himalayan range. the country area is divided into 6 climatic zones: tropical (up to 1,000 m a.s.l.), subtropical (1,000–2,000), temperate (2,000–3,000), subalpine (3,000–4,000), alpine (4,000–5,000) and nival (above 5,000 m a.s.l.). generally fi ve major seasons are distinguished: spring, summer, monsoon, autumn and winter. in the north, summers are cool and winters severe, while in the south summers are tropical and winters are mild. in the tarai, summer temperatures often exceed 37 °c, while winter temperatures range from 7 to 23 °c. in hilly central nepal and valleys, summers are temperate while winter temperatures can plummet under zero. in kathmandu the summer temperatures range between 19 and 35 °c and in winter between 2 and 12 °c. the average annual rainfall is 1,600 mm, but it varies by ecoclimatic zones, such as 5,500 mm in the windward slopes of annapurna himal to below 300 mm in the rain shadows of mustang. according to biogeographical division of takhtajan (1986) nepal belongs to the sino-japanese region (eastern himalayan province), irano-turanian region (tibetan provfig. 1. the study area in central nepal with main cities and biogeographical zones. weed communities of rice fields in nepal acta bot. croat. 75 (1), 2016 101 ince, western himalayan province), sudano-zambezian region (sindian province) and indian region (bengal and indochinese provinces). central nepal lies generally in the bengal province. the fl ora of nepal counts more than 6,000 fl owering plant species with ca. 250 endemics (press et al. 2000). this number is still being amended upwards (ferille and lachard 2013, nobis et al. 2014a, b). around 218 vascular plants were reported as weeds of rice paddy fi elds (moody 1989, bhatt et al. 2009). data and analyses during the study, in may 2013, 108 vegetation relevés were collected in rice fi elds in central nepal (fig. 1). the sampling sites were located mainly in the valleys of the rivers: trisuli, kali gandaki, rapti, madi, marsyangdi, anahi khola and tinau. the size of each vegetation relevé varied from 3 (some plots of water and mud communities) to 25 m2, depending on plant density, homogeneity of vegetation cover and the size of homogenous spatial units. in each relevé, all vascular plant species were recorded according to the cover-abundance scale of braun-blanquet (1964). the 7-degree scale was used (r, +, 1, 2, 3, 4, 5). all the relevés were stored in the juice program (tichý 2002). a twlnspan analysis (hill 1979) and detrended correspondence analyses (dca) were performed using the fl oristic data set (presence-absence data, no downweighting of rare species) to check the manual fl oristic-sociological classifi cation and to illuminate the relationships between the groups. for the ordination, canoco for windows 4.5 was used (ter braak and šmilauer 2002). the relevés data showed a clear unimodal response, with total inertia of ca. 5. vegetation classifi cation follows the sorted table approach of braun-blanquet (1964). in the analytic tables (tab. 1, on-line suppl. tabs. 1, 2), species constancies are tab. 1. associations of the potametea class in rice fi elds of central nepal during the study, in may 2013. ls – lemno-spirodeletum polyrrhizae; nm – najadetum minoris; cld – community of limnobium dubium; no – number. the cover-abundance scale of braun-blanquet (1964) was used: r = 1 or 5 individuals; + = few individuals (< 20) with cover < 5%; 1 = many individuals (20–100) with cover < 5%; 2 = 5%–25% cover; 3 = 25%–50% cover; 4 = 50%–75% cover; 5 = 75%–100% cover. successive number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 n o. o f o cc ur re nc e n o. o f o cc ur re nc e n o. o f o cc ur re nc eday/month 3/5 6/5 6/5 6/5 6/5 7/5 7/5 7/5 7/5 8/5 8/5 8/5 water depth (cm) 15 15 20 10 13 10 4 5 15 15 15 10 altitude (m) 568 217 217 217 217 205 190 190 205 377 377 377 cover of herb layer (%) 70 45 40 70 85 40 55 55 25 65 40 45 relevé area (m2) 25 25 25 25 25 25 25 25 25 3 3 3 ph 6.8 6.8 – 6.8 7.0 6.5 – 7.2 7.1 – 7.5 – number of weeds 10 2 3 4 3 3 6 6 2 4 4 3 rel. rel. rel. association/community ls ls ls nm nm nm nm nm nm cld cld cld 1–3 4–9 10–12 cultivated plant oryza sativa 3 3 4 3 2 3 2 3 3 2 2 2 ass. lemno-spirodeletum polyrrhizae spirodela polyrrhiza 3 3 2 1 + . . . . . . + 3 2 1 o. lemnetalia minoris et cl. lemnetea minoris azolla fi liculoides 1 . . . . . . . . . . . 1 – – ass. najadetum minoris najas minor . . . 4 5 3 3 2 2 + + . – 6 2 comm. limnobium dubium limnobium dubium + . . . . . . . . 4 3 3 1 – 3 accompanying species echinochloa colona . . . . . + + + 1 . . . – 4 – cyperus difformis + . . . . . + . . + + 1 1 2 alternanthera sessilis 1 . . . . . + 1 . . . . 1 2 – cyperus iria 1 . . . . . . . . + + . 1 – 2 centella asiatica . . . . . . 2 3 . . . . – 2 – polygonum barbatum . 1 2 . . . . . . . . . 2 – – elatine triandra . . . . . 1 + . . . . – 2 – echinochloa crus-galli . . . . . + . + . . . . – 2 – sporadic species: commelina diffusa 1(+); cynodon dactylon 5(+); dopatrium junceum 10(+); elatine ambigua 4(+); fimbristylis miliacea 4(+); lindernia anagallis 1(+); lindernia ciliata 3(+); rotala rotundifolia 1(1); veronica beccabunga 1(2). locality of relevé: 1 – (274548,3; 850244,4); 2, 3, 4, 5 – (273454,5; 842941,6); 6, 7, 9 – (273459,1; 842937,5); 8 – (273522,3; 842954,5); 10, 11, 12 – (274804,5; 845203,8). nowak a., nowak s., nobis m. 102 acta bot. croat. 75 (1), 2016 given in constancy classes (dierschke 1994). in the case of fewer than 8 relevés, the absolute number of species occurrences was specifi ed. some newly presented communities are described as associations according to the international code of phytosociological nomenclature (weber et al. 2000). the presented communities are arranged into a syntaxonomic overview at the end of the description. for documentation of basic habitat conditions governing the occurrence of the community, alkalinity was determined with an elmetron cp-105 ph meter. plant names were adopted mainly after the international plant names index (2012). the herbarium collection is hosted in opun (opole university, opole). results general fl oristic features the number of taxa recorded in the relevés totals 82, with 68 taxa exceeding 1% constancy and 47 taxa 5%. those with the highest constancy are: alternanthera sessilis (83 concurrencies), cyperus difformis (65), mazus pumilus (61), echinochloa colona (52), centella asiatica (45), cynodon dactylon (44), fimbristyllis miliacea (44), lindernia ciliata (39), cyperus iria (34), lindernia procumbens (31), veronica anagallis-aquatica (28), elatine triandra (26), bacopa procumbens (25) and marsilea minuta (21). the sampled plots of vegetation are almost exclusively composed of species originating from the oryzetea sativae class (miyawaki 1960). only a few species, like cynodon dactylon, veronica anagallis-aquatica, v. beccabunga, juncus prismatocarpus, ranunculus sceleratus, vicia hirsuta, chenopodium album, paspalum distichum and portulaca oleracea, often occur outside the hygrophilous vegetation of paddy fi elds. they occupy mostly drier segetal and ruderal habitats or are typical for rushes. cynodon dactylon has a wider ecological amplitude and was spotted in mud, meadow and rush communities. there is also a distinct group of obligatory water species such as spirodela polyrrhiza, limnobium dubium, azolla fi liculoides, nymphaea nouchalii and vallisneria spiralis. but the majority of the taxa found are related to muddy biotopes and have an amphibious character. numerical ordination the detrended correspondence ordination run for the entire data set distinctively separates the water plant communities which are set to the left part of the diagram. those are the limnobium dubium community, najadetum minoris and lemno-spirodeletum polyrrhizae (fig. 2). the gradients of water decrease and plots related to communities occurring on mud are located on the right part of the diagram. the associations of elatinetum triandro-ambiguae and rotaletum rotundifoliae are positioned in the bottom part of the graph. they prefer rainfed paddy terraces with clayey, brown muds as a soil substrate and intermediate period of water inundation. phytocoenoses concentrated in the central and right parts of the graph are similar in terms of habitat preferences. they occupy muddy soils with higher trophy and are generally composed of higher number of species despite the lower total cover of herb layer (figs. 2, 3). only the marsileetum minutae association is rather species poor and occurs in rice stands during the early phenological phase. the mazo pumili-lindernietum ciliatae association is considerably dispersed, showing the highest variability in fl oristic structure. several plots with a greater amount of sand fraction in the soil have been classifi ed as subassociation with frequent occurrence of caesulia axillaris as diagnostic species. the upper section of the fi gure is occupied by plots related to ammanietum pygmeae. this phytocoenosis is relatively species rich, however with low abundance of weeds. it develops on fertile muddy substrates in irrigated fi elds located in the warmest areas in the kali gandaki river valley. the association has a loose, open structure and was found on sites with low rice stands density. all of the defi ned plant associations are relatively distinct. only some small overlaps occur, because of habitat similarities and there are some widely distributed, non-specifi c species in common (alternanthera sessilis, cyperus difformis, cynodon dactylon, echinochloa colona). this lack of powerful discrimination between some associations in the dca could be also due to the relatively low number of species per relevé in rice fi eld phytocoenoses and to a low degree of fi delity for some rush or aquatic species. it is widely known, that although species of hygrophilous habitats display certain habitat preferences, different associations can thrive in the same pond (riis et al. 2000, hattonellis and grieve 2003, gacia et al. 2009). fig. 2. detrended correspondence analysis ordination for all samples (n = 108). weed communities of rice fields in nepal acta bot. croat. 75 (1), 2016 103 syntaxa of rice fi elds in central nepal 1. marsileetum minutae nowak a., nowak s., nobis m. 2015 (on-line suppl. tab. 2, rel. 43–48) diagnostic species: marsilea minuta this phytocoenosis has been found at a height of 200– 370 m a.s.l. in the valleys of the rivers seti and anahi khola. most of the plots of this association were found on wet mud or in shallow water inundation up to 10 cm (mean: approximately 5 cm). the community develops on rather fertile soils with close to neutral ph (7.3). the total herb layer cover is characterised by intermediate values in relation to other rice fi eld phytocoenoses. the mean cover in research plots was about 40%, ranging from ca. 5% to 50% (fig. 3). in central nepal, marsileetum minutae is a community composed of 3–7 species with a clear predominance of the diagnostic species, marsilea minuta. within the study sites the dwarf water clover clearly dominates, reaching up to 50% cover on plot surfaces. the most frequent accompanying taxa of the association are alternanthera sessilis, cyperus difformis and centella asiatica. fig. 3. species richness, shannon diversity index, cover of herb layer, water depth and altitudinal distribution of rice fi eld communities in central nepal. abbreviations: ls – lemno-spirodeletum polyrrhizae, nm – najadetum minoris, cld – community of limnobium dubium, rr – rotaletum rotundifoliae, eta – elatinetum triandro-ambiguae, ml – mazo pumili-lindernietum ciliatae, ap – ammanietum pygmeae, mm – marsileetum minutae, ca – mazo pumili-lindernietum ciliatae caesulietosum axillaris. whiskers present minimum and maximum observations within fences, block indicates fi rst and third quartile, circles the minimum and maximum value. outliers are shown as asterisks. nowak a., nowak s., nobis m. 104 acta bot. croat. 75 (1), 2016 2. najadetum minoris ubrizsy 1961 (tab. 1, rel. 4–9) diagnostic species: najas minor this phytocoenosis occurs rarely in shallow waters of irrigated rice paddies in the surrounding of sauraha village in rapti river valley. it has been noted at the lowest locations, around 200 m a.s.l. najadetum minoris grows in relatively small patches in whole paddy surface in dens as well as in loose rice stands. it prefers warm, shallow and neutral waters with depths of 5 to 15 cm (mean approx. 10 cm; fig. 3, tab. 1). the herb layer of this association is relatively abundant. the mean total cover of vascular plants is approximately 50%, ranging from 15% to almost 90%. however, this is not refl ected in species richness, which is one of the lowest, with a mean value of approx. 4 species per plot. apart from the diagnostic species, the greatest cover and constancy in this phytocoenosis is shared by echinochloa colona, centella asiatica and alternanthera sessilis (tab. 1). 3. elatinetum triandro-ambiguae ass. nova hoc loco (online suppl. tab. 2, rel. 1–17) nomenclatural type: on-line suppl. tab. 2, relevé 1 diagnostic species: elatine ambigua, e. triandra the elatinetum triandro-ambiguae was defi ned by the abundant occurrence of the diagnostic species in rainfed paddies of the kali gandaki, marsyangdi, trisuli and anahi khola rivers near putalibazar, aanbu khaireni, kurintar, bharatpur and siurenitar cities. it was found generally in dried-up or shallowly inundated rice fi elds with very scarce abundance of cultivated plants. the association was noted on sites with mean water depth value close to 0 cm (fig. 3) on loamy and clayey soils with ph ca. 7.0. the herb layer develops to moderate values of approximately 15–85% (mean: ca. 45%; fig. 3; on-line suppl. tab. 2). elatinetum triandro-ambiguae is a community of intermediate species richness, with 4 to 14 taxa per plot (mean approx. 10). centella asiatica, echinochloa colona and dopatrium junceum are the most frequent taxa of this association. 4. lemno-spirodeletum polyrrhizae koch 1954 (tab. 1, rel. 1–3) diagnostic species: spirodela polyrrhiza this phytocoenosis occurs with very rare frequency in central nepal. it was found only in two sites in the rapti river valley (near sauraha village) and in the trisuli river valley (near goganpani village). it was noted at heights of about 217 and 568 m a.s.l. the association of lemno-spirodeletum polyrrhizae develops in the study area in relatively deep waters (15–20 cm; fig. 3) on muddy ground with ph approx. 6.8. it was observed in moderately developed rice stands in early growth phases. the community is characterised by a moderate average herb layer cover. values of 25 to 65% were noted (mean ca. 40%; fig. 3). as in other parts of the association range, the plots are characterised by rather low species richness, however in one plot 10 taxa were noted. apart from the diagnostic plant only polygonum barbatum share was noticeable in research plots. 5. mazo pumili-lindernietum ciliatae ass. nova hoc loco (on-line suppl. tab. 1, rel. 1–39) nomenclatural type: on-line suppl. tab. 1, relevé 17 diagnostic species: lindernia ciliata, mazus pumilus this phytocoenosis appears to be the most frequent in central nepal, especially at lower elevations. as well as in the irrigated fi elds in kali gandaki and rapti river valleys near sauraha and bharatpur, it was noted near jarebagarm goganpani and kurintar in trisuli river valley. this association seems to be most typical for the mid-hills and lowlands of southern asia within the oryzetea sativae vegetation. the community has been noted mainly at elevations of approx. 200 m with the exception of the trisuli valley where the plots have altitudes of approx. 400–600 m a.s.l. (on-line suppl. tab. 1). vegetation patches dominated by lindernia ciliata and mazus pumilus develop on paddies with low water depths (0–10, mean 1 cm), on clayey or loamy muds with neutral reaction (ph approx. 7.0). values of total herb layer cover are moderate in comparison to other rice fi eld associations. the noted mean value was about 35%, ranging from approx. 15% to 65%. the mazo pumililindernietum ciliatae is an association with a relatively high average number of species per plot. approximately 11 plants were noted in each sample (range: 7 to 16). besides the diagnostic taxa, the predominant species are cyperus difformis, alternanthera sessilis, echinochloa colona, fimbristylis miliacea and lindernia procumbens. the subassociation mazo pumili-lindernietum ciliatae typicum is widespread in central nepal on clayey and loamy soils, inundated for a relatively long period by shallow water. the diagnostic species of the subassociation typicum and the nomenclatural type are identical with those of the association. 6. mazo pumili-lindernietum ciliatae caesulietosum axillaris subass. nova hoc loco (on-line suppl. tab. 1, rel. 40– 48) nomenclatural type: on-line suppl. tab. 1, relevé 40 diagnostic species: caesulia axillaris this subassociation appears to be typical for hilly landscapes with rainfed paddies on soils with a relatively high amount of the sand fraction. the plots of this syntaxon was found in the trisuli river valley at a few sites near chuwabote gaun. it was also sporadically noted in lowlands near sauraha in the rapti river valley. the phytocoenosis of mazo pumili-lindernietum ciliatae with the contribution of caesulia axillaris develops on wet, sandy muds without inundation (fig. 3). the soil ph reaction measured near chuwabote gaun was 7.8. the herbaceous layer was not abundant in plots, and had a mean value of approximately 30% (range: approx. 20%–40%). however, the phytocoenoses were relatively rich in species, having more than 10 species per plot on average. the most frequent and abundant taxa apart from the diagnostic ones were: alternanthera sessilis, bacopa procumbens, cyperus iria, scirpus juncoides, fimbristylis miliacea and echinochloa colona. weed communities of rice fields in nepal acta bot. croat. 75 (1), 2016 105 7. rotaletum rotundifoliae ass. nova hoc loco (on-line suppl. tab. 2, rel. 18–33) nomenclatural type: on-line suppl. tab. 2, relevé 20 diagnostic species: rotala rotundifolia the association occurs sporadically in central nepal in the trisuli and seti river valleys (cities of goganpani, aambu khaireni, dhaptar and damauli). it prefers higher elevations in mid-hill landscapes at altitudes of 360 to 400 m a.s.l. (fig. 3). rotaletum rotundifoliae is associated with loamy and clayey muds of ph approx. 6.5. the research plots were sampled in sites of moderate water depth, however some patches were quite deeply inundated. the mean water depth was approx. 4 cm. between 7 and 18 species were recorded in a single plot (mean approx. 9). the phytocoenoses are characterised by a moderate herb layer cover. the maximum cover of weed species in the phytocoenoses was about 75%. the mean cover of whole herbaceous layer was ca. 45%. (fig. 3). the diagnostic rotala rotundifolia has relatively high cover in all plots, reaching up to 50% maximum value (mean approx. 25%). among the most abundant and constant species in the phytocoenosis are alternanthera sessilis, centella asiatica, cynodon dactylon, dopatrium junceum and monochoria vaginalis. several rush species like veronica anagallis-aquatica were also noted in the sampled plots. 8. ammanietum pygmeae ass. nova hoc loco (on-line suppl. tab. 2, rel. 34–42) nomenclatural type: on-line suppl. tab. 2, relevé 34 diagnostic species: ammania pygmea, callitriche palustris var. oryzetorum, dichostylis micheliana the association is a very distinct from other segetal weed communities noted in rice fi elds in central nepal. it is associated mainly with relatively open, loose rice stands with small amounts of water, often completely dried up. in the research area the ammanietum pygmeae was noted in the lowest elevation in chitwan national park vicinity in the rapti river valley (near sauraha, ratnanagar and bharatpur) within the altitudinal range between 184 to 217 m a.s.l. (fig. 3). the association develops on wet and fertile muds in irrigated fi elds in the bottoms of the river valleys. the soil is neutral or slightly acidic (ph 6.4–7.1). plots of the association are relatively rich in species, having from 10–14 taxa (mean approx. 12). the total cover of weed species in the phytocoenoses rarely exceeds 60%, and in most cases it varies about 35% (fig. 3). typical of the observed plots was the insignifi cant cover of the diagnostic species – ammania pygmaea, which reaches the average value of approx. 3%. however this species as well as dichostylis micheliana and callitriche palustris var. oryzetorum contribute only to this phytocoenosis. among other species, the highest constancy is found in echinochloa colona, fimbristylis miliacea, lindernia procumbens, mazus pumilus and vernonia cinerea. the last species is typical rather for ruderal wet wastelands and its occurrence is related to high openness of the ammanietum pygmeae. 9. limnobium dubium community (tab. 1, rel. 10–12) diagnostic species: limnobium dubium (syn. hydrocharis dubia) this community was noted in only 3 spots in the trisulu river valley in the very early stages of rice cultivation. the community was noted at an elevation of ca. 380 m a.s.l. (fig. 3). the species is typical of various kinds of natural as well as artifi cial bodies of water in subtropical and tropical southern and south-eastern asia as well as northern australia (eflora 2014). due to the scarcity of phytosociological data as well as the extreme simplicity of the community, it cannot be defi ned as an association at present state of research. synopsis of syntaxa based on this study, we propose the following classifi cation for the weed communities of rice fi elds in central nepal: class: lemnetea minoris r. tx. 1955 o: lemnetalia minoris r. tx. 1955 all: lemnion gibbae r. tx. et a. schwabe 1974 in r. tx. 1974 1. lemno-spirodeletum polyrrhizae koch 1954 class: potametea klika in klika et novák 1941 o: potametalia koch 1926 all: potamion miljan 1933 2. najadetum minoris ubrizsy 1961 3. limnobium dubium community class: oryzetea sativae miyawaki 1960 o: cypero-echinochloetalia oryzoidis o. bolòs & masclans 1955 all: ludwigion hyssopifolio-octovalvis nowak a., nowak s., nobis m. 2015 4. elatinetum triandro-ambiguae ass. nova 5. mazo pumili-lindernietum ciliatae ass. nova 6. mazo pumili-lindernietum ciliatae caesulietosum axillaris subass. nova 7. rotaletum rotundifoliae ass. nova 8. ammanietum pygmeae ass. nova 9. marsileetum minutae nowak a., nowak s., nobis m. 2015 discussion the rather variable landscape of central nepal, the altitudinal amplitude, climate seasonality, habitat differentiation, various kinds of water supply of the fi elds, with different methods of rice cultivation, promote the development of several segetal phytocoenoses within paddy fi elds. our research reveals a relatively rich weed fl ora if compared to other studies on nepal crop agroceonoses which report ca. 40–60 species (e.g. manandhar et al. 2007, bhatt et al. 2009, sapkota et al. 2010). the richness of rice agrocoenoses is surely related to the extremely rich species pool nowak a., nowak s., nobis m. 106 acta bot. croat. 75 (1), 2016 in the whole country. nepal is known from its extremely rich vascular fl ora and was indicated as one of the world’s biodiversity hotspots (mittermeier et al. 2006). despite the ongoing processes of mechanisation and modernisation of agriculture in nepal including the system of rice intensifi cation (dobermann 2004, uprety 2010), the rice fi eld plots are still abundant and diverse in weed species. this allows phytosociological studies and brings credible data relating species composition and habitat preferences of weed species communities. then, the well developed associations could be easily defi ned and classifi ed in the hierarchical system of the oryztea sativae class (miyawaki 1960, nowak et al. 2013). however, the fi nal appearance of the syntaxonomical system of the class for southern and eastern asia still needs thorough and detailed studies in many regions, e.g. in burma, india, sri lanka, vietnam and other countries. our preliminary investigations in cambodia, southern thailand, malaysia, indonesia and philippines show, that the beta diversity of rice fi eld phytocoenoses is considerably high and reveals many syntaxa still to be described. this of course could cause changes in the diagnostic value of particular species and need of revision of the system. but even without comprehensive data for the whole sw asia region, after our studies we can be sure of the extreme richness of rice fi eld vegetation and fl ora in southern and south-eastern asia in comparison to phytocoenoses known from southern europe, middle asia and japan (e.g. bolòs and masclans 1955, miyawaki 1960, carretero 1989, nowak et al. 2013). obviously the rice phytocoenoses differ substantially in species composition to agrocoenoses known from mediterranean and temperate climates. despite several common taxa diagnostic generally on the order level (cypero-echinochloetalia oryzoidis o. bolòs & masclans 1955) like cynodon dactylon, cyperus difformis or echinochloa crus-galli, almost all other species are different. so, even if we compare relatively areas close to nepal such as tajikistan, the differences in fl oristic composition achieve the level of ca. 80%. but there is also considerable distinctiveness between nepal rice weed fl ora and tropical rice plant communities of southern thailand. the latter is characterised by frequent occurrence of leptochloa chinensis, ipomea aquatica, echinochloa crus-galii, sphenoclea zeylanica, ludwigia hyssopifolia, eclipta prostrata, ludwigia octovalvia, leersia hexandra, hymenachne acutigluma and aeschynomene indica. the only common and also frequent species for both areas are alternanthera sessilis, fimbristylis miliacea, cyperus difformis and echinochloa colona. this could suggest that the nepal rice association should be placed in separate alliance. to decide whether it is reliable to distinguish new alliance closely related to the provisionally proposed ludwigion hyssopifolio-octovalvis further data collection is needed from the whole area of nepal as well as from india, sri lanka, bangladesh and burma. at present the only constant and abundant species for the whole data set gathered in central nepal are mazus pumilus, lindernia ciliata and alternanthera sessilis. but these species have a wide geographical range and also occur in eastern and south-eastern asia, so they demonstrate considerable weaknesses as diagnostic taxa. that is why we decide to classify the described communities within the provisional alliance of ludwigion hyssopifolio-octovalvis proposed for southern thailand (nowak et al. 2015a) even though ludwigia octovalvis and l. hyssopifolia were very scarce in our data set from nepal. the detrended correspondence analysis ordination run for the entire data set clearly segregates the main syntaxa (fig. 2). the comparison of habitat requirements and fl oristic structures of the phytocoenoses found shows that the main dissimilarities in species compositions are due to water depth and trophy of the soil substrate. the left part of the diagram is occupied exclusively by plots of typically aquatic, permanently inundated habitats related to the limnobium dubium community, najadetum minoris and lemnospirodeletum polyrrhizae. in the bottom part of the diagram the samples of relatively species poor phytocoenoses of moderately fertile substrates are grouped. both associations, elatinetum triandro-ambiguae and rotaletum rotundifoliae, were found on brown, loamy and clayey soils of slightly acidic reaction. conversely, species rich phytocoenoses could be found in the upper part of the diagram. they prefer periodically inundated but sometimes dried-up fi elds located generally in lowland river valleys on muddy, fertile, close-to-neutral soils. despite the signifi cant species diversity, these communities, especially ammanietum pygmeae do not achieve high cover of weeds. this shows the close relation of that type of vegetation to early successional stages of associations belonging to the isoëto-nanojuncetea class. altitude is a crucial environmental variable infl uencing the species composition of weed phytocoenoses, particularly in mountainous areas (e.g. lososová et al. 2004, cimalová and lososová 2009, nowak et al. 2015b). although we did not explore in detail the variation of weed vegetation explained by altitude, looking at the species composition it is obvious that elevation above sea level, despite the homogenising effects of agricultural practices, is responsible for a considerable degree of the diversity of central nepal rice phytocoenoses. the higher elevations and hilly colline and montane belts are preferred by the associations of rotaletum rotundifoliae and elatinetum triandro-ambiguae. the apparent differences in climatic conditions between the lowland tropics and montane zone is clearly refl ected in species compositions. several species have found here their ecological optimum, among them cyperus pulcherrimus, lindernia antipoda, l. anagallis or dopatrium junceum. as regards the geography of the described plant communities, at the present state of research it is hardly possible to show their range limits. there is still a lack of credible data regarding the phytosociology and co-ocurrence of rice weeds in southern asia. undoubtedly, the lemno-spirodeletum polyrrhizae koch 1954 and najadetum minoris ubrizsy 1961 are plant communities distributed worldwide. other phytocoenoses noted in the research area have more unique character and presumably occupy subtropical and tropical zones of south-eastern asia. to state precisely to what extent and how frequently, further detailed studies are needed. weed communities of rice fields in nepal acta bot. croat. 75 (1), 2016 107 there is still a need to analyze the weed occurrence in rice paddy fi eld not only in context of agriculture (problems with yield losses, weeding effectiveness), but also considering the conservation issues. looking inside the fl oristic structure of rice phytocoenoses it is obvious that many weeds originated from former marshlands. after transformation of wetland habitats of river valleys into rice paddies, many species were able to adapt to new environments and regular human impact. the sump pond of rice fi elds, canals, drainage ditches could harbour many species known formerly exclusively from natural marshlands. many of them are regarded as rare and listed on the world red list of vascular plants (iucn 2013). generally they are assessed as of least concern, but with the demand for permanent monitoring and evaluation against iucn criteria. in central nepal we noted lindernia ciliata, l. antipoda, l. anagallis, ammannia baccifera, a. multifl ora, dopatrium junceum, ludwigia perennis, l. hyssopifolia, l. octovalvis, caesulia axilliaris, elatine ambigua, eleocharis retrofl exa, lippia no difl ora, cyperus difformis, c. diffusus, c. iria, c. michelianus, c. rotundus, marsilea minuta, monochoria vaginalis, najas minor, nymphaea nouchali in vegetation plots of rice fi elds. it seems to be important to fi nd the relation between the rice fi eld fl ora and natural marshland fl ora, their history, habitat requirements and adaptation potential. the dispute over the recruitment of the isoëto-nanojuncetea species and their spread in the northern hemisphere could be effectively supported by the outcomes of thorough analyses of rice fi eld vegetation and species diversity. then we can precisely ascertain the conservation value of rice paddies for rare and threatened fl ora, as it was suggested in earlier studies (barrett and seaman 1980, camenisch and cook 1996, bambaradeniya et al. 2004). references bajracharya, j., steele, k. a., jarvis, d. i., sthapit, b. r., witcombe, j. r., 2006: rice landrace diversity in nepal: variability of agro-morphological traits and ssr markers in landraces from a high-altitude site. field crops research 95, 327–335. bambaradenyia, c. n. b., edirisinghe, j. p., de silva, d. n., gunatilleke, c. v. s., ranawana, k. b., wijekoon, s., 2004: 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meshing mechanization with sri methods for rice cultivation in nepal. 28th international rice research conference, 8–12 november 2010, hanoi, vietnam. van dusen, e., gauchan, d., smale, m., 2007: on-farm conservation of rice biodiversity in nepal: a simultaneous estimation approach. journal of agriculture economy 58, 242–259. van elsen, t., berg, m., drenckhahn, d., dunkel, f. g., eggers, t., garve, e., kaiser, b., marquart, h., pilotek, d., rodi, d., wicke, g., 2006: karlstadter positionspapier zum schutz der ackerwildkräauter. zeitschrift für planzenkrankheiten und pfl anzenschutz 20, 527–533. weber, h. e., moravec, j., theurillat, j.-p., 2000: international code of phytosociological nomenclature. journal of vegetation science 11, 739–768. weed communities of rice fields in nepal acta bot. croat. 75 (1), 2016 o nlin e su pp l. ta b. 1 . c om m un iti es o f t he o ry ze te a sa tiv ae c la ss (p ar t i ) d ur in g th e st ud y, in m ay 2 01 3. m l – m az o pu m ili -l in de rn ie tu m c ili at ae , m l c -m az o pu m ili -l in de rn ie tu m c ili at ae c ae su lie to su m a xi lla ri s. t he c ov er ab un da nc e sc al e of b ra un -b la nq ue t ( 19 64 ) w as u se d : r = 1 o r 5 in di vi du al s; + = fe w in di vi du al s (< 2 0) w ith c ov er < 5 % ; 1 = m an y in di vi du al s (2 0– 1 00 ) w ith c ov er < 5 % ; 2 = 5 % –2 5% c ov er ; 3 = 2 5% –5 0% c ov er ; 4 = 5 0% – 75 % c ov er ; 5 = 7 5% –1 00 % c ov er . su cc es si ve n um be r o f r el ev é 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 constancy constancy d ay o f t he m on th 7 6 3 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 w at er d ep th (c m ) 0 0 10 0 0 1 0 0 0 1 1 1 2 2 0 1 1 3 2 4 0 0 0 0 3 4 5 2 0 0 0 0 0 2 2 1 3 2 2 0 0 0 0 0 0 0 0 0 a lti tu de (m ) 39 4 21 7 56 8 21 7 21 7 21 7 21 7 21 7 21 7 21 7 21 7 21 7 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 27 3 27 3 19 0 19 0 19 0 37 7 37 7 37 7 37 7 37 7 37 7 c ov er o f h er b la ye r ( % ) 55 60 35 45 60 60 20 55 40 45 30 60 30 45 50 35 70 55 20 30 60 50 55 35 35 30 40 20 40 50 60 70 70 40 50 30 20 70 70 30 30 50 20 35 40 60 15 45 r el ev é ar ea (m 2 ) 3 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 5 5 5 5 5 5 ph 6, 8 – – 7, 0 7, 2 – 7, 3 – – 7, 0 – 6, 9 7, 3 – 7, 2 7, 2 6, 9 – 7, 2 – 7, 0 6, 9 – – 7, 2 6, 9 – – – 6, 9 7, 2 – 7, 0 – 7, 2 7, 0 – – 7, 8 7, 8 7, 8 7, 8 – – 7, 6 – – n um be r o f w ee ds 10 9 8 9 11 11 12 13 10 11 12 13 11 11 9 11 8 9 7 9 9 8 9 8 13 12 11 11 11 14 11 10 9 9 10 12 11 15 16 11 14 14 11 11 11 10 13 12 re l. re l. a ss oc ia tio n/ c om m un ity m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l c m l c m l c m l c m l c m l c m l c m l c m l c 1– 39 40 –4 8 c ul ti va te d pl an t o ry za s at iv a 2 3 3 4 3 3 4 3 4 4 4 3 4 4 4 3 3 3 4 4 3 3 3 4 3 4 4 4 4 4 3 3 3 4 3 4 4 2 3 4 4 4 4 3 2 1 3 3 a ss . m az o pu m ili -l in de rn ie tu m c ili at ae m az us p um ilu s 1 3 + 2 2 1 1 1 1 1 + 1 1 1 3 2 1 1 + 1 2 3 3 2 1 1 + + . + 1 1 + 1 1 + + + 1 1 + + + + + 1 + . v v li nd er ni a ci lia ta . . . . + 1 1 3 2 3 2 1 1 1 + + 2 1 1 2 2 1 1 1 + . + 1 1 + + 1 2 1 1 + 1 1 + + + 1 + . . . + + v iv su ba ss . m az o pu m ili -l in de rn ie tu m c ili at ae c ae su lie to su m a xi lla ri s c ae su lia a xi lli ar is . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . 1 + 1 1 2 + 1 1 2 i v a ll. l ud w ig io n hy ss op ifo lio -o ct ov al vi s a lte rn an th er a se ss ili s + + 1 1 1 + + + 1 1 + . 1 2 + 1 2 3 1 + + 1 + 1 1 + 2 1 1 1 1 . . 1 + + 1 1 + 1 1 2 + 1 1 + + 1 v v f im br is ty lis m ili ac ea . + 1 1 + 1 + 1 + + 1 + . . . . 1 + . . . . . . . + . . 1 + + . . . . . . 1 1 + 2 1 1 1 + + . . ii i iv m ar si le a m in ut a . . + . . . . . . . + . . + . . . + . + . + + . . . . . . . . + + . . . . . . . . . . . . . . + ii i sp he no cl ea z ey la ni ca . . . . . . . . . . . . . . . . . . . . . . . . + + + 1 . . . . . 2 + 1 + . . . . . . . . . . . ii – e la tin e tr ia nd ra 1 . . . . . . . . . . . . . . . . . . . . . . . . . 1 + . . . . . + + . + . . . . . . . . . . . i – c yp er us k yl lin gi a . + 1 1 . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – lu dw ig ia p er en ni s . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . + . . + . . . . . . . . . . . . i – a lte rn an th er a ph ilo xe ro id es . . . . . . . . . . . . . . . . + . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . i – sp or ad ic s pe ci es : c al lit ri ch e pa lu st ri s va r. or yz et or um 3 8( +) . o . c yp er oe ch in oc hl oe ta lia o ry zo id is e t c l. o ry ze te a sa tiv ae c yp er us d iff or m is 1 2 2 1 2 1 1 1 + . . 3 + 1 1 1 2 1 + + 1 1 2 1 1 + + . + 1 1 . . 1 + 2 1 1 3 1 + + . . 2 2 . . v ii i e ch in oc hl oa c ol on a . . . . . . . . . . . + + 1 + 1 . . . + + . + + + 1 1 + 1 2 3 2 2 . . + + + 1 + + 1 . + + 1 + ii i iv c yp er us ir ia 1 . . . 1 + . . . + . . . . . . . . . . . . . . . . . . . 2 1 1 2 1 2 1 1 3 2 . + . + . 1 . 1 1 ii ii i sc ir pu s ju nc oi de s + . . . . . . + . . . . . . . . . . . . . . . . . . . . + . . . . . . . . 1 1 + . . + + 1 2 + 1 i iv a m m an ni a ba cc ife ra . . . . . . . . . 1 + 2 . . 1 + . . . . . . . . 1 + 1 1 . . . . . . . . . . . . . . . . . . + ii i a m m an ni a m ul tifl o ra . . . . . . . . . . . . . . . + . . . . . . . . + + + + . . . . . . . + . . + . + . . . . . . i ii m on oc ho ri a va gi na lis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . . . . 1 2 + + . + 1 . . . . . . . . i i sy ne dr el la n od ifl or a + . . . . . . . . . . . . . . + . . . . . . . + . . . . . . . . . . . . . . . . . . + + . . 1 + i ii i p hy lla nt he s fr at er ne s . . . . . . + . . + + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 . . . . . . i ii le pt oc hl oa c hi ne ns is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 + 3 2 . . + . . . . . . . . . . . . i – 1 nowak a., nowak s., nobis m. acta bot. croat. 75 (1), 2016 su cc es si ve n um be r o f r el ev é 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 constancy constancy d ay o f t he m on th 7 6 3 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 w at er d ep th (c m ) 0 0 10 0 0 1 0 0 0 1 1 1 2 2 0 1 1 3 2 4 0 0 0 0 3 4 5 2 0 0 0 0 0 2 2 1 3 2 2 0 0 0 0 0 0 0 0 0 a lti tu de (m ) 39 4 21 7 56 8 21 7 21 7 21 7 21 7 21 7 21 7 21 7 21 7 21 7 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 20 5 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 19 0 27 3 27 3 19 0 19 0 19 0 37 7 37 7 37 7 37 7 37 7 37 7 c ov er o f h er b la ye r ( % ) 55 60 35 45 60 60 20 55 40 45 30 60 30 45 50 35 70 55 20 30 60 50 55 35 35 30 40 20 40 50 60 70 70 40 50 30 20 70 70 30 30 50 20 35 40 60 15 45 r el ev é ar ea (m 2 ) 3 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 5 5 5 5 5 5 ph 6, 8 – – 7, 0 7, 2 – 7, 3 – – 7, 0 – 6, 9 7, 3 – 7, 2 7, 2 6, 9 – 7, 2 – 7, 0 6, 9 – – 7, 2 6, 9 – – – 6, 9 7, 2 – 7, 0 – 7, 2 7, 0 – – 7, 8 7, 8 7, 8 7, 8 – – 7, 6 – – n um be r o f w ee ds 10 9 8 9 11 11 12 13 10 11 12 13 11 11 9 11 8 9 7 9 9 8 9 8 13 12 11 11 11 14 11 10 9 9 10 12 11 15 16 11 14 14 11 11 11 10 13 12 re l. re l. a ss oc ia tio n/ c om m un ity m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l m l c m l c m l c m l c m l c m l c m l c m l c m l c 1– 39 40 –4 8 e cl ip ta p ro st ra ta . . . . . . . . . + + . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . + . . + i ii li nd er ni a an ag al lis . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 + . . . . . . . . . i – li nd er ni a hy ss op oi de s . . . . . . . . . . . . . . + . . . . . . . . + . . . . . . . . . . . . . . + . . . . . . . . . i – h ed yo tis d iff us a . . . . . . . . . . . . . . . . . . . + . . . . . + . . . + . . . . . . . . . . . . . . . . . . i – sp or ad ic s pe ci es : d op at ri um ju nc eu m 1 (3 ); e ch in oc hl oa c ru sga lli 3 0( +) ; p as pa lu m d is tic hu m 5 (+ ). a cc om pa ny in g sp ec ie s c yn od on d ac ty lo n . 1 + + + 1 + . . . . + . + . + . + 1 + 2 1 + . 1 . . . . . . 1 + . . . . . . . . . 1 + . . . . ii i ii ve ro ni ca a na ga lli saq ua tic a + 1 . + 1 2 + + 1 . + . . . . . . . + . . . . . . . + . . . . . . . . + . . . . . . 1 + 1 1 + 1 ii iv b ac op a pr oc um be ns . . . . . . + + 1 . . + + 1 . . . . . . + + . . + + . . + . . . . . . . . . . 1 . . + 1 + + + + ii iv li nd er ni a pr oc um be ns 1 1 . 2 2 2 1 1 + + 1 . . . . . . . . . . . . . . . . . . 1 1 . . . . . . + 1 . + 1 . . . . . . ii ii c en te lla a si at ic a . . . . . . . . . . . . . . 1 + + + + 1 + . + . 1 2 1 + . . . . . . . . . + + . + + . . . . . . ii ii c om m el in a di ffu sa . . . . . . . . . . . . . . . . . . . . . . . . + + 1 + . 1 + 1 1 . + . . . . . + + . . . . . . ii ii r an un cu lu s sc el er at us . + . + . + + 1 + + . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . ii – ve rn on ia c in er ea . . . . . . . . . . . + . . . . . . . . . . . . + . . . + . . . . . . . . 1 + . . . . + . + . . i ii sp ila nt he s ia ba di ce ns is . . . . . . + . . . . . 2 1 . . . . . . + . + . . . . . . + . . . . . . . . . . . . . . . . . . i – c ar yo ph yl la ce ae s p. . . . . . . . . . 1 + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . i ii vi ci a hi rs ut a . . . . . . . + + . . . + + . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . i – li pp ia n od ifl or a . . . . . . . . . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 1 i ii c he no po di um a lb um . . . . . . . . . . + . + . 1 + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – n aj as m in or . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . + + . + . . . . . . . . . . . i – sp ir od el a po ly rr hi za . . . . . . . . . . . + . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – p ol yg on um b ar ba tu m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 + . . . . . . . . . i – p lu ch ea in di ca . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 1 . . . . . . – ii li m no bi um d ub iu m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . i – e ch in oc hl oa g la br es ce ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + – ii sp or ad ic s pe ci es : c ol oc as ia e sc ul en ta 3 2( +) ; c yp er us o do ra tu s 30 (+ ); c yp er us ro tu nd us 1 3( +) ; j un cu s pr is m at oc ar pu s 5( +) ; n ym ph ae a no uc ha li 12 (+ ); p ol yg on um fl ac ci du m 3 (+ ); v er on ic a be cc ab un ga 8 (+ ). l oc al it y of r el ev é: 1 – (2 74 75 0, 2; 8 45 53 4) ; 2 , 4, 5 , 6 , 7 , 8 , 9 , 1 0, 1 1, 1 2 – (2 73 45 4, 5; 8 42 94 1, 6) ; 3 – (2 74 54 8, 3; 8 50 24 4, 4) ; 1 3, 1 4, 1 5, 1 6, 1 7, 1 8, 1 9, 2 0, 2 1, 2 2, 2 3, 2 4, 2 6, 2 8, 2 9, 3 1, 3 3, 3 5, 3 7, 4 1 – (2 73 45 9, 1; 8 42 93 7, 5) ; 2 5, 2 7, 3 0, 3 2, 3 4, 3 6, 4 0, 4 2 – (2 73 52 2, 3; 8 42 95 4, 5) ; 3 8 – (2 75 24 1, 2; 8 43 54 5, 5) ; 39 – (2 73 75 3, 4; 8 42 93 9, 7) ; 4 3, 4 4, 4 5, 4 6, 4 7, 4 8 – (2 74 80 4, 5; 8 45 20 3, 8) ; 2 weed communities of rice fields in nepal acta bot. croat. 75 (1), 2016 o nlin e su pp l. ta b. 2 . c om m un iti es o f t he o ry ze te a sa tiv ae c la ss (p ar t i i) d ur in g th e st ud y, in m ay 2 01 3. r r – r ot al et um ro tu nd ifo lia e, e ta – e la tin et um tr ia nd ro -a m bi gu ae , a p – a m m an ie tu m p yg m ea e, m m – m ar si le et um m in ut ae . t he c ov er -a bu nd an ce s ca le o f b ra un -b la nq ue t ( 19 64 ) w as u se d : r = 1 o r 5 in di vi du al s; + = fe w in di vi du al s (< 2 0) w ith c ov er < 5 % ; 1 = m an y in di vi du al s (2 0– 1 00 ) w ith c ov er < 5 % ; 2 = 5 % –2 5% c ov er ; 3 = 25 % –5 0% c ov er ; 4 = 5 0% –7 5% c ov er ; 5 = 7 5% –1 00 % c ov er . su cc es si ve n um be r o f r el ev é 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 constancy constancy constancy number of occurrence d ay o f t he m on th 5 5 5 5 5 5 7 7 7 7 7 7 7 7 7 7 7 7 7 3 3 3 3 3 3 3 3 3 3 3 3 3 3 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 w at er d ep th (c m ) 1 0 0 0 5 7 15 7 0 1 0 0 0 0 0 1 0 1 3 5 10 2 3 1 3 15 13 1 2 6 8 5 8 1 0 3 4 0 0 0 0 0 3 8 0 0 10 10 a lti tu de (m ) 46 8 46 8 46 8 46 8 46 9 46 9 27 3 27 3 27 3 27 3 35 0 35 0 39 4 39 4 39 4 27 3 27 3 39 4 39 4 56 8 56 8 36 8 36 8 36 8 36 8 36 5 36 5 36 5 36 5 36 5 36 5 36 5 36 5 21 7 21 7 21 7 21 7 21 7 18 4 18 4 18 6 18 6 20 5 20 5 37 7 37 7 20 5 20 5 c ov er o f h er b la ye r ( % ) 85 90 70 45 50 50 90 60 10 40 55 30 70 20 50 45 45 30 35 70 50 75 90 80 60 80 70 45 40 65 50 45 35 40 55 55 70 60 40 20 20 25 25 40 40 50 40 25 r el ev é ar ea (m 2 ) 20 20 15 25 25 25 25 25 3 3 3 3 3 3 3 3 3 5 5 25 25 25 25 25 25 25 25 25 25 25 25 25 25 10 10 5 10 5 20 15 10 25 25 25 5 5 5 5 ph 6, 8 7, 0 7, 2 7, 2 6, 8 7, 2 7, 0 6, 8 6, 8 7, 1 7, 2 7, 1 7, 0 7, 0 7, 2 7, 0 6, 8 6, 4 6, 7 6, 7 6, 4 6, 5 7, 0 – 6, 5 6, 5 6, 7 – 6, 8 6, 7 6, 6 6, 5 6, 4 6, 8 6, 4 7, 1 7, 0 6, 9 6, 9 6, 5 6, 5 – 7, 3 7, 3 7, 0 7, 0 7, 0 7, 0 n um be r o f w ee ds 9 12 13 13 13 14 10 8 7 9 10 10 9 10 8 4 4 9 12 16 11 11 11 15 18 9 8 9 7 8 8 9 10 13 13 14 12 14 11 11 11 10 7 3 5 5 3 3 re l. re l. re l. re l. a ss oc ia tio n/ c om m un ity e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r a p a p a p a p a p a p a p a p a p m m m m m m m m m m m m 1– 17 18 –3 3 34 –4 2 44 –4 8 c ul tiv at ed p la nt o ry za s at iv a 2 2 3 3 3 4 2 3 3 3 3 3 3 3 3 3 2 3 3 3 3 2 2 2 3 2 3 3 3 3 3 3 3 3 2 3 3 3 3 3 4 4 3 3 2 2 3 3 v a ss . e la tin et um tr ia nd ro -a m bi gu ae e la tin e tr ia nd ra 4 4 1 1 + . 4 3 1 1 3 2 2 1 2 3 3 + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i – – e la tin e am bi gu a 2 3 3 2 1 2 . . . 1 . . 1 + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii i – – – a ss . r ot al et um r ot un di fo lia e r ot al a ro tu nd ifo lia . . . . . . . . . . . . . . . . . 2 2 3 2 1 2 1 1 2 3 1 2 2 1 1 + . . . . . . . . . . . . . . . – v – – a ss . a m m an ie tu m p yg m ea e a m m an ia p yg m ae a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . 1 + + 1 1 + . . . . . . – – iv – d ic ho st yl is m ic he lia na . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 2 3 1 2 + . . . . . . . . . – – iv – c al lit ri ch e pa lu st ri s v ar . o ry ze to ru m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 + + + . . . + . . . . . . – – ii i – a ss . m ar si le et um m in ut ae m ar si le a m in ut a . . . . . . . . . . . . . . . . . . . 2 + . . + 1 . . . . . . + . . . . . . . . . . 2 3 3 3 3 2 ii – 6 a ll. l ud wi gi on h ys so pi fo lio -o ct ov al vi s a lte rn an th er a se ss ili s + + + 1 + 1 1 + + 1 + + 2 1 . + + 1 1 1 + . . + 1 + 1 1 1 . + . . . . . . . 2 1 1 1 + + + 1 . . v iv ii i 4 m az us p um ilu s . + . + . . . . . . . . . . + + . . . . . + 1 1 + . . + . . . . . + + 1 1 + 1 + + 1 . . . . . . ii ii v – f im br is ty lis m ili ac ea . . . + . . . . + + . . . . . . . . . + + + + . . . . . . + . . + . + + + 1 + 1 . . . . + 1 . . i ii iv 2 c yp er us k yl lin gi a . . . . . . . . . . . . . . . . . . . . + . . + + . . . . . . . . . . . . . . . + . . . . . . . – i i – lu dw ig ia p er en ni s . . . . . . . . . + . . . . . . . . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . i i – – li nd er ni a ci lia ta . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . i – i – sp or ad ic s pe ci es : l ud w ig ia h ys so pi fo lia 2 0( 1) ; l ud w ig ia o ct ov al vi s 20 (+ ). o . c yp er oe ch in oc hl oe ta lia o ry zo id is e t c l. o ry ze te a sa tiv ae c yp er us d iff or m is . . + . . . 1 2 . . 2 1 1 + 1 . . 1 1 1 1 . . 3 2 . . . . . . . . 2 + 1 . . . . + 1 1 + + . . . ii i ii ii i 3 e ch in oc hl oa c ol on a . . . . . . + . + . + + + + 1 + + . . . . . . . . . . . . . 1 2 1 1 1 1 + + . . . . . 1 + ii i i v 2 d op at ri um ju nc eu m + 1 1 + + + . . . . . . 1 + 2 . . . . . . . . + + 1 1 2 1 2 2 . . . . . . . . . . . . . . . . . ii i ii i – – m on oc ho ri a va gi na lis + + . . 3 2 . . . . . . . . . . . . . . . . . 1 + 1 + + + + + . . . . . . . + + . . . . . . ii ii i ii – c yp er us ir ia . . . . . + + . . . . . . + + . . + 1 + . . . . . . . . . . . . . . . . + 1 + . 1 1 . . . . . . ii i ii i – li nd er ni a an ag al lis . . . . . . + + 1 2 . . . . . . . . . + + 2 1 1 + . . . . . . . . . . . . . . . . . . . . . . . ii ii – – li nd er ni a an tip od a + 1 . + + . . . . . . . . . . . . . . . . . . . . . . . . + + 1 2 . . . . . . . . . . . . . . . ii ii – – e cl ip ta p ro st ra ta . . 1 + . . + + . . . . . . . . + . . . . . . . + . . . . . . . . . . . + + . . . . . . . . . . ii i ii – sc ir pu s ju nc oi de s . . + + 1 1 1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii – – – p hy lla nt he s fr at er ne s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + 1 . . + + . . + . . . . . – – ii i 1 3 nowak a., nowak s., nobis m. acta bot. croat. 75 (1), 2016 su cc es si ve n um be r o f r el ev é 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 constancy constancy constancy number of occurrence d ay o f t he m on th 5 5 5 5 5 5 7 7 7 7 7 7 7 7 7 7 7 7 7 3 3 3 3 3 3 3 3 3 3 3 3 3 3 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 w at er d ep th (c m ) 1 0 0 0 5 7 15 7 0 1 0 0 0 0 0 1 0 1 3 5 10 2 3 1 3 15 13 1 2 6 8 5 8 1 0 3 4 0 0 0 0 0 3 8 0 0 10 10 a lti tu de (m ) 46 8 46 8 46 8 46 8 46 9 46 9 27 3 27 3 27 3 27 3 35 0 35 0 39 4 39 4 39 4 27 3 27 3 39 4 39 4 56 8 56 8 36 8 36 8 36 8 36 8 36 5 36 5 36 5 36 5 36 5 36 5 36 5 36 5 21 7 21 7 21 7 21 7 21 7 18 4 18 4 18 6 18 6 20 5 20 5 37 7 37 7 20 5 20 5 c ov er o f h er b la ye r ( % ) 85 90 70 45 50 50 90 60 10 40 55 30 70 20 50 45 45 30 35 70 50 75 90 80 60 80 70 45 40 65 50 45 35 40 55 55 70 60 40 20 20 25 25 40 40 50 40 25 r el ev é ar ea (m 2 ) 20 20 15 25 25 25 25 25 3 3 3 3 3 3 3 3 3 5 5 25 25 25 25 25 25 25 25 25 25 25 25 25 25 10 10 5 10 5 20 15 10 25 25 25 5 5 5 5 ph 6, 8 7, 0 7, 2 7, 2 6, 8 7, 2 7, 0 6, 8 6, 8 7, 1 7, 2 7, 1 7, 0 7, 0 7, 2 7, 0 6, 8 6, 4 6, 7 6, 7 6, 4 6, 5 7, 0 – 6, 5 6, 5 6, 7 – 6, 8 6, 7 6, 6 6, 5 6, 4 6, 8 6, 4 7, 1 7, 0 6, 9 6, 9 6, 5 6, 5 – 7, 3 7, 3 7, 0 7, 0 7, 0 7, 0 n um be r o f w ee ds 9 12 13 13 13 14 10 8 7 9 10 10 9 10 8 4 4 9 12 16 11 11 11 15 18 9 8 9 7 8 8 9 10 13 13 14 12 14 11 11 11 10 7 3 5 5 3 3 re l. re l. re l. re l. a ss oc ia tio n/ c om m un ity e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta e ta r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r a p a p a p a p a p a p a p a p a p m m m m m m m m m m m m 1– 17 18 –3 3 34 –4 2 44 –4 8 c yp er us p ul ch er ri m us . + 2 1 + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii – – – f im br is ty lis s to lo ni fe ra . . . . . . . . . . . . . . . . . . . . . 1 + 1 + . . . . . . . . . . . . . . . . . . . . . . . – ii – – h ed yo tis d iff us a . . . . . + 1 + . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . i i – – sy ne dr el la n od ifl or a . . . . . . . . . . 1 + . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – a m m an ni a ba cc ife ra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . + + . . . . . . . . . . – – ii – a m m an ni a m ul tifl o ra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . + . . . . . . . . – – ii – h ym en ac hn e ac ut ig lu m a . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – sp or ad ic s pe ci es : e ch in oc hl oa o ry zo id es 2 1( +) ; e le oc ha ri s re tr ofl e xa 3 6( +) ; l ep to ch lo a ch in en si s 40 (+ ). a cc om pa ny in g sp ec ie s c en te lla a si at ic a 1 + 1 1 1 + 2 1 + 1 . . 1 1 . . . . . . . . . + + 3 2 1 + 2 2 2 1 . . . . . . . + + + . . . + 1 iv iv ii 3 c yn od on d ac ty lo n . + + + + + . . . . . . . . . . . . + . 1 2 3 + 1 + + + 1 . . + 1 + + + . . . . . . + . + + . . ii iv ii 3 li nd er ni a pr oc um be ns . . . . . . . . . . + + 2 1 1 . . + + . . . . . . . . . . . . . . . 1 + 2 1 1 + 1 1 . . . . . . ii i v – ve ro ni ca a na ga lli saq ua tic a . . . . . + . . . . . . . . . . . . . . . . . . + . . 1 1 1 + 1 1 + 1 . . . . . . . . . . . . . i ii i ii – ve rn on ia c in er ea . . . . . . . . . . . + . . + . . . + . . . . . . . . . . . . . . + + 1 . + 1 1 . . . . . + . . i i iv 1 ju nc us p ri sm at oc ar pu s . . + 1 1 + . . . . . . . . . . . . . . . 1 + 1 + . . + . . . . . . . . . . . . . . . . . . . . ii ii – – p ol yg on um v is co su m 1 + . . . . . . . . . . . . . . . . . . . . . + . . . . . + 1 1 + . . . . . . . . . . . . . . . i ii – – b ac op a pr oc um be ns . . . . . . . . . . + + . . . . . + + . . . . . . . . . . . . . . 1 . . + 1 . . . . . . . . . . i i ii – c om m el in a di ffu sa . . . . . . . . . . + + 1 + . . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii i – – ve ro ni ca b ec ca bu ng a . . . . . . . . . . . . . . . . . . . + 2 2 3 3 2 . . . . . . . . . . . . . . . . . . . . . . . – ii – – sp ila nt he s ia ba di ce ns is + . . . . . . . . . . . . . . . . . . . . + . . + + . . . . . 1 + . . . . . . . . . . . . . . . i ii – – p ol yg on um b ar ba tu m . . . . . . . . . + + 1 . . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i – – n aj as m in or . . . . . . . . . . . . . . . . . . . . . . . . + 1 1 . . . . . + . . . . . . . . . . . . . . . – ii – – p ol yg on um fl ac ci du m . . . . . . . . . . . . . . . . . . . + + . . . + . . . . . . . . . . . . . . . . . . . . . . . – i – – c ar yo ph yl la ce ae s p. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . + + . . . . . . – – ii – c yp er us d iff us us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 . . . . . . . . . . – – ii – a lg ae in de t. . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 . . . . . . . . . . . . . . . . . . . . . – i – – le er si a he xa nd ra . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – a re na ri a se rp yl lif ol ia . . . . . . . . . . . . . . . . . . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . . . . – i – – h yp er ic um ja po ni cu m . . . . . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – r um ex d en ta tu s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . + . . . . . . . . . . – – ii – vi ci a hi rs ut a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . . – – ii – e up ho rb ia h ir ta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . – – ii – sp or ad ic s pe ci es : a zo lla fi lic ul oi de s 20 (+ ); b lu m ea la ci ni at a 20 (+ ); c yp er us ro tu nd us 4 3( +) ; h ed yo tis r ac em os a 3( +) ; l im no ch ar is fl av a 20 (1 ); l in de rn ia v is co sa 2 (+ ); p or tu la ca o le ra ce a 11 (+ ); s pi ro de la p ol yr rh iz a 20 (1 ); v al lis ne ri a sp ir al is 2 4( +) . l oc al it y of r el ev é: 1 , 4 , 6 , 2 2, 2 3, 2 4, 2 5, 2 6, 2 9, 3 1, 3 3 – (2 75 44 3, 3; 8 43 11 2, 1) ; 2 , 3 , 5 – (2 80 63 7, 2; 8 35 23 5, 2) ; 7 , 1 0, 1 6, 4 1, 4 2 – (2 73 75 3, 4; 8 42 93 9, 7) ; 8 , 9 , 1 7 – (2 75 24 1, 2; 8 43 54 5, 5) ; 1 1, 1 2 – (2 74 84 9, 1; 8 44 71 3, 9) ; 1 3, 1 4, 1 5, 1 8, 1 9 – (2 74 75 0, 2; 84 55 34 ); 2 0, 2 1 – (2 74 54 8, 3; 8 50 24 4, 4) ; 2 7, 2 8, 3 0, 3 2 – (2 75 91 6, 1; 8 41 64 9, 9) ; 3 4, 3 5, 3 6, 3 7, 3 8 – (2 73 45 4, 5; 8 42 94 1, 6) ; 3 9, 4 0 – (2 73 61 6, 5; 8 43 03 8, 3) ; 4 3, 4 4, 4 7, 4 8 – (2 73 45 9, 1; 8 42 93 7, 5) ; 4 5, 4 6 – (2 74 80 4, 5; 8 45 20 3, 8) . 4 acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 43 acta bot. croat. 74 (1), 43–52, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 variability of pollen aperture heteromorphism in annual pansies (viola section melanium) sa ra magrini1,*, anna scoppola2 1 herbarium utv, tuscia university, via san camillo de lellis, i:01100 viterbo, italy 2 department of agriculture, forests, nature and energy (dafne), tuscia university, via san camillo de lellis, i:01100 viterbo, italy abstract – pollen heteromorphism is frequent in the section melanium of the genus viola, in which over 80% of the species produces pollen morphs with 3 to 6 apertures. some authors have pointed out that many factors can affect the proportion of the different pollen types in perennial species, and that this proportion can change among populations. this work focuses on the study of the polymorphic pollen assemblage of three annual pansies: viola arvensis, v. kitaibeliana, and v. hymettia, and on the assessment of its variability both within a population and within the same plant. in all the species, with both large and small fl owers, 3-, 4and 5-aperturate pollen grains were observed, with a large prevalence of 4-aperturate types. no pollen grains with 6 apertures were found. no signifi cant variability of the pollen assemblage among fl owers of the same plant was observed. in addition, in these three viola species the frequencies of the various pollen morphs are also fairly constant among plants of the same population. keywords: annual pansies, pollen morph, pollen heteromorphism, section melanium, variability, viola introduction viola l. section melanium ging. is a derived, monophyletic and morphologically welldefi ned group of about 80–100 perennial and annual species showing highly-reduced genetic divergence (yockteng et al. 2003). its geographical distribution extends over europe and westernmost asia, with a few species in northern africa and one disjointed and probably native species in north america (clausen et al. 1964, yockteng et al. 2003). pollen heteromorphism, defi ned as the production of several pollen grain morphs with different aperture numbers by the same plant (till-bottraud et al. 1995), occurs in over 30% of angiosperm species (mignot et al. 1994). it is particularly common in this section of the genus viola, in which over 80% of the species produce pollen morphs with 3 to 6 * corresponding author, e-mail: magrini@unitus.it copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. magrini s., scoppola a. 44 acta bot. croat. 74 (1), 2015 apertures (dajoz et al. 1993, till-bottraud et al. 1999, nadot et al. 2000). generally, 4or 5-aperturate pollen grains represent more than 85% and very often more than 95% (tillbottraud et al. 1999). the most frequent pollen type is always 4-aperturate for perennial pansies and for typically large-fl owered annuals like viola tricolor l., v. hymettia boiss. & heldr., and v. roccabrunensis espeut (clapham et al. 1987, till-bottraud et al. 1999, espeut 2004), as it is related to entomophilous pollination. on the other hand, in v. arvensis murray and v. kitaibeliana schult. 5-aperturate pollen grains prevail (pettet 1964, clapham et al. 1987, randall 2004), which is related to autogamous pollination strategy. still, 4-aperturate pollen grains were also observed by other authors (erdtman 1952, dajoz et al. 1995, espeut 1999). dajoz et al. (1991), mignot et al. (1994) and till-bottraud et al. (1999) pointed out that many genetic and environmental factors can affect the proportion of the different pollen types in heteromorphic perennial species, and that this can change among populations. on the other hand, to our knowledge, studies of the polymorphic pollen assemblage of annual pansies and the assessment of its variability within a population, or within the same plant, have not been previously reported in literature. furthermore, scoppola and lattanzi (2012), in accordance with kristofferson (1923) and erben (1985), observed an increasing reduction in the fl ower size from the lower to the upper parts of the specimen in all the analyzed species, both autogamous and heterogamous, and in v. arvensis and v. tricolor also from the main axis to the branches. as pollen heteromorphism is related to pollination strategy, can the main pollen type change within the same plant in such variable fl owers? the aims of this paper were: 1) to analyze the pollen assemblage of three annual pansies, viola arvensis, v. kitaibeliana and v. hymettia; 2) to assess its variability among fl owers within the same plant and among plants within the same population; and particularly, 3) to assess whether the main pollen type is directly correlated with the increasing reduction of the fl ower size in those species, like v. arvensis, that show both autogamous and heterogamous pollination, and in entomophilous species like v. hymettia. materials and methods study species viola arvensis (2n = 34), the fi eld pansy, is the most common of the annual species closely related to v. tricolor, the wild pansy. it is a mediterranean-eurasiatic element, regarded as an archaeophyte and widespread throughout almost the whole of europe and sw asia, from 0 to 1,500–1,800 m a.s.l., and as a weed linked to open shrubland and synanthropic habitats (gams 1926, valentine et al. 1968, marcussen and karlsson 2010, vollrath 2011). the corolla is shorter than or equal to the calyx (or, rarely, a little longer), usually pale creamy-yellow (fig. 1a). it is a mostly autogamous species with a very small stylar fl ap (labellum) and the entrance of the stigmatic cavity, in a front view, is obliquely forward directed (scoppola and lattanzi 2012). viola kitaibeliana (2n = 16), the dwarf pansy, is a mediterranean-caucasian species which extends to central europe where it is a component of early stages of grassland, stony slopes and screes, found also on sandy soils, fallow land, fi elds and other open places, from 0 to approximately 1,850 m a.s.l. (werner 1988, randall 2004, vollrath 2011, magrini variability of pollen heteromorphism in annual pansies acta bot. croat. 74 (1), 2015 45 and scoppola 2013). it is a small-fl owered and mostly autogamous species, with a corolla cup-shaped, not (or lightly) exceeding the calyx (fig. 1b); the stylar fl ap is absent and the entrance of the stigmatic cavity, in a front view, is obliquely forward directed (erben 1985, scoppola and lattanzi 2012). viola hymettia (2n = 16) is a se-european species which is widespread mainly in greece and the aegean islands, in central and southern italy, where it has a disjointed distribution, and in sicily; it is an early element of stony pastures, dry open habitat and scrub fringes, occurring from 200 to 800–1,000 m a.s.l. (merxmüller 1982, erben 1985, raus 1986, davis et al. 1988). this relatively large-fl owered species has a corolla distinctly exceeding its calyx, creamy and yellow colored, often suffused with violet (fig. 1c). the stigmatic opening, in a front view, is obliquely upwards directed (scoppola and lattanzi 2012). collection and study sites seedlings of v. arvensis and v. kitaibeliana were collected in wild populations in central italy, in february and march 2013, respectively (tab. 1). they were transplanted in pots, together with the soil collected in situ, and cultured ex situ until may 2013. viola hymet tia plants were studied in situ from january to april 2013. about 100 specimens belonging to these taxa were used for palynological investigations. samples of each species are deposited in herbarium utv, tuscia university (viterbo, italy). fig. 1. flowers of a) viola arvensis, b) v. kitaibeliana, and c) v. hymettia from wild populations of central italy. tab. 1. locations of the studied viola populations. species localities elevation coordinates habitat viola arvensis acque albule, tivoli (roma, italy) 66 m a.s.l. 41°57'47"n 12°42'53"e shrubby grassland in uncultivated land (calcareous soil) viola kitaibeliana le vigne, ofena (l’aquila, italy) 481 m a.s.l. 42°18'03"n 13°44'05"e arid and stony shrubby grassland (calcareous soil) viola hymettia riello, viterbo (viterbo, italy) 323 m a.s.l. 42°25'38"n 12°05'38"e uncultivated escarpment with pine trees and shrubs (volcanic soil) magrini s., scoppola a. 46 acta bot. croat. 74 (1), 2015 fig. 3. microphotographs of the observed pollen morphs: a) 3-aperturate, b) 4-aperturate, and c) 5-aperturate grains of viola hymettia (scale bar = 10 μm). palynological study pollen grains were sampled from 10–30 living plants per population that had been previously labeled and numbered. flowers were harvested possibly before full blooming to collect pollen from closed anthers, from 1 to 9 different fl owers per plant (only from the main axis) (fig. 2). all the fl owers were dissected immediately after harvesting to avoid pollen loss. the plant code (species and number) and the fl ower position were registered for each one. anthers were removed from the fl ower and put directly on a microscope slide, immersing them in a drop of lactic acid to let out all the pollen grains. observations of the entire pollen assemblage were carried out under a light microscope leitz hmlux3 with a magnifi cation of 100× (resolution: 0.25 μm) and the different pollen morphs were counted according to this scheme: • triangular shape = 3-aperturate grain, • squared shape = 4-aperturate grain, • pentagonal shape = 5-aperturate grain, • hexagonal shape = 6-aperturate grain, • round, elliptical, irregular shape = imma ture, aborted or unidentifi able grains. this usually entailed the counting of 100–1,600 grains, or even more. for each species, microphotographs of pollen grains were taken using a digital camera, fujifi lm finepix s2980. statistical analysis for statistical analysis only the sets of data with more than 150 pollen grains and with percentages of immature or aborted grains less than 20% were used. data were analyzed by one-way anova using graphpad prism 5.1, followed by tukey’s multiple comparison test fig. 2. sample of viola hymettia plant with the progressive numbering of the fl owers according to their arrangement from the lower to the upper parts of the stem (i–iv). the fourth fl ower is a bud suitable for pollen collection. variability of pollen heteromorphism in annual pansies acta bot. croat. 74 (1), 2015 47 to test the signifi cant differences among fl owers in each plant and among plants in the same population. pearson’s correlation test was performed to test for correlation between the percentages of the main pollen morph and the position of the fl owers at 95% confi dence interval. results pollen heteromorphism in the examined fl owers, 3-, 4and 5-aperturate grains were observed (fig. 3), while no 6-aperturate grains were found. in all the species, a statistically signifi cant prevalence of the 4-aperturate grains (on average more than 93%; p < 0.0001) over the other morphs was observed. particularly, in v. arvensis it ranges from 81% to 99% (93.58 ± 4.99%, mean ± standard deviation), with 1–18% of 5-aperturate grains (6.18 ± 5.11%) and with less than 3% of 3-aperturate ones (0.25 ± 0.57%) (fig. 4). in v. kitaibeliana almost all the grains observed were 4-aperturate (98.41 ± 1.13%) while 3and 5-aperturate grains accounted for fewer than 2% (0.47 ± 0.76% and 1.12 ± 1.13%, respectively) (fig. 4). v. hymettia showed the greater variability with about 77–98% of 4-aperturate grains (90.74 ± 6.55%), a little greater percentage of 3-aperturate grains than the other species, up to 15.5% (1.34 ± 3.53%), and with 5-aperturate grains that range from 0 to 21% (7.92 ± 6.16%) (fig. 4). fig. 4. frequencies of the different pollen morphs in all the analyzed fl owers of viola arvensis, v. kitaibeliana, and v. hymettia. data are ordered from the fi rst to the last fl owers. magrini s., scoppola a. 48 acta bot. croat. 74 (1), 2015 pollen heteromorphism variability no signifi cant differences in the proportions of the various morphs were observed among fl owers of the same plant in these three species (tabs. 2, 3, 4). viola hymettia showed the larger variability in the polymorphic pollen assemblage, independently of fl ower position (fig. 5, tab. 4), as no trend in the variation of the percentages of the different pollen morphs was observed. on the other hand, a certain variability in the pollen assemblage of the fi rst 2 fl owers was observed in v. arvensis, with 4-aperturate morphs ranging from about 81% to 100% and with 5-aperturate morphs from 0% to 18%. this variability was progressively and strongly reduced up to the last fl owers (fig. 5, tab. 2). viola kitaibeliana developed a maximum of 3 fl owers per plant showing less variability in the pollen assemblage, with 4-aperturate morphs ranging from about 96% to 100% and 5-aperturate from 0 to 4% (fig. 5, tab. 3). the results of anovas conducted on fl owers of different plants showed that, in v. arvensis, v. kitaibeliana, and v. hymettia, no signifi cant differences in the proportions of the different pollen morphs could be detected among individuals from the same population (p values vary from 0.0759 to 0.7834). in the three species, no statistically signifi cant correlatab. 2. polymorphic pollen assemblages of fl owers of viola arvensis. mean percentage ± standard deviation and results of one-way anova for three pollen morph, with statistically signifi cance at p < 0.05 (post hoc tukey’s test). flower position 3-aperturate 4-aperturate 5-aperturate i 0.25 ± 0.42% 94.05 ± 4.20% 5.70 ± 4.26% ii 0.16 ± 0.32% 92.77 ± 6.21% 7.07 ± 6.30% iii 0.69 ± 1.32% 93.98 ± 5.11% 5.34 ± 5.58% iv 0.03 ± 0.06% 96.11 ± 2.15% 3.86 ± 2.12% v 0.11 ± 0.02% 91.73 ± 0.04% 8.16 ± 0.05% vi – 91.94 ± 0.02% 8.06 ± 0.01% p 0.4292 0.8652 0.8491 f5,48 1.001 0.3717 0.3955 r2 0.1064 0.04237 0.04496 tab. 3. polymorphic pollen assemblages of fl owers of viola kitaibeliana. mean percentage ± standard deviation and results of one-way anova for three pollen morph, with statistically signifi cance at p < 0.05 (post hoc tukey’s test). flower position 3-aperturate 4-aperturate 5-aperturate i 0.29 ± 0.38% 98.13 ± 1.31% 1.59 ± 1.35% ii 0.88 ± 1.09% 98.41 ± 0.98% 0.70 ± 0.80% iii – 99.23 ± 1.09% 0.77 ± 1.10% p 0.3000 0.5309 0.4226 f2,13 1.361 0.6750 0.9400 r2 0.2140 0.1189 0.1582 variability of pollen heteromorphism in annual pansies acta bot. croat. 74 (1), 2015 49 tion was observed between the percentages of the main pollen morph and the position of the fl owers (pearson’s correlation test, two-tailed p values vary from 0.1174 to 0.9362). discussion in all three species, our results show a highly signifi cant prevalence of the 4-aperturate morph over the other two morphs. in v. hymettia the relative proportion of some pollen fig. 5. box-and-whisker graphs of the main pollen morphs in the fl owers of viola arvensis, v. kitaibeliana, and v. hymettia. the ordinal numbers of the fl owers, from i to ix, are reported according to their arrangement on the main stem from the lower to the upper parts of the plant. tab. 4. polymorphic pollen assemblages of fl owers of viola hymettia. mean percentage ± standard deviation and results of one-way anova for three pollen morph, with statistically signifi cance at p < 0.05 (post hoc tukey’s test). flower position 3-aperturate 4-aperturate 5-aperturate i 0.43 ± 0.40% 90.42 ± 4.30% 9.15 ± 4.69% ii 6.77 ± 7.64% 90.54 ± 11.27% 2.69 ± 3.71% iii 0.18 ± 0.18% 89.09 ± 7.33% 10.73 ± 7.20% iv 0.28 ± 0.24% 90.54 ± 6.08% 9.18 ± 6.31% v 0.22 ± 0.28% 90.12 ± 10.44% 9.67 ± 10.68% vi 0.27 ± 0.01% 92.25 ± 0.10% 7.48 ± 0.09% vii – 88.08 ± 0.01% 11.92 ± 0.02% viii 1.46 ± 0.07% 98.22 ± 0.02% 0.32 ± 0.02% ix 0.15 ± 0.02% 93.41 ± 0.10% 6.44 ± 0.08% p 0.0817 0.6869 0.1877 f8,19 2.174 0.7010 1.621 r2 0.4914 0.2376 0.4187 magrini s., scoppola a. 50 acta bot. croat. 74 (1), 2015 morphs varies quite considerably: in some fl owers, from sunny areas, there were no 3-aperturate pollen grains at all, while in more pigmented fl owers and in others with upper petals suffused with violet, in the shade of the trees, more than 15% were 3-aperturate pollen grains and only 77% 4-aperturate ones. variability in the color of the corolla related to light gradient is already known for v. tricolor, an annual pansy with creamy to bluish or violet large petals occurring in meadows and habitats linked to woodlands (pettet 1964). tillbottraud et al. (1999) proved that pollen-type proportions of a perennial pansy, v. calcarata l., vary among populations along altitudinal gradients. on the other hand, to our knowledge there is no information regarding pollen heteromorphism variability within the same population of annual pansies linked to gradient. the pollen assemblage of the two other species is more uniform than in v. hymettia. viola arvensis shows a certain variability in the percentages of the main morph, especially in the fi rst fl owers, where 4-aperturate grains range from 81% to 100%. a greater variability, ranging from 69% to 96%, was observed among those populations with mostly 5-aperturate grains (pettet 1964). in the pollen assemblage of v. kitaibeliana almost all the grains are 4-aperturate, in accordance with a recent study from iran (saeidi mehrvarz et al. 2014). by contrast, our recent unpublished data highlight a rather wide variability among other populations from central italy where 5-aperturate grains prevail (from 83 to 92%). the pollen assemblages of the last two species show less variability when 4-aperturate pollen grains predominate. our study was focused on the assessment of the pollen heteromorphism variability in these three viola species, taking into account differences within an individual and within a population. pollen heteromorphism is related to pollination strategy: in heterogamous plants there is a prevalence of pollen grains with a few apertures (generally 4 or less), while in the autogamous ones 5-aperturate morphs prevail (dajoz et al. 1993). so, we hypothesized a variability among fl owers related to the increasing reduction of their size from the lower to the upper parts of the plant in those species, like v. arvensis, that show both autogamous and heterogamous pollination. we even expected to observe the main pollen type changing among such variable fl owers within the same plant. on the other hand, we did not expect to fi nd this change in species like v. hymettia showing fl ower features so adapted to entomophilous pollination (large and fl attened corolla, scent, stigmatic opening up-wards directed, protruding labellum, etc.). our results have only confi rmed the last hypothesis. in fact, no signifi cant correlation was detected between the number of apertures and the position of the fl ower, either in v. hymettia or in two other species. no signifi cant differences were recorded even among individuals. our results are not in accordance with data reported by dajoz et al. (1995) for some v. arvensis populations where the percentages of the different pollen morphs vary signifi cantly among plants of the same population so much as to change the main morph. these confl icting fi ndings are probably due to differences in sampling methods that may often lead to biased estimates, especially the analysis of a maximum of only 200 pollen grains per fl ower without any indication about the developmental stage of the collected fl owers. there is a large difference in the number of pollen grains between fl owers in full bloom and fl ower buds (from about 60 to over 2,500). in fact, only in the closed anthers collected from buds can all the pollen assemblage be found, although with a variable percentage of unidentifi able grains. finally, according to our results, even when there is variability in the relative frequencies of the different pollen morphs, the same type always prevails within both a single plant variability of pollen heteromorphism in annual pansies acta bot. croat. 74 (1), 2015 51 and the population. then, it is possible to identify the main pollen morph by analyzing just a few fl owers per plant or per population. however, in order to avoid bias due to sampling methods, we suggest the collection of fl ower buds in well developed plants, with at least three or more fl owers, and the analysis of the whole pollen assemblage rather than a few hundred grains. acknowledgements thanks are due to e. chiummariello for assistance in plant collecting, in fl ower sampling and pollen grain recording, and to s. buono and t. kirin for images of viola hymettia and v. arvensis fl owers, respectively. we thank also the anonymous reviewers for their constructive comments. references clapham, a. r., tutin, t. g., moore, d. m., 1987: flora of the british isles. 3rd ed., 107– 112. cambridge university press, cambridge. clausen, j., channell, r. b., nur, u., 1964: viola rafi nesquii, the only melanium violet native to north america. rhodora 66, 32–46. dajoz, i., mignot, a., hoss, c., till-bottraud, i., 1995: pollen aperture heteromorphism is not due to unreduced gametophytes. american journal of botany 82, 104–111. dajoz, i., till-bottraud, i., gouyon, p. h., 1991: evolution of pollen morphology. science 253, 66–68. dajoz, i., till-bottraud, i., gouyon, p. h., 1993: pollen aperture polymorphism and gametophyte performance in viola diversifolia. evolution 47, 1080–1093. davis, p. h., mill, r. r., tan, k., 1988: viola l. in: davis, p. h., mill, r. r., tan, k. (eds.), flora of turkey and the east aegean islands (suppl. 1), vol. 10, 62–64. edinburgh university press, edinburgh. erben, m., 1985: cytotaxonomische untersuchungen an südosteuropeischen viola-arten der sektion melanium. mitteilungen der botanischen staatssammlung münchen 21, 339–740. erdtman, g., 1952: pollen morphology and plant taxonomy. almqvist & wiksell, stockholm. espeut, m., 1999: errata et addenda de l’article: »approche du genre viola dans le midi meditérranéen français«. le monde des plantes 467, 7–9. espeut, m., 2004: viola roccabrunensis sp. nov. le monde des plantes 482, 18–21. gams, h., 1926: viola l. in: hegi, g., illustrierte flora von mitteleuropa, vol. 5, 586–656. lehmanns verlag, münchen. kristofferson, k. b., 1923: crossings in melanium-violets. hereditas 4, 251–289. magrini, s., scoppola, a., 2013: is it possible to defi ne the real italian distribution area of the annual pansy, viola kitaibeliana? in: peccenini, s., domina, g. (eds.), contributi alla ricerca fl oristica in italia, 53–54. società botanica italiana, palermo. magrini s., scoppola a. 52 acta bot. croat. 74 (1), 2015 marcussen, t., karlsson, t., 2010: violaceae. in: jonsell, b., karlsson, t. (eds.), flora nordica, vol. 6, 12–52. royal swedish academy of sciences, stockholm. merxmüller, h., 1982: viola l. in: pignatti, s. (ed.), flora d’italia, vol. 2, 102–118. edagricole, bologna. mignot, a., hoss, c., dajoz, i., leuret, c., henry, j. p., dreuillaux, j. m., heberle-bors, e., till-bottraud, i., 1994: pollen aperture polymorphism in the angiosperms: importance, possible causes and consequences. acta botanica gallica 141, 109–122. nadot, s., ballard, h. e. jr, creach, j. b., dajoz, i., 2000: the evolution of pollen heteromorphism in viola: a phylogenetic approach. plant systematics and evolution 223, 155–171. pettet, a., 1964: studies on british pansies. ii. the status of some intermediates between viola tricolor l. and v. arvensis murr. watsonia 6, 51–69. randall, r. e., 2004: biological fl ora of the british isles no. 233: viola kitaibeliana schult(es). journal of ecology 92, 361–369. raus, t. h., 1986: viola l. in: strid, a. (ed.), mountain fl ora of greece, vol. i, 608–640. cambridge university press, cambridge. saeidi mehrvarz s., yousefi n., mohammadi m., marcussen t., 2014: pollen studies in the genus viola (violaceae) from iran. acta botanica croatica 73, 93–106. scoppola, a., lattanzi, e., 2012: viola section melanium (violaceae) in italy. new data on morphology of viola tricolor-group. webbia 67, 47–64. till-bottraud, i., mignot, a., de paepe, r., dajoz, i., 1995: pollen heteromorphism in nicotiana tabacum (solanaceae). american journal of botany 82, 1040–1048. till-bottraud, i., vincent, m., dajoz, i., mignot, a., 1999: pollen aperture heteromorphism. variation in pollen-type proportions along altitudinal transects in viola calcarata. comptes rendus de l’académie des sciences – series iii – sciences de la vie 322, 579–589. valentine, d. h., merxmüller, h., schmidt, a., 1968: viola l. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea, vol. 2, 270–282. cambridge university press, cambridge. vollrath, k. h., 2011: viola in nordostbayern. retrieved may 3, 2013 from http://www. regnitzfl ora.de/veilchen_20_12_10.pdf werner, k., 1988: viola kitaibeliana schultes auf der schwellenburg bei erfurt – ein neufund für die ddr. hercynia 25, 142–143. yockteng, r., ballard, h. e. jr, mansion, g., dajoz, i., nadot, s., 2003: relationships among pansies (viola section melanium) investigated using its and issr markers. plant systematics and evolution 241, 153–170. untitled acta bot. croat. 75 (1), 2016 s1 social news acta botanica croatica: editorial activity and scientometric analysis for the period 1998–2014 damir viličić, grozdana sirotić university of zagreb, faculty of science, department of biology, rooseveltov trg 6, 10000 zagreb, croatia abstract – an editorial report concerning and scientometric analysis of papers published in the journal acta botanica croatica for the period from 1998 to 2014 is presented. since 1998, original scientifi c papers have been written exclusively in english and the accessibility and communicability have increased. the journal is included in the citation base scopus (since 2007) and web of science – science citation index expanded (since 2008). beginning in 2011, a partnership with co-publishers versita – central european science publisher and walter de gruyter open publishing house, resulted in improved management, quality control, electronic publication and increasing visibility of the journal. the impact factor has been calculated since 2010 by journal citation reports and has increased from 0.435 to 0.839. acta botanica croatica is an international scientifi c journal published by the department of biology, faculty of science, university of zagreb, croatia; it is the traditional botanical journal of southern europe. the scope of the journal is fi eld (terrestrial and aquatic) and experimental botany (including plant viruses, fungi, bacteria, algae), from subcellular level to ecosystems. one decade after the end of the first world war, botanists in the department of botany of the faculty of philosophy of the university of zagreb of that time established a domestic botanical journal with the intention of improving communications with the international scientifi c community and exchanging it for other botanical journals (ilijanić 1992). the journal, named acta botanica instituti botanici regalis universitatis zagrebiennsis, was founded in 1925 by professor vale vouk (founder of the laboratory of plant physiology in zagreb university, organizer of oceanographic institutions in split, rovinj and dubrovnik). in the same period many other botanical journals were founded in europe. vale vouk was the fi rst editor of the journal, until his retirement in 1957, with an interval during world war ii, when the journal did not come out (tab. 1). in the period 1957–1968, during the editorship of professor stjepan horvatić whose research fi eld was the fl ora and vegetation of southwestern croatia and the adriatic islands), the name of the journal was changed to acta botanica croatica. it was the period of consolidation of the journal although it was not still regularly published. in the period 1969–1992, the journal was edited by professor ljudevit ilijanić whose scientifi c interest was in the fl ora, plant ecology, plant sociology and grassland vegetation in croatia. ljudevit ilijanić introduced a scientifi c (concise) style of writing, and achieved international revision of submitted manuscripts (one domestic and one international), as well as regular, annual publication of the journal. statistical analysis of the ten-year period from 1979 to 1988 reveals that papers from 10 botanical subjects were published; they may be divided into two main groups: group a dealing with phytosociology, phytogeography, ecology and taxonomy (59%), and group b with physiology, cytology, embryology, virology and molecular biology (41%) (jokić and borić 1992). the difference between these two groups of papers was in the citations, which were older in group a than in group b, indicating the diversifi cation of new scientifi c disciplines during that period. in the early post-war period in croatia, after the breakup of yugoslavia, professor ljerka marković took over the editorship of the journal, from 1993 to 1997 (vol. 56). the fi eld of scientifi c interest of professor ljerka marković was fl ora, expansion of neophytes, nitrophilous vegetation, riparian communities and grasslands. unfortunately, for a short period the journal experienced irregularity in its fi nancial support, resulting in a gap in publication in 1999, just before ljerka marković’s retirement. tab. 1. editors of acta botanica croatica. editor period number of issues edited volumes edited vale vouk 1925–1956 15 1–15 stjepan horvatić 1957–1968 12 16–26/27 ljudevit ilijanić 1969–1992 23 28–51 ljerka marković 1993–1997 5 52–55/56 damir viličić 1998–2013 32 57–72 branka salopek-sondi 2014– 73– s2 acta bot. croat. 75 (1), 2016 viličić d., sirotić g. in 1996 i, professor damir viličić, moved from the institute of oceanography and fisheries, dubrovnik, to the department of botany, faculty of science, university of zagreb where i established my position as full professor and founded the laboratory for marine phytoplankton. i was asked to take over the journal and i accepted this duty. here i give a short report of the editorial activity during my mandate which lasted from 1998 (vol. 57 which was published in 1999) until 2013 (vol. 72). beginning with volume 57 in 1998, acta botanica croatica improved requirements of scholarly publishing, such as editorial procedures and records, structure of (international) editorial board, regular publishing (two times yearly). the journal was published as an entirely english-language journal. we began to use the journal management and publishing software (online journal system). by fulfi lling the requirements for scholarly publishing (marušić and marušić 2005) the journal obtained fi nancial support from the croatian ministry of science again in 2000. impact factor was calculated in the scopus base from 1999, within the portal scimago journal ranking (fig. 1). in 2008, acta botanica croatica was listed in the directory of open access journals and responded to the invitation of the international study on quality assurance, organized by humboldt university, institute for library and information, berlin. after quality control, the journal was soon included in thomson reuters master journal list, and the same year acta botanica croatica was included in the web of science – science citation index expanded. the impact factor has been calculated since 2010 by journal citation reports. papers published in acta botanica croatica are more and more cited in highly competent journals, increasing the impact factor from 0.435 to 0.839 over the time window examined (fig. 1). beginning in 2011, partnership with co-publishers versita – central european science publisher and walter de gruyter open publishing house resulted in improved management, quality control, electronic publishing and increasing visibility in indexing and other services. since 2011 the number of libraries in europe and north america where acta botanica croatica content is disseminated increased to 624, i.e. 256 libraries in north america (according to eluna 2016) and 368 in europe (according to igelu 2016). metapress and springer provide »crosscheck service«. accepted publications are published »on-line fi rst« with digital object identifi er (doi). all these improvements led to the interest of authors in publishing in acta botanica croatica increasing exponentially during the last two decades (1997–2014) (fig. 2). the period between submission and acceptance after positive reviewer and editor decisions has been shortened. the authors of papers appearing in acta botanica croatica are dominantly from croatia, with ca 83% in the period 1991–1999 (jokić and sirotić 2002); however there has recently been an increase in the number of authors who are from other european (44%) and from non-european (37%) countries (tab. 2). one of the journal’s focuses is research into the karstic and arid areas of southern europe, resulting in an increasing number of publications coming from countries with arid environments, e.g. circum-mediterranean, african and asian countries. in april 2013, the editing process was improved by the introduction of a technical editor-in-chief, subject editors and technical editors with the intention of better operationalization of reviewing process and technical preparation of accepted papers. fig. 1. acta botanica croatica; impact factors (if) calculated by the scopus citation base (scimago journal ranking), and web of science – journal citation reports. fig. 2. exponential increase of submissions of manuscripts to acta botanica croatica in the last two decades. acta bot. croat. 75 (1), 2016 s3 acta botanica croatica: editorial activity and scientometric analysis 1998–2014 acta botanica croatica celebrated its 90 anniversary in 2015, and entered the 10th decade of publishing. high quality standards and modern scientifi c content should be a permanent editorial goal in order for the success of acta botanica croatica to be maintained. references eluna 2016: ex libris users of north america. retrieved february 1, 2016 from https://inpress.lib.uiowa.edu/eluna/list.php igelu 2016: the international group of ex libris users in europe. retrieved february 1, 2016 from https://identity.igelu. org/igelu/members/memberslist.php ilijanić, lj., 1992: časopis acta botanica croatica from the establishment until today (in croatian). acta botanica croatica 51, 177–187. jokić, m., sirotić, g., 2002: the communicability of the journal acta botanica croatica over the 1991–2000 period. acta botanica croatica 61, 221–230. jokić, m., borić, v., 1992: scientifi c research in botany according to acta botanica croatica (in croatian). acta botanica croatica 51, 169–176. marušić, m., marušić, a., 2005: possibilities of governmental quality support of scientifi c journals in croatia (in croatian). acta medica croatica 59, 285–296. tab. 2. affi liation of authors who published papers in acta botanica croatica in the period 1991–1999 (according to jokić and sirotić 2002) and 2008–2015, respectively. country 1991–1999 2008–2015 number of papers relative contribution (%) number of papers relative contribution (%) croatia 112 82.96 52 19.19 turkey 18 6.64 hungary 4 2.96 17 6.27 india 16 5.90 italy 12 8.89 16 5.90 slovenia 5 3.70 15 5.54 poland 10 3.69 usa 8 2.95 montenegro 7 2.58 pakistan 7 2.58 iran 6 2.21 serbia 6 2.21 austria 5 1.85 bulgaria 5 1.85 egypt 5 1.85 england 5 1.85 germany 5 1.85 greece 5 1.85 czech republic 4 1.48 russia 4 1.48 spain 4 1.48 sweden 4 1.48 bosnia herceg 3 1.11 brazil 3 1.11 japan 2 1.48 3 1.11 macedonia 3 1.11 saudi arabia 3 1.11 canada 2 0.74 denmark 2 0.74 mexico 2 0.74 morocco 2 0.74 nigeria 2 0.74 peoples r china 2 0.74 opce-str.vp acta bot. croat. 68 (1), 57–66, 2009 coden: abcra 25 issn 0365–0588 plate method for counting proteolytic sulphide-producing bacteria bo@idar stilinovi], jasna hrenovi]* university of zagreb, faculty of science, division of biology, rooseveltov trg 6, 10000 zagreb, croatia the proteolytic sulphide-producing bacteria (pspb) are widely distributed in water and sediment and are a good indicator of the ecological status of ecosystems. from the ecological point of view it is important to distinguish the physiological group of pspb from other bacteria producing h 2 s from sulphate, sulphite or thiosulphate. a new medium named peptone-cysteine-ammonium iron citrate agar (pca) was developed and tested. the medium regularly gave a higher number of colony forming units than the control media for detection of sulphide formation, indole production and motility (merck) and iron sulphite agar (oxoid). for the enumeration of pspb from the environment, the simultaneous incubation of samples in aerobic and anaerobic conditions is recommended and a higher number should be taken for interpretation. keywords: isolation, bacteria, counting, sediment, sulphide, peptone, proteins abbreviations: cfu – colony forming units; pca – peptone-cysteine-ammonium iron citrate agar; pspb – proteolytic sulphide-producing bacteria; sim – commercial medium for detection of sulphide formation, indole production and motility (merck); isa – commercial iron sulphite agar, oxoid introduction the processes of the decomposition of proteins by bacteria play an important role in the mineralization of organic matter. many bacteria decompose protein compounds to end products such as ammonia, hydrogen sulphide (h2s) and mercaptans. mineralization of proteins occurs in the water mass and in benthic sediments under aerobic and anaerobic conditions. the decomposition of putrescible organic matter by heterotrophic bacteria, both in water and sediment ecosystems, is an important step in the cycling of sulphur in the nature. during putrefaction of proteins, a particular amount of h2s originating from the sulphur-containing amino acids (organic sulphur) is liberated. under aerobic conditions the organic sulphur compounds are mineralized to sulphate, and in anaerobic conditions the result is the production of h2s (fenchel et al. 1998). acta bot. croat. 68 (1), 2009 57 * corresponding author, e-mail: jasnah@zg.biol.pmf.hr u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:42 color profile: disabled composite 150 lpi at 45 degrees there are many methods and formulations of culture media used for enumeration of classical sulphate-reducing bacteria, which reduce sulphate to h2s during anaerobic respiration (de bruyn and cloete 1993, skjerdal et al. 2004). in addition to the classical sulphate-reducing bacteria, a physiological group of sulphide-producing bacteria that produce h2s from sulphite or thiosulphate has been much investigated (stilinovic and hrenovic 2004a). however, some bacteria do not produce h2s from sulphate, sulphite or thiosulphate but only from the sulphur-containing amino acids (gram et al. 1987, skjerdal et al. 2004). the physiological group of sulphide-producing bacteria, which produce h2s from proteins, is less often referred to in the recent literature. from the ecological point of view it is important to distinguish the physiological group of proteolytic h2s producing bacteria (pspb) from other bacteria that produce h2s from sulphate, sulphite or thiosulphate. the bacterial production of h2s is generally detected in/on nutrient media by observing the blackening which it produces in the presence of salts of certain metals (such as lead, iron, bismuth) owing to the dark colour of the sulphide of these metals. standard nutrient media for the enumeration of h2s-producing bacteria contain sulphate, sulphite, thiosulphate or cysteine, and bacteria capable of forming h2s from either source of sulphur would appear as black colonies. it must be noted, that there is no generally accepted medium for the isolation and enumeration of the pspb. the goal of this study was to design an original medium for the isolation and enumeration of pspb from environmental samples. on the basis of previous investigations (stilinovi] and futa^ 1990), we started with the fact that the anaerobic condition will be effectuated in the middle of all bacterial colonies, growing aerobically on the surface of the solid nutrient medium during the incubation period. in the middle of the h2s producing colony, after a necessary time of incubation on the appropriate medium rich in amino acids containing sulphur, a black colour in the centre of a bacterial colony will appear soon. therefore, it will not be necessary to incubate the inoculated nutrient medium in anaerobic conditions. materials and methods environmental samples in total, 56 different environmental samples from the zagreb region were tested. of these, 28 samples were of fresh water from natural and artificial lakes, streams and rivers and 28 samples were of sediments from the corresponding fresh water. the subsurface water samples (10 cm) and corresponding surface layer of sediment (5 cm) were collected in sterile bottles, stored at 4 °c and analysed in the laboratory within 2 h of collection. the ph-values and dissolved oxygen concentration of water and interstitial water in sediment were measured with wtw 330 ph-meter and wtw oxi 330i oxygen meter in situ. media the medium peptone-cysteine-ammonium iron citrate agar (pca) was evaluated for the isolation and enumeration of pspb. the composition of pca medium was (in g l –1 of distilled water): peptone bacteriological (biolife) 5.0; proteose peptone (biolife) 5.0; l-cysteine (fluka) 0.25; nacl (kemika) 5.0; ammonium iron (iii) citrate (sigma-aldrich) 1.0; k2hpo4 (kemika) 0.3; agar (biolife) 15.0; ph 7.4±0.2. 58 acta bot. croat. 68 (1), 2009 stilinovi] b., hrenovi] j. u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:42 color profile: disabled composite 150 lpi at 45 degrees the merck commercial medium for the detection of sulphide formation, indole production and motility (sim) and the oxoid iron sulphite agar (isa) were used as standard media for comparison with the obtained results. the composition of sim medium was (in g l –1 of distilled water): peptone from casein 20.0; peptone from meat 6.6; ammonium iron (iii) citrate 0.2; sodium thiosulfate 0.2; agar 15.0; ph 7.3±0.2. the composition of isa medium was (in g l –1 of distilled water): tryptone 10.0; sodium sulphite 0.5; iron (iii) citrate 0.5; agar 15.0; ph 7.1±0.2. the ph of the medium was adjusted with 1 mol l –1 hcl or 1 mol l –1 naoh. the media were autoclaved (121 �c/20 min) and poured into petri plates. experimental methods fresh samples were analysed within 2 h of collection. serial dilutions (10 –1 to 10 –4 ) of one millilitre of water or one g of sediment were prepared. dilutions (0.1 ml) were plated by a spread plate method (apha 1995) in triplicate onto pca, sim and isa. these triplicates were directly incubated at 26±0.1 °c in order to obtain the number of aerobically grown pspb. another triplicate of inoculated pca, sim and isa was incubated (26±0.1 °c) in anaerocult a (merck) in order to obtain the number of anaerobically grown pspb. the number of developed black colonies after 3 d of incubation did not increase by incubation up to 5 d. therefore, the counted number of all developed colonies after 3 d of incubation was taken as final and cfu (colony forming units) of pspb per one ml of water or one g of sediment were calculated. as many different colony types of pspb as were visually distinguishable grown on pca, sim and isa were isolated on nutrient agar (biolife). pure cultures of pspb were stained by the gram method and examined in a light microscope (olympus, bx51) under the immersion objective at the magnification of 1000´. the isolates were identified according to the general characteristics given in bergey’s manual of determinative bacteriology (holt et al. 1994) and biochemical characteristics using the api 20e and api 50ch commercial kit. data analysis the statistical analyses were done using the computer program statistica (statsoft inc. 2006). the results were set up as cfu of pspb obtained on pca versus cfu of pspb obtained on sim or isa medium. data of this type are independent, and therefore the ordinary student’s t-test was performed. the null hypotheses tested by the analysis were: pca, sim and isa medium showed no difference in the final cfu. results were taken to be significant at the 5 % level (p=0.05). the correlation between variables was estimated using the pearson linear correlation. results after the inoculation of pca the first blackening of the colonies appeared after 24 h of incubation and increased by incubation up to 3 d, but did not increase further by incubation up to 5 d. sim and isa media showed the same trend in the development of black colonies. therefore, the final results considered for pca, sim and isa were 3 d after the sample inoculation. in all cases on pca, sim and isa media besides pspb (black colonies) the acacta bot. croat. 68 (1), 2009 59 proteolytic sulphide-producing bacteria u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:42 color profile: disabled composite 150 lpi at 45 degrees companied heterotrophic non-pspb were observed (pale colonies, fig. 1). these non-pspb bacteria constituted about 98% of total population, but did not disturb the recognition and enumeration of pspb. the non-pspb correlated significantly positively with the number of pspb on all three media (r=0.59, p�0.05). the cfu of pspb correlated significant positively with the cfu of total developed cfu on all media (r=0.62, p�0.05). the results of the evaluation of pca compared to the control sim medium for the isolation and enumeration of pspb from different environmental samples are shown in figures 2 and 3. a higher cfu of pspb was obtained by cultivation on pca than on sim. in water samples (fig. 2a) the cfu of aerobically grown pspb were on average 1.5 times higher (p�0.05) when cultivated on pca (310 ml –1 ) than on sim (210 ml –1 ). in the water samples (fig. 2b) the cfu of anaerobically grown pspb were on average 1.2 times higher (p�0.05) when cultivated on pca (149 ml –1 ) than on sim (125 ml –1 ). in the sediment samples (fig. 3a) the cfu of aerobically grown pspb were 2.9 times higher (p�0.05) when cultivated on pca (12,314 g –1 ) than on sim (4,286 g –1 ). in the sediment samples (fig. 3b) the average values of anaerobically grown cfu of the pspb were 3.2 times higher (p�0.05) when cultivated on pca (36,244 g –1 ) than on sim (11,165 g –1 ). the results of the evaluation of pca compared to the control isa medium for the isolation and enumeration of pspb are shown in figures 4 and 5. when testing different environmental samples, a higher cfu of pspb was always obtained by cultivation on pca than on isa medium. in the water samples (fig. 4a) the cfu of aerobically grown pspb were on average 2.6 times higher (p�0.05) when cultivated on pca (1,921 ml –1 ) than on isa (728 ml –1 ). in the water samples (fig. 4b) the cfu of anaerobically grown pspb were on average 1.9 times higher (p�0.05) when cultivated on pca (85 ml –1 ) than on isa (44 ml –1 ). in the sediment samples (fig. 5a) the cfu of aerobically grown pspb were 3.2 times higher (p�0.05) when cultivated on pca (2,349 g –1 ) than on isa (730 g –1 ). in the sediment samples (fig. 5b) the average values of anaerobically grown cfu of the pspb were 3.6 times higher (p�0.05) when cultivated on pca (26,690 g –1 ) than on isa (7,388 g –1 ). 60 acta bot. croat. 68 (1), 2009 stilinovi] b., hrenovi] j. fig. 1. black colonies of proteolytic sulphide-producing bacteria (pspb) and pale colonies of nonproteolytic sulphide-producing bacteria, cultivated on the peptone-cysteine-ammonium iron citrate agar medium. u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:44 color profile: disabled composite 150 lpi at 45 degrees in the samples of water the numbers of pspb cultivated aerobically were higher than those cultivated anaerobically (2.1 and 1.7 times higher on pca and sim, respectively, p�0.05 and 22.6 and 16.5 times higher on pca and isa, respectively, p�0.05). in the samples of sediment the numbers of anaerobically cultivated pspb were higher than those cultivated aerobically (2.9 and 2.6 times higher on pca and sim, respectively, p�0.05 and 11.4 and 10.1 times higher on pca and isa, respectively, p�0.05). this affinity of pspb population to grow in aerobic or anaerobic conditions is explained with the adaptation to the environmental conditions. namely, the concentration of dissolved oxygen was higher in water samples (6.9–9.1 mg o2 l –1 ), while the sediment samples were anoxic (0.3–2.0 mg o2 l –1 ). the average ph values of sediment samples (7.71±0.28) were significantly (p�0.05) lower than ph of the corresponding water samples (8.26±0.27). when summarising all results, the cfu of the pspb were significantly (p�0.05) higher when isolated on pca than on the sim or isa media. the average values of cfu of pspb were 3.1 times higher when cultivated on pca (12±23 ´ 10 3 ) than on sim (4±8 ´ 10 3 ) and acta bot. croat. 68 (1), 2009 61 proteolytic sulphide-producing bacteria aerobic, water 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 sample number -200 0 200 400 600 800 1000 1200 1400 1600 1800 mean sim mean±sd mean pca mean±sda anaerobic, water 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 sample number -100 0 100 200 300 400 500 600 700 800 mean pca mean±sd mean sim mean±sd b n u m b e r o f p s p b (c f u m l ) – 1 n u m b e r o f p s p b (c f u m l ) – 1 fig. 2. colony forming units (cfu ml–1) of aerobically (a) and anaerobically (b) grown proteolytic sulphide-producing bacteria (pspb) isolated from the samples of water on the pca and sim medium. u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:44 color profile: disabled composite 150 lpi at 45 degrees 3.5 times higher when cultivated on pca (8±23 ´ 10 3 ) than on isa (2±5 ´ 10 3 ). the correlation between pca and sim (r=0.72, p�0.05) and pca and isa (r=0.78, p�0.05) was significantly positive, which indicated that higher cfu were not the result of false positive results. the coefficient of variation of results on pca averaged 9.4%, which is not higher than for sim (10.4%) or isa (13.5%) and is satisfactory for a biological method such as cfu determination. the 64 isolated pure cultures of pspb belonged to the common microflora of freshwater and sediments. there was no appreciable difference in bacterial genera isolated on pca, sim and isa. microscopic examination of the isolated colonies of aerobically grown pspb confirmed gram-negative non-spore forming rod shaped cells and gram-positive sporogenic rod shaped cells in all cases. the common feature of isolates was gelatinolytic activity and facultative anaerobic type of metabolism. isolates mainly belonged to the genera aeromonas, shewanella, proteus, bacillus and in few cases erwinia and citrobacter. 62 acta bot. croat. 68 (1), 2009 stilinovi] b., hrenovi] j. aerobic, sediment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 sample number -5000 0 5000 10000 15000 20000 25000 30000 35000 mean pca mean±sda mean sim mean±sd anaerobic, sediment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 sample number -20000 0 20000 40000 60000 80000 100000 120000 140000 mean pca mean±sdb mean sim mean±sd n u m b e r o f p s p b (c f u g ) – 1 n u m b e r o f p s p b (c f u g ) – 1 fig. 3. colony forming units (cfu g–1) of aerobically (a) and anaerobically (b) grown proteolytic sulphide-producing bacteria (pspb) isolated from the samples of sediment on the pca and sim medium. u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:44 color profile: disabled composite 150 lpi at 45 degrees on anaerobically grown plates, beside facultative anaerobes, the obligate anaerobic bacteria from the genus clostridium were isolated. discussion the compound h2s is toxic to animals and plants and its formation in the environment occupies a great attention. the h2s in environmental samples could be the product of bacterial anaerobic decomposition of organic matter containing amino acids with sulphur such as methionine, cysteine and cystine (assimilatory sulphate reduction) or the product of reduction of sulphate, sulphite or thiosulphate (dissimilatory sulphate reduction) (bitton 2005). on the standard media for the detection of h2s-producing bacteria it is not possible to distinguish whether h2s is the product of the degradation of proteins or the reduction of thiosulphate (in the case of sim) or sulphite (in the case of isa). cysteine possesses the adacta bot. croat. 68 (1), 2009 63 proteolytic sulphide-producing bacteria aerobic, water 17 18 19 20 21 22 23 24 25 26 27 28 sample number -1000 0 1000 2000 3000 4000 5000 6000 a mean pca mean±sd mean isa mean±sd anaerobic, water 17 18 19 20 21 22 23 24 25 26 27 28 sample number -50 0 50 100 150 200 250 300 350 mean pca mean±sdb mean isa mean±sd n u m b e r o f p s p b (c f u m l ) – 1 n u m b e r o f p s p b (c f u m l ) – 1 fig. 4. colony forming units (cfu ml–1) of aerobically (a) and anaerobically (b) grown proteolytic sulphide-producing bacteria (pspb) isolated from the samples of water on the pca and isa medium. u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:44 color profile: disabled composite 150 lpi at 45 degrees vantage that it may be attacked with the liberation of h2s by certain bacteria which do not reduce sulphate, sulphite or thiosulphate. with this aim, in this study a medium containing only proteins as a source of sulphur has been designed. the designed pca medium always gave a higher number of pspb than the standard sim and isa media. since all media tested in this study (pca, sim and isa) contained proteins in the form of peptones, the sodium thiosulphate or sodium sulphite as supplements in sim and isa media can be eliminated in the explanation of the difference of obtained cfu of pspb. the higher cfu of pspb obtained on pca than on sim or isa media were probably the result of the composition of the peptones used (kahn 1924) and the initial cysteine content. the suitability of tested media for the isolation and enumeration of pspb can be given in order: pca, sim and isa. all the pure cultures of pspb isolated on pca grew and produced black colonies on sim and isa media. therefore, it can be summarised that the higher numbers of pspb on 64 acta bot. croat. 68 (1), 2009 stilinovi] b., hrenovi] j. aerobic, sediment 17 18 19 20 21 22 23 24 25 26 27 28 sample number -2000 0 2000 4000 6000 8000 10000 12000 mean pca mean±sda mean isa mean±sd anaerobic, sediment 17 18 19 20 21 22 23 24 25 26 27 28 sample number -20000 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 mean pca mean±sd b mean isa mean±sd n u m b e r o f p s p b (c f u g ) – 1 n u m b e r o f p s p b (c f u g ) – 1 fig. 5. colony forming units (cfu g–1) of aerobically (a) and anaerobically (b) grown proteolytic sulphide-producing bacteria (pspb) isolated from the samples of sediment on the pca and isa medium u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:45 color profile: disabled composite 150 lpi at 45 degrees pca is rather the result of better recovery of pspb from environmental samples than the result of isolation of more bacterial species. all the isolated pure cultures of pspb possessed the ability to liquefy gelatine. here it should be mentioned that at the same time the gelatinolytic activity (stilinovi] and hrenovi] 2004b) does not imply that they are pspb. the pspb may have facultative anaerobic or obligate anaerobic type of metabolism. therefore the question is raised in which condition of oxygen supply the environmental samples should be incubated. the designed medium in this study inoculated with the same samples was incubated in both aerobic and anaerobic conditions. the results showed that a higher cfu of pspb was grown in aerobic conditions of incubation in the water samples which had a higher dissolved oxygen concentration. in the anoxic sediment samples a higher cfu of pspb was grown in anaerobic than in aerobic conditions of incubation. therefore, for the enumeration of pspb from environmental samples the simultaneous incubation of the same samples in aerobic and anaerobic conditions is recommended and a higher number should be taken for interpretation. besides the lower dissolved oxygen concentration, the examined sediment samples had a lower ph values than the corresponding water samples, which indicated a higher intensity of the process of putrefaction. the processes of decomposition of organic matter in sediment are more intensive than in the water column, and this explains the higher numbers of pspb obtained in the sediment than in the water samples. the pspb could always be found in water known to receive sewage, human or animal wastes or stools (thompson 1921). therefore the isolation of pspb from environmental samples can be associated with the quality of the water and presence of readily biodegradable organic matter. conclusions the presented pca allows the isolation of higher cfu of pspb than the standard sim or isa media. the pca can be incubated in aerobic or anaerobic conditions in order to count most real cfu of pspb from environmental samples. the presented method could find an application in the detection of the consortia of pspb in the environment where the degradation of proteins and h2s production play an important role. acknowledgements this research was supported by the ministry of science, education and sports of the republic of croatia (project no. 119-1191155-1203). special thanks to r. horvat and v. ramljak for technical assistance during field and laboratory work. references apha, 1995: standard methods for the examination of water and wastewater, apha-awwa-wpcf, new york. bitton, g., 2005: wastewater microbiology. john wiley and sons, inc., new jersey. de bruyn, e. e., cloete, t. e., 1993: media for the detection of sulphide-producing bacteria in industrial water systems. journal of microbiological methods 17, 261–271. acta bot. croat. 68 (1), 2009 65 proteolytic sulphide-producing bacteria u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:45 color profile: disabled composite 150 lpi at 45 degrees fenchel, t., king, g. m., blackburn, t. h., 1998: bacterial biogeochemistry: the ecophysiology of mineral cycling. elsevier, amsterdam. gram, l., trolle, g., huss, h. h., 1987: detection of specific spoilage bacteria from fish stored at low (0 °c) and high (20 °c) temperatures. international journal of food microbiology 4, 65–72. holt, j. g., krieg, n. r., sneath, p. h. a., staley, j. t., williams, s. t., 1994: bergey’s manual of determinative bacteriology. williams and wilkins, baltimore. kahn, m. c., 1925: hydrogen sulphide production by anaerobic spore-bearing bacteria. journal bacteriology 10, 439–447. skjerdal, o. t., lorentzen, g., tryland, i., berg, j. d., 2004: new method for rapid and sensitive quantification of sulphide-producing bacteria in fish from arctic and temperate waters. international journal of food microbiology 93, 325–333. statsoft inc., 2006: statistica (data analysis software system) version 7.1. stilinovi], b., futa^, n., 1990: methods of study the bacterial production of hydrogen sulphide from proteins. biologia (bratislava) 45, 229–234. stilinovi], b., hrenovi], j., 2004a: rapid detection of sulfide-producing bacteria from sulfate and tiosulfate. folia microbiologica 49, 513–518. stilinovi], b., hrenovi], j., 2004b: percentage of gelatinolytic bacteria among heterotrophic bacteria as indicator of water qualty. folia microbiologica 49, 53–58. thompson, l. s., 1921: the group of sulphide producing bacteria. the journal of medical research 42, 383–389. 66 acta bot. croat. 68 (1), 2009 stilinovi] b., hrenovi] j. u:\acta botanica\acta-botan 1-09\stilinovic.vp 17. travanj 2009 10:02:45 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 76 (2), 2017 111 acta bot. croat. 76 (2), 111–119, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0001 issn 0365-0588 eissn 1847-8476 relation between plant species diversity and landscape variables in central-european dry grassland fragments and their successional derivates igor paušič, danijel ivajnšič, mitja kaligarič, nataša pipenbaher biology department, faculty of natural sciences and mathematics, university of maribor, koroška c. 160, si-2000 maribor, slovenia abstract – a systematic field survey of an area of 843 ha in the traditional central-european agricultural landscape of goričko nature park in slovenia revealed 80 fragments of dry semi-natural grasslands. vascular plant species diversity was studied in relation to landscape variables and to threat (slovenian red-listed species). our results show that fragment size does not affect plant species diversity. in addition, fragment shape index is not related to alpha diversity. higher alpha diversity was observed for abandoned grassland fragments. the lowest alpha diversity was perceived on more mesic fragments, where habitat specialists are much scarcer. it was confirmed that the highest diversity of specialists are in the driest fragments, both still mowed and abandoned. with an increase in the number of distinctly different bordering habitat types, the total number of species per fragment generally does not increase, except in the case of those fragments that are already in different succession stages. abandoned and typical dry grasslands are associated with a higher number of bordering habitats. typical dry grassland fragments and abandoned ones, which probably derived mostly from drier (less productive) grasslands, are found on lower altitude and have a lower shape index. habitat specialists sedum sexangulare, polygala vulgaris and spiranthes spiralis have higher frequency in fragments with a lower shape index. this means that these oligotrophic specialists occur in smaller fragments. but orchis morio has higher frequencies of occurrence on polygons with a higher shape index, which confirms the observation that this species occurs in larger and more irregular fragments, as well as close to houses and fields and along the roads. key words: goričko nature park, habitat specialists, ne slovenia, semi-natural dry grasslands, traditional agricultural landscape * corresponding author, e-mail: igor.pausic@um.si introduction over the past century, grasslands and other semi-natural plant communities in southern and temperate europe have suffered a dramatic decline in the area they cover, owing to land use changes (luoto et al. 2003). however, the primary driver of the current decrease in species densities (and their populations) and species loss is habitat fragmentation (fattorini and borges 2012). habitat fragmentation not only causes habitat loss or reduction, but also promotes habitat isolation, diminishes habitat quality, and increases edge effects (lomolino et al. 2010). the alteration of landscapes throughout the world has resulted in fragmentation of natural and semi-natural vegetation, imposing a great threat on numerous plant and animal species (kiviniemi and eriksson 2002). many researchers have concluded that habitat fragmentation poses an important additional threat to biodiversity (hanski 2005, janišova et al. 2014, zulka et al. 2014), but others claim that fragmentation per se is of secondary importance (fahrig 2003). one reason why the effect of connectivity (or fragmentation itself) might not be significant is the slow response of populations to environmental change (hanski 2005). one consequence of the intensification of agriculture in europe over the last half-century has been the loss and fragmentation of traditionally managed habitats, such as semi-natural grasslands (kiviniemi and eriksson 2002, cousins 2005). in fragmented landscapes, plant species persistence depends on functional paušič i., ivajnšič d., kaligarič m., pipenbaher n. 112 acta bot. croat. 76 (2), 2017 connectivity in terms of pollen flow to maintain genetic diversity within populations and seed dispersal to re-colonize habitat patches following local extinction (rico et al. 2011). habitat fragmentation is predicted to lead to area-related reductions in population size and decreasing colonization rates as a result of isolation (kiviniemi 2008). according to island biogeography theory, the species richness of patches is determined by their size and spatial isolation, while in conservation practice, it is patch quality that determines protection and guides management (zulka et al. 2014). dry grassland species-area relationships can be explained by two ecological hypotheses: the first predicts higher species richness due to the higher habitat heterogeneity and the second claims that colonisation (extinction) dynamics causes increasing species numbers with increasing habitat area independent of habitat heterogeneity (rosenzweig 1995, krauss et al. 2004). the area effect per se cannot be easily disentangled from the habitat diversity effect and so it is difficult to quantify the interrelationship (steinmann et al. 2011). previous studies have already reported a positive correlation between habitat heterogeneity and species diversity (rosenzweig 1995, ricklefs and lovette 1999). there are only a few studies dealing with habitat specialists in general while there are several studies of the frequency of particular grassland species in relation to fragment area, shape index and to habitat heterogeneity (paušič and kaligarič 2015). the landscape structure in goričko, i.e., the spatial distribution of ecotopes, is regarded as a mosaic of “frozen processes”. in other words, landscape structure is assumed to mirror the processes that have been going on in a landscape (forman 1995). small-scale patchiness and its diversity reveal the “typical” traditional agricultural landscape, which was fragmented for socio-economic reasons at the end of the 19th and at the beginning of the 20th century. goričko nature park was established in 2003 in order to protect this well-preserved, traditional central-european agricultural landscape. the mosaic structure of landscape and the variety of secondary habitats in goričko were caused by the simultaneous effect of natural and anthropogenic-geopolitical, economic and social factors (kaligarič et al. 2008). in the traditional agricultural landscape of goričko, there were basically four major land use (management) categories: arable fields, grasslands (including orchard grasslands), vineyards and gardens. during the past century, traditional land use changed drastically in central europe, and gradually also within the study area inside goričko nature park (škornik 2003). the area used for grasslands decreased mainly because of land abandonment and forest progression (cousins et al. 2014). the aim of this study was to investigate the relation between plant species diversity, fragment area and fragment shape index. the following methodological steps were undertaken: (1) analyse the differences in floristic composition of dry grassland fragments using twinspan software, (2) compare the plant composition/frequency in the fragments with selected landscape variables, (3) study the differences between twinspan groups and all three shannon-weaver diversity indices (alpha diversity, alpha diversity of habitat specialists and alpha diversity of habitat generalists), (4) test the relations between the landscape variables and frequency of single habitat specialists, and (5) find differences between threatened (red-listed) plant species and twinspan groups. material and methods study area and field methods the study area lies in ne slovenia within the borders of goričko nature park (fig.1), at approximately 46˚50’– 46˚52’ n, 16˚15’–16˚52’ e and covers 843 ha. the area has a central-european climate with a relatively dry winter. average annual rainfall is between 750 and 820 mm (arso 2014). the driest months are january and february, while the most rainfall occurs in july and august (arso 2014). mean annual temperature is between 9 and 10 °c (arso 2014). dry and semi-dry grasslands in goričko are not as rich in species as similar grasslands elsewhere in slovenia, owing to the low ph value of the soil (škornik 2003). we analyzed 800 plots on 80 dry grasslands fragments (including some earlier or middle successional stages after abandonment) systematically mapped in the study area. they mostly belong to the association hypochoerido-festucetum rupicolae or drier stands of the association ranunculo bulbosi-arrhenatheretum elatioris. all dry grassland fragments (80) were sampled and analysed on randomly dispersed square meter plots (10 per each fragment). species composition was recorded on 800 plots, where vascular plants were sampled using presence/absence data. for each plant species we calculate frequencies; 10 plot samples on 80 fragments. the vegetation composition data were collected in only one year (2013) by the first author. plant species occurring only in one sample were removed (we exclude 11 plant species), in order to exclude casual occurrences from the analyses. taxonomic nomenclature followed martinčič et al. (2007). plant species specialists and generalists were defined according to expert knowledge, by help of the syntaxonomic classification of the taxa. plant species characteristic for the alliances bromion erecti (and order brometalia erecti) and violion caninae represent species of drier, nutrient-poor neutral to acid soils of central european provenance and climate. the rest of the species were considered generalists, although some species from e.g. ruderal or forest habitats are also considered to be in this group. fig. 1. map of slovenia with bordering countries (a) and study area (b) with 80 dry grassland fragments. spatial pattern of dry grassland fragments acta bot. croat. 76 (2), 2017 113 species diversity shannon-weaver diversity index (h) was calculated for all species (called alpha diversity), and further indices were calculated only for species specialists (called alpha diversity specialists) and for species generalists (called alpha diversity generalists) (shannon and weaver 1963). we also identified threatened plant species using the red lists of the threatened flora of slovenia (anonymous 2002). of the 169 plant species included in the study, 7 were included in the red list and from this we calculated threatened species frequency (frk_red). threatened species frequency was noted as the number of threatened plant species recorded in 10 plots on each dry grassland fragment. landscape variables the spatial geometry (area and perimeter) of the grassland fragments was obtained using arcgis 9.3 spatial analyst tools (esri 2010) by digitalizing the plots – drawing polygons in vector format. the plots were first drawn on printed orto-photo imagery in 2009 (pixel size=0.5 m) (gurs 2010). later, the relation between fragment area and its perimeter – a straightforward “shape index”, perimeter-area ratio for each grassland fragments was calculated (para index, referred to as “shape index” later on in the text). all different bordering habitat types (polygons) (nofht) for each grassland fragment were determined using an existing 2012 habitat type map owned by the national institute for nature conservation (zrsvn) which is based on the physis typology (devillers and devillers-terschuren 1996), adopted and modified for slovenian conditions (jogan et al. 2004). the buffer analysis tool within arcgis 9.3 (esri 2010) was applied in order to quantify habitat types that share the border with the target grassland fragments. additionally, the above-mentioned habitat type map was used to calculate the distance from the settlement/houses variable (dis2h). in this regard, all polygons determined as buildings (houses) were selected and extracted from the initial habitat type map. the distance surfaces between all houses were calculated in the next step by using the euclidean distance algorithm in the arcgis spatial analyst tools (esri 2010). the mean distance from the settlement/houses for each grassland fragment in the study area was determined in the end with the zonal statistics tool in arcgis and was additionally log transformed for the purpose of statistical analysis. accordingly, a digital elevation model (dem) in a 5 m horizontal resolution (gurs 2010) served as the input data with a calculation of the mean height above sea level variable (hasl) for each grassland fragment and the application of the zonal statistics tool in arcgis 9.3 (esri 2010). all variables were z standardized for the purpose of landscape variables analysis. data analysis to explain the relations between alpha diversity of all 169 plant species and 46 habitat specialists the relations between fragment area and fragment shape index were tested separately with pearson’s correlation coefficient. the analysis was carried out in r (r development core team 2009). to classify grassland fragments according to their similarity we used two-way indicator species analysis (twinspan) (hill 1979). this analysis was run with frequencies data of 80 dry grassland fragments and 169 plant species. further, redundancy analysis (rda) ordination was conducted with 80 grassland fragments, to relate variation in the whole of the species data set (169) to landscape variables and threatened species richness. twinspan groups were added as supplementary variable without any effect on the analysis. ordination method (rda) and visualization of their results were carried out using the canoco and canodraw programs (ter braak and šmilauer 2002). additionally, a comparison was performed between the twinspan groups and all three shannon-weaver diversity indexes (alpha diversity, alpha diversity specialist and alpha diversity generalist) using one-way anova. oneway anova was also performed for twinspan groups and landscape variables. the analysis was carried out in r (r development core team 2009). we used poisson or quasipoisson regression (depending on the data dispersal) to test the effect of landscape variables (hasl, dis2h, nofht and shape index) on specialist species. we implemented poisson or quasipoisson regression within a generalized linear modelling (glm) framework, using a poisson link or quasipoisson link function. we tested only specialist species with frequencies higher than ten. the analysis was carried out in r (r development core team 2009). in order to present differences between threatened plant species frequency from the red list of slovenia we calculated sum of 10 plots on each dry grassland fragment. in addition we performed the kruskalwallis test to find possible differences between threatened species frequency (frk_red) and determined twinspan groups. results from 80 dry grassland fragments, we included in the dataset 169 vascular plant species (on-line suppl. tab. 1) with an average of 43 plant species per fragment. the total fragment area covers 25.56 ha. the area of the smallest fragment is 0.028 ha and the largest is 1.12 ha. from 169 vascular plant species, 46 were considered habitat specialists with an average of 13 plant specialists per fragment, mostly characteristic species of mesobromion and violion alliances. the average number of plant generalists per dry grassland fragment is 31 plant species. as the first step, the relations between the alpha diversity of all 169 plant species and 46 habitat specialists between fragment area and fragment shape index were tested separately. however, no significant relations between all mentioned parameters were found (results not shown). at the second step, differences in floristic composition were analysed with twinspan classification of matrix with 169 plant species and 80 dry grassland fragments (fig. 2). four groups were distinguished on the basic of this analysis. the first twinspan group represents the most typical extensively used oligotrophic and mostly dry grasslands fragments (referred to as dry), regularly mowed, with typpaušič i., ivajnšič d., kaligarič m., pipenbaher n. 114 acta bot. croat. 76 (2), 2017 ical and abundant species such as briza media, peucedanum oreoselinum and festuca rupicola. the second twinspan group represents also extensive and still dry, but relatively more mesic, grassland fragments (referred as mes) with typical and abundant species as sanguisorba officinalis, ranunculus bulbosus and centaurea jacea. the third twinspan group with the most typical and abundant species quercus petraea, prunus domestica (remnant of orchards), tanacetum vulgare, lychnis flos-cuculi, veronica camaedrys and hypericum peforatum represents abandoned grassland fragments (referred as aba), mostly already overgrown with young woody perennials. the fourth twinspan group is constituted by grassland fragments, which are ruderalised (disturbed) fragments (referred as rud), also containing weeds or ruderals as typical and abundant species: plantago major, cirsium arvense, setaria viridis or erigeron annuus. in the third step, we compared the plant composition/ frequency in the fragments with landscape variables. in the rda ordination of two matrixes: first one with 169 plant species and 80 dry grassland fragments and second one with 4 landscape variables (nofht, dis2h, hasl and shape index) plus threatened species richness was performed in order to differentiate the analysed dry grasslands fragment on the basic of selected landscape variables and their endangerment (threat) (fig. 3). the first two axes of rda analysis explained 33.1% of variation. the first axes were related positively to the shape of the fragments (shape index), altitude (hasl) and threatened species richness (frk_red). however, the second axes were related positively to the number of different bordering habitat types (nofht), threatened species richness (frk_red) and negatively to shape index, distance from the settlements/houses (dis2h) and altitude (hasl). furthermore, the dry and mes groups had more species from the red list than aba and rud groups and the dry and the aba group have highest frequencies of bordering habitat types than the mes and rud groups, in contrast to landscape variables shape index and altitude. additionally, as the fourth step, we were looking for the differences between twinspan groups and all three shannon-weaver diversity indexes (alpha diversity, alpha diversity specialist and alpha diversity generalist). we found significant differences for alpha diversity (all 169 species considered) in the first three twinspan groups – dry, mes and aba (p<0.001); only the rud group it is not significantly different from the dry and the mes group (fig 4a). very similar results were found when alpha diversity was calculated for generalists only. we found significant differences between mes and aba group (p< 0.001) and between the dry and the aba group (p<0.001). however, the alpha diversity, calculated for generalists does not differentiate dry from mes and aba groups. we found also no significant differences between the aba and rud group, when calculating the alpha diversity for generalists (fig. 4b). what is more, alpha diversity for specialists does not differentiate between the dry and the aba group or between the mes and rud group, but shows significant differences between mes and rud groups together and the aba group (fig. 4c). we found also significant differences in landscape variable “distance from the settlements/houses” (dis2h), but we have not found differences fig. 2. simplified twinspan classification tree (dendrogram) of matrix with 169 plant species and 80 dry grassland fragments. for each division number of groups, number of dry grassland fragment (in brackets) in group and indicator plant species are shown. grassland fragments were divided into four groups: dry – extensively used oligotrophic and mostly dry grasslands fragments, mes – more mesic grassland fragments, aba – abandoned grassland fragments mostly overgrown with young woody perennials, rud – ruderalised grassland fragments. abbreviation of plant species are explained in on-line suppl. tab. 1. fig. 3. redundancy analysis (rda) ordination diagram of two matrixes: first with 169 plant species and 80 dry grassland fragments and second matrix with 4 landscape variables, frequency of the species from the red list and 80 dry grassland fragments. eigenvalues: axis 1=0.158, axis 2=0.097, 25.5 % of variance in species explained by both axes. shown species have the highest weight. grassland fragments were divided into four groups with twinspan analysis: dry group, mes group, aba group, rud group; explained in fig. 2 in detail. hasl – height above sea level; shape – shape index; nofht – number of bordering habitat types, dis2h – distance from the settlements/houses, frk_red – threatened species richness. spatial pattern of dry grassland fragments acta bot. croat. 76 (2), 2017 115 in twinspan groups between any other landscape variables (fig 4d). significant differences in log landscape variable “distance from the settlements/houses” were found between the dry and the rud group (p<0.05). when testing the relations between the landscape variables and frequency of single habitat specialists, we found only some statistically significant correlations (results not shown). higher number of neighbouring habitats is correlated with higher frequency of trifolium montanum, spiranthes spiralis and centaurium erythrea. more distant from the settlements/houses are polygons with potentila argentea, carex caryophyllea and thymus pulegioides. relation with shape index gave opposite results: sedum sexangulare, polygala vulgaris and spiranthes spirales have higher frequency on plots with lower shape index; while orchis morio have higher frequencies of occurrence on polygons with higher shape index (on line-suppl. fig. 1). finally, we present differences between red listed (threatened) plant species frequency with highest frequency of species anacamptis morio and fragaria viridis (fig. 5). we fig. 5. threatened species frequency of seven plant species from red list of slovenia (left) and frequency of threatened species in each twinspan group (right). the abbreviations of species names: centaurea cyanus, daphne cneorum, fragaria viridis, ophioglossum vulgatum, orchis morio, orchis ustulata, spiranthes spiralis. abbreviations of twinspan groups are explained in fig. 2. fig. 4. differences between twinspan groups and all three shannon-weaver diversity indexes: alpha diversity (a), alpha diversity generalists (b) and alpha diversity specialists (c) and for twinspan groups and distance from the settlements/houses (dis2h) (d). twinspan groups: 1 – dry group, 2 – mes group, 3 – aba group, 4 – rud group; explained in fig. 2 in detail. letter in box-plot are from anova, duncan posthoc test. paušič i., ivajnšič d., kaligarič m., pipenbaher n. 116 acta bot. croat. 76 (2), 2017 were looking for differences between the twinspan groups and found the highest values for dry and mes twinspan group, but there were no significant differences among the four twinspan groups. discussion a large proportion of european biodiversity today depends on habitat provided by low-intensity farming practices, yet this resource is declining as european agriculture intensifies (sutcliffe et al. 2014). one consequence of the rationalization of agriculture in europe over the last half century has been the loss and fragmentation of traditionally managed habitats such as semi-natural grasslands (cousins 2001). in many agricultural landscapes, as also in the goričko area, only fragments have remained. the crucial question in the situation of the declining number and area of grasslands in the landscape is whether fragment size affects plant species diversity. this problem has received different answers in studies. however, our results show that the fragment size (large vs. small) does not affect species diversity of plants. this has been confirmed clearly already by helm et al. (2006) – no effect of current grassland size on plant species diversity in estonian alvar grasslands was confirmed. our results indicate that there is no relation between species diversity (here measured as alpha diversity of all species and habitat specialists only) and grassland area, which could be explained by the assumptions proposed by helm et al. (2006). long leaved perennial plants may also persist for relatively long time after the habitat has been degraded or converted to a later successional stage. in other words – since grassland fragments were assembled a long time ago and their structure is homogenous, these present remnants are still enough large to harbour more or less the entire species diversity of the previous (larger) area. the authors (e.g. cousins and eriksson 2002, eriksson et al. 2002, lindborg 2007, kuussaari et al. 2009, reitalu et al. 2010) summarized that it is not current but past grassland areas (prior to the drastic decline of grasslands) and the connectivity among them that affect the current species diversity in fragmented grasslands across europe. our study revealed that both fragment size and fragment shape index are not correlated to alpha diversity: the shapes of the remnant grasslands are different, in many cases elongated or irregular, but this does not influence alpha diversity. in semi-natural grasslands, the land use treatment (management) is one of the major ecological factors, known to affect plant reproductive success and vegetation patterns (lopez-marino et al. 2000, eriksson et al. 2002, lindborg et al. 2005, wissman 2006, gustavsson et al. 2007). sites with long land use continuity had higher plant species diversities than sites with shorter continuity (gustavsson et al. 2007). thus, we questioned if the fragments of dry grasslands are floristically homogenous. the twinspan classification revealed four twinspan groups, which show the influence of moisture/nutrient level (dryer – dry; more mesic – mes), abandonment (aba) and disturbance (ruderal – rud). in the next step we correlated the species composition in the fragments with some landscape variables. the influence of landscape context in habitat fragmentation studies is often ignored (wiens 1997, hanski 1999). high habitat diversity in the surroundings might enhance the species diversity of a local site. landscape context might affect gene flow via pollen and seed dispersal, the pressure of herbivory and the functional connectivity between habitat patches (ricketts 2001). the complex effect of landscape context on local plant species diversity revealed the importance of the bordering habitats in many studies (kollman and schneider 1999, metzger 2000, söderström et al. 2001). high landscape diversity in the surrounding matrix provides more distinct habitat types for generalists or species with other habitat preferences, supporting the prediction that landscape diversity enhances the number of generalists, especially at the edges, but hardly of the specialists (jonsen and fahrig 1997). in our study area, the fragments (thought to be bigger and more connected polygons in the past) are currently surrounded by numerous habitat types: arable fields, forest edges, mesic meadow orchards, intensive meadows, vineyards, gardens, roads and even settlements. the rda ordination diagram shows (fig. 3) that the aba and dry groups are associated with a higher number of different bordering habitats types (nofht). it is also worth mentioning that the most typical, dry fragments (dry) and abandoned ones (aba), which probably also derived mostly from drier (less productive) places, are found at lower altitudes and have lower shape indices. it has been established by tscharntke et al. (2002) that not all species depend equally on habitat area, isolation and landscape context. habitat specialists proved to be more affected by habitat loss than generalists (warren et al. 2001). the comparison of alpha diversity considering all species, habitat specialists only and generalists only between the rud groups revealed interesting results. higher alpha diversity was calculated for aba, because abandonment results in colonization of species from the later colonization stages, while grassland species persist. the lowest alpha diversity was perceived on more mesic fragments (mes), where habitat specialists are much scarcer. it was emphasized many times that early successional stages after abandonment represent the most species-rich assemblages (denslow 1980, balmer and erhardt 2000). a similar pattern was revealed, when only generalists were considered. interesting results were obtained when only habitat specialists were considered. it was confirmed again that the highest diversity of specialists are in the driest fragments, both still mowed (dry) and abandoned (aba), which means that among habitat specialists are mostly long-lived perennials which persist also during the earlier successional stages. long-lived plants decline slowly and may survive for decades after environmental change (eriksson 1996, dahlstrom et al. 2006, helm et al. 2006, lindborg 2007, hylander and ehrlén 2013). it could be concluded that, with a reduction in grassland fragment area, the majority of species still conform to previous distribution patterns, when grassland areas were larger. there are no habitat maps from past decades to support this with exact figures. however, spatial pattern of dry grassland fragments acta bot. croat. 76 (2), 2017 117 another study in the nw part of the goričko park revealed that at the beginning of the 20th century the area was dominated by open agricultural land, with a forest cover around 39%; a landscape composition today shows forests covering around 57% of the landscape (cousins et al. 2014). furthermore, 65% of the forests in nw goričko are between only 15 and 30 years old. young forests are primarily developed on former arable fields or grasslands (cousins et al. 2014). we claim that this is a strong proof that the grasslands in goričko area were much larger in the past, although exact spatial data on their distribution are lacking. high habitat diversity in the surroundings might enhance the species diversity of a local site. furthermore, the surrounding matrix provides more distinct habitat types for generalists or species with other habitat preferences, supporting the prediction that landscape diversity enhances the number of generalists, especially at the edges, but hardly of the specialists (jonsen and fahrig 1997). interesting results were obtained when the twinspan groups were confirmed on the basis of the distance of polygons from settlements/houses. it seems peculiar at first sight, but the shorter distance to the settlements/houses of the most dry and regularly mowed meadows in goričko can be explained by the fact that still mowed “best preserved” dry (oligotrophic!) fragments are close to the houses, due to the fact that houses are built on less productive soils in order to save better land for more productive (fertile) meadows and fields. when testing the relation between frequencies of single habitat specialists we found interesting results: sedum sexangulare, polygala vulgaris and spiranthes spirales have higher frequency on plots with a lower shape index. this means very small remnants of formerly larger polygons. but orchis morio has higher frequencies of occurrence on polygons with higher shape index, which confirm the observation that this species occurs on fragments of different complex shapes, also close to houses and fields and along the roads. semi-dry grasslands, the remnants of traditional agricultural landscape, are today threatened mainly due to quick overgrowing, but also due to intensification or conversion to arable land. disturbance in the form of ruderalisation (occasional fertilization or mulching) and the early stages of secondary succession emerged as much stronger drivers of community change than decreases in patch size per se. the selection of red-listed species on national level is not really supporting the conservation efforts for grasslands in goričko area; only a few such species are present in the area. it can only be concluded that in still mowed (dry and mes) fragments the number of red-listed species tends to be higher, although not significant and despite the relatively low number of such species. the dilemma whether biodiversity conservation should focus on a single or several large habitat fragments, or whether the protection of many small fragments (covering the same habitat area in a landscape) is of equal or even greater importance is still open (tscharntke et al. 2012). the dilemma is commonly referred as sloss (single large or 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0365-0588 eissn 1847-8476 early spring fl ora of the sub-pannonic steppic grassland (natura 2000 site) in bilje, northeast croatia tanja žuna pfeiffer1, dubravka špoljarić maronić1*, vanda zahirović1, filip stević1, milorad zjalić1, katarina kajan1, siniša ozimec2, melita mihaljević1 1 josip juraj strossmayer university of osijek, department of biology, cara hadrijana 8/a, hr-31 000 osijek, croatia 2 josip juraj strossmayer university of osijek, faculty of agriculture in osijek, kralja petra svačića 1d, hr-31 000 osijek, croatia abstract – the diversity of early spring vascular fl ora was studied in the sub-pannonic steppic grassland in the village of bilje, north eastern croatia. in all, 109 plant taxa within 35 families were found. the highest number of taxa belongs to the families poaceae, fabaceae, asteraceae, caryophyllaceae, lamiaceae and rosaceae. specifi c habitat conditions, characterized by moderately wet and moderately acidic soil with intermediate fertility and the effects of the continental climate favour the development of different plant life forms. out of the total recorded plant taxa, hemicryptophytes make up 59.6%, followed by therophytes (22.0%) and geophytes (13.8%). chorological analysis shows that the most numerous are plants of eurasian (33.9%), pontic-central-asian (21.1%) and central european (21.1%) fl oral elements. according to their status in the red list, three critically endangered (cr), one vulnerable (vu) and three nearly threatened (nt) plant species were found. altogether, the steppe-like grassland in bilje is a unique habitat rich in valuable plants of the croatian fl ora, including the critically endangered doronicum hungaricum, therefore it is of great importance to preserve it. important management tools include mowing and controlling the spreading of cultivated and invasive plant species. key words: chorological types, fl ora, life-forms, natura 2000, pannonian region, steppe-like grassland * corresponding author, e-mail: dspoljaric@biologija.unios.hr introduction grasslands, herbaceous communities mostly dominated by grasses (poaceae) or other graminoids, like cyperaceae and juncaceae (janišová et al. 2011), are among the largest ecosystems in the world. in europe there are various grassland types, ranging from humid grasslands in the north and north-west parts of the continent, through steppic and mesic grasslands to the near-desert types that occur in south-east spain (owen 2008). dry grasslands cover much smaller european areas than mesic and wet grasslands and most of them are semi-natural habitats, resulting from centuries of traditional land use, such as grazing, mowing, temporary abandonment of arable fi elds, and/or other disturbance events (dengler et al. 2014). dry grasslands involve a variety of grassland biotopes (rodwell et al. 2002) that are usually present on relatively dry and nutrient-poor soils. some of the most important dry grassland habitats are sub-pannonic steppic grasslands of the alliance festucion valesiacae (klika 1931) developed on rocky substrate and claysandy sedimentation layers enriched with gravels. through the centuries, many of sub-pannonic steppic grasslands have been destroyed by afforestation and ploughing, or by land abandonment which led to their degradation (ruprecht et al. 2009). steppe-like grasslands are dominated by festuca valesiaca schleich. ex gaudin, carex humilis leyss., chrysopogon gryllus (l.) trin., stipa species, but they are also important habitats for many rare and endangered plant species (cremene et al. 2005, buček et al. 2006, jones et al. 2010). therefore, these grasslands are considered biodiversity hotspots and included as an eu priority habitat type in the natura 2000 network, designated under code 6240*. the republic of croatia differs from most european countries with respect to its huge variety of habitats and great diversity of fl ora. grasslands cover approximately 17.6% of the croatian terrestrial area (anonymous 2006). sub-pannonic steppic grasslands are very rare and only few occur in the continental part of croatia. this study focused on the grassland in the village of bilje situated in northeast žuna pfeiffer t., špoljarić maronić d., zahirović v., stević f., zjalić m., et al. 158 acta bot. croat. 75 (2), 2016 croatia. the most signifi cant contribution to the fl oristic knowledge of the area was given by zahirović (2000), while there have been no recent scientifi c publications on surveying the steppe-like grassland fl ora of bilje. the aim of the current investigation was to investigate the diversity of spring steppe-like grassland fl ora with special reference to rare and endangered plant species. materials and methods the study was carried out at the grassland surface at the local cemetery in the village of bilje (45°36’n, 18°45’e), situated 5 km northeast of osijek (croatia, baranja region, fig. 1). the area is infl uenced by a continental climate. according to data from osijek meteorological station (1981– 2010), mean air temperature is 11.3 °c with an annual precipitation of 684 mm. geomorphologically, the study area mostly belongs to the lowland area built of quaternary sediments formed during the pleistocene and holocene (banak et al. 2012). the holocene sediments are mostly fl uvial sands and sandy clay (regional plan of the municipality of bilje 2005). steppe-like grassland covers 0.63 ha and it is among the last remnants of dry steppe grasslands of the alliance festucion valesiacae in croatia. thus, it is included in the national ecological network of croatia (designated with code hr2000728) which is a part of the eu natura 2000 network. in 2001, the area was protected as a natural monument. the research on the grassland fl ora was conducted weekly during eight fi eld surveys, carried out from the beginning of april to the end of may 2015. plant taxa were identifi ed according to javorka and csapody (1975), knežević and volenik (1981) and domac (1989) and arranged alphabetically and systematically. the nomenclature and interpretation of plant life forms (therophyta – t, chamaephyta – ch, hemicryptophyta – h, phanerophyta – p, nano-phanerophyta – n, geophyta – g) were adjusted according to the croatian flora checklist (nikolić 2016). phytogeographical analysis was interpreted according to josifović (1970–1977), zahirović (2000), and ranđelović et al. (2007). geoelements were described with the following numbers: 1 – pontic – central – asian, 2 – central european , 3 – sub-atlantic, 4 – sub-mediterranean, 5 – eurasian, 6 – sub-boreal, 7 – adventive plants and 8 – cosmopolites. the species were categorized by threat levels according to nikolić and topić (2005) and nikolić (2016), and invasive plant species according to nikolić et al. (2014). the diversity of environmental grassland parameters was determined according to ellenberg et al. (1994) and adjusted according to the croatian flora checklist (nikolić 2015). ellenberg indicator values (eiv) included the following indicators described by numerical scales ranging from 1–9: light (l), temperature (t), salinity (s), moisture (m), soil reaction (sr), and nitrogen (n). descriptive statistical analysis of environmental parameters was carried out using statistica software package (statsoft. inc., ver. 12). results a total of 109 vascular plant taxa (92 species and 17 subspecies) from 35 families were identifi ed during the study period (tab. 1). poaceae was the largest taxonomic group (represented by 12 taxa), followed by fabaceae (10 taxa), asteraceae (8 taxa) and caryophyllaceae (8 taxa). lamiaceae and rosaceae were present with six taxa, each. fig. 1. study area: sub-pannonic grassland in bilje (northeast croatia). tab. 1. list of vascular plant taxa occurring in steppe-like grassland in bilje in spring of 2015. life form: h – hemicryptophyta, t – therophyta, ch – chamaephyta, p – phanerophyta, n – nanophanerophyta, g – geophyta; chorological type: 1 – pontic – central – asian, 2 – central european, 3 – sub-atlantic, 4 – submediterranean, 5 – eurasian, 6 – sub-boreal, 7 – adventive plants and 8 – cosmopolites; threat levels: cr – critically endangered, vu – vulnerable, nt – nearly threatened; asterisk (*) denotes invasive species. list of taxa life form chorological types threat levels spermatophyta magnoliophytina magnoliatae apiaceae anthriscus sylvestris (l.) hoffm. h 5 peucedanum oreoselinum (l.) moench h 1 aristolochiaceae aristolochia clematitis l. h 4 asteraceae achillea millefolium l. h 5 ambrosia artemisiifolia l. * t 7 artemisia vulgaris l. h 8 doronicum hungaricum rchb. f. g 1 cr erigeron annuus (l.) pers.* h 7 inula hirta l. h 1 taraxacum offi cinale weber h 5 bellis perenis l. h 2 spring flora of the steppic grassland in bilje acta bot. croat. 75 (2), 2016 159 list of taxa life form chorological types threat levels boraginaceae anchusa offi cinalis l. h 2 myosotis discolor pers. subsp. discolor t 2 myosotis ramosissima rochel t 8 brassicaceae arabis glabra (l.) bernhardt h 8 arabidopsis thaliana (l.) heynh. t 5 capsella bursa-pastoris (l.) medik. h 8 erophila verna (l.) chevall. subsp. verna t 8 calepina irregularis (asso) thell. t 4 campanulaceae campanula patula l. h 2 campanula rapunculus l. h 5 caryophyllaceae cerastium semidecandrum l. h 2 dianthus carthusianorum l. h 4 holosteum umbellatum l. t 5 lychnis viscaria l. subsp. viscaria h 1 moenchia mantica (l.) bartl. subsp. mantica t 1 saponaria offi cinalis l. h 5 silene latifolia poir. subsp. alba (mill.) greuter & bourdet h 5 stellaria media (l.) vill. t 8 cistaceae helianthemum nummularium (l.) mill. subsp. nummularium ch 2 cichoriaceae hieracium pilosella l. subsp. pilosella h 2 hypochoeris maculata l. h 5 tragopogon pratensis l. subsp. orientalis (l.) čelak. h 5 clusiaceae hypericum perforatum l. h 5 crassulaceae sedum telephium l. subsp. maximum (l.) krock. h 5 euphorbiaceae euphorbia cyparissias l. h 5 fabaceae lotus corniculatus l. h 2 medicago minima (l.) bartal. t 1 medicago sativa l. subsp. sativa h 7 trifolium alpestre l. h 1 trifolium repens l. h 5 trifolium montanum l. h 1 vicia cracca l. h 5 vicia grandifl ora scop. t 1 vicia lathyroides l. t 2 vicia villosa roth subsp. villosa t 1 list of taxa life form chorological types threat levels fagaceae quercus robur l. p 2 geraniaceae erodium cicutarium (l.) ľ hér. h 5 geranium sanguineum l. h 1 geranium dissectum l. t 8 lamiaceae ajuga genevensis l. h 5 lamium purpureum l. t 2 mentha aquatica l. h 5 salvia pratensis l. h 2 thymus pulegioides l. ch 2 glechoma hederacea l. h 5 oleaceae syringa vulgaris l. n 7 papaveraceae papaver dubium l. t 8 papaver rhoeas l. t 5 plantaginaceae plantago lanceolata l. h 5 plantago media l. h 5 polygalaceae polygala comosa schkuhr h 5 polygonaceae rumex acetosa l. subsp. acetosa h 5 rumex acetosella l. g 8 ranunculaceae pulsatilla pratensis (l.) miller subsp. nigricans (störck) zamels h 1 cr ranunculus polyanthemos l. h 1 rosaceae filipendula vulgaris moench h 6 potentilla argentea l. h 1 potentilla cinerea chaix ex vill. h 1 potentilla heptaphylla l. h 2 prunus tenella batsch n 1 cr geum urbanum l. h 5 saxifragaceae saxifraga bulbifera l. h 4 scrophulariaceae verbascum phoeniceum l. h 1 veronica austriaca l. subsp. austriaca h 1 veronica chamaedrys l. h 2 veronica hederifolia l. t 2 veronica persica poir.* t 8 valerianaceae valerianella locusta (l.) laterrade t 4 violaceae viola arvensis murray t 5 tab 1. – continued žuna pfeiffer t., špoljarić maronić d., zahirović v., stević f., zjalić m., et al. 160 acta bot. croat. 75 (2), 2016 regarding the distribution of plant life forms (fig. 2a), hemicryptophytes were dominant (59.6%), followed by therophytes (22.0%) and geophytes (13.8%). according to phytogeographical analysis (fig. 2b) eurasian (33.9%), pontic-central-asian (21.1%), and central-european (21.1%) fl oral elements predominated. cosmopolites were represented with 13 (11.9%) taxa. ecological indicatory values (fig. 3) were defi ned for more than 90% of the plant taxa. the greatest number of plant taxa preferred half-light conditions (38 taxa, eiv l = 7) and relatively warm habitats (32 taxa, eiv t = 6) characterized by moderately wet (33 taxa, eiv m = 5) and moderately acid soils (41 taxa, eiv sr = 6) with intermediate fertility (24 taxa, eiv n = 5). almost none of the identifi ed taxa (93%) tolerate salty soils (eiv s = 0). according to threat status given in the red book of vascular flora of croatia, doronicum hungaricum rchb.f., pulsatilla pratensis (l.) miller subsp. nigricans (störck) zamels and prunus tenella batsch are critically endangered (cr), polygonatum latifolium (jacq.) desf., is vulnerable (vu) and carex praecox schreber, iris variegata l. and orchis morio l. are nearly threatened (nt). in the early list of taxa life form chorological types threat levels viola tricolor l. t 5 viola odorata l. h 2 liliatae amaryllidaceae allium sphaerocephalon l. subsp. sphaerocephalon g 1 allium vineale l. g 2 narcissus poeticus l. g 7 narcissus pseudonarcissus l. subsp. pseudonarcissus g 3 asparagaceae muscari botryoides (l.) mill. g 1 muscari comosum (l.) mill. g 4 ornithogalum umbellatum l. g 2 polygonatum latifolium (jacq.) desf. g 1 vu cyperaceae carex caryophyllea latourr. h 5 carex praecox schreb. h 5 nt iridaceae iris variegata l. g 1 nt iris sp. g 5 juncaceae luzula campestris (l.) dc. h 8 liliaceae gagea pratensis (pers.) dumort. g 5 orchidaceae orchis morio l. g 2 nt poaceae anthoxanthum odoratum l. h 5 apera spica-venti (l.) p. beauv. t 5 arrhenatherum elatius (l.) p. beauv. ex j. presl & c. presl h 2 briza media l. h 5 bromus inermis leyss. h 5 chrysopogon gryllus (l.) trin. h 1 dactylis glomerata l. h 5 festuca rupicola heuff. subsp. rupicola h 2 hordeum murinum l. t 8 koeleria pyramidata (lam.) p. beauv. h 2 poa bulbosa l. h 5 poa pratensis l. g 8 tab 1. – continued fig. 2. life form (a) and chorological spectrum (b) of steppe-like grassland fl ora occurring in bilje in spring 2015. fig. 3. box and whiskers plot for environmental variables. ellenberg indicator values (eiv): l – light, t – temperature, s – salinity, m – moisture, sr – soil reaction, n – nitrogen. spring flora of the steppic grassland in bilje acta bot. croat. 75 (2), 2016 161 spring fl ora, three invasive species (ambrosia artemisifolia l., erigeron annuus (l.) pers. and veronica persica poir) were found. discussion northeast croatia belongs to the pannonian region. in the period following the last ice age, forest steppes i.e. mosaic of grasslands and woodlands were parts of the original pannonian landscape and covered these areas for a long time (illyés et al. 2007). for centuries, the pannonian region was heavily infl uenced by humans and consequently, much of the area was converted to productive agricultural land. today, in the pannonian region, dry grasslands occur only as small fragments, mainly on sites not suitable for intensive cultivation and agricultural production (sundseth 2009). the steppe-like grassland in bilje remained preserved due to the specifi c location in the grounds of the local cemetery. the plant community indicates prevailing environmental conditions in certain habitats (schaffers and sýkora 2000). according to ellenberg indicator values the steppelike grassland in bilje is a well-illuminated, relatively warm habitat characterized by fresh soil of average moisture, moderately acid and moderately rich in nitrogen. the alliance festucion valesiacae was also found in warmer, drier, and less nutrient rich habitats in romania (dengler et al. 2012), serbia (aćić et al. 2015) as well as in the carpathians and the pannonian basin (dúbravková et al. 2010). dry grasslands are usually characterized by high species richness (dengler and boch 2008, purger and csiky 2008, zima and štefanić 2009, vassilev et al. 2011, vitasović kosić and britvec 2014, pirini et al. 2014). in comparison of the number of taxa with that of the steppelike grassland fl ora of the natura 2000 site near bapska, eastern croatia (79 taxa, šegota et al. 2015), we can conclude that plant species richness of the investigated grassland in bilje is relatively high, probably due to the anthropogenic infl uence. human impact may change habitats, leading to the increase in the number of plant taxa (alegro et al. 2006). the appearance of some species such as viola odorata, syringa vulgaris and iris sp., which were also cultivated on the surrounding graves, suggests that anthropogenic activities mediated their spreading along the grassland area. however, the early spring fl ora which accounts for more than 80% of the total grassland plant taxa found by the previous fl oristic investigation in bilje (zahirović 2000) indicates a well-conserved habitat. a specifi c characteristic of its fl ora is the prevalence of eurasian, ponticcentral-asian, and central-european fl oral elements with a mosaic complex of different steppic species which also were found in steppe-like grasslands in bulgaria (vasillev and apostolova 2013) and serbia (ranđelović et al. 2007). the family poaceae is represented with genera like festuca, chrysopogon, poa and koeleria, together with species from the fabaceae, asteraceae and caryophyllaceae families, which add certain specifi city to the grassland fl ora in bilje. these plant families usually characterize steppe fl ora around the world (türe and böcük 2007, matevski et al. 2008). hemicryptophytes made up the large majority of the recorded species. this life form is well represented in the european fl ora (ellenberg 1988) and usually characterizes grassland communities (šugar et al. 2005). furthermore, the high number of therophytes indicates the dryness of this region, the modifi cation of the climate (rauš and šegulja 1983, tomašević 2006) and considerable anthropogenic infl uence (mitić et al. 2007). some of the recorded plant species are listed in the red book of vascular plants of croatia (nikolić and topić 2005). the critically endangered pulsatilla pratensis subsp. nigricans grows at several locations in croatia (vrbek 2001, vitasović kosić et al. 2009, nikolić 2016), while the steppe-like grassland in bilje is one of the two reported habitats for doronicum hungaricum in the country (nikolić 2016). in northeast croatia, prunus tenella is reported as a very rare species (krčmar and ozimec 2004, purger and csiky 2008). it grows at the edge of the steppe-like grassland in bilje. besides in croatia, the endangered species doronicum hungaricum and prunus tenella, as well as the nearly threatened iris variegata and orchis morio, are also included in the national red lists of vascular flora of some other european countries (jackowiak et al. 2007, grulich 2012, turis et al. 2014). the steppe-like grassland was also marked by the presence of some woody (e.g. quercus, syringa) and invasive (ambrosia artemisiifolia, erigeron annuus, veronica persica) species. their occurrence could be an important threat for grassland fl ora. generally, trees and shrubs encroach on the sites and develop layers of high cover leading to a rapid decrease in species richness (dúbravková and hajnalová 2012). invasive species, due to the biological characteristics (lonsdale 1999), may outcompete native species for resources (brewer and cralle 2003), suppress their growth (jordan et al. 2008), and have a negative infl uence on species diversity (dogra et al. 2010). together with habitat loss, the abandonment of the traditional ways of land management and uncontrolled digging of plants have been generally recognized as the most important threats to plant diversity (young et al. 2005, nikolić 2016). in the investigated steppe-like grassland, plant diversity depends on the specifi c types of management. mowing is an especially important management tool to control the spread of woody plant species. considering the uncommon position of this steppe-like grassland within the local cemetery where the opening of new burial places is already limited, controlling the spread of cultivated and invasive plant species should be important steps toward long-term persistence of diverse steppe fl ora. since the plant diversity, characterized by rare and endangered plant species, represents an important and irreplaceable natural resource in these steppe-like grassland, conservation of this habitat is therefore of great importance. acknowledgements this study was supported by the environmental protection and energy effi ciency fund and the public institution for the management of protected natural areas in osijekbaranja county. we would like to thank željko zahirović, žuna pfeiffer t., špoljarić maronić d., zahirović v., stević f., zjalić m., et al. 162 acta bot. croat. 75 (2), 2016 msc, for his valuable suggestions and support. we also wish to thank the handling editor and anonymous 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(eds.), steppenlebensraume europas – gefahrdung, erhaltungsmassnahmen und schutz. pp. 191–200. vassilev, k., pedashenko, h., nikolov, s. c., apostolova, i., dengler, j., 2011: effect of land abandonment on the vegetation of upland semi-natural grasslands in the western balkan mts., bulgaria. plant biosystems 145, 654–665. vitasović kosić, i., britvec, m., ljubičić, i., maštrović pavičić, d., 2009: vascular fl ora of istria: endangered and rare taxa. agronomski glasnik 3, 199–214 (in croatian). vitasović kosić, i., britvec, m., 2014: floristic and vegetation characteristic of forest edges and grasslands of ćićarija (croatia). šumarski list 3–4, 167–184 (in croatian). vrbek, m., 2001: new sites of pulsatilla pratensis (l.) miller subsp. nigricans (störck) zamels in croatia. acta botanica croatica 60, 75–84. young, j., watt, a., nowicki, p., alard, d., clitherow, j., henle, k., johnson, r., laczko, e., mccracken, d., matouch, s., niemela, j., richards, c., 2005: towards sustainable land use: identifying and managing the confl icts between human activities and biodiversity conservation in europe. biodiversity and conservation, 14, 1641–1661. zahirović, ž., 2000: rare and endangered plant species of northeast croatia. master of science thesis. university of zagreb. faculty of science, department of biology, zagreb (in croatian). zima, d., štefanić, e., 2009: floristic characteristics of dry grasslands in the požega valley. agronomski glasnik 2, 141–150 (in croatian). acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 151 acta bot. croat. 74 (1), 151–157, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication small-fl owered bittercress, cardamine parvifl ora l. (brassicaceae), a new species of the croatian fl ora dragan prlić* donji meljani 92c, hr-33520 slatina, croatia abstract – cardamine parvifl ora l. was discovered in april 2014 during the study of vascular fl ora and habitats in the area of slatina (slavonia region). it was found in a fl ooded forest of narrow-leaved ash, in the vicinity of the villages medinci and novi senkovac. the species here grows in wet soil but partially submerged populations were also observed. it presents a new species of the croatian fl ora and expands the fl oristic inventory of slatina and its surroundings. other valuable taxa have also been recorded in the area, such as carex riparia curtis and ophioglossum vulgatum l. keywords: cardamine parvifl ora, fl ooded forest, slatina, vascular fl ora introduction the genus cardamine, one of the largest in the family brassicaceae, consists of annual to perennial herbs of approximately 200 species worldwide, with the tropical species confi ned to mountains (clapham et al. 1987, kučera et al. 2005). in the european fl ora it numbers a total of 54 species (lihova and marhold 2006), whereas in the croatian fl ora there are currently 18 species and 4 subspecies (nikolić 2014). the small-fl owered bittercress, cardamine parvifl ora l., occurs in numerous european countries, but is absent from certain parts of the balkan peninsula (jalas and suominen 1994, marhold 1995). it is already known from specifi c neighbouring countries of croatia, such as italy (pignatti 1982), hungary (javorka and csapody 1991, horváth et al. 1995) and serbia (josifović et al. 1972). so far there have been no records for slovenia (martinčič et al. 2010) or, considering the lack of a recent checklist, for bosnia and herzegovina (beck 1903). the euro+med plantbase (marhold 2011) notes the occurrence of this species in au(a) bu by cs es fe ga(f) ge hs(s) hu it la lt lu mo po rf(c e k n) rm sksr su uk(k u). cardamine parvifl ora is a short, erect, hairless annual, 7–22 (–40) cm. leaves are pinnate, the lower with 5–11 pairs, the upper with 7–13 pairs of linear to linear-oblong, cuneate, entire leafl ets. petals are 1.5–2.5 mm, obovate, white. siliqua 8–20 × 0.7–0.8 mm, erect * corresponding author, e-mail: prlicdragan@gmail.com copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. prlić d. 152 acta bot. croat. 74 (1), 2015 on patent pedicels (josifović et al. 1972, tutin et al. 1993). seeds elliptic, lateral compressed, apex rounded, base mostly truncate, 0.6–0.8 × 0.5–0.7 mm, narrowly winged (bojnansky and fargašova 2007). phytogeographically it belongs to the eurasian fl oral element (carlsen et al. 2009). in north america, populations of this species are sometimes treated as c. parvifl ora var. arenicola (britton) o. e. schulz or as a separate species, c. arenicola britton. in the recent account of the genus in the flora of north america, north american populations of c. parvifl ora are not treated as taxonomically different from eurasian ones (al-shehbaz et al. 2010). this species is not listed in plant determination handbooks for croatia (domac 1984, 2002) and it is absent from observational data and herbaria entries in the flora croatica database (nikolić 2014). since cardamine parvifl ora has not been previously identifi ed in croatia, it should, therefore, be treated as a new taxon of the vascular fl ora of croatia and included in the flora croatica database. material and methods field research is being conducted at several new localities within the city of slatina which is situated between the two natural borders, drava river to the north and foothills of mt papuk to the south. one of the localities being studied is the forest area »medinačko ražljevo«, spreading over the southeastern part of the villages medinci and novi senkovac (fig. 1). it is predominantly covered by pedunculate oak (quercus robur), with the exception of a few fl ooded sites with populations of black alder (alnus glutinosa) or narrowleaved ash (fraxinus angustifolia). the terrain elevation throughout the area is around 104 m. the climate is moderate continental with an average annual temperature of 11.3 °c and 726.4 mm of average annual precipitation measured in the period of 2000–2008 (meteorological and hydrological service). fig. 1. the location of cardamine parvifl ora l. (black dot) within the area of slatina (croatia). cardamine parviflora in croatia acta bot. croat. 74 (1), 2015 153 the literature used in determination of the plant material was josifović et al. (1972), pignatti (1982), rothmaler (2009) and tutin et al. (1993). the exact location of the species was recorded on-site using a handheld gps device modifi ed for the htrs96/tm national coordinate system. a corresponding central european mtb/64 quadrant is also given as a means of indirect mapping (nikolić 2006). the wider area around the subject species was fl oristically surveyed and the observational records will be added to the flora croatica database. samples of several plant specimens were collected and deposited in herbarium croaticum (za) and in a personal collection. results and discussion in april 2014, during the course of ongoing botanical research in the area of slatina (prlić 2012, 2013), the forest area »medinačko ražljevo« was surveyed and, as a result, an unknown plant species was recorded and identifi ed as cardamine parvifl ora l. (fig. 2). it was found in a fl ooded forest of narrow-leaved ash and summer snowfl ake (as. leucojofraxinetum angustifoliae glavač 1959), between the stream slatinska čađavica (denoted as »jova r.« on the recent topographic map) and an intermittent stream that cuts through the forest area. it is not very frequent in the investigated forest area and was only discovered in small interspersed populations. a representative population of the species can be found at the coordinates e598894 n5065374 which are located in the 0272.432 mtb/64 quadrant. a fairly rich ground layer with additional important plant species (anonymous 2013, nikolić 2014) was also observed at the site (tab. 1). tab. 1. list of plant species recorded in the study area. strictly protected and red list species are underlined. vegetation layer recorded plant species tree-layer acer campestre, fraxinus angustifolia, quercus robur, ulmus minor shrub-layer acer tataricum, cornus sanguinea, crataegus monogyna, frangula alnus, ligustrum vulgare, prunus spinosa, pyrus pyraster ground-layer ajuga reptans, alliaria petiolata, alopecurus pratensis, angelica sylvestris, athyrium fi lix-femina, caltha palustris, cardamine amara, cardamine fl exuosa, cardamine parvifl ora, cardamine pratensis, carex elata, carex elongata, carex remota, carex riparia, carex vulpina, cirsium canum, colchicum autumnale, cruciata laevipes, dryopteris fi lix-mas, eleocharis palustris, erigeron annuus, euphorbia palustris, galium aparine, galium palustre, glechoma hederacea, hedera helix, humulus lupulus, iris pseudacorus, lamium maculatum, lamium purpureum, leucojum aestivum, lychnis fl os-cuculi, lycopus europaeus, lysimachia nummularia, mentha aquatica, oenanthe fi stulosa, ophioglossum vulgatum, phragmites australis, ranunculus auricomus, ranunculus repens, rorippa amphibia, rosa canina, rubus caesius, scutellaria hastifolia, solidago gigantea, stellaria media, symphytum offi cinale, taraxacum offi cinale, tephroseris crispa, thlaspi alliaceum, urtica dioica, valeriana dioica, valeriana offi cinalis, veronica serpyllifolia prlić d. 154 acta bot. croat. 74 (1), 2015 fig. 2. cardamine parvifl ora l.: a) habitus, b) infl orescence and c) leaves. cardamine parviflora in croatia acta bot. croat. 74 (1), 2015 155 this species was found growing on wet soil and, in places, partially submerged in the water as the locality was lightly inundated at the time of the fi eld research. these are relatively open microhabitats not overgrown by other plants. such habitat conditions are particularly suitable because the species prefers periodically fl ooded and open lowland areas (zlinská 1990). the small-fl owered bittercress is already known from certain communities of alliances such as nanocyperion koch 1926, phalaridion arundiaceae kopecky 1961 or phragmition communis koch 1926 (zlinská 1992, marhold 1995). when the fi rst populations of cardamine parvifl ora were recorded in early april, these plants were already in fl ower and some of the specimens were even producing fruit at the time. this can be considered an early fl owering time as the literature indicates it starts fl owering in may, lasting until august in northern europe (marhold 1995, grey-wilson and blamey 2003). in serbia it is known to bloom a second time during the autumn (josifović et al. 1972). the extent of occurrence of this species in the area of slatina is restricted to less than 12 ha (0.12 km2) of narrow-leaved ash forest. a fi eld survey in the area has also recorded that the population consists of fewer than 500 adult individuals. consequently, according to the iucn standards (nikolić 2005), cardamine parvifl ora should be treated as a critically endangered (cr) species in croatia. although there are no known immediate threats to the habitat, further studies need to be conducted in order to monitor the population size. as an ephemeral annual plant, it is usually present in a wide area but changes particular, open, often fl ooded, microhabitats from year to year; therefore its preservation at a particular spot might require a certain form of active management. cardamine parvifl ora can possibly be confused with other members of the genus cardamine. in the croatian fl ora, it bears the closest resemblance to c. pratensis, however, the latter is different in its signifi cantly larger petals, usually grey-green leaves and orbicular basal leafl ets. there is also a possibility of confusion with c. impatiens which has marginally larger petals, but the leaves are auriculate at the base and its leafl ets are dentate or lobed. additionally, it may be mistaken for c. hirsuta which bears a distinctive basal rosette at anthesis. cardamine parvifl ora can be clearly distinguished in the fi eld by its small petals, emphasized leaf pinnation and entire leafl ets. as a newly recorded species with a very delimited area of occurrence, it is a valuable addition to the croatian fl ora. references al-shehbaz, i. a., marhold, k., lihová, j., 2010: cardamine linnaeus. in: flora of north america editorial committee (eds.), flora of north america: north of mexico, volume 7, magnoliophyta: salicaceae to brassicaceae, 464–484. oxford university press, inc., new york, oxford. anonymous, 2013: regulations on the strictly protected species (in croatian). narodne novine 144/13. retrieved june 12, 2014 from http://narodne-novine.nn.hr/clanci/sluzbeni/2013_12_144_3086.html beck, g., 1903: flora of bosnia, herzegovina and novopazarski sandžak, volume i: gymnospermae and monocotyledones (in serbo-croatian). zemaljska štamparija, sarajevo. bojnansky, v., fargašova, a., 2007: atlas of seeds and fruits of central and east-european fl ora, the carpathians mountains region. springer, dordrecht, the netherlands. prlić d. 156 acta bot. croat. 74 (1), 2015 carlsen, t., bleeker, w., hurka, h., elven, r., brochmann, c., 2009: biogeography and philogeny of cardamine (brassicaceae). annals of the missouri botanical garden 96, 215–236. clapham, a. r., tutin, t. g., moore, d. m., 1987: flora of the british isles, 3rd edition. cambridge university press, cambridge. domac, r., 1984: flora of croatia and neighbouring regions (in croatian). školska knjiga, zagreb. domac, r., 2002: flora of croatia (in croatian). školska knjiga, zagreb. grey-wilson, c., blamey, m., 2003: cassell’s wild fl owers of britain & northern europe. domino books, london. horváth, f., dobolyi, z. k., morschhauser, t., lőkös, l., karas, l., szerdahelyi, t., 1995: flora database 1.2. taxon-list and attribute data (in hungarian). mta ökologiai és botanikai kutatóintézete, vácrátót-budapest. jalas, j., suominen, j., 1994: atlas florae europaeae 10 – cruciferae (sisymbrium to aubrieta). committee for mapping the flora of europe, helsinki. javorka, s., csapody, v., 1991: iconography of the fl ora from the south-eastern part of central europe (in english, latin and hungarian). akadémiai kiadó, budapest. josifović, m., stjepanović, l., janković, m. m., gajić, m., kojić, m., diklić, n., 1972: flora of sr serbia iii (in serbian). srpska akademija nauka i umetnosti, beograd. kučera, j., valko, i., marhold, k., 2005: on-line database of the chromosome numbers of the genus cardamine (brassicaceae). biologia (bratislava) 60, 473–476. lihová, j., marhold, k., 2006: phylogenetic and diversity patterns in cardamine (brassicaceae) – a genus with conspicuous polyploid and reticulate evolution. in: sharma, a. k., sharma, a. (eds.), plant genome: biodiversity and evolution, vol. 1c, phanerogams (angiosperms–dicotyledons), 149–186. science publishers, enfi eld, new hampshire. marhold, k., 1995: taxonomy of the genus cardamine l. (cruciferae) in the carpathians and pannonia iii. folia geobotanica & phytotaxonomica 30, 397–434. marhold, k., 2011: brassicaceae. in: euro+medplantbase – the information resource for euro-mediterranean plant diversity. retrieved october 21, 2014 from http://ww2.bgbm. org/europlusmed/ptaxondetail.asp?namecache=cardamine%20parvifl ora&ptreffk=7200000 martinčič, a., wraber, t., jogan, n., podobnik, a., turk, b., vreš, b., ravnik, v., frajman, b., strgulc-krajšek, s., trčak, b., bačič, t., fischer, m. a., eler, k., surina, b., 2010: flora of slovenia – key to the identifi cation of ferns and fl owering plants, 4th ed. (in slovenian). tehniška založba slovenije, ljubljana. nikolić, t., 2005: assessment of threats (in croatian). in: nikolić, t., topić, j. (eds.), the red book of vascular fl ora of croatia, 24–34. ministry of culture, state institute for nature protection, zagreb. nikolić, t., 2006: flora – handbook for inventory and monitoring (in croatian). state institute for nature protection, zagreb. nikolić, t., 2014: flora croatica database. department of biology, faculty of science, university of zagreb. retrieved june 12, 2014 from http://hirc.botanic.hr/fcd pignatti, s., 1982: flora d’italia, vols. 1–3. edagricole, bologna. cardamine parviflora in croatia acta bot. croat. 74 (1), 2015 157 prlić, d., 2012: a contribution to the vascular fl ora of the slatina region. natura croatica 21, 21–48. prlić, d., 2013: phytogeographical characteristics of the slatina district (in croatian). ms thesis. subdepartment of quantitative ecology, department of biology, university of josip juraj strossmayer in osijek. rothmaler, w., 2009: exkursionsfl ora von deutschland, band 3, gefäβpfl anzen (atlasband). spektrum akademischer verlag, heidelberg. tutin, t. g., burges, n. a., chater, a. o., edmondson, j. r., heywood, v. h., moore, d. m., valentine, d. h., walters, s. m., webb, d. a., 1993: flora europaea, volume 1: psilotaceae to platanaceae, 2nd edition. cambridge university press, cambridge. zlinská, j., 1990: a new locality of cardamine parvifl ora l. in the záhorská nížina lowland (in slovakian). biologia (bratislava) 45, 71–72. zlinská, j., 1992: cardamine parvifl ora im inundationsgebiet des flusses morava. biologia (bratislava) 47, 593–595. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 287 acta bot. croat. 74 (2), 287–302, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0025 halophilic diatom taxa are sensitive indicators of even short term changes in lowland lotic systems zsuzsanna kókai1, istván bácsi2, péter török3, krisztina buczkó4, enikő t-krasznai1, csaba balogh1, béla tóthmérész3, viktória b-béres1* 1 environmental protection and nature conservation authority, trans-tisza region, debrecen, hatvan u. 16, h-4025 hungary 2 university of debrecen, department of hydrobiology, debrecen, egyetem tér 1, h-4032 hungary 3 mta-de biodiversity and ecosystem services research group, debrecen, egyetem tér 1, h-4032 hungary 4 hungarian natural history museum, budapest, könyves kálmán krt. 40. h-1476 hungary abstract – the occurrence and spread of halophilic diatom taxa in freshwater lotic ecosystems are infl uenced both by natural processes and anthropogenic pollution. diatom assemblages were regularly monitored in lowland lotic systems in hungary (central europe) during the unusually dry year of 2012. highly pronounced changes in diatom composition were observed from spring to autumn. halophilic taxa (especially nitzschia sensu lato species) appeared in the dry autumn. in addition, the total relative abundances of halophilic species also increased up to autumn. abundance of nitzschia cf. lorenziana and nitzschia tryblionella showed a positive correlation with chloride and phosphate concentration, while that of other taxa like tryblionella apiculata or tryblionella calida showed a positive correlation with the concentration of nitrate. our fi ndings clearly demonstrated that these halophilic and mesohalophilic diatom taxa were sensitive indicators of even short-term changes in lowland lotic ecosystems, such as the increasing salt concentration from spring to autumn caused by the lack of rainfall and/or environmental loads. keywords: cell size, dry year, freshwater river, nutrient load, salt concentration, taxa number, temporal dynamics introduction diversity, expressed often as the number of taxa, has an essential role in ecosystem processes and functioning, food chains, and ecosystem integrity (cottingham et al. 2001, * corresponding author, e-mail: beres.viktoria@gmail.com kókai z., bácsi i., török p., buczkó k., t-krasznai e., et al. 288 acta bot. croat. 74 (2), 2015 france and duffy 2006, smucker and vis 2011, borics et al. 2014). the structure and functioning of ecosystems can be altered by changes in species diversity (porter et al. 2013). biodiversity is infl uenced by biotic(vasas et al. 2013), or abiotic(ács et al. 2006), natural(lotter et al. 1997, buczkó et al. 2013, catalan et al. 2013) or human induced processes (bácsi et al. 2013). numerous anthropogenic factors cause shortor long-term changes in biodiversity (e.g. eutrophication, changes in some land-use processes, mining, and industry – tilman 1999, hooper et al. 2005, smucker and vis 2011). it is important to stress that aquatic assemblages respond much more sensitively to natural or anthropogenic disturbances than terrestrial communities (to changes in water fl ow conditions, precipitation, to the increase of nutrient concentrations or salinity – smucker and vis 2011, porter et al. 2013). benthic diatoms have a decisive structural and functional role in freshwater ecosystems (bolla et al. 2010, smucker and vis 2011). they are often the dominant photosynthetic algal group in the biofi lm (passy 2002, antoniades et al. 2004, kireta et al. 2012) and they have a crucial role in primary production (e.g. kireta et al. 2012). they are routinely used in biomonitoring assessments (e.g. berthon et al. 2011, várbíró et al. 2012), because they respond sensitively to the changes in the physical and chemical parameters of freshwater habitats (fl ow conditions, conductivity, ion composition, and nutrient conditions – ács et al. 2003, berthon et al. 2011, smucker and vis 2011, rusanov et al. 2012, stenger-kovács et al. 2013, b-béres et al. 2014). increased nutrient load or conductivity induce changes of species composition in diatom assemblages (e.g. ziemann et al. 2001, stengerkovács et al. 2013, b-béres et al. 2014) via the frequent appearance of new taxa (e.g. some halophilic, mesohalophilic eutraphenic taxa) while the abund ance of some others decreases (e.g. ziemann et al. 2001). structural parameters of diatom assemblages (i.e. the composition of size classes – berthon et al. 2011) can also change considerably, because of the increased interspecifi c competition caused by the immigrants. the occurrence and spread of halophilic and mesohalophilic diatom taxa in freshwater lotic ecosystems are caused both by natural processes (e.g. by the increase of nutrient concentrations caused by a decreased water discharge in lack of rainfall) and/or by anthropogenic pollution (e.g. fertiliser run off, atmospheric sedimentation, mining). in the spring of 2012 there was high precipitation, while the summer and autumn period was unusually dry in hungary, as revealed by the data of the hungarian meteorological service and the general directorate of water management. decreased water fl ow conditions occurred mainly because of the low precipitation input in the dry months in lowland rivers and channels (b-béres et al. 2014, kókai et al. 2014). diatom assemblages of 15 hungarian lowland small to large rivers and channels were studied during the humid spring and dry autumn period of 2012. in the present study we have focused on the changes in taxa composition, taxa number, cell size classes and the evenness of diatom assemblages with a special emphasis on halophilic/mesohalophilic taxa. we hypothesised the following: (i) taxa number and abundance of halophilic and mesohalophilic taxa increase from spring to autumn with the increase of conductivity and nutrient content; (ii) the ratio of small disturbance-tolerant taxa and large halophilic/mesohalophilic taxa increases from spring to autumn according to augmented conductivity and nutrient content and (iii) due to the increasing conductivity, the evenness of species diversity decreases from spring to autumn. halophilic diatom taxa in lowland lotic system acta bot. croat. 74 (2), 2015 289 materials and methods sampling setup samples were collected from 15 channels and small to large rivers of the trans-tisza region of hungary in 2012 (fig.1). altogether, 10 environmental factors were measured. conductivity (cond), concentration of dissolved oxygen (do), ph and water temperature (t) fig. 1. the study area on trans-tisza region of hungary: (a) localization of the study area (marked with grey borders); (b) sampling sites on the rivers and channels with eov coordinates, grey lines: borders of the study area, black lines: the rivers and channels. the abbreviations of the watercourses are the following: dióéri-főcsatorna (dio), dögös-kákafoki-főcsatorna (dka), hamvas-csatorna (ham), holt-sebes-körös (hsk), hortobágy (hor), hortobágyberettyó (hbe), keleti-főcsatorna (kfc), kígyósi-főcsatorna (kig), kis-körös-főcsatorna (kko), kösely (kos), kutas-főcsatorna (kut), nagy-ér (nag), nagytóti-toprongyoscsatorna (nto), sárréti-csatorna (sar), sebes-körös (skl). arrows indicate three representative sampling sites shown on fig. 2. a b kókai z., bácsi i., török p., buczkó k., t-krasznai e., et al. 290 acta bot. croat. 74 (2), 2015 were measured in the fi eld with a portable multiparameter digital meter (multi 350i-wtw, germany). water samples were kept at 4 °c in cooler bags during transportation to the laboratory for the measurement of the concentrations of hydrogen carbonate (hco3– – titrimetric method; msz 1987), chloride (cl– – argentometric method; msz 2009a), ammonium (nh4+ – spectrophotometric method; msz iso 1992), nitrite (no2–), nitrate (no3– – spectrophotometric method; msz 2009b), and phosphate (po43– – spectrophotometric method; msz en iso 2004). the year of 2012, especially the autumn period, was very dry, according to the data of the hungarian meteorological service. the amount of precipitation was only 427 mm in this year, which was 25% less than the hundred-year average (569 mm). sample collection and preparation benthic diatom samples were collected from macrophytes, samples were fi xed in lugol’s solution in the fi eld. diatom valves were prepared using the hot hydrogen-peroxide method (msz en 2003). naphrax synthetic resin was used for embedding (msz en 2003). a leica dmrb research microscope with leica pl fluotar objective with 100× magnifi cation and 1.30–0.60 aperture was used for the identifi cation of diatom taxa. at least 400 valves were counted (msz en 2004). krammer and lange-bertalot (1986, 1988, 1997a, b), krammer and lange-bertalot (1991a, b, 2004a, b), and potapova and hamilton (2007) were used during diatom identifi cation. the omnidia version 5.2 software package (lecointe et al. 1993) and algaebase (guiry and guiry 2014) were used for naming the diatom taxa. in each site three independent samples were collected. data processing and analyses diatoms were classifi ed into halophilic/mesohalophilic and non-halophilic taxa groups according to ziemann et al. 2001. diatom taxa were assigned to fi ve size classes (tab. 1) according to berthon et al. 2011. the biovolume of diatom taxa was calculated by using the omnidia version 5.2 software package (lecointe et al. 1993). to compare the taxa number of each size class with the total taxa number, we calculated the size class ratio (based on taxa numbers) using the following simple equation: size class ratio (based on taxa numbers) = = taxa number in a size class × total taxa number–1 furthermore, comparing halophilic taxa number of each size classes to the total halophilic taxa number, we calculated the size class ratio (based on halophilic taxa numbers) using the following simple equation: tab. 1. diatom size classes according to berthon et al. 2011. diatom size classes biovolume (μm3) s1 5 – 99 s2 100 – 299 s3 300 – 599 s4 600 – 1499 s5 ≥ 1500 halophilic diatom taxa in lowland lotic system acta bot. croat. 74 (2), 2015 291 size class ratio (based on halophilic taxa numbers) = = halophilic taxa number in a size class × total halophilic taxa number–1 principal component analysis (pca) was used to validate the importance of environmental factors according to lepš and šmilauer (2003) and stenger-kovács et al. (2013). to analyse the relationship between diatom taxa composition and validated environmental factors a detrended correspondence analysis (dca) was used, in which environmental factors were added by weighted averaging (lepš and šmilauer 2003, b-béres et al. 2014). results changes in environmental variables all of the 10 measured environmental variables were included into the pca (do, cond, ph, t and concentrations of hco3–, cl–, nh4+, po43–, no2–, no3–). the fi rst three axes of pca explained 93% of the total of species variance. the pca revealed that the factors with the highest correlation were with the fi rst axis conductivity (0.8627), concentrations of hco3– (0.7718), cl– (0.8469), and po43– (0.9569), with the second axis do (0.6450), and with the third axis the concentration of no3– (0.5022). conductivity showed a very high correlation with the concentration of hco3– (0.9033), cl– (0.9419), and po43– (0.9870), and hco3– and cl– both with po43– (0.8036 and 0.8744, respectively). thus, in the dca only the following validated factors were included: concentrations of no3–, hco3–, cl–, and do. the gradient lengths for the fi rst and second axis in dca were 4.186 and 3.026, while the cumulative percentage variances of species data were 21.6 and 28.9 for the fi rst and second axis, respectively. changes in total and halophilic taxa number – size classes and size class ratio (taxa number) highly pronounced changes in diatom composition were observed from the humid spring to the dry autumn. for better clarity, changes are plotted in fi gure 2 only for three representative sites: dögös-kákafoki-főcsatorna (dka), kis-körös-főcsatorna (kko) and kösely (kos) (marked with arrows on fig. 1b). taxa number increased up to autumn almost in all cases (fig. 2a), a lot of taxa appeared only in the dry autumn period (tab. 2). when size class ratios in spring were compared to those in autumn, there were clear increases only in the case of large taxa (s5 size class – 80% of all sampling sites; each representative site in fig. 2a) and small taxa (s1 or/and s2 size classes – 73% of all sampling sites; kos on fig. 2a). similar changes occurred also in halophilic taxa numbers. they increased from spring to autumn in two thirds of the sampling sites (each representative site in fig. 2b). from the identifi ed 31 halophilic and mesohalophilic diatom taxa, 13 taxa appeared only in the samples collected in autumn. the most important difference between the changes of total taxa number and the changes of halophilic taxa number was the distribution of changes in the size classes. while total taxa number increased in all size classes almost in all sites, a clear increase was detectable in halophilic taxa number only in cases of s2 (60%), s4 (80%) and s5 (53.3%) size classes. moreover, the increase of taxa numbers in the different size classes did not defi nitely mean the increase of the size class ratio. it increased only in the cases of kókai z., bácsi i., török p., buczkó k., t-krasznai e., et al. 292 acta bot. croat. 74 (2), 2015 s2 and s4 and/or s5 size classes (73% and 53% of all sampling sites, respectively; s2: each representative site, s4 and s5: dka and kos on fig. 2b). changes in total and halophilic taxon abundance – size classes increasing relative abundances of small taxa (s1 and/or s2 size classes) were observed from spring to autumn in almost every site (dka and kko on fig. 2c; tab. 3). nevertheless, not only the abundances of small taxa, but also the abundances of large taxa (s5 or/and s4 size classes) were increased (s4: dka and kos, s5: kko and kos on fig. 2c). however, the contribution of halophilic taxa to the increasing abundance of s4 size classes was fig. 2. (a) the total taxa number of different cell size classes and size class ratios based on total taxa number; (b) number of halophilic taxa of different cell size classes and size class ratios based on number of halophilic taxa; (c) relative abundances of different cell size classes and relative abundances of halophilic taxa of different cell size classes in spring and in autumn at three representative sampling sites. dka – dögös-kákafoki-főcsatorna, kko – kis-körösfőcsatorna, kos – kösely; ttn – total taxa number, htn – halophilic taxa number, scr – size class ratio and ra – relative abundances. halophilic diatom taxa in lowland lotic system acta bot. croat. 74 (2), 2015 293 tab. 2. diatom taxa appearing only in the dry autumn period. for explanation of size class types see tab. 1. taxa name type of size class fistulifera saprophila s1 nitzschia microcephala s1 sellaphora seminulum s1 nitzschia angustatula s2 cocconeis neodiminuta s2 encyonema minutum s2 navicula cf. kotschyi s2 nitzschia clausii s2 denticula tenuis s3 hippodonta s3 karayevia clevei s3 kolbesia ploenensis s3 encyonema caespitosum s4 luticola goeppertiana s4 nitzschia cf. lorenziana s4 nitzschia prolongata s4 surirella suecica s4 anomoeoneis sphaerophora s5 caloneis amphisbaena s5 cymbella lanceolata s5 craticula cuspidata s5 ctenophora pulchella s5 eunotia formica s5 gomphonema spp. s5 gyrosigma scalproides s5 nitzschia angustata s5 tryblionella calida s5 tab. 3. relative abundance of those small sized taxa the abundance of which increased from spring to autumn. for explanation of size class types see tab. 1. taxa name type of size class achnanthidium eutrophilum s1 eolimna minima s1 mayamaea permitis s1 eolimna subminuscula s2 mayamaea atomus s2 nitzschia paleacea s2 nitzschia pusilla s2 planothidium frequentissimum s2 kókai z., bácsi i., török p., buczkó k., t-krasznai e., et al. 294 acta bot. croat. 74 (2), 2015 much more pronounced (maximum 84%; each representative site in fig. 2c), while halophilic taxa only slightly (maximum 17%) contributed to the increasing abundance of small taxa in autumn. overall, relative abundances of halophilic taxa increased at 53% of all sampling sites from spring to autumn (each representative site on fig. 2c). while abundances of certain halophilic and mesohalophilic taxa (tab. 4) showed positive correlations with chloride and hydrogen carbonate concentrations, other taxa (tab. 5) correlated positively with nitrate (fig. 3). changes in evenness evenness of diatom assemblages also changed from spring to autumn, but inversely to the taxa number: the latter increased almost in every site, while in contrast, evenness decreased from spring to autumn (fig. 4a). the number and/or ratio of those taxa the relative abundances of which were under 0.01 clearly increased in autumn relative to total taxa number (fig. 4a). this was also was observed in the case of halophilic taxa (fig. 4b). evenness decreased from spring to autumn at 73.3% of the sampling sites (fig. 4b). the taxa number and the ratio of those halophilic taxa the abundances of which were under 0.01 tab. 4. halophilic diatom taxa correlating positively with chloride and hydrogen carbonate concentration. for explanation of size class types see tab. 1. taxa name type of size class diatoma tenuis s4 nitzschia cf. lorenziana s4 craticula buderi s5 craticula halophila s5 nitzschia tryblionella s5 tab. 5. halophilic diatom taxa correlating positively with nitrate. for explanation of size class types see tab. 1. taxa name type of size class nitzschia microcephala s1 nitzschia angustatula s2 nitzschia clausii s2 nitzschia frustulum s2 navicula schroeteri s4 nitzschia fi liformis s4 nitzschia prolongata s4 surirella suecica s4 anomoeoneis sphaerophora s5 caloneis amphisbaena f. subsalina s5 nitzschia angustata s5 halophilic diatom taxa in lowland lotic system acta bot. croat. 74 (2), 2015 295 (e.g. anomoeoneis sphaerophora, ctenophora pulchella, caloneis amphisbaena f. subsalina, tryblionella calida), increased (87% and 73.3%, respectively) in autumn relative to total halophilic taxa number. discussion changes in total and halophilic taxa number in spring there was a high amount of precipitation, while the summer and autumn was unusually dry in 2012 in hungary. decreasing water fl ow conditions were thus caused in these dry months in hungarian lowland rivers and channels (b-béres et al. 2014, kókai et al. 2014). we assumed that taxa number was primarily infl uenced by increase of conductivity (strongly connected with decreasing water fl ow conditions and increasing nutrient concentration and/or cl– content), because it has been found that there is a correlation between taxa number and certain ecological parameters (e.g. cl–, total nitrogen, chemical oxygen demand, conductivity) in small hungarian rivers (stenger-kovács et al. 2013). we hypothfig. 3. the relationship among halophilic diatom taxa composition and environmental factors displayed by a detrended correspondence analysis (dca). cumulative percentage variance of species data are 21.6 and 28.9, while gradient lengths are 4.186 and 3.026 for the fi rst and second axis, respectively. the concentrations of cl–, no3– and hco3–, and dissolved oxygen (do) are added as environmental variables using weighted averaging. halophilic species abbreviations are the followings: anomoeoneis sphaerophora – asph, caloneis amphisbaena fo. amphisbaena – camp, caloneis amphisbaena f. subsalina – cass, craticula buderi – crbu, craticula halophila – chal, diatoma moniliformis – dmon, diatoma tenuis – dite, entomoneis paludosa – epal, fragilaria pulchella – fpul, navicula recens – nrcs, navicula salinarum – nsal, navicula schroeteri – nshr, nitzschia angustata – nian, nitzschia angustatula – nzag, nitzschia capitellata – ncpl, nitzschia clausii – ncla, nitzschia fi liformis – nfil, nitzschia frustulum – nifr, nitzschia lorenziana – nlor, nitzschia microcephala – nmic, nitzschia prolongata – nprl, nitzschia pusilla – nipu, nitzschia thermaloides – nthe, nitzschia tryblionella – ntry, nitzschia umbonata – numb, surirella ovalis – sovi, surirella suecica – ssue, tryblionella apiculata – tapi, tryblionella calida – tcal, tryblionella hungarica – thun, tryblionella levidensis – tlev. kókai z., bácsi i., török p., buczkó k., t-krasznai e., et al. 296 acta bot. croat. 74 (2), 2015 esised that the number of those taxa that prefer higher nutrient concentration and/or conductivity would be higher in autumn than in spring. this fi rst hypothesis was confi rmed: in the samples collected in autumn just such new taxa appeared, those that prefer high nutrient concentrations or high levels of conductivity (e.g. anomoeoneis sphaerophora, caloneis amphisbaena f. subsalina, craticula spp., encyonema caespitosum, eolimna minima, fistulifera saprophila, gyrosigma scalproides, nitzschia clausii, nitzschia lorenziana, sellaphora seminulum – ziemann et al. 2001, zalat 2002, bolla et al. 2010, rimet 2012). however, increased ratio of taxa number in different size classes was pronounced only in the cases of small taxa (s1 and s2 classes) and large taxa (s5 class). new taxa appeared in autumn in small size classes (e.g. eolimna minima, fistulifera saprophila), are frequent in polluted water (beltrami et al. 2012, kelly and ector 2012, rimet 2012). in contrast, a high number of newly-appeared taxa of the s5 size class were halophilic or mesohalophilic (e.g. anomoeoneis sphaerophora, caloneis amphisbaena f. subsalina, nitzschia angustata, tryblionella calida – ziemann et al. 2001, zalat 2002). the number of mesohalophilic and halophilic taxa also showed a considerable increase in s4 size class (e. g. nitzschia cf. lorenziana, nitzschia prolongata – zalat 2002, carter and belcher 2010). fig. 4. (a) taxa number and evenness of whole diatom assemblages in spring and autumn; (b) taxa number and evenness of halophilic taxa in spring and autumn. the abbreviations of the samling sites are the following dióéri-főcsatorna (dio), dögös-kákafoki-főcsatorna (dka), hamvas-csatorna (ham), holt-sebes-körös (hsk), hortobágy (hor), hortobágy-berettyó (hbe), keleti-főcsatorna (kfc), kígyósi-főcsatorna (kig), kis-körös-főcsatorna (kko), kösely (kos), kutas-főcsatorna (kut), nagy-ér (nag), nagytóti-toprongyoscsatorna (nto), sárréti-csatorna (sar), sebes-körös (skl). halophilic diatom taxa in lowland lotic system acta bot. croat. 74 (2), 2015 297 changes in total and halophilic taxon abundance we hypothesised that the abundance of diatom taxa preferring or tolerating high nutrient contents would increase from spring to autumn, since nutrient concentration has a key role in shaping diatom assemblages (e.g. stevenson et al. 2008, stenger-kovács et al. 2013). we observed that the abundances of mobile and/or stalked taxa like planothidium frequentissimum, cocconeis placentula ssp., mayamaea permitis, cymbella lanceolata, encyonema caespitosum, eolimna minima, eolimna subminuscula, gomphonema spp., nitzschia paleacea highly increased from spring to autumn. these taxa can be frequent in water with high nutrient load and conductivity (rimet 2012, b-béres et al. 2014). the results supported our assumptions; an increase of conductivity is followed by the increase of the abundance of mesohalophilic and halophilic taxa (ziemann et al. 2001). most of the studied channels and rivers belonged to freshwater type (cl– < 100 mg l–1), according to the classifi cation of waters on the basis of cl– concentration (van dam et al. 2003). exceptions were the sampling sites hamvas-csatorna in spring and autumn (ham: fresh-brackish water, cl– concentration 274 mg l–1 and 223 mg l–1 respectively) and nagyér in autumn (nag: brackish-fresh water, cl– concentration 557 mg l–1). in these sampling sites diatom species of the kind known as halophilic taxa, preferring or tolerating high conductivity and nutrient contents were abundant (nitzschia frustulum, nitzschia fi liformis – hamvas-csatorna (ham) in spring; and craticula buderi –nagy-ér (nag) in autumn, respectively; blin and bailey 2001, ziemann et al. 2001, zalat 2002, bona et al. 2007, rimet 2012). in other sampling sites, belonging to freshwater types, increasing abundance of other halophilic taxa (e.g. tryblionella species, nitzschia cf. lorenziana, nitzschia pusilla and navicula schroeteri) occurred with increasing conductivity/cl– concentration and/or increasing nutrient content, confi rming the observations of kaštovský et al. (2010) and stenger-kovács et al. (2013). the composition of cell size classes in diatom assemblages includes useful information about the structure of the given assemblages and also about the pollution level and thereby about the physical and chemical composition of the water (berthon et al. 2011). we hypothesised that increasing nutrient content and/or conductivity cause conspicuous changes in composition of size classes; namely, the ratio of small disturbance-tolerant taxa and large halophilic/mesohalophilic taxa increases from spring to autumn. our results confi rmed this second hypothesis, the distribution of small size (s1 and/or s2 classes) and large size (s5 class) classes clearly changed. in accordance with berthon et al. (2011), an increasing abundance of s5 class was observed in relation to increasing nitrate and/or phosphate concentration and decreasing ammonium content. however, a clear increase in s1 size class was also observed in autumn, related to the increased nutrient contents and/or conductivity. we suggest that the reason for this was the composition of this size class, namely, 71% of the taxa were mobile (navicula and nitzschia spp.) or ruderal species (staurosira and pseudostaurosira spp.). generally, mobile taxa prefer enriched habitats, and their abundance and taxa number increase with increasing nutrient contents (fairchild et al. 1985, van der grinten et al. 2004, berthon et al. 2011). moreover, ruderal strategists prefer habitats with high amounts of nutrients (padisák 2001, passy 2007) kókai z., bácsi i., török p., buczkó k., t-krasznai e., et al. 298 acta bot. croat. 74 (2), 2015 changes in evenness the studied rivers and channels were exposed to both natural and anthropogenic impacts (lack of rainfall, agricultural pollution in the form of nutrient loads). these events caused an increased conductivity, and/or cl– and/or nutrient concentration in almost all sampling sites. the results supported our third hypothesis, that evenness of diatom assemblages decreases with increasing conductivity and/or nutrient content (nh4+, po43–), according to zalat (2002), ndiritu et al. (2006) and stenger-kovács et al. (2013). confi rming our fi ndings, they observed a decreasing diversity in diatom assemblages parallel to increasing salinity/conductivity, and/or nutrient (especially phosphate) contents. number of taxa with low abundances (under 0.01%) increased remarkably from spring to autumn. this was primarily due to the increasing number of halophilic taxa with low abundances and decreasing abundances of other taxa, which were sensitive to high conductivity or nutrient content (e.g. achnanthidium minutissimum, encyonopsis microcephala – de fabricius et al. 2003, ács et al. 2006, rusanov et al. 2009, rimet 2012, stenger-kovács et al. 2013). conclusions our results showed that even a one-year study can demonstrate such important changes in benthic diatom assemblages as decreasing diversity, loss of taxa, and increasing appearance of halophilic and mesohalophilic species caused by increasing conductivity and/or nutrient content. our results pointed out those diatom assemblages sensitively indicate short-term changes in lowland lotic ecosystem; we found that studying changes in the ratio of halophilic and mesohalophilic taxa is especially useful for detection of environmental changes caused by either anthropogenic or natural effects. knowledge based on such results provides an opportunity e.g. for rapid intervention for environmental protection (e.g. water supply in the case of smaller rivers or channels). our results could form a basis for a forthcoming comprehensive study on the primary or secondary salinisation in hungarian lowland medium or small rivers and channels. acknowledgements the authors are thankful for the support of the bolyai jános research scholarship of the hungarian academy of sciences, the internal research project of the university of debrecen (bácsi i.) and for the kind support of the hungarian scientifi c research found pd 100192 (török p.) during manuscript preparation. the work was supported by támop 4.2.1./b-09/1/konv-2010-0007, támop-4.2.2_b-10/1-2010-0024, támop 4.2.2.c-11/ 1/konv-2012-0010 and támop 4.2.4.a/2-11/1-2012-0001 »national excellence program – elaborating and operating an inland student and researcher personal support system«, projects (török p., t-krasznai e., b-béres v.). the támop projects are implemented through the new hungary development plan, co-fi nanced by the european social fund and the european regional development fund. the authors are thankful for measuring the physico-chemical parameters to the environmental protection and nature conservation authority, trans-tisza region, for rainfall data to the hungarian meteorological service. halophilic diatom taxa in lowland lotic system acta bot. croat. 74 (2), 2015 299 references ács, é., szabó, k., kiss, k. t., hindák, f, 2003: benthic algal investigations in the danube river and some of its main tributaries from germany to hungary. biologia 58, 545–554. ács, é., szabó, k., kiss, á. k., tóth, b., záray, g. y., kiss, k. t., 2006: 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system of rivers based on fi rst results of the hungarian phytobenthos surveillance monitoring. hydrobiologia 695, 125–135. vasas, g., farkas, o., borics, g., felföldi, t., sramkó, g., batta, g., bácsi, i., gonda, s., 2013: appearance of planktothrix rubescens bloom with [d-asp3, mdha7]mc–rr in gravel pit pond of a shallow lake-dominated area. toxins (basel) 5, 2434–2455. zalat, a. a., 2002: distribution and origin of diatoms in the bottom sediments of the suez canal lakes and adjacent areas, egypt. diatom research 17, 243–266. ziemann, h., kies, l., schulz, c., 2001: desalinization of running waters iii. changes in the structure of diatom assemblages caused by a decreasing salt load and changing ion spectra in the river wipper (thuringia, germany). limnologica 31, 257–280. opce-str.vp acta bot. croat. 68 (1), 153–158, 2009 coden: abcra 25 short communication issn 0365–0588 potentilla ´ aurulenta gremli (rosaceae), a nothospecies new to poland jeremi kolv odziejek* department of geobotany and plant ecology, university of lv ódzb, banacha 12/16, 90-237 lv ódzb, poland potentilla ´aurulenta gremli (= p. heptaphylla l. ´ p. tabernaemontani aschers.) is reported for the first time from poland. the morphological characteristics, ecological requirements, and distribution of the nothospecies are presented together with photographic documentation. keywords: potentilla ´aurulenta, taxonomy, poland introduction during an expedition to wzgórza trzebnickie, poland, in may 2004, the author found specimens of potentilla that differ from the congeners in poland. after a morphological study, literature searches (zimmeter 1884, wolf 1908, szafer and pawlv owski 1955, soják 1995, gerstberger 2002, kurtto et al. 2004) and examination of many species, the author concluded that it represents the hybrid of p. heptaphylla l. and p. tabernaemontani aschers. methods morphological features of the species potentilla ´aurulenta were described and compared with those of p. heptaphylla l. and p. tabernaemontani (tab. 1). basal leaves of potentilla ´aurulenta from herbarium material were rehydrated by boiling in water and detergent. two small pieces of leaves were cut from the lateral, right leaflet and subjected to critical point drying. for scanning electron microscopy (sem), samples were mounted on metal stubs, spattered with technical gold (pelco s.c. 6 coating system), examined and photographed using a tesla bs 340 scanning electron microscope. the hair types were analyzed on 5 photographs for each taxon. the phytocoenoses were analysed according to the central european zürich – montpellier school (braun-blanquet 1964). the positions of the localities of potentilla ´aurulenta were determined by gps garmin etrex vista. acta bot. croat. 68 (1), 2009 153 * corresponding address, e-mail: kolo@biol.uni.lodz.pl u:\acta botanica\acta-botan 1-09\kolodziejek2.vp 22. travanj 2009 12:52:59 color profile: disabled composite 150 lpi at 45 degrees nomenclature of taxa was used according to wolf (1908) and kurtto et al. (2004). the abbreviations of herbaria correspond with the index of herbaria (holmgren et al. 1990). results and discussion potentilla ´aurulenta gremli (= p. heptaphylla l. ´ p. tabernaemontani aschers.) (figs. 1, 3, 4) excurs.-fl. schweiz. 12.1884. – p. subopaca zimmeter, jahres.-ber. staats-ober.-realschule steyr. 14, 20. 1884. – synonymized by wolf (1908: 604). – p. explanata zimmeter, jahres.-ber. staats-ober.-realschule steyr. 14, 20. 1884. – synonymized by soják (1995: 313). – p. matzialekii domin, sitzber. böhm. ges. wiss. 25: 33. 1903. – synonymized by wolf (1908: 604). indicatio locotypica: schwitzerland, osterfingerbad bei schaffhausen. – original material: not traced. characteristic features (tab. 1): perennial with slender stock; lateral flowering stems up to 30–40 cm, mostly branched above middle with hairs borne on minute tubercles. basal 154 acta bot. croat. 68 (1), 2009 kolv odziejek j. tab. 1. comparison of main diagnostic characters among p. heptaphylla l., p. tabernaemontani aschers. and the hybrid potentilla ´aurulenta gremli. character p. heptaphylla p. tabernaemontani potentilla ´aurulenta stems with slender stock; lateral flowering stems up to 40 cm with numerous procumbent woody stems freely rooting at the nodes; lateral flowering stems not more than 10 cm high with slender stock; lateral flowering stems up to 30–40 cm leaflets ovate-lanceolate, dentate or creneate-dentate cuneate-oblanceolate, dentate or cuneate-dentate ovate-lanceolate, dentate stipules stipules of basal leaves ovate-lanceolate, acute stipules of basal leaves linear to linear-triangular stipules of basal leaves lanceolate, acute hairiness hairs on stems and leaves with soft hairs borne on minute tubercles at base, and shorter, often reddish multicellular glandular hairs hairiness consisting of simple curved hairs only hairiness consisting of mixture of simple curved and straight hairs borne on minute tubercles at base sepals ovate-lanceolate; epicalyx segments linear-lanceolate, as long as or shorter than sepals ovate, epicalyx segments lanceolate, obtuse, much shorter than sepals ovate, epicalyx segments lanceolate, obtuse, shorter than sepals petals 5–7 mm 6–10 mm 5–10 mm u:\acta botanica\acta-botan 1-09\kolodziejek2.vp 17. travanj 2009 11:19:47 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 155 potentilla ´ aurulenta a nothospecies new to poland fig. 1. representative specimens of potentilla ´aurulenta gremli. a b c d e f g a b c d e f g a b c d e f g a b c d e f g fig. 2. distribution of potentilla ´aurulenta gremli – cartographic square in poland atpol (zajac 1978). u:\acta botanica\acta-botan 1-09\kolodziejek2.vp 17. travanj 2009 11:19:49 color profile: disabled composite 150 lpi at 45 degrees 156 acta bot. croat. 68 (1), 2009 kolv odziejek j. fig. 3. upper surface of leaflets glabrous and at margin with soft simple straight hairs borne on minute tubercles at base, potentilla ´aurulenta gremli (sem). fig. 4. lower surface of leaflets on veins with simple curved hairs borne on minute tubercles at base; between veins with relatively sparse simple shorter, curved hairs, potentilla ´aurulenta gremli (sem). u:\acta botanica\acta-botan 1-09\kolodziejek2.vp 17. travanj 2009 11:19:50 color profile: disabled composite 150 lpi at 45 degrees and lower stem leaves digitate with 5–7 leaflets; stipules of basal leaves lanceolate, acute. terminal tooth at basal leaves equalling, slightly shorter or slightly longer than the adjacent lateral teeth. leaves on upper side glabrous and at margin with soft simple straight hairs borne on minute tubercles at base. lower surface of leaflets on veins and at margin with simple curved hairs borne on minute tubercles at base; between veins with relatively sparse simple shorter, curved hairs. flowers numerous, in cymes. 5-merous, yellow. sepals ovate-lanceolate; epicalyx-segments lanceolate shorter than sepals. style conical at basal, slightly clavate at apex. flowering: v–viii. specimens examined: poland, wzgórza trzebnickie: tarnowiec village near trzebnica 51°13’56”n, 16°46’01”e, 164 m (be37), 6.5.2004 j. kolvodziejek (lod). ecology: the plants grow in thermophilous xerothermic grasslands from the class festuco-brometea br.bl. et r.tx. phytosociological characteristic of the locality of potentilla ´aurulenta is documented by the following relevé: wzgórza trzebnickie: tarnowiec village near trzebnica 51°13’56”n, 16°46’01”e, 164 m, 6.5.2004, 5´5 m, ph in h2o = 7.6; d = 10%: hylocomium splendens 1, tortella tortuosa +; c = 90%: sedum sexangulare 3, potentilla ´aurulenta 2, fragaria viridis 1, pimpinella saxifraga 1, thymus serpyllum 1, acinos arvensis +, anthyllis vulneraria +, briza media +, centaurea scabiosa +, galium vernum +, g. verum +, leontodon hispidus +, linum catharticum +, poa pratensis +, polygala comosa +, potentilla tabernaemontani +, primula officinalis r, rhinanthus minor +, sanguisorba minor +, scabiosa ochroleuca +, vicia cracca r. acknowledgmens i would like to thank dr. thomas gregor (hochschule vechta, germany) for his help in identification of plants. references braun-blanquet, j., 1964: pflanzensoziologie. grundzüge der vegetationskunde. 3. aufl. springer verlag, wien. gerstberger, p., 2002: potentilla l. in: conert, h. j., hamann, a., schultze-motel, w., wagenitz, g. (eds), g. hegi. illustrierte flora von mitteleuropa 4(2c), 109–205. blackwell, berlin. holmgren, p. k., holmgren, n. h., barnett, l. c., 1990: index herbariorum, 1: the herbaria of the world. new york botanical garden, bronx. kurtto a., lampinen r., junikka l., 2004: rosaceae (spiraea to fragaria, excl. rubus). in: kurtto, a., lampinen, r., junikka, l. (eds.), atlas florae europaeae. distribution of vascular plants in europe 13. rosaceae (spiraea to fragaria, escl. rubus). the committee for mapping the flora of europe, societas biologica fennica, vanamo, helsinki. soják, j., 1995: potentilla l. in: slavik b. (ed.), flora of the czech republic 4, 283–314. academia, praha. acta bot. croat. 68 (1), 2009 157 potentilla ´ aurulenta a nothospecies new to poland u:\acta botanica\acta-botan 1-09\kolodziejek2.vp 22. travanj 2009 12:53:02 color profile: disabled composite 150 lpi at 45 degrees szafer, w., pawlv owski, b., 1955: potentilla l. in: szafer, w., pawlv owski, b. (eds), flora polska 7, 96–143. wydawnictwo naukowe pwn, warszawa-kraków. wolf, t., 1908: monographie der gattung potentilla l. bibliotheca botanica 71, 1–714. zajac, a., 1978: atlas of distribution of vascular plants in poland. taxon 27, 481–484. zimmeter, a., 1884: die europäischen arten der gattung potentilla. jahres-bericht der k. k. staats-ober-realschule in steyr 14, 1–31. 158 acta bot. croat. 68 (1), 2009 kolv odziejek j. u:\acta botanica\acta-botan 1-09\kolodziejek2.vp 17. travanj 2009 11:19:50 color profile: disabled composite 150 lpi at 45 degrees untitled acta bot. croat. 75 (1), 2016 53 acta bot. croat. 75 (1), 53–59, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0007 issn 0365-0588 eissn 1847-8476 alive and kicking, or, living on borrowed time? – microsatellite diversity in natural populations of the endangered ulmus minor mill. sensu latissimo from croatia marko zebec1*, marilena idžojtić1, zlatko šatović2, igor poljak1, zlatko liber3 1 university of zagreb, faculty of forestry, department of forest genetics, dendrology and botany, svetošimunska 25, hr-10000 zagreb, croatia 2 university of zagreb, faculty of agriculture, department of seed science and technology, svetošimunska 25, hr-10000 zagreb, croatia 3 university of zagreb, faculty of science, department of biology, division of botany, marulićev trg 9, hr-10000 zagreb, croatia abstract – the main objective of this research was to assess the genetic diversity of 5 natural fi eld elm populations in croatia. the study results suggest that the observed populations are characterized by a satisfactory amount of heterozygosity, and that the impact of the dutch elm disease on the amount of genetic diversity in the sampled populations is currently negligible. however, one population displayed a signifi cant excess of heterozygosity, implying a genetic bottleneck. the existence of a very clear genetic differentiation between the continental and the mediterranean populations of ulmus minor in croatia was noticed. keywords: bottleneck event, endangered species, genetic diversity, natural populations, nuclear microsatellites, ulmus minor * corresponding author, e-mail: mzebec@sumfak.hr introduction the majority of contemporary studies of elms are based on the need to preserve their genetic diversity and to resolve the complex situation in their classifi cation through research into genetic variability and morphological analyses (heimler et al. 1990, jeffers 1999, nielsen and kjær 2010). the reduction in genetic diversity of european elms is largely due to the anthropogenic-induced destruction of habitat, introduction of new elm species and spontaneous hybridization with ornamental species (u. pumila l.). likewise, the extremely high susceptibility of elms of the ulmus heybr. section to the ophiostoma novo-ulmi brasier pathogenic fungus, played an important role in the catastrophic wilting of adult elm trees through europe (collin et al. 2000). the dutch elm disease (ded) caused by the fungus was fi rst observed in france and the netherlands in around 1910 and rapidly spread worldwide afterwards (spierenburg 1921, buisman 1928). the european fi eld elm (ulmus minor mill. sensu latissimo) is a noble hardwood which, together with the european white elm (ulmus laevis pall.) and the wych elm (ulmus glabra huds.), belongs to the european segment of the ulmus l. genus. u. minor s.l. is present in most of europe except in northern areas, and also appears along the mediterranean coast, on most of the islands of the mediterranean sea, as well as in northern africa, asia minor, caucasus and transcaucasia (richens 1976). the natural populations of the fi eld elm are characterized by a very wide ecological tolerance, and therefore it occupies a wide range of habitats, from fl oodplain forests to dry mediterranean forest communities (richens 1983, namvar and spethmann 1985). the taxonomic status of the fi eld elm in europe could be viewed in two, mutually completely exclusive, ways. the fi rst, proposed by melville (1975, 1978) implies the existence of a larger number of small species (microspecies treatment). the second, suggested by richens (1968, 1980) affi rms the existence of only one collective species of the fi eld elm on the european continent, u. minor mill. sensu latissimo, as adopted in this paper. in contemporary research on fi eld elm genetic diversity, classical sampling methods, due to the small number of adult trees as a result of ded, have not been used by a majority of researchers. therefore, in recent studies, “a popuzebec m., idžojtić m., šatović z., poljak i., liber z. 54 acta bot. croat. 75 (1), 2016 lation” consists of set of trees, originating from the same geographic region and the researched samples include a mixture of trees derived from clonal plantations, ex situ collections as well as ded tolerant clones and trees from parks and hedges (machon et al. 1997, cogolludo-agustín et al. 2000). hence, the heterogeneity of sampled material is extremely great and yet the genetic diversity and structure of natural elm populations remains unknown. there is no dispute about the indigenous nature of the fi eld elm in croatia. this taxon occupies a well-defi ned ecological niche, with the possibility of hybridization with the wych elm reduced to a minimum. indeed, the only issue in croatia is the problem of assessing the variability of the taxon. the fi eld elm appears in the continental and the mediterranean part of croatia, where the ecological conditions of the distribution range are very different, which is manifested by the high variability of morphological traits (zebec 2010). pure stands of the fi eld elm are also frequent, particularly in anthropogenic habitats, as well as on abandoned farmland and pastures. a clear separation of the continental and the mediterranean populations of the fi eld elm according to leaf morphology was stated by zebec (2010), who conducted the fi rst extensive research on the entire area of this species’ spread in croatia. as microsatellite markers for the fi eld elm were not developed until 2004, most of the research on genetic diversity to date has been conducted using isozymes (machon et al. 1995, cogolludo-agustín et al. 2000), allozymes (machon et al. 1997), rapd technique and issrs (coleman et al. 2000, goodall-copestake et al. 2005) and cpdna pcrrflp markers (collin et al. 2004). microsatellites found their application in the resolving of the taxonomic status of u. procera salisb. (gil et al. 2004) and recently in assessing whether the fi eld elm is or is not indigenous to the balearic islands (fuentes-utrilla et al. 2014). the aim of this research was to assess the level of genetic diversity between and within the fi eld elm populations in croatia, and thus determine the degree of the negative impact of ded on the biodiversity of the species. there are several advantages of performing this research in croatia. firstly – since the balkan region is considered a glacial refugium, croatia by its geographical position remains a place of presumed high biodiversity. secondly – croatia combines aspects of both the continental and the mediterranean area of elm distribution. thirdly – in the southern area of its distribution (including croatia) the fi eld elm favors sexual reproduction and since there were a suffi cient number of individuals for the research to be performed, we defi ned the term “population” in this study in the standard biological way. material and methods plant material the quantifi cation of genetic diversity of the fi eld elm was conducted by an analysis of genotypes of 96 individuals from 5 populations (đurđevac, zagreb, pula, nin, neretva). the populations were carefully selected, in order to enable, in concert with their geographic location and climatic adaptation, precise insight into the genetic variability of the fi eld elm in croatia (fig. 1). taxonomical identifi cation of sampled plant material was confi rmed by dr. zlatko liber, a plant taxonomist of the department of botany of the university of zagreb and voucher specimens of plants (ids 37109 – 37113) have been deposited with the herbarium croaticum (za), university of zagreb, croatia (on-line suppl. tab. 1). dna extraction, pcr amplifi cation and microsatellite genotyping total genomic dna was extracted from silica-dried leaf terminal buds using a dneasy® plant mini kit (qiagen®) according to the manufacturer’s instructions. for this procedure 25 mg of dry tissue of single plants was used. the quality and concentration of the dnas were checked by electrophoresis in 0.8% (w/v) agarose gel and additionally by qubit 2.0® fluorometer using quant-ittm dsdna br assay kit (invitrogen®). five ssr markers were used in the study (whiteley et al. 2003, collada et al. 2004): ulm2 (ay300797), ulm8 (ay300800), ulmi1–21 (ay520827), ulmi1–98 (ay520829) and ulmi1–165 (ay520830) as presented in tab. 1. amplifi cation was performed in a geneamp pcr system 9700 (applied biosystems®). the pcr conditions described by collada et al. (2004) were used for the pcr amplifi cations of three microsatellite loci ulmi1–21 (ay520827), ulmi1– 98 (ay520829) and ulmi1–165 (ay520830), whereas the amplifi cation protocol proposed by whiteley et al. (2003) was used for microsatellite loci ulm2 (ay300797) and ulm8 (ay300800), respectively. amplifi cation products were separated and detected on an automated sequencer (abi prism 3130 dna sequencer, applied biosystems, foster city, ca, usa) at macrogen inc. (korea). fragment sizes were determined using the program genemapper® 4.0 (applied biosystems® 2005). fig. 1. geographical position of sampled populations. populations are indicated by following numbers: 1 – đurđevac; 2 – zagreb; 3 – pula; 4 – nin; 5 – neretva. genetic diversity of ulmus minor mill. sensu latissimo in croatia acta bot. croat. 75 (1), 2016 55 data analysis by means of descriptive statistical analysis, performed on original microsatellite data matrix using powermarker 3.23 software (liu 2002), the total number of alleles per locus (na), the observed heterozygosity (ho), the expected heterozygosity or gene diversity (he) and the polymorphism information content (pic) for each microsatellite locus as well as the average number of alleles nal, ho, he in each population across loci were calculated. in order to assess allelic richness (nar) and to estimate the signifi cance of genetic differentiation (fst) between population pairs we used the fstat 2.9.3.2 program package (goudet 1995). genepop 3.4 (raymond and rousset 1995) was used to test genotypic frequencies for each locus in each population for conformance to hardy-weinberg (hw) expectations as well as for calculation of the inbreeding coeffi cient (f) for each locus in each population following weir and cockerham (1984). the probability test was based on the markov chain method (guo and thompson 1992, rousset and raymond 1995) using 10,000 de-memorization steps, 100 batches and 5,000 iterations per batch. sequential bonferroni adjustments (holm 1979, rice 1989) were applied to correct for the effect of multiple tests using sas 8.02. the program bottleneck 1.2.02 (cornuet and luikart 1996, piry et al. 1999) was used to test for evidence of recent bottleneck events on the basis of this theoretical expectation. the gene diversity observed (he) was compared to the gene diversity expected at mutation-drift equilibrium (heq) and calculated from the observed number of alleles under different mutation models: infi nite allele model – iam (kimura and crow 1964), stepwise mutation model – smm (ohta and kimura 1973) and an intermediate twophase model – tpm (di rienzo et al. 1994). the tpm model was applied assuming 30% – tpm 1 (pascual et al. 2001, hoelzel et al. 2002, kuehn et al. 2003) and 5% multistep changes – tpm 2 (piry et al. 1999). based on the number of loci in our dataset, the wilcoxon sign-rank test (luikart et al. 1998) was chosen for the statistical analysis of heterozygote excess or defi ciency as recommended by piry et al. (1999). with the intention of depicting genetic relationships among elm trees visually, a factorial correspondence analysis (fca) was carried out using genetix 4.05 (belkhir et al. 2004). finally, pairwise nei’s standard genetic distances (nei 1972) were calculated and an unrooted phylogenetic tree was constructed using a neighbor-joining algorithm (saitou and nei 1987) with 10,000 bootstraps (felsenstein 1985) over microsatellite loci as implemented in the phylip 3.6b software package (felsenstein 2004). results microsatellite diversity in all, 54 alleles were detected by surveying 96 ulmus trees from 5 populations using 5 ssr markers. the number of alleles per ssr marker ranged from seven (ulmi1–98) to eighteen (ulmi1–165) with an average of 10.80 alleles per locus. pic values varied for a single locus from 0.468 to 0.858, with an average value of 0.628, by which it can be concluded that all microsatellite loci scored in this research, displayed a suffi cient level of polymorphism. overall observed heterozygosity values (per marker) ranged from 0.462 to 0.777, with a mean value of 0.594. the results showed some elevated levels of expected heterozygosity compared to those of observed heterozygosity, with values ranging from 0.487 to 0.871 and a mean value of 0.658 (tab. 1). intrapopulation diversity and hw equilibrium the mean number of alleles per locus in the populations ranged from 3.2 (nin) to 7.4 (đurđevac). the number of alleles per locus independent of sample size (the allelic richness) ranged from 3.06 (nin) to 6.80 (đurđevac). the observed heterozygosity per population varied from 0.530 (zagreb) to 0.709 (pula) and the expected heterozygosity per population varied from 0.418 (nin) to 0.642 (pula). expected heterozygosity values were higher than observed heterozygosity values in the đurđevac and zagreb populations or vice versa in the pula and nin populations. the neretva population had similar values of ho and he (tab. 2). signifi cant deviations from hw equilibrium using the inbreeding coeffi cient (f) were found for the pula population at most loci except ulm8 and also for the nin population but only at loci ulm2 and ulmi1–165. the inbreeding coeffi cient values were negative in the discussed cases, indicating excess of heterozygotes in a population compared to hw equilibrium, while other populations exhibited no signifi cant deviations from hw equilibrium (tab. 3). tab. 1. source, repeat motifs, size ranges, number of alleles (na), observed (ho) and expected heterozygosity (he) and polymorphic information content (pic) for fi ve microsatellite loci used in fi ve ulmus populations (n = 96). source locus repeat motif size range (bp) na ho he pic whiteley et al. (2003) ulm2 (ay300797) (cag)8 98–110 8 0.625 0.662 0.610 ulm8 (ay300800) (gct)12ctt(gct)2 cca. 180 10 0.472 0.487 0.468 collada et al. (2004) ulmi1–21 (ay520827) (ct)10 204–220 11 0.634 0.736 0.699 ulmi1–98 (ay520829) (ct)6n14(ct)7 124–156 7 0.462 0.533 0.507 ulmi1–165 (ay520830) (ct)9 128–166 18 0.777 0.871 0.858 mean 10.80 0.594 0.658 0.628 min 7 0.462 0.487 0.468 max 18 0.777 0.871 0.858 zebec m., idžojtić m., šatović z., poljak i., liber z. 56 acta bot. croat. 75 (1), 2016 test for mutation-drift equilibrium at all observed polymorphic loci under four mutation models in fi ve ulmus populations revealed a recent reduction in effective population size (genetic bottleneck) only in the pula population. accordingly, the pula population displayed signifi cant (p < 0.05) gene diversity excess (he > heq) on average across all 5 polymorphic loci under iam and tpm1, or across 4 polymorphic loci under tpm2 and smm, respectively. the đurđevac population showed signs of population expansion regarding signifi cant (p < 0.05) gene diversity defi ciency (he < heq) on average across all polymorphic loci under mutation models tpm1, tpm2 and smm. other populations did not show statistically signifi cant departures from optimal heterozygosity level, assumed by mutation-drift equilibrium (tab. 4). genetic differentiation among populations the results of fca are graphically presented by fig. 2, which delineates the projection of the individuals and population barycenters on the plane defi ned by the fi rst two fca axes, thus providing a better insight into genetic relationship between studied populations. the fi rst two axes accounted for 35.32% and 31.68% of the total inertia, respectively. clear differentiation between continental (đurđevac, zagreb) and mediterranean (nin, neretva) populations was notable along the fi rst axis, whilst the pula population located centrally with a barycenter closer to the continental group. there was some minor overlap in positioning of trees from the đurđevac and zagreb populations as well as nin and neretva. poor differentiation between continental (đurđevac, zagreb) and mediterranean (nin, neretva) populations was evident along the second fca axis, whereas the pula population occupied a discrete, distinct area of multivariate space. by means of nei’s standard genetic distance (dnei72) and index of genetic differentiation (fst) values, a similar pattern of differentiation among observed populations was expressed (tab. 5). the greatest dnei72 values were detected between pula and neretva (0.409), whilst the lowest values were observed between zagreb and đurđevac (0.106). intriguingly, lower dnei72 values were more remarkable between the pula and zagreb populations (0.278) than between those of pula and nin (0.371). relatively small genetic distance was determined among the mediterranean populations of nin and the neretva; likewise dnei72 values tab. 2. population, sample size and genetic variability estimates based on data from fi ve microsatellite loci in fi ve ulmus populations. no – number of populations; n – sample size; na – mean number of alleles; nar – number of alleles per locus independent of sample size (allelic richness); observed (ho) and expected heterozygosity (he). no. population n na nar ho he 1 đurđevac 20 7.4 6.80 0.566 0.619 2 zagreb 20 6.8 6.21 0.530 0.626 3 pula 20 3.8 3.74 0.709 0.642 4 nin 19 3.2 3.06 0.626 0.418 5 neretva 17 4.2 4.12 0.547 0.561 tab. 3. inbreeding coeffi cients (f) across fi ve microsatellite loci in fi ve ulmus populations. signifi cant deviations from hardy-weinberg equilibrium after sequential bonferroni corrections: ** – signifi cance at the 1% nominal level; * – signifi cance at the 5% nominal level; ns – non-signifi cant values. no – number of populations; a – monomorphic locus. no. population ulm2 ulm8 ulmi 1–21 ulmi 1–98 ulmi 1–165 1 đurđevac 0.177 ns –0.166ns –0.072 ns 0.215 ns 0.306 ns 2 zagreb 0.166 ns –0.048 ns 0.263 ns 0.230 ns 0.125 ns 3 pula –0.045** 0.111ns 0.209** –0.596** –0.099* 4 nin –0.615** –0.245 ns –0.397 ns –a –0.581* 5 neretva 0.491 ns –0.141 ns 0.032 ns –0.067 ns 0.017 ns tab. 4. number of loci showing heterozygosity defi ciency / heterozygosity excess. test for mutation-drift equilibrium at polymorphic loci in fi ve ulmus populations under four mutation models: infi nite allele model (iam), two phase model assuming 30% multistep changes (tpm1), two phase model assuming 5% multistep changes (tpm2), stepwise mutation model (smm). * – signifi cant (p < 0.05) gene diversity defi ciency (he < heq) on average across all polymorphic loci using wilcoxon’s test – sign of population expansion. # – signifi cant (p < 0.05) gene diversity excess (he > heq) on average across all polymorphic loci using wilcoxon’s test – sign of population bottleneck. no. population mutation model iam tpm1 tpm2 smm 1 đurđevac 3 / 2 5*/ 0 5*/ 0 5*/ 0 2 zagreb 1 / 4 2 / 3 3 / 2 3 / 2 3 pula 0 / 5# 0 / 5# 1 / 4# 1 / 4# 4 nin 2 / 2 2 / 2 3 / 1 3 / 1 5 neretva 2 / 3 2 / 3 3 / 2 3 / 2 fig. 2. factorial correspondence analysis (fca) of 96 ulmus trees belonging to fi ve populations. each individual genotype is indicated by a small sign, while the population barycenters are represented by larger ones. populations are depicted by following symbols: đur đe vac – fi lled squares; zagreb – fi lled rhombs; pula – un fi lled squares; nin – unfi lled rhombs; neretva – fi lled triangles. genetic diversity of ulmus minor mill. sensu latissimo in croatia acta bot. croat. 75 (1), 2016 57 among continental populations of zagreb and đurđevac were also low. correspondingly to the calculated dnei72 values for the 5 studied populations, it is evident that the distances between all population pairs within the same region were less than the genetic distance between pairs of populations belonging to different regions (continental/mediterranean). statistically signifi cant fst values were present between most population pairs, excluding đurđevac and zagreb. calculated signifi cant values ranged from 0.098 between zagreb and pula to 0.210 concerning nin and pula. according to the values of fst, pronounced genetic differentiation among populations belonging to different regions was apparent. interestingly, only pula stands out from this trend of population division (tab. 5). the unrooted neighbor-joining tree visually elucidates the pattern of genetic differentiation between the observed populations, suggesting the existence of a clear genetic differentiation between continental and mediterranean populations of the fi eld elm in croatia (fig. 3). the above mentioned differentiation is supported by high bootstrap values, resulting in grouping of populations based on an eco-geographical principle. bootstrap support value of 68% confi rmed the separation between continental (đurđevac, zagreb) and mediterranean (nin, neretva) populations, whilst the separation among geographically southern (nin, neretva) and northern (zagreb, đurđevac, pula) populations was verifi ed by even higher bootstrap support values of 99% (fig. 3). discussion this research revealed abundant allelic variation over fi ve loci and high overall genetic diversity in natural fi eld elm populations in croatia. the presented results are congruent with those obtained by studies of allozymes (machon et al. 1997), isozymes (machon et al. 1995, cogolludo et al. 2000), rapds and issrs (coleman et al. 2000, goodall-copestake et al. 2005) and ssrs (fuentes-utrilla et al. 2014). although studies of the fi eld elm on the european level suggest a high degree of intrapopulation genetic diversity, they also suggest conclusions concerning the small genetic differentiation between populations belonging to different geographic regions. as each individual author uses a different system of molecular markers and defi nes the term “population” differently, one should exercise a great deal of caution when comparing results. therefore, comparison of results obtained in this study with results from above mentioned studies in europe, is possible only for the purpose of orientation. the values of descriptive statistics parameters (na, nar, ho, he) suggest a native status of the fi eld elm in croatia (tab. 2). in continental populations (đurđevac, zagreb), higher values of nal are present, as well as of nar in relation to mediterranean populations (pula, nin, neretva). the đurđevac, zagreb and neretva populations did not deviate signifi cantly from hw equilibrium, unlike the pula and nin populations, where an excess of heterozygotes was found for specifi c loci (tab. 3). as elm populations in croatia do not demonstrate a statistically signifi cant deviation from hw equilibrium, it could be claimed that the danger of the appearance of inbreeding within observed populations is relatively small. the pula population somewhat deviates from the obtained results, which unequivocally suggest the separation of continental from mediterranean populations. the reasons for the deviations of the pula population from hw equilibrium, as well as for a signifi cant excess of heterozygosity should be sought in the recent bottleneck event (tab. 4). according to cornuet and luikart (1996), a recently bottlenecked population is expected to show fewer rare alleles, as they are lost most quickly. allelic diversity is reduced faster than heterozygosity during a bottleneck, because rare alleles are lost rapidly and have little effect on heterozygosity, thus producing a transient excess in heterozygosity relative to that expected in a population of constant size with the same number of alleles. namely, the pula locality is situated in the area of a former pure elm forest which was turned into an agro-biotope by intensive felling and clearing of land, which has a direct and negative impact on the gene diversity of this population. on the other hand, a lack of heterozygosity was found in the đurđevac population, which thus shows signs of expansion. the causes of tab. 5. nei’s standard genetic distance (above diagonal) and pairwise fst values (below diagonal) between fi ve ulmus populations. pairwise signifi cance after sequential bonferroni corrections: ** – signifi cance at the 1% nominal level, * – signifi cance at the 5% nominal level, ns – non-signifi cant values. no – number of populations. no. population 1 2 3 4 5 1 đurđevac 0.106 0.293 0.208 0.312 2 zagreb 0.029ns 0.278 0.332 0.406 3 pula 0.105** 0.098** 0.371 0.409 4 nin 0.140** 0.195** 0.210** 0.134 5 neretva 0.130** 0.160** 0.158** 0.102** fig. 3. unrooted neighbor-joining tree based on nei’s standard genetic distance between fi ve ulmus populations. numbers above branches indicate bootstrap support percentage over 50% in 10,000 pseudoreplicates. zebec m., idžojtić m., šatović z., poljak i., liber z. 58 acta bot. croat. 75 (1), 2016 population expansion can be various. as this is a larger forest complex, a stand in which economic measures are implemented, there is a possibility of a recent introduction of new individuals. the reasons for this could be to stimulate the natural renewal of the fi eld elm in that locality or improve the economic quality by forcing the planting of plus trees. if a population has been severely attacked by ded and consequently been under the infl uence of a genetic bottleneck, it will strive to establish as soon as possible a new, lower mutation-drift equilibrium by the process of expansion. although the incidence of ded in croatia is high, the parameters of genetic diversity of observed populations do not deviate signifi cantly from the ideal state of hw equilibrium. consequently, one could conclude that the disease has not had a signifi cant impact on the degree of genetic diversity, i.e. on the stability of genetic resources of the fi eld elm in croatia. however, if we take into account the fact that genetic diversity in natural populations of forest trees is very slowly decreasing, as well as the presence of a very small number of adult trees of the fi eld elm in the researched area, concern about the stability of this species’ genofund acquires a new dimension. this study, conducted in croatia, revealed a high genetic diversity of the fi eld elm at the intrapopulation level, but also discovered a signifi cant differentiation at the interpopulation level. although the separation of populations at such a high level could generate certain taxonomic implications, the fi nal decision about the taxonomic validity of hypothetical entities i.e. conservation units within the u. minor s. l. complex in croatia is possible solely if this issue is resolved on the european level. in such research, a systematic study of fructifi cation, modalities of dispersion and the monitoring of phenological phenomena over a certain time period would be necessary. acknowledgements this research was carried out with the fi nancial support of the ministry of science, education and sports of the republic of croatia. grant no. 068-0242108-2773 and partly grant no. 119-1191193-1232. references belkhir, k., borsa, p., chikhi, l., raufaste, n., bonhomme, f., 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0365-0588 eissn 1847-8476 short communication cardamine fi alae fritsch (brassicaceae) a new species in croatian fl ora mara vukojević1, ivana vitasović kosić2, antun alegro3, dmitar lakušić4, sandro bogdanović2* 1 croatian botanical society, rooseveltov trg 6, 10000 zagreb, croatia 2 university of zagreb, faculty of agriculture, department of agricultural botany, svetošimunska cesta 25, 10000 zagreb, croatia 3 university of zagreb, faculty of science, department of botany, marulićev trg 20, 10000 zagreb, croatia 4 botanical institute and garden, faculty of biology, university of belgrade, takovska 43, 11000 belgrade, serbia abstract – during a fl oristic survey of the northern slopes of matokit mountain in the surroundings of vrgorac in central dalmatia, a peculiar population of cardamine belonging to the c. maritima complex was found. because of its morphology, general habit, type of leaves and fruits, the collected plants were recognized as c. fi alae hitherto know only from bosnia and herzegovina. morphologically, c. fi alae is similar to the serbian endemic c. serbica with which it shares following characteristics: auricules at the base of lower cauline leaves, stem and rosette leaves bipinnate, margin of leafl ets serrate with hairy main stem and sepals, although it differs in having bigger petals and sepals, longer fi laments and 1–2 lateral stems. c. fi alae grows on lower altitudes in rocky ground within the vegetation of forest fringes of the sub-mediterranean zone. this is the fi rst report of the endemic species c. fi alae in croatian fl ora. key words: cardamine maritima complex, dalmatia, fl ora, matokit mountain, taxonomy, vrgorac * corresponding author, e-mail: sbogdanovic@agr.hr introduction the genus cardamine l. is one of the largest in the family brassicaceae and consists of approximately 200 species of annual to perennial herbs distributed on all continents except for mainland antarctica (kučera et al. 2005, 2010). in the european fl ora the genus is represented by ca. 54 species (lihova and marhold 2006), while in the croatian fl ora 26 taxa are currently known (kučera and marhold 2006, kučera et al. 2010, nikolić 2015). an interesting group of the amphi-adriatic plants is the cardamine maritima complex that has been studied recently from morphological and phylogenetic points of view (kučera et al. 2008, 2010). this complex has a diversity centre in the balkan peninsula and extends with one species beyond the adriatic. the complex includes seven endemic species with very restricted distributions, six of which occur in the balkan peninsula (c. maritima port. ex dc., c. adriatica jar. kučera, lihová & marhold, c. serbica pančić, c. fi alae fritsch, c. montenegrina jar. kučera, lihová & marhold and c. rupestris (o.e. schulz) k. malý) and one species on the apennine peninsula (c. monteluccii brilli-catt. & gubellini). within this complex an annual to biennial species has been described from klobuk in bosnia and herzegovina and named c. fi alae by fritsch (1897). in the past, some authors treated this taxon at different taxonomic levels, mainly at infraspecifi c level. trinajstić (1976) considered it a subspecies of c. maritima and sagorski (1911) a variety of c. maritima, but modern phylogenetic combined with morphological studies have shown that this taxon merits being treated as an independent species (kučera et al. 2008, 2010). hitherto, c. fi alae has been known only from three localities in bosnia and herzegovina: klobuk, ružići and grude (fritsch 1897, beck mannagetta 1903, kučera et al. 2008, 2010), which are only about 10 km north-east of the present localities. in april 2013 and 2015, during fl oristic mapping of matokit mountain (vukojević 2011, vukojević and vitasović kosić 2012) in the vicinity of vrgorac in central dalmatia, ten kilometres aerial distance from the locus classicus (klobuk in herzegovina), a population of c. maritima complex was found, and identifi ed as c. fi alae. this species is not listed in the plant identifi cation handbook of croatia (domac 2002) or in the flora croatica database (nikolić 2015). the euro+med plantbase (marhold 2011) note the occurrence and distribution of this species only for bosnia and herzegovina. since the endemic c. fi alae has not been previously recorded in croatia, it should be treated as a new species of the croatian vascular fl ora. vukojević m., vitasović kosić i., alegro a., lakušić d., bogdanović s. 214 acta bot. croat. 75 (2), 2016 materials and methods field research was carried out on matokit mountain and in the surroundings of the city of vrgorac in central dalmatia (fig. 1). the whole researched area has about 53 square km and belongs to the belt of sub-mediterranean deciduous forest of downy oak (quercus pubescens willd.) belonging to the alliance querco-carpinion orientalis horvatić 1939. the terrain elevation throughout the whole area is from 300–830 m a.s.l. the climate is a combination of sub-mediterranean and continental climate, with an average annual temperature of 14.3 °c and 1720 mm of average annual precipitation measured in the period of 1981–2010 for the city of vrgorac (vukojević 2011). identifi cation of c. fi alae regarding its morphology, general habit, shape of leaves and fruits, was done according to the identifi cation key for c. maritima complex proposed by kučera et al. (2010). the morphological description presented here follows beck mannagetta (1903), tri najstić (1976), šilić (1990), akeroyd and marhold (1993), kučera et al. (2010) and additional authors’ observations. herbarium vouchers of collected plants are digitized and deposited in the herbarium zagr; images are accessible through virtual herbarium zagr (bogdanović 2015). type specimens of c. fi alae (lectotype and isotypes form gzu and w) were also consulted and compared with croatian populations (abbreviations are according to thiers 2015). results and discussion cardamine fi alae fritsch, österr. bot. z. 47: 44 (1897) synonyms – c. maritima port. ex dc. subsp. fi alae (fritsch) trinajstić, suppl. fl. anal. jugosl. 4: 8 (1976); c. maritima port. ex dc. var. fi alae (fritsch) sagorski, österr. bot. z. 61: 18 (1911). lectotype was designated by kučera et al. (2010) in wu herbarium. morphological description – c. fi alae is an overwinter annual or rarely biennial plant, 12–30(50) cm high, whole plant is hairy. stems are erect and branched, on lower part usually purple-violet. rosette and stem leaves are bipinnate, with serrate margin of leafl ets, petiole of stem leaves auriculate. sepals hairy, winged, mucronate, 2 longer (5.1–) 5.4–8.3(–9.5) mm long. petals white, glabrous, (11.7–) 12.8–17.9(–19.7) mm long, with long ungues, limb obovate to obcordate. siliqua linear, glabrous, fl at, light brown when ripe, 40–55 (–60) mm long, 1.4–2.0(–2.5) mm wide, with 10–12 (–15) mm long beak, siliqua pedicel 10–15 (–20) mm long. seeds 4, brown, 5 mm long and ca 1.5 mm wide (figs. 2a–e). flowering time is from april to may. cardamine fi alae can possibly be confused with other members of the c. maritima complex. it differs from typical c. maritima in having auricules on the base of stem leaves, denser indumentum and bigger fl owers (figs. 2c, d). morphologically it is more similar to serbian endemic c. serbica with which it shares the following characteristics: auricules at the base of lower cauline leaves (fig. 2d), stem and rosette leaves bipinnate (fig. 2b), margin of leaflets serrate with hairy main stem and sepals, but it differs in having bigger petals and sepals (fig. 2c), longer fi laments and only 1–2 lateral stems. habitat and ecology – c. fi alae grows in limestone rocky ground within degraded forests of downy oak (q. pubescens) and oriental hornbeam (carpinus orientalis mill.) in the sub-mediterranean zone. it grows individually or in scattered groups on dry and shallow humose soils among rocks. on matokit mountain it grows on lower altitudes in rocky ground within the vegetation of forest fringes and secondarily also in ruderal places, in arable fi elds (fig. 2f), on shady screes and very rarely in rock crevices. these are relatively open microhabitats not overgrown by other plants. such semi-natural habitats and their conditions are particularly suitable for seeds to spread. when the fi rst population of c. fi alae was recorded in early april, the plants fig. 1. present distribution of cardamine fi alae fritsch in croatia and in bosnia and herzegovina. cardamine fialae in croatia acta bot. croat. 75 (2), 2016 215 were already in fl ower and some of the specimens were even producing fruits. this can be considered an early fl owering time as the literature indicates it starts fl owering in april or may, lasting until july or august (beck mannagetta 1903, šilić 1990). this endemic species can be classifi ed as an illyrian-adriatic fl oristic element according to the classifi cation of horvatić (1963a, b) and horvatić et al. (1967–1968). distribution in croatia and in bosnia and herzegovina – in april 2013 a population of c. fi alae was found on the northern slope of matokit mountain in turića dočići between a forest fringe and an arable fi eld, and is the biggest among all known populations. afterwards, in may and june 2015 it was found at another eight localities within different habitats, as follows. the three localities in bosnia and herzegovina are taken from kučera et al. 2008 (fig. 1): 1. vrgorac, northern slope of matokit mountain, turića dočići, between forest fringe and arable fi eld, representative population about 500 plants, 350 m a.s.l., 14.04.2013, 43°14’5.17’’ n, 17°19’19.83’’ e, 2. vrgorac, prapatnice, vegari, popriki dolac, on rocks in the forest, about 50 plants, 339 m a.s.l., 07.06.2015, 43°13’56.16’’ n, 17°19’39.04’’ e, 3. vrgorac, prapatnice, velika njiva, on deeper soil, very near rocks in the forest, about 20 plants, 319 m a.s.l., 06.06.2015, 43°13’54.29’’ n, 17°20’0.51’’ e, 4. vrgorac, prapatnice – stilja, put vukmira, on bare rocks of the forest edge, about 20 plants, 371 m a.s.l., 03.06.2015, 43°14’8.32’’ n, 17°19’4.74’’ e, 5. vrgorac, on the entry of the village gornje kašče, on bare rocks (without soil) of the oriental hornbeam forest edge, about 20 plants, 465 m a.s.l., 20.05.2015, 43°15’7.43’’ n, 17°19’56.25’’ e, 6. vrgorac, mijaca, within the walls of former barns on shallow soil, about 50 plants, 565 m a.s.l., 27.05.2015, 43°17’11.24’’ n, 17°18’44.17’’ e, 7. vrgorac, on the way to mijaca, near the road, on rocks of the forest edge, about 10 plants, 704 m a.s.l., 27.05.2015, 43°17’19.92’’ n,17°18’9.24’’ e, 8. vrgorac, hilly area orah, on the way to kućerina, on rocks of rocky pastures, about 20 plants, 317 m a.s.l., 19.05.2015, 43°14’20.47’’ n,17°24’15.62’’ e, 9. razdolje, transition to the southern side of the mountain trail toward ravča, bare rocks, about 10 plants, 830 m a.s.l., 30.05.2015, 43°13’14.94’’ n, 17°18’40.88’’ e. conservation status – according to the standards of iucn (2014) the endemic c. fi alae should be classifi ed in the category of vulnerable (vu) species, the criterion d2 being adopted. in april 2013 and may 2015, in all, approximately 900 mature individuals were counted within the complete range of the species including croatian and bosnian and herzegovinian locations. the threat status is based on number of mature individuals, which is below 1000, as well as on the area of occupancy (aoo) which is less than 20 km2, while extent of occurrence (eoo) is less than 90 km2. there are potential threats to the habitats (human impacts in agriculture, fi res and forest managements) and further studies need to be conducted in order to monitor the population size of c. fi alae. as a rare endemic species, c. fi alae should be protected by law and included in the national red list of croatia. conclusion – the area of dalmatian zagora e.g. vrgorac and matokit mountain belongs among regions that have been poorly botanically explored. all croatian localities of c. fi alae were found close to the border with bosnia and herzegovina (near the locus classicus) and potentially new localities of c. fi alae could be expected within similar habitats in this region. the newly recorded adriatic endemic species has very delimited distribution range, and the fi nding of c. fi alae is a valuable contribution to the croatian fl ora. acknowledgements we are grateful to curators of herbaria gzu and w for the examination of type material of cardamine fi alae. thanks to martina temunović, ph.d. (university of zagreb, faculty of forestry), for preparation of distribution map. fig. 2. cardamine fi alae fritsch: a) habitus, b) stem bipinnate leaf, c) fl owers, d) auricule, e) fruits and seeds, f) habitat (forest fringe) in turića dočići (photos by m. vukojević). vukojević m., vitasović kosić i., alegro a., lakušić d., bogdanović s. 216 acta bot. croat. 75 (2), 2016 references akeroyd, j. r., marhold, k., 1993: cardamine l. in: tutin, t. g., burges, n. a., chater, a., o., edmondson, j. r., heywood, v. h., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. 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(eds.), plant genome: biodiversity and evolution, vol. 1c, phanerogams (angiosperms–dicotyledons), 149–186. science publishers, enfi eld, new hampshire. marhold, k., 2011: brassicaceae. in: euro+med plantbase – the information resource for euro-mediterranean plant diversity. retrieved october 19, 2015 from http://ww2.bgbm.org/europlusmed/query.asp nikolić, t., (ed.) 2015: flora croatica database. university of zagreb, faculty of science, department of botany. retrieved october 21, 2015 from http://hirc.botanic.hr/fcd sagorski, e., 1911: üeber einigen arten aus dem illyrischen florenbezirk. österreichische botanische zeitschrift 61, 11–21. šilić, č., 1990: endemic plants. ip “svjetlost”, zavod za udž benike i nastavna sredstva, sarajevo-beograd (in croatian). thiers, b., 2015: index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden’s virtual herbarium. retrieved october 21, 2015 from http:// sweetgum.nybg.org/ih/ trinajstić, i., 1976: cardamine l. in: trinajstić, i. (ed.), analytical fl ora of yugoslavia vol. 2, 218–234. institute for botany, university of zagreb, zagreb (in croatian). vukojević, m., 2011: vascular fl ora of northern slopes of matokit mountain and the surroundings of vrgorac. graduation thesis, university of zagreb, faculty of agriculture. zagreb (in croatian). vukojević, m., vitasović kosić, i., 2012: mountain matokit and vrgorac city: a new localities of threatened and invasive plant taxa in croatia. journal of central european agriculture 13, 150–166. acta bot. croat. 77 (1), 2018 93 acta bot. croat. 77 (1), 93–96, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0001 issn 0365-0588 eissn 1847-8476 short communication chromosome numbers and karyotype features of phlomis olivieri benth. (lamiaceae) from iran hossein yousefi1, atefe amirahmadi2, reza naderi2*, morteza atri1 1 department of biology, faculty of sciences, bu-ali sina university, hamedan, iran 2 school of biology and institute of biological science, damghan university, damghan, 36716-41167, iran abstract – chromosome numbers were determined in ten accessions of phlomis olivieri benth. (lamiaceae). the seeds were collected from natural habitats in the west of iran. chromosome numbers of all accessions were 2n=2x=20. the chromosomes of accessions were metacentric or submetacentric, ranging in length from 2.66 to 8.604 μm. according to the average values of ten accessions, the karyotype of this species consists of 10 pairs of metacentric chromosomes. an ideogram was depicted for the species. this is the first report on the chromosome number and karyotype analysis of p. olivieri from iran. keywords: chromosome count, iran, karyotype feature, labiatae, phlomis * corresponding author, e-mail: rezanaderia@du.ac.ir introduction the genus phlomis l. is one of the largest genera of subfamily lamioideae (lamiaceae) with more than 100 recognized species distributed in asia, southern europe and northern africa, which have been divided into two main sections: phlomoides (moench) briq. and phlomis (rechinger 1982, albaladejo et al. 2005). the diagnostic character for separating the sections is corolla shape. species of the section phlomis, which have a corolla with a curved upper lip and trifid lower lip with large median and smaller lateral lobes, differs from species of section phlomoides that have corolla with straight upper lip and trifid lower lip with sub equal lobes (azizian and moore 1982). in iran, this genus is represented by 19 species (10 species are endemic) including p. olivieri benth. (sect. phlomis) which grows wild in north, west and central iran (rechinger 1982, jamzad 2012). recently, the taxonomic value of the indumentum as well as the anatomy and palynology of this species were investigated (yousefi et al. 2014). there is little cytological information on the genera, though there have been chromosome counts of some species of phlomis by workers such as wagner (1948), reese (1953), strid (1965), zhukova (1967), chuksanova and kaplanbekova (1971), azizian and cutler (1982), strid and andersson (1985), aparicio (1997), aparicio and albaladejo (2003), ghaffari (2006) and özdemir et al. (2014). the chromosome number of the species of phlomis varies from 2n=10 to 2n=46 (goldblatt and johnson 1979). phlomis olivieri is a perennial herb species distributed in iran and iraq. this species grows on mountainous regions, adjacent to rocky slopes, steppe vegetation and the overgrazed rangeland soils of the irano-turanian region and the hyrcanian district of iran (jamzad 2012) and could be considered one of important destroyed rangeland indicators together with stachys inflata benth. (mozaffarian 2005). the aim of this paper is to determine the chromosome number and karyotype features of p. olivieri for the first time. materials and methods plant samples of p. olivieri benth. were collected in wild populations in the west of iran. voucher specimens of all the materials studied were deposited in the herbarium of isfahan university, iran. the locality, collectors and dates of ten accessions are shown in table 1. root-tip meristems were obtained from wild collected seeds germinated on wet filter paper in petri dishes at room temperature in the dark. they yousefi h., amirahmadi a., naderi r., atri m. 94 acta bot. croat. 77 (1), 2018 were pretreated using an aqueous solution of colchi (0.05%) at room temperature for 3 h. the material was fixed in absolute ethanol and glacial acetic acid (3:1) for 12 h at room temperature and then stored in the fixative at 4 °c. samples were hydrolyzed in 1 n hcl for 2 min at 60 °c, stained in 1% aqueous aceto-orcein for 2–12 h at room temperature, squashed and mounted in a drop of 45% acetic acidglycerol (9:1). the best metaphase plates were photographed with a ziess axiostar photomicroscope and a canon digital camera. karyotype analysis was carried out according to the method described by levan et al. (1964). several parameters regarding the karyotypes symmetry/asymmetry including total length of chromosome (c), length of long arm (l), length of short arm (s), arm ratio (r: l/s), centromeric index (i: (s/c)×100) and centromeric position were calculated for each accession with the use of the dn2 microscopy image processing system. morphometric data regarding karyotypes were provided and an ideogram of the species was depicted based on the average of several accessions karyotypes which have approximately the same level of chromosome condensation. to assess the existence of published chromosome counts in the studied species we used the chromosome number database index to plant chromosome numbers (goldblatt and johnson 1979). results and discussion in the present paper, we determined chromosome numbers of p. olivieri. the chromosome numbers, karyotype analysis, and ideogram of p. olivieri are shown based on the tab. 1. localities and chromosome numbers of the investigated accessions in phlomis olivieri. no. location and collection number geographical character altitude (m) chromosome number date of collection 1 hamedan province: ganjnameh, before mishan plain, yousefi 19003. 34°46'03.89"n 48°25'43.67"e 2550 2n = 20 4 june 2011 2 hamedan province: asadabad, around taj abad sofla village, yousefi 19013. 34°52'49.62"n 48°12'44.58"e 1994 2n = 20 4 june 2011 3 hamedan province: malayer, lashkardar protected area, yousefi 19011. 34°12'45.14"n 48°58'46.25"e 2172 2n = 20 7 june 2011 4 hamedan province: nahavand, sarabe giyan, above farmland, yousefi 19005. 34°08'33.86"n 48°13'03.64"e 1717 2n = 20 5 june 2011 5 hamedan province: nahavand, sarabe giyan, yousefi 19006. 34°08'35.16"n 48°13'11.99"e 1698 2n = 20 5 june 2011 6 hamedan province: razan, boghaty mountains, yousefi 19012. 35°02'19.5"n 48°50'18.1"e 2350 2n = 20 12 june 2011 7 hamedan province: tuyserkan to malayer, yousefi 19010. 34°28'25.57"n 48°33'03.63"e 1864 2n = 20 7 june 2011 8 hamedan province: 5 km nahavand to malayer, left side of road, yousefi 19007. 34°12'45.29"n 48°24'09.62"e 1775 2n = 20 5 june 2011 9 hamedan province: nahavand to malayer, before kartilabad village, right side of road, yousefi 19009. 34°18'44.05"n 48°38'26.69"e 1697 2n = 20 5 june 2011 10 hamedan province: kabodarahang, around gholiabad village, yousefi 19014. 35°15'03.30"n 48°50'40.01"e 1906 2n = 20 12 june 2011 tab. 2. karyomorphological parameters of phlomis olivieri based on the average values of ten accessions. sd – standard deviation, m – metacentric. chromosome no. total length (μm)±sd long arm length (l) μm short arm length (s) μm arm ratio l/s centromeric index chromosome type 1 7.67±0.68 4.09 3.58 1.14 46.67 m 2 7.12±0.82 3.73 3.39 1.10 47.61 m 3 7.11±0.82 3.87 3.24 1.19 45.56 m 4 6.99±0.72 3.81 3.18 1.19 45.49 m 5 6.74±0.73 3.71 3.03 1.22 44.95 m 6 6.64±0.73 3.56 3.08 1.15 46.38 m 7 6.52±0.65 3.65 2.87 1.27 44.01 m 8 6.31±0.72 3.44 2.87 1.19 45.48 m 9 6.20±0.61 3.43 2.77 1.23 44.67 m 10 6.06±0.73 3.39 2.67 1.26 44.05 m 11 6.03±0.69 3.35 2.68 1.25 44.44 m 12 5.86±0.67 3.25 2.61 1.24 44.53 m 13 5.73±0.69 3.07 2.66 1.15 46.42 m 14 5.54±0.48 2.93 2.61 1.12 47.11 m 15 5.48±0.55 3.10 2.38 1.30 43.43 m 16 5.27±0.53 2.97 2.30 1.29 43.64 m 17 5.29±0.52 2.94 2.35 1.25 44.42 m 18 5.10±0.52 2.83 2.27 1.24 44.50 m 19 4.87±0.78 2.83 2.04 1.38 41.88 m 20 4.69±0.74 2.61 2.08 1.25 44.36 m chromosome number and karyotype of phlomis olivieri acta bot. croat. 77 (1), 2018 95 average values of ten accessions (tab. 1, tab. 2, fig. 1, and fig. 2). this is the first karyotype determination for this species. based on our results the basic chromosome number in the studied accessions was x=10. all accessions have the same chromosome number and represent diploid species with 2n=2x=20. chromosome sizes vary from 2.66 to 8.60 μm. the longest arm is 4.796 μm (accession 2) and the shortest arm is 1.16 μm (accession 9) (fig. 1.) different karyotypes have been found among the accessions of p. olivieri. generally, the studied karyotypes consist mostly of metacentric chromosomes with almost median centromere position (stace 1989). however, the karyotypes of accessions 1, 4, 6, 7 and 9 beside metacentric chromosomes also possess submetacentric chromosomes. such differences between accession karyotypes could be a result of different chromosome condensation due to the pretreatment procedure. this could be also a reason why secondary constrictions with satellites were not observed in the karyotype of this species. according to the average values of ten accessions, the karyotype of this species consists of 10 pairs of metacentric chromosomes (tab. 2). azizian and cutler (1982) revealed that species of the phlomis section phlomis are characterized by 2n=20 large chromosomes, while species of the phlomis section phlomoides possess 2n=22 and smaller chromosomes. özdemir et al. (2014) determined 2n=20 chromosomes for p. lunariifolia sm. and p. grandiflora h.s. thomps., two species of the genus phlomis sect. phlomis. the investigated species had 9 pairs of metacentric and 1 pair of submetacentric chromosomes. furthermore, the presence of 2n=20 chromosomes within the genus has been confirmed in several species such as p. cypria post var. cypria (yildiz and gücel 2006), p. lychnitis l., p. purpurea l., p. italica l. and p. herba-venti l. var. tomentosa boiss. (see mateu 1986). although it can be concluded that the presence of twenty somatic chromosomes in our study is consistent with the previous studies, karyotypic differences exist within or among species and alteration in chromosome symmetry may arise through translocations, pericentric inversions or fusion (levin 2002). our observations as well as comparison of photomicrographs obtained from previous reports also showed that phlomis chromosomes are the largest among species of the lamiaceae genera such as callicarpa l., salvia l., scutellaria l., sideritis l., stachys l., teucrium l. and thymus l. (jalas 1948, boşcaiu et al. 1998, yildiz and gücel 2006, yang et al. 2009, martin et al. 2011, contreras and ruter 2011, javadi et al. 2011). acknowledgement the authors would like to thank the graduate department of bu-ali sina university. a small part of the present study was financially supported by grant-in-aids for scientific research, no. 93035352 from insf (iran national science foundation). fig. 1. somatic metaphases in phlomis olivieri (accessions 1–10) with diploid chromosome number 2n=20. bar = 10 μm. fig. 2. ideogram of phlomis olivieri. scale in μm. yousefi h., amirahmadi a., naderi r., atri m. 96 acta bot. croat. 77 (1), 2018 references levin, d. a. 2002: the role of choromosomal change in plant evolution. oxford university press, oxford. martin, e., çetin, ö., akçiçek, e., dirmenci, t., 2011: new chromosome counts of genus stachys (lamiaceae) from turkey. turkish journal of botany 35, 671–680. mateu, i. 1986: revisión del género phlomis l. 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(ed.), plantarum in zonam polarem transportatio 2, 139– 149. leningrad. albaladejo, r. g., aguilar, j. f., aparicio, a., feliner, g. n., 2005: contrasting nuclear-plastidial phylogenetic patterns in the recently diverged iberian phlomis crinita and p. lychnitis lineages (lamiaceae). taxon 54, 987–998. aparicio, a., 1997: fitness components of the hybrid phlomis × margaritae aparicio & silvestre (lamiaceae). botanical journal of the linnean society 124, 331–343. aparicio, a., albaladejo, r. g., 2003: microsporogenesis and meiotic abnormalities in the hybrid complex of phlomis composita (lamiaceae). botanical journal of the linnean society 143, 79–85. azizian, d., moore, d. m., 1982: morphological and palynological studies in phlomis l., eromostachys bunge and paraphlomis prain (labiatae). botanical journal of the linnean society 85, 225–248. azizian, d., cutler, d. f., 1982: anatomical, cytological and phytochemical studies on phlornis l. and erernostachys bunge (labiatae). botanical journal of the linnean society 85, 249–281. boşcaiu, m., riera, j., estrelles, e., güemes, j., 1998: chromosome numbers of several lamiaceae from spain. folia geobotanica 33, 187–199. chuksanova, n. a., kaplanbekova, s. a., 1971: chromosome numbers in certain species of labiatae juss. and scrophulariaceae lindl. indigenous to the ussr. botanicheskii zhurnal (moscow & leningrad) 56, 522–528 (in russian). contreras, r. n., ruter, j. m., 2011: genome size estimates and chromosome numbers of callicarpa l. (lamiaceae). hortscience 46, 567–570. ghaffari, s. m., 2006: new or rare chromosome counts of some angiosperm species from iran. iranian journal of botany 11, 185–192. goldblatt, p., johnson, d. e., 1979: index to plant chromosome numbers. st. louis: missouri botanical garden. retrieved september 15, 2015 from http://www.tropicos.org/project/ ipcn jalas, j., 1948: chromosome studies in thymus. i. somatic chromosome numbers with special reference to the fennoscandian forms. hereditas 34, 414–434. jamzad, z., 2012: phlomis l. in: assadi, m., maassoumi, a. a., mozaffarian, v. (eds.), flora of iran, 76, 253–301. research institute of forest and rangelands, tehran (in persian). javadi, h., hesamzadeh hejazi, s. m., babayev, m. sh., 2011: chromosome reports on two species of thymus (lamiaceae). iranian journal of botany 18, 108–111. levan, a., fredga, k., sandberg. a. a., 1964: nomenclature for centromeric position on chromosomes. hereditas 52, 201–220. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 333 acta bot. croat. 74 (2), 333–343, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0031 diatoms in an extreme euxinic environment (rogoznica lake, eastern adriatic coast) nikola malešević1*, irena ciglenečki2, elvira bura-nakić2, marina carić3, iris dupčić3, enis hrustić3, damir viličić1, zrinka ljubešić1 1 university of zagreb, faculty of science, division of biology, rooseveltov trg 6, 10000 zagreb, croatia 2 ruđer bošković institute, division of marine and environmental research, bijenička cesta 54, 10000 zagreb, croatia 3 university of dubrovnik, institute for marine and coastal research, kneza damjana jude 12, 20000 dubrovnik, croatia abstract – the rogoznica lake marine system is a small, karstic, naturally eutrophic and euxinic marine environment. abundance and temporal distribution of phytoplankton was investigated in relation to environmental conditions in the period from 1998 to 2013. the 36 determined diatoms contributed 90% of the total phytoplankton abundance. the diatom composition is characterized by low species diversity and high single species abundance (up to 107 cells l–1). there were, on average, 2.6 diatom species per sample (maximum 14 diatom species per sample) refl ecting extreme environmental conditions. dominant diatoms thalassionema nitzschioides, cyclotella choctawhatcheeana, dactyliosolen fragilissimus and chaetoceros curvisetus occurred repeatedly and were alternately dominant in the lake during the whole research period. some diatoms were dominant only in limited period, like cyclotella choctawhatcheeana (evident since 2001), and pseudo-nitzschia spp. (evident in the period 2002 to 2009). it appears that the interplay of environmental conditions such as variability in thermohaline and redox conditions, nutrient and reduced sulphur concentration infl uence the phytoplankton development and abundance in the lake. keywords: diatoms, extreme environmental conditions, marine lake, phytoplankton introduction an increased level of reduced sulphur species (rss, including sulphide, polysulphide, elemental sulphur and thio-componds) and seasonal changes between oxic and anoxic conditions have long been recognized in the rogoznica lake marine system, central dalmatia (ciglenečki et al. 2005, 2013, kršinić et al. 2013). * corresponding author, e-mail: nikola712@gmail.com malešević n., ciglenečki i., bura-nakić e., carić m., dupčić i., et al. 334 acta bot. croat. 74 (2), 2015 the thermohaline conditions in this lake are mainly regulated by solar heating, evaporation and rainfall, which infl uence surface salinity variations (ciglenečki et al. 2005, 2013). surface water could be well oxygenated (oxygen saturation up to 300%), while hypoxia/ anoxia occurs in the bottom water layer (stipaničev and branica 1996, ciglenečki et al. 1998, 2005, 2013). the boundary between oxic and anoxic water layers is usually situated below the depth of 9 m (ciglenečki et al 2005, 2013). although phytoplankton, zooplankton nauplii and larvae of benthic organisms may enter the lake through fi ssures between the rocks (kršinić et al. 2000), few species are recorded in the lake. since the anoxic event in september of 1997, when the presence of hydrogen sulphide in the whole of the water column was detected, investigations of the lake have been intensive. this event, followed by massive mortality of all organisms, was taken as a zero point from which ecological recovery of the lake was monitored (barić et al. 2003, ciglenečki et al. 2005, šestanović et al. 2005, svensen et al. 2008, burić et al. 2009, žic et al. 2010). before the anoxic event in 1997, the dominant species in the phytoplankton was the diatom pseudo-nitzschia spp., which accounted for 88% of the phytoplankton community. the microplankton usually was composed of about 30 species, among which the most frequent and abundant were the rare microfl agellates prorocentrum arcuatum and hermesinum adriaticum (viličić et al. 1996/1997, burić et al. 2009) and diatoms (chaetoceros curvisetus and eunotia sp.) (ciglenečki et al. 2005, burić et al. 2009). recently published papers reported that diatoms took over the domination of phytoplankton assemblages after 2001, accounting for on average up to 94%, recognizing chaetoceros curvisetus and dactyliosolen fragilissimus as dominant species until the end of 2005 (kršinić et al. 2013). c. curvisetus and dinofl agellate ceratium furca were found as dominant species in the lake in summer 2004 (svensen et al. 2008). the calanoid copepod acartia italica is the only metazoan plankton species commonly present in rogoznica lake surviving after anoxia episodes and reaching high abundance in the lake (kršinić et al. 2000, 2013, svensen et al. 2008). before the disastrous anoxic event in 1997, top-down control governed the biological processes in the lake. this disappeared with anoxic stress conditions and it took several years for the full recovery of top-down control (kršinić et al. 2013). rogoznica lake due to its relative isolation, semi-closed nature, environmental characteristics and size (10300 m2 and maximum depth 15 m) can serve as natural laboratory for studying extreme environment and certain biogeochemical processes. in this paper changes of phytoplankton composition and low species diversity are studied in relation to physicochemical conditions in the lake. the special focus is on the diatoms, with the regularity of specifi c species domination being discussed. materials and methods study area rogoznica lake is situated on gradina peninsula (43°32’n, 15°58’e) in the vicinity of the settlement of rogoznica and the city of šibenik, on the eastern adriatic coast (fig. 1). it is a eutrophic, karstic lake with a surface area 10300 m2 and a maximum depth of 15 m. lake is round in shape and is surrounded by 4–24 m high porous karst cliffs with underground connections with the open sea visible through tides with a certain delay. diatoms in the extreme euxinic environment acta bot. croat. 74 (2), 2015 335 sampling and analyses investigation of phytoplankton was conducted in the period from december 1998 to september 2013 (tab. 1). samples for phytoplankton and nutrient analyses were collected in 5 l niskin bottles, from the water column in the center of the lake. samples were taken from various depths from surface to the bottom (0, 2, 5, 7, 8, 9, 10, 11, 12, 13 m). because fig 1. location of rogoznica lake (marked by arrow) in the eastern adriatic coast. tab. 1. sampling of phytoplankton conducted in the period from december 1998 to september 2013. sampling campaigns are marked with »+«. date jan feb mar apr may jun jul aug sep oct nov dec 1998 + 1999 2000 + + + + + + 2001 + + 2002 + + + + 2003 + + + 2004 + + + + + + 2005 + + 2006 + + + + 2007 + + + 2008 + + + 2009 + + + + + + 2010 + + + + + 2011 + + + + + + 2012 + + + + + + 2013 + + + malešević n., ciglenečki i., bura-nakić e., carić m., dupčić i., et al. 336 acta bot. croat. 74 (2), 2015 of the appearance of anoxic conditions at depths below 9 m, in this research these phytoplankton data were excluded. the samples were preserved in a 2% (fi nal concentration) neutralized formaldehyde solution. cell counts were obtained by the inverted microscope method according to utermöhl (1958). nutrient concentrations were measured by standard methods (strickland and parsons 1972, ivančić and degobbis 1984). software packages grapher 7 and statistica 12 were used for statistical and graphical analyses. results physico-chemical conditions in the investigated period, seasonal variations in vertical gradients of salinity and temperature, together with changes between oxic and anoxic conditions indicated the existence of surface (0–2 m) and bottom water layers (12 m) with higher salinity and temperature values (fig. 2). mixing of the water layers (marked by arrows in fig. 2) ending with the fig. 2. seasonal variations of salinity and temperature in the surface (0–2 m) and bottom waters (12 m) of rogoznica lake. mixing of water layers is marked by arrows, months are denoted by abbreviations: j for january, a for april, j for july and o for october. in all these mixing occasions, except october 2011, hypoxic conditions (1–3 mg l–1) within the whole water column with elevated concentrations of nitrite (up to 3.5 μm) and ammonium (31 μm) were recorded. in 2011 anoxia was present for more than 1 week in the whole water column of the lake. diatoms in the extreme euxinic environment acta bot. croat. 74 (2), 2015 337 formation of a chemically homogeneous water column, characterized by hypoxic or anoxic conditions, usually take place in autumn. all recorded mixing events in the investigated period except of the ones in october 2011, were characterized by hypoxic conditions (1–3 mg l–1) within the whole water column accompanied with elevated concentrations of nitrite (up to 3.5 μm) and ammonium (up to 31 μm). anoxic holomixis was present in the whole lake for more than a week in october 2011. the maximum concentration of nitrate (44.21 μmol l–1) was detected in january 2005 in the surface layer (0–1 m). phosphate concentration varied from 0.01 μmol l–1 to 33.1 μmol l–1 with the minimum recorded in may 2004 in the surface layer. silicate concentration varied from 0.34 μmol l–1 to 679 μmol l–1 (tab. 3). statistical analyses among nutrients showed a signifi cant positive correlation between nitrite and: nitrate (r = 0.15; p = 0.04), ammonium (r = 0.92; p = 0.00) and phosphate (r = 0.31; p = 0.00). phytoplankton composition the phytoplankton composition in the lake is characterized by low species diversity and high single species abundance (up to 107 cells l–1) (tab. 2). during the entire research period diatoms were dominant, accounting, on average, for 90% of the whole phytoplankton (fig. 3, tab. 3). in all, 36 diatoms were recorded in the lake with a low average diversity of 2.6 diatom species per sample (maximal number 14 diatom species per sample). dominant diatoms were: thalassionema nitzschioides, cyclotella choctawhatcheeana, dactyliosolen fragilissimus and chaetoceros curvisetus (tab. 2). those diatoms occurred repeatedly in the lake, and were alternately dominant in the phytoplankton assemblage over the research period (fig. 3). the diatom cyclotella choctawhatcheeana was found in the lake for the fi rst time in may 2004, and was a permanent constituent of the phytoplankton assemblage until the end of the investigated period. pseudonitzschia spp. was occasionally the dominant species from september 2002 to november 2009, and after that was not detected in lake (fig. 3). in the fi rst half of 2000, when stratifi cation of the water column was very weak, and concentration of rss increased in the upper layers the abundance of diatom species c. curvisetus decreased in the following period. in the summer of 2003, when thermohaline conditions in the lake water weakened again, the abundance of c. curvisetus and d. fragilissimus decreased, while the abundance of t. nitzschioides slightly increased. the next mixing of the water layers occurred in winter 2005 and probably triggered the decrease of abundance of all dominant species except of c. choctawhatcheeana. in the winter of 2007 mixing occurred again and consequently the abundance of the two dominant species d. fragilissimus and t. nitzschioides increased. in winter 2008 mixing resulted in a decreased abundance of the two dominant species c. curvisetus and t. nitzschioides, while the abundance of d. fragilissimus and of c. choctawhatcheeana increased. in autumn 2010 during isothermy in the lake, abundance of c. curvisetus and t. nitzschioides stayed almost the same, while the abundance of other two dominant species decreased (fig. 2, fig. 3). statistical analyses revealed a signifi cant positive correlation of d. fragilissimus with nitrite (r = 0.15; p = 0.03) and with ammonium concentration (r = 0.19; p = 0.01). there was no signifi cant correlation between the development of other dominant diatoms and nutrient concentration distribution. diatom abundance and development responded to mixing as shown in fig. 3. every mixing event had as consequence a change in the diatom development. furthermore, d. fragilissimus had positive correlation with the species pseudonitzschia spp. (r = 0.26; p = 0.00) and c. choctawhatcheeana with t. nitzschioides (r = 0.17; p = 0.02). malešević n., ciglenečki i., bura-nakić e., carić m., dupčić i., et al. 338 acta bot. croat. 74 (2), 2015 tab. 2. list of diatoms taxa found in the phytoplankton during the research period. max is for maximum abundance of cells per l and fr is frequency of appearance (382 samples is 100%). taxa fr (%) max cerataulina pelagica (cleve) hendey 2 137960 chaetoceros affi nis lauder 4 13300 chaetoceros compressus lauder 4 1122000 chaetoceros curvisetus cleve 51 1079090 chaetoceros danicus cleve 6 112530 chaetoceros decipiens cleve 2 28120 chaetoceros diversus cleve 2 6400 chaetoceros lauderi ralfs 2 1600 chaetoceros perpusilus cleve 2 560 chaetoceros rostratus lauder 2 800 chaetoceros spp. 11 1165510 cyclotella choctawhatcheeana prasad 30 7926200 cylindrotheca closterium (ehrenberg) reimann & j. c. lewin 1 760 dactyliosolen fragilissimus (bergon) hasle 30 2582210 eunotia sp. 3 170808 guinardia fl accida (castracane) h.peragallo 1 272790 guinardia striata (stolter.) hasle 6 109730 hemiaulus hauckii grunow ex van heurck 1 1520 hemiaulus sinensis greville 1 380 leptocylindrus danicus cleve 6 313480 leptocylindrus minimus gran 2 7090 licmophora sp. 3 1140 microtabella interrupta (ehrenberg) round 2 760 nitzschia longissima (brébisson) ralfs 10 63080 pleurosygma sp. 6 380 proboscia alata (brightwell) sundström 1 181060 pseudo-nitzschia delicatissima (cleve) heiden 1 2974060 pseudonitzschia spp. 6 622945 rhizosolenia calcar avis schultze 1 1890 rhizosolenia delicatula cleve 1 6400 rhizosolenia fragilissima bergon 1 4000 rhizosolenia imbricata brightwell 1 1140 striatella unipunctata (lyngbye) c. agardh 3 22800 thalassionema nitzschioides (grunow) mereschkowsky 30 1667885 thalassiosira sp. 10 100475 unidentifi ed pennate diatoms 25 812600 diatoms in the extreme euxinic environment acta bot. croat. 74 (2), 2015 339 tab. 3. maximum (max), minimum (min) and average (avg) of values of phytoplankton (during 1998–2013) and nutrients (during 2000–2008) in rogoznica lake. n – number of samples. parametar avg max min n total microphytoplankton (cells l–1) 461 400 8 138 480 0 382 diatoms (cells l–1) 451 360 7 926 200 0 382 dinofl agelates (cells l–1) 10 040 840 910 0 382 no3 (μmol l–1) 2.03 44.21 0 476 no2 (μmol l–1) 0.26 3.7 0 478 nh4 (μmol l–1) 14.84 315 0.23 457 total inorganic nitrogen (μmol l–1) 17.21 315.83 0.31 440 po4 (μmol l–1) 1.98 33.1 0.01 496 sio4 (μmol l–1) 5.5 679 0.34 497 fig 3. temporal distribution of: a) diatoms, total phytoplankton and cyclotella choctawhatcheeana; b) chaetoceros curvisetus, dactyliosolen fragilissimus and tthalassionema. nitzschioides in rogoznica lake for the period 1998–2013. diatom abundance is referring to average of 9 m water column. mixing events are marked by arrows. malešević n., ciglenečki i., bura-nakić e., carić m., dupčić i., et al. 340 acta bot. croat. 74 (2), 2015 discussion the diatoms thalassionema nitzschioides, cyclotella choctawhatcheeana, dactyliosolen fragilissimus, and chaetoceros curvisetus, were found to be the most frequent and abundant phytoplankton species, and appeared recurrently during annual plankton succession in the rogoznica lake. the maxima of species abundances in the lake are in the same order of magnitude as maxima of species abundances in other coastal regions of the eastern adriatic sea (cetinić et al. 2006, viličić et al. 2009, bosak et al. 2009, bužančić et al. 2012, marić et al. 2012, vidjak et al. 2012, godrijan et al. 2013). the extreme environmental conditions (high seasonal variations in salinity and temperature; redox conditions and nutrients; periodically high rss concentration in the whole water column) that characterize rogoznica lake govern the development of the above mentioned species that exist in the lake (ciglenečki et al. 2005, 2013, kršinić et al. 2013). during the research period (1998 – 2013) only 36 diatom taxa were recorded in the lake, as a signifi cant difference from other coastal and semi enclosed basins along the eastern adriatic coast; e.g. during one year of phytoplankton sampling in šibenik bay and kaštela bay in the middle adriatic, 61 and 80 diatom taxa were recorded, (bužančić et al. 2012) and 100 diatom taxa were found in the northern adriatic fjord-like lim bay (bosak et al. 2009). in comparison with other coastal regions of the adriatic sea, rogoznica lake is characterized by a high concentration of nutrients and their high variability within the water column (ciglenečki unpublished data). in potentially eutrophic coastal areas like the krka estuary and kotor bay, the recorded maximal concentrations of total inorganic nitrogen and silicates were up to 17.70 and 44.1 μmol l–1 (bosak et al. 2012; šupraha et al. 2014), while in the rogoznica lake these numbers are several times higher, 44.21 and 679 μmol l–1, respectively. however, registered mean values in the lake for total inorganic nitrogen, silicates and phosphate were 2.09 μmol l−1, 55.5 μmol l−1 and 1.98 μmol l−1, respectively. according to nutrient values rogoznica lake can be regarded as a highly eutrophic system. such a high concentration of phosphate was registered only in the most eutrophic environments along the eastern adriatic, e.g. in the krka estuary and kaštela bay (vidjak et al. 2012). during stratifi cation in the lake, the surface waters are poor in nutrients and phosphate limited (ciglenečki unpublished data). mixing events in the lake bring nutrient-rich bottom waters to the surface layer, which, accompanied by decomposition of dead organisms due to the anoxic stress, drastically change the concentration of nutrients and their ratios. it has a potential infl uence on the phytoplankton composition, abundance and diversity. however, it appears that the nutrients are not the main factor regulating the phytoplankton development in the lake. the impacts of redox conditions and light penetration might be crucial along with the mixing, concentration of nutrients and thermohaline conditions. changes in the thermohaline conditions in favor of lower salinity recorded after 2001 in the surface and middle water layers (fig. 2) (ciglenečki et al. 2013) coincide with a change in phytoplankton composition. after 2001 diatoms took over the domination with four dominant species that were alternately dominant. since d. fragilissimus abundance had a signifi cantly positive correlation with nitrite and ammonium, it could be speculated that d. fragilissiumus could be a possible indicator of the mixing in the lake. also, two dominant species c. choctawhatcheeana and t. nitzschioides had a positive correlation in the occurrence in the lake. diatoms in the extreme euxinic environment acta bot. croat. 74 (2), 2015 341 mixing of the water layers depends on meteorological conditions, which highly infl uence the intensity of the process (ciglenečki unpublished data). due to the oxidation of a relatively high concentration of sulphide, which is rapidly transported to the surface, a sudden consumption of oxygen and the occurrence of anoxic conditions in the whole water column may be seen. then the presence of rss (sulphide, polysulphide and colloidal sulphur formed by oxidation) can be found through the entire water column (ciglenečki et al. 2013). on the other hand, if the mixing is slow, oxygenated surface waters diffuse to the bottom of the lake, or the bottom waters, with plenty of sulphide and nutrients, diffuse to the surface layer of the lake (as was recorded in september of 2003 and november of 2006); the mixing process can last up to 7 days, while the surface layer remains rich in oxygen the whole time (ciglenečki unpublished data). in most cases when rss, i.e. sulphide, enters in the upper water layer, the abundance of phytoplankton species decreases because of the toxic effect which sulphur species could have on the phytoplankton community. on the other hand, those mixing events result in nutrient enrichment of the surface layer, stimulating phytoplankton development. variability in the stratifi cation conditions and the intensity of the mixing infl uence the phytoplankton development and domination of certain phytoplankton species. c. curvisetus usually reaches the highest abundance in spring during weak stratifi cation. after the mixing in autumn, d. fragilissimus takes over the dominant position, probably due to the absence of c. curvisetus which prefers lower temperatures and nutrient concentrations (burić et al. 2009). moderate phytoplankton abundance, extreme environmental conditions including high seasonal variations in salinity, temperature, mixing, periodically high rss and nutrient concentrations characterize rogoznica lake as highly eutrophic system which can be regarded as natural laboratory for monitoring of the selected biogeochemical processes. the changes in the phytoplankton composition and variability coincide with changes of the thermohaline conditions in the lake; only species with high adaptability to stress using encystment can survive in such an extreme environment. it can be speculated that the species which dominated in the lake during the research period were adjusted to these extreme conditions, while they were alternately dominant due to the adaptation of each species to the rapid changes of the specifi c environmental conditions. acknowledgements the croatian ministry of science, education and sports supported this study fi nancially through the projects 119-1191189-1228, 098-0982934-2717, 275-0000000-3186. financial support of croatian academy of science through the project »investigation of rogoznica lake, a unique anoxic environment on the adriatic coast related to recovering from catastrophic anoxia occurring in october 2011« is highly acknowledged. the authors thank z. roman, z. zovko, m. marguš, and m. čanković for help in sampling. references barić, a., grbec, b., kušpilić, g., marasović, i., ninčević, ž., grubelić, m., 2003: mass mortality event in a small saline lake (lake rogoznica) caused by unusual holomictic conditions. scientia marina 67, 129–141. malešević n., ciglenečki i., bura-nakić e., carić m., dupčić i., et al. 342 acta bot. croat. 74 (2), 2015 bosak, s., burić, z., djakovac, t., viličić, d., 2009: seasonal distribution of plankton diatoms in lim bay, northeastern adriatic sea. acta botanica croatica 68, 351–365. bosak, s., šilović, t., ljubešić, z., kušpilić, g., pestorić, b., krivokapić, s., viličić, d., 2012: phytoplankton size structure and species composition as an indicator of trophic status in transitional ecosystems: the case study of a mediterranean fjord-like karstic bay. oceanologia 54, 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eutrophic meromictic saline lake. fresenius environmental bulletin 14: 668–675. šupraha, l., bosak, s., ljubešić, z., mihanović, h., olujić, g., mikac, i., viličić, d., 2014: cryptophyte bloom in a mediterranean estuary: high abundance of plagioselmis cf. prolonga in the krka river estuary (eastern adriatic sea). scientia marina 78, 329– 338. utermöhl, h., 1958: zur vervollkommnung der quantitativen phytoplankton methodik. mitteilungen. internationale vereiningung für theoretische und angewandte limnologie 9, 1–38. vidjak, o., bojanić, n., matijević, s., kušpilić, g., ninčević gladan, ž., skejić, s., grbec, b., 2012: environmental drivers of zooplankton variability in the coastal eastern adriatic (mediterranean sea). acta adriatica 53, 243–262. viličić, d., djakovac, t., burić, z., bosak, s., 2009: composition and annual cycle of phytoplankton assemblages in the northeastern adriatic sea. botanica marina 52, 291– 305. viličić, d., marasović, i., kušpilić, g., 1996/1997: the heterotrophic ebridian microfl agellate hermesinum adriaticum zach in the adriatic sea. archiv für protistenkunde 147, 373–379. viličić, d., marasović, i., mioković, d., 2002: checklist of phytoplankton in the eastern adriatic sea. acta botanica croatica 61, 57–91. žic, v., carić-glunčić, m., viollier, e., ciglenečki, i., 2010: intensive sampling of iodine and nutrient speciation in naturally eutrophicated anchialine pond (rogoznica lake) during spring and summer seasons. estuarine, coastal and shelf science 87, 265–274. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 393 acta bot. croat. 74 (2), 393–406, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0029 the fi rst report on periphytic diatoms on artifi cial and natural substrate in the karstic spring bunica, bosnia and herzegovina anita dedić1*, anđelka plenković-moraj2, koraljka kralj borojević2, dubravka hafner1 1 department of biology, faculty of science and education, university of mostar, matice hrvatske bb, 88 000 mostar, bosnia and herzegovina 2 department of biology, faculty of science, university of zagreb, rooseveltov trg 6, 10 000 zagreb, croatia abstract – this study presents investigations of the periphytic diatoms on artifi cial (glass slides) and natural substrates in the karstic, limnocrene spring of bunica situated in the south of bosnia and herzegovina. investigations were performed in summer 2010. samples were collected every seven days for eight weeks. physical and chemical characteristics of water, temperature, oxygen saturation, dissolved oxygen, electric conductivity and nutrients as well as fl ow velocity at sample site, were measured simultaneously with each sampling. physical and chemical characteristics showed low temperature oscillations, good aeration and oligotrophic conditions. in general, greater diatom diversity was noted on natural substrate. a total of 104 diatom species were found on natural substrate and 82 on glass slides. the best represented genera on both types of substrate were gomphonema and navicula (each with eight species), nitzschia (with six species), and cocconeis (with fi ve species). achnanthidium exiguum, achnanthidium minutissimum, amphora pediculus, cymbopleura amphicephala and surirella minuta were recorded in all samples of natural substrate and gomphonema minutum in artifi cial substrate samples. key words: bosnia and herzegovina, bunica spring, glass slides, karst spring, periphytic diatoms introduction karst is a very specifi c geomorphological phenomenon, characterized by a rapid percolation of surface water into the underground as well as by the paucity of surface water fl ows. most fl ows occur below the surface. karst springs are a common occurrence in karst, as a special type of landscape, and they are places where groundwater comes to the surface * corresponding author, e-mail: dedic.anita@gmail.com dedić a., plenković-moraj a., kralj borojević k., hafner d. 394 acta bot. croat. 74 (2), 2015 creating a visible fl ow. limnocrene springs are the most widespread type of springs in calcareous substrates, such as karst (di sabatino et al. 1997). according to their hydrological, physical and chemical characteristics, karstic springs are attractive and interesting ecological sites. they are characterized by physico-chemical stability. almost all springs have a low water temperatures (8–12 °c) with small annual fl uctuations, high concentrations of oxygen (8–12 mg l–1) and high concentrations of carbon dioxide (up to 60 mg l–1) (blagojević 1974, cantonati et al. 2007). karst springs have high alkalinity, which is mainly due to carbonates, and high carbonate hardness (blagojević 1974, štambukgiljanović 1998). the concentration of nitrite is about 0.01 mg l–1 (cantonati et al. 2007) and concentrations of nitrate 0.4–8.0 mg l–1. sulphates are usually present with concentrations of a few tenths of mg l–1 while hundreds of mg l–1 can be found in springs fed by aquifers that also include evaporites or geological formations which contain gypsum (cantonati et al. 2007). phosphorus is also present in low concentrations (0.005–0.050 mg l–1) (blagojević 1974, werum 2001). silicates are very important for the growth of diatoms. underground waters in siliceous aquifers usually hold 3–10 mg l–1 and in carbonate aquifers about 1 mg l–1 sio2 (cantonati et al. 2007). a common characteristic of karstic springs, whether permanent or temporary, is the strong dependence of discharge on precipitation. as a consequence, the ratio between minimum and maximum discharge is great (1:60, or more). the minimum and maximum discharges registered for bunica spring were 0.72 and 207 m3 s–1, respectively (milanović 2006). all values of physico-chemical parameters during major rainfall may be increased. the periphytic communities of karstic springs in bosnia and herzegovina have not been suffi ciently studied; only few authors have addressed the issue (blagojević 1974, 1976, 1979, hafner 2009, hafner et al. 2010, dedić et al. 2012). algae, especially diatoms, in springs have received little attention in bosnia and herzegovina, despite the fact that springs provide specifi c conditions that cannot be found in any other freshwater ecosystem and notwithstanding their great importance in terms of general environmental changes. the aim of this study was to determine the qualitative and quantitative composition of periphytic diatoms on artifi cial (glass slides) and natural substrates in fast and slow water fl ow in the bunica spring. the results of this study give us preliminary results from the investigations of periphytic diatoms in springs and are a fi rst step towards a detailed elaboration of diatom distribution and taxonomy in this very poorly investigated area. materials and methods study area bunica spring is a deep siphonal limnocrene spring of the eponymous river bunica. it is located in the southeastern part of the mostar valley, near the towns of blagaj and mostar (fig. 1) in the south of bosnia and herzegovina. the bunica river is 6 km long and is the main left tributary of the river buna, a tributary of the river neretva. there are no direct hydrogeological connections between the buna and the bunica (milanović 2006). the outlet channel was researched to the depth of 73 m and over a length of 160 m (milanović 2006). bunica spring is the surface outlet of the underground part of the zalomka river, periphytic diatoms in karstic spring bunica acta bot. croat. 74 (2), 2015 395 subsequent to its sinking into the biograd ponor (fig. 1). the catchment area of bunica spring is estimated at approximately 160 km2 (not including the zalomka river) (milanović 2006). biograd ponor and bunica spring are directly connected with the karst channel which is one of the most direct underground connections in this part of dinaric karst (milanović 2006). bunica spring is located at the contact between karstifi ed carbonate rocks and miocene deposits. it is located in an area infl uenced by the mediterranean climate, which is characterized by long, hot summers and rainy winters with rare occurences of snow (galić 2011). the average annual air temperature is 14.6 °c. the highest daily average temperature is 23.2 °c in july and the lowest below 5.8 °c in january (data for the city of mostar, for the period 1971–2000, recorded by meteorological and hydrological service of bosnia and herzegovina). the total annual precipitation is about 1,515 mm. this area has approximately 2,291 hours of sunshine per year. average relative humidity is 69. water vegetation in bunica spring consists of algae and mosses, leaves, branches and roots of plants. sampling samples from artifi cial and natural substrate and water for physical and chemical analyses were collected in summer 2010 in bunica spring. summer minimum riverine water discharge and higher temperature conditions provide favorable and stable conditions for the development of periphytic diatoms (hillebrand and sommer 2000). diatom colonization was monitored after 7, 14, 21, 28, 35, 42, 49 and 56 days of exposure of artifi cial substrates (glass slides) that were horizontally placed and oriented parallel to the current velocity. two different microhabitats were defi ned with regard to the current velocity. current velocities of water were measured using a dostmann p 670 probe.the microhabitats were made of seven glass slides that were fi xed on the upper side of a brick. fig. 1. study area site of bunica spring located in the southeastern part of the mostar valley, near the towns of blagaj and mostar in the south of bosnia and herzegovina. dedić a., plenković-moraj a., kralj borojević k., hafner d. 396 acta bot. croat. 74 (2), 2015 glass slides were washed with distilled water before being placed into the spring and one or two slides were taken every seven days. samples from the artifi cial substrate were stored in a labeled bottle in the fi eld and upon return to the laboratory they were scraped with a razor blade and scalpel. simultaneously, samples from the natural substrate were collected applying the standard procedure of scraping sludgy material from the rock’s surface making sure that the total surface of all rocks is about 500 cm2 (european standard en 13946, 2003). rocks were chosen randomly according to standard procedures. identifi cation and morphological examination of diatoms all the samples were fi xed with 4% formaldehyde. permanent preparations of diatoms were prepared using the hustedt (1930) method. the species were identifi ed using the microscopes olympus bx53 and carl zeiss jena. different keys and guides were used for taxa determination: hustedt (1930), krammer and lange-bertalot (1986, 1988a, 1988b), krammer (1988, 2000, 2004, 2010), lange-bertalot (2001, 2002). the nomenclature of taxa is determined by the nomenclature set out in algae base (guiry and guiry 2014). measurement of physico-chemical parameters of spring water the physical and chemical parameters considered in this study were: water temperature, oxygen saturation, dissolved oxygen, electric conductivity and nutrients (nitrate, nitrite, silicate and orthophosphate) as well as fl ow velocity. water temperature, dissolved oxygen, oxygen saturation and conductivity were measured using the wtw probe (wissenschaftlich-technischewersätten gmbh & co. kg-weilheim) whereas nutrient concentrations (mg l–1) were determined using the standard spectrophotometrical method (apha, 1995) in public utility company in ljubuški. to test the differences between the fl ow regimes and different exposure periods, oneway anova with post hoc bonferoni test was employed. results physico-chemical parameters of bunica spring results of physico-chemical measurements of bunica spring water (temperature, conductivity, dissolved oxygen concentrations, oxygen saturation, nutrients and current velocities) are shown in table 1. data show low temperature oscillations, a low concentration of nutrients, good aeration and generally, oligotrophic conditions. the slow current velocity at the site had an average value of about 0.01 m s–1 and fast current velocity of about 0.9 m s–1. diatom community on artifi cial substrate in periphyton from artifi cial substrates a total of 82 diatom taxa were determined (tab. 2). the genus gomphonema dominates in terms of number of taxa (with 8 taxa), navicula is next (with 7 taxa), followed by cocconeis and nitzschia (each with 5 taxa). the most taxa, 77 of them, were recorded after 56 days of exposure of the artifi cial substrate in the slow fl ow, and the fewest, with only 2, after 21 days in the fast fl ow (fig. 2). periphytic diatoms in karstic spring bunica acta bot. croat. 74 (2), 2015 397 generally, abundance as well as diversity of diatom species was higher in the slow than in the fast fl ow throughout the monitored exposure period although the values were very similar in both types of sites (no statistically signifi cant differences, p > 0.05). the fi rst colonizers on the artifi cial biofouling substrate, regardless of the water current velocity were achnanthidium, amphora and cocconeis. gomphonema minutum, cocconeis placentula, cocconeis placentula var lineata and achnanthidium minutissimum were the most common taxa in biofouling from the artifi cial substrate. the aforementioned taxa were present in more than 68% of samples but with low abundance (fig. 3). taxa started to adhere to the artifi cial biofouling after 7 days of exposure in the slow fl ow (total number of cells per cm2 was 2854), and in the fast fl ow after 21 days (total number of cells per cm2 was 10) (fig. 2). in 28 to 56 days of exposure, more massive colonization of cymatopleura, epithemia, encyonema, gomphonema, meridion and nitzschia was recorded and they were more dominant in the slow fl ow. tab. 1. physical and chemical parameters of water at bunica spring. min. – minimum value, max. – maximum value, avg – average value, sd – standard deviation, f – fast fl ow, s – slow fl ow. parameter min. max. avg sd temperature (°c) 11.3 12.3 12.05 0.31 dissolved oxygen (mg l–1) 9.73 12.4 10.73 0.951 saturation (%) 91 115.6 99.828 9.805 conductivity (μs cm–1) 339 413 399.6 24.86 nitrite (mg l–1) 0.0002 0.0015 0.0009 0.0004 nitrate (mg l–1) 1.91 2.85 2.408 0.333 orthophosphate (mg l–1) 0.0093 0.0494 0.0288 0.0132 silicate (mg l–1) 2.06 2.53 2.475 0.136 current velocity f (m s–1) 0.58 1.44 0.85375 0.268 current velocity s (m s–1) 0 0.02 0.0175 0.007 fig. 2. abundance and diversity of periphytic diatoms on the artifi cial substrate on the 7th, 28th and 56th days of the exposure time in fast and slow fl ow in bunica spring. dedić a., plenković-moraj a., kralj borojević k., hafner d. 398 acta bot. croat. 74 (2), 2015 tab. 2. list of diatom taxa determined in the bunica spring samples. taxa frequency of occurrence (%) natural substrate artifi cial substrate achnanthes infl ata (kützing) grunow 5.9 0 achnanthidium exiguum (grunow) d. b. czarnecki 100 12.5 achnanthidium helveticum (hustedt) monnier, lange-bertalot & ector 5.9 6.3 achnanthidium minutissimum (kützing) czarnecki 100 68.8 achnanthidium pyrenaicum (hustedt) h. kobayasi 76.5 37.5 amphora ovalis (kützing) kützing 64.7 37.5 amphora pediculus (kützing) grunow ex a. schmidt 100 56.2 brebissonia boeckii (ehrenberg) e. o. meara 5.9 6.3 caloneis alpestris (grunow) cleve 23.5 6.3 caloneis bacillum (grunow) cleve 70.6 43.8 caloneis ventricosa(ehrenberg) f. meister 35.3 6.3 cocconeis neodiminuta krammer 11.8 25 cocconeis pediculus ehrenberg 52.9 50 cocconeis placentula var. lineata (ehrenberg) van heurck 64.7 68.8 coconeis placentula ehrenberg 82.4 75 coconeis placentula var. euglypta (ehrenberg) grunow 29.4 25 cyclotella meneghiniana kützing 29.4 6.3 cyclotella ocelatta pantoscek 41.2 12.5 cyclotella sp. 23.5 18.8 cymatopleura elliptica (brébissoni) w. smith 41.2 31.3 cymatopleura solea (brébissoni) w. smith 23.5 31.3 cymbella affi nis kützing 29.4 18.8 cymbella cymbiformis c. agardh 17.6 12.5 cymbella excise kützing 11.8 0 cymbella helvetica kützing 5.9 0 cymbella tumida (brébisson) van heurck 11.8 0 cymbopleura amphicephala (nägeli) krammer 100 12.5 cymbopleura naviculiformis (auerswald ex heiberg) k. krammer 5.9 12.5 denticula tenuis kützing 47.1 31.3 diatoma ehrenbergii f. capitulate (grunow) lange-bertalot 17.6 18.8 diatoma hiemalis (lyngbye) heiberg 17.6 18.8 diatoma mesodon (ehrenberg) kützing 35.3 25 diatoma vulgaris bory de saint-vincent 64.7 31.3 diploneis marginestriata hustedt 11.8 0 periphytic diatoms in karstic spring bunica acta bot. croat. 74 (2), 2015 399 taxa frequency of occurrence (%) natural substrate artifi cial substrate diploneis oblongella (nägeli ex kützing) cleve-euler 29.4 25 diploneis ovalis (hilse) cleve 11.8 18.8 ellerbeckia arenaria (moore ex ralfs) r. m. crawford 17.6 18.8 encyonema gracile rabenhorst 82.4 0 encyonema minutum (hilse) d. g. mann 58.8 50 encyonema prostratum (berkeley) kützing 5.9 0 encyonema silesiacum (bleisch) d. g. mann 82.4 50 encyonopsis microcephala (grunow) krammer 23.5 0 epithemia argus (ehrenberg) kützing 52.9 37.5 eunotia arcus ehrenberg 5.9 12.5 eunotia bilunaris (ehrenberg) schaarschmidt 23.5 6.3 eunotia pectinalis (kützing) rabenhorst 11.8 6.3 eunotia praerupta ehrenberg 11.8 31.3 fragilaria capucina desmazières 41.2 12.5 fragilaria capucina var. gracilis (oestrup) hustedt-unchecked 47.1 18.8 fragilaria capucina var. vaucheriae (kützing) lange-bertalot 29.4 6.3 fragilaria constricta ehrenberg 5.9 12.5 fragilaria construens (ehrenberg) grunow 64.7 0 frustulia vulgaris (thwaites) de toni 11.8 0 gomphonema acuminatum ehrenberg 23.5 6.3 gomphonema gracile ehrenberg 11.8 18.8 gomphonema intricatum kützing 82.4 50 gomphonema micropus kützing 52.9 6.3 gomphonema minutum (c. agardh) c. agardh 76.5 93.8 gomphonema olivaceum (hornemann) brébisson 41.2 18.8 gomphonema parvulum (kützing) kützing 94.1 31.3 gomphonema truncatum ehrenberg 23.5 12.5 gomphosphenia lingulatiformis (lange-bertalot & e. reichardt) lange bertalot 17.6 25 gyrosigma acuminatum (kützing) rabenhorst 52.9 31.3 gyrosigma attenuatum(kützing) rabenhorst 17.6 0 halamphora veneta (kützing) levkov 5.9 0 melosira sp. 11.8 0 melosira varians c. agardh 58.8 6.3 meridion circulare (greville) c. agardh 52.9 37.5 dedić a., plenković-moraj a., kralj borojević k., hafner d. 400 acta bot. croat. 74 (2), 2015 taxa frequency of occurrence (%) natural substrate artifi cial substrate navicula capitatoradiata germain 4.2 6.3 navicula cari ehrenberg 17.6 25 navicula cryptocephala kützing 58.8 31.3 navicula cryptotenella lange-bertalot 17.6 0 navicula exilis kützing 47.1 37.5 navicula radiosa kützing 35.3 6.3 navicula tripunctata (o. f. müller) bory de saint-vincent 88.2 37.5 neidium dubium (ehrenberg) cleve 5.9 0 neidium productum (w. smith) cleve 5.9 0 nitzschia amphibia grunow 41.2 0 nitzschia dissipata (kützing) rabenhorst 64.7 50 nitzschia linearis west 23.5 37.5 nitzschia palea (kützing) w. smith 76.5 62.5 nitzschia sigmoidea (nitzsch) w. smith 52.9 6.3 nitzschia sublinearis hustedt 82.4 6.3 placoneis clementioides (hustedt) cox 100 25 planothidium delicatulum (kützing) round&bukhtiyarova 29.4 6.3 planothidium ellipticum(cleve) round&bukhtiyarova 17.6 6.3 planothidium lanceolatum (brébisson ex kützing) lange-bertalot 11.8 18.8 planothidium rostratum (oestrup) lange-bertalot 5.9 31.3 psammothidium oblongellum (østrup) van de vijver 11.8 6.3 reimeria sinuata (gregory) kociolek&stoermer 5.9 0 rhoicosphenia abbreviata (c. agardh) lange-bertalot 70.6 12.5 rossithidium linearis(w. smith) round & bukhtiyarova 58.8 25 sellaphora pupula (kützing) mereschkovsky 17.6 6.3 stauroneis anceps ehrenberg 5.9 0 stauroneis smithii grunow 11.8 6.3 staurosirella pinnata (ehrenberg) d. m. williams & round 52.9 31.3 stephanodiscus hantzschii grunow 76.5 31.3 stephanodiscus sp. 41.2 6.3 surirella angusta kützing 11.8 6.3 surirella brebissonii krammer& lange-bertalot 5.9 0 surirella linearis w. smith 17.6 15.8 surirella minuta brébisson 100 0 ulnaria ulna (nitzsch) p. compère 58.8 43.8 periphytic diatoms in karstic spring bunica acta bot. croat. 74 (2), 2015 401 massive accumulation of taxa as well as number of cells was recorded from the 7th to the 35th day of periphyton colonization on the artifi cial substrate in both fast and slow fl ow. from 35th to 56th day the number of taxa was relatively constant but the number of cells was on the decrease (fig. 2). diatom community on natural substrate a total of 104 diatom taxa were determined in periphyton from natural substrates (tab. 2). in periphyton from natural substrates gomphonema and navicula were the most numerous genera (each with 8 taxa), followed by nitzschia (with 6 taxa) and cocconeis, cymbella and fragilaria (each with 5 taxa). achnanthidium exiguum (grunow) czarnecki, achnanthidium minutissimum (kützing) czarnecki, amphora pediculus (kützing) grunow ex a. schmidt, cymbopleura amphicephala (nägeli) krammer and surirella minuta brébisson were present in all samples during the research period. the highest number of taxa, 87 of them, were registered on the 2nd sampling in the slow fl ow, and the lowest, 55, on the 9th sampling in the fast fl ow (fig. 4). fig. 3. maximum abundance of the most common taxa on the artifi cial substrate on the 7th, 28th and 56th days of the exposure time in fast and slow fl ow in bunica spring. fig. 4. abundance and diversity of periphytic diatoms on the natural substrate at the 2th, 5th and 9th samplings. dedić a., plenković-moraj a., kralj borojević k., hafner d. 402 acta bot. croat. 74 (2), 2015 all the taxa recorded in the biofouling from the artifi cial substrate were present in the samples from the natural substrate as well. in the samples from the natural substrate there were 22 taxa, which were not found in the biofouling from the artifi cial substrate and these are achnanthes infl ata (kützing) grunow, cyclotella sp., cymbella excisa kützing, cymbella helvetica kützing, cymbella tumida (brébisson) van heurck, diploneis marginestriata hustedt, encyonema gracile rabenhorst, encyonema prostratum (berkeley) kützing, encynopsis microcephala (grunow) krammer, fragilaria construens (ehrenberg) grunow, frustulia vulgaris (thwaites) de toni, gyrosigma attenuatum (kützing) rabenhorst, halamphora veneta (kützing) levkov, melosira sp., navicula cryptotenella lange-bertalot, neidium dubium (ehrenberg) cleve, neidium productum(w. smith) cleve, nitzschia amphibia grunow, reimeria sinuate (gregory) kociolek & stoermer, stauroneis anceps ehrenberg, surirella brebissonii krammer & lange-bertalot and surirella minuta brébisson. the aforementioned taxa were present with low appearance frequency in samples, with the exception of encyonema gracile rabenhorst, fragilaria construens (ehrenberg) grunow and surirella minuta brébisson that were present in more than 50% of samples but with low abundance. total number of cells per cm2 was very similar in both fast and slow fl ow from 1st to 4th sampling, whereas on the 5th sampling there was a larger number of cells recorded in the fast fl ow than in the slow part of the fl ow, on the 9th sampling the number of cells sharply decreased in both fl ows (fig. 4). taxa and their frequency of appearance were similar in the fast as well as in the slow fl ow. discussion this paper presents research of periphytic diatoms sampled from natural substrate and artifi cial substrate in bunica spring. bunica spring showed low temperature oscillations, low concentration of nutrients, good aeration and oligotrophic conditions, which are characteristic of karstic springs (blagojević 1974) and the measured values correspond to values recorded in other springs (blagojević 1974, werum 2001). use of artifi cial substrates (glass slides) for quantifi cation of periphyton was recommended by wahl and mark (1999), albay and akcaalan (2003), liboriussen (2005) who consider them more favorable for experimental growth than natural substrates. glass slides are the most frequently used artifi cial substrates because of their uniform size and inert surface (àcs and kiss 1993, àcs et al. 2000, barbiero 2000). total surface area is known and the problems associated with the measurement of irregular natural substrates are eliminated (lamberti and resh 1985). in the early days of fouling, increased diversity can be related to the colonization of new taxa (hillebrand and sommer 2000). through development of fouling on artifi cial substrate, stages of growth and loss take turns. the growth stage is characterized by colonization and growth of algae whereas the loss stage can be related to emigration, dying, peeling off and grazing (biggs 1996). at the end of the exposure period, the recorded decrease of the taxa number was infl uenced by the weather conditions (42nd day) accompanied by the high current velocity, which caused fl ushing. slow fl ow showed a greater diversity and abundance of taxa possibly because it gives more favorable habitat for vegetation growth, which leads to greater colonization of epilithic taxa (falasco et al. 2012). periphytic diatoms in karstic spring bunica acta bot. croat. 74 (2), 2015 403 the relatively small number of taxa recorded in bunica spring, 104 on natural substrates and 82 on artifi cal substrates, matches the results in the reviewed papers (blagojević 1974, sabater and roca 1990, 1992, cantonati 1998, taxböck and preisig 2007, hafner et al. 2008, cantonati and spitale 2009, angeli et al. 2010, dedić et al. 2012, wojtal and sobezyk 2012). bunica spring also showed great similarity to the others with regard to the composition of diatom taxa. townsend and gell (2005) as well as cantonati and spitale (2009) argue that similar taxa occur on a large number of substrates. genera with the highest number of taxa on both types of substrate are gomphonema, navicula, nitzschia, achnanthidium, cocconeis, fragilaria, planothidium and cymbella, which is very similar to results of angeli et al. (2010). barbiero (2000) compared epilithic diatoms on natural and artifi cial substrates and concluded that if there was any difference between the substrates, the difference was insignifi cant. in this study samples from natural substrates showed greater species diversity. that was probably due to the fact that fouling on the natural substrate lasted for an unknown but probably longer time than on the artifi cial substrate, so the equilibrium was reached and competitive preferences of early colonizers were not limiting for the attainment of maximum richness under those conditions. achnanthidium minutissimum and cocconeis placentula were the most abundant taxa in this research, on both natural and artifi cial substrate. they were recorded as the fi rst colonizers of the artifi cial substrate in springs as well as of other habitats. the reason for their high abundance is their ability to respond well to disturbance and relatively high growth rates, which enable them to populate the surface before their competitors (barbiero 2000). wojtal and sobezyk (2012) carried out research into springs, fi nding that achnanthidium minutissimum was the most represented taxon. in both of the systems (fast and slow) and on the natural and the artifi cial substrate the same taxa were recorded. the most numerous taxa were similar as well; they had similar appearance frequency, which implies they are characteristic spring species. although numerous studies (munteanu and maly 1981, plenković et al. 2008) showed that fl ow velocity greatly infl uences diatom community, this study did not confi rm such fi ndings. that is probably caused by the small difference between the velocities (about 1 m s–1) or unrecorded changes in the fl ow regime between two sampling occasions. kralj borojević (2011) also recorded similar dominant and subdominant taxa in lothic and lenthic biotopes of the krka river. this study has shown that diatoms on an artifi cial substrate adequately represent diatoms on natural substrates, with respect both to taxa composition and abundance. acknowledgement the authors would like to thank zlatko grizelj for all the help during fi eldwork and measurement of physical and chemical parameters. references àcs, é., kiss, k. t., 1993: colonization processes of diatoms on artifi cial substrates in the river danube near budapest (hungary). hydrobiologia 269/270, 307–315. àcs, é., kiss, k. t., szabó, k., makk, j., 2000: short-term colonization sequence of periphyton on glass slides in a large river (river danube, near budapest). archiv für hydrobiologie supplement 136-algological studies 100, 135–156. dedić a., plenković-moraj a., kralj borojević k., hafner d. 404 acta bot. croat. 74 (2), 2015 albay, m., akcaalan, r., 2003: comparative study of periphyton colonisation on common reed (phragmites australis) and artificial substrate in a shallow lake, manyas, turkey. hydrobiologia 506–509, 531–540. angeli, n., cantonati, m., spitale, d., lange-bertalot, h., 2010: a comparison between diatom assemblages in two groups of carbonate, low-altitude springs with different levels of anthropogenic disturbances. fottea 10, 115–128. apha (american public health association) 1995: standard methods for the examination of water and wastewater. american public health association, washington, dc. barbiero, r. p., 2000: a multi-lake comparison of epilithic diatom communities on natural and artifi cial substrates. hydrobiologia, 438, 157–170. biggs, b. j. f., 1996: patterns in benthic algae of streams. in: stevenson, r. j., bothwell, m. l., lowe, r. l. 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plenković-moraj a., kralj borojević k., hafner d. 406 acta bot. croat. 74 (2), 2015 munteanu, i., maly, e. j., 1981: the effect of current on the distribution of diatoms settling on submerged glass slides. hydrobiologia 78, 273 – 282. plenković-moraj, a., kralj, k., gligora, m., 2008: effect of current velocity on diatom colonization on glass slides in unpolluted headwater creek. periodicum biologorum 110, 291–295. sabater, s., roca, j. r., 1990: some factors affecting distribution of diatom assemblages in pyrenean springs. freshwater biology 24: 493–507. sabater, s., roca, j. r., 1992: ecological and biogeographical aspects of diatom distribution in pyrenean springs. british phycological journal 27, 203–213. štambuk-giljanović, n., 1998: the waters of the neretva river and its basins. public health institute of split, croatia. taxböck, l., preisig, h. r., 2007: the diatom communities in swiss springs: a fi rst approach. proceedings of the 1st central european diatom meeting, berlin, 163–168. townsend, s. a., gell, p. a., 2005: the role of substrate type on benthic diatom assemblages in the daly and roper rivers of the australian wet/dry tropics. hydrobiologia 548, 101–115 wahl, m., mark, o., 1999: the predominantly facultative nature of epibiosis experimental and observational evidence. marine ecology progress series 187, 59–66. werum, m., 2001: die kieselalgengesellschaften in quellen. abhängigkeit von geologie und anthropogener beeinfl ussung in hessen (bundesrepublik deutschland). hessisches landesamt für umwelt und geologie. wojtal, a. z., sobezyk, l., 2012: the infl uence of substrates and physicochemical factors on the composition of diatom assemblages in karst springs and their applicability in water-quality assessment. hydrobiologia 695, 97–108. opce-str.vp acta bot. croat. 69 (1), 19–29, 2010 coden: abcra 25 issn 0365–0588 the lichen flora of risnjak national park (croatia) sini[a ozimec1*, ivica bo[kovi]1, tihomir florijan^i]1, dinko jelki]1, an\elko opa^ak1, zlatko pu[kadija1, irena labak2 1 university of j. j. strossmayer, faculty of agriculture, trg sv. trojstva 3, hr-31000 osijek, croatia 2 university of j. j. strossmayer, department of biology, trg lj. gaja 6, hr-31000 osijek, croatia this paper lists 80 lichen taxa recorded for risnjak national park. among the listed species, candelariella reflexa, chaenotheca brunneola, placynthiella icmalea, usnea diplotypus and usnea subfloridana have been already first reported for croatia. the field survey was carried out at 14 collection sites in the periods 1997–1998 and 2001–2002. floristic composition, life form spectrum and substrate preferences are described. the most numerous genera are cladonia, pertusaria, lecanora and peltigera. lichens growth on 16 various substrates, among which the deciduous trees, acer pseudoplatanus and fagus sylvatica dominate. the alliance lobarion pulmonariae, consisting of some rare and old-forest indicator species, is present within the area. key words: lichen, taxonomic list, life form, substrate, risnjak, croatia introduction the first botanical research in the area of risnjak and snje`nik mountains was undertaken more than 180 years ago. comprehensive botanical research including phytosociology and vegetation mapping in gorski kotar region was started by ivo horvat in 1927 (horvat 1930, 1931). the results of his thirty-five-year long investigation are described in the publication »vegetation of the mountains in western croatia« (horvat 1962). lichenological researches in croatia date back to the second part of the 19th century in the north adriatic coastal area and the nearby hinterland of gorski kotar (matkovi] 1879). a major contribution to the knowledge of lichen flora of rijeka and gorski kotar was made by johann schuler, who published a list of 329 taxa (schuler 1902). the lichens collected by schuler were discussed by zahlbruckner, whose important contribution is the description of several new lichen species (zahlbruckner 1905). the croatian natural scientist dragutin hirc, in a description of the path to mali risnjak recorded that the moss was densely overgrown by cetraria islandica (hirc 1898). during research into the vegetation acta bot. croat. 69 (1), 2010 19 * corresponding author: sinisa.ozimec@pfos.hr u:\acta botanica\acta-botan 1-10\ozimec.vp 13. travanj 2010 9:24:36 color profile: disabled composite 150 lpi at 45 degrees of the massifs of risnjak and snje`nik, horvat (1930, 1962) recorded the presence of cetraria islandica and cladonia sp. in phytocoenological relevés of the grassland alliance seslerion tenuifoliae. lichen vouchers collected during this research in 1927, 1948 and 1949 year are deposited in the ivo and marija horvat herbarium (zaho) in zagreb. the outstanding croatian lichenologist fran ku{an has visited risnjak and many other localities in gorski kotar in 1947 (bertovi] 1994). however, there is no evidence of specimens collected or any references in ku{an’s compilation on lichens from the former yugoslavia (ku[an 1953). lichenological research in the area of risnjak national park was renewed in the periods 1997–1998 and 2001–2002, as presented in this paper. study area lichenological research was carried out in gorski kotar, a mountainous region in western croatia, which is, with 60% forest cover, the most wooded part of the country. risnjak national park (fig. 1.) extends over 64 sq. km, including the massifs of risnjak (1528 m) and snje`nik (1506 m), part of the western branch of the dinaric mountains, and the source area of the kupa river up to the village hrvatsko (287 m). the area of the risnjak massif was proclaimed a national park in 1953, being extended in 1997 to snje`nik and the source of the kupa river. 20 acta bot. croat. 69 (1), 2010 ozimec s., bo[kovi] i., florijan^i] t., jelki] d., opa^ak a., pu[kadija z., labak i. fig. 1. map of the study area with indicated collection sites u:\acta botanica\acta-botan 1-10\ozimec.vp 9. travanj 2010 11:51:45 color profile: disabled composite 150 lpi at 45 degrees according to data from the meteorological station in lividraga (939 m), the climate is perhumid and moderately cold. mean annual temperature is 5.4 °c, the lowest in january (– 2.0 °c) and the highest, 14.2 °c, in july. rainfall averages 3770 mm per year, the highest amount occurring in november (488 mm), and lowest in august (166 mm). air humidity is high (94%). due to the prevailing limestone bedrock a typical karst relief with its specific morphology has developed. the vegetation of risnjak national park consists of around 30 plant communities, among which 14 are forest associations. due to their inaccessibility, some parts of the forests are still in primeval condition. mixed beech and fir forests of the dinaric vegetation zone (omphalodo-fagetum) cover the largest part of the area up to 1240 m (trinajsti] 1995). at the altitude of 1200–1400 m this zone is replaced by prealpine beech forest (ranunculo platanifolii-fagetum). the upper boundary of the forest vegetation above 1450 m makes a special dinaric association of mountain pine forest (lonicero borbasianae-pinetum mugi). the steep and warmer limestone blocks at 950–1350 m are covered by fir forests (calamagrosti-abietetum). in the lower parts of the park, subalpine spruce forest (listero-piceetum abietis) grows in wet and cold locations in the shallow depressions, and on the edge of dolines. acidophilous fir forest (blechno-abietetum) grows on acid soil at 680–800 m. the warmer climatic influence of the kupa valley favours the occurrence of thermophilous forest associations such as hop hornbeam forests (erico-ostryetum and ostryo-fagetum), while pure illyrian beech forests (lamio orvalae-fagetum) grow on heights between 450 and 700 m. on the western boundary of the park, in area of lazac and [egine, and in the leska valley, meadows of calcareous grasslands (bromo-plantaginetum and festuco-agrostietum) are surround by old forests. the peaks of risnjak and snje`nik are covered by alpine grasslands belonging to the endemic alliance seslerion tenuifoliae. material and methods lichen samples were collected in the periods 1997–1998, and 2001–2002 at 14 collection sites within the area of the risnjak national park, as listed below: 1. bijela vodica, 700 m, mtb 0554/3 (14.–15.11.1997, 13.8.1998, 10.6.2001, 27.9.2001) 2. leska, 700 m, mtb 0554/3 (15.–17. 11.1997, 13.8.1998) 3. podi, 960 m, mtb 0553/4 (17. 11. 1997) 4. markov brlog, 940 m, mtb 0553/4 (16. 11.1997) 5. smrekovac, 1140 m, mtb 0553/4 (16. 11.1997) 6. risnik, 800 m, mtb 0554/3 (15.11.1997) 7. vilje, 1180 m, mtb 0553/4 (4.10.1997) 8. cajtige, 1240–1380 m, mtb 0553/4 (4.10.1997) 9. lazac, 1100 m, mtb 0553/2 (12.8.1998) 10. japetova [egina, 940 m, mtb 0553/2 (12.8.1998) 11. medvje|a vrata, 1120–1300 m, mtb 0553/4 (4.10.1997, 16.11.1997) 12. veliki risnjak and ju`ni mali risnjak, 1360–1528 m, mtb 0553/4 (4.10.1997, 17.–18.11.1997, 23.7.1998, 30.4.2002) 13. razloge village, 500 m, mtb 0554/1 (24.6.1998, 11.8.1998, 10.6.2001) 14. between the source of the kupa river and village kupari, 321–500 m, mtb 0454/3, 0554/1 (24.6.1998, 11.8.1998, 2.5.2002) acta bot. croat. 69 (1), 2010 21 lichens of risnjak national park (croatia) u:\acta botanica\acta-botan 1-10\ozimec.vp 9. travanj 2010 11:51:45 color profile: disabled composite 150 lpi at 45 degrees identification in the field was made with a hand lens, and in the laboratory using a dissecting microscope, a light microscope and the usual spot test, according to the reference books: clerc (1998), purvis et al. (1994), vitikainen (1994) and wirth (1995). some specimens were analysed by thin-layer chromatography according to orange et al. (2001). the lichen collections from the herbaria: za, zaho, edi and gzu have been studied. data on locality and substrate for each species are given. nomenclature follows wirth (1995), suppan et al. (2000) and mayrhofer (2006). results according to literature sources, collection records at herbaria and field survey results, the currently known lichen flora of the risnjak national park comprises 80 species belonging to 53 genera. the lichen species are listed alphabetically (tab. 1). among the listed species, candelariella reflexa, chaenotheca brunneola, placynthiella icmalea, usnea diplotypus and usnea floridana were first reported for croatia by ozimec (2000). 22 acta bot. croat. 69 (1), 2010 ozimec s., bo[kovi] i., florijan^i] t., jelki] d., opa^ak a., pu[kadija z., labak i. tab. 1. the lichen species recorded in risnjak national park in the period 1997–1998, and 2001–2002. species site substrata identified lichen compounds arthonia radiata (pers.) ach 14 fraxinus excelsior bilimbia lobulata (sommerf.) hafellner et coppins 12 calcareous rock caloplaca cerina (ehrh. ex hedw.) th. fr. v. cerina 1 12 salix sp., acer pseudoplatanus fagus sylvatica caloplaca flavovirescens (wulfen) dalla torre et sarnth. 12 calcareous rock caloplaca herbidella (hue) h. magn. 11 14 acer pseudoplatanus prunus sp. candelariella reflexa (nyl.) lettau 1, 2, 4 acer pseudoplatanus cetraria islandica (l.) ach. risnjak and snje`nik; vauchers in zaho cetrelia olivetorum (nyl.) w. l. culb. et c. f. culb. 9 14 acer pseudoplatanus fagus sylvatica chaenotheca brunneola (ach.) müll. arg. 8 on the mosses cladonia coniocraea (flörke) spreng. 2 6 9 11 14 on the mosses abies alba fagus sylvatica, picea abies abies alba prunus sp. fumarprotocetraric acid u:\acta botanica\acta-botan 1-10\ozimec.vp 13. travanj 2010 9:26:09 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (1), 2010 23 lichens of risnjak national park (croatia) species site substrata identified lichen compounds cladonia fimbriata (l.) fr 1 on the mosses cladonia furcata (huds.) schrad. risnjak; vauchers in zaho fumarprotocetraric acid cladonia macilenta ssp. floerkeana (fr.) v. wirth 2 on the mosses cladonia pyxidata (l.) hoffm. 2 8 12 acer pseudoplatanus on the mosses, fagus sylvatica, pinus mugo on the soil, pinus mugo cladonia rangiferina (l.) weber ex f.h. wigg. risnjak; vauchers in zaho atranorin collema auriforme (with.) coppins et j.r. laundon 9 on the mosses collema cristatum (l.) f. h. wigg. 12 calcareous rock collema subflaccidum degel. 1 8 on the mosses calcareous rock degelia plumbea (lightf.) p. m. jørg. et p. james 1 tilia platyphyllos dermatocarpon miniatum (l.) w. mann 3,5 calcareous rock evernia prunastri (l.) ach. 1,10 14 acer pseudoplatanus prunus sp. flavoparmelia caperata (l.) hale 1 2 13 acer pseudoplatanus fagus sylvatica ulmus glabra fuscidea stiriaca (a. massal.) hafellner 2 fagus sylvatica graphis scripta (l.) ach. 2,4,6 6 14 acer pseudoplatanus corylus avellana fagus sylvatica, fraxinus excelsior, prunus sp. hypogymnia physodes (l.) nyl. 1, 3 9 14 acer pseudoplatanus picea abies prunus sp. hypogymnia tubulosa (schaer.) hav. 2 abies alba lecania cyrtella (ach.) th. fr. 1 acer pseudoplatanus lecanora argentata (ach.) malme 14 fraxinus excelsior lecanora carpinea (l.) vain. 4 acer pseudoplatanus tab. 1. – continued u:\acta botanica\acta-botan 1-10\ozimec.vp 13. travanj 2010 9:26:09 color profile: disabled composite 150 lpi at 45 degrees 24 acta bot. croat. 69 (1), 2010 ozimec s., bo[kovi] i., florijan^i] t., jelki] d., opa^ak a., pu[kadija z., labak i. species site substrata identified lichen compounds lecanora chlarotera nyl. 4, 6, 7, 11, 12 acer pseudoplatanus lecanora subcarpinea szatala 14 fagus sylvatica psoromic acid lecidella elaeochroma (ach.) massal. 1,12 4,12 12 14 salix sp. acer pseudoplatanus fagus sylvatica fraxinus excelsior lepraria incana (l.) ach. 8 11 12 fagus sylvatica abies alba acer pseudoplatanus, pinus mugo leptogium lichenoides (l.) zahlbr. 9 on the mosses lobaria amplissima (scop.) forssell 1 tilia platyphyllos lobaria pulmonaria (l.) hoffm. 2, 5, 10,14 14 acer pseudoplatanus fagus sylvatica, fraxinus excelsior lobarina scrobiculata (scop.) nyl. 1 acer pseudoplatanus megalaria laureri (th.fr.) hafellner 4 14 acer pseudoplatanus fagus sylvatica melanelixia fuliginosa (duby) o. blanco, a. crespo, divakar, essl., d. hawksw. et lumbsch 1 2 4,7 14 corylus avellana fagus sylvatica acer pseudoplatanus fraxinus excelsior, prunus sp menegazzia terebrata (hoffm.) a. massal. 2, 14 9 fagus sylvatica picea abies mycobilimbia lurida (ach.) hafellner et türk 8, 12 calcareous rock nephroma parile (ach.) ach. 12 fagus sylvatica normandina pulchella (borrer) nyl. 13 ulmus glabra ochrolechia androgyna (hoffm.) arnold 13 ulmus glabra gyrophoric acid pannaria conoplea (ach.) bory 13 ulmus glabra parmelia saxatilis (l.) ach. 1, 2, 4, 7, 9, 11, 12 abies alba, acer pseudoplatanus, fagus sylvatica, picea abies parmelia submontana hale 14 prunus sp. parmelia sulcata taylor 1, 4, 11, 12, 14 acer pseudoplatanus prunus sp., salix sp. tab. 1. – continued u:\acta botanica\acta-botan 1-10\ozimec.vp 13. travanj 2010 9:26:09 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (1), 2010 25 lichens of risnjak national park (croatia) species site substrata identified lichen compounds parmelina pastillifera (harm.) hale 1 2 11 prunus sp. fagus sylvatica acer pseudoplatanus parmeliopsis ambigua (wulfen) nyl. 12 picea abies parmotrema perlatum (huds.) m. choisy 13 14 ulmus glabra carpinus betulus, fraxinus excelsior stictic acid atranorin peltigera collina (ach.) schrad. 1 2 13 acer pseudoplatanus abies alba ulmus glabra peltigera horizontalis (huds.) baumg. 8, 12 fagus sylvatica peltigera polydactyla (neck.) hoffm. 14 acer campestre peltigera praetextata (sommerf.) zopf. 5, 8 6, 8, 10, 12, 14 on mosses acer pseudoplatanus fagus sylvatica pertusaria albescens (huds.) m. choisy et werner 1, 10, 11 acer pseudoplatanus allo-pertusaric acid dihydropertusaric acid pertusaria amara (ach.) nyl. 13 14 ulmus glabra prunus sp. picrolichenic acid protocetraric acid pertusaria hemisphaerica (flörke) erichsen 9 picea abies lecanoric acid pertusaria hymenea (ach.) schaer. 5 acer pseudoplatanus pertusaria pertusa (weigel) tuck. 4, 7, 11 acer pseudoplatanus phlyctis argena (spreng.) flot. 1 acer pseudoplatanus physcia aipolia (humb.) fürnr. 1 prunus sp., salix sp. physconia distorta (with.) j. r. laundon 1 prunus sp. placynthiella icmalea (ach.) coppins et p. james 11 abies alba platismatia glauca (l.) w.l. culb. et c.f. culb. 2 4 9,10 abies alba fagus sylvatica picea abies pleurosticta acetabulum (neck.) elix et lumbsch 1 acer pseudoplatanus tab. 1. – continued u:\acta botanica\acta-botan 1-10\ozimec.vp 13. travanj 2010 9:26:09 color profile: disabled composite 150 lpi at 45 degrees distribution of lichen life forms shows that the most numerous, 43%, are foliose, 36% are crustose, and 21% are fruticose lichens. the lichens were found living on 16 different organic (trees, shrubs, mosses) and inorganic (soil, calcareous rocks) substrata (fig. 2). ten deciduous trees and shrubs make 62.5% of all substrates, three conifers make 18.75%, while mosses, calcareous rock and soil make 6.25% each. the most frequent phorophytes are acer pseudoplatanus, which supports 36 species, fagus sylvatica with 26 species, prunus spp. with 14, and fraxinus excelsior and abies alba supporting 10 species each. the major habitats for lichens are solitary trees in valleys, and forest fringes with favourable microclimatic conditions. discussion the number of 80 species reported for the lichen flora of the risnjak national park is lower than that in the lichen flora of the neighbouring areas in the dinaric part of slovenia. although several rare microlichens have been found in the study area, a lot of especially crustose taxa are missing. for the sne`nik and javorniki area, which is situated in southern slovenia and bordering on croatia, prügger (2005) reported a total of 409 species, among 26 acta bot. croat. 69 (1), 2010 ozimec s., bo[kovi] i., florijan^i] t., jelki] d., opa^ak a., pu[kadija z., labak i. species site substrata identified lichen compounds pseudevernia furfuracea (l.) zopf. 1 4 2, 9 acer pseudoplatanus fagus sylvatica picea abies punctelia subrudecta (nyl.) krog 2 fagus sylvatica pyrenula nitida (weigel) ach. 14 fagus sylvatica ramalina farinacea (l.) ach. 10 13 acer pseudoplatanus ulmus glabra ramalina fastigiata (pers.) ach. 1 acer pseudoplatanus ramalina fraxinea (l.) ach. 14 malus sp., prunus sp. squamarina cartilaginea (with.) p. james 12 calcareous rock thelotrema lepadinum (ach.) ach. 2 4,14 acer pseudoplatanus, picea abies fagus sylvatica toninia sedifolia (scop.) timdal 12 on the soil usnea diplotypus vain. 1 acer pseudoplatanus usnic acid alectoronic acid usnea filipendula stirt. 1 2 acer pseudoplatanus abies alba usnea subfloridana stirt. 2 abies alba usnic acid salazinic acid xanthoria parietina (l.) th. fr. 1 prunus sp. tab. 1. – continued u:\acta botanica\acta-botan 1-10\ozimec.vp 13. travanj 2010 9:26:09 color profile: disabled composite 150 lpi at 45 degrees which 285 were described as being epiphytic. the area of trnovski gozd, located at the extreme northwest of the dinaric mountains near the italian-slovenian border, holds 209 lichen species (prügger et al. 2000). this comparison emphasizes the necessity for taking a further, comprehensive lichen inventory within risnjak national park, and in the dinaric region of croatia, too. most of the lichens recorded are indicators of montane and subalpine belts, whose climazonal forests vegetation prevails in the area. therefore, acer pseudoplatanus and fagus sylvatica are the common main trees supporting the lichens in the study area, as they are in sne`nik and javorniki in slovenia (prügger 2005). relief variety and climate characterized by high annual rainfalls, frequent fog and dew, and high air humidity enable the occurrence of lichen species classified into oceanic and suboceanic elements (schauer 1965). the currently known lichen flora of risnjak national park includes 28 oceanic and suboceanic species, which makes 39% of the total. this is a reflection of the broader geographical position of the gorski kotar and kvarner region along the eastern coast of the adriatic sea, which is more humid than the western adriatic coast. the scarcity of oceanic and suboceanic lichens in adriatic italy, with a percentage between 11.1 and 16.6% in total lichen flora, has been reported by nimis and tretiach (1999). a certain number of epiphytic lichens depend among other factors on the age of the trees. species from the lobarion pulmonariae alliance are long been recognised as key epiphytic species associated with ancient woodlands of high conservation interest across europe (wolseley and james 2000). endangered and rare species like degelia plumbea, lobaria amplissima, lobaria pulmonaria, lobarina scrobiculata and pannaria conoplea can still be found growing in good conditions on old sycamore, beech, linden and elm-trees within the risnjak national park. acta bot. croat. 69 (1), 2010 27 lichens of risnjak national park (croatia) 35 25 14 4 2 2 1 1 1 10 9 2 8 7 3 7 8 0 5 10 15 20 25 30 35 40 a ce r ps eu do pl at an us f ag us sy lv at ic a p ru nu s sp . u lm us gl ab ra f ra xi nu s ex ce ls io r s al ix sp . c or yl us av el la na ti lia pl at yp hy llo s a ce r ca m pe st re c ar pi nu s be tu lu s m al us sp . a bi es al ba p ic ea ab ie s p in us m ug o r oc k m os se s s oi l substrate n u m b e r o f s p e c ie s fig. 2. distribution of lichen species according to substrate u:\acta botanica\acta-botan 1-10\ozimec.vp 9. travanj 2010 11:51:46 color profile: disabled composite 150 lpi at 45 degrees acknowledgement we would like to thank dr brian j. coppins (royal botanic garden, edinburgh), and professor dr helmut mayrhofer (institute for plant sciences, karl-franzens university of graz), for their help with identification of lichen samples material and valuable comments. references bertovi], s., 1994: vegetation mapping in national park »risnjak« and gorski kotar (in croatian). proceedings 40 years of national park »risnjak« (1953–1993), 17–26. clerc, p., 1998: species concepts in the genus usnea (lichenized ascomycetes). lichenologist 30, 321–340. hirc, d., 1898: gorski kotar (reprint, 1996) (in croatian). tiskara rijeka. horvat, i., 1930: vegetation studies on croatian mountains, i. communities of mountain’s barren land. (in croatian). rad jugoslavenske akademije 238, 1–96. horvat, i., 1931: vegetation studies on croatian mountains, ii. communities of mountain’s rocks and screes. (in croatian). rad jugoslavenske akademije 241, 147–206. horvat, i., 1962: vegetation of the mountains in western croatia (in croatian). prirodoslovna istra`ivanja jugoslavenske akademije 30, acta biologica 2, 1–179. ku[an, f., 1953: the prodromus of lichen flora of yugoslavia (in croatian). jugoslavenska akademija znanosti i umjetnosti, zagreb, 1–595. matkovi], p., 1879: sulla flora crittogamica di fiume. programma della regia scuola media superiore di stato, 37–42. mayrhofer, h., 2006: additions and corrections to the catalogue of the lichenized and lichenicolous fungi of slovenia i. razprave 4 razreda sazu 47, 201–229. nimis, p. l., tretiach, m., 1999: itinera adriatica – lichens from the eastern part of the italian peninsula. studia geobotanica 18, 51–106. orange, a., james, p. w., white, f. j., 2001: microchemical methods for the identification of lichens. british lichen society, london. ozimec, s., 2000: five lichen species new to the croatian flora. natura croatica 9, 133–138. prügger, j., mayrhofer, h., bati^, f., 2000: beiträge zur flechtenflora von slowenien, 4. die flechten des trnovski gozd. herzogia 14, 113–143. prügger, j, 2005: the lichen flora of sne`nik and javornik (in slovenian). studia forestalia slovenica 122, 1–212. purvis, o. w., coppins, b. j., hawksworth, d. l., james, p. w., moore, d. m., 1994: the lichen flora of great britain and ireland. natural history museum and the british lichen society, london. schauer, t. (1965): ozanische flechten im nordalpenraum. portugaliae acta biologica (b) 8, 17–229. schuler, j., 1902: zur flechtenflora von fiume. mitthelungen des naturwissenschaftlichen clubs in fiume 6, 131–250. 28 acta bot. croat. 69 (1), 2010 ozimec s., bo[kovi] i., florijan^i] t., jelki] d., opa^ak a., pu[kadija z., labak i. u:\acta botanica\acta-botan 1-10\ozimec.vp 9. travanj 2010 11:51:46 color profile: disabled composite 150 lpi at 45 degrees suppan, u., prügger, j., mayrhofer, h., 2000: catalogue of the lichenized and lichenicolous fungi of slovenia. bibliotheca lichenologica 76, 1–215. trinajsti], i., 1995: plant geographical division of forest vegetation of croatia. annales forestales 20, 37–66. vitikainen, o., 1994: taxonomic revision of peltigera (lichenized ascomycotina) in europe. acta botanica fennica 152, 1–96. wirth, v., 1995: die flechten baden-württembergs, 1–2. verlag eugen ulmer, stuttgart. wolseley, p., james, p., 2000: factors affecting changes in species of lobaria in sites across britain 1986–1998. forest snow and landscape research 75, 319–338. zahlbruckner, 1905: vorarbeiten zu einer flechtenflora dalmatiens 3. osterreichische botanische zeitschrift 55, 1–6, 55–69. acta bot. croat. 69 (1), 2010 29 lichens of risnjak national park (croatia) u:\acta botanica\acta-botan 1-10\ozimec.vp 9. travanj 2010 11:51:46 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (2), 313–323, 2009 coden: abcra 25 issn 0365–0588 morphological variability of the planktonic diatom thalassiosira delicatula ostenfeld emend. hasle from the mexican pacific, in culture conditions david u. hernández-becerril1*, sara p. moreno-gutiérrez1, sofía a. barón-campis2 1 instituto de ciencias del mar y limnología, universidad nacional autónoma de méxico (unam), apdo. postal 70-305, méxico, d.f. 04510 méxico. 2 instituto nacional de pesca, pitágoras 1320, col. sta. cruz atoyac; méxico, d.f. 03310 méxico. cultures of marine microalgae have greatly contributed to the better understanding of the morphology, phylogeny, life cycles, physiology and ecology of these species. in this paper, the planktonic diatom thalassiosira delicatula ostenfeld emend. hasle, originally isolated and cultured (in f/2 medium, a light-darkness cycle of 12:12, and at temperature of 20 °c ± 2 °c) from material obtained from the tropical, southern mexican pacific, was studied by lm, sem and tem. general morphology of the species agrees well with previous descriptions, including the formation of chains and colonies embedded in mucilage, the chitin threads connecting cells in the chain, the shape of the cells (in girdle view rectangular with a concavity in the valve centre), and processes (with a large rimoportula between valve face and mantle, a marginal ring of occluded ones, and two or three marginal rings of fultoportulae) and areolae arrangement. however the shape and distribution of chloroplasts and threads produced by marginal fultoportulae in living cells, and the presence of more than one central fultoportula, close to a larger areola (two and three fultoportulae were detected in this study) had never been described. urn-shaped marginal fultoportulae were also very common in most specimens studied, whereas in the literature this character was found only once for the species. additionally, two rimoportulae were detected in at least one valve. this morphological variability is discussed. this species is a new record for the mexican pacific and also the first record of the northern pacific ocean. keywords: diatom, phytoplankton, thalassiosira, culture, morphology, ultrastructure, variability introduction cultures of marine microalgae have been and are still very useful to gain knowledge on several aspects of the morphology, phylogeny, life cycles, physiology and ecology of these species. some complex life cycles and cryptic species have been recently resolved and recognized from cultured material, notably marine planktonic dinoflagellates scrippsiella trochoidea (montresor et al. 2003), alexandrium taylori (figueroa et al. 2006) and diatoms pseudonitzschia (lundohlm et al. 2003) and skeletonema (sarno et al. 2005). acta bot. croat. 68 (2), 2009 313 * corresponding author: dhernand@cmarl.unam.mx u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:03 color profile: disabled composite 150 lpi at 45 degrees in mexico, historical studies on marine phytoplankton from the pacific ocean have yielded recognized diverse floras, with important tropical and subtropical components, and continuous findings of new records, especially of diatoms and dinoflagellates, and even new species (e.g. hernández-becerril and tapia peña 1995, aké-castillo et al. 1999, hernández-becerril and bravo-sierra 2004, hernández-becerril et al. 2008). however, it has also been recommended that future investigations should consider new and modern study methods and concepts (hernández-becerril 2003), including culturing of species and molecular studies. in this paper, the morphology and the morphological variation of the marine planktonic diatom thalassiosira delicatula were studied, based on cultured material obtained from the mexican pacific ocean. despite the wide geographic distribution of this species, this is its first record from the mexican pacific and also from the northern pacific ocean. material and methods cultures phytoplankton samples (either collected by 54 µm net and 1.5 l bottle) were obtained during the cruise tehua iv on board r/v »el puma«, from the tropical, southern mexican pacific (15° 07’ n, 93° 59’ w, in the gulf of tehuantepec, 5 september, 2006) and they were kept unfixed and living until observations in the laboratory. individual cells or short chains of thalassiosira delicatula were isolated from living samples, with a micropipette in an inverted microscope (invertoskope zeiss), and placed in a 24-well petri dish with 1.5 ml f/2 culture medium (guillard and ryther 1962). all wells were observed after some days to check growth and conditions of cells, and when cell density was high enough the cultures were transferred to plastic culture bottles and/or sterile plastic petri dishes with 15–25 ml of f/2 medium. unialgal cultures were obtained and studied. conditions of the cultures were a light-darkness cycle of 12:12, and room temperature of 20 °c ± 2 °c. study of the morphology specimens of thalassiosira delicatula were studied from a single culture, mainly in exponential growth, by light microscopy (lm), scanning electron microscopy (sem) and transmission electron microscopy (tem). living specimens were observed and measured in lm (olympus bx40), in fresh slides; cleaned material (acid treated, following recommendations by hasle 1978) was also observed by lm. for sem (jeol jsm-6360lv) cells were critically point dried, after rinsing and dehydration in an alcohol series, and/or acid treated to get only cleaned valves, with no organic remnant. this cleaning treatment was also followed for observations in tem (jeol tem1200 exii). results thalassiosira delicatula ostenfeld emend. hasle references: hasle 1980: 170, figs. 18–34; rivera 1981: 53, pls. 10–16; hallegraeff 1984: 507, figs. 24 a, b; herzig and fryxell 1986: 14; licea 1994: 319, figs. 33–36; harris et al. 1995: 123, figs. 13, 30; hasle and syvertsen 1997: 59, pl. 6, tab. 8; throndsen et al. 2007: 135. 314 acta bot. croat. 68 (2), 2009 hernández-becerril d. u., moreno-gutiérrez s. p., barón-campis s. a. u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:03 color profile: disabled composite 150 lpi at 45 degrees synonyms: thalassiosira chilensis krasske, thalassiosira coronata gaarder. hasle 1980: 170, figs. 18–34; rivera 1981: 53, pls. 10–16. description: living cells were most commonly joined in short to long chains (2–6 cells), separated from each other 2–3 times the length of cells (or even more), and united by chitin threads (figs. 1, 2). occasionally, cells formed chains with sibling cells connected by their valves, with no thread (fig. 8), as they are possibly recently divided cells as yet without any thread development; alternately, colonies were embedded in mucilage. cells in girdle view were cylindrical, rectangular in shape (1.5 to 3 times the diameter), 16–18.5 µm in pervalvar length, with rounded corners and a conspicuous concavity in the valve centre acta bot. croat. 68 (2), 2009 315 morphology of thalassiosira delicatula pl. 1. thalassiosira delicatula, lm. fig. 1. two living cells, connected by a chitin thread. fig. 2. three living cells, two in girdle view and the other in valve view, arranged in a typical chain. fig. 3. a single living cell in girdle view, with elongate and discoid chloroplasts and long marginal chitin threads. fig. 4. girdle bands of a frustule, from cleaned material (vc= valvocopula, c= copula, p= pleurae). fig. 5. a single valve with one central fultoportula, one marginal rimoportula (arrowed) and marginal processes. fig. 6. another valve with two central fultoportulae. fig. 7. a valve showing long occluded processes. u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:07 color profile: disabled composite 150 lpi at 45 degrees (figs. 1–3, 8–10). in valve view cells were completely circular, 8.2–13.9 µm in diameter (figs. 2, 5, 6). living cells also showed numerous and long chitin threads from the margin of the valves, directed in different angles to the main axis, but always oblique (figs. 1–3). various discoid to elongate chloroplasts were found all over the cell (figs. 1–3). 316 acta bot. croat. 68 (2), 2009 hernández-becerril d. u., moreno-gutiérrez s. p., barón-campis s. a. pl. 2. thalassiosira delicatula, sem. fig. 8 – two cells joined together, critical point drying (cpd). fig. 9 – a complete cell with remaining of chitin threads from marginal fultoportulae, cpd. fig. 10 – another complete cell showing concavity in the valve centre, marginal processes and structure of the girdle (same symbols as fig. 4), cpd. fig. 11 – valve with one central fultoportula, two marginal rings of fultoportulae and one ring of occluded processes. fig. 12 – another valve with two central fultoportulae. fig. 13 – valve with three central fultoportulae. u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:13 color profile: disabled composite 150 lpi at 45 degrees the valves are convex with a concavity in the center (figs. 10–14). areolae are fine (30–35 areolae in 10 µm) and displayed in a radial, fasciculate pattern (figs. 11–17), and in general they are very irregular, from oblong, rectangular, square, pentagonal to hexagonal in shape (figs. 13–20, 23). the valves usually have one central fultoportula (figs. 5, 10, 11, acta bot. croat. 68 (2), 2009 317 morphology of thalassiosira delicatula pl. 3. thalassiosira delicatula, sem and tem. fig. 14 – a single valve with details of areolae, central and marginal processes, cleaned material, sem. fig. 15 – another single valve, showing areolae and marginal processes, note concavity of valve, tem. fig. 16 – valve with areolae, central and marginal processes, the rimoportula is arrowed, cleaned material, tem. fig. 17 – valve in valve view, with a single central fultoportula and the marginal proceses, sem. fig. 18 – close up of a central fultoportula, next to a larger areola and details of the areolae, sem. fig. 19 – central fultoportula, also close to a larger areola and two satellite pores (arrows), tem. fig. 20 – two central fultoportulae, sem. u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:20 color profile: disabled composite 150 lpi at 45 degrees 14–17), adjacent to a larger areola (figs. 14, 16, 18, 19), but also two or three central fultoportulae can be found (figs. 12, 13, 20), with short to relatively long external protrusions and 4–5 satellite pores (fig. 19). this central fultoportula produces the chitin thread that connects cells in the chains (figs. 11, 18). 318 acta bot. croat. 68 (2), 2009 hernández-becerril d. u., moreno-gutiérrez s. p., barón-campis s. a. pl. 4. thalassiosira delicatula, sem and tem. fig. 21 – internal view of a single valve showing a central fultoportula, two scattered fultoportulae, two marginal rings of fultoportulae, and a rimoportula (arrowed), sem. fig. 22 – another valve from internal view, with three central fultoportulae, two scattered fultoportulae, two marginal rings of fultoportulae, and two rimoportulae (arrowed), sem. fig. 23 –. marginal processes of the valve: occluded ones (arrows) and urn-shaped fultoportulae (arrowheads), sem. fig. 24 – mantle of a valve and girdle bands: valvocopula (vc), copula (c) and pleurae (p), sem. fig. 25 – details of valvocopula (vc), copula (c) with ligula and one pleura (p), sem. fig. 26 – valvocopula with areolae, tem. fig. 27 – a single pleura with pores in vertical rows, tem. u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:26 color profile: disabled composite 150 lpi at 45 degrees some fultoportulae may be found scattered in the valve face (figs. 21, 22). there are also two or sometimes three marginal rings of fultoportulae between valve face and mantle or on the valve mantle (figs. 5, 6, 10–17, 23), with external tubes. many of these marginal fultoportulae have an urn shape and shorter external tubes, which were very common in most specimens studied (figs. 11, 12, 14, 15, 17, 23, 24). these fultoportulae were found with remnants chitin threads (fig. 9). a rimoportula (in one specimen two rimoportulae were found, fig. 22) is present between valve face and mantle (figs. 5, 16, 21, 22) with a fairly large external tube. additionally, there is a marginal ring of occluded processes, between valve face and mantle, with long tubes (figs. 9–17, 23). internally, the rimoportula shows a short neck and a slit orientated perpendicularly to the margin (figs. 21, 22). the girdle is composed of valvocopula, copula and a number of pleurae, of which there can be up to 10 (figs. 4, 9, 10, 24). the valvocopula is broad and has irregular areolae, arranged in vertical rows (4–5 areolae in 1 µm), which become smaller at the abvalvar margin (figs. 25, 26). the copula is less broad than the valvocopula. in the case of smaller areolae that become smaller at the abvalvar margin, a ligula was present (fig. 25). pleurae have very small pores arranged in vertical rows (figs. 24, 27). discussion morphology and morphological variation almost all morphological details depicted here have been previously described for thalassiosira delicatula: formation of chains connected by chitin threads, shape of cells in girdle and valve views, details of valves, with their convex shape and a concavity in the centre, areolae arrangement and different processes (one central fultoportulae, one or two marginal rings of fultoportulae, one ring of occluded processes, and one rimoportula between valve face and mantle or on the mantle) (e.g. hasle 1980, rivera 1981). many details by em were provided by hasle (1980), so that led her to amend the original descriptions by ostenfeld and also to consider thalassiosira coronata gaarder, conspecific to thalassiosira delicatula, and hence a synonym of the latter. later, the morphological variability showed in specimens of thalassiosira delicatula from chilean material and revisions made on the type material, led rivera (1981) to synonymize another species, thalassiosira chilensis krasske. however, no living material had been studied, to show the shape and distribution of the chloroplasts, which indeed are similar to other species of the genus (horner 2002), and the long chitin threads or fibers that not only join the cells in the chains, but are also present in numbers at the margin of the valves (also a common character in many species of the genus). complete cells in chains from fixed and mounted material show remains of the protoplasm with only traces of chloroplasts and no marginal threads (hasle 1980, rivera 1981). here we illustrate and depict both elongate to discoid chloroplasts and long marginal threads in living cells. the central chitin thread is obviously associated to the chain formation, by joining cells together in the chain, and the marginal chitin threads are related to the decrease of cell sinking velocity by increasing form resistance, as in another species of the genus, thalassiosira fluviatilis hustedt (walsby and xypolyta 1977). all living cells observed in culture had acta bot. croat. 68 (2), 2009 319 morphology of thalassiosira delicatula u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:26 color profile: disabled composite 150 lpi at 45 degrees long marginal threads and were suspended in the medium within the petri dish, whereas dead cells, with very short, smaller threads or no threads were found sunk at the bottom of the petri dish. almost intact cells (critically point dried) are also shown here in sem, with some of the sibling valves joined together, with no threads. these complete cells show the marginal rings of fultoportulae with the remains of chitin threads, occluded processes, and a significant number of pleurae (up to 10 in some specimens) in the girdle bands. rivera (1981) mentioned 3–4 pleurae with no particular structure, but we found some small pores arranged in vertical rows (fig. 27). the presence of more than one central fultoportula, close to a larger areola had never been described: all available descriptions illustrate and depict only one fultoportula at the centre. we found valves with two and three fultoportulae in this study. this feature cannot be explained easily but we can speculate that those cells showing this condition were not in exponential growth or they were in rather old cultures experiencing significant variations or even producing theratological forms, either by deficiency in nutrients that could affect »normal« morphogenesis or because of incomplete cell division. structure and function of fultoportulae (also called strutted process) were discussed by schmid (1984), regarding wall morphogenesis of thalassiosira eccentrica (ehrenberg) cleve, indicating that »locally exerted stress« affects valve patterns, occurring »only during morphogenesis«, in this case we could appeal this reason in the formation of »extra« fultoportulae in the valve centre, but see also below. the marginal fultoportulae were usually urn-shaped, with short external tubes, a character that was illustrated for this species only once in the literature (rivera 1981: pl. 16, fig. 103). the presence of this kind of fultoportulae has been very rarely reported, although fryxell (1978) found these in thalassiosira punctigera (castracane) hasle (synonym, thalassiosira angstii (gran) makarova). it is very difficult to relate this shape to any possible function or culture conditions, and it is probably not a useful, consistent taxonomic character, but future experimental investigations on environmental conditions should be conducted. kaczmarska et al. (2006) have made a hypothesis that central and marginal fultoportulae are non-homologous and they have different origins; this was also largely discussed by theriot (2008). we detected two rimoportulae in at least one valve, but it is possible that other valves may also develop more than one rimoportula per valve. no clear explanation exists, but we may also consider different culture conditions that cause this variation, or as in the case of numerous central fultoportulae, theratological forms. interestingly, the valve we found with two rimoportulae had in addition three central fultoportulae (fig. 22). in cultures of species of the »pennate« genus pseudonitzschia, cells display a different chain arrangement than usual, possibly because »the raphe of these cells has apparently stopped functioning« (lundholm et al. 2002); unusual forms have been found in »old cultures« (e.g. cells with one or two prominences at their margins, personal unpublished data). in other diatoms, development of abnormal structures or absence of the normal, usual number of structures, related to the frustules, are found even in field samples, not only in cultures: e.g. terminal or intercalary setae in the »centric« planktonic diatom chaetoceros similis cleve (hernández-becerril 2009). a minor source of variation was detected in the sizes of cells we studied. whereas we found relatively smaller specimens (8.2–13.9 µm in diameter), other authors have reported 320 acta bot. croat. 68 (2), 2009 hernández-becerril d. u., moreno-gutiérrez s. p., barón-campis s. a. u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:26 color profile: disabled composite 150 lpi at 45 degrees larger cells in diameter: 9–25 µm (rivera 1981), 9–30 µm (hasle 1980, hasle and syvertsen 1997). the pervalvar length was 16–18.5 µm (this study), against 11–26 µm detected by hasle (1980). areolae density was higher in our study: 30–35 areolae in 10 µm, than reported elsewhere: 24–33 (rivera 1981), 22–26 (hasle 1980), and 20–33 µm (licea 1994). the presence of occluded processes is a unique character occurring only in species of the family thalassiosiraceae (including the genus lauderia cleve), although even in this group there are not many species possessing this character. fryxell (1978) listed 8 species having occluded processes, without including thalassiosira delicatula. her study was preceded that of hasle (1980), where the presence of occluded processes in the species was shown. fryxell (1978) had already discussed some hypotheses to explain the presence and possible functions of occluded processes, according to (1) valve size (larger valves in some species do not have occluded processes), (2) salinity ranges in relation to the number of these processes, (3) physical balance, (4) »initial step toward multiple labiate processes«, and (5) »defense mechanism against grazers«. hypotheses 3 and 5 are more convincing to us, although we have to test them. the character of possession of occluded processes was not considered »a consistent feature taxonomically« by fryxell (1978) and not a regular pattern for the genus by hasle and syvertsen (1997). distribution and ecology thalassiosira delicatula is now considered to be a cosmopolitan species, excluding to polar regions (hasle and syvertsen 1997). it was originally found in the benguela current and since then it has been reported from all around the world (also as thalassiosira chilensis and t. coronata), as in the atlantic ocean: north east atlantic ocean, norwegian west coast (ca. 60° n) and east coast of north america (hasle 1980), scotland (56° 32’ n) (harris et al. 1995), gulf stream and sargasso sea (herzig and fryxell 1986), southern gulf of mexico (licea 1994), and the southwestern atlantic ocean and argentinean waters (lange 1985, sar 1996, sar at al. 2002), as in the pacific ocean: southern pacific ocean, chilean coasts (rivera 1981), sidney, australia (hallegraeff 1984). here we recognize the first record of the species from the mexican pacific and also from the northern pacific ocean. this species has been found mainly in coastal systems, and we can consider it a coastal species. no high abundances have been recorded for the species. acknowledgements we are grateful to yolanda hornelas (sem service, icmyl, unam) for her skilled assistance at sem. partial support for this work was provided by papiit (dgapa, unam) (project in223206-2). references aké-castillo, j. a., hernández-becerril, d. u., meave del castillo, m. e., 1999: species of the genus thalassiosira (bacillariophyceae) from the gulf of tehuantepec. botanica marina 42, 487–503. figueroa, r. i., bravo, i., garcés, e., 2006: multiple routes of sexuality in alexandrium taylori (dinophyceae) in culture. journal of phycology 42, 1028–1039. acta bot. croat. 68 (2), 2009 321 morphology of thalassiosira delicatula u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:26 color profile: disabled composite 150 lpi at 45 degrees fryxell, g. a., 1978: the diatom genus thalassiosira: t. licea sp. nov. and t. angstii (gran) makarova, species with occluded processes. botanica marina 21, 131–141. guillard, r. r. l., ryther, g. h., 1962: studies of marine planktonic diatoms. i. cyclotella nana hustedt and detonula confervecea (cleve) gran. canadian journal of microbiology 8, 229–239. hallegraeff, g. m., 1984: species of the diatom genus thalassiosira in australian waters. botanica marina 27, 495–513. harris, a. s. d., medlin, l. k., lewis, j., jones, k. j., 1995: thalassiosira species (bacillariophyceae) from a scottish sea-loch. european journal of phycology 30, 117–131. hasle, g. r., 1978: diatoms. in: sournia, a. (ed.), phytoplankton manual, 136–142. unesco, paris. hasle, g. r., 1980: examination of thalassiosira type material: t. minima and t. delicatula (bacillariophyceae). norwegian journal of botany 27, 167–173. hasle, g. r., syvertsen, e. e., 1997: marine diatoms. in: tomas, c. r. (ed.), identifying marine phytoplankton, 5–385. academic press, san diego. hernández-becerril, d. u., 2003: la diversidad del fitoplancton marino de méxico: un acercamiento actual. in: barreiro, m. t., meave, m. e., signoret, m., figueroa, m. g. (eds.), planctología mexicana, 1–17. sociedad mexicana de planctología (sompac) y universidad autónoma metropolitana, méxico, d. f. hernández-becerril, d. u., 2009: morphological variability of the marine planktonic diatom chaetoceros similis (bacillariophyceae). cryptogamie, algologie 30, 125–134. hernández-becerril, d. u., bravo-sierra, e., 2004: new records on planktonic dinoflagellates (dinophyceae) from the mexican pacific ocean. botanica marina 47, 417–423. hernández-becerril, d. u., tapia peña, m. i., 1995: planktonic diatoms from the gulf of california and coasts off baja california: species of the genus thalassiosira. botanica marina 38, 543–555. hernández-becerril, d. u., ceballos-corona, j. g. a., esqueda-lara, k., tovarsalazar, m. a., león-álvarez, d., 2008: marine planktonic dinoflagellates of the order dinophysiales (dinophyta) from coasts of the tropical mexican pacific, including two new species of the genus amphisolenia. journal of the marine biological association of the united kingdom 88, 1–15. herzig, w. n., fryxell, g. a., 1986: the diatom genus thalassiosira cleve in gulf stream warm core rings: taxonomy, with t. intrannula and t. lineoides, spp. nov. botanica marina 29, 11–25. horner, r. a., 2002: a taxonomic guide to some common marine phytoplankton. biopress limited, bristol. kaczmarska, i., beaton, m., benoit, a. c., medlin, l. k., 2006: molecular phylogeny of selected members of the order thalassiosirales (bacillariophyta) and evolution of the fultoportula. journal of phycology 42, 121–138. lange, c., 1985: spatial and seasonal variations of the diatom assemblages off the argentinean coast (south western atlantic). oceanologica acta 8, 361–370. 322 acta bot. croat. 68 (2), 2009 hernández-becerril d. u., moreno-gutiérrez s. p., barón-campis s. a. u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:26 color profile: disabled composite 150 lpi at 45 degrees licea, s., 1994: thalassiosira species from the southern gulf of mexco. proceedings 11 international diatom symposium, san francisco, 311–335. lundholm, n., daugbjerg, n., moestrup, ø., 2002: phylogeny of the bacillariaceae with emphasis on the genus pseudo-nitzschia (bacillariophyceae) based on partial lsu rdna. european journal of phycology 37, 115–134. lundholm, n., moestrup, ø., hasle, g. r., hoef-emden, k., 2003: a study of the pseudonitzschia pseudodelicatissima/cuspidata complex (bacillariophyceae): what is p. pseudodelicatissima? journal of phycology 39, 797–813. montresor, m., sgrosso, s., procaccini, g., kooistra, w. h. c. f., 2003: intraspecific diversity in scrippsiella trochoidea (dinophyceae): evidence for cryptic species. phycologia 42, 56–70. rivera, p., 1981: beiträge zur taxonomie und verbeitung der gattung thalassiosira cleve (bacillariophyceae) in den küstengewässern chiles. bibliotheca phycologica 56. j. cramer, vaduz. sar, e. a., 1996: flora diatomológica de bahía san antonio (prov. de río negro, argentina). centrales i. revista del museo, la plata (n.s.) 14, botánica 106, 365–400. sar, e. a., sunesen, i., lavigne, a. s., 2002: the diatom genus thalassiosira: species from the northern san matías gulf (río negro, argentina). nova hedwigia 74, 373–386. sarno, d., kooistra, w. h. c. f., medlin, l., percopo, i., zingone, a., 2005: diversity in the genus skeletonema (bacillariophyceae). ii. an assessment of the taxonomy of s. costatum-like species with the description of four new species. journal of phycology 41, 151–176. schmid, a. m. m., 1984: wall morphogenesis in thalassiosira eccentrica: comparison of auxospore formation and the effect of mt-inhibitors. proceedings 7 international diatom symposium, philadelphia, 47–70. theriot, e. c., 2008: application of phylogenetics principles to testing evolutionary scenarios: a comment on kaczmarska et al. »molecular phylogeny of selected members of the order thalassiosirales (bacillariophyta) and evolution of the fultoportulae«. journal of phycology 44, 821–833. throndsen, j., hasle, g. r., tangen, k., 2007: phytoplankton of norwegian coastal waters. almater forlag as, oslo. walsby, a. e., xypolyta, a., 1977: the form resistance of chitan fibres attached to the cells of thalassiosira fluviatilis hustedt. british phycological journal 12, 215–223. acta bot. croat. 68 (2), 2009 323 morphology of thalassiosira delicatula u:\acta botanica\acta-botan 2-09\hernandez.vp 6. listopad 2009 10:53:26 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (2), 211–220, 2009 coden: abcra 25 issn 0365–0588 middle miocene record of pliocaenicus changbaiense sp nov. from changbai (jilin province, china) katarzyna stachura-suchoples*, regine jahn freie universität berlin, botanic garden and botanical museum berlin-dahlem, königin-luise str. 6–8, 14195 berlin, germany the first record of the genus pliocaenicus from middle miocene deposits is presented in this paper. this record from changbai (jilin province, china) places the origin of the genus further back into the middle miocene. the genus has been so far reported from the late miocene/early pliocene till recent. the population from changbai belongs to the group of pliocaenicus species possessing complex alveolae, such as p. cathayanus wang, p. jilinensis wang and p. omarensis (kuptsova) stachura-s. et khursevich. both p. cathayanus and p. jilinensis have been described from china (pliocene deposits), and are only known from the type locality. the third species, p. omarensis is reported from eurasia and africa (age range: late miocene-pleistocene). in this report we describe p. changbaiense, a new species from china, and focus on those characters having the potential for developing further an evolutionary and taxonomical concept in pliocaenicus. we anticipate that these will contribute to our understanding of the driving forces of diatom dispersal. key words: diatom, pliocaenicus changbaiense, ultrastructure, alveolae, miocene, china introduction the relatively newly established genus pliocaenicus round and håkansson emend. khursevich and stachura-suchoples was erected to accommodate two fossil species of cyclotelloid diatoms (round and håkansson 1992). four further species were transferred at the same time. today the genus contains nine species, both fossil and extant (flower et al. 1998, wang 1999, tanaka and nagumo 2004, stachura-suchoples and khursevich 2007, stachura-suchoples et al. 2008, this paper). the genus description, biogeographical distribution of the species and a key for identification have been published elsewhere (khursevich and stachura-suchoples 2008). in this study, we focus on a middle miocene population of pliocaenicus species from china. these specimens represent the oldest known record of the genus and place the oriacta bot. croat. 68 (2), 2009 211 * corresponding author, e-mail: k.stachura@bgbm.org u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:27 color profile: disabled composite 150 lpi at 45 degrees gin of the genus further back to ca. 13 ma. for china, this is the record of the third representative of the genus pliocaenicus. the first observations were done by wang (1999), who described p. cathayanus and p. jilinensis from fossil deposits lacking sufficient geological data, however pliocene? is suggested. both species are so far only known from the type locality (jilin province, china). besides p. cathayanus and p. jilinensis, from asia are reported: (i) extinct, p. nipponicus tanaka et nagumo and p. omarensis, and extant: p. costatus (loginova, lupikina et khursevich) flower, ozornina et kuzmina and p. seczkinae stachura-s., genkal et khursevich. moreover, recent studies indicate that the distribution of the genus pliocaenicus is restricted to the northern hemisphere, and the living populations are reported from asian arctic and mountain zones exclusively (khursevich and stachura-suchoples 2008, stachura-suchoples et al. 2008). here we present ultrastructural observations of pliocaenicus specimens from changbai shan and focus on characters that might have the potential to be of importance for further understanding of evolutionary processes and for the development of the taxonomical concept of the genus. in addition, this will contribute to diatom biogeographical studies. materials and methods the investigated samples were collected from a deposit in badaogou, changbai, jilin province (china) situated near the border with north korea. changbai shan – changbai mountains (china) or baekdu mountains (korea) – is a mountain range on the border between china and north korea (41°41’ to 42°51’ n; 127°43’ to 128°16’ e). there, plant-bearing diatomites of the manshancun formation were intercalated between basalt flows. in the 1980s, radiometric dating of olivine-basalts indicated ca. 13.4 ma. for the basalts (kovar-eder and ge sun 2009). the diatomites yield foliage with excellent cuticle preservation. the flora is a mixture of deciduous and evergreen angiosperm taxa and conifers. also, the pollen flora is very well preserved. herbaceous plants occur in low abundances (kovar-eder and ge sun 2009). the diatom samples were cleaned in 30% h202 solution, and then washed several times with distilled water. the permanent diatom slides were mounted in naphrax. light microscope (lm) observations were made using a zeiss axioplan microscope. for scanning electron microscope (sem) observations specimens were mounted on aluminum stubs and sputter-coated with gold-palladium. the sem observations were made with sem philips 515 at the bgbm, berlin-dahlem. the terminology follows khursevich and stachurasuchoples (2008). results pliocaenicus changbaishanense stachura-suchoples and r.jahn sp. nov. holotype: b 400040653 as represented in figure 11, deposited at the herbarium of the botanical museum berlin-dahlem (b). isotype: b 400040653a, deposited at the research center of paleontology and stratigraphy of jilin university in changchun, china. original material: b 400040654, mixed sample from type locality. type locality: a deposit in badaogou, changbai county (jilin province, china). 212 acta bot. croat. 68 (2), 2009 stachura-suchoples k., jahn r. u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:27 color profile: disabled composite 150 lpi at 45 degrees age and distribution: miocene, known only from the type locality. etymology: the species name refers to the type locality, changbai in china. diagnosis: pliocaenicus changbaiense differt a p. cathayanus et p. jilinensis positione fultoportularum marginalium in costis crassioribus et absentia alveolarum simplicium. pliocaenicus changbaiense a p. omarensis differt absentia alveolarum simplicium, costis secundariis male evolutis hyalinis, et aperturis fultoportularum marginalium ad basin interstriarum latiorium. diagnosis: valves round, diameter 10–36 mm. valve face more or less transversely undulated; smooth to colliculate. areolae in single rows radiating from valve centre to mantle, 10–12 along the radius, no distinct interstriae in between. valve face areolae with internal domed cribra. mantle puncta smaller than those on the valve face, arranged in fascicles divided by v-like shape interfascicles. fultoportulae possess three satellite pores. valve face fultoportulae (from 3 to15) set out in circular pattern; externally, open by smaller puncta than areolae. mantle fultoportulae on each second-third, really on each or on each fourth costa. externally, mantle fultoportulae open on the base of wide hyaline strips; internally, located on each second-third thick costa, rarely on each one or each fourth. alveolae complex with secondary poorly developed secondary costae (folds), restricted to the valve mantle. a single raised rimoportula located in the submarginal zone of the valve face. externally, the rimoportula opening difficult to detect. on the valve surface granules can be present. the frustules are round (figs. 1–4). the diameter varies from 10 to 36 mm. number of costae ranges from 7 to 8 in 10 mm. the valve face is usually transversely undulated. areolae, c. 10–12 along the radius, are usually arranged in single rows without fascicles, an irregular pattern can be observed (fig. 4). valve usually round, transversely undulated (figs. 5, 6). externally, relief of the external valve face smooth or colliculate (figs. 6–7); areolae loculate, arranged in radiate striae, sometimes irregular (figs. 5, 6, 8); on the mantle, the vertical rows of fine puncta are acta bot. croat. 68 (2), 2009 213 pliocaenicus from miocene, china pl. 1. pliocaenicus changbaiense specimens from middle miocene, changbai, china. lm, scale bar= 10 mm. u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:28 color profile: disabled composite 150 lpi at 45 degrees grouped in fascicles divided by hyaline interfascicles that do not go to the valve edge (figs. 7, 8); pseudofenestral-like structures on the valve face/mantle junction (figs. 7, 8); valve face fultoportulae lacking external tubuli (fig. 5), mantle fultoportulae open at the base of wide hyaline strips (figs. 7, 8). internally, areolae with domed cribra (fig. 14). alveolae complex with secondary thin poorly developed costae (folds) (figs. 11, 12). the marginal fultoportulae located on each second-third primary thick costa, rarely on each or each fourth (figs. 9–12, 14). valve face fultoportulae (from 3 to15 in the valve face) positioned in circular pattern (figs. 9–11). mantle and valve face fultoportulae have three satellite pores (figs. 9–11, 14). a single, raised rimoportula (figs. 10, 11, 13, 14) located in the marginal zone of the valve face. 214 acta bot. croat. 68 (2), 2009 stachura-suchoples k., jahn r. pl. 2. pliocaenicus changbaiense specimens from middle miocene, changbai (china), external valve view. scale bars: 10 mm (fig. 6), 7 mm (fig. 5), 6 mm (fig. 7), 4 mm (fig. 8). the transversely undulated valves, relief of the valve face smooth (fig. 5) or colliculate (fig. 6). areolae loculate, irregular or arranged in radiate striae. note external openings of fultoportulae (fig. 5): valve face (arrow vf) and mantle, on the base of wide hyaline strips (arrow pcf), and shorter hyaline strips without openings of fultoportulae. the valve mantle with vertical rows of fine puncta arranged in fascicles, divided by v-like shape hyaline strips that do not go to the valve edge :shorter without opening of fultoportulae (arrow pc) or longer with opening of mantle fultoportulae at the base of (arrow pcf) (fig. 7). the pseudofenestral-like structure on the valve face/ mantle junction. the girdle band without ornamentation (fig. 8). u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:30 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 215 pliocaenicus from miocene, china pl. 3. pliocaenicus changbaiense specimens from middle miocene, changbai (china), internal valve view. the internal view of the valve surface showing the valve face (arrow vf) and mantle fultoportulae with three satellite pores located on thick costae (arrow pcf), and rimoportula (arrow r) (figs. 9–11). holotype b 4000 (fig. 11). note: structure of complex alveolae. details on complex alveolae structure, note costae bearing fultoportulae (arrow pcf), costae non-bearing fultoportulae (arrow pc), and poorly developed secondary costae (arrow sc) (fig. 12). the valve surface with a single raised rimoportula (arrow r) located in the submarginal zone of the valve (figs. 13, 14). valve face fultoportulae (arrow vf) and mantle fultoportulae with three satellite pores on thick costae (arrow pcf), areolae with internal domed cribra (fig. 14). scale bars: 9 mm (fig. 10), 8 mm (fig. 9), 6 mm (fig. 11), 1 mm (figs. 12–14). u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 9. listopad 2009 13:07:14 color profile: disabled composite 150 lpi at 45 degrees discussion the genus pliocaenicus is defined by seven characters as proposed by khursevich and stachura-suchoples (2008), see also round and håkansson (1992): (i) areolae not fasciculate on valve face; (ii) external mantle fasciculate with opening of fultoportulae on interfascicle; (iii) external openings of mantle fultoportulae lacking tubuli; (iv) external openings of valve face fultoportulae lacking tubuli on the valve surface; (v) external openings of rimoportula lacking tubuli; (vi) marginal costae with centrifugal 'roofing over'; (vii) internal valve face with domed cribra. the specimens observed possess all characters to be accommodated in pliocaenicus. additionally, ultrastructural observations on p. changbaiense (figs. 5–14) revealed typical characters for the group of pliocaenicus cathayanus-jilinensis-omarensis (khursevich and stachura-suchoples 2008). these species can be characterized as having: (i) both, simple and complex alveolae; or only complex (as in p. changbaiense: see figures 11, 12) (ii) externally, narrow interfascicles (costae) on the mantle that do not go to the valve edge; (iii) internally, mantle fultoportulae located on thick or thin recessed costa; (iv) valve diameter of 5–47 mm. all these characters are observed in p. changbaiense. the comparison of morphometric and morphological characters is given in table 1 and 2 respectively (see also key for taxonomic identification in khursevich and stachura-suchoples 2008). in general, the range of the valve diameter of our population varies significantly and overlaps with all the species forming the complex alveolae group. for example, the smallest measured specimen of p. changbaiense is 3.6 times smaller than the biggest observed valve. the number of areolae in 10 mm and the number of puncta rows in the mantle fascicles in specimens from changbai is slightly lower than in the other three species. at this point it is necessary to stress that, in the case of less pronounced hyaline areas without external opening of fultoportula (e.g. p. jilinensis) or very narrow interstriae (e.g. p. omarensis) the measurement of 216 acta bot. croat. 68 (2), 2009 stachura-suchoples k., jahn r. tab. 1. morphometric data and information on pliocaenicus species group with complex alveolae. valve diameter [mm] areolae in 10 mm costae in 10 mm rows of puncta in mantle fascicle valve face fultoportulae p. cathayanus1) 14–44 14–16 c. 6 7–9 several (8–12) p. jilinensis1) 7–19 c.124 c. 64 15–20; c. 7* several p. omarensis2) 5–47 10–28 7–15 c. 11; c. 7* several (2–8) p. changbaiense3) 10–36 10–12 c. 7–8 c. 6–7 several (3–15) after 1 – wang (1999), 2 – sec. khursevich and stachura-suchoples (2008), 3 – this paper, 4 – tanaka and nagumo (2004), * – from reference papers. u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:33 color profile: disabled composite 150 lpi at 45 degrees number of rows of puncta between interstriae can give misleading results (for illustration see e.g. figures 5–8, see also table 1). in p. changbaiense the number of costae in 10 mm is similar to p. cathayanus and p. jilinensis, and in the lowest range to p. omarensis. all species posses low number of valve face fultoportulae. the above presented comparison of morphometric data indicates that fine structural observations are necessary for identification on the species level. the main diagnostic character in the species of the group ii.2. (pliocaenicus cathayanus-jilinensis-omarensis) is the occurrence of both simple and complex alveolae (khursevich and stachura-suchoples 2008). in p. cathayanus and p. jilinensis the mantle fultoportulae are located on recessed costae (wang 1999). this character is indicated in the diagnosis of p. cathayanus (wang 1999: 126). in the case of p. jilinensis in the diagnosis it is written. »every second, third or fourth costa bearing a fultoportula (wang 1999: 127), however later on wang (1999: 128) added:...« those fultoportula-bearing costae are also recessed like those of p. cathayanus«. in p. changbaiense, mantle fultoportulae are located externally at the base of wide hyaline strips (figs. 5–8), and internally on thick costae (figs. 9, 10). the alveolae cross section observations indicate that the secondary costae are located between the primary costae (figs. 11, 12). the secondary costae are poorly developed (folds) and are located on the valve mantle. these characters differentiate p. changbaishanense from p. cathayanus and p. jilinensis. complex alveolae as observed in p. omarensis are restricted by thick bearing mantle fultoportulae costae, while between them the much thinner well developed secondary costae are located (khursevich and stachurasuchoples 2008). this character differentiates p. omarensis from p. changbaiense. besides, the specimens observed possess only complex alveolae, in contrast to p. cathayanus, p. jilinensis and p. omarensis, which have both simple and complex alveolae (as suggested by khursevich and stachura-suchoples 2008). however, this character should be further investigated, for example in p. cathayanus secondary costae can be seen between primary costae (wang 1999: fig. 17). therefore, additional investigation on complex alveolar structures of other species can reveal new important data. unfortunately, we were not able to re-investigate type material of p. cathayanus and p. jilinensis. furthermore, as suggested by e.g. loseva (1981) and khursevich and stachurasuchoples (2008) additional observations on worldwide reported populations of p. omarensis are required. in all three species from china the external openings of mantle fultoportulae are positioned at the base of wider hyaline strips, while shorter hyaline strips reflect the internal costae non-bearing fultoportulae. in p. omarensis the hyaline strips are narrow. additionally, different patterns of valve face fultoportulae are observed in the case of p. changbaiense, p. cathayanus and p. jilinensis in contrast to p. omarensis (circular: wang 1999: figs. 16–18, 24; this paper: fig. 5; arc: khursevich and stachura-suchoples 2008: figs. 30–33). the number of valve face fultoportulae of all species from the complex alveolae group is three. in the case of mantle fultoportulae in p. cathayanus, wang (1999) wrote that they probably possess no satellite pores, however khursevich and stachurasuchoples (2008) suggested that they can have three satellite pores. for p. jilinensis wang (1999) did not mention the number of satellite pores of mantle fultoportulae, khursevich and stachura-suchoples (2008) also suggested the presence of three satellite pores. the presence of a fenestrate structure is a diagnostic character in p. cathayanus; a similar structure called a pseudo-fenestrate structure, is a character observed in p. acta bot. croat. 68 (2), 2009 217 pliocaenicus from miocene, china u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:33 color profile: disabled composite 150 lpi at 45 degrees jilinensis (terminology after wang 1999). the pseudofenestral structure is also observed in p. costatus, p. omarensis and p. seczkinae. similar to the pseudofenestral structure a cluster of less orderly organized areolae is located at the valve face/mantle junction in p. changbaiense. now, the key for the identification of pliocaenicus species can be extended (for details see khursevich and stachura-suchoples 2008). we suggest that p. changbaiense is the closest related to p. omarensis. here, we also added the position of the rimoportula on the valve face as one of the diagnostic character for the species accommodated in the group ii.2. extention of the key for identifying of pliocaenicus changbaiense within group ii. 2 (khursevich and stachura-suchoples 2008). 218 acta bot. croat. 68 (2), 2009 stachura-suchoples k., jahn r. tab. 2. ultrastructural features of pliocaenicus species group with complex alveolae. alveolae areolae valve face fultoportulae mantle fultoportulae rimoportula structure arrangement a) structure, b) location a) structure, b) position, c) localisation a) structure, b) localisation p. cathayanus1,2 simple and complex radiate, single rows a) three-(four) satellite pores b) ring in central area a) none (?), three (?) satellite pores b) internally, on thin recessed costae a) single, sessile or raised b) near the base of an internal costa p. jilinensis1,2 simple and complex radiate, single rows a) three satellite pores b) ring in central area a) three? satellite pores b) internally, on recessed costae, c) a) single, raised b) near the base of an internal costa p. omarensis2 simple and complex radiate, single rows a) three satellite pores b) arc within central depression a) three satellite pores b) internally, on thick costae, c) a) single, sessile or raised b) near or at the base of an internal costa p. changbaishanense3 complex irregular, radiate, single rows a) three satellite pores b) ring in central area a) three satellite pores b) internally, on thick costae, c) on each (1)2–3(4) costa a) single raised b) near the base of an internal costa or in submarginal zone after 1 – wang (1999), 2 – khursevich and stachura-suchoples (2008), 3 – this paper u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:34 color profile: disabled composite 150 lpi at 45 degrees group ii. valves round, rarely elliptical, transversely undulate. alveolae simple or both simple and complex. valve face and mantle fultoportulae with two or three satellite pores. one sessile or raised rimoportula near or at the base of an internal costa or in the chambered region. 2. alveolae both simple and complex or only complex. one sessile or raised rimoportula on the valve face (submarginal zone). diameter of valves 5–47 mm. externally interstriae on the mantle do not go the valve edge. internally, mantle fultoportulae located on thick or thin recessed costa. a. internally, mantle fultoportulae located on thick costae. a. both simple and complex (with thin recessed costae) alveolae . . . . . . p. omarensis b. complex alveolae with poorly developed secondary costae . . . . . . p. changbaiense b. internally, mantle fultoportulae located on thin recessed costae. our results indicate that detailed investigations of alveolae, especially cross sections would be valuable, helping to develop further the concept in pliocaenicus species possessing complex alveolae. moreover, as revealed by stachura-suchoples et al. (2008), detailed ultrastructural observations on valve face fultoportulae have already differentiated pliocaenicus seczkinae – the species reported so far only from holocene and recent populations from lake el’gygytgyn (chukotka, russia) – from p. costatus, which is widely distributed and known from the late miocene to recent. independently, previous studies on the genus pliocaenicus also documented morphological variability on population/species level observed in p. costatus (e.g. genkal et al. 2001, stachura-suchoples 2006), p. omarensis (gasse 1980, see also khursevich and stachura-suchoples 2008) and p. seczkinae (stachura-suchoples et al. 2008). in the population of p. changbaishanense variable characters are e.g.: a valve face relief (smooth: figs. 5, 8 to colliculate: figs. 6, 7) and presence (fig. 5) or absence (figs. 6–8) of granules. in conclusion, this study extends the biostratigraphical, biogeographical and evolutionary knowledge on the genus pliocaenicus. the oldest record of the genus pliocaenicus, according to a preliminary dating of the deposit in changbai (which needs to be reconfirmed in the future), places the origin of the genus further back into the middle miocene. this would be similar to the origin of other freshwater genera in the family thalassiosiraceae lebour emend. hasle such as, cyclostephanos round, mesodiction theriot et bradbury, stephanodiscus ehrenberg or conticribria stachura-suchoples et d. m. williams (stachurasuchoples and williams in press). further, detailed ultrastructural observations on representatives of pliocaenicus will contribute to a refined species concept of the genus. they will also contribute to our understanding of evolutionary processes at the generic and inter-generic levels and diatom biogeography. acknowledgements we are grateful to johanna kovar-eder (staatliches museum für naturkunde stuttgart, germany) and ge sun (normal university shenyang, china) who provided us with material and information from the deposit. we warmly thank galina khursevich for her kindness and useful comments. harrie sipman kindly helped us to prepare the latin diagnosis. part of this research received support from the synthesys project: http//www.synthesys.info/ acta bot. croat. 68 (2), 2009 219 pliocaenicus from miocene, china u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:34 color profile: disabled composite 150 lpi at 45 degrees which is financed by european community research infrastructure action under the fp 6 »structuring the european research area« programme (to k.s., applications gb-taf-3994, nhm london and at-taf-4573, nhm vienna). references flower, r. j., ozornina, s. p., kuzmina, a., round, f. e., 1998: pliocaenicus taxa in modern and fossil material mainly from eastern russia. diatom research 13, 39–62. gasse, f., 1980: les diatomées lacustres plio-pléistocenes du gabeb (étiopie). systématique, paléoécologie, biostratigraphie. revue algologique mémoire hors-série 3, 1–360. genkal, s. i., popovskaya, g. i., bondarenko, n. a., 2001: about morphology and taxonomy of pliocaenicus costatus (log., lupik. et kurs.) flower, ozornina et kuzmina (bacillariophyta) (in russian with english summary). biology of inland waters 2, 53–64. khursevich, g., stachura-suchoples, k., 2008: the genus pliocaenicus round håkansson (bacillariophyta): morphology, taxonomy, classification and biogeography. nova hedwigia 86, 419–444. kovar-eder, j., ge sun, 2009: the neogene flora from badaogou of changbai, ne china – nearest living relatives of selected taxa and relations to the european record. review of palaeobotany and palynology. in press. loseva, e, 1981: the valve ultrastructure of some fossil cyclotella species. proceedings 6 international diatom symposium, budapest, 15–26. round f., håkansson, h., 1992: cyclotelloid species from a diatomite in the harz mountains, germany, including pliocaenicus gen. nov. diatom research 7, 109–125. stachura-suchoples, k., 2006: morphological variability of recent population of pliocaenicus costatus sensu lato from the verkhojansk mountains, and its relationship to pliocaenicus costatus var. costatus flower, ozornina et kuzmina. proceedings 18 international diatom symposium, miedzyzdroje, 363–370. stachura-suchoples, k., khursevich, g., 2007: on the genus pliocaenicus round and håkansson (bacillariophyceae) from the northern hemisphere. proceedings 1 central european diatom meeting, berlin, 155–158. stachura-suchoples, k., genkal, s., khursevich, g., 2008: pliocaenicus seczkinae sp. from lake el’gygytgyn, chukotka (ne russia). diatom research 23, 171–184. stachura-suchoples, k., williams, d. m., in press: description of conticribria tricircularis, a new genus and species of thalassiosirales, with a discussion on its relationship to other continuos cribra species of thalassiosira cleve (bacillariophyta) and its freshwater origin. european journal of phycology. tanaka, h., nagumo, t., 2004: pliocaenicus nipponicus sp. nov., a new freshwater fossil diatom from central japan. diatom 20, 105–111. wang, g., 1999: pliocaenicus cathayanus sp. nov. and p. jilinensis sp. nov. from a diatomite of jilin province, northeast china. proceedings 14 international diatom symposium, tokyo, 125–133. 220 acta bot. croat. 68 (2), 2009 stachura-suchoples k., jahn r. u:\acta botanica\acta-botan 2-09\stachura-suchoples.vp 5. listopad 2009 14:45:34 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2016 za web.indd acta bot. croat. 75 (2), 2016 253 acta bot. croat. 75 (2), 253–259, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0023 issn 0365-0588 eissn 1847-8476 the effect of biostimulant and fertilizer on “low input” lettuce production slavica dudaš1, ivana šola2,*, barbara sladonja3, renata erhatić4, dean ban3, danijela poljuha3 1 polytechnic of rijeka, poreč agricultural department, karla huguesa 6, poreč, croatia 2 department of biology, faculty of science, university of zagreb, horvatovac 102a, zagreb, croatia 3 institute of agriculture and tourism, karla huguesa 8, poreč, croatia 4 križevci college of agriculture, milislava demerca 4, križevci, croatia abstract – lettuce production in the winter on the adriatic coast, especially in a non-heated plastic tunnel, requires longer cultivation and is characterised by lower head mass and yield. in these conditions, the effect of biostimulant bio-algeen s-90 and fertilizer megagreen on the production of the traditional winter lettuce cultivar ‘four seasons’ was tested. both treatments showed a positive effect on the growth and total yield of winter lettuce, and decreased the share of non-marketable yield. bio-algeen s-90 treatment increased the plant height by 61.5%, and foliar treatment with megagreen by 60.9%, as compared to the control treatment. equally, both treatments resulted in higher leaf numbers (47.7% for bio-algeen s-90 and 37.2% for megagreen). the head mass of lettuce treated with bio-algeen s-90 and megagreen was 30.3% and 25.0% higher than in the control treatment, respectively. megagreen contributed more to chlorophyll and carotenoid content, while bio-algeen s-90 elevated the amount of vitamin c and dry matter. the ph value of lettuce juice decreased after bio-algeen s-90, while the mineral content (n, p and k) did not differ between the tested treatments. lower nitrate content was detected after both treatments. the obtained results elucidate the effect of bio-algeen s-90 and megagreen on “low input” lettuce production. key words: bio-algeen s-90, carotenoids, chlorophyll a and b, megagreen, mineral content, non-heated plastic tunnel, vitamin c * corresponding author, e-mail: ivana.sola@biol.pmf.hr slavica dudaš and danijela poljuha have contributed equally to this work. introduction growing vegetables such as winter lettuce, rocket, leek, onion, leafy brassicas or radishes, often of local types and traditional cultivars, in semi-mediterranean climatic conditions during the winter period with poorer light and milder temperature conditions, extends the production season, provides fresh vegetables for local markets and ensures additional income for local producers. such production is commonly carried out in non-heated high plastic tunnels without heating and lightning. traditional varieties are today in increasing demand not only in produce markets but also in hotels and in the local gastronomy for tourist purposes, while worldwide they are being increasingly included in revitalisation and protection programmes for growing traditional varieties, for regional supply as niche products (lissek-wolf et al. 2009), but also in larger variety trials (wenz and wenger 2012) and in organic farming. traditional cultivars are cultivated in istria (northern coastal croatia) on small areas in response to the needs of local markets, where this type of cultivar is highly appreciated, recognizable and achieves good prices. one of the widely spread traditional lettuce cultivars in istria is ‘four seasons’. this cultivar belongs to the class of butterhead lettuce (lactuca sativa var. capitata l.), which is predominantly well suited for protected cultivation during winter because of its ability to develop heads of acceptable quality and mass in poorer light and temperature conditions. its leaves are dark reddish-brown, and it is one of the darkest butterhead types of lettuce when grown in optimal growing conditions, though the colour intensity as well as the compactness of the heads depends on the weather and temperature (wenz and wenger 2012). the ‘four seasons’ cultivar has a long growing tradition in organic farming in early spring or for autumn and winter harvest, due to its good cold-hardiness, and as its name says, it can be cultivated all year round. however, the heat and water shortage in summer, as well as the defi ciency of light and emergence of frost in the winter on the northern adriatic coast restrict the optimal yield of the lettuce. dudaš s., šola i., sladonja b., erhatić r., ban d., poljuha d. 254 acta bot. croat. 75 (2), 2016 the use of biostimulants and fertilizers to enhance plant yield nowadays is a regular farming practice. biostimulants include diverse substances and microorganisms that enhance plant growth (calvo et al. 2014). the effects of several biostimulants on plant growth from spring until autumn in the mediterranean region have been investigated so far (vernieri et al. 2002, amanda et al. 2009, lucini et al. 2015). regarding the winter season, johnstone et al. (2005) and tahsin (2010) investigated the effect of preplant phosphorous and nitrogen application, respectively, on lettuce yield, but this was not in the mediterranean region. organic extracts of seaweed bioactive substances have been used for ages to enhance soil fertility and crop productivity. from among biostimulants prepared from seaweed extracts, crouch et al. (1990) investigated the effect of ecklonia maxima (osbeck) extract application on l. sativa l. cv. winter crisp. one of the most commercial seaweed extracts is made from brown seaweed ascophyllum nodosum (l.) le jolis. however, the extract composition may vary according to the a. nodosum source, the season of collection and the extraction process used (calvo et al. 2014). the a. nodosum extract often used in croatia is bio-algeen s-90. mineral fertilizers are products containing nutrients essential for the normal growth and development of plants (isherwood 2000). for some period in croatia npk (nitrogen, phosphorus, potassium) were the most used fertilizers; however new preparations with other elements alongside n, p and k emerged and gave promising results as well. one such fertilizer is megagreen, a ca fertilizer with micronutrients. bio-algeen s-90 and megagreen, which are both implemented in this study, are on the list of permitted fertilizers and soil conditioners for organic farming (ministry of agriculture rc 2013). so far, bio-algeen s-90 has been applied on several crops, among them: cereals (kolbe and blau 1998), soybean (redžepović et al. 2007), basil (kwiat kowski and juszczak 2011) and tomato (mikiciuk and dobromilska 2014). the effect of megagreen was also studied on several cultures, like potatoes (horvat et al. 2014) and sugar beet (artyszak et al. 2014). both products were revealed to have a positive effect on the investigated crops. as far as we know, the possible application of these two preparations in the winter production of leafy vegetables, including lettuce has not been tested so far. as for the production of lettuce, it has been proven that organic fertilizers are more suitable than inorganic ones in lettuce cultivation in river sand (masarirambi et al. 2010), that toxic nitrate concentration in romaine-type lettuce increases after fertilization with sewage sludge (castro et al. 2009), and that mineral fertilization increases the nitrate concentration in the lettuce cultivar mantecosa (premuzic et al. 2001). the aim of this study was to examine the effects of bioalgeen s-90 biostimulant and the megagreen fertilizer on winter production of the traditional lettuce cultivar ‘four seasons’. for that purpose we determined the effects of both treatments on: a) plant height, leaf number, head mass, marketable and non-marketable yield of lettuce, b) chlorophyll a and b content, c) carotenoid content, d) vitamin c content, e) ph of lettuce leaf juice, f) nitrate content, and g) mineral content (n, p, k). materials and methods plant material and growth condition the experiment was carried out during 2012 – 2013 in a non-heated high plastic tunnel at the agricultural department in poreč, polytechnic of rijeka, croatia. the tra ditional cultivar of lettuce ‘four seasons’ (seeds franchi, italy) was sown in klasmann 1 horticultural substrate (klas manndeilmann, germany) and transplanted into 60 cell fl ats (532 mm x 323 mm x 60 mm, koplast, croatia). the experiment included three treatments: double watering with 1.0% bio-algeen s-90 (200 ml per plant), double foliar treatment with 0.3% megagreen (100 ml m–2) and control. the basic unit in the experiment was the plot, 3.5 m long and 2.5 m wide (8.75 m2), and each treatment was represented by three replicates, i.e. three plots. plots were arranged according to a random schedule. the experiment consisted of 9 plots (in total, 78.75 m2), the distance between plots (the track) was 0.5 m, while the total area of the experiment amounted 99 m2. in total, 112 lettuce plants per plot were planted at a distance of 0.30 m x 0.30 m, and the total number of plants in the trial was 1008. the soil was terra rossa whose nutrient content and ph values were determined according to standard soil analysis methods. soil was additionally enriched with 2.5 kg per 10 m2 of pelleted organic compost hortyfl or stallatico (fomet, italy). all the treatments were carried out manually, using small bottles with sprayers and a container for watering with a dosing cup. every plant in the plot was treated separately, watered or foliar treated according to the planned trials and treatments. the fi rst application was done 6 days after planting and the second 14 days later. during frost, all variants of lettuce treatments and the control variants were additionally protected with agrotextile (17 g m–2). bio-algeen s-90 and megagreen preparations bio-algeen s-90 (shulze & hermsen gmbh, germany) is an organic biostimulant derived from marine alga ascophyllum nodosum, a natural algae extract, with macroand micronutrients, containing natural chemical compounds including vitamins, amino acids and alginic acid. according to the label, megagreen (velebit inf. international d.o.o., croatia) is a ca fertilizer with micronutrients for foliar implication: 44.1% cao, 2.2% mgo, 1.2% fe2o3, 0.7% al2o3, 9.1% sio2, 0.11% so4, 132 mg kg–1 mn, 60 mg kg–1 zn, 22.5 mg kg–1 cu, 11.5 mg kg–1 pb, 3.3 mg kg–1 ni, 3.25 mg kg–1 cr, 0.8 mg kg–1 cd and hg in traces. analysis of qualitative and quantitative yield the following parameters were monitored 82 days after the second application: plant height, leaf number, head mass, total yield, marketable yield, the share (%) of nonmarketable yield (head mass lower than 100 g, visible symptoms of diseases and/or physiological disorders), chlolettuce biostimulation and fertilization acta bot. croat. 75 (2), 2016 255 rophyll a and b content, carotenoid content, nitrate content in fresh leaves, ph value of fresh lettuce juice, vitamin c content in fresh leaves, dry matter content of leaves, and total n, p and k content in leaves. chlorophyll a, chlorophyll b and carotenoid content were analysed spectrophotometrically by measuring absorbance at 665, 649 and 480 nm, respectively, according to wellburn (1994). nitrate content was determined spectrophotometrically according to cataldo et al. (1975). the ph value was determined in fresh lettuce juice using a ph meter (sartorius croatia – libra elektronik d.o.o., zagreb, croatia), vitamin c content using 2,6-dichloroindophenol titrimetric method and dry matter content gravimetrically at 105 °c until the mass became constant. total n, p, k contents were determined according to standard iso 11261 (2004), iso 6878 (1998) and iso 9964-3 (1993) procedures, respectively. statistical analysis obtained data were statistically analysed using anova and post hoc tukey test for multiple comparisons between groups on the basis of p ≤ 0.05. due to homogeneity of error variance, a synthesis of the results of two year studies was carried out. the obtained data was processed using spss v. 17.0, and using ibm spss statistics software version 22.0 for principal component analysis. results climatic conditions in the cultivation period at the location in poreč were characterised by mild temperatures and the emergence of frost, mostly of short duration. the average air temperature was in the range 4.8 – 7.8 °c, while soil temperature was between 5.0 and 8.7 °c (fig. 1). nutrient content and ph values of terra rossa soil are presented in tab. 1. the soil contained approximately 69 mg kg–1 k2o, 50 mg kg–1 p2o5 and 4 mg kg–1 caco3. the ph value of soil water was slightly alkaline, approximately 7.8; and of kcl approximately 7.3. the share of humus in the soil was around 4.0%. double watering at the start of cultivation with bio-algeen s-90 increased the plant height of the lettuce by 61.5%, and double foliar treatment with megagreen by 60.9%, compared to the control treatment (tab. 2). equally, the lettuce treated with bio-algeen s-90 and megagreen yielded a signifi cantly higher leaf number compared to the control treatment (increase by 47.7% and 37.2%, respectively). the difference in leaf number between bio-algeen s-90 and megagreen applications was on average 7.1% and was not signifi cantly confi rmed. lettuce reached a head mass between 255 and 330 g (tab. 2). application of either bio-algeen s-90 or megagreen signifi cantly increased lettuce head mass. the highest mass of lettuce head was reached after bio-algeen s-90 treatment. it was 30.3% higher than in the control treatment, while megagreen treatment resulted in head mass increase of 25.0%. there was no signifi cant difference between bio-algeen s-90 and megagreen effects on head mass (tab. 2). biostimulant and fertilizer treatment signifi cantly affected total yield as well as marketable yield and share (%) of non-marketable lettuce heads, compared to the control variant. control treatment had a signifi cantly lower total yield and marketable yield as well as a signifi cantly higher percentage of non-marketable heads in total yield (tab. 3). the infl uence of biostimulant and fertilizer on pigment content is shown in the tab. 4. chlorophyll a and b and carotenoid content varied signifi cantly between bio-algeen s-90 and megagreen treatments. compared to control, chlorophyll b level was signifi cantly higher after megagreen treatment, but chlorophyll and carotenoids did not differ signifi cantly. bio-algeen s-90 treatment did not change signifi cantly the amount of any of the tested pigments. fig. 1. average air and soil temperature for cultivation period on location in poreč, croatia (december – march), (croatian meteorological and hydrological service, 2013). sd – standard deviation. tab. 1. analysis of minerals (p2o5, k2o and caco3), ph value and humus content in terra rossa soil. results are mean values of three replicates ± standard deviation (sd). depth 0–30 cm values±sd p2o5 (mg kg–1) 49.98±0.45 k2o (mg kg–1) 68.80±1.21 caco3 (mg kg–1) 3.61±0.70 ph h2o 7.77±0.01 ph kcl 7.26±0.01 humus (%) 4.06±0.29 tab. 2. the effect of bio-algeen s-90 and megagreen treatments on plant height, leaf number and head mass of ‘four seasons’ lettuce. results are mean values of three replicates ± standard deviation (sd). different letters in the same column indicate signifi cant difference (tukey’s test, p ≤ 0.05). lsd – least signifi cant difference. treatment plant height (cm) leaf number head mass (g) bio-algeen s-90 10.87±2.97 a 19.20±4.52 a 332.10±30.87 a megagreen 10.83±3.19 a 17.83±3.43 a 318.74±29.33 a control 6.73±2.11 b 13.00±4.07 b 254.92±21.71 b lsd, p ≤ 0.05 3.21 4.53 35.96 dudaš s., šola i., sladonja b., erhatić r., ban d., poljuha d. 256 acta bot. croat. 75 (2), 2016 the amount of nitrates in lettuce fresh leaves signifi cantly decreased after both bio-algeen s-90 and megagreen treatment (fig. 2) and was 47.5% and 34.0%, respectively of the value in control plants. there was no signifi cant difference in treatments with bio-algeen s-90 and megagreen preparations with respect to their effectiveness on the level of nitrate decrease. the ph value of fresh lettuce juice was signifi cantly decreased only after bio-algeen s-90 treatment (tab. 5). vitamin c and dry matter content increased signifi cantly after both of the treatments (tab. 5); however a higher increase was caused by bio-algeen s-90 treatment. the content of n, p and k minerals was not affected by either of the treatments (tab. 5). a biplot constructed by two principal components showing treatments and tested yield components is presented in fig. 3. principal component 1 (pc1) and principal component 2 (pc2) provided signifi cant indications of 100% of the total variance in the data and showed clear separation of the treatments into three groups (fig. 3). discussion agricultural growing practices have been evolving towards organic, sustainable or environmentally friendly systems. fertilizers and lately biostimulants have increasingly been used as a tool with the potential to enable a more sustainable agriculture production (bulgari et al. 2015). the global market for plant biostimulants and fertilizers is growing fast; according to the study mentioned by calvo et al. (2014) the largest market for biostimulants in 2012 was europe. the greatest increase in fertilizer consumption in europe occurred in the second half of the 20th century (isherwood 2000). about ten years ago the biostimulant biofig. 3. biplot of the principal component analysis based on ten tested compounds and three yield components. fig. 2. nitrate content (ppm) in fresh leaves of lettuce treated with bio-algeen s-90 and megagreen. the means labelled by different letters are signifi cantly different (tukey’s test, p ≤ 0.05). tab. 3. the effect of bio-algeen s-90 and megagreen treatments on yield, marketable and non-marketable yield of ‘four seasons’ lettuce. *non-marketable lettuce heads with head mass lower than 100 g, visible symptoms of diseases and/or physiological disorders like loose heads or bolting. results are mean values of three replicates ± standard deviation (sd). different letters in the same column indicate signifi cant difference (tukey’s test, p ≤ 0.05). lsd – least signifi cant difference. treatment yield (kg m–2) marketable yield (kg m–2) non-marketable yield* (%) bio-algeen s-90 3.1±0.12 a 2.54±0.32 a 18.02±0.61 a megagreen 2.9±0.08 a 2.31±0.11 a 20.22±1.11 a control 2.0±0.07 b 1.26±0.13 b 36.91±1.19 b lsd, p ≤ 0.05 0.56 0.99 12.30 tab. 5. the effect of bio-algeen s-90 and megagreen treatment on ph value of fresh lettuce juice, vitamin c (vit c) content, percentage of dry matter and mineral content in ‘four seasons’ lettuce leaves. results are mean values of three replicates ± standard deviation (sd). different letters in the same column indicate signifi cant difference (tukey’s test, p ≤ 0.05). treatment ph juice vit c mg100 g–1fm dry matter % n % p2o5 % k2o % bio-algeen s-90 6.27±0.05 b 17.38±2.45 a 5.93±0.82 a 3.76±0.75 a 1.09±08 a 8.42±1.67 a megagreen 6.43±0.03 a 15.48±1.11 b 4.43±1.41 b 4.24±0.83 a 1.23±04 a 6.66±1.55 a control 6.40±0.08 a 13.90±0.51 c 3.93±2.12 c 4.13±0.09 a 0.98±01 a 4.85±1.39 a tab. 4. the effect of bio-algeen s-90 and megagreen treatments on chlorophyll a (chl a), chlorophyll b (chl b) and carotenoid (car) contents in ‘four seasons’ lettuce leaves. results are mean values of three replicates ± standard deviation (sd). different letters in the same column indicate signifi cant difference (tukey’s test, p ≤ 0.05). fm – fresh mass. lsd – least signifi cant difference. treatment chl a chl b car mg g–1fm bio-algeen s-90 0.725±0.08 b 0.670±0.09 b 0.155±0.03 b megagreen 1.011±0.02 a 1.038±0.05 a 0.216±0.00 a control 0.849±0.01 ab 0.829±0.07 b 0.182±0.02 ab lsd, p ≤ 0.05 0.167 0.199 0.043 lettuce biostimulation and fertilization acta bot. croat. 75 (2), 2016 257 algeen s-90 and the fertilizer megagreen appeared on the market; however there are no data about their effects on the qualitative and quantitative yields of leafy vegetables, including lettuce. therefore, in this work the biological effects of the two preparations on “low input” lettuce cultivation in winter season were investigated. the obtained positive effect of both bio-algeen s-90 and megagreen on winter production of lettuce is in accordance with similar studies on lettuce. wenz and wenger (2012) accordingly have obtained a positive effect of the organic chitin fertilizer biosol®, produced on the basis of penicillium chrysogenumon, on lettuce head mass, and also reported the relationship between head mass and seed origin in the same cultivar. the application of npk-based fertilizer one® in the study of bulgari et al. (2014) was as effi cient in plant weight increase of lettuce as the applications we investigated. in the experiment conducted on lettuce cv. mathilda, sternecker and balas (2014) achieved a head mass increase of 31.0% by using the biostimulant plantasalva, extract from 21 plant species with associated lactobacillus and yeast, which is in good accordance with our results. amanda et al. (2009) confi rmed the increase of baby leaf lettuce yield after treatment with actiwave® biostimulant with betaine, alginic acid, and caidrine, a derivative of vitamin k1. recently, lucini et al. (2015) have shown that l. sativa l. cv. regina di maggio treated with trainer, a biostimulant with plant (l) amino acids, was more resistant to yield and biomass reduction caused by stress conditions. treatments with both bio-algeen s-90 or megagreen increased plant height and number of leaves. since both preparations affect plant height almost twice as much as head mass and leaf number, we assume that their effects could be associated with synthesis, transport and/or accumulation of auxins in lettuce. other studies also confi rmed the positive effect of biostimulants on plant growth. vernieri et al. (2002) showed that the application of the biostimulant radifarm, a complex of plant extracts with polysaccharides, aminoacids, betains and enriched in vitamins and micronutrients, had a positive effect on plant growth in lettuce and spinach and improved the root/shoot ratios. in lettuce, the biostimulant strongly stimulated the root growth and showed also an increase of the leaf area. we found that bio-algeen s-90 and megagreen effects on chlorophyll and carotenoid contents differ signifi cantly. namely, megagreen yielded higher amounts of pigments than bio-algeen s-90 treatment. this is not surprising if we take into account that the core of each chlorophyll molecule is the mg2+ ion for which megagreen is a far better source than bio-algeen s-90 (megagreen contains 2.2% mgo, but bio-algeen s-90 only 0.021% mg; see http://www.agroklub.com/gnojiva/shulze-i-hermsen-gmbh-284/bio-algeens-92-313/). compared to control plants, only plants treated with megagreen had increased chlorophyll b content. therefore, we would recommend the use of megagreen for the production of lettuce with higher pigment content. with regard to the treatments with megagreen and bio-algeen s-90, there are no comparable results in the literature for lettuce production. however, some other studies revealed the effect of the same preparations on pigment content in other cultures, e.g. the effect of the fertilizer megagreen on potato (horvat et al. 2014) and the effect of the seaweed extract bio-algeen s-90 on tomato (mikiciuk and dobromilska 2014). although megagreen treatment did not infl uence total chlorophyll content, it increased the photosynthetic intensity and larger sized tuber yield. mikiciuk and dobromilska (2014) proved the increased chlorophyll and carotenoid content in small-sized tomato leaves after multiple spraying with bio-algeen s-90 during summer cultivation in a high plastic tunnel. it also contributed to early harvest and increased the clusters’ length and the number of tomato fruits in the cluster. although results from tomato cultivation are not directly comparable with lettuce, they can serve as an orientation. furthermore, they are not in accordance with our results observed in winter cultivation, probably due to different light conditions. as previously mentioned, the chlorophyll content depends on the environmental conditions, especially light quality and quantity (dias et al. 2007, kosma et al. 2013). in suboptimal conditions, those environmental factors could infl uence the chlorophyll content more than the treatment with biostimulants. other reasons for the different results obtained could be the method and frequency of biostimulant application. the presence of nitrates in vegetables, water and other food is a serious threat for human health, especially the part converted to the more toxic nitrites (santamaria 2006), which account for approximately 5.0% of all ingested nitrates (spiegelhalder et al. 1976, pannala et al. 2003) but can reach to 20% for individuals with a high rate of conversion (thomson et al. 2007, correia et al. 2010). in our experiment, we found that nitrate content in the control plants, with a value of 6,446 ppm, exceeded the permissible limit of nitrate in leafy vegetables (commission regulation 1258/ 2011). however, after either bio-algeen s-90 or megagreen treatment of fresh lettuce leaves, the amount of nitrates was signifi cantly decreased, to 34.0 and 47.5% of the control value, respectively (fig. 2). since in lettuce l. sativa, protected or open grown, harvested in the period from october 1 to march 31 the limit amount of nitrates is 4,500 ppm expressed on the fresh weight basis, bio-algeen s-90 or megagreen are obviously great treatments for nitrate-related toxicity reduction of lettuce. compared to the sewage sludge used by castro et al. (2009), and mineral (94% ca(no3)2 + 6% nh4no3) and organic (biostabilised compost) fertilizers used by premuzic et al. (2001), in terms of nitrate concentration, megagreen fertilizer would be a better choice for lettuce production. the ph value of fresh lettuce juice, as expected, varied within the slightly acidic range, between 6.27 and 6.43. us fda/cfsan (2007) indicates approximate ph values for lettuce in general between 5.80 – 6.15, noting that ph values of vegetable juices can vary according to the cultivar, season, growing conditions or processing methods. in our experiment, the ph value of fresh lettuce juice was decreased after bio-algeen s-90 treatment, and we presume this could be due to amino acids and alginic acid present in this preparation (see “materials and methods” section), as well as due to a signifi cantly higher amount of vitamin c (ascorbic acid) detected after this treatment (tab. 5). dudaš s., šola i., sladonja b., erhatić r., ban d., poljuha d. 258 acta bot. croat. 75 (2), 2016 both of the treatments signifi cantly increased the amounts of vitamin c and dry matter content, however a higher increase was caused by bio-algeen s-90 treatment. this was expected since bio-algeen s-90 preparation already contains natural chemical compounds, including vitamins. thus, even though after bio-algeen s-90 and megagreen treatments the total yield increased similarly, in case of need for higher dry mass, we would recommend the use of bio-algeen s-90 preparation. while vitamin c differed signifi cantly between the tested treatments, mineral contents in lettuce leaves did not change (tab. 5) and were in the range indicated in literature (ismail and fun 2003, koudela and petříková 2008, masamba and nguyen 2008). since bio-algeen s-90 preparation contains amino acids, we expected that this treatment would increase the concentration of n in lettuce leaves; however, this was not the case. we speculate this could be due to limited root absorption of amino acids from the soil and/or retention of amino acids in the lettuce root. compared to the result of premuzic et al. (2001) who found that n or biostabilised compost fertilization does not change lettuce vitamin c content, we found that megagreen fertilizer increased vitamin c content in lettuce and in this term suggest it as a better choice for lettuce treatment. the principal component analysis (pca) based on ten tested compounds and three yield components enabled visualization of correlations between analysed data. principal component 1 (pc1) and principal component 2 (pc2) analyses provided signifi cant indications of 100% of the total variance in the data and showed clear separation of the treatments into three groups (control, bio-algeen s90 and megagreen). based on the obtained results we conclude that application of either bio-algeen s-90 or megagreen in winter cultivation conditions showed a signifi cant positive effect on the growth, head mass and yield of lettuce fresh leaves compared to the control treatment. the content of lettuce chlorophylls a and b and carotenoids was signifi cantly higher after megagreen treatment compared to bio-algeen s-90 treatment; however, compared to control plants, megagreen treatment signifi cantly increased only chlorophyll b amount. the content of lettuce vitamin c and dry matter was signifi cantly increased after both treatments (more after bio-algeen s-90 and less after megagreen treatment). both treatments signifi cantly decreased toxic nitrate content and the share of non-marketable yield in total lettuce yield. the ph value of lettuce juice decreased after bio-algeen s-90 treatment. mineral content (n, p, k) of lettuce leaves was not affected by either biostimulant or fertilizer application. finally, since the potent biostimulant bio-algeen s-90 is a preparation derived from the northern atlantic ocean alga a. nodosum, and croatia is a maritime state with various types of algae in its adriatic sea, we propose investigation of adriatic algae extracts as possible biostimulants. acknowledgements the present research was not funded by any national or international institution, but the authors are grateful to the polytechnic of rijeka, poreč agricultural department, to the institute of agriculture and tourism in poreč, department of biology at faculty of science, university of zagreb and to the college of agriculture in križevci for material and infrastructural support given during the research. references amanda, a., valagussa, m., piaggesi, a., ferrante, a., 2009: effect of biostimulants on quality of baby leaf lettuce grown under plastic tunnel. acta horticulturae 807, 407–412. artyszak, a., gozdowski, d., kucińska, k., 2014: the effect of foliar fertilization with marine calcitein sugar beet. plant, soil and environment 60, 413–417. bulgari, r., cocetta, g., trivellini, a., vernieri, p., ferrante, a., 2015: biostimulants and crop responses: a review. biological agriculture and horticulture 31, 1–17. bulgari, r., podetta, n., cocetta, g., piaggesi, a., ferrante, a., 2014: the effect of a complete fertilizer for leafy vegetables production in family and urban gardens. bulgarian journal of agricultural science 20, 1361–1367. calvo, p., nelson, l., kloepper, j. w., 2014: agricultural uses of plant biostimulants. plant soil 383, 3–41. castro, e., manas, m. p., de las heras, j., 2009: nitrate content of lettuce (lactuca sativa l.) after fertilization with sewage sludge and irrigation with treated wastewater. food additives and contaminants 26, 172–179. cataldo, d. a., maroon, m., schrader, l. e., youngs, v. l., 1975: rapid colorimetric determination of nitrate in plant tissues by nitration of salicylic acid. communication in soil science and plant analysis 6, 71–80. commission regulation (eu) no 1258/2011 amending regulation (ec) no 1881/2006 as regards maximum levels for nitrates in foodstuffs from 2 december 2011. offi cial journal of the european union 320, 15–17. correia, m., barroso, â., barroso, m. f., soares, d., oliveira m. b. p. p., delerue-matos, c., 2010: contribution of different vegetable types to exogenous nitrate and nitrite exposure. food chemistry 120, 960–966. croatian meteorological and hydrological service, 2013. retrieved june 10, 2014 from http://www.dhmz.htnet.hr/ (in cro atian). crouch, i. j., beckett, r. p., van staden, j., 1990: effect of seaweed concentrate on the growth and mineral nutrition of nutrient stressed lettuce. journal of applied phycology 2, 269–272. dias, j., pimenta, j. a., medri, m. e., tores boeger, m. r., toledo de freitas, c., 2007: physiological aspects of sun and shade leaves of lithraea molleoides (vell.) engl. 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(uso di biostimolanti nella produzione di piantine da orto.). colture protette 31, 75–79. wellburn, a. r., 1994: the spectral determination of chlorophyll a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. journal of plant physiology 144, 307–313. wenz, j., wenger, m., 2012: die überwinterung von gartensalat (lactuca sativa l.) im freiland. university of applied science, hochschule weihenstephan-triersdorf, diplomarbeit. acta botanica 2-2016 za web.indd 236 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 236–243, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0031 issn 0365-0588 eissn 1847-8476 aquatic plant trapa natans l. as bioindicator of trace metal contamination in a freshwater lake (skadar lake, montenegro) dragana petrović1*, dejan jančić2, martina furdek3, nevenka mikac3, slađana krivokapić1 1 university of montenegro, faculty of natural science, department of biology, podgorica, montenegro 2 center for ecotoxicological research of montenegro, podgorica, montenegro 3 ruđer bošković institute, division for marine and environmental research, zagreb, croatia abstract – skadar lake is the largest shallow lake in southeastern europe. it is located within a national park, and is included in the ramsar list of international important wetlands, so its preservation and protection from pollution is very important. the aim of this study was to investigate bioaccumulation of the ecotoxic metals cd, pb and cr from sediments of skadar lake in the aquatic macrophyte trapa natans l. samples of sediment and plants were collected at nine locations covering all major water inputs to the lake as well as locations where contamination could be expected. the obtained results indicate that sediments from the skadar lake are only locally contaminated with cd (0.03–1.18 mg kg–1), generally contaminated with cr (15.8–180 mg kg–1), the concentrations of both elements frequently exceeding sediment quality guidelines, while concentrations of pb were low (2.7–17.4 mg kg–1). the highest bioaccumulation of all metals from sediment to trapa natans l. was observed in the root, with accumulation effi ciency decreasing in the order cd > cr > pb. translocation from root to stem was also higher for cd than for cr and pb, while the translocation from stem to leaf was comparable for all three metals. from the three investigated metals cd showed the highest mobility. the results indicate that trapa natans l. may be a very promising bioindicator of trace metal contamination in skadar lake. key words: bioaccumulation, bioindicator, cadmium, chromium, lead, sediment, skadar lake, trapa natans l. * corresponding author, email:draganap2104@gmail.com introduction metals are introduced into aquatic systems from both natural and anthropogenic sources and today contamination with metals is a widespread problem due to increasing industrialization and urban development. upon introduction to the aquatic environment metals are deposited in the bottom sediments and from there may be transferred to plants and the aquatic food chain (vardanyan and ingole 2006, rai 2009, mazey et al. 2010). some aquatic plants have the ability to accumulate metals and other contaminants and can be used as bioindicators of environmental contamination (whitton and kelly 1995, cardwell et al. 2002, kumar et al. 2006). plants that can both accumulate metals and have high tolerance to them are important in phytoremediation strategies of contaminated aquatic systems (clemens et al. 2002, wang at al. 2002, marchand et al. 2010). uptake from sediment and distribution of metals in plants depends on many factors: the chemical characteristics of the metal, the plant species used and the environmental conditions (baldantoni et al. 2004, kumar et al. 2006). the availability of trace metals in sediment is related to their chemical forms in pore water and their affi nity for particulate matter, which depends on different factors such as ph, redox potential or organic matter content (guilzzoni 1991). some of the sediment phases (exchangeable cations, organic phases, carbonates) contain metals in a form that can be easily released into the pore water and thus made available for uptake by plants. aquatic macrophytes differ both in their capacity to take up metals in root and in the proportion of metals transferred to the above-ground parts (baldantoni et al. 2004, vardanyan and ingole 2006, mazey and germ 2009). trapa natans l. is an aquatic, fl oating macrophyte, typical for natural wetlands. trapa natans l. is also known to live within a wide range of nutrient levels and metal concentrations (rai et al. 1996, rai and sinha 2001, kumar at al. 2002, sweta et al. 2015). as it fl oats on the water surface, it is exposed, in addition to uptake of contaminants bioaccumulation of trace metal in the trapa natans acta bot. croat. 75 (2), 2016 237 from sediment, to their uptake from water and the atmosphere. the large biomass of this species and its ability to accumulate metals makes it suitable for the monitoring and even for the phytoremediation of contaminated aquatic ecosystems (kumar at al. 2002, sweta et al. 2015). skadar lake is the largest shallow lake in southeastern europe. it is a national park, included in the ramsar list of international important wetlands, so its preservation and protection from pollution is very important. however, intensive industrial and urban development in the region exposed skadar lake to anthropogenic pollution by organic and inorganic contaminants, including metals (stešević et al. 2007). the largest tributaries of the skadar lake are the morača river and the crnojevića river which fl ow through industrial and urban settlements and transport pollutants to the lake. previous studies demonstrated that lake sediments are contaminated by metals, mostly ni and cr (stešević et al. 2007, vemić et al. 2014). uptake of metals from water and sediment into two types of aquatic macrophytes from the skadar lake (phragmites communis, ceratophyllum demersum) was also studied (kastratović et al. 2013, 2014). the aim of this work was to investigate the bioaccumulation of the ecotoxic metals cd, pb and cr from sediments of the skadar lake in the aquatic macrophyte trapa natans l. cadmium and lead are important as they are on the eu priority list of pollutants that should be regularly monitored, while chromium is especially important for the skadar lake as it was suggested that elevated concentrations of this metal caused toxic effects to the aquatic plants from the lake stešević et al. (2007). the study includes determination of the level of these three metals in lake sediments, study of their bioaccumulation from sediment to the root of trapa natans l., and their further transfer to the aboveparts (stem and leaf) of the plant. the main goal of this study is to evaluate if trapa natans l. could be a good bioindicator of contamination of the skadar lake with cd, pb and cr, thus providing a tool for monitoring these metals in the future. materials and methods sample collection sediment and plant materials were collected from may to june 2012 at nine locations in the skadar lake (fig. 1). description of sampling locations and their positions are given in on-line suppl. tab. 1. sampling locations cover all major water inputs to the lake (morača river, crnojevića river, raduš underwater spring) as well as locations where potential local contamination with metals (like small villages or ports) can be expected. possible anthropogenic input of metals into the ecosystem of the skadar lake comes from industries located in the vicinity of the lake as well as from the use of agricultural fertilizers and pesticides. in the small town of reka crnojevića, which lies on the river from which its name derives, untreated industrial (fi sh processing plant) and municipal wastewaters are discharged into the crnojevića river. after a preliminary survey of areas where the plant trapa natans l. can be found in suffi cient abundancy, 3–4 complete healthy plants of similar size, shape and weight were sampled at each sampling location, over an area of about 25 m2. plants were collected by hand, packed in polyethylene bags and transferred to the laboratory. sediment samples were taken from the same place as the plant material. sediment samples (1 kg) were taken using an ecman-type dredge and the layer of 0–20 cm was collected. metal analysis in the laboratory plant material was washed thoroughly with deionized water to remove detritus and periphyton. samples of plants were divided into roots, stems and leaves and dried at 75 °c for 48 hours. dry samples were ground into a fi ne powder and homogenized in an electric blender. prepared samples (0.5 g) were mineralized with a milestone microwave ethos 1, with a mixture of hno3 and h2o2 (3:1). after digestion, the solution was diluted with deionized water to a fi nal volume of 50 ml. sediment samples were fi rst dried in air and then in an oven at 75 °c for 48 hours. dry sediment samples were ground in an agate mortar and sieved through a 1.5 mm sieve. approximately 0.5 g of the sample was mineralized by microwave digestion with a mixture of hcl:hno3 (3:1). after mineralization, solutions were diluted with 2 m hno3 to a fi nal volume of 100 ml. concentrations of metals in plant (cd, pb, cr) and sediment (cd, pb, cr, fe) samples were determined by inductively coupled plasma optic emission spectroscopy (icp-oes) technique on a spectro acros instrument. working standards for measurements of elements were prepared from fig. 1. map of skadar lake with sampling locations. t1 – infl ow of the morača river, t2 – small lake at the right branch of the morača river, t3 – kamenik, t4 – milovića bay, t5 – underwater spring raduš, t6 – infl ow of the morača river, t7 – infl ow of the plavnica river, t8 – crnojevića river near the small town of reka crnojevića, t9 – the village karuč. petrović d., jančić d., furdek m., mikac n., krivokapić s. 238 acta bot. croat. 75 (2), 2016 sigma aldrich solutions of 1000 mg dm–3 each. the reliability of the analytical method was evaluated by analysis of certifi ed standard reference materials ncs dc73348 (bush branches and leaves) and ncs dc70312 (tibet sediment) from the china national analysis center for iron and steel, beijing. all results are expressed on a dry weight basis. calculation of bioconcentration factor and translocation ability transfer of metals from sediment to plant (metal phytoavailability) was estimated by the bioconcentration factor from root to sediment (bcf = metalroot/metalsediment). higher bcf implies greater phytoaccumulation ability. transfer of metals within the plant (from root to stem and from stem to leaf) was estimated by the translocation ability (ta), which was calculated as the ratio of concentration of metal between the individual parts of the plant, from lower to the upper part of the plant (ta = metal root or stem/metalstem or leaf). a higher ta means a smaller translocation ability. statistical analysis experimental data were analyzed using the statistical software program statistica 7.1. (statsoft inc., 2006). since the data did not show a normal distribution, the statistically signifi cant differences between groups were tested using the nonparametric kruskal wallis test (p < 0.05), followed by the post hoc tukey test (p < 0.05). results distribution of metals in sediments and plants distributions of cd, pb, cr and fe concentrations in sediment at nine investigated locations are presented in fig. 2. concentrations of cd (fig. 2a) showed the greatest variations (from 0.03 to 1.18 mg kg–1) and were the highest at locations t8 and t9. locations t3 and t4 demonstrated medium cd concentrations (from 0.5 to 0.7 mg kg–1), while at the remaining locations the cd level was below 0.2 mg kg–1. concentrations of pb (fig. 2b) varied from 2.7 to 17.4 mg kg–1 and pb distribution in sediment showed some similarities with cd distribution, as the highest pb values were found at locations t9, t3 and t4 and the lowest at locations t2 and t7. concentrations of cr (fig. 2c) were also quite variable (15.8 to 180 mg kg–1), but showed a very different distribution to those of cd and pb, with the highest concentration at location t1 and the lowest at location t8, while remaining locations showed medium cr levels. concentrations of fe (fig. 2d) varied between 9.1 and 51 g kg–1, covering the whole range between typical fe contents in limestone and shale (15 and 48 g kg–1, respectively, wedepohl 2004). this indicates that the abundance of the fi ne sediment fraction (rich in fe) at investigated locations is very variable, being the lowest at locations t8 and t2 and the highest at location t3. distributions of cd, pb and cr concentrations in individual parts of trapa natans l. at nine investigated locations are presented in fig. 3. concentrations of cd in different parts of the plants varied between 0.03 and 1.05 mg kg–1. at all locations the cd concentration was highest in the root, but differences between concentrations in root, stem and leaf were not large, except at location t8 and partly t9, where the highest cd levels in the plant were observed. for pb, concentrations were in the range from 0.03 to 3.68 mg kg–1 and the difference in concentrations between root and the upper parts of the plants was much larger than for cd at most locations. the highest concentrations of pb in the plant were obtained in the root at locations t9 and t8, as in cd, while pb concentrations in stem and leaf were low at all locations. distributions of cd and pb in the plant (especially in the root) were similar to the distribution of these elements in sediment, as the highest concentrations in both media were obtained at locations t8 and t9. concentrations of cr varied between 0.37 and 15.8 mg kg–1 and were also the highest in the root, showing, as for pb, a large fig. 2. concentration of cd (a), pb (b), cr (c) and fe (d) in sediment samples from different locations (see fig. 1 for explanation). bioaccumulation of trace metal in the trapa natans acta bot. croat. 75 (2), 2016 239 difference between levels in root and the upper parts of the plant. however, cr distribution in the root was very different from distribution in sediment, as at location t1, where the highest level of cr was observed in sediment, while the concentration in the root was rather low. transfer of metals in the sediment/plant system root/sediment bioconcentration factors of metals at nine investigated locations are presented in fig. 4a, whereas translocation abilities of metals between root/stem and stem/leaf compartments are presented in figs. 4b and c. calculated bcfs indicated a much higher uptake from sediment to root for cd (bcf = 0.1–3.7) than for pb and cr, which demonstrated similar mobility (bcf = 0.01–0.5 and 0.02–0.5, respectively). the highest transfer from sediment to root for all three metals was observed at locations t7, t8 and t9 and for cd also at location t2. at remaining locations bcf for cd was lower than 0.7 and for pb and cr lower than 0.1. translocation ability from root to stem was also highest for cd (taroot/stem = 1.0–3.2) and showed a decreasing trend from cr (taroot/stem = 1.1–12.6) to pb (taroot/ stem = 1.3–72). the highest values of taroot/stem for all three metals were observed at locations t3, t8 and t9 and for pb also at locations t4 and t6. translocation ability from stem to leaf demonstrated much lower variations both among the three metals and among the different locations. except two higher tastem/leaf values for cr at locations t3 and t4 all other tastem/leaf values were lower than 2. discussion evaluation of sediment contamination by cd, pb and cr in addition to the metals of natural origin, sediments may also accumulate elements from anthropogenic sources. concentration of elements of natural origin is usually a function of the abundance of the fi ne sediment fraction, as this fraction has the highest ability to adsorb and bind trace elements. thus, normalization of trace elements concentrations to some of the main components of the fi ne sediment fraction, such as al or fe, which are usually conservative and not affected by anthropogenic infl uence, may help to distinguish if elements are coming from natural or anthropogenic sources (boes et al. 2011). the relationship between fe and concentrations of cd, cr and pb in investigated sediments is presented in fig. 5. elevated concentrations of cd at locations t8, t9 and t4 suggest that some anthropogenic source of cd exists at these locations. the highest level of cd is obtained at location t8 (figs. 2 and 5), which is probably related to the discharge of untreated industrial and municipal waste waters of the town of reka crnojevića placed downstream from this location. lead showed elevated concentrations at the same locations as cd (figs. 2 and 5), suggesting identical contamination source for both metals, but the extent of contamination was lower for pb, especially at location t8. both distribution of cr in sediment (fig. 2) and fig. 4. bioconcentration factors (bcf root/sed) (a) and translocation ability (ta root/stem (b) and ta stem/leaf (c)) for cd, pb and cr at different sampling locations (see fig. 1 for explanation). fig. 3. concentrations of cd (a), pb (b) and cr (c) in trapa natans parts sampled from different locations (see fig. 1 for explanation). petrović d., jančić d., furdek m., mikac n., krivokapić s. 240 acta bot. croat. 75 (2), 2016 its relationship to fe (fig. 5) indicate that cr is transported to skadar lake by the morača river. previous investigations also demonstrated that cr is one of the most signifi cant pollutants in the montenegrin part of the skadar lake and that the principal origin of cr is waste waters from an aluminum processing plant located near podgorica (ste šević et al. 2007). ranges of cd, pb and cr concentrations obtained in this work were similar to previous measurements in the same area (on-line suppl. tab. 2). comparison with data from other remote freshwater lakes in europe (on-line suppl. tab. 2) indicates similar ranges of concentrations for cd and pb, but much higher levels of cr in skadar lake than in the pristine plitvice lakes (croatia). in order to evaluate possible ecotoxic effect of metal concentrations in sediments we compared the measured concentrations with the most frequently used sediment quality criteria for freshwater sediment (macdonald at al. 2000), which defi ne tec (threshold level concentration) as a lower limit below which toxic effect is not probable, and pec (probable effect concentration) as an upper limit above which toxicity to aquatic organisms can be expected (on-line suppl. tab. 2). according to such criteria, all pb and most of cd concentrations can be considered as non-toxic to aquatic organisms, as they were lower than tec, except cd level at location t8 which was higher than tec, but lower than pec. however, in the case of cr, only concentration at location t8 (crnojevića river) was lower than tec; the majority of concentrations were between tec and pec, and at locations t1 and t3 they were even higher than pec, indicating that some toxic effects of cr may be expected. stešević et al. (2007) indeed demonstrated that the content of metals in sediments from skadar lake inhibited growth of myriophyllum aquaticum and attributed this toxic effect to the elevated cr concentrations. bioaccumulation of cd, pb and cr in the trapa natans l. distribution of metals in aquatic plants depends primarily on the plant species, plant organs and the type of metal (guilzzoni 1991). some metals are accumulated mostly in the root, because of the existence of a physiological barrier to their transport into the above-ground parts of plants, while others can be easily transported to the branches (kumar et al. 2006, baldantoni et al. 2004). in our study the highest concentrations of all three investigated metals (cd, pb and cr) were found in the root of the trapa natans l. (fig. 3) and for all metals the average concentration in the root was signifi cantly higher (p < 0.05) than in the stem or leaf (fig. 6). furthermore, considering plants from all locations, variations in the concentration of cd, pb and cr in the root were much larger than in the stem and leaf (fig. 6). slightly higher concentrations of cd and cr could be noticed in the stem, but they were not signifi cantly higher than fig. 6. box-plot graphs of cd (a), pb (b) and cr (c) concentrations in the individual parts of trapa natans at nine sampling locations (box-plot boundaries indicate average value, standard deviations, and minimum and maximum value). the statistically signifi cant differences among groups according to post hoc tukey’s test (p < 0.05) are indicated by different letters. fig. 5. correlation of cd, pb and cr with fe concentration in sediment at different sampling locations (see fig. 1 for explanation). bioaccumulation of trace metal in the trapa natans acta bot. croat. 75 (2), 2016 241 in the leaf (fig. 6). it is interesting to note that the concentration of pb was slightly higher in the leaf than in the stem (fig. 6). this is due to higher pb levels in the leaf at locations t1 and t9 (fig. 3), which could be a consequence of pb absorption from the atmosphere through the leaf surface (schreck et al. 2012). some other aquatic plants also demonstrated consistently higher metal concentrations in root than in stems or leaves (cardwell et al. 2002, baldantoni et al. 2004, mazej and germ 2009). we further compared the concentrations of cd, cr and pb in the parts of trapa natans l. obtained in this work with the content of cd and pb in trapa natans l. from some other water environments. we also compared concentrations for all three metals in trapa natans l. with data for two other macrophytes from skadar lake (on-line suppl. tab. 2). the average concentration of cd was lower in skadar lake than in other areas, but at location t8, which was contaminated with cd (fig. 3), concentrations of cd in all plant parts were similar to those in areas contaminated with metals (sawidis et al. 1995, sweta et al. 2015). in all plant species from skadar lake accumulation of metals decreased in the order cr > pb > cd, following the levels of these metals in lake sediments. at all locations, concentrations of all three metals were higher in sediments than in the roots of the plant (figs. 2 and 3), with the exception of cd at location t7, where very low cd concentration in sediment was observed. bioaccumulation ability of metals from sediment to root, estimated by bioconcentration factor, bcfroot/sed (fig. 4a), varied greatly among themetals and decreased in the order cd > cr > pb. the bcf values for cd at all locations (fig.7a) were signifi cantly higher (p < 0.05) than for pb and cr (the average concentration for cd was about 6 times higher), thus indicating much higher root uptake of cd than pb and cr. the availability of trace metals for plants is related to their chemical forms in pore waters and to their availability in particulate matter (guilzzoni 1991). different factors such as ph, redox potential, organic matter content and microbial activity infl uence metal distribution between pore water and sediment particles and thus their availability to aquatic macrophytes (guilzzoni 1991, mazej and germ 2009). metals investigated in this study show different chemical behavior in sediment and affi nity to the main sediment components, such as carbonates, fe-mn oxides, organic matter and aluminosilicates, and their mobility in sediment decrease in the order cd > pb > cr (filgueiras et al. 2004), which explains the much higher bcfroot/sed for cd than for pb and cr. the same order of bioavailability of these three elements was found in other aquatic plants in skadar lake (kastratović et al. 2013, 2014) and also in the publications of other authors (mazej and germ 2009). translocation factors between root and stem (fig. 4b) decreased in the order pb > cr > cd and were the highest for pb at all locations. however, due to the large variations for pb values (fig. 7b), a statistically signifi cant difference (p < 0.05) between taroot/stem for various metals could be confi rmed only between cd and cr. very low values of taroot/stem for cd (in average 10 times lower than for pb), indicate that cd is very mobile within the plant, and its translocation from root to stem, after its uptake from the sediment, is very effective. on the other hand, pb is mostly retained in the root after its accumulation from sediment, while cr shows medium mobility from root to stem. however, there was no signifi cant difference in the translocation of the three investigated metals from stem to leaf (fig. 7c), since the determined values of tastem/leaf mostly varied between 1 and 2 (except for cr at location t3 and t4 where slightly higher values are noticed, fig. 3). this leads us to assume that metals translocated from root to stem are easily further transported to the leaves. other studies on translocation of metals in macrophytes also demonstrated the relatively high mobility of cd and the comparably low translocation of cr and pb, which was explained by the existence of the physiological barrier for the transport of cr and pb to the above ground parts of the plant (baldantoni et al. 2004, mazej and germ 2009). trapa natans l. as biondicator of ecotoxic metals contamination a comparison of cd distribution in sediment and plant (figs. 2 and 3) shows that, both in sediments and plants, the highest cd concentrations were found at locations t8 and t9, thus showing that effective cd accumulation occurs at these sites. actually, taking into account data from all nine locations, signifi cant correlation (pearson; r = 0.77; p < 0.05) can be observed between the content of cd in sedifig.7. box-plot graphs of bcfroot/sed (a), taroot/stem (b) and tastem/leaf (c) values for cd, pb and cr at nine sampling locations (box-plot boundaries indicate average value, standard deviations, and minimum and maximum value). the statistically signifi cant differences among groups according to post hoc tukey’s test (p < 0.05) are indicated by different letters. petrović d., jančić d., furdek m., mikac n., krivokapić s. 242 acta bot. croat. 75 (2), 2016 ment and its concentration in the root (fig. 8). it is evident that the highest concentration of cd in sediment at location t8 is refl ected in the signifi cantly higher level of this metal in the root of the plant. this leads us to assume that trapa natans could be a potential biondicator for cd contamination. furthermore, low taroot/stem values for cd at all locations indicate effective translocation of accumulated cd to the above ground plant parts. high accumulation of cd in trapa natans was also demonstrated in ponds in industrial areas in india, and this plant was proposed as a suitable candidate for the phytoremediation of metals from aquatic ecosystems (sweta et al. 2015). regarding pb and cr, no correlation between content in sediment and root was established in skadar lake, and bioaccumulation to the plant was much lower than for cd. however, in india, in ponds highly contaminated with metals (including cr and pb), where this plant is cultivated as a source of food, high accumulation of cr and pb in trapa natans fruit was found, indicating that, in polluted areas, accumulation of these metals may also take place (rai and sinha 2001). if we compare the metal bioaccumulation ability of trapa natans with two other plants which were studied in skadar lake (phragmites australis – kastratović et al. 2013 and ceratopgyllum demersum – kastratović et al. 2014) we can notice that the same order of metal bioaccumulation effi ciency (cd > pb > cr) from sediment was found for all three macrophytes. however, trapa natans showed the highest bioconcentration factors from sediment to root for cd and cr, thus further indicating that it may be a promising biondicator for metal contamination in the skadar lake. acknowledgement financial supports from the ministries of science and education of croatia and montenegro within the bilateral collaboration, as well as from the croatian science foundation under the project »transport and chemodynamics of trace elements in freshwater and coastal sedimentary systems« (hrzz7555) are gratefully acknowledged. fig. 8. correlation between cd content in sediment and in the root of the plant. references baldantoni, d., alfani a., di tommasi p., bartoli g., de santo a.v., 2004: assessment of macro and microelement accumulation capability of two aquatic plants. environmental pollution 130, 149–156. boes, x., rydberg, j., martinez-cortizas, a., bindler, r., renberg, i., 2011: evaluation of conservative lithogenic elements (ti, zr, al, and rb) to study anthropogenic element enrichments in lake sediments. journal of paleolimnology 46, 75–87. camarero, l., botev, i., muri, g., psenner, r., rose, n., stuchlik, 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and prospects of wetland plants. acta biotechnologica 22, 199–208. wedepohl, k.h., 2004: the composition of earth’s upper crust, natural cycles of elements, natural resources. in: merien, e., anke, m., inhat, m., stoeppler, m. (eds.) elements and their compounds in the environment, vol 1., general aspects, 3–16. wiley-vhc verlag gmbh and co. kgaa, weinheim. whitton, b., kelly, m., 1995: use of algae and other plants for monitoring rivers. australian journal of ecology 20, 45–56. acta botanica 2-2016 za web.indd 226 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 226–235, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0021 issn 0365-0588 eissn 1847-8476 long-term cropland abandonment does not lead per se to the recovery of semi-natural herb communities deemed habitats of community interest natalia troiani1, federico maria tardella2*, luca malatesta3, marcello corazza1, carlo ferrari1, andrea catorci2 1 university of bologna, department of biological, geological and environmental sciences, botanic garden via irnerio 42, 40126, bologna, italy; natalia.troiani2@unibo.it, marcello.corazza@unibo.it, carlo.ferrari@unibo.it 2 university of camerino, school of biosciences and veterinary medicine, via pontoni 5, 62032, camerino, mc, italy; dtfederico.tardella@unicam.it, andrea.catorci@unicam.it 3 university of camerino, school of advanced studies, via lili 55, 62032, camerino, mc, italy; e-mail: luca.malatesta@unicam.it abstract – abandoned croplands can be considered a new category of »scattered elements« of mountain landscapes. to gain a deeper understanding of the conservation status (sensu eec directive 92/43) of abandoned cropland in the northern apennines, we used the concept of the social behavior type (sbt) of plant communities. sbts refer to the behaviour and ecological attributes of species at a given observation level and allow the understanding of the plant community conservation status. we found that topographic and soil conditions drive species assemblage in pastures after crop abandonment, but that long-term abandonment does not lead per se to the recovery of the semi-natural grassland communities deemed worthy of conservation in the eec directive. it was mainly the lack of appropriate disturbance regimes that allowed the spread of dominant tall herbs, which, in turn, reduced site suitability for subordinate plants. moreover, their spread fostered the presence of elements such as ruderals and fringe species. we concluded that these abandoned croplands had a good potential to develop into a habitat as defi ned in the eu directive but without appropriate management plans they would remain of low representativeness. keywords: abandonment, croplands, ellenberg’s indicator values, grassland recovery, management, protected habitats, social behaviour types abbreviations: awc – available water capacity, cvre – cross-validation relative error, dca – detrended correspondence analysis, eiv – ellenberg’s indicator value, isa – indicator species analysis, iv – indicator value, m – soil moisture, mrt – multivariate regression tree, n – soil nutrients content, r – soil chemical reaction, rda – redundancy analysis, sbt – social behaviour type, t – air temperature. * corresponding author, e-mail: dtfederico.tardella@unicam.it introduction in a large part of europe, the abandonment of traditional extensive farming activities has led to successional changes toward forest (poschlod and wallisdevries 2002), and the abandonment of hilly and mountain croplands to the formation of new herbaceous communities (peroni et al. 2000); these abandoned areas can be viewed as new »scattered elements« in areas where forest recovery and expansion are underway as well as in agricultural landscapes, which are generally undergoing processes of intensifi ed land use. in fact, in both cases, socio-economic and natural processes are threatening biodiversity and leading to homogeneous landscapes (robinson and sutherland 2002). the conservation and management of these new open ecosystems are key elements within the european agricultural policies (rieger 2000, rounsevell et al. 2005). currently, the restoration of grassland on former croplands is a high priority of nature conservation (stadler et al. 2007) and is one of the most frequent habitat restoration actions in central and northern europe (cramer et al. 2008). habitat (sensu 92/43/eec directive) protection entails regular monitoring, but before that, the target habitats must grassland recovery after long-term cropland abandonment acta bot. croat. 75 (2), 2016 227 be properly defi ned and their conservation status assessed (ejrnæs et al. 2004). however, annex i of the directive, building on an extensive classifi cation of habitats, fails to specify how to discriminate between protected and non protected habitats along the continuum from natural to cultural, and there is not much help in the scientifi c literature in this regard (ejrnæs et al. 2008). signifi cant contributions to solving this problem may be found in the phytosociological assessment of vegetation, since the phytosociological composition of a plant community (that is the presence of species belonging to different phytosociological units, such as classes, orders, etc.) reveals both its ecological and its dynamic conditions (biondi 2011). moreover, since the 92/43/ eec directive defi ned habitats in phytosociological terms, the phytosociological assessment of vegetation would be a logical tool for habitat interpretation. another useful approach could be species grouping by social behavior types (sbts) (moola and vasseur 2004, gondard et al. 2006). sbts derive from species behaviour and ecological attributes at a given observation level (borhidi 1995). social behaviour can be defi ned as the role that a plant species plays in the community considering species in regard to their auto-ecology, morphology and physiological performances (alard and poudevigne 2000). therefore, assessment of an sbt provides information on the mechanisms underlying species assemblage and helps to clarify the ecological meaning of the species pool characterizing a certain plant community (catorci et al. 2011a). since the higher levels of phytosociological classifi cation (class and order) group species that share wide ecological needs and dynamic features, they can be useful indicators of sbts (catorci et al. 2011a). in this way, it is possible to couple the phytosociological approach with the sbt assessment of a plant community, thus gaining a deeper understanding of its conservation status. we focused on semi-natural dry grasslands that had taken over abandoned croplands in the hilly landscape of the tuscan-emilian apennines, since this territory is undergoing strong processes of cropland abandonment and the reforestation of mountain slopes (piussi and pettenella 2000). in addition, only few studies have examined grassland recovery after fi eld abandonment in a sub-mediterranean climate in relation to conservation status as eu habitats. such an assessment of how biotic and abiotic features affect the species assemblage might also provide key information for managing these ecosystems in order to achieve a favorable conservation status. it has been argued that succession of abandoned fi elds leads to the formation of semi-natural communities whose fl oristic and structural features depend on such factors as resource availability (ejrnæs et al. 2008), time since abandonment, and availability of seeds in neighbouring habitats (pywell et al. 2002, ruprecht 2006). moreover, altitude and land form (burrascano et al. 2013), soil features (catorci and gatti 2010), land use history (catorci et al. 2011a) and disturbance type and intensity (peco et al. 2006, de bello et al. 2007, kramberger and kaligarič 2008, catorci et al. 2012, ribeiro et al. 2012) have proved to be crucial factors in determining grassland species composition. in particular, grazing animals facilitate the dispersal of seeds, create gaps for colonization (gibson and brown 1992, bruun and fritzbøger 2002) and foster species with resistance strategies to herbivory (grime 2001). thus, we expected that in former cropland (abandoned nearly 20 years ago and previously ploughed every year) dynamic processes would lead to the formation of herbaceous communities with different species composition depending on the different abiotic conditions, but these processes are not suffi cient per se to transform abandoned crops into semi-natural grassland communities included in annex i of the habitats directive. we also postulated that the absence of disturbance allows the spread of competitive tall grasses and shrubs and thus affects the species composition, hindering the achievement of a favourable conservation status. the specifi c research questions were: i) which environmental factors drive the species assemblage of plant communities after crop abandonment? ii) how do environmental features infl uence species composition of plant communities? iii) does the evaluation of the coenological composition of plant communities after crop abandonment provide insight signifi cant for the assessment of their dynamic state and conservation status according the standards defi ned by the 92/43/eec directive? materials and methods study area the study area (fig. 1) is located in the south-eastern and south-western part of bologna province (italy), between 200 and 500 m a.s.l. from a bioclimatic viewpoint, the area is included in the sub-mediterranean variant of the temperate continental and oceanic bioclimates (pesaresi et al. 2014). the mean annual temperature is 13–14 °c. the annual rainfall is 800–900 mm. a period of drought stress occurs in summer; july is the driest month (average rainfall 40–50 mm), and winter cold stress occurs between early december and late february. the geological substratum consists of a mosaic of clays with chaotic structure (debris fl ow – mudfl ow), stone material, conglomerates and gravel, fig. 1. location of the study area in the south-eastern and southwestern part of the bologna province (italy). troiani n., tardella f. m., malatesta l., corazza m., ferrari c., catorci a. 228 acta bot. croat. 75 (2), 2016 chalks, sandstones, marly, silty and fl aky clays. soils are generally deep (30–60 cm) with neutro-basic ph (6.5–7.5), mainly with sandy-loam and silty-clayey textures. the actual natural potential vegetation is referred to quercus pubescens s.l. woods of the peucedano cervariae-quercetum pubescentis association and hop-hornbeam woods of the ostryo-aceretum opulifolii (blasi 2010). the studied sites encompass former croplands (with wheat and alfalfa grown in rotation), where agricultural or pastoral practices have been absent at least for the last 20 years. all the study sites are characterized by herbaceous communities that are currently unmanaged. sampling design and data collection we collected data in june-july 2013 in order to observe both the spring and the summer fl owering species. using a gis, we overlaid the study area with a grid composed of 100 × 100 m macro-plots, each further divided into 100 plots (10 × 10 m). in each macro-plot we randomly selected one 10 × 10 m plot, excluding those that fell completely or partly on woods, brush and shrub vegetation, as well as those that fell in a buffer of 50 m from these vegetation types to avoid the edge effect. in all, we surveyed 100 plots. in each plot we recorded species cover values (percent values, visually estimated), altitude (m a.s.l.), slope aspect (azimuth degrees), slope angle (vertical degrees), outcropping rock cover and litter cover (percent values, visually estimated), soil depth (cm, fi ve measurements per plot) and collected fi ve soil sub-samples, taken within a depth of 30 cm and combined in one bulked sample. to better characterize the plant communities from an ecological viewpoint, we assessed the ellenberg indicator values (eivs). we gathered eivs from pignatti (2005) and guarino et al. (2012). eivs proved to be useful in analyzing the drivers of vegetation change (i.e. mccollin et al. 2000, klaus et al. 2012) especially when they are considered for comparison on a local scale (godefroid and dana 2007). in particular, we assessed air temperature (t), soil moisture (m), soil nutrient content (n), and soil chemical reaction (r). to assess the dynamic and conservation state of the plant communities, we grouped species in sbts. species sbts were assessed with respect to their regional synecology, following the most widely accepted phytosociological placement of each species (ubaldi 2013, biondi et al. 2014, biondi and pesaresi 2004, biondi et al. 2005, čarni 2005). we considered the following sbts: sbt1, species of perennial semi-natural grassland usually undergoing herbivory (characteristic of festuco-brometea br.-bl. et tx. 1943 class); sbt2, species of xeric grasslands (characteristic of tuberarietea guttatae br.-bl., roussine & nègre 1952 and sedoscleranthetea br.-bl. 1955 classes); sbt3, species of meadows (characteristic of molinio-arrhenatheretea tx. 1937 class) usually undergoing soil nitrogen enrichment and annual cutting; sbt4, species of fringe habitats (i.e. species usually growing at the border of woody ecosystems and of forest clearings, characteristic of trifolio-geranietea sanguinei müller 1962 class); sbt5, successional species (characteristic of rhamno-prunetea rivas goday & borja ex tüxen 1962, calluno-ulicetea br.-bl. & tüxen ex klika & hadac 1944 and querco-fagetea br.-bl. & vlieger in vlieger 1937 classes); and sbt6, pioneer, ruderal, nitrophilous and semi-nitrophilous species growing in cultivated and uncultivated lands (characteristic of artemisietea vulgaris lohmeyer, preising & tüxen ex von rochow 1951 and stellarietea mediae tüxen, lohmeyer & preising ex von rochow 1951 classes). in this way we were able to assess the dynamic status of the plant communities (considering for instance sbts 1, 2, and 3 versus sbts 4 and 5) or the management-related impact on species composition (i.e. by assessing the abundance of sbts 5 and 6), besides the relationship between environmental constraints and species composition. to assess the state of conservation of plant communities according the standards defi ned by the 92/43/eec directive, we consulted as a reference manual the »italian interpretation manual of habitats of the 92/43/ eec directive« (biondi et al. 2009). species nomenclature followed conti et al. (2005). data analysis aspect azimuth was fi rst converted from the 0–360 compass scale to a linear (0–180) scale, giving northerly aspect a value approaching 0 and southerly aspect a value approaching 180. this transformation also converted east and west azimuth degrees so that they were equally distant from north. moreover, as south-south-west-facing slopes are the warmest aspect, the aspect azimuth was shifted to a minimum on north-north-east slopes (22.5°) and a maximum on south-south-west slopes (202.5°). data on soil depth were averaged for each plot. soil samples were analysed in the laboratory of the university of bologna to determine the percentages of skeleton, sand, loam, and clay. in order to have a proxy for the soil water regimes, for each plot we calculated the soil available water capacity (awc) using the software developed in microsoft offi ce excel 2000 by armiraglio et al. (2003). awc represents the maximum amount of available water the soil can provide. it mainly depends on soil texture and depth and, secondly, on soil specifi c weight and organic matter content (mcrae 1991). the input variables processed were soil depth (cm), soil texture (percentage of sand, clay, loam, and percentage of skeleton), and a coeffi cient of water available for plants under a pressure of 0.05–15.00 bars, obtained from tabulated values (mcrae 1991). even though the outputs obtained are imprecise because they lack data on organic matter, nonetheless they offer a good proxy for comparing plots, because the estimation was based on soil depth and texture, and thus was independent of vegetation structure (i.e. vegetation cover, tiller density, canopy height, litter decomposition rate, etc.), which might alter the soil organic matter content. to transform sbt binary data (presence/absence) into quantitative data (i.e. aggregated cover values of each sbt), we multiplied the »relevés × species cover« matrix by the »species × sbt« matrix to provide a »relevés × sbt cover« matrix, which formed the basis for the following analyses. grassland recovery after long-term cropland abandonment acta bot. croat. 75 (2), 2016 229 i) environmental features driving the species assemblage after cropland abandonment to assess the infl uence of macro-environmental variables on species assemblage, a constrained clustering using multivariate regression tree (mrt) analysis (de’ath 2002) with 100 iterations was executed on the »relevés × species« matrix constrained by the environmental variables (altitude, slope aspect, slope angle, awc, outcropping rock cover, and litter cover). prior to mrt analysis, species data were transformed using chord transformation (legendre and gallagher 2001). an overall error statistic (cross-validation relative error, cvre) was computed for each test group and partition size (number of groups), to choose the optimal size of the tree. for each partition size, the mean and standard error of all cvre estimates were computed. to provide a general overview of the ecological characteristics of the plant communities highlighted by mrt, we calculated mean, standard deviation, median, and 1st and 3rd quartiles for each environmental variable. we used the shapiro-wilk test to test the distribution normality of data. as data were not normally distributed, we ran the non-parametric wilcoxon exact rank test to evaluate differences between groups at each step of clustering. mean species cover values and species relative frequency in each group are reported in the on-line suppl. tab. 1. to further assess the environmental features characterizing the groups of relevés emerging from mrt analysis, we first calculated the unweighted mean of t, m, n and r eivs of the species present within each plot. following zelený and schaffers (2012), we fi tted mean eivs, calculated for samples, onto a detrended correspondence analysis (dca) ordination employing a permutation test that corrects the bias in the relationship between mean species eivs and sample scores along ordination axes, introduced by the fact that mean eiv inherits compositional similarity among samples. finally, we calculated the mean and median eiv for each group of relevés. to perform mrt analysis, shapiro-wilk tests, wilcoxon exact rank tests, and dca we used the r software (version 2.15.2, r core team 2012) packages mvpart version 1.6-1 (mvpart function), stats version 2.15.2 (shapiro.test function), exactranktests version 0.8-27 (wilcox.exact function), and vegan version 2.0-10 (decorana function), respectively. the r function used for projection of mean eivs onto an ordination, with modifi ed permutation test was envfi t.iv (zelený and schaffers 2012). ii) how do environmental features infl uence species and coenological composition of plant communities? to understand how abiotic factors infl uence the species composition of plant communities, we performed indicator species analysis (isa) to highlight the indicator species set (i.e. the species pool showing a preferential distribution among clusters) of each group (dufrêne and legendre 1997). to identify the indicator sbts (aggregated cover values of species sharing the same sbt) for each group identifi ed by mrt, we executed isa on the »relevés × sbt cover« matrix using membership of mrt groups as grouping variable. to perform isa we used labdsv version 1.6-1 (indval function) r-package. finally, to better assess the status of grassland communities, we calculated the relative abundance of each sbt in every plant community considered (cover percent value of the considered sbt out of the total cover value of all the recorded sbts in the plant community). results i) environmental features driving the species assemblage after cropland abandonment mrt analysis generated a 3-leaved mrt with cvre = 0.88 and standard error = 0.056 (fig. 2), which was frequently selected as the best solution during the cross-validation iterations. the basic descriptive statistics of the environmental variables for each group highlighted by mrt and the statistical signifi cance of differences between groups at each partition are shown in tab. 1. the variable discriminating between the two branches in the fi rst node of the 3-leaved mrt was rockiness. the relevés of the third leaf (t3.3) were characterized by the presence of outcropping rocks, while in the other groups rockiness was absent. moreover, this group had lower awc mean values and higher litter cover and altitude mean values than the group composed of t3.1 and t3.2. differences between the two groups with regard to these variables were statistically signifi cant (tab. 1). the second partition was defi ned by altitude (fig. 2). group t3.1 included relevés located at altitudes higher than 290 m a.s.l. (mean value 418 m), with low fig. 2. the 3-leaved multivariate regression tree for the species data set, constrained by the explanatory variables data set. rockiness (%) and altitude (m a.s.l.) discriminated the two branches of the tree. the threshold values shown for each partition of the tree correspond to the mean of the two limit values of the considered variables at the break between the branches. the relative abundances of the species are shown in histograms at the tips of the branches, with the species in the same order as in the input fi le. under each histogram, the sum of squared errors for the group and the number of relevés in the leaf (n) are indicated. below the tree, the residual error (error), the cross-validation error (cv error) and standard error (se) are indicated. troiani n., tardella f. m., malatesta l., corazza m., ferrari c., catorci a. 230 acta bot. croat. 75 (2), 2016 slope angles, while altitudes lower than 290 m, slightly higher awc values and lower litter covers characterized relevés belonging to group t3.2 (tab. 1). the fi rst two axes of dca based on the »relevés × species« matrix using mean randomized eiv values as constraining variables explained 77% of the constrained variance (52% explained by axis 1 and 25% by axis 2). soil chemical reaction was strictly related to axis 1, while temperature was related to both axes. the combination of these two variables explained the separation of the three groups of plots, as indicated by the dca scatterplot (fig. 3). the differences in mean and median values of eivs are in line with dca results (tab. 2). the t3.1 group was positively correlated with eiv soil reaction (i.e. with soils with higher ph), and characterized by a mean value greater than groups t3.2 and t3.3. the t3.2 group was positively correlated with eiv air temperature and characterized by higher mean t value than the other groups. moisture and nutrient values showed very small differences among the three communities (tab. 2). ii) how do environmental features infl uence species and coenological composition of plant communities? we recorded 209 species in the total set of relevés. the indicator species identifi ed by isa for groups highlighted by mrt analysis, as well as their mean abundances, are shown in tab. 3. the fi rst and second groups of the 3-leaved mrt included ten indicator species each; instead, the third was characterized by 17 indicator species. bromus erectus huds. dominated group t3.1, which also showed a high cover value of brachypodium rupestre (host) roem. & tab. 1. descriptive statistics of the environmental variables for the groups highlighted by the multivariate regression tree analysis (t3.1, t3.2, and t3.3) and statistical signifi cance of differences, as indicated by the results of wilcoxon exact rank test (2-tailed), between the groups segregated in the 3-leaved multivariate regression tree. aspect azimuth was converted from the 0–360 compass scale to a linear scale, giving northerly aspect a value approaching 0 and southerly aspect a value approaching 180; then it was shifted to a minimum on north-north-east slopes (22.5°) and a maximum on south-south-west slopes (202.5°). sd – standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. – not signifi cant. group statistics altitude (m a.s.l.) slope aspect (azimuth degree) slope angle (vertical degree) rockiness (%) litter (%) available water capacity (mm) t3.1 mean 418.17 129.00 14.40 0.00 24.30 108.80 sd 56.50 46.80 6.46 0.00 32.60 4.06 1st quartile 384.00 67.50 10.00 0.00 0.00 105.92 median 430.00 157.50 15.00 0.00 10.00 107.96 3rd quartile 460.00 157.50 20.00 0.00 30.00 110.00 t3.2 mean 205.80 155.25 18.00 0.00 8.00 109.00 sd 43.30 7.10 7.80 0.00 16.80 0.55 1st quartile 201.00 157.50 15.00 0.00 0.00 107.60 median 201.00 157.50 15.00 0.00 0.00 108.70 3rd quartile 238.50 157.50 25.00 0.00 0.00 108.80 t3.3 mean 474.70 143.00 19.90 14.40 53.10 104.07 sd 56.20 24.05 6.70 14.48 28.02 6.10 1st quartile 454.00 112.50 15.00 5.00 30.00 100.96 median 494.00 157.50 20.00 10.00 60.00 105.24 3rd quartile 513.00 157.50 25.00 20.00 80.00 108.80 t3.1–t3.2 vs. t3.3 *** *** n.s. *** *** *** t3.1 vs. t3.2 *** *** n.s. n.s. *** ** fig. 3. scatterplot of the detrended correspondence analysis (dca) ordination executed on mean randomized ellenberg indicator values and relevés data set. sites are represented with different symbols to highlight the groups identifi ed by multivariate regression tree analysis (circles – group t3.1; triangles – group t3.2; crosses – group t3.3). only signifi cant vectors (p < 0.05) have been represented (t – air temperature; r – soil chemical reaction). grassland recovery after long-term cropland abandonment acta bot. croat. 75 (2), 2016 231 schult. the t3.2 group had higher values of avena sterilis l. and triticum ovatum (l.) raspail, while bromus erectus and sulla coronaria (l.) medik. dominated group t3.3 (tab. 3, on-line suppl. tab. 1). isa identifi ed several coenological differences among groups (tab. 4). sbt1, 3, 4, and 5 were indicators of the t3.1 community. sbt1, 2, and 6 were indicators of t3.2 community. sbt6 was indicator of t3.3 community (tab. 4). the abundance (cover percentage) of each sbt with respect to the total cover value in the three groups is shown in tab. 5. discussion i) plant community composition and site ecology mrt analysis highlighted three main plant communities, linked to different environmental conditions (fig. 2, tabs. 1–2). this is consistent with previous research, which showed that in the sub-mediterranean climate, landforms are key factors in determining plant species and communities distribution (arévalo et al. 2012, burrascano et al. 2013, catorci et al. 2014b). in fact, sub-mediterranean regions are characterised by summer drought stress with different intensities, depending on the elevation and landform factors (somot et al. 2008). the main ecological factor behind these variables is the total solar radiation amount per unit area (biondi et al. 2011). on south-facing slopes, the greater radiation in summer dramatically reduces the soil water content (joffre and rambal 1993), posing one more stress factor faced by plants in mountain areas (catorci et al. 2014b). landforms affect soil features (i.e. soil depth) as well (cerdà et al. 1995). in detail, the t3.1 cluster grouped plots located on moderately steep slopes with southerly aspects (from east to west) (tab. 1) and is dominated by competitive caespitose species, i.e. bromus erectus (occurring in about 96% of plots with mean cover of 40.4%) and brachypodium rupestre (occurring in 77.8% of plots with mean cover of about 23%). this community spreads in conditions that are not very dry, as highlighted by the lowest temperature eiv and the slightly higher awc values (tabs. 1–2). it is also noteworthy that the t3.1 group was positively correlated with soil reaction eiv and characterized by a mean value greater than the t3.2 and t3.3 groups, namely by the most basic ph among the considered clusters (tab. 2, fig. 3). in accordance with catorci and gatti (2010), these conditions allow the spread of species of sbt1 and sbt3 (tab. 4), indicating the affi nity of these plant communities with the sub-mediterranean grasslands of the festab. 2. mean and median ellenberg indicator values (eivs) for air temperature, soil moisture, soil reaction, and soil nutrients of each group (t3.1, t3.2, and t3.3) emerging from the multivariate regression tree analysis. ellenberg indicator values group t3.1 t3.2 t3.3 mean median mean median mean median air temperature 5.98 5.68 7.72 7.67 6.92 6.93 soil moisture 3.58 3.44 3.45 3.56 3.47 3.46 soil reaction 7.05 7.28 5.64 5.67 5.68 5.75 soil nutrients 3.75 3.68 3.73 3.62 3.54 3.52 tab. 3. indicator species of the relevé groups highlighted by indicator species analysis for the 3-leaved multivariate regression tree and their respective indicator values (calculated by multiplying the relative abundance and the relative frequency of species in a group) and mean cover percentages, as highlighted by the indicator species analysis. only signifi cant indicator values (p < 0.05) higher than 30 are shown. max group: group with maximum indicator value; p: proportion of indicator values obtained by randomized trials, equal to or exceeding the observed indicator value. the statistical signifi cance (p) of the observed maximum indicator values was tested using permutation tests with 4,999 iterations. max group indicator species indicator value p mean cover (%) t3.1 brachypodium rupestre 0.66 0.00 22.9 achillea collina 0.53 0.00 1.0 cota tinctoria 0.49 0.01 1.5 bromus erectus 0.44 0.01 40.4 vicia sativa 0.39 0.01 0.5 l otus corniculatus 0.35 0.01 0.6 fraxinus ornus 0.35 0.01 3.5 rubus ulmifolius 0.34 0.00 1.9 securigera varia 0.33 0.01 0.3 inula salicina 0.32 0.01 0.9 t3.2 triticum ovatum 0.77 0.00 14.9 avena sterilis 0.64 0.00 32.1 scorpiurus subvillosus 0.47 0.00 2.7 hippocrepis comosa 0.46 0.00 0.5 calamintha nepeta 0.41 0.01 2.2 gladiolus italicus 0.38 0.00 0.1 potentilla recta 0.38 0.00 0.3 phalaris canariensis 0.35 0.00 0.4 trifolium campestre 0.35 0.04 1.5 crepis pulchra 0.30 0.02 0.3 t3.3 linum bienne 0.85 0.00 3.7 hypochaeris achyrophorus 0.74 0.00 11.5 sonchus asper 0.68 0.00 0.7 picris hieracioides 0.67 0.00 0.7 allium schoenoprasum 0.65 0.00 0.4 sulla coronaria 0.63 0.00 19.0 potentilla hirta 0.54 0.00 0.6 daucus carota 0.50 0.00 0.9 althaea hirsuta 0.49 0.00 0.7 geranium dissectum 0.44 0.01 1.2 bartsia trixago 0.40 0.01 0.4 genista tinctoria 0.40 0.00 1.1 sonchus oleraceus 0.40 0.00 0.5 thymus longicaulis 0.39 0.02 0.7 ononis masquilleri 0.36 0.01 1.6 crepis vesicaria 0.33 0.02 0.3 filago pyramidata 0.31 0.03 0.4 troiani n., tardella f. m., malatesta l., corazza m., ferrari c., catorci a. 232 acta bot. croat. 75 (2), 2016 tuco-brometea class (see biondi and galdenzi 2012), referred to the habitat »semi-natural dry grasslands and scrubland facies on calcareous substrates (festuco-brometalia)« (habitat code 6210, important orchid sites)]. according to the italian interpretation manual of the 92/43/eec directive habitats (biondi et al. 2009), this habitat includes perennial species-rich secondary grasslands dominated by hemicryptophytic grasses, from xeric to semi-mesophylous, mainly spread along the apennine ridge. in the study case, however, as postulated by grime (2001), the absence of disturbance allows a relatively high litter cover value (tab. 1). likewise, as stated by bonanomi et al. (2013), the lack of disturbance enhances the spread of b. rupestre (tab. 3, online suppl. tab. 1). this species is a dominant tall grass whose competitive success is related to its high tiller density and branching frequency (pottier and evette 2011), as well as to its capacity for clonal growth and clonal integration strategy (de kroon and bobbink 1997). these traits allow a rapid spread in the absence of disturbance, especially in productive conditions (grime 2001). the invasion of brachypodium species has a strong impact on the species composition of plant communities (catorci et al. 2011a, b) and gives rise to the spread of fringe and successional species (catorci et al. 2011a). actually, sbt4 (species of fringe habitat) and sbt5 (successional species) are indicators of the t3.1 group (tab. 4). vegetation of the t3.1 community is also undergoing dynamic processes, as indicated by the presence among the indicator species of successional species such as fraxinus ornus l. and rubus ulmifolius schott (tab. 3), potentially leading to the formation of woody communities and to the loss of open habitats. plots of the t3.2 group are located at the lowest altitudes and on markedly south-facing slopes (tab. 1). the most abundant and frequent species are avena sterilis (mean cover 32.1%, occurring in 90.0% of plots), triticum ovatum (mean cover 14.9%, in 90.0% of plots), and b. erectus (mean cover 12.6%, in 80.0% of plots) (see on-line suppl. tab. 1). this community was positively correlated with temperature eiv and showed its highest mean value (tab. 2). these harsh conditions may explain why sbt2 (species of xeric, grasslands) emerged as indicators. the indication of sbt6 (ruderal species) as well as their great cover values (tab. 5) is consistent with the indication by isa (tab. 4) of several species of croplands and abandoned fi elds (e.g. t. ovatum, phalaris canariensis l., a. sterilis, crepis pulchra l., and scorpiurus subvillosus l.). it indicates the incomplete evolution of the considered communities towards the kind of semi-natural grasslands indicated in the eu directive. in particular, this herbaceous community seems to have a potential affi nity for the *6220 habitat [pseudo-steppe with grasses and annuals (thero-brachypodietea) because it hosts several species of the tuberarietea guttatae class, even if the total cover value of sbt2 is not very high. the manual of interpretation of eu habitats includes in this habitat mediterranean and sub-mediterranean xerophilous, mostly open, short-grass annual grasslands rich in therophytes and therophyte communities of oligotrophic soils on base-rich, often calcareous substrates. according to the italian interpretation manual of the 92/43/eec directive (biondi et al. 2009), this habitat encompasses vegetation types that differ considerably in physiognomy, fl oristic composition, ecological and structural features, sometimes very valuable from a naturalistic viewpoint, but more often trivial and very common in mediterranean sectors of italy. the t3.3 cluster includes undisturbed plots of the steepest hilly southerly slopes, characterized by the highest rockiness and litter cover, and the lowest awc values (tab. 1), as well as by low soil moisture eivs. the most abundant and frequent species were bromus erectus (mean cover 25.9%, occurring in 97.8% of relevés), sulla coronaria (mean cover 19.5% in 95.6% of relevés), and hypochaeris achyrophorus l. (mean cover 11.8% in 93.3% of relevés). the xerophilous group of indicator species such as filago pyramidata l., h. achyrophorus, thymus longicaulis c. presl, and potentilla hirta l. (tab. 3) suggests the presence of patches with shallow soils. on the other hand, the indicatab. 4. indicator social behaviour types (sbts) of the relevés groups (t3.1, t3.2, and t3.3) calculated by multiplying the relative abundance and the relative frequency of sbts in a group, highlighted by indicator species analysis for the 3-leaved multivariate regression tree. only signifi cant (p < 0.05) indicator values higher than 30 are shown. sbt1 – species characteristic of perennial pastures; sbt2 – species of xeric grasslands; sbt3 – species of meadows; sbt4 – species of fringe habitats; sbt5 – successional species; sbt6 – ruderal species. max group: group with maximum indicator value; p – proportion of indicator values obtained by randomized trials, equal to or exceeding the observed indicator value. the statistical signifi cance of the observed maximum indicator values was tested using permutation tests with 4,999 iterations. social behaviour type max group indicator value p sbt1 t3.1 0.546 0.000 t3.2 0.471 0.002 sbt2 t3.2 0.540 0.003 sbt3 t3.1 0.500 0.006 sbt4 t3.1 0.390 0.012 sbt5 t3.1 0.342 0.004 sbt6 t3.2 0.729 0.000 t3.3 0.497 0.015 tab. 5. percent abundance of each social behavior type (sbt) in each group of relevés (t3.1, t3.2, t3.3) highlighted by the multivariate regression tree analysis. sbt1 – species characteristic of perennial pastures; sbt2 – species of xeric grasslands; sbt3 – species of meadows; sbt4 – species of fringe habitats; sbt5 – successional species; sbt6 – ruderal species. social behaviour type group t3.1 t3.2 t3.3 sbt1 62.7 18.6 28.3 sbt2 6.3 20.7 16.7 sbt3 3.7 1.9 1.4 sbt4 1.4 0.7 0.6 sbt5 10.2 6.2 5.4 sbt6 15.7 51.9 47.6 grassland recovery after long-term cropland abandonment acta bot. croat. 75 (2), 2016 233 tion by isa of sulla coronaria, sonchus asper (l.) hill, s. oleraceus l., geranium dissectum l., crepis vesicaria l., daucus carota l., as well as of sbt6 (pioneer, ruderal, nitrophilous and semi-nitrophilous species) (tabs. 3–4), highlights both the past use of these areas as croplands and the absence of disturbance, suggested also by the highest litter cover value (grime 2001). in fact, the abundance of these species is reduced by both grazing and mowing (grime 2001). moreover, litter can infl uence the processes of species assemblage within plant communities (catorci et al. 2011b), reducing seed germination, establishment of individuals (eriksson 1995) and growth of established plants (facelli and pickett 1991). on the other hand, the low cover value of dominant tall grasses (tab. 3) such as brachypodium rupestre, usually fostered by abandonment, may be due to the harsh condition of sites (vitasović kosić et al. 2011). the spread of geophytes, such as orchids (i.e. anacamptis pyramidalis (l.) rich., orchis morio l., o. coriophora l.), as well as the relatively high cover value of sbt1 (28%) and of sbt2 (17%) that are typical of semi-natural dry grasslands belonging to the scorzonero-chrysopogonetalia (brometalia erecti) order and the festuco-brometea class (biondi and galdenzi 2012), suggests the existence of ecological conditions that potentially could lead to xeric bromus erectus-dominated community (which could be referred to the 6210 habitat). finally, the not negligible cover value of successional species (sbt5 – 5–6%) in the t3.2 and t3.3 groups indicates the presence of dynamic processes leading to forest recovery and, therefore, the future loss of these open habitats. ii) conservation status of plant communities and management implications the three communities highlighted by mrt analysis showed a different contribution of sbts to the species pool (tab. 5). it also emerged that after fi eld abandonment, different environmental conditions drive the dynamic processes towards herbaceous communities that are likely to develop into eu habitats. however, in only one community (t3.1) did we observe a fl oristic composition clearly dominated by species of permanent pastures (namely, sbt1) as happens in the semi-natural dry grasslands of the italian peninsula. in fact, in these communities the average cover value of festuco-brometea and/or molinio-arrhenatheretea species generally exceeds 70% (see biondi et al. 2005). in the other groups (t3.2 and t3.3), species of semi-natural grasslands had a lower mean cover value (ranging from 41 to 46%), with a conspicuous contribution (17–21%) to the total abundance of species belonging to tuberarietea guttatae and sedo-scleranthetea classes, especially in cluster t3.2, while species of artemisietea vulgaris and stellarietea mediae classes had a cover ranging from 48 to 52%. as a matter of fact, only grasslands of the t3.1 and t3.3 groups seem to have the potential to develop into dry grasslands belonging to the festuco-brometea class (6210 habitat), with inclusions, in grasslands belonging to the t3.3 group, of patches of the tuberarietea guttatae class (6220 habitat). however, an open issue is the assessment of their degree of representativeness in comparison with the standards defi ned by the italian manual of interpretation of eu habitats (biondi et al. 2009). in fact, the studied grasslands spread on non-calcareous substrates instead of on limestone. on the other hand, it should be considered that ph values are quite neutro-basic, as indicated by the manual of interpretation.with regard to the plant community of the t3.2 group, it is diffi cult to evaluate its representativeness with respect to aspects of greater conservation interest of the 6220 habitat. we found that the abundance of sbt 4, 5 and 6 (encompassing species of fringe habitat, successional species, and pioneer, ruderal, nitrophilous and semi-nitrophilous species, respectively) is still high in any condition. time after abandonment is considered a key issue in recovery of seminatural grassland (pywell et al. 2002, ruprecht 2006), but probably these species (generally tall plants with ruderallike ecological needs) are also able to persist in abandoned fi elds due to the absence of disturbance (grime 2001).this means that the herbaceous plant communities considered fail to meet the fl oristic standards needed to ascribe them to the habitat sensu the 92/43/eec directive. because of this, we can argue that the achievement of a favourable conservation status requires careful and deliberate management plans. in particular, sheep or cows should graze on grasslands belonging to the t3.3 group during the late spring and summer periods to reduce the abundance of dominant tall plants and enhance that of species with avoidance and tolerance strategies, which are species of the festuco-brometea class (grime 2001). sheep, in particular, are known as dispersal vectors for most of the species of the festuco-brometea class (fischer et al. 1995), and thus they can facilitate the processes related to species spread in managed grasslands (kaligarič et al. 2006). grasslands of the t3.1 and t3.2 groups should be mown once a year in early summer, since late mowing allows the conservation of the maximum species richness (catorci et al. 2014a). moreover, fresh phytomass should be removed to avoid litter deposition and summer grazing can be useful to achieve a higher level of representativeness. in conclusion, we found that the environmental factors driving the species assemblage in the sub-mediterranean hilly landscape of the tuscan-emilian apennines after crop abandonment are linked to topographic (rockiness and altitude) and soil (ph and awc) conditions, which mainly determine the intensity and duration of water scarcity. moreover, our results indicated that not all former croplands, notwithstanding their having undergone the same land use history, have the potential to achieve a high representative status of the eu directive habitats (sometime because of unsuitable soil ph) and that long-term abandonment of croplands does not lead per se to the recovery of the seminatural herb communities of the eu habitats directive in sub-mediterranean climate. in line with smith et al. (2000), the main reason of such a result may be found in the lack of appropriate disturbance regimes. the absence of disturbance has a further double effect. it allows the spread of woody successional species (shrubs and pioneer trees) and thus leads to forest recovery as well as the permanence of plants with ruderal-like ecological needs. likewise, the absence of disturbance allows the spread of competitive species which, in turn, reduces the site suitability for non-dominant herbaceous plants (such as small-sized and early fl owering species – grime 2001), which are key elements troiani n., tardella f. m., malatesta l., corazza m., ferrari c., catorci a. 234 acta bot. croat. 75 (2), 2016 of the grasslands referred to the 6210 priority habitat protected by the 92/43/eec directive. moreover, the spread of competitive grasses (i.e. brachypodium rupestre) fosters the presence of elements, such as fringe species, that are not characteristic of semi-natural disturbed grasslands (catorci et 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via mattioli 4, i-53100, siena, italy 2 department of physical, earth and environmental sciences, university of siena; strada laterina 8, i-53100, siena, italy abstract – in this study we investigated the seasonal variations in the intracellular content of 14 trace elements (al, as, ba, cd, ce, cr, cu, fe, mn, ni, pb, pd, sb, zn) and physiological parameters (namely chlorophyll a, chlorophyll b, ergosterol, photosynthetic efficiency, cell membrane integrity) in the thalli of the lichen evernia prunastri (l.) ach. exposed to an urban environment (siena, central italy). lichen thalli were collected before each exposure period from an unpolluted area and transplanted to 16 sites; every 3 months the thalli were retrieved and replaced with new ones. exposed-to-control ratios of trace elements revealed a marked intracellular accumulation of cd in summer and autumn, and of sb in autumn and spring, possibly as a result of vehicular traffic pollution. however, considering the low absolute concentrations of these elements, the intracellular fraction of depositions may hardly have caused an impairment of physiological parameters. as a matter of fact, indicators of photobiont vitality (content of chlorophylls a and b and photosynthetic efficiency) did not show any fluctuation across seasons, while changes in the indicators of mycobiont vitality (cell membrane damage and ergosterol content) overall did reflect some seasonal changes and/or lichen growth. keywords: air pollution, antimony, bioaccumulation, biomonitoring, cadmium, epiphytes, heavy metals * corresponding author, e-mail: andrea.vannini@unisi.it introduction it is generally accepted that elements accumulated in lichen thalli reflect deposition of elements from the atmosphere. lacking specialized structures, with metabolism depending on mineral uptake from the atmosphere, lichens are very efficient in trapping trace elements from the surrounding environment, tending to reach an equilibrium between the elemental content in the thallus and the respective environmental levels (loppi and paoli 2015). basically lichens have three uptake mechanisms: 1) particulate trapping onto the thallus surface and/or within intercellular spaces, 2) extracellular cation exchange, and 3) intracellular accumulation through selective uptake mechanisms (bačkor and loppi 2009). element uptake starts with wet or dry depositions on the thallus surface and may continue with the transfer into the thalli according to their chemical binding affinities for the cell wall and plasma membrane components (bačkor and loppi 2009). thus, airborne trace elements mainly occur in particulate material deposited onto the lichen surface or between intercellular spaces; many cations can be trapped by reversible binding to negatively charged anionic sites in the cell wall or the outer layer of the plasma membrane (bačkor and loppi 2009). these two fractions represent the extracellular components of the trace element depositions in lichen thalli. finally, cations may enter and accumulate inside mycobiont and photobiont cells through energy-dependent and plasma membrane controlled systems (bačkor and loppi 2009). this fraction represents the intracellular component originating from trace element depositions. with the transplant technique, healthy lichen thalli are taken from a relatively clean site, transplanted to the study site(s) and their response recorded (bargagli and mikhailova 2002). at polluted sites lichens will adapt their metabolism according to the new environment and accumulate airborne elements to levels far above their metabolic requirements (garty 1993). however, exposure to a polluted environment can lead to physiological damage to sensitive species as a result of vannini a., paoli l., nicolardi v., antonello di lella l., loppi s. 172 acta bot. croat. 76 (2), 2017 pollution intensity, and hence of elemental saturation (bergamaschi et al. 2007, bačkor and loppi 2009, maslać et al. 2016). according to godinho et al. (2008), the elemental content of lichen transplants does not unequivocally represent the average or cumulative environmental availability during the exposure period, because it is also dependent on the physiological status of the samples. the same authors suggested that by the introduction of a physiological parameter into a mathematical model, good correlations between total lichen elemental contents and bulk deposition may be obtained. nevertheless, it should be borne in mind that in terms of pollutants, physiological alterations are mainly caused by the intracellular fractions of these elements (branquinho et al. 1999). in addition, elemental content and physiological parameters may strongly vary across seasons, due to changes in both chemical and meteorological conditions in the environment to which the lichens are exposed (malaspina et al. 2014). field applications of lichens as bioaccumulators of trace elements have mainly focused on total concentrations, generally disregarding element localization at extracellular or intracellular level. this latter point is very important, since the intracellular fraction of depositions is responsible for the toxic effects at physiological level, and may thus in turn influence total concentrations. the purpose of this study was to investigate the seasonal variation of the intracellular content of selected trace elements in lichen thalli and its interaction with selected physiological parameters, taken as markers of photobiont and mycobiont vitality. materials and methods experimental design the lichen evernia prunastri (l.) ach. is a fruticose (shrub-like) species with high surface-volume ratio and is commonly used in biomonitoring studies of trace element depositions (ayrault et al. 2007, loppi et al. 1998). from june 2012 to june 2013, every 3 months, thalli were collected and exposed for 3 months at ca 2 m above ground at 16 sites within the urban area of siena (tuscany, italy). ten thalli were taken before each exposure and used as control (pre-exposure). siena (55,000 inhabitants) is located at an elevation of 322 m a.s.l., the climate is humid sub-mediterranean with an average annual temperature of 13.9 °c and an annual rainfall of about 850 mm. lacking industrial activities, the urban area is characterized by limited air contamination, and the main source of atmospheric pollution is from vehicular traffic (loppi et al. 2002), that is maximal in spring and summer due to massive tourism. counting workers and tourists, the daily traffic flow in spring is about 26,000 cars: 17,000 enter the town in the morning and 9,000 exit (data of the municipality of siena). meteorological and environmental data for each season during the experiment are summarized in table 1. trace elements lichen samples were cleaned with nylon tweezers under a binocular microscope to remove extraneous material. samples were then washed with deionized water to remove elements simply deposited as particles and then washed with na2-edta-0.2 m to remove the fraction accumulated extracellularly (branquinho and brown 1994). air dried samples were then homogenized under liquid nitrogen with a ceramic mortar and pestle. about 300 mg of the homogenized material were mineralized with a 6:1 v/v mixture of concentrated hno3, h2o2 and hf at 280 °c and pressure of 55 bars, in a microwave digestion system (milestone ethos 900). intracellular trace element (al, as, ba, cd, ce, cr, cu, fe, mn, ni, pb, pd, sb, zn) concentrations were determined by inductively coupled plasma mass spectrometry (icpms, perkinelmer sciex 6100) and expressed on a dry weight basis. analytical quality was checked by the standard reference material iaea-336 ‘lichen’. precision of analysis was estimated by the variation coefficient of four replicates and was found to be within 10% for all elements. photosynthetic pigments about 20 mg of lichen material was dissolved in 1 ml of dimethylformamide and homogenized with ultraturrax for 1 minute. the homogenate was centrifuged at 10,000 rcf for 10 minutes and 50 µl of the resulting supernatant was analysed by high performance liquid chromatography (hplc) (waters lc i plus) using an ascentis supelco c18 column (250×4.6 mm, porosity 5 μm). concentrations of photosynthetic pigments were determined according to suzuki et al. (1993), using water/methanol/acetone as mobile phase with a flow rate of 1 ml/minute. runs were monitored at 440 nm. quantification of chlorophyll a and chlorophyll b was performed using calibration curves of standards from sigma-aldrich (usa). three replicates were measured for each sample. photosynthetic efficiency chlorophyll a fluorescence emission was analysed to assess the quantum efficiency of open photosystem ii (psii) reaction centres, a measure of photosynthetic efficiency tab. 1. meteorological and pollution data within the urban area of siena (tuscany, italy) during the study period (june 2012 – june 2013). pm10 – particulated matter less than 10 µm; no2 – nitrogen dioxide; sd – standard deviation. meteorological parameters summer autumn winter spring rainfall (mm) 206 424 475 247 number of rainy days 7 38 36 47 number of stormy days 4 12 7 8 mean relative humidity (%) 53 76 76 65 mean temperature (°c) 23 10 7 17 average (±sd) pm10 (µg m–3) 30±9 29±10 34±11 29±8 average (±sd) no2 (µg m–3) 35±19 32±17 37±18 30±19 evernia prunastri and heavy metals acta bot. croat. 76 (2), 2017 173 (maxwell and johnson 2000), using the physiological indicator fv/fm. prior to the measurements, lichen samples were fully hydrated and maintained in a dark chamber (20 °c, relative humidity (rh) 99%) for 24 hours. after the hydration, samples were dark-adapted for 30 minutes and then exposed to light for 1 second with a saturating light pulse (3000 µmol m–2 s–1). integrity of cell membranes the plasma membrane integrity can be easily evaluated by placing a piece of lichen thallus in deionized water and measuring the variation in electric conductivity (marques et al. 2005). prior to the measurement, lichen thalli were placed in a humid chamber (rh>90%) for 24 hours to stabilize electrolyte leakage (buck and brown 1979). about 50 mg of lichen material was rinsed 3 times for 5 seconds in deionized water to remove particles deposited onto the lichen surface (garty 1993). samples were then soaked for 1 hour in 50 ml of deionized water and thereafter fully hydrated thalli were placed overnight at –80 °c to generate mechanical breakage of all cell membranes, in order to have a theoretical maximum cell membrane damage. samples were again soaked in 50 ml of deionized water and the final results were expressed as relative electric conductivity. ergosterol about 20 mg of lichen material was homogenized in 1 ml of absolute ethanol with ultraturrax for 2 minutes and then centrifuged at 10,000 rcf for 20 minutes. 200 μl of the supernatant was added to 1 ml of a 1 m naoh solution in 90% methanol and 10% of water and then placed in a thermostatic cell at 37 °c for 2 hours under stirring. for each sample, 2 ml of n-hexane was added to extract ergosterol and the supernatant was processed in a thermostatic centrifuge at room temperature to promote the volatilization of n-hexane. the dried product was then resuspended in 1 ml of absolute ethanol for about 2 minutes in a vortex, to promote the complete resuspension of ergosterol. samples were analysed by hplc (agilent 1100 series). ergosterol was separated with an agilent c18 column (25×4.6 mm, porosity 5 µm) using methanol as mobile phase at the flow rate of 1 ml/minute and then read at 280 nm (dahlman et al. 2002). the calibration curve was prepared with standard solutions of ergosterol (sigma-aldrich, 96%) from 1 to 20 µg/ml in absolute ethanol (hplc grade). statistical analysis since the intracellular content of some elements also fluctuated in pre-exposure control samples, for data interpretation the data were normalized by calculating the ratio between the values after each exposure and that prior to each exposure (exposed-to-control ratio, ec ratio) as suggested by frati et al. (2005). a one-way anova was run to check for differences in physiological parameters across seasons, using the tukey test (p<0.05) for post hoc comparisons. correlation analysis was used to find significant relationships between element concentrations and physiological parameters, using the pearson correlation coefficient (p<0.05). prior to analysis, data not matching a normal distribution (shapiro-wilk w test at the 95% confidence interval) were log-transformed. results according to the ec ratios, expressed as % variation (fig. 1), a significant intracellular uptake (ec ratio>175%, as suggested by frati et al., 2005) occurred for cd in summer (p<0.01) and autumn (p<0.05), and for sb in autumn and spring (p<0.05). to a lesser extent, intracellular uptake (ec ratio about 150%) occurred also for pb and zn in autumn and spring. the values of ecophysiological parameters at the end of each exposure season are shown in table 2. markers of photobiont vitality (content of chlorophyll a and b, photosynthetic efficiency) did not show any seasonal variation, while markers of mycobiont vitality showed seasonal fluctuations: cell membrane damage was higher during dry periods (spring and summer) and lower during humid periods (autumn and winter), while the content of ergosterol was higher in winter. correlation analysis did not show any significant relationship between intracellular trace elements and physiological parameters (data not shown). discussion a lichen bioaccumulation study by monaci et al. (1997) revealed that vehicular traffic was the main source of air pollution in the urban area of siena. a subsequent lichen bioindication study by loppi et al. (2002) showed a general improvement in air quality over time, and this trend towards the improvement of conditions was confirmed 10 years later by paoli et al. (2013a). this latter study showed that the diversity of epiphytic lichens increases with dis-epiphytic lichens increases with distance from traffic emissions, and that traffic is still the most important source of air pollution by heavy metals in the area. fig. 1. intracellular accumulation of metals, calculated as exposedto-control (ec) ratio, in thalli of the lichen evernia prunastri (l.) ach. at the end of the four seasonal exposures in the urban environment of siena. vannini a., paoli l., nicolardi v., antonello di lella l., loppi s. 174 acta bot. croat. 76 (2), 2017 intracellular element uptake has been calculated according to the ec ratio, since this ratio allows the accumulation degree of elements in transplanted lichens to be defined, corrected for their concentration in control samples (frati et al. 2005). our results indicated the intracellular accumula-our results indicated the intracellular accumulation for cd and sb, although in different seasons. in a previ-in a previous study carried out in siena, loppi and paoli (2015) showed that total element concentrations in the thalli of e. prunastri exposed and retrieved monthly reflected the characteristics of bulk deposition, and traffic was identified as the main source of sb deposition. on the other hand, wear of tires, brakes, engine, and vehicle components is a well known source of cd and sb atmospheric pollution (cadle et al. 1997, beckerman et al. 2008). however, it should be considered that intracellular concentrations of cd and sb measured in the present study (range of seasonal average: cd=0.03–0.08 µg g–1 dw, sb=0.22–0.46 µg g–1 dw) were low and in line with intracellular values reported for lichens from unpolluted areas (bačkor et al. 2010, paoli et al. 2013b). nevertheless, assuming sb as a main tracer of traffic emissions, positive correlations emerged between sb and ba (r=0.52, p<0.05), cr (r=0.65, p<0.001), cu (r=0.66, p<0.001), fe (r=0.37, p<0.05) and zn (r=0.70, p<0.001), suggesting also that the intracellular content of these elements was partially associated with vehicular traffic. the content of trace elements in lichen thalli is greatly influenced by season (corapi et al. 2014). rain may change the elemental composition of the particles adsorbed onto the thallus surface through the partial removal of the adhering particles, forming leachates enriched in soluble elements (lang et al. 1976, brown and brown 1991). in fact, rain after dry periods may cause displacement of cations and modifications of the elemental composition of the cell wall (bargagli 1998, reis et al. 1999), by removing cations from the thalli or even making them available for intracellular uptake, hence stimulating cellular metabolism (brown and brown 1991, nash and gries 1995). the uptake of metals can be substantially considered a passive process, in which elements move from more to less concentrated positions, e.g., from extracellular to intracellular spaces in the lichen thallus (kularatne and de freitas 2013). similarly, the loss of metals from the intracellular sites can be considered the consequence of the balance between the concentrations at intracellular and extracellular binding sites, and at once, between the whole lichen and the surrounding environment (wolterbeek et al. 2003). changes in ecophysiological parameters of transplanted lichen samples depend especially on the different levels of pollutants (godinho et al. 2004, ra et al. 2005, lackovičová et al. 2013), but might also be influenced by the different environmental and microclimatic conditions at the exposure sites (rydzak 1968). cell membrane damage was higher during dry periods (spring and summer) and lower during humid periods (autumn and winter). although seasons with the highest cell membrane damage correspond with the periods of heaviest traffic, and accumulation of heavy metals is known to cause cell membrane damage (garty et al. 1993, 1998; paoli et al. 2011), no correlation emerged with trace elements accumulated intracellularly, and it is thus possible that a limited water availability, or a combination of air pollution and unfavourable water conditions, may have negatively affected this parameter. paoli et al. (2013b) showed that sb accumulated at intracellular level caused deleterious effects on membrane integrity in the lichen xanthoria parietina, but these sb values were much higher than those measured in the present study. similarly, bačkor et al. (2010) found that intracellular cd accumulation at values similar to those found in the present study hardly caused cell membrane damage in the lichens peltigera rufescens and cladina arbuscula subsp. mitis. the content of ergosterol, the principal sterol of the plasma membrane of fungi, was significantly higher in winter than in the other seasons, reflecting the amount of metabolically active cells in the mycobiont, and hence, mycelial growth (sung et al. 1995). this is consistent with much higher total lipid content in winter found in the lichen x. parietina (piervittori et al. 1995). interestingly, in winter, intracellular accumulation of heavy metals was limited, and probably ergosterol increased in response to meteorological and environmental conditions favourable for lichen metabolism. conclusions exposed-to-control ratios of trace elements revealed intracellular accumulation of cd and sb in varying seasons, possibly as a result of vehicle traffic pollution. however, considering the low absolute concentrations of these elements, the intracellular fraction of deposition can hardly have caused any impairment of physiological parameters. as a matter of fact, indicators of photobiont vitality (content of chlorophylls a and b, and photosynthetic efficiency) tab. 2. mean values (±standard deviations) of physiological parameters in the thalli of the lichen evernia prunastri (l.) ach. at the end of the four seasonal exposures in the urban environment of siena. different letters indicate statistically significant differences (p<0.05). fv/fm – photosynthetic efficiency; el.c (%) – relative electric conductivity. summer autumn winter spring chlorophyll a (µg mg–1) 1.05±0.19 1.13±0.23 1.18±0.24 1.08±0.23 chlorophyll b (µg mg–1) 0.22±0.04 0.25±0.04 0.22±0.05 0.23±0.05 fv/fm 0.67±0.05 0.75±0.10 0.78±0.14 0.66±0.05 el.c. (%) 59.6±4.5 (a) 48.9±6.6 (b) 51.7±7.9 (b) 60.9±10.7 (a) ergosterol (µg mg–1) 0.43±0.10 (b) 0.42±0.10 (b) 0.72±0.15 (a) 0.54±0.15 (ab) evernia prunastri and heavy metals acta bot. croat. 76 (2), 2017 175 did not show any fluctuation across seasons, while changes in the indicators of mycobiont vitality (cell membrane damage and ergosterol content) overall did reflect some seasonal changes and/or lichen growth. further investigation is necessary to shed light on the role of intracellular trace element content in lichens, in order to allow comparison of these data with other biomonitoring studies and get an insight into the ecophysiological effects of air pollution. references ayrault, s., clochiatti, r., carrot, f., daudin, l., bennett, j. p., 2007: factors to consider for trace element deposition biomonitoring surveys with lichen transplants. science of the total environment 372, 717–727. bačkor, m., kováčik., j., piovár, j., pisani, t., loppi s., 2010: physiological aspects of cadmium and nickel toxicity in the lichens peltigera rufescens and cladina arbuscula subsp. mitis. water, air and soil pollution 207, 253–262. bačkor, m., loppi, s., 2009: interactions of lichens with heavy metals. biologia plantarum 53, 214–222. bargagli, r., 1998: trace elements in terrestrial plants. an ecophysiological approach to biomonitoring and biorecovery. springer, berlin. bargagli, r., mikhailova, i., 2002: accumulation of inorganic contaminants. in: nimis, p. l., scheidegger, c., wolseley, p. a. 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(eds.), bioindicators & biomonitors. principles, concepts and applications. trace metals and other contaminants in the environment, 377–419. elsevier. acta botanica 2-2016 za web.indd a word from the guest editors dear readers, the sixth balkan botanical congress (bbc) held in rijeka (september 14–18, 2015) was one in a series of successful meetings of botanists devoted to the plant life of the balkan peninsula and wider area of southeastern europe and the near east. it was preceded by congresses in thessaloniki (greece, 1997), istanbul (turkey, 2000), sarajevo (bosnia and herzegovina, 2003), sofi a (bulgaria, 2006) and belgrade (serbia, 2009) with a slightly prolonged temporal gap due to a congress planned for montenegro in 2012 that failed to materialize. the problem of disrupted continuity is linked to the specifi c situation of there being no international organization responsible for coordination of the balkan botanical congresses. after each congress, the scientifi c board becomes somehow responsible for selection of proposals for the next one. thus topic was discussed at one of the round tables of the 6th bbc and we concluded that at least an informal umbrella organization to serve as a network for balkan or see botanical societies and similar ngos is needed. there were several reasons why rijeka had been selected as a meeting place: in addition to the practical touristic reasons, the availability of a modern campus, and its position in nw croatia (the main organizers were rijeka natural history museum, croatian botanical society and botanical society of slovenia). two centuries ago one of the most prominent balkan botanists, josip pančić (1814–1888), was born in the vicinity of rijeka, in ugrini, part of the village of bribir. later his dynamic life brought him to serbia, where after an orthodox wedding he changed his name to josif pančič. and under that name he became famous as one of the most productive students of serbian, but also the wider balkan fl ora. in 2014 two exhibitions dedicated to josif pančić were put on in belgrade, one of which was opened in rijeka natural history museum in a modifi ed form at the time of the 6th bbc. on behalf of congress participants, a small delegation visited a monument to josif pančić in crikvenica and the house where he was born in bribir. comparing to previous bbcs, in rijeka there were slightly fewer active participants but we could all agree that the level of excellence was high, with an obvious positive trend comparable to past congresses. all in all, there were 50 oral (four plenary lectures) and 160 poster presentations in rijeka with the total number of over 500 participating authors and co-authors from 19 countries. oral and poster presentations were grouped in different topics covering the areas of plant anatomy and morphology, physiology, taxonomy and systematics, fl oristics, invasive species, phylogeography and phylogeny, conservation botany, vegetation and ecology, ethnobotany, archaeobotany, algology, mycology, lichenology and botanical collections. in the fi ve congress days four were spent on the exchange of scientifi c information and the middle day was devoted to two parallel excursions, one to the northern part of velebit mountain range and the other to the island of krk. in addition to that, a post-congress daily excursion went to snežnik (slovenia). two round tables were organized with the topics “do we still need classical botany?” and “do we need an umbrella ngo in the balkans?” with conclusions that offered a strongly positive answers to the title questions. a small selection of the best presentations (ten articles) from the 6th bbc is represented in this volume of acta botanica croatica, all of them having passed the standard peer review (pages from 157–216). at the end, in the name of organizers of 6th bbc, we can all thank the participants for shaping up a pleasant, informative and fruitful congress with an exchange of up-to-date results from various fi elds of botany. and we have to express our gratitude to the tourist agency pbz card ltd. which took the responsibility for all the complex logistics behind the congress. and fi nally, but not the least, we have to thank the organizing consortium of local organizers: rijeka natural history museum and the university of rijeka, which offered us a comfortable space in their campus, and the active members of the two botanical associations croatian botanical society (hbod) and botanical society of slovenia (bds). the call for organizers of the next balkan botanical congress was informally opened at the end of our meeting in rijeka; later a circular formal invitation letter was sent to all the 6th bbc boards’ members and to additional potential organizers that already expressed their willingness before. in this issue, we have also hosted publications from the fi eld of plant biology presented at the 12th croatian b iological congress (cbc) held in sveti martin na muri (september 18–23, 2015). for more than thirty years the cbc has offered biologists from different scientifi c disciplines from croatia and abroad a unique opportunity to gather together. we are looking forward to meeting you at the next bbc, as well as at the cbc. sandro bogdanović and nejc jogan presidents of 6th bbc scientifi c committee guest editors of acta botanica croatica 75(2) 2016 zagreb, september 2016 untitled acta bot. croat. 75 (1), 2016 45 acta bot. croat. 75 (1), 45–52, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0005 issn 0365-0588 eissn 1847-8476 species delimitation and genetic diversity analysis in salvia with the use of issr molecular markers masoumeh safaei1, masoud sheidai1*, behnaz alijanpoor2, zahra noormohammadi3 1 faculty of biological sciences, shahid beheshti university, tehran, iran 2 iran national botanic garden, tehran, iran 3 department of biology, school of basic sciences, science and research branch, islamic azad university, tehran, iran abstract – thirty-nine plant specimens of six salvia species were collected from different localities of the alborz mountain region in iran and studied for morphological and genetic variability and species relationship. inter simple sequence repeats (issr) molecular markers showed a high degree of within-species and interspecifi c genetic variability in salvia. analysis of molecular variance and hickory tests showed signifi cant molecular difference among the studied populations. a principal coordinate analysis plot of morphological characters grouped the species into two distinct groups, supporting their taxonomic treatment. this was partly supported by issr networking. the mantel test did not show a correlation between genetic distance and the geographical distance of the studied populations. structure and reticulation analyses revealed some degree of gene fl ow among the species. the present study showed that issr molecular markers could be used in salvia species delimitation along with morphological study. key words: issr, network, population structure, salvia * corresponding author, e-mail: msheidai@yahoo.com introduction the genus salvia l. is the largest genus of plants in the mint family lamiaceae, which contains about 900 species distributed throughout the old and new world growing in temperate and subtropical areas (standley and williams 1973, özdemir and senel 1999). within the lamiaceae, salvia is member of the tribe mentheae within the subfamily nepetoideae. it is one of several genera commonly referred to as sage. western asia and mediterranean regions have been considered as the original centers of the distribution of salvia (wu and li 1982). salvia has undergone marked species radiations in three regions of the world: central and south america (500 spp.), central asia/mediterranean (250 spp.), and eastern asia (90 spp.) (walker et al. 2004). salvia species are herbaceous, suffruticose or shrubby perennials, rarely biennial or annual, often strongly aromatic. these species are of horticultural, commercial and medicinal value. they contain monoterpene with antiseptic characteristics (özdemir and senel 1999) and the compounds obtained from these species decrease dna synthesis in the cell, an important feature in the diagnosis and treatment of cancer. many species of the lamiaceae are aromatic and are often used as herbs, spices, folk medicines and fragrances. in addition, salvia species are grown in parks and gardens as ornamental plants (özdemir and senel 1999). for example, s. nemorosa l., which is also included in the present study, is a popular herbaceous perennial species with both seed and vegetative propagated cultivars. s. spinosa l., another taxon discussed in the present work, contains essential oil in its aerial parts with antibacterial properties (salehi-sormaghi et al. 2006). there have been about 70 salvia species reported from flora iranica (hedge 1982) with 40% endemism. these species are distributed in subtropical, temperate, sub-arctic and arctic areas as well as in the tropical regions of iran (hedge 1982, sheidai et al. 2010). some of these species are very distinct while others show close affi nity to each other, and some of the species are in a state of evolutionary fl ux (hedge 1982). interspecifi c hybridization is suspected to be operative in this genus leading to a great morphological diversity (hedge 1982). the occurrences of both naturally formed interspecifi c hybrids as well as artifi cial crosses have been reported in salvia. successful crosses usually occur between closely related species. these hybrids usually have intermediate morphological features (tychonievich and warner 2011, radosavljević et al. 2012). different molecular markers have been used in different investigations related to salvia taxa: dna barcoding methsafaei m., sheidai m., alijanpoor b., noormohammadi z. 46 acta bot. croat. 75 (1), 2016 od (rpob, rbcl, matk and trnh-psba) (de mattia et al. 2011), combination of chloroplast and nuclear ribosomal dna sequences and allozyme (sudarmono and okada 2008), random amplifi ed polymorphic dna (rapd) and inter simple sequence repeats (issr) markers (wang et al. 2011, sepehry-javan et al. 2012), chloroplast dna regions of rbcl and trnl-f (walker et al. 2004), nrdna its sequences (zhan et al. 2012), pcr-rflp (karaca et al. 2008) and simple sequence repeats (ssr) markers (radosavljević et al. 2011, 2012). the present study was performed to investigate genetic diversity and morphological variation in six salvia species with the aim of producing data regarding their interand intra-population genetic structure and possible interspecies gene fl ow. such information will be important in the conservation of these medicinal plants and also in the provision of information about the evolution of the genus. we used issr molecular markers for genetic diversity analysis and for studying the species relationship, as these molecular markers were reported to be suitable for such investigations (wang et al. 2011, sepehry-javan et al. 2012). material and methods plant material thirty-nine accessions of six salvia species were collected from natural habitats in iran (tab. 1). sampling was done in the central alborz region during 2013. salvia species studied are: s. hypoleuca benth., s. limbata c. a. mey., s. nemorosa l., s. xanthocheiala boiss. ex. benth. (from the species group 1), and s. spinosa l., s. reuterana boiss. (from the species group 3). the voucher specimens are deposited in herbarium of shahid beheshti university (hsbu) (tab. 1). morphological studies morphological characters studied are: pedicel length, calyx length, stem leaf length, stem leaf width, bract length, fi lament length, anther length, corolla length, nut length, nut width, basal leaf length, basal leaf width, corolla color, corolla shape, bract shape, seed color, seed shape, bract color, corolla latex, leaf surface, calyx shape, and basal leaf shape. dna extraction and issr assay fresh leaves were collected randomly from plant specimens and dried in silica gel powder. genomic dna was extracted using the cetyltrimethyl ammonium bromide (ctab) activated charcoal protocol (sheidai et al. 2013). the quality of extracted dna was examined by running on 0.8% agarose gel. ten issr primers; (agc)5gt, (ca)7gt, (agc)5gg, ubc810, (ca)7at, (ga)9c, ubc807, ubc811, (ga)9a and (gt)7ca custom synthesized by ubc (the university of british columbia) were used. pcr reactions were performed in a 25 μl volume containing 10 mm tris-hcl buffer at ph 8, 50 mm kcl, 1.5 mm mgcl2, 0.2 mm of each dntp (bioron, germany), 0.2 μm of a single primer, 20 ng genomic dna and 3 u of taq dna polymerase (bioron, germany). the amplifi cation reactions were performed in a techne thermocycler (germany) with the following program: a 5 min initial denaturation step at 94 °c, 30 s at 94 °c; 1 min at 50 °c and 1 min at 72 °c. the reaction was completed with a 7 min extension step at 72 °c. the amplifi cation products were visualized by running on 2% agarose gel, followed by the ethidium bromide staining. the fragment size was estimated by using a 100 bp molecular size ladder (fermentas, germany). data analyses principal coordinate analysis (pcoa) was performed to group the plant specimens according to morphological characters and a principal components analysis (pca biplot) was used to identify the most variable morphological characters among the studied species (podani 2000). morphological data were standardized (mean = 0, variance = 1) for these analyses (podani 2000). issr bands obtained were coded as binary characters (presence = 1, absence = 0). genetic diversity parameters were determined in each population. these parameters were nei’s gene diversity (h), shannon information index (i), number of effective alleles, and percentage of polymorphism (weising 2005, freeland et al. 2011). nei’s genetic distance was determined among the studied populations and used for clustering (freeland et al. 2011, weising 2005). for grouping of the plant specimens, the neighbor joining (nj) clustering method and the neighbor-net method of networking were performed after bootstrapping 100 times (huson and bryant 2006, freeland et al. 2011). the mantel test was performed to check correlation between the geographical distance and the genetic distance of the studied species (podani 2000). past ver. 2.17 (hamer et al. 2001), darwin ver. 5 (2012) and splitstree4 v4.13.1 (2013) programs were used for these analyses. signifi cant genetic differences among the studied populations and provinces were determined by: 1 – analysis of molecular variance (amova) test (with 1000 permutations) with the use of genalex 6.4 (peakall and smouse 2006), and 2 – nei’s gst analysis of genodive ver.2 (2013) (meirmans and van tienderen 2004). furthermore, populations’ genetic differentiation was studied by g’st_est = standardized measure of genetic differentiation (hedrick 2005), and d_ est = jost measure of differentiation (jost 2008). in order to overcome potential problems caused by the dominance of issr markers, a bayesian program, hickory (ver. 1.0) (holsinger et al. 2003), was used to estimate parameters related to genetic structure (theta b value). three runs were conducted with default sampling parameters (burn-in = 50,000, sample= 250,000, thin = 50) to ensure consistency of results (tero et al. 2003). the genetic structure of geographical populations and provinces was studied with two methods. first we carried out a bayesian based model structure analysis (pritchard et al. 2000). for this analysis, data were scored as dominant markers (falush et al. 2007). an evanno test genetic diversity in salvia acta bot. croat. 75 (1), 2016 47 was performed on the structure result to fi nd the proper number of k by using delta k value (evanno et al. 2005). secondly, we performed k-means clustering as performed in genodive ver. 2. (2013). in this analysis, the optimal clustering is the one with the smallest amount of variation within clusters. this is calculated by using the within-clusters sum of squares. the minimization of the within-groups sum of squares that is used in k-means clustering is, in the context of a hierarchical amova, equivalent to minimizing the among-populations-within-groups sum of squares, ssdap/wg (meirmans 2012). we used two summary statistics to present k-means clustering, 1 pseudo-f (caliński and harabasz 1974), and 2bayesian information criterion (bic) (schwarz 1978). the clustering with the highest value for pseudo-f is regarded as providing the best fi t, while clustering with the lowest value for bic is considered to provide the best fi t (meirmans 2012). similarly, non-metric multidimensional scaling (mds) tab 1. salvia species studied, their locality and voucher number. species locality latitude longitude height voucher number s. hypoleuca benth. porkan, chalous 51.04.03 36.56.05 1613 hsbu 2012131 s. hypoleuca polur1, mazandaran 52.25.12 35.50.43 2273 hsbu 2012132 s. hypoleuca polur2, mazandaran 52.25.12 35.50.43 2273 hsbu 2012133 s. hypoleuca chanar gharb, firoozkooh 52.85.12 35.39.35 1572 hsbu 2012134 s. hypoleuca dizin1, tehran 51.25.19 36.00.58 3476 hsbu 2012135 s. hypoleuca dizin2, tehran 51.25.19 36.00.58 3476 hsbu 2012136 s. hypoleuca jagrod1, alborz 51.47.09 35.35.32 1235 hsbu 2012137 s. hypoleuca jagrod2, alborz 51.47.09 35.35.32 1235 hsbu 2012138 s. nemorosa l. polur, mazandaran 52.25.12 35.50.43 2273 hsbu 2012139 s. nemorosa damavand1, alborz 52.32.50 35.50.46 2221 hsbu 2012140 s. nemorosa damavand2, alborz 52.32.50 35.50.46 2221 hsbu 2012141 s. nemorosa khojir1, firoozkooh 51.43.19 35.39.71 1300 hsbu 2012142 s. nemorosa khojir2, firoozkooh 51.43.19 35.39.71 1300 hsbu 2012143 s. nemorosa dizin1,tehran 51.25.19 36.00.58 3476 hsbu 2012144 s. nemorosa dizin2,tehran 51.25.19 36.00.58 3476 hsbu 2012145 s. nemorosa chanargharb1, firoozkooh 52.85.12 35.39.35 1572 hsbu 2012146 s. limbata c.a.mey chanargharb2, firoozkooh 52.85.12 35.39.35 1572 hsbu 2012147 s. limbata khojir1, firoozkooh 51.43.19 35.39.71 1300 hsbu 2012148 s. limbata khojir2, firoozkooh 51.43.19. 35.39.71 1300 hsbu 2012149 s. limbata tochal1,tehran 51.24.02 35.49.14 1857 hsbu 2012150 s. limbata tochal2,tehran 51.24.02 35.49.14 1857 hsbu 2012151 s. limbata gulahak, firoozkooh 52.07.35 35.39.31 2022 hsbu 2012152 s. xanthocheila boiss. laar1, alborz 53.03.22 35.51.19 2163 hsbu 2012153 s. xanthocheila laar2, alborz 53.03.22 35.51.19 2163 hsbu 2012154 s. xanthocheila dizin1,tehran 51.25.19 36.00.58 3476 hsbu 2012155 s. xanthocheila dizin2,tehran 51.25.19 36.00.58 3476 hsbu 2012156 s. xanthocheila emamzadeh hashem, mazandaran 52.20.21 35.46.48 2699 hsbu 2012157 s. spinosa l. khojir1, firoozkooh 51.43.19 35.39.71 1300 hsbu 2012158 s. spinosa khojir2, firoozkooh 51.43.19 35.39.71 1300 hsbu 2012159 s. spinosa chitgar1, tehran 51.12.29 35.46.47 1285 hsbu 2012160 s. spinosa chitgar2, tehran 51.12.29 35.46.47 1285 hsbu 2012161 s. spinosa hesar1, chalous 51.01.53 35.49.24 1394 hsbu 2012162 s. spinosa hesar2, chalous 51.01.53 35.49.24 1394 hsbu 2012163 s. reuteriana boiss. tochal1,tehran 51.24.02 35.49.14 1857 hsbu 2012164 s. reuteriana tochal2,tehran 51.24.02 35.49.14 1857 hsbu 2012165 s. reuteriana darakah, tehran 51.23.48 48.25.45 1697 hsbu 2012166 s. reuteriana porkan1, chalous 51.04.03 36.56.05 1613 hsbu 2012167 s. reuteriana porkan2, chalous 51.04.03 36.56.05 1613 hsbu 2012168 s. reuteriana chanar gharb, firoozkooh 52.85.12 35.39.35 1572 hsbu 2012169 safaei m., sheidai m., alijanpoor b., noormohammadi z. 48 acta bot. croat. 75 (1), 2016 (podani 2000) was performed to study the genetic distinctness of the provinces. recently frichot et al. (2013) introduced the statistical model called “latent factor mixed models (lfmm)”, which tests correlations between environmental and genetic variation, while estimating the effects of hidden factors that represent background residual levels of population structure. we used this method to check if the issr markers used here show any correlation with the environmental features of the species studied. the analysis was done with the lfmm program version: 1.2 (2013). results morphometry the analysis of variance (anova) test showed a signifi cant difference (p < 0.05) for quantitative morphological characters among salvia species. moreover, pcoa plot of both quantitative and qualitative morphological characters separated the studied species into two distinct groups (fig. 1). s. reuterana and s. spinosa of the species group 3, were placed close to each other and much separated from the other studied species of the species group 1 according to flora iranica (hedge 1982). within the species group 3, s. hypoleuca showed morphological similarity with s. nemorosa, while, s. limbata and s. xanthocheila, were placed closer to each other. pca analysis revealed that the fi rst 3 components comprised about 86% of total morphological variability. in the fi rst pca components with about 45% of total variation, characters like calyx length, bract length, seed shape, calyx shape, and basal leaf shape showed the highest positive correlation (> 0.80). these morphological characters mainly separated s. spinosa and s. reuterana (species group 3) from the other species (fig. 2). the nut width, basal leaf length, seed color, and leaf surface showed the highest positive correlation (> 0.70) with the second pca component. these characters separated s. hypoleuca and s. limbata from s. nemorosa and s. xanthocheila of the species group 1 (fig. 2). issr analysis we obtained reproducible bands from almost all issr primers used and fi nally, a data matrix was formed. the detrended correspondence analysis (dca) plot revealed (not shown) scattered distribution of the issr loci studied, which indicated these loci are not linked and are suitable for population genetic structure analysis. genetic diversity parameters determined in 6 studied species (tab. 2) revealed that s. limbata had the highest level of genetic polymorphism (57.14%), while the lowest level of genetic polymorphism (28.57%) occurred in s. reuterana. s. limbata also had the highest values for effective number of alleles (ne = 1.256) and shannon information index (i = 0.25). the amova test revealed signifi cant molecular differences (p = 0.01) among the studied species. it also revealed that 21% of total genetic variability occurred among the studied populations while 79% occurred within these species. furthermore, pair-wise amova test as well as nonmetric mds analysis revealed that most of the paired samples comparisons differed signifi cantly from each other (p = 0.01) (fig. 3). tab. 2. genetic diversity parameters determined in salvia species. na – no. of different alleles, ne – no. of effective alleles, i – shannon’s information index, he – expected heterozygosity, uhe – unbiased expected heterozygosity, %p – percentage of polymorphic loci. species n na ne i he uhe %p s. hypoleuca 8 0.78 1.119 0.14 0.083 0.088 38.46% s. nemorosa 7 0.96 1.183 0.20 0.125 0.134 47.25% s. limbata 7 1.15 1.256 0.26 0.163 0.175 57.14% s. xanthocheila 5 1.00 1.192 0.21 0.132 0.146 49.45% s. spinosa 6 0.87 1.237 0.22 0.145 0.158 42.86% s. reuterana 6 0.67 1.169 0.15 0.098 0.107 28.57% fig. 1. principal coordinate analysis plot of morphological characters, separating two salvia species groups from each other. fig. 2. principal components analysis biplot of morphological characters in salvia species. character numbers: 1) pedicel length, 2) calyx length, 3) stem leaf length, 4) stem leaf width, 5) bract length, 6) fi lament length, 7) anther length, 8) corolla length, 9) nut length, nut width, 10) basal leaf length, 11) basal leaf width, 12) corolla color, 13) corolla shape, 14) bract shape, 15) seed color, 16) seed shape, 17) bract color, 18) corolla latex, 19) leaf surface, 20) calyx shape, and 21) basal leaf shape, respectively. genetic diversity in salvia acta bot. croat. 75 (1), 2016 49 the hickory test we used also produced a theta b value of 0.25, which is a signifi cant value. gst value (0.18, p = 0.001), and hedrick, standardised fi xation index (g’st = 0.23, p = 0.001) as well as the jost, differentiation index (d-est = 0.06, p = 0.001) showed that salvia species are genetically differentiated. nei’s genetic identity and the genetic distance determined among the studied species are presented in on-line suppl. tab. 1. the results showed that the highest degree of genetic similarity (0.989) occurred between s. hypoleuca and s. limbata and then between s. limbata and s. spinosa (0.981). the lowest degree of genetic similarity occurred between s. nemorosa and s. reuterana (0.877). nj tree based on nei,s genetic distance (fig. 4), showed that s. reuterana differed genetically from the other studied species, as it stands far from them. this dendrogram showed close genetic affi nity between s. hypoleuca and s. nemorosa, while s. xanthocheila joined them with some distance. the neighbor-net network obtained for all plant specimens revealed more detailed information about intraspecies genetic variability as well as the genetic affi nity between the studied species (fig. 5). it showed that all plant specimens of s. reuterana were placed close to each other. this holds true for plant specimens of s. limbata. however, specimens studied from the other species showed genetic variability and were placed in different clusters. the network also showed genetic affi nity between specimens of s. hypoleuca and s. nemorosa (coded 1 and 2 respectively), and between s. xanthocheila and s. spinosa (coded 4 and 5 respectively). these results are in agreement with the nj tree result. fig. 4. neighbor joining tree of inter simple sequence repeats data in the studied salvia species. fig. 5. neighbornet tree of inter simple sequence repeats data in the studied salvia species. numbers at the tip of splits are bootstrap values. fig. 3. multidimentional scaling plots showing genetic distinctness of salvia species. safaei m., sheidai m., alijanpoor b., noormohammadi z. 50 acta bot. croat. 75 (1), 2016 a mantel test did not produce signifi cant correlation (r = 0.01, p = 0.63) between geographical distance and genetic distance of these species and therefore, no isolation by distance (ibd) exists between them. salvia species studied are placed in two different species groups (namely species group 1 and 3 in flora iranica). in order to check if we have also two distinct genetic structures, we performed k-means clustering and bayesian based method of structure analysis. k-means clustering result (on-line suppl. tab. 2), showed that the optimum number of genetic groups (number of k) present in our data according to calinski & harabasz’ pseudo-f is k = 2 (the highest value of pseudo-f = 7.218). on the other hand, according to bayesian information criterion, k = 3 (the lowest value of bic = 235.0). structure analysis followed by evanno test also produced delta k = 3 (on-line suppl. fig. 1). therefore, we do have at least 2–3 genetic groups in the studied species. structure plot (on-line suppl. fig. 2), which is based on bayesian analysis, recognized 3 distinct genetic: 1specimens having mostly blue colored segments (allelic combination) including s. hypoleuca and s. limbata; 2 specimens with blue and red segments, including s. nemorosa and s. xanthocheila; and 3specimens having mostly green colored segments, including s. spinosa and s. reuterana. this genetic grouping is in agreement with the delta k of the evanno test and k-means clustering. reticulation analysis (on-line suppl. fig. 3) showed that some degree of inter-specifi c gene fl ow occurs among members of the studied species and not a single species is entirely isolated from all other studied species. the analysis done by lfmm program produced a very low value of -log10 (p-value) not higher than 0.20 which were all non-signifi cant values (p >0.05). these results are summarized in a manhattan plot, which is presented in online suppl. fig. 4. discussion morphological analyses of the studied salvia species showed that they are well differentiated from each other both in quantitative measures (the anova test result) and qualitative characters (the pcoa plot result). moreover, the present study supports the taxonomic treatment of these taxa and the placing of them in two different species groups in flora iranica (hedge 1982). in addition, pca analysis suggests that characters like calyx length, bract length, seed shape, calyx shape, and basal leaf shape, could be used in species groups delimitation. amova test revealed signifi cant molecular difference (p = 0.01) among the studied species. it also revealed that 21 % of total genetic variability occurred among the studied populations while, 79% occurred within these species. the possible reason for high within-species genetic variability is due to new mutations that occur and gradually accumulate within populations of the certain species. similar results were reported in salvia miltiorrhiza bunge (song et al. 2009). both issr and srap molecular markers revealed the presence of a greater proportion of total genetic variation within s. miltiorrhiza populations, rather than among populations. the signifi cant pair-wise amova test, as well as the non-metric mds result, revealed the genetic distinctness of salvia species studied. however, neighbor-net network and nj tree results indicated a higher degree of genetic uniformity within s. reuterana and s. limbata than in the other studied species. an interesting result was obtained from k-means clustering and structure analysis, which showed that 2–3 genetic groups are present among the species studied. this is in agreement with species groups identifi ed in salvia and also supports the amova result showing genetic distinctness of the studied taxa. as we presented before, morphological and issr groupings separated s. spinosa, and s. reuterana of the species group 3 from the other studied species of the species group 1. however, the issr based grouping showed closer affi nity between s. hypoleuca and s. limbata, while a morphological grouping showed closer similarity between s. hypoleuca and s. nemorosa. differences between the two groupings might be due to various reasons, such as the gene expression, environment, and gene introgressions as also reported in s. miltiorrhiza accessions and its varieties (wang et al. 2011). similar studies in populations of s. japonica and some other salvia species (sudarmono and okada 2008) did not show correlation between morphological variations and allozyme and dna sequences. it was concluded that s. japonica is still at the early stage of speciation process. sympatry or co-occurrence of closely related species can either result from a sympatric speciation process or from secondary contact due to range expansion after speciation. under the allopatric scenario, genetic variation tends to be uniform across the genome due to a large proportion of the genome changing through a combination of divergent selection, differential response to similar selective pressures and genetic drift (see for example strasburg et al. 2012). in contrast, in the extreme case of sympatric speciation, gene fl ow between the incipient species can homogenize most of the genome, except for loci that experience strong divergent selection pressures or regions that are tightly linked with these loci (see for example, strasburg et al. 2012, via 2012). different mechanisms, including isolation by distance, lack of gene fl ow, local adaptation, and genetic drift followed by strong selection pressure, are responsible for species/population differentiation and genetic divergence (tero et al. 2003, freeland et al. 2011, frichot et al. 2013). in the present study, a mantel test did not produce a signifi cant correlation between the geographical and the genetic distance of these species and therefore, no isolation by distance (ibd) exists between them. moreover, the structure result indicated that a high degree of infra-specifi c genetic variability is present in salvia species due to change in the allelic frequency and gene fl ow/admixture. however, a manhattan plot did not show correlation between issr loci and environmental features of salvia taxa. these regenetic diversity in salvia acta bot. croat. 75 (1), 2016 51 sults indicate that salvia species differentiation is not solely due to genetic isolation and lack of gene fl ow. however, we are not sure about the main evolutionary mechanism for salvia species diversifi cation at present and further investigation is needed and possibly in many cases 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(2nd ed.), crc press, boca rayton, fl., usa. wu, c. y., li, h. w., 1982: on the evolution and distribution in labiateae. acta botanica yunnanica 4, 97–118. zhan, l., zhao, h. x, fan, x., wang, m., ding, c. b., yang, r. w., yin, z. q., xie, x. l., zhou, y. h., wan, d. g., 2012: genetic diversity among salvia miltiorrhiza bunge and related species inferred from nrdna its sequences. turkish journal of biology 36, 319–326. genetic diversity in salvia acta bot. croat. 75 (1), 2016 on-line suppl. tab. 1. nei’s genetic identity (above diagonal) and genetic distance (below diagonal). population id: 1 – salvia. hypoleuca, 2 – s. limbata, 3 – s.nemorosa, 4 – s. xanthocheila, 5 – s. spinosa, 6 – s. reuterana. population 1 2 3 4 5 6 1 0.000 0.989 0.965 0.978 0.972 0.889 2 0.010 0.000 0.969 0.981 0.977 0.892 3 0.035 0.031 0.000 0.967 0.966 0.877 4 0.021 0.018 0.033 0.000 0.973 0.888 5 0.027 0.022 0.033 0.027 0.000 0.921 6 0.117 0.113 0.130 0.117 0.081 0.000 on-line suppl. tab. 2. k-means clustering of inter simple sequence repeats (issr) data. clustering statistics from k = 2 to k = 6. asterisk (*) denotes best clustering according to calinski & harabasz’ pseudo-f: k = 2. symbol “&” denotes best clustering according to bayesian information criterion: k = 3. k ssd(t) ssd(ac) ssd(wc) r-squared pseudo-f bic 2* 410.974 67.085 343.889 0.163 7.218 235.100 3& 410.974 98.270 312.704 0.239 5.657 235.056 4 410.974 121.766 289.208 0.296 4.912 235.673 5 410.974 142.352 268.623 0.346 4.504 236.457 6 410.974 162.407 248.567 0.395 4.312 237.094 on-line suppl. fig. 1. delta k value of evanno test. on-line suppl. fig. 2. structure plot of salvia species showing interspecifi c genetic variability and admixture. 1 safaei m., sheidai m., alijanpoor b., noormohammadi z. acta bot. croat. 75 (1), 2016 on-line supplement fig. 3. reticulogram of salvia species. species numbers are: 1-8 = s. hypoleuca, 9-15 = s. limbata, 16-22 = s. nemorosa, 23-27 = s. spinosa, 28-33 = s. xanthocheila, 34-39 = s. reuterana, respectively. on-line supplement fig. 4. manhattan plot of inter simple sequence repeats (issr) data. 2 acta botanica 2-2016 za web.indd acta bot. croat. 75 (2), 2016 217 acta bot. croat. 75 (2), 217–225, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0027 issn 0365-0588 eissn 1847-8476 the effect of agricultural landscape type on fi eld margin fl ora in south eastern poland małgorzata wrzesień1*, bożena denisow2 1 department of geobotany, institute of biology and biochemistry, maria curie-skłodowska university, 19 akademicka str., 20-033 lublin, poland 2 department of botany, laboratory of horticultural plants biology, university of life sciences in lublin 15 akademicka str., 20-950 lublin, poland abstract – plant species diversity is threatened in many agricultural landscapes due to the changes it has to undergo. although the modifi cation of the agricultural landscape pattern is observed across europe, both extensive and intensive agricultural landscapes still co-exist in poland. the objective of the study was to examine the fl ora in fi eld margins in intensively and extensively managed agricultural landscapes, located across three regions in se poland. the fl ora was compared with respect to species richness, diversity, and evenness indices. detrended correspondence analysis was employed to characterise variation in species composition. agricultural landscape type made a higher contribution than the topography or geology to species richness and composition in fi eld margins. field margins function as important habitats for general vascular plant species diversity and are useful for the conservation of rare, threatened, endangered or bee plants. a signifi cant decline in species diversity was observed over a distance of 1000 m from the habitat elements. plants growing on fi eld margins are mainly perennials; however participation of annuals clearly increases in intensive landscapes. the participation of wind-dispersed species decreased in an open-spaced intensive landscape. animal-dispersed plants predominated in an extensive landscape with forest islands. irrespective of landscape type, native species predominated. however, these habitats create the biota and corridors for alien-invasive species as well. keywords: extensive landscape, intensive landscape, invasive species, vascular plant biodiversity * corresponding author, e-mail: mseptember@tlen.pl introduction agricultural landscape constitutes ca 47% of the area of the european union (eurostat 2015) and ca 60% of land in poland (central statistical offi ce 2014). since 1970s, the vegetation of the agricultural landscape has been under increased anthropogenic pressure in europe (stoate et al., 2009, andreasen and andresen 2011). agricultural landscape structure i.e. ‘a heterogeneous land area composed of a cluster of interacting ecosystems’ is affected by large-scale changes, i.e. the fragmentation of habitats is rising signifi cantly and the decline of heterogeneity of semi-natural habitats is observed (baudry et al., 2000, forman and baudry 1984, liira et al. 2008). as a result, a variety of small biotopes – woodlots, hedgerows, ditches or fi eld boundaries – have largely disappeared from agricultural landscape (robinson and sutherland 2002, reif et al. 2008). the occurrence of non-crop habitats within cultivated fi eld systems is particularly important. these structures are a buffer against run-off of chemicals from the fi eld into water, serve to reduce soil erosion, fl oods and pesticide drift, provide breeding and shelter sites, extend food niches for a variety of animals (marshall and moonen 2002, delattrea et al. 2010), and also provide seed banks of many taxa (duelli and obrist 2003, dajdok and wuczyński 2008). on a landscape level, non-crop habitats ensure linkages between habitats, maintain landscape diversity (vickery et al. 2009), and have positive aesthetic effects (marshall and moonen 2002). among non-crop habitats, fi eld margins are of economic interest for farmers, because these structures harbour organisms such as pollinators and predators of pests, (herzon and o’hara 2007, denisow and wrzesień 2007, 2015a, wrzesień and denisow 2007, morelli 2013). in western european countries, fi eld margins have been reduced drastically (robinson and sutherland 2002). by contrast, in central and eastern europe with more extensive farming the network of fi eld margins is richer (reif et al. 2008). since 1990s, radical economic reforms and changes in agriculture sector have occurred in poland. currently, both intensive (market oriented) and extensive (self-suffi wrzesień m., denisow b. 218 acta bot. croat. 75 (2), 2016 cient, family-run) farms can be distinguished. the economic changeovers led to environmental changes that are refl eced in the modifi cation of the agricultural landscape pattern (wuczyński et al. 2014). one of the specifi c features of the polish agricultural landscape structure is the co-existence of both extensive (related to more traditional) and intensive (related to modern) landscapes. the aim of the study was to analyse fi eld margin fl ora on the large-scale landscape level in both intensively and extensively managed agricultural landscapes across three regions of poland. the more specifi c goal was to investigate how habitat elements (forests and meadows) have an impact on the species composition, richness and diversity. we established fi eld margins at increasing distances from habitat elements to measure effects of isolation on species diversity. to make the survey more complex we analysed lifespan, dispersal mechanism, geographical status, and synecological groups of species in fi eld margins. material and methods study area the survey was conducted in the lublin province (se poland) with about 68.4% of the area (1657.3 ha) covered by farmlands (central statistical offi ce 2014). the study area included the three regions selected due to variability in agricultural landscape types (kondracki 2002). in each region both extensively and intensively managed landscapes as well as various habitat elements (grasslands, forests) are present. the regions are similar in climatic conditions, yet slightly differ in topography (fig.1). the hrubieszów basin (hb) (50°48’n, 23°53’e) has an almost fl at to gently undulating topography, with elevations of generally less than 220 m above sea level, average annual temperature is 7.3 °c, annual precipitation is 600 mm. the soils are brown and chernozem. the natural vegetation of the area is composed of grasslands (festuco-brometea, molinio-arrhenatheretea) and mesophilous forests (tiliocarpinetum). the agricultural landscape is a mosaic of small-acreage fi elds and modern large-acreage fi elds and mean fi eld acreage is 9.2 ha. the main crops are wheat, sugar beets and legume crops. giełczew elevation (ge) (51°5’n, 22°58’e) is a plain with gently undulating topography, often with hills > 300 m above sea level, the average annual temperature is 7.5 °c and the annual precipitation is 630 mm. the main soil types are brown and grey-brown or rendzina. the area has an intensive agricultural landscape dominated by ≥ 6 ha fi elds (~83% of region area) and only small areas of grassland (molinietalia, arrhenatheretalia) and forests (tilio -carpinetum). the common crops are wheat, potatoes, herbs. lubartów high plain (lhp) (51°28’n, 22°38’e) is an undulating area, 170–180 m above sea level, average annual temperature is 7.4 °c, annual precipitation is 590 mm. soils are mainly podzolic composed of sands and clays. landscape structure is dominated by small-scale farming, mean fi eld acreage is 6.3 ha. forests (peucedano-pinetum and querco-pinetum), and grasslands (molinio-arrhe na the retea) occur in the landscape matrix. rye and other cereals predominate among crops. data collection the fi eld survey was conducted in 2010 and 2011 from late june to mid august. field margins were defi ned as homogeneous linear structures with vegetation occurring on the outer border of fi elds. for each landscape type, we randomly selected 15 transect plots (300 m long and 1.3 to 2.8 meters width), i.e. in each region 45 transect plots have been investigated. in total 135 transect plots were explored. transect plots were designed according to dajdok and wuczyński (2008). the geographic position of each transect plot was recorded with a differential gps. the transect plots were categorized based on the type of agricultural landscape. according to the habitat types in the surrounding of fi eld margins in each region three agricultural landscapes were selected (1) intensively managed with absence of habitat elements at > 1000 m distance from fi eld margins (i); (2) extensively managed with grasslands in the surrounding of fi eld margins (eg); and (3) extensively managed with forests in the surrounding of fi eld margins (ef). the grassland and forest habitats were located at < 1000 m distance from transect plots. vascular plant species were identifi ed in each transect plot. the abundance of plant species was estimated on the basis of the braun-blanquet scale (van der maarel 1979). the syntaxonomic units were described according to matuszkiewicz (2001), and the nomenclature of vascular plants was based on mirek et al. (2002). to make the description of fi eld margin fl ora more complex we analyzed how landscape type interacts with species characteristics. we compared the distribution patterns of: lifespan (annuals, biennials, perennials) and dispersal mechanism (animal, wind, auto). in addition, geographical status (natives, archaeophytes, i.e. those alien species that arrived prior to 1500, neophytes, i.e. those alien species that fig. 1. map of province in se poland, showing the study area; a – the location of regions: 1 – lubartów high plain (lhp), 2 – giełczew elevation (ge), 3 – hrubieszów basin (hb). type of agriculture landscape: b – intensively managed, c – extensively managed with grasslands, d – extensively managed with forests. field margin flora in se poland acta bot. croat. 75 (2), 2016 219 arrived after 1500), synecological groups (grassland species, forest species, synanthropic species) have been considered. all of these categories are hereafter called ‘traits’ in this paper. the relevant data concerning the species characteristics were obtained from the leda traitbase (kleyer et al. 2008) and bioflor database (klotz et al. 2002). some species were assigned to more than one trait of a set of multistate categorical traits. data analyses the vegetation on the transect plots was compared with respect to three types of indices, focusing on (i) species richness s = ni, where ni = species i; (ii) species diversity with the shannon-wiener index – h’ = −∑pi log2pi, where pi = frequency of the species i; (iii) species evenness with the pielou index– j’ = h’/lns, defi ned as the ratio of the observed diversity to the maximum diversity, where: s = the number of species and h max= lns. j’ is constrained between 0 and 1; the less variation in communities between the species, the higher j’ is. the mvsp package was used to calculate the indices (kovach 2005). the mean and sd (standard deviation) were computed and the values obtained were compared by the kruskal-wallis non-parametric test to reveal the signifi cance of differences in the above-mentioned indices (stanisz 2007). to characterize the general pattern of variation in species composition within the entire data set of vegetation we used an indirect ordination method, detrended correspondence analysis (dca), from canoco ver. 5 (ter braak and šmilauer 2012). the strength of the relation between species diversity and the distance from habitat elements was measured with the pearson’s correlation coeffi cient (r). the statistica software package version 10 developed by statsoft krakow was used for these analyses. results a total of 376 vascular plant species, belonging to 36 families was recorded on fi eld margins within the three regions and agricultural landscape types (fig. 2). the most abundant were asteraceae (63 species – 16.7%), fabaceae (35 species – 9.3%), poaceae (31 species – 8.2%), rosaceae (25 species – 6.6%), lamiaceae (22 species – 5.8%), ca ry ophyllaceae (17 species – 4.5%), accounting for 51.3% of species. the most frequent were 32 species (noted on > 80% of transects), e.g. dactylis glomerata, elymus repens, hypericum perforatum, knautia arvensis, veronica chamaedrys, alopecurus pratensis, berteroa incana, euphorbia cyparissias, achillea millefolium. the next most frequent 57 species were noted in 50–80% transects, 251 species were present in 10–50% transects, and 36 species were recorded with a frequency lower than 10% in agricultural landscape. a few rare weeds – e.g. anchusa arvensis, cerinthe minor, consolida regalis, fumaria vaillantii, lathyrus tuberosus, salvia verticillata, stachys annua, herniaria hirsuta, agrosthemma githago, neslia paniculata, euphorbia falcata, and bromus secalinus were found (noted on < 5% of transects), including seven species from the red list of the vascular plants in poland (zarzycki and szeląg 2006) (adonis aestivalis, bromus secalinus, cerasus fruticosa, ely mus hispidus, muscari comosum, myosurus minimus, potentilla rupestris). native species (274 species – 72.83%) predominated on fi eld margins under consideration (fig. 2). out of total vascular fl ora recorded, 102 species – 27.17% were alien species. the number of alien species was similar among regions (kruskal-wallis test: h = 5.83, p = 0.122), but differed by the type of agricultural landscape (kruskal-wallis test: h = 15.33, p = 0.0005). the highest number of alien species was recorded within traditional landscape with forests in the surrounding of fi eld margins. most of the alien species were identifi ed as archaeophytes (68 species –18.05%), only 34 neophytes (species – 9.08%) were noted. most of the archaeophytes were identifi ed as segetal weeds (e.g. thlaspi arvense, consolida regalis, geranium dissectum, viola arvensis, fumaria offi cinalis) and they were almost exclusively found in the peripheral zones of margins (= adjacent to fi elds). we recorded 19 invasive plant species among neophytes (tab. 1). the most frequent were galinsoga ciliata, erigeron annuus, conyza canadensis, galinsoga parvifl ora, setaria pumila, solidago gigantea, echinochoa crus-galli, ama ranthus retrofl exus. the most abundant neophytes, with > 30% of cover were solidago gigantea and amaranthus retrofl exus. the species composition was similar among the regions, out of 80% of fi eld margin fl ora was recorded in all regions, however the species composition differed considerably among landscape types (fig. 3). the number of species in the particular transect plots was variable (mean = 97 ± 29.7 sd; ranging from 36 to 160). species richness noted in fi eld margins differed among the types of agricultural landscape (kruskal-wallis test: h = 9.83, p = 0.032), however was similar across regions (krusfig. 2. the number of native and alien (archaeophyte and neophyte) species recorded in various type of agricultural landscape, located in se poland (mean from three regions). vertical bars indicate standard deviation (+ sd); the values indicated with different small letters are signifi cantly different between types of landscapes according to the kruskal-wallis test. wrzesień m., denisow b. 220 acta bot. croat. 75 (2), 2016 kal-wallis test: h = 5.83, p = 0.176). the number of species on fi eld margins located in extensively managed landscapes with forest (ef) was approx. 30% higher than in margins located in extensively managed landscapes with grasslands (eg), and approx. 50% higher than in fi eld margins located in intensive landscapes (i) (fig. 4). no signifi cant relation was observed between species diversity and the habitat elements (forests, meadows) under a distance of 700 m (fig. 5). at larger distances, over 1000 m from habitat elements, species diversity decreased signifi cantly with increasing distance from the habitat islands. the ratio of perennials to biennials to annuals was approximately 5:1:2 (averaged 74 ± 7.3 and 34 ± 5.59 and 30 ± 18.15 species in the total fl ora, respectively). the share of perennials, biennials and annuals was related to landscape type but not the region (kruskal−wallis test for landscape effect: h = 9.32, p = 0.035; for region: h = 1.65, p = 0.43). the highest participation of annuals was recorded in intensively managed landscape (fig. 6). there was no difference in the share of perennial plant species between extensively managed landscapes (ef vs. eg). the dispersal type of species noted within fi eld margins was related to the types of agricultural landscape (kruskalwallis test: h = 0.32, p = 0.035). the number of wind-dispersed species was the lowest in the modern landscape. animal-dispersed plants predominated in the traditional landscape with forest islands (fig. 6). taking into consideration the synecological groups, the participation of grassland species (molinio-arrhenatheretab. 1. list of invasive plant species occurring in fi eld margins in se poland. the frequency of invasive species in transects depending on landscape type (i – intensively managed landscape, eg – extensively managed landscape with grasslands, ef – extensively managed landscape with forests) and the habitats under threat. n – total number of transects, a – habitats created by humans, s – habitats partly transformed, n – communities of a natural character, ar – archaeophyte, ne – neophyte. asterisk (*) denotes potentially invasive species. species n % type of habitats colonized geographicalhistorical groupi eg ef amaranthus retrofl exus 48 43.75 20 43.75 a ne aster x salignus 13 6.25 17.5 5.55 a,s ne bunias orientalis 31 12.25 20 37.53 a,s ne conyza canadensis 98 56.25 86.66 75 a ne echinochloa crus-galli 57 37.55 40 50 a,s ar echinocystis lobata 14 6.25 13.3 12.54 a,s,n ne erigeron annuus 100 75 53.33 93.8 a,s ne galinsoga ciliata 121 81.25 86.66 100 a ne galinsoga parvifl ora 97 62.55 53.33 100 a ne geranium sibiricum* 6 – – 12.55 a ne helianthus tuberosus 6 – 6.67 6.25 a,s,n ne heracleum sosnovsky 3 – 5.66 – a,s,n ne lupinus polyphyllus 28 6.25 6.67 50 a,s,n ne rosa rugosa 17 12.5 13.33 12.5 a,s,n ne rumex confertus 14 12.5 13.33 6.25 a,s ne setaria pumila 68 75 33.33 43.75 a ar setaria viridis 27 16.25 13.33 31.25 a ar solidago gigantea 66 31.25 40 75 a,s,n ne vicia grandifl ora 11 12.6 6.67 5.8 a,s ne fig. 3. ordination diagram of the detrended correspondence analysis (dca) based on species matrix comprising the fl ora occurring in fi eld margins within three regions in se poland. each point refers to the fi eld margins location within agriculture landscape. white points correspond to intensively managed landscape (i), grey points – extensively managed landscape with grasslands (eg) and black points – extensively managed landscape with forests (ef). regions: hb – hrubieszów basin, ge – giełczew elevation, lhp – lubartów high plain. eigenvalues: axis 1 – 0.102, axis 2 – 0.066. the diagram explains 18.2% of total variance. field margin flora in se poland acta bot. croat. 75 (2), 2016 221 tea, festuco-brometea), coniferous and mesophilous broadleaved forest species (vaccinio-piceetea, querco-fagetea) and synanthropic communities species (artemisietea, stellarietea mediae) were recorded. irrespective of the agricultural landscape type, grassland species predominated and accounted for 40 – 43%. discussion the fi eld margins in the agricultural landscape of se poland function as important habitats for general vascular plant species diversity, which is typifi ed by our research in which 376 vascular plant species were identifi ed, i.e. approximately 1/3 of the regional fl ora (fijałkowski 2003). this is consistent with a study conducted in mediterranean region (bassa et al. 2011) or finland (tarmi et al. 2009) and indicates the essentiality of fi eld margins as hotspots of plant species richness in agricultural landscape, irrespective of geographic regions, climatic types or fl ora history. it is well documented that the species richness in fi eld margins is particularly important for wildlife conservation in a cropland surrounding. it is crucial for agronomic reasons, e.g. many plants that grow in fi eld margins are hosts for insects and spiders that are benefi cial to agriculture by controlling the number of crop pests, i.e. aphids (marshall 2004). the diversity of species in fi eld margins refl ected the occurrence of grassland and forest islands in their vicinity. the species diversity declined signifi cantly over the 1000 m distance from habitat elements indicating that mid-fi eld islets are valuable sources of diversity in the landscape. landscape heterogeneity is one of the landscape factors most adequate to explain plant diversity in non-crop habitats of agricultural landscapes (andreasen and andresen 2011). in several studies the species diversity declined signifi cantly with increasing distance from the nature reserves; however different distances for such a decline have been reported. for example, kohler et al. (2008) documented a drastic decline in forb species in fi eld margins in the fi rst 75 m from habitat elements. marshal and arnold (1995) demonstrated that fi eld margin fl ora is strongly infl uenced by fig. 4. species richness, shannon-wiener diversity index (h’), and pielou evenness index (j’) calculated for fl ora in fi eld margins located in intensively and extensively managed agricultural landscapes. vertical bars indicate standard deviation (+ sd); the values indicated with different small letters are signifi cantly different between type of landscapes according to the kruskal-wallis test. fig. 5. pearrson’s correlation between the diversity of species within fi eld margins located in extensively managed (a) and intensively managed (b) agricultural landscapes. wrzesień m., denisow b. 222 acta bot. croat. 75 (2), 2016 location and documented a variety of species from adjacent woodlands. the plant communities of fi eld margins are determined by colonization along these linear structures (marshall and moonen 2002). maintenance of diversity requires continuous colonization and our results suggest that beyond 1000 m colonization of species from the habitat islands can no longer compensate disappearance. here, only species well adapted to the intensive management practices in the agricultural landscape are able to survive. the species composition across regions was similar, indicating that local topography, geology and environmental conditions had far less signifi cance for fi eld margin fl ora than the type of agricultural landscape. according to aavik et al. (2008) the effects of agricultural landscape structure on fi eld margin fl ora is particularly important. it is accepted that fi eld margin fl ora refl ects the specifi city of habitat, e.g. the soil eutrophication, the increase in nitrogen and other nutrient levels (kleijn and verbeek 2000) or the physical disturbance of the soil environment related to agricultural practices (bassa et al. 2011). we noted a great number of nitrophilous weeds (urtica dioica, amaranthus retrofl exus, artemisia vulgaris, cirsium arvense, glechoma hederacea) and frequent occurrences of disturbance-tolerant generalists were also recorded (e.g. poa pratensis, rumex acetosa, achillea millefolium, elymus repens, equisetum arvense, artemisia vulgaris). interestingly, regardless of the region and the landscape type we found a relatively high share of grassland specialists in fi eld margins. lindborg et al. (2014) reported that high grasslands species richness found in linear structures (fi eld margins, road verges) across agricultural landscapes is partly related to transformation of grasslands to cropfi elds. indeed, the process has been continuing since the 1960s in the study area (fijałkowski 2003). studies from fig. 6. boxplots displaying various traits of fi eld margin fl ora depending on the type of agriculture landscape located in three regions in se poland (hb – hrubieszów basin, ge – giełczew elevation, lhp – lubartów high plain). vertical bars indicate standard deviation (± sd); the values indicated with different small letters are signifi cantly different between type of landscapes according to the kruskal − wallis test. field margin flora in se poland acta bot. croat. 75 (2), 2016 223 other parts of europe (estonia, switzerland) have also revealed the role of fi eld margins as alternative habitats for grassland species (aavik and lira 2010). we documented a high participation of perennials in fi eld margins; however their number declined in intensively managed landscape. in poland the margins are allowed to regenerate naturally, therefore the occurrence of long-lived plants refl ects the intermediate stages of ecological succession. many data have demonstrated the benefi cial effects of perennials on the diversity of many organisms, i.e. insects (szymkowiak et al. 2014), including pollinators (faring et al. 2015), butterfl ies (delattrea et al. 2010), birds (vickery et al. 2009), small mammals (żurawska-seta and barczak 2012). the effects are due to repeatable food niches, i.e. vegetative organs feed insects, seeds and fruits are suitable for birds (vickery et al. 2009), nectar and pollen enhance pollinators (denisow and wrzesień 2015b). among perennials we noted, i.e. ranunculus acris, hypericum perforatum, berteroa incana, euphorbia cyparissias, pastinaca sativa, potentilla argentea, geranium pratense, species regarded as particularly important for pollinators (denisow 2011). indirectly, the nectar and pollen producing perennials observed near entomophilous crops may have positive effects on their yields, as wild fl ower abundance increases the sizes of wild pollinator populations (meek et al. 2002, denisow and wrzesień 2015a). in some eu countries, fi eld margins are exploited in the agri-environmental programs for sowing fl ower-rich seed mixes to counteract the unprecedented decline in pollinators (potts et al. 2010). therefore, we assume that the occurrence of wild bee-fl ora in the surrounding of crops should be regarded as important in consideration of these habitats as playing a role in the conservation of pollinators. the proportion of annual weeds in fi eld margins correlated with the type of agricultural landscape. an analogous result was reported by petersen et al. (2006), liira et al., (2008) and lindborg et al. (2014), who found that more annuals are present in fi eld margins located in intensely managed modern agricultural landscape than in those that are extensively managed. the relationship may refl ect the differences in farm management, agricultural operations, or differences in herbicide applications followed by largescale and small-scale farmers. for example, disturbance of fi eld margins, reported from many european countries is more common in modern, intensive farming (marshall and moonen 2002). the habitat perturbance can create background, i.e. gaps for colonization of annuals, the rstrategist (sensu grime, 1974). these species possess the ability to use resources rapidly for successful establishment in changing environmental conditions. the absence of differences in the participation of annual species between fi eld margins located in extensive landscape indicates that the number of annual weeds was effectively reduced by competition from perennials. the signifi cance of perennial species for the limitation of annual weeds was highlighted by aavik (2008). we documented that the type of dispersion was signifi cantly related to landscape type. in accordance with lindborg et al. (2014), we found that the share of animaland auto-dispersed-species increased signifi cantly in extensively managed landscapes with mid-fi eld vegetation islets. presumably, directional dispersal by biotic agents (animal or self-dispersal), which delivers seeds less randomly is more effective to enhance colonization in an extensive landscape with different vegetation patches. however, in contrast to our expectations, we noted the lowest share of wind-dispersed species in an open-spaced intensively managed landscape. the phenomenon needs more empirical study to be explained. we recorded 3–4 fold more native than alien species. predominance of native species in fi eld margins was also recorded in agricultural landscapes in other parts of poland (dajdok and wuczyński 2008). among aliens, the prevalence of archaeophytes (i.e., those aliens that arrived prior to 1500), over neophytes (i.e., those aliens that arrived after 1500) has been found in our study. the majority of archaeophytes were identifi ed as segetal weeds. according to dajdok and wuczyński (2008), weed archaeophytes are noted most frequently in the peripheral areas of fi eld margins, i.e. in zones that adjoin fi elds, and therefore fi eld margins play a minor role in the re-dispersion of weeds into crops. notwithstanding their positive impact on general species richness, fi eld margin habitats also create corridors for migration of alien-invasive species. we observed that some of neophytes formed dense patches. invasive alien species have a particularly devastating impact on native biota and are responsible for the decline of species richness or even extinctions (vilà et al. 2010). in the regions studied, the calcareous species (e.g. adonis aestivalis, fumaria vaillantii, stachys annua, thlaspi perfoliatum, valerianella dentata) are considered at high risk from invasive plants (haliniarz and kapeluszny 2014). in poland, neophytes from asia and north america are particularly disadvantageous for native biodiversity (tokarska-guzik et al. 2012). among them, we noted bunias orientalis and solidago gigantea. due to the attractive fl oral reward (nectar and pollen), these species lure a variety of pollinators (denisow 2011). therefore, in addition to negative effects on local plant species biodiversity such species may induce the collapse of pollination webs and disrupt pollination services of entomophilous crops. we observed strong competition for apis mellifera between bunias orientalis and oilseed rape (brassica napus). we frequently noted amaranthus retrofl exus, setaria pumila and galinsoga parvifl ora. these species are known to invade various habitats (ditch banks, grasslands, wood edges) as well as fi elds, vineyards, pastures, orchards in many parts of the world, not only in europe (tokarskaguzik et al. 2012, daisie 2015). among the species the geographical distribution of which has expanded and the number of stations substantially increased (approx. 40% since 1970; latowski et al. 2010, wrzesień 2010) we recorded vicia grandifl ora and geranium sibiricum. our results confi rm the fi ndings that fi eld margins are useful for the conservation of biodiversity in the agricultural landscape, as well as for plant species currently considered rare, threatened or endangered. in the 1970s, most of these species were common weeds associated with crops. radical changes in cropping methods and chemical appliwrzesień m., denisow b. 224 acta bot. croat. 75 (2), 2016 cations are responsible for the disappearance of segetal weed species or even a risk of their extinction (haliniarz and kapeluszny 2014, wuczyński et al. 2014). therefore disappearance of weed species, mainly archaeophytes, is nowadays a common trend in many regions of poland (zając et al. 2009) and in europe (pinke et al. 2011), where fi eld margins are also recognized as refugial habitats (hamre et al. 2010, fahrig et. al. 2015). the presence of rare or red list species has been suggested as an alternative indicator for the evaluation of diversity in agricultural landscapes (weibull and östman 2003). however, our observations indicate that only few rare, endangered or protected species occurred in fi eld margins and consequently, the idea that rare species might indicate the biodiversity in agroecosystems seems to be untenable. acknowledgements this research was supported fi nancially by the ministry of science and higher education of poland (project okb/ ds/2) as a part of the statutory activities of department of geobotany, institute of biology and biochemistry, maria curie-skłodowska university and the department of botany, university of life sciences in lublin. we are grateful to anna wesołowska-zoń for reading the text and for making linguistic corrections. references aavik, t., liira, j., 2010: quantifying the effect of organic farming, fi eld boundary type and landscape structure on the vegetation of fi eld boundaries. agriculture ecosystems and environment 135, 178–186. aavik, 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(2), 199–209, 2009 coden: abcra 25 issn 0365–0588 fossil diatom flora from the marine paleogene stratigraphic key section of northeast kamchatka, russia andrey yu. gladenkov* geological institute, russian academy of sciences, pyzhevskii per. 7, moscow 119017, russia the oligocene diatom flora from the marine paleogene stratigraphic key section of northeast kamchatka is described for the first time. the fossil flora contains a number of taxa of moderate to poor preservation and low abundance. analysis of stratigraphic occurrence of diatoms throughout the section has been conducted. two diatom assemblages of different ages are recognized in different parts of the 900 m-thick alugivayam formation. the younger assemblage is characterized by the presence of cavitatus sp. cf. jouseanus and odontella sawamurae. co-occurrence of these taxa may indicate the age of enclosing sediment not older than ~31 ma (the early oligocene). the older assemblage from the basal part of the alugivayam formation lacks the mentioned taxa but contains stephanopyxis species such as st. grunowii and st. marginata. most likely, its age is at the earliest oligocene. it is shown that in the studied paleogene stratigraphic section diatoms appear just above the eocene/oligocene boundary. data obtained on diatoms and diatom biomarkers may indicate increased diatom productivity in the sea basin after the eocene/ oligocene transition. the first data on diatom flora from stratigraphically well-controlled samples support the oligocene age of the alugivayam formation in the il’pinskii peninsula section and contribute to regional correlations of the oligocene strata in kamchatka. key words: diatom, biostratigraphy, paleogene, oligocene, pacific, kamchatka introduction the stratigraphic section on the il’pinsky peninsula, northeast kamchatka (fig. 1), is a unique key section for the marine paleogene of northeast asia with a practically continuous and complete sequence composed of 2500 m-thick paleocene through oligocene sediments. moreover, it is one of the northernmost known places in the north pacific region where planktic foraminifera and calcareous nannofossils of the paleocene and eocene have been documented from different stratigraphic levels (beniamovskii et al. 1992, volobueva et al. 1994, and others). numerous mollusk-bearing horizons are also typical of the section. the assemblages of planktic foraminifera and coccolithophorids that are correlative with acta bot. croat. 68 (2), 2009 199 * corresponding e-mail address: agladenkov@ilran.ru u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:11 color profile: disabled composite 150 lpi at 45 degrees their analogues from standard paleogene zones have been used to subdivide the host sedimentary succession and determine the age of established stratigraphic units. however, until recently the two upper formations (the gailkhavilanvayam and alugivayam, 1150 m thick in total) have been considered to be barren of both calcareous and siliceous planktic marine microorganisms, including diatoms. the initial data on the first finds of diatom remains from the alugivayam formations were reported only two years ago (a. gladenkov and y. gladenkov 2007). meanwhile, fossil diatoms are one of the primary biostratigraphic tools for the dating and correlation of the post-eocene marine sediments in the middle to high latitudes of the north pacific (the current north pacific oligocene to quaternary diatom zonation includes more than 20 zones). the oligocene age and position of its lower boundary in the il’pinsky peninsula stratigraphic section were determined based on assemblages of benthic foraminifera and mollusks. in general, the alugivayam formation was previously attributed to the oligocene, while the underlying gailkhavilanvayam formation was referred to the upper eocene. the present study of diatoms from the alugivayam formation is the first description of cenozoic diatom flora from the section. materials and methods in order to collect additional geological and paleontological material, fieldwork in the western part of the il’pinskii peninsula was conducted recently by the joint russian-japanese scientific group. in particular, the work was aimed at collecting samples for diatom analysis from the gailkhavilanvayam and alugivayam formations. the gailkhavilanvayam formation (~250 m thick) is composed of alternating tuffaceous siltstones, tuffaceous argillites, and tuffstones with thin interbeds of silicic tuffs (fig. 2). the formation contains abundant concretions of variable sizes and shapes. the base of the formation is corresponds to the laparelam lithologic marker »horizon« 7.5 m thick composed of light-colored silicic ash tuff. the overlying alugivayam formation (900 m thick) is composed of tuffaceous siltstones and shales with interbeds and lenses of tuffstones. the deposits con200 acta bot. croat. 68 (2), 2009 gladenkov a. yu. fig. 1. location of the il’pinskii peninsula (shown by black arrow) in kamchatka u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:14 color profile: disabled composite 150 lpi at 45 degrees tain shapeand size-variable carbonate concretions either isolated or clustered into layers inclusive. besides, there are glendonites and »floating« pebbles in the rocks. the formation is subdivided into the lower (120 m thick) and upper (approximately 780 m thick) subformations. the mulatkhan lithologic marker »horizon« (10 m thick) at the base of the upper alugivayam subformation is composed of tuffstones, gravelstones, and tuffaceous siltstones. the contact with the underlying gailkhavilanvayam formation is conformable. acta bot. croat. 68 (2), 2009 201 oligocene diatoms from northeast kamchatka fig. 2. generalized stratigraphic column of the alugivayam formation and underlying strata of the il’pinskii peninsula stratigraphic section, northeast kamchatka, with indicated position of diatom-bearing samples, and defined beds with diatoms (modified from a. gladenkov and y. gladenkov 2007). 1 – shale, tuffaceous shale; 2 – siltstone, tuffaceous siltstone; 3 – sandstone, tuffstone. 4 – silicic tuffs; 5 – carbonate concretions (a) and glendonites (b); 6 – the laparelam (l) and mulatkhan (m) lithologic marker »horizons«; 7 – samples from interiors of mollusk shells; 8 – samples from carbonate concretions; fo – the first occurrence. u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:17 color profile: disabled composite 150 lpi at 45 degrees samples of carbonate concretions and the interiors of fossil mollusk shells were collected for diatom analysis during fieldwork. collection of such material was critical as diatoms were absent in the enclosing matrix as shown by previous studies. the concretions and interiors of mollusk shells were sampled because in some cases diatoms are present in fine sediment within concretions or shells and are thereby protected from mechanical and chemical abrasion in the course of sedimentation or during fossilization and catagenesis (barron and mahood 1993, gladenkov 2003, 2007). due to the induration of the sediments and the low concentration of diatoms, a procedure including a treatment by acetic acid and sodium pyrophosphate and subsequent centrifugation with heavy liquid was used to process the samples (gladenkov 2003). strewn slides were prepared on 18 ´18 mm cover glasses and mounted in naphrax mounting medium (index of diffraction = 1.74) on glass slides. the slides were examined in their entirety under a jeneval (zeiss) light microscope at 400´, with identifications routinely checked at 1000´. because of low abundance of diatoms, all valves were encountered on a slide and all of the taxa were tabulated for each strewn slide. the preservation of diatoms is listed as m (moderate), and p (poor) depending on the degree of destruction and dissolution of valves. the relative abundance is evaluated as f (few, more than 100 valves), r (rare, 10–100 valves), and vr (very rare, less than 10 valves). numerical ages, geological epochs, subepochs and periods are used herein according to the cenozoic geochronologic and geomagnetic polarity scales (berggren et al. 1995). results all 10 samples collected from the gailkhavilanvayam formation are barren of siliceous microfossils. fossil diatoms (tab. 1) were found in the 24 examined samples (16 samples are fossil mollusks and 8 are carbonate concretions) from the alugivayam formation (figs. 2, 3, tab. 1). overall, diatoms are rare to very rare and moderately to poorly preserved. the first occurrence of diatoms is in sample gin 70/44 from the basal part of the lower alugivayam subformation. in particular, the appearance of stephanopyxis spp., st. grunowii and st. marginata is documented at this level (7 m above the base of the alugivayam formation). the stratigraphically lowest sample yielding cavitatus sp. cf. jouseanus and odontella sawamurae is sample gin 70/5 from the lower alugivayam subformation (about 215 m above the base of the alugivayam formation). both these taxa range to the top of the section in sample gin 70/10 (880 m). ikebea tenuis occurs from sample gin 70/19 (260 m) to sample gin 70/9 (840 m), and paralia sulcata from sample gin 70/3 (105 m) to sample gin 70/9. chaetoceros spp. spores range from sample gin 70/4 (145 m) to the top of the section, and trochosira sp. cf. spinosa from sample gin 70/18 (300 m) to the top of the section in sample gin 70/10 (880 m). samples gin 70/43 (50 m) and gin 70/19 (260 m) yield kisseleviella sp., while sample gin 70/3 (105 m) contains pyxilla sp. stellarima microtrias occurs from sample gin 70/14 (370 m) to sample gin 70/15 (620 m), while hemiaulus polymorphus from sample gin 70/19 (260 m) to sample gin 70/1 (760 m). the typical diatom flora from the alugivayam formation is characterized by marine neritic-planktic and sublitoral taxa. low abundance of diatoms and their poor to moderate preservation may probably indicate abrasion and dissolution of valves during sedimentation and/or diagenesis. 202 acta bot. croat. 68 (2), 2009 gladenkov a. yu. u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:17 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .68 (2),2009 203 o l ig o c e n e d ia t o m s f r o m n o r t h e a s t k a m c h a t k a tab. 1. stratigraphic occurrence and relative abundance of marine diatoms from the alugivayam formation, the il’pinskii peninsula stratigraphic section, northeast kamchatka. preservation: m – moderate, p – poor. numbers of valves of diatoms counted in each sample are shown. relative abundance: f – few; r – rare; vr – very rare. partial boxes within the table indicate the first observations of biostratigraphically important taxa in the alugivayam formation. above a base of the alugivayam formation (m) 7 50 105 145 180 215 260 300 335 370 410 450 490 520 550 585 620 655 695 730 760 795 840 880 sample #gin70/ 44 43 3 4 42 5 19 18 41 14 40 13 17a 17 16 39 15 6 7 12 11 8 9 10 relative abundance f vr r vr r r r r r vr r r r r r vr r vr r vr vr r vr vr preservation p p p p m p p p p p p p p p p p p p arachnoidiscus sp. 1 azpeitia sp. 1 cavitatus sp. cf. jouseanus (sheshukova) williams 3 4 3 2 2 2 1 3 4 1 3 1 2 1 2 1 1 chaetoceros spp. spores 1 2 2 5 3 3 2 3 16 21 11 3 2 9 5 5 3 4 3 3 2 cocconeis spp. 1 2 1 1 1 1 1 coscinodiscus argus ehrenberg 1 c. marginatus ehrenberg 1 1 coscinodiscus spp. 1 1 1 3 1 genus et species indet. 1 1 4 3 3 1 1 1 1 hemiaulus polymorphus grunow 2 1 1 2 1 1 hemiaulus spp. 2 4 1 1 hyalodiscus sp. 1 ikebea tenuis (brun) akiba 3 1 2 1 3 1 2 1 1 kisseleviella sp. 1 1 odontella sawamurae akiba 2 1 2 8 1 paralia sulcata (ehrenberg) cleve 1 2 1 2 3 1 1 1 1 1 ploiaria sp. 2 m –p m –p m –p m –p m –p m –p u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ g l a d e n k o v . v p 9 . l i s t o p a d 2 0 0 9 1 3 : 0 6 : 1 7 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s 204 a c t a b o t .c r o a t .68 (2),2009 g l a d e n k o v a .y u . above a base of the alugivayam formation (m) 7 50 105 145 180 215 260 300 335 370 410 450 490 520 550 585 620 655 695 730 760 795 840 880 sample #gin70/ 44 43 3 4 42 5 19 18 41 14 40 13 17a 17 16 39 15 6 7 12 11 8 9 10 relative abundance f vr r vr r r r r r vr r r r r r vr r vr r vr vr r vr vr preservation p p p p m p p p p p p p p p p p p p pyxilla sp. 1 rhabdonema sp. 1 sceptroneis sp. 1 stellarima microtrias (ehrenberg) hasle et sims 1 1 1 stephanopyxis grunowii grove et sturt 6 1 1 1 st. marginata grunow 3 1 1 1 st. spinosissima grunow 1 st. superba (greville) grunow 1 st. turris (greville et arnott) ralfs 2 2 3 1 7 6 1 2 stephanopyxis spp. 88 3 2 4 19 4 trochosira sp. cf. spinosa kitton 2 1 2 1 3 1 1 xanthiopyxis panduraeformis pantocsek 1 xanthiopyxis sp. cf. ovalis lohman 2 1 1 2 xanthiopyxis sp. 4 1 1 tab. 1. – continued m –p m –p m –p m –p m –p m –p u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ g l a d e n k o v . v p 9 . l i s t o p a d 2 0 0 9 1 3 : 0 6 : 1 7 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s discussion initial analysis of molluscan and diatom assemblages from the alugivayam formation made it possible to distinguish the »beds with mollusks« and »beds with diatoms« (a. gladenkov and y. gladenkov, 2007). analysis of diatoms from different stratigraphic levels allows recognition of the beds with stephanopyxis spp. in the lower part of the alugivayam ranging approximately from the base of the formation to level of 215 m above this base, and the beds with cavitatus cf. jouseanus – odontella sawamurae in the higher stratigraphic interval (fig. 2). a base of the former beds is characterized by appearance of diatoms in the section, and a base of the latter beds is marked by the first occurrences of cavitatus cf. jouseanus and odontella sawamurae. determining a precise age for the defined units has proven difficult owing to the absence of most of age-diagnostic cenozoic marine taxa. the most biochronologically-important taxa found are cavitatus cf. jouseanus and odontella sawamurae. in particular, their occurrence is of some significance for estimating the lower age limit of the beds characterized by the presence of these taxa. it is known that cavitatus is the extinct cosmopolitan marine genus including a number of planktonic species. cavitatus jouseanus is the oldest species within this genus and widespread in oligocene to miocene sediments. according to available data, in the north pacific region the first representatives of cavitatus (c. jouseanus) appear in the early oligocene (akiba et al. 1993, gladenkov and barron 1995, and others). their first occuracta bot. croat. 68 (2), 2009 205 oligocene diatoms from northeast kamchatka fig. 3. diatoms from the alugivayam formation. 1, 2 – cavitatus sp. cf. jouseanus (sheshukova) williams; 3–5 – odontella sawamurae akiba; 6 – stephanopyxis marginata grunow; 7 – st. turris (greville et arnott) ralfs; 8 – st. grunowii grove et sturt; 9 – ikebea tenuis (brun) akiba; 10 – chaetoceros sp. (spore); 11 – stephanopyxis sp.; 12 – trochosira sp. cf. spinosa kitton. scale bars = 10 mm (a: for 1–2; b: for 3–12). u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:21 color profile: disabled composite 150 lpi at 45 degrees rence is within the early oligocene rhizosolenia oligocaenica zone of the north pacific diatom zonation (fig. 4). the first occurrence of cavitatus jouseanus marks a base of the subzone b within the rhizosolenia oligocaenica zone at ~31 ma (gladenkov 1998, 1999). a top of the rhizosolenia oligocaenica zone defined by the first occurrence of 206 acta bot. croat. 68 (2), 2009 gladenkov a. yu. fig. 4. the north pacific oligocene through early miocene diatom zones (after barron and gladenkov 1995, gladenkov and barron 1995, gladenkov 1998, 1999) correlated to the geochronologic and geomagnetic polarity time scales of berggren et al. (1995). fo – the first occurrence; fco – the first common occurrence; a–c – subzones; mid. – middle. u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:23 color profile: disabled composite 150 lpi at 45 degrees rocella vigilans is at the level of 30.2 ma (gladenkov and barron 1995, gladenkov 1998). thus, the finds of cavitatus cf. jouseanus may indicate an age not older than ~31 ma for diatom flora from the upper part of the alugivayam formation. on the other hand, the presence of odontella sawamurae is also important. this extinct marine species is typical of the northwestern pacific marginal sediments and is documented from the oligocene to early miocene deposits of japan, sakhalin, and kamchatka (morita et al. 1996, gladenkov at al. 2000, oreshkina 2009, and others). among these localities, the oldest finds of odontella sawamurae are known from hokkaido island and confined to the early oligocene rocella vigilans zone (30.2 to 29.6 ma) (morita et al. 1996). however, in sequences from the island slope of the kuril-kamchatka trench odontella sawamurae ranges from an older interval of the early oligocene corresponding to the rhizosolenia oligocaenica zone (tsoi 2002). these data suggest that the 30.2 to 29.6 ma age for the first occurrence of odontella sawamurae from hokkaido may not apply in more northern regions, such as northeast kamchatka, where this datum level is maybe older. thus, the presence of odontella sawamurae and its co-occurrence with cavitatus cf. jouseanus do not conflict with a lower possible limit for the early oligocene enclosing sediments of not more than ~31 ma. it follows that diatom assemblage from the underlying beds with stephanopyxis spp. lacking both cavitatus cf. jouseanus and odontella sawamurae, and ikebea tenuis, is older and perhaps its age may be dated as the earliest oligocene. an early oligocene age for the diatom assemblages is supported by data obtained on benthic fossil groups. particularly, a transition from the late eocene plectofrondicularia packardi – caucasina eocaenica kamtschatica benthic foraminifera assemblage to the oligocene haplophragmoides laminatus – melonis chimokiensis assemblage is documented at the boundary between the gailkhavilanvaym formation and alugivayam formation (beniamovskii et al. 1992, volobueva et al. 1994). the recent data on magnetostratigraphy indicate that a base of the normal-polarity chron 13n (at 33.5 ma) is located just above a boundary between the gailkhavilanvaym and alugivayam formations, and the early oligocene reverse-polarity chron 12r is compressed in the alugivayam formation (minyuk and gladenkov 2007). the latest data on highly branched isoprenoids (the typical diatom biomarkers) from the il’pinskii peninsula section should be especially emphasized. it has revealed that concentrations of isoprenoids increase drastically just above the boundary between the gailkhavilanvaym and alugivayam formations (shiine et al. 2008). this means these concentrations in the lower oligocene sediments are clearly higher than those from upper eocene, indicating a higher diatom productivity after the eocene/oligocene boundary (shiine et al. 2008). conclusions the alugivayam formation of the il’pinskii peninsula stratigraphic section, northeast kamchatka, contains oligocene marine diatom assemblages of moderate to poor preservation and low abundance. the presence of cavitatus cf. jouseanus and odontella sawamurae in the younger diatom assemblage may indicate the early oligocene age with a lower possible limit for the enclosing sediments of not more than ~31 ma. an age of the older assemblage from the underlying strata of the alugivayam formation is inferred as the acta bot. croat. 68 (2), 2009 207 oligocene diatoms from northeast kamchatka u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:23 color profile: disabled composite 150 lpi at 45 degrees earliest oligocene. the data on diatoms from stratigraphically well-controlled samples support and refine the oligocene age of the aluginskaya formation in the il’pinskii peninsula section. information obtained on the age of diatom-bearing deposits contributes to regional correlations of the oligocene strata in kamchatka. data on diatoms together with the latest data on diatom biomarkers from the section indicate increased diatom productivity in the sea basin after the eocene/oligocene transition. acknowledgements i thank the organizing committee of the 20th international diatom symposium and its chairman – prof. nenad jasprica from the institute for marine and coastal research, university of dubrovnik, croatia, for his invitation to write this paper for acta botanica croatica. i am also grateful to two anonymous reviewers for their critical reviews of the manuscript and helpful comments. the study was supported by the russian foundation for basic research, project nos. 09-05-00015 and 09-05-92101-jf. references akiba, f., hiramatsu, c., yanagisawa, y., 1993: cenozoic diatom genus cavitatus williams: an emended description of two new biostratigraphically useful species, c. lanceolatus and c. rectus from japan. bulletin of the natural museum of tokyo, ser. c 19, 11–39. barron, j. a., gladenkov, a. y., 1995: early miocene to pleistocene diatom stratigraphy of leg 145. proceedings of the ocean drilling program, scientific results 145. college station, 3–20. barron, j. a., mahood, a. d., 1993: exceptionally well-preserved early oligocene diatoms from glacial sediments of prydz bay, east antarctica. micropaleontology 39, 29–45. beniamovskii, v. n., fregatova, n. a., spirina, l. v., boyarinova, m. e., volobueva, v. i., gladenkov, y. b., tariverdieva, t. n. 1992: zones of planktonic and benthic foraminifers in the paleogene of eastern kamchatka (in russian). izvestia rossiiskoi akademii nauk, seria geologicheskaya 1, 100–113. berggren, w. a., kent, d. v., swisher, c. c., iii, aubry, m.-p., 1995: a revised cenozoic geochronology and chronostratigraphy. sepm special publication 54, 129–212. gladenkov, a. y., 1998: oligocene and lower miocene diatom zonation in the north pacific region. stratigraphy and geological correlation 6, 50–64. gladenkov, a. y., 1999: a new oligocene zone for the north pacific diatom scale. proceedings 14 international diatom symposium, tokyo, 581–590. gladenkov, a. y., 2003: diatom biostratigraphy of the neogene milky river formation, alaska peninsula, southwestern alaska. proceedings of the california academy of sciences 54, 27–64. gladenkov, a. y., 2007: late cenozoic detailed stratigraphy and marine ecosystems of the north pacific region (based on diatoms) (in russian). geos, moscow. 208 acta bot. croat. 68 (2), 2009 gladenkov a. yu. u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:24 color profile: disabled composite 150 lpi at 45 degrees gladenkov, a. y., barron, j. a., 1995: oligocene and early middle miocene diatom biostratigraphy of hole 884b. proceedings of the ocean drilling program, scientific results 145. college station, 21–41. gladenkov, a. y., gladenkov, y. b., 2007: new data on paleontological characteristic of the oligocene in the il’pinskii peninsula, northeastern kamchatka. stratigraphy and geological correlation 15, 231–235. gladenkov, a. y., white, l., gladenkov, y. b., blueford, j. r., 2000. cenozoic biostratigraphy of the pogranichnyi region, eastern sakhalin, russia. palaeogeography palaeoclimatology palaeoecology 158, 45–64. minyuk, p. s., gladenkov, y. b., 2007: magnetostratigraphy of paleogene deposits in kamchatka. stratigraphy and geological correlation 15, 100–111. morita, r., titova, l. v., akiba, f., 1996: oligocene-early miocene mollusks and diatoms from the kitami-tsubetsu area, eastern hokkaido, japan. science reports of the tohoku university, sendai, second series (geology) 63, 55–213. oreshkina, t. v., 2009: new data on diatoms from the marine paleogene deposits of western kamchatka. stratigraphy and geological correlation 17, 331–345. shiine, h., suzuki, n., motoyama, i., hasegawa, s., gladenkov, a. y., gladenkov, y. b., ogasawara, k., 2008: diatom biomarkers during the eocene/oligocene transition in the il’pinskii peninsula, kamchatka, russia. palaeogeography palaeoclimatololy palaeoecology 264, 1–10. tsoi, i. b., 2002: oligocene diatom assemblages from the island slope deposits of the kuril-kamchatka trench. oceanology 42, 252–265. volobueva, v. i., gladenkov, y. b., beniamovskii, v. n., vitukhin, d. i., minyuk, p. s., muzylyov, n. g., oleinik, a. e., sinel’nikova, v. n., sokolova, z. s., titova, l. v., fregatova, n. a., shchiraya, o. a., 1994: reference section of the marine paleogene in the northern far east (the il’pinskii peninsula) (in russian). svknii dvo ran, magadan. acta bot. croat. 68 (2), 2009 209 oligocene diatoms from northeast kamchatka u:\acta botanica\acta-botan 2-09\gladenkov.vp 9. listopad 2009 13:06:24 color profile: disabled composite 150 lpi at 45 degrees untitled acta bot. croat. 75 (1), 2016 149 acta bot. croat. 75 (1), 149–152, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0006 issn 0365-0588 eissn 1847-8476 short communication alyssum desertorum stapf (brassicaceae), new for the italian fl ora fabrizio bartolucci*, fabio conti school of biosciences and veterinary medicine, university of camerino – floristic research center of the apennine, national park of gran sasso and laga mountains, san colombo, 67021 barisciano (l’aquila), italy abstract – the occurrence of alyssum desertorum, a species belonging to a. sect. alyssum, is reported for the fi rst time in italy. it was found in abruzzo (central italy) in the territory of national park of gran sasso and laga mountains and surrounding areas. morphological similarities with the other taxa recorded in italy belonging to a. sect. alyssum are briefl y discussed. information about the typifi cation of the name, habitat, phenology and distribution in italy are also provided. keywords: abruzzo, alyssum, central apennine, distribution, typifi cation, vascular fl ora * corresponding author, e-mail: fabrizio.bartolucci@gmail.com introduction the genus alyssum l. (brassicaceae) consists of about 195 species distributed in europe, asia, northern africa and northern america (warwick at al. 2008, rešetnik et al. 2013, li et al. 2014). this genus was divided into six sections by dudley (1964): alyssum sect. alyssum, a. sect. gamosepalum (hausskn.) t. r. dudley, a. sect. meniocus (desv.) hook. f., a. sect. psilonema (c. a. mey.) hook. f., a. sect. tetradenia (spach) t. r. dudley, and a. sect. odontarrhena (c. a. mey.) w. d. j. koch. some years later the species of a. sect. tetradenia were transferred into the genus hormathophylla & t. r. dudley (kupfer 1974). however, recent molecular phylogeny studies indicate that alyssum is polyphyletic and that the current taxonomic circumscription of the genus needs to be revised (rešetnik et al. 2013, li et al. 2014). thirteen alyssum taxa are currently recorded in italy (conti et al. 2005, 2007, španiel et al. 2011a, b, 2012; magauer et al. 2014): a. alyssoides (l.) l., a. cuneifolium ten. subsp. cuneifolium, a. diffusum ten. subsp. diffusum, a. diffusum subsp. calabricum španiel, marhold, n.g. passal. & lihová, a. diffusum subsp. garganicum španiel, marhold, n.g.passal. & lihová, a. minutum schltdl. ex dc., a. orophilum jord. & fourr, a. repens baumg. (doubtfull presence), a. siculum jord., a. simplex rudolphi, a. strigosum banks & sol., a. wulfenianum bernh. subsp. wulfenianum and a. wulfenianum subsp. ovirense (a. kern.) magauer, schönsw. & frajman. only a. diffusum is endemic to italy (španiel et al. 2012, peruzzi et al. 2014, peruzzi et al. 2015). alyssum montanum l. has been excluded from the fl ora of italy (španiel et al. 2011a, b, 2012), a. ligusticum breistr. has to be transferred to the genus hormathophylla as h. halimifolia (boiss.) p. küpfer (küpfer 1974, warwick 2008) and fi nally all the species belonging to a. sect. odontarrhena (c. a. mey.) w. d. j. koch (a. argenteum all., a. bertolonii desv., a. tavolarae briq., a. alpestre l., a. nebro dense tineo) have recently been transferred to the genus odontarrhena c. a. mey. (cecchi and selvi 2013). during the last few years, many papers concerning the taxonomic and fl oristic knowledge of the vascular fl ora of the central apennines and abruzzo were published (e.g., conti and peruzzi 2006, conti et al. 2006, peruzzi and bartolucci 2006, bartolucci and peruzzi 2007, conti 2007, 2010, peruzzi et al. 2007, 2013, conti et al. 2008, 2011, di pietro et al. 2008, minutillo et al. 2010, conti and tinti 2012, conti et al. 2012, bartolucci and conti 2013, 2014, conti et al. 2015). in this context, we have also studied the vascular fl ora of the national park of gran sasso and laga mountains over the last ten years (bartolucci et al. 2007, conti and tinti 2008, bartolucci et al. 2012). during the fi eld research undertaken in 2013 concerning this project, we found an annual alyssum with glabrous silicles and caducous sepals not corresponding to any recorded taxa for the italian fl ora (conti et al. 2005). during the editorial process of this manuscript, alyssum desertorum was recorded as casual alien species from northern italy, based on a single individual found in a camping in trentino alto adige (bertolli and prosser 2014). bartolucci f., conti f. 150 acta bot. croat. 75 (1), 2016 materials and methods according to the relevant literature and some european fl oras (dudley 1962, 1964, 1965, ball and dudley 1996, hartvig 2002, schneeweiss 2000, plazibat 2009), we were able to identify the plant collected, and housed in herbarium apenninicum (app), as alyssum desertorum. the protologue (stapf 1886) and the original material (w, wu, je, studied from digital images) were also examined as well as the material from fi, fiaf and ge, in order to confi rm the preliminary identifi cation. acronyms are according to thiers (2015). results and discussion alyssum desertorum, belongs to a. sect. alyssum, is native to central and south-eastern europe, and central-west asia, and it is considered naturalized in northern america (dudley 1964, 1965, 1968, cheo et al. 2001, hartvig 2002, plazibat 2009). according to greuter et al. (1986) and marhold (2011), previous indications of this species in italy (fiori 1924 as alyssum minimum willd.) are erroneous. fiori (1924) reported it from croatia (istria a castelvenere), where the species was recently excluded (plazibat 2009), and from genova (north-western italy). we searched for the specimens cited by fiori (fi, fiaf, ge) but none was found. hence, the occurrence in italy of alyssum desertorum is reported here for the fi rst time. all the species belonging to a. sect. alyssum occurring in italy (a. minutum, a. simplex, a. strigosum, a. wulfenianum, a. repens, a. diffusum, a. cuneifolium, a. orophilum) have pubescent silicles with the exception of a. minutum. this latter species, quoted from southern italy, sicily and sardinia (conti et al. 2005, arrigoni 2010), is characterized by styles hairy at the base, glabrous silicles with sparse indumentum on the upper margins only when young and persistent sepals. in contrast, a. desertorum has styles glabrous, silicles always glabrous and sepals deciduous (fig. 1). dudley (1962) recognized three varieties for a. desertorum on the basis of the fruit trichomes and the habitus: var. desertorum (erect or decumbent habitus with racemes elongated up to 10 cm), var. himalayensis t.r. dudley (with minute stellate hairs on the silicles margin), and var. prostratum t.r. dudley (with reduced and prostrate habitus, and racemes condensed up to 2 cm). a fourth variety, a. desertorum var. socolacicum plazibat, was recently described from macedonia (plazibat 2009), and it is characterized by luxuriant habitus and racemes over 30 cm in length. the plants we have collected belong to a. desertorum var. desertorum. alyssum desertorum stapf, denkschr. kaiserl. akad. wiss., wien. math.-naturwiss. kl. 51(2): 302. 1886 var. desertorum lectotype (first step: dudley 1965. second step: rechinger 1968): [azerbaijan] jelizabethapol, 1882, pichler t. s.n. ex iter persicum d.ris j.e. polak (w 1904-0002633!, image is available at http://herbarium.univie.ac.at/database/ detail.php?id=548215; isolectotypes: wu-0043213! image is available at http://herbarium.univie.ac.at/database/de-tail. php?id=119430, wu-0043214! image is available at http:// herbarium.univie.ac.at/data-base/detail.php?id=119431, je-00003062!, k). description: annual, stems erect or decumbent up to 25 cm tall; indument (stem, leaves, fruiting pedicels, sepals and petals) with appressed, sessile, 6–20-rayed stellate trichomes. cauline leaves subsessile or attenuate at the base, linear to oblanceolate-linear with apex acute, 0.5–2.5 cm × (0.5)1–3(4) mm. racemes elongate and cylindrical up to 10 cm. fruiting pedicels ascending or subdivaricate, straight, 1.5–3 (3.5) mm. sepals oblong, 1.4–1.8(2) × 0.4–0.5 mm, deciduous. petals yellow, oblanceolate. fruit ovate to orbicular, glabrous, 3–4(4.5) mm, apex emarginate; valves not veined, uniformly infl ated at the centre, broadly fl attened at margin; style 0.3–0.7(1) mm, slender, glabrous. seeds often 2 per locule, ovate, 1.2–1.5 × 0.9–1.1 mm, slightly compressed, narrowly winged. habitat: disturbed sites, roadsides, arid fi elds, rocky slopes, 700–1.200 m a.s.l.; usually grows together with a. simplex and a. alyssoides. phenology: fl owering from april to june. notes on the typifi cation: a. desertorum was described from material collected during an expedition to persia in 1882 (stapf 1886): in collibus prope baku (13.iv); in deserto prope jelisabethpol in consortio a. linifolii (5.iv); inter tifl is et baku (1.iv). dudley (1962) indicated a specimen at w as syntype: “persia: in desertis prope jeliza bethpol, iter polak, 5 apr. 1882, pichler (w)”. three years later he designated isolectotypes (dudley 1965), and not a lectotype as he wrote, indicating duplicate specimens of a single gathering preserved in two different herbaria: “caucasus, azerbaydzhan, in deserto prope jelizabethpol [kirovabad], 5.iv.1882, pichler (w, k)”. according to art. 9.17 of the icn (mcneill et al. 2012) the dudley’s lectotypifi cation must be accepted (fi rst-step) but may be narrowed to a single one of the specimens of the gathering (second-step). rechinger (1968) selected the specimen housed in w as a lectotype. fig. 1. alyssum desertorum: a – infl orescence, b – raceme whit gla brous silicles and caducous sepals (photo by: a – f. conti, b – f. bartolucci). new alyssum in the italian flora acta bot. croat. 75 (1), 2016 151 distribution in italy (fig. 2): abruzzo region (central italy), municipality of l’aquila in the territory of national park of gran sasso and laga mountains and surrounding areas. the previous record from genova (north-western italy) (fiori 1924 as a. minimum willd.) is erroneous and no specimens from this area were found. specimina visa: italy: abruzzo. orto botanico di s. colombo (barisciano, l’aquila), incolti aridi, 1100 m, 18 april 2013, f. bartolucci s.n. (app n. 52960); da fonte vedice lungo la strada sterrata per filetto (barisciano, l’aquila), incolto arido, 1200 m, 20 april 2013, f. conti s.n. (app n. 55127); presso barisciano in loc. la cona, incolti al margine stradale, 860 m, 20 april 2013, f. conti s.n. (app n. 55194); loc. urràino (caporciano, l’aquila), campi, 730 m, 19 april 2013, f. conti s.n. (app.n. 55197). additional specimens examined: [azerbaijan] baku, 13.iv.1882, pichler t. s.n. ex iter persicum d.ris j. e. polak (wu-0043211! image is available at http://herbarium.univie.ac.at/database/detail.php?id=119433, wu-0043212! image is available at http://herbarium.univie.ac.at/database/ detail.php?id=119432). acknowledgements we would like to thank gabriele galasso (natutal history museum of milan, msnm) and jochen müller (friedrich-schiller university of jena, je) for nomenclatural advices. many thanks are due to the directors and curators of the following herbaria: fi, fiaf, ge, je, k and w. references arrigoni, p. v., 2010: flora dell’isola di sardegna, ii. carlo delfi no editore, sassari. ball, p. w., dudley, t. r., 1996: alyssum l. in: tutin, t. g., heywood, v. h., burges, n. a., valentine, d. h., moore, d. m. 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ribosomal its dna sequences. canadian journal of botany 86, 15–336. acta botanica 2-2016 za web.indd 186 acta bot. croat. 75 (2), 2016 acta bot. croat. 75 (2), 186–193, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0034 issn 0365-0588 eissn 1847-8476 the impact of cadmium on photosynthetic performance and secondary metabolites in the lichens parmelia sulcata, flavoparmelia caperata and evernia prunastri ana maslać1, maja maslać2, mirta tkalec1* 1 university of zagreb, faculty of science, department of biology, rooseveltov trg 6, hr-10000 zagreb, croatia 2 oikon ltd. institute of applied ecology, trg senjskih uskoka 1–2, hr-10000 zagreb, croatia abstract – lichens are one of the most common air quality bioindicators. airborne heavy metal pollution causes various physiological changes in lichens, but sensitivity to metal pollution is species specifi c. in this research, three lichen species (parmelia sulcata, flavoparmelia caperata and evernia prunastri) were exposed to cadmium (50 mg l–1) in laboratory conditions. photosynthetic effi ciency of photosystem ii and content of secondary metabolites were determined after one, three and eight days of exposure. in all investigated species treatment of lichen thalli with cadmium signifi cantly changed fv/fm and rfd only after eight days of exposure. quantifi cation of metabolites showed a decreased content of the medullary depsidones salazinic acid (in p. sulcata) and protocetraric acid (in f. caperata) but increased content of cortical depside atranorin (in p. sulcata) and dibenzofurane usnic acid (in f. caperata) after cadmium exposure. however, no changes in secondary metabolites were found in e. prunastri. results show that investigated species are relatively resistant to short-term cadmium-exposure and that secondary metabolites could have an important role in the protection of primary metabolism from negative cadmium impacts, at least in some species. key words: air pollution, heavy metal, hplc, photosynthesis * corresponding author, e-mail: mtkalec@zg.biol.pmf.hr introduction lichens are perennial, slow-growing organisms that have the ability to take in all necessary nutrients and water directly from the air. due to the absence of cuticle and roots, they absorb pollutants over their entire surface. moreover, lichens have effi cient mechanisms that take in more nutrients than they need. it has been found that lichens are able to accumulate various metals (cadmium, lead, zinc, etc.) at high levels (garty 2001, aprile et al. 2010). ever since the industrial revolution in the 1800s lichens have been recognized as bioindicators of air quality and, nowadays, they are commonly used to indicate the presence of air pollutants. more recently, lichens have also been used as biomonitors, in experiments measuring the lichen physiological responses to atmospheric pollution over time and providing additional information about the amount and intensity of the exposure (garty et al. 2003, bačkor and loppi 2009, paoli et al. 2015a). so far, they have been used as biomonitors of atmospheric pollution from different sources (loppi et al. 2004, branquino et al. 2008, lackovičová et al. 2013, paoli et al. 2015b). signifi cant correlations between metal concentrations in the lichen thalli and in their environment have been found in many studies (garty 2001, bačkor et al. 2003, carreras et al. 2005, dzubaj et al. 2008, stamenković et al. 2013). however, physiological responses of lichens to metals and their tolerance mechanisms are species-specifi c, ranging from relative resistance to high sensitivity. exposure of lichens to metals can affect membrane integrity (garty et al. 2003, pisani et al. 2010), chlorophyll content (bačkor and zetíková 2003, carreras and pignata 2007), photosynthetic performance (karakoti et al. 2014, paoli et al. 2015a) and secondary metabolites (hauck et al. 2013, gauslaa et al. 2016). among heavy metals, cadmium (cd) is considered to be particularly toxic for various lichen species causing adverse physiological changes (sanità di toppi et al. 2005). increased cd content in the environment can be attributed to human activities such as battery production, industrial metallurgical processes, combustion of fossil fuels and emissions from motor vehicles (pacyna 1998). lichens produce various secondary metabolites that have multiple functions in the interactions of the lichens with the the impact of cadmium on lichens acta bot. croat. 75 (2), 2016 187 environment. some of them are located in the upper cortex, while most are located in the medulla (solhaug et al. 2009). these compounds have antiherbivore, antimicrobial and larvicidal effects, and can protect thalli from high uv irradiation and oxidative stress. they might also have important role in metal homeostasis and lichen tolerance to pollution, although the biochemical mechanisms are mostly unknown (molnár and farkas 2010). nevertheless, it is known that lichen substances function in vitro as chelators of cations, including heavy metals (bačkor and loppi 2009). few studies have compared metal pollution and secondary compound concentration, and showed different responses. levels of medullary compounds in hypocenomyce scalaris (lecanoric acid) and cladonia furcata (fumarprotocetraric acid) were increased by metal pollution (pawlikskowrońska and bačkor 2011). białonska and dayan (2005) found that levels of atranorin, a cortical compound, and medullary compounds physodic and hydroxyphysodic acid decreased, while medullary physodalic acid increased after transplantation of hypogymnia physodes to the polluted area. on the other hand, paoli et al. (2015a) showed that the medullary compound caperatic acid decreased and the cortical compound usnic acid increased in flavoparmelia caperata located close to the landfi ll. the aim of this study was to investigate the effects of cd-exposure on photosynthetic performance and content of secondary metabolites in three widely distributed epiphytic lichen species parmelia sulcata, flavoparmelia caperata and evernia prunastri in a short-term laboratory experiment. materials and methods lichen material the foliose lichens parmelia sulcata taylor (fig. 1a) and flavoparmelia caperata (l.) hale (fig. 1b), and the fruticose lichen evernia prunastri (l.) ach. (fig. 1c) were collected from old branches of quercus sp. in maksimir park (45°49’45”n, 16°01’17”e, 160 m above sea level) in zagreb in october, 2014. lichens were placed on several layers of fi lter paper wetted with distilled water, and then acclimated in a growth chamber for a week. for each species individual thalli were sprayed with 5 ml of cd solution in a concentration of 50 mg l–1, which should cause toxic effects in a short time. lichens sprayed only with distilled water were used as control. all lichens were kept in the growth chamber under fl uorescent light (60 μmol photons m–2 s–1, photoperiod of 16 h day/8 h night) at 22 ± 2 °c. chlorophyll fl uorescence and secondary metabolite content were determined after one, three and eight days of cd-exposure. all treatments were done in triplicate. chlorophyll fl uorescence parameters light-induced chlorophyll fl uorescence parameters at continuous saturating white light were measured using a chlorophyll fl uorometer (qubit systems inc., canada) in 30 min pre-darkened thalli. low intensity red light was used to determine the minimal fl uorescence level (f0), and then continuous saturating light ca. 1500 μmol m–2 s–1 was applied. upon irradiation, the fl uorescence increased from f0 to the maximum fm and then declined to steady state fl uorescence (fs) during seven minutes. maximum quantum yield of psii (fv/fm), a widely used indicator of photosynthetic effi ciency of photosystem ii, was calculated as (fm – f0)/fm (maxwell and johnson 2000). additionally, the fl uorescence decrease ratio (rfd), a parameter directly related to the rate of photosynthesis (lichtenthaler et al. 2005), was calculated as (fm – fs)/fs, according to lichtenthaler et al. (2005). high-performance liquid chromatography lichen secondary metabolites were extracted from lyophilised thalli (18 mg) which were suspended in 1.5 ml acetone and incubated for 1 h at 4 °c. the samples were centrifuged for 15 min at 4 °c and 15 000 g. the supernatant was separated with a pipette into a separate tube and centrifuged again for 30 min at 4 °c and 30 000 g. the supernatants were then transferred with a pipette into dark vial glass bottles. secondary metabolites were analysed by high-performance liquid chromatography (hplc) using a perkin elmer series 200 system with a uv/vis diode-array detector. analytes were separated on a reverse-phase c18 brownlee speri-5 ods column (5 μm, 250 × 4.6 mm, perkin elmer, usa) with pre-column (5 × 4.6 mm). the elution program was modifi ed according to feige et al. (1993). the mobile phase consisted of 1% (v/v) phosphoric acid (a) and 100% methanol (b). for p. sulcata the elution program was: 0.6 min equilibration with 30% b, 11 min linear gradient from 30% to 70% b, 4 min linear gradient from 70% to 100% b, fig. 1. lichens used in the experiment: (a) parmelia sulcata, (b) flavoparmelia caperata and (c) evernia prunastri. maslać a., maslać m., tkalec m. 188 acta bot. croat. 75 (2), 2016 10 min isocratic with 100% b and 6 min re-equilibration with 30% b. for e. prunastri and f. caperata the linear gradient from 70% to 100% b was 8 min instead of 4 min. the fl ow rate was 0.8 ml min–1, and elution was moni tored at 245 nm. the identifi cation of lichen metabolites was made by comparing retention times in combination with uv spectral data with known chromatographic data (feige et al. 1993, huneck and yoshimura 1996). quantifi cation was performed using calibration curves of individual compounds isolated from authentic-source lichens and the results were expressed as mg per gram of dry weight (mg g–1dw). statistical analysis results were shown as average ± standard error. determination of photochemical effi ciency and quantifi cation of secondary compounds content was performed in triplicate. for processing data microsoft excel 2010 and statistica 10 (statsoft inc., sad) were used. the results were compared by analysis of variance (anova) and post hoc tukey’s test. differences between means were considered statistically signifi cant at p ≤ 0.05. results fluorescence parameters in control lichen species the values of fv/fm parameter were around 0.625 during the experiment, except in f. caperata which had a slightly lower value (0.56) at the end of the experiment. however, the difference was not signifi cant (p > 0.05) compared to the values measured on other days (fig. 2). lichens treated with cd did not show signifi cant differences (p > 0.05) from corresponding controls after one and three days of exposure, while after eight days of exposure the fv/fm values in all species were signifi cantly lower than the values in untreated lichens measured on the same day (p < 0.01) and those measured in cd-treated lichens on the other days (p ≤ 0.01). in untreated lichens, the highest rfd value ( 2.4) was measured in f. caperata, but it decreased signifi cantly (p ≤ 0.05) to 1.5 after eight days of experiment (fig. 3). in the other two species, the rfd values were lower (1.4 for e. prunastri and 1.7 for p. sulcata) and no signifi cant change (p < 0.05) in values was observed during the course of the a b c ab ab ab b ab a ab c 0 0.2 0.4 0.6 0.8 1 0 1 3 8 f v/ f m days flavoparmelia caperata control cadmium ab ab ab b ab a ab c 0 0.2 0.4 0.6 0.8 1 0 1 3 8 f v/ f m days evernia prunastri control cadmium a a a a a a a b 0 0.2 0.4 0.6 0.8 1 0 1 3 8 f v/ f m days parmelia sulcata control cadmium a b c a a a b a a a b 0 1 2 3 4 0 1 3 8 r f d days flavoparmelia caperata control cadmium ab ab ab a ab ab ab b 0 1 2 3 4 0 1 3 8 r f d days evernia prunastri control cadmium ab ab ab a ab ab ab b 0 1 2 3 4 0 1 3 8 r f d days parmelia sulcata control cadmium fig. 2. maximum effi ciency of psii (fv/fm) in lichens parmelia sulcata (a), flavoparmelia caperata (b) and evernia prunastri (c) before (0 day), and after one, three and eight days of the exposure to cadmium in a concentration of 50 mg l–1. different letters above the bars denote signifi cantly different results (p ≤ 0.05). fig. 3. fluorescence decrease ratio (rfd) in lichens parmelia sulcata (a), flavoparmelia caperata (b) and evernia prunastri (c) before (0 day), and after one, three and eight days of the exposure to cadmium in a concentration of 50 mg l–1. different letters above the bars denote signifi cantly different results (p ≤ 0.05). the impact of cadmium on lichens acta bot. croat. 75 (2), 2016 189 experiment. in lichens treated with cd, the rfd values were not signifi cantly different (p > 0.05) from those of unexposed controls after one and three days of exposure. however, the values signifi cantly decreased in e. prunastri (p = 0.03) and p. sulcata (p = 0.05) eight days after cd-exposure compared to the corresponding control values measured on the same day. in f. caperata, the rfd of cd-treated thalli was signifi cantly lower (p < 0.01) than rfd values obtained for treated lichens earlier in the experiment, but it was not signifi cantly different (p > 0.05) from the control value measured on the same day. secondary metabolites in p. sulcata acetone extracts we successfully separated two secondary metabolites and identifi ed them according to their retention times and uv spectral data (fig. 4). the major metabolite was depsidone salazinic acid (peak at 24 min) while the minor metabolite was depside atranorin (peak at 36 min). in untreated samples, the content of salazinic acid slightly decreased during the exposure experiment, from 2.04 to 1.76 mg g–1dw, while the content of atranorin did not signifi cantly change, with values around 0.3 mg g–1dw (tab. 1). in cd-treated p. sulcata, the content of salazinic acid was lower than in the corresponding control, but signifi cantly (p < 0.01) only after three days of exposure when it decreased to 1.10 mg g–1dw. in contrast, the content of atranorin was signifi cantly higher (p < 0.01) than in the corresponding controls after one and eight days of cd-exposure, amounting 0.58 and 0.62 mg g–1dw, respectively. in f. caperata samples, hplc analysis revealed depsidone protocetraric acid (peak at 20 min), as a major metabolite, and dibenzofurane usnic acid (peak at 35–36 min), as a minor metabolite (fig. 5). in control lichens, the contents of metabolites slightly increased during the experiment, from 0.77 to 0.99 mg g–1 dw for protocetraric acid and from 0.22 to 0.52 mg g–1dw for usnic acid (tab. 1). in cd-treated lichens, the content of protocetraric acid was slightly higher fig. 4. chromatogram of the acetone extract of parmelia sulcata at 245 nm. identifi ed peaks: acetone (5 min), salazinic acid (24 min) and atranorin (36 min). uv spectral data of metabolites quantifi ed in the experiment are also shown. tab. 1. content of secondary metabolites quantifi ed in untreated (control) and cadmium-treated lichens parmelia sulcata, flavoparmelia caperata and evernia prunastri after one, three and eight days of exposure. different letters in superscript denote signifi cantly different results (p ≤ 0.05). dw – dry weight. lichen metabolites (mg g–1dw) parmelia sulcata flavoparmelia caperata evernia prunastri salazinic acid atranorin protocetraric acid usnic acid evernic acid atranorin treatment days control 1 2.04±0.01a 0.27±0.06b 0.77±0.09b 0.22±0.00b 1.08±0.09a 2.11±0.07a 3 1.95±0.23a 0.35±0.07b 0.97±0.09ab 0.53±0.02ab 1.16±0.10a 2.03±0.31a 8 1.76±0.07a 0.26±0.03b 0.99±0.02a 0.52±0.05ab 1.07±0.11a 2.02±0.24a cadmium 1 1.73±0.03a 0.58±0.02a 0.92±0.01ab 0.29±0.04b 1.05±0.06a 2.01±0.11a 3 1.10±0.18b 0.35±0.08b 0.80±0.02ab 0.42±0.00ab 1.15±0.11a 2.14±0.04a 8 1.57±0.22ab 0.62±0.06a 0.76±0.10b 0.75±0.19a 1.10±0.12a 1.89±0.09a maslać a., maslać m., tkalec m. 190 acta bot. croat. 75 (2), 2016 (0.92 mg g–1dw) than in the corresponding control after one day of cd-exposure. however, it decreased to 0.80 mg g–1dw after three days of cd-exposure and then to 0.76 mg g–1dw after eight days of exposure, which was signifi cantly lower (p < 0.05) than in corresponding control. content of usnic acid in cd-treated f. caperata was mostly similar to control values after one and three days of exposure, but the value increased signifi cantly (p < 0.01) up to 0.75 mg g–1dw after eight days of exposure. in samples of e. prunastri we found depsides evernic acid (peak at 31 min) and atranorin (peak at 36–37 min), as major metabolites, and chloroatranorin (peak at 37–38 min) as a minor metabolite (fig. 6). we further quantifi ed only the two major metabolites. in control samples, the contents of evernic acid and atranorin did not change during experimental period, with the values amounting 1.10 for evernic acid and 2.05 mg g–1dw for atranorin (tab. 1). treatment with cd did not cause any signifi cant change in the content of the investigated metabolites. fig. 5. chromatogram of the acetone extract of flavoparmelia caperata at 245 nm. identifi ed peaks: acetone (5 min), protocetraric acid (19.5 min) and usnic acid (36 min). uv spectral data of metabolites quantifi ed in the experiment are also shown. fig. 6. chromatogram of the acetone extract of evernia prunastri at 245 nm. identifi ed peaks: acetone (5 min), evernic acid (31 min), usnic acid (35.5 min), atranorin (36.5 min) and chloroatranorin (37.5 min). uv spectral data of metabolites quantifi ed in the experiment are also shown. the impact of cadmium on lichens acta bot. croat. 75 (2), 2016 191 discussion lichens, due to their morphology and physiology, receive all nutrients from the atmosphere, including heavy metals. in this study, the effects of short-term cd exposure on photosynthetic performance and secondary metabolites content were studied using the epiphytic lichen species p. sulcata, e. prunastri and f. caperata. these lichen species had already been used as bioindicators and/or biomonitors of atmospheric pollution from different sources (loppi et al. 2004, lackovičová et al. 2013, stamenković et al. 2013). in the last 20 years, chlorophyll fl uorescence measurements were successfully employed in various lichen studies investigating heavy metal pollution (bačkor et al 2010, karakoti et al. 2014, paoli et al. 2015a). the maximum quantum yield of psii (fv/fm), besides being an important indicator of photosynthetic effi ciency, can indicate the vitality of a lichen photobiont (paoli et al. 2015a). in all three lichen species investigated in our study, the fv/fm values of untreated thalli were mostly in accordance with values characteristic for lichens (0.63–0.76), which are lower than those found in plants (0.74–0.83) (jensen 2002). in cdtreated thalli signifi cantly lower fv/fm values, indicating damage of the photosynthetic apparatus, were observed. however, a decrease was observed only eight days after the exposure suggesting the relative resistance of the lichen photobiont to short-term exposure to high cd concentration. bačkor et al. (2010) also reported relatively low toxicity of cd, compared to other metals, e.g. cu in lichens peltigera rufescens and cladina arbuscula 24 h after the exposure. karakoti et al. (2014) showed that the thallus of lichen pyxine cocoes, which contained different amounts of various metals did not show a decrease in fv/fm. in contrast, a decrease of fv/fm value was observed in the epiphytic fruticose lichen ramalina lacera containing higher amounts of ba, ni, s, v and zn after exposure to anthropogenic pollution (garty et al. 2000). it seems that sensitivity of photosynthetic effi ciency to different metals varies between different lichen species. a delay in the cd-effect on photosynthetic performance observed in our study might suggest that metal toxicity effect could depend on time of exposure. it has been found that prolonged exposure to lead (pb) leads to an additional decrease of fv/fm in flavoparmelia caperata (garty 2002). in our study, we also employed a fl uorescence decrease ratio (rfd), a parameter which, when measured at saturation irradiance is directly correlated to the net co2 assimilation rate (lichtenthaler et al. 2005). the rfd values in control lichens were mostly similar to rfd values found in lichen anaptychia ciliaris (valladares et al. 1995). in cd-treated lichen species, rfd showed the same trend as fv/fm parameter, confi rming tolerance of a photosynthetic process to cd in the investigated species. interestingly, prolonged time in laboratory conditions caused a decrease of both, fv/fm and especially rfd in untreated thalli of f. caperata, suggesting that this species is sensitive to the environmental conditions that are not natural to it. most secondary compounds combinations in lichens are species-specifi c and therefore are widely used in lichen taxonomy and systematics (molnár and farkas 2010). in lichen species analysed in this study almost all lichen substances specifi c for particular species (nash et al. 2002) were successfully detected: salazinic acid and atranorin in p. sulcata; evernic acid, atranorin and chloroatranorin in e. prunastri; and protocetraric acid and usnic acid in f. caperata. we did not fi nd only two compounds, consalazinic acid, a minor metabolite in p. sulcata and caperatic acid, a minor metabolite in f. caperata. caperatic acid is an aliphatic acid which is not detectable by the hplc method used in this study. lichen metabolites play an important role in tolerance of lichens to metal pollution (bačkor and loppi 2009). some reports say that compounds located in the medulla (e.g. depsidones) might be chelators of cations (solhaug et al. 2009). in this study, we found that exposure to cd in foliose lichens decreased content of the medullary depsidones salazinic acid (in p. sulcata) and protocetraric acid (in f. caperata), but increased the content of cortical depside atranorin (in p. sulcata) and dibenzofurane usnic acid (in f. caperata), whereas metabolites (all depsides) of fruticose lichen e. prunastri did not change after cd treatment. these results are in accordance with lackovičová et al. (2013) and paoli (2015a) suggestion that the ratio between cortical and medullary secondary metabolites can increase in lichen samples in polluted environment. however, there are some opposite fi ndings that report increased medullary compounds and decreased cortical compounds content after heavy metal exposure. for example, białonska and dayan (2005) found increased levels of medullary depsidone physodalic acid but decreased levels of cortical depside atranorin in hypogymnia physodes transplanted to industrial areas with high emissions of heavy metals. also, pawlik-skowrońska and bačkor (2011) obtained higher amounts of the medullary compounds, i.e. depside lecanoric acid in hypocenomyce scalaris and depsidone fumarprotocetraric acid in cladonia furcata at a mining site polluted with pb and zn. results of several studies suggested that lichen metabolites control metal homeostasis by promoting the uptake of certain metal cations and/or reducing the adsorption of others that could possibly be toxic (molnár and farkas 2010). for example, physodalic acid from hypogymnia physdodes, increase the fe2+ uptake and decrease the uptake of cu2+, mn2+ and na+ (hauck and huneck 2007, hauck 2008). dibenzofurane usnic acid and divaricatic acid were both found to increase the intracellular uptake of cu2+ in evernia mesomorpha and ramalina menziesii (hauck et al. 2009), but reduce the mn2+ uptake. recently, uv spectroscopic studies and x-ray diffraction analyses showed that the complexation of metal ions with lichen substances is widespread (bačkor and fahselt 2004, hauck et al. 2009). the most recent study by gauslaa et al. (2016) reported that medullary metabolites in fruticose (ramalina farinacea, usnea dasypoga) were reduced in polluted sites, but were not in foliose lichens (parmelia sulcata, lobaria pulmonaria) whereas cortical metabolites did not change in any species. moreover, l. pulmonaria experienced strong reduction in viability in polluted sites despite increased content of medullary compound stictic acid which has a possibility of heavy metal chelating. all these results taken together might suggest that the function of lichen secondary maslać a., maslać m., tkalec m. 192 acta bot. croat. 75 (2), 2016 metabolites in metal homeostasis depend on several factors including chemical structure of compounds (depsides/depsidones/dibenzofuranes), their location (cortex/medulla) as well as lichen form of growth (foliose/fruticose) and type of metal. moreover, valencia-islas et al. (2007) suggested that increased content of cortical usnic acid in ramalina asahinae could contribute to the antioxidant protection against air pollution. it seems that although our knowledge about the importance of lichen metabolites has increased in the last few years, their biological roles and interactions have not yet been entirely understood. in conclusion, our results show that the investigated species are relatively resistant to short-term cd exposure and that secondary metabolites might have an important role in protection against negative cd impacts, at least in some species. references aprile, g. g., di salvatore, m., carratù, g., mingo, a., carafa, a. m., 2010: comparison of the suitability of two lichen species and one higher plant for monitoring airborne heavy metals. environmental monitoring and assessment 162, 291–299. bačkor, m., zetíková, j., 2003: effects of copper, cobalt and mercury on the chlorophyll content of lichens cetraria islandica and flavocetraria cucullata. journal of the hattori botanical laboratory 93, 175–187. bačkor, m., fahselt, d., 2004: using edx-microanalysis and xray mapping to demonstrate metal uptake by lichens. biologia 59, 39–45. bačkor, m., loppi, s., 2009: interactions of lichens with heavy metals. biologia plantarum 53, 214–222. bačkor, m., paulíková, k., geralská, a., davidson, r., 2003: monitoring of air pollution in košice (eastern slovakia) using lichens. polish journal of environmental studies 12, 141–150. bačkor, m., kováčik, j., piovár, j., pisani, t., loppi, s., 2010: physiological aspects of cadmium and nickel toxicity in the lichens peltigera rufescens and cladina arbuscula subsp. mitis. water, air, and soil pollution 207, 253–262 białońska, d., dayan, f. e., 2005: chemistry of the lichen hypogymnia physodes transplanted to an industrial region. journal of chemical ecology 31, 2975–2991. branquinho, c., gaio-oliveira, g., augusto, s., pinho, p., máguas, c., correia, o., 2008: biomonitoring spatial and temporal impact of atmospheric dust from a cement industry. environmental pollution 151, 292–299. carreras, h., wannaz, e., perez, c., pignata, m. l., 2005: the role of urban air pollutants on the heavy metals accumulation performance of usnea amblyoclada. environmental research 97, 50–57. carreras, h., pignata, m. l., 2007: effects of the heavy metals cu2+,ni2+,pb2+, and zn2+ on some physiological parameters of the lichen usnea amblyoclada. ecotoxicology and environmental safety 67, 59–66. dzubaj, a., bačkor, m., tomko, j., peli, e., tuba, z., 2008: tolernace of the lichen xanthoria parietina (l.) th. fr. to metal stress. ecotoxicology and environmental safety 70, 319–326. feige, g. b., lumbsch, h. t., huneck, s., elix, j. a., 1993: identifi cation of lichen substances by a standardized high-performance liquid chromatographic method. journal of chromatography a 646, 417–427. garty, j., 2001: biomonitoring atmospheric heavy metals with lichens: theory and application. critical reviews in plant sciences 20, 309–371. garty, j., 2002: biomonitoring heavy metal pollution with lichens. in: kranner, i., beckett, r. p., varma a. k. 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phenolic secondary metabolites in lichens exposed to chronic oxidant air pollution from mexico city. journal of chemical ecology, 33, 1619–1634. valladares, f., sanchez-hoyos, a., manrique, e., 1995: diurnal changes in photosynthetic effi ciency and carotenoid composition of the lichen anaptychia ciliaris: effects of hydration and light intensity. bryologist 98, 375–382. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 211 acta bot. croat. 74 (2), 211–232, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0017 applied use of taxonomy: lessons learned from the fi rst german intercalibration exercise for benthic diatoms mirko dreßler1*, geurt verweij2, sonja kistenich1, maria kahlert3, petra werner4 1 university of rostock, institute of bio-science, department for botany and botanical garden, wismarsche str. 8, d-18057 rostock, germany 2 koeman en bijkerk, oosterweg 127, 9751 pe haren, the netherlands 3 swedish university of agricultural science, department of aquatic sciences and assessment, box 7050, lennart hjelms väg 9, 750 07 uppsala, sweden 4 diatoms as bioindikators, grainauer str. 8, 10777 berlin, germany abstract – the fi rst german intercalibration exercise for benthic diatoms was conducted to improve the application of the implementation of the european water framework directive for running waters and lakes in germany. the intercalibration exercise revealed several taxonomic problems. among others, considerable problems occurred with identifying and differentiating species of the following four groups: (i) amphora indistincta levkov and a. pediculus (kützing) grunow, (ii) cocconeis placentula var. euglypta ehrenberg and c. placentula var. lineata (ehrenberg) van heurck, (iii) navicula cryptotenella lange-bertalot and n. cryptotenelloides lange-bertalot and (iv) n. reichardtiana lange-bertalot and n. caterva hohn & hellermann. the taxonomic problems that emerged occurred due to both insuffi cient use of given taxonomic details (by limnologists) and ambiguous species descriptions and documentation (by taxonomists). thus, we recommend to the applied limnologist to use the mandatory identifi cation literature and to document any ambiguous valves during routine counts. also, it would be desirable to further investigate certain species by taxonomists and, in general, to provide more basic data with species descriptions or in identifi cation manuals. these measures will improve the use of diatoms as bioindicators and consequently benefi t both applied limnologists and taxonomists. key words: amphora, benthic diatoms, cocconeis, intercalibration exercise, navicula, quality control, taxonomy * corresponding author, e-mail: mirko.dressler@gmx.de dreßler m., verweij g., kistenich s., kahlert m., werner p. 212 acta bot. croat. 74 (2), 2015 introduction benthic diatoms are well-established, robust bioindicators (smol and stoermer 2010) that are widely used, e.g. in paleolimnology (hübener et al. 2009, dreßler et al. 2011) or when implementing the european water framework directive (eu-wfd 2000) (prygiel 2002, werner and dressler 2007, kelly 2013). in various european countries intercalibration exercises and taxonomic workshops are conducted: – to ensure the comparability of diatom counting results among diatomists when implementing the water framework directive, – to facilitate a uniform approach for dealing with taxonomic problems, and – to perform the basic quality control for diatom counting results (kelly 2001, prygiel et al. 2002, kahlert et al. 2012). in germany, the fi rst intercalibration exercise for benthic diatoms was conducted in 2011 and 2012 to improve the application of the german instruction protocol that assesses the water quality of lakes and rivers using diatoms according to the european water framework directive (schaumburg et al. 2006, 2007, 2011). the 37 participants of this german intercalibration exercise came from germany, belgium, the czech republic, france, ireland, italy, the netherlands, slovakia, spain and sweden and counted benthic diatom samples from two rivers and two lakes according to the german method (schaumburg et al. 2006, 2007, 2011). the counting results of three auditors of these four samples were used as references. the auditor results were a statistically reliable reference for three of the four samples and provided the diatom assemblage to which the participant results were compared to and assessed with. nine of the 37 participant results deviated signifi cantly to the results of all three auditors in at least one sample. these signifi cant differences identifi ed taxonomic problems within twelve genera. the aim of this study was a detailed description and analysis of four identifi ed taxonomic problems to recommend measures that may improve the applied use of diatoms as bioindicators. these four problems apparent from the fi rst german intercalibration exercise were the identifi cation and differentiation of (i) some small amphora-taxa (mainly a. indistincta levkov and a. pediculus (kützing) grunow); (ii) the varieties of cocconeis placentula ehrenberg (mainly c. placentula var. euglypta ehrenberg and c. placentula var. lineata (ehrenberg) van heurck); (iii) navicula cryptotenella lange-bertalot and n. cryptotenelloides lange-bertalot and (iv) navicula reichardtiana lange-bertalot and navicula caterva hohn & hellermann. material and methods the fi rst german intercalibration exercise was based on four samples of benthic diatoms: 1. lake krossinsee, northern germany, lowland lake, calcareous, polymictic 2. lake geneva, switzerland, alps/alpine foothills lake, calcareous, dimictic 3. river klepelshagener bach, northern germany, lowland river, calcareous 4. river drau, austria, alps/alpine foothills river, siliceous benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 213 the taxonomic problems discussed here occurred only in sample lake krossinsee (northern germany) and in sample lake geneva (switzerland). thus, we will only address the two lake samples in the following. lake krossinsee (52°36'31.5"n; 13°69'46.5"e) was sampled on 12th july 2010 from various stones and various stalks (below water surface) of phragmites australis (cavanilles) trinius ex steudel and typha latifolia l. lake geneva (46°22'31.3"n; 6°15'18.0"e) was sampled on 7th august 2011 from various stones. the selected and here presented taxonomic problems concern the differentiation and identifi cation of the following taxa: 1. amphora indistincta and a. pediculus 2. cocconeis placentula var. euglypta and var. lineata 3. navicula cryptotenella and n. cryptotenelloides 4. navicula reichardtiana and n. caterva diatom samples were taken according to the german instructions for implementing the european water framework directive (schaumburg et al. 2006, 2007, 2011). only naturally prevalent substrates were sampled (stones and macrophytes in lake krossinsee and stones in lake geneva) in at least 20 cm water depth. periphyton was scratched from at least 15 stones and plants per site into a 500 ml bottle and alcohol was added. diatom samples were oxidised and prepared with hcl, h2o2, h2so4, kmno4 and c2h2o4 following modifi ed kalbe and werner (1974). dried slurries containing the diatoms were then mounted with naphrax® (refraction index 1.73) onto slides. each slide from the two samples was prepared by one person and then sent to the participants. the slides were labelled with the diatom water body type, i.e. participants knew the region and lake-type. however, participants did not know which lake exactly they were counting during the intercalibration exercise. the slides for the auditors were taken from the same batch of prepared slides as for the participants. the auditors are diatom-specialists with more than 20 years of experience of analysing diatom slides. all samples were counted three times on three different slides by the following auditors: (1) dr. gabriele hofmann: three lake samples (one sample twice, i.e. two slides from one sample) and two running water samples, (2) dr. thomas hübener: three lake samples (one sample twice) and two running water samples, (3) dr. peter pfi ster: two running water samples. thus, the here discussed lake samples were counted by two auditors and the running water samples (which are not part of this paper) by three auditors. accordingly, each lake sample was counted twice by one auditor using two prepared slides to enable an assessment of the variability among slides per sample. participants and auditors were instructed to base their counts on schaumburg et al. (2006, 2007) (both in english) and additionally the new and relevant changes of instructions given in schaumburg et al. (2011), which were translated and send to the participants in the letter accompanying the samples. the standard identifi cation literature was hofmann et al. (2011). additionally, identifi cation had to be based on the supplementary books krammer and lange-bertalot (1986–91, 2004), lange-bertalot (1993, 2001), langebertalot and moser (1994), lange-bertalot and metzeltin (1996), krammer (1997a, 1997b, 2000, 2002, 2003), reichardt (1999), witkowski et al. (2000) and levkov (2009). participants and auditors entered the diatom counting results via the eqat-webpage (www.planktonforum.eu) into an entry mask using the laboratory code that was given to each person with the slides. the analyses and presentation of the results are solely based on the laboratory codes. dreßler m., verweij g., kistenich s., kahlert m., werner p. 214 acta bot. croat. 74 (2), 2015 diatom photos were taken with a progres® speedxtcore3 (jenoptik) camera attached to an axioplan light microscope (zeiss) (differential interference contrast (dic), 100x oilimmersion objective plan-apochromat, aperture 1.4) at an overall magnifi cation of 1000x. valves were measured using the software analysis® (soft imaging system gmbh). results small amphora-species in lake krossinsee the most abundant amphora species in the sample from lake krossinsee were a. indistincta (pl. 1: 1–4) and a. pediculus (pl. 1: 8–12; fig. 1). additionally, valves of a. cf. indistincta (pl. 1: 5–7), a. cf. pediculus (pl. 1: 13–15) and amphora spec. (pl. 1: 16–17) occurred in low abundances in this sample. rare amphora-taxa (< 0.2%) were amphora copulata (kützing) schoeman & archibald, a. cf. copulata, amphora eximia carter, amphora inariensis krammer, amphora cf. subatomus levkov, amphora minutissima w. smith, a. cf. minutissima and amphora lange-bertalotii levkov & metzeltin, which are not further discussed here. the valves 1–4 depicted in pl. 1 perfectly fi t the ranges given for a. indistincta and 8–12 (pl. 1) for a. pediculus (see levkov 2009 or hofmann et al. 2013). thus, they are named without problems. the valves 5–7 (pl. 1) were labelled a. cf. indistincta, because their habita morphologically corresponded to a. indistincta, but some traits differed from the species description in levkov (2009). the valve widths were too small (1.9–2.9 μm instead of 3–4 μm) and dorsal striae density was too high (24–25 instead of 18–22 striae in 10 μm). the valves 13–15 (pl. 1) were labelled a. cf. pediculus, because they mainly correspond to a. pediculus as even the dorsal areolae are visible using light microscopy despite their small size. however, the valves are too small (5.8–6.9 μm long instead of 7–15 μm and 1.9–2.3 μm wide instead of 2.5–4.0 μm) and the striae are too dense (e.g. dorally 25 and more striae in 10 μm instead of 18–24 in 10 μm). pl. 1. light microscopic pictures of amphora indistincta (1–4), a. cf. indistincta (5–7), a. pediculus (8–12), a. cf. pediculus (13–15) and amphora spec. (16–17) from lake krossinsee, germany. benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 215 the valves 16–17 (pl. 1) distinctly deviate from the species descriptions of a. indistincta and a. pediculus. thus, it is even more diffi cult to allocate an appropriate name to these valves, which were thus labelled amphora spec. overall, the sum of small amphora-species were more or less similar among participants (1.1–17.6%, average 7.2%) and auditors (6.0–8.7%, average 7.5%) in the sample of lake krossinsee (fig. 1a). just the results of laboratory 10 (1.1%) and laboratory 15 (17.6%) deviated distinctly from the rest. however, distinct differences occurred when each species is looked at by itself. all participants (0.8–17.6%, average 6.6%) and auditors (average 3.3%) identifi ed a. pediculus (fig. 1b). however, relative abundances of a. pediculus of the three auditors varied distinctly with 7.8%, 1.2% and 0.9%, respectively. complementary, the auditors identifi ed none, 7.3% and 3.8% of a. indistincta (fig. 1c). of the 37 participants only fi ve detected a. indistincta in sample lake krossinsee (fig. 1c). despite the occurrence of some small amphora-valves in lake krossinsee (see pl. 1: 5–7 and 13–17) that do not fi t any amphora-taxa description in hofmann et al. (2011) or fig. 1. relative abundance of small amphora-species from lake krossinsee, germany, based on the results of the fi rst german intercalibration exercise for benthic diatoms. a: sum of a. inariensis, a. indistincta, a. cf. indistincta, a. pediculus, a. cf. pediculus and amphora spec. b: a. pediculus, c: a. indistincta, d: amphora spec., a. cf. indistincta and a. cf. pediculus. dark grey bars: participants; light grey bars: auditors. dreßler m., verweij g., kistenich s., kahlert m., werner p. 216 acta bot. croat. 74 (2), 2015 fig. 2. relative abundance of cocconeis placentula ehrenberg from lake krossinsee, germany, based on the results of the fi rst german intercalibration exercise for benthic diatoms. a: sum of c. placentula var. lineata, c. placentula var. euglypta, c. placentula var. placentula and c. placentula-aggregate, b: c. placentula var. lineata, c: c. placentula var. euglypta, d: c. placentula var. placentula and e: c. placentula-aggregate, dark grey bars: participants; light grey bars: auditors. levkov (2009) only four laboratories and two auditors called some valves amphora spec., a. cf. pediculus or a. cf. indistincta (fig. 1d), i.e. they were uncertain about the identity of some valves from this taxonomic group. cocconeis placentula in lake krossinsee cocconeis placentula was one of the dominant taxa in the lake krossinsee sample (fig. 2). one main problem was the differentiation of c. placentula var. placentula (pl. 2: 19–22), c. placentula var. lineata (plate 2: 23–27) and c. placentula var. euglypta benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 217 pl. 2. light microscopic pictures of the rapheless valves of cocconeis placentula-varieties from the intercalibration exercise sample lake krossinsee, germany, labelled according to the morphological concept of krammer and lange-bertalot (2004). c. placentula var. placentula (19–22), c. placentula var. lineata (23–27), c. placentula var. euglypta (28–31) and rapheless valves of c. placentula with a striae density of ~24–26 in 10 μm (32–45). these latter valves are c. placentula var. placentula based on their striae density according to krammer and lange-bertalot (2004). however, the arrangement and shape of the areolae better fi t with c. placentula var. euglypta (32–39) or c. placentula var. lineata (40–45) according to the concept of krammer and lange-bertalot (2004). dreßler m., verweij g., kistenich s., kahlert m., werner p. 218 acta bot. croat. 74 (2), 2015 (pl. 2: 28–31) according to the results of this intercalibration exercise. the remaining cocconeis-taxa in lake krossinsee (c. placentula var. tenuistriata geitler, c. neothumensis krammer, c. pseudolineata (geitler) lange-bertalot and c. pediculus ehrenberg) were either only present in low abundances (< 1.0%) or were taxonomically not problematic according to the results of this intercalibration exercise. thus, they are not presented or discussed below. various morphological concepts exist for differentiating the varieties of c. placentula (see e.g. hustedt 1930, geitler 1982, krammer and lange-bertalot 2004, jahn et al. 2009, romero and jahn 2013), which are currently in practical use simultaneously. the german instruction protocol (schaumburg et al. 2006, 2007, 2011) stipulates using hofmann et al. (2011) as standard identifi cation literature for counting diatoms. hofmann et al. (2011) refer to krammer and lange-bertalot (2004) for differentiating the varieties of c. placentula. thus, we identifi ed the varieties according to krammer and lange-bertalot (2004). all 37 participants identifi ed c. placentula including the varieties euglypta, lineata or placentula or c. placentula-aggregate (12.9–44.3%, average 24.4%) (fig. 2a). 19 participants detected c. placentula var. lineata (0.5–31.8%, average 9.9%) (fig. 2b) and 22 participants identifi ed c. placentula var. euglypta (0.2–42.8%, average 14.8%) (fig. 2c). eleven participants identifi ed c. placentula var. placentula (0.18–29.7%, average 7.6%) (fig. 2d) of which two (laboratories 15 and 23) did not detect any other varieties of c. placentula. 14 participants detected c. placentula-aggregate (2.8–33.9%, average 21.9%) (fig. 2e). the results of the auditors suggest that c. placentula was dominated by the varieties euglypta and/or lineata in sample lake krossinsee. two auditors exclusively identifi ed c. placentula var. lineata (25.5% and 23.5%, respectively) (fig. 2b). the third auditor detected 21.7% c. placentula var. euglypta (fig. 2c) and 2.2% c. placentula var. lineata (fig. 2b). navicula cryptotenella and n. cryptotenelloides in lake krossinsee and lake geneva in the sample from lake krossinsee the genus navicula bory de saint-vincent was dominated by navicula cryptotenella, while n. cryptotenelloides occurred with less than 0.2% (see pl. 3: 47–68). in contrast, in the sample from lake geneva the auditors identifi ed n. cryptotenelloides as the dominant taxon within the genus navicula, while n. cryptotenella was very rare (< 1%) but present as well (pl. 3: 47–68). in both samples other navicula-taxa occurred too, such as n. antonii lange-bertalot, n. reichardtiana and n. tripunctata (o.f. müller) bory de saint-vincent. however, they will not be further discussed here. in the lake krossinsee sample the three auditors identifi ed n. cryptotenella with 5.9– 10.0% (average 8.3%) (fig. 3b), while 33 of the 37 participants detected n. cryptotenella with 1.4–8.9% (average: 5.4%) (fig. 3b). n. cryptotenelloides was detected in two auditor samples with 0.4% and 0.6% (average: 0.5%) and in 24 of 37 participant samples with 0.2–3.1% (average: 0.9%) (fig. 3c). especially the results of the participants 15, 19, 22 and 28 expose taxonomic problems. these laboratories did not detect any n. cryptotenella in sample lake krossinsee (fig. 3b). laboratories 15 and 28 only identifi ed n. antonii and were the only participants that did not fi nd any evidence of n. cryptotenella or n. cryptobenthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 219 tenelloides (see fig. 3a). additionally, laboratories 13, 19, 22 and 25 detected a considerable percentage of the taxa discussed here with ambiguity (labelled with cf.). similarly to the participants, the results of the auditors suggest taxonomic diffi culties of these naviculataxa. for example, auditor 40 named some counted objects as n. cf. cryptotenelloides (1.4%). in the lake geneva sample 35 of 37 participants detected n. cryptotenelloides (including cf.) and/or n. cryptotenella (including cf.) (1.4–11.8%, average: 7.0%) (fig. 3d). laboratories 28 and 36 did not identify any n. cryptotenelloides or n. cryptotenella (fig. 3d). in contrast to single taxa abundances (figs. 3e–f) the sum of n. cryptotenelloides and/or n. cryptotenella were uniform among auditors (average: 7.0%) (fig. 3d). n. cryptotenelloides was detected by 31 of 37 participants (1.4–11.0%, average: 6.4%) and by two of three auditors (average: 7.3%) (fig. 3e). n. cryptotenella was not detected by any auditor in this sample (fig. 3f). in contrast, 25 of the 37 participants identifi ed n. cryptotenella (0.2–6.1%, average: 1.6%) (fig. 3f). one auditor (no. 40) counted solely n. cf. cryptotenella. similarly, four participating laboratories counted n. cf. cryptotenella (laboratories pl. 3. light microscopic pictures of navicula cryptotenella (47–55), n. cryptotenelloides (56–61) and valves that can not be identifi ed with certainty with a valve width between 4.2 and 5.0 μm (62–68) from the lake krossinsee sample, germany (47–55 and 61, 63–65 and 67), and lake geneva sample, switzerland (56–60, 62, 66 and 68). dreßler m., verweij g., kistenich s., kahlert m., werner p. 220 acta bot. croat. 74 (2), 2015 fig. 3. relative abundance of navicula cryptotenella and n. cryptotenelloides from samples lake krossinsee, germany (a–c), and lake geneva, switzerland (d–f), based on the results of the fi rst german intercalibration exercise for benthic diatoms. a: sum of navicula cryptotenella and n. cryptotenelloides (including cf.), b: n. cryptotenella, c: n. cryptotenelloides, d: sum of n. cryptotenella and n. cryptotenelloides (including cf.), e: n. cryptotenelloides, f: n. cryptotenella, dark grey bars: participants; light grey bars: auditors. benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 221 1 and 24) or n. cf. cryptotenelloides (laboratories 16 and 34). the results of participants 10, 16, 19, 26, 28 and 36 particularly reveal taxonomic problems. laboratories 28 and 36 did not detect either n. cryptotenelloides or n. cryptotenella (fig. 3d). laboratories 10, 16, 19 and 26 did not detect any n. cryptotenelloides (fig. 3e). laboratories 10, 19 and 26 solely identifi ed n. cryptotenella (figs. 3e–f). laboratory 16 counted mainly n. cf. cryptotenelloides. similarly, the results of auditor no. 40, who solely identifi ed n. cf. cryptotenella, emphasize the taxonomic problems within the n. cryptotenella and n. cryptotenelloidesgroup. besides general problems with identifying n. cryptotenella and n. cryptotenelloides, there is an additional problem that led to distinct diffi culties with differentiating n. cryptotenella and n. cryptotenelloides. according to lange-bertalot (1993, 2001) the two taxa can always and certainly be distinguished by their width, as the given width for n. cryptotenelloides is 3.7–4.2 μm and for n. cryptotenella is 5.0–7.0 μm. overlapping valve width (4.2–5.0 μm) is not supposed to occur (lange-bertalot 1993, 2001). in contrast, valves with a valve width of 4.2–5.0 μm occurred regularly in the samples examined here. for elucidating this problem of overlapping valve width 38 valves of the n. cryptotenella and n. cryptotenelloides-group were measured and photographed in each of the lake krossinsee and lake geneva samples (fig. 4). of the 38 measured valves from lake geneva 23 valves had a width of 3.6–4.2 μm (fig. 4), corresponding to n. cryptotenelloides. interestingly, all 23 valves had a striae density of 18.5–21.0 in 10 μm (fig. 4), which consistently exceeds 16–18 striae in 10 μm, the given range for n. cryptotenelloides (lange-bertalot 2001, hofmann et al. 2011). one of the 38 lake geneva valves had a width of 5.5 μm and 17 striae in 10 μm. even though the striae density is slightly too high according to lange-bertalot (2001) and hofmann et al. (2011), this valve is probably n. cryptotenella. the remaining 14 valves (36.8%) of lake geneva had a width between 4.2–5.0 μm (fig. 4), contrasting the details given in langebertalot (1993, 2001), i.e. the width were in the range between n. cryptotenella and n. cryptotenelloides. these 14 valves had a striae density between 17.3 and 21.0 in 10 μm and were thus more similar to n. cryptotenelloides than to n. cryptotenella. however, it remains disputable which taxon these valves belong to. thus, we suggest labelling these valves as cf. fig. 4. striae density and valve width from valves of the navicula cryptotenella and n. cryptotenelloides–complex from 38 valves from lake geneva, switzerland (black diamonds), and 38 valves from lake krossinsee (grey circles). vertical dashed lines denote the valve width of n. cryptotenelloides (3.7–4.2 μm) and n. cryptotenella (5.0–7.0 μm), horizontal dashed line denotes the differentiating striae density of 16 striae in 10 μm according to lange-bertalot (2011). dreßler m., verweij g., kistenich s., kahlert m., werner p. 222 acta bot. croat. 74 (2), 2015 of the 38 measured valves from lake krossinsee two valves had a width of ~3.7 and 4.0 μm and a striae density of ~18 and 20 in 10 μm, respectively (fig. 4). correspondingly, these valves were probably n. cryptotenelloides. 13 valves had a width > 5.0 μm (fig. 4), matching n. cryptotenella. of these 13 valves 10 valves had ~14–16 striae in 10 μm (fig. 4) and are thus probably n. cryptotenella. three of these 13 valves had ~17–18 striae in 10 μm (fig. 4) and are therefore only ambiguously n. cryptotenella. the remaining 23 valves (60.5%) from lake krossinsee had a width between 4.2–4.9 μm (fig. 4), i.e. in a range between the width of n. cryptotenella and n. cryptotenelloides. similarly, the striae density ranged between 15.5 and 19.9 in 10 μm (fig. 4). thus, it remains unclear if the valves belong to n. cryptotenella or n. cryptotenelloides and should also best be labelled as cf. despite these distinct deviations from the description of n. cryptotenella and n. cryptotenelloides of most valves from lake krossinsee and lake geneva (fig. 4), only four of the 37 participants and one auditor labelled any valves with cf for lake krossinsee and only four other participants and the same auditor used cf. for these valves from lake geneva. navicula reichardtiana and n. caterva in lake geneva navicula reichardtiana was detected by 25 of the 37 participants and two auditors in the lake geneva sample. one additional participant and the third auditor identifi ed n. cf. reichardtiana. n. caterva was detected by fi ve participants and no auditor. the sum of all n. reichardtiana and n. caterva from all participants and auditors was below 2.5%. thus, not listing these taxa does not necessarily suggest an identifi cation mistake or problem. as two auditors and most participants unambiguously identifi ed n. reichardtiana and no auditor and only fi ve participants identifi ed n. caterva, we could assume that the latter are misidentifi cations. however, with the data at hand a misidentifi cation cannot be verifi ed. more taxonomic examinations would be necessary. the workshop that was conducted subsequent to the intercalibration exercise identifi ed complicated taxonomic problems instead of simple misidentifi cation leading to some participants determining n. caterva instead of n. reichardtiana and to two participants identifying both taxa in the same sample. similarly, one auditor only identifi ed n. cf. reichardtiana from this complex. in the following we will demonstrate these problems exemplary by describing the identifi cation approaches of two participants (a and b) when identifying n. reichardtiana and n. caterva (fig. 5). both participants measured the striae density along the margin of the axial area, beginning at the end of the central area. participant a counted the striae along a 5 μm scale, while participant b used a shorter scale (fig. 5). both extrapolated to striae density in 10 μm (tab. 1). these different interpretations of striae counting methods and consequently varying measuring methods only led to small differences in the extrapolated number of striae in 10 μm (16.2 and 15.1, respectively; tab. 1), but have a great impact on the identifi cation process (see below). interpretation and species identifi cation by participant a: participant a argues that the width of the valve in the given example (fig. 5) fi ts the range of n. caterva (4.2–5.5 μm according to lange-bertalot 2001) and is too small for n. reichardtiana (5–6 μm according to lange-bertalot 2001). the striae density (16.17 in 10 μm) only slightly exceeds the range for n. reichardtiana (14–16 in 10 μm according to lange-bertalot 2001) and fi ts the range of n. caterva ((16)18–21 in 10 μm according to benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 223 lange-bertalot 2001). participant a denotes the central area as small, which characterises both species (lange-bertalot 2001). the striae orientation changes only once abruptly (bottom right in fig. 5) and three times gradually, which participant a thinks fi ts better to n. caterva than to n. reichardtiana (see discussion, i.e. remarks about discrepancies between the text that describes the species and the fi gures that depict the species in langebertalot 2001 and hofmann et al. 2011). in summary, participant a identifi es the valve in fig. 5 as n. caterva. interpretation and species identifi cation by participant b: participant b argues that the width of the valve in the given example (fig. 5) only so slightly exceeds the range of n. reichardtiana that this valve may still be identifi ed as n. reichardtiana. striae density (15.14 in 10 μm) fi ts the range of n. reichardtiana and is outside the range of n. caterva. similar to participant a, participant b denotes the central area as small, which characterises both species. the striae orientation changes abruptly once (bottom right, see fig. 5), which distinctly characterises n. reichardtiana according to participant b. in summary, participant b argues that the valve in fig. 5 more resembles n. reichardtiana than n. caterva. however, as the width is slightly outside of the range of n. reichardtiana and the striae orientation changes only gradually on three sides, participant b identifi es the valve in fig. 5 as n. cf. reichardtiana. fig. 5. example of different striae-counting approaches (vertical lines) from participant a (left) and b (right) on a valve from the navicula reichardtiana and n. caterva-group from sample lake geneva. scale bar (horizontal line) = 5 μm. tab. 1. results of measurements of participant a and b of the valve shown in fig. 5 of the navicula reichardtiana and n. caterva-group from sample lake geneva. participant a measured directly in μm while participant b measured in pixels and then converted into μm. in this given example 0.0549 pixels convert to 1 μm. participant length (μm) width (μm) striae/μm (pixel) striae/ 10 μmtop left top right bottom left bottom right a 15.93 4.78 8.25/5 8/5 8/5 – 16.17 b 16.36 4.78 5/3.3 (60) 5/3.4 (62) 5/3.3 (60) 4/2.6 (47) 15.14 dreßler m., verweij g., kistenich s., kahlert m., werner p. 224 acta bot. croat. 74 (2), 2015 both participants considered very carefully all traits when identifying th e valve in fig. 5. still, they arrived at different names for the very same valve. based on the different approaches of the two participants it is not surprising that they also named many other valves differently from each other. for example, when identifying 15 different valves from the n. reichardtiana and n. caterva-complex, participants a and b agreed only in fi ve cases and differed in the naming of the remaining ten valves. discussion the taxonomic problems that occurred in the intercalibration exercise for benthic diatoms and that we discuss in the following sections based on a few examples represent only a fraction of the problems that occur during routine diatom-counts in practise. interviews and discussions during the workshop of the intercalibration exercise identifi ed several reasons for deviating counting results. sometimes participants differed in the interpretation of the species specifi cations in the identifi cation literature or their results differed due to insuffi cient use of taxonomic specifi cations. in other cases the results differed due to partly ambiguous species descriptions and to misleading recommendations about which traits to use for species identifi cation or differentiation in the identifi cation literature and also due to currently insuffi cient taxonomic and ecological knowledge of some diatom taxa. 1. using different identifi cation literature the problem of using different identifi cation literature was especially apparent when identifying the small amphora-species and the varieties of cocconeis placentula. for example, amphora pediculus and a. indistincta occurred roughly with similar abundances in lake krossinsee. still, a. indistincta was detected only by fi ve of the 37 participants and by two auditors. one important reason for this discrepancy is the fact that some participants did not use the current and mandatory identifi cation literature (e.g. levkov 2009, hofmann et al. 2011). some participants only used krammer and lange-bertalot (1986–2004), which do not incorporate a. indistincta. thus, these participants did not fi nd any a. indistincta. instead a. indistincta valves from lake krossinsee were counted as a. pediculus or a. inariensis. this problem can be resolved fairly easily by using the new identifi cation literature (e.g. levkov 2009, hofmann et al. 2011 or 2013). similarly, participants used various identifi cation literatures when identifying the varieties of cocconeis placentula. this is especially problematic, because several morphological concepts exist for distinguishing the varieties of c. placentula (see e.g. hustedt 1930, geitler 1982, krammer and lange-bertalot 2004, jahn et al. 2009, romero and jahn 2013) that are concurrently in practical use. thus, in this case counting results can only be comparable, if the same literature is used or if the used literature is detailed and the concept is documented with pictures and a comprehensive and detailed accompanying text. 2. morphological variability another problem apparent from the intercalibration exercise is the relatively frequent presence of valves from certain diatoms that are outside the given morphometric ranges of the species in one or more traits. thus, identifi cation without ambiguity is not always possible. this phenomenon occurred commonly in all four taxonomic groups presented here benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 225 from the lake krossinsee and lake geneva samples. for example, the valves labelled a. cf. indistincta (pl. 1: 5–7) and a. cf. pediculus (pl. 1: 13–15) were distinctly too small and had striae that were too dense compared to their description in levkov (2009). most rapheless valves from the cocconeis placentula-aggregate from lake krossinsee had a striae density of ~24–26 in 10 μm (pl. 2: 32–45), corresponding to c. placentula var. placentula according to krammer and lange-bertalot (2004). however, the arrangement and the shape of the areolae rather correspond to c. placentula var. euglypta or c. placentula var. lineata according to krammer and lange-bertalot (2004). also, about 37% of the valves of the navicula cryptotenella and n. cryptotenelloides-complex from lake geneva had a width between 4.2–5.0 μm and ~ 61% of the lake krossinsee valves from this complex (pl. 3: 62–68, fig. 4). this width is exactly between the width of n. cryptotenella and n. cryptotenelloides and should not occur according to lange-bertalot (1993, 2001). on the contrary, the width is supposed to be a very good criterion to discriminate the two species (lange-bertalot 1993, 2001). additionally, most valves of the n. cryptotenella and n. cryptotenelloides-complex had a higher striae density than specifi ed in lange-bertalot (2001) (see fig. 4). at least two reasons may be responsible for the frequent occurrence of diatom-valves that are outside the given morphological description in some traits but that otherwise fi t well to the species specifi cations: (1) some described species have a greater morphological range than in the given species specifi cations (see e.g. wolf et al. 2002, dressler and hübener 2006). (2) some species specifi cations describe species-groups instead of species, i.e. they include several similar species that have not been examined suffi ciently neither taxonomically nor ecologically so far (see e.g. evans et al. 2008, jüttner et al. 2013). both reasons indicate some challenges for taxonomists that will improve diatom identifi cation in practise. one problem is that some type material for species descriptions contains only a few valves and that species descriptions are often only based on one or few sites or species populations. thus, some species descriptions include just a small part of the true morphologic variability and ecological range of a species. consequently, it would be desirable if the species descriptions and details in the identifi cation literature included measures that enable the applied limnologist and other diatomists to better assess the morphological and ecological variability of a taxon. for example, it would be desirable to know on how many valves from how many populations, samples and inland waters a species description is based on. 3. ambiguous taxa descriptions and documentations in the identifi cation literature besides the morphological variability of taxa described above, diatom-identifi cation is further complicated by ambiguous or relatively short taxa-descriptions in some cases. for example, in the german phylib-method for assessing water quality (schaumburg et al. 2006, 2011) hofmann et al. (2011) is listed as the mandatory and main identifi cation literature. they (hofmann et al. 2011) refer to krammer and lange-bertalot (2004) for the differentiation of the cocconeis placentula varieties. additionally, they recommend not differentiating the c. placentula varieties for now, when assessing the water quality with the german phylib-method (except cocconeis pseudolineata (geitler) lange-bertalot). thus, this latter comment could be responsible for just listing c. placentula in the intercalibration dreßler m., verweij g., kistenich s., kahlert m., werner p. 226 acta bot. croat. 74 (2), 2015 exercise in lake krossinsee by 14 laboratories, next to the morphological problems. however, when using the german phylib-method (schaumburg et al. 2006, 2011) a separation of the varieties is essential, as the indicator values of the c. placentula-varieties differ from one another (german water quality assessment software phylib, version 4.1; october 2012). thus, using the mandatory german water quality assessment software, the lumping or splitting of c. placentula (and varieties) will affect the results of the ecological water quality assessment. according to krammer and lange-bertalot (2004) the differentiation of the varieties should be based on the fi ne structures of the rapheless valves that are visible in the light microscope. accordingly, the varieties placentula and tenuistriata can be easily distinguished based on the striae density (krammer and lange-bertalot 2004). problems occur with the most common varieties, i.e. lineata and euglypta, as the striae density and number of areolae per stria of euglypta (19–22 striae in 10 μm and 3–5 areolae per stria) are entirely within the range of lineata (16–23 striae in 10 μm and 3–10 areolae per stria) (krammer and lange-bertalot 2004). additionally, the areolae of euglypta are supposed to be more robust compared to lineata, whereas the striae of lineata are supposed to appear distinctly dotted (krammer and lange-bertalot 2004). however, the differences between robust and distinct areolae remain unclear. the variety lineata is supposed to often have slit-like areolae which appear somewhat isolated from one another and an irregular or zigzag pattern of the longitudinal lines of areolae (areolae along the apical axis) (krammer and lange-bertalot 2004). overall, it remains diffi cult to clearly distinguish the varieties euglypta and lineata based on these rather vague traits. thus, these vague descriptions probably explain some of the inconsistent results of the intercalibration exercise with respect to the euglypta and lineata varieties. another problem is the mismatch between pictures and species description in the identifi cation literature for the varieties euglypta and lineata (e.g. krammer and lange-bertalot 2004). for example, the valves on plate 53, figures 17–18 (page 354) depict c. placentula var. euglypta in krammer and lange-bertalot (2004), but show distinctly more than fi ve areolae per stria and a pattern of the longitudinal lines of areolae that can be called irregular or an arrangement in a zigzag pattern (which should both be typical for lineata not euglypta according to krammer and lange-bertalot 2004). based on the currently inadequate state of information about the taxonomy and ecology of the varieties of c. placentula, it is diffi cult to make recommendations for the practical use (except: document your choices with pictures and text). further taxonomic and ecological work is necessary for a distinct differentiation of the c. placentula varieties. during the identifi cation of valves from the n. reichardtiana and n. caterva-complex two participants (a and b) carefully considered the whole combination of characters (see tab. 1) to identify the valve in fig. 5. however, they differ in their identifi cation of the valve in fig. 5 due to different approaches and interpretation, especially of the traits »striae density" and »changes of striae orientation towards the poles". it is diffi cult or even impossible to decide, which participant is correct, as there are some ambiguities that cannot be resolved. it is not documented in suffi cient detail in which way the describing authors counted the striae density. thus, no decision can be made about which measuring approach is more appropriate. ultimately, striae density should be measured in the same manner as the describing authors. however, this is often not known or documented. benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 227 the text of lange-bertalot (2001) and hofmann et al. (2011) describe that the striae orientation towards the poles of n. reichardtiana changes abruptly and of n. caterva gradually. however, the pictures in lange-bertalot (2001) and hofmann et al. (2011) do not always correspond to this description. for example, hofmann et al. (2011) depict valves of n. caterva with abruptly changing striae orientation towards the poles (pl. 31, fig. 38, striae top left and bottom left). the same valve is also depicted in lange-bertalot (2001) (fig. 7, pl. 33) and also named n. caterva. similarly, valves of n. reichardtiana are depicted with gradually changing striae orientation towards the poles in hofmann et al (2011) (pl. 31, figure 29 and 30, for both: top striae) and in lange-bertalot (2001) (pl. 13 fig. 26) (gradual 3-times except bottom right). thus, for identifying n. reichardtiana and n. caterva the question arises what to do with the mismatch between describing text and describing pictures in the identifi cation literature. currently, there is a rather wide range of possibilities to interpret the trait »striae orientation towards the poles«, which partly explains the different approaches of participants a and b (see tab. 1). again, it would be key to know how the describing authors interpreted this trait. there are more mismatches of pictures and species descriptions similar to the c. placentula-varieties and to n. reichardtiana / caterva in the identifi cation literature. for example, the areolae on the dorsal striae of a. pediculus should typically be visible in a light microscope according to levkov (2009) and hofmann et al. (2011), but are not discernable on most valves labelled a. pediculus in hofmann et al. (2011; page 785). overall, we recommend always using all three: the pictures, the measurable dimensions (e.g. width, length, striae density) and also the verbal species descriptions when identifying diatoms. in case of mismatching pictures and descriptions the applied limnologist should document with text and pictures the approach and reasoning taken to identify the diatom. the taxonomist may correct the current mismatches and avoid them in future by choosing the pictures more carefully or by providing appropriate explanations. 4. marking identifi cation uncertainties another substantial problem became apparent in the groups of navicula cryptotenella / n. cryptotenelloides, n. reichardtiana / n. caterva and the small amphora-species. for example, 37% (lake geneva) and 61% (lake krossinsee) of the measured 38 valves had a width between the width of n. cryptotenella and n. cryptotenelloides. still, only eight of the 37 participants and one auditor labelled the navicula cryptotenella / n. cryptotenelloides results with uncertainties by using cf. in either lake. similarly, part of the amphoravalves in the lake krossinsee sample deviated distinctly in their morphology from the descriptions in hofmann et al. (2011) and levkov (2009) (see pl. 1: 5–7, 13–15, 16–17). however, only four laboratories and two auditors indicated any uncertainty about the identifi ed small amphora-valves by using »spec.« and »cf.«. overall, it seems very likely that a. indistincta and a. pediculus have wider morphological ranges than given in the identifi cation literature (levkov 2009, hofmann et al. 2011). thus, for example the valves 5–7 (pl. 1) (a. cf. indistincta) and 13–15 (pl. 1) (a. cf. pediculus) may actually belong to their respective species. however, as long as further taxonomic and morphological examinations do not clarify the dimensional ranges, these and other valves that are outside their species specifi cations, should be counted with a »cf.« and be documented with a picture. dreßler m., verweij g., kistenich s., kahlert m., werner p. 228 acta bot. croat. 74 (2), 2015 one reason for submitting results with more certainty than actually present was identifi ed during the workshop of the intercalibration exercise. several participants reported that they noted deviations of some valves from the descriptions without labelling them with a cf. they reasoned that the german method for implementing the water framework directive (schaumburg et al. 2006, 2011) only allows a maximum of 5.0% diatom objects that could not be determined (sp., spp.) and/or could not unambiguously be determined (cf.). otherwise the diatom-indices are assumed to be unreliable and the sample cannot contribute to the water quality assessment. thus, the participants wanted to avoid the exclusion by indicating ambiguity (using cf.) as little as possible. as a consequence of this german method it is common practise to pool certain ambiguous taxa with other taxa to avoid labelling objects with »spec.« or »cf.« for the ecological water quality assessment to allow for a seemingly reliable assessment according to some participants. ultimately, this issue is also a psychological problem. a contractor may increase (or think to increase) the chances of future assignments without further quality control (such as participating in an intercalibration exercise), if he or she always delivers »good, clean and reliable results« and who seemingly identifi es all or almost all diatoms in a sample with certainty. thus, there is the danger that a low number of ambiguous taxa becomes an alleged quality attribute, especially for the german method. nonetheless, ambiguous taxa should be labelled with »cf.« or »spec.« also in practise (i.e. by the applied limnologist) and be documented with pictures to facilitate or allow a later comparison and verifi cation of counting results. in principle, quality assessments of counting results are very useful. however, the < 5%-cf cut-off is problematic, as there are samples in practise with less than 5% ambiguous taxa that contain only very few indicative taxa (werner and dressler 2007). thus, they would still be considered reliable according to the german method (schaumburg et al. 2006, 2007). in this respect a new quality assessment was introduced for lakes in the german method, whereby samples with < 60% indicative taxa are deemed unreliable (schaumburg et al. 2011). however, it is problematic that they retained the < 5%-cf cut-off in this context, because samples are still deemed unreliable that may have a high abundance (>> 60%) of indicative taxa. 5. ecological references and species identifi cation another problem for the applied limnologist is the recommendation in the identifi cation literature to use the ecological preferences of a taxon as an additional criterion for taxa identifi cation for some taxa. for example, in contrast to a. pediculus, amphora indistincta is supposed to live in nutrient-poor waters (hofmann et al. 2011). this may lead to the preclusion or disregard of certain taxa during the identifi cation process. however, in some cases the ecological preferences are not verifi ed suffi ciently. for example, a. indistincta and a. pediculus lived concurrently in lake krossinsee, despite their different ecological amplitude described in hofmann et al. (2011). diatom assemblages are used to infer the ecological class status of a water body in the routine counts for the european water framework directive. it would thus be a circular argument to use ecological preferences for identifying the diatom taxa, which in turn, are used to determine the water quality. thus, taxa that are not supposed to occur in a certain water body or lake/river-type should not be disregarded during the identifi cation process. benthic diatoms in germany: applicable approach acta bot. croat. 74 (2), 2015 229 conclusions the fi rst german intercalibration exercise for benthic diatoms identifi ed the following fi ve major issues and thus generated the subsequent recommendations. 1) use of different identifi cation literature led to different species names of the same taxa. 2) some diatom valves of the intercalibration exercise had morphological dimensions outside the given species ranges to a certain degree in one or more traits. 3) participants and auditors also allocated different names to the same taxa due to ambiguous species descriptions and certain recommendation of traits that should be used to differentiate species in the mandatory identifi cation literature. also, the pictures do not always correspond to the species description in the literature, which led to further deviation among counting results. 4) some taxa were reported with more certainty than actually present, i.e. despite morphologic deviations from the defi ned ranges in the identifi cation literature the valves were not labelled with a »cf.«. 5) insuffi cient knowledge about the ecology of some species with misleading recommendations based on this ecology for some diatoms led to different naming. recommendation for diatom counts by applied limnologists: during routine diatom counts in practise we recommend to carefully consider all traits defi ning a species and to use the mandatory identifi cation literature. also, for problematic taxa or ambiguous taxa the actually used traits for identifi cation should be specifi ed and the used identifi cation books should be specifi ed to the contracting authority, as it may make a difference if you, for example, only use hofmann et al. (2011) or if you can additionally use krammer (1997a, b) when identifying encyonopsis ssp. or encyonema ssp.. additionally, we recommend a photographic documentation of ambiguous taxa (spec., aff., cf.) for a better comparability of counting results among diatomists and for the possibility of a later adjustment of the results according to new research results. ambiguous diatom valves should be labelled with »spec.«, »cf.« or »aff.« in the counting results. in the german method for water quality assessment using diatoms (schaumburg et al. 2011) the relative abundance of 5% ambiguous taxa (or more) should not make the results of a count unreliable. instead, this measure should be replaced by other measures, such as the percentage of indicative taxa in a sample that contributed to the water quality assessment that should be above a certain threshold. recommendations for taxonomists: when describing new diatom-taxa and when compiling identifi cation books the taxonomist should keep in mind that his or her work will also be used by the applied limnologist and not just by fellow scientists. accordingly, ambiguous documentations should be avoided, i.e. all traits necessary for identifi cation should be depicted in detail and without ambiguity and according to unambiguous text descriptions. for an assessment of the possible morphological variability of a taxon the taxonomist needs to provide basic data, i.e. on how many valves are the given ranges in the identifi cation literature based on, from how many samples and/or populations and from how many different inland waters (sites). similarly, data about the ecology of each taxon should be part of the basic data (if available). also important is the method description that was used to generate the measured ranges, espedreßler m., verweij g., kistenich s., kahlert m., werner p. 230 acta bot. croat. 74 (2), 2015 cially of striae density. surely, these recommendations have often already been followed, but this intercalibration exercise demonstrates that there is room for improvement. overall, the recommendations for the applied limnologist may help to reduce the error when assessing the water quality with diatoms. an improved method that already works well will remain an important tool for water managers and may thus be used even more often. also, the intercalibration exercise revealed the need for funding fundamental diatom research, as the taxonomist can help to further improve the use of diatoms as bioindicators by investigating species specifi cations. acknowledgements our thanks go to all 37 participants of the fi rst german intercalibration exercise, the auditors (gabriele hofmann, thomas hübener and peter pfi ster), sven adler for the statistical analyses and andreas meybohm for the administrative operation of the intercalibration exercise and for providing the internet platform (http://www.planktonforum.eu/). references dressler, m., hübener, t., 2006: morphology and ecology of cyclostephanos delicatus (genkal) casper & scheffl er (bacillariophyceae) in comparison with c. tholiformis stoermer, håkansson & theriot. nova hedwigia 82, 409–434. dressler, m., schwarz, a., hübener, t., adler, s., scharf, b. w., 2011: use of sedimentary diatoms from multiple lakes to distinguish between past changes in trophic state and climate: evidence for climate change in northern germany during the past 5,000 years. journal of paleolimnology 45, 223–241. eu-wfd 2000: european water framework directive 2000, directive 2000/60/ec of the european parliament and of the council of 23 october 2000 establishing a framework for the community action in the fi eld of water policy. offi cial journal l 327. evans, k. m., wortley, a. h., simpson, g. e., chepurnov, v. a., mann, d. g., 2008: a molecular systematic approach to explore diversity within the sellaphora pupula species complex (bacillariophyta). journal of phycology 44, 215–231. geitler, l., 1982: die infraspezifi schen sippen von cocconeis placentula des lunzer seebachs. algological studies 30, 1–11. hofmann, g., werum, m., lange-bertalot, h., 2011: diatomeen im süßwasser-benthos von mitteleuropa. bestimmungsfl ora kieselalgen für die ökologische praxis. über 700 der häufi gsten arten und ihre ökologie. a.r.g. gantner verlag k.g., rugell. hofmann, g., werum, m., lange-bertalot, h., 2013: diatomeen im süßwasser-benthos von mitteleuropa. bestimmungsfl ora kieselalgen für die ökologische praxis. über 700 der häufi gsten arten und ihre ökologie. 2. korrigierte aufl age. koeltz scinetifi c books, königstein. hustedt, f., 1930: bacillariophyta (diatomeae). in: pascher, a. 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2000: diatoma fl ora of marine coasts. iconographia diatomologica 7. koeltz scientifi c books, königstein. wolf, m., scheffler, w., nicklisch, a. 2002: stephanodiscus neoastraea and stephanodiscus heterostylus (bacillariophyta) are one and the same species. diatom research 17, 445–451. acta bot. croat. 77 (1), 2018 1 acta bot. croat. 77 (1), 1–9, 2018 coden: abcra 25 doi: 10.1515/botcro-2017-0017 issn 0365-0588 eissn 1847-8476 comparative analysis of specialized metabolites and antioxidant capacity in vitro of different natural populations of globularia spp. maja friščić1*, semir maslo2, rade garić3, željan maleš1, kroata hazler pilepić 1 1 department of pharmaceutical botany, faculty of pharmacy and biochemistry, university of zagreb, zagreb, croatia 2 lundåkerskolan, gislaved, sweden 3 institute for marine and coastal research, university of dubrovnik, dubrovnik, croatia abstract – total phenolic, flavonoid, condensed tannin and iridoid content, as well as antioxidant capacity in vitro, were determined spectrophotometrically in methanolic extracts of different plant parts of the mediterranean medicinal plant globularia alypum l. and three widespread european species of the same genus: g. cordifolia l., g. meridionalis (podp.) o. schwarz and g. punctata lapeyr. in order to consider possible environmental influences on the production of specialized metabolites, each species, except g. alypum, was collected from three different natural populations. great variations in the amounts of specialized metabolites were observed among different plant parts and species. for example, total phenolic content ranged from 10.13 (g. punctata, flowers) to 44.90 (g. cordifolia, flower stems) mg gallic acid equivalent g–1 dry weight. moreover, great differences, attributed to location-specific environmental factors, were observed among different populations of the same species. for example, a strong positive correlation was observed among mean monthly temperatures and total phenolic contents in the leaves of studied globularia spp. (r = 0.75, p = 0.019). however, despite these differences, all species were rich in bioactive substances when compared to g. alypum, especially in their aerial parts. a very good positive correlation was observed between total phenolic content and dpph radical scavenging capacity (r = 0.86, p < 0.001)/abts radical scavenging capacity (r = 0.83, p < 0.001). the results obtained show that g.  cordifolia, g. meridionalis and g. punctata are rich in bioactive substances, providing support for their pharmaceutical utilization. further investigations are needed to verify the possibility of their medicinal use. keywords: antioxidant activity, environmental factors, flavonoids, globularia, iridoids, polyphenols, proanthocyanidins, secondary metabolites * corresponding author, e-mail: mfriscic@pharma.hr introduction plants have been used as healing agents since ancient times; many bioactive compounds isolated from herbal sources have been used as drugs or served as lead compounds in drug development. in europe, herbal medicines in the crude forms of teas and decoctions are often used as supportive therapy, while standardized herbal preparations are a popular alternative to synthetic drugs. finally, about 80% of the world population (primarily in developing countries) still uses herbal medicine in the treatment of different diseases and in maintaining health (gurib-fakim 2006). the old world genus globularia l., recently included in the plantaginaceae family (albach et al. 2005), consists of perennials, subshrubs and small shrubs. some members of the genus, mainly globularia alypum l., g. arabica jaub. & spach and g. trichosantha fisch. & c.a. mey., are used in the traditional medicine of countries such as spain, italy, morocco, algeria, tunisia, libya, egypt and turkey (leporatti and ghedira 2009, altundag and ozturk 2011, carrió and vallès 2012, de natale and pollio 2012, bouzabata 2013, eissa et al. 2014, el abbouyi et al. 2014). they are traditionfriščić m., maslo s., garić r., maleš ž., hazler pilepić k. 2 acta bot. croat. 77 (1), 2018 ally used as hypoglycaemic agents, purgatives, depuratives (de natale and pollio 2012, bouzabata 2013), antiparasitic and antifungic agents (altundag and ozturk 2011, de natale and pollio 2012), tonics and diuretics, for wound healing and in the treatment of insomnia, fits of epilepsy, gastrointestinal disorders, intermittent fever (leporatti and ghedira 2009, de natale and pollio 2012, eissa et al. 2014), arthritis and rheumatism (de natale and pollio 2012). phenolic compounds and iridoids are the main specialized (secondary) metabolites of globularia species (kirmizibekmez et al. 2008, kirmizibekmez et al. 2009, tundis et al. 2012b). g. alypum, the most widely used member of the genus globularia, was shown to be especially rich in phenolic compounds in comparison with some other medicinal plants (djeridane et al. 2006, djeridane et al. 2010, amessis-ouchemoukh et al. 2014, el guiche et al. 2015). phenolic compounds possess a wide range of biological activities and thus may contribute to the healing properties of globularia preparations. radical scavenging activity, also attributed to plant phenolics, is one of the possible protective mechanisms against cancer, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases (rice-evans et al. 1997). it was noted that g. alypum extracts possess high antioxidant capacity both in vitro (djeridane et al. 2010, amessis-ouchemoukh et al. 2014) and in vivo (taleb-dida et al. 2011). antioxidant activity was also recently reported in g. meridionalis (tundis et al. 2012a). the aim of the present study was to analyse and compare the total phenolic content, including flavonoid and condensed tannin content, iridoid content and antioxidant capacity in four members of the genus globularia l. three of these, namely g. cordifolia l., g. meridionalis (podp.) o. schwarz and g. punctata lapeyr., are less-investigated, although they have a relatively wide distribution in europe (tutin 1972). in order to examine their therapeutic potential with regard to the content of bioactive substances, their results were compared to those of the well-investigated medicinal plant g. alypum l., which is distributed mainly in the mediterranean area. since synthesis and distribution of specialized metabolites is complex and differs between tissues and organs (boudet 2007), the study was focused on different plant parts. the analysis was done on material collected from three different locations in order to consider possible ecological influences on the production of the specialized metabolites investigated. exceptionally, g. alypum was collected from the only location at which it grows wild in croatia and was sampled without underground parts because of its near threatened status. obtained phytochemical data were correlated with environmental factors of different habitats, enabling the interpretation of obtained results from both medicinal and ecological perspectives. materials and methods plant material and extraction a total of ten samples of four globularia taxa were collected during the phenophase of blooming from seven locations in croatia and one in bosnia and herzegovina (tab. 1). voucher specimens are deposited in the herbarium of the department of pharmaceutical botany, faculty of pharmacy and biochemistry, university of zagreb, croatia. the identity of plant material was verified by prof. kroata hazler pilepić. meteorological data were obtained from the nearest meteorological stations (meteorological and hydrological service tab. 1. collection and geographical data for plant material of the investigated globularia species; ga – globularia alypum, gc – g. cordifolia, gm – g. meridionalis, gp – g. punctata. species (voucher no.) location geographical latitude geographical longitude elevation (m) habitat type harvest time ga (16 020) dubrovnik area (konavle cliffs) 42°30´50˝n 18°19´07˝e 15 limestone cliffs by the sea march 2013 gc (1) (16 032) northern velebit area (alan) 44°43´15˝n 14°58´05˝e 1340 calcareous grasslands may 2013 gc (2) (16 031) middle velebit area (baške oštarije) 44°31´41˝n 15°08´38˝e 917 calcareous grasslands may 2012 gc (3) (16 030) mostar area (the neretva banks) 43°21´41˝n 17°48´20˝e 57 rocky grasslands along a river may 2012 gm (1) (16 041) istrian peninsula (mala učka) 45°17´59˝n 14°11´27˝e 926 limestone cliffs may 2012 gm (2) (16 040) rijeka area (grobničko polje) 45°22´39˝n 14°30´53˝e 308 karst field may 2013 gm (3) (16 043) middle velebit area (baške oštarije) 44°31´41˝n 15°08´38˝e 917 calcareous grasslands may 2012 gp (1) (16 051) istrian peninsula (vižintini) 45°23´36˝n 13°51´20˝e 386 calcareous grasslands may 2012 gp (2) (16 056) rijeka area (grobnik field) 45°22´39˝n 14°30´53˝e 308 karst field may 2013 gp (3) (16 057) žumberak area (slapnica canyon) 45°44´29˝n 15°29´26˝e 325 limestone cliffs may 2013 specialized metabolites of globularia spp. acta bot. croat. 77 (1), 2018 3 of croatia and the federal hydrometeorological institute of bosnia and herzegovina) (tab. 2). dried and powdered plant parts (2.5 g) were subjected to ultrasound-assisted extraction (bandelin sonorex super, germany) at room temperature for 30 min with 25 ml of methanol. the residue after filtration was extracted again for 30 min with 25 ml of methanol, filtered and the final volume was adjusted to 50 ml. four classes of metabolites were measured spectrophotometrically using a varian cary 50 bio uv-vis spectrophotometer (usa). determination of plant specialized metabolites the total phenolic content was determined using folinciocalteu’s reagent according to the method of singleton and rossi (1965). the reaction mixture was prepared by mixing 0.5 ml of methanolic extract with 2.5 ml of 10% (v/v) folin-ciocalteu’s reagent (diluted in distilled water). after 5 min, 2 ml of 7.5 g/100 ml sodium carbonate decahydrate solution were added and the mixture was incubated for 1 h at room temperature. after incubation, the absorbance was read at 765 nm against a distilled water blank. gallic acid was used for the construction of the calibration curve. the total phenolic content was expressed as mg gallic acid equivalent (gae) g–1 dry weight (dw). the flavonoid content was measured using the dowd method (arvouet-grand et al. 1994). briefly, 1 ml of appropriately diluted extract was mixed with 1 ml of 2 g/100 ml alcl3 solution in pure methanol. the absorbance was measured after 15 min at 415 nm against a sample blank which consisted of 1 ml of extract and 1 ml of methanol. a standard calibration curve was plotted using quercetin as a reference standard. the results were expressed as mg quercetin equivalent (qe) g–1 dry weight (dw). the condensed tannin (proanthocyanidin) content was determined using the vanillin assay (broadhurst and jones 1978, sun et al. 1998) as described previously by toda (2005), but with a slightly modified reaction temperature. for the preparation of the sample 2 ml of 1 g/100 ml vanillin in 7 m sulfuric acid were added to a test tube containing 1 ml of diluted extract and the mixture was incubated for 15 min in a water bath with the temperature set at 30±1 °c. the reaction was performed in normal laboratory daylight. after the incubation, the absorbance was recorded at 500 nm against a sample blank which consisted of 1 ml of extract and 2 ml of 7 m sulfuric acid, which was incubated under the same conditions as the sample. (+)–catechin was chosen as a standard for the calibration curve. the levels of total condensed tannin content were expressed as mg catechin equivalent (ce) g–1 dry weight (dw). the iridoid content was measured using the trimhill reagent (trim and hill 1952) as adapted by tundis et al. (2012a). to 200 µl of diluted extract 2 ml of trimhill reagent (glacial acetic acid:0.2% copper (ii) sulfate pentahydrate:concentrated hydrochloric acid at a ratio of 10:1:0.5, v:v:v) were added and the mixture was heated in a boiling water bath for 5 min. after that, absorbance of the prepared solution was read at 609 nm with methanol used as blank. the concentration of iridoids in the samples was calculated based on an aucubin calibration curve and the results were presented as mg aucubin equivalent (ae) g–1 dry weight (dw). evaluation of antioxidant capacity antioxidant capacity of the extracts was evaluated by the blois method (1958) using 2,2-diphenyl-1-picrylhydrazyl (dpph) radical solution according to the procedure of thetsrimuang et al. (2011) with some modifications. a 0.1 mm stock solution of dpph in methanol was prepared. the working solution was obtained by diluting the stock solution with methanol to obtain an absorbance of 0.7±0.02. to 2 ml of this solution, 10 µl of properly diluted extract were added and the decrease in absorption of the radical was measured at 517 nm after 30 min incubation in the dark against a methanol blank. a calibration curve was obtained by ustab. 2. records of mean monthly temperatures and monthly precipitation amounts from meteorological stations close to the locations where globularia species were collected (for details see tab. 1). records are shown for the month in which the plant material was harvested (m), the three previous months (m-3, m-2, and m-1) as well as their average; ga – globularia alypum, gc – g. cordifolia, gm – g. meridionalis, gp – g. punctata. plant species mean monthly temperature (°c) monthly precipitation amounts (mm) meteorological station (elevation (m)) m-3 m-2 m-1 m average m-3 m-2 m-1 m average ga 9.6 10.1 9.5 11.2 10.1 283.2 205.2 238.1 214.0 235.1 dubrovnik (52) gc (1) –6.4 –3.1 3.8 5.6 0.0 284.6 293.9 152.7 253.4 246.2 zavižan (1594) gc (2) –5.0 7.3 9.8 13.4 6.4 58.2 1.0 116.2 102.0 69.4 gospić (564) gc (3) 1.7 13.5 13.2 18.6 11.8 202.7 0.3 266.6 92.3 140.5 mostar (99) gm (1) 0.7 8.9 11.7 15.5 9.2 30.1 1.3 54.2 104.4 47.5 letaj (120) gm (2) 5.1 8.0 14.3 16.4 11.0 210.1 386.9 80.0 228.3 226.3 rijeka (120) gm (3) –5.0 7.3 9.8 13.4 6.4 58.2 1.0 116.2 102.0 69.4 gospić (564) gp (1) 0.2 8.8 11.4 15.3 8.9 11.9 0.0 61.4 105.6 44.7 pazin (291) gp (2) 5.1 8.0 14.3 16.4 11.0 210.1 386.9 80.0 228.3 226.3 rijeka (120) gp (3) 1.5 4.4 12.9 15.8 8.7 93.7 121.1 50.2 107.0 93.0 maksimir (128) friščić m., maslo s., garić r., maleš ž., hazler pilepić k. 4 acta bot. croat. 77 (1), 2018 ing different concentrations of gallic acid and the results were expressed as mg gallic acid equivalent (gae) g–1 dry weight (dw). the 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) radical scavenging capacity was determined following the method of re et al. (1999) with some modifications. an activated solution of abts˙+ radical cation was prepared by mixing abts and potassium peroxodisulfate solutions so that the final concentrations in the mixture were 7 mm and 2.45 mm, respectively. the solution was held at room temperature (22±2 °c) for at least 16 h before use. the working solution was obtained by diluting the stock solution with distilled water to obtain an absorbance of 0.7±0.02. the fall of absorbance was measured at 734 nm against a distilled water blank 1 min after mixing 10 µl of properly diluted extract with 2 ml of activated radical. abts radical scavenging activity was calculated from a gallic acid calibration curve and presented as mg gallic acid equivalent (gae) g–1 dry weight (dw). statistical analysis all measurements were performed in triplicate and the results are presented as means ± standard deviations. a twoway analysis of variance (anova) followed by the bonferroni post-hoc test was carried out on averaged results for plant parts of each species, to determine significant differences among the same plant parts of different species and different plant parts of the same species. pearson’s correlation coefficient (r) was used to determine the association among parameters, more precisely, among the contents of particular groups of specialized metabolites (total phenolics, flavonoids, condensed tannins and iridoids) in all samples (altogether 48 samples) and their antioxidant capacities evaluated by two different assays (dpph and abts) or among the contents of particular groups of specialized metabolites in specific plant parts and averages of mean monthly temperatures and monthly precipitation amounts of individual locations (maximum of ten samples per analysis), with a significance level α = 0.05. the statistical analysis was carried out using graphpad prism 5.03 for windows (graphpad software, san diego, usa). results content of plant specialized metabolites all four investigated species were found to be rich in phenolic compounds, with observed differences among plant parts (tab. 3). the content of total phenolics ranged from 10.13 (g. punctata (3), flowers) to 44.90 (g. cordifolia (1), flower stems) mg gae g–1 dw. high amounts of polyphenols were observed in leaves of all species, with no significant differences noticed between g. alypum and related species. leaves and flowers of g. alypum were the richest plant parts for this species. on the other hand, it was observed that flower stems of g. cordifolia and g. meridionalis, as well as woody stems and underground parts of g. punctata, also contained high amounts of phenolic compounds. flowers of g. punctata were the poorest of all the tested samples (p < 0.05). the flavonoid content ranged from 0.84 (g. cordifolia (3), woody stems) to 17.77 (g. punctata (2), leaves) mg qe g–1 dw (tab. 3). when different plant parts between species were compared, it was observed that the leaves contained the highest amounts of flavonoids. g. punctata leaves and flower stems contained more flavonoids than those of other species (p < 0.05), while there were no significant differences among the species for flowers and underground parts. the lowest amounts of flavonoids were observed for woody stems and underground parts. condensed tannin (proanthocyanidin) content varied from 0.19 (g. meridionalis (2), underground parts) to 9.77 (g. cordifolia (3), flower stems) mg ce g–1 dw (tab. 3). taking into account all plant parts, condensed tannin content was especially high in the population collected from mostar. green aerial parts contained more tannins than woody stems and underground parts. in g. cordifolia, leaves and flower stems contained the highest amounts of tannins (p < 0.05), while for other species no significant differences were observed among plant parts. the content of iridoids varied from 3.94 (g. punctata (3), underground parts) to 143.29 (g. punctata (1), leaves) mg aucubin equivalent (ae) g–1 dw (tab. 3). high amounts of iridoids were found in g. cordifolia, g. meridionalis and g. punctata, especially in their leaves and flower stems (p < 0.05). no significant differences in iridoid content were noticed between different organs of g. alypum. correlations between phenolic/iridoid content and environmental factors in order to verify the influence of specific environmental factors on the variability of the compounds determined, results obtained for each plant part were correlated with mean monthly temperatures and monthly precipitation amounts of different stands and seasons (tab. 2). averages for the month in which the plant material was collected and the three previous months were correlated with the amounts of investigated compounds. during evaluation of the influence of temperature, the population of mala učka had to be excluded due to the great elevation difference between the location of sampling (926 m) and the nearest meteorological station (120 m), from which meteorological data were available, which made it impossible to accurately predict the mean monthly temperatures for this location. taking this into account, however, a strong positive correlation was observed between mean monthly temperatures and total phenolic contents in the leaves of investigated species (r = 0.75, p = 0.019), together with a strong negative correlation in the flower stems (r = –0.88, p = 0.002). in spite of the significant variability discovered in the iridoid contents among populations, no correlation with mean monthly temperatures (p > 0.05) was found. also, no significant correlation between precipitation and amounts of bioactive substances was observed (p > 0.05). specialized metabolites of globularia spp. acta bot. croat. 77 (1), 2018 5 tab. 3. total phenolic, flavonoid, condensed tannin and iridoid contents as well as radical scavenging capacities obtained by 2,2-diphenyl-1-picrylhydrazyl (dpph) and 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) assays, in different plant parts of four globularia species collected from different locations (for details see tab. 1). values are means ± sd, n = 3; statistically significant differences (p < 0.05) between the same plant parts of different species are indicated by different superscript lower case letters (a > b > c > d) and between different plant parts of the same species in different superscript capital letters (a > b > c > d); * – flowers of the plant are carried by woody stems; ga – globularia alypum, gc – g. cordifolia, gm – g. meridionalis, gp – g. punctata; l – leaves, f – flowers, fs – flower stems, ws – woody stems, up – underground parts; n.m. – not measured; gae – gallic acid equivalent, dw – dry weight, qe – quercetin equivalent, ce – catechin equivalent, ae – aucubin equivalent. plant species plant part polyphenols (mg gaeg–1 dw) flavonoids (mg qe g–1 dw) tannins (mg ce g–1 dw) iridoids (mg ae g–1 dw) dpph (mg gae g–1 dw) abts (mg gae g–1 dw) ga l 37.58±0.85aa 9.23±0.39ba 0.76±0.03ba 9.38±0.55ca 18.29±2.64aa 12.35±0.81aa f 35.39±0.78aa 5.46±0.04ab 0.77±0.01aa 6.18±0.08aa 20.48±2.03aa 14.41±0.56aa fs = ws* 21.16±0.72cb 4.74±0.04cb 0.34±0.03ba 5.06±0.04ba 11.67±0.37ab 8.09±0.37bb up n.m. n.m. n.m. n.m. n.m. n.m. gc (1) l 27.67±0.82 9.08±0.36 2.55±0.07 77.34±1.54 9.98±0.97 8.18±0.02 f 25.68±0.70 7.12±0.08 2.30±0.10 24.67±0.81 7.96±0.68 7.52±1.02 fs 44.90±1.18 9.56±0.15 4.17±0.07 57.56±2.25 14.70±1.43 12.52±1.98 ws 22.22±0.51 2.00±0.01 0.28±0.01 8.37±0.48 9.12±0.96 7.91±0.43 up 22.15±1.03 1.96±0.07 0.21±0.00 6.03±0.14 9.91±0.22 7.56±0.10 gc (2) l 34.42±0.94 9.24±0.15 1.92±0.02 132.43±1.18 11.28±1.90 11.53±0.14 f 20.06±0.45 4.55±0.02 1.40±0.05 28.74±3.16 7.22±2.27 7.86±0.28 fs 33.31±0.32 6.73±0.09 3.24±0.08 51.04±0.63 11.45±0.52 8.95±0.19 ws 14.55±0.10 0.93±0.01 0.33±0.00 6.64±0.01 5.19±0.04 7.27±0.16 up 16.54±0.90 1.36±0.13 0.26±0.01 4.92±0.47 5.96±0.26 6.47±0.13 gc (3) l 33.33±0.96 9.50±0.27 8.01±0.17 98.25±2.47 10.36±0.49 10.37±0.32 f 18.88±0.28 3.73±0.09 4.53±0.04 40.56±0.98 5.44±0.44 5.83±0.15 fs 23.66±0.52 7.59±0.07 9.77±0.20 100.51±1.31 9.80±0.08 8.73±0.33 ws 13.61±0.56 0.84±0.01 0.75±0.01 10.51±0.04 4.53±1.17 6.24±0.13 up 20.37±0.72 1.45±0.02 0.66±0.01 9.95±0.25 8.71±0.91 8.30±0.16 gc l 31.81±3.62aab 9.27±0.21ba 4.16±3.35aa 102.70±27.81aba 10.54±0.67ba 10.03±1.70aa average f 21.54±3.63bcbc 5.13±1.77ab 2.74±1.61aab 31.32±8.25ac 6.87±1.30ba 7.07±1.09ba fs 33.96±10.63aa 7.96±1.45ba 5.73±3.53aa 69.70±26.88ab 11.98±2.49aa 10.07±2.13ba ws 16.79±4.72cc 1.26±0.65dc 0.45±0.26bb 8.51±1.94bc 6.28±2.48ba 7.14±0.84ba up 19.69±2.87bbc 1.59±0.32ac 0.38±0.25ab 6.97±2.64ac 8.19±2.03aa 7.44±0.92ba gm (1) l 23.03±0.29 6.96±0.08 1.38±0.00 85.89±5.76 10.16±0.22 9.47±0.34 f 21.69±0.78 5.16±0.03 1.74±0.02 37.65±2.01 9.89±0.08 8.33±0.16 fs 29.50±0.55 6.07±0.03 2.12±0.02 62.61±1.42 14.07±0.85 11.36±0.11 ws 17.56±0.04 1.02±0.02 0.34±0.00 8.54±0.12 5.76±0.82 7.68±0.07 up 26.05±0.68 2.25±0.04 0.39±0.00 15.98±0.53 9.91±0.18 10.01±0.28 gm (2) l 35.18±0.22 11.32±0.17 1.77±0.03 70.50±0.77 12.08±1.01 9.95±0.67 f 18.28±0.29 5.35±0.07 1.86±0.12 20.50±1.55 7.04±0.61 4.62±0.10 fs 29.38±0.45 6.37±0.24 2.78±0.09 40.94±3.89 8.19±1.06 8.50±0.31 ws 20.00±0.17 1.71±0.09 0.24±0.00 9.29±0.49 6.34±0.10 5.67±0.16 up 17.13±0.53 1.08±0.01 0.19±0.01 6.20±0.22 5.73±1.20 5.38±0.15 gm (3) l 30.81±0.45 8.40±0.10 1.71±0.05 114.08±7.72 9.49±1.08 10.02±0.18 f 27.25±0.85 4.09±0.03 1.60±0.02 25.19±1.60 6.82±2.20 6.66±0.24 fs 37.76±1.22 7.86±0.09 3.55±0.11 49.46±4.80 13.19±0.17 9.96±0.92 ws 16.80±0.54 0.90±0.02 0.41±0.01 8.27±0.13 6.59±0.29 7.99±0.30 up 15.86±0.19 1.40±0.09 0.26±0.01 5.04±0.15 5.25±0.25 5.25±0.22 gm l 29.67±6.15aab 8.89±2.22ba 1.62±0.21aba 90.16±22.10ba 10.58±1.34ba 9.81±0.30aa average f 22.41±4.53bab 4.87±0.68ab 1.73±0.13aa 27.78±8.86abc 7.92±1.71ba 6.54±1.86ba fs 32.21±4.80aba 6.77±0.96bcab 2.82±0.72aba 51.00±10.92ab 11.82±3.17aa 9.94±1.43ba ws 18.12±1.67cb 1.21±0.44dc 0.33±0.09ba 8.70±0.53bc 6.23±0.43ba 7.11±1.26ba up 19.68±5.55bb 1.58±0.60ac 0.28±0.10aa 9.07±0.60ac 6.96±2.56aa 6.88±2.71ba gp (1) l 35.31±1.31 14.46±0.20 1.68±0.04 143.29±7.15 15.32±1.04 12.22±0.24 f 14.02±0.25 6.76±0.05 0.84±0.02 15.26±0.41 4.18±0.42 4.34±0.01 fs 24.60±0.59 13.93±0.25 0.97±0.04 86.75±1.62 12.91±0.57 7.53±0.29 ws 34.96±0.57 3.71±0.04 0.58±0.02 8.53±0.18 16.94±1.09 18.95±0.50 up 32.68±0.59 2.47±0.02 0.43±0.01 4.37±0.33 11.38±0.77 12.53±0.47 friščić m., maslo s., garić r., maleš ž., hazler pilepić k. 6 acta bot. croat. 77 (1), 2018 correlations between phenolic/iridoid content and antioxidant capacity in this study, all tested samples showed antioxidant activity, which was in very good correlation with observed amounts of total phenolic compounds (r = 0.86, p < 0.001 for dpph; r = 0.83, p < 0.001 for abts). poor correlation was observed between flavonoid content and dpph radical scavenging capacity (r = 0.32, p < 0.05) (tab. 4). g. alypum leaves and flowers showed higher antioxidant activity than those of related species in the dpph assay, while in the abts assay only flowers were significantly different (p < 0.05) (tab. 3). discussion comparison to previous studies of g. alypum and evaluation of obtained results in the present study, the medicinal plant g. alypum served as a control species, with which all other investigated species were compared. the reason for this was its broad and well-documented medicinal use together with a number of studies highlighting the high contents of its specialized metabolites and pronounced antioxidant activity. determination of specialized metabolites was conducted according to the same procedures as those used in previous studies of g. alypum and related species (djeridane et al. 2006, khlifi et al. 2011, tundis et al. 2012a, amessis-ouchemoukh et al. 2014, taghzouti et al. 2016, touaibia and chaouch 2016) to enable a more reliable comparison with literature data. amounts of total phenolics and flavonoids observed for g. alypum in this study were comparable to those in previously reported studies (djeridane et al. 2006, djeridane et al. 2010, chograni et al. 2012), in which they were expressed in the same manner as in our study, per g dry weight of plant (djeridane et al. 2006), not per g dry extract, as in some other studies. the latter, unsurprisingly, resulted in several times higher values (khlifi et al. 2011, amessis-ouchemoukh et al. 2014, taghzouti et al. 2016, touaibia and chaouch 2016). the present study also shows that green aerial parts of globularia species contain higher amounts of specialized metabolites, with the exception of total phenolics, which were also observed to be high in woody stems and underground parts of g. punctata. leaves of globularia species were rich in all investigated bioactive substances. this could explain why they are frequently used plant parts in folk medicine (de natale and pollio 2012, bouzabata 2013, el abbouyi et al. 2014). however, it is important to notice that the use of flowers (bouzabata 2013) and aerial parts (de natale and pollio 2012) is also known, which could be explained by the high amounts of polyphenols found in g. alypum flowers and their high antioxidant activity as observed in this study, as well as their recently reported high catalpol content (sertić et al. 2015) and various biological activities such as antioxidative, anti-inflammatory and acetylcholinesterase inhibitory activity (amessis-ouchemoukh et al. 2014). two of the most commonly used assays for estimating radical scavenging activity are the 2,2-diphenyl-1-picrylhydrazyl (dpph) assay and the 2,2´-azino-bis(3-ethylbenplant species plant part polyphenols (mg gaeg–1 dw) flavonoids (mg qe g–1 dw) tannins (mg ce g–1 dw) iridoids (mg ae g–1 dw) dpph (mg gae g–1 dw) abts (mg gae g–1 dw) gp (2) l 36.10±0.45 17.77±0.87 1.49±0.04 125.83±1.79 11.71±0.23 10.59±0.15 f 11.19±0.13 5.52±0.24 1.16±0.07 8.02±0.43 3.44±0.06 2.36±0.12 fs 19.50±0.27 12.55±0.21 1.55±0.07 72.79±1.47 5.12±0.22 5.50±0.26 ws 30.79±0.27 2.78±0.00 0.30±0.01 6.84±0.28 9.65±0.57 12.05±0.06 up 30.87±0.47 2.71±0.01 0.30±0.00 4.76±0.02 9.90±0.22 12.07±0.18 gp (3) l 30.46±1.57 14.46±0.58 2.38±0.02 110.32±1.14 10.33±0.71 13.24±1.40 f 10.13±0.23 5.57±0.07 0.56±0.03 9.44±0.29 3.38±0.54 2.20±0.14 fs 22.38±0.69 11.65±0.13 2.11±0.07 75.59±1.03 8.92±0.08 5.45±0.96 ws 44.77±0.49 2.84±0.11 0.60±0.00 9.95±0.52 18.33±1.05 16.24±1.37 up 35.20±0.83 2.75±0.07 0.41±0.01 3.94±0.08 12.88±0.89 9.39±0.50 gp l 33.96±3.05aab 15.56±1.91aa 1.85±0.47aba 126.50±16.49aa 12.45±2.58bab 12.02±1.34aab average f 11.78±2.01cc 5.95±0.70ac 0.85±0.30aa 10.91±3.84ac 3.67±0.45bc 2.97±1.19cc fs 22.16±2.56bcbc 12.71±1.15ab 1.54±0.57ba 78.38±7.39ab 8.98±3.90abc 6.16±1.19cc ws 36.84±7.18aa 3.11±0.52cdd 0.49±0.17ba 8.44±1.56bc 14.97±4.66aa 15.75±3.48aa up 32.92±2.18aab 2.64±0.15ad 0.38±0.07aa 4.36±0.41ac 11.39±1.49aab 11.33±1.70ab tab. 3. continued tab. 4. pearson’s correlation coefficients between total phenolic, flavonoid, condensed tannin and iridoid content and 2,2-diphenyl1-picrylhydrazyl (dpph) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) radical scavenging capacity in different plant parts of four globularia species collected from different locations. n = 48; statistically significant correlations are indicated by an asterisk (*) for p < 0.05 and three asterisks (***) for p < 0.001. dpph abts polyphenols 0.86*** 0.83*** flavonoids 0.32* 0.17 tannins 0.08 0.05 iridoids 0.22 0.20 specialized metabolites of globularia spp. acta bot. croat. 77 (1), 2018 7 zo (abts) assay (sánchez-moreno 2002). both assays were previously used by different authors for estimating the antioxidant capacity of g. alypum and showed the species to be a good source of antioxidants (djeridane et al. 2006, essafi et al. 2007, djeridane et al. 2010, khlifi et al. 2011, chograni et al. 2012). strong positive correlations between the total phenolic content and antioxidant capacity, similar to those observed in the present study, were also found in earlier studies (djeridane et al. 2006, khlifi et al. 2011, chograni et al. 2012). however, the antioxidant activity observed for g. alypum leaves and flowers was not statistically different (p > 0.05) from that of previous reports (chograni et al. 2012, amessis-ouchemoukh et al. 2014). djeridane et al. (2006) noticed higher antioxidant activity in g. alypum and other plant extracts in which phenolic acids predominated in comparison to those extracts containing only flavonoids. this could explain why only a poor correlation between flavonoid content and dpph radical scavenging capacity was found in our study. evaluation of medicinal potential of g. cordifolia, g. meridionalis and g. punctata the high amounts of polyphenols and iridoids found in g. cordifolia, g. meridionalis and g. punctata suggest these species have potentially beneficial health effects similar to those of g. alypum. however, it should be noted that the method used for iridoid determination, could only detect the aucubin and asperuloside type iridoids (trim and hill 1952), while catalpol derivatives, which are the predominant iridoid glycosides of g. alypum (chaudhuri and sticher 1981), could not be estimated (harborne 1998). however, the three species were recently also shown to possess higher amounts of aucubin than g. alypum, with g. punctata having higher catalpol contents as well (sertić et al. 2015). high iridoid content in the green aerial parts of g. cordifolia, g. meridionalis and g. punctata observed in this study could be explained by the presence of various aucubin and asperuloside derivatives, which have been previously isolated from these species (chaudhuri and sticher 1980, kirmizibekmez et al. 2003, tundis et al. 2012b). indeed, much higher asperuloside amounts have been observed in g. cordifolia, g. meridionalis and g. punctata than in g. alypum (friščić et al. 2016). similarly to our results, lower amounts of iridoids were previously reported for g. meridionalis underground parts in comparison to its aerial parts (tundis et al. 2012a). although in most cases, there were no significant differences observed in the amounts of specialized metabolites of the three species, g. punctata seems to contain more polyphenols in the woody parts and more flavonoids in the leaves and flower stems. a high catalpol content in the flowers of this species was also recently observed (sertić et al. 2015). on the other hand, g. cordifolia and g. meridionalis gave similar results in all performed assays (i.e., no statistically significant differences between the two species were observed). this is not surprising considering their close relationship (tutin 1972). although these species could not be distinguished based on the used spectrophotometric assays, it should be noted that the comparison of the proanthocyanidin content indicated a somewhat characteristic chemical composition of the g. cordifolia population collected from mostar. the same phenomenon was observed after an analysis of the essential oil composition of different globularia populations (crkvenčić et al. 2016). according to our study, the antioxidant activity of all four species is in good correlation with their total phenolic content. because g. cordifolia, g. meridionalis and g. punctata are more widely distributed in europe, it would be interesting to investigate if they have other biological activities that are similar to those of g. alypum. some researches in this field have already yielded promising results. for example, g. meridionalis was recently shown to possess inhibitory activity on acetylcholinesterase and butyrylcholinesterase, enzymes representing the targets for the symptomatic treatment of alzheimer’s disease (tundis et al. 2012a). iridoid glucoside globularifolin, found both in g. cordifolia (chaudhuri and sticher 1980) and g. meridionalis (tundis et al. 2012b), was also recently reported to possess immunomodulatory activity (sipahi et al. 2014). production of plant specialized metabolites with respect to environmental factors our results confirmed the assumption that phenolic and iridoid concentrations can vary significantly between populations of the same species collected from different locations (tab. 3) and thus provided a justification for including more populations of each species when comparing different species harvested from natural habitats. as mentioned before, g. alypum was presented with only one population. however, the amounts of its specialized metabolites could be compared to numerous literature data. it is known that environmental factors have a major influence on the synthesis of plant specialized metabolites causing notable differences in the yields of biologically active compounds among different populations. the correlations observed in this study indicate a possible shift in the polyphenolic production and/or translocation of phenolic compounds from the flower stems to the leaves under the influence of increased temperature. on the other hand, the higher tannin content found in all plant parts of g. cordifolia from mostar area could be connected with its location on the humid neretva river banks, having in mind that all other samples were collected from well-drained areas with full sun exposure. it was previously reported that tannin-rich plants grew mostly on infertile soils with poor drainage (kraus et al. 2003). knowing that iridoids play a major role in defending plants against pathogens or herbivores (dobler 2011), stimuli other than those considered in the present study seem to be more important for their production. bearing in mind that, along with the genetically determined variations of metabolite production, plant specialized metabolites represent a way of adaptation to environmental factors (ghasemzadeh and ghasemzadeh 2011), the yield of bioactive compounds can be significantly different among friščić m., maslo s., garić r., maleš ž., hazler pilepić k. 8 acta bot. croat. 77 (1), 2018 populations. this fact is very important for users of native medicinal plants. investigations of specialized metabolites with the aim of checking or predicting the medicinal potential of a given plant species should therefore be conducted on several plant populations, when possible. unfortunately, this 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(eds.), flora europaea (vol. 3), 282–283. cambridge university press, cambridge. 80 acta bot. croat. 77 (1), 2018 acta bot. croat. 77 (1), 80–87, 2018 coden: abcra 25 doi: 10.1515/botcro-2017-0012 issn 0365-0588 eissn 1847-8476 in vitro multiplication, micromorphological studies and ex vitro rooting of hybanthus enneaspermus (l.) f. muell. – a rare medicinal plant mahipal s. shekhawat1*, m. manokari2 1 biotechnology laboratory, department of plant science, m. g. g. a. c. mahe, puducherry, india 2 department of botany, kanchi mamunivar centre for postgraduate studies, puducherry, india abstract – hybanthus enneaspermus is a rare medicinal plant. we defined a protocol for micropropagation, ex vitro rooting of cloned shoots and their acclimatization. surface-sterilized nodal segments were cultured on murashige and skoog (ms) medium with different concentrations of 6-benzylaminopurine (bap) and kinetin (kin). medium supplemented with 1.5 mg l–1 bap was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. shoots were further proliferated maximally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on ms medium augmented with 1.0 mg l–1 each of bap and kin within 4–5 weeks. the shoots were rooted in vitro on half strength ms medium containing 2.0 mg l–1 indole-3 butyric acid (iba). the cloned shoots were pulse-treated with 300 mg l–1 of iba and cultured on soilrite® in a greenhouse. about 96% of the iba-pulsed shoots rooted ex vitro in soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. the ex vitro rooted plantlets showed a better rate of survival (92%) in a field study than in vitro rooted plantlets (86%). a comparative foliar micromorphological study of h. enneaspermus was conducted to understand the micromorphological changes during plant developmental processes from in vitro to in vivo conditions in terms of variations in stomata, vein structures and spacing, and trichomes. this is the first report on ex vitro rooting in h. enneaspermus and the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant. key words: conservation, ex vitro rooting, hybanthus enneaspermus, micromorphology, micropropagation, rare medicinal plant * corresponding author, e-mail: smahipal3@gmail.com burning sensations, urinary infections, leucorrhoea, dysuria and sterility (tripathy et al. 2009). the plant is also valued for its antimicrobial (retnam and britto 2007), antiplasmodial, antiarthritic (subramoniam et al. 2013), antimalarial, antirheumatic, emmenagogic, sedative, antispasmodic, antiasthmatic, anti-infertility (nathiya and selvi 2013), antibacterial, anticonvulsant, antidiabetic, antifungal (arumugam et al. 2011), anti-allergic and analgesic, antinociceptive, antioxidant and aphrodisiac properties (kumar et al. 2013). this plant contains various important phytochemicals like dipeptide alkaloids, aurantiamide acetate, isoarborinol and βsitosterol, flavonoids, steroids, triterpenes, phenols, tannin, glycosides etc. (krishnamoorthy et al. 2014). there is no commercial cultivation of h. enneaspermus and the plants are collected from wild sources. it is facing genetic threat due to sporadic distribution, poor germinaintroduction hybanthus enneaspermus (l.) f. muell. (formerly ionidium suffruticosum ging.), belongs to the family violaceae. it is a rare multipotent herb, endemic to the deccan peninsula in india with various invigorating properties (prakash et al. 1999, sudeesh 2012). it is a small suffrutescent perennial herb found in india, sri lanka, tropical asia, africa and australia (anand and gokulakrishnan 2012). the plant grows up to 15–30 cm in height with many diffused branches (kirtikar and basu 1991). h. enneaspermus is traditionally known as padmavati, lakshmisheshta, padmacharini or purusharathna in india and considered a valuable healing herb in the indian systems of medicine (satheeshkumar 2011). it is well documented in the folklore medicine of india for its aphrodisiac and stimulant activity (awobajo et al. 2009). h. enneaspermus has therapeutic applications. moreover, the whole plant is used to treat diarrhea, painful dysentery, and strangury, mailto:smahipal3@gmail.com micropropagation of hybanthus enneaspermus acta bot. croat. 77 (1), 2018 81 tion of seeds, anthropogenic activities, overgrazing and over exploitation by herbal drug manufacturers (arunkumar and jayaraj 2011, verma and singh 2011). it has been disappearing from the large area of the western ghats of india due to widespread cultivation of rubber in the natural habitat of this plant (joseph et al. 2000). h. enneaspermus is conventionally propagated through seeds. the seeds show poor viability and germination in the wild (arunkumar and jayaraj 2011). conventional propagation methods are unable to meet the demand of the pharmaceutical industries and drug research. therefore, it is necessary to develop a non-conventional method for propagation to fulfill the demands of the drug market (rathore et al. 2008). in vitro propagation methods offer a powerful tool for conservation of germplasm and mass-multiplication of threatened plant species (murch et al. 2000). they can support the in situ and ex situ conservation of this rare genotype. since natural propagation is unable to support the demand, in vitro methods could be viable options. some in vitro work on h. enneaspermus is available in the literature (arunkumar and jayaraj 2011, velayutham et al. 2012, premkumar et al. 2013, sudharson et al. 2014). the present work is more effective in terms of number of multiple shoots regenerated per explant. survival of plantlets in field conditions is the major constraint in the micropropagation of h. enneaspermus. an ex vitro rooting method could help in better acclimatization which increases the chances of field adaptation of plantlets in the natural environment. improved rooting and acclimatization can be achieved simultaneously with ex vitro rooting of in vitro propagated shoots (baskaran and van staden 2013).this was found to reduce time, labor, energy involved and the cost factor of micropropagated plantlets (patel et al. 2014). therefore, the aim of the present study is to establish in vitro methodologies for mass production of this rare plant species using ex vitro rooting and to evaluate the optimum conditions for in vitro development of plantlets. this is the first report on ex vitro rooting of in vitro regenerated shoots in h. enneaspermus. the widespread application of in vitro regeneration technologies is restricted by the difficulties during transfer of plantlets to the field conditions (pospíšilová et al. 1999). this is due to the sudden change in the culture environment to relatively harsh environments. the ultimate success of micropropagation depends on successful hardening and field transfer of plantlets. the plants micropropagated in a culture vessel are partially heterotrophic; they acquire some developmental changes to make them fully autotrophic after being transferred to the field. the present study also aimed to investigate the foliar epidermal micromorphological changes during transfer of plantlets from an in vitro to a field environment. materials and methods plant material and surface sterilization hybanthus enneaspermus was selected from the coromandel coast (kanchipuram, villupuram, puducherry, cuddalore, nagapattinam and karaikal districts) of india for the present study. slender young emerging stems were used as the source of explants. the nodal segments (approximately 3.0 cm in length) were harvested from two months old field-grown plant using sterilized surgical scissors. these explants were sterilized with a systemic fungicide (0.1% bavistin; basf india ltd., india) and then under laminar air flow bench with 0.1% hgcl2 (w/v) for 4–5 min. the sterilized explants were washed with autoclaved double distilled water 5–6 times to remove the adhered traces of hgcl2. medium and culture conditions murashige and skoog medium (murashige and skoog 1962) augmented with 3% sucrose as carbon source and 50 mg l–1 of ascorbic acid and 25 mg l–1 each of arginine, adenine sulphate and citric acid were incorporated in the culture medium as additives to initiate the cultures. culture medium was solidified by 0.8% agar (hi-media, india) to support the proper position of the plant material in the medium. the ph of the medium along with plant growth regulators was adjusted to 5.8±0.02 prior to autoclaving. the cultures were maintained at 25±2 °c under a 12 h photoperiod light regime with a light intensity of 40–50 μmol m−2 s−1 photosynthetic photon flux density (ppfd) implemented by cool white fluorescent lamps (philips, india). culture initiation and multiple shoot induction to establish cultures in vitro, stout, green nodal explants were inoculated on ms medium containing different concentrations of cytokinins (6-benzylaminopurine, bap; kinetin, kin) (hi-media, india) ranging from 0.5–3.0 mg l–1 to induce bud break. cultures showing bud break were further multiplied by subsequent transfer of in vitro regenerated axillary shoot clumps (5–7 shoots) with mother explants by subculturing onto fresh ms medium. the medium was supplemented with additives, bap and kin (0.1 to 2.0 mg l–1) alone or combinations of optimized concentrations. subculturing was performed at 4 weekly intervals. in vitro rooting shoots can be harvested after 3–4 subcultures for rooting experiments. for root induction under in vitro conditions, multiplied shoots longer than 4–5cm were separated individually from shoot clumps and transferred to different strengths of ms media (full ms, ½ ms and ¼th ms) with various concentrations of auxins (indole-3 acetic acid, iaa; indole-3 butyric acid, iba; α-naphthalene acetic acid, naa and naphthoxy acetic acid, noa) (hi-media, india) (1.0 to 4.0 mg l–1). the cultures were initially incubated under diffused light conditions (20–25 μmol m−2 s−1) for 2–3 days for in vitro root induction and thereafter transferred to an in vitro culture environment, and maintained at a light intensity of 40–45 μmol m−2 s−1 ppfd with a 12 h photoperiod per day. ex vitro rooting of in vitro regenerated shoots experiments were carried out for ex vitro root induction from in vitro-produced shoots. basal end (4–6 mm) of in vishekhawat m. s., manokari m. 82 acta bot. croat. 77 (1), 2018 tro-raised shoots were treated with different concentrations of auxins (iaa, iba, naa and noa) (50–400 mg l–1) for 5 min and transferred to eco-friendly paper cups containing sterile soilrite® (a combination of perlite with peat moss and exfoliated vermiculite procured from kelperlite, bangalore, india) and moistened with one fourth strength of ms basal salts. the cups were kept in the greenhouse for maintenance at 25±2 °c with 80–90% relative humidity (rh). after 4–5 weeks, the rooted plantlets were carefully taken out from the paper cups and transplanted to the nursery polybags in a greenhouse. acclimatization and field transfer of regenerated plantlets in vitro rooted plantlets were taken out cautiously from the culture tubes and rinsed with distilled water to remove adhered nutrients and agar. they were transferred to autoclaved soilrite® in bottles, moistened with 1/4th strength of ms basal salts and maintained in the greenhouse. the ex vitro rooted plantlets were acclimatized in paper cups which were covered with transparent polythene cups to provide enough space for gas exchange. the in vitro rooted plantlets were acclimatized by gradual loosening and then completely removing the transparent cup of the bottles. these plantlets were subsequently transferred to nursery polybags containing soilrite®, garden soil and organic manure (1:1:1) in the greenhouse for further acclimatization process. plantlets were transferred to the field after 5 weeks of acclimatization. foliar micromorphological studies of in vitro and field transferred plantlets experiments were conducted to study foliar micromorphological developments of veins (vein density and venation pattern), stomata types and density, and trichomes in leaves of plants grown in vitro after 4th subculture in multiplication phase and in those transferred to the field after 6th week. plants were randomly selected from both the environments. the entire foliar apparatus (leaves) (10 from each stage of plantlets) third to seventh leaves from the base were excised manually for all the experiments. to observe the changes in structure and functioning of developing stomata, epidermal peels were separated manually by the traditional method (johansen 1940) from the leaves. the leaves were fixed primarily in formalin–acetic acid–ethyl alcohol, faa (1:1:3) and cleared in 70% ethanol (v/v) until the chlorophyll was removed (12–24 h), bleached with 5% (w/v) naoh for 24–48 h, and rinsed three times in distilled water (sass 1940). the leaves were then stained with 1% safranine (loba chemie, india) aqueous solution for 4–8 min and rinsed carefully in water to remove excess stain and then mounted in distilled water and examined under microscope (labomed ivu 3100, usa). experimental design, data collection and statistical analysis the experiments were performed with 20 replicates per treatment and repeated thrice. data were subjected to analysis of variance and the significance of differences was calculated by duncan’s multiple range test using spss software (version 16.0). observations were noted at 4 weeks interval. results establishment of cultures and multiplication of cultures in vitro the fresh and light green colored nodal segments responded better than old and dark colored explants. cultures were initially placed in diffused light (20–25 μmol m−2 s−1) to induce bud breaking, and further transferred to a culture room with a higher light intensity (40–50 μmol m−2 s−1) for proper establishment of culture. the ms medium supplemented with 1.5 mg l–1 bap was observed suitable for bud breaking and 97% of the explants responded with 6.4±0.69 shoots from each nodal explants (fig. 1a, tab. 1). maximum tab. 1. effect of different concentrations of cytokinins, 6-benzylaminopurine (bap) and kinetin (kin) on bud breaking from nodal explants of hybanthus enneaspermus. significant variation between the concentrations was studied using duncan’s multiple range test at 0.5% level, sd – standard deviation. cytokinins (mg l–1) response (%) number of shoots (mean ± sd) shoot length (cm) (mean ± sd) 0.00 0 0.0±0.00a 0.00±0.00a bap 0.50 32 2.3±1.06abc 3.12±0.31bcd 1.00 56 4.2±0.28bcd 3.40±0.18cd 1.50 97 6.4±0.69d 5.60±0.49e 2.00 76 4.9±1.02cd 4.16±0.38de 2.50 50 4.1±0.57bcd 3.28±0.33cd 3.00 37 2.3±1.09abc 2.06±0.12bc kin 0.50 26 2.1±0.54abc 1.32±0.43ab 1.00 41 2.9±0.76bc 2.07±0.64bc 1.50 68 3.0±0.43ab 2.78±0.21bcd 2.00 59 3.6±0.47bc 3.08±0.30bcd 2.50 50 2.0±0.53ab 3.12±0.36bcd 3.00 40 2.1±0.49ab 2.17±0.17bc fig. 1. micropropagation of hybanthus enneaspermus: induction of shoots from the nodal segments with 6-benzylaminopurine (a); multiplication of shoots on ms medium (b); in vitro rooting of the excised shoots on ms medium with indole-3 butyric acid (c); ex vitro rooted plantlets (d), scale bars = 2 cm. micropropagation of hybanthus enneaspermus acta bot. croat. 77 (1), 2018 83 tab. 2. effect of different concentrations and combinations of cytokinins 6-benzylaminopurine (bap), kinetin (kin), and combination of bap + kin on shoot multiplication of hybanthus enneaspermus. significant variation between the concentrations was studied using duncan’s multiple range test at 0.5% level, sd – standard deviation. cytokinins (mg l–1) number of shoots (mean±sd) shoot length (cm) (mean±sd) 0.00 0.0±0.00a 0.00±0.00a bap 0.10 14±0.10ab 4.0±0.54b 0.50 25±0.93abc 6.4±0.32hi 1.00 42±1.51d 6.6±0.23i 1.50 40±7.37d 5.3±0.17ef 2.00 34±6.24cd 6.4±0.62hi kin 0.10 19±8.91bc 5.0±0.44de 0.50 22±6.74bc 4.3±0.33bc 1.00 36±5.13cd 4.6±0.63cd 1.50 29±7.11abc 5.1±1.70def 2.00 20±8.83bc 4.9±0.54de bap + kin 0.10 98±8.76e 5.6±1.30fg 0.50 172±9.27g 5.9±1.05gh 1.00 228±10.3h 7.5±0.43j 1.50 121±10.1f 6.1±0.79ghi 2.00 93±9.22e 5.1±0.43def ex vitro rooting of in vitro produced shoots the basal end of in vitro-produced micro-shoots was treated (5 min) with root-inducing growth regulators and transferred to a greenhouse environment. the lower part (4– 6 mm) of in vitro-regenerated shoots evaluated with 300 mg l–1 iba exhibited about 96% rooting (tab. 4). a maximum of 12.5±0.54 roots per shoot with 5.10±0.62 cm length was observed within 4 weeks (fig. 1d). poorer rooting than with iba was recorded with all the concentrations of iaa, naa, and noa in this study. acclimatization and field transfer of regenerated plantlets in vitro and ex vitro rooted plants were acclimatized efficiently in a greenhouse (figs. 2a–2c). a profusely branched root system was observed in ex vitro rooted plantlets during transfer to the field. it resembled the conventional root system obtained under natural conditions. the hardened plantlets were successfully transferred to the field with 92% survival rate (fig. 2d) but the survival rate of in vitro rooted plantlets was only 86%. micromorphological studies of micropropagated plantlets the plants developed under in vitro conditions possessed normal leaves with hairs and denticulate margins. the midtab. 3. effect of auxins indole-3 acetic acid (iaa), indole-3 butyric acid (iba), α-naphthalene acid acid (naa), and naphthoxy acetic acid (noa) on in vitro root induction, number and length of roots on half strength ms medium. significant variation between the concentrations was studied using duncan’s multiple range test at 0.5% level, sd – standard deviation. auxins (mg l–1) response (%) number of roots (mean±sd) length of root (cm) (mean±sd) 0.00 0 0.00±0.00a 0.0±0.00a iaa 1.0 26 12.9±0.16d 2.98±0.83h 2.0 40 16.6±10.75h 3.00±0.38h 3.0 34 16.0±1.56gh 2.35±0.33f 4.0 31 14.8±0.40f 2.05±0.20de iba 1.0 63 18.5±3.43i 2.15±0.26e 2.0 98 25.7±3.90k 6.21±0.78k 3.0 71 21.0±2.37j 4.18±0.39j 4.0 59 13.7±1.70e 3.24±0.49i naa 1.0 29 10.5±0.80b 1.78±0.13c 2.0 33 13.6±0.75de 2.90±0.23h 3.0 49 15.9±0.23gh 1.90±0.91cd 4.0 32 11.5±0.10c 1.95±0.26cd noa 1.0 30 11.9±1.43c 2.71±0.83g 2.0 42 13.6±1.90de 3.32±0.38i 3.0 54 15.3±2.37fg 2.93±0.33h 4.0 50 10.5±1.20b 1.43±0.20b response of the explants was observed on ms medium augmented with bap rather than with kin in present study. the maximum number of shoots (228±10.3 shoots with 7.5±0.43 cm length) was regenerated on a subculture of the in vitro regenerated shoot clumps on ms medium fortified with 1.0 mg l–1 each of bap and kin within 4–5 weeks (tab. 2). the shoot number and shoot length was increased by repetitive subculturing up to 3–4 passages onto fresh medium (fig. 1b). six fresh shoots from a single explant transferred to fresh medium yielded a maximum of 228 shoots (228/6=38) within 4–5 weeks. the rate of shoot multiplication increased more than 35 fold in 4–5 weeks. cream colored callus was observed from the basal part of the cultures if the medium was augmented with auxins (iaa and iba) along with cytokinins. in vitro rooting of the shoots among the different strengths of ms medium and auxins experimented for in vitro root induction, ½ strength ms medium supplemented with 2.0 mg l–1 iba was observed best for in vitro rooting (fig. 1c). maximum number of roots (25.7±3.90) with highest length (6.21±0.78 cm) were recorded with this medium combination (tab. 3). poor response with smaller root number and root length was reported on media fortified with iaa, naa and noa. shekhawat m. s., manokari m. 84 acta bot. croat. 77 (1), 2018 rib was fairly prominent projecting equally on both the sides, but bluntly conical on the adaxial side and hemispherical on the abaxial side. veins and vein-islets were fewer in in vitro than in field transferred plantlets (figs. 3a and 3b). the vein density and distinct vein-islets were increased during the hardening period, and became distinct, rhomboidal and rectangular in shape after field transfer of the plantlets. the stomata were more frequent in the inter-coastal areas than in the coastal areas, facing all directions with irregular distribution. the stomatal frequency was greater in the in vitro environment than in the field transferred plants with anisocytic stomata predominating, but these were nonfunctional as they were always in open condition (figs. 3c and 3d). anisocytic (cruciferous), paracytic (rubiaceous), diacytic (caryophyllaceous), anomocytic (rununculaceous), anisotricytic, isotricytic, tetracytic, staurocytic, desmocytic and pericytic stomata were observed in the in vitro produced leaves. anomocytic and pericytic stomata were occasionally observed in these leaves. the field-transferred plants possessed the aforementioned stomatal types except for the anomocytic and pericytic. anisocytic and paracytic stomata were prominent but diacytic and desmocytic stomata were rare in this plant. trichomes were simple, unicellular or uniseriate emerging from the epidermis. under in vitro conditions, the trichomes were unicellular, less frequent and underdeveloped but these were fully developed in field transferred plants after 6 weeks (fig. 3e). the uniseriate and unicellular hairs were frequent but the bicellular and tricellular hairs were occasionally observed after plantlets were transferred to the field (fig. 3f). adaxial surface possessed numerous shaggy trichomes, and the trichome density was found maximum in field transferred plantlets compared to fig. 2. hardening of hybanthus enneaspermus plantlets in the greenhouse (a-c), and in vitro raised plantlets under field conditions (d). fig. 3. micromorphological studies of hybanthus enneaspermus: venation pattern in leaves of in vitro shoots (a); and field plant (b); stomatal pattern in leaves of in vitro shoots (c), and field plant (d); and trichomes in leaves of in vitro shoots (e), and field plant (f). tissues were stained with 1% safranine aqueous solution. scale bars = 100 µm. tab. 4. effect of auxins indole-3 acetic acid (iaa), indole-3 butyric acid (iba), α-naphthalene acid acid (naa), and naphthoxy acetic acid (noa) on ex vitro root induction from in vitro raised shoots. significant variation between the concentrations was studied using duncan’s multiple range test at 0.5% level, sd – standard deviation. auxins (mg l–1) response (%) number of roots (mean±sd) length of root (cm) (mean± sd) 0.00 2 0.43±0.13a 1.21±0.32b iaa 50 30 0.45±0.30a 1.19±0.54b 100 45 0.62±0.11e 2.56±0.36h 200 51 1.93±0.15f 4.01±0.44m 300 50 3.22±0.32f 3.92±0.19l 400 43 2.07±0.28cd 2.05±0.30g iba 50 35 0.96±0.21c 3.15±0.73k 100 42 2.78±0.59h 3.84±0.61l 200 56 4.89±0.41i 4.10±0.49n 300 96 12.5±0.54l 5.10±0.62p 400 84 5.83±0.49j 4.25±0.53o naa 50 23 0.39±0.33a 1.03±0.69a 100 35 0.73±0.21b 1.41±0.74d 200 47 2.73±0.19h 2.05±0.21g 300 44 6.21±0.48l 3.91±0.28l 400 39 6.10±0.30k 3.01±0.35j noa 50 33 0.11±0.45c 1.33±0.30c 100 47 0.29±0.29d 1.49±0.49e 200 56 2.39±0.16g 1.90±0.24f 300 41 3.26±0.72h 2.94±0.92j 400 38 2.30±0.64d 2.70±0.62i micropropagation of hybanthus enneaspermus acta bot. croat. 77 (1), 2018 85 the in vitro grown leaves. mucilaginous cells were also observed in field transferred plants but these were totally absent in the in vitro grown plantlets. discussion the success of tissue culture experiments basically depends on the selection of starting material. the mature explants responded later than the fresh and light green colored nodal segments under diffused light conditions in the present experiment. bap induced more shoots on ms medium than kin. similar results were reported by many researchers recently in a number of plant species (panwar et al. 2012, premkumar et al. 2013, rathore et al. 2013a, sudharson et al. 2014). shoot multiplication was achieved by repetitive transfer of mother explants with regenerated shoots onto fresh medium and by subculturing of freshly regenerated shoots isolated from the mother explants. this approach of shoot multiplication has been used in several plant species (rai et al. 2010, patel et al. 2014, shekhawat and manokari 2016). the higher rate of shoot multiplication during repeated transfer may be due to inhibition of apical dominance which stimulates the basal dormant meristematic cells to produce young shoots (phulwaria et al. 2013). a maximum of 228 shoots were induced per culture vessel within 4–5 weeks in this study. premkumar et al. (2013) induced the most (52.3) shoots, when the regenerated shoots were subcultured on ms medium containing iaa along with kin and bap. contrary to this report, callus formation was observed when the medium was supplemented with iaa and iba along with bap and kin in the present study. sudharson et al. (2014) reported maximum of 11.8 shoots, when the cultures of h. enneaspermus were inoculated on ms medium supplemented with 2.0 mg l–1 bap. maximal 90 shoots were reported in this plant by velayutham et al. (2012) from the callus cultures on ms medium augmented with bap and kin. this supports our findings where the most shoots were regenerated on bap and kin, but our results were far better than the earlier reports in multiple shoot formation. the shoots were rooted maximally on half strength ms medium augmented with iba. the half strength ms salts and sucrose in medium was appropriate for in vitro rooting and supports many authors’ findings in different plant species (rai et al. 2010, premkumar et al. 2013, patel et al. 2014). we report more roots (25.7±3.90) per shoot in this study than were found in earlier works on h. enneaspermus. maximum 2.8 roots per shoot was reported by prakash et al. (1999), 5–8 roots by velayutham et al. (2012) and 21.3 roots by premkumar et al. (2013) in this plant species. the superiority of iba over other auxins for root induction has been recognized by several researchers in a number of plants (barreto and nookaraju 2007, rai et al. 2010, rathore et al. 2013b). plants rooted under ex vitro environment were better suited to natural conditions and reported easy to harden (yan et al. 2010). it has been reported that ex vitro rooted plants are better suited to tolerate environmental stresses (pospíšilová et al. 1999, tiwari et al. 2002, shekhawat et al. 2015a). about 96% shoots were rooted with iba with maximum 12.5 roots per shoot in this report. iba is more effective than naa and noa in ex vitro root induction in many plant species, and applied economically worldwide (debergh et al. 1992, yan et al. 2010, ranaweeraa et al. 2013, shekhawat et al. 2015b). this is the first report on ex vitro rooting of in vitro regenerated shoots in h. enneaspermus with maximum rate of survival under natural conditions. the rooted plantlets were hardened in greenhouse with development of profusely branched root system in ex vitro rooted plantlets. about 92% ex vitro rooted and 86% in vitro rooted plantlets survived in the field conditions. ex vitro rooting reduced the time, energy of production of plantlets and mortality during hardening and field transfer. normal flowering and fruiting was observed in the field transferred plantlets. these micromorphological studies of micropropagated plantlets were performed to understand the developmental changes in the leaves of plantlets, when they were transferred to field conditions. the stomata were present on both surfaces of the leaf but the frequency was less on the adaxial surface (narayanaswamy et al. 2006, retnam and britto 2007) therefore, the abaxial surface was further considered for the study. the specific artificial conditions in vitro are responsible for the structural changes occurring in micropropagated plantlets. the lesser stomatal density under field conditions may help to check the rate of transpiration and prevent water loss (singh et al. 2003). transitional types of stomata between anisocytic and paracytic are also present in h. enneaspermus (inamdar 1969). anisotricytic and isotricytic stomata could be the transitional form between anisocytic and paracytic types of stomata. unicellular, less frequent and underdeveloped trichomes were observed under in vitro conditions but fully developed trichomes were reported in field-transferred plants. the mucilaginous cells were not observed with the in vitro leaves but found in field-transferred plants. our findings are supported by the results of various researchers (chandra et al. 2010, rathore et al. 2013b, lodha et al. 2015). understanding the changes in foliar micromorphology of in vitro grown and hardened plantlets could be useful in improvement of in vitro clonal propagation protocols and for large scale production of plants. conclusion an efficient in vitro propagation protocol has been developed using various plant growth regulators for successful conservation of this rare plant species. excellent rate of shoot multiplication was achieved in vitro. ex vitro rooting has been successfully demonstrated in h. enneaspermus, which could save time, labor and energy in production of plantlets. the hardened plantlets were successfully transferred to the field with a 92% survival rate. the results of foliar micromorphological studies could help in understanding the response of plantlets under field conditions. the data could contribute significantly to meeting the market demand for this multipotent healing herb and conservation of this valuable genotype through biotechnological interventions. shekhawat m. s., manokari m. 86 acta bot. croat. 77 (1), 2018 acknowledgements the authors are grateful to the university grants commission, new delhi, government of india and the department of science, technology and environment, government of puducherry for providing financial support 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new fern species for the fl ora of montenegro danijela stešević1*, christian berg2 1 faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81 000 podgorica, montenegro 2 institute of plant sciences, karl-franzens-university graz, holteigasse 6, 8010 graz, austria abstract – in this short communication we report a new fern for montenegro – botrychium matricariifolium (döll) a. braun ex w.d.j. koch, known as the chamomile grapefern. up to now, the only botrychium species in the fl ora of montenegro was b. lunaria (l.) sw., a typical species for montane to alpine grasslands and meadows. one individual of b. matricariifolium was found in a forest opening on the mountain babji zub. b. matricariifolium was accompanied by numerous individuals of b. lunaria. these species are clearly distinguished by the lamina, which is in b. matricariifolium 2-pinnatifi d, while in b. lunaria it is 1-pinnate with trapezoid to fl abellate pinnae. although chamonile grapefern has a large range of distribution, it is everywhere a rare species that has some kind of protection status in the most european countries. in order to defi ne the protection status of the species in montenegro, further investigation is needed. keywords: botrychium, ferns, montenegro introduction the genus botrychium belongs to the fern family ophioglossaceae, easily recognizable by separate sporophore and trophophore leaf structures, limited secondary growth in the rhizome, sheathing leaf bases, circular bordered pits, subterranean and non-chlorophyllous gametophytes, and the absence of circinate venation, root hairs, and sclerenchyma (wagner 1990, dauphin et al. 2014). in narrow sense the genus contains 30 species (hauk et al. 2003, dauphin et al. 2014), distributed throughout europe, north america, asia, australia, africa (atlas mountains), the pacifi c islands, new zealand, and patagonia (south america) (mayer and horvatić 1967, ellis 2014). it mainly inhabits grasslands, meadows, forests and is often associated with light to moderate disturbance (farrar and johnson-groh * corresponding author, e-mail: danijela.denist@gmail.com copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. stešević d., berg c. 182 acta bot. croat. 74 (1), 2015 1990). classifi cation of botrychium has been complicated by the high incidence of polyploidy, which has resulted from hybridization among members of the twelve diploids (dauphin et al. 2014). species of the genus are considered of interest not only to phylogeny (clausen 1938, kato 1987, dauphin et al. 2014) but also to conservation. out of 7 species represented in europe (b. boreale milde, b. lanceolatum (s. g. gmel.) ångstr., b. lunaria (l.) sw., b. matricariifolium (döll) a. braun ex w. d. j. koch, b. multifi dum (s. g. gmel.) rupr., b. simplex e. hitchc. and b. virginianum (l.) sw. (euro+med 2006), three species are on the bern convention appendix i. in the european red list of vascular plants, the following status is given: nt (near threatened) b. matricariifolium and b. simplex, and dd (data defi cient) b. multifi dum (bilz et al. 2011). materials and methods the species was identifi ed using rothmaler (1964), mayer and horvatić (1967), farrar (2006). due to the fact that only one individual was found, no herbarium specimen was collected. the species was photographed and accordingly documented and presented in this paper (fig. 2). site geocoding was done by gps device, garmin e-trex vista c. in identifi cation of the vegetation type, lakušić (1966) was used. results and discussion according to rohlena (1942) and mayer and horvatić (1967) the only botrychium species in the fl ora of montenegro is b. lunaria (l.) sw. this species is quite widespread on the meadows and grasslands from the montane to the alpine zone. during a fi eld trip on the mountain babji zub on july the 21th, 2014, in a forest opening at the locality katunine (n42.87436°, e19.38503°, fig. 1), we recorded one individual of botrychium matricariifofig. 1. position of the site katunine (mt babji zub), where botrychium matricariifolium was found (base map, utm grid map of montenegro), and image of the site (photo: d. stešević). botrychium matricariifolium in montenegro acta bot. croat. 74 (1), 2015 183 lium. this rather small forest opening (ca. 12,000 m²) is located in the beech-fi r zone on the bottom of the steep mountain slopes of gradišta and babji zub, at the elevation of ca 1,440 m. toward the e, the plateau of katunine is open to the lipovo valley and faces onto savine grede. under the köppen climate classifi cation the area belongs to the dfs"bx" subtype of cold temperate climate or the boreal climate or humid taiga climate without arid period. the air temperature in the coldest month is below –3 °c, while the average air temperature of the warmest month is up to 22 °c. total average annual precipitation is 1,500 mm. the primary precipitation maximum is in the autumn and the secondary is in the spring (burić et al. 2014). on the brownized rendzina on the limestone crust, shallow (fuštić and đuretić 2000) vegetation of mesophyllic meadows is developed. with its fl oristic composition and signifi cant participation of endemic species like pancicia serbica vis., silene sendtneri boiss., scrozonera rosea waldst. & kit., gentianella crispata (vis.) holub the meadow fi ts into the description of the endemic alliance pancicion lakušić 1964 of the order arrhenatheretalia pawl. 1928 (lakušić 1966). in the past, when cattle breeding was more intensive, the forest opening at katunine was frequently used for grazing. nowadays, cattle breeding has been signifi cantly reduced. that has allowed the regeneration of the woody vegetation and growth of pioneer saplings of acer heldreichii ophr. subsp. visianii k. maly and rhamnus alpina subsp. fallax (boiss.) maire & petitmengin. katunine is also popular for camping, but the camping area is rather small, placed near the forest edge and recognizable by the almost monodominant cover of plantago major s. l. the single individual of b. matricariifolium was found on the open and sunny part of the meadow hidden among numerous b. lunaria individuals, associated with lotus corniculatus l., luzula multifl ora fig. 2. the image of botrichium lunaria (a) and botrychium matricariifolium (b) (photo: d. stešević). stešević d., berg c. 184 acta bot. croat. 74 (1), 2015 (retz.) lej., thesium alpinum l., alchemilla xanthochlora rothm., carex humilis leyss., silene sendtneri, pancicia serbica, armeria canescens (host) boiss., lilium albanicum griseb., polygonum viviparum l., poa alpina l. subsp. badensis (haenke ex willd.) beck, polygala major jacg., ranunculus bulbosus l., dianthus carthusianorum l., galium verum l., viola tricolor l., viola montana l., allium oleraceum l., etc. these two botrychium species can be distinguished clearly by the lamina and location of the sterile leaf part. in b. lunaria it is pinnate with pinnae being trapezoid to fl abellate and is located in the middle of the aerial stem, while in b. matricariifolium it is 2-pinnate with pinnae and pinnulae ovate to oblong, and is located clearly above the middle of the aerial stem (fig. 2). b. matricariifolium is an amphi-atlantic species the distribution area of which covers the eastern part of north america (farrar 2006) and in europe albania, austria, bulgaria, corsica, czech republic, croatia, denmark, estonia, finland, france, germany, switzerland, netherlands, spain, hungary, italy, latvia, lithuania, norway, poland, central european russia, northern european russia, northwest european russia, romania, slovakia, slovenia, serbia, sweden, ukraine (euro+med 2006). the species is considered as a target species – species of european concern (ozinga and schaminée 2005) and in different european countries it has different threat and protection status: i) the species is considered cr (critically endangered) in austria (witowski et al. 2003), bulgaria (petrova and vladimorov 2009), croatia (borovečki-voska 2011), czech republic (klaudisová and pohlová 2004), estonia (ryttäri et al. 2003), hungary (engyel 2009), norway (kålås et al. 2006), serbia (zlatković et al. 2009), en (endangered) in germany (ludwig and schnittler 1996), poland (jakowiak et al. 2007), vu (vulnerable) in finland (rassi et al. 2001), sweden (gärdenfors 2005), ukraine (witowski et al. 2003), dd (data defi cient) in slovenia (skoberne 2004), re (regionally extinct) in, switzerland (moser et al. 2002), ii) it is under protected of the bern convention appendix i, iii) and it is under statutory protection in the czech republic (klaudisová and pohlová 2004), germany (jäger 2011), poland (jackowiak et al. 2007) and many other countries. the newly reported population of b. matricariifolium in montenegro is considered very poor, thus it requires urgent conservation measures. in order to precisely determine its conservation status, further investigation is needed. it is possible that our and the recent record of b. matricariifolium in croatia will after 30 years allow an optimistic view of the situation of the species in the western balkan peninsula. according to the analytical flora of yugoslavia (mayer and horvatić 1967), in the fl oras of neighboring countries following botrychium species were recorded: b. simplex (in herzegovina) and b. multifi dum (serbia); thus both species could be expected in the fl ora of montenegro. in the near future, more attention should be paid to this genus, its diversity and distribution in montenegro. acknowledgements authors would like to thank mountaineer boris čelebić for help in geopositioning of katunine. references bilz, m., kell, s. p., maxted, n., lansdown, r. v., 2011: european red list of vascular plants. luxembourg: publications offi ce of the european union. botrychium 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(eds.), pteridophytes and gymnosperms. the families and genera of vascular plants 1. berlin: springer-verlag. 193–197. witkowski, z. j., król, w., solarz, w., 2003: carpathian list of endangered species. wwf and institute of nature conservation, polish academy of sciences, vienna-krakow. zlatković, b., tomović, g., ranđelović, v., vukojičić, s., niketić, m., 2009: distribution and conservation status of several new and neglected vascular plants in serbia. phytologia balcanica 15, 95 – 105. acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 103 acta bot. croat. 76 (1), 103–106, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0050 issn 0365-0588 eissn 1847-8476 short communication microsatellite allele length variations in inter-specifi c hybrids of eucalyptus murugan sumathi, yasodha ramasamy* institute of forest genetics and tree breeding coimbatore 641 002 tamilnadu, india abstract – the genus eucalyptus encompasses several species of industrial importance. many of these species have been subjected to genetic characterization using different kinds of dna markers. more than 1000 microsatellites have been identifi ed from the genome of eucalypts and they are highly amenable for cross species transferability. during cross amplifi cation of microsatellites, homoplasy is reported in many species in which although the allele size might be the same, the sequences are not. thus, it is essential to ascertain the dna sequence homology with source and target microsatellite repeats. accordingly, fifty five alleles from six microsatellite loci (ecc1, ecc2, eg61, embra100, embra1468 and embra2002) were amplifi ed in two inter-specifi c hybrid populations (eucalyptus tereticornis × e. grandis and e. tereticornis × e. camaldulensis) and sequenced. the results showed that all the microsatellite loci were amplifying the target repeat types except for the loci eg61 and embra2002. the locus eg61 has target repeat of (caa)(gat) but the sampled alleles had either (caa)(gat) or (gat) alone. similarly, the embra2002 locus was targeting interrupting repeats of (cca)..(cca), but the sequenced alleles had repeats of (cca) with or without interruption. nevertheless, the allele size estimated in electrophoresis for hybrids was in conformity with that of the parent alleles. this study suggests the need for validation of the repeat characteristics of microsatellites by sequencing of the alleles particularly in cross species amplifi cation. keywords: allele sequences, eucalyptus, inter-specifi c hybrids, microsatellites, repeat size * corresponding author, e-mail: yasodharaja@yahoo.com introduction the species of the genus eucalyptus are widely planted in tropical and temperate regions of the world and are an important commercial tree species for paper pulp, bioenergy and timber production. several members of eucalyptus have been genetically characterized because of the ease of generating inter-specifi c crosses and vegetative propagation, its unique biological properties, fast growing nature and comparatively small genome size. various dna markers have been applied for the genetic characterization of the eucalyptus genome. microsatellites or simple sequence repeats (ssrs) are the dna marker system of choice, for it is highly conserved and easily transferrable across species. many of these ssrs were shown to be potentially involved in growth, wood quality and stress responsive pathways (gion et al. 2015). ssr based genetic linkage map has a signifi cant place in eucalyptus genetic improvement because the high cross species transferability led to the integration of linkage maps across different genetic backgrounds. genetic maps in eucalypts were used in mapping quantitative trait loci (qtl), synteny and collinearity analysis and genome sequence assembly (hudson et al. 2012, bartholome et al. 2015). recently, grattapagalia et al. (2014) showed that 84% of the mapped microsatellites had colinearity between linkage map and physical position in the assembled e. grandis genome. although to date about 1200 ssrs have been characterized in different species of eucalypts, only about 535 ssrs have been utilized for genetic mapping studies. hence, to generate high density genetic map in new combinations of inter-specifi c hybrids requires the addition of a higher number of ssr loci. so far, the majority of the ssrs mapped (both genomic and expressed sequence tag-derived ssrs) were developed from e. grandis and e. urophylla and have been transferred to other species. during cross transfer, it is essential to verify the allele variations by sequencing the pcr fragments, wherein the structural variants in the form type of repeats, insertion or deletion generated by the ssrs are accurately detected. in eucalypts, 240 est-ssrs were confi rmed for their sequence homology with the original est sequences, however, no information on repeat size variations were reported (zhou et al. 2014). recently, it was emphasized that the sequence verifi cation sumathi m., ramasamy y. 104 acta bot. croat. 76 (1), 2017 of ssr alleles is critical for interpretation of the ssr polymorphism for genetic analysis (barthe et al. 2012). further, size homoplasy is commonly reported among the ssr alleles (curtu et al. 2004, javed et al. 2014), hence, information on dna sequence is essential before the utilization of ssr loci variations. thus, in the present study, attempts were made to sequence the ssr-pcr amplicons generated in the inter-specifi c hybrid populations, e. tereticornis × e. camaldulensis and e. tereticornis × e. grandis. materials and methods plant material and dna isolation the two inter-specifi c hybrid populations used in this study were e. tereticornis (et86) × e. grandis (eg9) and e. tereticornis (et217) × e. camaldulensis (ec17). these species belong to the subgenus (symphyomyrtus) and the section exsertaria (e. tereticornis and e. camaldulensis) and lato angulatae (e. grandis). the number indicated for each species represents the parent clone identity. the hybrid populations were generated by controlled pollination between selected parents. f1 seeds generated from controlled pollination were established in a fi eld trial at panampally, kerala. total genomic dna was extracted using dna isolation kit (qiagen ltd, uk) from the freshly collected leaves of two individual hybrid plants in each population. the dna of parents was isolated from leaf samples of trees employed in controlled pollination. dna quality and quantity was assessed by electrophoresis on 0.8% agarose gel with lambda dna (bangalore genei, bangalore, india) as the standard and spectrophotometry (nanodrop 8000; thermo scientifi c, wilmington, de, usa). ssr amplifi cation three hundred and twenty ssrs (sumathi et al. 2014) were tested between the parents of two hybrid populations for the selection of polymorphic loci. for sequence analysis and repeat motif identifi cation of the parents and hybrids, 6 loci (4 genomic ssrs – ecc1, ecc2, eg61, embra100, and 2 est-ssrs -embra1468, embra2002) were randomly selected to represent different repeat motif types and source species such as e. camaldulensis (da silva et al. 2009) and e. globulus (thamarus et al. 2002) and e. grandis (brondani et al. 2006, faria et al. 2010). details on allele size range, annealing temperature and repeat motif and ncbi accession number of each ssr loci are presented in tab. 1. ssr amplifi cation was carried on 40 μl of reaction containing 4 μl of 10× buffer (100 mm tris–hcl (ph 8.3), 500 mm kcl and 15 mm mgcl2), 125 μm of dntps, 0.4 μm of each forward and reverse primer, 4 u of taq dna polymerase, and 20 ng of template dna of 2 parents and 2 f1 individuals of the hybrid populations. the pcr amplifi cation was carried out for initial denaturation 5 min at 94 °c for 1 cycle and 30 cycles of 1 min at 94 °c, 30 s at the primer specifi c annealing temperature (tab. 1), 1 min at 72 °c, fi nal extension for 15 min at 72 °c. pcr products were size separated using 5% denaturing polyacrylamide (page) gels of size 21×50 cm (sequi-gen gt system, bio-rad, usa) containing 7 m urea and 1× tris-borate-edta buffer. a dna ladder of 50 bp (mbi, fermentas, usa) was included during electrophoresis of samples in the fi rst and last well of the electrophoresis system. the gels were run at 61 w constant power for 3 h and visualized by silver staining. sequencing of pcr fragments and sequence analysis to isolate the specifi c bands of ssrs for sequencing, the pcr amplifi ed products were separated on a 3.5% metaphor agarose gel (cambrex bio science, rockland, me, usa) in 1× tbe buffer and visualized with ethidium bromide staining. only the best amplifi ed bands for each ssr primer combination was removed from the gel and purifi ed using qiaquick gel extraction kit (qiagen, crawley, uk). these dna fragments were sequenced at eurofi ns biotechnologies pvt ltd, bangalore, india using standard procedures on an abi3700 dna analyser (applied biosystems, usa) by employing the specifi c forward or reverse ssr primer. the sequences obtained were checked for their homology with the ssr source sequence in ncbi and the genome sequence of eucalyptus grandis in phytozome (https://phytozome.jgi.doe.gov/pz/portal.html). all the dna sequences of ssr locus along with the source ssr sequence and e. grandis genomic region harbouring ssr were aligned using clustal omega (http://www.ebi.ac.uk/tools/msa/clustalo/) to identify the structural variations in microsatellites. results and discussion microsatellites are well known to generate high polymorphism across species due to their mutational characteristics (bhargava and fuentes 2010). cross transferability of these markers across genera and species enabled the use of microsatellites, where the dna sequence is not available. tab. 1. informat ion on the six microsatellite loci used in this study. sa m pl e ssr code repeat motif annealing temperature (ºc) allele size linkage group reference ncbi accession no.original optimized et86 × eg9 optimized et217 × ec17 1 ecc1 (tc)18 60–45 56 58 157–192 1 da silva et al. 2009 gq302860.1 2 ecc2 (ga)9tata(ga)6 60–46 57 57 238–255 5 da silva et al. 2009 gq302861.1 3 eg61 (gaa)9(gat)6 64 56 61 302–316 2//7 thamarus et al. 2002 eu699745.1 4 embra100 (ct)26 56 58 56 195 1 brondani et al. 2006 bv682837.2 6 embra1468 (tc)12 60 57 59 181 8 faria et al. 2010 gf101903.1 5 embra2002 (cca)8…(cca)8 60 56 64 263 1 faria et al. 2010 gf101907.1 microsatellite allele length variations in eucalyptus acta bot. croat. 76 (1), 2017 105 hence, it is essential to confi rm the repeat characteristics of ssrs to utilize them as markers for dna fi ngerprinting, genetic linkage map generation and quantitative trait loci identifi cation. in order to evaluate the variation in repeat region of ssr alleles, six ssr loci were sequenced in two different inter-specifi c hybrid populations of eucalyptus. the lengths of the sequenced fragments varied between 206–338 bp. out of 87 bands (alleles), fi fty fi ve bands showing high intensity in the agarose gel were selected for sequencing and the dna sequences were analyzed to estimate the degree of sequence divergence among equally sized alleles, repeat number variations and repeat composition. the information on allele size, repeat motif for the sequenced alleles is presented in tab. 2. the ssr locus, ecc1 (gq302860.1) was developed from e. camaldulensis targeting (tc) repeats which had amplifi ed same type of repeats in all the samples. the parents et86 and eg9 generated four different alleles (247 and 297 bp; and 263 and 275 bp, respectively) for the ecc1 locus while their hybrids generated allelic combination of 275/297 bp (h1) and 247/263 bp (h2). sequencing of all the four alleles showed repeat number variations from 16 to 25 (tab. 2) and no direct relationship between repeat number and allele size could be established, because the allele size variation could be due to the variations in the fl anking regions also. the parent et217 amplifi ed two different alleles of size 263 and 297 bp and the parent ec17 amplifi ed a homozygous allele of size 263 bp. sequencing was done only for the 263 bp allele from all the four individuals and all the sequences showed same repeat number. the 263 bp allele sequence of parent et217 showed 19 (tc) repeats followed by an insertion of one (gt) repeat and 6 (tc) repeats. such insertions or interruptions within the motif sequence were documented in vitis (masi et al. 2004), citrus (barkley et al. 2009) and rice (dong et al. 2013). sequence data on microsatellite variation in birds indicated that size homoplasy exists with no sequence similarity (anmarkrud et al. 2008). in tree species, barthe et al. (2014) showed the high amount of variations in fl anking regions. the similarity of repeat regions across the genome sequence of e. grandis in phytozome, the ssr source sequence and the allele sequences for all the loci analyzed are given in on-line suppl. material. the locus ecc2 produced heterozygous alleles in parents et86 (241/247 bp) and et217 (235/250 bp) and homozygous alleles in parents eg9 and ec17 (238 bp). in the original e. camaldulensis sequence of ecc2 (gq302861.1), ga repeat was interrupted with tata and was not observed in any of the sequences analyzed including the genome sequence of e. grandis. instead, all the sequences had (ga) repeats in uninterrupted form and the number varied from 16–19. similarity across the sequences was also very low except in the repeat region. the eg61 ssr locus generated heterozygous alleles in both the parents (et86 and eg9). original microsatellite sequence of eg61 (eu699745.1) and e. grandis genome sequence showed compound ssr (gaa) (gat) repeats. the parents et86 and eg9 showed only (gat) repeats and their hybrid individuals showed (tat) and (aat) repeats and this could be attributed towards the error during pcr amplifi cation or sequencing. ta b. 2 . d et ai ls o n al le le s iz e an d re pe at ty pe fo r t he s ix m ic ro sa te lli te lo ci s eq ue nc ed fr om th e pa re nt s an d th ei r h yb ri ds o f e uc al yp t s pe ci es (a lle le s in b ol d le tte rs w er e se qu en ce d) . a st er is k (* ) d en ot es se qu en ce fr om w hi ch th e ss r s ar e re po rt ed . sample pl an t i de nt ity e c c1 e c c2 e g6 1 e m br a 10 0 e m br a 14 68 e m br a 20 02 a lle le si ze r ep ea t ty pe a lle le si ze r ep ea t ty pe a lle le si ze r ep ea t ty pe a lle le si ze r ep ea t ty pe a lle le si ze r ep ea t ty pe a lle le si ze r ep ea t ty pe 1 e . g ra nd is – (t c )1 8 – (g a )1 8 – (g a a )6 (g a t )8 – (c t )2 6 – (t c )2 3 – (c c a )7 … .(c c a )8 2 so ur ce s eq ue nc e* – (t c )1 8 – (g a )9 ta ta (g a )6 – (g a a )9 (g a t )9 – (c t )1 9 – (t c )1 2 – (c c a )8 … .(c c a )8 3 e t8 6 24 7/ 29 7 (t c )1 6/ (t c )2 5 24 1/ 24 7 (g a )1 8 32 2/ 32 8 (g a t )6 25 3/ 26 9 (c t )2 5 20 6/ 21 8 (t c )2 0 26 3/ 27 2 (c c a )3 4 e g9 26 3/ 27 5 (t c )1 7/ (t c )1 9 23 8/ 23 8 (g a )1 6 32 5/ 33 8 (g a t )7 23 8/ 24 7 (c t )2 0 21 8/ 22 5 (t c )2 1 27 5/ 27 8 (c c a )6 (c c a )7 5 e te gh 1 27 5/ 29 7 (t c )2 5/ (t c )1 9 23 8/ 24 1 (g a )1 7 32 2/ 33 8 (t a t )3 24 7/ 26 9 (c t )2 3 21 8/ 22 5 (t c )2 2 26 3/ 27 8 (c c a )4 6 e te gh 2 24 7/ 27 5 (t c )1 6/ (t c )1 7 23 8/ 24 7 (g a )1 7 32 2/ 33 8 (a a t )4 23 8/ 26 9 (c t )1 8 20 6/ 21 8 (t c )1 8 26 3/ 27 5 (c c a )3 7 e t2 17 26 3/ 29 7 (t c )1 9 23 5/ 25 0 (g a )1 8 32 5/ 32 5 (g a a )6 (g a t )8 25 3/ 26 9 (c t )1 8 22 5/ 22 5 (t c )1 8 26 3/ 26 8 (c c a )4 8 e c1 7 26 3/ 26 3 (t c )1 9 23 8/ 23 8 (g a )1 5 31 3/ 32 5 (g a t )5 23 8/ 24 4 (c t )2 9 20 6/ 21 8 (t c )1 3 27 8/ 27 8 (c c a )9 9 e te ch 1 26 3/ 29 7 (t c )1 9 25 0/ 23 8 (g a )1 9 31 3/ 32 5 (g a t )5 24 4/ 25 3 (c t )2 0 22 5/ 21 8 (t c )1 8 26 3/ 26 3 (c c a )4 10 e te ch 2 26 3/ 26 3 (t c )1 9 23 5/ 23 8 (g a )1 7 31 3/ 32 5 (g a t )5 23 8/ 25 3 (c t )2 2 22 5/ 21 8 (t c )1 3 26 8/ 26 8 (c c a )7 sumathi m., ramasamy y. 106 acta bot. croat. 76 (1), 2017 the et217 parent had (gaa and (gat) motifs and ec17 and the hybrids showed only (gat) motifs. the ssr loci embra100 (bv682837.2) was present in the e. grandis gene sequence protein transparent testa12 (eucgr.a02047), which had 18 repeat units of the target motif (ct). this locus was heterozygous in both the crosses analyzed. alleles from the individuals sequenced showed (ct) repeats varying from 18 to 25. the est-ssr locus embra1468 (gf101903.1) showed similarity with the gene sequence acetyl-coa c-acetyltransferase / acetoacetylcoa thiolase (eucgr.h00849). it had heterozygous alleles in et86, eg 9 and ec17 whereas the et217 showed homozygous alleles. the original sequence consisted of 12 repeat units of tc and all the alleles analyzed had 13–22 repeat units. the ssr locus embra2002 (gf101907.1) sequence showed similarity with auxin responsive protein (auxin_inducible) of e. grandis (eucgr.a01420). except ec17 all the parents had heterozygous alleles and the target repeat of (cca) was present in varying repeat numbers in interrupted and uninterrupted form (tab. 2). this study demonstrated the successful cross species amplifi cation of several eucalypt ssr loci within the genus. microsatellite allele sequencing results of parents and their hybrids show almost same repeat type, corroborating the earlier results in eucalypt species (ochieng et al. 2007, he et al. 2012). variations in fl anking regions and repeat numbers refl ect the occurrence of mutational processes in both the repeat and fl anking regions. similar observations were made in vitis (masi et al. 2004) and shorea (javed et al. 2014) and insisting the requirement of information on underlying dna sequence. however, with the availability of genome sequence of e. grandis, the primer sequence similarity search can be applied to fi nd the primer binding regions. loci with more than one similar site may be avoided to limit any discrepancy during genotyping. although microsatellites are proven to be highly informative markers and continue to be used along with ngs based markers, though they throw up lot of challenges during genotyping and allele calling in the form of homoplasy, which demands enough care while employing the microsatellites for linkage mapping and qtl studies. acknowledgements this research was supported by a research grant from the department of biotechnology (dbt), new delhi, india. references anmarkrud j. a., kleven o., bachmann l., lifjeld j. t., 2008: microsatellite evolution: mutations, sequence variation, and homoplasy in the hypervariable avian microsatellite locus hru10. bmc evolutionary biology 8, 138. barthe s., gugerli f., barkley n. a., maggia l., cardi c., scotti i., 2012: always 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tajikistan) arkadiusz nowak1,2*, sylwia nowak1, marcin nobis3,4, agnieszka nobis3 1 department of biosystematics, laboratory of geobotany and plant conservation, opole university, oleska st. 22, 45-052 opole, poland 2 department of biology and ecology, university of ostrava, 710 00 ostrava, czech republic 3 department of plant taxonomy, phytogeography and herbarium, institute of botany, jagiellonian university, kopernika st. 27, 31-501 kraków, poland 4 laboratory of systematics and phylogeny of plants, tomsk state university, 36 lenin prospekt, tomsk, 634050, russia abstract – the paper presents the results of phytosociological researches on rocky slope vegetation in tajikistan with the aim to establish a comprehensive syntaxonomical classifi cation system. field studies were conducted in 2010–2014 in pamir alai ranges and pamirian plateau. nearly 500 relevés documenting rock and scree vegetation were made according to the braun-blanquet method. numerical analyses of selected 58 relevés representing dwarf shrub vegetation on rock ledges made it possible to distinguish: ephedro glaucaespiraeion baldschuanicae and ephedrion regeliano-fedtschenkoi alliances, as well as spiraeetum baldschuanicae, rhamnetum coriaceae, pentaphylloidetum parvifoliae and pentaphylloidetum dryadanthoidis associations, community of ephedra glauca and community of rhamnus minuta. the classifi cation of vegetation of dwarf shrubs on rock walls occurring in the pamir alai mts is proposed. because of the species composition, physiognomy and microhabitat conditions, the plant communities were included into artemisio santolinifoliae-berberidetea sibiricae class ermakov et al. 2006. the main factors determining the species composition of the classifi ed associations seem to be the elevation above sea level.the newly described syntaxa are largely defi ned by species restricted to very narrow ranges in middle asia. keywords: alpine vegetation, campanuletalia incanescentis, chasmophytes, saxicolous communities, syntaxonomy * corresponding author, e-mail: anowak@uni.opole.pl introduction rocks and screes with their niche diversity create habitats for many specialized plant species (favarger 1972, kazakis et al. 2006, nowak et al. 2011). rock faces can serve as dry islands, while the crevices, fi ssures or deep clefts are better supplied with water. rocky niches can also considerably differ from each other in amount of soil, which is usually not very fertile. the extreme habitat differentiation and uniqueness of petrophytic fl ora is refl ected in the great variety of plant communities developing on rocky walls. many phytosociological studies on rupicolous vegetation have been recently conducted in mountainous areas of europe (e.g., valachovič et al. 1997, sanda et al. 2008, chytrý 2009, tzonev et al. 2009), especially in the mediterranean region (e.g., carmona et al. 1997, deil 1998, parolly 1998, onipchenko 2002, ermolaeva 2007, deil et al. 2008, terzi and d’amico 2008) as well as arid zones of asia (e.g. ermakov et al. 2006, deil 2014). despite the considerable areas of mountain ranges and their great diversity in terms of altitudinal amplitude, rock types and climatic conditions, the rock and scree communities in asia have not yet been studied in detail. the mountain ranges in tajikistan, especially those with the highest amounts of precipitation (i.e. the hissar mts and alichur mts), constitute a refuge for a considerable number of stenochoric plant species sensitive to climate change (kazakis et al. 2006, baettig et al. 2007, fay and patel 2008). the chasmophytic fl ora of tajikistan comprises many taxa geographically restricted to this country or to middle asia (e.g. scutellaria megalodonta, s. shugnanica, s. zaprjagaevii, achoriphragma darvazicum, dionisia involucrata, viola majchurensis, asperula fedtschenkoi, andrachne fedtschenkoi, callipeltis cucullaris, trichodesma incanum, hypericum scabrum, silene brahuica and many others). within the project of the phytosociological survey of tajik vegetation (e.g. nowak and nobis 2012, 2013, nowak et al. 2013a, 2013b) research on chasmophytic vegetation started in 2009. although a number of papers focused nowak a., nowak s., nobis m., nobis a. 110 acta bot. croat. 75 (1), 2016 on typical rock vegetation (asplenietea trichomanis) have been presented in recent years, still little is known regarding the scree and talus vegetation or the transitional microhabitats of much eroded rocks or stabile screes (nowak et al. 2014a, 2014b, 2014c). the problem of classifi cation of dwarf-shrub dominated plots on rocky slopes and rock ledges in middle asia needs to be investigated and the relation of these phytocoenoses to both asplenietea trichomanis and artemisio santolinifoliae-berberidetea sibiricae class fi nally settled. the paper is a part of our survey on rock vegetation in tajikistan with the fi nal intention of building up the syntaxonomical system of all types of rupicolous environments within the country. particularly it aims at completing the knowledge of the diversity of rock and scree vegetation in tajikistan as well as presenting the last successional stage of saxicolous communities here – the dwarf shrub communities which should be considered as a pedoclimax vegetation for this kind of habitat. material and methods study area tajikistan covers an area of ca. 143000 km2 and is located between 36°40’–41°05’e and 67°31’–75°14’n in middle asia (fig. 1). according to recent studies about 4550 vascular plant species occur in tajikistan with ca. 30% generally accepted as endemics (nowak et al. 2011). this number is still incomplete, as new investigations regularly add new species to the fl ora of tajikistan (e.g. nobis 2013, nobis et al. 2014a, 2014b, 2014c). an alpine landscape of high mountains dominates the country. more than 50% of the country’s area is located above 3000 m. as typical for the mediterranean type of climate, the area has generally high solar radiation, as well as a low percentage of cloud cover, high-amplitude annual temperatures, low humidity and precipitation (with the exception of the spring period, when there is a considerable amount of rainfall, fig. 2) . in the south-western regions of tajikistan, the average june temperatures rise to 30 °c. in the temperate zone and alpine elevations the average temperatures in mid-summer are between 9.7 °c and 13.5 °c. annual precipitation ranges in tajikistan from ca. 70 mm (in the mountainous deserts of eastern pamir and south-western lowlands of the country) to ca. 600 mm (on the southern slopes of the hissar range). the limit of perpetual snow is at an altitude of 3500–3600 m in the western pamir alai mts, rising to about 5800 m a.s.l. in the highest elevations of eastern pamir (latipova 1968, narzikulov and stanjukovich 1968). the study was situated in different mountain ranges. they include: the zeravshan mts, hissar mts, hazratishokh mts, darvaz mts, rushan mts, vanch mts, turkestan mts, peter i mts, yazgulem mts, karateginian mts, alichur mts, shugnan mts and the sarikol mts. all of them belong to the pamir alai mountain system (fig. 3). studies on the geology of tajikistan are still scarce and a bit outdated (e.g. nedzvedskiy 1968). the middle and fig. 1. the location of tajikistan in middle asia. fig. 2. climatic characterisation of the study area according to the dushanbe weather station (850 m a.s.l., n38°34’47’’; e68°42’27’’, average year temperature 12 °c, yearly precipit ation 575 mm. dwarf shrub vegetation on rocks in tajikistan acta bot. croat. 75 (1), 2016 111 higher parts of the hissar mts are largely composed of extrusive rocks, mainly granite, granitoid and syenite (e.g. varzob valley). some igneous outcrops also occur in the darvaz mts, kuraminian mts and in the western pamir ranges. in the zeravshan and turkestan mts, cambrian and silurian sediments predominate. the rocks here are generally limestone (micrite limestone, bituminous limestone, marly limestone and dolomitic coral limestone), marble, dolomite, dolomitic shale, clay shale, phyllitic schist and argillaceous slate. also, several metamorphic rocks are present within the study area. the most common are migmatic gneiss, conglomerates and metamorphic mudstones. in eastern pamir, carboniferous sediments dominate, mainly granite, granodiorite and diorite. the rupicolous vegetation of tajikistan is for now classifi ed into 32 associations, 2 alliances and one order (nowak et al. 2014a, 2014b, 2014c). for rock communities of the alpine zone developing on solid rock faces, crevices and ledges the campanuletalia incanescentis nobis et al. 2013 order and asperulo albifl orae-poion relaxae nobis et al. 2013 were proposed (nobis et al. 2013, nowak et al. 2014a, 2014b). plots representing these phytosociological units are characterised by high constancy and abundance of petrophytic taxa with an irano-turanian distributional range. the most frequent species contributing to the phytocoenoses are campanula incanescens, c. lehmanniana, poa relaxa, artemisia rutenica and sergia regelii. due to signifi cant differences in habitat conditions (e.g. inclination, insolation, crevice size, soil amount) and species composition, two main groups of plant communities within asperulo albifl orae-poion relaxae were distinguished. the fi rst group includes communities inhabiting fi ne fi ssures and tiny crevices on rock faces (representing the campanulenion lehmannianae suballiance) and the second group comprises communities developing on larger clefts and small ledges with considerable amounts of soil (representing pentanemenion albertoregeliae suballiance). for the montane and colline zone, the caricion koshevnikovii alliance defi ning the phytocoenoses dominated by acidophilous species like scutellaria hissarica, s. schugnanica, s. zaprjagaevii, s. baldshuanica, tylosperma lignosa and dionysia involucrata was proposed (nowak et al. 2014c). the scree and talus vegetation still needs further investigations. the fi rst insights into the vegetation of colluvial cones and sampling of ca. 300 relevés were obtained in 2014 by the team of authors. however the research is still at the beginning stage and the data set will have to be completed and thoroughly analyzed. data and analyses the research was conducted in 2010–2014. we sampled vegetation plots on mountain cliffs, slopes and terrace walls. the studied vegetation patches were located between 890 and 4280 m a.s.l. the vegetation plot size was delimited in such a way as to represent the full fl oristic composition of the phytocenoses. it varied from 3 to 5 m2 depending on plant density and the homogeneity of vegetation cover. the sampling procedure covers all altitudinal zones, variety of bedrocks, inclinations and exposures. for each vegetation plot all vascular plants and cryptogams were recorded. epilithic lichens have not been considered, as non-specifi c and insignifi cant in defi ning the associations. plant species were recorded according to the braun-blanquet method as the most relevant in the analysis of vegetation variability (braun-blanquet 1964). the 7-degree cover-abundance scale was transformed into percentage cover in the juice program: r = 0.1%; + = 1.0%; 1 = 2.5%; 2 = 15.0%; 3 = 37.5%; 4 = 62.5%; 5 = 87.5% (tichý 2002). the rock type was determined by analyzing the lithology, pore geometry, mineralogical components, texture, permeability, hardness and ph by a professional geologist (see acknowledgments). the phytocoenoses were developed on different types of rock substrate, with a range of ph reaction between 6.2 and 8.8. hydrogen ion concentrations were measured in aqueous rock solution using the elmetron cp-105 ph meter. during fi eld surveys, 488 phytosociological relevés documenting patches representing the asperulo-poion (nobis et al. 2013, nowak et al. 2014a, 2014b), caricion koshevnikovii nowak et al. 2014 (nowak et al. 2014c) as well as fern associations on rock clefts and crevices and the association of dionysietum involucratae nowak et al. 2014 (nowak et al. 2014d) were taken. additionally ca. 300 relevés were sampled in scree and talus vegetation to fi nd out the relation of the plots to the main vegetation classes (asplenietea rupestria and artemisio santolinifoliae-berberidetea sibiricae ermakov et al. 2006) which should eventually include the samples investigated. all the relevés were stored in a database using the juice program (tichý 2002). a modifi ed twlnspan analysis was conducted (roleček et al. 2009) to get the initial idea of the data structure and resolution. we applied the pseudospecies cut levels of 0%, 2%, 5% and 10%. the sampled data showed a unimodal response, allowing us to use a detrended correspondence analysis (dca) with the fl oristic data set (no down-weighting of rare species) to check the fl oristic-sociological classifi cation and to show the relationships between the groups. for the ordination, canoco for windows 4.5 was used (ter braak and šmilauer 2002). affig. 3. the area of tajikistan with main mountain ranges, cities and lakes. nowak a., nowak s., nobis m., nobis a. 112 acta bot. croat. 75 (1), 2016 ter grouping the samples, 58 relevés were identifi ed as suitable for the description of dwarf shrub vegetation on rock ledges. vegetation classifi cation follows the sorted table approach of braun-blanquet (1964). in the analytic tables (tab. 1, on-line suppl. tab. 1), species constancies are given in class i–v (dierschke 1994). in a case in which a particular species was noted in fewer than 8 relevés, the absolute number of species occurrences was specifi ed in the tables (communities of ephedra glauca and rhamnus minuta). newly presented syntaxa, described as order, alliance or associations were proposed according to the international code of phytosociological nomenclature (weber et al. 2000). while distinguishing and ranking the association the works of nowak et al. (2014a, 2014b, 2014c) were taken into account. the association concept follows willner (2006). plant material collected during fi eld studies is deposited in the herbarium of the middle asia mountains, housed in opun (opole university, poland) and kra (jagiellonian university, poland). species nomenclature follows czereptab. 1. plant communities of the ephedrion regeliano-fedtschenkoi in pamir alai mts in tajikistan, in 2013. locations of samples (according to the numbers of relevés): 1, 3, 7 – to the south from karasu (375754,3; 735421); 2, 4 – to the south-west from murgab (375541,6; 735202,4); 5, 8 – chatyr-tash (380632,1; 735308,3); 6, 9 – chatyr-tash (375000,9; 733413,7); 10, 11 – to the davaz pass (383747,6; 704301,6). relevé number 1 2 3 4 5 6 7 8 9 10 11 c onstancy n um ber of occurrence day/month 24/8 24/8 24/8 24/8 24/8 24/8 24/8 24/8 24/8 20/6 20/6 ph 8 8.4 7.8 8.4 8.8 8 7.6 7.7 8 8.4 8.4 aspect se nw se nw se ne se se ne w w inclination (degrees) 65 60 65 65 55 55 65 50 65 55 60 altitude (m) 3909 4217 3909 4217 3811 4280 3909 3811 4275 3150 3150 cover of shrub layer (%) 60 30 35 35 55 35 35 30 20 45 30 cover of herb layer (%) 10 15 10 10 5 2 10 3 2 15 25 relevé area (m2) 3 3 3 3 3 3 3 3 3 3 3 rel. rel. number of species 8 8 11 7 5 6 8 8 7 7 10 1−9 10−11 diagnostic species ass. pentaphylloidetum dryadanthoidis pentaphylloides dryadanthoides b 4 3 3 3 4 3 3 3 2 . . v – community of rhamnus minuta rhamnus minuta b . . . . . . . . . 3 2 – 2 all. ephedrion regeliano-fedtschenkoi ephedra fedtschenkoi b + 1 1 + . + . . . . . iii – o. ephedretalia gerardianae et cl. asplenietea trichomanis melissitus pamiricus + + 1 1 1 . . + + . . iv – poa relaxa . . + 1 + + + + . . + iv 1 paraquilegia anemonoides 1 1 + . . . + + + . . iv – artemisia rutifolia . 1 + + . . + + + . . iv – onosma dichroantha . + . + + + + . + . . iv – potentilla malacotricha 1 1 + + . . . + . . . iii – allium tianschanicum + . + . . . + . . . . ii – asperula strizhoviae . . . . . . . . . 1 2 – 2 psychrogeton leucophyllus . . . . . . . . . + + – 2 sporadic species: campanula incanescens 11; eritrichium subjacquemonti 9. others roegneria czimganica + + + . + . 1 + + . . iv – draba lanceolata . . . . . . . . . 1 + – 2 pseudosedum fedtschenkoanum . . . . . . . . . + 1 – 2 rosularia lutea . . . . . . . . . + 1 – 2 oxytropis chiliophylla . . + . . + . . . . . ii – youngia diversifolia . . + . . . . + . . . ii – stipa glareosa . . . . . + + . . . . ii – sporadic species: androsaceae lehmanniana 1; cerasus verrucosa b 11; ephedra sp. b 11(1); tulipa turkestanica 10. dwarf shrub vegetation on rocks in tajikistan acta bot. croat. 75 (1), 2016 113 anov (1995) with exception of ephedra fedtschenkoi the name of which has been adapted after international plant names index (www.ipni.org). results general fl oristic features and habitat characteristics the number of taxa recorded in all relevés totals 133, ranging from 3 to 18 species in particular plots (mean ca. 8). more than 59 taxa exceed 5% constancy. those with the highest constancy are: spiraea baldshuanica (18 occurrences), artemisia rutifolia (17), poa relaxa (17), campanula incanescens (15), rhamnus coriacea, stipa caucasica (13), bromus tectorum, carex koshevnikovii, pentaphylloides parvifolia (12) and callipeltis cucullaris (11). most of the contributing species are typical chasmophytes adapted to extreme rocky habitats and almost exclusively restricted to eroded rocks. however, there are also a number of taxa with wide ecological amplitude known from other vegetation types. in some studied plots we found e.g. bromus tectorum, poa bulbosa, phleum graecum, conringia planisiliqua and bromus oxyodon as species frequently sampled earlier in segetal and disturbed habitats of urbanized areas in tajikistan. the group of plants that inhabits mainly screes was also numerous in the data set. the most frequent were: silene brahuica, centaurea squarrosa, veronica rubrifolia, aulacospermum roseum, atraphaxis pyrifolia. some of the species, e.g. impatiens parvifolia or leptorhabdos parvifl ora, are related to the forest communities. others have come over from the neighboring rock swards (e.g. sedum ewersii, rosularia lutea) or xerothermophilous swards (e.g. haplophyllum latifolium, ixiolirion tatarica). typically for rock crevice vegetation, moss species also contribute to the sampled plots, however with very low frequency. the most common were bryum argenteum, b. caespiticum and grimmia pulvinata. detrended correspondence analysis dca run for the entire data set clearly segregates relevés representing associations described for the fi rst time: spiraeetum baldschuanicae, rhamnetum coriaceae, pentaphylloidetum parvifoliae and pentaphylloidetum dryadanthoidis (fig. 4). also well-distinct are samples representing two new alliances: ephedro glaucae-spiraeion baldschuanicae (left-bottom part of the diagram) occurring in the western pamir alai ranges and ephedrion regeliano-fedtschenkoi (upper-right part of the graph) confi ned to the uppermost elevations of eastern pamir. this is due to essential differences in the fl oristic composition and structure of individual phytocoenoses that accompany considerably different climatic conditions in those two areas. it seems that the vertical gradient of the graph is related to altitude with plots of rhamnus minuta community and pentaphylloidetum dryadanthoidis in the upper part and samples of rhamnetum coriaceae and spiraeetum baldshuanicae in the bottom part. from left to the right the environmental variable which controls the gradient is not so evident. to some extent the fl oristic differentiation could be related to climate continentality and associated humidity which affect greater share of species originated in central asia and tibetan plateau. within the group of western pamir alai plots are those described as ephedra glauca community, showing the close relation to the spiraeetum baldschuanicae and pentaphylloidetum parvifoliae. syntaxa of dwarf shrub vegetation of rock ledges and clefts in the pamir alai mts in tajikistan alliance: ephedro glaucae-spiraeion baldschuanicae all. nova hoc loco holotypus: spiraeetum baldshuanicae hoc loco diagnostic species: ephedra glauca (syn. e. heterosperma), e. intermedia, spiraea baldschuanica. distribution and ecology: the phytocoenoses of the ephedro glaucae-spiraeion baldschuanicae were recorded in the western pamir alai mts, mainly in the hissar, zeravshan and turkestan ranges and less frequently in the darvatab. 2. principal ecological characteristics of the typifi ed association habitats. soil amount: m – medium, l – low; rock type: c – calcareous, n – neutral, a – acidophilous; insolation (insol.): h – high, m – moderate, l – low. exposition: w – western, s – southern, e – eastern, n – northern; altitude: h – high, m – medium, l – low. community so il r oc k ty pe in so l. e xp os iti on a lti tu de spiraeetum baldschuanicae m a h/m w l rhamnetum coriaceae m c h se m pentaphylloidetum parvifoliae l a h n,nw m/h pentaphylloidetum dryadanthoidis l a h se h fig. 4. detrended correspondence analysis for all samples of rocky dwarf shrub communities in study area (n = 58). nowak a., nowak s., nobis m., nobis a. 114 acta bot. croat. 75 (1), 2016 sian and peter i ranges. the communities plots occur between (1200–)1500–2500(–3000) m a.s.l. the phytocoenoses of ephedro glaucae-spiraeion baldschuanicae consist on average of moderate number of taxa per plot. apart from the taxa diagnostic for the alliance, the group of high-abundance species includes: artemisia rutifolia, poa relaxa, campanula incanescens, rhamnus coriacea, stipa caucasica, pentaphylloides parvifolia, callipeltis cucullaris, asperula albifl ora and clypeola jonthlaspi. the alliance is intrinsically heterogeneous. there are distinct communities found on slopes and walls with high precipitation and those with lower amount of rainfall. the hissar and babatag ranges are inhabited mainly by spiraeetum baldschuanicae while the more arid mountains like zeravshan serve as habitat for rhamnetum coriaceae and pentaphylloidetum parvifoliae. 1. spiraeetum baldschuanicae ass. nova typus relevé: on-line suppl. tab. 1, rel. 2, holotypus hoc loco diagnostic species: spiraea baldshuanica the phytocoenoses of spiraeetum baldschuanicae have been found in several stations in the hissar mts, mainly on the eastern and southern slopes of the varzob, takob, khondara, maychura, sorbo and sarday-myena river valleys. spiraea baldschuanica is an endemic species of the western pamir alai mts distributed in the south-western sections of hissar range at an altitude of 1100–2300 m a.s.l. (ovchinnikov 1975). the altitudinal distribution of the association is compliant with this amplitude. the samples were taken in the colline zone at the altitudes of 1000 to 1950 m a.s.l. (mean approx. 1500; fig. 5, on-line suppl. fig. 5. species richness, plot diversity, cover values of herb layer, altitudinal distribution, cliff inclinations and ph of the researched communities: spiraeetum baldschuanicae (sb), rhamnetum coriaceae (rc), pentaphylloidetum parvifoliae (pp), pentaphylloidetum dryadanthoidis (pd) and communities of ephedra glauca (ceg) and rhamnus minuta (crm). whiskers present minimum and maximum observations within fences, block indicates fi rst and third quartile, circles the minimum and maximum value. outliers are shown as asterisks. dwarf shrub vegetation on rocks in tajikistan acta bot. croat. 75 (1), 2016 115 tab. 1). the association inhabits granite and granodiorite rocks (mean ph 6.6), heavily eroded and generally loose. its plots were found mainly on western and southern expositions with inclinations of mean value approx. 75°. they are characterised by a moderate abundance of vegetation cover which corresponds to the amount of rain and soil deposit on rock ledges. the total cover of the herb layer generally was between 25 and 100% with a mean value of more than 50% (on-line suppl. tab. 1, fig. 5). the phytocoenosis is characterised by a moderate number of species as rupicolous vegetation is concerned, having in one relevé from 4 to 15 taxa (mean value approx. 8). mosses contribute quite signifi cantly to the association in comparison to other communities from the alliance. the most abundant were bryum caespiticium and schistidium apocarpum. among the vascular plants the highest values of constancy and abundance were found in: carex koshevnikovii, poa bulbosa, p. relaxa, campanula incanescens and bromus tectorum. 2. rhamnetum coriaceae ass. nova typus relevé: on-line suppl. tab. 1, rel. 19, holotypus hoc loco diagnostic species: rhamnus coriacea rhamnus coriacea is an endemic species to middle asia, distributed in western pamir alai and western tianshan. in tajikistan it occurs mainly in the zeravshan, turkestan and southern part of darvasian mountain ranges at the altitude of 1300–2600 m a.s.l. (ovchinnikov 1981). during our research the association defi ned by rhamnus coriacea was found in a few locations of the iskander-daria river valley in the zeravshan mts. the phytocoenoses were found almost exclusively on limestone, rarely on dolomite shales (ph 7.4–8.0). the association prefers slope rocks with south-eastern sunny expositions and inclinations of 20°–70° with a mean of approx. 60°. the phytocoenosis develops on moderate elevations within the lower alpine belt. mean elevation value for research plots was approximately 1750 m a.s.l. rhamnetum coriaceae is an association with moderate plant cover value per plot. the observed variation of this feature was 27%–67% with a mean of 46% (fig. 5, on-line suppl. tab. 1). the species number per plot reaches one of the highest values between the dwarf shrub communities. from 7 to 17 species were noted in a single relevé. on average 9 species were observed in a sample. mosses were not observed on eroded slopes within the patches. the most abundant and constant vascular plant species within the phytocoenoses of rhamnetum coriaceae are: stipa caucasica, artemisia rutifolia, centaurea squarrosa, ephedra glauca and silene brahuica. 3. pentaphylloidetum parvifoliae ass. nova typus relevé: on-line suppl. tab. 1, rel. 40, holotypus hoc loco diagnostic species: pentaphylloides parvifolia pentaphylloides parvifolia is a chasmophytic species with wide geographical range, known from middle asia, the altai mts, siberia, mongolia and western china (ovchinnikov 1975). in tajikistan the species occurs along the main zeravshan ridge at elevations of approximately 1600 to 3000 m a.s.l, reaching here the southern limits of its natural range. during the research, populations of pentaphylloides parvifolia were observed in western and central sections of zeravshan range in pastrud-daria, iskanderdaria, veshan and zeravshan river valleys. the phytocoenosis was found exclusively on limestone and marble rocks of solid structure with coarse crevices and ledges (ph 7.5– 8.4). the phytocoenosis develops on relatively high elevations in the alpine zone with a cool microclimate. the observed altitudinal range of the community was between 1850 to 3000 m a.s.l. (mean approx. 2250 m). the association develops on sloping rock ledges and terraces with the inclination value ranging from 55° to 80° (mean approximately 60°, fig. 5). pentaphylloidetum parvifoliae was generally found on shady, northern and north-western slopes. the mean value of total herb cover was relatively high and exceeds 45%, ranging from 45 to 70% (fig. 5). mosses do not contribute to the association. apart from the diagnostic species, the most abundant and constant species of vascular plants were: callipeltis cucullaris, campanula incanescens, veronica capillipes and v. rubrifolia. 4. community of ephedra glauca on-line suppl. tab. 1, relevés 44–47. ephedra glauca has the optimum of its occurrence in high, alpine elevations and in arid areas, e.g. in zeravshan and turkestan ranges. the collected sample contains a few relevés representing dwarf shrub vegetation with domination of ephedra glauca. all of the relevés were taken in the central part of the zeravshan mts (1600–2200 m a.s.l.). despite the fact that it seems to us that this species can not be used as diagnostic for any association, we decide to depict these plots and show them on the diagram. further survey is needed to check whether it should be regarded only as diagnostic for the alliance or it can also serve as a species defi ning its own association with central position within the alliance. alliance: ephedrion regeliano-fedtschenkoi all. nova hoc loco holotypus: pentaphylloidetum dryadanthoidis hoc loco diagnostic species: ephedra regeliana, e. fedtschenkoi, e. gerardiana. distribution and ecology: the plots of the ephedrion regeliano-fedtschenkoi were recorded almost exclusively in the highest elevations of eastern pamir (in the eastern part of tajikistan). few samples representing the community with rhamnus minuta were also recorded in the central part of the darvaz range. it is possible that the patches representing ephedrion regeliano-fedtschenkoi occur also in arid plateau of eastern pamir, in western pamirian ranges (e.g. vanch, rushan and shugnan mts) as well as in more humid darvazian range. the plots were sampled at altitudes of 3150–4275 m a.s.l. the phytocoenoses of ephedrion regeliano-fedtschenkoi had low numbers of taxa per plot, approximately 8 on average (fig. 5). apart from the taxa diagnostic for the alliance, the group of species with highest abundance and frequency includes: pentaphylloides dryadanthoides, artemisia rutifolia, melissitus pamiricus, poa relaxa, paraquilegia anemonoides and roegneria czimganica. defi ned by the distributional ranges of both ephedra species, the alliance could be delimited to the pamir, tianshan, karakorum and western himalayan ranges. it includes plant communities adapted to the harshest conditions in the arid, nival zone of the central asian mountains. nowak a., nowak s., nobis m., nobis a. 116 acta bot. croat. 75 (1), 2016 5. pentaphylloidetum dryadanthoidis ass. nova typus relevé: tab. 1, rel. 1, holotypus hoc loco diagnostic species: pentaphylloides dryadanthoides pentaphylloides dryadanthoides distribution is confi ned to the highest elevations in the pamirian plateau and in the surrounding mountains, in tajikistan and western china (ovchinnikov 1975). the species was found in several sites in central part of eastern pamir, in murgab and alichur river valleys, in muzkol and psharskyi ranges. the phytosociological research confi rmed that the species builds its own association developing on relatively fi rm and solid limestone rocks (ph 7.6–8.8). the association prefers elevations in high alpine and nival zones (fig. 5), within the altitudinal range between 3800 and 4275 m a.s.l. (mean approximately 4000 m). the phytocoenosis develops on rocks with small or medium-sized crevices, on sloping walls, rock faces or on rock tops. the noted inclination values varied signifi cantly between 50° and 65° (fig. 5). pentaphylloides dryadanthoides prefers generally south-eastern, fully insolated expositions (fig. 6). in the sample plots, between 6 and 11 taxa were noted (mean approx. 8), so as majority of the rupicolous vegetation, the association should be classifi ed as moderately rich in species. as well as by scarcity of species, the association is characterized by the moderate value of total cover of vascular plants in the plots. it could reach up to 70% with the mean value of approximately 45 % (fig. 5). no moss species were found within the recorded patches. the group of species with the highest constancy includes: artemisia rutifolia, ephedra fedtschenkoi, melissitus pa miricus, onosma dichroantha, paraquilegia anemonoides and roegneria czimganica. 6. community of rhamnus minuta tab. 1, relevés 10–11. rhamnus minuta is a rare species in tajikistan with its range restricted to a few mountain ranges in the eastern tajikistanian and pamirian geobotanical regions. the species is known also from kashgaria in western china. habitat preferences of rhamnus minuta, especially its altitudinal amplitude and bedrock type suggest that the community should be included in ephedrion regeliano-fedtschenkoi. this was clearly confi rmed by the results of the numerical ordination (fig. 4). plots of the community were found on fig. 6. the exposition preferences of the plant associations researched. spiraeetum baldschuanicae (sb), rhamnetum coriaceae (rc), pentaphylloidetum parvifoliae (pp), pentaphylloidetum dryadanthoidis (pd). dwarf shrub vegetation on rocks in tajikistan acta bot. croat. 75 (1), 2016 117 few limestone outcrops near the khaburabot pass at the altitude of approximately 3150 m a.s.l. they develop on solid limestone rocks with western expositions and moderate inclinations (ca. 65°). within the recorded vegetation plots a considerable contribution of rock swards taxa were noted, among others pseudosedum fedtschenkoanum and rosularia lutea. unfortunately, the survey in other regions of the rhamnus minuta range did not confi rm its presence nowadays. so, at this stage of the research we have decided to present the phytocoenosis as a community. synopsis of syntaxa based on this study, we propose the following classifi cation of vegetation of dwarf shrubs on rock walls in the pamir alai mts in tajikistan: class: artemisio santolinifoliae-berberidetea sibiricae erma kov et al. 2006 order: hyperico scabri-lactucetalia orientalis nom. prov. (for explanation see discussion) alliance: ephedro glaucae-spiraeion baldschuanicae a. nowak, s. nowak, m. nobis et a. nobis all. nova 1. spiraeetum baldschuanicae a. nowak, s. nowak, m. nobis et a. nobis ass. nova 2. rhamnetum coriaceae a. nowak, s. nowak, m. nobis et a. nobis ass. nova 3. pentaphylloidetum parvifoliae a. nowak, s. nowak, m. nobis et a. nobis ass. nova 4. community of ephedra glauca order: ephedretalia gerardianae nom. prov. (for expla nation see discussion) alliance: ephedrion regeliano-fedtschenkoi a. nowak, s. nowak, m. nobis et a. nobis all. nova 5. pentaphylloidetum dryadanthoidis a. nowak, s. nowak, m. nobis et a. nobis ass. nova 6. community of rhamnus minuta discussion position of the described association in relation to other types of rupicolous vegetation in tajikistan and central asia comparing the microhabitat and climatic conditions of pamir alai (middle asia) to other areas in eurasia, the most similar must occur in central asia. in particular, the eastern pamir plateau, turkestan, zeravshan and the peter i range seem to be closely related in terms of precipitation, average temperatures and continentality. only the southern slopes of the hissar range are remarkably more humid, amounts of precipitation and its distribution throughout a year being mediterranean rather than continental. although some authors stressed that the tajikistan is infl uenced by a mediterranean-type bioclimate, it is rather an oro-mediterranean subtype overlain by extreme and harsh alpine conditions (rivas-martinez et al. 2011). of course there are considerable differences in diurnal temperature oscillations if we compare the valley bottoms and alpine or nival elevations. that is why in our opinion, the climatic conditions of the research area could be compared with the central asiatic. another important issue when analyzing the classifi cation of the studied vegetation is the environmental characteristic of the plots inhabited by dwarf-shrub vegetation. the researched phytocoenoses occupy the transitional habitats between typical asplenietea trichomanis rock faces with crevices and fi ssures and scree habitats with mobile gravel deposits. this type of microhabitat could be defi ned as considerably eroded rocks or relatively well stabilized screes. a refl ection of this can be seen in the species composition of plant communities with many plants typical for scree vegetation and many coming over from neighboring typical rock habitats. a thorough analysis of the fl oristic structure of plant communities convinced us to include the rupicolous dwarf shrub vegetation of pamir alai into artemisio santolinifoliae-berberidetea sibiricae. this class was proposed for the taluses and screes of the western sayan mts and the altai range (ermakov et al. 2006). as in central asia, in tajikistan too many typical scree species contribute to the researched plots: callipeltis cucullaris, stipa caucasica, silene brahuica, clypeola jonthlaspi, veronica rubrifolia, galium spurium, aulacospermum roseum and others. also the shrubbery physiognomy, lack of moss layer, very scarce soil content on the sites and the gentle inclination of slopes are fairly similar to those in altai and sayan ranges. however, because of the transitional character and the vicinity of rock walls, typical asplenietea species also contribute to the plant communities. plots representing ephedro glaucae-spiraeion baldschuanicae are characterized by a considerable share of diagnostic species of the campanuletalia incanescentis, mainly campanula incanescens, poa relaxa and carex koshevnikovii. despite the habitat and structural similarities, there are some important differences in species composition of the researched vegetation if compared to phytocoenoses from southern siberia. only a few species were found in common with one that had a considerable abundance and frequency – artemisia rutifolia. that is why we propose to include the plant communities of dwarf shrubs in pamir alai mts into a new order within the artemisio santolinifoliaeberberidetea sibiricae. provisionally we suggest hyperico scabri-lactucetalia orientalis with hypericum scabrum, lactuca (scariola) orientalis, callipeltis cucullaris as diagnostic taxa. all of them are distributed in middle asia, northern pakistan, northern afghanistan, iran and some parts of near east. as we know from our preliminary studies in tajikistan, this type of vegetation occurs in semi-arid mountainous areas in colline and alpine belts in western sections of the pamir alai ranges. the considerable share of petrophytic species of irano-turanian distributional type is typical for this kind of vegetation (nowak et al. 2014c). despite some similarities in species composition (i.e. the presence of asperula albifl ora and poa relaxa), the physiognomy as well as habitat requirements of the communities from the ephedro glaucae-spiraeion baldschuanicae are clearly different from those of the associations from the caricion koshevnikovii and asperulo-poion relaxae alliances. the phytocoenoses of ephedro glaucae-spiraeion baldschuaninowak a., nowak s., nobis m., nobis a. 118 acta bot. croat. 75 (1), 2016 cae occupy much-eroded walls with coarse ledges and stabilized screes with moderate inclinations (on-line suppl. tab. 1). such conditions are conducive to soil accumulation, increase the fertility of the habitat and allow shrubby vegetation to thrive. shrubs are the climax vegetation in those biotopes. the shrubby vegetation seems not to be so diverse and species rich as vegetation dominated by herbs. that is why we have decided to include all dwarf shrub associations of rock ledges and clefts in one alliance. it should be stressed, however, that the communities distinguished within the alliance differ in habitat requirements and altitude range. spiraeetum baldshuanicae certainly inhabits more humid, lower and warmer areas than phytocoenoses dominated by pentaphylloides parvifolia and rhamnus coriacea. we had also expected further variation of shrubby rock vegetation in latitudinal gradient, so we explored the south-western part of tajikistan with the warmest climatic conditions. in that area we were looking for communities with the domination of rhamnus baldshuanica and zygophyllum gontscharovii. phytocoenoses dominated by the latter species were noted in several locations during our survey in 2013 but only on landslides with inclination of ca. 20–30 degrees, so we could not insert them to dwarf shrub vegetation on screes and rocks. the situation in the communities in eastern tajikistan is different. the arid conditions of the highly elevated pamirian plateau and also of some “rock islands” in eastern tajikistanian geobotanical region (peter i and central section of darvaz ranges) are rather similar to those prevailing in the arid and semi-arid ranges of tibet, karakorum and western himalayas. as it is shown on the ordination diagram, the difference between those two groups of relevés is considerable. that is why we have decided to classify phytocoenoses from ephedrion regeliano-fedtschenkoi to a distinct order of vegetation. however, the scarcity of phytosociological studies and the areas in china that are diffi cult of access allow us to propose only a provisional name for that order. this group of plant communities probably includes the ephedretum gerardiane association described for the fi rst time from the western himalayas (kojima 1990). although the ephedra species in the arid areas of himalaya, tibet and karakorum are not restricted to rock habitats but also grow on screes, degraded pastures and stony river beds, their communities can be included in provisional ephedretalia gerardianae order because of climatic and species composition similarities. our observations done in pamir alai indicate that along with increasing continentality and aridisation of climate, petrophytic species, which occur almost exclusively on rock walls, spread also into the neighbouring screes and gravel habitats. the patches of petrophytic vegetation in eastern tajikistan are really clearly distinct from those occurring in the western areas of the country. preliminary studies of these communities show signifi cant differences in species composition. as examples of abundant species inhabiting the eastern margins of tajikistan, hippolytia darvasica, h. shugnanica, ajania tibetica, a. gracilis, inula schmalhauseni, parrya shugnanica, corydalis tenella and waldheimia glabra can be mentioned. further studies should specifi cally identify the species composition of herb and shrub vegetation in dry areas of the highest elevations in central asian mountains allowing for the preparation of the fi nal classifi cation of rock and scree vegetation. it should be also stressed that our analysis is constrained by the ambiguities in treating the species from ephedra genus, especially in ephedra glauca and e. intermedia complexes. several other taxa like e. heterosperma, e. microsperma, e. intermedia, e. tibetica, e. valida were described making the fi eld plant determination diffi cult during the fi eld studies. species composition, chorology and habitat of pamir alai petrophytic dwarf shrubs the pamir alai rupicolous vegetation is highly diverse in terms of endemism, habitat preferences and physiognomy of phytocoenoses. to date, more than 30 plant associations have been defi ned (nobis et al. 2013, nowak et al. 2014a, 2014b, 2014c). this is due to extreme diversity of ecological niches caused by differences in altitudinal amplitude, bedrock type, exposition, inclination, crevice type, amount of soil and water supply within the biotope. the species also respond to geographical barriers responsible for increasing endemism rate and separateness of the fl oristic structure. it is commonly known from other mountainous areas that highly elevated mountain ridges fasten the speciation and make the rupicolous fl ora distinct (favarger 1972, médail and verlaque 1997). the dca based on fl oristic composition of sampled plots revealed some considerable differences within the dwarf shrub vegetation in the pamir alai mts (fig. 4). the main discrimination factor seems to be longitudinal gradient which is related to altitude, precipitation and temperature. in eastern pamir, the climatic conditions are harsh (arid and cold), so the distinctiveness of the fl ora is clearly marked in comparison to other regions of middle asia. this holds true also in the group of unique species which are almost all confi ned to those geobotanical subregions (nowak et al. 2011). these climatic conditions are responsible for the considerable difference between the plots from the western pamir alai mts and the eastern ranges, although the range of peter i and central and northern sections of the darvaz mts could be regarded somehow as transitional area (fig. 3). the species composition is signifi cantly different in samples from those two areas, with allium tianschanicum, asperula strizhoviae, pentaphylloides dryadanthoides, ephe dra fedtschenkoi, melissitus pamiricus, oxytropis chiliophylla, potentilla malacotricha, rhamnus minuta, roegneria czimganica, stipa glareosa and youngia diversifolia occurring exclusively in eastern part of the surveyed area. the evident difference between spiraeaetum baldshuanicae and rhamnetum coriaceae could be explained by the bedrock type. spiraea baldshuanica prefers acidophilous rocks while rhamnus occurs only on limestones (tab. 2). the plots sampled in shrubby vegetation in tajikistan clearly differ from the corresponding rock vegetation communities known from distant ranges in southern europe or southern asia (dimopoulos et al. 1997, hein et al. 1998, parolly 1998, ermolaeva 2007). only shrubby ephedretum gerardianae described from nepal can be compared with dwarf shrub vegetation on rocks in tajikistan acta bot. croat. 75 (1), 2016 119 communities recorded in eastern pamir (kojima 1990). however, apart from a few widely distributed taxa like convolvulus arvensis, chenopodium botrys or ch. fi cifolium, in the patches of these communities almost no species from our study area were noted. this separateness might be attributed again to the unique habitat conditions in rupicolous environments and the related fl oristical uniqueness. in result the plant communities are also distinct and the beta diversity of mountainous areas increases (valachovič et al. 1997, deil et al. 2008, deil 2014). the newly described plant associations are largely defi ned by species restricted to very narrow ranges (e.g. spiraea baldshuanica known only from w tajikistan, anaphallis darvazica distributed in the southern part of the country, atraphaxis seravshanica known exclusively from zeravshan mts, dionysia involucrata occur ring in western section of hissar range or onosma atrocyanea scattered only in western pamir alai). this type of stenochory of petrophytic plant species and communities is also observed in many other mountainous areas, especially in those with mediterranean-type climates, such as the bokkoya mts in northern morocco (deil 1994, deil and hammoumi 1997), in gibraltar (galán de mera et al. 2000), in crete and mainland greece (dimopoulos et al. 1997), in bulgaria (mucina et al. 1990), in the caucasus and the mountains of central asia (agakhanjanz and breckle 2002, ermolaeva 2007), in the taurus mts in turkey 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2000: international code of phytosociological nomenclature. 3rd edition. journal of vegetation science 11, 739–768. willner, w., 2006: the association concept revisited. phytocoenologia 36, 67–76. dwarf shrub vegetation on rocks in tajikistan acta bot. croat. 75 (1), 2016 o nlin e su pp l. ta b 1. a ss oc ia tio ns o f t he e ph ed ro g la uc ae -s pi ra ei on b al ds ch ua ni ca e in p am ir a la i m ts in t aj ik is ta n. l oc at io ns o f s am pl es (a cc or di ng to th e nu m be rs o f r el ev es ): 1 – r om it (4 13 05 1, 6; 7 21 15 9, 1) ; 2 – v ar zo b (3 85 40 6, 4; 6 84 94 6, 9) ; 3 – v is to n (4 15 23 1, 2; 7 15 85 3, 2) ; 4 – t ak ob (3 85 01 8, 7; 6 85 50 5, 7) ; 5 , 6 , 8 – v ar zo b (3 84 65 1, 3; 6 84 91 4, 8) ; 7 , 9 – t ak ob (3 85 03 8; 6 85 01 4) ; 1 0 – m ay ch ur apo st (3 90 13 9, 4; 6 84 70 5) ; 1 1 – v ar zo b (3 85 62 0, 1; 68 48 11 ,8 ); 1 2 – e fu ch ( 38 49 41 ,7 ; 7 01 04 4, 2) ; 1 3, 1 4 – r om it (4 00 95 8, 9; 7 33 21 5, 2) ; 1 5, 1 6 – k ho nd ar a (4 00 95 8, 9; 7 33 21 5, 2) ; 1 7 – v is to n (4 13 05 1, 6; 7 21 15 9, 1) ; 1 8 – ta ko b (3 85 01 8, 3; 6 85 50 4, 6) ; 1 9 – m ar zi ch ( 39 11 26 ,9 ; 68 44 20 ,5 ); 2 0 – k ha yr on be d (3 90 92 1, 2; 6 82 94 5, 1) ; 2 1, 2 7, 2 9 – be tw ee n sa rv a nd k ha yr on be d (3 90 83 8, 9; 6 82 84 9, 9) ; 2 2, 2 3 – z er av sh an ii (3 91 01 7, 8; 68 30 44 ,7 ); 2 4, 2 5, 2 6, 2 8 – sa rv od a (3 91 00 7, 1; 6 83 04 1) ; 3 0, 3 1 – k ha yro nb ed (3 90 64 5, 3; 6 82 45 5, 1) ; 3 2, 3 4 – c ho re (3 91 85 0, 5; 6 82 62 6, 9) ; 3 3, 3 5, 3 6 – 4t h l ak e (3 91 05 0, 1; 6 75 01 1, 8) ; 3 7 – v es ha n (3 92 33 7, 2; 6 81 54 9, 5) ; 3 8 – ja gn ob g or ge (3 91 11 1, 6; 6 85 42 2, 2) ; 3 9, 4 3 – m az ar ish ar if (3 92 23 8, 2; 67 50 39 ,3 ); 4 0, 4 1 – k an te ( 39 14 55 ,4 ; 6 82 92 7, 5) ; 4 2 – po ym az or ( 39 18 50 ,1 ; 6 82 62 2, 9) ; 4 4 – ta kf on ( 39 11 32 ,5 ; 6 84 00 7, 2) ; 4 5 – a nz ob ( 39 10 20 ,5 ; 6 84 73 2, 7) ; 4 6 – a nz ob ( 39 09 56 ,2 ; 6 85 11 9, 1) ; 4 7 – z er av sh an i i (3 91 22 7, 7; 69 31 59 ). r el ev é nu m be r 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 constancy constancy constancy number of occurrence d ay 8 17 9 8 8 8 8 8 8 18 17 18 6 6 6 6 8 14 30 21 17 10 10 17 17 17 17 17 17 17 17 19 19 19 19 19 19 19 19 29 29 11 13 9 9 9 8 d at e: m on th 6 8 6 6 6 6 6 6 6 6 8 6 6 6 6 6 6 6 6 6 8 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 6 6 8 6 6 6 6 6 y ea r 13 13 13 12 12 12 12 12 12 13 13 13 13 13 13 13 13 12 14 14 13 12 12 13 13 13 13 13 13 13 13 12 12 12 12 12 12 12 12 14 14 12 12 12 12 12 12 ph 6, 6 6, 2 7, 4 6, 2 6, 4 6, 5 7 6, 3 6, 5 7, 8 6, 2 7, 5 6, 5 6, 4 6, 8 6, 3 6, 4 6, 2 8 7, 7 7, 8 7, 7 7, 7 7, 6 7, 6 7, 6 7, 8 7, 4 7, 8 7, 8 – 8 8, 2 8, 2 7, 9 7, 9 7, 5 8, 4 8, 2 8, 4 8, 4 8, 1 8, 2 7, 7 7, 8 7, 7 7, 5 a sp ec t w w w sw w w s w s n e sw sw w w w w w n w s se e se se se se se se e se w w n w n w n n n w n n n w e se n n s s se sw in cl in at io n (d eg re es ) 85 85 70 60 75 80 75 75 10 0 80 80 75 85 85 50 70 80 25 70 20 35 35 40 60 60 60 60 45 65 70 70 60 60 55 55 60 55 55 60 75 80 80 60 70 75 65 85 a lti tu de (m ) 89 0 14 01 19 09 15 60 10 85 10 85 12 36 10 85 12 50 19 31 15 86 13 50 19 28 19 28 19 25 19 25 89 0 15 90 20 92 17 78 17 70 17 43 17 43 17 14 17 14 17 14 17 75 17 13 17 75 19 61 19 61 29 13 18 74 29 13 18 74 18 74 18 56 22 15 18 78 24 82 24 82 29 59 18 97 18 25 21 10 21 81 16 50 c ov er o f s hr ub la ye r ( % ) 30 60 65 35 20 20 5 45 5 30 45 20 30 40 60 35 30 5 35 30 30 25 30 40 20 15 20 40 30 20 55 55 60 55 45 40 55 55 40 35 30 30 15 70 60 70 3 c ov er o f h er b la ye r ( % ) 15 15 20 15 35 55 30 10 20 25 15 15 20 20 25 25 15 25 15 40 10 15 10 20 25 10 10 15 3 40 10 5 3 3 2 5 3 3 3 10 15 10 40 10 5 5 45 c ov er o f m os s la ye r ( % ) 5 15 – – – – – – – – – – – 15 15 10 10 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – r el ev é ar ea (m 2 ) 5 3 5 4 3 3 3 3 3 3 3 3 5 5 5 5 5 3 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 4 re l. re l. re l. re l. n um be r o f s pe ci es 8 7 6 8 9 7 4 10 4 8 5 5 5 10 8 11 14 6 8 11 8 7 12 7 9 9 10 17 11 7 7 7 6 8 7 8 10 8 8 7 6 6 18 4 5 4 3 1− 18 19 −3 1 32 −4 3 44 −4 7 d ia gn os tic s pe ci es a ss . s pi ra ee tu m b al ds ch ua ni ca e sp ir ae a ba ld sh ua ni ca b 3 4 4 3 2 3 1 3 1 3 3 2 3 3 4 3 3 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v – – – sp ir ae a ba ld sh ua ni ca c . . . . . . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – a ss . r ha m ne tu m c or ia ce ae r ha m nu s co ri ac ea b . . . . . . . . . . . . . . . . . . 3 3 3 3 3 3 2 2 2 3 3 2 4 . . . . . . . . . . . . . . . . – v – – a ss . p en ta ph yl lo id et um p ar vi fo lia e p en ta ph yl lo id es p ar vi fo lia b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 4 3 3 4 4 3 3 3 3 2 . . . . – – v – c om m un it y of e ph ed ra g la uc a e ph ed ra g la uc a b . . . . . . . . . . . . . . . . . . . . . . . . + + + + + . . . . . . . . . . . . . . 4 4 4 1 – ii – 4 a ll. e ph ed ro g la uc ae -s pi ra ei on b al ds ch ua ni ca e et o . h yp er ic o sc ab ri -l ac tu ce ta lia o ri en ta lis c al lip el tis c uc ul la ri s . + . . . . . . . . . . . . . . . + . . . . + . + . . 1 . . . 1 . + + 1 . + . . . . 1 . . . . i ii ii i – c l. a rt em is io s an to lin ifo lia eb er be ri de te a si bi ri ca e st ip a ca uc as ic a + . . . . . . . . . . . . . . 1 . . . . + 1 1 1 2 1 1 . + 1 1 . . . . . + . . . . . . . . . . i iv i – si le ne b ra hu ic a . . . . . . . . . . . . . . . . . . . . . + + . . + + . . . . . . . . + + . . . . . + . . . . – ii ii – c ly pe ol a jo nt hl as pi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + . + + + . . . . . . . . . – – ii i – g al iu m s pu ri um . . + . . . . . . . . . . + . . . . . . . . + . . . . . . . . . . . . . . . . . . . + . . . . i i i – a ul ac os pe rm um ro se um . . . . . . . . . . . . . + . . . . . . . . . . + + . + . . . . . . . . . . . . . . . . . . . i ii – – st ip a or ie nt al is . . . . . . . . . . . . . . 1 . . . . . + . . . . . . . . 1 . . . . . . . . . . . . . . . . . i i – – a tr ap ha xi s py ri fo lia b . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . + . . . + . . . . . . . . . . – i i – p se ud os ed um fe dt sc he nk oa nu m + . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . i – i – 1 nowak a., nowak s., nobis m., nobis a. 2 acta bot. croat. 75 (1), 2016 r el ev é nu m be r 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 constancy constancy constancy number of occurrence d ay 8 17 9 8 8 8 8 8 8 18 17 18 6 6 6 6 8 14 30 21 17 10 10 17 17 17 17 17 17 17 17 19 19 19 19 19 19 19 19 29 29 11 13 9 9 9 8 d at e: m on th 6 8 6 6 6 6 6 6 6 6 8 6 6 6 6 6 6 6 6 6 8 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 6 6 8 6 6 6 6 6 y ea r 13 13 13 12 12 12 12 12 12 13 13 13 13 13 13 13 13 12 14 14 13 12 12 13 13 13 13 13 13 13 13 12 12 12 12 12 12 12 12 14 14 12 12 12 12 12 12 ph 6, 6 6, 2 7, 4 6, 2 6, 4 6, 5 7 6, 3 6, 5 7, 8 6, 2 7, 5 6, 5 6, 4 6, 8 6, 3 6, 4 6, 2 8 7, 7 7, 8 7, 7 7, 7 7, 6 7, 6 7, 6 7, 8 7, 4 7, 8 7, 8 – 8 8, 2 8, 2 7, 9 7, 9 7, 5 8, 4 8, 2 8, 4 8, 4 8, 1 8, 2 7, 7 7, 8 7, 7 7, 5 a sp ec t w w w sw w w s w s n e sw sw w w w w w n w s se e se se se se se se e se w w n w n w n n n w n n n w e se n n s s se sw in cl in at io n (d eg re es ) 85 85 70 60 75 80 75 75 10 0 80 80 75 85 85 50 70 80 25 70 20 35 35 40 60 60 60 60 45 65 70 70 60 60 55 55 60 55 55 60 75 80 80 60 70 75 65 85 a lti tu de (m ) 89 0 14 01 19 09 15 60 10 85 10 85 12 36 10 85 12 50 19 31 15 86 13 50 19 28 19 28 19 25 19 25 89 0 15 90 20 92 17 78 17 70 17 43 17 43 17 14 17 14 17 14 17 75 17 13 17 75 19 61 19 61 29 13 18 74 29 13 18 74 18 74 18 56 22 15 18 78 24 82 24 82 29 59 18 97 18 25 21 10 21 81 16 50 c ov er o f s hr ub la ye r ( % ) 30 60 65 35 20 20 5 45 5 30 45 20 30 40 60 35 30 5 35 30 30 25 30 40 20 15 20 40 30 20 55 55 60 55 45 40 55 55 40 35 30 30 15 70 60 70 3 c ov er o f h er b la ye r ( % ) 15 15 20 15 35 55 30 10 20 25 15 15 20 20 25 25 15 25 15 40 10 15 10 20 25 10 10 15 3 40 10 5 3 3 2 5 3 3 3 10 15 10 40 10 5 5 45 c ov er o f m os s la ye r ( % ) 5 15 – – – – – – – – – – – 15 15 10 10 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – r el ev é ar ea (m 2 ) 5 3 5 4 3 3 3 3 3 3 3 3 5 5 5 5 5 3 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 4 re l. re l. re l. re l. n um be r o f s pe ci es 8 7 6 8 9 7 4 10 4 8 5 5 5 10 8 11 14 6 8 11 8 7 12 7 9 9 10 17 11 7 7 7 6 8 7 8 10 8 8 7 6 6 18 4 5 4 3 1− 18 19 −3 1 32 −4 3 44 −4 7 a tr ap ha xi s se ra vs ha ni ca b . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . + . . . . . . . . i – i – b er be ri s in te ge rr im a b . . . . . . . . . . . . . . . . . . . + . . . . . . . + . . + . . . . . . . . . . . . . . . . – ii – – p ol yg on um p ar on yc hi oi de s . . . . . . . . . . . . . . . . . . . . . . . . . . + + + . . . . . . . . . . . . . . . . . . – ii – – zi zi ph or a pa m ir oa la ic a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . + . + . . . . . . . . – – ii – p se ud os ed um c on de ns at um . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . i – – 1 c er as us v er ru co sa b . . . . . . . . . . . . . 1 . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i – – r os a m ar ac an di ca b . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . + . . . . . . . . . . . . . . . . – i – – p ip ta th er um la tif ol iu m . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . 1 . . . – i – 1 o ri ga nu m ty tta nt hu m + . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – st ep to rh am ph us c ra m bi fo liu s . + . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – c ep ha lo rh yn ch us s on go ri cu s . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . – i – – sc hr en ki a va gi na ta . . . . . . . . . . . . . . . . . . . . . . . . . + . + . . . . . . . . . . . . . . . . . . . – i – – sp or ad ic s pe ci es : a lli um o sc ha ni ni i 2 5; a ra bi s ko ka ni ca 4 3; b ol bo sa po na ri a in tr ic at a 47 (3 ); c ol ut ea p au ls en ii b 20 ; h ap lo ph yl lu m la tif ol iu m 2 6; o no sm a at ro cy an ea 4 0; p ac hy pt er yg iu m b re vi pe s 36 ; p ip ta th er um k ok an ic um 4 3; p yr et hr um p yr et hr oi de s 40 ; r os ul ar ia h is sa ri ca 1 8( 2) ; s an gu is or ba al pi na 1 2( 1) ; s ca nd ix s te lla ta 2 0; s ile ne lo ng ic al yc in a 43 ; s tr og an ov ia p an ic ul at a 33 ; t ri ch od es m a in ca nu m 2 0. c l. a sp le ni et ea tr ic ho m an is c am pa nu la in ca ne sc en s . 1 . . . . + + + . + . . . . . . . . . . . . + . . . . . 1 . + . + . . . . . 1 + + . + + . . ii i ii i 2 c ar ex k os he vn ik ov ii . 1 2 2 2 3 2 1 . + . 1 . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . + + . . . . . ii i i i – a rt em is ia r ut ifo lia . . . . . . . . . . . . 1 2 2 2 . . 1 2 . . . . . . . + + + . . . . . . . . . . 1 . . . . 1 . ii ii i 1 p oa re la xa . . . . . . . . . 2 1 1 1 + . . . . . . . . + . . . . . . . . . . . . . . . + . . + 1 1 . . . ii i ii 1 a sp er ul a al bi fl o ra . . . . . . . . . + . . 1 . . + . . . . . . . . . . . . . . 1 . . . . . . . . 1 1 1 1 . . . . i i ii – p ar ie ta ri a ju da ic a . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . 2 + . . . + . . . . . . . . . . . . – ii i – st ip a ze ra vs ch an ic a . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . + 1 1 . . . . . – i ii – sc hi st id iu m a po ca rp um d 1 2 . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – in ul a gl au ca . . + . . . . . . 1 + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – a sp er ul a la ev is . . . . . . . . . . . . . . . . . . . . . . . 1 + + . . . . . . . . . . . . . . . . . . . . . – ii – – sc ut el la ri a m eg al od on ta . . . . . . . . . . . . . . . . . . . . . . . + + . . . . 1 . . . . . . . . . . . . . . . . . – ii – – g ri m m ia p ul vi na ta d . . . . . . . . . . . . . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – p en th at he m a al be rt or eg el ia . . . . . . . . . . . . . . . . . . . . . . . + . . . . + . . . . . . . . . . + . . . . . . . – i i – dwarf shrub vegetation on rocks in tajikistan acta bot. croat. 75 (1), 2016 r el ev é nu m be r 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 constancy constancy constancy number of occurrence d ay 8 17 9 8 8 8 8 8 8 18 17 18 6 6 6 6 8 14 30 21 17 10 10 17 17 17 17 17 17 17 17 19 19 19 19 19 19 19 19 29 29 11 13 9 9 9 8 d at e: m on th 6 8 6 6 6 6 6 6 6 6 8 6 6 6 6 6 6 6 6 6 8 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 6 6 8 6 6 6 6 6 y ea r 13 13 13 12 12 12 12 12 12 13 13 13 13 13 13 13 13 12 14 14 13 12 12 13 13 13 13 13 13 13 13 12 12 12 12 12 12 12 12 14 14 12 12 12 12 12 12 ph 6, 6 6, 2 7, 4 6, 2 6, 4 6, 5 7 6, 3 6, 5 7, 8 6, 2 7, 5 6, 5 6, 4 6, 8 6, 3 6, 4 6, 2 8 7, 7 7, 8 7, 7 7, 7 7, 6 7, 6 7, 6 7, 8 7, 4 7, 8 7, 8 – 8 8, 2 8, 2 7, 9 7, 9 7, 5 8, 4 8, 2 8, 4 8, 4 8, 1 8, 2 7, 7 7, 8 7, 7 7, 5 a sp ec t w w w sw w w s w s n e sw sw w w w w w n w s se e se se se se se se e se w w n w n w n n n w n n n w e se n n s s se sw in cl in at io n (d eg re es ) 85 85 70 60 75 80 75 75 10 0 80 80 75 85 85 50 70 80 25 70 20 35 35 40 60 60 60 60 45 65 70 70 60 60 55 55 60 55 55 60 75 80 80 60 70 75 65 85 a lti tu de (m ) 89 0 14 01 19 09 15 60 10 85 10 85 12 36 10 85 12 50 19 31 15 86 13 50 19 28 19 28 19 25 19 25 89 0 15 90 20 92 17 78 17 70 17 43 17 43 17 14 17 14 17 14 17 75 17 13 17 75 19 61 19 61 29 13 18 74 29 13 18 74 18 74 18 56 22 15 18 78 24 82 24 82 29 59 18 97 18 25 21 10 21 81 16 50 c ov er o f s hr ub la ye r ( % ) 30 60 65 35 20 20 5 45 5 30 45 20 30 40 60 35 30 5 35 30 30 25 30 40 20 15 20 40 30 20 55 55 60 55 45 40 55 55 40 35 30 30 15 70 60 70 3 c ov er o f h er b la ye r ( % ) 15 15 20 15 35 55 30 10 20 25 15 15 20 20 25 25 15 25 15 40 10 15 10 20 25 10 10 15 3 40 10 5 3 3 2 5 3 3 3 10 15 10 40 10 5 5 45 c ov er o f m os s la ye r ( % ) 5 15 – – – – – – – – – – – 15 15 10 10 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – r el ev é ar ea (m 2 ) 5 3 5 4 3 3 3 3 3 3 3 3 5 5 5 5 5 3 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 4 re l. re l. re l. re l. n um be r o f s pe ci es 8 7 6 8 9 7 4 10 4 8 5 5 5 10 8 11 14 6 8 11 8 7 12 7 9 9 10 17 11 7 7 7 6 8 7 8 10 8 8 7 6 6 18 4 5 4 3 1− 18 19 −3 1 32 −4 3 44 −4 7 sc ut el la ri a hi ss ar ic a . . . . . 1 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – a sp le ni um r ut am ur ar ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . + . . . . . . . . . – – i – sp or ad ic s pe ci es : a na ph al lis d ar va zi ca 1 1( 1) ; a sp er ul a cz uk av in ae 4 3; d io ny si a in vo lu cr at a 9( 2) ; g al iu m v er tic ill at um 1 7; s yn tr ic hi a ru ra lis d 1 7( 1) . o th er s b ro m us te ct or um 1 . . + . 1 . . . . . . . . + + + . . 1 . 1 1 . . . . . . . . . . . . . . + + . . . . . . . + ii ii i 1 p oa b ul bo sa . + . . 2 1 . + . 1 . . . . . . . + . . . . . . . . . + . . . . . . . . . . . . . . 1 . . . . ii i i – c en ta ur ea s qu ar ro sa . . . . . . . . . . . . . . . . . . . 2 1 1 + . 1 + . . . . . . . . . . . . . . . . . . . . . – ii i – – p hl eu m g ra ec um . . . . . + . . . . . . . . . . + . . . . . . . . . . . . . . + + . . + . . + . . . . . . . . i – ii – c on ri ng ia p la ni si liq ua . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . + . . . . + . + . . . . . . + . – i ii 1 ve ro ni ca c ap ill ip es . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . + + + . . . + . . . . . . . . . – i ii – ve ro ni ca r ub ri fo lia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + . . . . . . . . . – – ii i – b ro m us o xy od on . . . + . 1 . + . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . i i – – vu lp ia c ili at a . . . . . . . + . . . . . . . . . + . . . . . . . . . . . . . . . . . + + . . . . . . . . . . i – i – b ry um c ae sp iti ci um d . . . . . . . . . . . . . 1 2 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – th ym us z er av sh an ic us . . . . . . . . . . . . . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . . . 2 . . . . i – i – b ry um a rg en te um d . . . . . . . . . . . . . 1 . 1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – b ro m us s qu ar ro su s . . . . . . . . . . . . . . . . . . . 1 1 . . . . . . . + . . . . . . . . . . . . . . . . . . – ii – – im pa tie ns p ar vi fl o ra . . + . . . . . . . . . . . . + 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – c re pi s pu lc hr um . . . . + . . + . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – r oe gn er ia in te rr up ta . . . . . . . . . + . . . . . . . . . . + . . . . . . . + . . . . . . . . . . . . . . . . . . i i – – se du m e w er si i . . . . . . . . . . . . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – si sy m br iu m b ra ss ic ifo rm e . . . . . . . . . . . . . . . . + . . . . . . . . . . + . . + . . . . . . . . . . . . . . . . i i – – b ot ri oc hl oa is ch ae m um . . . . . . . . . . . . . . . . . . + . + . . . . . + . . . . . . . . . . . . . . . . . . . . – ii – – a ly ss um d es er to ru m + . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . i i – – e re m ur us s te no ph yl lu s . . 1 . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – c le m at is o ri en ta lis . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . 1 . . – i – 1 g er an iu m d iv ar ic at um . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – 3 nowak a., nowak s., nobis m., nobis a. 4 acta bot. croat. 75 (1), 2016 r el ev é nu m be r 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 constancy constancy constancy number of occurrence d ay 8 17 9 8 8 8 8 8 8 18 17 18 6 6 6 6 8 14 30 21 17 10 10 17 17 17 17 17 17 17 17 19 19 19 19 19 19 19 19 29 29 11 13 9 9 9 8 d at e: m on th 6 8 6 6 6 6 6 6 6 6 8 6 6 6 6 6 6 6 6 6 8 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 6 6 8 6 6 6 6 6 y ea r 13 13 13 12 12 12 12 12 12 13 13 13 13 13 13 13 13 12 14 14 13 12 12 13 13 13 13 13 13 13 13 12 12 12 12 12 12 12 12 14 14 12 12 12 12 12 12 ph 6, 6 6, 2 7, 4 6, 2 6, 4 6, 5 7 6, 3 6, 5 7, 8 6, 2 7, 5 6, 5 6, 4 6, 8 6, 3 6, 4 6, 2 8 7, 7 7, 8 7, 7 7, 7 7, 6 7, 6 7, 6 7, 8 7, 4 7, 8 7, 8 – 8 8, 2 8, 2 7, 9 7, 9 7, 5 8, 4 8, 2 8, 4 8, 4 8, 1 8, 2 7, 7 7, 8 7, 7 7, 5 a sp ec t w w w sw w w s w s n e sw sw w w w w w n w s se e se se se se se se e se w w n w n w n n n w n n n w e se n n s s se sw in cl in at io n (d eg re es ) 85 85 70 60 75 80 75 75 10 0 80 80 75 85 85 50 70 80 25 70 20 35 35 40 60 60 60 60 45 65 70 70 60 60 55 55 60 55 55 60 75 80 80 60 70 75 65 85 a lti tu de (m ) 89 0 14 01 19 09 15 60 10 85 10 85 12 36 10 85 12 50 19 31 15 86 13 50 19 28 19 28 19 25 19 25 89 0 15 90 20 92 17 78 17 70 17 43 17 43 17 14 17 14 17 14 17 75 17 13 17 75 19 61 19 61 29 13 18 74 29 13 18 74 18 74 18 56 22 15 18 78 24 82 24 82 29 59 18 97 18 25 21 10 21 81 16 50 c ov er o f s hr ub la ye r ( % ) 30 60 65 35 20 20 5 45 5 30 45 20 30 40 60 35 30 5 35 30 30 25 30 40 20 15 20 40 30 20 55 55 60 55 45 40 55 55 40 35 30 30 15 70 60 70 3 c ov er o f h er b la ye r ( % ) 15 15 20 15 35 55 30 10 20 25 15 15 20 20 25 25 15 25 15 40 10 15 10 20 25 10 10 15 3 40 10 5 3 3 2 5 3 3 3 10 15 10 40 10 5 5 45 c ov er o f m os s la ye r ( % ) 5 15 – – – – – – – – – – – 15 15 10 10 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – r el ev é ar ea (m 2 ) 5 3 5 4 3 3 3 3 3 3 3 3 5 5 5 5 5 3 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 4 re l. re l. re l. re l. n um be r o f s pe ci es 8 7 6 8 9 7 4 10 4 8 5 5 5 10 8 11 14 6 8 11 8 7 12 7 9 9 10 17 11 7 7 7 6 8 7 8 10 8 8 7 6 6 18 4 5 4 3 1− 18 19 −3 1 32 −4 3 44 −4 7 a lli um s p. . . . + . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i – – – c le m at is s on ga ri ca . . . . + . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . i i – – ta en ia te ru m c ri ni tu m . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . i – i – k oe lp in ia li ne ar is . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . + . . . . – i i – le pt or ha bd os p ar vi fl o ra . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . – i – – zi zi ph or a te nu io r . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . + . . . . . . . . . – i i – p er ov sk ia v ir ga ta . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . + . . . . – i i – a ul ac os pe rm um d ic ho to m um . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . . – – i – sp or ad ic s pe ci es : a ve na tr ic ho ph yl la 5 ; b ro m us p se ud od an th no ni ae 4 3; b un iu m s er av sh an ic um 4 3( 1) ; c ap pa ri s sp in os a 20 ; c re pi s m ul tic au lis 2 8; d es cu ra in ia s op hi a 45 ; f er ul a sp . 5 ; f es tu ca v al es ia ca 2 8; h an de lia tr ic ho ph yl la 8 ; i xi ol ir io n ta ta ri ca 1 5; k ra sc he ni nn ik ov ia c er at oi de s 29 ; k ud ry as ch ew ia ja ku bi 2 9; l ap pu la c on sa ng ui ne a 27 ; m in ua rt ia m ey er i 2 7; p ar ie ta ri a se rb ic a 46 ; s ca bi os a so ng ar ic a 1( 1) ; s id er iti s m on ta na 2 8; s tr ig os el la a fr ic an a 29 ; t ar ax ac um s p. 4 3; t ha lic tr um a lp in um 1 7( 1) ; t hl as pi p er fo lia tu m 4 3. acta botanica 2-2016 za web.indd acta bot. croat. 75 (2), 2016 173 acta bot. croat. 75 (2), 173–178, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0037 issn 0365-0588 eissn 1847-8476 micromorphological, anatomical and cytogenetical studies in endemic crepis macropus boiss. & heldr. (asteraceae) from turkey huseyin inceer*, nursen aksu kalmuk, kemal vehbi imamoglu, ozge duman, sema hayirlioglu-ayaz, gokhan arslan karadeniz technical university, faculty of sciences, department of biology, 61080 trabzon, turkey abstract – in the present study, the micromorphological structure of achene, pappus and style using scanning electron microscope (sem), stomatal characteristics, anatomy of stem and achene together with chromosome number and nuclear dna content of the turkish endemic crepis macropus boiss. & heldr. are provided in order to expand knowledge of its taxonomy. the sem studies in this species show that dense spiny cells are found on the achene surface, the pappus bristle has 3–5 spikes and the style possesses slender papillae. the stem structure is composed of epidermis, collenchyma, parenchymatous cortex and pith. the species has anomocytic stomata in both the upper and the lower surface of the leaves. the pericarp of the achene is mainly composed of several layers of sclerenchymatous cells. in this species, the chromosome number is 2n = 2x = 8, karyotype consists of two submetacentric and six subtelocentric chromosomes and nuclear dna content (2cvalue) is 12.96 pg. these data are presented here for the fi rst time and their taxonomic values are discussed. keywords: anatomy, chromosome, crepis, endemic, micromorphology, nuclear dna content, turkey. * corresponding author, e-mail: inceer@ktu.edu.tr introduction the genus crepis l. belongs to the tribe cichorieae of the asteraceae family and comprises over 200 species (bremer 1994). its species are distributed throughout the northern hemisphere with single species occurring in south east asia. some species also occur in different regions of africa, the canary islands and madeira (enke 2008). it is thought that the origin of the genus crepis is in the altai/ tien shan region in the central asia (babcock 1947a). the genus presently has its highest species diversity in the circum-mediterranean area (enke 2008). in the turkish fl ora, the genus is represented by 42 taxa and eight taxa of them are endemic to turkey (ekim 2012). crepis is a problematic genus from a taxonomical point of view and it is notorious for its lack of discriminating characters (enke 2008). polymorphism is common in the genus and many taxonomic characters vary more within a species than between closely related species, and this often leads to unclear species-specifi c boundaries. additionally, some species of crepis are especially similar to hieracium and lapsana in their morphological characteristics and are also similar to many other cichorieae in their habits. therefore, they have been confused both taxonomically and nomenclaturally with each other and other cichorieae genera. the achenes of cichorieae are in many cases indispensable for the identifi cation of the genera and species and provide the systematically most valuable features on all taxonomic levels (kilian et al. 2008). as studies in the cichorieae have shown before (pak and kawano 1990, pak 1993, pak et al. 2001, zhu et al. 2006, sennikov and illarionova 2008), achene anatomy is helpful in the generic delimitation and infrageneric classifi cation of crepis (enke 2008, 2009, jana and mukherjee 2012). in general the achene surface features are also taxonomically valuable, mainly at species level, and more rarely concur with supraspecifi c delimitation (kilian et al. 2008). the pappus has always been an important feature for the discrimination of groups on all taxonomic levels in the cichorieae (kilian et al. 2008). the style morphology has been an important morphological character in major clade delimitation of the asteraceae (bremer 1996). some taxonomic value of micromorphology of the pappus and style has been reported in crepis (enke 2008, 2009). stem and leaf anatomy are considered as diacritical character in some groups of the tribe cichorieae (carlquist 1967), but there are rare data on stem and leaf anatomy of crepis in the literature (metcalfe and chalk 1979). the relevance of cytological and cytogenetical information to knowledge of the taxonomy and evolution of crepis inceer h., aksu kalmuk n., vehbi imamoglu k., duman o., hayirlioglu-ayaz s., arslan g. 174 acta bot. croat. 75 (2), 2016 was noted long ago. karyotype and genome size similarities and differences, especially chromosome number have been used as criteria to infer species relationships in crepis (babcock 1947a, b, siljak-yakovlev and cartier 1986, siljak-yakovlev and wraber 1988, kamari et al. 1991, godelle et al. 1993, dimitrova and greilhuber 2000, 2001, siljakyakovlev et al. 2010, enke et al. 2011, 2015). most of the micromorphological, anatomical and cytogenetical studies conducted in crepis have concentrated on common species, with some work having been interested in endemic species (kamari et al. 1991, kamari 1992, enke 2009, enke et al. 2011, siljak-yakovlev and peruzzi 2012). to our knowledge, except the chromosome counting of c. dioritica (2n = 8, davis et al. 1988) in turkey, no micromorphological, anatomical and cytogenetic studies have been reported for turkish endemic species of crepis. crepis macropus is an endemic species that belongs to section berinia (babcock 1947a, b, enke 2009). this endemic species is distributed in central, north and north-west anatolia. it grows on chalky, dry, stony or rocky slopes, steppe and fi eld sides at altitudes from 300 to 1600 m (babcock 1947b, lamond 1975). crepis macropus exhibits closer similarities to its balkan relatives, c. turcica and c. albanica, than to those of anatolia, but it is more like c. turcica based on its habit, involucres, fl orets and achenes (babcock 1947b). according to babcock (1947a), c. macropus is an “intermediate” type with regard to its phylogenetic position in the genus crepis, and there is strong positive correlation between this advanced type and adaptation to low altitude and arid environment, and it is relatively young species. thus, c. macropus might be neo-endemic and according to the terminology siljak-yakovlev and peruzzi (2012), it can be considered schizoendemic. according to the relevant literature records, except for molecular analysis based on its and chloroplast matk sequences (enke and gemeinholzer 2008, enke 2009), the micromorphological, anatomical and cytogenetical characteristics of c. macropus have not yet been studied. we aimed to give a detailed account of the micromorphological structure of achene, pappus and style, leaf epidermal characteristics, achene and stem anatomy, karyotype and nuclear dna content for this turkish endemic species. material and methods plant materials plant samples were collected from natural populations in 2013 and 2014 from ankara (beynam forest, rocky slopes, 1320 m a.s.l., june, 11th, 2013, achene and pappus for micromorphological observations; leaf, stem and achene for anatomical observations; achene for cytogenetic analysis: inceer 1009), çankırı (near çakmak village, rocky slopes, 885 m a.s.l., june, 27th, 2014, leaf for cytogenetic analysis: aksu 196) and nevşehir (ürgüp national park, 1095 m a.s.l, july, 1st, 2013, capitulum for micromorphological observations of style: inceer 1015) provinces. vouchers are deposited in the herbarium at the karadeniz technical university, department of the biology (ktub). scanning electron microscopy micromorphological structure of achene, pappus and style was analyzed on the scanning electron microscope (sem). dry and mature specimens were mounted directly on the stubs using double-sided adhesive tape, and then observed in agilent fesem 8500 scanning electron microscope. the micromorphological characterization was performed according to enke (2008). anatomical studies anatomical observations were performed in the leaf, stem and achene. for this purpose, peripheral sections of upper (adaxial) and lower (abaxial) epidermis of the leaves were taken by hand using commercial razor blades from fi xing materials in faa (5% formaldehyde : 5% glacial acetic acid : 70% ethyl alcohol). semi-permanent slides were mounted in glycerine. stomatal index was assessed in both the upper and lower epidermis (meidner and mansfi eld 1968). transverse sections from middle parts of the stem were taken by hand using commercial razor blades from fi xing materials in formaldehyde-glacial acetic acid-ethyl alcohol and were stained with safranin and then mounted in entellan. transverse sections of achene were carried out with the paraffi n method (algan 1981) and stained with hematoxylin and then mounted in entellan. three slides obtained from three individuals were prepared and the anatomical characters were measured using an ocular micrometer under the light microscope. the photographs were taken using a leica dm 4000 microscope and a leica dfc 490 digital camera. illustrations were drawn with a lucida camera attached to a light microscope. cytogenetic analyses the root tip meristems obtained directly from germinated achenes were used for chromosome analysis. the root tips were pre-treated with 0.05% aqueous colchicine solution for 3–5 h at room temperature and then fi xed in absolute ethanol-glacial acetic acid (3:1) for at least 24 h at 4 °c (inceer and hayirlioglu-ayaz 2007). they were hydrolyzed in 1 n hcl at 60 °c for 12–15 minutes. staining was carried out in 1% lacto-propionic orcein for 12–18 h at room temperature and squash preparations were made in 45% acetic acid. the best metaphase plate of each individual was photographed with a leica dm 4000 microscope with a leica dfc 490 digital camera. five well-spread metaphase plates of different fi ve individuals were used for chromosome analysis and idiogram building. karyotype analysis was performed according to levan et al. (1964). idiograms were prepared based on the average measurements of each chromosome pair. chromosomes were classifi ed on the basis of arm ratio in accordance with stebbins (1971). the intrachromosomal asymmetry index (a1) was calculated by use of the formula proposed by romero zarco (1986), and the interchromosomal asymmetry index (a2) was measured as the ratio of chromosome length/mean chromosome length. in haploid idiograms, chromosomes were arranged according their length. studies on crepis macropus from turkey acta bot. croat. 75 (2), 2016 175 the young leaves were taken from three specimens in natural population for fl ow cytometric analysis. the leaves of lycopersicon esculentum cv. ‘swanson’ (2 pg/2c) potted and grown were used as an internal standard. nuclear dna content was assessed by fl ow cytometry as follows. leaf fragments of the sample plant and the standard plant were chopped using a razor blade in 1 ml of woody plant buffer (loureiro et al. 2007; 0.2 m tris hcl, 4 mm mgcl2×6h2o, 2 mm na2edta×2h2o, 86 mm nacl, 10 mm potassium metabisulphite, 1% pvp-10, 1% (v/v) triton x-100, ph 7.5) supplemented with 50 μg ml–1 propidium iodide and 50 μg ml–1 dnase-free rnase, fi ltered through a 30 μm mesh and stored on ice, in the dark, until measurement. three independent samples were extracted, fi ltered and measured on the same day. the measurements were made three consecutive days using bd accuri™ c6. usually 10,000 nuclei per sample were analyzed for nuclear dna content. results micromorphology in this species, all achene surfaces have prominent spiny cells (figs. 1a, b). the pappus bristles are slender and made up of 3–5 cells in diameter. the pappus bristles have 3–5 spikes per 100 μm, diameter is 21–44 μm (fig. 1c). the style and the style branches consist of papillae. the papillae are type a and more densely arranged in the style. the style diameter is 102.23 ± 2.91 μm, and the diameter of its arm is 119.43 ± 12.72 μm (figs. 1d, e). anatomy in the leaves of c. macropus, there is a single layered isodiametric epidermis with wavy walls on both the adaxial and the abaxial surface. adaxial epidermal cells are more or less equal to the abaxial ones. stomata are present on both surfaces of the leaf (amphistomatic) and anomocytic type (stomata without subsidiary cells). they lie more or less at the epidermis level. the guard cells are oval-shaped. the stomata on both the adaxial and abaxial sides are almost the same size. on the adaxial side, the size of stomata is 26.43 ± 0.65 × 37.66 ± 2.17 μm, on the abaxial side, 26.68 ± 0.80 × 37.74 ± 1.37 μm (figs. 2b, c). however, the stomatal index on the abaxial side is 18.06, whereas it is 14.80 on the adaxial side. the stem of c. macropus is more or less round in the transverse sections. in its stem anatomy, the epidermis consists of single layered, roundish-ovate, frequently arranged cells and is surrounded by a more or less thick cuticle layer. cells dimensions are 15.86 ± 03.02 × 13.42 ± 01.29 μm. collenchyma is located below the epidermis. it has 2–3 layers at the margins and its thickness is 183.20 ± 10.04 μm. cortex is composed of 7–8 rows, parenchymatous and oval cells. its thickness is 183.20 ± 10.04 μm. vascular bundles are collateral, scattered in a circular order in the ground tissue. phloem and xylem members are clear. the phloem thickness is 95.36 ± 07.81 μm, whereas the xylem thickness is 194.18 ± 15.29 μm. width of the vascular bundle is 113.66 ± 14.89 μm. the cambium formation is distinguishable in the vascular bundles. the pith of the stem consists of large, round parenchymatic cells (fig. 2a). the achene of the species has a rounded outline with 12 ribs consisting of sclerenchymatous cell bundles in the transverse sections. the ribs are prominent without inter rib furrows. pericarp consists of exocarp, mesocarp and endocarp. exocarp is one layered, with a thick outer cell wall, but the cells are collapsed. mesocarp only consists of sclerenchymatic bundles and cells. endocarp is two layered and collapsed. the pericarp thickness is 91.09 ± 17.84 μm, and its width is 96.79 ± 19.74 μm. testa is circular. its thickness is 2.44 ± 0.14 μm. endosperm consists of two layered cells. the thickness of the endosperm is 10.30 ± 0.47 μm. embryo is formed by more or less oval or round, parenchymatic cells. the orientation of cotyledons is at right angles to the axis of achene (fig. 2d). cytogenetics the chromosome number of c. macropus is 2n = 2x = 8. the two chromosome types in its karyotype are distinfig. 1. scanning electron microscope micrographs of crepis macropus: a, b) achene (inceer 1009), c) pappus (inceer 1009), d) style (inceer 1015), e) style arm (inceer 1015), scale bars: a, d) = 100 μm; b, e) = 10 μm; c) = 40 μm. fig. 2. anatomical structure of crepis macropus (inceer 1009): a) stem, cl – collenchyma, cr – cortex, e – epidermis, ph – phloem, xy – xylem, scale bar = 100 μm, b) abaxial surface in the leaves, c) adaxial surface in the leaves, scale bars (b–c) = 50 μm, d) achene, ct – cotyledons, es – endosperm, sc – sclerenchyma, t – testa, scale bar = 50 μm. inceer h., aksu kalmuk n., vehbi imamoglu k., duman o., hayirlioglu-ayaz s., arslan g. 176 acta bot. croat. 75 (2), 2016 guished: one submetacentric and three subtelocentric chromosome pairs. the chromosome length ranges from 5.26 to 7.64 μm. the relative length ranges from 20.25 to 29.42. (tab. 1, figs. 3a, b). the classis of karyotype asymmetry is placed in 4a, the index of a1 is 0.69 and the index of a2 is 0.15. the nuclear dna content (2c-value) of this endemic species is 12.96 ± 0.11 pg. its monoploid genome size (1cvalue) is 6.48 pg (fig. 4). discussion the results obtained from micromorphological, anatomical and cytogenetical studies on c. macropus are presented in tab. 1 and figs. 1–4. our fi ndings are in agreement with the previous results on the other species of the genus (babcock 1947a, b, metcalfe and chalk 1979, enke and gemeinholzer 2008, enke 2009, enke et al. 2011, 2015, yildirim et al. 2011). the additional morphological, anatomical and cytogenetical characters supporting systematic delimitation of the genus have been used because the molecular analyses by enke (2009) could not support the current taxonomic sections (babcock 1947b). enke (2008) pointed that the surface features of achene, pappus and style are taxonomically valuable, mainly at species level in crepis. the present morphological analyses show that the achenes of c. macropus have prominent spiny cells on its surface. the pappus is comparatively homogenous in the species and it confi rms the general description presented by enke (2008). additionally, enke (2009) reported three papillae types as “type a, type b and type c” in style and style branches. the papillae type in the style and style branches of c. macropus is type a in this species. similar results are reported by enke (2008) for c. leontodontoides and c. zacintha. many anatomical characters of the leaves are useful for systematics, particularly the epidermal cells and stomata (metcalfe and chalk 1979, dickison 2000, araújo et al. 2010, inceer and ozcan 2011). paradermal sections taken from the leaves show that c. macropus has isodiametric epidermal cells together with anomocytic stomata. the epidermal cell walls are also of the same structure, with anticlinal undulate walls on both the adaxial and abaxial surface. these characteristics agree with the results previously reported by metcalfe and chalk (1979) and inceer and ozcan (2011) for the family asteraceae. metcalfe and chalk (1979) reported that vascular bundles are taxonomically important and cambium can occur in the member of asteraceae family. the stem anatomy of c. macropus is composed of epidermis, cortex, corticular vascular bundles with distinguishable cambium formation and parenchymatic pith. the present results are in agreement with previous data of metcalfe and chalk (1979). the achene anatomy has proven so far to be of some relevance for classifi cation of the crepis species. enke (2009) reported four different achene anatomy types, “type fig. 3. somatic metaphase of crepis macropus (inceer 1009): a) microphotograph of mitotic chromosome plate (2n = 8), b) haploid idiogram, scale bar = 10 μm. fig. 4. flow cytometry histograms: a) peak of standard lycopersicon esculentum cv. ‘swanson’ (2c = 2.00 pg), b) peak of crepis macropus, aksu 196 (2c = 12.96 pg). tab. 1. morphometric data of karyotype of crepis macropus. data are arithmetic mean ± standard deviation, n = 5. s – short arm length, l – long arm length, t – total length, l/s – arm ratio, sat – satellite, ci – centromeric index, rl – relative length, sm – submetacentric, st – subtelocentric. chromosome pairs s l t l/s sat ci rl chromosome type(μm) (μm) (μm) (μm) 1 5.28±0.48 2.36±0.25 7.64±0.53 2.24 – 30.89 29.42 sm 2 5.16±0.14 1.53±0.52 6.69±0.54 3.37 – 22.87 25.76 st 3 4.95±0.26 1.43±0.32 6.38±0.59 3.46 – 22.41 24.57 st 4 4.32±0.10 0.94±0.25 5.26±0.26 4.60 – 17.87 20.25 st studies on crepis macropus from turkey acta bot. croat. 75 (2), 2016 177 i, type ii, type iii and type iv” in crepis. we observed that c. macropus possesses type iv in its achene anatomy. according to enke (2009), in type iv, exocarp can be collapsed and the costae are very prominent with deep or no intercostal furrows. crepis is a model group for genetic studies explaining evolution and speciation in higher plants because it has low chromosome numbers. therefore, many chromosomal studies on crepis have been published so far (babcock 1947a, b, siljak-yakovlev and cartier 1982, dimitrova and greilhuber 2000, 2001). different basic chromosome numbers x = 3, 4, 5 and 6, are present in the genus crepis (babcock 1947a, b, kamari 1992, dimitrova and greilhuber 2000, 2001, enke 2008) and karyotypes can be symmetrical or asymmetrical (babcock 1947b). crepis macropus is a diploid species with 2n = 2x = 8 chromosomes. this species has the same chromosome number with the members of the section berinia such as c. turcica, c. merxmuerlleri, c. sibthorpiana, c. sonchifolia (nazarova 1984, kamari et al. 1991, kamari 1992, constantinidis et al. 2002, enke 2008). there is a minor difference in the chromosome morphology between c. macropus and c. turcica (kamari et al. 1991). the chromosome size values in c. macropus range from 5.26 to 7.64 μm, whereas the chromosome size values range from 4.7 to 6.7 μm in c. turcica (kamari et al. 1991). the karyotypes in both species also consist of long chromosomes. crepis macropus has an asymmetrical karyotype. it is widely accepted that the evolutionary trend is toward an increase in karyotype diversity (stebbins 1971, liu et al. 2006, inceer et al. 2012). it was also reported that increasing karyotype asymmetry could occur via a shift of the centromere position from median to subterminal or terminal chromosome regions, or through accumulation of differences in relative size between individual chromosomes (liu et al. 2006, inceer et al. 2012). despite the impressive amount of genome size studies seen the last few years (bennet and leitch 2005, enke et al. 2011, garcia et al. 2013), taxonomic coverage remains very restricted, with c-values assessed for less than 40% of the crepis species. the nuclear dna content of c. macropus is presented with a histogram in fig. 4, and its genome size (1c-value) is 6.48 pg. the genome size (1c-value) in c. turcica is 6.41 pg (enke 2009), and thus there is slight difference in genome size between c. macropus and c. turcica. according to leitch et al. (1998) and soltis et al. (2003), genome sizes can be assigned to a series of distinct categories: very small (1c ≤ 1.4 pg), small (1.4 < 1c ≤ 3.5 pg), intermediate 3.5 < 1c < 14 pg), large (14 ≤ 1c < 35 pg) and very large (1c ≥ 35 pg). in these terms, the endemic c. macropus has an intermediate genome. similar results are reported in other species of crepis (godelle et al. 1993, siljak-yakovlev et al. 2010, enke et al. 2011). the similarities in the nuclear dna content as well as karyotype and external morphology of c. macropus and its relative suggest that their genomes evolved from a common ancestral genome and underwent some structural differentiation of the genomes. in conclusion, the present results obtained from micromorphological, anatomical and cytogenetical analyses will increase taxonomic information about c. macropus in the literature. furthermore, features of achene, pappus, style, karyotype and nuclear dna value can be used as taxonomic characters for this turkish endemic species. acknowledgements the authors thank the scientifi c and technological research council of turkey (tubitak project no.112t132) for fi nancial support; anonymous reviewers and dr. sandro bogdanović for comments that improved the manuscript considerably. the corresponding author wish to thank dr. jaroslav doležel and dr. jana cizkova from the institute of experimental botany as cr for facilities in centre of plant structural and functional genomics; council of higher education, turkey, for the grant in centre of plant structural and functional genomics of institute of experimental botany as cr. references algan, g., 1981: bitkisel dokular icin mikroteknik. fırat university science faculty press, i̇stanbul (in turkish). araújo, j. s., azevedo, a. a., silva, l. c., meira, r. m. s. a., 2010: leaf anatomy as an additional taxonomy tool for 16 species of malpighiaceae found in cerrado area (brazil). plant systematics and evolution 286, 117–131. babcock, e. b., 1947a: the genus crepis. part one: the taxonomy, phylogeny, distribution and evolution of crepis. university of california publications 21. university of california press, berkeley, los angeles. babcock, e. b., 1947b: the genus crepis. part two: systematic treatment. university of california publications 22. university of california press, berkeley, los angeles. bennett, m. d., leitch, i. j., 2005: the evolution of the genome. in: gregory, t. r. 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(asteraceae) from east anatolia, turkey. nordic journal of botany 29, 14–19. zhu, s. x., qin, h. n., shih, c., 2006: achene wall anatomy and surface sculpturing of lactuca l. and related genera (compositae: lactuceae) with notes on their systematic signifi cance. journal of integrative plant biology 48, 390–399. 146 acta bot. croat. 76 (2), 2017 acta bot. croat. 76 (2), 146–153, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0009 issn 0365-0588 eissn 1847-8476 allium cepa root meristem cells under osmotic (sorbitol) and salt (nacl) stress in vitro agnieszka kiełkowska institute of plant biology and biotechnology, faculty of biotechnology and horticulture, university of agriculture in krakow, al. 29-listopada 54, 31-425 krakow, poland abstract – the effects of various concentrations of sorbitol (100, 200 and 360 mm) and nacl (100, 200 and 300 mm) on root meristem cells of in vitro-cultured allium cepa l. were analyzed after 10 and 20 days. both root meristem cell cross-section area and nuclear volume decreased under osmotic and salt stress. the osmotic component of applied stresses had a greater impact on cell shrinkage, while ionic stress perturbed cell functioning, resulting in cell cycle arrest and various aberrations, affecting nucleus integrity. a concentration of 300 mm of nacl in the culture medium caused complete inhibition of mitotic activity in onion root tip cells after 20 days of exposure. analysis of the action of iso-osmotic concentrations of nacl (200 mm) and sorbitol (360 mm) showed stronger mitodepressive effects of salt stress in comparison to osmotic stress. keywords: aberrations, cell size, mitotic index, nuclear volume, phase index, stress * corresponding author, e-mail: kielkowska@ogr.ur.krakow.pl introduction to study the effects of abiotic stress on plants, in vitro cultures are suitable, providing aseptic conditions, rigorous control of the physical environment and nutrition in the relatively small space needed for culture (verslues et al. 2006, kiełkowska et al. 2012). the stress in tissue culture can be induced by the addition of specific compounds to the growth medium. based on their physiochemical properties, stress inducers can be divided into categories of ionic and cell penetrating (nacl, kcl); non-ionic and penetrating (mannitol, sorbitol); and non-ionic and non-penetrating (polyethylene glycol (peg)) (gangopadhyay et al. 1997). usually, non-ionic osmotic and water stresses are simulated by adding peg, mannitol or sorbitol. non-ionic osmotic stress refers to changes in any activity or integrity of plant cells due to an increase or decrease in the concentration of non-ionic solutes changing the water potential of the cellsurrounding solution (ahmad et al. 2007). ionic stress might be induced by nacl or kcl, producing specific ionic toxicities; however, both salts also induce osmotic stress, due to the rise in salt concentration outside the cells, leading to inhibition of water uptake (claeys et al. 2014). osmotic stress under salinity affects plants immediately after the increase in salt concentration, while ionic stress develops later, together with the excessive accumulation of ions (munns and tester 2008, deinlein et al. 2014). research on osmotically mediated water deficit has been focused mainly on the evaluation of its effects on germination, plant morphology, biomass production and biochemical processes (bhargava and paranjpe 2004, grzesiak et al. 2006, ahmad et al. 2007), and little attention has been devoted to its action at the cytological level. the effect of nacl-induced stress has been investigated mostly in cereals, and results show that concentrations greater than 300 mm cause serious damage at the cellular level and lead to cell death (munns and tester 2008, yumurtaci et al. 2009, tabur and demir 2010). lower concentrations of nacl reduced root growth, which was also reflected in the reduction of the size of the root meristem (hanif and davies 1998, zadeh 2007). recently, a reduction in root meristem size was also reported for wheat under peg-mediated osmotic stress (ji et al. 2014). however, the mechanism by which the stress affects the meristem length is not fully understood. it is related to the inhibition of cell division under stress conditions. west et al. (2004) suggested that stress rapidly blocks cell cycle progression to prevent entry into stages where the cell is vulnerable to damage, allowing the cellular defense system to be activated. stress-induced inhibition of cell division leads to fewer cells being produced, which results in reduction in meristem size. kimball et al. (1975) reported that mannitol and sorbitol caused a reduction in the size of soybean cells in suspension cultures. similar results were reported for epidermal onion root meristem under osmotic and salt stress acta bot. croat. 76 (2), 2017 147 cells of maize after nacl treatment (zörb et al. 2015). however, it is unclear whether the root meristem cell size is also affected under osmotic/salt stress conditions and whether the affected cells maintain the ability to divide. to evaluate these effects, it is necessary to perform observations for an extended period of time. such an approach will ensure enough time for several rounds of cell cycle progression in stress conditions. most available reports show the effects of salt/osmotic shock rather than stress, as results were collected within minutes to hours after application of stress conditions (shavrukov 2013, deinlein et al. 2014). onion is an important crop worldwide and is considered salt sensitive (shannon and grieve 1999). there are single studies covering the reaction of onion to salt stress at the cellular level (bennici and tani 2009, chatterjee and majumder 2010). such information is needed to gain better knowledge concerning stress responses and stress tolerance mechanisms in glycophytes. this information has both fundamental and economic importance, as all vegetables are included in this group. the goal of this study was to evaluate the cytogenetic effects of nacl-mediated salt stress and sorbitol-mediated osmotic stress on onion root meristem cells. to reveal the effects of applied stresses on mitosis and cell and nuclear size, experiments were conducted for extended periods of 10 and 20 days. analysis of the effects of iso-osmotic solutions of nacl (200 mm) and sorbitol (360 mm) allows comparison of the impact of the osmotic and ionic compounds of the applied stresses on the tested features. materials and methods seeds of allium cepa cv. majka f1, (plantico, kraków, poland) were surface-disinfected in 70% (v/v) ethanol for 5 min and in a 20% chloramine t (biochemie poland, katowice, poland) solution (w/v) for 20 min and then washed three times in sterile water, each for 5 min. for germination, seeds were placed in 500-ml plastic culture boxes (pakler lerka, kraków, poland) containing 80 ml of basal ms medium (murashige and skoog 1962) without growth regulators. basal ms medium was prepared using a powdered ms salt mixture including vitamins (duchefa, unimarket, poznań, poland) with 30 g l–1 sucrose and 0.8% (w/v) agar (biocorp, warszawa, poland), ph 5.7−5.8. cultures were maintained at 25±2 °c in darkness for germination. boxes with seedlings were kept at 25±2 °c (16-h days, 55 μmol m–2 s–1). ten-day-old plants were placed in boxes with 80 ml of culture medium consisting of basal ms medium with addition of 100, 200 or 300 mm of nacl and 100, 200 or 360 mm sorbitol. solution of 360 mm sorbitol is iso-osmotic to solution of 200 mm nacl. plants grown in basal ms medium were used as controls. plants were exposed to the media with given concentrations of nacl and sorbitol for 10 or 20 days. at the end of exposure period, plants were removed from the culture boxes and prepared for observations. roots from in vitro-cultured plants were fixed in 96% ethanol/glacial acetic acid (3:1 v/v) and hydrolyzed with 1 n hcl for 10 min at 60 °c. roots were then stained with schiff’s reagent (sigma aldrich ltd., poznań, poland) for 1 h at room temperature. from roots, approximately1-mmlong root tips were excised and then squashed in 50 µl of acetocarmine. observations of slides were performed under a nikon eclipse (nikon instruments inc., japan) light microscope. microscopic analyses included measurements of interphasic cell area, interphasic nuclear volume (vn), determination of the mitotic (mi) and phase (pi) indices, chromosomal aberration score, and nuclear abnormalities. for the calculations of vn and cell area, image analysis of interphase cells using nikon imaging software nis elements documentation was performed. cell cross-section area was measured for 2000 interphase cells for each treatment. results were expressed as the mean cell cross-section area in square micrometers. for vn calculations, the radius (r, µm) of 2000 nuclei for each treatment was measured. the vn was then calculated according to methods of mesi and kopliku (2013). to study mi and aberrations, approximately 2000 cells per each of five slides per sample were analyzed. the mi was calculated as the number of cells in mitosis divided by the total number of cells × 100%. pi was calculated as the percentage of cells in a certain mitotic phase of division (prophase, metaphase, anaphase and telophase). in addition, the values of the metaphase/ana-telophase (m/a + t) ratios were calculated. data were analyzed using a factorial analysis of variance (anova), and mean separations were conducted using a post-hoc student−newman−keuls (snk) test at p ≤ 0.05. all results were expressed as the mean±standard error (se) of the mean. results interphasic cell cross-section area the average cross-section area of in vitro-grown onion root meristem cells was 407−413 µm2, and it did not change significantly during the experiment (tab. 1). a decrease in the cell cross-section area with increasing concentrations of stress agents during the time of exposure was observed. after the 10-day treatment, root meristem cells of plants grown on medium supplemented with 100 mm of sorbitol were 16% smaller than control cells during the same period of time, while after 20 days of exposure cells were 28% smaller. a similar tendency was observed in the roots of plants grown on the media with higher concentrations of sorbitol. at day 10 of the experiment, the average cross-section area of meristem cells in onion roots cultured on medium with 100 mm of nacl was 375.9±10.8 µm2, which represented 92% of the area of control cells. at the same time, the cross-section area of meristem cells in roots cultured on medium with 300 mm of nacl was reduced to a size representing 78% of the area of control cells. after 20 days of exposure to the salt stress, the decrease in cell area continued in parallel with salt concentration, and the meristem cells in roots grown on medium with 300 mm of nacl were approximately half of the size of control cells. kiełkowska a. 148 acta bot. croat. 76 (2), 2017 after 10 days of exposure of onion roots to iso-osmotic concentrations of sorbitol (360 mm) and nacl (200 mm), a greater decrease in cell area was observed in sorbitol-treated roots; however, after 20 days of exposure, the mean areas of sorbitoland salt-treated interphasic cells were similar: 218.6±7.0 µm2 and 201.1±13.1 µm2, respectively. interphasic nuclear volume the mean vn of onion root meristem cells in the control treatment was approximately 1440 µm3 (tab. 2) and was relatively constant during the experiment. sorbitol at a concentration of 100 mm had a minor effect on interphasic vn in root tip cells; however, higher concentrations caused a decrease in vn. the nuclear volume of root meristem cells exposed to 200 mm of sorbitol for 20 days was approximately 20% smaller than that of control cells. exposure of root meristems to increasing concentrations of nacl resulted in a decrease of vn of treated cells compared to that of controls. the average vn of root tip cells after 10-day exposure to medium with 100 mm of nacl was similar to that of controls; however, the vn decreased gradually with increasing salt concentration. the nuclei of root meristem cells exposed to 300 mm of nacl represented 46% of the volume of nuclei in control cells. after 20 days of exposure to 360 mm of sorbitol, the volume of average nuclei in onion root meristem cells was 1045.0±29.0 µm3 (73% of vn of control cells), while in the roots cultured on medium with iso-osmotic concentration of nacl the volume of the nuclei was significantly reduced to 660.1±18.7 µm3 (51% of vn of control cells). mitotic activity mitosis in the roots of control plants was normal and undisturbed (tab. 3, figs. 1a−e). alterations in mi were observed in the roots of treated plants depending upon the type of stress agent, its concentration and length of exposure. in the roots of plants grown on media with the addition of sorbitol, a decrease in mitotic activity compared to the controls was observed. during the entire experiment, the mi of sorbitol-treated cells was approximately 50% lower than the mi observed for control cells. although not statistically significant, a decrease in mi with increasing concentration of sorbitol in the culture media and time of exposure was observed. addition of nacl to the culture medium caused a more severe decrease in mitotic activity of onion root tip cells. on medium with 100 mm of nacl, the average number of mitotic figures was 25−60% lower than that of the controls and decreased with increasing concentration of salt and time of exposure. twenty-day exposure to 300 mm of nacl completely arrested cell divisions. the mi in onion roots grown for 10 and 20 days on medium with 360 mm of sorbitol was 14.8±1.6% and 16.4±3.2%, tab. 1. mean cross-section area of allium cepa root meristem cells exposed to sorbitol and nacl in vitro. means followed by the same letter are not significantly different (p≤0.05, student−newman−keuls test). treatment (mm) exposure (days) 10 20 cell area (µm2 ± se) % to ctrl cell area (µm2 ± se) % to ctrl control (ctrl.) 407.7±15.0 a 100 413.7±15.9 a 100 sorbitol 100 341.5±8.4 c 84 296.5±11.0 de 72 200 295.3±10.2 de 72 283.3±9.1 ef 68 360 241.8±6.1 gh 59 218.6±7.0 hi 53 nacl 100 375.9±10.8 b 92 258.7±27.1 fg 63 200 335.7±16.5 c 82 238.0±12.4 gh 58 300 319.6±72.0 cd 78 201.1±13.1 i 49 tab. 2. nuclear volume (vn) of allium cepa root meristem cells exposed to sorbitol and nacl in vitro. means followed by the same letter are not significantly different (p≤0.05, student−newman−keuls test). treatment (mm) exposure (days) 10 20 vn (µm3 ± se) % to ctrl vn (µm3 ± se) % to ctrl control (ctrl) 1449.4±51.0 a 100 1428.3±46.1 a 100 sorbitol 100 1458.1±55.0 a 101 1327.4±54.9 ab 93 200 1189.8±45.2 bc 82 1147.2±43.4 bc 80 360 987.1±39.4 c 68 1045.0±29.0 c 73 nacl 100 1400.1±60.0 a 97 1106.0±36.6 bc 77 200 1126.3±28.5 bc 78 660.1±18.7 d 51 300 673.4±37.4 d 46 721.8±16.2 d 46 tab. 3. mitotic (mi) and phase (pi) indices in allium cepa root meristem cells exposed to sorbitol and nacl in vitro. means followed by the same letter are not significantly different (p≤0.05, student−newman−keuls test). p – prophase, m – metaphase, a + t – anaphase and telophase. treatment (mm) e xp os ur e (d ay s) mi pi (% ± se) % to ctrl p m a+t m/ a+t control (ctrl) 10 31.0±2.9 a 100 57.7 21.9 20.4 1.1 20 28.2±1.4 a 100 53.7 23.6 22.7 1.0 so rb ito l 100 10 16.9±3.7 bcd 55 41.0 30.2 28.8 1.1 20 15.1±1.2 bcd 53 49.8 25.8 24.4 1.1 200 10 16.4±2.3 bcd 53 43.1 24.0 32.8 0.7 20 17.7±2.9 bcd 63 46.7 24.6 28.8 0.9 360 10 14.8±1.6 bcd 48 52.2 19.0 28.8 0.7 20 16.4±3.2 bcd 58 42.0 25.7 32.3 0.8 n ac l 100 10 23.3±2.3 b 75 44.9 19.7 35.3 0.6 20 12.0±2.1 cde 42 62.0 10.8 27.3 0.4 200 10 10.4±1.3 cde 34 52.6 14.2 33.2 0.4 20 7.5±1.6 df 27 37.8 15.2 47.0 0.3 300 10 4.3±1.0 ef 14 41.7 16.7 41.7 0.4 20 0.0±0.0 f 0 0.0 0.0 0.0 0.0 onion root meristem under osmotic and salt stress acta bot. croat. 76 (2), 2017 149 respectively, while in roots of plants grown on medium with iso-osmotic concentration of nacl (200 mm) the mi was 10.4±1.3% and 7.5±1.6%, respectively. microscopic observations showed that in the meristematic cells of roots exposed to 200 mm and 300 mm of nacl, nuclei were shifted toward the cell wall (fig. 1r). moreover, in the roots of plants grown on medium with 300 mm of nacl, in some of the observed meristem cells, despite staining, no nucleus was visible. in the control roots, the ratios of different mitotic phases after 10 and 20 days of growth were similar (tab. 3). most (54−58%) of the dividing root tip cells were in prophase, 22−24% were in metaphase and 20−23% were in ana/telophase, and the m/a + t ratio was approximately 1. similar observations were made for roots treated with 100 mm of sorbitol. in the meristems of roots grown on media with higher concentrations of sorbitol, occurrences of anaphase and telophase were more numerous compared to those of metaphase, and the m/a + t ratio was 0.7−0.9. in the meristems of roots treated with nacl, the m/a + t ratio was very low and ranged from 0.3−0.6. in this treatment, cells in prophase were prevalent; however, the number of cells in metaphase decreased. abnormalities in control plants, most root tip cells underwent a typical course of mitosis without disturbance (tab. 4). in roots of onion cultured on media with sorbitol, the total number of aberrant cells did not exceed 20%. the number of abnormal cells increased with sorbitol concentration in the medium and time of exposure. in this treatment, the most frequent abnormalities were nuclear budding (1.8−8.1%; fig. 1o) and nuclear fragmentation (0.2−6.8). addition of 100 mm of nacl to culture medium resulted in the appearance of chromosomal aberrations such as sticky chromosomes (12.7%; fig. 1f) as well as chromosomal bridges and fragments (10.1%; figs. 1g−j). the highest percentage (46.5± 8.1) of aberrant cells was recorded in roots of plants treated with 200 mm of nacl for 20 days. in this treatment, considerable frequency of meristem cells with lagging chromosomes (9.5%); nuclear budding (5.5%); nuclear disintegration and fragmentation (13.3%; fig. 1p); and micronuclei (8.9%; figs. 1k−n) was recorded. in roots exposed to isoosmotic solutions of sorbitol, total percent of aberrant cells was lower, and nuclear budding and fragmentation were mainly observed. fig. 1. allium cepa root tip cells exposed in vitro to sorbitol and nacl and control cells. typical stages of mitosis in control roots: interphase (a), prophase (b), metaphase (c), anaphase (d) and telophase (e). chromosomal aberrations after nacl treatment: stickiness in metaphase (f) laggards, chromosome breaks and losses (g, h, i), chromosome bridge (j). nuclear aberrations: micronuclei (k, l, m) and nuclear budding (n) in root tip cells treated with 200 mm nacl; nuclear budding (o) in root tip cells treated with 360 mm sorbitol; nuclear disintegration (p) in root tip cells treated with 200 mm nacl. interphasic root tip cells after 20 days of treatment with 300 mm nacl (r). scale bar = 10 µm. kiełkowska a. 150 acta bot. croat. 76 (2), 2017 discussion cell size is controlled by a hierarchy of regulatory systems that senses the overall organ size and the total mass of the organism (kondorosi et al. 2000). the maintenance of cell size requires homeostasis between macromolecule synthesis and degradation. maintenance is also linked to nutrient and growth factor availability and is influenced by the environment (zonia and munnik 2007, osakabe et al. 2013). changes in root meristem cell size under abiotic stress have not been deeply investigated. in this study, both sorbitol and nacl caused a decrease in root meristem cell area, which confirms previous findings in other types of cells (kimball et al. 1975, zörb et al. 2015). a greater decrease in interphasic cell cross-section area was observed after salt treatment; however, comparison of the effects of iso-osmotic solutions of sorbitol and nacl showed similar results, and the area of treated cells was reduced to approximately half the size of the control cells after 20 days of treatment. the decrease in cell size might mainly be due to the reduction in cytoplasmic volume, the loss of cell turgor being a consequence of osmotic outflow of intracellular water (rhodes and samaras 1994, mahajan and tuteja 2005). the decrease in cell cross-section area was observed from the beginning of the experiment and progressed gradually with stress agent concentration. during the entire experiment, meristem cells in roots of plants cultured on media with sorbitol and 100 and 200 mm of nacl were mitotically active. this implies that the onion meristem cells that were reduced in size still undergo mitosis and produce new cells. previously reported reductions in meristem size under salt and osmotic stress (hanif and davies 1998, zadeh 2007, ji et al. 2014), which suggested as being due to a decrease in mitotic activity, might be also partially due to smaller size of newly produced meristem cells. microscopic observations also reveal that in salt-treated onion root tip cells (200 and 300 mm of nacl) the nucleus was shifted toward the cell periphery. during salt stress, excessive ions accumulate in the vacuole to avoid ion toxicity but also to increase cellular osmolarity to counter osmotic stress (garbarino and dupon 1988). mimura et al. (2003) reported that in bruguiera sexangula protoplasts subjected to nacl an increase in vacuolar volume was observed. although vacuole volume was not measured here, this process might explain the observed polarization of meristem cell nuclei after nacl treatment. there are very few reports covering interphasic vn changes in plant cells under salt and osmotic stress. the effects of salt on vn were reported for wheat (yumurtaci et al. 2009) using sensitive and tolerant genotypes. the results showed a reduction in vn in roots of salt-sensitive wheat cultivars exposed to 300−500 mm of nacl for 24 h. on the other hand, there are also studies reporting an increase in the size of nuclei under salt stress. interestingly, such observations were made for the halophyte atriplex prostrata (katembe et al. 1998) and for a salt-tolerant cultivar of wheat (yumurtaci et al. 2009). the present study showed that in a. cepa, both sorbitol and nacl cause a decrease in the vn of root tip cells. comparison between the effects of iso-osmotic solutions of nacl and sorbitol showed that the decrease was more severe after salt treatment. such results suggest that osmotic stress resulting from sorbitol and nacl treatment influences the vn of root meristem cells; however, the excessive accumulation of ions in the root tips of plants cultured on media with 200 and 300 mm nacl increases the adverse effects. the observed decrease in the vn under nacl-induced stress might be from nuclear deformation and subsequent nuclear degradation as a consequence of specific na+ and cl– ion toxicity, which are responsible for alterations in dna and fragmentation of chromatin (katsuhara and kawasaki 1996, yazdani and mahdieh 2012). the observed decrease in vn under osmotic stress is probably due to changes in hydration of the nucleus (mitchell and van der ploeg 1982, dogan et al. 2012). sorbitol does not produce any ion toxicity; however, it has a dose-dependent ability to lower the water potential of an external solution, causing hypertonic stress, which could explain the observed decrease in both cell size and nuclear volume. the mi in untreated (control) root tips of a. cepa collected from roots of small bulbs (3−4 g) placed in glass bottles filled with water ranged from 6% (nefic et al. 2013) to 15.4% (mesi and kopliku 2013), while in similar experiments with large bulbs (20−60 g) the mi in root tips was close to 60% (sehgal et al. 2006, kumari et al. 2009). in experiments where roots were produced from small onion bulbs (1.5−2 cm in diameter) placed in boxes with sand moistened with water, the reported mi was approximately 60% (ateeq et al. 2002, pandey et al. 2014); however, in roots produced from small bulbs grown in hoagland’s solution, the mi was 12−21% (glińska et al. 2007, zou et al. tab. 4. cytogenetic abnormalities in allium cepa root meristem cells exposed to sorbitol and nacl in vitro. means in column followed by the same letter are not significantly different (p≤0.05, student−newman−keuls test). lg – laggard chromosomes, br/fr – bridges and fragments, stc – sticky chromosomes, nb – nuclear budding, nf – nuclear fragmentation, mn – micronuclei. treatment (mm) e xp os ur e (d ay s) chromosome aberrations (%) nuclear aberrations (%) total aberrant cells (%±se) lg br/ fr stc nb nf mn control 10 0.0 0.0 0.0 0.0 0.0 0.0 0.0±0.0 e 20 0.0 0.0 0.1 0.0 0.0 0.0 0.1±0.0 e so rb ito l 100 10 0.8 0.0 0.8 8.7 0.2 0.3 10.7±3.0 ed 20 1.2 0.7 4.1 1.8 5.3 0.2 13.3±4.4 cde 200 10 0.3 0.0 4.5 5.7 2.0 1.3 13.8±4.5 cde 20 2.1 2.2 1.7 4.5 5.5 1.8 17.7±4.8 bcd 360 10 1.6 0.0 2.1 3.6 3.0 0.1 10.4±1.8 ed 20 1.8 1.3 0.0 8.1 6.8 0.6 18.6±6.7 bcd n ac l 100 10 8.1 5.1 3.3 1.6 0.5 0.4 19.1±4.6 bcd 20 6.9 10.1 12.7 0.0 1.5 1.1 32.3±6.0 ab 200 10 12.1 4.2 2.8 1.4 3.4 2.4 26.2±2.8 bc 20 9.5 5.0 4.3 5.5 13.3 8.9 46.5±8.1 a 300 10 4.4 1.1 2.6 0.6 4.0 2.8 15.5±0.5 bcde 20 0.0 0.0 0.0 0.1 0.2 0.1 0.4±0.0 e onion root meristem under osmotic and salt stress acta bot. croat. 76 (2), 2017 151 2012). in experiments where root tips were collected from onion seeds germinated in dishes with moistened paper, the mi was 17−25% (fernandes et al. 2007, leme et al. 2008). in the present study, mitotic divisions were scored in roots obtained from seeds germinating and growing in sterile culture media. mitosis in the roots of control plants was normal and undisturbed and nearly 30% of observed cells undergo mitosis, which ranks among the higher limits reported for a. cepa mi index values, very probably a consequence of used plant material (seeds) and applied growth conditions (tissue cultures). applied stress affected mitotic activity in onion roots. the total absence of mitotic activity was observed after a 20-day exposure to the highest concentration (300 mm) of nacl, while approximately 16% of the meristematic cells in roots growing on media with the highest concentration (360 mm) of sorbitol still underwent divisions during this period. such results point to differences in the mitodepressive effect of ionic and non-ionic stress. during root growth, the number of dividing cells in meristematic tissue is related to the duration of mitosis in the cell cycle. the number of cells in a division phase is proportional to the time spent by that phase in relation to the total length of mitosis (gonzález-bernáldez et al. 1968). in roots treated with sorbitol, the proportion of certain mitotic phases was similar to that of the controls, while nacl treatment significantly altered the phase ratio. nacl treatment decreased the number of cells in the metaphase and increased the number of anaphases and telophases, while in the controls almost equal proportions of metaphase and ana/telophase cells were observed. the accumulation of anaphase and telophase, which is evident from the low (0.4) m/a + t ratio, may be a consequence of the weakened chromosome separation, probably by stickiness of chromosomes (tajbakhsh et al. 2006). in addition, after stress treatment the cells entered mitosis, but they were arrested in prophase, and the cell cycle often did not progress further, as was observed in control cells. these results suggest an increase in duration of the cell division cycle under applied stress conditions and a stronger mitodepressive effect of salt stress in comparison with sorbitol-mediated osmotic stress. in the roots of salt-tolerant genotypes, no significant changes in mi between treated and control samples were observed (radić et al. 2005). thus, the level of mitotic activity reduction in root meristem cells under stress might be a specific marker for discrimination between salt-sensitive and salt-tolerant genotypes. detailed cytological analysis of sorbitoland nacl-treated onion root tip cells allowed for characterization of numerous abnormalities in interphase and during cell division. the frequency of aberrant cells differed with the stress agent and its concentration. in salt-treated roots, the highest percent of aberrations (approximately 46%) was observed in roots of plants grown on medium treated with 200 mm of nacl. in those cells, considerable frequencies of chromosomal aberrations, nuclear fragmentation and micronucleated cells were observed. in root meristems of plants exposed to iso-osmotic solutions of sorbitol, the percent of abnormalities was lower and accounted mainly for nuclear aberrations. observed chromosome fragmentation and chromosome breaks indicate clastogenic action, while chromosome stickiness may be a result of inter-chromosomal linkages coupled with excessive formation of nucleoproteins (leme and marin-morales 2009). lagging chromosomes resulted most likely from the failure in organization of spindle apparatus (tabur and demir 2009, utani et al. 2010). the formation of micronuclei, observed here as prevalent in nacl-treated cells, may be a consequence of chromosome fragments or lagging chromosomes failing to incorporate into the daughter nuclei (yi and meng 2003). in conclusion, the effects of different osmotica on plant tissues depend on the physical and chemical nature of stress-inducing agents. sorbitol is an osmoticum that does not induce ion toxicity, although influences the water balance within the cell. results obtained in this study suggest that onion root meristem cell size is mostly affected by changes in extracellular osmolarity, while nuclear size is affected both by ionic and osmotic components of applied stresses. both stress agents caused a decrease in mitotic activity and increased the number of mitotic abnormalities in root tip meristems of onion, albeit with various doseand exposure-dependent frequencies, which points to differences in their cytotoxicity. comparison of effects of iso-osmotic nacl (200 mm) and sorbitol (360 mm) concentrations showed that the ionic rather than the osmotic component of salt stress had more detrimental effects on onion root meristems. acknowledgement this research was financed by polish ministry of science and higher 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90–97. zörb, c., mühling, k. h., kutschera, u., geilfus, c. m., 2015: salinity stiffens the epidermal cell walls of salt-stressed maize leaves: is the epidermis growth-restricting? plos one 10(3), e0118406. zou, j., yue j., jiang w., liu d., 2012: effects of cadmium stress on root tip cells and some physiological indexes in allium cepa var. agrogarum. acta biologica cracoviensia series botanica 54, 129–141. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 253 acta bot. croat. 74 (2), 253–264, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0027 first report of navicula jakovljevicii hustedt (bacillariophyta) from hungary: distribution, comparative morphology and a related species viktória b-béres1*, istván bácsi2, enikő t-krasznai1, zsuzsanna kókai1, krisztina buczkó3 1 environmental protection and nature conservation authority, trans-tisza region, debrecen, hatvan u. 16. h-4025 hungary 2 university of debrecen, department of hydrobiology, debrecen, egyetem tér 1. h-4032 hungary 3 hungarian natural history museum, budapest, könyves kálmán krt. 40. h-1476 hungary abstract – in hungary navicula jakovljevicii was fi rstly recorded in biofi lm of elodea nuttallii in 2005 in an oxbow of the catchment area of the river danube. subsequently, in 2006, n. jakovljevicii was also found in the same oxbow on reed stems as well. in the following years it appeared in another oxbow, suggesting an expanding distribution in the tributaries of the danube in hungary. the hungarian population can be characterised as having mixed morphological features in comparison with other known n. jakovljevicii populations of europe. when the morphological study was expanded, a similar, but 'giant form' was detect ed in fossil material. we found similarities and a possible connection between n. jakovljevicii and navicula lucida, a diatom taxon described from a neogene deposit in the carpathian basin. despite the morphological similarities in the shape, apices, striae pattern and raphe structure of these two species, there are signifi cant differences in valve dimensions: the valves of n. lucida are larger and more heavily silicifi ed than n. jakovljevicii. keywords: biofi lm, danube, elodea nuttalli, navicula jakovljevicii, navicula lucida, oxbow, pantocsek collection introduction navicula jakovljevicii hustedt (1945: 931) was described from the balkan peninsula in 1945 by friedrich hustedt (hustedt 1945). for a long time it seemed that the distribution of this diatom species was restricted to south europe (plenković-moraj 1995). the taxo* corresponding author, e-mail: beres.viktoria@gmail.com b-béres v., bácsi i., t-krasznai e., kókai z., buczkó k. 254 acta bot. croat. 74 (2), 2015 nomical position of the species is uncertain. according to the earlier published morphological details (reichardt 1992, lange-bertalot 2001, levkov et al. 2005) neither the raphe structure (raphe fi ssures weakly lateral, slightly curved) nor the striae pattern (the striae consist of poroids that are elongated externally but alveolate internally) are the same as the members of navicula s. str. furthermore a hole can be detected on the valve apex, which is an unusual feature. lange-bertalot (2001) indicated in the monograph of diatoms of europe, that some fossil species from the upper tertiary period bear the same structures as navicula jakovljevicii. he mentioned köpecz (căpeni, romania today) material, and published two pictures (plate 62 fi gs 1–2) as navicula (?nov.) spec. from this locality. the legends of these fi gures are the following: köpecz, southern carpatians. the structural pattern conforms to the recent navicula jakovljevicii, and appears like a 'giant form' of this species (langebertalot 2001 p. 360). the hungarian natural history museum holds the józsef pantocsek diatom collection, where – among many others – diatom samples from the carpathians are wel l represented, including samples form köpecz (pantocsek 1892, 1905, buczkó 2012). an obvious endeavour was to compare the recent navicula jakovljevicii with other ancient navicula taxa from the köpecz material and try to fi nd this 'giant form'. due to the hydrological impacts of the bős/gabčikovo dam, a low water fl ow condition was formed both in the main river channel of the danube and in the smaller arms (othelova and valachovic 2002, ráth et al. 2003, hajósy 2012). based on these facts, a macro and microfl ora monitoring project (including diatom monitoring also) was conducted between 1994 and 2009 on the river danube, on its river branches and on the oxbows. the aim of that study was to follow up the effects of the bős/gabčikovo dam on the macroand microfl ora assemblages of the so-called szigetköz region (hajósy 2012). it was observed, that the changing water discharge and fl ow conditions allowed appearances and distribution of adventive and invasive macrophytes, as elodea canadensis michaux (1803: 20) and elodea nuttallii (planchon 1848: 86) h. st john (1920: 29) (ráth et al. 2003, othelova and valachovic 2003, király et al. 2007, kočić et al. 2014). although macrophytes could serve as ‘spreading transport substrate’ for invasive species, diatoms among them (horváth and lamberti 1997), the fi rst hungarian observation of a characteristic diatom taxa during the counting procedure in 2005 was surprising. this diatom taxa was identifi ed as navicula jakovljevicii. the main aims of the present study were (i) to report for the fi rst time observation of navicula jakovljevicii in hungary; (ii) to compare the morphological features of the hungarian n. jakovljevicii population with other known populations of the species; (iii) to report the observation of fossil analogues of navicula jakovljevicii in miocene material and (iv) to give a possible explanation of the recent observation and spread of n. jakovljevicii in hungary. material and methods sampling localities szigetköz is a major island of the danube, harbouring a complex system of habitats. it is situated in northwest hungary, where the river enters the carpathian basin (fig. 1). here, the fast fl owing river slows down, deposits the gravel carried from the alps, divides into many smaller arms turning into a braided channel. diatom samples were collected between distribution and comparative morphology of navicula jakovljevicii acta bot. croat. 74 (2), 2015 255 1994 and 2009 on the river danube and in its catchment area twice a year (fig. 1). samples were collected from different macrophytes of sixteen sampling locations (fig. 1). during the sixteen years, more than 1300 samples were collected and analysed. nearly 300,000 valves were identifi ed from the samples. sample preparation diatom samples were prepared using standard digestion procedures (battarbee 1986). aliquot-evaporated suspensions were embedded in zrax. at least 400 valves were counted from each sample using a light microscope (lm) (leica dm lb2 with 100 hcx plan apo objective). light microscopic pictures were taken by fujifi lm digital camera finepix s2 pro and later by vsi–3.om(h) digital camera. for scanning electron microscopic (sem) analyses, cleaned samples were air–dried on an aluminium stub. coating with gold– palladium was accomplished using a xc7620 mini sputter coater for 120 s at 16 ma. a hitachi s–2600n sem operated at 20 kv and 5–8 mm distance was used. morphological terminology follows barber and haworth (1981) and round et al. (1990). valve measurements were made from digital images using the camera software. the following materials were examined and presented in this paper: raw materials and permanent slides of the hungarian natural history museum bp 2007/72; bp 2007/73; pantocsek diatom collection köpecz material (new permanent slides and stubes for sem were made from dried diatom sample no bp-88, and bibarczfalva no bp-22). results in hungary navicula jakovljevicii was fi rst recorded in autumn of 2005 in an oxbow at ásványráró (fig. 1). the substrate of the biofi lm was the waterweed elodea nuttallii. elodea nuttallii was present also in the following three years in this oxbow en masse. in 2006 n. jakovljevicii was also found on reed stems in the same oxbow, as well as on the elodea fig. 1. the sample locations of hydrobiological monitoring and the occurrences of navicula jakovljevicii (arrows) in the szigetköz section of the river danube. ásványráró (eov x: 535520 eov y: 279130; and eov x: 535570 eov y: 278990); cikolasziget (eov x: 525150 eov y: 290300). b-béres v., bácsi i., t-krasznai e., kókai z., buczkó k. 256 acta bot. croat. 74 (2), 2015 species. the diatom appeared in another oxbow in szigetköz on e. nuttallii in 2008. although the diatom species had moved upwards in the catchment area of danube, its relative abundance was low in every sample, never reaching 3%. altogether n. jakovljevicii was found only at three sampling sites from the sixteen sites, it was recorded continuously from 2005 until the end of the monitoring in 2009. the recent distribution of the species is shown on on-line supplement fig. 1. a detailed morphological description about this characteristic species for comparison with other known populations (hustedt 1945, reichardt 1992, levkov et al. 2005) is given below. morphological observations of navicula jakovljevicii light microscopy valves are lanceolate to elliptic-lanceolate with obtusely to broadly rounded ends, 32– 53 μm long, 7–8.7 μm broad (pl. 1, figs. 1–7). raphe fi ssures are weakly lateral. axial area is narrow, linear, central area is small, weakly asymmetrically rounded. striae are moderately lateral, parallel to weakly convergent at the ends; there are 14–17 striae in 10 μm (pl. 1, figs. 1–7). scanning electron microscopy raphe fi ssures are distinctly lateral, narrower to the secondary side of the valve, slightly expanded at the proximal part. the proximal raphe endings externally are forked (pl. 2, figs. 1–4; pl. 3, figs. 1, 3), a silicate tongue divides the raphe terminal. lineolae are narrow, elongated, 29–32 in 10 μm. the alveoli are partly occluded on the valve interior (pl. 2, fig. 5). the distal raphe ends straight (pl. 2, figs. 1–2) or bent to the secondary side (pl. 2, fig. 4) and has a well close to the valve ends (pl. 2, figs. 1–4; pl. 3, fig. 4). internally, a hole can be detected on the valve apex (pl. 2, fig. 5; pl. 3, fig. 2). internally, the distal raphe ends are lateral, close to each other (pl. 3, fig. 5). the mantle is narrow (pl. 3, fig. 6). pl. 1. figs.1–7: navicula jakovljevicii light microscopy (lm) micrographs of type population in the ásványráró section of the danube, collected from elodea nuttallii (sample bp 2007/73). scale bar represents 10 μm. distribution and comparative morphology of navicula jakovljevicii acta bot. croat. 74 (2), 2015 257 a related navicula species from tertiary diatom deposits (navicula lucida pantocsek 1892: tab. 18, fi g. 264) based on the indication of lange-bertalot (2001) the köpecz and bibarczfalva materials were reinvestigated and some valves of a characteristic, long diatom were found in the samples that are reminiscent of the published pictures in the monograph of diatoms of europe (lange-bertalot 2001 plate 62 fi gs 1, 2). we identifi ed this diatom species as navicula lucida pantocsek (1892, 1905: 73; plate 4, fi g.1). this species was very rare, altogether twelve valves having been documented. the latin diagnosis (pantocsek 1905) refers to only one valve that is 192 μm long and 19 μm wide. the striae number is 15–16 in 10 μm; they are slightly radial in the middle but almost through parallel along the valves. it was found in the tertiary, in the miocene, in fresh water, at localities close to köpecz and bodos by pantocsek. morphological observations of navicula lucida light microscopy valves are lanceolate, gradually tapering to the apex. the ends of these valves are obtusely rounded. the lengths are 110–200 μm (n = 10), the breadths are 15–22 μm. raphe is pl. 2. figs. 1–5: navicula jakovljevicii scanning electron micrographs (sem) of the population from elodea nuttallii (sample 2007/73); figs. 1–2: external view of entire valves; fig. 3: oblique position showing the girdle bands; fig. 4: broken valve; fig. 5: internal detail of a broken valve. scale bars represent 10 μm. b-béres v., bácsi i., t-krasznai e., kókai z., buczkó k. 258 acta bot. croat. 74 (2), 2015 distinctly lateral, outer fi ssure curved distinctly towards the central pores. axial area is moderately narrow and linear, central area is slightly elliptic. striae are almost parallel, 14–16 in 10 μm; lineolae hardly distinct in lm (pl. 4, fig. 2). scanning electron microscopy raphe is lateral; externally the central raphe endings are distinctly forked (pl. 4, fig. 2; pl. 5, fig. 1) just as in n. jakovljevicii (pl. 2, figs. 1–4; pl. 3, figs. 1, 3). lineolae are narrow, elongated, 34–38 in 10 μm. the alveoli are partly occluded on the valve interior (pl. 4, fig. 4). externally, the distal raphe ends turn to the girdle (pl. 4, fig. 3; pl. 5, fig. 3). internally, the proximal raphe endings are close to each other (pl. 5, fig. 2). internally, there is an expansion on the proximal part of the raphe fi ssure (pl. 5, fig. 2). there is a hole on the apex close to the valve end (pl. 5, fig. 4). discussion taxonomical remarks on navicula jakovljevicii and its distribution in the original description by hustedt (1945), n. jakovljevicii was characterized by 32–60 μm in length, 8–11 μm in width and 16–18 striae in 10 μm. reichardt (1992) found pl. 3. figs: 1–6: navicula jakovljevicii scanning electron micrographs (sem) of the population from elodea nuttallii (sample 2007/73); figs. 1, 3: external detail of the central area with the proximal raphe endings and the typical ornamentation, note the forked central raphe ends; fig. 2: internal detail of a valve apex showing a hole; fig. 4: external detail of a valve apex showing the turning raphe end; fig. 5: internal detail of the central area; fig. 6: external detail of a frustulum apex in oblique view showing the mantle elements. scale bars represent 2 μm. distribution and comparative morphology of navicula jakovljevicii acta bot. croat. 74 (2), 2015 259 pl. 4. figs. 1–4: navicula lucida; fig. 1: iconotype, reprint from pantocsek 1892; fig. 2: light microscopy (lm) micrograph from köpecz (romania), miocene; figs. 3–4: scanning electron micrographs (sem), fig. 3: external but oblique view of a whole frustule, fig. 4: internal view. scale bar for lm is 10 μm; on sem bars are 50 μm. pl. 5. figs. 1–4: details of navicula lucida valves. fig. 1: external view of central area with the characteristic forked central raphe endings; fig. 2: internal details of central area. fig. 3: external view of a valve apex. fig. 4: internal view of a valve apex with hole. scale bars represent 5 μm. b-béres v., bácsi i., t-krasznai e., kókai z., buczkó k. 260 acta bot. croat. 74 (2), 2015 three, slightly different, populations in lake zug, in the river krka and in the plitvice lakes. the population found in lake zug was slightly thinner (8.4–9.7 μm in width) and smaller (32.9–53.0 μm in length), moreover there were fewer striae (14–15 in 10 μm) than in the form described by hustedt. in contrast, valves from the river krka were characterized by being 39–85 μm in length and 8.8–11.7 μm in width. the characteristics of the valves from populations originating in the the plitvice lakes were completely in the middle range. subsequently, levkov et al. (2005) published a detailed morphometric analysis of n. jakovljevicii populations from nidze mountain and lake ohrid. they found that at least two different populations could be distinguished: the fi rst, n. jakovljevicii morphotype 1, is similar to hustedt’s (1945) description as regards the number of striae in 10 μm (16–18) and the presence of a longitudinal band, but has a greater length (50–80 μm). the second, n. jakovljevicii morphotype 2, has smaller valves (30–50 μm), an indistinct longitudinal band and considerably fewer striae in 10 μm (12–14). the lengths of the valves of the hungarian populations were partly similar to the transitional forms described by hustedt (1945), and partly to the populations from lake zug (reichardt 1992). the numbers of striae of individuals from hungary were the closest to the original description (hustedt 1945). navicula jakovljevicii was primarily known from southern, south-east, and central europe (lake ohrid – jurilj 1954, lake zug, river krka and plitvice lakes – reichardt 1992, france – coste and ector 2000, transylvania in fossil deposit – lange-bertalot 2001, nidze mountain and lake ohrid – levkov et al. 2003, macedonia – smith and smith 2003, river trnovcica – levkov et al. 2005, apennine mountains – torrisi and dell’uomo 2008, river raška – vidakovic et al. 2014; on-line supplement fig. 1). recently n. jakovljevicii was reported from the northern region of europe (the netherlands, 2010, on-line supplement fig. 1). only few data are available regarding to the substrate of this diatom taxon. samples containing n. jakovljevicii were collected from limestone (lake zug – reichardt 1992, apennine mountains – torrisi and dell’uomo 2008), branches (river krka – reichardt 1992) and encrusted moss (plitvice lakes – reichardt 1992). a related species: navicula lucida we proved the presence of the 'giant form' of navicula jakovljevicii in tertiary deposits that was mentioned by lange-bertalot (2001). this large diatom was described by józsef pantocsek as navicula lucida (pantocsek 1982). all of the main valve features (shape, apices, striae pattern, raphe) of these two species are the same, but there are signifi cant differences in valve dimensions. in our observation the valves belonging to n. lucida are large and heavily silicifi ed. this is not surprising, since most diatomite in central europe is connected to volcanisms (e.g. hajós 1986). high silicate concentration in the lake water, related to volcanic activity, probably facilitated the development of strong diatom cell walls during the miocene (witkowski et al. 2011). all characteristic features are in agreement with n. jakovljevicii and n. lucida, but probably the forked raphe terminals are the most remarkable. we can only speculate about the connection between the extinct and the extant navicula species. it might be possible that these two species are close relatives. n. lucida could be the ancestor of n. jakovljevicii, the large form gradually diminishing in parallel with the restricted silica supply because of the reduced volcanic activity. but maybe they developed independently from each other. due to the sporadic occurrences of diatom dedistribution and comparative morphology of navicula jakovljevicii acta bot. croat. 74 (2), 2015 261 posits, the lack of a continuous profi le makes the answer to this question diffi cult. navicula lucida was found in neogene fossil deposits – köpecz (căpeni), bodos (bodoş) and bibarczfalva (baraolt) – in romania. furthermore, the presence of this taxon was detected in the kichevo basin macedonia (ognjanova-rumenova and dumurdzhanov 2008 as brachysira sp. on plate 2 fi g. 6). perspectives notwithstanding the intensive studies on the biogeography of diatoms, our knowledge about their distribution pattern is very limited. detailed taxonomical studies have often revealed several new taxa (potapova and hamilton 2007, novais et al. 2009), but changes in species’ distribution are poorly known. recently, attention has been paid to invasive and expansive diatom taxa (coste and ector 2000, blanco and ector 2009, kaštovský et al. 2010, jellyman et al. 2011, beltrami et al. 2012, t-krasznai et al. 2014), but there are only a few taxa with suffi cient available data. the species mostly in the focus of attention are those that have caused drastic ecological and/or economic events (blanco and ector 2009). the incorrect identifi cation of certain taxa (especially closely related-taxa, or centric diatoms) could create diffi culties in the interpretation of the appropriate biogeographical data in some cases (kaštovský et al. 2010). navicula jakovljevicii has even been regarded as a rare species (coste and ector 2000, vidakovic et al. 2014). its occurrence in the hungarian part of the danube catchment area might be related to the invasive elodea nuttallii, which may refer to the changes of the habitat. affected by the bős/gabčikovo dam, water fl ow conditions have changed and water discharge has decreased in the danube and its catchment area (ráth et al. 2003). these changes in hydrological circumstances primarily led to the spread of adventive and/or invasive species, like the waterweeds elodea canadensis and e. nuttallii (ráth et al. 2003). a lot of abiotic and biotic factors control the appearance of non-native species in new habitats (b-béres et al. 2012, porter et al. 2013, t-krasznai et al. 2014). we speculated that the new observations of n. jakovljevicii in the hungarian fl ora were primarily due to the spread of e. nuttallii. these macrophytes could be regarded as the transport substrate of n. jakovljevicii. but it is pertinent to emphasize that this ‘transport substrate assumption’ requires a lot of further studies. the improving taxonomical skills of diatomologists and the available fl ora books (e.g. diatoms of europe) could also contribute new data of the presence and ecological demands of n. jakovljevicii. conclusion appearance of new taxa in diatom-fl ora, especially a rare species with little information about its ecological relevancy, requires a lot of care. even if the abundance of the given taxa is low, its effects on the local microfl ora are unknown and unpredictable. our results were the fi rst report about the distribution of navicula jakovljevicii in hungary. there are only a few data about its distribution in europe. in order to predict the effects and the directions of the current expansion of the taxon, every single item of data has a major importance. our result suggests that n. jakovljevicii might appear in the further danubian catchment area, not only in hungary but in adjacent countries as well. b-béres v., bácsi i., t-krasznai e., kókai z., buczkó k. 262 acta bot. croat. 74 (2), 2015 acknowledgements we thank the hungarian scientifi c fund for the fi nancial support (otka 83999). furthermore, the authors are thankful for the support of támop 4.2.4. a/2-11-1-2012-0001 »national excellence program – elaborating and operating an inland student and researcher personal support system«, projects (t-krasznai e., b-béres v.). the támop projects are implemented through the new hungary development plan, co-fi nanced by the european social fund and the european regional development fund. the authors are thankful for the support of the bolyai jános research scholarship of the hungarian academy of sciences (bácsi i.) and the internal research project of the university of debrecen (bácsi i.) during manuscript preparation. references barber, h. g., haworth, e. y., 1981: a guide to the morphology of the diatom frustule with a key to the british freshwater genera. freshwater biological association scientific publication 44, 112. battarbee, r. w., 1986: diatom analysis. in: berglund, b. e. 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nagy-tapolcsány, buchdrucherei von julius platzko. pantocsek, j., 1905: beiträge zur kenntniss der fossilen bacillarien ungarns. iii. beschreibung der auf tafel 1–4 abgebilden arten, pozsony. 1–118, pls. 1–42. retrieved from http://dx.doi.org/10.5962/bhl.title.14916. b-béres v., bácsi i., t-krasznai e., kókai z., buczkó k. 264 acta bot. croat. 74 (2), 2015 planchon, 1848: the annals and magazine of natural history. plenković-moraj, a., 1995: diatoms (bacillariophyceae) of the croatian freshwater. acta botanica croatica 54, 22–33. porter, e. m., bowman, w. d., clark, c. m., compton, j. e., pardo, l. h., soong, j. l., 2013: interactive effects of anthropogenic nitrogen enrichment and climate change on terrestrial and aquatic biodiversity. biogeochemistry 114, 93–120. potapova, m., hamilton, p. b., 2007: morphological and ecological variation within the achnanthidium minutissimum (bacillariophyceae) species complex. journal of phycology 43, 561–575. ráth, b., janauer, g. a., pall, k., 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(eds.), 2003: country study for biodiversity of the republic of macedonia (fi rst national report). ministry of environment and physical planning, skopje. st john, h., 1920: flora of north america. rhodora. journal of the new england botanical club. cambridge, ma. t-krasznai, e., b-béres, v., kókai, zs., buczkó, k., balogh, cs., török, p., 2014: adatok kilenc adventív vagy invazív alga hazai előfordulásához. kitaibelia – journal of pannonian botany 19, 11–21. torrisi, m., dell’uomo, a., 2008: benthic diatoms of springs and streams in the central apennines (italy). abstract book of central european diatom meeting, trento, italy. vidakovic, d., krizmanic, j., sovran, s., 2014: new taxa of the genus navicula (bacillariophyceae) in the diatom fl ora of serbia. oceanological and hydrobiological studies 43, 185–190. witkowski, j., harwood, d. m. chin, k., 2011: taxonomic composition, paleoecology and biostratigraphy of late cretaceous diatoms from devon island, nunavut, canadian high arctic. cretaceous research 32, 277–300. opce-str.vp acta bot. croat. 68 (2), 381–399, 2009 coden: abcra 25 issn 0365–0588 diatom preservation: differential preservation of sedimentary diatoms in two saline lakes roger j. flower1*, david b. ryves2 1 environmental change research centre, department of geography, university college london, gower street, london wc1e 6bt, uk 2 department of geography, loughborough university, loughborough le11 3tu, uk the integrity of all sedimentary diatom assemblages is influenced to some degree by taphonomic processes. recognising these processes with regard to preservation pathways for diatom assemblages and for individual species can be instructive for interpreting sediment core diatom records. diatoms deposited in saline lakes are usually particularly exposed to both chemical and physical processes that promote poor preservation. aspects of diatom preservation are explored in two markedly different saline lakes (one in north america and one in egypt) and observations are used to make some initial inferences about diatom preservation. by applying dissolution indices to evaluate differences in valve preservation states between assemblages and between species in an objective manner, sedimentation processes and valve characteristics are indicated important implications for interpreting sedimentary diatom records. it is further argued that, by taking account of diatom dissolution states, both qualitative and quantitative inferences about past environments can be extended. keywords: diatom, dissolution, preservation, biogenic silica, ultrastructure, saline lake, sedimentation, taphonomy, climate change abbreviations: ddi – diatom dissolution index, introduction assuring the integrity of sedimentary diatom records in aquatic sediments, either freshwater or saline/marine, is a prime concern if qualitative or quantitative information contained in the fossil assemblages is to be used for palaeo-environmental reconstructions. yet in aquatic environments, diatom communities are subject to a variety of environmental processes before, during and after incorporation into sediments that can highly modify resultant sedimentary assemblages. such modifications can influence the effectiveness of palaeo-environmental reconstruction methods. the value of recognising the influences of processes involved in the transformation of organismal remains into fossils was recognised by the palaeontologist i. a. efremov over 60 years ago. he used the term 'taphonomy' to acta bot. croat. 68 (2), 2009 381 * corresponding author: r.flower@geog.ucl.ac.uk u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:18 color profile: disabled composite 150 lpi at 45 degrees describe the transformation processes (efremov 1940). some specific taphonomic criteria that are particularly relevant to diatoms were discussed by cameron (1995) where the issue of representativity of fossil diatom assemblages is dealt with in some detail. the 'faithfulness' of sedimentary records can be assessed in a number of ways that include comparisons with archived material and with source communities as well as by using diatom preservation experiments and indices (barker 1992, ryves et al. 2001). taphonomy is an issue for diatom valves in both modern and sedimentary environments as well as for archival and other diatom procedures. in the environment, prior to and during the accumulation of diatom valves in sediments, breakage and dissolution can cause differential preservation. indeed, these processes are not mutually exclusive but are often closely correlated (e.g. straub 1993, ryves et al. 2006). excessive valve breakage is typical of high energy environments (beyens and denys 1982) but also results from grazing effects (turner 1991) and, after deposition, by compaction and drying of sediments (flower 1993). diatom dissolution will always occur when diatom remains are exposed to water with low (below saturation) concentration of dissolved silica. on a longer time scale, diatoms incorporated into sediment can undergo diagenesis involving the crystallization or mineral replacement of the diatom silica (williams and crerar 1985, barker et al. 1994, leng and barker 2006). the literature describes a variety of specific processes and conditions that are known to affect diatom dissolution. in many cases, it is the ph, salt concentration and temperature of surrounding media (lewin 1961, lawson et al. 1978, hurd and birdwhistell 1983, barker et al. 1994, bidle et al. 2002, ryves et al. 2006) that are particularly important in influencing valve dissolution. water permanence, depth and meromixis (reed 1998, ryves et al. 2006) can affect preservation, as can diatom sedimentation or sinking rates in the water column (conley and schelske 1989, shimada et al. 2003, battarbee et al. 2005). sediment texture, grazing and (bio) turbation (hecky and kilham 1973, turner 1991, flower 1993) are all influential external factors. diatom structure and surface area (lewin 1961, flower 1993, ryves et al. 2001) and diatom concentration (bradshaw et al. 2005, swann and mackay 2006) together with bacterial action (patrick and holding 1985, bidle and azam 1999) all have further implications for diatom preservation. this paper examines some common features of diatom dissolution and evaluates sedimentary diatom sequences from two different lakes where current salinity is relatively low (spiritwood lake, usa) and relatively high (lake qarun, egypt). by taking a direct approach to investigating preservation in situ we hope to refine interpretation techniques for the occurrence and abundance of poorly preserved sedimentary diatom sequences. rather than taking an experimental approach (e.g. lewin 1961, barker et al. 1994, ryves et al. 2001), characteristics of particular diatom facies are investigated by using objective methods of dissolution assessment. assessing diatom dissolution there are two principal ways of tackling preservation problems in sediment cores. firstly, the various diatom dissolution studies described above can be consulted and used to infer possible causes of poor preservation. to aid this approach the results of many studies addressing diatom preservation are summarized in table 1 so that principal processes or conditions that are known to favour good or poor diatom valve preservation (either in the 382 acta bot. croat. 68 (2), 2009 flower r. j., ryves d. b. u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:18 color profile: disabled composite 150 lpi at 45 degrees water column or in surficial sediments) are identified. this simple checklist of preservation characteristics and conditions is derived mainly from experimental studies, observations on sinking material and from sediment profiles. so, although table 1 indicates some of the main processes and conditions that affect diatom preservation, it does not take into account the interactions between dissolution processes. hence, its value for interpreting lake sediment records from any specific site is limited. any particular lake will display strongly differing patterns of preservation through time where past process interactions and phasing are unknown. a second approach that is more objective is based on measuring the quality of specific assemblages by applying a quantitative method of assessment. dissolution indices (e.g. ryves et al. 2001, 2006) can be used to compare proportions of part dissolved and undissolved (pristine) valves and there are essentially two simple ways to do this. one, the f index (flower and likhoshway 1993, ryves et al. 2001), is based on the ratio of (partially) dissolved to pristine valves, where fi = f index of sample i, n = pristine valves of species j in sample i, n = sum of pristine + dissolved valves (girdle views are excluded): f n n i ij j m ij j m = ∑ ∑ (1) values range from 0 (all valves visibly dissolved under lm) to 1 (no valves show signs of dissolution). the other index is the diatom dissolution index (ddi; ryves et al. 2006) which compares diatoms in an assemblage across all dissolution states (categorised into 2 to 4 dissolution »stages«, depending on species, and denoted s). dissolution patterns (stages) are illustrated for a number of major diatom morphologies in ryves et al. (2009). for an assemblage i, ns = sum of valves of all taxa in each stage s, n = total number of valves classified acta bot. croat. 68 (2), 2009 383 diatom preservation in saline lake sediments tab. 1. a simple check-list of some factors known to influence diatom preservation in modern aquatic environments and in sediments (see text for relevant references). process/environmental characteristic preservation poor good ph high low conductivity (high base cations) high low water depth (and permanence) shallow (ephemeral) deep (permanent) sinking rate (in water) low high sediment accumulation rate low high sediment texture coarse fine grazing and bioturbation (including bacteria) high low diatom structure delicate robust diatom concentration low high temperature high low u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:18 color profile: disabled composite 150 lpi at 45 degrees (again, girdle views are excluded), and smax is the theoretical end point for dissolution in the assemblage (i.e. largest value of s recognised for any significant component of the assemblage; generally smax = 3 or 4): ddi n s n s s s s max = • − • − = = ∑ ( 1) ( 1) 1 4 (2) ddi indices range from 0 to 1, but here with 0 indicating excellent preservation (all valves in stage 1, the undissolved or »pristine« state), to 1 (very poor preservation, with valves in the most dissolved state recognised). both indices (f and ddi) can be applied to individual taxa or whole assemblages, and enable quite diverse and different assemblages to be compared. diatom preservation in sediments of two saline lakes diatom distributions and preservation states were explored in sediment core material from two lakes that have experienced fluctuating salinity across the freshwater/saline threshold. the first is spiritwood lake in north dakota, usa, (area 1.67 km2) where salinity has varied between around 1 and 4 g l–1 in the past 100 years (ryves et al. 2009). the second is from a much more complex lake, lake qarun, located in the faiyum depression of middle egypt at the edge of the western desert. this fairly large lake (around 40 x 10 km) is fed via a regulated branch of the river nile and its salinity has varied considerably, by between around 7 and 30 g l–1, during the past nearly 100 years (see flower et al. 2006). spiritwood lake is a relatively deep (zm = 16 m), dimictic meso/eutrophic lake developed in a glacial meltwater channel, largely filled with glaciofluvial sediments. diatom analysis was performed on core sw1 (82 cm long), collected close to the lake centre in august 1991 from c. 14 m depth, and dated using 210pb (appleby 2001). full details of the diatom stratigraphy and dating methods are given in ryves et al. (2009). lake qarun is now a shrunken remnant of a much larger lake (see hassan 1986) and currently has a maximum depth of around 9 m (flower et al. 2006). following initial coring in 1997 (flower et al. 2006), an 8.3 m core (qaru2) was collected from near the centre of this lake in 2004 where water depth was 8.4 m. the core spans approximately the last 2000 years (see foster et al. 2008) and the availability of only one date (from near the core base) prevented calculation of sediment accumulation rate variations. methods methods for preparing sediment samples from both lake cores were similar and involved treatment with hot, 30% hydrogen peroxide and concentrated hydrochloric acid (after battarbee et al. 2001) before washing and mounting on cover slips in naphrax diatom mountant. diatom valve concentrations in the lake qarun sediment core samples were estimated by adding microspheres (battarbee and kneen 1982). diatom inferred salinities or conductivities were calculated using separate diatom-salinity models developed from regional training sets: for spiritwood lake, the original north american great plains 384 acta bot. croat. 68 (2), 2009 flower r. j., ryves d. b. u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:18 color profile: disabled composite 150 lpi at 45 degrees (ngp) model of fritz (1990), initially unadjusted for diatom dissolution (see ryves et al. 2009) and for lake qarun, the north african training set of the european diatom database initiative (http://craticula.ncl.ac.uk/eddi/jsp/). results spiritwood lake: diatom analysis of the short core from this plankton-dominated lake showed major changes both in the proportions of dissolved values of two stephanodiscus species (s. niagarae and s. minutulus-complex) and cyclotella meneghiniana (fig. 1) and in the proportions of the species themselves (ryves et al. 2009). diatom preservation of assemblages (as f index) is weakly inversely correlated with dissolution-unadjusted inferred salinity (fig. 2a, r2 = 0.18, p = 0.05) and more strongly, positively with sediment accumulation rate in cm yr–1 (fig. 2b, r2 = 0.51, p < 0.001), with weak correlation with sediment flux rate (as g–1 dry wt cm–2; r2 = 0.24, p = 0.03, not shown). pair-wise comparisons of the three main species using ddi (fig. 3) show markedly different patterns of dissolution behaviour, with s. niagarae and c. meneghiniana following the same pattern (fig. 3a), but the dissolution behaviour of s. minutulus is only weakly correlated to that of s. niagarae acta bot. croat. 68 (2), 2009 385 diatom preservation in saline lake sediments fig. 1. sem micrographs of diatoms common in the spiritwood lake short core, across a range of preservation states (»dissolution stages«). (a)–(c): stephanodiscus niagarae (a: pristine = stage 1, b: dissolved valve, stage 2, c: dissolved valve, stage 3). (d)–(e): stephanodiscus minutulus (d: pristine, e: dissolved valve, stage 3). (f): cyclotella meneghiniana, dissolved valve: stage 2. adapted from supplementary material in ryves et al. (2009). u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:21 color profile: disabled composite 150 lpi at 45 degrees (fig. 3b), with the smaller, less robust valves of s. minutulus being better preserved on average than the larger, silica-rich s. niagarae, contrary to expectation from gross valve morphology and dissolution experiments (barker et al. 1994, ryves et al. 2001). lake qarun: in the lake sediment core qaru2, diatoms were present throughout but showed major changes in concentration, with the section between 6 and 7.5 m containing much higher diatom concentrations than above or below. diatom assemblages were dominated by aulacoseira granulata but cyclostephanos dubius and cyclotella meneghiniana were also common. thalassiosira faurii was also abundant but only within the depth interval 6–7.5 m. differential diatom preservation in the core is demonstrated in figure 4 with campylodiscus (fig. 4c) and particularly cyclostephanos (at 150 cm depth) showing sig386 acta bot. croat. 68 (2), 2009 flower r. j., ryves d. b. fig. 2. spiritwood lake short core sw1. scatter plots of (a) assemblage f index and diatom-inferred salinity (using the original ngp model of fritz, 1990, fritz et al., 1993, not adjusted for dissolution data) and (b) assemblage f index and accumulation rate (cm yr–1). a b u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:21 color profile: disabled composite 150 lpi at 45 degrees nificant valve damage (fig. 4b) but with thalassiosira faurii (at 700 cm depth) showing pristine valve condition (fig. 4a). the distribution and extent of diatom dissolution was assessed by applying the f index, both to the whole assemblage (for those taxa present at frequencies > 5%) and to individual taxa. according to the f index evaluated for the whole assemblages (fig. 5a), good preservation was principally confined to between 6 and 7.5 m depth in the core. in this zone the f acta bot. croat. 68 (2), 2009 387 diatom preservation in saline lake sediments fig. 3. correlations between ddi (diatom dissolution index) scores for the 3 dominant taxa from spiritwood lake short core sw1. increasing values indicate poorer preservation for ddi (in contrast to the f index). the dotted line shows the 1:1 line of equal preservation. (a) stephanodiscus niagarae against cyclotella meneghiniana; (b) stephanodiscus niagarae against stephanodiscus minutulus complex. b a u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:22 color profile: disabled composite 150 lpi at 45 degrees index was generally between 0.3–0.65. above 6 m depth, preservation was generally very poor with most f index values being between 0.1 and 0.2 and only exceeding 0.3 at around 375, 220, 160 and 150 cm point depths. below 750 cm depth, the assemblages were again very poorly preserved with index values of 0.2 or less. indices for the whole assemblage were generally lower than those for the most common diatom, aulacoseira granulata (fig. 5c). the other two common planktonic diatoms, c. meneghiniana and t. faurii (fig. 5b and d), were less uniformly distributed within the core. c. meneghiniana was present from above 800 cm depth and occurred sporadically. t. faurii was restricted to between 775 and 597 cm depth in the core. preservation of both taxa exceeded around 0.3 only in the lower core section but several levels displayed exceptional preservation for t. faurii with f index » 1 (almost all valves were pristine). preservation of c. meneghiniana never exceeded an f index value of 0.63. 388 acta bot. croat. 68 (2), 2009 flower r. j., ryves d. b. fig. 4. sem micrographs of diatoms extracted from a sediment core from lake qarun (qaru2, see text). (a): a near pristine valve of thalassiosira faurii sampled from around 670 cm depth in the core; (b): a valve of cyclostephanos dubius (though identification is hampered by dissolution) from around 420 cm in the core with extensive dissolution features; (c): a campylodiscus valve from around 420 cm depth in the core showing marginal dissolution features (note, the cyclotella meneghiniana valve central area in the top right of this image; dissolution stage 4). u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:24 color profile: disabled composite 150 lpi at 45 degrees the relative preservation of individual taxa can be demonstrated more clearly by plotting the index scores against one another (fig. 6). three patterns emerge and there is a fairly good correlation (r2 = 0.79; p = < 0.0001, cf. fig. 5a and b) between the f index values of a. granulata and of the whole assemblage (as expected as this is the dominant diatom in the core). however, the whole assemblages are relatively less well preserved than are the aulacoseira assemblages. when c. meneghiniana and a. granulata are compared (fig. 6a) there is a positive correlation between f index values of the two taxa (r2 = 0.18, p < 0.001). however, the relationship shows a strong and consistent skew in favour of relatively better preserved a. granulata. when t. faurii and a. granulata are compared (fig. 6b) there is no significant relationship between the valve preservation states of these two taxa (r2 = 0.11, p > 0.1). although some of the sample points indicate better preservation of a. granulata, several samples of t. faurii were much better preserved than those of a. granulata and in two core samples (fig. 5d) the valves of t. faurii were in near pristine condition (as verified by sem, fig. 4a). comparing total valve concentration with whole assemblage f index values indicated no trends (fig. 7a) other than that the diatoms in most samples with low diatom concentrations tended to be poorly preserved. however, the data suggested a causal positive link between the f index and diatom concentrations from around 1 to 20 x 106 valves g–1 dry sediment (r2 = 0.51, p < 0.005, fig. 7a). considering the diatom-inferred palaeosalinity values for the core assemblages, no correspondence was identified between these and whole assemblage preservation f index values except that the highest diatom concentrations occurred when inferred salinities were < 2500 µs cm–1 (fig. 7b). similarly, no clear relationacta bot. croat. 68 (2), 2009 389 diatom preservation in saline lake sediments fig. 5. graphs a–d show the down-core (qaru2) variations in f index values for the (a) total diatom assemblage, (b) cyclotella meneghiniana, (c) aulacoseira granulata, and (d) for thalassiosira faurii, respectively (see text). solid bars indicate core sections with good valve preservation. ba c d u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:25 color profile: disabled composite 150 lpi at 45 degrees ship between inferred salinity and diatom concentration was observed, although no samples with an inferred salinity value > 1000 ìs cm –1 exceeded 25 x 106 valves g–1 dry sediment. discussion diatom preservation is a major issue in saline lake palaeolimnology, yet by recognising that dissolution is dependant on a variety of processes and conditions and by applying an objective assessment methods, interpretation of damaged valves can be improved. dissolution of diatoms is a progressive process that seems to differ according to whether the valves are exposed to sub aerial, aquatic or sub-aquatic sedimentary conditions. dissolution sequences are relatively well known for a range of diatoms in aquatic or sub-aquatic sedimentary environments (barker 1992, flower et al. 2006, warnock et al. 2007, ryves et al. 2009) but much less is known around the combinations of environmental factors that promote dissolution in a particular lake. 390 acta bot. croat. 68 (2), 2009 flower r. j., ryves d. b. fig. 6. correlations between the f index scores for dominant taxa from the lake qarun core qaru2. decreasing values indicate poorer preservation for the f index (in contrast to ddi). the dotted line shows the 1:1 line of equal preservation. (a) aulacoseira granulata against cyclotella meneghiniana; (b) aulacoseira granulata against thalassiosira faurii. the significant correlation between f index scores in (a) and the trend (solid) line is indicated. a b u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:25 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (2), 2009 391 diatom preservation in saline lake sediments fig. 7. (a) the relationship between sedimentary diatom concentrations against depth in core qaru2 over the entire range of diatom concentrations, 1 – 20 x106 valves g–1 dry sediment. preservation and diatom concentration appear to be correlated at lower values (between 1 and < 20 x106 valves g–1 dry sediment and the regression is plotted over this range). (b) the relationship between assemblage f index scores and diatom-inferred conductivity values for qaru2. fig. 8. diatom concentrations in qaru2 plotted on a log scale to show four (a–d) very low diatom abundance zones (tab. 2) which fall below the range where valve concentration and poor preservation are related (see fig. 7). the solid bar indicates the core section with good valve preservation. a b u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:26 color profile: disabled composite 150 lpi at 45 degrees sedimentary diatom dissolution: the sedimentary diatom records for spiritwood lake (sw1) and lake qarun (qaru2) reflect different time scales with the former extending a little over 150 years and the latter around 2000 years. the lakes also differ in size, present-day salinity, stratification pattern, morphology, climatic setting (and sensitivity) and have different human impacts. for example, spiritwood lake is ice-covered for several months of the year, and is dimictic, while the water temperature of lake qarun rarely falls below 16 ºc and is monomictic. these differences will particularly affect kinetic rates of dissolution and biologically-mediated taphonomic processes, which makes direct comparisons between the two sites inappropriate. they are markedly different aquatic ecosystems characterized by different diatom species. in recent spiritwood lake sediments, stephanodiscus is the most common genus while in lake qarun sediment aulacoseira granulata has been generally most abundant. nevertheless, taphonomic processes strongly influence the integrity of the sedimentary diatom record in both lakes. further, it is likely that in both lakes the preservation states of diatom valves result mainly from water column and surface sediment conditions at the time of diatom deposition (cf. ryves et al. 2003, flower et al. 2006). using diatom dissolution indices (e.g. f index and ddi), however, the quality and taphonomy of these sediment records as diatom archives can be directly and fruitfully, compared. preservation status of diatoms in the spiritwood lake core (sw1) in this core, sediment accumulation, as both flux (g dry sediment cm–2 y–1) and as rate (cm y–1) increase towards the core top. while f index values also increase towards the top of sw1, as preservation improved, there was a weak, yet significant, correlation between f index values and reconstructed salinity. a stronger agreement between both the sediment accumulation rates and f index was seen, possibly indicating that sedimentary diatom ac392 acta bot. croat. 68 (2), 2009 flower r. j., ryves d. b. tab. 2. lake qarun sediments. four levels (around 377, 478, 560 and 820 cm depths) with very low diatom abundances (a–d) in sediment core qaru2 are selected to indicate diatom assemblage f index values, diatom valve concentrations (dc, in million valves g–1 dry sediment weight), sediment characteristics (co3 = carbonate, om = organic matter and bms = bulk magnetic susceptibility), the common diatom taxa and the presence of ostracod remains. some taphonomic and environmental aspects are indicated on the right of the table. concentration depth minimum f index dc (x10*6) sediment characteristics microfossil assemblage taphonomy and environment a (c. 820 cm) 0.06 0.8 higher bms higher co3 high % dry wt. a. granulata c. dubius (few ostracods) near desiccated lake, sediment reworking b (560 cm) 0.11 0.2 higher om higher co3 a. granulata c. meneghiniana (many ostracods) rapid sediment redeposition and focusing c (478 cm) 0.05 0.5 higher om higher co3 a. granulata c. meneghiniana (few ostracods) rapid desiccation and sediment reworking d (377 cm) 0.31 0.2 higher bms higher co3 surirella cf. smithii (few ostracods) low lake level u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:26 color profile: disabled composite 150 lpi at 45 degrees cumulation and diatom abundance promotes preservation in this core. the possible diatom accumulation and preservation relationship is yet only one factor (see below) and the dissolution indices of individual taxa showed that while preservation states between some species showed good correspondence (i.e. s. niagarae and c. meneghiniana), those between s. niagarae and s. minutulus do not. this may reflect differences in the pathways by which valves of particular diatom species are incorporated into the sediment record (cf. ryves et al. 2009). in the northern great plains, s. minutulus is a summer blooming species, reaching peak abundance at a time when the lake is strongly stratified and the hypolimnion is anoxic. in contrast, s. niagarae (and c. meneghiniana) tend to be autumn blooming species (e.g. holland 1969, baker and baker 1981), forming seston which sinks through a less strongly stratified water column. anoxia has been linked with enhanced preservation, as bioturbation is reduced, and organic coatings are less likely to be mineralized (ryves et al. 2006). preservation status of diatoms in the lake qarun core (qaru2) only one date is available for qaru2 and so calculating diatom accumulation rates is inappropriate. nevertheless, the concentration of sedimentary diatoms shows strong down-core variations with high values (> 20 x 106 valves g–1) only in the deeper part of the core (around 600–760 cm depth). while diatom concentration shows no overall relationship with the total diatom assemblages dissolution states, a positive link between valve preservation and concentration (over the range 1 to 20 x 106 valves g–1) does hint at a diatom concentration-mediated preservation effect, although other factors clearly influence diatom abundance within and beyond this range (tab. 1). concerning individual taxa, the preservation state of a. granulata, overall the most common sedimentary diatom, unsurprisingly corresponds most closely with the total assemblage preservation. however, other taxa do not show this relationship and cyclotella meneghiniana is relatively and consistently more poorly preserved compared to a. granulata, an observation which confirms the relative susceptibility of the former to dissolution from experiment and observation (flower et al. 2006). due to its robust central area, c. meneghiniana can remain identifiable despite considerable dissolution (cf. barker 1992) and it has a long »dissolution half-life« (see terminology of ryves et al. 2001), in common with other species of this genus (ryves et al. 2009). thalassiosira faurii preservation, on the other hand, is unrelated to the preservation state of a. granulata (fig. 6b) and this indicates that the former has undergone a different sediment entrainment pathway. the almost perfect preservation of many valves of this species at several levels in the core indicates either a more rapid mode of valve incorporation into the sediment, mass abundances of growing source populations, or sedimentation at a time of year when limnological conditions were extremely favourable to good preservation (e.g. during summer anoxia of deeper waters and lack of bioturbation). this unusually good preservation of t. faurii, as for s. minutulus (above), is contrary to expectations based on gross valve morphology (sa:v ratio; see barker et al. 1994). interpreting diatom dissolution for lake qarun, diatom-inferred (di) salinity shows no relationship with valve concentration or with the f index but sedimentary diatom concentration in qaru2 was always acta bot. croat. 68 (2), 2009 393 diatom preservation in saline lake sediments u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:26 color profile: disabled composite 150 lpi at 45 degrees relatively low when di salinity exceeded c.1000 ìs cm –1. this seems to indicate that, although high salinity is inimical to diatom valve preservation (cf. ryves et al. 2006), other processes also control sedimentary diatom concentrations. placing diatom abundances in qaru2 on a log scale clearly picks out four abundance minima (fig. 8, a–d) and for at least three of which poor preservation is implicated. these minima seem to have different causes which can be at least partly identified by taking a multi-proxy approach and using information provided by other sediment characteristics such as carbonate content, magnetic minerals, organic matter and ostracod remains (from foster et al. 2008, k. keatings, pers. comm.). additional stratigraphic information is added for each of the four levels with low diatom concentrations (tab. 2). each level is associated with elevated sedimentary carbonates, and minimum d seems to be associated with a low lake level stage (benthic surirella spp. exceed 20% abundance at this depth). assemblage preservation in this level is highest of the 4 minima (f = 0.31), suggesting that low diatom abundance is (partly) reflecting low productivity. minima a and c probably indicate periods of falling lake level since the f index values are low but not as low as in levels b and d. lake desiccation is inferred for minimum a (and possibly c) since ostracods are very few (and broken) and sediment density is high. for minimum b, many ostracods and higher organic matter could indicate sediment reworking as well as shallower water. there is no direct, simple correspondence between the diatom concentration minima and di-salinity. diatom productivity and ecology, low lake level, sediment in-wash and sediment reworking all must influence lake qarun's sedimentary diatom record, both quantitatively and qualitatively. identifying the combined causes of diatom concentration minima is made particularly difficult because variations in sediment accumulation rate remain unknown. clearly however there are different factors operating during the accumulation of qaru2 but, despite the lack of sediment flux data, considerable information around particular events can be gleaned by taking a multi-proxy approach. interrogating the detail of the stratigraphic record at points of change by using preservation states as well as multi-proxy evidence offers a promising way of extracting more palaeolimnological information. contrary to its importance in surface sediments across the ngp lakes (ryves et al. 2006) and over the last around 120 years for devils lake (ryves et al. 2009), salinity only appears to have a muted role in diatom preservation in spiritwood lake. in part, this may reflect the lower salinities this lake has experienced over the last around 150 years, during which it has generally remained subsaline (0.5–3 g l–1 tds), although dissolution is known to be driven by salinity even across short gradients (e.g. in west greenland lakes below 3 g l–1, ryves et al. 2006). additionally, it may also reflect inaccuracies within the original ngp diatom model itself (fritz 1990, fritz et al. 1993), where dissolution within the training set and fossil data may alter model performance and inferred values (ryves et al. 2009). applying two dissolution-adjusted models to the spiritwood lake data (one where dissolution stages are considered as »dissolution taxa« in their own right, and another where percentages are re-calculated according to individual species' f values; see ryves et al. 2009) did improve salinity-f index correlations here (r2 = 0.25 and 0.35 respectively; p < 0.02; not shown). even given some slight potential effect for involving dissolution data in the process of generating salinity inferences as well as fossil f index scores, this does appear to strengthen the argument that salinity has some role on preservation at spiritwood lake. 394 acta bot. croat. 68 (2), 2009 flower r. j., ryves d. b. u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:26 color profile: disabled composite 150 lpi at 45 degrees the role of salinity is however often subsidiary to that of physical processes, and the f index remains more strongly correlated with sediment accumulation rate (cm yr–1, fig. 2b). this suggests that diatom preservation is controlled more by the rate of volumetric accumulation of fine material that buries diatom valves within a matrix. this further suggests that the rapid build up of saturation silica levels in pore water may be important, preventing recently sedimented diatom valves from dissolving further and reducing diffusion of silica to undersaturated upper sediments and overlying water (perhaps especially important when the lake bed is rarely oxic with infrequent bioturbation and reduced mineralization of organic valve coatings). there is clearly an interplay between accumulation rate and initial sedimentary diatom concentration, by which pore waters become saturated with dissolved silica. the strong relationship between diatom concentration and preservation observed at low diatom abundances in qarun sediments (fig. 7a) may be, in part, the taphonomic (post-dissolution) imprint of such an interaction. sedimentation rate may thus be an important factor affecting diatom preservation across a range of freshwater and subsaline lakes where high salinity, or extreme lake level fluctuations and even periodic desiccation, are not the primarily drivers of diatom taphonomy. implications of diatom dissolution assessments undertaking an objective assessment of the preservation states of sedimentary diatoms in these two markedly different lakes provides taphonomic insights into the preservation of diatom species and of diatom assemblages within sediment cores and between samples. using an index to measure dissolution allows quantification of valve damage but making generalizations will always present problems because: (1) dissolution pathways can vary between species and between sites; (2) down-core changes in diatom dissolution and diatom abundance result from a variety of interacting contemporary processes; (3) exceptional but different events such as high salinity episodes, sediment in-wash and sediment redeposition conspire to obfuscate simple interpretation of stratigraphic signals; and (4) the nature of individual diatom species, their seasonal productivity and sedimentation characteristics all influence the sedimentary diatom record. multi-proxy sedimentary data are often required to help explain diatom dissolution and abundance relationships in sediment cores. diatom dissolution information can aid qualitative and quantitative palaeo-environmental inferences, however. for example, in surface sediment diatom inferred salinity models, the addition of dissolution data alone can reduce both apparent and prediction error by 10–15% (ryves et al.2009). conclusions exploring diatom preservation in sedimentary sequences from two saline lakes, spiritwood lake and lake qarun, indicates that poor preservation is often associated with low sedimentary diatom concentrations (as cause and effect) and/or higher (di) salinity, even when salinity changes are small. in the qarun core, however, neither diatom concentrations nor preservation indices were high when di-lake salinity was high. although 3 of the 4 down-core diatom concentration minima in core qaru2 are linked with very poor diatom preservation, there are clearly different contributory factors that reflect a variety of proacta bot. croat. 68 (2), 2009 395 diatom preservation in saline lake sediments u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:26 color profile: disabled composite 150 lpi at 45 degrees cesses that differ according to environmental conditions operating at particular points in time. the higher resolution study permitted at spiritwood lake highlights the role that seasonal diatom succession and stratification pattern has on the taphonomic pathway of different species. the lake qarun record, on the other hand, reveals broader taphonomic processes from larger-scale climatic and lake level changes (though salinity changes appear significant for preservation at both lakes). spiritwood lake further provides evidence that bulk sedimentation rate is an important control on diatom preservation over shorter time scales, though further examples from a range of different lake types and time periods are needed to assess this critically. assessing dissolution can improve diatom based inferences about past environments, both qualitatively (by inferring planktonic diatom seasonality and sinking characteristics, for example) and quantitatively (by improving diatom inferred models and calibrating sedimentary diatom frequencies and accumulation rates; e.g. mackay et al. 2005, battarbee et al. 2005, flower et al. 2006, ryves et al. 2006, 2009). the mechanisms, conditions and interactions that influence diatom dissolution states in saline lakes (especially those in which lake level and salinity fluctuate markedly) require further study especially since predicted climate change (influencing for example salt balances, lake levels and stratification intensity) will likely affect diatom dissolution patterns and pathways and therefore, potentially, inferences based on existing training sets. acknowledgements for this work the authors are grateful for grants from the british academy (rjf; under grant lrg-6045) and nerc (dbr; under grants gr9/02033 and gt4/90/als/28). we thank sheri fritz and kate laird for fieldwork assistance and project support at spiritwood lake. for lake qarun we are indebted to f. a. hassan, k. keatings, m. hamdan and others for advice and fieldwork help and to r. niederreitter who led the coring team that collected subaquatic sediment cores from this lake in 2004. we also thank two anonymous reviewers for thoughtful and useful comments on the manuscript. thanks to hong yang for help with figure presentations. references appleby, p. g., 2001: chronostratigraphic techniques in recent sediments. in: last, w. m., smol, j. p. 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1985: silica diagenesis ii. general mechanisms. journal of sedimentary petrology 55, 312–321. acta bot. croat. 68 (2), 2009 399 diatom preservation in saline lake sediments u:\acta botanica\acta-botan 2-09\flower.vp 9. listopad 2009 13:32:26 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 78 (1), 2019 35 acta bot. croat. 78 (1), 35–45, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0001 issn 0365-0588 eissn 1847-8476 distribution of taraxacum microspecies along soil property gradients in salt and brackish meadows on the polish baltic coast beata bosiacka1*, helena więcław1, paweł marciniuk2, marek podlasiński3 1 department of plant taxonomy and phytogeography, faculty of biology, university of szczecin, wąska 13, 71-415 szczecin, poland 2 department of botany, university of podlasie, prusa 12, 08-110 siedlce, poland 3 department of land recultivation and environmental chemistry, west pomeranian university of technology, słowackiego 14, 71-434 szczecin, poland abstract – the vegetation of protected salt meadows along the baltic coast is fairly well known; however, dandelions have been so far treated as a collective species. the aim of our study was to examine the microspecies diversity of the genus taraxacum in polish salt and brackish coastal meadows and to analyse soil property preferences of the dandelion microspecies identified. in addition, we analysed the relations between soil properties and vegetation patterns in dandelion-supporting coastal meadows (by canonical correspondence analysis). the salt and brackish meadows along the polish baltic coast we visited were found to support a total of 27 dandelion microspecies representing 5 sections. analysis of vegetation patterns showed all the soil parameters (c:n ratio, organic matter content, ph, concentration of mg, p, k, electrolytic conductivity of the saturated soil extract ece) to explain 32.07% of the total variance in the species data. the maximum abundance of most dandelion microspecies was associated with the highest soil fertility, moderate ph values and organic matter content, and with the lowest magnesium content and soil salinity. the exceptions were t. latissimum, t. stenoglossum, t. pulchrifolium and t. lucidum the occurrence of which was related to the lowest soil fertility and the highest salinity. in addition, several microspecies (t. leptodon, t. gentile, t. haematicum, t. fusciflorum and t. balticum) were observed at moderate c:n ratios and ece. four other microspecies (t. infestum, t. cordatum, t. hamatum, t. sertatum) occurred at the lowest ph and organic matter content. the information obtained increases the still insufficient body of knowledge on ecological spectra of individual dandelion microspecies, hence their potential indicator properties. keywords: coastal grasslands; dandelion microspecies; plant species composition, soil properties * corresponding author, e-mail: bebos@univ.szczecin.pl introduction the genus taraxacum (asteraceae, cichorioideae) comprises about 2800 microspecies with various reproductive systems, which can be grouped – based on their morphology and ecology – into about 60 sections (kirschner and štĕpánek 1996, kirschner et al. 2015). most dandelion microspecies belong to the polyploid-agamospermous hybrid complexes. in addition, the genus includes some primarily diploid and sexual taxa. parthenogenic seed production, definitely a dominant seed production mode in the genus, has resulted in maintenance of genetic differences, even small ones, among the microspecies (kirschner and štĕpánek 1996). these genetic differences underpin the morphological, biochemical and ecological variability of dandelions (van dijk 2003). due to reproductive isolation, many agamospermous microspecies can coexist in the same area (uhlemann 2001). despite the growing interest in the supraspecific taxonomy of taraxacum, knowledge about the dandelion flora is still far from complete. reports documenting the presence of single dandelion microspecies are relatively frequent (e.g. lundevall and øllgaard 2006, uhlemann et al. 2007, trávniček bosiacka b., wiȩcław h., marciniuk p., podlasiński m. 36 acta bot. croat. 78 (1), 2019 et al. 2008, marciniuk et al. 2012). on the other hand, comprehensive studies of the dandelion flora from larger areas in europe are rather rare. the identification guide to dandelions of great brittan and ireland (dudman and richards 1997) includes keys to different recognized taxa representing nine sections and provides information on the biology, preferred habitats, distribution and status of the genus in the british isles. trávníček et al. (2010) described the dandelion flora known to date from the czech republic; their description contains identification keys and considers the morphology, ecology, coenology and distribution of 179 dandelion taxa. some monographic studies focus on taraxacum section palustria, one of the most threatened dandelion groups. kirschner and štěpanek (1998) described the morphology, most common habitats and geographical distribution of 127 dandelion taxa of the section palustria in europe. schmid (2002) reported on the morphology, ecology, coenology, distribution and risk status of 24 dandelion taxa of the section palustria in southern germany. the same dandelion group was examined in poland and described by marciniuk (2012) whose monograph is complete with a key, a description of the morphological characteristics, habitat requirements and distribution of 23 taxa of the section palustria. dandelions are plants typical of open grassland ecosystems. at the section level, they are assigned to habitats of varying moisture and fertility (e.g. kirschner and štěpanek 1998, trávníček and vašut 2011). as demonstrated by some more detailed observations, individual microspecies within a section may be indicative of the grassland management type and shortterm environmental improvement or deteriorations (oosterveld 1978, 1983). grassland ecosystems are under threat because traditional management practices, such as mowing and grazing, have been largely abandoned, giving rise to progressive succession (muller 2002, isselstein et al. 2005). particularly threatened are coastal salt grasslands, as they shrink or completely disappear, especially when natural factors (e.g. low seawater salinity) act in concert with anthropogenic effects. this is the case along the coast of the baltic sea, a microtidal (to almost atidal) sea of a naturally low salinity (emeis et al. 2003). in poland, the geomorphological structure of a coastline dominated by sandy dunes and cliff sections is additionally unfavourable to seawater incursions. marsh areas favouring the development of halophytic vegetation, are found on the coast in only a few locations, such as the shores of estuaries and shallow embayments (herbich 2004, bosiacka 2012). salt grasslands along the baltic coast have developed mainly from brackish reed beds as a result of their centuries-long traditional, extensive use for grazing and mowing (dijkema 1990). hulisz et al. (2016) found that the seacoast vegetation patterns along the baltic sea shore were inconsistent with the salinity gradient of the open sea water, but were significantly dependent on specific, local conditions, e.g. factors that determined long-term seawater stagnation and intensive evaporation (resulting in soil salinity increase) and grassland management. intensive livestock grazing produces a low turf, homogeneous and species-poor. on the other hand, cessation of hay-making or grazing leads to the development of homogeneous, tall vegetation and to the withdrawal of heliophytic species (bakker 2012).the polish coastal salt grasslands are assigned to the eu habitat type 1330 atlantic salt meadows (interpretation manual, 2003). this habitat is protected under the natura 2000 network (habitats directive 92/43/eec). although the vegetation of salt meadows along the baltic coast is fairly well known (dijkema 1990, wanner 2009, bosiacka 2012, hulisz et al. 2016), dandelions have been so far treated as a collective species taraxacum officinale agg. our research is meant to contribute to the filling of this gap and, moreover, to find out whether different taraxacum microspecies prefer different microhabitats. the aim of our study was to examine the microspecies diversity of the genus taraxacum in polish salt and brackish coastal meadows and to analyse soil property preferences of the dandelion microspecies identified. in addition, we analysed the relations between soil properties and the vegetation patterns in dandelion-supporting coastal meadows. materials and methods study area our study encompassed all the salt and brackish meadows along the polish coast, of which there are currently few. due to natural and anthropogenic factors limiting the extent of this habitat type along the polish baltic sea coast, salt and brackish meadows occupy a small area compared to that along the south-western coast of the baltic sea or the north sea coast. at present, short grasslands with halophytes cover as little as a few hundred hectares in northern poland (bosiacka 2012). marsh areas conducive to seawater incursions are found in the north-western part of poland on the shores of szczecin lagoon, as well as on dozens of islands in the river świna storm delta, and in the eastern part of the polish coast, i.e. in puck bay. nevertheless, a large part of the habitat potentially amenable to salt meadow presence in those areas is overgrown by beds of reed (phragmitetum australis). in addition, brackish meadows supporting some halophytes have developed around several coastal lakes in the central part of the polish baltic coast. in nw poland, sites located close to the seashore (3–8 km away from it) show the presence of several salt marshes resulting from the ascending brine (bosiacka et al. 2011). the origin of brine in north-western poland is associated with the culmination of the mid-polish trough (krzywiec 2009). the cenozoic groundwater salinity is mainly a result of the ascending mesozoic relic seawater. in addition, the groundwater salinity in the anticline areas located in a close proximity to the seacoast may be in part a remnant of the juvenile relic water left by the littorina sea (kaczor 2006). not all the sites of the salt and brackish meadows along the polish baltic coast were observed to support dandelions. in fact, they were recorded at as few as eight sites only (fig. 1). the dandelion flora of the polish coastal grasslands acta bot. croat. 78 (1), 2019 37 field sampling field data were collected in april and may of 2013 and 2014. in all, 32 plots were sampled (all located in patches supporting dandelions). the plots (relevés) were 2 m x 2 m in size. the species cover in each plot was estimated using a nine-grade scale (van der maarel 1979). both vascular plants and bryophytes were recorded. the vascular plant nomenclature follows mirek et al. (2002), the system of ochyra et al. (2003) being followed for the bryophyte nomenclature. dandelion specimens were collected from each plot as vouchers and were deposited in the university of szczecin herbarium (szub). for each relevé, three soil samples from the plant root zone (0–25 cm) were collected with egner's soil sampler. the three samples were blended to form a single sample, representative of a given relevé, to be used in chemical analyses. laboratory analyses soil samples were dried at room temperature and sieved to remove fractions larger than 1 mm. the following properties were determined in the sieved material: (1) the organic matter content, as loss on ignition at 550 °c, (2) soil ph, potentiometrically in 1 m kcl, (3) electrolytic conductivity of the saturated soil extract (ece), conductometrically, (4) the content of available forms of potassium (k2o) in 0.5 m hcl, by atomic absorption spectroscopy, aas (american society of agronomy method), (5) the content of available forms of magnesium (mgo) in 0.5 m hcl, by aas, (6) the content of available forms of phosphorus (p2o5) in 0.5 m hcl, colorometrically, (7) the total carbon and nitrogen contents in air-dried triturated soil samples, using a chns analyser (costech analytical technologies inc.). the soil salinity was determined based on the conductivity of the saturated soil extract (ece). the following scale was used to classify salinity ranges obtained: non-saline soils (0–2 ds m–1); slightly saline soils (2–4 ds m–1); moderately saline soils (4–8 ds m–1); strongly saline soils (8–16 ds m–1); very strongly saline soils (>16 ds m–1) (richards 1954). data analysis relationships between plant species composition and soil properties were determined using the canoco v. 4.5 software package (ter braak and šmilauer 2002). plant species distribution patterns in relation to soil properties were determined by the canonical correspondence analysis (cca), after detrended correspondence analysis (dca) had detected a unimodal structure of the species data (the gradient length represented by the first ordination axis exceeded 3 sd). the data were not transformed. tests of significance of the first and all canonical axes were performed for the statistical assessment of the relation between plant species composition and environmental variables (monte carlo test: 499 permutations under the reduced model). the monte carlo permutation test was further applied to determine the statistical significance of environmental variables in explaining the plant species composition. for this purpose, the stepwise “forward selection” of explanatory variables (available in canoco) was used. the procedure started with selection of the best explanatory variable (a variable that best explains the total data variance), and the sequence of other variables was determined according to their decreasing importance in explaining the total variance in the data set, together with the previously selected variables. to this end, an “extra fit” (lambda a) value was calculated, the value representing a change in the sum of all the cca eigenvalues when another variable is added. additionally, the statistical significance of each variable was determined. variation in the plant species composition explained by environmental variables included in the analysis was expressed as a percentage representing the ratio of the sum of all canonical eigenvalues to the value of total variance (total inertia). variation in the species composition explained by individual variables was calculated from the ratio of lambda a to the total variance (total inertia), expressed as a percentage. the basic statistics (interquartile ranges of values, medians, outlier values, extreme values) were calculated for each soil property associated with individual dandelion microspefig. 1. study area; 1-8 sites in polish salt and brackish coastal meadows supporting the dandelion growth. bosiacka b., wiȩcław h., marciniuk p., podlasiński m. 38 acta bot. croat. 78 (1), 2019 cies. ranges of those properties were illustrated by individual box and whisker plots. relationships between the presence of individual dandelion microspecies (for 6 microspecies occurring in more than 2 plots only) and soil parameters as well as between the number of dandelion microspecies per plot and soil parameters were examined using spearman’s rank correlation test (statistica statsoft v. 10.0). plant communities were distinguished in the set of phytosociological relevés by the hierarchical divisive cluster analysis performed with twinspan v. 2.3 software (hill and šmilauer 2005). results the salt and brackish meadows along the polish baltic coast visited were found to support a total of 27 microspecies representing 5 sections: 1 microspecies of the section palustria (t. balticum, an agamospermous tetraploid which does not produce pollen), 2 microspecies of the section celtica (t. nordstedtii, an agamospermous hexaploid which does not produce pollen and t. gelertii, a pollen-producing agamospermous triploid), 6 microspecies of the section hamata (t. fusciflorum, t. infestum, t. hamatum, t. hamatiforme, t. kernianum, t. lancidens, all pollen-producing agamospermous triploids), 1 microspecies of the section borea (t. ostenfeldii, an agamospermous triploid which does not produce pollen) and 17 microspecies of the section taraxacum (ruderalia), all pollen-producing agamospermous triploids. the number of dandelion taxa at a site varied from 2 to 10 (in individual relevés: from 1 to 6 microspecies per 4 m2). the number of microspecies per plot was positively correlated with soil fertility, expressed as the c:n ratio (inversely proportional to soil fertility) and negatively correlated with soil salinity, k and mg concentrations (tab. 1). no dandelions were found in the most waterlogged salt meadows (where stagnant water is observed even in april and may) or in salt meadows abandoned for a long time (which often experience encroachment of the robust common reed phragmites australis). most of the identified dandelion microspecies occurred in 1–2 plots. only six microspecies were more frequent in coastal meadows and occurred in larger numbers: t. nordstedtii (in 12 plots, in the western part of the coast only), t. haematicum (in 10 plots), t. balticum (in 8 plots, in the western part of the coast only), t. hamatiforme (in 7 plots), t. gelertii (in 5 plots) and t. sellandii (in 4 plots). analysis of vegetation patterns in the dandelion-supporting coastal meadows (cca) showed all the environmental variables (soil parameters) to explain 32.07% of the total variance in the species data. the first axis and all the canonical axes were significant as tested by the unconstrained monte carlo permutation test (p = 0.002). results of the stepwise forward selection of variables revealed five out of the seven variables considered (c:n, organic matter, ph, mg, ece,) to be statistically significant and explain 26.45% of the total variance in the plant species composition. the largest amount of the total variance (7.31%) was explained by soil fertility (tab. 2). the maximum abundance of most dandelion microspecies was associated with the highest soil fertility (soil fertility is inversely proportional to the c:n ratio), moderate ph values and organic matter content, and with the lowest magnesium content and salinity (fig. 2). the exceptions were taraxacum latissimum, t. stenoglossum, t. pulchrifolium and t. lucidum the occurrence of which was related to the lowest soil fertility (i.e. the highest c:n ratios) and the highest salinity. in addition, several microspecies (t. leptodon, t. gentile, t. haematicum, t. fusciflorum and t. balticum) were observed at moderate c:n ratios and ece. four other microspecies (t. infestum, t. cordatum, t. hamatum, t. sertatum) occurred at the lowest ph and organic matter content. fig. 3 shows in detail the ranges of soil properties in relation to the different dandelion microspecies. with retab. 1. results of spearman’s rank correlation test between the number and occurrence of taraxacum microspecies and soil parameters, * denotes p < 0.05, ece – electrolytic conductivity. taxon organic matter content [%] c:n ece [ds m–1] ph p [mg kg–1] k [mg kg–1] mg [mg kg–1] t. balticum 0.452* 0.175 0.373* 0.159 0.192 0.164 0.334 t. gelertii –0.241 –0.255 –0.367* 0.052 –0.049 –0.278 –0.111 t. haematicum –0.369* –0.098 –0.006 0.259 0.158 0.016 –0.131 t. hamatiforme –0.048 –0.492* –0.417* –0.159 0.168 –0.349* –0.467* t. nordstedtii –0.147 –0474* –0.413* –0.125 0.128 –0.209 –0.267 t. sellandii 0.076 –0.169 –0.159 –0.142 0.054 –0.082 0.005 no. dandelion microspecies –0.169 –0.552* –0.582* –0.126 0.034 –0.389* –0.361* tab. 2. forward selection of explanatory variables with the significance test for variables (soil parameters) explaining variance in the taraxacum species composition; * denotes p < 0.05, org. mat. – organic matter content, ece – electrolytic conductivity. variables lambdaa (variance) explained data variance [%] f-ratio p-value c:n 0.29* 7.31 2.37 0.002 org. mat. 0.21* 5.29 1.77 0.002 ph 0.20* 5.04 1.77 0.010 mg 0.18* 4.53 1.60 0.006 ece 0.17* 4.28 1.48 0.028 p 0.11 2.77 1.20 0.452 k 0.11 2.77 1.14 0.546 the dandelion flora of the polish coastal grasslands acta bot. croat. 78 (1), 2019 39 gard to the soil salinity classes identified, slightly saline soils (2–4 ds m–1) supported the following microspecies: t. balticum, t. fusciflorum, t. gelertii, t. gentile, t. haematicum, t. hamatiforme, t. lucidum, t. nordstedtii, t. sellandii, t. stenoglossum, while moderately saline soils (4–8 ds m–1) were found to support t. balticum, t. haematicum, t. latissimum, t. leptodon and t. pulchrifolium. other dandelion microspecies were recorded mainly in non-saline soils, in phytocoenoses developing on the edges of salt meadow patches. results of spearman’s rank correlation tests applied to the six dandelion microspecies occurring in more than 2 plots (tab. 1) showed significant direct association between t. balticum and the organic matter content and soil salinity, as well as a significant positive correlation between t. nordstedtii and t. hamatiforme and soil fertility expressed as the c:n ratio (inversely proportional to soil fertility). negative correlations were observed between t. haematicum and the organic matter content; t. hamatiforme and soil salinity and k and mg concentrations; t. nordstedtii and soil salinity. the hierarchical divisive cluster analysis identified, in the first division, two groups of plant communities (tab. 3). cluster i showed the following species as indicators: carex nigra, cardamine pratensis and taraxacum nordstedtii, cluster ii being typified by eleocharis uniglumis, plantago maritima and phragmites australis. this division largely coincides with the diversity of communities found in the western (sites 1–5) and eastern (sites 6–8) parts of the polish coast. the following species are indicative of the further division of cluster i (dominant phytocoenoses in the western part of the coast): juncus effusus, holcus lanatus, taraxacum hamatiforme (cluster iii) and triglochin maritimum, calliergonella cuspidata, taraxacum balticum (cluster iv). taraxacum nordstedtii was recorded only in the western part of the coast, both in the meadow phytocoenoses from cluster iii, growing on slightly saline or non-saline edges of salt marshes, and in the phytocoenoses from cluster iv, developing on lowor moderate salinity soils. cluster iii phytocoenoses seldom supported halophytes, while the frequent taraxacum hamatiforme dominated among other dandelion microspecies. the following microspecies occurred exclusively in this group, but only in single plots: t. hamatum, t. infestum, t. kernianum, t. laticordatum, t. ostenfeldii, t. piceatum and t. sertatum. phytocoenoses from cluster iv were characterized by a higher contribution of halophytes. apart from t. nordstedtii, also frequent was t. balticum (in the western part of the coast only). the following species occurred exclusively in this group, but only in single plots: t. copidophyllum, t. fagerstroemii, t. hemicyclum, t. sinuatum and t. sublaeticolor. the following species are indicative of the further division of cluster ii (phytocoenoses dominant in the eastern part of the coast, except for relevés/plots 4, 17, 20, 21): glaux maritima, plantago maritima (cluster v) and phragmites australis, rumex crispus (cluster vi). phytocoenoses from cluster v were characterized by the abundance of halophytes and a low contribution of phragmites australis. t. stenoglossum and t. cordatum occurred exclusively in this group, but fig. 2. diagrams of species of polish coastal meadows and soil properties ordination along the first two cca axes: a) taraxacum microspecies with the most common accompanying plant species, b) taraxacum microspecies visible only; eigenvalues of axis i and axis ii: 0.361 and 0.229 respectively; sum of all eigenvalues (total inertia): 3.971; sum of all canonical eigenvalues: 1.271; * denotes statistically significant variables. abbreviations of species names consist of the first three letters of a generic name and the first three letters of a species name (see tab. 3), with exceptions: tar.hme – taraxacum hamatiforme, tar.hum – taraxacum hamatum, tar.lco – taraxacum laticordatum, tar. lsi – taraxacum latissimum, car.dist – carex distans, car.dch – carex disticha; ece electrolytic conductivity. bosiacka b., wiȩcław h., marciniuk p., podlasiński m. 40 acta bot. croat. 78 (1), 2019 fig. 3. the ranges of soil property values related to individual taraxacum microspecies of polish coastal meadows: a) c:n ratio, b) organic matter content, c) ph, d) mg concentration, e) electrolytic conductivity (ece) of the saturated soil extract, f ) p concentration, g) k concentration. grey boxes indicate 25-75% of the interquartile ranges of values, black boxes – the medians (with the exception of microspecies found in only one sample), white circles – outlier values, asterisk – extreme values. the dandelion flora of the polish coastal grasslands acta bot. croat. 78 (1), 2019 41 tab. 3. plant communities in polish salt and brackish coastal meadows separated by the hierarchical divisive cluster analysis. species indicative of the division into clusters are underlined. no. of stand (see fig. 1) 3 3 3 6 5 5 1 5 5 5 1 4 4 1 3 3 3 3 5 5 5 6 6 6 6 2 6 8 7 7 7 7 no. of relevé/plot  1 2 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 2 2 3 3 8 9 1 3 5 6 3 4 9 8 2 2 3 1 0 5 6 7 7 0 1 2 5 6 7 4 4 2 8 9 0 1 no. of clusters i ii iii iv v vi taraxacum balticum 1 1 3 4 3 3 2 3 taraxacum copidophyllum 2 taraxacum cordatum 2 taraxacum fagerstroemii 1 taraxacum fusciflorum 3 taraxacum gelertii 3 2 3 1 3 taraxcaum gentile 3 taraxacum haematicum 2 3 2 2 3 2 3 3 2 3 taraxacum hemicyclum 1 taraxacum hamatiforme 3 3 2 3 2 3 2 taraxacum hamatum 2 taraxacum infestum 2 taraxacum kernianum 1 taraxacum lancidens 2 3 taraxacum laticordatum 1 taraxacum leptodon 3 3 taraxacum latissimum 2 taraxacum lucidum 1 taraxacum nordstedtii 3 3 4 2 3 2 3 1 2 2 2 3 taraxacum ostenfeldii 1 taraxacum piceatum 1 taraxacum pulchrifolium 3 taraxacum sellandii 2 2 2 4 taraxacum sertatum 1 taraxacum sinuatum 2 taraxacum stenoglossum 3 taraxacum sublaeticolor 1 1 agrostis stolonifera 4 4 5 5 5 6 6 6 4 3 6 6 6 6 6 7 6 7 7 4 5 6 7 7 6 7 6 alopecurus geniculatus 2 7 alopecurus pratensis 3 6 1 4 2 3 anthoxanthum odoratum 5 6 2 2 1 2 aster tripolium 2 3 2 atriplex prostrata ssp. 1 2 berula erecta 2 blysmus compressus 1 4 2 3 3 4 3 5 brachythecium rutabulum 3 3 3 5 briza media 2 caliergonella cuspidata 3 8 9 9 9 9 9 caltha palustris 3 2 2 2 2 carex cuprina 3 2 3 2 carex disticha 6 3 6 4 2 3 2 6 3 carex distans 3 4 carex hirta 2 2 carex nigra 5 6 4 3 7 5 6 6 4 4 3 6 3 6 3 3 2 3 3 3 carex panicea 3 3 3 2 2 2 3 3 3 cardamine pratensis 3 2 2 3 3 2 3 2 2 2 2 3 3 2 2 2 2 centaurea jacea 3 4 4 cerastium holosteoides 2 2 1 2 1 bosiacka b., wiȩcław h., marciniuk p., podlasiński m. 42 acta bot. croat. 78 (1), 2019 tab. 3. continued no. of stand (see fig. 1) 3 3 3 6 5 5 1 5 5 5 1 4 4 1 3 3 3 3 5 5 5 6 6 6 6 2 6 8 7 7 7 7 no. of relevé/plot  1 2 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 2 2 3 3 8 9 1 3 5 6 3 4 9 8 2 2 3 1 0 5 6 7 7 0 1 2 5 6 7 4 4 2 8 9 0 1 no. of clusters i ii iii iv v vi chenopodium rubrum 2 cirsium arvense 2 2 2 2 2 dactylorhiza incarnata 2 1 dactylorhiza majalis 3 2 3 4 1 2 1 deschampsia caespitosa 4 5 4 4 7 3 eleocharis palustris 4 3 3 3 eleocharis uniglumis 4 3 3 4 3 4 2 3 3 3 4 4 6 6 elytrygia repens 5 5 5 3 3 3 2 1 3 3 2 3 eriophorum angustifolium 2 2 3 2 festuca arundinacea 2 3 festuca rubra 2 3 4 5 6 3 6 4 5 3 4 7 7 7 8 2 6 4 6 galium palustre 2 2 2 5 glaux maritima 2 3 4 3 4 4 3 4 2 3 holcus lanatus 3 4 4 4 2 hydrocotyle vulgaris 3 2 2 2 juncus articulatus 2 3 juncus compressus 3 3 juncus effusus 6 4 3 3 5 3 2 2 4 2 3 2 juncus gerardi 4 2 5 7 6 5 5 5 3 5 6 5 3 kindbergia praelonga 6 leontodon autumnalis 2 2 2 3 3 3 2 2 2 3 2 3 2 3 leptodictyum riparium 4 lotus tenuis 4 lotus uliginosus 2 2 2 2 2 2 3 luzula multiflora 3 2 lychnis flos-cuculi 2 3 2 2 2 2 2 1 lysimachia nummularia 3 3 2 mentha aquatica 2 2 2 2 phragmites australis 2 3 4 3 3 4 5 7 6 6 plantago lanceolata 3 4 3 3 2 1 2 plantago maritima 4 6 3 3 5 4 2 2 2 2 plantago winteri 2 2 2 3 2 2 3 3 2 poa pratensis 3 2 potentilla anserina 5 3 6 5 4 5 5 3 3 3 4 5 3 4 4 3 5 4 4 3 3 3 4 5 4 5 4 4 ranunculus acer 2 2 2 3 1 2 2 2 2 2 2 2 3 2 2 2 2 2 ranunculus flammula 2 1 2 2 2 2 ranunculus repens 3 6 2 3 4 3 3 3 3 3 2 rhitidiadelphus squarrosus 3 6 9 rumex acetosa 2 3 2 2 2 2 2 2 rumex crispus 2 1 2 2 2 2 2 1 2 schoenoplectus tabernaemonatni 3 2 3 sonchus arvensis 3 2 2 tortula truncata 3 trifolium fragiferum 4 5 3 3 3 4 3 3 2 4 3 3 3 2 3 3 6 2 trifolium repens 4 5 4 3 4 4 4 4 5 4 4 3 4 4 5 3 triglochin maritimum 2 4 3 3 2 5 3 3 3 3 5 6 7 5 5 4 5 6 3 4 3 1 triglochin palustre 2 2 2 4 valeriana dioica 2 3 3 2 the dandelion flora of the polish coastal grasslands acta bot. croat. 78 (1), 2019 43 only in single plots. phytocoenoses from cluster vi showed the highest contribution of phragmites australis (albeit of reduced vitality) and the lowest number of species, including t. fusciflorum, t. gentile and t. pulchrifolium occurring only in this group. discussion dandelion microspecies frequently occur sympatrically, which is probably the rule in the whole area supporting agamospermous forms. due to their reproductive isolation, obligately agamospermous microspecies can be constant coexisting taxa (uhlemann 2001). a possible underlying cause of the coexistence may be a small-scale environmental heterogeneity. unfortunately, details of dandelion microspecies’ habitat requirements are still scarce and are mainly of a phytosociological nature or represent ellenberg’s intermediate indication values (e.g. sterk et al. 1983, schmid 2002). moreover, as suggested by comparative studies on the seed germination ecology of some taraxacum microspecies, ecological differentiation is commonly among the microspecies and plays an important role in the maintenance of the taxonomic diversity within the genus (van loenhoud and duyts 1981). the genotype-environment interactions in apomictic dandelion populations have been studied (without microspecies identification) in recent years, some of the studies showing very small-scale local ecological adaptations (vellend et al. 2009, drumond et al. 2012, mcleod et al. 2012). in poland, the genus taraxacum is represented by three very rare sexual diploids and by about 400 apomictic species from 12 sections (mirek et al. 2002). we recorded a relatively high dandelion flora diversity (27 microspecies) in the coastal salt and brackish meadows visited. the typical low-sward phytocoenoses representative of the habitat currently occupy a small (less than 400 hectares) area along the polish baltic coast (bosiacka 2012). the salt grasslands were reduced in size mainly due to cessation of grazing and mowing. research on the dandelion flora conducted in the netherlands demonstrated that the number of microspecies depends on the intensity of grassland management: the highest densities of dandelion taxa (up to 19 microspecies per 125 m2) were typical of the most intensively fertilized and grazed pastures, while extensively managed, ungrazed and unfertilized grasslands support much lower numbers of taraxacum microspecies. this effect concerns mainly the section taraxacum (ruderalia), dandelions in which the number of specimens as well as the number of microspecies increase when more intensive farming is practiced; however, the total number of dandelions (microspecies and individuals) decreases rapidly under constant management (oosterveld 1983, sterk et al. 1983). our results correspond with the positive effect of soil fertility on the number of dandelion microspecies, observed in the studies referred to above. dandelions of each section are found under a specific set of environmental conditions; for example, microspecies from the section palustria are almost exclusively confined to natural and semi-natural habitats and are usually found in periodically flooded sites, with an accompanying effect of new minerals available from the sediments (kirschner and štěpanek 1998, schmid 2002, marciniuk 2012); microspecies from the section celtica prefer plots under relatively low organic manuring and are found in meadows, pastures, as well as along roads in urban areas (horn et al. 1996, trávníček et al. 2010); microspecies from the section taraxacum (ruderalia) prefer regularly fertilized grasslands and various anthropogenic habitats; as the summer habitat dry-out is probably the main factor limiting their occurrence, they are often found in areas affected by the maritime climate (sterk et al. 1983, trávníček et al. 2010); microspecies from the section hamata are usually found in moist meadows, preferably with lower ph, an intermediate mineral content and a partly disturbed vegetation, as well as in shaded places in urban areas (øllgaard 1983, trávníček and vašut 2011). oosterveld (1983) conducted one of the few direct studies on the ecological preferences (involving soil properties) of individual dandelion microspecies. he examined soil phosphate concentrations in the immediate vicinity of the root system of the microspecies co-occurring in a pasture. his results allowed the presumption that the high variability within taraxacum may be associated with increasing rate of phosphate availability. he also classified the dandelion microspecies in relation to management invariability (oosterveld 1978). in another detailed study, bosiacka et al. (2016) determined soil properties and identified plant communities associated with marsh dandelions in polish and estonian coastal grasslands. soil salinity was found to correlate moderately strongly albeit significantly with all the three marsh dandelion microspecies found (positive correlation for taraxacum balticum and negative correlation for t. suecicum and t. decolorans). in addition, t. suecicum was inversely correlated with the organic matter content and was positively correlated with soil ph. a much higher number of t. balticum stands (22 stands, 36 samples) was found on the estonian coast than in the polish coastal grasslands. the taxon occurred there in the widest ranges of all soil properties considered and in all types of phytocoenoses studied: in salt and brackish meadows, in coastal alvar grasslands and in transitional areas. all the dandelion-supporting meadows visited along the polish baltic coast are currently, or had been until recently (in the previous decade), extensively used. usually, the meadows showed a mosaic of wet halophytic and glycophytic vegetation patches on a peat substrate. at some sites, small areas of mineral inclusions were scattered among peat deposits. in addition, many sites showed evidence of wild boar rooting. therefore, the spatial and taxonomic structure of the vegetation was locally very heterogeneous. the overall contribution of dandelions to coastal meadow phytocoenoses was low. analysis of relationships between the species composition of the meadows studied and the soil properties revealed a few statistically significant variables, including soil fertility as the most important one, followed by the organic matter content, ph, available magnesium content, and salinity. the available phosphorus content proved non-significant, as opposed to the study of oosterveld (1983). most of the dandelion microspecies we found were associated with relatively fertile and lowest-salinity soils. only four microspebosiacka b., wiȩcław h., marciniuk p., podlasiński m. 44 acta bot. croat. 78 (1), 2019 cies of the section taraxacum (ruderalia): t. latissimum, t. stenoglossum, t. pulchrifolium and t. lucidum were found in single patches with the lowest fertility and the highest salinity. the ecological spectrum of those species is very wide; they occur in wet meadows and in miscellaneous anthropogenic grassy habitats, including roadsides and highly salted urban lawns (trávníček et al. 2010). a few microspecies (t. infestum, t. cordatum, t. hamatum, t. sertatum) occurred on soils with the lowest organic matter content and the lowest ph. among them, t. cordatum is known to prefer sandy soil, and can penetrate even a very dry dune environment which is the domain of the section erythrosperma (sterk et al. 1983). our specimens, often buried in sand, were found in a brackish meadow adjacent to coastal dunes, the only site of this type among the ones we surveyed. taraxacum balticum was found during our fieldwork in phytocoenoses with a high contribution of halophytes. kirschner and štěpanek (1998) define the microspecies as one of the most halophilous dandelions, its distribution range being closely connected with the baltic coast. like other taxa of the section palustria, it is becoming less and less common. none of our sites supporting the microspecies has been previously reported in the literature. apart from the coast, t. balticum has been reported from only one inland salt grassland and one chalk meadow in central poland (marciniuk 2012). taraxacum nordstedtii was the most common microspecies in our study in the western part of the coast, and occurred both in typically halophytic phytocoenoses and along their edges, among glycophytes. the microspecies has the widest ecological spectrum of all the species of the section celtica (sterk et al. 1983, kirschner and štěpanek 1984, horn et al. 1996). based on qualitative inventories of grasslands under different management conditions in the netherlands, and according to the classification of dandelion microspecies associated with low to highly dynamic habitats, t. nordstedtii is a low-dynamic species and is the last to disappear under stable mowing conditions (oosterveld 1978, 1983). in poland, it is a rare microspecies, occurring at the eastern limit of its range; 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(asteraceae, lactuceae) in germany. feddes repertorium 112, 15–35. uhlemann, i., kirschner, j., øllgaard, h., štěpánek, j., 2007: four new species of taraxacum sect. ruderalia (asteraceaecichorieae) from central europe and scandinavia. phyton 47, 103–121. van dijk, p. j., 2003: ecological and evolutionary opportunities of apomixis: insights from taraxacum and chondrilla. philosophical transactions of the royal society of london b 358, 1113–1121. van loenhoud, p. j., duyts, h., 1981: a comparative study of the germination ecology of some microspecies of taraxacum wigg. acta botanica neerlandica 30, 161–182. vellend, m., drummond, e. b. m., muir, j. l., 2009: ecological differentiation among genotypes of dandelions (taraxacum officinale). weed science 57, 410–416. wanner, a. 2009: management, biodiversity and restoration potential of salt grassland vegetation of the baltic sea: analyses along a complex ecological gradient. – phd thesis, university of hamburg, hamburg. acta bot. croat. 77 (2), 2018 135 acta bot. croat. 77 (2), 135–140, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0010 issn 0365-0588 eissn 1847-8476 photosynthetic performance of two freshwater red algal species tamás pálmai1*, beáta szabó2, katalin eszter hubai1, judit padisák1,2 1 department of limnology, university of pannonia, egyetem u. 10, veszprém 8200, hungary 2 mta-pe limnoecology research group, egyetem u. 10, veszprém 8200, hungary abstract – photosynthetic performances of two freshwater red algal populations from freshwaters of the carpathian basin were measured in this study. populations were collected from different habitats: bangia atropurpurea from lake balaton and batrachospermum gelatinosum from the tapolca stream. their photosynthesis was studied in a wide range of temperature (5–35 °c) and light intensity (0–1150 µmol m–2 s-1) in a photosynthetron. we found both species’ photosynthesis maxima at 25 °c but b. atropurpurea had significantly higher photosynthetic production. low and medium values were calculated for the species’ photoadaptation parameters. compensation light intensities determined in this study were similar to those obtained in previous studies. both species utilized light efficiently; photoinhibition was detected only at two measuring temperatures for bangia and at four measuring temperatures for batrachospermum. p-t characteristics of the species revealed that both have temperature optima at 25 °c under high and medium light intensities but there are no such remarkable optima at low irradiance. the biomass specific respiration of both species increased with increasing temperature. we confirmed the good light utilization of these red algal species but found temperature optima higher than reported previously. keywords: bangia atropurpurea, batrachospermum gelatinosum, ecophysiology, light, photosynthetic characteristics, temperature abbreviations: ps – maximal production obtained in the absence of photoinhibition, pbmax – biomass specific maximal photosynthetic rate, ik – photoadaptation parameter, ic – compensation light intensity, α – initial slope of the curve, light utilization, β – photoinhibition parameter, rb – biomass specific respiration. * corresponding author, e-mail: palmait@almos.uni-pannon.hu introduction red algae are mostly marine macroscopic organisms. besides the 5000–5500 marine rhodophyta species there are about 180 species living in fresh waters; most are limited to running waters. they are often considered as indicators of good water quality and clean habitats as they prefer very clear, transparent habitats (skuja 1938, sheath 1984). bangia atropurpurea (mertens ex roth) c. agardh and batrachospermum gelatinosum (linnaeus) de candolle are filamentous red algae with worldwide distribution, commonly growing attached to the surface of shoreline rocks of lakes and streams. bangia atropurpurea is an unbranched filamentous red alga that grows along rocky shorelines. the species occurs both in freshand seawater. it was often mentioned under the name b. fuscopurpurea (dillw.) till geesink (1973) concluded that these two taxa are conspecific. b. atropurpurea was described in north america, europe, the middle east, and in japan (belcher 1960, geesink 1973, sheath and cole 1980, araki et al. 1994, barinova 2013). barinova (2013) found b. atropurpurea in a wide range of temperature (15.5– 35 °c), ph (6.6–8.0), and conductivity (0.82–4.0 ms cm–1). temperature and day length dependence of the growth rate, maturation and size of the species were also reported (sommerfeld and nichols 1973). monospore germination of b. atropurpurea is light-dependent (charnofsky et al. 1982). optimum conditions for the photosynthesis were found at 20 °c and 750 µmol m–2 s–1 (graham and graham 1987). batrachospermum gelatinosum is widely distributed in the running waters of north america and europe. popupálmai t., szabó b., hubai k. e., padisák j. 136 acta bot. croat. 77 (2), 2018 lations, as red algae usually, occur at cold temperatures (0–22 °c) in circumneutral (ph 6–8.5) waters with low conductivity (10–490 µs cm–1) (kremer 1983, vis et al. 1996, vis and sheath 1997, carmona et al. 2011, chiasson et al. 2014, drerup and vis 2014). b. gelatinosum was also described in central and south america, australia and in the middle east under similar conditions (vis and entwisle 2000, jiménez et al. 2004, vis et al. 2008, barinova 2013). besides the distribution, the physiology of some batrachospermum species (b. ambiguum, b. delicatum, b. gelatinosum, b. helminthosum and b. vogesiacum) were also studied. it was shown that low irradiance is advantageous for the growth of red algae species. species-specific temperature optima of the growth were established by zucchi and necchi (2001) and drerup et al. (2015). our knowledge about the hungarian red algae flora is quite limited. the total number of taxa recorded in northern european countries is higher than that in centraland south-europe. in hungary, nine taxa were reported (kwandrans and eloranta 2010), two of these (batrachospermum ectocarpum sirodot and b. moniliforme roth) were found in the springs of the northern bakony mountains (kol 1968). additionally, some species (audouinella ciolacea (kütz.) hamel, audouinella chalybea, chroodactylon ornatum (ag.) basson, hildebrandia rivularis (lieben) ag., porphyridium purpureum (bory) drew et ross, thorea hispida) were reported from the two main rivers (danube and tisza) (kiss and pelyhe 2004, szemes 1967, uherkovich 1957). extended stands of bangia atropurpurea were described from the shoreline rocks of lake balaton (tamás 1959) but, as a consequence of eutrophication, the species was suppressed in the last decades (kiss and pelyhe 2004). as a consequence of restoration measures (istvánovics et al. 2007), b. atropurpurea reappeared in early spring, 2015 offering an opportunity to study its photosynthetic performance. because of our scarce knowledge about the hungarian red algae and a lack of physiological data, measurements were carried out to determine not only the temperature and light intensity optima but also the tolerance range of the populations. for this reason, we carried out experiments in a wide range of temperature and light conditions. materials and methods two populations of freshwater red algae were investigated in this study. samples were collected during the period of highest abundance from surfaces of stones then were kept in filtered (0.45 µm pore size membrane filter) stream or lake water depending on sampling site. bangia atropurpurea was collected from shoreline rocks of lake balaton in february 2015 (water temperature was 4.9 °c) (46°54'53.9928" n, 17°53'34.4148" e). lake balaton is the largest shallow lake in central europe, with slightly alkaline water (ph~8.3). the dominant ions of the lake are ca2+, mg2+ and hco3–, and secchi transparency rarely exceeds 1 m. long sections of the lake’s shoreline are artificially covered by large red sandstone bricks to protect the shoreline constructions against wave action. these rocks provide suitable habitats for b. atropurpurea. batrachospermum gelatinosum was collected from the tapolca stream (46°51'1.224" n, 17°25'17.8248" e) in april 2015. the stream is fed by hot springs. its temperature varies between 9.6 and 21 °c during the year and it cools down only slightly in winter. during the sampling, water temperature was 16.1 °c. small and medium size stones (5–15 cm) cover the streambed; red algae grew attached to the surface of this substrate. the following physical parameters were recorded during samplings: temperature, ph, dissolved oxygen concentration, oxygen saturation and conductivity with hq40d portable multi-parameter meter (hach lange) and intellical™ phc101 rugged gel filled ph electrode, intellical™ cdc401 rugged conductivity probe, intellical™ ldo101 rugged luminescent/optical dissolved oxygen (ldo) (tab. 1). photosynthesis measurements were carried out in a photosynthetron developed by üveges et al. (2011), which allows measurements at nine different light intensities at the same time at a given temperature. light intensities were set between 0–1150 µmol m–2 s-1 (0, 15, 35, 75, 150, 300, 600, 800, 1000 µmol m–2 s–1 were used for bangia and 0, 10, 30, 75, 130, 200, 420, 1150 µmol m–2 s–1 were used for batrachospermum), par was provided by daylight tubes (tungsram f74). light intensities in the different cells were measured with a spherical (4 π) quantum sensor. a wide range of temperature was used to determine not only the temperature optima but also the tolerance ranges of the species. incubation temperature was increased by 5 °c increments in the temperature range 5–35 °c. permanent temperature was provided by neslab rte-211. red algal samples were filtered onto 1.2 µm pore size gfc filters, and then fresh weight was measured with 0.1 mg accuracy. known fresh weights of the field samples were placed into karslruhe-flasks, which were then filled with fresh filtered (0.4 µm pore size mixed cellulose-ester membrane filter) stream or lake water before each measurement. prior to the experiments, samples were pre-incubated at least for 2 h at each measuring temperature. photosynthetic tab. 1. environmental data measured at sampling sites. tapolca-stream lake balaton collected species batrachospermum gelatinosum bangia atropurpurea date 20 april 2015 16 february 2015 location 46°51'1.224"n 17°25'17.8248"e 46°54'53.9928"n 17°53'34.4148"e temperature (°c) 16.1 4.9 ph 7.61 8.5 dissolved oxygen (mg l–1) 9.19 12.17 oxygen saturation (%) 93.7 94.9 conductivity (µs cm–1) 704 565 photosynthesis of two rhodophyta species acta bot. croat. 77 (2), 2018 137 the increase of the pbmax was about 75–80% for both species and both had maxima at 25 °c. a remarkable difference was found between the photosynthetic production levels of the species. the highest pbmax of batrachospermum was 0.683 µg c µg–1 fw h–1 in contrast to bangia, which exhibited a photosynthetic production higher by an order of magnitude (pbmax = 8.171 µg c µg–1 fw h–1). at 35 °c, the highest experimental temperature, both species’ photosynthetic activity dropped. the pbmax of bangia decreased at 35 °c but it was still remarkable. the drop in the photosynthetic activity of batrachospermum was about 95%, and gross photosynthetic activity at 35 °c was detected only at five light intensities. ps values of bangia were calculated only at 5 and 10 °c because at higher temperatures photoinhibition was not observed, and the obtained data were similar to pbmax. in the ps values of batrachospermum, differences were found: contrary to pbmax, ps values increased with temperature and reached the highest value at 30 °c (1.195 µg c µg–1 fw h–1). a decrease was observed only at the highest temperature (35 °c) but this drop was remarkable. photoadaptation parameters (ik) of bangia varied between 61.6 and 275.1 µmol m–2 s–1. they increased with the increasing temperature till 25 °c. at higher temperatures a slow decrease was observed in the ik values. ik values of batrachospermum were lower and ranged from 32 to 165.8 µmol m–2 s–1. the highest value was found at 30 °c. light utilization (α) of bangia decreased with increasing temperature and changed between 1.39 × 10–2 and 3.26 × 10–5 µg c µg–1 fw h–1 (µmol m–2 s–1)–1, which means that the efficiency of light utilization of the species decreased with increasing temperature. α parameters of batrachospermum also decreased along the temperature gradient: it varied from 6.8 × 10–3 to 9 × 10–4 µg c µg–1 fw h–1 (µmol m–2 s–1)–1. photoinhibition (β) was not observed at any of the applied temperatures. bangia showed photoinhibition at a lower part of the temperature range (5–10 °c): β was 6 × 10–4 and 1.5 × 10–3 µg c µg–1 fw h–1 (µmol m–2 s–1)–1. in contrast, batrachospermum had β values at almost each measuring activity of the species were followed by measuring dissolved oxygen concentration (do) with an intellical™ ldo101 sensor (hach lange). after the pre-incubation period, do was measured at the beginning, then after 1 h and 2 h. the special design of the karlsruhe-flasks prevents gas exchange with the environment. in the case of both species, measurements were conducted in three replicates at each of the nine light intensities. respiration, grossand net photosynthesis were determined according to wetzel and likens (2000). two equations were used to determine the following photosynthetic parameters of the species: biomass specific maximal photosynthetic rate (pbmax), maximal production obtained in the absence of photoinhibition (ps; without photoinhibition it is equal to pbmax), photoadaptation parameter (ik), compensation light intensity (ic), initial slope of the curve (α), biomass specific respiration (rb). in the absence of photoinhibition, photosynthetic parameters were calculated according to webb et al. (1974). if photoinhibition was observed, photoinhibition parameter (β) was also calculated according to platt et al. (1980). curves were fitted using grafit software (leatherbarrow 2009). to determine the compensation light intensity (ic), a reordered form of the equation of webb et al. (1974) was used in each case: i p r p c s b s� � � � � � ���� � � ln 1 � where ic is the light intensity at which photosynthetic production becomes equal to respiration. results photosynthesis-light intensity characteristics the biomass specific maximal production (pbmax) of the species increased in parallel with the temperature (figs. 1, 2). fig. 1. gross photosynthesis–irradiance (p–i) curves of bangia atropurpurea measured along a temperature gradient. data are average mean ± sd, n= 3. fig. 2. gross photosynthesis–irradiance (p–i) curves of batrachospermum gelatinosum measured along a temperature gradient. data are average mean ± sd, n= 3. pálmai t., szabó b., hubai k. e., padisák j. 138 acta bot. croat. 77 (2), 2018 temperature including the higher temperature range (20–35 °c). they varied between 4.6 × 10–5 and 1.1 × 10–3 µg c µg–1 fw h–1 (µmol m–2 s-1)–1. respiration of both species increased with temperature. this increase was higher than 90% for both species. although bangia had a higher biomass specific respiration, these values for the two species were much more similar than their biomass specific productions. photosynthesis-temperature characteristics plotting the mean values of photosynthetic activity as a function of the temperature (p–t), it is possible to examine the temperature dependence of the photosynthesis at each light intensity. in the 75–1000 µmol m–2 s–1 light intensity range biomass specific productions of bangia atropurpurea increased with the temperature and reached a maximum at 25 °c (fig. 3.). besides, at the highest temperatures (30 and 35 °c) a decrease was found. the highest level of photosynthesis was observed at 600 µmol m–2 s–1. at lower light intensities (35 and 15 µmol m–2 s–1) outliers were found at 10 °c. at light intensities between 1150 and 130 µmol m–2 s–1, the biomass specific maximal production of batrachospermum showed normal distribution and had maxima at 20 and 25 °c (fig. 4.). at 75 µmol m–2 s–1, pbmax of the species reached the highest value at 15 °c and after a slight decrease a remarkable drop was found at 35 °c. at low light intensities (30–10 µmol m–2 s–1), pbmax maxima were detected at the lowest temperature (5 °c) and a slight decrease was observed. under low light conditions at the highest measuring temperature (35 °c), grossand net photosynthetic activity were not observed. discussion physiological optima of different red algal species are thoroughly studied worldwide. temperature and light intensity optima of the different species were determined in several studies (on-line suppl. tab. 1). adaptation to low light intensity and temperature has been reported both for batrachospermum gelatinosum and for bangia atropurpurea (sommerfeld and nichols 1973, geesink 1973, necchi and zucchi 2001, necchi and alves 2005). since previous studies applied many different and inconvertible units, direct comparisons are difficult. however, it is possible to compare trends. in contrast to many other red algae, bangia atropurpurea is a well-studied species. low temperature and light intensity are considered the optimal conditions for the species. because rhodophyta species are commonly found at low temfig. 3. gross photosynthesis–temperature (p–t) curves of bangia atropurpurea measured at high (a) and low (b) incubation light intensities. data are average mean ± sd, n= 3. fig. 4. gross photosynthesis–temperature (p–t) curves of batrachospermum gelatinosum measured at high (a) and low (b) incubation light intensities. data are average mean ± sd, n= 3. photosynthesis of two rhodophyta species acta bot. croat. 77 (2), 2018 139 peratures and light intensities, most previous experiments were limited to low temperature and light intensity ranges (on-line suppl. tab. 1). in most cases, these values varied between 9–20 °c and 4–200 µmol m–2 s–1 (belcher 1960, geesink 1973, sommerfeld and nichols 1973, sheath and cole 1980, charnofsky et al. 1982). graham and graham (1987) carried out laboratory experiments on growth and reproduction of bangia in a wide range of temperatures and light intensities. their finding that bangia has a temperature optimum at 20 °c and a light intensity optimum at 750 µmol m–2 s–1 differs from previous findings as well as from our observations. the extremely high light optimum should be a result of the different data analysis. our results regarding temperature preferences are similar to those of graham and graham (1987): we found a temperature optimum for photosynthetic production at 25 °c. this temperature is much higher than the usual cultivating temperatures and other findings reported previously. as to light preferences, we found a maximum of 275 µmol m–2 s–1 in ik values at 25 °c. light utilization of the species decreased with the increasing temperature, which suggested an acclimation to low light intensity and temperature in agreement with the previous findings (sheath 1984). photoinhibition was found only at low temperatures (5–10 °c) in contrast to graham and graham (1987). even though they did not do the calculations, their results suggest that bangia sp. had photoinhibition at elevated temperatures (20–30 °c). batrachospermum gelatinosum, like red algae in general, occurs in cold, clean running waters (7–14 °c), kremer (1983), vis et al. (1996), vis and sheath (1997), drerup and vis (2014) and we also found it under such conditions (tab. 1). several experiments were carried out on the photosynthesis of different batrachospermum species. in accordance with our results, kremer (1983) found a temperature optimum at 20–25 °c for the photosynthetic production of batrachospermum sp. when a short temperature adaptation time was used before measurement; however, kremer (1983) found a lower temperature optimum (15 °c) if the adaptation time was longer, and suggested that the use of a longer adaptation time is needed. necchi and zucchi (2001), zucchi and necchi (2001), necchi and alves (2005) and drerup et al. (2015) investigated rhodophyta species, including batrachospermum species (on-line suppl. tab. 1). zucchi and necchi (2001) found growth optimum for batrachospermum species at 20 °c along with a preference for low light intensity (65 µmol m–2 s–1). necchi and zucchi (2001), necchi and alves (2005) and drerup et al. (2015) conducted photosynthesis measurements at 20 °c and found differences between batrachospermum species light optima for photosynthetic production. ik values varied between 6 and 76 µmol m–2 s–1 which is lower than our findings at this temperature (122 µmol m–2 s–1). as to compensation light intensity, ic values varied between 2 and 16 µmol m–2 s–1 , which was similar to data by necchi and zucchi (2001), necchi and alves (2005) and drerup et al. (2015). confirming previous publications, we also found a high level of light utilization and photoinhibition at 20 °c (necchi and zucchi 2001, necchi and alves 2005, drerup et al. 2015). in comparison to ecophysiological data of species other than rhodophyta, it is apparent that most species have photosynthesis maxima at higher temperature (e.g. üveges et al. 2012, pálmai et al. 2013, lengyel et al. 2015, pálmai et al. 2016) than these two rhodophyta populations. similar ik values were found for microcystis aeruginosa, merismopedia tenuissima and oscillatoria sp. (coles and jones 2000), porphyra species (lin et al. 2008), aphanizomenon flos-aquae (üveges et al. 2012), nitzschia frustulum (lengyel et al. 2015), but there are also species with lower (picocystis salinarum (pálmai et al. 2013), and with higher photoadaptation parameters, like arthrospira fusiformis (pálmai et al. 2013), microcystis flos-aquae (pálmai et al. 2016). similarly, efficient light utilization was reported for rhodophyta species (necchi and zucchi 2001, necchi and alves 2005, drerup et al. 2015) but also good utilization was determined for aphanizomenon flos-aquae (üveges et al. 2012) and for picocystis salinarum (pálmai et al. 2013). acknowledgement this research was supported by the hungarian national research, development and innovation office (nkfih k-120595). references araki, t., hayakawa, m., tamaru, y., yoshimatsu, k., morishita, t., 1994: isolation and regeneration of haploid protoplasts from bangia atropurpurea (rhodophyta) with marine bacterial enzymes. journal of phycology 30, 1040–1046. barinova, s., 2013: diversity, ecology and survivor of freshwater red algae in israel. natural resources and conservation 1, 21–29. belcher, j. h., 1960: culture studies of bangia atropurpurea (roth) ag. new phytologist 59, 367–373. carmona, j., sánchez-díaz, e., perona, e., loza, v., 2011: morphological and ecological characterization of batrachospermales (rhodophyta) in the jarama basin, iberian peninsula. limnetica 30, 117–128. charnofsky, k., towill, l. r., sommerfeld, m. r., 1982: light requirements for monospore germination in bangia atropurpurea (rhodophyta). journal of phycology 18, 417–422. chiasson, w. b., vis, m. l., 2014: new collections of freshwater red algae (batrachospermales, rhodophyta) from historically important areas in france. cryptogamie algologie 35, 303–316. coles, j. f., jones, r. c., 2000: effect of temperature on photosynthesis-light response and growth of four phytoplankton species isolated from a tidal freshwater river. journal of phycology 36, 7–16. drerup, s. a., vis, m. l., 2014: varied phenologies of batrachospermum gelatinosum gametophytes (batrachospermales, rhodophyta) in two low-order streams. fottea 14, 121–127. drerup, s. a., gonzalez, d. a., vis, m. l., 2015: photosynthetic characteristics of some common temperate freshwater red algal taxa (rhodophyta). phycologia 54, 609–616. pálmai t., szabó b., hubai k. e., padisák j. 140 acta bot. croat. 77 (2), 2018 garwood, p. e., 1982: ecological interactions among bangia, cladophora, and ulothrix along the lake erie shoreline. journal of great lakes research 8, 54–60. geesink, r., 1973: experimental investigations on marine and freshwater bangia (rhodophyta) from the netherlands. journal of experimental marine biology and ecology 11, 239–247. graham, j. m., graham, l. e., 1987: growth and reproduction of bangia atropurpurea (roth) c. ag. 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phycological research 49, 103–114. acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 123 acta bot. croat. 74 (1), 123–142, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 physiological responses of peanut (arachis hypogaea l.) cultivars to water defi cit stress: status of oxidative stress and antioxidant enzyme activities koushik chakraborty*, amrit l. singh, kuldeep a. kalariya, nisha goswami, pratap v. zala directorate of groundnut research (icar), junagadh – 362001, gujarat, india abstract – from a fi eld experiment, the changes in oxidative stress and antioxidant enzyme activities were studied in six spanish peanut cultivars subjected to 25−30 days of water defi cit stress at two different stages: pegging and pod development stages. imposition of water defi cit stress signifi cantly reduced relative water content, membrane stability and total carotenoid content in all the cultivars, whereas total chlorophyll content increased at pegging stage but decreased at pod developmental stage. chlorophyll a/b ratio increased under water defi cit stress in most of the cultivars suggesting a greater damage to chlorophyll b rather than an increase in chlorophyll a content. oxidative stress measured in terms of h2o2, superoxide radical content and lipid peroxidation increased under water defi cit stress, especially in susceptible cultivars such as drg 1, ak 159 and icgv 86031. relationship among different physiological parameters showed that the level of oxidative stress, in terms of production of reactive oxygen species, was negatively correlated with activities of different antioxidant enzymes such as superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and glutathione reductase. in conclusion, the study shows that water defi cit stress at pod development stage proved to be more detrimental than at pegging stage. the higher activities of antioxidant enzymes in the tolerant cultivars like icgs 44 and tag 24 were responsible for protection of oxidative damage and thus provide better tolerance to water defi cit stress. keywords: antioxidant enzymes, arachis hypogaea l., lipid peroxidation, oxidative stress, peanut, reactive oxygen species abbreviations: apx – ascorbate peroxidase, cat – catalase, das – days after sowing, fw – fresh weight, gr – glutathione reductase, gssg – oxidized glutathione, msi – membrane stability index, pod – peroxidase, ros – reactive oxygen species, rwc – relative water content, sod – superoxide dismutase, sor – superoxide radical, tbars – thiobarbituric acid reactive substances * corresponding author, e-mail: koushik_iari@rediffmail.com copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 124 acta bot. croat. 74 (1), 2015 introduction water defi cit stress is one of the major environmental constraints limiting agricultural productivity and plays a major role in the distribution of plant species across different types of environments (ashraf 2010). two-thirds of the potential yields of major crops are usually lost due to adverse growing environments (chaves et al. 2009). drought or water defi cit condition can be defi ned as the absence of adequate moisture necessary for normal plants to grow and complete their life cycle (zhu 2002). the lack of adequate moisture leading to water stress is the most common phenomenon in rain-fed areas, where erratic rainfall distribution and poor irrigation facilities prevails (wang et al. 2005). peanut is an important legume crop grown in tropical and sub-tropical semi-arid regions of the world in rain-fed areas; the yield level is severely affected by shortage of soil moisture. in india, rainfall accounts for over 50% of variance in yield (challinor et al. 2003) and the average peanut yield in our country is very low because of moisture stress faced at various growth stages, irrespective of the other factors of the crop production package (singh et al. 2013). plants differ in their responses to water defi cit stress. those that are tolerant try to overcome the stress by modifying morpho-physiological and biochemical characters, while susceptible plants develop symptoms of stress (dhruve et al. 2009). under water defi cit stress the production of reactive oxygen species is invariably increased, often leading to damage of the cellular and sub-cellular components in plants (shao et al. 2008). oxidative stress occurs when the defensive capacity of plants is broken by the formation of free radicals. further down, in the fenton/haber-weiss pathway, toxic hydroxyl radical (•oh) is produced inside the plants, which ultimately destabilizes the membrane lipids via lipid peroxidation leading to membrane injury (mittler 2002). under limited water supply, the photosynthetic process in the plant slows down considerably and the constant accumulation of photo-reducing power causes an excess of electrochemical energy in membranes. this extra energy is canalized through the mehler reaction, which generates reactive oxygen species (ros) mainly superoxide anion (o2•–) and hydrogen peroxide (h2o2) (hernandez et al. 2001), ultimately provoking the oxidative stress syndrome (kholova et al. 2009). these ros are cytotoxic and highly detrimental to the cellular lipids, nucleic acids and proteins and the genotypes that are able to maintain a low steady state of the ros are better adapted to tolerate stress conditions (bajji et al. 2001). damage to cellular membranes and chlorophylls are reliable indicators for determination of the extent of damage to the plants due to oxidative stress (sairam et al. 2000). plants generally scavenge and dispose of these reactive substances by the use of antioxidant defence enzymes like superoxide dismutase (sod), catalase (cat), peroxidase (pod), ascorbate peroxidase (apx), glutathione reductase (gr) and other associated enzymes involved in the cellular detoxifi cation mechanism. the sod constitutes the fi rst line of defence via detoxifi cation of superoxide radicals to hydrogen peroxide; cat and pod degrade hydrogen peroxide (santos and almeida 2011). cellular accumulation of h2o2 is restricted by its reduction to the non-toxic h2o molecule, either through the action of catalase or ascorbate-glutathione cycle. finally, nadph-dependent reduction of oxidized glutathione (gssg) is performed by glutathione reductase to replenish the cellular pool of reduced glutathione (gsh) (noctor et al. 2002). physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 125 the protective roles of the antioxidant enzymes during drought stress have been reported in a number of crop plants including wheat (agarwal et al. 2005, sairam and saxena 2005) and rice (sharma and dubey 2005), and also in peanut under simulated water defi cit stress condition using various concentrations poly-ethylene glycol (celikkol-akcay et al. 2010). however, studies related to stage-specifi c response of peanut crop due to water defi cit stress under fi eld conditions are lacking. hence, the objectives of this study were to quantify the levels of oxidative stress faced by peanut cultivars under water defi cit stress at two different growth stages and to ascertain the role of antioxidant enzymes in imparting tolerance to drought. materials and methods plant material and growth condition a fi eld experiment was conducted during the summer season of 2011 (february–june) using six spanish (bunch type) peanut cultivars namely sg 99, icgs 44, icgv 86031, tag 24, ak 159 and drg 1 with a maturity period of 110–120 days, at the research farm of the directorate of peanut research, junagadh, gujarat (21º31’n, 70º36’e), india on a site of black clay soil (ph 7.8). preliminary trials (data not shown here) showed that out of these cultivars, icgs 44 and tag 24 are tolerant, while sg 99 behaves moderately and rest of the cultivars are susceptible to water defi cit stress. the experiment was laid out in split plot design with six cultivars and water defi cit stress treatment and corresponding controls. the net plot size was 4 × 3 m with 9 rows per plot at 45 cm row to row and 10 cm plant to plant spacing. for the present study, water defi cit conditions were created by withholding irrigation at two different stages in two different plots, which were compared with the control (nonstressed) plot in each case. in the present study, the fi rst sample was drawn from 60-day old plants which were subjected to water defi cit stress by withholding irrigation for the previous 30 days (30−60 das), corresponding to the r1−r5 stage in peanut (boote 1982) denoted as the ‘pegging stage’ (beginning of blooming to the beginning of seed formation). similarly, the samples were collected from another set of 85-day old plants, subjected to water defi cit stress for the previous 25 days (60−85 das), corresponding to the r5−r7 stage in peanut (boote 1982) and denoted as the ‘pod development stage’ (beginning of seed formation to beginning of maturity). however, the control plot was irrigated to replenish 100% cumulative pan evaporation at weekly intervals. the stages selected for the present study were considered to be most critical in terms of water requirement in peanut (nautiyal et al. 2012). to characterize the level of imposed stress, soil moisture content was measured from each plot at every stage by the gravimetric method [(fresh weight-dry weight)/dry weight × 100] (black 1965). recommended plant protection measures were followed to maintain a healthy crop stand. sampling for different physiological parameters, oxidative stress and antioxidant enzyme activities was done at 24–48 h before the withdrawal of stress at both the stages, from the third fully-matured leaf in control and stressed plants by randomly collecting samples in triplicate from 3 replicated plots. analysis of physiological parameters specifi c leaf area was calculated by dividing total leaf area by total leaf weight in at least 5 plant samples (nageswara rao et al. 2001). leaf relative water content (rwc) was chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 126 acta bot. croat. 74 (1), 2015 estimated by recording the fresh and turgid weight, and dry weight (weatherley 1950). membrane stability index (msi) was estimated by measuring the electrical conductivity of leaf samples (100 mg) in 10 ml double distilled water by heating at 40 °c for 30 min and 100 °c for 10 min according to sairam et al. (1997). for total chlorophyll and carotenoid estimation 50 mg leaf material was extracted in 10 ml dimethylsulfoxide (hiscox and israelstam 1979), incubated at 65 °c for 4 h, and then cooled to room temperature. absorbance of the extract was recorded at 470, 645 and 663 nm. chlorophyll and carotenoid content was calculated according to arnon (1949), and lichtenthaler and wellburn (1985). determination of oxidative stress superoxide radical (sor) content was estimated by its capacity to reduce nitroblue tetrazolium chloride (nbt) and the absorption of end product was measured at 540 nm (chaitanya and naithani 1994). briefl y, one gram leaf tissue was homogenized in 10 ml of pre-cooled phosphate buffer (0.2 m, ph 7.2) containing 1 mm diethyldithiocarbamate to inhibit sod activity and 10 μm diphenylene iodonium chloride. the homogenate was centrifuged at 10,000 × g for 10 min at 4 °c and supernatant was immediately used for the measurement of superoxide radical. the reaction mixture contained 0.25 ml supernatant, 0.075 mm nbt, 25 mm na2co3, 0.1 mm ethylenediaminetetraacetic acid (edta), 13.33 mm l-methionine and water to make the volume of 3 ml. the reaction mixture was incubated at 30 °c for 10 min and absorbance was recorded at 540 nm. superoxide radical content was calculated according to its extinction coeffi cient ε = 12.8 mm–1 cm–1. hydrogen peroxide (h2o2) was measured through formation of the titanium-hydro peroxide complex (rao et al. 1997). one gram leaf material was ground with liquid nitrogen and the fi ne powdered material was mixed with 10 ml cooled acetone in a cold room (10 °c). mixture was fi ltered through whatman no.1 fi lter paper followed by the addition of 4 ml titanium reagent and 5 ml ammonium solution to precipitate the titanium-hydro peroxide complex. reaction mixture was centrifuged at 10,000 × g for 10 min at 4 °c. precipitate was dissolved in 10 ml of 2 m h2so4 and then recentrifuged. absorbance of the supernatant was taken at 415 nm against blank. hydrogen peroxide contents were calculated by comparison with a standard curve drawn with known hydrogen peroxide concentrations. the level of lipid peroxidation was measured in terms of thiobarbituric acid-reactive substances (tbars) content (heath and packer 1968). leaf sample (0.5 g) was homogenized in 10 ml of 0.1% (w/v) trichloroacetic acid (tca). the homogenate was centrifuged at 15,000 × g for 15 min. to a 1.0 ml aliquot of the supernatant, 4.0 ml of 0.5 % (w/v) thiobarbituric acid (tba) in 20% tca was added and the mixture was heated at 95 °c for 30 min and then cooled in an ice bath. after centrifugation at 10,000 × g for 10 min the absorbance of the supernatant was recorded at 532 nm. the tbars content was calculated according to its extinction coeffi cient ε = 155 mm–1 cm–1. the values for non-specifi c absorbance at 600 nm were subtracted. antioxidant enzyme assays enzyme extract for sod, apx, gr, pod and cat was prepared by fi rst freezing the weighed amount of leaf samples (1 g) in liquid nitrogen to prevent proteolytic activity followed by grinding with 10 ml extraction buffer (0.1 m phosphate buffer, ph 7.5, containing 0.5 mm edta in case of sod, gr, pod, cat and 1 mm ascorbic acid in the case of physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 127 apx). extract was passed through 4 layers of cheesecloth and fi ltrate was centrifuged for 20 min at 15,000 × g and the supernatant was used as enzyme. the total sod (ec 1.15.1.1) activity was measured by the inhibition of the photochemical reduction of nbt by the enzyme (dhindsa et al. 1981). the 3 ml reaction mixture contained 13.33 mm methionine, 75 μm nbt, 0.1 mm edta, 50 mm phosphate buffer (ph 7.8), 50 mm sodium carbonate, 0.1 ml enzyme extract and water to make a fi nal volume of 3.0 ml. the reaction was started by adding 2 mm ribofl avin (0.1 ml) and placing the tubes under two 15 w fl uorescent lamps for 15 min. illuminated and non-illuminated reaction mixture without enzyme were used for calibration. the absorbance was recorded at 560 nm, and one unit of enzyme activity was taken as that amount of enzyme that reduced the absorbance reading to 50% in comparison with tubes lacking enzyme per unit time. the catalase (ec 1.11.1.6) activity was assayed by measuring the disappearance of h2o2 (aebi 1984) in a reaction mixture (3 ml) consisting of 0.5 ml of 75 mm h2o2 and 1.5 ml of 0.1 m phosphate buffer (ph 7) on the addition of 50 μl of diluted enzyme extract. the decrease in absorbance at 240 nm was observed for 1 min in a uvvisible spectrophotometer. enzyme activity was computed by calculating the amount of h2o2 decomposed. the initial and fi nal contents of h2o2 were calculated by comparison with a standard curve drawn with known concentrations of h2o2. the peroxidase (ec 1.11.1.7) activity was measured in terms of increase in absorbance due to the formation of tetraguaiacol at 470 nm, and the enzyme activity was calculated as per extinction coeffi cient of its oxidation product, tetraguaiacol ε = 26.6 mm–1 cm–1 (castillo et al. 1984). the reaction mixture contained 50 mm phosphate buffer (ph 6.1), 16 mm guaiacol, 2 mm h2o2 and 0.1 ml enzyme extract. the mixture was diluted with distilled water to make up fi nal volume of 3.0 ml. enzyme activity is expressed as μmol tetraguaiacol formed per min per mg protein. the ascorbate peroxidase (ec 1.11.1.11) activity was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm (nakano and asada 1981). the 3 ml reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0), 0.5 mm ascorbic acid, 0.1 mm edta, 0.1 mm h2o2, 0.1 ml enzyme and water to make a fi nal volume of 3.0 ml in which 0.1 ml of h2o2 was added to initiate the reaction. decrease in absorbance was measured spectrophotometrically and the activity was expressed by calculating the decrease in ascorbic acid content using a standard curve drawn with known concentrations of ascorbic acid and expressed as μmol oxidised ascorbate mg–1protein min–1. the glutathione reductase (ec 1.8.1.7) activity was assayed using the method of smith et al. (1988). the reaction mixture containing 66.67 mm potassium phosphate buffer (ph 7.5) and 0.33 mm edta, 0.5 mm 5,5-dithiobis-(2-nitro) benzoic acid in 0.01 m potassium phosphate buffer (ph 7.5), 66.67 μm nadph, 666.67 μm oxidized glutathione (gssg) and 0.1 ml enzyme extract. the mixture was diluted with distilled water to make up a fi nal volume of 3.0 ml. the reaction was started by adding 0.1 ml of 20.0 mm gssg. the increase in absorbance at 412 nm was recorded spectrophotometrically and the activity was expressed as micromole of gssg reduced per mg protein per min. total soluble protein was determined according to the method of bradford (1976), with bovine serum albumin as a calibration standard. chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 128 acta bot. croat. 74 (1), 2015 statistical analyses all the data recorded were the mean values of at least three independent assays with three replications each. the data was subjected to analysis of variance appropriate to the experimental design. the least signifi cant differences at 5% probability level (lsdp=0.05) were considered statistically signifi cant (gomez and gomez 1984). correlations (pearson correlation coeffi cient) among different physiological parameters (rwc, msi, total chlorophyll and carotenoid content), oxidative stress build-up (h2o2 and sor production, and lipid peroxidation) and antioxidant enzyme (sod, cat, pod, apx, gr) activities under water defi cit stress condition were analyzed. all the 14 physiological parameters analysed in the present study was further categorized by multivariate statistics i.e. principal component analysis (pca) by spss program (version 16.0). results intensity of water defi cit stress and soil moisture status water defi cit stress imposed by the withholding irrigation for 30 days at pegging stage, reduced soil moisture content by 45% at 0−15 cm soil depth and 44% at 15−30 cm soil depth compared to irrigated control plot where optimum moisture level was maintained throughout the crop growth period. similarly, at pod development stage the extent of stress was more severe as it recorded 69 and 50% decrease in soil moisture content at 0−15 and 15−30 cm soil depth, respectively (fig. 1). physiological parameter the relative water content (rwc) of leaves decreased in all the peanut cultivars with the imposition of water defi cit stress during pegging as well as pod development stage (tab. 1). fig. 1. changes in soil moisture content at different depths in response to water defi cit stress at pegging and pod development stages. values are mean of three replicates ± se. lsd0.05 values for pegging and pod development stages are 0.86 and 1.14, respectively, where lsd0.05 value represents least signifi cant difference value at 5% probability level. physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 129 ta b. 1 . c ha ng es in re la tiv e w at er c on te nt (r w c ), m em br an e st ab ili ty in de x (m si ) a nd s pe ci fi c le af a re a (s l a ) o f p ea nu t l ea ve s at d iff er en t d ev el op m en ta l st ag es in re sp on se to w at er d efi c it st re ss . v al ue s ar e m ea n of th re e re pl ic at es ± s e . t – tr ea tm en t; c – c ul tiv ar ; c × t – c ul tiv ar × tr ea tm en t i nt er ac tio n, n s – no nsi gn ifi ca nt . l sd (0 .0 5) – le as t s ig ni fi c an t d iff er en ce s at 5 % p ro ba bi lit y le ve l. r w c (% ) m si (% ) sl a (c m 2 ) c ul tiv ar pe gg in g st ag e po d de ve lo pm en t st ag e pe gg in g st ag e po d de ve lo pm en t st ag e pe gg in g st ag e po d de ve lo pm en t st ag e c on tr ol st re ss c on tr ol st re ss c on tr ol st re ss c on tr ol st re ss c on tr ol st re ss c on tr ol st re ss sg 9 9 94 .2 ± 2. 6 91 .7 ± 0. 3 90 .2 ± 2. 7 87 .2 ± 0. 2 89 .4 ± 0. 6 80 .7 ± 2. 3 71 .4 ± 2. 8 62 .2 ± 1. 6 14 6. 0± 1. 7 12 5. 9± 2. 8 15 8. 8± 2. 8 13 9. 2± 1. 6 ic g s 44 90 .9 ± 3. 1 90 .3 ± 3. 3 91 .9 ± 1. 8 86 .8 ± 1. 9 91 .2 ± 1. 2 83 .8 ± 0. 8 68 .3 ± 0. 8 61 .9 ± 0. 7 14 8. 3± 2. 1 13 2. 6± 2. 6 15 3. 2± 0. 8 13 5. 8± 0. 7 ic g v 8 60 31 91 .7 ± 1. 9 85 .7 ± 3. 2 91 .2 ± 1. 1 83 .5 ± 1. 8 83 .8 ± 1. 8 73 .3 ± 2. 5 73 .3 ± 3. 7 63 .2 ± 0. 9 12 9. 1± 1. 8 10 9. 7± 2. 4 14 4. 4± 3. 7 11 8. 7± 0. 9 ta g 2 4 91 .2 ± 2. 2 88 .1 ± 1. 5 88 .7 ± 1. 3 86 .1 ± 0. 8 85 .8 ± 2. 1 82 .4 ± 0. 5 73 .4 ± 2. 1 65 .3 ± 0. 9 14 9. 7± 3. 4 13 5. 3± 3. 4 16 0. 5± 2. 1 14 0. 5± 0. 9 a k 1 59 91 .3 ± 2. 6 87 .7 ± 2. 1 91 .6 ± 1. 5 83 .9 ± 2. 9 84 .5 ± 1. 4 81 .0 ± 0. 6 66 .7 ± 1. 8 54 .3 ± 1. 5 14 1. 4± 2. 9 12 4. 7± 3. 2 15 3. 0± 1. 7 13 3. 6± 1. 5 d r g 1 91 .8 ± 1. 6 86 .4 ± 2. 2 92 .1 ± 0. 9 82 .8 ± 1. 2 82 .0 ± 0. 3 73 .9 ± 2. 0 67 .2 ± 2. 0 52 .3 ± 2. 2 14 4. 0± 6. 8 12 1. 6± 2. 7 15 8. 1± 2. 1 13 4. 3± 2. 2 l sd 0. 05 t 1. 74 2. 56 n s 3. 24 1. 88 8. 58 l sd 0. 05 c 2. 39 2. 71 4. 28 4. 31 3. 43 9. 93 l sd 0. 05 c × t n s 3. 82 6. 05 6. 09 10 .1 1 14 .0 4 chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 130 acta bot. croat. 74 (1), 2015 though the difference in rwc within the cultivars at pegging stage was not signifi cant, at pod development stage the differences were signifi cant and the cultivar tag 24 showed the least reduction in rwc compared to non-stressed plants. the average reduction in leaf rwc at pod developmental stage was 6.5%, which resulted in reduced growth and partial wilting of leaves in stressed plants (data not shown). similarly, membrane stability index (msi) was also reduced due to imposition of water defi cit stress and the interaction effect of stress and peanut cultivars was found to be signifi cant for all cultivars at both stages (tab. 1). the cultivar icgv 86031 and drg 1 showed highest reduction in msi at pegging and pod development stages, respectively. imposition of water defi cit stress signifi cantly decreased specifi c leaf area (sla) at both the stages (tab. 1). the reduction in sla was higher (> 15%) in icgv 86031 and dgr 1 than in the rest of the cultivars at both pegging and pod development stages, whereas it was only 11% in icgs 44. interestingly, the total chlorophyll content (mg g–1 fresh weight) in peanut leaves increased when water defi cit stress was imposed at pegging stage. it was reduced in all the cultivars when stress was imposed at pod development stage. the highest reduction in total chlorophyll content under stress at pod development stage was observed in drg 1 (14.4%), while the cultivar icgs 44 showed the least reduction (8.9%) (fig. 2a). however, the total carotenoid content reduced in all the cultivars under water defi cit stress both at pegging and pod development stages with higher reduction at later stage. the cultivar sg 99 showed the highest reduction in total carotenoid content at both the stages followed by icgv 86031 and ak 159 at pegging and pod development stages, respectively (fig. 2b). chlorophyll a/b ratio increased under water defi cit stress in most of the cultivars except in icgs 44 at pegging stage and tag 24 and ak 159 at pod development stage (fig. 2c). oxidative stress water defi cit stress resulted in increased levels of oxidative stress in all the peanut cultivars. the h2o2 content increased signifi cantly at both pegging and pod development stages, but, increased progressively at a later stage (86.5%). the cultivar icgs 44 showed the least increase in h2o2 content from 0.99 (in the control) to 1.40 μmol g–1 fw (in stressed plants) at pegging stage and from 1.14 (in the control) to 1.72 μmol g–1 fw (in stressed plants) at pod development stage, followed by tag 24 and sg 99 (fig. 3a). superoxide radical (sor) content also increased signifi cantly under water defi cit stress with more pronounced effect in cultivars drg 1 and icgv 86031, at both, pegging and pod development stages. on the other hand, tag 24 (19.7%), icgs 44 (19.9%) and ak 159 (24.5%) showed a smaller increase in sor content at pod development stage (fig. 3b). similarly, lipid peroxidation, measured in terms of tbars content, increased signifi cantly under water defi cit stress in all the cultivars, with higher increase at pod development stage (39%) than at pegging stage (28%) (fig. 3c). the least increase in lipid peroxidation was observed in tag 24 (15.6%) followed by icgs 44 (17.7%), compared to non-stressed plants, while the highest tbars content (18.9 nmol mg–1 fw) was found in drg 1 at pod development stage. antioxidant enzyme activities water defi cit stress, especially at later stages of growth signifi cantly increased the activities of all the antioxidant enzymes. under water defi cit conditions, the highest increase physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 131 fig. 2. changes in (a) total chlorophyll content (mg g–1 fresh weight); (b) total carotenoid content (mg g–1 fresh weight); (c) chlorophyll a/b ratio in leaves of different cultivars of peanuts in response to water defi cit stress at pegging and pod developmental stages. values are mean of three replicates ± se. lsd0.05 (c × t) values for pegging and pod development stages are 0.23 and 0.32 for (a), 0.22 and 0.16 for (b), and 0.52 and 0.43 for (c), respectively, where lsd0.05 (c × t) value represent least signifi cant difference value of cultivar and treatment interaction at 5% probability level. chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 132 acta bot. croat. 74 (1), 2015 fig. 3. changes in (a) h2o2 content (μmol g–1 fresh weight); (b) superoxide radical content (δa540 g–1 fresh weight); (c) lipid peroxidation (nmol of tbars content mg–1 fresh weight) in leaves of peanut cultivars in response to water defi cit stress at pegging and pod developmental stages. values are mean of three replicates ± se. lsd0.05 (c × t) values for pegging and pod developmental stages are 0.13 and 0.20 for (a), 1.80 and 0.91 for (b), and non-signifi cant and 1.42 for (c), respectively, where lsd0.05 (c × t) value represent least signifi cant difference value of cultivar and treatment interaction at 5% probability level. physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 133 fig. 4. changes in (a) sod activity (unit mg–1protein min–1); (b) cat activity (μmolh2o2 reduced mg –1 protein min–1); (c) pod activity (μmoltetra-guaiacol formed mg–1protein min–1) in leaves of peanut cultivars in response to water defi cit stress at pegging and pod developmental stages. values are mean of three replicates ± se. lsd0.05 (c × t) values for pegging and pod developmental stages are 0.11 and 0.20 for (a), 0.09 and 0.11 for (b), and non-signifi cant and 0.97 for (c), respectively, where lsd0.05 (c × t) value represent least signifi cant difference value of cultivar and treatment interaction at 5% probability level. chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 134 acta bot. croat. 74 (1), 2015 in total sod activity was observed in icgs 44 (1.12 unit mg–1protein min–1), tag 24 (0.99 unit mg–1protein min–1) and ak 159 (0.74 unit mg–1 protein) in comparison to the respective controls (0.68, 0.72 and 0.61 unit mg–1protein min–1, respectively) (fig. 4a) at pegging stage. however, water defi cit stress during pod development stage increased sod activity in all the cultivars and the highest increase (75%) was observed in icgs 44 and tag 24. the cat activity also followed the similar trend as of sod. imposition of water defi cit stress at pegging stage increased cat activity in relatively tolerant cultivars tag 24 (58.4%) and icgs 44 (47.7%), but reduced in susceptible cultivars icgv 86031 and drg 1 (fig. 4b). at pod development stage, cat activity increased in all the cultivars under water defi cit stress, and in tag 24 it was more than double than in non-stressed plants. the fig. 5. changes in (a) apx activity (μmolascorbate oxidized mg–1protein min–1); (b) gr activity (μmolgssg reduced mg–1protein min–1) in leaves of peanut cultivars in response to water defi cit stress at pegging and pod developmental stages. values are mean of three replicates ± se. lsd0.05 (c × t) values for pegging and pod developmental stages are 2.95 and 1.98 for (a), and 2.71 and 2.82 for (b), respectively, where lsd0.05 (c × t) value represent least signifi cant difference value of cultivar and treatment interaction at 5% probability level. physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 135 pod activity increased with imposition of water defi cit stress at both early and later stages of growth and icgs 44 and tag 24 recorded the highest increase (62%), followed by sg 99 (44%) at pegging stage (fig. 4c). the pod activity was almost doubled in stressed plants of icgs 44 and tag 24 compared to the non-stressed plants at pod development stage. imposition of water defi cit stress at both pegging and pod development stage invariably increased the activity of apx in all the cultivars, but the extent of change varied from cultivar to cultivar. compared to non-stressed plants, the cultivar icgs 44 and tag 24 showed maximum increase (63%) in apx activity at pegging stage, while icgs 44 recorded maximum increase (37%), from 10.7 μmol oxidized ascorbate per min per mg of protein in control to 14.7 μmol oxidized ascorbate per min per mg of protein, at pod development stage (fig. 5a). similarly, the activity of gr increased at both pegging and pod development stages in all the cultivars under water defi cit stress. however, the changes were higher when the stress was imposed at pod development stage than at pegging stage. the cultivar icgs 44 showed higher gr activity than other cultivars with 10.6 and 12.4 μmol gssg reduced per minute per mg of protein at pegging and pod development stages, respectively (fig. 5b). relationship among physiological parameters, oxidative stress and antioxidant enzyme activities under stress signifi cant negative correlations (–0.900, –0.735 and –0.881) were observed between msi and h2o2, sor production and lipid peroxidation, and msi and lipid peroxidation, respectively (tab. 2), suggesting more damage to the membrane structure with rising levels of oxidative stress under water defi cit condition. in general, negative correlations among h2o2, sor production and lipid peroxidation and antioxidant enzyme activities indicated fig. 6. results of principal component (pc) analysis of different physiological parameters under water defi cit stress in peanut cultivars, where rwc – relative water content, msi – membrane stability index, sla – specifi c leaf area, chl – total chlorophyll content, car – total carotenoid content, chlab – chlorophyll a/b ratio, lp – lipid peroxidation, apx – ascorbate peroxidase, cat – catalase, gr – glutathione reductase, pod – peroxidase, sod – superoxide dismutase, sor – superoxide radical. chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 136 acta bot. croat. 74 (1), 2015 ta b. 2 . r el at io ns hi p (p ea rs on c or re la tio n co ef fi c ie nt ) be tw ee n di ff er en t ph ys io lo gi ca l pa ra m et er s, o xi da tiv e st re ss a nd a nt io xi da nt e nz ym e ac tiv iti es i n pe an ut le av es u nd er w at er d efi c it st re ss . a px – a sc or ba te p er ox id as e, c a t – ca ta la se , g r – g lu ta th io ne re du ct as e, m si – m em br an e st ab ili ty in de x, po d – p er ox id as e, r w c – re la tiv e w at er c on te nt , s o d – s up er ox id e di sm ut as e, s o r – s up er ox id e ra di ca l, l p – lip id p er ox id at io n. * a nd * * re pr ese nt le ve l o f s ig ni fi c an ce a t 5 % a nd 1 % p ro ba bi lit y, re sp ec tiv el y. r w c m si c h l c a r h 2o 2 so r l p so d c a t po d a px g r r w c 1. 00 0 m si 0 .8 09 ** 1. 00 0 c h l 0 .6 29 * 0 .9 04 ** 1. 00 0 c a r 0 .6 11 * 0 .8 83 ** 0 .9 27 ** 1. 00 0 h 2o 2 –0 .7 81 ** –0 .9 00 ** –0 .8 56 ** –0 .8 27 ** 1. 00 0 so r –0 .8 10 ** –0 .7 35 ** –0 .5 01 ** –0 .5 80 ** 0 .7 70 ** 1. 00 0 l p –0 .8 60 ** –0 .8 81 ** –0 .7 26 ** –0 .7 49 ** 0 .9 27 ** 0 .9 39 ** 1. 00 0 so d –0 .0 87 0 –0 .4 34 0 –0 .6 05 ** –0 .3 80 0 0. 28 8 –0 .1 54 0 0. 05 0 1. 00 0 c a t –0 .0 56 0 –0 .4 03 0 –0 .5 34 ** –0 .3 75 0 0. 25 8 –0 .1 37 0 0. 05 2 0 .8 91 ** 1. 00 0 po d 0. 33 2 0. 06 2 –0 .1 48 0 0. 11 0 –0 .1 86 0 –0 .5 69 ** –0 .4 18 0 0 .8 09 ** 0 .7 61 ** 1. 00 0 a px 0. 44 6 0. 35 6 0. 15 2 0. 33 1 –0 .4 39 0 –0 .6 33 ** –0 .6 23 ** 0 .6 20 * 0. 48 8 0 .7 80 ** 1. 00 0 g r 0. 31 3 0. 02 8 –0 .1 70 0 –0 .0 67 0 –0 .2 04 0 –0 .5 18 ** –0 .4 07 ** 0 .7 68 ** 0 .8 41 ** 0 .8 22 ** 0 .7 75 ** 1. 00 0 physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 137 the role of antioxidant enzymes in lowering the level of oxidative stress in peanut cultivars under water defi cit condition. three principal components (pcs) of eigenvalue more than 1.0 were identifi ed (fig. 6). among these pc 1 screened most variable parameters as h2o2 content (0.927), sod activity (0.864), and rwc (–0.843) under imposition of water defi cit stress. however, the pc 2 selected chlorophyll content (0.772), apx activity (0.626), and carotenoid content (0.591) as another set of parameters showing more variability as compared to rest of the parameters. the pc 3-screened sensitive parameters towards stress were chlorophyll a/b ratio (–0.747), sla (0.681), and cat activity (0.597) showing maximum variability as detected by pca. discussion water defi cit stress induces several physiological and biochemical changes in plants depending upon the time, intensity and duration of stress (pattangual and madore 1999). general effect of water defi cit stress in plants is manifested in terms of osmotic stress which results in lower leaf water potential, turgor potential and stomatal conductance (boote and ketring 1990). this study demonstrated that water defi cit stress led to differential responses in peanut cultivars when it was imposed at different stages of crop growth. the extent of reduction in rwc, msi and sla due to water defi cit stress at the pegging stage was lower than at pod development stage. dhruve et al. (2009) also reported decrease in leaf water content and membrane stability under water defi cit stress in peanut. interestingly, in the present study, the total chlorophyll content of the leaf increased in most of the cultivars when drought was imposed at the initial stage. but subsequent drought at a later stage reduced it signifi cantly in the all the cultivars. water defi cit stress destroys the chlorophyll and prevents its biosynthesis as well (lessani and mojtahedi 2002), which was reported by researchers in many crops, including peanut (sharada and naik 2011, arjenaki et al. 2012). it was also reported that drought increased chlorophyll content in sesame at the initial stage and that later it remained unchanged (mensah et al. 2006). ability to synthesize more chlorophyll under water defi cit condition at initial stage of growth is a good measure of the ability of peanut genotypes to cope with drought stress during initial stages of growth (arunyanark et al. 2008). here, the tolerant cultivars tag 24 and icgs 44 showed higher increase in chlorophyll content at pegging stage indicating their tolerance to water defi cit stress. water defi cit stress in general reduced the total carotenoid content, but this reduction was relatively less in tolerant cultivars icgs 44 and tag 24. as a part of light-harvesting complex carotenoids act as cellular protector of chlorophyll pigments and membrane structure by quenching triplet chlorophyll and removing oxygen from the excited chlorophylloxygen complex (young 1991). carotenoids also play a protective role as molecular antioxidant in the cell by scavenging of singlet oxygen (knox and dodge 1985) and hence their comparative levels in cultivars are of much importance in determining relative tolerance. decrease in carotenoid content under water stress in the present investigation probably contributed to greater ros production and further destruction of photosynthetic pigments. increased chlorophyll a/b ratio with decreasing total chlorophyll content under water defi cit stress signifi es greater damage to chlorophyll b rather than an increase in chlorophyll a (mafakhri 2010). the present study showed a comparatively lower chlorophyll a/b ratio in tolerant than in susceptible cultivars, which implies more degradation to chlorophyll b in chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 138 acta bot. croat. 74 (1), 2015 the latter than increased synthesis of chlorophyll a per se. increased chlorophyll a/b ratio under water defi cit condition suggests the greater vulnerability of the chlorophyll b molecule under stress. the chlorophyll a/b ratio is an indicator of the antenna sizes of ps i and ps ii. the core antenna contains only chlorophyll a, whereas the outer antenna contains both chlorophyll a and b (taiz and zeiger 2006). the lower chlorophyll a/b ratio in tolerant genotypes probably was obtained due to lesser damage to ps i and ps ii, leading to better capacity to photosynthesize even under stressed condition. increase in chlorophyll a/b ratio due to imposition of drought is also reported in other legumes (mafakheri 2010). water defi cit stress increases production of ros that inactivate enzymes, damage cellular components (shao et al. 2008) and diminish the defense capacity of plants (erice et al. 2010). plants possess antioxidant defense system against ros, operative through the changes in activities of sod, cat, pod and enzymes of ascorbate and glutathione redox cycle (noctor and foyer 1998). in this study, oxidative stress (measured as sor and h2o2 production) varied in the peanut cultivars facing water defi cit stress at various stages; however the extent varied with the cultivars. the net production of h2o2 and sor in the tolerant cultivars tag 24 and icgs 44 was relatively lower than in the susceptible ones due to better balance maintained between ros production and the capacity of the defense mechanisms to protect the plant under severe stress. severe drought depresses the activity of enzymes devoted to detoxify h2o2, which is in conformity with the greatest display of h2o2 concentration and peroxidation to membrane lipids in stressed leaves (fini et al. 2012). sor and h2o2 accumulated due to water defi cit stress resulted in membrane lipid peroxidation and destabilization of the cellular and subcellular membrane via production of toxic hydroxyl radical (mittler 2002). in the present study, the plants faced water stress, showing greater accumulation of sor and higher lipid peroxidation than the plants grown under non-stressed condition. here, the tolerant cultivar tag 24 and icgs 44 accumulated less sor and also showed the least lipid peroxidation. studies revealed positive correlation between leaf dehydration and increase in h2o2 and sor concentration under sequential drought (jubany-marí et al. 2010). the sod constitutes the primary step of cellular defence and dismutates superoxide radical to h2o2 and o2. further, the accumulation of h2o2 is restricted through the action of catalase in mitochondria, by the ascorbate glutathione cycle in chloroplast, where ascorbate peroxidase reduces it to h2o. finally, glutathione reductase catalyzes the nadph-dependent reduction of oxidized glutathione to the reduced glutathione (noctor et al. 2002). in this study, the sod activity increased with imposition of water defi cit stress during pod developmental stage with higher activities recorded in tag 24 and icgs 44 correlating well with relatively lower production of superoxide radical in these cultivars under water defi cit stress. this might be the primary reason of low oxidative stress built-up in these cultivars leading to water defi cit stress tolerance. higher activity of sod, in roots and leaves has been identifi ed as an indicator of a redox status change under drought (schwanz and polle 2001). downstream to sod, cat and pod are the most important enzymes involved in regulation of intracellular levels of h2o2 (prasad et al., 1995). here, we observed relatively higher activities of cat and pod in the tolerant cultivars, whereas hardly any induction was noticed in susceptible cultivars like drg 1 and ak 159. this result is in accordance with the values for oxidative stress parameters, which were quite high in the susceptible cultivars. sharifi et al. (2012) reported that the tolerant wheat lines revealed high activity of physiological responses of peanut cultivars to water deficit stress acta bot. croat. 74 (1), 2015 139 pod and cat enzyme under drought conditions with higher yields, thus showing positive correlation between enzyme activity and yield in drought conditions. the enzymes apx and gr complete the detoxifi cation cycle of ros inside cell via the halliwell-asada pathway and the activity of these enzymes determine the rate of dissipation of toxic substances (mittler 2002). the present study has shown higher activities of these enzymes under water stress, especially in tolerant cultivars, which is a desirable trait that may be associated with water defi cit stress tolerance in peanut. antioxidants such as ascorbate and glutathione are involved in scavenging h2o2 in conjunction with monodehydro ascorbate reductase (mdar) and gr, which regenerate ascorbate (horemans et al. 2000). in conclusion, the study shows that water defi cit stress in peanut imparted different physiological and biochemical responses at different growth stages. as a general response, the peanut cultivars showed decrease in rwc, membrane stability, specifi c leaf area and chlorophyll and carotenoid contents, but the extent of reduction varied with cultivars and developmental stage of the crop. at the initial stage, the impact of water defi cit stress was lower, but with the advancement of crop growth stage a more pronounced effect was observed. the production of various reactive oxygen species and lipid peroxidation also supports this conclusion, showing a greater degree of oxidative stress when water defi cit stress is imposed at pod developmental stage than at pegging stage. higher activities of sod, cat, pod, apx and gr, as a whole, contributed to less oxidative stress in tolerant cultivars like icgs 44 and tag 24, establishing the role of antioxidant defence system in the ability of the peanut to tolerate water defi cit stress. acknowledgements authors gratefully acknowledge the director, dgr, for providing facilities, and mr. c.b. patel for technical support in raising the crop during the conduct of the experiment. the authors are also grateful to dr. d. bhaduri for her support rendered in statistical analysis. referenc es aebi, h., 1984: catalase in vitro. methods in enzymology 105, 121–126. agarwal, s., sairam, r. k., srivastava, g. c., meena, r. c., 2005: changes in antioxidant enzymes activity and oxidative stress by abscisic acid and salicylic acid in wheat genotypes. biologia plantarum 49, 541–550. arjenaki, f. g., jabbari, r., morshedi, a., 2012: evaluation of drought stress on relative water content, chlorophyll content and mineral elements of wheat (triticum aestivum l.) varieties. international journal of agriculture and crop sciences 4, 726–729. arnon, d. i., 1949: copper enzymes in isolated chloroplasts. polyphenol oxidase in beta vulgaris. plant physiology 24, 1–15. arunyanark, a., jogloy, s., akkasaeng, c., vorasoot, n., kesmala, t., rao, r. c. n., wright, g. c., patanothai, a., 2008: chlorophyll stability is an indicator of drought tolerance in peanut. journal of agronomy and crop science 194, 113–125. ashraf, m., 2010: inducing drought tolerance in plants. biotechnological advances 28, 169–183. chakraborty k., singh a. l., kalariya k. a., goswami n., zala p. v. 140 acta bot. croat. 74 (1), 2015 bajji, m., lutts, s., kinet, j. m., 2001: water defi cit effects on solute contribution to osmotic adjustment as a function of leaf ageing in three durum wheat (triticum durum desf.) cultivars performing differently in arid conditions. plant science 160, 669–681. black, c. a., 1965: methods of soil analysis: part i physical and mineralogical properties. american society of agronomy, madison, wisconsin, usa. boote, k. j., 1982: growth stages of peanut (arachis hypogaea l.). peanut science 9, 35−40. boote, k. j., ketring, d. l., 1990: peanut. in: stewart b. a., nielson d. r. 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defi cit in leaves. new phytologist 49, 81–87. young, a. j., 1991: the photo-protective role of carotenoids in higher plants. physiologia plantarum 83, 702–708. zhu, j. k., 2002: salt and drought stress signal transduction in plants. annual reviews of plant biology 53, 247–273. untitled acta bot. croat. 75 (1), 2016 89 acta bot. croat. 75 (1), 89–98, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0013 issn 0365-0588 eissn 1847-8476 dry grasslands of hippocrepido glaucae-stipion austroitalicae in the pollino massif (calabria, italy) massimo terzi1*, franceso s. d’amico2 1 institute of bioscience and bioresource – cnr, via amendola 165, bari, italy 2 dept. of biology, botanical garden museum, university of bari, via orabona 4, italy abstract – rocky pastures dominated by stipa austroitalica in the south-east of italy were classifi ed within an endemic alliance, hippocrepido glaucae-stipion austroitalicae, originally assigned to a balkan order (scorzoneretalia villosae). actually, the distribution area of s. austroitalica extends further westwards and large patches are found on the south-east side of the pollino massif. this study aims to describe and characterise the plant communities dominated by s. austroitalica in this area and analyse their fl oristic and chorological relationships with other associations of hippocrepido-stipion. moreover, their syntaxonomy is discussed in the context of the italian and south european dry grasslands biogeography. the grasslands were studied on the basis of 19 phytosociological relevés. a larger data set, including 185 relevés with s. austroitalica, was used to visualise the relationships among the associations through nonmetric multi-dimensional scaling ordination. the results allowed the description of a new association, bupleuro gussonei-stipetum austroitalicae, classifi ed within hippocrepido-stipion. as a consequence, the alliance synrange was extended up to the pollino massif. the hip pocrepido-stipion, together with cytiso spinescentis-bromion erecti, was arranged in euphorbietalia myrsinitidis, an endemic order of the italian peninsula. the proposed scheme upgrades the syntaxonomy and nomenclature of the dry grasslands vegetation of central and southern italy. keywords: calcareous grasslands, euphorbietalia myrsinitidis, pollino national park, scorzoneretalia villosae, stipa austroitalica, syntaxonomy. abbreviation: art. – article of the 3rd edition of the international code of phytosociological nomenclature * corresponding author, e-mail: massimoterzi@ibbr.cnr.it introduction the alliance hippocrepido glaucae-stipion austroitalicae forte et terzi 2005 was originally conceived to describe the mediterranean steppe-grasslands of two of the largest karst areas in the south-east of italy, murge hill and gargano promontory, and a few other sites in the molise region (fanelli et al. 2001, forte et al. 2005). the grasslands are characterised by a high fl oristic richness, with a remarkable phytogeographical value. the dominant species, stipa austroitalica martinovský, is an endemic species of the south of italy and is considered a priority species for european biodiversity conservation strategies (annex ii of directive 92/43/eec). as a consequence, plant communities of hippocrepido-stipion are worthy of the highest safeguarding and attention (paura et al. 2014). besides its importance for biodiversity conservation, hippocrepido-stipion is also of particular concern for the biogeography of mediterranean vegetation in the european context. in fact, due to the presence of many taxa with a north-eastern mediterranean and south-eastern european distribution, the alliance was originally classifi ed within a balkan order, scorzonero villosae-chrysopogonetalia grylli horvatić et horvat in horvatić 1963 nom. illeg. – whose correct name is scorzoneretalia villosae kovacević 1959 (cf. terzi in press) – of which it would constitute the southwestern outpost (forte et al. 2005). actually, the syntaxonomic position of hippocrepido-stipion raised one more time the question on the relationship between grassland vegetation of the italian peninsula and the western balkans, after it has been debated for nearly 40 years (lakušić 1969, horvat et al. 1974, bonin 1978, royer 1991, biondi et al. 1995, fanelli et al. 2001, terzi et al. 2010, biondi and galdenzi 2012, terzi and di pietro 2013, di pietro and wagensommer 2014, biondi et al. 2014a). although the largest phytocoenoses with s. austroitalica can be found along the southern part of the italian adriatic border, the species was recorded in many other sites in the south of italy, with large patches also on the south-eastterzi m., d’amico f. s. 90 acta bot. croat. 75 (1), 2016 ern side of the pollino mountain range, in calabria region (moraldo 1986, brandmayr et al. 2002). it is interesting to observe that bonin (1978) – who fi rst extended the distribution area of scorzoneretalia villosae up to southern italy – based his hypothesis on a phytosociological study on the grasslands of the pollino massif. bonin’s relevés (bonin 1978) were carried out from nearly 800 m above sea level (asl) upward and, even if they include some records of stipa mediterranea trin. et rupr., it seems improbable that they could be referred to s. austroitalica, whose grasslands develop mainly at lower altitudes, in an area around castrovillari and nearby towns, in the province of cosenza (fig. 1). this area has been widely surveyed for both fl ora and fauna (i.e., terraciano 1890, gavioli 1936, bernardo and maiorca 1996, brandmayr et al. 2002, bernardo et al. 2011) whereas, despite its importance, it has not been studied from a phytosociological standpoint nor has its vegetation been compared to other s. austroitalica associations. it is worth mentioning that these phytocoenoses are also partially included within the pollino national park and the proposed site of community importance ‘la petrosa’. thus, the aim of this study is threefold: 1) to describe and characterise the plant communities dominated by s. austroitalica in the southern side of the pollino massif; 2) to analyse their fl oristic and chorological relationships with other s. austroitalica grasslands; 3) to discuss their syntaxonomy in the context of italian and south european mediterranean grasslands. material and methods study area the pollino massif is located on the border between the regions of calabria and basilicata, in the south of italy. its main peaks, pollino mountain (2248 m) and serra dolcedorme (2267 m), are the highest in southern italy (fig. 1). the core area of the massif is made of limestone and dolomitic rocks, cut by faults and deep gorges. in the calabrian side, around the towns of castrovillari and frascineto, the relief degrades southand eastwards to the sibari plain and the ionian sea, roughly following the route of the coscile, the most important river of the area. the geological structure of the area is quite complex with mesozoic calcareousdolomite reliefs, quaternary soils of the river basin, sediments of miocene and pliocene-pleistocene deposits, outcrops of palaeozoic rocks (sericite schists, phyllites, calcareous schists) (giannini et al. 1963). the stipa austroitalica communities develop mainly on limestone, often outcropping, in an area roughly between 300–800 m asl. the bioclimate of the study area, deduced from the thermo-pluviometric data of the meteorological station of castrovillari, is mediterranean pluvioseasonal-oceanic with a meso-mediterranean thermotype and a subhumid ombrotype (fig. 2, tab. 1). the bioclimatic indexes (rivas-martinez 2008, without compensation for the altitude) were calculated on the basis of thermo-pluviometric data available on the institutional web sites of arpa-calabria (http:// www.cfd.calabria.it/) and protezione civile-apulia (http:// www.protezionecivile.puglia.it/). vegetation analysis rocky pastures dominated by stipa austroitalica in the area surrounding castrovillari and nearby towns (fig. 1) were sampled by 16 phytosociological relevés, according to the standard method of the zürich–montpellier school (braun-blanquet 1932, westhoff and van der maarel 1978). we sampled plant communities with s. austroitalica occurring with cover values greater than 30% (i.e., 3, 4 or 5 on the braun-blanquet abundance-dominance scale). the plot sizes ranged between 70–100 m2, which is nearly equal to the mean plot size calculated for the hippocrepido-stipion (see below). the altitudinal range was between 430 and 700 m asl. moreover, to take into account the altitudinal variations of grassland vegetation, three additional relevés were carried out near campotenese at nearly 1000 m asl where s. austroitalica is replaced by stipa dasyvaginata martinovský subsp. apenninicola martinovský et moraldo (online suppl. tab. 1, on-line suppl. appendix 1). fig. 1. (a) map of the south of italy displaying the mountain range of murge, gargano and pollino. (b) south-eastern part of the national park of pollino (shaded area). the study area is indicated by vertical lines. fig. 2. ecological climate diagram for castrovillari, in the province of cosenza (italy). the dashed line indicates mean monthly temperature (°c); the solid line indicates mean monthly precipitation (mm). hippocrepido-stipion in the pollino massif (italy) acta bot. croat. 75 (1), 2016 91 ordination to visualise the fl oristic, chorological and life-form relationships among the studied pastures and those already assigned to the hippocrepido glaucae-stipion austroitalicae or to other grasslands with s. austroitalica, a larger data set (185 relevés) was carried out by adding relevés already published and assigned to the “community with ephedra nebrodensis and scorzonera villosa subsp. columnae (gargano)” and the following associations: chamaecytiso spine scentis-stipetum austroitalicae terzi et forte 2005 (murge of matera), irido pseudopumilae-scorzoneretum columnae di pietro, misano et terzi 2010 (south-eastern murge), convolvulo elegantissimi-stipetum austroitalicae biondi et guerra 2008 (south-eastern murge), centaureo apulae-andropogonetum distachyi biondi et guerra 2008 (art. 44, south-eastern murge), stipo austroitalicae-hyparrhenietum hirtae biondi et guerra 2008 (south-eastern murge), cardopato corymbosi-brometum erecti biondi et guerra 2008 (south-eastern murge), chamaeleono gummiferi-stipetum austroitalicae brullo, scelsi et spampinato 2001 (aspromonte), sideritido italicae-stipetum austroitalicae fanelli, lucchese & paura corr. terzi, di pietro et d’amico 2010 (gargano), stipo austroitalicae-seslerietum juncifoliae di pietro et wagensommer 2014 (gargano), phagnalo illyricistipetum frentanae terzi, di pietro et d’amico 2010 (molise), polygalo mediterraneae-stipetum austroitalicae terzi, di pietro et d’amico 2010 (dauno sub-apennine), acino suaveolentis-stipetum austroitalicae forte & terzi, 2005 (north-western murge), anthemido columnae-stipetum austroitalicae fascetti, pirone et rosati 2013 (maddalena mts.) (brullo et al. 2001, fanelli et al. 2001, forte et al. 2005, biondi and guerra 2008, di pietro and wagensommer 2008, terzi et al. 2010, fascetti et al. 2013, di pietro and wagensommer 2014, on-line suppl. appendix 2). species abundance-dominance values were transformed to the ordinal scale proposed by van der maarel (1979). the plot sizes of relevés vary from 2 to 350 m2, with an average value of nearly 80 m2. only relevés with a plot size of 10– 200 m2 were retained together with the nomenclatural type relevés; the types were kept even if their plot sizes exceeded the given thresholds. the resulting data set (consisting of 164 relevés) was ordinated by nonmetric multi-dimensional scaling (nmds, kruskal 1964, mather 1976) after outlier relevés had been removed. outlier analysis was performed by pc-ord software 6.11 (mccune and mefford 2011), considering as outliers those relevés that were more than 2.00 standard deviation units away from the mean. nmds was carried out by using the ‘thorough and slow’ option of the pc-ord autopilot mode, with the relative sørensen distance (mccune and grace 2002). chorotypes and life-forms refer to pignatti (1982). the following chorotypes were used: circum-adriatic; atlantic, including also european-atlantic and sub-atlantic taxa; circumboreal; endemic, including also subendemic species; eurosiberian; eurasiatic; european; mediterraneanatlantic; eury-mediterranean; steno-mediterranean; mediterranean-montane, including also the south and south-east european orophytes; paleotemperate; wide distribution taxa, including the remaining types (e.g., cosmopolitan, sub-cosmopolitan). within the previous chorological types, pignatti (1982) recognizes some “eastern” taxa (i.e., east steno-mediterranean, east eury-mediterranean, east mediterranean-montane, south-east european and circum-adriatic taxa). to evaluate their infl uence on the associations, the percentages of these “eastern” taxa were separately calculated and summarised in another additional type (referred as “est” in table 3). the strengths of the relationships between life-forms and chorotypes and nmds ordination scores were visualised throughout joint plots with an r2 cutoff arbitrarily set at 0.30. as summarising data, life-form and chorological spectra were also calculated on the basis of species frequency in the original diagnoses of the associations (tab. 3). nomenclature taxonomic nomenclature refers to euro+med plantbase and subordinately to conti et al. (2005) and recent taxonomic monographs (gonzalo et al. 2013, quintanar and castroviejo 2013, gallo 2014). regarding s. austroitalica, three subspecies were identifi ed in the study area (moraldo tab. 1. bioclimate of stations representative of some stipa austroitalica associations. alt. – altituide; me.po – mediterranean pluvioseasonal oceanic; te.oc/sm – temperate oceanic, submediterranean variant; te (%) – percentage of temperate (hydrologic) years in the time range, me (%) – percentage of mediterranean (hydrologic) years; mme – mesomediterranean; tme – thermomediterranean; mte – mesotemperate; sme – supramediterranean; sec – dry; shu – subhumid; hum – humid; i – lower belt; s – upper belt; cont. – continentality type; euoc – euoceanic; smct – semicontinental; ic – index of continentality; io – ombrothermic index; tp – yearly positive temperature; itc – compensated thermicity index. (1): from fascetti et al. (2013). stations alt. (m) time range bioclimate te (%) me (%) thermo type ombro type cont. ic io tp itc manfredonia 2 1950–2010 me.po 11.7 88.3 mme.i sec.i euoc 16.5 2.4 1869.4 314.4 reggio calabria 15 1950–2010 me.po 0.0 100.0 tme.i sec.i euoc 14.8 2.7 2218.7 422.2 crispiano 265 1950–2010 me.po 25.0 75.0 mme.i sec.s smct 17.3 3.0 1923.2 320.5 castrovillari 353 1950–2010 me.po 26.7 73.3 mme.i shu.i smct 17.5 4.6 1831.8 297.8 matera 401 1950–1999 me.po 36.7 63.3 mme.i sec.s smct 18.2 3.1 1814.1 286.5 altamura 461 1950–2010 me.po 31.7 68.3 mme.s sec.s smct 18.4 3.2 1759.4 271.1 s. giovanni rotondo 557 1950–2010 te.oc/sm 68.3 31.7 mte.i shu.s smct 17.9 5.0 1654.9 249.0 moliterno (1) 879 1926–1987 me.po sme hum.i smct 18.4 terzi m., d’amico f. s. 92 acta bot. croat. 75 (1), 2016 1986) but, as claimed by bernardo et al. (2011), and we agree with them, their distinction is diffi cult on the basis of the indicated diagnostic traits. a recent dissertation on the issue (gonzalo et al. 2013) recognises only two subspecies of s. austroitalica, one (s. a. subsp. sicula) being endemic of sicily. following this last revision, all the records from the study area were referred to s. austroitalica subsp. austroitalica. for s. dasyvaginata, we referred to the revision of moraldo (1986) who observed that when moving away from the locus classicus (simbruini mountains), it becomes diffi cult to fi nd the typical traits of the taxon. the taxonomic relationship between s. dasyvaginata and stipa eriocaulis borbás subsp. eriocaulis is in fact uncertain and molecular analyses are needed to clarify it (gonzalo et al. 2013). for koeleria, taxonomic nomenclature refers to the recent revision paper by quintanar and castroviejo (2013). for the phytosociological nomenclature, the rules of the 3rd edition of the international code of phytosociological nomenclature (icpn, weber et al. 2000) were followed. correct names of syntaxa were quoted throughout the text even if they replace other names commonly used in scientifi c literature. these latter were listed as synonyms in the syntaxonomic scheme below. results vegetation grasslands dominated by stipa austroitalica in the south-east of the pollino massif include some taxa that have been already considered diagnostic for hippocrepido glaucae-stipion austroitalicae (e.g., s. austroitalica, thymus spinulosus ten., scorzonera villosa scop. subsp. columnae (guss.) nyman, hippocrepis glauca ten., melica transsilvanica schur subsp. transsilvanica) or have been recorded with high frequencies in its communities, such as bromopsis erecta (huds.) fourr., koeleria splendens c. presl, teucrium capitatum l. subsp. capitatum, anthyllis vulneraria l., avena barbata link, eryngium campestre l., galium corrudifolium vill., briza maxima l., dasypyrum villosum (l.) p. candargy (cf. terzi et al. 2010). the physiognomy of the phytocoenoses is mainly due to the high abundance-dominance value of s. austroitalica and other caespitose hemicryptophytes (b. erecta, brachypodium retusum (pers.) p. beauv., dactylis glomerata l. subsp. hispanica (roth) nyman) as well as chamaephytes, such as t. spinulosus, euphorbia spinosa l. or t. capitatum subsp. capitatum. notwithstanding the general fl oristic and structural similarities with other s. austroitalica communities, those from the pollino area are fl oristically quite differentiated by some species, among which are the steno-endemic bupleurum gussonei (arcang.) snogerup & b. snogerup (cf. snogerup and snogerup 2001) and the south european euphorbia rigida m. bieb. these two species are considered as diagnostic of the new association bupleuro gussonei-stipetum austroitalicae ass. nov. (tab. 2, on-line suppl. tab. 1, rel. 1–16). e. rigida has been already considered as diagnostic for euphorbion rigidae brullo et spampinato 1990 which develops in different ecological conditions, being typical of chamaephytic vegetation on incoherent substrata in hilly tab. 2. the bupleuro gussonei-stipetum austroitalicae ass. nov. hoc loco: holotypus. date and geographic coordinates: 19/05/2010, 39° 49’ 01’’, 16° 17’ 13’’ altitude: 445 m a.s.l. exposition: 290° slope: 30 % cover: 100 % plot size: 100 m2 species: stipa austroitalica martinovský subsp. austroitalica (5), bromopsis erecta (huds.) fourr. (3), brachypodium retusum (pers.) p. beauv. (2), scorzonera villosa scop. subsp. columnae (guss.) nyman (2), thymus spinulosus ten. (2), dactylis glomerata l. subsp. hispanica (roth) nyman (1), festuca circummediterranea patzke (1), hypochaeris achyrophorus l. (1), nigella damascena l. (1), rostraria cristata (l.) tzvelev (1), seseli tortuosum l. (1), aira caryophyllea l. (+), anagallis arvensis l. (+), anisantha madritensis (l.) nevski (+), anthyllis vulneraria l. (+), asterolinon linum-stellatum (l.) duby (+), avena barbata link (+), bellardia trixago (l.) all. (+), briza maxima l. (+), bromus intermedius guss. (+), bupleurum baldense turra (+), bupleurum gussonei (arcang.) snogerup & b. snogerup (+), campanula erinus l. (+), carduus nutans l. subsp. perspinosus (fiori) arènes (+), carlina corymbosa l. (+), centaurea deusta ten. (+), centaurium erythraea rafn (+), cephalaria leucantha (l.) roem. & schult. (+), clinopodium nepeta (l.) kuntze subsp. glandulosum (req.) govaerts (+), convolvulus cantabrica l. (+), convolvulus elegantissimus miller (+), coronilla scorpioides (l.) w. d. j. koch (+), crepis rubra l. (+), crupina crupinastrum (moris) vis. (+), cytisus spinescens c. presl (+), dasypyrum villosum (l.) p. candargy (+), echium vulgare l. (+), elaeoselinum asclepium (l.) bertol. (+), eryngium amethystinum l. (+), eryngium campestre l. (+), erysimum pseudorhaeticum polatschek (+), euphorbia exigua l. (+), euphorbia rigida m. bieb. (+), galium corrudifolium vill. (+), gastridium ventricosum (gouan) schinz & thell. (+), koeleria splendens c. presl (+), linum strictum s.l. (+), linum tryginum l. (+), malva cretica cav. (+), matthiola fruticulosa (l.) maire (+), micromeria graeca (l.) benth. (+), micromeria graeca (l.) benth. subsp. fruticulosa (bertol.) guinea (+), odontites luteus (l.) clairv. (+), ophrys tenthredinifera willd. (+), orchis coriophora l. (+), ornithogalum montanum cirillo (+), phleum hirsutum honck. subsp. ambiguum (ten.) tzvelev (+), poa bulbosa l. (+), polygala monspeliaca l. (+), potentilla pedata wild (+), reichardia picroides (l.) roth (+), salvia verbenaca l. (+), sanguisorba minor scop. (+), scandix pecten-veneris l. (+), serapias vomeracea (burm. f.) briq. (+), sherardia arvensis l. (+), sixalix atropurpurea (l.) greuter & burdet (+), stachys germanica l. subsp. salvifolia (ten.) gams (+), stipa capensis thunb. (+), teucrium capitatum l. subsp. capitatum (+), teucrium chamaedrys l. (+), tragopogon porrifolius l. (+), trifolium campestre schreber (+), trifolium scabrum l. subsp. scabrum (+), trifolium stellatum l. (+), trigonella gladiata m. bieb. (+), tyrimnus leucographus (l.) cass. (+), urospermum dalechampii (l.) f. w. schmidt (+), valeriana tuberosa l. (+), valeriana tuberosa l. (+), valerianella muricata (stev. ex m. bieb.) j.w. loudon (+), vincetoxicum hirundinaria medik. (+), vulpia ciliata dumort. (+), xeranthemum inapertum (l.) mill. (+). hippocrepido-stipion in the pollino massif (italy) acta bot. croat. 75 (1), 2016 93 and mountainous river valleys of sicily and southern italy (brullo and spampinato 1990). the euphorbion rigidae has been classifi ed within the thlaspietea rotundifolii br.-bl. 1948 (scrophulario bicoloris-helichrysetalia italici brullo 1984) whose character species are rather rare or absent in bupleuro-stipetum, where e. rigida acts as a differential species. the new association describes the rocky pastures dominated by s. austroitalica, developing on calcareous substrata of the pollino massif, between nearly 300 and 800 m above sea level, under a semi-continental mediterranean pluvioseasonal-oceanic bioclimate, in the meso-mediterranean thermotype (fig. 2, tab. 1). these pastures are often juxtaposed with other xerothermic grasslands dominated by hyparrhenia hirta (l.) stapf, with which they share many species. as a consequence, many frequent taxa of bupleuro-stipetum, and among them many annuals, are ingressives from thero-brachypodietea s.l. their presence has been observed in nearly all the associations of hippocrepido-stipion and some of them can be considered as differential species of the alliance. towards higher altitudes, the sociological importance of s. austroitalica decreases until it is substituted for by stipa dasyvaginata subsp. apenninicola in quite different communities (on-line suppl. tab. 1, rel. 17–19) with seseli tommasinii rchb. f., helictochloa versicolor (vill.) romero zarco subsp. praetutiana (arcang.) romero zarco, sideritis italica (mill.) greuter & burdet, crepis lacera ten., cerastium tomentosum l., euphorbia myrsinites l., brachypodium rupestre (host) roem. & schult. and others. these communities are clearly related to cytiso spinescentis-bromion erecti bonin ex bonin 1978. it is interesting to notice that some taxa that frequently inhabit the s. austroitalica grasslands on the adriatic side of the italian peninsula (e.g., euphorbia myrsinites in the acino-stipetum of murge, sideritis italica in the sideritido-stipetum of gargano) are rare or absent in bupleuro-stipetum while on the contrary they can be found in the upper vegetation belt. nmds ordination the three-axis solution of the nmds ordination attained a minimum stress of 14.83. the three axes explained nearly 80% of total variation, being 40.4%, 25.0% and 13.6% the proportion of variance represented by each axis respectively. only the fi rst two axes are shown (fig. 3) and discussed below. axis 1 roughly represents an altitudinal gradient from the more xerothermic associations of lower altitude (chamaeleono-stipetum, convolvulo-stipetum, centaureo-andropogonetum, stipo-hyparrhenietum and iridoscorzoneretum), with a high percentage of steno-mediterranean species, to associations of higher altitudes on the left, with a greater percentage of chamaephytes, mediterranean montane and european taxa (anthemido-stipetum, sideritido-stipetum, stipo-seslerietum and the relevés 17–19 of tab. 2). increasing altitude is also associated with a higher percentage of ‘eastern’ species (fig. 3). the associations originally assigned to hyparrhenietalia hirtae rivas-martinez 1978 (brullo et al. 2001, biondi and guerra 2008, see also terzi et al. 2010) are situated in the upper right hand side of the diagram. among them, cha maeleono-stipetum is well differentiated for the low number of species, the very low percentage of therophytes and the highest amount of steno-mediterranean taxa. in the central-lower part of the diagram, there are associations characterised by higher percentages of therophytes (except for cardopato-brometum, tab. 3) and eury-mediterranean species (polygalo-stipetum, chamaecytiso-stipetum, acino-stipetum, cardopato-bromion, bupleuro-stipetum, irido-scorzoneretum and the ephedra nebrodensis and scorzonera villosa subsp. columnae community). the bupleuro-stipetum showed high percentage of both stenoand eury-mediterranean taxa, on the whole at nearly 70% (tab. 3). the association is among those with the highest percentage of therophytes that roughly equals hemicryptophytes, followed by chamaephytes. similar life-form spectra characterise also the other associations of the hippocrepido-stipion, such as the acino-stipetum of the murge hill (tab. 3). in few associations, such as stipo-seslerietum or anthemido-stipetum, hemicryptophytes are numerically dominant whereas chamaephytes increase and therophytes decrease. these associations share many species with the cytiso-bromion and mark the transition from the hippocrepido-stipion to the upper vegetation belt. the same general pattern was also observed for the life-form spectra weighted with species cover values (data not shown). fig. 3. nonmetric multi-dimensional scaling ordination. the square symbols represent relevés of the bupleuro-stipetum austroitalicae, the circular ones those of the other associations. the relevés 17–19 of tab. 2 are indicated by rhombus symbols. filled squares/circles indicate the nomenclatural types of the associations, whose abbreviations are written next to them (cf., tab. 3). for the “community with ephedra nebrodensis and scorzonera villosa subsp. columnae”, the abbreviation is placed next to the fi rst relevé of the original table (di pietro and wagensommer 2008). mes – steno-mediterranean (r2 = 0.54), t – therophytes (r2 = 0.35), med – eury-mediterranean (r2 = 0.33), mon – montane (r2 = 0.59), eur – european (r2 = 0.48), est – eastern taxa (r2 = 0.34), ch – chamaephytes (r2 = 0.37), end – endemics (r2 = 0.33). terzi m., d’amico f. s. 94 acta bot. croat. 75 (1), 2016 from a bioclimatic standpoint, the bioclimate of castrovillari turned out to be intermediate among those of crispiano (province of taranto), matera and altamura (province of bari), except for the higher mean annual precipitations (tab. 1). in fact, in the ordination diagram, the bupleurostipetum is placed near chamaecytiso-stipetum (matera), acino-stipetum (nw-murge, altamura) and cardopatobromion (crispiano). the bioclimate of reggio calabria – in the south-western part of the italian peninsula, where chamaeleono-stipetum develops – stands out as the most mediterranean, with a thermo-mediterranean thermotype and a dry ombrotype. in the south of gargano, at the same altitude, manfredonia, in the province of foggia, shows a higher continental tendency and a meso-mediterranean thermotype and a subhumid ombrotype, highlighting the bioclimatic differences between the eastern and western sides of the italian peninsula. near manfredonia and san giovanni rotondo, in the gargano promontory – where the bioclimate is submediterranean temperate – sideritido-stipetum and stiposeslerietum were described. these two associations are placed in the ordination diagram near anthemido-stipetum, which develops near moliterno, in the province of potenza, under a mediterranean climate within the supra-mediterranean thermotype and humid ombrotype. in a nutshell, the represented associations develop along a bioclimatic gradient that goes from the bioclimates of reggio calabria and manfredonia to those of moliterno and s. giovanni rotondo. discussion the presence within bupleuro-stipetum of numerous characters and frequent taxa of hippocrepido glaucaestipion austroitalicae proves the ecological relationship between the new association and the other ones already assigned to the alliance. the relationship is also highlighted by the life-form and chorological standpoints (fig. 3, tab. 3). the alliance hippocrepido-stipion occupies a vegetation belt intermediate between the thermo-mediterranean vegetation of the class thero-brachypodietea s.l. and the typical apennine vegetation of the cytiso-bromion (festuco-brometea br.-bl. et tx. ex klika et hadač 1944). this vegetation belt is well represented in the south-east of italy (murge and gargano) while it is thinner and restricted to local patches westand southwards. bupleuro-stipetum develops in the lower part of the gradient so that, together with species of festuco-brometea, such as bromopsis erecta, eryngium amethystinum and koeleria splendens, it also includes many species from thero-brachypodietea s.l. the classifi cation within hippocrepido-stipion is due to the sociological role of species such as s. austroitalica, s. villosa subsp. columnae, t. spinulosus, h. glauca, that, especially in the south-east of italy, show their preference for festucobrometea. in fact, within other classes they lose their importance and are generally recorded with lower cover values. similar situations, intermediate between thero-brachypodietea s.l. and festuco-brometea, have been observed ta b. 3 . l ife fo rm s an d ch or ol og ic al s pe ct ra , w ei gh te d by s pe ci es fr eq ue nc y, o f t he a ss oc ia tio ns in cl ud ed in th e da ta s et . l ife fo rm s: c h – c ha m ae ph yt es ; g – g eo ph yt es ; h – h em ic ry pt op hy te s; p – p ha ne ro ph yt es ; t – t he ro ph yt es . c ho ro ty pe s: a dr – c irc um -a dr ia tic ; a tl – a tla nt ic ; c br – c irc um bo re al ; e nd – e nd em ic ; e si – e ur os ib er ia n; e ua – e ur as ia tic ; e ur – e ur op ea n; m ea – m ed ite rr an ea na tla nt ic ; m ed – eu ry -m ed ite rr an ea n; m es – s te no -m ed ite rr an ea n; m on – m ed ite rr an ea nm on ta ne ; p al – p al eo te m pe ra te ; w id – o th er ty pe s; e st – p er ce nt ag e of “ ea st er n” ta xa , a lre ad y in cl ud ed in th e pr ev io us ty pe s. a ss oc ia tio ns c od e l if e fo rm s (% ) c ho ro ty pe s (% ) e st (% ) c h g h p t a dr a tl c br e nd e si e ua e ur m ea m ed m es m on pa l w id a ci no s ua ve ol en tis -s tip et um a us tr oi ta lic ae a cst 10 .5 8. 7 37 .7 1. 0 42 .1 0. 0 0. 1 0. 0 11 .6 2. 0 1. 4 5. 5 0. 2 36 .1 31 .1 4. 4 5. 8 1. 8 7. 5 a nt he m id o co lu m na est ip et um a us tr oi ta lic ae a nst 22 .5 15 .0 50 .8 1. 3 10 .4 0. 8 1. 7 0. 4 12 .9 2. 1 7. 9 12 .9 0. 4 25 .4 10 .0 13 .3 10 .8 1. 3 11 .7 b up le ur o gu ss on ei -s tip et um a us tr oi ta lic ae b ust 10 .9 7. 4 38 .4 3. 3 40 .0 0. 9 0. 0 0. 0 8. 6 2. 4 0. 9 5. 5 1. 1 36 .0 33 .5 2. 7 5. 2 3. 0 8. 4 c ar do pa to c or ym bo si -b ro m et um e re ct i c ab r 7. 3 12 .1 59 .3 3. 6 17 .7 0. 0 0. 8 0. 0 5. 2 3. 6 7. 3 9. 3 2. 4 40 .7 14 .1 3. 2 12 .5 0. 8 9. 7 c en ta ur eo -a nd ro po go ne tu m d is ta ch yi c ea n 4. 6 16 .1 54 .0 0. 0 25 .3 0. 0 0. 0 0. 0 19 .5 4. 6 1. 1 2. 3 0. 0 24 .1 31 .0 4. 6 11 .5 1. 1 6. 9 c ha m ae cy tis o sp in es ce nt is -s tip et um a us tr oi ta lic ae c hst 10 .9 6. 3 48 .0 1. 6 33 .2 3. 3 2. 0 0. 0 10 .9 0. 0 0. 3 10 .2 0. 7 29 .9 27 .0 4. 3 10 .5 1. 0 11 .2 c ha m ae le on o gu m m ife ri -s tip et um a us tr oi ta lic ae c gst 16 .0 17 .2 59 .5 1. 2 6. 1 0. 0 0. 0 0. 0 9. 8 0. 6 0. 0 5. 5 0. 0 15 .3 58 .3 0. 0 4. 9 5. 5 0. 6 c on vo lv ul o el eg an tis si m ist ip et um a us tr oi ta lic ae c ost 8. 9 15 .7 45 .1 1. 3 28 .9 0. 9 0. 0 0. 0 14 .0 0. 9 0. 9 4. 3 1. 3 27 .7 35 .7 0. 9 9. 8 3. 8 7. 2 ir id o ps eu do pu m ila esc or zo ne re tu m c ol um na e ir -s c 6. 1 13 .1 27 .5 4. 4 48 .9 0. 5 0. 0 0. 0 7. 6 0. 6 0. 8 3. 1 1. 5 37 .7 37 .7 0. 3 6. 4 3. 7 4. 7 p ha gn al o ill yr ic ist ip et um fr en ta na e ph -s t 21 .8 8. 6 44 .3 5. 2 20 .1 2. 3 0. 0 0. 0 10 .3 4. 0 0. 0 4. 6 0. 0 38 .5 29 .3 3. 4 5. 2 2. 3 6. 9 p ol yg al o m ed ite rr an ea est ip et um a us tr oi ta lic ae po -s t 8. 2 6. 6 47 .5 1. 4 36 .3 0. 8 0. 0 0. 6 7. 8 3. 6 2. 1 8. 0 0. 7 37 .8 27 .0 2. 2 7. 5 1. 9 7. 5 si de ri tid o ita lic ae -s tip et um a us tr oi ta lic ae si -s t 16 .8 12 .4 46 .4 0. 5 23 .9 0. 4 0. 0 0. 0 10 .6 2. 1 4. 6 12 .0 0. 9 29 .2 25 .0 7. 6 5. 7 1. 9 8. 0 st ip o au st ro ita lic ae -h yp ar rh en ie tu m h ir ta e st -h y 11 .3 14 .7 44 .1 2. 3 27 .7 0. 0 0. 0 0. 0 10 .2 1. 7 0. 6 2. 3 0. 6 24 .3 46 .3 1. 1 7. 3 5. 6 4. 0 st ip o au st ro ita lic ae -s es le ri et um ju nc ifo lia e st -s e 29 .2 4. 6 55 .1 3. 2 7. 8 7. 0 1. 0 0. 0 19 .9 1. 0 3. 2 14 .1 0. 4 21 .9 12 .5 13 .3 3. 8 1. 8 17 .1 c om m un ity w ith e ph ed ra n eb ro de ns is a nd sc or zo ne ra v ill os a su bs p. c ol um na e e psc 6. 6 10 .7 21 .1 3. 7 57 .9 0. 0 0. 0 0. 0 9. 1 2. 5 2. 9 3. 3 0. 0 39 .7 32 .6 1. 7 3. 3 5. 0 3. 7 hippocrepido-stipion in the pollino massif (italy) acta bot. croat. 75 (1), 2016 95 along the entire mediterranean edge of europe (royer 1991, apostolova et al. 2014, pirini et al. 2014). in the western mediterranean, barbero and loisel (1972) included these vegetation types within the brachypodio-brometalia barbero et loisel 1972 which in their opinion should replace the balkan scorzoneretalia villosae and its southeastern vicariant, astragalo onobrychidis-potentilletalia micevski 1971. the brachypodio-brometalia originally included two suborders: astragalo-festucenalia barbero et loisel 1972 and brachypodienalia phoenicoidis (braunblanquet ex moliner 1934) barbero et loisel 1972. the latter is more often considered with its original rank of order. the syntaxonomic positions of scorzoneretalia villosae, brachypodietalia phoenicoidis and astragalo-potentilletalia, have not been unanimously interpreted as they were classifi ed both within the thero-brachypodietea s.l. or festuco-brometea. the ‘uncertain’ syntaxonomic position can be easily detected comparing many large scale revisions or synopses where the orders are placed in different classes (e.g., blečić and lakušić 1976, lakušić et al. 1978, royer 1991, redžić 1999, rodwel et al. 2002, bardat et al. 2004, trinajstić 2008, biondi et al. 2014b). moreover, the scorzoneretalia villosae was also divided into two orders that were arranged within thero-brachypodietea s.l. and festuco-brometea respectively (horvatić 1973). for the adriatic side of the balkan peninsula, a third and intermediate class, called brachypodio-chrysopogonetea horvatić 1963, was proposed (horvatić 1958, 1963). the same idea was then extended to the southern european margin under the illegitimate name brachypodio-brometea barbero et loisel 1972 nom. illeg. (art. 29c, barbero and loisel 1972, terzi in press). however, this proposal has not been the subject of any wide consensus in more recent scientifi c literature. some recent papers on the issue considered the orders quoted above within festuco-brometea (rodwell et al. 2002, pirini et al. 2014, apostolova et al. 2014, terzi in press). in the south of italy, hippocrepido-stipion was originally classifi ed within scorzoneretalia villosae and festuco-brometea (cf. fanelli et al. 2001, forte et al. 2005). therefore, it was separated at the order level from the apennine dry grasslands of the cytiso-bromion and instead arranged within the brometalia erecti koch 1926 (royer 1991, biondi et al. 1995). ubaldi (1997) proposed to differentiate the xeric grasslands of the brometalia erecti within a new order that was validated some years later under the name artemisio albae-brometalia erecti ubaldi ex mucina et denggler (mucina et al. 2009). other authors (biondi and galdenzi 2012), following the syntaxonomic arrangement of bonin (1978), reunited both hippocrepido-stipion and cytiso-bromion (sub phleo ambigui-bromion erecti biondi et al. ex biondi et galdenzi 2011 nom. illeg.) under the same order, scorzoneretalia villosae. by contrast, ubaldi (2011) assigned hippocrepido-stipion to thero-brachypodietea s.l. and the apennine grasslands to several new other orders and alliances, almost all invalidly described, of festuco-brometea. summarising, the two italian alliances, hippocrepido-stipion and cytiso-bromion, have been classifi ed in two different classes (ubaldi 2011), in different orders of festuco-brometea (forte et al. 2005) or within the same balkan order (biondi and galdenzi 2012). from the above, it is clear that a defi nitive syntaxonomic scheme should be obtained by a large scale study (yet lacking), at least on a european scale, which will clarify through modern statistical methods the fl oristic relationships among all the high rank syntaxa involved and evaluate the different interpretations given up to now. for the circum-adriatic area, the preliminary results of a revision comparing artemisio-brometalia and scorzoneretalia villosae highlighted important differences in the frequencies of some species along the opposite side of the adriatic sea (terzi and di pietro 2013). moreover, taxa such as chrysopogon gryllus (l.) trin., festuca illyrica markgr.-dann., festuca valesiaca schleich. ex gaudin, stipa eriocaulis borbás, satureja subspicata bartl., scorzonera villosa subsp. villosa are frequent or dominant in the sub-mediterranean balkan grasslands while others prevail on the italian side (e.g., festuca circummediterranea patzke, phleum hirsutum honck. subsp. ambiguum (ten.) tzvelev, s. austroitalica, thymus spinulosus, crepis lacera ten., sideritis italica (mill.) greuter & burdet, scorzonera villosa subsp. columnae) (see also di pietro and wagensommer 2014). despite some circum-adriatic taxonomic similarities, that have been observed for a very long time, the presence of many endemic taxa suggested a syntaxonomic scheme with three orders: with an endemic italian peninsular order differentiated from the artemisio-brometalia (central europe and nw italy) and scorzoneretalia villosae (western balkan and ne italy) (terzi and di pietro 2013). the italian order would include at least the two alliances cytiso-bromion and hippocrepido-stipion. similar observations led biondi et al. (2014a) to defi ne a new endemic order for the italian peninsula. however, to the best of our knowledge, the earlier available valid name for such an order is euphorbietalia myrsinitidis ubaldi 2011, whereas other names are to be listed as synonyms. the considerations given above led to an upgrade of the syntaxonomy and nomenclature currently used in the italian literature (cf. biondi et al. 2014). the following scheme is here proposed: class: festuco-brometea br.-bl. et tx. ex klika et hadač 1944 ord.: euphorbietalia myrsinitidis ubaldi 2011 [holotypus: sideritidion italicae (biondi, ballelli, allegrezza et zuccarello 1995) ubaldi 2011; synonyms: brometalia caprini ubaldi 1997 nom. inval. (art. 2b, 3o), festuco-seslerietalia nitidae 2003 nom. inval. (art. 2b, 8), asphodelino liburnicae-brometalia erecti ubaldi 2011 nom. inval. (art. 2b), phleo ambigui-brometalia erecti biondi, allegrezza, blasi et galdenzi in biondi, allegrezza, casavecchia, galdenzi, gasparri, pesaresi, vagge et blasi 2014). all.: cytiso spinescentis-bromion erecti bonin ex bonin 1978 (lectotypus: lavandulo angustifoliae-asphodelinetum luteae bonin 1978; synonyms: cytiso-bromion caprini bonin in barbero et bonin 1969 nom. inval. (art. 2b, 3b), cytiso-bromion caprini bonin 1969 nom. inval. (art. 2b), crepido lacerae-phleion ambigui biondi et blasi 1982 nom. inval. (art. 3o), phleo ambigui-bromion erecti biondi et terzi m., d’amico f. s. 96 acta bot. croat. 75 (1), 2016 blasi ex biondi, ballelli, allegrezza et zuccarello 1995 nom. inval. (art. 30), seslerio nitidae-caricion macrolepidis ubaldi 1997, valeriano tuberosae-festucion circummediterraneae ubaldi 2003 nom. inval. (art. 8), sideritidion italicae (biondi et al. 1995) ubaldi 2011, knautio calycinae-bromion caprini ubaldi 2011 nom. inval. (art. 2b, 8), violo pseudogracilis-bromopsion caprini terzi 2011, phleo ambigui-bromion erecti biondi, balleli, allegrezza et zuccarello ex biondi et galdenzi 2012) all.: hippocrepido glaucae-stipion austroitalicae forte et terzi 2005 in forte, perrino et terzi 2005 [holotypus: acino suaveolentis-stipetum austroitalicae forte et terzi 2005 in forte, perrino et terzi 2005] ass.:bupleuro gussonei-stipetum austroitalicae ass. nov. hoc loco nomenclature notes the name cytiso-bromion has been considered not effectively published (art. 1) but at least the ‘tableaux et fi gures’ (tables and fi gures) of bonin’s thesis were effectively published, given the indication of the ‘centre regional de documentation pedagogique – service d’impression’ on their cover page (bonin 1978, di pietro 2011). if the name cytiso-bromion was validated by bonin (1978), most of the scientifi c literature that referred to it failed to provide the place of publication correctly. the earliest lectotypifi cation in accordance with the rules of icpn gave the lavandulo angustifoliae-asphodelinetum luteae bonin 1978 as lectotypus of the alliance (di pietro 2011). regarding the rank of order, the name asphodelino liburnicae-brometalia erecti ubaldi 2011 nom. inval. was invalidly published because of the lack of an unambiguous reference to the cytiso-bromion (art. 2b, note 3). the brometalia caprini ubaldi 1997 nom. inval. was invalidly published for the lack of a nomenclatural type of the next subordinate principal rank (art. 17). the festuco-seslerietalia nitidae 2003 nom. inval. was instead not validly published for the lack of an unambiguous reference to an earlier original diagnosis (art 2b) and because character, differential or diagnostic taxa were not explicitly indicated (art. 8). the last article can be quoted also for the invalid publication of the valeriano tuberosae-festucion circummediterraneae ubaldi 2003 nom. inval. and knautio calycinae-bromion caprini ubaldi 2011 nom. inval. some other valid alliance names are here considered as syntaxonomic synonyms of the cytiso-bromion. however, some of them (e.g., seslerio-caricion) could be dealt with as autonomous alliances (cf. terzi and di pietro 2013), depending on results of a more in-depth analysis of italian peninsular grasslands vegetation. conclusion the results obtained with this study extend the synrange of hippocrepido glaucae-stipion austroitalicae westwards up to the pollino massif where rocky pastures dominated by stipa austroitalica were described by the new association bupleuro gussonei-stipetum austroitalicae. it seems reliable that the distribution area of the alliance is even wider, following the distribution area of its diagnostic species. bupleuro-stipetum was characterized by the predominance of mediterranean taxa and nearly equal percentages of therophytes and hemicrytophytes. similar life-form and chorological spectra were observed in other associations of hippocrepido-stipion. in the western part of the range, the ecological space of hippocrepido-stipion is scattered in local patches because it is compressed between the more xerothermic vegetation of thero-brachypodietea s.l. and the higher altitude vegetation of cytiso-bromion. hippocrepid o-stipion and cytiso-bromion are provisionally classifi ed within an endemic xerophytic order of the italian peninsula, whose correct name is euphorbietalia myrsinitidis. nonetheless, this paper highlighted the need for a syntaxonomic revision at european level of dry grassland vegetation at the southern border of the festuco-brometea to clarify the fl oristic relationships with the more xerothermic vegetation of thero-brachypodietea s.l. in fact, many proposals have been put forward without having been verifi ed by means of modern statistical analyses and in the context of the european mediterranean grasslands framework. acknowledgments we wish to thank l. bernardo (university of calabria), r. di pietro (university la sapienza, rome) and b. 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(ed.), classifi cation of plant communities. junk, the hague. hippocrepido-stipion in the pollino massif (italy) acta bot. croat. 75 (1), 2016 o nlin e su pp l. ta b. 1 : t he b up le ur o gu ss on ei -s tip et um a us tr oi ta lic ae a ss . n ov . h oc lo co (r el ev és 1 –1 6, h ol ot yp us re le vé s 3) ; v eg et at io n of th e c yt is ob ro m io n (r el ev és 1 7– 19 ). r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 b up le ur o gu ss on ei -s tip et um a us tr oi ta lic ae a ss . n ov . e up ho rb ia r ig id a m . b ie b. + + + + . 1 + + + + . + + + + + . . . b up le ur um g us so ne i ( a rc an g. ) s no ge ru p & b . s no ge ru p . . + 2 + + + . . + + 1 . 2 + + . . . h ip po cr ep id o gl au ca est ip io n au st ro ita lic ae st ip a au st ro ita lic a m ar tin ov sk ý su bs p. a us tr oi ta lic a 5 5 5 4 5 5 5 4 4 3 4 4 4 3 3 3 . . . c on vo lv ul us e le ga nt is si m us m ill er + + + + + + + + + + + + + + + + . . + th ym us s pi nu lo su s te n. + + 2 . + + + + 1 1 1 3 2 1 3 + + 2 2 sc or zo ne ra v ill os a sc op . s ub sp . c ol um na e (g us s. ) n ym an 1 + 2 + + 2 + + . + + + + + 1 + . . . c re pi s ru br a l . + + + + + + + . 1 + + . . + + + . . + e up ho rb ia s pi no sa l . . . . 3 . 2 . + 2 . + 2 1 1 2 + . . . st ac hy s ge rm an ic a l . s ub sp . s al vi fo lia (t en .) g am s + + + + . + + . . + . + . . + + . . . r ha m nu s sa xa til is j ac q. s ub sp . i nf ec to ri a (l .) p. f ou rn . + . . + . . . . + + + + . 1 + + . . . h ip po cr ep is g la uc a te n. . . . + + . . + + . + + + + + . . 2 1 a sp ho de lu s m ac ro ca rp us p ar l. . . . . . 3 . . 3 1 1 + . 2 2 + . . . m el ic a tr an ss ilv an ic a sc hu r s ub sp . t ra ns si lv an ic a . . . . . . . + + 1 + + . + 2 1 . . + p ol yg al a m on sp el ia ca l . + 1 + + + + + . . . . . . . . . . . . c ar du us n ut an s l . s ub sp . p er sp in os us (f io ri ) a rè ne s + + + + . + + . . . . . . . . . . . . p ot en til la d et om m as ii te n. . . . . + . . . . + . . . . . . . . 1 o no sm a ec hi oi de s (l .) l . s ub sp . a ng us tif ol ia (l eh m .) pe ru zz i & n . g . p as sa l. . . . + . . . + . . . . . . . . . . . o no br yc hi s ae qu id en ta ta (s m .) d’ u rv . . . . + . . . . . . . . . . . . . . . c yt is o sp in es ce nt is -b ro m io n er ec ti st ip a da sy va gi na ta m ar tin ov sk y su bs p. a pe nn in ic ol a m ar tin ov sk y & m or al do . . . . . . . . . . . . . . . . 1 2 3 sc ab io sa h ol os er ic ea b er to l. . . . . . . . + . . . . . . . . + 1 + si de ri tis it al ic a (m ill .) g re ut er & b ur de t . . . . . . . . . . . . . . . . + + + h el ic to ch lo a ve rs ic ol or (v ill .) r om er o z ar co s ub sp . p ra et ut ia na (a rc an g. ) r om er o z ar co . . . . . . . . . . . . . . . . + + + p ol yg al a m aj or j ac q. . . . + . . . 1 . . . . . . . . + + + se se li to m m as in ii r ch b. f. . . . . . . . . . . . . . . . . + + + 1 terzi m., d’amico f. s. acta bot. croat. 75 (1), 2016 r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 h el ia nt he m um o el an di cu m (l .) d um . c ou rs . s ub sp . i nc an um (w ill k. ) g . l óp ez . . . . . . . . . . . . . . . . . 2 1 h el ic hr ys um it al ic um (r ot h) g . d on . . . . . + . . . . . . . . . . . + 1 c er as tiu m to m en to su m l . . . . . . . . . . . . . . . . . 1 . + p te ri di um a qu ili nu m (l .) k uh n . . . . . . . . . . . . . . . . . 1 + c re pi s la ce ra t en . . . . . . . . . . . . . . . . . + + . a sp ho de lin e lu te a (l .) r ch b. . . . . . . . . . . . . . . . . . . + lo m el os ia c re na ta (c ir ill o) g re ut er & b ur de t . . . . . . . 2 . . . . . . . . . . . e up ho rb ie ta lia m yr si ni tid is k oe le ri a sp le nd en s c . p re sl + . + . . + . 4 + + + 3 1 + 1 . 2 1 + c ep ha la ri a le uc an th a (l .) r oe m . & s ch ul t. . . + . . . . + 2 . + 1 . 1 1 1 + . . c en ta ur ea d eu st a te n. + . + + . + . + + . . . . + + . + . . c yt is us s pi ne sc en s c . p re sl + + + 2 3 . . + . . . . 2 . . . . 2 . a sp ho de lin e lib ur ni ca (s co p. ) r ch b. . . . . . . . . . 1 . . 2 + . 1 . 1 + f es tu ca c ir cu m m ed ite rr an ea p at zk e 2 . 1 . . . . . . . . . . . . . 2 2 2 p hl eu m h ir su tu m h on ck . s ub sp . a m bi gu um (t en .) t zv el ev . . + . . . . . . . . . . . + . 3 1 + e la eo se lin um a sc le pi um (l .) b er to l. + . + . . + + . . . . . . . . . . . + e ry si m um p se ud or ha et ic um p ol at sc he k + + + . + . . . . . . . . . . . . . . se du m o ch ro le uc um c ha ix s ub s. m ed ite rr an eu m g al lo . . . . . + . . . + . . + . . . + . . a et hi on em a sa xa til e (l .) w . t . a ito n . . . . . . + . . + . . + . . . + . . e up ho rb ia m yr si ni te s l . . . . . . . . . . . . . . . . . + + + m at th io la fr ut ic ul os a (l .) m ai re + . + . . . . + . . . . . . . . . . . p im pi ne lla tr ag iu m v ill . . . . . . . . . . . . . . . . . 2 1 . th ym us s tr ia tu s v ah l . . . . . . . . . . . . . . . . . + . ju ri ne a m ol lis (l .) r ch b. . . . . . . . + . . . . . . . . . . . a ly ss um d iff us um t en . . . . . . . . . . . . . . . . . + . . li na ri a pu rp ur ea (l .) m ill . . . . . . . . . . . . . . . . . + . . f es tu co -b ro m et ea sa ng ui so rb a m in or s co p. 2 + + . + 1 + + 1 + + 1 + 1 1 + 1 1 1 te uc ri um c ap ita tu m l . s ub sp . c ap ita tu m + + + + + + + 1 + + + 2 1 1 1 + . + 1 2 hippocrepido-stipion in the pollino massif (italy) acta bot. croat. 75 (1), 2016 r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 g al iu m c or ru di fo liu m v ill . + + + 2 + 1 1 . + + + + + + 1 + + + + b ro m op si s er ec ta (h ud s. ) f ou rr . 3 3 3 2 4 . + + . 2 2 4 2 3 3 2 4 4 5 p et ro rh ag ia s ax ifr ag a (l .) l in k ss p. g as pa rr in ii (g us s. ) g re ut er & b ur de t . + . + + . 1 + + + 1 + + + 1 1 1 + + a nt hy lli s vu ln er ar ia l . + . + + + + 1 3 . + . . + 1 + + 4 3 2 p hl om is h er ba -v en ti l . + 2 . + + . + . + + 1 1 1 + + + . . . e ry ng iu m c am pe st re l . . 1 + 2 + + 1 . + + 2 + . . + . . . + te uc ri um c ha m ae dr ys l . + + + + . . + . . + . . + + + + . + + e ry ng iu m a m et hy st in um l . + . + + . + . 1 + . . . . . + . 1 1 1 c on vo lv ul us c an ta br ic a l . . + + + + 2 1 . + . + . . . . + . . . d ia nt hu s lo ng ic au lis t en . . . . . . . . + . 1 . + + + + + . + + c ar ex fl ac ca s ch re b. s ub sp . s er ru la ta (s pr en g. ) g re ut er . 1 . + 2 . + . . . . 1 . + + . . . 1 tr ag op og on p or ri fo liu s l . + + + + + + . . . . + . . + . . . . . a na ca m pt is p yr am id al is (l .) r ic h. + + . + . + 1 . . . . . . . . . + + . se ra pi as v om er ac ea (b ur m . f .) b ri q. + + + + + + + . . . . . . . . . . . . p ot en til la p ed at a w ild . + + + . . + . . . . 1 . . + . . . . o rc hi s co ri op ho ra l . + + + . + + + . . . . . . . . . . . . p la nt ag o ho lo st eu m s co p. . . . . . . . 2 . . + + . . . . + 1 . o no ni s pu si lla l . . . . . + . . + . . . . + + . . . + . a sp er ul a ar is ta ta l . f s ub sp . s ca br a (j . p re sl & c . p re sl ) n ym an . . . . . . . . . + . . . . + + + . . h el ia nt he m um a pe nn in um (l .) m ill . s ub sp . a pe nn in um . . . . . . . . . . . . + . . . . 2 2 b ra ch yp od iu m r up es tr e (h os t) r oe m . & s ch ul t. . . . . . . . . . . . . . . . . + 2 + se du m a m pl ex ic au le d c . s ub sp . t en ui fo liu m (s m . i n si bt h. & s m .) g re ut er . . . . . . . + . . . . . . . . 1 . + li nu m te nu ifo liu m l . . . . . . + . + . . . . . . . . . + . lo tu s co rn ic ul at us l . . . . . . . 1 . . . . . . . . . + . . m ed ic ag o fa lc at a l . . . . + + . . . . . . . . . . . . . . c lin op od iu m a ci no s (l .) k un tz e . . . . . . . + . . . . + . . . . . . si le ne o tit es (l .) w ib el . . . . . . . + . . . . . . . . . + . h yp oc ha er is c re te ns is (l .) b or y & c ha ub . + . . . . . . . . . . . . . . . . + . a ra bi s hi rs ut a (l .) sc op . . . . . . . . . . . . . . . . . + + . 3 terzi m., d’amico f. s. 4 acta bot. croat. 75 (1), 2016 r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 th ym us lo ng ic au lis c . p re sl . . . . . . . . . . . . . . . . . 3 . . te uc ri um m on ta nu m l . . . . . . . . . . . . . . . . . . 2 . e up hr as ia s tr ic ta j . f . l eh m . . . . . . . . . . . . . . . . . . 1 . e ch in op s ri tr o l . . . . . . . . + . . . . . . . . . . . p ol yg al a ni ca ee ns is w .d .j . k oc h s. l. . . . . . . . . . . . + . . . . . . . st ac hy s he ra cl ea a ll. . . . . . . . . . . . . + . . . . . . st ac hy s re ct a l . s ub sp . l ab io sa (b er to l.) b ri q. . . . . . . . . . . . + . . . . . . . st ac hy s of fi c in al is (l .) tr ev is . . . . . . . . . . . . + . . . . . . . o rc hi s m or io l . . . . . . . . . . . . . . . . . + . . m ed ic ag o lu pu lin a l . . . . . . . . . . . . . . . . . + . . se ra pi as c or di ge ra l . . . . . . . . . . . . . . . . . . + . h yp oc ha er is r ad ic at a l . . . . . . . . . . . . . . . . . . . + tr ifo liu m p ra te ns e l . . . . . . . . . . . . . . . . . + . . p ilo se lla p ilo se llo id es (v ill .) so já k . . . . . . . . . . . . . . . . . + . h im an to gl os su m h ir ci nu m (l .) sp re ng . . . . . . . . . . . . . . . . . + . . va le ri an a tu be ro sa l . . . + . . . . . . . . . . . . . . . . r hi na nt hu s al ec to ro lo ph us (s co p. ) p ol lic h . . . . . . . . . . . . . . . . + . . ly ge ost ip et ea d ac ty lis g lo m er at a l . s ub sp . h is pa ni ca (r ot h) n ym an 1 1 1 1 + + + . 1 + 2 + 1 + 2 + . . + b ra ch yp od iu m re tu su m (p er s. ) p . b ea uv . + . 2 + 2 . 2 1 1 2 1 + 2 2 2 2 . . . si xa lix a tr op ur pu re a (l .) g re ut er & b ur de t + + + + + . . + . . + + + + + + . + + m ic ro m er ia g ra ec a (l .) b en th . + + + . . + . + + + . . . . 1 1 + + + h yp ar rh en ia h ir ta (l .) st ap f . 2 . 2 2 2 1 + + + + . . . . 2 . . . d ri m ia p an cr at io n (s te in h. ) j . c . m an ni ng & g ol db la tt . + . + + . + . + + + . . + + + . . . u ro sp er m um d al ec ha m pi i ( l .) f. w . s ch m id t + 1 + 1 . + + + . . . + . . . . . . . b itu m in ar ia b itu m in os a (l .) c . h . s tir t. . . . + 1 2 2 . + . + . . . . + . . . p al le ni s sp in os a (l .) c as s. s ub sp . s pi no sa . + . + . + + . . . . + + . . + . . . a m pe lo de sm os m au ri ta ni cu s (p oi r.) t . d ur an d & s ch in z . . . . + . + . . . . . . . + . . . . c lin op od iu m n ep et a (l .) k un tz e su bs p. g la nd ul os um (r eq .) g ov ae rt s . . + . . . . . . . . . . . . . . . . hippocrepido-stipion in the pollino massif (italy) acta bot. croat. 75 (1), 2016 r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 h el ia nt he m et ea g ut ta ti tr ifo liu m c am pe st re s ch re be r + + + + + + + . + + + + + + + + 1 . 1 h yp oc ha er is a ch yr op ho ru s l . 1 + 1 + + 1 + . + + + . + + + + . . . b ri za m ax im a l . + + + + . + + . + + + . + + + + . . . li nu m s tr ic tu m s .l. + + + + + + + + . . 1 . + + 1 . . . . c at ap od iu m r ig id um (l .) c . e . h ub b. + + . + . + + . + + + . + + 1 + . . . li nu m tr yg in um l . . + + . + . . + . + + + + . . 1 . . . tr ifo liu m s te lla tu m l . + + + + + . + . . . . . . + . . + . . e up ho rb ia e xi gu a l . + + + + + + + . . . . . . . . . . . . f ila go p yr am id at a l . + + . . . . + . . . . . + + + . . . + a st er ol in on li nu m -s te lla tu m (l .) d ub y + + + . + 1 + . . . . . . . . . . . . h el ia nt he m um s al ic ifo liu m (l .) m ill . + + . + + . . . . + . . . . + . . . . o no ni s re cl in at a l . . + . . . . . + . + + . . . + + . . . m ed ic ag o m in im a (l .) l . . . . + + + + . + . . . . . . . . . + o no br yc hi s ca pu tga lli (l .) l am . . + . + + . . . + . . . . . + + . . . a ir a ca ry op hy lle a l . + + + . . . . . . . . . . . + + . . + si de ri tis ro m an a l . s ub sp . r om an a . . . . . + . . + + . . . . . + . . + c re pi s ne gl ec ta l . s ub sp . n eg le ct a 2 . . . . + + . + . . . . . . . . . . tr ac hy ni a di st ac hy a (l .) l in k . . . . . + . . + . . . + . 1 . . . . c ru pi na v ul ga ri s c as s. . . . . . . . . . . . . . . + + + + . lo tu s or ni th op od io id es l . . . . + . . + . . . + . . . . + . . . p la nt ag o be lla rd ii a ll. + + . . . + . . . . . . . . . . . . . a ly ss um s im pl ex r ud ol ph i . . . . . . + . . + + . . . . . . . . vu lp ia m yu ro s (l .) c . c . g m el . . . . . . . . . . + . . + + . . . . . sc or pi ur us m ur ic at us l . . . . . + . . . . . . . . . . + . . . la gu ru s ov at us l . + . . . . . . . . . . . . . . . . . . h ip po cr ep is b ifl or a sp re ng . . . . . . . . . . . . . . . + . . . . h ip po cr ep is c ili at a w ill d. . . . . . . . . . . . . . + . . . . . . tr ig on el la m on sp el ia ca l . . . . . . . + . . . . . . . . . . . . 5 terzi m., d’amico f. s. acta bot. croat. 75 (1), 2016 r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 o th er s pe ci es c ar lin a co ry m bo sa l . + + + + + + + + + + + + + + + . . + . b el la rd ia tr ix ag o (l .) a ll. + + + + + + + + + + + + + + + + . . . b up le ur um b al de ns e tu rr a . + + + . . + . 1 + + + + + 3 + + + + c ru pi na c ru pi na st ru m (m or is ) v is . + + + + + + + + + + + + + + . . . . + av en a ba rb at a l in k + + + + + + . . + + 2 + . + + + . . + r ei ch ar di a pi cr oi de s (l .) r ot h + . + . + + + . + + . + + + + + . . + sh er ar di a ar ve ns is l . + + + + + + + . + . . . + + + + . . + ty ri m nu s le uc og ra ph us (l .) c as s. + + + + + . + . . + + + + + + + . . . n ig el la d am as ce na l . + 1 1 + + . + . + + + . . . + + . . . tr ifo liu m s ca br um l . s ub sp . s ca br um . + + + + . + . + . + . . . + + + . + e ch iu m v ul ga re l . + . + + + . . . + + . . + + + + . . + d as yp yr um v ill os um (l .) p. c an da rg y + + + 1 + . + . + + + . . . . . . . + to rd yl iu m a pu lu m l . . + . + . + + . + + + + . + + . . . . tr iti cu m o va tu m (l .) r as pa il . . . + + + + . . . + + . . + . . + + a sp ar ag us a cu tif ol iu s l . + + . . + + + . . + + . . . + + . . . m ic ro m er ia g ra ec a (l .) b en th . s ub sp . f ru tic ul os a (b er to l.) g ui ne a + . + + . + + . . . + + + + . . . . . p is ta ci a te re bi nt hu s l . s ub sp . t er eb in th us . + . + + + . . 1 + . . . 2 . + . . . vu lp ia c ili at a d um or t. 1 . + 1 + + + . . . + . . . + . . . . c en ta ur iu m e ry th ra ea r af n + + + . . + . . . . . 1 + . . . . + + th es iu m h um ifu su m d c . 1 . . + 1 . 1 + . . . + . + . . . . . se se li to rt uo su m l . + + 1 . . + . . . . + . . + + . . . . a na ga lli s ar ve ns is l . + + + + + + + . . . . . . . . . . . . c or on ill a sc or pi oi de s (l .) w . d . j . k oc h + . + . + + 2 . . . . . . . + . . . . a ni sa nt ha m ad ri te ns is (l .) n ev sk i + 1 + + + + . . . . . . . . . . . . . m ed ic ag o di sc ifo rm is d c . . + . 1 + + + . . . + . . . . . . . . g as tr id iu m v en tr ic os um (g ou an ) s ch in z & t he ll. + . + . . . . . . + . + . + + . . . . tr ig on el la g la di at a m . b ie b. + . + + + + + . . . . . . . . . . . . b ro m us in te rm ed iu s g us s. + + + . + . . . . . + + . . . . . . . 6 hippocrepido-stipion in the pollino massif (italy) acta bot. croat. 75 (1), 2016 r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 c yn os ur us e ch in at us l . . . . + + + . . + . + . . . + . . . . c ar do pa tiu m c or ym bo su m (l .) pe rs . . 1 . + + . . . . . + 3 . . . . . . . o do nt ite s lu te us (l .) c la ir v. . . + + + + . 1 . . . . . . . . . . . p oa b ul bo sa l . + . + . . . . . . . . . . + . . + + . o rn ith og al um m on ta nu m c ir ill o + + + + + . . . . . . . . . . . . . . st ip a ca pe ns is t hu nb . . 1 + + . . + . . . . . . . . . . . . c ar th am us la na tu s l . . . . + . . . . . . 1 + . + . . . . . o ph ry s te nt hr ed in ife ra w ill d. 1 + + . + . . . . . . . . . . . . . . a lli um s ph ae ro ce ph al on l . . . . . . . . + . . + . . . + + . . . p la nt ag o se rr ar ia l . + + . . . . + . . . . + . . . . . . . tr ifo liu m a ng us tif ol iu m l . . . . . . . + . + . . . . . + + . . . b la ck st on ia p er fo lia ta (l .) h ud s. . + . . . + . + . . . . + . . . . . . sa lv ia v er be na ca l . . + + . . . . . . . + + . . . . . . . a lth ea h ir su ta l . . . . . . . . . . + . . + . + + . . . c am pa nu la e ri nu s l . . + + . . . . . . + . . + . . . . . . p til os te m on s te lla tu s (l .) g re ut er . . . . . . . . . + + + + . . . . . . f um an a th ym ifo lia (l .) sp ac h ex w eb b . . . + . . . 3 + . . . . . . . . . . r os tr ar ia c ri st at a (l .) t zv el ev 1 1 1 . . . . . . . . . . . . . . . . a lli um s ub hi rs ut um l . 1 . . + . + . . . . . . . . . . . . . k la se a fl a ve sc en s (l .) h ol ub . . . . . . . . . . + 1 . + . . . . . a ch na th er um b ro m oi de s (l .) p. b ea uv . . . . . . . . . 1 . . . . . + + . . . vu lp ia m ur al is (k un th ) n ee s . 1 . . . . . . . . + . . . . . . . + g yp so ph ila a rr os tii g us s. . . . . . . . + + . . . . . . 1 . . . h yp er ic um p er fo ra tu m l . . . . . . . . + . . . . . . . . + . + u ro sp er m um p ic ro id es (l .) f. w . s ch m id t . + . + + . . . . . . . . . . . . . . li nu m b ie nn e m ill . + . . . . . . + . . . . . . . . . + . d or yc ni um h ir su tu m (l .) se r. . . . + + . + . . . . . . . . . . . . o sy ri s al ba l . . . . . . . . . . + + + . . . . . . . x er an th em um in ap er tu m (l .) m ill . . . + . . . + . . . . . . . . . . . + 7 terzi m., d’amico f. s. acta bot. croat. 75 (1), 2016 r el ev és 1 2 3* 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a lti tu de (m a .s .l. ) 43 3 45 4 44 5 49 8 44 0 54 6 48 0 67 3 53 0 55 0 63 0 65 0 68 3 53 6 55 0 70 0 10 28 98 0 10 10 e xp os iti on (° ) 31 0 90 29 0 0 0 80 24 0 0 0 27 0 20 0 20 0 24 0 28 0 32 0 16 0 34 0 26 0 70 sl op e (% ) 25 10 30 0 0 10 20 20 0 40 40 20 20 15 25 35 10 40 25 c ov er (% ) 10 0 10 0 10 0 95 10 0 90 90 10 0 95 90 95 10 0 75 90 70 80 10 0 95 90 pl ot s iz e (m 2 ) 10 0 10 0 10 0 70 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 80 10 0 10 0 10 0 10 0 10 0 c ar du us n ut an s l . s ub sp . n ut an s . . . . . . . . . . . . + . . . . + + a sp le ni um c et er ac h l . . . . . . . . . . + . . + . . . + . . sp ar tiu m ju nc eu m l . . . . . + . . . . + . + . . . . . . . lo nc om el os n ar bo ne ns is (t or n. in l .) r af . . . . + . + + . . . . . . . . . . . . sp or ad ic s pe ci es 4 4 5 5 3 4 3 1 1 7 6 4 4 1 2 1 8 2 3 sp or ad ic s pe ci es : a ju ga c ha m ae pi ty s g us s. 1 3 (+ ); a lli um fl av um l . 1 3 (+ ); a m m oi de s pu si lla (b ro t.) b re is tr. 1 9 (+ ); a nt he m is a rv en si s l . 1 1 (+ ); a sp ho de lu s al bu s m ill . 1 (+ ); 1 2 (+ ); a st ra ga lu s se sa m eu s l . 7 (1 ); 9 (+ ); b el lis p er en ni s l . 1 8 (+ ); c is tu s cr et ic us l . s ub sp . e ri oc ep ha lu s (v iv .) g re ut er & b ur de t 8 (1 ); c is tu s m on sp el ie ns is l . 6 (+ ); 1 4 (+ ); c le m at is fl am m ul a l . 5 (+ ); c ra ta eg us m on og yn a ja cq . 1 9 (+ ); c re pi s fo et id a l . 6 (+ ); 1 5 (+ ); c ru ci an el la a ng us tif ol ia l . 1 7 (+ ); c yn og lo ss um c he ir ifo liu m l . s ub sp . c he ir ifo liu m 2 (+ ); d el ph in iu m h al te ra tu m s m . s ub sp . h al te ra tu m 1 (+ ); d ia nt hu s br ac hy ca ly x h ue t e x b ac ch et ta , b ru llo , c as ti et g iu ss o 17 (+ ); e up ho rb ia p ep lu s l . 1 (+ ); g er op og on h yb ri du s (l .) sc h. b ip . 4 (+ ); h ed yp no is r ha ga di ol oi de s (l .) f. w . s ch m id t 6 (+ ); h es pe ri s la ci ni at a a ll. 1 7 (+ ); k na ut ia in te gr ifo lia (l .) b er to l. 1 (+ ); 3 (+ ); l eo nt od on ro sa ni i ( te n. ) d c . 1 1 (+ ); l om el os ia b ra ch ia ta (s m .) g re ut er & b ur de t 4 (+ ); 5 (+ ); m al op e m al ac oi de s l . 1 1 (+ ); 1 2 (+ ); m al va c re tic a c av . 3 (+ ); m ar ru bi um v ul ga re l . 1 (+ ); 1 3 (+ ); m ed ic ag o ri gi du la (l .) a ll. 2 (+ ); 4 (+ ); m el ic a ci lia ta l . 4 (+ ); o do nt ite s vu lg ar is m oe nc h 17 (+ ); o no ni s na tr ix l . 1 1 (+ ); 12 (+ ); o ri ga nu m v ul ga re l . 1 2 (+ ); p ar en tu ce lli a vi sc os a (l .) c ar ue l 1 8 (+ ); p ic ri s hi er ac io id es l . 1 1 (+ ); p is ta ci a le nt is cu s l . 1 5 (+ ); 1 6 (+ ); p la nt ag o af ra l . 1 1 (2 ); p la nt ag o la nc eo la ta l . 1 7 (+ ); sa lv ia p ra te ns is l . s .l. 2 (+ ); s ca nd ix p ec te nve ne ri s l . 3 (+ ); 1 7 (+ ); s co rz on er a hi rs ut a l . 1 (+ ); s co rz on er a hi sp an ic a su bs p. n ea po lit an a (g ra nd e) g re ut er 1 (+ ); 4 (+ ); s ed um a cr e l . 1 7 (+ ); s ed um da sy ph yl lu m l . 1 (+ ); s ile ne n oc tu rn a 6 (+ ); s ile ne v ul ga ri s (m oe nc h) g ar ck e 1 (+ ); t ha lic tr um m in us l . 1 (+ ); 1 3 (+ ); t or ili s no do sa (l .) g ae rt n. 2 (+ ); t ri po di on te tr ap hy llu m (l .) fo ur r. 5 (+ ); 7 (+ ); va le ri an a tu be ro sa l . 3 (+ ); v al er ia ne lla m ur ic at a (s te v. e x m . b ie b. ) j .w . l ou do n 3 (+ ); v er ba sc um m ac ru ru m t en . 1 (+ ); 1 9 (+ ); v ic ia c ra cc a l . 1 7 (+ ); v ic ia s at iv a l . 7 (+ ); v in ce to xi cu m h ir un di na ri a m ed ik . 3 (+ ). o nlin e su pp l. a pp en di x 1: d at e an d ge og ra ph ic c oo rd in at es o f r el ev és in th e o nlin e su pp l. ta b. 1 : r el . 1 , 1 9/ 05 /2 01 0, 3 9° 4 9’ 0 8’ ’, 16 ° 17 ’ 1 6’ ’; r el . 2 , 1 9/ 05 /2 01 0, 3 9° 4 9’ 0 6’ ’, 16 ° 17 ’ 1 8’ ’; r el . 3 , 19 /0 5/ 20 10 , 3 9° 4 9’ 0 1’ ’, 16 ° 17 ’ 1 3’ ’; r el . 4 , 1 9/ 05 /2 01 0, 3 9° 4 9’ 1 6’ ’, 16 ° 18 ’ 3 6’ ’; r el . 5 , 1 9/ 05 /2 01 0, 3 9° 4 9’ 1 7’ ’, 16 ° 17 ’ 4 1’ ’; r el . 6 , 1 9/ 05 /2 01 0, 3 9° 5 0’ 1 7’ ’, 16 ° 10 ’ 5 1’ ’; r el . 7 , 1 8/ 05 /2 01 0, 3 9° 49 ’ 3 8’ ’, 16 ° 16 ’ 4 6’ ’; r el . 8 , 2 6/ 06 /2 00 9, 3 9° 5 0’ 1 5’ ’, 16 ° 08 ’ 0 5’ ’; r el . 9 , 2 6/ 06 /2 00 9, 3 9° 5 0’ 0 4’ ’, 16 ° 10 ’ 4 6’ ’; r el . 1 0, 2 5/ 06 /2 00 9, 3 9° 5 0’ 3 8’ ’, 16 ° 12 ’ 4 ’’; r el . 1 1, 2 5/ 06 /2 00 9, 3 9° 5 0’ 3 5’ ’, 16 ° 12 ’ 21 ’’; r el . 1 2, 2 5/ 06 /2 00 9, 3 9° 5 0’ 3 6’ ’, 16 ° 12 ’ 2 2’ ’; r el . 1 3, 2 5/ 06 /2 00 9, 3 9° 5 0’ 5 1’ ’, 16 ° 12 ’ 3 2’ ’; r el . 1 4, 2 5/ 06 /2 00 9, 3 9° 5 0’ 3 6’ ’, 16 ° 11 ’ 0 3’ ’; r el . 1 5, 2 4/ 06 /2 00 9, 3 9° 5 0’ 2 9’ ’, 16 ° 11 ’ 2 8’ ’; r el . 1 6, 24 /0 6/ 20 09 , 3 9° 5 0’ 3 5’ ’, 16 ° 11 ’ 2 1’ ’; r el . 1 7, 2 5/ 06 /2 00 9, 3 9° 5 1’ 3 9’ ’, 16 ° 05 ’ 5 3’ ’; r el . 1 8, 2 6/ 06 /2 00 9, 3 9° 5 2’ 4 1’ ’, 16 ° 02 ’ 5 5’ ’; r el . 1 9, 2 6/ 06 /2 00 9, 3 9° 5 2’ 3 7’ ’, 16 ° 03 ’ 0 0’ ’. o nlin e su pp l. a pp en di x 2: r el ev és a nd a ss oc ia tio ns in cl ud ed in th e da ta se t: ta b. 2 fr om f an el li et a l. (2 00 1: s id er iti do it al ic ae -s tip et um a us tr oi ta lic ae ); t ab . 9 6 fr om b ru llo e t a l. (2 00 1: c ha m ae le on o gu m m ife ri -s tip et um a us tr oi ta lic ae ); t ab . 3 a nd 4 fr om f or te e t a l. (2 00 5: a ci no s ua ve ol en tis -s tip et um a us tr oi ta lic ae a nd c ha m ae cy tis o sp in es ce nt is -s tip et um a us tr oi ta lic ae ); t ab . 1 3, 1 4, 1 5 an d 16 fr om b io nd i a nd g ue rr a (2 00 8: c on vo lv ul o el eg an tis si m ist ip et um a us tr oi ta lic ae , c ar do pa to c or ym bo si -b ro m et um e re ct i, c en ta ur eo a pu la ea nd ro po go n e tu m d is ta ch yi a nd s tip o au st ro ita lic ae -h yp ar rh en iet um h ir ta e) ; t ab . 4 (r el . 1 -7 ) f ro m d i p ie tr o an d w ag en so m m er (2 00 8, p . 1 96 : “ a gg r. a e ph ed ra n eb ro de ns is e s co rz on er a vi llo sa s ub sp . c ol um na e” ); t ab . 2 fr om t er zi e t a l. (2 01 0: p ol yg al o m ed ite rra ne ae -s tip et um a us tr oi ta lic ae , i ri do p se ud op um ila esc or zo ne re tu m c ol um na e an d p ha gn al o ill yr ic ist ip et um fr en ta na e) ; t ab . 1 f ro m d i p ie tr o an d w ag en so m m er ( 20 14 : s tip o au st ro ita lic ae -s es le ri et um ju nc ifo lia e) ; t ab . 1 1 fr om f as ce tti e t a l. (2 01 3: a nt he m id o co lu m na est ip et um a us tr oi ta lic ae ). 8 acta bot. croat. 78 (1), 2019 99 acta bot. croat. 78 (1), 99–101, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0006 issn 0365-0588 eissn 1847-8476 short communication setaria adhaerens (forssk.) chiov. (poaceae), a new alien species in the croatian flora semir maslo primary school, lundåkerskola, södra storgatan 45, 332 33 gislaved, sweden abstract – setaria adhaerens (forssk.) chiov. a pantropical plant species present in some parts of the european continent has been recorded for the first time in croatia in two anthropogenic habitats in dalmatia and one in kvarner bay. the paper presents a short morphological description and photographs of the new alien species, as well as its distribution. a determination key is given for the setaria taxa most frequently found in europe. keywords: alien plants, dalmatia, distribution, morphology, naturalization, setaria corresponding author, e-mail: semmas@edu.gislaved.se introduction the genus setaria p. beauv. belongs to the tribe paniceae, subfamily panicoideae and family poaceae. there are 115160 species worldwide occurring in tropical, subtropical and temperate regions. a total of 66 species and five varieties are present in the old world (morrone et al. 2014). in the european vascular flora eight species have been recorded according to valdés and scholz (2009), among which seven are present in croatia: native s. pumila (poir.) roem. & schult., s. verticillata (l.) p. beauv., s. verticilliformis dumort., s. viridis (l.) p. beauv., and three alien species, s. faberi r. a. w. herrm. (dujmović purgar and hulina 2004), s. italica (l.) p. beauv. and s. parviflora (poir.) kerguélen (milović et al. 2010). s. adhaerens has been reported in europe as native in corse, cyprus, greece, spain and turkey (valdés and scholz 2009), and as introduced in belgium with luxembourg, germany, portugal, sardegna, sweden and ukraine (karlsson 1987, valdés and scholz 2009). material and methods field work was undertaken in 2000, 2009 and 2017. digital photographs and gps coordinates were taken in the field. identification of the specimens was done according to henrard (1940), rominger (1962), belo-correia and costa (1986), karlsson (1987) and amigo et al. (1991). the nomenclature follows the euro-med checklist (euro+med 2006). collected plant specimens were deposited in herbarium croaticum (za) of the faculty of science in zagreb (za 44544 and za 44545). results and discussion the species setaria adhaerens has been discovered in the territory of the republic of croatia in the coastal part of dalmatia and in kvarner bay. among european species, s. adhaerens is most similar to s. verticillata, which is native in most of europe (valdés and scholz 2009). they differ from all the other european setaria species by the setae covered with retrorse prickles. to identify this new species, an adjusted key based on rominger (1962), karlsson (1987) and verloove (2016) is presented here. 1. bristles below spikelets retrorsely barbed, strongly catching to clothes, animal fur, and with other spikes; inflorescence axis scabrous with minute spinules; annual grasses .................................................................................................2 1. bristles below spikelets antrorsely barbed, not catching; inflorescence axis hairy or scabrous; annual or perennial grasses .....................................................................................3 2. margin of leaf sheaths glabrous; inflorescence narrowly conical, blades more or less loosely hairy, spikelets 1.5–2.0 mm long ..................................................................... s. adhaerens maslo s. 100 acta bot. croat. 78 (1), 2019 2. margin of leaf sheaths with long hairs; inflorescence cylindric, blades glabrous, spikelets 2.0-2.2 mm long ........... ........................................................................... s. verticillata 3. upper lemma strongly transversely rugose; bristles usually yellowish, always at least four below each spikelet ..........4 3. upper lemma smooth to rugose; bristles very rarely yellowish, usually 3 below each spikelet ................................5 4. annual grasses; spikelets 3.0-3.5 mm long .........s. pumila 4. perennial grasses; spikelets 2.0-3.0 mm long ...................... ..............................................................................s. parviflora 5. inflorescence axis scabrous with minute spinules .............. ......................................................................s. verticilliformis 5. inflorescence axis hairy ........................................................6 6. leaf blades softly long hairy adaxially; upper glume ca 3/4 as long as upper lemma; upper lemma rugose; panicle nodding from near the base at maturity ................s. faberi 6. leaf blades glabrous; upper glume 3/4 to as long as upper lemma; upper lemma smooth or rugose; panicle erect or nodding from near the base at maturity ............................7 7. upper lemma smooth and shining, exposed at maturity, not falling off at maturity; spikelets up to 3 mm; panicle often nodding from near the base at maturity, often very large and tick, sometimes branched ...................... s. italica 7. upper lemma rugose and dull, not exposed at maturity, falling off at maturity; spikelets up to 2.2 mm; panicle erect, rarely slightly nodding, small to moderately large .. .................................................................................... s. viridis setaria adhaerens (forssk.) chiov., in nuovo giorn. bot. ital., nov. ser., 26: 77 (1919) (syn.: s. verticillata subsp. aparine (steud.) t. durand & schinz), (fig. 1). annual, stem branched at the base, 25-60 cm tall, the culms often geniculate below. the nodes glabrous, leaf sheaths glabrous, the hyaline margins glabrous to the summit. ligule a ring of stiff, white hairs, 1-2 mm long. leaf blades flat, 5-10 mm wide, usually less than 10 cm long, strigose on both surfaces, bearing papillose hairs 1 mm long. panicle strongly verticillate, yellowish-green or purplish, tapering above, 2-6 cm long, the axis retrorsely scabrous-hispid on the angles. spikelets clustered on short branchlets, bristles about 5 mm long, retrorsely scabrous to the base. spikelets 1.5-2 mm long, oblong-elliptic, first glume about ½ as long as the spikelet, obtuse, second glume nearly as long as the spikelet (rominger 1962). according to valdés and scholz (2009) the s. verticillata complex presently contains three species: s. adhaerens, s. verticillata and s. verticilliformis. s. verticilliformis differs most conspicuously from other two in the complex by the presence of antrorsely barbed bristles. the taxonomical status of the species s. adhaerens and s. verticillata has been the subject of several discussions. some authors recognize s. adhaerens and s. verticillata as one species while others recognize them as two. according to clayton (1980), veldkamp (1994) and morrone et al. (2014) s. adhaerens is only one of a number of variants of s. verticillata that do not seem to merit specific rank, while for other authors (henrard 1940, rominger 1962, scholz 1985, belo-correia and costa 1986, karlsson 1987, amigo et al. 1991, danin and scholz 1997, valdés and scholz 2009, verloove 2016) they are clearly separate species. the more temperate s. verticillata has ciliate sheath-margins, glabrous blades, elongated and cylindrical inflorescence and spikelets over 2 mm long. the more tropical s. adhaerens has glabrous sheath-margins, strigose blades, shorter and clear conical inflorescence (obviously widest at base) and spikelets under 2 mm long. s. adhaerens also has a shorter panicle, shorter leaves, and a shorter culm height than s. verticillata. plants with antrorsely barbed bristles are occasionally found and described as s. adhaerens var. font-queri calduch (amigo et al.1991). recent genetic researches within the setaria genus indicate that these are two distinct species. wang et al (2009) pointed out that diploid counts for s. verticillata are most likely misidentified s. adhaerens plants. the authors suggest that diploid s. verticillata should be named s. adhaerens and the tetraploid form be kept the commonly accepted name of s. verticillata. the first findings of this species for croatia are from southern dalmatia, at veliki zaton near dubrovnik in 2000, where it was found in flowerbeds and ruderal places near the local church (42°41'30" n, 18°02'23" e) and independentfig. 1. setaria adhaerens in the vicinity of the former dugi rat carbide and ferroalloys factory near split, a) habitat, b) upper part of plant, c) panicle, d) leaf sheath (photos by semir maslo). setaria adhaerens in croatia acta bot. croat. 78 (1), 2019 101 ly in the same year from the vicinity of senj in kvarner bay (44°57'6" n, 14°55'41" e); at the motel kalič; ruderal places, leg. n. jogan, 19.9.2000 (n. jogan pers. comm.). subsequently, the species was also recorded in 2009 in the vicinity of dugi rat near split (43°26'18" n, 16°38'36" e) (fig. 2). about twenty specimens were recorded growing on waste places in the area of the former carbide and ferroalloys factory, together with some other alien grasses such as: ceratochloa cathartica (vahl) herter, paspalum dilatatum poir. and setaria parviflora (poir.) kerguélen. upon re-visit of the site in july 2017, i observed that the number of individuals was almost unchanged. in my opinion s. adhaerens has good prospects of spreading further in croatia, especially in dalmatia. probably it already occurs in the country more widely than the new records indicate, but it is most likely to have been overlooked by collectors because of its similarity to other setaria taxa, especially s. verticillata. it is not entirely clear if the species is originally native there, but in any case there is an appreciable invasion in southern europe (amigo et al. 1991). according to richardson et al. (2000), the observation period is too short to understand and declare a state of naturalized species. therefore it can be considered a casual alien, probably not yet naturalized, waiting for further field investigations to achieve the proper status attribution. acknowledgements i would like to thank nejc jogan for reporting his own unpublished record, aldin boškailo for the mapping of distribution of species as well as jessica andersson for improving the english of this paper.fig. 2. the distribution of setaria adhaerens in croatia. references amigo, j., bujan, m., romero, i., 1991: révision taxonomique du genre setaria (gramineae) dans la péninsule ibérique. bulletin de la société botanique de france, lettres botaniques 138, 155–165. belo-correia, a. l., costa, m. f., 1986: setaria verticillata (l.) p. beauv. e setaria adhaerens (forssk.) chiov.-i. revista de biologia 13, 117–143. clayton, w. d., 1980: setaria beauv. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea. vol. 5, 263–264. cambridge university press, cambridge. danin, a., scholz, h., 1997: on the occurrence of two taxa of the setaria verticillata complex in israel and the sinai. willdenowia 27, 177–179. dujmović purgar, d., hulina, n., 2004: vineyard weed flora in the jastrebarsko area (nw croatia). acta botanica croatica 63, 113–123. henrard, j. th., 1940: notes on the nomenclature of some grasses. blumea 3, 411–480. karlsson, t., 1987: setaria adhaerens and s. faberi new species to sweden. svensk botanisk tidskrift 81, 305–311. (in swedish). milović, m., mitić., b., alegro, a., 2010: new neophytes in the flora of croatia. natura croatica 19, 407–431. morrone, o., aliscioni, s. s., veldkamp, j. f., pensiero, j. f., zuloaga, f. o., kellogg, e. a., 2014: revision of the old world species of setaria (poaceae: panicoideae: paniceae). systematic botany monographs 96, 161. richardson, d. m., pyšek, p., rejmánek, m., barbour, m. g., panetta, f. d., west c. j., 2000: naturalization and invasion of alien plants: concepts and definitions. diversity and distributions 6, 93–107. rominger, j. m., 1962: taxonomy of setaria (gramineae) in north america. illinois biological monographs 29, 1–132. scholz, h. 1985: setaria p. beauv. in: davis p. h. (ed.), flora of turkey and east aegean islands. vol. 9, 597–600. edinburg. valdés, b., scholz, h., 2009: poaceae (pro parte majore). in: euro+med plantbase – the information resource for euromediterranean plant diversity. retrieved september 2017 from http://ww2.bgbm.org/europlusmed/ veldkamp, j. f., 1994: miscellaneous notes on southeast asian gramineae. ix. setaria and paspalum. blumea 39, 373–384. verloove, f., 2016: setaria beauv. in: manual of the alien plants of belgium. botanic garden of meise, belgium. retrieved september 2017 from alienplantsbelgium.be. wang, y., zhi, h., li, w., li, h., wang, y., huang, z., diao x., 2009: a novel genome of c and the first autotetrapoid species in the setaria genus identified by genomic in situ hybridization. genetic resources and crop evolution 56, 843–850. acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 159 acta bot. croat. 74 (1), 159–164, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication echinochloa colona (l.) link (poaceae), a new species in the fl ora of croatia dario hruševar1*, božena mitić1, dubravka sandev1, antun alegro2 1 university of zagreb, faculty of science, department of botany with the botanical garden, marulićev trg 9 a, hr10000, zagreb, croatia. 2 university of zagreb, faculty of science, department of botany with the botanical garden, marulićev trg 20/ii, hr10000, zagreb, croatia. abstract – during fl oristic research into the eastern parts of mt medvednica (nw croatia), in the period from 2007 to 2010, the neophyte taxon echinochloa colona (l.) link was found. since e. colona is not included in plant identifi cation handbooks, a new determination key for the two closely related taxa of genus echinochloa, presented in croatia, has been prepared, and the biology of this new alien plant is briefl y discussed. keywords: croatia, echinochloa colona, medvednica, neophyte, new species introduction the genus echinochloa beauv. includes about 50 species that are widespread in both tropical and temperate regions of the world in dry or water-fl ooded soils (yabuno 1966, michael 1983). the most widespread species of the genus are echinochloa crus-galli (l.) p. beauv. and echinochloa colona (l.) link (yabuno 1983), two of the worst weeds in crop fi elds (holm et al. 1977). echinochloa colona (l.) link (name accepted from michael 2009) is a grass plant native to india and now widespread, especially beyond 30°n and 30°s latitude (michael 1983). as a cosmopolitan weed, it is known under many synonyms: panicum colonum l., syst. nat. ed. 10, 2: 870 (1759); milium colonum (l.) moench, methodus: 202 (1794); oplismenus colonus (l.) kunth in f. w. h. von humboldt, a. j. a. bonpland and c. s. kunth, nov. gen. sp. 1: 108 (1816); echinochloa zonalis (guss.) parl., fl. panorm. 1: 119 (1839); brachiaria longifolia gilli, ann. naturhist. mus. wien 69: 39 (1966) (clayton et al. 2002) and common names: jungle rice, little barnyard grass, corn panic grass, deccan grass, jungle rice grass, millet rice, southern cockspur, swamp grass (cabi 2012). in europe, the species is established in france, greece, italy, spain, cyprus, the european part of turkey and on the mediterranean islands like balearics, corsica and * corresponding author, e-mail: dario.hrusevar@biol.pmf.hr copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. hruševar d., mitić b., sandev d., alegro a. 160 acta bot. croat. 74 (1), 2015 sicily (pirola 1965, daisie 2012). according to nobanis (2012), the species is also known from sweden, where it was fi rst recorded in 1924 and it is listed in the fl ora of czech republic as a casual neophyte (pyšek et al. 2012). materials and methods study area mt medvednica is 42 km long and approximately 9 km wide (poljak 2007), stretching in a ne-sw direction in northwest croatia, in the continental biogeographical region (cf. radović et al. 2009). here, on only 0.4% of state territory, 1,205 taxa are present, which represent 22% of total croatian fl ora (nikolić and kovačić 2008). in order to protect the fl ora and vegetation, especially the forest habitats, medvednica nature park was proclaimed in 1981 and currently covers an area of 17,938 ha (anonymous 2009b). the researched area (fig. 1), which is heavily anthropogenically infl uenced, occupies 5.5 km2 outside the nature park’s borders. it is located between four settlements – čučerje on the west and vugrovec on the east, the northern border is the road via goranec which connects two places mentioned above, and the southern border is near šimunčevec. although the massif is mainly built of palaeozoic and triassic metamorphic rocks (predominantly green schist) (šikić 1995), the study area is characterised by carbonate and dolomite bedrock covered with limestone-dolomite black soil (kalkomelanosol) and brown soil on the limestone and dolomite (kalkokambisol) (anonymous 2011). according to the climate classifi cation by köppen, mt medvednica belongs to the temperate c climate (warm-temperate rainy clifig. 1. upper left corner – position of mt medvednica in croatia. larger image – medvednica nature park is marked with black line and the studied area is indicated as a white oval circle. echinochloa colona in croatia acta bot. croat. 74 (1), 2015 161 mate – cfb) (bertić et al. 2005) with 1,238 mm of annual precipitation on sljeme (nikolić and kovačić 2008). plant identifi cation and mapping plant identifi cation was done by using relevant the determination key for european vascular fl ora (clayton 1980) and checked again by two additional keys (michael 1983, felger 1990). collected plant specimens were deposited in the zagreb herbarium (za). geocoding of the site was performed with the use of a gps device. results and discussion a small population of echinochloa colona with fewer than ten individuals was found in the area of vugrovec (45°52'52.49"n, 16°06'25.41"e). plants were grown in the ditch alongside the road, in the edge of crop fi eld, mixed with other ruderal species such as artemisia vulgaris l., ambrosia artemisiifolia l. and festuca arundinacea schreb. after being recorded for the fi rst time in the area of mt medvednica (hruševar 2009), the species was also recorded in zagreb (bastijančić 2010). even though the continental climate of croatia is not quite suitable for e. colona, special attention should be given to its potential to become naturalized, especially should it occur in the mediterranean region, where it could become invasive. it is also possible that e. colona was misidentifi ed in previous research, due to its similarity with some unawned varieties of e. crus-galli (michael 1983). until the fi nding of e. colona, there was only one barnyard grass species occurring in croatia: echinochloa crus-galli. this species is well known weed and common grass in the fl ora of croatia (hulina 1998, nikolić 2012), where it forms the alliance panico-setarion sissingh in westhoff et al. 1946 (anonymous 2009a). according to mareković et al. (2009), it is the second most widespread grass species in medvednica nature park. even though e. colona and e. crus-galli are hexaploids, they differ in genome constitution so their f1 hybrids are sterile (yabuno 1962). echinochloa colona (fig. 2) is an annual or occasionally perennial grass, up to 60 cm high. culms are stout, usually reddish-purple, erect, ascending or decumbent, often branching from the base, and rooting at the lower nodes. sheath is 3–7 cm long, compressed, keeled and glabrous. ligule is absent. leaf blade is light green, sometimes with transverse purple bands, fl at, glabrous, elongate, 4–10 cm long, 3–8 mm wide, with occasionally scabrous margins, apex is acute. panicle is erect or nodding, green or purple-tinged, 5–15 cm long. racemes are numerous, 2–4 cm long, spreading, ascending, sometimes branched, the lower ones up to 1 cm apart, the upper ones crowded. spikelets are green tinged with purple, crowded, arranged in circa 4 rows, about 3 mm long, rarely with a short point up to 1 mm long. first glume is 1.2–1.5 mm long, 3-nerved, nearly half as long as the spikelet; second glume is 2.5-3 mm long, 7-nerved; the fi rst lemma is similar to the second glume, fi rst palea ovate, circa 2 mm long, glabrous; second lemma, broadly ovate, circa 2 mm long, glossy. caryopsis is whitish, broadly ovate, 1.7–2 mm long, fl at on one side, convex on the other (wagner et al. 1999). chromosome number is 2n = 6x = 54 (yabuno 1962). e. colona inhabits cultivated areas, waste grounds, ditches and fi elds (cabi 2012), from sea level to the height of 2000 m (holm et al. 1977, lazarides 1980). we supposed that jungle rice was introduced with the import of crop seeds or with the transport of soil, which is possible due to the signifi cant human impact in the researched area, such as traffi c, agrihruševar d., mitić b., sandev d., alegro a. 162 acta bot. croat. 74 (1), 2015 cultural and construction activities. one plant of e. colona can produce from a few thousand (holm et al. 1991, chauhan and johnson 2010) to more than 40,000 seeds (mercado and talatala 1977). the fl owering period starts 3–4 weeks after germination, quickly followed by fructifi cation. the fi rst seeds mature 45 days after fl owering. the minimum, optimum and maximum temperature for germination temperatures are 15, 30 and 40 °c (uremis and uygur 1999). it propagates mostly by seeds but also vegetatively, by rooting at its nodes. the seeds are spread by farm machinery, in crop seed, in irrigation canals, on the feet, fur, feathers, and skin of rodents, birds, and larger animals, including humans (holm et al. 1991). e. colona has a wide ecological niche, it is adapted to full sunlight or partial shade, and grows on loam, silt and clay soils (manidool1992). according to holm et al. (1977), e. colona is associated with 35 crops in more than 60 countries. determination key for echinochloa species in croatia 1. spikelets 1.5–3 mm, regularly arranged in 4 rows. first glume regularly half the length of the spikelet. sterile lemma and second glume often with a few hairs or short spines but not beset with harsh spines. long bristles mostly absent from main axis and branches of infl orescence, occasionally a few scattered along the branches and clustered at the nodes. anthers purple. caryopses whitish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . echinochloa colona 2. spikelets mostly more than 3 mm, irregularly arranged. first glume about one-third the length of spikelet. sterile lemma and second glume with harsh spines. long bristles along main axis and branches of infl orescence present or absent. anthers brown or yellow. caryopses brownish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . echinochloa crus-galli fig. 2. habitus of echinochloa colona (photo by dario hruševar). echinochloa colona in croatia acta bot. croat. 74 (1), 2015 163 acknowledgements we wish to thank our colleague igor boršić for providing a map of the researched area and valuable comments on the manuscript. references anonymous, 2009a: annex i of the ordinance on amendments to the ordinance on habitat types, habitat map, threatened and rare habitat types and measures for conserving habitat types (in croatian). offi cial gazette 119/09. anonymous, 2009b: law amendments to the act to designate the western part of the medvednica nature park (in croatian). offi cial gazette 25/09. anonymous, 2011: field course in pedology – medvednica (in croatian). faculty of forestry, university of science, zagreb. bastijančić, m., 2010: urban fl ora of the zagreb’s settlements volovčica and ferenščica (in croatian). diploma thesis. department of biology, faculty of science, university of zagreb, 1–77. bertić, i., šehić, d., šehić, d., 2005: geographical atlas of croatia (in croatian). europapress holding, zagreb. cabi, 2012: invasive species compendium. retrieved september 2012 from http://www. cabi.org/ chauhan, b. s., johnson, d. e., 2010: implications of narrow crop row spacing and delayed echinochloa colona and echinochloa crus-galli emergence for weed growth and crop yield loss in aerobic rice. field crops research 117, 177–182. clayton, w. d., 1980: echinochloa beauv. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. 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rice research institute and international weed science society, manila. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 303 acta bot. croat. 74 (2), 303–316, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0028 diatom composition of the rheoplankton in a rhithral river system ágnes bolgovics1*, éva ács2, gábor várbíró3, keve tihamér kiss2, balázs a. lukács3, gábor borics3 1 university of eötvös loránd, 1/a, pázmány péter str. h-1117 budapest, hungary 2 mta centre for ecological research, danube research institute, jávorka s. u. 14, h-2131 göd, hungary 3 mta centre for ecological research, danube research institute, department of tisza research, 18/c., bem square, h-4026 debrecen, hungary abstract – diatom composition of the rheoplankton (phytoplankton) in the sajó-hernád river system (slovakia and hungary) was studied. forty two sample sites were designated on the watershed from source to mouth of the two rivers and their tributaries. samples were taken in july 2012. altogether, 258 diatom taxa were identifi ed. the microfl ora was dominated by tychoplanktonic elements. according to the relative abundance of the occurring taxa, four groups could be distinguished. differentiation of these groups was confi rmed by differences in the habitat characteristics, viz. altitude, width of watercourse, macrophyte coverage and river bed material. diversity of diatom taxa in the phytoplankton was also studied. a positive relationship was found between the macrophyte coverage and the simpson and the shannon indices. in contrast, a negative relationship was shown between the macrophyte coverage and berger-parker diversity, in which metric the role of the dominant taxa is emphasized. although the phytoplankton in rhithral rivers is infl uenced by stochastic events, our results reveal that geographical and hydromorphological characteristics of the rivers and coverage of macrophytes can also play role in shaping the composition and diversity of the phytoplankton. keywords: diversity, hydromorphological variables, rhithroplankton introduction in parallel with the physical constraints, structural and functional characteristics of a stream, communities show considerable changes in rivers moving from source to mouth. the biota of the upper sections consists primarily of benthic elements and their survival is essentially based on non-native matter and energy input (vannote et al. 1980). in the lower, * corresponding author, e-mail: bolgovics.agnes@okologia.mta.hu bolgovics á., ács é., várbíró g., kiss k. t., lukács b. a., borics g. 304 acta bot. croat. 74 (2), 2015 potamal sections of the rivers the native primary production of phytoplankton communities becomes dominant and provides carbon sources for the decomposers (tamás-dvihally 1993, vörös et al. 2000, thorp and delong 2002). the composition of phytoplankton communities shows continuous changes from headwaters to alluvial sections, which can be demonstrated by the ratio of tychoand euplanktonic algae (vannote et al. 1980). investigating the phytoplankton of the tisza river (hungary) uherkovich (1966 a, b) identifi ed three algal-based river regions: rheon, rheoplankton and plankton. the rheon section is free of algae, in the rheoplankton section tychoplanktonic elements dominate, while in the plankton section euplanktic algae prevail. it has been also demonstrated that the longitudinal variation of the algal assemblages strongly depends on current hydrological conditions (uherkovich 1971). during fl oods the regions shift downward, and the planktonic region may disappear. this concept is not restricted to the tisza river catchment, but can be applied to other river systems (stanković et al. 2012, abonyi et al. 2014). in middle and low discharge periods in the lower sections of alluvial rivers high biomass phytoplankton assemblages might develop, dominated mostly by chlorococcaleans and centric diatoms (schmidt 1994, schmidt et al. 1994, kiss and genkal 1996, kiss and schmidt 1998, várbíró et al. 2007, kiss et al. 2012). it has been also shown that concerning the dominant algae, the phytoplankton of large potamal rivers is similar to that of shallow turbid lakes (reynolds et al. 1994). the potamoplankton of large rivers has been extensively studied (kiss 1987, kiss and genkal 1996, reynolds and descy 1996, kiss et al. 2002, kiss and ács 2009), but the phytoplankton of the upper river segments has received much less attention. what we know from the sporadic studies on stream phytoplankton is that it is dominated by benthic elements, mostly by diatoms (szemes 1948, 1967a, 1967b, váncsa 1974, 1976, 1977). the low number of studies dealing with stream phytoplankton can be explained by the fact that most algological investigations are aimed at assessing the ecological state of water bodies, and since quality assessment in the upper sections of the rivers is based on benthic communities, phytoplankton is generally not considered. however, phytoplankton composition in the lower river segments should be strongly infl uenced by the inocula conveyed by the upper tributaries, which necessitates the investigation of the phytoplankton of these less studied systems. in previous studies (uherkovich 1971, rojo et al. 1994) only the basic types of river phytoplankton assemblages were described; viz. rheoplankton dominated by tychoplanktonic taxa and potamoplankton in which euplanktonic elements prevail. investigating the diversity of phytoplankton in these two river types borics et al. (2014) demonstrated that diversity trends are determined by different underlying mechanisms. in rhithral river systems stochastic processes shape the diversity patterns, while in potamal rivers the role of competition seems to be of great importance. diversity trends were also dependent on the metrics used for diversity calculations. much less attention has been paid to the detailed description of the phytoplankton in rhithral rivers (blum 1954, 1957) than to the potamoplankton of the lower river segments. therefore, the aim of the present study was to explore the composition and diversity of the phytoplankton in a rhithral river system. since previous results (váncsa 1976, pozderka et al. 2014) suggested that diatoms are major components of the rheoplankton, we focused exclusively on this group. we hypothesized that (i) planktonic diatom assemblages are not just stochastic mixtures of species, but are tightly coupled to stream types; (ii) diversity of the planktonic assemrheoplankton in rhithral rivers acta bot. croat. 74 (2), 2015 305 blages is infl uenced by the hydro-morphological types of the rivers, and (iii) increases with the size of the water bodies. material and methods study area all rivers sampled in this study belong to the sajó river watershed (central europe, slovakia and hungary). sajó is the second largest right-side tributary of the tisza river containing streams of 1st to 6th orders (stenger-kovács et al. 2014). the river rises at stolické vrchy 1,280 m a.s.l. (slovakia), and enters the tisza at 95 m a.s.l. in hungary, where the hungarian great plain meets the foothills of the bükk mountains. the river’s catchment area is 12,708 km2, its length 223 km, average discharge at the river mouth is 60 m3 s–1. the mean water residence time according to leopold et al. (1995) and soballe and kimmel (1987) is 14.9 days. annual mean precipitation in the watershed is 600–1,250 mm, annual mean temperature is 4.5–11.0 °c. the upper sections of the river sajó and its tributaries are typical mountain rivers and although the lower river enters the northern part of the hungarian lowland the river keeps its rhithral character, with prevailing coarse substrates (macro and mezolithal). sampling samples were collected from 42 sampling sites, which covered the whole watershed (fig. 1) in july 2012. the sampling points were selected to include the main sections of the sajó and hernád rivers and the relevant tributaries. twenty liters of water was fi ltered through plankton net (mesh size 10 μm) and concentrated to 50 cm3. samples were taken from the thalweg. the samples were fi xed with formaldehyde (applying 4% fi nal concentration) and stored in plastic containers (cen 2003). environmental variables (water temperature, ph, dissolved oxygen, electrical conductivity) were measured on the spot with a fig.1. watershed of the sajó river and the sampling sites designated. dashed line marks the hungarian-slovakian border. identical symbols indicate sampling sites which belong to the same cluster. h – hungary, s – slovakia. bolgovics á., ács é., várbíró g., kiss k. t., lukács b. a., borics g. 306 acta bot. croat. 74 (2), 2015 hach-lange hq40d water quality fi eld kit. other variables (water depth, width of the river bank, relative frequency of dominant substrates and percentage cover of the main life forms of macrophytes (euhydrophytes and helophytes) were also estimated in parallel with the samplings. for these parameters depending on the size of the rivers 500–1,000 m long river sections were surveyed. the relative abundance of the various sediment types provide useful information on the velocity of water currents, and help in characterising the various types of rhithral river systems. sample processing to study the diatom components of the microfl ora we prepared permanent slides. for the removal of organic matter the samples were digested using h2o2 in a water bath (60 °c), and a drop of hcl was also added to the samples to remove calcium carbonate (cen 2003). after fi nishing digestion the frustules were washed in distilled water and mounted in cargille meltmount mounting medium (kelly et al. 1998) (refracting index = 1.704). cleaned diatoms were identifi ed and counted under oil immersion at a magnifi cation of 1000× with the application of differential interference contrast (dic). to equalize the counting effort 400 valves were counted in each sample. identifi cation of diatom species was performed according to krammer and lange-bertalot (1986–1991), krammer (2003) and hofmann et al. (2011). data analysis a cluster analysis based on euclidean distance and using ward’s (1963) agglomeration algorithm was applied to phytoplankton data with a view to identifying distinct, empirical clusters. a kruskal-wallis anova was used to test the signifi cance of the relationship between environmental variables and diversity indices. indicator value analysis (indval) (dufrene and legendre 1997) was used to identify those species that can be considered to be indicators of the groups identifi ed by the cluster analysis. the value of the indval index reaches its maximum (1.0) if all individuals of a species are found in one defi nite group of sites (specifi city), and if the species can be found in all sites of that group (fi delity) (dufrene and legendre 1997). we used a sample-based species accumulation curve (colwell et al. 2004) for the prediction of species richness which implements the analytical solution known as »mao tau«, with standard deviation. the species accumulation curve and the cluster analysis were made with the past software package (hammer 2001). in the various metrics used for characterizing diversity of biotic communities different weights are given to the dominant taxa, and thus, the metrics capture different aspects of diversity. to describe all relevant aspects of diversity tóthmérész (1998) proposed the application of special cases of rényi’s entropy (eq. 1). the higher the value of the scale parameter (α) the higher weighting the given to the most abundant taxa. hr0 is the logarithm of species richness; hr1 is the shannon diversity (eq. 3); hr2 is the simpson diversity; hr∞ is the berger-parker index (eq. 5). eq. 1 where α ≥ 0 and α ≠ 1 s i i 1 1 hr log p 1       rheoplankton in rhithral rivers acta bot. croat. 74 (2), 2015 307 eq. 2 eq. 3 eq. 4 eq. 5 pearson’s (1897) correlation coeffi cient was applied to explore the relationship between environmental variables and diversity metrics. family-wise bonferroni corrections were used to decrease the risk for a type i error in pairwise comparisons. results phytoplankton associations based on diatom composition four distinct groups could be distinguished (fig. 2). the results of the indval also supported the presence of the four groups identifi ed by the cluster analysis. the indval analysis showed that several species had signifi cant (p < 0.05) specifi city for and fi delity to the groups (tab. 1). tryblionella levidensis, cocconeis neodiminuta, didymosphaenia geminata, hannaea arcus, navicula splendicula, placoneis elginensis were characteristic for the fi rst group. in the second group cymatopleura elliptica, fallacia 0hr log s s 1 i i1 i 1 lim hr hr p log p     s 2 2 i i 1 hr log p    ihr log(max p ) fig. 2. dendrogram of sampling sites based on diatom composition of the rhithroplankton. bolgovics á., ács é., várbíró g., kiss k. t., lukács b. a., borics g. 308 acta bot. croat. 74 (2), 2015 subhamulata, navicula antonii had the highest fi delity values. gyrosigma attenuatum, aulacoseira granulata and gomphonema parvulum had high indicator and low p values in the third group. in the fourth cluster nitzschia capitellata and thalassiosira lacustris were the most characteristic elements. the relationship between the groups identifi ed by the cluster analysis and the relevant physicochemical and hydromorphological variables and macrophyte coverage was also studied (fig. 3). high-altitude rivers were characteristic of the fi rst group. small middle tab. 1. species considered as indicators of the river clusters (1–4) by the indval analysis. indicator values (ind.val.) and p–values are shown. groups ind.val. p tryblionella levidensis 1 36.7 0.074 cocconeis neodiminuta 1 36.4 0.045 didymosphaenia geminata 1 27.3 0.052 hannaea arcus 1 28.2 0.137 navicula splendicula 1 22.1 0.104 placoneis elginensis 1 36.4 0.054 amphora veneta 2 37.1 0.056 cymatopleura elliptica 2 49.4 0.008 fallacia subhamulata 2 41.7 0.035 navicula antonii 2 41.8 0.033 rhopalodia gibba 2 23.7 0.119 nitzschia dissipata 3 32.9 0.129 sellaphora bacillum 3 30.4 0.118 tryblionella constricta 3 34.8 0.100 gyrosigma acumium 3 40.2 0.065 gyrosigma attenuatum 3 58.5 0.012 fragilaria ulna v. acus 3 38.3 0.066 gomphonema parvulum 3 42.1 0.022 nitzschia gracilis 3 31.8 0.098 nitzschia inconspicua 3 37.3 0.106 nitzschia intermedia 3 37.7 0.040 aulacoseira granulata 3 53.5 0.012 handmannia balatonis 3 22.8 0.122 fragilaria delicatissima 4 23.1 0.117 gomphonema angustatum 4 36.8 0.061 navicula erifuga 4 21.9 0.117 nitzschia capitellata 4 57.8 0.015 surirella bifrons 4 21.6 0.118 thalassiosira lacustris 4 49.3 0.011 rheoplankton in rhithral rivers acta bot. croat. 74 (2), 2015 309 alti tude rivers with coarse substrates constituted the second river cluster. in the third group middle altitude rivers (200–300 m) with relatively high macrophyte abundances were found. large lowland rivers with fi ne sediments constituted the fourth cluster. diversity the calculated species diversity metrics showed a very poor relationship with the measured environmental variables (tab. 2). the value of the pearson correlation coeffi cient was low in the cases of all the indices. relatively high values (> 0.3) were found between the macrophyte coverage and shannon and simpson indices. a negative relationship was found between the macrophyte coverage and the berger-parker index of dominance (–0.31). regarding their diversity, the four river clusters showed remarkable differences. in the case of the high altitude rivers the species richness was high. however, occasionally some species occurred in high relative abundance in the samples, which is refl ected in the high values of the berger-parker dominance index. in the second river group the low species numbers were associated with high dominance index values. a high species number could be observed in the third river group. however the occurrence of species was well balanced in this group, which was clearly illustrated by the low value of the berger-parker dominance index. similarly to the fi rst group, in the fourth river group both richness and dominance values were high (fig. 4). fig. 3. the distribution of the relevant environmental variables in the four river clusters (1–4). the line graphs indicate mean values ± standard errors; same letters indicate homogenous groups according to kruskall–wallis anova (p < 0.05). bolgovics á., ács é., várbíró g., kiss k. t., lukács b. a., borics g. 310 acta bot. croat. 74 (2), 2015 the observed number of species was related to the very high species diversity of the watershed. however, we also wanted to know how large the potential species pool of the sajó river’s watershed is; therefore, a species accumulation curve was used to characterize the relationship between the sample number and species richness (fig. 5). the relationship could be described by a power function: y = 52.816 × x0.4326; where x is the sample number and y is the species richness of the watershed. the lack of asymptote means that in case of additional samplings increase in the number of taxa is expected. discussion algae suspended in lotic systems are commonly referred to as potamoplankton (kalff 2002). however in recent studies this term has been applied to the plankton of large potamal rivers (stoyneva 1994, gosselain et al. 1998). since the potamoplankton consists pritab. 2. correlation matrix of the river’s attributes and diversity indices. bolded values indicate signifi cant relationships. cpom – coarse particulate organic matter, fpom – fi ne particulate organic matter. river’s attributes taxa simpson shannon berger-parker width of fl oodplain (m) –0.18 –0.05 –0.07 0.06 maximal width of watercourse (m) –0.05 0.07 0.07 –0.03 maximal water depth (m) –0.10 0.03 0.03 0.03 width of watercourse (actual) (m) –0.07 0.03 0.05 0.01 average water depth (actual) (m) –0.24 –0.23 –0.17 0.24 temperature (°c) 0.08 0.02 0.11 0.00 ph –0.12 0.04 0.05 0.05 conductivity (μs cm–1) 0.24 0.28 0.29 –0.21 megalithal > 40 cm 0.01 –0.16 –0.09 0.24 natural macrolithal > 20–40 cm –0.07 –0.05 –0.08 0.07 artifi cial macrolithal > 20–40 cm 0.00 0.11 0.07 –0.14 mezolithal > 6–20 cm –0.03 –0.15 –0.15 0.23 microlithal > 2–6 cm –0.11 –0.02 –0.10 –0.11 akal > 2 mm–2 cm 0.01 0.13 0.11 –0.15 psammal > 6 μm–2 mm 0.21 0.21 0.26 –0.25 argyllal < 6 μm –0.16 –0.27 –0.25 0.25 macro-algae (%) 0.23 0.04 0.07 –0.05 micro-algae (%) –0.15 0.11 0.04 –0.15 submerged macrophytes (%) 0.25 0.22 0.30 –0.24 emerged macrophytes (%) 0.23 0.31 0.36 –0.35 living terrestrial macrophytes (%) 0.21 0.15 0.19 –0.17 xylal (%) –0.08 –0.17 –0.19 0.15 cpom (%) 0.15 0.16 0.15 –0.22 fpom (%) –0.05 0.21 0.16 –0.28 rheoplankton in rhithral rivers acta bot. croat. 74 (2), 2015 311 marily of euplanktonic elements, using this term for the plankton of the upper river segments where tychoplanktonic elements prevail seems ambiguous. therefore, we propose to use the term rhithroplankton for the planktonic communities of the upper, rhithral rivers. fig. 4. the distribution of the diversity indices in the four river clusters (1–4). the line graphs indicate mean values ± standard errors; same letters indicate homogenous groups according to kruskall–wallis anova (p < 0.05). fig. 5. species accumulation curve: relationship between the number of collected samples and the predicted number of diatom taxa in the sajó river watershed. dashed lines indicate 95% confi dence interval. bolgovics á., ács é., várbíró g., kiss k. t., lukács b. a., borics g. 312 acta bot. croat. 74 (2), 2015 phytoplankton associations várbíró et al. (2007) differentiated 8 riverine phytoplankton assemblages including one benthic type and seven others, mostly transitional and typical potamal assemblages, and impacted ones. in this study the so called »benthic type« was investigated in high spatial resolution. the presence of the four well delineated phytoplankton groups shown in this study clearly demonstrates that rhithroplankton assemblages cannot be considered as a simple stochastic co-occurrence of benthic species. the fi rst bifurcation in the dendrogram was strongly supported by the altitudinal differences of the rivers in the two groups. although the additional bifurcations in the group of middle and low altitude rivers were also supported by some hydrological and/or biological variables, in these groups spatial effects occasionally overcame the environmental effects. this was evidenced by the fact that spatial proximity of sampling sites sometimes was associated with similarity in species composition. this kind of spatial autocorrelation was also demonstrated for other microscopic systems (heino et al. 2010). the spatial effect was not characteristic of the fourth group. the three sampling points belonging to this group were situated far from each other. however, the hydromorphological characteristics of the groups were similar. the fi ne substrate (argillaceous) indicates low relief of the river valleys. two points were at the lowest part of the tributary, while the third point can be found in the upper, impounded stretch of the hernád river. in these sampling sites as well as the planktonic forms (aulacoseira granulata, thalassiosira lacustris) several benthic taxa (cymatopleura elliptica, gyrosigma attenuatum, nitzschia capitellata) had high indval values. this can be explained by the fact, that these species frequently occur in lentic environments (szabó et al. 2005), which are more characteristic of the lower parts of the rivers’ watershed. diversity although the values of diversity metrics are exposed to disturbances entering the systems (borics et al. 2013), these indices are the most frequently used quantitative descriptors of community properties (hacker and gaines 1997). high diversity values might refer to complexity, stability, or to the ecological state of the systems. however, phytoplankton of the rhithral rivers is not a community in the traditional sense of the term. it can be considered an eclectic mixture of benthic and euplanktonic species, which are entrained into the suspension from various habitats. thus, the plankton integrates the species arriving from benthic substrates, ponds, impoundments, pools and shallows of the rivers (stoyneva et al. 1994, borics et al. 2007). since the rhithroplankton diversity refl ects the habitat diversity of the river catchment (borics et al. 2014), artifi cial modifi cation of the watershed contributes to the increase of the phytoplankton diversity. the high number of the euplanktonic species observed in the samples is partly attributed to this impact. occurrence of the species cyclotella meneghiniana, aulacoseira granulata or aulacoseira muzzanensis can be considered natural in rivers’ phytoplankton (várbíró et al. 2007). these taxa can fl ourish even in benthic environments (istvánovics et al. 2011). however, occurrence of other centrics (thalassiosira lacustris) refers to the presence of large lentic habitats, or a slightly saline environment, which was not characteristic for the natural catchment of the sajó river. in the middle sections of the sajó watershed main channel impoundments and several offriver reservoirs were established which might serve as potential sources of algal inocula. rheoplankton in rhithral rivers acta bot. croat. 74 (2), 2015 313 as to the diversity, our expectation was that diversity would increase in parallel towards the larger rivers, thus, variables like water depth, width of the water-course should correlate with the increase of diversity metrics. in contrast, we found that the only variable that showed signifi cant relationship with diversity metrics was the abundance of macrophytes. emergent and submerged macrophytes provide prominent substrate for benthic diatoms (passy 2007), therefore the presence of macrophytes largely determines the species composition of rhithroplankton. this suggests that the local increase in benthic habitat diversity can play important role in shaping rhithroplankton species diversity. phytoplankton diversity is highly sensitive to environmental disturbances (sommer et al. 1993) and thus shows considerable spatial and temporal variability both in rivers and lakes. it is indisputable that detailed description of diversity needs long-term monitoring data for the phytoplankton. these kinds of data have been restricted to the potamal sections of the rivers; for the rhithral sections only sporadic data are available. in conclusion, the high spatial resolution snapshot survey performed on the sajó-hernád river system provided 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(hyacinthaceae) taxa from serbia and hungary and their taxonomic implication andrijana m. andrić*, milica m. rat, lana n. zorić, jadranka ž. luković department of biology and ecology, faculty of science, university of novi sad, trg dositeja obradovića 2, 21000 novi sad, serbia abstract – anatomical characters of two morphologically similar ornithogalum taxa, o. umbellatum and o. divergens, were investigated. an analysis of leaf and scapus cross-sections was performed on plants from ten populations from serbia and hungary, using light microscopy. the aim of this research was to give data about the qualitative and quantitative anatomical characteristics of these taxa, in order to evaluate their taxonomic signifi cance and single out distinctive anatomical features, as well as to contribute to the knowledge of the genus ornithogalum in the studied region. on the basis of the variability of anatomical characters, similar populations formed two clusters, joining the plants previously determined as o. divergens and o. umbellatum. the two taxa signifi cantly differed for most of the quantitative leaf and scapus characters. since only quantitative differences were recorded in this research, anatomical characters could not be solely used to separate these two taxa. however, the results of anatomical investigations are consistent with the results of previous morphological and genetic analyses; therefore anatomical parameters could be useful as additional taxonomic characters. key words: anatomy, leaf, ornithogalum, scapus * corresponding author, e-mail: andrijana.andric@dbe.uns.ac.rs introduction genus ornithogalum l. is one of the most abundant in species and taxonomically the most interesting in the family hyacinthaceae batsch ex borckhausen 1797 (order asparagales bromhead, subfamily ornithogaloideae) (manning et al. 2009, martínez-azorín et al. 2011). the defi nition of taxa within this group has troubled taxonomists for a long time (martínez-azorín et al. 2011); an accurate number of belonging species is still a controversial issue. ornithogalum is native in europe, asia (as far as afghanistan in the east) and africa, where it is widely distributed (martínez-azorín et al. 2010). the mediterranean and south africa are considered to be the centers of distribution (zahariadi 1980, cullen 1984). natural habitats of these bulbous monocots are very heterogeneous; they are in bloom in spring and summer and the fl owers are entomophilous. these plants are important hosts for insects, especially for some groups of hoverfl ies, whose adults feed on pollen and nectar and larvae develop in the bulbs of these geophytes (vujić et al. 2012). some species of the genus are of importance in the fl ower trade, being grown as cut fl ower or potted plant crops, while some are poisonous to livestock and considered weeds (obermeyer 1978, littlejohn and blomerus 1997). eleven subgenera and 34 species of the genus ornithogalum have been described in the flora europaea (zahariadi 1980) and ornithogalum umbellatum l. 1753 and ornithogalum divergens boreau 1887 are given as separate species. thirteen ornithogalum species were recorded in the flora of serbia (diklić 1975), whilst eight were recorded in hungarian fl ora (soó 1973). o. divergens was described as a subspecies of o. umbellatum in both of them. ornithogalum umbellatum is a type species of the genus ornithogalum. it is widespread in europe, north africa and southwestern asia and was introduced in north america (europlusmed 2006, efloras 2008). the species rank of this taxon is not questionable; however, the intraspecifi c systematics are often interpreted differently. the taxonomic position of ornithogalum divergens has had lots of different interpretations (martínez-azorín et al. 2009); it was considered a hexaploid form of the polyploid complex of o. umbellatum (moret et al. 1991, moret 1992), a subspecies, a variety and it has also been widely misidentifi ed as andrić a. m., rat m. m., zorić l. n., luković j. ž. 68 acta bot. croat. 75 (1), 2016 o. umbellatum. nevertheless, martínez-azorín et al. (2009, 2010) presented its nomenclature and taxonomy as well as its ecology and distribution and described this species as clearly different from o. umbellatum s. s., primarily for its ploidy level, morphology of bulbils and infl orescence structure. the need for redefi nition of the infrageneric taxa of the genus ornithogalum was suggested by garbari et al. (2003, 2008) in italy. herrmann (2002) deals with ornithogalum umbellatum s. l. in central europe. a biometric study of o. umbellatum s. l. was done by moret et al. (1991) in france and moret (1992) investigated ploidy levels using numerical taxonomy within this genus, concluding that species from o. umbellatum s. l. from france exhibit considerable variability. systematic studies had been made previously on this complex by van raamsdönk (1986) who singled out o. umbellatum as the most widespread species among european representatives of the genus and a variable wide-ranging taxon with populations showing different morphological character combinations. the nomenclature and taxonomy of o. umbellatum s. l. continue to be investigated in recent years in spain (martínez-azorín et al. 2009), and taxonomic revision of subgenus ornithogalum was also done for the spanish species including ornithogalum divergens (martínez-azorín et al. 2010). many authors consider the genus ornithogalum inconvenient from the standpoint of systematics stating that its morphology is insuffi ciently connected with variations in karyotype (martínez-azorín et al. 2010). since this additionally complicates taxonomic delimitations, anatomical data could make a particularly useful contribution to the solution of taxonomical problems (meriç et al. 2011). studies regarding anatomical characteristics referring to these taxa have not been numerous. a comprehensive view on leaf anatomy in relation to the systematics of the whole hyacinthaceae family was given by lynch et al. (2006). morpho-anatomical variability of the leaves among different taxa of the genus ornithogalum was also investigated by peruzzi et al. (2007). differences in leaf anatomy between ornithogalum nutans l. and ornithogalum narbonense l. from bulgaria were presented by popova and anastasov (1997). morphological and anatomical analyses were conducted for leaf and scapus of o. nutans and ornithogalum boucheanum (kunth) aschers from turkey (meriç et al. 2011). comparative anatomical studies of leaf and scapus features of twelve ornithogalum species, belonging to the subgenera ornithogalum and beryllis, were recently done in central anatolia (öztürk et al. 2014). types of crystals are especially interesting from a taxonomic point of view and tilton and lersten (1981) described idioblasts with calcium oxalate crystals as a type of specialized cells in the gynoecium of ornithogalum caudatum aiton. although the examined taxa have been research subjects in many fi elds worldwide, detailed investigations and publications on their anatomy in particular are very scarce, especially in this part of europe. the main goal of this study is to contribute to the knowledge of the qualitative and quantitative anatomical characteristics of leaf and scapus of two ornithogalum taxa, from several populations from serbia and hungary, with the aim of evaluating their taxonomic signifi cance and singling out distinctive anatomical features potentially useful as diagnostic characters. materials and methods plant sampling was carried out during the fl owering period, in april and may (2008–2013). specimens for analysis were collected from ten different native populations in serbia and hungary (tab. 1, fig. 1). plants were determined and voucher specimens deposited in the herbarium buns (2-1632 – 2-1641) at the department of biology and ecology, faculty of science, university of novi sad. ten specimens from each population were fi xed in 50% ethanol. cross-sections of the middle parts of leaf and scapus, 60 μm thick on average, were made using a leica cm 1850 cryostat at a temperature of – 20 °c. anatomical characters were observed by light microscopy, using the image analyzing system motic 2000. ten cross-sections were examined for each population. in total, 31 quantitative characters were measured and compared, and relative proportions were calculated. the total cross-section area of leaf and scapus was measured and percentages of other parameters were determined considering total cross-section area as 100%. palisade cell indices were calculated as the ratio of their height and width. data were statistically processed using statistica for windows version 12.0 (statsoft, 2014). the signifi cance of differences in the analysed parameters among all tab. 1. analysed populations of ornithogalum umbellatum and o. divergens in serbia (srb) and hungary (hun). abb – abbreviation. taxon sampling locality abb coordinates sampling date voucher o. umbellatum deliblatska peščara, srb de n44°59’16” e20°56’42” 18.04.2008 2-1632 o. umbellatum vršačke planine, srb vr n45°07’22” e21°19’38” 26.04.2009 2-1633 o. umbellatum zlotska klisura, srb zl n44°01’45” e21°57’28” 10.05.2009 2-1634 o. umbellatum valjevo, srb va n44°25’43” e19°54’35” 29.04.2012 2-1635 o. umbellatum mecsek, hun me n46°05’56” e18°13’07” 21.04.2013 2-1636 o. umbellatum szeged, hun sz n46°15’10” e19°38’36” 22.04.2013 2-1637 o. divergens titelski breg, srb ti n45°17’01” e20°14’29” 20.04.2012 2-1638 o. divergens susek, srb su n45°13’51” e19°28’38” 19.04.2012 2-1639 o. divergens novi sad, srb ns n45°14’51” e19°49’43” 26.04.2013 2-1640 o. divergens hódmezövásárhely, hun ho n46°50’42” e20°31’47” 22.04.2013 2-1641 ornithogalum anatomy acta bot. croat. 75 (1), 2016 69 the populations was established using duncan’s test (p ≤ 0.05), and between ornithogalum umbellatum and ornithogalum divergens applying t-test (p ≤ 0.05). the general structure of sample variability was determined by principal component analysis (pca), based on a correlation matrix. multivariate discriminant function analysis (mda) was done in order to test the hypothesis that the analysed sample was composed of groups which differed from each other according to anatomical features of leaf and scapus. any characters that were not signifi cantly different between the two taxa, according to the results of t-test, were not included in mda. results leaf anatomy cross section of the leaf is u-shaped, slightly curved towards the dorsal side (fig. 2). it is signifi cantly smaller in ornithogalum umbellatum, with an average total area of 1.2–2.2 mm2, compared to 2.8–3.7 mm2 in ornithogalum divergens. adaxial side is smooth while the abaxial has 6–8 or 8–10 more or less prominent ribs, in o. umbellatum and o. divergens, respectively. epidermis is single layered, covered with a thin cuticle. epidermal cells are round in shape, or slightly elongated, smaller and more elongated on the abaxial side. the leaves are amphistomatic, with fewer stomata on the adaxial side, positioned at the same level as epidermal cells. there are no trichoma on the epidermis. mesophyll is composed of chlorenchyma differentiated into palisade and spongy tissue. palisade tissue is single layered and present on both adaxial and abaxial sides. however, it is partly missing adaxially due to presence of large lacunae. chlorenchyma cells are absent in the region of the main vein, and broad lacunae appear subepidermally, interspersed with irregularly shaped parenchyma cells. palisade tissue cells are elongated, narrower towards the the adaxial side, perpendicular to lamina surface (fig. 3). chloroplasts are more numerous closer to the cell walls that surround intercellulars. spongy tissue cells are round or irregular in shape, with fewer chloroplasts than palisade cells. intercellulars are larger in spongy tissue (figs. 2, 3). the cells in the middle part of the mesophyll are larger, and do not contain chloroplasts. some of the large mesophyll cells contain bundles of needle-shaped caox raphides (figs. 3 a, c). vascular bundles are arranged in two rows, the bigger ones centrally located, and the smaller ones towards abaxial side. ornithogalum umbellatum has 7–10 vascular bundles of both types, whilst they are more numerous in ornithogalum divergens (10–11 abaxial and 10–12 central vascular bundles). vascular bundles are surrounded with one to two layers of parenchyma cells (figs. 2, 3). large lacunae are present in mesophyll, between the bundles (figs. 3 a, b). scapus anatomy cross section of the scapus is round to oval in shape, with slightly wavy edges and larger total cross-section area in ornithogalum divergens (8.7–10.4 mm2) than in ornifig. 1. localities of the analysed populations in serbia and hungary. for abbreviations of sample localities see tab. 1. fig. 2. leaf cross section: a) ornithogalum umbellatum (vršačke planine), b) o. umbellatum (zlotska klisura), c) o. divergens (susek), d) o. divergens (novi sad). abbreviations: ri – rib. scale bars = 100 μm. fig. 3. leaf cross section: a) ornithogalum umbellatum (deliblatska peščara), b) o. divergens (titelski breg), c) o. umbellatum (szeged), d) o. divergens (novi sad). abbreviations: ade – adaxial epidermis, abe – abaxial epidermis, sto – stomata, pt – palisade tissue, st – spongy tissue, cvb – central vascular bundle, avb – abaxial vascular bundle, la – lacunae, cc – calcium oxalate crystals. scale bars = 100 μm. andrić a. m., rat m. m., zorić l. n., luković j. ž. 70 acta bot. croat. 75 (1), 2016 thogalum umbellatum (3.0–7.7 mm2) (figs. 4 a, b). epidermis is one-layered, with round shaped cells in cross section, glabrous, with thin cuticle and a few stomata (figs. 4 c, d). cortex consists of 4–5 layers of spherical parenchymatous cells, some of which contain chloroplasts. idioblasts with caox raphide crystals are sometimes observed in cortex, beneath epidermis. pith is predominantely composed of parenchymal tissue. in its peripheral part 1 to 3 layers of sclerenchyma occur. small vascular bundles are located within sclerenchyma and just below it. their number is very variable, (12–27 in o. umbellatum and 17–27 in o. divergens), depending on total cross-section area and the area of sclerenchyma, in sense that they are more numerous if these areas are larger. larger, colateral vascular bundles, 10 to 20 of them in o. umbellatum and 17–24 in o. divergens, surrounded with parenchyma sheath, are randomly distributed in the pith. their number is usually smaller than the number of small sclerenchyma bundles. the bundles become larger towards the central part of the scapus. interpopulation variability of the anatomical characters duncan’s test and t-test showed that the plants previously determined as ornithogalum divergens (populations ho, ns, ti and su) signifi cantly differed from those determined as ornithogalum umbellatum (de, vr, zl, va, me and sz), for most of the quantitative characters (tab. 2, online suppl. tabs. 1, 2). considering leaf characters, the two taxa had a similar number of stomata, percentage of vascular tissue and size of adaxial epidermal cells. o. umbellatum had s signifi cantly smaller leaf cross-section area, with better developed palisade tissue, composed of signifi cantly larger and more elongated cells, than o. divergens. although o. umbellatum had a signifi cantly lower number of vascular bundles, the total percentage of vascular tissue was not statistically different between the two taxa. comparative analysis of scapus characters revealed that o. umbellatum had a signifi cantly smaller scapus cross-section area, with better developed sclerenchyma and vascular tissue, than o. divergens. however, the percentage of pith parenchyma was lower in o. umbellatum and parenchyma tissue was composed of signifi cantly smaller cells. signifi cant differences between the two taxa were not recorded in percentages of scapus cortex and pith, size of epidermal cells and the number of small vascular bundles. the variation of the anatomical parameters was examined by pca (on-line suppl. tab. 3). the fi rst principal component accounted for 25.86% of total variation. parameters that contributed the most to the total variability were percentages of palisade and spongy tissue, scapus crosssection area and percentage of scapus epidermis. the second component represented 18.91% of variation and was defi ned by the percentage of scapus cortex. the cumulative contribution percentage of the fi rst three pcs was 53.74%. the projection of the cases of the fi rst two components demonstrated that the investigated specimens could be separated into groups according to the variability of anatomical parameters (fig. 5). although the groups representing populations were heterogeneous, ornithogalum divergens and ornithogalum umbellatum populations clustered, but were not completely distinctive from each other. characters showing no signifi cant differences between the two taxa were not included in mda. the results of the mda (on-line suppl. tab. 4) indicated similarities in anatomical characters between the populations from the same group. ornithogalum divergens and ornithogalum umbellatum populations were clearly separated along the fi rst discriminant axis (fig. 6). the parameter that contributed most to the discrimination of the populations, as well as of the two taxa, was leaf cross-section area, which was signifi cantly higher in o. divergens. percentages of palisade and spongy tissue and percentages of scapus epidermis and pith parenchyma singled out as characters that also had a share in separation of populations and taxa. fig. 4. scapus cross section: a) ornithogalum umbellatum (valjevo), b) o. divergens (titelski breg), c) o. umbellatum (zlotska klisura), d) o. divergens (titelski breg). abbreviations: ep – epidermis, co – cortex, sc – sclerenchyma, svb – small vascular bundle, lvb – large vascular bundle, pp – pith parenchyma. scale bars: 100 μm. fig. 5. the projection of the specimens of the fi rst two components of the principal component analysis based on anatomical characteristics. ellipses indicate populations of different taxa (right – ornithogalum umbellatum, left – o. divergens) and represent 95% of each sample. for abbreviations of sample localities see tab. 1. ornithogalum anatomy acta bot. croat. 75 (1), 2016 71 discussion in order to contribute to currently insuffi cient knowledge about the genus ornithogalum in the studied region, leaf and scapus anatomical characteristics of ornithogalum umbellatum and ornithogalum divergens were analysed. this type of research represents an addition to previous and a foundation for future ornithogalum studies, especially when it comes to problematic taxa such as these; however there are different opinions on the potential taxonomical importance of this approach. peruzzi et al. (2007) concluded that leaf anatomical characteristics of ornithogalum are useful for grouping similar species, while they are usually not suffi cient for the characterization of every taxon individually. thus they are the most valuable for general phylogenetic recapping and as a supplement to other types of analyses. investigating leaf and scapus anatomy of species fig. 6. the projection of the populations of the fi rst two factors of the multivariate discriminant analysis based on anatomical characteristics. for abbreviations of sample localities see tab. 1. tab. 2. anatomical characteristics of ornithogalum umbellatum and o. divergens: mean value ± standard error and coeffi cient of variation % (in parenthesis). asterisk (*) indicates signifi cant differences between the two taxa and “ns” stands for “not signifi cant”, according to t-test (p ≤ 0.05). character / taxon o. umbellatum o. divergens t-test total leaf cross-section area (mm2) 1.7 ± 0.1 (34) 3.2 ± 0.1 (27) * % leaf epidermis 11.9 ± 0.3 (20) 8.7 ± 0.3 (19) * % palisade tissue 20.7 ± 0.5 (20) 13.0 ± 0.7 (36) * % spongy tissue 64.3 ± 0.8 (9) 75.8 ± 0.8 (7) * % vascular tissue 2.7 ± 0.2 (44) 2.5 ± 0.1 (18) ns cross-section area of adaxial epidermis cells (μm2) 704 ± 34.5 (38) 784 ± 38.7 (31) ns cross-section area of abaxial epidermis cells (μm2) 504 ± 21.4 (33) 600 ± 25.2 (27) * cross-section area of adaxial palisade tissue cells (μm2) 1499 ± 49.2 (25) 1179 ± 95.4 (51) * cross-section area of abaxial palisade tissue cells (μm2) 1578 ± 40.4 (20) 1243 ± 100 (51) * leaf thickness (mm) 0.6 ± 13.2 (16) 0.7 ± 16.6 (14) * adaxial palisade cells index 2.5 ± 0.1 (26) 1.5 ± 0.1 (40) * abaxial palisade cells index 3.1 ± 0.1 (25) 1.9 ± 0.1 (40) * number of ribs on abaxial side 7 ± 0.2 (18) 9 ± 0.3 (23) * number of abaxial vascular bundles 9 ± 0.2 (14) 10 ± 0.3 (18) * number of central vascular bundles 9 ± 0.2 (15) 11 ± 0.3 (18) * number of stomata on adaxial epidermis on cross-section 7 ± 0.4 (45) 7 ± 0.4 (38) ns number of stomata on abaxial epidermis on cross-section 12 ± 0.5 (34) 13 ± 0.7 (38) ns total scapus cross-section area (mm2) 5.7 ± 0.3 (45) 9.6 ± 0.5 (32) * % scapus epidermis 4.8 ± 0.2 (31) 3.1 ± 0.2 (32) * % scapus cortex 20.3 ± 0.9 (33) 20.5 ± 0.6 (19) ns % scapus pith 74.8 ± 1.0 (10) 76.4 ± 0.7 (6) ns % scapus sclerenchyma 6.3 ± 0.2 (26) 4.4 ± 0.2 (22) * % scapus small vascular bundels 0.6 ± 0.03 (33) 0.3 ± 0.02 (29) * % scapus large vascular bundles 2.6 ± 0.1 (26) 2.2 ± 0.1 (24) * % scapus pith parenchyma 65.3 ± 1.1 (13) 69.5 ± 0.7 (7) * cross-section area of scapus epidermal cells (μm2) 725 ± 26.7 (29) 738 ± 38.2 (33) ns cross-section area of scapus cortex parenchyma cells (μm2) 1176 ± 39.2 (26) 1493 ± 55.1 (23) * cross-section area of pith parenchyma cells (μm2) 5001 ± 196 (30) 6652 ± 269 (26) * number of scapus cortex cell layers 4 ± 0.1 (17) 5 ± 0.1 (12) * number of small vascular bundles (in sclerenchyma) 20 ± 1.0 (37) 22 ± 1.0 (26) ns number of large vascular bundles (in parenchyma) 16 ± 0.7 (35) 20 ± 0.9 (30) * andrić a. m., rat m. m., zorić l. n., luković j. ž. 72 acta bot. croat. 75 (1), 2016 belonging to two different subgenera (ornithogalum and beryllis), öztürk et al. (2014) came to a similar conclusion. one of the latest anatomical researches of ornithogalum was done by meriç et al. (2011) on ornithogalum boucheanum and ornithogalum nutans in turkey. it is diffi cult to distinguish these two species morphologically, thus anatomical characters could be particularly useful. they discovered no differences in scapus cross-sections, except in total sectional area and in the number of vascular bundles, features that were not considered of anatomical importance (meriç et al. 2011). similarly, öztürk et al. (2014) found no signifi cant variability on interspecies level, although they noted that plants belonging to subg. beryllis had larger scapus diameter than members of subg. ornithogalum, as well as more numerous vascular bundles. our research has shown that o. umbellatum has a smaller scapus total crosssection area than o. divergens, with better developed sclerenchyma and vascular tissue, and a lower percentage of parenchyma tissue. however, no signifi cant qualitative distinctions were found among o. umbellatum and o. divergens samples regarding scapus characteristics. on the other hand, it has been recorded that leaves differ in their anatomy in different species of this genus. namely, leaves of o. nutans and ornithogalum narbonense are presented as epistomatic (popova and anastasov 1997), while the o. umbellatum and o. divergens studied here have amphistomatic leaves, as was shown for all 12 species investigated by öztürk et al. (2014), including o. umbellatum. mesophyll of o. narbonense consists of two types of cells, which form palisade tissue and spongy parenchyma, while o. nutans mesophyll is composed of three cell types of palisade cells: elongated, rounded and irregularly shaped (popova and anastasov 1997). meriç et al. (2011) have found mesophyll of o. nutans to be thinner, unifacial and to consist of monotypic chlorenchyma cells, while o. boucheanum has thicker equifacial mesophyll differentiated into palisade and spongy tissue, with large lacunae between vascular bundles and spongy tissue cells (meriç et al. 2011). leaves of the o. umbellatum and o. divergens studied here, like those of o. narbonense (popova and anastasov 1997), o. boucheanum (meriç et al. 2011) and various members of ornithogalum and beryllis subgenera (öztürk et al. 2014), have chlorenchyma differentiated into palisade and spongy tissue. palisade tissue is one-layered and present on both adaxial and abaxial sides of the o. umbellatum and o. divergens leaves analysed in our study. uniseriate upper and lower palisade parenchyma was also shown in some species belonging to subg. ornithogalum, including o. umbellatum, while members of subg. beryllis had only abaxial palisade tissue layer (öztürk et al. 2014). well defi ned, large lacunae appear in spongy tissue. popova and anastasov (1997) noted that the adaxial and abaxial epidermis become contiguous marginally at the edges of leaf blade in o. narbonense and considered this a typical feature of the species. o. umbellatum and o. divergens have cells of the upper and lower epidermis clearly separated by palisade tissue to the very edge, as shown in o. nutans (popova and anastasov 1997). in most of the representatives of the subfamily ornithogaloideae vascular bundles in the leaf are distributed in two rows and bigger alternate with smaller (lynch et al. 2006), which is also the case in our taxa. there is a wide range of variations regarding shapes of crystals in the hyacinthaceae family. prychid and rudall (1999) noted that different calcium oxalate crystal types, their presence or absence in monocotyledons, may represent useful taxonomic characters in certain groups, due to their specifi city and constancy. they observed the presence of raphides in this family. lynch et al. (2006) found solitary styloides in leaves, which are considered to be a diagnostic feature for certain families; they noted raphides and transitional forms between styloids and raphids, as well as crystal druses. in ornithogalum nutans and ornithogalum boucheanum, only raphides, immersed in the mucus of the parenchyma cells of scapus and leaves, were detected (meriç et al. 2011). the presence of needle-like crystals in parenchyma cells of leaves and scapus, singly as well as in small groups of a few, was observed in ornithogalum umbellatum and ornithogalum divergens in our study. they are considered to be transitional forms between styloides and raphides, already mentioned in related taxa (prychid and rudall 1999, lynch et al. 2006). it has been noted that raphides and styloids could be mutually exclusive, but if both types are present then intermediate forms occur, with two or three crystals per cell (prychid and rudall 1999). chiappini (1962) recorded raphides in the parenchymatous tissues of the leaf and in several parts of the fl oral region of o. caudatum. crystals, their presence in plants, their morphology and distribution, were noted to be very important features (franceschi and nakata 2005), thus constancy of the type and characteristics of crystals might be considered potential taxonomic characters. variety of shapes of calcium oxalate crystals seems to be repeatable throughout the generations, illustrating consistency of genetic and physiological parameters controlling them (prychid and rudall 1999) and further studies of this aspect could be usefull for systematic analysis of this group. statistical analyses pointed out that most of the analysed anatomical characters have shown signifi cant differences among observed populations. observed differences were mostly quantitative, not qualitative. the most variable scapus characters among populations were total cross-section area and percentages of epidermis and cortex. among the leaf characters, percentages of palisade and spongy tissue contributed most to the total interpopulational variability. the type of variability of these characters separated specimens that belong to two taxa, ornithogalum umbellatum and ornithogalum divergens, into different groups. the size of leaf cross-section area proved to be the most important discriminative character between the two taxa. moreover, the percentage of palisade tissue is signifi cantly higher in ornithogalum umbellatum and that of spongy tissue in ornithogalum divergens; these are the leaf characters that contribute to discrimination of taxa with somewhat lower proportions. according to the pca and mda results, the ten populations could be classifi ed into two groups: one joining the plants previously determined as ornithogalum divergens and the other one comprising ornithogalum umbellatum populations. t – test showed signifi cant differences between acta bot. croat. 75 (1), 2016 73 ornithogalum anatomy the two taxa in most of the analysed characters. these results are consistent with the studies of morphological charac teristics and ploidy levels (garbari et al. 2003, 2008, martí nez-azorín et al. 2009, 2010). however, anatomical characters alone could not be used to separate these morphologically similar taxa, due to the fact that only quantitative, not qualitative anatomical differences were recorded between them. these parameters could be a useful additional tool in resolving taxonomical problems, yet not for the characterization of a single species. they enabled a successful grouping of similar populations, but still remained insuffi ciently reliable for precise delimitation of o. divergens from o. umbellatum. acknowledgements the authors would like to thank tijana nikolić, sonja mudri-stojnić and dušanka krašić, colleagues who helped us with this work. the study was supported by the ministry of education, science and technological development, republic of serbia, grant no. 173002. references chiappini, m., 1962: osservazioni carioembriologiche in ornithogalum di sardegna: nota i. – sporogenesi e sviluppo dei game tofi ti in ornithogalum caudatum ait. giornale botanico italiano 69, 91–102. cullen, j., 1984: ornithogalum l. in: davis, p. h. 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(eds.), flora europaea 5, 35–40. cambridge university press, cambridge. ornithogalum anatomy acta bot. croat. 75 (1), 2016 o nlin e su pp l. ta b. 1 . a na to m ic al c ha ra ct er is tic s of th e le af p re se nt ed a s m ea n va lu e, a nd c oe ffi c ie nt o f v ar ia tio n % (i n br ac ke ts ). c ha ra ct er s: 1 – to ta l c ro ss -s ec tio n ar ea (m m 2 ) , 2 – % le af e pi de rm is , 3 – % p al is ad e tis su e, 4 – % s po ng y tis su e, 5 – % v as cu la r t is su e, 6 – c ro ss -s ec tio n ar ea o f a d ax ia l e pi de rm is c el ls (μ m 2 ) , 7 – c ro ss -s ec tio n ar ea o f a ba xi al e pi de rm is c el ls (μ m 2 ) , 8 – c ro ss -s ec tio n ar ea o f ad ax ia l p al is ad e tis su e ce lls (μ m 2 ) , 9 – c ro ss -s ec tio n ar ea o f a ba xi al p al is ad e tis su e ce lls (μ m 2 ) , 1 0 – le af th ic kn es s (m m ), 11 – a da xi al p al is ad e ce lls in de x, 1 2 – ab ax ia l p al is ad e ce lls in de x, 1 3 – nu m be r of ri bs o n ab ax ia l s id e, 1 4 – nu m be r o f a ba xi al v as cu la r b un dl es , 1 5 – nu m be r o f c en tr al v as cu la r b un dl es , 1 6 – nu m be r o f s to m at a on a da xi al e pi de rm is o n cr os sse ct io n, 1 7 – nu m be r o f s to m at a on a bax ia l e pi de rm is o n cr os sse ct io n. d iff er en t s up er sc ri pt s in di ca te th at d iff er en ce s be tw ee n lo ca lit ie s ar e si gn ifi ca nt a cc or di ng to d un ca n’ s te st (p ≤ 0 .0 5) . o . u m be lla tu m o . d iv er ge ns v rš ač ke pl an in e z lo ts ka k lis ur a d el ib la ts ka pe šč ar a v al je vo m ec se k sz eg ed su se k n ov i s ad ti te ls ki b re g h ód m ez ö vá sá rh el y 1 2. 2c (2 9) 1. 6c d (2 9) 1. 9c (2 4) 1. 9c (3 3) 1. 2d (1 5) 1. 3d (1 5) 3. 4a b (1 9) 3. 7a (3 5) 3. 0b (1 6) 2. 8b (2 1) 2 12 .1 bc (1 7) 15 .0 a ( 11 ) 11 .4 bc d ( 16 ) 12 .5 b ( 17 ) 10 .8 cd e ( 10 ) 9. 5e fg (1 6) 8. 8f gh (1 2) 8. 2g h ( 15 ) 10 .3 de f ( 20 ) 7. 7h (1 4) 3 17 .3 c ( 17 ) 19 .6 bc (2 0) 20 .7 b ( 17 ) 20 .7 b ( 14 ) 25 .1 a ( 19 ) 20 .7 b ( 13 ) 18 .2 bc (1 8) 13 .3 d ( 17 ) 12 .3 d ( 33 ) 8. 3e (3 1) 4 66 .6 de fg (6 ) 59 .9 h ( 18 ) 64 .6 ef g ( 6) 64 .8 fg (6 ) 62 .3 gh (9 ) 67 .7 de f ( 4) 70 .7 cd (6 ) 76 .4 b ( 3) 74 .8 bc (5 ) 81 .2 a ( 4) 5 4. 0a (3 4) 3. 5a b ( 31 ) 3. 3b c (2 3) 2. 0f (3 5) 1. 8f (2 5) 1. 9f (2 3) 2. 3d ef (1 5) 2. 1e f ( 18 ) 2. 7c de (1 5) 2. 8b cd (1 0) 6 81 4a bc (3 7) 88 4a b (2 8) 82 4a bc (3 1) 74 6b c (2 4) 49 6d e (2 3) 45 9e (3 3) 10 08 a ( 30 ) 66 8b cd (2 1) 66 2c d ( 26 ) 79 8a bc (2 1) 7 60 6a b (2 2) 48 6b c (2 4) 62 8a b (3 0) 53 6a b (2 6) 39 4c (4 2) 37 8c (1 9) 66 5a (1 7) 65 8a (2 6) 49 8b c (2 0) 58 1a b (3 3) 8 18 83 a ( 18 ) 14 96 b ( 24 ) 17 15 ab (7 ) 15 92 ab (1 6) 97 2c (2 4) 13 37 b ( 9) 17 57 ab (2 7) 14 95 b ( 32 ) 61 3d (5 7) 85 2c d (2 4) 9 18 80 a ( 17 ) 14 30 b ( 17 ) 16 26 ab (1 8) 17 16 ab (1 7) 13 58 bc (1 7) 14 57 b ( 13 ) 18 56 a ( 16 ) 16 41 ab (3 5) 65 8c (5 1) 81 8c (2 4) 10 0. 7b (1 8) 0. 6c (2 0) 0. 7b (1 4) 0. 7b (1 4) 0. 6c (8 ) 0. 6c (9 ) 0. 8a (9 ) 0. 8a (1 1) 0. 6c (1 0) 0. 7b (1 0) 11 2. 2b (2 1) 2. 4a b (3 2) 2. 8a (1 6) 2. 6a b (1 9) 2. 2b (2 5) 2. 6a b (3 5) 2. 1b (1 3) 1. 7b c (3 9) 1. 2c (3 0) 1. 0c (2 8) 12 2. 5c (1 5) 2. 5c (2 3) 3. 0b c (1 1) 3. 8a (1 6) 3. 2b (2 1) 3. 6a b (2 6) 2. 8b c (1 0) 2. 1c (3 1) 1. 5d (3 7) 1. 3d (4 6) 13 7b (1 8) 8b (1 9) 7b (2 2) 8b (9 ) 6b (1 0) 6b (1 3) 10 a ( 12 ) 10 a ( 28 ) 10 a ( 24 ) 8b (1 7) 14 9b c (1 0) 10 b ( 9) 10 b ( 9) 9b c (1 2) 8c (8 ) 7d (1 5) 10 b ( 15 ) 11 a ( 15 ) 11 a ( 19 ) 10 b ( 16 ) 15 10 bc (1 0) 10 bc (1 0) 9c (8 ) 8c d (1 1) 8c d (1 3) 7d (1 3) 10 bc (1 7) 11 b ( 16 ) 12 a ( 14 ) 10 bc (2 0) 16 8a b (6 0) 8a (3 6) 8a bc (3 3) 8a (3 1) 5c (2 5) 5b c (4 1) 7a bc (4 1) 8a b (3 3) 6a bc (4 4) 6a bc (3 4) 17 13 ab (3 2) 12 ab (2 8) 13 ab (3 5) 15 a ( 26 ) 8c (2 4) 10 bc (3 2) 14 ab (3 9) 14 ab (3 2) 12 ab (3 9) 10 bc (4 0) 1 andrić a. m., rat m. m., zorić l. n., luković j. ž. 2 acta bot. croat. 75 (1), 2016 o nlin e su pp l. ta b. 2 . a na to m ic al c ha ra ct er is tic s of th e sc ap us p re se nt ed a s m ea n va lu e, a nd c oe ffi c ie nt o f v ar ia tio n % (i n br ac ke ts ). c ha ra ct er s: 1 – to ta l c ro ss -s ec tio n ar ea (m m 2 ) , 2 – % s ca pu s ep id er m is , 3 – % s ca pu s co rt ex , 4 – % s ca pu s pi th , 5 – % s ca pu s sc le re nc hy m a, 6 – % s ca pu s sm al l v as cu la r b un de ls , 7 – % s ca pu s la rg e va sc ul ar b un dl es , 8 – % s ca pu s pi th p ar en ch ym a, 9 – c ro ss -s ec tio n ar ea of s ca pu s ep id er m al c el ls (μ m 2 ) , 1 0 – cr os sse ct io n ar ea o f s ca pu s co rt ex p ar en ch ym a ce lls (μ m 2 ) , 1 1 – cr os sse ct io n ar ea o f s ca pu s pi th p ar en ch ym a ce lls (μ m 2 ) , 1 2 – nu m be r o f s ca pu s co rt ex c el l l ay er s, 13 – n um be r of s m al l v as cu la r bu nd le s (i n sc le re nc hy m a) , 1 4 – nu m be r of la rg e va sc ul ar b un dl es ( in p ar en ch ym a) . d iff er en t s up er sc ri pt s in di ca te th at d iff er en ce s be tw ee n lo ca lit ie s ar e si gn ifi ca nt a cco rd in g to d un ca n’ s te st (p ≤ 0 .0 5) . o . u m be lla tu m o . d iv er ge ns v rš ač ke pl an in e z lo ts ka k lis ur a d el ib la ts ka pe šč ar a v al je vo m ec se k sz eg ed su se k n ov i s ad ti te ls ki b re g h ód m ez ö vá sá rh el y 1 7. 2c de (3 0) 3. 0g (4 8) 6. 6d e ( 29 ) 4. 1f g ( 25 ) 5. 7e f ( 30 ) 7. 7b cd e ( 42 ) 8. 7a bc d ( 43 ) 10 .1 ab (1 2) 10 .4 a ( 22 ) 9. 2a bc (4 8) 2 3. 7c d (2 0) 6. 3a (2 7) 4. 4b c (1 7) 6. 3a (1 4) 4. 6b (2 6) 3. 7c d (2 3) 4. 4b c (1 6) 3. 0d e (1 2) 2. 2e (2 8) 2. 8d e (2 2) 3 18 .9 bc (1 4) 27 .7 a ( 18 ) 19 .9 bc (1 5) 27 .4 a ( 16 ) 12 .8 e ( 23 ) 15 .4 de (3 1) 21 .7 b ( 11 ) 25 .0 a ( 11 ) 18 .3 cd (1 3) 17 .2 cd (1 5) 4 77 .5 b ( 4) 66 .0 e ( 9) 75 .7 bc (4 ) 66 .3 e ( 7) 82 .6 a ( 4) 80 .9 ab (7 ) 73 .9 cd (3 ) 72 .0 d ( 4) 79 .5 ab (4 ) 80 .0 ab (4 ) 5 6. 2a b (1 5) 6. 9a (2 3) 6. 5a b (1 9) 5. 6b (3 4) 7. 3a (2 4) 5. 2b c (2 7) 5. 5b (2 0) 3. 8d (1 6) 4. 1c d (1 4) 4. 2c d (1 8) 6 0. 8a (1 5) 0. 6a b (5 3) 0. 6a b (3 3) 0. 5b c (2 7) 0. 7a b (3 0) 0. 6a b (2 1) 0. 4c d (2 4) 0. 4c d (1 8) 0. 4c (2 8) 0. 3d (2 3) 7 3. 4a (1 8) 2. 8b (2 4) 2. 8b c (2 2) 2. 1d (1 9) 2. 1d (1 9) 2. 5b cd (1 3) 2. 3c d (3 6) 2. 0d (2 1) 2. 3b cd (1 9) 2. 1d (1 5) 8 67 .1 b ( 4) 55 .6 c ( 13 ) 65 .8 b ( 5) 58 .1 c ( 10 ) 72 .5 a ( 6) 72 .6 a ( 9) 65 .9 b ( 5) 65 .9 b ( 4) 72 .7 a ( 5) 73 .5 a ( 4) 9 57 0c (2 2) 71 1b c (2 9) 77 8b (2 2) 96 4a (2 5) 73 4b c (1 3) 59 4c (1 8) 97 4a (3 2) 77 3b (2 0) 61 3b c (1 8) 59 3c (1 9) 10 12 22 bc (2 1) 12 07 bc (2 9) 14 05 b ( 24 ) 13 49 bc (1 4) 94 8d (2 0) 92 6d (1 3) 17 51 a ( 14 ) 17 44 a ( 14 ) 13 27 bc (1 8) 11 50 cd (1 8) 11 40 35 ef (2 1) 36 09 f ( 26 ) 47 88 de (2 0) 56 38 cd (2 1) 54 58 cd (2 3) 64 81 bc (2 8) 53 34 cd (1 8) 83 86 a ( 20 ) 54 89 cd (1 3) 74 03 ab (1 2) 12 5a (1 8) 4b cd (1 4) 4a bc (1 9) 4c d (0 ) 4d (1 3) 4d (1 7) 4a bc (1 2) 5a (9 ) 4a bc (1 2) 5a b (1 5) 13 27 a ( 32 ) 12 e ( 33 ) 25 ab (2 4) 15 cd e (1 2) 19 cd (1 8) 24 ab c (2 8) 17 cd e (2 2) 21 bc (2 2) 27 a ( 12 ) 22 bc (2 6) 14 20 b ( 26 ) 10 d ( 26 ) 19 bc (2 7) 12 d ( 15 ) 14 cd (2 4) 18 bc (2 8) 17 bc (3 7) 18 bc (1 4) 24 a ( 10 ) 20 b ( 41 ) ornithogalum anatomy acta bot. croat. 75 (1), 2016 on-line suppl. tab. 3. principal component analysis: factor coordinates of the variables based on correlations. only variables with factor values > 0.7, which signifi cantly contribute to total variation, were presented. character factor 1 factor 2 factor 3 percentage of palisade tissue 0.724 –0.213 0.363 percentage of spongy tissue –0.779 0.113 –0.082 total scapus cross-section area –0.775 –0.109 0.359 percentage of scapus epidermis 0.829 0.260 –0.173 percentage of scapus cortex 0.307 0.718 –0.365 % total variance 25.86 18.91 8.97 on-line suppl. tab. 4. multivariate discriminant analysis: standardized coeffi cients for canonical variables. only variables with root values > 0.7, which signifi cantly contribute to total variation, were presented. character root 1 root 2 root 3 total leaf cross-section area 1.346 0.456 –0.416 percentage of palisade tissue 0.071 0.132 –1.336 percentage of spongy tissue 0.264 0.088 –1.105 percentage of scapus epidermis –0.400 –0.190 –0.815 percentage of scapus pith parenchyma –0.477 0.999 –0.058 percentages of the vectors 41.89 18.97 16.48 3 acta bot. croat. 79 (1), 2020 95 acta bot. croat. 79 (1), 95–97, 2020 coden: abcra 25 doi: 10.37427/botcro-2020-004 issn 0365-0588 eissn 1847-8476 short communication physcomitrium eurystomum sendtn., a new moss species in the bryophyte flora of montenegro danijela stešević*, branko anđić, milica stanišić-vujačić faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81000 podgorica, montenegro abstract – in a vegetation survey conducted in the northeast part of montenegro, physcomitrium eurystomum sendtn. was collected. this is a new moss species for the bryophyte flora of montenegro. the species has a wide temperate-tropical distribution, but its populations are rather scattered. in most european countries, the species is included on the relevant national red lists and most recently, it was added to the european red list of mosses, liverworts and hornworts. in order to expand our knowledge of its distribution in montenegro, and to assess its iucn threat status, further investigations are needed. keywords: biodiversity, conservation, funariaceae. * corresponding author e-mail: danijela.stesevic@ucg.ac.me introduction bryological research in montenegro has been intensified over the last two decades, and this has resulted in a significant number of new publications and records (anđić et al. 2018). nevertheless, because some regions still remain completely unexplored, our knowledge of the diversity of the bryophyte flora of montenegro cannot be considered complete and new discoveries can be expected. during vegetation research in the gorge of bukovička rijeka (in the northeast part of montenegro), one new moss species belonging to the bryophyte flora of montenegro was collected – physcomitrium eurystomum sendtn. although, this temperate-tropical example of bryophyte flora (dierßen 2001) is distributed across europe, tropical africa, and southern, southeastern and southwestern parts of asia, its populations are rather scattered (porley 2013, hodgetts 2015). the european scope of the range includes: to the north denmark, to the west great britain, in the south france and italy, in central areas austria, belgium, the czech republic, germany, hungary, the netherlands, poland, slovakia and switzerland, in the southeast bulgaria, romania, slovenia, serbia and turkey, and in the east belarus, the crimea, latvia, northwest russia and ukraine (papp et al. 2013, eccb 2016). in the majority of these countries it has a defined status category, and it was recently included in the european red list of mosses, liverworts and hornworts (hodgetts et al. 2019). in this paper, we present the details of the first record of p. eurystomum in montenegro; however, in order to extend our knowledge of its distribution in the whole country, and to define its conservation status, further investigations are needed. material and methods the study area bukovička rijeka, located in the northeast part of montenegro, is a left tributary of the ibar river; it is formed by two arms that originate at 1500 and 1600 m a.s.l. after 20 km and a drop of ca. 600–700 m, it flows into the ibar river at 865 m a.s.l. although the river is water poor, it has strong erosive energy; thus, it has carved a narrow, 200 m deep and 5 km long gorge in the middle and lower part of its flow. this gorge is very narrow; in some areas, the two sides of the gorge are just 5m from each other. the geological substrate consists of lower and middle triassic limestone. the climate fits into the cold alpine type with long and snowy winters, and short and fresh summers. the gorge is characterized by an inversion of both temperature and vegetation, which means, for example, that the mesophilous beech forests (fagion moesiacae blečić et lakušić 1970) are developed in lower positions along the gorge, and thermophilous oak and hornbeam forests (ostryion carpinifoliae lakušić 1975) in the upper ones (markišić and martinović 1998). stešević d, anđić b, stanišić-vujačić m 96 acta bot. croat. 79 (1), 2020 sampling specimens of physcomitrium eurystomum were collected in june 2018 during a vegetation survey of bukovička rijeka, from the muddy soils alongside the stream. the species was identified according to smith (2004), while the nomenclature follows ros et al. (2013). specimens were deposited in the herbarium collection at the university of montenegro (tgu), voucher number 1419038. during the field trip, notes on the relevant habitats, substrates and associated bryophytes were taken. results and discussion physcomitrium eurystomum sendtn. specimen examined: montenegro; gorge of bukovička rijeka, 42°55'22'' n, 20°15'31'' e, 970 m a.s.l.; date: june 23, 2018; leg. et det.: d. stešević, b. anđić, m. stanišić-vujačić; taxonomic confirmation: beata papp; herbarium number (tgu): 1419038. in the muddy soil deposits in the gorge at a distance of ca. 4 m from the water body, two turfs of physcomitrium eurystomum were found (fig. 1), of an approximate size of 5 cm2 each. on both patches, fruiting gametophytes were noticed. the p. eurystomum was accompanied by marchantia polymorpha l., bryum pseudotriquetrum (hedw.) p. gaertn., weissia controversa hedw. and riccia sp., all growing in the shadow of petasites hybridus (l.) g. gaertn., b. mey. et scherb. physcomitrium eurystomum is considered to be one of the representative species for habitat type c3.5b – periodically exposed shore with stable, mesotrophic sediments with pioneer or ephemeral vegetation (deil 2005), which is quoted in the european red list of habitats. at this rather remote site in the gorge of bukovička rijeka, human impact is completely absent; thus, the only possible threat to the species might be a change in the watercourse or the configuration of the riverbank. up to now, the bryophyte flora of montenegro has included only one species of the genus physcomitrium – the cosmopolitan species p. pyriforme, which, according to sabovljević et al. (2004), is classified as lr (low risk) on the bryophyte red list of serbia and montenegro. so far the species has been reported in 22 european countries (papp et al. 2013, eccb 2016), while in 15 it has a defined status category: endangered in great britain (hodgetts 2011) and hungary (papp et al. 2010); highly endangered in austria (eccb 2016); vulnerable in germany (eccb 2016), the czech republic (kučera and váňa 2003), slovakia (šoltés et al. 2002), switzerland (bafu 2011), and estonia (ingerpuu et al. 2018); threatened in belarus (maslovsky 2005), belgium (eccb 2016), bulgaria, romania and turkey (sabovljević et al. 2001); susceptible in the netherlands (eccb 2016) and data deficient in slovenia (martinčić 2016). in the balkan peninsula, the species is reported in serbia (papp et al. 2013), bulgaria, and slovenia (sabovljević et al. 2008), but due to the presence of similar habitat types in other parts of the peninsula, new records are expected. more recently, the p. eurystomum was included on the european red list of mosses, liverworts and hornworts (hodgetts et al. 2019), within the following iucn red list categories: europe vu b2b(ii,iii)c(iii,iv) and eu 28 (28 european union countries) en b2b(ii,iii)c(iii,iv). considering its status in montenegro, based on what is currently limited knowledge, it is not possible to assess its iucn conservation status. as such, further investigations are needed and recommended. taking into account its international status category, we consider the species to be a good potential candidate for the new and supplemented list of protected plants in montenegro. acknowledgements this study was financially supported by the montenegrin ministry of science within the bilateral project with austria “vegetation of the rock crevices with special focus to the moltkeetalia petraeae order”. we would like to thank to the anonymous reviewers for their careful reading of our manuscript and many valuable suggestions and comments. fig. 1. habitus 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cambridge university press, cambridge. šoltés, r., kubinská, a., janovicová, k., 2002: extinction risk to the bryophytes in slovakia, reasons and evaluation. portugaliae acta biologica 20, 57–63. acta bot. croat. 77 (2), 2018 161 acta bot. croat. 77 (2), 161–171, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0018 issn 0365-0588 eissn 1847-8476 the impact of spatio-temporal changes in flora attributes and pollen availability on insect visitors in lamiaceae species jacek jachuła1, małgorzata wrzesień2, monika strzałkowska-abramek1, bożena denisow1* 1 department of botany, laboratory of horticultural plants biology, university of life sciences in lublin, 15 akademicka str., 20-950 lublin, poland 2 department of geobotany, institute of biology and biochemistry maria curie-skłodowska university, 19 akademicka str., 20-033 lublin, poland abstract – there is growing evidence that food, in particular pollen, limitation is the strongest factor in pollinator decline. we have considered the potential effects of diversity in plant-community attributes as well as variations in the pollen and energy amount on the abundance and frequency of insect visitors to the lamiaceae species salvia pratensis l., s. verticillata l., thymus serpyllum l., betonica officinalis l. syn. stachys officinalis (l.) trevis., and origanum vulgare l. the species were grown in two different habitat types (dry grassland vs. railway embankment) in the lublin upland, poland. we found significant inter-species, inter-habitat, and inter-year disparities in the pollen mass and total energy amount per unit area. canonical correspondence analysis (cca) revealed that the blossom cover, species richness, and diversity noted at the plant community level significantly influenced the distribution of insect visitors to lamiaceae species. the pollen caloric value and pollen abundance (but not the protein content in the pollen) had a considerable impact on the abundance and frequency of honeybees, bumblebees, and solitary bees in lamiaceae flowers. butterflies, beetles and flies did not respond to these factors. the model including all variables explained 66.4% of the observed variance. the studied lamiaceae species, due to abundant flowering and good pollen nutritional value should be considered in the protocols to improve food resources, especially for social bees; however, disparities in pollen quantity and energy amount should not be ignored. keywords: dry grasslands, pollen caloric value, pollen mass, pollinators, railway embankments * corresponding author, e-mail: bozena.denisow@up.lublin.pl introduction human activities are having an impact on ecosystems globally to an unprecedented degree (hoekstra et al. 2005) and species differ tremendously in their responses to environmental changes. in particular, pollinators are undergoing severe population declines following multi-directional environmental transformations and destruction of pollinator-friendly habitats (hülsmann et al. 2015). the role of pollinator service in general biodiversity conservation (lonsdorf et al. 2009), functioning of biocenoses and ecosystems (kunin 1997, steffan-dewenter 2003, lázaro et al. 2009), development of the agricultural sector (klein et al. 2007), and indirectly in human health is unquestionable (gallai et al. 2009). therefore, the pollinator decline phenomenon has been described as a ‘pollination crisis’ and has attracted attention globally from science, business, and even politicians (ghazoul 2005). the ‘pollination crisis’ is evident in declines and damage to webs of plant-pollinator interaction (lázaro et al. 2013). these declines are usually attributed to multiple interacting causes, rather than one single cause (vanbergen 2013). it has been suggested that nesting, breeding, and food niches as well as the accessibility of refugia for the avoidance of chemical exposure regulate the size of pollinator populations (roulston and goodell 2011). however, there is growing evidence that food limitation (decreased dietary diversity and reduced food abundance) is the strongest factor in pollinator decline (garibaldi et al. 2013, gonzález-varo et al. 2013, ollerton et al. 2014, vaudo et al. 2015). as pollinator nutrition entirely relies on the nectar and pollen available in flowers, the main conservation issue is to develop and implement tools aimed at improving flower-rich habitats (dicks et al. 2015). one of the concepts is the protecjachuła j., wrzesień m., strzałkowska-abramek m., denisow b. 162 acta bot. croat. 77 (2), 2018 tion of existing natural habitats identified as drivers of pollinator biodiversity (albrecht et al. 2007). recently, there has been increasing interest in proper management of alternative (man-made) habitats (lowenstein et al. 2015). this concept has arisen from the current expansion of both urban and open man-made habitats and the growing body of evidence showing that these habitats fulfil a significant role in supporting a high level of pollinator diversity and population size (banaszak-cibicka and żmihorski 2012, moroń et al. 2014). however, the provided data indicate that pollinator taxa respond differently to the natural-to-urban gradient and to the surrounding land use, i.e. rural-agricultural sites are friendlier to hoverfly species, while urban sites support wild bees (e.g. verboven et al. 2014). numerous methods can be applied to estimate habitat quality, from the level of individual species (flowers) through patches to biotopes and landscape (szigeti et al. 2016). in europe, semi-natural dry grasslands (steppe-like ‘warm-stage refugia’) are among the most species-rich plant communities. these sites create refuges for rare and endangered plants and invertebrates (wallisdevries et al. 2002), as well as for a variety of nectariferous and polleniferous plants (wrzesień and denisow 2006) in a modern rural landscape. there is evidence that the value of man-made habitats for pollinators varies considerably, e.g. the level of diversity and abundance of spontaneous bee forage flora is higher in railway embankments along lowthan along high-traffic volume tracks (wrzesień et al. 2016). pollen is considered to be an important resource (müller et al. 2006), as it is a main source of protein, vitamins, mineral salts, organic acids, and hormones (pacini 2000, szczęsna 2006). it is obvious that inadequate pollen resources usually lead to disorders in physiological processes at different stages of insect life cycles and are particularly detrimental for developing larvae and young bees (alaux et al. 2010, nicolson 2011). in the case of apis mellifera, protein deficiencies have an indirect effect on the income of apiaries and reduce honey yields (keller et al. 2005). it has been proposed that the restoration and management of pollinator-friendly habitats should begin with an inventory of the resources provided by particular species and alternative habitats (e.g. fussell and corbet 1992, denisow 2009, garbuzov and ratnieks 2014, denisow and wrzesień 2015b). lamiaceae species are important components of plant communities in europe (petanidou and vokou 1990, petanidou and smets 1995). in poland, lamiaceae species are frequently found in xerothermic swards and on ruderal sites, e.g. along railway embankments (wrzesień and denisow 2006a, b). they form dense patches and flower abundantly and by that means potentially could support floral resource for pollinators (e.g. bożek 2003b). in this study, we evaluated how the composition, abundance, and richness of the flora as well as the flowering spectrum at the community level can influence the abundance and frequency of insect visitors to several lamiaceae species. within the study area, five lamiaceae species (salvia pratensis l., s. verticillata l., thymus serpyllum l., betonica officinalis l. syn. stachys officinalis (l.) trevis., and origanum vulgare l.) have been found to occur both in dry grasslands and in plant communities developed on railway embankments, therefore we tried to assess how the potential quantity and quality of pollen resources of the same species can vary among habitat types (dry grassland vs. railway embankment). we also assessed the importance of the pollen mass, pollen energy available, and protein content in lamiaceae pollen for bees and other insects by investigation of the spectrum of insect visitors. materials and methods study sites in situ observations were performed in 2013–2014. the study area was located on the lublin upland, south-eastern poland (50°54′24″n, 23°09′12″e) (on-line suppl. fig. 1). the floristic inventory of nectarand/or pollen-yielding flora (hereinafter called forage flora) was made in (1) festufig. 1. changes in seasonal pattern of flowering of forage and non-forage plants in the dry grassland (dg) and the railway embankment (re) habitats in se poland. the percentage of plant species in bloom at each study point was established based on the cover of plant species in particular transects (n = 5 per habitat). mean from 2013-2014. insect visitors in lamiaceae acta bot. croat. 77 (2), 2018 163 co-brometea br.-bl. & tüxen ex soó 1947 dry grassland and (2) man-made habitat along the railway embankment (line no. 69 rejowiec-hrebenne), situated near the grassland area. the dry grassland occupies the edge of the river valley and is located on the se slope (195 m a.s.l., inclination 50°). since 2008, the festuco-brometea site has been included in the natura 2000 network of nature protection areas in the territory of the european union. the railway embankment includes se and sw slopes (200 m a.s.l., inclination 42°). the vegetation is composed of diverse flowering plant species, i.e. natural, ruderal, and segetal. a temperate climate with oceanic and continental influences is characteristic for the lublin upland (annual temperature averaged 7.4 °c, annual precipitation averaged 630 mm) (kaszewski 2008). vegetation survey the method of phytosociological relevés was employed to characterize the flora in the dry grassland and railway embankment habitats. the frequency and abundance of each vascular plant species was recorded according to braun– blanquet (1964). each experimental patch was randomly selected and was 200 × 5–6 m in the dry grassland (n = 5) and 300 × 2 m in the railway embankment (n = 5). the total coverage for each species was estimated visually and recorded using a cover-abundance scale within seven cover classes, i.e.: 1 to 5 individuals; +: a few individuals (< 20) with cover < 5%; 1: many individuals (20–100) with cover < 5%; 2: 5%–25% cover; 3: 25%–50% cover; 4: 50%–75% cover; 5: 75%–100% cover (van der maarel 1979). this survey allowed us to analyse the flora composition, richness, and abundance in each habitat type. the geographic position of each experimental plot was recorded with a differential gps. the plant nomenclature followed mirek et al. (2002). flowering phenology to establish the seasonal spectrum of forage species and non-forage species in bloom for every vegetation type, forage species have been defined according to literature data and own observations (wrzesień and denisow 2006a,b, denisow and wrzesień 2007). the experimental patches were monitored 5 times per year, i.e. in spring (2–10 may), early summer (5–10 june), summer (20–30 june and 15–30 july), and late summer (15–25 august). to assess flowering phenology, we followed the instruction of denisow (2011). the coverage of the species in bloom was estimated with a coverabundance scale (in %) as a percentage of the total area. for each time point, the species in bloom have been counted. the date of the onset of flowering and the duration of the flowering phase were documented for each species. the species full bloom was recorded as the period when > 75% of flowers in the population were in bloom (point 5 in the coverabundance scale). we also estimated the cover of non-forage plants. species with low cover (< 5%) or singly noted have been excluded from the graphs and only the main flowering species are presented. the estimates were made on the base of 5 transect per each habitat. pollen production, energy amount and protein content in pollen due to the time-consuming character of the analyses, only five species (representatives from the lamiaceae family) were selected for the study of pollen production. the observations were made and samples collected in two habitats (dry grassland and railway embankment) for each species. the following species: salvia pratensis l., s. verticillata l., thymus serpyllum l., betonica officinalis l. syn. stachys officinalis (l.) trevis., and origanum vulgare l. were chosen for the experiment. pollen production was assessed using the ether-ethanol method described in detail by denisow (2011). during each year, we randomly selected well-developed flower buds (n = 30–70) at the full flowering phase of each species. buds were collected from different individuals (n = 10–15), placed in plastic containers, and transported to the laboratory in a portable cooler. in the laboratory, the anthers before dehiscence were dissected from the flower buds and placed in tarred vessels (n = 100–200 anthers per sample; i.e. 25–50 flowers). we collected 4 samples per species and habitat (in total n = 40 samples per year). the samples were transferred into a dryer (elcon cl 65) at ca 33 °c for approx. 20–30 days. pure ether (2–3 ml) was used to rinse pollen from the anthers. next, 70% ethanol (3–4 ml) was used to separate pollen from the anther tissues. pollen production was expressed per flower and per 1 m2 for each species and habitat type. the pollen amount per unit area was determined using the detailed data of the flowering abundance established for every lamiaceae species. we counted the number of flowers produced per unit area; all buds, flowers, and set fruits were counted in randomly chosen stems (n = 14–20). next, the number of stems was determined using the circular frame (36.7 cm in diameter); n = 15–20 counts for each study site were made. the data was converted to the number of flowers per 1 m2. the pollen energy amount (= pollen caloric value) was calculated using a value of 5.69 kcal g–1 (petanidou and vokou 1990) and was expressed in kcal per 1 m2. the tests for the n content in pollen were performed according to the kjeldahl method in 2013 for each species and habitat. the crude protein content was calculated by multiplying 5.60 by % n in 100 g of pollen (rabie et al. 1983). insect visitors to assess the structure of insect visitor groups for each lamiaceae species, we monitored the patches simultaneously in the two different habitats (dry grassland vs. railway embankment). the insect monitoring was made in the same patches where floristic surveys had been conducted. the observations were made in 7–15-day intervals during the fullbloom phase of each species. in each habitat, we randomly chose 1.0 m2 patches (n = 3) and recorded the insect visitors that collected nectar and/or pollen from flowers of the studied lamiaceae plants. insect visitor activity was monitored between 9.00–17.00 h (gmt + 2 h). earlier and later obserjachuła j., wrzesień m., strzałkowska-abramek m., denisow b. 164 acta bot. croat. 77 (2), 2018 vation hours have been excluded, as according to literature, the peak insects activity to lamiaceae species are morning/ mid-day hours (bożek 2000, 2003 a,b, 2008). records (5–10 min. per census) were made at each plot at two-(three)-hour intervals. the observations were made when weather conditions were favourable for the activity of insect visitors, i.e. the daily temperature was 23–28 °c, with low wind speed, and without rainfall. all flower-visiting insects were recorded and grouped into their taxonomic categories: honeybees = apis mellifera, bumblebees = bombus spp., solitary bees = hymenoptera, flies = diptera, beetles = coleoptera. the procedure facilitated the apportionment of the proportion of insect categories to particular species as well as visitation frequency. data analysis we used a variety of approaches to analyse the data. the vegetation within the dry grassland and the railway embankment was described by the species richness, shannon diversity index (h'), and evenness index (pielou’s j′ evenness). the value of h' ranges from 0 to 1, with higher values representing more even distributions in abundance among species. j' is constrained between 0 and 1. the lesser the variation in communities among the species, the higher the j' index is. the mann-whitney non-parametric test was used to compare the values of indices obtained for the dry grassland and the railway embankment (stanisz 2007). the multivariate statistical package (mvsp) was used for these analyses (kovach 2005). to identify the general pattern of variation in the species composition within the entire data set of flora, an indirect ordination method (detrended correspondence analysis, dca) was used (a unimodal response model; environmental gradient > 3) (canoco 5.0, ter braak and šmilauer 2012). subsequently, canonical correspondence analysis (cca) was used to visualize and establish the relationship between the floristic composition of the vegetation plots (relevés) and the spatial distribution of insect visitors and between the insect visitor pattern and lamiaceae species traits. for each type of vegetation, the data from the study periods were pooled. seven environmental variables were analysed. two groups of variables (1) flora attributes, i.e. species richness (richness), species diversity (diversity), ‘blossom cover’ i.e. flower abundance (cover), flowering season, (flowering), and (2) lamiaceae species traits, i.e. pollen amount (pollen a), energy content in pollen (= pollen caloric value; pollen e), and protein content in pollen (pollen p) were tested. analysis of variance (anova) was applied to assess the difference in the mean values of the analysed traits among the populations and within the populations among the habitats and the years of the study. the normality of the data was evaluated prior to the analysis. if significant differences were detected, tukey’s hsd test was applied. pearson’s correlation (r) was established between the total pollen output and the studied criteria (abundance of blooming and mass of pollen in flowers). the level of statistical significance to measure the differences between the means for all the analyses was at p = 0.05. statistical tests were performed with statistica (statsoft, inc., krakow), version 10.0. results nectar and pollen yielding plants the total number of plant species found was 185, of which 151 species (81.6%) were identified as visited by insects (on-line suppl. tab. 1). in the dry grassland, 135 (112 forage species = 82.9%) were documented, while 115 species (90 forage species = 78.2%) were noted on the railway embankment. the species richness, species diversity, and species evenness was significantly higher on the dry grassland (u-test, tab. 1). the number of forage species per relevé ranged from 10 to 48 (mean = 39.7 ± 5.2 in the grassland; mean = 19.9 ± 5.8 in the railway embankment). species yielding both nectar and pollen predominated (138 species = 91.3%). pollen as floral reward (= no nectar) was offered by 13 species (8.6%) of the bee forage flora. community-level changes in the flowering spectrum the pattern of the flowering of forage plants slightly differed between the dry grassland and the railway embankment sites (fig. 1). in spring, more abundant flowering of forage plants was noted in the dry grassland. here, the flowering peak was observed during the summer (june/july). in the summer period, the lamiaceae species contributed substantially to the food availability for pollinators. on the railway embankment, the flowering of the forage plant species was shifted towards the end of the summer. tab. 1. comparison of plant forage species richness (s), the values of diversity indices (h’, j’), and the mean frequency of insect individuals to lamiaceae flowers in the dry grassland and the railway embankment. means ± sd (standard deviation) are shown. the values indicated by the same small letter are not statistically different between the types of habitats (mann-whitney test has been applied; z – statistic value, p – probability).   type of habitat z pdry grassland railway embankment   mean ± sd mean ± sd species richness (s) 33.70b 5.21 19.80a 5.77 5.35 0.000 diversity (h') 1.50b 0.08 1.24a 0.12 5.48 0.000 evenness (j’) 0.984b 0.011 0.974a 0.013 2.52 0.013 insects frequency per flower/per season 0.005a 0.002 0.022b 0.01 4.16 0.004 insect visitors in lamiaceae acta bot. croat. 77 (2), 2018 165 pollen production, energy content, and pollen protein species, habitat, and year-to-year disparities were found for the mass of pollen produced in the flowers of lamiaceae species (tabs. 2, 3, 4). the lowest amount of pollen was found in the flowers of thymus serpyllum and the highest in betonica officinalis. for the same species, the mass of pollen produced per flower differed between the habitats. a significantly higher mass of pollen was produced in the flowers of salvia pratensis (df = 1, p = 0.033), s. verticillata (df = 1, p = 0.031), and origanum vulgare (df = 1, p = 0.028) growing in the dry grassland compared to those from plants in the railway habitat. in turn, t. serpyllum and b. officinalis produced similar pollen mass in the flowers of plants in both the dry grassland and the railway embankment (df = 1, p = 0.122; df = 1, p = 0.242, respectively). a higher amount of pollen per flower was produced in 2014: on average, 4-fold higher than in 2013. a significant positive correlation was found between the pollen yield per 1 m2 and the mass of pollen per flower (r = 0. 431, p = 0.033) and between the pollen yield per 1 m2 and the number of flowers per 1 m2 (r = 0. 844, p = 0.014). the number of lamiaceae flowers per unit area was related to the species (df = 4, p = 0.035) (tab. 2). the lowest number of flowers, i.e. only 1.32 thousand per 1 m2, was noted in s. verticillata, while the highest number was recorded for o. vulgare – on average 201.61 thousand per 1 m2 (tabs. 2, 3). the number of flowers developed per unit area was significantly higher in the dry grassland for four out of the five species studied. only o. vulgare bloomed more abundantly on the railway embankment. for the same species, differences between study years were also established for the abundance of flowering (tabs. 3, 4). on average, more flowers were produced in 2013. the estimated total pollen production per 1 m2 and the total pollen energy available per 1 m2 differed among the species, between the populations developed in the dry vs. railway habitats (habitat effect), and among the years of the study (tabs. 2, 3, 4). the lowest and the highest pollen yield and pollen energy amount were recorded for t. serpyllum (mean = 0.08 g per 1 m2, 0.46 kcal per m2) and o. vulgare (mean = 8.87 g per 1 m2, 44.83 kcal per m2), respectively. on average, s. pratensis and s. verticillata produced similar pollen mass and energy amount per 1 m2. comparing habitats, the total pollen yield and the energy amount were higher in the dry habitat than in the railway habitat. only o. vulgare produced more pollen and energy amount (approx. 10%) on the railway plots, compared to the dry patches. the mean pollen mass and energy in 2013 (2.09 g per 1 m2, and 11.89 kcal per 1 m2, average) was relatively low compared to that established for 2014–2.89 g per 1 m2 and 15.88 kcal per 1 m2 (tab. 3). there were evident effects of the species (df = 4, p = 0.001) and habitat (df = 1, p = 0.003) on the protein content in pollen (tabs. 2, 4). the pollen of t. serpyllum, b. officinalis, tab. 2. the number of flowers per 1 m2, the mass of pollen produced, pollen energy available, protein content in pollen, and insect frequency in lamiaceae species depending on habitat (dg – the dry grassland, and re – the railway embankment). means are calculated from 2013-2014. standard deviations (±sd) are given. anovas were performed separately for each of analyzed feature. means followed by the same small letter are not significantly different between habitats within species and that followed by the same capital letter are not different among species, according to tukey hsd test. species site no. flowers per 1 m2 (thousand) mass of pollen per flower (mg) mass of pollen per 1 m2 (g) pollen energy per 1 m2 (kcal) protein (%) insect visitors frequency per flower/per season mean ±sd mean ±sd mean ±sd mean ±sd mean ±sd mean ±sd salvia pratensis dg 7.11b 2.33 0.02b 0.01 0.14b 0.04 0.81b 0.16 28.8a 5.1 0.003a 0.0002 re 3.42a 1.21 0.01a 0.00 0.03a 0.00 0.19 a 0.01 26.0a 2.6 0.013b 0.0001 mean 5.27b 0.02b 0.09a 0.50a 27.4b 0.008c salvia verticillata dg 1.92b 0.61 0.12b 0.05 0.23b 0.13 1.31a 0.49 27.3a 3.2 0.015a 0.0024 re 0.71a 0.14 0.03a 0.01 0.02a 0.01 0.12b 0.11 25.8a 3.6 0.080b 0.0035 mean 1.32a 0.08cd 0.13a 0.72a 26.6b 0.047d thymus serpyllum dg 12.83b 5.08 0.01a 0.00 0.13b 0.02 0.73b 0.19 22.7b 2.1 0.006a 0.0005 re 7.30a 2.04 0.01a 0.00 0.07a 0.01 0.42a 0.07 17.6a 1.5 0.011b 0.0361 mean 10.07c 0.01a 0.10a 0.57a 20.2a 0.009c betonica officinalis dg 9.09b 3.26 0.09a 0.04 0.82b 0.25 4.65b 1.64 23.9b 2.2 0.002a 0.0011 re 5.19a 1.54 0.11a 0.07 0.57a 0.38 3.25a 1.52 19.7a 2.0 0.005b 0.0023 mean 7.14b 0.10d 0.69b 3.95b 21.8a 0.003b origanum vulgare dg 122.00a 7.02 0.06b 0.03 7.32a 4.81 41.65a 19.18 28.7b 2.5 0.001a 0.0042 re 281.22b 107.78 0.03a 0.02 8.44b 5.32 48.00b 30.21 18.8a 2.5 0.001a 0.0001 mean 201.61d 0.05c 7.88c 44.83c 23.8c 0.001a mean for habitat dg 30.59x 51.25 0.06y 0.05 1.63x 3.14 9.83x 17.86 26.3y 2.8 0.004 0.0056 re 59.57y 123.93 0.04x 0.04 1.83y 3.70 10.40y 21.06 21.6x 4.0 0.022 0.0328 jachuła j., wrzesień m., strzałkowska-abramek m., denisow b. 166 acta bot. croat. 77 (2), 2018 and o. vulgare accumulated more protein in the individuals located in the dry habitat. the protein content in the pollen of s. pratensis and s. verticillata was similar, irrespective of the habitat. insect visitors the cca model revealed that the blossom cover and the species richness noted in the study patches had a significant impact on the distribution of insect visitors to the lamiaceae species (fig. 2, tab. 5). the pollen caloric value and pollen abundance also considerably influenced the structure of the insect visitors to the lamiaceae flowers. the model including all variables explained 66.4% of the observed variance. the eigenvalues were 0.226 for axis 1 and 0.137 for axis 2. according to the cca model, the abundance of pollinators to the lamiaceae species was statistically significant, with the exception of butterflies, beetles and flies. in total, 2214 insect individuals were found during our survey (dry grassland vs. railway embankment – 629 vs. 1585). the participation of flower visitors differed among the species; however, the main visitors to the lamiaceae species were bees, with a predominance of honeybees (fig. 3). bumblebees were observed most abundantly in o. vulgare flowers (mean = 28%) tab. 3. year-to-year disparities in the number of flowers, mass of pollen, and pollen energy available (mean for lamiaceae species studied and study sites). anovas were performed separately for each of analyzed variable. means followed by the same small letter are not significantly different between years, according to tukey hsd test. variable 2013 2014 mean from years mean ±sd mean ±sd no. flowers per 1 m2 (thous.) 73.52b 9.98 24.94a 4.26 49.23 mass of pollen per flower (mg) 0.03a 0.01 0.12b 0.02 0.07 mass of pollen (g m–2) 2.09a 0.36 2.89b 0.86 2.49 pollen energy (kcal m–2) 11.89a 7.90 15.88b 5.36 13.89 fig. 2. canonical correspondence analysis (cca) of the relationship between the floristic composition of the vegetation plots (relevés) and the spatial distribution of insect visitors and between the insect visitor pattern and lamiaceae species traits. grey points correspond to the dry grassland, black points to the railway embankment. eigenvalues: axis 1 – 0.41, axis 2 – 0.38. the diagram explains 66.4 % of total variance. explanations: species richness (richness), species diversity (diversity), the ‘blossom cover’ i.e. flower abundance (cover), flowering season (flowering), amount of pollen (pollen a), energy available in pollen (pollen e), and protein content in pollen (pollen p). tab. 4. anova’s of the effects of the species, the habitat, and the years of study on the studied variables in lamiaceae species in south-eastern poland. variable effect df f p – value flowers per 1 m2 (number) species 4 150.1 0.000 habitat 1 62.2 0.001 year 1 21.3 0.022 dry weight of anthers (mg) species 4 46.1 0.000 habitat 1 4.6 0.038 year 1 11.7 0.041 mass of pollen per flower (mg) species 4 40.2 0.000 habitat 1 71.9 0.000 year 1 0.4 0.020 pollen energy (kcal m–2) species 4 24.5 0.015 habitat 1 4.7 0.045 year 1 17.2 0.026 protein content in pollen (%) species 4 84.5 0.001 habitat 1 205.2 0.003 tab. 5. simple effects and conditional effects obtained from the summarize effects of explored variables in cca model. explanations: species richness (richness), species diversity (diversity), flower abundance (cover), flowering season (flowering), amount of pollen (pollen a), energy available in pollen (pollen e), protein content in pollen (pollen p),*p<0.05, ns – non significant. variable simple effects conditional effects bumblebees 16.6* 16.6* pollen e (kcal m–2) 16.5* 12.5* honeybees 15.5* 8.6ns pollen a (g m-2) 14.4* 7.6ns solitary bees 12.4* 7.4ns cover (%) 12.3* 10.6* flowering 10.2* 10.2* richness 10.4* 10.1* butterflies 9.2ns 8.0ns diversity 8.4ns 6.9ns beetles 7.9ns 5.0ns pollen (%) 6.0ns 4.5ns flies 4.0ns 3.2ns insect visitors in lamiaceae acta bot. croat. 77 (2), 2018 167 and solitary bees in b. officinalis (mean = 25.5%). flies were found in the flowers of all the studied species, but accounted for only 1.5% to 13.0% of visits, on average. beetles were observed (mean=2.1%) only in the flowers of b. officinalis. for all the studied species, the general pattern of insect groups observed in the lamiaceae flowers was quite similar between the habitats. however, the frequency of insect visitors to the flowers of the same lamiaceae species differed significantly between the habitats and was higher on the railway embankments (approx. 4-fold) (tab. 1). the relationship between the number of flowers and the frequency of insect visitors was not significant (r = –0.271, p = 0.488). during sunny weather, insect visitors collected nectar (approximately between 9.00–16.00) and pollen (approximately between 11.00– 15.00). this pattern was similar for all the species studied. discussion our study is a step towards quantifying the floral resources for pollinators. we found differences in flora attributes (plant species composition, species richness, species diversity, blossom cover, flowering spectrum) as well as the pollen mass and pollen energy available between the dry grassland and railway embankment biotopes. these disparities were intimately linked with the relative abundance and frequency of pollinators to the lamiaceae species. in our study, the blossom cover (= flower abundance) within vegetation patches significantly determined the insect visitor pattern to the lamiaceae species, explaining 12.3% of the variance. it is accepted that abundantly flowering vegetation patches have a strong community-level influence on the activity of insect visitors to particular plant species (kevan 1999). similarly, potts et al. (2003) documented the fact that greater numbers of individual insect foragers were simply recruited to plant species in floral community patches when floral resource availability progressively increased. insects are sensitive to energy stress; therefore, energy intake plays an important role in their foraging modes. there is a positive correlation between the blossom cover and daily energy availability; therefore, flower-dense patches that offer higher energy reward are simply preferred to minimize the costs of search (e.g. heinrich and raven 1972, heinrich 1975, roberts and harrison 1998, waddington 2001, cnaani et al. 2006). in our study, both the pollen abundance and the pollen energy content significantly influenced the pattern of insect visitors to the lamiaceae species (30.9% of the variance). pollen abundance determines the structure of bee fauna in the landscape of mediterranean ecosystems (potts et al. 2003). also, the pollen energy content can considerably influence pollinator abundance (petanidou and vokou 1990). in general, pollen abundance is positively correlated with the amount of pollen produced per flower and the number of flowers produced per unit area. lamiaceae species usually form dense patches and bloom abundantly, irrespective of the habitat and climatic zone (petanidou and vokou 1990, denisow and bożek 2008, bożek 2008, denisow 2011) and the quantity of floral reward offered in lamiaceae species is undoubtedly of particular importance for the foraging economics of pollinators. the protein content in pollen was not important for determination of the activity of insect visitors to the lamiaceae species. inconsistent results have sometimes been obtained concerning insect preferences to the protein content in pollen. roulston et al. (2000) found that protein content in pollen did not affect foraging preferences of pollinators. fig. 3. percentage participation of insect visitors on lamiaceae species studied in dry grassland (dg) and railway embankment (re). mean values from the years 2013-2014 are given, n = total number of insect individuals recorded during observations. jachuła j., wrzesień m., strzałkowska-abramek m., denisow b. 168 acta bot. croat. 77 (2), 2018 by contrast, hanley et al. (2008) noted that the protein content could be a selective factor in the choice of plant species foraged by the honeybee, since the pollen collectors clearly gathered larger amounts of pollen rich in protein. possibly, the variation in the protein content (mean from 20.2–27.4%) recorded for the lamiaceae representatives in our study was too small to be noticed by the entomofauna. however, it is still not clear which pollen property is particularly important to insect visitors. pollen is an almost exclusive source of the protein and non-carbon elements critical for the nourishment of larvae and development of adults (keller et al. 2005, müller et al. 2006, nicolson 2011). as suggested by lunau (2000), castellanos et al. (2006), or hanley et al. (2008), the scent released from the surface of pollen grains is more important than quality-driven pollen properties in determining insect food choice. according to baker and baker (1990), both protein content and the carbohydrate:lipid ratio influence the plant pollinator relationship and floral choices. although the protein content in the pollen (on average more than 20%) was not important for the activity of insect visitors to the lamiaceae species, it was considered high (roulston et al. 2000, denisow 2011). according to different authors (e.g. genissel et al. 2002, vanderplanck et al. 2014), high protein content in pollen is attractive to pollen-collecting insects and positively correlates with the development of bee larvae (roulston et al. 2000, vaudo et al. 2015). therefore, our view is that lamiaceae species should be considered while suitable plant species to restore and/or develop a pasture for pollinators are being chosen. we observed significant recruitment of honeybees, bumblebees, and solitary bees to the lamiaceae species in our study patches. the preference of social bees (apis mellifera, bombus spp.) for lamiaceae species has been evidenced by many authors in mediterranean ecosystems (e.g. petanidou and vokou 1990, petanidou et al. 2000). different species from the lamiaceae family have been classified as honeybee, bumblebee, or solitary bee species in poland (bożek 2000, 2003ab, denisow and bożek 2008, denisow 2011). in contrast, butterflies, flies and beetles did not respond to the traits of lamiaceae species. flies and beetles show high preferences for whitish or yellow flowers with open, i.e. dish-, or bowlshaped, corollas (glover 2014). these features do not fit our lamiaceae species, which developed deep, pink, purple, or violet corollas. it is generally accepted that flower morphology (e.g. corolla deepness, flower size) and features of floral reward (e.g. composition of nectar sugars, nectar concentration, scent released from the surface of pollen grains) exert an impact on the behaviour of insect visitors and the guild structure in plant species (petanidou et al. 2000, nicolson and thornburg 2007, denisow and wrzesień 2015a). contrary to our expectations, the lamiaceae species occurring in the homogeneous patches situated in the railway embankments received more insect visitors than the individuals in the heterogeneous ones. our findings conflict with results presented by e.g. fussell and corbet 1992, corbet 2000, ebeling et al. 2008 or wrzesień et al. 2016, who reported the highest frequency of pollinators for species occurring in high species-rich patches. a variety of factors affect insect visitor abundance and their frequency. at the community level, the size of the floral display (abundance of blooming), the flower attractiveness (i.e. flower morphology and traits of floral reward; discussed above), the co-flowering components of phytocoenoses, and the insect interactions with other individuals might be important in determining the flower choice by pollinators and their abundance and visitation frequency (kunin 1997, steffan-dewenter 2003, lázaro et al. 2009). moreover, the insect population size and the number of nests in the immediate neighbourhood strongly shape the abundance and rates of insect visitors (goulson 1999) and might be expected to have a dominant influence on the variation in the relative abundance of insect visitors to lamiaceae flowers. at the level of individuals, the floral visitation frequency decreases linearly with the total number of flowers (see, e.g., fussell and corbet 1992, steffan-dewenter 2003). no relation between the number of flowers and insect visitor frequency was established in our study. the above reasons indicate that the recruitment of insect visitors to particular species in floral patches is likely to be a complex phenomenon. as revealed by moroń et al. (2014), due to the occurrence of bare ground, railway embankments constitute a particularly valuable habitat for diverse ground-nesting bees and wasps and support populations of butterflies and hoverflies. presumably, the higher insect species richness and/or abundance in the railway biotope influence their number in our study species. however, a limitation of our study is that it does not include measurements of the insect diversity and population size between the habitats. for the same species, pollen productivity differed in the spatio-temporal pattern. first, the space-varying growth conditions (e.g. nutrients and/or water availability) between the dry grassland and the railway embankment are presumably responsible for the diversity of inter-habitat pollen resources noted in our study. the habitat type has been documented to affect the pollen resource levels in flowers; hence, optimal habitat and /or environmental conditions are necessary to ensure efficient pollen resources (e.g. denisow and wrzesień 2015a, denisow 2009, 2011). second, significant differences in the amount of pollen produced in flowers between years are supportive of a strict relationship between changeable external environmental factors (weather conditions) and the mass of produced pollen. the considerable impact of the weather parameters during the differentiation of generative buds or microsporogenesis on the mass of pollen was emphasized in many studies (e.g. bożek 2003b, aguilera and valenzuela 2013). in extremely unfavourable weather conditions (e.g. temperature drops or prolonged shortage of precipitation), plants even fail to produce pollen (denisow 2011). the year-to-year fluctuations in the total pollen yield and pollen energy available per unit area obtained from particular lamiaceae species are understandable; they result from different factors that can occur at the same time, e.g. flora dynamics, disparity in pollen output per flower, and varying abundance of flowering in the patches of plant communities. their effects on the variations in food insect visitors in lamiaceae acta bot. croat. 77 (2), 2018 169 resources (mass and energy) in plant communities was also described by parrish and bazzaz (1979), tanacs and gulyas (1986), petanidou and vokou (1990), petanidou and smets (1995), or denisow (2011). our data provide strong evidence that insect abundance in lamiaceae species vary with flowering phenology (10.2% of variance). we have demonstrated community-level differences in the seasonal blooming spectrum between the dry grassland and the railway embankment. abundant flowering begins earlier in the season in the dry grassland habitat, while railway embankment flora ensures resource availability at the end of the season. abundant spring flowering and a decrease during the summer-drought period is a distinct feature of the flora in dry grassland communities in europe and relate to the predominance of perennial life forms (klimešová et al. 2008). a scarcity of plants flowering in spring and a shift in flowering abundance towards the end of the season was highlighted for alternative man-made habitats (denisow 2011, denisow and wrzesień 2015b, wrzesień et al. 2016). therefore, to provide pollinators with a balanced diet throughout the vegetation season, diverse habitats (semi-natural and man-made) are required in the landscape to supply a complementary bloom pattern and ensure continuity of food resources. high values of landscape heterogeneity have been proposed to increase the pollinator biodiversity (β-diversity hypothesis; steffan-dewenter et al. 2002, tscharntke et al. 2012). in conclusion, due to their abundant blooming, the amount of pollen resources provided, the pollen caloric value, and the high protein content in the pollen, lamiaceae species attract a variety of insect pollinators. therefore, lamiaceae species should be considered in the protocol to improve food resources for pollinators to guarantee constant and high quality of food resources. however, the substantial interand intra-species disparities in pollen quality should not be ignored, especially if we intend to provide sufficient floral resources for pollinators on a landscape scale (winfree 2010, nicolson 2011). acknowledgements the material from the species growing within the protected xerothermic grassland areas was collected in compliances with polish law under permit from the regional nature conservator in lublin. our thanks go also to michał wrzesień, who supported the logistics. we are grateful to mrs anna wesołowska-zoń for improving our english. research was supported financially by the ministry of science and higher education of poland as a part of the statutory activities of department of botany (project okb/ds/2), university of life sciences in lublin and department of geobotany, institute of biology and biochemistry, maria curie-skłodowska university in lublin. references castellanos, m. c., wilson, p., keller, s. j., wolfe, a. d., 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(eds.), cognitive ecology of pollination, 41–60. cambridge university press, cambridge. wallisdevries, m. f., poschlod, p., willems, j. h., 2002: challenges for the conservation of calcareous grasslands in northwestern europe: integrating the requirements of flora and fauna. biological conservation 104, 265–273. winfree, r., 2010: the conservation and restoration of wild bees. annals of the new york academy of sciences 1195, 169–97. wrzesień, m., denisow b., 2006a: the share of nectariferous and polleniferous taxons in chosen patches of thermophilous grasslands of the lublin upland. acta agrobotanica 59, 213– 221. wrzesień m., denisow b., 2006b. the usable taxons in spontaneous flora of railway areas of the central-eastern part of poland. acta agrobotanica 59, 95-108. wrzesień, m., jachuła, j., denisow, b., 2016: railway embankments – refuge areas for food flora, and pollinators in agricultural landscape. journal of apicultural science 60, 39–51. acta botanica 1-2017 za web.indd acta bot. croat. 76 (1), 2017 9 acta bot. croat. 76 (1), 9–14, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0040 issn 0365-0588 eissn 1847-8476 floristic composition of vegetation in habitats suitable for erigeron × huelsenii (asteraceae) artur pliszko*, małgorzata jaźwa department of taxonomy, phytogeography and paleobotany, institute of botany, jagiellonian university, 31 kopernika street, 31-501 kraków, poland abstract – the paper provides data on the fl oristic composition of vegetation in anthropogenic habitats suitable for erigeron × huelsenii, a spontaneous hybrid between the alien e. canadensis and native e. acris s. str. the study is based on 21 phytosociological relevés (vegetation plots) of 5×5 m size made in 2013–2014 in the polish part of the lithuanian lakeland (north-eastern poland) using the braun-blanquet method. the plots were taken on a roadside verge, a roadside slope, in abandoned sand and gravel pits, and on arable fi elds with grass-legume mixtures. there are 91 species of vascular plants, four species of bryophytes, three species of lichens, and one species of cyanobacteria. vegetation is represented by populations of species typical of plant communities from the classes stellarietea mediae, artemisietea vulgaris, koelerio-corynephoretea, festucobrometea and molinio-arrhenatheretea. two fl oristic groups recognized from hierarchical cluster analysis and principal component analysis correspond with ruderal and segetal habitats. the study reveals that arable fi elds with grass-legume mixtures on sandy soils can be very suitable for e. × huelsenii. keywords: anthropogenic habitat, braun-blanquet method, erigeron acris, erigeron canadensis, invasive species, multivariate analysis, plant hybridization * corresponding author, e-mail: artur.pliszko@uj.edu.pl introduction habitats disturbed by human activities play a major role in the process of hybridization between alien and native plant species (vilà et al. 2000, daehler and carino 2001, blair and hufbauer 2010, guo 2014). it has long been recognized that disturbance can bring two parental species into close proximity, increasing rates of cross-pollination and hybridization. moreover, disturbance often creates intermediate habitats that are especially suitable for hybrids (anderson 1948, daehler and carino 2001). hybridization involving alien and native species used to be viewed as one of the indirect effects of biological invasions (vilà et al. 2000). nowadays, it is an impact mechanism taken into consideration in the classifi cation of alien species (blackburn et al. 2014). spontaneously occurring hybrids between alien and native plants have been well-documented (vilà et al. 2000, daehler and carino 2001, bleeker et al. 2007, lehman et al. 2014, stace et al. 2015), and according to pyšek et al. (2004) they must be understood as alien plant species. in europe, erigeron canadensis l. (≡ conyza canadensis (l.) cronquist) (asteraceae), one of the commonest alien plants of north american origin (weaver 2001, lambdon et al. 2008), hybridizes with a native congener e. acris l. s. str. giving a hybrid known as e. × huelsenii vatke (≡ × conyzigeron huelsenii (vatke) rauschert). the hybrid has been reported from the united kingdom, belgium, germany, the czech republic, slovakia and poland (wurzell 1995, šída 2000, bleeker et al. 2007, danihelka et al. 2012, verloove and lambinon 2014, pliszko 2015, stace et al. 2015). it is characteristically found as a single plant or in small numbers together with the parental species in disturbed areas, and is considered to be completely sterile (stace et al. 2015). the alien parent e. canadensis is a species of ruderal and segetal communities of the class stellarietea mediae, and the native parent e. acris s. str. is a species of semi-natural dry grassland communities of the classes koelerio-corynephoretea and festuco-brometea and ruderal communities of the class artemisietea vulgaris (mucina 1997, šída 2004, pliszko 2015). both parental species are light-demanding plants and occur on mineral, mesotrophic, usually dry soils (zarzycki et al. 2002). due to its ephemeral occurrence and intermediate morphological features the hybrid is easily overlooked during fl oristic and ecological fi eld surveys. in consequence, phytosociological studies on habitats suitable for e. ×huelsenii have not been undertaken. in this paper we aimed to compare the fl oristic composition of vegetation in disturbed places in pliszko a., jaźwa m. 10 acta bot. croat. 76 (1), 2017 poland where the spontaneous hybridization between e. canadensis and e. acris s. str. occurs. materials and methods study area the study was conducted in seven newly discovered localities of erigeron × huelsenii in the polish part of the lithuanian lakeland, north-eastern poland (on-line suppl. fig. 1, tab. 1). this lowland area is located in a transitory temperate climate zone with some infl uence from the continental climate. in the period 1971–2000 the average annual air temperature was about 6.5 °c, and the average annual precipitation was 550–600 mm (lorenc 2005). the native vegetation is represented mainly by nemoral forest communities with boreal and subboreal infl uences (szafer and zarzycki 1972), however, the area is highly deforested and has an agricultural character. the soils in the studied sites developed from the glacial-fl uvial sands and gravels deposited during the vistula glaciation (ber 1981). sampling a total of 21 phytosociological relevés (vegetation plots) of 5×5 m size were made in 2013–2014 in open anthropogenic habitats, including one plot on a sandy roadside verge, one plot on a sandy roadside slope, eight plots in abandoned sand and gravel pits, and 11 plots on sandy arable fi elds with grass-legume mixtures (tab. 1). visual estimation of cover-abundance in plots was based on the widely applied braun-blanquet method (braun-blanquet 1964, westhoff and van der maarel 1973). names of taxa followed mirek et al. (2002), wójciak (2003), and siemińska and wołowski (2003). taxonomic treatment of erigeron followed the concept proposed by greuter (2003). names of syntaxa and diagnostic species followed mucina (1997). taxa were identifi ed using morphological features given by wójciak (2003), rutkowski (2004), keshari et al. (2015), and stace et al. (2015). geographical-historical status of vascular plants is according to mirek et al. (2002) and tokarska-guzik et al. (2012). hybrids between alien and native plant species are treated as alien species (pyšek et al. 2004). voucher specimens of e. × huelsenii collected during the fi eld studies are deposited in the herbarium of the institute of botany of the jagiellonian university in kraków (kra). data analysis cover-abundance values of the braun-blanquet scale were transformed to the numerical 1–9 scale (van der maarel 1979). the vegetation plots were analyzed by multivariate methods. data analyses were performed using mvsp version 3.1 (multivariate statistical package) (kovach 1999). the jaccard similarity index was used to estimate the species composition similarity between the vegetation plots. the dendrogram was prepared using the unweighted pair group method with arithmetic mean (upgma). the scatter diagram was prepared using the procedure of principal component analysis (pca). results all vegetation plots together contain 91 species of vascular plants (excluding microspecies of the genus taraxacum), four species of bryophytes, three species of lichens, and one species of cyanobacteria. there are 15 alien species and three species of uncertain geographical-historical status in the polish fl ora. the plots sampled on arable fi elds include fi ve species cultivated as fodder crops (grass-legume mixtures), namely dactylis glomerata l., lolium perenne l., medicago sativa l., phleum pratense l. and trifolium pratense l. (on-line suppl. tab. 1). the number of species per plot is 24.62 ± 6.20. the cover of individual species in plots is low, including erigeron ×huelsenii and its parental species, and only in three cases it reaches 75% (i.e. calamagrostis epigejos (l.) roth, dactylis glomerata and ceratodon purpureus (hedw.) brid.). vegetation is represented by populations of species typical of plant communities from the classes stellarietea mediae, artemisietea vulgaris, koelerio-corynephoretea, festuco-brometea and molinioarrhenatheretea (on-line suppl. tab. 1). fig. 1. dendrogram made on the basis of cluster analysis (unweighted pair group method with arithmetic mean and the jaccard index) showing fl oristic similarity between the vegetation plots. numbers (1–21) indicate respective relevés described in tab. 1 and on-line supplement tab. 1. vegetation in habitats suitable for erigeron × huelsenii acta bot. croat. 76 (1), 2017 11 from the hierarchical cluster analysis (upgma) and principal component analysis (pca) two main fl oristic groups were clearly recognized (figs. 1–2). these groups correspond with ruderal and segetal habitats. the plots taken on a roadside verge (5), a roadside slope (2), and in abandoned sand and gravel pits (1, 6–9, 13–15) represent a group of ruderal habitats, whereas the plots taken on arable fi elds with grass-legume mixtures (3, 4, 10–12, 16–21) represent a group of segetal habitats. the herb layer has a lower cover in plots sampled in ruderal habitats than those sampled in segetal habitats, but the moss layer has a higher cover in ruderal than in segetal habitats. moreover, in seven plots taken on arable fi elds there is no moss layer (on-line suppl. tab. 1). discussion based on this study spontaneous hybridization between erigeron canadensis and e. acris s. str. occurs in two fl oristically different types of anthropogenic habitats in poland. tab. 1. location, date of sampling and habitat description of plots sampled for the study. according to the atpol cartogram method (zając 1978) the capital letters indicate 100-km square, whereas the numbers indicate 10-km square; m.a.s.l. – meters above sea level. plot no. locality gps coordinates altitude (m.a.s.l.) atpol cartogram unit habitat date 1 suwałki near sobolewo 54°4′ 43.8″ n 22°57′ 30.18″ e 159 fb18 abandoned sand and gravel pit 7 august 2013 2 filipów czwarty 54°9′ 54.24″ n 22°36′ 57.54″ e 195 fb06 sandy roadside slope 9 august 2013 3 filipów pierwszy 54°9′ 49.32″ n 22°37′ 6.84″ e 185 fb06 sandy arable fi eld with grass-legume mixture 16 august 2013 4 filipów pierwszy 54°9′ 49.44″ n 22°37′ 5.4″ e 186 fb06 sandy arable fi eld with grass-legume mixture 16 august 2013 5 kamionka stara near bakałarzewo 54°4′ 46.2″ n 22°40′ 48.18″ e 164 fb17 sandy roadside verge 18 august 2013 6 suwałki near sobolewo 54°4′ 43.8″ n 22°57′ 25.8″ e 158 fb18 abandoned sand and gravel pit 6 august 2014 7 suwałki near sobolewo 54°4′ 44.64″ n 22°57′ 28.56″ e 157 fb18 abandoned sand and gravel pit 6 august 2014 8 suwałki near sobolewo 54°4′ 11.64″ n 22°58′ 5.88″ e 159 fb18 abandoned sand and gravel pit 6 august 2014 9 suwałki (former żwirownia pkp) 54°6′ 26.58″ n 22°53′ 55.8″ e 173 fb08 abandoned sand and gravel pit 8 august 2014 10 mieruniszki 54°8′ 57.96″ n 22°34′ 19.5″ e 196 fb06 sandy arable fi eld with grass-legume mixture 10 august 2014 11 mieruniszki 54°8′ 58.26″ n 22°34′ 21.36″ e 195 fb06 sandy arable fi eld with grass-legume mixture 10 august 2014 12 mieruniszki 54°9′ 2.34″ n 22°34′ 21.24″ e 196 fb06 sandy arable fi eld with grass-legume mixture 10 august 2014 13 suwałki near sobolewo 54°4′ 14.64″ n 22°57′ 57.12″ e 162 fb18 abandoned sand and gravel pit 18 august 2014 14 suwałki near sobolewo 54°4′ 14.58″ n 22°58′ 0.6″ e 157 fb18 abandoned sand and gravel pit 18 august 2014 15 suwałki near sobolewo 54°4′ 14.46″ n 22°58′ 2.04″ e 158 fb18 abandoned sand and gravel pit 18 august 2014 16 ostrowo near pluszkiejmy 54°16′ 55.26″ n 22°27′ 11.58″ e 180 fa85 sandy arable fi eld with grass-legume mixture 7 september 2014 17 ostrowo near pluszkiejmy 54°16′ 56.34″ n 22°27′ 12.24″ e 180 fa85 sandy arable fi eld with grass-legume mixture 7 september 2014 18 filipów czwarty 54°10′ 5.52″ n 22°36′ 52.02″ e 195 fb06 sandy arable fi eld with grass-legume mixture 8 october 2014 19 filipów czwarty 54°10′ 5.82″ n 22°36′ 52.2″ e 195 fb06 sandy arable fi eld with grass-legume mixture 8 october 2014 20 filipów pierwszy 54°9′ 50.16″ n 22°37′ 6.18″ e 187 fb06 sandy arable fi eld with grass-legume mixture 9 october 2014 21 filipów pierwszy 54°9′ 49.32″ n 22°37′ 4.68″ e 186 fb06 sandy arable fi eld with grass-legume mixture 9 october 2014 pliszko a., jaźwa m. 12 acta bot. croat. 76 (1), 2017 these types concern ruderal and segetal areas. it has been reported that e. × huelsenii occurs mainly in places of a ruderal character such as sand pits, roadside verges, disused ironworks, railway areas (wurzell 1995, crompton and preston 2001, verloove and lambinon 2014, stace et al. 2015), but its occurrence in segetal habitats is poorly recognized. what is interesting is that the vegetation in all plots sampled in ruderal habitats represents an early stage of secondary succession where there are no tree and shrub layers, and the cover values of the herb and moss layers are generally low. moreover, the early stage of secondary succession in these habitats can be characterized by the presence of epigeic species of cyanobacteria, lichens and bryophytes, as was pointed out by langhans et al. (2009). in segetal habitats sampled for the study, in contrast, the process of secondary succession is hindered by the regular cutting of grass-legume crops for hay, thus there are no woody plant species. nevertheless, many herbaceous plants can thrive in arable fi elds with grass-legume mixtures as weeds (sanderson et al. 2013), especially on poor sandy soils where the canopy of cultivated plants is usually not dense or there are gaps in the canopy. the results of principal component analysis (fig. 2) show that the group of ruderal habitats is fl oristically more heterogeneous than the group of segetal habitats. this can be explained by the fact that in the early stages of secondary succession, abandoned sand and gravel pits are readily colonized by various plant species typical of ruderal, grassland and meadow communities (řehounková and prach 2006). in the vegetation plots sampled for the study most of the associated species with the highest constancy are light-demanding plants and occur usually on dry, mesotrophic or oligotrophic, sandy soils (e.g. artemisia campestris l., hieracium pilosella l. and sedum acre l.), like e. canadensis and e. acris (zarzycki et al. 2002). wurzell (1995) suggested that persistence of e. × huelsenii in ruderal habitats depends on competition from more vigorous wayside and wasteland perennial plants. moreover, it appears that the changes in the light conditions caused by the overgrowth of trees and shrubs can limit the occurrence of the hybrid and its light-demanding parents, especially in abandoned sand and gravel pits where some woody plants are already present in the herb layer (i.e. betula pendula roth, malus domestica borkh., pinus sylvestris l. and populus tremula l.). for all of the aforementioned reasons, we assume that e. × huelsenii occurs in ruderal and segetal habitats due to favourable light conditions and to the presence of canopy gaps in which the hybrid seedlings can survive the competition from other plants. in this paper the fl oristic composition illustrates the summer and autumn aspects of vegetation. the late time of plot sampling is a consequence of the phenology of e. × huelsenii. it is hard to fi nd and identify vegetative individuals of the hybrid in spring. besides, in the case of arable fi elds with grass-legume mixtures, when there is the second cutting of crops, the hybrid is visible only after regrowth. it is also worth mentioning that in plot number 17 (located in an arable fi eld with grass-legume mixture) two spontaneous hybrids between alien and native plants were recorded, namely e. × huelsenii and solidago × niederederi khek, which seems to be a very unusual situation (on-line suppl. tab. 1). in poland, e. canadensis is treated as a nationally invasive alien species (tokarska-guzik et al. 2012). it is able to appear in large numbers, mainly in anthropogenic habitats, and hybridization with a native relative e. acris s. str. is one of its neglected impacts. although e. canadensis and e. acris s. str. are commonly distributed in the country (zając and zając 2001), and often share habitats, the hybrid e. × huelsenii is rarely found (pliszko 2015). its rarity is a refl ection of the fact that it is usually overlooked by taxonomists and ecologists during fi eld surveys. fig. 2 scatter diagram presenting the results of principal component analysis (pca) for the studied plots. numbers (1–21) indicate respective relevés described in tab. 1 and on-line supplement tab. 1. selected species are represented by arrows. vegetation in habitats suitable for erigeron × huelsenii acta bot. croat. 76 (1), 2017 13 the present study reveals that, aside from the various ruderal habitats, arable fi elds with grass-legume mixtures on sandy soils can be very suitable for the hybrid between e. canadensis and e. acris s. str. however, there is a need for further observations on the persistence of e. × huelsenii in such specifi c habitats. acknowledgements the fi eld studies were fi 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(eds.), flora of the czech republic, vol. 7, 140–153. academia, praha (in czech). tokarska-guzik, b., dajdok, z., zając, m., zając, a., urbisz, a., danielewicz, w., hołdyński, c., 2012: alien plants in poland with particular reference to invasive species. generalna dyrekcja ochrony środowiska, warszawa (in polish). verloove, f., lambinon, j., 2014: the sixth edition of the nouvelle flore de la belgique: nomenclatural and taxonomic remarks. dumortiera 104, 7–40. vilà, m., weber, e., d’antonio, c. m., 2000: conservation implications of invasion by plant hybridization. biological invasions 2, 207–217. pliszko a., jaźwa m. 14 acta bot. croat. 76 (1), 2017 weaver, s. e., 2001: the biology of canadian weeds. 115. conyza canadensis. canadian journal of plant science 81, 867–875. westhoff,v., maarel, e. van der, 1978: the braun-blanquet approach. in: whittaker, r. h. (ed.), classifi cation of plant communities, 287–399. dr w. junk bv publishers, the hague. wójciak, h., 2003: lichens, bryophytes, pteridophytes. flora of poland. multico ofi cyna wydawnicza, warszawa (in polish). wurzell, b., 1995: × conyzigeron huelsenii in east london. bsbi news 68, 32–33. zając, a., 1978: atlas of distribution of vascular plants in poland (atpol). taxon 27, 481–484. zając, a., zając, m., (eds.) 2001: distribution atlas of vascular plants in poland. laboratory of computer chorology, institute of botany, jagiellonian university, kraków. zarzycki, k., trzcińska-tacik, h., różański, w., szeląg, z., wołek, j., korzeniak, u., 2002: ecological indicator values of vascular plants of poland. w. szafer institute of botany, polish academy of sciences, kraków. 126 acta bot. croat. 77 (2), 2018 acta bot. croat. 77 (2), 126–134, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0007 issn 0365-0588 eissn 1847-8476 the flora and vegetation of the ne mediterranean islet with centuries-long human influences nenad jasprica1, katija dolina1*, milenko milović2 1 institute for marine and coastal research, university of dubrovnik, p.o. box 83, hr-20000 dubrovnik, croatia 2 "antun vrančić" grammar school, put gimnazije 64, hr-22000 šibenik, croatia abstract – this paper is based on the main results of an analysis of spontaneous flora and vegetation on the small islet of vrnik (0.281 km2), on which there are some abandoned limestone quarries, on the eastern adriatic coast. the investigations were carried out from 2014 to 2016. altogether, 251 vascular plant taxa (species and infraspecific units) were recorded on the islet. a total of 11 plant associations, one subassocation and two stands within 10 vegetation classes were identified. due to high anthropogenic influences during the last centuries, quarrying in particular, the islet investigated showed a relatively low variety of vascular plant taxa. in addition, clear signs of fragmentation of the forest vegetation were observed. keywords: croatia, eastern adriatic, phytosociology, syntaxonomy, vascular plant diversity * corresponding author, e-mail: katija.dolina@unidu.hr introduction the karstic islet of vrnik on the eastern adriatic coast (wgs84: 42°56′9″n 17°10′9″e; or according to croatian terrestrial reference system 1996 (htrs96): x=554623, y=4755335) is part of the korčula archipelago situated in the channel between the pelješac peninsula and the island of korčula in southern croatia (fig. 1). the islet is the second largest in the whole archipelago which comprises 19 islets and five rocks. exploitation of mineral resources (architectural building stone) has a long tradition in the archipelago, including the islet of vrnik in particular, dating back to the roman empire (krklec et al. 2011). the senonian limestone was used fig. 1. geographical position of the islet of vrnik in south croatia, eastern adriatic (abbreviations: slo: slovenia; hr: croatia; bih: bosnia and herzegovina; mne: montenegro; i: italy). flora and vegetation of the mediterranean islet acta bot. croat. 77 (2), 2018 127 for the construction and decoration of villas and residences in the region of dalmatia, the city of dubrovnik in particular, but it was also exported to the orient (e.g., constantinople) and italy (gjivoje 1969, krklec et al. 2011). the finding of the lumbarda psephisma, the most important monument written in greek in croatia, carved into a stone pillar which was found near to the village of lumbarda on the island of korčula, suggests that the islet was populated at the beginning of the 4th or 3rd century bc (kršinić-šove 1971, solarić and solarić 2009). during the last six centuries or so around six hundred people lived there and worked in thirty quarries (dunda et al. 2003). quarrying on the island continued until the late 1950s and cessation of it coincides with the depopulation of the island (galić et al. 1999). the population in the middle of the 20th century was almost 200; numbers then gradually diminished and nowadays there are only a few permanent inhabitants. nowadays, it is very popular as a tourist destination in the summer time having a population in excess of about 200 people in a very small settlement (the village of vrnik). the modern village of vrnik occupies the land between the quarry-face and the northern coast, an area previously quarried away. abandoned quarry workers' cottages cluster in the eastern part of the modern village. in the mediterranean basin the flora of small and very small islands has long been a subject of interest in relation both to basic floristic knowledge and also in order to assess the impact of land use on the land cover (jasprica et al. 2015, and references therein). in the northern section of the mediterranean sea, the adriatic basin forms its most important part. the adriatic sea has over 1,300 islands and isles, mostly located along its eastern, croatian, coast, which are considered among the most diverse in the mediterranean region. generally, the dalmatian coast could be also defined as a hotspot, but the data on its flora are still incomplete (médail and quézel 1997). an estimation using the species-area relationship analysis (sar) for 106 adriatic islands (nikolić et al. 2008), shows that 1,807 plant taxa grow on the croatian islands, providing a heritage of biodiversity that must be bequeathed to future generations as a ‘reservoir’ available for the processes of biological evolution and for their ecological value. however, there naturally remain islands and islets that have not yet been floristically and ecologically investigated. generally, all islets in the korčula archipelago remain completely botanically unknown, and only a few botanical studies have been made of the rest of the islets (e.g. bogdanović and brullo 2015, jasprica and milović 2016). the aim of this study was to present results of investigations into the flora and vegetation of the islet of vrnik. our hypothesis was that vegetation cover on the islet is mostly the result of temporary natural factors. study area the islet has an area of 0.281 km2 with a maximum length of 900 meters, a width of 450 m, a maximum altitude of 46 m and a distance from the island of korčula of 100 m (fig. 1). the predominant soil type is calcocambisol (korolija et al. 1977). the coast is low and rocky and has a total length of 2.3 km. sea depths around the islet are between 2 and 15 m. quarrying activity was focused at the northern part of the islet (fig. 2). a near continuous quarry face runs across this area for just over 500 m from midway along the northeast side of the islet to midway along its northwest side; a second quarry-face runs from this point down the western side of the islet for at least 250 m (russell and glicksman 2015). the climate of the islet is mediterranean, with mild winters and hot summers. the reference thermo-pluviometric station is korčula. the average annual air temperature is 16.8 °c, precipitation ranges from 1000 – 1250 mm yr–1 and there are 2,400 hours of sunshine a year (krklec et al. 2011). overall the bioclimate of the area can be defined as lower mesomediterranean (sensu rivas-martínez et al. 2011, jasprica and milović 2016). the islet belongs, as a part of the island of korčula, to the important plant areas (ipas) in croatia (jasprica 2010). in addition, the islet is part of the natura 2000 european ecological network of sites important for birds (site code hr1000036: middle dalmatian islands and the pelješac peninsula; official gazette of the republic of croatia 2013b, 2015). unfortunately, there are currently no plans for natura 2000 network management of the islet. fig. 2. quarry-face on the northern side of the islet of vrnik in the late 1950s. materials and methods flora the study was carried out from 2014 to 2016 in different seasons (early and late spring, early summer and autumn). herbarium specimens are deposited in the herbarium collection of the laboratory for terrestrial flora and fauna of the university of dubrovnik (www.imp-du.com). collections were made only if ten or more individuals were present in a plant population. coordinates were listed according to the croatian terrestrial reference system 1996 (htrs96) based on the european terrestrial reference system 1989 (etrs89) with geodetic reference system 1980 (grs80) level-ellipjasprica n., dolina k., milović m. 128 acta bot. croat. 77 (2), 2018 soid as a mathematical and physical approximation of earth’s shape. data on the surface area and perimeter of the islands were obtained from the croatian programme for the protection and use of small, both inhabited and uninhabited, islands and the surrounding sea (ministry of the sea, transport and infrastructure 2007). the highest altitude was determined using topographic maps to the scale 1:25000 (tk25, topo hr). in the list (on-line suppl. tab. 1), species are given in alphabetical order. for each taxon, life form and chorological element are reported. the life form follows the raunkiaer system (raunkiaer 1934) as proposed by pignatti (1982). regarding chorological form, reference was made to jasprica et al. (2016a), milović et al. (2016) and references therein. taxonomic nomenclature follows the flora croatica database (nikolić 2017). were separated into associations/stands on the basis of the diagnostic and/or dominant species, in line with the traditional syntaxonomic system of the communities. the syntaxonomical system (the eurovegchecklist) proposed by mucina et al. (2016), and followed by škvorc et al. (2017) was applied. classification of the vegetation units distinguished into habitat types of annex i of the habitats directive 92/43/eec was made according to the list of natura 2000 habitats declared by the croatian government (european commission 1992, 2013; official gazette of the republic of croatia 2014). priority habitats are denoted by an asterisk (*) as shown in the syntaxonomic scheme. results flora two hundred and fifty-one (251) vascular plant taxa (species and infraspecific units) were recorded on the islet of vrnik (on-line suppl. tab. 1). altogether, 70 families and 186 genera were noted. among them, the families most represented were: poaceae (30 taxa, 12%), fabaceae (28, 11.2%), cichoriaceae (16, 6.4%), asteraceae (13, 5.2%), lamiaceae (11, 4.4%), rubiaceae and caryophyllaceae (eight taxa in each, 3.2%), and apiaceae, brassicaceae, euphorbiaceae and ranunculaceae (six taxa in each, 2.4%). four taxa can be considered as endemic: anthyllis vulneraria ssp. weldeniana, limonium dictyophorum, lolium subulatum and seseli tomentosum. from the croatian red list, papaver hybridum is considered to be a critically endangered (cr) taxon. endangered taxa (en) were carex extensa, delphinium staphisagria and glaucium flavum, while ophrys sphegodes, orchis simia, parapholis incurva and salsola soda have been classified as vulnerable (vu). in addition, nine taxa (adiantum capillusveneris, anacamptis pyramidalis, cyclamen repandum, elymus pycnanthus, limonium dictyophorum, matthiola incana, seseli tomentosum, sternbergia lutea and teucrium fruticans) were classified as near threatened (nt). ruscus aculeatus was found to be of least concern (lc). seven taxa (brachypodium phoenicoides, ecballium elaterium, echinaria capitata, lolium subulatum, raphanus raphanistrum ssp. landra, trifolium echinatum and posidonia oceanica) have been classified as data deficient (dd). in total, 16 taxa were strictly protected (spt) by croatian law: anacamptis pyramidalis, anthyllis vulneraria ssp. weldeniana, carex extensa, delphinium staphisagria, echinaria capitata, gladiolus illyricus, glaucium flavum, limonium dictyophorum, lolium subulatum, ophrys sphegodes, orchis simia, papaver hybridum, parapholis incurva, posidonia oceanica, salsola soda and seseli tomentosum. among the recorded taxa, ailanthus altissima, carpobrotus acinaciformis, conyza canadensis and c. sumatrensis are considered to be invasive (ias). carpobrotus acinaciformis covers larger areas on the northern slopes of the islet. the analysis of plant life forms showed that the vrnik flora is dominated by therophytes (35.4%) and hemicryptophytes (23.9%), respectively (tab. 1). tab. 1. life-form spectra on the islet of vrnik. life forms no. (%) therophytes (t) 89 (35.4) hemicryptophytes (h) 60 (23.9) phanerophytes (p) 49 (19.5) chamaephytes (ch) 27 (10.8) geophytes (g) 25 (10.0) hydrophytes (hy) 1 (0.4) total taxa 251 (100.0) taxa listed in the red book of vascular flora of croatia (nikolić and topić 2005, nikolić 2017) are marked with their corresponding iucn status (iucn 2016). taxa considered to be endemic are denoted according to nikolić et al. (2015). in addition, strictly protected taxa (spt) as defined by croatian law are also denoted (official gazette of the republic of croatia 2013a, c). any invasive alien taxa (ias) have been defined according to nikolić et al. (2014) and nikolić (2017). taxa were determined using the standard determination keys, books and guides: bonnier (1911–1935), fiori (1923– 1929), hayek (1924–1933), hegi (1936–1987), horvatić and trinajstić (1967–1981), tutin et al. (1968–1980, 1993), javorka and csapody (1975), trinajstić (1975–1986), pignatti (1982), delforge (1995, 2006), etc. vegetation vegetation was studied in accordance with the principles of the braun-blanquet approach (braun-blanquet 1964), adopting the international code of phytosociological nomenclature (weber et al. 2000, see also dengler et al. 2008). altogether, 26 phytosociological relevés were collected on the islets. the plot size used to sample vegetation was established so as to represent the full floristic composition, depending on plant density and homogeneity of vegetation cover. geographical coordinates using the croatian terrestrial reference system 1996 (htrs96), elevation above sea level, aspect and slope inclination were noted for each relevé. the relevés flora and vegetation of the mediterranean islet acta bot. croat. 77 (2), 2018 129 limonietum anfracti ilijanić and s. hećimović 1982 helichrysetosum italici jasprica 2015 [habitat code 1240 – vegetated sea cliffs of the mediterranean coasts with endemic limonium spp.] saginetea maritimae westhoff et al. 1962 parapholis incurva community [habitat code 1310 – salicornia and other annuals colonising mud and sand] cymbalario-parietarietea diffusae oberd. 1969 tortulo-cymbalarietalia segal 1969 galio valantiae-parietarion judaicae rivas-mart. ex o. de bolòs 1967 oxalido-parietarietum judaicae (br.-bl. 1952) segal 1969 cymbalario-asplenion segal 1969 linario cymbalariae-parietarietum ramiflorae pignatti 1952 (=cymbalarietum muralis görs 1966) [habitat code 8210 – calcareous rocky slopes with chasmophyic vegetation] lygeo sparti-stipetea tenacissimae rivasmart. 1978 nom. conserv. propos. (=thero-brachypodietea ramosi br.-bl. in br.-bl. et al. 1947) cymbopogono -brachypodietalia ramosi horvatić 1963 cymbopogono-brachypodion ramosi horvatić 1963 piptatheretum miliaceae horvatić (1956) 1958 [habitat code 6220* – pseudosteppe with grasses and annuals therobrachypodietea] papaveretea rhoeadis s. brullo et al. 2001 nom. conserv. propos. aperetalia spicae-venti j.tx. et tx. in malato-beliz et al. 1960 nom. conserv. propos. cynodon dactylon community chenopodietea br.-bl. in br.-bl. et al. 1952 brometalia rubenti-tectorum (rivas goday et rivas-mart. 1973) rivas-mart. et izco 1977 nom. conserv. propos. hordeion murini br.-bl. in br.-bl. et al. 1936 hordeetum leporini br.-bl. 1936 quercetea ilicis br.-bl. ex a. bolòs et o. de bolòs in a. bolòs y vayreda 1950 pinetalia halepensis biondi, blasi, galdenzi, pesaresi et vagge in biondi et al. 2014 pistacio lentisci-pinion halepensis biondi, blasi, galdenzi, pesaresi et vagge in biondi et al. 2014 pistacio lentisci-pinetum halepensis de marco, veri et caneva 1984 querco ilicis-pinetum halepensis loisel 1971 [habitat code 9540 – mediterranean pine forests with endemic mesogean pines] pistacio lentisci-rhamnetalia alaterni rivas-mart. 1975 the mediterranean floral element (60.6 %), followed by a considerable proportion of south european plants (15.5%) and cosmopolitans (13.1%), dominated on the islet (tab. 2). tab. 2. floral elements on the islet of vrnik. floral elements no. (%) mediterranean (med) 152 (60.6) south european (seu) 39 (15.5) cosmopolitan (wisp) 33 (13.1) eurasian (euas) 13 (5.2) european (euro) 6 (2.4) cultivated and adventive plants (cuad) 6 (2.4) illyrian-balkanic (ilba) 1 (0.4) southeast european (seeu) 1 (0.4) total taxa 251 (100.0) vegetation in all, 11 plant associations, one subassocation and two stands within 10 vegetation classes were identified on the islet. these communities are described below and discussed in the same order of the syntaxonomical scheme. their syntaxonomic scheme is as follows (corresponding natura 2000 habitat codes are added in square brackets): phragmito-magnocaricetea klika in klika et novák 1941 oenanthetalia aquaticae hejný ex balátovátuláčková et al. 1993 eleocharito palustris-sagittarion sagittifoliae passarge 1964 eleocharitetum palustris savič 1926 zosteretea pignatti 1953 posidonietalia oceanicae den hartog ex mucina in mucina et al. 2016 posidonion oceanicae br.-bl. ex molinier 1960 posidonietum oceanicae (funk 1927) molinier 1958 [habitat code 1120* – posidonia beds (posidonion oceanicae)] cakiletea maritimae tx. et preising in tx. ex oberd. 1952 thero-atriplicetalia pignatti 1953 euphorbion peplidis tx. ex oberd. 1952 euphorbio pineae-glaucietum flavi horvatić 1934 [habitat code 1210 – annual vegetation of drift lines (euphorbion peplidis)] crithmo-staticetea br.-bl. in br.-bl. et al. 1952 crithmo-staticetalia molinier 1934 limonion anfracti-cancellati (horvatić 1934) mucina in mucina et al. 2016 jasprica n., dolina k., milović m. 130 acta bot. croat. 77 (2), 2018 oleo-ceratonion siliquae br.-bl. ex guinochet et drouineau 1944 myrto communis-pistacietum lentisci (molinier 1954) rivas-mart. 1975 erico manipuliflorae-calicotometum infestae horvatić 1958 [habitat code 9320 – olea and ceratonia forests] short description of the associations and stands eleocharitetum palustris savič 1926. this monospecific or species-poor community develops in small standing water bodies, e.g. shallow depressions with water depths of 10–50 cm, which are fed by rainfall (fig. 3). these habitats remain slightly wet in summer, but in some years the community can also survive periods of soil desiccation (e.g. šumberová 2011). in the case of vrnik, eleocharis palustris (5) formed stands with the co-occurrence of chara vulgaris (1) [rel.: 15.6.2016., htrs96 x=554620, y=4755266, plot size 16 m2, altitude 40 m, vegetation cover 100%]. the association has also been reported for the continental and mediterranean regions of croatia (stančić 2008), other parts of the balkans (tzonev et al. 2009, šilc and čarni 2012, laketić et al. 2013), and also in central and south europe (šumberová 2011, landucci et al. 2013). posidonietum oceanicae (funk 1927) molinier 1958. it occurs on various stations around the islet and develops on the moving seabed in depths between 1 and 15 m, and represents the breeding site for many species. extensive descriptions of the structure and the functioning of posidonia beds have been produced by buia et al. (2000) and den hartog (2003). euphorbio pineae-glaucietum flavi horvatić 1934 (online suppl. tab. 2, rels. 1–2). on the islet of vrnik this is represented by the therophytic halo-nitrophilous vegetation that has colonised the sandy pebbled beach, rich in organic matter. the association is very common along the whole eastern adriatic in the mid-littoral zone on sandy and gravel substrates (horvatić 1963, jasprica et al. 2016b). limonietum anfracti ilijanić and s. hećimović 1982 helichrysetosum italici jasprica 2015 (on-line suppl. tab. 2, rels. 3–7). the rocky shores are home to the limonietum anfracti plant association, characterised by limonium dictyophorum, a species endemic to the southern coast of the eastern adriatic (nikolić et al. 2015), which forms dense low-spreading formations that colonise the cracks in the rocks. parapholis incurva community. [rel.: 15.6.2016., htrs96 x=554795, y=4755453, plot size 6 m2, altitude 1 m, vegetation cover 70%]. this therophytic community is rare on the islet and it occurs within the village at sunny sites at the edge of paths. soils are very dry, usually with a sand admixture. in the case of vrnik, the floristic composition was as follows: parapholis incurva (4), dorycnium hirsutum (+), cakile maritima (+), silene vulgaris ssp. angustifolia (1), helichrysum italicum (+), pinus halepensis (r), anagalis arvensis (+), ailanthus altissima (r). on the islet wall vegetation was only found at a few sites within the village. the association oxalido-parietarietum judaicae (on-line suppl. tab. 3, rels. 1–3) is a sciaphilous and nitrophilous association mainly occurring on n-facing or shady walls (brullo and guarino 1998). in its typical aspect it forms a belt in the lower part of the wall. the presence of scaligeria cretica (=s. napiformis), a rare taxon of croatian flora, expands our knowledge of its chorology on the eastern adriatic (skelin et al. 2014). in the linario cymbalariaeparietarietum ramiflorae (on-line suppl. tab. 3, rel. 4), cymbalaria muralis contributed significantly. additionally, quarry faces are covered with inula verbascifolia, adianthum capillusveneris, pinus halepensis, cupressus horizontalis, calamintha nepetoides and hedera helix (on-line suppl. 3, rels. 5–6). the thermophilic chasmophytic vegetation of the asplenietea trichomanis class, at least in part, is not developed on the islet. piptatheretum miliaceae horvatić (1956) 1958. [rel.: 15.6.2016., htrs96 x=554334, 4755425, plot size 8 m2, altitude 1 m, vegetation cover 100%]. this association is rare and mostly localized on sites within the village, and sporadically on deep and wet soil within aleppo pine wood. the floristic composition on the islet was as follows: pipatherum miliaceum (5), verbascum sinuatum (1), calamintha nepetoides (+), conyza sumatrensis (+), lagurus ovatus (+), lavatera arborea (+), asphodelus fistulosus (+), parietaria judaica (+), euphorbia peplus (+), rubia peregrina (+), ailanthus altissima (+), hedera helix (+), bituminaria bituminosa (+), sonchus asper ssp. glaucescens (+), geranium sp. (+). cynodon dactylon community. [rel.: 15.6.2016., htrs96 x=554367, 4755533, plot size 12 m2, altitude 1 m, vegetation cover 90%]. it occurs at trampled sites with sandy dry soils in the village. the floristic composition on the islet was as follows: cynodon dactylon (5), hordeum murinum ssp. leporinum (1), silene vulgaris ssp. angustifolia (1), melilotus officinalis (1), trifolium scabrum (+), galium lucidum (+), medicago truncatula (+), anthemis arvensis (+), arenaria leptoclados (+), desmazeria rigida (+), lophochloa cristata (+), lagurus ovatus (+), lolium perenne (+), malva sylvestris (+), spergularia salina (+). hordeetum leporini br.-bl. 1936. this nitrophilous vegetation occurs in sunny sites at the edge of paths or on aban-fig. 3. eleocharitetum palustris savič 1926. flora and vegetation of the mediterranean islet acta bot. croat. 77 (2), 2018 131 doned land where the soil formation is embryonic and human disturbance is a significant factor. it is characterized by species of predominantly annual spring development. the floristic composition on the islet [rel.: 26.4.2014., htrs96 x=554369, y=4755536, plot size 25 m2, altitude 1 m, vegetation cover 90%] was as follows: hordeum murinum ssp. leporinum (2), malva sylvestris (+), plantago coronopus (1), anthemis arvensis (1), bromus madritensis (+), bromus racemosus (+), carduus pycnocephalus (+), crepis rubra (+), euphorbia helioscopia (+), galium aparine (+), geranium molle (+), lagurus ovatus (+), medicago polymorpha (+), melilotus officinalis (3), mercurialis annua (+), rhagadiolus stellatus (+), setaria viridis (+), sonchus oleraceus (+), trifolium tomentosum (+). the association has already been noted for ruderal sites of the eastern and western adriatic coasts and also reported for the w. mediterranean (jasprica et al. 2016b, and references therein). among the wood vegetation, querco-pinetum halepensis occupies the majority of the area of the islet of vrnik with a vegetation cover of 90–100%. it occurs exclusively on the southern slopes. it has value from an aesthetic and ecological point of view (on-line suppl. tab. 4, rels. 9–13). quercus ilex is developed in the brush layer and pinus halepensis is the dominant taxon. by contrast, pistacio-pinetum halepensis develops on the eastern and south-eastern part of the islet, where pistacia lentiscus contributes significantly (on-line suppl. tab. 4, rels. 5–8). among macchia, the myrto-pistacietum association has developed as low (mostly between 1 and 1.5 m) and dense shrub formations, and forms a strip between halophytic vegetation and the islet’s central area (online suppl. tab. 4, rels. 1–3). the erico-calicotometum, with a height of 3–4 m, occupies a very limited surface area on the southern side of the islet, and has the greatest number of taxa (on-line suppl. tab. 4, rel. 4). in general most of the companions of these associations were treated as characteristic species of the festuco-brometea and lygeo-stipetea classes. among companions, brachypodium retusum has the highest frequency. discussion the predominance of the poaceae, cichoriaceae, asteraceae and fabaceae families, a clear dominance of therophytes in the life-form spectrum and the mediterraneans in the chorological spectrum include the islet in the mediterranean context. the presence of the endemic taxa and those occurring in the national red list, including strictly protected flora, confirm the peculiarity of the surveyed area from the phytogeographic point of view (nikolić et al. 2008). the islet investigated showed a relatively low variety of vascular plant taxa. this is, at least partly, in accordance with the findings of pandža and milović (2015) for some uninhabited eastern adriatic islets with surface areas of less than one km2. however, the differences in the floristic diversity among the islets are very difficult to evaluate due to variations in the topography and the degree of human presence on the islets (pasta et al. 2014, jasprica et al. 2016b, and references therein). in general, this is not a rare pattern on such islets, which often represent ‘unbalanced biota’ (jasprica et al. 2015). the currently low number of alien species to be found in the islet may be related to the low level of human activity. however, habitat modification or space occupation by carpobrotus acinaciformis might represent a threat (brundu 2013, celesti-grapow et al. 2016). the risk of invasion may increase if more species are directly introduced to the islet, and if anthropogenic disturbance and a changing climate make conditions more favourable for alien species (lloret et al. 2005, pretto et al. 2012, cardona pons et al. 2013, valaoras 2014). nevertheless, quarrying operations caused ecosystem disturbance and profound modifications of the substratum and the topographical profile of the islet (khater and arnaud 2007). in our case, fragmentation of the forest vegetation was observed. parts of the open quarry-faces have become overgrown during the last few decades (fig. 4), and, in general, on such heavily disturbed areas, spontaneous colonization is slow (whisenant et al. 1995). this survey revealed the presence on quarry cliffs of ‘true’ rupicolous species (e.g. campanula pyramidalis, etc.), which have been recorded for fig. 4. vegetation cover on the islet during the second world war (a) and in 2016 (b). jasprica n., dolina k., milović m. 132 acta bot. croat. 77 (2), 2018 the neighbouring islet (jasprica and milović 2016). generally, the scientific literature lacks suitable references highlighting the ecological value of abandoned quarries. arnal (1993) and vela (2002) confirmed the presence in quarries of particular plant species, some of them being exclusive to this type of ecosystem and some rare and/or protected species spotted in abandoned quarries have been reported beyond their natural distribution area. these were not found in our case. regarding vegetation, the islet shows a relatively low variety of terrestrial plant associations, and most of these are fragmentary. however, some of them (e.g. parapholis incurva and cynodon dactylon communities) are little known on the eastern adriatic and further comparable research is required for a more complete understanding of these stands. in conclusion, we confirm our hypothesis that the vegetation cover on the islet is in general a result of temporary natural factors. however, the environmental impact from past quarrying activities is observable. the study also emphasizes the importance of continuous floristic and phytosociological investigations on the croatian islands and islets, as local and foreign authors have done for other sites in the adriatic basin and in some other mediterranean countries. eight habitattypes have been included in the 92/43/eec directive annex i. we wish to stress that these habitats deserve correct management which should be grounded on a deep knowledge on the croatian island and islets. acknowledgements the authors thank smiljana matijaca, nebojša jeričević and neven fazinić for providing the photographs (figs. 2 and 4a,b), steve latham (uk) for improving the english, and two anonymous reviewers whose observations improved the quality of the final version of the manuscript. thanks also are extended to milka batistić, mirjana jeričević and nebojša jeričević for their support during the feld research, and dr rade garić for drawing figure 1. references arnal, m. g., 1993: la reconquête par la végétation spontanée des carrières de fontainebleau. approche de la dynamique végétale. recommandation pour une valorisation écologique. l'aménagement et la réhabilitation écologique des carrières sèches. association francaise des ingénieurs ecologues, beaune. bogdanović, s., brullo, s., 2015: taxonomic revision of the limonium cancellatum group (plumbaginaceae) in croatia. phytotaxa 215, 1–87. bonnier, g., 1911–1935: flore compléte illustrée en couleurs de france, suisse et belgiqe. delachaux et niestlé, neuchâtel; librairie générale 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(eds.), current environmental issues and challenges, 207–216. springer science+bussines media, dordrecht. vela, e., 2002: biodiversité des milieux ouverts en région méditerranéenne. le cas de la végétation des pelouses du luberon (provence calcaire). thèse de doctorat. ecologie, université de droit, d'économie et des sciences d'aix marseille (aix marseille iii), maseille. weber, h. e., moravec, j., theurillat, j. p., 2000: international code of phytosociological nomenclature. journal of vegetation science 11, 739–768. whisenant, s. g., thurow, t. l., maranz, s. j., 1995: initiating autogenic restoration on shallow semiarid sites. restoration ecology 3, 61–67. appendix 1. syntaxa quoted in the text and tables (in alphabetical order), but not in scheme adiantetea br.-bl. et al. 1952 ammophiletea br.-bl. et tx. ex westhoff et al. 1946 artemisietea vulgaris lohmeyer et al. in tx. ex von rochow 1951 asplenietea trichomanis (br.-bl. in meier et br.-bl. 1934) oberd. 1977 carpino-fagetea sylvaticae jakucs ex passarge 1968 epilobietea angustifolii tx. et preising ex von rochow 1951 festuco-brometea br.-bl. et tx. ex soo 1947 isoëto-nanojuncetea br.-bl. et tx. in br.-bl. et al. 1952 juncetea maritimi br.-bl. in br.-bl. et al. 1952 ononido-rosmarinetea br.-bl. in a. bolòs y vayreda 1950 quercetea pubescentis doing-kraft ex scamoni et passarge 1959 salicornietea fruticosae br.-bl. et tx. ex a. bolòs y vayreda et o. de bolòs in a. bolòs y vayreda 1950 untitled acta bot. croat. 75 (1), 2016 153 acta bot. croat. 75 (1), 153–156, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0012 issn 0365-0588 eissn 1847-8476 short communication first italian record of paspalum notatum flüggé (poaceae) and its typifi cation adriano stinca1*, gabriele galasso2, enrico banfi 2 1 department of agriculture, university of naples federico ii, via università 100, 80055 portici (naples), italy 2 sezione di botanica, museo di storia naturale di milano, corso venezia 55, 20121 milan, italy abstract: in the present work the presence of paspalum notatum flüggé (poaceae) in italy was reported for the fi rst time. it is a neophyte native to america, known for applications in phytoremediation. its typifi cation, ecology and invasiveness status are also presented. keywords: alien species, invasiveness, phytoremediation, vascular fl ora * corresponding author, e-mail: adriano.stinca@unina.it introduction the genus paspalum l. (poaceae) comprises about 330 species (zuloaga and morrone 2005) and is chiefl y distributed in tropical and temperate regions of america (zuloaga et al. 2003). it includes small to robust perennial, rarely annual, herbs that are usually cespitose, but may creep via rhizomes or stolons, and have filiform to lanceolate leaf blades, which may be flat, plicate, or involute. the infl orescence is racemose, unilateral, with solitary or paired spikelets. the lower glume is commonly absent, the upper one and the lower lemma are herbaceous to membranous, and the upper anthecium is indurate to membranous. in the european vascular fl ora 10 species have been recorded (valdés et al. 2009–2014), among which 6 are present in italy, all aliens (celesti-grapow et al. 2009a, b, soldano and verloove 2015): p. dilatatum poir., p. distichum l., p. exaltatum j. presl & c. presl, p. paucispicatum vasey, p. quadrifarium lam. and p. vaginatum sw. from recently, another two alien species are in course of reporting in italy: p. dasypleurum kunze ex desv. and p. thunbergii kunth ex steud. (banfi and galasso 2015) in this paper the neotropical neophyte p. notatum flüggé (bahiagrass) is recorded for the fi rst time in italy. its typifi cation, ecology and invasiveness status are also presented. materials and methods field research in southern italy was undertaken from 2013 to 2015. species was identifi ed according to zuloaga et al. (2004), zuloaga and morrone (2005), and allen and hall (2002). the flüggé’s (1810) protologue was also examined. collected specimens are kept in herbarium porticense (porun, acronym according to thiers 2011) and museo di storia naturale di milano (msnm). geocoding of the italian locality of the plant was performed with the use of a portable gps device (gps map 60csx, garmin, usa), calibrated beforehand (geographic system utm wgs84). italian literature was examined to detect previous indications of the species in italy (e.g. fiori 1923, zangheri 1976, pignatti 1982, conti et al. 2005, 2007, celestigrapow et al. 2009a, b, 2010). the degree of naturalization (status) was defi ned according to richardson et al. (2000) and pyšek et al. (2004) through the monitoring of the italian population. in detail was observed the presence of the species and its ability to spread in the fi eld in three consecutive years (2013–2015). results and discussion paspalum notatum flüggé (sub paspalus notatus), gram. monogr., paspalum: 106(–108, 207–208). 1810 [5.v.1810]. typus, hic designatus: united states virgin islands, st. thomas, e. p. ventenat s.n., 1802; lectotypus in bm-ex herb. nolte (picture in si!), isolectotypus in us-2855762! (fragm. ex bm). we were not able to fi nd the duplicates kept in b, kiel, mo, p-la, presumably being destroyed during the second world war. the present typifi cation is a consequence of the fact that koning and sosef (1985: 313) indicated isolectotypi on a set of several herbaria, while zuloaga and morrone (2005: 180) did not establish a valid typifi cation as they didn’t use the phrase “designated here” (hic designatus) or an equivalent (art. 7.10 of icn, mcneill et al. 2012). stinca a., galasso g., banfi e. 154 acta bot. croat. 75 (1), 2016 = paspalum notatum var. saurae parodi, revista argent. agron. 15(1): 55 (fi g. 1b). 1948. ≡ paspalum saurae (parodi) parodi, darwiniana 15(1– 2): 106. 1969 [30.i.1969]. typus: argentina, entre ríos: concepción del uruguay, l. r. parodi 12670, january 1937; holotypus in baa, isotypus in us-1721333! fig. 1. distribution of paspalum notatum in europe (including azores and canary islands) with the fi rst italian record (star) and previous reports (circles: 1 – scholz 2002; 2 – pyke 2003; 3 – verloove 2003; 4 – verloove 2005; 5 – böhling and scholz 2004; 6 – silva et al. 2005; 7 – sánchez gullón et al. 2006; 8 – valdés et al. 2007; 9 – verloove and reyes-betancort 2011; 10 – siverio núñez et al. 2013). fig. 2. italian specimen of paspalum notatum preserved at porun. paspalum notatum in italy acta bot. croat. 75 (1), 2016 155 p. notatum is common in pastures along the belt warm and temperate american regions, from central mexico to uruguay (e.g. quarín et al. 1984). it was introduced in tropical and subtropical areas of the world for purposes of turf establishment, forage crop growth, erosion control and slope stabilization (e.g. busey 1992, ogura et al. 2005). in recent years, this grass has been found effective in renaturation of vegetation of mined lands, uptake of heavy metals and even removal of radioactive elements (phytoremediation) (e.g. xia 2004, huang et al. 2009). in addition, laboratory experiments have shown that pollen of this plant cause allergic respiratory diseases (davies et al. 2012). p. notatum was collected at scalea (province of cosenza) in southern italy. the site is located on the tyrrhenian coast of the calabria region in the northern sector of the lao river plain, at 4–5 m a.s.l. (utm wgs84: 33s 567494 e – 4407547 n, 33s 567436 e – 4407488 n, and 33s 567427 e – 4407484 n). climate (thermopluviometric data from fiumefreddo meteorological station, 220 m a.s.l., about 65 km from the studied site) belongs to the mediterranean type characterized by average annual temperature of 16.7 °c, average annual rainfall of 1,141 mm, and drought summer period ranging from may to august (brandmayr et al. 1991). p. notatum was found in very disturbed grassland sites. this alien species was probably introduced via seeds to create lawns and therefore subsequently spread.the discovered population covers an area of approximately 300 m2. it shares the place with avena barbata pott ex link, cynodon dactylon (l.) pers., dactylis glomerata l. subsp. glomerata, oxalis corniculata l., parietaria judaica l., reichardia picroides (l.) roth, solanum nigrum l., s. villosum mill., sonchus oleraceus l., and with other aliens, i.e. chamaesyce maculata (l.) small, erigeron sumatrensis retz., paspalum dilatatum poir., p. distichum l., that emphasize the human impact. today in europe (fig. 1) p. notatum has been reported in greece (scholz 2002), spain (böhling and scholz 2004, sánchez gullón et al. 2006, valdés et al. 2007), and azores islands in portugal (silva et al. 2005). in spain (pyke 2003, verloove 2003, 2005, verloove and reyes-betancort 2011) and canary islands (siverio núñez et al. 2013, fi nding confi rmed by verloove and reyes-betancort 2011) the var. saurae (synonym by p. notatum according to allen and hall 2002) was also recorded, and it presents the same morphological features of the new italian population. in italy p. notatum was detected for three consecutive years (2013–2015), showing a noticeable vegetative propagation. according to richardson et al. (2000) and pyšek et al. (2004), the observation period is too short to understand the real success of vegetative propagation and declare a state of naturalized species. therefore it must be considered an alien casual, waiting for further fi eld investigations to achieve the proper status attribution. specimina visa italy: calabria, scalea (cosenza), park between corso mediterraneo and via michele bianchi (utm wgs84: 33s 567494 e – 4407547 n), mowed lawn, 5 m a.s.l., no exp., 14 aug 2013, leg. a. stinca, det. e. banfi & g. galasso (porun-3063, msnm-44681); calabria, scalea (cosenza), park between corso mediterraneo and via ruggiero di lauria (utm wgs84: 33s 567436 e – 4407488 n), grassy site, 4 m a.s.l., no exp., 20 aug 2014, leg. a. stinca, det. a. stinca, e. banfi & g. galasso (porun-3064-3065, msnm-45057) (fig. 2); calabria, scalea (cosenza), park between corso mediterraneo and via ruggiero di lauria (utm wgs84: 33s 567427 e – 4407484 n), grassy site, 4 m a.s.l., no exp., 16 aug 2015, leg. a. stinca & m. ravo, det. a. stinca (porun-3577). usa: united states virgin islands, st. thomas, e. p. ven tenat s.n., 1802 [bm (picture in si, us-2855762 (fragm. ex bm)]. references allen, c. m., hall, d. w., 2002: 25.26 paspalum l. in: flora of north america editorial committee (eds.), flora of north america north of mexico, 25 (magnoliophyta: commelinidae (in part): poaceae, part 2), 566–599. oxford university press, new york, oxford. banfi , e., galasso, g., 2015: paspalum (poaceae), aggiornamento per la fl ora italiana. in: peruzzi, l., domina, g. 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botany monographs 71, 1–75. 164 acta bot. croat. 78 (2), 2019 acta bot. croat. 78 (2), 164–168, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0014 issn 0365-0588 eissn 1847-8476 evaluation of trace elements and particulate matter deposition on plant foliage exposed to vehicular pollution aasawari a. tak, umesh b. kakde* department of botany, the institute of science, 15-madame cama road, fort, mumbai – 400032, maharashtra, india abstract – in recent times, highly developed metropolitan cities have progressively used biodiverse roadside plants as an eco-sustainable tool for the mitigation of air pollution. the present study has been designed to scrutinize the impact of particulate matter (pm) deposition and heavy metal accumulation in roadside plants. some of the common roadside plants found along the national highway at thane (mumbai) region that were selected for this study are alstonia schlolaris, bauhinia variegata, ficus benghalensis, ficus religiosa, cassia fistula, and mangifera indica. the inductively coupled plasma atomic emission spectroscopy (icp-aes) analysis was carried out for five heavy metals, namely cr, cu, fe, mn and zn. the particulate matter deposition was observed to be the highest in ficus benghalensis (1.14 mg kg–1), while it was found to be the lowest in bauhinia variegata (0.71 mg kg–1). the present study revealed that the accumulation of heavy metals in plants inversely varies with the deposition of dust on the surface of leaves. the tolerant species of plants can serve as natural biofilters that can alleviate environmental pollution to certain extent. keywords: heavy metal, inductively coupled plasma atomic emission spectroscopy analysis, national highway, particulate matter, road traffic *corresponding author e-mail: drumeshkakde@gmail.com introduction the plant physiology, anatomy, and its biochemical constituents are significantly influenced by specific climatic factors and certain air pollutants. it is a well-known and wellestablished fact that biodiversity in metropolitan cities is considerably affected by modern civilization, industrialization, economic growth, and urbanization. one such example is the city of thane that has been known for its ecologically sensitive and rich flora and fauna; however, the severe stress of air pollution has brought the city on the verge of an environmental disaster. green plants function as natural filters for air pollution as they continuously accumulate and absorb pollutants from the air. the plants that exhibit high tolerance towards air pollution are generally recommended for green belt development in urban and industrial areas (tak and kakde 2017a). heavy metals are stable elements (tokalioglu and kartal 2006) that cannot be destroyed or degraded, and are known as the essential natural elements of the earth’s crust. these elements enter into the environment due to several anthropogenic activities such as agriculture, industrial processes, energy production, and burning of fossil fuel. in urban green forests, these metals are released into the environment in the form of air particulates that scatter in solid or liquid states, with sizes of about 1 µm (sawidis et al. 2011). vehicular emissions in urban regions are considered to be the primary source of pollution caused by these elements (duong and lee 2011). some of the other anthropogenic sources of heavy metals include traffic, mining processes, combustion of petrochemical byproducts, diffuse and smelter sources (piping), human activities, and industrial sources (al-khashman et al. 2011). it was recorded by thorpe and harrison (2008), the high emission rate of automobile exhaust affects the roadside plants. friction of brake linings and tires and corrosion and erosion of vehicular metal parts over time contribute to the emission of various heavy metal pollutants onto the roadside environment. brake linings are generally made up of iron (fe) and copper (cu). the application of brakes results in the release of iron and copper due to frictional heat (luhana et al. 2004). iron is also used in the construction of roads, bridges, and welding work, and corrosion of iron parts over time leads to release of iron in the environment. during the manufacturing of rubber, zinc is used as an important additive short communication trace elements and dust deposition on roadside plants acta bot. croat. 78 (2), 2019 165 for vulcanization of rubber. hence, erosion and corrosion of tires leads to release of zn, (hjortenkrans et al. 2007). therefore, the present study is based on analyzing the accumulation of five heavy metals, namely cr, cu, fe, mn, and zn on plant foliages, and considers the amount of dust deposition. dust or particulate matter and gaseous pollutants can have adverse effects on plants as well as human health (raabe 1999, rai et al. 2013). since, the leaves of plants are sensitive; they show visible damages on their surfaces during the overall developmental stages when exposed to these pollutants (steubing et al. 1989). some of the most common and high severity air pollutants include so2, no2, and co2. these air pollutants also leave a negative impact on the morphology, physiology, and biochemistry of plants. at the same time, the use of tolerant plant species to mitigate air pollution is widely accepted. by assessing the morphological, physiological, and biochemical parameters of plants, an early evaluation of urban air quality can be arrived at. several studies were carried out to analyze and understand the impacts of air pollution on selected plants in polluted urban regions (tak and kakde 2017b). on the other hand, no systematic analysis has been performed in an urban section of an ecologically sensitive region such as thane, india. in addition to this, a plant’s response may alter under varying levels of pollution stress (tak and kakde 2017b). plants are usually readily available and can be modified more easily compared to other organisms such as fungi, algae, lichens, or mosses (berlizov et al. 2007). in the light of the above-mentioned discussion, the present study deals with the quantification of dust particles and accumulation of heavy metals on the leaves of different roadside plant species in the thane (mumbai) region. material and methods study area national highway 48 (nh 48) is a national highway of india that starts at delhi and ends at chennai and goes through jaipur, udaipur, vadodara, mumbai, pune, and bengaluru, traversing through six states of india. the present investigation was carried out at national highway 48 from teen haat naka (co-ordinates 19°11’17.4192”n 72°57’49.9932”e) to kasarvadavali (co-ordinates 19°16’14.1348”n 72°57’53.604”e) on ghodbunder road, thane, maharashtra. the selected area is a high traffic area and the national highway 48 passes through it. thousands of vehicles such as cars, bikes, trucks, and tractors continuously pass through this area, which is a source of high level of heavy metals pollution. in addition, there is a greater intensity of dust accumulation on highway roads. hence, the plant foliage along the highway was selected for the analysis of dust and heavy metals. the yeoor hill forest (co-ordinates 19°13'56.5464 n 72°56'48.066”e) was selected as non-polluted site (control). sampling the plant species that were selected include alstonia scholaris, bauhinia variegata, ficus benghalensis, ficus religiosa, cassia fistula, and mangifera indica. these plants are commonly available and grow naturally. for the dust and heavy metal analysis, the leaf samples of these roadside plants were taken from the height of 2 to 3 m from ground level, where high amount of dust particles settle on the surface of foliage. the foliage facing towards roadside was collected for sampling early in the morning from 8.00 a.m. to 10.00 a.m. through random selection. three replicates of leaf samples were put in separately labeled zip lock polythene bags. all the leaf samples were carefully transferred to laboratory for further analysis. dust analysis the amount of dust deposited on the leaf surface area was calculated by taking the initial and final weight of beaker in which the leaf samples were washed. the following formula was used for the calculation: w = w2 – w1/a, where w = dust content (mg cm–2), w1 = weight of beaker without dust, w2 = weight of beaker with dust and a = total area of leaf in cm2. heavy metal analysis inductively coupled plasma atomic emission spectroscopy (icp-aes) was used for analyzing the presence of heavy metals, i.e., chromium, copper, iron, manganese, and zinc, which are commonly and abundantly found in the dust samples collected from the roadside foliage. using the closedvessel microwave system and the wet method, the collected samples were processed in a fusion of nitric acid and hydrogen peroxide. a high performance microwave digestion system multiwave 3000 (anton paar) was applied for microwave digestion of the samples. perkin elmer elan drc ii inductively coupled plasma mass spectrometer was used for the determination of cr, cu, fe, mn and zn. to begin with, 0.1 g (dry mass) of plant sample was weighed into the digestion vessels. then, 5.0 ml of concentrated hno3 and 1.0 ml of 30% h2o2 were added to each sample. all the chemicals used for the digestion process were of analytical grade. the samples were then pre-digested overnight (for 16 hours) in a fume hood at room temperature. since temperature control was required during the entire decomposition, a temperature sensor was used in one vessel. the plant samples were digested according to the specifications of the following optimized program (power in w/time in min): 1000/28, ventilation for 20 min. during the last step and ventilation, the internal temperature was limited to 240 °c. after cooling, the entire digest was transferred in portions into 60 ml plastic bottles and each bottle was diluted with 50 ml of double deionized water. similarly, reagent blanks were prepared for the samples. the entire sample solution was clear and diluted 10 times before analysis. the icp-aes system was calibrated by the method that involved external standards with rh, and internal standards with re. the reagent blank solution contained 1% of concentrated hno3. using the reagent blank solutions, a mixed standard solution was prepared that contained all five elements, i.e., cr, cu, fe, tak a.a., kakde u.b. 166 acta bot. croat. 78 (2), 2019 mn and zn. the background interferences from the plasma gases, air entrainment, and solvent were corrected by subtraction of signals from reagent blank solutions. statistical analysis the correlation coefficient between dependent and independent variables was calculated using the analysis toolpack in excel. the degree of correlation was determined by the pearson correlation coefficient. in the present investigation, the independent variable was dust deposition and the dependent variables were heavy metals. for all primary data, n = 12, and significance was tested at a 5% level (that is, p < 0.05). data interpretation was executed by one-way anova, followed by the tukey kramer test to check the significance of dust accumulation and heavy metal absorption by plant leaves for selected study sites at (p < 0.05). results and discussion accumulation of heavy metals the heavy metals released from vehicular emissions directly get deposited in soil, and are thus translocated in plants via the root system (shparyk and parpan 1990). accumulation of selected heavy metals at control and polluted sites has been depicted in figure 1. the elements with metallic properties and atomic number more than 20 are usually referred to as heavy metals. heavy metal pollution is a significant environmental issue as these metals are likely to have toxic effects with increased concentration or accumulation in plants. the accumulation of heavy metals in plants leads to physiological toxicity in plants, animals, and microorganisms. due to the accumulation of heavy metals, the leaves may get damaged and cause serious ecological and health problems if the heavy metals enter into the food chain. heavy metal pollution does not undergo biodegradation and has a harmful effect on biological systems. in the present investigation, accumulation of chromium (cr; fig. 1a) was recorded to be maximum in cassia fistula (16.4 mg kg–1) and bahunia variegata (11.1 mg kg–1), while the minimum accumulation of this metal was recorded in ficus religiosa and ficus benghalensis (1.0 mg kg–1). according to pais and jones (1997), cr contents in plants ranges from 0.02–0.2 mg kg–1 with phytotoxicity at >10 mg kg–1. cr content in air ranges from 0.001–1.0 mg m–3, but in industrial regions, it reaches up to 30–50 mg m–3. in this investigation, the concentration of cr was recorded to be higher in plants than the values recorded when the plants are under normal conditions. the presence of cr in polluted areas is found to be toxic for the growth of plants. higher concentration of cr in plants results into chlorosis of foliage, necrotic spots, and injured rot growth (kabata-peadias et al. 1994). the accumulation of cu was recorded to be the highest in bahunia variegata (39.6 mg kg–1) while it was found to be the lowest in ficus religiosa (10.8 mg kg–1). the normal range of cu in plant leaves is from 3–7 mg kg–1 of the dry matter, while toxicity is in the range of 20–30 mg kg–1 fig. 1. accumulation of heavy metals in leaves of alstonia scholaris, bauhinia variegata, ficus benghalensis, ficus religiosa, cassia fistula, and mangifera indica at control and polluted sites. a – accumulation of chromium (cr), b – accumulation of copper (cu), c – accumulation of iron (fe), d – accumulation of manganese (mn), e – accumulation of zinc (zn). data are mean values (n=3), the error bars indicate standard deviations. different letters denote statistically different means according to the tukey kramer post ad-hoc test (p < 0.05). control plants were collected from the yeoor hill forest (non polluted area). trace elements and dust deposition on roadside plants acta bot. croat. 78 (2), 2019 167 (kabata-peadias and pendias 1994). it is observed that the concentration of cu in the leaf samples was higher than the normal limits. the accumulation of iron (fe) was recorded to be the highest in bahunia variegata (7.963 mg kg–1), while it was recorded to be the lowest in ficus religiosa (1.081 mg kg–1). the fe content in plants ranges from 10–1000 mg kg–1 in dry matter. fe is the most important element in protein constituent of plants, which helps in n2 fixation, and is also known as a catalyst in chlorophyll formation caselles et al. (2002). the maximum accumulation of manganese (mn) was recorded in cassia fistula (213.6 mg kg–1), while the lowest was in ficus benghalensis (38.3 mg kg–1) (figure 1). an excess accumulation of mn by plants leads to older leaves with brown spots (horiguchi 1987). in this analysis, the mn concentration was found to be below the range of toxicity. the accumulation of zinc (zn) was recorded to be the highest in bahunia variegata (103.4 mg kg–1), while it was the lowest in ficus religiosa (45.0 mg kg–1). high accumulation of zn in plants results in older leaves as it has high bioaccumulation index (aleksandra et al. 2017). accumulation of dust by plants the accumulation of dust deposition was found to be the highest in ficus benghalensis (1.14 mg kg–1), while it was the lowest in bauhunia variegata (0.71 mg kg–1). an analysis of dust accumulation indicated insignificant correlation with the deposition of toxic heavy metals such as cr, cu, fe, mn, and zn at the polluted site. a decreasing sequence of heavy metal contamination in plant foliage was recorded as follows: ficus benghalensis > mangifera indica > cassia fistula > alstonia scholaris > bauhunia variegata > ficus religiosa. figure 2 depicts the dust deposition pattern on foliage of selected plants. to analyze the correlation between accumulated heavy metals and dust, a correlation matrix was plotted and is presented in tab. 1. a significant positive correlation was observed between mn-cr (0.961), cu-fe (0.995), and zn-mn (0.822). correlation between cu-cr, fe-cr and fe-mn was not significant, but it was found to be positive. all selected heavy metals and dust were observed to be negatively correlated. one-way anova test for heavy metals and dust revealed that all the heavy metals were significant among control and experimental sites. the accumulation or absorption of dust depends on the surface geometry, external climatic changes, phyllotaxy, leaf area, hairs, cuticle, height, and canopy structure of plant species. plants with broad leaf area and wide branches tend to absorb more dust. the parameters such as height and phyllotaxy of leaf surfaces are considered to cause a reduction in the particulate matter or dust deposition on leaf surfaces (thambavani and sabitha 2011). the effect on dust deposition and heavy metals accumulation in plants foliage in the present investigation due to these parameters was observed to be insignificant. conclusion from the present study, some useful observations were made regarding the dust absorbing capacity and the extent of heavy metal accumulation of different plants. the vehicular emission, known to be hazardous to human health, is found to negatively impact the life cycle of plants as well. as the heavy metals are released in soil and in air, plants accumulate and absorb these into their system either by root translocation system or through photosynthesis and respiration.it was observed that plants, namely ficus benghalensis, mangifera indica, cassia fistula, alstonia scholaris, bauhunia variegata, and ficus religiosa, have considerable tolerance toward pollutants as they accumulated some high toxic metals in their system. these plants are suggested to be planted along roadside where vehicular emission is more. as plants function as biofilters of air pollution, they also accumulate some toxic metals into their root system, making the soil less toxic. to sum up, urban roadside plantation can serve as biomarkers or natural biofilters of air pollution for the development of greenbelts. tab. 1. pearson correlation of accumulated heavy metal concentration (chromium, copper, iron, manganese and zinc) and dust in leaves of different roadside plant species (ficus benghalensis, mangifera indica, cassia fistula, alstonia scholaris, bauhunia variegata, and ficus religiosa). significance was tested for p < 0.05 and significance values were highlighted by *. positive values in the matrix indicate positively correlated, while negative (–) values indicate inversely correlated results. dust cr cu fe mn zn dust 1.000 cr –0.357 1.000 cu –0.645 0.493 1.000 fe –0.592 0.477 0.995* 1.000 mn –0.564 0.961* 0.652 0.637 1.000 zn –0.619 0.770 0.805 0.764 0.822* 1.000 fig. 2. dust deposition on foliage of alstonia scholaris, bauhinia variegata, ficus benghalensis, ficus religiosa, cassia fistula and mangifera indica. control plants were collected from the yeoor hill forest (non polluted area). data are mean values (n=3). the error bars indicate standard deviations. error bars with different alphabets have statistically different mean inter specific as per the tukey kramer post ad-hoc test (p < 0.05). tak a.a., kakde u.b. 168 acta bot. croat. 78 (2), 2019 references aleksandra, n.s., marta, k.c., michał, t., gabriela, b., 2017: air pollution tolerance index and heavy metal bioaccumulation in selected plant species from urban biotopes. chemosphere 183, 471–482. al-khashman, o.a., al-muhtaseb, a.h., ibrahim, k.a., 2011: date palm (phoenix 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(eitzinger et al. 2003, singh et al. 2013). seed germination is critical in the early stage of growth in most plants and may determine the success of crop production (finch-savage and bassel 2016). drought is an important environmental factor that negatively affects seed germination and seedling growth in dryland soil (singh et al. 2013, khayatnezhad and gholamin 2011). the effects of drought stress can be mimicked resorting to polyethylene glycol (peg). peg is a non-ionic water polymer that is not expected to penetrate the plant tissue rapidly, thus compromising water uptake (almansouri et al. 2001). genetic diversity analysis and fingerprinting in cereals have been successfully carried out by start codon targeted (scot) markers (cabo et al. 2014, pour-aboughardareh et al. 2017). these are highly polymorphic functional markers based on the short-conserved region in plant genes surrounding the atg translation start codon (collard and mackill 2009). ancient wheat cultivars present an important group of genetic resources and may present abiotic stress tolerance characteristics (martins-lopes et al. 2009, carvalho et al. 2010). nonetheless, possible stress tolerance characteristics within elite crop germplasm should not be disregarded (gilliham et al. 2017). our objective was to quantify early drought stress resistance and evaluate genetic diversity of four winter wheat cultivars – three modern elite germplasm lines developed in the iberian peninsula (‘antequera’, ‘jordão’ and ‘roxo’) and an ancient portuguese bread wheat cultivar (‘mestiço’). short communication pavia i., rocha l., moutinho-pereira j., lima-brito j., correia c. 170 acta bot. croat. 78 (2), 2019 materials and methods seeds of durum wheat (triticum durum desf.) ‘core’ and bread wheat (triticum aestivum l.) ‘antequera’, ‘jordão’, ‘roxo’ and ‘mestiço’ were used in this study. seeds of durum wheat and modern (elite) bread wheat ‘antequera’, ‘jordão’ and ‘roxo’ were stored at the plant breeding station, elvas (portugal). seeds of ‘mestiço’ were obtained from the plant germplasm bank, at the department of genetics and biotechnology, university of tras-os-montes and alto douro, vila real (portugal). seeds of t. aestivum were disinfected using a solution of sodium hypochlorite 1% for 3 min and then washed with distilled water. twenty seeds per cultivar/treatment (in 3 repetitions) were germinated in 90 mm petri dishes with filter paper moistened with polyethylene glycol solutions (peg6000) of 0, –0.33, –0.75, –1 and –1.5 mpa water potential in the dark at 25 °c. germination was annotated daily during eight days. peg solutions were changed at the fourth day. germinability (g), mean time of germination (mt), mean germination rate (mr) and synchrony of germination (z) were calculated according to ranal et al. (2009). five days after seed germination, longest root and coleoptile growth as well as dry and fresh weight were measured in fifteen seedlings per cultivar × water potential combination. water content was calculated based on these values. vigor index (vi) was calculated based on coleoptile length and germinability according to kumari et al. (2014). logit transformation was performed in data expressed in proportion/percentage (g and water content) prior to statistical analysis; original values are shown. anova and tukey post-hoc analysis were performed in ibm spss statistics v23. dna was extracted from bulks of seven seedlings from each cultivar using dneasy plant mini-kit (qiagen) according to the manufacturer’s instructions. dna integrity was checked by electrophoresis on 1.5% agarose gels, quantified on nanodrop nd-1000, and diluted to 30 ng μl–1. dna amplification was performed with 8 scot primers (fig. 1a) developed by collard and mackill (2009). reaction mixture was made as reported by cabo et al. (2014) and pcr conditions were as follows: initial denaturation of 3 min at 94 °c followed by 35 cycles of 1 min at 94 °c, 1 min at 50 °c and 2 min at 72 °c; final extension was of 5 min at 72 °c. bands were scored as described by cabo et al. (2014). genetic similarity among cultivars was accessed by an unweighted pair group method using arithmetic averages (upgma) dendrogram of genetic similarity constructed with the software “ntsys-pc, numerical taxonomy system” (rohlf, 1998). results and discussion dynamics of seed germination wuest and lutcher (2012) reported critical water potentials for 75% germination of several dryland wheat cultivars to be between –1.1 and –1.6 mpa. by contrast, singh et al. (2013) achieved 75% germination only at –0.75 mpa and no germination at –1.5 mpa in five commonly rainfed winter wheat cultivars. here, although all cultivars’ germinability (g) decreased at –1.5 mpa, we did not observe complete germination inhibition. g of modern cultivars reached near 100% from 0 till –1 mpa, while that of mestiço was around 80% (tab. 1). the low g value registered in mestiço might not reflect a characteristic of this cultivar. mestiço was not stored at the same location as the remaining cultivars, which might have significantly influenced germination performance (wuest and lutcher, 2012). germinability was only affected when the higher stress level was applied (tab. 1). at –1.5 mpa, overall g dropped to 64.2%. in antequera and jordão it dropped to 71.7 and 78.3%, respectively, while in mestiço it fell to 58.3%. in roxo it dropped steeply from 95% at –1 mpa till 48.3% at –1.5 mpa. mean time of germination (mtg) steady increased with lower water potentials in all cultivars from near 1 till over 4 days (tab. 1). mean germination rate (mr) was similar in all cultivars, with the exception of mestiço, and decreased with the decrease of the water potential (tab. 1). synchrony of germination (z) was overall higher in antequera and jordão (tab. 1). the observed delay in germination with the decrease of water potential is consistent with the findings of other authors (almansouri et al. 2001, singh et al. 2013, kumari et al. 2014). early biomass production drought highly affected fresh weight, while dry weight variation was only observed between cultivars (fig. 2a). at 0 mpa all cultivars showed similar water content, ranging from 84.67 till 89.45% (fig. 2b). mestiço showed the lowest variation in water percentage between 0 and –1.5 mpa, with 89.45 and 45.81% respectively. the remaining cultivars dropped to values lower than 40% at –1.5 mpa (fig. 2b). khayatnezhad and gholamin (2011) found that the effect of peg on leaf area of 10-day-old plants was greater than the effect on fresh weight, leading us to believe that mestiço, which showed the lowest water percentage variation across water potentials (fig. 2b), will also be the least affected by the future growing reduction due to drought stress. drought stress decreased both root and shoot size (fig. 3a). field and laboratory experiments indicate that cultivars with long coleoptiles have superior emergence on deep seeding (singh et al. 2013). the highest coleoptile growth was verified in mestiço at 0 mpa, with an average of 8.22 cm. coleoptile length coupled with the ability of seeds to germinate at low water potentials are important factors controlling winter wheat seedling emergence (singh et al. 2013). in contrast to the modern cultivars, mestiço is a tall wheat variety (maçãs, b. and coutinho, j., unpublished data). modern varieties assuredly have reduced height (rht) genes which have been associated with reduced coleoptile size (ellis et al. 2004). this is a possible explanation for the reduced coleoptile size in modern cultivars when compared with mestiço. vigor index (vi) that considers both coleoptile length and germinability may present an important clue regarding cultivars ability to withstand drought stress. vi values ranged from 10.47 till 786.67 (fig. 3b). vi was influenced by both cultivars and drought stress, as reported by others (kumari et al. 2014). the highest values were observed, for all cultivars, at 0 mpa and the lowest at –1.5 mpa. the highest drought resistance during germination of iberian wheats acta bot. croat. 78 (2), 2019 171 averaged vi values across the different water potentials were observed in mestiço and jordão. ancient wheat varieties as a source of genetic diversity ancient bread wheat cultivars show a high genetic diversity (carvalho et al. 2010) and interesting agronomic traits, such as aluminum resistance (martins-lopes et al. 2009). although the limitations of bulk analysis are recognized, low intra-cultivar variability is expected and it allows assessment of genetic variation among cultivars. scot markers showed an average of 64.6% of polymorphic bands (fig. 1a). the genetic profile of each of the studied cultivars, based on the dna amplification with scot markers, is presented in figure 1b. mestiço showed a higher genetic difference than the fig. 1. results of the genetic diversity analysis: (a) sequence, number of bands and polymorphism percentage of each start codon targeted (scot) marker, (b) genetic profile, based on dna amplification with scot markers, of bread wheat (triticum aestivum l.) ‘antequera’, ‘jordão’, ‘roxo’ and ‘mestiço’ and durum wheat (triticum durum desf.) ‘core; and (c) unweighted pair group method with arithmetic mean dendrogram of genetic similarity of the studied wheat cultivars; mw – molecular weight in base pairs. pavia i., rocha l., moutinho-pereira j., lima-brito j., correia c. 172 acta bot. croat. 78 (2), 2019 elite cultivars (fig. 1c). this was expected as mestiço is an ancient portuguese wheat cultivar, not subjected to a modern breeding program. such a distinction is also observed in the germination (tab. 1) and growth responses (figs. 2, 3). among the studied cultivars, mestiço may be the least affected by any future growing reduction due to drought stress (khayatnezhad and gholamin 2011). introgression of alleles from ancient cultivars, such mestiço, into modern ones can be useful when breeding for suboptimal environments, such as those prevalent in the mediterranean basin (soriano et al. 2018). furthermore, we propose future research into rht genes able to maintain reduced height in modern cultivars without affecting early growth, such as rht8 (ellis et al. 2004). screening for drought resistance using peg assays development of stress-tolerant varieties is a core objective of many breeding programs. still, success has been limited due to lack of adequate screening techniques (kumari et al. 2014). due to the vast number of ancient cultivars stored in germplasm banks, most are still to be agronomically characterized. nonetheless, the use of ancient wheat cultivars in breeding programs may be useful when breeding for suboptimal environments such as those prevalent in the mediterranean basin (soriano et al. 2018). we believe that peg experiments, such as the one described here, are suitable for screening drought tolerance prior to field trials. this screening could significantly reduce the overall cost and manpower tab. 1. germination parameters in wheat cultivars on drought stress. data are mean values ± sd, n = 60. g ‒ germination percentage; mt ‒ mean time of germination; mr ‒ mean germination rate; z ‒ synchrony of germination. different letter (a, b, c) correspond to statistical differences among results by the tukey-test (p < 0.05) cultivar (cv) g (%) mt (day) mr (day–1) z antequera 92.00 ± 11.46 a 2.48 ± 1.42 a,b 0.531 ± 0.271 a 0.656 ± 0.216 a jordão 94.00 ± 9.30 a 2.28 ± 1.15 a 0.549 ± 0.260 a 0.683 ± 0.223 a mestiço 80.67 ± 14.00 b 2.69 ± 0.98 b 0.419 ± 0.057 b 0.490 ± 0.159 a,b roxo 88.67 ± 21.42 a 2.48 ± 1.33 a,b 0.519 ± 0.265 a 0.597 ± 0.247 b water potential (w, in mpa) 0 95.83 ± 6.34 a 1.16 ± 0.23 a 0.884 ± 0.140 a 0.804 ± 0.209 a –0.33 97.08 ± 4.98 a 1.77 ± 0.37 b 0.586 ± 0.112 b 0.557 ± 0.123 b –0.75 95.83 ± 5.97 a 2.15 ± 0.19 c 0.469 ± 0.041 c 0.748 ± 0.171 a –1 91.25 ± 9.08 a 2.79 ± 0.31 d 0.363 ± 0.044 d 0.523 ± 0.202 b,c –1.5 64.17 ± 14.72 b 4.56 ± 0.40 e 0.221 ± 0.019 e 0.400 ± 0.113 c cv × w antequera × 0 96.67 ± 5.77 1.05 ± 0.05 0.954 ± 0.045 0.904 ± 0.095 antequera × –0.33 96.67 ± 5.77 1.59 ± 0.25 0.640 ± 0.112 0.523 ± 0.075 antequera × –0.75 96.67 ± 2.89 1.98 ± 0.16 0.506 ± 0.040 0.838 ± 0.103 antequera × –1 98.33 ± 2.89 2.86 ± 0.31 0.352 ± 0.037 0.554 ± 0.151 antequera × –1.5 71.67 ± 5.77 4.93 ± 0.39 0.204 ± 0.016 0.459 ± 0.192 jordão × 0 100.00 ± 0.00 1.03 ± 0.03 0.968 ± 0.027 0.933 ± 0.058 jordão × –0.33 100.00 ± 0.00 1.53 ± 0.12 0.655 ± 0.052 0.472 ± 0.040 jordão × –0.75 100.00 ± 0.00 2.10 ± 0.13 0.477 ± 0.029 0.835 ± 0.205 jordão × –1 91.67 ± 7.64 2.49 ± 0.40 0.408 ± 0.062 0.718 ± 0.159 jordão × –1.5 78.33 ± 2.89 4.23 ± 0.33 0.238 ± 0.019 0.459 ± 0.043 mestiço × 0 86.67 ± 2.89 1.52 ± 0.13 0.660 ± 0.057 0.477 ± 0.051 mestiço × –0.33 91.67 ± 5.77 2.26 ± 0.34 0.450 ± 0.072 0.632 ± 0.141 mestiço × –0.75 86.67 ± 2.89 2.38 ± 0.11 0.420 ± 0.018 0.541 ± 0.125 mestiço × –1 80.00 ± 8.66 3.00 ± 0.06 0.334 ± 0.007 0.437 ± 0.266 mestiço × –1.5 58.33 ± 14.43 4.31 ± 0.02 0.232 ± 0.011 0.363 ± 0.071 roxo × 0 100.00 ± 0.00 1.05 ± 0.05 0.954 ± 0.045 0.904 ± 0.095 roxo × –0.33 100.00 ± 0.00 1.70 ± 0.28 0.599 ± 0.095 0.602 ± 0.177 roxo × –0.75 100.00 ± 0.00 2.12 ± 0.10 0.473 ± 0.024 0.779 ± 0.050 roxo × –1 95.00 ± 5.00 2.80 ± 0.22 0.359 ± 0.028 0.384 ± 0.084 roxo × –1.5 48.33 ± 10.41 4.76 ± 0.25 0.211 ± 0.011 0.320 ± 0.064 p-values cv p < 0.001 p < 0.001 p < 0.001 p = 0.001 w p < 0.001 p < 0.001 p < 0.001 p < 0.001 cv × w p = 0.044 p = 0.003 p < 0.001 p = 0.006 drought resistance during germination of iberian wheats acta bot. croat. 78 (2), 2019 173 fig. 2. mean values of (a) fresh and dry weigh, and (b) water content of 5-day old seedlings of four iberian cultivars at five water potentials, n = 15. results of two-way anova showed p-values for cultivar (cv), water potential (w) and the interaction of both factors (cv × w) for each parameter. p-values for fresh weight: cv = 0.015, w < 0.001, cv × w = 0.275; p-values for dry weight; cv < 0.001, w = 0.047, cv × w = 0.933; p-values for water content: cv < 0.001, w < 0.001, cv × w = 0.049. fig. 3. mean values of (a) root and coleoptile length, and (b) vigor index of 5-day old seedlings of four iberian cultivars at five water potentials, n = 15. results of two-way anova showed p-values for cultivar (cv), water potential (w) and the interaction of both factors (cv × w) for each parameter. p-values for root length: cv < 0.001, w < 0.001, cv × w < 0.001; p-values for coleoptile length: cv < 0.001, w < 0.001, cv × w < 0.001; p-values for vigor index: cv < 0.001, w < 0.001, cv × w = 0.003. pavia i., rocha l., moutinho-pereira j., lima-brito j., correia c. 174 acta bot. croat. 78 (2), 2019 required for field trials, as reported previously by others (singh et al. 2013, kumari et al. 2014, gilliham et al. 2017). conclusion this study reinforces the importance of research into ancient wheat cultivars, such mestiço. this cultivar showed the highest coleoptile size, high vi values, as well as the lowest variation in water content due to stress conditions. all these features lead us to believe it may be able to germinate in extremely dry soil and may be the least affected by any future growing reduction due to drought stress. still, to be relevant to breeders, research into improving stress tolerance should focus on elite cultivars. paradoxically, breeding programmes have been traditionally focused on maximising yield, while disregarding abiotic stress tolerance. among the modern cultivars, jordão revealed particularly good germination characteristics, such g and z as well as vi values across water potentials. this elite cultivar, which has shown high and stable yield in various environments (coco, 2013), presents a likely choice for future studies under drought stress conditions. acknowledgements we thank eng. coutinho (iniav-elvas) for supplying seeds of durum wheat ‘core’ and bread wheat ‘antequera’, ‘jordão’ and ‘roxo’ stored at the plant breeding station at elvas (portugal). the authors ip (pd/bd/113611/2015) and lr (pd/bd/113612/2015) acknowledge their doctoral grants funded by the fct (portuguese foundation for science and technology) under the doctoral program “agricultural production chains – from fork to farm” (pd/00122/2012). this work was also supported by national funds by fct, under the project uid/agr/04033/2019. references almansouri, m., kinet, j.m., lutts, s., 2001: effect of salt and osmotic stresses on germination in durum wheat (triticum 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vienna university of technology getreidemarkt 9/134, 1060 wien, austria 3 institute of microengineering and nanoelectronics, universiti kebangsaan malaysia 43600 ukm, bangi, selangor, malaysia 4 ac2t austrian center of competence for tribology, viktor kaplan-straße 22700 wiener neustadt, austria diatoms are single-celled organisms with rigid parts in relative motion at the microand nanometer length scales. some diatom species form colonies comprising many cells. in this manuscript, the results of a two-dimensional finite element computer model are presented. this model was established to discover if diatom colonies start to exhibit some kind of »pumping« behaviour when subjected to water flow. to analyze this computationally, a model diatom colony with continuously repeated units of ten cells is investigated in a fluid dynamic simulation. in this first simple model, undisturbed fluid flow is allowed for between the single cells. the cells do not move actively, and are solely moved by the water. the initial fluid velocity is assumed between 0.01 m s–1 and 1 m s–1. a computational result that does not change anymore with time is called a steady state solution. such a steady state solution is reached in all calculations performed. the steady state for a chain where initially all diatoms are spaced equally (equidistant spacing) has forces that encourage the formation of cell pairs with less distance between one another. these forces result from the flow of the surrounding fluid. the steady state for a chain with initially paired cells shows the opposite effect: the pairs tend to un-pair and head for the equidistant state again. the mutual change in forces between these two states, i.e., paired and unpaired formations, suggests that these two steady states lead into each other: the computer simulations suggest that a diatom colony subjected to water flow exhibits some kind of oscillatory movement. such movement might facilitate nutrient uptake of the diatom colony. keywords: diatom chain, fluid dynamics, hydroelastics, nutrient uptake, computer simulation acta bot. croat. 68 (2), 2009 431 * corresponding author: gebeshuber@iap.tuwien.ac.at u:\acta botanica\acta-botan 2-09\gebeshuber2.vp 9. listopad 2009 13:43:08 color profile: disabled composite 150 lpi at 45 degrees introduction diatoms are single-celled organisms with rigid parts in relative motion at the microand nanometer length scales. the diatom frustule is a very rigid structure. this has been revealed by force measurements (hamm et al. 2003, hamm 2005, losic et al. 2007). adhesives produced by diatoms are modular, self-healing and extraordinarily tough (gebeshuber et al. 2002, 2003; higgins et al. 2002, 2003a, 2003b; dugdale et al. 2005). such properties are the reason for increasing interest in diatoms for nanoand microtechnological applications (gebeshuber et al. 2005, in press; gordon et al. 2005, 2009; crawford and gebeshuber 2006; gebeshuber and crawford 2006; gebeshuber 2007, gordon et al. 2009). some diatom species form colonies comprising many cells in the form of long chains (round et al. 1990). rutilaria philipinnarum is an example of such a species (ross 1995). r. philipinnarum is a fossil colonial diatom thought to have lived in inshore marine waters. in this species, the single diatoms connect by linking spines and by a complex siliceous structure termed the periplekton (fig. 1). these linking structures on the one hand keep the cells together, but on the other hand also keep a distance between the cells. the shape of the spines allows expansion of the chain to a certain maximum distance and compression to a minimum distance, in which case there is still some fluid between the cells. the links allow movement of single cells in the chain against or from each other in a rather one-dimensional way (gebeshuber and crawford 2006). such elaborated linking mechanisms inspired the question what would happen to such a diatom colony if it were subjected to water flow. a diatom chain subjected to fluid flow orthogonal to its long axis is soon moved with the flow as a whole. however, in situations where the direction or velocity of flow changes, the inertia of the whole diatom chain prevents immediate acceleration according to new flow conditions. during the situation of acceleration, water flows through the gaps between the single cells creating relative motion between water and the chain. to analyze the problem, the method of computational fluid dynamics (cfd) is used. cfd is one of the branches of that uses and algorithms to solve and analyze problems that involve fluid flows. the governing equations that need to be solved consider the conservation of mass, momentum, pressure and turbulence (landau and lifshitz 1987). indeed, these equations are so closely coupled and difficult to solve that it was not until the advent of modern digital computers in the 1960s and 1970s that they could be resolved for real flow problems within reasonable timescales. nowadays, cfd has become an indispensable part of the aerodynamic and hydrodynamic design process for planes, trains, automobiles, rockets, ships, submarines; and indeed any moving craft or manufacturing process that mankind has devised. a basic introduction to fluid mechanics for the interested reader is given by chorin and marsden (1990). numerical methods used to solve the governing equations in fluid mechanics can be found in leveque (2005). the computer simulations presented here could inspire biologists working on diatoms to perform experiments validating the results, and thereby initiate interdisciplinary research involving groups from technical and biological backgrounds (gebeshuber and drack 2008). 432 acta bot. croat. 68 (2), 2009 srajer j., majlis b. y., gebeshuber i. c. u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 15:44:21 color profile: disabled composite 150 lpi at 45 degrees materials and methods to prove the principle, a simple two-dimensional finite element model of a diatom chain has been created. this model chain does not contain linking spines or periplekton or surface textures of the diatoms, but solely concentrates on primary aspects concerning the boundary conditions of multiple gaps (fig. 2). in the model, the diatom cell size is assumed to be 140 mm x 34 mm, and the distance between the cells can achieve a value from 10 mm to 20 mm. the colony is assumed to be comprised of single units of 10 cells each that are reacta bot. croat. 68 (2), 2009 433 colonial diatoms and oscillatory movement fig. 2. the white boxes counted from a1 to a10 symbolize the model diatoms. the model diatoms have one degree of freedom of movement, namely in the ± y direction, indicated by the arrows in cells a5 and a6. the xand y-axes of the coordinate system used are given on the right. the size of each model diatom cell is 140 µm x 34 µm, the distance between them is 15 µm. in the gaps between the boxes water flows in the direction marked with the bold arrows. the brighter area in the middle of the figure represents the »unit cell« of the endlessly repeated sequence, repeated units being indicated by the darker areas to the left and the right. fig. 1. schematic drawing of a chain of r. philipinnarum, illustrating three single diatoms with the linking structures and the periplektum with its screw-like structure. the long spines arranged in a ring ensure a certain minimum distance between the single cells while connecting them to each other. u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 15:44:42 color profile: disabled composite 150 lpi at 45 degrees peated endlessly. this simplifies calculations, but does not provide any information about possible influences of the ends of the chain. the chain of model-diatoms is surrounded by water. in all calculations, stationary solutions are sought. if the length of a model-diatom is chosen as a characteristic length scale, the reynolds number (re) turns out to be very low. re = r v l h–1 here r describes the density (1000 kg m–3), v the velocity (varied between 0.01 m s–1 and 1 m s–1) h the dynamic viscosity (1.003 x 10–3 pa s) of the surrounding water and l is the assumed characteristic length (140 x 10–6 m). therefore the maximum reynolds number turns out to be ~ 140. this allows us to assume that the problem is laminar. in this simple isothermal model (i.e. constant temperature), undisturbed fluid flow is allowed for between the single cells. the simulated fluid is water with a temperature of 293.15 k. the cells do not move actively, and are solely moved by the water. to simulate the fluid flow, boundary conditions are necessary. the inlet velocity of the fluid is varied between 0.01 m s–1 and 1 m s–1 in steps of an order of magnitude. as an outlet condition, an averaged static pressure of one bar is assumed. assuming the diatom colony to comprise much more than ten cells justifies the other two boundary conditions as to be symmetry. in this way, the four boundary conditions needed for the two dimensional model are defined, and the problem is ready to be solved. for solving the problem, the software package ansys cfx 11.0 is used. similar to further commercial software packages ansys uses the finite-volume method, which is a standard technique where the equations are solved on discrete control volumes. the models consist of up to 308283 volumes. this mesh of elements is created in ansys icem cfd 11.0. results in the simulations, steady state solutions (i.e. a computational result that does not change anymore with time) are calculated for two different arrangements of the model diatom chain (fig. 2). there are two different possible directions of the fluid to flow: along the long axis of the chain in the y-direction or across the chain through the gaps in the x-direction. all other possible fluid flows are simple superpositions of these two cases. the first case, when the fluid flows along the chain, only reveals a simple elongation of the chain. this is the reason why this case has not been investigated any further. on the other hand, fluid flow orthogonal to the long axis of the chain is worth examining in more detail: the first pre-set configuration investigated has equidistant model diatom cells (i.e. all model diatom cells have the same distance to their neighbouring cells, fig. 3). this represents a balanced position of the diatoms in the chain. the second pre-set configuration investigated has alternating distances of the model diatom cells (dmin between a1 and a2, dmax between a2 and a3, dmin between a3 and a4, dmax between a4 and a5, and so on, with dmin being 10 µm and being dmax 20 µm). this arrangement is one single example for an imbalanced condition of the chain and is shown in figure 4. the resulting acting forces grow proportionally with the velocity but do not change in direction with the 434 acta bot. croat. 68 (2), 2009 srajer j., majlis b. y., gebeshuber i. c. u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:42 color profile: disabled composite 150 lpi at 45 degrees different velocities investigated (0.01 m s–1–1 m s–1). therefore, the influence of different incoming flow velocities turns out to be not significant for the specific behaviour investigated. even though the acting forces in the flow direction (x-direction, fig. 2, tab. 1) are about a hundred times larger than the forces orthogonal to the flow direction (y-direction, fig. 2, tab. 1) for the equidistant state, it is mainly results for the forces orthogonal to the flow direction that are presented. the reason for this is that the solution for the forces in the x-direction is trivial: the whole chain of model diatoms accelerates in the direction of the flow. forces on the cells strongly correlate with the static pressure of the surrounding fluid, which again corresponds to the velocity of the fluid in the gaps due to bernoulli’s principle. in fluid dynamics, bernoulli’s principle states that for an in-viscous flow, an increase in the speed of the fluid occurs simultaneously with a decrease in pressure (e.g. lamb 1994). bernoulli’s principle is named after its developer daniel bernoulli (1700–1782). equidistant model diatoms: figure 3 shows the result for an inlet flow-velocity of 0.1 m s–1 in the positive x-direction. the white boxes represent the model diatoms. the fluid velocity is coloured from blue (slow) to red (fast).the remarkably small range of velocity in this figure was chosen to clarify the following statement: even though all gaps between the cells are equal, water velocity differs. this behaviour correlates to the forces acting on acta bot. croat. 68 (2), 2009 435 colonial diatoms and oscillatory movement fig. 3. the model diatoms (white boxes) are in the equidistant state, i.e. the distances between the single model diatoms are all the same. in this state, the stationary solutions of the computer simulation are velocities – coloured from red (fast) to blue (slow) in a range from 0.1 m s–1 to 0.11 m s–1 – that are not the same in all the gaps between the model diatoms, although the distances are the same. this result is caused by interference phenomena (similar to patterns on a river after a bridge with equidistant pylons). u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:49 color profile: disabled composite 150 lpi at 45 degrees single diatoms. table 1, which includes diatoms counted from a1 to a10, shows the specific forces acting on each diatom in the xand y-direction. note that the ends (a1 and a10) are not the real ends of the chain, because the chain itself has infinite length (periodic boundary conditions). so these ends only build the bounds of an endlessly repeated sequence. to assure the same height of all model diatoms in the simulated chain, the first (a1) and the last (a10) diatom are modelled with half the height (because of the periodic boundary conditions). the velocity distribution in the gaps shows interferential phenomena (as can be seen from the different sizes of the red coloured areas. the velocities in the gaps between the model diatoms are not equal for every gap even though all the gaps are the same (fig. 3). forces: as can be seen from table 1, the force acting on diatom a1 is positive (fa1 = 830.60 x 10–11 n) and the force acting on diatom a2 is also positive (fa2 = 0.52 x 10 –11 n). this means that if the model diatoms were allowed to move freely a1 would move in a positive direction and a2 would also move in a positive direction, but less, because the absolute value of the force is smaller. therefore, the gap between these two model diatom cells would decrease, since fa2–fa1 (0.52 x 10 –11 n – (830.60 x 10–11 n)) gives a negative value for the increase of the gap. the force acting on diatom a3 is negative (fa3 = –5.77 x 10 –11 n), and the force on model diatom a4 is positive (fa4 = 1.41 x 10 –11 n), and forces the cell into a positive y-direction. therefore, the gap between these two model diatom cells would increase, since fa4–fa3 gives a positive value. in this way, the changes in gap widths (if the model diatoms were allowed to move) can readily be calculated. the tendency if one performs these calculations (with data from table 1) is that the single gaps nearly alternately increase and decrease. however, as the simulation shows, attraction and repulsion do not always alternate, but there are some cases where more than two neighboured gaps act the same way: e.g. the gaps a1/a2 and a2/a3 decrease, also the gaps a4/a5 and a5/a6 do not alternate but both increase. the reason for the remarkable 436 acta bot. croat. 68 (2), 2009 srajer j., majlis b. y., gebeshuber i. c. tab. 1. forces acting on model diatom cells numbered from a1 to a10 for incoming flow conditions of 0.01 m s–1 in the positive x-direction. due to symmetric boundary conditions the first and the last diatom of the chain (a1 and a10) are only designed half so that forces in the x-direction are also half of the actual value. diatoms force fai in y-direction [10–11 n] force faj in x-direction [10–9 n] a1 830.60 12.62 a2 0.52 25.24 a3 –5.77 25.24 a4 1.41 25.24 a5 3.30 25.24 a6 3.46 25.24 a7 1.68 25.24 a8 –6.10 25.24 a9 0.59 25.24 a10 –829.50 12.62 u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:49 color profile: disabled composite 150 lpi at 45 degrees increase of force on a1 and a10 is unknown (tab. 1). perfect alternating increase and decrease of neighbouring gaps would represent the highest possible frequency of the oscillatory system the model diatom chain is proposed to be. this leads to the second case investigated, paired model diatoms. paired model diatoms: in the second case investigated, the distance between the cells is fixed and alternates between the minimum (10 mm) and the maximum distance (20 mm) the model diatoms can achieve. again, there is an inlet flow of 0.1 m s–1 in the positive x-direction. also in this case, interference phenomena in the velocity distribution appear, but this time they are negligible compared to the effect of the velocity in the larger gaps being much larger than the velocity in the smaller gaps (fig. 4). therefore the static pressure is much lower in the larger gaps and acting forces tend to readjust the chain into a balanced equidistant state. table 2 shows the acting forces in two different directions. the alternating algebraic sign for forces directed in the y-direction easily shows the described behaviour, if one performs the calculation for gap-distance as above. the fact that an imbalanced state tends to establish the balanced state is, however, a more effect than the first case investigated. acta bot. croat. 68 (2), 2009 437 colonial diatoms and oscillatory movement fig. 4. the model diatoms (white boxes) are in the paired state, i.e. the distances between two model diatoms are fixed and are alternately small or large. a stationary solution of the computer simulation is calculated. velocity – coloured from red (fast) to blue (slow) in a range from 0.1 m s–1 to 0.7 m s–1 – is distinctively larger in the large gaps compared to the velocity in the small gaps. this velocity distribution leads to higher static pressures in the small gaps. furthermore, the cells are forced to reduce the distance of the larger gaps while enlarging the smaller ones. u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:57 color profile: disabled composite 150 lpi at 45 degrees discussion this work is a numerical simulation. experimental verification of the results is highly desirable. even though there is a high grade of convergence in the simulations, numerical failure can never be excluded. the following interpretation only refers to the results from the numerical approximations as described above. the stationary result of the equidistant state forces the chain into imbalance of distance between cells. the stationary result of a non-equidistant state forces the equilibrium as shown in the other exemplary solution. this leads to the conclusion that there may be an additional oscillatory movement to the expected acceleration in the flow direction during the time of relative motion between chain and fluid. it is a behaviour that is comparable to the so-called flutter mechanism in turbine flows (nowinski and panovsky 2000), even though the flutter mechanism is mainly described as an aero-elastic phenomenon and therefore relevant in gas-flows. in the examples described here, it would be an example of hydroelasticity that is concerned, with the motion of bodies through (bishop and price 1979). the assumption of a diatom colony with exactly the same distance between all neighbouring cells is an idealistic one. in real diatom chains the gaps are not exactly the same, i.e., the chain is imbalanced. according to the computer simulations such a chain aspires to reach the »balanced« configuration with equidistant cells. however, the state with perfectly equidistant cells will never be reached and therefore oscillations occur as long as there is a non-zero relative velocity between the chain and the surrounding fluid. supposing that the natural habitat of the diatom colony is the open sea with its unsteady flow conditions such relative motion between diatoms and water is likely, and diatoms may have evolved a mechanism to utilize such flow changes, i.e., structure leading to function: oscillatory movement increases the advective diffusion through the surface of the diatoms and therefore increases nutrient supply in a homogeneous nutrient solution (pahlow et al. 1997). 438 acta bot. croat. 68 (2), 2009 srajer j., majlis b. y., gebeshuber i. c. tab. 2. forces acting on diatoms numbered from a1 to a10 for incoming flow conditions of 0.01 ms–1 in the positive x-direction. due to symmetric boundary conditions, the first and the last diatom of the chain (a1 and a10) are only depicted as halves, so that forces in the x-direction are also half of the actual value. diatoms force in y-direction [10–9 n] force in x-direction [10–9 n] a1 74.36 22.28 a2 9.99 50.36 a3 –9.70 50.46 a4 10.29 50.44 a5 –10.34 50.46 a6 10.26 50.46 a7 –10.25 50.44 a8 10.22 50.48 a9 –10.45 50.39 a10 –74.46 17.65 u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:57 color profile: disabled composite 150 lpi at 45 degrees the question of how a linked diatom chain gets the signal to build end valves is a question long discussed in the diatom community (e.g. fryxell 1976, crawford 1979, davey and crawford, 1986, crawford and sims, 2007). the computer simulation results presented here indicate various velocities of the water in the gaps and variance in the forces acting on the model diatom cells. this might provide a needed »signal« for building end valves (kooistra pers. comm.). a comparison of the results with a turbulent computation with the same boundary conditions shows the same effect but with forces ten times stronger than the laminary solution. the existence of a similar interference in a laminar calculation also shows that the modelled effects are not only turbulence phenomena, but inherently have to do with the inner elasticity of water. more detailed modelling approaches as well as experimental corroboration of the modelling results are needed to verify if real diatom chains exposed to conditions as described above also exhibit the oscillatory movement that results from this modelling study. acknowledgements the authors thank r. willinger and w. leitner for their expert introductions to fluid dynamic simulations, j. beigelboeck from the central computer services (zid) of tu vienna for expert help concerning the software package, dick crawford for continuous empathic support concerning all biologic and taxonomic questions concerning the diatoms as well as literature supply, w.h.c.f. kooistra for visionary possible applications of this model in diatom science and d. williams for literature supply. furthermore, the authors thank j.f. harper for discussion of this microfluid model. part of the present work has been funded by the 'austrian kplus-program' via the 'austrian center of competence for tribology', ac2t research gmbh, wiener neustadt. references bishop, r. e. d., price, w. g., 1979: hydroelasticity of ships. cambridge university press. chorin, a. j, marsden, j. e., 1990: a mathematical introduction to fluid mechanics. springer, new york. crawford, r. m., sims, p. a., 2007: some principles of chain formation as evidenced by the early diatom fossil record. nova hedwigia 133, 171–186. crawford, r. m. 1979: filament formation in the diatom genera melosira c.a. agardh and paralia heiberg. nova hedwigia 64, 121–133. crawford, r. m., gebeshuber, i. c., 2006: harmony of beauty and expediency. science first hand a good journal for inquisitive people 5, 30–36. davey, m. c., crawford, r. m., 1986: filament formation in the diatom melosira granulata. journal of phycology 22, 144–150. dugdale, t. m., dagastine, r., chiovitti, a., mulvaney, p. wetherbee, r., 2005: single adhesive nanofibers from a live diatom have the signature fingerprint of modular proteins. biophysical journal 89, 4252–4260. acta bot. croat. 68 (2), 2009 439 colonial diatoms and oscillatory movement u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:57 color profile: disabled composite 150 lpi at 45 degrees fryxell, g. a., 1976: the position of the labiate process in the diatom genus skeletonema. british phycological journal 11, 93–99. gebeshuber, i. c., thompson, j. b., del amo, y., stachelberger, h., kindt, j. h., 2002: in vivo nanoscale atomic force microscopy investigation of diatom adhesion properties. materials science and technology 18, 763–766. gebeshuber, i. c., kindt, j. h., thompson, j. b., del amo, y., stachelberger, h., brzezinski, m., stucky, g. d., morse, d. e., hansma, p. k., 2003: atomic force microscopy study of living diatoms in ambient conditions. journal of microscopy 212, 292–299. gebeshuber, i. c., stachelberger, h., drack, m., 2005: diatom bionanotribology – biological surfaces in relative motion: their design, friction, adhesion, lubrication and wear. journal of nanoscience and nanotechnology 5, 79–87. gebeshuber, i. c., crawford, r. m., 2006: micromechanics in biogenic hydrated silica: hinges and interlocking devices in diatoms. journal of engineering tribology 220, 787–796. gebeshuber, i. c., 2007: biotribology inspires new technologies. nano today 2, 30–37. gebeshuber, i. c., drack, m., 2008: an attempt to reveal synergies between biology and engineering mechanics. journal of mechanical engineering science 222, 1281–1287. gebeshuber, i. c., aumayr, m., hekele, o., sommer, r., goesselsberger, c. g., gruenberger, c., gruber, p., borowan, e., rosic, a., aumayr, f., in press: bacilli, green algae, diatoms and red blood cells – how nanobiotechnological research inspires architecture. in: yong zhou (ed.), bio-inspired nanomaterials and nanotechnology. nova science publishers, new york. gordon, r., sterrenburg, f., sandhage, k., 2005: a special issue on diatom nanotechnology. journal of nanoscience and nanotechnology 5, 1–178. gordon, r., losic, d., tiffany, m. a., nagy, s. s., sterrenburg, f. a. s., 2009: the glass menagerie: diatoms for novel applications in nanotechnology. trends in biotechnology 27, 116–127. hamm, c. e., merkel, r., springer, o., jurkojc, p., maier, c., prechtel, k., smetacek, v., 2003: architecture and material properties of diatom shells provide effective mechanical protection. nature 421, 841–843. hamm, c. e., 2005: the evolution of advanced mechanical defenses and potential technological applications of diatom shells. journal of nanoscience and nanotechnology 5, 108–119. higgins, m. j., crawford, s. a., mulvaney, p., wetherbee, r., 2002: characterization of the adhesive mucilages secreted by live diatom cells using atomic force microscopy. protist 153, 25–38. higgins, m. j., sader, j. e., mulvaney, p., wetherbee, r., 2003a: probing the surface of living diatoms with atomic force microscopy: the nanostructure and micromechanical properties of the mucilage layer. journal of phycology 39, 722–734. higgins, m. j., molino, p., mulvaney, p., wetherbee, r., 2003b: characterizing the structure and nanomechanical properties of the adhesive mucilage that mediates diatom-substratum adhesion and motility. journal of phycology 39, 1181–1193. 440 acta bot. croat. 68 (2), 2009 srajer j., majlis b. y., gebeshuber i. c. u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:57 color profile: disabled composite 150 lpi at 45 degrees lamb, h., 1994: hydrodynamics. cambridge university press. landau, l. d., lifshitz, e. m., 1987: fluid mechanics, 6. butterworth-heinemann, oxford, uk. leveque, r. j., 2005: numerical methods for conservation laws. birkhäuser, basel. losic, d., short, k., mitchell, j. g., lal, r., voelcker, n. h., 2007: afm nanoindentations of diatom biosilica surfaces. langmuir 23, 5014–5021. nowinski, m., panovsky, j., 2000: flutter mechanism in low pressure turbine blades. journal of engineering for gas turbines and power 122, 82–88. pahlow, m., riebesell, u., wolf-gladrow, d. a., 1997: impact of cell shape and chain formation on nutrient acquisition my marine diatoms. limnology and oceanography 42, 1660–1672. purcell, e. m., 1977: life at low reynolds number. american journal of physics 45, 3–11. ross, r., 1995: a revision of rutilaria greville (bacillariophyta). bulletin of the natural history museum botany series 25, 1–93. round, f. e., crawford, r. m., mann, d. g., 1990: the diatoms: biology and morphology of the genera. cambridge university press. acta bot. croat. 68 (2), 2009 441 colonial diatoms and oscillatory movement u:\acta botanica\acta-botan 2-09\gebeshuber.vp 6. listopad 2009 13:18:57 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 76 (2), 2017 177 acta bot. croat. 76 (2), 177–182, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0008 issn 0365-0588 eissn 1847-8476 genetic diversity of potamogeton pectinatus l. in iran as revealed by issr markers shabnam abbasi1, saeed afsharzadeh2*, hojjatollah saeidi3 department of biology, university of isfahan, 81746-73441, isfahan, iran abstract – potamogeton pectinatus l. is a widespread aquatic species distributed widely in aquatic ecosystems of iran. in this study, inter simple sequence repeat (issr) markers were used to assess the genetic diversity of 35 accessions and 175 individuals of p. pectinatus collected from different regions of iran. in total, 123 polymorphic dna fragments were amplified from five combinations of issr primers. the issr based principle coordinate analyses (pcoa) demonstrated four different groups mostly corresponding with their geographic origins (north, kerman/fars, centre and southwest). the most variable populations were found in the central region of iran possibly as a consequence of the larger number of samples from that region. the result of molecular variance (amova) attributed 11% of the total genetic variation among and 89% within population variation. the results showed high levels of intra-regional and low inter-regional gene flow between clones, although the northern accessions were clearly differentiated from the others. there was a low correlation between genetic distance and geographic distance of accessions. the results of structure analysis suggested the presence of three genetic groups of this species in iran, mostly adapted to different ecological conditions. our results cover one of the gaps of different studies worldwide. in addition, our results confirm high levels of genetic diversity of p. pectinatus in iran. key words: genetic diversity, iran, issr, potamogeton pectinatus, potamogetonaceae * corresponding author, e-mail: s.afshar@sci.ui.ac.ir introduction potamogeton l. (potamogetonaceae) comprises about 100 species and 50 interspecific hybrids worldwide (wiegleb 1988, sculthorpe 1967), 14 species of which occur in iran (dinarvand 2009, 2011, abbasi et al. 2015). among them, p. pectinatus l. is one of the most diverse submerged aquatic species (sandsten et al. 2005, wiegleb and kaplan 1998). this species harbors many useful physiological traits such as tolerance to a wide range of nutrients, ability to grow in oligotrophic to eutrophic waters (triest et al. 2010), capacity to improve water quality by absorbing nutrients (lone et al. 2013) and immunomodulatory activity (kumar et al. 2012). these traits have made the species a potentially important organic tool for cleaning up polluted waters by absorption of heavy metals (demirezen and aksoy 2004, ren et al. 2006). potamogeton pectinatus reproduces both sexually and vegetatively through propagules emerging from the rhizomes (van wijk 1989) and waterfowl probably have an important role in the species dispersal (green et al. 2002). accessions of p. pectinatus have been analysed using isozyme (hettiarachchi and triest 1991), rapd (mader et al. 1998, hangelbroek et al. 2002), issr (king et al. 2002) and aflp (han et al. 2014) markers. in most cases there was a geographic pattern of diversity and also a correlation between genetic diversity and migration pathways of water birds (mader et al. 1998, hangelbroek et al. 2002). triest and fenart (2013) indicated a correlation between genetic structure of clones and habitat type in this species. it has been shown that morphological variation within this species is mostly correlated with ecological conditions (kaplan 2002), and therefore morphological characters are not precise indicators for evaluation of a population’s genetic structure. of the several molecular markers developed so far, inter simple sequence repeat (issr) markers developed by zietkiewicz et al. (1994) have been widely used to detect genetic similarities in plants (zietkiewicz et al. 1994, mousavifard et al. 2015, akhavan et al. 2015). due to high polymorphism, only a few issr loci, (as few as five to seven primer pairs) are sufficient to obtain reliable information about genetic diversity (matesanz et al. 2011). in the present study, we used issr markers to evaluate genetic diversity within iranian germplasm of p. pectinatus abbasi s., afsharzadeh s., saeidi h. 178 acta bot. croat. 76 (2), 2017 and elucidate the patterns of diversity in contrast to geographic distribution and dispersal mechanisms. regarding the progressive drought in iran which can result in loss of the genetic diversity of this species, we hope that the results of this study will provide useful information for design of more efficient conservation strategies. materials and methods plant material a total of 35 accessions of p. pectinatus (each composed of five individuals) were collected from different regions of iran during july – october 2014. the herbarium voucher specimens are deposited in the herbarium of the university of isfahan. accessions were morphologically identified according to flora iranica (dandy, 1971); flora europaea (dandy 1980), flora of turkey (uotila 1984), flora of iraq (dandy 1985), flora palaestina (feinbrun-dothan 1986), monograph of wiegleb and kaplan (1998). geological features and ecological conditions of collecting sites accessions were divided into four geographic groups (center, north, southwest, kerman/fars). accessions code, locality and other details regarding the plant materials used in this study are provided in table 1. tab. 1. geographical information of the studied potamogeton pectinatus accessions in iran; n – north, c – center, kf – kerman-fars, swsouthwest. accession code population locality location coordinates elevation (m) pot-p1 n mazandaran, tonekobon toward ramsar river n:36, 47 e:50, 55 –20 pot-p8 n mazandaran, zaghmarz river n:36, 29 e:52, 53 –3 pot-p7 n mazandaran, valasht lake n:36, 27 e:51, 32 983 pot-p11 n gilan, siahrood river n:36, 18 e:52, 53 62 pot-p25 n delijan, delijan river n:30, 06 e:52, 55 1674 pot-p18 n gilan, langrood river n:44, 53 e:47, 45 76 pot-p28 n gilan, sefidrood river n:37, 22 e:50, 10 –2 pot-p16 n gilan, amirkelaie river n:37, 16 e:50, 13 76 pot-p32 n gilan, siahrood river n:36, 18 e:52, 53 62 pot-p2 c isfahan, hojatabad river n:32, 30 e:50, 50 1902 pot-p3 c isfahan, hamzeali river n:31, 50 e:51, 04 2303 pot-p4 c isfahan, chamaseman wetland n:32, 24 e:21, 22 1750 pot-p5 c chaharmahal va bakhtiari, boroujen river n:31, 57 e:51, 19 2128 pot-p6 c isfahan, chamheidar river n:32, 27 e:50, 59 1780 pot-p9 c chaharmahal va bakhtiari, gandoman wetland n:31, 48 e:51, 05 2254 pot-p12 c chaharmahal va bakhtiari, chaghakhor wetland n:31, 55 e:50, 55 2320 pot-p15 c isfahan, jarghoie river n:32, 09 e:52, 37 1413 pot-p19 c isfahan, cheshmedimeh spring n:32, 30 e:50, 12 2133 pot-p21 c isfahan, polechoom river n:32, 35 e:51, 46 1578 pot-p33 c chaharmahal va bakhtiari,, lordegan, barm spring n:31, 34 e:51, 12 1635 pot-p10 kf kerman, gogher river n:29, 29 e:56, 38 2740 pot-p14 kf kerman, yaschaman river n:29, 27 e:56, 37 2824 pot-p20 kf fars, hasanabad river n:29, 39 e:53, 20 1621 pot-p30 kf fars, komjan wetland n:29, 40 e:53, 08 1625 pot-p31 kf fars, komjan wetland n:29, 40 e:53, 08 1625 pot-p34 kf fars, sivand river n:30, 06 e:52, 55 1717 pot-p35 kf kerman, yaschaman river n:29, 27 e:56, 37 2824 pot-p13 sw shushtar, pole bande mizan river n:32, 03 e:48, 51 127 pot-p17 sw khuzestan, izeh toward lordegan river n:32, 03 e:48, 51 1307 pot-p22 sw shushtar, shushtar river n:32, 03 e:48, 51 127 pot-p23 sw khuzestan, abadan, minoo island n:30, 20 e:48, 13 128 pot-p24 sw khuzestan, shadegan wetland wetland n:30, 15 e:48, 19 137 pot-p26 sw khuzestan, miangaran wetland n:31, 52 e:49, 52 925 pot-p27 sw khuzestan, abadan toward ahwaz lake n:30, 26 e:48, 11 110 pot-p29 sw khuzestan, susangerd river n:31, 47 e:47, 55 113 genetic diversity of potamogeton pectinatus l. acta bot. croat. 76 (2), 2017 179 dna extraction and pcr from each sample, total dna was extracted following the method developed by gawel and jarret (1991) from the leaves of each accession. in order to perform issr analysis, forty combinations of issr primers (blair et al. 1999) were tested, from which five primer pairs (tab. 2) amplifying detectable and polymorphic dna fragments were selected for further analysis from genomic dna of p. pectinatus accessions. the pcrs were carried out in a 15 µl volume with 250 nm of each primer (tab. 2), 0.2 mm of each dntp, 1.5 mm mgcl2, 1 u taq polymerase, and 50–100 ng of genomic dna. after 4 min at 95 °c, pcr was followed by 45 cycles of 1 min at 95 °c, 1 min at annealing temperature (50–56 °c; tab. 3), 2 min at 72 °c, followed by a final extension step of 10 min at 72 °c. pcr products were detected by 2% agarose and ethidium bromide staining under uv light. data analysis the presence (1) or absence (0) of each band (dna fragment amplified in pcr) was scored and genetic similarity was calculated based on jaccard (1908) similarity coefficients. correlation between genetic distances and geographic distances (r) was measured using mantel test statistics (mantel, 1967) implemented in genalex software (ver 6.5; peakall and smouse 2006). to assess genetic diversity, basic parameters including nei`s gene diversity index (h), shannon index (i), percentage of polymorphic loci (ppl) and mean expected heterozigosity (he) were calculated from the data using popgene software ver. 1.32 (yeh et al. 2000). analysis of molecular variance (amova) was performed to calculate the proportion of intra-accession and inter-accession genetic diversity using genalex 6.5 software. the principal coordinates analysis (pcoa) was performed using genalex 6.5. to infer the genetic structure of sampled populations, structure software was used. results in the present study, genetic diversity was examined in potamogeton pectinatus based on issr markers. in total, 5 issr primer combinations (tab. 2) amplified 123 dna fragments from genomic dna of 35 accessions of p. pectinatus. the number of bands per primer ranged from 18 to 30 with an average of 24.6 bands per primers pair. the highest and the lowest numbers of bands were produced from the primer combinations issr 823 (30 bands) and issr 811 – issr 812 combination (18 bands), respectively. in pcoa 2d plot (fig. 1), grouping mainly followed geographic origin. in the pcoa 2d plot, accessions collected from the north were grouped and the southern accessions were also loosely grouped. accessions collected from isfahan, kerman and fars however showed no grouping related to their geographic origin. in the structure analysis, accessions were divided into three clusters (k=3, red, green and blue in fig 2, which can be regarded as genetic populations). accessions collected from the north of iran and accessions collected from the south (kerman-fars) were more uniform than those of the center and southwest (fig. 2). mantel test showed a low correlation between genetic distance and geographic distance (r = 0.240, p = 0.02). in analysis of molecular variance (amova), 11% of total genetic variation was attributed to the between and 89% to the within population differentiations and the amount of phipt was 0.11. the amount of gene flow between populations (geographic regions) was indirectly calculated from phipt as nm = 0.25[(1/phipt)–1] = 2. in calculation of shannon index, which readily translates into heterozygosity, the highest observed heterozygostab. 2. sequences and annealing temperatures of primers (blair et al. 1999). primer id sequence (5’→ 3’) tm issr 807 agagagagagagagagt 50 ubc 872 gatagatagatagata 38 issr 823 tctctctctctctctcc 52 issr 826 acacacacacacacacc 52 issr 811 gagagagagagagagac 52 issr 812 gagagagagagagagaa 50 ubc 873 gacagacagacagaca 48 tab. 3. primer combinations, annealing temperature (ta), percentage of polymorphism, total (t.b.) and average (a.b.) number of bands produced by primers used for inter simple sequence repeat (issr). p.b. – number of polymorphic bands; p.p. – percentage of polymorphism; b.s. – band size range (bp); pic -polymorphism information content. primer combination ta (°c) t.b. p.b. p.p. b.s. a.b. pic issr807+issr872 52.1 28 28 100 250–2000 7 0.33 issr823 45.7 30 30 100 200–3000 7.5 0.19 issr826 44 22 22 100 310–1500 5.5 0.22 issr811+issr812 48.7 18 18 100 150–700 4.5 0.21 issr811+issr873 53.9 25 25 100 100–2000 6.25 0.14 total 123 123 1.09 average 24.6 24.6 0.218 abbasi s., afsharzadeh s., saeidi h. 180 acta bot. croat. 76 (2), 2017 ity (0.230) was calculated within the isfahan (c) population and the lowest one (0.193) was calculated within the kerman-fars (kf) population. the highest polymorphism ratio was calculated between germplasm collected from the center of iran (66.67%) and the lowest one (49.59%) from the south (kf) (tab. 4). genetic structure of accessions is shown in fig. 2. fig. 1. principle coordinate analysis (pcoa) 2d plot based on inter simple sequence repeat (issr) data, the numbers are accession numbers (1–35) of potamogeton pectinatus from iran, divided into 4 geographic regions: c – central, n – north, kf – kerman/ fars, s – south west. fig. 2. map of the collection site and population structure of the 35 accessions of potamogeton pectinatus from iran, grouped in 4 geographic regions (1 – north, 2 – center, 3 – south, 4 – southwest) analyzed using inter simple sequence repeat (issr) markers. structure analysis results revealed three clusters (green = cluster 1, red = cluster 2, blue = cluster 3) for 4 regions for the wild iranian p. pectinatus gene pool. each vertical column represents one accession. tab. 4. genetic diversity within populations of potamogeton pectinatus in iran revealed by inter simple sequence repeat (issr) data. n.a. – number of accessions; p – percentage of polymorphism at population level; ae – mean effective number of alleles; ho – mean observed heterozygosity (shannon index); he – mean expected heterozygosity (unbiased); h – nei’s gene diversity. population n.a. p (%) ae ho he h center 10 66.67 1.201 0.230 0.137 0.13 north 9 56.91 1.201 0.218 0.134 0.13 kerman/fars 7 49.59 1.172 0.193 0.117 0.11 southwest 9 58.54 1.193 0.221 0.134 0.13 average 57.93 0.215 0.130 genetic diversity of potamogeton pectinatus l. acta bot. croat. 76 (2), 2017 181 discussion potamogeton pectinatus is distributed along zagros and alborz mountains in iran, in rivers, wetlands, springs, lakes and swamplands characterized by a vast range of environmental conditions. our sampling covered ecologically different habitats of the species in iran. regarding the species geographic distribution in different ecological conditions, some kind of adaptation-based variation was expected within the iranian gene pool of this species that may not be well reflected in terms of morphological characters. as wang et al. (2012) have noted, the mixed mode of reproduction has an important effect on the genetic structure of p. pectinatus. asexual reproduction is likely to be responsible for short-distance gene flow, while sexual reproduction is expected to be long-distance dispersal (van wijk 1989). therefore, the pattern of diversity in this species could not be influenced by only ecological adaptations. we used issr markers to demonstrate the patterns of diversity and test these hypotheses. the results of this study showed that the iranian gene pool of p. pectinatus harbors a high level of heterozygosity (ho = 0.25, the highest amount of heterozygosity for dominant markers are 0.50). as freshwater and brackish water ecosystems are usually geographically separated, one may expect that aquatic plants in these regions are genetically isolated by distance (see for example triest et al. 2010). in the case of p. pectinatus, this species grows both in fresh water, brackish water and rivers in a vast range of ecological conditions. therefore it has probably experienced different adaptations which possibly resulted in higher genetic diversity. accessions collected from the center showed higher genetic diversity than those collected from other regions. this region is frequently visited by migratory birds coming from northern and southern regions (sehhatisabet and khaleghizadeh 2013), which can easily bring seeds that are characterized by different genotypes to this region, resulting in higher genetic diversity. the northern accessions of the species were closely clustered and showed low genetic diversity (fig 1). this lower genetic diversity could be the result of lower levels of ecological variation present in this region. such a situation is also observed in southern (kf) accessions, those that grouped together in dendrogram and pcoa 2d plot. hangelbroek et al. (2002) indicated that seeds rather than vegetative structures are responsible for gene flow within populations of p. pectinatus. this species is an important food source for many herbivorous waterfowls and water birds (triest et al. 2010). as seeds can be easily transported by water birds, it can be concluded that seeds are more probably responsible for long distance gene flow. iran is on the migration way of birds coming from the northern and southern territories. therefore they have probably an important role in gene flow among isolated populations as in the central region of iran. as shown in pcoa 2d plot, accessions were clearly grouped in correspondence to their geographical origin. this can be interpreted as an indication for establishment of specific genotypes in different regions with high intra-regional and low inter-regional gene flow between clones. the clones sampled in the north are genetically more similar than the ones from the other regions and such finding can be a result of a relative uniformity of the ecological conditions in the northern region. the structure diagram divided accessions into three clusters (red, blue and green, fig. 2). the results corresponded with the grouping in pcoa. the genetic structuring suggested that there are three genetic groups of the species in iran, which presumably adapted to different ecological conditions. the result of mantel test, which showed no significant correlation between genetic and geographic distance, indicates that dispersal occurs in different directions and probably with different mechanisms (sexual and asexual) involved in the dispersal of the species (wang et al. (2012). the results of present study were partly in contrast with the results of mader et al. (1998) which showed a correlation between genetic distance and geographic distance. in that study, the analyzed accessions were collected from europe, north africa, north and south america, whereas our collection originated from a smaller region (iran), in which habitats are frequently visited by migratory birds. therefore, it can be concluded that on a geographically small scale, as in the present case, inter populations gene flow occurs more frequently than on a larger scale, as in the collection of mader et al. 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(potamogetonaceae). folia geobotanica 33, 241– 316. yeh, f. c., yang, r., boyle, t. j., ye, z., xiyan, j. m., 2000: popgene 32: microsoft windows-based freeware for population genetic analysis. edmonton: molecular biology and biotechnology center, university of alberta. zietkiewicz, e., rafalski, a., labuda, d., 1994: genome fingerprinting by simple sequence repeat (ssr) anchored polymerase chain reaction amplification. genomics 20, 176–183. acta bot. croat. 77 (2), 2018 181 acta bot. croat. 77 (2), 181–188, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0022 issn 0365-0588 eissn 1847-8476 genetic structure of populations of several endangered and endemic dianthus species revealed by microsatellite markers anca butiuc-keul1,2*, cornelia crăciunaș1,2, irina goia3,2, anca farkas1,2, liliana jarda4, victoria cristea4,2 1 department of molecular biology and biotechnology, faculty of biology and geology, babeş-bolyai university, 400084 cluj-napoca, romania 2 laboratory of plant biology, genetics, microbiology and biotechnology, center of systems biology, biodiversity and bioresources, babeş-bolyai university, 400006 cluj-napoca, romania 3 department of taxonomy and ecology, faculty of biology and geology, babeş-bolyai university, 400015 cluj-napoca, romania 4 alexandru borza botanical garden, babeş-bolyai university, 400015 cluj-napoca, romania abstract – in order to develop a proper conservation programme for several endangered, rare or endemic species of dianhtus from romania, molecular characterization by simple sequence repeat (ssr) markers has been accomplished. amplification of ssr loci in individuals belonging to different populations of d. callizonus, d. glacialis ssp. gelidus, d. henteri, d. nardiformis and d. tenuifolius revealed 23 polymorphic alleles. d. callizonus and d. tenuifolius showed particular sets of ssr alleles. d. glacialis ssp. gelidus, d. henteri and d. nardiformis proved to share almost the same alleles in most of the loci. the highest genetic diversity was observed in d. glacialis ssp. gelidus and d. tenuifolius in locus ms-dinmadsbox. allelic patterns across dianthus species indicate that the mean number of different alleles was highest in d. glacialis ssp. gelidus, while the number of effective alleles was highest in d. tenuifolius. there are no particular differences in individuals belonging to the same species. genetic diversity is generally low, ranging from 0.18 (d. callizonus) to 0.44 (d. henteri). regarding the genetic diversity within populations of the same species, no differences were revealed by the use of the ssr markers tested in the present study. key words: dianthus, endemic species, genetic structure, microsatellites, ssr markers, polymorphism * corresponding author, e-mail: anca.keul@ubbcluj.ro introduction in the last decades, habitat modifications induced by climate change and human activity led to the extinction of many plant species. species conservation programs targeting large areas should focus on the most valuable populations and so require a better understanding of species biology and ecology as well as knowledge of the genetic diversity distribution within and among populations. genetic diversity is important because it influences the populations’ ability to adapt to a changing environment conditions. a characteristic of rare or endemic plants is the maintenance of low levels of genetic variation. limited genetic diversity has been reported for many rare plant species (hamrick et al. 1991, frankham 1996, leimu et al. 2006, szczecińska et al. 2016). the genetic diversity within an endangered population is lost due to relatively faster genetic drift, which is exacerbated by limited gene flow. this can lead to inbreeding depression and higher homozygosity, which results in reduced adaptive potential (reed and frankham 2003, johansson et al. 2007). genetic diversity is often correlated with plant fitness (leimu et al. 2006, ilves et al. 2013). genetic structure of plant populations, the level of genetic polymorphism within and among populations could offer information of value in the development of proper strategies for their conservation. the aim of conservation programs is to preserve the highest number of populations, as well as their genetic structure and variability. the genetic structure and variability of endemic and rare plant populations were butiuc-keul a., crăciunaş c., goia i., farkas a., jarda l., cristea v. 182 acta bot. croat. 77 (2), 2018 intensively studied by izoenzymes (gitzendanner and soltis 2000) and nuclear dna markers (pritchard et al. 2000, cruzan 2001, kang et al. 2005, °cuan et al. 2006, breinholt el al. 2007). among these markers, simple sequence repeats (ssr) are frequently used to investigate genetic structure and variability in plant populations because of their cost effectiveness (zietkiewicz et al. 1994, varshney et al. 2005). there are many previous data regarding the usefulness of ssr in genetic studies in rare and endangered plants (katoh et al. 2007, sosa et al. 2010). molecular characterization of the carnation was previously reported by sequence-related amplified polymorphism (srap) and inter simple sequence repeat (issr) markers (fu et al. 2008). ssr markers have been also used for genotyping of carnation varieties (smulders et al. 2003). dianthus is one of the most diverse mediterranean-type plant genera; more than 300 species are distributed throughout eurasia and africa. over 100 species of dianthus species occur in europe, and more than 70 are endemic (valente et al. 2010). in romania, 58 dianthus taxa have been recorded (ciocârlan 2009), of which 8 are endemic. recent works have analysed the genetic variability of two endemic and endangered species of dianthus from romania (d. spiculifolius schur; d. giganteus d'urv. subsp. banaticus (heuff ) (cristea et al. 2014, jarda et al. 2014, gabel et al. 2017). in this paper we investigate the genetic diversity of 5 endemic and threatened romanian dianthus species using ssr markers. d. callizonus schott & kotschy, a dacian element, is strictly endemic to the curvature carpathian mountains. previously classified as vulnerable (vu) by oprea (2005), it is currently considered as low risk (lr) species in the red book of vascular plants from romania (dihoru and negrean 2009). d. callizonus grows on alpine and subalpine calcareous rocks. d. glacialis haenke ssp. gelidus (schott, nyman & kotschy) tutin is a taxon endemic to the eastern and southern carpathians (oprea 2005). it is sporadic in alpine communities on calcareous rocks. d. henteri heuff. ex griseb. & schenk is endemic to the southern carpathians. this species grows on rocky soils, in the beech and spruce belt (sârbu et al. 2013). d. nardiformis janka grows on rocky areas, in the lower danube basin and in the dobrudja region. this species is considered vu (oprea 2005, dihoru and negrean 2009). d. tenuifolius schur is an endemic species in the romanian carpathians and grows in meadows, on skeletal soil. it is considered a least concern (lc) species by oprea (2005). all taxa are presumably diploids (2n=30), according to carolin (1957), a ploidy feature that may allow an easy interpretation of their ssr patterns. all the above mentioned taxa are perennial and were identified in different natura 2000 sites from romania. besides d. nardiformis, which is found not only in eastern romania but also in northern bulgaria, all the other 4 species are found only in romania and are strictly endemic to the above mentioned areas. materials and methods plant material the plant material was collected from different populations of the studied dianthus taxa from romania (fig. 1). five individuals were collected from each location. thus, d. callizonus was collected from 2 locations from piatra craiului mountains: p1-spîrlea refuge and zăplaz (45º31'42.60" n, 25º12'00.17" e) and diana refuge and padina popii (45°33'29.18" n, 25°14'29.75" e); d. glacialis ssp. gelidus from 3 populations from bucegi mountains: p1omu peak (45°22'51.00" n, 25°30'40.00" e), p2-bâlea lake, făgăraş mountains (45°31'40.00" n, 24°44'26.00" e), p3obârşia, bucegi mountains (45°22'50.00" n, 25°30'39.00" e); d. henteri from 3 populations from vâlcea county: p1-cornet (45°23'19.82" n, 24°18'28.54" e), p2-călineşti valley (45°22'19.34" n, 24°17'23.23" e), p3-jiului gorge (45°16'46.00" n, 23°23'19.00" e); d. nardiformis from 3 populations from tulcea county: p1-allah bair (44°29'01.42" n, 28°13'24.88" e), p2-consul hill (45°10'55.19" n; 28°16'15.77" e), p3-măcin (45°14'22.71" n, 28°35'01.01" e); d. tenuifolius from 2 populations from suceava county: p1-stânişoarei mountains (47°21'50.87" n, 25°36'10.88" e), p2-bistriţei mountains (47°23'30.00" n, 25°30'16.65" e). ssr analysis genomic dna was isolated from leaves using the ctab method described by doyle and doyle (1987). for ssr analysis, a total of five primer pairs (on-line suppl. tab. 1) were used (smulders et al. 2000). pcr amplifications were performed in a 0.2 ml tube containing 2 mm mgcl2, 1 μm of each primer, 200 μm of each dntp, 1.5 u of taq (fermentas) and 25 ng of genomic dna in a final volume of 25 μl. dna amplification was performed according to the following program: 1. t = 94 °c, 5 min; 2. t = 94 °c, 45 s; 3. primer annealing at 55 °c, 45 s; 4. elongation t = 72 °c, 45 s; steps 2–4 were repeated 35 times. amplicons were separated on 1.5% agarose gel, stained with 0.5 μg ml–1 ethidium bromide. at least 2 independent pcr amplifications were performed for each primer. data analyses several genetic diversity parameters were calculated for each locus and per dianthus species using powermarker 3.25 (liu and muse 2005). these included gene diversity (expected heterozygosity), observed heterozygosity (referred fig. 1. sampling locations for dianthus populations in romania. genetic structure of populations of some dianthus species acta bot. croat. 77 (2), 2018 183 to as heterozygosity), and polymorphism information content (pic) value. these parameters take into account allele frequencies. within species, richness and evenness were also explored using genalex6.5 (peakall and smouse 2006, 2012). the number of alleles per species (arithmetic mean across loci), the number of alleles with a frequency greater than 5%, the effective number of alleles, the number of private alleles and the number of locally common alleles occurring in less than 50% of the populations were explored. genetic diversity was assessed via shannon’s information index, on a single-locus basis, based on natural logarithmic allele frequencies. the evolutionary history was inferred using the neighbour-joining method (saitou and nei 1987). the optimal tree with the sum of branch length = 100.13433838 was constructed. the tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances were provided by the user. evolutionary analyses were conducted in mega 6 (tamura et al. 2013). principal coordinate analysis (pcoa) was used to explore multivariate tab. 1. genetic characterization of dianthus species based on ssr alleles. different types of alleles: a-bsy-150, b-bsy-100, c-bsy-75, dbsy-50, e-dia-200, f-dia-175, g-dia-100, h-dia-75, i-dia-50, j-dinca-200, k-dinca-150, l-dinca-100, m-dinca-50, n-dingsta-200, o-dingsta-175, p-dingsta-100, r-dingsta-75, s-box-300, t-box-175, u-box-150, v-box-100, x-box-75, z-box-50, absence of allele. sp ec ie s/ po pu la tio n lo ca tio n n o. o f i nd iv id ua ls m sd c a m c r b sy m sd c d ia 30 m sd in c a r a c c m sd in g st a m sd in m a d sb o x d. callizonus p1 spîrlea refuge and zăplaz, diana refuge and padina popii, piatra craiului mountains 5 1-2 alleles: 100:75 (-b, bc) 2 alleles: 100:75 (gh) 1 allele: 100 (l) 1 allele: 100 (p) 1 allele: 100 (v) d. glacialis ssp. gelidus p1 omu peak, bucegi mountains 5 0-1 alleles: 100 (b-) 1-2 alleles: 200:50 (ei, -i) 1-2 alleles: 200:50 (jm, -m) 1 allele: 75 (r) 0-2 alleles: 150:100:75 (vx, ux, --, x-) p2 bâlea lake, făgăraş mountains 5 1-2 alleles: 100:75 (b-, bc) 1-2 alleles: 200:50 (ei, -i) 1-2 alleles: 200:50 (jm, -m) 1 allele: 75 (r) 1-2 alleles: 150:75:50 (uz, x-) p3 obârşia, bucegi mountains 5 1-2 alleles: 150:50 (ad, b-) 1-2 alleles: 200:50 (-i, ei) 1-2 alleles: 200:50 (jm, -m) 1 allele: 75 (r) 1-2 alleles: 150:75:50 (uz, -z, x-) d. henteri p1 cornet, vâlcea county 5 2 alleles: 100:50 (bd) 1-2 alleles: 200:75 (eh, -h) 2 alleles: 200:150:100 (jk, kl) 2 alleles: 200:75 (nr) 1-2 alleles: 150:100 (uv, -v) p2 călineşti valey, vâlcea county 5 2 alleles: 100:50 (bd) 1-2 alleles: 200:75 (eh, -h) 2 alleles: 200:150:100 (jk, kl) 2 alleles: 200:75 (nr) 1-2 alleles: 150:100 (uv, -v) p3 jiului gorge 5 2 alleles: 100:50 (bd) 0-1 alleles: 75 (-h) 2 alleles: 150:100 (kl) 1-2 alleles: 200:75 (nr, -r) 0-1 alleles: 100 (-v) d. nardiformis p1 allah bair, tulcea county 5 0-1 alleles: 100 (-b, --) 1-2 alleles: 200:75 (-h, eh) 1-2 alleles: 150:100 (-l, kl) 1-2 alleles: 200:100 (n-, np) 1-2 alleles: 150:75 (-u, ux) p2 consul hill, tulcea county 5 0-1 alleles: 150:100 (a-, -b) 1-2 alleles: 200:75 (-h, eh) 1-2 alleles: 150:100 (kl, k-) 1-2 alleles: 200:100 (n-, np) 1-2 alleles: 150:75 (-u, ux) p3 măcin, tulcea county 5 0-1 alleles: 100 (a-) 1-2 alleles: 200:75 (-h, eh) 1-2 alleles: 150:100 (-l, kl) 1-2 alleles: 200:100 (-p, np) 1-2 alleles: 150:75 (-x, ux) d. tenuifolius p1 stânişoarei mountains, suceava county 5 0-1 alleles: 100 (a-) 2 alleles: 175:75 fh 0-1 alleles: 100 (l, --) 1-2 alleles: 200:175:75 (or, r-, no) 1-2 alleles: 300:150:75 (-x, ux, su) p2 bistriţei mountains, suceava county 5 1-2 alleles: 150:75 (a-, ac, -c) 0-1 alleles: 75 (-h) 0-1 alleles: 100 (l, -) 1-2 alleles: 175:75 (or, -r) 2 alleles: 300:150 (su) butiuc-keul a., crăciunaş c., goia i., farkas a., jarda l., cristea v. 184 acta bot. croat. 77 (2), 2018 relationships among inter-individual genetic distances within each species and among dianthus populations. additionally, divergence statistics were computed using the analysis of molecular variance (amova) implemented in arlequin 3.5 (excoffier and lischer 2010). results genetic polymorhism all loci amplified well in nearly all individuals tested. a total of 23 polymorphic alleles were amplified. the number of alleles per locus and the frequency in each species is shown in on-line suppl. tab. 2. the average number of alleles was 4.6. the genetic variation within populations is generally low. almost all the individuals belonging to a population share the same alleles. the allelic composition of each ssr locus based on the results of all five dianthus species is detailed in tab. 1. the most frequent allele in the locus bsy was b in all species except d. tenuifolius and the allele d had the lowest frequency, being present only in d. henteri individuals. in the locus dia the most frequent allele was h, while the alleles f and g had the lowest frequencies. in the locus dinca the most frequent allele was l and the least frequent was m. the most frequent allele in the locus dingsta was r and the lowest frequency was observed for the allele o. in the locus box the highest frequency was observed for allele x and the lowest frequency for allele z. the alleles b, l and v were present in most of the species, while the allele g was present only in d. callizonus. individuals of d. glacialis ssp. gelidus showed a particular combination of alleles in most of the loci, the alleles i and m. other alleles, j and r, were present only in some individuals of d. henteri and d. tenuifolius. the individuals belonging to d. henteri species showed the particular allele d and the allele v which was present in d. callizonus as well. d. tenuifolius showed a particular combination of alleles, the allele a which was also present only in d. nardiformis and the allele c also present in d. callizonus. the alleles f, o and s, were present only in d. tenuifolius. in our study the gene diversity is low, ranging from 0.18 to 0.44, the lowest gene diversity being observed in d. callizonus (tab. 2). the proportion of the heterozygous individuals (observed heterozygosity) ranged between 0.3 and 0.57, the lowest value being observed again in d. callizonus. the distribution power of the ssr marker, estimated by the polymorphism information content (pic) value ranged between 0.14 (d. callizonus) and 0.34 (d. henteri) (tab. 2). high similarity among dianthus species collected from romania was observed in this study. allelic patterns across dianthus species, as revealed in fig. 2, indicate that the mean number of different alleles was highest in d. glacialis ssp. gelidus, while the number of effective alleles was found to be higher in d. tenuifolius. the amount of alleles with a frequency equal to or higher than 5% was similar in all five species, excepting for d. callizonus, where it was slightly lower. except for d. nardiformis, private alleles were detected in all dianthus populations. the strongest genetic divergence was observed among d. callizonus and d. tenuifolius. the number of locally common alleles with a frequency higher than 5% revealed that d. henteri and d. nardiformis share almost the same alleles in most of the loci. shannon’s information index (i) reveals low levels of genetic diversity in all populations, especially in d. callizonus (i = 0.251). d. henteri was characterized by the highest genetic diversity (i = 0.626). moreover, shannon’s index was closely related to the expected heterozygosity. a high similarity in allelic patterns was observed in case of d. henteri and d. tenuifolius. genetic structure and evolutionary analysis a neighbour-joining tree showed the evolutionary relationships of taxa (fig. 3). thus, some of the individuals belonging to d. henteri, d. glacialis and d. callizonus clustered independently while an overlapping distribution was observed in case of d. nardiformis and d. tenuifolius and other individuals of previous species. this distribution was confirmed by principal coordinate analysis (pcoa). this analysis shows a relatively high diversity of patterns in d. nardiformis and d. glacialis ssp. gelidus. consistent with the low levels of genetic diversity, a low variety of allelic profiles was observed in d. callizonus population (fig. 4). the first, the second and the third principal coordinates accounted for 32.27%, 18.69% and 11.99%, respectively, explaining for 62.96 of the total genetic variation across individuals belonging to different dianthus species. tab. 2. mean gene diversity (he), heterozygosity (ho) and polymorphism information content (pic) values of the 5 studied dianthus species. species he ho pic d. callizonus 0.18 0.3 0.14 d. glacialis ssp. gelidus 0.31 0.32 0.26 d. henteri 0.44 0.57 0.34 d. nardiformis 0.35 0.32 0.28 d. tenuifolius 0.4 0.47 0.32 fig. 2. distribution of allelic patterns across dianthus species. na – number of different alleles; na freq ≥ 5% – number of different alleles with a frequency ≥ 5%; ne – number of effective alleles; i – shannon's information index; no. private alleles – number of alleles unique to a single species; no. lcomm alleles – number of locally common alleles with a frequency ≥ 5% found in 50% or fewer species; he – expected heterozygosity. error bars represent standard deviations. genetic structure of populations of some dianthus species acta bot. croat. 77 (2), 2018 185 firmed the results of the overall amova and revealed that the greatest amount of genetic variance occurred among all five dianthus species (50.88%). genetic variance found within dianthus species was 49.12% (tab. 3). tab. 3. results of analysis of molecular variance (amova) based on fst from data of five microsatellite loci and gini-simpson index of dianthus species. source of variation degrees of freedom sum of squares variance components percentage variation (p < 0.001) amova analysis for all loci among species 4 66.876 0.748 51.48 within species 105 74.042 0.705 48.52 locus by locus amova among species 83.317 0.961 50.88 within species 94.336 0.927 49.12 fig. 3. evolutionary relationships of dianthus taxa in romania. fig. 4. a two-dimensional plot of the principal coordinate analysis (pcoa) of ssr data showing the clustering of five dianthus species in romania. analysis of molecular variance (amova) for all loci showed that the highest amount of genetic variance (51.48%) occurred among dianthus species and a variance of 48.52% was found within the species. locus-by-locus amova condiscussion genetic diversity of romanian dianthus taxa molecular investigation of dianthus species from europe has recently aroused interest in several aspects. analysis of the genetic diversity and structure of populations of endangered, rare or endemic species of dianthus are useful tools in conservation planning. molecular analysis could aid especially in the case of plant species capable of clonal propagation as dianthus. in many cases, endangered, rare or endemic species with large populations can be genetically similar or identical. in spite of the high number of individuals, the low genetic diversity variation is due to clonal multiplication or bottlenecks. thus, data about genetic structure and variability should be useful before the development of proper conservation strategies. there are few data about molecular analysis of rare or endemic dianthus species (cristea et al. 2014, jarda et al. 2014) despite the numerous data regarding the conservation of these species. in romania these plant species were preserved by in vitro cultures (butiuc-keul et al. 2001, miclăuş et al. 2003, cristea et al. 2006, 2010, marcu et al. 2006, jarda et al. 2011, 2014). our study revealed the genetic diversity structure of 5 endangered or endemic species of dianthus found in different sites in romania. most of these locations have been designated natura 2000 sites. by the use of ssr markers developed with the 5 primer pairs tested in this study, 10–11 alleles from a total of 23 polymorphic alleles amplified were identified in most of the species. the population of d. callizonus showed the lowest diversity, probably as a result of limited distribution (this is a local endemic species). according to these results, d. callizonus conservation should be a priority for the administration of the piatra craiului natural park. a single allele was identified in each of the loci ms-dincaracc, ms-dingsta and ms-dinmadsbox and 2 alleles were identified in loci ms-dcamcrbsy and ms-dcdia30. the highest diversity was revealed in the populations of d. glacialis ssp. gelidus, especially in locus ms-dinmadsbutiuc-keul a., crăciunaş c., goia i., farkas a., jarda l., cristea v. 186 acta bot. croat. 77 (2), 2018 box (4 from the 6 alleles observed). this highest diversity can be explained by its larger distribution area. regarding the genetic diversity among populations of the same species, no differences were revealed by ssr markers tested in the present study. the highest genetic diversity was observed in d. glacialis ssp. gelidus and d. tenuifolius in locus ms-dinmadsbox. the genetic diversity is generally low in the dianthus species studied here. this may be related to several factors. mountain and alpine plants have to cope with harsh environmental conditions, e.g. short vegetation periods, unstable and low-fertility soils, desiccating winds, and high solar radiation, thus a population with low genetic diversity has limited possibilities to fight against harsh environmental conditions. in the short term, a loss of genetic diversity can reduce plant performance and population viability and may limit the potential for further adaptive evolution (ellstrand and elam 1993). low genetic diversity could also be explained by the clonal propagation of many plants, as in dianthus species grown in severe environmental conditions (stöcklin 1992). clonal propagation usually produces a rapid, but spatially limited spread of genotypes. along an altitudinal gradient, the vegetative growth is increased, because reproduction by seeds may be hampered by the harsh alpine conditions (young et al. 2002, gabel et al. 2017). the low values of gene diversity, heterozygosity and pic found in our study are in accord with other data showing low diversity and polymorphism of dianthus species from the iberian peninsula (balao et al. 2010) and iran (farsi et al. 2013). these data seem to suggest that dianthus species may be in general very young and the romanian species are not an exception to the pattern. it is well known that small populations isolated in fragmented habitats contain less genetic diversity than larger populations because over time, individuals will become more homozygous due to the low amounts of available genetic diversity within the population and increased inbreeding (duminil et al. 2007). there are also complex interactions between small population size, genetic diversity and individual fitness (aguilar et al. 2008) with several consequences, such as inbreeding depression (jolivet et al. 2013). human activity and land use changes was followed by smallscale fragmentation of grassland habitats in most regions of central europe, thus small populations are facing extinction due to local maladaptation in remnant habitats (busch et al. 2016). in romania, populations of these endangered species of dianthus seems to be also such small populations with low adaptation to the habitats. on the other hand in the case of d. gratianopolitanus, genetic variation within populations seems to be much more affected by population density and size than by isolation. thus, genetic variation decreases with increasing population density across all studied populations, which may be due to the effect of gene flow within populations, which should be stronger in dense populations. genetic variation was higher among populations of d. gratianopolitanus in germany than in switzerland. in the german region with a higher magnitude of isolation, genetic variation within populations depended on population size. in the swiss study region, with a lower magnitude of isolation, the higher genetic variations may be attributed to the gene flow among populations even within smaller populations (putz et al. 2015). other studies showed that the historical landscape structure may be more important for genetic diversity than present habitat conditions. thus, populations that were persisting in abandoned grassland fragments significantly influence the genetic variability of those species even under deteriorating habitat conditions. all of these studies may lead to the conclusion that individuals from different populations should be included in approaches to preserve the genetic variation of such plant species (reisch et al. 2017). evolutionary framework and genetic variation of the studied carnations dianthus genus is characterized by its extensive morphological and genetic variability (erhardt 1990, 1991, friedman et al. 2001, bloch et al. 2006). many phylogenetic studies revealed that evolutionary radiations have occurred in dianthus (balao et al. 2010, valente et al. 2010). polyploidy, hybridization, and genome duplication, are common evolutionary forces in plants and act as potential drivers of plant radiation (otto 2007, paun et al. 2009, soltis and soltis 2009). although interspecific hybridization is feasible in several clades of dianthus (collin and shykoff 2003), it seems, however, that the incomplete lineage sorting and/or ancestral polymorphism processes have played a prominent roles in its recent evolutionary history (balao et al. 2010, valente et al. 2010). hybridization is exceptionally high in dianthus compared to many genera, partly in consequence of the recent origin of most species. high rates of hybridization have possibly contributed to fast diversification by providing evolutionary potential via lineage fusions and may explain the shared alleles. one of the phylogenetic studies of the genus revealed that rates of diversification in mediterranean dianthus have been exceptionally high (valente et al. 2010) and suggest that a combination of polyploidy and geographical speciation may have driven cladogenesis in the group. the major patterns of variation within and among populations of particular species on mountains have not been clearly described to date, and thus it has been difficult to discuss our results of specific case studies in relation to general patterns. however a recent study based on morphological and molecular data and analyses suggest that dianthus binaludensis, distributed on binalud mountain, northeast iran, also displays a local morphological divergence (farsi et al. 2013). in conclusion, the gene diversity is low in all of the five endemic or rare species of dianthus. the genetic structure is similar in their different populations, d. callizonus and d. tenuifolius show particular sets of ssr alleles, the other 3 species of d. glacialis ssp. gelidus, d. henteri and d. nardiformis share almost the same alleles in most of the loci. our results of evolutionary neighbour-joining analysis show overlapping distribution in the case of d. nardiformis, d. tenuifolius and several individuals of d. henteri, d. glacialis and d. callizonus. genetic structure of populations of some dianthus species acta bot. croat. 77 (2), 2018 187 this phenomenon could occur from the evolutionary processes in recently differentiated species such as dianthus species (balao et al. 2010, valente et al. 2010). from these data we suggest that d. henteri, d. glacialis have not yet fully diverged. although these two species clustered independently, several individuals clustered together with other species. acknowledgments this work was supported by the projects 31-008/2007 and 71/2014, partnerships in priority areas – pn ii, financed by romanian ministry of national education (cnmp and 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(asteraceae). biological conservation 106, 71–78. zietkiewicz, e., rafalski, a., labuda, d., 1994: genome fingerprinting by simple sequence repeat (ssr)-anchored polymerase chain reaction amplification. genomics 20, 176–183. opce-str.vp acta bot. croat. 69 (1), 47–64, 2010 coden: abcra 25 issn 0365–0588 comparative study of salvia limbata c.a. and s. palaestina bentham (sect. aethiopis bentham, labiatae) from east anatolia, turkey ahmet kahraman*, musa dogh an middle east technical university, department of biological sciences, 06531 ankara, turkey morphological characteristics of stems, leaves, bracts, calyces and corollas are taxonomically discriminating characters in salvia. in this paper we present morphological, anatomical and ecological features of s. limbata c.a. meyer and s. palaestina bentham as well as micromorphological characteristics of their pollen grains and nutlets using scanning electron microscopy. anatomical characters such as size of cortex and vascular tissue, number of palisade parenchyma rows and vascular bundles are found to be important species specific characters. pollen grains in the species are different in shape and size, but they look similar in their exine sculpturing; shape, size and ornamentation of nutlets are found to be different. the two investigated salvia species grow on clayey-loamy and loamy soils, with ph 7.6–7.9, with 0.4–2.1% of organic matter, 0.006–0.026% of total salt content, 4.2–21.0 mg kg–1 phosphorus and 87.0–445.8 mg kg–1 potassium. key words: morphology, anatomy, ecology, palynology, nutlet, salvia limbata, salvia palaestina introduction the family labiatae includes 250 genera and about 7000 species distributed all over the world (thorne 1992). it is the third largest family in turkey with 45 genera and 574 species, 256 of which are endemic. the rate of endemism is 44.5% in this family (davis 1965–1985, güner et al. 2000). the genus salvia l. represents a cosmopolitan assemblage of nearly 1000 species, distributed in three regions of the world: central and south america (500 species), western asia (200 species) and eastern asia (100 species) (walker and sytsma 2007). the lever mechanism regulates pollination, selection and evolution in the group (claßen-bockhoff et al. 2004). the genus has been the subject of a number of studies on morphology (hedge 1982), anatomy (metcalfe and chalk 1972, kahraman et al. 2009a, 2010a, b), palynology (erdtman 1945, cantino et al. 1992) and nutlet micromorphology (marin et al. 1996). acta bot. croat. 69 (1), 2010 47 * corresponding author, e-mail: ahmetk@metu.edu.tr u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:20 color profile: disabled composite 150 lpi at 45 degrees as many as 107 species of salvia has been recognized in the flora orientalis, 75 of which were recorded from turkey (boissier 1875). these species have been placed under seven sections using bentham’s (1833) sectional delimitation. these sections are as follows: eusphace benth., hymenosphace benth., aethiopis benth., plethiosphace benth., horminum benth., drymosphace benth. and hemisphace benth. after that sect. eusphace was changed to sect. salvia (hedge 1972). salvia limbata and s. palaestina were placed in the section aethiopis (bentham 1833, boissier 1875). section aethiopis comprises biennial or perennial herbs or chamaephytes. the characteristic features of the section are tubular or campanulate calyx, more or less falcate upper lip of corolla and not annulate corolla tube. staminal connectives are longer than filaments, arms are unequal, the sterile shorter and more or less flattened distally. the first revision of salvia in turkey was made by hedge (1982), who recognized 87 species, one of them however being doubtful. between 1982 and 2005, four more species, s. nydeggeri (huber-morath 1982), s. aytachii (vural and adigüzel 1996), s. hedgeana (dönmez 2001), s. anatolica (hamzaogh lu et al. 2005), were published from turkey. since 2005, as a part of a revision of the genus salvia in turkey, the authors have carried out extensive field studies and collected a large number of specimens. population size, habitat, phenological and ecological properties of the species were observed and their gps coordinates and photographs were taken in the field. the studies have revealed two new species, s. marashica (i · lçi · m et al. 2009) and s. ekimiana (celep and dogh an 2010) and new records of s. macrosiphon boiss. (kahraman et al. 2009b) and s. viscosa jacq. (celep et al. 2009). salvia limbata and s. palaestina grow in eastern anatolia and have similar morphological features and confused taxonomy. in this paper for the first time we give descriptions of s. limbata and s. palaestina, their comparative root, stem, leaf and petiole anatomy, palynology and nutlet micromorphology. materials and methods specimens of s. limbata and s. palaestina were collected from six localities in turkey (tab. 1, fig. 1). the specimens have been stored in the middle east technical university (metu) department of biological sciences and ankara university herbarium (ank). morphological studies were carried out on living as well as herbarium materials. 25 individuals of each species and their 25 characters were studied and measured so as to determine their morphological characteristics using a leica s8ap0 stereomicroscope. minimum and maximum ranges of measured characters are presented. anatomical studies were carried out on specimens kept in 70 % alcohol. the paraffin method was used for the transverse sections of the root, the stem, the leaf and the petiole, and surface sections of leaves. the specimens were embedded in paraffin and then sectioned with a leica rm2125rt rotary microtome. all sections were stained with safranin and fast green and then mounted in canada balsam or entellan (johansen 1944). measurements and photographs were taken using a leica dm1000 binocular light microscope and a leica dfc280 camera. for palynological investigations, pollen material was obtained from herbarium samples. the pollen slides were prepared according to wodehouse (1935) technique. measurements and observations were made using the leica dm1000 binocular light micro48 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:20 color profile: disabled composite 150 lpi at 45 degrees scope and leica dfc280 camera. polar length, equatorial length, colpus length, exine and intine thickness for 30 pollen grains were measured under the light microscope (magnification of 1000´). pollen grain exine sculpturing was observed using a jeol-6060 scanning electron microscope. the pollen terminology of faegri and iverson (1975) has been used. the nutlets were examined using the leica s8ap0 stereomicroscope to ensure their size and maturity. in order to determine the average seed sizes, 30 mature nutlets were measured. for sem, the mature nutlets were placed on stubs directly and covered with gold (dogh an 1988). after that, they were observed and photographed with a jeol jsm-6400 sem. for ecological studies, soil samples were taken from suitable habitats of s. limbata and s. palaestina. all soil samples were analyzed at the soil, fertilizer and water resources central research institute, ankara. soil texture, organic matter, total salt, ph, caco3, p and k analysis were made using standard techniques (bayrakli 1987) and the results have been evaluated according to kaçar (1972). acta bot. croat. 69 (1), 2010 49 a comparative study of salvia sect. aethiopis (labiatae) tab. 1. collection data of salvia limbata and s. palaestina from turkey. collection data and collector number s. limbata b8 erzurum: ilica to erzurum, 5 km to erzurum, 38° 55’ 00’’ n 41° 12’ 42’’ e, 1817 m, 10.7.2006, akahraman 1293. b9 van: bahçesaray to van, near naren village, 38° 09’ 568’’ n 43° 01’ 452’’ e, 2281 m, 9.7.2007, akahraman 1437. c10 hakkari: hakkari to van, 1 km to van city border, 37° 48’ 015’’ n 44° 05’ 274’’ e, 1839 m, 7.6.2008, akahraman 1571. s. palaestina b6 malatya: kangal to hekimhan road, 39° 02’ 132’’ n 37° 44’ 240’’ e, 1460 m, 2.6.2008, akahraman 1528b. b8 diyarbakir: lice to diyarbakir, 65 km to diyarbakir, 38° 18’ 675’’ n 40° 31’ 895’’ e, 789 m, 4.6.2008, akahraman 1533a. c9 şirnak: 5 km from şirnak to cizre, 37° 30’ 891’’ n 42° 25’ 613’’ e, 1090 m, 5.6.2008, akahraman 1544. fig. 1. distribution map of salvia limbata (�) and s. palaestina (�) in turkey. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:20 color profile: disabled composite 150 lpi at 45 degrees results morphological characteristics stems of salvia limbata c.a. meyer (synonym: s. chrysadenia freyn) are 30–120 cm. the stem indumentum is retrorsely scabridulous below and with sessile glands above. leaves are (3.5–) 5–16 ´ (2–) 4–10 cm, ovate-oblong and erose-dentate. the leaf indumentum is glabrous and glandular-punctate below, and sparsely pilose above. petiole is 2–12 cm. inflorescence is 15–80 cm. verticillasters are 2–6 (–8)-flowered. bracts are 2–7 mm, pale green. pedicels are 2–6 mm. calyces are ovate-campanulate, 6–10 mm in flower and 11–13 mm in fruit. the calyx indumentum is eglandular scabrid or densely pilose with hairs with many sessile glands. its upper lip is shortly tridentate and recurved. corolla is 15–25 mm and white with pale yellow lip. corolla tube is 5–9 mm and squamulate. filaments are 3–4 mm. fertile anthers are 3–4 mm. upper thecae are 15–20 mm and lower thecae are 1–2 mm. style is 35–40 mm (fig. 4). stems of salvia palaestina bentham (synonym: s. lorentii hochst.) are 20–65 cm. the stem indumentum is hirsute with long flattened eglandular hairs below and densely glandular pilose (sometimes with long flattened eglandular hairs) above. leaves are 4.5–15 (–20) ´ 1.5–7 (–9) cm, oblong to ovate and erose. the leaf indumentum is tomentose. petiole 2.5–15 cm. inflorescence is 10–50 cm. verticillasters are (2–) 3–6-flowered. bracts are 15–25 ´ 10–20 mm, often tinged pink or purple. pedicels are 2–5 mm. calyces are almost tubular, often pink or purple (rarely green), 12–16 mm in flower and 17–25 mm in fruit. the calyx indumentum is papillose-glandular with some longer hairs. its upper lip is equally tridentate and spinulose. calyx teeth are 2–4 mm, corolla 20–35 mm long, lilac or whitish-lilac. corolla tube is 10–20 mm long and not squamulate. filaments are 2–3 mm long. fertile anthers are 3–4 mm. upper thecae are 12–15 mm and lower thecae are 1–2 mm. style is 25–45 mm (fig. 5). 50 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. fig. 2. cross-sections of root. a–b. s. limbata, c–d. s. palaestina. p – periderm, c – cortex, sc – sclerenchyma, ph – phloem, x – xylem, pr – pith ray, pi – pith. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:23 color profile: disabled composite 150 lpi at 45 degrees anatomical characteristics root anatomy: cross-sections taken from the root of salvia limbata have revealed that the periderm layer on the outermost surface of the root is thin and its cells are irregular. a multilayered parenchymatic cortex is present under the periderm. there are several sclerenchyma groups above the phloem. the xylem (800–1300 mm) is composed of vessels and tracheids. those cells underlying the xylem are thick-walled and narrower. pith rays comprise 2–6-rowed rectangular cells. the pith consists of polygonal or orbicular parenchymatous cells (fig. 2a–b, tab. 2). cross-sections taken from the root of salvia palaestina have showed that the multilayered periderm layer on the outermost surface of the root is thick and its cells are squashed or breaking up. under the periderm, there is a parenchymatic cortex with a few layers. phloem and xylem are distinguished from one another by sclerenchyma and ground tissue. phloem is embedded in sclerenchyma tissue as several groups. the xylem (1500–2000 mm) consists of regular vessels and tracheids. pith rays are composed of 1–8(–10)-rowed rectangular cells. the pith comprises polygonal, oval or orbicular parenchymatous cells (fig. 2c–d, tab. 2). stem anatomy: cross-sections taken from the stem of salvia. limbata have exhibited a monolayer epidermis covered by an undulate cuticle. the epidermis is composed of oval, acta bot. croat. 69 (1), 2010 51 a comparative study of salvia sect. aethiopis (labiatae) tab. 2. comparative anatomy of root, stem, leaf and petiole of s. limbata and s. palaestina. s. limbata s. palaestina width (mm) length (mm) width (mm) length (mm) min. – max. min. – max. min. – max. min. – max. root periderm cell 15–50 10–45 30–100 30–70 cortex cell 30–50 10–20 25–50 15–20 pith ray 15–20 15–40 10–35 15–60 stem cuticle 4–7 5–7 epidermis cell 15–55 10–20 20–45 10–20 cortex cell 30–120 25–80 30–105 20–65 trachea cell 20–40 30–50 25–85 25–110 pith cell 50–180 50–150 45–150 50–150 leaf cuticle 3–7 1.5–4 upper epidermis cell 25–65 15–35 15–55 10–25 lower epidermis cell 10–30 7–25 15–50 10–25 palisade parenchyma 11–16 25–60 10–15 20–50 spongy parenchyma 10–16 15–20 8–20 10–20 petiole abaxial epidermis 15–25 10–20 15–25 15–20 adaxial epidermis 10–20 10–20 15–30 15–20 cortex cell 30–105 30–115 30–125 30–130 u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:23 color profile: disabled composite 150 lpi at 45 degrees ovate or rectangular cells. underneath the epidermis, multilayered collenchyma cells (60–90 mm) are located at the corners and there are 1–2 rows of chlorenchyma cells between them. the cortex tissue (250–400 mm) consists of 7–10 layers of oval, ovate or orbicular parenchymatous cells. the phloem (80–120 mm) is surrounded by more or less sclerenchymatous fibers. cambium is distinguishable. the xylem (500–1150 mm) considerably bulges at ridges. the pith comprises hexagonal or orbicular parenchymatous cells with intercellular spaces (fig. 3a, tab. 2). 52 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. fig. 3. cross-sections of stem. a. s. limbata, b. s. palaestina. e – epidermis, gh – glandular hair, eg – eglandular hair, co – collenchyma, c – cortex, sc – sclerenchyma, ph – phloem, x – xylem, pi – pith. fig. 4. general appearance of s. limbata. scale bar: 2 cm. fig. 5. general appearance of s. palaestina. scale bar: 2 cm u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:28 color profile: disabled composite 150 lpi at 45 degrees cross-sections taken from the stem of s. palaestina display a monolayer epidermis covered by an undulate cuticle. the epidermis consists of oval or rectangular cells. multilayered collenchyma cells (70–130 mm) are located at the corners. there are 1–3 rows of chlorenchyma (20–30 mm) with a large number of chloroplasts between the corners. the cortex (150–200 mm) is composed of 4–7 layers of oval or orbicular parenchymatous cells. the phloem with less or more sclerenchymatic fibers measures 25–55 mm. cambium is not clearly distinguishable. size of the xylem is 90–300 mm and it immediately bulges at the ridges. the pith region comprises large hexagonal, polygonal or circular parenchymatous cells (fig. 3b, tab. 2). leaf anatomy: cross-sections of the lamina and surface sections of upper and lower epidermises of s. limbata have showed that both epidermises are covered with eglandular hairs and sessile glands and they consist of uniseriate rectangular or oval cells with undulate cuticles. cells of the upper epidermis are clearly larger than the lower. the leaf is equifacial and amphistomatic, with diacytic stoma type. stomata on the lower surface show higher frequencies than those on the upper surface. on the upper surface, the length of stomata varies from 26–29 mm while the width of stomata ranges from 15–20 mm. on the lower surface, the length of stomata varies from 22–30 mm whereas the width of stomata ranges from 16–20 mm. mesophyll (135–220 mm) comprises elongated palisade and isodiametric spongy parenchyma cells. palisade parenchyma is 2–3-rowed under the upper and lower epidermis. spongy parenchyma is 1–2-rowed in the middle. the midrib region, which forms a projecting part, comprises 1–3 layers of collenchyma adjacent the epidermal cells. a single large vascular bundle is located in the center (fig. 6a–d, tab. 2). cross-sections of the lamina and surface preparations of both epidermises of s. palaestina have revealed that the upper and lower epidermises are covered with glandular and eglandular hairs and they are composed of uniseriate rectangular or oval cells with undulate cuticles. cells of the upper epidermis cells are nearly equal to the lower. the equifacial leaf is amphistomatic, with diacytic stoma type. number of stomata on the lower surface is nearly equal to that of stomata on the upper surface. on the upper surface, the length of stomata varies from 38–45 mm while the width of stomata ranges from 30–36 mm. on the lower surface, the length of stomata varies from 34–43 mm whereas the width of stomata ranges from 23–30 mm. mesophyll region (60–180 mm) consists of 1–2 layers of elongated palisade parenchyma cells found on both sides of 1–2 layers of isodiametric spongy parenchyma cells, occupying a small part in the middle. the midrib region, which forms a projecting part, comprises 2–3 collenchyma layers below the epidermal cells. there is a single large vascular bundle in the center (fig. 6e–h, tab. 2). petiole anatomy: in cross-sections taken from the petiole of s. limbata it has been observed that the epidermal cells of both surfaces are oval, rectangular or squarish epidermal cells. the adaxial epidermis cells are slightly larger than the abaxial epidermis cells. there are several layers of collenchyma cells under the epidermis. four broad vascular bundles are located in the middle. also, there are four small vascular bundles in each of the petiolar wings. vascular bundles are of collateral type. the sclerenchyma tissue is well developed outside of the phloem and the xylem (fig. 7a–b, tab. 2). cross-sections taken from the petiole of s. palaestina showed that the epidermal cells of both surfaces are oval, rectangular or squarish epidermal cells. adaxial and abaxial epidermis cells are equal in size. 2–5-layered collenchyma are located under the epidermis. acta bot. croat. 69 (1), 2010 53 a comparative study of salvia sect. aethiopis (labiatae) u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:28 color profile: disabled composite 150 lpi at 45 degrees two or three broad vascular bundles are located in the middle. moreover, there are two small vascular bundles in each of the petiolar wings. the vascular bundle tissue surrounded by parenchymatic cells seems as a shallow crescent and collateral. there are several sclerenchymatous cells on the phloem (fig. 7c–d, tab. 2). 54 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. fig. 6. cross and surface sections of leaf. a–d. s. limbata, e–h. s. palaestina. cu – cuticle, eg – eglandular hair, ue – upper epidermis, le – lower epidermis, pc – parenchyma cell, x – xylem, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:29 color profile: disabled composite 150 lpi at 45 degrees ecological characteristics both salvia limbata and s. palaestina prefer clayey-loamy and loamy soils that are slightly alkaline. the organic matter and caco3 content of the soil of s. limbata are 2.07–2.10% (a middle level) and 0.94–16.90% (a very small to a high level), whereas the organic matter and caco3 content of the soil of s. palaestina are 0.44–2.04% (a very low to middle level of organic matter) and 3.89–72.84% (a small to a very high level) (tab. 3). acta bot. croat. 69 (1), 2010 55 a comparative study of salvia sect. aethiopis (labiatae) fig. 7. cross-sections of petiole. a–b. s. limbata, c–d. s. palaestina. ad – adaxial epidermis, ab – abaxial epidermis, co – collenchyma, pc – parenchyma cell, vb – vascular bundle, x – xylem, ph – phloem, sc – sclerenchyma. tab. 3. soil features of s. limbata and s. palaestina. parameter s. limbata s. palaestina sand (%) 43.9–46.6 23.0–42.8 silt (%) 25.9–30.5 31.3–38.2 clay (%) 22.9–30.2 25.9–38.8 texture clayey-loamy and loamy clay loam and loamy ph 7.56–7.93 7.60–7.82 organic matter (%) 2.07–2.10 0.44–2.04 total salt (%) 0.014–0.016 0.006–0.026 total caco3 (%) 0.94–16.90 3.89–72.84 p (ppm) 9.36–16.00 4.21–21.01 k (ppm) 113.55–296.31 87.00–445.84 u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:31 color profile: disabled composite 150 lpi at 45 degrees salvia limbata shares its habitat with mainly herbaceous plants including s. nemorosa l., s. poculata náb, s. xanthocheila boiss. ex bentham, s. macrochlamys montbret et aucher ex bentham, s. atropatana bunge, s. syriaca l., s. multicaulis vahl, s. aethiopis l., s. virgata jacq., s. verticillata l. subsp. verticillata and amasiaca (freyn et born.) bornm., senecio sp., euphorbia sp., vicia sp., dactylis sp., verbascum sp., hordeum sp. and centaurea sp. other species growing in association with s. palaestina are s. cryptantha montbret et aucher ex bentham, s. euphratica montbret et aucher ex bentham var. euphratica and var. leiocalycina (rech. fil.) hedge, s. candidissima vahl subsp. candidissima, s. ceratophylla l., s. syriaca l., s. multicaulis vahl, s. aethiopis l., s. virgata jacq., s. verticillata l. subsp. amasiaca (freyn et born.) bornm., tripleouspermum sp., crupina sp., psorolea sp., phlomis sp., crepis sp., tragopogon sp., avena sp., trigonella sp., torillis sp., aegilops sp., mellilotus sp., alopecurus sp., scabiosa sp. and alkanna sp. pollen characteristics shape of pollen grains of s. limbata is oblate-spheroidal (fig. 6a–b). polar axis is 52.41 ± 4.69 mm and its equatorial axis is 59.82 ± 4.89 mm. the ratio of p/e is 0.88. colpus length is 49.25 ± 4.78 mm and colpus width is 7.57 ± 1.61 mm. the exine thickness is 1.33 ± 0.20 mm and the intine thickness is 0.55 ± 0.09 mm (tab. 4). the exine sculpturing is bireticulate-perforate (fig. 8c–d). shape of pollen grains of s. palaestina is prolate-spheroidal (fig. 9a–b). polar axis is 47.72 ± 3.85 mm and equatorial axis is 45.40 ± 3.73 mm. the ratio of p/e is 1.05. colpus length is 42.07 ± 3.99 mm and colpus width is 3.79 ± 0.72 mm. the exine thickness is 1.37 ± 0.21 mm and the intine thickness is 0.75 ± 0.03 mm (tab. 4). the exine sculpturing is bireticulate-perforate (fig. 9c–d). 56 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. tab. 4. pollen morphological data of s. limbata and s. palaestina. parameter s. limbata s. palaestina polar axis* 41.52–65.03 52.41 ± 4.69 40.00–51.96 47.72±3.85 equatorial axis* 50.16–74.10 59.82 ± 4.89 38.35–48.99 45.40±3.73 p/e 0.88 1.05 colpus length* 39.61–59.15 49.25 ± 4.78 33.08–46.24 42.07±3.99 colpus width* 5.25–11.78 7.57 ± 1.61 3.00–5.21 3.79±0.72 exine thickness* 1.03–1.67 1.33 ± 0.20 1.07–1.70 1.37±0.21 intine thickness* 0.42–0.70 0.55 ± 0.09 0.71–0.81 0.75±0.03 shape oblate-spheroidal prolate-spheroidal exine surface bireticulate-perforate bireticulate-perforate * range, mean and standard deviation u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:31 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (1), 2010 57 a comparative study of salvia sect. aethiopis (labiatae) fig. 8. lm and sem micrographs of the pollen grains of s. limbata. a–b equatorial and polar view in lm micrographs, c–d equatorial view and exine sculpturing sem. fig. 9. lm and sem micrographs of the pollen grains of s. palaestina. a–b equatorial and polar view in lm micrographs, c–d equatorial view and exine sculpturing in sem. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:34 color profile: disabled composite 150 lpi at 45 degrees nutlet micromorphology the nutlets of salvia limbata are ovate to oblong in their outline. mature nutlets of s. limbata are 2.5–3.0 mm long and 1.8–2.1 mm wide. their hilum diameter is 0.40–0.55 mm. the nutlet surface is glabrous and has irregular protuberances and undulating ridges (fig. 10 a–b). the mature nutlet is light brown. the nutlets of salvia palaestina are spherical in their outline. mature nutlets of s. palaestina are 2.4–2.8 mm long and 2.2–2.6 mm wide. their hilum diameter is 0.23–0.38 mm. the nutlet surface is glabrous and smooth to slightly papillaes (fig. 10 c–d). the mature nutlet is light brown. habitat and phenology salvia limbata grows on stony slopes, roadsides, steppe and cornfields between 1060– 2400 m. it flowers in june to august. salvia palaestina grows on limestone and igneous rocky slopes, in quercus scrub, vineyards, fallow fields and roadsides between 300 and 1460 m. it flowers in april to july. discussion the main morphological characters useful for salvia species determination are stem, leaf and calyx indumentum, bract and calyx length, bract and corolla colour. the stem texture of s. limbata is retrorsely scabridulous below and with sessile glands above, whereas 58 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. fig. 10. sem photos of the nutlets of salvia limbata and s. palaestina. a–b general appearance and surface of the nutlets of s. limbata, c–d general appearance and surface of the nutlets of s. palaestina. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:34 color profile: disabled composite 150 lpi at 45 degrees that of s. palaestina is hirsute with long flattened eglandular hairs below and often glandular pilose above. in s. limbata the leaf texture is glabrous and glandular-punctate below and sparsely pilose above, but in s. palaestina the leaf indumentum is tomentose. the calyx texture is eglandular scabrid or densely pilose in s. limbata but papillose-glandular in s. palaestina. the length of bracts and calyces in s. limbata are shorter than that of bracts and calyces in s. palaestina. in s. limbata bract is pale green and corolla is white with pale yellow lip whereas in s. palaestina bract colour is pink or purple and corolla colour is lilac or whitish-lilac. s. limbata has taller corolla tube, upper thecae and filaments than s. palaestina. morphological characters of s. limbata and s. palaestina are compared in tab. 5. our findings usually agree with the description of the flora of turkey (hedge 1982), but some differences were found here and so their descriptions were emended and expanded. it is reported that the stem length of s. limbata was 30–100 cm, its leaf size was 10–16 cm in length and 6–10 cm in width, its bract size was 2–6 mm, its corolla length was c. 25 mm, its calyx length was 8–10 mm in flower and to c. 12 mm in fruit while the stem length of s. palaestina was 30–60 cm, its leaf size was 5–13 (–20) cm in length and 1.5–7 (–8.5) cm in width, its bract size was c. 15 ´ 18 mm, its corolla length was c. 25 mm, its calyx length was c. 15 mm in flower and to c. 17 mm in fruit (hedge 1982). according to our study, the stem length of s. limbata was 30–120 cm, its leaf size was (3.5–) 5–16 cm in length and (2–) 4–10 cm in width, its bract size was 2–7 mm, its corolla length was 15–25 mm, its calyx length was 6–10 mm in flower and 11–13 mm in fruit, but the stem length of s. palaestina was 20–65 cm, its leaf size was 4.5–15 (–20) cm in length and 1.5–7 (–9) cm in width, its bract size was c. 15–25 ´ 10–20 mm, its corolla length was 20–35 mm, its calyx length was 12–16 mm in flower and 17–25 mm in fruit. additionally, inflorescence, corolla tube, filament, fertile anther, style, upper thecae and lower thecae features are presented the first time in this study. acta bot. croat. 69 (1), 2010 59 a comparative study of salvia sect. aethiopis (labiatae) tab. 5. diagnostic morphological features of salvia limbata and s. palaestina. character s. limbata s. palaestina stem indumentum retrorsely scabridulous below and with sessile glands above hirsute with long flattened eglandular hairs below and densely glandular pilose above leaf indumentum glabrous and glandular-punctate below and sparsely pilose above tomentose bract length (mm) 2–7 15–25 bract colour pale green pink or purple calyx length (mm) 6–10 in flower, 11–13 mm in fruit 12–16 in flower, 17–25 in fruit calyx indumentum eglandular scabrid or densely pilose hairs papillose-glandular corolla colour white with pale yellow lip lilac or whitish-lilac corolla length (mm) 15–25 20–35 corolla tube length (mm) 5–9 10–20 corolla tube shape squamulate not squamulate upper theca length (mm) 15–20 12–15 filament length (mm) 3–4 2–3 u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:34 color profile: disabled composite 150 lpi at 45 degrees root anatomy of the family labiatae (metcalfe and chalk 1972) is characterized by pith rays of roots composed of 2–12 or more rowed cells. our study shows that pith rays of s. limbata consist of 2–6 rowed cells while those of s. palaestina comprise 1–8 (–10) rowed cells. it was reported that s. macrochlamys boiss. et kotschy (sect. salvia) has mainly 1–4 rays of cells (kahraman et al. 2010a) and s. ballsiana (rech. fil.) hedge has 1–3 (–4) rays of cells (kahraman 2010b). the number of pith ray rows may be used to distinguish salvia species at the sectional level. in addition, xylem size is a diagnostic character in the two species. in s. limbata xylem size in is 800–1300 mm while in s. palaestina it is 1500–2000 mm. the stems of labiatae are rectangular, have well-developed collenchyma covering a broad area at the corners and have a sclerenchymatic tissue surrounding the vascular bundle tissue (metcalfe and chalk 1950). we found the same anatomical features in the stems of s. limbata and s. palaestina and also detected a few layers of chlorenchyma cells below the epidermis between the corners. however, vascular and cortex size are different in the two species. in s. limbata phloem is 80–120 mm in width while in s. palaestina phloem is 25–55 mm in width. xylem is 500–1150 mm in s. limbata, however, xylem is 90–300 mm in s. palaestina. the cortex tissue of s. limbata (250–400 mm) is larger than that of s. palaestina (150–200 mm). in addition, s. limbata has a 7–10-layered cortex while s. palaestina has a 4–7-layered cortex. mesophyll is completely parenchymatic and midrib is surrounded by collenchyma cells in salvia species (metcalfe and chalk 1972). we observed the same characteristics in the leaves of s. limbata and s. palaestina. however, the number of palisade parenchyma rows in the two species is different. the mesophyll of s. limbata is composed of 2–3 layers of palisade parenchyma cells on both sides while that of s. palaestina consists of 1–2 layers of palisade parenchyma cells. cells of the upper epidermis of s. limbata are clearly larger than the lower epidermis, but those of the upper epidermis of s. palaestina are almost equal to the lower epidermis. additionally, stomata of s. limbata (22–30 mm in length and 15–20 mm in width) are smaller than stomata of s. palaestina (34–45 mm in length and 23–36 mm in width) on both surfaces of leaves. the structure of the vascular bundles in the petiole structure of the family labiatae is a taxonomically significant character (metcalfe and chalk 1950). in the petiole of salvia limbata, there are four broad vascular bundles in the middle and also there are four small vascular bundles in each of the petiolar wings. in the petiole of s. palaestina, there are two or three large vascular bundles in the middle and also there are two small vascular bundles in each of the petiolar wings. s. ballsiana (kahraman 2010b) has a broad vascular bundle in its middle part of the petiole and four or six small bundles on its wings, and s. macrochlamys (kahraman et al. 2010a) has a single large vascular bundle in the center of the petiole and two small bundles its wings. to sum up, number of vascular bundles in the petiole can be used to distinguish the species. salvia limbata and s. palaestina grow on clayey loamy and loamy soils with slightly alkaline (ph 7.56–7.93) and a low or medium level of organic matter (0.44–2.10%). the amounts of p, k and total salt present vary between 4.21–21.01 ppm (enough to very much), 87.00–445.84 ppm (very much) and 0.006–0.026% (saltless to slightly salty), respectively. however, the soils of s. limbata (0.94–16.90%) are less limy than that of s. palaestina (3.89–72.84%). both the species generally shares their habitats with herbaceous plants. 60 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:34 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (1), 2010 61 a comparative study of salvia sect. aethiopis (labiatae) fig. 11. boxplots of the nutlet length, width and hilum diameter of salvia limbata and s. palaestina. nutlet length (a), width (b) and hilum diameter (c) of s. limbata. nutlet length (d), width (e) and hilum diameter (f) of s. palaestina. tab. 6. comparison of anatomical, palynological and nutlet characters of salvia limbata and s. palaestina. character s. limbata s. palaestina root xylem size in (mm) 800–1300 1500–2000 stem phloem size (mm) 80–120 25–55 xylem size (mm) 500–1150 90–300 cortex size (mm) 250–400 150–200 cortex layer 7–10-layered 4–7-layered leaf number of palisade parenchyma rows 2–3 1–2 size of stomata (mm) 22–30 ´ 15–20 34–45 ´ 23–36 upper epidermis versus lower epidermis large equal petiole number of vascular bundles 4 large in the center and 4 small in each wing 2–3 large in the center and 2 small in each wing pollen size (mm) 52.41± 4.69 ´ 59.82 ± 4.89 47.72 ± 3.85 ´ 45.40 ± 3.73 shape oblate-spheroidal prolate-spheroidal nutlet size (mm) 2.5–3.0 ´ 1.8–2.1 2.4–2.8 ´ 2.2–2.6 shape ovate to oblong spherical hilum diameter (mm) 0.40–0.55 0.23–0.38 ornamentation irregular protuberances and undulating ridges smooth to slightly papillaes u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:35 color profile: disabled composite 150 lpi at 45 degrees pollen characteristics of the family labiatae have considerable taxonomic importance (erdtman (1945). the classification of genera in labiatae has been revised (cantino et al. (1992), with salvia placed within the subfamily nepetoideae because it had hexacolpate pollen. light microscope observations in this study show pollen size and shape in different species is different. however, scanning electron micrographs reveal that their exine sculpturing is similar to each other. salvia limbata has a pollen size of 52.41 ± 4.69 ´ 59.82 ± 4.89 mm while s. palaestina has a pollen size of 47.72 ± 3.85 ´ 45.40 ± 3.73 mm (tab. 4). in addition, pollen shape is oblate-spheroidal in s. limbata but it is prolate-spheroidal in s. palaestina. the exine sculpturing of the two species are bireticulate-perforate. pollen size, shape and sculpturing of s. indica l. (section aethiopis) are 44.98 ± 4.22 ´ 52.24 ± 4.41 mm, suboblate and bireticulate-perforate, respectively (kahraman 2009a). s. anatolica hamzaoglu et a. duran and s. bracteata banks et sol. were observed to be oblate-spheriodal shaped pollen grains. the sculpturing in s. anatolica is euryreticulate, however the sculpturing in s. bracteata is suprareticulate (hamzaogh lu et al. 2005). seed surface micromorphology was found to have systematic value at generic and specific levels (hedge 1970, marin et al. 1996). the nutlets of s. sclarea were observed to be rounded-trigonous, c. 3 ´ 2 mm (hedge 1982) and its surface had protuberances (marin et al. 1996). the nutlets of s. verticillata l. (sect. hemisphace) were analyzed to be c. 2.2 ´ 1.3 mm (hedge 1982) and its surface had reticulo-papillosae (marin et al. 1996). according to our study, the nutlets of s. limbata are ovate to oblong, 2.5–3.0 mm long and 1.8–2.1 mm wide while the nutlets of s. palaestina are spherical, 2.4–2.8 mm long and 2.2–2.6 mm wide. additionally, hilum diameter in the former species is 0.40–0.55 mm, however in the latter species it is 0.23–0.38 mm. the nutlet surface of s. limbata has irregular protuberances and undulating ridges in s. palaestina it is smooth to slightly papillaes. their seed length, width and hilum diameter are illustrated in figure 11. consequently, seed size, shape and ornamentation characters appear to have significant taxonomic value in distinguishing s. limbata and s. palaestina. diagnostic anatomical, palynological and nutlet characteristics of the two species are summarized (tab. 6). acknowledgements we wish to thank the scientific and technological research council (tübi · tak -tbag-104 t 450) for their financial assistance, professor sevil pehlivan and dr. birol baser for taking sem pictures and mr. ferhat celep making valuable suggestions. references bayrakli, f., 1987: analysis of soil and plant. ondokuz mayis university agricultural faculty publications, 17. university of samsun, samsun. bentham, g., 1833: labiatarum genera et species. ridgway, london. boisseir, e. p., 1875: flora orientalis. composees 3, 151–883. cantino, p. d., harley, r. m., wagstaff, s. j., 1992: genera of labiatae: status classification. in: harley, r. m., reynolds, t. (eds.), advanced in labiatae science. royal botanical gardens, kew. 62 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:35 color profile: disabled composite 150 lpi at 45 degrees celep, f., dogh an, m., 2010: salvia ekimiana (lamiaceae), a new species from turkey. annales botanici fennici 47, 63–66. celep, f., dogh an, m., duran, a., 2009: a new record for the flora of turkey: salvia viscosa jacq. (labiatae). turkish journal of botany 33, 57–60. claben-bockhoff, r., speck, t., tweraser, e., wester, p., thimm, s., reith, m., 2004: the staminal lever mechanism in salvia l. (lamiaceae): a key innovation for adaptive radiation?. organism diversity and evolution 4, 189–205. davis, p. h. 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(eds.), salvia l., 3, 188–192. cambridge university press, cambridge. hedge, i. c., 1982: flora of turkey and the east aegean islands. in: davis, p. h., edmondson, j. r., mill, r. r., tan, k. (eds.), salvia l., 7, 400–461. edinburgh university press, edinburgh. huber-morath, a., 1982: salvia nydeggeri hub.-mor. nova species section eusphace bentam. bauhinia 7, 181. i · lçi · m, a., celep, f., dogh an, m., 2009: salvia marashica (lamiaceae), a new species from turkey. annales botanici fennici 46, 75–79. johansen, d. a., 1944: plant microtechnique. mcgraw-hill, new-york. kaçar, b., 1972: plant feeding practical manual. ankara university agricultural faculty publications, ankara. kahraman, a., celep, f., dogh an, m., 2009a: morphology, anatomy and palynology of salvia indica l. (labiatae). world applied sciences journal 6, 289–296. kahraman, a., celep, f., dogh an, m., 2009b: a new record for the flora of turkey: salvia macrosiphon boiss. (labiatae). turkish journal of botany 33, 53–55. acta bot. croat. 69 (1), 2010 63 a comparative study of salvia sect. aethiopis (labiatae) u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:35 color profile: disabled composite 150 lpi at 45 degrees kahraman, a., celep, f., dogh an, m., 2010a: morphology, anatomy, palynology and nutlet micromorphology of salvia macrochlamys (labiatae) in turkey. biologia 65, 219–227. kahraman, a., dogh an, m., celep, f., akaydin, g., koyuncu, m., 2010b: morphology, anatomy, palynology and nutlet micromorphology of the rediscovered turkish endemic salvia ballsiana (lamiaceae) and their taxonomic implications. nordic journal of botany 28, 91–99. marin, p. d., duleti], s., petkovi], b., 1996: nutlet ornamentation in selected salvia l. species (lamiaceae). flora mediterranea 6, 203–211. metcalfe, c. r., chalk, l., 1972: anatomy of the dicotyledons, 2. clarendon press, oxford. thorne, r. f., 1992: classification and geography of the flowering plants. botanical review 58, 225–348. walker, j. b., sytsma, k. j., 2007: staminal evolution in the genus salvia (lamiaceae): molecular phylogenetic evidence for multiple origins of the staminal lever. annals of botany 100, 375–391. wodehouse, r. r., 1935: pollen grains. mcgraw-hill, new york. vural, m., adigüzel, n., 1996: a new species from central anatolia: salvia aytachii m. vural et n. adigüzel (labiatae). turkish journal of botany 20, 531–534. 64 acta bot. croat. 69 (1), 2010 kahraman a., dogh an m. u:\acta botanica\acta-botan 1-10\kahraman.vp 9. travanj 2010 12:26:35 color profile: disabled composite 150 lpi at 45 degrees 36 acta bot. croat. 77 (1), 2018 acta bot. croat. 77 (1), 36–44, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0002 issn 0365-0588 eissn 1847-8476 physiological performance of sunflower genotypes under combined salt and drought stress environment muhammad umar, zamin shaheed siddiqui* stress physiology lab., department of botany, university of karachi, karachi – 75270, pakistan abstract – the physiological performance of some sunflower genotypes (s.28111, sf0049, hysun-33, hysun-39) under salt, drought stress separately and in combination was examined. salt, drought and a combination of these stresses were applied to plants by gradual increments. the plants were exposed to stress for two weeks. relative water content, osmotic potential, stomatal conductance, performance index, dark adapted quantum yield and chlorophyll contents were reduced upon salinity and drought stresses. however, when plants were subjected to a combination of these stresses, a greater reduction in all tested attributes was observed. proline and carotenoid contents in drought stress were elevated compared to salt stress. superoxide dismutase (sod) and catalase (cat) showed the highest activity in individual salt and drought stress with less accumulation of h2o2. combined stress reduced the activity of antioxidant enzymes which ultimately decreased the physiological performance of sunflower plants. however, among the tested genotypes, s.28111 and sf0049 were found to be more tolerant to drought, salt and combined stress than both hysun genotypes. the physiological performance of genotypes against salinity and drought individually and in combination is discussed in detail. key words: combined stress, genotypes, photosynthesis, screening, single stress, sunflower * corresponding author, e-mail: zaminss@uok.edu.pk introduction the sunflower (helianthus annuus) is the most important source of edible oil and fourth largest oilseed crop in the world and its production is still expanding. it is planted over an area of 319,743 hectares producing 420,487 tons annually in pakistan (amanullah and khan 2011). sunflower is moderately tolerant to water stress, but its growth and production are limited in drought and salt stress environments (aziz et al. 2013). the growth and yield of sunflower are adversely affected by abiotic stress and its yield is reduced up to 60% (mazahery-laghab et al. 2003). it was observed that the general effects of salt and drought stress on plant are restricted to separate exposure of these stresses. however, physiological assessment of sunflower genotypes in a combination of salt and drought stress is rather scarce. further, the effect of combined stresses on sunflower genotypes and the basic physiological and biochemical mechanisms are still unclear. therefore, it is necessary to study the physiological performance of sunflower plants in conditions of combined salt and drought stresses. further, a selection of sunflower cultivars with better physiological responses to combined abiotic stresses would provide novel options to growers under different environmental conditions, as in nature plants are exposed to a combination of several stresses at a time. in the last few decades drastic climatic changes have been observed, probably due to global warming, which has brought about not only a serious environmental threat but also substantial reductions in the yield and the crop quality. plants are frequently exposed to many extremes such as drought stress, low/high temperature, salt stress, flooding stress, and heavy metal toxicity while growing in nature. among these, drought and salinity are the major environmental constraints that lead to detrimental effects on a plant’s life and hence crop productivity. about 33% of the world’s arable land has to face crop reductions due to cyclic or unpredictable drought, whereas salinity, a huge and worldwide problem, has affected about one billion hectares of land (wicke et al. 2011). salt and drought mailto:zaminss@uok.edu.pk plant response against combined stress acta bot. croat. 77 (1), 2018 37 stresses affect many morphological features and alter the physiological processes that are associated with plant growth and development (siddiqui et al. 2008). it was reported that in drought stress, plant growth retardation is linked with photosynthesis, respiration, and nutrient metabolism. further, production of reactive oxygen species (ros) in extreme environments like high salt concentration adversely affects plant growth and development (munns and tester 2008). to improve agricultural productivity, it is necessary to develop resistant varieties by an understanding of the physiological and biochemical mechanisms or selecting better genotypes capable of performing well in stress conditions. it was reported that changes undergone by plants as a result of combined stresses are markedly different to responses to individual stresses (grzesiak et al. 2016). different plant species and even genotypes may have varying responses to salt and drought stresses. therefore, the objectives of the present study were: 1) to assess the physiological performance of sunflower genotypes in salt and drought stresses separately or in combination, and 2) to identify a suitable genotype for a stress environment on the basis of its physiological and antioxidant performance. in this study, physiological and biochemical parameters used in the screening of salt or drought stress were selected (siddiqui et al. 2016) and their correlations are presented. this approach may be useful to identify physiologically tolerant genotypes of sunflower under water limited, salinity prone and combined stress environments. materials and methods plant material and experimental operation the experiment was carried out in a greenhouse at the department of botany, university of karachi, pakistan. plastic pots (15 × 18 cm) were filled with 1.5 kg of air dried soil. the soil types used in this trial were sandy loam, ph 7.5. seeds of helianthus annuus (sunflower cultivars: s.28111, hysun-33, hysun-39, and sf0049) were collected from the seed certification department of the government of pakistan. s.28111 and sf0049 are new sunflower genotypes originated by arysta life science and fmc corporation. physiological performance of these genotypes under stress environment was not available. however, genotypes hysun-33 and hysun-39 were known to moderately tolerate drought and salt without the physiological explanation for this having been reported. seeds were surface sterilized with 2% sodium hypochlorite for five minutes. five seeds per pot were sown and 12 days after sowing (das) pots were thinned to three seedlings per each pot. salinity and drought treatments were initiated 30 das and lasted till about 44 das. control plants were irrigated with tap water and soil was kept humid (more that 70% water holding capacity) until harvesting. for salinity, 175 mm nacl concentration (almost 17 ds m–1) was applied in gradual increments. to achieve the desired salt concentration, aliquots of the following nacl solutions were used in order: 75 mm, 50 mm and 50 mm (tab. 1). later, soil was kept humid, retaining more than 70% water holding capacity throughout the experimental periods (table 1). drought stress was imposed by withholding water until soil moisture content (smc) reached 20% and then maintained throughout the experiment. combined salt and drought stress was achieved by progressive imposition of 175 mm nacl i.e. 1st salt application was 75 mm, 2nd 50 mm and 3rd 50 mm and then withholding water until smc fell to 20%. soil moisture, electrical conductivity and ph were measured using soil sensor (sdi-12 hydra probe ii, stevens water, usa) and ph meter (adwa ad111, romania), respectively (tab. 1). the experiment was arranged in a randomized block design with four replicates for each treatment. at the end of the experiment, physiological measurements, biochemical quantification, and phenotypic characters were studied. chlorophyll fluorescence and stomatal conductance the stomatal conductance (gs) and chlorophyll fluorescence were recorded on the youngest fully expanded leaf between 9:00 am – 11:00 am using a steady state diffusion porometer, model sc-1 (decagon devices) and chlorophyll fluorescence meter (os-30p+, opti-science, usa) respectively. for chlorophyll fluorescence leaves were dark-adapted using clips. after half an hour of dark adaptation, the chlorophyll fluorescence parameters ratio of variable florescence to maximum florescence (fv/fm), which reflected maximum photochemical efficiency of ps-ii, and photosynthetic performance index (piabs) were recorded (maxwell and johnson 2000). afterwards, plants were harvested and biomass production, relative water content, free proline quantification, h2o2 content and antioxidant enzymes activity were examined. photosynthetic pigments fresh leaf samples (500 mg) were used for the extraction of pigments in 10 ml of 96% methanol, which was then centritab. 1. soil parameters: soil moisture contents (smc) and electrical conductivity (ec) during gradual increment of the soil salinity/ drought treatments. drought stress was imposed by withholding water. parameters of the last treatment were maintained until the time of harvesting. ± se indicates the standard error of mean. bold values represent the final parameters values of each treatment at the time of harvesting. stress type salt application nacl (mm) day smc (%) ec (ds m–1) ph salinity 1st 75 1st 80–90 7.4±0.08a 7.98 2nd 50 3rd 80–90 12.0±0.15b 8.04 3rd 50 5th 80–90 16.9±0.05c 8.09 drought --2nd 60–65 1.8±0.014a 8.01 --4rd 35–40 2.0±0.013ab 8.08 --6th 20–25 2.3±0.034b 8.13 salinity + drought 1st 75 1st 80–90 7.4±0.08a 8.01 2nd 50 3rd 80–90 12.0±0.15b 8.05 3rd 50 5th 80–90 16.9±0.05c 8.11 --7th 60–70 19.4±0.08d 8.18 --9th 40–50 21.1±0.12e 8.19 --12th 20–25 24.2±0.17f 8.24 umar m., siddiqui z. s. 38 acta bot. croat. 77 (1), 2018 fuged at 4000 rpm for 10 min. total chlorophyll, chlorophyll a, chlorophyll b and carotenoid contents were determined according to lichtenthaler (1987). the absorbance was read at 666, 653 and 470 nm using a spectrophotometer. the amounts of these pigments were calculated and expressed in µg mg–1 fresh weight (fw) (lichtenthaler and wellburn 1985): ca = 15.65 × a666 – 7.340 × a653 cb = 27.05 × a653 – 11.21 × a666 carotenoids = (1000 × a470 – 2.860 × ca – 129.2 × cb) / 245 where a = absorbance, ca = chlorophyll a content and cb = chlorophyll b content leaf water status leaf water status was evaluated as the leaf relative water content (rwc). rwc was determined from the youngest fully expanded leaves. the leaves were weighed immediately after harvesting to obtain the fresh weight (fw). the same leaves were then subsequently rehydrated in distilled water for 4 h to obtain the turgid weight (tw), and finally dried for 48 h at 72 °c to obtain the dry weight (dw) (barrs and weatherley 1962). rwc was calculated by the following formula with slight modification and expressed in percent (smart and bingham 1974): rwc = fw – dw / tw – dw × 100 osmotic potential (ψs) three randomly selected fully developed leaves of each treatment and control were taken and frozen in liquid nitrogen to measure ψs. the frozen leaves were thawed at room temperature and centrifuged at 12,000 g. finally the osmolarity of extracted sap was recorded using an osmometer type 6 (loser messtechnik, berlin, germany) and expressed in bars. hydrogen peroxide (h2o2) content h2o2 content was measured according to the procedure of velikova et al. (2000). 100 mg of fresh leaf samples were homogenized with 3 ml of 0.1% (w/v) trichloroacetic acid and then centrifuged at 12,000 g for 15 minutes. later, 0.5 ml of 10 mm potassium phosphate buffer (ph 7.0) and 1 ml of 1 m potassium iodide were added to 0.5 ml of the supernatant. finally, the absorbance was recorded at 390 nm. the amount of h2o2 was calculated using extinction coefficient (43.6 mm cm–1) and expressed as µmol g–1 fw. proline analysis proline was estimated according to the method of bates et al. (1973). fresh leaf samples (500 mg) were homogenized in 10 ml of sulphosalicylic acid (3% w/v) and the extract was filtered through whatman’s no. 2 filter paper. in a 2 ml aliquot, 2 ml of acid ninhydrin and 2 ml of glacial acetic acid were added and the contents were boiled for 1 h at 100 °c in a water bath. the reaction mixture was further extracted with 2 ml of toluene by mixing thoroughly with vigorous stirring for 15 to 20 sec. chromophore containing toluene was separated from the aqueous phase. later absorbance was recorded at 520 nm against toluene blank. proline content in sample was estimated by referring to a standard curve made from known concentrations of proline using the following formula: µmoles proline g–1 fw = {(µg proline / ml × ml toluene) / 115.5 µg / µmol} / (g sample) / 5 enzyme extraction 500 mg of leaf samples were collected and crushed in liquid nitrogen and then homogenized at 4 °c in 10 ml protein extraction buffer containing 100 mm tris-hcl (ph 6.8), 50 mg polyvinylpyrrolidone (pvp), and 0.1 mm edta. the contents were centrifuged at 14095 ×g for 15 min using smart r-17, hanil centrifuge. total protein was estimated by the method of bradford (1976). catalase (cat) activity was estimated by the method of patterson et al. (1984). the decomposition of h2o2 was measured at 240 nm taking the optical density at 240 nm as 43.6 mm cm–1 (extinction coefficient). reaction mixture (3.0 ml) consisted of 10.5 mm h2o2 in 0.05 m potassium phosphate buffer (ph 7.0) and the reaction was initiated after the addition of 0.1 ml enzyme extract at 25 °c. the decrease in absorbance at 240 nm was used to calculate the activity. one unit of cat activity is defined as the amount of enzyme that catalyzes the conversion of 1 mm of h2o2 min–1 at 25 °c. the assay for superoxide dismutase (sod) activity was performed by following the method of beyer and fridrovich (1987). the reaction mixture (27.0 ml) with ph 7.8 was prepared using 23.75 ml of potassium phosphate buffer (0.05 m), 1.5 ml of l-methionine (300 mg per 2.7 ml), 1.0 ml of nitroblue tertrazolium salt (14.4 mg per 10 ml), 0.75 ml of triton x-100. one ml aliquots of assay mixture were transferred into small glass tubes, followed by the 20 µl enzyme extract and 10 µl of riboflavin (4.4 mg per 100 ml) were added. the cocktail was mixed and then illuminated for 15 minutes in an aluminum foil-lined box, containing 25 w fluorescent tubes. in a control tube the sample was replaced by 20 µl of buffer and the absorbance was measured at 560 nm. the reaction was stopped by switching off the light and placing the tubes in the dark. increase in absorbance due to formation of formazan was measured at 560 nm. under the described conditions, the increase in absorbance in the control was taken as 100% and the enzyme activity in the samples was calculated by determining the percentage inhibition per minute. one unit of sod is the amount of enzyme that causes a 50% inhibition of the rate for reduction of nitro blue tetrazolium salt under the conditions of the assay. statistical analysis statistical analysis of the collected data was performed using the duncan (1955) multiple range test (p ≤ 0.05) and pearson’s correlation with the help of the personal computer software packages ibm spss statistics (version 20). to test the differences among mean values, duncan’s test was computed and the resultant values were expressed in a bar graph as alphabets. difference in means and their f-values were calculated and are given in table 2. plant response against combined stress acta bot. croat. 77 (1), 2018 39 results the application of single stress (drought and salt separately) and combined stress showed significant reduction in biomass production of each tested sunflower genotype. relative water content was substantially decreased in all treated genotypes under single and combined stress environment (fig. 1). however, in combined stress, 45, 56, 49 and 37% reduction were observed in s.28111, husun-33, hysun-39 and sf0049, respectively (tab. 2). leaf area of each plant was decreased in all treatments compared to control. reduction in leaf area was recorded up to 58, 59, 67 and 51% under combined stress in s.28111, hysun-33, hysun-39 sf0049, respectively (tab. 2). reduction in leaf number was the highest in hysun-33, whereas the lowest reduction was observed in sf0049 (fig. 1). salt and drought stress significantly reduced the height of all sunflower genotypes in comparison to corresponding controls. it was observed that s.28111 is the least affected among the genotypes (fig. 1). higher reduction in shoot height was observed in hysun-33 and hysun-39, of about 42 and 38% as compared to their controls, respectively. free proline production was significantly affected in the tested sunflower genotypes. it was observed that proline concentrations were higher in combined stress as compared to drought or salt stress (fig. 1). the order of proline accumulation in genotypes under combined stress varied for instance from the maximum in hysun-39>hysun-33>s.28111>sf0049 (minimum). sunflower genotypes exhibited a substantial decrease in piabs in all stresses as compared to control. combined stress significantly reduced the piabs in all the genotypes as compared to control and individual stresses (fig. 2). however, the highest decreased in piabs was shown in hysun-33 under tab. 2. effects of double stress on performance index (piabs), quantum yield (fv/fm ratio), stomatal conductance (gs), relative water content (rwc), total chlorophyll content (t.chl), leaf area (la), osmotic potential (ψs), catalase activity (cat), hydrogen peroxide (h2o2), photochemical quenching (qp), carotenoids contents (car), superoxide dismutase activity (sod) in sunflower genotypes expressed as increment or decline percentage over control. highest increment/decline is presented in bold. * denotes p-value <0.01, ** denotes p-value <0.001, ***denotes p-value <0.0001, ns – not significant. decline % (–) genotypes piabs f-value fv/fm f-value gs f-value rwc f-value t.chl f-value la f-value s.28111 31.8 16.4** 10.6 20.5*** 80.4 225*** 44.9 42.8*** 12.4 20.2*** 58.4 188*** hysun-33 52.1 20.6** 21.0 35.2*** 75.0 110** 55.6 30.9** 63.1 1169* 59.2 202*** hysun-39 32.8 14.3** 13.8 40.2*** 76.9 252* 49.6 125*** 32.8 58.0*** 67.4 74.4*** sf0049 21.8 11.6* 07.4 15.7** 77.8 452*** 37.2 13** 31.1 32.7*** 54.4 239*** increment % (+) genotypes ψs f-value cat f-value h2o2 f-value qp f-value car f-value sod f-value s.28111 81.8 393** 50 38.4*** 43.2 109** 80.4 1.43ns 10.6 149*** 20.4 26.3*** hysun-33 70.5 171*** 25 13.2** 48.9 83.8*** 75.6 15.8** 22.9 70.2*** 26.6 21.7*** hysun-39 70.9 112** 40.3 25.0*** 49.2 57.9*** 79.4 0.33 ns 06.4 40.5* 21.2 18.5* sf0049 81.9 210*** 45 20.7*** 44.6 99.5** 34.1 5.88* 20.2 19.6** 17.1 18.8** fig. 1. effects of salt (s), drought (d) and combined (s + d) stress on shoot height, number of leaves, relative water content and free proline content of sunflower genotypes. bar represents mean ± se (n = 4). same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. umar m., siddiqui z. s. 40 acta bot. croat. 77 (1), 2018 combined stress. a marginal decline was detected in maximum photochemical efficiency of photosystem ii (fv/fm ratio) when sunflower plants were treated alone with salt and drought stress. a significant reduction was observed in fv/fm ratio under combined stress, of 21% in hysun-33 whereas only a 7.4% reduction was found in sf0049. in both individual and combined stresses photochemical quenching (qp) was not significantly affected. the stomatal conductance of each genotype was reduced by the salt and drought stress (fig. 2). the stomatal conductance under salt and drought stress was higher in sf0049 than in the other genotypes. it was observed that both hysun genotypes had the lowest stomatal conductance in the individual stress conditions (fig. 2). the plants showed a substantial reduction in total chlorophyll content in stress environments (fig. 3). greater reduction was found in husun-33 in combined stress conditions than in the other genotypes. the total carotenoid concentration was increased in stressful environment when genotypes were treated with single or combined stresses. the highest carotenoid concentration was shown in hysun-33 under combined stress whereas the lowest concentration was recorded in s.28111. osmotic potential (ψs) of each genotype decreased in drought and salt stressed plants as compared to unstressed (control) plants. a greater decline in osmotic potential was observed during combined stresses (fig. 4). antioxidant enzymes like sod and cat were examined in genotypes when plants were exposed to a single (drought/salt) and to combined (salt + drought) stresses. plants treated with drought, salinity or in combination, showed significant increases in sod and cat as compared to control. it was observed that fig. 2. effects of salt, drought and combined (s + d) stress on fv/fm ratio, piabs, qp and gs of sunflower genotypes. values are mean ± se (n = 4). same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. fig. 3. effects of salt, drought and combined (s + d) stress on contents of photosynthetic pigments of sunflower genotypes. values are mean ± se (n = 4). same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. plant response against combined stress acta bot. croat. 77 (1), 2018 41 cat and sod activities were increased in all genotypes under single stress as compared to control (fig. 5). more antioxidant activities were observed in s.28111 and sf0049 than in hysun-33 and hysun-39; however, sod activity did not significantly differ in single stresses, drought or salinity. h2o2 is a known stress indicator in plants and its concentration increases in abiotic stress. the data showed that the concentration of h2o2 increased in all genotypes under both drought and salt stress. however, h2o2 contents increased much more under drought stress than under salt stress, concentrations getting higher still under combined stress. it was observed that production of h2o2 contents were reduced in s.28111 and sf0049 more than in other genotypes. table 2 showed that both hysun genotypes had the highest decline in rwc, leaf area, total chlorophyll and piabs under combined stress compared to their control, whereas s.28111 and sf0049 had the highest osmotic potential and cat activity. the outcome of correlation analysis is given in table 3. proline, carotenoids and h2o2 are negatively correlated with all the parameters and are positive with each other. catalase activity was negatively correlated with piabs, fv/fm ratio, rwc, gs, and chlorophyll content. the correlation of cat is found to be significant with gs, carotenoid, h2o2, ψs and sod. piabs significantly correlated with sod and non-significantly with cat whereas gs was non-significantly correlated with cat and significantly with sod. discussion reduction in plant biomass in response to drought and salt stress is a common phenomenon. substantial reductions in number of leaves and height of plants were recorded in hysun-33 and hysun-39, more so than in s.28111 and sf0049. rwc of each sunflower genotype was reduced in both single fig. 4. effects of salt, drought and combined (s + d) stress on activity of antioxidant enzymes cat and sod (expressed as unites mg–1 proteins), h2o2 content and osmotic potential of sunflower genotypes. values are mean ± se (n = 4). same letter above the bars denotes that among each genotype the difference between means is not significant at p = 0.05. tab. 3. pearson correlation among performance index (piabs), maximum quantum yield (fv/fm ratio), relative water contents (rwc), stomatal conductance (gs), leaf area (la), total chlorophyll content (t.chl), carotenoids content (car), proline, hydrogen peroxide (h2o2), osmotic potential (ψs), and superoxide dismutase (sod). ** denotes p<0.01 (2-tailed), * denotes p<0.05 level (2-tailed), ns – not significant. piabs fvfm rwc gs la t.chl car proline h2o2 ψs sod cat piabs 1 .735** .886** .700** .742** .594* –.654** –.836** –.847** .763** –.520* –.413 ns fv/fm 1 .820** .748** .830** .822** –.630** –.890** –.840** .529* –.160 ns –.279 ns rwc 1 .841** .857** .599* –.606* –.942** –.929** .718** –.395 ns –.465 ns gs 1 .920** .598* –.516* –.850** –.910** .777** –.474 ns –.611* la 1 .609* –.583* –.918** –.912** .712** –.441 ns –.589* t-chl 1 –.715** –.640** –.712** .529* –.224 ns –.297 ns car 1 .551* .666** –.627** .413 ns .612* proline 1 .918** –.673** .340 ns .391 ns h2o2 1 –.846** .533* .611* ψs 1 –.629* –.604* sod 1 .830** cat 1 umar m., siddiqui z. s. 42 acta bot. croat. 77 (1), 2018 stress and combined stress. however, maximum reduction was observed in hysun-33 and minimum reduction found in sf0049 and s.28111. rwc is a better indicator of leaf water status and is successfully used to determine stress resistance or tolerance in many crop plants (siddiqui et al. 2014). many reports reveal that rwc is reduced under drought and salinity (masoumi et al. 2010) but those plants that maintain high rwc under extreme stress are supposed to be more stress tolerant. hence it is assumed that sunflower genotypes sf0049 and s.28111, which maintained substantial water in leaf under combined stress, could be more stress tolerant than other genotypes. it is suggested that reduced shoot height, leaf area and number of leaves in sensitive genotypes may be due to their leaves having less rwc. the accumulation of proline in a single stress and a combined stress environment was observed. s.28111 accumulated a sufficient amount of proline under a combined stress environment. the accumulation of compatible osmolytes such as proline has been regarded as a basic strategy for the protection and survival of plants under abiotic stress (alia et al. 2001). the better plant height, number of leaves, leaf area and rwc in s.28111 under combined stress could be due to the higher accumulation of proline. relative water content and proline content are negatively correlated (r = ‒0.942, table 3). therefore, it may be suggested that an increase in proline concentration might be due to the reduction in rwc under osmotic stress, indicating that synthesis of proline became higher as soon as rwc declined. results show that hysun-33 had the highest decline in fv/fm (21%) and piabs (53%) of all the genotypes, while sf 0049 showed better results having 7.4% decline in fv/fm ratio and 31% in piabs in a single stressor in a combination (tab. 2). chlorophyll fluorescence is a sensitive indicator that often fluctuates in stress environments (weng et al. 2008). in stress sensitive plants, fv/fm ratio decreases under limited water conditions indicating chronic photo-inhibition (zlatev 2009). the vitality of the plant could be characterized by the piabs, which is more sensitive to abiotic stresses (oukarroum et al. 2007). combined stress significantly reduced the piabs in all the genotypes as compared to control and single stresses. however, the highest decrease in piabs was shown in hysun-33 under combined stress reflecting poor functionality of both psi and psii under stress (strasser et al. 2004). a very small decline was observed in sf0049 under s + d as compared to control indicating better performance under stress. the piabs showed significant correlation with all the tested parameters except catalase. rwc were highly correlated with piabs (0.886). this correlation showed that the reduction in performance index might be due to the decreased rwc. the stomatal conductance of each sunflower genotype was reduced by the salt and drought stress. the maximum reduction in stomatal conductance was observed in s.28111 and sf0049 under combined stress. during salt and drought stress, less water is available for the plants to facilitate their metabolism smoothly. in osmotic stress, an imbalance between water uptake by roots and water loss by transpiration causes the plant to wilt (lafitte 2002). therefore, water use efficiency may be an important strategy to increase fitness under osmotic stress environment. reports have shown that in some stress tolerant genotypes aba is synthesized and accumulated which leads to stomatal closure thus avoiding unnecessary transpiration to maintain substantial cellular osmotic potential. drought tolerant plants maximize fitness by decreasing stomatal conductance in response to the shortage of water (ares et al. 2000). the genotypes s.28111 and sf0049 showed 80 and 78% decline in stomatal conductance under combined stress compared to control, respectively (tab. 2). it is suggested that these two genotypes tolerate salt and drought by decreasing stomatal conductance and thereby increasing water use efficiency. levels of plant sensitivity, tolerance, and response timing against water stress fluctuate among genotypes / species. for example, slow-growing plant species have been found to be more sensitive than fast-growing species (munns 2002). it was observed that tolerant plants adapt two different strategies during abiotic stress: long-living annuals and perennials decrease their leaf size and/or stomatal conductance (siddiqui et al. 2014), while shorter-living annuals maximize fitness by increasing stomatal conductance (lessening water-use efficiency) to increase carbon gain and avoid stress. this tactic lets them grow speedily, flower early and increase yield before the start of substantial soil drying (mckay et al. 2003). thus, it is thought genotypes s.28111 and sf0049 might adopt the early developmental strategy and enhance stress tolerance whereas both hysun genotypes adopt the late developmental strategy and thereby have less tolerance to salt, drought and combined stress. genotype s.28111 showed the highest chlorophyll contents in a combined stress environment of all the tested genotypes. further, abiotic stress causes ros production that damages the chloroplast and as a result a reduction in chlorophyll contents occur (smirnoff 1995). reduction in chlorophyll under drought and salinity stresses has been reported in many plant species including sunflower (manivannan et al. 2007). higher reduction in chlorophyll contents in hysun genotypes might be due to imbalance in ros production and scavenging mechanism. the non-enzymatic antioxidants like carotenoids and proline contents were negatively correlated with photosynthetic parameters and their correlation with piabs, fv/fm and total chlorophyll were highly significant. this negative correlation might be due to the protective strategy of plants under harmful environmental conditions in which chlorophyll contents are reduced and carotenoids increased. sunflower plants treated with drought and salinity singly or in combination, showed a significant increase in sod and cat compared to control. it was observed that cat and sod activities were increased under stresses compared to control but combined stress reduced the activity of both enzymes in all the genotypes. however, more antioxidant activities were observed in s.28111 and sf0049 than in hysun-33 and hysun-39. it has been reported that plant tolerance can be maintained by controlling the ros production through non-enzymatic mechanisms, including carotenoids, proline and phenolic compounds (jaleel et al. 2009) or to prevent plants from oxidative damage by antioxidant enzymes plant response against combined stress acta bot. croat. 77 (1), 2018 43 activity like cat and sod (quan et al. 2008). the present study showed that cat and sod activities of sunflower genotypes were negatively correlated with h2o2 concentrations. it is presumed that the lower h2o2 production in drought stress may be due to enzyme activities or it may be related to higher carotenoid synthesis, which may lower h2o2 production in chloroplasts, thus avoiding oxidative damages. cat is one of the enzymes that detoxify h2o2 in plants and it is mostly located in peroxisomes. cat use h2o2 as a substrate and changes it to h2o and o2 as products. it is clear that s.28111 and sf0049 genotypes showed better photosynthetic performance than the other genotypes under combined stress (tab. 2). both genotypes showed minimum decline in fv/fm ratio, performance index and total chlorophyll. this minimum decrease under combined stress environment showed that s.28111 and sf0049 showed better physiological performance than either of the hysun genotypes. the maximum increment in ψs and cat activity and minimum increment in h2o2 contents also showed that s.28111 and sf0049 have better tolerance than the hysun genotypes in individual and combined stress. the parameters used in this trial are significantly correlated (tab. 3). despite combined salt and drought stress these parameters are useful to identify genotypes tolerant to water-limited and salinity-prone environments. the present study has shown that s.28111 and sf0049 exhibited a greater degree of tolerance than the hysuns in terms of better physiological performance against salinity, drought and their combination. this might be due to their early development strategy, as compared to both hysun genotypes. it was based on the evaluation of genotypes responding to stress tolerance, which is shown as percentage increase (+) /decrease (–) over control and illustrated in table 2. the genotypes s.28111 and sf0049 showed the lowest decline in piabs, fv/fm ratio, rwc and chlorophyll content. however, in all treatments the highest increase in osmotic potential, proline contents, qp, carotenoids contents and antioxidant enzymes activities was recorded. results of the present investigation showed that sf0049 and s.28111 genotypes have better tolerance than either of the hysun genotypes. genotypes s.28111 and sf0049 not only showed better photosynthetic performance but also reduced the concentration of h2o2 in all stresses. therefore, it may be suggested that genotypes s.28111 and sf0049 could be used in field trials for a more comprehensive study. however, more detailed molecular work 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(1993) reported somatic embryogenesis from in vitro leaf explants. induction of somatic embryos from ovary explants of ginger and its regeneration were described by nirmal babu et al. (1996). guo and zhang (2005) established somatic embryogenic cultures of four ginger cultivars from shoot tip explants. a protocol for plant regeneration via somatic embryogenesis from vegetative bud-derived callus cultures of ginger was reported by suma and keshavachandran (2005). due to lack of seed set, the embryological studies in ginger are still in the initial stage. the histological studies of somatic embryos in the present study revealed the origin, evolution and structure of ginger embryos. barring few studies on the histology of indirect somatic embryogenesis in ginger (kackar et al. 1993, suma and keshavachandran 2005), there are no reports on direct somatic embryogenesis and its ontogeny in ginger. the present work reports both direct and indirect somatic embryogenesis in ginger including the histological and developmental changes during somatic embryogenesis. materials and methods in vitro and in planta aerial stem explants taken from two varieties of ginger (zingiber officinale rosc.), var. jamaica and var. varada were used in this study. in planta explants collected from 3–7 months old disease-free plantswere thoroughly washed in tap water with 5% detergent solution (teepol) for 20 minutes followed by 2–3 washes in sterile distilled water. the explants were cut into convenient sizes after removal of the leaf sheaths. the cut pieces were surface sterilized with 0.1% hgcl2 for 10 minutes and rinsed 4–5 times with sterilized double distilled water and then trimmed to 1.0–1.5 cm size. these explants were transferred onto half strength ms medium (murashige and skoog 1962) supplemented with 2, 4-d (2, 4-dichlorophenoxy acetic acid) at different concentrations (0.5, 1.0, 2.0 mg l–1). the in vitro aerial stem explants, collected from 3–4 months old plantlets cultured on a medium containing bap and naa in different concentrations, were cut into 1.0 –1.5 cm length and transferred to the same medium. explants were maintained in an air conditioned room at 22±2 �c and photoperiod regime of 16 hr light and 8 hr dark with a light intensity of 3000 lux, provided by philips cool white fluorescent tubes. callus cultures (about 2–2.5 g) derived from the aerial stem explants were subcultured onto ms medium containing 2,4-d + bap in different combinations (0.2: 0.2, 0.5: 0.2, 1.0: 0.5, 2.0: 0.5, 1.0: 1.0 mg l–1). these cultures were subjected to stress by keeping them without sub culturing for 40 – 60 days to induce embryogenic calli. the embryogenic calli were then transferred onto ms medium containing bap in different concentrations (0.2, 0.5, 1.0, 2.0, 5.0 mg l–1) for the induction of somatic embryos. 94 acta bot. croat. 68 (1), 2009 lincy a. k., remashree a. b., sasikumar b. u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:22 color profile: disabled composite 150 lpi at 45 degrees mature somatic embryos were transferred onto ms medium with bap + naa (a – naphthalene acetic acid) in different combinations (0.2: 0.2, 1.0: 0.5, 1.0: 1.0, 2.0: 0.2, 2.0: 1.0 mg l –1 ). single explants were inoculated onto ms medium supplemented with tdz and iba (thidiazuron and indole, 3-butyric acid) at different concentrations (0.5: 0.5; 1.0: 0.5; 1.0:1.0 mg l –1 ) and tdz alone (0.2, 0.5, 1.0 mg l –1 ). standard procedures (johansen 1940) were followed to carry out histological studies. specimens at different developmental stages were fixed in faa (5 ml formalin: 5 ml glacial acetic acid: 90 ml 70% alcohol) for 24 hour, then washed under tap water for 5 hours and dehydrated in an ethyl alcohol – tertiary butyl alcohol (tba) series. the specimens were processed for gradual infiltration and embedded in melted paraffin wax (56 – 58 °c, qualigens, mumbai). the specimens were serially sectioned with an ernst leitz wetzlar gmbh (germany) microtome at 10μm thickness and stained with papanicolous staining solution (harries hematoxyline solution, qualigens) for 20 minutes and washed in water. the slides were then dehydrated through tba – xylene series. representative sections were photographed with an olympus bx 50 microscope. well-rooted plantlets of 3–4 months old were removed from the culture flasks, washed in tap water to remove the adhering medium and transplanted in disposable polythene cups (250 ml) containing sterile sand and coir dust and kept at room temperature for further growth. the experiments were set according to completely randomized design. observations recorded include days taken for morphogenic response from ten random tubes, percentage of culture responded, nature of callus – callus colour and texture, type of callus response (induction of somatic embryos, rhizogenesis and callus proliferation) besides number of shoots and roots regenerated from each somatic embryo culture. all experiments were performed in triplicate. ten culture tubes constituted an experimental unit. two factor anova was performed to test the significance of treatment effect. statistical procedures were performed using mstat c. results induction of callus from aerial stem – effect of explants and genotypes the in vitro aerial stem produced callus by the second week of culturing in both the varieties. however, the in planta aerial stem took 32 days in the var. jamaica and 25 days in the var. varada for callus induction. the in vitro aerial stem of the var. jamaica produced profuse callusing in ms with 2,4-d (2 mg l –1 ) and moderate callusing in 2,4-d (1 mg l –1 ). the in planta aerial stem of this variety showed only moderate callusing with 2,4-d (2 mg l –1 ). in the var. varada, the in vitro aerial stem showed profuse callusing in 2,4-d (1 mg l –1 and 2 mg l –1 ), while the in planta aerial stem produced moderate callusing in 2,4-d (2 mg l –1 ) (tab. 1). color and texture of callus – effect of explants the quality and type of the callus produced from the different explants varied. callus induced from the in vitro aerial stem was hard, nodular and yellowish in colour. the surface acta bot. croat. 68 (1), 2009 95 somatic embryogenesis in zingiber u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:22 color profile: disabled composite 150 lpi at 45 degrees of the callus was covered with small hairy structures (hereafter referred to as type i callus) while the in planta aerial stem produced soft, sticky callus with pale white or creamy colour of friable nature (hereafter referred to as type ii callus). no genotypic differences were observed. induction of somatic embryos the type ii calli showed callus proliferation in all cultures containing 2,4-d and bap in different concentrations, in both the varieties. these proliferated calli, when subjected to stress for 40 to 60 days without subculturing, became desiccated due to the depletion of water and nutrients. these desiccated calli started to produce good quality, white friable callus and on subculturing onto the medium containing 2 mg l–1 bap produced somatic embryos. the other concentrations of bap resulted in callus proliferation only. the percentage of somatic embryo induction showed variation with the different varieties. in the var. jamaica, 100% of cultures produced somatic embryos by 30–35 days of culturing and in the var. varada, only 50% of cultures resulted in somatic embryo induction after 45 days of culturing. the type i callus as well as type ii callus that were not subjected to stress did not become embryogenic on repeated subculturing but produced roots in all the cultures, in both the varieties. the embryogenic calli produced localized groups of cells, which differentiated into embryogenic forms on the peripheral region of the callus and latter differentiated into white globular embryoids (fig. 1 a) after 30 days of culture. this process continued in the subsequent subculturing in the same medium leading to the production of oval shaped embryo (fig. 1 b). after 45 days, these somatic embryos differentiated into a more mature, elongated club shaped or cylindrical stage, and cotyledon initiation, as well as signs of scutellum initiation, occurred at this stage (fig. 1 c). after 60 days the coleoptile became green and the shoot completely differentiated with one or two leaf plumules. at this stage, the embryos started germinating and developed shoot. the pattern of embryo development was the same in both the varieties, though in case of the var. jamaica the events occurred a little faster. secondary somatic embryogenesis was observed when the embryogenic calli with somatic embryos were transferred onto fresh medium with bap (2.0 mg l–1) or without any growth hormones (fig. 1 d). the presence of bap hastened the secondary somatic embryo induction. 96 acta bot. croat. 68 (1), 2009 lincy a. k., remashree a. b., sasikumar b. tab. 1. effect of explants and genotypes on callus induction sl. no hormone mg l –1 (2,4-d) jamaica varada in vitro aerial stem % of callus induced cultures in planta aerial stem % of callus induced cultures in vitro aerial stem % of callus induced cultures in planta aerial stem % of callus induced cultures 1 2 3 0.5 1.0 2.0 + + + + + + 30 70 90 – + + + – 20 60 + + +++ +++ 40 60 100 – + + + – 40 70 + low callusing; + + moderate callusing; + + + profuse callusing (observations based on amount of callus in 10 cultures). u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:23 color profile: disabled composite 150 lpi at 45 degrees germination of somatic embryos the germination of embryos was obtained by the second week of culturing in the regeneration medium containing bap and naa in different combinations. the mature embryos showed a green color after 12 days of culturing (fig. 1 e, f). then these embryos produced shoots and roots simultaneously (fig. 1 g). germination of somatic embryos was observed in all the hormone combinations tried and the percentage of germination varied from 60 to 100% in both the varieties (tab. 2). among the five hormone combinations tried, the optimum concentration for shoot regeneration was 2 mg l –1 bap + 0.2 mg l –1 naa in the var. jamaica and 2 mg l –1 bap + 1 mg l –1 naa in the var. varada. the effects of hormones on shoot regeneration were signifacta bot. croat. 68 (1), 2009 97 somatic embryogenesis in zingiber fig. 1. callus proliferation in ginger. a – white globular embryoids on the peripheral region of the callus cultured on medium supplemented with bap 2 mg l–1. b – globular and oval shaped embryos, c – club shaped embryo with cotyledon and scutellum initiation, d – secondary somatic embryogenesis, e – mature embryos showed green color after 12 days of culturing (1.0 bap + 1.0 naa mg l–1), f – close-up view of somatic embryo germination, g – shoot and root proliferation from somatic embryos cultured on 2 bap + 0.2 naa mg l–1, h – irregular and aberrant formation of somatic embryos, i – in planta aerial stem explant of var. jamaica showing direct somatic embryo induction (0.5 tdz + 1.0 iba mg l–1), j – leaf base explant of var. varada showing direct somatic embryo induction (0.5 tdz mg l–1), k – acclimatization of plantlets). u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:26 color profile: disabled composite 150 lpi at 45 degrees icant. maximum root regeneration was observed in bap and naa (1:0.5 mg l –1 ) in the var. jamaica and bap and naa (1:1 and 2:1 mg l –1 ) in the var. varada (tab. 2). some well developed somatic embryos (20–40%) failed to germinate and formed irregular and aberrant structures (fig. 1 h). induction of direct somatic embryos in planta aerial stem explants of the var. jamaica produced somatic embryos directly from 30% of explants in the cultures containing tdz + iba (0.5 + 1.0 mg l –1 ) (fig. 1) with 30 – 40 days of culturing. in the var. varada, direct somatic embryos were induced from the in planta leaf base explants (20%) cultured on the medium containing tdz (0.5 mg l –1 ) after 45 days of culturing (fig. 1 j). the other hormone combinations showed no morphogenic responses. however, no plantlet regeneration was observed in the direct regenerated somatic embryos in either of the varieties. histological studies – origin and development of somatic embryos anatomical sections prepared from the embryogenic cultures of ginger containing somatic embryos at different developmental stages revealed the ontogeny of somatic embryos. the histological sections showed groups of meristamatic cells formed at the peripheral region of embryogenic callus, which can be clearly identified from the normal non embryogenic calli. these meristamatic cells were small, compact and densely cytoplasmic with distinct nuclei. the embryos developed from single cells, which underwent continuous divisions and formed two, four, six and eight celled stages. after that, it formed a glo98 acta bot. croat. 68 (1), 2009 lincy a. k., remashree a. b., sasikumar b. tab. 2. in vitro shoot and root regeneration from somatic embryos on ms medium supplemented with different growth hormones. values for each treatment are means of three replicas; ten culture tubes constituted one experimental unit. data analyzed using two-way anova. ns – non significant; cd – critical difference. growth regulators in basal medium (mg l –1 ) jamaica varada mean bap : naa % of shoot inducing cultures no. of shoots no. of roots % of shoot inducing cultures no. of shoots no. of roots no. of shoot no. of root 0.2 : 0.2 1.0 : 0.5 1.0 : 1.0 2.0 : 0.2 2.0 : 1.0 mean cd (p <0.05) variety hormone variety x hormone 100 60 100 100 80 4.4 0.8 1.8 8.2 2.0 3.4 ns 0.74 1.04 6.4 12.6 9.0 9.0 7.8 9.0 ns ns ns 60 100 80 100 100 1.4 2.2 1.2 2.8 9.0 3.3 8.2 7.6 10.2 5.2 10.2 8.3 2.9 1.5 1.5 5.5 5.5 7.3 10.1 9.6 7.1 9.0 u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:26 color profile: disabled composite 150 lpi at 45 degrees bular mass (proembryoids) that showed a distinct epidermis. further differentiation of these structures led to the formation of globular, oval shaped embryos on the surface of the callus (fig. 2 a). the embryo was attached to the embryogenic callus with a prominent multicellular stalk, the suspensor. at the cylindrical stage of somatic embryo, the embryo primordium started to grow as a broad outgrowth and a notch arose at terminal region of the embryo (fig. 2 a). the scutellum developed as a hump opposite the notch where the shoot apex is organized and the coleoptile developed as a circular primordium around the shoot apex. the shoot apex had a lateral position and the root apex developed opposite the shoot apex. thus the somatic embryos had a scutellum, coleoptile, shoot apex and coleorhiza with an independent vascular system that is not connected to the maternal tissue (figs. 2 b, c). the primary and secondary somatic embryos at the peripheral region of the callus had a bunch-like appearance (fig. 2 d). secondary somatic embryos were developed due to the proliferation of cells in the coleorhizal part of mature primary somatic embryos (fig. 2 d). somatic embryos were induced directly from the primary thickening meristem of in planta aerial stem explant and from the epidermal tissues of leaf base explant (figs. 2 e, f) with distinct scutellum, coleoptile, shoot apex and root apex (fig. 2 g). hardening and establishment of plantlets well developed plantlets were transferred to polythene cups containing sterile sand and coir dust and 85 – 90% of the plantlets could be hardened (fig. l k). after 2 weeks of hardening the plantlets were transferred to field or experimental shed. discussion somatic embryos were induced indirectly from in planta aerial stem derived callus and directly from aerial stem as well as leaf base explants of ginger. the callus induced from the in vitro aerial stem explants of the two varieties was shown to be inappropriate for somatic embryo induction in the present study. the type of callus was also different with regards to different explant types. source of explants and genotypes are considered to be important factors in the induction of callus and somatic embryo (george 1993). guo and zhang (2005) reported variation over different genotypes on somatic embryogenesis in ginger as we observed in the present study. ma and gang (2006) reported that the rate of callus formation in ginger varied dramatically depending on the tissues used. in our study, the calli induced in medium containing 2,4-d were further proliferated in a medium with 2,4-d + bap. the calli become embryogenic only in the presence of bap. it is observed that complete removal of 2,4-d from the medium favoured somatic embryogenesis. earlier workers in zingiber officinale rosc. and kaempferia galanga l. observed the same results (guo and zhang 2005, vincent et al. 1992). a period of stress was found favorable to induce somatic embryogenesis from callus cultures of ginger in the present study. stress-induced somatic embryogenesis has already been reported in carrot (harada et al. 1990). different stresses (osmotic, heavy metal ion and dehydration stress) are implicated in somatic embryo induction in arabidopsis thaacta bot. croat. 68 (1), 2009 99 somatic embryogenesis in zingiber u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:26 color profile: disabled composite 150 lpi at 45 degrees liana (iwai et al. 2003). probably, each stress treatment induces expression of some factors (genes) that control the start of somatic embryogenesis (iwai et al. 2003). successful plantlet regeneration from somatic embryos was observed in the present study with bap and naa. guo and zhang (2005) reported that bap was effective for regeneration of somatic embryos also, besides induction of somatic embryos. kackar et al. 100 acta bot. croat. 68 (1), 2009 lincy a. k., remashree a. b., sasikumar b. fig. 2. globular and oval shaped embryos on the surface of the callus (a). arrow mark indicating the scutellar notch at the terminal region of the embryo. b – somatic embryo with scutellum, coleoptile, shoot apex and coleorhizae, c – somatic embryo with an independent vascular system, d – primary and secondary somatic embryos – secondary somatic embryos originating from the coleorhizal part of primary somatic embryo, e – direct somatic embryo induction from aerial stem explants, f – direct somatic embryo induction from leaf explant – globular stage, g – single somatic embryo with shoot apex and root apex. cl – coleoptile, cr – coleorhiza, ge – globular embryo, pe – primary embryo, ra – root apex, sa – shoot apex, sc – scutellum, se – secondary embryo, sn – scutellar notch, vs – vascular system. bars denote 0.5 mm. u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:30 color profile: disabled composite 150 lpi at 45 degrees (1993) also reported that inclusion of bap (0.5–1.5 mg l–1) in the regeneration medium hastened the germination of somatic embryos in ginger. the somatic embryos directly induced were not regenerated in the present study. long exposure of explants to tdz inhibited the capacity of shoot regeneration and multiplication. gisbert et al. (2006) reported that the development of shoots decreased when the concentration of tdz increased. tdz is known to inhibit shoot proliferation, especially in solid cultures (amutha et al. 2006). the somatic embryos in ginger followed typical developmental stages described for other monocot systems: globular embryo stage, club shaped cotyledon initiation stage and the plumule initiation stage. shah (1982) reported that the monocotyledon embryo is cylindrical and shoe shaped with a slightly pointed distal end and broad blunt coleorhizal end. vasil et al. (1984), guiderdoni and demarly (1988), smith and krikorian (1991) also reported similar pattern of embryo development in other monocots like maize, sugar cane and day lily, respectively. smith and krikorian (1991) reported that embryogenic callus of day lily yielded various ’neo morph-like’ structures (developmentally abnormal or poorly developed pseudoembryonic forms that did not yield plantlets and eventually died) comprising a root and a poorly developed or altogether lacking shoot tip meristem. germination without plantlet recovery, i.e. the formation of root but not an epicotyl, may be due to malformation of the apical shoot meristem or else to the embryo being damaged during its isolation for transfer to germination medium (valladares et al. 2006). histological studies of somatic embryogenesis in ginger and other monocotyledons were reported by earlier workers. kackar et al. (1993) observed the presence of nodular structures containing richly cytoplasmic cells delimited by a single layered epidermis. these authors also observed stalked somatic embryos with scutellum, coleoptile and coleorhiza in ginger. swamy (1982) observed that the quadrant and octant stages are invariable and necessary stages in the embryogenesis of both dicotyledons and monocotyledons. wardlaw (1955) reported that in a monocotyledonous embryo, the shoot apex occupies a lateral position in the somewhat cylindrical embryo and the cotyledon is terminal. bechtel and pomeranz (1978) and batygina (1969) observed the presence of scutellum and coleoptile in the monocot embryo. secondary somatic embryos were originated from the coleorhizal part of the developed somatic embryos (primary somatic embryos). kuo et al. (2005) reported that secondary somatic embryogenesis occurred at the basal part of primary embryos and originated from outer cell layers, in phalaenopsis, an orchid species. direct somatic embryogenesis from epidermal tissues of leaf explants was also reported in phalaenopsis (chen and chang 2006; kuo et al. 2005). in the present study, we established a reliable and reproducible protocol for indirect somatic embryogenesis from aerial stem explants and direct somatic embryogenesis from aerial stem as well as leaf base explants of ginger. the results of the study revealed the origin and development of direct and indirect somatic embryogenesis. we were able to induce somatic embryos repeatedly from primary somatic embryos as well (secondary somatic embryogenesis). such a system for cyclic somatic embryogenesis is useful in the production of large scale somatic embryo induction, which can be used for the genetic transformation of ginger. acta bot. croat. 68 (1), 2009 101 somatic embryogenesis in zingiber u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:30 color profile: disabled composite 150 lpi at 45 degrees acknowledgments the first author is grateful to the indian society for plantation crops (ispc), kasaragod for financial assistance in the form of research fellowship to undertake the study as a part of her ph d program. the authors thank the director, indian institute of species research, calicut, kerala, for providing all the necessary facilities for the present work. references amutha, s., muruganantham, m., ganapathi, a., 2006: thidiazuron – induced high frequency axillary and adventitious shoot regeneration in vigna radiata (l.) wilczek. in vitro cellular and developmental biology plant 42, 26–30. batygina, t. b., 1969: on the possibility of separation of a new type of embryogenesis in angiosperms. revue de cytologie et de biologie végétales 32, 335–341. bechtel, d. b., pomeranz, y., 1978: ultra structure of the mature ungerminated rice (oriza sativa) caryopsis. the germ. american journal of botany 61, 78–85. chen, j. t., chang, w. c., 2006: direct somatic embryogenesis and plant regeneration from leaf explants of phalaenopsis amabilis. biologia plantarum 50, 169–173. george, e. f. 1993: plant propagation by tissue culture, 2. exegetics ltd, edington. guiderdoni, e., demarly, y., 1988: histology of somatic embryogenesis in cultured leaf segments of sugarcane plantlets. plant cell tissue and organ culture 14, 71–88. gisbert, c., prohens, j., nuez, f., 2006: efficient regenration in two potential new crops for subtropical climates, the scarlet (solanum aethiopicum) and gboma (s. macrocarpon) explants. new zealand journal of crop and horticultural science 34, 55–62. guo, y., zhang, z. x., 2005: establishment and plant regenerations of somatic embryogenic cell suspension cultures of the zingiber officinale rosc. scientia horticulturae 107, 90–96. harada, h., kiyosue, t., kamada, h., kobayashi, t., 1990: stress induced carrot somatic embryogenesis and its application to synthetic seed. in: sangwan, r. s., sangwan norreel, b. s. (eds), the impact of biotechnology in agriculture, 129–157. kluwer academic publishers, dordrecht. iwai, m. i., umehara, m., satoh, s., kamada, h., 2003: stress induced somatic embryogenesis in vegetative tissues of arabidopsis thaliana. the plant journal 34, 107–114. johansen, d., 1940: plant micro techniques. mc graw hill publication, new york. kackar, a., bhat, s. r., chandel, k. p. s., malik, s. k., 1993. plant regeneration via somatic embryogenesis in ginger. plant cell tissue and organ culture 32, 289–292. kantharajah, a. s., golegaonkar, p. g., 2004: somatic embryogenesis in egg plant. scientia horticulturae 99, 107–117. kuo, h. l., chen, j. t., chang, w. c., 2005: efficient plant regeneration through direct somatic embryogenesis from leaf explants of phalaenopsis ’little steve’. in vitro cellular and developmental biology plant 41, 453–456. ma, x., gang, d. r., 2006: metabolic profiling of in vitro micropropagated and conventionally green house grown ginger (zingiber officinale). phytochemistry 67, 2239– 2255. 102 acta bot. croat. 68 (1), 2009 lincy a. k., remashree a. b., sasikumar b. u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:30 color profile: disabled composite 150 lpi at 45 degrees murashige, t., skoog, f., 1962: a revised medium for rapid growth and bioassays with tobacco tissue culture. physiologia plantarum 15, 473–497. nirmal babu, k., samsudeen, k., rathnambal, m. j., ravindran, p. n., 1996: embryogenesis and plant regeneration from ovary derived callus cultures of ginger (z. officinale rosc.). journal of spices and aromatic crops 5, 134–138. shah, c. k., 1982: morpho-histochemical and sem studies of some monocotyledonous embryos. phytomorphology 32, 211–221. smith, d. l., krikorian, a. d., 1991: growth and maintenance of an embryogenic cell culture of day lily (hemerocallis) on hormone free medium. annals of botany 67, 443–449. suma, b., keshavachandran, r., 2005: somatic embryogenesis and plantlet regeneration from callus cultures of ginger (z. officinale rosc.). proceedings 17 science congress, kerala, 29–31. swamy, b. g. l., 1982: embryogenesis in sagittaria sagittaeflia. phytomorphology 30, 204–212. valladares, s., sanchez, c., martinez, m. t., ballester, a., vieitez, a. m., 2006: plant regeneration through somatic embryogenesis from tissue of mature oak trees: true to type conformity of plantlets by rapd analysis. plant cell reports 25, 879–886. vasil, v., vasil, i. k., lu, c., 1984: somatic embryogenesis in long term callus cultures of zea mays l. (gramineae). american journal of botany 71, 158 – 161. vincent, k. a., hariharan, m., mathew, k. m., 1992: embryogenesis and plantlet formation in tissue culture of kaempferia galanga l. – a medicinal plant. phytomorphology 42, 253–256. wardlaw, c. w., 1955: embryogenesis in plants. methuen, new york. acta bot. croat. 68 (1), 2009 103 somatic embryogenesis in zingiber u:\acta botanica\acta-botan 1-09\lincy izmjena.vp 22. travanj 2009 12:24:30 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 377 acta bot. croat. 74 (2), 377-392, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0026 development of periphytic diatoms on different artifi cial substrates in the eastern adriatic sea tina nenadović*1, tena šarčević2, hrvoje čižmek2, jelena godrijan3, daniela marić pfannkuchen3, martin pfannkuchen3, zrinka ljubešić1 1 university of zagreb, faculty of science, department of biology, rooseveltov trg 6, 10000 zagreb, croatia 2 »20.000 leagues« marine explorers society, l. sorkočevića 3, 23000 zadar, croatia 3 ruđer bošković institute, centre for marine research, g. paliaga 5, 52210 rovinj, croatia abstract – the settling of diatoms as fouling organisms on a certain substrate is greatly infl uenced by substrate characteristics and the preferences of a diatom community and diatom species. a distinction among substrates can be made by analysing the specifi c abundance and composition of diatoms on different substrates. in this study, 11 different artifi cial substrates were exposed to a marine environment for a period of 30 days. abundance and taxonomic composition of periphytic diatoms was determined on each of the substrates and on shoots of the marine seagrass posidonia oceanica. the aim was to compare diatom community structure on different newly colonized surfaces. on all surfaces examined, periphytic diatoms were the pioneering organisms with differences in quantitative and qualitative composition on the different substrates. taxonomic analysis of diatom communities on the substrates examined revealed 41 diatom taxa, with the dominant genera cylindrotheca, amphora, nitzschia, cocconeis and navicula. given that all the examined artifi cial substrates were solid materials, differences in the abundance and species composition of diatoms found between the materials point to the substrates’ physical and chemical characteristics as a major infl uence on the fi nal settling of diatoms. knowledge from investigating the settlement of fouling organisms on anthropogenic substrates can have future use in management of waste materials that end up in the marine environment. key words: adriatic sea, anthropogenic materials, artifi cial substrates, diatoms, fouling, marine litter, periphyton introduction anthropogenic solid materials often end up in the marine environment and are commonly known as marine litter. in their exposure to sea water, those materials undergo chem* corresponding author, e-mail: nenadovictina@gmail.com nenadović t., šarčević t, čižmek h., godrijan j., marić pfannkuchen d., et al. 378 acta bot. croat. 74 (2), 2015 ical and biological conditioning known as the fouling process. chemical conditioning produces a molecular fi lm comprising compounds including glycoproteins, humic material, proteins, lipids, nucleic acids, polysaccharides, aromatic amino acids and/or unspecifi ed macromolecules (garg et al. 2009 and the references therein), whereas biological conditioning supersedes with the accumulation of prokaryotic and eukaryotic unicellular organisms (railkin 2004) incorporated in the matrix of extracellular polymers (donlan 2002). a newly formed conditioning matrix, or biofi lm, is mostly composed of bacteria and diatoms. other unicellular organisms, like fl agellates, ciliates, yeasts and protozoans contribute less than 1% of the total cell count in the biofi lm (railkin 2004). the presence of bacteria and unicellular algae in the biofi lm can enhance further colonization of the substrate by plants and animals (totti et al. 2007 and the references therein). the initial colonizing biomasses, along with bacteria, are diatoms (wetherbee et al. 1998), eukaryotic microalgae, which constitute the periphyton community on immersed substrates. diatom adhesion is mediated by the physico-chemical properties of the substrate and the diatom cell properties. the surface charge of diatom cells is dependent on their cell wall. although the cell wall is siliceous and therefore negatively charged, it is covered with an organic layer of polysaccharides, proteins and glycoproteins (hecky et al. 1973, staats et al. 1999), which allows cell surface potential to vary. extracellular polymeric substances (eps), secreted through numerous openings in the cell wall and the raphes is recognized as the extracellular adhesive which provides diatoms with the ability of permanent adhesion and motility on various substrates (wang, et al. 1997, wustman et al. 1997). in the investigation of the diatoms’ cell surfaces and their ability of adhesion many techniques have been successfully used. scanning electron microscopy (sem) and transmission electron microscopy (tem) are traditional and the most commonly used techniques preformed on dried samples. since diatoms form the bulk of initial colonizing biomass on immersed substrates, researchers have used artifi cial substrates to understand the preference of diatoms for a certain substrate (rodrigues and bicudo 2001, danilov and ekelund 2002, tank and dodds 2003, townsend and gell 2005). the fi rst recorded study using an artifi cial substrate for periphyton settling was done by hentschel in 1915. the advantage of newly introduced artifi cial substrates in the marine environment is the opportunity to monitor initial development and the succession of diatoms in the periphyton. comparative research on nutrient concentration of periphyton on living substrates (macrophytes, shells), epilithion (danilov and ekelund 2002, kahlert and pettersson 2002) and the periphyton on inorganic (glass, plastic and rock) and organic substrates (wood, leaves) showed that living and organic substrates act as additional source of nutrients for attached communities (danilov and ekelund 2002, tank and dodds 2003). some studies pertaining to the structural variations between artifi cial and natural substrates showed a signifi cant difference between artifi cial and natural substrates (townsend and gell 2005) and others little (rodrigues and bicudo 2001) or no structural difference (lane et al. 2003). also, the development of periphyton on man-made structures in the marine environment has become a widespread issue and the literature about periphyton development has been rapidly growing since the 1980s (bhosle et al. 1989, railkin 2004, yebra et al. 2004, shultz et al. 2011). recent periphytic studies in the adriatic sea have focused on epiphytic diatoms of the northern adriatic (munda 2005) and the toxic bloom of benthic dinofl agellates ostreopsis (totti et al. 2007, pfannkuchen et al. 2012), whilst in the middle adriatic research is focused on the ecology and taxonomy of periphytic diatoms in the estuary of rivers, like the river zrmanja (burić et al. 2004, caput et al. 2005). periphytic diatoms on different artificial substrates acta bot. croat. 74 (2), 2015 379 in this research, we examined the initial colonization of diatoms in the periphytic community on a wide array of immersed artifi cial substrates with various physico-chemical properties. the abundance of diatoms on a newly formed biofi lm has been studied, as has the diatom genera composition. the seagrass posidonia oceanica in a nearby meadow was also sampled to compare the abundance and composition of the diatom community from artifi cial substrates with an already established community on a natural substrate. the aim of this study was to determine the difference in abundance and composition of diatom community on various artifi cial substrates and to discuss the difference in terms of diatom affi nity to a specifi c artifi cial material. the relevance of investigating the affi nity of diatoms as a major fouling community to a specifi c artifi cial material is to get an insight into the outcome of debris in the marine environment in order to systematically work towards alleviating this initially negative impact on the marine environment. materials and methods study area the experiment was carried out in the puntamika peninsula near zadar, croatia, coastal area of the central adriatic sea (fig. 1). the adriatic sea is an elongated basin, the northernmost part of the mediterranean. the eastern coastal region is infl uenced by the ingoing current from the ionian sea, characterized by high salinity and low content of nutrients, and freshwater discharge from oligotrophic karstic rivers. the investigated station is situated in a region defi ned according to the physicochemical conditions as a region similar to the open waters of the middle adriatic sea (vidjak et al. 2012). artifi cial substrates were exposed to the marine environment at a depth of 12 m for the period of one month (march 7th to april 6th 2012). eleven different materials were used as artifi cial substrates: asbestos, painted iron, wood, concrete, glass, plastic, unpainted iron, rubber, ceramics, stone and aluminium. materials used as substrates were of variable shapes and sizes and were acquired from the local waste deposit site. description of the materials is as follows: glass – transparent glass used as a window, asbestos – plates used as roofi ng material, ceramic – smooth, glazed ceramic indoor tiles, rubber – rubber inner tube of a bicycle tire, wood – processed, smoothed wooden bar, plastic – hard non-transparent plastic fig.1. study area at puntamika, zadar, croatia. nenadović t., šarčević t, čižmek h., godrijan j., marić pfannkuchen d., et al. 380 acta bot. croat. 74 (2), 2015 plate, aluminium – fl at plate, iron – fl at plate, painted iron – painted fl at plate, rock – karstic rock, concrete – building block. two replicates of each substrate were used in case of eventual loss of one or more substrates. substrates were submerged with the use of scuba diving to the depth of 12 m, which provided enough light for the development of periphyton, and protection from the infl uence of surge. temperature of the sea while submerging was taking place was 11 °c, and 12 °c during the retrieval of the substrates. all substrates were placed on the seabed and arranged at 45º angle, leaning against a plastic pipe that followed the seabed at the location. the plastic pipe is an outlet pipe that has been there for a substantial amount of time. plastic and rubber substrates were additionally anchored with materials (stone, glass jar) found at the location. sampling after 30 days’ exposure, scuba divers retrieved one replicate of each substrate. upon retrieval, the substrates were enclosed, each in a separate plastic bag to minimize the effect of the phytoplankton to the substrates. upon retrieval, a 36 cm2 area of the mucous biofi lm covering the surface of the substrates was scraped from both the top and bottom sides of each substrate with a scalpel and brush. scraped surface of each substrate was rinsed with distilled water into marked, wide-mouthed sample plastic vials and preserved in a 4% formaldehyde solution. two shoots of the seagrass posidonia oceanica l. (delile) from a nearby seagrass meadow were also sampled. the top parts of the shoots were cut, placed in a 0.5 l wide-mouthed sample container and preserved in 4% formaldehyde solution. in all, 22 samples were collected, 20 from the studied substrates and two shoots of p. oceanica. in two substrates, ceramics and concrete, sampling of the bottom side was not possible, due to roughness of the bottom surface of the materials and the inability to sample properly. cell abundance analysis fixed samples were stirred gently in plastic vials until they became homogeneous. subsamples were taken with a pipette and poured into 10 cm3 sedimentation chambers. samples containing posidonia oceanica shoots were shaken vigorously and the surfaces of the shoots gently rubbed to remove the remaining periphytic diatoms and allowed to detach from the shoot surfaces into the suspension. after 24 hours of sedimentation, samples were analyzed in sedimentation chambers following the standard utermöhl (1958) method using an inverted microscope (zeiss axiovert 200, zeiss gmbh). due to the substantial amount of organic matter secreted by the cells and the small size of the cells, it was not possible to determine diatoms under the light microscope, with the exception of cylindrotheca closterium (ehrenberg) reinmann. c. closterium cell abundance was counted separately, due to its high dominance in the samples and the specifi city of the cells, which allowed easy determination. taxonomic analysis for a more detailed taxonomic analysis using scanning electron microscopy (sem), samples needed to be cleaned from cellular residue and organic matter. the cleaning process is based on the oxidation of organic matter with the method of strong acid oxidation. samples were treated according to the von stoch method (hasle and syvertsen 1997) usperiphytic diatoms on different artificial substrates acta bot. croat. 74 (2), 2015 381 ing nitric acid (65%) and sulfuric acid (97%) in the quantity 1:1 and 3:1 in proportion to the sample volume respectively. the sample was stirred and heated to boiling point, which made the sample clear. after the sample was cooled, it was rinsed fi ve times with distilled water to remove the acid. after acid cleaning of the samples, samples were prepared for examination under the sem. a drop of the cleaned diatom material was mounted on aluminium stubs, air dried and gold coated with a sputter coater (s150a sputter coater; edwards ltd., crawley, uk). observations were made with a philips 515 sem (fei co.). diatoms were identifi ed to the genus level according to the classifi cation system of round et al. (1990) using standard determination keys. results upon retrieval, after 30 days of exposure, a visible light brown macroscopic biofi lm could be observed on all submerged substrates with uniform distribution. there were no apparent visual differences between the biofi lms covering the substrates. microscopic analysis showed that the biofi lm consisted mostly of diatoms, with dinofl agellates, microscopic green algae (chlorophyta) and brown algae (phaeophyta) appearing sporadically. a marked difference in abundance of diatoms between different materials was recorded. abundance of diatoms on artifi cial substrates the highest abundance of diatoms (6948 cells cm–2) was recorded on asbestos. diatom abundance on other materials is as follows from greatest to least: painted iron (4622 cells cm–2), wood (4186 cells cm–2), concrete (3666 cells cm–2), posidonia oceanica (2769 cells cm–2), glass (2266 cells cm–2), plastic (2261 cells cm–2), unpainted iron (1180 cells cm–2), rubber (1100 cells cm–2), ceramics (839 cells cm–2), stone (777 cells cm–2) and the lowest recorded abundance was on aluminium (216 cells cm–2) (fig. 2). the mean abundance of diatom cells on studied substrates was 2569 cells cm–2. artifi cial substrates were placed at an angle of 45 º on the sea bed, so the bottom sides of all the substrates, except concrete and ceramics, were sampled and abundance analyzed. the highest abundance on the bottom side of the substrates was on glass (2225 cells cm–2), followed by wood (1550 cells cm–2), rubber (1329 cells cm–2), plastic (653 cells cm–2), asbesfig. 2. abundance of diatoms on top sides of examined substrates. nenadović t., šarčević t, čižmek h., godrijan j., marić pfannkuchen d., et al. 382 acta bot. croat. 74 (2), 2015 tos (466 cells cm–2), unpainted iron (455 cells cm–2), stone (360 cells cm–2), aluminium (50 cells cm–2) and the least was on painted iron (46 cells cm–2) (fig. 3). mean abundance of diatom cells on bottom sides of studied substrates was 793 cells cm–2. after preliminary analysis of the samples using light microscopy, it was noted that the diatom cylindrotheca closterium was the dominant species of diatoms present in all the samples. therefore, in addition to the analysis of the abundance of all diatoms on investigated substrates, the abundance of the diatom c. closterium was analyzed independently. the highest abundance of c. closterium was recorded on wood (3091 cells cm–2), then painted iron (1161 cells cm–2), concrete (861 cells cm–2), plastic (569 cells cm–2), glass (424 cells cm–2), ceramics (383 cells cm–2), asbestos (238 cells cm–2), stone (161 cells cm–2), unpainted iron (140 cells cm–2), p. oceanica (137 cells cm–2), aluminium (16 cells cm–2) and the lowest on rubber (11 cells cm–2). the mean of c. closterium diatoms on the substrates studied was 599 cells cm–2. abundance of c. closterium on the bottom sides of the substrates was also analyzed. the highest was on glass (132 cells cm–2), followed by wood (100 cells cm–2), stone (60 cells cm–2), plastic (50 cells cm–2), rubber (17 cells cm–2), asbestos (16 cells cm–2), painted iron (9 cells cm–2), unpainted iron (5 cells cm–2) and none was recorded on aluminium. the mean abundance of diatoms c. closterium on the bottom sides of studied substrates was 43 cells cm–2. taxonomic analysis taxonomic analysis revealed 41 diatom genera (tab. 1), of which ten were identifi ed as planktonic genera sporadically present on several substrates. the dominant diatom species recorded on all substrates were cylindrotheca closterium, identifi ed and counted with light microscopy, and diatom genera amphora, identifi ed with sem. the iron substrate had the highest diversity (20 taxa), the same as the diversity recorded in natural periphytic assemblages on posidonia oceanica (20 taxa). the lowest diatom diversity was recorded on plastic (4 taxa), concrete (4 taxa) and rubber (2 taxa) (tab 1). other diatom genera with frequent occurrence noted in the samples were nitzschia (8 substrates + p. oceanica) (fig. 4b), cocconeis (7 substrates + p. oceanica) (fig. 4c) and navicula (7 substrates + p. oceanica) (fig. 4d). fig. 3. cylindrotheca closterium abundance on top sides of examined substrates. periphytic diatoms on different artificial substrates acta bot. croat. 74 (2), 2015 383 tab. 1. diatom diversity recorded on all examined substrates. asterisk presents planktonic genera, p.o. – posidonia oceanica, ir. – iron, gls. – glass, al. – aluminium, cer. – ceramics, p.ir. – painted iron, as. – asbestos, st. – stone, w. – wood, pls. – plastic, crt. – concrete, rub. – rubber. substrate p.o. ir. gls. al. cer. p.ir. as. st. w. pls. crt. rub. genus amphora spp. × × × × × × × × × × × × coconeis spp. × × × × × × × × nitzschia spp. × × × × × × × × × navicula spp. × × × × × × × × haslea spp.* × × × × entomoneis spp. × × × × × × fallacia spp. × × × × lyrella spp. × × × × licmophora spp. × × × striatella spp. × × × striatella spp. × × × grammathopora spp. × × × tabularia spp. × × × ardissonia spp. × × campylodiscus spp. × × campyloneis spp. × × actinoptychus spp. × × plagiogrammopsis spp. × dimeregramma spp. × microtabella spp. × paralia spp. × × opeophora spp. × × pleurosygma spp.* × × climaconeis spp. × × diploneis spp. × fragilaria spp. × thalassiosira spp.* × thalassionema spp.* × auricula spp. × parlibellus spp. × toxarium spp. × rophalodia spp. × planktioniella spp*. × astreromphalus spp.* × bactreiastrum spp.* × nenadović t., šarčević t, čižmek h., godrijan j., marić pfannkuchen d., et al. 384 acta bot. croat. 74 (2), 2015 discussion difference in quantitative and qualitative composition in the diatom community recorded on the substrates investigated impliess the preference of diatoms for specifi c substrates. quantitative values of diatom abundance were recorded on the top sides of all the substrates and shoots of the seagrass posidonia oceanica from a nearby meadow. materials of substrates used in this study vary in different physico-chemical properties, e.g. surface roughness, hydrophobicity, surface energy, solubility etc. with the substrates being placed at 45º angle, the top sides of the substrates had expectedly higher abundance of diatoms than the bottom side of the substrates due to the availability of the light diatoms use for fig. 4. scanning electron microscope microphotographs of dominant genera of diatoms found on various substrates: a) amphora sp. on glass substrate, b) nitzchia sp. on ceramic tile substrate, c) cocconeis sp. on aluminium substrate, and d) navicula sp. on painted iron substrate. substrate p.o. ir. gls. al. cer. p.ir. as. st. w. pls. crt. rub. genus membraneis spp.* × chaetoceros spp.* × delphineis spp.* × mastogloia spp. × berkeleya spp. × psammodictyon spp. × proschkinia spp. × tab. 1. – continued periphytic diatoms on different artificial substrates acta bot. croat. 74 (2), 2015 385 photosynthesis. the interest in recording the diatom abundance on the bottom sides of substrates, where light is much more reduced, was in investigating whether any of the substrates acts as a nutrient source for the diatom community. the highest abundance of diatoms in this study was recorded on the asbestos substrate. high abundance of diatoms on asbestos suggests that this substrate is a favorable habitat for diatoms. asbestos is composed of silicate fi bers, and as such, is chemically inert and resistant to biodegradation. fibers could provide high protection from grazers and the space between the fi bers can serve as a nutrient trap. due to its porosity, it absorbs water and prolonged exposure of asbestos in water leads to slow, progressive rinsing of metal and silicate compounds. fibers of the most commonly used chrysotile asbestos (asbestos fi bers are divided into chrysotile and amphiboles) are coated with a brucite layer (mgoh2) which dissolves relatively fast in water. the highly polar surface of chrysotile promotes adsorption of various organic and inorganic substances (speil and leinerveber 1969). as the brucite layer of chrysotile asbestos dissolves, magnesium ions (mg2+) are rinsed into the surrounding medium as mgoh2 (chissick 1985). high concentration of mg2+, as shown by (song and leff 2006) in a study with pseudomonas fl uorescens migula 1895, can augment the production of exopolisaccharids (eps) in bacteria, stabilize the structure of biofi lms and facilitate further surface settlement (costerton et al. 1995). further investigation is needed to ascertain if asbestos used in this research releases mg2+ to the surrounding water and what effect mg2+ has on biofi lm formation. painted iron had the second highest abundance of diatoms, after asbestos. the paint covering the surface of the iron makes the substrate’s surface very smooth, so the high abundance of diatoms on such a smooth surface is unexpected. in their study on microbial colonization, characklis et al. (1990) recorded an increase in the extent of microbial colonization with substratum surface roughness. common physico-chemical properties of metal substrata that could infl uence the settlement of organisms are their high surface energy and hydrophilicity. high surface energy of metal surfaces could promote adhesion, but is soon after immersion reduced by the adsorption of organic particles (kinloch 1990; caillou et al. 2008). hydrophilicity of metal surfaces, as shown in several previous studies (pedersen 1990; becker and wahl 1991; becker 1996), makes adhesion of diatoms more diffi cult as opposed to adhesion on hydrophobic surfaces. the composition of the paint covering with its specifi c, yet undetermined, chemical characteristics could have facilitated the adhesion and settlement of the diatoms and been responsible for the high abundance recorded on coloured iron. abundance of diatoms on wood was similar to that on painted iron. the high abundance of the diatom cylindrotheca closterium on wood substrate greatly contributed to the high overall abundance of diatoms on wood. our study showed that wood has proven to be a very favorable substrate for the diatom c. closterium. wood can pose as a source of nutrients, as suggested in a study by vadeboncoeur and lodge (2000) which concluded that the availability of nitrogen and phosphorus in the water column can depend on the level of wood degradation, and zhang et al. (2013) proposed that wood is a potential source of nitrogen due to saprophyte domination. furthermore, scholz and boon (1993) showed that wood can be a substantial source of carbon for periphytic algae due to bacterial and fungal decomposition. a high abundance of diatoms was also recorded on a concrete substrate. concrete was indicated to be an excellent substrate and habitat for the settling of periphytic organisms in nenadović t., šarčević t, čižmek h., godrijan j., marić pfannkuchen d., et al. 386 acta bot. croat. 74 (2), 2015 »guidelines for marine artifi cial reef materials« by lukens and selberg (2004). it is known that macroalgae can use some elements from the concrete (calcium, aluminium, iron and potassium) that they need for metabolism (berton 2004).the high roughness of concrete can trap detritus in the surface cracks which can serve as an additional source of nutrients (taniguchi and tokeshi 2004), as well as a protection from grazers (bergey and weaver 2004). diatom abundance on posidonia oceanica was similar to the mean abundance recorded on studied artifi cial substrates, but the diversity of diatoms was the same as the highest diversity recorded on the unpainted iron substrate. seagrass is a natural substrate for periphyton and the diatom community on shoots of seagrass was already a settled, more stable community. macrophytes generally serve as a nutrient source for attached periphyton community (cattaneo and amireault 1992). also, epiphytes on shoots of seagrass have better availability to light, as well as to nutrients from the water column and the substrate (hutchinson 1975). additionally, macrophytes as an elastic substrate can sustain wave energy and reduce turbulent effects on the biofi lm surface, which could promote biofi lm formation (pfannkuchen et al. 2012). glass and plastic substrates showed very similar diatom abundance, around the mean value. glass and plastic panels have been widely used as artifi cial substrates for the settlement of diatoms in both marine and fresh water environments by many researchers (cooksey et al. 1984, wahl and mark 1999, caput et al. 2005, nayar et al. 2005, webster and negri 2006, reisser et al. 2014). although both materials are inert in the marine environment, glass and plastic have different physico-chemical properties. glass is a high-energy hydrophilic surface, while plastic is a low-energy hydrophobic surface, and as reported by many studies, diatoms adhere more successfully to hydrophobic surfaces (fletcher 1988, becker and wahl 1991, becker 1996). the shared property of both substrates is the smoothness of the substrate, which could be the cause of the similar diatom abundance. the unpainted iron substrate showed low diatom abundance, but the highest diatom diversity. the hydrophilicity of iron makes the adhesion of diatoms on metal surfaces diffi cult. this is also attributed to the low surface energy caused by organic layer, which is effective in preventing adhesion (townsin and anderson 2009) as mentioned before. metals in sea-water form hydroxides biologically unavailable for uptake by algae (lewandowska and kosakowska 2004). most studies of diatoms, and biofi lm in general, on metal surfaces have investigated diatom biofi lms on hydrodynamic drag on vessels (bohlander 1991, shultz et al. 2011) and the biofi lm development on stainless steel surfaces (schneider 1996, landoulsi et al. 2011). diatom abundance on a rubber substrate was as low as that on unpainted iron. although rubber is inert in the marine environment, some research has focused on leaching of heavy metals and organic compounds from rubber to surrounding water. heavy-metal leaching in seawater could be effective in preventing the colonization of the substrate by fouling organisms, as reported by jelić-mrčelić (2006) in tests with heavy-metal leaching antifouling (af) coatings. zinc, as a major toxicant that rubber leaches into the water environment, was identifi ed by collins et al. (1995) and gualtieri et al. (2005), but according to their study, the amount of zinc being released in that way does not have any signifi cant effect on most marine organisms. the settling of fouling communities on rubber substrates should be studied in more detail, since rubber is one of the most common materials used for artifi cial reefs. also, the disposal of rubber tires has become a widespread issue as large amounts of car tires are being disposed of in the marine environment (personal observation), especially for imperiphytic diatoms on different artificial substrates acta bot. croat. 74 (2), 2015 387 plementing artifi cial reefs and often without any prior investigation of the infl uence of tires on the marine environment and their suitability for the settling of marine fouling organisms. the extremely smooth surface of the glazed ceramic tile could be the reason for low diatom abundance. the results of murdock and dodds (2007) in their study with benthic microalgae showed higher abundance of algae on unglazed than on glazed ceramic tiles. studies by characklis et al. (1990), investigating the adhesion of bacteria on surfaces of different roughness, and woods and fletcher, (1991), investigating adhesion of diatoms on rough and smooth surfaces, came to the same conclusion – that the adhesion for both bacteria and diatoms was more successful on rougher surfaces because of the bigger surface for attachment in comparison with smooth surfaces. low abundance on stone substrata was surprising given that stone is a natural substrate for the periphyton, and has a rough surface texture. periphyton on stone substrates can acquire nutrients from the surrounding water column or microbial regeneration inside the periphyton matrix (stevenson and glover 1993). aluminium substrates had the lowest diatom abundance of all the substrates investigated. the very smooth surface of aluminium can make adhesion of diatoms diffi cult and facilitate detachment of cells from the surface. also, it is possible that aluminium and aluminium oxide could have a toxic effect on periphytic organisms. several studies showed that aluminium dissolved in sea-water inhibits the growth of some planktonic species by reducing the amount of available dissolved phosphorus (dickson 1978, driscoll 1985) or with direct toxic effects (folsom et al. 1986). the interest in recording the abundance of bottom sides of the substrates where light is much more reduced was in investigating whether any of the substrates acts as a nutrient source for the diatom community. the bottom side of the glass substrate had the highest abundance of diatoms. the transparency of the glass plate used in this study made photosynthesis available to the diatom community on the bottom side of the substrate. high diatom abundance on the bottom side of wood substrate could be attributed to wood being a nutrient source for the periphyton community, as mentioned before. rubber substrate showed high abundance of diatoms and was the only substrate that showed higher abundance of diatoms on the bottom side of the substrate. the reason for this could be in the method the rubber substrate was anchored to the sea bed. the rubber used in this research was a black bicycleinner tube anchored around a glass jar found on the location, so the bottom side of the rubber was in direct contact with the glass jar from which the already established diatom community could migrate to the rubber substrate. plastic, asbestos, unpainted iron and stone showed similar low abundances, that can be explained by insuffi cient light availability. aluminium and painted iron had extremely low abundances, which could be attributed to the antimicrobial effects of metals (jain 1990), as well as to the low light intensity on the bottom side of the substrates. abundance of the diatom cylindrotheca closterium, due to its specifi city and high abundances in the preliminary examination of substrates, was recorded for the top and bottom sides of all substrates. by far the highest abundance was on wood substrate, followed by painted iron, concrete, plastic, glass, ceramics, asbestos, p. oceanica, unpainted iron, stone, and the the lowest was on aluminium and rubber. the abundance of the bottom side followed the same trend, with the exception of the glass substrate, which showed the highest abundance of c. closterium due to its transparency and the availability of light. it can be concluded that the wood substrate is the most favorable substratum for the diatom c. closterium. nenadović t., šarčević t, čižmek h., godrijan j., marić pfannkuchen d., et al. 388 acta bot. croat. 74 (2), 2015 this study recorded cylindrotheca closterium and the genera navicula, nitzschia, cocconeis and amphora as dominating diatoms in the periphyton community on the artifi cial substrates studied. it has been shown that pennate diatoms dominate diatom communities in biofi lms (patil and anil 2005, and included citations) with frequently identifi ed pennate genera navicula, nitzschia, cocconeis, amphora (railkin 2004), as was recorded in this study. the majority of studies showed the dominance of pennate genera amphora, navicula, nitzchia (khatoon et al. 2007) and cylindrotheca (molino et al. 2009; dobretsov and thomason 2011; briand et al. 2012) on different artifi cial substrates as well as on fouling release coated substrates (cassé and swain 2006; zargiel et al. 2011). the recorded genera of diatoms in this study are therefore typical periphytic diatoms in biofi lms. in conclusion, this study recorded a qualitative and quantitative difference of diatom abundance in periphyton community among different artifi cial substrates. it was proven that the settlement of diatom community on a specifi c substrate is dependent on light availability, surface roughness and properties. we hypothesize that some substrates, like wood and concrete, serve as a nutrient source to the periphyton community. hydrophobicity of the substrates was not shown to have a major infl uence on diatom settlement. as mentioned in the discussion, there have been many efforts at fi nding favorable artifi cial reef materials that promote the biodiversity of marine organisms. development of periphyton is the fi rst step towards the settlement of higher organisms, and, provided with a suitable substrate, the biodiversity could be enhanced. thus, this initially negative impact that the disposal of litter has on the marine environment can be alleviated with proper management and further research. acknowledgments this work was supported through projects of ministry of science, education and sports of the republic of croatia no. 119-1191189-1228 and 098-0982705-2731. the authors would like to thank ana car, phd for her help in diatom determination and maya sertić, b. sc., for the english language revision. references becker, k. 1996: exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions. microbial ecology 32, 23–33. becker, k., wahl, m. 1991: infl uence of substratum surface tension on biofouling of artifi cial substrata in kiel bay (western baltic): in situ studies. biofouling 4, 275–291. bergey, e. a., weaver, j. 2004: the infl uence of crevice size on the protection of epilithic algae from grazers. freshwater biology 49, 1014–1025. berton, a., escadeillas, g., duchesne, j. 2004: cement paste alteration by liquid manure organic acids: chemical and mineralogical characterization. cement and concrete research, 1823–1835. bohlander, g. s. 1991: biofi lm effects on drag: measurements on ships. proceedings of »polymers in a marine environment«, 1–34. bhosle, n. b., nandakumar, k., venkat, k., dhople, v. m., sawant, s. s., wagh, a. b., kelkar, p. g. 1989: biological and biochemical characterization of microfouling on alperiphytic diatoms on different artificial substrates acta bot. croat. 74 (2), 2015 389 uminium panels placed in the arabian sea. proceedings of the indian national science academy b55 no. 1, 51–56. briand, j. f., djeridi, i., jamet, d., coupé, s., bressy, c., molmeret, m. 2012: pioneer marine biofi lms on artifi cial surfaces including antifouling coatings immersed in two contrasting french mediterranean coast sites. biofouling 28, 453–463. burić, z., caput, k., viličić, d. 2004: distribution of the diatom cocconeis scutellum in the karstic estuary (zrmanja, eastern adriatic sea). biologia 59, 1–8. caillou, s., gerin, p. a., nonckerman, c. j., fleith, s., dupont-gillain, c. c, landoulsi, j., pancera, s. m., genet, m. j, rouxhet, p. g. 2008: enzymes at solid surfaces: nature of the interfaces and physico-chemical processes. electrochimica acta 54, 116–122. caput mihalić, k., viličić, d., ahel, m., burić, z., carić, m. 2005: periphytic algae development in the upper reach of the zrmanja estuary (eastern adriatic coast). vie et milieu-life and environment, 58, 203–2013. cassé, f., swain, g. w. 2006: the development of microfouling on four commercial antifouling coatings under static and dynamic immersion. international biodeterioration & biodegradation 53, 179–185. cattaneo, a., amireault, m. c. 1992: how artifi cial are artifi cial substrata for periphyton? journal of the north american benthological society 11, 244–256. characklis, w. g., mcfeters, g. a., marshall, k. c. 1990: physiological ecology in biofi lm systems. in: characklis, w. g., marshall, k. c. 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sediment acta bot. croat. 79 (2), 2020 1 on-line suppl. tab. 1. results of principal component analysis. pc1 pc2 pc3 eigenvalues 3.50 2.61 1.75 % variance explained 31.9 23.7 15.9 cumulative % explained 31.9 55.6 71.5 eigenvectors secchi depth –0.153 –0.085 –0.527 turbidity –0.238 –0.332 0.347 salinity 0.088 0.403 –0.261 tds 0.095 0.477 –0.269 temperature –0.425 0.265 0.178 ph –0.267 –0.130 –0.423 do2 0.351 –0.406 –0.155 o2 % –0.314 –0.328 –0.014 n –0.428 0.071 –0.112 p 0.040 0.350 0.445 sio2 –0.495 0.077 –0.001 cyanobacterial assemblages in coastal waters and sedimentkolda a, ljubešić z, gavrilović a, jug-dujaković j, pikelj j, kapetanović d 2 acta bot. croat. 79 (2), 2020 acta bot. croat. 79 (2), 2020 3 on-line suppl. fig. 1. cladogram displaying grouping of 100 cyanobacterial asvs, with addition of multi value bar chart of sample frequencies in different asvs (ca – central adriatic, sa – southern adriatic, aquaculture – site type under the influence of fish farms, control – control site type; à sea water sample type, o – sediment sample type). leaf labels are automatically assigned by silva database (black letters), or ncbi genbank database blast search hit results (blue letters). acta bot. croat. 78 (1), 2019 9 acta bot. croat. 78 (1), 9–16, 2019 coden: abcra 25 doi: 10.2478/botcro-2018-0023 issn 0365-0588 eissn 1847-8476 responses of phytochelatin and proline-related genes expression associated with heavy metal stress in solanum lycopersicum dursun kısa bartın university, faculty of science, department of molecular biology and genetics, 74110, bartın, turkey abstract – the expression of stress related-genes against adverse environmental conditions has essential importance for plants. this study, using rt-qpcr, determined the expression of p5cs and pcs genes to investigate their roles in the leaves of tomato plants grown under heavy metal conditions. the expression of the pcs1 gene is significantly induced under such conditions. transcript expression of p5cs1, a gene responsible for proline synthesis, changed depending on heavy metal doses; treatments of cu (20 and 50 ppm), cd and pb (10 and 20 ppm) remarkably increased p5cs1 expression. however, the p5cs1 gene expression at 10 ppm dose of cu and 50 ppm doses of pb and cd was not significantly different from that in control plants. the metal-chelating potency of the extract of tomato leaves exposed to pb and cd was higher than that of untreated plants. the proline content as assessed in the leaves of stressed plants was significantly increased by applications of 10 and 20 ppm of cd and pb, and high doses of cu. in addition, the results showed that the proline content had a positive correlation with the p5cs1 gene expression in tomato leaves under application of these tree heavy metals and that there was a positive relation between the pcs1 gene expression and metal-chelating ability of cd-stressed plants. keywords: gene expression, heavy metal, metal chelating activity, phytochelatin synthase gene (pcs), pyrroline-5-carboxylate synthetase gene corresponding author, e-mail: drsn57@hotmail.com introduction plants sustain their lives under varying external conditions and they respond to environmental stimuli by synthesizing amino acids and peptides such as proline, phytochelatins (pcs), and metallothioneins (mts) (chen et al. 1997). heavy metals are major environmental pollutants for plants, and their accumulation of them in agricultural areas influences the growth, development, and productivity of the plants. although plants need to some heavy metals such as cu, zn and mn for various biochemical pathways, which aid in their growth and development, the excesses quantities of these metals and the presence of other non-essential heavy metals such as cd and pb reduce the plant life and degrade yield quality. heavy metals can interact with thioyl and histidyl groups of proteins, induce the production of reactive oxygen species (ros) production, which and eventually causes the toxicity in plants (sharma and dietz 2009, nagajyoti et al. 2010). the accumulation of heavy metals changes the plant’s molecular, biochemical and physiological responses and plants have developed advanced mechanisms to withstand the abiotic stress factors. plant defense mechanisms in response to heavy metal toxicity include immobilization, exclusion, chelation, compartmentalization and synthesis of stress proteins (toppi and gabbrielli 1999, lee et al. 2002, rellán-álvarez et al. 2006, hossain and komatsu 2012). the overexpression of metal-binding proteins, such as pcs and mts increases the metal-binding ability and tolerance in plants. these proteins are enzymatically derived and synthesized when plant exposed to metal ions (mejáre and bülow 2001). the potential toxicity of heavy metals requires rigorous control to maintain the cell homeostasis by chelation of metal ions with pcs which represent one of the general mechanisms and tolerance. pcs are post-translationally synthesized cytosol proteins. pcs are cysteine-rich and comprise the amino acids glutamine, cysteine, and glycine, and pcs are particularly synthesized in response to heavy metals, including cd, cu, ni, pb, and zn. pcs have the general structure (ɣ-glutamylcysteinyl)n-glycine (n = 2–11) and are synthesized from reduced glutathione (gsh) by the enzymatic reaction of phytochelatin synthase (pcs) (cazalé and clemens 2001, gupta et al. 2004, shen et al. 2010, shanmugaraj et al. 2013, tripathi et al. 2013). moreover, pcs biosynthetic pathway is regulated by a series of mechanisms one of which kisa d. 10 acta bot. croat. 78 (1), 2019 is the regulation of gsh biosynthesis and pcs biosynthesis has been correlated with the expression of gsh (ben ammar et al. 2008). glutathione is synthesized from its constituent amino acids — cysteine, glycine, and glutamate — catalyzing ɣ-glutamylcysteine synthase (ɣ-ecs) and glutathione synthase (gs). the pc synthase (pcs) catalyzes the conversion of glutathione (gsh) into phytochelatin (mejáre and bülow 2001). metal-chelating potency of plants indicates the ability of chelating compounds to bind the metal ions; this ability is dependent on the amount of phytochelatins and metallothioneins, unique phenolic structure of plants and location of the hydroxyl groups in the plant phenolic compounds (wang et al. 2009, flora and pachauri 2010, tito et al. 2011). the adaptation of plants to abiotic stress is achieved by the synthesis of the compatible solutes, such as proline and osmotin. proline accumulation is considered as an adaptive role in response to environmental stresses and this accumulation relies mainly upon the increased synthesis and reduced degradation (verbruggen and hermans 2008). proline has important roles in protecting the cellular homeostasis from stress-induced damage (li et al. 2015). plants have two different precursors involved in proline biosynthesis glutamate (glu) and ornithine (orn). the glu pathway usually occurs in the cytosol and chloroplast under the abiotic stress, whereas the orn pathway is associated with seedling development. proline is synthesized from glutamate via gsa (glutamate-semialdehyde) and p5c (δ1-pyrroline-5carboxylate); the first reaction is catalyzed to gsa by p5cs (δ1 -pyrroline-5carboxylate synthetase) and it spontaneously converted to p5c. then, p5c is reduced to proline by p5cr (δ1 -pyrroline-5-carboxylate reductase) (verbruggen and hermans 2008, wang et al. 2015). the p5cs activity regulates the generation of proline in the biosynthetic pathway, which is controlled by the p5cs transcription. the p5cs gene has two copies: p5cs1 and p5cs2. the p5cs1 gene is believed to be ubiquitously expressed in both vegetative and reproductive organs and is induced by stress, whereas pc5s2 is preferentially expressed in mature plants and in response to incompatible interactions (turchetto-zolet et al. 2009). environmental stress to plants is a global concern and leads to product loss by causing the injuries in plants. although several molecular studies on have been done to study abiotic stress in plants, such as arabidopsis, tomato, bean, rice, and chickpea, many studies are intensively focused on the biochemical and physiological parameters, particularly antioxidant enzymes and total phenolic content (gupta et al. 2004, mattioli et al. 2009, tripathi et al. 2013, chen et al. 2016, singh et al. 2016). in the current study, to understand the molecular basis of solanum lycopersicum under the heavy metals stress, the expression analysis of two selected genes (pcs and p5cs) encoding the key enzymes responsible for phytochelatin and proline biosynthesis respectively are investigated in the leaves of tomato exposed to different doses of cd, cu, and pb. in addition, the proline content which is considered the main index under abiotic stress, and metalchelating capacity depending on the hydroxyl groups in chelating compounds were determined in the leaves of tomato cultivated in heavy metal-polluted boxes. materials and methods plant material and growth conditions the cultivars of tomato (solanum lycopersicum cv. çiko f1) provided by the agricultural faculty of gaziosmanpasa university were cultivated in plastic boxes containing an 11-kg mixture of peat and garden soil (1:1) under unheated glasshouse conditions. the entire experiment was performed as a randomized plot design with 16:8 photoperiod, 25 ± 2 °c, and n (150 ppm), p (80 ppm), k (100 ppm) and b (20 ppm) were applied in sufficient quantities for growth and development. the characteristics of plant soil were ph 7.65, mg = 6.86 ppm, ca = 55.25 ppm, fe = 10.83 ppb, mn = 417.35 ppb, zn = 79.26 ppb, cu = 30.55 ppb, pb = 12.13 ppb, and cd = 0.95 ppb. after the plants had sufficiently grown in approximately 3 weeks, the exposure concentrations of cd, cu and pb from cdcl2, cuso4, and pb(no3)2 as sources were 10, 20 and 50 ppm (mg kg–1). the application of heavy metals was performed three times with an interval of 2 days and this addition was done as a single dose to the pots containing soil, in the early hours of the day. the leaves of tomato were harvested for sampling two weeks after the treatments, and these samples of tomato were kept at –80 °c until rna isolation and analysis of proline and metalchelating capacity. three biological replicates were used for analysis of these assays. isolation of total rna and rt-qpcr analysis tomato leaves were ground into fine powder in liquid n2; the tissue powder obtained was used for rna extraction. the total rna was extracted by using the plant rna mini-preps kit with the ez-10 spin column according to the manufacturer’s protocols (bio basic, ontario, canada). the obtained rna was dissolved in rna-free water, and rna concentration was then quantified by using a µdrop plate (thermo scientific, forssa, finland). the first-strand cdna was synthesized from the isolated total rna; the relative expression levels of the selected genes of tomato were detected by quantitative real-time pcr (rt-qpcr) with the one-step quantitect sybr green rt-pcr kit (qiagen) according to the manufacturers’ protocols. the capillary tubes comprised the master mix 10 μl, primers 2 × 2 μl, rt mix 0.3 μl, and template rna 3 μl; the reaction volume was brought up to 20 μl with rnase-free water. the quantitect sybr green rt-pcr master mix was included in pre-optimized rox as the passive reference dye for fluorescence normalization. the genes sequences for pcs and p5cs were obtained from the ncbi (http://www.ncbi.nlm.nih.gov/nucleotide) databank and they also were verified by the sol genomics network (http://solgenomics.net/feature/17933775/details) using previous studies (ouziad et al. 2005; sangu et al. 2015). gene-specific primers were designed by using the primer3 software (version 4.0) based on the ncbi genbank and the % gc and tm values of primers were checked with an oligonucleotide properties calculator (http://www.basic.northwestern.edu/biotools/oligocalc.html). the following primers were used: pcs1 for 5ˈ– tgctagcatttgttgccaag –3ˈ phytochelatin and proline-related genes in tomato in heavy metal stress acta bot. croat. 78 (1), 2019 11 were considered significant at p < 0.05. the relations among the proline content, p5cs1 expression, pcs1 expression and metal-chelating activity in the leaves of tomato in response to heavy metal stress was analyzed with bivariate (pearson’s) correlation using the individual values of the results. results the effect of heavy metals on the pcs1 gene expression in the leaves of tomato plants exposed to the different concentrations of cu, cd, and pb is shown in fig. 1. compared with control plants, the leaves of treated plants showed significantly higher accumulation of pcs1 transcript due to the heavy metal treatment (p < 0.05). the most evident expressions were observed in plants given the high doses of heavy metals, with the highest expression observed in the plant exposed to 50 ppm of pb. moreover, cd and pb which are not essential for plant development, induced the expression of pcs1 more than cu did. the transcript level of p5cs1 in the leaves of tomato subjected to cu, cd, and pb varied with depending on the heavy metals and their doses (fig. 2). the application of cu to the plants through soil growth medium increased the expression of p5cs1 at the 20 and 50 ppm; however, the expression was not significantly different from that in controls at the low dose of cu (10 ppm) (p < 0.05). the expression level of the p5cs1 gene significantly increased with the applica(forward), 5ˈ– acgtagggaccagaacatcg -3ˈ (reverse); p5cs1: 5ˈ ctgttgtggctcgagctgat –3ˈ (forward), 5ˈ– gacgaccaacacctacagca -3ˈ(reverse) and actin 5ˈ– gggatggagaagtttggtggtgg-3ˈ (forward), 5ˈ– cttcgaccaagggatggtgtagc-3ˈ(reverse). these nucleotide sequences have been deposited in the embl nucleotide sequence database under accession numbers aw154892 and, ay897574 for pcs1 and p5cs1, respectively. the quantitative real-time pcr (rt-qpcr) was performed in triplicate on a light-cycler 1.5 pcr machine (roche) under the following conditions of the qiagen protocols with minor modifications: the reverse transcription step 20 min at 50 °c, the pcr initial activation step 15 min at 95 °c, followed by 50 cycles, denaturation 15 s at 94 °c, annealing 30 s at 54 °c, extension 30 s at 72 °c, cooling 20 s at 40 °c. the fold expression of target genes was normalized with actin as an internal control. the relative transcript expression levels of the selected genes were quantified using the 2−δδct method (livak and schmittgen 2001). determination of proline content the free proline content was estimated using with the ninhydrin reaction (bates et al. 1973). the tomato leaves were crushed into liquid n2, homogenized with 4 ml 3% (w/v) sulfosalicylic acid, and the solution was then filtered with filter paper. the filtrate (1 ml) was reacted with 1 ml of acid-ninhydrin and 1 ml of glacial acetic acid in the test tubes for 1 h at 100 °c, and the reaction was stopped by using ice bath. the reaction mixture was extracted with toluene (2 ml), and the absorbance of the chromophore was read at 520 nm. the proline content was calculated using the calibration curve obtained from pure proline and expressed as mg g–1 fw. metal-chelating ability assay the metal-chelating capacity of the leaf extract was determined by the previously described ferrous ion chelating assay (hsu et al. 2003). tomato leaves (2 g) were grounded into liquid n2, extracted with 10 ml of methanol-chloroform (4:1), and the obtained homogenates were sonicated in the ultrasonic bath for 30 min at ambient temperature. the filtered extract (120 µl) was dissolved in methanol and mixed with 50 µl of 2 mm fecl2, and 200 µl of 5 mm ferrozine. after the vortexing, the mixture was incubated for 10 min at room temperature and then the absorbance was measured at 562 nm. the metal-chelating capacity was calculated by using the following equation with edta as the control: chelating capacity (%) = [1 – (a562 nm, sample/a562 nm, control)] × 100. statistical analysis data analysis was conducted with one-way analysis variance, followed by duncan multiple tests (spss 20.0) to detected the differences between control and treated groups. standard deviation was calculated by using the results of the 2−δδct method. the results are presented as mean ± sd. differences fig. 1. the expression level of pcs1 in leaves of tomato subjected to 0, 10, 20, 50 ppm of cuso4 (a), pb(no3)2 (b) and cdcl2 (c). the gene expression was normalized with the housekeeping actin transcript. bars represent means values ± sd from independent experiments (n = 3). values with the different letters indicate significant difference between means at p ≤ 0.05. fig. 2. the expression level of p5cs1 in leaves of tomato subjected to 0, 10, 20, 50 ppm of cuso4 (a), pb(no3)2 (b) and cdcl2 (c). the gene expression was normalized with the housekeeping actin transcript. bars represent means values ± sd from independent experiments (n = 3). values with the different letters indicate significant difference between means at p ≤ 0.05. r el at iv e ex pr es si on r el at iv e ex pr es si on copper doses (ppm) lead doses (ppm) cadmium doses (ppm) copper doses (ppm) lead doses (ppm) cadmium doses (ppm) 3 2 1 0 6 4 2 0 5 4 3 2 1 0 5 4 3 2 1 0 4 3 2 1 0 0 10 20 50 0 10 20 50 0 10 20 50 5 4 3 2 1 0 a a b b c b a b b c c a b c c 0 10 20 50 0 10 20 50 0 10 20 50 a a a c b b a b b a c a c b a kisa d. 12 acta bot. croat. 78 (1), 2019 they are known to respond to stress by synthesizing various molecules and compounds. the synthesis of proline and pcs are considered as a metabolic response of plants when subjected to the heavy metal stress, and several genes responsible for their synthesis are induced by environmental stimuli. the genes associated with a the plant’s response to stress may lead to a series of biochemical and physiological changes (zhang et al. 2005, goel et al. 2010, patade et al. 2013). in this current study, the transcript expression patterns of pcs1 and p5cs1 genes, proline content, and metal chelating potency were investigated in leaves of tomato plants when grown under heavy metal-containing soil. the results of the present study indicated that the transcript expression of pcs1 is obviously stimulated in the leaves of tomato cultivated in the cu, cd and pb-containing soils. the increases varied with heavy metals and their doses; pb treatment at 50 ppm resulted in a strong increase in the transcript expression of pcs1 compared with control plants. treatment with cu, tion of pb and cd except for the expression at 50 ppm of pb and cd, which showed expression not significantly different from that in the controls. the free proline content in tomato leaves varied with the application of heavy metals, and these results are shown in fig. 3. the proline content in the leaf extract significantly increased with the addition of 10 and 20 ppm of pb and cd, but significantly decreased at high doses (50 ppm) (p < 0.05). the maximum increase in proline content relative to controls was seen with 10 ppm doses of pb. however, compared to control plants, 20 ppm and 50 ppm of cu increased and 10 ppm of cu decreased the proline content in the leaves of tomato. the metal-chelating potency of tomato leaves is shown in fig. 4. the metal-chelating activity was higher in plants cultivated in pb and cd-containing soils than in control plants (p < 0.05). however, the leaf extracts of tomato grown in cucontaining medium showed different chelating potency; the plants grown in soil containing 10 ppm of cu showed lower chelating ability than control plants; however, those grown in soils containing 50 ppm of cu showed higher activity than control plants. the proline content is indicated to have a positive correlation with p5cs1 transcript expression when the tomato plant is subjected to the three heavy metals (tab. 1). the metal-chelating activity has a positive correlation with the pcs1 transcript expression under the cd application. moreover, the metal-chelating activity has a positive correlation with proline content under cu and pb treatments. in addition, p5cs1 transcript expression and the metal-chelating potency were positively correlated in the leaves of tomato plants grown in the pb containing soil (tab. 1). discussion plants have been exposed to the different biotic and abiotic stresses because of changing of environmental conditions, and they respond to these situations with several complex physiological, biochemical and molecular mechanisms for survival, growth, and productivity. the responses of the plants to environmental stresses are quite sophisticated, and fig. 3. the free proline content in leaves of tomato subjected to 0, 10, 20, 50 ppm of cuso4 (a), pb(no3)2 (b) and cdcl2 (c). bars represent means values ± sd from independent experiments (n = 3). values with the different letters indicate significant difference between means at p ≤ 0.05. fig. 4. the metal chelating activity of the leaves extract of tomato subjected to 0, 10, 20, 50 ppm of cuso4 (a), pb(no3)2 (b) and cdcl2 (c). bars represent means values ± sd from independent experiments (n = 3). values with the different letters indicate significant difference between means at p ≤ 0.05. tab. 1. correlation coefficients (r values) among the proline, transcript levels of p5cs1 and pcs1 genes, and metal chelating activity in the leaves of tomato under treatments with cu, pb, and cd. correlation is significant at the 0.01 (**) and 0.05 (*) level (2-tailed). applications p5cs1 pcs1 proline metalchelating copper p5cs1 1 pcs1 0.161 1 proline 0.689* –0.250 1 metalchelating 0.378 0.187 0.884** 1 lead p5cs1 1 pcs1 –0.169 1 proline 0.854** –0.338 1 metalchelating 0.733** 0.400 0.717** 1 cadmium p5cs1 1 pcs1 0.137 1 proline 0.645* –0.132 1 metalchelating 0.201 0.976** 0.064 1 c on te nt o f p ro lin e (m g g –1 f w ) copper doses (ppm) lead doses (ppm) cadmium doses (ppm) 0 10 20 50 0 10 20 50 0 10 20 50 copper doses (ppm) lead doses (ppm) cadmium doses (ppm) 0 10 20 50 0 10 20 50 0 10 20 50 m et al c he la ti ng c ap ac it y (% )300 225 150 75 0 750 600 450 300 150 0 375 300 225 150 75 0 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 a b a c bc b b d c a c b c c a a b a b c a b c b b c a b c bc phytochelatin and proline-related genes in tomato in heavy metal stress acta bot. croat. 78 (1), 2019 13 an essential element for plant growth and development, induced the transcript expression of pcs1, albeit only marginally compared with the other two non-essential elements. it was observed that the leaf extract of tomato plants grown in pb and cd-containing soils exhibited the higher metal chelating ability than control plants; however, the cu influence on the chelating potency of leaves was dose-specific, with highest activity observed at 50 ppm of cu compared to plants grown unpolluted soils. a study on lycopersicon esculentum reported that after the tomato plants were exposed to different concentrations of cdcl2, pcs1 transcript expression was improved in both leaves and roots, and mrna accumulation of pcs1 was significantly higher in roots than in leaves. however, no expression changed was observed in leaves and roots under any concentration of cuso4 treatment (hui et al. 2010). it was stated that in the leaves of tomato, the content of pcs increased on treatment of 1, 5 and 10 µm cd, but it decreased at higher concentrations. in addition, the level of pcs increased in the roots of tomato, and the production of pcs was higher in roots than leaves (ben ammar et al. 2008). the content of pcs in the leaves of solanum nigrum linearly enhanced with the increasing cd concentrations (10, 25, 50 and 100 µg g–1), whereas the content of pcs in the leaves of s. melongena increased under the cd exposures of 25 µg g–1 and evidently declined obviously at higher concentrations (sun et al. 2007). the pcs1 transcript expression in rice cultivars in response to arsenite showed that compared to the control plants, the transcript expression was significantly increased in the roots of the tolerant cultivar but not in the sensitive cultivar. pcs activity showed an important positive correlation with total pc, and pcs activity pc2, pc3, pc4 increased on treatment with arsenite (tripathi et al. 2013, begum et al. 2016). pcs transcript expression was reportedly induced with increasing doses of cdcl2 in brassica cultivars (shanmugaraj et al. 2013). a study performed on the roots of allium sativum l. stated that pcs1 transcript expression was significantly induced by cd, as, and heat shock not by cu and salt stress. however, in a study cu induced evident accumulation of mrna transcript of pcs1 in the leaves of garlic seedlings (zhang et al. 2005). in addition, rt-qpcr analysis showed no significant change in pcs1 transcript expression in roots of alfalfa in respond to cdcl2 and k2sio3 stresses (kabir et al. 2016). another study on metal stress noted that cd and as rapidly increased the formation of pcs in the roots, but other metals, such as cu, zn, co, and ni, did not induce any such change. however, none of these metals initiated any evident production of pcs in the leaves of chickpea (gupta et al. 2004). pcs are the primary binding peptides involved in chelating heavy metal ions in plants (guo et al. 2008). in the current study, the transcriptional profile of pcs1 was investigated in the leaves of tomato subjected to cu, cd, and pb, and the expression of pcs1 was induced by these heavy metals. it is considered that plants increase their hm-binding capacity through chelators to avoid the harmful effect of hm by synthesizing various metal-binding peptides/proteins to withstand heavy metal stress. it has been suggested that one of the possible detoxification and tolerance mechanism is tight control of metal ions, and the expression of the pcs gene and the accumulation of pcs may play a general role in metal homeostasis (cazalé and clemens 2001, begum et al. 2016). the results of proline content and gene expression of p5cs1, a gene responsible for proline synthesis in the present study indicated comparable induction by the application of all heavy metals, except for the induction obtained with 10 ppm doses of cu and 50 ppm doses of cd; however, the level of increase did not occur likewise in the leaves of tomato. the application of pb and cd at 10 and 20 ppm doses resulted in clear increases of p5cs1 transcript expression, and cu at 20 and 50 ppm increased the expression of p5cs1 gene in leaves. further, compared with untreated plants, no significant change was observed in plants treated with a high dose (50 ppm) of pb and cd. compared with the control plants, the free proline content was increased in treated plants by adding 10 ppm and 20 ppm concentrations of pb and cd in plant growth medium; the application of 20 ppm and 50 ppm doses of cu moderately increased free proline content in leaf samples. heavy metal applications, other than mentioned above, decreased the content of proline content when compared with control plants. a previous study showed that cd-induced stress increased the proline content in leaves of tomato treated with 0.2 and 1 mm cdcl2 (gratao et al. 2012). the concentration of free proline in s. melongena reportedly reached the highest level when plants were treated with 10, 25 and 50 µg g–1 cd and declined to the level of control when plants were exposed to the highest cd doses (100 µg g–1) (sun et al. 2007). a previous study on lycopersicon esculentum reported that the expression of p5cs gene significantly increased on 2 h of cold exposure in transgenic tomato; however, in response to a 24-h exposure, the expression level of p5cs decreased to significantly lower levels than in the control plants. however, in wild type tomato plants subjected to 2 or 24 h of cold stress, the expression of p5cs was significantly reduced as compared to that in the untreated control plants. in addition, the proline content in the same work reduced significantly in the wild type plant on 2 and 24-h cold exposure, while the proline accumulation was significantly higher in transgenic tomato plants exposed to cold for 2 h than untreated plants or those exposed for 24 h (patade et al. 2013). the transcript pattern of p5cs1 of barley genotypes was mainly up-regulated in response to drought stress at all exposures (guo et al. 2009). it was stated that the p5cs gene was up-regulated in the heat tolerant tomato genotypes cln1621l (at the mature stage) and cln5915-93d (at the meiosis stage) whereas it was down-regulated in the heat-sensitive genotypes ca4 and cln2498e (sangu et al. 2015). in addition, it was indicated that the proline content increased in tomato exposed to different stress conditions, such as nacl stress, cold stress and nutrient deficit (claussen 2005, zhao et al. 2009, goel et al. 2010, singh et al. 2016). the transcript expression of the p5cs1 gene was slightly increased under the low salt stress (100 mm) and it was significantly induced by higher salt doses (200 and 300 mm); however, the gene expression reduced again slightly at 400 mm nacl in the kisa d. 14 acta bot. croat. 78 (1), 2019 leaves of kosteletzkya virginica. the proline content remarkably increased in roots, leaves and stems after the nacl stress (wang et al. 2015). another study reported that compared to control plants, in leaves of nicotiana benthamiana, water deficit and cuso4 increased the gene expression of p5cs and proline content; however, the water deficit was more responsible than cu for the increases in p5cs gene expression and proline accumulation (ku et al. 2012). the accumulation of stress metabolites, such as proline, is induced by adverse environmental conditions, and proline content varies with the plant species and varieties (verbruggen and hermans 2008). in the current study, p5cs1 transcript expression and the free proline content were examined, and levels of p5cs1 and proline fluctuated according to heavy metal concentrations. however, it can be interpreted from the results that p5cs1 transcript expression and the proline content generally increased except for when the plants were grown under high doses of heavy metals. the gene expression analysis of p5cs1 revealed differential transcript regulation in the leaves of tomato on heavy metal exposures. proline has been considered an important osmo-protectant, and it can play a critical role in adapting to abiotic stress by increasing the osmotic potential of cells. plants alter the transcriptional pattern of stress-responsive genes, such as the p5cs gen involved in the synthesis of functional amino acid and peptides, such as proline (chen et al. 2010). proline accumulates in various plants in response to environmental stress, and its accumulation can affect stress tolerance in multiple ways. proline has various roles, such as that of a protective molecule as an osmolyte or a molecular chaperone as well as an antioxidant molecule working as ros scavenger; it supports to the maintenance of redox balance by contributing to the reductions of free radicals. although proline accumulation is usually considered as a response to stress conditions, it is assumed that the accumulation can change depending on the plant species (szabados and savoure 2009). the production of metal-pc complexes is the evidence of plant metal tolerance, and pc synthesis is considered as a protective mechanism used by plants to protects heavy metal-sensitive enzymes (citterio et al. 2003). in this study, the expression of genes associated with proline and pc was examined, and it is indicated that the expression of p5cs1 has positive correlation with free proline content under heavy metal stress. in addition, there was a positive correlation between the data of pcs1 gene and metal-chelating activity in the leaves of tomato exposed to cd. this study also revealed a strong correlation between metal-chelating potency and proline content in cu and pb applications. it has been previously found that free proline chelates cd ions in plant tissues and converts them into non-toxic cd-proline complexes (sharma et al. 1998). the lack of correlation between pcs1 and metal-chelating activity for pb and cu applications could be compensated by proline, since metalchelating potency and proline showed a strong correlation for these treatments. besides the upregulation of pcs1 in all treatments, proline could play a role as a potential metal chelator for plants growing under to cu and pb presence, thus protecting them from deleterious effects. plants have responded to biotic and abiotic stress factors with a series of mechanisms through the transcriptional regulation of stress responsive, regulatory, and effector genes (patade et al. 2013). by this study, the gene expressions of pcs1 and p5cs1 related with the pcs and proline content, respectively were investigated in the leaves of tomato under heavy metal stress. to further define and elucidate the responses of plants under stress conditions, the expression of genes and their corresponding enzymes associated with pcs and proline metabolisms, such as p5cr (pyrroline-5-carboxylate reductase), p5cdh (p5c dehydrogenase), pdh (pro dehydrogenase), pox (proline oxidase), prot (proline transporter), ɣecs (ɣ-glutamylcysteine synthetase), and pc synthase, should be studied together to reveal the depth of responses in tomato subjected to abiotic stresses, such as heavy metals. acknowledgements this study was conducted with equipment’s in the previous projects which stocked in molecular biology laboratory at gaziosmanpaşa university and by previous project (no: 0315.tgsd.2015) from the ministry of science, industry and technology in the republic of turkey. the author thanks to prof. dr. şaban tekin (genetic engineering and biotechnology 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(lamiaceae) from sinite kamani natural park, eastern balkan range neli h. grozeva1*, mariya a. gerdzhikova2, dimitar h. pavlov2, galia d. panayotova2, mima h. todorova2 1 trakia university, faculty of agriculture, department of biology and aquaculture, stara zagora, bulgaria 2 trakia university, faculty of agriculture, department of plant growing, stara zagora, bulgaria abstract – four populations of betonica bulgarica degen et neič. at sinite kamani natural park were morphologically tested. intrapopulation and interpopulation variabilities were established. the rеlationship between morphological variability, number, area and ecological appurtenance of the studied populations were explored. the results demonstrated that the main source of phenotype variation is intrapopulation variability, mainly due to the age structure of populations. the most variable traits are height of stem and dimensions of leaves. the registered interpopulation variability was affected by the differences in altitude, soil type and differences in environmental conditions and soil properties. indumentum and morphology of generative organs had taxonomic signifi cance for distinguishing b. bulgarica from the other species in the genus, including the species that were morphologically most similar to it – betonica offi cinalis l. keywords: betonica bulgarica, morphology populations, variations * corresponding author, e-mail: grozeva@uni-sz.bg introduction endemic plants are an emblematic symbol of the bulgarian fl ora and one of the most sensitive and vulnerable units in the country’s nature ecosystems (anchev 2011). they comprise 12.8% of the members of the fl ora of bulgaria and demonstrate the specifi city and genetic features of its fl ora (petrova et al. 2005). the bulgarian endemic betonica bulgarica degen et neič. (syn. stachys bulgarica hayek), described by the hungarian botanist a. v. degen and the bulgarian botanist i. neichev in 1906 from materials collected in the central balkan range, grows on open, stony and sandy ground in the oak and beech forest belts in the balkan range (central, eastern) and the thracian lowland (velchev et al. 1992, petrova 2006, genova 2011). the species is morphologically similar to betonica offi cinalis l. (syn. stachys offi cinalis (l.) trevis.). although b. bulgarica is a protected species by the biological diversity act of bulgaria (anonymous 2002) and most of its populations are found in protected territories – the central balkan national park, sinite kamani natural park and in the protected areas of natura 2000 – recent expert evaluations defi ne it as endangered (genova 2011). to this day, the species has not been subjected to detailed morphological studies. the polyphenol content in roots and above-ground parts has been studied (bankova et al. 1999). data about the state of b. bulgarica populations on the territory of balkan range are contained in the management plans of the central balkan national park (yankov et al. 2001) and sinite kamani natural park (grozeva et al. 2004). b. bulgarica was fi rst registered in the eastern balkan range by neichev (1906) but materials were not deposited in bulgarian scientifi c herbaria (som, so, soa). nowadays the species locality specifi ed by neichev is part of the territory of the sinite kamani natural park. grozeva et al. (2004) registered its population in the ablanovo area of the park. currently, the species populations have been registered in three other areas of the park territory – near the microyazovir in karandila area, on slancheva polyana and near upper lift station (grozeva et al. 2014). their state was assessed and their requirements as to soil fertility were studied (grozeva et al. 2014). the objective of the present study was to establish the morphological population variability of b. bulgarica on the territory of sinite kamani natural park, to identify the traits that have the greatest taxonomic value and to seek the relagrozeva n. h., gerdzhikova m. a., pavlov d. h., panayotova g. d., todorova m. h. 82 acta bot. croat. 75 (1), 2016 tionship between the degree of morphological variability, area, number and ecological conditions of the studied populations. materials and methods the subject of this survey were the four currently known populations of b. bulgarica in the territory of sinite kamani natural park (tab. 1). observations were carried out during the vegetation periods of 2013 and 2014, and morphometric tests were performed during the vegetation period of 2014. in each population the sites for taking morphological measurements were selected at the beginning of vegetation according to the presence of suffi cient population density and the presence of b. bulgarica and at least four sites were identifi ed. they were found along the diagonals in both directions of the population and areas in the central parts were deliberately included. the number of plants measured at each point was at least ten with a total of 50 plants used from each population. morphometric measurements were conducted eight times during the vegetation period from april 5th to september 5th. the morphological analysis included 35 quantitative traits (on-line suppl. tabs. 1 and 2). a total of 21 qualitative traits were also analyzed (tab. 2). due to lack of variability in the studied 21 quantitative traits, the data obtained from the four investigated populations are presented in a summarized form in tab. 2. flower and fruit dimensions, specifi cs of leaf epidermis and pollen morphology were recorded in laboratory conditions for fl owers, fruit and leaves collected earlier from the population. ripe fruits collected in isolation bags were measured. after the measurements, nutlets were sown within the borders of each population. all other characteristics were recorded directly on site. to study pollen and nutlet morphology and for a more detailed study of leaf epidermis and type of trichomes the electronic microscope method was used. studies were conducted on dry plant parts in the laboratory at the faculty of chemistry and pharmacy in st. kliment ohridski university of sofi a. at least twenty pollen grains and ten nutlets and leaves from each population were studied. pollen terminology followed erdtman (1952) and punt et al. (1994). data from the microscopic measurements of pollen were presented in generalized form for the species due to lack of signifi cant variability within and between populations. the results obtained for the parameters related to the morphological characteristics were statistically processed with statistica for windows 10. the statistical reliability of differences among various parameters, the interpopulation and intrapopulation variations of traits were established through anova. cluster analysis was applied to group populations by similarity of 35 of the quantitative morphological traits. to allocate populations into groups by strength and direction of infl uence of the basic traits (from the included 35 morphological traits) principal component analysis (pca) was conducted. results the data on the mean arithmetic values of the studied quantitative traits and the variance values of each trait are presented in on-line suppl. tabs. 1 and 2. interpopulation variability was traced based on variance values. the values differed both by individual traits of each population and among populations. the following traits were the most variable among all the populations: height of stem, basal leaf petiole length and stem leaf petiole length. in the populations from ablanovo and slancheva polyana a higher amtab. 1. studied population of betonica bulgarica degen et neič. locality ecological conditions population ablanovo area, n 42°42.638’, e 26°17.262’, 540 m a.s.l. an open meadow on the edge of a mixed deciduous forest comprising carpinus betulus l., quercus robur l., ulmus minor mill., fraxinus ornus l. and crataegus monogyna jacq. the grassland community is dominated by betonica bulgarica.the terrain is very slightly sloped (3°–4°), non-eroded, facing south-east. the bedrock is limestone, the soil type – chromic luvisols (wrbsr, 2006). the area of the population is 1600 m2 and it numbers 440 specimens, incl. 310 fl owering and 50 juvenile ones. upper lift station, n 42°43.100´, е 26°21.619´, 1015 m a.s.l. an open meadow on the edge of a forest consisting of fagus sylvatica l. ssp. moesiaca (k. maly) hjelmquist, picea abies (l.) karst. and pinus sylvestris l. the grassland community is dominated by cereal species.the terrain has a slope of 7°, slightly eroded, dry, facing west. the bedrock is limestone, the soil type is rendzinas (wrbsr, 2006). the plant community is dominated by cereal species. the area of the population is 150 m2 and it numbers 140 specimens, incl. 92 fl owering and 48 juvenile ones. microyazovir area n 42°42.852´, e 26°22.654´, 945 m a.s.l. an open meadow near the cliffs east of the dams. the terrain is very sloped 11°–20°, highly eroded, dry, facing north. the grassland community is dominated by sesleria latifolia degen.the bedrock is quartz porphyry, the soil type – eutric cambisols. due to the reported high level of soil erosion in karandila area, east of the dam, the soil in the studied area was determined as regosols, too (wrbsr, 2006). the population area is 950 m2 and it numbers 95 specimens, incl. 74 fl owering and 21 juvenile ones. slancheva polyana area n 42°43.252´, e 26°21.668´, 1001 m a.s.l. an open meadow on the edge of a mixed forest consisting of fagus sylvatica ssp. moesiaca, pinus silvestris, fraxinus ornus, prunus cerasifera ehrh., acer campestre l., with some bushes of juniperus communis l., crataegus monogyna, rosa canina l., rubus canescens dc. in the grass layer festuca valesiaca schleich. ex gaudin is dominant. the terrain has a slope of up to 11°, non-eroded, dry, facing west. the bedrock is limestone, the soil type – eutric cambisols. the area of the population is 724 m2 and it numbers 215 specimens, incl. 155 fl owering and 60 juvenile ones. bulgarian endemic betonica bulgarica degen et neič. morphology acta bot. croat. 75 (1), 2016 83 plitude of variability was registered for other traits as well: stem leaf petiole length, raceme and branch length. the least variable in all the populations were the following traits: basal leaves length/width ratio, raceme leaves petiole length, fi rst raceme leaves width, third raceme leaves width, corolla width base and those characterizing seed (length, width, length/width ratio). in the ablanovo population a low level of variability was registered for other traits as well: corolla length/width top and calyx length, in the population at slancheva polyana – second raceme leaves petiole length, in the population at upper lift station – raceme branch length and third raceme leaves length, and for that at microyazovir – raceme branch length, second raceme leaves petiole length and calyx length/width ratio. in the population at slancheva polyana no variability was found for third raceme leaves length and third raceme leaves length/width ratio or in the population at the microyazovir for third raceme leaves length/width ratio. the anova results (on-line suppl. tabs. 1 and 2) showed that intrapopulation variability was dominant in the total variability. higher interpopulation variability was found only for petiole length of basal leaves. the data from the unweighted pairgroup average (upga) cluster analysis based on morphological pairwise similarities (euclidean distances between population centroids) showed the greatest similarity in the entire complex of qua litative traits among specimens from the populations at slancheva polyana and microyazovir (fig. 1). the greatest differences were recorded for the population from the ablanovo area. factor scores based on the principal component analysis of the distribution of populations according to the 35 morphological traits studied showed that the population at ablanovo had positive values for both factor 1, and factor 2 (on-line suppl. tab. 3). the populations at microyazovir and slancheva polyana had positive values for the second tab. 2. studied qualitative features of betonica bulgarica degen et neič. feature 1. colour of stem light green. 2. indumentum of stem non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; peltate glandular trichomes. 3. shape of leaf lamina rounded to elliptic and lanceolate, with a blunt tip, concave base and scalloped margin for annual plants developed from seeds; lanceolate with a blunt tip, concave or asymmetric base and scalloped edge at the biennial and perennial plants developed from seeds and rhizomes. 4. colour of leaf lamina green to light green. 5. indumentum of leaf lamina adaxial and abaxial leaf surface – non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; peltate glandular trichomes. 6. shape of epidermal cell of leaf lamina adaxial and abaxial leaf surface – elongated, polygonal, with varied size. 7. type of stomata аbaxial leaf surface – anomocytic, diacytic, paracytic. 8. indumentum of leaf petiole non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; peltate glandular trichomes. 9. type of infl orescence flowers gathered in fl ower vertebrae, forming tight cyme. the bottom vertebra is often separated at a distance from the cluster. 10. shape of bracts lanceolate, with smooth edge, awned peak. persists in the fruit. 11. indumentum of bracts adaxial and abaxial leaf surface – non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes along the edge and the primary vein; peltate glandular trichomes. 12. shape of calyx segments sepals fused in a tube, calyx teeth styliform, almost equal to the tube. calyx retained in the fruit. 13. colour of calyx segments light green to dark purple. 14. indumentum of calyx segments calyx tube – non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; non glandular stellate multicellular trichomes with 2–3 rays; peltate glandular trichomes. calyx teeth – non glandular multicellular, simple, uniseriate, hard, with smooth surface and peltate glandular trichomes. 15. shape of corolla segments corolla tube narrow, ± curved, upper lip entire, sometimes binary, lower lip ternary with wavy middle part and egg-shaped side parts. 16. colour of corolla segments light purple 17. indumentum of corolla segments non glandular multicellular, simple, uniseriate with micropapillae cultural surface trichomes; non glandular stellate multicellular trichomes with 2–3 rays; peltate glandular trichomes. 18. colour of anthers upon opening of the fl ower – yellow, during fl owering – purple. 19. shape of nutlet triangular, elongated, the outside almost fl at, the edges with narrow wings, which at the top edge go into irregularly toothed membranous appendage. 20. colour of nutlet dark brown 21. nutlet surface smooth formed by oblong cells variable in size grozeva n. h., gerdzhikova m. a., pavlov d. h., panayotova g. d., todorova m. h. 84 acta bot. croat. 75 (1), 2016 factor but negative ones for the fi rst one. the population at the upper lift station area was negative for both factors. the population from ablanovo exceeded the other populations by 11 out of a total of 35 studied traits. height of plants, length, width of basal leaves, petiole length of basal leaves, length and width of stem leaves were signifi cant. the data related to the qualitative traits studied are presented in tab. 2; figs. 2, 3a–b and 4. in the four studied populations no signifi cant variability in the types of trichomes and stomatas, pollens and nutlet morphology was established. the epidermal surfaces of all examined plants showed both non-glandular and glandular trichomes (tab. 2). stem, leaves, bract, calyx and corolla were more or less covered with peltate glandular trichomes (figs. 2 and 3a–b). on the fresh parts of b. bulgarica small capilate trichomes were found, practically invisible on the herbarised plant parts due to their small size. stem, leaf petioles, bracts and the calyx tube were covered with non-glandular multicellular, simple, uniseriate trichomes with micropapillae cuticular surface (figs. 2a–e and 3a). the calyx and corolla tube of b. bulgarica were covered with non-glandular stellate multicellular trichomes with two to three rays (figs. fig. 1. dendrogram of the cluster analysis of four betonica bulgarica degen et neič. populations based on 35 traits of vegetative and generative morphological variability (see on-line suppl. tabs. 1 and 2). fig. 2. scanning electron micrographs of betonica bulgarica degen et neič.: a) stem surface; b) abaxial leaf surface; c, d) adaxial leaf surface; e) petiole surface; f) adaxial bract surface; g) abaxial bract surface; h) calyx tube surface; a – non-glandular multicellular uniseriate trichomes, b – peltate glandular trichomes; c – non-glandular stellate multicellular trichomes; d – paracytic stomata; e – anomocytic stomata; f – diacytic stomata. fig. 3. scanning electron micrographs of betonica bulgarica degen et neič.: a) calyx teeth surface; b) corolla surface; c–d) pollen grains; e) stigmas; f) whole nutlet dorsal face; g) whole nutlet ventral face; h) details of nutlet surface; a – non-glandular multicellular uniseriate trichomes, b – peltate glandular trichomes; c – non-glandular stellate multicellular trichomes; d – muri; e – lumina. bulgarian endemic betonica bulgarica degen et neič. morphology acta bot. croat. 75 (1), 2016 85 2h and 3b). by the calyx teeth non-glandular multicellular hard non-branched trichomes of up to 2 mm and branched ones with three to fi ve rays were found (fig. 3a). three types of stomata were established in the survey – anomocytic, diacytic and paracytic, the anomocytic being observed most frequently (tab. 2 and figs. 2b–d, f–g). among the four studied populations no signifi cant variability in pollen morphology was established. the pollens of b. bulgarica were monad, medium sized, and 3-zonocolpate (figs. 3c–d). polar axis (p) was between 30.2 and 38.5 μm, equatorial axis (e) 20.4–24.2 μm and p/e rate 1.3–1.6. the coloration was reticulate. the width of muri was 0.3– 0.6 μm (fig. 3d). the luminas were slightly elongated with length ranging within 0.8–1.6 μm and width – 0.4–1.2 μm. the nutlets of b. bulgarica were dark brown, triangular, elongated, the outside almost fl at, the edges were with narrow wings, which at the top edge went into irregularly toothed membranous appendage (tab. 2 and figs. 3f–h). the three sides of the nutlets had even surfaces formed by oblong cells variable in size and usually with no trichomes (fig. 3g); peltate trichomes were only found on the membranous appendage of single nutlets (fig. 3f). discussion intrapopulation variability was dominant in the phenotypic variability of b. bulgarica (on-line suppl. tabs. 1 and 2). our studies showed that a possible reason for this was the age structure of the population. due to the perennial type of the species and the possibilities for seed and vegetative (through roots) reproduction within each population, well-developed perennials were observed. there were also annuals that propagated through seeds and during the fi rst year formed only a leaf patera, as well as biennials that propagated through seeds, which did not always reach fl owering and fruiting. in addition, there were vegetatively propagated plants in which the basal leaves were often reduced. morphometric analyses found that the size of vegetative organs in each of these groups of plants varied signifi cantly within the range of each of the four populations studied. a certain intrapopulation variability was also registered for the size of generative organs in each of the age groups. this determined the dominant role of intrapopulation within total variability along the entire complex of morphological traits. the height of stem in the different groups in each population varied to a great extent, due to the greatest registered trait variance values (on-line suppl. tab. 1). the competitive relations which arise among the specimens of b. bulgarica and those of other species in the community, infl uenced the high variability of that trait. in the populations of upper lift station, microyazovir and slancheva polyana, the uneven distribution of moisture due to the ground slope had an additional effect. it was visible that the specimens located on the steepest parts of the population had shorter stems than those located on even ground. higher variability was registered for the traits petiole length of basal and stem leaves (on-line suppl. tab. 1). a typical property of the species is that basal leaves always have petioles longer than the laminae, stem leaves have petioles shorter than the laminae and the upper pair are sessile (koeva 1989). the data from morphometric measurements showed that in some of the specimens the petioles of basal leaves were up to 7 times longer than the lamina and for a period of 20 days their dimensions increased manifold. since the vegetative development of b. bulgarica lasts from the end of march to the fi rst half of april, when the majority of the dominant species are well developed, a rapid extension of leaf petioles results from the competitive relations and provides the leaf laminae with appropriate access to light. dominant for this trait in the common variability was the interpopulation one (on-line suppl. tab. 1) due to the varied composition of communities in the four populations and the different competitive relationships. the greatest amplitude of variability was registered for the population at the ablanovo area, where the altitude was the lowest and b. bulgarica dominated in the grass community (tab. 1). in specimens that had competitive relations with dominant species of that population, the length of leaf petioles increased up to 2–3 times during a period of 20 days. at the same time in plants that were neighbours to other specimens of b. bulgarica, also at the beginning of their vegetative development, the length of leaf petioles and the growth rate were far smaller. the registered high amplitude of variability led to the higher average values of the petiole length of basal and stem leaves for this population. the conducted two-year study showed that in all populations that were in competitive relations with cereals or other plant specimens of b. bulgarica, leaf petioles of basal and stem leaves had a greater size. regardless of the registered variability, the variation in the size of leaf petioles of the studied specimens in the four populations was within the species variability as stem leaves always have shorter petioles than the basal ones and the upper pair of stem leaves are sessile. variability was also found for the dimensions of leaf laminae within each population. plants developing from seeds during the fi rst year had smaller, rounded, elliptic to lanceolate laminae with a blunt tip, scalloped edge and concave base (figs. 4a–b). biennial plants produced from seeds (figs. 4c–d), as well as the other perennial specimens, which reproduced from seeds or vegetatively (figs. 4e–f), had lanceolate lamina, obtuse apex, asymmetric or concave bases and their dimensions varied. the leaf laminae of biennial plants developed from seeds were always smaller than those of the others (fig. 4). regardless of the registered variability in the dimensions of leaf laminae, the ratio between their length and width was one of the traits that had lower variability. with annual specimens that developed from seeds the ratio between length and width of leaf laminae was 1–1.5, and in the other age groups – from 1.5 to 4.5. regardless of the differences in their size and shape, leaf laminae are always covered with trichomes. the laminae of annual specimens developed from seeds had the thickest trichome cover of all the populations. in specimens from the other age groups no regularities were found in the degree of trichome covering. variability of basal leaves was established for another species from the genus – b. offi cinalis (dušek et al. 2010). grozeva n. h., gerdzhikova m. a., pavlov d. h., panayotova g. d., todorova m. h. 86 acta bot. croat. 75 (1), 2016 the dimensions of fl oral parts (fig. 5 and on-line suppl. tab. 2) in each of the four populations varied slightly and corresponded to those mentioned for the species by koeva (1989) and genova (2011). certain differences were found in the colour of anthers. according to data by koeva (1989) the stamens of the species have yellow anthers. our observations showed that the anthers were yellow only immediately after opening of the fl ower and after that they were purple. the greatest similarity out of the entire set of quantitative traits (fig. 1) was registered between the populations at slancheva polyana and microyazovir. in these two populations, the values of the greater part of the studied quantitative traits were very close (see on-line suppl. tabs. 1 and 2). most probably the similarity found was infl uenced by the similarity in soil type and soil reaction that had contributed to the formation of similar ecological conditions within their boundaries. the soil type in both areas is eutric cambisol. the soil is characterized by sandy clay loam soil texture, weakly structured with granular structure and acidic soil reaction (grozeva et al. 2014). a certain similarity of the populations from microyazovir and slancheva polyana was found with the population from upper lift station, which has an intermediate altitude compared to the other two populations, signifi cantly smaller area and a different soil type (fig. 1 and tab. 1). domifig. 4. variation of leaf lamina of betonica bulgarica degen et neič.: a–b) annual plants developing from seeds; c–d) biennial plants developing from seeds; e–f) perennial plants. fig. 5. betonica bulgarica degen et neič. – branch with upper leaves and infl orescence (photo m. srebreva). bulgarian endemic betonica bulgarica degen et neič. morphology acta bot. croat. 75 (1), 2016 87 nant within the community in all three populations were cereal species, and moisture availability of the soil was lower during the greater part of vegetation. the greatest difference compared to the populations of microyazovir, upper lift station and slancheva polyana was observed for the ablanovo population (fig. 1), where b. bulgarica was the dominant species. the area of the population was considerably bigger and the altitude signifi cantly lower (tab. 1). the soil type also as differed and throughout the entire vegetation soil moisture was moderate. according to our previous investigation (grozeva et al. 2014), the soil type in that area is chromic luvisol (anonymous 2006). the studied soil is characterized as moderately structured, with crumb structure in the upper surface horizon and a slightly acidic soil reaction with values of ph up to 6.6. the differences in ecological conditions most probably led to the appearance of the registered morphological differences in the species of that population from those of the other three populations. dušek et al (2010) also evaluated the morphological characteristics between plant populations of different origin from another species of genus betonica – b. offi cinalis. the most conservative traits in all populations were those characterizing trichomes, stomata, pollen and nutlet morphology (figs. 2 and 3). the taxonomic value of the indumentum and its importance in systematic and phylogenetic relationships was well known in lamiaceae (abu-assab and cantino 1994, navarro and el oualidi 2000, dinç and őztűrk 2007, giuliani et al. 2008). trichomes are among the most useful taxonomic characters for distinguishing the species of genus betonica and genus stachys. the epidermal surfaces of all examined plants of b. bulgarica had both non-glandular and glandular trichomes. the peltate and capillate glandular trichomes observed on stem, leaves, bract, calyx and corolla were registered by giuliani and maleci bini (2012) in other species of genus stachys and genus betonica. the peltate trichomes established for b. bulgarica (figs. 2a, c, h, figs. 3a–b) had the morphological characteristics of the trichomes specifi ed by giuliani and maleci bini (2012) as type a, whereas the capillate trichomes observed in fresh plants were specifi ed as type b. according to giuliani and maleci bini (2012) type a peltate trichomes consist of one basal epidermal cell, one neck cell and a multicellular head with a large subcuticular space where the secretion is accumulated, whereas type b capillate trichomes consist of one basal epidermal cell, one stalk cell and a head of two to four cells with a thin subcuticular space where secretion is accumulated. type a trichomes are mainly essential oil producers, whereas type b have an exclusive or prevalent polysaccharide content (giuliani et al. 2008). the non-glandular multicellular, simple, uniseriate trichomes observed on stem, leaf petioles, leaves, bracts and calyx tube of b. bulgarica were also found by naidu and shan (1981) on b. offi cinalis. the non-glandular stellate multicellular trichomes with two to three rays found on calyx and corolla tube on b. bulgarica (fig. 2h, fig. 3b) were similar to the trichomes on calyx and corolla surface of b. offi cinalis and b. scardica as pointed out by giuliani and maleci bini (2012), where, the rays were two to eight. the non-glandular multicellular hard non-branched trichomes up to 2 mm long and branched with three to fi ve rays typical of the calyx teeth of b. bulgarica (fig. 3a) were not mentioned by koeva (1989) for the calyx teeth of the morphologically closest bulgarian species – b. offi cinalis. salmaki et al. (2009) observed numerous types of trichomes of iranian stachys taxa (incl. betonica) that showed considerable variability among different species, but were constant among different populations of one species, and therefore afford valuable characters in the delimitation of sections and species. stomata were described on the leaves of some lamiaceae by different researchers (el-gazza and watson 1970, inambad and bhatt 1972, naidu and shan 1981). from their study of this family el-gazza and watson (1970) reported that the stomata were predominantly diacytic, predominantly anomocytic, predominantly anisocytic and a mixture of anomocytics and anisocitycs. inambad and bhatt (1972) found that in 33 lamiaceae species diacytic was the most frequent type of stomata. naidu and shan (1981) pointed out that out of 34 lamiaceae species the diacytic was the most frequent type, and the anomocytic was the next most abundant type. our studies confi rmed the data known from literature since in all studied plants from the four populations 3 types of stomata have been found – anomocytic, diacytic and paracytic, anomocytic being the most frequent (figs. 2b–d, f–g). stomata were evenly distributed throughout the epidermis of abaxial surface without any defi nite pattern of orientation. according to naidu and shan (1981) the anomocytic is the most frequent type in stachys offi cinalis (syn. betonica offi cinalis), s. palustris and s. recta. investigations of pollen morphology in lamiaceae were essential from the point of view of improving the classifi cation within this family (abu-assab and cantino 1994). according to dinç and őztűrk (2007) the pollen morphology of the genus stachys (incl. betonica) is not well known. palynological studies on the bulgarian betonica taxa have not been conducted so far. pollen morphology of b. bulgarica (figs. 3c–d) exhibited the features specifi ed by moore et al. (1991) as stachys sylvatica type. typical for that type are: trizonocolpate pollens with reticulate ornamentation and lumina more or less uniform in size. moore et al. (1991) included in the group with stachys sylvatica type pollen eight of the species studied by them, b. offi cinalis among them. the systematic importance of nutlet morphology in genus stachys and genus betonica was underlined by demissew and harley (1992), martin mosquero et al. (2000), etc. in a comparison of the nutlet characteristics of b. bulgarica with those published by various researchers (demissew and harley 1992, martin mosquero et al. 2000, dinç and doĝan 2006, salmaki et al. 2008, satil et al. 2012) for other species in the genus, the greatest similarity according to nutlet shape was established with b. offi cinalis. regardless of the similarity, nutlets of b. bulgarica differed from those of b. offi cinalis by their size (on-line suppl. tab. 2) as well as by lack of differences in the reticulate pattern among the three sides of the nutlet surface and the lack of non-glandular trichomes on it (figs. 3f–h). in conclusion, the results from the present study demonstrated that the registered intrapopulation variability of the grozeva n. h., gerdzhikova m. a., pavlov d. h., panayotova g. d., todorova m. h. 88 acta bot. croat. 75 (1), 2016 four studied populations of the bulgarian endemic b. bulgarica was based mainly on their age structure, while the established interpopulation variability derived from differences in altitude, soil type and differences in environmental conditions and soil properties. the most taxonomic signifi cance for distinguishing b. bulgarica from the other species in the genus, including from the species that is morphologically most similar to it – b. offi cinalis – was found in the indumentum and morphology of generative organs, especially calyx segments and nutlets. acknowledgement this work was fi nancially supported under contract no opos 2-36 / 24.07.2013 to project no 5103020-15-658 for sinite kamani natural park, funded under contract no 5103020-с-002 operational programme “environment 20072013”. references abu-assab, m. s., cantino, p. d., 1994: systematic implications of pollen morphology in subfamilies lamioideae and pogostemonoideae (labiatae). annals of the missouri botanical garden 81, 653–686. anchev, m., 2011: general characteristics of bulgarian fl ora and fl oristic regions in bulgaria. in: peev, d. 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a,bdifferences among parameters in the points mentioned with equal letters are not statistically signifi cant. ablanovo area slancheva polyana upper lift station microyazovir ssv, % 1. height of stem, cm 73.93 50.18 60.84 46.39 58.43 224.24 158.79 88.32 196.51 2. number of basal leaves 5.66 6.22 6.47 6.02 97.46 2.09 3.34 3.59 5.17 3. basal leaves length, cm 11.12 8.33 7.40 8.01 77.83 9.70 10.23 6.16 7.16 4. basal leaves width, cm 5.75 4.40 4.14 4.00 77.58 1.91 2.10 1.87 2.14 5. basal leaves length/width ratio 1.95ab 1.90a 1.86b 2.09 96.60 0.16 0.15 0.17 0.27 6. basal leaves petiole length, cm 26.30 13.11 12.29 11.20 41.50 46.83 21.88 21.38 32.53 7. stem leaves length, cm 6.03 5.10 5.28 4.79 95.79 5.73 4.95 4.14 4.12 8. stem leaves width, cm 3.18 2.91 2.92 2.59 97.46 2.15 1.64 1.45 1.26 9. stem leaves length/width 2.04 1.87 1.90 1.957 98.68 0.37 0.39 0.25 0.3 10. stem leaf petiole length cm 8.14 5.77 6.66 5.29 94.40 31.31 14.07 19.90 14.15 11. raceme length, cm 12.81 7.82 10.45 6.08 82.07 31.14 13.98 71.91 7.58 12. first racemes leaves length, cm 2.64 2.41 2.88 2.19 95.16 1.22 1.05 1.37 0.85 13. first racemes leaves width, cm 1.01 0.89 1.14 0.83 94.86 0.27 0.18 0.27 0.22 14. first racemes leaves length/width ratio 2.91а 2.99а 2.68 2.98а 99.02 1.09 3.39 0.40 1.34 15. raceme leaves petiole length, cm 0.64 0.39 0.48 0.31 85.41 0.13 0.02 0.10 0.02 16. branch length, cm 13.03 5.03 12.86 4.94 69.61 70.88 7.20 20.98 2.78 17. raceme branch length, cm 4.00 1.80 2.66 1.44 73.91 7.03 2.04 0.20 0.24 18. second raceme leaves length, cm 1.35 1.34 2.08 1.37 84.86 0.68 0.57 0.55 0.39 19. second raceme leaves width, cm 0.44 0.53 2.36 0.53 77.85 0.09 0.11 9.29 0.08 20. second raceme leaves length/width ratio 3.19 2.75a 2.21 2.71a 84.81 0.43 0.61 1.37 0.26 21. second raceme leaves petiole length, cm 0.00 0.21 0.33 0.99 98.18 0.00 0.02 0.49 0.01 22. third raceme leaves length, cm 1.36 1.00 1.41 1.98 58.69 0.47 0.00 0.19 7.35 23. third raceme leaves width, cm 0.46 0.30 0.46 0.98 59.76 0.05 0.00 0.01 0.02 24. third raceme leaves length/width ratio 3.05a 3.33a 3.14a 3.00a 99.28 0.77 0.00 0.51 0.00 1 grozeva n. h., gerdzhikova m. a., pavlov d. h., panayotova g. d., todorova m. h. acta bot. croat. 75 (1), 2016 on–line suppl. tab. 2. mean (upper numbers in each row), variance value (lower numbers in each row, %) and percentage of intrapopulation variation (ssv, %) in the overall morphological variation of betonica bulgarica degen et neič. populations for each of the 11 observed generative traits; a,bdifferences among parameters in the points mentioned with equal letters are not statistically signifi cant; l/w – length/width ratio. ablanovo area slancheva polyana upper lift station mikroyazovir ssv, % 25.calyx length, cm 7.78a 7.72a 7.93a 7.81a 99.24 0.25 0.46 1.61 0.75 26. calyx width, cm 2.80a 3.20b 2.89ab 3.15ab 99.24 0.75 0.56 0.33 0.55 27. calyx l/w 2.72a 2.47b 2.74ab 2.43b 93.21 0.36 0.18 0.43 0.21 28. corolla length, cm 10.73 11.64a 11.88a 11.83a 89.42 2.11 1.55 2.51 1.44 29. corolla width base, cm 1.06a 1.09a 1.09a 1.10a 97.49 0.03 0.04 0.05 0.04 30. corola l/w base 10.27 10.64a 10.89a 10.74a 88.63 4.08 4.63 13.15 5.16 31. corolla width top, cm 5.45a 5.02a 4.06 5.06a 89.17 2.52 2.59 1.08 2.77 32. corolla l/w top 1.95a 2.31a 2.92a 2.33a 96.51 0.13 5.94 0.21 5.94 33. nutlets length, mm 3.93a 3.71a 3.82a 3.85 76.05 0.10 0.11 0.11 0.18 34. nutlets width, mm 1.75a 1.61b 1.69ab 1.77a 90.66 0.058 0.030 0.029 0.044 35. nutlets length/width ratio 2.24a 2.38a 2.27a 2.22a 96.82 0.206 0.154 0.176 0.289 on–line suppl. таb. 3. factor scores based on principal component analysis and correlations of all betonica bulgarica degen et neič. populations – average of 1600 measurements for 35 traits. factor 1 factor 2 factor 3 ablanovo 1.434345 0.3975 –0.18615 slancheva polyana –0.38116 0.16364 1.441506 upper lift station –0.17819 –1.43334 –0.40469 microyazovir –0.875 0.87221 –0.85067 2 82 acta bot. croat. 78 (1), 2019 acta bot. croat. 78 (1), 82–90, 2019 coden: abcra 25 doi 10.2478/botcro-2019-0010 issn 0365-0588 eissn 1847-8476 a cytogenetic and pollen study of annual medicago species from soummam valley (northeastern of algeria) linda djafri-bouallag*, malika ourari, mohamed sahnoune laboratory of ecology and environment, department of environment biological sciences, faculty of nature and life sciences, université de bejaia, targa ouzemmour, 06000 bejaia, algeria abstract – this paper reports a cytogenetic study of eight medicago l. species sampled from the soummam valley (northeastern algeria). chromosome numbers and meiosis irregularities during microsporogenesis were explored. pollen viability rate and pollen size were also examined. the studied taxa are diploid and display bivalent pairing and regular chromosome segregation during meiosis. although meiosis appears regular, some anomalies were detected in relatively high cumulated rates (14.66%–26.14%). the most common meiotic abnormality examined here is related to cytomixis (from 14.66% in m. littoralis to 25.83% in m. laciniata). other anomalies were also detected, including chromatic bridges, asynchronous divisions, micronuclei and multipolar cells. consequently, the species exhibited varying percentages of pollen viability (from 70.11% in m. laciniata to 99.14% in m. littoralis). pollen viability was negatively correlated with meiotic abnormalities (pearson correlation coefficient r = −0.72, p = 0.043). the pollen grains were also heterogeneous in size. medicago truncatula gaertn. and m. laciniata (l.) miller presented the most variable pollen size (relative standard deviation exceeding 19%). medicago littoralis is distinguished from other species by possessing homogeneous and large sized pollen (relative standard deviation rsd = 6.73 %). the cytogenetic and pollen data provided by this study are discussed in the context of species systematics and in the perspective of genetic improvement. keywords: chromosome numbers, medicago, meiotic abnormalities, microsporogenesis, pollen grain area, pollen viability, soummam valley * corresponding author, e-mail: djafribouallaglinda@gmail.com introduction medicago l. is one of the largest genera in the fabaceae family with 87 species including shrubs and herbaceous plants (small 2011). medicago species are characterized by flowers exhibiting a unique explosive pollination mechanism (small 2011). taxonomically, this genus belongs to the trifolieae tribe and to the trigonellinae sub-tribe. basing their work on two nuclear genes (cngc5, β-cop) and one mitochondrial dna sequence (rps 14-cob), maureira-butler et al. (2008) estimated the divergence time between medicago and its sister clade trigonella as having been ca 15.9 million years ago. the ancestral medicago forms originated from the western asia and mediterranean regions and are supposed to be perennial and preferentially allogamous (lesins and lesins 1979). the derived annual species would have differentiated six to seven million years ago (prosperi et al. 1995) and now have highly variable distributions depending of human interference. annual species such as m. truncatula, m. polymorpha, m. orbicularis or m. minima are considered colonizers (olivieri et al. 1991, prosperi et al. 1995) and they are widely distributed in mediterranean-type climates. other species including m. coronata are rare or endemic and restricted to particular regions. with their ability to fix atmospheric nitrogen, medicago species are of real interest in improving soil fertility, in mainly arid and semiarid areas. these species are also used in crop rotation and pest management plans to maintain a productive soil. in addition to their agricultural and ecological interests, medicago species are used for animal nutrition (forage) and human consumption due to their high protein level. medicago is recognized to be one of the most important genera of pasture plants in the world (heyn 1963). moreover, according to small (2011), medicago has potential for the production of biopharmaceuticals, bioplastics, cellulose and biofuel. medicago truncatula is used as model plant for legume biology as a result of varimicrosporogenesis and pollen in medicago l. acta bot. croat. 78 (1), 2019 83 ous biological characteristics (cannon et al. 2006) including diploidy, small genome size, self-pollination, and rapid reproductive cycle. the systematics of the genus is based on morphological and floral (heyn 1963, lesins and lesins 1979, small 2011) and biochemical (classen et al. 1982, jurzysta et al. 1992) traits. pollen morphology (small 2011) and interspecific hybridization (simon 1965, simon and millington 1967, lesins and lesins 1979) were also studied. recent phylogenetic studies involved chloroplast (matk, trnk/matk), mitochondrial (rps14-cob) and nuclear (ga3ox1, its, ets, cngc5, β-cop) dna sequences (steele et al. 2010, hu et al. 2014, de sousa et al. 2016). however, homonyms and similarities still make species delineation and subspecies identification confusing. in algeria, studies on this genus are scarce, being mainly conducted with regard to autoecology (abdelguerfi et al. 1988b), physiology (refoufi 1988, amouri and fyadlamèche 2012) morphology and biochemistry (medoukali et al. 2015, moulai and fyad-lamèche 2017), and rhizobiamedicago symbiotic interactions (cheriet et al. 2014). the genus still remains under-investigated with regard to cytogenetics, largely due to both small chromosome size and technical difficulties encountered during staining procedures. most of the studies conducted to date remain restricted to mitotic chromosome counts (heyn 1963, simon and simon 1965, lesins and lesins 1979, schlarbaum et al. 1984, mariani et al. 1996). very few karyological studies have involved algerian medicago populations (abdelgeuerfi and guittonneau 1979, abdelguerfi et al. 1988a, fyad-lamèche et al. 2016) and even fewer have considered the haploid phase of these plants, as is generally the case in the whole medicago genus worldwide. medicago species exhibit significant diversity in vegetative morphology as a result of interspecific hybridization (heyn 1963). meiotic analyses may provide valuable information regarding the genetic or genomic causes of this diversity. such studies could also provide significant insight into the cytological evolution of species that can be used in genetic improvement and in the conservation of genetic resources (kumar and singhal 2011). in the present study, we analyzed natural populations of eight annual medicago species sampled from different localities of the soummam valley in northeastern algeria. the main aim of this paper, in addition to chromosome counts, is to examine the male meiotic course and the subsequent effects of meiotic abnormalities encountered during different stages of meiosis i and ii on pollen grain size and pollen viability. materials and methods eight annual species, namely m. truncatula gaertn., m. littoralis rohde ex lois., m. intertexta (l.) miller, m. ciliaris (l.) krocker, m. arabica (l.) huds., m. polymorpha l., m. laciniata (l.) miller and m. minima (l.) bart. were sampled from wild populations of the soummam valley and neighborhoods (northeastern algeria). the geographical characteristics of the sampling sites are given in tab. 1. species identification was performed according to lesins and lesins (1979) and voucher specimens were deposited at ensa herbarium of the botany department of the national high school of agriculture in algiers (dpt.botanique@ensa.dz; www.ensa.dz). flower buds at different stages of development before anthesis were collected, fixed in situ in carnoy’s fixative (glacial acetic acid – chloroform – absolute ethanol, 1:3:6) and preserved at 4 °c in a refrigerator until used. cytological analysis was performed on pollen mother cells (pmcs). young flower buds were hydrolyzed in 1 n hcl at 60 °c in a water bath for three minutes. under a binocular stereomicroscope, the removed immature anthers were immediately excised on a slide in a drop of 1% lactopropionic orcein and squashed under a coverslip. all stages of meiosis were analyzed in order to determine chromosome numbers, to study meiotic behavior and to evaluate meiotic abnormality frequencies. about fifty individuals per population were harvested and around 1000 pmcs per species were scrutinized. pollen viability was determined by staining mature flowers before anthesis using cotton blue in lactophenol (1% blue aniline in lactophenol) according to mertens and hammersmith (1998). flowers were dissected to free up pollen grains from the anther and mounted in a drop of cotton blue. the staining takes from 20 to 30 minutes. viable pollen grains are stained uniformly dark blue, whereas defective grains are weakly stained and appear vacuolated, plasmolysed, and atypical in size and shape. photomicrographs were taken using a digital camera connected to an optika b-350 microscope. about 15 flowers were prepared for each population and up to 1000 pollen grains per flower were analyzed for evaluating pollen viability. an average pollen viability rate was estimated for each species. the pollen grain cell area was measured for 100 viable pollen grains per sample using imtab. 1. geographical characteristics of the sampling sites of the algerian medicago l. species. taxa sampling site latitude, longitude altitude (m) m. truncatula gaertn. vahloul 36°32'03,65"n, 4°35'46,23"e 238 m. littoralis rohde ex lois. allaghane 36°23'42,76"n, 4°27'43,13"e 206 m. intertexta (l.) miller ideraken (timzrit) 36°37'48,78"n, 4°46'29,97"e 75 m. ciliaris (l.) krocker el ghaba (smaoune) 36°36'53,79"n, 4°50'30,86"e 305 m. arabica (l.) huds. taddart tamokrante (amizour) 36°37'6.30"n 4°59'57.80"e 511 m. polymorpha l. allaghane 36°23'42,76"n 4°27'43,13"e 206 m. laciniata (l.) miller allaghane 36°23'42,76"n 4°27'43,13"e 206 m. minima (l.) bart. aftis 36°23'7.85"n 4°27'23.45"e 217 djafri-bouallag l., ourari m., sahnoune m. 84 acta bot. croat. 78 (1), 2019 agej 1.48v image processing and analysis in java (rasband 2016). pearson correlation coefficient (r) between meiotic abnormalities frequency and pollen viability rate as well as the relative standard deviation (rsd) of pollen grain area were estimated for each species sample. the relative standard deviation formula is: rsd = 100 s / x-; where s is the sample standard deviation, xis the sample mean. fisher’s exact test was applied to compare frequencies of meiotic abnormalities and pollen viability rates; and the least significant difference (lsd) test was used to check the homogeneity of pollen viability rates and pollen grain area mean values across samples. before running lsd test, raw data were divided by sd in order to homogenize their variances. statistical treatments were performed using statistica 8.0 (statsoft 2008). results meiotic chromosome counts revealed that the m. truncatula gaertn., m. littoralis rohde ex lois., m. intertexta (l.) miller, m. ciliaris (l.) krocker, m. arabica (l.) huds, m. laciniata (l.) miller and m. minima (l.) bart. populations were all diploid with a basic number x = 8 chromosomes. m. polymorpha l. proved also diploid but the basic chromosome number was x = 7 (figs. 1, a-h). our samples showed no tetraploid or aneuploid cytotypes. the studied taxa displayed regular bivalent pairing and chromosome segregation at meiosis. the analysed pmcs show exclusively bivalent associations (figs. 1, a-h). no multivalent or univalent associations were observed. numerous anomalies in relatively high rates (≈ 25%) were detected, including cytomixis, the most common abnormality, chromatic bridges, asynchronous divisions, micronuclei and multipolar cells (tab. 2). m. littoralis is distinguished by its lowest abnormality rate, restricted to cytomixis (14.66%). the samples show cytomixis rates varying from 14.66% in m. littoralis to 25.83% in m. laciniata (tab. 2). m. arabica, tab. 2. chromosome numbers and meiotic abnormality rates in the medicago l. species from northeastern algeria. abbreviations: pmc – pollen mother cells; cm – cytomixis; cb – chromatic bridges; ad – asynchronous divisions; mn – micronuclei; mc – multipolar cells; ca – cumulative abnormality frequency; pv – pollen viability; sd – standard deviation; pga – pollen grain area; rsd – relative standard deviation; n – number of individuals (for each species). for each column separately, values with different letters are significantly different as revealed by fisher’s exact test (small letters) and lsd test (capital letters) at p = 0.05. species chromosome numbers and meiotic abnormalities pollen data pmc 2n cm (%) cb (%) ad (%) mn (%) mc (%) ca (%) pv±sd (%) (n=15) pga±sd (102 µm2) (n=100) pga rsd (%) (n=100) m. truncatula 1170 16 18.29b 1.2c 0 1.88c 2.56c 23.93 0.79±0.16b 6.37±1.24c 19.56 m. littoralis 921 16 14.66a 0 0 0 0 14.66 0.99±0.01g 10.75±0.72f 6.73 m. intertexta 1137 16 15.04a 1.06c 1.32c 0.79b 0.79b 19.00 0.88±0.21b 7.01±0.99e 14.15 m. ciliaris 1035 16 14.78a 1.45c 0 0 0 16.23 0.96±0.05e 6.39±0.99d 15.58 m. arabica 1124 16 24.11cd 0 0 0 0 24.11 0.97±0.02f 6.81±0.95e 13.91 m. polymorpha 1323 14 22.45c 0.68b 1.13c 0 0 24.26 0.81±0.12c 5.01±0.53a 10.75 m. laciniata 1270 16 25.83d 0 0.31b 0 0 26.14 0.70±0.30a 7.22±1.37c 19.04 m. minima 1437 16 22.13c 0.63b 0.84c 0.42b 0 24.01 0.87±0.05d 6.34±0.73b 11.63 m. polymorpha, m. laciniata and m. minima (sect. leptospirae) exhibited cytomixis rate values exceeding 22.13%, significantly higher than those of the remaining species (tab. 2). the occurrence of cytomixis in our samples was recorded from early prophase i to microspore stage (figs. 2, a-e). the chromatin transfer is performed either by one or by several strands in all the analysed species. it also occurs by direct contact between two or more cells (figs. 2, a-b). another abnormality recorded in m. truncatula, m. intertexta, m. ciliaris, m. polymorpha and in m. minima was chromatic bridges at rates ranging from 0.63% in m. minima to 1.45% in m. ciliaris (tab. 2, figs. 2, f ). asynchronous divisions were observed at anaphase ii in m. intertexta, m. polymorpha, m. laciniata and in m. minima (fig. 2, g). the rate recorded for this irregularity in the analysed samples varies from 0.31% in m. laciniata to 1.32% in m. intertexta (tab. 2). micronuclei were found in m. truncatula (1.88%), m. intertexta (0.79%), and in m. minima (0.42%) (figs. 2, h-i). the last anomaly observed in the analysed taxa is the form of multipolar cells. in this case, the ultimate meiotic product corresponds to polyads instead of tetrads (fig. 2, j-l). this abnormality was observed only in m. truncatula (2.56%) and m. intertexta (0.79%) (tab. 2). pollen viability rates were variable among and within populations (fig. 3). while m. littoralis appears to be invariable with a high percentage of viability (99.14%), m. laciniata shows significant variability ranging from 0 to 98.75% with a relatively low mean value (70.11%). pollen viability rates seem to be inversely proportional to the cumulative rate of meiotic anomalies, since the pearson coefficient correlation value is − 0.72 with p = 0.043. pollen average area varies from 501.21 µm2 ± 53.86 in m. polymorpha to 1074.93 µm2 ± 72.32 in m. littoralis (figs. 4, 5). m. truncatula and m. laciniata reveal heterogeneous pollen grain area; with a calculated relative standard deviation of pollen grain area (rsd) of around 19%. it is worth noting that m. littoralis is distinguished from microsporogenesis and pollen in medicago l. acta bot. croat. 78 (1), 2019 85 fig. 1. micromeiocyte metaphase plates in the medicago l. species from northeastern algeria. a) m. truncatula (8 bivalents, 2n=2x=16); b) m. littoralis (8 bivalents, 2n = 2x = 16); c) m. intertexta (8 bivalents, 2n = 2x = 16); d) m. ciliaris (8 bivalents, 2n = 2x = 16); e) m. arabica (8 bivalents, 2n = 2x = 16); f ) m. laciniata (8 bivalents, 2n = 2x = 16); g) m. minima (8 bivalents, 2n = 2x = 16); h) m. polymorpha (7 bivalents, 2n = 2x = 14). scale bars = 10 µm. fig. 2. abnormal meiotic behaviour in the studied medicago l. species from northeastern algeria. a) cytomixis between two micromeiocytes at prophase i; b-d) cytomixis between several micromeiocytes at different stages; e) cytomixis between two microspores; f ) chromatic bridge (arrow); g) asynchronous division; h-i) micronucleus (arrow); j-l) multipolar cells. scale bars = 10 µm. djafri-bouallag l., ourari m., sahnoune m. 86 acta bot. croat. 78 (1), 2019 other species by the large size and the homogeneity of its pollen (rsd = 6.73 %) (fig. 5). discussion in this article, chromosome numbers, meiotic behaviour and meiotic abnormalities were performed by analysis of dividing pollen mother cells on natural populations in northeastern algeria. pollen viability analyses were also carried out. the present results represent the first haploid phase cytogenetic observations reported for algerian populations of medicago species. the encountered basic chromosome numbers (x = 8, x = 7) were previously reported in the genus medicago by heyn (1963), simon (1965) and by lesins and lesins (1979). according to steele et al. (2010), the basic chromosome number of x = 8 would be plesiomorphic in the trifolieae tribe. the basic chromosome number of x = 7 was interpreted as an innovation deriving from x = 8 following chromosome rearrangements (lesins and lesins 1979). based on the phylogeny of plastid and nuclear sequences (trnk / matk, ga3ox1), the apomorphic base number x = 7 arose four times in the genus. furthermore, this phylogeny showed that m. polymorpha and m. murex form a monophyletic group called 'polymorpha clade' (steele et al. 2010). as in m. murex, m. polymorpha genome would have arisen from a translocation of chromosome 8 on chromosome 3 by losing the centromeric region and thus forming de novo a long satellite (lesins and lesins 1979). the chromosome counts 2n = 2x = 14 and 2n = 2x = 16, obtained for the analysed species are in agreement with those made by several authors (heyn 1963, simon and simon 1965, lesins and lesins 1979, schlarbaum et al. 1984, mariani et al. 1996). however, some authors reported deviating counts with different chromosome basic numbers. in fact, humphries et al. (1978) and vogt and aparicio (1999) found 2n = 14 in m. truncatula gaertn. in m. polymorpha, several authors (see rani et al. 2012) found 2n = 16 with x = 8. in this taxon, the deviating count 2n = 16 is considered to be due to either species misidentification or presumably to the number over-estimation attributed to the presence of a pair of chromosomes with large satellites (kamari et al. 1996). the absence of tetraploid or aneuploid cytotypes is consistent with the results obtained by the majority of authors for these species (simon and simon 1965, fig. 3. pollen viability in the medicago l. species from northeastern algeria. max – maximum, m – mean; se – standard error, min – minimum. tru – m. truncatula, lit – m. littoralis, int – m. intertexta, cil – m. ciliaris, ara – m. arabica, pol – m. polymorpha, lac – m. laciniata, min – m. minima. different capital letters above bars denote significantly different results as revealed by lsd test (p = 0.05). fig. 4. pollen grains of the medicago l. species from northeastern algeria stained by cotton blue in lactophenol. a) m. truncatula (heterogeneous pollen); b) m. littoralis (homogeneous pollen); c) m. intertexta (homogeneous pollen); d) m. ciliaris (homogeneous pollen); e) m. arabica (homogeneous pollen); f ) m. polymorpha (homogeneous pollen); g) m. laciniata (heterogeneous pollen); h) m. minima (homogeneous pollen). arrows indicate defective pollen grains. scale bars = 10 µm. microsporogenesis and pollen in medicago l. acta bot. croat. 78 (1), 2019 87 lesins and lesins 1979, schlarbaum et al. 1984, mariani et al. 1996, small 2011, sadeghian and hesamzadeh hejazi 2014). however, polyploid levels were reported for two species (m. intertexta, m. polymorpha) by some authors. as reported by fernandes and santos de fatima (kamari et al. 1996) but not confirmed by lesins and lesins (1979), the individual of m. intertexta (l.) miller from portugal was tetraploid (2n = 32). recently, in m. polymorpha l., a tetraploid level with 2n = 4x = 32 was reported from india by rani et al. (2014) in a general survey on angiosperms. the few deviating chromosome counts should be verified since polyploidy is relatively rare among the annual species of medicago. indeed, according to small (2011), only m. ovalis, m. rugosa and m. scutellata are certainly tetraploid. aneuploid forms were also reported for m. ciliaris (l.) krocker by some authors. early on, in 1956, heyn found in a middle east accession of m. ciliaris a diploid chromosome number 2n = 18 (2n = 16 + 2b). however, clement (1962) and lesins and lesins (1979) are reluctant to accept this possibility and consider the “supernumerary chromosomes” as detached satellites, not countable as complete chromosomes. later, abdelguerfi and guittonneau (1979) in an algerian population and kamari et al. (1996) in a tunisian population again found this count of 2n = 18. analysis of meiotic chromosome behaviour in metaphase i is essential to formulate a breeding strategy. our samples show exclusively bivalent associations. these chromosome pairings were a prerequisite for a balanced segregation (da ines et al. 2014). they also generate new combinations of parental alleles that increase genetic diversity (naranjo 2012). the natural hybridizations reported within the complex m. littoralis m. truncatula m. tornata (heyn 1963, lesins and lesins 1979) appear to be absent in the analysed littoralis population since meiosis seems regular. the low rate of meiotic anomalies could explain its morphological homogeneity and therefore, the studied population may correspond to the “pure” form of m. littoralis. on the other hand, although the studied m. truncatula population appears to be morphologically homogeneous, it showed numerous meiotic anomalies suggesting potential natural hybridization with some other related species, such as m. littoralis or m. tornata. the most common meiotic abnormality observed in all the studied samples is cytomixis. this is related to the migration of chromatin between meiocytes through cytoplasmic channels. this chromatin transfer is under genetic control but can be influenced by environmental conditions, as pointed out by ranjbar et al. (2011a) in trigonella spruneriana boiss. the analysed samples show variable cytomixis rates. in astragalus, thecytomixis rate was relatively low, varying from 0% to 3.82% (ranjbar et al. 2011b). in contrast, this rate can reach 45% in lotus (sheidai and jalilian 2006). in medicago l., the few cytomictic studies reported have often dealt with polyploid taxa. the cytomixis rate recorded for the tetraploid species m. sativa reached 46% (bellucci et al. 2003). ranjbar et al. (2011b) reported that cytomixis in astragalus seem to be more prevalent among polyploid taxa than in their diploid counterparts. in contrast, in solanaceae, singhal and kumar (2008) have observed higher rates in diploids than in polyploids. in general, cytomictic activity is maximal during early prophase i due to the parallel transfer of trophic factors and signal molecules ensuring the synchronisation of meiosis (mursalimov et al. 2013). in m. sativa, chromatin transfer is mostly detected in prophase i and rarely in later stages (bellucci et al. 2003). in the analysed samples, the cytomixis was recorded from early prophase i to microspore stage. this report was similar to those observed in astragalus cyclophyllos (ranjbar et al. 2011b), lotus corniculatus (jeelani et al. 2012) and in nepeta govaniana (kaur and singhal 2014). the chromatin transfer performed either by one or by several strands and by direct contact between two or more cells in all the analysed species is also reported by several authors for other taxa such as medicago sativa, lotus corniculatus and astragalus cyclophyllos (bellucci et al. 2003, sheidai and jalilian 2006, ranjbar et al. 2011b). the chromatic bridges recorded in m. truncatula, m. intertexta, m. ciliaris, m. polymorpha and in m. minima samples have also been reported in some species such as onobrychis chorassanica (ranjbar et al. 2010), trigonella spruneriana (ranjbar et al. 2011a) and calamagrostis emodensis (saggoo and kumari 2013) at variable frequencies. in the genus passiflora (passifloraceae), this rate can reach 26% (souza and pereira 2011). chromatic bridges could be explained either by chromosomal rearrangement such as heterozygous paracentric inversions (ruvalcaba-ruiz and rodríguez-garay 2002, elrod and stansfield 2003) or by unrepaired double-strand breaks induced by spo11 enzyme (horlow and doutriaux 2003). the observed asynchronous divisions in m. intertexta, m. polymorpha, m. laciniata and in m. minima samples result from a mutation affecting the spindle “checkpoint” (risso-pascotto et al. 2003). in this case, only one cell of the dyad progresses in the cell cycle while the defective one stays blocked at metaphase ii. the rate recorded for this irregularity was variable in the analysed samples. variable rates are reported in many leguminosae species. for reference, the rates vary from 0.41% to 0.79% in trigonella and from 0 fig. 5. pollen grain area in the medicago l. species from northeastern algeria. max – maximum, m – mean; se – standard error, min – minimum. tru – m. truncatula, lit – m. littoralis, int – m. intertexta, cil – m. ciliaris, ara – m. arabica, pol – m. polymorpha, lac – m. laciniata, min – m. minima. different capital letters above bars denote significantly different results as revealed by lsd test (p = 0.05). djafri-bouallag l., ourari m., sahnoune m. 88 acta bot. croat. 78 (1), 2019 to 24.98% in astragalus (ranjbar et al. 2011a, ranjbar and jahanian 2013, jahanian and sarpoushi 2014). micronuclei, the other anomaly recorded in our samples, would be caused by the condensation of the chromatin originating from cytomixis or fragments of chromatic bridges. these micronuclei can then be removed by cytokinesis or by budding producing thus sterile microcytes (baptista-giacomelli et al. 2000, kiihl et al. 2011). a literature overview reveals that in angiosperms the micronuclei rate varies according to the ploidy level. thus, in diploid taxa, it ranges from 0% in trigonella spruneriana to 1.66% in astragalus ebenoides (ranjbar et al. 2011a, ranjbar and jahanian 2013). however, in tetraploid taxa, this rate varies from 0% in astragalus carmanicus to 5.52% in vicia pallida (singhal and kaur 2011, jahanian and sarpoushi 2014). in the octoploid alchornea triplinervia (euphorbiaceae) tree species, this rate can reach 9.61% (godoy et al. 2012). the observed polyads in m. truncatula and in m. intertexta were the result of several mutations such as ms28 and ms17, which affect the bipolar spindle formation (albertsen and phillips 1981, golubovskaya and distanova 1986). another hypothesis, put forward by caetano-pereira and pagliarini (2001), refers to the role of transposable elements. spindle orientation abnormalities have been reported in several species: in agropyron cristatum, carthamus tinctorius, fuchsia, thunbergia mysorensis, aloysia lycioides, zea mays (caetano-pereira and pagliarini 2001 and references therein). this abnormality is reported in many leguminosae species at variable rates (e.g. 0.13% in onobrychis chorassanica, 0.32% in astragalus carmanicus, 10.98% in vicia pallida) (ranjbar et al. 2010, singhal and kaur 2011, jahanian and sarpoushi 2014). the pollen viability showed an important intraand interspecific variation. a significant variation in viability rate was reported within populations and among species in medicago (simon and millington 1967, lesins and erac 1968, singh and lesins 1972). sometimes, hybrids show rates similar to those of the parental taxa (simon and millington 1967). our pollen samples also showed an important interspecific variation in size. there are no data in the literature about pollen grain size in medicago. however in other species, some authors, such as kumar et al. (2010), explain this pollen size heterogeneity by the presence of meiotic abnormalities. in conclusion, the eight annual medicago populations investigated in the soummam valley (northeastern algeria) showed regular meiosis with the presence of exclusively 8 bivalents in m. truncatula gaertn., m. littoralis rohde ex lois., m. intertexta (l.) miller, m. ciliaris (l.) krocker, m. arabica (l.) huds, m. laciniata (l.) miller, m. minima (l.) and 7 bivalents in m. polymorpha l. at metaphase i revealing the diploid nature of these species. the observed meiotic abnormalities related to cytomixis, multipolar cells, micronucleus, asynchronous divisions and chromatic bridges seem to be correlated with pollen size and pollen viability. these meiotic anomalies would be potential mechanisms at the origin of the genetic variability in natural populations of the medicago l. genus. medicago species, especially annuals because of their potential source of germplasm for alfalfa breeding programs, can be 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acta bot. croat. 75 (1), 2016 31 acta bot. croat. 75 (1), 31–38, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0018 issn 0365-0588 eissn 1847-8476 physiological responses of two halophytic grass species under drought stress environment zamin shaheed siddiqui1*, huda shahid1, jung-il cho2*, sung-han park2, tae-hun ryu2, soo-chul park2 1 stress physiology lab., department of botany, university of karachi, karachi – 75270, pakistan 2 national academy of agricultural sciences, rural development administration, suwon 441-707, republic of korea abstract – the physiological responses of two halophytic grass species, halopyrum mucronatum (l.) staph. and cenchrus ciliaris (l.), under drought stress were evaluated. biomass accumulation, relative water content, free proline, h2o2 content, stomatal conductance, photosynthetic performance and quantum yield (fv/fm ratio) were studied. under drought conditions, these halophytic plants expressed differential responses to water defi cit. stomatal conductance and free proline content were higher in h. mucronatum than in c. ciliaris, while h2o2 content in h. mucronatum was substantially lower than in c. ciliaris. performance index showed considerable sensitivity to a water defi cit condition, more so in c. ciliaris than in h. mucronatum. results were discussed in relation to comparative physiological performance and antioxidant enzymes activity of both halophytic grasses under drought stress. keywords: antioxidant enzymes activity, cenchrus ciliaris, chlorophyll a fl uorescence, drought stress, halopyrum mucronatum, photosystem ii * corresponding author, e-mail: zaminss@uok.edu.pk, jungilcho@korea.kr introduction drought and salinity have long been known as the most prevalent abiotic stresses inhibiting the growth and productivity of many wild and domestic plant species across the world (qadir 2008, naz et al. 2010). in locations with limited water resources and an increasing human population, conventional crop production might not be able to meet food demands. in this distressing situation, effective measures are adopted not only to minimize crop losses but also to fi nd alternate means of food production. the only economic solution considered in the present circumstance is the use of halophytic plants as an alternate source of food and therefore their growth performance should be tested in arid and saline habitats (khan and duke 2001, nedjimi 2011). for that, focus on developing halophytes as cash crops in the future should be amplifi ed (breckle 2009). in the sub-continent, the salt range and large coastal area enable a large number of halophytes or salt tolerant plant species to grow. among them, halopyrum mucronatum l. staph. and cenchrus ciliaris l. are important and widely occurring halophytic grasses. most of these halophytic grasses have been investigated and physiological explanations regarding their salt tolerance have been provided (khan and ungar 1999, saini et al. 2007, siddiqui and khan 2011). however, the effects of drought stress on these plants and their physiological mechanism have not been examined. generally, c. ciliaris l. is considered to be an important pasture grass and is being used for cattle and sheep production in arid and semiarid regions (khan and ungar 1999, saini et al. 2007, sidiqui and khan 2011). h. mucronatum is an excellent salt-tolerant grass species, and is used as fodder (siddiqui and khan 2011). furthermore, it was also reported that these two grasses might have the ability to tolerate long dry seasons under varying soil conditions indicating some degree of drought tolerance (ayerza 1981, de leon 2004). it was reported that some of the metabolic reactions triggered by drought and salinity are similar. among them, osmotic adjustment, changes in relative water content, maximum quantum yield and dry mass accumulations are well known (munns et al. 2002, kwon et al. 2009, siddiqui et al. 2014). early responses to drought or salt stress are generally the same, apart from acting as water stress either qualitatively (saline) or quantitatively (amount of water) and the specifi c ion effect. therefore, it is hypothesized that those halophytic grasses that showed salt stress tolerance in saline habitats may also exhibit drought tolerance in dry envi ronments. hence, the physiological performance and siddiqui z. s., shahid h., cho j.-i., park s.-h., ryu t.-h., park s.-c. 32 acta bot. croat. 75 (1), 2016 antioxidant enzymes activity of two known salt tolerant grasses in drought stress environment have been examined. materials and methods plant materials the two halophytic grass species: h. mucronatum (l.) staph. and c. ciliaris l were used for experiments. caryopses were collected at maturity from these plants growing on dunes of hawks bay beach, karachi, pakistan. they were collected on december 2013. hulled seeds were cleaned and stored in a refrigerator prior to use. germination, growth and treatments the seeds were surface sterilized in 0.52% sodium hypochlorite solution for one minute and rinsed thoroughly with sterilized distilled water. seeds were pre-soaked in distilled water for 4 h. ten seeds were placed in 90 mm sterilized petri plate. plants were allowed to grow in a growth chamber (hotpack usa) at 25–28 ± 2 °c day / night temperature with 69–80% humidity. light intensity varied between 2000 and 2300 μmol m–2 s–1. after 20 days, equalsized seedlings were then transferred to pots with a diameter of 30.48 cm. there were fi ve plantlets in each pot with 6 replications for each treatment, i.e., one for control and other for the drought treatment. the plantlets were grown up to four leaf stage and then drought was induced up to 7 days when soil moisture content reached 15%. after 7 days, the stomatal conductance (gs) and chlorophyll fl uorescence was recorded on the youngest fully expanded leaf between 9:00 – 11:00 am using a steady state diffusion porometer, model sc-1 (decagon devices) and a chlorophyll fl uorescence meter (os-30p+, opti-science, usa) respectively. afterwards, plants were harvested and biomass production, relative water content, free proline quantifi cation, h2o2 content and antioxidant enzymes activity were examined. chlorophyll fluorescence after half an hour of dark adaptation, the chlorophyll fluorescence parameters, minimal chlorophyll fl orescence (f0) and maximal fluorescence (fm), were measured in order to determine the maximum quantum yield (fv/fm ratio) (maxwell and johnson 2000) of ten fully expanded leaves using a portable fluorometer, model os-30p+ (opti-science, usa). pigment analysis leaf samples (500 mg) were ground in 10 ml of 96% methanol and then centrifuged at 4000 rpm for 10 min. total chlorophyll (chl a+b), chlorophyll a (ca), chlorophyll b (cb) and total carotenoid (cx+c) contents were determined (lichtenthaler 1987). the supernatant was separated and the absorbance was read at 666, 653 and 470 nm in uv – vis spectrophotometer (shimadzu), respectively. later the pigments were quantifi ed according to the following formulas: ca = 15.65 × a666 – 7.340 × a653 cb = 27.05 × a653 – 11.21 × a666 cx+c = 1000 × a470 – 2.860 × ca – 129.2 × cb/245 free proline content free proline content was estimated according to bates et al. (1973). fresh leaf samples (500 mg) were homogenized in 10 ml of sulphosalicylic acid (3% w/v). later, the extract was fi ltered through whatman no. 2 fi lter paper. to 2 ml of the aliquot, 2 ml of acid ninhydrin and 2 ml of glacial acetic acid were added and the contents were boiled at 100 °c for an hour. the mixture was further extracted with 2 ml of toluene by mixing thoroughly with vigorous stirring for 15 to 20 s. the upper layer was separated from the aqueous phase and absorbance was read at 520 nm against toluene blank. hydrogen peroxide content hydrogen peroxide (h2o2) was estimated by the procedure of sergiev et al. (1997). fresh leaf samples (500 mg) were homogenized in 5 ml 0.1% (w/v) trichloroacetic acid (tca) using ice bath. afterwards, the homogenate was centrifuged at 12,000 g for 15 min. to 0.5 ml supernatant, 0.5 ml of 10 mm potassium phosphate buffer and 1 ml of 1 m potassium iodide (ki) were added. the absorbance was read at 390 nm. the h2o2 contents were estimated using a standard curve. relative water content four leaf strips of 4 cm2 were excised randomly and fresh weights (fw) were determined. for the measurement of turgid weight (tw), leaves were left in distilled water for 24 h under low irradiance conditions. samples were then oven-dried at 80 °c for 48 h and dry weight (dw) was determined. relative water content (rwc) was calculated according to barrs and weatherley (1962) according the formula: relative water content = (fw – dw / tw – dw) × 100 enzyme assays leaf samples (500 mg) were randomly collected and crushed in liquid nitrogen at 4 °c and homogenized in 10 ml protein extraction buffer containing tris-hcl ph 6.8, 50 mg polyvinylpyrrolidone, 0.05 mm ethylenediaminetetraacetic acid (edta). the contents were centrifuged at 12,000 rpm in a refrigerated micro centrifuge (smart r-17, hanil) for 10 min. total protein was estimated by the method of bradford (1976). catalase (cat; ec 1.11.1.6) activity was estimated by the method of patterson et al. (1984). the decomposition of h2o2 was measured at 240 nm taking δε as 43.6 mm cm–1. reaction assay (3.0 ml) consisted of 10.5 mm h2o2 in 0.05 m potassium phosphate buffer (ph 7.0) and the reaction was initiated after the addition of 0.1 ml enzyme extract at 25 °c. the decrease in absorbance at 240 nm was used to calculate the activity. one unit of cat activity is defi ned as the amount of enzyme that catalyzes the conversion of 1 mm of h2o2 min–1 at 25 °c. halophytic grasses halopyrum and cenchrus under drought acta bot. croat. 75 (1), 2016 33 ascorbate peroxidase (apx; ec 1.11.1.11) activity was performed by the method of nakano and asada (1981). the reaction mixture (2.0 ml) contained 50 mm potassium phosphate buffer (ph 7.0), 0.2 mm edta, 0.5 mm ascorbic acid and 0.25 mm h2o2. the reaction was started after the addition of 0.1 ml enzyme extract at 25 °c. the decrease in absorbance at 290 nm for one minute was recorded and the amount of ascorbate oxidized was calculated from the extinction coeffi cient 2.8 mm cm–1. the unit of activity is expressed as micromole of ascorbic acid oxidized min–1 at 25 °c. superoxide dismutase (sod; ec 1.15.1.1) activity was performed by the method of beyer and fridovich (1987). the reaction mixture consisted of 27.0 ml of 0.05 m potassium phosphate buffer (ph 7.8), 1.5 ml of l-methionine (300 mg per 2.7 ml), 1.0 ml of nitroblue tetrazolium salt (14.4 mg per 10 ml), and 0.75 ml of triton x-100. aliquots (1.0 ml) of this mixture were delivered into small glass tubes, followed by the addition of 20 ml enzyme extract and 10 ml of ribofl avin (4.4 mg per 100 ml). the cocktail was mixed and then illuminated for 15 minutes in an aluminium foil-lined box, containing 25 w fl uorescent tubes. in a control tube the sample was substituted for by 20 ml of buffer and the absorbance was measured at 560 nm. the reaction was stopped by switching off the light and placing the tubes in the dark. increase in absorbance due to the formation of formazan was measured at 560 nm. under the described conditions, the increase in absorbance in the control was taken as 100% and the enzyme activity in the samples were calculated by determining the percentage inhibition per minute. one unit of sod is the amount of enzyme that causes a 50% inhibition of the rate for reduction of nitroblue tetrazolium salt under the conditions of the assay. statistical analysis all data from treated and control were subjected to statistical analysis using spss 17.0 (ibm, usa). the values were expressed as mean and standard errors. t-test (p = 0.05) was computed between drought treatments and corresponding controls for each species separately. levels of signifi cance were expressed on bar graph with different letters. results drought stress signifi cantly reduced leaf fresh and dry mass in both halophytic grass species (fig. 1). fresh weight in both species was lower than in the control. however, decrease in turgid weights was non-signifi cant within a species. on the other hand, dry weights in both the species were substantially lower under drought stress conditions than in the control. in comparison with their respective controls, a greater decrease was observed in h. mucronatum than in c. ciliaris. subsequently, relative water content in leaves was slightly higher in h. mucronatum than in c. ciliaris in a drought stress environment. halophytic grasses subjected to drought showed a decrease in chlorophylls a and b and total chlorophyll that was signifi cant compared to control (fig. 2). however, c. ciliaris showed higher decrease than h. mucronatum under drought stress conditions. similarly, total carotenoid content decreased in both species, although more so in c. ciliaris. 0.00 0.02 0.04 0.06 0.08 0.10 b io m as s (g ) 0.00 0.02 0.03 0.05 0.06 0.08 0.09 0.11 0.12 control drought b io m as s (g ) 0.00 0.01 0.02 0.03 0.04 0.05 r el at iv e w at er c on te nt ( % ) 0 20 40 60 80 100 plant species h. mucronatum c. ciliaris h. mucronatum c. ciliaris a ab ab b a b b ab ab a a b a b b b fw tw dw c hl or op hy ll b ( μ g m g -1 f w ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 control drought (6 d) t ot al c hl or op hy ll ( μ g m g1 f w ) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 t ot al c ar ot en oi ds ( μ g m g -1 f w ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 c hl or op hy ll a ( μ g m g -1 f w ) 0.00 0.30 0.60 0.90 1.20 1.50 1.80 2.10 plant species h. mucronatum c. ciliaris h. mucronatum c. ciliaris a b a b a b a b a b a b a a a b fig. 1. biomass and relative water content of halopyrum mucronatum (l.) staph. and cenchrus ciliaris l. under drought stress. bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separately, according to paired “t” test at p < 0.05. vertical lines on bar graphs represent mean ± standard error. fw – fresh weight, tw – turgid weight, dw – dry weight. fig. 2. total chlorophyll and carotenoid content of halopyrum mucronatum (l.) staph. and cenchrus ciliaris l. under drought stress. bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separately, according to paired “t” test at p < 0.05. vertical lines on bar graphs represent mean ± standard error. siddiqui z. s., shahid h., cho j.-i., park s.-h., ryu t.-h., park s.-c. 34 acta bot. croat. 75 (1), 2016 a noticeable reduction was observed in performance index (piabs) and photochemical quenching (qp) in both the species under drought stress environment (fig. 3). however, results showed that a higher reduction in piabs and qp was recorded in c. ciliaris than in h. mucronatum under drought stress (fig. 3). a marked reduction in stomatal conductance (gs) was observed in two tested halophytic grass species due to drought stress. however, reduction in stomatal conductance was greater in c. ciliaris than in h. mucronatum. the level of drought stress damage was examined in terms of free proline and hydrogen peroxide (h2o2) production (fig. 4). leaf proline concentration of both grasses increased due to drought stress. however, h. mucronatum accumulated more proline than c. ciliaris under drought stress condition. on the other hand, h 2 o 2 content in c. ciliaris increased signifi cantly as compared to h. mucronatum under stress. activity of antioxidant enzymes like superoxide dismutase (sod), ascorbate peroxidase (apx) and catalase (cat) was measured under drought stress against control and is illustrated in fig. 5. observations revealed that sod and cat activities of treated samples increased signifi cantly in h. mucronatum as compared to c. ciliaris. however, antioxidant activities of all tested enzymes were higher in treated samples of both halophytes compared to control. among the treated samples h. mucronatum showed an increase that was substantial in sod and cat as compared to c. ciliaris discussion the physiological responses of two halophytic grass h. mucronatum (l.) staph., and c. ciliaris l., were evaluated under drought conditions. it was observed that biomass production was reduced in the two tested species under drought stress. it is well known that drought stress conditions cause substantial reduction in biomass and growth in many plant species (mahiwal and sutaria 1992, ashraf et f v/ f m r at io 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 p er fo rm an ce in de x (p i a bs ) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 control drought p ho to ch em ic al q ue nc hi ng ( q ) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 s to m at al c on du ct an ce , g s (m m ol m -2 s1 ) 0 40 80 120 160 200 240 280 320 plant species h. mucronatum c. ciliaris h. mucronatum c. ciliaris a a a a a b a b a a a a a b a b h 2 o 2 ( m m ol g -1 f w ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 control drought plant species f re e ro li ne c on te nt ( μ m ol g -1 f w ) 0 5 10 15 20 25 30 h. mucronatum c. ciliaris h. mucronatum c. ciliaris a b a b a a a b apx 0 1 2 3 4 5 6 control cat sp ec if ic a ct iv ity (u ni t m g1 o f p ro te in ) 0 1 2 3 4 5 6 h. mucronatum c.ciliaris 0 50 100 150 plant species sod a b a b a b a b a b a b fig. 3. maximum quantum yield (fv/fm ratio), performance index (piabs) and photochemical quenching (qp), and stomatal conductance (gs) of halopyrum mucronatum (l.) staph. and cenchrus ciliaris l. under drought stress. bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separately, according to paired “t” test at p < 0.05 level. vertical lines on bar graphs represent mean ± se. fig. 4. total free proline and h2o2 content of halopyrum mucronatum (l.) staph. and cenchrus ciliaris l. under drought stress. bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separately, according to paired “t” test at p < 0.05 level. vertical lines on bar graphs represent mean ± se. fig. 5. antioxidant enzyme activity of halopyrum mucronatum (l.) staph. and cenchrus ciliaris l. under drought stress. bars followed by the same letter denote no signifi cant difference between drought treatment and control, for each species separately, according to paired “t” test at p < 0.05 level. vertical lines on bar graphs represent mean ± se. halophytic grasses halopyrum and cenchrus under drought acta bot. croat. 75 (1), 2016 35 al. 1998, karsten and macadam 2001, tavakol and pakniyat 2007, siddiqui 2013). in the present study, rwc of both the halophytic species was signifi cantly reduced under drought conditions compared to control (unstressed) plants. however, the reduction of rwc in c. ciliaris was higher than that in h. mucronatum. hence, it can be suggested that h. mucronatum has a better drought tolerance through the maintenance of higher water content in leaf under drought. furthermore, it was observed that rwc in crop plants and the tolerance of the plants to stress are directly related (schonfeld et al. 1988, merah 2001, siddiqui et al. 2014). therefore, it may be suggested that h. mucronatum may have a better ability to regulate intracellular water relations through biomass accumulation than c. ciliaris under drought stress conditions. it is well documented that decline in rwc is related to cell membrane properties and its adaptability to environmental changes such as drought (katerji et al. 1997, el hafi d et al. 1998, de pereira-neto et al. 1999, liu et al. 2002, molnar et al. 2002, blokhina et al. 2003). however, spatial differences among the species cannot be ruled out as water relation characteristics refl ect the physiological differences among species and cultivars. nevertheless, rwc is a good indicator of drought tolerance or adaptation in various plant species (ashraf et al. 1994, siddiqui et al. 2014). photosynthetic performances of the two halophytic grasses under drought conditions were examined in terms of their components, such as maximum quantum yield (fv/ fm ratio), photochemical quenching (qp), performance index and photosynthetic pigments analysis. drought stress caused a signifi cant reduction in total chlorophyll and carotenoid content. however, a higher decrease was observed in c. ciliaris as compared to h. mucronatum. photosynthetic pigments like chlorophylls and carotenoids are responsible for converting energy and/or trapping it in chemical forms for almost all green plants. it was observed that plant metabolism is clearly linked with photosynthetic pigment and adversely affected by abiotic stress like drought (li et al. 2012, siddiqui et al. 2013, reza and hassan 2014). the decrease in chlorophyll under water stress is primarily a result of injury in chloroplasts caused by reactive oxygen species, which are usually elevated as a consequence of drought (smirnoff 1995, siddiqui et al. 2014). in this study the performance index and quenching substantially were reduced from the maximum yield in both the species under drought stress. observations showed that greater reductions in stomatal conductance, piabs and qp, were recorded in c. ciliaris than in h. mucronatum under drought. the fv/fm ratio, characterizes the maximal effi ciency yield of excitation energy captured by “open” photosystem ii reaction centres. this suggests that the photosynthetic activity of c. ciliaris might be decreased due to inhibition in chlorophyll synthesis and their quenching ability which may have an effect on the performance index (lutts et al. 1996, tijen and ismail 2006, siddiqui 2013, siddiqui et al. 2014). photosystem ii (psii) in photosynthetic response is related to chlorophyll and carotenoids concentration and it was varied against fl uctuating environmental (baker 1991). therefore, changes in photosynthesis under water stress conditions are to be expected. likewise, performance index (piabs) is an excellent indicator that showed plant fi tness and provides useful quantitative information about photosynthetic apparatus (strauss et al. 2003, xia et al. 2004, oukarroum et al. 2007, mehta et al. 2010, stefanov et al. 2011). likewise, photosynthetic pigments and maximum quantum yield are important physiological parameters refl ecting the photosynthetic ability of plants in stressful environments. (colom and vazzana 2002, parida et al. 2003, waseem et al. 2006, siddiqui et al. 2008). h. mucronatum accumulated more proline and less hydrogen peroxide content than c. ciliaris. a greater accumulation of proline in response to drought stress is well documented in many plants and maintains homeostasis in leaf (abdel-nasser and abdel-aal 2002, parida et al. 2007, slama et al. 2007, mostajeran and rahimi-eichi 2009, kumar et al. 2011). it was suggested that an amino acid like proline might play a highly valuable role in plants exposed to various stress conditions. besides acting as an excellent osmolyte, proline plays three major roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a signalling molecule, which results in substantial reduction in ros activity (hayat et al. 2012). three possibilities can be predicted from the results: (1) h. mucronatum may have a certain chloroplast protein against oxidative damages, (2) high carotenoid concentrations and proline enhance the antioxidant ability of the h. mucronatum (3) the increased chlorophyll in h. mucronatum as compared to c. cilliaris provides a continuous and substantial energy supply to maintain quantum yield in drought stress. it has been suggested that the sensitivity, tolerance, and response timing of plants to drought vary among species. for example, slow-growing species have been found to be more sensitive than fast-growing species (waseem et al. 2006, munns 2002). this was seen in water stress; some drought-tolerant plants developed fi tness by reducing leaf area and stomatal conductance to transpiration (nativ et al. 1999, ares et al. 2000). thus, plants might adapt physiologically to drought conditions by reducing stomatal conductance to water vapour, increasing their water-use effi ciency (wue). tolerant plants have been observed to adapt two different strategies during drought: long-living annuals and perennials decrease their leaf size and/or stomatal conductance (geber and dawson 1997, querejeta et al. 2003), while shorter-living annuals maximize fi tness by increasing stomatal conductance (decreasing wue) to increase carbon gain and avoid drought stress. this strategy lets them grow rapidly, fl ower early, and increase yield before the start of substantial soil drying (geber and dawson 1997, mckay et al. 2003). when drought is experienced at later developmental stages, selection should favour decreased stomatal conductance (high wue) and smaller leaves, whereas when plants experience drought at early developmental stages, increased stomatal conductance (low wue) should be selected for and leaf size may be of no adaptive value. it was observed that antioxidant enzyme activity like sod and cat were higher in h. mucronatum than in c. ciliaris in a drought stress environment however, compared to control, both halophytes showed substantial increases in siddiqui z. s., shahid h., cho j.-i., park s.-h., ryu t.-h., park s.-c. 36 acta bot. croat. 75 (1), 2016 antioxidant enzymes activities. it was reported that antioxidant enzymes like sod, cat, apx and gr played a signifi cant role in combating drought stress and maintaining substantial growth rate under stress (siddiqui 2013, vujčić and radić brkanac 2014). it was observed that under stress, two different defensive mechanisms are provoked: a) an antioxidant non-enzymatic system such as a synthesis of osmolytes and phenols, and b) antioxidant enzyme systems such as synthesis and activity of enzymes like sod, apx, cat etc. (siddiqui et al. 2014) in conclusion, through higher photosynthetic performance, photo-quenching and lower stomatal conductance, maintaining substantial higher relative water content, h. mucronatum may be considered to have more drought tolerance than c. ciliaris. furthermore, it was concluded that h. mucronatum maintains a substantially better performance index and lower h2o2 contents than c. ciliaris, which may be due to greater antioxidant enzyme activity, such as 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salinity stress on psii in ulva lactuca as probed by chlorophyll fl uorescence measurements. aquatic botany 80, 129–137. acta bot. croat. 77 (2), 2018 209 acta bot. croat. 77 (2), 209–213, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0011 issn 0365-0588 eissn 1847-8476 short communication taxonomic notes on the genus chaenorhinum (plantaginaceae) in turkey golshan zare1*, barış özüdoğru2, gökhan ergan2,3, çağatay tavşanoğlu2 1 department of pharmaceutical botany, faculty of pharmacy, hacettepe university, 06100, sıhiyye, ankara, turkey 2 department of biology, faculty of science, hacettepe university, 06800, beytepe, ankara, turkey 3 eastern mediterranean research association, 182, yağca st., yağca, döşemealtı, antalya abstract – chaenorhinum gerense is an eastern mediterranean species with rare distribution and a large variety in plant and seed morphology. in this study, the accuracy of the taxonomic status of this species, which was initially reported by p.h. davis as c. rubrifolium from south eastern turkey, is discussed and the typical representatives of c. rubrifolium were collected for the first time for turkey from muğla province, southwestern anatolia. c. gerense is closely related to the c. rubrifolium, from which it differs by having a small and cream corolla with red blotch, capsule as long as or smaller 1/2-calyx teeth and triangular or rectangular blunt crest on seed. detailed descriptions and identification keys for these two taxa are provided. key words: chaenorhinum rubrifolium, chaenorhinum gerense, mediterranean basin, plantaginaceae, turkey * corresponding author, e-mail: golshanzare@gmail.com introduction the genus chaenorhinum (dc.) rchb. (1828: 123) was described as a section of linaria by de candolle (1815: 410) and then raised by reichenbach (1828) to the generic level. the taxonomy of the genus especially c. rubrifolium (robill. & castagne ex dc.) fourr. species complex is rather problematic, because of considerable intraspecific variation, insufficient distribution data, confusing synonymies, incorrectly cited names, poorly described species and the distinction of numerous subspecies, varieties and forms (fourreau 1869, fernandes 1971, speta 1980, sutton 1988, benedígonzález 1991). the genus consists of ca. 20 species mainly distributed in southwest europe, north africa and south west asia (benedí and güemes 2009). davis (1978a) reported six species for the flora of turkey and then, c. semispeluncarum yıldırım, kit tan, şenol & pirhan and c. yildirimlii yıldırım, kit tan, şenol & pirhan have been described as new species (yıldırım et al. 2010). consequently, five (ca. 50%) of the eight chaenorhinum species present in turkey are endemics. despite the high endemism ratio and species diversity, the genus is poorly known throughout the mediterranean basin, and especially in turkey. during field studies (march 2014 and june 2016) in a study on post-fire recovery of mediterranean pine forests in muğla province (turkey), some chaenorhinum materials were collected. comparisons with present materials in turkish herbaria (ank, gazi, hub and iste) and the flora of turkey (davis 1978a) yielded no matching identification. further herbarium studies and detailed micro-morphological investigations clearly showed that these materials belong to the common mediterranean species c. rubrifolium, which was reported initially from the eastern part of anatolia by davis in his account (davis 1978b). more detailed fieldwork and micro and macro-morphological investigations of the east anatolian materials confirmed sutton’s decision (1988) to place these taxa belong in c. gerense (stapf ) speta (1980: 27). therefore, c. rubrifolium is introduced here as a new record for the flora of turkey. materials and methods fresh plant materials were collected from the field during excursions in 2014 and 2015. the specimens are deposited in hacettepe university herbarium (hub). herbarium specimens of chaenorhinum in ank, b, bc, e, gazi, hub, iste, msb and w (acronyms according to thiers 2016) were also examined. for scanning electron microscopy (sem), dry seeds were mounted directly on stubs (plaza et al. 2004) and coated with gold in a sputter coater prior to observation with a jeol jsm-6490 lv scanning electron microscope. zare g., özüdoğru b., ergan g., tavşanoğlu ç. 210 acta bot. croat. 77 (2), 2018 results and discussion chaenorhinum gerense (stapf ) speta in stapfia 7: 27 (1980) basionym: linaria gerensis stapf, bull. misc. inform. kew (3): 75 (1906) type: persia: in rupestribus prope pagum gere inter abuschir et schiras, 23.3.1842, th. kotschy 92, holotype k; isotypes g, g-bois, je, m, p, w!. morphological description: erect, slender, annual, 2.5– 12(–25) cm, glandular-pubescent. stem flexuous, simple or branched, adpressed to rocks. basal rosette leaves elliptical to ovate, 8–12 × 3–4 mm, petiolate, green rarely purple below, glabrous, median cauline leaves elliptical to lanceolate, subsessile, glandular-pubescent. raceme lax, flower patent. pedicels ascending or patent in fruit, 5–10 mm, as long as or longer than leaf-like bracts. calyx lobes 4–7 mm, linear to linear-spathulate, obtuse, usually patent or recurved. corolla creamy, suffused with red or pink batch on the upper side, 5–7 (9) mm (included spur); spur tapering, 1.5–2 mm, subacute. capsule spherical, 2–3 mm, 2–3× as short as calyx teeth. seed ovoid-ellipsoid, small, 0.30–0.50 × 0.30–0.35 mm, dark brown, with prominent longitudinal crest, discontinues, triangular or rectangular blunt and obtuse apex, testa of the intercostal spaces covered by papilla (figs. 1,2). flowering and fruiting time: april-may. habitat: crevices of limestone rocks and walls from 350 to 1200 m altitude. general distribution: eastern mediterranean, cyprus, iran, iraq, pakistan, turkey. examined specimens: turkey: c8 mardin: c. 11–13 km from mardin to savur, may 1957, davis 28529 (e!), 4 km e. of mardin, 1200 m, 25 may 1957, davis 28577 (e!), e of mardin, eskikale village, limestone rocks, 1114 m, 37°18'29" n 40°45'53" e, 06 june 2015, g. zare & g. ergan, gz1093 (hub!), c9 mardin: cizre, 350 m, walls of mosque, 09 may 1966, davis 42705 (e!). chaenorhinum rubrifolium (robill. & castagne ex dc.) fourr. in ann. soc. linn. lyon nov. ser. 17: 127 (1869) basionym: linaria rubrifolia robill. & castagne ex dc., fl. fr. 5: 410 (1815) lectotype: described from s. france, cette plante croît sur les collines rocailleuses des environs de marseille, notamment près le fort de n.-d.-de-la-garde du côté de la mer [lectotype designated by c. benedí in collect. bot. (barcelona) 20: 72 (1991): g-dc] morphological description: erect, slender, annual, 4 ̶22 cm high, glandular-pubescent often tinged purple. stem flexuous, simple or branched. basal leaves broadly obovate or spathulate, 7 ̶25 × 5 ̶11 mm, dark green above, purple below, subglabrose; cauline leaves narrowly elliptical, subsessile, glabrous, elliptical to lanceolate. flower in elongating lax raceme. pedicels erect-patent, slender, 5–15 mm, as long as or longer than leaf-like bracts. calyx lobes 5–9 mm, linear to linear-spathulate, obtuse, usually patents. corolla white, suffused with violet on the upper lip, 8.5–10.5 mm; spur tapering, 2 ̶3 mm, subacute. capsule spherical, 4.5–6 mm long, shorter than calyx teeth. seeds ovoid-ellipsoid, 0.65–0.70 × 0.45–0.50 mm, dark brown to black, with prominent longitudinal crest, discontinuous, clearly echinate, testa of the intercostal spaces covered by papilla (figs. 2,3). flowering and fruiting time: may-june. habitat: recently burned pinus brutia forest from 100 to 200 m altitude. examined specimens: turkey: c1 muğla: ören, between çamlıca and kumluca villages, recently burned pinus brutia forest, 150 m, 37°03'28.50" n, 27°57'34.26" e, 09 may 2014, g. zare & g. ergan, gz963 (hub!) greece: arkadhia, ep. mandinias, nw artemisi, acker, krautflurent, 600 m, 37°41'04'' n, 22°21'41'' e, jaunery 2007, fg 27, no 93.138 (b!) fig. 1. chaenorhinum gerense: a) habitat; b) a general appearance; c) fruits and flowers (gz1093). fig. 2. comparison of seed ornamentations by sem. a, b) chaenorhinum gerense (gz1093); c, d) c. rubrifolium (gz963). taxonomic notes on chaenorhinum in turkey acta bot. croat. 77 (2), 2018 211 forms depending upon the treatment of different authors (fernandes 1971, speta 1980, sutton 1988, benedí-gonzález 1991). these taxonomic problems are mainly based on the distribution area of these taxa and the lack of sufficient collections of materials belonging to this species. in fact, these species are relatively rare despite their wide range from sw europe and nw africa to sw asia (speta 1980, sutton 1988, benedí-gonzález 1991). our research indicated that seeds of this species have a high-level of dormancy, and they germinate only when some fire-related germination cues are applied (tavşanoğlu et al. 2017). this germination behaviour of this annual species can explain why it is scarce, and can be found in recently burned mediterranean sites (céspedes et al. 2014, tavşanoğlu et al. 2017). fernandes (1971) defined three subspecies and two forms for european c. rubrifolium, and following his treatment, sutton (1988) divided the species into four subspecies: subsp. rubrifolium, subsp. formenterae (gand.) r. fernandes (1971:227), subsp. raveyi (boiss.) r. fernandes (1971:227) and subsp. gerense (stapf ) d.a.sutton (1988:114), while some authors have treated all these subspecies as separate species (benedí and güemes, 2009). davis (1978b) in his account on turkish chaenorhinum species listed five species of the genus and he did not include c. rubrifolium. in the flora of turkey and east aegean islands (davis 1978a), later, he reported this species from two closely located sites in se anatolia based on his collections (mardin, coll. numb. 28577, coll. numb. 28529 and cizre, coll. numb. 42705). davis (1978a) mentioned differences in seed characteristics and non-persistent basal rosette leaves at the flowering time of these samples with west mediterranean materials but treated these samples as c. rubrifolium. we assume that he evaluated the differences in these specimens as intraspecific variation and did not believe that these characteristics were sufficient for these taxa to be considered separate species. sutton (1988), during the revision of the genus identified these materials as c. rubrifolium subsp. gerense. in the more recently published flora iranica by podlech and iranshahr (2015), the authors followed the sutton classification and evaluated this taxon as c. rubrifolium subsp. gerense. our detailed work on se anatolian samples revealed that the indumentum, branching pattern and long calyx teeth of c. gerense resemble those of c. rubrifolium, but it is remarkably different from this species by the small, cream corolla with red blotch, ascending or patent pedicels in fruit, calyx teeth two times longer than the capsule and triangular or rectangular with blunt seed ornamentation type (tab. 1, figs. 1–3). considering all the above, we confirmed the sutton decision about materials collected from se anatolia by davis, but we believe these specimens should be treated as a separate species, c. gerense. consequently, these two species have increased the species number of chaenorhinum in turkey to nine, in which five are endemic. in any case, further research is still needed to clarify the relationships among chaenorhinum taxa at inter and intra-species levels. fig. 3. chaenorhinum rubrifolium: a) habitat; b) a general appearance; c, d) flowers (gz963). cyprus: aradippou, sw of village, eastern side of motorway s of kalo chorio/aradippou exit open gypsum slopes, c.100 m, 34°56'32'' n 33°33'49'' e, 07 may 2009, r. hand & c.s. christodoulou (b!) morocco: prov. taza, s of road azrou-fes, lake dayat aoua, n-side; 1480 m, 23 june 1996, a.chhal el kadmiri & e. vitek 96-0889 (w!) italy: dorgali, sudhang des mte. bardia oberh. cala gonone. ca. 450 m, 20 april 1966, h. merxmuller & f. oberwinkler 21129 (msb!) spain: almeria, sierra alhamilla, cerca del cortijo 1000 m, 14 april 1921, e gros 19/27 (w!, bc!); formentera, cap de berberia, ca. 2 km sudl. von can fita, bodenaushubflache am wege, 8 jun 1972, h. kuhbier u. g. finschow (msb!); aranjuez (madrid), 15 may 1945, rivas goday et monasterio (msb!); la rioja, leza de rio leza. 30twm4988, 500 m., 28 june 1985, a.s. rodriguez, (msb!, bc!); cadiz: sierra del pinar, ad 1550 m., 1.julay.1925, p.font, i. quer & e. gros, (msb!, bc!); toledo: yepes, 30svk4717, 700 m., 11 june 1984, amich y ehas. (bc!); yebra de basa, mont serraton, au nord du village, 970 m, 30tyn2308, 28 may 1988, p. montserrat n jaca 112388 (msb!, bc!) general distribution: algeria, baleares, greece, france, italy, morocco, spain, tunisia and turkey. chaenorhinum rubrifolium is a species with a complex taxonomy consisting of many subspecies, varieties and zare g., özüdoğru b., ergan g., tavşanoğlu ç. 212 acta bot. croat. 77 (2), 2018 identification key to the genus chaenorhinum in turkey 1a. flowers subsessile; calyx lobes 8–15 mm; corolla 14–20 mm (excl. spur); seeds cuneate-oblong, appearing pitted between main ribs …………………………………………………………………………………………………………2 1b. flowers conspicuously pedicellate; calyx lobes 2–6 mm; corolla 6–10 mm (excl. spur); seeds ovoid-ellipsoid to broadly oblong, not appearing pitted………………………………………………………………………………………………………3 2a. corolla spur 2–3 mm …………………………………………………………………………………………calycinum 2b. corolla spur 9–12 mm …………………………………………………………………………………..huber-morathii 3a. capsule longer than calyx lobes; seeds 0·5–1 mm, seeds with distinctly broad crest; plant not chasmophytic ……………………………………………………………………………………………………………………………4 3b. capsule shorter or as long as calyx lobes, spherical; seeds 0·3–0.7 mm, echinate or with discontinues crest; plants chasmophytic …………………………………………………………………………………………………………………5 4a. seeds 0.5–0.7 mm; capsule 3–5 mm, slightly longer than broad; branches usually flexuous …………………….minus 4b. seeds 0.8–1 mm; capsule longer than 5 mm; spherical; branches ± straight ………………litorale subsp. pterosporum 5a. plant with basal leaves; median leaves elliptic to lanceolate ……………………………………………………………………6 5b. plant without basal leaves; leaves all ovate……………………………………………………………………………………7 6a. leaves petiolate; capsule 2–3 mm; seeds 0.3–0.5 mm, cresta discontinues, triangular or rectangular with blunt apex ……………………………………………………………………………………………………………………gerense 6b. leaves subsessile; capsule 4.5–6 mm; seeds 0.6–0.7 mm, cresta echinate…………………………………...rubrifolium 7a. calyx lobes 4–5 mm; seeds shallowly ribbed …………………………………………………………semispeluncarum 7b. calyx lobes 2–3 mm; seeds distinctly ribbed…………………………………………………………………………………8 8a. inflorescence secund; pedicels recurved in fruit; seeds 0.5–0.6 mm……………………………………………yildirimlii 8b. inflorescence multilateral, pedicels erect in fruit; seeds 0.7–0.9 mm…………………………………………cryptarum table 1. morphological comparison of chaenorhinum gerense with c. rubrifolium. characters c. rubrifolium c. gerense height (cm) 4-22 2.5-12 (25) basal leaves leaves subsessile, ovate to ovate-oblong leaves petiolate, elliptic-ovate basal leave size (mm) 7-25 × 5-11 8-12 × 3-4 pedicels (mm) 5-15, erect 3-10, ascending flower erect patent corolla colour white with violet upper lip cream with red batch in upper lip corolla size (mm) 8.5-10.5 5-7(9) spur (mm) 2-3 1.5-2 calyx teeth (mm) 6-8 unequal slightly longer than capsule 5-7 unequal 2 × longer than capsule capsule (mm) 4.5-6 2-3 seed ornamentation crest echinate (spiny) crest triangular or rectangular with blunt and obtuse apex seed size (mm) 0.65-0.70 × 0.45-0.50 0.30-0.50 × 0.3-0.35 acknowledgements the rufford foundation [rsg 13663-1] financially supported the field work and g. ergan. g. zare thanks to the scientific and technical research council of turkey (tübi̇tak-bi̇deb 2216) for their financial support. references benedí, c., güemes, j., 2009: chaenorhinum. in: c. benedi, e. rico, j. güemes, alberto herrero (eds.), flora iberica. plantas vasculares de la península ibérica e islas baleares. vol. xiii, plantaginaceae-scrophulariaceae, 167–198. real jardín botánico, madrid, spain. benedí-gonzález, c., 1991: taxonomy of chaenorhinum rubrifolium aggr. (scrophulariaceae) in western mediterranean area. collectanea botanica (barcelona) 20, 35–77. céspedes, b., torres, i., pérez, b., luna, b., moreno, j. m., 2014: burning season does not affect post-fire regeneration but fire alters the balance of the dominant species in a seeder-dominated mediterranean shrubland. applied vegetation science 17, 711–725. davis, p. h., 1978a: chaenorhinum. in: davis, p. h. (ed.), flora of turkey and the east aegean islands. vol. 6, 650–654. edinburgh university press. taxonomic notes on chaenorhinum in turkey acta bot. croat. 77 (2), 2018 213 davis, p. h., 1978b: chaenorhinum. in: davis, p. h. (ed.), materials for a flora of turkey xxxv: primulaceae and scrophulariaceae, 36, 1–22. notes from the royal botanic garden, edinburgh. de candolle, a. p., 1815: flore francaise. vol. 5. desray, paris. endlicher, s., 1839: genera plantarum secundum ordines naturales disposita part 9. vienna. fernandes, r. b., 1971: notes taxonomiques sur le genre chaenorrhinum (dc.) reichenb. in: v. h. heywood (ed.), flora europaea. notulae systematicae ad hora europaeam spectantes n. 9. botanical journal linnean society 64, 215–229. fourreau, m., 1869: catalogue des plantes du cours du rohne, annales de la societe linneenne de lyon 17, 89–201. plaza, l., fernandez, i., juan, r., pastor, j., pujadas, a., 2004: micromorphological studies on seeds of orobanche species from the iberian peninsula and the balearic islands, and their systematic signi cance. annals of botany 94, 167–178. podlech, d., iranshahr, m., 2015: chaenorhinum. in: rechinger, k.h. (ed.), flora iranica. vol. 180, 4–11. naturhistorischen museums wien. reichenbach, h.g.l. 1828: conspectus regni vegetabilis. cnobloch, leipzig. speta, f., 1980: die gattungen chaenorhinum (dc.) reichenb. und microrrhinum (endl.) fourr. im östlichen teil ihrer areale (balkan bis indien). stapfia 7, 1–72. stapf, o., 1906: plantarum novarum in herbario horti regii conservatarum. decades xl, xli. bulletin of miscellaneous information kew 3, 71–78. sutton, d. a., 1988: a revision of the tribe antirrhineae. oxford university press. london & oxford. tavşanoğlu, ç., ergan, g., çatav, ş.s., zare, g., küçükakyüz, k., özüdoğru, b., 2017: multiple fire-related cues stimulate germination in chaenorhinum rubrifolium (plantaginaceae), a rare annual in the mediterranean basin. seed science research 27, 26–38. thiers, b., 2016+ [continuously updated]: index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden's virtual herbarium. retrieved november, 2016 from http://sweetgum.nybg.org/science/ih/. yıldırım, h., tan, k., şenol, s. g., pirhan, a. f., 2010: chaenorhinum semispeluncarum sp. nov. and c. yildirimlii sp. nov. (scrophulariaceae) from east anatolia, turkey. nordic journal of botany 28, 457–464. acta bot. croat. 79 (1), 2020 s1 social news thirty-ninth eadsve meeting will be held in dubrovnik, croatia, in 2021. at the end of the 1950s vegetation scientists erwin aichinger from austria, sandro pignatti from italy and maks wraber from slovenia met and decided to found a society to stimulate cross-border cooperation in studies of the botanically interesting area of the eastern alpine and dinaric region. in 1960, the first meeting was held in klagenfurt. the eastern alpine and dinaric society for vegetation ecology (eadsve) was established in 1960 with the objective of fostering cross-border cooperation in studies of vegetation. the society organises meetings every second year. the meeting is open not only to members, but also to other researchers interested in the flora and vegetation of the region. the society cultivates contacts between scientists of central and south-eastern europe and it pays particular attention to encouraging young scientists to present themselves and their work with presentations and posters. invitations for discussions and field trips were also organised as additional activities. to date, 38 meetings have been organised. in the last ten years they were in pörtschach, austria (2009), camerino, italy (2011), ohrid, north macedonia (2013), osijek, croatia (2015), prizren, kosovo (2017) and colfiorito, italy (2019). during the last eadsve meeting in colfiorito, and according to a proposal of professors nenad jasprica and željko škvorc from the croatian side, it was decided that the next meeting would be organised by the croatian botanical society and the university of dubrovnik in dubrovnik, croatia, from april 14th to april 17th, 2021. dubrovnik, the pearl of the adriatic, is listed as a unesco world heritage site and is an old city on the adriatic sea coast in the southernmost part of croatia. its population was 43,700 in 2015. phytogeographically, it lies within the class quercetea ilicis br.-bl. ex a. bolòs et o. de bolòs in a. bolòs y vayreda 1950. the south adriatic coast is a unique location in croatia with wonderful opportunities to enjoy the natural world, including the world famous dalmatian karst, as well as unique cultural opportunities, and many gastronomic and horticultural riches. a wonderful place to bring your family. in addition, april is the best time to visit dubrovnik, and is the last month before peak season truly gets under way. in 2021, the meeting of eadsve will be held on the campus of the university of dubrovnik. all scientists, including graduate and undergraduate students, are invited to present their research results at the meeting, either orally or as posters. field trips will require advance registration. more detailed information is available at http://www.eadsve.org/. željko škvorc and nenad jasprica, vegetation ecology section of the croatian botanical society https://www.tripadvisor.com/restaurants-g29998-ashland_oregon.html https://www.localharvest.org/ashland-or/farms https://www.localharvest.org/ashland-or/farms https://associations.sou.edu/2019ashland/families19.html http://www.eadsve.org/ acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 471 acta bot. croat. 73 (2), 471–480, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication least adder’s-tongue (ophioglossum lusitanicum l.) in croatia – distribution, ecology and conservation slavko brana1, nina vuković2*, mitja kaligarič3,4 1 istrian botanical society, trgovačka 45, hr-52215 vodnjan, croatia 2 university of zagreb, faculty of science, division of biology, department of botany with botanical garden, marulićev trg 20, hr-10000 zagreb, croatia 3 university of maribor, faculty of natural sciences and mathematics, department of biology, institute of biology, ecology and nature conservation, koroška cesta 160, si-2000 maribor, slovenia 4 university of maribor, faculty of agriculture and life sciences, pivola 10, si-2311 hoče, slovenia abstract – the presence of the least adder’s tongue (ophioglossum lusitanicum) in croatia is sparsely documented in scientifi c literature, with only a few records to date. after fi ndings from the 19th century, the species was not confi rmed in the fi eld for a whole century, and was consequently considered extinct in the croatian fl ora. it has been recently confi rmed in southern istria (croatia), in habitats with moderate anthropogenic impacts. keywords: croatia, distribution, endangered species, fl ora, istria, ophioglossum lusitanicum introduction the genus ophioglossum l. belongs to ophioglossaceae, one of the most distinctive families among ferns, distinguished from ‘modern’ ferns by many atypical anatomical and morphological features (wagner 1990, pryer et al. 2004). the genus comprises approximately 25–30 species distributed worldwide, but is most commonly found in warmer parts of the world. altogether four species of ophioglossum are registered in the european fl ora (derrick et al. 1987, akeroyd 1993), three of them in croatia and istria: the adder’s-tongue (ophioglossum vulgatum l.), least adder’s-tongue (o. lusitanicum l.) and small adder’stongue (o. azoricum presl) (hršak 1994, nikolić and topić 2005, brana and rešetnik * corresponding author, e-mail: nina.vukovic@biol.pmf.hr copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. brana s., vuković n., kaligarič m. 472 acta bot. croat. 73 (2), 2014 2005, brana 2010, nikolić 2013). o. lusitanicum is a cosmopolite species, widely distributed in europe in the mediterranean/western european part (clausen 1938, jalas and suominen 1972, derrick et al. 1987, akeroyd 1993). the least adder’s-tongue was fi rst recorded in croatia in the middle of the 19th century, during the earliest botanical studies of this area. according to wraber (2001), the earliest note dates from january 1843, relating to a herbarium example collected in istria (islet of veruda) and deposited in the natural history museum of trieste (tsm). the label on this example, written by mutius (muzio) tommasini, indicates that it was collected by checco, who was for 24 years collecting for tommasini (wraber 2001). the fi rst published note on ophioglossum lusitanicum in istria is from tommasini (1873), providing february 1st 1846 as the date of the record in the surroundings of premantura. later freyn (1877) published several localities in the surroundings of pula, while petter (1852) and visiani (1852) record o. lusitanicum in hvar archipelago. the record from hvar archipelago was quoted later by several botanists (schlosser and vukotinović 1869, hirc 1905, trinajstić 1993), but remains unconfi rmed to date. in spite of the many botanists who studied the fl ora of potential localities of ophioglossum lusitanicum on the eastern adriatic coast (neugebauer 1875, rossi 1930, mezzena 1986, topić and šegulja 2000) no additional localities of this species were discovered. only some reviews of yugoslavian fl ora contained notes on o. lusitanicum, presumably adopted from historical literature (janchen 1952, mayer and horvatić 1967). the fi rst fi eld confi rmation, a hundred years after the historical data, was made by slovenian and other botanists in 1974, who recorded o. lusitanicum several times between 1974 and 2001 in the surroundings of ribnjak in southern istria (volme), but published it only in 2001 (wraber 2001). for this reason, o. lusitanicum was overlooked and listed as extinct in the fi rst complete index of the croatian fl ora (hršak 1994) and the fi rst red book of plant species of the republic of croatia (marković 1994). however, the species re-appeared in the new red book of vascular fl ora of croatia as a critically endangered species (cr), partly supported by our personal observations and discovery of new localities (marković 2005). regarding the eastern adriatic coast in croatia and slovenia, the least adder’s-tongue was never recorded north of southern istrian localities (pospichal 1897, martinčič 1999). although the species is noted for the fl ora of italy, there are no known localities in northern italy (pignatti 1982), except the records for friuli (fiori 1943), which have not been confi rmed. ecologically, ophioglossum lusitanicum prefers disturbed habitats in their early to middle successional stage, and its development strongly depends on mycorrhizal fungi (wagner 1990, merckx 2013). overall, the species has a winter growing season, with above-ground parts disappearing in spring. accordingly, the sporulation occurs during winter, although different authors provide different periods: december–april (freyn 1877), october–february (mayer and horvatić 1967), april–may (schlosser and vukotinović 1869), and august–december (pignatti 1982). taking into consideration the defi ciency of data on ophioglossum lusitanicum in croatia, the aim of this paper is to: 1) provide details on historic and recent studies of this species and reveal its currently known distribution in croatia, 2) elaborate in more detail the distributional/ecological information used for assessing its current threat status and 3) provide further considerations on its conservation. ophioglossum lusitanicum in croatia acta bot. croat. 73 (2), 2014 473 materials and methods intensive fi eld work started in the period from 2000 to 2002, as a part of the project »endemic, rare & threatened taxa of istrian vascular fl ora« and was continued throughout the following years. the search included istrian localities from the literature, as well as many other sites where ophioglossum lusitanicum might occur. in addition, the islands of krk, cres, lošinj and partly rab were thoroughly searched for this species. when found, the species was photographed, and in some cases herbarium specimens were collected and stored in herbarium croaticum (za). since the discovery, istrian localities have been visited yearly in the winter period, with the last visit in 2013. we have also searched the istrian peninsula for ophioglossum vulgatum and o. azoricum to record the emergence of leaves and duration of the sporulation period and make comparisons with the phenology of o. lusitanicum. since most localities of o. lusitanicum are situated in south western istria, the observation of the phenology of all three species was carried out on the populations ranging mostly between pula and rovinj, and the average between those meteorological stations was used as a climatic data. in 2005, co-occurring species were recorded at fi ve localities where o. lusitanicum grows most abundantly (sveta foška, kamenjak, cintinere, barbariga and vela gospa) and their abundance was estimated, to determine the species composition at sites where o. lusitanicom occurs in great numbers. the determination of species was carried out using standard determination keys and guides (jávorka and csapody 1975, tutin et al. 1968–1980, tutin et al. 1993, domac 1994, rothmaler 1995) and the nomenclature is in accordance with nikolić (2013). all localities, except the unconfi rmed historical records, were associated with corresponding gauss-krueger coordinates (geocoded with a gps device garmin e-trex). the current known records on the distribution of the species in istria are presented using the mtb 1/64 grid, according to the proposed national standard (nikolić et al. 1998), and a distribution map is prepared with esri arcgis 9.2 software. since we were not able to geocode two historical records (štinjan and mt. pero) into mtb 1/64 fi elds, those records are not presented on the map. results and discussion distribution although many localities in the northern part of the eastern adriatic were examined, the species was confi rmed only in istria, with the fi rst fi nding in 2000 on cape kamenjak. during the following years, several more spots with ophioglossum lusitanicum were registered on kamenjak, which nowadays represents one of the richest croatian localities of this species. altogether, we registered its occurrence in 23 localities (fig. 1, tab. 1). the number of individuals in most of the recorded populations is extremely small (sometimes only few individuals). the largest populations are recorded in sveta foška, kamenjak and cintinere, comprising several hundred (up to more than a thousand) specimens, while the populations in barbariga and vela gospa are also among larger ones (tens of individuals, up to one hundred). apart from the diffi culties in the detection of the species, estimation of its population size is additionally problematic due to the vegetative reproduction by adventitious roots. brana s., vuković n., kaligarič m. 474 acta bot. croat. 73 (2), 2014 our study shows that the least adder’s-tongue nowadays appears only in southern istria, most abundantly in the area kamenjak–marlera. in spite of a very detailed search, we could not confi rm most records from the wider surroundings of pula from freyn (1877). we conclude that the species probably disappeared from those localities due to habitat loss. the record from pakleni otoci (visiani 1852) remains unconfi rmed. considering the time elapsed, it is possible that the species is actually lost on this locality. however, o. lusitanicum is a very small, non-distinctive plant, easily overlooked in the grassland habitat. its leaves appear above ground at the same time when young leaves of other plants start to emerge and develop and thus it could be easily mistaken for some other species. most importantly, it grows in the winter period, i.e. outside the main vegetation season. due to the above-mentioned diffi culties, the species is rarely found during regular fl oristic studies. a detailed, specifi c search is necessary to be more certain about the loss from pakleni otoci, but to our knowledge, no such search was ever undertaken. habitat and phenology regarding soil types, the eolian sands of southern istria seem to be optimal for this species, especially on spots subjected to aerosol during late autumn. in our case, the least adder’s-tongue mostly grows on disturbed habitats, e.g. pathways, passages and edges of paths in the anthropogenically infl uenced garrigue vegetation, where trampling, grazing and fi re occur (fig. 2c), confi rming that this species is favoured by a certain level of disturbance, as previously indicated by wagner (1990). in addition, the composition of other species indicates that the largest populations of least adder’s-tongue are found in anthropogenically infl uenced, heterogeneous habitats, mostly transitions between pasture and garrigue vegetation. species such as eragrostis minor, anagallis arvensis, euphorbia cyparissias, muscari comosum, plantago spp., trifolium angustifolium and tragus racemosus clearly show anfig. 1. main map: distribution of ophioglossum lusitanicum l. in istria, presented in mtb 1/64 fi elds. lower right corner: location of all known records of o. lusitanicum in croatia. black dots – confi rmed and new fi ndings, crosses – examined but unconfi rmed historical fi ndings, black square – unexamined historical fi nding from pakleni otoci. for further explanation of the nomenclature of mtb fi elds see nikolić et al. (1998). ophioglossum lusitanicum in croatia acta bot. croat. 73 (2), 2014 475 thropogenic infl uence in the habitat (on-line supplement tab 1.). presuming that the largest populations refl ect the optimal environmental conditions for this species, these observations are also in agreement with wagner (1990), who associates all ophioglossaceae with early to middle succession. tab. 1. records of ophioglossum lusitanicum l. in istria presented in mtb 1/64 fi elds. unconfi rmed records are in bold. for further explanation of the nomenclature of mtb fi elds see nikolić et al. (1998). mtb 1/64 code mtb basic fi eld name locality reference of previous fi ndings 0948/324 bale črnibek 0948/411 bale vela gospa 0948/433 bale betiga 0950/332 labin rebići 0950/422 labin prklog 1048/214 fažana barbariga-komunal 1048/222 fažana mednjan-kanestrin 1048/223 fažana sveta foška 1048/241 fažana sveta foška 1048/421 fažana peroj 1049/323 vodnjan monteki 1148/2 štinjan štinjan freyn (1877) 1148/244 štinjan fort max freyn (1877) 1149/1 pula mt. pero freyn (1877) 1149/113 pula pta. aguzzo freyn (1877) 1149/134 pula bat. corniale freyn (1877) 1149/142 pula jadreški 1149/144 pula šikići 1149/213 pula jadreški 1149/231 pula jadreški 1149/313 pula veruda freyn (1877) 1149/322 pula čampanož 1149/341 pula gospa od volam wraber (2001) 1149/341 pula kunfi ni 1149/432 pula cintinere 1149/434 pula cintinere 1249/124 premantura kamenjak tommasini (1873) 1249/142 premantura kamenjak 1249/213 premantura kamenjak 1249/231 premantura kamenjak brana s., vuković n., kaligarič m. 476 acta bot. croat. 73 (2), 2014 it is noteworthy that habitats of cape kamenjak, one of the richest localities of least adder’s-tongue in croatia, are regularly disturbed by grazing, tourist visits and even military exercises (performed yearly in autumn). a recent study on the effect of trampling on some rare species on the french island quessant has revealed that o. lusitanicum favours lowmedium intensity of trampling (kerbiriou et al. 2008). it has been found that for successful germination, spores of o. lusitanicum require variable periods of darkness (whittier 1981). therefore, pushing the spores deeper into the soil by trampling could account for the benefi cial effects of this type of disturbance on this species. all ophioglossum species are believed to be dependent on mycorrhizal relationships in both gametophyte and sporophyte generations (wagner 1990). due to the occurrence of heterotrophic life stages, the least adder’s-tongue shares many of the habitat preferences with other myco-heterotrophic plants such as orchids, which are indeed abundantly present in the same localities (e.g. on kamenjak, vuković et al. 2011, 2013). for the same reason, it is not rare to fi nd several species of ophiglossum growing in a mixed population, a so-called »genus community«, also usual for some other ophioglossaceae (wagner and wagner 1983). in some cases, we have found o. lusitanicum and o. azoricum in such communities, where their different phenology served as a distinctive feature, along with some other (sometimes very subtle) differences. regarding phenology, freyn (1877) remarks that the least adder’s-tongue appears in great numbers, but with a small number of fertile individuals. our observations of the phenology have revealed that the above-ground parts (figs. 2a, b) emerge during winter and sporulation occurs during november-january, while the two other ophioglossum species in croatia show spring/summer reproductive seasons (fig. 3). we have noticed that the number of fertile leaves can differ from year to year, sometimes even extremely, possibly due to the difference in the climate conditions. for example, we found approximately 1,000 fertile leaves in sveta foška in 2003, but only 30 fertile leaves in 2009. we observed the highest number of fertile leaves in winters with high moisture and mild temperatures, while during harsh winters fertile leaves appear in considerably smaller numbers. however, we did not compare exact climatic data with the number of fertile leaves. in our opinion, the appearance of fertile leaves should not be ascribed to one factor exclusively, and some other factors such as moisture in the soil and/or the condition of the micorrhyzal fungi could also be important in this context. fig. 2. habitus (a), sporangia (b) and habitat of ophioglossum lusitanicum l. on kamenjak (c) (photo: s. brana). ophioglossum lusitanicum in croatia acta bot. croat. 73 (2), 2014 477 conservation considerations cytological observations suggest that the genus ophioglossum has undergone repeated cycles of polyploidy, and nowadays represents a group of species at the very edge of extinction, a possible evolutionary »dead end« (khandelwal 1990). in croatia, o. lusitanicum is estimated as critically endangered (marković 2005) and accordingly legally strictly protected (anonymous 2013). the species is mainly threatened by anthropogenic infl uence in the habitat: pasture, tourism and infrastructure development (marković 2005). however, our experience of more than 10 years of observations suggests that this is not the case. on the contrary, according to our observations, moderate trampling and grazing are in fact benefi cial, and o. lusitanicum is favoured by such disturbance, which is in accordance with observations of wagner (1990) and kerbiriou et al. (2008). in our opinion, active conservation measures are necessary to prevent the loss of this species from the national fl ora, and this especially applies to the conservation of habitats. ophioglossum lusitanicum requires special conditions in the environment (disturbance and the presence of fungi), while at the same time its habitats are threatened by succession. within this context, we recommend the development and implementation of an action plan for this species, which would include measures against natural succession, while keeping trampling at a medium level. in addition, we strongly support legal protection of the largest fi nding sites of o. lusitanicum. fig. 3. emergence of leaves (black arrows) and sporulation (white circles) of ophiglossum lusitanicum l., o. azoricum presl and o. vulgatum l. compared to climate in the period 1985–2005. the data for rainfall (solid line) and temperature (dashed line) throughout the year represent the average values between pula and rovinj meteorological stations. brana s., vuković n., kaligarič m. 478 acta bot. croat. 73 (2), 2014 acknowledgments the study was undertaken as part of the project »endemic, rare & threatened taxa of istrian vascular fl ora«, funded by the public institution natura histrica. the study was also funded by slovenian research agency, through program groups p1-0164 and »ladiks«. references akeroyd, j. r., 1993: ophioglossum l. in: tutin, t. g., burges, n. a., chater, a. o., edmondson, j. r., heywood, v. h., moore, d., m., valentine, d., h., walters, s., m., webb, d. a. 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(apiaceae) – a pleistocene in situ survivor nina šajna1*, mirjana šipek1, jelka šuštar – vozlič2, mitja kaligarič1 1 university of maribor, faculty of natural sciences and mathematics, koroška c. 160, si-2000 maribor 2 agricultural institute of slovenia, hacquetova ulica 17, si-1000 ljubljana abstract – the present-day diversity of european flora in temperate mountain ranges was mainly formed by the negative effects of pleistocene glaciation, which caused extinctions, restricted survival in situ, and subsequent re-colonization. only rarely can we find species that have retained the molecular information of in situ survival. one such example is the extremely rare h. pastinacifolia rchb., a monotypic genus and a narrow endemic of a mountain plateau south-east of the julian alps (slovenia). we investigated the germination behavior and dispersal indications, which are often closely related to rarity and persistence and thus valuable for species conservation. additionally, results about h. pastinacifolia help us to understand better what kind of species survived glaciations in europe in situ. our results show that these seeds have an underdeveloped embryo and require an ecologically relevant moist chilling period of about 100 days. the temperature sequence of the colder period that h. pastinacifolia seeds received in nature was 20/15 °c (52 days), 10/5 °c (40 days), 5/0 °c (65 days), 10/0 °c (45 days), 15/5 °c (21 days). the germination rate was high, significantly increased by scarification, and therefore prevented long-term seed bank establishment. we found cryptic seed differences expressed by two types of dormancy, each related to the order of the umbel: simple and complex morphophysiological dormancy for the lateral and the main umbel, respectively. seed dispersal was very much influenced by precipitation in autumn. the germination and dispersal characteristics could explain the rarity and at the same time the persistence of h. pastinacifolia. keywords: apioideae, seed dormancy, embryo: seed ratio, temperate region, endemic, pleistocene survivor, persistences * corresponding author e-mail: nina.sajna@um.si introduction the trnovski gozd karst plateau, a mountainous area in western slovenia with elevations up to 1500 m, has an important biogeographic position, representing a meeting zone between the southern sub-mediterranean and dinaric biomes, and the northern alpine one. it represents an orographic barrier characterized by precipitation that is unusually high compared to other, nearby, regions of slovenia, reaching above 3000 mm yearly in 120 precipitation days, mainly as snow, which can last up to 6 months (melik 1960). the precipitation peak falls in october and november; however, because of high oscillations in october, in some years precipitation is absent (pristov 1997). because of the jurassic and cretaceous limestone bedrock dominating the plateau (orehek and ogorelec 1979), it is strongly karstified, without surface water bodies (grom 1969, kranjc 2006). biogeographic location and the specific environmental factors contribute to extremely high plant species diversity with many endemics, even though much of the plateau is covered by dense forest stands, except for steep overhangs on the southern and northern edges, representing continuously open vegetation. during the pleistocene, a local glacier was present in this region (perko 2001). however, the highest peaks remained un-glaciated, constituting a small nunatak. one species that could be connected to survival in situ is the endemic monotypic hladnikia pastinacifolia rchb. with a distinct taxonomic position within the apiaceae lindl. (umbelliferae juss.) family (šajna et al. 2012) – a species found only on trnovski gozd and nowhere else in the world. the species was discovered šajna n., šipek m., šuštar-vozlič j., kaligarič m. 108 acta bot. croat. 78 (2), 2019 as late as 1819 and was from the very beginning regarded as an ancient paleo-endemic and tertiary relict species (mayer 1960, pawlowski 1970). this has also been confirmed by recent molecular studies showing that all known populations of h. pastinacifolia are genetically depauperated as a consequence of survival during pleistocene glaciation in situ on trnovski gozd karst plateau (šajna et al. 2012). in situ survival theories have rarely been used to explain the phylogeographic patterns in temperate mountain ranges, despite numerous phylogeographic studies, since rare species which lack the potential for rapid range expansion and re-colonization are the ones most likely to retain the molecular information of in situ survival (westergaard et al. 2011). in cases in which, because of re-colonization, in situ survivors and conspecific populations from nonglaciated peripheral refugia have met, the genetic information was lost by interbreeding. besides molecular information, survival in situ should also be recognizable from the current distribution pattern and species ecology (schneeweiss and schönswetter 2011). the distribution area of h. pastinacifolia is extremely narrow (less than 4 km2; čušin 2004), fragmented and lying in the near proximity of an area believed to have remained non-glaciated (šajna et al. 2012). the species’ occurrence is restricted to the southern and northern, non-forested edges of the plateau and the upper belt of their steep overhangs. hladnikia pastinacifolia is considered an in situ survivor, not only because of its narrow range size, distribution pattern and genetic diversity, but also on account of its endemism, temporal persistence, rarity and taxonomic distinctness (šajna et al. 2012). however, its rarity is somewhat surprising, since this saxicolous species is not a habitat specialist occupying a narrow ecological niche. it can be found growing in various habitats, like stony grassland, rock crevices and screes (šajna et al. 2012). most habitats are sloped; in the northern distribution margin, both populations are restricted to vertical rock outcrops. in general, habitats of h. pastinacifolia are characterized by high environmental stress and a medium disturbance regime, preventing shading and rapid succession. lack of specific habitat preference also explains why h. pastinacifolia can be found in very variable plant communities (wraber 1990), which have been well described phytosociologically (martinčič 1961, poldini 1978, kaligarič 1997, kaligarič and poldini 1997, dakskobler 1998, dakskobler 2006). in h. pastinacifolia habitats at the southern edge of trnovski gozd, grazing has resulted in semi-natural vegetation belonging to the alliance satureion subspicatae, with many species characteristic for the subalpine alliance seslerion albicantis (kaligarič 1997). in rock crevices, h. pastinacifolia is found among other chasmophytic plants of the potentilletalia caulescentis syntaxon (kaligarič 1997, dakskobler 1998). in the northern section, the area with open vegetation at the edge of the plateau is much narrower, ending abruptly towards the trebuša valley. there, h. pastinacifolia is found growing only in patches of caricetum firmae grasslands, which are more similar to plant communities from the southeastern alps than from the northern dinaric mountains (surina and dakskobler 2005). according to its protected status and a recognized level of general concern, we have considerable knowledge of the species’ life history, ecology and biology. hladnikia pastinacifolia is a spring-germinating monocarpic species. the development of the flowering plant takes several vegetation seasons (šajna et al. 2014). before the flowering, plants form only basal rosettes of numerous glossy, leathery leaves. the white flowers are bisexual and protandrous. frequent visits by insects and a high pollen-ovule ratio indicate cross-pollination by less successful pollinators (šajna et al. 2014). the fruit is a typical schizocarp, at maturity splitting into two mericarps (hereafter, seeds) 4 mm long and 2 mm wide. on average, 60% of schizocarps are developed intact, and both mericarps are fertile (šajna et al. 2014). seed testa and fruit pericarp are connected, without any structures for better dispersal, whether by wind or animals. however, we still lack information about the germination strategy that has enabled the survival of h. pastinacifolia, and that should be of prime importance for understanding the rarity and persistence of any rare species. representatives of the apiaceae family typically form seeds with abundant endosperm and an underdeveloped embryo (martin 1946), which is true for h. pastinacifolia as well (šajna et al. 2014). in such species, the fully developed seed cannot germinate until the embryo growth reaches the critical length needed for germination (baskin and baskin 2004). seed morphology is often the cause behind a specific type of dormancy break. in various apiaceae species from the temperate region, 4 types of dormancy have been observed (sensu nikolaeva in baskin and baskin 1998): morphological dormancy – md (apium graveolens l., conium maculatum l., pastinaca sativa l.), non-deep simple morphophysiological dormancy – mpd (selinum carviflora (l.) l., angelica sylvestris l., vandelook et al. 2007), nondeep complex mpd (osmorhiza longistylis (torr.) dc, baskin and baskin 1998), and deep complex mpd (heracleum sphondylium l., stokes 1952, anthriscus sylvestris (l.) hoffmann, baskin et al. 2000, aegopodium podagraria l., phartyal et al. 2009). dormancy type and germination can differ in seeds from a single individual if seed heteromorphism is present (thomas et al. 1979, baskin and baskin 1998), especially if exhibited by morphological and anatomical differences between seeds from the main and lateral umbel in some apiaceae (thompson 1984, moravcová et al. 2005, jurkonienė et al. 2016). in h. pastinacifolia at first, only one main umbel (the first order umbel) is formed. approximately one month later, when rosette leaves begin to senesce, the stem branches form several lateral umbels (the second order umbel). therefore, different order umbels are visited by different pollinators (own observation) and we expected seeds from different umbels to differ in germination. this is especially likely because seed heteromorphism can be cryptic (venable 1985) when seeds exhibit different germination behavior even though they are morphologically similar, e.g. they respond differently to cold stratification and water availability (leverett and jolls 2014). we aimed to investigate the species’ germination behavior, since h. pastinacifolia is extremely rare, geographically germination behavior of endemic hladnikia pastinacifolia acta bot. croat. 78 (2), 2019 109 restricted, and at the same time believed to be one of the rare in situ survivors. germination behavior is often in close relation to rarity, if germination rate is low. on the other hand, it can enable persistence, if dormant seeds are able to survive in the soil seed bank for long time. in this regard, germination behavior, together with seed dispersal potential and seedling establishment, is an important knowledge base for species conservation. we examined (1) embryo growth during after-ripening, (2) critical embryo length, (3) the germination rate in artificial and natural conditions, and (4) spatial distribution of seedlings in the natural habitat as an indication of dispersal. materials and methods seed collection, embryo growth and germination collection of ripe seeds was restricted in accordance with sampling permission from the institute of the republic of slovenia for nature conservation. we were allowed to sample only a few plants and their parts in a natural h. pastinacifolia population at predmeja (45°56’33’’n, 13°52’50’’e; 894 m a.s.l.). this population has been recognized as the one where sampling and other studies would have the least negative effect (šajna et al. 2011). we collected randomly 40 seeds originating from the main and lateral umbels separately. at the beginning of november 2006 and september 2008, we buried seeds immediately after their collection on site and in 2008 we collected additional seeds at the same location for germination studies in the laboratory. after the end of the study, seedlings were used to start a tissue culture (ambrožič dolinšek et al. 2016, ciringer et al. 2018) and the remaining seeds were returned to the natural site. seeds for the two laboratory tests were dry stored at room temperature until the beginning of the study in february 2009. we used only fully-developed seeds, as established by slightly pinching each one with forceps to see whether seeds contained a firm endosperm (baskin and baskin 1998). in the first experiment the embryo length to seed length ratio, the e:s ratio, was determined for imbibed seeds during after-ripening at 4 °c and 23 °c in the dark for seeds originating from two umbel orders by longitudinal hand-sections of 5 intact imbibed seeds each time (in total, 15 times in a 150 day period). length was measured with an ocular micrometer. the critical embryo length and the critical e:s ratio were recorded at the time of seed coat rupture without radicle protrusion (vandelook et al. 2007). in each germination study of the second experiment, 20 unsterilized seeds were allowed to germinate on two layers of filter paper moistened with 10 ml deionized water in a petri dish (ø = 10 cm). we first tested under which conditions seeds germinate, and whether germination differed according to umbel origin. therefore, both seed types were exposed to low temperature at 4 °c in the dark, or to 2 conditions in the growth chamber (photoperiod 12/8 h or total darkness, both at 23 °c). seeds were either scarified (slight incision in the testa with a sterilized razor, not damaging the embryo) or left intact. the second part of the germination study was determination of the final maximal germination rate. we used the unscarified main and lateral umbel seeds from the study above, which were held at 4 °c for 106 days (until may 15th), when we transferred them to the growth chamber. to test how seeds respond to environmental conditions in the natural site, we performed a burial experiment in the field. in the autumn of 2006 and 2008, we buried nylon mesh bags containing 40 intact seeds, from the main and lateral umbel separately, 2 cm deep in the soil of predmeja pasture. in 2006 we additionally put seeds under a large stone in a stone wall, and in 2008 we additionally buried seed in the roadside gravel at another location near golobnica gorge (location details in šajna et al. 2009). the bags that we had buried on 5th of november 2006 were collected on 21st of april 2007, and the bags buried on 4th of september 2008 were collected on 16th of may 2009. together with the seeds, we each time buried a temperature sensor (stowaway tidbit xt; onset computer corporation, bourne, usa), which recorded values every 12 minutes. temperature data was evaluated with boxcar pro 4 (onset computer corporation, bourne, usa) software. seedling distribution in the microhabitat to test whether seed dispersal in a wet autumn is minimized by rain, which we expected would beat down the seeds on the site of the dead mother plant (šajna et al. 2014), we evaluated seedling density and spatial occurrence at the microhabitat level. we selected four distinct habitats and/or localities where h. pastinacifolia was present. we did not include rock crevices, since the establishment of seedlings there is much more mediated by crevice density and availability than by dispersal itself. in the study habitats, plots (70 × 70 cm) were installed, which were divided by a grid into 25 cells – subplots (14 × 14 cm). we assessed seedling establishment at peak occurrence in june and august 2005 in predmeja (2 permanent plots) and only in august on the gravel in golobnica and on poldanovec peak by counting the number of seedlings per subplot. for better understanding of the spatial distribution of h. pastinacifolia, we counted non-flowering rosettes and flowering plants on plots, as well. because fine-scale spatial environmental heterogeneity can influence plant establishment (fenner and thompson 2005), we measured the following microhabitat conditions in each subplot: soil temperature (once, in the middle of the subplot), soil depth (3 times per subplot), vegetation height and soil humidity (once in the middle of the subplot if substrate depth allowed). connections between occurrences of h. pastinacifolia life stages and the measured plot characteristics were evaluated for significant correlations (at p < 0.05). we expected that, in the event of low dispersal, seedlings would have a clustered distribution. therefore, the density in a selected grid cell would correspond strongly to the density of a neighboring cell. for describing the spatial distribution of seedlings, we calculated whether the possibilities of seedling occurrence in a given subplot were higher if the numšajna n., šipek m., šuštar-vozlič j., kaligarič m. 110 acta bot. croat. 78 (2), 2019 bers of seedlings in the neighboring subplot were also high and vice versa. therefore, we arranged the subplots according to seedling abundance and calculated for all 25 subplots the ratio in which their neighboring subplots contained 0, 1, 2, 3, …, n seedlings. for defining neighboring subplots, we applied the 8-cell neighbor rule (next-nearest neighbor), by which two subplots are considered neighbors if they share sides along either a cardinal or a diagonal direction. results embryo growth hladnikia pastinacifolia seeds have an underdeveloped embryo, which is of the linear axile type. cotyledons are evident even though underdeveloped. at the time of dispersal, the embryo of h. pastinacifolia seeds measured about 0.4 mm and needed to reach the critical length for germination (fig. 1). before we began to evaluate embryo growth in an imbibed seed, we measured the dry seeds as well, and this data is included in fig. 2 as time zero. un-imbibed seeds had, on average, an e:s ratio of 0.24 ± 0.07 (for the main umbel) and 0.20 ± 0.03 (seeds from the lateral umbel). because of the seed’s water uptake during imbibition, the e:s ratio decreased over the next few days. we noticed the first significant changes in embryo length only with seeds exposed to 4 °c, where seeds from the lateral umbel already showed changes after 50 days, and seeds from the main umbel much later – after 92 days. however, we observed the first sporadic seedlings from the main umbel emerge sooner than from the lateral umbel. therefore, we estimated that 150 days at 4 °c is the maximum time when embryos in most mericarps reached their critical length above 3.6 mm, whether originating from the main or the lateral umbel. we determined that the critical e:s ratio for the start of germination was above 0.84. germination in the laboratory to obtain representative germination results which we could later compare to the results from the natural site, we fig. 1. cross-sections of hladnikia pastinacifolia seeds during germination: under-developed embryo (a), its elongation on the 90th day (b), and embryo reaching the critical length inside the mericarp (c). fig. 2. ratio between embryo length and seed length (e:s ratio) of hladnikia pastinacifolia imbibed seeds during after-ripening at 4 °c and 23 °c: for mericarps of the lateral umbel at 4 °c (a), for mericarps of the lateral umbel at 23 °c (b), for mericarps of the main umbel at 4 °c (c), for mericarps of the main umbel at 23 °c (d). germination behavior of endemic hladnikia pastinacifolia acta bot. croat. 78 (2), 2019 111 did not sterilize the seeds. in the growth chamber, all seeds from the main umbel were already overgrown by fungus (fusarium sp.) after 4 days, and none germinated, while the first germinated seeds from the lateral umbel were observed after 102 days, with a germination rate (g%) of 8%. the first germinated seeds at 4 °c emerged after 92 days when originating from the main umbel and after 116 days in the case of lateral umbel seeds. after 116 days, we transported seeds originating from each umbel type from 4 °c to 23 °c. the final g% after 175 days was 20% for seeds from the main umbel and 28% for seeds from the lateral umbel. seeds from the main umbel reached a germination peak between the 144th and 162nd day (between 22nd of june and 10th of july), followed by seeds from the lateral umbel from day 160 onwards. scarification increased the germination rate for the main umbel seeds to 92%, and for lateral umbel seeds, to 76%. during further seedling development, the pericarp was first ruptured by the radicle, and at a radicle length of 0.9 cm, the hypocotyl protruded from the pericarp (fig. 3). at that time, the hypocotyl was bent like a hairpin (loop sensu kondo et al. 2006). cotyledons separated from the rest of the pericarp when the total seedling length measured between 1.6 and 2.5 cm. the first leaf developed when the seedlings reached 4.5 cm. for main umbel seeds and 13% for lateral umbel seeds buried in the stone wall. most of the seeds in bags buried in stony pasture soil for 253 days (from september 2008 until may 2009), germinated before their collection. however, the seedlings were still alive. the germination rate for main umbel seeds was 96%, and 80% for lateral umbel seeds. on the other hand, all seedlings from seeds buried in the roadside gravel of the golobnica gorge died of drought. under the dissecting microscope, we were able to reconstruct g% reached, which was 63% for the main umbel seeds and 20% for the lateral umbel seeds, while the un-germinated mericarps were full of fusarium macroconidia. the temperature conditions to which buried seeds were exposed (fig. 4) in stony pasture on the southern part of the h. pastinacifolia distribution (predmeja) during winter 2006/07 showed a daily fluctuation of 7 °c from september to november, and of about 9 °c from mid-february onwards. temperatures below 0 °c prevailed for only one week at the end of december, reaching a minimum of –1.4 °c for one week in the beginning of february. additionally, natural chilling conditions between 0 and 4 °c were first recorded on the 2nd of november 2006, and finally on the 9th of april 2007. during this 5-month period, chilling temperatures occurred on 91 days, with the longest series of 33 days in a row from the 9th of december until the 10th of january. fig. 3. germination of hladnikia pastinacifolia: radicle protruding (a), enlarged view (b), growth of the radicle (c) – (e), bent hypocotyl (black arrows indicating the loop) (f ), cotyledons separated from the pericarp (h). burial experiments in both burial experiments, the seeds were germinated at the time of excavation. buried seeds were kept constantly in total darkness, especially in the case where the bag was placed under a stone. seeds buried in november 2006 germinated best in the soil of the pasture – reaching a germination rate (g%) of 38% for the main umbel and 30% for the lateral umbel seeds after 167 days, on 21st of april. seeds from bags placed in a man-made stone wall had lower g%: main umbel seeds 30%, lateral umbel seeds 8%. we were able to transport these seeds into the laboratory, where the final g% on 2nd july, 2007 was as follows: 65% for main umbel seeds and 43% for lateral umbel seeds buried in pasture soil; 53% distribution of various h. pastinacifolia developmental stages in relation to microhabitat characteristics fine-scale spatial environmental heterogeneity of stony pasture habitat in june showed the following significant correlation with ontogenetic stage abundance: for seedlings, a negative correlation with soil humidity (r = –0.2957, p = 0.0371); for the cumulative number of h. pastinacifolia individuals, a negative correlation with soil humidity (r = –0.3043, p = 0.0317) and a positive one with soil depth (r = 0.2683, p = 0.0596). abundance was never significantly correlated with soil temperature in the pasture. for the man-made stone wall, we could only test for correlations with soil temperature, showing that the abundance of non-flowering rosettes was negatively correlated with soil temperature (r = –0.1906, p = 0.3614). fig. 4. daily maximum, minimum and mean temperatures to which buried hladnikia pastinacifolia seeds were exposed in natural habitat in predmeja from september 2006 to april 2007. šajna n., šipek m., šuštar-vozlič j., kaligarič m. 112 acta bot. croat. 78 (2), 2019 when we evaluated the same plots in august, these correlations were consistent: a negative correlation between seedling abundance and soil humidity (r = –0.3282, p = 0.02) and a negative correlation between the cumulative number of h. pastinacifolia individuals and soil humidity (r = –0.3210, p = 0.0230), since seedlings represent the majority of individuals. at the same time, the cumulative number of individuals as well as the number of non-flowering rosettes was positively related to soil depth in the pasture (r = 0.0115, p = 0.937 and r = 0.3503, p = 0.0126, respectively). however, in august a significant correlation was found between seedling occurrence and temperature (r = 0.3106, p = 0.0281). the abundance of seedlings between june and august varied – decreasing or increasing by about 20%, suggesting that seedling emergence occurred throughout the vegetation period. however, because of the contradictory effect of seedling emergence and mortality, we cannot use these results for seedling survival predictions. in a similar habitat (stony grassland) on poldanovec peak at the northern distribution margin, no correlations were found for either developmental stage. this was also true for the scree habitat in the golobnica gorge. spatial distribution of seedlings suggesting dispersal characteristics spatial distribution of seedlings in various habitats (fig. 5), calculated according to the possibility for seedling occurrence in a given subplot and in the neighboring subplot, showed that in each habitat, a greater abundance of seedlings in a given subplot was more likely if the neighboring subplot contained more seedlings. all graphs showed an increase in seedling abundance more or less in the direction of the x = y line. discussion consistently with other apiaceae members, hladnikia pastinacifolia forms seeds with underdeveloped embryos and abundant endosperm. the linear axile embryo is differentiated into cotyledons and a hypocotyl with radicle; however, the embryo is underdeveloped in terms of size at the time of dispersal. seeds with such embryos exhibit dormancy related to the time required for the embryo to grow to the critical length when the radicle can protrude from the seed testa (larcher 2003). if, additionally, specific temperature conditions for further embryo development are needed, germination is prevented by mpd (baskin and baskin 1998, baskin and baskin 2004, vandelook et al. 2007). both types, seeds from the main and from the lateral umbel, were morphologically dormant because of an underdeveloped embryo. according to germination results, the after-ripening during cold stratification at a constant temperature of 4 °c ended sooner in seeds from the main umbel and took 92 days, while germination from lateral seeds was observed 24 days later. in h. pastinacifolia, seeds of both types germinated immediately fig. 5. spatial distribution of hladnikia pastinacifolia seedlings in a microhabitat: stony pasture (predmeja) (a), man-made stone wall (predmeja) (b), stony grassland (poldanovec mountain peak) (c), gravel – scree (golobnica gorge) (d). colors show the proportion of seedlings present, where the data was optimized according to the distance weighted least squares method. for details see materials and methods. germination behavior of endemic hladnikia pastinacifolia acta bot. croat. 78 (2), 2019 113 after completion of embryo growth. the embryo in seeds from the main umbel grew only at low temperatures, indicating that the seeds have complex mpd. however, seeds from the lateral umbel also germinated in the growing chamber exposed to light, thus indicating simple mpd. as stressed by baskin and baskin (2004), to further evaluate which type of complex mpd (nondeep, intermediate, deep) is exhibited by seeds from the main umbel, we would have to test the response of the seeds to gibberellic acid (ga3). from laboratory studies, we have estimated that the ecologically relevant moist chilling period is about 100 days. the burial experiment enabled us to obtain the actual temperature sequence of the colder period which h. pastinacifolia seeds would receive in nature (averaged and pulled data from fig. 4): 20/15 °c (52 days), 10/5 °c (40 days), 5/0 °c (65 days), 10/0 °c (45 days), and finally 15/5 °c (21 days). according to phartyal et al. (2009), who studied the impact of temperature regimes on the germination of aegopodium podagraria, the temperature of 0 °c was more favorable for the dormancy break than 5 °c. in nature, h. pastinacifolia seeds were cold stratified for 110 days at 0 °c (more precisely from –2.5 to 2.5 °c), a period of time similar to that needed in the laboratory studies conducted at 4 °c. in the laboratory, g% was increased by an additional 72% and 48%, with scarification for seeds from the main and lateral umbel, respectively. in the natural scree habitat such scarification could easily happen when seeds fall into the gravel substrate. in the natural site, buried seeds’ g% for main umbel seeds was 96% and 80% for lateral umbel seeds, very similar to the g% reached in the laboratory with scarification. in monocarpic species, plant establishment is critical for population persistence. they can recover population from a soil seed bank (e.g. campanula cervicaria l., often 1999, eisto et al. 2000). dormancy would assure that at least some seeds would germinate over a longer period; however, this was not the case in h. pastinacifolia. the high germination rate in natural conditions did not allow the establishment of a persistent soil seed bank, while at the same time, european apiaceae generally have short-lived seeds (walters et al. 2005). variability of fruits and seeds on a single plant can be a source of dispersal and of functional plasticity, increasing the recruitment potential of seedling establishment in space and time, especially under variable environmental conditions (imbert 2002). seed heteromorphism in h. pastinacifolia has to be understood as differences in germination characteristics related to the order of the umbel on which seeds occurred on the plant. this is a form of cryptic seed heteromorphism, shown in germination rate and dormancy type, but not exhibited by the seed size. true and cryptic heteromorphism is often associated with species growing in extreme, highly stressful, and/or unpredictable environments like deserts and fire-prone or open arid areas. however, heterodiaspory also occurs in weedy annuals of disturbed habitats even in temperate regions, as in the common apiaceae member apium graveolens (gutterman 1992). most habitats of h. pastinacifolia are sloped, while the most isolated population occupies a vertical rock outcrop. in all habitats, high environmental stress is present, combined with medium to strong disturbance. such habitats, when they exhibit high irradiation and simultaneous gaps in vegetation cover, are characterized by the ability to host species with weak competitive potential because of slow succession (černý et al. 2006). similarly, the stenoendemic obligate chasmophyte campanula tommasiniana (surina 2013) is expected to have low biotic competitiveness, but this is in a way compensated with species’ remarkable plasticity to tolerate various abiotic conditions (surina and martinčič 2014). the first indication that h. pastinacifolia has low competitive and low establishment potential in its habitats is the previously mentioned high germination rate of both seed types, which prevents seed accumulation in the soil seed bank to ensure persistence of the species over time. the second one is the strong correlation between seedling occurrence and soil humidity, especially since seedlings have a clustered distribution in space. high seedling density generally has a strong negative effect on seedling survival (koelewijn 2004). less clustered distribution of h. pastinacifolia seedlings was observed on the scree than in the pasture. this could be explained by the instability of the scree substrate rather than by seed dispersal. the scree habitat was also recognized as the most suitable habitat for h. pastinacifolia from morphological analyses (šajna et al. 2012). according to the chasmophytes on screes in the close vicinity (surina and martinčič 2012), h. pastinacifolia could also be a niche edge left-over because of low dispersal. adaptations for marginal persistence in the expected niche could be attributed to several traits involving drought tolerance (šajna et al. 2014). therefore, environmental constraints, including the presence of unoccupied gaps, are more crucial for the survival of relic species in such habitats than the species composition itself (černý et al. 2006). we believe the same explanation can apply to h. pastinacifolia’s occurrence and persistence in various plant communities, even though numerous attempts were made to describe typical h. pastinacifolia associations in the past. open, unshady areas, like the karstic pasture in our study, seem to provide alternative open habitats. acknowledgements the authors are grateful to p. kušar for valuable commentary and to two anonymous reviewers for valuable suggestions improving the manuscript. we thank the republic of slovenia institute of nature conservation for issuing the sampling permit. the study and 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(apiaceae). seed science research 17, 283–291. venable, d.l., 1985: the evolutionary ecology of seed heteromorphism. the american naturalist 126, 577–595. walters, c., wheeler, l.m., grotenhuis, j.m., 2005: longevity of seeds stored in a genebank: species characteristics. seed science research 15, 1–20. westergaard, k.b., alsos, i.g., popp, m., engelskjøn, t., flatberg, k.i., brochmann, c., 2011: glacial survival may matter after all: nunatak signatures in the rare european populations of two west-arctic species. molecular ecology 20, 376–393. wraber, t., 1990: čaven, ein botanisch beruhmter berg in slowenien. carinthia 180, 195–210. 70 acta bot. croat. 77 (1), 2018 acta bot. croat. 77 (1), 70–79, 2018 coden: abcra 25 doi: 10.1515/botcro-2017-0011 issn 0365-0588 eissn 1847-8476 media composition affects seed dormancy, apical dominance and phenolic profile of knautia sarajevensis (dipsacaceae), bosnian endemic erna karalija1,2, sanja ćavar zeljković3,4, petr tarkowski3,4, edina muratović1, adisa parić1,2 1 university of sarajevo, faculty of science, department of biology, laboratory for plant physiology, zmaja od bosne 33–35, 71000 sarajevo, bosnia and herzegovina 2 university of sarajevo, faculty of science, department of biology, laboratory for research and protection of endemic resources, zmaja od bosne 33–35, 71000 sarajevo, bosnia and herzegovina 3 centre of the region haná for biotechnological and agricultural research, central laboratories and research support faculty of science, palacky university, šlechtitelů 27, 78371 olomouc, czech republic 4 centre of the region haná for biotechnological and agricultural research, department of genetic resources for vegetables, medicinal and special plants, crop research institute, šlechtitelů 29, 78371 olomouc, czech republic abstract – knautia sarajevensis is an endemic plant of the dinaric alps and is mainly distributed on bosnian mountains. due to the quite large flower heads and easy maintenance, this plant has a potential use as a substitute ornamental plant for k. arvensis in perennial beds. the current study evaluated the germination process in different treatments in an attempt to suppress dormancy and increase germination rate, and to develop a successful protocol for micropropagation. an over 60% germination rate was achieved through cultivation of seeds on ms basal medium with reduced mineral nutrient composition and the absence of sucrose. on the other hand, a below 10% germination rate was achieved with untreated seeds. suppression of apical dominance was achieved through application of high concentrations of kinetin, apical shoot decapitation or cultivation of shoots in liquid media. overall, liquid cultures were more successful as a micropropagation system for this plant. shoots spontaneously developed roots on multiplication treatments and were successfully acclimatized. moreover, phenolic compound profile was analysed in the light of the possible medicinal potential of this plant. variable amounts of total phenolic compounds as well as individual phenolics were recorded, according to treatment and solidification of media. an increase in rosmarinic acid content was reported for kinetin treatments and acclimatized plants comparing to mother plants in natural habitat. the present study shows that choice of cytokinin concentration, explant type as well as culture type influences not only shoot proliferation and apical dominance suppression but also in vitro production of phenolics. keywords: apical dominance, acclimatisation, endemic plant, knautia sarajevensis, phenolics, seed dormancy * corresponding author, e-mail: erna.k@pmf.unsa.ba; erna.karalija@gmail.com introduction knautia sarajevensis (beck) szabó is an endemic species of the dinaric alps that can be found in wood margins and woodland meadows in bosnia. as an endemic plant species it is included in the red list of flora for the federation of bosnia and herzegovina and it is mainly found on mountains around sarajevo (mt. igman, bjelasnica, trebevic, jahorina, ozren; đug et al. 2013). all of these mountains are subject to great anthropogenic impact due to tourism and to their role as popular recreational sites. these regions are also subject to constant deforestation and neglect, which affects habitat characteristics and plant life, especially plants like k. sarajevensis. knautia sarajevensis is a clonal species and its reproduction is achieved through branching of vegetative organs, while seed germination and establishment of fully grown plants is considered to be rare and usually is linked to population establishment at new sites. poor germination is probably an effect of seed dormancy, elaiosome presence and obligatory light induced germination (mayer and svoma 1998). since k. arvensis is a popular ornamental plant used in perennial beds (hartmann et al. 2010) k. sarajevensis too mailto:erna.k@pmf.unsa.ba mailto:erna.karalija@gmail.com micropropagation process affects phenolic profile acta bot. croat. 77 (1), 2018 71 must have a potential use as an ornamental and could replace k. arvensis due to its larger flower heads and number of flowers than k. arvensis. usually k. arvensis is propagated by dormant crown division or stem cuttings. in vitro culture research into the family dipsacaceae is very scattered, and only micropropagation protocols for scabiosa columbaria producing an average of 3.0 shoots per explant (romeijn and van lammeren 1999) and s. caucasica (hosoki and nojima 2004) are available. apical dominance is a term that signifies apical shoot growth and the inhibition of axillar shoot growth in perennial plants (cline and sadeski 2002), and can be a problem in the micropropagation of these species. in its natural habitat k. sarajevensis shows apical dominance of flowering over rosette formation and in the first year of growth, formation of a ground rosette takes place, while shoots bearing the flower heads are produced the following year. the reason for this probably lies in the importance of accumulation of a critical mass for flowering, as already recorded for succisa pratensis (jongejans et al. 2006). emergence of apical dominance in in vitro culture of k. sarajevensis is probably the result of a lack of critical mass accumulation and must be supressed in order to produce shoots in vitro. the potential medicinal properties of the knautia genus are still not well explored although there are data about knautia arvensis plants as remedies for various skin disorders, and tea made from flowers and leaves of this plant can be used for many lung problems (grieve 1931); this species is listed as a relaxant and blood purifier (mattalia et al. 2013). knautia bidens is listed as a rich source of phenolic compounds comparable to some salvia species (alali et al. 2007). dipsacus genus has been broadly investigated for its medical properties, and there is evidence about its cytotoxic and anticomplementary activities (oh et al. 1999; hung et al. 2005). since dipsacus and knautia genera are closely related, they could share the same or similar medicinal potentials, so the importance of a micropropagation protocol development lies not only in the need for conservation but also in the potential medicinal uses of the genus knautia. accordingly, the aim of this study was to develop a successful micropropagation protocol, through plant regeneration from seeds, for conservation purposes. also production of phenolic compounds was taken under consideration in relation to concentrations of phenolics in mother plants. materials and methods aseptic seed germination fully ripped fruits of knautia sarajevensis were collected from a mother plant from a population located on mt igman. a voucher specimen of the seeds was marked and stored in the herbarium of the national museum of bosnia and herzegovina (no. 51413). all fruits were cleaned, elaiosomes were removed as was epicalyx. seeds were counted and separated into groups of 250 seeds for further treatments (tab. 1). prepared seeds were washed with 70% (v/v) ethyl alcohol for 30 sec, followed by 20 min submergence in 20% (v/v) solution of commercial sodium hypochlorite (4% active chlorine), then rinsed five times with sterile-distilled water. the experiment was conducted through a randomized design with 10 petri dishes containing 20 ml of media, and in each petri dish 25 seeds were inoculated for each treatment. two sets of ten petri dishes for evaluation of germination rate were used, one set for a 30 day, and other set for a 60 day period. all media were prepared according to murashige and skoog (1962) (ms) basal salt composition, ph was adjusted to 5.8 prior to agar addition (0.8%) and were sterilised in an autoclave (1 bar, 121 °c, for 20 minutes). sucrose (3.0%) was added before ph adjustment to all treatments except ks11 and ks12 (tab. 1). gibberellic acid (ga) was filter-sterilised and added after sterilisation of the media for appropriate treatments. all cultures were kept at 23 °c in growth chambers (70% humidity, 2000 lux light intensity, 16 h tab. 1. breaking dormancy and germination rate of knautia sarajevensis seeds. ms – media composition according to murashige and skoog (1962), ga – gibberelic acid. mean values not sharing the same letter(s) within one column are significantly different (p=0.01) according to newman-keuls test. pre-treatment treatment incubation temperature % germination 30 days % germination 60 days ks1 – ms; 3% sucrose; 0 mg l–1 ga 23 °c 9.78±0.01h 24.51±0.00i ks2 – ms; 3% sucrose; 0.15 mg l–1 ga 23 °c 9.95±0.01h 31.94±0.00g ks3 30 days at +4 °c ms; 3% sucrose; 0 mg l–1 ga 23 °c 14.59±0.02g 41.15±0.00e ks4 30 days at +4 °c ms; 3% sucrose; 0.15 mg l–1 ga 23 °c 19.62±0.01e 61.13±0.01a ks5 30 days at +4 °c ms; 3% sucrose;0 mg l–1 ga 48 h +4 °c; +23 °c 30.15±0.00c 48.55±0.00c ks6 30 days at +4 °c ms; 3% sucrose; 0.15 mg l–1 ga 48 h +4 °c; +23 °c 33.05±0.01b 60.54±0.00b ks7 30 days at +4 °c; part of endosperm removed ms; 3% sucrose; 0 mg l–1 ga 23 °c 17.39±0.01f 40.61±0.00f ks8 30 days at +4 °c; part of endosperm removed ms; 3% sucrose; 0.15 mg l–1 ga 23 °c 19.14±0.00e 48.15±0.00d ks9 30 days at +4 °c; isolated embryo ms; 3% sucrose; 0 mg l–1 ga 23 °c 2.26±0.00 j 3.03±0.01l ks10 30 days at +4 °c; isolated embryo ms; 3% sucrose; 0.15 mg l–1 ga 23 °c 7.63±0.00i 7.90±0.00k ks11 – ½ ms; 0% sucrose 0 mg l–1 ga 23 °c 27.06±0.02d 48.20±0.00 j ks12 – ½ ms; 0% sucrose 0.15 mg l–1 ga 23 °c 65.60±0.01a 90.60±0.01h karalija e., ćavar zeljković s., tarkowski p., muratović e., parić a. 72 acta bot. croat. 77 (1), 2018 photoperiod). obtained seedlings were further used for the establishment of the k. sarajevenisis shoot cultures. establishment of the shoot cultures and apical dominance suppression roots were removed from all germinated seedlings prior to cultivation on shoot culture media. the obtained shoots were decapitated and apical and nodal parts were separately cultivated on media containing cytokinins (kinetin, 6-benzyladenin and zeatin respectively) and indole-3-butyric acid (iba). cytokinins were added in 3 different concentrations, i.e. 0.1, 1.0 and 10.0 mg l–1 alone or in combination with 0.1 mg l–1 of iba. treatments without plant growth regulators (pgr free) were used as control. each treatment was represented by 5 erlenmeyer flasks in three independent replications, and in each flask 5 explants were inoculated. all cultures were kept at 23 °c in a growth chamber (70% humidity, 2000 lux light intensity; 16 h photoperiod). after 21 days of cultivation the multiplication rate (number of reactive explants) (mr), multiplication index (average number of produced shoots per explant) (mi), presence of roots and presence of callus (tab. 2) were recorded. evaluation of the media consistency effect on apical dominance suppression in order to evaluate the media consistency effect on apical dominance suppression and biomass production, shoots about 5 cm in length with minimum of two pairs of leaves were cultivated in solid and liquid media with the addition of 0.1, 1.0 and 10.0 mg l–1 of kinetin (kin). treatment without pgr was used as control. all media contained 3.0% of sucrose, were prepared according to the ms procedure, and had 0.8% agar (only solid cultures). media were sterilised in an autoclave, and all cultures were kept at 23 °c in a growth chamber (70% humidity, 2000 lux light intensity; 16 h photoperiod). all treatments were incubated in a shaker (65 rpm), and comprised 5 erlenmeyer flasks per treatment, with three independent replicates for all treatments. every erlenmeyer flask contained 50 ml of media and 5 explants (shoots; 75 per treatment). after 21 days of cultivation for all treatments the multiplication index, and the presence of roots and callus were recorded. plantlet acclimatisation during shoot multiplication, good rooting was achieved and no additional rooting was necessary. plants with developed roots were washed in tap water to remove the agar and then transferred into pots containing a mixture of soil and sand (3:2). all pots were kept under a 16 h photoperiod (2000 lux light intensity), and constant humidity (70%) and temperature (+23 °c). during the period of 15 days plants were gradually exposed to lower humidity (40%) and larger temperature fluctuation (±10 °c). after 20 days, the development of new leaves was noticed and plants were transferred in greenhouse. spectrophotometric analysis of phenolic compounds chemical analysis was done for mother plants and regenerated in vitro plants as well as acclimatized plants (5 plants per sample). phenolics were isolated from the aerial parts of plants by maceration in 80% methanol (hplc grade) by incubation for 24 h at 4 °c. extracts were centrifuged at 2000 rpm for 15 min, and supernatants were collected for further analysis. total phenolic content was analysed according to wolfe et al. (2003) using folin-ciocalteu reagent. quantification was done according to calibration curve of gallic acid and expressed as mg of gallic acid equivalent per g of dry weight (mg gae/g dw). total flavonoid content was done using the aluminium chloride method (ordoñez et al. 2006). concentration of flavonoids was estimated using the calibration curve of catechin. results were expressed as mg of catechin equivalent per g of dry weight (mg ce/g dw). total flavanol content was analysed according to modified method of gadzovska et al. (2007) using 1% dmaca (w/v) reagent (p-dimethyl aminocinnamaldehyde in hcl:ch3oh, 8:92) and using calibration curve of catechin. results were expressed as mg of catechin equivalent per g of dry weight (mg ce/g dw). total proanthocyanidins content was analysed using vanillin-hcl method (wettstein et al. 1977) and tab. 2. effects of decapitation and kinetin application on the multiplication rate and index of knautia sarajevensis cultivated in solid media. mr – multiplication rate (percentage of explants with formatted shoots), mi – multiplication index (average number of formatted shoots per explant), kin – kinetin, iba – indole-butyric acid. mean values not sharing the same letter(s) within one column are significantly different (p=0.01) according to newman-keuls test. kin (mg l–1) iba (mg l–1) nodal explants apical explants mr mi roots callus mr mi roots callus 0.0 0.0 45.15 3.10±0.10c – – 0.00 1.00±0.00b – – 0.1 0.0 52.13 5.08±0.35a good, long – 0.00 1.00±0.00b small, underdeveloped – 0.1 0.1 43.15 2.80±0.33c good, long – 0.00 1.00±0.00b short – 1.0 0.0 42.34 2.40±0.07c short, thick – 0.00 1.00±0.00b short small, with indirect root regeneration 1.0 0.1 32.15 2.05±0.01c longer – 0.00 1.00±0.00b short 10.0 0.0 45.56 3.70±0.08b – – 1.00 2.00±0.05a – – 10.0 0.1 44.43 3.30±0.01c – – 0.00 1.00±0.00b – with shoot regeneration micropropagation process affects phenolic profile acta bot. croat. 77 (1), 2018 73 quantified according to the calibration curve of catechin. results were expressed as mg of catechin equivalent per g of dry weight (mg ce/g dw). samples with the highest total phenolic content were further analysed by hplc-uv technique. hplc analysis of phenolic compounds separation and analysis of flavonoids and phenolic acids (hydroxycinnamic and hydroxybenzoic acids) were performed on a shimadzu lc-2010c hplc using phenomenex kinetex c18 (2.6 µm id, 150 × 4.6 mm) column. the mobile phase included component a (20 mm formic acid in water) and component b (acetonitrile) in a defined gradient (0 min 5% b; 4 min 5% b; 54 min 40% b; 60 min 40% b; 60, 5 min 5% b; 70 min 5% b; 70 min stop) at flow rate 0.4 ml/min. hplc was equipped with uv detector, and absorbance was monitored at 270 nm. concentration of flavonoid components was calculated in relation to calibration curves of standards: myricetin, quercetin, naringenin, apigenin, kaempherol, chrisin, pinocembrin and galangin. for phenolic acids standards of chlorogenic, caffeic, sinapic, ferulic, rosmarinic, gallic, 4-hydroxybenzoic, vanillic, syringic and salicylic acids were used (concentration range: 1×10–7 mol l–1 – 1×10–3 mol l–1). all standards used were purchased from sigma and were of at least analytical grade. statistical analysis all results were expressed as the mean values (±standard deviation; stdev) of the three independent replicates. analysis of variance of parametric data was done according to anova test using newman-keuls test as a post hoc analysis. differences between treatments were evaluated at p<0.01. correlation coefficient was calculated according to pearson product-moment correlation coefficient at p<0.05. all statistical testing was done using statistica 10.0 software (copyright© statsoft. inc. 1984–2011). results aseptic seed germination seeds of k. sarajevensis have elaiosomes and the removal of elaiosomes and seed coat was necessary for dormancy suppression. isolation of embryos, one of the methods for dormancy suppression, was also conducted. different cold pre-treatments for elaiosome-free seeds were used for improvement of the germination rate in combination with gibberellic acid application. the efficiency of gibberellin stimulation of seed germination in k. sarajevensis depended upon the pre-treatment and cultivation conditions. seedlings developed a pair of leaves 15 days after cultivation but germination time was unsynchronized within one treatment. when no cold pre-treatment was applied maximum germination rate was prolonged up to 2 months even with ga application. incubation of non-imbibed seeds at +4 °c for 30 days increased germination percentage. further incubation at 4 °c after imbibition of cold pre-treated seeds (ks5 and ks6 treatments) significantly increased seed germination especially in combination with ga application and maximum germination rate was achieved in the first 30 days (tab. 1). removal of sucrose in combination with reduction of basal salts in the media significantly induced germination and reduced duration of germination period, especially when ga was applied (tab. 1), moreover up to 65% of the seeds germinated in first 30 days. establishment of the shoot cultures and apical dominance suppression in preliminary research (data not shown), seedlings were cultivated on media containing 0.1, 1.0 and 10.0 mg l–1 ba (6-benzyladenine), zea (zeatin) and kinetin (kin) in solid media. during cultivation, emergence of apical dominance was noticed and no shoot formation was recorded. shoots cultivated on solid media containing ba and zea showed signs of chlorosis and root formation was very low, while kinetin induced good root formation, and no chlorosis was noted but apical dominance was present. for further multiplication and apical dominance suppression seedlings from which apical and nodal explants were derived were used. apical parts of the shoots and nodal segments were separately cultivated on media containing different concentrations of kinetin alone or in combination with 0.1 mg l–1 iba. cultivation of explants in kinetin-containing media provided different responses depending upon kinetin concentration and explant type. apical explants did not produce any shoots except when the highest concentration of kin was applied (10 mg l–1), while nodal explants produced shoots in different frequencies. the highest multiplication rate and index were reported for treatment with 0.1 mg l–1 of kin using nodal explants (tab. 2, fig. 1). high concentrations of kin in this study also induced callus formation in the basis of nodal explants with indirect shoot regeneration (tab. 2, fig. 1). addition of iba to the media had inhibitory effects on shoot and root formation (tab. 2, fig. 2). plants with good roots were acclimatized in greenhouse conditions with a survival rate over 70%. all plants formed good ground rosettes comprising up to 10 leaves with no shoot formation demonstrating a 2 year vegetation period as recorded in the natural habitat. evaluation of liquid media effect on apical dominance suppression in solid culture shoots were short (5–7 cm) and no elongation was noticed and apical dominance suppression was only possible by removal of shoot apex or by application of high kinetin concentrations. in contrast, the highest concentration of kin (10 mg l–1) was unfavourable in liquid system and plants decayed within 3 days and this treatment was not further analysed. shoots cultivated in the liquid media containing 0.1 mg l–1 of kin demonstrated a 100% multiplication rate and multiplication was achieved mainly through proliferation of several axillary buds (fig. 3a). also, development of elongated shoots was reported (fig. 3b). apical dominance was suppressed and production of up to 5.4 shoots per explant was achieved using a low concentration of kinetin. karalija e., ćavar zeljković s., tarkowski p., muratović e., parić a. 74 acta bot. croat. 77 (1), 2018 fig. 1. effects of decapitation and kinetin application on apical dominance suppression in knautia sarajevensis; nodal explants cultivated in solid media containing kinetin: (a) 0.1 mg l–1, (b) 1 mg l–1, (c) 10 mg l–1, d) 0 mg l–1; apical explants cultivated in media containing kinetin: (e) 0.1 mg l–1, (f) 1 mg l–1, (g) 10 mg l–1, (h) 0 mg l–1. bars = 1 cm. fig. 2. effects of decapitation and kinetin (kin) application in combination with indole-3-butyric acid (iba) on apical dominance suppression in knautia sarajevensis; nodal explants cultivated in solid media containing: (a) 0.1 mg l–1 kin and 0.1 mg l–1 iba, (b) 1 mg l–1 kin and 0.1 mg l–1 iba, (c) 10 mg l–1 kin and 0.1 mg l–1 iba, (d) 0 mg l–1 kin and 0 mg l–1 iba; apical explants cultivated in solid media containing: (e) 0.1 mg l–1 kin and 0.1 mg l–1 iba, (f) 1 mg l–1 kin and 0.1 mg l–1 iba, (g) 10 mg l–1 kin and 0.1 mg l–1 iba, (h) 0 mg l–1 kin and 0 mg l–1 iba. bars = 1 cm. micropropagation process affects phenolic profile acta bot. croat. 77 (1), 2018 75 higher concentrations of kin (1 mg l–1 and 10 mg l–1) had negative effects on shoot proliferation and induced vitrification of the plants. in this study the use of a small amount of the medium (50 ml) and a low kinetin concentration were enough for the avoidance of vitrification during multiplication (fig. 3c). moreover, developing shoots were not submerged in the media because explants floated due to the large leaves and the hairs on the leaves in which air can be trapped. phenolic compounds profile analysis of total phenolic content in k. sarajevensis showed a high concentration of phenols and flavonoids in the aerial part of mother plants (tab. 3). hplc analysis showed that around 30% of phenolic profile is composed of phenolic acids (gallic, 4-hydroxybenzoic, vanillic, salicylic, chlorogenic, caffeic, sinapic, ferulic and rosmarinic acids), (tab. 4) and some flavonoids: myricetin as a most abundant (2.72 nmol mg–1), and apigenin and kaempherol in traces. similar profile with lower concentration of individual components was found in in vitro cultivated plants as well as in acclimatized plants. elevation of rosmarinic acid concentab. 3. phenolic contents in knautia sarajevensis shoots after cultivation in solid and liquid media. k – kinetin, 01 – 0.1 mg l–1, 1 – 1.0 mg l–1, s – solid culture, l – liquid culture, pgr– no plant growth regulators added, mp – mother plant, ap – acclimatised plant. mean values not sharing the same letter(s) within one column are significantly different (p=0.01) according to newman-keuls test. treatment phenolics (mg g–1 dw–1) total phenols total flavonoids flavanols proanthocyanidins mp 121.04±8.97a 154.51±16.11a 6.19±0.03a 33.46±3.86a ap 33.16±8.00c 165.13±4.38a 0.94±0.00cb 21.50±0.77b pgr s 31.14±3.68cd 63.27±11.71b 0.83±0.12cd 2.09±0.04e pgr l 21.98±0.15e 27.86±1.70cd 0.53±0.11cd 14.63±0.07c 01k s 20.31±3.55e 25.57±2.48cd 0.31±0.01d 3.46±0.04e 01k l 24.488±3.62de 34.24±4.84cd 0.80±0.01cd 9.40±0.42d 1k s 26.02±2.52d 33.88±3.18cd 0.46±0.02cd 3.73±0.02e 1k l 56.50±2.67b 49.24±4.02bc 1.51±0.03b 4.16±0.46e tration was reported for acclimatised plants and some kin treatments (tab. 4); in addition, the synthesis of syringic acid under kin treatment was noticed. discussion natural populations of endemic knautia sarajevensis are subject to anthropogenic impacts and it is important to develop a micropropagation protocol in order to give opportunities for the growth of this species in new habitats. micropropagation of endangered species enables plant propagation regardless of the season from a small number of mother plants (mikulík 1999). for conservation purposes it is recommended to use seed for establishment of the cultures as they have a broader genetic base (chua and henshaw 1999). removal of elaiosomes alone was not sufficient for dormancy removal in k. sarajevensis seeds, and different cold pre-treatments combined with gibberellic acid application were used for improvement of the germination rate. gibberellic acid alone or in combination with cold treatment can break dormancy and promote germination as recorded for fig. 3. apical dominance suppression and multiplication of knautia sarajevensis shoots in liquid culture: a) bud clusters, b) shoot multiplication, and c) liquid media cultivation. bars = 1 cm. karalija e., ćavar zeljković s., tarkowski p., muratović e., parić a. 76 acta bot. croat. 77 (1), 2018 tab. 4. hplc analysis of phenolic compounds in knautia sarajevensis shoots cultivated in solid and liquid media. k – kinetin, 1 – 1.0 mg l–1, s – solid culture, l – liquid culture, pgr– no plant growth regulators added, mp – mother plant, ap – acclimatised plant, nd – not detected. hydroxybenzoic acid (nmol mg–1 of extract) treatment mp ap pgr s pgr l 1k s 1k l gallic acid 8.71 4.75 2.88 1.23 6.15 nd 4-hydroxybenzoic acid 46.05 11.07 14.40 7.16 16.41 2.72 vanillic acid 0.38 0.62 0.12 0.37 4.87 1.35 syringic acid nd 0.36 nd 0.19 3.09 0.94 salicylic acid 20.59 11.40 12.75 7.19 18.97 4.18 chlorogenic acid 1.67 0.18 1.30 0.43 0.32 2.49 caffeic acid 0.031 0.59 0.32 0.35 0.20 nd sinapic acid 53.65 1.98 0.61 0.63 0.95 0.10 ferulic acid 57.69 2.59 0.56 nd 0.94 0.27 rosmarinic acid 2.61 12.71 13.86 5.01 11.66 2.61 myricetin 2.72 0.47 nd 0.80 0.77 nd sum 194.11 45.89 46.81 23.39 64.27 14.66 many different genera (nau 1996, raeber and lee 1991). efficiency of gibberellin stimulation of seed germination in k. sarajevensis depended upon the pre-treatment and cultivation conditions used, but was usually pronounced when imbibed seeds were additionally exposed to cold treatment as previously recorded for other species (yamauchi et al. 2004, okamoto et al. 2006). endosperm plays an important role in seed germination (yan et al. 2014), synthesis of hydrolytic enzymes, sensing the red and far-red light necessary for germination (lee et al. 2012) and controlling embryo growth (lee et al. 2010). removal of part of the endosperm or isolation of embryos can be a useful tool for seed dormancy removal (yan et al. 2012) since seed dormancy can be caused through an impermeable seed coat or other dormancy factors from the endosperm (nonogaki, 2014). in this study such treatments did not stimulate seed germination in k. sarajevensis even when ga was applied, as compared to other treatments. an explanation for this could lie in the composition of k. sarajevensis endosperm. seeds of the knautia genus contain oils as a reserve of nutrients (mayer and svoma 1998, tonguç and erbaş 2012). for germination and energy gain, oils from the endosperm must be catabolised though gluconeogenesis in order to be passed to the embryo in the form of sucrose (penfield et al. 2004). endosperm removal excludes the reserve oils in which case embryos are under stress since they have no energy source. it is recorded that embryos in suboptimal growth conditions experience restriction of the gluconeogenic pathway (rylott et al. 2003), which could lead to reduction in the growth and germination rate. removal of sucrose in combination with a reduction of basal salts in the media was the most successful treatment for elaiosome-free seeds of k. sarajevensis. presence of sucrose in media can induce osmotic stress and inhibit seed germination. also, growth of the seedlings can be inhibited by sucrose hydrolytic products, which are formed during media sterilisation process (sawyer and hsiao 1992, pan and van staden 1999). elevation of germination rate by reduction of media mineral composition is recorded also for other species as well (mishra et al. 2013). apical dominance of ground rosette over flowering stems is present in natural habitats of k. sarajevensis, and this phenomenon has previously been recorded for succisa pratensis (jongejans et al. 2006), and it is probably the reason for the recorded apical dominance emergence in shoot cultures in this study. choice of an adequate cytokinin and a concentration that stimulates shoot induction and elongation varies depending upon plant species and must be optimised for each particular plant species (jana et al. 2013). successful multiplication was achieved by low kinetin concentrations and such a treatment has been recorded for a relatively small number of species (deo et al. 2014). elevation of kinetin concentration induced callus formation, which has been reported in scrophularia takesimensis cultures (ding and chen 2007), with the possibility for regeneration from petiole and leaf (wang et al. 2013); similar results were also reported for other scabiosa species (hosoki and nojima 2004). decapitation was relatively successful as a tool for apical dominance suppression in k. sarajevensis shoot cultures. apical shoot removal was previously recorded as an effective method for apical dominance suppression (podwyszynska 1997). also it is considered that the endogenous auxin to cytokinin ratio suppresses bud outgrowth and an exogenous supply of cytokinins can change this ratio (hillman 1984), which in turn stimulates bud outgrowth. senescence of older leaves was observed for all apical explants and control treatment (no plant growth regulators added), which probably impaired successful multiplication (mensual-sodi et al. 2007). kinetin application reduced senescence in nodal explants, and the role of kinetin in senescence has been previously demonstrated (mukharjee and kumar 2007). negative effects of exogenous auxins on multiplication have been previously reported. single shoot development and callus induction was demonstrated due to apical dominance resulting from increased auxin concentration (buah et al. 2010). in this study micropropagation process affects phenolic profile acta bot. croat. 77 (1), 2018 77 high endogenous auxin levels were responsible for root formation on media containing only kinetin while additional exogenous auxins had a negative effect on rhizogenesis. the negative effect of high auxin concentration on root formation has been well documented with reference to a number of species (buah et al. 2010, north et al. 2010). high survival rate during acclimatisation has been reported for some scabiosa species (hosoki and nojima 2004, wang et al. 2013), but data regarding knautia species are scattered. since suppression of apical dominance is possible through the use of liquid cultures (mehrotra et al. 2007), in the next step we used a liquid culture using the same kinetin concentrations that were favourable for multiplication in a solid culture with no signs of callus formation to ensure that only shoots by direct regeneration were obtained. development of bud clusters and mechanical separation of shoots in liquid systems provide an efficient delivery system in micropropagation (levin et al. 1997, ziv et al. 1998). production of buds enables the production of a large number of plants (takayama and misawa 1981). the contact of the explants with the medium facilitates the uptake of nutrients and plant growth regulators, which lead to promotion of shoot and root formation (sandal et al. 2001). forced aeration, due to continuous shaking of the medium, provides oxygen supply to the whole explant, which stimulates growth (mehrotra et al. 2007). there are many studies suggesting that agar elimination causes vitrification of tissue during micropropagation (john 1986, kevers et al. 1987). the problem of asphyxiation is a common problem in liquid cultures (mehrotra et al. 2007). in this study reduction of the media amount and low kinetin concentration were enough for avoidance of vitrification during multiplication. moreover, developing shoots were not submerged in the media because explants tended to float due to their large leaves and the hairs on the leaves among which air can be trapped. the use of surface tension and floating properties of an explant is very useful for the avoidance of submergence of tissues and vitrification phenomena in a liquid medium (debergh et al. 1992). since closely related members of the dipsacaceae family have been demonstrated to have medicinal properteis (oh et al. 1999, hung et al. 2005, mattalia et al. 2013), prospects for the micropropagation of k. sarajevensis as a potential medicinal plant also included phytochemical screening as the first step towards such a use of the plant. there are no available data about the phenolic compounds found in k. sarajevensis, and also very little is known about the genus knautia, in general; some of the identified components are reported for the first time. according to the available data apigenin, swertijaponin, and giganteoside a were found in k. montana (movsumov et al. 2011); cryptochlorogenic and chlorogenic acid and isovitexin 7-β-d-glucopyranoside were detected in k. arvensis (moldoch et al. 2011), while a high concentration of polyphenolics in k. bidens has been reported and the plant is considered a rich source of phenolic compounds (alali et al. 2007). kinetin is considered to be a promoter of secondary metabolite synthesis (klessig and malamy 1994, kim et al. 2009), as demonstrated in this study. there is less synthesis than in the mother plants, but some components that are not recorded for mother plants have been identified in kinetin-treated in vitro plants, which is in accordance with other studies (rao and ravishankar 2002, luczkiewicz and gold 2005). manipulation of pgr concentration and culture conditions can provide changes in secondary metabolites (rao and ravishankar 2002, luczkiewicz and gold 2005, lucchesini et al. 2009) as also shown in this study. use of shoots instead of callus is more convenient and it is also considered that more differentiated tissues produce more metabolites (luczkiewicz and gold 2005, nath and buragohain 2005, sood and chauhan 2010), and use of liquid systems gives better results (savio et al. 2011) as demonstrated in this study. to conclude, this study provides a successful micropropagation protocol for knautia sarajevensis and accordingly a useful tool in the establishment of new population sites as a method for conservation of this endemic species and for its potential use as an ornamental plant, replacing k. arvenisis in perennial beds. successful micropropagation can be performed by germination of seeds on media without sucrose and reduced basal salt concentration. apical dominance can be overcome by decapitation of shoots or cultivation in liquid media. due to the high endogenous concentration of auxin, rooting media is not necessary and roots develop in multiplication media with successful acclimatization of rooted plants. 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breaking and salt tolerance in hordeum spontaneum seeds. the russian journal of plant physiology 50, 423–427. ziv, m., ronen, g., raviv, m., 1998: proliferation of meristematic clusters in disposable presterlized plastic bioreactors for large scale micropropagation of plants. in vitro cellular and developmental biology – plant 34, 152–158. acta bot. croat. 77 (2), 2018 109 acta bot. croat. 77 (2), 109–118, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0014 issn 0365-0588 eissn 1847-8476 review bryophytes and heavy metals: a review jelena d. stanković, aneta d. sabovljević, marko s. sabovljević* institute of botany and botanical garden, faculty of biology, university of belgrade, takovska 43, 11000 belgrade, serbia abstract – bryophytes, a group of terrestrial plants widely used in biomonitoring, are reviewed for their relation to heavy metals. in the present article, we summarized the knowledge on heavy metals pollution and accumulation effects on bryophytes. mechanisms of tolerance and resistance are given as well. key words: liverworts, metals, mosses, relationship, toxicity * corresponding author, e-mail: marko@bio.bg.ac.rs introduction heavy metals naturally occur in earth's crust, from which they are released into the atmosphere and the water bodies (nagajyoti et al. 2010). some of them are essential for the normal metabolic functioning of organisms, but if deficient, or present in excess, they can lead to physiological stress and have detrimental consequences (nagajyoti et al. 2010, krzesłowska 2011). others, like pb, cd, al, and hg are harmful at all concentrations (krzesłowska 2011). although men have used heavy metals for thousands of years (järup 2003), reckless human behaviour, associated with urbanization and industrial development, drastically altered the previous distribution and geochemical cycles of heavy metal (singh et al. 2011). as a consequence, numerous potentially hazardous metals were introduced into the environment or their concentrations increased substantially in areas in which they were previously present only in small quantities (nagajyoti et al. 2010, varela et al. 2013). heavy metals are particularly significant as pollutants. once introduced into the environment, they are hard to remove and tend to accumulate in the tissues of plants and other organisms through the food chains (lee and von lehmden, 1973, maevskaya et al. 2001). the steadily increasing contamination requires continuous monitoring of heavy metal concentrations in the environment and their influence and effects on ecosystems (markert and weckert 1989). due to their widespread distribution and the ability to accumulate great amounts of heavy metals, bryophytes have been used as an important biological monitoring system for heavy metal pollution since 1968 (tremper et al. 2004). also, the phenomenon that some bryophytes tend to grow on substrates containing certain heavy metals led to their use as bioindicators that could imply the presence of a specific metal in that particular environment (shaw 1987). additionally, the relative simplicity of these plants (markert and weckert 1989, reski 1998) makes them an important model for the investigation of morphological and genomic alterations in plants due to heavy metal toxicity (carginale et al. 2004, choudhury and panda 2005). finally, the key phylogenetic position of bryophytes in plant evolution, connecting the terrestrial and aquatic mode of life (shaw and renzaglia 2004, shaw et al. 2011, strotbek et al. 2013), and the fact that they are the most conservative group of land plants (reski 1998), emphasize their importance for the studies of the evolution of plant resistance mechanisms to this type of environmental pollution. classification of heavy metals although there have been a lot of research works regarding heavy metals and their effects on the environment, there is still no broad consensus on which factors define an element as a heavy metal. one of the most accepted definitions is that heavy metals are metals with a specific density of more than 5 g cm–3 (järup 2003). the problem with this definition is that it includes the alkali metals, alkaline earth metals, lanthanides and the actinides that in the chemical sense are not considered “heavy”, while it excludes some other elements, such as arsenic, that is usually considered a heavy metal because of its chemical-ecological effects (martin and coughtrey 1982, agarwal 2009). one of the classifications that can combine these properties and also account for the similarities in the heavy metal toxicity mechanisms among different organisms is based on the equilibrium constants stanković j. d., sabovljević a. d., sabovljević m. s. 110 acta bot. croat. 77 (2), 2018 that describe the formation of the metal ion-ligand complexes. according to this, there are three categories of metals with different binding preferences. elements with an affinity for ligands containing oxygen comprise class a, elements with preferences for ligands containing nitrogen or sulphur are in class b, while elements that have an intermediate character with the similar preference for bonding to o-, s-, or n containing ligands are referred to as borderline (nieboer and richardson 1980). the elementusually considered as heavy metals in terms of their effects on the environment are all in the class b and the borderline group (martin and coughtrey 1982, choudhury and panda 2005). metals in the borderline group (as, cd, co, cr, cu, ni, sn and zn) are less toxic than those in group b, and some of them, like copper and zinc, are even essential micronutrients in plant physiology (choudhury and panda 2005). they can be cofactors and activators of enzymes, take part in redox reactions and electron transfer or have structural functions in nucleic acid metabolism (nagajyoti et al. 2010). conversely, metals in the b group (e.g. au, ag, hg, pb), are toxic to plants at all concentrations, they are not a part of any enzyme and their effect is more pronounced with an increase in class b character. biomonitoring of heavy metal pollution using bryophytes monitoring of heavy metal pollution in the environment is a very complex process, particularly when it comes to airborne pollutants. based on the known point sources it is possible to postulate some general theoretical principles of pollutant dispersion, but it is virtually impossible to make an exact estimation due to the complexity of the microclimate and the topography around each sampling site. dispersion simulations are particularly hard to devise in complex surveys of airborne pollution that include a large number of sampling sites (little and martin 1974). conversely, field receptor measurements that include sophisticated sampling techniques and instrumentation can indicate the presence of additional sources of heavy metals, give a precise and reliable estimation of their distribution and validate the dispersion models (wolterbeek 2002). however, these measurements are associated with high expenses for equipment and manpower, and usually are extremely time-consuming, because they involve long-term sampling at a large number of sampling sites (little and martin 1974, wolterbeek 2002). additionally, this approach does not reveal the amounts of metals that are accumulated by the vegetation in the monitored area, or their effects on these biological systems (wolterbeek, 2002). thus, the use of biological systems capable of absorbing heavy metals in such a way that their tissue loads reflect the concentrations in the environment (fernández et al. 2013) and their distance from the sources could give quantitative information about heavy metal pollution in the environment and account for its effects on the biosphere (onianwa 2001, chakrabortty and paratkar 2006). in this sense, bryophytes, especially mosses, are very important (markert and weckert 1989, boquete et al. 2014). bryophytes were the first green plants to colonize the terrestrial environment (nickrent et al. 2000), and as such had to evolve mechanisms to cope with the much greater amounts of heavy metals present on land than in the water (degola et al. 2014). these mechanisms resulted in the ability of many bryophytes to be consistent colonizers of metal contaminated environments (shaw et al. 1989), or to accumulate large amounts of heavy metals, in extremely polluted areas without any visible negative effect on their growth and development (sassmann et al. 2010). this is one of the prerequisites for their use as biomonitors (zechmeister et al. 2007). bryophytes are usually divided into three large phyla: the liverworts (marchantiophyta), mosses (bryophyta), and hornworts (anthocerophyta) (shaw et al. 2011). due to their morpho-physiological properties, mosses (berg and steinnes 1997, zechmeister et al. 2007, zvereva and kozlov 2011), and recently, liverworts too (carginale et al. 2004, tipping et al. 2008), have been widely used as excellent systems for the monitoring of heavy metal pollution in both terrestrial and aquatic environments. the absence of a root system indicates the ability of these plants to absorb heavy metals over the entire surface (berg and steinnes 1997, degola et al. 2014). the lack of the cuticle layer, which makes their cell walls easy accessible for metal ions (choudhury and panda 2005, koz and cevik 2014), pronounced ion-exchange properties (little and martin 1974) and a large surface-to-weight ratio also significantly contribute to this ability (sun et al. 2009). consequently, they can react to and reflect the changes in the heavy metal concentrations faster than most vascular plants (zvereva and kozlov 2011). on the other hand, due to the absence of specialized conducting tissues (onianwa 2001) and the slow growth rate (chakrabortty and paratkar 2006), moss growth segments can give the information about the integrated exposure to heavy metals over longer periods of time, and not just about the current state, which is particularly important in the areas where levels of introduced heavy metals change rapidly. advantages of bryophyte-performed monitoring, compared to conventional measurements, are cost-effectiveness and easier sampling that results in much higher sampling density and a larger number of sites that can be included in the survey (berg and steinnes 1997, schröder et al. 2010). due to the great capacity of bryophytes to absorb and retain heavy metals in high concentrations, it is also easier to perform chemical analysis and there are fewer contamination problems (berg et al. 1995, berg and steinnes 1997). they also provide information on the interactions between different heavy metals and their effects on living systems, which cannot be obtained using instrumental measurements (tremper et al. 2004). due to all these traits, bryophytes have been successfully used for decades, not only in monitoring studies of the airborne metal pollution, where they are of immense importance (zechmeister et al. 2003), but also in the monitoring of heavy metal pollution in aquatic environments (kelly et al. 1987). however, these surveys do not give the absolute concentrations of elements that accumulate in the environment during a particular period (berg et al. 1995, berg and steinnes 1997). to obtain that information, it is essential to establish and maintain the linear correlation between the concentrations in bryophyte tissue and the concentrations of metals to which it is exposed, accounting for bryophytes and heavy metals acta bot. croat. 77 (2), 2018 111 all the factors that could disturb this relationship (boquete et al. 2014). in this sense, the toxicity of heavy metals could also cause the alternation of the metal accumulation characteristics of the bryophytes, which could affect their reliability as biomonitoring systems (wolterbeek 2002) or result in an effective physiological markers of heavy metal pollution (sun et al. 2009). investigations on bryophytes’ relation with heavy metals in strictly controlled condition like in vitro culture are still in very short supply (e.g. vukojević et al. 2004, sabovljević et al. 2014). the accumulation of heavy metals by bryophytes bryophytes accumulate heavy metals by several mechanisms, but the initial and frequently limiting step is reversible adsorption on the cell surface (gonzález and pokrovsky 2014). adsorbed metals can be trapped as particulate matter within the surface layer, dissolved in liquids or deposits surrounding cells (intercellular fraction), bound in exchangeable form to exchange or chelating sites on the cell wall and outer surface of the plasma membrane (extracellular fraction) or transported inside the cells and held in soluble or insoluble form (intracellular fraction) (vazquez et al. 1999, salemaa et al. 2004, castello 2007, gonzález and pokrovsky 2014). the extracellular accumulation of heavy metals is mediated by the ion exchange process (wells and brown 1990) and the formation of complexes between the metals and the organic functional groups in the cell walls of bryophytes (shakya et al. 2008). the great binding capacities of mosses for some heavy metals are often attributed to the functional groups of polygalacturonic acid and related polymers in the cell walls (tipping et al. 2008). experiments exploring the acid-base properties of the mosses resulted in the detection of several possible functional groups involved in the binding of heavy metals. these include phosphodiester, carboxyl, phosphoryl and amine groups, as well as polyphenols. considering the organic composition of the cell walls of mosses, carboxyl and phosphoryl groups could be regarded the dominant metal-binding groups forming the complexes with heavy metals at the surface of moss cells. other groups, such as sulfhydryl and amine, could be determinants in the presence of small amounts of heavy metals or under extreme ph conditions (gonzález and pokrovsky 2014). greater amounts of uronic acids (containing carboxyl groups) in the cell walls compared to cellulose and hemicellulose (having hydroxyl groups) could explain the higher heavy metal binding affinity of the plants cultured in the laboratory than that of the field-grown mosses observed by wells and brown (1987). the dominance of carboxyl and phosphoryl groups in the cell walls of different mosses could also explain the similar patterns of heavy metal adsorption in different bryophyte species seen in some studies (vazquez et al. 1999, tremper et al. 2004, gonzález and pokrovsky 2014). however, it has been observed that adsorbing properties and uptake efficiencies for the same metals may vary significantly between the mosses and liverworts (shakya et al. 2008). this could be a result of the different cell wall composition of these two bryophyte groups, where uronic acid is a characteristic component of the cell wall of mosses and mannuronic acid in that of the liverworts. other studies, performed by rühling and tyler (1970) and vazquez et al. (1999), have shown that different heavy metals may follow the same order in maximum concentrations reached in the extracellular fractions regardless of the moss species, suggesting that this property depends mainly on the type of the metal. on the other hand, the affinity of extracellular binding sites for different metals may vary significantly among the species (vazquez et al. 1999). heavy metals adsorbed on the moss surface can reach the interior of the cell by specific membrane transport proteins or via channels present in the cell membrane (basile et al. 2012). while the extracellular fraction of heavy metals in mosses is usually easily exchangeable and tends to reflect the current environmental conditions and sporadic peaks in contamination, the intracellular fraction is usually a result of the integration of metals during the longer period of time and thus represents the average situation in the environment (fernández et al. 2006). it has been shown, as in other organisms, that the intracellular metal ion uptake by bryophytes displays saturation kinetics (wells and brown, 1990, basile et al. 2012). though it is hypothesised that uptake is a slow metabolically-controlled process (vazquez et al. 1999), the study of fernandez et al. (2006) revealed that when the bioavailability of heavy metals in the environment is high, intracellular uptake can be rather quick, leading to an accumulation of large amounts of the metals inside the cell in a short period of time. nevertheless, in this study, the high velocity of heavy metal accumulation inside the cells of the aquatic moss fontinalis antipyretica hedw. resulted in a quick onset of the release of the same elements into the exterior, suggesting the existence of saturating concentrations inside cells (fernández et al. 2006). interestingly, in the study of basile et al. (2012) on different mosses, intracellular concentrations of heavy metals that act as micronutrients, such as cu and zn, remained rather constant regardless of their extracellular concentrations, while the accumulation of the elements with no metabolic function, such as pb and cd, increased with increasing metal supply in the environment. a similar relationship between the extracellular and intracellular concentrations of cd was also observed in the moss pseudoscleropodium purum (hedw.) m. fleisch. by fernández et al. (2013). one of the potential reasons for the lack of control of non-essential metals input could be the absence of the specific transporters for these metals (pérez-llamazares et al. 2011). instead, they could be using channels and transporters of the plasma membrane that normally function in the uptake of essential ions (wells et al. 1995, choudhury and panda 2005), which leads to the increase of their intracellular concentration independently of the previously existent intracellular concentration (choudhury and panda 2005). sources and factors influencing the accumulation of heavy metals by bryophytes there are numerous sources and factors that can influence the contents of heavy metals in bryophytes (berg et al. stanković j. d., sabovljević a. d., sabovljević m. s. 112 acta bot. croat. 77 (2), 2018 1995, berg and steinnes 1997, schröder et al. 2010). metals from the atmosphere can reach the surface of terrestrial bryophytes in solution (precipitation) or in the form of dry deposition that can later be solubilised or washed away (couto et al. 2004, fernández et al. 2013). even though terrestrial bryophytes take most of the substances from the atmosphere, soil contributes significantly to the heavy metal contents (berg and steinnes 1997). this is particularly accentuaed during the rainy seasons or snowmelt when many substances from soil can be transported in the form of solutes, wetting the plant (salemaa et al. 2004, klos et al. 2012). the bioavailability and mobility of heavy metals in soil are strongly correlated to its acidity, the amount of organic matter, and the chemical composition (salemaa et al. 2004, klos et al. 2012). windblown particles from the ground containing heavy metals can also influence the amounts of heavy metals in bryophytes (berg et al. 1995, berg and steinnes 1997, salemaa et al. 2004, chakrabortty and paratkar 2006). the retention of these particles on moss surface depends on the particle size and the surface structure (chakrabortty and paratkar 2006). the study of klos et al. (2012) has shown that the contribution of each of the two mechanisms of metal transport from soil to bryophyte depends mainly on the local climatic conditions. besides these, other sources, such as natural trace element cycling processes and leaching of the heavy metals that were previously accumulated in vascular plants through their root system, may also contribute to the heavy metal content in bryophytes (berg et al. 1995). water also has a significant role in the heavy metal uptake by bryophytes. for the aquatic bryophytes, it is their living environment and the primary source of all the minerals, including the heavy metals (claveri et al. 1994). in the case of terrestrial bryophytes, water can bring or dissolve particles that are already deposited on the bryophyte surface facilitating the uptake of heavy metals by the plant, but it can also wash out the deposited pollutants and lower down the uptake of these elements (fernández et al. 2013). the quantity, intensity, and the duration of the precipitation determine the amount of accumulated and leached heavy metals from the terrestrial bryophytes (chakrabortty and paratkar 2006). the study of čeburnis and valiulis (1999) on two moss species (hylocomium splendens (hedw.) schimp. and pleurozium schreberi (brid.) mitt.) has shown that the heavier the rain, the less efficient is the uptake process for different heavy metals. they have also found that leaching can significantly influence the uptake of almost all investigated heavy metals. while the uptake efficiencies for metals such as pb and ni remain generally stable, leaching process may influence the uptake efficiencies for metals such as cd, cu and zn or even be a dominant factor in the case of mn and cr. however, maevskaya et al. (2001) and couto et al. (2004) have shown using different bryophyte species that elements that are already accumulated in intracellular or extracellular particulate fractions cannot be easily leached under normal conditions. the chemical composition of the medium in contact with the bryophyte surface dominantly influences which heavy metal and what amount of it is going to be absorbed and retained by the plant. different heavy metals differ in their affinities for the binding sites of the cell walls of bryophytes (rühling and tyler 1970), indicating that competition effects may significantly alter the uptake kinetics of a specific heavy metal (wolterbeek 2002). the phenomenon of cation exchange capacity (cec) is widely known to be extraordinarily high in peat-mosses (sphagnum) so that they can acidify their environment by exchanging tissue-bound protons for basic cations. however, sphagnum cec seems to be similar to that of other bryophyte species (soudzilovskaia et al. 2010). wells and brown (1987, 1990) and wells et al. (1995) showed that different cations, depending on their binding affinity and the amounts in the environment, could exclude or prevent the binding of heavy metals to cation exchange sites in cell walls or membranes of different bryophytes (couto et al. 2004). these findings are in agreement with the hypothesis that different sea-salt cations in the marine areas could also interfere with the uptake and retention of heavy metals (berg and steinnes 1997, wolterbeek 2002). however, the concentrations of metals in the environment are usually not high enough to cause the occupation of the majority of the extracellular exchange sites. conversely, the concentration of protons in strongly acidic environments is high enough, and may prevent the binding of different heavy metals by bryophytes or even lead to the leaching of different heavy metals from their cell wall (couto et al. 2004). wells and brown (1990) have shown that in the moss rhytidiadelphus squarrosus (hedw.) warnst. lowering of ph not only reduces the extracellular binding of cd but it also affects its intracellular uptake. while the first could be due to the protonation and occupation of the available extracellular anionic binding sites, the second could be a result of the protoninduced conformational changes of transporting proteins in the bryophyte membranes. thus, the type (soil, air, or water) and chemical composition of the media, and its acidity are probably the most important factors determining which metal and what amount of it is going to be accumulated by different bryophyte species. the effects of heavy metals on bryophytes though growth and development are commonly used parameters for the assessment of the heavy metal toxicity in plants, negative effects of heavy metal pollution could be detected before the alteration of these two parameters become obvious (wolterbeek 2002). these effects include ultrastructural changes as well as the changes in the plant physiological processes and characteristics (sun et al. 2009, canivet et al. 2015). ultrastructural changes seen in bryophytes under heavy metal stress may include alternations of the chloroplast shape and thylakoid organization (choudhury and panda, 2005) as well as the appearance of the stromal plastoglobules in them (basile et al. 2009). in the moss scorpiurum circinatum (brid.) fleisch. & loeske, basile et al. (2012) have shown that the appearance of these traits was metal-specific. for metals such as pb and cd, dose-dependence was also observed, while the other two metals tested (cu and zn) showed similar effects at all concentrations. the dose-debryophytes and heavy metals acta bot. croat. 77 (2), 2018 113 pendent effect of cd on the cell structure of bryophytes was also confirmed in the study by carginale et al. (2004), where it led to the changes in the appearance of the membranes of the different organelles (chloroplasts and mitochondria). besides these changes, the presence of cytoplasmic vesicles, multivesicular bodies, electron dense bodies and lipid droplets was also observed in the cytoplasm of heavy metalstressed bryophytes (basile et al. 2009, basile et al. 2012). in the liverwort lunularia cruciata (l.) dumort. treated with cd, degola et al. (2014) also found numerous small vacuoles containing electron-dense precipitates, which were absent in the control plants. the additional analysis showed that cd and sulphur co-localized in these vacuoles indicating an important role of sulphates in the sequestration of intracellular heavy metals. along with the ultrastructural changes, heavy metals may also disrupt various metabolic processes and lead to physiological stress in bryophyte cells (shakya et al. 2008, sun et al. 2010). these negative effects could be explained by the high affinity of heavy metals for sulfhydryl groups in various proteins, which can lead to inhibition of the enzyme activity (boquete et al. 2014) or to conformational modifications of the proteins. the alternations in different cell processes could also be a result of the displacement and thus deficiency of an essential element by a specific heavy metal (zengin and munzuroglu 2005). the chlorophyll content is an often-used parameter for assessment of the physiological status and biological activity (photosynthetic capacity) of plants (tremper et al. 2004, zengin and munzuroglu 2005, rau et al. 2007). however, there have not been many studies investigating the relationship between the presence of different heavy metals in bryophytes and the chlorophyll concentration. additionally, comparable results are limited to laboratory experiments (varela et al. 2013). nevertheless, most of these studies have revealed the reduction in total chlorophyll content as a specific response to heavy metal stress, though the degree of it may vary between the species and the metals tested (choudhury and panda 2005, shakya et al. 2008, sun et al. 2009). in the study of tremper et al. (2004) on rhytidiadelphus squarrosus, of the three metals investigated (cu, zn, and pb) only copper caused a significant decline in the total chlorophyll. the dominant effect of copper and the smallest influence of zn on this physiological parameter was also confirmed by shakya et al. (2008) using the mosses, thuidium delicatulum (hedw.) schimp. and t. sparsifolium (mitt.) a. jaeger, and the leafy liverwort, ptychanthus striatus (lehm. et lindenb.) nees. additionally, in all three species under heavy metal stress, the amount of chlorophyll b was greater than that of the chlorophyll a, indicating that in these bryophytes heavy metals could induce the conversion of chlorophyll-a to chlorophyll-b (shakya et al. 2008). another study, involving he moss hypnum plumaeforme wilson, showed that pb and ni, single or combined, at higher concentrations, can also lead to a strong decline in total chlorophyll concentration (sun et al. 2009). the highly negative effect of pb on total chlorophyll content was also demonstrated in the moss taxithelium nepalense (schwaegr.) broth. (choudhury and panda 2005). additionally, rother et al. (2006) demonstrated that cd too can have a negative effect on chlorophyll content and lead to subsequent loss of vitality in the tested physcomitrella patens (hedw.) bruch & schimp. the decline in total chlorophyll content in all the investigated bryophytes under heavy metal stress could be a result of the heavy metal interference with chlorophyll synthesis either through the direct inhibition of an enzymatic step or by inducing the deficiency of an essential nutrient (zengin and munzuroglu 2005). the differences in the reduction of chlorophyll content under various heavy metals could be explained by the different uptake and action mechanisms for these metals (bruns et al. 2001; shakya et al. 2008). since nitrogen is an essential component of amino acids, it has been hypothesised that heavy metals may disturb the biochemical and physiological processes in plant cells through the alteration of the nitrogen metabolism (sutter et al. 2002). the exposure of the moss fontinalis antipyretica to increasing concentrations of cd, pb and zn led to a concentration-dependent decrease of nitrogen incorporation into amino acids, and also to an additional concentration-related inhibition of protein biosynthesis. from these observations it can be inferred that the effects were independently influenced by heavy metals at different phases of nitrogen assimilation. the initial reduction of nitrogen incorporation into amino acids may be a consequence of the lowered nitrogen uptake due to the plasma membrane damage, while the discrepancies between the amino acid amounts and the protein abundance may be a result of a concentration-dependent inhibition of protein biosynthesis (sutter et al. 2002). the influence of heavy metals on nitrogen metabolism in bryophytes was also confirmed by panda and choudhury (2005), who found that under cr, zn or cu stress, nitrate reductase of polytrichum commune hedw. was inhibited. apart from the direct alteration of biological structures and processes in plant cells, heavy metals can also induce reactive oxygen species like hydrogen peroxide (h2o2), superoxide radicals (o2-), and hydroxyl radicals (oh–) that could react with lipids, proteins, pigments and nucleic acid, resulting in lipid peroxidation, membrane damage or enzyme inactivation (choudhury and panda 2005, panda and choudhury 2005). in the moss hypnum plumaeforme exposed to increasing concentrations of pb and ni, single or combined, a dose-dependent increase of two ros species, h2o2 and o2–, was observed. the increase of the free radicals was more pronounced when the two metals were applied together, indicating the synergistic effect on ros production and accumulation (sun et al. 2009, sun et al. 2010). the study of choudhury and panda (2005) of the moss taxithelium nepalense revealed a similar trend of ros accumulation under pb and cr. additionally, the increase of h2o2 and o2– in the moss cells was demonstrated to be dependent on the duration of the metal treatment. further, the effect of these heavy metals on lipid peroxidation and membrane distortion through the generation of ros was investigated by analysing malondialdehyde (mda), a cytotoxic product of lipid peroxidation (choudhury and panda 2005, sun et stanković j. d., sabovljević a. d., sabovljević m. s. 114 acta bot. croat. 77 (2), 2018 al. 2009). the increase in mda content in bryophytes was observed in relation to all metals tested in these two studies, as well as in the study of panda and choudhury (2005), who investigated the effects of cu, zn, and cr on the moss polytrichum commune. this confirms that heavy metals lead to oxidative stress in bryophytes, which can result in lipid peroxidation, mda accumulation and consequently the loss of membrane integrity and cell damage (sun et al. 2009). additionally, the observation that h2o2 and mda accumulated under all metal treatments (choudhury and panda 2005, panda and choudhury 2005, sun et al. 2009) proportionally with the increase in duration and concentration of the treatment, implies that these parameters could be used as markers of oxidative stress induced by heavy metals, even when there are no changes in the appearance of the bryophytes (sun et al. 2009). mechanisms of bryophyte resistance to heavy metal pollution the toxic effects of heavy metals in bryophyte cells are predominately caused by the intracellular fraction while the metals outside the cells do not have immediate effects on cellular metabolism (fernández et al. 2006, shakya et al. 2008, basile et al. 2012). thus, the strategies used by bryophytes in response to heavy metal stress may include both the avoidance and the tolerance of this type of abiotic stress. avoidance includes all the processes preventing the entrance of the heavy metals into the protoplast (krzesłowska et al. 2013), and in that sense, the cell wall plays a crucial role (basile et al. 2009). modification of any of the characteristics influencing its retention and cation exchange capacities, or the activity of metal transporters in plasma membrane could lead to exclusion of heavy metals (boquete et al. 2014). for example, differences in the cell wall chemical composition between the mosses and liverworts or in different species in a group, could explain the differences in the uptake of different metals and thus the differences in their sensitivity to these pollutants observed in shakya et al. (2008). the significance of the cell wall in avoiding heavy metal stress in bryophytes has also been demonstrated by wells et al. (1995). they have shown that the degree of tolerance to cadmium may be influenced by the cell wall binding of different non-toxic cations naturally occurring in the cells or the environment, which then can create unfavourable conditions for the binding of heavy metals around the plasma membrane and prevent their entrance into the cytoplasm. in contrast to avoidance mechanisms, tolerance to heavy metal stress involves neutralisation of the metals or their toxic effects as well as translocation of these metals from the cytoplasm to compartments such as the vacuole and cell wall. chelating of heavy metals is one of the strategies involved in maintaining heavy metal homoeostasis and metal detoxification inside the plant cells (krzesłowska et al. 2013). in the process of intracellular heavy metal chelation in plants, low molecular weight thiols such as glutathione (gsh) and cysteine play the crucial role. gsh is a major transport and storage form of reduced sulphur and it may be directly involved in the binding of the heavy metals or indirectly as a substrate for the synthesis of the phytochelatins (pcs) that have a particularly high affinity for some heavy metals. the formed complexes between the heavy metal ions and pcs can then be transported into the vacuole, decreasing the concentration of metals in the cytoplasm and protecting the plants from their deleterious effects (yadav 2010). these mechanisms also operate in bryophytes as has been demonstrated by carginale et al. (2004) and degola et al. (2014) in studies on the liverwort lunularia cruciata exposed to cadmium stress. the results have shown that cd is accumulated in the vacuoles of the cd-stressed liverwort and that this is accompanied by an increase of sulphur concentration in this organelle. more importantly, it has also been found that most of the intracellular cd is bound to the thiol-rich compounds of similar weight such as phytochelatins, indicating that, as in other plants, these compounds may constitute the principal mechanism for heavy metal sequestration. further, degola et al. (2014) have unambiguously confirmed that the compounds found in the l. cruciata are phytochelatins and that some of them (such as pc2) may constantly be present in bryophyte cells managing the homeostasis of the micronutrients. however, testing the effects of different heavy metals on the induction of phytochelatin synthesis, and comparing the results of this study with those from a study of arabidopsis thaliana (l.) heynh., led to the conclusion that bryophyte pcs and their synthases have a narrower function, involved only in the regulation of the fe/zn homeostasis and detoxification of cd. conversely, phytochelatin synthases and phytochelatins of a. thaliana are more responsive and involved in the effective detoxification of many different heavy metals, indicating that other mechanisms may have greater significance for the detoxification of these elements in bryophytes. thus, mechanisms other than complexation of heavy metals with phytochelatins have a more important part to play in bryophyte detoxification of heavy metals. this has been additionally confirmed by bruns et al. (2001). during their study performed on the moss f. antipyretica and 19 other bryophyte species, no phytochelatins could be detected in any of the tested species regardless of the metal and concentration applied. during that time, however, an increase of gsh content, primarily under cd treatment, could be observed. the findings of this study and the studies performed by carginale et al. (2004) and degola et al. (2014) showed that intracellular cd is primarily stored in the vacuoles of bryophyte cells. here, high amounts of s and p were also observed, which led to the conclusion that one of the dominant mechanisms of bryophyte tolerance to heavy metals, at least to cd, is the formation of cytoplasmatic gsh/cd complexes and their subsequent transport into vacuoles, where they can be degraded and the cd accumulated as phosphate. apart from the need of bryophytes to neutralise or remove heavy metals from the cells to avoid harmful effects on cellular structures and processes, they also have to possess an antioxidative system to deal with the overproduction of reactive oxygen species caused by heavy metals. this system bryophytes and heavy metals acta bot. croat. 77 (2), 2018 115 comprises numerous enzymes and compounds of low molecular weight (zengin and munzuroglu 2005). sod is one of the most important enzymes in the protection of plant cells against oxidative stress since it transforms superoxide radicals into less destructive h2o2 that can further be removed by peroxidase (pod), catalase (cat), or ascorbate peroxidase (apx). additionally, low molecular weight compounds such as ascorbic acid (asa), glutathione, non-protein thiol, cysteine, proline and others could directly interact and detoxify these reactive species (sun et al. 2009). sun et al. (2009, 2010) treated the moss h. plumaeforme with different concentrations of pb and ni, singly or combined, to investigate the activity of the ros scavenging system under heavy metal stress in bryophytes. they discovered that the predominant enzyme involved in the bryophyte protection against the oxidative stress induced by heavy metals was pod, with its activity being dose-dependent on the concentrations of the applied metals. additionally, a synergistic effect of these metals with pod activity was observed. the activity of other enzymes (apx and sod) was only slightly increased, while the catalase activity actually decreased. on the other hand, the accumulation of both of the investigated components of the non-enzymatic antioxidative system (asa and proline) has been detected, with pb and ni displaying a synergistic effect on their accumulation. this indicated that both asa and proline could be important superoxide anion scavengers in bryophyte cells, with a significant role in the reduction of the damage to cell membranes under heavy metal stress. the accumulation of these two low molecular weight substances in response to the stress induced by metals other than ni and pb has been also observed in other plants, indicating that they could represent significant heavy metal tolerance constituents in other bryophytes (zengin and munzuroglu 2005). it has been hypothesised that the successful survival of different species in polluted environments is a consequence of their high reproductive potential. thus, the production of large amounts of spores or gemmae could explain the widespread distribution of some bryophytes in the areas with high amounts of heavy metals (leblanc and rao 1974). since the inhibition of sexual reproduction in many bryophytes in a heavy metal-polluted environment has often been detected (leblanc and rao 1974, shaw 1987), vegetative reproduction as an alternative strategy could explain their success in these disturbed sites (carginale et al. 2004). they showed that the gemma cups from the cadmium-treated gametophytes of l. cruciata produced normal gemmae that germinated at the same rate as those from the controls when transplanted into fresh medium. vegetative reproduction is one of the important bryophyte strategies under the conditions of heavy metal stress. this is emphasized by the fact that in these plants, high amounts of cadmium were found in the gemma cups, while only minute quantities reached the gemmae themselves. in general, the importance of the reproductive potential for bryophyte survival in heavy metalpolluted environments has also been confirmed by basile et al. (2001). they demonstrated that gametophyte and sporophyte tissues of bryophytes accumulate heavy metals differently, with gametophytes containing much higher concentrations of metals. further analysis showed that the placenta between them is responsible for this unequal distribution. it disturbs the apoplastic continuity between the two generations and sequesters toxic metals or toxic concentrations of micronutrient metals, preventing their accumulation in the sporogenous tissue and spores. this way, cells are protected during meiosis from the harmful effects of heavy metals in polluted environments. conclusions heavy metals are extremely toxic and may cause many alterations in the physiology and morphology of bryophytes, modifying the way they integrate, retain and release these pollutants. such metals also induce different exclusion mechanisms in bryophytes that reduce heavy metal toxicity by preventing the entry of these elements into the tissues (wolterbeek 2002, boquete et al. 2014). thus, the accuracy and reliability of information obtained using these plants as biomonitors depend on the understanding of the mechanisms, factors and bryophyte species responses (fig.1) that fig. 1. main factors influencing bryophyte heavy metal content. stanković j. d., sabovljević a. d., sabovljević m. s. 116 acta bot. croat. 77 (2), 2018 can influence the uptake and the linearity of the relationship between dose and tissue content (berg et al. 1995, berg and steinnes 1997). though there are studies about the effects of heavy metals on bryophyte physiological parameters, there are only a few examining the consequences of these changes to dose–response relations (wolterbeek 2002). additionally, detection of the heavy metal dose-dependent effects and physiological responses in bryophytes at a cellular level could result in the establishment of markers, which could be used for bioindication or biomonitoring of heavy metal pollution in the environment (rau et al. 2007, sun et 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environment 301, 55–65. zengin, f. k., munzuroglu, o., 2005: effects of some heavy metals on content of chlorophyll, proline and some antioxidant chemicals in bean (phaseolus vulgaris l.) seedlings. acta biologica cracoviensia series botanica 47, 157–164. zvereva, e. l., kozlov, m. v., 2011: impacts of industrial polluters on bryophytes: a meta-analysis of observational studies. water, air & soil pollution 218, 573–586. acta botanica 2-2015.indd acta bot. croat. 74 (2), 2015 195 acta bot. croat. 74 (2), 195–210, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 doi: 10.1515/botcro-2015-0018 review paper how to defi ne a diatom genus? notes on the creation and recognition of taxa, and a call for revisionary studies of diatoms john patrick kociolek1*, david m. williams2 1 museum of natural history and department of ecology and evolutionary biology, university of colorado, boulder, co 80309 usa 2 department of life sciences, the natural history museum, cromwell road, london, sw7 5bd, u. k. abstract – we highlight the increase in the number of diatom genera being described, and suggest that their description be based on the concept of monophyly. that is, a new genus will contain the ancestor and all its descendants. past criteria or guidance on how to circumscribe genera are reviewed and discussed, with conceptual and actual exemplars presented. while there is an increase in the rate of genus descriptions in diatoms, and there are many journal and series dedicated to facilitating this important activity, we call for revisionary works on diatom groups, to assess and establish monophyletic groups at all levels of hierarchy in the diatom system. keywords: bacillariophyta, cladistics, genera, monophyly, phylogeny, revisionary studies, systematics introduction during the last 15 years of studies in diatom taxonomy, over 80 new genera have been described (fourtanier and kociolek 2011). exploring the period from 1930–1969, it took some 40 years to describe a comparable number of genera in the 20th century (fourtanier and kociolek 2011). in a previous review, we noted that over a much longer period, 1805– 1975 (documented in 5 year periods), there was an average of between 3 and 4 new generic descriptions per year (williams and kociolek 2011: table 14). the number of diatom genera being described is increasing. in 2013 alone the journal »phytotaxa«, dedicated to publishing descriptions of new botanical taxa, accounted for seven new diatom genera. as editors of the diatom section of »phytotaxa«, as well as being working diatom taxonomists * corresponding author, e-mail: patrick.kociolek@colorado.edu kociolek j. p., williams d. m. 196 acta bot. croat. 74 (2), 2015 ourselves, we have been frequently asked by authors whether the specimens encountered in any study constitute a new genus, and if so, on what characters could (should) they base the description? ultimately, the reviewer community has helped with those decisions – but when pressed for an answer by those who ask whether they should propose a new genus of diatoms, our reactions have not necessarily been satisfying to them. the question has been addressed a number of times in the diatom community (e.g. round 1997a, b, kociolek 1997, 1998, williams 2009, williams and kociolek 2011) but we return to it here in an attempt at clarifi cation. in short, genera should be monophyletic; indeed, ideally all taxonomic categories should be monophyletic. the study and discovery of monophyly is often associated with a theory of systematics called cladistics. when cladistics was fi rst mooted as a way of understanding the living world, its primary focus was on phylogenetic relationships and how to discover them. ushered in as a reasoned critique of the old palaeontological approach to phylogeny (»… if only i had enough fossil evidence then all would become clear …«), its baggage was a rather copious supply of new words and terms, most, but not all, coming directly from willi hennig (1913–1976), often considered to be the founder of cladistics (schmitt 2013). with time, cladistics was understood to apply more generally to problems of taxonomy rather than simply being a refi ned version of phylogenetics. in a previous series of papers we attempted to show how many of the early diatomists followed what would amount to a cladistic understanding of their data and the taxa so discovered: agardh (1824), pfitzer (1871), petit (1877), and mereschkowsky (1902, 1903a) would be among those to whom this approach might be applied. the approach is quite simple: how do data relate to conclusions? put another, more specifi c way, how do characters relate to taxa? what do we mean by character? cladistics offered a solution to the problem of ‘characters’ by subdividing them into different ‘kinds’: synapomorphies, symplesiomorphies and convergences. the latter term might be viewed as straightforward, an old term, perhaps differently used in a modern sense, but nonetheless an old term (haas and simpson 1946). but the fi rst two words, synapomorphies and symplesiomorphies, both from hennig, are of some signifi cance – and while some systematists still consider them new (and their occurrence in the pages of journal »diatom research« still relatively rare), they are over 60 years old (hennig 1953: 14). rather than the terms synapomorphy and symplesiomorphy, the most often encountered words are ‘similar’ or ‘similarity’, which are applied to both characters and taxa. one might, then, re-phrase the above concerning characters: cladistics offered a solution to the problem of ‘similarity’ by subdividing it into different ‘kinds’. interestingly enough, one might also fi nd scattered in the same diatom literature reference to taxa (or characters) being ‘related’ or ‘closely related’ and sometimes one gets the impression that ‘similar’ and ‘related’ or ‘closely related’ (even ‘closely similar’) are all meant to mean much the same thing. yet they are not: synapomorphies, symplesiomorphies and convergences might all be construed as kinds of similarity – but only synapomorphies depict, or characterize, monophyletic groups. the importance of monophyly it is almost universally accepted today that the only groups of species that should be recognized (named) are monophyletic groups (e.g. donoghue 1985, mishler and brandon 1987, mishler and theriot 1997). why monophyly? it might be said that, given the reladiatom genus: a revision acta bot. croat. 74 (2), 2015 197 tionship between monophyly and synapomorphy, the only groups that can be recognized are monophyletic groups; these groups can be discovered – they have characters of their own (kociolek et al. 1989, williams and kociolek 2011, but see below). monophyletic groups represent a specifi c part of evolutionary history: they are collections of species that are more closely related amongst themselves rather than to anything else. for example, all species recognized as belonging in a genus are (presumed) most closely related amongst themselves and (presumed to) share a common ancestor. the same holds for any other generic group. that proposition reveals another useful parameter of monophyletic groups: they are predictive. that is, one expects to fi nd more characters (synapomorphies) congruent with those already known; they will specify the same group or, at the very least, will not contradict it. monophyletic groups, then, are the basis for any natural classifi cation. they are the groups that should receive formal identity and a name. of course, some of these groups, with further study, may turn out not to be monophyetic. but this refl ects the process of systematics, its scientifi c aspect, if you will: sampling characters and testing them against known (named) groups. other types of classifi cation systems a classifi cation of monophyletic groups is not the only way to represent organisms. there are many ways to classify. one might choose a functional approach and group, say, all organisms that are autotrophs, contrasting them with heterotrophs, or all organisms that fl y, contrasted with all those that do not. this is not uninteresting but, by defi nition, refl ects only one property and constitutes a very special kind of classifi cation, one that is based on a specifi cally defi ned character or characters rather than characters that are discovered as properties of the taxa. alternatively, one might choose to classify organisms on the basis of some defi ned organizational criteria. diatoms, like other organisms, have many groups like this, often referred to as grades, evolutionary grades (huxley 1959). examples of grades are ‘centric’ diatoms, defi ned by their symmetry (hustedt 1927); ‘araphid’ diatoms, also defi ned by their symmetry along with their lack of a raphe (round et al. 1990); ‘monoraphid’ diatoms, defi ned by their possession of one valve per frustule having a raphe, the other lacking it (patrick and reimer 1966). these groups refl ect a kind of organization rather than any specifi ed relationships. these two kinds of classifi cations, functional groups and grades, may collectively be referred to as examples of artifi cial classifi cations. this does not imply they are necessarily wrong – just that they refl ect something imposed rather than discovered. what does it take to describe a genus? some traditional responses the diatom literature speaks to the idea that in some way creating a genus of diatoms is a task that should be done with care, and that a substantive body of evidence must be demonstrated to support its creation (e.g. van heurck 1896). it may be, however, that diatomists have been – and still are – too cautious in their readiness to erect genera. for example, »the catalog of fishes« (eschmeyer 1988a, b, online version 2014, see also eschmeyer et al. 2010) treats just over 64,000 names ascribed to extant fi sh species, which is about the kociolek j. p., williams d. m. 198 acta bot. croat. 74 (2), 2015 same number of names treated by the »catalogue of diatom species« (fourtanier and kociolek 2011). but ichthyologists have sorted those ca. 64,000 taxon names into 12,000 genera, while diatomists have recognized only ca. 1,200 diatom genus names (fourtanier and kociolek 2011). this situation is likely to become worse if we assume that there still remains a signifi cantly higher proportion of diatom species diversity yet to describe versus that already known in fi shes. other examples that might suggest our descriptions of diatom genera may be too conservative. for example, very few freshwater diatom genera have narrow (specifi c) biogeographic circumscriptions. most that come to mind have only recently been described: gomphocymbella o. f. müller (1905, c. 15 species, mostly in africa, excluding fossils); tibetiana li et al. (2010, 1 species, so far, from china); eunophora vyverman et al. (1998, 5 species from new zealand and tasmania); perinotia metzeltin and lange-bertalot (2007, 1 species from south america) and tetralunata hamsher et al. (accepted, 19 taxa from indonesia). also, the hawaiian islands, which occupies ca. 6,400 square miles, comparable in area to los angeles county in california, has nearly 80% of the diatom genera found in the entire north american flora (kingston 2003, kociolek and spaulding 2003a, b, lowe 2003, stoermer and julius 2003) even though the number of species present are less than 1% of the fl ora of north america (kociolek 1997, personal observations). below we address some of the commonly understood criteria: 1. a genus requires many features: suppose we have a genus a diagnosed by two characters (two synapomorphies). that genus consists of three species (b, c, d), each diagnosed by a single character (fig. 1a). study of more specimens yields a further species (e) with the same two characters (two synapomorphies), suggesting it too belong to genus a. it is recognized as an additional species as it has four unique characters of its own. it would be tempting to promote species e to a genus as it has many (four) characters of its own, relative to the single character for each of the other included species, making e readily distinguishable. the consequences of that action, however, are profound. if e is recognized as a genus (fig. 1b) then it will render genus a undiagnosable as it cannot now be diagnosed by the two generic synapomorphies as species e (now genus e), also has them also; it also renders genus a a collection of species where only some of the most closely related species are included (e is now excluded). thus, under these circumstances, species e is best included in genus a, albeit an unusual – in the sense of having four distinctive characters – member of it. fig. 1. a) conceptual model of relationship between taxa as part of a genus (genus a). b) results of relationships with the addition of a highly derived taxon (e) recognized as a separate genus, making genus a, with taxa b, c and d, non-monophyletic. c) conceptual model of relationships, with taxa a, b, and c as new genera, required by identifying taxon e as a separate (new) genus. diatom genus: a revision acta bot. croat. 74 (2), 2015 199 further, imagine that as our investigations proceed we discover yet another species, e, with the four characters that diagnose species e thus making them most closely related relative to all the other species in genus a (fig. 1c). this presents a problem of representation. species e and e1 now share a number of characters amongst themselves only. it could be named a genus. as a consequence, b, c and d have unclear relationships and could now be considered three monotypic genera. the meaning being that these three have no specifi ed relationships outside the immediate group of a—e1. while this example may be unusual, the point here is that classifi cation at the genus level may have consequences elsewhere. many of these consequences can be seen in the sub-divisions made of larger diatom genera, such as eunotia. 2. homogeneity of features: round (1996) called for very narrow circumscriptions of diatom genera, suggesting that with this approach there would be homogeneity within a genus, which would yield a more natural classifi cation. initially this would seem to make sense. except, as kociolek (1998) demonstrated, there are occasions where two things might look very similar but may actually be more closely related to other taxa that, due to many derived features, look unlike their closest allies. even in this case, we would still argue for the recognition of monophyletic groups rather than groups based on »overall similarity«, a concept that was discredited some time ago because it lacks the properties of monophyletic groups outlined above. 3. good characters: cleve (1895) summarized his thoughts on a wide variety of features, indicating his experience in the use of different features at different levels of taxonomic hierarchy, and which ones are ‘useful’ for classifi cation. hustedt (1928) suggested that the raphe is a good guide to genera (and that other attributes could be used to diagnose species). cox (1890) also reviewed features he thought were helpful at the level of genus. throughout the years a wide range of features have been thought of as ‘good« characters, including symmetry, sigmoid shape, and possession of specialised structures (costae, for example kützing 1833, schütt 1896, karsten 1928, hustedt 1928, among many others). of course, over the years, and based on the experience of individual researchers, there have been differing ideas about the effi cacy of certain features and their ability to explain or support placement of taxa within groups. and, as cox (2010) and others have indicated, ideas about whether symmetry or special features have changed over time, and what was thought to be ‘good characters’ in the past have been shown to not refl ect evolutionary descent. one example of the use of apparent ‘good’ or, what is sometimes referred to as, ‘conservative’ characters for the recognition of diatom genera is amphoroid symmetry. the combination of different valve mantle heights between the dorsal and ventral margins, combined with the number and structure of the girdle elements, provide an easily recognizable symmetry. possession of those features was used to unite a diversity of forms, with differing valve morphologies, ecologies, biogeographies and cytoplasmic features (cleve 1895, mereschkowsky 1903b). cleve stated »the asymmetrical form is not a suffi cient characteristic for a natural family, but is merely a facies, which may occur in groups of very different types and seem to depend on the method of growth, amphorae occurring attached to algae and other objects« (cleve 1895: 99). amphora was retained because »if the above named large groups of amphorae were admitted as distinct genera, which i believe they ought to be, the synonymy would be still more intricate than it is at present. i propose for this reason, that the species of the different groups should retain their generic name amphora, which in kociolek j. p., williams d. m. 200 acta bot. croat. 74 (2), 2015 all cases signifi es that they are asymmetrical naviculae. this will also afford an opportunity of testing my views, which are entirely new, before admitting the proposed new genera« (cleve 1895: 100) but regardless of his point of view, the group of species remained within amphora for over a century (see hustedt 1930, schoeman and archibald 1986). levkov (2009) separated out one group from amphora, recognizing the subgenus halamphora (cleve) levkov at the level of genus, due to its possession of a dorsal raphe ledge, fi nely biseriate striae on the dorsal portion of the valve and uniseriate striae on the ventral section of the valve. a phylogenetic analysis of 30 species of the diatom genus amphora sensu lato using 3 genes (ssu rdna, rbcl and psbc) within the context of the raphid diatoms showed the origin of ‘amphoroid’ symmetry in six different lineages (stepanek and kociolek 2014). amphoroid species were retained in biremis (round et al. 1990) and lyrella (mann and stickle 1997), and analysis of the morphology of amphora scabriuscula cleve & grove in cleve and a new species, navicula petrovii nevrova et al. in witkowski et al. (2014) suggests they, too, have amphoroid symmetry as a homoplasy (independently derived), and that this species is better placed within navicula bory (witkowski et al. 2014). this is just one example, of many, related to a feature that was deemed a »good« or »conservative« that has been shown to be homoplasious across several lineages of diatoms. cox (2010) also has examples where this concept can be applied, and suggests that we do not know a priori which features should be used to diagnose lineages, but rather those will be identifi ed through formal analyses. can one feature diagnose a genus? yes, why not? after all, each character provides evidence for or against a particular grouping of species. at the moment, the only synapomorphy to diagnose ulnaria is their closed bands, at the very least a closed valvocopula; no other synapomorphy (or character) has yet been suggested to contradict that grouping of species. morales et al. (2014), following van de vijver and cocquyt (2009), suggested »the absence of spines and lack of ribbon-like colonies holding cells together by spines« also characterise species of ulnaria. such characters do indeed form part of its description but as they are both absences they hardly constitute additional evidence for the genus ulnaria – but neither do they contradict it. does every taxon in the genus (or group) have to possess all the features used to diagnose that genus (group)? initially this may seem an uninteresting question. by virtue of identifying features and using them to diagnose groups, it would follow that all the taxa to be included in that group should have the diagnostic feature(s). but some recently published papers in diatoms argue against this conclusion. for example, the monotypic diatom genus diprora main was described as endemic from caves in hawaii (main 2003). this diatom has valves with typical pennate symmetry, but bears no hint of a raphe system at any stage during the ontogeny of the valve (kociolek et al. 2013). because of its symmetry and it lack of a raphe, main suggested that it must be an ‘araphid’ diatom. it is worth remembering here that ‘araphid’ diatoms are not a natural group, so to place it in such a group is the same as declaring it to be unknown. a phylogenetic analysis of pennate diatoms using three genes (ssu rdna, rbcl, psbc) showed diprora haenaensis main to be nested deep within the raphid pennate diatoms, being closely related to sellaphora, fallacia and pinnularia (fig. 2, see also kociolek et al. 2013). thus, while never exhibiting the feature of a raphe system, we would still assign this genus to the raphid diatoms. its phylogenetic position within this group is supported by non-morphological (molecular) data, and its lack of a raphe is due to secondary loss. diatom genus: a revision acta bot. croat. 74 (2), 2015 201 a similar situation of secondary loss was described for some of the most derived species within the genus gomphoneis cleve, where these smaller species have secondarily lost internal laminae of silica (marginal lamina, axial plate), features used to help diagnose the fig. 2. phylogenetic position of diprora nested deep within the raphid diatoms. after kociolek et al. (2013). kociolek j. p., williams d. m. 202 acta bot. croat. 74 (2), 2015 genus (kociolek and stoermer 1989). therefore, a phylogenetic context can help us understand certain cases where species within a genus (or a genus within a family or order) may not possess the features used to diagnose a genus (or group). current state of diatom taxonomy and systematics discovery and description estimates suggest that for life in its entirety roughly 16,000–18,000 new taxon descriptions are added each year (mora et al. 2011, fontaine et al. 2012). diatom taxonomy adds roughly 200+ to that total, around 1.2% of newly described biodiversity (table 1). tab. 1. numbers of new taxon records per year (data derived from the online catalogue of diatom names, fourtanier and kociolek 2011). * indicates numbers for entries up until 19th september 2011, a number we suspect is lower than it should be. the average number of descriptions is recorded to include 2011 (225) and to exclude it (252). year number of records number of publications 2005 244 39 2006 195 67 2007 414 42 2008 971 50 2009 370 61 2010 194 65 2011 62* 26* average number 225 [252*] 50 [54*] remarkably, there is a working taxonomist – horst lange-bertalot – who has described nearly as many species (> 1,400; 1,145 in de clerck et al. 2013; on lange-bertalot’s contribution see kusber and jahn (2003)) as the early pioneers in the fi eld, such as ehrenberg (1795–1876, 2,055 names, this fi gure includes all microalgae); kützing (1807–1893, 2,636 names, this fi gure includes all microalgae), grunow (1826–1914, 1,251 names), hustedt (1886–1968, 1,219 names; all fi gures taken from de clerck et al. 2013: table 1), the fi rst three all of whom worked a century or more before him. many journals are dedicated, solely or in part, to publishing new species descriptions (phytotaxa, nova hedwigia, diatom research, iconographia diatmologica, bibliotheca diatomologica, diatom monographs, algological studies, phytokeys). species description is important work as it is the baseline for documenting the diversity on, or once inhabiting, the planet. but it is not the only approach to capturing diatom diversity. kinds of description certain questions arise concerning the specifi cs of diatom descriptions (questions that may, indeed, be generalised as applicable to all groups of taxa), such as: how many specimens were used to formulate the description that appears in the protologue (defi ned as »… diatom genus: a revision acta bot. croat. 74 (2), 2015 203 everything associated with a name at its valid publication, i.e. description or diagnosis, illustrations, references, synonymy, geographical data, citation of specimens, discussion, and comments«.)? how were the specimens examined? where are those specimens now? were additional data sought, such as breeding behaviour, observations on live material, dna barcodes, and so on? of course, no provisions or guidelines are provided in the codes of nomenclature (in the case of diatoms, now the international code of nomenclature for algae, fungi, and plants, http://www.iapt-taxon.org/nomen/main.php, for the online version; mcneill et al. 2012 for the printed version). nor should there be: the quantity of data required to support the description of any new taxon (regardless of rank) is a scientifi c question, related to the discovery of defi ning character(s), rather than a question pertaining to nomenclature, the naming of any particular taxon once discovered. traditionally, new diatom taxa were illustrated with line drawings, and since the 1970s line drawings have given way to photographs taken with the light microscope. since the introduction of the scanning electron microscope as a standard in the investigation of diatom morphology, photographs derived from that source have become almost standard. should new diatom taxa be described in the absence of evidence from scanning electron microscopy? this question requires some consideration. before we offer some thoughts on the question above, let us fi rst focus on how diatomists actually behave when they record and present their data (here we ignore the inclusion of ‘additional’ forms of data – breeding behaviour, live material, dna bar-codes – for the simple reason that the capture of the fi rst two primarily requires light microscopy only, and capture of dna sequence data involves technology other than a microscope). for simplicity, we focus on just one year, 2008 (as it happens, other years in the fi ve year period documented in table 1 have more or less the same fi gures as 2008). in 2008, 97 new diatom nomenclatural acts were made, appearing in 50 publications, diverse in content, ranging from those dedicated to diatom studies (e.g. diatom research, diatom and the proceedings of the international diatom symposium), those dedicated to phycology (e.g. phycologia, phycological research), those dedicated to botany (e.g. botany, iheringia, séries botânica), through to journals with a much broader scope (e.g. мікробіологія і біотехнологія = microbiology and biotechnology). of the 97 names introduced, 28 were new combinations and one was proposed as a substitute name, leaving 68 new taxa described for that year. it is the last group that is of interest here. practically all 68 new names published in 2008 were documented with observations gained from both light and electron microscopy (although the numbers of each kind of image varied; on occasion, light micrographs out-numbered electron micrographs) – two names were accompanied with only light micrographs, two with only scanning electron micrographs and one with a combination of light, scanningand transmission electron microscopy. thus, if a consensus perspective was to be adopted, one might argue that as a standard for descriptions of new diatom taxa they should have, at the very least, both lightand electron microscope images of relevant structures as supporting evidence. documenting the exact specimens photographed on a slide or stub (by giving coordinates of the specimens) should be considered by diatomists as a ‘best practice’. the counter view relates to two specifi c viewpoints, one practical, the other philosophical. the practical issue concerns the availability of specimens, their rarity, a problem particularly pertinent when fossil specimens are the primary source of evidence (but the problem does not relate exclusively to fossils, e.g. kulikowsky et al. 2012, thessen et al. 2012 kociolek j. p., williams d. m. 204 acta bot. croat. 74 (2), 2015 for relevant discussion with a non-diatom example). in general, when only a few specimens are available (restrictions on collecting, availability of herbarium material, access to regions, and so on) and those specimens have only been found on glass slides, it would appear useful, if not necessary, to still document them using the light microscope rather than delay and err on the side of caution in the expectation that the future will yield further relevant specimens. for example, by the mid-1950s synedra berolinensis, known for nearly half a century, only had a handful of drawings, most copied from one fl ora to another (williams, 2013). given the ever-changing environmental conditions as well as the disturbing frequency of man-made disasters, further specimens may never be found, as the areas harbouring the original specimens may be altered (or even destroyed) beyond their original condition. without publication of the original record (details of the original specimens), a part of life on earth that would have been documented will now disappear forever. second, if species names are supposed to represent simple hypotheses about order in nature, then such hypotheses require further examination when more data comes to light, regardless of how well documented any particular taxon was in the fi rst place. to be sure, this may not be generally true for the past where, in some cases, a rather poor line drawing is all that was offered – and all that is left. however, advocating as least light microscopy means that a permanent slide has to be made and it should follow that the preservation of that slide (specimens) is also of signifi cance. taxonomy, as a science, has an especially close relationship with its past for these two very good reasons. given the arguments above concerning rarity, light microscopy alone may be offered but justifi cation for adopting such a procedure would be secured by listing in the protologue all specimens examined (including, of course, the single gathering used for the holotype of a new species) a practice up to now not universally adopted by diatomists (but see ross 1995, for an exemplary account) but is by most other botanists. phylogenetics and revisionary monographs both of these approaches, phylogenetic and revisionary studies, are of signifi cance as this is the process whereby both known and unknown taxa are treated in a specifi ed framework to understand their placement in the »tree of life« (their phylogenetic placement), yielding a classifi cation system derived from formal analysis of their relationships. some associate this kind of work exclusively with molecular data (see mann and evans 2007), which have indeed provided dramatic insights and unprecedented contributions to our understanding of diatom phylogeny (sims et al. 1996, alverson 2008, ruck and theriot 2011, lundholm et al. 2002, kemarrec et al. 2011, rimet et al. 2011, stepanek and kociolek 2014). but these contributions have come to the fore because they are methodologically set up to provide ready, formal analysis of relationships. these methods are fraught with challenges similar to those that occur in formal studies of phylogeny based on morphology (making of certain assumptions, baker and gatesy 2002). there are studies with formal analyses of morphological data (see for example williams 1985, 1990, kociolek and stoermer 1986, 1988, 1989, 1993, julius and tanimura 2001, edgar and theriot 2004, ruck and kociolek 2004). regardless of the discovery of new sources of data, a purely morphological approach remains necessary for the analysis of fossil diatoms, which at the level of genus and species may include more formally described taxa than all the recent taxa combined. diatom genus: a revision acta bot. croat. 74 (2), 2015 205 a call for revisionary studies and formal phylogenetics whatever the approach, molecular, morphological, or both, there is a pressing need for genusand family-level revisionary studies. many diatom genus names in everyday use have never had a systematic revision; the same is true for many higher levels of classifi cation. for example, barely any of the 11 subclasses, 36 new orders, 4 new suborders 42 new families and 19 genera proposed in round et al. (1990) have never been analyzed in any formal, systematic way. just as molecular data have turned several assumptions about diatom relationships on their head, to have far-reaching implications for our understanding of diatom evolutionary relationships and requiring a new approach to the way we talk about and teach students about the group (williams and kociolek 2011), we believe that such will be the case once formal analyses of genusand higher level groups commences in earnest. we are agnostic as to the types of data that should be used in these formal analyses, and in fact, as stated previously, morphology will be necessary for revisionary and phylogenetic analyses for many extinct diatom taxa. it is time for those involved in diatom studies to engage in this important work. without these formal analyses it will be nearly impossible to advance our knowledge of the many facets of diatom biology and the practical applications of these remarkable organisms. references agardh, c. a., 1824: systema algarum. pp. [i]-xxxvii, [1]-312. lundae: literis berlingianis. alverson, a. j., 2008: molecular systematics and the diatom species. protist 159, 339–353. baker, r. h., gatesy, j., 2002: is morphology still relevant? in: desalle, r, giribit, g., wheeler, w. 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(bacillariophyceae), a naviculoid diatom with amphoroid symmetry and its relationship to navicula sensu stricto and other naviculoid genera. nova hedwigia beihefte 143, 469–484. untitled acta bot. croat. 75 (1), 2016 25 acta bot. croat. 75 (1), 25–30, 2016 coden: abcra 25 doi: 10.1515/botcro-2016-0017 issn 0365-0588 eissn 1847-8476 impact of nickel on grapevine (vitis vinifera l.) root plasma membrane, ros generation, and cell viability ján pavlovkin1*, roderik fiala1, milada čiamporová1, michal martinka1,2, vladimír repka1 1 institute of botany, slovak academy of sciences, dúbravská cesta 9, sk-84523, bratislava, slovak republic 2 department of plant physiology, faculty of natural sciences, comenius university, mlynská dolina b-2, sk-84215 bratislava, slovak republic abstract – the present study investigated the impact of nickel (ni2+) on trans-membrane electrical potential (em) and permeability properties of plasma membrane (pm) in epidermal cells of adventitious grapevine roots. the relationship between disturbances of membrane functionality and the production of superoxide anion, hydrogen peroxide and cell viability after the exposure of roots to ni2+ was also studied. treatments with 0.1–5 mmol l–1 nicl2 induced a concentration-dependent transient pm depolarization, which was recovered to the initial resting potential within 50–70 min in the presence of ni2+. longer (up to 24 h) exposure of roots to 1 mmol l–1 of ni2+ hyperpolarized the em by approximately 17 mv. application of the highest 5 mmol l–1 concentration of ni2+ during longer treatments (up to 48 h) resulted in the increase of membrane permeability; however the em, cell viability, and superoxide content remained unaffected. the increase in the formation of hydrogen peroxide was timeand concentrationdependent and maximum production was recorded after 180 min of ni2+ treatment. we can conclude that oxidative stress resulting from an imbalance in the generation and/ or removal of hydrogen peroxide in the root tissues of grapevine was the major cause of ni2+ toxicity. keywords: cell viability, grapevine, nickel trans-membrane electrical potential, oxidative stress, roots * corresponding author, e-mail: jan.pavlovkin@savba.sk introduction among different environmental heavy-metal pollutants, nickel (ni2+) has gained considerable attention in recent years, because of its rapidly increasing concentrations in soil, air, and water in different parts of the world. most agricultural soils contain ni2+ in average of 25 mg kg–1 soil dry weight (dw) but its content is often substantially increased up to 26,000 mg kg–1, by human activities such as mining, emission of smelters, coal and oil burning, sewage, phosphate fertilizers and pesticides (holmgren et al. 1993). many plants that naturally grow on such contaminated soils contain ni2+ in concentrations exceeding 1000 mg g–1 dw in their tissues (gonnelli et al. 2001) but they generally possess mechanisms allowing them to tolerate ni2+ and to develop without phytotoxic problems (gabbrielli et al. 1990). however, many of agriculturally important crops contain less than 5 μg of ni g–1 dw, and the symptoms of phytotoxicity often become apparent at soil ni2+ concentrations as low as 25–30 μg g–1 (khalid and tinsley 1980). nickel is now considered an essential mineral nutrient (brown et al. 1987, seregin and kozhevnikova 2006), which in low concentrations fulfi ls a variety of essential roles in plants. therefore, ni2+ defi ciency produces an array of effects on growth and metabolism of plants, including reduced growth, and induction of senescence, leaf chlorosis, alterations in nitrogen metabolism, and reduced iron uptake (ahmad and ashraf 2011). according to bollard (1983) iron defi ciency could explain part of the symptoms induced by ni2+. in addition, ni2+ is a constituent of several metalloenzymes such as urease (brown et al. 1987). on the other hand excess of ni2+ in the medium alters various physiological processes, resulting in detrimental effects on plants and causing diverse toxicity symptoms (sharma and dhiman 2013). among these, iron defi ciency that leads to chlorosis and foliar necrosis and inhibition of nutrient absorption by roots have been widely found in different plant species (pandey and sharma 2002, chen et al. 2009). ouzounidou et al. (2006) working with wheat plants reported, that long exposure with 1 mm ni2+ reduced iron content leading to iron and manganese defi ciency. additionally, excess ni2+ also can retard shoot and root growth, impair plant metabolism, inhibit photosynthesis and transpiration, and cause ultrastructural modifi cations, which are well documented in the review by sharma and dhiman (2013). pavlovkin j., fiala r., čiamporová m., martinka m., repka v. 26 acta bot. croat. 75 (1), 2016 other symptoms observed in ni2+-treated plants are related to oxidative stress (gajewska et al. 2006, sharma and dietz 2009). when generation of reactive oxygen species (ros) such as superoxide and peroxide exceeds their removal, injuries may occur in the cells (agrawal et al. 2013). the most common indicator of oxidative stress is lipid peroxidation resulting in disturbances of membrane integrity and consequently its enhanced permeability (lukatkin et al. 2013). baccouch et al. (2006) and hao et al. (2006) showed enhanced lipid peroxidation in ni2+-treated wheat plants. by contrast, ni2+ effects on wheat plants were not caused by lipid peroxidation (gajewska et al. 2006). the changes in lipid and protein composition might disturb plasma membrane (pm) functions and, consequently the ion balance in the cytoplasm (llamas et al. 2008). several authors have shown that impairment of nutrient balance may result not only from the changes of plasmalemma lipid composition, but also that ni2+ affected plasma membrane h+-atpase activity (morgutti et al. 1981). llamas et al. (2008) and sanz et al. (2009) reported that ni induced a concentration-dependent pm depolarization in rice and barley but the activity of the pm h+-atpase was not affected. however, in the long term experiments a drastic loss of potassium was recorded. the fi rst plant organ facing the elevated ni2+ concentrations is the root system and especially the root cell plasma membrane being not only the site of ni2+ entry but also a target of its toxic impact. the use of electrophysiological technique allowed us to record instant changes in electrophysiological parameters of individual epidermal cells of the adventitious grapevine roots during ni2+ treatment. the trans-membrane electrical potential (em) and permeability measurements have been supplemented with determination of superoxide and hydrogen peroxide generation as well as cell viability in the roots exposed to various ni2+ concentrations for up to 24 h. material and methods plant material and growth conditions grapevine (vitis vinifera l., cv. limberger) shoot cuttings were taken from the production vineyards of the region rúbaň, slovakia. after stratifi cation in cold room (4 °c) for one month, nodal explants (10 cm) with single axillary bud were used for hydroponic cultivation. the explants were grown in magenta jars fi lled to 60 ml with aerated half strong ms medium (murashige and skoog 1962) at 25 ± 1 °c under 14 h photoperiod. ni2+ treatments based on published research of other authors (pandey and sharma 2002, llamas et al. 2008), and our preliminary experiments, the concentrations of nicl2 that have caused the clear effects within short (48 h) treatments were chosen herein (1–5 mmol l–1). although such concentrations are not common in vineyards soils, they were not lethal for vitis root cells. two-month old explants with adventitious roots were transferred to aerated solutions containing 0.1 mmol l–1 kcl and 0.1 mmol l–1 cacl2, ph 5.8 supplemented with 0 (control), 1, 2 or 5 mmol l–1 nicl2 for 24 h in the concentration-dependence experiments, and with 0 (control) and the highest concentration 5 mmol l–1 nicl2 for 10, 30, 60 or 180 min in the time-dependence experiments. the roots were then stained for confocal microscope analyses as described later. electrophysiology measurements of trans-membrane electrical potential (em) were performed on single epidermal cells within the root zone located 0.5–0.9 mm from the root tip of 20 mm long root segments. the root epidermal cells being in direct contact with the environment are more sensitive comparing to the internal root tissues as shown in our previous research on grapevine root cells exposed to cadmium (fiala et al. 2015). the em was measured using standard microelectrode technique as described in detail by kenderešová et al. (2012). after rinsing with 0.5 mmol l–1 caso4, the roots were mounted in a 5-ml volume plexiglas chamber and constantly perfused (5 ml min–1) with bathing solution containing 0.1 mmol l–1 kcl and 0.1 mmol l–1 caso4. the initial maximum depolarization (δem) induced by 0.1– 5 mmol l–1 ni2+ concentrations was measured by addition of nicl2 to the perfusion solution. subsequently, the effect of short-term treatments with ni2+ was registered after the cells attained the resting potential with an equimolar cacl2 solution by exchanging cacl2 with nicl2 in the perfusion solution, to avoid the effect of the counterion. the em of roots treated for several hours (up to 24 h) with ni2+ was also measured using the same solution, containing 1 and 5 mmol l–1 ni2+. membrane permeability changes in electrical conductivity of the solution surrounding the adventitious roots of 12 nodal explants were measured to assess the changes of membrane permeability caused by ni2+ treatments. the 0.5 cm long apical segments of approximately 2 cm long roots (0.4–0.5 g) were transferred to 0.5 mmol l–1 caso4 solution for 2 h in order to wash out the nutrient solution from the apoplast. after this time the segments were transferred into 10 ml of distilled water or 1 and 5 mmol l–1 nicl2 and incubated in a shaking water bath in the dark at 25 °c. effl ux of electrolytes from roots was determined within 48 h by conductivity meter ok-109-1 (radelkis, hungary). the conductivity was expressed in μs cm–1 g–1 fresh weight (fw). the changes in conductivity were expressed as differences between the values of particular conductivity measured, and the initial conductivity. confocal laser scanning microscopy propidium iodide (pi, fluka, switzerland) was used to counterstain the cell wall and nuclei of ruptured cells. 2’, 7’-dichlorodihydrofl uorescein diacetate (h2dcfda, serva) was used as indicator for hydrogen peroxide accumulation in cells. to monitor real time superoxide production in the root tips we used superoxide detection kit (enzo life sciences, usa). apical 0.5 cm long root segments were nickel effects on grapevine roots acta bot. croat. 75 (1), 2016 27 stained 2 min with 10 μg ml–1 pi in water, 15 min with 50 μmol l–1 h2dcfda in 50 mmol l–1 phosphate buffer ph 7.5 or 15 min with superoxide staining solution (following the manufacturer’s manual), washed for 2 min in distilled water (for superoxide just briefl y) and observed in confocal microscope olympus fv1000 (olympus, japan). pi and h2dcfda were excited at 488 nm and fl uorescence was detected using emission barrier fi lters 560–660 nm for pi or 505–550 nm for h2dcfda. the superoxide stain excitation wavelength was 543 nm and the emission was detected using 560–660 nm barrier fi lter. the confocal microscopy images represent at least three roots and were selected from at least three different images of each root. fluorescence signal intensity (ctcf, corrected total cell fl uorescence) was measured and calculated with the open source analysing software image-j2/fiji (http://imagej.net/fiji). statistical analysis each experiment was repeated at least three times. data on em and membrane conductivity were expressed as mean ± standard deviation (sd) with the number of samples (n). data on cell viability, hydrogen peroxide accumulation and superoxide production were analysed using one-way anova with p < 0.05 (prism 5, graphpad software inc.). means and standard deviations were calculated from three independent experiments (n = 10 apical root segments). as for confocal microscopy, only representative images are shown. results trans-membrane electrical potential under control conditions the em values of epidermal cells located within the distance of 0.5–0.9 mm from the root tip varied between –121 and –133 mv (–126 ± 5.9 mv, mean ± sd, n = 39). in short term experiments (up to 70 minutes) the application of different ni2+ concentrations induced immediate changes in the em values of root epidermal cells (fig. 1). both ions, ni2+ and cl–, contributed equally to the em depolarization. thus, for adequate balancing of high concentrations of ni2+ and cl–, the resting potential was fi rst measured at corresponding concentrations of cacl2, which were then replaced by the same concentrations of nicl2, with simultaneous presence of 0.1 mmol l–1 caso4. the ni2+-induced rapid and transient depolarization was concentration-dependent (fig. 2). the initial em depolarization induced with different ni2+ concentrations repolarized to the initial resting potential values within 50–70 min (fig. 1). after transient depolarization, the ni2+ applied at fi nal 1 mmol l–1 concentration caused a slow membrane hyperpolarization. its magnitude reached the maximum value 8 h after the metal application and, in comparison with the values of control cells it was more negative (by ∆mv 17.6 ± 3.3 mv, mean ± sd, n = 13). after withdrawal of ni2+ from the perfusion solution the em repolarized to the value of control cells. compared to control, the em was more negative after 1 mmol l–1 ni while there was no difference after 5 mmol l–1 ni2+ treatment (fig. 3). fig. 1. changes of the trans-membrane electrical potential (em) induced by increasing ni2+ concentrations in epidermal cells of grapevine adventitious roots. the time of ni2+ application to the perfusion solution is indicated by arrow. representative curves of individual cells (n = 2–4) are shown. fig. 2. initial trans-membrane electrical potential depolarization (δem, mv) induced in epidermal cells of grapevine adventitious roots, by increasing concentrations of nicl2 added to the perfusion solution bathing the roots. results are shown as mean values ± standard deviations, n = 3. fig. 3. transmembrane potential difference (∆em, mv) recorded in epidermal cells of grapevine adventitious roots treated with 1 and 5 mmol l–1 nicl2 for 0–24 h. results are shown as mean values ± standard deviations, n = 3–8. pavlovkin j., fiala r., čiamporová m., martinka m., repka v. 28 acta bot. croat. 75 (1), 2016 membrane permeability the treatment of roots with 1.0 mmol l–1 ni2+ concentration did not change the root cell membrane permeability. only the exposure to the highest ni2+ concentration 5 mmol l–1 for 24 and 48 h resulted in membrane permeability increase comparing to control roots (fig. 4). cell viability, superoxide, hydrogen peroxide in all experiments only a weak pi fl uorescence was localized in the walls of the root tip cells indicating that the cells were viable, with intact cell membranes. superoxidespecifi c staining did not reveal an increased orange fl uorescence at any ni2+ concentration or time used in the experiments (figs. 5a, 6a). only slightly elevated superoxide level occurred in the roots treated with 5 mmol l–1 ni for 30 min (fig. 6a). the dynamics of hydrogen peroxide accumulation monitored in the grapevine root cells with h2dcfda showed concentration-dependence of the ni-induced hydrogen peroxide formation as indicated by green cytoplasmic staining (figs. 5a, b). the time course of hydrogen peroxide accumulation in grapevine root cells treated with 5 mmol l–1 ni demonstrated that the highest hydrogen peroxide production occurred mainly after 180 min (figs. 6a, b). discussion structural, physical and chemical properties of the pm itself as well as any effects of metal ions at the cell surfaces in general have an impact on transport processes. alterations of the pm-h-atpase activity can be assessed by fig. 4. time-course of electrolyte leakage, measured as electrical conductivity, from the segments of grapevine adventitious roots treated with 1 and 5 mmol l–1 nicl2. results are shown as mean values ± standard deviations, n = 3. fig. 5. concentration-dependent effects of ni-treatment on cell viability, superoxide and hydrogen peroxide accumulation in grapevine 0.5 cm apical root segments demonstrated with confocal microscope (a) and expressed using fl uorescence signal intensity (ctcf) value (b). bar represents 1 mm. different letters indicate signifi cant differences at 5% level. results are shown as mean values ± standard deviations, n = 3. fig. 6. time-dependent responses to 5 mmol l–1 nicl2 of cell viability, superoxide and hydrogen peroxide accumulation in grapevine 0.5 cm apical root segments demonstrated with confocal microscope (a) and expressed using fl uorescence signal intensity (ctcf) value (b). bar represents 1 mm. different letters indicate signifi cant differences at 5% level. results are shown as mean values ± standard deviations, n = 3. nickel effects on grapevine roots acta bot. croat. 75 (1), 2016 29 studying changes in the em. according to our results, the effect of ni on em of grapevine adventitious root epidermal cells differed from that reported for the other divalent cation, cadmium (llamas et al. 2000, pavlovkin et al. 2006, fiala et al. 2015) and mercury (repka et al. 2013). in shortterm experiments, ni induced rapid and concentration-dependent transient depolarization of the pm in the grapevine roots indicating its entry into the cells. but after this initial depolarization the em of ni-treated roots reached the values similar or slightly higher than those of the control in less than 70 min. llamas et al. (2008) working with rice plants suggested that such effect may be due to a stimulation of h+ effl ux, as demonstrated for ni in maize roots (morgutti et al. 1981). according to these authors, the entry of ni into the cells occurs downhill the electrochemical gradient by a mechanism of uniport, inducing an immediate h+ effl ux for charge compensation, followed by k+ effl ux. their results may explain the more negative values of em in comparison to the control, in the grapevine roots treated 24 h with 1 mmol l–1 ni. in addition to the initial effect on the active component of em, the effect of ni2+ on the passive component of em cannot be ruled out. there is evidence that the pm permeability alterations might be involved in plant heavy metal tolerance (llamas et al. 2008) and its disruption can be a consequence of increased peroxidation of unsaturated fatty acids in the cell membranes (lukatkin et al. 2013). according to our results, no signifi cant changes of the membrane permeability comparing to controls were found in grapevine roots treated with 0.5 mmol l–1 ni2+ up to 24 h. however, when the roots were treated with 5 mmol l–1 ni2+, a progressive increase in membrane permeability was measured after 16 h of treatment. taken together, the changes induced by ni2+ stress were not signifi cant in em (figs. 1–3), superoxide production or cell viability (figs. 5b, 6b). however, hydrogen peroxide concentration signifi cantly increased in the adventitious roots of grapevine exposed to 1, 2, and 5 mmol l–1 ni2+ (figs. 5b, 6b). at the highest 5 mmol l–1 concentration of ni2+ a signifi cant increase in membrane permeability occurred (fig. 4), which could be responsible for the changes in water content, as was determined by llamas et al. (2008). according to gajewska et al. (2006), ni2+ stress in roots is more related to the accumulation of hydrogen peroxide in root tissues than to enhanced lipid peroxidation. furthermore, the elevated levels of hydrogen peroxide may be a consequence of its inappropriate removal. in line with this statement, a signifi cant decrease of catalase activity was observed in ni-treated wheat leaves (gajewska and sklodowska 2007). our results suggest than ni2+ does not directly affect plasma membrane atpase and superoxide production. however, it increases plasma membrane permeability and hydrogen peroxide production, which can be related with ni2+ toxicity in grapevine roots. acknowledgement the work was supported by slovak grant agency vega, project 02/0023/13. references agrawal, b., czymmek, k. j., sparks, d. l., bais, h. p., 2013: transient infl ux of nickel in root mitochondria modulates organic acid and reactive oxygen species production in nickel hyperaccumulator alyssum murale. journal of biological chemistry 8, 7351–7362. ahmad, m. s., ashraf, m., 2011: essential roles and hazardous effects of nickel in plants. review of environmental contamination and toxicology 214, 125–167. baccouch, s., chaoui, a., el ferjani, e., 2006: nickel toxicity induces oxidative damage in zea mays roots. journal of plant nutrition 24, 1085–1097. bollard, e. g., 1983: involvement of unusual elements in plant growth and nutrition. in: läuchli, a., bieleski r. l. (eds.), encyclopedia of plant physiology, new series, vol. 15b, inorganic plant nutrition, springer, pp. 695–744. brown, p. h., welch, r. m., cary, e. e., 1987: nickel: a micronutrient essential for higher plants. plant physiology 85, 801– 803. chen, c., huang, d., liu, j., 2009: functions and toxicity of nickel in plants: recent advances and future prospects. clean soil, air, water 37, 304–313. fiala, r., repka, v., čiamporová, m., martinka, m., pavlovkin, j., 2015: early cadmium-induced effects on reactive oxygen species production, cell viability and membrane electrical potential in grapevine roots. vitis 54, 175–182. gabbrielli, r., pandolfi ni, t., vergnano, o., palandri, m. r., 1990: comparison of two serpentine species with different nickel tolerance strategies. plant and soil 122, 271–277. gonnelli, c., galardi, f., gabbrielli, r., 2001: nickel and copper tolerance and toxicity in three tuscan populations of silene paradoxa. physiologia plantarum 113, 507–514. gajewska, e., slaba, m., andrzejewska, r., skłodowska, m., 2006: nickel-induced inhibition of wheat root growth is related to h2o2 production, but not to lipid peroxidation. plant growth regulation 49, 95–103. gajewska, e., skłodowska, m., 2007: effect of nickel on ros content and antioxidative enzyme activities in wheat leaves. biometals 20, 27–36. hao, f., wang, x., chen, j., 2006: involvement of plasma-membrane nadph oxidase in nickel-induced oxidative stress in roots of wheat seedlings. plant science 170, 151–158. holmgren, g. g. s., meyer, m. w., chaney, r. l., daniels, r. b., 1993: cadmium, lead, zinc, copper and nickel in agricultural soils of the united states of america. journal of environmental quality 22, 335–348. khalid, b. y., tinsley, j., 1980: some effects of nickel toxicity on rye grass. plant and soil 55, 139–144. kenderešová, l., staňová, a., pavlovkin, j., ďurišová, e., nadubinská, m., čiamporová, m., ovečka, m., 2012: early zn2+induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive arabidopsis species. annals of botany 110, 445–449. llamas, a., ullrich, c. i., sanz, a., 2000: cd2+ effects on transmembrane electrical potential difference, respiration and pavlovkin j., fiala r., čiamporová m., martinka m., repka v. 30 acta bot. croat. 75 (1), 2016 membrane permeability of rice (oryza sativa l) roots. plant and soil 219, 21–28. llamas, a., ullrich, c. i., sanz, a., 2008: ni2+ toxicity in rice: effect on membrane functionality and plant water content. plant physiology and biochemistry 46, 905–910. lukatkin, a. s., kashtanova, n. n., duchovskis, p., 2013: changes in maize seedlings growth and membrane permeability under the effect of epibrassinolide and heavy metals. russian agricultural sciences 39, 307–310. morgutti, s., ferrari-bravo, p., marre, m. t., cocucci, s. m., 1981: effects of ni2+ on proton extrusion and related transport processes and on the transmembrane electrical potential in maize roots. plant science letters 23, 123–128. murashige, t., skoog, f., 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum 15, 473–97. ouzounidou, g., moustakas, m., symeonidis, l., karataglis, s., 2006: response of wheat seedlings to ni stress: effects of supplemental calcium. archives of environmental and contamination toxicology 50, 346–352. pandey, n., sharma, c. p., 2002: effect of heavy metals co2+, ni2+, and cd2+ on growth and metabolism of cabbage. plant science 163, 753–758. pavlovkin, j., luxová, m., mistríková, i., mistrík, i., 2006: short and long-term effects of cadmium on transmembrane electric potential (em) in maize roots. biologia 61, 109–114. repka, v., fiala, r., čarná, m., pavlovkin, j., 2013: membrane potential differences and viability of grapevine root cells treated with hgcl2. plant, soil and environment 59, 353–358. sanz, a., llamas, a., ullrich, c. i., 2009: distinctive effects of cd and ni on membrane functionality. plant signalling and behaviour 4, 980–982. seregin, i. v. kozhevnikova, a. d., 2006: physiological role of nickel and its toxic effects on higher plants. russian journal of plant physiology 53, 257–277. sharma, s. s., dietz, k. j., 2009: the relationship between metal toxicity and cellular redox imbalance. trends in plant science 14, 43–50. sharma, a., dhiman, a., 2013: nickel and cadmium toxicity in plants. journal of pharmaceutical and scientifi c innovation 2, 20–24. acta bot. croat. 80 (2), 2021 117 acta bot. croat. 80 (2), 117–124, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-011 issn 0365-0588 eissn 1847-8476 effects of two synthetic pyrethroids on arthrospira platensis gomont growth and antioxidant parameters hatice tunca1*, kübra hödük1, feray köçkar2, ali doğru1, tuğba ongun sevindik1 1 university of sakarya, faculty of arts and science, department of biology, sakarya, turkey 2 university of balıkesir, faculty of arts and science, department of molecular biology and genetics, balıkesir, turkey abstract – the transport of pesticides from application areas to other areas results in pesticide contamination and this sort of contamination has led to unexpected environmental problems worldwide. it is important to determine the responses of phytoplanktonic organisms to these chemicals for an understanding of the effects of pesticides on aquatic ecosystems. in this study, arthrospira platensis gomont cyanobacteria were exposed to different concentrations of the pesticides cypermethrin (cyp; 0–50 µg ml–1) and deltamethrin (dlm; 0–2 µg ml–1). changes in chlorophyll-a concentration, the absorbance at od560, antioxidant parameters (superoxide dismutase – sod, ascorbate peroxidase – apx, glutathione reductase – gr, malondialdehyde – mda, h2o2, and proline) were determined under the pesticide exposure. our results showed that there is a decrease in od560 absorbance and chlorophyll-a content proportional to the increase of pesticide levels. sod activity decreased with cyp and dlm application in a. platensis cultures. gr activity also decreased with cyp applications but did not change with dlm application. apx activity increased with cyp treatments but did not change with dlm applications. although mda and hydrogen peroxide contents did not change with cyp applications, they increased with dlm applications. proline contents increased with cyp applications but decreased with dlm applications. in conclusion, deltamethrin was more toxic than cypermethrin in the concentrations applied. keywords: antioxidant enzymes, arthrospira platensis, cypermethrin, deltamethrin, growth introduction many chemicals, including pesticides, are released through modern industrial and agricultural activities and have reached a high level in the environment (burkiewicz et al. 2005). pesticides affect not only target organisms but also non-target organisms in the aquatic biota (tremolada et al. 2004). these organisms play an important role in biological processes such as biogeochemical cycling, production, separation, and interaction with other organisms. pesticides disturb the balance of water ecosystems with their direct action on plants and animals or with their bioaccumulation and transfer abilities in the food chain (netrawali and gandhi 1990, burkiewicz et al. 2005). planktonic algae display a fundamental role as primary producers (burkiewicz et al. 2005) and a decrease in algal density and species composition affects the aquatic ecosystem directly by reducing biodiversity and primary production (li et al. 2005). they are sensitive indicators that allow testing of the different effects of chemicals released into the water (burkiewicz et al. 2005). for this reason, microalgae are frequently used in various bioassays (li et al. 2005). agricultural chemicals inhibit the growth rate, biomass, and pigment content of freshwater algae by contaminating surface waters in agricultural areas (netrawali and gandhi 1990). pyrethroids are a class of synthetic insecticides designed and optimized based on pyrethrin structure (elliott 1995). these pesticides are effective insecticides that are widely used to control agricultural and healthcare pests. after use, they are released into the environment and enter water resources (mittal et al. 1994). cypermethrin (cyp) and deltamethrin (dlm) have increased toxicity, especially to aquatic organisms with increasing life expectancy (johri et al. 1997). megharaj et al. (1987) observed that cyp has inhibitory effects on scenedesmus bijugatus kützing. xiong et al. (2002) and li et al. (2005) found similar results for scenedesmus * corresponding author e-mail: htunca@sakarya.edu.tr tunca h., hödük k., köçkar f., doğru a., sevindik o. t. 118 acta bot. croat. 80 (2), 2021 pesticide application. responses given by phototrophic microorganisms to pesticides will help us to have an idea of how the communities are affected by the pesticides in the water ecosystems. material and methods algae culture and treatment arthrospira platensis, strain m2, was obtained from the soley microalgae institute (california, usa) (culture collection no: slsp01). algae were grown in spirulina medium (aiba and ogawa 1977) under axenic conditions. 20 ml algal cultures were inoculated onto 180 ml culture medium in an erlenmeyer flask and were allowed to grow under full-spectrum lamps providing 93 µmol photons m–2 s–1 photosynthetically available radiation in a 12:12 h light/dark cycle at 30 ± 1 °c during 10 days. at the end of 10 days, cultures were renewed and, all the flasks contained 50 ml algal culture. stock solutions of the commercial formulation of cyp and dlm (200 g l–1 and 25 g l–1, respectively; ec, sakarya, turkey) were prepared with distilled water and were then diluted for all bioassays. various final concentrations of cypermethrin (10, 20, 30, 40, 50 µg ml–1) and deltamethrin (0.125, 0.25, 0.5, 1, 1.5, 2 µg ml–1) were in the culture medium. the range of concentrations was determined with preliminary range-finding bioassays according to ec50 value for growth parameters and ic50 value for enzyme activity assays and at least five concentrations were selected under these values. cell growth and chlorophyll-a assay optical densities (ods) at 560 nm were measured spectrophotometrically over 7 days under control and stressed conditions. . chlorophyll-a content was estimated by methanol extraction and measured spectrophotometrically over 7 days (mackinney 1941). antioxidant enzyme activities on the 7th day of the study, 2 ml culture solutions from the control and treated samples were centrifuged at 14,000 rpm for 20 min at 4 °c and the resulting pellets were kept at –20 °c until enzyme activity was measured. pellets were ground with liquid nitrogen and suspended in specific buffers with proper ph values for each enzyme. the protein concentrations of algal cell extracts were determined according to bradford (1976), using bovine serum albumin (bsa) as a standard. the superoxide dismutase activity was determined by the method of beyer and fridovich (1987), based on the photoreduction of nbt (nitroblue tetrazolium). extraction of pellets (0.2 g) was performed in 1.5 ml homogenization buffer containing 100 mm k2hpo4 buffer (ph 7.0), 2% pvp, and 1 mm na2edta. after centrifugation at 14,000 rpm for 20 min at 4 °c, the resulting supernatants were used to measure sod activity. the reaction mixture consisted of 100 mm k2hpo4 buffer (ph 7.8) containing 9.9 × 10–3 m methionine, 5.7 × 10–5 m nbt, 1% triton x-100, and enzyme obliquus kützing. wang et. al. (2011) carried out a growth inhibition test on skeletonema costatum cleve, scrippsiella trochoidea (f.stein) a.r.loeblich iii and chattonella marina (subrahmanyan) y.hara et m.chihara during 96 h. sáenz et al. (2012) studied cyp toxicity to scenedesmus quadricauda chodat, scenedesmus acutus meyen, chlorella vulgaris beyerinck (beijerinck) and pseudokirchneriella subcapitata (korshikov) f.hindák. wang et al. (2012) tested cyp effetcs on scenedesmus obliquus. these references show that cyp has toxic effects on various groups of microalgae. there are also some studies on dlm toxicity to aquatic microorganisms. baeza-squiban et al. (1987) have shown that the growth of dunaliella sp. teodoresco and chlamydomonas sp. ehrenberg was inhibited by dlm application. caquet et al. (1992) observed dlm effects on phytoplankton communities in freshwater mesocosms and found dlm to disappear rapidly from the aquatic ecosystem. antioxidants are compounds that reduce the harmful effects of oxidation via inhibiting free oxygen formation or eliminating free radicals (baublis et al. 2000). superoxide dismutase (sod: ec 1.15.1.1) is a class of metalloprotein that catalyses superoxide to oxygen and hydrogen peroxide (h2o2) (valentine et al. 1998). ascorbate peroxidase (apx: ec 1.11.1.11) converts hydrogen peroxide to water by using ascorbate as an electron donor and thereby accumulations of h2o2 toxic levels are prevented in photosynthetic organisms (chew et al. 2003). glutathione reductase (gr: ec 1.6.4.2), is a member of nadph-dependent oxidoreductases, found in both prokaryotic and eukaryotic cells. gr catalyses the reduction of oxidized glutathione (gssg) to reduced glutathione (gsh) together with the oxidation of nadph and plays an important role in the cellular defence systems against reactive oxygen metabolites by creating a reduced gsh pool (anjum et al. 2010). malondialdehyde is a metabolite that occurs with peroxidation of lipids including three or more double bonds. lipid peroxidation causes malondialdehyde formation (goel and sheoran 2003). it is known that proline content increases under various stress factors and proline accumulation protects cell parts and cell contents such as enzymes, membranes and polyribosomes (kishor et al. 2005). the enzymatic and non-enzymatic antioxidant defence systems create the response to oxidative stress and reflect the tolerance and susceptibility of algae to pesticide exposure. however, although there is some information about algal antioxidant systems, which are either a response to the environmental conditions or a defensive system (mallick and mohn 2000) studies about the harmful or activator effects of pyrethroids on algal antioxidant systems are limited. the aims of our study are: (i) to determine metabolic damage due to cypermethrin and deltamethrin toxicity and (ii) to measure the intracellular level of antioxidant responses for pesticide detoxification in arthrospira platensis-m2 strain. for this purpose, the alterations of some parameters such as growth rate (od560), chlorophyll-a content, superoxide dismutase (sod), ascorbate peroxidase (apx), glutathione reductase (gr), malondialdehyde (mda), hydrogen peroxide (h2o2) and proline have been investigated during oxidative damage of pesticide on arthrospira platensis acta bot. croat. 80 (2), 2021 119 extract. the reaction was started by the addition of 0.9 µm riboflavin and the mixture was exposed to light with an intensity of 375 µmole m–2 s–1. after 15 min, the reaction was stopped by switching off the light, and absorbance was read at 560 nm. the sod activity was calculated by a standard graphic and expressed as u mg–1 protein. the ascorbate peroxidase activity was determined according to wang et al. (1991) by estimating the decreasing rate of ascorbate oxidation at 290 nm. apx extraction was performed in 50 mm tris–hcl (ph 7.2), 2% pvp, 1 mm na2edta, and 2 mm ascorbate. the reaction mixture consisted of 50 mm k2hpo4 buffer (ph 6.6), 2.5 mm ascorbate, 10 mm h2o2, and enzyme-containing 100 µg protein in a final volume of 1 ml. the enzyme activity was calculated from the initial rate of the reaction using the extinction coefficient of ascorbate (e = 2.8 mm cm–1 at 290 nm). the glutathione reductase activity was measured with the method of sgherri et al. (1994). extraction was performed in 1.5 ml of suspension solution containing 100 mm k2hpo4 buffer (ph 7.0), 1 mm na2edta, and 2% pvp. the reaction mixture (total volume of 1 ml) contained 100 mm k2hpo4 buffer (ph 7.8), 2 mm na2edta, 0.5 mm oxidised glutathione (gssg), 0.2 mm nadph and enzyme extract containing 100 µg protein. the decrease in absorbance at 340 nm was recorded. correction was made for the non-enzymatic oxidation of nadph by recording the decrease at 340 nm without adding gssg to assay mixture. the enzyme activity was calculated from the initial rate of the reaction after subtracting the non-enzymatic oxidation using the extinction coefficient of nadph (e = 6.2 mm cm–1 at 340 nm). determination of malondialdehyde and hydrogen peroxide the malondialdehyde content was determined by the method of heath and packer (1968). 0.2 g of pellet was homogenized in 3 ml of 0.1% tca (4 °c) and centrifuged at 4100 rpm for 15 min and the supernatant was used in the subsequent determination. 0.5 ml of 0.1 m tris–hcl ph 7.6 and 1 ml of tca–tba–hcl reagent (15% w/v) (trichloroacetic acid–0.375% w/v thiobarbituric acid–0.25 n hydrochloric acid) were added to the 0.5 ml of the supernatant. the mixture was heated at 95°c for 30 min and then quickly cooled in an ice bath. to remove suspended turbidity, the mixture was centrifuged at 4100 rpm for 15 min, then the absorbance of the supernatant at 532 nm was recorded. non-specific absorbance at 600 nm was measured and subtracted from the readings recorded at 532 nm. the mda content was calculated using its extinction coefficient of 155 mm−1 cm−1. for determination of the hydrogen peroxide content, 0.5 ml of 0.1 m tris–hcl (ph 7.6) and 1 ml of 1 m ki were added to 0.5 ml of supernatant. after 90 min, the absorbance was recorded at 390 nm. the proline content determination the proline content was determined by the method of weimberg et al. (1982) where 0.1 g of pellet was homogenized in 10 ml of 3% aqueous sulphosalicylic acid and homogenates were incubated in a hot water bath at 95 °c for 30 minutes. the samples were cooled and centrifuged at 4,100 rpm for 10 min. two ml of the extract reacted with 2 ml of acid–ninhydrine and 2 ml of glacial acetic acid for 1 h at 100 °c. the reaction mixture was extracted with 4 ml toluene. the chromophore containing toluene was separated and the absorbance was recorded at 520 nm. statistical analysis the differences between the means of control and treated samples were analysed by one-way analysis of variance (anova), taking p < 0.05 as significant according to lsd with two degrees of freedom. three replicate cultures were used for each treatment. the mean values ± standard error (se) are shown in figures. results biomass and chlorophyll-a content biomass production and chlorophyll-a content of arthrospira platensis-m2 cells were significantly decreased by cyp and dlm application to the culture medium for 7 days (p < 0.05, figs. 1, 2). the most significant reductions were observed at the highest pesticide treatments, (50 µg ml–1 for cyp and 2 µg ml–1 for dlm) in biomass production and fig. 1. biomass values, as absorbance at 560 nm (a), and chlorophyll-a concentration (b) of arthrospira platensis treated with 0–50 µg ml–1 cypermethrin during 7 days. data are the means ± se of three replicates. *significantly different from control, p ˂ 0.05 (lsd analysis). tunca h., hödük k., köçkar f., doğru a., sevindik o. t. 120 acta bot. croat. 80 (2), 2021 chlorophyll-a content. cyp and dlm toxicity affected the biomass production and chlorophyll-a content of a. platensis-m2 cells, which increased in a time-dependent manner during the 7 days (figs. 1a,b, 2a,b). the ec 50 values of cyp and dlm applications were 53.12 ± 1.16 µg ml–1 and 1.76 ± 0.07 µg ml–1 respectively for 7th day, based on biomass production. the antioxidant parameters the effect of cyp applications on sod activity in a. platensis-m2 cells is presented in fig. 3a. all cyp concentrations (5, 10, 20, 30, 40, 50 µg ml–1) caused an increase in sod activity on a. platensis-m2 cells as compared to control. the maximum inhibition in sod activity (96%) occurred at 40 µg ml–1 cyp concentration. apx activities increased at the same concentrations (p < 0.05, fig. 3b). on the other hand gr activities also decreased at all concentrations except 5 µg ml–1 cyp application (p < 0.05, fig. 3c). ten, 20, 30, 40, 50 µg ml–1 cyp concentrations caused 17, 66, 47, 70, 85% decrease in gr activity, respectively. mda and h2o2 contents (fig. 4a,b) did not change significantly, but proline contents (fig. 4c) increased at all cyp concentrations (p > 0.05). the effect of dlm applications on sod activity in a. platensis-m2 cells is presented in fig. 5a. sod activities decreased by 37, 47, 21, 39, 36, 52% (p < 0.05) at all dlm concentrations, respectively; apx and gr activities did not display significant alteration (p > 0.05, fig. 5b,c). mda contents increased at 0.5, 1, 1.5 µg ml–1 dlm applications 74, 85, 78%; respectively (p < 0.05, fig 6a); however h2o2 content increased with 1, 1.5 and 2 µg ml–1 dlm applications (p < 0.05, fig. 6b). proline content decreased significantly at 2 µg ml– 1 dlm concentration (p < 0.05, fig. 6c). discussion in this study, we have found that exogenous addition of different concentrations of two synthetic pyrethroids (cypermethrin and deltamethrin) showed varying degrees of toxicity to the growth of arthrospira platensis m2 strain. in a study of scenedesmus bijugatus, nostoc linckia bornet ex bornet et flahault, synechococcus elongatus (nägeli) nägeli and phormidium tenue gomont, megharaj et al. (1987) found that cypermethrin inhibited the growth of scenedesmus bijugatus at 10–50 μg ml−1 concentrations. xiong et al. (2002) applied cypermethrin to scenedesmus obliquus during a 96 h period and found that the ec50 was 112 mg l–1. li et al. (2005) reported that cypermethrin inhibited the growth of scenedesmus obliquus at 50–250 mg l–1 concentrations during 96 h, and the chlorophyll-a and carotenoid content of scenedesmus obliquus decreased with the application. li et al. found the ec50 value of cypermethrin as 112 ± 9 mg l–1. wang et. al. (2011) carried out a growth inhibition test about cypermethrin toxicity on skeletonema costatum, scrippsiella trochoidea, and chattonella marina during 96 h and they determined ec50 values as 71.4 μg l−1, 205 μg l−1 and 191 μg l−1, respectively. sáenz et al. (2012) study cypermethrin toxicity to scenedesmus quadricauda, scenedesmus acutus, chlorella vulgaris and pseudokirchneriella subcapitat and the inhibited growth concentrations are 0.3–5 mg l–1, 0.6 mg l–1, 0.15 – 2.5 mg l–1 and 0.075 mg l–1, respectively. in another study, wang et al. (2012) determine the toxic effects of commercial cypermethrin on s. obliquus and measure ec50 value of 2.37 mg l–1. the data and the concentrations used (10–50 µg ml–1) of megharaj et al. (1987) are the closest to the results of our study. as was shown in the literature, cyp has an inhibitory effect on algal growth and chlorophyll-a content. as with the toxicity of cyp, there are some studies about the effect of dlm on algae. in the study of burkiewicz et al. (2005), the cell density of scenedesmus subspicatus chodat decreased with 2.5, 5 and 10 mg l–1 dlm applications during 24 h and 1.25 mg l–1 dlm application during 72 h. lutnicka et al. (2014) observed that dlm has inhibited the growth of chlorella vulgaris by 13% at the end of 14 days with a 0.02 μg l–1 dlm application. although the dlm concentrations in the study of lutnicka et al. (2014) were lower than in our applications, the concentrations of burkiewicz et al. (2005) were similar to those in our studies’. in our study, sod activities of a. platensis decreased at all cyp applications except 5 µg ml–1 and at all dlm applifig. 2. biomass values, as absorbance at 560 nm (a), and chlorophyll-a concentration (b) of arthrospira platensis treated with 0–2 µg ml–1 deltamethrin during 7 days. data are the means ± se of three replicates. *significantly different from control, p ˂ 0.05 (lsd analysis). oxidative damage of pesticide on arthrospira platensis acta bot. croat. 80 (2), 2021 121 cations according to control. in our study, cyp and dlm pesticides also caused significant reductions in chlorophylla, and thus loss of photosynthetic metabolism may have caused significant reductions in sod activity. wang et al. (2011) also reported that cyp pesticide in high concentrations (> 50 μg l–1) inhibited sod activity in skeletonema costatum, scrippsiella trochoidea and chattonella marina, and they pointed out the inactivation of sod activity may have been due to growth being inhibited by cypermethrin. one or more of the suggested reasons in these articles may be the main mechanism of cyp and dlm toxicity on the sod enzyme activity in our study. gr and glutathione are effective on the halliwell / asada pathway in the inactivation of h2o2 in plant cells (bray et al. 2000). gr catalyzes the last step of the ascorbate-glutathione pathway. gr activity significantly decreased at 10, 20, 30, 40, and 50 µg ml–1 cyp applications. sáenz et al. (2012) found that cyp concentrations causing algicidal effects, and thereby inhibitory effects of gr enzyme activity due to oxidative stress damage on p. subcapitata. the reductions in gr enzyme activities may have been related to loss of cell viability or deterioration of enzyme structure and enzyme reactions. in contrast, gr activity did not change in dlm application in our study. dewez et al. (2005) treated a fungicide fludioxonil to scenedesmus obliquus and indicated that gr activity was not significantly affected and the oxidized glutathione pool may have also been used by other enzymes. apx enzyme activity statistically increased at all concentrations in cyp treatment. apx use ascorbic acid for the elimination of detrimental h2o2 (verma and dubey 2003). studies on plants showed that apx activity increased in various stress conditions (hideg and vass 1996, verma and dubey 2003). teisseire and vernet (2001) supported the conclusion that gr enzyme activity was related to the apx enzyme activity. in our study, it was concluded that the absence of alteration in gr activity at the application concentrations of dlm promoted the absence of alteration in apx enzyme at fig. 3. total superoxide dismutase (sod; a), ascorbate peroxidase (apx; b) and glutathione reductase (gr; c) activities of arthrospira platensis treated with 0–50 µg ml–1 cypermethrin. data are the means ± se of three replicates. *significantly different from control, p ˂ 0.05 (lsd analysis). fig. 4. malondialdehyde (mda; a), hydrogen peroxide (h2o2; b) and proline (c) contents of arthrospira platensis treated with 0–50 µg ml–1 cypermethrin. data are the means ± se of three re plicates. *significantly different from control, p ˂ 0.05 (lsd analysis). tunca h., hödük k., köçkar f., doğru a., sevindik o. t. 122 acta bot. croat. 80 (2), 2021 similar concentrations because the ascorbate pool was counterbalanced by the gr enzyme. in our study, mda content in a. platensis cultures treated with dlm significantly increased at 0.5 μg ml–1, 1 μg ml– 1, and 1.5 µg ml–1 concentrations. the changes in mda content were parallel with the changes of h2o2 content for dlm pesticide. the increase of h2o2 content caused the formation of oh radicals by the haber-weis reaction and therefore lipid peroxidation was increased (goel and sheoran 2003). moreover, the inhibition of sod activity caused accumulation of o2– in the medium in deltamethrin application. it is known that lipid peroxidation is linked to the amount of o2– (choudhary et al. 2007). also, baruah and chaurasia (2020) reported that alpha-cypermethrin decreased the photosynthetic pigment content and it increased the mda content of chlorella sp. at 6–48 mg l–1 during 96 h bioassay. the reduction of photosynthetic pigments also increases lipid peroxidation (chen et al. 2008). a significant reduction of chlorophyll-a content may take place with lipid peroxidation at dlm application. it was reported that mda content, an indicator of lipid peroxidation, was increased by the endosulfan application (kumar et al. 2008). wang et al. (2011) found that cypermethrin enhanced mda content on skeletonema costatum, scripsiella trochoidea and chatonella marina. there was no significant change in the mda content at the cyp application and it was supported by the results of h2o2 content. moreover, the increase in the proline content may have prevented the cell membrane damage and the amount of mda. siripornadulsil et al. (2002) reported that the cadmium application did not alter the amount of mda in the proline overproducing transgenic chlamydomonas reinhardtii p.a.dangeard, and they suggested that proline may have inhibited free radical damage due to its antioxidant action. the amount of h2o2 in a. platensis cultures exposed to the dlm significantly increase with addition of 1 μg ml–1, 1.5 μg ml–1, and 2 μg ml–1 dlm. it was expected that the application of dlm pesticide would result in a decrease in the h2o2 content with the increase of sod enzyme activity, but the h2o2 content likely increased due to the increased activity of oxidases such as glycolate oxidase, glucose oxidase, amino acid oxidase and sulfite oxidase in plants (asada 1999). fig. 6. malondialdehyde (mda; a), hydrogen peroxide (h2o2; b) and proline (c) contents of arthrospira platensis treated with 0–2 µg ml–1 deltamethrin. data are the means ± se of three replicates. *significantly different from control, p ˂ 0.05 (lsd analysis). fig. 5. total superoxide dismutase (sod; a), ascorbate peroxidase (apx; b) and glutathione reductase (gr; c) activities of arthrospira platensis treated with 0–2 µg ml–1 deltamethrin. data are the means ± se of three replicates. *significantly different from control, p ˂ 0.05 (lsd analysis). oxidative damage of pesticide on arthrospira platensis acta bot. croat. 80 (2), 2021 123 the amount of h2o2 in all concentrations of cultures of a. platensis exposed to cyp (except 1 μg ml–1 and 5 μg ml–1) was not changed by increased apx enzyme activity. the decrease in the amount of h2o2 is likely due to the increase in enzymes consuming the h2o2 content, such as the apx enzyme (mallick and mohn 2000). the free proline amount of a. platensis cultures exposed to cyp was statistically significantly increased at all concentrations compared to the control. proline accumulation has been reported in plants exposed to heavy metal stress (saradhi and saradhi 1991). proline is an effective singlet oxygen scavenger and regulates the cell redox potential (saradhi and saradhi 1991, alia et al. 2001). proline acts as an osmotic regulator and scavenger of oh radical in cells and thus it interacts with cell macromolecules such as dna, protein, and lipids and consequently stabilizes the structure and function of these molecules (kavir et al. 2005). the increase of proline content may be an adaptive response under extreme stress conditions (fatma et al. 2007, kumar et al. 2014). proline reduces free radical production under stress conditions (alia 1993). fatma et al. (2007) studied the effects of environmental pollution by evaluating proline content on westiellopsis prolifica janet cyanobacterium and found that alphamethrin pesticide and heavy metals increased proline accumulation. the free proline content of a. platensis cultures exposed to the dlm effect was significantly reduced at 2 µg ml–1 concentration. ewald and schlee (1983) found that sulfide decreased the proline content by inhibiting proline synthesis. likewise, dlm may have inhibited the proline synthesis or may have disrupted proline configuration in our dlm application. in conclusion, in this study, the decrease in biomass and chlorophyll-a was related to the concentration of cyp and dlm. antioxidant enzyme activities and parameters were affected by different degrees according to the particular pesticides and their concentrations. these differences arose from the ros producing capacity of cyp and dlm. also, deltamethrin is more toxic than cypermethrin according to concentrations. since these pesticides have a toxic effect on aquatic organisms, care should be taken when they are used with all necessary precautions to prevent their release into the aquatic ecosystem. acknowledgement this study was supported by sakarya university research projects under grant no. bap 2017-02-20-001 and bap 2016-02-20-002. references aiba, s., ogawa, t., 1977: assessment of growth yield of a bluegreen alga: spirulina platensis in axenic and continuous culture. journal of general microbiology 102, 179–182. alia, s.p.p., 1993: suppression in mitochondrial electron transport is the prime cause behind stress induced proline accumulation. biochemical and biophysical research communications 193, 54–58. alia, s.p.p., mohanty, p., matysik, j., 2001: effect of proline on the production of singlet oxygen. amino acid 21, 195–200. anjum, n.a., umar, s., chan, m.t., 2010: ascorbate-glutathione pathway and stress tolerance in plants. in: pang, c.h., wang, p.s. 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ex lam. (rubiaceae) – a new species for the flora of ukraine pavel novák1*, dominik zukal1 1 department of botany and zoology, faculty of science, masaryk university, kotlářská 2, cz 611-37 brno, czech republic abstract – galium divaricatum pourr. ex lam. is an annual plant species occurring mainly in dry grasslands. the centre of its distribution range is situated in the mediterranean region, while in central europe it has been known only from a few isolated sites in slovakia and hungary. in 2016 we discovered this species in open dry grassland of the class sedo-scleranthetea on the upper edge of a basaltic andesite quarry near the village of siltse, irshavskyi district, zakarpatska region, as a new species for the ukrainian flora. the site is situated in the transitional region between the pannonian basin and the eastern carpathians. the new locality represents the northernmost recent occurrence of this species. keywords: carpathians, mediterranean floral element, pannonian basin, phytogeography, sedo-scleranthetea, zakarpatska region * corresponding author, e-mail: pavenow@seznam.cz introduction galium divaricatum pourr. ex lam. is a member of the galium parisiense group, which contains annual species of open dry habitats. besides g. divaricatum, two other species of this group occur in central europe, g. parisiense and g. tenuissimum. all three of these species are sparsely distributed and occur mainly in the pannonian basin and in the warm foothills of the carpathians (paucă 1961, řehořek 2007, bartha et al. 2015). of this group, only g. tenuissimum has been known in ukraine (mosyakin and fedoronchuk 1999), where it is limited to crimea (yena 2012). this paper focuses on g. divaricatum which was discovered in the zakarpatska region (w ukraine) as a new plant for the ukrainian flora in june 2016. galium divaricatum was described in france and is considered to be a mediterranean element of the european flora. the centre of its distribution range is in the near east and southern europe. it is distributed in the eastern mediterranean basin including lebanon, syria, turkey and cyprus and in the whole of the balkan (e.g. paucă 1961, ehrendorfer and schönbeck-temesy 1982) and italian (pignatti 1982) peninsulas. it is also known in southern france (royer and tison 2014). the westernmost localities of its distribution range are located in the iberian peninsula (ortega olivencia and devesa 2007). g. divaricatum is also reported as a neophyte in various regions of the world (lipscomb and nesom 2007). there are several records of g. divaricatum in central europe (fig. 1). it is known only in the south-eastern fig. 1. distribution map of galium divaricatum in central europe based on literature records (circles) and the new finding in ukraine (asterisk). novák p., zukal d. 194 acta bot. croat. 77 (2), 2018 part of this region. its northern distribution limit has been assumed to occur in southern slovakia (řehořek 2007) and western hungary (bartha et al. 2015). there are historical records from volcanic bedrock at the southern edge of the western carpathians in slovakia. it has been reported there in five sites near the villages of blhovce, fiľakovské kováče, plášťovce, šiatorská bukovinka and tešmák (řehořek 2007). the second site represents its northernmost occurrence in its native distribution range (48°17' n). however, it is currently supposed to be an extinct species of the slovak flora (eliáš et al. 2015). there are also several localities mentioned in romanian transylvania (paucă 1961, pop and hodişan 1964). nevertheless, it has not been known in ukraine until now (cf. mosyakin and fedoronchuk 1999), when it was found in the western part of the country (zakarpatska region). the zakarpatska region has been investigated by botanists for more than one hundred years. however, their research (e.g. thaisz 1911, stojko et al. 1982) has focused mainly on the higher altitudes of this area and on a few sites with thermophilous vegetation in the volcanic carpathian foothills (fodor 1974, votkalchuk 2012). in contrast, the flora of the flat lowland part of the region and the lower volcanic hills has not been sufficiently studied, as is reflected by several recent findings of new vascular plant species in this area (e.g. felbaba-klushyna 2015). g. divaricatum prefers open nutrient-poor dry grassland vegetation developed on substrates of various ph (paucă 1961, royer and tison 2014). to our knowledge, the only phytosociological relevé with g. divaricatum recorded in central europe was published in the above mentioned paper from romanian transylvania (pop and hodişan 1964). it occurs there in dry grassland dominated by festuca valesiaca with the occurrence of several annual species indicating disturbance. this grassland probably belongs to the alliance festucion valesiaceae klika 1931. the aim of this study is to report the occurrence of g. divaricatum in ukraine, to describe the new site and to put this finding into a broader phytogeographical context. materials and methods the zakarpatska region is located in the transitional zone between the pannonian and carpathian biogeographical regions (fekete et al. 2016) which makes its position quite unusual for ukraine. its low-altitudinal south-western part, including the locality of g. divaricatum, belongs to the distinctive pannonian province of the forest-steppe geobotanical zone (didukh and shelyag-sosonko 2007). the mean annual temperature of the surroundings of the g. divaricatum site is around 9 °c and annual precipitation is around 800 mm (andó 1999). the climate supports the development of forest vegetation on zonal sites with deep soils on flysch or loess. therefore, remnants of dry grasslands are limited to small patches with shallow dry soils situated on sunny slopes of the volcanic hills that are scattered in the lower part of the region (fodor 1974). g. divaricatum was determined using identification literature (e.g. paucă 1961, ehrendorfer and schönbeck-temesy 1982, royer and tison 2014) and herbarium vouchers stored in the herbarium of masaryk university, brno, czech republic (brnu). the determination was confirmed by an expert in the g. parisiense group vladimír řehořek (masaryk university, brno). the herbarium specimens of g. divaricatum from the new site have been deposited in brnu. the nomenclature of plants in the article follows euro+med plantbase (2006–). fig. 2. galium divaricatum (left) and its site in a basaltic andesite quarry (right) above the village of siltse near irshava taken from the south. the place in which the species occurs is indicated by an arrow (photo p. novák, 10 june 2016). galium divaricatum in ukraine acta bot. croat. 77 (2), 2018 195 results and discussion during phytosociological research into dry grassland vegetation near the village of siltse (irshavskyi district) on 10 june 2016, we discovered several dozen individuals of an annual plant species belonging to the galium parisiense group. it was subsequently recognised as g. divaricatum, a new species for the ukrainian flora (cf. mosyakin and fedoronchuk 1999). the new locality of g. divaricatum is a basaltic andesite quarry on a south-facing slope of a hill above the village of siltse (fig. 2). the hill is part of the vihorlat–gutâi volcanic belt (andó 1999). g. divaricatum grows on the upper edge of the quarry near a footpath to the hilltop (48°17'36.1" n, 22°59'17.6" e) at an altitude of 200 m. it was recorded in open grassland containing mainly thermophilous acidotolerant therophytes such as anthemis arvensis, arenaria serpyllifolia, bromus hordeaceus, filago arvensis, polycnemum arvense, scleranthus annuus and vulpia myuros. some perennial species occurred there as well, e.g. centaurea stoebe, chondrilla juncea and rubus sp. this temporary pioneer vegetation type may be classified within the class sedo-scleranthetea br.-bl. 1955. remnants of dry grasslands dominated by festuca valesiaca s.l. (class festuco-brometea br.-bl. et tüxen ex soó 1947) were developed on the hilltop above the quarry. however, g. divaricatum was not found in this vegetation. the soil ph of the rocky dry grassland near the site with g. divaricatum was 5.6 (measured in a distilled water suspension). the flora of the region contains many thermophilous species of both subcontinental (e.g. koeleria glauca and stipa pulcherrima) and sub-mediterranean (e.g. fraxinus ornus, quercus cerris, q. pubescens, ventenata dubia) floral elements. the co-occurrence of these elements is highly characteristic for the region (fodor 1974) as well as for the whole of the pannonian basin (fekete et al. 2016). therefore, the new occurrence of g. divaricatum fits well into the character of the regional flora. since the species is considered extinct in slovakia (eliáš et al. 2015), the newly discovered site in ukraine represents the northernmost outpost of its current occurrence. the origin of this newly discovered population is unclear. it cannot be ruled out that it was brought to the site by human activities; this hypothesis is supported by the fact that the species was not observed in the surrounding dry grassland vegetation during our field survey. nevertheless, it is considered to be a native species both in hungary (bartha et al. 2015) and slovakia (řehořek 2007). if the native status of the new population is confirmed, it should be classified as a critically endangered species (cr) of the ukrainian flora with the application of criteria b1a + b2ab (iii, v) of the iucn standards (2016). acknowledgements we would like to thank vladimír řehořek who kindly revised the herbarium specimen of galium divaricatum from ukraine, kryštof chytrý and kateřina píšťková for taking care of the field herbarium, martin večeřa for preparing the distribution map, lubov borsukiewicz and iveta škodová for providing some regional literature and milan chytrý and veronika kalníková for valuable comments on the manuscript. the study was supported by the czech science foundation (project 14-36079g). references andó, m., 1999: hydrogeographical features of the upper tisa river-system. tiscia 4, 135–172. bartha, d., király, g., schmidt, d., tiborcz, v., barina, z., csiky, j., jakab, g., lesku, b., schmotzer, a., vidéki, r., vojtkó, a., zólyomi, sz. (eds.), 2015: distribution atlas of vascular plants of hungary. nyugat-magyarországi egytem kiadó, sopron (in hungarian). didukh, ya. p., shelyag-sosonko, yu. r., 2007: vegetation. in: rudenko, l. h. (ed.), national atlas of ukraine, 440. dnvp cartography, kyiv. ehrendorfer, f., schönbeck-temesy, e., 1982: 6. galium l. in: davis, p. h. (ed.): flora of turkey and the east aegean islands, 767–849. edinburgh university press 7, edinburgh. eliáš, p., dítě, d., kliment, j., hrivnák, r., feráková, v., 2015: red list of ferns and flowering plants of slovakia, 5th edition (october 2014). biologia 70, 218–228. euro+med plantbase, 2006–: euro+med plantbase – the information resource for euro-mediterranean plant diversity. retrieved january 22, 2017 from http://ww2.bgbm.org/ europlusmed/. fekete, g., király, g., molnár, z., 2016: delineation of the pannonian vegetation region. community ecology 17, 114–124. felbaba-klushyna, l. m., 2015: utricularia intermedia (utriculariaceae), a new species for the flora of transcarpathia. ukrainskyi botanichnyi zhurnal 72, 237–240 (in ukrainian). fodor, s. s., 1974: flora of transcarpathia. vysha shkola, lviv (in ukrainian). iucn, 2016: iucn standards and petitions subcommittee. 2016. guidelines for using the iucn red list categories and criteria. version 12. prepared by the standards and petitions subcommittee. retrieved february 12, 2017 from http://www. iucnredlist.org/documents/redlistguidelines.pdf. lipscomb, b. l., nesom, g. l., 2007: galium anglicum (rubiaceae) new for texas and notes on the taxonomy of the g. parisiense/ divaricatum complex. journal of the botanical research institute of texas 1, 1269–1276. mosyakin, s. l., fedoronchuk, n. m., 1999: vascular plants of the ukraine. a nomenclatural checklist. kholodny institute of botany, national academy of sciences of the ukraine, kiev. ortega olivencia, a., devesa, j. a., 2007: 8. galium l. in: castroviejo, s. (ed.), flora iberica: plantas vasculares de la península ibérica e islas baleares, vol. 15, rubiaceae–dipsacaceae, 56– 162. real jardín botánico, madrid. paucă, a., 1961: fam. 100. rubiaceae b. juss. in: săvulescu t. (ed.), flora of the romanian people's republic, vol 8, 524–589. editura academiei republicii populare române, bucureşti (in romanian). pignatti, s., 1982: flora d’italia. 2. edagricole, bologna. pop, i., hodişan, i., 1964: floristic and vegetation studies of the craciunesti gorge (hunedoara region, brad district). studia universitatis babeş-bolyai seria biologia 1, 7–24 (in romanian). novák p., zukal d. 196 acta bot. croat. 77 (2), 2018 royer, j.-m., tison, j.-m., 2014: galium l. in: tison, j.-m., de foucault, b. (eds.), flora gallica. flore de france, 1020–1028. biotope, mèze. řehořek, v., 2007: g. divaricatum and galium tenuissimum in slovakia. bulletin slovenskej botanickej spoločnosti 29, 97– 98 (in slovak). stojko, s. m., tasjenkevych, l. o., milkina, l. i., malynovskyj, k. a., tretjak, p. r., manko, m. p. et al., 1982: flora and vegetation of the carpathian reserve. naukova dumka, kyiv (in ukrainian). thaisz, l., 1911: contribution to the flora of the bereg county. magyar botanikai lapok 10, 38–64 (in hungarian). votkalchuk, k. a., 2012: analysis of raritetly fitogenofond of volcanic carpathians. naukovyj visnyk uzhorodskoho universytetu seriya biologiya, vypusk 33, 52–63 (in ukrainian). yena, a. v., 2012: spontaneous flora of the crimean peninsula. n. orianda, simferopol. opce-str.vp acta bot. croat. 69 (1), 65–70, 2010 coden: abcra 25 issn 0365–0588 achene surface features in potentilla subarenaria borbás ex zimmeter and p. intermedia l. non wahlenb. (rosaceae) jeremi ko£odziejek university of £ódÿ, department of geobotany and plant ecology, banacha 12/16, 90-237 £ódÿ, poland achenes morphology in potentilla l., i.e. p. subarenaria borbás ex zimmeter and p. intermedia l. non wahlenb. was examined with stereoscope and scanning electron microscope (sem). achenes of these taxa varied in shape, size, colour, the surface sculpture and in the dimensions of dorsal ridge and ribs. sem analyses allowed distinguishing two morphological types of seed coats pattern: ruminate-reticulate sculpture due to well preserved epidermal cells in p. subarenaria and tuberculate sculpture in p. intermedia. the main taxonomic features of these two taxa are: the microstructure, size, shape and colour of achenes. key words: rosaceae, potentilla, achenes, sem introduction fruit in potentilla l. named also achene is dry, not dehiscent and monospermous, small in size. morphological description of achenes of selected species from the genus potentilla were given by many authors before: clark and fletcher (1909), bresinsky (1963), rousi (1965), ockendon and walters (1970), werner and soule (1976), leht (1989), soják (1987, 1993), andenberg (1994), ko£odziejek and gabara (2007). within this genus, kelley (1953) described the fruit morphology for 11 taxa and included a key for their determination, werner and soule (1976) described it in 3 taxa, anderberg (1994) in 28 taxa, leht (1989) in 24 taxa. however, among these authors, only leht (1989), ko£odziejek and gabara (2007) presented sem analysis of selected taxa of potentilla. it is known that fruit and seeds are very useful in identification and classification of plant taxa (murley 1951, karcz 1996, ko£odziejek and gabara 2007, özcan 2004, fagúndez and izco 2004). description of achenes in p. subarenaria and p. intermedia is limited to their length, shape and colour (wolf 1908, juzepczuk 1941, kelley 1953, szafer and paw£owski 1955, soják 1995). these features, however, are not sufficient to identify species. nobody has studied achenes of p. intermedia and p. subarenaria based on scanning electron microscope (sem); only andenberg (1994) examined both species acta bot. croat. 69 (1), 2010 65 * corresponding author, e-mail: kolo@biol.uni.lodz.pl u:\acta botanica\acta-botan 1-10\kolodziejek1.vp 9. travanj 2010 12:37:08 color profile: disabled composite 150 lpi at 45 degrees based on light microscopy (lm). this author described achenes of this taxa as medium in size between 0.8–1.3 mm long, 0.7–0.9 mm wide and 0.4–0.6 mm thick and medium buff and orange-brown in colour. many european authors consider p. intermedia as hybridogenous species, with p. argentea l. and p. norvegica, or even other allied species, as the parents. p. subarenaria is commonly interpreted as an apomictic species possibly of relatively recent hybrid origin from p. tabernaemontani ascherson and p. incana p. gaertner, b. meyer et scherb. (kurtto et al. 2004). the aim of the study is to present a morphological analysis of achenes in two potentilla species using a scanning electron microscope (sem) and to estimate the usefulness of shape, size and fruit surface for the taxonomy of investigated species. materials and methods nomenclature of potentilla subarenaria borbás ex zimmeter and p. intermedia l. non wahlenb. was used according to wolf (1908). plant material was obtained from natural populations collected by the author in the area of poland. analysis of qualitative and quantitative characteristics was performed on ripe, fully developed achenes. some achenes, even at the ripe stage, are distinctly smaller than typical ones and are also deformed – such achenes were deformed and distinctly smaller than typical ones and such achens were not measured. colour of the achenes was determined in the day light on the basis of colour scale recommended by berggren (1969). dimensions – length, width and thickness of the achenes, width of the aril and width and height of dorsal ridge were measured according to the description presented in figure 1. morphometric analysis of the achenes, except aril and rib 66 acta bot. croat. 69 (1), 2010 ko£odziejek j. fig. 1. achene of potentilla sp.: ventral (a) and lateral (b) view. 1 – length, 2 – width, 3 – thickness, 4 – dorsal ridge, 5 – aril around scare attachment, 6 – style scare. u:\acta botanica\acta-botan 1-10\kolodziejek1.vp 9. travanj 2010 12:37:10 color profile: disabled composite 150 lpi at 45 degrees dimensions, was made by a stereomicroscope nikon smz 800. at least 30 achenes from five plants were analyzed for each taxon. for scanning electron microscopy (sem) samples were mounted on metal stubs, sputtered with technical gold (pelco s.c 6 coating system), examined and photographed using a tesla bs 340 scanning electron microscope. shape of achenes, dimensions of aril and ribs as well as the seed surface pattern were analyzed on 5 photographs for each taxon. the carpological documentation includes sem figures which represent: outline of the achenes from the lateral side and the seed surface structure from the central part of the lateral surface. results achenes in subarenaria and p. intermedia are bilateral, shape ovate, laterally flattened, apex obtuse, curved, base obtuse (fig. 2). fruit thickness measured in a straight line perpendicular to the symmetry plane ranges from 0.61 (± 0.02) mm in p. subarenaria to 0.85 (± 0.02) mm in p. intermedia. the shape of fruit is described to some extent also by the elongation and flattening coefficients given in table 1. achenes are elongated, i.e. from 1.19 (p. intermedia) to 2.04 (p. subarenaria) times longer than wide. flattening of the achenes is usually bilateral, rarely the achenes are ± equally thick as wide. the most flattened are achenes of p. subarenaria (width/thickness ratio = 1.31), the least flattened ones – the achenes of p. intermedia (ratio = 1.18). two types of achenes are distinguished: large, present in p. subarenaria and small, present in p. intermedia (tab. 1). achene colour varies from red-brown in p. intermedia to nut-brown in p. subarenaria. scare attachment, i.e. the point of achene attachment to the receptacle is surrounded by an aril slightly visible in p. subarenaria or clearly – in p. intermedia, with their respective widths being 20 mm and 40 mm (tab. 2). a clear dorsal ridge about 40 mm wide and 40 mm thick was present in achenes of p. intermedia (tab. 2), while unclear one, about 10 mm wide and 10 mm thick was observed in p. subarenaria (fig. 2a left). ribs seen at achenes surfaces are brown in colour (tab. 1). distinct ribs, very sharp in shape were seen in achenes of p. intermedia (figs. 2a, b right), or oval in shape – in p. subarenaria (figs 2a, b left). width of ribs varied from 20 mm in p. subarenaria up to 80 mm in p. intermedia (tab. 2). similarly rib height was the lowest (20 mm) in p. subarenaria, while the largest (40–60 mm) was in p. intermedia. acta bot. croat. 69 (1), 2010 67 achene surface features in potentilla tab. 1. colour and dimension of achenes (in mm) in taxons of p. subarenaria borbás ex zimmeter and p. intermedia l. non wahlenb. taxon lengh width thicknes lengh/ width width/ thicknes colour achenes ribs p. intermedia 1.19±0.02 1.00±0.02 0.85±0.02 1.19±0.02 1.18±0.02 red-brown pale p. subarenaria 1.63±0.01 0.80±0.01 0.61±0.02 2.04±0.02 1.31±0.02 nut-brown brown u:\acta botanica\acta-botan 1-10\kolodziejek1.vp 9. travanj 2010 12:37:10 color profile: disabled composite 150 lpi at 45 degrees 68 acta bot. croat. 69 (1), 2010 ko£odziejek j. tab. 2. characteristics of achenes in potentilla subarenaria borbás ex zimmeter and p. intermedia l. non wahlenb. (sem). taxon aril dorsal ridge surface sculpture ribs (mm)width width thickness mm mm width heigth p. intermedia conspicuous 40 conspicuous 80 40 tuberculate 80 40–60 p. subarenaria iconspicuous 20 inconspicuous 10 10 ruminatereticulate 20 20 fig. 2. surface sculpture of the achenes of potentilla subarenaria borbás ex zimmeter (left a, b, c figures) and p. intermedia l. non wahlenb. (right a, b, c figures), at different magnifications; ´200 (a), ´1000 (b) and ´3000 (c). arrow indicates dorsal ridge. u:\acta botanica\acta-botan 1-10\kolodziejek1.vp 9. travanj 2010 12:37:11 color profile: disabled composite 150 lpi at 45 degrees based on the features of the arrangement of cells and cell outline, two types of seed coats pattern were identified: – ruminate-reticulate sculpture; among distinct and sharp ribs well preserved epidermal cells, hexagonal in shape were visible. seed coats of this type were characteristic of p. subarenaria (figs 2b, c left); – tuberculate sculpture, characterized by wrinkles and ridges irregular, most of them running in one direction. this type of seed coats was typical for p. intermedia (figs 2b, c right). discussion scanning electron microscopic analysis of achenes from p. subarenaria and p. intermedia allowed to distinguish new additional features such as aril, dorsal ridge and aril dimensions, useful in taxonomy of this taxa. these features of achenes in addition to the morphology of leaves (wolf 1908, juzepczuk 1941, ball et al. 1968, soják 1995) proved to be of high systematic importance in taxonomy of potentilla species. the most useful diagnostic characteristic in determination of p. subarenaria and p. intermedia is surface sculpture. my investigations in taxa from potentilla l., i.e. p. subarenaria and p. intermedia. revealed differences in colour and shape of achenes, as well as in their sizes. according to my measurements the lengths of achenes p. intermedia, 1.19 mm, were similar to those described by soják (1995) and andenberg (1994), 0.8–1.3 mm. the identification of immature achenes is hardly practical. many of the species whose achenes are prominently ridged or wrinkled when mature are completely smooth when young. on the other hand, the style scar is more likely to be easily located in young achenes, and in many species the style may still persist. sites and collection of p. subarenaria and p. intermedia: p. subarenaria – œl¹sk prov., góry towarne hills near kusiêta village (wy¿yna czêstochowska upland) 50°45’n/19°16’e, xerothermic grassland; p. intermedia – £ódÿ prov., nowosolna village near £ódÿ 51°42’n/19°37’e, gravel-pit; œl¹sk prov., olsztyn village near czêstochowa (wy¿yna czêstochowska upland) 50°46’n/ 19°16’e, roadside. acknowledgement the author would like to thank dr krzysztof polañski (institute of physics, university of £ódÿ) for taking the photographs. references andenberg, a. l., 1994: atlas of seeds and small fruits of northwest-european plant species with morphological descriptions. resedaceae-umbelliferae 4, 1–281. swedish museum of natural history, stockholm. acta bot. croat. 69 (1), 2010 69 achene surface features in potentilla u:\acta botanica\acta-botan 1-10\kolodziejek1.vp 9. travanj 2010 12:37:11 color profile: disabled composite 150 lpi at 45 degrees ball, p. w., paw£owski, b., walters, s. m., 1968: potentilla l. in: tutin, t. g., heywood, v. h., borgesm, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea 2, 36–47. cambridge university press, cambridge. berggren, g., 1969: atlas of seeds 2, 3–124. swedish natural scientific research council, stockholm. bresinsky, a., 1963: bau, entwicklungsgeschichte und inhaltsstoffe der elaiosomen. bibliotheca botanica 126, 1–54. clark, g. h., fletcher, j., 1909: farm weeds of canada. canada department of agriculture seed branch, ottava. fagundez, j., izco, j., 2004: seed morphology of calluna salisb. (ericaeae). acta botanica malacitana 29, 215–220. juzepczuk, s., 1941: potentilla l. in: shishkin, b. k., juzepczuk, s. v. (eds), flora urss (in russian) 10, 78–223. academiae scientarum urss, moskow, leningrad. karcz, j., 1996: scanning electron microscope in carpological studies. wiadomosci botaniczne 40, 55–65 (in polish). kelley, w. r., 1953: study of seeds identification and seed germination of potentilla app. and veronica spp. memoirs of the cornell university agricultural experiment station 317: 3–31. ko£odziejek, j., gabara, b., 2007: characteristic of achenes in potentilla collina group (rosaceae). acta societatis botanicorum poloniae 76, 35–42. kurtto, a., lampinen, r., junikka, l., (eds) 2004: atlas florae europaeae. distribution of vascular plants in europe. rosaceae (spiraea to fragaria, excl. rubus) 13, 195–203. the committee for mapping the flora of europe and societas biologica fennica vanamo, helsinki. leht, m., 1989: the genus potentilla in the baltic republics. leaf epidermis (in russian). proceedings of the estonian academy of scences, biology 38, 33–39. murley, m. r., 1951: seeds of the cruciferae of northeastern north america. american midland naturalist 46, 1–81. ockendon, d. j., walters, s. m., 1970: studies in potentilla anserina l. watsonia 8, 134–144. özcan, t., 2004: analysis of the fruit surface in bupleurum l. (umbelliferae) with sem. plant systematics and evolution 247, 61–74. rousi, a., 1965: biosystematic studies on the species aggregate potentilla anserina l. annales botanici fennici 2, 47–112. soják, j., 1987: notes on potentilla paradoxa and p. supina. preslia 59, 271–272. soják, j., 1993: taxonomische bemerkungen zu einigen mediterranen potenilla-sippen. preslia 65, 117–130. soják, j., 1995: potentilla l. (in czech). in: slavik, b. (ed.), flora of the czech republic 4, 283–314. academia, praha. szafer, w., paw£owski, b., 1955: potentilla l. in: szafer, w., paw£owski, b. (eds), flora of poland 7, 96–43. panstwowe wydawnictwo naukowe, warszawa. werner, p. a., soule, l. d., 1976: the biology of canadian weeds. 18. potentilla recta l., p. norvegica l. and p. argentea l. canadian journal of plant science 56, 591–603. wolf, t., 1908: monographiae der gattung potentilla l. bibliotheca botanica 16, 1–714. 70 acta bot. croat. 69 (1), 2010 ko£odziejek j. u:\acta botanica\acta-botan 1-10\kolodziejek1.vp 9. travanj 2010 12:37:11 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 69 (1), 93–105, 2010 coden: abcra 25 issn 0365–0588 trichome micromorphology in drupe of amygdalus l. (rosaceae) from iran mahnaz vafadar1*, farideh attar1, hosein maroofi2 1 central herbarium of university of tehran, school of biology, faculty college of science, university of tehran, po. box: 14155-6455, tehran, iran. 2 research center of agriculture and natural resources, kurdistan province, po. box: 66169-36311-714, sanandaj, iran. for delimitation of species and systematic resolution, pericarp indumentum in drupes of 17 species and two hybrids of the genus amygdalus l., representing two subgenera and two sections distributed in iran, were studied using scanning electron microscopy (sem) in order to assess whether in this genus, pericarp micromorphological characters are of taxonomic value. the pericarp indumentum type is velutinous with different density of trichomes. glabrous pericarp was observed solely in a. reticulata runemark ex khatamsaz. all trichomes are simple. two basic types of trichomes were distinguished on the surface of the pericarp: tubular and flattened. among these, tubular trichomes are more frequent than other trichomes in most species except for a. kotschyi boiss. et hohen., a. eburnea spach, a. spinosissima bge. subsp. spinosissima and a. lycioides spach var. lycioides. density of trichomes was variable among the studied taxa. in the first subgenus (subgen. amygdalus) with two sections, there was enough difference between sections. while density of trichomes in sect. spartioides spach was very low, most of species in the another section, sect. amygdalus showed dense pericarp indumentum. regarding density, the second subgenus, subgen. dodecandra (spach) browicz showed dense indumentum. two hybrids studied, a.´keredjensis browicz and a.´kamiaranensis khatamsaz et assadi showed different pericarp indumenta, dense and sparse respectively. in conclusion, micromorphological investigation of pericarp indumentum in drupes is a useful tool for distinguishing taxa in some cases, especially those of the two sections in the first subgenus in amygdalus. key words: amygdalus, pericarp, drupe, indumentum, micromorphology introduction the genus amygdalus l. with approximately 40–45 species in the world is distributed mainly in southwest asia, central asia and middle east, although a few species in this genus are distributed in the east of asia (china and mongolia) as well as southeast europe (browicz and zohary 1996). the main phytogeographical region for amygdalus distribution is the irano-touranian region. the genus amygdalus is classified in the tribe amygacta bot. croat. 69 (1), 2010 93 * corresponding author, e-mail: vafa@khayam.ut.ac.ir u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:23 color profile: disabled composite 150 lpi at 45 degrees daleae, subfamily spiraeoideae, former subfamily amygdaloideae (cronquist 1981, takhtajan 1997), in family rosaceae (potter et al. 2007). classification of amygdalus has changed over the course of time. in most classic classifications, amygdalus was considered as a subgenus or as a section in the genus prunus. for example, rehder (1940) considered five subgenera in prunus: prunus, amygdalus, cerasus, laurocerasus and padus (lee and wen 2001). this treatment is the most acceptable classifications in the world. phylogenetic classifications treated a new state for this genus. according to phylogeny and classification of rosaceae (potter et al 2007), amygdalus has been inserted within the genus prunus with armeniaca, cerasus, laurocerasus, padus, pygeum and maddenia in the tribe amygdaleae, subfamily spiraeoideae. in the flora of iran, in persian, amygdalus is treated as a single genus in the subfamily prunoideae. we say that there are sufficient morphological differences between amygdalus and prunus for them to be considered different genera. according to the study of browicz (1969), there are 15 species and two hybrids of amygdalus in iran. however, according to studies of khatamsaz (1992), in persian, 21 species and six hybrids of this genus occur in iran. among those, seven species, one variety and all the hybrids are endemic elements for the flora of iran (khatamsaz 1992). this genus includes two subgenera, subgen. amygdalus and subgen. dodecandra (browicz 1969, khatamsaz 1992). the former subgenus includes 15 species as trees or shrubs in two sections, sect. amygdalus and sect. spartioides spach. the second subgenus includes six species (khatamsaz 1992). regarding vegetative features, two sections of the former subgenus are different from each other as follows. the main morphological feature of sect. amygdalus is the presence of brachyblast (short shoots) whereas species in sect. spartioides lack brachyblast and exhibit junciform feature (these species include shoots that show them as juncus-like form). the second subgenus, subgen. dodecandra, is completely different from the first, the species in this subgenus being spiny and shrubby with thick spines (browicz 1969, khatamsaz 1992). in fact, this genus is one of the most important elements of the elburz and zagros mountains, steppes, rocky places and semi-arid habitats in iran. amygdalus is one of the most problematic genera of the family rosaceae. this genus involves numerous taxonomic problems including a high degree of morphological variation even in different populations of one species and interspecific hybridization; so boundaries between species are not clear and delimitation of species in this genus is not possible based on morphological characters alone. in addition, we need more evidence including micromorphological, anatomical, molecular and phytochemical information to elucidate the taxonomic complexity in this problematic genus. micromorphological studies are useful in resolving taxonomic problems because of high efficiency and reliability as well as the detailed data they provide. plant hairs (trichomes) are of great interest to descriptive and experimental botanists and data on these and indumenta are routinely included in many types of studies (juri[i] grube[i] et al. 2007). scanning electron microscopy (sem) provides useful data on surface indumentum ultrastructure of seed, fruit, leaf and stem. several researchers have focused on hair micromorphology of different families and genera including eugenia l., myrtaceae (fontenelle et al. 1994), ranunculus l., ranunculaceae (xuhan and van-lammeren 1994), cordia l. and onosma l., boraginaceae (rapisarda et al. 1997, akcin 2007), rosaceae and sanguisorba l. (dowidar et al. 2003, latif 2004), astragalus l. and arbus adanson, fabaceae (zarre 2003, agbagwa and okoli 2006), verbascum l., scrophula94 acta bot. croat. 69 (1), 2010 vafadar m., attar f., maroofi h. u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:23 color profile: disabled composite 150 lpi at 45 degrees riaceae (attar et al. 2007), teucrium l. and nepeta l., lamiaceae (juri[i] grube[i] et al. 2007, kaya et al. 2007) and isatis l., brassicaceae (moazzeni et al. 2007). so far, micromorphology of pericarp indumentum in the drupe of amygdalus has not been studied and this study was conducted to investigate the pericarp indumentum of iranian species of amygdalus. regarding the drupe, among allied genera in the tribe amygdaleae, subfamily spiraeoideae, or as formerly classified in the subfamily amygdaloideae, amygdalus has a unique feature as its mesocarp is thin and completely dried after fruit maturation and together with pericarp is separated from the endocarp, which is stony with various surface ornamentations. generally, the surface of the pericarp is hairy in amygdalus. the main aims of this study are to document a pericarp micromorphology of iranian species of amygdalus and then to use this feature for evaluating taxonomic relationships in this genus. material and methods matured, dried and clean drupes of 17 species and two hybrids of iranian species of amygdalus were selected and their pericarp was removed. the specimens are deposited in the central herbarium of the university of tehran (tuh) and the voucher specimens are presented in table 1. a piece of pericarp was mounted on a 12.5 mm diameter aluminium stub with double sided adhesive and then was coated in a sputter coater with approximately 25 nm of gold-palladium at an accelerating voltage of 10–15 kv. the specimens were examined and photographed with leo sem-440i and tscan sem-vega mostly at magnifications of 300–400´. the terminology for indumentum description follows that of harris and harris (1994). results the main features of pericarp indumentum of the studied species of iranian amygdalus are presented in table 2 and sem micrographs in plates 1–3 (figs. 1–23). the pericarp indumentum type is velutinous. except for a. reticulata runemark ex khatamsaz with glabrous pericarp (fig. 11), all other examined taxa were hairy. the trichomes could be studied with respect to different aspects including density, shape and length. regarding density, trichomes were subdivided into four subtypes: dense, dense-sparse, sparse and very sparse. all trichomes are simple but two basic types of shape were found in the pericarp: tubular and flattened. trichome length differs among the studied species. the length of tubular trichomes showed a diverse range from approximately 100–200 mm in a. communis l. and a. carduchorum bornm. to more than 1000 mm in a. korshinskyi (hand.-mazz.) bornm., a. haussknechtii (c. k. schneider) bornm. var. pubescens bornm., a. lycioides spach var. horrrida (spach) browicz and a. ´kamiaranensis khatamsaz et assadi. the length of flattened trichomes was in the range of 100–150 mm in a. ´kamiaranensis to an approximately 800–900 mm or more in a. kotschyi boiss. et hohen., a. lycioides spach var. lycioides and then followed by a. arabica spach, a. spinosissima bge. subsp. spinosissima. tubular trichomes were more frequent than other trichome in most species except for a. kotschyi (subgen. amygdalus) and the three studied taxa of subgen. dodecandra (spach) browicz including a. eburnea spach, a. spinosissima bge. subsp. spinosissima and a. lycioides var. lycioides (figs. 8, 17, 19, 21). acta bot. croat. 69 (1), 2010 95 trichome micromorphology in drupe of amygdalus u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:23 color profile: disabled composite 150 lpi at 45 degrees 96 a c t a b o t .c r o a t .69 (1),2010 v a f a d a r m .,a t t a r f .,m a r o o f ih . tab. 1. voucher specimens of iranian species of amygdalus used in the drupe pericarp indumentum study collectors collection data species attar, maroofi & zamani iran, kurdistan: ca. 30 km to ghorveh, 36323-tuh a. arabica olivier attar, maroofi & zamani iran, kurdistan: kamiaran to marivan, takht-zangi village, 36333-tuh a. communis l. maroofi & naseri iran, kurdistan: marivan, dezli, 6285-kurdistan herbarium a. carduchorum bornm. mobayen iran, fars: road of nourabad, 8621-tuh a. eburnea spach attar & zamani iran, fars: pass after dasht-e ardzan, 36286-tuh a. elaeagnifolia spach subsp. elaeagnifolia attar & zamani iran, east azerbayjan: after kaleibar, 37212-tuh a. fenzliana (fritsch) lipsky attar & zamani iran, fras: kotal pir-e zan, 36299-tuh a. glauca browicz attar, maroofi & zamani iran, kurdistan: ca.15 km to kamiaran, morvarid pass, 36330-tuh a. haussknechtii (c. k. schneider) bornm. var. pubescens bornm. attar, maroofi & zamani iran, kurdistan: dezli pass, 36343-tuh a. korshinskyi (hand.-mazz.) bornm. attar, maroofi & zamani iran, kurdistan: ca. 30 km from saqqez to baneh, nakarouz mountain, 363673-tuh a. kotschyi boiss. et hohen. mobayen iran, tehran: kiasman, fasham mountain, 19425-tuh a. lycioides spach var. horrida (spach) browicz attar & zamani iran, esfahan: fereidoonshahr, vahdatabad village, pishkooh mountain, 36319-tuh a. lycioides spach var.lycioides attar & zamani iran, east azerbayjan: after ahar to tabriz, 37219-tuh a. nairica fed. et takht. attar, khatamsaz & sheikh iran, fars: shiraz, bamou national park, 20390-tuh a. reticulata runemark ex khatamsaz attar & zamani iran, fars: dasht-e ardzan, 36285-tuh a. scoparia spach ghahreman, attar, okhovvat & mehdigholi iran, khorasan: mashhad to torbat-e heidariyeh, robat-e sang, 27289-tuh a. spinosissima bge. subsp. spinosissima attar, maroofi & zamani iran, kurdistan: takht-zangi village, 36331-tuh a. trichamygdalus (hand.-mazz.) woronow mirtadzadini iran, kerman: bam, mij, 37820-tuh a. wendelboi freitag attar, maroofi & zamani iran, kurdistan: ca. 35 km from sanandaj to kamiaran, 36322-tuh a. ´ kamiaranensis khatamsaz et assadi rieben iran, tehran: karaj, koohdashteh, 8618-tuh a. ´ keredjensis browicz u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 0 \ v a f a d a r . v p 9 . t r a v a n j 2 0 1 0 1 3 : 1 4 : 2 3 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s amygdalus haussknechtii var. pubescens contained only tubular hairs (fig. 7). in three studied taxa including a. kotschyi, a. arabica and a. lycioides var. lycioides, flattened trichomes were folded (figs. 8, 12, 13, 21). most studied species of sect. amygdalus (subgen. acta bot. croat. 69 (1), 2010 97 trichome micromorphology in drupe of amygdalus plate 1. sem micrographs of drupe pericarp indumentum of iranian species of amygdalus. 1, 2 – a. communis, 3 – a. trichamygdalus, 4 – a. wendelboi, 5 – a. korshinskyi, 6 – a. fenzliana, 7 – a. haussknechtii var. pubescens, dense tubular trichomes, 8 – a. kotschyi, frequent flattened trichomes. u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:28 color profile: disabled composite 150 lpi at 45 degrees amygdalus) except for a. reticulata contained indumentum with dense trichomes including a. communis l., a. trichamygdalus (hand.-mazz.) woronow, a. wendelboi freitag, a. korshinskyi, a. fenzliana, a. haussknechtii var. pubescens, a. kotschyi and a. elaeagnifolia spach subsp. elaeagnifolia (figs. 1, 3, 4, 5, 6, 7, 8, 10) but in a. carduchorum, indumentum was dense-sparse (fig. 9). in contrast to the former section, three species of sect. spartioides spach showed very sparse indumentum with tubular and flattened trichomes (figs. 12, 15, 16). the density of trichomes was very low and trichomes were very sparse. examined species of subgen. dodecandra including a. spinosissima subsp. spinosissima, a. 98 acta bot. croat. 69 (1), 2010 vafadar m., attar f., maroofi h. tab. 2. features of pericarp indumentum of iranian species of amygdalus. abbreviations: f. t. – flattened trichomes; t. t. – tubular trichomes. species trichome density tubular trichomes flattened trichomes dominance of trichomes subgen. amygdalus sect. amygdalus a. communis dense + + t. t. a. trichamygdalus dense + + t. t. a. wendelboi dense + + t. t. a. korshinskyi dense + + t. t. a. fenzliana dense + + t. t. and f. t. a. haussknechtii var. pubescens dense + – t. t. a. kotschyi dense + + f. t. a. carduchorum dense-sparse + + t. t. a. elaeagnifolia subsp. elaeagnifolia dense + + t. t. a. reticulata glabrous – – – sect. spartioides a. arabica very sparse + + both rare a. glauca very sparse + + both rare a. scoparia very sparse + + both rare subgen. dodecandra a. spinosissima subsp. spinosissima dense + + f. t. a. eburnea dense + + f. t. a. nairica dense + + t. t. and f. t. a. lycioides var. horrida dense + + t. t. and f. t. a. lycioides var. lycioides dense + + f. t. hybrids a. ´keredjensis dense + + t. t. a. ´kamiaranensis sparse + + both few abbreviations: f. t.: flattened trichomes; t. t.: tubular trichomes. u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:28 color profile: disabled composite 150 lpi at 45 degrees eburnea, a. nairica and a. lycioides (both varieties) showed dense indumentum (figs. 17, 18, 19, 20, 21). two varieties of a. lycioides showed different portions of trichomes as follows: in a. lycioides var. horrida, both tubular and flattened trichomes were frequent while in another variety, var. lycioides, flattened trichomes were more frequent (figs. 20, 21) acta bot. croat. 69 (1), 2010 99 trichome micromorphology in drupe of amygdalus plate 2. sem micrographs of drupe pericarp indumentum of iranian species of amygdalus. 9 – a. carduchorum, 10 – a. elaeagnifolia subsp. elaeagnifolia, 11 – a. reticulata, showing the glabrous pericarp, 12–14 – a. arabica, 15 – a. glauca, 16 – a. scoparia. u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:33 color profile: disabled composite 150 lpi at 45 degrees (tab. 2). two studied hybrids, a.´keredjensis and a.´kamiaranensis showed different indumentum types, dense and sparse respectively (figs. 22, 23). worthy of note, these two hybrids showed different details of trichomes, as well (tab. 2). 100 acta bot. croat. 69 (1), 2010 vafadar m., attar f., maroofi h. plate 3. sem micrographs of drupe pericarp indumentum of iranian species and hybrids of amygdalus. 17 – a. eburnea, 18 – a. nairica, 19 – a. spinosissima subsp. spinosissima, 20 – a. lycioides var. horrid, 21 – a. lycioides var. lycioides, 22 – a. ´keredjensis, 23 – a.´kamiaranensis. u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:38 color profile: disabled composite 150 lpi at 45 degrees discussion in the present study, we studied pericarp indumentun in the drupe of iranian amygdalus. trichome micromorphology has been the subject of several studies for taxonomic goals in different plant families which include trichomes in various parts especially leaf, stem, and seed-fruit coat (fontenelle et al. 1994, xuhan and van-lammeren 1994, zarre 2003, agbagwa and okoli 2006, akcin 2007, attar et al. 2007, juri[i] grube[i] et al. 2007, kaya et al. 2007, moazzeni et al. 2007). reports that have assessed hair micromorphology of the family rosaceae are rare. these studies deal with surface of seed and achene coat that is mostly glabrous and possesses different seed coat pattern including reticulate, faviulariate, ruminate, coolleculate, honey-combed, different cell wall features and different shapes of epidermal cells (dowidar et al. 2003, latif 2004). in the following sections, we will summarize the pericarp indumentum of the drupe of iranian taxa of amygdalus. subgen. amygdalus a. sect. amygdalus in this section, there are two vegetative groups regarding habit, tree (three species) and shrub (nine species). tree species are: a. communis, a. trichamygdalus and a. wendelboi (khatamsaz 1992). the first two species are distributed in the west and northwest of iran but the third, a. wendelboi, an endemic species for the flora of iran (khatamsaz 1992), has a distribution range far from the two former localities and is found in the south of iran. a. communis also has been found in some other areas including turkey (browicz 1972), russia (shishkin and yuzepchuk 1941) and china (lu and bartholomew 2003). amygdalus trichamygdalus is also distributed in turkey (browicz 1972). regarding pericarp indumentum micromorphology, except the length of tubular trichomes, longer in a. trichamygdalus than in the two closely related species, these three species show similarities and homogeneity in pericarp trichomes including dense indumentum and frequent tubular trichomes (figs. 1, 3, 4). it seems that in this subgroup, different ecological conditions including semi-humid to semi-arid environments in the west and northwest iran in zagros region and arid environments in the south iran do not affect the indumentum density of the pericarp. this finding is in contrast to findings concerning the indumentum of other plant parts including leaf or stem showing that their trichomes are dependent on habitat conditions; in arid environments, the indumentum is dense for adaptation to severe conditions (zarrinkamar 1993, zarrinkamar and dinarvand 2006). nine shrubby species are included in sect. amygdalus (khatamsaz 1992). among them, seven species were investigated for pericarp indumentum study including a. korshinskyi (west and northwest iran), a. fenzliana (northwest iran), a. haussknehtii var. pubescens (west and central iran), a. kotschyi (west and northwest iran), a. carduchorum (west and northwest iran), a. elaeagnifolia subsp. elaeagnifolia (central and south iran) and a. reticulata (south iran) (browicz 1969, khatamsaz 1992). among those, three species including a. haussknechtii, a. elaeagnifolia and a. reticulata, are endemic species for the flora of iran (browicz 1969, khatamsaz 1992). some species of this subgroup are found in neighboring countries especially in turkey (browicz 1972). regarding pericarp indumentum, this shrubby subgroup is the most heterogeneous group with emphasis on indumentum type, density and portion of trichomes (tab. 2). except for a. reticulata from acta bot. croat. 69 (1), 2010 101 trichome micromorphology in drupe of amygdalus u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:38 color profile: disabled composite 150 lpi at 45 degrees the south of iran with glabrous pericarp (fig. 11), other species have dense or dense-sparse indumentum (figs. 5, 6, 7, 8, 9, 10). a. haussknechtii var. pubescens and a. korshinskyi possessed the longest tubular trichomes among the studied species of this subgroup (figs. 5, 7) and a. kotschyi possessed the longest flattened trichomes (fig. 8). in a. korshinskyi and a. fenzliana, two related species, we observed a number of differences (tab. 2, figs. 5, 6). also, two closely related species (according to morphological evidence) including a. kotschyi and a. carduchorum exhibit extreme difference regarding to density and portion of trichomes as follows: as against most of the species, in a. kotschyi with dense indumentum, the flattened hairs were long and more frequent (fig. 8), while in a. carduchorum, the tubular trichomes were short and more frequent (fig. 9). it is worthy of mention that pericarp indumentum in this species is dense-sparse. pericarp indumentum heterogeneity is observed in the last closely related species of this subgroup including a. elaeagnifolia and a. reticulata. in a.reticulata, the pericarp was completely glabrous, a unique feature among the studied species (fig. 11) but in a. elaeagnifolia subsp. elaeagnifolia, it is dense (fig. 10). worthy of note, like the findings in former subgroup, in this subgroup, different ecological conditions could not affect the pericarp indumentum and as seen above, most species from the west and northwest iran and south iran exhibit dense indumentum except for the a. reticulata. as discussed above, as against the first subgroup in subgen. amygdalus, our findings of pericarp indumentum show considerable variation in this shrubby subgroup even between closely related species. it is interesting that this subgroup is the most problematic subgroup in the genus amygdalus from a morphological point of view. a. sect. spartioides this section includes three junciform species: a. arabica, a. glauca and a. scoparia. the main morphological feature of this section is the absence of brachyblast (short shoots) (browicz 1969, khatamsaz 1992). among those, a. glauca is an endemic element for the flora of iran (browicz 1969, khatamsaz 1992). amygdalus arabica is mainly distributed in the west of iran as well as turkey (browicz 1972). amygdalus scoparia is distributed in many different localities in iran except for the west and northwest and is one of the most widespread species of amygdalus in iran. it is found in russia as well (shishkin and yuzepchuk 1941). amygdalus glauca is found in a limited region in the south of iran in fars province (browicz 1969, khatamsaz 1992). regarding pericarp indumentum, there was significant difference between this and the former section. in contrast to sect. amygdalus with dense indumentum in most studied species, all species in sect. spartioides exhibited very sparse indumentum with more or fewer similarities (figs. 12, 15, 16). subgen. dodecandra this subgenus is completely different from the former regarding its habit feature, because in this subgenus there are six shrubby species with thick spiny shoots (browicz 1969, khatamsaz 1992). species in this subgenus are found in different ecological conditions in the west, northwest, east, northeast, central, north and south iran. some of them are found in the neighboring countries (shishkin and yuzepchuk 1941, browicz 1969, 1972). from six species in this subgenus, we studied four species. among them, a. lycioides is widely distributed in iran. in all studied species, the pericarp indumentum was dense (figs. 17, 18, 19, 20, 21). studied species in this subgenus show differences in pericarp trichomes in shape and trichome portions (tab. 2). two varieties of a. lycioides exhibit significant difference. a. lycioides var. lycioides possessed the longest flattened trichomes and a. 102 acta bot. croat. 69 (1), 2010 vafadar m., attar f., maroofi h. u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:38 color profile: disabled composite 150 lpi at 45 degrees lycioides var. horrida the longest tubular trichomes (figs. 20, 21). while in a. lycioides var. horrida, an endemic variety for the flora of iran (khatamsaz 1992), both tubular and flattened trichomes were frequent (fig. 20), in another variety, var. lycioides, flattened trichomes were more frequent and they were folded as well (fig. 21). according to results in this subgenus, the density of trichomes in the pericarp is independent of ecological conditions. hybrids among amygdalus hybrids, two hybrids were examined for pericarp indumentum study. both hybrids are endemic elements for the flora of iran (khatamsaz 1992). these two hybrids show different features regarding to pericarp trichomes (tab. 2). amygdalus ´keredjensis from the north of iran showed dense indumentum. tubular hairs were more frequent (fig. 22). amygdalus ´kamiaranensis, a hybrid from the west of iran, showed sparse indumentum (fig. 23). also, amygdalus ´kamiaranensis, together with a few other species, possessed the longest tubular trichomes. amygdalus ´ keredjensis is a hybrid resulting from a hybridization process between a. scoparia and a. lycioides (browicz 1969, khatamsaz 1992). comparison of pericarp features between this hybrid and its parents showed that regarding pericarp indumentum, a. ´ keredjensis was related to a. lycioides (figs. 20, 22). also according to khatamsaz (1992), a. ´ kamiaranensis is a hybrid between a. haussknectii and a. scoparia, but based on our studies on iranian amygdalus, two other species are its parents: a. arabica and a. lycioides, because a. ´ kamiaranensis is a hybrid that found in the west of iran but a. scoparia is found in different localities in iran except for the west and northwest. the presence of thin spiny shoots in this hybrid shows that one of its parents is a spiny species from the second subgenus as well; so a. haussknechtii could not be another parent. khatamsaz’ hypothesis is based on morphological evidence as is the hypothesis presented in this paper. unfortunately, she did not introduce the exact parents because she did not consider the distribution of parent species and morphological features exactly, whereas evidence for the present hypothesis was shown above. we have collected numerous specimens of this hybrid from the distribution localities in the west iran. amygdalus scoparia is not distributed in the west of iran and based on presence of thin spiny shoots in this hybrid, one spiny species should be another parent. unfortunately there are no crossing studies about these hybrids in iran. a molecular phylogenetic study on iranian species and hybrids of amygdalus is currently being undertaken. perhaps this investigation will be able to throw light on the real parents of a. ´ kamiaranensis. a comparison of pericarp results only showed some similarities to a. lycioides (figs. 20, 23). conclusion with an emphasis on pericarp indumentum micromorphological findings, it is clear that in some cases, these results confirm classic classifications in the genus amygdalus. for example, two sections of subgen. amygdalus were easily distinguished from each other. but two subgenera were not separated according to pericarp indumentum results. within sect. amygdalus there was enough variation even between two closely related species in the shrubby subgroub such as a. elaeagnifolia and a. reticulata. regarding our findings in this study, it is clear that the density of trichomes in pericarp is not dependent on habitat condiacta bot. croat. 69 (1), 2010 103 trichome micromorphology in drupe of amygdalus u:\acta botanica\acta-botan 1-10\vafadar.vp 9. travanj 2010 13:14:39 color profile: disabled composite 150 lpi at 45 degrees tions. as there has been no molecular phylogenetic study about iranian species of amygdalus and since molecular investigations on genus prunus s. l. include only a few species of amygdalus, with only one species being found in iran (lee and wen 2001, bortiri et al. 2001, wen et al. 2008), the phylogenetic importance of micromorphological studies could not be evaluated here. acknowledgements we are grateful to dr. khezri in the research center of agriculture and natural resources in kurdistan province. also we thank mrs. eshghi in the scanning electron microscopy division of islamic azad university, science and research branch and mr. rahmani in razi metalorgy research institute for providing photos. this investigation received support from the research council at the 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abstract – the genus eucalyptus occurs in a wide range of environmental conditions, including rainforests, subalpine, arid/semi-arid and moist temperate zones. it includes species with the capacity to cope with extremely low water potential. this study aims to screen water stress tolerance in two eucalyptus species under nursery conditions. inter-specific variation in morphological, physiological, biochemical and molecular parameters in two eucalyptus species (e. tereticornis and e. camaldulensis) with contrasting levels of tolerance to progressive short term water-deprived condition was evaluated. water stress reduced growth measured in terms of root:shoot ratio and specific leaf area (sla), photosynthetic parameters, leaf water potential and relative water content (rwc) in both genotypes. biochemical parameters including total sugars, phenol, phytohormones (indole acetic acid and abscisic acid) and proline were found to significantly increase during stress in both genotypes. water responsive transcripts like osmotin and dreb/cbf registered significant expression variation in the two genotypes, suggesting their key role in regulating water stress tolerance in eucalyptus. keywords: drought stress, leaf temperature, osmotin, sugars, transcription factors * corresponding author e-mail: ghoshm@icfre.org introduction the genus eucalyptus with over 900 species forms a dominant canopy of woodland ecosystems (boland et al. 2006). the ecological range of eucalyptus species encompasses high rainfall, temperate, semi arid and extreme arid conditions (merchant et al. 2006). several species belonging to the subgenus symphyomyrtus are adapted to prolonged water deficit conditions, determining the species distribution (adams 1996). eucalypts are widely introduced as a plantation crop due to their rapid growth, adaptability and superior wood properties for the paper and pulp industries. eucalyptus grandis, e. urophylla, e. globulus, e. camaldulensis, and their hybrids account for 80% of plantations globally. seven species, e. camaldulensis, e. grandis, e. globulus, e. pellita, e. tereticornis, e. urophylla and corymbia citriodora, were reported to be suitable for indian agro-climatic conditions and are widely planted in the subcontinent (kallarackal and somen 1997, kallarackal et al. 2002). the high potential productivity of eucalyptus has resulted in the deployment of genetically improved planting stock in mono-specific plantations to maximize harvesting performance (forrester et al. 2010). however, this practise renders the plantations vulnerable to abiotic and biotic stresses that affect establishment and growth (white et al. 2009). further, the potential productivity in eucalypts is governed by stem growth, biomass allocation and water availability (stape et al. 2008). response to water-deprived conditions in eucalyptus species has been extensively studied. significant changes in osmotic potential, gas-exchange parameters, cell turgor adjustment (white et al. 1996), stomatal function (costa e silva et al. 2004), chlorophyll and carotenoid contents (michelozzi et al. 1995) in leaf tissues are reported during water-deprived conditions. other parameters used to assess water stress tolerance included changes in leaf temperature, photosynthetic rate, leaf water potential, and transpiration rate (dye 1996, amrutha s., parveen a.b.m., muthupandi m., sivakumar v., nautiyal r., ghosh dasgupta m. 126 acta bot. croat. 78 (2), 2019 roden and ball 1996, rolando and little 2003, stokes 2004). concurrently, reduction in plant growth (chaves et al. 2003, bedon et al. 2012), photosynthesis (warren et al. 2007) and hormone production (granda et al. 2011) have also been reported during water stress conditions. the institute of forest genetics and tree breeding, coimbatore, india initiated its program on the genetic improvement of eucalyptus in 1997 in association with csiro, australia. productive clones with superior pulping quality were tested under multi-environment conditions and subsequently released and deployed in plantation programs. in the present study two eucalyptus species were evaluated for their response to water deficit conditions and variations in growth, physiological, biochemical and transcript expression patterns were documented to identify key parameters governing water stress tolerance in the species studied. materials and methods plant material the two species used in the present study are second generation clones derived from the eucalyptus breeding program implemented by the institute of forest genetics and tree breeding, coimbatore, india. eucalyptus tereticornis (et88) was selected from the seed production area established and maintained by the institute at pudukottai, tamil nadu, india. eucalyptus camaldulensis (ec226) is of katherine, northern territory, provenance and was selected from the seed orchard at satyavedu, andhra pradesh, india, which was established and maintained by the institute. the selection of the genotypes in the present study was based on a preliminary phenotypic screening of 60 clones for water stress tolerance (data not shown). eucalyptus camaldulensis (ec226) was used as the susceptible genotype, while e. tereticornis (et88) served as the tolerant genotype. experiment design and water stress imposition coppice cuttings of both species were rooted and maintained in polybags containing a soil mixture of pallam soil, river sand and red earth in the ratio of 2:1:0.5. the cuttings were fortified with hoagland’s media (hoagland and arnon 1950) supplemented to its field capacity (fc) (30 ml) on alternate days for a period of 5 days prior to being subjected to water stress conditions. subsequently, six month old cuttings were transferred to a controlled environmental chamber and maintained at 32 ± 3 °c with relative humidity at 70–80 ± 5%. fifteen ramets of each genotype were used for the study in a randomized design. six ramets per genotype were subjected to a well-watered (ww) regime wherein the cuttings were watered to 100% fc every day, while nine ramets were subjected to progressive water stress (ws) regime. the stress treatment was initiated by complete water withdrawal for a period of 15 days and the soil moisture content was recorded everyday using a tensiometer. on day 6 when soil moisture content was 0.005 ± 0.0011 mpa, the susceptible genotype ec226 showed symptoms of leaf curling and hence all parameters except root:shoot ratio were assessed on days 5 and 6 after stress imposition. the root:shoot ratio was documented on day 15 after the imposition of water stress treatment with soil moisture content of 0.0215 ± 0.0017 mpa. all parameters were recorded in three randomly selected ramets and used as biological replicates. physiological parameters root:shoot ratio analysis of clones subjected to ww and ws conditions was conducted on oven dried samples after completion of the experiment on day 15 post stress imposition. specific leaf area (sla) was determined from scanned images of 4–5 leaves and the area was determined using image j software (schindelin et al. 2015). digital images of leaves (with ruler) were uploaded in the software and the scale was fixed using the ‘set scale’ option in the tool. subsequently, under the option ‘image’, colour threshold was adjusted to shade the leaf area followed by measuring the area using the wand tool. sla was calculated as area divided by dry weight determined by oven drying of leaf material at 45 °c for 2 days and represented as cm–2 g–1 leaf dry weight (ldw). the leaf surface temperature and chlorophyll content were measured on day 6 after initiation of stress, since complete leaf curling was recorded after day 6 of stress imposition in the susceptible genotype ec226. temperature was manually recorded in mature leaves each day between 11.00 a. m. – 12.00 p. m. using an infrared thermometer (gaby instruments, india) at 3 zonal levels of the plant (apex, midlevel and basal zone). the ambient temperature of the chamber was also recorded. three fully expanded leaves of each ramet were selected for estimating the chlorophyll content. the chlorophyll content index (cci) was recorded using the chlorophyll concentration meter mc-100 (apogee instruments inc., ut, usa) for both the ww and ws regime. photosynthetic rate (a), stomatal conductance (gs), intercellular co2 concentration (ci) and transpiration rate (e) were measured using an lci-sd photosynthesis system (adc bioscientific ltd. uk). measurements were taken between 10 a. m. to 12 p. m. in mature leaves on day 6 after imposition of stress in both ww and ws plants. relative water content of the leaf (% rwc) was determined from detached leaf segments of 1 – 2 cm length and their fresh weight (fw) was recorded on day 6 after imposition of water stress. the leaf segments were placed in deionised water for a period of 4 h and turgid weight (tw) was recorded. subsequently, leaves were dried at 60 °c overnight and dry weight was determined (dw). rwc was estimated according to morgan (1984) using the formula 100 × (fwdw)/(tw-dw) . mid-day leaf water potential (ψw) was assessed from a single mature leaf from three different ramets using the psy1 stem psychrometer (ict international, australia) on day 5 response of eucalyptus species under water stress condition acta bot. croat. 78 (2), 2019 127 after imposition of stress. the psychrometer chamber was vacuum fixed over the non-cuticularized leaf surface and water potential was measured in mpa. biochemical parameters leaf tissues from both clones subjected to ww and ws conditions were assayed for total phenols, flavonoids, total sugars, proline content, free aba and iaa content. leaves were collected from three ramets each for both conditions on day 6 after imposition of water stress and processed independently for biochemical estimation. fifty mg of leaf tissues were ground in liquid nitrogen and subsequently used for biochemical assay. total phenols were estimated using the protocol described by malik and singh (1980), while total sugars were quantified by anthrone method (hedge and hofreiter 1962). total flavonoids were estimated by aluminium chloride method (kiranmaiet et al. 2011). the phytohormones, aba and iaa were estimated using the protocol described by unyayar et al. (1996), while leaf proline was determined using the procedure described by ábrahám et al. (2010). expression profiling of water stress responsive transcripts total rna was isolated from the leaves of ww and ws plants of both genotypes on day 6 of stress imposition. single leaves from three independent ramets were harvested for rna isolation and maintained as three biological replicates. rna was isolated using rnaqueous®-micro total rna isolation kit (thermo scientific, usa). the concentration and purity of rna were evaluated using nanodrop nd-1000 uv–vis spectrophotometer (thermo scientific, usa). approximately 1 µg of total rna was treated with dnase i and used to synthesize the first strand cdna with revertaid mmulv reverse transcriptase (thermo scientific, usa) following the manufacturer’s protocol. in the present study, 12 drought responsive transcripts were selected according to their role in water stress response. transcripts from hormone signaling pathways (ar, ger3, lea6); osmoprotectant pathways (gols2, sip, osm34); sucrose pathway (tppb); antioxidant (gpx6); membrane intrinsic protein (pip) and drought related transcription factors (dreb, cbf1c, cbf2) were selected. transcript selection was based on an earlier study in e. grandis (ghosh dasgupta and dharanishanthi 2017). the expression of peg induced water stress responsive transcripts were analysed by quantitative real time (qrt-pcr ; on-line suppl. tab. 1). qrtpcr was performed using abi prism 7500 step one plus sequence detection system (applied biosystems, usa) using the following program: one cycle of 95 °c for 10 min; 40 cycles of 95 °c for 15 s and 60 °c for 1 min. the qrt-pcr reactions (10 µl) included 200 ng of cdna, 5.0 ml sybr green jumpstarttaq readymix (sigma, st. louis, tab. 1. effect of short-term progressive water stress on different parameters in eucalyptus tereticornis (et88) and eucalyptus camaldulensis (ec226). data presented are mean of triplicate values ± sd (standard deviation). ww – well watered condition; ws ‒ water stress condition. sla ‒ specific leaf area; ldw ‒ leaf dry weight; cci ‒ chlorophyll content index; rwc ‒ relative water content; aba ‒ abscisic acid; iaa ‒ indole acetic acid; fw ‒ fresh weight. clone id et 88 ec 226 treatments ww ws ww ws sla (cm–2 g–1 ldw) 46.56±9.28 45.09±7.99 47.04±1.50 43.47±4.58 root : shoot ratio 0.42±0.06 0.45±0.08 0.76±0.4 0.98±0.2 leaf temperature (°c) 30.70±0.70 31.78±0.39 30.52±0.69 31.60±0.31 cci 11.12±1.94 13.36±1.69 9.63±0.98 15.61±1.50 rwc (%) 82.78±2.68 78.79±3.50 76.09±3.35 40.07±5.64 ψw (mpa) –2.34±0.57 –4.25±0.05 –1.68±0.26 –2.94±0.08 transpiration rate (mmol m–2 s–1) 0.85±0.09 0.80±0.12 0.82±0.14 0.42±0.07 stomatal conductance (cm s–1) 0.11±0.01 0.10±0.02 0.13±0.03 0.06±0.02 photosynthetic rate (µmol m–2 s–1) 22.39±4.84 17.18±1.71 21.21±11.8 8.39±3.46 total carbohydrate (mg g–1 fw) 2.04±0.23 5.28±1.65 4.23±3.05 9.04±1.59 flavonoids (mg g–1 fw) 7.83±1.58 11.31±2.69 11.28±0.43 10.01±1.19 phenols (mg g–1 fw) 0.40±0.14 0.97±0.43 0.67±0.01 1.13±0.18 proline (μmol g–1 fw) 4.86±1.83 9.03±3.89 3.56±0.10 8.44±5.76 aba (mg g–1 fw) 0.04±0.01 0.09±0.06 0.03±0.01 0.06±0.00 iaa (mg g–1 fw) 0.29±0.18 0.60±0.08 0.24±0.15 0.50±0.03 amrutha s., parveen a.b.m., muthupandi m., sivakumar v., nautiyal r., ghosh dasgupta m. 128 acta bot. croat. 78 (2), 2019 reduction in rwc in ws condition when compared to et88 (tab. 1). mid-day leaf water potential (ψw) was also found to reduce in ws condition in both et88 and ec226 when compared to ww condition (tab. 1). the photosynthetic parameters including transpiration rate, stomatal conductance and photosynthesis were recorded and a decrease in ws conditions in both genotypes was registered (tab. 1). however, the decrease documented in ec226 for transpiration rate, stomatal conductance and photosynthetic rate was high as compared to et88 (tab. 1). biochemical parameters six biochemical parameters including total sugars, flavonoids, phenols, proline, aba and iaa content were recorded after 6 days of water stress imposition. all parameters were found to increase under ws condition in both genotypes except flavonoid content. increase in total sugars was documented in clone ec226 in ws condition as compared to the ww condition. a similar trend was observed in et88 (tab. 1). a decrease in the flavonoid content was recorded in ec226 under ws condition, while it increased in et88 (tab. 1). significant differences across species and treatments were found in nested anova for each measured parameter, at least in one of the factors (treatment or genotype or both) (tab. 2). leaf temperature and total phenol content were significantly different only for the treatment facmo, usa), 200 nm each of forward and reverse primer and 0.4 µl of 20 mg ml–1 bsa. all reactions were conducted in three biological replicates. the melting curve was generated to ensure product specificity and to differentiate between the product and primer dimer. actin related protein 3 (arp3) was used as reference gene for normalization of data (karpaga raja sundari and ghosh dasgupta 2012). the relative quantification value (rq) was calculated using the formula 2–δδct (livak and schmittgen 2001) and the fold decrease was calculated as the reciprocal of the fold change (peirson et al. 2003). statistical analyses all parameters were determined for three independent ramets and the data was represented as mean ± sd. statistical analysis for morphological, physiological and biochemical parameters was performed using statistica version 7.0. the effect of species, treatment and their interaction on all parameters was tested using nested anova. ramets were nested within treatment and treated as random effect in analyses, while the water stress treatment and species were considered fixed factors. the post hoc test with bonferroni correction was conducted when an effect was found to be significant. in transcript expression studies, delta ct for ww and ws conditions across species were statistically analyzed using t-test implemented in graphpad prism® version 7 and the difference between the conditions were considered statistically significant when p < 0.05. results the effect of water stress on two species of eucalyptus was evaluated and the variations in growth, physiological, biochemical and transcript expression were recorded in ww and ws conditions. physiological parameters percent leaf curling was recorded in both genotypes under ww and ws conditions. in tolerant genotype et88, 5.71% ± 0.48 leaf curling was documented, while susceptible genotype ec226 recorded 96% ± 2.71 leaf curling under ws conditions (fig. 1). the root:shoot ratio was variable between the genotypes and ec226 showed a decrease in shoot growth upon stress imposition, while no difference was registered in et88 (tab. 1). sla was recorded in both genotypes under ww and ws conditions and a marginal decrease was documented in et88, while a decrease in sla was recorded in ec226 under ws conditions when compared to ww condition (tab. 1). leaf temperature showed a marginal increase under ws condition in both genotypes tested, while cci was found to increase in ec226 under ws condition when compared to ww condition. an increase in cci in clone et88 was also recorded (tab. 1). percent rwc was found to vary in both genotypes subjected to ws condition, where ec226 showed a fig. 1. effect of progressive water stress in six month old rooted cuttings of (a) eucalyptus tereticornis (et88) and (b) eucalyptus camaldulensis (ec226); ww ‒ well watered condition; ws ‒ water deprived condition. response of eucalyptus species under water stress condition acta bot. croat. 78 (2), 2019 129 tor, whereas significant differences for the species were recorded for root:shoot ratio. significant differences in rwc, leaf water potential and total sugars content were found for both treatment and species. expression profiling of water stress responsive transcripts relative quantification (rq) of twelve transcripts related to water deficit condition was determined using qrt-pcr in both genotypes. osmotin (osm34), dehydration responsive element binding proteins (dreb), c-repeat/dre-binding factor (cbf1c and cbf2), glutathione peroxidase (gpx6), and raffinose synthase family protein (sip) showed significant up-regulation in et88 when compared to ec226 (fig. 2). maximum expression was recorded for osm34 with et88 registering a 54-fold increase compared to the 5-fold increase in ec226. further, ec226 showed down regulation of all the transcripts profiled except osm34 (6.31) and sip (2.10). down regulation of cbf2 (-22-fold), cbf1c (-11-fold) and dreb (-10-fold) was documented in ec226 when compared to their expression level in et88 which was up-regulated 20-fold, 28-fold and 15-fold, respectively (fig. 2). discussion a growing concern in commercial plantation programs has been the depletion in water resources and area of cultivation. managing tree plantations to maximize wood production from available water resources is the critical parameter in tree breeding programs. in eucalyptus, the concept of plantation water productivity (pwpwood) is gaining importance in the provision of social license for wood production from plantations (white et al. 2014). hence, several species of eucalypts have been screened for their water stress tolerance and variations in response to low water potential are reported among species (myers and neales 1986) among provenances (li 1998) and between clones (pita and pardos 2001). the interand intraspecific variation in response to changes in osmotic potential has been used as a selection criterion in genetic improvement programs in eucalypts (van der moezel et al. 1991, lemcoff et al. 1994). the imposition of a water-deprived condition elicits multi-dimensional responses at morphological, physiological, biochemical and molecular levels (granda et al. 2014). in eucalyptus species and their hybrids, adjustments during water stress conditions are mediated by hormonal balance (granda et al. 2011), variation in pigment content (shvaleva et al. 2006, 2008), osmotic changes (merchant et al. tab. 2. statistical analysis of some morphological, physiological and biochemical parameters, in eucalyptus tereticornis (et88) and eucalyptus camaldulensis (ec226) exposed to water stress, across treatment and species. df ‒ degrees of freedom; ss ‒ sum of squares; ms ‒ mean of squares; f ‒ f value. level of significance: *p < 0.01, **p < 0.05. parameter species treatment leaf temperature df 1 2 ss 0.11 3.40 ms 0.11 1.70 f 0.61 9.18* rwc df 1 2 ss 1428.41 2043.93 ms 1428.41 1021.96 f 816.91* 583.95* leaf water potential df 1 2 ss 2.90 7.80 ms 2.90 3.90 f 28.47* 3.905* total carbohydrates df 1 2 ss 2658.55 5036.65 ms 2658.55 2518.32 f 7.24** 6.85** phenols df 1 2 ss 7.79 124.199 ms 7.79 62.099 f 0.58 4.61** root : shoot df 1 2 ss 0.562 0.076 ms 0.562 0.038 f 8.32** 0.56 fig. 2. expression profiling of water stress responsive transcripts in leaves of eucalyptus tereticornis (et88) and eucalyptus camaldulensis (ec226). values are mean of data from three biological replicates and the error bars represent sd. gene ids are given in on-line suppl. tab. 1. asterisk indicates statistical significance between well watered and water stressed plants when p < 0.05. amrutha s., parveen a.b.m., muthupandi m., sivakumar v., nautiyal r., ghosh dasgupta m. 130 acta bot. croat. 78 (2), 2019 2006, warren et al. 2007, callister et al. 2008), photosynthesis adjustment (warren et al. 2007) and growth modifications (drew et al. 2009). in general, water stress reduces growth rate in all species by modifying the biomass allocation in above and below ground tissues, which determines the morphological plasticity of the genotype (maseda and fernández 2016). in eucalypt species, reduction in growth and biomass under water stress conditions is reported in e. globulus (costa e silva et al. 2004, correia et al. 2014, coopman et al. 2008), e. camaldulensis (maseda and fernández 2016) and e. microtheca (li 1998). in the present study, the genotype (et88) showed marginal reduction in sla and root:shoot ratio during stress imposition, while ec226 documented significant reduction in both parameters, revealing that the tissue biomass was significantly reduced in ec226 during stress conditions. the photosynthetically active leaf is the plant part that is critically affected by water limitation, which causes reduction in leaf emergence/expansion and shrinkage/curling (burling et al. 2013, scoffoni et al. 2014). the maintenance of balance between the areas involved in transpiration and absorption is a key determinant of survival and productivity as reported in e. globulus (marron et al. 2003, costa e silva et al. 2004). hence, reduction in leaf area during water stress condition is a response in all eucalypt species as reported in e. globulus (costa e silva et al. 2004, maseda and fernández 2016), e. camaldulensis (lemcoff et al. 2002, maseda and fernández 2016) and e. microtheca (susiluoto and berninger 2007). specific leaf area (sla), which essentially describes the distribution of leaf biomass relative to area, is a major trait that correlates with whole plant growth (cheng et al. 2016). it links carbon allocation and water status and thus is a critical parameter to assess soil moisture content (pierce et al. 1994). in a recent study by wellstein et al. (2017) in grasses and forbs from temperate and sub-mediterranean ecosystems, it was hypothesized that phenotypic adjustments like sla show variable response during short-term water stress and reduced sla was associated with enhanced water use efficiency, while increased sla indicated better growth performance. hence, it was concluded that the differential response of sla during drought conditions had to be interpreted according to the evolutionary history and environmental niche of the species. in agreement with this hypothesis, sla was reported to decrease in e. globulus (costa e silva et al. 2004, coopman et al. 2008) and increase in e. microtheca (susiluoto and berninger 2007) subsequent to stress imposition. in another study, no significant difference in sla was reported in either e. camaldulensis or e. globulus seedlings during water stress (maseda and fernández 2016). in the present study, decrease in sla was documented in both e. camaldulensis and e. tereticornis. leaf relative water content (rwc) is a key indicator of plant water status since it reflects the balance between water availability and transpiration rate (lugojan and ciulca 2011). the general trend of decrease in rwc during water-deprived conditions is documented in several eucalypt species including eucalyptus camaldulensis (maseda and fernández 2016), e. globulus (pita and pardos 2001), e. oblique, e. camaldulensis, e. rubida, e. polyanthemos, e. tricarpa, e. cladocalyx (merchant et al. 2007) and eucalyptus grandis × e. urophylla (valadares et al. 2014). in the present study, a decrease in rwc was recorded in both genotypes but the level of reduction in ec226 was significantly higher than in the clone et88. accumulation of solutes like inorganic cations, organic acids, carbohydrates and free amino acids in response to water stress is generally reported in most plant species. they contribute to osmotic adjustments, thus maintaining a balance between sucrose synthesis and translocation (morgan 1984, akinci and lösel 2012). accumulation of soluble sugars in water deprived condition was reported in eucalyptus species (quick et al. 1992, warren et al. 2011). it was also reported that e. camaldulensis and e. globulus from a ‘mesic’ environment underwent osmotic adjustments through increased sugar accumulation, while species from dry (‘xeric’) environments adjusted through increased concentration of quercitol (merchant et al. 2006). in agreement with the earlier report, significant increase in the accumulation of total sugars was documented in both species, which occupy the ‘mesic’ environment. leaf/canopy temperature is an indicator of stomatal closure in response to soil water stress (jones et al. 2009, 2002). during water stress condition, closure of stomata to maintain inner moisture content is an immediate response, resulting in increased leaf temperature, decreased transpiration and reduced photosynthesis, which adversely affect productivity (luquet et al. 2003, jones 2004, feller 2016). leaf temperature, water status and stomatal opening are closely connected (valladares and pearcy 1997, feller 2006, reynolds-henne et al. 2010, feller and vaseva 2014, teskey et al. 2015) and hence determination of leaf/canopy temperature as a measure of stomatal conductance using infrared thermometry was suggested as early as the 1980s (jarvis 1981) to facilitate irrigation scheduling. it was reported that maintenance of lower leaf surface temperature in tolerant genotypes during stress conditions could lead to favourable water status, sustained transpiration and increased photosynthetic efficiency, resulting in improved yields (kumar 2005). using leaf temperature as drought index has been reported in potato (gerhards et al. 2016), maize (carroll et al. 2017), sorghum (blume et al. 1978), wheat (fischer et al. 1998, talebi 2011), sugarcane (silva et al. 2007), rice (hirayama et al. 2006) and firmiana platanifolia (yu et al. 2015). however, in woody perennials leaf surface temperature as water stress index is of limited use. in a recent study on pinus taeda and populus deltoides × nigra, it was reported that leaf temperature had a direct effect on stomatal opening and an increase in leaf temperature was documented under water deprived condition in both genus irrespective of leaf morphology, xylem structure and physiology (urban et al. 2017). the effect of leaf temperature on stomatal limitation to photosynthesis, intercellular co2 concentration and net photosynthesis was demonstrated in the study. similarly, an increase in leaf temperature was documented in both species in the present study. response of eucalyptus species under water stress condition acta bot. croat. 78 (2), 2019 131 transcriptional regulation of different pathways to maintain cellular homeostasis during water stress is well documented in plant systems (reviewed by kaur and asthir 2017). several drought responsive genes including dehydrins, molecular chaperones, water channel proteins, transporters and biosynthetic enzymes for compatible solutes, growth regulators, enzymes that detoxify reactive oxygen species (bartels and sunkar 2005, wang et al. 2006), transcription factors, protein phosphatases, phospholipid metabolic proteins and protein kinases (shinozaki and yamaguchi-shinozaki 2007, morran et al. 2011) are reported to play a pivotal role in cellular protection and stress alleviation. in eucalyptus, extensive studies on morpho-physiological and biochemical response to water stress condition is reported, while molecular response is limited to transcriptome-wide response in e. globulus and e. cladocalyx (spokevicius et al. 2017), e. camaldulensis (thumma et al. 2012), e.alba hybrid and e. urophylla × e. grandis (villar et al. 2011), and e. grandis (ghosh dasgupta and dharanishanthi 2017). osmotin and osmotinlike proteins (olps) are known to act as osmotic modulators by compartmentalizing solutes or regulating structural and metabolic alterations (chowdhury et al. 2017). biosynthesis of osmotin/olps was reported to depend on water status of cells, revealing their key role in osmotic adaptations (weber et al. 2014, patade et al. 2013). in transgenic systems, the role of osmotin as an osmoprotectant gene has been extensively reported in crops (reviewed by khan et al. 2015, das et al. 2011). expression of osmotin during water deficit condition is reported in e. cladocalyx (spokevicius et al. 2017) and e. grandis (ghosh dasgupta and dharanishanthi 2017). in the present study, a significantly high expression of osmotin (osm34) was registered in the clone et88 (54-fold) as compared to ec226 (fivefold), indicating that level of osmotin can act as an indicator in screening water-responsive genotypes. the role of transcription factors during dehydration stress has been extensively reviewed in crop plants (nakashima et al. 2014, singh and laxmi 2015). in eucalyptus, the major transcription factors which regulate water stress include zinc finger transcription factor family protein (villar et al. 2011), myb, nac, erf, hb12 (thumma et al. 2012) and hsfs, dreb2a, dear3 (ghosh dasgupta and dharanishanthi 2017). the aba independent dehydration responsive element binding/c-repeat binding factors (dreb/cbf) belonging to the ethylene responsive factor/apetala2 (erf/ ap2) family (shinozaki and yamaguchi-shinozaki 2007) are major regulators governing drought response (agarwal and jha 2010). genome-wide analysis in e. grandis revealed the presence of 17 dreb1/cbf genes and 6 dreb2. further, it was reported that dreb1/cbf was cold-inducible, while dreb2 was induced during water stress (cao et al. 2015). in another study, ectopic expression of egucbf1a⁄b from e. gunnii in a eucalyptus hybrid conferred drought and freezing tolerance (navarro et al. 2011). an earlier study in e. globulus also revealed that drought tolerant genotypes exhibited better cold acclimation due to the accumulation of soluble sugars (costa e silva et al. 2009). in the present study, downregulation of cbf2, cbf1c and dreb in the clone ec226, in contrast to their significant up-regulation in et88, revealed the crucial role of dreb/cbf in conferring drought tolerance in eucalypts. in a similar study in hevea, a strong correlation between fold expressions of dreb with drought tolerance was reported by luke et al. (2015). in the present study, several morpho-physiological, biochemical and molecular parameters were assessed to document their response during progressive water stress in two species of eucalypts with contrasting levels of tolerance. leaf temperature, total phenol, root:shoot ratio, rwc, leaf water potential and total sugars showed significant variations across the two genotypes. water responsive transcripts like osmotin and dreb/cbf registered significant expression variation in the two genotypes, indicating their key role in regulating water stress tolerance. hence, the reported physiological (leaf temperature, rwc, leaf water potential and root:shoot ratio), biochemical (total sugars) and molecular (osmotin and dreb/cbf) parameters can be used as an explanatory variables in determining the selection of eucalypts under progressive short term water stress in nursery condition. acknowledgements the authors acknowledge department of biotechnology, government of india for funding the research work (project no. bt/pr10539/pbd/16/1064/2013). funding support in the form of research fellowships was provided to sa, amp and mm by the department of biotechnology, government of india. references ábrahám, e., hourton-cabassa, c., erdei, l., szabados, l., 2010: methods for determination of proline in plants. methods in molecular biology 639, 317–331. adams, m. a., 1996: distribution of eucalypts in australian landscapes: landforms, soils, fire and nutrition. in: attiwill, p.m., adams, m.a. 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efficient wood production in eucalyptus globulus plantations. forest ecology and management 331, 272–280. yu, m.h., ding, g.d., gao, g.l., zhao, y.y., yan, l., sai, k., 2015: using plant temperature to evaluate the response of stomatal conductance to soil moisture deficit. forests 6, 3748–3762. 154 acta bot. croat. 76 (2), 2017 acta bot. croat. 76 (2), 154–162, 2017 coden: abcra 25 doi: 10.1515/botcro-2016-0054 issn 0365-0588 eissn 1847-8476 physiological responses of crop plants against trichoderma harzianum in saline environment roomana yasmeen, zamin shaheed siddiqui* stress physiology phenomics centre, department of botany, university of karachi, karachi 75270, pakistan abstract – the physiological response of crop plants against trichoderma harzianum (th-6) in a saline habitat was studied. trichoderma harzianum (th-6) is an endophytic fungus that shows salt tolerance and establishes a symbiotic relationship with a host plant. to evaluate the role of trichoderma harzianum (th-6) in mitigating the consequences of salinity stress on crop plants, seeds of maize and rice were coated with trichoderma before sowing and salt treatment. later, after germination, twenty-one day old seedlings were subjected to nacl concentrations (50, 100 and 150 mm). salinity negatively affected all investigated physiological parameters in both crops. treatment of seeds with trichoderma improved plant growth and th-treated plants exhibited substantial physiological adjustment in a saline environment compared to th-untreated plants. the th-treated plants under salt stress showed higher relative water content and stomatal conductance, better photosynthetic performance and higher pigment concentrations, as well as higher catalase and superoxide dismutase activities. moreover, proline content in salt stress environment was higher in th-treated plants, while h2o2 content declined. the physiological role of trichoderma harzianum in mitigating the salt related consequences of both crop plants is discussed. keywords: antioxidant enzymes activity, maize, physiological performance, rice, salinity, trichoderma harzianum * corresponding author, e-mail: zaminss@uok.edu.pk introduction plants are often subjected to unfavorable changes in their environment. abiotic stresses play a major role in reducing crop production around the globe (bybordi 2012). among them, salinity is one of the most important abiotic stresses that are widely distributed in both irrigated and non-irrigated areas of the world. research on salinity in plants has produced a vast literature showing its negative influence on crop plant productivity (mahajan and tuteja 2005, oliveira et al. 2013). most salt sensitive crops cannot tolerate a high concentration of nacl, especially in the soil (prasad et al. 2000). this results in poor germination, growth and biomass allocation (neumann 2008, ahmad and prasad 2012). studies have shown that plants at vegetative and reproductive phases are more sensitive to soil salinity (hu and schmidhalter 2005, lauchli and grattan 2007, siddiqui et al. 2014). prolonged exposure to salinity causes specific ion toxicity, nutritional and hormonal imbalance, reduced water potential and so on (ahmad et al. 2010, siddiqui and khan 2013) trichoderma sp are beneficial endophytic plant symbionts that are widely used as biocontrol agents against fungal diseases in crop plants (harman 2011, afzal et al. 2013). however, some studies have reported that trichoderma induces tolerance to biotic and abiotic stresses in plants (monnet et al. 2001, evelin et al. 2009, mastouri et al. 2010, shoresh et al. 2010, estrada et al. 2013). treatment of seed with trichoderma spp in many cereals and vegetable crops has a positive impact on plant growth, improving hormone performance (howell 2003, harman 2006), which could enhance tolerance to salinity stress (gachomo and kotchoni 2008, rawat et al. 2011, hashem et al. 2014). however, the role of trichoderma harzianum in crop plant tolerance to abiotic stress like salinity and drought and the physiological mechanism involved needs to be closely monitored. therefore, the present study was designed to examine the physiological responses of two crops plants, maize and rice, in a saline environment following seed treatment with trichoderma harzianum (th-6). some physiological attributes of salt tolerance were selected for this study. physiological responses of crops against endophytes acta bot. croat. 76 (2), 2017 155 materials and methods seed selection seeds of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel were obtained from the department of plant protection, karachi, pakistan. seeds were surface sterilized in 10% sodium hypochlorite solution for 3 minutes and rinsed thoroughly with distilled water then air dried. culture collection and treatment the pure strain of trichoderma harzianum (th-6) was obtained from the plant pathology laboratory, department of botany. first, an experiment was conducted in petriplates containing potato dextrose agar at different nacl concentrations (25, 50, 100, 150, 200 mm) together with a trichoderma harzianum (th-6) disc for 8 days. the salt concentration was selected according to the optimum growth achieved by trichoderma harzianum in saline media (fig. 1). seeds of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel were treated with trichoderma harzianum using 2% gum arabic as sticker. the colony forming unit (cfu) was 67.3 conidia 10–3 of trichoderma. later, seeds were sown on a pot filled with 500 g soil each (one plant per pot). autoclaved (1 hour at 80 °c) soil was used for the experiment with the following composition: sand particles; 80.5, silt; 7.1, clay; 8.1, organic carbon; 0.20, nitrogen. ph 7.5 and ec 1.8 ds m–1 were recorded according to dahnke and whitney (1988) by a cmd 500 wpa conductivity meter, linton cambridge u.k). maize and rice crops were allowed to grow at an average daynight temperature (26±4 °c and 18±3 °c). salt stress was applied to twenty-one-day old seedlings each day by using 25 mm nacl to achieve the desired level (gorham et al. 1987) and moisture contents were maintained with tap wa-and moisture contents were maintained with tap water. three nacl concentrations (50, 100 and 150 mm nacl) were applied. plant treated with tap water served as control. each treatment and control was replicated four times. root, shoot length, biomass, physiological parameters and antioxidant enzyme activities were examined. relative water content four leaf strips of 4×2 cm2 from the mid-veins and the edge section of leaves were cut with scissors from each treatment of rice and 4 1.2 cm2 discs of maize were excised and fresh weights (fw) were determined. for the measurement of turgid weight (tw), leaves were left in distilled water for 24 h under low irradiance condition. samples were then dried at 80 °c for 48 h in oven and dry weight (dw) was determined. relative water content (rwc) was calculated by the fresh leaf sample method described by barrs and weatherley (1962) and modified as: rwc=(fw–dw/tw–dw)×100 quantum yield psii and stomatal conductance measurements of chlorophyll fluorescence emission from the 20 randomly selected leaves were monitored with a fluorescence monitoring system (handy pea) in the pulse amplitude modulation mode. a leaf adapted to dark conditions for 30 min using leaf clips, was initially exposed to the modulated measuring beam of far-red light (led source with typical peak at wavelength 735 nm). the original (f0) and maximum (fm) fluorescence yields were measured under weak modulated red light (0.5 μmol m–2 s–1) with 1.6 s pulses of saturating light (6.8 μmol m–2 s–1 par). the variable fluorescence yield (fv) was calculated by the equation fm–f0. the ratio of the variable to maximum fluorescence (fv/fm) was calculated as the dark-adapted quantum yield of psii photochemistry and performance index and non-photochemical quenching were calculated as described by maxwell and johnson (2000). likewise, the stomatal conductance (gs) of 20 randomly selected leaves of each treated and control plant was examined using a leaf porometer (model sc-1, decagon). photosynthetic pigment extraction and estimation leaf samples (0.5 g) were ground in 10 ml of 96% methanol and then centrifuged at 4000 rpm for 10 min. total chlorophyll [chl(a+b)], chlorophyll a (ca), and chlorophyll b (cb) contents were determined according to lichtenthaler (1987). the supernatant was separated and the absorbances were read at 666, 653 and 470 nm on spectrophotometer. the amount of these pigments was calculated according to the following formulas: ca=15.65×a666–7.340×a653 cb=27.05×a653–11.21×a666 cx+c=1000×a470–2.860×ca–129.2×cb/245 where, ca – chlorophyll a, cb – chlorophyll b, cx+c – total carotenoids chlorophyll contents of leaf tissues were expressed as µg mg−1 fw. proline content proline content was measured according to the procedure of bates et al. (1973). leaf samples (0.5 g) were homogenized with 5 ml sulphosalicylic acid (3% w/v) and the homogenate was filtered on whatman no. 1 filter paper. fig. 1. trichoderma harzianum was cultured in potatoes dextrose agar containing 100 mm nacl. yasmeen r., shaheed siddiqui z. 156 acta bot. croat. 76 (2), 2017 then, 2 ml of extract in test tube was taken and 2 ml of glacial acetic acid and 2 ml of acid ninhydrin where added. the mixture was heated in a boiling water bath at 100 °c for an hour. a brick red color developed. after cooling of the reaction mixture, 4 ml toulene was added and mixed vigorously for 15 to 20 seconds. chromophore containing toluene was separated from the aqueous phase. then the mixture was allowed to reach room temperature. the absorbance was recorded at 520 nm against a toluene blank. proline content in sample was estimated by referring to a standard curve made from known concentrations of proline by taking following formula: µmol proline g–1fw=(µg proline/ml×ml toulene)/ /115.5 µg/µmol)/(g sample)/5 h2o2 production hydrogen peroxide content was measured according to the procedure of velikova et al. (2000). freshly harvested leaf samples (100 mg) were homogenized with 3 ml of 0.1% (w/v) trichloroacetic acid in an ice bath and the homogenate was centrifuged at 12,000 g for 15 min. later, 0.5 ml of 10 mm phosphate buffer (ph 7.0) and 1 ml of 1 m potassium iodide (ki) were added to 0.5 ml of the supernatant. the absorbance of the supernatant was read at 390 nm. the amount of h2o2 was calculated using a standard curve and expressed as µmol g–1 fw. enzyme assays leaf samples (500 mg) were crushed and homogenized in 10 ml protein extraction buffer containing tris-hcl ph 6.8, 50 mg polyvinylpyrrolidone and 0.05 mm ethylenediaminetetraacetic acid (edta). whole contents were centrifuged at 12,000 rpm for 10min in a smart r-17, hanil centrifuge. the supernatant was collected and used to determine the activities of catalase and superoxide dismutase. total protein was estimated by the method of bradford (1976). catalase (cat; ec 1.11.1.6) activity was estimated by method of patterson et al. (1984). the decomposition of h2o2 was measured at 240 nm taking δε as 43.6 mm cm–1. the reaction mixture (3.0 ml) consisted of 10.5 mm h2o2 in 0.05 m potassium phosphate buffer (ph 7.0) and the reaction was initiated after the addition of 0.1 ml enzyme extract at 25 °c. the decrease in absorbance at 240 nm was used to calculate the activity. one unit of cat activity is defined as the amount of enzyme that catalyzes the conversion of 1 mm of h2o2 min–1 at 25 °c. superoxide dismutase (sod; ec 1.15.1.1) activity was recorded according to the method of beyer and fridovich (1987). the reaction mixture consisted of 27.0 ml of 0.05 m potassium phosphate buffer (ph 7.8), 1.5 ml of l-methionine (300 mg per 2.7 ml), 1.0 ml of nitrobluetetrazolium salt (14.4 mg per 10 ml), and 0.75 ml of triton x-100. aliquots (1.0 ml) of this mixture were delivered into small glass tubes, followed by the addition of 20 ml enzyme extract and 10 ml of riboflavin (4.4 mg per 100 ml). the cocktail was mixed and then illuminated for 15 minutes in an aluminum foil-lined box, containing 25 w fluorescent tubes. in a control tube the sample was substituted for by 20 ml of buffer and the absorbance was measured at 560 nm. the reaction was stopped by switching off the light and placing the tubes in the dark. increase in absorbance due to the formation of formazan was measured at 560 nm. under the described conditions, the increase in absorbance in the control was taken as 100% and the enzyme activity in the samples were calculated by determining the percentage inhibition per minute. one unit of sod is the amount of enzyme that causes a 50% inhibition of the rate for reduction of nitrobluetetrazolium salt under the conditions of the assay. statistical analysis statistical analysis was carried out using the personal computer software packages spss version 20. all data were subjected to spss and two-way anova was performed. 1 1 2 3 fig.1. trichoderma harzianum was cultured in potatoes dextrose agar containing 100 mm 4 nacl. 5 0 50 100 150 0 50 100 150 r o o t (c m ) 5 10 15 20 without trichoderma with trichoderma nacl mm s h o o t (c m ) 0 10 20 30 40 r o o t (c m ) 2 4 6 8 10 12 14 16 without trichoderma with trichoderma nacl mm s h o o t (c m ) 0 5 10 15 20 25 a a a b c d a b c d a b c d maize rice b b c c d d a b c d a b c d a b c d * * * * * * * * * * * * * * * ns fig.2. effect of t. harzianum seed treatments on seedling growth of maize (zea mays l.) var. 7 nt6621 and rice (oryza sativa l.) var. kernel in saline environment . vertical lines on bars 8 graph stand for means standard error (±). same alphabets on the bar graphs showed non-9 significant difference within each treatment (*) stand for significant and (ns) for non-10 significant difference among the treatments (with and without th). 11 fig. 2. effect of trichoderma harzianum seed treatment on seedling growth of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment. results are expressed as means±standard errors. same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without t. harzianum. physiological responses of crops against endophytes acta bot. croat. 76 (2), 2017 157 results the results showed that shoot and root length significantly declined with the increase in salinity concentration in the soil (fig. 2). however, seed treated with trichoderma harzianum (th) has shown substantial increase in plant growth. shoot and root length increased significantly in thtreated maize and rice plants subjected to 50, 100 and 150 mm nacl treatments as compared to those plants that were not treated with trichoderma (fig. 2). relative water content (rwc) of maize and rice significantly decreased at all salinity concentrations (50, 100, 150 mm) (fig. 3). however, in th-treated plants, the adverse effects of salinity were alleviated, showing a substantial increase in the rwc of both crop plants over th-untreated plants. 2 12 0 50 100 150 0 50 100 150 nacl mm r e la ti v e w a te r c o n te n t (% ) 0 20 40 60 80 100 without trichoderma with trichoderma nacl mm r e la ti v e w a te r c o n te n t (% ) 0 20 40 60 80 100without trichoderma with trichoderma a b c d a a b b c c d d maize rice a b c a* * * ** ns ns ns fig.3. effect of t. harzianum seed treatments on relative water content and biomass 14 accumulation of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel in 15 saline environment. vertical lines on bars graph stand for means standard error (±).same 16 alphabets on the bar graphs showed non-significant difference within each treatment (*) stand 17 for significant and (ns) for nonsignificant difference among the treatments (with and without 18 th). 19 20 21 22 23 fig. 3. effect of trichoderma harzianum seed treatment on relative water content and biomass accumulation of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment. results are expressed as means±standard errors. same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without t. harzianum. 3 00 50 100 150 50 100 150 c h l b (  g m g -1 f w ) 1 2 3 4 5 6 7 t o ta l c h l ( g m g -1 f w ) 2 4 6 8 10 12 14 nacl mm c a ro te n o id s (  g m g -1 f w ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 c h l a (  g m g -1 f w ) 2 4 6 8without trichoderma with trichoderma c h l b (  g m g -1 f w ) 1 2 3 4 5 6 7 t o ta l c h l ( g m g -1 f w ) 2 4 6 8 10 12 nacl mm c a ro te n o id s (  g m g -1 f w ) 0.0 0.2 0.4 0.6 0.8 1.0 a a b b c c d d a a b b c d c d a a b c d b c d a a b b c d a b c d a b c d a b c d c d a b c d a b d c a b c d c h l a (  g m g -1 f w ) 2 4 6 8 without trichoderma with trichoderma a a b b c c d d maize rice * * * * * * * * * * * * * * * * * * * * * * * * ns ns ns * ns ns ns ns fig.4. effect of t. harzianum seed treatments on photosynthetic pigments of maize (zea mays 25 l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment . vertical lines 26 on bars graph stand for means standard error (±).same alphabets on the bar graphs showed 27 non-significant difference within each treatment (*) stand for significant and (ns) for non28 significant difference among the treatments (with and without th ) 29 30 fig. 4. effect of trichoderma harzianum seed treatments on photosynthetic pigments of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment. results are expressed as means±standard errors. same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without t. harzianum. yasmeen r., shaheed siddiqui z. 158 acta bot. croat. 76 (2), 2017 salinity caused a considerable decrease in pigment content but th treatment mitigated the salinity affect and improved pigment concentration during all nacl treatments (fig. 4). the values of photosynthetic attributes like dark-adapted quantum yield (fv/fm ratio), performance index (piabs), photochemical quenching (qp) and stomatal conductance (gs) were reduced in all salt treatments but th-treated plants showed higher values than th-untreated plants (fig. 5). in the present study, free proline and h2o2 contents of both crop plants were measured at different nacl concentrations (50, 100, and 150 mm) with and without the trichoderma inoculum (fig. 6). results revealed that proline and h2o2 contents were significantly influenced by the presence of trichoderma in a saline environment. however, in comparison between the crop plants, free proline content was significantly higher in maize than in rice. seeds that were treated with trichoderma showed maximum accumulation of proline content in all saline treatments (0, 50, 100, 150 mm) as compared to those treatments without trichoderma. however, maximum proline content was recorded at 150 mm nacl (fig. 6). h2o2 production was elevated at all salinity levels in plants that were not treated with trichoderma (fig. 6). however, h2o2 production significantly lowered with the increasing concentration of nacl in thtreated plants. activity of antioxidant enzymes like sod and cat were measured at different nacl concentrations with or without the trichoderma inoculum (fig. 7). observations revealed that the sod and cat activities of both maize and rice plants were substantially increased with increased nacl concentration. however, in a comparison between the crop plants, a maize plant showed greater antioxidant activity than rice (fig. 7). it was observed that the presence of trichoderma in a saline environment additionally increased the activity of antioxidant enzymes as compared to plants in saline medium without trichoderma. discussion salinity is a major abiotic factor that restricts plant growth and productivity, which not only causes osmotic stress but also alters physiological and biochemical mechanism in plants. crops such as rice and maize (poaceae) are sensitive or moderately sensitive to salinity. they are unable to tolerate a higher amount of salt in soil. results showed that the application of trichoderma to the crop plants enhances tolerance to a high concentration of nacl. trichoderma is an endophytic symbiont, as its inoculation 4 31 32 0 50 100 150 0 50 100 150 p e rf o rm a n c e i n d e x (p i a b s ) 0.5 1.0 1.5 2.0 2.5 3.0 p e rf o rm a n c e i n d e x (p i a b s ) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 d a rk a d a p te d q u a n tu m y ie ld (f v /f m r a ti o ) 0.5 1.0 1.5 2.0without trichoderma with trichoderma p h o to c h e m ic a l q u e n c h in g (q p ) 1 2 3 4 5 p h o to c h e m ic a l q u e n c h in g (q p ) 0.5 1.0 1.5 2.0 2.5 nacl mm s to m a ta l c o n d u c ta n c e (m m o l m -2 s -1 ) 0 100 200 300 400 nacl mm s to m a ta l c o n d u c ta n c e (m m o l m -2 s -1 ) 0 100 200 300 400 a b c d a b b c a a b c d b c d a a b c d b c d a a b c d b c d a b c d a b c d a a b b c d c d a a b c d b c d d a rk a d a p te d q u a n tu m y ie ld (f v /f m r a ti o ) 0.5 1.0 1.5 2.0 2.5 3.0 without trichoderma with trichoderma a a b b c c d d maize rice * * * * * * * * * * * * ns * * * * * * * * ns ns ns ns ns ns ns ns ns ns ns fig.5. effect of t. harzianum seed treatments on photosynthetic attributes of maize (zea mays 34 l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment . vertical lines 35 on bars graph stand for means standard error (±).same alphabets on the bar graphs showed 36 non-significant difference within each treatment (*) stand for significant and (ns) for non37 significant difference among the treatments (with and without th ) 38 39 fig. 5. effect of trichoderma harzianum seed treatments on photosynthetic attributes of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment. results are expressed as means±standard errors. same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without t. harzianum. physiological responses of crops against endophytes acta bot. croat. 76 (2), 2017 159 has antagonistic properties and therefore enhances the systemic tolerance to salt stress in plants (harman et al. 2004). it was observed that salinity caused a substantial reduction in growth and biomass of those plants without a th treatment. the application of th mitigates salt-related consequences in plants, which results in considerable increases in growth and biomass production. it was reported that the application of th in a saline habitat improved biomass and growth parameters (moud and maghsoudi 2008, rasool et al. 2013, ahmad et al. 2014). the increase in growth relative water content and biomass production with a th application may be due to its ability to produce phytohormones like gibberellins and cytokine, which may not only promote the plant growth but also increase some degree of tolerance in a saline environment (harman 2000, benitez et al. 2004, iqbal and ashraf 2013, ahmad et al. 2015). relative water content decreased in maize and rice crops under nacl stress but increased due to the application of th. the maintenance of a substantial amount of relative water content in leaves is a main strategy for maintaining optimal growth of plants under salinity (siddiqui et al. 2014). it was reported that t. harzianum provides better ability to regulate additional intracellular water relations due to biomass accumulation resulting from the uptake of more water under salt stress (rawat et al. 2011, hashem et al. 2014). 6 c a t ( u n it m g -1 o f p ro te in ) 20 40 60 80 100 120 140 160 180 without trichoderma with trichoderma c a t ( u n it m g -1 o f p ro te in ) 20 40 60 80 100 120 140 160 without trichoderma with trichoderma nacl mm s o d ( u n it m g -1 o f p ro te in ) 0 20 40 60 80 100 120 140 160 nacl mm s o d ( u n it m g -1 o f p ro te in ) 0 20 40 60 80 100 120 140 160 0 50 100 150 0 50 100 150 a a b b c c d d a a b b c c d d a a b b c c d d a a b b c c d d maize rice * * * * * * * * * * * * * * * * 49 fig.7. effect of t. harzianum seed treatments on catalase (cat) and superoxide dismutase 50 (sod) antioxidant enzyme activity of maize (zea mays l.) var. nt6621 and rice (oryza 51 sativa l.) var. kernel in saline environment. vertical lines on bars graph stand for means 52 standard error (±).same alphabets on the bar graphs showed non-significant difference within 53 each treatment (*) stand for significant and (ns) for nonsignificant difference among the 54 treatments (with and without th 55 56 57 provide please all figures as tiff or pdf with resolution of 300 dpi 58 fig. 6. effect of trichoderma harzianum seed treatments on proline and hydrogen peroxide content of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment. results are expressed as means±standard errors. same alphabets show nonsignificant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without t. harzianum. fig. 7. effect of trichoderma harzianum seed treatments on catalase (cat) and superoxide dismutase (sod) antioxidant enzyme activity of maize (zea mays l.) var. nt6621 and rice (oryza sativa l.) var. kernel in saline environment. results are expressed as means±standard errors. same alphabets show non-significant difference within each treatment, (*) stands for significant and (ns) for non-significant difference among the treatments with and without t. harzianum. yasmeen r., shaheed siddiqui z. 160 acta bot. croat. 76 (2), 2017 photosynthetic attributes like fv/fm ratio, piabs, qp and gs of th-treated plants were increased in all nacl treatments compared to th-untreated plants. it has been demonstrated that content of plant photosynthetic pigments like chl a, chl b, total chlorophyll and carotenoids generally reduces under nacl (parida and das 2005, sairam et al. 2002). the result of the decrease in carotenoid contents under salt stress might be due to decrease in β-carotene and zeaxanthin formation (sultana et al. 1999). salt stress directly affects the chloroplast function, degrading enzymes which results in substantial reduction in photosynthesis of plants (siddiqui et al. 2014). however, in the present study, the application of trichoderma in a saline environment produced a considerable increase in all the tested photosynthetic attributes. it was reported that th stimulates the synthesis of chlorophyll enzymes and phytohormones under different biotic stress in plants (rawat et al. 2011, zhang et al. 2013, hashem et al. 2014). there was a maximum amount of photosynthetic pigments present in th-treated plants in maize and rice crops as compared to untreated plants under nacl condition. further, these results are in accordance with the results of mishra and salokhe (2011) who reported that inoculation of seed by trichoderma enhanced pigment system psii performance and produced a higher rate of transpiration in plants under salt stress conditions. in plants subjected to salinity stress, photosynthetic rate and stomatal conductance might be disturbed due to the higher amount of na+ ions accumulation which disturb the electron transport chain during photosynthesis (kanwal et al. 2011). from the present investigation, it was clearly observed that the dark adapted quantum yield (fv/fm ratio), performance index (piabs), photochemical quenching (qp) and stomatal conductance (gs) were decreased with the increase in salinity concentration but increased by th application. this could be due to an increase in chlorophyll concentration by the application of trichoderma in saline environment (sheng et al. 2008). results showed a substantial decrease of proline and an increase of h2o2 contents in untreated plants subjected to salt stress, but increased proline with decreased h2o2 production in th-treated plants was observed in both crops. it was suggested that proline accumulation provided protection to the cell through balancing osmolyte concentration under salt stress conditions in tolerant plants (greenway and munns 1980). in an abiotic stress like salinity, elevated h2o2 production damages protein and lipid molecules (siddiqui et al. 2014). it is presumed that the application of trichoderma lowers h2o2 production in salt stress due to proline accumulation. moreover, it was reported that trichoderma enhances antioxidant enzymes such as glutathione s-transferases (gsts) and peroxidase (pod) activities and lowers ros production (hajiboland et al. 2010, wu et al. 2010, alqarawi et al. 2014). it was reported that maximum quantum yield and photosynthetic performance control the production of ros (siddiqui and khan 2013, siddiqui et al. 2014). photosynthetic performance and quantum yield are inter-related and are often decreased in abiotic stress and under elevated ros level. fluctuating response with respect to quantum yield and performance index in stress environment are diversified and specific for some plant species (behera et al. 2002). substantial photosynthetic performance index and quantum yield in maize treated with trichoderma indicate that maize could develop better symbiotic relationship with th in saline environment compared to rice. in our study, it was detected that the presence of trichoderma in a saline environment increased the activity of antioxidant enzymes as compared to those saline media that did not have trichoderma. earlier, it was reported that the activities of antioxidant enzymes like cat, sod, pod and ascorbate peroxidase (apx) were increased in a saline environment (siddiqui 2013). it is presumed that the presence of trichoderma in a saline environment diminished h2o2 production due to elevated antioxidant enzyme activities as well as proline production. it was reported that antioxidant enzymes, production of osmolytes and polyols like proline, sorbitol etc. are important physiological strategy for coping with the consequences of abiotic stress and maintaining ion homeostasis (sairam et al. 2002, siddiqui and khan 2011, rasool et al. 2013). further it was observed that a trichoderma inoculation enhanced antioxidant enzyme activities and decreased salt stress in plants (hajiboland et al. 2010, hashem et al. 2014, ahmad et al. 2015). it can be concluded that application of trichoderma harzianum enhances salt tolerance of maize and rice through higher antioxidant activities and high proline content. treatment with trichoderma harzianum not only enhanced some physiological parameters but also lowered the h2o2 concentration reducing the damaging effect of ros within plants. acknowledgments the authors are grateful to the rural development administration, suwon, republic of korea 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0365-0588 eissn 1847-8476 diversity and gradients of vegetation of sivrihisar mountains (eskişehir-turkey) neslihan balpinar1*, ali kavgaci2, m. ümit bingöl3, osman ketenoğlu3 1 mehmet akif ersoy university, faculty of arts and science, department of biology, 15030, burdur, türkiye 2 southwest anatolia forest research institute, 07022 antalya, türkiye 3 ankara university, faculty of science, department of biology, 06100, ankara, türkiye abstract – this study was carried out to determine the plant communities and understand the main topographical driving factors of floristic differentiation in the sivrihisar mountains (eskişehir province). vegetation samplings were carried out according to the braun-blanquet approach. the relevés were stored in the turboveg database management program. hierarchical classification was carried out in pc-ord program with ward’s method and euclidean distance as a resemblance measure. the diagnostic species were identified by a fidelity measure in the juice program. the results of the classification were visualized by ordination techniques in the canoco package by using principal component analysis. in conclusion, except for the degraded forest community, all the 7 steppe and 1 scrub plant communities studied were identified and described as new associations. also, a syntaxonomical scheme for the vegetation of sivrihisar mountains was suggested. keywords: classification, numeric analysis, ordination, phytosociology, steppe vegetation * corresponding author, e-mail: nerdogan@mehmetakif.edu.tr introduction steppes, which face habitat loss, fragmentation and degradation, are among the most endangered biomes of the world. treeless vegetation dominated by perennial xerophilous grass and herbs is typical of these types of formation (török et al. 2016). the central anatolian plateau has a distinctive plant cover on the account of its isolation, due in turn to the high surrounding mountains and special nature (aksoy and hamzaoğlu 2006). steppe vegetation is the main vegetation type, as it is in east and parts of south anatolia, as a result of precipitation insufficient for the growth of trees under arid and semi-arid variants of mediterranean climate (atalay and efe 2010). numerous genera such as acantholimon, astragalus, centaurea, cousinia or verbascum have their evolutionary origin and main centre of diversity in this region (kürschner and parolly 2012). the central anatolian steppe is generally typically surrounded by steppic woods and forest formations (hamzaoğlu and duran 2004). although the floristic richness in the central anatolian steppe vegetation is very high, showing high levels of endemism, it is threatened by overgrazing pressure, intensive farming, and other management practices (aydoğdu et al. 2004, kurt et al. 2006, kaya et al. 2011). even though this steppe, forming the central anatolian province of irano-turanian region, has long been studied by many botanists (louis 1939, krause 1940, walter 1956, birand 1961, 1970; çetik 1985, akman et al. 1985, ketenoğlu et al. 1983, 2000; hamzaoğlu and duran 2004), much of its ecological diversity is understudied and more studies are needed. this study was carried out in this context, to determine plant communities and their structural components, to define ecological and geographical gradients and to examine the floral diversity in the sivrihisar mountains in turkey. we expect that this classification will guide future phytosociological studies and will serve as a base for various prospective studies. materials and methods study area sivrihisar mountains is a mountain chain extending from the sakarya arc in the southeast corner of eskişehir province, in the se-nw direction towards kaymaz district, located between 39° 28' 00" latitude and 31° 34' 60" longitude (fig. 1). the study area is mainly covered by steppe vegetamailto:nerdogan@mehmetakif.edu.tr vegetation of sivrihisar mountains acta bot. croat. 77 (1), 2018 19 tion but also by degraded shrub and forest vegetation. the vegetation in the study area was intensely affected by overgrazing, agricultural activities and the expansion of stone and marble quarrying. the altitude ranges from 1000 m a.s.l. to 1415 m a.s.l. the study area, within the irano-turanian phytogeographical region and placed in the b3 square according to the grid system of davis (1965–1985), is represented by the “eastmediterranean precipitation regime type ii” according to emberger’s classification (akman 1990) (see on-line suppl. tab. 1). the area does not have any protection status. largely the paleozoic rocks are exposed in sivrihisar mountains in eskişehir, while mesozoic and tertiary outcrops are also encountered. vegetation samplings and data analysis the field work was carried out between 2003 and 2005. homogenous sampling plots with an area of 50 m2 were selected for steppe vegetation, the dominant vegetation type in the area, 100 m2 for scrub vegetation and 150 m2 for the degraded forest vegetation. the protocol for each plot included general, topographic and other data of individual plots, such as altitude, inclination, aspect, vegetation cover (total and of individual layers) and a list of all vascular plants, in which a cover value was assigned to each species according to the nine degree braun-blanquet scale (braun-blanquet 1964, westhoff and van der maarel 1973). the samples (hereinafter relevés) were stored in the turboveg database management program (hennekens and schaminée 2001). hierarchical classification of the data set was carried out in the computer program pc-ord (mc cune and meffords 2006). the euclidean distance was used as a resemblance measure for analysis and ward’s method for dendrogram construction. various levels of division were accepted in the dendrogram, resulting in nine clusters interpretable in terms of ecology. additionally, the diagnostic species of the accepted clusters were identified by a fidelity measure in the juice program (tichý 2002). the threshold of the phi value was subjectively selected at 0.50 for a species to be considered as diagnostic (chytrý et al. 2002). besides, the constant and dominant species of the clusters were defined in juice. species that had a more than 50% occurrence frequency for a given community were defined as constant species, while species attaining a cover higher than 30% in more than 70% of the relevés were accepted as dominant species. the results of the classification were visualized by ordination techniques in the canoco 4.5 package (ter braak and šmilauer 2002). principal component analysis (pca), which is an indirect ordination method assuming a unimodal response of species to the environment, was run due to the high heterogeneity in the matrix of species (lepš and šmilauer 2003). topographical variables were passively projected on the ordination plane. we also calculated the spectra of geo-elements according to davis (1965–1985) and davis et al. (1988) and life forms in accordance with raunkiaer (1934) and they were also passively projected on the ordination diagram. correlations between pca releve scores and topographical variables, geoelement and growth form were calculated by using the non-parametric kendall coefficient in statistica (anonymous 2007). the nomenclature of plant species follows flora of turkey (davis 1965– 1985, davis et al. 1988) and new syntaxa were described in accordance with the international code of phytosociological nomenclature (weber et al. 2000). syntaxonomical interpretations of taxa for the forest vegetation were made according to quézel (1973), akman et al. (1978a, b, 1979), and quézel et al. (1978, 1980). classification of the taxa belonging to the steppe communities was made according to the studies carried out by akman et al. (1984, 1985), ketenoğlu et al. (1983), and quézel et al. (1992). results classification the classification analysis resulted in nine main clusters representing clear floristic and ecological differences of vegetation in the sivrihisar mountains (fig. 2). while seven of these clusters represent steppe vegetation, the other two clusters represent the scrub and forest vegetation in the study area. diagnostic, constant and dominant species of these clusters are as follows: cluster 1: artemisia scoparia-dominated steppe diagnostic species: agrostemma gracilis, artemisia scoparia, astrodaucus orientalis, bupleurum gerardii, campanula argaea, coronilla varia ssp. varia, hordeum bulbosum, fig. 1. the geographical position of the sivrihisar mountains in turkey. dots show the places of the relevés. balpinar n., kavgaci a., bi̇ngöl m. ü., ketenoğlu o. 20 acta bot. croat. 77 (1), 2018 melica ciliata ssp. ciliata, parietaria judaica, pimpinella tragium ssp. polyclada, rubus sanctus, silene squamigera ssp. squamigera, thymus sipyleus ssp. sipyleus var. sipyleus, trifolium arvense var. arvense. constant species: astragalus angustifolius ssp. angustifolius var. angustifolius, convolvulus holosericeus ssp. holosericeus, galium verum ssp. verum, koeleria cristata, poa bulbosa. dominant species: artemisia scoparia cluster 2: thymus leucostomus var. argillaceusdominated steppe diagnostic species: alyssum strigosum ssp. strigosum, bromus japonicus ssp. japonicus, b. squarrosus, ebenus hirsuta, hypericum aviculariifolium ssp. depilatum var. bourgaei, ornithogalum oligophyllum, scandix iberica, silene dichotoma ssp. dichotoma, thymus leucostomus var. argillaceus. constant species: androsace maxima, anthemis cretica ssp. anatolica, astragalus vulnerariae, eryngium campestre var. virens. dominant species: thymus leucostomus var. argillaceus. cluster 3: astragalus microcephalus-dominated steppe diagnostic species: astragalus microcephalus, chrysopogon gryllus ssp. gryllus, cichorium intybus, cirsium arvense ssp. vestitum, daucus broteri, echium angustifolium, elymus hispidus ssp. hispidus, helianthemum nummularium ssp. ovatum, inula montbretiana, lolium perenne, teucrium orientale var. orientale, thymelaea passerina, thymus longicaulis ssp. longicaulis var. subisophyllus. constant species: acantholimon acerosum var. acerosum, astragalus condensatus, centaurea virgata, dianthus zonatus var. zonatus, eryngium campestre var. virens, euphorbia anacampseros var. anacampseros, festuca valesiaca, filago pyramidata, marrubium parviflorum ssp. oligodon, scabiosa argentea, teucrium polium, xeranthemum annuum. dominant species: astragalus microcephalus cluster 4: astragalus angustifolius ssp. angustifolius var. angustifolius-dominated steppe diagnostic species: alkanna orientalis var. orientalis, alyssum murale var. murale, anthemis tinctoria var. pallida, arabis nova, astragalus angustifolius ssp. angustifolius var. angustifolius, ceratocephalus falcatus, erodium hoefftianum, erophila verna ssp. verna, geranium lucidum, hypecoum procumbens, lamium amplexicaule, lamium garganicum ssp. reniforme, lathyrus cicera, medicago minima var. minima, m. sativa ssp. sativa, moenchia mantica ssp. caerulea, ranunculus isthmicus ssp. stepporum, r. reuterianus, rumex acetosella, valerianella costata, v. pumila, veronica triphyllos, vicia lathyroides, viola parvula, crataegus monogyna ssp. azarelle, constant species: anthemis tinctoria var. tinctoria, artemisia santonicum. dominant species: astragalus angustifolius ssp. angustifolius var. angustifolius cluster 5: artemisia santonicum-dominated steppe diagnostic species: allium flavum ssp. tauricum var. tauricum, allium sieheanum, alyssum sibiricum, artemisia santonicum, asperula stricta ssp. latibracteata, bothriochloa ischaemum, consolida thirkeana, erysimum crassipes, phlomis pungens var. laxiflora, sideritis lanata, s. montana ssp. montana, verbascum uschakense, centaurea solstitialis ssp. solstitialis. constant species: achillea coarctata, astragalus lydius, bromus tomentellus, centaurea virgata, cruciata taurica, eryngium campestre var. virens, festuca valesiaca, marrubium parviflorum ssp. oligodon, minuartia anatolica var. arachnoidea, onobrychis armena, paronychia kurdica ssp. kurdica var. kurdica, scabiosa argentea, stachys lavandulifolia var. lavandulifolia, stipa holosericea. dominant species: artemisia santonicum, sideritis montana ssp. montana cluster 6: astragalus condensatus-dominated steppe diagnostic species: astragalus condensatus, fumana arabica var. arabica, globularia orientalis, linum nodiflorum, medicago rigidula var. rigidula, muscari neglectum, phleum subulatum ssp. subulatum, polygala anatolica, scorzonera eriophora, silene lydia, stachys cretica ssp. anatolica, thymus argaeus, valerianella coronata, veronica pectinata var. glandulosa, paronychia condenseta, poa timoleontis, silene macrodanta. constant species: hypericum origanifolium, ornithogalum alpigenum, phlomis armeniaca, scutellaria orientalis ssp. pectinata. dominant species: astragalus condensatus cluster 7: astragalus plumosus-dominated steppe diagnostic species: adonis aestivalis ssp. aestivalis, alyssum thymops, anchusa undulata ssp. hybrida, astragalus plumosus var. plumosus, bromus cappadocicus ssp. cappadocicus, bungea trifida, centaurea triumfettii, erodium cicutarium ssp. cicutarium, hedysarum varium, moltkia coerulea, salvia cryptantha, scandix australis ssp. grandiflora, scorzofig. 2. hierarchical classification diagram of relevés from sivrihisar mountains: 1. artemisia scoparia-dominated steppe, 2. thymus leucostomus var. argillaceus-dominated steppe, 3. astragalus microcephalus-dominated steppe, 4. astragalus angustifolius ssp. angustifolius var. angustifolius-dominated steppe, 5. artemisia santonicum-dominated steppe, 6. astragalus condensatus-dominated steppe, 7. astragalus plumosus-dominated steppe, 8. cistus laurifolius-dominated scrubland, 9. quercus pubescens-dominated forest. vegetation of sivrihisar mountains acta bot. croat. 77 (1), 2018 21 nera suberosa ssp. suberosa, sisymbrium altissimum, tragopogon coloratus. constant species: adonis flammea, astragalus lydius, eryngium campestre var. virens, hypericum origanifolium, scutellaria orientalis ssp. pectinata, teucrium polium. dominant species: astragalus plumosus var. plumosus, salvia cryptantha cluster 8: cistus laurifolius-dominated scrubland diagnostic species: alyssum murale var. murale, berberis crataegina, campanula lyrata ssp. lyrata, cistus laurifolius, colutea cilicica, cotoneaster nummularia, daphne oleoides ssp. oleoides, fritillaria armena, globularia trichosantha ssp. trichosantha, helichrysum plicatum ssp. plicatum, hypericum linarioides, h. scabrum, juniperus oxycedrus ssp. oxycedrus, lathyrus digitatus, minuartia juniperina, plantago lanceolata, prunella laciniata, prunus spinosa ssp. dasyphylla, pyracantha coccinea, pyrus elaeagnifolia ssp. elaeagnifolia, ranunculus illyricus ssp. illyricus, rosa canina, sanguisorba minor ssp. muricata, silene italica, sorbus umbellata var. umbellata, trifolium physodes var. physodes, trigonella spruneriana var. spruneriana, ziziphora taurica ssp. taurica. constant species: koeleria cristata. dominant species: cistus laurifolius cluster 9: quercus pubescens-dominated forest diagnostic species: aegilops umbellulata ssp. umbellulata, alkanna pseudotinctoria, alyssum minus var. micranthum, briza humilis, bupleurum odontites, centaurea pichleri ssp. pichleri, ceratocephalus testiculatus, conringia perfoliata, ephedra major, erysimum sintenisianum, geranium tuberosum ssp. tuberosum, holosteum umbellatum var. umbellatum, jasminum fruticans, juniperus excelsa, j. oxycedrus ssp. oxycedrus, lamium garganicum ssp. reniforme, linum cariense, matthiola longipetala ssp. bicornis, poa timoleontis, quercus pubescens, ranunculus gracilis, rhamnus rhodopeus ssp. anatolicus, rhamnus thymifolius, salvia cadmica, s. syriaca, trifolium pannonicum ssp. elongatum, veronica grisebachii, vinca herbacea, viola kitaibeliana. constant species: astragalus lydius, cruciata taurica, eryngium campestre var. virens, hypericum origanifolium, ornithogalum alpigenum, sedum acre, teucrium polium. dominant species: quercus pubescens ordination the pca ordination of relevés for the nine clusters (fig. 3) shows that there is a clear gradient along the both axes of the ordination. these gradients show the ecological differences among the communities in the study area. the gradient of the ordination mainly results from the differentiation of juniperus oxycedrus-cistus laurifolius and quercus pubescens forest. these clusters are characterized by the high appearance of phanerophytic species (fig. 3) which has a strong positive correlation with both of the ordination axes and the altitude (tab. 1). these two clusters are also formed on the highest elevation zones of the study area (fig. 3 and tab. 1). all topographical variables used in the study have strong effects on the floristic differentiation of the vegetation. however, altitude has the highest effect (tab. 2). due to the strong differentiation of j. oxycedrus-c. laurifolius and q. pubescens forest, representing the higher vegetation types (scrub and forest) in the study area, these two communities were excluded from further ordination analysis to show clearly the differentiation between the others. for the lower vegetation types fig. 3. indirect ordination of the vegetation relevés from the sivrihisar mountains carried out by principal component analysis. tab. 1. kendal correlation coefficients (weighted correlation) between first two principal component analysis axes and topographical variables, geo-element and life form properties for the nine plant communities in the sivrihisar mountains. legend: *** means p<0.001, ** means p<0.01, * means p<0.05. a lti tu de a sp ec t in cl in at io n ir an otu ra ni an m ed ite rr an ea n eu ro -s ib er ia n w id e di st ri bu tio n th er op hy te c ry pt op hy te h em ic ry pt op hy te c ha m ap hy te ph an er op hy te axis 1 0.321*** 0.110 –0.162* 0.154* 0.112 –0.174** –0.133* –0.059 0.249*** –0.255*** –0.160* 0.254*** axis 2 0.199** 0.002 0.131* –0.407*** 0.176* 0.269*** 0.245*** –0.311*** –0.026 –0.018 0.000 0.423*** balpinar n., kavgaci a., bi̇ngöl m. ü., ketenoğlu o. 22 acta bot. croat. 77 (1), 2018 (steppe communities), ordination also shows clear gradients along two axes (fig. 4) and there are significant topographical differences between steppe communities (tabs. 3, 4). discussion ecological differences the effects of topographical variables on species richness and diversity, like the other ecological components affecting the biological mechanisms, were clearly shown (burke et al. 1989, sebastiá 2004). in our case, it was also seen that topographical variables (altitude, aspect, inclination) have explicit driving effects on community differentiation of steppe vegetation. these kinds of effects of topography were also shown for different vegetation types in turkey before like forests, scrublands and grasslands (fontaine et al. 2007, kavgacı et al. 2010a, b; özkan 2009, özkan et al. 2009, 2010). the clear gradients of these topographical factors also correspond to the geo-elemental structure. the communities at higher altitudes have a higher proportion of euro-siberian plant species in comparison with the other communities. the communities at lower altitudes are clearly represented by irano-turanian plants. as is known, higher elevations are associated with higher precipitation (basist and bell 1994) and the phytogeographical differences in the study area can be a result of these local climatic differences. plant communities show clear growth form differences at local, regional and global scales by depending on the changing environmental and ecological conditions (rowe and speck 2005). in this context, clear growth form differences among the communities in the study area were also observed. the communities at lower altitudes and with northern aspects are mostly represented by the therophytes and cryptophytes. the high proportion of therophytes may indicate the dry site conditions at these areas. chamaephytes are more common and characteristic for higher zones and southern slopes, which is probably the result of the human pressure at these sites. syntaxonomy there are nine different plant communities in the study area. two of them are cistus laurifolius-dominated scrub tab. 2. conditional effects of topographical variables on species composition for nine plant communities in the sivrihisar mountains, carried out by canonical correspondence analysis. the table is with the additional variances that each variable explains (lambda a) and the significance of the variables (p-value) together with their test statistics (f-value). variable var. n lambdaa p f altitude 1 0.43 0.002 4.62 slope 3 0.32 0.002 3.41 aspect 2 0.31 0.002 3.42 fig. 4. indirect ordination of the relevés from steppe vegetation in the sivrihisar mountains. see fig. 3 for the explanations of the symbols. tab. 3. conditional effects of topographical variables on species composition for seven steppe communities carried out by canonical correspondence analysis. the table is with the additional variances that each variable explains (lambda a) and the significance of the variables (p-value) together with their test statistics (f-value). variable var. n lambdaa p f altitude 1 0.21 0.002 2.42 slope 3 0.33 0.002 3.62 aspect 2 0.34 0.002 3.73 tab. 4. kendal correlation coefficients (weighted correlation) between first two two principal component analysis axes and topographical variables, geo-element and life form properties for the seven steppe communities in the sivrihisar mountains. legend: *** means p<0.001, ** means p<0.01, * means p<0.05. a lti tu de a sp ec t in cl in at io n ir an otu ra ni an m ed ite rr an ea n eu ro -s ib er ia n w id e di st ri bu tio n th er op hy te c ry pt op hy te h em ic ry pt op hy te c ha m ap hy te ph an er op hy te axis 1 –0.016 0.019 0.161* –0.339*** 0.104 0.300*** 0.198** –0.224** –0.164* 0.119 0.162* 0.225** axis 2 0.375*** 0.394*** 0.036 0.081 –0.129 –0.301*** 0.027 –0.100 –0.144* –0.096 0.359*** 0.160* vegetation of sivrihisar mountains acta bot. croat. 77 (1), 2018 23 and quercus pubescens-dominated degraded forest while the rest are lower steppe communities. from the phytosociological point of view, the central part of the anatolian steppe has lowland steppe characteristics and is included in the order onobrychido armenae-thymetalia leucostomi of the astragalo-brometea class (akman et al. 1985). all of the steppe communities which were identified in the research area were classified under the alliance phlomido armeniacae-astragalion microcephali since this alliance represents the communities on radiolarite, flysch, marly and serpentine rocks at altitudes ranging from 750 m to 1350 m a.s.l. (akman et al. 1984). artemisia scoparia-dominated community occurs at approximately the middle elevation zone of the study area. it is mostly seen on inclined slopes. the phanerophytic species, rubus sanctus and amygdalus orientalis join to the floristic composition of this community locally. the dominance of euro-siberian plants is very clear in this community and it is also characterized by the more frequent appearance of widely distributed plants. the diagnostic species of the community are clearly differentiated from the previously described a. scoparia-dominated communities from central anatolia (akman et al. 1991, kurt 2002). due to this fact, it is classified as thymo sipylei-artemisetum scopari ass. nova hoc loco. the nomenclatural type is relevé number 16 in on-line suppl. tab. 2 (holotypus hoc loco: on-line suppl. tab. 2/16). the thymus leucostomus var. argillaceus-dominated community is distributed on the lower elevation belts with northern aspects. it is represented by the highest occurrences of therophytic and cryptophytic species. the high occurrence of therophytes, generally indicating a typically desert spectrum vegetation, may be a result of over-grazing in this community (jankju et al. 2011). none of the geo-elements shows clear dominance in this community. the previously described t. leucostomus var. argillaceus-dominated communities in central anatolia are found on gypsum bedrock (akman 1990, kurt et al. 1999) and classified under the alliance astragalo karamasici-gypsophylion eriocalycis which is clearly different from our case. due to this fact, the thymus leucostomus var. argillaceus-dominated community is classified as alysso strigosi-thymetum argillacei ass. nova hoc loco. the nomenclatural type is relevé number 65 in on-line suppl. tab. 2 (holotypus hoc loco: on-line suppl. tab. 2/65). the astragalus microcephalus-dominated community is placed on the lower vegetation belts with northern aspects. the site of this community is highly inclined. the community can be characterized by the relatively high proportion of chamaephytes and low proportion of therophytes. due to the wide distribution of a. microcephalus, many a. microcephalus communities are described in anatolia (çetik 1963, akman 1974, 1976; akman and ketenoğlu 1976, düzenli 1976, akman et al. 1983, kılınç 1985, akman 1990, akman et al. 1991, ocakverdi and ünal 1991, ocakverdi and oflas 1999, aydoğdu et al. 1994, tatli et al. 1994, hamzaoğlu 1999, kurt et al. 1999, kurt 2000, 2002). however, our community shows clear differences from these communities because of the different diagnostic species, and it is classified as thymo longicauli-astragaletum microcephali ass. nova hoc loco. the nomenclatural type is relevé number 129 in on-line suppl. tab. 2 (holotypus hoc loco: on-line suppl. tab. 2/129). the astragalus angustifolius ssp. angustifolius var. angustifolius-dominated community is placed on a very narrow vegetation belt at the lower elevation zone. the site of this community is formed on gentle slopes. the number of iranoturanian plants and chamaephytes is the highest in this community. there are several a. angustifolius dominated communities described from the different parts of anatolia which were classified under different alliances (schwarz 1936, quézel 1973, akman 1974, 1976; akman and ketenoğlu 1976, düzenli 1976, kılınç 1985, akman 1990, akman et al. 1991, ocakverdi and ünal 1991, hamzaoğlu 2000, şanda et al. 2000). but in our case, there is a clear floristic difference and the a. angustifolius ssp. angustifolius var. angustifolius-dominated community is classified as anthemo tinctoriae-astragaletum angustifoli ass. nova hoc loco. the nomenclatural type is relevé number 11 in on-line suppl. tab. 2 (holotypus hoc loco: on-line suppl. tab. 2/11). the lowest distribution zone of the study area is formed by the artemisia santonicum-dominated community. these sites have mostly northern oriented slopes with relatively high inclinations. this community can also be characterized by the relatively higher appearance of euro-siberian plants than the other communities. the number of hemicryptophytes, cryptophytes and therophytes is also high in this community. a. santonicum-dominated communities are described by ocakverdi and ünal (1991), aydoğdu et al. (2001), aydoğdu et al. (1994), kurt (2002) and grouped under different alliances. the a. santonicum-dominated community in sivrihisar mountains represents a different floristic composition from the previous studies, and it is classified as sideritido montani-artemisetum santonici ass. nova hoc loco. the nomenclatural type is relevé number 26 in on-line suppl. tab. 2 (holotypus hoc loco: on-line suppl. tab. 2/26). the majority of the study area, covered by lower communities, is formed by an astragalus condensatus-dominated community and an astragalus plumosus var. plumosus-dominated community. these communities, showing similar environmental characteristics, are distributed mostly on southern slopes. they are dominated by chamaephytes differently from the other communities and characterized by the highest appearances of irano-turanian plants, which indicate a higher steppic character (kurt et al. 2006) in comparison with the other steppe communities. akman et al. (1984) describes an astragalus condensatus-dominated community under the alliance salvio tchihatcheffii-hedysarion varii. but the a. condensatus-dominated community in the sivrihisar mountains represents a very different floristic composition from this community, and it is classified as globulario lumose-astragaletum condensati ass. nova hoc loco. the nomenclatural type is relevé number 58 in on-line suppl. tab. 2 (holotypus hoc loco: on-line suppl. tab. 2/58). the communities dominated by the spiny taxon astragalus plumosus var. plumosus are described by akman (1976), akman and ketenoğlu (1976), akman (1990) and yurdakulol et al. (1990). however, the community at sivrihisar mountains is formed by different floristic composition than those combalpinar n., kavgaci a., bi̇ngöl m. ü., ketenoğlu o. 24 acta bot. croat. 77 (1), 2018 munities and is classified as salvio cryptanthae-astragaletum plumosi ass. nova hoc loco. the nomenclatural type is relevé number 90 in on-line suppl. tab. 2 (holotypus hoc loco: online suppl. tab. 2/90). the dominant components of the peripheral vegetation around central anatolia have been included in the class quercetea pubescentis (akman et al. 1984). cistus laurifolius is an important element of this vegetation type and appears intensively especially in the transitional zone between inner anatolia and the mediterranean, aegean and marmara regions. it mostly occurs as the remnants of pinus nigra forests as a result of a regressive succession process. in the initial phase of the succession, it is often found as mixed communities with oak species. the c. laurifolius-dominated shrub community in the study area is named junipero oxycedri cistetum laurifoli ass. nova hoc loco. the nomenclatural type is relevé number 21 in on-line suppl. tab. 2 (holotypus hoc loco: on-line suppl. tab. 2 /21). it is grouped under quercion anatolicae alliance belonging to querco-carpinetalia orientalis order due to lack of congruence with pinocistion laurifolii alliance characteristics which is accepted as the alliance of c. laurifolius dominated communities in the other parts of anatolia (akman and ketenoğlu 1976, çetik and vural 1979, hamzaoğlu and duran 2004). the high frequency of characteristic species of astragalo-brometea and onobrychido-thymetalia in this community may indicate a conversion through steppe vegetation. the steppe vegetation of central anatolia has resulted from the reduction or extinction of former primary forest vegetation, which has disappeared due to biotic factors (kılınç 1979, kurt et al. 2006). the higher scrub and wood communities in central anatolia represent their degraded forms (kurt et al. 2006), and these communities are under high anthropogenic pressure today. quercus pubescens dominated forests in the study area are one of the remnants of those forests. although forest species such as trifolium pannonicum ssp. elongatum, vicia cracca ssp. stenophylla, coronilla varia ssp. varia, pyrus elaeagnifolia, juniperus oxycedrus ssp. oxycedrus are found as accompanying plants in the degraded q. pubescens forests in the area, the high coverage by steppic species may indicate that the formation is a transitional phase. although several q. pubescens forest communities are described from anatolia (akman and ketenoğlu 1976, ketenoğlu and akman 1982, akman et al. 1983, akman and aydoğdu 1986), the q. pubescens forest community in thesivrihisar mountains is not identified at association level due to its highly degraded structure. in conclusion, in addition to the q. pubescens forest community, 7 steppe and 1 scrub associations are identified and described as new associations in this study. the degraded structure of forest and scrub vegetation indicates the severe anthropogenic effects in the region. however, the presence of various plant associations in such a small area reflects the high floristic and ecological diversity. this emphasizes the importance of the region in terms of nature conservation. so, the richness and diversity of the region should be taken into consideration during the preparation of a management plan for the region, especially from the restoration point of view. in accordance with these assessments, the syntaxonomical scheme of communities and nomenclature type relevés of the newly described syntaxa can be suggested as follows: class: astragalo microcephali-brometea tomentelli quézel 1973 order: onobrychido armenae-thymetalia leucostomi akman, ketenoğlu, quézel 1985 alliance: phlomido armeniacae-astragalion microcephali akman, ketenoğlu, quézel et demirörs 1984 association: thymo sipylei-artemisetum scopari ass. nova association: alysso strigosi-thymetum argillacei ass. nova association: thymo longicauli-astragaletum microcephali ass. nova association: anthemo tinctoriae-astragaletum angustifoli ass. nova association: sideritido montani-artemisetum santonici ass. nova association: globulario lumose-astragaletum condensati ass. nova association: salvio cryptanthae-astragaletum plumosi ass. nova class: quercetea pubescentis (oberd. 1948) doing kraft 1955 order: querco cerridis-carpinetalia orientalis quézel, barbéro et akman 1980 alliance: quercion anatolicae akman, barbéro et quézel 1979 association: junipero oxycedri-cistetum laurifoli ass. nova quercus pubescens community 1. the nomenclature type of the association thymo sipylei-artemisetum scopari ass. nov. holotypus hoc. loco: n. balpınar (10.06.2003). plot size: 50 m2, altitude:1275 m, slope: 40%, sw: 39°22.504´ n, 031°40.130´ e, cover herb layer: 80%. herb layer: thymus sipyleus ssp. sipyleus var. sipyleus: 2, artemisia scoparia: 3, astragalus angustifolius ssp. angustifolius var. angustifolius: +, alyssum murale var. murale: +, alyssum sibiricum: 1, paronychia condensata: +, helianthemum canum: +, convolvulus holosericeus ssp. holosericeus: 2, centaurea urvillei ssp. stepposa: +, astragalus vulnerariae: +, onobrychis hypargyrea: 1, carduus nutans ssp. trojanus: +, poa bulbosa: +, minuartia hirsuta ssp. falcata: +. 2. the nomenclature type of the association alysso strigosi-thymetum argillacei ass. nov. holotypus hoc. loco: n. balpınar (12.06.2004). plot size: 50 m2, altitude:1240 m, slope: 25%, nw: 39°31.030´ n, 031°18.442´ e, cover herb layer: 80%. herb layer: alyssum strigosum ssp. strigosum: +, bromus japonicus ssp. japonicus: +, ornithogalum oligophyllum: +, scandix iberica: +, thymus leucostomus var. argillaceus: 3, hypericum aviculariifolium ssp. depilatum: +, bromus squarrosus: +, lolium perenne: +, astragalus condensatus: +, stachys cretica ssp. anatolica: +, centaurea triumfettii: +, briza humilis: +, astragalus lydius: +, phlomis armeniaca: 2, centaurea virgata: 1, onobrychis armena: +, ziziphora tenuior: +, onobrychis hypargyrea: +, eryngium campestre var. vivegetation of sivrihisar mountains acta bot. croat. 77 (1), 2018 25 rens: +, cruciata taurica: +, bromus tectorum: +, minuartia anatolica var. arachnoidea: 1, festuca valesiaca: +, androsace maxima: +, alyssum minus var. micranthum: +, adonis flammea: +, filago pyramidata: +, ajuga chamaepitys ssp. chia var. chia: +, bromus tomentellus: 1, globularia trichosantha ssp. trichosantha: +, chardinia orientalis: +, centaurea depressa: +, scleranthus annuus ssp. annuus: +, allium paniculatum ssp. paniculatum: +, thlaspi perfoliatum: +, elymus lazicus ssp. divaricatus: +, crupina crupinastrum: +, erysimum kotschyanum: +. 3. the nomenclature type of the association thymo longicauli-astragaletum microcephali ass. nov. holotypus hoc. loco: n. balpınar (14.07.2004). plot size: 50 m2, altitude:1175 m, slope: 45%, nw: 39°27.548´ n, 031°39.139´ e, cover herb layer: 80%. herb layer: thymus leucostomus var. argillaceus: 1, hypericum aviculariifolium ssp. depilatum: +, thymelaea passerina: +, teucrium orientale var. orientale: 1, astragalus microcephalus: 4, elymus hispidus ssp. hispidus: +, helianthemum nummularium ssp. ovatum: 1, inula montbretiana: +, daucus broteri: +, lolium perenne: +, thymus longicaulis ssp. longicaulis var. longicaulis: 3, phlomis armeniaca: 1, centaurea virgata: 1, dianthus zonatus var. zonatus: 2, hypericum origanifolium: +, centaurea urvillei ssp. stepposa: +, scabiosa argentea: 2, euphorbia macroclada: 2, acantholimon acerosum var. acerosum: 1, teucrium polium: +, festuca valesiaca: 2, xeranthemum annuum: 1, euphorbia anacampseros var. anacampseros: +, ornithogalum alpigenum: +, chardinia orientalis: +, achillea setacea: +, stipa pontica: 2, acanthus hirsutus: +. 4. the nomenclature type of the association anthemo tinctoriae-astragaletum angustifoli ass. nov. holotypus hoc. loco: n. balpınar (09.06.2003). plot size: 50 m2, altitude:1190 m, slope: 30%, n: 39°27.346´ n, 031°32.387´ e, cover herb layer: 80%. herb layer: artemisia scoparia: +, astragalus microcephalus: +, viola parvula: +, geranium lucidum: +, alkanna orientalis var. orientalis: 2, astragalus angustifolius ssp. angustifolius: 3, erophila verna ssp. verna: +, lamium garganicum ssp. reniforme: +, crataegus monogyna ssp. azarelle: +, ranunculus reuterianus: +, moenchia mantica ssp. caerulea: +, vicia lathyroides: +, arabis nova: +, rumex acetosella: +, veronica triphyllos: +, anthemis tinctoria var. pallida: +, lamium amplexicaule: +, lathyrus cicera: +, erodium hoefftianum: +, alyssum murale var. murale: +, artemisia santonicum: 1, minuartia juniperina: +, veronica grisebachii: +, centaurea pichleri ssp. pichleri: +, potentilla recta: +, paronychia kurdica ssp. kurdica var. kurdica: +, onobrychis armena: 1, eryngium campestre var. virens: +, cruciata taurica: +, alyssum minus var. micranthum: +, euphorbia anacampseros var. anacampseros: +, koeleria cristata: +, anthemis cretica ssp. anatolica: +, anthemis tinctoria var. tinctoria: 3, thlaspi perfoliatum: +, stipa pontica: +, holosteum umbellatum var. umbellatum: +, myosotis ramosissima ssp. ramosissima: +, lotus aegaeus: +. 5. the nomenclature type of the association sideritido montani-artemisetum santonici ass. nov. holotypus hoc. loco: n. balpınar (26.06.2003). plot size: 50 m2, altitude:1120 m, slope: 45%, n: 39°32.168´ n, 031°13.365´ e, cover herb layer: 80%. herb layer: thymus leucostomus var. argillaceus: +, consolida thirkeana: +, asperula stricta ssp. latibracteata: +, allium sieheanum: +, phlomis pungens var. laxiflora: +, sideritis montana ssp. montana: 3, verbascum uschakense: 1, artemisia santonicum: 4, erysimum crassipes: +, alyssum sibiricum: +, bromus cappadocicus ssp. cappadocicus: +, dianthus zonatus var. zonatus: +, marrubium parviflorum ssp. oligodon: +, paronychia kurdica ssp. kurdica var. kurdica: +, onobrychis armena: +, convolvulus holosericeus ssp. holosericeus: +, centaurea urvillei ssp. stepposa: +, scabiosa argentea: +, cruciata taurica: +, teucrium polium: +, minuartia anatolica var. arachnoidea: +, carduus nutans ssp. trojanus: +, stipa holosericea: 3, stachys lavandulifolia var. lavandulifolia: +, anthemis tinctoria var. tinctoria: +, pistacia terebinthus ssp. palaestina: 1. 6. the nomenclature type of the association globulario lumose-astragaletum condensati ass. nov. holotypus hoc. loco: n. balpınar (29.05.2004). plot size: 50 m2, altitude:1360 m, slope: 45%, se: 39°20.559´ n, 031°42.122´ e, cover herb layer: 80%. herb layer: thymus longicaulis ssp. longicaulis var. longicaulis: +, erysimum crassipes: +, phleum subulatum ssp. subulatum: +, poa timoleontis: +, medicago rigidula var. rigidula: +, fumana arabica var. arabica: +, thymus argaeus: +, scorzonera eriophora: +, astragalus condensatus: 5, paronychia condensata: +, stachys cretica ssp. anatolica: +, veronica pectinata var. glandulosa: +, silene macrodanta: +, silene lydia: +, linum nodiflorum: +, globularia orientalis: 3, polygala anatolica: +, juniperus oxycedrus ssp. oxycedrus: 1, salvia cadmica: +, phlomis armeniaca: +, teucrium chamaedrys ssp. syspirense: +, hypericum origanifolium: +, acantholimon acerosum var. acerosum: 1, cruciata taurica: +, bromus tectorum: +, androsace maxima: +, euphorbia anacampseros var. anacampseros: +, adonis flammea: +, anthemis cretica ssp. anatolica: +. 7. the nomenclature type of the association salvio cryptanthae-astragaletum plumosi ass. nov. holotypus hoc. loco: n. balpınar (09.06.2003). plot size: 50 m2, altitude:1190 m, slope: 35%, w: 39°31.466´ n, 031°14.444´ e, cover herb layer: 80%. herb layer: astragalus microcephalus: 1, hypecoum procumbens: +, asperula stricta ssp. latibracteata: +, globularia orientalis: 1, tragopogon coloratus: +, scorzonera suberosa ssp. suberosa: +, sisymbrium altissimum: +, alyssum thymops: +, anchusa undulata ssp. hybrida: +, erodium cicutarium ssp. cicutarium: +, astragalus plumosus var. plumosus: 4, bungea trifida: +, salvia cryptantha: 3, moltkia coerulea: +, hedysarum varium: +, astragalus lydius: +, potentilla recta: +, paronychia kurdica ssp. kurdica var. kurdica: +, lappula barbata: +, aubrieta anamasica: +, eryngium campestre var. virens: +, poa bulbosa: +, adonis flammea: +, ajuga chamaepitys ssp. chia var. chia: +, anthemis tinctoria var. tinctoria: +, iris schachtii: +, anchusa leptophylla ssp. incana: +, orobanche cilicica: +, alyssum hirsutum var. hirsutum: +. 8. the nomenclature type of the association junipero oxycedri-cistetum laurifoli ass. nov. holotypus hoc. loco: n. balpınar (10.06.2003). plot size: 100 m2, altitude:1350 m, slope: 45%, ne: 39°23.453´ n, 031°42.182´ e, cover shrub layer: 75%, cover herb layer: 80%. balpinar n., kavgaci a., bi̇ngöl m. ü., ketenoğlu o. 26 acta bot. croat. 77 (1), 2018 shrub layer: cistus laurifolius: 5, daphne oleoides ssp. oleoides: 1, prunus spinosa ssp. dasyphylla: 1, cotoneaster nummularia: 1, berberis crataegina: 1, juniperus oxycedrus ssp. oxycedrus: 2, amelanchier parviflora var. parviflora: 2. herb layer: hypericum linarioides: 1, lathyrus digitatus: 1, prunella laciniata: 1, silene italica: 1, pyracantha coccinea: 1, sanguisorba minor ssp. muricata: 1, globularia trichosantha ssp. trichosantha: 1, trigonella spruneriana var. spruneriana: 1, trifolium physodes var. physodes: 1, alyssum murale var. murale: 1, ranunculus illyricus ssp. illyricus: 1, hypericum scabrum: 1, plantago lanceolata: 1, rosa canina: 2, minuartia anatolica var. arachnoidea: 1, koeleria cristata: 1, crataegus orientalis var. orientalis: 2, veronica multifida: 1, achillea setacea: 1. acknowledgements some data used in this study is obtained from corresponding author's ph.d. thesis, which was partially financed by research fund of ankara university (project id: 200407-05-085). references akman, y., 1974: etude phyto-écologique de la région de 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(ed.), handbook of vegetation science 5, ordination and classification of communities, 617– 726. junk, the hague (in dutch). yurdakulol, e., aydoğdu, m., çetin, b., 1990: a study of the steppe vegetation surrounding area of kırıkkale-kalecik-kırşehir from the phytosociological aspect. doğa, turkish journal of botany 14, 215–234 (in turkish). opce-str.vp acta bot. croat. 68 (2), 421–430, 2009 coden: abcra 25 issn 0365–0588 variability of the pennatae diatom gomphonema ventricosum gregory from far eastern lakes sakiko yoshitake1*, hiroshi fukushima2, tsutomu kimura2, ekaterina v. lepskaya3, tuyako ko-bayashi2 1 shonan junior college, 82 inaoka-cho yokosuka, kanagawa, japan 2 institute of phycology, 2-3-10 uraga kanagawa, japan 3 kamchatka institute for fisheries research and oceanography, peteropavlovsk-kamchatsky, russia, 683000 the present study examined by light microscope the morphological variations of 625 specimens of gomphonema ventricosum gregory collected from lake hövsgöl (mongolia), lake baikal (russia) and lake kurilskoye (kamchatka, russia). we classified the specimens into five types with respect to valve outline; (1) type a (type form of gomphonema ventricosum), (2) type b (lake baikal type), (3) type c (lake karluk type), (4) type d (gomphoneis septa type) and (5) type e (g. ventricosum f. curta type). based on the relative frequencies of these five types, specimens from lake baikal were shown to be different from those of the other two lakes. specimens from lake baikal are remarkably smaller both in length and breadth; specimens from the other two lakes are longer than those from lake baikal, though specimens from lake kurilskoye are wider than those from lake hövsgöl. thus, clear differences in morphological features are recognized between the specimens from these three lakes. keywords: diatom, gomphonema ventricosum, morphology, variability, lake hövsgol, lake baikal, lake kurilskoye, mongolia, russia introduction gomphonema ventricosum was first described by gregory (1856) from the river sprey in scotland. later, kociolek and stoermer (1987) provided a list of localities where this taxon had been reported. the distribution of this taxon is restricted to northern latitudes between 47°n and 68°n in eurasia, essentially in a longitudinal transect from kamchatka to the british isles. on the north american continent, it was observed between 36 °n and approximately 68 °n, in a longitudinal transect from massachusetts to the aleutian islands (kociolek and stoermer 1987). thus, the geographic distribution of this taxon is limited to the northern hemisphere and has been considered a northern alpine or cold water species (hustedt 1930, krasske 1932, tynni 1978, foged 1981, kociolek acta bot. croat. 68 (2), 2009 421 * corresponding author: yositake@shonan.ac.jp u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:42 color profile: disabled composite 150 lpi at 45 degrees and stoermer 1987, bahls 2005). kociolek and stoermer (1987) attempted to compare the type specimens with other populations from eurasia and concluded that the specimens from eurasia were conspecific. they also investigated specimens from the north american continent and considered that most of the population identified as gomphonema ventricosum (except for specimens from the alaskan mainland) belonged to the genus gomphoneis. specimens from washington are identical to those of gomphonema ventricosum (bahls 2005), which suggests that this taxon is more widely distributed over the north american continent. the purpose of the present study is to compare the morphological characteristics (such as length, breadth and striae density as well as valve outline) of g. ventricosum from three far eastern lakes in eurasia. materials and methods attached algae were sampled in lake hövsgöl in august 2005, in lake baikal in september 2006 and lake kurilskoye in august 2005 (fig. 1a). in lake baikal, the ph value, water temperature and electrical conductivity were 8.5, 12.0 °c and 93 µs cm–1; in lake kurilskoye 6.9, 9.0 °c and 149 µs cm–1, respectively. physicochemical data for the samples from lake hövsgöl cannot be shown as they were provided by another investigator. the specimens were fixed in 10% formalin and subsequently cleaned with sulphuric acid and hydrogen peroxide, rinsed several times with distilled water and then embedded in pleurax. observations were made with a carl zeiss axioscope differential interference contrast microscope. diatom identification was performed using a 100 x objective and microphotographs. microphotographs of 625 valves (lake hövsgöl: 145 valves, lake baikal: 280 valves and lake kurilskoye: 200 valves) were analyzed in this study. 422 acta bot. croat. 68 (2), 2009 yoshitake s., fukushima h., kimura t., lepskaya e. v., ko-bayashi t. fig. 1. location of sampling sites of far eastern lakes. h – lake hövsgöl (mongolia), b – lake baikal (russia), k – lake kurilskoye (kamchatka, russia) u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:43 color profile: disabled composite 150 lpi at 45 degrees results type classification by valve outline gomphonema ventricosum was classified according to valve shape, with reference to the microphotographs as well as illustrations published by many investigators. referring to the microphotographs of kociolek and stoermer (1987), we classified the specimens of g. ventricosum into five types based on valve outline (tab. 1), as follows: type a is the type form of g. ventricosum (figs. 1–6). the valves are swollen in the central portion and taper gently to a broadly rounded headpole, while tapering very considacta bot. croat. 68 (2), 2009 423 gomphonema ventricosum gregory from far eastern lakes plate 1. light microphotographs of gomphonema ventricosum gregory. figs. 1, 2 – type a specimens from lake hövsgöl; figs. 3, 4 – type a specimens from lake baikal; figs. 5, 6 – type a specimens from lake kurilskoye; figs. 7, 8 – type b specimens from lake hövsgöl; figs. 9, 10 – type b specimens from lake baikal; figs. 11, 12 – type b specimens from lake kurilskoye. u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:45 color profile: disabled composite 150 lpi at 45 degrees erably to the footpole. in our study, the samples from lake hövsgöl and lake kurilskoye contain high relative frequencies of this type (tab. 1). although lake hövsgöl is connected to lake baikal, the valve outlines of specimens from the two lakes are not so similar. type b is the lake baikal type (figs. 7–12). the valves of this type lack a protracted headpole while tapering considerably towards the footpole. in lake baikal this type has the highest relative frequency (tab. 1). type c is the lake karluk (kodiak island, north america) type (figs. 13–18). the valve outline resembles that of specimens from manguin’s material (manguin 1960). the central part of the valve is obviously tumid and steeply concave towards the headpole, 424 acta bot. croat. 68 (2), 2009 yoshitake s., fukushima h., kimura t., lepskaya e. v., ko-bayashi t. plate 2. light microphotographs of gomphonema ventricosum gregory. figs. 13, 14 – type c specimens from lake hövsgöl; figs. 15, 16 – type c specimens from lake baikal; figs. 17, 18 – type c specimens from lake kurilskoye; figs. 19, 20 – type d specimens from lake hövsgöl; figs. 21, 22 – type d specimens from lake baikal, fig. 23 – girdle view. u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:46 color profile: disabled composite 150 lpi at 45 degrees forming a protruding round end, while the footpole tapers towards a rounded end. in lake kurilskoye, the relative frequency of this type is 19.5%, which is the highest among the three lakes (tab. 1). type d is the gomphoneis septa type (figs. 19–22). specimens of this type resemble gomphoneis septa (mogh.) kociolek, stoermer et bahls. valves gradually narrow to a headpole forming a broadly rounded end, and the headpole is more elongate than the footpole which abruptly tapers towards the end. although specimens of this type rarely appear in our samples, the highest relative frequency (3.3%) of this type is found in lake hövsgöl. this type was rarely observed in lake kurilskoye (tab. 1). acta bot. croat. 68 (2), 2009 425 gomphonema ventricosum gregory from far eastern lakes tab. 1. type classification by valve outline and relative frequency (%) of each type of gomphonema ventricosum in the three lakes. type of valve outline lakes l. hövsgöl l. baikal l. kurilskoye type a g. ventricosum type form 86.7 6.2 78.1 type b lake baikal type 1.6 52.0 2.4 type c lake karluk type 8.4 16.0 19.5 type d gomphoneis septa type 3.3 0.8 0.0 type e g. ventricosum f. curta type 0.0 25.0 0.0 plate 3. light microphotographs of gomphonema ventricosum gregory. figs. 24–26 – type e specimens from lake baikal; figs 27–29 – initial valves of g. ventricosum; figs. 27, 28 – specimens from lake hövsgöl; fig. 29 – specimens from lake kurilskoye. u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:47 color profile: disabled composite 150 lpi at 45 degrees type e is the g. ventricosum f. curta type (figs. 24–26). the valve outline resembles g. ventricosum f. curta skvortzov et k. i. meyer. the upper half of the valve is elliptical and its lower half is wedge-shaped with a somewhat acute end. the valve length of this type is the shortest among the five types and in lake baikal it had a relative frequency of 25%. morphometric data morphometric characters such as length, breadth and striae density are used in this study (tab. 2), as well as the data of kociolek and stoermer (1987). specimens from lake baikal are shorter and narrower than those from the other two lakes. specimen lengths from lake hövsgöl and lake kurilskoye have a similar distribution of relative frequency (fig. 30); however, specimens from lake kurilskoye exhibit a broader valve 426 acta bot. croat. 68 (2), 2009 yoshitake s., fukushima h., kimura t., lepskaya e. v., ko-bayashi t. tab. 2. measurement of parts of valve in gomphonema ventricosum gregory. numbers in brackets refer to average values author sampling sites length (mm) breadth (mm) striae density 10mm–1 type material from kociolek & stoermer scotland 28–48 10–12.5 11–12 eurasia 23–78 9–15 9–13 north american continent 25–75 9–13 9–14 this study lake hövsgöl 27.5–61 (36.8) 8.5–12.5 (10.4) 13–16 (14.3) lake baikal 18.5–54.5 (32.2) 7.5–11.5 (9.3) 12–15.5 (13.9) lake kurilskoye 27–51 (37) 9.5–13.5 (11.3) 13–18 (15.2) 0 5 10 15 20 25 30 35 40 45 <2 0 20 –2 3. 5 24 –2 7. 5 28 –3 1. 5 32 –3 5. 5 36 –3 9. 5 40 –4 3. 5 44 –4 7. 5 48 –5 1. 5 52 4l valve length (µm) r e la ti v e fr e q u e n c y (% ) l. hövsgöl l. baikal l. kurilskoye fig. 30. relative frequency histogram of valve length of gomphonema ventricosum in the three lakes. u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:47 color profile: disabled composite 150 lpi at 45 degrees breadth than those from lake hövsgöl (fig. 31). specimens from lake baikal are shorter and narrower than those of the other two lakes. these tendencies are also clear from the microphotograph data (figs. 1–18). initial valves of g. ventricosum (figs. 27–29) were observed from lake hövsgöl and lake kurilskoye. initial valves from these lakes are, as mentioned by kociolek and stoermer (1987), tumid in their central portion and taper gently towards both poles, appearing almost naviculoid in shape. initial valves appear to be larger than valves of normal acta bot. croat. 68 (2), 2009 427 gomphonema ventricosum gregory from far eastern lakes 0 5 10 15 20 25 30 35 40 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 valve breadth (µm) r e la ti v e fr e q u e n c y (% ) l . hövsgöl l. baikal l. kurilskoye fig. 31. relative frequency histogram of valve breadth of gomphonema ventricosum in the three lakes. 0 5 10 15 20 25 30 35 40 45 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 l . hövsgöl l. baikal l. kurilskoye r e la ti v e fr e q u e n c y (% ) striae density (number 10µm ) of valve–1 fig. 32. relative frequency histogram of striae density of gomphonema ventricosum in the three lakes. u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:47 color profile: disabled composite 150 lpi at 45 degrees vegetative cells. valve size is in general closely related to the frequency of auxospore formation and this coincides well with our data, in which initial valves were rarely observed in the population from lake baikal. with respect to striae densities, the lowest densities of 12 striae per 10µm were recorded in specimens from lake baikal. higher striae densities were observed in specimens from lake kurilskoye (fig. 32) and the average density is 15.2 in 10 µm, while the densities specimens of lake hövsgöl and lake baikal are 14.2 and 13.9, respectively (tab. 2). with regard to lineolae densities, specimens from lake baikal have a lower density while higher densities were recognized in specimens from lake kurilskoye (fig. 33). discussion specimens of gomphonema ventricosum collected from lake hövsgöl, lake baikal and lake kurilskoye are classified into five types based on valve outline and morphometric differences between specimens from the three lakes, including valve length, breadth, striae density and lineolae density. kociolek and stoermer (1987) reported that illustrations of g. ventricosum from central europe (hustedt 1922, 1930), and those from scandinavia (cleve-euler 1955, tynni 1978) are very similar to type a and moreover to the type specimen of h. l. smith (slide #670 from christiana, sweden). this type is common in lake hövsgöl and lake kurilskoye, representing 86.7% and 78.1% of the populations, respectively. specimens of type b appear most frequently in the population from lake baikal. in specimens from lake baikal, valves with a weakly protruding headpole resemble the specimens in the lake baikal sample of kociolek and stoermer (1987, figs. 20–23). in their study, specimens from lake baikal exhibited a high degree of variability in valve shape; however, the specimens in our population show less variation. three microphotographs from kamchatka (schmidt 1899) show a great similarity to type b. type c was first described in karluk lake on kodiak island (manguin 1960). specimens from karluk lake 428 acta bot. croat. 68 (2), 2009 yoshitake s., fukushima h., kimura t., lepskaya e. v., ko-bayashi t. 0 5 10 15 20 25 30 35 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 lineorae density (number 10µm ) of valve–1 r e la ti v e fr e q u e n c y (% ) l. hövsgöl l. baikal l. kurilskoye fig. 33. relative frequency histogram of lineolae density of gomphonema ventricosum in the three lakes. u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:47 color profile: disabled composite 150 lpi at 45 degrees have a rounded central area and resemble gomphoneis eriense (grun.) skv. et meyer. regarding type e, skvortzow and meyer (1928) reported specimens of this type from lake baikal as g. ventricosum f. curta, later skvortzow (1937, pl. 14, fig. 22) named this type g. ventricosum. with respect to valve outline, specimens from lake hövsgöl and lake kurilskoye resemble the type form and those from lake baikal differ from those from the other two lakes. this applies also to valve length. in the lake hövsgöl and lake kurilskoye specimens, the mode of valve length appears to be longer than those from lake baikal (fig. 30). fig. 31 shows that specimens from each lake represent different ranges in valve breadth. skvortzow (1937) and dawson (1973) indicated coarse stria densities, as few as nine striae per 10µm in specimens from lake baikal; however, in our study the striae densities per 10µm ranged from 12.0 to 18.5. it is clear g. ventricosum appears to have a wide range of striae densities. in lake baikal skvortzow and meyer (1928) illustrated g. ventricosum and g. ventricosum f. curta, but skvortzow (1937) later showed four illustrations including the descriptions of g. ventricosum from lake baikal. recently, it has become difficult to find any data about g. ventricosum f. curta. judging from his four illustrations, these specimens include types b, c and e, apart from types a and d. this is in agreement with our lake baikal data. although negoro and ikuta (1989) showed six illustrations of gomphoneis from lake baikal, including three specimens of gomphoneis eriense var. eriense and three of g. eriense var. rostrata, these specimens are all considered to be g. ventricosum judging from the shape of the central area and location of the stigma. we classified these six specimens as types b and e. in this case, specimens of types a and d are rarely observed, as reported by skvortzow (1937). a comparison of the g. ventricosum specimens from the three lakes indicates that differences in valve outline can be recognized between specimens from lake baikal and those from the other two lakes as well as valve length, breadth and striae density. acknowledgements the authors are thankful to prof. genki inou, dsc, (otsuma women’s university) who collected and provided the samples from lake hövsgöl, and to aleksey maslov for helping to sample lake kurilskoye. references bahls, l. l., 2005: ecology of the diatom community of the upper east gallatin river, montana, with in situ experiments of the effect of current velocity of features of the aufwuchs. in: bahls, l.l. (ed.), northwest diatoms 2, 1–20 + 1–448. cleve-euler, a., 1955: die diatomeen von schweden und finnland. kungl svenska vetenskaps akademiens handlinger 5, 1–232. dawson, p. a., 1973: observations on some species of the diatom genus gomphonema c. a. agardh. british phycological journal 8, 413–423. foged, n., 1981: diatoms in alaska. bibliotheca phycologica 53, 1–317. gregory, w., 1856: notice of some new species of british fresh-water diatomaceae. quarterly journal of microscopical science 4, 1–14. acta bot. croat. 68 (2), 2009 429 gomphonema ventricosum gregory from far eastern lakes u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:47 color profile: disabled composite 150 lpi at 45 degrees hustedt, f., 1922: die bacillariaceen-vegetation des lunzer seegebietes (nieder-österreich). internationale revue der gesamten hydrobiologie und hydrographie 10, 40– 112, 233–270. hustedt, f., 1930: bacillariophyta (diatomeae). in: pascher, a. (ed.), die süsswasser-flora mitteleuropas, 10. gustav fischer, jena. kociolek, j. p., stoermer, e. f., 1987: geographic distribution and variability of the diatom (bacillariophyceae) gomphonema ventricosum gregory. nova hedwigia 45, 223– 236. krasske, g., 1932: beiträge zur kenntnis der diatomeenflora der alpen. hedwigia 72, 92–134. manguin, e., 1960: contribution à la flore diatomique de l’alaska: lac karluk, espèces critiques ou nouvelles. revue algologique, nouvelle series 5, 266–288. negoro, k., ikuta, m., 1989: a glimpse of the diatom – vegetation at some stations of the southern littoral zone of lake baikal. journal of the hiraoka environmental science laboratory 2, 1–17. schmidt, m., 1899: tafel 216. in: schmidt, a. (ed.), 1874–1959, atlas der diatomaceen-kunde. r. reisland, leipzig. skvortzow, b. w., 1937: bottom diatoms from olhon gate of baikal lake, siberia. philippine journal of science 62, 293–377. skvortzow, b. w., meyer, c. i., 1928: a contribution to the diatoms of baikal lake. proceedings of the sungaree river biological station 1, 1–55. tynni, r., 1978: über finnlands rezente und subfossile diatomeen, 10. geological survey of finland bulletin 296, 1–55. 430 acta bot. croat. 68 (2), 2009 yoshitake s., fukushima h., kimura t., lepskaya e. v., ko-bayashi t. u:\acta botanica\acta-botan 2-09\yoshitake.vp 6. listopad 2009 13:00:47 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 76 (2), 2017 b1 morfologija biljaka. razvoj, građa i uloga biljnih tkiva, organa i organskih sustava. by toni nikolić. 2017, 569 pp. isbn 978-953-297-805-6. publisher: alfa d.d., zagreb. at the beginning of the development of botany as a scientific discipline morphological data was almost the only type in use. even today, most of the information about plants we have is actually morphological data. in spite of numerous new biological disciplines, morphology still plays a central role in biology (kaplan 2001) and still remains fundamentally relevant to practically all biological disciplines, such as molecular genetics, physiology, ecology, evolutionary biology and systematics (sattler and rutishauser 1997). for that reason, there is a need for quality, comprehensive books that will give an overview of all aspects of plant morphology. a new textbook, in croatian, entitled „plant morphology. development, structure and role of plant tissues, organs and organic systems” by the author tony nikolić is presented here. in this textbook, the author gives a modern presentation of all scientific aspects of plant morphology as a fundamental botanical discipline. plant morphology is interpreted in a broad sense, not only the external macro morphological features but also the form and structure at all organizational levels (whole plants, organs, tissues, cells and molecules). the book has 569 pages divided into 17 main chapters. the text is accompanied by 480 numbered illustrations (often very complex including microand macro-photographs, diagrams and hand drawings) and 30 tables. in the introduction the author defines morphology, and refers to the history of morphology from its founding to today’s modern concepts. he then continues with a chapter on basic morphological terminology (prefixes and suffixes, names of surface forms and bodies, colour and symmetry) as a necessary prerequisite for the transfer of knowledge about the richness of forms and morphological phenomena among botanists. he then continues with a chapter on alternations of plant generations in which he talks about sporophytes and gametophytes, fertilization and embryogenesis. in the chapter anatomy basics, he presents plant tissues involved in the formation of plant organs. as the author himself states, only a brief overview of the basic types of tissue is given here and a deeper knowledge of the anatomy of plants requires reference to more extensive literature. however, this part of the textbook is very useful and will be of value primarily to teachers, professors and students because there are very few texts on plant anatomy available in croatian. in the chapter on morphology of vegetative organs, root, shoot and leaf are elaborated in detail, as well as the morphological adaptation of plants to different abiotic conditions. understandably, the greatest part of the textbook is book review b2 acta bot. croat. 76 (2), 2017 dedicated to the chapter on morphology of reproductive organs. considering the alternations of plant generations, it processes the structures of dual origin. thus, in the introductory parts he presents structure, development and evolution of sporangia and sporophylls as well as gametophyte. following that, the angiosperm flower morphology with its parts such as receptacle, perianth, stamen, pollen, carpel and nectary, are elaborated in detail. finally, there is a chapter on the origin, structure and classification of the inflorescences. as already mentioned, the morphology of plants is here understood broadly, so that the morphology of reproductive organs is understood in terms of generative propagation. thus the structures and processes related to generative reproduction of plants are discussed in several chapters. firstly, come two extensive chapters on pollination, fertilization and embryogenesis. then chapters on seeds and fruits follow logically, where the author has detailed and illustrated the classification of fruits based on the fundamental morphology of the carpel. in this part of the textbook, a number of examples of morphology and fruit anatomy are described, including, among other things, the economically most important fruits such as citrus, apple, tomato, banana, coconut and others. in separate chapters, the dispersion of propagules and dispersion strategy in plants, as well as germination and seedling morphology are shown. the last chapters cover vegetative propagation in plants, as well as teratology, chimeras and other morphological forms. at the end of the book the author gives an extensive list of references and an index. of particular value in the book are the 30 frames with excellent texts which give some morphological topics in more detail (e.g. leaf type classification, shapes of lamina apex, margin and base, floral diagrams, etc.) with some interesting specificities of the world of plant morphology (e.g. pollination in the common fig, recognition of forms, colours and patterns, seeds as food, grain in human consumption, the strange balausta fruit – punica granatum, etc.). the book is generally presented in a highly systematic, clear and understandable way. this was not an easy task given the scope of the material, and also the fact that plant morphology is treated here as a multidisciplinary area that draws on a knowledge of numerous branches of biology. it offers the reader not only an overview of the structure and appearance of plant varieties, but also an understanding of the role and manner of functioning and the origins of certain plant organs and organisms as a whole. considerable attention is devoted to issues of significance in applied botanical disciplines such as agriculture, forestry, pharmacy, horticulture etc. it is of particular value that the textbook proposes unambiguous croatian terms for certain plant organs, parts of organs and various processes. this will greatly help standardize croatian terminology in plant morphology as a basic botanical discipline. the croatian literature in this field is scarce and our botanical terminology comprises a great number of terms that overlap or are not clearly defined, as well as synonyms of latin, greek, german, croatian and other origins. all in all, this publication is an excellent example of a textbook that incorporates the latest scientific knowledge in the field of plant morphology. it can be concluded that such an original and up to date textbook will be a major contribution not only to the improvement of university botanical teaching in croatia but also to the advancement of various scientific and professional disciplines relying on the morphology of plants. željko škvorc faculty of forestry, university of zagreb, croatia references kaplan, d. r., 2001: the science of plant morphology: definition, history, and role in modern biology. american journal of botany 88, 1711–1741. sattler, r., rutishauser r., 1997: the fundamental relevance of morphology and morphogenesis to plant research. annals of botany 80, 571–582. book review acta bot. croat. 79 (1), 2020 87 acta bot. croat. 79 (1), 87–94, 2020 coden: abcra 25 doi: 10.37427/botcro-2020-011 issn 0365-0588 eissn 1847-8476 optimizing irrigation and determining the most sensitive development stage to drought in barley (hordeum vulgare l.) in a semi-arid environment leila romdhane1, nicola dal ferro2, amor slama3*, leila radhouane1 1 university of carthage, national institute of agronomic research of tunisia, hédi karray street, 2049 ariana, tunisia 2 university of padova, department of agronomy, food, natural resources, animals and environment, viale dell’università 16, 35020 legnaro, padova, italy 3 university of carthage, faculty of science, 7021 jarzouna-bizerte, tunisia abstract – rising temperatures and increasing water scarcity, which are already important issues, are expected to intensify in the near future due to global warming. optimizing irrigation in agriculture is a challenge. understanding the response of crop development stages to water deficit stress provides an opportunity for optimizing irrigation. here we studied the response of two barley varieties (rihane, martin), to water deficit stress at three development stages (tillering, stem elongation, and heading) by measuring water status and grain yield components in a field experiment in tunisia. the three stages were selected due to their importance in crop growth and grain development. water deficit stress was initiated by withholding water for 21 days at the three stages with subsequent re-watering. water deficit led to a progressive decrease in leaf water potential. in both varieties, heading was the stage most sensitive to water deficit. leaf water potential measurements indicated that water deficit stress was more severe during heading, which to some extent may have influenced the comparison between growth stages. during heading, the number of ears per plant and weight of a thousand grains were reduced by more than 70% and 50%, respectively compared with stress at tillering. comparison of yield components showed differences between the two barley varieties only when the water deficit was produced during the tillering stage. keywords: barley, climate change, crop development stage, grain yield components, leaf water potential, water deficit. * corresponding author e-mail: slamaamor@yahoo.fr nicola dal ferro and amor slama contributed equally in writing and reviewing of this paper. introduction climate change has detrimental impacts on agriculture worldwide (stevanović et al. 2016). the mediterranean region, described as the hot-spot of climate change (rochdane et al. 2014), is vulnerable to changes in climate, with a predicted decrease in crop production(olesen et al. 2011). several models envisaged a shorter growing season, increased heat, and water deficit stress in southern mediterranean regions, which will reduce harvestable yields in both spring (4-40%) and winter crops (4-17%), counteracting the gains due to increased atmospheric co2 concentration (giannakopoulos et al. 2009, moriondo et al. 2011, gammans et al. 2017). the main rainfed crops grown in tunisia are cereals (mainly wheat – triticum durum desf. – and barley – hordeum vulgare l.), occupying about two-thirds of total cultivated areas and accounting for 16% of the agricultural production value. cereal yields can vary significantly from year to year due to unpredictable and largely irregular rainfall patterns (deghaïs et al. 2007). this situation will become more critical against the background of a changing climate, which already impacts agricultural production around the globe. the production of cereals, which are the most strategic and vital crops in tunisia, is projected to be severely compromised by the combined effects of high temperature and water deficit (perniola et al. 2015). recent studies have shown that already by 2020, rainfall is expected to drop by between 5 and 20% in tunisia, while by 2100 temperatures could rise between 2 and 4 °c (mougou et al. 2011). these changes could contribute to soil degradation as a result of drought and other restrictive factors (sultan 2012, balkovič romdhane l, dal ferro n, slama a, radhouane l 88 acta bot. croat. 79 (1), 2020 et al. 2018). in agronomy, to solve the issues of water shortage we have to cultivate low water requiring crops or apply less water to the crop (bashir et al. 2017). in addition to being affected by scarce rainfall and its patchy distribution during the cereal season, yield depends on fertilization and the ability of selected varieties or species to cope with water deficits. under water deficit conditions, plants present several morpho-physiological and biochemical changes as part of their strategies to reduce water deficit stress effects (slama et al. 2018), which has led farmers and researchers to look for alternative varieties, such as the old ones, in which lower yields can be offset with better adaptation to the environment (malek and verburg 2018). barley is the second most cultivated cereal crop in tunisia after durum wheat. barley yield components depend on the different development stages (vegetative period, heading, anthesis, and post-anthesis), on the availability of assimilates, on genotype, and on the amount of water supplied (tambussi et al. 2005, al-ajlouni et al.2016).water deficit stress can be mitigated by supplementary irrigation, especially during the critical development phases (meng et al. 2017, wang 2017). moreover, the response of crop development stages to water deficit stress is apparently more important than the quantity of water supplied (morison et al. 2008). it is important to adopt a water-saving management regime (zhang et al. 2019). in this context, attention is increasingly being paid to old varieties as alternatives to recent ones. it is suggested that their genetic heritage could be a resource for actual and future adaptation to changing climatic conditions. however, to our knowledge only a few have studied the growth behavior of recent and old varieties under water deficit stress (cattivelli et al. 2011). in this context, the aim of this study was to determine the development stage most sensitive to water deficit conditions and to assess whether the sensitive period differs among varieties in relation to drought. two barley varieties, an old (martin) and a recent one (rihane) that are both recognized as being drought-tolerant, were selected and submitted to episodic water deficit stress and then re-irrigated at different development stages: (i) to determine the most critical period under water deficit, (ii) to quantify the effect of this constraint on the recent and old variety. materials and methods study area the study was conducted at the experimental farm of the national institute of agronomic research of tunisia (inrat) at ariana (36°51' n, 10°11' e), in northern tunisia. the local climate is semi-arid, with a mean annual rainfall of about 320 mm and a mean annual temperature of 20.2 °c, ranging from the lowest average monthly temperature in january/february, 11.5 °c, to the highest in july-august, 27 °c (fig.1). the soil is a silt loam (sand 23.5%, silt 55.1%, clay 21.4%), with aph of 8.2 and soil organic matter content of 2%. the soil water retention capacity is 0.30 kg kg–1 at –0.05 mpa, and 0.20 kg kg–1 at -1.5 mpa. additional information was already reported in romdhane et al. (2016). plant growth and water deficit stress the experiment was conducted in the 2015-2016 cropping season. two barley (hordeum vulgare l.) varieties, martin and rihane, were selected for the study. these varieties cover roughly 35% of total barley cultivation in tunisia (ouji et al. 2018). martin is an old six-row variety developed in algeria in 1931. martin is early maturing, has moderate yield (2.7 t ha–1), good resistance to water deficit stress (deghaïs et al. 2007, ouji et al. 2018). rihane is also a six-row variety. it was released by the international center for agricultural research in the dry areas (icarda) and introduced in tunisia in 1987. rihane is widely cultivated in tunisia because of its early maturity, high yield (3.56 t ha–1), and resistance to water deficit stress and fungal diseases. permanent wilting point in barley ranges from -1.8 mpa to -1.5 mpa (mansouri and radhouane 2015, ouji et al. 2018). the seedbed was prepared in the same way for both barley varieties, with autumn ploughing at 0.3 m depth, followed by harrowing at 0.2 m just before sowing. sowing was on november 15, 2015. each plot was planted in four 2m-long rows, spaced 0.25 m apart, and an area of 2 m2 perplot at a density of 400 seeds m–2 in four replicates. fertilization consisted of two applications of ammonium-nitrate (n = 33%): 100 kg n ha–1 at sowing and 100 kg n ha–1 at four leaf stage, during tillering. the experiment was conducted in two adjacent plotsas completely randomized design for each trial: water deficit stress and control (no water deficit) trials. each trial was organized in 24 plots (2 varieties × 3 stages × 4 replicates), separated 1.5 m apart on each side to minimize the effect of lateral water movement. water deficit was initiated by withholding irrigation for 21 days during tillering (zadoks scale = 24), stem elongation (zadoks scale = 32), and heading (zadoks scale = 51) (fig. 1). the twovarieties have similar growth stages and water deficit treatment was not influenced by differences in development. at the end of the stress, fig. 1. daily mean of air temperature (°c) and rainfall (mm) during the crop season (from november 2015 to june 2016). response of barley to drought acta bot. croat. 79 (1), 2020 89 plots were re-watered to the control level. control plots were maintained at 0.30 kg kg–1of soil water content (about -0.05 mpa) throughout the experiment. manual irrigation was applied by assessing soil moisture in the 0–50 cm soil layer, which was measured every week by gravimetric method. if rain seemed likely, all plots were covered with transparent plastic film to prevent variations in moisture content.as a consequence, radiation, air humidity and temperature also differed from natural atmospheric conditions during the periods of soil and vegetation cover. soil moisture and plant water status soil moisture and leaf water potential were measured on day 1 and every week thereafter during the 21-day stress (d1: 1 day of dry, d7: 7 days of dry, d14: 14 days of dry, d21: 21 days of dry) (figs. 2 and 3). to measure soil moisture, four bulk samples were taken from different areas in the middle of each plot along the 0–50 cm depth, wrapped in bags, and immediately transferred to the laboratory. then, 50 g fresh soil of each replicate was placed in aluminum cans and dried in an oven at 110 °c for 72 hours for dry weight determination. leaf water potential was determined by the pressure chamber method according to scholander et al. (1961), and expressed in mpa. it was measured with a model 1000 pressure chamber (pms instrument company, albany, usa) on the different dates (d1, d7, d14 and d21). measurements of leaf water potential were always conducted on the fully developed flag leaf on four randomly selected plants of the two middle rows and from the two water treatments, between 10:00 a.m. and 12:00 noon. at maturity, number of ears per plant (nep), number of grains per ear (nge), and thousand grains weight (wtg) were measured in both control and water deficit plots. statistical analysis changes in soil moisture and leaf water potential between the two varieties were evaluated using repeated measure anova. measurements taken over the four time points (1, 7, 14, 21 days) during the 21-day stress period were compared in the two varieties and the three developmental stages (tillering, elongation, and heading). in addition, nep, nge, and wtg, were compared using two-way anova, by treating variety and developmental stage as fixed factors. anova assumptions of normality and homogeneity of variance were tested for all parameters using the shapiro-wilk test and the bartlett test, respectively. significant differences between means were differentiated with a post-hoc student-newman-keuls test (p< 0.05). statistical analyses were performed using stat-itcf version 5 software. results variation of soil moisture and leaf water potential the decreases in soil moisture throughout the drying cycle are shown in fig. 2. for both cultivars rihane and martin, soil moisture evolved differently depending on the development stage that was affected by water deficit stress. in particular, soil moisture during stem elongation was generally higher than in both tillering and heading until d14 (i.e., after 14 days of dry conditions), whereas the differentiation was reduced at d21 when significant differences were observed between heading and the other two development stages, i.e. tillering and stem elongation, under water deficit conditions. in contrast, no significant differences were observed in soil moisture between varieties (tab. 1). since conditions were similar for both barley varieties (e.g., soil, evaporative surface, sowing density), the differences that were observed in terms of soil moisture were mainly due to the development stage and transpiration of each variety. indeed, both for rihane and martin varieties, soil moisture changes differed depending on development stage. although the water deficit period was the same for all stages, the permanent wilting point (-1.5 mpa, 0.20 kg kg–1) was reached only during the heading stage. wilting point was earlier for martin (before d14), while the rihane variety reached it after d14 (fig. 2). both varieties appeared to absorb more water at the tillering stage than at the elongation stage during the first 15 days of water deficit stress. after this first period, the decrease in soil moisture was similar for the two stages, although more pronounced during stem elongation. the leaf water potential in the control treatment was similar between varieties, being -0.65 mpa (±0.05 s.e.)and -0.70 mpa (±0.03 s.e.) in rihane and martin at d1 tillering, and slightly decreasing until minimum values of -1.1 mpa (±0.05 s.e.) and -0.7 mpa (±0.00 s.e.) at d21, respecfig. 2. gravimetric soil moisture (%) dynamics in water deficit stress treatments according to days of dry (d1, d7, d14, d21) at different development stages in rihane (a) and martin (b) barley varieties. (variety × stage ×days of dry; p< 0.05, four replicates). error bars ± standard error. d1:1 day of dry, d7: 7 days of dry,d14: 14 days of dry, d21: 21 days of dry. romdhane l, dal ferro n, slama a, radhouane l 90 acta bot. croat. 79 (1), 2020 tively. similar dynamics were observed at elongation, when rihane and martin slightly decreased leaf water potential from –1.1 (±0.03 s.e.) at d1 to –1.2 (±0.04 s.e.) at d21, on average, as well as at heading, when on average both varieties reduced it as follows: –1.8 mpa (±0.05 s.e.) at d1 > –2.0 mpa (±0.08 s.e.) at d7 > –2.2 mpa (±0.03 s.e.) at d14 > –2.3 mpa (±0.04 s.e.) at d21. the decrease in leaf water potential (ψh) according to soil drying conditions for both varieties and during the different stages are shown in fig. 3. leaf water potential at the tillering stage was significantly higher (p< 0.001, tab. 1) than at the other two stages. martin and rihane showed average values of –0.92 and –0.89 mpa, respectively, which decreased to –1.36 and –1.23 mpa, and –2.93 and –2.66 mpa, during elongation and heading. despite differences being observed in barley growth stages, similar ψh dynamics were observed in tillering and elongation. in contrast, a strong ψh reduction was found during heading, highlighting firstly its gradual decrease at soil moisture >20%, thereafter a sharp reduction at values close to wilting point when leaf water potential reached minimum values of –4.10 and –3.88 mpa in martin and rihane, respectively. the regressions highlight similar crop response in the two varieties, especially during tillering and elongation (fig. 4). increasing differences were observed at heading stage (fig. 4c), when the martin variety reached similar ψh to those recorded in rihane, but in drier soil conditions. for instance, during tillering and elongation a slight leaf water potential reduction was observed at increasing drought: a 10% decrease in soil moisture (from 30% to 20%) induced only 0.5 mpa ψh average changes (fig.4a, 4b), whereas the same difference in soil moisture caused ψh to be reduced to 1.2 mpa in heading. these results were also emphasized by large variations between maximum and minimum leaf water potential values during the water deficit stress treatment, which showed: (i) higher variations in heading, followed by tillering and finally elongation, (ii) negligible changes between varieties (tab. 2). tab. 1.analysis of variance for soil moisture and leaf water potential. abbreviations: df = degree of freedom, ss = sum of squares, ms = mean square, f = fisher test. development stages: tillering, stem elongation, and heading. cultivars: rihane and martin. days of dry: d1: 1 days of dry, d7: 7 days of dry, d14: 14 days of dry, d21: 21 days of dry. source df ss ms f p-value soil moisture main effects variety 1 3.20 3.20 1.8 0.224 stage 2 176.44 88.22 50.6 < 0.001 days of dry 3 889.35 296.45 236.8 < 0.001 interaction variety × stage 2 14.39 7.20 4.1 0.008 variety × days of dry 3 1.94 0.65 0.5 0.677 stage × days of dry 6 70.77 11.80 9.4 < 0.001 variety × stage × days of dry 6 6.39 1.06 0.8 0.548 leaf water potential main effects variety 1 0.48 0.48 102 < 0.001 stage 2 63.30 31.65 6703 < 0.001 days of dry 3 15.42 5.14 851 < 0.001 interaction variety × stage 2 0.23 0.11 24 < 0.001 variety × days of dry 3 0.22 0.08 12 < 0.001 stage × days of dry 6 8.70 1.45 240 < 0.001 variety × stage × days of dry 6 0.44 0.07 12 < 0.001 fig. 3. leaf water potential (mpa) dynamics in water deficit stress treatments according to days of dry (d1, d7, d14, d21) at different development stages in rihane (a) and martin (b) barley varieties. (stage × days of dry; p< 0.05, four replicates). error bars ± standard error. d1:1 day of dry, d7: 7 days of dry, d14: 14 days of dry, d21: 21 days of dry. response of barley to drought acta bot. croat. 79 (1), 2020 91 variation of yield components at the end of each drying cycle, barley was re-irrigated and yield components were measured at harvest. barley yield has two major components, i.e. grain number and grain weight (al-ajlouni et al. 2016); it is hence critical to study the number of ears per plant, number of grains per ear, and weight of a thousand grains. analysis of variance indicated that both variety and stage had significant effects (p< 0.05) on ear number per plant (nep) (tab. 3), which ranged between 1.10 in martin at heading and 5.00 in rihane at tillering. however, the main differences were observed between water deficit stress stages that always differed from one another, whereas varieties at the same stage of water deficit stress showed differences only at tillering. the variation in nep, according to the time of water stress application (development stage) and variety, showed that when water deficit was induced at a later stage, nep was lower; the heading stage thus appeared to be the most sensitive to water deficit stress, which can be quantified as –76% with respect to the control treatment. mean grain number per ear (nge) was similar for the two varieties (tab. 3), although with a tendency to higher nge in martin in the control treatment and with water deficit stress at tillering only. however, significant differences in water deficit stages were observed between tillering (37.0, on average) and both elongation and heading (17.2, on average). as a result, it can be stated that nge was more affected when the water deficit stress was induced late, during the elongation and heading stages, than in the early period, during tillering. the period least sensitive to water deficit was the tillering stage, with nge values twice as high as those for the heading stage. a significant reduction in weight of a thousand grains (wtg)was also observed according to the stage of the water deficit reached. when water deficit stress was induced at a later stage, the wtg reduced significantly from 42.4 g at tillering, to 28.9 g at elongation, and 20.55 at heading. as also observed for other parameters, the heading stage appeared more sensitive than the tillering stage, with a reduction of 50%. contrarily to nep, wtg was significantly higher in martin (44.4 g) than in rihane (40.4) when water deficit stress was induced during tillering (tab. 3). tab. 2. percentage decrease rate (1 – (ψhmin/ψhmax) × 100) of leaf water potential (ψh) between maximum and minimum values for two barley varieties, rihane and martin. stages varietyrihane martin tillering 49.1% 47.0% elongation 27.0% 27.0% heading 57.4% 55.0% fig. 4. regressions between soil moisture and leaf water potential for the two barley varieties (martin, rihane) at different stages of water deficit stress: tillering (a), elongation (b), and heading (c). tab. 3. comparison of number of ears per plant (nep) and grains per ear (nge), and weight of a thousand grains (wtg) (two-way anova, varieties × stage) for two barley varieties, rihane and martin. different letters (a, b, c, d) indicate significant differences according to student-newman-keuls test at p < 0.05 ± standard error. control treatment (without water deficit stress) was not included in the statistical analysis. control tillering elongation heading nep rihane 6.25±0.37 5.00±0.36a 2.40±0.056c 1.40±0.15d martin 4.35±0.15 3.6±0.16b 2.20±0.17c 1.10±0.06d nge rihane 35.25±1.44 35.60±1.36a 17.5±0.28b 17.9±2.18b martin 42.25±2.17 38.4±3.25a 14.4±1.83b 18.9±3.79b wtg rihane 40.25±0.85 40.4±0.48b 28.8± 0.89c 20.0±0.40d martin 46.50±0.65 44.4±0.25a 29.0±0.83c 21.1±0.36d romdhane l, dal ferro n, slama a, radhouane l 92 acta bot. croat. 79 (1), 2020 discussion water deficit is one of the most constraining factors for the growth, development and yields of plants in arid and semi-arid regions of the world (ben naceur et al. 2018). in this study, water deficit stress induced a decrease in leaf water potential. this reduction depends on the development stage and plant growth conditions. the exposure of barley to a drying cycle showed a highly significant variation between the three development stages and two studied varieties. according to previous studies, plant behavior depends on the variety, duration and intensity of the water deficit stress, but also its timing (tardieu 2013). leaf water potential decreased proportionally with reductions in soil moisture, although some decrease was also observed in the control treatment that may depend on climatic conditions (lösch et al. 1992). however, a sharp decrease in ψh (down to 58%) was observed only at the heading stage during the 21-day water deficit test, which was due to high plant water requirement. indeed, soil moisture reached values far below 20%, which is characterized by -1.5 mpa of matrix potential and can be considered the permanent wilting point. in contrast, at both tillering and stem elongation soil moisture never reached values below 20% so that barley varieties did not undergo critical water deficit stress. of the investigated development stages, heading was the most sensitive to water deficit stress, while stem elongation was the least affected. plant water potential is a good indicator of water deficit stress and its magnitude is influenced by both soil water deficit as well as evaporative demand. this could be seen during heading, where the plant water potential was much lower than during tillering and elongation at similar soil water content values. the measured leaf water potentials are in good agreement with the results of lösch et al. (1992), who found that leaf water potential of barley could decrease to 2.5 mpa in fully irrigated treatments under warm sunny conditions and to values of -4.0 mpa in drought stressed treatments. ben naceur et al. (1999) revealed a leaf water potential of -0.8 mpa and a humidity of 18% under well-watered conditions, buta leaf water potential and a humidity rate of -2.2 mpa and close to 12%, respectively, at boot swollen and anthesis stages, for plots subjected to water deficit stress. reduced leaf water potential has a primary role in osmoregulation and maintenance of plant tissue water status. under water stress, abscisic acid is produced in shoot and root tissue and starts ionic regulation of water status at cell and tissue levels. therefore, it is a combination of osmotic stress, hormonal metabolism and ionic regulation that maintains plant water status at the cost of plant growth and leads to osmoregulation. nonetheless, osmoregulation at the cost of plant growth is acceptable in dry environments to make crops drought tolerant and water use efficient even with reduced yield (farooq et al. 2019). nevertheless, maintaining a relatively constant leaf water potential when the soil is drying may be associated with a mechanism to avoid tissue dehydration (chaves et al. 2002, abid et al. 2018). this mechanism, observed at the tillering stage, could be explained by a high capacity to extract water from the soil and an efficient control of transpiration losses (tardieu 2013, fahad et al. 2017). regarding the varieties, similar behavior was observed in rihane and martin at late water deficit stress conditions, whereas greater water use and reductions in moisture and leaf water potential were observed in martin than rihane. the study of al-ajlouni et al. (2016) showed a huge impact of pre-anthesis water deficit on barley yield components due to water deficit stress during tillering and stem elongation stages, whereas in the mediterranean zone cereal crops are exposed to frequent post-anthesis water deficit. our results indicate that water deficit stress affects yield components to varying degrees, according to the development stages. in general, all treatments negatively affected yield parameters, but water deficit stress during the heading stage had the most detrimental effects. this constraint at the heading stage led to the largest difference in crop development parameters. several authors have reported the detrimental effect of water deficit stress at the anthesis period and its consequences for grain number per ear (al-ajlouni et al. 2016, mahrookashani et al. 2017) and grain weight and size (maiti and satya 2014, mahrookashani et al. 2017). ben naceur et al. (1999) in a wheat crop study demonstrated that water deficit stress caused a reduction of the yield parameters. when the water deficit occurred at the tillering stage, it mainly reduces the number of ears by surface unit, quantified in about 40%. moreover, it was 33% and 17% respectively at swelling and anthesis stages, thereby reducing the weight of final yield. consequently, whatever the stage during which the water deficit occurs, it affects both growth and yield. however, when it occurs just before heading (boot swollen), its consequences are the most harmful. during this period the ear is already formed, but the organ of flowering can be seriously damaged. therefore, supplemental irrigation during this period is pivotal to mitigate the effects of water deficit stress. the decrease in nge would be one of the most significant effects of water deficit stress (honsdorf et al. 2017, senapati et al. 2019). our results are supported by a previous study by saedi et al. (2012), which showed that water deficit during the grain filling stages significantly reduced wtg and thus grain yield, especially in more sensitive varieties. breeders tend to breed barley varieties that can cope with water deficit during the most critical development stage before the beginning of grain filling, which is essential for the determination of grain number (francia et al. 2013). our study indicated that nge, nep and wtg were reduced by more than 50% under water deficit conditions in the later stages. it should be noted that the same decrease in leaf water potential and in grain yield was recorded during the heading stage (50%) underwater deficit. these reductions could be explained by sterile pollen or decreased pollen numbers reducing the final grain number per ear (fahad et al. 2017, honsdorf et al. 2017), or by the high accumulation of abscisic acid in the ear (dong et al. 2017). according todong et al. (2014), water deficit induces pollen sterility and, consequently, reduces the number of grains. this effect can be hypothesized in both martin and rihane. response of barley to drought acta bot. croat. 79 (1), 2020 93 at the tillering stage, water deficit stress did not significantly affect the plants, suggesting that the studied varieties could overcome water-deficit periods and resume growth after rehydration. moreover, a greater adaptation to water deficit stress at tillering was observed in martin than in rihane, which was quantified in both higher nep and nge. farooq et al. (2019) reported that significant genetic variation exists among crops or within genotypes of the same crop for water use efficiency, suggesting the need to tailor more water-efficient genotypes. when the water deficit stress period ceased, even small amounts of water could have significant impacts on plant physiological functions (tambussi et al. 2005). in this context, abid et al. (2018) and boguszewska-mańkowska et al. (2018) showed that drought-tolerant plants, as opposed to drought-sensitive plants, were able to produce a high quantity of dry matter after rehydration. thus, in the case of severe water deficit at the tillering and elongation periods, irrigation is recommended for farmers. conclusions the aim of this study was to investigate the effect of water deficit and its timing on barley yields. water deficit stress led to a gradual decline in leaf water potential and a decrease in yield components. however, plant responses depended on the time of stress application (stage) and its intensity (number of dry days). the effects of water deficit on yield were more pronounced when it occurred at the heading stage of the two studied varieties, an old (martin) and a recent (rihane) one, as suggested by the lowest water potential reduction at the heading stage under soil moisture conditions comparable with the tillering and elongation ones. a leaf water potential reduction of 50% at the heading stage during 21 days of water deficit stress generated the same reduction in grain yield (50%). this may be explained by the low capacity of the plant to recover growth when the water deficit stress occurred during a late plant development phase. the stage considered the most sensitive – heading – is the one when the leaf area was expectedly the largest. additionally, at this time of the cycle plants are more advanced and may suffer from increased temperatures during the late growing season,which might partially compromise a full comparison between development stages. irrigation in this period can help to increase the resistance of barley cultivars to water deficit conditions and improve grain yield in the mediterranean zone. this work shows that if it is possible to reduce and to manage the amount of irrigation water in the semi-arid zone, irrigation will be economically more gainful because supplemental irrigation during dry periods is more efficient at the heading stage. the use of less water through thecereal cropping season helps countries to save their water. however, additional experiments under fully controlled conditions will provide a better understanding of the plant response to water stress conditions. martin barley, which is an old variety, performed better than rihane in terms of weight of a thousand grains and has had traits of tolerance to water deficit since its creation. these characteristics improved over time because it has increasingly adapted to a semi-arid environment. these results highlight the fact that old varieties should be promoted as a means to preserve their genetic heritage, and used for improving adaptation to changing climatic conditions. acknowledgments authors are grateful to dr. hatem cheikh m'hamed, national institute of agronomic research of tunisia. this study was supported by the national institute of agronomic research – tunisia (inrat). references abid, m., ali, s., qi, l.k., zahoor, r., tian, z., jiang, d., snider, j.l., dai, 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cetinjski put bb., 81000 podgorica, montenegro 2 department of biology, faculty of science, university of novi sad, trg dositeja obradovi}a bb., 21000 novi sad, serbia 3 institute of marine biology, p.o. box 69, 85330 kotor, montenegro nutrients and chlorophyll a concentrations were analysed at five sampling stations in the inner part of the boka kotorska bay (southern adriatic sea), in the period september 2003 to august 2004. maximum concentrations of nitrates (14.4 mmol l–1), nitrites (3.5 mmol l–1), phosphates (0.84 mmol l–1), and silicates (4.5 mmol l–1) preceded maximum phytoplankton biomass (5.07 to 6.47 mg m–3 chlorophyll a) in december. chlorophyll a was positively correlated with oxygen, nitrites and silicates, but negatively correlated with temperature, salinity, transparency and nitrates. kotor bay is the most eutrophied environment in the eastern coastal adriatic sea. key words: chlorophyll a, nutrients, seasonality, eutrophication, adriatic sea introduction eutrophication in the mediterranean sea has been mainly related to local extensive use of the littoral zone, maritime activities and the increasing development of tourism. the frequency of high chlorophyll a concentrations, blooms of toxic algae, occurrences of anoxia, hypoxia and changes in the composition of planktonic and benthic communities steadily increases along the mediterranean coast, especially in coastal embayments into which elevated nutrient loads are discharged (vili^i], 1989, vollenweider 1992, le pape et al. 1995, degobbis et al. 2000, moncheva et al. 2001, puigserver et al. 2002, de jonge et al. 2002). the boka kotorska bay is located in the south eastern part of the adriatic sea, comprising a part of the montenegrin coast. in the last decade, the effects of anthropogenic impact have been evident in its inner part. the bay is greatly influenced by the great influx of fresh water from numerous streams and submarine springs. the scope of this paper is to present acta bot. croat. 68 (1), 2009 45 * corresponding author, e-mail: sladjana69@yahoo.com u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:23 color profile: disabled composite 150 lpi at 45 degrees spatial and temporal changes in physicochemical parameters and phytoplankton abundance and biomass (chlorophyll a concentrations) in the inner part of boka kotorska bay. materials and methods boka kotorska bay (42° 31’ 00’’ n – 42° 23’ 32’’ s; 18° 46’ 32’’ e – 18° 30’ 29’’ w) occupies an area of 87.3 km 2 , a volume of 2.4 � 10 6 m 3 , and a coastline length of 105.7 km. the inner part of boka kotorska bay comprises 27% of the area and 26% of the volume of the whole bay. seawater samples were taken with a hydrobios sampler, at five stations in the inner part of the bay (fig. 1), which comprises three coves: morinj cove in the west, risan cove in the north and kotor cove in the east. the sampling was performed on a monthly basis from september 2003 to august 2004. at the innermost stations near the institute of marine biology (1), orahovac (2) and morinj (5), samples for physical, chemical and biological analyses were taken from three depths: 0, 4, and 8 m. in the central stations of kotor (3) and risan (4) coves, samples were taken from five depths: 0, 5, 10, 20, and 30 m. temperature and salinity were measured in situ by multi line p4 – universal meter. oxi-guard handy gamma was use for measuring oxygen concentrations in situ. transparency was determined with a white secchi disk. the nutrient samples were taken with niskin bottles and stored in polyethylene bottles. determination of nutrients needed to be done the same day after sampling, or the else nutrients would freeze. nitrates, nitrites, phosphates and silicates were determined at sea by using standard methods (strickland and parsons 1972). the absorbance readings were made on a perkin elmer uv/vis spectrophotometer (lambda 2), at at different wavelength for every nutrient. all samples for chlorophyll a measurement were pre–filtered through a 330 μm mesh net to remove large zooplankton. after filtration through whatman gf/f, pigment extraction was performed in 90% acetone, and chlorophyll a concentrations were determined by measurement of absorbance with perkin – elmer uv/vis spectrophotometer, and calcula46 acta bot. croat. 68 (1), 2009 krivokapi] s., stankovi] @., vuksanovi] n. fig. 1. location of the sampling sites u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:24 color profile: disabled composite 150 lpi at 45 degrees tion according to jeffrey et al. (1997). abundance of phytoplankton was determined according to utermöhl (1958). statistica 7 was used for statistical analyses. principal component analysis (pca) was carried out according to splus program (getting started with s-plus for windows, insightful corporation, seattle, wa). results maximum monthly mean temperature was found in august, the minimum in january (fig. 2). the maximum salinity values appeared in july to september (fig. 3a). extremely low salinity of 3.9 was measured in january after heavy rains. as a result of the influence of freshwater, statistically significant differences of mean salinity values between stations 1, 2 and 5, as well as between stations 3 and 4 were found (fig. 3b). oxygen concentrations ranged from 7.21 mg l –1 in august to 8.48 mg l –1 in april (fig. 4a). the minimum oxygen concentrations were determined at stations 3 and 4 (fig. 4 b). acta bot. croat. 68 (1), 2009 47 seasonal variations of phytoplankton biomass median 25%–75% min-max outliers extremes9 10 12 1 2 3 4 5 6 7 8 month 4 6 8 10 12 14 16 18 20 22 24 26 t e m p e ra tu re (° c ) fig. 2. seasonal distribution of temperature values at the sampling sites in the inner part of boka kotorska bay. the centre horizontal line in each box is the median value. fifty percent of the data points lie within each box. the whiskers above and below the box indicate the 75% and the 25% percentiles. 0 5 10 15 20 25 30 35 40 45 s a li n it y ( ) ‰ 1 2 3 4 5 station 9 10 12 1 2 3 4 5 6 7 8 month a) 0 5 10 15 20 25 30 35 40 45 s a li n it y ( ) ‰ b) fig. 3. temporal (a) and spatial (b) distribution of salinity values in the inner part of boka kotorska bay. for explanation of box plots, see fig. 2. u:\acta botanica\acta-botan 1-09\krivokapic.vp 22. travanj 2009 11:26:10 color profile: disabled composite 150 lpi at 45 degrees 48 acta bot. croat. 68 (1), 2009 krivokapi] s., stankovi] @., vuksanovi] n. o x ig e n c o n c e n tr a ti o n (m g l ) – 1 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 9 10 12 1 2 3 4 5 6 7 8 month 1 2 3 4 5 station o x ig e n c o n c e n tr a ti o n (m g l ) – 1 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 a) b) fig. 4. temporal (a) and spatial (b) distribution of mean oxygen concentration value in the inner part of boka kotorska bay. for explanation of box plots, see fig. 2. 2 4 6 8 10 12 14 16 s e c c h i d e p th (m ) 2 4 6 8 10 12 14 16 s e c c h i d e p th (m ) 1 2 3 4 5 station 9 10 12 1 2 3 4 5 6 7 8 month a) b) fig. 5. temporal (a) and spatial (b) distribution of mean transparency in the inner part of boka kotorska bay. for explanation of box plots, see fig. 2. tab. 1. maximum (max), minimum (min), average (avg), standard deviation (sd), and number of samples (n) for physico-chemical and biological parameters in the inner part of boka kotorska bay. variables average min max sd n temperature (c°) 16.9 6.5 25 4 198 salinity (psu) 23.9 3.9 39.7 1 198 o2 (mg l –1 ) 7.9 5.84 10 1 198 secchi visibility (m) 7.3 4 15 3 198 no3 – (mmol l –1 ) 3.24 0 14.4 2.85 198 no2 – (mmol l –1 ) 0.24 0 3.5 0.35 198 po4 3– (mmol l –1 ) 0.08 0 0.23 0.11 198 sio2 (mmol l –1 ) 0.57 0 4.5 0.61 198 chlorophyll a (mg l –1 ) 2.6 0.06 10 2 198 phytoplankton abundance (10 3 cell l –1 ) 999 48 15055 1587 198 u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:25 color profile: disabled composite 150 lpi at 45 degrees the transparency ranged between 4.39 m in march (fig. 5a) and 11.83 m in september. a significant difference in transparency was found between station 1 and other stations (fig. 5b). nutrient concentrations were low (tab. 1) during the investigation period. maximum concentrations of nitrates (14.4 mmol l –1 ), nitrites (3.5 mmoll –1 ), phosphates (0.84 mmol l –1 ), and silicates (4.5 mmol l –1 ) were detected in the period september to december. the minimal monthly mean value for nitrate concentration was 0.2 mmol l –1 in october and the maximal of 8.06 mmol l –1 came in september (fig. 6a left). a significant difference acta bot. croat. 68 (1), 2009 49 seasonal variations of phytoplankton biomass 0 2 4 6 8 10 12 14 16 n it ra te s (ì m o l l ) – 1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0 1 2 3 4 5 1 2 3 4 5 station 9 10 12 1 2 3 4 5 6 7 8 month 0 2 4 6 8 10 12 14 16 n it ra te s (ì m o l l ) – 1 1 2 3 4 5 station 9 10 12 1 2 3 4 5 6 7 8 month 9 10 12 1 2 3 4 5 6 7 8 month 9 10 12 1 2 3 4 5 6 7 8 month n it ra te s (ì m o l l ) – 1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 n it ra te s (ì m o l l ) – 1 p h o s p h a te s (ì m o l l ) – 1 s il ic a te s (ì m o l l ) – 1 c) d) b) a) fig. 6. temporal and spatial distribution of nutrient concentrations: nitrates (a); nitrites (b); phosphates (c); silicates (d). for explanation of box plots, see fig. 2. u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:26 color profile: disabled composite 150 lpi at 45 degrees was found between the mean value of nitrate concentration at site 3 and the other stations (fig. 6a right). the minimal monthly value for nitrites (fig 6b left) was determined in february (0.1 mmol l –1 ), and the maximal in january (0.7 mmol l –1 ). a statistically significant difference was found between the mean values of nitrite concentration at site 1 (0.43 mmol l –1 ) and the other researched sites (fig. 6b right). the mean monthly value of phosphate concentration (fig. 6c) ranged from a minimal 0.01 mmol l –1 in april to 0.22 mmol l –1 in october. in the case of silicates, the maximal monthly mean value of 1.2 mmoll –1 was determined in december (fig. 6d). in july and august the concentration of silicates was below the detection limit. 50 acta bot. croat. 68 (1), 2009 krivokapi] s., stankovi] @., vuksanovi] n. sampling station 1 0 2 4 6 8 10 12 0 2 4 6 8 10 c h lo ro p h y ll (m g m ) a – 3 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 c h lo ro p h y ll (m g m ) a – 3 c h lo ro p h y ll (m g m ) a – 3 c h lo ro p h y ll (m g m ) a – 3 0 2 4 6 8 10 12 c h lo ro p h y ll (m g m ) a – 3 9 10 12 1 2 3 4 5 6 7 8 month 9 10 12 1 2 3 4 5 6 7 8 month 9 10 12 1 2 3 4 5 6 7 8 month 9 10 12 1 2 3 4 5 6 7 8 month 9 10 12 1 2 3 4 5 6 7 8 month sampling station 2 sampling station 3 sampling station 4 sampling station 5 fig. 7. chlorophyll a concentrations on sampling sites 1, 2, 3, 4 and 5, in the period september 2003–august 2004. for explanation of box plots, see fig. 2. u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:27 color profile: disabled composite 150 lpi at 45 degrees phytoplankton biomass (chlorophyll a concentration) ranged from 0.47 mg m –3 (august, site 5) up to 6.47 mg m –3 (december, site 1) (fig. 7). chlorophyll biomass provided bimodal seasonality with annual maxima mostly determined in october – december, and march – july (figs. 7, 8). the phytoplankton abundance was characterised by spring maxima in march – april (2.41 ´ 10 6 cells l –1 ) (fig 9a). the maximum biomass and highest abundance were determined at station 1, and this station was significantly different from the other investigated stations (figs. 8b, 9b). the pca analysis provided 11 principal components, the first two (fig. 10) had eigen values > 1 and explained 88.6% of the variance of the original data set. pc1 accounted for 69.38 %, pc2 19.19 %, and pc3 6.28% of total variance within data set. salinity, nitrate and nitrite are positively correlated with first main component (tab. 3), while oxygen concentration, oxygen saturation, silicate, phytoplankton abundance and chlorophyll a are negatively correlated with first main component. acta bot. croat. 68 (1), 2009 51 seasonal variations of phytoplankton biomass 0 2e6 4e6 6e6 8e6 1e7 1,2e7 1,4e7 1,6e7 0 2e6 4e6 6e6 8e6 1e7 1,2e7 1,4e7 1,6e7 1 2 3 4 5 station 9 10 12 1 2 3 4 5 6 7 8 month a) b) p h y to p la n k to n a b u n d a n c e (c e ll d m ) – 3 p h y to p la n k to n a b u n d a n c e (c e ll d m ) – 3 fig. 9. temporal (a) and spatial (b) distribution of phytoplankton abundance in the inner part of boka kotorska bay. for explanation of box plots, see fig. 2. 0 2 4 6 8 10 12 c h lo ro p h y ll (m g m ) a – 3 0 2 4 6 8 10 12 c h lo ro p h y ll (m g m ) a – 3 1 2 3 4 5 station 9 10 12 1 2 3 4 5 6 7 8 month a) b) fig. 8. temporal (a) and spatial (b) distribution of chlorophyll a in the inner part of boka kotorska bay. for explanation of box plots, see fig. 2. u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:27 color profile: disabled composite 150 lpi at 45 degrees discussion the bimodal phytoplankton seasonality is characterized by the maximum development in winter and the minimum in the warmer period of the year, as already indicated in the adriatic sea (nin^evi] and marasovi] 1998). high concentrations of nitrites and phosphates flushed by heavy rains preceded the appearance of maximum chlorophyll a concentration in winter. the mean monthly values of chlorophyll a concentrations were high in comparison to published data elsewhere in the mediterranean, such as the bay of trieste (tedesco et al. 2005), ionian sea and morocco (claustre et al. 2004), the north eastern coastal adriatic (precali et al. 2001, vili^i] et al. 2008), but lower than values from the west port of alexandria (dorgham et al. 2004), and local areas of the island of mallorca (puigerver et al. 2002). the high chlorophyll a concentrations occasionally appeared during low abundances, probably due to different photosynthetic activities in different cell size fractions, different composition of phytoplankton, and the different physiological state of cells (nin^evi] and marasovi] 1998). in summer, the inner part of boka kotorska bay is characterized by low concentrations of nutrients, high light transparency and absence of phytoplankton blooms, which suggests summer oligotrophication, as in other eastern adriatic nutrient enriched environments (svensen et al. 2007, vili^i] et al, 2008). chlorophyll a is positively correlated with phytoplankton abundance (phy. ab.), oxygen, nitrites and silicates (tab. 2), and negatively correlated with temperature, salinity, transparency and nitrates. high values of silicate concentration occurred during the winter-spring period, and its negative correlation with salinity indicate higher freshwater influence and its estuarine character. nitrate concentration was positively correlated with salinity, indicating low content of nitrates in fresh waters discharging into the kotor bay. in contrast, salinity and nitrates show a negative correlation in other eastern adriatic estuaries (legovi] et al. 1994, cetini] et al. 2006, buri] et al. 2007). 52 acta bot. croat. 67 (2), 2008 krivokapi] s., stankovi] @., vuksanovi] n. tab. 2. correlation matrix for environmental variables in the sampling sites (p<0,05; ns-not significant). temp. sal. conc.o2 trans. n-no3 – n-no2 – p-po4 3– si-sio2 chl.a phy.ab temp. 1,00 sal. 0,35 1,00 conc.o2 –0,61 –0,67 1,00 trans. 0,61 0,23 –0,48 1,00 n-no3 – 0,28 0,48 –0,35 0,29 1,00 n-no2 – –0,15 ns –0,15 –0,28 –0,26 1,00 p-po4 3– –0,16 ns ns –0,16 ns 0,19 1,00 si-sio2 –0,37 –0,57 0,47 –0,16 –0,27 ns 0,17 1,00 chl.a –0,20 –0,25 0,15 –0,24 –0,24 0,17 ns 0,36 1,00 phy.ab. ns –0,33 0,38 –0,19 –0,23 ns ns 0,19 0,23 1,00 u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:27 color profile: disabled composite 150 lpi at 45 degrees water streams and submarine springs are the main external sources of silicates in the bay. the input of nutrients in spring and autumn was the main cause of increased abundance of phytoplankton and chlorophyll a increase, while temperature was not a limiting factor for phytoplankton growth. salinity is an important factor that regulates phytoplankton development (dorgham et al. 2004), although the relationship could be negative, as in this study and elsewhere (puigserver et al.. 2002). according to chlorophyll a concentration and the criteria of håkanson (1994), the area could be described as eutrophic to hypereutrophic during winter. it seems that heavy rains contribute to the increased content of nutrients in the bay during winter and consequently to the higher phytoplankton biomass. acta bot. croat. 68 (1), 2009 53 seasonal variations of phytoplankton biomass columns temp. sal oxy. conc. oxy. sat trans no 3 no 2 po 4 sio 2 chl a phy. ab. -0,6 -0,3 0,0 0,3 0,6 0,9 -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 pc 1 p c 2 fig. 10. position of physical, chemical and biological parameters of boka kotorska bay in space defined by principal components 1 and 2. tab. 3. principal component analysis. variables pc1 loading pc2 loading pc3 loading temp. (c°) 0.544 0.800 –0.205 sal. (%0) 0.938 0.341 0.035 ox sat. (%) –0.930 0.231 0.201 con. o2 (mg l –1 ) –0.865 0.427 0.172 tran. (m) 0.593 –0.522 0.602 no3 – (mmol l –1 ) 0.954 –0.018 –0.197 no2 – (mmol l –1 ) 0.936 –0.234 0.059 po4 3– (mmol l –1 ) –0.527 0.693 0.309 sio2 (mmol l –1 ) –0.908 –0.405 –0.106 hl a (mg m –3 ) –0.768 –0.376 –0.252 ph. ab. (cell dm –3 ) –0.993 –0.079 –0.058 pc, principal component loading of the individual variables along the pc1, pc2 and pc3. u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:28 color profile: disabled composite 150 lpi at 45 degrees boka kotorska bay is an outstanding dynamic system with extremely variable ecological factors, where natural is more important than anthropogenic eutrophication. therefore, for protection of boka kotorska bay, a particular care should be taken of effluent discharge from the town of kotor. references buri], z., cetini], i., vili^i], d., caput mihali], k., cari], m., oluji], g., 2007: spatial and temporal distribution of phytoplankton in a highly stratified estuary (zrmanja, adriatic sea). marine ecology 28, 169–177. cetini], i., vili^i], d., buri], z., oluji], g., 2006: phytoplankton seasonality in a highly stratified karstic estuary (krka, adriatic sea). hydrobiologia 555, 31–40. claustre, h., hooker, s. b., van heukelem, l., berthon, j. 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1-09\krivokapic.vp 16. travanj 2009 11:31:28 color profile: disabled composite 150 lpi at 45 degrees precali, r., djakovac, t., smodlaka, n., ivan^i], i., 2001: long-term changes of nutrient concentration and phytoplankton biomass in the northern adriatic sea. rapports du congres de la ciesm 36, 156. puigserver, m., ramon, g., moýa, g., martínez-taberner, a., 2002: planktonic chlorophyll a and eutrophication in two mediterranean littoral systems (mallorca island, spain). hydrobiologia 475/476, 493–503. svensen, c., vili^i], d., wassman, p., arashkevich, e, ratkova, t., 2007: plankton distribution and vertical flux of biogenic matter during high summer stratification in the krka estuary (eastern adriatic). estuarine coastal and shelf science 71, 381–390. strickland, j. d. h., parsons, t. r., 1972: a practical handbook of seawater analysis. bulletin of fisheries research board of canada 167, 1–310. tedesco, l., socal, g., bianchi, f., acri, f., veneri, d., vichi, m., 2007: nw adriatic sea variability in relation to chlorophyll-a dynamics in the last 20 years (1986–2005). biogesciences 4, 651–685. utermöhl, h., 1958: zur vervollkommung der quantitativen phytoplankton methodik, mitteilungen. internationale vereiningung fuer theoretische und angewandte limnologie 9, 1–38. vili^i], d., 1989: phytoplankon population density and volume as indicators of eutrophication in the eastern part of the adriatic sea. hydrobiologia 174, 117–132. vili^i], d., terzi], s., ahel, m., buri], z., jasprica, n., cari], m., caput mihali], k., oluji], g., 2008: phytoplankton abundance and pigment biomarkers in the oligotrophic, eastern adriatic estuary. environmental monitoring and assessment 142, 199– 218. vollenweider, r. a., 1992: costal marine eutrophication: principles and control. proceedings of the symposium on marine coastal eutrophication, bologna, 1–20. acta bot. croat. 68 (1), 2009 55 seasonal variations of phytoplankton biomass u:\acta botanica\acta-botan 1-09\krivokapic.vp 16. travanj 2009 11:31:28 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 80 (1), 2021 s1 social news the croatian national diatom collection the croatian national diatom collection was established on 1st march 2018 at the university of zagreb, faculty of science (department of biology, division of botany) and registered in the index herbariorum of the new york botanical garden (http://sweetgum.nybg.org/science/ih/ herbarium-details/?irn=252706). it was officially presented to the scientific and general public on 9th december 2019 in the croatian academy of sciences and arts. it is the first institutional collection of permanent microscopic specimens of diatoms in croatia, currently containing 1000 microscopic specimens and 200 wet samples and 16 type specimens (fig. 1). most importantly, the collection contains 16 type specimens of newly discovered species in croatia, which are no longer sent to other european collections, but kept in croatia as a treasure of national biodiversity. diatoms are an exceptionally diverse group of mostly photosynthetic microorganisms and have a significant role in primary production and the fixation of atmospheric carbon. they are present in both plankton and benthos, often as dominant organisms in both marine and freshwater ecosystems. as an integral part of the planktonic and benthic water communities, they serve as a biological indicator and an essential factor in assessing the ecological status of water. biodiversity is the total variety of living organisms in a certain ecosystem, and croatia is considered to be one of the most biodiverse countries in europe. this variety stems from the specific geographic location of croatia, which entails exceptional ecological, climactic and geomorphological conditions. all scientific knowledge about its organisms – from the biggest and publicly most accessible plants and animals to the smallest microorganisms – is extremely important for croatia. the diversity of diatoms in croatia has occupied the interest of many world-famous scientists in history (adolf steuer, friedrich hudstedt, anto jurilj), which has resulted in world-famous publications (viličić and ljubešić 2017, and references therein). croatian algologist, professor anto jurilj (1910-1981), although he actively researched diatoms in croatia, is remembered in science for a description of 44 new diatom taxa from lake ohrid in n. macedonia. equal interest in the study of diatoms in croatia has been shown by scientists today, who have recently described a significant number of new species (gligora et al. 2009, mejdandžić et al. 2017, 2018, gligora udovič et al. 2018, caput mihalić et al. 2019, majewska et al. 2019, 2020, alhandal et al. 2020, mucko et al. 2020, van de vijver et al. 2020). a systematic inventory of the diatoms of the krka river from source to mouth has been conducted, resulting in an impressive diatom collection. newly described diatom species in croatia, the diatom collection of the krka river together with the historical diatom collection of the professor anto jurilj, provided the impetus for the croatian national diatom collection in 2018. fig. 2. croatian national diatom collection logo by nikola koletić. fig. 1. permanent slides hosted in the croatian national diatom collection. s2 acta bot. croat. 80 (1), 2021 ciae sp. nov. a new species from crveno jezero, croatia. phytotaxa 351, 229–238. gligora, m., kralj, k., plenković-moraj, a., hinz, f., acs, e., grigorszky, i., cocquyt, c., van de vijver, b., 2009: observations on the diatom navicula hedinii hustedt (bacillariophyceae) and its transfer to a new genus envekadea van de vijver et al. gen. nov. european journal of phycology 44, 123–138. majewska, r., ashworth, m.p., bosak, s., goosen, w.e., nolte, c., filek, k., van de vijver, b., taylor, j.c., manning, s.r., nel, r., 2020: on sea turtle‐associated craspedostauros (bacillariophyta), with description of three novel species. journal of phycology, doi:10.1111/jpy.13086 majewska, r., bosak, s., frankovich, t.a., ashworth, m.p., sullivan, m.j., robinson, n.j., lazo-wasem, e.a., pinou, t., nel, r., manning, s.r., van de vijver, b., 2019: six new epibiotic proschkinia (bacillariophyta) species and new insights into the genus phylogeny. european journal of phycology 54, 609–631. mejdandžić, m., bosak, s., nakov, t., ruck, e., orlić, s., gligora udovič, m., peharec štefanić, p., špoljarić, i., mršić, g., ljubešić, z., 2018: morphological diversity and phylogeny of the diatom genus entomoneis (bacillariophyta) in marine plankton: six new species from the adriatic sea. journal of phycology 54, 275–298. mejdandžić, m., bosak, s., orlić, s., gligora udovič, m., peharec štefanić, p., špoljarić, i., mršić, g., ljubešić, z., 2017: entomoneis tenera sp. nov., a new marine planktonic diatom (entomoneidaceae, bacillariophyta) from the adriatic sea. phytotaxa 282, 1–18. mucko, m., bosak, s., mann, d.g., trobajo, r., wetzel, c.e., peharec štefanić, p., ljubešić, z., 2020: a polyphasic approach to the study of the genus nitzschia (bacillariophyta): three new planktonic species from the adriatic sea. journal of phycology, doi:10.1111/jpy.13085 van de vijver, b., robert, k., witkowski, a., bosak, s., 2020: majewskaea gen. nov. (bacillariophyta), a new marine benthic diatom genus from the adriatic sea. fottea 20, 112–120. viličić, d., ljubešić, z., 2017: razvoj metoda istraživanja fitoplanktona u jadranskom moru. hrvatske vode: časopis za vodno gospodarstvo 99, 49–58. today, the croatian national diatom collection is, in both professional and scientific terms, a testimony to the high diatom diversity in croatia and is an invaluable database as well as a testament of our time for the future generations. collections like these offer an invaluable abundance of information on biological research and offer insights into the diversity of diatom taxonomy, biodiversity of a certain area, variety distribution and changes in time and space, which all together enable effective protection of the water ecosystems in croatia. supported by the faculty of science, it is now open to both experts and the public. it is recognizable by its official herbarium code – hrndc and the official logo, which shows a combination of centric diatoms in the middle, three black semicircles representing c for croatian, c for collection and d for diatom, and stylized croatian interlacing which represents the word national (fig. 2). the croatian national diatom collection acquires materials for the permanent collection and all the information can be accessed on the website www. diatoms.biol.pmf.hr. assoc. prof. marija gligora udovič assoc. prof. zrinka ljubešić university of zagreb faculty of science department of biology references al-handal, a.y., mucko, m., wulff, a., 2020: entomoneis annagodhei sp. nov., a new marine diatom (entomoneidaceae, bacillariophyta) from the west coast of sweden. diatom research 35, 269–279. caput mihalić, k., gligora udovič, m., galović, i., stanković, i., šušnjara, m., žutinić, p., kulaš, a., špoljarić, i., levkov, z., 2019: tetramphora croatica sp. nov. a new brackish-water species from lake vransko, croatia. phytotaxa 401, 276–286. gligora udovič, m., žutinić, p., kavre piltaver, i., kulaš, a., ozimec, r., tofilovska, s., 2018: gomphosphenia plenkoviopce-str.vp acta bot. croat. 69 (2), 249–257, 2010 coden: abcra 25 issn 0365–0588 effect of seed age and soil texture on the germination of some ludwigia species (onagraceae) in nigeria matthew oziegbe1*, julius o. faluyi1, abimbola oluwaranti2 1 department of botany, obafemi awolowo university, ile ife, nigeria. 2 department of crop production and protection, obafemi awolowo university, ile ife, nigeria. seed germination in ludwigia was greatly influenced by seed age and soil type. in ludwigia abyssinica germination was not influenced by seed age and soil texture. freshly shed seeds and six month old seeds of ludwigia decurrens variety b showed a very low percentage germination on all the germination media, and six month old seeds germinated significantly earlier than freshly shed seeds. some soil types could significantly reduce germination of freshly shed seeds of l. hyssopifolia, l. erecta, l. leptocarpa and l. octovalvis var linearis. key words: germination, seed, soil, ludwigia introduction the genus ludwigia has existed at least 50 million years and is considered one of the largest and least specialized genera of the family onagraceae (peng 1988). the genus is pantropical; it includes some 82 species distributed among 23 sections. twenty five of these species occur in the old world, including 8 of its 23 sections. a very diverse assemblage of ludwigia species occur in south america, where 45 of the 82 species occur with most primitive species; this may have been the centre of origin for the genus and family the onagraceae (ramamoorthy and zardini 1987, peng 1989). in west africa, they are represented by fourteen species (sixteen taxa) and nine species (eleven taxa) of these are found in nigeria (wogu and ugborogho 2000). a few ludwigia species are predominantly aquatic but all of the species grow in wet places where they concentrate around coastal regions, lakes, lagoons, canals, rivers, streams, seas, gutters and water logged areas. the genus contains both herbaceous and woody species. many aquatic species are phenotypically plastic, such that their growth forms vary under different environmental conditions, which often complicates species identification and has led to a number of fluctuations in their taxonomic classifications (dutartre et al. 2004). seed dispersal and germination are phases in the reproductive cycle that are typically of great importance for species fitness. variations in seed dispersal efficacy or germination percentage are often interpreted as reflecting acta bot. croat. 69 (2), 2010 249 * corresponding author, e-mail: matthewoziegbe@yahoo.com u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:48 color profile: disabled composite 150 lpi at 45 degrees adaptations to specific ecological conditions (venable and lawlor 1980, grime et al. 1981, martin et al. 1995, nishitani and masuzawa 1996). clearly, the characteristics of the microsite occupied by a seed may strongly influence its probability of germination and subsequent survival, including germination capacity (schaal 1980, dolan 1984, hendrix 1984, stanton 1985, marshall 1987, naylor 1993), resistance to intra and interspecific competition (wulff 1986, mazer 1989, houssard and escarre 1991), dormancy period (stamp 1990), distance dispersed with respect to the mother plant (augspurger and franson 1986) and seedling survival and/or growth (schaal 1980, howe and richter 1982, gross 1984, stanton 1984). ludwigia peruviana seeds can remain dormant for two years, seeds are able to germinate while afloat, 20 percent of seeds are dormant, propagation can also be done by young stems; seed production is of 450000 seeds m2, 65000 seeds m2 in soil seed bank and 30000 seeds m2 for seeds that remained on old stems (jacobs et al. 1994). it has been reported that the capacity of seed germination in ludwigia species is not known (ruaux et al. 2009). fruits of many ludwigia species remain on the parent stem when ripened while some are detached immediately at ripening. some seeds are dispersed freshly from fruits on parent stems in to water as fruits dehisce irregularly while some seed remain on the fruits for several months before they are dispersed. fruits of l. adscendens and l. leptocarpa are detached from the parent stem immediately they are ripened and remain afloat in water while it takes several weeks and months for their seeds to be dispersed from the fruits. in l. hyssopifolia which possesses both non-endocarp and endocarp seeds, non-endocarp seeds are first dispersed at ripening of fruit while endocarp seeds remain on the parent plant for several weeks and months before they are eventually dispersed into water. studies have reported the germination of some ludwigia seeds in the field (ruaux et al. 2009) but there are few studies on seed germination capacity in relation to soil types (wogu and ugborogho 2000). as the fruits of most ludwigia species remain on the parental plant (i.e. not dispersed), it was important to study the seed aging by comparing fresh seeds with seeds stored over maternal plants for six months and determine their germination on soil types. this study intends to determine the effect seed age and soil types on germination of seven ludwigia species. materials and methods seeds used for this study were collected from ludwigia species grown in the garden from march 2006 to december 2006 at the department of botany, obafemi awolowo universty, ile ife. germination study was carried out on nine ludwigia seed samples which belonged to seven ludwigia species: l. abyssinica a. rich, l. adscendens (linn) hara. ssp. diffusa (forssk.) raven, l. decurrens var a walter, l. decurrens var b walter, l. hyssopifolia (g.don) excell, l. erecta (linn) hara, l. leptocarpa (nutt.) hara, l. octovalvis var linearis (jacq.) raven and l. octovalvis var brevisepala. l. decurrens var a has big ovate lanceolate leaves, the stem is prominently winged and 5angled, it initiates flowering within (64–70) days after germination; the number of petals ranges from (4–5). it is mostly found as monotypic stands in most aquatic habitats in ile–ife. l. decurrens var b has narrow lanceolate leaves, the stem is narrowly winged and 4 angled, it initiates flowering within (30–40) days after germination, the number of petals is fixed at (4). it is found growing sparsely among l. decurrens var a and rarely recognized. l. octovalvis var brevisepala has short lin250 acta bot. croat. 69 (2), 2010 oziegbe m., faluyi j. o., oluwaranti a. u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:48 color profile: disabled composite 150 lpi at 45 degrees ear leaves, stem slightly bent, densely villous and the number of petals is fixed at (4). l. octovalvis var linearis has long narrow linear leaves, stem erect, subglabrous and petal number ranges from (4–5). one hundred freshly ripened seeds and six month old seeds of each species were seeded into petri dishes of diameter 8.00 cm (4 replicates of every treatment) lined with whatman filter-papers no 1. also 100 seeds of freshly ripened seeds and six month old seeds of each species were seeded separately on soil surfaces into 8.00cm diameter plastic cups filled with clayey, sandy and loamy soil with four replicates for every treatment. the petri dishes and cups were watered regularly with distilled water. germination was recorded when the radicule emerges from the seeds every day for a period of 30 days for each treatment. days to initial germination (appearance of radicule) and percentage germination were recorded for each of the treatments. germinated seeds were removed from the petri dishes and cups during each count. the experiment was carried out in the screen house of the department of botany, obafemi awolowo university, ile-ife under conditions of natural photoperiod and ambient temperature (20–25 oc). soil samples used were collected from obafemi awolowo university campus located within (latitude 7o 30 n to 7o 35 n and longitude 4o 30 and 4o 35o e). soil textures were determined at department of soil science, obafemi awolowo university, ile ife, using a mechanical analysis method. duncan’s multiple range test (dmrt) was used to compare sets of means obtained for germination regimes, at a probability level of 0.05, using system analysis software (sas) version 9.2. results the study on ludwigia species reveals that percentage germinations of six months seed were very high and varied significantly from the very low percentage germinations observed in freshly shed seeds on the three types of soils with the exception of l. abyssinica, l. hyssopifolia, l. erecta and l. leptocarpa (tabs. 1, 2). the soil media also significantly reduced the percentage germination of freshly shed seeds of (l. hyssopifolia, l. erecta, l. leptocarpa and l. octovalvis var linearis) when compared to their control. in l. adscendens, l. decurrens variety a, l. decurrens variety b, l. hyssopifolia, l. erecta and l. octovalvis var linearis six months old seeds germinated significantly earlier than freshly shed seeds; but in l. abyssinica and l. octovalvis var brevisepala there was no significant difference in days to initial germination between six month old seeds and freshly shed seeds on any of the germination media (tab. 2). ludwigia abyssinica percentage germination of new seeds and six month seeds of l. abyssinica were very high in all the germination media with no significant differences in the percentage germinations (tab. 1). seeds germinated very early in all the media and there was no significant difference in days to initial germination in any of the media (tab. 2). ludwigia adscendens very high percentage germination in six month seeds of l. adscendens was significantly different from the very low percentage germination of new seeds on all the germination media. seed age affected days to initial germination, six month old seeds germinated sigacta bot. croat. 69 (2), 2010 251 germination of ludwigia species (onagraceae) u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:48 color profile: disabled composite 150 lpi at 45 degrees nificantly earlier than freshly shed seeds but the soil types had no significant effect on days to initial germination (tab. 2). ludwigia decurrens the high percentage germination (93.4) of six month seeds observed in the control of l. decurrens var a is significantly different from the very low percentage germination of freshly shed seeds in control (5.20), loamy (1.60), clayey (1.40) and sandy (13.4) soils; there were significant differences in percentage germinations of six month seeds on the three types of soils (tab. 1). seed age affected days to initial germination: six month old seeds germinated significantly earlier than freshly shed seeds but the soil types had no significant effect on days to initial germination (tab. 2). the percentage germination of l. decurrens var b was very low in six month and freshly shed seeds in all germination media; freshly shed seeds did not germinate on the control and loamy soil, with no significant difference in percentage germination observed on the other freshly shed seeds media and six month seeds media (clayey and sandy); highest percentage germination (39.60) of six month seed was observed in the control, which is significantly different from the 34.20 observed on loamy soil (tab. 1). age and soil type affected days to initial germination, and six month seeds germinated significantly earlier than freshly shed seeds (tab. 2). ludwigia hyssopifolia there was no significant difference in the high percentage germination observed in six month and freshly shed seeds of the control; six month seeds in the soil media showed a significantly higher percentage germination than freshly shed seeds on the soil media (tab. 1). seed age and soil type affected days to initial germination; six month old seeds germinated significantly earlier than all other species throughout the experiment (tab. 2). ludwigia erecta there was no significant difference in percentage germination of six month seeds and freshly shed seeds in the control; the high percentage germination of six month seeds varied significantly from the low percentage germination of new seeds on the three soil media. seed age affected days to initial germination, six month old seeds germinated significantly earlier than freshly shed seeds. soil type had no significant effect on days to germination (tab. 2). ludwigia leptocarpa there was no significant difference in percentage germination of six month seeds of control, loamy, clayey soils and the control of freshly shed seeds, which varied significantly from the low percentage germination of freshly shed seeds in loamy, clayey and sandy soils (table1). seed age and soil type affected days to initial germination, and six months old seeds germinated significantly earlier than freshly shed seeds (tab. 2). ludwigia octovalvis in l. octovalvis var linearis the high percentage germination (88.60) in six month seeds of the control is significantly different from the 74.20 percentage germination in the freshly 252 acta bot. croat. 69 (2), 2010 oziegbe m., faluyi j. o., oluwaranti a. u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:49 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (2), 2010 253 germination of ludwigia species (onagraceae) tab. 1. media growth mean values of percentage germination of freshly shed seeds and six month old seeds on germination media. freshly shed seeds six months old seeds control loamy clayey sandy control loamy clayey sandy ludwigia abyssinica 94.40a 90.60a 94.20a 94.20a 92.20a 93.40a 92.80a 92.80a l. adscedens 1.80c 2.40c 1.00c 1.00c 90.80a 90.00a 89.20a 85.20b l. decurrens var a 5.20f 1.60f 1.40f 13.40f 93.40a 63.20b 38.00c 27.40d l. decurrens var b 0.00c 0.00c 1.00c 1.00c 39.60a 34.20b 1.60c 1.20c l. hyssopifolia 93.20a 5.20d 3.40d 32.40c 96.00a 91.60b 91.20b 90.20b l. erecta 94.20a 16.00e 28.00d 44.00c 94.00a 94.80a 93.80a 86.60b l. leptocarpa 89.40a 33.80d 34.80d 59.60c 84.40a 86.80a 82.40a 71.80b l. octovalvis var linearis 74.20b 5.40e 11.80d 15.20d 88.60a 85.80a 77.40b 68.60c l. octovalvis var brevisepala 32.20c 37.40c 35.20c 34.80c 91.40a 82.40b 85.60a 83.20b means in each row followed by the same letter(s) are not significantly different at 5% level of significance tab. 2. media growth mean values of days to initial germination of freshly shed seeds and six month old seeds on germination media. freshly shed seeds six months old seeds control loamy clayey sandy control loamy clayey sandy ludwigia abyssinica 4.20a 4.20a 4.0a 4.40a 4.00a 4.00a 4.20a 4.00a l. adscedens 17.6a 16.60a 17.00a 17.60a 3.80b 4.20b 4.40b 4.80b l. decurrens var a 12.60a 12.60a 12.80a 12.40a 3.40b 4.00b 4.20b 3.80b l. decurrens var b 0.00d 0.00d 7.40a 7.60a 4.60c 4.60c 6.60b 6.80b l. hyssopifolia 10.20c 11.60b 12.60a 12.80a 3.20ef 4.00d 3.80ed 3.00f l. erecta 6.40a 6.40a 6.40a 6.60a 4.00b 3.80b 3.80b 4.00b l. leptocarpa 5.40bc 6.00ba 6.60a 6.60a 4.40d 4.80dc 4.40d 5.40bc l. octovalvis var linearis 6.00b 6.60ba 6.60ba 6.80a 3.40d 4.40c 4.00dc 4.20c l. octovalvis var brevisepala 3.60a 3.60a 3.80a 3.80a 3.60a 3.60a 3.80a 3.80a means in each row followed by the same letter(s) are not significantly different at 5% level of significance u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:49 color profile: disabled composite 150 lpi at 45 degrees shed seeds of the control; high percentage germinations (85.80, 77.40 and 68.60) in six month seed on the soil media also varied significantly from the very low percentage germinations (5.40, 11.80 and 15.20) in freshly shed seeds of the soil media. the situation is quite similar in l. octovalvis var brevisepala in which a high percentage germination (91.40) of old seeds in the control is significantly different from low percentage germination (32.20) in the control of freshly shed seeds; high percentage germinations (82.40, 85.60 and 83.20) in six month seeds on soil media also varied significantly from the very low percentage germinations (37.40, 35.20 and 34.80) in freshly shed seeds on the soil media (tab. 1). seed age and soil type affected days to initial germination in l. octovalvis var linearis, and six month old seeds germinated significantly earlier than all other species throughout the experiment with the exception of l. hyssopifolia. (tab. 2). discussion the very high percentage germination shown by six month old seeds as compared to freshly shed seeds of ludwigia adscendens, l. decurrens var a and b and l. leptocarpa reveals that freshly shed seeds of these species exhibit some degree of dormancy and the dormancy was relieved at six months, because the seeds germinated early showing a very high percentage germination. the high percentage germination of six month old seeds and freshly shed seeds of l. abyssinica, l. hysopifolia, l. leptocarpa, and l. octovalvis in the control indicate there is no dormancy. the very low percentage germination of freshly shed seeds can be regarded as primary dormancy according to (karssen 1982); this is a state in which freshly shed seeds do not germinate under any set of environmental condition until dormancy is relieved. six month old seeds are expected to have higher desiccation than freshly shed seeds. depending on the weed species, desiccation of seeds either improves germination or does not affect germination at all (karssen et al. 1988). reduced germination at high densities may be a population maintenance mechanism. a weed species can produce seeds copiously under favourable conditions, while having only a few germinating at any time, thus maintaining a seed reservoir over extended periods (palmblad 1968, muoghalu and chuba 2005). higher percentage germination in dense arrays will result in greater competition. it is also possible that when several seeds germinate together the combined force of several roots growing simultaneously may help them to penetrate a hard soil. many plants however have specific requirements for germination and subsequent establishment (yan 1976). soil type might not be a critical factor in the distribution of six month old seeds of ludwigia species because they showed very high percentage germination on all the three types of soils except in l. decurrens var a and b in which percentage germination was very low (tab. 1). the significantly very low percentage germination of freshly shed seeds of (l. hyssopifolia, l. erecta, l. leptocarpa and l. octovalvis var linearis) on the three soils indicates that the higher percentage of freshly shed seeds of these ludwigia species will not germinate immediately on dispersal in their natural habitat. seed dormancy observed in some ludwigia species will ensure that they survive adverse conditions in their environment as dormant seeds only germinate when the environmental conditions favour the survival of their seedlings. but the very high percentage germination of freshly shed seeds of l. abyssinica indicates that the seeds will germinate readily on three types of soils and the 254 acta bot. croat. 69 (2), 2010 oziegbe m., faluyi j. o., oluwaranti a. u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:49 color profile: disabled composite 150 lpi at 45 degrees plant is not likely to have a soil seed bank. influence of soil factor on germination of seeds is based on transmittance of light through the soil, which includes particle size, moisture content, particle colour and presence of organic matter (tester and morris 1987). studies have noted the decrease in soil transmittance with decreased particle size (bliss and smith 1985). however, there have been no detailed measurements of the penetration of light through soil mixtures of widely different particle sizes, as would often be found in field situations. depending on the soil type, moisture content either increases or decreases the light transmittance of the soil. when sand is saturated the transmittance will increase, whereas saturation of clay and loam decreases the transmittance of light. this difference is probably attributed to a reduction in the reflection of light by the soil particles. when the particles are translucent, as in sand, transmission can increase through the particles; but in dark soil, reduced reflection only leads to increased absorption by the particles (bliss and smith 1985). the darker particles are thought to absorb the light. another explanation is the increased reflection by particles of the paler coloured soils, whereas the reflection in dark soils is lower (tester and morris 1987). six month old seeds are likely to contribute more to plant population in ludwigia species, in which they germinated significantly earlier than freshly shed seeds. seed age and soil type have been found to affect germination in the ludwigia species studied. six month old seeds of ludwigia species are more likely to contribute to plant populations than the freshly shed seeds germination media investigated. retention of fruits and seeds on ludwigia stems at maturity might be a strategy for maintaining plant population density. there is a need for further investigation of variations because of the very significant higher percentage germination than freshly shed seeds shown by l. species on growth media as well as seed germination at one year to determine seed contribution to plant populations at this age. references augspurger, c. k., franson, s. e., 1986: wind dispersal of artificial fruits varying in mass, area, and morphology. ecology 68, 27–42. bliss, d., smith, h., 1985: penetration of light into soil and its role in the control of seed germination. plant cell and environment 8, 475–483. dolan, r. w., 1984: the effect of seed size and maternal source on individual size in a population of ludwigia lectocarpa (onagraceae). american journal of botany 71, 1302–1307. dutartre, a., dandelot, s., haury, j., lamber, e., le goff, p., menozzi, m-j., 2004: les jussies: caractérisation des relations entre sites, populations et activités humaines. in: implications pour la gestion. rapport intermédiaire programme invabio, cemagref, bordeaux, 44. grime, j. p., mason, g., curtis, a. v., rodman, j., band, s. r., mowforth, m. a. g., neal, a. m., shaw, s., 1981: a comparative study of germination characteristics in a local flora. journal of ecology 69, 1017–1059. gross, k. l., 1984: effects of seed size and growth form on seedling establishment of six monocarpic perennial plants. journal of ecology 72, 369–387. acta bot. croat. 69 (2), 2010 255 germination of ludwigia species (onagraceae) u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:49 color profile: disabled composite 150 lpi at 45 degrees hendrix, s. d., 1984: variation in seed mass and its effects on germination in pastinaca sativa l. (umbelliferae). american journal of botany 71, 795–802. houssard, c., escarre, j., 1991: the effects of seed weight on growth and competitive ability of rumex acetosella from two successional old-fields. oecologia 86, 236–242. howe, h. f., ritcher, w., 1982: effects of seed size on seedling size in virola surinamensis: a within and between tree analysis. oecologia 53, 347–351. jacobs, s. w. l., perrett, f., jacobs, b. j., 1994: ludwigia peruviana (onagraceae) in the botany wetlands near sydney, australia. australian journal of marine and freshwater research 45, 1481–1490. karssen, c. m., 1982: seasonal patterns of dormancy in weed seeds. in: khan, a. a. (ed.), the physiology and biochemistry of seed development, dormancy and germination, 243–270. elsevier biomedical press, amsterdam karssen, c. m., derkx, m. p. m., post, b. j., 1988: study of seasonal variation in dormancy of spergula arvensis l. seeds in a condensed annual temperature cycle. weed research 28, 449–457. marshall, d. l., 1987: effects of seed size on seedling success in three species of sesbania (fabaceae). american journal of botany 73, 457–464. martin, a., grzeskowiak, v., puech, s., 1995: germination variability in three species in disturbed mediterranean environments. acta oecologica 16, 479–490. mazer, s. j., 1989: ecological, taxonomic, and life history correlates of seed mass among indiana dune angiosperms. ecological monographs 59, 153–175. muoghalu, j. i., chuba, d. k., 2005: seed germination and reproductive strategies of tithonia diversifolia (hemls.) gray and tithonia rotundifolia (p. m.) blake. applied ecology and environmental research 1, 39–46. naylor, r. e. l., 1993: the effect of parent plant nutrition on seed size, viability and vigour, and on germination of wheat and triticale at different temperatures. annals of applied biology 123, 379–390. nishitani, s., masuzawa, t., 1996: germination characteristics of two species of polygonum in relation to their altitudinal distribution on mt. fuji, japan. arctic and alpine research 28, 104–110. palmblad, i. g., 1968: competition in experimental populations of weeds with emphasis on the regulation of population size. ecology 49, 26–34. peng, c. i., 1988: the biosystematics of ludwigia sect. microcarpium (onagraceae). annals of the missouri botanical garden 75, 970–1009. peng, c. i., 1989: the systematics and evolution of ludwigia section microcarpium (onagraceae). annals of the missouri botanical garden 76, 221–302. ramamoorthy, t. p., zardini, e., 1987: the systematics and evolution of ludwigia sect. myrtocarpus sensu lato (onagraceae). monographs in systematic botany from the missouri botanical garden 19, 1–120. ruaux. b., sabine, g., jacques, h., jean-pierre, b., 2009: sexual reproduction of two alien invasive ludwigia (onagraceae) on the middle loire river, france. aquatic botany 90, 143–148. 256 acta bot. croat. 69 (2), 2010 oziegbe m., faluyi j. o., oluwaranti a. u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:49 color profile: disabled composite 150 lpi at 45 degrees schaal, b. a., 1980: reproductive capacity and seed size in lupinus texensis. american journal of botany 67, 703–709. stamp, n. e., 1990: production and effect of seed size in a grassland annual (erodium brachycarpum, geraniaceae). american journal of botany 77, 874–882. stanton, m. l., 1984: seed variation in wild radish: effect of seed size on components of seedling and adult fitness. ecology 65, 1105–1112. stanton, m. l., 1985: seed size and emergence time within a stand of wild radish (raphanus raphanistrum l.): the establishment of a fitness hierarchy. oecologia 67, 524–531. tester, m., morris, c., 1987: the penetration of light through soil. plant cell and environment 10, 281–286. venable, d. l., lawlor, l., 1980: delayed germination and dispersal in desert annuals: escape in space and time. oecologia 46, 272–282. wogu, a., ugborogho, r. e., 2000: seed morphology, germination and seedling characters in ludwigia species (onagraceae) in nigeria as aids to identification. seed science technology 28, 657–697. wulff, r. d., 1986: seed size variation in desmodium paniculatum, 3. effects on reproductive yield and competitive ability. journal of ecology 74, 115–121. yan, b. l., 1976: density dependent seed germination strategies in colonizing versus non – colonizing plant species. journal of ecology 64, 375–380. acta bot. croat. 69 (2), 2010 257 germination of ludwigia species (onagraceae) u:\acta botanica\acta-botan 2-10\293-oziegbe.vp 11. listopad 2010 13:39:49 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (1), 99–107, 2011 coden: abcra 25 issn 0365–0588 lichen flora of @umberak-samoborsko gorje nature park, nw croatia anamarija partl state institute for nature protection, trg ma`urani}a 5, hr 10000 zagreb, croatia abstract – during 2007 and 2008 epiphytic and terrestrial lichen communities were surveyed in the @umberak-samoborsko gorje nature park (nw croatia); 84 taxa were recorded including, lecanora thysanophora, which was new to croatia, and four, bryoria fuscescens, lobaria pulmonaria, usnea subfloridana and usnea hirta, which are red data species in croatia. keywords: lichen, check list, @umberak, samoborsko gorje, croatia introduction most research into lichens in croatia was done in the late 19th and the first part of the 20th century, and summarised in ku[an (1953). few works were published until the 1990s after recommendations for lichenological research were made (christensen 1987, 1988; christensen and hansen 1994; ozimec 2000; partl and asta 2003; ozimec et al 2009). in 2007 the first check list and red list of croatian lichens were published, later revised in 2009 (ozimec and partl 2009a, b). the distribution of most species in croatia is still not well known. this paper is an attempt to contribute to a broader understanding of lichen flora in the north croatian mountain region. this area had previously been visited once before (ku[an 1928), resulting in the recording of 67, mostly saxicolous, taxa. study area the lichen survey was carried out in the @umberak-samoborsko gorje nature park. this is situated in nw croatia, on the border with slovenia. it includes parts of @umbera~ko and samoborsko gorje mountains, between the rivers sava and kupa, in croatia, and the river krka in slovenia. altitudes range from 180m in the kupa river valley increasing to 1178m at the summit of sveta gera, which is the highest peak of @umbera~ko gorje mountain range. in this paper, previous lichen records for the mountains are also mentioned, and all the localities visited are marked on the enclosed map (fig.1). acta bot. croat. 70 (1), 2011 99 * corresponding address, e-mail: anamarija.partl@dzzp.hr copyright ® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:48 color profile: disabled composite 150 lpi at 45 degrees most rock exposures are limestone and dolomites that weather to form a karst landscape. locally, other rock type outcrops are present, such as sandstone and clay, whilst magmatic rocks can be found in the southeast part of the park. over three hundred freshwater springs are recorded in the region and most of the permanent streams run in rather deep valleys. climate is moderate-continental, with a mediterranean influence. average annual temperature is from 7–10 °c, and average annual precipitation in the region exceeds 1100mm, and may reach 1300mm in the highest parts. the area is mostly covered with forests in which the major types are beech (fagus silvatica) forests, and, in the lower parts, oak (quercus spp) and hornbeam (carpinus betulus) forests. semi-natural and anthropogenic habitats include planted conifers, old orchards, and meadows. materials and methods the lichens found during this survey were collected from 24 sites (fig. 1). old records, referenced by toponyms (ku[an 1928, 1953), were grouped and recorded from 7 additional sites. these should be considered as approximate locations as in the original studies the records were from quite wide areas. stations indicated by numbers were those examined in this research; old records by ku[an (1928) are indicated by letters. 100 acta bot. croat. 70 (1), 2011 partl a. fig. 1: map of the study area in croatia with sampling sites. u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees lichens were collected in paper bags labelled with site number and substratum. after drying, specimens were identified using both stereo and compound microscopes, and by chemical spot tests. several keys were used (clauzade and roux 1985, dobson 1992, purvis et al 1992, wirth 1995). nomenclature mostly follows nims and martellos (2008). results lichen taxa recorded in this survey, together with older records, are listed alphabetically (tab. 1), showing stations where they were found. in the present research done during 2007 and 2008, 84 taxa were recorded; previous research from 1928 recorded 67 taxa. only 10 taxa from 1928 were re-discovered during the present survey. this is probably due to the focus of the earlier survey being on saxicolous lichens, whilst this study concentrated on epiphytic and terrestrial taxa. in further analyses, only taxa recorded in this research were used, as old records could not be properly geo-referenced or verified. acta bot. croat. 70 (1), 2011 101 lichens from @umberak and samoborsko gorje, croatia tab. 1: lichens from @umberak-samoborsko gorje national park species stations amandinea punctata (hoffm.) coppins et scheid. 2, 6, 7, 8 anaptychia ciliaris (l.) körb. 7 arthonia spadicea leight. e aspicilia calcarea (l.) mudd g bacidia bagliettoana (a.massal. et de not.) jatta e baeomyces rufus (huds.) rebent. c bagliettoa limborioides a.massal. c bagliettoa parmigera (j.steiner) vìzda et poelt d bilimbia sabuletorum (schreb.) arnold e bryoria fuscescens (gyeln.) brodo et d. hawksw. 1, 19, 21, 23 buellia leptocline (flot.) a. massal. e caloplaca cerinella (nyl.) flagey 7 caloplaca ferruginea (huds.) th. fr. 7 caloplaca flavovirescens (wulfen) dalla torre et sarnth. c caloplaca holocarpa (ach.) a. e. wade 18, 19 candelaria concolor (dicks.) stein 12 candelariella reflexa (nyl.) lettau 10 candelariella vitellina (hoffm.) müll. arg. e candelariella xanthostigma (ach.) lettau 1, 2, 4, 7, 10, 12, 17, 18, 19, 21 catillaria lenticularis (ach.) th. fr. c cetrelia olivetorum (nyl.) w. l. culb. et c. f. culb. 4, 5, 7, 12 cladonia caespiticia (pers.) flörke c cladonia coniocraea (flörke) spreng. 1, 10, e, g u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees 102 acta bot. croat. 70 (1), 2011 partl a. species stations cladonia digitata (l.) hoffm. e, g cladonia fimbriata (l.) fr. 1, g cladonia furcata (huds.) schrad. e, g cladonia macilenta hoffm. subsp. macilenta c cladonia pyxidata (l.) hoffm. 15, c, e, f cladonia rangiferina (l.) f. h. wigg. c, g cladonia rangiformis hoffm. f cladonia squamosa hoffm. var. squamosa e, g clauzadea monticola (schaer.) hafellner et bellem. d collema flaccidum (ach.) ach. 2, 13 collema fuscovirens (with.) j. r. laundon 13 collema tenax (sw.) ach. e, f dermatocarpon intestiniforme (körb.) hasse d dermatocarpon miniatum (l.) w. mann 13, a dibaeis baeomyces (l.) rambold et hertel d diploschistes muscorum (scop.) r. sant. e diplotomma alboatrum (hoffm.) flot. g evernia prunastri (l.) ach. 1, 2, 7, 12, 17, 18, 20, 21, 22, 23, 24 farnoldia jurana (schaer.) hertel subsp. jurana e flavoparmelia caperata (l.) hale 1, 4, 6, 8, 10, 18, 19, 20, 22, 24, e fuscidea stiriaca (a.massal.) hafellner 8, e graphis scripta (l.) ach. 2, 4, 8, 10, b gyalecta hypoleuca (ach.) zahlbr. e gyalecta jenensis (batsch) zahlbr. c hypocenomyce scalaris (ach.) m. choisy 1 hypogymnia physodes (l.) nyl. 1, 3, 7, 10, 12, 17, 18, 19, 20, 21, 22, 24 hypogymnia tubulosa (schaer.) havar. 1, 7, 10, 12, 17, 18, 19, 20, 21, 23 lecanora allophana nyl. 2, 7, 10 lecanora argentata (ach.) malme 7, 10 lecanora carpinea (l.) vain. 10, 12, 18 lecanora chlarotera nyl. 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 17 lecanora conizaeoides cromb. 4 lecanora dispersa (pers.) sommerf. d lecanora expallens ach. 7 lecanora leptyrodes (nyl.) degel. 4, 9 lecanora thysanophora r. c. harris 10 widctlparlecanora saligna (schrad.) zahlbr. 1, 10 lecidella elaeochroma (ach.) m. choisy 1, 2, 4, 5, 7, 8, 10, 11, 20 lepraria nivalis j. r. laundon 5 leptogium lichenoides (l.) zahlbr. b tab. 1 – continued u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (1), 2011 103 lichens from @umberak and samoborsko gorje, croatia species stations leptogium saturninum (dicks.) nyl. 2, 14 lobaria pulmonaria (l.) hoffm. 2, 7, 10, c melanelia elegantula (zahlbr.) essl. 7 melanelia exasperata (de not.) essl. 1, 7, 18, 10, 21 melanelia exasperatula (nyl.) essl. 6 melanelia fuliginosa (duby) essl. subsp. glabratula 1, 2, 4, 5, 7, 8, 10, 14, 17, 18, 19, 20, 22, 23, 24 melanelia glabra (schar.) essl. 1 melanelia subaurifera (nyl.) essl. 2, 7, 10, 12 menegazzia terebrata (hoffm.) a. massal. e micarea peliocarpa (anzi) coppins et r. sant. g mycobilimbia lurida (ach.) hafellner et türk f ochrolechia turneri (sm.) hasselrot 5, 7 opegrapha calcarea sm. d opegrapha rupestris pers. d, e parmelia omphalodes (l.) ach. g parmelia saxatilis (l.) ach. 1, 2, 4, 7, 10 parmelia sulcata taylor 1, 2, 4, 6, 7, 8, 9, 10, 12, 17, 18, 18, 20, 21, 24 parmelina pastillifera (harm.) hale 4, 6, 7, 10 parmelina quercina (willd.) hale 18 parmelina tiliacea (hoffm.) hale 1, 2, 4, 6, 7, 8, 17, 18, 19, 20, 21, 22, 23, 24 parmeliopsis ambigua (wulfen) nyl. 1, 3, 10 parmotrema crinitum (ach.) m. choisy g parmotrema perlatum (huds.) m. choisy 7, 10, 14 peltigera canina (l.) willd. e peltigera didactyla (with.) j. r. laundon 16 peltigera horizontalis (huds.) baumg. 2, 15, g peltigera membranacea (ach.) nyl. 2 peltigera polydactyla (neck.) hoffm. 2, 10, 13, 16 peltigera praetextata (sommerf.) zopf 14, 15, 16, e peltigera rufescens (weiss) humb. 2 peltigera venosa (l.) hoffm. d pertusaria albescens (huds.) m. choisy et werner 2, 4, 7, 10, 22 pertusaria amara (ach.) nyl. 7, 22 pertusaria coccodes (ach.) nyl. 7, 10 pertusaria corallina (l.) arnold g pertusaria flavida (dc.) j. r. laundon 20 pertusaria leioplaca dc. e tab. 1 – continued u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees 104 acta bot. croat. 70 (1), 2011 partl a. species stations pertusaria pertusa (weigel) tuck. 2, 4, 5, 7, 8, 10 phaeophyscia orbicularis (neck.) moberg 1, 7, 18 phlyctis argena (spreng.) flot. 4, 7 physcia adscendens (fr.) h. olivier 2, 7 physcia stellaris (l.) nyl. 1, 3, 7, 12, 18, 19, 20, 21, 23 physcia tenella (scop.) dc. 1, 5, 7, 18, 21 physcia tribacia (ach.) nyl. 7 physconia distorta (with.) j. r. laundon 1 physconia grisea (lam.) poelt subsp. grisea 7 physconia perisidiosa (erichsen) moberg 1, 2 placynthium nigrum (huds.) gray d platismatia glauca (l.) w. l. culb. et c. f. culb. 1, 7, 10, 19, 21, 24 pleurosticta acetabulum (neck.) elix et lumbsch 7 porina lectissima (fr.) zahlbr. c porpidia albocaerulescens (wulfen) hertel et knoph c porpidia crustulata (ach.) hertel et knoph d porpidia macrocarpa (dc.) hertel et a. j. schwab e protoblastenia rupestris (scop.) j. steiner d pseudevernia furfuracea (l.) zopf var. furfuracea 1, 7, 10, 12, 17, 18, 19, 20, 21, 24 punctelia subrudecta (nyl.) krog 2 ramalina calicaris (l.) fr. 7, 9 ramalina farinacea (l.) ach. 1, 2, 4, 6, 7, 9, 10 ramalina fastigiata (pers.) ach. 7, 20 ramalina fraxinea (l.) ach. 1, 2, 7, 8 rhizocarpon reductum th. fr. e rinodina confragosa (ach.) körb. e rinodina oxydata (a.massal.) a. massal. e sarcogyne regularis körb. var. regularis d solorina saccata (l.) ach. d, f usnea hirta (l.) f. h. wigg. 1, 12 usnea subfloridana stirt. 1, 7, 10, 12, 17, 18, 19, 20, 21, 22, 23, 24 verrucaria margacea (wahlenb.) wahlenb. e verrucaria muralis ach. d verrucaria nigrescens pers. c verrucaria saprophila (a. massal.) trevis. d vulpicida pinastri (scop.) j.-e. mattsson 1, 19 xanthoparmelia conspersa (ach.) hale e xanthoria candelaria (l.) th. fr. 12, 18 xanthoria parietina (l.) th. fr. 1, 3, 7, 12, 18, 19, 20, 21, 23, 24, c xanthoria polycarpa (hoffm.) rieber 7, 19, 21 tab. 1 – continued u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees discussion lecanora thysanophora is the first croatian record for this species, and out of 84 taxa recorded during the survey 74 are new records for the area. most lichens were epiphytes on birch (betula pendula), oak (quercus sp.) and, at the higher altitudes, beech (fagus silvatica). of particular importance as porophytes were veteran fruit trees in grassland orchards and planted larch trees at location 12. foliose lichens were the most abundant (42 species), followed by crustose taxa (23) and then fruticose lichens (13). other growth forms were not common, and comprised three composite taxa (cladonia type), two leprose, and one squamose lichen. the most important habitats for lichens were open forests, old orchards, and old forests undisturbed by forestry management. in terms of response to light intensity, most of the species recorded preferred well lit situations but were also able to tolerate shade. orchards and open forests were the favoured habitats for such species. no extreme skiophytes were found, and very few species requiring direct solar irradiation. species recorded from old forests favoured semi-shaded to shaded habitats. as regards temperature tolerance, most of the lichens recorded were those favouring submontane to montane regions, which is consistent with the climate and terrain in the area. extremely thermophilous lichens were not recorded. tolerance to cold is a survival factor for a few montane to high montane species, such as bryoria fuscescens, cetrelia olivetorum, hypogymnia tubulosa, melanelia exasperata, parmeliopsis ambigua, platismatia glauca and usnea subfloridana and one highmontane to subalpine species – vulpicida pinastri, which inhabited valleys where cold air persists, or snow lays for longer periods. oceanity shows the tolerance of lichens to extreme climate changes during the year – oceanic species prefer milder climate with fewer variations and are more common in western europe: such are the most numerous in the region if we exclude species that are widespread. no continental species were found, which suggests the climate is mild continental with no extremes. no extreme hygrophytes nor xerophytes were found – the 1000 mm yearly precipitation experienced by the area being sufficient to satisfy the water requirements of the species recorded. lichens are sensitive to ph and the substrata, which makes them good indicators of environmental quality. a number of species are epiphytes on certain tree types. many species in this area grow on mildly acid substrata, but a few are able to tolerate more acid conditions: they can either tolerate pollution resulting from acid rain or prefer trees with more acid bark such as conifers and birch (which is the case in the study area). for epiphytic lichens a further limiting factor is the nutrition value of the substratum. some tree species have higher concentrations of important minerals in bark than others, such as ash (fraxinus excelsior) and maples (acer sp.) an absence of epiphytes on trees does not therefore necessarily indicate environmental pollution. most of the lichens found in the study area were epiphytes on trees that indicated low nutrition requirements, with a smaller number (presumably requiring a higher mineral requirement) found on naturally richer bark. a number of species were usually recorded from the base of trees, where acta bot. croat. 70 (1), 2011 105 lichens from @umberak and samoborsko gorje, croatia u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees eutrophication is caused by animal excrements or by exhaust gases of vehicles at the roadsides. because of their sensitivity to certain anthropogenic chemicals lichens have long been used as indicators of environmental pollution. the presence of toxitolerant species does not necessarily signify pollution, but the appearance of sensitive taxa is a good signal. in the study area most of the species recorded a fall somewhere in the middle of the scale of tolerance; three species extremely sensitive to environmental pollution– leptogium saturninum, lobaria pulmonaria and melanelia exasperata – and another 12 moderately sensitive taxa were recorded. this indicates that the area has experienced excellent air quality over a long time period. lobaria pulmonaria, which is an old forest indicator, was found in several localities, showing that this area has remained undisturbed or minimally managed for some considerable time a factor that makes its protection of significant importance. to summarise; the presence of old forest indicators, together with species sensitive to environmental pollution indicates that this area should be protected. old orchards within the study area, in which agro chemicals have never been used, support high numbers of otherwise rare and sensitive species, including usnea subfloridana, u. hirta and bryoria fuscescens, which makes their continued, traditional management as grassland orchards of vital importance. some areas shown a high number of interesting species, and should be protected as special reserves: budinjak is a still a thriving village, and the nearby birch and oak forest, that have been minimally managed, should be conserved. in particular the locations where lobaria pulmonaria was found, especially areas near the peak on the slovenian border, should be protected as special forest reserves and left unmanaged or managed minimally. this lichen does not survive intensive commercial forestry, particularly when growing outside its normal oceanic climate range (which is the case within the park). a number of rare species have been recorded near the summit in slovenia, in two special forest reserves, trdinov vrh and ravna gora (ho^evar 1985). any future study should be performed in terms of various environmental factors, such as light requirements, temperature, oceanity, water requirements, ph and nutrition values of the substratum and toxitolerance, and should also compare relationships with other studies (wirth 1992). acknowledgements i would like to thank prof. helmut mayrhofer (austria) for doing the tlc analysis and determination of leprose lichens, ivan pedley (england) for help with determination of some samples and english language revision, prof. franc bati~ (slovenia) for providing me needed literature, and dr. matija frankovi} (croatia) who accompanied me in most of my fieldwork in @umberak – samoborsko gorje nature park and provided information on the location of lichen rich habitats. 106 acta bot. croat. 70 (1), 2011 partl a. u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees references christensen, s. n., 1987: contribution to the lichen flora of yugoslavia. acta botanica croatica 46,161–171. christensen, s. n., 1988: contribution to the lichen flora of istria, yugoslavia. acta botanica croatica 47,127–134. christensen, s. n., hansen, e. s., 1994: lichens from croatia. acta botanica croatica 53, 101–113. clauzade, g., roux, c., 1985: lichens of the western europe (in esperanto). bulletin de la societe botanique du centre-ouest, nouvelle serie, no. special 7, 1–893. dobson, f. s., 1992: lichens. an illustrated guide to the british and irish species. richmond publishing, slough. ho^evar, s., 1985: dinaric old montane forests (in slovenian). strokovna in znanstvena dela 76, 7–262. ku[an, f., 1928: contribution to the lichen flora of croatia (in croatian). acta botanica instituti botanici universitatis zagrebensis 3, 1–40. ku[an, f., 1953: lichen check list of yugoslavia (in croatian). jugoslavenska akademija znanosti i umjetnosti, zagreb. nims, p. l., martellos, s., 2008: italic – the information system on italian lichens. version 4.0. univesity of trieste, department of biology, in4.0/1. (http://dbiodbs. univ.trieste.it/) ozimec, s., 2000: five new lichen species to the croatian flora. natura croatica 9, 133–138. ozimec, s., florijan^i], t., opa^ak, a., pu[kadija, z., topi], j., 2009: lichen mycota from the island of krk (northern adriatic sea, croatia). natura croatica 18, 367–385. ozimec, s., partl, a., 2009a: red list of lichens of croatia (in croatian). dr`avni zavod za za{titu prirode, zagreb. ozimec, s., partl, a., 2009b: check list of lichens of croatia (in croatian). dr`avni zavod za za{titu prirode, zagreb. partl, a., asta, j., 2003: epiphytic lichen flora on mountain medvednica and in northern zagreb: bioindication of environmental factors. periodicum biologorum 105, 337–343. purvis, o. w., coppins, b. j., hawksworth, d. l., james, p. w., moore, d., 1992: the lichen flora of great britain and ireland. natural history museum publications in association with the british lichen society, london. wirth, v., 1992: bioindication values of lichens (in german). scripta geobotanica 18, 215–237. wirth, v., 1995: flechten aus baden wuerttenberg, 1–2. ulmer, stuttgart. acta bot. croat. 70 (1), 2011 107 lichens from @umberak and samoborsko gorje, croatia u:\acta botanica\acta-botan 1-11\partl.vp 28. o ujak 2011 16:29:49 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 77 (1), 2018 s1 social news “lost worlds of archaic gardens” – 2018 exhibition in the botanical garden of the faculty of science, university of zagreb acta botanica croatica is an international scientific journal published by the department of biology, faculty of science, in the 2018 season, the zagreb botanical garden of the faculty of science will present an exhibition created by the garden curators, dr sanja kovačić, dr dubravka sandev and dr vanja stamenković, in cooperation with the faculty’s botanists, palaeontologists, geographers and chemists. the exhibition, named “lost worlds of archaic gardens” (in croatian: izgubljeni svjetovi ‒ pradavni vrtovi devona, karbona i krede), will present the evolution of the plant kingdom on our planet through the imaginary “gardens” of three well-known periods in the geological time scale: early devonian (palaeozoic), late carboniferous (palaeozoic) and mid-cretaceous (mesozoic). the exhibition is intended for schoolchildren and students primarily, but will also be of interest to the widest audience (pl. 1a). through three dioramas – fictional garden beds from the prehistoric times – the visitors will be able to learn how and when the plants conquered land and through hundreds of millions of years finally brought us to the world we know today. the diorama from the early devonian (–400 mya) will be arranged around a painting of the english author richard bizley (lyme regis, dorset, uk). this well-known and often repeated scenery, which depicts a famous rhynie chert site in scotland, will be accompanied by models of some of the long-vanished first plants on earth (rhynias, cooksonias) and some of the living specimens from the botanical garden collections, resembling (if not related to) these ancient organisms (e.g. whisk ferns, pl. 1b, and spike mosses, pl. 1c). the visitors will learn what scientists believe today, why and how the earliest plants inhabited the land, still without soil. the diorama from the late carboniferous (–300 mya) will be designed around a computer graphic by another famous palaeo-artist, the american walter b. myers (chicago, usa). the largest forests on earth, dominated by such primitive plants as clubmosses and horsetails (pl. 1d) but of enormous dimensions, reigned our planet throughout that period. even today, many descendants of the early carboniferous plants grow around the world, such as cycads and araucarias (pl. 1e). how did the vast coal deposits form and why would that be impossible today…? finally, the backdrop for the mid-cretaceous diorama (–100 mya) will be custom-made by the croatian artist berislav kržić, recognized for his dinosaur paintings and graphics. the forests of that period are much more familiar to us than any before. the ferns were present, the wellknown gymnosperms (for example, sequoias and ginkgos, pl. 1f), together with some early angiosperms (such as magnolias and water lilies, pl. 1g), already well linked with their insect-pollinators. but what happened? what caused these vast woods to vanish? why did the mass extinction events wipe out most of the life on earth at least five times through the geological history of our planet…? the city of zagreb, department of biology (faculty of science), croatian botanical society and “kolding” ltd. will financially support the exhibition. entrance will be free of charge to all visitors. dr sanja kovačić, senior garden curator botanical garden, department of biology, faculty of science, university of zagreb, marulićev trg 9a, hr-10000 zagreb, croatia e-mail: sanja.kovacic@biol.pmf.hr s2 acta bot. croat. 77 (1), 2018 plate 1. a) exhibition logo “flora palaeozoica” by dr vanja stamenković, senior garden curator; b) whisk fern (psilotum nudum (l.) p. beauv.) from zagreb botanical garden collection (courtesy of the botanical garden of eötvös university, budapest). still resembling the earliest plants from the late silurian, these primitive ferns are actually of more recent ancestry; c) several species of spikemosses (selaginella spp.) are grown in the temperate glasshouse collections of the garden. that primitive family (selaginellaceae) of vascular plants evolved in the palaeozoic; d) the recent “great” horsetail (equisetum telmateia ehrh.) could reach up to 2.5 meters in height. it is a successor to the large carboniferous equisetums, such as the famous calamites, that were more than 30 meters tall; e) norfolk island pine (araucaria heterophylla (salisb.) franco) represents the araucariaceae family distributed in ancient gondwanaland. zagreb botanical garden holds several species of this ancient family; f) the maidenhair tree (ginkgo biloba l.) is the single living descendant of the ginkgoaceae family of gymnosperms, which appeared in the mesozoic. it is commonly grown in parks and gardens around the world; g) water lilies (nymphaeaceae), such as this nymphaea (‘marliacea rubra’ cultivar) from the garden collection, evolved in the cretaceous. it is thought that they are one of the earliest angiosperm families on earth still present today. https://en.wikipedia.org/wiki/carl_linnaeus https://en.wikipedia.org/wiki/palisot_de_beauvois https://en.wikipedia.org/wiki/palisot_de_beauvois https://en.wikipedia.org/wiki/jakob_friedrich_ehrhart https://en.wikipedia.org/wiki/richard_anthony_salisbury 62 acta bot. croat. 77 (1), 2018 acta bot. croat. 77 (1), 62–69, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0005 issn 0365-0588 eissn 1847-8476 effects of carrageenan as elicitor to stimulate defense responses of basil against cuscuta campestris yunck effat ahmadi mousavi *1, khosrow manochehri kalantari1, fatemeh nasibi1, hakimeh oloumi2 1 biology department, faculty of science, shahid bahonar university of kerman, kerman, iran 2 ecology department, institute of science and high technology and environmental sciences, graduate university of advanced technology, kerman, iran abstract – cuscuta campestris is a holostemparasitic plant that obtains its resources from its hosts. sweet basil is an important commercial plant, widely cultivated in many countries. it is a common host for c. campestris. generally, c. campestris has negative effect on the growth of infected plants and its infestation is difficult to control. therefore, environmental friendly control of c. campestris seems to be useful. in this work, the relationship between c. campestris and its host, sweet basil, and effects of κ-carrageenan on protection against c. campestris and suppression of its invasion were studied. basil was sprayed with a solution of carrageenan at a final concentration of 1 g l–1, once a week, 3 times in total. infection of basil with c. campestris was performed 2 days after the last carrageenan treatment and the plants were collected two weeks after c. campestris attachment. in this study, phenylalanine ammonia-lyase activity (pal), phenolic, flavonoids and antioxidant content increased remarkably in the basil plants parasitized with c. campestris, and therefore it seems that the parasitic plant induced a defense response in the host plants. treatment with carrageenan significantly increased shoot length and leaf area of basil and decreased c. campestris infestation by about 26%. carrageenan treatment caused a significant increase in pal activity, phenols, antioxidant and lignin content in basil. thus, the present observation suggested the phenylpropanoid pathway was activated and defense responses were stimulated. our results showed that carrageenan spraying induced beneficial effects in plants, corresponding to growth stimulation and defense compound synthesis. thus carrageenan treatment is recommended as a natural biostimulator for the protection of plants against c. campestris. keywords: carrageenan, defense against parasites, field dodder, ocimum basilicum * corresponding author, e-mail: effatmousavi@yahoo.com introduction among plants with medicinal value, plants of the genus ocimum (lamiaceae) are very important for their therapeutic potentials (ramasubramania 2012). sweet basil (ocimum basilicum l.) is an important species of the genus ocimum that grows in different parts of the world (tewari et al. 2012). sweet basil is an annual plant native to asia that is cultivated as a field crop in the mediterranean countries and is widely used in food (for flavour) and in oral care. the essential oil of the plant is additionally used in perfumery (tewari et al. 2012). leaves and flowers of basil are traditionally used as digestive, antispasmodic, stomachic, carminative, galactagogue, aromatic and tonic agents (gülçin et al. 2007). antimicrobial and antiviral activities of this plant have also been reported (chiang et al. 2005). sweet basil is a common host for the stem-parasitic plant cuscuta campestris yunck. with the common name of field dodder. this parasite is one of the major constraints that limit the productivity of basil (behbahani 2014) and therefore the parasitic interaction of c. campestris with its hosts seems interesting. c. campestris is a well known member of the cuscuta genus, of which it is the most widespread species. it is a holostemparasitic plant without leaves and roots, but it can produce absorptive haustoria that provide physical and physiological bridges between the parasite and its host (kushan et al. 2006). it can infect various host species (dawson et al. 1994), self-parasitize and hyper-parasitize (liao et al. 2005). upon successful creation of vascular connections with the host, cuscuta becomes a strong sink, withdrawing water, ions, sugars, amino acids and other nutrients. it obtains its resources entirely from its host plants (dawson et al. 1994). cuscuta-infested plants gradually become weak, resulting in decline in growth and yield (aly 2013). in addition to its impact on ornamentals, native plants and forage crops, it has been reported that cuscuta can suppress or kill https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&sqi=2&ved=0ahukewidleol7onoahvkomakhqobdjaqfggcmaa&url=https%3a%2f%2fen.wikipedia.org%2fwiki%2fphenylalanine_ammonia-lyase&usg=afqjcnfn3bypkis3rtl9bwitqbgkm6ydeg&sig2=4s6htxdxwzzq53hpj4fgga&bvm=bv.127984354,d.bgg mailto:effatmousavi@yahoo.com carrageenan stimulates defense of basil against cuscuta acta bot. croat. 77 (1), 2018 63 vegetables and weeds (fathoulla and duhoky 2008). control of cuscuta spp. is extremely difficult, thus it causes huge losses in agriculture (shen et al. 2007). natural extracts from plants and other organism for protecting crops from pathogens and pests have recently been reported. they protect plants without negatively affecting the growth and yield (kapooria et al. 2007, aly 2013).an effective method of achieving crop protection is inducing resistance by activating natural protection system with elicitors that are environmentally safe. it has been reported that this strategy is effective and was used against a vast range of pathogens, pests and parasites, including fungi, bacteria, viruses, insects and herbivores (halim et al. 2004), but there is no report of such method being used for the control of parasitic plants, especially c. campestris. the major elicitors described in literature are varied in nature and include oligosaccharides, polysaccharides, glycopeptides, lipids, peptides and proteins and are derived from cell wall, culture filtrate and cytoplasm of different parasitic and nonparasitic plant pathogens (rao et al. 1996, halim et al. 2004). in most cases natural elicitor activity is related with the polysaccharide fraction of the varied preparations. other elicitors derived from plants can be linear or ramified oligosaccharides and polysaccharides (creelman and mullet 1997). it is well documented that polysaccharides purified from algae as well as derived oligosaccharides have the capacity to trigger plant defense responses (potin et al. 1999, khan et al. 2009). liquid extracts obtained from seaweeds have been reported to stimulate the growth of plants, improve resistance to diseases and pests, amend resistance to abiotic stresses (e.g., temperature extremes, salinity, drought and heavy metal stress), increase yield and quality, enhance mineral uptake from soil and antioxidant properties (zhang et al. 2003, bi et al. 2008, khan et al. 2009, craigie 2011, naeem et al. 2012, hashmi et al. 2012). the best-characterized seaweed elicitors that have been reported are laminarin (from the brown seaweeds laminaria digitata) and carrageenans (from species of red seaweeds of the class rhodophyceae) that have the potential to activate disease resistance in plants and animals (gonzàlez et al. 2013b). carrageenans are the major polysaccharides existing in many red macroalgae (jatinder et al. 2011). these gel-forming polysaccharides have a linear structure of d-galactose residues joined with alternating α-(1,3) and ß-(1,4) linkages which are substituted for by one (kappa (κ)-carrageenan), two (iota (ɩ)-carrageenan), or three (lambda (λ)-carrageenan) ester-sulphonic groups per di-galactose repeating unit (mercier et al. 2001, gonzàlez et al. 2013a). mercier et al. (2001) reported that ĸ-carrageenan elicited numerous plant defense responses, probably through the effect of its high sulphate content, and induced signaling and defense gene expression in tobacco leaves. since carrageenans have been shown to activate plant defense responses against pathogens (bi et al. 2011, shukla et al. 2016), it is plausible that carrageenans might stimulate resistance in plants to parasitic plants like cuscuta. however, the effect of carrageenans on plant resistance in the face of c. campestris is not known. most elicitors derived from plants or algae stimulate plant defense responses, mainly through the activation of the phenylpropanoid pathway (la caméra et al. 2004). the stimulation of the phenylpropanoid pathway produces phenolic compounds such as phytoalexins, lignins and salicylic acid, with potential defensive activities (lattanzio et al. 2006). thus, the purpose of this study was to investigate how carrageenan acts as elicitor to stimulate growth and defense responses to c. campestris infestation in sweet basil plants. materials and methods plant material and treatments sweet basil (ocimum basilicum l.) seeds were sterilized by immersing them in 0.1% (w/v) sodium hypochlorite solution for 5 min and then washed extensively with distilled water and finally rinsed with sterile water. pots (14 cm diameter × 12 cm height) were filled with a homogenous mixture of soil and organic manure (4:1). physicochemical characteristics of the soil were: texture – sandy loam, ph (1:2) (soil:water, v:v) 7.2 and electrical conductivity (1:2) (soil:water, v:v) 0.43 ds m−1 (hanlon 2015). basil and cuscuta campestris grow best under conditions of high light, moisture and temperature, these conditions are provided in a natural environment, and therefore the pot experiment was conducted in natural conditions on june, 2015. all experiments were repeated twice. the repetition of cultivation and experiments was conducted in the following year (2016). basil seeds were planted at 2 cm depth in the center of each pot. most seedlings emerged above the soil surface 4 days after planting and were kept then thinned to 8 plants per pot and the pots were watered when required. pure κ-carrageenan (sigma–aldrich, usa) was dissolved in hot deionized water. a concentration of carrageenan solution (1 g l−1) was finally prepared for foliar spray treatments. the first carrageenan treatments were applied to the basil plants 14 days after sowing, when the plants were at the 2 true leaf stages. carrageenan solution was sprayed on the upper and lower surface of basil leaves and on the stem, while the 2nd and 3rd sprays were done one and two weeks after the first treatment, respectively. control plants were sprayed with deionized water. c. campestris is simply transferable to host plants by vegetative propagation. significant changes in the amount of metabolites at the apical region of filament indicate that this region is most active during haustorium development for parasitization (furuhashi et al. 2012). placement of actively growing cuscuta spp. stems on host plants usually results in the formation of haustoria and thus causes a new c. campestris plant (hong et al. 2011). therefore, threadlike stems (120 mm from tip) of c. campestris without haustorium were separated and placed on the top of 30-day-old basil stem. the basil plants were treated two days in advance with carrageenan before c. campestris inoculation. the c. campestris was allowed to attach to plants. usually, c. campestris stem attaches to basil shoot one day after inoculation. the threadlike stem of c. campestris and parasitized basil leaves mousavi e. a., manochehri kalantari k., nasibi f., oloumi h. 64 acta bot. croat. 77 (1), 2018 (third row of opposite leaves counted from the cotyledon node) were collected 2 weeks after c. campestris attachment, the carrageenan treated and un-treated healthy basil leaves (third row of opposite leaves counted from cotyledon) were harvested 44 days after sowing. performance of the plant was assessed in terms of growth attributes (infestation percentage, the plant height and leaf area), and biochemical parameters (with three biological replications). infestation percentage of c. campestris for analysis of defense against c. campestris invasion and measuring of infestation of basil plants by c. campestris, the percentage of c. campestris tight coupling (its haustorium penetration) to basil shoot was determined 14 days after the infection. the experiments comprised twenty replications and were repeated twice. plant growth parameters the height of a plant is the vertical distance from the soil at its base to the highest point reached with all parts in their natural position. the height of the basil plant in its natural position was measured. the experiments comprised twenty replications and were repeated. the methods for leaf area measurement include weighing, copying the shape of the 5th leaf counted from cotyledon on a fragment of paper and weighing the copy. so leaf area is estimated by the following equation: la = w / c, where la is the leaf area, w is the weight of the paper and c is the coefficient of the paper (weight of unit area). the experiments comprised twenty replications and were repeated twice. phenolic content analysis in order to measure total phenolic content, fifty milligrams of plant material of each sample (the 5th basil leaf counted from the cotyledon node and threadlike stem of c. campestris) were ground and dissolved in 1 ml of 80% ethanol, using pestle and mortar . the homogenate was kept at 25° c for 24 h in the dark. then it was brought to a volume of 5 ml with ethanol and centrifuged at 2000 g for 10 min. soluble phenolic content was analyzed by the method of gao et al. (2000), using the folin-ciocalteu reagent. one hundred µl of extract was mixed with folin-ciocalteu reagent (200 µl) and distilled water (2 ml) and incubated at room temperature for 3 min. after incubation, a sample of aqueous sodium carbonate (20% w/w, 1 ml) was added to the mixture. the phenolics were measured after one hour of incubation at room temperature. the absorbance of the resulting blue color was determined at 765 nm. gallic acid was used as a standard and the results were expressed in milligrams of gallic acid equivalent per gram fresh weight of leaf. the experiments comprised three replications and were repeated twice. in order to measure total flavonoids, nine hundred milligrams of plant material of each sample (the 5th basil leaf counted from the cotyledon node and the threadlike stem of c. campestris) were ground and dissolved in 3 ml of 80% methanol, using mortar and pestle. the homogenate was kept at 25 °c in the dark. then it was brought to a volume of 3 ml with methanol and centrifuged at 2000 g for 10 min. total flavonoid content was determined according to the aluminium chloride colorimetric method (chang et al. 2002). two ml of extracts (0.3 g ml–1) in methanol were mixed with 0.1 ml of 10% aluminium chloride hexahydrate, 0.1 ml of potassium acetate (1 m) and 2.8 ml of distilled water. after 40 min incubation at room temperature, the absorbance of the reaction solution was determined spectrophotometrically at 415 nm. quercetin was chosen as a standard (standard concentration range: 12 to 200 mg l–1) and the results were expressed in milligrams of quercetin equivalent per gram fresh weight of leaf. anthocyanin content was determined according to a modified wagner (1979) method. the plant material of each sample (0.1 g of the 5th basil leaf counted from the cotyledon node and the threadlike stem of c. campestris) was crushed in 10 ml acidified methanol [methanol:hcl (99:1, v:v)]. the tissues were soaked and incubated at room temperature for 24 h in the dark. the extracts were then centrifuged at 4000 g for 10 min at 25 °c. the absorption of the supernatant was read spectrophotometrically at 550 nm. to calculate the amount of anthocyanins, the extinction coefficient 33000 l mol–1 cm–1 was used and it was expressed as μmol per gram fresh weight of leaf. antioxidant activity the antioxidant activity of plant material of each sample was assessed by a modified girennavar method (2007), using the stable free radical 2, 2-diphenyl-1-picrylhydrazyl radical (dpph) which forms a violet solution and reacts with antioxidants by losing color. ten µl of each extract was mixed with 100 µm dpph in methanol in a final volume of 1 ml. the changes were monitored over 20 min. a control was prepared as described above without samples or standards. methanol was used for the baseline correction. the changes in the absorbance of all the samples and standards were measured at 517 nm. radical scavenging activity was expressed as the inhibition percentage and was calculated using the following formula: percent of radical scavenging activity = (control optical density-sample optical density/control optical density) × 100. ascorbic acid was chosen as a standard antioxidant and extract activity is expressed in µmol of ascorbic acid equivalents per gram fresh weight of leaf. lignin content analysis protein and other uv-absorbing materials removal protocols were essential to avoid the measurement of these constituents together with lignin at 280 nm. five hundred milligrams of the plant material of each sample (the same age stem of basils based on leaf number and threadlike stem of the dodders) were homogenized in 2 ml water using a pestle and mortar and then transferred into a screwcap centrifuge tube and centrifuged at 1000 g for 10 min and the supernatant was removed. the pellet was washed two times with ethanol, then the pellet was soaked and incubated at room https://www.google.com/search?q=quercetin&start=0&spell=1&biw=1366&bih=610 https://www.google.com/search?q=quercetin&start=0&spell=1&biw=1366&bih=610 carrageenan stimulates defense of basil against cuscuta acta bot. croat. 77 (1), 2018 65 temperature for 24 h in methanol: chloroform (1:2), the supernatant was removed and rinsed with acetone. the pellet was dried in an oven (60 °c, 24 h) and cooled in a vacuum desiccator. the dry matter obtained was defined as the protein-free cell wall fraction (hatfield et al. 1999). lignin content was determined according to a modified iiyama and wallis method (1990). protein-free cell wall sample (20 mg) was placed into a screwcap centrifuge tube containing 0.5 ml of 25% acetyl bromide (v/v in glacial acetic acid) and incubated at 70 °c for 30 min. samples also contained 100 µl of perchloric acid to aid in the total dissolution of the wall material. after complete digestion, the sample was quickly cooled in an ice bath, and mixed with 0.9 ml of 2 m naoh and 6 ml of glacial acetic acid sufficient for complete solubilization of the lignin extract, and then samples were diluted to 25 ml with acetic acid. after centrifugation at 1000 g for 5 min, the absorbance of the supernatant was measured at 280 nm. guaiacol was used as a standard and the results were expressed in milligrams of guaiacol equivalent per gram fresh weight of basil stem. phenylalanine ammonia lyase (ec 4.3.1.5) enzyme assay three hundred milligrams of plant material of each sample (the 5th basil leaf counted from the cotyledon and threadlike stem of c. campestris) was homogenized in an ice cold mortar using 50 mm tris-hcl buffer (ph=8.8) containing 10 mm 2-β-mercaptoethanol, 1 mm ethylenediaminetetraacetic acid (edta) and 2.5% polyvinylpyrrolidone (pvp). the mixture was centrifuged at 20000 g for 20 min and the clear supernatant was desalted in aliquots using an amicon ultra-15 centrifugal filter units with ultracel-50 membrane (merck millipore, germany) and assayed for pal activity. pal activity was determined according to the method of şirin et al. (2016). the enzyme reaction mixture contained 400 µl of reaction buffer (100 mm tris-hcl, ph 8.8) and 200 µl of substrate (40 mm l-phenylalanine, 100 mm tris-hcl, ph 8.8) and a 200 µl aliquot of the sample filtrate (or 200 µl of deionised water used as a blank). the reaction was carried out at 37 °c for 30 min and terminated by the addition of 50 μl of 4 m hcl, and the cinnamic acid concentration was measured spectrophotometrically by the absorbance at 290 nm. one unit of pal activity is equal to 1 μmol of cinnamic acid produced per min. the enzyme activity was expressed in u per milligram protein. protein content was determined according to the method of bradford (1976) using bovine serum albumin as standard. data analysis the experiments were performed by a factorial arrangement, based on complete randomized design. obtained data were the average of the replications and two repetitions of each experiment, because there was no interaction. results were determined using analysis of variance (anova) via statistical analysis software (sas, version 9.4, sas institute inc., cary, nc, usa). the univariate procedure of sas was used to test for normality of residuals. each column value in figures represents mean ± standard deviation (sd). means were compared using fisher’s protected least significant differences (lsd) test. differences at p ≤ 0.05 were considered to be significant. results in the present study, we observed that the infestation percentage of c. campestris in basil that was not treated with carrageenan was 60.48%, but in carrageenan-treated basil plants a significantly lower attachment of c. campestris, 33.93%, was recorded. these findings showed that the infestation of basil plant treated with carrageenan by c. campestris decreased by about 26% (tab. 1), and therefore spraying with carrageenan significantly increased the resistance of basil plants to c. campestris invasion. c. campestris attachment occurred along the shoot of control plants (fig. 1), but in basils which were sprayed with carrageenan, it was observed only at shoot apical part with newly formed organs (stem, leaves and petioles). tab. 1. effect of foliar application of carrageenan (1 g l–1) and cuscuta campestris invasion on the growth parameters of ocimum basilicum (basil) and percentage of cuscuta infestation. b0 – basil sprayed with water (control); b1 – basil sprayed with carrageenan; b0+d – basil parasitized by c. campestris; b1+d – basil sprayed with carrageenan and parasitized by c. campestris. values are mean ± sd of twenty replications. the different letters in the same column indicate significant difference at p ≤ 0.05. treatments shoot length (cm) leaf area (cm2) cuscuta infestation (%) b0 22.05b±1.56 5.91b±0.43 – b1 24.325a±0.99 8.43a±0.95 – b0+d 13.625d±0.54 4.06c±0.73 60.48a±2.69 b1+d 16.375c±0.60 6.09b±0.71 33.93b±2.23 fig. 1. infestation of basil plants ocimum basilicum by cuscuta campestris: a) control and b) carrageenan treated basil plant. mousavi e. a., manochehri kalantari k., nasibi f., oloumi h. 66 acta bot. croat. 77 (1), 2018 infestation of c. campestris significantly decreased the shoot length and leaf area of basil, and the carrageenan treatment significantly increased the growth parameters in comparison with the carrageenan-untreated set of basil (tab. 1). therefore, it seems that the foliar application of carrageenan significantly alleviated the negative effect of the parasite (c. campestris) on the growth of infested host plants (tab. 1). the carrageenan treatment alone caused a significant increase of pal activity and phenolic and antioxidant content of basil. moreover, basil plants parasitized by c. campestris showed significantly higher pal activity, phenolic, flavonoids and antioxidant content (fig. 2). the lignin content of the carrageenan treated-basil did not show significant changes compared to the control set but the carrageenan treatment significantly increased the lignin content of basil plants that were parasitized by c. campestris (fig. 2). in this study, we did not observe any significant change in phenolics, flavonoids, antioxidant, anthocyanin and lignin content of the c. campestris attached to the control group of basil plants and c. campestris from basil hosts treated with carrageenan (fig. 2). discussion besides microbial pathogens and herbivorous arthropods, plants can also be parasitized by other plants. cuscuta species is recognized to cause serious economic losses in crop plants. in this study, infestation by c. campestris significantly decreased the shoot length and leaf area of basil. generally, with regards to the infected plant species, cuscuta invasion has been reported severely to affect the growth and reproduction of its host (lanini and kogan 2005). some researchers have shown that parasitic flowering plants of the genus cuscuta, most especially c. campestris yunck., c. australis r. br. and c. chinensis lam., can significantly reduce their host’s growth (zan et al. 2003, lanini and kogan 2005). also farah and al-abdulsalam (2004) showed that fig. 2. effect of foliar application of carrageenan and cuscuta campestris invasion on the biochemical parameters of basil leaves and cuscuta stems: a) phenol content, b) total flavonoid content c) antioxidant content, d) phenylalanine ammonia-lyase activity (pal), e) total anthocyanin content, f ) lignin content. values represent mean ± standard deviation (n=3). different letters above bars indicate significant difference at p ≤ 0.05. b – basil; b+d – basil parasitized by c. campestris; d – c. campestris carrageenan stimulates defense of basil against cuscuta acta bot. croat. 77 (1), 2018 67 c. campestris caused variable reductions in the vegetative (plant height, dry weights of shoot and root systems, number of leaves per plant) and reproductive (number of pods per plant and number of flowers per plant) traits of numerous legume crops. although cuscuta contains a few chloroplasts and slight chlorophylls, and thus possesses an extremely low photosynthetic activity, it is believed to be absolutely dependent on its host for nutrient sources (birschwilks et al. 2006, furuhashi et al. 2014) and will severely inhibit the growth of the host by removing its resources for photosynthesis, growth and leaf production. in addition, cuscuta spp. forms a dense, thick mat over the host, which decreases the sun’s radiation and consequently photosynthesis and growth (dawson et al. 1994). the specific pathways involved in defense against parasitic plants are unknown. in this research we showed that c. campestris induced pal activity and increased phenolic and flavonoid content in the basil plants. it has been well documented that responses of host plants to attack by c. reflexa include a hypersensitive-like response and phytoalexin production (borsics and lados 2002). best studied among host plant defenses against cuscuta sp. is the resistance responses of tomato plants to c. reflexa, in which elongation of hypodermal host cells, accumulation of phenolics and peroxidases at binding site create a mechanical barrier that can block haustorial formation (sahm et al. 1995). our results confirmed that c. campestris infestation increased the flavonoid levels in host plants. the application of herbicides to manage weed populations is very common but is not nature friendly and is not suitable for all situations. therefore, other ways of control, such as biological control, more friendly to the environment and humans, are necessary (rosskopf et al. 1999). carrageenans are recognized to elicit defense responses in plants and animals against various plant pathogens and mammalian viruses (khan et al. 2009). the effect of carrageenans on plant resistance to weeds is not known, and in this research we investigated the effects of ĸ-carrageenan on sweet basil resistance to cuscuta campestris infestation. in this study, the application of external carrageenan enhanced shoot length and leaf area of basil and alleviated the negative effect of the parasite on the growth of infested plants. it is known that κ, λ and ɩ-carrageenans at a concentration of 1 mg ml–1 increased shoot height and leaf biomass in tobacco plants by increasing the net photosynthesis, rubisco activity, the glutamate dehydrogenase activity involved in nitrogen assimilation, basal metabolism, and cell proliferation (castro et al. 2012, gonzàlez et al. 2013a). also, there is a report showing that seaweed extracts improved nutrient uptake by roots, thereby causing enhanced plant growth (mancuso et al. 2006). in this research, the anti-infestation activities of carrageenan were observed. infestation by c. campestris of basil plants treated with carrageenan decreased by about 26%. carrageenantreated basil plants attracted fewer c. campestris as compared to the control. it has been reported that the chemical reactivity of carrageenans is mostly due to their half-ester sulfate groups that are extremely anionic (necas and bartosikova 2013). these negatively charged compounds perform their inhibitory effect by interacting with the positive charges on the cell wall surface of parasite plants and thereby prevent the prehaustorium attachment and entrance into the host cells. in addition, the negative charge and antioxidant properties (yuan et al. 2006) of carrageenan probably cause inhibition of hydrolysis enzymes inside the cell wall of c. campestris, causing the inhibition of prehaustorium penetration in the host cell wall. moreover, it seems likely that κ-carrageenan induces several biochemical pathways in the development of plant resistance. in this work, c. campestris attachment occurred along the stem of control group of basil plants, but in basil with foliar spray of carrageenan few infections occurred in shoot apical parts not affected by carrageenan, because these organs were formed after carrageenan treatment. a recent study has shown that seaweed extracts can be used as an important source of plant defense elicitors (khan et al. 2009). besides influencing the physiology and metabolism of plants, it has been reported that ĸ-carrageenan is very effective and induces maximum browning and high level of secondary metabolites (pathogen resistance compounds) in various crop plants like chickpea, carrots and potatoes (bi et al. 2008). in addition ĸ-carrageenan is an effective substance for induction of defensive genes in tobacco leaves (mercier et al. 2001). we demonstrated that plant defense response was induced by ĸ-carrageenan. carrageenan treatment caused a significant increase in the pal activity and phenolic compound of basil, but flavonoid and anthocyanin content did not show considerable changes. induction of pal activity, the first and key enzyme of the phenylpropanoid pathway (vera et al. 2012) induces the accumulation of some phenyl propanoid compounds (ppcs) in tobacco plants. it has been reported that κ-, λand ɩ-carrageenans increase protection against fungal, viral, and bacterial infection in tobacco plants, which was due, at least in part, to the accumulation of ppcs (vera et al. 2011). also, it has been reported that oligo-carrageenans increased growth and activated defensive mechanisms against pathogens by enhancing the amount of some ppcs in eucalyptus trees (gonzàlez et al. 2013a, b). we observed that in basil, ĸ-carrageenans elicited pal enzyme activities, with the consequence of a higher phenolic content. the phenolics are implicated in plant defense through scavenging reactive oxygen species (cho and lee 2015). phenolic compounds are also involved in the defense response through reinforcement of the cell wall by biopolymer deposition near parasite infestation sites. in accordance with this explanation, the present study showed carrageenan treatment increased the lignin content of a basil plant parasitized by c. campestris, which is a desirable trait that may be associated with resistance of basil against c. campestris invasion. deposition of lignin has been hypothesized to interfere with the enzymatic hydrolysis and mechanical penetration of host plant tissue by cuscuta and may also weaken the movement of water and diffusible molecules between host and cuscuta and help to starve the parasite (lattanzio et al. mousavi e. a., manochehri kalantari k., nasibi f., oloumi h. 68 acta bot. croat. 77 (1), 2018 references 2006). as pointed out, the ability of c. campestris haustorium to penetrate from a looser site of the basil stem was the cause of the lower lignin content in the parasitized basil plant than in the control. this research demonstrates the ability of carrageenan to modulate the resistance of basil to c. campestris. the mechanism of this suppression is not clearly known, but we believe that the activation of natural plant resistance mechanisms and the ability of carrageenan to stimulate the synthesis of compounds (secondary metabolites) with antiinfestation activities will be the cause. importantly, this study is the first indication that carrageenan treatment can directly suppress infestation of the basil plant by c. campestris. in this study, antioxidant content increased remarkably in basil plants with c. campestris infestation and carrageenan treatment. the antioxidative effect is mainly due to phenolic components, which can act as reducing agents, hydrogen donors, and singlet oxygen quenchers. they may also have a metal chelating potential (niccholson 1992). basil plants possess valuable antioxidant properties that may be associated with lower incidence and lower mortality rates of cancer in several human populations (gülçin et al. 2007). our findings support the idea that treatment of basil plants with carrageenan stimulated the activation of the phenylpropanoid pathway, which may lead to the accumulation of phenolic compounds. thus, carrageenan foliar treatment of host plants may be directly and indirectly involved in the resistance mechanism to parasitic plants. because carrageenans are 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acta bot. croat. 77 (1), 2018 45 acta bot. croat. 77 (1), 45–50, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0003 issn 0365-0588 eissn 1847-8476 salicylic acid-induced germination, biochemical and developmental alterations in rye (secale cereale l.) fatma yanik1, özlem aytürk2, aslihan çetinbaş-genç1, filiz vardar1* 1 marmara university, science and arts faculty, biology department, göztepe campus, 34722 i̇stanbul, turkey 2 maltepe university, fine and arts faculty, gastronomy and culinary department, marmara eğitim köyü, i̇stanbul, turkey abstract – salicylic acid (sa) is one of the endogenous plant growth regulators that modulate various metabolic and physiological events. to evaluate the exogenous sa-induced germination, biochemical and developmental alterations, different concentrations (10, 100, 500 and 1000 µm) of sa were applied to rye (secale cereale l.) seeds in hydroponic culture conditions for 15 days. the observations revealed that seed germination and root elongation were stimulated in 10 µm sa treatment, however they were inhibited in higher concentrations (100 and 500 µm) of sa. furthermore, there was no germination in 1000 µm sa. the analysis of antioxidant enzymes revealed that although superoxide dismutase activity increased, catalase activity decreased in comparison to control. besides, lipid peroxidation and peroxidase activity increased in 10 µm sa, whereas they decreased in higher concentrations. similarly total chlorophyll content increased in 10 µm sa, but it decreased in 100 and 500 µm sa treatments. moreover anthocyanins and carotenoids increased after sa treatment. in conclusion, exogenous sa application causes developmental and biochemical alterations in rye. key words: antioxidant enzyme, lipid peroxidation, photosynthetic pigments, salicylic acid * corresponding author, e-mail: filiz.vardar@gmail.com introduction agricultural crops and wild flora are faced with a variety of intense environmental stress factors causing considerable economic losses worldwide (tuzhikov et al. 2011). plant defense is controlled through various type of protein and nonprotein signaling molecules. most phytohormones, such as ethylene, abscisic acid, jasmonic acid and salicylic acid (sa), have important roles as defensive molecules in the signaling pathways (vicente and plasencia 2011, war et al. 2011). sa is one of the endogenous plant growth regulators that belong to a diverse group of plant phenolics (pandey et al. 2013). it affects metabolic and physiological events in relation to growth and development in plants. it has long been known that sa regulates seed germination, adventitious root formation, photosynthetic reactions, cellular respiration, thermogenesis, flower formation and anthesis, seed production and senescence (raskin 1992, singh and usha 2003, khodary 2004, vicente and plasencia 2011). numerous researches indicated that sa content increases under various types of oxidative stress conditions in plants (larkindale and knight 2002, war et al. 2011). sa mediates the recognition of pathogens and activation of defense pathways from local to distal infected tissue inducing systemic acquired resistance (an and mou 2011). it also modulates plant defense under abiotic stress conditions, being an important signaling molecule (borsani et al. 2001, muñoz-sanchez et al. 2013). sa regulates the activity of enzymatic antioxidants such as superoxide dismutase (sod), polyphenol oxidase (ppo) and peroxidase (pod) in response to excessive production of reactive oxygen species (ros). however, sa inhibits the activity of some enzymatic antioxidants, such as ascorbate peroxidase (apx) and catalase (cat), leading to excess ros accumulation as signaling molecule (hayat et al. 2008). based on the regulatory and defensive role of sa in multiple developmental processes, exogenous application of sa has attracted attention in induction of plant defense against biotic and abiotic stress factors. however, sa controls plant growth and development, depending on the applied dose and species. according to previous research, a broad range of exogenous applied sa doses (10 nm to 10 mm) was effective in different plant species. (janda et al. 1999, shakirova et al. 2003, vicente and plasencia 2011). it has been reported yanik f., aytürk ö., çetinbaş-genç a., vardar f. 46 acta bot. croat. 77 (1), 2018 that higher concentrations may cause inhibitory effects on plant growth and development (hayat et al. 2008). the objective of the study is to investigate the effects of different concentrations of sa on growth, antioxidant enzymes and photosynthetic pigment content in rye (secale cereale l.), an important crop worldwide. material and methods the rye (secale cereale l. cv aslım 95) seeds were provided from the bahri dağdaş international agricultural research (konya, turkey). the surface-sterilized seeds were germinated in petri dishes with nutrient solution with or without sa (10, 100, 500 and 1000 µm) in a plant growth room set at 23±2 °c temperature, 45-50% relative humidity and a light intensity of 70 µmol (photon) m–2 s–1 (day/night: 16/8). the nutrient solution was a modified version of hoagland’s solution (ph 6.0) including 5 mm ca(no3)2, 5 mm kno3, 2 mm mgso4, 1 mm kh2po4, 30 µm fe(iii)-edta and standard hoagland micronutrients (hoagland and arnon 1950). all experiments were conducted three times and twenty five seeds were used for each repetition and experimental groups. the germinated control and sa treated seeds were counted and germination index was calculated. at the end of 15 days primary root length was measured and relative root growth was calculated according to schildknecht and de campos vidal (2002). hydrogen peroxide (h2o2) content was assayed according to junglee et al. (2014). control and sa treated roots (300 mg) were homogenized with 2 ml of the extraction buffer (0.1% trichloroacetic acid, 1 m ki, 10 mm phosphate saline buffer) and centrifuged at 12000 g for 15 min at 4 °c (sigma 3k18). the supernatants were incubated in total darkness for 20 min and then measured at 390 nm, spectrophotometrically. the standard curve was used to calculate the h2o2 content. control and sa treated roots (100 mg) were homogenized with 1 ml of cold sodium-phosphate buffer (pbs, 50 mm, ph 7.0). homogenates were centrifuged at 14000 rpm for 20 min at +4 °c. the supernatant was stored in ice for enzymatic assays. superoxide dismutase (sod) activity was determined by the method of cakmak and marschner (1992). the reaction mixture containing 2 ml of substrate buffer (100 mm ph 7.0 pbs, 2 m na2co3, 0.5 m edta, 300 mm lmethionin, 7.5 mm nitro blue tetrazolium, 0.2 mm riboflavin) and 2 µl of the supernatant was incubated under 15 w fluorescent lamps for 10 min and measured at 560 nm, spectrophotometrically. peroxidase (pod) activity was evaluated by the method of birecka et al. (1973). the reaction mixture containing 1.5 ml of substrate buffer (0.1 m pbs ph 5.8, 5 mm h2o2, 15 mm guaiacol) and 10 µl of enzyme extract was measured immediately for 2 min at 470 nm, spectrophotometrically. catalase (cat) activity was assayed as described by cho et al. (2000). the reaction mixture containing 1 ml of substrate buffer (20 mm pbs ph 7.0, 6 mm h2o2) and 25 µl of enzyme extract was measured by the decrease in absorbance for 2 min at 240 nm, spectrophotometrically. lipid peroxidation (lpo) was evaluated by determining the concentration of malondialdehyde (mda) (cakmak and horst 1991). control and sa treated roots (200 mg) were homogenized with 1 ml tca (0.1%) and centrifuged at 12000 g for 20 min at +4 °c. the reaction mixture containing 1 ml of substrate buffer (0.6% thiobarbituric acid in 20% tca) and 250 µl of enzyme extract was incubated for 30 min at 95 °c. the mixture was cooled immediately on ice and centrifuged at 12000 g for 10 min. the supernatant was measured at 532 and 600 nm. total chlorophyll, chlorophyll a, b and carotenoid contents were determined according to arnon (1949). control and sa treated leaves (0.5 g) were homogenized with 15 ml acetone (80%) and centrifuged at 3000 g for 10 min at +4 °c. the supernatant was measured at 470, 645 and 663 nm, spectrophotometrically. the anthocyanin content, one of the non-enzymatic antioxidants, was assayed according to rabino and mancinelli (1986). control and sa treated leaves (0.5 g) were extracted in 10 ml methanol:hcl (99:1, v/v) and centrifuged at 12000 rpm for 10 min at +4 °c. the supernatant was measured at 530 and 657 nm, spectrophotometrically. statistical analysis was performed using one way analysis of variance (anova), (spss 21.0 software). the significance of the applications was designated at the p < 0.05 level using the tukey's test. all data presented are means ± sd. results to determine the dose-dependent effects of salicylic acid (sa), rye seeds were germinated in hoagland solution with or without of sa (10, 100, 500 and 1000 µm). according to our results the seed germination percentages were 26.67% in control, 41.33% in 10 µm, 22.67% in 100 µm and 17.33% in 500 µm (fig. 1a). no germination was recorded at the highest concentration of 1000 µm. the germination percentages revealed that higher concentrations of sa reduced the seed germination; however 10 µm stimulated the germination in comparison to control. similarly, relative root elongation was decreased at higher concentrations, but it was stimulated at 10 µm sa. root elongation was 10.28% in control, 13.66% in 10 µm, 5.93% in 100 µm and 3.73% in 500 µm sa after 15 days (fig. 1b). to assess the oxidative stress after sa application hydrogen peroxide (h2o2) content and some antioxidant enzyme activities were evaluated after 15 days. based on our results, the content of h2o2, generated after univalent reduction of superoxide radicals was reduced by 25.5% in 10 µm sa. however, it was increased by 2.5% in 100 µm and 16.7% in 500 µm sa with regard to control (fig. 2a). one of the antioxidant enzymes, sod is responsible for catalyzing the reduction of superoxide anions into h2o2. after sa application sod activity increased by 8.1% in 10 µm, 49.3% in 100 µm and 61.9% in 500 µm with respect to control after 15 days (fig. 2b). according to our results, after sa application significant reduction was observed in cat activities suggesting the inhibition of h2o2 breakdown to water (fig. 3a). the reduction was 60% in 10 µm, 50% in 100 µm and 35% in 500 µm as compared to control. effects of salicylic acid on secale cereale acta bot. croat. 77 (1), 2018 47 lipid peroxidation (lpo) was monitored by the malondialdehyde (mda) level. whereas 500 µm sa application decreased the mda content by 46.2%, the lower concentrations increased the mda content by 53.9% in 10 µm and 7.7% in 100 µm referring to lipid peroxidation (fig. 3b). although 10 µm sa application increased pod activity by 56.3%, it was decreased by 31.3% in 100 µm and 37.5% in 500 µm compared to untreated control (fig. 4a). to assess the possible effects of sa on photosynthetic pigments, they were quantified after 15 days (tab. 1). the results revealed that sa application increased chlorophyll a content. based on our results the most significant increase was observed by 5.3% in 10 µm sa solution. however, chlorophyll b content was decreased dose-dependently. correlated with chlorophyll b reduction, total chlorophyll decreased in 100 and 500 µm sa applications. conversely, after 10 µm sa application, total chlorophyll increased by 3%. in addition, the content of carotenoids, which are lipid soluble antioxidants functioning in oxidative stress tolerance, increased in all sa applications. one of the non-enzymatic antioxidants, anthocyanin, in the class of flavonoids increased by 36% in 10 µm, 2.4 fold in 100 µm and 2.9 fold in 500 µm, compared to control (fig. 4b). fig. 1. seed germination (a) and root elongation (b) of rye treated with different concentrations of sa (10, 100 and 500 µm) after 15 days. the data with different letters are significantly different according to tukey's test at p < 0.05 for independent samples. results are expressed as mean ± sd. fig. 2. h2o2 content (a) and sod activity (b) of rye roots after 15 days of treatment with different concentrations of sa (10, 100 and 500 µm). the data with different letters are significantly different according to tukey's test at p < 0.05 for independent samples. results are expressed as mean ± sd. fig. 3. catalase (cat) activity (a) and lipid peroxidation (b) of control and sa-treated (10, 100 and 500 µm) rye roots after 15 days of treatment. the data with different letters are significantly different according to tukey's test at p < 0.05 for independent samples. results are expressed as mean ± sd. yanik f., aytürk ö., çetinbaş-genç a., vardar f. 48 acta bot. croat. 77 (1), 2018 discussion salicylic acid (sa), which is one of the phytohormones, has various regulatory roles in plant metabolism (raskin 1992, popova et al. 1997). it has been proposed that sa has an important role in bioproductivity, defense and resistance in plants (hayat and ahmad 2007). it has been reported that application of sa promoted seed germination, root and shoot growth in soybean, wheat, gloxinia, violet and brassica juncea plants in a dose dependent manner (gutiérrezcoronado 1998, fariduddin et al. 2003, shakirova et al. 2003, hayat et al. 2005, martín-mex et al. 2015). this situation can be explained by sa treatment stimulating plant growth by stimulating mitotic activity (shakirova et al. 2003). although most of the studies mentioned that the applied doses between 10–10 and 10–5 µm stimulated growth and development, the higher doses caused inhibitory effects (pancheva et al. 1996, pancheva and popova 1998). in the presented study 10 µm sa stimulated seed germination and root growth but the higher concentrations (100 and 500 µm) were inhibitory after 15 days, consistently with the previous results. moreover we observed no seed germination in the highest concentration of 1000 µm in rye. under appropriate conditions, ros generation and scavenging are balanced in the cells. cellular homeostasis is managed by complex signal transduction pathways (dutilleul et al. 2003). it has been reported that under biotic and abiotic stresses endogenous sa is accumulated and this accumulation indicates that in stressed plants sa plays a crucial role as a signaling molecule in the management of cellular redox homeostasis (apel and hirt 2004). oxidative stress induces the generation of ros such as superoxide anion (o2•−), hydrogen peroxide (h2o2), and hydroxyl radical (ho•) in plant cells. the ros scavengers include enzymatic and non-enzymatic antioxidants operating in the different cellular organelles (noctor and foyer 1998, hernandez et al. 2001). h2o2 is generated from reduction of o2•− by superoxide dismutase (sod) which is one of the key enzymatic antioxidants. excess h2o2 causes oxidative stress and catalase (cat) can directly catalyzes its decomposition into o2 and h2o (quan et al. 2008). as we presented in our study, although 10 µm sa application induced a slight increase in sod activity, it was increased progressively in 100 and 500 µm sa. consistent with the sod activity results, h2o2 levels were increased in 100 and 500 µm sa. although cat levels increased depending on the sa dose, all activities remained lower than that of the control suggesting the inhibition of h2o formation. it is widely known that sa restricts cat activity and stimulates an increase in h2o2 level as has been shown (chen et al. 1993, janda et al. 2003). there is also evidence for a complicated relationship between h2o2 and sa, which stimulate each other (rao et al. 1997, dat et al. 2000). peroxidase (pod) activity is a common response to various types oxidative stress factors. numerous research results indicated that sa-controlled reduction in h2o2 levels is related to elevated pod activities (wang et al. 2004). our results revealed that pod activity increased in 10 µm sa, inducing the reduction of h2o2, but decreased in higher concentrations. this situation suggests that higher concentrations may inhibit pod enzyme activity via alterations in enzyme and/or biochemical pathways. on the other hand sod may control the integrity of membrane structures of the cell cultab. 1. total chlorophyll, chlorophyll a, b and carotenoid contents (mg ml–1) in rye leaves treated with different concentrations of sa (10, 100 and 500 µm) after 15 days. the data with different letters are significantly different according to tukey's test at p < 0.05 for independent samples. values represent means ± sd. treatment chlorophyll a (mg ml–1) chlorophyll b (mg ml–1) chlorophyll a/b total chlorophyll (mg ml–1) total carotenoids (mg ml–1) control 302.32c±1.04 167.1a±1.07 1.81 469.41ab±1.58 89.53b±0.7 10 µm sa 318.36a±1.09 163.59a±1.28 1.95 481.95a±1.41 95.90ab±0.35 100 µm sa 304.69c±1.04 159.73ab±0.79 1.91 450.30b±1.61 97.42a±0.24 500 µm sa 308.62b±0.53 146.37b±0.89 2.11 454.99b±1.12 94.96ab±0.42 fig. 4. peroxidase activity (a) and total anthocyanin (b) of control and sa-treated (10, 100 and 500 µm) rye roots after 15 days of treatment. the data with different letters are significantly different according to tukey's test at p < 0.05 for independent samples. results are expressed as mean ± sd. effects of salicylic acid on secale cereale acta bot. croat. 77 (1), 2018 49 minating in the processes of lipid peroxidation (lpo) deactivation (zenkov et al. 2001). in the present study, lpo increased in 10 µm sa because of slight sod activity. however, in higher sa concentrations lpo decreased according to the increased sod activity. horváth et al. (2007) also indicated that exogenous sa application causes a rapid transient increase of ros including superoxide anions (o2•−). considering our results, inefficient sod activity, which is responsible for reduction of o2•− to h2o2, may result o2•− accumulation in 10 µm sa culminating in lpo. a widely known effect of sa is that it raises the photosynthetic pigment levels (khodary 2004). in the present study total chlorophyll content was increased in 10 µm sa, but it was decreased in higher concentrations, suggesting a decrease in photosynthetic capacity. however total carotenoids were increased, representing close rates in applied concentrations. similarly, pancheva et al. (1996) reported that chlorophyll content decreased after different concentrations (100 µm to 1 mm) of sa application in barley leaves. in addition, moharekar et al. (2003) indicated that different concentrations of sa (5, 10, 50, 100 and 200 mg kg–1) activated the synthesis of carotenoids and xanthophylls but reduced the level of total chlorophyll in wheat and moong. according to szepesi et al. (2009) 10–4 m sa did not cause any reduction in total chlorophyll, but the amount of carotenoids was slightly increased in solanum lycopersicum. higher sa concentrations may decrease the chlorophyll content due to the sa-induced excess ros accumulation and the consequent inhibition of plant growth and development (ma et al. 2013). in photosynthetic organisms, carotenoids serve as ros scavenger photoprotectants, having a role in ros scavenging and lpo suppression (gill and tuteja 2010). anthocyanins are flavonoid pigments located in vacuoles responsible for coloring fruits and flowers (grotewold 2006). they are considered to be non-enzymatic antioxidants under stress conditions (kovinich et al. 2012). the function of stress-induced anthocyanins is thought to be that of ros scavengers (hatier and gould 2009, agati et al. 2012). szepesi et al. (2008) indicated sa pre-treatments increased the accumulation of anthocyanins in both the presence and absence of salt stress. according to our results sa application induced an increase in anthocyanin content as well in carotenoids due to sa-induced ros accumulation. conclusion some progress in understanding the effects of sa has been obtained under normal and stressful conditions. although the pathway of signal regulation under biotic-abiotic stress factors in plant tolerance is still complicated, it is known that sa behaves as a signaling molecule, triggering a cascade of protective reactions. according to our results, exogenous sa application causes alterations to germination, antioxidant enzymes and photosynthetic pigment content in rye, depending on the dose. at low concentration sa increased the germination rate, the elongation of roots and enhanced the photosynthetic pigment contents. higher concentrations resulted in h2o2 accumulation due to increased sod and decreased cat activities and concomitantly they decreased the germination rate and root growth. this comprehensive study may help to improve our knowledge of the complex mechanism of sa action, principally in relation to the doses applied. in conclusion exogenous sa application at an appropriate dose may be a possible approach for the control of growth and environmental stress response considering enzymatic and non-enzymatic antioxidant activity. references agati, g., azzarello, e., pollastri, s., tattini, m., 2012: flavonoids as antioxidants in plants: location and functional significance. plant science 196, 67–76. an, c., mou, z., 2011: salicylic acid and its function in plant immunity. journal of integrative plant biology 53, 412–428. apel, k., hirt, h., 2004: reactive oxygen species: metabolism, oxidative stress, and signal transduction. annual review of plant biology 55, 373–399. arnon, d. i., 1949: copper enzymes in isolated chloroplasts, polyphenoxidase in beta vulgaris. plant physiology 24, 1–15. birecka, h., briber, k. a., catalfamo, j. l., 1973: comparative studies on tobacco pith and sweet potato root isoperoxidases in relation to injury, indoleacetic acid, and ethylene effects. plant physiology 52, 43–49. borsani, o., valpuesta, v., botella, m. a., 2001: evidence for a role of salicylic acid in the oxidative damage generated by nacl and osmotic stress in arabidopsis seedlings. plant physiology 126, 1024–1030. cakmak, i., horst, j. h., 1991: effects of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (glycine max). physiologia plantarum 83, 463–468. cakmak, i., marschner h., 1992: magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. plant physiology 98, 1222–1227. chen, z., silva, h., klessig, d. f., 1993: active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. science 262, 1883–1886. cho, y. w., park, e. h., lim, c. j., 2000: glutathione s-transferase activities of s-type and l-type thiol transferase from arabidopsis thaliana. journal of biochemistry and molecular biology 33, 179–183. dat, j. f., lópez-delgado, h., foyer, c. h., scott, i. m., 2000: effects of salicylic acid oxidative stress and thermotolerance in tobacco. journal of plant physiology 156, 659–665. dutilleul, c., garmier, m., noctor, g., mathieu, c., chetrit, p., foyer, c. h., de paepe, r., 2003: leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation. plant cell 15, 1212–1226. fariduddin, q., hayat, s., ahmad, a., 2003: salicylic acid influences net photosynthetic rate, carboxylation efficiency, nitrate reductase activity, and seed yield in brassica juncea. photosynthetica 41, 281–284. gill, s. s., tuteja, n., 2010: reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. plant physiology and biochemistry 48, 909–930. http://www.elsevier.com/locate/issn/09819428 http://www.elsevier.com/locate/issn/09819428 yanik f., aytürk ö., çetinbaş-genç a., vardar f. 50 acta bot. croat. 77 (1), 2018 grotewold, e., 2006: the genetics and biochemistry of floral pigments. annual review of plant biology 57, 761–780. gutie´rrez-coronado, m. a., trejo-lo´pez, c., larque´-saavedra, a., 1998: effects of salicylic acid on the growth of roots and shoots in soybean. plant physiology and biochemistry 36, 563–565. hatier, j. h. b., gould, k. s., 2009: anthocyanin function in vegetative organs. in: winefield, c., davies, k., gould, k. s. 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acta bot. croat. 79 (1), 26–34, 2020 coden: abcra 25 doi: 10.37427/botcro-2020-008 issn 0365-0588 eissn 1847-8476 somatic embryogenesis as a tool for virus elimination in croatian indigenous grapevine cultivars nenad malenica1, mateja jagić1, bruno pavletić2, nataša bauer1, darko vončina3,4, goran zdunić5, dunja leljak levanić1* 1 division of molecular biology, department of biology, faculty of science, university of zagreb, horvatovac 102a, zagreb, croatia 2 graduate programme molecular biology, faculty of science, university of zagreb, horvatovac 102a, zagreb, croatia 3 department of plant pathology, faculty of agriculture, university of zagreb, svetošimunska 25, zagreb, croatia 4 centre of excellence for biodiversity and molecular plant breeding (crop-biodiv), svetošimunska cesta 25, zagreb, croatia 5 institute for adriatic crops and karst reclamation, put duilova 11, split, croatia abstract – most croatian indigenous grapevine cultivars and vineyards are infected with a few dominant viruses. the goal of this study was to establish somatic embryogenesis as an efficient method for virus elimination from valuable croatian cultivars and creating a reliable source of healthy plants. somatic embryogenesis was induced from immature anthers and somatic embryogenesis-derived plantlets for seven indigenous cultivars were successfully regenerated. this procedure led to the elimination of viruses gflv, glrav-1, glrav-3 and gfkv that were initially detected in the field-grown cultivars ‘plavac mali’ and ‘babica’ with an elimination success of at least 30%. the described method has the potential for production of virus-free rooted plantlets for all economically important cultivars or superior cultivar clones and for the establishment of a steady source of certified virus-free planting material. keywords: babica, croatian grapevine cultivars, plavac mali, rt-pcr, somatic embryogenesis, virus elimination, virus-free plants, vitis vinifera l. * corresponding author e-mail: dunja@zg.biol.pmf.hr introduction grapevine (vitis vinifera l.) is one of the world’s most important fruit species and in croatia more than a hundred indigenous varieties are an important part of the natural heritage. the national collection of croatian indigenous cultivars contains 130 accessions, which are the result of a long tradition of grape growing in this area (maletić et al. 2015). almost all cultivars are critically endangered since they are, as a rule, grown in very narrow areas, rarely or hardly propagated and represented by fewer than 1000 individuals (maletić et al. 2015). furthermore, most croatian indigenous cultivars and vineyards are infected with at least a few dominant viruses due to the lack of systematic selection and control, as well as long-term propagation of infected materials. virus-infected vineyards, besides having a shorter life span, show a decrease in quality and quantity of wine produced. for example, the productive life of vineyards infected by grapevine fanleaf virus (gflv) is 15–20 years shorter than would otherwise be expected (andret-link et al. 2004) and the grapes, must and wine have lower sugar concentration and increased overall acidity, causing economic losses of 80% (andret-link et al. 2004), or even 100% (raski et al. 1983). in addition, croatia as an eu member state has to adopt the use of certified virus-free planting material. therefore, establishing an efficient method for virus elimination from valuable croatian cultivars and providing a stable source of healthy plants is urgently needed. today, about 65 viruses are known to be infective for grapevine, but only a few cause significant economic damage (san pedro et al. 2017). in europe, the most damaging virus strains are listed in the classification of the european and mediterranean plant protection organization (eppo 2019). the virus elimination in grapevine acta bot. croat. 79 (1), 2020 27 most widespread are grapevine leafroll-associated virus 1, 2 and 3 (glrav-1, glrav-2 and glrav-3), arabis mosaic virus (armv), grapevine fleck virus (gfkv) and grapevine fanleaf virus (gflv) (vončina et al. 2017, xiao et al. 2018). according to the propositions of eppo, certified virus-free vines should be free of these 5 viruses: armv, gflv, glrav-1, glrav-3 and gfkv (only rootstocks). several approaches for virus sanitation have been tested on different grapevine cultivars. the most important elimination technique for viruses and other pathogens from a large number of plant species is apical meristem culture, but it has a limited efficiency in grapes. in general, plant meristem consists of small actively dividing cells located in the central part of the meristematic zone without peripheral leaf primordia. these cells are not connected by the vascular system to the rest of the plant. active plant cell division and lack of vascular tissue prevent the spread of the virus to the most apical region of the meristem. success in the isolation of this small virusfree area determines virus elimination efficiency. therefore, apical meristem isolation is very demanding, since the size of the explant must be as small as possible (< 0.5 mm for v. vinifera) in order to reduce the risk of contact with the vascular system and the virus (sim and golino 2010). moreover, the low survival rate of such small explants in tissue culture (10-30%), additionally decreases the overall success of the method (sim 2006). apical meristem isolation might be combined with exposure of plants to high temperatures (thermotherapy) or application of chemicals (chemotherapy), both slowing down the spread of the viruses. the therapies are commonly used in combination and substantially increase the efficacy of the apical meristem culture-based virus elimination (wang et al. 2018), but they are frequently connected with new sets of unwanted effects such as phytotoxicity. moreover, some viruses cannot be eliminated by any of the described meristem-based procedures (sim 2006). an alternative method used for virus elimination is somatic embryogenesis (se). in this process somatic cells give rise to somatic embryos, which develop in a way a way similar to that of their zygotic counterparts. the power of se as a propagation tool becomes especially apparent when the goal is to generate large numbers of propagules (barlass and skene 1978). successful se, subsequent maintenance and plantlet regeneration have been established for many species including v. vinifera (gray and meredith 1992). it was first demonstrated in ‘cabernet sauvignon’ with the use of nucellar tissue (mullins and srinivasan 1976) and later on confirmed on a variety of grape explants such as leaf segments (robacker 1993), zygotic embryos (emershad and ramming 1994), shoot tips (barlass and skene 1978), tendrils (salunkhe et al. 1999), anthers (stamp and meredith 1988, salunkhe et al. 1999), ovaries (gray and mortensen 1987) and seeds (san pedro et al. 2017). the potential of se in virus elimination is based on the fact that somatic embryos develop from just one somatic cell or a few-celled cluster, where embryos develop separately from the mother plant’s vascular system (newton and goussard 1990). as in the apical meristem, fast cell divisions during the onset of embryogenesis and postponed development of vascular system inhibit virus replication, propagation and spread through the newly developed embryo. somatic embryogenesis could be effective in elimination of almost all viruses (gambino et al. 2009, 2011) including the recalcitrant “rugose wood” complex (gribaudo et al. 2006), which gives se an important advantage when compared to other methods. on the other hand, se has some limitations during the induction step as well as the embryo-to-plant conversion step (goebel-tourand et al. 1993, lópez-pérez et al. 2006). the overall success varies from 0% in some cultivars to up to 83% in others (xu et al. 2005). also, explant type, composition of the medium, physiological status of donor plant and culture conditions have a remarkable impact (martinelli et al. 2001, prado et al. 2010). in conclusion, the standardization of the se protocol for particular cultivar is rather difficult (dhekney et al. 2009). in this work we report the results of a study in which se was initiated and standardised for a set of seven indigenous croatian cultivars. field-grown donor plants and se-derived plantlets were inspected for the presence of 6 typical viruses gflv, armv, glrav-1 -2, -3 and gfkv. the results showed that viruses gflv, glrav-1, -3 and gfkv present in donor plants were successfully eliminated by se. this is also one of only a few reports that includes detection of viruses in croatian cultivars by rt-pcr (vončina et al. 2017), a method that has a higher sensitivity than the routinely used serological elisa (gambino and gribaudo 2006). material and methods plant material plant material was collected from the national collection of indigenous grape varieties of the republic of croatia located in the experimental field at jazbina (faculty of agriculture, university of zagreb). for explant isolation, inflorescences from vitis vinifera l. ‘babica’, ‘plavac mali’, ‘babić’, ‘pošip’, ‘malvazija dubrovačka’, ‘ljutun’ and ‘teran’ were collected in may and june, 2014 and 2015, approximately 2-3 weeks before anthesis. alternatively, explants were isolated irrespective of the season, from dormant vine cuttings (approx. 30 cm in length), induced to form inflorescence by immerging their basal side into distilled water and exposing them to a 16 h day/8 h night photoperiod (daylight florescent tube 40 w, 400-700 nm 17 wm–2) at 26 °c. identical conditions were used to obtain young leaf material for phenotype and rtpcr analysis of donor plants. sterilization and inoculation of explants inflorescences used as explant donors were incubated for 1 minute in 70% ethanol followed by 2-3 washes in sterile distilled water. they were then incubated for 10 minutes in 50% sodium hypochlorite solution (1.5% available chlorine final concentration, kemika), 0.1% mucasol® (merz consumer care gmbh) and 0.1% tween with constant agitation. inflorescences were rinsed three times with sterilized malenica n, jagić m, pavletić b, bauer n, vončina d, zdunić g, leljak levanić d 28 acta bot. croat. 79 (1), 2020 distilled water in a sterile air-flow cabinet and kept 1-3 days on a wet filter-paper in parafilm-sealed petri dishes at 4 °c prior to explant isolation and culturing. anthers were isolated from young buds (ø 1.5 mm), and used as explants (fig. 1a). at that stage, anthers were 0.8 – 1.2 mm long with yellowish and translucent locules and clear cell walls, representing the most reactive stage for induction of embryogenesis (dhekney et al. 2009). individual flower buds were aseptically removed from inflorescence and opened by cutting the basal side of the bud. filaments were cut at their bases using a thin medical needle (diameter 0.25 mm) under the stereomicroscope and together with the attached anthers placed with the adaxial side on top of the solid growth medium. explants were cultivated at 26 ºc in the dark. twenty to 25 explants were inoculated per small petri dish (ø 30 mm). somatic embryo induction and development in this study we used ms medium (murashige and skoog 1962), nn (nitsch and nitsch 1969) and piv medium (franks et al. 1998), all described as embryo inductive (newton and goussard 1990, morgana et al. 2004, xu et al. 2005, cadavid-labrada et al. 2008, dhekney et al. 2009). media were supplemented with three plant growth regulators (pgrs): 6-benzylaminopurine (ba), naphthoxyacetic acid (noa) and 2,4-dichlorophenoxyacetic acid (2,4-d) in different combinations and ratios (dhekney et al. 2009). in addition, some modifications have been applied to all of the listed media (patent pending, application number rh p20190444, will be published in september 2020). in general, 12 media formulas have been tested in order to identify the most effective combination for the majority of the croatian grapevine varieties tested. all the media were supplemented with 2% (w/v) sucrose and 7% (w/v) agar and ph was adjusted to 5.8 before sterilization. media were sterilized at 121 ºc and 103 kpa for 15 minutes. media for embryo development were of the same composition as those used for embryogenesis induction, with standard ms nitrogen and without pgrs. all media were supplemented with activated charcoal (0.25%). mass of proembryogenic structures, globular and hearth stage embryos were spread individually onto the appropriate medium and cultured in a 16 h day/8 h night photoperiod (daylight florescent tube 40 w, 400-700 nm 17 wm–2) at 26 °c. subculturing was done at 4to 6-week intervals. plant regeneration torpedo stage embryos (apical basal length 2 – 3 mm) were induced to germinate on the embryo germination (eg) medium supplemented with 10 µm indole-3-acetic acid (iaa) and 1 µm gibberellic acid (ga3) (lópez-pérez et al. 2005). five embryos were placed per petri dish and cultivated vertically in normal gravitropic root-shoot orientation in the following growth chamber conditions: 26 °c and 16/8 h photoperiod. plantlets with well-developed root and green leaves were subsequently transferred to glass test tubes (14 × ø 2.5 cm with 20 ml of eg medium) in order to grow more robust plantlets. after the plantlets developed 5-10 cm shoot (> 5 true leaves), they were planted in small pots (7 × 7 cm) filled with a 5:1 soil substrate and sand mixture (steckmedium, klassman-deilmann, germany) and transferred to magenta m-7 boxes for acclimatization. acclimatisation was performed during a 7–10 day period. immediately after transferring the plantlets to soil, the lids of the magenta boxes were tightly closed in order to develop high humidity conditions. starting on the 3rd day, the lid was slightly loosened till day 5. then the lid was fully removed and very loosely returned on top of the magenta box leaving a small space for direct air circulation between the box and the growth chamber. from day 6 to day 10 the air circulation was gradually increased by moving the lid position. on day 11 the lid was fully removed. well acclimatized plantlets showed vigorous leaf growth during the next 1-2 weeks. phenotypic analysis of plants used as source material for somatic embryogenesis colour, chlorotic mottling and leaf deformations were analysed on autumn leaves of naturally growing plants that were used as explant donor and on young leaves induced from dormant cuttings (collected in january 2018). rna isolation and rt-pcr for total rna isolation, 50 mg of leaf tissues including leaf veins were collected from both virus-infected and virusfree plants (regenerated through se) and immediately frozen in liquid nitrogen. leaves of regenerated plantlets were harvested one year after their acclimatisation to ambient conditions in the growth chamber. nitrogen frozen tissue was homogenized for 3 minutes in retsch mm 200 homogenizer at 30 hz using four metal beads (ø 3 mm). total rna was extracted from the samples using a thermo scientific magjet plant rna kit following the manufacturer’s instructions for manual plant total rna purification. lysis buffer from the kit was supplemented with dithiotreitol (1,4-dithiothreit, carl roth) at final concentration of 0.4 m and polyvinylpyrrolidone (pvp40, sigma-aldrich) at a 2% (w/v) final concentration. modification was made regarding the dnase treatment: 2 µg of isolated rna was treated with dnase i (amplification grade, invitrogen) and thereafter the enzyme was inactivated following manufacturer’s protocol. rna purity and concentrations were determined using nanodroptm 1000 spectrophotometer (thermo scientific). isolated total rna was used for virus detection by a twostep rt-pcr reaction, using six virus-specific primer pairs and a primer pair for 18s ribosomal rna internal control (tab. 1) (gambino and gribaudo 2006). first-strand cdna was synthesised using 1 µg of isolated rna, 200 units of revertaid h minus reverse transcriptase, 1 × reaction buffer (thermo scientific), 50 units of rnase inhibitor (ribolock, thermo scientific), 0.5 mm dntps (sigma-aldrich) and 1.25 µm random nonamers (sigma-aldrich). rt reaction mix (20 µl) was incubated for 50 min at 37 °c. virus elimination in grapevine acta bot. croat. 79 (1), 2020 29 tion of se in other croatian indigenous cultivars: ‘malvazija dubrovačka’ (fig. 2a), ‘teran’ (fig. 2b), ‘babica’ (fig. 2c), ‘pošip’ (fig. 2d), ‘babić’ (fig. 2e) and ‘ljutun’ (fig. 2f). the highest efficiency of somatic embryogenesis was obtained in ‘babica’ (70%) while, next to ‘plavac mali’ the lowest efficiency of 16% was obtained in ‘malvazija dubrovačka’ (tab. 2). development of somatic embryos beside the appearance of proembryogenic mass, during the cultivation on the induction medium nmm, some embryogenic structures developed further into globular and hearth stage embryos. pronounced embryo maturation was established after removal of pgrs from the nmm composition and the addition of 0.25% activated charcoal. approxifor each target virus or gene, pcr reaction mix (25 µl) was prepared using 2 µl of cdna, 0.2 µm of each virus-specific primer, 1 unit of gotaq flexi dna polymerase, 1 × gotaq flexi buffer (promega), 0.2 mm dntps (sigma-aldrich) and 2,5 mm mgcl2 (thermo fisher scientific). cycling conditions for all primer sets were as follows: initial denaturation at 95 °c for 2 min, 35 cycles at 95 °c for 30 s, 57 °c for 30 s and 72 °c for 1 minute, ending with a final elongation at 72 °c for 5 minutes. reaction products were analysed by electrophoresis on a 2.5% agarose gel (1 × tae buffer) and visualised by uv light after staining in ethidium bromide. results induction of somatic embryogenesis in this study, ‘plavac mali’ has been chosen as a priority cultivar for virus elimination because it belongs to the group of the most endangered croatian cultivars (maletić et al. 2015) and has a high importance for vine production. immature anthers in developmental stage 2 and 3 (fig.1a) were cultivated on ms (murashige and skoog 1962), nn (nitsch and nitsch 1969) and piv (franks et al. 1998) media. unfortunately, none of them could induce se in ‘plavac mali’. we further tested different modifications of ammonium and nitrate composition (leljak and jelaska 1995) and different 2,4-d, noa and ba ratios to determine the most inductive formula. eventually, the appropriate media composition, further designated as nmm medium (patent pending), enabled good explant reactivity in which 18/200 (9%) explants developed embryogenic calli within the first month in culture (5 explants) or later (13 explants) (fig. 1b-d, tab. 2). the explant response in ‘plavac mali’ was visible as unorganized growth along the filament as soon as 2 weeks after the explant inoculation (fig. 1b). four weeks later, embryogenic structures were formed (fig. 1c), while after 6-8 weeks of culturing, a sufficient amount of embryos were obtained and used for the induction of next developmental stages (fig. 1d). the same procedure was applied for the inductab. 1. primer sequences and expected rt-pcr product sizes. target primer sequences 5’– 3’ product size (bp) 18s (internal control) forward cgcatcattcaaatttctgc 844 reverse ttcagccttgcgaccatact glrav-2 forward ggtgataaccgacgcctcta 543 reverse cctagctgacgcagattgct armv forward tgacaacatggtatgaagcaca 402 reverse tatagggcctttcatcacgaat glrav-3 forward tacgttaaggacgggacacagg 336 reverse tgcggcattaatcttcattg glrav-1 forward tctttaccaaccccgagatgaa 232 reverse gtgtctggtgacgtgctaaacg gfkv forward tgaccagcctgctgtctcta 179 reverse tggacagggaggtgtaggag gflv forward atgctggatatcgtgaccctgt 118 reverse gaaggtatgcctgcttcagtgg fig. 1. time course of somatic embryo induction on immature anther explants of ‘plavac mali’ cultivated on a new medium formula designated nmm. immature anthers of stage 2 (a, left) and 3 (a, right). formation of callus on stamen filament after 2 weeks of culturing (b). appearance of embryogenic structures after 4 weeks: proembryogenic masses (arrow) and globular embryos (c, arrowhead). embryogenic callus dominantly consist of globular embryos 8 weeks after explant inoculation (d). size bar: 1 mm. malenica n, jagić m, pavletić b, bauer n, vončina d, zdunić g, leljak levanić d 30 acta bot. croat. 79 (1), 2020 mately two weeks after subculturing of the embryogenic calli onto embryo development medium, globular, heart and torpedo stage embryos were predominantly formed (fig. 2cf). some embryos sporadically developed well-formed cotyledons. after removing a single torpedo stage embryo from the underlying callus, a narrowed point of attachment reminiscent of suspensor was apparent (fig. 3a) as previously described by jayasankar et al. (2003). the embryos were not attached to each other, so their separation was simple and did not damage their structures. for transfer to germination medium, we used 1-3 mm torpedo stage embryos in the apical-basal orientation (fig. 3a) or early cotyledonary embryos (fig. 3b). plantlets regeneration after transferring torpedo or cotyledonary stage embryos onto eg medium, they were able to form embryogenic root and cotyledons on the opposite poles, acquiring a clear root-shoot axis followed by hypocotyl elongation and development of green cotyledons. development was continued until the emergence of the first leaves (fig. 3c) when they were transferred from petri dishes to glass test tubes. after 3 to 4 weeks, they reached a height of approximately 2-3 cm (fig. 3d, e). for each cultivar ten somatic embryo-derived plantlets were transferred from petri dishes to test tubes, magenta boxes and finally acclimatised to grow in growth chamber conditions (fig. 3f, g). fig. 2. embryogenic calli of different cultivars during the cultivation on induction medium (a, b) and medium for embryo development (c – f). ‘malvazija dubrovačka’ (a), ‘teran’ (b), ‘babica’ (c), ‘pošip’ (d), ‘babić’ (e) and ‘ljutun’ (f). embryogenic callus dominantly consists of globular embryos (a, b) or torpedo stage embryos (c – f). size bars: 1 mm (a, b), 5 mm (c, d) and 2 mm (e, f). tab. 2. efficiency of somatic embryogenesis induction in different croatian indigenous vitis vinifera l. cultivars expressed as a percentage of reactive explants. fast embryogenic response includes appearance of embryogenic structures within first month of cultivation while later appearance of embryogenic callus is considered as prolonged. cultivar no. of explants no. of explants with embryogenic response fast/prolonged % of explants with embryogenic response no. of regenerated plantlets plavac mali 200 5/13 9 10 babić 50 14/13 54 3 babica 50 17/18 70 10 ljutun 50 12/21 66 3 malvazija dubrovačka 50 0/8 16 3 pošip 50 18/12 6 3 teran 50 15/14 58 3 fig.3. regeneration of somatic embryos in cultivar ‘plavac mali’. torpedo (a) and cotyledonary (b) stage embryos. young plantlets with first leaf formed (c). plantlets with well developed shoot and root of approximately 6 cm (d, e). acclimatised plants grown in pots: growth chamber (f) or outside (g). size bar: 1 mm. virus elimination in grapevine acta bot. croat. 79 (1), 2020 31 sanitary status of outdoor growing plants used as explant donors sanitary statuses of vines used as explant donors for cultivars ‘babica’ and ‘plavac mali’ were analysed. clearly visible phenotypic characteristics of infection (martelli 1993, andret-link et al. 2004, meng et al. 2017) were obvious on leaves of both cultivars (fig. 4a-d). in ‘babica’ the area around the leaf veins was darker and the edges were twisted downwards (fig. 4a). in mature leaves anthocyanin accumulation was pronounced (fig. 4b). although a widely used method for virus detection is based on serological tests (enzyme-linked immunosorbent assay – elisa), here we used the more sensitive reverse transcription polymerase chain reaction – rt-pcr (gambino and gribaudo 2006) to test the presence of six dominant grapevine viruses: gflv, armv, glrav-1 -2, -3 and gfkv. the results showed that the phenotype of ‘babica’ described was provoked by the presence of glrav-3 and gflv viruses, since both were detected in donor plants (fig. 4e). the presence of a symptomless gfkv (bota et al. 2014) was also detected by rt-pcr. the mature leaves of v. vinifera ‘plavac mali’ showed the most pronounced disease symptoms. such results were confirmed by rt-pcr analysis. namely, in all v. vinifera ‘plavac mali’ plants with significant phenotypic symptoms of infection, among six viruses tested for, three (glrav-1, glrav-3 and gflv) were detected. as described above, the twisted edges and darkening of the vein area in young and older leaves of ‘plavac mali’ (fig. 4c, d) are symptoms characteristic of viruses from the leafroll complex (glravs). rtpcr analysis demonstrated that the symptoms of v. vinifera ‘plavac mali’ are likely to be caused by glrav-1 and glrav-3. similarly, the pronounced chlorosis of mature leaves (fig. 4d) was caused by gflv infection. sanitary status of somatic embryogenesis derived plantlets three out of ten se-derived plantlets of cultivars ‘babica’ and ‘plavac mali’ were randomly selected for sanitary status analysis. virus presence in se-derived plants was determined by rt-pcr with the same parameters already used for explant donor plants. the results showed that for ‘babica’ two out of three plantlets were completely free of three viruses (fig. 5a) previously detected in donor plants. the third plant was still infected with gflv (fig. 5b). similar results were obtained with the cultivar ‘plavac mali’, in which one plant was completely virus-free (fig. 5c) while two plants were still infected by gflv (fig. 5d). discussion somatic embryogenesis and plantlets regeneration there are several reports on se regeneration system in vitis showing that a series of factors such as the genotype, the developmental stage and type of explant, physiological and sanitary status of donor plants, culture media and pgr composition all affect efficiency of the method (martinelli and gribaudo 2001). immature anthers at developmental stage 2 and 3, used in our work as explants, were previously shown as the most reactive explant type regarding se success in different grapevine cultivars (cadavid-labrada et al. 2008, dhekney et al. 2009). moreover, ms (murashige and skoog 1962), nn (nitsch and nitsch 1969) and piv (franks et al. 1998) media were previously described as embryo-inductive for the majority of tested v. vinifera cultivars (newton and goussard 1990, morgana et al. 2004, xu et al. 2005, cadavid-labrada et al. 2008). none of them was embryoinductive for the croatian cultivars tested. consequently, we fig. 4. phenotypic and rt-pcr-based analysis of virus infection in ‘babica’ and ‘plavac mali’. (a) young leaf developed from winter buds of ‘babica’ with obvious fan-like morphology and downward leaf distortion (circled), dark green vein area (arrowhead) with vein yellowing (boxed) and mosaic yellow chlorosis (arrow). (b) mature leaf from field-grown ‘babica’ with anthocyanin accumulation (arrow) and darkened veins (arrowhead). (c) young leaf developed from winter buds of ‘plavac mali’ with obvious fan-shaped edges (circled), darkened vein area (arrowhead) and yellow mosaic chlorosis (arrow). (d) mature leaf from field-grown ‘plavac mali’ with prominent chlorosis (arrow) and darkened vein area (arrowhead). rt-pcr analysis of grapevine leafroll-associated viruses (glrav-1, glrav-2 and glrav-3), arabis mosaic virus (armv), grapevine fleck virus (gfkv) and grapevine fanleaf virus (gflv) in ‘babica’ (e) and ‘plavac mali’ (f). nc – negative control: 18s rdna – positive control. bends with numbers are amplified viruses. numbers are fragments size in bp. malenica n, jagić m, pavletić b, bauer n, vončina d, zdunić g, leljak levanić d 32 acta bot. croat. 79 (1), 2020 tested different modifications of listed media regarding ammonium and nitrate composition (leljak and jelaska 1995) as well as different 2,4-d, noa and ba ratios to determine the most inductive formula for the seven croatian grapevine cultivars. the composition of nitrogen sources and pgrs in newly established nmm medium enabled a rather low somatic embryo induction in ‘plavac mali’ and ‘malvazija dubrovačka’, while in other tested cultivars explant reactivity was higher than 50%. the obtained high levels of embryogenesis induction in most of the cultivars are in agreement with somatic embryogenesis efficiency higher than 80% or 27% as described in cadavid-labrada et al. (2008) and morgana et al. (2004), respectively. morgana et al. (2004) suggest stigma/style explants as a more favourable type for somatic embryogenesis while cadavid-labrada et al. (2008) and dhekney et al. (2009) emphasize anthers as the most advantageous type of explant. dhekney et al. (2009) demonstrated in their work that anthers are more suitable than pistils; embryogenesis was successfully induced in 25 out of the 26 cultivars when anthers were used compared to 22 reactive cultivars in experiments on pistils, thus rejecting hypotheses that female organs are the most responsive explants (kikkert et al. 2005). in our work with ‘plavac mali’, embryogenesis was not obtained when ovules or pistils were used (data not shown). while in previous reports induction medium is adjusted for each particular cultivar (eg. ‘sugraone’ in morgana et al. 2004; ‘carménére’ in cadavid-labrada et al. 2008), we succeeded in developing only one medium formula for seven cultivars. it should be noted that in our work we used as explants the most reactive developmental stages of immature anthers. this additionally increased the rate of the reactivity, compared to those obtained in dhekney et al. (2009). the onset of embryogenesis was characterised with tissue proliferation along anther filaments which coincide with the results of faure et al. (1996) who showed that grapevine anther cultures usually produce embryogenic tissue from diploid cells of filament connective tissues. this somatic origin of embryogenic tissue is important for enabling progeny genetically identical to the mother plant (faure et al. 1996) thus preserving cultivar identity. finally, our goal was to find a medium with potential for the induction of se in most of the tested croatian cultivars. in conclusion, our newly established formula (nmm) enables induction of embryogenesis in all tested cultivars. after removal of pgrs, pronounced maturation was induced, and globular, heart and torpedo stage embryos were predominantly formed. further development of embryos includes hypocotyl elongation and development of green cotyledons (cadavid-labrada et al. 2008). since germination and plantlet regeneration were higher than 80%, this was not a limiting step in our report, or in the reports of others (cadavid-labrada et al. 2008). interestingly, in contrast to the previously reported need for dormancy breaking treatment like stratification (jayasankar et al. 2003), in our work the addition of ga3 to germination media was sufficient for direct germination without a stratification period (pearce et al. 1987). sanitary status of field-grown plants used as explant donors phenotypic analysis was done on leaves induced from dormant vine cuttings as well as on autumn leaves of fieldgrown ‘babica’ and ‘plavac mali’ that were used as explant donors. the clearly visible phenotypic characteristics of infection were a darker area around the leaf veins and downward-twisted leaf edges (martelli 1993, andret-link et al. 2004, meng et al. 2017). both symptoms are characteristic of red varieties infected with the most widespread and damaging viruses (alliaume et al. 2018, sabella et al. 2018) of the leafroll complex (meng et al. 2017). in the advanced phase of infection, the viruses of the leafroll complex cause anthocyanin accumulation. presence of yellow mosaic chlorosis, fan-like morphology and vein yellowing (oliver and fuchs 2011) indicate presence of gflv. rtpcr analysis confirmed that described phenotypes were fig. 5. rt-pcr analysis of grapevine leafroll-associated viruses (glrav-1, glrav-2 and glrav-3), arabis mosaic virus (armv), grapevine fleck virus (gfkv) and grapevine fanleaf virus (gflv) in on ‘babica’ and ‘plavac mali’ regenerants obtained by somatic embryogenesis. healthy plants of ‘babica’ (a) and ‘plavac mali’ (c). gflv-infected regenerants of ‘babica’ (b) and ‘plavac mali’ (d). visible unlabelled fragments do not correspond to the expected virus specific sizes and are products of unspecific amplification. nc – negative control, 18s rdna – positive control. numbers represent a fragment size in bp. virus elimination in grapevine acta bot. croat. 79 (1), 2020 33 provoked by presence of glrav-3 and gflv. the presence of gfkv is not connected to the visible symptoms since it is an symptomless virus for grape (bota et al. 2014). by se regeneration, we obtained at least one completely virus-free plantlet out of three tested while the other plants remained infected only with gflv. gflv is a soil-borne nepovirus and the main agent of the most damaging and widespread viral diseases affecting grapevine (gambino et al. 2009). in sensitive cultivars this nepovirus can cause rapid death of young plants or a progressive decline over several years (martelli 1993). in contrast to phloem-limited viruses, gflv and other nepoviruses readily invade meristems, embryogenic calli (lacking vascular system) and embryo-derived plantlets. neither the meristem culture nor se are efficient enough for its eradication without additional thermotherapy (goussard and wiid 2017). in contrast, gambino et al. (2009) showed that gflv elimination is possible without thermotherapy, which according to the authors was enabled by prolonged exposure of explants and calli to auxins and cytokinins that have detrimental effects on some viruses (clarke et al. 1998). in our study, the exposure to pgrs was shortened to provide maximum cytological and genetic stability during cultivation (bennici et al. 2004, morgana et al. 2004). therefore, we can assume that complete virus elimination is possible in other cultivars, but it is necessary to carry out further testing on a wider number of regenerated plants. taken together, our protocol enabled regeneration of seven croatian cultivars through somatic embryogenesis within 10 to 12 weeks. in addition, for two tested cultivars we obtained at least one plantlet cleaned of all viruses that were initially detected in the donor plants. by using our virus elimination protocol in combination with nodal segment propagation, it is possible to obtain a high number of 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tayyip erdoğan university, faculty of agriculture and natural sciences, department of field crops, rize, turkey abstract – in this study, the chemical and morphological diversity among eleven wild populations of hypericum aviculariifolium jaub. et spach subsp. depilatum (freyn et bornm.) n. robson var. depilatum, an endemic turkish species was studied. these populations were investigated for their contents of hypericin, pseudohypericin, hyperforin, the chlorogenic, neochlorogenic, caffeic and 2,4-dihydroxybenzoic acids, hyperoside, quercitrin, isoquercitrin, avicularin, 13,118 biapigenin, (+)-catechin and (–)-epicatechin as well as for their morphological traits, including density of leaf light and dark glands, leaf area, leaf length/width ratio and plant height. the top two-thirds of the plants representing thirty individuals was harvested at full flowering from eleven sites and analyzed for the content of bioactive compounds by high-performance liquid chromatography after being dried at room temperature. morphological characterization of the wild populations was performed on twenty randomly selected individuals from each plant-growing locality. the content of the tested compounds, except for caffeic acid and avicularin, and some morphological traits, namely, the density of leaf translucent glands and black nodules and leaf area varied significantly with the investigated populations. it was observed that hypericin and pseudohypericin contents were connected positively with leaf black nodule density, but negatively with leaf area and the contents of hyperforin, quercitrin and 13,118-biapigenin were correlated positively with leaf translucent gland density. data presented here could be useful in determining future targets for further wide-ranging studies on this endemic species as well as in identifying superior germplasm in terms of high chemical content. keywords: 13,118-biapigenin, chemical and morphological diversity, hyperforin, hypericin, hypericum aviculariifolium, phenolic acids, pseudohypericin, quercitrin * corresponding author e-mail: kalinor27@gmail.com introduction hypericum genus (hypericaceae) includes over 400 species with world-wide distribution and is one of the 100 largest genera including twenty two percentage of angiosperm variety (carine and christenhusz 2010). among its members, only hypericum perforatum l. has been investigated widely so far. this species, herbal preparations of which have been utilized largely as a medicine in the treatment of mild to moderate depression, especially over the last three decades (ng et al. 2017), is considered officinal. hypericum species are well-known medicinal plants and have been used for centuries as traditional healing agents owing to their large number of pharmacological activities. all these species have traditionally been used for sedative, wound healing, disinfectant and spasmolytic preparations in turkish folk medicine with the local names of “sarıkantaron, askerotu, kılıç otu, kanlıot and kuzu kıran”. turkey is a centre of great extensity for the hypericum genus and according to güner et al. (2012) there are a total of 96 hypericum taxa in the turkish flora, of which 46 are endemic. hypericum aviculariifolium jaub. et spach subsp. depilatum (freyn et bornm.) n. robson var. depilatum [syn. hypericum origanifolium var. depilatum (freyn et bornm.) n. robson, sensu wfo 2019] is one of these endemic species (davis 1988), growing wild in arid, stony and limy areas of northern turkey. the distri mailto:kalinor27@gmail.com chemical diversity among hypericum aviculariifolium populations acta bot. croat. 79 (1), 2020 79 bution range of this endemic species is very localized by its exogenously dormant seeds (cirak et al. 2007a). its shoots are up to 30 cm in length with yellow inflorescence and typical dark glands on all aerial parts (fig. 1). results of recent studies documenting the antibacterial (gül et al. 2017) and antioxidant (maltas et al. 2013) properties of h. aviculariifolium subsp. depilatum var. depilatum indicate that this endemic species can be a substitute for widely known h. perforatum l. naphthodianthrones, principally represented by hypericin and psudohypericin, the phloroglucinol derivatives adhyperforin and hyperforin, flavonoids such as rutin, hyperoside, quercetin and quercitrin, phenolic acids and essential oils with a wide range of bioactivities are considered to be the principal constituents of hypericum plant taxa (zhao et al. 2015). in the past, hypericins were indicated as the main chemicals responsible for the antidepressant activity of hypericum extracts; however, recent studies have proved that antidepressant activity is revealed synergistically by both hypericins and hyperforin (nabavi et al. 2018). hyperforin and its derivatives were also reported to induce antitumor, antiangiogenic and neuroprotective activities (ma et al. 2018). although hyperforin and hypericin have been indicated as providing essential support to the pharmacological activities of hypericum-derived products, some other ingredients such as chlorogenic acid, quinic acid, hyperoside, rutin, quercitrin, quercetin and amentoflavone were also reported significantly to promote the antidepressant (tusevski et al. 2018), neuroprotective (silva et al. 2008), antioxidant and antimicrobial (zorzetto et al. 2015) activities. a great number of hypericum species have been subjected to studies, documenting their chemical content/composition from turkish flora as well as other growing localities of the world such as brazil, iran, jordan, serbia, italy, portugal, tunisia, peru and lithuania (cirak et al. 2016, and references therein). results from the former works revealed significant differences attributed to concentrations of the ingredients among the various hypericum species from several taxa (cirak et al. 2016); diversified populations of the same species from various geographic regions (nogueira et al. 2008), various phenological stages of the same species (abreu et al. 2004) and between various shoots as well, regenerated from the same in vitro culture (cellarova et al. 1994). however, the precise pattern of bioactive compound accumulations inside and among members of hypericum genus is not fully understood. it is not explained to what extent the chemical content and composition bear upon specific genotypes within species. it is also not clarified how far plant geographic origin affects the spectrum of phytochemicals. in our previous works, we reported h. aviculariifolium subsp. depilatum var. depilatum to include hypericins, hyperforins, various flavonoids and phenolics as hyperoside, quercetine, chlorogenic acid, rutin, isoquercetine and quercitrine (cirak et al. 2007b, 2013). however, population variability of the chemical compounds as well as of morphologic traits has not yet been studied with respect to the endemic species. hence, in the present work, our intention has been to specify for the first time the regional variability in the content of hypericin, pseudohypericin, hyperforin, the chlorogenic, neochlorogenic, caffeic and 2,4-dihydroxybenzoic acids, hyperoside, quercitrin, isoquercitrin, avicularin, (+)-catechin, (–)-epicatechin and 13,118-biapigenin as well as five morphological traits including light and dark gland density on leaves, leaf area, leaf length/width ratio and plant height as well as the correlations between the chemical and morphological data among h. aviculariifolium subsp. depilatum var. depilatum populations from eleven localities in the middle black sea geographic region of northern turkey. in addition, neochlorogenic, caffeic and 2,4-dihydroxybenzoic acids, 13,118-biapigenin, isoquercitrin, avicularin, (+)-catechin and (–)-epicatechin were not detected previously in this endemic species. hereby, we also report the first occurrence of the corresponding compounds in h. aviculariifolium subsp. depilatum var. depilatum. fig. 1. hypericum aviculariifolium subsp. depilatum var. depilatum plant flowering in its native habitat (a), and its aerial parts with typical dark glands, namely leaves and stems (b), floral buds (c) and flowers (d, e). dark and translucent glands on leaf under dissecting microscope (f ). scale bars = 5 cm (a) and 1 cm (b–f ). cirak c, özcan a, yurteri e, kurt d, seyis f 80 acta bot. croat. 79 (1), 2020 materials and methods plant materials the plant materials were described in our previous studies (see cirak et al. 2013, cirak and bertoli 2013). the species were identified by dr. samim kayikci, mustafa kemal university, faculty of arts and sciences, department of biology, turkey. voucher specimens were deposited in the herbarium of ondokuz mayis university vocational high school of bafra and the numbers of the voucher specimens are given in tab. 1. experimental procedures the aerial parts of h. aviculariifolium subsp. depilatum var. depilatum plants exemplify 30 shoots were harvested at flowering stage from eleven localities in middle black sea geographic region of northern turkey (tab. 1). the top two thirds of the plants was reaped between 14:00 pm and 15:00 pm. conditions on the day of collection were clear and sunny at all sites and temperatures varied between 28 and 30 °c. the plant materials were dried at room temperature (20 ± 2 °c), and subsequently analyzed for chemical in gredients by hplc. morphological characterization of plants was made, as described previously in our previous study (cirak et al. 2007b), on 20 randomly selected plants from each growing locality according to plant height, leaf dark and translucent gland density, leaf area, and leaf length/width ratio. plant height was measured from the flowering crown of the primary stem to the base of the plant. leaf area, leaf length/ width ratio and the number of dark and light spheroid nodules, were measured on 10 leaves of each selected plant from 11 different sites. the number of leaf dark and translucent glands was counted using a dissecting microscope (fig. 1). for leaf area and leaf length/width ratio calculations, leaves were placed on aphotocopier, held flat and secure and copied onto an a3 sheet (at 1:1 ratio). placom digital planimeter (sokkisha planimeter inc., model kp-90) was utilized to measure the actual leaf area of the copy. leaf width (cm) was measured from tip-to-tip at the widest part of the lamina and leaf length (cm) was measured from lamina tip to the point of petiole intersection along the midrib. preparation of plant extracts air-dried plant material was mechanically ground using a laboratory mill to obtain a homogeneous drug powder. samples of about 0.1 g (weighed with 0.0001 g precision) were extracted in 10 ml of 100% methanol by ultrasonication at 40 °c for 60 min in an ultrasonic bath. the prepared extracts were filtered through a membrane filter with a pore size of 0.22 µm (carl roth gmbh, karlsruhe, germany) and kept in a refrigerator at 4 °c until analysis. the extracts for naphthodianthrones analyses were exposed to light under xenon lamp (765 w/m2) for 8 min for the photoconversion of protohypericins into hypericins. hplc analyses and quantification separation of the flavanoids and phenolic acids tested was carried out by using an rp-18 (5 µm, 250  4.0 mm) column in a shimadzu lc-2030c-3d hplc device equipped with a dad detector. the binary gradient elution method was used for detection of corresponding compounds. the mobile phase a consisted of water acidified with 0.3% phosphoric acid as eluent a and acetonitrile containing 0.3% phosphoric acid as eluent b. the elution profile was used as following: 0-10 min 10% b, 10-30 min 25% b, 30-38 min 60% b, 38-45 60% b and 45-45.01 min 1% b. flow rate was 0.6 ml min–1 at 25 °c column temperature. the extract injection volume was 10 µl. the calibration of components was obtained at 203 – 280 – 320 – 360 nm wavelengths using 5, 10, 20, 50, 100 and 200 ppm standard solutions. for hypericin, pseudohypericin and hyperforin, the same device, shimadzu lc-2030c-3d hplc equipped with a dad detector, was used. separation of these chemicals was carried out using an rp-18 (5 µm, 250  4.0 mm) column. the mobile phase of isocratic solution consisted of ethyl acetate, aqueous 0.1 m sodium dihydrogen phosphate solution was adjusted to ph 2.0 by using phosphoric acid and methanol (39:41:160 v/v). the flow rate was 1 ml min–1 at 40 °c column temperature. the volume of extract injected was 20 µl. the calibration of components was obtained at wavelengths 207 and 589 nm using 1, 5, 10, 20, 50 and 100 ppm standard solutions. analytical standards used for hplc analysis and validation values of the method are shown in on-line suppl. tab. 1. the standards are also described in on-line suppl. tab. 2. tab. 1. geographical data and annual climatic conditions of hypericum aviculariifolium subsp. depilatum var. depilatum-growing localities from northern turkey. bmyo stands for “bafra meslek yüksekokulu”, vocational high school of bafra, turkey; popul. population; latit latitude; long longitude elev elevation, t mean annual temperature; p mean annual precipitation popul. collection date voucher no. latit (n) long (e) elev (m) t (°c) p (mm) habitat 1 june 03, 2018 bmyo # 27/1 40° 54΄ 35° 25΄ 1053 08.78 765 rocky and open slopes 2 june 03, 2018 bmyo # 27/2 40° 54΄ 35° 38΄ 1075 08.52 782 rocky and open slopes 3 june 03, 2018 bmyo # 27/3 40° 55΄ 35° 25΄ 1293 08.07 821 rocky and open slopes 4 june 03, 2018 bmyo # 27/4 40° 55΄ 35° 24΄ 1452 07.52 875 rocky and open slopes 5 june 03, 2018 bmyo # 27/5 40° 50΄ 35° 09΄ 952 09.29 922 igneous slopes and rock ledges 6 june 04, 2018 bmyo # 27/6 40° 50΄ 35° 10΄ 882 11.53 937 pinus woodland 7 june 04, 2018 bmyo # 27/7 40° 49΄ 35° 09΄ 989 10.64 932 arid pasturelands 8 june 04, 2018 bmyo # 27/8 40° 45΄ 35° 08΄ 1243 09.11 856 stony riverside 9 june 04, 2018 bmyo # 27/9 40° 45΄ 35° 07΄ 1373 08.77 872 stony riverside 10 june 04, 2018 bmyo # 27/10 40° 45΄ 35° 08΄ 1262 08.92 727 stony riverside 11 june 04, 2018 bmyo # 27/11 41° 25΄ 36° 58΄ 441 12.64 982 igneous slopes and rock ledges chemical diversity among hypericum aviculariifolium populations acta bot. croat. 79 (1), 2020 81 hydroxybenzoic acid and quercitrin were accumulated at significantly higher levels by plants of population-8 (0.58 and 7.10 mg g–1 dm, respectively). the highest accumulation level of hyperoside and isoquercitrin was reached in plants of population 3 and population 2 (0.73 and 0.63 mg g–1 dm, respectively) (tab. 2). the present results also indicate that h. aviculariifolium subsp. depilatum var. depilatum accumulates lower concentrations of hyperforin, hypericin, psedohypericin, neochlorogenic acid, hyperoside, isoquercitrin, (+)-catechin and (-)-epicatechin, comparable concentrations of avicularin and 13,118-biapigenin and higher concentrations of chlorogenic acid, caffeic acid, 2,4-dihydroxybenzoic acid and quercitrin when compared to h. perforatum, a well known commercial source of the compounds examined (tab. 3). significant variations (p < 0.01) were also observed in mean values of leaf dark and translucent gland density and leaf area among the investigated populations; however, leaf length/width ratio and plant height did not vary with plant growing localities (tab. 4). results of correlation analysis indicated an evident connection between leaf dark gland density/leaf area and hypericin/pseudohypericin contents of plants and leaf translucent gland density and hyperforin, quercitrin and 13,118-biapigenin contents of plants. no significant correlation was determined among the rest of the morphological traits and secondary metabolites tested (on-line suppl. tab. 3). the number of translucent glands and black nodules on leaf and leaf area varied considerably with the investigated populations. leaf dark gland density was significantly higher in plants of the populations 10, 9 and 8 whose hypericin and pseudoypericin contents were also found to be significantly higher. in a similar way, population 11, which accumulated the highest hyperforin, quercitrin and 13,118-biapigenin contents, was found to be superior to the others with respect to leaf translucent gland density. positive and significant relationships were determined between leaf dark gland density and hypericin (r2 = 0.86, p < 0.01) / pseudohypercin data analysis data of secondary metabolites contents and morphological characters of plant material were subjected to one-way analysis of variance (anova) and significant differences among mean values were tested with the duncan multiple range test (p < 0.01). correlation analysis was performed to clarify the relationship between the chemical and morphological data, and principal component analysis (pca) was carried out to elucidate the relationship of investigated populations regarding the chemical and morphological diversity they exhibited by using the statistical software package xlstat2010 trial version. pca analysis is the twodimensional visualization of the position of investigated accessions relative to each other. the principal components represent the axes which are the orthogonal projections for the values representing the highest possible variances in the case of pc1 and pc2. the obtained data were used to create scatter plot diagrams (backhaus et al. 1989). therefore, a factor analysis was performed, whereby each variable was used to calculate relationships between variable and investigated factors. based on the obtained data the cluster dendrogram was created. results results of the present study indicate that the contents of hypericin, pseudohypericin, hyperforin, the chlorogenic, neochlorogenic and 2,4-dihydroxybenzoic acids, hyperoside, quercitrin, isoquercitrin, (+)-catechin, (-)-epicatechin and 13,118-biapigenin in plants differed greatly by populations (p < 0.01) whereas caffeic acid and avicularin were accumulated at similar levels in all growing localities. plants from population-11 supplied the highest accumulation level of hyperforin, chlorogenic acid, neochlorogenic acid, 13,118-biapigenin, (+)-catechin and (–)-epicatechin (1.63, 21.71, 0.85, 2.09, 2.42 and 1.82 mg g–1 dm, respectively) whereas hypericin and pseudohypericin were yielded in the highest level by plants of population-10 (0.58 and 4.89 mg g–1 dm, respectively). 2,4-ditab. 2. mean contents (mg g–1 dm) of different compounds: hypericin (a), pseudohypericin (b), hyperforin (c), chlorogenic acid (d), neochlorogenic acid (e), caffeic acid (f ), 2,4-dihydroxybenzoic acid (g), 13,118-biapigenin (h), hyperoside (i), isoquercitrin (j), quercitrin (k), avicularin (l), (+)-catechin (m), (–)-epicatechin (n) in hypericum aviculariifolium subsp. depilatum var. depilatum populations (popul.) located in northern turkey. values are means of three replications and those, followed by different small letters in each column are significantly different (p < 0.01) according to duncan’s multiple range test. se = standard errors popul. compounds a b c d e f g h i j k l m n 1 0.14 d 1.17 d 0.25 c 12.13 b 0.47 c 0.25 0.26 b 1.30 b 0.11 c 0.51 a 2.53 e 0.65 1.38 b 1.05 b 2 0.28 c 1.93 c 0.21 c 9.91 c 0.69 b 0.55 0.42 a 1.46 b 0.24 b 0.63 a 2.97 e 0.65 1.26 b 0.79 c 3 0.39 b 2.10 c 0.07 de 7.64 c 0.45 c 0.26 0.09 c 1.29 b 0.73 a 0.38 b 3.65 d 0.65 1.00 bc 0.69 c 4 0.31 c 3.53 b 0.11 d 2.11 e 0.19 e 0.32 0.14 c 1.09 b 0.01 c 0.15 d 2.16 e 0.64 0.47 c 0.21 d 5 0.27 c 1.67 d 0.15 d 3.45 de 0.27 d 0.23 0.11 c 1.58 b 0.01 c 0.16 d 2.34 e 0.64 0.62 c 0.11 d 6 0.30 c 2.16 c 0.03 e 4.47 d 0.23 de 0.25 0.09 c 1.21 b 0.36 b 0.29 c 5.93 c 0.65 0.68 c 0.19 d 7 0.44 b 2.64 c 0.01 e 4.56 d 0.31 d 0.27 0.09 c 0.98 c 0.01 c 0.24 c 5.00 c 0.64 0.69 c 0.12 d 8 0.53 a 4.16 a 0.27 c 11.81 b 0.38 c 0.24 0.58 a 1.59 b 0.38 b 0.42 b 5.42 c 0.66 1.65 b 1.94 a 9 0.55 a 4.85 a 0.34 c 3.31 d 0.18 e 0.26 0.12 c 2.01 a 0.13 c 0.22 c 3.85 d 0.66 0.68 c 0.21 d 10 0.58 a 4.89 a 0.65 b 8.86 c 0.42 c 0.28 0.29 b 1.97 a 0.44 b 0.41 b 6.57 b 0.66 1.10 b 1.11 b 11 0.31 c 2.16 c 1.63 a 21.71 a 0.85 a 0.27 0.22 b 2.09 a 0.29 b 0.45 b 7.10 a 0.66 2.42 a 1.82 a se 0.042 0.393 0.140 1.720 0.063 0.027 0.048 0.179 0.068 0.046 0.534 0.002 0.173 0.202 cirak c, özcan a, yurteri e, kurt d, seyis f 82 acta bot. croat. 79 (1), 2020 (r2 = 0.92, p < 0.01) contents and leaf translucent gland density and hyperforin (r2 = 0.75, p < 0.05), quercitrin (r2 = 0.71, p < 0.05) and 13,118-biapigenin (r2 = 0.77, p < 0.05) contents. as for the leaf area, the highest and lowest values were detected in population-1 and population-10 (11.38 and 4.82 cm2, respectively) yielding the highest and lowest levels of hypericin and pseudohypericin accumulations. likewise, the populations producing higher amounts of hyperforin, quercitrin and 13,118-biapigenin had lower values of leaf area. leaf area was found to be negatively correlated with the hypericin (r2 = 0.81, p < 0.01) and pseudohypericin (r2 = 0.86, p < 0.01) contents of plant material. tab. 3. comparison of the chemical content (mg g–1 dm) in hypericum aviculariifolium subsp. depilatum var. depilatum (in the present study) and hypericum perforatum, globally known commercial species of hypericum genus (compiled from various relevant sources). compound h. aviculariifolium subsp. depilatum var. depilatum* h. perforatum references hyperforin 0.21–1.63 8.35–11.50 greeson et al. 2001, maggi et al. 2004, couceiro et al. 2006 hypericin 0.14–0.58 0.01–2.77 sirvent et al. 2002, southwell and bourke 2001, bagdonaite et al. 2010 psedohypericin 1.17–4.89 0.05–6.75 ayan and cirak 2008, bagdonaite et al. 2010, büter and büter 2002, bagdonaite et al. 2012 chlorogenic acid 2.11–21.71 1.11–2.19 maggi et al. 2004, cirak et al. 2007b, c neochlorogenic acid 0.42–0.85 3.34–4.25 jürgenliemk and nahrstedt 2002 caffeic acid 0.23–0.55 <0.01 patocka 2003, nahrstedt and butterweck 1997 2,4–dihydroxybenzoic acid 0.09–0.58 trace jürgenliemk and nahrstedt 2002 hyperoside 0.01–0.73 2.07–7.69 maggi et al. 2004, bagdonaite et al. 2012 quercitrin 2.16–7.10 0.05–4.77 martonfi and repcak 1994, radusiene et al. 2004 isoquercitrin 0.22–0.63 3.19–6.99 jürgenliemk and nahrstedt 2002 avicularin 0.64–0.66 0.32–0.96 wu et al. 2002, wei et al. 2009 13,118–biapigenin 1.21–2.09 1.78–2.65 jürgenliemk and nahrstedt 2002, nahrstedt and butterweck 1997 (+)–catechin 0.62–2.42 1.41–8.70 ploss et al. 2001, kalogeropoulos et al. 2010 (–)–epicatechin 0.11–1.94 20.6–118.9 ploss et al. 2001, kalogeropoulos et al. 2010 *the lowest and highest contents of the corresponding compounds, observed in the present study. fig. 2. principal component analysis biplot showing populations (1–11) and vectors of the chemicals and morphological traits based on 20 samples for each population. a two-dimensional (2d) visualization of the relative position of the phytochemicals tested was created by using the values of the principal components (the bioactive compounds examined here) relative to the investigated populations. this was provided by utilizing the principal component analysis (pca). a biplot was created to see the correlations between samples and the investigated traits. results of biplot analysis revealed that the investigated populations could clearly be differentiated according to their chemical contents and morphological traits. populations 3, 4, 5, 6, 7 and 9 were different in plant height and populations 1 and 2 were different with regards to caffeic acid content and leaf chemical diversity among hypericum aviculariifolium populations acta bot. croat. 79 (1), 2020 83 area. with the first two principal components, 68.19% of the present variation could be explained (fig. 2). further, the investigated populations were differentiated into two main groups namely, group a including populations 1, 2, 3, 4, 5, 6, 7, 9 and group b consisting of populations 8, 10, 11, with respect to their chemical contents and morphological traits on the dendogram, created by biplot analysis. as shown in fig. 3, populations 1 and 2 and populations 4 and 7 from group a were found to be similar chemically and morphologically. heterogeneity of hypericum plants from different origins is reported to influence the pharmacological activity of plant extracts significantly and to pose a great risk to the standardization of final hypericum-derived products (costa et al. 2016). hence, there have been many investigations regarding population variability of bioactive compounds from hypericum species. in h. perforatum l., the most common and commercially recognized species of the genus, wild populations of turkey (cirak et al. 2007c), canada, australia, armenia and lithuania (bagdonaite et al. 2010, and references therein) are shown to yield significantly different amounts of hyperforin, pseudohypericin and hyperforin. essential oil composition is reported to differ significantly in accordance with the geographic origin of wild accessions of h. pulchrum l., h. humifusum l., h. perfoliatum l. and h. linarifolium vahl. (nogueira et al. 2008). considerable differences were determined in concentrations of hypericins, hyperforin and various phenolics such as rutin, hyperoside, amentoflavone and quercetin in the four wild accessions of h. triquetrifolium turra from turkey. in a similar way, eleven populations of h. orientale l. and five wild populations of h. montbretii spach and h. lydium boiss. are reported to yield different quantities of hypericins, hyperforins, phenolic acids and several flavonoids such as hyperoside, quercetin, amentoflavone, rutin, avicularin isoquercitrin and quercitrin (cirak et al. 2015, and references therein). results of previous studies indicated the geographic origin of plants as a distinct factor influencing the observed chemical variation among wild hypericum populations. in a similar way, we observed significant differences in accumulation levels of 14 bioactive compounds among h. aviculariifolium subsp. depilatum var. depilatum from eleven geographic origins in the present work. two populations of this endemic species are also reported to yield quantitatively and qualitatively different amounts of essential oil (cirak and bertoli 2013). the investigated populations varied with the main environmental factors creating different growing conditions as they were located in different places of northern turkey as shown in table 1. the wide variation observed in accumulation levels of the bioactive compounds tested among the populations could somewhat be attributed to adaptive fig. 3. dendrogram showing the differentiated groups of hypericum aviculariifolium subsp. depilatum var. depilatum populations (group a represents populations 1, 2, 3, 4, 5, 6, 7 and 9; group b represents populations 8, 10 and 11) regarding their chemical contents and morphological traits. tab. 4. mean values of the morphological characters evaluated in hypericum aviculariifolium subsp. depilatum var. depilatum populations located in northern turkey. values are means of three replications and those, followed by different small letters in each column are significantly different (p < 0.01) according to duncan’s multiple range test. se = standard errors. population dark glands (per mm2) light glands (per mm2) leaf area (cm2) leaf length/width plant height (cm) 1 0.25 e 5.81 c 11.38 a 2.20 35 2 0.30 e 5.01 c 10.39 b 2.71 40 3 0.32 e 4.20 d 8.33 c 2.27 37 4 0.48 c 4.83 d 6.35 e 2.29 38 5 0.30 d 5.07 c 10.62 b 2.11 36 6 0.48 c 4.34 d 7.99 c 2.36 42 7 0.47 c 3.42 e 6.88 e 2.33 41 8 0.64 b 6.42 b 7.59 d 2.04 35 9 0.66 b 6.51 b 5.67 f 2.36 42 10 0.84 a 6.84 b 4.82 g 2.04 39 11 0.49 c 7.30 a 7.69 d 2.30 34 se 0.055 0.371 0.631 0.056 0.878 discussion among the factors contributing to the variations in the phytochemical accumulation in the hypericum genus, the geographic origin of plants is of considerable importance as the main environmental factors of a plant-growing habitat such as altitude, temperature, soil etc. influencing synthesis and accumulation of a given bioactive compound were diverse mainly according to the growing sites. the chemical cirak c, özcan a, yurteri e, kurt d, seyis f 84 acta bot. croat. 79 (1), 2020 strategies of wild plants to changing environmental factors. it is also possible to evaluate the observed chemodiversity among populations as a result of genetic distinctness, but data on the connection between the genetic and phytochemical structures in hypericum spp. is scant and results are typically contradictory. for example, he and wang (2013) reported only a partial correlation between chlorogenic acid, quercetin, rutin and hyperoside concentrations and genetic data of 12 wild h. perforatum populations from china. however, tonk et al. (2011) detect significant connections between hypericin content and random-amplified polymorphic dna (rapd) data for 19 field-grown h. perforatum clones indicating the necessity for further chemical and molecular researches on the genus hypericum to differentiate exactly the genetic and environmental effects on the monitored chemical variation among wild populations. the comparison of eleven wild populations of h. aviculariifolium subsp. depilatum var. depilatum revealed an intraspecific diversity in the distribution of light and dark glands corresponding with the accumulation of hypericins, hyperforin, quercitrin and 13,118-biapigenin in the present study. hypericum plants are categorized generally by three types of secretory structures namely, translucent glands, black or dark nodules and secretory canals (kimáková et al. 2018). among hypericum chemicals, hypericins are reported to accumulate most extensively in the black nodules of aerial parts (kornfeld et al. 2007) and previous results have proved the localization of hypericin and pseudohypericin in the dark glands of plant aerial parts in all species producing hypericins (kusari et al. 2015, kuchariková et al. 2016a, b). besides, the absence of dark glands in aerial parts is described as an accurate indication of the absence of hypericins in several species of hypericum such as h. brasiliense choisy, h. caprifoliatum cham. et schltdl., h. carinatum griseb. (ferraz et al. 2002), h. androsaemum l., h. kouytchense levl., h. monogynum l., h. stellatum n. robson, and h. canariense l. (kuchariková et al. 2016a). in previous researches, a close relationship is observed between dark gland number of leaf and total hypericin content of plants in h. perforatum (southwell and campbell, 1991) and h. lydium (cirak 2006). in accordance with the previous results, we observed a positive and significant relationship with high r2 values between leaf dark gland density and the plant content of hypericin and pseudohypercin in the present work. we also detected that plant content of hypericins is negatively correlated with leaf area, as reported by cirak et al. (2007c) for h. perforatum. it may be speculated that in the enlargement of leaf area concludes in a decrease of dark gland density and that the inverse connection between leaf area and the content of hypericins might be attributed to this decrease. as for hyperforin, soelberg et al. (2007) report that concentrations of the bioactive compound in translucent glands of leaves surpassed that of original leaves by more than 100% in h. perforatum. based on these results, the authors indicate translucent glands as the main site of hyperforin accumulation as confirmed latterly by kusari et al. (2015). hyperforin, besides, is reported to accumulate primarily in translucent glands of leaves in h. stellatum, h. annulatum moris, h. androsaemum, h. kouytchense, h. monogynum, h. kalmianum l., h. balearicum l. and h. canariense (kuchariková et al. 2016b). however, no attempt has been undertaken so far to investigate the correlation between number of translucent glands and content of hyperforin. we report here for the first time the significant and positive relationship between leaf translucent gland number and hyperforin accumulation levels which can be useful to explain sites of hyperforin synthesis and function of the bioactive compound within the genus hypericum. as opposed to hypericins and hyperforin, it may not be feasible to ascertain a pattern of localization for flavonoids as data on the localization of them on the aerial parts are discrepant and vary with species. hyperoside, isoquercitrin, quercetin and quercitrin were accumulated mainly in leaf dark glands of h. olympicum l., h. perforatum, and h. rumeliacum boiss. and rutin was accumulated only in leaf black nodules of h. maculatum crantz and h. erectum thunb. however quercetin, the most prevalent flavonoid is reported to localize mainly in leaf translucent glands of h. rumeliacum (kusari et al. 2015, kuchariková et al. 2016a). hyperoside and isoquercitrin, interestingly, are also reported to accumulate mainly in leaf translucent glands in h. kalmianum (kuchariková et al. 2016a). by contrast, hyperoside, isoquercitrin and quercitrin are accumulated primarily in both dark and translucent glands in h. humifusum leaves and quercetin and quercitin were accumulated principally in translucent glands and non-secretory structures in leaves of the species, namely, h. androsaemum, h. kouytchense, h. monogynum, h. stellatum (kuchariková et al. 2016a). in the present study, we detected a positive and significant connection between leaf translucent gland density and the content of quercitrin and 13,118-biapigenin indicating translucent glands as the main site for the accumulation of corresponding compounds in h. aviculariifolium subsp. depilatum var. depilatum. principal component and cluster analyses are favored means for characterization of genotypes and their grouping on similarity. pca is a beneficial statistical tool for the differentiation of plant materials, giving information on the variation in chemical content/composition of several species. a combination of the two statistical tools provides broad information of the traits making significant contributions to genetic diversity in crops. biplot is another widely utilized procedure for graphical display of accession groups with the aim of searching for the relationships among agro-morphological characters in several cultivars (malik et al. 2014). in the present study, we used the above mentioned statistical tools to evaluate the chemical and morphological data of eleven h. aviculariifolium subsp. depilatum var. depilatum populations from turkish flora. in conclusion, chemical and morphologic characterizations of wild plant populations seem to be first step to define superior germplasm and to provide improved chemical profiles. results of the present study indicate substantial correlations between hypericin, pseudohypericin, hyperforin, quercitrin and 13,118-biapigenin contents and densichemical diversity among hypericum aviculariifolium populations acta bot. croat. 79 (1), 2020 85 ty of leaf dark and translucent glands and leaf area. thus, these morphological characters could be utilized as selection criteria in identifying germplasm that is superior with regard to high contents of the corresponding compounds. data presented here could also be useful in determining the forthcoming goals for further wide-ranging studies on this endemic species as well as enriching data of current literature on hypericum chemistry. references abreu, i.n., porto, a.l.m., marsaioli, a.j. mazzafera. p., 2004: 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0365-0588 eissn 1847-8476 breaking of dormancy in the narrow endemic jasione supina sieber subsp. supina (campanulaceae) with small seeds that do not need light to germinate gürcan güleryüz1*, serap kırmızı2, hülya arslan1, elif güleryüz1 1 bursa uludağ university, faculty of science and arts, department of biology, görükle, bursa, turkey 2 bursa uludağ university, gemlik asım kocabıyık graduate vocational school, programme of horticulture, gemlik, bursa, turkey abstract – the germination properties of jasione supina sieber subsp. supina (campanulaceae), which is endemic to mount uludağ, bursa, turkey, were determined. in this study, we investigated the effects of ga3, the combination of hormone series and short-term moist chilling (1-month), and long-term moist chilling (4-month) on the germination percentage and mean germination time in relation to seed dormancy breaking. all treatment series were incubated under continuous dark (20 °c, 24 h) and light/dark (20/10 °c, 12/12 h) conditions. seeds were collected from specimens widespread on the alpine and subalpine grasslands and dwarf shrubs of mount uludağ (1800–1900 m a.s.l.). depending on the concentration, ga3 and the combination of hormone and moist chilling treatments promoted seed germination in both dark and light/dark conditions. after 4-month-moist chilling treatment, seeds germinated 27% in a light/dark and 80% in a dark regime. hormone and moist chilling treatments reduced the mean germination time. our results showed that j. supina seeds have physiological dormancy, require prolonged times of moist chilling and preferentially complete germination in darkness. keywords: alpine belt, endemic, germination, jasione supina, seed dormancy introduction alpine environments have high levels of endemic species, as a result of extreme environmental conditions including low temperatures, frost, and wind erosion (körner 2003). the diversity ofmicrohabitats has resulted in a range of germination strategies and dormancy types among alpine species. thus, it is difficult to address a common germination strategy for alpine plants (körner 2003, schwienbacher et al. 2011). studies of the germination strategies and factors regulating seed dormancy of rare or endangered species are important for conservation efforts and establishing ex situ collections (maunder et al. 2004, havens et al. 2006). dormancy is a process that helps seeds to adjust their germination timing to environmental conditions. the germination time is crucial for seedling survival in alpine environments (baskin and baskin 1998, schwienbacher et al. 2011). many alpine species evince deep physiological dormancy, though others can be non-dormant (schwienbacher et al. 2011). many researches have recently been focused on the effect of global climate change on alpine plant species as their phenology, seed germination, seedling establishment, and distribution areas are negatively affected (for details see briceño et al. 2015, giménez-benavides et al. 2018). light requirement for seed germination especially small size seeds is an important parameter for evaluating the germination properties of plants (fenner and thompson 2005). since small sized seeds probably have a more restricted burial capacity in soil than large sized seeds, they can remain on or close to the surface of the soil (milberg et al. 2000). according to milberg et al. (2000), there is a negative correlation between seed size and light requirement and this relationship is considered coevolved for adaptation. besides, bu et al. (2017) reported that the germination is more stimulated by light in smaller-seeded species than in larger-seeded ones. although the germination of many genera belong* corresponding author e-mail: gurcan@uludag.edu.tr breaking of dormancy in jasione supina subsp. supina acta bot. croat. 80 (1), 2021 13 ing to campanulaceae has attracted well-informed discussion (koutsovoulou et al. 2014), studies regarding the germination requirements on the genus jasione are less worthy of attention. a consideration of seed germination requirements for jasione shows that the seeds of j. montana, j. heelreichii, j. montana subsp. montana, and j. orbiculata are light-germinating and non-dormant (pegtel 1988, peco et al. 2006, koutsovoulou et al. 2014). it has been found that jasione cavanilesii has a low germination percentage in light and is considered to be dormant (fernández-pascual et al. 2017). jasione supina sieber subsp. supina is an endemic perennial species, caespitose with numerous procumbent or ascending stems. basal leaves are oblong, spathulate and obtuse, and ciliated at the base. cauline leaves are ovate to lanceolate and sessile. calyx lobes are linear to lanceolate. flowers are blue (fig. 1). its flowering time is between june and august (davis 1967). seed maturation and dispersion periods of species are between july and august. this taxon is confined to mount uludağ and it has rare and narrow distribution from 1600 to 2300 m. in 1997, it was classified as a rare taxon by the international union for conservation of nature (walter and gillett 1998). mount uludağ was established as a national park in 1961 and is categorized as one of the important plant areas (ipas) of turkey (güleryüz et al. 2010). it also however, hosts important winter sport centres with hotels and other facilities whose activities have damaged plant diversity (güleryüz et al. 2010, 2011). nowadays, these activities continue to increase, threatening the habitats of many plants as apart from j. supina. plant conservation requires a deeper knowledge of a plant’s life cycle, and seed germination is a critical stage. in this study, we aim to understand the germination properties of j. supina in order to provide information for possible ex situ conservation efforts. material and methods seed material seed sampling sites were selected from six different populations among grasslands, hard cushion and dwarf shrub communities to represent the subalpine and alpine belts of mount uludağ. mature seeds were collected from at least 100 plants with dry fruits of each population between 1800– 2000 m a.s.l. (40°05’54” n and 29°09’40” e) in august 2010. then, seeds were extracted from the dry fruits with the help of forceps and stored under laboratory conditions (25 °c, 50% relative humidity). all experiments were started within 1 month of collection. mean seed mass was determined to be 48.25 ± 0.01 µg seed–1 (n=1000). seed germination treatments sterile, plastic, 9-cm petri dishes were used for seed germination. surfaces were sterilised using a 5% sodium hypochlorite solution for 5 minutes, and then seeds were rinsed with tap water. seeds were placed on sterilised filter paper moistened with 4 ml of distilled water (control) or ga3 (potassium salt) solution. ga3 at concentrations of 250, 500 and 1000 mg l–1 alone or in combination with short-term moist chilling (1 month, +4 °c in a refrigerator) was tested. seeds were imbibed for 24 hours in ga3 solutions before sowing. in addition, seeds were maintained in a fridge at +4 °c in petri dishes with distilled water for 4 months for long-term moist chilling. to reduce evaporation, petri dishes were wrapped with stretch film during photoperiods and with aluminium foil for dark incubations. we evaluated two different temperature regimes for the germination experiments: 20 °c (24 h dark), 20/10 °c (12 h / 12 h light/dark, approx. 30 µmol m–2 s–1 photosynthetic photon flux density during the light phase provided by phillips tld 30w/54764 cool fluorescent tubes). the variable temperature regimes stimulated the expected diurnal changes in temperature in the natural habitat. namely, the climate in mount uludağ is included in the first family of the eastern mediterranean climatic group (akman 1990); the lower parts reflect a mediterranean climate, and at higher altitudes, a rainy, micro-thermic, ice-filled winter climate. annual mean temperature of mount uludağ is 4.6 °c, and average annual precipitation is 1483 mm, according to data of the meteorological station in zirve (1877 m). annual mean number of snowy days at the top of mount uludağ is 66.7, total number of snow covered days being 179.2, and maximum snow depth 430 cm (güleryüz 1992). four replications of 25 seeds per petri dish were used. seeds were considered to have germinated when the radicle emerged from the testa. germinated seeds were counted and removed every day for up to 25 days. seeds incubated in darkness were checked under filtered red light. germination parameters the final percentage of germinated seeds was calculated as follows: (i) g (%) = (a/b) × 100, where a represents the total number of seeds that germinated within 25 days, and b represents the total number of seeds tested (25 seeds). for all experiments, the final germination percentage (mean ± standard error) and mean germination time (mgt, mean in days ± standard error) were calculated. the latter reprefig. 1. habit of jasione supina sieber subsp. supina (campanulaceae). güleryüz g., kirmizi s., arslan h., güleryüz e. 14 acta bot. croat. 80 (1), 2021 sented the average length of time in days it took seeds to germinate, calculated by the formula: mgt= ∑dn/∑n; where d is the number of days counted from the date of sowing and n is the number of seeds germinated on day d. the final germination percentages were arcsine square-root transformed. statistical analysis final germination (arcsine-transformed) and mgt were analysed by two-way anova and differences between groups were performed by tukey hsd test using spss ver. 22 for windows. independent factors were ga3 concentration, moist chilling, and their interaction (moist chilling × ga3). non-transformed data (percentage values) are presented in table 1. all tests were analysed at a significance level of α= 0.05. results germination significantly increased in the ga3 and moist chilling treatments at both darkness and photoperiod conditions (tab. 1). our results indicated that seed germination was possible in dark conditions. moreover, germination was enhanced in dark conditions compared to light/ darkness conditions (tab. 1). the germination percentages of j. supina seeds in distilled water were 19% under darkness and 10% under light/darkness conditions. short-term moist chilling provided increasing rates of germination: 30% in dark and 17% at light/dark in distilled water-treated seeds (tab. 1). long-term moist chilling increased the germination to the level of 80% in the dark and 27% in light/ dark conditions in distilled water treatments (tab. 1, fig. 2). ga3 treatment resulted in full germination at a concentration of 1000 mg l–1 for all treatments except that of shortterm moist chilling incubated under light/dark conditions. tab. 1. final germination percentages and mean germination time (mgt) of jasione supina seeds subjected to different ga3 concentrations in dark (20 °c) and light/dark conditions (20/10 °c; 12/12 h) with or without short-term moist chilling treatment for 25 days. values are mean ± standard error (n = 4). capital letters are used to compare results (p < 0.05) between hormone series and combination of short-term moist chilling and hormone series, while lower-case letters are used for comparison among different series (short-term moist chilling x hormone series, and hormone series), for two main treatments (light/dark and continuous dark-incubated seeds, separately. the variance analysis and tukey hsd test were not applied to long-term moist chilling treatments. treatment ga3 (mg l1) germination (%) mgt (days) continuous dark (20 °c) hormone series 0 19.0 ± 6.0ce 8.2 ± 1.8ab 250 92.0 ± 6.5bb 4.7 ± 1.8bc 500 100.0 ± 0.0aa 5.8 ± 1.2bc 1000 100.0 ± 0.0aa 4.9 ± 0.3bc short-term moist chilling (1-month, +4 °c) and hormone series 0 30.0 ± 2.3dd 11.8 ± 1.6aa 250 54.0 ± 8.3cc 9.1 ± 1.7bb 500 80.0 ± 0.0bb 2.5 ± 0.2cd 1000 100.0 ± 0.0aa 2.2 ± 0.9cd light/dark (20/10 °c;12/12 h) hormone series 0 10.0 ± 5.1ce 15.1 ± 4.5aa 250 69.0 ± 6.8bb 8.4 ± 0.3bb 500 79.0 ± 15.1bb 8.1 ± 0.4bb 1000 100.0 ± 0.0ab 7.6 ± 0.5bb short-term moist chilling (1-month, +4 °c) and hormone series 0 17.0 ± 3.8cd 11.8 ± 1.6aa 250 54.0 ± 11.5bc 9.5 ± 2.6bb 500 55.0 ± 7.5bc 6.3 ± 1.7bb 1000 92.0 ± 16.0aa 3.9 ± 1.0ccd long-term moist chilling (4-month, +4 °c) continuous dark (20 °c) 0 80.0 ± 3.2 7.5 ± 0.4 light/dark (20/10 °c; 12/12 h) 0 27.0 ± 13.6 11.6 ± 1.3 fig. 2. cumulative germination percentage of jasione supina subsp. supina seeds after 4-months-chilling of seeds at 4 °c. the moist chilled seeds were then incubated under dark (20 °c) and photoperiod (20/10 °c, 12 h dark/12 h light, respectively) conditions in distilled water during 25 days. vertical lines indicate standard error (n=4). breaking of dormancy in jasione supina subsp. supina acta bot. croat. 80 (1), 2021 15 the mean germination time (mgt) was delayed from 8.2 days to 11.8 days after a short-term moist chilling of seeds in distilled water in darkness. however, the mgts of short-term moist chilling and light/darkness-incubated seeds in distilled water were reduced from 15.1 days to 11.8 days (table 1). mgt values were decreased depending on hormone concentrations. in darkness as well as light/dark conditions, the combination of hormone and short-term moist chilling treatment was more efficient in reducing mgt than treatment with hormone alone. for example, the most striking decrease of mgt was found at 1000 mg l–1 ga3 and short-term moist chilling combination treatment under darkness conditions (2.2 days). the 4-month-moist chilling also decreased mgt; it was 7.47 days for darkness and 11.6 days at light/darkness compared to distilled water controls (tab. 1). with respect to germination percentage, two-way anova showed significant differences among all treatment series (p < 0.05) except short-term moist chilling treatment under light/dark conditions (tab. 2). the effects of moist chilling on mgt were not significant under both dark and light/dark conditions (p > 0.05) (tab. 2). however, ga3 alone and ga3 × moist chilling combination interaction effects were significant under both dark and light/dark conditions (tab. 2). discussion our results indicate that the seeds of j. supina were dormant since we could not determine the germination above 50 percentage in any distilled water treatments under dark and light/dark conditions except for four-month-moist chilling treatments. interestingly, long-term moist chilling that extended to four-month clearly increased the germination in darkness. this can be related to the role of long-term moist chilling in seed germination behaviour of j. supina seeds in natural conditions under the snow cover along the mean 179.2 days in a year (güleryüz 1992). several studies have reported that cold stratification is effective at breaking dormancy (baskin et al. 2001, kırmızı et al. 2010, garcíafernández et al. 2015, schütz and rave 1999). however, other studies have found no cold stratification requirement in some campanulaceae members such as lobelia boninensis (marikoco and kachi 1995) and some lobelioid shrubs from this family (baskin et al. 2005). fernández-pascual et al. (2017) suggested that alpine plant species have two types of germination niche (warm and cold stratification) depending on habitat characteristics. among jasione species, j. cavanilesii was found to be dormant among 22 species of subalpine and alpine grasslands of the cantabrian mountains of spain and germinated after warm stratification (at 25 °c) (fernández-pascual et al. 2017). seeds of j. crispa, which is a high mountain species from the mediterranean region, germinate at an average of 59% at warm temperatures and thus are not considered to be dormant (giménez-benavides et al. 2005). in addition, j. montana seeds germinate easily over a relatively wide range of temperatures (10–25 °c), including relatively low temperatures (pegtel 1988). treatments with ga3 and the combination of ga3 and one-month-moist chilling stimulated the germination percentage up to 100% and reduced mgt (tab. 1). these results indicate that ga3 can substitute for long-term moist chilling and that j. supina seeds have physiological dormancy (pd). ga3 is known as a germination stimulator that helps to overcome seed dormancy. ga3 has been shown to induce the germination of many dormant species such as some allium (kırmızı et al. 2017), campanula glomerata subsp. hispida (gülbağ and özzambak 2017) and allium stracheyi (payal et al. 2014). light is thought to induce germination in plant species with small seeds (pérez-garcía and gonzales-benito 2006, probert 2000). given the size, the seeds of j. supina can be counted as small seed mass with a weight of 48 µg seed–1 (jaganathan et al. 2015). however, our results revealed that j. supina seeds can germinate in dark conditions (tab. 1, fig. 2). wu et al. (2013) found that there was a negative correlatab. 2. two-way anova results of main effects (short-term moist chilling treatment and ga3 treatments) for the arcsine-transformed germination percentage and mean germination time under dark and light/dark conditions for jasione supina seeds. values are two-way anova f-ratios. values in bold indicate significant (p < 0.05) differences. factor germination percentage mean germination time df f p df f p continuous dark (20 °c) short-term moist chilling 1 159.57 <0.0001 1 1.20 0.283 ga3 3 292.10 <0.0001 2 37.15 <0.0001 short-term moist chilling × ga3 2 40.59 <0.0001 2 17.29 <0.0001 error 21 18 light/dark (20/10 °c; 12/12 h) short-term moist chilling 1 3.83 0.062 1 1.52 0.231 ga3 2 129.52 <0.0001 2 10.50 <0.0001 short-term moist chilling × ga3 2 6.04 0.003 2 9.08 <0.0001 error 18 18 güleryüz g., kirmizi s., arslan h., güleryüz e. 16 acta bot. croat. 80 (1), 2021 tion between seed mass and light requirement in smallseeded alpine meadow species of the qinghai-tibetan plateau. moreover, koutsovoulou et al. (2014) studied the germination of 131 species with small mass seeds from campanulaceae, including j. helreichii, j. montana subsp. montana and j. orbiculata, finding that seeds require light for germination. they argued that plant species with small seeds can germinate only if found on the soil surface or at shallow depths. in other studies, the seeds of j. montana have also been found to require light for germination and do not have dormancy (peco et al. 2006, pegtel 1988). some researchers have reported that requirements for darkness and light in seeds can play an important role in delaying germination and contribute to seed persistence (fenner and thompson 2005, koutsovoulou et al. 2014). there have been only two studies reporting darkness germination in campanulaceae. one was concerned with wahlenbergia stricta (willis and groves 1991), whereas the other was focused on howellia aquatilis (lesica 1992). however, koutsovoulou et al. (2014) hypothesised that willis and groves (1991) examined dark incubations under short periods of light, which was sufficient for seed light requirements. mgt is a parameter that is used for monitoring the speed of germination. a shortened mgt may provide support for the establishment of seedlings at the beginning of the next vegetation period. in our study, under both dark and light/dark conditions, moist chilling and ga3 combination reduced the mgt of the seeds. in this way, species can survive by maintaining themselves in accordance with the vegetation period in an alpine environment. generally, campanulaceae members that have small seed mass are considered to have a light requirement for germination. however, j. supina with small seeds did not have a light requirement for germination (fig. 2). this characteristic seems to be consistent with its habitat characteristics. it is spread among juniperus communis dwarf shrub and festuca cyllenica, f. punctoria, and acantholimon ulicinum hard-cushion plant communities that provide shade to soil surfaces and dark environmental conditions to germinate. moreover, the breaking of dormancy with longterm moist chilling in this taxon may be evidence that its seeds are germinated in the spring after staying under snow cover during the winter. our results can be important for possible ex situ or in situ conservation efforts. acknowledgements this study was part of a research project funded by the turkish scientific and research council (no: 107t494). we also thank mehmet adanur (bursa uludağ university, english lecturer) for linguistic and editing help. references akman, y., 1990: climate and bioclimate types (bioclimate methods and climate in turkey). palme publication distribution, ankara (in turkish). baskin, c.c., baskin, j.m., 1998: seeds: ecology, biogeography, and, evolution of dormancy and germination. academic press, san diego. baskin, c.c., milberg, p., andersson, l., baskin, j.m., 2001: seed dormancy-breaking and germination requirements of drosera anglica, an insectivorous species of the northern hemisphere. acta oecologica 22, 1–8. baskin, c.c., baskin, j.m., yoshinaga, a. 2005: morphophysiological dormancy in seeds of six endemic lobelioid shrubs (campanulaceae) from the montane zone in hawaii. botany 83, 1630–1637. briceño, v.f., hoyle, g.l., nicotra, a.b., 2015: seeds at risk: how will a changing alpine climate affect regeneration from seeds in alpine areas? alpine botany 125, 59–68. bu, h., ge., w., zhou, x., qi, w., liu, k., xu, d., wang, x., du, g., 2017: the effect of light and seed mass on seed germination of common herbaceous species from the eastern qinghai-tibet plateau. plant species biololgy 32, 263–269. davis, p.h., 1967: flora of turkey and the east aegean islands. vol. 2. edinburg university press, edinburg. fenner, m, thompson, k., 2005: the ecology of seeds. cambridge university press, cambridge. fernández-pascual, e., jiménez-alfaro, b., bueno, á., 2017: comparative seed germination traits in alpine and subalpine grasslands: higher elevations are associated with warmer germination temperatures. plant biology 19, 32–40. garcía-fernández, a., escudero, a., lara-romero, c., iriondo, j.m., 2015: effects of the duration of cold stratification on early life stages of the mediterranean alpine plant silene ciliate. plant biology 17, 344–350. giménez-benavides, l., escudero, a., pérez-garcia, f., 2005: seed germination of high mountain mediterranean species: altitudinal, interpopulation and interannual variability. ecological research 20, 433–444. giménez-benavides, l., escudero, a., garcía-camacho, r., garcía-fernández, a., iriondo, j.m., lara-romero, c., morentelópez, j., 2018: how does climate change affect regeneration of mediterranean high-mountain plants? an integration and synthesis of current knowledge. plant biology 20, 50–62. gülbağ, f., özzambak m.e., 2017: effect of light, temperature, and different pre-treatments on seed germination of campanula glomerata l. subsp. hispida (witasek) hayek (campanulaceae). propagation of ornamental plants 17, 120–125 güleryüz, g., kırmızı, s., arslan, h., kondu yakut, e., 2011: alterations of the nitrogen mineralization rates in soils of forest community depending on the ski run constructions (mount uludağ, bursa, turkey). journal of mountain science 8, 53–61. güleryüz, g., malyer, h., kaynak, g., özhatay, n., 2010: uludağ a2 (a) bursa. in: byfield, a., atay, s., özhatay, n. (eds.), important plant areas in turkey: 122 key turkish botanical sites, 77–79. wwf turkey, i̇stanbul. güleryüz, g., 1992: the studies on the nutrient turnover and productivity at some plant communities of the uludağ alpine region. phd thesis. university of uludağ, bursa. havens, k., vitt, p., maunder, m., guerrant, e.o., dixon k., 2006: ex situ plant conservation and beyond. bioscience 56, 525–531. jaganathan, g.k., dalrymple, s.e., liu, b., 2015: towards an understanding of factors controlling seed bank composition and longevity in the alpine environment. the botanical review 81, 70–103. kırmızı, s., güleryüz, g., arslan, h., sakar, f.s., 2010: effects of moist chilling, gibberellic acid, and scarification on seed dormancy in the rare endemic pedicularis olympica (scrophulariaceae). turkish journal of botany 34, 225–232. breaking of dormancy in jasione supina subsp. supina acta bot. croat. 80 (1), 2021 17 kırmızı, s., güleryüz, g., arslan, h., 2017: effects of environmental and storage conditions on the germination of allium species. fresenius environmental bulletin 26, 3470–3478. körner, c., 2003: alpine plant life: functional plant ecology of high mountain ecosystems. springer-verlag, berlin, heidelberg. koutsovoulou, k., daws, m.i., thanos, c.a., 2014: campanulaceae: a family with small seeds that require light for germination. annals of botany 113, 135–143. lesica, p., 1992: autecology of the endangered plant howellia aquatilis: implications for management and reserve design. ecological applications 2, 411–421. marikoco, s., kachi, n. 1995: seed ecology of lobelia boninensis koidz. (campanulaceae), an endemic species in the bonin islands (japan). plant species biology 10, 103–110. maunder, m., havens, k., guerrant, e.o., falk, d., 2004: ex situ methods: a vital but underused set of conservation resources. in: guerrant, e.o., havens, k., maunder, m. (eds.), ex situ plant conservation: supporting species survival in the wild, 3–20. island press, washington dc. milberg, p., andersson, l., thompson, k., 2000: large-seeded species are less dependent on light for germination than small-seeded ones. seed science research 10, 99–104. payal, k., maikhuri, r.k., rao, k.s., kandari, l.s., 2014: effect of gibberellic acid-and water-based pre-soaking treatments under different temperatures and photoperiods on the seed germination of allium stracheyi baker: an endangered alpine species of central himalaya, india. plant biosystems 148, 1075–1084. peco, b., lopez-merino, l., alvir, m., 2006: survival and germination of mediterranean grassland species after simulated sheep ingestion: ecological correlates with seed traits. acta oecologica 30, 269–275. pegtel, d.m., 1988: germination in declining and common herbaceous plant populations co-occuring in an acid peaty heathland. acta botanica neerlandica 37, 215–223. pérez-garcía, f., gonzales-benito, m.e., 2006: seed germination of five helianthemum species: effect of temperature and presowing treatments. journal of arid environments 65, 688– 693. probert, r.j., 2000: the role of temperature in the regulation of seed dormancy and germination. in: fenner, m. (ed.), seeds: the ecology of regeneration in plant communities, 261–292. cab international, wallingford. schütz, w., rave, g., 1999: the effect of cold stratification and light on the seed germination of temperate sedges (carex) from various habitats and implications for regenerative strategies. plant ecology 144, 215–230. schwienbacher, e., navarro-cano, j.a., neuner, g., erschbamer, b., 2011: seed dormancy in alpine species. flora 206, 845–856. walter, k.s., gillett h.j. (eds.), 1998: 1997 iucn plants red list of threatened plants. iucn, gland, cambridge. willis, a.j., groves, r.h., 1991: temperature and light effects on the germination of seven native forbs. australian journal of botany 39, 219–228. wu, g.-l., du, g.-z., shi, z.-h., 2013: germination strategies of 20 alpine species with varying seed mass and light availability. australian journal of botany 61, 404–411. acta bot. croat. 77 (2), 2018 197 acta bot. croat. 77 (2), 197–202, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0012 issn 0365-0588 eissn 1847-8476 short communication physiological and growth responses of sour cherry (prunus cerasus l.) plants subjected to short-term salinity stress ioannis e. papadakis1,2,*, georgia veneti2, christos chatzissavvidis2,3, ioannis therios2 1 laboratory of pomology, department of crop science, agricultural university of athens, iera odos 75, 118 55 athens, greece 2 laboratory of pomology, school of agriculture, aristotle university, 54124 thessaloniki, greece 3 department of agricultural development, democritus university of thrace, 68200 orestiada, greece abstract – the gradual response of cab-6p sour cherry (prunus cerasus l.) plants to nacl-induced salinity stress (60 mm nacl) was investigated in a short-term hydroponic experiment, based on parameters relating to the growth, water relations, chlorophyll and mineral nutrition. the results showed that cab-6p plants are very sensitive to salinity stress because their growth and leaf chlorophyll concentration were both affected negatively from the 3rd and 5th day, respectively, after incurring salinity stress. since root growth was suppressed more severely than shoot growth, the shoot to root ratio was significantly increased under saline conditions. the concentrations of na in leaves and stem of nacl-treated plants were much lower than those measured in roots, suggesting na exclusion mechanism from the shoot. the opposite trend was observed for cl, indicating cl inclusion mechanism to leaves. as regards the concentrations of ca, mg, p, k, na, fe, zn and mn, they were not changed in higher salinity conditions, apart from k, concentrations of which in leaves and roots were significantly increased and decreased, respectively (k translocation to leaves). salinity further reduced k/na ratio in root and stem as well as leaf water and osmotic potentials, whereas leaves of control and nacl-treated plants presented similar turgor potential and k/na ratio. these data add very important information to our knowledge about the physiological events occurring in sour cherry plants after even short-term exposure to salinity. keywords: chlorophyll, cab-6p rootstock, cl inclusion, ion concentrations, na exclusion, osmotic adjustment, water relations * corresponding author, e-mail: papadakis@aua.gr introduction an excess of soil salinity affects the performance of hundreds of crop species worldwide and may be particularly deleterious to most fruit tree crops, which are usually saltsensitive (ziska et al. 1990). it is also well documented that various prunus species are included in the group of salt sensitive species (andreu et al. 2011). although, there are many published papers dealing with the effects of salinity on growth, antioxidants, photosynthesis, nutritional status and several other parameters of prunus species (ziska et al. 1990, papadakis et al. 2007, chatzissavvidis et al. 2008, andreu et al. 2011), there are no detailed studies regarding the changes and/or possible adaptations occurring just after exposure to saline environments. such data would add valuable information to our knowledge about the initial events that take place during the exposure of fruit trees to salinity. in fact, salinity is not a real problem in the main world’s regions where sweet and sour cherries are traditionally cultivated, since these areas have usually high levels of rainfall and, therefore, high availability of good quality irrigation water. nowadays, however, there are some new low-chill cherry varieties, and, of course, more will be produced in the future. these new varieties can be cultivated in warmer/more arid areas of the world, including lowlands and coastal ares, where, at least during the summer months, irrigation water of poor quality containing high levels of salts is commonly used. according to tattini et al. (1995), the maintenance of plant growth and papadakis i. e., veneti g., chatzissavvidis c., therios i. 198 acta bot. croat. 77 (2), 2018 productivity is very important for perennial crops that are subjected to transitional periods of saline conditions. on the other hand, rootstocks possess a critical role for the sustainable cultivation of plants, and are a tool with which farmers can alleviate salinity and/or other environmental constraints in their orchards (papadakis 2016). therefore, the current study aimed to investigate the growth and physiological responses (plant growth, chlorophyll concentration, water relations and nutrient status) of the well-known sour cherry genotype cab-6p, which is widely used as a rootstock for both sour and sweet cherry cultivars, under salt stress. materials and methods one-year-old, uniform, self-rooted plants of the clone cab-6p (prunus cerasus l.) were transplanted in black plastic bags filled with 3 l of a sand/perlite mixture (50/50, v/v). in fact, the use of small-asexually propagated plants of about 25 cm in height was aimed at determining even the initial, tiny changes caused by the salinity stress. the plants were irrigated with good quality tap water for seven weeks before nacl treatment was imposed. afterwards, the plants were irrigated, thrice a week, with 300 ml of the well-known hoagland nutrient solution no 2 (hoagland and arnon 1950) containing either 0 or 60 mm nacl. the concentration of 60 mm nacl is within the range of nacl concentrations usually observed in irrigation waters of the mediterranean basin (flowers 1999). the experiment was carried out in a glasshouse, with temperatures ranging between 18 and 30 °c, and lasted 13 days. three plants per treatment were randomly selected, every two days after salinity imposition, for measuring water (ψw), osmotic (ψπ) and turgor (ψp) potentials as well as chlorophyll concentration in fully expanded apical leaves. finally, the plants were separated into root, stem and leaves. after estimating the fresh and dry weights of all these plant portions, they were further analyzed for na, cl, k, n, p, ca, mg, fe, mn and zn. all the methods, procedures and equipments used for the aforementioned measurements and determinations have already been described in our previously published paper (papadakis et al. 2007). the experimental design was completely randomized. in all, 36 plants were used: 18 were controls (0 mm nacl) and 18 were nacl-treated (60 mm nacl). from the third day (3rd day) after salinity imposition until the last day of experimentation (13th day), 3 plants from each treatment were harvested every two days, i.e., 2 treatments (control, salinity) × 3 plants per sampling day and treatment × 6 sampling days (3rd, 5th, 7th, 9th, 11th and 13th). for each parameter and sampling day, means of control and nacl-treated plants were compared with each other using the student’s t-test (p ≤ 0.05). results and discussion salinity reduced the weights of leaves (fig. 1a), stems and roots (fig. 1b), and therefore the total weight of cab6p plants (fig. 1c), even from the 3rd day of the experiment. furthermore, nacl-treated plants presented significantly higher values of shoot to root ratio (on a dry weight basis) than the control plants, from the 7th day of the experiment and thereafter (fig. 1d). a decline in dry weight of leaves and stems was also observed in two cherry cultivars fig. 1. dry weight (d.w.) of leaves (a), root (b), and entire plant (c); shoot to root ratio (d) of control (0 mm nacl) and salinity-treated (60 mm nacl) prunus cerasus plants at different days (3, 5, 7, 9, 11 or 13) after nacl imposition. results are expressed as mean values ± se. for each parameter and day after salt stress imposition, asterisks indicate significant differences between control and nacl-treated plants, at p ≤ 0.05 (*), p ≤ 0.010 (**) or p ≤ 0.001 (***); n.s. indicates non-significant differences. salinity stress response in prunus cerasus acta bot. croat. 77 (2), 2018 199 subjected to salinity conditions (papadakis et al. 2007). the osmotic stress leads to an instant decline in cell expansion of young roots and leaves, causing stomatal closure as well. enhanced resistance to osmotic stress would be beneficial for leaf growth and stomatal conductance, but the resulting increased leaf area would be productive only if plants had adequate soil water (munns and tester 2008), such as under the current experimental conditions. the latter consideration could possibly explain the fact that the root growth of cab-6p plants was inhibited by salinity to a greater extent than shoot growth (increased values of shoot/root ratio). in other words, the root of cab-6p plants is more sensitive to salinity than the shoot, at least in the first two weeks after the imposition of nacl stress. this finding is not in accordance with the results of other researchers working with many different crops, like tomato (albacete et al. 2008). more specifically, the shoot to root ratio in salt-treated tomato plants was decreased (an important adaptive response) due to the rapid inhibition of shoot growth (which limits plant productivity) while root growth was maintained (albacete et al. 2008). the reduction of shoot to root ratio is one of the most usual symptoms of plants subjected to salinity stress resulting in improvement of water use efficiency within the plant. it was suggested that the plants subjected to high salinity save water by minimizing their shoot growth and by keeping a normal water status for growth (sohan et al. 1999). in contrast to these reports, our results are in agreement with those stated by bernstein and coworkers (2004), who found that the shoot growth of avocado plants was less sensitive to salinity than root growth. the strong inhibition of root growth is expected to affect the entire plant and thus the root growth could be considered an important criterion for rootstocks’ tolerance to nacl (bernstein et al. 2004). overall, since the root growth of cab-6p plants was dramatically affected by salinity, even from the first days of the current short-term experiment, the use of cab-6p as rootstock has to be avoided when sweet or sour cherry varieties are going to be cultivated in areas having low quality irrigation water. genotypes with high capabilities to control cl and na absorption by roots and/or cl and na movement from roots to stems and especially to leaves, present high tolerance to salinity (storey and walker 1999). ion exclusion and compartmentation at the root level regulates ion concentration in the xylem sap, preventing thus the accumulation of potentially toxic ions in the aboveground plant parts. this mechanism is effective at levels of salinity up to 50 mm nacl, but it considerably impedes plant growth. the accumulation of na in roots provides a mechanism for many crops, such as olive, to cope with salinity in the root zone and may indicate the existence of an inhibitory mechanism of na transport to leaves (chartzoulakis 2005). in the current study, the concentrations of na in the shoots (leaves and stems) of nacl-treated plants (fig. 2a-b) were much lower than those in the roots (fig. 2c). moreover, lower na concentrations were found in leaves (fig. 2a) than in stems (fig. 2b). therefore, cab-6p sour cherry plants must have had the capability to control the absorption of na and/or its transport to stems and leaves. in general, na exclusion from the shoot has often been reported as a crucial mechanism for plants to tolerate high salinity (jha et al. 2010). on the other hand, the increase of cl concentration in the nacl-treated plants was simultaneous in all plant parts (leaves, stems and roots), from the 9th day of nacl imposition and thereafter (figs. 2df). furthermore, higher cl concentrations were determined in leaves of nacl-treated plants (fig. 2d) than in the stems (fig. 2e) and roots (fig. 2f), especially after the 11th day of the experiment, which indicates that a cl inclusion mechanism (export to leaves) was implicated. therefore, it could be concluded that cab-6p sour cherry plants are very sensitive to salinity, since the mechanism controlling (restricting) cl absorption by roots collapsed quickly (from the 9th day of plants’ exposure to salinity and thereafter), although the concentration of nacl (60 mm) in the nutrient solution was relatively low. in other words, a high susceptibility of cab-6p plants to cl absorption when they were subjected to saline conditions was revealed. leaf cl concentration has been commonly used as a reliable criterion for rating plant species according to their efficiency under saline conditions (antcliff et al. 1983, storey and walker 1999). in short, the sensitivity of sour cherry plants to salinity is closely correlated with cl concentration in various plant parts (mainly in the leaves). this criterion could be applied for future screening studies of other sour cherry genotypes for their relative sensitivity to saline conditions. in a previous study where the effects of nacl in the mineral nutrition of two sweet cherry varieties were investigated, it was found that the concentrations of all nutrients, except for n and mg, in leaves, stems and roots of both cultivars were altered under the treatment of 50 mm nacl. in addition, the root was the main plant organ whose nutrient content was mostly affected by nacl, probably because it is the plant part that salts enter first (papadakis et al. 2007). based on the concentrations of various mineral elements (n, p, mg, ca, mn, zn and fe) determined in roots, stems and leaves of cab-6p plants, no one significant difference was found between control and nacl-treated plants (data not shown). this finding could be mainly ascribed to the short duration of the salinity stress imposition which was not enough to affect the overall nutritional status of sour cherry plants. the only exception was potassium (k) which was strongly affected by nacl (fig. 2g-i). liu and van staden (2001) suggested that a constant or increased level of internal k is of major importance for a better response of entire plants and/or plant tissues to salinity stress. in the current study, although control and nacl-treated cab-6p plants had similar concentrations of k in their stems (fig. 2h), salinity decreased or increased k concentrations in roots (at 11th and 13th day) (fig. 2i) and leaves (at 13th day) (fig. 2g), respectively. a possible explanation could be the existence of a mechanism that removes k from roots to aerial plant parts, especially to leaves (romero and marañón 1994). furthermore, the relatively high salt tolerance is usually correlated with relatively high values of k/na ratio in plant tissues (liu and van staden 2001). giri et al. (2007) experimenting with the mycorrhizal papadakis i. e., veneti g., chatzissavvidis c., therios i. 200 acta bot. croat. 77 (2), 2018 acacia nilotica under saline conditions found that elevated k/na ratios in root and shoot may prevent k-mediated enzymatic processess from disruption. in contrast, our data have shown that stem and root k/na ratios in nacl-treated plants were much lower than those of control plants, from the 3rd (root) or 5th (stem) day after salinity stress imposition (figs. 2k-l). regarding the leaf k/na ratio (fig. 2j), no significant difference between control and salinity stressed plants during the experiment was found. moreover, the k/na ratio in leaves of cab-6p plants (fig. 2j) was much higher than in stems (fig. 2k) and roots (fig. 2l), irrespective of the nacl treatment and/or sampling day. this latter finding was due to the relatively high concentrations of k and low concentrations of na found in cab-6p leaves, a fact that was governed by the parallel existence of an na-exclusion mechanism from the shoot and a k-inclusion (transport) mechanism to the leaves. in other words, salt-treated cab-6p plants were capable of maintaining an appropriate (similar to that of control plants) leaf k-to-na ratio, probably for protecting the most actively growing portion of plants (leaves). in accordance with the results of other studies (chartzoulakis 2005, and references therein), the diminution of k concentrations in roots of nacl-treated plants, which resulted in too low k/na ratios, may also provide a mechanism by which cab-6p plants achieved ionic balance following the high na absorption by their roots. fig. 2. concentrations of na (a, b, c), cl (d, e, f) and k (g, h, i) in leaves (a, d, g), stem (b, e, h) and root (c, f, i); k/na ratio in leaves (j), stem (k) and root (l) determined in control (0 mm nacl) and salinity-treated (60 mm nacl) prunus cerasus plants at different days (3, 5, 7, 9, 11 or 13) after nacl imposition. results are expressed as mean values ± se. for each parameter and day after salt stress imposition, asterisks indicate significant differences between control and nacl-treated plants, at p ≤ 0.05 (*), p ≤ 0.010 (**) or p ≤ 0.001 (***); n.s. indicates non-significant differences. salinity stress response in prunus cerasus acta bot. croat. 77 (2), 2018 201 fig. 3. total leaf chlorophyll concentration (a); leaf water potential (ψw) (b), osmotic potential (ψπ) (c) and turgor potential (ψp) (d) determined in control (0 mm nacl) and salinity-treated (60 mm nacl) prunus cerasus plants at different days (3, 5, 7, 9, 11 or 13) after nacl imposition. results are expressed as mean values ± se. for each parameter and day after salt stress imposition, asterisks indicate significant differences between control and nacl-treated plants, at p ≤ 0.05 (*), p ≤ 0.010 (**) or p ≤ 0.001 (***); n.s. indicates non-significant differences. in a previous in-vitro experiment with cab-6p explants treated with 60 mm nacl, one of the main effects of the high leaf cl concentration (4.2-5.6 % d.w.) was the decline in chlorophyll concentration of leaves (chatzissavvidis et al. 2008). similarly, in another study with prunus salicina, leaf chlorophyll concentration decreased when leaf chloride exceeded 0.25 mol kg-1 d.w. (about 0.9 % d.w.) (ziska et al. 1990). in the current short-term study, the total leaf chlorophyll concentration was also reduced by the salinity, from the 5th day of the experiment and thereafter (fig. 3a). however, this decrease in leaf chlorophyll could not mainly be ascribed to either the toxic effects of accumulated na (ranged between 0.07 % d.w. -5th dayand 0.19 % d.w. -13th day-) and cl (ranged between 0.67 % d.w. -5th dayand 1.63 % d.w. -13th day-) ions in leaves or to deficiencies of other mineral nutrients (similar concentrations were found in salt-treated and control plants). alternately, it might rather have been due to nacl-induced changes in the overall physiological status of plants (low water potential, possible closure of stomata, a decrease of photosynthetic performance, oxidative damages, etc.). the lower values of water (fig. 3b) and osmotic (fig. 3c) potentials found in the leaves of nacl-treated sour cherry plants, compared with those of control plants, resulted in turgor potential values (fig. 3d) similar to those of control plants. this latter observation in combination with the increased concentrations of na, k and cl found in leaves of nacl-treated plants leads to the conclusion that cab-6p plants are osmoregulated by the accumulation of inorganic ions. in any case, however, the possible contribution of some compatible solutes in the osmotic adjustment of sour cherry plants could not be excluded since relevant measurements were not carried out. therefore, since (a) it is nearly impossible for a cell to adjust osmotically using only inorganic ions, and (b) prunus species produce and accumulate significant amounts of sorbitol and proline under dehydration stress (jiménez et al. 2013, zrig et al. 2016), which may work as a membrane and as metabolism stabilizers and antioxidants, the contribution of soluble carbohydrates and other compatible organic solutes in the osmoregulation of sour cherry plants subjected to salinity stress has to be further investigated. overall, cab-6p sour cherry plants are very sensitive to salinity stress since their growth and leaf chlorophyll concentration were both affected negatively from the 3rd and 5th day, respectively, after the imposition of salinity. moreover, the results of this short-term study revealed some interesting responses of sour cherry plants under saline conditions. firstly, their root growth was affected more than their shoot growth (dry weight basis). secondly, an na-exclusion mechanism from the shoot (stems and leaves) was found to take place. thirdly, the existence of cland k-inclusion mechanisms to leaves were also a fact. therefore, cab-6p plants were able to control the uptake of na and/or its transport from roots to leaves more appropriately than they could cl. fourthly, plants had the capability of osmoregulation, a fact that apparently limited the detrimental effects of salinity. finally, from a pomological point of view, the use of the cab-6p sour cherry clone as rootstock for sweet 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j. r., dejong, t. m., 1990: salinity induced limitations on photosynthesis in prunus salicina, a deciduous tree species. plant physiology 93, 864–870. zrig, a., mohamed, h. b., tounekti, t., khemira, h., serrano, m., valero, d., vadel, a. m., 2016: effect of rootstock on salinity tolerance of sweet almond (cv. mazzetto). south african journal of botany 102, 50–59. acta bot. croat. 79 (2), 2020 193 acta bot. croat. 79 (2), 193–200, 2020 coden: abcra 25 doi: 10.37427/botcro-2020-022 issn 0365-0588 eissn 1847-8476 growth inhibition of the toxic cyanobacterium cylindrospermopsis raciborskii by extremely low-frequency electromagnetic fields zakaria mohamed1*, fadel ali2, medahat abdel-lateef3, asmaa hosny3 1 sohag university, faculty of science, department of botany and microbiology, sohag, egypt 2 cairo university, faculty of science, biophysics department, cairo, egypt 3 sohag university, faculty of science, department of physics, sohag, egypt abstract – this study investigates the effects of extremely low-frequency electromagnetic fields (elf-emfs) on the growth and antioxidant defence enzymes of the toxic cyanobacterium cylindrospermopsis raciborskii (woloszynska) seenayya et subba raju. to determine resonance frequency of growth inhibition of c. raciborskii, cells were subjected to elf square amplitude modulated waves (qamw) with a range of frequencies (0.1, 0.3, 0.5, 0.7, 0.9 hz) at single intensity of 100 v m–1 for 30 minutes. the results revealed that the highest growth inhibition of cylindrospermopsis occurred upon exposure to 0.7 hz qamw for 30 min. elf-emf-exposed cultures exhibited a marked decrease in cell number, chlorophyll-a content and activity of antioxidant enzymes compared to control cultures, and this effect increased with the prolongation of exposure time. moreover, elf-emf induced morphological changes in cylindrospermopsis cells upon exposure to 0.7 hz qamw for 120 min, including shrinking and disintegration of cytoplasmic contents, and thickening of the cell wall. changes in dielectric properties, as a measure of interaction of cellular constituents (e.g., plasma membrane, cell wall and cytoplasm), with electromagnetic fields were also observed for treated cells. our results provide a new possibility for using elf-emfs to eliminate toxic cyanobacteria from drinking and recreational water sources. keywords: cyanobacteria, cylindrospermospsis, electromagnetic fields, growth inhibition, water treatment * corresponding author e-mail: mzakaria_99@yahoo.com introduction environmental exposure to non-ionizing, non-thermal, extremely low frequency (<300 hz) electromagnetic fields (elf-emf) is becoming increasingly widespread in many countries due to the frequent use of electric appliances, electronic devices, communication systems and electric transmission lines (zhu et al. 2016, bodewein et al. 2019). despite their extremely low frequency and low energy, elf-emfs have a number of different biological effects and bring about cellular changes (sienkiewicz et al. 2005, santini et al. 2009). many studies have reported the effects of elf-emfs on the growth and cell functions of microorganisms including bacteria (inhan-garip et al. 2011, fadel et al. 2014, oncul et al. 2016), yeasts (malıkova et al. 2015) and fungi (potenza et al. 2012). however, the results are still controversial. the inhibitory or stimulatory effect of elf-emfs on microorganisms has been shown to be dependent on the strength and frequency of the electromagnetic field applied, and strain used (justo et al. 2006). therefore, many studies are required to evaluate the influence of different emf frequencies and intensities on different species. however, little is known about the effects of elf-emfs on cyanobacteria (fadel et al. 2018). cyanobacteria are prokaryotic oxygen-evolving photosynthetic microorganisms that live in a wide range of marine and freshwater habitats (zanchett and oliveira-filho 2013). although they serve as a food source for most organisms in the aquatic environment (mohamed and al-shehri 2013), they represent an ecological problem when their numbers increase and they form harmful blooms under eutrophic conditions (i.e. high nutrient concentrations) (mohamed 2016a). these blooms can cause the formation of hypoxic dead zones in lakes, leading to the suffocation of aquatic animals (zhu et al. 2015). furthermore, many mohamed z, ali f, abdel-lateef m, hosny a 194 acta bot. croat. 79 (2), 2020 species of cyanobacteria can produce different kinds of toxins (hepatotoxins, neurotoxins, irritants and gastrointestinal toxins) that adversely affect animal and human health, particularly in drinking water sources (codd et al. 2005). such toxigenic species in drinking water sources can impair the water quality (dittmann and wiegand 2006, mohamed and al-shehri 2009) and should thus be eliminated properly in drinking water treatment plants. different conventional methods have been applied to remove cyanobacterial cells from drinking water including chlorination, coagulation, sand filtration and sonication (de la cruz et al. 2011). however, the main drawbacks of these methods are that they are expensive, complex and cause cell lysis leading to the release of intracellular toxins into the surrounding water (zanchett and oliveira-filho 2013, mohamed et al. 2015). most studies on the biological effects of electromagnetic fields yielded promising results for the application of elf-emfs as potential means for control or killing pathogenic and toxic microorganisms (bodewein et al. 2019). our previous study tested the potential inhibitory effect of elf-emf on the growth of the cyanobacteria microcystis aeruginosa (kützing) kützing and anabaena circinalis rabenhorst ex bornet & flahault (fadel et al. 2018). the results revealed different effects of elf-emf on the two species, strongly inhibiting the growth of a. circinalis with no remarkable effect on m. aeruginosa. cylindrospermopsis raciborskii (woloszynska) seenayya et subba raju is among the toxic cyanobacterial species frequently found in drinking water sources and linked to harmful blooms in freshwater sources over the world (rzymski and poniedzialek 2014). it has been associated with the production of water-soluble toxins including the hepatotoxic alkaloid, cylindrospermopsin (cyn), and the neurotoxic alkaloid, saxitoxin (stx) (carneiro et al. 2013). therefore, the present study investigates in details the effects of elf-emfs on growth and antioxidant defence system of the toxic cyanobacterium c. raciborskii. the results of this study could be useful for controlling the growth of toxic cyanobacteria in drinking and recreational water sources. materials and methods elf-emf exposure system square amplitude modulated waves (qamw) were generated by an arbitrary function generator (bk precision 4085 40 mhz) as described with sketch diagram by fadel et al. (2018). the carrier frequency was a 10 mhz sine wave with amplitude ± 10 vpp, and modulation depth of ± 2 vpp. cyanobacterial cultures in sterilized tubes were exposed to qamw through two parallel copper electrodes connected to the output of the generator. the temperature inside the coil was maintained at 30 ± 0.5 °c (the organism's optimal growth temperature). experimental organism and culture conditions cylindrospermopsis raciborskii strain (lc107906) used in this study was obtained from the microalgal culture collection of botany and microbiology department, faculty of science, sohag university, sohag, egypt. this species was isolated from fish ponds in sohag region, egypt, and reported as cylindrospermopsin producer (mohamed 2016b). the strain was grown in bg-11 liquid medium with low nitrogen (3% nano3, ph = 8) (ripka et al. 1979) under controlled conditions of light (50 µmol m–2 s–1) and temperature (30 ± 0.5 °c) in a climate-controlled chamber. based on the growth curve, the doubling time of this strain was 15.33 h (fig.1). one ml of exponentially growing c. raciborskii cells (8 × 105 cells ml–1) was then inoculated into 25 ml-glass test tubes containing modified bg-11 medium (i.e. without nitrogen), and used to determine resonance frequency of growth inhibition of c. raciborskii. the inoculated cells were further grown for 24 h for adaptation in the same growth chamber. thereafter, 30 tubes with cyanobacterial cells were connected to output of the generator at a single intensity of 100 v m–1 and different frequencies (0.1– 1.0 hz) for 30 minutes. other 3 tubes were used as control and kept in the same conditions as the exposed ones (at 50 µmol m–2 s–1 light intensity and temperature of 30 °c), but outside the emf generating instrument (i.e. without field application). to further confirm the effect of elf-emfs on the c. raciborskii, additional experiments were conducted by exposing cyanobacterial cells to the electromagnetic field at the frequency showing the strongest inhibitory effect (0.7 hz) in the former experiment, for different exposure times (15, 30, 60, 120 min). the experiment for each exposure time was run in 9 tubes, 3 tubes were incubated for 4 h, 3 tubes incubated for 24 h and 3 tubes for 48 h. three tubes were used as a control for each incubation period. both treated and control cultures of c. raciborskii were incubated under controlled conditions of light (50 µmol m–2 s–1) and temperature (30 ± 0.5 °c) in a climate-controlled chamber. an aliquot (2 ml) from the cultures was taken under aseptic conditions for analysis. subsamples of treated and control cultures were collected after 4, 24 and 48 h for antioxidant enzyme activities and after 24 and 48 h for analysis of cell number and chlorophyll-a. each experiment was done in triplicate. fig. 1. the growth curve of cylindrospermopsis raciborskii under optimal growth conditions. each value is presented as mean ± standard deviation of three measurements. inhibitory effects of electromagnetic fields on cyanobacteria acta bot. croat. 79 (2), 2020 195 cell abundances cell abundances (cells ml–1), as an estimator of cylindrospermopsis raciborskii growth, were estimated using a sedgewick rafter chamber under a light microscope following the method of hötzel and croome (1999). the morphotype of the strain is a coiled trichome with an average length of 180 µm. therefore, c. raciborskii was first counted as trichomes. the total abundance of c. raciborskii in the samples was calculated by multiplying the number of trichomes (trichomes ml–1) by the average number of cells per trichome (22 cells) estimated in our study. chlorophyll-a determination chlorophyll-a concentration (chl-a, pg cell–1) as a measure of biomass and an indicator of the physiological status of c. raciborskii, was determined spectrophotometrically at 665 and 750 nm (appota 6200 uv/vis spectrophotometer) in a methanol extract prepared by homogenizing cyanobacterial cells of a known volume (2 ml) of cultures according to tailing and driver (1963). chl-a was calculated using the equation: chl-a = 13.9 (a665-a750) × v/v.n, where a665 and a750 correspond to the absorbance of methanol extracted supernatant at 665 nm and 750 nm wavelength with a 1 cm pathway cuvette, 13.9 is the extinction coefficient, v is volume of extract (ml), v is volume of culture sample (ml) and n is the number of cells present in the culture sample. assay of antioxidant enzymes to determine the activity of antioxidant enzymes, samples of treated and control cultures (2 ml) of c. raciborskii were harvested by centrifugation at 10,000 g for 10 min at 4 °c and the pellets obtained were washed with 10 mm na2edta (ethylene diamine tetraacetic acid), then twice with distilled water. the algal pellets were added to 3 ml of 50 mm phosphate buffer (na2po4/k2hpo4), ph = 7.0, containing 0.1 mm na2-edta and 1% of polyvinylpyrrolidone (pvp) and ground by sonication in ice-water bath. the homogenate was centrifuged at 13,500 g for 20 min at 4 °c. the supernatant was re-centrifuged at 13,500 g for 15 min at 4 °c, and the resultant supernatant was collected and stored at –20 °c for analysis of catalase (cat) and peroxidase (pod). cat activity was measured according to the method of aebi (1984) with minor modifications. the reaction mixture (3.0 ml) consisted of 50 mm phosphate buffer (ph = 7.0), 0.1 mm edta, 45 mm h2o2 and 50 μl enzyme extract). the reaction was started by addition of the extract. the activity of catalase was estimated by the decrease of absorbance at 240 nm for 1 min as a consequence of h2o2 consumption. catalase activity was calculated based on an extinction coefficient 36 m–1 cm–1 and expressed as nmole h2o2 min–1 cell–1. total pod activity was determined as described by macadam et al. (1992) by the oxidation of guaiacol in the presence of h2o2 in a reaction mixture (3.0 ml) containing 0.1 m phosphate buffer (ph = 6.5), 0.1 mm edta, 0.2 m guaiacol, 0.03 m h2o2 and 50 μl enzyme extract. the increase in the absorbance due to oxidation of guaiacol was measured at 470 nm. the enzyme activity was calculated in terms of nmole of tetraguaiacol oxidized per min per cell at 25 ± 2 °c using the absorbance coefficient 26.6 mm–1 cm–1.all spectrophotometric analyses of enzyme activities were performed on a spectrophotometer appota 6200 uv/vis spectrophotometer. dielectric measurements the dielectric parameters were determined for cyanobacterial cultures exposed to 0.7 hz square amplitude modulated waves for 2 hours, after a 24 h incubation period. these measurements were carried out in subsamples of these cultures containing a fixed cell number of c. raciborskii (105 cells ml–1), which was monitored using a sedgewick rafter chamber as described above. the cell number was adjusted by the dilution of the subsamples in sterile deionized water. cyanobacterial cells in the subsamples (1 ml) were separated from the medium by centrifugation at 14,000 g at 4 °c for 15 min. the pellet was re-suspended in a 1 ml volume of sterile deionized water. this step was repeated twice. the dielectric measurements were carried out in cyanobacterial cell suspensions in the frequency (f) range 42–100000 hz using a loss factor meter (lcr hi tester 3532, hioki, ueda, nagano, japan) with a sample cell (pw 9510/60, philips weisshausstrasse, aachen, germany) according to fadel et al. (2017). the relative permittivity ɛo, dielectric constant ε, conductivity σ and relaxation time (t) of the samples were calculated according to the equations outlined by fadel et al. (2017). cylindrospermopsin analyses to detect and quantify cylindrospermopsin (cyn) toxin within cylindrospermopsis cells as well as toxin potentially released into the medium, an aliquot (2 ml) of 48 h cultures (i.e. control and elf-treated cells) was filtered through gf/c filters. the filters with retained cells were extracted in methanol (50%) for intracellular toxin, while the filtrate was used for the determination of extracellular cyn potentially released into the medium. cyn concentration was determined by elisa using commercial kits for this toxin purchased from abraxis (54 steamwhistle drive warminster, pa 18974). briefly, a 100 µl culture filtrate or toxin extract, toxin standard, calibrator or negative control was added to 96-microplate wells and incubated for 30 min at room temperature. a 100 µl aliquot of a microcystin-enzyme conjugate solution was then added and incubated for another 30 min at room temperature. the wells were emptied and washed four times with phosphate buffer saline and distilled water. a 100 µl aliquot of substrate was added to each well and incubated for 30 min at room temperature. the absorbance of the colour generated from the transformation of a substrate by the enzyme was measured spectrophotometerically at 450 nm using the microplate reader. the concentration of cyn toxin in the culture filtrate was directly calculated from standard curves drawn with the standard solutions provided in kits. detection limit for cyn according to the kit's manufacturer is 0.04 ng ml–1. mohamed z, ali f, abdel-lateef m, hosny a 196 acta bot. croat. 79 (2), 2020 statistical analysis differences in growth and physiological parameters between treated and control cultures of c. raciborskii were determined by one-way anova (p < 0.05) and tukey’s posthoc multiple range test using the software spss ver.16.0 (spss inc. released 2007). results the growth response of cylindrospermopsis raciborskii exposed to 0.1–1.0 hz amplitude modulating frequencies for 30 min is shown in fig. 2. the exposure of c. raciborskii to 0.7 hz for 30 min after 24 h of incubation caused a more significant decrease (f = 201.2, p = 0.001) of cell number than other frequencies (fig. 2). the effect of exposure time at 0.7 hz on the growth and physiological functions of the microorganism was also evaluated. the results showed that after 24 h of incubation, a significant reduction in the cell number in treated cultures was observed compared to control, and that reduction varied significantly (f = 177.7, p = 0.0003) based on the exposure time of the cultures to the electromagnetic field (fig. 3). the increase in the exposure time led to a sharp reduction in microbial growth (i.e. low cell number). however, at the incubation period of 24 h, no complete inhibition of cyanobacterial growth was found at different exposure times tested. on the other hand, at 48-hour incubation, the exposure to the electromagnetic field reduced the cyanobacterial growth sharply in comparison to 24-hour incubation, and caused complete death for 2-hour exposure cells (fig. 3). the cyanobacterial cell number also showed a decrease in control cultures at 48 h incubation period, indicating that the cells entered the decline phase at that period based on growth curve (fig .1). alongside the cell density of c. raciborskii, concentration of chl-a, was also influenced by an elf-emf at 0.7 hz. chl-a content of treated cells exhibited a significant decrease (f = 9.2, p = 0.002) in comparison to control at 24 hours incubation period (fig. 4), and this decrease was exposure time-dependent. two-hour exposure to electromagnetic field completely reduced chl-a content at 48 hours incubation period (fig. 4). the activity of antioxidant enzymes (cat and pod) in c. raciborskii responded differently, when exposed to elfemfs compared to that of control cultures (fig. 5). the activity of pod in the cells of 2 h-exposure to the electromagnetic field was enhanced after the first 4 h of incubation period (fig. 5). this activity decreased sharply in 2-h expofig. 2. the change in cell number of cylindrospermopsis raciborskii exposed to square amplitude modulated waves at different frequencies (0.1–1.0 hz) for 30 minutes and grown for 24 hours. each value is the average of three replicates ± standard deviation. asterisk indicates significant differences (one-way anova at p ≤ 0.05) in cell number between different exposure times and control. fig. 4. the change in chlorophyll-a concentration of cylindrospermopsis raciborskii exposed to 0.7 hz for different exposure periods, and grown for 24 and 48 h. each value is the average of three replicates ± standard deviation. different uppercases letters indicate significant differences (one-way anova at p = 0.05) in chlorophyll-a concentrations between incubation periods (24 and 48 h) for the same exposure time. different lowercases letters indicate significant differences (one-way anova at p ≤ 0.05) in chlorophyll-a concentrations among different exposure times for the same incubation period. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 control 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 sllec( reb mun lle c x 10 6 m l– 1 ) frequency (hz) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 control 15 30 60 120 c hl or op hy lla (p g ce ll1 ) exposure time (min) 24 hours 48 hours aa aa ab ab ac aa bb ac bd be fig. 3. the growth inhibition of cylindrospermopsis raciborskii exposed to 0.7 hz square amplitude modulated waves for different exposure periods, and grown afterwards for 24 and 48 h. each value is the average of three replicates ± standard deviation. asterisks indicate significant differences (one-way anova at p ≤ 0.05) in cell number between incubation periods (24 and 48 h) for the same exposure time. different letters indicate significant differences (one-way anova at p ≤ 0.05) in cell number among different exposure times for each incubation period. inhibitory effects of electromagnetic fields on cyanobacteria acta bot. croat. 79 (2), 2020 197 sure cultures after 48 h incubation period. on the other hand cat activity in treated cells increased along the whole incubation period, except cells of 1-h and 2-h exposure, which showed a marked reduction in cat activity at 48 h incubation period. morphological changes were also noted in cells exposed to elf-emf at 0.7 hz for 2 h. these changes began with shrinking of cytoplasmic contents forming amorphous aggregates, and thickening of the cell wall. they eventually ended with the disintegration of cytoplasmic material, but without apparent lysis in the cell wall (data not shown). changes in dielectric properties, as a measure of interaction of cellular constituents (e.g. plasma membrane, cell wall and cytoplasm) with electromagnetic fields, were evident in the present study. the data shown in table 1 revealed a significant decrease in the dielectric increment (∆ɛ) and conductivity (s) for exposed cyanobacteria cells to elf-emf at 0.7 hz for 2 hours and incubated for 24 h, as compared with control (f = 9, p = 0.04 and f = 12.1, p = 0.02, respectively). the results of elisa for cyn toxin revealed that cyn concentrations released naturally into the medium of control cultures were 0.2 ± 0.03 µg l–1 while the toxin was not detectable in the medium of elf-treated cultures. conversely, the toxin concentrations within cells of elf-treated cultures (26.1–27.3 pg cell–1) were higher than those in cells of control cultures (4.31–6.25 pg cell–1). discussion the present study determined the effects of the different frequencies of elf-emf on the growth of cylindrospermopsis raciborskii. maximum growth inhibition and decrease in chl-a content of c. raciborskii were observed at a frequency of 0.7 hz. the results also provided evidence for the inhibitory effect of elf-emfs at 0.7 hz on the physiological functions and antioxidant system of the toxic cyanobacterium c. raciborskii. this is in agreement with the results of our previous study, which found that the growth inhibition of anabaena circinalis by elf-emfs occurred at a resonance frequency 0.7 hz (fadel et al. 2018). for heterotrophic bacteria, the resonance frequency for the growth inhibitory effects of elf-emf seems to vary among species. for instance, the growth of salmonella typhi was highly inhibited by an elfemf at a resonance frequency of 0.8 hz (fadel et al. 2014), while the inhibiting resonance frequency of elf-emf was 1.0 hz for agrobacterium tumefaciens (fadel et al. 2017), and 50 hz for escherichia coli and pseudomonas aeruginosa (segatore et al. 2012). the inhibition effect is due to the interference of the elf-emfs according to the frequency with the bioelectric signals generated from the physiological functions of microbial cells (zanchett and oliveira-filho 2013). in this respect, fadel et al. (2014) reported that the bioelectric signals generated during metabolic activities of cells are usually in the extremely low frequency range. therefore, the applied electromagnetic wave could have a similar frequency, interfering with these signals. our data also revealed that cell proliferation of c. raciborskii decreased sharply when exposed to the electromagnetic field compared to control cultures, and this decrease was dependent on the exposure time. our results are thus in accordance with the results of previous studies demonstrating that the inhibitory effect of elf-emfs on the growth of bacteria (gaafar et al. 2006) and cyanobacteria (fadel et al. 2018) increased with the length of exposure. meanwhile, there was a decrease in the cell number of c. raciborskii in tab. 1. dielecteric parameters for cylindrospermopsis raciborskii exposed to 0.7 hz square amplitude modulated waves for 2 hours, as compared with the control (untreated). asterisks indicate significant difference (one-way anova at p ≤ 0.05) in the parameters between control and treated cultures (n=3). relaxation time (τ µs) conductivity 𝜎(s m–1) at 100 khz dielectric increment ∆ɛ(ɛ˳–ɛ͚) control 14.05 ± 1.0 (6 ± 1.2) × 10-3 1721 ± 512.6 0.7 hz 12.9* ± 0.97 (4.8* ± 2.1) × 10-3 1347* ± 165.3 fig. 5. the change in the activities of the antioxidant enzymes peroxidase (a) and catalase (b) of cylindrospermopsis raciborskii exposed to 0.7 hz for different exposure periods, and grown for 4, 24 and 48 h. each value is the average of three replicates ± standard deviation. different uppercases letters indicate significant differences (one-way anova at p = 0.05) in enzyme activity among different exposure times for each incubation period. different lowercases letters indicate significant differences (one-way anova at p ≤ 0.05) between incubation periods (4, 24 and 48 h) for the same exposure time. 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 4 24 48 p er ox id as e (n m ol m in -1 ce ll1 ) a control 15min. 30min. 60min. 120min. aa ac aa ab ab bc ba bb cc ca cb dc da db ec 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 4 24 48 c at al as e ac tiv ity (n m ol m in -1 ce ll1 ) incubation time (hours) b aa aa ab aa ab bc aa bb cc aa cb dc ba db ec 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 4 24 48 c at al as e ac tiv ity (n m ol m in -1 ce ll1 ) incubation time (hours) b aa aa ab aa ab bc aa bb cc aa cb dc ba db ec mohamed z, ali f, abdel-lateef m, hosny a 198 acta bot. croat. 79 (2), 2020 control cultures at 48 h of incubation period. this decrease may be due to the high initial cell density of inoculum we used in our experiments (fig.1), which promoted the rapid growth of cyanobacterial cells leading to nutrient consumption, shortness of cell proliferation time and early decline phase. this agrees with the finding of cheng et al. (2018) reporting that high inoculum density of algal cells can shorten the time of cell proliferation and promote the rapid growth of cells. in the present study, elf-emf also reduced the concentration of chl-a of this cyanobacterium. this may be due to the decrease in the cyanobacterial cell number or the inhibition of chl-a synthesis. therefore, chl-a is a useful indicator of the phototrophic biomass and physiological status of the organism, and has been used in bioassays of environmental stresses (borowiak et al. 2015). the damaging effect of elfemf on photosynthetic pigments may occur through active oxygen mediated peroxidation (zeeshan and prasad 2009). in this respect, numerous hypotheses have been proposed to explain the mechanisms of the biological effects of electromagnetic fields on cells and organisms. the main theories are based on fact that elf-emf promotes the formation of free radicals (e.g. reactive oxygen species, ros) through changing the level of ionization of water molecules (fernie and reynolds 2005), which affect the synthesis of macromolecules and relevant metabolic processes (cabsicol et al. 2000). the other possible effects are that elf-emf changes the permeability of the ionic channels in the cell membrane (oncul et al. 2016), and thus affect ion transport into the cells resulting in biological changes in the organism. our data revealed an increase in the dielectric properties of cyanobacterial cell constituents of treated cultures, indicating that damage and change in the permeability of cell membrane occurred. notably, the electrical conductivity and relaxation time are directly related to the electric dipole moment of the microbial cell, and the decrease in these parameters is mainly due to changes in the charge distribution upon the protein molecules of the cell membrane. therefore these two parameters can be indicators for structural changes in the cell membrane of an organism upon exposure to stress such as elf-emfs (fadel et al. 2014). therefore, our study supports the hypothesis that the biological effect of elf-emfs is due the change in the permeability of the ionic channels in the membrane (oncul et al. 2016). in this regard, it has been reported that dielectric properties of a biological system reflect information about the morphology and permeability of the cellular membrane, and about possible changes in structure and composition of biological macromolecules such as protein, dna, amino acids (dopp et al. 2000). additionally, our study found that elf-emfs induced morphological changes in c. raciborskii cells including shrinking and disintegration of cytoplasmic material. such morphological changes in addition to filament fracture and coils dissociation were also observed for anabaena circinalis when exposed to elf-emf (fadel et al. 2018). inhan-garip et al. (2011) demonstrated shrinking and disintegration of cell contents, and cell wall lysis of gram-negative bacteria induced by elf-emfs. however, the cell wall lysis of elf-emf-exposed gram-negative bacteria observed by inhan-garipet al. (2011) did not occur for the cyanobacterium c. raciborskii during our study (not shown). our observation is similar to that obtained for gram-positive bacteria cell wall being apparently notdisrupted upon exposure to elf-emfs (inhan-garip et al. 2011). the discrepancy between gram negative bacteria and cyanobacteria can be explained by the fact that although cyanobacteria are gram-negative bacteria, their cell wall contains features of gram-positive bacteria. these include: the peptidoglycan layer found in cyanobacteria is considerably thicker than that of most gram-negative bacteria, the degree of cross-linking between the peptidoglycan chains within the murein is more similar to that of gram-positive bacteria (56% to 63%), and the cyanobacterial peptidoglycan is complexed with specific polysaccharides (litzinger and mayer 2010). what is interesting in this study is that unlike naturally occurring in control cultures (i.e. toxin release), cyn toxin was found within the cells of elf-treated cultures but it was not detectable at all in the medium of these cultures during the whole incubation period (i.e. not just a decrease in its concentration). therefore, the presence of cyn toxin within treated cells and its absence in the medium indicates that these cells could not release the toxins into the medium. these results confirm that elf did not lyse cyanobacterial cell wall but increased its thickness so that cytoplasmic contents including toxins cannot be released from the cells. however, the presence of cyn in the medium of control cultures can be explained by the fact that cyn is often naturally released from living cells into the surrounding water (rücker et al. 2007). furthermore, increased activities of cat and pod enzymes in c. raciborskii cells exposed to the electromagnetic field during the first 24 hours incubation period in comparison to control, provide evidence that elf-emf induces oxidative stress and promotes the formation of ros. pod reduces h2o2 to water using various substrates as electron donors, and cat catalyzes the breakdown of h2o2 into water and oxygen (wang et al. 2009). this finding supports the hypothesis that reactive oxygen species (ros) mediate the effects of elf-emfs on living organisms (fernie and reynolds 2005). in this respect, it is well known that ros are continuously produced and eliminated by living organisms normally maintaining them at certain steady-state levels (lushchak 2011). most organisms including cyanobacteria have developed an antioxidant defence system comprising enzymes such as cat and pod that scavenge the resultant ros and maintain the balance between ros generation and elimination (ganapathy et al. 2015). however, under some circumstances (e.g., stress intensity and duration, rate of ros production, capacity of ros scavenging), this balance is disturbed, leading to enhanced ros level and oxidative damage (lushchak 2011). accordingly, our results showed a sharp decrease in the activity of antioxidant enzymes (cat and pod) of cells with 1-h and 2-h exposure time after the initial increase in the activity. decreases in cat and pod inhibitory effects of electromagnetic fields on cyanobacteria acta bot. croat. 79 (2), 2020 199 activities at higher exposure times (1h and 2h) to elf-emf may have been caused by high concentrations of free radicals generated by electromagnetic field leading to the inhibition of protein synthesis (luna et al. 1994).the alterations in the activity of antioxidant enzymes upon exposure to elf-emf have been previously documented in plants (shashurin et al. 2017), human cells (mahmoudinas et al. 2016) and bacteria (fadel et al. 2017). conclusion in conclusion, the results of this study demonstrated that the 2-h exposure to an elf-emf at 0.7 hz inhibited the growth of the toxic cyanobacterium c. raciborskii. the reduction in cell number was associated with induction and subsequent decrease in the antioxidant defense enzymes, leading to changes in cell 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role of spermidine in overwintering of cyanobacteria. journal of bacteriology 19, 2325–2334. https://www.scopus.com/sourceid/19705?origin=recordpage https://www.scopus.com/sourceid/25076?origin=recordpage https://www.scopus.com/sourceid/25076?origin=recordpage acta bot. croat. 78 (1), 2019 s5 social news professor emeritus ljudevit ilijanić: an appreciation on the occasion of his ninetieth birthday professor ljudevit ilijanić was the editor-in-chief of the journal acta botanica croatica in the period from 1969 to 1992, when, thanks to his dedication, the journal was the main source of botanical information from croatia for botanists in this country and as well the wider world. ljudevit ilijanić was born on september 27, 1928 in the village of slapno near the town of ozalj (karlovac county), where he completed his elementary education. he finished at the higher real gymnasium in the city of karlovac in 1947 and graduated in biological sciences at the faculty of science of the university of zagreb in 1952 with the theme 'investigating flora of ozalj and its surroundings'. in 1953, after graduation obtained an assistant's position in the botany division of the faculty of science, university of zagreb. professor ilijanić spent his entire professional work at this faculty where he has been an assistant professor since 1963, associate professor since 1969, and a full professor since 1974 until retiring in 1993. he defended his phd thesis 'ecological and phytosociological studies on the lowland meadows of croatia' in 1960, and then spent a year of postdoctoral training at the station internationale de géobotanique méditerranéenne et alpine (sigma) in montpellier (france) with phytosociologist josias braun-blanquet (1884-1980). sabbaticals in former czechoslovakia, poland, russia, switzerland, germany and the usa helped to establish and maintain his international contacts. professor ilijanić served as head of the department of biology in the period 1970-1971, and vice-dean of the faculty of science in the academic years 1974-1975 and 19751976, and the head of the botany division in the period 1987-1989. in 2000 the senate of the university of zagreb gave ljudevit ilijanić the special recognition of professor emeritus. as a teacher, professor ilijanić took particular pride in his role in creating the curriculum for the first students of ecology at the university of zagreb. he taught several courses in the field of plant ecology and geobotany in undergraduate and postgraduate studies, and also set up and organized an ecological practicum program. he supervised more than 50 msc theses, and a fair number of phd theses. the purely scientific work of professor ljudevit ilijanić has been oriented to the flora and vegetation of croatia. his numerous updates to the croatian flora constitute an important contribution to plant distribution and ecology. notably, he was the first to start extensive ecological research into grassland vegetation in croatia. further published work relates to swamp, peat and halophilous plant communities, emphasizing phytosociological and autecological approaches. he particularly encouraged studies on vegetation dynamics and succession. ljudevit ilijanić, as an author or with his co-authors, described new syntaxonomic units: the alliance trifolion pallidi ilijanić 1969, and associations e.g. serratulo-plantaginetum altissimae ilijanić 1967, ventenato-trifolietum pallidi ilijanić 1967, limonietum anfracti ilijanić et s. hećimović 1982, and several more subassociations. his concept of the wet meadows vegetation of the humid continental regions of the north-central balkans was also included in the recent comprehensive work 'vegetation of europe', colloquially known as eurovegchecklist (mucina et al. 2016). in total, professor ilijanić’s research oeuvre published in national and international journals comprises about a hundred papers, some of which laid the very foundations to the study of plant communities in croatia. in addition, he authored more than 40 popular science papers and 250 articles and entries in encyclopedias of the croatian lexicographic institute. a lists and analysis of the scientific papers authored by professor ljudevit ilijanić have previously been published in the acta botanica croatica (marković 1993, regula and regula 2000). professor ljudevit ilijanić during the investigation of vegetation on the dinara mt. in 2008 (photo: j. topić). s6 acta bot. croat. 78 (1), 2019 i list here his papers published after 1999 in chronological order: 1. tomašević, m., samardžić, i., ilijanić, lj., 1999: new localities of the species doronicum orientale hoffm. (asteraceae) in croatia. natura croatica 8, 87–93. 2. ilijanić, lj., topić, j., 2000: cyperaceae. in: nikolić, t. (ed.) flora croatica: index florae croaticae. pars 3. natura croatica 9, suppl. 1, 123–129. 3. ilijanić, lj., topić, j., 2000: poaceae. in: nikolić, t. (ed.) flora croatica: index florae croaticae. pars 3. natura croatica 9, suppl. 1, 130–149. 4. ilijanić, lj., topić, j., 2002: eriophorum vaginatum l. (cyperaceae), new and critically endagered plant species in croatia. razprave iv razreda sazu 4, 217–225. 5. ilijanić, lj., andreškić, a., cigić, p., 2005: aeluropus littoralis (gouan) parl. in: nikolić, t., topić, j. (eds.), red book of vascular flora of croatia: categories ex, re, cr, en and vu, 104–105. ministarstvo kulture republike hrvatske, državni zavod za zaštitu prirode, zagreb (in croatian). 6. ilijanić, lj., cigić, p., nikolić, t., 2005: lathyrus ochrus (l.) dc. in: nikolić, t., topić, j. (eds.), red book of vascular flora of croatia: categories ex, re, cr, en and vu, 192–193. ministarstvo kulture republike hrvatske, državni zavod za zaštitu prirode, zagreb (in croatian). 7. ilijanić, lj., rešetnik, i., nikolić, t., 2005: plantago indica l. in: nikolić, t., topić, j. (eds.), red book of vascular flora of croatia: categories ex, re, cr, en and vu, 221–222. ministarstvo kulture republike hrvatske, državni zavod za zaštitu prirode, zagreb (in croatian). 8. ilijanić, lj., rešetnik, i., nikolić, t., 2005: puccinellia distans (l.) parl. ssp. distans. in: nikolić, t., topić, j. (eds.), red book of vascular flora of croatia: categories ex, re, cr, en and vu, 230–231. ministarstvo kulture republike hrvatske, državni zavod za zaštitu prirode, zagreb (in croatian). 9. ilijanić, lj., andreškić, a., cigić, p., 2005: scirpus mucronatus l. in: nikolić, t., topić, j. (eds.), red book of vascular flora of croatia: categories ex, re, cr, en and vu, 241–242. ministarstvo kulture republike hrvatske, državni zavod za zaštitu prirode, zagreb (in croatian). 10. ilijanić, lj., alegro, a., praljak, i., 2005: sporobolus pungens (schreb.) kunth. in: nikolić, t., topić, j. (eds.), red book of vascular flora of croatia: categories ex, re, cr, en and vu, 247–248. ministarstvo kulture republike hrvatske, državni zavod za zaštitu prirode, zagreb (in croatian). 11. ilijanić, lj., nikolić, t., andreškić, a., cigić, p., 2005: cyperus rotundus l. in: nikolić, t., topić, j. (eds.), red book of vascular nflora of croatia: categories ex, re, cr, en and vu, 300–301. ministarstvo kulture repubprofessor nenad jasprica university of dubrovnik, institute for marine and coastal research editor in chief acta botanica croatica like hrvatske, državni zavod za zaštitu prirode, zagreb (in croatian). 12. topić, j., ilijanić, lj., tvrtković, n., nikolić, t., 2006: staništa: priručnik za inventarizaciju, kartiranje i praćenje stanja. državni zavod za zaštitu prirode, zagreb. 13. ilijanić, lj., topić, j., anić, i., gottstein, s., kušan, v., peternel, h., hima, v., ivaštinović, d. (eds.), 2008: priručnik za kartiranje i upravljanje staništima u parku prirode lonjsko polje. javna ustanova park prirode lonjsko polje, krapje. 14. mitić, b., topić j., ilijanić, lj., jasprica, n., milović, m., ruščić, m., pandža, m., bogdanović, s., dolina, k., 2009: kartiranje flore dalmacije. prioritetna područja: otok pag, estuarij krke, otok vis i pučinski otoci, pelješac i mljet, tok cetine. očuvanje i održivo korištenje biološke raznolikosti na dalmatinskoj obali. coast, undp, split; prirodoslovno-matematički fakultet sveučilišta u zagrebu. currently professor ilijanić continues to be a member of editorial board of the scientific journals: acta botanica croatica, natura croatica and hacquetia. professor ilijanić is a member of several scientific or professional societies: international association for vegetation science – iavs, eastern alpine and dinaric society for vegetation ecology – eadsve, croatian society of natural sciences, croatian ecological society, croatian biological society, croatian botanical society, etc. for achievements in his work and endeavours in botanical sciences, professor ilijanić has received several commendations. more recently, due to his considerable contribution to croatian botanical science, the croatian botanical society in 2016 declared professor ilijanić an honorary member. today, plant scientists throughout the world appreciate and respect him both as a colleague and as a friend. professor ljudevit ilijanić has acted as 'an ambassador of croatian botany'. we greatly appreciate his numerous contributions to the scientific community and his years of dedicated and skilled services as editor of acta botanica croatica. i would like to congratulate professor ljudevit ilijanić on his ninetieth birthday, on behalf of all of his colleagues, friends and students, and to pay tribute to him for his outstanding contributions during more than half a century of dedicated work. we all wish him good health and many years of successful scientific work for the benefit of croatian botany. acta bot. croat. 78 (1), 2019 s7 references aguiar, c., freitag, h., hennekens, s. m., tichý, l., 2016: vegetation of europe: hierarchical floristic classifcation system of vascular plant, bryophyte, lichen, and algal communities. applied vegetation science 19, suppl. 1, 3–264. regula, i., regula, lj., 2000: professor ljudevit ilijanić dr. sc. on the occasion of his seventieth birthday. acta botanica croatica 59, 1–3. marković, lj., 1993: prof. dr. ljudevit ilijanić (prigodom 65. godišnjice života). acta botanica croatica 52, 155–166. mucina, l., bültmann, h., dierßen, k., teurillat, j. p., raus, t.,čarni, a., šumberová, k., raus, t., di pietro, r., gavílan garcía, r., chytrý, m., iakushenko, d., schaminée, j. h. j., bergmeier, e., santos guerra, a., daniëls, f. j. a., ermakov, n., valachovič, m., pignatti, s., rodwell, j. s., pallas, j., capelo, j., weber, h. e., lysenko, t., solomesh, a., dimopolous, p., acta bot. croat. 78 (2), 2019 155 acta bot. croat. 78 (2), 155–163, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0013 issn 0365-0588 eissn 1847-8476 phytosociology and biodiversity patterns of annual wetland communities of pyriatynskyi national nature park (poltava oblast, ukraine) oleksii kovalenko*, mariia kalista department of botany, national museum of natural history of nas of ukraine, bohdana khmelnytskoho 15, 01607 кyiv, ukraine abstract – the results of a comparative structural analysis of the annual wetland herb vegetation syntaxa (class isoëto-nano-juncetea) of pyriatynskyi national nature park (poltava oblast, ukraine) are presented. the systematic, biomorphological, ecological and geographical structures of syntaxa were studied using cluster, discriminant and factor analysis. the principal conformity of the floristic similarity dendrogram and the previously developed classification scheme were analysed. it was revealed that the leading factor of coenosis development is the soil moisture, while most parameters of these ecotopes are constant. the critical differentiation of nano-cyperion and eleocharition ovatae alliances and the legitimacy of the recognition of the radiolion linoidis alliance and polygono recti-juncetum juzepczukii association as separate syntaxa of the main ranks are emphasized. keywords: analysis of syntaxa, annual wetland, herb vegetation, isoëto-nano-juncetea vegetation, syntaxonomy * corresponding author e-mail: corydalis@ukr.net, crambe@ukr.net introduction one of the problems of modern phytosociology is the harmonization of regional systems of classification and monographic processing of some classes of vegetation. this fully applies to dynamic and compositionally complex communities of the annual wetland herb vegetation belonging to the class isoëto-nano-juncetea br.-bl. et tx. ex br.-bl. et al. 1952. despite more than a century of research in western and central europe (ellenberg 1982), as well as recent intensive research in siberia and eastern europe (taran 1993, 1995, 2001, senchylo and goncharenko 2008a, b, shapoval 2006, golub et al. 2007, kovalenko 2014), there is considerable confusion in the definition and interpretation of the main associations and alliances of this class. to build a stable hierarchical system of isoëto-nano-juncetea, we propose to use a complex comparative and structural analysis of determined syntaxa as multiparameter systems (senchylo and goncharenko 2008a). materials and methods we studied the annual wetland herb vegetation of the pyriatynskyi national nature park (nnp) (poltava oblast, ukraine) in 2010–2012. we have already proposed its syntaxonomical scheme and described 2 associations as new for science (kovalenko 2014). the floristic similarity of the syntaxa determined was counted using the index of inclusion of kulchytsky (semkin and komarova 1977). peculiarities of ecological differentiation of communities of the annual wetland herb vegetation were assessed by the method of synphytoindication (didukh and pliuta 1994), using the ecological scales of didukh (2011). the comparative and structural analysis of the syntaxa was carried out according to 5 groups of parameters: taxonomical, biomorphological (the main type of biomorph, the character of seasonal vegetation, the structure of the above-ground and underground shoots, the type of the root system, the type of pubescence, the methods of pollination and dissemination), the chorological (type of range, chorionic element and activity), ecological and coenotical (tolerance for climatic and edaphic factors, ecological strategy) block and the block of transformation of flora (tolerance for anthropogenic factors, resistance to conditions of urbanization and hemeroby). the basic principles of the allocation of these categories are justified in the works of novosad and krytska (2010) and also senchylo and goncharenko (2008a). kovalenko o., kalista m. 156 acta bot. croat. 78 (2), 2019 in our analysis we used the parameter “purity of syntaxon”, which means the percentage ratio of the diagnostic species to the general list of flora. it indicates a role of the ephemeral annuals in the composition of communities that are critically important for the determination of the syntaxa of the isoëto-nano-juncetea class. for factor analysis, we used 121 non-discrete parameters of the systematic, biomorphological, ecological-coenotical and geographical structures. this data set includes floristic information and results of ordination by the phytoindication method. the data obtained were used for cluster, canonical discriminant and factor analysis in the statistica 7.0 program (statsoft). results annual wetland communities of pyriatynskyi national nature park are presented by 1 order, 4 alliances and 7 associations: cl. isoëto-nano-juncetea br.-bl. et tx. ex br.-bl. et al. 1952 ord. nano-cyperetalia klika 1935 all. 1. nano-cyperion flavescentis koch ex libbert 1932 ass. 1. cyperetum flavescentis koch ex aichinger 1933 2. juncetum bufonii felföldy 1942 all. 2. eleocharition ovatae philippi 1968 ass. 3. cyperetum micheliani horvatić 1931 4. eleocharito acicularis–limoselletum aquaticae wendelberger-zelinka 1952 all. 3. radiolion linoidis pietsch 1973 ass. 5. psammophiliello–juncetum nastanthi kovalenko 2014 all. 4. verbenion supinae slavnič 1951 ass. 6. polygono recti–juncetum juzepczukii kovalenko 2014 7. eragrostidetum suaveolentis golub et al. 2007 the analysis of the floristic similarity of syntaxa (online suppl. tab. 1) with subsequent clustering is followed by dendrogram (fig. 1). the branching of trees did not change significantly depending on the chosen binding parameters. the first clade was formed by associations of verbenion supinae and radiolion linoidis alliances. the associations of verbenion supinae are the most similar to each other by floristic criteria. psammophiliello–juncetum nastanthi association is well-separated from this alliance. the second cluster of the dendrogram was formed by associations belonging to two alliances (nano-cyperion flavescentis and eleocharition ovatae). the basic coeofloristic proportions are the parameter that shows the systematic richness of the syntaxa (number of genera and species per one family). this parameter is similar for the determined syntaxa of isoëto-nano-juncetea (tab. 1). tab. 1. coenofloristic proportions of the syntaxa of the isoëto-nano-juncetea of pyriatynskyi national nature park.the basic proportions are parameters that indicate an average number of species in families and average number of genera in families in floristic composition. generic coefficient is an average number of species in all genera. magnoliopsida to liliopsida ratio is a ratio of monocots to dicots. species richness of coenoses is a parameter that indicates a number of species in one relevé with average standard deviation as numbers. the “purity of syntaxon” is a ratio of diagnostic species within the floral composition (first number indicates absolute number of community diagnostic species; the second number indicates a percentage contribution). syntaxon basic proportions generic coefficient magnoliopsida to liliopsida ratio species richness coenoses «purity syntaxon» (%) cyperetum flavescentis 1 : 1.47 : 1.73 1.18 2 11.08±3 10; 38.46 juncetum bufonii 1 : 1.27 : 1.64 1.29 2.6 6.64±3 13; 72.22 cyperetum micheliani 1 : 1.38 : 1.75 1.27 3.67 10.64±7.81 21; 75 eleocharito-limoselletum 1 : 1.76 : 2.44 1.39 2.42 7.55±5.45 16; 26.23 psammophiliello-juncetum 1 : 1.08 : 1.33 1.23 2.2 9.26±3.74 12; 75 polygono-juncetum 1 : 2.4 : 3.2 1.33 3.2 9±5 13; 40.63 eragrostidetum suaveolentis 1 : 1.62 : 1.77 1.10 3 8.46±2.54 17; 73.91 nano-cyperion 1 : 1.39 : 1.94 1.4 1.5 8.96±4.04 17; 48.57 eleocharitionovatae 1 : 1.71 : 2.54 1.48 2.63 7.88±7.62 23; 32.39 radiolion linoidis 1 : 1.08 : 1.33 1.23 2.2 9.26±3.74 12; 75 verbenion supinae 1 : 2.38 : 2.93 1.33 2.73 9.12±3.78 18; 43.9 nano-cyperetalia and isoëto-nano-juncetea 1 : 2.41 : 3.89 1.62 1.94 8.74±8.63 41; 39.05 fig. 1. dendrogram of the floristic similarity of associations of the annual wetland herb vegetation of pyriatynskyi nnp: 1 – cyperetum flavescentis; 2 – juncetum bufonii; 3 – cyperetum micheliani; 4 – eleocharito acicularis–limoselletum aquaticae; 5 – psammophiliello–juncetum nastanthi; 6 – polygono recti–juncetum juzepczukii; 7 – eragrostidetum suaveolentis. annual wetland communities of pyriatynskyi national park acta bot. croat. 78 (2), 2019 157 the percentage ratio of diagnostic species to the general list of flora (“purity of syntaxon”) negatively correlates with the duration of vegetation of communities, reaching the minimum values for the most trivial association of eleocharito-limoselletum (on-line suppl. fig. 1). the genus juncus dominates in the annual wetland herb vegetation of pyriatynskyi nnp (6 species, 5.7%). this also corresponds to the phytocoenotic role of its representatives in characteristic coenoses. the carex genus in the communities studied is represented by 5 species (4.77%), but with no typical ephemerals among them. the third position is occupied by the temperate-tropical persicaria genus (4, 3.81%), whose representatives are particularly active in the coenoses of the eleocharition ovatae alliance. also, four species in the phytocoenoses of the annual wetland herb vegetation belong to the polygonum genus, which has the highest constancy in the verbenion supinae alliance. among the morphological traits (tab. 2) of the floristic composition of the class isoëto-nano-juncetea, monoecious plants (including facultative ones) take the first place, slightly ahead of the relative number of herbaceous perennial plants. an almost equal ratio of these two life forms is characteristic for the communities cyperetum flavescentis and eleocharito-limoselletum, and also in general for the unions of nano-cyperion and eleocharion ovatae, while the remaining syntaxa have a considerable dominance of annuals. perennial plants, as a rule, do not undergo a full cycle of development and are represented mainly by pregenerative age states in communities of the annual wetland herb vegetation. according to the type of above-ground shoots, the main group is composed of erosulate plants; only in the association cyperetum flavescentis do semirosette forms have the main position. a distinctive feature of the communities isoëto-nano-juncetea is the high share of species with an ephemeral type of vegetation. with decreasing humidity of specific habitats, the relative participation of summer-green plants increases, reaching the maximum values in the communities of the verbenion supinae. the plants with fibrous root systems are more common in mesohygrophytic conditions, whereas with increasing xerophyticity the specific gravity of the tap root species increases. in the type of underground shoots, plants without rhizomes predominate, although the involvement of the short rhizome species is noticeable in the composition of the vegetative cover of hygrophytic and mesophytic alliances of nano-cyperion, eleocharion ovatae and radiolion linoidis. most annual wetland herbs are insect-pollinated or wind-pollinated plants (fig. 2a), however, self-pollination has a significant part in the phytocoenosis of the class. the specific gravity of the latter increases in the most extreme habitats typical of the associations juncetum bufonii and psammophiliello-juncetum. the leading ways of diasporas dispersal are anemochory, barochory, zoochory (mainly epizoochory) and hydrochory (fig. 2b). the ratio of representatives of these dissemination strategies does not change significantly in each individual syntaxon. seasonal fluctuations of moisture conditions from hygrophilic to xeromesophilic are reflected in the distribution of species according to the degree of pubescence (fig. 2c). in all coenoses, the group of non-pubescent plants is dominant, and in communities with the shortest period of flooding (radiolion linoidis and verbenion supinae), the proportion of heavily pubescent species is increased, while the prolonged phase of moistening promotes the groups of medium-inferior species. tab. 2. morphological traits structure of the annual wetland herb vegetation syntaxa of pyriatynskyi national nature park. syntaxon life form %) aboveground shoots (%) root system (%) phenological groups (%) underground shoots (%) a nn ua ls pe re nn ia ls tr ee s r os et te se m ir os et te eo su la te te ta pr oo t fi br ou se ep he m er al s su m m er -g re en su m m er -w in te rgr ee n w ith ou t r hi zo m e lo ng -r hi zo m e sh or t r hi zo m e c au de x cyperetum flavescentis 50.0 50.0 0.0 11.5 53.9 34.6 38.5 61.5 62.3 29.2 38.5 50.0 19.2 23.1 7.7 juncetum bufonii 77.8 22.2 0.0 16.7 38.9 44.4 66.7 33.3 66.7 22.2 11.1 72.2 0.0 22.2 5.6 cyperetum micheliani 78.6 21.4 0.0 7.1 32.1 60.8 60.7 39.3 71.4 21.4 7.1 78.5 3.6 14.3 3.6 eleocharito-limoselletum 55.7 42.7 1.6 9.8 39.4 50.8 50.0 50.0 31.1 42.7 26.2 47.6 18.0 24.6 9.8 psammophiliello-juncetum 75.0 25.0 0.0 18.8 25.0 56.2 50.0 50.0 43.8 37.5 18.7 75.0 0.0 25.0 0.0 polygono-juncetum 71.8 21.9 6.3 0.0 58.5 41.5 53.1 46.9 40.6 501 9.3 78.1 3.1 9.4 9.4 eragrostidetum suaveolentis 87.0 8.7 4.3 0.0 43.5 56.5 60.9 39.1 56.5 34.8 8.7 91.4 4.3 4.4 0.0 nano-cyperion 54.3 45.7 0.0 11.4 48.6 40.0 48.6 51.4 42.9 25.7 31.4 54.2 14.3 22.9 8.6 eleocharition ovatae 51.4 47.1 1.5 58.6 35.7 45.7 50.0 50.0 30.0 45.7 24.3 52.8 15.7 22.9 8.6 radiolion linoidis 75.0 25.0 0.0 18.8 25.0 56.2 50.0 50.0 43.7 37.5 18.8 75.0 0.0 25.0 0.0 verbenion supinae 77.8 17.8 4.4 0.0 44.5 55.5 66.7 33.3 37.8 48.9 13.3 82.2 4.4 6.7 6.7 nano-cyperetalia and isoëto-nano-juncetea 55.8 42.3 1.9 6.7 48.2 45.1 51.0 49.0 29.8 26.0 44.2 58.7 14.4 20.2 6.7 kovalenko o., kalista m. 158 acta bot. croat. 78 (2), 2019 soil humidity has the leading value among factors characterizing edaphic conditions (didukh 2011). according to it, the highest range of amplitude has eleocharito acicularis– limoselletum aquaticae. communities of these associations are common for the national park and forms in habitats with different regimes of humidity. associations cyperetum flavescentis, juncetum bufonii and cyperetum micheliani have similar values of these factors. the vegetation of the alliances radiolion linoides and verbenion supinae are presented on the most xeric soils. the communities of eragostidetum suaveolentis are clearly different from polygono-juncetum according to their humidity regime (fig. 3a). the variability of moisture is very important factor for annual wetland communities. all associations excluding eragostidetum suaveolentis and polygono-juncetum have a similar range of values. dry soils in the habitats of verbenion supinae are the main reason for the invasion of species with adaptation to hydrocontrastophily (irregular wetting of soil layer) in characteristic communities (fig. 3b). according to the relations of associations to soil acidity and total salt regime we can clearly see the differentiation of cyperetum flavescentis due to existing soils with sodium sulphate salting (figs. 3c, d). this association has a higher rank of carbonate content in the soil too (fig. 3e). also, radiolion linoides communities grow on chernozems, enriched enough with carbonates. almost all associations have similar ranks of nitrogen content in soil. the most nitrophilic syntaxa are cyperetum flavescentis and eleocharito acicularis–limoselletum aquaticae. on the other hand, communities of eragostidetum suaveolentis include a lot of plants that grow on poor mineralnitrogen oligotrophic soils (fig. 3f ). soil aeration limits the distribution of many ephemeral annual herbs. the highest ranking (9.37) is eleocharito acicularis–limoselletum aquaticae, the lowest rank (6.44) is characteristic for eragostidetum suaveolentis. other syntaxa are very similar (fig. 4a) according to this parameter (7.33–8.67). according to the thermal climate and humidity, all of the associations demonstrated no significant value of variation (figs. 4b, c). other climate parameters are the source of differentiation of isoëto-nano-juncetea syntaxa. in juncetum bufonii and associations of radiolion and verbenion subcontinental species prevail. in the other syntaxa, we determined the dominance of hemi-oceanic elements (fig. 4d). by the parameter of the cryo-climate, we identified cyperetum flavescentis as the most cryophilic association due its northern area of distribution (fig. 4e). the regimes of light in characteristic habitats of the annual wetland vegetation are similar. most syntaxa have strict ranges of amplitude by this parameter. only communities of polygono-juncetum have lower value of light due their development in partly woody areas (fig. 4f ). the canonical discriminant analysis showed a clear dispersion of the cyperetum flavescentis, cyperetum micheliani, eleocharito-limoselletum and eragrostidetum associations (fig. 5). the remaining associations from three different alliances demonstrated a significant overlap in the amplitudes of the whole set of ecological parameters studied, as already mentioned above. the chorological core of the class in the national park is wide-ranging progressive species with holarctic (22; 31.73%), cosmopolitan (22, 21.15%) and palaearctic (12, 11.53%) types of areas. the proportion of geographic elements has a similarity in all syntaxa, with the exception of the verebenion. in this alliance, cosmopolitan species dominate. anthropogenic transformation of communities of the annual wetland vegetation ephemeretum is manifested in the significant representation in the general list of flora of euhemerobes (27; 25.96%) and mesohemerobes (40; 38.46%). euhemerobes are particularly active in the coenoses of the association eragrostidetum suaveolentis, but in more humid conditions they are rare. fig. 2. the types of pollination (a), dissemination (b) and pubescence (c) of syntaxa isoëto-nano-juncetea plants. 1 – cyperetum flavescentis; 2 – juncetum bufonii; 3 – cyperetum micheliani; 4 – eleocharito acicularis–limoselletum aquaticae; 5 – psammophiliello–juncetum nastanthi; 6 – polygono recti–juncetum juzepczukii; 7 – eragrostidetum suaveolentis; all.1 – nano-cyperion; all.2 – eleocharition ovatae; all.3. – verbenion supinae. contribution of different species (%) are presented. annual wetland communities of pyriatynskyi national park acta bot. croat. 78 (2), 2019 159 the ratio of urbanophiles, urbanoneutral and urbanophobic species is correlated with the previous indicator. among the apophytes, the prevalent hemiapophytes (29; 54.71%), euapophytes and random apophytes in all syntaxa are represented uniformly. the lowest percentage of alien species was noted for the alliances of nano-cyperion and radiolion lionoidis (5.50% and 6.25% respectively), while similar indicators for the alliances eleocharition ovatae (12.86%) and verbenion supinae (21.95%) are significantly higher. the absolute majority of alien species are coenophytes (10, 66.67%) predominantly of north american origin (8, 53.33%). the factor analysis by the principal components method (fig. 6) showed, on one hand, a significant affinity for the associations cyperetum micheliani and juncetum bufonii, which are discrete in floral, physiognomic and ecologifig. 3. box-whisker plot for distribution of associations of the class isoëto-nano-juncetea by: a) soil humidity (hd); b) moisture (fh); c) acidity (rc); d) total salt regime (sl); e) carbonate content in soil (ca); f ) nitrogen content in soil (nt). 1 – cyperetum flavescentis; 2 – juncetum bufonii; 3 – cyperetum micheliani; 4 – eleocharito acicularis–limoselletum aquaticae; 5 – psammophiliello–juncetum nastanthi; 6 – polygono recti–juncetum juzepczukii; 7 -eragrostidetum suaveolentis. box-whisker plot: centre line denotes median value, the box encloses the inner two quartiles (25th and 75th percentile), and the whiskers display the 10th and 90th percentile. kovalenko o., kalista m. 160 acta bot. croat. 78 (2), 2019 cal relationships while on the other hand it clearly delineated the associations of the verbenion supinae association, which were close by the same criteria. discussion results of cluster analysis of syntaxa correspond strongly to the obtained scheme of classification. however, the association juncetum bufonii did not unite with the cyperetum flavescentis, as might be expected according to the syntaxofig. 4. box-whisker plot for distribution of associations of the class isoëto-nano-juncetea by: a) aeration (ae); b) thermal climate (tm); c) humidity (om); d) continental climate (kn); e) cryoclimate (cr); f ) light in community (lc). 1 – cyperetum flavescentis; 2 – juncetum bufonii; 3 – cyperetum micheliani; 4 – eleocharito acicularis–limoselletum aquaticae; 5 – psammophiliello–juncetum nastanthi; 6 – polygono recti–juncetum juzepczukii; 7 – eragrostidetum suaveolentis. box-whisker plot: centre line denotes median value, the box encloses the inner two quartiles (25th and 75th percentile), and the whiskers display the 10th and 90th percentile. nomic position. juncetum bufonii has a high level of floristic similarity with associations of the eleocharition ovatae alliance. on one hand, this result confirms the need for the recognition of the separate alliance juncion bufonii philippi 1968 or, by contrast, a wider understanding of the nano-cyperion alliance, as suggested by a number of authors (borhidi 2003, sanda et al. 2008), but without inclusion in its communities of radiolion linoidis. in our opinion, this discrepancy between the dendrogram and the proposed syntaxonomic scheme is due to the regional difference of juncetum bufonii annual wetland communities of pyriatynskyi national park acta bot. croat. 78 (2), 2019 161 communities with their main distribution area in the north, as well as their spatio-functional contacts with the phytocoenoses of the eleocharion ovatae alliance. coenofloristic proportion diagnoses a rather low overall level of species diversity, which is due to the unique ecological parameters of the characteristic communities of the class. due to the increase of the moisture gradient, the coenotic role and representation of monocots in the communities also increase. the spectrum of the leading families of the annual wetland herb vegetation differs significantly from analogous relationships for other types of vegetation. in addition, if the dominance of the representatives of asteraceae and poaceae is the background for most of the coenofloras of the holarctic temperate zone, and the increasing role of cyperaceae is typical for communities of phragmiti-magno-caricetea and scheuchzerio-caricetea nigrae classes, then the high positions of polygonaceae and juncaceae are a distinctive feature for isoëtonano-juncetea. the ecological analysis of isoëto-nano-juncetea vegetation showed the clearest differentiation of associations according to the soil moisture factor. the nano-cyperion alliance is traditionally considered the most hygrophytic. however, we find communities of cyperetum micheliani and eleocharito-limoselletum associations in conditions with higher soil moisture. the answer to whether this is a regional specificity, or yet more evidence in favour of an expanded understanding of the nano-cyperion will be given by detailed comparative studies of the ecological amplitude of the union throughout the range. according to substrate chemistry, most associations did not show significant differences. in the general background, only the cyperetum flavescentis association, genetically linked to weakly saline ecotopes, stands out. concerning the factors of thermo-regime and continentality, the association eragrostidetum suaveolentis, described from the volga-akatubinsk annual wetland herb vegetation, is clearly differentiated, and found its western boundary of distribution here. the coenotic differentiation of the syntaxa of annual wetland vegetation is formed mainly due to the moisture factor against the background of the constancy of the edaphic and climatic conditions of the habitats. the communities of class isoëto-nano-juncetea consist predominantly of species that have a wide eco-coenotic amplitude (generalists and semigeneralists according to novosad and krytska (2010)). the main fragments of their coenotic distribution are in the space of classes phragmiti-magno-caricetea, molinio-arrheneteretea and scorzonero-juncetea. it should also be noted that in communities of the annual wetland vegetation, such species are represented either by special forms or by the pregenerative age stages. the role of species with narrow coenotic amplitude (specialists and semi-specialists) increases in the spatially restricted communities of the alliances nano-cyperion and verbenion. the low value of total coverage and the mofig. 5. differentiation of phytocoenoses of the annual wetland herbs vegetation in the space of two discriminant functions according to the data of ecological analysis. fig. 6. distribution the annual wetland herb vegetation associations in the multifactorial space. kovalenko o., kalista m. 162 acta bot. croat. 78 (2), 2019 saic of the grass stand and the weakly expressed dominance in the studied communities contribute to the active introduction of alien species (alien species account for 14.42%), among which the most constant are bidens frondosa, conyza canadensis and iva xantiifolia. some of the alien species have a high coenotic value and are therefore considered as diagnostic species, for example, juncus tenuis for the alliance, nano-cyperion, epilobium adenocaulon and digitaria ischaemum for verbenion supinae. it is interesting that such a status for them is also characteristic within the primary areas (brullo and minisalle 1998, šumberová 2011). this analysis confirms the vulnerability of the annual wetland communities to the factor of human pressure, as well as the abundant evidence of the radical transformation and elimination of isoëto-nano-juncetea phytocoenoses in western and central europe (korneck 1960, philippi 1968, pietsch 1973, popiella 1996, šumberová 2011). the results of factor analysis are additionally against synonymization of polygono-juncetum with eragrostidetum suaveolentis as some authors proposed (dubyna et al. 2016). the problem of determining the ceonoses of nano-cyperion, radiolion and eleocharition is obvious, at least at the regional level. the peculiarity of the association eleocharito-limoselletum is explained by the largest representation, and as a consequence, by the diversity of its communities on the territory of pyriatynskyi nnp. conclusions analysis of the syntaxa of the annual wetlands vegetation as multiparameter systems revealed a complex picture of their differentiation in a system of floral, ecological and geographic coordinates. the dendrogram of the floristic similarity of phytocoenes largely coincides with the early classification of vegetation of the class isoëto-nano-juncetea developed for pyriatynskyi nnp. the systematic and biomorphological structure of the determined syntaxa is quite original due to the ecological and phenological uniqueness of the studied communities. according to the data of the synphytoindication, the leading factors of the variety of annual wetland communities are the soil, humidity and to a lesser extent, salt regime against the background of the constancy of the remaining ecological parameters. the geographic structure of the lower-level syntaxa is quite conservative and coincides with that for the class. anthropogenic pressure on the communities of isoëto-nano-juncetea cases increasing role of anthropophilic elements in the composition of the vegetation cover. as a consequence, the development of special measures for the protection of this unique type of vegetation is necessary. a complex comparative study of isoëtonano-juncetea syntaxa analysis pointed on one hand to the critical nature of the distinction between the nano-cyperion and eleocharion ovatae alliances and on the other hand confirmed the acceptability of the radiolion linoidis as a separate syntaxon, and the legitimacy of distinguishing associations eragrostidetum suaveolentis and polygono-juncetum. in view of the above, we think that a large-scale study of the syntaxa of the annual wetland vegetation allows us to make a stable system of the isoëto-nano-juncetea class and sheds light on many theoretical and practical problems in the classification of vegetation. references borhidi, a., 2003: magyarország növénytársulásai. akadémiai kiadó, budapest. brullo, s., minisalle, p., 1998: considerazioni sintassonomische sulla classe isoëto-nanojuncetea. itinerra geobotanica 11, 263–290. didukh, y.p., 2011: ecological scales for the species of ukrainian flora and their use in synphytoindification. phytosociocentre, kyiv. didukh, y.p., pliuta, p.h., 1994: phytoindication of ecological factors. naukova dumka, kyiv (in ukrainian). dubyna, d.v., dziuba, t.p., iemelianova, s.м., davydov, d.a., 2016: contemporary state and actual tasks of protection of pioneer vegetation in ukraine. ukrainian botanical journal 73, 11–20 (in ukrainian). ellenberg, h., 1982: vegetatiom mitte leuropas mit den alpen in ökologischer sicht. stuttgart, 794–802. golub, v.b., dubyna, v.b., kuzmina, e.v., 2007: communities eragrostidetum suaveolentis ass. nova in the valley of the lower volga. samara bend 16, 532–537 (in russian). korneck, d., 1960: beobachtungen an zwergbinsengesellschaften im jahre 1959. beiträge zur naturkundlichen forschung in südwestdeutschland 19, 101–110. kovalenko, o.a., 2014: syntaxonomy of flood-plain ephemerous vegetation communities (isoëto-nano-juncetea br.-bl. et tx. ex br.-bl. et al. 1952) of national nature park “pyriatynskyi” (poltava oblast, ukraine). botanical journal 99, 34–60 (in russian). novosad, v.v., krytska, l.i., 2010: phytoand flora diversity of the middle dniester banks vascular plants, 1. phyton, kyiv. (in ukrainian). philippi, g., 1968: zur kenntnis der zwergbingesellschaften (ordnung der cyperetalia fusci) des oberrheingebiet. veroff. landestelle natursch. u. landschaftspfelge baden-württemberg 3, 68–130. pietsch, w., 1973: beitrag zur gliederung der europäischen zwergbinsengesellschaften (isoëto-nanojuncetea br.-bl. et tx. 1943). vegetatio 28, 401–438. popiella, a., 1996: occurrence of isoeto-nanojuncetea associations in poland. fragmenta floristica et geobotanica polonica 3, 289–310 (in polish). sanda, v., öllerer, k., burescu, p., 2008: phytocoenoses in romania: syntaxonomy, structure, dynamics and evolution. ars docendi, bucharest (in romanian). semkin, b.i., komarova, t.a., 1977: analysis of phytocoenotic descriptions using inclusion measures (using plant communities in the valley of the amgoumi river in chukotka). botanical journal 62, 54–36 (in russian). senchylo, o.o., goncharenko, i.v., 2008a: isoeto-nanojuncetea of the alluvial sand outcrops of the forest-steppe dnipro. bulletin of donetsk national university. series a: natural sciences 5, 334–343 (in ukrainian). senchylo, o.o., goncharenko, i.v., 2008b: methodology of characterizing of the syntaxes as multiparameter systems. bulletin of donetsk national university. series a: natural sciences 2, 344–356 (in ukrainian). annual wetland communities of pyriatynskyi national park acta bot. croat. 78 (2), 2019 163 shapoval, v. v., 2006: on syntaxonomy of vegetation depressions of the left bank of the lower dnipro. classes: isoëto-nanojuncetea br.-bl. et r. tx. ex westhoff et al. 1946, molinio-arrhenateretea r. tx. 1937 and festuco-brometea br.-bl. et r. tx. in br.-bl. 1949. news biosphere reserve “askania nova” 8, 15–48 (in ukrainian). šumberová, k., 2011: class isoëto-nano-juncetea. in: chytrý m. (ed.), vegetation of the czech republic 3. aquatic and wetland vegetation, 312–315. academia, praha (in czech). taran, g.s., 1993: on syntaxonomy of black irtysh floodplain ephemerous vegetation. siberian journal of biology 5, 79–84 (in russian). taran, g.s., 1995: a little-known vegetation class of the former ussr-flood plain ephemeretum (isoëto-nanojuncetea br.-bl. et tx. 1943). siberian journal of ecology 2, 373–382 (in russian). taran, g.s., 2001: association cypero-limoselletum (oberd. 1957) korneck 1960 (isoëto-nanojuncetea) in the floodplain of the middle ob river. vegetation of russia 1, 43–56 (in russian). social news the sixth croatian botanical symposium, held in zagreb, croatia (august 30–september 1, 2019) the sixth croatian botanical symposium, organized by the croatian botanical society, was held in the esplanade hotel in zagreb, croatia, from 30th august to 1st september 2019. the program included 26 oral and 40 poster presentations, with the total number of authors being 205. alongside croatian authors, scientists from other countries (austria, albania, hungary, kosovo, serbia, slovenia and the united kingdom) also participated. abstracts of all presentations are included in the book of abstracts which can be found on the symposium web-page. our invited plenary speakers were professor marko sabovljević from belgrade (serbia) and drs. sanja kovačić, ivana rešetnik, jadranka stojanovski and dunja šamec from zagreb. oral presentations by the croatian phycology section were organized within the 7th european phycological congress led by professor zrinka ljubešić and the croatian botanical society (zagreb, august 25–30, 2019). the greatest number of presentations belonged to the fields of taxonomic and plant systematic studies and floristic and vegetational studies. contributions included floristic mapping, plant recolonization, notes on rare and alien flora and taxa new to science, phylogeography of plant taxa in the amphi-adriatic area and the balkans, vegetation of forests, dry grasslands, tufa and saltern, archaeobotanical research and themes covered in the disciplines of didactics. presentation of the flora croatica (vols. 1–3: vascular flora of the republic of croatia; vol. 4: the excursion flora) by professor toni nikolić, faculty of science, university of zagreb, and published by alfa d.d, zagreb, was also organized. this is a long-awaited opus which, as a complete presentation of croatian vascular flora, will have a valuable impact on education and both the popular and professional fields of science. the symposium was accompanied by several events aimed at addressing the current state and future perspective of the flora croatica database. the round table collections was also organized to highlight the problems of managing collections and their financing in croatia and neighbouring countries. finally, during the closing remarks, the focus was on the broad topics of excellence, research, training, skills and mobility. all participants of the sixth croatian botanical symposium enjoyed a one-day trip to the mrežnica river on 1st september 2019. professor nenad jasprica croatian botanical society, president participants of the sixth croatian botanical symposium, zagreb, croatia (august 30–september 1, 2019). acta bot. croat. 78 (2), 2019 s1 acta bot. croat. 78 (1), 2019 91 acta bot. croat. 78 (1), 91–94, 2019 coden: abcra 25 doi: 10.2478/botcro-2018-0009 issn 0365-0588 eissn 1847-8476 a new addition to the alien flora of tunisia, amaranthus spinosus l. (amaranthaceae s.l.), with notes on a. diacanthus raf. duilio iamonico1*, ridha el mokni2,3 1 university of rome sapienza, department pdta, section environment and landscape, via flaminia 72, 00196 rome, italy 2 laboratory of botany and plant ecology, faculty of sciences of bizerta, university of carthage, jarzouna-7021, bizerta, tunisia 3 faculty of pharmacy of monastir, university of monastir, bp. n° 207, avenue avicenna, monastir-5000, tunisia abstract – amaranthus spinosus l. (amaranthaceae s.l.), a species native to the neotropics, has been found in four localities (bizerta, bir bouregba, hammamet, and nabeul) of n. tunisia. our discovery represents the first record at national level, and the second one for n. africa. morphological characters and ecological data are given. nomenclatural notes are provided for the name a. diacanthus, which was regarded by some authors as heterotypic synonym of a. spinosus. a neotype is designated in the present paper based on a specimen preserved at lsu. keywords: alien species, amaranthus, neophyte, typification * corresponding author, e-mail: d.iamonico@yahoo.it introduction the genus amaranthus l. (amaranthaceae juss.) comprises about 70 mostly annual, monoecious and dioecious species, of which approximately 40 are native in america (see e.g., costea et al. 2001, iamonico 2015a). some species of amaranthus are used as ornamentals and are able to escape from cultivation, having an economic impact mainly on agricultural systems (reduction of productivity and crop quality), while other taxa have an ecological impact on native flora and vegetation [e.g. a. tuberculatus (moq.) j.d.sauer in a riparian habitat] (see e.g., iamonico 2015b). the infrageneric classification of amaranthus still remains unresolved, lacking a comprehensive molecular study (see e.g., hernández-ledesma et al. 2015). on the basis of morphology and chorology, mosyakin and robertson (1996) recognized three subgenera: subg. acnida (l.) aellen ex k.r. robertson with three sections, subg. albersia (kunth) gren. et godr. with four sections, and subg. amaranthus, with three sections and two subsections. according to mosyakin and robertson (1996: 278), amaranthus spinosus l. is the only species belonging to amaranthus subg. amaranthus sect. centrusa griseb. the flora of africa (see sanbi 2012 and literature therein, iamonico 2015c and literature therein) currently includes amaranthus spinosus, which is recorded in many countries of central and southern parts of the continent, while in the north it occurs only in egypt. as part of an ongoing nomenclatural and taxonomic study on the genus amaranthus (see e.g., iamonico 2014a, 2014b, 2015a, 2015c, 2016a, 2016b, 2016c, iamonico and das 2014) and the tunisian amaranthaceae s. lat. (see e.g., iamonico and el mokni 2017, sukhorukov et al. 2016), some populations have been found that certainly refer to a. spinosus. the number of tunisian amaranthus species has thereby been increased, since this finding represents the first record for the flora of tunisia and the second for the flora of n. africa. moreover, the name amaranthus diacanthus, which some authors suggest be regarded as a synonym of a. spinosus (e.g., rojas-sandoval 2015, wunderlin et al. 2017), is here investigated from the nomenclatural point of view, as it is still untypified. materials and methods the work is based on an extensive analysis of literature, and examination of the specimens kept in the herbaria fi, hfla, linn, lsu, and ro (acronyms according to thiers 2019+), and in the herbarium of the faculty of pharmacy of short communication iamonico d., el mokni r. 92 acta bot. croat. 78 (1), 2019 monastir and of the faculty of sciences of bizerta (not listed in index herbariorum), where the personal collections of r. el mokni are kept. the articles cited through the text agree with the shenzen code, icn (turland et al. 2018). results and discussion nomenclatural notes on amaranthus diacanthus rafinesque (1817: 31–32) described a. diacanthus and provided a short diagnosis. rafinesque (l.c.) also cited the flore louisinaise by robin (1807). the latter author (robin 1807: 372) listed an atriplex taxon, but he did not report any epithet. as a consequence, rafinesque (l.c.), probably on the basis of the detailed description made by robin (l.c.), proposed the plants occurring in louisiana (s-usa) as a new species. we have not been able to trace rafinesque’s material, which could be useful for typification purposes. we looked at material collected by c. c. robin in louisiana, which could be observed and used by c. s. rafinesque, to describe a. diacanthus. unfortunately, no robin specimen was traced [moreover, stafleu and cowan (1985: 812) reported “herbarium and types: unknown”]. since no original material seems to be extant, no lectotype can be designed, and neotypification is required (see arts. 9.2, 9.3, and 9.7). accordingly, we selected a specimen preserved at lsu (catalog number 83081) and collected in louisiana, in webster county. the lsu-83081 is here designated as the neotype of the name amaranthus diacanthus. concerning the identity of a. diacanthus, note that the diagnosis by rafinesque (1817) and the description of atriplex sp. made by robin (1807) include an important morphological character [“...glomerulis bispinosis...” by rafinesque (1807), and “...les paques de fleurs des sisselles accompagnés de deux épines divergentes...” by robin (1807)]: the occurrence of a spine-like feature at the bases of the floral glomerules [according to costea and tardif (2003) these structures are actually the bracts of the first flower in the first cyme of the glomerule which metamorphoses in the early phase of the onthogeny]. this structure was the basis for the selection of the neotype, representing a unique feature in the genus amaranthus, characterizing the species currently known as a. spinosus (see e.g., bojian et al. 2003, mosyakin and robertson 2003, iamonico 2015a). as a consequence, these two names can be treated as heterotypic synonyms, the linnaean having nomenclatural priority (1753 vs. 1817). taxonomic treatment amaranthus spinosus l., sp. pl., 2: 991. 1753. lectotype (designated by fawcett and redle 1914: 103): herb. linnaeus, no. 1117.27 (linn!). image of the lectotype available at http://linnean-online.org/11653/). = amaranthus diacanthus raf., fl. ludov.: 31-32. 1817. neotype (here designated): u.s.a., louisiana, webster county, camp minden, 1.2 km e of jim davis cemetery, 0.3 km nw of richardson cemetery, 14 july 2003, l. blay, t. pazzaglia et n. hastings 906 (lsu-83081!). image of the neotype available at http://data.cyberfloralouisiana.com/lsu/). description – herbs 6–15 dm tall, monoecious, annual (therophyte). stems erect, glabrous brownish, branched. leaves green to reddish (on the upper surface), ovate to lanceolate, rhomboidal [(2.0–)2.5–6.5(–7.5) × (1.0–)2.5–4.0(–3.5) cm], with entire margins, apex obtuse or acute, mucronate, base cuneate, glabrous, petioled (petiole 1.5–8.5 cm long), associated to 2 spine-like structures (modified bracts), 7.0– 8.0 mm long. synflorescences terminal, panicle-like, the main florescence up to 15 cm long, usually green and axillary glomerules. floral bracts lanceolate (3.0–4.0 × 0.5−1.0 mm) as long as or shorter than the perianth, acute, awned, margin entire, glabrous. staminate flowers with 5 tepals, ovatelanceolate; stamens 5. pistillate flowers with 5 tepals, lanceolate–spathulate (3.5–4.0 × 0.9–1.0 mm), with usually obtuse and mucronate apex; stigmas 3–4. fruit brown or yellowish, ellipsoidal (2.0–2.1 × 1.4–1.5 mm) as long as or slightly shorter than the perianth, smooth below, rugose on top, dehiscent. seed lenticular (about 0.9 mm in diameter), brown. phenology – flowering and fruiting time october-december. habitat – roadsides, public gardens, and cultivated areas with trees (e.g. citrus sp.), at 6–41 m a.s.l. fig. 1. habit of amaranthus spinosus l. (bar = 1 cm) at nabeul region in tunisia with detail of spine-like structures (left corner; bar = 4 mm) (photos by r. el mokni). amaranthus spinosus in tunisia acta bot. croat. 78 (1), 2019 93 distribution in tunisia – four populations (16-18 individuals in total) were found in the following localities: bizerta [three individuals (each about 150 cm tall) covering an area of 9 m2], bir bouregba [two individuals (each about 70 cm tall) covering 2 m2], hammamet [three-four individuals (each about 90 cm tall covering 4 m2], and nabeul [eight-nine individuals (each about 60 cm tall) covering 25 m2]. native range and status of naturalization in tunisia – a. spinosus is a neophyte species native to the neotropics, while it is an alien in many countries of europe (iamonico 2015c), c. and s. africa (sanbi 2012), n. and e. australia (atlas living of australia 2016), and asia (see e.g., townsend 1974, bojian et al. 2003). concerning n. africa, this species was previously recorded only in egypt (see boulos 2009: 56, iamonico 2015c). the four populations of a. spinosus discovered in tunisia (see above “distribution in tunisia”) were observed from 2012 to 2016. although the plants seem to self-replace without direct human intervention, we prefer to consider a. spinosus as casual alien species in tunisia at the moment. chromosome number – a. spinosus is a diploid species with 2n = 34 (see e.g., greizerstein and poggio 1992, song et al. 2002, sheidai and mohammadzdeh 2008). specimina visa – tunisia: bizerta (north-bizerta, corniche), 37°19'31" n, 09°51'52" e, uncultivated land, 06 m a.s.l., 27 october 2013, r. el mokni (hfla, herb. univ. bizerta); nabeul (north-nabeul, cap-bon), 36°26'26" n, 10°42'25" e, roadsides, 08 m a.s.l., 09 december 2015, r. el mokni (herb. univ. monastir); nabeul (north-hammamet, cap-bon), 36°24'35" n, 10°36'06" e, cultivated land, 22 m a.s.l., 10 december 2016, r. el mokni (herb. univ. monastir); nabeul (bir bouregba, cap-bon), 36°25´39" n, 10°34'56" e, roadsides, 41 m a.s.l., 09 december 2015, r. el mokni (herb. univ. monastir). acknowledgements thanks to the directors and curators of all quoted official herbaria and colleagues who own the cited personal herbaria for their support during our visits, loan of specimens, photographs or for providing interesting information. references atlas of living australia 2016+: amaranthus spinosus l. retrived march 15, 2017 from [http://bie.ala.org.au/species/http:// id.biodiversity.org.au/node/apni/2886429]. bojian, b., clemants, s. e., borsch, t., 2003: amaranthus l. in: wu, z. y., raven p. h., hong, d. y. 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(eds.) 2018: international code of nomenclature for algae, fungi, and plants (shenzhen code) adopted by the nineteenth international botanical congress, shenzhen, china, july 2017. regnum vegetabile 159. koeltz botanical books, glashütten, germany. wunderlin, r. p., hansen b. f., franck a. r., essig, f. b. 2017. atlas of florida plants. retrieved april 13, 2017 from http://florida. plantatlas.usf.edu/ plant.aspx?id=1453. acta bot. croat. 80 (1), 2021 35 acta bot. croat. 80 (1), 35–42, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-010 issn 0365-0588 eissn 1847-8476 expression of genes for selected plant aminoacyl-trna synthetases in the abiotic stress jurica baranašić1#, anita mihalak1, ita gruić-sovulj1, nataša bauer2, jasmina rokov-plavec1* 1 university of zagreb, faculty of science, department of chemistry, division of biochemistry, horvatovac 102a, hr-10000 zagreb, croatia 2 university of zagreb, faculty of science, department of biology, division of molecular biology, horvatovac 102a, hr-10000 zagreb, croatia # current address: laboratory for advanced genomics, division of molecular medicine, ruđer bošković institute, bijenička 54, hr-10000 zagreb, croatia abstract – plants, as sessile organisms, have evolved intricate mechanisms to adapt to various environmental changes and challenges. because various types of stress trigger significant decrease in global translation rates we examined the stress-related expression of aminoacyl-trna synthetases (aarss), enzymes that participate in the first step of protein biosynthesis. we have analyzed promoters of genes encoding cytosolic seryl-trna synthetase (serrs), cytosolic aspartyl-trna synthetase (asprs) and cytosolic cysteinyl-trna synthetase (cysrs) in arabidopsis thaliana l., and examined serrs, asprs and cysrs gene expression in seedlings exposed to different abiotic stressors. although global translation levels are repressed by stress, our results show that plant aarss expression is not decreased by osmotic, salt and heavy metal/cadmium stress. moreover, during exposure to stress conditions we detected increased asprs and cysrs transcript levels. serrs gene expression did not change in stress conditions although participation of serrs in stress response could be regulated at the protein level. expression of the examined aars genes under stress correlated well with the length of their predicted promoters and the number of available binding sites for the stress related transcription factors. it thus appears that during stress it is important to keep steady state levels of aarss for translation of specific stress-related mrnas and furthermore to rapidly continue with translation when stress conditions cease. importantly, increased levels of plant aarss during stress may serve as a pool of aars proteins that can participate directly in stress responses through their noncanonical activities. keywords: aminoacyl-trna synthetase, abiotic stress, gene expression, heavy metal stress, osmotic stress, plant, salt stress, translation introduction as sessile organisms, plants have evolved elaborate mechanisms to adapt to adverse environmental changes and challenges. the molecular responses in plants to abiotic stresses are probably more advanced and prominent than in animals (qin et al. 2011). research on how plants adjust to various environmental stresses is crucial not only to plant biologists but also to agronomists, because abiotic stress has a particularly negative impact on crop production (cramer et al. 2011). the need to understand the mechanisms involved in plant abiotic stress responses and the generation of plants tolerant to stress has accordingly gained much attention in recent years (qin et al. 2011). in plants, as in all eukaryotes, gene expression reprogramming plays a pivotal role in the response to abiotic stress. this reprogramming is a highly complex process, including transcription, mrna processing, mrna transport and stability, translation or protein turnover; each participating but having a different weight in the final protein levels (yángüez et al. 2013). one of the earliest plant metabolic responses to abiotic stresses is the inhibition of protein biosynthesis and an increase in * corresponding author e-mail: rokov@chem.pmf.hr baranašić j., mihalak a., gruić-sovulj i., bauer n., rokov-plavec j. 36 acta bot. croat. 80 (1), 2021 synthetase (asprs) as a new master regulator of resistance to pathogens induced by beta-aminobutyric acid (baba). this non-proteinaceous amino acid is a well-known priming agent that protects many plants from various biotic and abiotic stresses. baba interferes with asprs canonical activity resulting in the activation of cellular defense mechanisms (luna et al. 2014). high throughput proteomic screen for proteins involved in the early responses of arabidopsis thaliana l. to oxidation stress due to cadmium exposure identified, among other proteins, several aminoacyl-trna synthetases (sarry et al. 2006). prompted by findings that various types of stress significantly decrease global translation rates (merchante et al. 2017, zhao et al. 2019) we sought to determine stress related expression levels of aarss, the crucial enzymes in protein biosynthesis. we have analyzed promoters of genes encoding cytosolic seryl-trna synthetase (serrs), cytosolic asprs and cytosolic cysteinyl-trna synthetase (cysrs), and examined serrs, asprs and cysrs gene expression in a. thaliana seedlings exposed to different abiotic stressors for 3, 6, 8 and 24 hours. although stress induces global translation repression, our results show that expression of plant aars genes is not decreased. moreover, during exposure to stress conditions we detected increased asprs and cysrs transcript levels, while serrs gene expression did not change. this indicates that aarss are required for translation of specific stress-related mrnas, as well as for resumption of translation after stress. furthermore, plant aarss may participate directly in plant stress responses through their noncanonical activities. materials and methods analysis of transcription factor binding sites in promoter region the occurrence of transcription factor (tf) binding site motifs in the promoter of a gene may be used to predict whether the gene is regulated by a specific tf. here we analyzed promoter sequences of genes for cytosolic serrs (at5g27470), cytosolic asprs (at4g31180) and cytosolic cysrs (at5g38830). epd, agris atcisdb and athamap databases were used for promoter analysis and detection of tf binding sites (yilmaz et al. 2011, hehl et al. 2016, dreos et al. 2017). the databases are suitable for a comprehensive analysis of genomic sequences for potential tf binding sites and provide information about experimentally validated cis-regulatory elements, as well as predicted motifs present in a specific promoter. plant material and stress conditions wild-type seeds of arabidopsis thaliana (ecotype columbia) were surface-sterilized for one minute in 70% (v/v) ethanol and then 10 minutes in 1% (w/v) izosan (pliva, croatia), rinsed 5 times with distilled water. seeds were planted on half-strength murashige and skoog (ms5519, sigma, usa), 0.5% (w/v) sucrose and 0.8% (w/v) agar medium. afchaperone levels controlling protein folding and processing. repression of global protein biosynthesis is often accompanied with selective translation of mrnas encoding proteins that are vital for cell survival and recovery from stress (liu and qian 2014, merchante et al. 2017). in the first step of protein biosynthesis, cells need to aminoacylate trnas with their respective amino acids in a process catalyzed by aminoacyl-trna synthetases (aarss) (perona and gruic-sovulj 2014). during protein biosynthesis on the ribosome, amino acid is transferred from the trna onto a growing peptide according to the genetic code. aminoacyl-trna synthetases are therefore housekeeping proteins that play an important role in the expression of genes to create proteins. besides that, aarss may be involved in a myriad of other cellular processes exerting their noncanonical function beyond translation (guo and schimmel 2013, mocibob et al. 2016). it is well documented that aarss participate in cellular stress responses in bacteria and metazoa. for example, vertebrate tyrosyl-trna synthetase has been shown to translocate to the nucleus during oxidative stress and protects against dna damage by activating transcription factor e2f1, which promotes expression of dna repair genes (wei et al. 2014, cao et al. 2017). during oxidative stress, phosphorylated methionyl-trna synthetase (metrs) enhances the mischarging of methionine on non-methionyl trnas. methionine carried by noncognate trnas is incorporated into growing polypeptides during translation and is used as scavanger of reactive oxygen species, protecting cells from oxidative damage and apoptosis (lee et al. 2014). during oxidative stress, oxidized phenylalanyl-trna synthetase (phers) exhibits increased proofreading activity against cytotoxic tyrosine isomers, noncognate amino acids that are increased during oxidative stress. increased proofreading of phers maintains high fidelity of translation even under conditions of oxidative stress (steiner et al. 2019). research on plant aarss has revealed their cellular localization (rokov-plavec et al. 2008, duchêne et al. 2009, kekez et al. 2016), substrate specificity (rokov et al. 1998, rokov and weygand-durasevic 1999, rokov-plavec et al. 2002, 2004, aldinger et al. 2012), fidelity (rokov-plavec et al. 2013, lee et al. 2016, hoffman et al. 2019) and protein interactors (yang et al. 2018, kekez et al. 2019). thus far, only one crystal structure of plant aars has been reported (kekez et al. 2019). as enzymes essential for protein biosynthesis in the cytosol, chloroplast and mitochondria, plant aarss appeared important for plant growth and development, and disruption of their function was shown to be either lethal or cause severe defects early in plant development (berg et al. 2005, kim et al. 2005, kitagawa et al. 2019, zheng et al. 2019). participation of aarss in plant stress responses is poorly characterized. a few reports have indicated aars involvement in plant responses to stress, but the exact molecular mechanisms were not revealed. wheat metrs was shown to be highly expressed during biotic stress imposed by the fungal pathogen fusarium graminearum resulting in significant resistance of the plant to this pathogen (zuo et al. 2016). a recent genetic screen identified aspartyl-trna plant aminoacyl-trna synthetases in abiotic stress acta bot. croat. 80 (1), 2021 37 ter three days of stratification, seeds were grown for eight days in a growth chamber under 16 h light / 8 h dark conditions (120 to 130 μmol m-2 s-1) at 24 °c. half of the eight-dayold seedlings were planted on the same medium as described above for control and on the medium with added stressor (200 μm cdcl2, 200 mm mannitol or 200 mm nacl). after 8 h and 24 h the seedlings were collected and snap-frozen. the other half of the eight-day-old seedlings were placed in liquid half-strength murashige and skoog medium with 0.5% (w/v) sucrose as control and in liquid half-strength murashige and skoog medium with 0.5% (w/v) sucrose and added stressor (200 μm cdcl2, 200 mm mannitol or 200 mm nacl). after 3 h and 6 h the seedlings were collected and snap-frozen. to evaluate stressor effect on seedlings we applied short term exposure in liquid medium, and long term exposure on solidified medium. treatment in liquid medium is more reliable and reproducible since seedlings are completely immersed in the stressor. however, long term incubation of a. thaliana seedlings in a liquid medium per se triggers stress response. to avoid this stress-related effect of liquid medium, for any longer exposure of seedlings to stressors we used a solidified medium. for each stressor, the entire experiments were performed three times using different batches of seedlings (three biological replicates). rna isolation, quality control and cdna synthesis to isolate the total rna, 50 mg of plant material was frozen with liquid nitrogen and homogenized with glass beads. isolation of total rna using rnazol (sigma-aldrich, usa) was performed according to the manufacturer’s protocol. total rna was resuspended in 50 μl of depctreated water. the integrity of rna was checked by measuring the absorbance using nanodrop nd-1000 spectrophotometer (nanodrop technologies) and using agarose gel-electrophoresis. dna was removed with turbo dnafreetm kit (ambion) according to the manufacturer’s protocol. up to 12.5 µl of total rna was incubated with 2 u of turbo dnase for 30 minutes at 37 °c in 1x turbo dnase buffer, followed by inactivation of turbo dnase. the integrity of dnase-treated rna was checked by measuring the absorbance using a nanodrop nd-1000 spectrophotometer and using agarose gel-electrophoresis. cdna was synthesised using high capacity cdna reverse transcription kit (ambion) according to the manufacturer’s protocol. reverse transcription was performed with 1 µg of dnasetreated total rna in a thermal cycler (eppendorf) under the following conditions: 10 minutes at 25 °c, 120 minutes at 37 °c and 5 minutes at 85 °c. real-time pcr and data analysis gene-specific primers were designed using ncbi primer-blast (ye et al. 2012) and are listed in tab. 1. quantitative pcr was performed in an optical 96-well plate with 7500 fast real-time pcr system (applied biosystems) and universal cycling conditions (10 min 95 °c, 40 cycles of 15 s at 95 °c and 60 s at 60 °c) followed by the generation of a dissociation curve to check for specificity of amplification. reactions contained sybr green master mix (applied biosystems), 300 nm of a gene-specific forward and reverse primer and 2 μl of 5x diluted cdna in each 20 μl reaction. no template controls (ntc) contained 2 μl rnase-free water instead. in qpcr, samples from various biological replicates were run in triplicate (technical replicates). pcr amplification efficiencies were calculated using linregpcr software (ruijter et al. 2009). pcr amplification efficiencies for reference gene and genes of interest were comparable and are shown in tab. 1. the relative quantification of gene expression was calculated using the comparative δδct method with actin 2 gene (at3g18780) as reference gene. the relative expression of specific genes was calculated by the following formulas: δct=ct specific gene ct reference gene and δδct=δct treated sample δct untreated sample. the relative expression level was calculated as 2-δδct. statistical analysis data analysis was conducted with student’s t-test to detect the differences between control and treated groups (yuan et al. 2006). standard deviation (sd) was calculated with the use of the results of the 2−δδct method. the results are presented as mean ± sd from three independent experiments. differences were considered significant at p < 0.05. asterisk symbols (*) indicate significant differences: **p < 0.01 and *p < 0.05. tab. 1. primer pairs used for quantitative real-time pcr. act2 primer sequences were taken from czechowski et al. 2005 and remans et al. 2008. gene strand sequence pcr efficiency act2 (at3g18780) forward 5’ cttgcaccaagcagcatgaa 3’ 1.886 ± 0.007 reverse 5’ ccgatccagacactgtacttcctt 3’ serrs (at5g27470) forward 5’ agcccgtagttgctgatacc 3’ 1.898 ± 0.008 reverse 5’ aatttcaagaaaacagaagagtcgt 3’ asprs (at4g31180) forward 5’ tcccagaagtcttggagcaac 3’ 1.875 ± 0.014 reverse 5’ caaatccaccgtgtagaggc 3’ cysrs (at5g38830) forward 5’ cgcagctagagagttcgtca 3’ 1.888 ± 0.004 reverse 5’ tccaccatcttcagacaatgc 3’ baranašić j., mihalak a., gruić-sovulj i., bauer n., rokov-plavec j. 38 acta bot. croat. 80 (1), 2021 results we examined genomic regions upstream of the genes encoding cytoslic serrs, asprs and cysrs using the a. thaliana cis-regulatory element database atcisdb, which considers a promoter to be an intergenic region upstream of the gene of interest, excluding any coding region of upstream genes (yilmaz et al. 2011). according to the atcisdb database, upstream intergenic regions of all three aars genes were identified as predicted promoters, since no experimentally validated binding of tfs to these regions is documented so far. analysis using atcisdb revealed that the predicted promoter of serrs gene is only 194 base pairs long and that transcription factors of wrky family are predicted to bind to serrs promoter and regulate transcription of serrs gene. the promoter sequence of asprs gene is predicted to be 1039 bp long, with putative binding sites for transcription factor families bhlh, homeobox, bzips and abi3vp1, while cysrs promoter is the longest (2391 bp) and contains binding motifs for transcription factor families bhlh, homeobox, wrky, mads, myb, bzip, abi3vp1 and lfy. furthermore, using athamap database (hehl et al. 2016) we examined the region from -1,000 bp to +200 bp relative to the transcription start of a gene considering that, in general, majority of gene regulatory sequences (86%) are found inside this area (yu et al. 2016). analysis showed that this region in serrs, asprs and cysrs genes contains many potential places for binding of tfs belonging to major stress-related tf protein families ap2/erf, nac, myb, bzip and wrky (baillo et al. 2019) (tab. 2). altogether, bioinformatics analysis showed that promotors of three aars genes contain many potential binding sites for stress-related tfs, implying that abiotic stress may influence expression of the aars genes examined. in order to determine the impact of abiotic stress conditions on the expression of selected aars genes, seedlings of a. thaliana were exposed to osmotic stress imposed with 200 mm mannitol, salt stress imposed with 200 mm nacl or heavy metal stress imposed with 200 µm cadmium. expression of serrs, asprs and cysrs genes was measured at four different time points. the transcript levels in control and treated samples were measured using rt-qpcr and normalized by using the expression of actin2 gene as endogenous control. the effect of 200 mm mannitol is shown in fig. 1. the serrs gene expression did not differ significantly from serrs gene expression in control, untreated seedlings (fig. 1a). on the other hand, in certain conditions osmotic stress significantly enhanced asprs and cysrs gene expression. expression of asprs gene was significantly elevated 8 hours after osmotic stress application (fig. 1b), while cysrs gene expression rose immediately after mannitol application and remained significantly increased for 3, 6 and 8 hours (fig. 1c). the effect of 200 mm nacl is shown in fig. 2. the serrs gene expression did not significantly differ from that in controls (fig. 2a), while the expression level of the asprs gene significantly increased 8 hours after addition of nacl and stayed induced even 24 hours following salt application (fig. 2b). salt stress imposed with 200 mm nacl significantly enhanced cysrs expression 6 and 8 hours after application, while after prolonged exposure (24 hours) cysrs gene expression decreased (fig. 2c). protein levels of serrs, asprs and cysrs were shown to be increased during early responses of a. thaliana due to cadmium exposure (sarry et al. 2006). in order to examine whether accumulation of these proteins is a result of enhanced transcript accumulation we tested the gene expression of selected aarss after exposure to cadmium for 3, 6, 8 and 24 hours. the effect of 200 µm cadmium on aarss gene expression in the a. thaliana seedlings is shown in fig. 3. compared with control seedlings, the treated seedlings did not show a statistically significant change in serrs transcript levels during different exposure times (fig. 3a). the same results were obtained during analysis of asprs and cysrs transcript levels (fig. 3b, c). fig. 1. expression of cytosolic seryl-trna synthetase (serrs), cytosolic aspartyl-trna synthetase (asprs) and cytosolic cysteinyltrna synthetase (cysrs) under osmotic stress induced by 200 mm mannitol. the expression level for each gene in stress condition was calculated relative to its expression in the control, untreated sample. the gene expression was normalized with the housekeeping actin 2 transcript. bars represent mean values ± sd from independent experiments (n = 3). asterix indicate significant difference between treated and control samples, *p < 0.05, **p < 0.01. fig. 2. expression of cytosolic seryl-trna synthetase (serrs), cytosolic aspartyl-trna synthetase (asprs) and cytosolic cysteinyl-trna synthetase (cysrs) under salt stress induced by 200 mm nacl. the expression level for each gene in stress condition was calculated relative to its expression in the control, untreated sample. the gene expression was normalized with the housekeeping actin 2 transcript. bars represent mean values ± sd from independent experiments (n = 3). asterix indicate significant difference between treated and control samples, *p < 0.05. plant aminoacyl-trna synthetases in abiotic stress acta bot. croat. 80 (1), 2021 39 discussion land plants are anchored in one place for most of their life cycle and therefore must constantly be able to adapt their growth and metabolism to abiotic stresses such as changes in light intensity and temperature, high salinity conditions, heavy metal exposure and the lack of water and essential minerals (floris et al. 2009). transcription factors are key regulators of gene expression that control cellular and developmental processes and the response to environmental stimuli. the number of transcription factor genes in a. thaliana genome is >1500 which is 3 times more than in animals with similar genome size (drosophila melanogaster (diptera) and caenorhabditis elegans (nematode); riechmann et al. 2000), which suggests that transcriptional regulation plays a more important role in plants then in animals. hence, plants’ survival depends on their ability to rapidly regulate gene expression in order to adapt their physiology to their environment. to gain more insight into aarss in stress response we analyzed promoters of genes encoding cytosolic serrs, asprs and cysrs. the rationale was to reveal whether promoters of these genes contain binding site motifs for the transcription factors important in plant stress response. in addition, we determined stress related expression levels of selected aars genes. we exposed a. thaliana seedlings to osmotic, salt and heavy metal stress and analyzed transcript expression patterns of selected aars genes (serrs, asprs and cysrs) 3, 6, 8 and 24 hours after the application of various stressors. our results show that expression of none of the investigated aars genes changes during stress induced by exposure to cadmium (fig. 3). this observation is surprising considering that previous proteomic investigation showed that protein levels of all three investigated aarss are increased during early responses of a. thaliana cells to oxidation stress due to cadmium exposure (sarry et al. 2006). the discrepancy between previous proteomic data and our gene expression study may be related to the increased stability of aars proteins or mrnas, as well as increased translation of aars mrnas on ribosomes during stress. it is known that increased protein levels are not always correlated to transcript levels and selective mrna translation is documented especially during exposure to stress (sablok et al. 2017). it is interesting to note that results of the mentioned proteomic inv e s t i g a t i o n i n d i c a t e d t h a t s e rr s u n d e r g o e s posttranslational modification during cadmium stress. we have recently solved crystal structure of a. thaliana serrs and identified the disulfide link in this cytosolic protein (kekez et al. 2019). considering that cadmium induces oxidative stress in cells, it is plausible that cysteines in serrs are oxidized and form a disulfide link, thus creating a new, posttranslationally modified form of serrs. this oxidized form of serrs may possess higher stability due to introduction of a covalent disulfide link and may participate in plant stress response mechanisms. in this work we did not observe a statistically significant change in serrs transcript levels during exposure to osmotic, salt and heavy metal stress, although promoter analysis revealed many potential binding sites for stress related transcription factors in the region from -1,000 bp to +200 bp relative to the transcription start site (tab. 2). although this region is regulatory for many a. thaliana genes (yu et al. 2016), it appears that regulation of serrs gene is controlled by its short promoter (196 bp) with only few putative tf binding sites (as predicted with atcisdb database) and that expression of serrs gene is not responsive to stress fig. 3. expression of cytosolic seryl-trna synthetase (serrs), cytosolic aspartyl-trna synthetase (asprs) and cytosolic cysteinyl-trna synthetase (cysrs) under heavy metal stress induced by 200 µm cadmium. the expression level for each gene in stress condition was calculated relative to its expression in the control, untreated sample. the gene expression was normalized with the housekeeping actin 2 transcript. bars represent mean values ± sd from independent experiments (n = 3). tab. 2. number of potential transcription factor (tf) binding sites in region -1000 bp to +200 bp from transcription start site of serrs, asprs and cysrs genes. analysis was performed using athamap and plant transcription factor database (plntfdb, pérez-rodríguez et al. 2010). tf family serrs asprs cysrs tf family function (according to plntfdb) ap2/erf 88 30 46 plant-specific transcription factors, key regulators of floral organ identity determination and of leaf epidermal cell identity, key regulators of several abiotic stresses and respond to multiple hormones. nac 68 51 47 plant-specific transcription factors, key regulators of stress perception and developmental programmes. myb 56 24 43 involved in the control of the cell cycle, involved in control of plant secondary metabolism, as well as the identity and fate of plant cells. bzip 21 1 8 regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. wrky 3 1 0 play significant roles in responses to biotic and abiotic stresses, and in development. baranašić j., mihalak a., gruić-sovulj i., bauer n., rokov-plavec j. 40 acta bot. croat. 80 (1), 2021 conditions used in this work. however, as explained above, serrs expression in stress can be regulated posttranscriptionally, at the mrna or protein level. the expression level of the asprs gene significantly increased during osmotic and salt stress, induced by the application of 200 mm mannitol or 200 mm nacl, respectively. the increase in asprs gene expression was observed 8 hours after stress application, and increased levels of asprs mrna were present even after 24 hours in the case of salt stress. the observed increased asprs gene expression is in agreement with our detailed analysis of asprs promoter which indicated 51, 30 and 24 binding site motifs for tfs belonging to nac, ap2/erf and myb families, respectively (tab. 2). these tf families are important regulators of stress response (nuruzzaman at al. 2013, baillo et al. 2019). it was previously shown that transcription of a. thaliana asprs gene was enhanced during biotic stress imposed by pathogen hyaloperonospora arabidopsidis (peronosporaceae) infection and that the enzyme can switch from canonical asprs activity to noncanonical defense activity upon pathogen infection (luna et al. 2014). here we show that the level of asprs transcripts increases during osmotic and salt stress indicating that asprs also participates in abiotic stress responses. therefore, it appears that asprs can serve as a general stress factor in plants. the expression level of the cysrs gene significantly increased for 3, 6 and 8 hours after the application of 200 mm mannitol and with the application of 200 mm nacl in duration for 6 and 8 hours. our analysis showed that the promoter of cysrs gene contains 47, 46 and 43 possible binding sites for nac, ap2/erf and myb transcription factors, respectively (tab. 2). this high number of stress-related tf binding sites and large promoter size (2391 bp) is in accordance with prompt and enhanced cysrs gene expression after exposure to stress. it is interesting to note that an increase in free cysteine levels has been reported as a response to various abiotic stress factors, including cadmium, salt and drought stress (zagorchev et al. 2013). cysteine is mainly needed for the biosynthesis of sulfur-rich compounds with anti-stress activity, such as gsh, phytochelatins (oligomers of gsh) and stress-related proteins (zagorchev et al. 2013). if cysteine is excessively used for synthesis of antistress compounds, cysteine levels are presumably not sufficient to support translation and therefore cysrs is overproduced to mitigate that effect. on the other hand, recent studies have shown that bacterial and human cysrs use cysteine to produce persulfides, such as cysssh or longer chain sulfur compounds (polysulfides, including cyss/gs– (s)n–h) (akaike et al. 2017). these persulfides have antioxidative effects. it is plausible to assume that plant cysrs participates in similar processes through its noncanonical function. although stress triggers a rapid downregulation of general protein biosynthesis (merchante et al. 2017), our results show that expression of plant aarss that participate in translation is not decreased. moreover, during exposure to stress conditions we detected increased asprs and cysrs transcript levels, while others reported increased serrs, asprs and cysrs protein levels (sarry et al. 2006). considering that during stress, translation of stress related proteins is enhanced we may assume that the levels of aarss are kept at steady-state to pursue translation of specific mrnas encoding proteins that are essential for stress resistance and survival. also, when stress conditions cease and global translation starts again, it may be advantageous to have sufficient levels of aarss to sustain translation. finally, increased protein levels of plant aarss during stress may serve as a pool of aars proteins that can participate directly in stress responses through their noncanonical activities. further research on plant aars role in stress is required to reveal the underlying molecular mechanisms that link aarss to plant stress response. acknowledgments the authors thank vlatka zoldoš for use of qpcr instrument. this work was supported by the grants from the croatian science foundation (ip-2016-06-6272), the foundation of croatian academy of sciences and arts and university of zagreb. references akaike, t., ida, t., wei, f.y., nishida, m., kumagai, y., alam, m.m., ihara, h., sawa, t., matsunaga, t., 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localized protein and protects plants against fusarium pathogens and mycotoxins. phytopathology 106, 614–623. acta bot. croat. 80 (2), 2021 221 acta bot. croat. 80 (2), 221–224, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-023 issn 0365-0588 eissn 1847-8476 short communication species introductions through coconut fibre: dactyloctenium aegyptium and glinus oppositifolius, new records for the balearic islands, spain marcello dante cerrato1*, arnau ribas-serra1, carles cardona1,2, lorenzo gil1 1 university of the balearic islands, interdisciplinary ecology group, department of biology. rd. valldemossa, km 7,5. 07122 palma, spain 2 balearic islands forestry centre (cefor), balearic institute for nature, gremi corredors, 10 (pol. son rossinyol), 07009 palma, spain abstract – based on plant material collected in the forest nursery of the balearic island forestry center (cefor) for autochthonous plant production and the university of the balearic islands experimental facilities, two new plant records are presented for the mediterranean island of mallorca. dactyloctenium aegyptium, an invasive grass previously recorded in other areas of the mediterranean basin, and glinus oppositifolius, a new record for the european flora. in both cases the species are presumed to have arrived through contaminated batches of the coconut fibre substrate used in both facilities. keywords: balearic islands, coconut fibre, exotic species, substrate, plant invasions introduction allochthonous species are recognized as one of the main threats worldwide to biodiversity. plant nurseries and other facilities dedicated to plant production are known to be an important centre of alien plant introductions. in this sense, recent studies have focused specifically on propagules contained in substrates, pointing out the specific role of coconut fibre substrate as a possible vector of long-distance weeds. this pattern has been extensively recorded in at least two regions: in new zealand (popay et al. 2008) and spain, valencian community (verloove et al. 2014 and references therein). in both cases, subtropical plants have been recorded and traced to their presumed origin in sri lanka or india, where coconut fibre is produced. some of the recorded species have been already observed under natural environments (muñoz et al. 2019), which confirms the danger of this substrate, especially when it is used for restoration purposes (verloove et al. 2014). based on a series of findings during experimental trials in the university of the balearic islands, and during autochthonous plant production in the centre forestal de les illes balears (cefor), we report on two new taxa for the balearic flora, one of them also being a novelty for the european flora. both taxa are presumed to have arrived through contaminated batches of the coconut substrate used in both facilities. considering the importance of plant introduction, the aim of this study is to assess for the first time in a european insular ecosystem this specific channel for plant introduction. materials and methods plants were recorded in both cefor and the university of the balearic islands experimental facilities, and voucher specimens were collected and preserved. species identification and formal description was done with respect to dactyloctenium aegyptium (l.) willd. by consultation of the online world grass flora grassbase (clayton et al. 2006-2020). in the case of glinus oppositifolius (l.) aug. the lack of any unified work for the genera necessitated the consultation of several studies (gagnepain 1914, hauman 1949, jeffrey 1961, gonçalves 1978, short 2002, vincent 2003) which are provided with a synthetic key to * corresponding author e-mail: marcellocerrato@hotmail.com cerrato m. d., ribas-serra a., cardona c., gil l. 222 acta bot. croat. 80 (2), 2021 species identification. a formal description can be found in flora zambesiaca (gonçalves 1978). plant distribution was assessed by consulting global online species database as cabi (2020) and gbif (2020). names follow the accepted names in gbif. when collected, herbarium vouchers were deposited in the public herbarium of the university of the balearic islands. codes are indicated for each species. results and discussion dactyloctenium aegyptium (l.) willd. in enum. pl. 2:1029. (1809). synonym – cynosurus aegyptius l., sp. pl.: 72 (1753) specimens examined – spain. balearic islands. palma. university of the balearic islands, 39°38’10.4” n 2°38’31.3”e, 90 m.a.s.l. date: 10/08/2020. leg. et det.: m.d. cerrato, l. gil & a. ribas; herbarium code: herbariuib 16860. (fig. 1). spain. balearic islands. escorca. menut, centro forestal cefor, 39°49’44.9” n 2°54’01.3”e, 568 m.a.s.l. date: 10/10/2020. obs. pers.: c. cardona (no herbarium voucher recorded). distribution in the balearic island – two individuals growing in different pots of experimental plants located outdoors of the university. individuals observed in some pots with plants for forestry purposes, and individuals growing at the margins of the cefor nursery. native range and status of naturalization – native in africa and asia (clayton et al. 2006-2020), its current distribution covers tropical and subtropical areas from africa, asia, australia and america, and there are also some dispersed observations in europe (cabi 2020, gbif 2020). in the mediterranean basin, it has been indicated to be expanding with records in morocco, italy and cyprus (muñoz et al. 2019). records in spain were indicated for the first time in barcelona, and afterwards on several occasions in valenfig. 1. herbarium specimen of dactyloctenium aegyptium (l.) willd. collected in experimental pots on the univeristy of the balearic islands in 2020 (herbariuib 16860). species introduction through coconut fibre acta bot. croat. 80 (2), 2021 223 cia and andalusia (muñoz et al. 2019). to our knowledge this record can be considered the first evidence of the presence of d. aegyptium in the balearic islands. based on previous comments and on observed naturalization events in cefor nursery, it is highly possible that future individuals will be recorded colonizing natural areas. glinus oppositifolius (l.) aug. dc, in bull. herb. boiss., sér. 2, 1: 552, 559 (1901). synonym – mollugo oppositifolia l., sp. pl.: 89 (1753) specimens examined – spain. balearic islands. palma. university of the balearic islands, 39°38’10.4” n 2°38’31.3”e, 90 m.a.s.l. date: 03/10/2019. leg. et det.: m.d. cerrato & a. ribas; herbarium code: herbariuib 16859. (fig. 2). balearic islands. escorca. menut, centro forestal cefor, 39°49’44.9” n 2°54’01.3”e, 568 m.a.s.l. date: 10/09/2018. obs. pers.: c. cardona (no herbarium voucher recorded). distribution in the balearic island – two individuals growing in three different pots of experimental plants located in the outdoor premises of the university in two consecutive years. individuals observed in some pots with plants for forestry purposes in the cefor nursery. native range and status of naturalization – glinus oppositifolius covers tropical and subtropical areas, with its presence recorded as native in central and south africa, south and south-east asia and north australia and as allochthonous in new zealand (popay et al. 2008). in the case of the balearic islands and the european continent, due to the lack of any record (gbif 2020), we consider its presence a novelty. individuals were observed to successfully flower and produce seeds, but died during the summer, for which reason we consider the species to be of limited invasiveness. key to species identification for the glinus genera (based on references indicated in material and methods): fig. 2. herbarium specimen of glinus oppositifolius (l.) aug. dc. collected in experimental pots on the university of the balearic islands in 2019 (herbariuib 16859). cerrato m. d., ribas-serra a., cardona c., gil l. 224 acta bot. croat. 80 (2), 2021 1. pedicellate flowers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1. short pedicellate (< 3 mm) or sessile flowers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. (3)5 to 10 stamens per flower. perianth segments 3.5 to 5 mm long. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. oppositifolius 2. more than 10 stamens per flower. perianth segment up to 8 mm long . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. glabrous plants. obovate sub-succulent leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .g. orygioides 3. young parts pubescent with multicellular prickly hairs. lanceolate or oblanceolate leaves . . . . . . . . . . . . . .g. bainesii 4. 2 stamens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. sessiliflorus 4. more than 2 stamens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5. more than 5 stamens per flower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5. 3-5 stamens per flower. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6. 0-8 staminodes white-green. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. lotoides 6. 9-18 staminodes yellow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. setiflorus 7. leaves 3-5 mm length x 2-3 mm width. leaf blade obovate obtuse, slightly attenuated towards apex. petiole 0.5-1 mm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. microphyllus 7. bigger leaves in length (variable in g. radiatus). leaf blade obovate or elliptic to spatulate with rounded or acute apex. petiole 1-7 mm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8. leaves nearly glabrous. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. herniarioides 8. leaves densely pubescent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 9. seeds smooth. sepal apex long acuminate or attenuate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. radiatus 9. seeds papillate. sepal apex rounded to slightly mucronate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. lotoides there is finally one taxon described as endemic to egypt, g. runkewitzii. we could not access the formal description or any reference with morphological data. references cabi. 2020: dactyloctenium aegyptium. invasive species compendium. wallingford, uk: cab international. retrieved september 9, 2020 from http://www.cabi.org/isc clayton wd, vorontsova ms, harman kt, williamson h. 20062020: grassbase. the online world grass flora. retrieved september 23, 2020 from http://www.kew.org/data/grassesdb.html/ gbif.org. 2020: gbif home page. retrieved december 23, 2020 from: https://www.gbif.org/ gagnepain f. deux gisekia et mollugo nouveaux d’indo-chine. notulae systematicae. herbier du museum de paris. 1914;3:367–8. gonçalves ml. 1978: molluginaceae. in: launert, e. (ed.), flora zambesiaca, vol. 4. rosaceae-cornaceae, 537-541. royal botanic gardens, kew, london. hauman l. notes sur quelques aizoacées et sur un chenopodium du congo belge. bulletin du jardin botanique de l’état bruxelles. 1949;19:443–8. https://doi.org/10.2307/3666834 jeffrey c. 1961: aizoaceae. in: hubbard, c.e., milne-redhead, e. (eds.), flora of tropical east africa, 1-37. crown agents, london. muñoz af, gullón es, devesa ja. dactyloctenium aegyptium (l.) willd. (poaceae), novedad para andalucía (españa). acta bot malacit. 2019;44:71–2. https://doi.org/10.24310/ abm.v44i0.5335 popay ai, james tk, sarty m, dickson m, bullians ms. 2008: pineapple leaves and coconut husks: closing biosecurity pathways to prevent further infiltration. in: froud k.j., popay a.i., zydenbos, s.m. (eds.), surveillance for biosecurity: pre-border to pest management, 45-49. the new zealand plant preotection society, pihia. short ps. a new species of glinus l. (molluginaceae) from the northern territory, australia. telopea (syd). 2002;9:761–3. https://doi.org/10.7751/telopea20024014 verloove f, laguna e, ferrer-gallego pp. some potentially weedy cyperaceae new to spain. flora mediterr. 2014;24:197– 205. vincent ma. 2003: molluginaceae rafinesque in: flora of north america editorial committee (eds.), flora of north america north of mexico, vol. 4. magnoliophyta: caryophyllidae, 509-512. oxford university press, new york and oxford. 152 acta bot. croat. 77 (2), 2018 acta bot. croat. 77 (2), 152–160, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0017 issn 0365-0588 eissn 1847-8476 subalpine vegetation in giresun mountains (turkey) rena hüseyinova1, erkan yalçin2* 1 şebinkarahisar school of applied sciences, giresun university, giresun, turkey 2 department of biology, faculty of arts and science, ondokuz mayıs university, samsun, turkey abstract – in this study, the subalpine vegetation in the giresun mountains of northern turkey was investigated. the study area included northand south-facing slopes at altitudes ranging between c. 2000 and 2500 meters. for vegetation classification and for describing the relationships between vegetation and environment, traditional braun-blanquet methods and multivariate analysis techniques were used. the vegetation mainly consisted of subalpine grasslands and coniferous cushion scrubs. caricetea curvulae and astragalo microcephali-brometea tomentelli were found to be dominant syntaxa in the vegetation of the study area. land topography, soil physical and chemical factors and species richness have important impacts on the development of subalpine vegetation according to the results of multivariate analysis. three associations and two subassociations were newly determined and classified. hemicryptophytes, chamaephytes and geophytes participated in the floristic composition of these syntaxa. eunis habitat code and names for described syntaxa were also proposed. keywords: environment, grasslands, life form, phytosociology, species richness, syntaxonomy * corresponding author, e-mail: eryalcin@omu.edu.tr introduction mountainous areas are mainly located in the northern hemisphere (vogiatzakis 2012) and orographic, edaphic and climatic factors have drawn an alpine borderline in these mountainous areas (holtmeier and broll 2005). alpine and subalpine belts of mountains form specific ecological conditions for vascular flora and vegetation due to geographical isolation, glaciation, the existence of microhabitats, climatic and historical changes (uysal et al. 2011). subalpine vegetation is greatly influenced by different types of natural and anthropogenic disturbance (tappeiner et al. 1998) and is rather sensitive to global climate change (cannone et al. 2007). it is expected that the subalpine plant composition will be changed by upward species migration due to ongoing global climate changes. these changes have been observed by the gloria monitoring network (gloria-europe = global observation research initiative in alpine environments and gloria-worldwide) in high mountain regions. subalpine and alpine grasslands are considered biodiversity hotspots and included in the natura 2000 network as an eu priority habitat type (pfeiffer et al. 2016). syntaxonomic and ecological investigations on alpine and subalpine belts have been carried out by many researchers in europe and asia. the first vegetation investigation on alpine vegetation in western and southern anatolia was done by quézel and pamukçuoğlu (1970) and quézel (1973). rehder et al. (1994) and hein et al. (1998) researched new alpine plant syntaxa in uludag-bursa province from turkey. in these studies, the alpine vegetation was mostly included in the class astragalo microcephali-brometea tomentelli quézel 1973 em. parolly and its five orders. these orders include following vegetation types; 1astragalo microcephali-brometalia tomentelli quézel 1973: oreal to subalpine xerophytic grasslands, dwarf-shrub and the thorny-cushion communities of anatolia, 2drabo-androsacetalia quézel 1979: alpine to subnival mat-forming communities, 3hyperico linarioidisthymetalia skorpilii akman, quézel, yurdakulol, ketenoğlu, demirörs 1987: xerophytic grasslands, dwarf-shrub and thorny-cushion communities of the west and middle black sea mountains, 4onobrychido armenae-thymetalia leucostomi akman, ketenoğlu, quézel & demirörs 1984: xerophytic grasslands, dwarf-shrub and the thorny-cushion communities of central anatolia, 5trifolio anatolici-polygonetalia arenastri quézel 1973: hygrophytic to mesophytic vegetation of dolines, snow-patch and the melt water communities of the taurus range (hamzaoğlu 2006). subalpine vegetation in giresun mountains acta bot. croat. 77 (2), 2018 153 turkish phytosociologists and ecologists have mostly concentrated on the forest and steppe vegetation in mountainous areas in turkey (parolly 2004). alpine and subalpine vegetation studies were mostly carried out under the leadership of foreign researchers in turkey. however, some of the turkish botanists such as akman et al. (1987), tatlı (1987), düzenli (1988), kılınç and karakaya (1992), vural (1996), uysal et al. (2011) partly focused on alpine and subalpine vegetation. vural (1996) published a comprehensive syntaxonomic scheme about the alpine belts of mountains of the eastern black sea, while hein (1998) and parolly (1998, 2004) did important revision for some of the syntaxa in the alpine vegetation of the west and eastern taurus mountains. although the syntaxonomic classification of vegetation is very important, the relationships between vegetation and environment have not been sufficiently investigated for alpine and subalpine vegetation in turkey. the phytosociological approach of vegetation classification considered that each community is evolved by a site-specific range of environmental factors leading to differences in species composition (vonlanthen et al. 2006). the aim of this study was to describe syntaxa and the relationships between vegetation and environmental properties of the subalpine communities in giresun mountains from northern turkey. materials and methods study area the study area is situated at a7 according to the grid system of davis (1965, 1985), and is approximately located between n 40°29'27'' and e 38°25'41''. the study area is surrounded by high mountains. in the study area, the subalpine belt extends from c. 2000 to 2500 m upwards on south and north-facing slopes. giresun mountains are a system of mountains that extend up to the peaks on mt karadağ, 3391 m, in the east and on the karagöl plateau with 3095 m in the west. the study area is located in the north of şebinkarahisar, between the eastern pontide metallogenic belt and the north anatolian fault system (yavuz et al. 2008). the turkish pontides comprise the cretaceous to eocene igneous record of the convergence between eurasia and gondwana (boztuğ et al. 2004) the nearest district to the study area (şebinkarahisar) has a mediterranean type of climate with 525 mm mean annual precipitation (p), and a drought period is observed in july with 0.5 mm precipitation according to the şebinkarahisar state meteorological station. the mean annual temperature is 11.3 °c. summer rainfall (pe) is 37 mm. the mean maximum for the hottest month (m) and mean minimum for the coldest month (m) are 30.3 and -16.1 °c, respectively. index of xericity (s=pe/m) is 1.8. pluviometric quotient (q= 2000 p/ [(m + m + 546.4) * (m-m)] is 40.7, and the precipitation regime is sub-mediterranean (spring, autumn, winter, summer). northeast winds predominate, thus north facing slopes are partly windward, whereas the south facing slopes are leeward. the north facing slopes are snow covered for a longer time than the south facing slopes. the vegetation mostly consists of festuca-dominated grasslands and traditionally has been grazed by sheep, cows and goats since the 19th century. however, grazing pressure has gradually decreased over the last two decades due to declining livestock production. vegetation sampling the taxonomy of vascular plants follows davis (1965, 1985), davis et al. (1988), tutin et al. (1964, 1980), güner et al. (2000) and güner et al. (2012), respectively. all plant samples were preserved in the herbarium of the faculty of the arts-sciences of ondokuz mayıs university (omub). non vascular plants were omitted. for sampling procedure, we considered the study area according to the north and the south facing slopes. both of them occupied altitudes ranging between 2000 and 2500 m. five floristically and environmentally homogeneous sample plots were established with 100 m altitudinal intervals on each slope. plot size was selected as 20 and 50 m2 (van der maarel 2005). in total, 60 plots were obtained during the spring and summer months of 2015 and 2016. phytosociological records were carried out by using the cover/abundance values proposed by braun-blanquet (1964) in each plot. in each plot, the slope degrees (%), altitude (meters above the sea level), aspect and gps coordinates were also logged. aspect in degrees was transformed using the formula of beers et al. (1966). plant associations were named according to the international code of phytosociological nomenclature (icpn) (weber et al. 2000). syntaxonomic interpretations were made by using the available phytosociological literature (quézel 1973, akman et al. 1987, vural 1996, mucina 1997, parolly 1998 and 2004, onipchenko 2002, mucina et al. 2016). plant life forms were identified according to mueller-dombois and ellenberg (2002). eunis habitat code and names were identified according to davies et al. (2004) and eunis habitat type hierarchical view (eea 2017). soil sampling and analysis from each plot, soil samples were taken at a depth of 20 cm from the topsoil. soil samples were then air dried and sieved through a 2 mm mesh prior to analysis. soil organic matter (%) was determined using the walkley and black method (black et al. 1965). soil texture was determined using the bouyoucus hydrometer method, and the clay content was expressed as %. the ph values were measured in deionized water (1:1) and the soil nitrogen (%) was determined by the micro kjeldahl method. the soil phosphorus (ppm) was determined spectrophotometrically following the extraction by ammonium acetate. soil potassium (cmol(+)/ kg) was determined using a petracourt pfp-7 flame photometer after nitric acid wet digestion. the caco3 (%) concentrations were determined using a scheibler calcimeter. for the determination of soil moisture, freshly weighed soil samples were air dried for 48 h to calculate wet to dry mass ratios. hüseyinova r., yalçin e. 154 acta bot. croat. 77 (2), 2018 these values were used to calculate soil moisture (gravimetric method) (kacar 2009). data analysis prior to data analysis, 10 vegetation plots were subjectively excluded due to high heterogeneity and their uncertain positions. the resulting dataset of 50 plots and 160 species was classified by the pc-ord program (mccune and grace 2002), using ward’s method and the jaccard similarity index as a resemblance measure. “crispness of the classification” was used for the optimal number of clusters (botta-dukát et al. 2005). diagnostic taxa for each group were defined in the juice program (tichý 2002) by calculating the fidelity of each species to each group (chytrý et al. 2002) using the φ-coefficient as the fidelity measure. species with a fidelity of above 50 (φ > 50) were considered as diagnostic. to detect gradients in species composition and speciesenvironment relations, canonical correspondence analysis (cca) was performed by using the ecom 2.1.3.137 version software programme (seaby and henderson 2007). canonical correspondence analysis includes two matrices, one of which has 50 plots, classified into syntaxa by phytosociological analysis, × 160 species (average % cover values of species corresponding to the transformations of the braun-blanquet scale as proposed by van der maarel (1979) were r = 1, + = 2, 1 = 3, 2m = 4, 2a = 5, 2b = 6, 3 = 7, 4 = 8, 5 = 9) and second matrix 50 plots × 7 environmental parameters (soil moisture (sm), soil ph, soil clay content (clay), total soil nitrogen ratio (n), aspect, altitude and slope). the interpretation of the results was made by the first two axes because only these two axes were statistically significant. statistically significant variables that derived from cca were shown by a bold number. species diversity was calculated as the shannon–wiener index at log base 10 (magurran 2004) performed by using biodiversity version 2.0 (mcalleece et al. 1997) software programme, respectively. moreover, the comparison of growth form and species diversity parameters for the described communities was visualized by a box-whisker diagram prepared in spss 21.0 version (ibm corp. 2012). the data of the şebinkarahisar (giresun) state meteorological station, the nearest meteorology station to the study area, were used to explain the climatic properties. results cluster analyses yielded that vegetation of eğribel pass was shown by four main clusters (fig. 1). crispness of classification value verified that the data set was classified in four clusters. the most prominent split within the four-clusters concerned the cluster c, which is clearly separated from the other clusters due to syntaxonomic distinction on the basis of the summed up average cover values of diagnostic species. we defined two grassland associations and subassociations dominated by festuca, and a cushion juniperus scrub on the different slopes. the south facing slopes had drier soil conditions than the north facing slopes. cluster a contained the plots sampled in the southern slopes in the study area. festuca pinifolia var. pinifolia, bunium microcarpum subsp. bourgaei, veronica peduncularis and lotus corniculatus var. alpinus were constant. we defined two subassociations from the cluster. subassociations were formed by different environmental variables such as altitude, slope, soil organic matter and nitrogen ratios (tab. 1). we propose the following name for the association and subassociations: bunio microcarpae-festucetum pinifoli ass. nova hoc loco (holotypus: plot 14 in on-line suppl. tab. 1), bunio microcarpae-festucetum pinifoli subass. oxytropetosum lazicae subass. nova hoc loco (holotypus:plot 10 in on-line suppl. tab. 1) and bunio microcarpae-festucetum pinifoli subass. verbascetosum froedinii subass. nova hoc loco (holotypus: plot 16 in on-line suppl. tab. 1), respectively. oxytropis lazica and verbascum froedinii were dominant taxa in the subassociations. oxytropis lazica spread on mesic slopes while verbascum froedinii occupied drier slopes. cluster b included plots where the vegetation was dominated by festuca amethystina subsp. orientalis var. turcica, lotus corniculatus var. corniculatus and scorzonera cana var. radicosa in northern slopes. we named cluster b lotus corniculati-festucetum turcici ass. nova hoc loco (holotypus:plot 33 in on-line suppl. tab. 1). bunio microcarpae-festucetum pinifoli and lotus corniculati-festucetum turcici were differentiated by the bulk of caricetea curvulae br.-bl. 1948 and caricetalia curvulae br.-bl. in br.-bl. et jenny 1926 species in these clusters puts them in these syntaxa. at the alliance level, their affiliation is clearly with agrostio lazicae-sibbaldion parviflorae vural 1996 owing to the species diagnostic of alliance. moreover, molinio-arrhenatheretea r. tx. 1937 and astragalo microcephali-brometea tomentelli quézel 1973 em. parolly species participated in the floristic composition of both of them. cluster c which was coniferous cushion scrublands comprised of the plots sampled in the southern slopes in fig. 1. dendrogram of vegetation plots (50 plots and 160 species) of the giresun mountains. a: bunio microcarpae-festucetum pinifolii, 1: oxytropetosum lazicae, 2: verbascetosum froedinii, b: lotus corniculati-festucetum turcici, c: alysso pseudo-mouradici-juniperetum saxatilis stands. subalpine vegetation in giresun mountains acta bot. croat. 77 (2), 2018 155 tab. 1. the mean values environmental, soil parameters and species richness in the associations and their comparison with anova test. the difference in letters indicates significant difference (p < 0.05) between means according to tukey’s (hsd) test among associations and subassociations. bg – between groups, wg – within groups; bfol – bunio microcarpae-festucetum pinifoli subass. oxytropetosum lazicae, bfvfi – bunio microcarpae-festucetum pinifoli subass. verbascetosum froedinii, lf – lotus corniculati-festucetum turcici, aj – alysso pseudo-mouradici-juniperetum saxatilis. parameter associations mean±std. error sum of squares df mean square f p aspect bfol 0.68±0.21 b bfvfi 0.32±0.09 b 15.27 (bg) 3(bg) 5.09(bg) 17.13 <0.01 lf 1.62±0.08 a 15.16 (wg) 51(wg) 0.29(wg) aj 1.44±0.14 a slope bfol 24.33±2.22 b bfvfi 42.00±0.81 a 2794.05(bg) 3(bg) 931.35(bg) 17.92 <0.01 lf 27.75±1.96 b 2649.58(wg) 51(wg) 51.95(wg) aj 39.50±0.89 a altitude bfol 2290.86±16.69 a bfvfi 2113.10±14.16 b 206244.59(bg) 3(bg) 68748.19(bg) 6.19 <0.01 lf 2257.70±35.55 a 566279.33(wg) 51(wg) 11103.51(wg) aj 2245.50±10.13 a species richness bfol 23.13±0.63 a bfvfi 26.90±1.90 a 929.97(bg) 3(bg) 309.99(bg) 16.85 <0.01 lf 17.50±1.09 b 937.73(wg) 51(wg) 18.38(wg) aj 27.30±0.86 a soil moisture bfol 6.23±0.96 ab bfvfi 3.59±1.06 b 396.25(bg) 3(bg) 132.08(bg) 10.71 <0.01 lf 9.32±0.93 a 628.87(wg) 51(wg) 12.33(wg) aj 2.54±0.09 b ph bfol 4.65±0.12 b bfvfi 5.10±0.19 b 7.22(bg) 3(bg) 2.40(bg) 11.21 <0.01 lf 4.76±0.10 b 10.95(wg) 51(wg) 0.21(wg) aj 5.65±0.01 a silt bfol 27.86±1.35 ab bfvfi 27.73±1.06 b 527.07(bg) 3(bg) 175.69(bg) 8.04 <0.01 lf 32.69±1.24 a 1113.96(wg) 51(wg) 21.84(wg) aj 24.39±2.17 b sand bfol 59.30±1.34 b bfvfi 58.41±1.73 b 738.76(bg) 3(bg) 246.25(bg) 7.86 <0.01 lf 55.42±1.54 b 1596.41(wg) 51(wg) 31.30(wg) aj 65.91±0.64 a caco3 bfol 2.79±0.24 a bfvfi 2.47±0.29 ab 5.97(bg) 3(bg) 1.99(bg) 2.88 0.04 lf 2.57±0.19 ab 35.13(wg) 51(wg) 0.68(wg) aj 1.82±0.06 b organic matter bfol 11.18±1.32 b bfvfi 4.78±0.53 c 1123.63(bg) 3(bg) 374.54(bg) 28.98 <0.01 lf 15.65±0.82 a 659.14(wg) 51(wg) 12.92(wg) aj 5.45±0.19 c total soil nitrogen bfol 0.54±0.06 b bfvfi 0.27±0.04 c 2.27(bg) 3(bg) 0.75(bg) 24.66 <0.01 lf 0.75±0.03 a 1.56(wg) 51(wg) 0.03(wg) aj 0.27±0.09 c the study area and, dominated by juniperus communis var. saxatalis, alyssum pseudo-mouradicum and dianthus zederbaueri. cluster c is named as alysso pseudo-mouradicijuniperetum saxatilis ass. nova hoc loco (holotypus: plot 58 in on-line suppl. tab. 1). in the association, astragalo microcephali-brometea tomentelli quézel 1973 em. parolly, hyperico linarioidis-thymetalia scorpilii akman, quézel, yurdakulol, ketenoğlu, demirörs in all, 1987 species are present. the diagnostic species of peduicularo comosaasterion alpini akman, quézel, yurdakulol, ketenoğlu, demirörs 1987 were represented in this association such as aster alpina and jasione supina subsp. pontica. hüseyinova r., yalçin e. 156 acta bot. croat. 77 (2), 2018 the syntaxonomic scheme for syntaxons in the study area is as follows: class: astragalo microcephali-brometea tomentelli quézel 1973 em. parolly order: hyperico linarioidis-thymetalia scorpilii akman, quézel, yurdakulol, ketenoğlu, demirörs 1987 alliance: peduicularo comosa-asterion alpini akman, quézel, yurdakulol, ketenoğlu, demirörs 1987 association: alysso pseudo-mouradici-juniperetum saxatilis ass. nova hoc loco class: caricetea curvulae br.-bl. 1948 order: caricetalia curvulae br.-bl. in br.-bl. et jenny 1926 alliance: agrostio lazicae-sibbaldion parviflorae vural 1996 association 1: bunio microcarpae-festucetum pinifoli ass. nova hoc loco subassociation 1.1: oxytropetosum lazicae subass. nova hoc loco subassociation 1.2: verbascetosum froedinii subass. nova hoc loco association 2: lotus corniculati-festucetum turcici ass. nova hoc loco hemicryptophytes were the dominant life form in all syntaxa, while chamaephytes and geophytes also contributed to the life form composition (fig. 2). alysso pseudo-mouradici-juniperetum saxatilis had the highest species richness and most diverse syntaxon while lotus corniculati-festucetum turcici was the syntaxon with lowest species richness and the least diverse syntaxon (figs. 3 a,b). the environmental factors and species richness of syntaxa were statistically different (tab. 1). lotus corniculati-festucetum turcici contained species related to plots with more humid, nitrogenous, silty soil, rich in organic matter, while alysso pseudo-mouradicijuniperetum saxatilis had species related to plots with sandy soil and plots of greater inclination. the first two cca axes were significant (p < 0.001) according to the monte-carlo permutation test. eigenvalue of axis 1 was 0.70 and explained 13.75% of total variance, while eigenvalue of axis 2 was 0.51 and explained 23.87% of total variance (on-line suppl. tab. 2). according to cca ordination axes, aspect, slope, altitude, soil moisture, clay and nitrogen contents were important environmental parameters for the development of different vegetation types in the study (on-line suppl. tab. 2). the slope, proportion of soil moisture, clay and nitrogen were negatively correlated with the first and second cca axes, while aspect and altitude were positively correlated with the second axis (fig. 4). in the cca ordination, alysso pseudo-mouradici-juniperetum saxatilis preferred higher soil moisture, clay and nitrogen fig. 2. life form spectra for associations and subassociations. bfol – bunio microcarpae-festucetum pinifoli subass. oxytropetosum lazicae, bfvfi – bunio microcarpae-festucetum pinifoli subass. verbascetosum froedinii, lf – lotus corniculati-festucetum turcici, aj -alysso pseudo-mouradici-juniperetum saxatilis. fig. 3. species richness (a) and shannon diversity index values (b) for associations and subassociations. the difference in letters indicates the significant difference (p < 0.05) between means according to tukey’s (hsd) test among associations and subassociations. abbreviations for associations and subassociations are as in fig. 2. subalpine vegetation in giresun mountains acta bot. croat. 77 (2), 2018 157 content in spite of the spread of lotus corniculati-festucetum turcici on the highest altitude slopes. caricetea curvulae br.bl. 1948 plots were located in a separate position towards the right margin of the ordination space and showed high correlations with topographical factors such as altitude, slope and aspect (fig. 4). bunio microcarpae-festucetum pinifoli and alysso pseudo-mouradici-juniperetum saxatilis spread on the south facing slopes in spite of lotus corniculati-festucetum turcici occupying the north facing slopes. the plots of bunio microcarpae-festucetum pinifoli subass. oxytropetosum lazicae spread on the landscapes that had higher altitudes and lower slopes than bunio microcarpae-festucetum pinifoli subass. verbascetosum froedinii plots. discussion in the eastern black sea region, detailed research dealing with vegetation of the highest mountains above the timberline began in the 1980s. in the first studies, turkish authors (düzenli 1988, kılınç and karakaya 1992, vural 1996) recorded a number of vegetation plots from different alpine and subalpine communities. düzenli (1988) investigated the vegetation in the alpine zone of mount tiryal (artvin). he represented the alpine vegetation as associations alchemillo-campanuletea tridentate quézel et düzenli 1979 and alchemillo-campanuletalia tridentatae quézel et düzenli 1979, respectively. vural (1996) studied the high mountain vegetation of rize. in his paper, subalpine vegetation was considered within one class and two orders. kılınç and karakaya (1992), suggested that subalpine vegetation in the orduçambaşı plateau should be included in the class molinio-arrhenatheretea r. tx. 1937, order arrhenatheretalia and class alchemillo retinervis-sibbaldietea parviflorae vural 1996. aspect, slope and altitude influence the plant communities and environmental conditions in the giresun mountains. consequently, in this study, different plant associations and subassociations were defined. vural (1996), in his study, formerly described alchemillo retinervis-sibbaldietea parviflorae vural 1996, alchemillo retinervis-sibbaldietalia parviflorae vural 1996 and swertio ibericae-nardetalia strictae vural 1996. but parolly (2004) proposed that all of them should be included in caricetea curvulae br.-bl. 1948 and caricetalia curvulae br.-bl. in br.-bl. et jenny 1926. onipchenko (2002) supported a similar syntaxonomic scheme for the subalpine vegetation of the teberda reserve, the northwestern caucasus. we agreed with this viewpoint and firstly classified the subalpine festuca grasslands within these syntaxa in turkey. at the alliance level, agrostio lazicae-sibbaldion parviflorae vural 1996 was accepted by parolly (2004) for subalpine grassland vegetation from northern turkey. we also considered that alliance for the syntaxa dominated by festuca in this study. a coniferous cushion scrub community spread at 2200-2300 meters altitude was dominated by juniperus communis var. saxatilis in this study. akman et al. (2014) previously reported a similar community on ilgaz mountain. in the present study, it was found that the bulk of astragalo microcephali-brometea tomentelli and hyperico linarioidis-thymetalia scorpilii species in the floristic composition of the coniferous cushion scrub association was dominated by juniperus communis var. saxatilis. at the alliance level, the association affiliated with peduicularo comosa-asterion alpini akman, quézel, yurdakulol, ketenoğlu, demirörs 1987. cluster groups in dendrogram of plots that also reflected a floristic assemblage characterized by a mixture of species of caricetea curvulae and astragalo microcephali-brometea tomentelli. hemicryptophytes were dominant in accordance with general characterisation of subalpine vegetation in this study. hemicryptophytes were fairly common life form in alpine and subalpine landscape, which showed adaptations to snowbed environmental conditions by the strong persistence via plurennial stocks or dense turfs, above-ground renewal buds over winter, lateral spreading over short distances and generalistic diaspore dissemination (komac et al. 2015, scheepens et al. 2015). nevertheless, considerable percentages of particular plant types (like therophytes, various kinds of chamaephytes, succulents, evergreens and berryproducers) generated a highly diversified alpine belt (illa et al. 2006). chamaephytes also presented low percentages in the floristic composition that were related to various stress conditions in this study. these plants mainly grew in infertile soils and were able to persist for many years with small above-ground lignified perennial structures (sanz‐elorza et al. 2003, schweingruber 2007). in this study, the environmental factors influenced vegetation differentiation at different physiognomic and syntaxonomic levels (onipchenko and semenova 1995, onipchenko 2002). etzold et al. (2016) reported that altitude, slope and aspect were also the main topographical factors and formed different syntaxa in subalpine and alpine grassland vegetation in the northeastern greater caucasus of azerbaijan. noroozi et al. (2010) also reported that slope and aspect were also the main driving topographical factors on vegetation differentiafig. 4. canonical correspondence analysis ordination diagram of the dendrogram groups related to environmental factors in the study area. abbreviations for associations and subassociations are as in fig. 2. sm – soil moisture ratio, n – total soil nitrogen ratio. hüseyinova r., yalçin e. 158 acta bot. croat. 77 (2), 2018 tion in the high alpine vegetation of the tuchal mountains (central alborz, iran). likewise, new plant associations and subassociations were described depending on altitude and aspect gradients, in our study. the position of caricetea curvulae br.-bl. 1948 probably reflected a moisture gradient in terms of the longer persistence of snow. altitude decreases temperature while increases precipitation that indirectly influence vegetation (walther et al. 2005). aspect could indirectly alter soil moisture and mineralization due to their effect on the solar radiation (winkler et al. 2016). therefore, we found that different plant associations grew on different slopes and humid soils. gottfried et al. (1998) and pauli et al. (1999) showed increasing elevation limits of alpine grassland from south-western to south-eastern slopes on schrankogel in the central eastern alps. besides, this study showed that south-exposed mountain slopes favour local-scale species richness, compared to the northern sides of the same mountains. this is consistent with the species-energy hypothesis (wright 1983) and other temperature-related diversity hypotheses (currie et al. 2004), suggesting temperature-driven processes as decisive determinants of vascular plant species richness (winkler et al. 2016). cca reflected soil clay content being one of the important environmental factors for the floristic composition of subalpine vegetation. the soil particle size is determined by microtopography and fine-textured soils (mainly clay and silt) have higher water-holding capacity (michalet et al. 2002). zanelli et al. (2007) hypothesised that vegetation change has led to changes in soil chemistry and soil mineralogy. clay minerals are often a weathering product of the near-surface that is dependent on the precursor minerals and the surrounding environmental conditions (egli et al. 2008). in addition to this, the clay contents in soils are favourable for the immobilization of nutrient ions and enzymes. subalpine soils have high contents of total nitrogen, which also explains the lower activities and microbial biomass in these soils. nitrogen is generally a limiting factor for soil biological processes (margesin et al. 2009). we also found that soil clay and nitrogen content had an impact on vegetation differentiation in subalpine belts in the black sea mountains. new subalpine syntaxa were identified in the black sea mountains in turkey. they were included in two classes caricetea curvulae br.-bl. 1948 and astragalo microcephalibrometea tomentelli quézel 1973 em. parolly. astragalo microcephali-brometea tomentelli quézel 1973 em. parolly was widespread on the different altitudes and bedrocks in eastern and inner anatolia. this study revealed that this class penetrated into the black sea high mountain chains. it also showed that caricetea curvulae br.-bl. 1948 and astragalo microcephali-brometea tomentelli quézel 1973 em. parolly were distributed together on different altitudes and aspects in the subalpine belt in black sea mountains in turkey. however, these classes diverged in some environmental parameters such as altitude, slope, aspect, soil nitrogen, moisture and clay contents. the eunis habitat classification is important for species, habitat types and designated sites compiled in the framework of natura 2000 (davies et al. 2004, özüdoğru and duygu 2009). festuca-dominated grasslands could be included in pontic alpine grasslands as eunis habitat code e4.441. the juniperus communis dominated syntaxa were classified in eunis by the habitat code and name f3.16 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(ed.), mediterranean mountain environments,1–8, wiley-blackwell publishing company, uk. vonlanthen, c. m., bühler, a., veit, h., kammer, p. m., eugster, w., 2006: alpine plant communities: a statistical assessment of their relation to microclimatological, pedological, geomorphological, and other factors. physical geography 27, 137– 154. vural, m., 1996: the high mountaine vegetation of rize. turkish journal of botany 20, 83–102. walther, g. r., beißner, s., burga, c. a., 2005: trends in the upward shift of alpine plants. journal of vegetation science 16, 541–548. weber, h. e., moravec, j., theurillat, j. p., 2000: international code of phytosociological nomenclature. 3 rd edition. journal of vegetation science 11, 739–768. winkler, m., lamprecht, a., steinbauer, k., hülber, k., theurillat, j. p., breiner, f., choler, p. ertl, s., girón, a. g., rossi, g., vittoz, p., akhalkatsi, m., bay, c., alonso, j. l. b., bergström, t., carranza, m. l., corcket, e., dick, j., erschbamer, b., calzado, r. f., fosaa, a. m., gavilán, r. g., ghosn, d., gigauri, k., huber, d., kanka, r., kazakis, g., klipp, m., kollar, j., kudernatsch, t., larsson, p., mallaun, m., michelsen, o., moiseev, p., moiseev, d., molau, u., mesa, j. m., di cella, u. m., nagy, l., petey, m., pușcaș, m., rixen, c., stanisci, a., suen, m., syverhuset, a. o., tomaselli, m., unterluggauer, p. ursu, t., villar, l., gottfried, m., pauli, h., 2016: the rich sides of mountain summits–a pan‐european view on aspect preferences of alpine plants. journal of biogeography 43, 2261–2273. wright, d. h., 1983: species-energy theory – an extension of species-area theory. oikos, 41, 496–506. yavuz, f., fuchs, y., karakaya, n., karakaya, m. ç., 2008: chemical composition of tourmaline from the asarcık pb–zn– cu±u deposit, şebinkarahisar, turkey. mineralogy and petrology 94, 195–208. zanelli, r., egli, m., mirabella, a., giaccai, d., abdelmoula, m., 2007: vegetation effects on pedogenetic forms of fe, al and si and on clay minerals in soils in southern switzerland and northern italy. geoderma 41, 119–129. acta bot. croat. 80 (2), 2021 215 acta bot. croat. 80 (2), 215–216, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-017 issn 0365-0588 eissn 1847-8476 short communication bromus diandrus (poaceae), an addition to the bulgarian flora georgi kunev sofia university “st. kliment ohridski”, faculty of biology, department of botany, dragan tzankov” blvd. no. 8 “, sofia 1164, bulgaria abstract – the current report discusses bromus diandrus, a new species in the flora of bulgaria. a concise description of its morphological features with an emphasis on the characters that distinguish it from the already known representatives of bromus sect. genea in the country is presented. the newly established locality is characterized floristically and ecologically. the probabilities of the native or alien origin of the studied population are also discussed. keywords: anisantha diandra, bromus, dune vegetation, grate brome, native species introduction bromus diandrus roth (syn.: anisantha diandra (roth) tutin ex tzvelev) belongs to the infrageneric sect. genea dumort., which in europe comprises 8–11 taxa with species or subspecies rank (smith 1980, sales 1993, valdés et al. 2009). the section accommodates mostly ruderal species with annual or biennial life cycles. three taxa from the section, b. sterilis l., b. madritensis l. and b. tectorum l. have previously been confirmed from bulgaria (georgiev 1963, assyov and petrova 2012). the similar b. rigidus roth (reported as b. maximus desf.) was noted by stojanov et al. (1967) as having been erroneously recorded in bulgarian flora without any reference. the aim of the current report is to provide a brief description of the species, underlining the characters distinguishing it from other representatives of the section, to comment on aspects of its environmental requirements, habitat affiliation and the probable origin of the studied population. materials and methods the species was observed during field research into the coastal sands at irakli on the black sea coast, about 6 km south of the city of obzor, on 15 august, 2020. the distinctive features were noted in the field and after examination of the collected specimens under a stereomicroscope. to verify the identification, the collected specimens were compared with herbarium materials kept at so (nos. 07440, 82367) and som (nos. 113297, 137672, 140540) or as digital collections from e, p, b, wu and mw herbaria, available at gbif (2019). the taxonomy of the species follows smith (1980). exsiccata are deposited at so (nos. 108028–108030) and som (nos. 177047, 177048). habitat description is based on the author’s personal observations. the names of the accompanying species or subspecies are quoted after assyov and petrova (2012) and/or valcheva et al. (2020). results and discussion bromus diandrus is distinctive by reason of its robust appearance; lax and nodding one-sided panicles (fig. 1a); hirsute panicle axis and stem below the panicle (fig. 1b); larger glumes, lemmas, lemma lobes and awns as compared with the other representatives of sect. genea from bulgaria (fig. 1c). from b. sterilis it differs also by the general outline shape of the inf lorescence, the usually shorter panicle branches, which are not arched-pendulous, as well as the narrow and longer callus scar. from the similar b. rigidus, it is distinguished mainly by the callus scar, ovate, rounded at the end (vs. elliptic, pointed at the end), and by the presence of conspicuous constriction at the base of lemma (fig. 1d). for more comprehensive description of the representatives of the sect. genea refer to sales (1993). bromus diandrus is a winter annual species native to the irano-turanian, pontic and mediterranean regions but introduced elsewhere as an alien weed (cabi 2020). currently it has global distribution and occupies coastal sands or various types of anthropogenic habitats such as crop fields, waste places, roadsides, etc. (gbif 2019, cabi 2020). several locations are known from the coastline of the black sea and its surroundings crimea, southwestern russia, georgia and turkey (gbif 2019). most probably, the closest documented locality within the native range of the species to * corresponding author e-mail: gikunev@uni-sofia.bg kunev g 216 acta bot. croat. 80 (2), 2021 the one at irakli is in the vicinity of edirne, european turkey (webb 1966). more recently, the species was reported for the flora of north macedonia, preliminarily accepted as native (kostadinovski et al. 2019). the site of bromus diandrus at irakli has the approximate coordinates in wgs84: 42°44’53.22”n, 27°53’21.44”e. the habitat type is identified as b1.324 pontic white dunes (eunis 2007). the vegetation constitutes a narrow (5-15 m wide), sparsely vegetated linear strip, between a non-vegetated sand beach and the foot of sea cliff slope. the species composition is typical for the dune vegetation from the western pontic region. most abundant of the recorded species were leymus racemosus (lam.) tzvelev subsp. sabulosus (m.bieb.) tzvelev, elymus farctus (viv.) runemark ex melderis subsp. bessarabicus (savul. et rayss) melderis, eryngium maritimum l., cakile maritima scop. subsp. euxina (pobed.) nyár., salsola tragus l. subsp. tragus and vulpia fasciculata (forsskal) r.m. fritsch. much less abundant or present as single individuals were ammophila arenaria (l.) link, peucedanum obtusifolium sm., stachys maritima gouan, euphorbia peplis l., lactuca tatarica (l.) c.a.mey., xanthium orientale l. subsp. italicum (moretti) greuter, crambe maritima l. and pancratium maritimum l. in addition, some species typical for dry grassland communities were also recorded: bromus tectorum l., hypochaeris radicata l., centaurea salonitana vis., allium guttatum steven, silene dichotoma ehrh., alyssum hirsutum m.bieb., carthamus lanatus l., pyrus communis l. subsp. pyraster (l.) ehrh. at its first bulgarian site, bromus diandrus grows in more or less dense patches unevenly distributed over an area of less than 0.1 ha. between 15-120 culms per m² were counted, while the overall area covered by the species alone was approximately 200 m². considering the number of individuals and the population density, probably the species occurred in this area a long time ago. the rapid growth of tourism and its supporting infrastructure during the last 20 years has affected many natural sites along the bulgarian black sea coast. as a consequence, tourism can be expected to have affected the dune vegetation at the studied locality in terms of the introduction of untypical native or alien f lora. however, excluding xanthium orientale subsp. italicum, no other alien species were observed. this is in agreement with the evaluation of valcheva et al. (2020), who reported limited diversity and abundance of alien (incl. invasive) species in the coastal vegetation of bulgaria. in conclusion, it can be stated that the locality of bromus diandrus at irakli is insignificantly affected by trampling, unregulated campsites, campfires or other type of disturbances, thus its current state could be evaluated as close to natural. moreover, the species is found within its native range, the conditions correspond to its ecological requirements while the state of its population proves that it is well adapted to the environment at this locality. therefore, b. diandrus could be presumably regarded as native for the bulgarian flora. however, more studies are needed, particularly in the regions of the country influenced by more or less typical dry mediterranean climate in order to determine the status of the taxon reported here accurately. references assyov, b., petrova, a. (eds.), 2012: conspectus of the bulgarian vascular flora. distribution maps and floristic elements, ed. 4. bbf, sofia. cabi, 2020: bromus diandrus (great brome), invasive species compendium. wallingford, uk: cab international. retrieved november 21, 2020 from https://www.cabi.org/isc/ datasheet/10024 eunis, 2007: eunis habitat classification 2007, revised 2012, amended 2019. retrieved november 21, 2020 from https:// www.eea.europa.eu/data-and-maps/data/eunis-habitat-classification gbif, 2019: bromus diandrus roth in gbif secretariat (2019), gbif backbone taxonomy, checklist dataset https://doi. org/10.15468/39omei. retrieved november 21, 2020 from https://www.gbif.org/species/2703760 georgiev, t., 1963: bromus l. in: jordanov, d. (ed.), flora reipublicae popularis bulgaricae, vol. 1, 420–433. in aedibus academiae scientiarum bulgaricae, serdicae (in bulgarian). kostadinovski, m., ćušterevska, r., matevski, v., 2019: anisantha diandra (roth) tutin and ochlopoa infirma (kunth) h. scholz – new species of poaceae family in republic of macedonia. contributions. section of natural, mathematical & biotechnical sciences 40, 273–276. sales, f., 1993: taxonomy and nomenclature of bromus sect. genea. edinburgh journal of botany 50, 1–31. smith, p.m., 1980: bromus l. in: tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a., (eds.), flora europaea, vol. 5, 182–189. cambridge university press, cambridge. stojanov, n., stefanov, b., kitanov, b., 1967: bromus l. in: flora of bulgaria, ed. 4, vol. 2, 132–137. nauka i izkustvo, sofia (in bulgarian). valcheva, m., sopotlieva, d., apostolova, i., 2020: current state and historical notes on sand dune flora of the bulgarian black sea coast. flora 267, 151594. valdés, b., scholz, h., von raab-straube, e., parolly, g., 2009: poaceae (pro parte majore). euro+ med plantbase-the information resource for euro-mediterranean plant diversity. retrieved december 28, 2020 from http://ww2.bgbm.org/europlusmed/ webb, d.a., 1966: the flora of european turkey. in: proceedings of the royal irish academy. section b: biological, geological, and chemical science, pp. 1–100. royal irish academy. fig. 1. distinguishing features of bromus diandrus: a – one-sided semi-nodding panicle, b – upper stem and panicle axis, c – glumes and first lemma with up to 60 mm long awn, d – ovate callus scar with notable constriction at lemma’s base. acta bot. croat. 78 (2), 2019 175 acta bot. croat. 78 (2), 175–180, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0015 issn 0365-0588 eissn 1847-8476 phenolic profiles of quince (cydonia oblonga mill.) leaf extracts obtained by different extraction methods martina persic*, robert veberic, maja mikulic-petkovsek agronomy department, chair for fruit, vine and vegetable growing, biotechnical faculty, university of ljubljana, ljubljana, slovenia abstract – extracts from quince leaves are a well-known home remedy used for treating diverse health problems. most of the beneficial properties of quince leaf extracts may be assigned to their high content of phenolic compounds, particularly tannins. in this research, we have evaluated the efficiency of various methods for phenolic extraction from quince leaves and determined detailed phenolic profiles of different extracts. the results indicated that leaf drying is a suitable pretreatment for enhancing the extraction of phenolic compounds. higher extraction of phenolics was achieved at higher temperatures (i.e. infusion or decoction). phenolic profiles of quince leaf extracts differed among the extraction solvents and time of extraction. flavanols prevailed in extracts obtained by decoction and ethanolic maceration, while extracts obtained by maceration in water and infusion were rich in phenolic acids. a highly concentrated quince leaf extract was attained by ethanolic maceration, using a standard ratio of solvent and leaf material. keywords: decoction, infusion, maceration, phenolics, quince, tannins * corresponding author e-mail: martina.persic@bf.uni-lj.si introduction quince (cydonia oblonga mill.) trees are traditionally grown in private mediterranean gardens as ornamental and utilitarian trees. the quince fruit resembles an apple or a pear in its shape, is highly aromatic, firm and rich in tannins (lim 2012). the latter make the fruit inedible in its raw stage and therefore quince fruit is usually used for jam, marmalade, compote and in the production of juices, liqueurs and schnapps. additionally, quince fruit, seeds and leaves possess healing and health beneficial properties (khoubnasabjafari and jouyban 2011). in comparison to the fruit, quince leaves are characterized by fivefold higher content of total phenolics, a threefold higher content of polymeric proantocyanidins, a sixfold higher content of mono-, diand oligomeric flavan-3-ols, a 14-fold higher content of phenolic acids and a 13-fold higher content of flavonols (teleszko and wojdyło 2015). in traditional medicine, an infusion or decoction of dry quince leaves is often used to relieve problems of the gastrointestinal tract. the use of decoctions, hydro-ethanolic extracts and infusions of quince leaves is also recommended for their antidiabetic, antioxidant, antihemolytic and antihyperglycemic properties (sajid et al. 2015). the antidiarrheal properties of quince leaf infusion, widely used in home remedies, can be attributed to the exceptionally high tannin content (11%) of quince leaves (lim 2012). tannins are usually defined as phenolics with a high degree of polymerization. condensed tannins are oligomers and polymers of flavanols linked with c-c bonds (schofield et al. 2001) that are often found in the peel and pulp of different fruits. a shared feature of condensed and hydrolyzed tannins (so-called “true tannins”) is the precipitation of proteins. the group of “pseudo tannins” encompasses molecules of lower molecular weight (< 500 g mol–1) with low or no protein precipitating properties (theisen et al. 2014). the term “pseudo-tannins” derives from the astringent, tannin-like taste of these compounds (okuda et al. 1985). this group is formed from esters of hydroxycinnamic acids (chlorogenic, caffeic, ferulic and p-coumaric acid), monomeric flavanols ((epi)catechin, (epi)gallocatechin) and building blocks of hydrolyzed tannins (gallic and ellagic acid). moreover, “pseudo-tannins” can be speciesor genusspecific tannins (e.g. hamamelitannin from hamamelis virginiana l. or ipecacuanhic acid from carapichea ipecacuanha (brot.) (theisen et al. 2014). short communication persic m., veberic r., mikulic-petkovsek m. 176 acta bot. croat. 78 (2), 2019 tannins have both positive and negative effects on human health. the consumption of tannin-rich foods could decrease the nutrient conversion efficiency in the digestive system (chung et al. 1998). on the other hand, studies report antimutagenic, anticarcinogenic, anti-inflammatory and other health-beneficial properties of tannins (dai and mumper 2010). in this research detailed phenolic profiles of various quince leaf extracts were evaluated. extracts were obtained with decoction, maceration and infusion and all were prepared in a traditional way from fresh and dried quince leaves. cold, ultrasound assisted extraction was used as a standard reference method for laboratory extraction. the extracts were compared based on their phenolic profiles, as phenolics represent the main bioactive, health-beneficial compounds in quince leaf extracts. to our knowledge this is the first study that compares the detailed phenolic profiles of various extracts of quince leaf produced from uniform material in uniform conditions. materials and methods materials leaves were sampled from five-year-old 'champion' cultivar quince trees grown in a private garden in rijeka, croatia. leaves were collected on the 25th of july 2016, transported in a portable icebox to the laboratory of the biotechnical faculty in ljubljana, slovenia and immediately processed. methods prior to final assessment, test extractions were performed. different durations of maceration, decoction and infusion were tested. the adequate solvent to material ratios were acquired from the examination of traditional recipes and european pharmacopoeia (council of europe 2007). a portion of fresh quince leaves was dried in an oven at a constant temperature of 40 °c for 24 h. average water content of dry material for infusion and decoction was 28.2%. the remaining part of fresh quince leaves was used for fresh leaf extraction. maceration fresh quince leaves were de-stemmed and cut into small pieces. a mass of 2 g fresh quince leaves was extracted in 25 ml of 100% ethanol (mac etoh) or water (mac h2o), respectively. maceration was carried out at room temperature in sealed containers for 24 h. infusion and decoction half of gram of dried quince leaves was infused in 100 ml of distilled water. water was heated to the boiling point and immediately poured over the dried material in 250 ml beakers, which were then covered with petri dish. the leaves were left to extract for 15 minutes. decoction was conducted on 1 g of fresh (dec f) and 0.5 g of dried (dec d) material. the material was boiled for 15 minutes in 100 ml (dry leaves) or 150 ml (fresh leaves) of distilled water. after decoction, the extract was left to cool down for 5 minutes. during boiling, the volume of water evaporated to 50% of the initial level. cold, ultrasound assisted extraction the procedure was carried out according to the standard protocol for polyphenolic extraction in our laboratory (persic et al. 2018a; persic et al. 2017) with slight modifications. fresh quince leaves were de-stemmed and cut into small pieces. exactly 2 g of leaf material was placed into 10 ml centrifuge tubes and topped up with 25 ml of ethanol (etoh us) or 25 ml of water (h2o us). tubes were subsequently deposited into an ice filled sonis 4 gt ultrasonic water bath (iskra pio, šentjernej, slovenia) and extracted for 1 h at 50 hz. phenolic compounds analysis by hplc-ms after various extraction procedures, the samples were centrifuged and filtrated through 0.20 μm chromafil ao20/25 polyamide filters (macherey-nagel, düren, germany) into vials. the samples were further analyzed by the protocol described in persic et al. (2018). the determination of individual phenolics was carried out on a thermo finnigan surveyor high-performance liquid chromatography system (hplc, thermo fisher scientific, waltham, usa). phenolics were further identified on a mass spectrometer (ms, thermo fisher scientific) with electrospray ionization (esi) operating in negative ion mode. the concentration of individual phenolic compounds was calculated from corresponding calibration curves of standard solutions; procyanidin b1, catechin, epicatechin, p-coumaric acid, caffeic acid, 3-caffeoylquinic acid (3cqa), 4-caffeoylquinic acid (4cqa), 5-caffeoylquinic acid (5cqa), quercetin-rutinoside, quercetin-galactoside, quercetin-glucoside, quercetin-rhamnoside, kaempferol-glucoside and isorhamnetin-glucoside. all extractions were carried out in six replicates (five leaves per replicate). the concentration of phenolic compounds in samples was calculated per dry matter in order to enable accurate comparison of extracts processed from partially dry (inf and dec d) and fresh material (etoh us, h2o us, mac etoh, mac h2o and dect f). ultrasound assisted extraction was used as a reference method for efficient extraction of phenolic compounds. statistical analysis the statgraphics plus 4.0 program (manugistics. inc.; rockville, maryland, usa) was used for data analysis. significant differences in the concentration of individual phenolics, phenolic groups, total analyzed phenolics and total phenolics concentration among extracts were tested using one way analysis of variance (anova). duncan’s test was used to calculate significant differences in the concentration of phenolics among the different extraction procedures. p-values lower than or equal to 0.05 were considered statistically significant. in the graphs, significant differences among values are denoted by different letters. for graphic interpretation of the results, a heat map was plotted in r-commander using phenolic profile of quince (cydonia oblonga mill.) leaves acta bot. croat. 78 (2), 2019 177 the gplot package (r formation for statistical computina, anckland, new zeland) based on standardised data (µ = 0, σ = 1). ward’s method based on the square euclidian distance was used for hierarchical cluster analysis and grouping of varieties according to their individual phenolic compounds. results and discussion the optimum maceration time was 24 h and the optimum duration for quince leaf infusions and decoctions was 15 minutes. in all, twenty-nine phenolic compounds, belonging to the groups of flavanols, flavonols and phenolic acids, were identified and quantified in the extracts of quince leaves analyzed. in all quince extracts analyzed, the group of flavonols is composed of seven derivatives of kaempferol, four derivatives of quercetin, and isorhamnetin pentoside (fig. 1, on-line suppl. tab. 1). quercetin-rutinoside is most dominating flavonol in all quince leaf extracts except dec f, whose flavonol profile is dominated by quercetin-galactoside (fig. 2). the distinct grouping of ethanolic and water extract based on ward’s method with squared euclidean distance is also observable in fig. 1. additionally, it is noticeable that the decoction of fresh material (dec f) has the most distinctive flavanol profile with quercetin-galactoside, kaempferolglucoside and kaempferol-rhamnosylhexoside iv as its main constituents. the distinctive grouping of ethanolic extracts is probably due to the lower content of all kaempferol-rhamnosylhexoside isomers in comparison to other extracts. the overall highest concentration of flavonols from quince leaves was achieved by the decoction of dry quince leaves (5.4 ± 0.5 g kg–1 dw) and by ethanolic maceration (4.7 ± 0.3 g kg–1 dw) (fig. 2, on-line suppl. tab. 1). significantly lower concentration of flavonols was detected in samples acquired with us in ethanol (3.1 ± 0.1 g kg–1 dw), infusion (3.5 ± 0.3 g kg–1 dw) and decoction of fresh material (2.7 ± 0.2 g kg–1 dw). the lowest concentration of flavonols was determined in extracts prepared with cold (h2o us) and room temperature (mac h2o) extractions in water. high flavonol levels are typical of phenolic extracts from leaves since these compounds play an important role in protection against excessive uv radiation (jakopic et al. 2009). similar results in alcoholic extraction of phenolics from apple leaves were acquired by jakopic et al. (2009). the group of phenolic acids in quince leaves is composed of seven caffeoylquinic (cqa) derivatives and five coumaric acid derivatives (fig. 3, on-line suppl. tab. 2). in contrast to earlier findings, we have identified five additional derivatives of coumaric acid in this species’ leaves; additional isomers of 3cqa, 5cqa, 5-coumaryilquinic acid and p – cumaric acid hexoside (costa et al. 2009, oliveira et al. 2007, teleszko and wojdyło 2015). 5cqa is the most abundant phenolic acid in all extracts except dec f. it is interesting that combined content of 3cqa and 5cqa i represented as much as 85% of all analyzed phenolic acids in water infusion. furthermore, caffetannins in quince leaf infusion encompass 3cqa, 4cqa, 5cqa i, fig. 1. the flavonol profile in various quince leaf extracts; ultrasound extraction in water (h2o us), ultrasound extraction in ethanol (etoh us), water maceration (mac h2o), ethanolic macerate (mac etoh), water infusion (inf ), decoction of dry material (dec d) and decoction of fresh material (dec f). the data are standardised (µ = 0, σ = 1), low values are presented with light colors, higher values are presented with dark colors. q-quercetin, k-kaempferol. fig. 2. concentration of the phenolic groups (flavonols, phenolic acids (pa) and flavanols) in various extracts from quince leaves; ultrasound extraction in water (h2o us), ultrasound extraction in ethanol (etoh us), water maceration (mac h2o), ethanolic macerate (mac etoh), water infusion (inf ), decoction of dry material (dec d) and decoction of fresh material (dec f). the comparison was made regarding phenolic group, for each extract separately. lowercase letters (a, b, etc.) indicate significant differences among concentration of flavonols in various extracts, bold lowercase letters (a, b, etc.) indicate significant differences among concentration of pa in various extracts, while uppercase letters (a, b, etc.) stand for significant differences among the concentration of flavanols in various extracts; determined by the duncan test (p < 0.05). standard error is presented with error bar. persic m., veberic r., mikulic-petkovsek m. 178 acta bot. croat. 78 (2), 2019 5cqa ii and dicaffeoylquinic acid i, and represent 95% of all identified phenolic acids and more than 50% of total analyzed phenolics in all analyzed extracts from quince leaves. grouping of various extracts regarding the profile of phenolic acids showed significant separation of dec f and etoh us extracts. the separation of dec f from the rest of analyzed extracts is due to its lower concentration of 5cqa and significantly higher concentration of 4cqa. meanwhile, the separation of extract obtained by ultrasound extraction in ethanol (etoh us) is mainly based on the higher concentration of isomers of dicaffeoylquinic acid and lower concentration of the derivatives of coumaroylquinic and coumaric acid. regarding the specific extractability of individual phenolic acids in various solvents, 5cqa ii was better extracted in ethanol, while 3cqa was better extracted in water. overall, the highest total concentrations of analyzed phenolic acids were measured in samples prepared with water infusion inf (9.9 ± 1.0 g kg–1 dw) followed by dec d (7.3 ± 0.8 g kg–1 dw), dec f (4.0 ± 0.3 g kg–1 dw), both macerations (mac etoh and mac h2o) and finally, us extractions (etoh us, h2o us) (fig. 2, on-line suppl. tab. 2). additionally, the highest ratio of phenolic acids in comparison to other phenolic groups was detected in water extractions preformed at temperatures below the boiling point. fig. 3. the profile of phenolic acids in various extracts from quince leaves; ultrasound extraction in water (h2o us), ultrasound extraction in ethanol (etoh us), water maceration (mac h2o), ethanolic macerate (mac etoh), water infusion (inf ), decoction of dry material (dec d) and decoction of fresh material (dec f). the data are standardised (µ = 0, σ = 1), low values are presented with light colors, higher values are presented with dark colors. cqa – caffeoylquinic acid. fig. 4. the profile of flavanols in various extracts from quince leaves; ultrasound extraction in water (h2o us), ultrasound extraction in ethanol (etoh us), water maceration (mac h2o), ethanolic macerate (mac etoh), water infusion (inf ), decoction of dry material (dec d) and decoction of fresh material (dec f). the data are standardised (µ = 0, σ = 1), low values are presented with light colors, higher values are presented with dark colors. meanwhile, the highest concentration of flavanols was achieved using a decoction of dry material (dec d 20.8 ± 0.6 g kg–1 dw), followed by maceration in ethanol (mac etoh 7.2 ± 0.3 g kg–1 dw), decoction of fresh leaves (dec f 6.1 ± 0.5 g kg–1 dw) and water infusion (inf 4.2 ± 0.7 g kg–1 dw) (fig. 2). significant variability in the efficiency of extraction of individual flavanols was detected among the extraction methods. epicatechin and procyanidin trimer ii accounted for more than 50% of all analyzed flavanols in the various analyzed extracts from quince leaves. extractability of procyanidin trimer ii was generally higher in water than in ethanol, and at lower rather than at higher temperatures (fig. 4, online suppl. tab. 3). extraction of catechin from quince leaves was higher at higher temperatures, while catechin was dominant only in extracts acquired by the decoction of dry material. these results are compatible with previous findings on the solubility of phenolic compounds described by mikulicpetkovsek et al. (2015). the higher concentration of epicatechin in inf, dec d, etoh us, and in mac etoh can be explained through degradation of phenolic compounds during drying of plant material before extraction for inf and dec d, while ethanol could play an important part in the degradation of oligomeric to monomeric flavanols in etoh us and mac etoh. the grouping of extracts using ward’s method with square euclidian distance showed separation of analyzed extracts based on the content of epicatechin (fig. 4). the highest level of total analyzed phenolics was detected in samples prepared with decoction of dry material (dec d 34.7 ± 1.2 g kg–1 dw) (suppl. fig. 1). a lower content of total analyzed phenolics was measured in extracts in the following order: infusion of dried leaves inf (17.6 ± 1.9 g kg–1 dw) > mac etoh (14.6 ± 2.2 g kg–1 dw) > dec f (12.8 ± 0.8 g kg–1 dw) > mac h2o (5.8 ± 2.7 g kg–1 dw) ≈ etoh us (6.8 ± 0.2 g kg–1 dw) > h2o us (3.3 ± 0.2 g kg–1 dw). as a rule, all extraction techniques (with the exception of water macerate) were more effective in extraction of phenolic compounds than standard laboratory methods (us extracphenolic profile of quince (cydonia oblonga mill.) leaves acta bot. croat. 78 (2), 2019 179 tions). lower content of phenolic compounds in water macerate can partially be ascribed to the oxidation and degradation of phenolic compounds during 24 h extraction at room temperature (khachatryan et al. 2005). among the analyzed extracts, mac etoh is the most concentrated in terms of mass of phenolic compounds per liter of extract (951.9 ± 8.5 mg l–1) (on-line suppl. fig. 1). the lowest concentration of total analyzed phenolics was detected in inf (66.0 ± 6.9 mg l–1) and dec f (107.0 ± 6.7 mg l–1). the decoction obtained from dry material (dec d 230.8 ± 24.7 mg l–1) was twice as concentrated as the decoction obtained by fresh quince leaf processing (91.6 ± 5.5 mg l–1). the difference in water content between fresh leaves and partially dried material was only 5.8% and therefore, lower concentration of phenolics in fresh leaf extracts cannot solely be explained by the variation in the initial water content. however, the higher concentration of phenolic compounds in dec d than in dec f may be explained by pretreatment of the material, i.e. 24 h drying at 45 °c. additionally, mokrani and madani (2016) confirmed the significant effect of solvent type, temperature and time of maceration on the extraction of total phenolics. because of the differences in the extractability of phenolic compounds, substantial differences in the ratio among flavanols, phenolic acids and flavonols were detected in quince leaf extracts. the most noticeable differences in phenolic profiles were recorded between inf and dec d samples. the flavonols: phenolic acids: flavanols ratio in infusion was 1:3:1 and the ratio in dec d was 1:1:3 (fig. 2). in this research, the specific solubility of phenolic compounds and even isomers of the same molecule was observed in various solvents. this could be due to different polarity, which affects the solubility of isomeric molecules (gaikar and phatak 1999). in addition to phenolic profiling of various quince leaf extracts, the aim of this research was to evaluate various extracts based on their phenolic profile. ethanolic extract (mac etoh) was approx. 14-fold more concentrated than quince leaf water infusion. additionally, the phenolic profiles of these two extracts differ significantly. quince leaf infusion consists of 58% phenolic acids, 19% flavonols and 23% flavanols but, by contrast, an ethanolic macerate consists of 21% phenolic acids, 31% flavonols and 48% flavanols. on the other hand, water macerate (mac h2o) is comparable to quince leaf infusion (inf ). the former was characterized by an almost 6-fold more concentrated phenolic composition per liter of extract and a similar phenolic profile to that from the infusion. as little as 17 ml of water macerate (mac h2o) could replace 100 ml of infusion (inf ). according to our results, pretreatment (i.e. drying) had a significant positive effect on the extractability of phenolic compounds from quince leaves. abascal et al. (2005) reviewed the impact of drying on the extraction of phenolic compounds from various plant samples and concluded that there is no uniform effect of drying on phenolic extraction or on altered phenolic profiles of the extracts. to our knowledge, no previous findings report the differences in phenolic extraction from dried and fresh quince leaves. in conclusion, our results demonstrate the poor efficiency of ultrasound assisted extraction for the extraction of quince leaf phenolic compounds compared to other examined methods. drying followed by decoction of quince leaves stands out as the most favorable method for the extraction of polyphenolic compounds. acknowledgements this work is part of the program horticulture p40013-0481, supported by the slovenian research agency (arrs). references abascal, k., ganora, l., yarnell, e., 2005: the effect of freeze‐drying and its implications for botanical medicine: a review. phytotherapy research 19, 655–660. chung, k.t., wong, t.y., wei, 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1847-8476 coastal sand dune vegetation of velika plaža (montenegro) danijela stešević1, filip küzmič2, đorđije milanović3, milica stanišić-vujačić1, urban šilc2* 1 faculty of natural sciences and mathematics, university of montenegro, džordža vašingtona bb, 81000 podgorica, montenegro 2 research centre of the slovenian academy of sciences and arts, institute of biology, novi trg 2, 1000 ljubljana, slovenia 3 faculty of forestry, university of banja luka, s. stepanovića 75a, 78000 banja luka, bosnia and herzegovina abstract – velika plaža (ulcinj, montenegro) is the largest sandy beach along the coast of the eastern adriatic that still has well-developed sand-dune vegetation. although the characterization of the flora and vegetation of velika plaža has been addressed by many authors, knowledge on its vegetation remained poor. we made a phytosociological study of sandy beach vegetation comprising both dunal and wetland areas to provide a comprehensive survey of sand dune vegetation and habitat typology of velika plaža. based on 149 relevés (both from literature and recent field work), and with numerical classification (flexible beta) and ordination (non-metric multidimensional scaling) our results show that the vegetation of velika plaža is much more diverse than previously known. altogether, 19 plant communities from 6 vegetation classes were identified. among them we described two new associations: cuscuto cesatianae-phyletum nodiflorae and onobrychido caput-galli-vulpietum fasciculatae. keywords: classification, endangered, habitat types, plant communities, psammophytic, sandy beach, syntaxonomy * corresponding author e-mail: urban@zrc-sazu.si introduction sand dunes are one of the most extreme ecosystems due to abiotic environmental factors, and among the most endangered, due to various kinds of human impact (maun 2009). these two factors account for the high research interest of the remaining sandy beaches and their biota. thirty coastal habitats are included in the european red list of habitats as they are experiencing decline in extent and quality (janssen et al. 2016). in fact it is estimated that about 70% of dune ecosystems of european coasts were lost during the last century due to urbanization (brown and mclachlan 2002). velika plaža (fig. 1) is the largest sandy beach along the eastern adriatic coast with still well-developed vegetation. coasts along the ne adriatic are mainly rocky and steep (see šilc et al. 2016a), and anthropogenic impact on many sandy beaches has destroyed or depauperated the vegetation, so that in some locations (nin and velika saplunara, croatia) only fragments are still developed. surveys of sand dune vegetation (marcenò et al. 2018, šilc et al. 2016a) show gaps of vegetation data along the eastern adriatic coast and our study contributes to filling them. characterization of flora and vegetation of velika plaža have been researched by many authors (bubanja 2016, bubanja et al. 2019, and references therein). recently several studies for different purposes were conducted to present the impact of alien species (stešević et al. 2017a, šilc et al. 2019), trampling (šilc et al. 2017), and litter on vegetation (šilc et al. 2018) as well as human impact in general (šilc et al. 2016b), but a complete overview of vegetation communities has never been made. according to several authors (trinajstić 1989a, mijović 1994, mijović et al. 2006, 2012,) only two plant communities were reported for velika plaža: cakilo-xanthietum italici and euphorbio paraliae-agropyretum junceiformis; however, recent studies (šilc et al. 2019, stešević et al. 2017b) have suggested that the vegetation of velika plaža is much more diverse. the aim of our study was to make a comprehensive survey of sand dune vegetation of the largest sand beach system in eastern adriatic with own field work and literature data, 44 acta bot. croat. 79 (1), 2020 stešević d, küzmič f, milanović đ, stanišić-vujačić m, šilc u and to present our results also in the view of habitat typology of habitat directive (european communities council 1992). materials and methods study area velika plaža in ulcinj (montenegro) is considered to be the northernmost and longest beach on the eastern adriatic coast (ca. 12 km), with still well preserved sand dune vegetation. since 1968, the beach has been recognized as a monument of nature (official gazette of the socialist republic of montenegro, srcg 30/68). according to the spatial plan of special purpose for the coastal zone, one spatial unit on the east side of velika plaža is recognized as a newly protected nature reserve, with forests, marshes and meadows. the area is protected from trampling or grazing with a wooden fence (jpmd 2015). however, the rest of the beach, which represents more than 75% of the natural coast and 97% of whole sand dune system, is heavily impacted by tourism, illegal dumping, sand extraction and urbanization (petrović and karaman 2009). eleven natura 2000 habitats are reported for the beach and its hinterland: annual vegetation of drift lines (1210), embryonic shifting dunes (2110), shifting dunes along the shoreline with ammophila arenaria (white dunes, 2120), fixed coastal dunes with herbaceous vegetation (grey dunes, 2130*), humid dune slacks (2190), dunes with euphorbia terracina (2220), mediterranean salt meadows (juncetalia maritimae) (1410), brachypodietalia dune grasslands with annuals (2240), wooded dunes with pinus pinea and/or pinus pinaster (2270*), mediterranean temporary ponds (3170), and salix alba and populus alba galleries (92a0) (petrović et al. 2012). the site is included in the list of ipa areas (petrović and karaman 2009). sampling and data analysis in the period of 2012–2019 we recorded 93 relevés of all the vegetation types found on the sand dune system according to the standard central european method (braun-blanquet 1964, van der maarel 2005). the new relevés made on sand dunes (on-line suppl. tabs. 1 and 2) and some from the literature (trinajstić 1989a, mijović 1994, mijović et al. 2006, 2012,) were entered into the turboveg 2 database (hennekens and schaminée 2001). for the first step, numerical classification, we used a larger (149 original and published relevés) and a smaller (93 original relevés) dataset to find out the general structure. as both analyses revealed similar pattern, for further numerical analysis we used only the original relevés as those from the literature had large plot sizes, were not made for syntaxonomical study or were frequently transitional. nevertheless we used them in ordination analysis of whole dataset. numerical classification (flexible beta (β = -0.25) and relative sørensen) was performed on the 93 original relevés. some of the transitional relevés were later classified according to expert knowledge. diagnostic species for the plant communities were determined in the juice program (tichý 2002) by their fidelity values (chytrý et al. 2002). the size of all groups was standardized to equal sizes (tichý and chytrý 2006), and the fisher’s exact test (p < 0.05) was applied. species with phi-coefficient values higher than 0.20 were considered as diagnostic. unweighted average ecological indicator values (pignatti et al. 2005) of relevés were passively projected onto a non-metric multidimensional scaling (nmds) biplot (bray-curtis distance measure used) to show ecological relationship among these relevés and to explain environmental gradients underlying the main ordination axes. log transformed cover data were used as the input data and all 149 relevés were used in this analysis. analysis was performed in program r (r development core team 2012) using the vegan package in the juice program (tichý 2002). the nomenclature of taxa follows euro+med (2006). syntaxonomical concepts and nomenclature of higher syntaxa follow mucina et al. (2016). for the crosswalk between vegetation syntaxa and habitat types, we refer to the catalogue of habitat types of montenegro important for the europe (petrović et al. 2012). results we compiled a set of 149 relevés (93 original) and identified 19 plant communities classified into six vegetation classes. they are presented in the syntaxonomical scheme and described in brief. the first classification, using the whole dataset of 149 relevés (not shown), identified two main clusters separating sand dune and wetland vegetation on the sandy beach. in the next step we classified each of these two clusters separately (figs. 2, 3). fig. 1. sandy beach velika plaža in montenegro. acta bot. croat. 79 (1), 2020 45 coastal sand dune vegetation of velika plaža tab. 1. shortened synoptic table of studied syntaxa. diagnostic species with fidelity index phi higher than 0.20 are presented. columns: 1 – juncetum maritimo-acuti, 2 – limonio narbonensis-juncetum gerardii, 3 – cuscuto cesatianae-phyletum nodiflorae, 4 – eriantho-schoenetum nigricantis, 5 – holoschoenetum romani, 6 – cakilo-xanthietum strumarii, 7 –euphorbio paraliae-agropyretum junceiformis, 8 – medicagini marinae-ammophiletum australis, 9 – cutandia maritima community, 10 – euphorbio terracinae-silenetum nicaeensis, 11 – onobrychido caput-galli-vulpietum fasciculatae, 12 – scirpetum maritimo-litoralis, 13 – phragmitetum communis, 14 – typhetum angustifoliae, 15 – scirpetum lacustris, 16 – cladietum marisci, 17 – cyperetum longi, 18 – cyperetum flavescentis, 19 – cypero-paspaletum distichi. column 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 number of relevés 4 2 12 7 4 8 7 6 5 7 7 7 4 1 1 7 1 1 2 juncus maritimus 57.6 ----25.6 3.5 ----------------------------juncus acutus 42 ------------------------------------juncus gerardi --83.4 ----3 ----------------------------phyla nodiflora ----49 ----------------12.5 --------------cuscuta australis ssp. cesatiana ----43 --------------------------------centaurium spicatum ----21.7 --------------------------------polypogon maritimus ----24.7 --------------------------------bidens frondosus ----24.2 --------------------------------schoenus nigricans ------74.5 ------------------------------tripidium ravennae ------75.2 ------------------------------rubus ulmifolius ------28.5 ------------------------------linum bienne ------24.5 ------------------------------serapias vomeracea ------20.7 ------------------------------scirpoides holoschoenus ----22.3 7.8 30.3 ----------------------------carex distans --------20.3 ----------------------------lysimachia vulgaris --------20.3 ----------------------------xanthium orientale ssp. italicum ----11.7 ----12.2 --------------------------cakile maritima ----------17.7 10.3 ------------------------salsola kali ----------18.2 ----23.8 --------------------cyperus capitatus ------------35.3 19.6 ----------------------echinophora spinosa ----------12 29.9 13.6 ----------------------eryngium maritimum ----------11 29 13.6 ----------------------pancratium maritimum ------------22.7 --7.1 --9.7 ----------------equisetum ramosissimum ------------26.5 ------------------------anisantha madritensis ------------24.6 ------------------------ammophila arenaria --------------66.8 ----------------------cutandia maritima ----------------57 --------------------phleum arenarium ----------------40.8 --------------------anisantha sterilis ----------------23 --21.8 ----------------medicago littoralis --------------9.7 20.5 10.2 11.6 ----------------calystegia soldanella --------------8.6 20.3 --------------------euphorbia terracina ------------------57.3 ------------------artemisia campestris ------------9.5 ----40.6 ------------------medicago marina ------------12.1 --7.4 42.6 ------------------vulpia fasciculata --------------3 --39.3 47.2 ----------------alkanna tinctoria ------------------33.3 27.8 ----------------hedypnois rhagadioloides ------------------25.5 ------------------valerianella sp. ------------------20.7 ------------------onobrychis caput-galli ----------------5.3 --70.6 ----------------hypochaeris glabra --------------------30.9 ----------------lagurus ovatus ------------9.7 ------28 ----------------typha latifolia --------------------24.9 ----------------dasypyrum villosum --------------------20.7 ----------------bolboschoenus maritimus --4.6 ------------------48.1 --------------juncus compressus ----------------------22.1 --------------phragmites australis ------------------------62.8 ------------baldellia ranunculoides ------------------------21 ------------nymphaea alba ------------------------20.3 ------------butomus umbellatus ------------------------29.3 ------------schoenoplectus lacustris ssp. glaucus --------------------------31.6 ----------potamogeton lucens --------------------------26 20.6 --------utricularia vulgaris --------------------------27.3 27.3 --------schoenoplectus litoralis ----------------------11.6 ----68.6 --------cladium mariscus ------------------------------86.7 ------gratiola officinalis ------------------------------20.4 ------cyperus longus --------------------------------96.7 ----potentilla reptans --------------------------------52.6 ----oenanthe pimpinelloides ----3.8 --------------------------33.7 ----poa trivialis ------7.5 ------------------------31.9 ----calystegia sepium --------------------------------52.2 ----rumex conglomeratus --------------------------------35.1 ----vicia villosa ssp. varia --------------------------------36.7 ----verbena officinalis --------------------------------32.4 ----carex hirta --------------------------------40.7 ----holcus lanatus --------------------------------40.7 ----daucus carota --------------------------------36.7 ----quercus robur --------------------------------36.7 ----crepis vesicaria --------------------------------36.7 ----cyperus flavescens ----------------------------------90.4 --plantago major ssp. intermedia ----------------------8 ----------30.4 --paspalum distichum ----1.8 ------------------------------85.6 paspalum dilatatum ------------------------------------38.8 46 acta bot. croat. 79 (1), 2020 stešević d, küzmič f, milanović đ, stanišić-vujačić m, šilc u syntaxonomical scheme juncetea maritimi br.-bl. in br.-bl. et al. 1952 juncetalia maritimi br.-bl. ex horvatić 1934 juncion maritimi br.-bl. ex horvatić 1934 juncetum maritimo-acuti horvatić 1934 cuscuto cesatianae-phyletum nodiflorae ass. nova hoc loco holotypus: tab. 2, relevé no. 10 limonio narbonensis-juncetum gerardii géhu et biondi 1994 agropyro-plantaginion maritimi horvatić 1934 holoschoenetum romani tchou 1948 cakiletea maritimae tx. et preising ex br.-bl. et tx. 1952 thero-atriplicetalia pignatti 1953 euphorbion peplidis tx. ex oberd. 1952 cakilo-xanthietum strumarii (beg. 1941) pignatti 1958 (syn. xanthio-cakiletum maritimae, salsolo kali-cakiletum maritimae costa et manz. corr. riv.-mart. et al. 1992) ammophiletea br.-bl. et tx. ex westhoff et al. 1946 ammophiletalia br.-bl. et tüxen ex westhoff et al. 1946 ammophilion br.-bl. 1921 euphorbio paraliae-agropyretum junceiformis tüxen in br.-bl. et tüxen 1952 corr. darimont, duvigneaud et lambinon 1962 (syn. sporobolo-elymetum farcti (gehu et al.) gehu 1984, agropyretum mediterraneum (kuhn.) br.-bl. 1933, echinophoro-elymetum farcti gehu 1988, eryngio-cyperetum capitati dmitar lakušić nom.nud. 2011) medicagini marinae-ammophiletum australis br.-bl. 1921 corr. f. prieto et t.e. díaz 1991 (syn. ammophiletum australis r. lakušić 1965 inedit) cutandia maritima community isoëto-nanojuncetea br.-bl. et tx. in br.-bl. et al. 1952 nanocyperetalia klika 1935 nanocyperion koch 1926 cyperetum flavescentis w.koch ex aichinger 1933 verbenion supinae slavnić 1951 cypero-paspaletum distichi horvatić 1954 phragmito-magnocaricetea klika in klika et novák 1941 bolboschoenetalia maritimi hejny in holub et al. 1967 scirpion maritimi dahl et hadač 1941 scirpetum maritimo-litoralis (br.-bl. in br.-bl., roussine et nègre 1952) o. de bolòs 1962 phragmitetalia koch 1926 phragmition communis koch 1926 phragmitetum communis savič 1926 typhetum angustifoliae soo 1927 scirpetum lacustris chouard 1924 saccharetalia ravennae biondi, blasi et casavecchia in biondi et al. 2014 imperato cylindricae-saccharion ravennae br.-bl. et o. de bolos 1958 eriantho-schoenetum nigricantis (pignatti 1953) géhu in géhu et al. 1984 magnocaricetalia pignatti 1953 magnocaricion elatae koch 1926 cladietum marisci allorge 1921 cyperetum longi micevski 1957 helianthemetea guttati rivas goday et rivas-mart. 1963 vulpietalia pignatti 1953 (syn. cutandietalia) laguro-vulpion fasciculatae géhu et biondi 1994 euphorbio terracinae-silenetum nicaeensis lavrentiades 1964 onobrychido caput-galli-vulpietum fasciculatae ass. nova hoc loco holotypus: tab. 3, relevé no. 3 acta bot. croat. 79 (1), 2020 47 coastal sand dune vegetation of velika plaža diagnostic species obtained by fidelity coefficient can only be considered as local because of the limited study area and many transitional stands (tab. 1). diagnostic species contain both differential and character species, some of which can be diagnostic for two plant communities. salt marshes juncetum maritimo-acuti is dominated by tall rushes (juncus maritimus and j. acutus) and forms large stands in velika plaža. it thrives in the back of the dunes on sand-silty substrate and is periodically flooded. scirpetum maritimo-litoralis is a community dominated by bolboschoenus maritimus, which occupies flooded areas with brackish water. cuscuto cesatianae-phyletum nodiflorae ass. nova is found in flooded depressions on sand dunes that develop between higher parts of the dunes or behind them or along smaller streams of sub-saline water, additionally sometimes also on heavily trampled surfaces (tab. 2). the soil is often silty and becomes compacted during summer droughts. the diagnostic species is cuscuta australis ssp. cesatiana, known as a frequent inhabitant of sandy habitats, parasitizing on polygonum maritimum, xanthium orientale ssp. italicum and other psammopyhtes (pignatti 1982). limonio narbonensis-juncetum gerardii is a helophytic community that thrives in stagnant brackish water that dries out during summer. stands are species poor, dominated by juncus gerardi. holoschoenetum romani thrives in depressions most commonly in the backdunes. these are the lowest sites in the dune system and are flooded for the longest period of time. fig. 2. classification of sand dune vegetation of velika plaža. clusters: 1 – cakilo-xanthietum strumarii, 2 – euphorbio paraliaeagropyretum junceiformis, 3 – cutandia maritima community, 4 – medicagini marinae-ammophiletum australis, 5 – euphorbio terracinae-silenetum nicaeensis, 6 – onobrychido caput-galli-vulpietum fasciculatae. fig. 3. classification of wetland vegetation of velika plaža. clusters: 1 – juncetum maritimo-acuti, 2-4 – holoschoenetum romani, 5 – cyperetum flavescentis, 6 – cyperetum longi, 7 –limonio narbonensisjuncetum gerardii, 8 – cypero-paspaletum distichi, 9-11 – scirpetum maritimo-litoralis, 12 – cuscuto cesatianae-phyletum nodiflorae, 13 – phragmitetum communis, 14 – typhetum angustifoliae and scirpetum lacustris, 15 – cladietum marisci, 16 – eriantho-schoenetum nigricantis. sand dunes cakilo-xanthietum strumarii is found on the sand deposition zone, the first part of the beach following the aphytic zone. it is a species poor community, strongly influenced by sea waves, intense sand movement and salt water spraying. it occupies the nutrient rich drift line. the association is characterized by the presence of an alien species xanthium orientale subsp. italicum, which is very abundant on velika plaža in the first and the second vegetation zone. the following plant communities on the sand dunes in the sea-inland zonation are still subjected to strong wind, waves, salt spray, and drought. embryonic dunes are occupied by the stands of euphorbio paraliae-agropyretum junceiformis. this vegetation type is very common on velika plaža and occupies flat, low sand dunes. in general sand dunes on velika plaža are low compared to those in albania or italy. the next community in the zonation is the medicagini marinae-ammophiletum australis, where elytrigia juncea is replaced by ammophila arenaria, which further stabilizes the sand and makes these shifting dunes higher than the embryonic dunes. ammophila arenaria stands are small and patchy on velika plaža, forming loosely connected “dune islands” with steeper slopes. wind and water erosion are more pronounced and the development of these stands is a result of micro topography and edaphic conditions and they are rare on velika plaža. on the other hand embryonic, semi-fixed and fixed dunes are syndynamically connected and represent successional series (fig. 4). 48 acta bot. croat. 79 (1), 2020 stešević d, küzmič f, milanović đ, stanišić-vujačić m, šilc u tab. 2. phytosociological table of the cuscuto cesatianae-phyletum nodiflorae from velika plaža (ulcinj, montenegro). * – nomenclatural type. relevé number 1 2 3 4 5 6 7 8 9 10* 11 12 relevé number in on-line suppl. tab. 1 7 8 9 10 11 12 13 14 15 16 17 18 relevé area (m2) 4 4 4 4 4 4 4 4 4 4 4 2 cover herb layer (%) 70 60 70 45 60 40 80 70 70 70 50 80 diagnostic species phyla nodiflora 4 4 4 3 3 3 4 4 4 1 2 4 cuscuta australis subsp. cesatiana . . 3 . 2 1 + + 1 1 1 2 juncetea maritimi scirpoides holoschoenus 1 + . 1 1 + 3 2 3 3 2 1 juncus maritimus . . . . . . 2 1 + 1 . . polypogon maritimus 1 + . . . . . 1 + + . . oenanthe pimpinelloides . . . . . . . . . . + + samolus valerandi . + . . . . + . . . . . phragmitetea phragmites australis + . . + + + + . . . . . typha angustifolia . . . + . + 2 . . . . . bolboschoenus maritimus . 1 . . . 1 1 . . . . . ammophiletea tripolium pannonicum subsp. tripolium + . . + 1 . . . + 1 + . echinophora spinosa . . . . . . . . . . r r cynanchum acutum . . . . . . . . + . . . molinio-arrhenatheretea mentha aquatica . . . 1 1 + + . . + . . juncus articulatus . + + 1 . . + . . . . . lythrum salicaria . + + . . + . . . . . . lotus corniculatus + + . . . . . . . . . . gratiola officinalis . . + . 1 . . . . + . . juncus compressus . . . . . + . . . . . . other species xanthium orientale subsp. italicum + . 1 1 2 1 1 . 2 2 3 1 bidens frondosus . . . + 1 . . . . + + + centaurium spicatum + . . . . . + 1 1 2 + . paspalum distichum + + 1 . . 1 + . . . . . dittrichia viscosa + . . + . . . + . . + . euphorbia maculata + . 1 . 1 . . . . . . . ranunculus sp. . . . . . . . . . . + + atriplex prostrata . . . . . . . . . . r r amorpha fruticosa . . . . . . r . . . r . salix alba . . . + . . . . . . r . lythrum hyssopifolia . . 1 . . . + . . + . . centaurium erythrea . + . . . . . . . . . + species present only in one relevé: tamarix dalmatica 1: +; rumex conglomeratus 1: +; hypochaeris radicata 2: +; parapholis incurva 3: +; vitex agnus-castus 4: +; baldellia ranunculoides 4: +; leontodon sp. 5: +; persicaria maculosa 5: 1; cynodon dactylon 7: 1; medicago littoralis 8: +; periploca graeca 9: +; plantago major ssp. intermedia 9: +; limbarda crithmoides 10: +; populus alba 11: +. fig. 4. scheme of typical vegetation zonation on the sand beach velika plaža. acta bot. croat. 79 (1), 2020 49 coastal sand dune vegetation of velika plaža semi-fixed dunes with euphorbia terracina are found behind the embyonic shifting dunes and are the next stage in the succession. this asssociation can be placed floristically between euphorbio-agropyretum and onobrychidi-vulpietum, although it is more similar to the latter association. we classified it as euphorbio terracinae-silenetum nicaeensis. fixed dunes are the last in the zonation before the onset of woody vegetation. the most widespread community is the ass. nova dominated by vulpia fasciculata and onobrychis caput-galli, which are also the diagnostic species of the association (tab. 3). stands occupy the greatest part of sand dunes, which are predominantly flat, compared to the previous dune zones with consistent (even if small) slopes, and are in a mosaic with ammophiletea communities. grasslands have the most closed vegetation cover which becomes more open due to human impact. fragmentarily, on stabilized dunes, there are also stands of cutandia maritima, representing the first stages of development towards onobrychido-vulpietum. typical sand dune vegetation is best preserved in the south-eastern part of the beach, which is so far less touristically developed. dune slacks among other sand dune communities a great diversity of usually monodominant plant communities appears in wet dune slacks and depressions. their floristic composition depends on the salinity of the water and period of flooding. phragmitetum communis is dominated by phragmites australis and is widely distributed in the area, mostly in the back of the dunes where fresh water is present for a longer period. typhetum angustifoliae is present in patches within phragmites australis stands or around ponds in the hinterland. scirpetum lacustris is found in brackish water and is connected to phragmites stands but flooded for longer periods. eriantho-schoenetum nigricantis stands are developed in the depressions between the dunes with tripidium ravennae and schoenus nigricans as edificators of community. the site is flooded by subsaline water. cladietum marisci occupies large areas with cladium mariscus as the dominant species. cyperetum longi thrives on wet soils, often near channels and is dominated by the tall sedge cyperus longus. tab. 3. phytosociological table of the onobrychido caput-galli-vulpietum fasciculatae from velika plaža (ulcinj, montenegro). * – nomenclatural type. relevé number 1 2 3* 4 5 6 7 relevé number in on-line suppl. tab. 1 63 64 65 66 67 68 69 relevé area (m2) 20 15 25 15 15 16 8 cover herb layer (%) 70 80 100 60 80 85 80 diagnostic species vulpia fasciculata 3 2 5 3 3 3 2 onobrychis caput-galli 2 3 4 3 4 4 2 alkanna tinctoria 2 + 3 . 1 . 1 helianthemetea guttati medicago littoralis + . 3 1 . + + hypochaeris glabra . 1 2 . 1 + 1 lagurus ovatus . . + 2 + + 1 ammophiletea pancratium maritimum 1 2 . . + + + cyperus capitatus 1 1 . . 2 2 . echinophora spinosa + + . . + + . medicago marina 3 2 . + . . . elytrigia juncea . . . + + + . eryngium maritimum + . . . 1 . + calystegia soldanella . . . . . . . euphorbia terracina . . . . . 2 + other species crepis foetida 1 + . 1 1 + + anisantha sterilis . . . 1 . + 3 erigeron annuus . + . . + + . chondrilla juncea . . . 1 . + + phleum arenarium . . . 1 . . . xanthium orientale ssp. italicum . . . r . . . dasypyrum villosum . . . . + . 1 silene conica . . + . 1 . . verbascum sinuatum . . . . . + + petrorhagia saxifraga . . . . . + 1 oenothera sp. + 2 . . . . . other species: pseudorlaya pumila 1: +; linum trigynum 1: +; euphorbia sp. 2: 1; anagallis arvensis 2: +; linum bienne 3: +; plantago lanceolata 3: r; cutandia maritima 3: r; avena barbata 4: +; cynodon dactylon 7: +; imperata cylindrica 7: +; carthamus lanatus 7: +; tragopogon porrifolius 7: +; plantago bellardii 7: +; scirpoides holoschoenus 7: +; oenothera biennis agg. 50 acta bot. croat. 79 (1), 2020 stešević d, küzmič f, milanović đ, stanišić-vujačić m, šilc u two dwarf cyperaceous communities developed on small surfaces are cyperetum flavescentis and cypero-paspaletum distichi. they are found on wet, often flooded soils. two paspalum species dominate the stands; they are often trampled and their occurrence could be anthropogenic. moisture, salinity and nutrients are the most important ecological factors influencing the vegetation composition and spatial distribution of sand dunes (fig. 5). these variables are also correlated to the first axis and salinity is correlated to the second axis of the nmds plot. on the left side of the ordination diagram vegetation types of marshes and swamps in the centre are periodically flooded communities and on the right side communities of sand dunes are grouped. the centroid of group 2 (limonio-juncetum gerardii) is ecologically very similar to isoëto-nanojuncetea communities. plant communities identified on velika plaža can be translated into eight habitat types according to habitat directive (tab. 4). tab. 4. classification of syntaxa (plant associations) on velika plaža into habitat directive habitat types (european communities council 1992). marceno et al. 2018 natura 2000 habitat type (annex i 92/43/eec) syntaxa zone without vegetation zone without vegetation mudflats and sandflats not covered by seawater at low tide 1140 deposition zone (drift line zone) strand line annual vegetation of drift lines 1210 cakilo-xanthietum strumarii   embryonic dune embryonic dune embryonic shifting dunes 2110 euphorbio paraliae-agropyretum junceiformis white dune foredune shifting dunes along the shoreline with ammophila arenaria 2120 medicagini marinae-ammophiletum australis fixed dune dunes with euphorbia terracina 2220 euphorbia terracinae-silenetum nicaeensis malcolmietalia dune grasslands 2230 onobrychido-vulpietum fasciculatae dune slack dune slack mediterranean salt meadows (juncetalia maritimae) 1410 juncetum maritimo-acuti cuscuto cesatianae-phyletum nodiflorae limonio narbonensis-juncetum gerardii scirpetum maritimo-litoralis phragmitetum communis typhetum angustifoliae scirpetum lacustris cladietum marisci humid dune slacks 2190 eriantho-schoenetum nigricantis cyperetum flavescentis cypero-paspaletum distichi cyperetum longi holoschoenetum romani fig. 5. non-metric multidimensional scaling (nmds) ordination spider-plot of the vegetation of velika plaža. numbers refer to centroids of vegetation types and dots represent relevés linked to the corresponding centroid (tab. 1). ecological indicator values are represented as arrows (moist-moisture, react-soil reaction, light-light, nutr-nutrients, cont-continentallity, temp-temperature). plant communities found in similar site conditions are outlined. stress value in two dimensions was 0.12. acta bot. croat. 79 (1), 2020 51 coastal sand dune vegetation of velika plaža discussion the vegetation of the sandy beach velika plaža is very diverse in vegetation types and many of them have not previously been recorded there or anywhere else in montenegro. the transitional geographic position of velika plaža between the adriatic and ionian seas makes clear syntaxonomic classification of the plant communities of sand dunes difficult. the vegetation of velika plaža was frequently studied since the first mentions by blečić and lakušić (1976). trinajstić (1989a) recorded the association sporobolo-elymetum farcti and later mijović (mijović 1994, mijović et al. 2006, 2012) studied the zonation of sand dune vegetation, but mentioning only cakilo-xanthietum italici and echinophoro-elymetum farcti (=agropyretum mediterraneum). all these authors identified only two plant communities: one on the drift line and the second on the dunes, without dividing it into different associations based on zonation and the stability of the dunes (doing 1985, marcenò et al. 2018). the dunes at velika plaža are still well developed and the ideal zonation of dunes is still recognizable (fig. 4) although human impact is present (šilc et al. 2016a). we have confirmed several marsh and swamp associations, which have already been reported for ulcinj and montenegro in general but without relevé material (blečić and lakušić 1976). additionally, horvatić (1963) reported the presence of euphorbio paraliae-agropyretum for budva-sutoran area in montenegro, which does not exist anymore. certain similarities can be found with the results of the vegetation study of the buna river protected landscape in albania (fanelli et al. 2015), which is a natural continuation of velika plaža to the south. however, in the sand dune vegetation on velipoje beach today only drift line vegetation is present. the reasons are mainly erosion, but also the fast tourism development and intensive human impact on coastal areas in albania. still in the mid-1980s mullaj (1989) found complete zonation on the sand dunes of velipoje with cakilo-xanthietum italici, agropyretum mediterraneum, ammophiletum arundinaceae and sporoboletum. in croatia sand dunes can be found fragmentarily in a few places with more or less depauperated plant composition of typical sand dune associations (horvatić 1963, trinajstić 1989b, trinajstić and jasprica 1998, šilc et al. 2016b). horvatić (1963) reports agropyretum mediterraneum, euphorbio-glaucietum petrosum, and also cyperetum flavescentis and cypero-paspaletum distichi. on the velika plaža we described a new plant association cuscuto cesatianae-phyletum nodiflorae. the species phyla nodiflora (=lippia n.) was first recorded for ex-yugoslavia in port-milena channel, near ulcinj (bajić 1963). in the literature ph. nodiflora occurs in four associations as a diagnostic species: lippio nodiflorae-panicetum repentis o. de bolòs 1957, lippio nodiflorae-paspaletum vaginati galán de mera et al. 2009, fimbrystylo cymosae-lippietum nodiflorae de foucault 1987 and kyllingo peruvianae-phyletum nodiflorae vanden berghen 1990 (de foucault 1987, vanden berghen 1990). only the first is present in europe, while the others are typical for peru, guadalupe and senegal, respectively. the association from velika plaža is very similar to lippio nodiflorae-panicetum repentis, especially to stands from sicily (brullo and sciandrello 2006), where ph. nodiflora is a co-dominant species, while in spain these are short, dense grass carpets of panicum repens sometimes even without ph. nodiflora (royo pla 2006). syntaxonomical classification of lippio-panicetum varies among different authors. de bolòs (1957) originally classified it into the alliance trifolio fragiferi-cynodontion dactyli, and the classification is followed by royo pla (2006) and ninot et al. (2012). brullo and sciandrello (2006) classified it into paspalo-agrostion semiverticillati of the class molinio-arrhenatheretea. mucina et al. (2016) classified the alliance into bidentetea tripartitae. taking into account the ecological features of the habitat of the new association cuscuto cesatianae-phyletum nodiflorae and its floristic structure and composition, we classify it into the alliance juncion maritimi, order juncetalia maritimi, and the class juncetea maritimi. in the hinterland of velika plaža, the species ph. nodiflora thrives within stands of juncus maritimus. such a community is also reported for the viluni lagoon in albania in the vicinity of velika plaža. it develops in retrodunal depressions characterized by brackish water and strong disturbance by erosion and grazing by cattle (fanelli et al. 2015). due to the presence of similar habitats along the albanian coast, it is expected that the association cuscuto cesatianae-phyletum nodiflorae will have a wider and more southwards distribution, although the species is not mentioned in the coastal flora of albania or in the list of albanian plant communities (mullaj 1989, dring et al. 2002). classification of the newly described association onobrychido-vulpietum ass. nova into higher syntaxa presents a particular challenge. stands show a floristic composition distinct from other plant communities in the zonation and a description of a new association is therefore justified. there are no literature references of a similar plant community from neighbouring croatia or albania (šilc et al. 2016b). there are two similar associations reported from italy: onobrychido caput-galli-malcolmietum ramosissimae brullo, scelsi et spampinato 2001 and silene nicaeensis-vulpietum fasciculatae (paradis and piazza 1991) géhu et biondi 1994, but their floristic composition is different and characteristic species are not present in velika plaža (díez-garretas et al. 2003). therophytic stands on semi-stabilised sand dunes in europe are classified into different alliances in addition to the different synsystems present. mucina et al. (2016) introduced two ephemeral therophytic orders on sand dunes of the class helianthemetea guttati: vulpietalia under salt-spray influence and malcolmietalia without it. within the first one three alliances from the balkan peninsula area are classified: psammo-vulpion from the north adriatic coasts, vulpio-lotion along balkan-illyric coast and maresion nanae on north aegean sand dunes. inclusion of the newly described association into vulpio-lotion would be geographically most eligible but stands lack many characteristic species, mainly of the genus trifolium (horvat et al. 1974). in fact, typical vul52 acta bot. croat. 79 (1), 2020 stešević d, küzmič f, milanović đ, stanišić-vujačić m, šilc u pio-lotion therophytic grasslands are found in the hinterland of velika plaža and are not under the influence of saltspray. in montenegro in general grasslands of vulpio-lotion are developed mainly on inland lowlands and not on the sand dunes (pers. obser.). a different classification was previously presented by biondi et al. (2014) for italy including two orders within the helianthemetea guttati on sand dunes. cutandietalia (a synonym for vulpietalia) comprises two alliances alkanno-maresion nanae and laguro-vulpion. the main difference between these alliances is in the disturbance of sand dunes (brullo et al. 2001). the latter includes annual, xerophytic, pioneer communities of mediterranean and thermo-atlantic dunes in an early state of alteration owing to both natural and anthropic causes. characteristic species (vulpia fasciculata, lagurus ovatus) are also present in the stands of onobrychido-vulpietum, also under constant anthropogenic pressure, which corresponds to the alliance description. according to some authors, alkanno-maresion nanae and laguro-vulpion could be merged together with some additional alliances due to a lack of differential species, which would yield one alliance, spanning the whole northern mediterranean coast (díez-garretas et al. 2003). on the other hand the alliance laguro-vulpion fasciculatae was put into the chenopodietea class by mucina et al. (2016) indicating anthropogenic influence. in our opinion onobrychido-vulpietum should be classified into the alliance laguro-vulpion as an additional part of the vulpietalia order. we classified the stands dominated by euphorbia terracina into the existing association euphorbio-silenetum nicaeensis, described from greece. it is found on more stabilised dunes (lavrentiades 1964) and was classified into the crucianellion maritimae alliance (sýkora et al. 2003). two out of three characteristic species according to lavrentiades (1964) are present (euphorbia terracina and hedypnois cretica), but we are of the opinion that these stands should also be classified into the therophytic alliance laguro-vulpion instead of the chamaephyte-dominated alliance crucianellion. the floristic similarity of euphorbio-silenetum nicaeensis and onobrychido-vulpietum is well supported by the dendrogram (fig. 2) and ordination (fig. 5). classification of paspalum distichum (=p. paspalodes) dominated stands is dubious. at velipoje they are classified as paspalo-agrostidetum br.-bl. 1936 (1952) (fanelli et al. 2015) but in nearby bosnia and herzegovina (hutovo blato) as cypero-paspaletum distichi horvat 1954 (jasprica et al. 2003). these communities are classified in different classes, molinio-arrhenatheretea and isoëto-nanojuncetea, respectively. our stands fit better into the latter class with characteristic ephemeral, annual vegetation of flooded sites, although they are fragmentarily developed, and with many character and differential species (veronica anagalloides, lotus tenuis, bidens tripartitus, inula britannica, persicaria lapathifolia) missing. fanelli et al. (2015) classified the association eriantho-schoenetum nigricantis into the class molinio-arrhenatheretea, but we are of the opinion that it should be classified into the alliance imperato cylindricae-saccharion ravennae and in the class phragmito-magnocaricetea, as suggested by mucina et al. (2016). association holoschoenetum romani was traditionally classified into molinio-holoschoenion (molinio-arrhenatheretea), but recently mucina et al. (2016) described this alliance as seasonally flooded meadows on subsaline soils of the western mediterranean. on the other hand, biondi et al. (2014) list the alliance agrostio stoloniferae-scirpoidion holoschoeni (a synonym of molinio-holoschoenion) for italy. we are the opinion that this community should be classified into the juncetea class. limonio narbonensis-juncetum gerardii is traditionally classified into juncion maritimae alliance (juncetea maritimi), but in our case it shows high floristic similarity with the class isoëto-nanojuncetea (fig. 5). cyperus longus appears as the dominant species in two communities: sparganio-cyperetum longi horvatić 1939 and cyperetum longi micevski 1957 (hadžiablahović 2018). the latter is found on velika plaža in depressions, usually in contact with holoschoenetum vulgaris stands (landucci et al. 2013). although habitat types occurring on velika plaža have already been reported (petrović et al. 2012) we can now list them with the corresponding species composition. it should be pointed out that some of the plant communities were sampled for the first time on this beach. for protection of the sand dune system a prerequisite is to have a survey of plant communities and their translation into habitat typology (tab. 3). some of the listed habitats, however, should be translated into new types in the future: dunes along the mediterranean shoreline with ammophila arenaria (new code 2280) and mediterranean embryonic dunes (new code 2290) as proposed by feola et al. (2011). in the first report of habitat types in montenegro cakilo-xanthietum strumarii was classified into habitat type 2110 (petrović et al. 2012), but it should be distinguished as a particular habitat type, annual vegetation of drift lines 1210. habitat type 2220 with euphorbia terracina was placed separately but our analysis shows high floristic similarity with onobrychido-vulpietum fasciculatae (habitat type 2230) and an analysis on a larger scale is needed to confirm the existence of this habitat type on velika plaža. the most stable dunes were previously classified into fixed coastal dunes with herbaceous vegetation (grey dunes) (2130*), but they should be now translated into malcolmietalia dune grasslands (2230) (biondi et al. 2012, petrović et al. 2012, prisco et al. 2012). a plant community with euphorbia terracina was not confirmed in a previous survey of sand dune vegetation along the eastern adriatic (šilc et al. 2016b). our results confirm for the first time the presence of plant communities of laguro-vulpion (and corresponding habitat types) along the north-eastern adriatic coast and additional survey of such sand dunes is needed particularly in albania to record any further stands to get a more comprehensive view on the vegetation type in the area. transitional sand dune habitats (e.g. 2230 and 2210) are those with the highest acta bot. croat. 79 (1), 2020 53 coastal sand dune vegetation of velika plaža diversity of phytosociological associations (prisco et al. 2012). important habitat types that are present on velika plaža but not sampled in our study are 2270* wooded dunes with pinus pinea and/or pinus pinaster and 91e0* alluvial forests with alnus glutinosa and fraxinus excelsior (alno-padion, alnion incanae, salicion albae). although pine forests in velika plaža are planted they are an important habitat type and may maintain the “valuable” mediterranean coastal biodiversity pool (bonari et al. 2017). riparian forests are under-researched in south-eastern europe (douda et al. 2016) and many of them have already been destroyed in the vicinity in albania (kárpáti and kárpáti 1961). there is a potential for the existence of habitat type 2240 brachypodietalia dune grasslands with annuals, which might be confirmed in the further vegetation sampling. acknowledgement the research (d.s., u.š., f.k.) was partly financed through the rufford project (14048-1), programme p1-0236 (arrs), and bilateral grant (bi-me/16-17-018). d.m. acknowledges support by daphne – inštitút aplikovanej ekológie. authors are grateful to mihailo jovičević and danka petrović for help during field work and discussions. references bajić, d., 1963: species l. nodiflora rich new to flora of yugoslavia. radovi poljoprivrednog fakulteta univerziteta u sarajevu 12, 209–211 (in bosnian). biondi, e., blasi, c., allegrezza, m., anzellotti, i., azzella, m.m., carli, e., casavecchia, s., copiz, r., del vico, e., facioni, l., galdenzi, d., gasparri, r., lasen, c., pesaresi, s., poldini, l., sburlino, g., taffetani, f., vagge, i., zitti, s., zivkovic, l., 2014: plant communities of italy: the vegetation prodrome. plant biosystems 148, 728–814. biondi, e., burrascano, s., casavecchia, s., copiz, r., del vico, e., galdenzi, d., gigante, d., lasen, c., spampinato, g., venanzoni, r., zivkovic, l., blasi, c., 2012: diagnosis and syntaxonomic interpretation of annex i habitats (dir. 92/43/eec) in italy at the alliance level. plant sociology 49, 5–37. blečić, v., lakušić, r., 1976: list of plant communities of montenegro. glasnik republičkog zavoda za zaštitu prirode i prirodnjačkog muzeja u titogradu 9, 57–98 (in montenegrin). bonari, g., acosta, a.t., angiolini, c., 2017: mediterranean coastal pine forest stands: understorey distinctiveness or not? 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and commercial biocide based on silver ions and hydrogen peroxide željko d. savković, miloš č. stupar*, milica v. ljaljević grbić, jelena b. vukojević university of belgrade, faculty of biology, institute of botany and botanical garden “jevremovac”, studentski trg 16, 11000 belgrade, serbia abstract – the antifungal activities of origanum vulgare essential oil (eo) and of a biocide based on silver and hydrogen peroxide (sanosil s003) against seven aspergillus species isolated from different substrata (stone, brick, silk and paper) of cultural heritage objects in serbia were evaluated. microdilution, agar dilution and microatmosphere methods were used to determine minimal fungistatic and minimal fungicidal concentrations (mic and mfc), and light microscopy to determine structural abnormalities. mic and mfc values for o. vulgare eo ranged from 0.2 to 5 mg ml−1 and for sanosil s003 from 5 to 250 mg ml−1. aspergillus sp. sect. fumigati was the most susceptible isolate, where mic and mfc values were achieved at 0.5 mg ml−1 for o. vulgare eo, while mic and mfc values for sanosil s003 were achieved at 5 and 10 mg ml−1, respectively. morpho-physiological changes were documented in all isolates, including lack of sporulation, depigmentation of conidiogenous apparatus and conidia, and presence of aberrant fungal structures. o. vulgare eo exhibited stronger anti-aspergillus activity than sanosil s003, as demonstrated by the higher mic and mfc values and fewer morpho-physiological changes observed in the tested sanosil s003 concentrations. o. vulgare eo could be an excellent alternative to commercial biocides, with high potential in the fi eld of cultural heritage conservation. keywords: antifungal activity, aspergllus spp., cultural heritage, essential oil, origanum vulgare, sanosil s003 abbreviations: cya – czapek yeast extract agar, eo – essential oil, mea – malt extract agar, meb – malt extract broth, mic – minimal inhibitory concentration, mfc – minimal fungicidal concentration * corresponding author, e-mail: smilos@bio.bg.ac.rs introduction aspergillus is a genus of fungi, including more than 180 recognized species with worldwide distribution. several species have attracted attention as human and animal pathogens or because of their ability to produce various mycotoxins (samson et al. 2010). species of this genus are considered one of the most frequent infestation agents of cultural heritage objects (florian 2002, hu et al. 2013) and are capable of producing masses of airborne conidia and can grow and reproduce on many different carbon sources, including a variety of materials of which cultural heritage artifacts are composed (paper, textile, frescoes etc.) (garg et al. 1995, florian 2002). they are also involved in the degradation of a broad range of organic substrata (goldman and osmani 2007) due to their proteolytic, cellulolytic and amylolytic activity (borrego et al. 2010, 2012a, eida et al. 2011). a vast number of microfungi, including aspergillus species, are able to produce different organic and inorganic acids during their metabolic activities on monument surfaces (farooq et al. 2015). this leads to biodeterioration of stone substrata (such as sandstone, limestone, marble, mortar, brick etc.). furthermore, aspergilli are able to produce different pigments and contribute to the formation of biofi lms, which diminish the esthetic value of cultural heritage artifacts and accelerate their biodeterioration (vivar et al. 2013). prevention of mold growth is nowadays a signifi cant challenge for restorers, conservators and architects since chemical treatments must be non-destructive and non-toxic (sterfl inger 2010). in the fi eld of the safeguarding of the cultural heritage, both synthetic and natural biocides are applied. although substances with biocidal activities extracted from plants are commonly used in medicine and food preservation, their savković ž. d., stupar m. č., ljaljević grbić m. v., vukojević j. b. 122 acta bot. croat. 75 (1), 2016 use in the control of mold growth on cultural heritage objects is little reported (de saravia et al. 2008). oregano (origanum vulgare l., lamiaceae) is a widely known aromatic plant that has been used in agricultural, pharmaceutical and cosmetic industries (şahin et al. 2004). essential oil (eo) of this plant is recognized as an excellent natural source of substances with biological activities, such as terpens and phenolic compounds (sivropoulou et al. 1996). the antimicrobial, antioxidative and cytotoxic activities of o. vulgare eo have already been demonstrated by sivropoulou et al. (1996) and şahin et al. (2004), while its antifungal activity has been documented in numerous studies (e.g. adam et al. 1998, clef et al. 2013, delić et al. 2013) along with several studies concerning anti-aspergillus activity (e.g. viuda martos et al. 2007, carmo et al. 2008, souza et al. 2010). commercial biocides include numerous organic compounds, metals, oxidizing agents and various synthetic products that have been widely used in the fi eld of cultural heritage conservation (warscheid and braams 2000). silver and its compounds are known to be one of the most effective antimicrobial agents (russel 1994). hydrogen peroxide is an oxidizing agent capable of transforming numerous organic molecules (petri et al. 2011). it has been documented that this compound exhibits antibacterial and antifungal properties (baldry 1983). there are several studies concerning the antifungal activity of biocides based on silver ions and hydrogen peroxide (tasić 2009, abdel-mageed et al. 2012) but, to our knowledge, very few regarding aspergillus species (e.g. nabizadeh et al. 2008). the aim of this study was to evaluate the in vitro antifungal activity of origano vulgare eo, a natural product, and also of a synthetic biocide based on silver ions and hydrogen peroxide, on selected aspergillus species isolated from cultural heritage objects in serbia. materials and methods essential oil origanum vulgare l. eo (frey+lau, ulzburg, germany), a commercial sample from the collection of the institute for medicinal plant research “dr josif pančić” in belgrade, was used in the experiment. the chemical composition of the tested eo was reported by stupar et al. (2014). the main components were carvarcrol (64.06%), linalool (17.56%), p-cymene (4.44%) and thymol (3.86%). according to the manufacturer’s guidelines, the quality of the tested eos corresponds to european pharmacopeia 6 (2007). commercial biocide the commercial biocide sanosil s003 (sanosil ltd.) was obtained, as a water solution of fi nal concentration 2.7% (silver nitrate, 0.2%; and hydrogen peroxide, 2.5%), from the institute for protection of cultural monuments in serbia. aspergillus isolates seven aspergillus species used in this study were isolated from different substrata of cultural heritage objects in serbia: aspergillus sp. 1 sect. flavi (isolated from stone sculpture in the museum of contemporary art, belgrade); aspergillus sp. 2 sect. flavi (brick wall of arača church, novi bečej); aspergillus sp. sect. circumdati and aspergillus sp. sect. nigri (silk icon from central institute for conservation in belgrade); aspergillus sp. sect. terrei and aspergillus sp. sect. fumigati (archive paper from central institute for conservation in belgrade); aspergillus sp. sect. nidulantes (stone wall, church of the stara pavlica monastery, raška). all tested isolates were identifi ed to section level using identifi cation keys: raper and fennell (1965) and samson et al. (2010). isolated fungi were deposited in the mycotheca of the department for algology, mycology and lichenology, institute of botany, faculty of biology, university of belgrade. isolates were maintained on malt extract agar (mea), and czapek yeast extract agar (cya), stored at 4 °c and subcultured once a month. antifungal assays the antifungal activities of the selected eo and the biocide sanosil s003 were investigated using three different methods: microdilution, agar dilution and microatmosphere methods. microdilution and microatmosphere methods were used for testing the antifungal activity of eo, while the biocide sanosil s003 was tested using microdilution and agar dilution methods. microatmosphere method the test was performed in sterile petri plates (85 mm dia.) containing 20 ml of mea (maruzzella and sicurella 1960). tested aspergilli were inoculated at the center of the mea medium, using a sterile needle under a stereomicroscope (stemi dv4, zeiss), after which the petri plates were turned over. a sterilized fi lter paper disc, soaked with various concentrations of o. vulgare eo, was placed in the center of the petri plate lid interior. concentrations of tested oil ranged from 0.1 to 5 mg ml−1. plates were incubated for 21 days at 25 °c, during which the fungal growth was monitored weekly. after cultivation period, minimal inhibitory concentrations (mics), defi ned as the lowest concentration of added eo with no visible fungal growth on mea, were determined. minimal fungicidal concentrations (mfcs) were determined by re-inoculation of treated inoculums onto sterile mea. the lowest concentrations of eo giving no visible growth after re-inoculation were regarded as mfcs. agar dilution method the modifi ed mycelia growth assay with mea was used to investigate the antifungal activity of the biocide sanosil s003 (ishii 1995). the stock solution of biocide (2.7%) was further diluted in melted mea in petri plates to make fi nal concentrations of the biocide ranging from 10 to 250 mg ml−1. the fungi were inoculated at the center of the mea. plates were incubated for 21 days at 25 °c. mic and mfc values were determined in the same manner as described above. antifungal activity of origanum vulgare l. essential oil and sanosil s003 acta bot. croat. 75 (1), 2016 123 microdilution method to determine the antifungal activity of o. vulgare eo and the commercial biocide, the modifi ed microdilution technique was used (hanel and raether 1998, daouk et al. 1995). conidial suspensions of selected aspergilli were prepared by washing conidia from the surface of the 7 days old mea slants with sterile saline (nacl 0.85%, hemofarmhospitalogica) containing 0.1% tween 20 (sinex laboratory). using a hemocytometer (reichert, warner-lambert technologies) conidia were counted on a 1 mm2 surface and the concentration of conidia in the suspensions were calculated per formula: number of conidia / mm2 × 104 × dilution the conidia suspension was adjusted to a concentration of approximately 1.0 × 107 in a fi nal volume of 100 μl per each well. the inocula were stored at –20 °c for further use. dilutions of all aspergilli inocula were transferred onto solid mea to verify contamination absence and in order to check the validity of the inocula. determination of the mics was performed by a serial dilution technique using 96-well microtiter plates. different volumes of investigated eo and biocide sanosil s003 were dissolved in malt extract broth (meb) medium with aspergilli inoculums (10 μl) to make the same fi nal concentrations, as those used in microatmosphere and agar dilution methods. the microtiter plates were incubated for 72 h at 28 °c. the lowest concentrations of tested biocides without visible growth under a binocular microscope were defi ned as the concentrations that completely inhibited aspergilli growth (mics). the minimum fungicidal concentrations (mfcs) were determined by serial subcultivation of inocula (2 μl) into microtiter plates containing 100 μl of meb medium. the lowest concentration with no visible growth was defi ned as the mfc, indicating 99.5% killing of the original inoculum. microscopic analysis after the incubation period, a sample of mycelium was taken from the periphery of a colony grown on mea enclosed with evaporated eo (microatmosphere method) or on mea enriched with different concentrations of sanosil s003 (agar dilution method). a sample of mycelium was also taken from microwell of a microtiter plate. the samples were dyed and fi xed with glycerol and observed under a light microscope (zeiss axio imager m.1, with axiovision release 4.6 software) to examine structural abnormalities. samples from the control plate without oil were also stained and observed. additional observation was done 7 days after the incubation period in order to assess sustainability of morphological changes. results fungal susceptibility to essential oil high fungistatic and fungicidal activity of o. vulgare eo was demonstrated with low mic and mfc values. mic values ranged between 0.2 and 2.5 mg ml−1 and mfc values varied between 0.5 and 5 mg ml−1, obtained in both methods used (fig. 1). aspergillus sp. sect. fumigati was the most susceptible isolate to eo treatments and fungicidal effect was achieved at 0.5 mg ml−1. the most resistant isolates were aspergillus sp. sect. nigri and aspergillus sp. sect. terrei. for these isolates, fungicidal effect in microdilution method was achieved only with the highest tested eo concentration (5 mg ml−1). furthermore, lower mic and mfc values were obtained in microatmosphere method for most isolates. fungal susceptibility to commercial biocide the biocide sanosil s003 exhibited moderate antifungal activity, signifi cantly lower than o. vulgare eo. mic values from 5 to 250 mg ml−1 and mfc values from 10 to 250 mg ml−1 were obtained in both methods used (fig. 1). aspergillus sp. sect. fumigati was the most susceptible isolate to biocide treatments and fungicidal effect was achieved at 10 mg ml−1. in the agar dilution method, fungicidal effect for both isolates from flavi section and for aspergillus sp. sect. circumdati was obtained only with the highest tested biocide concentration (250 mg ml−1). lower mic and mfc values were obtained in the microdilution method for most isolates. fig. 1. minimal inhibitory concentration (mic) and minimal fungicidal concentration (mfc) values of origanum vulgare essential oil and sanosil s003 on tested aspergillus isolates obtained by: a) microatmosphere and agar dilution methods, b) microdilution method. values in y axis are shown in logarithmic scale. savković ž. d., stupar m. č., ljaljević grbić m. v., vukojević j. b. 124 acta bot. croat. 75 (1), 2016 morpho-physiological changes morpho-physiological changes, such as depigmentation of conidiogenous apparatus and conidia, lack of sporulation and presence of aberrant fungal structures, in addition to slower mycelial growth, were observed at eo concentrations lower than mics obtained for each isolate (tab. 1). aberrant formation of conidial heads, including changes in shape and color, was observed in aspergillus sp. sect. nigri colonies grown at eo concentration of 0.1 mg ml−1 (fig. 2, tab. 1). vesicles had a squashed appearance and were sometimes depigmented. metulae were not developed – phialides were directly formed on vesicles in contrast to the biseriate conidial heads observed in control sample. additionally, hyphae of aspergillus sp. sect. nigri had swellings and apical buddings (on-line suppl. fig. 1). pigmentation loss and shape change in reproductive structures were observed in aspergillus sp. 1 sect. flavi colonies grown at eo concentration of 2.5 mg ml−1 (on-line suppl. fig. 2). in biocide concentrations lower than the mics obtained in the agar dilution and microdilution methods, fewer morpho-physiological changes were documented in the tested isolates (tab. 1). these included lack of sporulation and depigmentation of conidiogenous apparatus and conidia. no morpho-physiological changes were documented in any of the tested concentrations of the biocide in the agar dilution method, although slower mycelial growth was observed in concentrations lower than the mics obtained for each isolate. tab. 1. observed morpho-physiological changes in tested aspergillus isolates at different concentrations of origanum vulgare essential oil and sanosil s003, in microdilution method (after 72 h) and in microatmosphere and agar dilution method (after 21 day). legend: ma – microatmosphere method, md – microdilution method, ad – agar dilution method, (+) – growth without morpho-physiological changes, (–) – no growth, dp – depigmentation of conidiogenous apparatus, ls – lack of sporulation, ac – aberrant conidiogenous apparatus development isolates concentrations of o. vulgare eo (mg ml−1) concentrations of biocide sanosil s003 (mg ml−1) 0.1 0.2 0.5 1 5 10 20 40 ma md ma md ma md ma md ad md ad md ad md ad md aspergillus sp. 1 sect. flavi + dp, ls + dp, ls dp – – – + + + + + dp, ls + – aspergillus sp. 2 sect. flavi dp dp, ls dp dp, ls dp – – – + + + + + + + dp, ls aspergillus sp. sect. circumdati + dp, ls dp, ls dp, ls – dp, ls – – + dp + dp + dp, ls + – aspergillus sp. sect. nigri dp, ac ls – dp, ls – – – – + + + + + + + – aspergillus sp. sect. terrei + dp, ls dp, ls dp, ls dp dp, ls – dp, ls + dp, ls + dp, ls + dp, ls + – aspergillus sp. sect. fumigati + dp, ls dp, ls dp, ls – – – – + – + – + – + – aspergillus sp. sect. nidulantes + dp, ls – dp, ls – – – – + dp, ls + dp, ls + – + – fig. 2. morphological changes of aspergillus sp. sect. nigri reproductive structures observed in microatmosphere method (essential oil in concentration of 0.1 mg ml−1): a) normal conidiogenous apparatus (control); b–d) aberrant conidiogenous apparatus. scale bars = 10 μm. antifungal activity of origanum vulgare l. essential oil and sanosil s003 acta bot. croat. 75 (1), 2016 125 discussion the results of the microdilution and microatmosphere methods showed that various concentrations of o. vulgare eo exhibited relatively strong fungistatic and fungicidal activity against the tested aspergillus isolates. the antifungal potential of this eo was demonstrated in several other studies. antifungal activity of o. vulgare ssp. vulgare eo against 15 fungal isolates was documented, including a. fl avus and a. variecolor (şahin et al. 2004) and strong antifungal activity of the same eo against a. niger and a. fl avus was shown by viuda-martos et al. (2007). the latter authors showed that o. vulgare eo exhibited stronger antifungal activity than the eos of thymus vulgaris and syzigium aromaticum. the antifungal activity of o. vulgare eo against 6 aspergillus species was demonstrated by carmo et al. (2008), who noted that some concentrations of eo exhibited stronger antifungal activity than the tested antifungicals (amphotericine b and ketoconasole). some authors documented the antifungal activity of o. vulgare eo against several fungi isolated from different substrates of cultural heritage objects, including a. niger and a. ochraceus (stupar et al. 2014). these authors pointed out that o. vulgare eo exhibited antifungal activity similar to that of the tested biocide benzalkonium chloride but stronger compared than the eos of rosmarinus offi cinalis and lavandula angustifolia. the strong antifungal activity of o. vulgare essential oil can be attributed to its high content of some phenolic compounds, mostly carvacrol and thymol (viuda-martos et al. 2007, clef et al. 2013). although the thymol content in the eo used in this study was relatively low (3.86%), carvacrol was the main component (64.06%). phenolic compounds, in appropriate concentrations, are reported to be effective against some microorganisms. their mechanism of antimicrobial activity is related to disruption of microbial cell membrane and precipitation of cellular proteins (burt 2004). it is also suggested that presence of an aromatic nucleus with oh group is responsible for making hydrogen bonds with active sites of target enzymes (velluti et al. 2003). therefore, it is possible to suppose that these groups are responsible for antimicrobial activity. it has been shown that eos are able to cause morphophysiological changes in aspergillus species including loss of sporulation and pigmentation and aberrant development of both hyphae and reproductive structures (de billerbeck et al. 2001). morpho-physiological changes were documented in all tested isolates in our study, such as lack of sporulation, depigmentation of conidiogenous apparatus and conidia, terminal and intercalar swellings and apical buddings. as a result of o. vulgare eo activity, morphophysiological changes in a. fl avus were showed by souza et al. (2010). authors reported loss of cytoplasm content, depigmentation and distorted development of hyphae with swellings and apical budding. furthermore, the absence of conidiation was noted. some authors used four different eos in an antimicrobial vapor assay and documented morpho-physiological alterations of several fungal isolates, including a. niger (ferdeş and ungueranu 2012). they reported absence of phialides on vesicles and reduced conidial formation. degenerative changes in hyphal morphology were reported as well. sporulation is an essential part of the fungal life cycle and melanin is responsible for the survival and endurance of fungal spores (wheeler and bell 1988). depigmentation of spores and conidial heads is probably due to the inhibition of melanin synthesis. melanin is an important virulence factor for pathogenic fungi (tsai et al. 1999) and, therefore, its loss can signifi cantly reduce pathogenicity. this may be of great importance when aspergillus species are considered, since some of them are well known human and animal pathogens (e.g. a. fumigatus), and producers of mycotoxins (e.g. a. fl avus, a. parasiticus, a. ochraceus) (samson et al. 2010). it is reported that application of eos may result in retraction of cytoplasm and interaction of eo components with fungal cell wall (carmo et al. 2008). there also may be interference in enzymatic reactions of cell wall synthesis, which affects fungal growth and morphogenesis (de billerbeck et al. 2001, souza et al. 2010). the fact that structural changes were observed in all fungal isolates tested in this study indicates that o. vulgare eo can affect the morphological development of different aspergillus species and probably other fungi as well. results of the microdilution and agar dilution method showed that different concentrations of the biocide sanosil s003 exhibited moderate fungistatic and fungicidal activity. antifungal studies of biocides based on silver ions and hydrogen peroxide are scarce and there are only few studies concerning anti-aspergillus activity. according to the manufacturer (sanosil ltd), antifungal properties of the biocide have been proven against numerous microorganisms, including a. niger as one of the tested fungal species, but there is no information about mic or mfc values. it was shown that the biocide sanosil super 25 in a concentration of 40 ppm destroys the blastospores of candida albicans (tasić 2009). the same biocide also inhibits mycelial growth and sclerotal formation of botrytis cinerea and sclerotinia sclerotiorum (abdel-mageed et al. 2012). antifungal activity of the biocide based on silver and hydrogen peroxide (nanosil) was investigated by nabizadeh et al. (2008). according to these authors, bacterial species were more sensitive to tested biocide than fungal species, where a. niger was the most resistant tested microorganism in the study. antifungal activity of sanosil s003 could be ascribed to synergistic activity of silver ions and hydrogen peroxide. hydrogen peroxide is an inorganic compound that belongs to the group of reactive oxygen species (ros). this molecule is relatively stable under physiological conditions and can oxidize lipids, proteins, dna and other organic molecules (bienert et al. 2007). it is known that silver ions are highly reactive and can interfere with numerous biological processes in microorganisms, including structural and functional alterations of cell membrane and cell wall (jo et al. 2009, rai et al. 2012). silver ions can inhibit replication by binding to dna molecule (landsdown 2002, castellano et al. 2007). moreover, silver ions have the capacity to react with thiol groups of proteins which leads to the inactivation of enzymes (feng et al. 2000) and they can also cause prosavković ž. d., stupar m. č., ljaljević grbić m. v., vukojević j. b. 126 acta bot. croat. 75 (1), 2016 tein inactivation by binding to their functional groups (sondi and salopek-sondi 2004). in this way, silver ions block aquaporin-mediated water diffusion in saccharomyces cerevisiae cells (bienert et al. 2007). since silver ions can interfere with structural components of the cell, cellular metabolism and reproduction, it is clear that they possess strong antifungal activity. it has been shown that combination of silver ions and hydrogen peroxide exhibits a synergistic mode of action that can sometimes be a thousand-fold higher than the sum of the separate agents (pedahzur et al. 2000, tasić 2009). the aim of this study was to compare the antifungal activity of o. vulgare eo, which is a mixture of various organic compounds, and sanosil s003, a commercial product that is a solution of inorganic compounds. according to the results presented in this study, it is evident that o. vulgare eo exhibits stronger antifungal activity than sanosil s003 on the tested aspergillus isolates. in some cases, the mic and mfc values obtained with sanosil s003 were several hundred times higher than those obtained with o. vulgare eo. weaker antifungal activity of sanosil s003 was confi rmed with fewer observed morpho-physiological changes in the microdilution method, while none were observed in the agar dilution method. the global commercial supply of conventional biocides is well established. however, biocides containing eos are typically used in small niche product applications due to limited effi cacy and therefore are only sold in small amounts (browne et al. 2012). additionally, eos have only recently been recognized as a special class of biocides by eu biocide legislation (since 2011). eos have seldom been tested in vitro against fungi isolated from cultural heritage objects. examples include isolates from the documentary heritage (borrego et al. 2012a), royal tomb paintings (sakr et al. 2012), stone and wooden artifacts (stupar et al. 2014). reports regarding the implementation of natural products in the fi eld of conservation of cultural heritage are scarce. however, rakotonirayni et al. (2005) demonstrated preventive and curative action of linalool, as a main component of some eos, on inoculated books in a glass test chamber. gatenby and townley (2003) showed the effi ciency of melaleuca alternifolia eo, in vapor form, in reducing spore number in castle hill museum (sydney) storage rooms. according to chung et al. (2001), volatile extracts of some eos have no negative effects on organic materials and as such can be used as an alternative source in control of biodeterioration of cultural heritage. also, plant products with antimicrobial activities, such as eos, have little negative impact on the environment or human health (borrego et al. 2012b). since o. vulgare eo demonstrated stronger antifungal activity than a commercial biocide against aspergillus species isolated from cultural heritage objects the high potential of eos as alternative agents in conservation of cultural heritage is indicated. further studies are required to develop appropriate methods to apply eos in the conservation of cultural heritage objects. conclusions the results of this study show the effi cacies of o. vulgare eo and of the commercial biocide sanosil s003. the anti-aspergillus potential of o. vulgare eo was higher than that of sansoil s003. this claim was substantiated by the observed morpho-physiological changes in all tested isolates. this study presents o. vulgare eo as an excellent alternative to commercial biocides and suggests it can be usefully applied in the fi eld of the conservation of cultural heritage. acknowledgements this research was carried out as part of the project no.173032, fi nancially supported by the ministry of education, science and technological development of the republic of serbia. references abdel-mageed, m. h., mohamed, f. g., soltan, h. h., hafez, m. s. abdel-rahman, f. a., 2012: controlling the grey mold and white rot diseases on bean pods under storage conditions using some safely chemical compounds. journal of biological chemistry and environmental sciences 7, 617–634. adam, k., sivropoulou, a., kokkini, s., lanaras, t. arsenakis, m., 1998: antifungal activities of origanum vulgare subsp. hirtum, mentha spicata, lavandula angustifolia, and salvia fruticosa essential oils against human pathogenic fungi. journal of agricultural and food chemistry 46, 1739–1745. baldry, m. g. c., 1983: the bactericidal, fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid. journal of applied microbiology 54, 417–423. bienert, g. p., møller, a. l., kristiansen, k. a., schulz, a., møller, i. m., schjoerring, j. k. jahn, t. p., 2007: specifi c aquaporins facilitate the diffusion of hydrogen peroxide across membranes. journal of biological chemistry 282, 1183–1192. borrego, s., guiamet, p., de saravia, s. g., batistini, p., garcia, m., lavin, p., perdomo, i., 2010: the quality of air at archives and the biodeterioration of photographs. international biodeterioration and biodegradation 64, 139–145. borrego, s., lavin, p., perdomo, i., gómez de saravia, s., guiamet, p., 2012a: determination of indoor air quality in archives and biodeterioration of the documentary heritage. isrn microbiology. doi:10.5402/2012/680598 borrego, s., valdés, o., vivar, i., lavin, p., guiamet, p., battistoni, p. borges, p., 2012b: essential oils of plants as biocides against microorganisms isolated from cuban and argentine documentary heritage. isrn microbiology. doi:10.5402/2012/ 826786 browne, b. a., geis, p., rook, t., 2012: conventional vs. natural preservatives. cspa preservative defense task force. retrieved october 15, 2012 from http://www.dow.com/microbial/pdfs/6974natpreserve0512.pdf burt, s., 2004: essential oils: their antibacterial properties and potential applications in foods – a review. international journal of food microbiology 94, 223–253. carmo, e. s., lima, e. o., souza, e. l., 2008: the potential of origanum vulgare l. 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(ed.), current topics in medical mycology, 338–387. springer-verlag, new york. antifungal activity of origanum vulgare l. essential oil and sanosil s003 acta bot. croat. 75 (1), 2016 1 on-line suppl. fig. 1. morpho-physiological changes of aspergillus niger hyphae observed in microatmosphere method (essential oil in concentration 0.1 μl ml−1): ab – apical budding; sw – swelling; scale bar = 10 μm. on-line suppl. fig. 2. morpho-physiological change s of aspergillus fl avus reproductive structures observed in microatmosphere method (essential oil in concentration 2.5 μl ml−1): a) normal conidiogenous apparatus (control), b) depigmented conidiogenous apparatus. scale bars = 10 μm. 176 acta bot. croat. 80 (2), 2021 acta bot. croat. 80 (2), 176–183, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-020 issn 0365-0588 eissn 1847-8476 dna barcoding of marine algae from malta: new records from the central mediterranean angela g. bartolo1,2*, gabrielle zammit2,3, hannah russell1, akira f. peters1,4, frithjof c. küpper1,5 1 university of aberdeen, school of biological sciences, cruickshank building, st. machar drive, aberdeen ab24 3uu, scotland, uk 2 university of malta, laboratory of applied phycology, centre for molecular medicine & biobanking, fourth floor, biomedical sciences building, msida, malta 3 university of malta, microbiology laboratory, department of biology, second floor, biomedical sciences building, msida, malta 4 bezhin rosko, 40 rue des pêcheurs, 29250 santec, brittany, france 5 university of aberdeen, department of chemistry, marine biodiscovery centre, aberdeen ab24 3ue, scotland, uk abstract – the heterokont benthic multicellular algae schizocladia ischiensis e.c. henry, k. okuda et h. kawai (schizocladiophyceae), hecatonema terminale (kützing) kylin and striaria attenuata (greville) greville (phaeophyceae) are reported for the first time from the waters around the maltese islands in the central mediterranean. they were identified through algal isolation from incubated natural substrata, coupled with dna barcoding targeting the biomarkers coi and rbcl plus the rubisco spacer. for three additional brown algae, colpomenia sinuosa (mertens ex roth) derbès et solier, asperococcus bullosus j.v.lamouroux and sphacelaria sp., dna sequences confirmed previous morphology-based records from malta. this paper also provides an updated literature-based species list of the marine macroalgae present in malta. keywords: dna barcoding, germling emergence, macroalgae, malta, mediterranean sea, phaeophyceae, schizocladiophyceae introduction during the past 25 years, only seven studies have been published about the diversity of marine macroalgae found around the maltese islands, and these were entirely based on morphological identification (borg et al. 1998, lanfranco et al. 1999, schembri et al. 2005, evans et al. 2015, bonnici et al. 2018, era 2020). of all these studies, the only publication focusing solely on macroalgae was a checklist by cormaci et al. (1997), which reported ‘199 rhodophyceae, 63 fucophyceae and 57 chlorophyceae’, making up a total of 319 macroalgal species in malta. to date, no dna studies have been conducted specifically to identify maltese macroalgae, and indeed, few such studies have been carried out in the mediterranean area as a whole (bartolo et al. 2020). molecular tools have challenged the idea that marine species have wide geographical ranges. instead, they have demonstrated that some marine macroalgal ‘species’ actually consist of several geographically restricted cryptic species, i.e. species which are classified as one due to a lack of or only few morphological differences (payo et al. 2012). broad distribution ranges of many algae can be attributed to pervasive cryptic diversity (tronholm et al. 2012). moreover, molecular assessment of the diversity of macroalgal species has demonstrated that morphological species identification underestimates the diversity in a given location (payo et al. 2012, vieira et al. 2017). for the present study, substrata around the maltese islands were sampled to reveal macroalgal biodiversity from cryptic life stages, including species with microscopic thalli. we used the germling emergence (ge) method in combination with dna barcoding of the 5’-end of the mitochondrial cytochrome c oxidase subunit 1 gene (coi) and * corresponding author e-mail: angiebartolo@gmail.com dna barcoding of marine algae from malta acta bot. croat. 80 (2), 2021 177 the plastid-encoded large subunit of ribulose-1,5-bisphosphate carboxylase (rbcl) markers to identify algal species. the study of macroalgal microstages and microscopic species in situ is a challenging task, which was overcome by the germination and isolation of microscopic algal stages and microscopic species in vitro. this ge method has shown a potential for increasing the biogeographic and taxonomic knowledge on macroalgae (peters et al. 2015). in fact, here we present three macroalgal species that were previously unreported from the maltese islands and confirm the presence of another three algal species. materials and methods substratum samples, including small pebbles and shell fragments, as well as posidonia oceanica (linnaeus) delile and padina pavonica (linnaeus) thivy fragments, were collected from four sites in the maltese islands (tab. 1). algal germlings were isolated from the substratum using the ge method (peters et al. 2015), which involves the incubation of the substratum in a herbivore-free and nutrient-rich environment. the samples were cultured in 90 mm petri dishes filled with 35 ml of provasoli-enriched natural autoclaved seawater (starr and zeikus 1993, coelho et al. 2012), incubated at 18 ºc and exposed to natural light. clonal strains of filamentous algae were isolated after 1-3 months by cutting fragments of emerging algae under the stereomicroscope and transferring them into new dishes. monoeukaryotic strains (tab. 1) were obtained by sub-isolating fewcelled thallus fragments. the isolates were studied via light microscopy (nikon eclipse ti-s inverted microscope connected to a nikon digital ds-fi 1 camera). the keys in cormaci et al. (2012) were used for morphological identification of the species. dna was extracted from each specimen using the dneasy plant mini kit (qiagen, hilden, germany) according to the manufacturer’s protocol modified with a ctab pre-treatment according to gachon et al. (2009). the dna was quantified using a nanodrop 2000 spectrophotometer. partial coi and rbcl genes, as well as the rubisco spacer, were amplified using the primer pairs listed in tab. 2. pcr amplifications were performed in a total volume of 50 μl, containing approximately 100 ng of dna, a deoxynucleoside triphosphate mixture (0.2 mm each), supplemented to give a final concentration of 1.8 mm mgcl2, 0.625 u of onetaq quick load 2× master mix with standard tab. 1. provenance of strains including spatial data collected by means of a hand-held garmin 78s marine global positioning system (gps) device. all samples were found submerged in seawater. isolate number location coordinates site description depth (m) mt17-026 saint paul’s bay, malta 35°56.976’ n beneath wignacourt tower, 1 14°24.056’ e on posidonia oceanica leaf mt17-059 cirkewwa, malta 35°59.162’ n near desalination plant outfall, 1.5 14°20.305’ e on hard substratum mt17-068 cirkewwa, malta 35°59.162’ n near desalination plant outfall, 1.5 14°20.305’ e on large stone mt17-092 dwejra, gozo 36°03.185’ n blue hole, on hard substratum 18.4 14°11.283’ e mt17-099 dwejra, gozo 36°03.185’ n collapsed rock debris, 16.9 14°11.283’ e fresh colonisation mt17-100 marsascala, malta 35°52.036’ n close to wreck, 22 14°34.421’ e from soft substratum tab. 2. list of primers used in this study, including the target biomarker, name and sequence for each. biomarker primer name primer no. sequence reference coi gazf2 1 ccaaccayaaagatatwggtac lane et al. 2007 gazr2 2 ggatgaccaaaraaccaaaa lane et al. 2007 dumr1 3 aaaaaycaraataaatgttga saunders 2005 rbcl and rubisco spacer rbclp2f/ rbcl40df 4 gawcgractcgawtwaaaagtg kawai et al. 2007 rbcs139r 5 agaccccataattcccaata peters and ramírez 2001 rbcl rbcl1273f 6 gtgcgacagctaaccgtg peters et al. 2010 rbcs139r 7 as above as above bartolo a. g., zammit g., russell h., peters a. f., küpper f. c. 178 acta bot. croat. 80 (2), 2021 buffer (new england biolabs, inc.), 0.5 pmol of each primer and of 21 μl nuclease-free water. amplifications were carried out in a geneamp thermocycler pcr system 2700 (applied biosystems, foster city, ca, usa) or t3000 thermocycler (biometra, jena, germany) according to the pcr programmes listed in tab. 3. pcr products were verified on 1% (w/v) agarose gel. pcr products were purified using a qiaquick pcr purification kit (qiagen, hilden, germany) and sequenced via a bigdye terminator v3.1 cycle sequencing kit on an abi 3730xl dna analyser (applied biosystems, foster city, california, usa) at eurofins genomics (germany). the sequences were manually checked for correctness by inspecting the chromatograms and were compared to published sequences by the basic local alignment search tool (blast) housed at the united states national center of biotechnology information (zhang et al. 2000). the nucleotide sequences obtained during this study were deposited in the ddbj/genbanktm/ebi data bank and accession numbers are listed in tab. 4. the biomarkers obtained were then analysed to arrive at the taxonomic identity of the algae. taxonomic identities of algae based on molecular studies are highly dependent on the correct identification of dna sequences in molecular databases, the degree of representation of the species concerned, and the percentage identity between the sequences being compared. the resolving power as specieslevel cut-off used for coi in the ectocarpales was 1.8% ( peters et al. 2015). this barcode gap, previously identified empirically by peters et al. (2015), was confirmed to range from 0.011 to 0.037 k2p pair-wise genetic distance in ectocarpus (montecinos et al. 2017), i.e. the equivalent of 1.1% to 3.7%. in fact, for all coi sequences in this study the species-level cut-off applied was more conservative, at 0.6%. in the case of the rbcl gene, a more conservative approach was applied, taking into consideration that the rbcl is less variable (camacho et al. 2019), with the highest species-level cut-off used being 0.4%. this ensured that all species and genera presented in this study were identified only to the level at which there is high-level confidence. a literature review was also conducted on google scholar to provide an updated macroalgal species list for malta. the following terms were combined in the search: (“macroalgae” or “marine algae” or “seaweeds” or “algae” or “brown algae” or “phaeophyceae” or “rhodophyta” or “chlorophyta” or “green algae” or “red algae” or “alien algae”) and (“maltese islands” or “malta” or “gozo” or “comino”). this resulted in seven publications (cormaci et al. 1997, borg et al. 1998, lanfranco et al. 1999, schembri et al. 2005, evans et al. 2015, bonnici et al. 2018, era 2020). further searches were conducted using the ‘distribution’ feature on algaebase (guiry and guiry 2020). moreover, algaebase (guiry and guiry 2020) was also used to update the species names in the compiled list to reflect revisions in taxonomy. results in this paper, we report 14 sequences based on surveys in the maltese islands using coi, rbcl and the rubisco spacer. the results include four coi, five rbcl and five rubisco spacer barcodes. tab. 5 provides the results of the blast searches including the length of sequence, the percentage identity with the closest hits, as well as the percentage query cover. the blast searches resulted in five strains being identified up to species-level and one strain up to genus-level as follows: schizocladia ischiensis e.c. henry, k. okuda et h. kawai (schizocladiophyceae), hecatonema terminale (kützing) kylin, striaria attenuata (greville) greville, colpomenia sinuosa (mertens ex roth) derbès et solier, asperococcus bullosus j.v.lamouroux and sphacelaria sp. schizocladia ischiensis is the only taxonomically accepted species in the genus schizocladia (guiry and guiry 2020), tab. 3. pcr programme conditions used for each primer pair in this study. primer pairs initial amplification (temperature in °c) final extension reference cycles denaturation annealing elongation 1 and 2 4 min at 94 38 1 min at 94 30 s at 50 1 min at 72 7 min at 72 lane et al. 2007 1 and 3 1 min at 94 35 1 min at 94 1.5 min at 50 1 min at 72 5 min at 72 peña et al. 2015 4 and 5 3 min at 95 30 30 s at 95 30 s at 55 2 min at 72 7 min at 72 muñoz 2016 6 and 7 3 min at 95 30 30 s at 95 30 s at 55 1 min at 72 7 min at 72 muñoz 2016 tab. 4. list of sequences produced in this study, with the corresponding ncbi accession number. isolate number identity rbcl + rubisco spacer coi mt17-026 sphacelaria sp. – mw580390 mt17-059 colpomenia sinuosa mw659855 mw580391 mt17-068 hecatonema terminale mw659856 mw580392 mt17-092 striaria attenuata mw659857 mt17-099 asperococcus bullosus mw659858 mw580393 mt17-100 schizocladia ischiensis mw659859 – dna barcoding of marine algae from malta acta bot. croat. 80 (2), 2021 179 and there are four rbcl sequences in genbank representing the species. the rbcl (tab. 5: 1006 bp) and rubisco spacer (tab. 5: 82 bp) produced values of 99.8% and 100% identity respectively to the sequence with genbank accession number mn996275 (rizouli et al. 2020). this species identification was determined with a high level of confidence. the genus hecatonema currently includes 11 species (guiry and guiry 2020) and there are 42 coi and three rbcl sequences in genbank representing this genus. the coi sequence (tab. 5: 633 bp) produced a high identity (100%) with the sequence having genbank accession number lm995391 (peters et al. 2015, as hecatonema maculans) and this was determined with a high level of confidence. in addition, the rbcl and rubisco spacer further confirmed this conclusion since the closest hit in genbank was to an unpublished sequence of hecatonema sp. (accession no. af207802). currently, there are 10 species that are accepted taxonomically in the genus colpomenia (guiry and guiry 2020) and these are represented by 41 coi and 116 rbcl sequences in genbank. the rbcl (tab. 5: 194 bp) and rubisco spacer (tab. 5: 189 bp) provided 100% and 97.4% identity, respectively, to the published c. sinuosa sequence with genbank accession number af385839 (cho et al. 2001), and the species identification was determined with a high level of confidence. the coi sequence (tab. 5: 538 bp) provided the closest hit (97.3% identity) to a sequence of c. sinuosa with accession number kf281125 (mcdevit and saunders 2017). the coi marker did not provide species identity. striaria attenuata is the only taxonomically accepted species in the genus (guiry and guiry 2020) and there is only one rbcl sequence in genbank representing it. the rbcl (tab. 5: 194 bp) and rubisco spacer (tab. 5: 181 bp) provided 100% and 98.3% identity respectively to the published s. attenuata sequence having genbank accession number af055415 (siemer et al. 1998). there are 10 species currently accepted taxonomically in the genus asperococcus, with six coi and 10 rbcl sequences in genbank representing this genus. the coi sequence (tab. 5: 625 bp) resulted in an identity of 99.8% to the a. bullosus sequence having genbank accession no mn1184505 (bringloe et al. 2019). in addition, the rbcl provided supporting information with a 99.6% level identity to the published a. bullosus sequence having genbank accession number lc016509 (kawai et al. 2016). algaebase currently lists 39 taxonomically accepted species for the genus sphacelaria (guiry and guiry 2020), but only nine coi sequences are available in genbank to represent these. the coi sequence (tab. 5: 608 bp) gave a 99.3% identity to the sphacelaria sp. sequence having genbank accession number lm994971 (peters et al. 2015). this genus-level identification was determined with high confidence. it is evident that coi and rbcl together with the rubisco spacer reference sequences are not always available in genbank, and when found, they are not always defined up to species-level. another result of this study is the updated marine algal species list for malta, given in the on-line suppl. tab. 1. the species list now consists of 69 phaeophyceae, 1 member of the schizocladiophyceae, 194 florideophyceae, 4 bangiophyceae, 3 compsopogonophyceae, 1 palmophyllophyceae, 3 stylonematophyceae and 63 ulvophyceae. there are a total of 338 species, also including the new records discovered in this work. discussion through the combination of the ge method, isolation of strains and dna barcoding targeting the cytoplasmic markers coi and rbcl plus the rubisco spacer, the heterokont benthic multicellular algae schizocladia ischiensis tab. 5. results of blast searches including the length of sequence, percentage identity, query cover and details of the closest hit. species name strain marker length (bp) % identity % cover accession species name and locality colpomenia sinuosa mt17-059 rbcl 194 100 100 af385839 colpomenia sinuosa, korea, cho et al. 2001 colpomenia sinuosa mt17059 spacer 189 97.4 100 af385839 colpomenia sinuosa, korea, cho et al. 2001 colpomenia sp. mt17059 coi 538 97.3 95 kf281125 c. sinuosa, australia, mcdevit & saunders, 2017 sphacelaria sp. mt17026 coi 608 99.3 99 lm994971 sphacelaria sp., greece, peters et al. 2015 hecatonema terminale mt17068 coi 633 100 98 lm995391 h. maculans, greece, peters et al. 2015 hecatonema terminale mt17068 rbcl 1403 99.9 100 af207802 hecatonema sp., unpublished hecatonema terminale mt17068 spacer 207 99.5 99 af207802 hecatonema sp., unpublished schizocladia ischiensis mt17100 rbcl 1006 99.8 100 mn996275 schizocladia ischiensis, italy, rizouli et al. 2020 schizocladia ischiensis mt17100 spacer 82 100 100 mn996275 schizocladia ischiensis, italy, rizouli et al. 2020 striaria attenuata mt17092 rbcl 194 100 100 af055415 striaria attenuata, chile, siemer et al. 1998 striaria attenuata mt17092 spacer 181 98.3 100 af055415 striaria attenuata, chile, siemer et al. 1998 asperococcus bullosus mt17099 rbcl 1427 99.6 96 lc016509 asperococcus bullosus, japan, kawai et al. 2016 asperococcus bullosus mt17099 spacer 178 91.2 100 ay095321 asperococcus fistulosus, uk, cho et al. 2003 asperococcus bullosus mt17099 coi 625 99.8 99 mn184505 a. bullosus, norway, bringloe et al. 2019 bartolo a. g., zammit g., russell h., peters a. f., küpper f. c. 180 acta bot. croat. 80 (2), 2021 (schizocladiophyceae), hecatonema terminale and striaria attenuata (phaeophyceae) are being reported for the first time from the waters around the maltese islands in the central mediterranean. for three additional brown algae, colpomenia sinuosa, asperococcus bullosus and sphacelaria sp., dna sequences confirmed previous morphology-based records in malta (cormaci et al. 1997, borg et al. 1998). all the species and genera presented in this study are identified only to the level at which there is high-level confidence. schizocladia ischiensis (fig. 1) was germinated from a substratum sample collected at marsascala at a depth of 22 m. the thallus was made up of branched filaments of 3–7 μm diameter, each containing one or two brown parietal plastids. the zoospores, which have a teardrop-shape and an eyespot (kawai et al. 2003), were not examined in this study. molecular phylogenies indicate a close relationship to phaeophyceae; however, schizocladia belongs to a different class since it lacks cellulose and plasmodesmata in the cell wall and the presence of a flagellar transitional helix (kawai et al. 2003). the class schizocladiophyceae and the species s. ischiensis were originally described from a single strain (ku-333) isolated from substratum collected off the island of ischia near naples in italy; the diagnosis was based on photosynthetic pigment analysis, morphology, and molecular phylogenies (kawai et al. 2003). the rbcl and rubisco spacer sequences obtained for the maltese isolate are almost identical to those from a s. ischiensis strain from naples (tab. 5: rbcl 99.8% identity and rubisco spacer 100% identity with mn996275, rizouli et al. 2020), but slightly different from strain rh15-53 (rbcl 99.4% identity and rubisco spacer 97.6% identity to lc521905), a recent record off the greek island of rhodes (rizouli et al. 2020). a germling of h. terminale (fig. 2) emerged from a stone fragment collected from cirkewwa, malta, at the outfall of a desalination plant. species of the genus hecatonema are conf luent microscopic tufts that could also be solitary ( parente et al. 2010). they consist of a monostromatic basal layer, which in some places could be distromatic, from which unbranched or sparsely branched filaments arise (fletcher 1987). hecatonema terminale is abundant in brittany and has been reported in the mediterranean from ischia and naples in italy, korinthiakos gulf, korinthos in greece ( peters et al. 2015, as hecatonema maculans), as well as from sicily (giaccone et al. 1985). the family hecatonemataceae (tribu hecatonematees in loiseaux, 1967) are currently placed within the chordariaceae (peters and ramıirez 2001). coi sequences suggest that this clade might form a separate family (peters et al. 2015), but this is yet to be confirmed by multi-gene phylogenies. the comparison with coi sequences deposited in genbank shows that the sequence obtained for the maltese isolate is identical to that of strain gr11-52b from greece (tab. 5: 100% identity to lm995391, peters et al. 2015). colpomenia sinuosa (fig. 3) was isolated from a pebble collected at a depth of 1.5 m at the outfall of the same desalination plant in cirkewwa. preliminary morphological identification indicated the strain belonged to c. sinuosa, the type species of this genus, which was then confirmed through sequencing of the rbcl and rubisco markers, which gave a high percentage identity to a strain from jeju, korea (tab. 5: rbcl 100% identity and rubisco spacer fig. 3. light micrograph of colpomenia sinuosa (mertens ex roth) derbès et solier strain from malta. fig. 2. light micrograph of the hecatonema terminale (kützing) kylin strain from malta. fig. 1. light micrograph of schizocladia ischiensis e.c. henry, k. okuda et h. kawai strain from malta. dna barcoding of marine algae from malta acta bot. croat. 80 (2), 2021 181 97.4% identity to af385839, cho et al. 2001). the coi gene provided a 97.3% identity to c. sinuosa (tab. 5: kf281125, mcdevit and saunders, 2017). there are only eight coi sequences for c. sinuosa in genbank and they all originate from korea (two sequences) or australia (six sequences). the comparison with coi sequences deposited in genbank shows that the maltese isolate could be a cryptic species. cryptic speciation in c. sinuosa has been studied through the use of the rbcl and cox3 gene, which have shown that there are three main genetic groups (lee et al. 2013). the rbcl of the maltese isolate provided the highest identity (99.6, 100 and 100% respectively) to ay398468, ab022234, ab578988, i.e. c. sinuosa group 1 in lee et al. (2013). group 1 is the most diverse group and includes five subgroups from both temperate and tropical waters. however, it is probable that there are no coi sequences in genbank for this group. further molecular investigations are thus required for c. sinuosa, especially to sequence the coi gene from specimen growing in different areas including the type locality in cadiz, spain (guiry and guiry, 2020), as well as from different areas in the mediterranean sea. colpomenia sinuosa occurs intertidally and subtidally (cho et al. 2009) and is widespread in temperate and warm waters, penetrating boreal waters (guiry and guiry, 2020). colpomenia sinuosa and c. peregrina sauvageau, both have a spherical and saccate appearance and both occur around malta. the main difference between the two is that c. sinuosa has plurilocular sporangial punctate sori with a cuticle and four to six layers of medullary cells, as opposed to extensive sori without a cuticle and a thinner thallus wall of three to four layers of colourless medullary cells in c. peregrina (toste et al. 2003). for this study, s. attenuata and a. bullosus specimens were collected in gozo from the blue hole at dwejra. previously, the presence of s. attenuata had been recorded in different mediterranean locations including sicily (giaccone et al. 1985) and karpasia in cyprus (tsiamis et al. 2014), but it had never been identified from the maltese islands. on the other hand, a. bullosus had been morphologically identified in the north-eastern coast of malta (borg et al. 1998). the analysis of the new biomarkers of s. attenuata obtained in this study resulted in a high percentage identity to strain sat 49 from chile (tab. 5: rbcl 100% identity and rubisco spacer 98.3% identity to af055415, siemer et al. 1998). the sequences obtained for a. bullosus gave a high percentage identity to strain ku-570 from japan and strain gws040819 from norway (tab. 5: rbcl 99.6% identity to lc016509, kawai et al. 2016 and coi 99.8% identity to mn184505, bringloe et al. 2019). the sphacelaria sp. isolate collected from an algal tuft on padina sp. in st paul’s bay had a high percentage identity to strain gr11-34 (tab. 5: coi 99.3% identity to lm994971, peters et al. 2015) collected from kavouri (greece). in this case, the species identity is not obvious, possibly due to the dearth of sphacelariales coi sequences in the public databases that are attributable to primer mismatches (peters et al. 2015). in fact, there are only nine coi sequences available in genbank representing the genus sphacelaria, which is a highly limited number compared to the 39 species that currently make up this genus (guiry and guiry 2020). thus, further molecular investigations are urgently required for the genus sphacelaria. other species of sphacelaria that have been previously recorded from the maltese islands on the basis of morphology include s. cirrosa (roth) c.agardh, s. fusca (hudson) s.f.gray, s. plumula zanardini, s. rigidula kützing and s. tribuloides meneghini (cormaci et al. 1997). for the phaeophyceae, our results confirm that the rubisco spacer is more variable than rbcl (tab. 5) and that this spacer, in combination with other biomarkers, such as cox2-3, could be used to study intraspecific groups in biogeographic studies (cho et al. 2007). it is important to note that only c. sinuosa, a. bullosus and sphacelaria sp. were recorded through the application of morphological surveys and the ge method coupled with dna barcoding. thus, without the latter part, our study would have overlooked s. ischiensis, s. attenuata and h. terminale. thus, our results indicate that algal isolation and culturing in combination with dna barcoding is a useful unbiased tool to reveal overlooked biodiversity. it also shows that sediment and other substrata, such as pebbles, represent an unexplored environment that harbours countless cryptic microstages of macroalgae with potential for the detection of species. this same method could also be used to detect new introductions of non-indigenous species to the mediterranean at an early stage. the method also suggests that ‘eradicating’ non-indigenous species by removing the macrothalli is impractical since most algae may exist as microstages in the sediment itself. the ge method certainly has a strong potential to enhance algal biodiversity checklists and is both cost-effective with a low environmental impact in comparison to shipor rov-based surveys, such as those targeting deep-water / circalittoral algal communities in the eastern mediterranean (küpper et al. 2019). finally, this study provides an updated checklist of marine macroalgal species present in maltese waters (on-line suppl. tab. 1). this was important as it was a challenge to search records of maltese macroalgae, because these had not been revised since 1997 (cormaci et al. 1997). species names were updated to reflect revisions in taxonomy. for instance, previous mentions of aglaothamnion byssoides and a. tenuissimum have now been recorded as one species in the updated list, a. tenuissimum (bonnemaison) feldmannmazoyer. moreover, any references to misidentified algae, such as asparagopsis armata, which does not occur in malta (evans et al. 2015), were removed. acknowledgements this research is partially funded by the endeavour scholarship scheme (malta)group b – national funds. agb was supported by the mars network for a mars travel award, which offered an exciting opportunity to develop this research project at an early stage. appreciation is bartolo a. g., zammit g., russell h., peters a. f., küpper f. c. 182 acta bot. croat. 80 (2), 2021 due to the environment & resources authority (malta), the total foundation (paris) and the marine alliance for science and technology for scotland pooling initiative (masts), the latter funded by the scottish funding council (grant reference hr09011) and contributing institutions. thanks are due to eleni kytinou for diving assistance during 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genus cleome l. (cleomaceae) from pakistan sana riaz1, rubina abid1*, syed abid ali2, iqra munir2, muhammad qaiser1 1 department of botany, university of karachi, karachi-75270, pakistan 2 hej research institute of chemistry, international centre for chemical and biological sciences (iccbs), university of karachi, karachi–75270, pakistan abstract – a new species of the genus cleome l. from pakistan is described and illustrated. the new species is described under the name c. karachiensis sp. nov. and compared with two closely related species i.e., cleome brachycarpa and c. viscosa in terms of morphology, palynology, seed morphology and seed protein profile. a key to the species of genus cleome l. from pakistan is also provided. keywords: cleomaceae, cleome, pakistan, species novum, seeds * corresponding author, e-mail: rubinaku@yahoo.com introduction the genus cleome l. was first described by linnaeus (1753) with 8 species under the family capparidaceae. since then many workers have treated this genus under the family capparidaceae (de candolle 1824, bentham and hooker 1862, boissier 1867, oliver 1868, hooker 1875, hedge and lamond 1970, jafri 1973). pax (1891) divided the family capparidaceae into two subfamilies viz. capparoideae and cleomoideae, and placed the genus under the subfamily cleomoideae. airy shaw (1965) raised the status of cleomoideae to the family level, cleomaceae. similarly, the apg iv system (2016) based on dna studies also accepted cleomaceae as an independent family. the genus cleome l. is the largest genus of the family cleomaceae, with 250 species, distributed all over the world (mabberley 2008). jafri (1973) in his treatment of the family capparidaceae for the flora of pakistan recognized 9 species of cleome and one subspecies i.e., c. heratensis subsp. pakistanica jafri. later on, the status of subspecies c. heratensis subsp. pakistanica was raised to specific level on the basis of morphological differences i.e., habit, leaf, style and petal apex (khatoon and perveen 2003). during a field survey of the vegetation of karachi university campus the authors observed a small population of plants resembling cleome viscosa l. and c. brachycarpa vahl. ex dc., which at first sight appears to be a miniature of c. viscosa. however, detailed studies showed that they were not only different from both species but also showed no similarity to any other known species of cleome. so the species is reported here as a new addition to the genus cleome. in order to confirm the authenticity of the new species whether it is a biological species or not, the new species has been compared with both the closely related species i.e., cleome brachycarpa and c. viscosa using the following parameters: i) pollen morphology ii) seed morphology iii) protein and peptide fingerprinting along with overall general morphology. materials and methods study sites herbarium specimens (kuh) along with fresh material collected from karachi university campus and hub river road, karachi district were examined (on-line suppl. appendix i). pollen morphology pollen grains were collected from mature anthers. for light and scanning electron microscopy, pollen grains were prepared following the standard method of erdtman (1952). pollen grains were mounted in unstained glycerine jelly for new species of the genus cleome l. acta bot. croat. 78 (1), 2019 103 light microscopy. for scanning electron microscopy, pollen material was directly mounted with capillary tubes on metallic stubs using double adhesive tape, dried at room temperature then gold plated in a sputtering chamber and observed under scanning electron microscope. the observations were based on 5-10 specimens of c. brachycarpa, c. viscosa, and the new taxon. pollen size, shape, colpi length and tectum surface were observed. the terminologies used are in accordance with erdtman (1952) and hoen (2011). leaf trichome and styles leaves and styles of all the three taxa were studied under scanning electron microscope (jsm-6380 a). for scanning electron microscopy, mature dried 1 cm2 parts of leaves and full length styles were mounted on metallic stubs using double adhesive tape, then gold plated in a sputtering chamber for a period of 6 minutes and observed under scanning electron microscope. the terminology used is in accordance with metcalfe and chalk (1957). seeds morphology mature and healthy seeds of the three taxa were collected and studied under stereomicroscope (nikon xn model) and scanning electron microscope (jsm-6380 a). for scanning electron microscopy, the mature dried seeds were mounted on metal stubs using double adhesive tape, then gold plated in a sputtering chamber for a period of 6 minutes and observed under scanning electron microscope. seed characters like seed shape, colour and surface were studied. the terminology used is in accordance with lawrence (1970), radford et al. (1974) and stearn (1983) with slight modifications. protein analysis crude seed extracts (20 µg) of the three taxa were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) analysis under dissociating and denaturing conditions (ph 8.8 in the presence of sds and β-mercaptoethanol) for protein and peptide fingerprinting. the proteins and peptides were separated in 14% resolving and 5% stacking gels at 140 v for 1.5 h. and at the end of electrophoresis the gel was stained with 0.2% coomassie brilliant blue g-250. in addition to the sds-page, the same samples (100 µg) were also subjected to reversed phase fast protein liquid chromatography (akta-design amersham biosciences, buckinghamshire, uk) using a reversed phase fplc column (µrpc c2/c18, st 4.6/100; amersham biosciences, uk). the columns were equilibrated in 0.1% trifluoroacetic acid (tfa)-h2o (buffer-a) and eluted with a slight linear gradient (60% b in 20 min.) of 100% acetonitrile containing 0.1% tfa (buffer-b). the flow rate was maintained at 1 min/ml and the elution was observed at 280 nm. automated software unicorn 5.0 (amersham biosciences, uk) was used for data analysis. results and discussion taxonomic treatment cleome karachiensis s. riaz, r. abid and m. qaiser sp. nov. (fig. 1). annual herb, stem erect, soft, unbranched, 4–17 cm tall, glandular hairy, leaves alternate, petioled, palmately compound, 3–5 foliate, leaflets entire, obovate–oblanceolate, acute, base cuneate, 6–16 × 3–9 mm, capitate glandular, stofig. 1. scheme of some characteristics of cleome species. cleome karachiensis s. riaz, r. abid and m. qaiser: habit (a), flower (b), seed (c), type specimen (f); c. brachycarpa: seed (d), c. viscosa: seed (e). riaz s., abid r., ali s. a., munir i., qaiser m. 104 acta bot. croat. 78 (1), 2019 mata actinocytic. flower yellow, complete, 5–8 mm across, pedicel 0.5–1.5 cm; sepals, 4, green, dorsally glandular, oblong–obovate, acute, base truncate, 2–4 × 0.5–1 mm; petals 4, yellow, oblanceolate, obtuse, cuneate, attenuate base, 2.5–6 × 1–2.5 mm. stamens 6–8. ovary glandular; style 0.5–1 mm long; capsule linear, many seeded, 10–29 × 1–2 mm (fig. 1); seeds light brown, myrtle green, retortiform with long groove, groove narrow towards the edges, surface concentrically ridged, foveate with appressedly colliculate, hilum lateral (on-line suppl. fig. 3). holotype: g-4 pakistan, karachi dist.: near department of visual studies, karachi university campus, 28-8-2014, sana 8 (kuh) (fig. 1f). paratype: g-4 pakistan, karachi dist.: near department of visual studies, karachi university campus, 28-8-2014, sana 9 (kuh); near department of visual studies, karachi university campus, 4-9-2014, sana & rubina abid 61, 64, 65 (kuh); near department of visual studies, karachi university campus, 16-8-2015, sana 101, 102, 103 (kuh); near department of visual studies, karachi university campus, 8-92015, sana108 (kuh); hub river road, 28-9-2017, sana & rubina abid 149 (kuh). etymology: the species is named after the city of karachi, from which the type specimen was collected. distribution: known only from the district of karachi (pakistan). ecology: the species grows in the sandy and dry plains in association with cleome brachycarpa, c. viscosa, prosopis juliflora, calotropis procera, abutilon spp., peristrophe bicalyculata and various grasses. flowering period: augustoctober. c. viscosa l. erect herb, 15–100 cm tall, branched or unbranched, hairy with glandular hairs. leaves compound, alternate, tri–pentapalmate, petiole 5–43 mm. leaflets elliptic, broad elliptic, oblanceolate, lanceolate or obovate, acute or obtuse, base cuneate, attenuate, glandular, 14–32 × 3–17 mm. flower complete, 7–15 mm across, yellow, pedicel 0.8–1.5 cm. sepals 4, green, glandular, linear–oblong, oblong or oblong–lanceolate, acute, 4–6 × 1–2 mm. petals 4, oblanceolate, oblong–lanceolate or oblong–elliptic, obtuse, base attenuate,6–10 × 1–3 mm. stamens 10–20, free, 3–10 mm long, unequal in length. ovary glandular, 2.5–8.5 mm long, style persistent, 0.5–1 mm long, gynophore absent. capsule linear, acute, base cuneate, glandular, striate, 37–80 × 3 mm. seeds many, glabrous, rust brown or dark brown. c. brachycarpa vahl. ex dc. semi erect herb, 10–35 cm tall, branched from base, glandular. leaves alternate, compound, tri–pentapalmate, glandular, petiole 2–30 mm. leaflets elliptic, obovate, oblanceolate, oblong or orbicular, acute or obtuse, base cuneate, 6–20 × 2–8 mm. flower complete, 0.8–1.2 cm across, yellow, pedicellate. sepals 4, green, glandular, oblong, lanceolate, elliptic or oblong–elliptic, acute, 1.5–2.5 mm rarely up to 4 × 0.5–2 fig. 2. scaning electron micrographs of styles in cleome species: cleome karachiensis (a), c. brachycarpa (b), c. viscosa (c); scale bars: a, c = 200 µm; b = 1 mm. fig. 3. analysis of seed proteins of cleome species. protein and peptide finger printing using 14% polyacrylamide gel electrophoresis under dissociating and denaturing conditions (a), protein liquid chromatography of the same samples on µrpc c2/c18 column (b). new species of the genus cleome l. acta bot. croat. 78 (1), 2019 105 mm. petals 4, obovate or obovate–elliptic, acute, base cuneate, 3–7 × 1–3 mm. stamens 6, free, 2–6 mm long. ovary glandular, 1–5 mm, ovules many, style persistent, 1–4.5 mm, gynophore absent. capsule 5–10 × 1.5–2 mm, oblong, glandular. seeds many, glabrous, rust brown or maroon. cleome karachiensis sp. nov. shows close affinities with c. viscosa and c. brachycarpa in having 3-5 foliate, glandular leaves with acute apex and cuneate base, dorsally glandular sepals, yellow petals (jafri 1973) and tricolporate pollen (sanchez-acebo 2005), non-angular seeds and a common protein band at 21.5 kda. besides the morphological affinities all the three species are growing simultaneously and occupying the same habitat. the new species is restricted in distribution, although other species are widely distributed. c. karachiensis sp. nov. seems to be the closest to c. viscosa in having capitate glandular trichomes on leaves (on-line suppl. fig. 1), obtuse petals, short style, subprolate pollen grains (on-line suppl. fig. 2), linear capsule and retortiform seeds (fig. 1). on the other hand, c. viscosa remains distinct by having 10-20 stamens (hedge and lamond 1970), rust brown or dark brown seeds, concentrically ridged, foveate without appressedly colliculate seed surface and reticulate– rugulate pollen tectum. the new species is also distinct from c. brachycarpa which is characterized by having peltate glandular trichomes on leaves (on-line suppl. fig. 1), acute petals, long style (fig. 2), 6 stamens (hedge and lamond 1970, jafri 1973), oblong capsule and elliptic–pyriform and ovoid seeds (tab. 1), larger colpi, spinulose tectum (on-line suppl. fig. 2). however, the new species remains distinct from both of the allied species by having a soft herbaceous and 4–17 cm stem; 6–8 stamens (fig. 1), shorter colpi (11.5–13.8 µm), verrucate tectum (on-line suppl. fig. 2). the protein profile is also distinct from c. viscosa and c. brachycarpa in having only two protein bands between 14.4–21.5 kda zone and a single band between 31–45 kda zone. in contrast, in c. viscosa and c. brachycarpa there were 5 bands between 14.4– 21.5 kda and 5 bands at 31–45 kda zones (fig. 3a). similarly, rp-fplc data also support the findings of sds-page and all the three species can be distinguished from each other at the end of elution profile by protein fraction (retention time 5–20) at the peak pattern (fig. 3b) key to the species of cleome l. from pakistan 1 leaves simple ........................................................................2 leaves compound ................................................................7 2 undershrub, leaves scanty and very small ......................... ......................................................................... c. pakistanica herbs, leaves many and comparatively large ...................3 3 fruit linear ............................................................................4 fruit oblong ..........................................................................5 4 stem and leaves glabrous, flowers yellow, 1 cm across, seeds hairy......................................................... c. oxypetala stem and leaves hairy glandular, flowers pale white or pinkish, 3–4 mm across, seeds glabrous ........... c. scaposa 5 stamens 6, fruits 50 mm long, seeds hairy ......c. rupicola stamens 4, fruits 12–30 mm long, seeds glabrous ..........6 6 leaf base truncate or cordate, seeds angular ..................... .........................................................................c. dolichostyla leaf base obtuse or cuneate, seeds not angular ................. .............................................................................c. fimbriata 7 petals linear-oblong, oblong, obovate, obovate-elliptic, stamens 6 ...............................................................................8 petals oblanceolate, oblong-lanceolate, oblong-elliptic, stamens 6–20 ......................................................................10 8 stem semi erect, branched from base, flowers yellow ...... ........................................................................c. brachycarpa stem erect, branched but not from base, flowers white or pinkish ...................................................................................9 9 leaves 3 foliate, flowers 3–5 mm across, filaments 1–6 mm long ............................................................................c. ariana leaves 5–7 foliate, flowers 25 mm across, filaments 25 mm long .......................................................................... c. spinosa tab. 1. distinguishing characteristics of cleome karachiensis sp. nov., cleome viscosa and cleome brachycarpa. data in brackets indicate the minimum to maximum values. characters c. viscosa c. karachiensis c. brachycarpa habit erect erect semi erect plant height (cm) 37.2 ± 23.90 10.76 ± 3.91 22.3 ± 9.28 (15–100) (4–17) (10–35) branching branched/unbranched unbranched branched from base leaf trichomes capitate glandular capitate glandular peltate glandular petal apex obtuse obtuse acute no. of stamens 10–20 6–8 6 style length (mm) 0.75 ± 0.26 0.75 ± 0.26 2.55 ± 1.16 (0.5–1) (0.5–1) (1–4.5) capsule linear linear oblong seeds retortiform retortiform elliptic-pyriform or ovoid pollen shape subprolate subprolate prolate tectum reticulate-rugulate verrucate spinulose colpi length (µm) 19.6 ± 0.2981 12.5 ± 0.86 21.9 ± 0.1943 (19–20.1) (11.5–13.8) (21.6–22.2) riaz s., abid r., ali s. a., munir i., qaiser m. 106 acta bot. croat. 78 (1), 2019 10 stamens 6–8, fruit 10–29 mm long, seed surface appressedly colliculate ..................................... c. karachiensis stamens 10–20, fruit 37–80 mm long, seed surface not appressedly colliculate .......................................... c. viscosa acknowledgements we are thankful to the director, centre for plant conservation for providing the herbarium facilities. thanks are also due to mr. abrar ali for illustration and photographic enhancement. references airy shaw, h. k., 1965: diagnosis of new families, new names etc., for the seventh edition of willis’s “dictionary”. kew bulletin 18, 256. apg (angiosperm phylogeny group) iv. 2016: an update of the angiosperm phylogeny group for the orders and families of flowering plants. botanical journal of the linnean society 181, 1–20. bentham, g., hooker, j. d., 1862: genera plantarum ad exemplaria imprimis in herbariis kewensibus servata definite 1. l reeve & co., london. boissier, e., 1867: flora orientalis. vol. 1, 410–416. reimpression facsimilec a. asher and co., geneva. de candolle a. p., 1824: prodromus systematics naturalis regni vegetabilis, vol. 1, 237–241. sumptibus sociorum treuttel et wurtz, paris. erdtman g., 1952: pollen morphology and plant taxonomy, angiosperms, chronica botanica co., waltham. hedge i., lamond j., 1970: capparidaceae. in: rechinger k. h. (ed.), flora iranica, vol. 68, 13–30. academische druck-u– verlagsanstalt graz austria. hoen p., 2011: glossary of pollen and spore terminology (2nd ed.), laboratory of paleobotany and palynology, utrecht, nl. hooker j.d., 1875: flora of british india, vol. 1, 168–170. l. reeve & co. ltd., london. jafri s.m.h., 1973: capparidaceae. in: nasir, e., ali s. i. (eds.), flora of pakistan, vol. 34, 20–32. the herbarium, dept. of botany, university of karachi, karachi, pakistan. khatoon s., perveen a., 2003: cleome pakistanica (capparidaceae) – a new endemic species from pakistan. pakistan journal of botany 35, 145–146. lawrence g.h.m., 1970: taxonomy of vascular plants. the macmillan company, colliermacmillan canada, ltd., toronto, ontario, new york. linnaeus c., 1753: species plantarum, vol. 2, 671–672. british museum (natural history) london. mabberley d.j., 2008: mabberley’s plant-book (3rd ed.), 194. cambridge university press, cambridge. metcalfe c.r., chalk, l., 1957: anatomy of the dicotyledons, ii. calerendon press, oxford. oliver d., 1868: flora of tropical africa, vol. 1, 74–81. l. reeve & co. ltd., ashford, kent. pax., 1891: capparidaceae. in: engler, a., prantl, k. (eds.), pflanzenfam iii. 2, 220. radford a. e., dickison w.c., massey j.r., ritchie bell c., 1974: vascular plants systematics. harper and row, new york, evanston, san francisco, london. sanchez-acebo, l., 2005: a phylogenetic study of the new world cleome (brassicaceae, cleomoideae). annals of the missouri botanical garden 92, 179–201. stearn t.w., 1983: botanical latin (3rd ed.), david & charles, uk. 18 acta bot. croat. 80 (1), 2021 acta bot. croat. 80 (1), 18–28, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-007 issn 0365-0588 eissn 1847-8476 the synergistic effects of hydro and hormone priming on seed germination, antioxidant activity and cadmium tolerance in borage parisa sheikhzadeh*, nasser zare, fatemeh mahmoudi university of mohaghegh ardabili, faculty of agriculture natural resources, department of agronomy and plant breeding, ardabil, iran abstract – this research investigated the effects of priming with water for 24 hours and hormone-priming with different concentrations of gibberellic acid (ga3) (50, 100, 150 mg l–1) for 24 hours and hydro and hormone priming techniques either alone or in combination on the germination, growth and biochemical properties of borage seedlings under cadmium stress conditions. the results showed that cadmium stress reduces seed germination and seedling growth indices. seed priming led to a significant increase in the germination percentage and rate, seedling dry weight and length, seedling vigor indices, as well as the catalase and peroxidase activity of borage seedlings under both cadmium stress and non-stress conditions. among the priming treatments, the combination of hydro and hormone priming showed the greatest effects on the improvement of germination and seedling growth under cadmium conditions, significantly greater than those achieved with the individual uses of hydro or hormone priming. at all levels of cadmium stress, utilization of combined hydro and hormone priming led to a 0.9 to 11.53-fold, and 0.95 to 2.63-fold increase in seedling dry weight as compared with the control treatment and individual use of hydro or hormone priming, respectively. the highest activity of catalase and peroxidase enzymes in borage seedlings was obtained from seeds primed with the combination of hydro priming with 150 mg l–1 ga3, which was significantly higher than those of the other treatments. generally, at all levels of cadmium stress, combined hydro and hormone (specially 150 mg l–1 ga3) priming had the most positive effects on seed germination, growth and biochemical properties of borage seedlings. keywords: borago officinalis, gibberellic acid, heavy metal, seed priming technique, seedling growth introduction heavy metals are the most hazardous environmental pollutants and their toxicity is a significant problem, for ecological, evolutionary, nutritional and environmental reasons. today, soil and water pollution with heavy metals, and their accumulation in agricultural products are one of the most critical environmental issues that threaten human and plant life due to the adverse effects on crop growth and food safety (hasan et al. 2009, jaishankar et al. 2014). cadmium (cd) due to its significant solubility in water and high toxicity is one of the most important heavy metal pollutants of the environment and agricultural products. cadmium gets easily and rapidly absorbed by roots and quickly translocates to the aerial parts of plants (hasan et al. 2009). although other heavy metals are also released to the environment from various other industrial and agricultural sources, including sewage treatment, plating, mining and the production of plastic as well as pest control (jaishankar et al. 2014), it is cadmium that has the most adverse effects on many physiological, biochemical and metabolic processes such as seed germination and seedling growth, photosynthesis, respiration, plant water relations and function of stomata in plant cells (hasan et al. 2009). it has been shown that cadmium decreases co2 fixation and consequently, photosynthesis in the plants by reducing chlorophyll synthesis and the inhibition of rubisco. in addition, cadmium stress induces the production of various reactive oxygen species * corresponding author e-mail: sheikhzadehmp@gmail.com borage seed priming enhanced cadmium tolerance acta bot. croat. 80 (1), 2021 19 mination process in two ways. first, ga3 increases embryo growth through stimulating expression of the genes involved in cell expansion, which causes the weakness of seed cover. second, endogenous increase of ga3 level or exogenous application of ga3 stimulate the expression, activation and secretion of hydrolytic enzymes including α-amylase which degrade the seed food reserves (proteins, carbohydrates and lipids) into the soluble amino acids, sugars and other products that are necessary for embryo growth (gupta and chakrabarty 2013, kumar et al. 2014). the accumulation of toxic heavy metals in agricultural soils can affect the production of plants, especially medicinal plants through the reduction of seed germination, seedling growth, yield, and quality of these plants (munzuroglu and zengin 2006, mahmoudi et al. 2017). therefore, recognizing the effects of cadmium stress on germination and growth of borage seedlings and the development of suitable treatments to improve borage seed germination and seedling growth under cadmium stress conditions is highly valuable. regarding the medical importance of the borage plant and weak seed germination this plant, this study aimed to investigate the effects of hydro and hormone priming techniques, either alone or in combination, on the germination and growth characteristics, and biochemical properties of borage seedlings under cadmium-free and cadmium stress conditions. materials and methods plant material and seed priming matured borage seeds were obtained from the medicinal plants research centre of the academic center for education, culture & research. in order to apply hydro and hormone priming, first the borage seed lots were washed with distilled water, then the seeds were soaked in distilled water (hydro-priming) or solutions containing 50, 100 or 150 mg l–1 of ga3 (hormone priming) for 24 hours at 10 °c. for sequential application of hydro and hormone priming of borage seeds, firstly, the seeds were treated for 24 hours in distilled water at 10 °c, and then, these seeds were treated with different concentrations of ga3 (50, 100, 150 mg l–1) at 10 °c for 24 hours. the primed seed lots were rinsed with distilled water, and then dried at room temperature (20 to 22 °c) until they reached the initial moisture content (mcdonald, 2000). unprimed borage seed lots were used as the control. germination test in order to perform the germination test, borage seeds (primed and non-primed) were placed on filter paper (whatman no. 1) in 10 cm diameter petri plates. three replicates with 25 seeds per replicate were used for each treatment. the filter paper was moistened with 4 ml of water solution containing different concentrations of cadmium (0, 10, 50 and 100 mg l–1 cdcl2); this was repeated every 2 days. the cdcl2 (0, 10, 50 and 100 mg l–1 cdcl2) solutions were prepared by dissolving appropriate amounts of cdcl2 in 100 ml distilled (ros) and oxidative stress, which cause peroxidation of the lipids, cell membrane damage and disruptions to cell growth and function (mittler 2002, tiryakioglu et al. 2006). it has been shown that in the germination and emergence stage, the presence of cadmium reduces the percentage and rate of germination and seedling growth (munzuroglu and zengin 2006, mahmoudi et al. 2017). borage (borago officinalis l.) is one of the most important medicinal plants, having many applications in the pharmaceutical industries and traditional medicine. the flowers of borages are usually prescribed as an anti-inflammatory, and were once widely used for the prevention of colds, bronchitis and respiratory infections as well as digestive and cardiovascular disorders. furthermore, oleic acid and palmitic acid from borage flowers have on the effect of lowering blood cholesterol (gupta and singh 2010). seeds of most medicinal plant species have uneven and weak germination, which limits the cultivation and production of these plants. several methods and approaches have been suggested for the improvement of seed germination and seedling growth, as well as for decreasing the adverse effects of environmental stresses. seed priming is a simple, efficient, and environmental-safe method that improves seed vigor and germination under favorable and unfavorable conditions (mcdonald 2000, ghassemi golezani et al. 2008). in this technique, the seeds are treated with distilled water (hydro-priming), hormones or other chemical materials for a specified period, so that the germination processes are started, without the radicle emerging from the seed. among the priming methods, hydro-priming is a very simple and economical as well as the most commonly used method. seed priming was suggested as an approach for improving seed vigor, seed germination, seedling emergence and establishment, and resistance to environmental and biotic stress, and consequently improving product quantity and quality (mcdonald 2000, ghassemi golezani et al. 2008). also, seed priming, by increasing the activity of antioxidant enzymes such as catalase, peroxidase, superoxide dismutase, glutathione reductase, and other enzymes, leads to the elimination of reactive oxygen species (ros) (gill and tuteja 2010). in general, the beneficial effects of seed priming on germination and seedling establishment and growth of barley seeds (munzuroglu and zengin 2006), pigeon pea (sneideris et al. 2015) and chickpea (ghassemi golezani et al. 2008) subjected to heavy metals stress have been reported. plant hormones include various groups of regulatory and signaling molecules which are synthesized in small quantity in different plant cells, and work in a complex signaling network to regulate various aspects of plant growth, development, fruit ripening, reproduction and response to environmental stresses. gibberellic acid (ga3) is one of the growth-promoting hormones that play a crucial role in the seed germination process through stimulating the degradation of seed storage materials into usable products for the embryo and consequently triggering the germination process (kumar et al. 2014). gibberellic acid is one of the growth-promoting hormones that influence the seed gersheikhzadeh p., zare n., mahmoudi f. 20 acta bot. croat. 80 (1), 2021 water. the experiment was carried out in the dark in an incubator at 20 °c. the number of germinated seeds was counted daily for 10 days, and the emergence of a 2 mm rootlet was considered as the criterion for seed germination (ista, 2017). the seed germination rate was calculated using the following equation (ellis and roberts 1981). r n d n = ∑ ∑ in this formula, r is the average seed germination rate, d the number of days from the beginning of the experiment, and n is the number of seeds germinated during the day (d). seedling dry weight and vigor index at the end of the germination test (after 10 days), the percentage of seed germination) was calculated, and the length of seedlings was measured. dry weight of seedlings was measured after drying at 80 °c in an oven until a constant weight was recorded. seed vigor is the sum of those seed attributes that characterize the level of activity and performance of the seed lot during germination and seedling emergence. seed vigor is an important quality parameter, which supplements germination and viability tests to achieve insights into the seed lot performance in the field or in experimental condition. it can be evaluated by various methods, such as seedling vigor indices. seedling vigor index i and ii were calculated as follows (vashisth and nagarajan 2010): seedling vigor index i = germination (%) × mean seedling length (cm) seedling vigor index ii = germination (%) × mean seedling dry weight (mg) enzyme activity assay crude protein extract from seedlings (10 days old) was prepared using the chang and koa (1988) method. briefly, 0.8 g of seedlings were thoroughly ground into fine powder in liquid nitrogen using a mortar and pestle and then homogenized in 6 ml of 0.2 m potassium phosphate buffer, (ph 7.0 and containing 0.1 mm edta). the homogenate was filtered and centrifuged at 15000 × g for 20 min at 4 °c, and the supernatant fraction was used as a crude extract for protein and antioxidant enzyme activity assays. total protein content was estimated according to the bradford (1976) method using bovine serum albumin as the standard. catalase (ec 1.11.1.6) activity was determined based on the consumption of h2o2 and the decrease in absorbance at 240 nm for 1 min (aebi 1984). briefly, one ml of reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0) and 10 mm h2o2 and 50 µl of enzyme extract. the activity of catalase was calculated using the extinction coefficient of h2o2 (40 mm−1 cm−1 at 240 nm). enzyme activity was expressed in terms of mmol of h2o2 min–1 g–1 fw. peroxidase activity was determined based on the conversion of guaiacol to tetraguaiacol in the presence of h2o2 (fielding and hall 1978). briefly, 1 ml of reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0), 7.2 mm guaiacol, 11.8 mm h2o2, and 50 µl of enzyme extract. the reaction was initiated by adding enzyme extract, and the increase in absorbance at 470 nm was measured for 1 min. the activity of peroxidase was calculated using the extinction coefficient of tetraguaiacol (22.6 mm−1 cm−1 at 470 nm) and expressed as micromole of guaiacol oxidized per min. the proline content of the seedlings was estimated according to the method of bates et al. (1973). seedling samples (0.5 g) were ground in liquid nitrogen and extracted with 3% sulfosalicylic acid. after centrifugation (10,000 rpm, 10 minutes), 2 ml of the supernatant was taken and 2 ml of glacial acetic acid and 2 ml of ninhydrin reagent (2.5% of ninhydrin in glacial acetic acid and 6 m orthophosphoric acid mixture) was added and incubated in boiling water for 60 min. the reaction was terminated by placing the tubes on ice. then, 4 ml of toluene was added to the tubes, vigorously mixed for 20 s and incubated at room temperature for 5 min. the absorbance of the upper layer was read at 520 nm, and the proline content was determined using the l-proline standard curve and expressed as μmol g–1 fresh weight. statistical analysis in order to investigate the effect of hydro-priming and hormone priming on the germination, growth and biochemical activities of borage (borago officinalis l.) seedlings under cadmium stress, a factorial experiment based on a completely random design (crd) was carried out with three independent replications and 25 seeds per replication. the experimental factors include cadmium stress [0 (control), 10, 50 and 100 mg l–1] and different seed priming (hydro-priming for 24 hours, priming with different concentration of gibberellic acid (ga3) (50, 100, 150 mg l–1) for 24 hours, sequential hydro-priming and hormone priming and control seeds (non-primed). distribution of data sets for all traits was analyzed using different normality tests including kolmogorov-smirnov, skewness and kurtosis, and the results showed that the data sets follow a normal distribution. all data were subjected to anova followed by duncan’s multiple range test (parametric methods) and a nonparametric kruskal-wallis test for group comparison (p < 0.05). given that the kruskal-wallis test for group comparison showed a pattern of significance similar to those of anova and duncan’s multiple range test, with very little and negligible changes, here we report the results of the parametric analysis method. analyses were performed using spss ver. 16 (spss inc, chicago, il.) and sas ver. 9 (sas institute, cary, nc, usa) software and the graphs were produced using microsoft office excel 2010. all values are presented as mean ± se (standard error) and with significance at p ≤ 0.05. borage seed priming enhanced cadmium tolerance acta bot. croat. 80 (1), 2021 21 results seed germination data analysis indicated that seed germination and rate were significantly influenced by cadmium stress and seed priming and their interaction. both the percentage and rate of borage seed germination were decreased with an increase in the intensity of cadmium stress. therefore, the highest and lowest seed germination percentage and rate were observed under non-stress (without cadmium stress) and 100 mg l–1 cadmium treatments, respectively (fig. 1 and 2a). as shown in fig. 1, seed priming significantly improved the percentage of seed germination in both non-stress and stress conditions. furthermore, the seed germination rate of primed seeds was significantly higher than that of nonprimed seeds (fig. 2b). these results indicate that the sequential application of hydro and hormone priming of seed led to an increase in seed germination rate (fig. 2b). under cadmium stress, the highest percentage of germination percentage was obtained from seeds primed with distilled water fig. 1. the effects of cadmium (cd) stress (10, 50, 100 mg l–1) and seed priming (non-priming, hydro-priming, hormone priming (50, 100, 150 mg l–1 ga3) and sequential application of hydro and hormone priming (hydro-priming + ga3) (50, 100, 150 mg l–1) for 24 hours) on the germination percentage of borage (borago officinalis l.) seeds. the values present mean of data from three technical replicates (n=3) and 25 seeds per replicate. the error bars show standard error (se). values followed by different letters are statistically different (duncan’s multiple range test, p <0.05). fig. 2. the effects of cadmium stress (10, 50, 100 mg l–1) (a) and seed priming (non-priming, hydro-priming, hormone priming (50, 100, 150 mg l–1 ga3) and sequential application of hydro and hormone priming (hydro-priming + ga3) (50, 100, 150 mg l–1) for 24 hours) (b) on the germination rate of borage (borago officinalis l.) seeds. the values represent mean data from three technical replicates (n=3) and 25 seeds per replicate. the error bars show standard error (se). values followed by different letters are statistically different (duncan’s multiple range test, p <0.05). sheikhzadeh p., zare n., mahmoudi f. 22 acta bot. croat. 80 (1), 2021 (hydro-priming) + 150 mg l–1 ga3 and with distilled water (hydro-priming) + 100 mg l–1 ga3, which was significantly higher than that of the non-primed seeds and other treatments. on the other hand, as shown in fig. 1, in non-stress conditions there were no considerable differences among the different priming methods (hydro-priming, hormone priming and sequential hydro and hormone priming), but at the 10 mg l–1 cadmium stress condition, the percentage of seed germination observed for the seeds primed with the combination of hydro-priming with 100 or 150 mg l–1 ga3 (distilled water + 100 or 150 mg l–1 gibberellic acid) was significantly higher than that of either hydro or hormone-priming treatments, as well as that of non-primed seeds. seedling growth characteristics seedling growth characteristics, including seedling dry weight and length, and seedling vigor indices (vigor index i and ii), were significantly influenced by cadmium stress, seed priming, and their interaction. as shown in figs. 3, 4 and 5a,b, with increasing cadmium concentration, seedling growth characteristics were significantly decreased, so that the lowest and the highest growth of borage seedlings were related to the treatment with 100 mg l–1 cadmium and the control (cadmium-free), respectively (figs. 3, 4, 5a,b). at non-stress and lower (10 mg l–1) cadmium stress conditions, the highest seedling dry weight and vigor index ii were obtained from hydro-priming and its combination with 50, 100 or 150 mg l–1 gibberellin (distilled water + (50, 100 or 150 mg l–1 ga3)), which were significantly higher than those of the other treatments (figs. 3 and 5b). however, at higher concentrations of cadmium (50 and 100 mg l–1), seedling dry weight and vigor index ii obtained from sequential hydro and hormone priming treatments were higher than those of either hydro or hormone priming and as well as non-primed seeds (figs. 3 and 5b). in non-stress conditions, the longest seedlings and highest vigor index i were obtained from seeds primed with distilled water + 100 or 150 mg l–1 ga3, which were significantly higher than those from other priming treatments and non-primed seeds. at lower level (10 mg l–1) of cadmium stress, seedling length and vigor index i of seedlings obtained from hormone priming and its combination with hydro-priming were significantly higher than those of hydro-primed and non-primed seeds, but there were no significant differences between ga3 alone and ga3 in combination with hydro-priming treatments (figs. 4, 5a). however, at higher concentrations (50 and 100 mg l–1) of cadmium, sequential application of hydro and hormone priming resulted in a statistically significant increase in seedling length and in vigor index i as compared with only hormone or hydro-priming treatments as well as with nonprimed seeds (figs. 4, 5a). these results indicate the synergistic effects of hormone and hydro-priming techniques on the improvement of seedling growth and performance, especially under cadmium stress conditions. antioxidant enzymes activity and proline content according to the results of variance analysis, the activity of catalase and peroxidase enzymes and proline content in borage seedlings were significantly influenced by cadmium stress, seed priming, and their interaction. cadmium stress caused a significant decrease in the activity of catalase fig. 3. the effects of cadmium stress (10, 50, 100 mg l–1) and seed priming (non-priming, hydro-priming, hormone priming (50, 100, 150 mg l–1 ga3) and sequential application of hydro and hormone priming (hydro-priming + ga3) (50, 100, 150 mg l–1) for 24 hours)) on the seedling dry weight of borage (borago officinalis l.). the values represent mean data from three technical replicates (n=3) and 25 seeds per replicate. the error bars show standard error (se). values followed by different letters are statistically different (duncan’s multiple range test, p <0.05). borage seed priming enhanced cadmium tolerance acta bot. croat. 80 (1), 2021 23 and peroxidase enzymes, but seed priming treatments significantly increased the activity of these enzymes in both non-stress and cadmium stress environments. on the other hand, under both non-stress and cadmium stress conditions, the activity of catalase and peroxidase enzymes in seedlings obtained from primed seeds was significantly higher than that of seedlings from non-primed seeds (fig. 6a, b). under both non-stress and cadmium stress conditions, the highest activity of catalase was observed in the seedlings obtained from the seeds primed with the combination of hydro and hormone, which was significantly higher than that of seedlings obtained from non-primed seeds and other seed priming treatments (fig. 6a). however, the highest activity of peroxidase enzyme was observed in seedlings obtained from seeds primed only with distilled water (hydro-priming) and 150 mg l–1 ga3 under both non-stress and cadmium stress conditions, which was significantly higher than its activity in seedlings obtained from other treatments, especially from seedlings obtained from sequential hydro and hormone priming treatments (fig. 6b). the proline content of borage seedlings significantly increased after applying cadmium stress and seed priming, and the amount of enhancement was varied depending on seed priming treatments and cadmium stress level (fig. 7). as shown in fig. 7, the proline content of seedlings significantly increased with an increase in the severity of cadmium stress, so that, at all seed priming treatments the highest amount of proline in borage seedlings was related to the 100 mg l–1 cadmium stress, which was significantly higher than the other levels of cadmium stress. the highest amount of proline was recorded under severe cadmium (100 mg l–1) stress in seedlings obtained from seeds primed with distilled water and distilled water (hydro priming) +150 mg l–1 ga3 treatments, which was significantly higher than that of the other treatments. discussion seed germination generally, cadmium stress negatively affected the germination of borage seeds (fig. 1). these adverse effects could be attributed to the accumulation of cadmium in the cell and its inhibition effects on nutrient uptake and translocation as well as cellular metabolism and photosynthesis (gill et al. 2013). furthermore, it has been shown that cadmium uptake and accumulation in the cells lead to the destruction of protein and enzyme structures or inhibit their function and activity (hasan et al. 2009, hussain et al. 2016) as well as their synthesis. these enzymes and structural proteins are needed in cell growth and division, and germination processes (tiryakioglu et al. 2006, dar et al. 2016). nevertheless, seed priming significantly reduces these adverse effects of cadmium; and leads to an increase in the percentage of borage seed germination under cadmium stress conditions (fig. 1). the improved and accelerated germination of borage seeds under both non-stress and cadmium stress conditions could be attributed to the improving effects of seed priming, especially of combined hydro and hormone priming, on biochemical processes in the plant cell. it has been shown that seed priming induces the cytoplasmic membrane and dna damage repairing systems, dna, rna and protein synthesis and activity of starch-degrading enzymes such as alpha-amylase and reduces the leakage of metabolites. these fig. 4. the effects of cadmium (cd) stress (10, 50, 100 mg l–1) and seed priming (non-priming, hydro-priming, hormone priming (50, 100, 150 mg l–1 ga3) and sequential application of hydro and hormone priming (hydro-priming + ga3) (50, 100, 150 mg l–1) for 24 hours)) on the seedling length of borage (borago officinalis l.). the values represent mean data from three technical replicates (n=3) and 25 seeds per replicate. the error bars show standard error (s.e.). values followed by different letters are statistically different (duncan’s multiple range test, p <0.05). sheikhzadeh p., zare n., mahmoudi f. 24 acta bot. croat. 80 (1), 2021 improved cellular and molecular processes lead to an increase in the cell division and embryo growth, and finally improved seed germination and vigor (mcdonald 2000, hussain et al. 2016). the improved percentage and rate of seed germination through hydro or hormone priming have also been reported in chickpea (ghassemi golezani et al. 2008), pigeon pea (sneideris et al, 2015), in borage (mahmoudi et al. 2017), and maize (li et al. 2017). in borage, the seed priming increases varied from 1.1-1.2-fold under control (without cadmium) condition and 1.03-1.3-fold under cadmium stress conditions, as compared with non-primed seeds (fig. 1). seedling growth characteristics the superiority of primed seeds in terms of the enhanced borage seedling growth under non-stress and cadmium stress conditions can be attributed to the higher germination rate (fig. 2b) of these seeds. seed priming treatments improved the seedling growth characteristics in both non-stress and cadmium stress conditions, so at all levels of cadmium stress, dry weight and length, and seedling vigor indices of seedlings obtained from primed seeds were significantly higher than those obtained from nonprimed seeds (figs. 3, 4, 5a, b). the fact that primed seeds had a higher germination rate than non-primed seeds fig. 5. the effects of cadmium (cd) stress (10, 50, 100 mg l–1) and seed priming (non-priming, hydro-priming, hormone priming (50, 100, 150 mg l–1 ga3) and sequential application of hydro and hormone priming (hydro-priming + ga3) (50, 100, 150 mg l–1) for 24 hours)) on the seedling vigor index ӏ (a) and seedling vigor index ii (b) of borage (borago officinalis l.). the values represent mean data from three technical replicates (n=3) and 25 seeds per replicate. the error bars show standard error (se). values followed by different letters are statistically different (duncan’s multiple range test, p <0.05). borage seed priming enhanced cadmium tolerance acta bot. croat. 80 (1), 2021 25 means a quicker seed germination and the production of higher biomass than in non-primed seeds. it has been shown that seed priming increases the activity of α-amylase in maize (li et al. 2017) and rice (hussain et al. 2016) seeds, which could increase the germination rate and finally growth characteristics of the seedlings. therefore, seed priming through the reduction of embryonic growth barriers and increasing seed vigor improves seed germination and growth of seedlings, which leads to the production of more vigorous seedlings. however, there were significant differences among the various seed priming treatments in terms of the abovementioned growth characteristics. our results indicated that in the non-stress condition, combined hydro and hormone priming led to about 0.9 to 4.41-fold increases in the borage seedling weight as compared with seedlings obtained from the non-primed seeds. under cadmium stress conditions, the combined hydro and hormone priming caused a 5.80–11.53 and a 1.02-2.63-fold increase in the seedling dry weight in comparison with the seedlings obtained from non-primed seeds and seeds primed using either hydro or hormone, respectively (fig. 3). as shown in fig. 4, under severe cadmium stress (100 mg l–1), the combination of hydro and hormone priming increased the borage seedling length by 3.75–4.58 and 1.5–2.03 times in comparfig. 6. the effects of cadmium stress (cd) (10, 50, 100 mg l–1) and seed priming (non-priming, hydro-priming, hormone priming (50, 100, 150 mg l–1 ga3) and sequential application of hydro and hormone priming (hydro-priming + ga3) (50, 100, 150 mg l–1) for 24 hours)) on the catalase (a) and peroxidase (b) enzymes activity of borage (borago officinalis l.) seedlings. the values represent mean data from three technical replicates (n=3) and 25 seeds per replicate. the error bars show standard error (se). values followed by different letters are statistically different (duncan’s multiple range test, p <0.05). sheikhzadeh p., zare n., mahmoudi f. 26 acta bot. croat. 80 (1), 2021 ison with that of the seedlings obtained from non-primed seeds and either hydro or hormone primed seeds, respectively. in other words, the sequential application of hydro and hormone priming on borage seeds enhances the positive effects of each of the techniques on the seedling growth characteristics such as seedling length (fig. 4) and seedling vigor index i (fig. 5a). these results are consistent with the findings of tiryakioglu et al. (2006) in barley, mahmoudi et al. (2017) in borage. antioxidant enzymes activity and proline content as shown in fig. 6a, the activity of catalase in borage seedlings from the seeds primed with the combination of distilled water and ga3 were significantly higher than those of the seeds primed with alone distilled water or ga3, as well as non-primed seeds. these results emphasize the synergistic effects of hydro and hormone priming techniques, which lead to improvement of the biochemical and antioxidant potential of borage seedlings. it has been shown that environmental stresses such as cadmium stress induces the production of ros in plant cells, which attack biological molecules, including proteins and enzymes at the cellular and subcellular levels, and finally reduce or stop cell growth and the proliferation process (mittler 2002, tiryakioglu et al. 2006). the plant cells adapted various enzymatic and non-enzymatic mechanisms that scavenge ros molecules and protect the biological molecules and cells from the damaging effects of environmental stress. catalase and peroxidase, the most important antioxidant enzymes of plant cells, play a crucial role in the ros scavenging process. these enzymes catalyze the conversion of h2o2 (hydrogen peroxide) to h2o and o2 in plant cells (gill and tuteja 2010). on the other hand, the balance between ros production and activity of antioxidant systems may determine the plant’s responses to stressful environments, such as cadmium stress, and their damaging effects. it could be concluded that hydro and hormone priming of borage seeds enhanced the activity of catalase and peroxidase enzymes in the borage seedlings, which in turn enhance the antioxidant capacity of the cells and their ability for germination (fig. 1) and growth (figs. 3–5b) under cadmium stress. for example, the sequential application of hydro and hormone priming led to 2.42–2.57 and 2.93– 18.72 fold increase in the activity of catalase enzyme under non-stress and cadmium stress conditions, respectively, as compared with those of non-primed seeds (fig. 6a). the relationship between reduced damaging effects of environmental stresses and enhanced activity of antioxidant enzymes have been reported in several plants, including rice (guo et al. 2006, hussain et al. 2016) response to drought and chilling, sugar beet (bor et al. 2003) response to salinity, and mung bean (tiryakioglu et al. 2006) response to heavy metal. on the other hand, it could be concluded that these seed priming techniques mitigate the damaging effects of cadmium in borage seedlings through enhancing the activity of antioxidant systems and reducing adverse effects of cadmium-induced oxidative stress in the cells (fig. 6a,b). among amino acids, proline biosynthesis and catabolism in plant cells play vital roles in physiological responses and plant adaptation to environmental and biotic stresses (dinakar et al. 2008). it has been shown that this amino acid acts as a free radical scavenger and chemical chaperone that stabilizes proteins in their native conformation, cellufig. 7. the effects of cadmium stress (cd) (10, 50, 100 mg l–1) and seed priming (non-priming, hydro-priming, hormone priming (50, 100, 150 mg l–1 ga3) and sequential application of hydro and hormone priming (hydro-priming + ga3) (50, 100, 150 mg l–1) for 24 hours) on proline content of borage (borago officinalis l.) seedlings. the values represent mean data from three technical replicates (n=3) and 25 seeds per replicate. the error bars show standard error (s.e.). values followed by different letters are statistically different (duncan’s multiple range test, p < 0.05). borage seed priming enhanced cadmium tolerance acta bot. croat. 80 (1), 2021 27 lar and subcellular membranes, especially under stressful conditions (hossain et al. 2014). in borage, the proline content of seedlings from primed seeds was significantly higher than that of seedlings obtained from non-primed seeds, but the amount of increase in proline content varied from a 1.06 to a 1.45-fold change depending on cadmium stress level and seed priming treatments (fig. 7). this elevated level of proline improves the cellular and physiological processes in borage seedlings and facilitates the activation of detoxification pathways (dar et al. 2016), cytoplasmic osmotic adjustment and the balancing of cell redox reactions (hossain et al. 2014). on the other hand, the content of proline in borage seedlings was increased 1.45 and 1.37 times through combined hydro and 150 mg l–1 ga3 priming treatment under 10 mg l–1 and 100 mg l–1 cadmium stress, respectively, as compared to the non-primed seeds (fig. 7). this regulation of proline concentration in the plant cells is achieved through its biosynthesis, catabolism, and transport between cells and subcellular organelles (man et al. 2011, hossain et al. 2014), and these processes are strongly influenced by various factors, especially plant genotype, environmental conditions and hormone balance in the plant cells (dar et al. 2016). accumulation of free proline in plant cells and tissues in response to the heavy metal and other abiotic and biotic stresses has been reported in a wide range of plant species. for example, increased levels of proline content was found to occur in arachis hypogaea l. seedlings (dinakar et al., 2008) and ocimum basilicum l. leaves (georgiadou et al., 2018) in response to heavy metal stress, and in tall fescue (man et al. 2011) in response to drought stress. conclusions there is little information in the literature concerning the relationship between seed priming techniques and cadmium toxicity and tolerance in plants. in the present study, for the first time, we demonstrated that the hydro and hormone seed priming techniques effectively mitigated the damaging effects of cadmium stress and improved seed germination and seedling growth under both non-stress and cadmium stress conditions. among the seed priming treatments, combined hydro and hormone seed priming improved borage seed germination and seedling growth under stressful environments. this improved germination and seedling growth of primed borage seeds was associated with an increased activity of antioxidant enzymes (catalase and peroxidase) and an elevated proline content in borage seedlings. references aebi, h., 1984: catalase in vitro. methods in enzymology 105, 121–126. bates, l.s., waldren, r.p., teare, i.d., 1973: rapid determination of free proline for water-stress studies. plant and soil 39, 205–207. bor, m., ozdemir, f., turkan, i., 2003: the effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet beta vulgaris l. and wild beet beta maritima l. plant science 164, 77–84. bradford, m.m., 1976: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analytical biochemistry 72, 248–254. chang, c.j., koa, c.h., 1988: h2o2 metabolism during senescence of rice leaves: changes in 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(eds.), seed technology and its biological basis, 287–325. sheffield academic press, sheffield. mittler, r., 2002. oxidative stress, antioxidants and stress tolerance. trends in plant science 7, 405–410. munzuroglu, o., zengin, f.k., 2006: effect of cadmium on germination, coleoptile and root growth of barley seeds in the presence of gibberellic acid and kinetin. journal of environmental biology 27, 671–677. sneideris, l.c., gavassi, m.a., campos, m.l., damico-damiao, v., carvalho, r.f., 2015: effects of hormone priming on seed germination of pigeon pea under cadmium stress. anais da academia brasileira de ciencias 87, 1847–1852. tiryakioglu, m., eker, s., ozkutlu, f., husted, s., cakmak, i., 2006: antioxidant defense system and cadmium uptake in barley genotypes differing in cadmium tolerance. journal of trace elements in medicine and biology 20, 181–189. vashisth, a., nagarajan, s., 2010: effect on germination and early growth characteristics in sunflower (helianthus annuus) seeds exposed to static magnetic field. journal of plant physiology 167, 149–156. opce-str.vp acta bot. croat. 69 (2), 199–214, 2010 coden: abcra 25 issn 0365–0588 flowering phenology and airborne pollen occurrence of corylus and castanea in trieste (italy), 1991–2004 loredana rizzi longo*, marialuisa pizzulin sauli department of life sciences, university of trieste, faculty of science, via l. giorgieri 10, 34127 trieste, italy the flowering season and the pollen season of corylus and castanea were analyzed and compared in trieste over a period of 14 years (1991–2004). a great variability in the flowering phenophase progression was found both among plants and from year to year. the pollen seasons of corylus and castanea were longer than the respective flowering seasons. a lack of correlation between the maximum flowering and the maximum pollen concentration was observed, the highest airborne pollen counts occurring two or more weeks after the maximum flowering. given the geographical complexity of the area around trieste, the onset of the flowering of corylus and castanea does not occur at the same time everywhere, and grains coming from different local and extra-local sources, characterized by a late blooming, were also found. medium-long range transport from slovenia should also be considered. keywords: aerobiology, corylus, castanea, flowering, phenology, italy, trieste introduction phenology is the study of the timing of the biological phases recurring in the annual cycles of plants and animals. as regards plants, the biological events include sprouting, flowering and fruit ripening. phenological models are considered suitable for studying the response of plants and ecosystems to climate changes and global warming (menzel 2000, sparks et al. 2000, ahas et al. 2002, menzel et al. 2006); in order to evaluate a possible response of plants to the recent increase in temperature, the climatic impact on plant flowering has also been studied by using the data from pollen monitoring in correlation with temperature data (jäger et al. 1996; emberlin et al. 1997, 2002, 2007; frei 1998, tedeschini et al. 2006, bonofiglio et al. 2009, orlandi et al. 2009). studies on flowering phenophases may also be useful for interpreting aerobiological data and for discovering the sources of airborne allergenic pollen; for this reason, phenological studies have been carried out in order to evaluate the relationships between the flowering of allergenic plants and the occurrence of airborne pollen (puppi branzi and zanotti 1992; latorre 1997, 1999; latorre and bianchi 1998; jato et al. 2002). in italy, the phenological working group of the italian association of aerobiology founded a phenological network in 1991, with the purpose of comparing acta bot. croat. 69 (2), 2010 199 * corresponding author, e-mail: rizzi@units.it u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:41 color profile: disabled composite 150 lpi at 45 degrees the phenophase progression of castanea, corylus and gramineae in different italian sites (zanotti et al. 1998). in trieste, aerobiological monitoring and phenological observations have been carried out since 1978 (mandrioli et al. 1980; rizzi longo and cristofolini 1987; rizzi longo 1990, 2003; puppi et al. 1994; rizzi longo et al. 2007). the distribution and frequency of pollen sources as well as the sensitization to some allergenic taxa in trieste have also been investigated (larese et al. 1992, 1998, 2000; pizzulin sauli et al. 1992; poldini et al. 1997/1998; rizzi longo and martini 2000; martini et al. 2002; rizzi longo et al. 2003). trieste is located at the northern end of the adriatic sea (fig. 1), on the border between the mediterranean and the continental climate. the annual mean temperature in trieste is 15.1 °c, with monthly means ranging from 6.4 in january, to 24.9 in august (tab. 1), and the total precipitation is about 1000 mm (stravisi 2006). local coastal sea (nw) and land breezes (e) prevail during the warm season and make weather conditions stable (stravisi 1977). the mesoscale flow is dominated by the continental dry wind the »bora« (ene), which blows at higher speed and frequency during the cold season; secondary events are represented by moist se winds across the adriatic sea, mainly the »sirocco« (fig. 2). the town lies on sandstone hills, which are surrounded by the steep slope of the karst plateau, with a mean elevation of 250 m a.s.l. woody coenoses, mainly oak woods, are frequent in the area of trieste. the aim of this study was the analysis of the flowering phenological behaviour and the occurrence of airborne pollen in two allergenic taxa in trieste: corylus and castanea. corylus avellana l., a winter-flowering taxon, is frequent in shrub thickets and in karst 200 acta bot. croat. 69 (2), 2010 rizzi longo l., pizzulin sauli m. fig. 1. study area and location of the pollen sampling site in trieste (�); location of the phenological stations for castanea (�) and corylus (�), where phenological surveys were performed. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:42 color profile: disabled composite 150 lpi at 45 degrees dolines; castanea sativa mill., a spring-summer flowering tree, grows in oak woods (poldini 1989). the relationships between the flowering phenophases and the pollen curves were also investigated. acta bot. croat. 69 (2), 2010 201 phenology and aerobiology of corylus and castanea in trieste fig. 2. trieste (1990–2004): mean distributions of the annual wind duration (in hours) and wind run (in kilometers). tab. 1. monthly temperatures for trieste (1991–2004). years jan feb mar apr may jun jul aug sept oct nov dec mean 1991 5.7 4.7 12.0 12.5 14.3 20.5 25.1 24.8 21.9 14.1 10.3 5.6 14.4 1992 5.5 6.6 9.0 13.2 19.4 21.2 24.0 26.4 20.9 15.0 12.3 7.7 15.1 1993 5.7 6.8 7.5 13.3 20.0 22.5 23.0 24.8 19.5 15.9 8.3 8.5 14.7 1994 8.9 6.5 12.2 13.4 17.9 21.9 26.8 25.9 20.8 14.9 13.1 8.1 15.9 1995 6.1 8.6 8.9 12.7 17.4 19.9 25.9 23.2 18.5 17.2 10.6 7.2 14.7 1996 6.7 5.1 7.6 13.7 18.4 22.4 22.9 23.3 17.4 15.6 12.2 6.6 14.4 1997 7.4 8.1 11.6 11.2 18.4 21.9 23.4 24.0 21.2 14.6 11.1 8.3 15.1 1998 7.3 9.1 9.3 13.2 18.3 22.6 24.8 25.7 19.6 15.7 8.9 6.2 15.1 1999 6.3 5.7 10.3 13.9 18.9 22.4 24.5 24.5 22.0 16.1 9.6 6.8 15.1 2000 4.8 7.7 9.9 15.0 19.8 23.5 22.9 25.3 20.8 17.3 13.1 9.7 15.8 2001 7.9 8.7 11.9 13.0 20.2 21.3 24.7 26.2 18.0 18.3 10.4 4.9 15.5 2002 5.4 7.6 11.7 13.6 18.8 23.5 24.7 23.8 19.5 16.4 14.0 7.9 15.6 2003 5.8 4.4 10.1 13.0 20.3 25.8 26.1 27.9 19.8 13.8 12.2 8.5 15.7 2004 5.1 5.7 8.7 13.6 16.4 21.6 24.3 24.6 20.7 16.8 11.7 9.0 14.9 mean 6.4 7.0 10.2 13.2 18.5 22.1 24.4 24.9 20.0 15.9 11.3 7.4 15.1 u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:42 color profile: disabled composite 150 lpi at 45 degrees materials and methods flowering phenophase monitoring phenological observations of the flowering period of corylus and castanea were carried out in trieste over a 14-year period, from 1991 to 2004. during the whole period two survey stations were observed (fig. 1): one for castanea (in vicolo delle rose, at 150–170 m) and one for corylus – named ts1 – (in via commerciale, at 260 m). the examined plants of these survey stations grow in disturbed oak woods, together with quercus petraea liebl., robinia pseudacacia l. and sambucus nigra l. from 1992 to 1995, another survey station (named ts2) was considered in order to study the flowering of corylus. ts2 was located in bosco vignano near to muggia, in the south-eastern area of the province of trieste, on the border with slovenia, at 70 m a.s.l. (fig. 1): here corylus plants grow in a semi-natural oak wood together with carpinus betulus l., castanea sativa and quercus petraea. the phenophase progression was monitored according to the standard method adopted by the italian phenological network (zanotti et al. 1998). phenological observations were carried out once a week; a sample of five plants was used in each population. only the following steps of the phenological cards, corresponding to some of the stages of the phenological key proposed by marcello (1935), were considered in this paper: lengthened catkins with closed anthers (stage +00); catkins with some open anthers releasing pollen (stage ++0); catkins with some exhausted anthers (stage +++); catkins with open and exhausted anthers (stage 0++); catkins with only exhausted anthers (stage 00+). the stage +++ corresponds to the stage 65 of the extended bbch-scale (meier 1997). the beginning of the flowering season was defined as the moment when at least one of the five examined plants showed long catkins with closed anthers (stage +00); the time when all the examined plants displayed catkins with closed, open and exhausted anthers (stage +++) was considered the moment of full flowering; the end of the flowering season was defined as the moment when all the examined plants showed catkins with only exhausted anthers (stage 00+). airborne pollen sampling daily airborne pollen records of corylus and castanea were collected over a 14-year period (1991–2004). during this period, a hirst type 7-day volumetric spore trap (burkard until 1999, vpps 2000 lanzoni from 2000) was operating at about 20 m above ground level on the bastione fiorito of san giusto castle, in the town centre (fig. 1). sampling method, slide preparation and data interpretation were performed according to the standard method adopted by the italian aeroallergen network (mandrioli 1990). the pollen counts were expressed as pollen grains per cubic meter of air (p/m3). for each year, the main pollen season (mps) of each taxon was determined by taking 90% of the annual total pollen concentration, using cumulative values (nilsson and persson 1981). the start date of the mps was set in correspondence to the day when the sum of the daily concentrations reached 5% of the total sum; the end date of the mps was made to correspond to the time when that sum reached 95%. for each year, the total annual pollen amount, the peak day and the peak value were also considered. since phenological observations were carried out once a week, daily pollen counts of 7-day periods were summed in order to compare the phenological and the aerobiological 202 acta bot. croat. 69 (2), 2010 rizzi longo l., pizzulin sauli m. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:42 color profile: disabled composite 150 lpi at 45 degrees data. in order to compare these two sets of data, the full flowering in the survey stations was considered correspondent to the time of maximum pollen production. the period from the start of the season to the maximum pollen production was considered the ascendant phase, whereas the period from the maximum pollen production to the end of the season was considered the descendant phase. in the graphic presentation, the weekly pollen variations of each year have been presented from january to late june for corylus, from late april to the end of september for castanea. these periods were chosen on account of the fact that very small amounts of pollen grains of the examined taxa were found during the rest of the year (rizzi longo et al. 2005, 2007). moreover, both the weekly sums of the daily pollen concentrations calculated for each year and the phenological data were averaged out over the 14 years (1991–2004). results corylus flowering season during the 14-year period, several differences in the timing of the flowering phenophases were recorded for hazel plants observed in the survey station named ts1 (tab. 2). the earliest start of the flowering of corylus was recorded in 1994 and in 2001 (2nd january), the latest one was recorded in 1995 and in 1996 (28th january), with a difference of 26 days. the earliest end of the flowering time was observed in 1994 (13th february), the latest one in 2003 (30th march), with a difference of 45 days. the flowering season lasted on average seven weeks, the longest one was recorded in 2003 (63 days) and the shortest one in 2000 (35 days). the course of the flowering of hazel plants observed in the survey station named ts1 differed greatly over the years (fig. 3). in several years, corylus began flowering in the fourth week of january and finished flowering between late february and late march. the full flowering usually occurred in february. in some years, the full flowering lasted two weeks. a long descendant flowering phase was frequently observed and attributed to the irregular course of meteorological parameters, which brought about an interruption and a subsequent restarting of the blooming (stravisi 2006). in 1994, the flowering of corylus was anomalous: both the beginning and the full flowering occurred within the first week of january, catkins being not yet lengthened in the previous week and already almost exhausted in the following one. the flowering behaviour of corylus was examined from 1992 to 1995 in survey station ts2, too. in these years, the flowering phenophase progression differed between the two studied populations (ts1 and ts2) and showed a constant delay of the flowering season in ts2 in comparison with ts1 (fig. 3). corylus began flowering 10 to 15 days earlier in ts1 than in ts2 in 1992, 1994 and 1995, and a month earlier in 1993 (tab. 3). the flowering season was longer in ts2 than in ts1 in 1992 and 1994. pollen season during the 14-year period, significant differences were recorded in the mps dates and in the annual pollen totals of corylus (tab. 2). the mean start date of the main pollen season (mps) fell at the beginning of february; an early start was recorded in 1994 (8th january), a late one in 1996 (1st march). the main pollen season was very short in 1991 and in 1992 (35 days), and very long in 1994 (84 days). on average, the mps of corylus lasted acta bot. croat. 69 (2), 2010 203 phenology and aerobiology of corylus and castanea in trieste u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:42 color profile: disabled composite 150 lpi at 45 degrees about two months, from early february to early april. different annual totals of corylus were recorded over the years, the lowest in 2000 (197), and the highest in 1998 (1796). the highest daily pollen values also varied over the years. the earliest daily peak occurred in 2001, on 6th february, and the latest one was recorded in 1996, on 28th march. the mean daily maximum was 83 p/m3, with the highest value of 324 in 1998, and the lowest value of 24 in 2000. the course of the weekly pollen concentrations was rather different over the years (fig. 3). in 1991, 1992, 1998 and 2000, only one peak was recorded; in 1995, 1996, 1997, 1999, 2001 and 2004, two or more peaks (variable as for the pollen amount) were observed. in the other years (1993, 1994, 2002, 2003), no evident peak was found. long ascendant phases were observed, particularly in 1992, 1999 and 2004. the weekly peaks usually occurred in late february or in march. in 1998, an exceptionally high weekly amount of hazel airborne grains was recorded. 204 acta bot. croat. 69 (2), 2010 rizzi longo l., pizzulin sauli m. tab. 3. corylus flowering seasons mean data at stations ts1 and ts2 (1992–1995). years flowering season start end length ts1 ts2 ts1 ts2 ts1 ts2 1992 19/1 3/2 8/3 30/3 49 56 1993 24/1 20/2 14/3 10/4 49 49 1994 2/1 11/1 13/2 10/3 42 58 1995 28/1 8/2 11/3 22/3 42 42 tab. 2. flowering and pollen seasons of corylus in trieste at station ts1 (1991–2004). flowering season column data refer to mean data; the pollen season column data refer to 90% pollen concentration method. the start of the pollen season was chosen as the day when the cumulated daily counts reached 5% of the annual sum; the end as the day when the cumulated daily counts reached 95%. years flowering season pollen season annual total peak value (p/m3) peakdatestart end length start end length 1991 20/1 10/3 49 19/2 26/3 35 518 65 26/2 1992 19/1 8/3 49 13/2 19/3 35 803 137 1/3 1993 24/1 14/3 49 28/1 30/3 61 347 28 19/3 1994 2/1 13/2 42 8/1 2/4 84 816 39 4/3 1995 28/1 11/3 42 5/2 19/4 73 577 38 22/2 1996 28/1 17/3 49 1/3 24/4 53 829 108 28/3 1997 26/1 16/3 49 2/2 5/4 62 692 71 4/3 1998 4/1 1/3 56 12/2 5/4 52 1796 324 15/2 1999 10/1 28/2 49 2/2 8/4 65 1244 125 14/3 2000 23/1 27/2 35 1/2 2/4 61 197 24 28/2 2001 2/1 26/2 55 16/1 6/4 80 758 87 6/2 2002 27/1 10/3 42 10/2 5/4 54 432 36 23/2 2003 26/1 30/3 63 17/2 24/4 66 333 25 12/3 2004 25/1 22/3 57 19/2 28/4 67 928 56 17/3 mean 19/1 9/3 49 6/2 8/4 61 734 83 4/3 u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:42 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (2), 2010 205 phenology and aerobiology of corylus and castanea in trieste fig. 3. airborne pollen counts (weekly sums of the daily pollen concentrations) and mean flowering phenophases of corylus in trieste during 1991–2004. the horizontal black lines show the flowering period. for the years from 1992 to 1995, two horizontal black lines are presented, the upper line showing the flowering phenophases for ts1, the lower one showing the flowering phenophases for ts2. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:45 color profile: disabled composite 150 lpi at 45 degrees flowering-pollen relationships hazel airborne pollen was usually recorded in coincidence with or some days after the start of the flowering season of corylus avellana, and several days after its end. on average, the start and the end of the main pollen season of corylus occurred 18 days after the beginning of the flowering season and one month after its end, respectively (tab. 2). the weekly airborne pollen peaks rarely corresponded to the full flowering of hazel plants; on the contrary they were usually recorded one or even several weeks after (fig. 3). taking into consideration the whole period (1991–2004), pollen grains of corylus were detected in the air of trieste from january to mid may, and refloated grains were detected also later (fig. 4). in the mean weekly pollen curve the descendant phase appears longer than the ascendant one. on average, the mean flowering season of corylus lasted nearly three months, from january to mid march, with the maximum falling in february. on average, the highest concentration of airborne pollen was found when the flowering of corylus began to diminish. the weekly pollen sums and the phenological data collected in the two survey stations (ts1 and ts2) were averaged out over the period from 1992 to 1995 (fig. 5). the flowering started and ended earlier in ts1 than in ts2: the full blooming usually occurred in early february in ts1, and in late february, with a delay of two weeks, in ts2. the mean pattern of the weekly pollen concentrations was bimodal, the first peak corresponding to the full blooming in ts1, and the second one occurring a month later, and not corresponding to the full flowering either in ts1 or in ts2. 206 acta bot. croat. 69 (2), 2010 rizzi longo l., pizzulin sauli m. fig. 4. mean variation of pollen counts (weekly sums of the daily pollen concentrations) and flowering phenophases of corylus in trieste (1991–2004). the horizontal black line shows the mean flowering period. fig. 5. mean pattern of pollen counts and flowering phenophases of corylus at stations ts1 and ts2, in the period 1992–1995. the horizontal black lines show the mean flowering periods. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:46 color profile: disabled composite 150 lpi at 45 degrees castanea flowering season the timing of flowering phenophases of castanea varied over the years (tab. 4). the earliest start of flowering was recorded in 1993 (15th may), the latest one in 1991 (2nd june), with a difference of 18 days. in most cases, castanea began flowering in late may and finished flowering between late june and early july. the earliest end of the flowering was observed in 2000 (17th june), the latest one in 1991 (14th july), with a difference of 27 days. the duration of the flowering season varied between 28 and 42 days. the flowering of castanea usually lasted about six weeks, with a flowering course rather similar over the years (fig. 6). long ascendant and descendant flowering phases were observed in some years; in 1999, the full flowering of castanea became suddenly exhausted. pollen season over the years some differences emerged in the pollen season of castanea. during the 14-year period, the start of the main pollen season varied between 3rd june – in 2000 – and 25th june – in 1991 (tab. 4). the longest pollen season was recorded in 1994, the shortest in 1995. the annual sums of the daily pollen concentrations were rather variable, with the lowest total in 2002 (261), and the highest one in 2004 (1452). the highest daily pollen values were usually recorded in late june or at the beginning of july, with the earliest peak occurring on 12th june 2003, and the latest on 21st july 1991. the mean daily maximum was equal to 62 p/m3, with the highest value of 180 grains in 2004, and the lowest value of 23 acta bot. croat. 69 (2), 2010 207 phenology and aerobiology of corylus and castanea in trieste tab. 4. castanea flowering and pollen seasons in trieste (1991–2004). flowering season column data refer to mean data; main pollen season column data refer to 90% method. the start of the pollen season was chosen as the day when the cumulated daily counts reached 5% of the annual sum; the end as the day when the cumulated daily counts reached 95%. years flowering season pollen season annual total daily peak (p/m3) peak datestart end length start end length 1991 2/6 14/7 42 25/6 8/8 44 577 69 21/7 1992 16/5 27/6 42 7/6 31/7 54 417 36 27/6 1993 15/5 26/6 42 11/6 1/8 51 580 33 17/6 1994 27/5 2/7 36 9/6 13/8 65 392 26 3/7 1995 27/5 8/7 42 21/6 22/7 31 598 49 30/6 1996 29/5 3/7 35 12/6 26/7 44 638 44 1/7 1997 25/5 29/6 35 13/6 23/7 40 571 56 21/6 1998 24/5 28/6 35 12/6 20/7 38 1081 110 11/7 1999 23/5 20/6 28 10/6 22/7 42 511 36 22/6 2000 20/5 17/6 28 3/6 12/7 39 940 72 5/7 2001 20/5 1/7 42 10/6 18/7 38 619 47 16/6 2002 26/5 23/6 28 8/6 20/7 42 261 23 22/6 2003 17/5 21/6 35 8/6 18/7 40 983 93 12/6 2004 22/5 3/7 42 20/6 23/7 33 1452 180 9/7 mean 23/5 29/6 37 12/6 25/7 43 687 62 29/6 u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:46 color profile: disabled composite 150 lpi at 45 degrees 208 acta bot. croat. 69 (2), 2010 rizzi longo l., pizzulin sauli m. fig. 6. airborne pollen counts (weekly sums of the daily pollen concentrations) and mean flowering phenophases of castanea in trieste in the period 1991–2004. the horizontal black lines indicate the flowering periods. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:50 color profile: disabled composite 150 lpi at 45 degrees grains in 2002. the course of the weekly pollen concentrations (fig. 6) showed some differences over the years. in some years only one peak was found; in some cases we are dealing with a clearly recognizable peak (1998, 2001, 2003, 2004), whereas for 1995 and 1999 the peak is hardly identifiable. for the remaining years, at least two peaks were observed, though different with regard to the pollen amount; the lower peak was generally recorded two or more weeks after the main peak, less frequently before it. flowering-pollen relationships the mean start date of the pollen season of castanea occurred 20 days after the mean start date of the flowering season, and the mean end date of the pollen season fell 26 days after the mean end date of the flowering season (tab. 4). the peaks of the weekly airborne pollen concentrations never corresponded to the full flowering of the observed trees, but usually occurred 2 or 3 weeks after (fig. 6). taking into account the whole studied period (1991–2004), the mean weekly pollen curve displays a symmetrical pattern, in which the ascendant and the descendant phases are similar in length (fig. 7). the pollen season of castanea lasted from early june to late july, with the highest weekly peak occurring in the first week of july. on average, the flowering season of castanea lasted six weeks, from mid may to late june, with the maximum in mid june; on average, the highest concentrations of airborne pollen were detected three weeks later. castanea pollen was usually recorded in the air before the start of the flowering season of the examined trees, and for a long time after its end. discussion the flowering phenological behaviour of corylus avellana and castanea sativa in trieste varied greatly over the years (1991–2004). on average, the flowering season of corylus lasted from late january to early march, with the full bloom occurring in february. castanea flowered later, from late may to late june, with the full bloom occurring in mid june. interannual differences were observed both for the timing and for the length of the examined phenophases, as observed for betula by jato et al. (2002), and for alnus, corylus and betula by kasprzyk (2003). phenological differences in the timing of the flowering were recorded more often for corylus than for castanea. corylus flowered at the beginning of the year, when the weather is very unstable; castanea flowered in late spring – acta bot. croat. 69 (2), 2010 209 phenology and aerobiology of corylus and castanea in trieste fig. 7. mean variation of pollen counts (weekly sums of the daily pollen concentrations) and flowering phenophases of castanea in trieste (1991–2004). the horizontal black line shows the mean flowering period. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:50 color profile: disabled composite 150 lpi at 45 degrees early summer, when the weather is less changeable from day to day, as well as from year to year. meteorological parameters, particularly temperature, are known as the main variable that influences the timing of the flowering (rodríguez-rajo et al. 2006, emberlin et al. 2007). in trieste and especially in winter, daily temperatures are very changeable, because of the peculiar climate of this area, strongly influenced by two dominant winds, the »bora« (ene) and the »sirocco« (se).the »bora« causes sudden drops in temperature, whereas the »sirocco« brings about significant increases in temperature (stravisi 2006). given the remarkable effect of chilling and post chilling temperatures on the flowering of corylus (frenguelli et al. 1992, 1997), the flowering behaviour of this plant was very variable over the years, in accordance with the course of the temperature during late autumn and early winter (stravisi 2006). between the two examined populations of corylus, differences in the flowering period were also observed: a constant delay was found in the full bloom of the plants growing in the colder and more humid site ts2. as already observed by chuine et al. (2000), who found significant differences in the phenological response to temperature among corylus populations, the phenological behaviour of this temperate woody taxon seems to adapt locally to microclimate. in ts2, a fifteen-day delay in the start of flowering was recorded, on average. the earliest floral phenophase was recorded in ts1 in 1994, when corylus showed both start and full bloom within the first week of january, since the chilling requirement necessary to break dormancy had already been achieved and the following heat accumulation was quickly reached. in each population, differences among plants were also found; these differences depend on the age and the size of the plants, and on microclimatic and edaphic factors, since the flowering patterns are different in each individual due to different micro-environmental features. as a matter of fact, young plants started blooming earlier than old plants, in the same way that plants growing in protected and sunny sites bloomed earlier than plants growing in more windy or shady sites. taking into consideration the occurrence of airborne pollen in the studied years, the main pollen season of corylus lasted on average from early february to early april, with the daily peak usually occurring in late february or early march. the main pollen shedding of castanea usually occurred between early june and late july, and the daily peak was recorded around late june or at the beginning of july. the course of the weekly pollen concentrations varied greatly over the years, especially for corylus, since the timing and the behaviour of the pollen release were strongly influenced by the weather. on average, the pollen curves showed a symmetrical pattern in the case of castanea, with the ascendant and descendant phases similar in length, and a partly symmetrical pattern for corylus, with a descendant phase longer than the ascendant one. refloated grains were frequently found after the end of the pollen season. the flowering patterns of corylus and castanea were compared with the respective curves of the airborne pollen. the pollen seasons of corylus and castanea grains were usually found, sometimes even in great amounts, outside the flowering period and the pattern outlined by the pollen curves is hardly ever in accordance with that of the flowering phenophases. as already noticed by latorre (1999), airborne pollen is not suitable for indicating the exact length of the flowering season, above all when we consider the final stage of the flowering, when a clear discrepancy emerges between the flowering and the end of the presence of airborne pollen. a lack of correlation between the maximum flower210 acta bot. croat. 69 (2), 2010 rizzi longo l., pizzulin sauli m. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:50 color profile: disabled composite 150 lpi at 45 degrees ing and the maximum pollen concentration was observed, the highest airborne pollen counts occurring two or more weeks after the maximum flowering. because of the geographical complexity of the area around trieste, the flowering of corylus and castanea do not occur at the same time everywhere, and grains coming from plants growing at different altitudes and at various distances from the pollen trap were also found. these pollen grains may come from extra-local sources when specific weather conditions allow a medium/long range transport from slovenia or from the julian pre-alps (rizzi longo et al. 2005). the discontinuous occurrence of pollen observed every year seems, in its turn, to be related to the late flowering of both local and extra-local trees. the phenological data collected in the survey stations do not permit a correct comparison of flowering behaviour with airborne pollen occurrence in this area. the peculiar geographical conformation of the area around trieste, together with the direction of the two dominant winds (ene, se) in relation to the position of the trap and to the location of the survey stations, might represent the main cause of this lack of correlation. to remedy this, other pollen sources could be considered, such as plants growing on the north-eastern outskirts of the town or on the karst plateau and in farther sites. according to jato et al. 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59, 17–42. rizzi longo, l., pizzulin sauli, m., martini, f., larese filon, f., 2003: the allergenic flora of trieste (ne italy). annales, annals for istrian and mediterranean studies, series historia naturalis 13, 265–280. rizzi longo, l., pizzulin sauli, m., ganis, p., 2005: aerobiology of fagaceae pollen in trieste (ne italy). aerobiologia 21, 217–231. rizzi longo, l., pizzulin sauli, m., stravisi, f., ganis, p., 2007: airborne pollen calendar for trieste (italy), 1990–2004. grana 46, 98–109. rodríguez-rajo, f. j., fernández-gonzález, m. d., vega-maray, a. m., suárez, f. j., valencia-barrera, r. m., jato, v., 2006: biometeorological characterization of the winter in north-west spain based on alnus pollen flowering. grana 45, 288–296. acta bot. croat. 69 (2), 2010 213 phenology and aerobiology of corylus and castanea in trieste u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:50 color profile: disabled composite 150 lpi at 45 degrees sparks, t. h., jeffree, e. p., jeffree, c. e., 2000: an examination of the relationship between flowering times and temperature at the national scale using long-term phenological records from the uk. international journal of biometeorology 44, 82–87. stravisi, f., 1977: il regime dei venti a trieste (1951–1975). bollettino della società adriatica di scienze 61, 87–104. stravisi, f., 2006: la meteorologia a trieste. in: cortemiglia, g.c. (ed.), la variabilità del clima locale relazionata ai fenomeni di cambiamento climatico locale, 245–288. patron, bologna. tedeschini, e., rodríguez-rajo, f. j., caramiello, r., jato, v., frenguelli, g., 2006: the influence of climate changes in platanus spp. pollination in spain and italy. grana 45, 222–229. zanotti, a. l., puppi, g., mandrioli, p., sirotti, m., caramiello, r., zerboni, r., manfredi, m., 1998: monitoraggio fenologico su graminacee, castagno e nocciolo. notiziario aerobiologico, bollettino di informazione dell’associazione italiana di aerobiologia 4, 1–75. 214 acta bot. croat. 69 (2), 2010 rizzi longo l., pizzulin sauli m. u:\acta botanica\acta-botan 2-10\301 rizzi longo.vp 11. listopad 2010 12:18:50 color profile: disabled composite 150 lpi at 45 degrees social news professor emeritus damir viličić: an appreciation on the occasion of his seventieth birthday damir viličić is professor emeritus at department of biology, faculty of science, university of zagreb. he was born on may 27, 1948, in zagreb, where he grew up and has finished his primary education. on faculty of science, university of zagreb he obtained his bsc. degree in biology in 1973, msc in 1977 and phd in 1983. professor viličić has started his scientific career in the institute of oceanography and fisheries in dubrovnik in 1975 where he has achieved his position of senior scientist in 1991. in autumn 1996 he moves back to zagreb and continues his career as full professor on department of biology, faculty of science, university of zagreb. on 2014 he retires and in 2015 is promoted to professor emeritus. his main research focus is on ecology and taxonomy of marine phytoplankton. he has published 113 peer reviewed scientific papers (43 in top rated scientific journals), two university text books and one book. his work has been cited 944 times (wos), 1059 times (scopus), 1385 times in google scholar with h-index 20 and 22 respectively. during his fruitful career he has been trained at different laboratories – laboratorio contaminazione marina – c.n.e.n., fiascherino (la spezia), italy; dunstaffnage marine research laboratory, oben, institute of aquaculture university of stirling stirling, scotland, the institute of marine environmental research and the marine biological association laboratory, plymouth, uk; universite des sciences et tecniques du languedoc, montpellier, france; stazione zoologica anthon dohrn napoli, italy. he has been actively involved in teaching of the courses protists, pelagial microbiology, microbiology of the ecosystem, biological oceanography and marine biology. he had supervised 25 master and five phd students. professor viličić was principal investigator of projects of the ministry of science of the republic of croatia and was collaborator on international projects and research in the adriatic sea (rv knorr, dolcevita project). his scientific contributions were recognized by the award of the croatian academy of science and art for scientific achievements in croatia in 2003, and since 2006 he is associate member of the croatian academy of science and art. viličić was secretary (1995-2011) and president (2012-2015) of the council for the scientific research of the adriatic sea of the croatian academy of science and art. from 1998 to 2014 he was editor-in-chief of acta botanica croatica. on this important birthday, we wish professor viličić many happy returns, to stay positive and active, to keep the good health and vital strength that is such an inspiration to us all. his career is a proof that all problems can be overcome, that if the door closes, to look for a window and most important never to give up, never stop observing and critically question all that you observe. we look forward for all the good times and discussions with professor that are in front of us. assoc. prof. zrinka ljubešić university of zagreb faculty of science department of biology professor viličić, winter 2008 expedition in velebit channel, on the break during the night sampling. acta bot. croat. 78 (1), 2019 s7 s8 acta bot. croat. 78 (1), 2019 list of chosen publications books 1. viličić, d., 2002: fitoplankton jadranskoga mora. biologija i taksonomija. školska knjiga, zagreb. 2. viličić, d., 2003: fitoplankton u ekološkom sustavu mora. školska knjiga, zagreb. 3. viličić, d., 2014: ecology and composition of phytoplankton in the adriatic sea. koeltz scientific books, koenigstein, germany. 15 most cited publications 1. mihanović, h., vilibić, i., carniel, s., tudor, m., russo, a., bergamasco, a., bubić, n., ljubešić, z., viličić, d., boldrin, a., malačić, v., celio, m., comici, c., raicich, f., 2013: exceptional dense water formation in the adriatic shelf in the winter of 2012. ocean science 9, 561–572 2. viličić, d., đakovac, t., burić, z., bosak, s., 2009: composition and annual cycle of phytoplankton assemblages in the northeastern adriatic sea. botanica marina 52, 291–305. 3. viličić, d., terzić, s., ahel, m., burić, z., jasprica, n., carić, m., caput-mihalić k., olujić, g., 2008: phytoplankton abundance and pigment biomarkers in the oligotrophic, eastern adriatic estuary. environmental monitoring and assessment 142, 199–218. 4. burić, z., cetinić, i., viličić, d., caput mihalić, k., carić, m., olujić, g., 2007: spatial and temporal distribution of phytoplankton in a highly stratified estuary (zrmanja, adriatic sea). marine ecology 28 (suppl. 1), 169–177. 5. svensen, c., viličić, d., wassmann, p., arashkevich, e., ratkova, t., 2007: plankton distribution and vertical flux of biogenic matter during high summer stratification in the krka estuary (eastern adriatic). estuarine coastal and shelf science 71, 381–390. 6. cetinić, i., viličić, d., burić., z., olujić, g., 2006: phytoplankton seasonality in a highly stratified karstic estuary (krka, adriatic sea). hydrobiologia 555, 31–40. 7. ciglenečki, i., carić, m., kršinić, f.,viličić, d., ćosović, b., 2005: the extinction by sulfide-turnover and recovery of a naturally eutrophic, meromictic seawater lake. journal of marine systems 56, 29–44. 8. batistić, m., kršinić, f., jasprica, n., carić, m., viličić, d., lučić, d., 2004: gelatinous invertebrate zooplankton of the south adriatic: species composition and vertical distribution. journal of plankton research 26, 459–474. 9. viličić, d., marasović, i., mioković, d., 2002: check list of phytoplankton in the eastern adriatic sea. acta botanica croatica 61, 57–91. 10. kršinić, f., carić, m., viličić, d., ciglenečki, i., 2000: the calanoid copepod acartia italica steuer, phenomenon in the small saline lake rogoznica (eastern adriatic coast). journal of plankton research 22, 1441–1464. 11. ćosović, b., ciglenečki, i., viličić, d., ahel, m., 2000: distribution and seasonal variability of organic matter in a small eutrophicated salt lake. estuarine coastal and shelf science 51, 705–715. 12. viličić, d., leder, n., gržetić, z., jasprica, n., 1995: microphytoplankton in the strait of otranto (eastern mediterranean). marine biology 123, 619–630. 13. viličić, d., 1989: phytoplankton population density and volume as indicators of eutrophication in the eastern part of the adriatic sea. hydrobiologia 174, 117–132. 14. viličić, d., legović, t., žutić, v., 1989: vertical distribution of phytoplankton in a stratified estuary. aquatic sciences 51, 31–46. 15. viličić, d., 1985: an examination of cell volume in dominant phytoplankton species of the middle and southern adriatic waters. internationale revue der gesamten hydrobiologie 70, 829–843. 116 acta bot. croat. 78 (2), 2019 acta bot. croat. 78 (2), 116–124, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0018 issn 0365-0588 eissn 1847-8476 effects of exogenous nitric oxide on cadmium toxicity in black poplar (populus nigra): physiological approaches yakup cikili1, semsettin kulac2, halil samet3, ertugrul filiz4* 1 department of soil science and plant nutrition, faculty of agriculture, çanakkale onsekiz mart university, çanakkale, turkey 2 department of forest engineering, faculty of forestry, duzce university, duzce, turkey 3 vocational school of food and agriculture, department of crop and animal production, kocaeli university, kocaeli, turkey 4 cilimli vocational school, department of crop and animal production, duzce university, duzce, turkey abstract – cadmium (cd) is a highly toxic metallic contaminant that negatively affects plant metabolism and causes reductions in productivity. nitric oxide (no) is a signaling molecule that regulates various physiological processes and is involved in response to biotic/abiotic stresses. this work investigated the effects of exogenous sodium nitroprusside (snp), a nitric oxide (no) donor, application on cd toxicity in black poplar (populus nigra). black poplars were exposed to individual/combined cdcl2 and snp treatments for 21 days by complete randomized design with three replications. cd concentrations increased in leaves, bark, and roots at cd treatments, whereas cd + snp applications had alleviative effects on cd exposures, except for leaves. photosynthetic pigments (chlorophyll a, b, a + b and carotenoids) reduced with cd treatments in leaves, while they increased in cd + snp applications. similarly, plant biomass was reduced with cd treatments, but cd + snp application prevented these reductions. snp also alleviated malondialdehyde (mda) and hydrogen peroxide (h2o2) accumulation in leaves under cd treatments. catalase (cat, ec 1.11.1.6) and ascorbate peroxidase (apx, ec 1.11.1.11) activities were also affected by cd and cd + snp applications. cd exposure also decreased zn2+, fe2+ and mn2+ levels in leaves, bark and roots, while it increased cu2+ level in leaves and roots. this study concludes that cd toxicity caused a reduction of plant growth and mineral nutrition parameters. however, snp indicates great potentials for improving the growth under cd toxicity in p. nigra. keywords: antioxidant enzyme, growth inhibition, heavy metal stress, mineral nutrition, sodium nitroprusside * corresponding authors e-mails: yakupcikili@gmail.com, ertugrulfiliz@gmail.com introduction cadmium (cd) is a non-essential trace metal and it is also one of the most toxic elements adversely affecting plant growth and development. it is accumulated in soil as a result of anthropogenic activities such as traffic emissions, mining, and agricultural practices like waste water irrigation, excessive fertilizer use and pesticide application to sewage sludge (white and brown 2010, tran and popova 2013). cd is toxic even at very low concentrations; it is taken up by roots with similar charged metal (fe, zn, mn) ion transporters and loaded into xylem for transport to leaves (schützendübel and polle 2002, verbruggen et al. 2009). three major mechanisms are involved in plant response to cd toxicity, such as the production of free radicals and reactive oxygen species (ros), thiol groups contained in cysteine residues of proteins, and the competition among cd and some mineral nutrients containing similar chemical properties (schützendübel and polle 2002, paradiso et al. 2008). its toxicity causes damage to root structure, limits nutrient absorption and translocation, decreases chlorophyll content and plant biomass, leads to leaf chlorosis and withering, and negatively affects stomatal opening, conductance and root water conductivity (vandecasteele et al. 2003, benavides et al. 2005, wang et al. 2008). on the other hand, to reduce the toxic effects of cd, plants employ two major mechanisms: (i) limiting cd uptake by binding and sequestrating it to biomolecules, and (ii) increasing antioxidant enzyme activities such as superoxide dismutase (sod; ec 1.15.1.1), catalase (cat, ec 1.11.1.6), ascorbate peroxidase (apx, ec 1.11.1.11), effects of nitric oxide on cadmium toxicity in populus nigra acta bot. croat. 78 (2), 2019 117 glutathione peroxidase (gpx, ec 1.11.1.9), monodehydroascorbate reductase (mdhar, ec 1.6.5.4) and glutathione reductase (gr, ec 1.6.4.2) (dixit et al. 2001, gill and tuteja 2010). in plants, thiol-containing chelating ligands such as glutathione (gsh) and phytochelatins (pcs) are used for transport of cd-complexes into the vacuole or in the apoplast (wolf et al. 1996, cobbett 2000). other low-molecularweight cysteine (cys) rich peptides, such as metallothioneins (mts), also play important roles in the binding of metal ions like cd and thus contribute to cd detoxification from the cytosolic environment (cobbett 2000). nitric oxide (no) is a hydrophobic gaseous free radical and plays important roles in cell signaling, various physiological processes and response to biotic/abiotic factors (arasimowicz and floryszak-wieczorek 2007). plants have four types of enzymes involved in no production (gill et al. 2013), such as cytosolic nitrate reductase (nr, ec 1.6.6.1), plasma-membrane (pm)-nitrite: no reductase (ni:nor), nitric oxide synthase (nos, ec 1.14.13.39) and xanthine dehydrogenase (xdh, ec 1.1.1.204). no regulates antioxidant enzyme machinery at gene expression and activity levels, resulting in an increase or decrease of the cellular redox status of cells under stress conditions (groß et al. 2013). no also interacts with other signaling molecules and plant hormones, including jasmonic acid (ja), salicylic acid (sa), abscisic acid (aba), the ethylene signaling pathway and hydrogen peroxide (h2o2; neill 2007, gill et al. 2013). the genus populus includes five sections with about 30– 40 species distributed all around the worldwide, but mainly in the northern hemisphere (polle and douglas 2010). previous studies projected that different clones of the same poplar species may show different heavy metal tolerance due to genetic variations (castiglione et al. 2009). for example, he et al. (2013) reported various cd tolerance levels and physiological changes among six poplar species. gaudet et al. (2011) reported intraspecific variations of physiological and molecular response under cd-stressed p. nigra trees. in another paper, lomaglio et al. (2015) reported physiological variations at photosystem ii (psii) quantum yield, h2o2 generation and hormone levels from short-term cd-stressed p. nigra trees. accordingly, in this work an endeavour was made to understand the effects of exogenous sodium nitroprusside (snp) that provides nitric oxide (no) at physiological activities and mineral nutrition of black poplar (p. nigra) under different cd treatment levels. materials and methods plant materials and treatments cuttings (~30 cm length × 1 cm diameter) from black poplar (p. nigra genotype gazi) were obtained from the poplar and fast growing forest trees research institute, izmit, turkey. at least 18 cuttings with sprouts were planted and rooted in perlite medium. after 10 weeks, these cuttings were transplanted at a rate of one plant per pot filled with 3 liters of perlite. to enhance acclimation in root zone, modified hoagland solution was used for watering the plants. for this purpose, the poplar cuttings were grown two weeks by using one-quarter strength (1/4 rate dilution of full-strength modified hoagland solution), one week by one-half strength (dilution of 1/2 rate of full-strength modified hoagland solution) and one week full-strength modified hoagland solutions each day. the full-strength modified hoagland solution consisted of 5 mm ca(no)2×4h2o, 5 mm kno3, 2 mm mgso4×7h2o, 1 mm kh2po4, 45.5 µm h3bo3, 44.7 µm feso4×7h2o, 30.0 µm nacl, 9.1 µm mnso4×h2o, 0.77 µm znso4×7h2o, 0.32 µm cuso4×5h2o, 0.10 µm (nh4)2mo7o244h2o and 54.8 µm na2edta×2h2o adjusted to ph 6.0. for cd treatment, 18 acclimatized poplar cuttings with similar heights were selected and divided into two groups, including with and without sodium nitroprusside (snp) (sigma aldrich st. lois, mo, usa) treatments. poplar cuttings were exposed to six treatments during 21 days, including (i) control (full-strength modified hoagland solution), (ii) addition of 100 µm cdcl2, (iii) addition of 500 µm cdcl2, (iv) addition of 100 µm snp, (v) addition of 100 µm cdcl2 and 100 µm snp, and (vi) addition of 500 µm cdcl2 and 100 µm snp dissolved in full-strength modified hoagland solution. the experiment was designed as a complete randomized factorial design with three replications. three weeks after treatments, leaves in cuttings were carefully harvested and washed in tap water, after which samples were rinsed three times with de-ionized water. similarly, root and bark in cuttings were removed, cleaned and washed. all leaf, bark and root tissues were oven-dried at 70 °c for at least three days, and dry weight (dw) was immediately determined. photosynthetic pigment analysis fresh leaf samples were taken from the youngest fully expanded leaves before harvest. then, they (500 mg) were homogenized using a homogenizer (heidolph, diax 900) with added 10 ml of acetone (90% v v–1). the absorbance of extracts was measured at 663, 645 and 470 nm wavelengths using a spectrophotometer (shimadzu uv-1201; tokyo). chlorophyll a, b, a + b, and carotenoid amounts and the chlorophyll a/b ratio were calculated according to the lichtenthaler (1987) formula. cd and nutrient ion analysis all leaf, bark and root tissues were digested using the dry-ashing method in a muffle furnace at 500 °c for 6 hours (miller 1998). then, the concentrations of cd and metal ions, iron (fe), zinc (zn), manganese (mn), and copper (cu) in leaf, bark and root tissues were measured by using inductively coupled plasma optical emission spectrometry (icp-oes, perkin elmer optima 2100 dv, waltham, ma). h2o2 and lipid peroxidation estimation h2o2 levels in fresh leaf samples were calorimetrically estimated as described by mukherjee and choudhuri (1983). firstly, fresh leaf samples were extracted with cold acetone. an aliquot (3 ml) of extracted solution was mixed with 1 ml cikili y., kulac s., samet h., filiz e. 118 acta bot. croat. 78 (2), 2019 statistical analyses statistical analysis was done with anova using the minitab package program (minitab corp., state college, pa). multiple means comparisons between cd treatments were analyzed using duncan’s multiple range test at significance level (α = 0.05). the levels of significance are represented as * at p < 0.05, ** at p < 0.01, *** at p < 0.001, and ns – non-significant. results biomass and cd accumulations in this study, cd exposure mostly negatively affected leaf and bark dry weights (dw) in black poplar trees, while 100 μm cd + snp and 500 μm cd + snp applications caused an increase in leaf biomass compared to cd treatments as well as 500 μm cd + snp application in bark biomass (tab. 1). snp treatment increased dws in plant leaves by 40.5% at 100 µm cd and by 36.4% at 500 µm cd following 100 μm cd + snp and 500 μm cd + snp treatments, respectively. the reduction in root dws under cd exposure was not found statistically significant, except at 500 μm cd + snp treatment. in all plant tissues, cd levels were significantly increased depending on applied concentrations (tab. 1). their increases in leaves, bark and roots respectively were about 35-, 16 and 215-fold at 100 µm cd, and 39-, 15and 242-fold at 500 µm cd treatments in comparison to controls. however, the levels in bark and roots decreased by 29.0% and 5.3% respectively at 100 µm cd + snp compared to 100 µm cd treatment, and by 51.7% and 21.6% at 500 µm cd + snp treatments compared to 500 µm cd treatment. on the other hand, leaf cd content increased by 77.8% at 100 µm cd + snp treatment in comparison to 100 µm cd treatment, and by 86.3% at 500 µm cd + snp treatment in comparison to 500 µm cd treatment. although snp application alleviated cd content in p. nigra barks and roots, it was not effective in the plant leaves. 0.1% titanium dioxide in 20% (v:v) h2so4. subsequently, the mixture was centrifuged at 6000 x g for 15 min. the intensity of yellow supernatant was measured at 415 nm. the concentration of h2o2 was calculated from a standard curve plotted with the range of 100–1000 nmol h2o2 and expressed as µmol g–1, fresh weight. the amount of lipid peroxidation in fresh leaves was identified by measuring malondialdehyde (mda), a major thiobarbituric acid reactive species (tbars) and product of lipid peroxidation (hodges et al. 1999). mda concentration was calculated by means of an extinction coefficient of (ε = 155 mm–1 cm–1). extraction and assaying enzyme activities all enzymatic measurements were performed at 0–4 °c. fresh leaf samples were homogenized with a homogenizer (heidolph, diax 900) in 5 ml of 100 mm potassium phosphate buffer (ph 7.5), including 1 mm edta-na2 and 0.5 mm ascorbate. then, homogenate was centrifuged at 10,000 × g for 5 min. supernatant was used as crude enzyme extract to analyze the activities of catalase (cat, ec 1.11.1.6) and ascorbate peroxidase (apx, ec 1.11.1.11). all colorimetric measurements with enzyme activities were made at 25 °c using a spectrophotometer (shimadzu uv/vis1201). enzyme activities were expressed as units per gram fresh weight of tissue. apx activity was determined by measuring the decrease in absorbance at 290 nm for 1 min in 2 ml reaction mixture, including 50 mm potassium phosphate buffer (ph 7.0), 1 mm edta–na2, 0.5 mm ascorbic acid, 0.1 mm h2o2 and 50 ml crude enzyme extract (nakano and asada 1981). apx activity was calculated using extinction coefficient of (2.8 mm–1 cm–1). cat activity was determined by measuring the decrease in absorbance at 240 nm for 1 min following decomposition of h2o2 (cakmak et al. 1993). the reaction mixture (3 ml) consisted of 50 mm phosphate buffer (ph 7.0), 15 mm h2o2 and 50 m l crude enzyme extract. cat activity was calculated using extinction coefficient (40 mm–1 cm–1) for h2o2. tab. 1. changes in biomass accumulation and total cd content in leaf, bark and roots of black poplar plants exposed to individual cd, sodium nitroprusside (snp) and combined cd + snp treatments for 21 days. values are the mean of three replicates (means ± se, n= 3). different letters in the same column denote significantly different means according to duncan’s multiple range test (α = 0.05). for f-test the levels of significance are represented as * at p < 0.05, ** at p < 0.01, *** at p < 0.001 dw – dry weight. snp (µm) cd (µm) dry weight (g plant−1) cd content (µg g−1 dw) leaf bark root leaf bark root 0 0 3.37±0.16 ab 0.98±0.03 a 0.91±0.05 a 1.2±0.1 d 2.9±0.2 d 3.35±0.20 c 100 2.58±0.15 cd 0.90±0.07 ab 0.69±0.06 ab 42.7±0.6 c 47.3±2.3 a 721.7±18.9 ab 500 2.14±0.07 d 0.70±0.03 b 0.70±0.02 ab 46.8±0.9 c 43.5±3.2 a 811.0±59.5 a 100 0 3.40±0.11 ab 1.03±0.07 a 0.88±0.12 a 2.7±0.2 d 3.0±0.1 d 5.27±0.42 c 100 3.62±0.28 a 1.11±0.11 a 0.75±0.07 ab 75.9±3.0 b 33.6±4.3 b 683.5±39.9 b 500 2.92±0.17 bc 0.99±0.07 a 0.57±0.05 b 87.2±2.4 a 21.0±2.3 c 635.9±47.7 b f-test significance 10.8*** 4.1* 3.2* 477.7*** 58.1*** 105.9*** effects of nitric oxide on cadmium toxicity in populus nigra acta bot. croat. 78 (2), 2019 119 posures respectively. however, its levels also decreased by 13.1% and 22.8% at 100 and 500 µm cd + snp treatments in comparison to 100 and 500 µm cd, respectively. so, it appeared that snp treatment attenuates the deleterious effect of cd exposure in poplar plants. in parallel to mda, h2o2 levels also increased under cd exposure in black poplars (fig. 1b). however, h2o2 content decreased by 14% with 500 µm cd + snp exposure in comparison to 500 µm cd treatment. antioxidative enzyme activities in this study, cat activity notably increased at 500 µm cd exposure; however, the changes in apx activity with all levels of cd exposure were not found significant statistically (fig. 2). besides, cat activity in black poplar was significantly decreased by 100 and 500 µm cd + snp applications compared to 100 and 500 µm cd treatments (fig. 2a). however, the apx activity was notably enhanced in leaves, 2.34and 1.91-fold at 100 and 500 µm cd + snp treatments in comparison to 100 and 500 µm cd applications, respectively (fig. 2b). mineral nutrition the concentrations of mineral nutrients fe, zn, mn and cu in poplar leaves, bark, and roots were influenced by cd photosynthetic pigments chlorosis is a common symptom of cd toxicity. in this work, content of photosynthetic pigments was notably decreased upon cd exposure (tab. 2). the reductions in chlorophyll a, b, a + b, and carotenoid were 53.4%, 61.8%, 55.1% and 53.9% in 100 µm cd-treated plants, and 65.7%, 72.9%, 67.2% and 63.3% in 500 µm cd-treated plants, respectively. however, with 500 µm cd + snp application, chlorophyll a, b, a + b and carotenoid contents significantly increased as 2.50-, 2.87-, 2.56and 2.35-fold at 500 µm cd exposure, respectively. also, chlorophyll a, b, a + b and carotenoid contents were decreased with 100 µm snp exposure in comparison to the controls as a result of being toxic to plants depending on the level in growth media. increasing the level of cd toxicity remarkably enhanced the chlorophyll a/b ratio (tab. 2). moreover, chlorophyll a/b was decreased with 500 µm cd + snp application in comparison to 500 µm cd treatments. in this study, snp exposures were noted to have ameliorative effects on the photosynthetic pigments of cdexposed p. nigra. mda and h2o2 content in this work, mda contents were increased at all levels of cd exposures (fig. 1a). lipid peroxidation significantly elevated by 17.3% and 34.3% at 100 and 500 µm cd extab. 2. changes in contents of photosynthetic pigments in leaves of black poplar plants exposed to individual cd, sodium nitroprusside (snp) and combined cd + snp treatments for 21 days. values are the mean of three replicates (means ± se, n= 3). different letters in the same column denote significantly different means according to duncan’s multiple range test (α = 0.05). for f-test the levels of significance are represented as * at p < 0.05, ** at p < 0.01, *** at p < 0.001 fw – fresh weight snp (µm) cd (µm) chlorophyll a chlorophyll b chlorophyll a+b carotenoid chlorophyll a/b ratio(mg g−1 fw) 0 0 0.959±0.024 a 0.251±0.009 a 1.211±0.033 a 0.603±0.016 a 3.82±0.08 d 100 0.447±0.023 d 0.096±0.003 d 0.544±0.024 d 0.278±0.007 d 4.64±0.27 ab 500 0.329±0.003 e 0.068±0.001 e 0.397±0.002 e 0.221±0.004 e 4.79±0.07 a 100 0 0.886±0.003 b 0.211±0.004 b 1.097±0.007 b 0.561±0.008 b 4.19±0.07 cd 100 0.792±0.012 c 0.175±0.001 c 0.968±0.011 c 0.488±0.002 c 4.52±0.09 abc 500 0.823±0.026 c 0.195±0.013 bc 1.018±0.040 c 0.519±0.002 c 4.25±0.15 bcd f-test significance 200.3*** 103.1*** 189.4*** 189.9*** 6.2** fig. 1. malondialdehyde (mda; a) and hydrogen peroxide (h2o2; b) contents in leaves of black poplar exposed to cd (0, 100, and 500 µm), sodium nitroprusside (snp) and combined cd + snp (100 µm +100 µm or 500 µm +100 µm) for 21 days. bars indicate means of three replicates ± se. different letters on the bars indicates significant differences according to duncan’s multiple range test (α = 0.05). cikili y., kulac s., samet h., filiz e. 120 acta bot. croat. 78 (2), 2019 exposure and snp treatments, except for cu concentrations in bark (tab. 3). cd treatments significantly reduced fe, zn and mn levels in leaves, bark and roots but increased cu levels in leaves and roots. however, fe concentrations in leaves, bark, and roots, mn concentrations in bark and roots were remarkably enhanced with cd + snp applications in comparison to cd treatments. cd exposure notably decreased zn concentrations in all plant parts, and also zn concentrations in leaves, bark, and roots were significantly reduced with cd + snp treatments in comparison to cd treatments. in comparison with control, cu concentrations in root were increased 100 and 500 µm cd treatments while the increments in leaf cu concentration were found significantly with only 500 µm cd treatments. otherwise, cu concentration in leaves decreased with 500 µm cd + snp treatment compared to 500 µm cd treatment, but cu concentration in fig. 2. catalase (cat; a) and ascorbate peroxidase (apx; b) activities in leaves of black poplar exposed to cd (0, 100, and 500 µm), sodium nitroprusside (snp) and combined cd + snp (100 µm + 100 µm or 500 µm + 100 µm) for 21 days. bars indicate means of three replicates ± se. different letters on the bars indicates significant differences according to duncan’s multiple range test (α = 0.05). tab. 3. changes in concentrations of fe, zn, mn and cu in leaf, bark and roots of black poplar plants exposed to individual cd, sodium nitroprusside (snp) and combined cd + snp treatments for 21 days. values are the mean of three replicates (means ± se, n= 3). different letters in the same column denote significantly different results according to duncan’s multiple range test (α = 0.05). for f-test the levels of significance are represented as * at p < 0.05, ** at p < 0.01, *** at p < 0.001, and ns – non-significant. snp (µm) cd (µm) concentrations (µg g–1) fe zn mn cu leaves 0 0 79.09±3.11 d 30.65±0.73 a 42.59±1.29 ab 8.08±0.84 c 100 56.79±1.57 e 21.08±0.18 c 36.41±1.41 c 7.02±1.29 c 500 49.32±0.24 f 27.12±0.32 b 37.60±0.86 bc 24.61±2.16 a 100 0 116.96±1.55 a 28.07±0.69 b 47.33±2.70 a 9.05±0.15 c 100 106.00±0.09 b 19.12±0.35 d 41.93±1.25 abc 4.00±0.22 c 500 99.88±0.48 c 18.85±0.90 d 40.96±2.91 bc 17.69±3.37 b f-test significance 324.7*** 73.8*** 4.2* 19.7*** bark 0 0 29.32±1.80 c 30.49±1.03 a 18.33±0.47 a 5.14±0.37 100 20.51±0.64 d 21.86±0.70 b 12.21±0.05 c 4.81±0.26 500 25.08±0.47 cd 19.20±0.05 c 12.13±0.48 c 5.53±0.30 100 0 53.39±0.30 a 21.98±0.22 b 14.38±0.34 b 5.24±0.11 100 54.22±0.77 a 15.24±0.32 d 15.34±0.49 b 5.51±0.47 500 44.31±4.16 b 14.02±0.40 d 9.64±0.17 d 4.63±0.24 f-test significance 59.6*** 111.9*** 65.3*** ns root 0 0 447.4±12.2 c 44.64±2.25 a 49.10±1.52 c 10.35±0.19 c 100 461.0±18.3 c 27.36±1.17 c 36.93±0.35 d 13.38±0.57 b 500 302.3±3.5 d 35.70±1.11 b 33.90±2.35 d 12.74±0.79 b 100 0 1011.4±7.1 a 28.88±0.84 c 64.21±0.96 a 8.57±0.11 d 100 942.3±27.8 a 17.65±0.19 d 60.23±5.32 ab 11.02±0.86 c 500 736.1±103.0 b 29.55±0.69 c 53.18±1.15 bc 15.09±0.18 a f-test significance 42.7*** 54.7*** 23.3*** 18.4*** effects of nitric oxide on cadmium toxicity in populus nigra acta bot. croat. 78 (2), 2019 121 roots increased while changes in cu concentrations in bark were not significant. discussion in the current study, it was aimed to reveal alleviated effects of snp exposure on cd toxicity in black poplar. for this purpose, the black poplar was exposed to only cd, only snp, and combined cd + snp treatments. in this context, biomass accumulation was adversely affected by cd exposure; in contrast, snp treatment with cd increased the biomass. in a similar study, total root dw in p. nigra was markedly lower at 200 μm cd exposure (he et al. 2013). dai et al. (2013) showed that cd treatments decreased leaf, bark and root dws at four cd levels such as 10, 30, 50 and 70 μm. in two p. nigra genotypes, cd treatment reduced leaf dw for genotype 58–861 whereas genotype poli was not affected (gaudet et al. 2011). no plays crucial roles in ros and hormone regulation (lamattina et al. 2003). it also regulates gene expression patterns in signal transduction, transport, defense, cell death and ros production/degradation (palmieri et al. 2008). besides, snp, as no donor, stimulates the electron transport through photosystem (ps) ii (ederli et al. 2009). leaf biomass increments under snp exposure may thus prove the crucial roles of no in response to cd toxicity. it is thought that increased biomass is closely correlated with the increase of chlorophyll content which promotes the photosynthesis. cd contamination is considered a major environmental problem due to chemical fertilizers, sewage wastewater irrigation, and rapid industrialization (sanità di toppi and gabbrielli 1999). in this study, cd exposure increased the cd amounts in all plant tissues while snp application generally decreased cadmium amounts. in populus × canescens plantlets, cd levels in roots, bark, wood, and leaves increased with different cd exposures were found as roots > wood > bark > leaves (dai et al. 2013). in another similar work, cd amounts in populus × canescens roots, wood, bark and leaves exposed to 50 μm cdso4 were increased for 1, 10 or 20 days (he et al. 2013). gaudet et al. (2011) reported 22.5 and 33.9 mg kg–1 cd levels at 50 µm treatment in leaves of the p. nigra poli and 58–861 genotypes respectively. those reported cd concentrations were not significantly different than the findings of the present study. in p. nigra and salix alba (willow), comparisons of cd content at 50 µm revealed that black poplar roots contained ~2.4 fold higher cd levels than those of willow, while by contrast, s. alba leaves had ~16 fold higher cd concentrations than those of poplar (zacchini et al. 2011). in six poplar species, mean cd concentrations at 200 µm cd exposure were found increased by 43-, 9-, 23-, and 25-fold in roots, wood, bark and leaves respectively (he et al. 2013). in this paper, cd concentrations were also found significantly increased in root, leaves and bark without snp applications, suggesting that these variations in different poplar species may be attributed to genomic background. many studies also showed that endogenous no plays a key role in regulation of cd toxicity (besson-bard et al. 2009, de michele et al. 2009, gill et al. 2013). in this study, cd + snp exposures significantly decreased cd accumulations in bark and roots compared to cd treated plants. so, this reduction in cd concentrations may be due to the positive signalling effects of no in cellular metabolism. cd is considered one of the heavy metals most toxic to plant metabolic pathways. when plants are exposed to cd toxicity, growth inhibition, necrosis, chlorosis, leaf rolling or drying symptoms are generally observed (cosio et al. 2006, amani 2008). baszyński et al. (1980) showed that cd disturbs the biosynthesis of chlorophyll and carotenoids. it negatively affects chloroplast metabolism, including chloroplast replication and cell division (prasad, 1995, baryla et al. 2001). in populus × canescens plantlets, chlorophyll a, b, a + b and carotenoid were reported to be decreased at 50 µm cd exposure (he et al. 2013). the same authors reported that upon 200 µm cd exposure, chlorophyll a and b decreased in three poplar species but also found no statistical significance. these reports were also in parallel with the present findings. in arabidopsis, snp treatments induced the expression of 614 genes out of which 579 genes were upregulated, while 35 genes were down-regulated. most of the over expressed genes play important roles in biotic and abiotic stress responses (ahlfors et al. 2008). in our study, the recovery of photosynthetic pigments could be affected by gene expression related with abiotic stress responses by no induced signaling in black poplar. mda, which is an indicator for membrane lipid oxidation, is used to evaluate plant tolerance to abiotic stresses. he et al. (2013) reported that mda concentrations are not significantly affected by cd exposure but some species-specific differences were present in six poplar species. in the same study, the highest mda concentrations were also reported in p. nigra bark and leaves. in another study, mda was increased by cd treatments but no significant difference was detected in plants treated for four days, whereas eight and 12 day p. yunnanensis plants showed high mda levels (yang et al. 2015). it can be proposed that genotypic variations and the ameliorative effect of no may cause a reduction of mda amounts in response to oxidative stress. hydrogen peroxide (h2o2), a form of reactive oxygen species, is considered a common cellular metabolite. it is continually generated in enzyme and non-enzyme pathways in plants. also, it plays important roles in cell signaling, including various physiological and biochemical processes in plants (barba-espín et al. 2011). he at al. (2013) reported that h2o2 concentrations in leaves were elevated by 31% in p. cathayana and by 41% in p. nigra under cd treatment. in p. yunnanensis, h2o2 levels were elevated with an increase of exposure days (yang et al. 2015). in plantlets of populus × canescens (p. tremula × p. alba), h2o2 and mda levels were significantly increased in root, followed by wood, bark and leaf (dai et al. 2012). these results were corroborated by the findings of this work that h2o2 and mda levels were increased under cd exposure. kopyra and gwóźdź (2003) reported that exogenous no reduces the devastating effect of heavy metals, ethylene and herbicides on plants. so, ros cikili y., kulac s., samet h., filiz e. 122 acta bot. croat. 78 (2), 2019 productions are suppressed and oxidative damage is limited during stress conditions. also, no may directly prevent the production of the superoxide anion (o2˙ –) which results in an abrogation of toxic effects by the conversion of o2˙ – into peroxonitrite (onoo–; neill et al. 2003). in this study, the reductions in mda and h2o2 levels by snp treatment in comparison to control may prove the efficacy of the physiological functions of no in black poplar. ros as toxic by-products are considered to cause oxidative damage in cells (suzuki and mittler 2006). ros can be generated by different pathways such as imbalance of the electron transport chains in both chloroplasts and mitochondria (hernandez et al. 1995). anti-oxidative enzymes play crucial roles in ros detoxification under various abiotic and biotic stress conditions (pandhair and sekhon 2006). he et al. (2013) reported that apx activity was markedly enhanced, by 59%, in p. deltoides wood but apx was significantly inhibited by 43–49% in leaves of four poplar species (populus × euramericana, p. alba × p. glandulosa, p. nigra and p. popularis) under cd exposure. cat activities were found higher in poplar roots and bark but lower in leaves exposed to 200 μm cd. in hybrid poplar (p. nigra × maximowitzii × p. nigra var. italica), cat and sod activities were significantly higher at a 10–5 m cd treatment, whereas the increase was not significant at a 10–4 m cd treatment (nikolić et al. 2008). in populus × canescens, cat activities were increased after 1 day cd exposure in roots and leaves, and then decreased more strongly for 10 and 20 days in leaves. apx activities declined during cd exposure in roots and leaves for 1, 10 or 20 days (he at al. 2013). in hybrid poplar (populus × canescens), cat and apx activities in leaves were significantly enhanced at 10 μm cd treatment but also declined significantly according to the level of cd exposures (30, 50, or 70 μm cd) at 28 days (dai et al. 2012). yang et al. (2015) reported significant increases in apx, cat and sod activities under increasing cd stress durations (0, 4, 8, and 12 days) in p. yunnanensis. in p. tremula, apx activity increased slightly on days 7 and 14 with cd exposure in leaves, but it decreased rapidly at day 28 and reached a low on day 56 (kieffer et al. 2009). to alleviate/eliminate cd toxicity, plants induce many regulatory networks, including modulation of transcription factors, activation of metal transporters and biosynthesis of chelating compounds (dalcorso et al. 2010). in the light of the present findings about antioxidative enzyme activities and other available reports, it could be suggested that no as signaling molecule by snp treatment may induce different scavengers, including protein or non-protein antioxidants for ros balance under cd toxicity in p. nigra. in the soil-plant relationship, cd may mainly affect physiological processes and biochemical mechanisms by changing the concentration and functions of mineral nutrients. the interaction of cd with the nutrient elements has been shown (khan et al. 2007). sarwar et al. (2010) reported that chemical similarity between zn and cd is a major reason for cd toxicity in higher plants due to their interactions with each other. in this study, the decline in the zn level may be attributed to the interactions of these metals. several plant transporters are known to be responsible for the metal transport into the cytosol, such as zinc regulated transporter (zrt), iron-regulated transporter (irt)-like protein (zip), natural resistance-associated macrophage protein (nramp) family and oligo peptide transporters (opts) family. zrt and zip families also transport other divalent metals, including zn2+, mn2+ and cd2+ (colangelo and guerinot, 2006, vatansever et al. 2015, vatansever et al. 2016). overall, decline in divalent metal ions under cd exposure may result from competition between cd and other divalent ions, where cd could suppress the influx of available divalent metal ions into cells. in addition, it has been determined that cd + snp treatment generally improves mineral nutrition in black poplar, demonstrating the positive effects of snp for mineral nutrition. conclusion the present work has investigated the effects of exogenous snp treatments on cd toxicity in p. nigra. it was revealed that cd exposure causes reductions in growth parameters and inhibits physiological activities. leaf and bark dry weights and photosynthetic pigments (chlorophyll a, b, a + b, and carotenoids) were decreased by cd exposure. in addition, levels of mda and h2o2 content, as stress indicators, were increased under cd treatments and antioxidative enzyme activities such as those of cat and apx also increased. the treatment also causes a decline in the influx of divalent metal ions like fe, zn and mn. however, snp applications alleviated the symptoms of cd toxicity on growth, mineral nutrition and physiological activities, and enhance plant stress tolerance. exogenous snp application, accordingly, could be an efficient tool to cope with cd toxicity in black 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1996: degradation of glutathione s-conjugates by a carboxypeptidase in the plant vacuole. febs letters 384, 31–34. yang, y., li, x., yang, s., zhou, y., dong, c., ren, j., sun, x., yang, y., 2015: comparative physiological and proteomic analysis reveals the leaf response to cadmium-induced stress in poplar (populus yunnanensis). plos one 10(9), e0137396. zacchini, m., iori, v., scarascia-mugnozza, g., pietrini, f., massacci, a., 2011: cadmium accumulation and tolerance in populus nigra and salix alba. biologia plantarum 55, 383–386. acta bot. croat. 80 (2), 2021 217 acta bot. croat. 80 (2), 217–220, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-019 issn 0365-0588 eissn 1847-8476 short communication aristida oligantha – a new alien species on the eastern adriatic coast danijela stešević1*, đorđije milanović2, milica stanišić-vujačić1, urban šilc3 1 university of montenegro, faculty of natural sciences and mathematics, džordža vašingtona bb, 81000 podgorica, montenegro 2 university of banja luka, faculty of forestry, s. stepanovića 75a, 78000 banja luka, bosnia and herzegovina 3 research centre of the slovenian academy of sciences and arts zrc sazu, institute of biology, novi trg 2, 1000 ljubljana, slovenia abstract – an alien and potentially invasive species of north american origin aristida oligantha michx. was found in the hinterland of velika plaža, a sandy beach in the town of ulcinj (montenegro), the second known record of this plant in europe. here we describe the aristida oligantha community. further monitoring is suggested in order to evaluate its invasiveness and to plan appropriate eradication measures. keywords: aristida oligantha community, invasive species, northeastern mediterranean, sandy beach, south montenegro introduction genus aristida l. is one of the exotic grasses in the european flora. it belongs to the subfamily aristidoideae, which consists of three genera: aristida l., stipagrostis nees and sartidia de winter, being easily recognisable by oneflowered spikelets, with 3-awned lemmas, involute margins, a sharp pointed callus, and a line of hairs for a ligule ( cerros tlatilpa et al. 2011). so far, stipagrostis and sartidia have not been reported in europe, while the genus aristida has been noted as having two representatives: a. adscensionis l. and a. oligantha michx. (euro+med 2006, rakaj and pagad 2020). the first was been recorded in spain, italy, greece (euro+med 2006), belgium (desmet et al. 2020) and france (thevenot et al. 2020), while the second was recently reported in central part of albania (rakaj and pagad 2020). within its native area of distribution, which includes north america, aristida oligantha grows on waste or bare ground, old fields and dry hills (allred 1986). it is considered an extremely aggressive weed species, which is rather dangerous for cattle, since its awns and sharp callus cause injuries to the mouth, nostrils, and eyes. it disperses in two ways, by wind or by attachment to passing animals thanks to its retrorsely-barbed, pointed callus (owensby and launchbaugh 1977). finding of the species in the hinterland of the velika plaža sandy beach in the town of ulcinj (montenegro) represents its first record along the eastern adriatic coast. aim of this paper is to warn of the newly discovered alien species aristida oligantha, to present the new records, as well as the species composition and structure of the aristida oligantha plant community. material and methods the collected plant material was deposited in the herbarium collection at the university of montenegro, podgorica, montenegro (tgu), under the voucher number: tgu 1570528. the specimens were identified according to allred (1986). phytosiociological relevés were recorded according to the method of braun-blanquet (1964), stored in a tubroveg (hennekens and schaminée 2001) and incorporated into the vegetation database of montenegro (eu-me-001, http:// www.givd.info /id /eu-me-001). the nomenclature of the taxa follows the euro+med (2006). * corresponding author e-mail: danijela.stesevic@ucg.ac.me stešević d., milanović ð., stanišić-vujačić m., šilc u. 218 acta bot. croat. 80 (2), 2021 results and discussion aristida oligantha (fig. 1) was recorded in full bloom during the autumn field survey at sandy beach of velika plaža in town of ulcinj (montenegro), just along the narrow road that passes through the hinterland. the plant builds almost monodominant stands in rather anthropogenized but poorly trampled sites, at which brachypodietalia dune grasslands with annuals (annex i habitat type, code 2240) dominate during the spring. from the roadside, aristida stands are mainly connected with a narrow line of trampled vegetation dominated by cynodon dactylon, and on the opposite side there is either forest vegetation of planted maritime pines or the association eriantho-schoenetum nigricantis (pignatti 1953) géhu in géhu et al. 1984 within which aristida colonizes the area between tussocks of schoenus nigricans (tab. 1). the substrate is mostly sandy; exceptionally it is of crushed stones, which are used in this area as construction material for both car parks and paths. the total vegetation cover is mostly greater than 90%. in the aristida oligantha community, the most frequent taxa were cynodon dactylon, erigeron canadensis (100%), artemisia campestris, tragus racemosus, petrorhagia saxifrage and verbascum sinuatum (77.7%, tab. 1). the combination of late summer and autumn species, notably cynodon dactylon, tragus racemosus and euphorbia maculata define the tab. 1. phytosociological table of the aristida oligantha community (rels. 1-9) and the eriantho-schoenetum nigricantis association (rel. 10). see appendix for place of the relevés. relevé no. 1 2 3 4 5 6 7 8 9 10 plot size (m2) 10 25 25 10 25 25 25 25 25 25 vegetation cover (%) 95 100 90 97 95 90 50 97 90 100 aristida oligantha 5 4 5 5 4 4 3 5 5 2 cynodon dactylon 2 2 1 1 1 1 1 + + 1 erigeron canadensis 1 + + + + + + + 1 + tragus racemosus 1 . 1 1 + 2 2 1 . . verbascum sinuatum + 1 . + . + 1 + 1 . petrorhagia saxifraga 1 + . 1 + + . + + . plantago lanceolata + + 1 + 1 + . . . 2 tortella tortuosa 2 + 2 1 1 1 . . . 1 euphorbia maculata 1 + 1 . 2 2 1 . . . artemisia campestris 1 3 1 + 2 . . 2 1 . prospero autumnale + 1 + + . + . . . + scirpoides holoschoenus . . 1 1 1 + + . . 1 crepis foetida 1 + 1 . . . . + + + trifolium lappaceum 1 + . + 1 1 . . . . tripidium ravennae . . + 1 + . + + . . sanguisorba minor subsp. muricata . . . + 1 . + 1 1 . hypericum perforatum subsp. veronense 1 + + . + . . . . . bothriochloa ischaemum 1 . + . + . . + . . alkanna tinctoria 1 . . 1 . . + 1 + . lomelosia argentea + . + + + . . . . . dittrichia graveolens . . + . + . + . + . dittrichia viscosa . + + . . . . . . + teucrium capitatum 1 . . . . . . . + . asphodelus ramosus . . . + + . . . . . echium plantagineum . . . + . . . + . . digitaria ciliaris . . . . . + + . . . oenothera biennis aggr. . . . . . . + . . + rubus ulmifolius . . . . . . + . . 1 helianthemum jonium . . . . . . . + + . schoenus nigricans . . . . . . . . . 5 sporadic taxa: rel. 1 – salsola kali (1), setaria viridis (1), ajuga chamaepitys (+), rel. 2 – trifolium stellatum (+), odontites vernus ssp. serotina (+), scolymus hispanicus (+), rel. 3 – plantago arenaria (+), hypochaeris radicata (+), rel. 6 – medicago minima (1). aristida oligantha in montenegro acta bot. croat. 80 (2), 2021 219 syntaxonomic affiliation of this community to the alliance eragrostion of the order eragrostietalia and class digitario sanguinalis-eragrostietea minoris. aristida oligantha was recently reported in albania as a new alien species in european flora (rakaj and pagad 2020). actually, the species was recorded from the banks of the osum river, near the town of berat, south albania, in 2013 (marash rakaj, pers. comm.). as the sites are far apart, the introduction is probably independent. nevertheless, due to the extremely close connection between the municipality of ulcinj and albania (trade, construction, tourism, and vigorous cross-border traffic), it is possible that species was imported from albania. introduction of species via construction material is a common means of spreading invasive species in montenegro, as has been demonstrated by the spread of ambrosia artemisiifolia (stešević et al. 2014). in autumn, in the hinterland of the velika plaža beach, a. oligantha was recorded in monodominant stands – a. oligantha community (tab. 1), as well as within the association eriantho-schoenetum nigricantis, which represents the vegetation type equivalent of natura 2000 habitat 2190 humid dune slack. taking into consideration the great invasive potential of this species, we propose further monitoring and assessment of its invasive status and the planning of eradication measures. in order to deepen our knowledge on the synecology of this species beyond the boundaries of its natural range, as well as to describe any new association that might arise, it is necessary to do more detailed phytocenological research within the boundaries of the secondary range, which up to now covers only albania and montenegro in europe. acknowledgements the authors would like to thank kelly allred for confirmation of the identification of a. oligantha and branko anđić for confirmation of the identification of the moss material. the research was financed by jp morsko dobro (through the project: welcome). urban šilc was supported by programme p1-0236 (arrs) and bilateral grant (bime/16-17-018) of arrs. appendix date and coordinates (wgs84) of the relevés (tab. 1). all relevés were collected at 1 m a.s.l. rel. 1 – 2019/10/06, 41.90632 n, 19.26582 e; rel. 2 – 2019/10/06, 41.89915 n, 19.2901 e; 3 rel. – 2019/10/06, 41.90543 n, 19.26919 e; rel. 4 – 2019/10/06, 41.90363 n, 19.27509 e; rel. 5 – 2019/10/06, 41.90321 n 19.27589 e; 6 rel. – 2019/10/06, 41.90544 n, 19.27069 e; rel. 7 – 2019/10/06, 41.90127 n 19.28215 e; rel. 8 – 2019/10/06, 41.90242 n, 19.27777 e; rel. 9 – 2019/10/06, 41.90249 n, 19.27758 e; rel. 10 – 2019/10/06, 41.90542 n 19.26927 e. references allred, k.w., 1986: studies in the genus aristida (gramineae) of the southeastern united states. iv. key and conspectus. rhodora 88, 367–387. braun-blanquet, j., 1964: pflanzensoziologie. grundzüge der vegetationskunde. springer verlag, wien. cerros-tlatilpa, r., columbus, j.t., barker, n.p., 2011: phylogenetic relationships of aristida and relatives (poaceae, arisfig. 1. aristida oligantha michx.: a – habitus, b – spikelets stešević d., milanović ð., stanišić-vujačić m., šilc u. 220 acta bot. croat. 80 (2), 2021 tidoideae) based on noncoding chloroplast (trnl-f, rpl16) and nuclear (its) dna sequences. american journal of botany 98, 1868–1886. desmet, p., reyserhove, l., oldoni, d., groom, q., adriaens, t., vanderhoeven, s., pagad, s., 2020: global register of introduced and invasive species belgium. version 1.8. invasive species specialist group issg. checklist dataset https://doi. org/10.15468/xoidmd accessed via gbif.org on 2020-03-29. euro+med, 2006: euro+med plantbase the information resource for euro-mediterranean plant diversity. retreived march 16, 2020 from http://ww2.bgbm.org/europlusmed/ hennekens, s.m., schaminée, j.h.j., 2001: turboveg, a comprehensive data base management system for vegetation data. journal of vegetation science 12, 589–591. owensby, c.e., launchbaugh, j.l., 1977: controlling prairie threeawn (aristida oligantha michx.) in central and eastern kansas with fall burning, journal of range management 30, 337–339. rakaj, m., pagad, s., 2020: global register of introduced and invasive species albania. version 1.4. invasive species specia l ist group issg. check l ist dataset ht t ps://doi. org/10.15468/km1q9p accessed via gbif.org on 2020-03-29. stešević, d., latinović, n., caković, d., 2014: invasive alien plant species in montenegro, with special focus on ambrosia artemisiifolia. in: uludağ, a., trichkova, t., rat, m., tomov, r. (eds.), proceedings of the 4th esenias workshop: international workshop on ias in agricultural and non-agricultural areas in esenias region, 17–31. çanakkale onsekiz mart university, çanakkale. thevenot, j., albert, a., collas, m., de massary, j., dupont, p., masse, c., moutou, f., poulet, n., roques, a., souty-grosset, c., vincent, b., jenna wong, l., pagad, s., 2020: global register of introduced and invasive species france. version 1.2. invasive species specialist group issg. checklist dataset https://doi.org/10.15468/up1tr5 accessed via gbif.org on 2020-03-29. 184 acta bot. croat. 80 (2), 2021 acta bot. croat. 80 (2), 184–190, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-021 issn 0365-0588 eissn 1847-8476 one new species of cymbella c.a. agardh (bacillariophyta) from high altitude lakes in the hengduan mountains of southwest china qi liu1, han wu1, yan-ling li1,*, john patrick kociolek2 1 institute for ecological research and pollution control of plateau lakes, school of ecology and environmental science, yunnan university, kunming 650500, p. r. china 2 museum of natural history and department of ecology and evolutionary biology, university of colorado, boulder, colorado – 80309, usa abstract – this paper describes a new species of the genus cymbella c.a. agardh from an alpine lake in the hengduan mountains of southwestern china. a detailed morphological description of the new species, named cymbella luoyulanae sp. nov., is presented; the description is based on light and scanning electron microscopy. the main features of c. luoyulanae are strongly dorsiventral valves with strongly reverse-lateral raphe branches near the proximal ends, a large central area occupying approximately a half of the width with the valve and slit-like areolae comprising striae that may be unseriate or partially biseriate. the new species has morphological characteristics that resemble those of c. heihainensis y.li et gong, c. modicepunctata krammer and c. asiatica metzeltin, lange-bertalot et y.li, but it differs from these three species in details of size, valve shape, striae density, central area, and number of stigmata. keywords: china, cymbella, diatom, new species, taxonomy, sem introduction diatoms from the yunnan plateau have been only cursorily studied in the past (skuja 1937, zhu and chen 1994, li et al. 2007a,b). cymbella c.a. agardh (1830: 1) is one of the largest diatom genera, representing nearly two thousand described taxa (kociolek et al. 2020a). however, in the past 30 years, the family cymbellaceae has been split by some researchers more finely (kociolek and stoermer 1987, krammer 1997a,b, lange-bertalot and genkal 1999, krammer 2002, 2003, jüttner et al. 2010a, bahls 2015, kulikovskiy et al. 2014, 2015, kulikovskiy and kuznetsova 2016, kapustin et al. 2017, 2020, kociolek et al. 2017, glushchenko et al. 2019, kezlya et al. 2020). as a result of this recent work about 20 different genera are recognized (tab. 1). since the revision of cymbella, a number of other new species have been described from china (summarized in kociolek et al. 2020b, see also li et al. 2019, liu et al. 2020, zhang et al. 2020). in southwestern china, specifically, li et al. (2003a,b) described new species of cymbella respectively from northwest tibet and qinghai province. gong and li (2011) described a new species of cymbella from the yunnan plateau. hu et al. (2013) described three new species of cymbella from the high altitude lakes of the hengduan mountains region. in the present paper, we describe one new cymbella species from the hengduan mountains area based on light and scanning electron microscopical investigations and compare its morphology with similar species. material and methods samples were taken using a kajak-type gravity core (renberg and hansson 2008) from the surface sediments of lake shadecuo (29°44′35.8′′ n, 101°21′39.8′′ e, tab. 2), located in the eastern hengduan mountains region of southwest china. the ph and specific conductivity of the lake water were measured in the field using a ysi 650 multi-parameter display system (650 mds, ysi incorporated 1700/1725 brannum lane, yellow springs, oh 45387 usa) with a 600 xl probe. water samples were taken from 50 cm under the water surface. transparency was estimated using a secchi disk. total nitrogen (tn) and total phosphorus * corresponding author e-mail: yanlingli@ynu.edu.cn new species of diatom from china acta bot. croat. 80 (2), 2021 185 (tp) were measured by a shimadzu uv 2450 ultravioletvisible spectrophotometer at nanjing institute of geography and limnology, chinese academy of sciences, using the alkaline potassium persulfate digestion-uv spectrophotometric method (nydahl 1978) and the ammonium molybdate spectrophotometry method (ebina et al. 1983), respectively. diatom samples were kept under 4 °c in the refrigerator before laboratory treatments. in the laboratory, the diatoms were treated with hcl and h2o2 (battarbee 1986). permanent slides were made from cleaned materials and mounted in naphrax® for observation with light microscopy (lm; olympus, bx-51, dic). relative abundances of diatoms in the samples were determined with a count of 300 valves. cleaned materials were investigated with a leo 1530 scanning electron microscope (sem). samples and slides are preserved in the herbarium of nanjing institute of geography and limnology, chinese academy of sciences, china. results taxonomy class bacillariophyceae haeckel 1878 subclass bacillariophycidae d.g. mann in round et al. 1990 order cymbellales d.g. mann in round et al. 1990 family cymbellaceae greville 1833 genus cymbella c.a.agardh 1830 cymbella luoyulanae y.li sp. nov. fig. 1 a-e; fig. 1c is the holotype lm (fig. 1): valves moderately dorsiventral. dorsal margin strongly convex. ventral margin concave, slightly tumid in middle. valve ends slightly truncated and broadly rounded. length 59-82 μm, width 16.0-18.5 μm, maximum length/ width ratio ca. 4.5. axial area moderately broad. central area elliptical, about 1/4-1/3 of width of valve. raphe distinctly lateral, abruptly reverse-lateral near proximal ends. striae slightly radiate, becoming more radiate towards valve ends. 3-5 stigmata, some larger than others, occur ventrally from central nodule, distant from middle ventral striae. striae 7-8/10 μm at centre becoming 10-12/10 μm towards apices, with 20-22 areolae in 10 μm. in sem external valve view (fig. 2): striae uniseriate, partly biseriate with elliptical and transapically oriented areolae. areolae slit-like with apically-orientated openings in transapical striae (fig. 2 a-f). stigmata rounded, separated from the areolae at central area on ventral side. small round depressions may occur on the dorsal side of the central area (fig. 2 c, d). raphe centrally located on the valve face in a tab. 1. a chronological listing of genera based on species formerly in the genus cymbella or closely related genera (zhang et al. 2021). * indicates the genus may be more closely related to freshwater gomphonemoid diatoms. genera references encyonema kützing (1833: 589) kützing 1833 *reimeria kociolek et stoermer (1987: 457) kociolek and stoermer 1987 pseudencyonema krammer (1997: 156) krammer 1997a encyonopsis krammer (1997: 156) krammer 1997b cymbellopsis krammer (1997: 157) krammer 1997b cymbopleura krammer (1999: 292) krammer 2003 navicymbula krammer (2003: 123) krammer 2003 delicata krammer (2003: 110; recently renamed as delicatophycus m.j. wynne 2019: 1) krammer 2003 gomphocymbellopsis krammer (2003: 127) krammer 2003 *afrocymbella krammer (2003: 129) krammer 2003 krsticiella levkov in levkov et al. (2007: 14–15) levkov et al. 2007 oricymba jüttner et al. (2010: 408) jüttner et al. 2010a ochigma kulikovskiy, lange-bertalot et metzeltin (2012: 214) kulikovskiy et al. 2012 khursevichia kulikovskiy, lange-bertalot et metzeltin (2012: 157) kulikovskiy et al. 2012 kurtkrammeria bahls (2015: 6) bahls 2015 celebesia kapustin, kulikovskiy et kociolek (2017: 153) kapustin et al. 2017 karthickia kociolek, glushchenko et kulikovskiy (2019:606) glushchenko et al. 2019 tab. 2. physical and chemical parameters in lake shadecuo. lake shadecuo latitude (°n) 29°44′35.8′′ longitude (°e) 101°21′39.8′′ altitude (m a.s.l.) 4428 maximum depth (m) 8 ph 7.66 secchi disk depth (m) 4.8 total nitrogen (mg·l–1) 0.288 total phosphorus (mg·l–1) 0.004 conductivity (μs·m–1) 33 liu q., wu h., li y.-l., kociolek j. p. 186 acta bot. croat. 80 (2), 2021 narrow hyaline axial area with straight to slightly dorsally arched proximal ends (fig. 2 a, b). external distal raphe ends deflected dorsally (fig. 2 e, f); external proximal raphe ends dilated (fig. 2 c, d). the apical pore fields present and comprised by a group of porelli, almost entirely on the valve mantle (fig. 2 e, f). in sem internal valve view (fig. 3): striae with internal areolae openings lack any occlusions (fig. 3 a, b). the central area distinct, projecting internally, and lacking an intermissio. stigma opening round, with a slight expanded depression evident on the central nodule. stigmata lack any ingrowths or occlusion (fig. 3 c, d). distal raphe endings bent slightly towards the dorsal margin, terminating in helictoglossae. (fig. 3 e, f). type: – china. sichuan province: kangding city, lake shadecuo, 29°44′35.8′′ n, 101°21′39.8′′ e, elevation 4428 m a.s.l., samples collected by dr. yulan, luo, 14th october 2017. holotype shadecuo 1-1 in coll. li yanling, yunnan university., kunming, china. fig. 1 is of the holotype. etymology: –the specific epithet ‘luoyulanae’ refers to the collector of the sample on which these observations are based. fig. 1. light microscopy micrographs of the type population of cymbella luoyulanae sp. nov. in lake shadecuo, china. a-e – valve views, showing the valve variability of the holotype population. fig. 2. external valve view of cymbella luoyulanae sp. nov. by scanning electron microscope. a, b – external view of an entire valve. c, d – external view of valve center, the proximal raphe endings and the very large central area with 4-5 stigmata. e, f – valve apices, striae with slit-like areolae, some of them biseriate, and pore field separated by the distal raphe fissures. new species of diatom from china acta bot. croat. 80 (2), 2021 187 ecology cymbella luoyulanae has been observed in the surface sediment sample from lake shadecuo. in lake shadecuo, this species was associated with cyclotella ocellata pantocsek (1902: 134) (20%), achnanthidium minutissimum ( kützing) czarnecki (czarnecki 1994: 157) (15%), c. comensis grunow in van heurck (1882, pl. 93, fig. 3 e, f) (14%), pseudostaurosira pseudoconstruens (marciniak) williames et round (1987: 275) (15%), and staurosirella pinnata ( ehrenberg) d.m. williames et round (1987: 274) (5%). discussion the features found in cymbella luoyulanae are compared to all other known species of the genus in tab. 3. the taxon most similar to c. luoyulanae is c. heihainensis. the valves of this species are wider, have a larger central area, drop-like proximal raphe ends, and a higher number of areolae in 10 µm, distinguishing it from c. luoyulanae. cymbella modicepunctata has larger valves with a more distinct central area, and fewer striae in 10 µm, distinguishing it from c. luoyulanae. cymbella luoyulanae has stigmata that may vary in size along the ventral margin, while c. modicepunctata has uniformly-sized stigmata on the ventral side. the same stigmata feature of c. luoyulanae is also found in c. schimanskii krammer and its variety var. excelsa ( meister) krammer. in sem view, c. schimanskii has y-shaped areolae openings in the middle of the valve, which is different from the biseriate of c. luoyulanae, and they differ from c. luoyulanae by having larger valves, a smaller central area, fig. 3. internal valve views of cymbella luoyulanae sp. nov. by scanning electron microscope. a, b – internal view of entire valve. c, d – showing the valve centre and four big and one small stigmata as elongated furrows connected to the striae. e, f – internal view of valve apex with prominent helictoglossa deflecting to the dorsal side. liu q., wu h., li y.-l., kociolek j. p. 188 acta bot. croat. 80 (2), 2021 and in the shape of the central pores (rounded vs expanded as in c. luoyulanae). cymbella luoyulanae can be distinguished from c. asiatica by valve size and striae density. cymbella asiatica varies between 50 and 105 µm in length and 14-18 µm in breadth, has no stigmata-like depressions on the dorsal side, and only 6-7 striae in 10 µm. cymbella luoyulanae is also similar to c. arctissima metzeltin, which can be distinguished from our new species which has dorsally deflected proximal raphe endings, is shorter (98-105 µm vs. 59-94 µm), and has a broader central area (2/3 width vs. 1/2 width). another member of the genus cymbella, c. distalebiseriata bing liu et d.m. williams in liu et al. (2018: 41), was described from hunan province, china. cymbella distalbiseriata shares the feature of having striae with uniseriate and partially biseriate striae. the only other species of cymbella exhibiting (a few) biseriate striae include c. yakii jüttner et van de vijver in jüttner et al. (2010b), c. duplopunctata krammer 2002 and c. buechleri krammer 2002 (both described from a fossil deposit in western north america). three of the five species exhibiting biseriate striae are found in asia, and two have been described from china. the other two taxa with biseriate striae are known from a fossil deposit in western north america (apparently of miocene age, see krammer 2002, p. 109). these are distinct from one another in size, shape and other morphological features (krammer 2002, jüttner et al. 2010b, liu et al. 2018). it is unclear if the possession of biseriate striae is a homologous feature for this group or not; formal phylogenetic analysis is required to understand character evolution in this regard. however, if the taxa with biseriate striae are indeed closely related among themselves and their distribution remains between asia and western north america, it supports the notion, noted by ehrenberg over 180 years ago (ehrenberg 1849) and supported by additional diatom examples (kociolek and stoermer 1989, kociolek et al. 2013, 2015, genkal and kulikovskiy 2016, kociolek 2019) as well as some genera of higher plants (xiang and soltis 2001, nie et al. 2006, kadereit and baldwin 2012), of there being a close relationship between the floras of these two areas. acknowledgments this work was supported by the projects of the national sciences and foundation of china (grant no. 41877296, 41877434, and 42077424). references agardh, c.a., 1830: conspectus criticus diatomacearum. part 1. litteris berlingianis, lund. bahls, l.l., 2015: kurtkrammeria, a new genus of freshwater diatoms (bacillariophyta, cymbellaceae) separated from encyonopsis. nova hedwigia 101, 165–190. battarbee, r.w., 1986: diatom analysis. in: berglund, b.e. (ed.), handbook of holocene palaeoecology & palaeohydrology, 527–570. john wiley and sons ltd., chichester, uk. tab. 3. morphological characteristics of cymbella luoyulanae sp. nov. and cymbella species sharing similar morphological features. species /feature c. luoyulanae c. heihainensis li et gong c. modicepunctata krammer c. asiatica metzeltin, lange-bertalot et li, y. c. schimanskii krammer c. arctissima metzeltin reference this study hu et al. (2013) krammer (2002) metzeltin et al. (2009) krammer (2002) krammer (2002) valve length, µm 59-94 82-100 102-140 50-105 100-200 98-105 valve width, µm 16.0-19.5 19.0-22.5 21-22 14-18 29-36 17-18 length/ width ratio 4.8 max 4.7 max 6.4 max 5.8 max 5 max 5.8 striae in 10 µm 7-10 7-10 5.5-6 6-7 7-12 7-10 areolae in 10 mm 20-22 14-16 14-16 18-22 10-16 18-21 areolae slit-like, biseriate slit-like, y-, xshaped lineolate not known slit-like, y-shaped slit-like stigmata ventral side: 3-5 big and some smaller, dorsal side: 0-3 ventral side: 7-10 big and some smaller, dorsal side: 0–5 ventral side: 7-10 ventral side: 4-6 ventral side: 6-10 big and some smaller, dorsal side: sometimes some ventral side: 6-8 central area 1/2 width 1/2 width 2/3-3/4 width 1/2 width 1/3 width 2/3 width central pores of raphe expanded drop-like, slightly ventrally deflected expanded drop-like, slightly ventrally deflected distinct, very slightly reverse-lateral distinct, abruptly reverse-lateral small, slightly reverse-lateral small, dorsally deflected new species of diatom from china acta bot. croat. 80 (2), 2021 189 czarnecki, d.b., 1994: the freshwater diatoms culture collection at loras college, dubuque, iowa. in: kociolek, j.p. 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(eds), compilation of report on survey of algal resources in southwestern china, 79–130. science press, beijing. opce-str.vp acta bot. croat. 68 (1), 29–44, 2009 coden: abcra 25 issn 0365–0588 phytoplankton composition and biomass of the northern adriatic lagoon of stella maris, croatia neda fanuko1*, marko val^i]2 1 faculty of arts and sciences, 51000 rijeka, omladinska 14, croatia 2 faculty of maritime studies, 51000 rijeka, studentska 2, croatia this study provides information on the seasonality of phytoplankton abundance, biomass expressed as cell volume and cell carbon, as well as species composition, in the small, shallow, brackish northern-adriatic lagoon of stella maris near umag (croatia). the lagoon is permanently connected with the adjacent sea. wide seasonal temperature and salinity excursions regulate phytoplankton assemblages. unlike other adriatic lagoons, the lagoon of stella maris showed moderate phytoplankton abundance, cell volume and carbon content and a high number of species. the specific diatom volumes from the stella maris lagoon were higher than those found in other adriatic lagoons, whereas the specific volumes of dinoflagellates were in the same range. diatoms represented 55% of all the species found, but there was a considerable contribution of nanoplankton and dinoflagellates in the annual outbursts. keywords: phytoplankton, taxonomy, cell volume, cell carbon, coastal lagoon, adriatic sea introduction the lagoons of the northern adriatic sea are characterized by shallowness, strong influence from the adjacent land and considerable fluctuations in hydrographic conditions. the lagoons of the northwest adriatic coast have been studied with much attention for over two centuries (nardo 1847, ninni 1906, babi] 1911, kiesselbach,1936, brunetti et al.1983, orel et al. 2001, covelli et al. 2005), particularly with respect to lagoon phytoplankton (vatova 1940, 1961; marchesoni 1954; tolomio 1982; tolomio and bullo 2001; facca et al. 2002, 2003; socal et al. 2006). however, on the eastern coast the few lagoons have been investigated only sporadically (zanon 1941; malej et al.1979; fanuko 1979, 1984; de menech 2005; fanuko et al. 2008). this paper provides information on phytoplankton assemblages, their species composition, abundance, cell volume and carbon in the small, shallow brackish lagoon of stella maris near umag (croatia). acta bot. croat. 68 (1), 2009 29 * corresponding author, e-mail: fanukon@ffri.hr u:\acta botanica\acta-botan 1-09\fanuko.vp 22. travanj 2009 11:20:19 color profile: disabled composite 150 lpi at 45 degrees materials and methods study area the study area is a small, natural macro-tidal northern adriatic lagoon (45°27’06.35’’n, 13°30’59.80’’e), only 15,000 square meters large and 2 m deep in most parts, permanently connected with the adjacent sea by a narrow channel, 6m wide and 40 m long (fig. 1). the climate of the region is sub-mediterranean with an average annual air temperature of 16.4 °c and a rainfall up to 1,000 mm per year, distributed mostly over autumn and winter. in the lagoon there are several submarine springs, active mostly during late autumn and winter. the level and water exchange inside the lagoon is influenced generally by the tidal range of up to 2.04 m, while the prevailing weak winds from west and southwest probably represent an additional forcing factor. the euphotic zone comprises the whole water column. the water temperature varies in a wide range, from 4.2 °c in january to 30.2 °c in july and the salinity, ranging from 29 to 37, is directly influenced by daily events: rainfall and subsurface spring activities, with the highest values, above 33, observed in summer. the lagoon is located in the middle of a tourist resort, where bungalows are inhabited only during spring and summer. in 1979 the lagoon and the channel were deepened, a pier and lateral quays were erected, transforming the lagoon into a small marina, equipped with water and electricity supply, accessible to vehicles, with one hundred moorings for smaller boats anchoring between april and october, reaching the maximum number in august. during the cold part of the year the lagoon is entirely abandoned and the only human activity inside is sporadic fishing. 30 acta bot. croat. 68 (1), 2009 fanuko n., val^i] m. fig. 1. location of stella maris lagoon with sampling site u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:33 color profile: disabled composite 150 lpi at 45 degrees the sandy to muddy sediments are populated by eelgrass cymodocea nodosa which dominates the macroalgae chaetomorpha sp. and cystoseira sp. in winter and spring and padina pavonica in summer. in autumn and winter, when anthropogenic influence is sparse, the lagoon becomes a habitat for 3 species of water birds: tachybaptus ruficollis, aythya fuligula and larus genei. occasionally from may to september a mucilage phenomenon extending from surface to bottom is observed, in the same days but to a greater extent than in the outside sea. phytoplankton from september 2004 to september 2005, with the exception of october, january and february, the sampling was carried out once or twice a month at the 1 m deep station. the phytoplankton samples for microscopic analysis were preserved with buffered formaldehyde (1.5% final concentration) and the subsamples (50 ml) were settled overnight in sedimentation chambers. the entire bottom chamber plate area was counted at 250´ magnification for cells larger than 10 mm, whereas for smaller cells (< 10 mm) one transect of the chamber bottom was scanned at 500´ magnification. the species were identified and classified according to streble and krauter (1984) for cyanobacteria, throndsen (1997) for naked flagellates, heimdal (1997) for coccolithophorids, peragallo and peragallo (1908), hustedt (1930), hendey (1964) and hasle and syvertsen (1997) for diatoms, steidinger and tangen (1997) for dinoflagellates. cells of approximately 2 mm in size that were hard to identify were reported as minute nanoplankton. during each count, linear measurements of cell size, made by ocular micrometer, were made for 3 to 5 specimens of perennial species and every specimen of rare species. these values were converted to specific average biovolume using the geometric formula of either a sphere, a parallelepiped, a cylinder, a cone or truncated cone, an ellipsoid or two composite geometric bodies. the average cell volume was converted to cell carbon using the conversion factor of 0.13 pg c mm –3 for armoured dinoflagellates and 0.11 pg c mm –3 for other phytoplankton groups (andersson and rudehäll 1993). results species composition and phytoplankton successions the phytoplankton assemblage of the stella maris lagoon was composed of 151 taxa (tab. 1). diatoms were the dominant group (55% of all the species found), followed by dinoflagellates (28%) and prymnesiophytes (7%). the shallow lagoon assemblage was characterized by 21 genera of pennate diatoms that appeared throughout the year in low but steady number and were obviously well adapted to the fluctuating abiotic variables. the microscopic observations revealed that the winter specimens of these pennate diatoms had larger chloroplasts, which were more abundant and more intense in colour than those observed in the cells of the same species that appeared in summer. the outbursts of abundance, cell volume or phytoplankton carbon were caused by other groups. in march 2005, when the sea temperature was 12 °c and the salinity 34.4, the coccolithophorid acanthoica aculeata reached its maximum of 1.52´10 5 cells l –1 , while in may, when water temperature and salinity rose over 20 °c and 33 respectively, dinoflaacta bot. croat. 68 (1), 2009 31 phytoplankton in stella maris lagoon u:\acta botanica\acta-botan 1-09\fanuko.vp 22. travanj 2009 11:20:20 color profile: disabled composite 150 lpi at 45 degrees 32 acta bot. croat. 68 (1), 2009 fanuko n., val^i] m. tab. 1. list of the phytoplankton species found in the stella maris lagoon, their average cell volume and carbon content t a x o n cell volume (mm³) cell carbon content (pgc) c y a n o b a c t e r i a aphanizomenon gracile lemmermann 25 3 dactylococcopsis acicularis lemmermann 157 17 oscillatoria sp. 19 2 phormidium faveolarum montagne ex gomont 6 1 synechococcus aeruginosus nägeli 462 51 c r y p t o p h y c e a e hillea fusiformis schiller 14 2 c h r y s o p h y c e a e dictyocha fibula ehrenberg 2094 230 meringosphaera tenerrima lohmann 268 29 mesocena polymorpha lemmermann 3534 389 uroglena volvox ehrenberg 28 3 p r y m n e s i o p h y c e a e acanthoica aculeata kamptner 133 15 calyptrosphaera oblonga lohmann 1047 115 calciosolenia murrayi gran 209 23 emiliania huxleyi (lohmann) hay et mohler 268 29 michaelsarsia adriatica (schiller) manton, bremer et oates 335 37 ophiaster formosum gran 34 4 ophiaster hydroideus (lohmann) lohmann 26 3 prymnesium parvum carter 56 6 rhabdosphaera stylifera lohmann 524 58 syracosphaera pulchra lohmann 717 79 b a c i l l a r i o p h y c e a e c e n t r a l e s biddulphia biddulphiana (smith) boyer 395640 43520 biddulphia titiana grunow 339120 37303 cerataulina pelagica (cleve) hendey 44179 4860 chaetoceros affinis lauder 15708 1728 chaetoceros brevis schütt 3402 374 chaetoceros compressus lauder 2650 291 chaetoceros curvisetus cleve 2011 221 chaetoceros decipiens cleve 282743 31102 chaetoceros peruvianus brightwell 261979 28818 chaetoceros simplex ostenfeld 34 4 chaetoceros tetrastichon cleve 942 104 chaetoceros tortissimus gran 877 96 chaetoceros wighami brightwell 1356 149 coscinodiscus excentricus ehrenberg 9770 1075 u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:33 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 33 phytoplankton in stella maris lagoon t a x o n cell volume (mm³) cell carbon content (pgc) coscinodiscus perforatus ehrenberg 1286 141 guinardia flaccida castracane (peragallo) 1781283 195941 hemiaulus hauckii grunow 80592 8865 leptocylindrus danicus cleve 7853 864 leptocylindrus minimus gran 125 14 melosira nummuloides agardh 785 86 melosira sulcata (ehrenberg) kützing 1155 127 odontella mobiliensis (bailey) grunow 196250 21587 proboscia alata (brightwell) sundström 6283 691 pseudosolenia calcar avis (schultze) sundström 1178097 129591 rhizosolenia styliformis brightwell 105029 11553 skeletonema sp. 2356 259 thalassiosira decipiens (grunow) jørgensen 17671 1944 p e n n a l e s achnantes brevipes agardh 117810 12959 achnantes longipes agardh 376991 41469 amphiprora sulcata o’meara 4385 482 amphora crassa gregory 2880 317 amphora hyalina kützing 3240 356 amphora marina (w smith) van heurck 78540 8639 amphora ostrearia brébisson 165360 18190 amphora ovalis kützing 180 20 amphora sulcata (brébisson) cleve 5000 550 amphora sp. 2880 317 auricula adriatica peragallo 19250 2117 auricula insecta (grunow) cleve 24000 2640 campylodiscus adriaticus grunow 28260 3109 cocconeis scutellum ehrenberg 943 104 cylindrotheca closterium (ehrenberg) reimann et lewin 524 58 diploneis bombus ehrenberg 6250 687 diploneis crabro ehrenberg 14400 1584 entomoneis paludosa (w. smith) reimer 11025 1213 fragilaria crotonensis kitton 707 78 grammatophora marina (lyngbye) kützing 8000 880 grammatophora oceanica ehrenberg 16000 1760 licmophora communis (heiberg) grunow 1600 176 licmophora flabellata (carmichael) agardh 12087 1330 licmophora lyngbyei (kützing) grunow 27500 3025 licmophora paradoxa (lyngbye) agardh 6480 713 licmophora quadriplacata mereschkowsky 126 14 licmophora remulus grunow 30000 3300 licmophora sp. 6480 713 tab. 1. – continued u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:33 color profile: disabled composite 150 lpi at 45 degrees 34 acta bot. croat. 68 (1), 2009 fanuko n., val^i] m. t a x o n cell volume (mm³) cell carbon content (pgc) lioloma pacificum (cupp) hasle 5655 622 mastogloia asperula grunow 6000 660 mastogloia citrus cleve 2400 264 navicula cancellata donkin 3000 330 navicula lyra ehrenberg 3900 429 navicula spp. 6000 660 nitzschia incerta grunow 6000 660 nitzschia longissima (brébisson) ralfs 3351 369 pleurosigma angulatum (quekett) w. smith 255563 28112 pleurosigma balticum smith 180000 19800 pleurosigma elongatum w. smith 144000 15840 pleurosigma formosum w. smith 194000 21340 podocystis adriatica kützing 73476 8082 pseudo-nitzschia sp. 1 147 16 pseudo-nitzschia sp. 2 1800 198 striatella unipunctata (lyngbye) agardh 252500 27775 surirella fluminensis grunow 15000 1650 synedra crystallina (agardh) kützing 19110 2102 synedra fasciculata (agardh) kützing 4500 495 synedra hennedyana gregory 22973 2527 synedra tabulata (agardh) kützing 5655 622 synedra toxoneides castracane 1050 115 synedra sp. 5655 622 thalassionema nitzschioides (grunow) mereschkowsky 120 13 thalassionema frauenfeldi (grunow) hallegraeff 3750 412 toxarium undulatum bailey 22973 2527 tropidoneis lepidoptera (gregory) cleve 60000 6600 e u g l e n o p h y c e a e euglena viridis (o.f. müller) ehrenberg 3142 346 eutreptia lanowii steuer 1571 173 d i n o p h y c e a e alexandrium minutum halim 3462 450 ceratium furca (ehrenberg) claparéde et lachmann 36559 4753 ceratium fusus (ehrenberg) dujardin 9739 1266 ceratium macroceros (ehrenberg) vanhöfen 39270 5105 ceratium massiliense (gourret) e.g. jørgensen 188495 24504 ceratium tripos (müller) nitzsche 150795 19603 dinophysis caudata seville-kent 104720 13614 dinophysis fortii pavillard 111910 14548 dinophysis hastata stein 85910 11168 dinophysis schroederi pavillard 91630 11912 goniodoma polyedricum (pouchet) jorgensen 38288 4977 tab. 1. – continued u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:33 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 35 phytoplankton in stella maris lagoon t a x o n cell volume (mm³) cell carbon content (pgc) goniaulax polygramma stein 22725 2954 gymnodinium simplex (lohmann) kofoid et swezy 589 65 gymnodinium sp. 589 65 gyrodinium fusiforme kofoid et swezy 21206 2333 gyrodinium sp. 21206 2333 oxytoxum longiceps schiller 1571 204 oxytoxum tesselatum (stein) schütt 1140 148 oxytoxum variabile schiller 697 91 phalacroma rotundatum (claparede et lachmann) kofoid et michener 6936 902 prorocentrum arcuatum issel 29438 3827 prorocentrum balticum (lohmann) loeblich 173 22 prorocentrum compressum (bailey) abé ex dodge 22808 2965 prorocentrum dactylus (stein) dodge 18850 2450 prorocentrum gracile schütt 2566 334 prorocentrum lima (ehrenberg) dodge 14158 1841 prorocentrum micans ehrenberg 13090 1702 prorocentrum minimum (pavillard) schiller 2545 331 prorocentrum scutellum schröder 20944 2723 prorocentrum triestinum schiller 785 102 protoperidinium crassipes (kofoid) balech 174411 22673 protoperidinium depressum (bailey) balech 184103 23933 protoperidinium diabolus (cleve) balech 150795 19603 protoperidinium divergens (ehrenberg) balech 110733 14395 protoperidinium globulus (stein) balech 2617 340 protoperidinium kofoidi fauré-fremiet 233674 30378 protoperidinium leonis (pavillard) balech 11641 1513 protoperidinium pallidum (ostenfeld) balech 102108 13274 protoperidinium solidicorne (mangin) diwald 43422 5645 protoperidinium steinii (jörgensen) balech 63617 8270 protoperidinium tuba (schiller) balech 3393 441 protoperidinium sp. 63617 8270 scripsiella trochoidea (stein) loeblich 6283 817 dinoflagellate cyst 1 8831 971 dinoflagellate cyst 2 14130 1554 c h l o r o p h y c e a e carteria marina diesing 188 21 chlamydomonas sp. 385 42 dunaliella sp. 198 22 tetraselmis sp. 385 42 minute nanoplankton 6 1 incertae sedis 785 86 tab. 1. – continued u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:33 color profile: disabled composite 150 lpi at 45 degrees 36 acta bot. croat. 68 (1), 2009 fanuko n., val^i] m. 0 2 4 6 8 10 12 14 16 2004 2005 sampling days total phytoplankton minute nanoplankton 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se pa b u n d a n c e (1 0 c e ll s l ) 5 – 1 fig. 2. annual variation of phytoplankton abundance 2004 2005 sampling days 160 250 0 10 20 30 40 50 60 70 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se p diatoms 0 0.010 0.020 0.030 0.040 0.050 silicoflagellates 150 0 1 2 3 4 5 6 7 coccolithophoridae 0 4 8 12 16 18 armoured dinoflagellates 0 200 400 600 800 1000 1200 1400 minute nanoplankton 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se pa b u n d a n c e (1 0 0 0 c e ll s l ) – 1 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se p 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se p 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se p 2004 2005 sampling days fig. 3. seasonality of some phytoplankton groups u:\acta botanica\acta-botan 1-09\fanuko.vp 22. travanj 2009 11:20:21 color profile: disabled composite 150 lpi at 45 degrees gellates (goniaulax, gymnodinium, prorocentrum, protoperidinium, scripsiella) became more abundant though their number never exceeded 5´10 4 cells l –1 . in july and august the two centric diatoms were blooming: skeletonema sp. and chaetoceros simplex with 1.57´10 5 cells l –1 and 2.33´10 5 cells l –1 respectively. skeletonema sp. appeared when the temperature and salinity conditions were among the highest registered (30.2 °c and 36.2). in terms of abundance the minute nanoplankton cells were the most conspicuous group throughout the year (fig. 2), contributing up to 91% of the average annual phytoplankton abundance. seasonality in diatoms showed bimodal annual pattern and they were most abundant in summer and autumn; the silicoflagellates appeared in modest abundances in autumn, the coccolithophorids appeared from march to august, while the armoured dinoflagellates were most abundant in may (fig. 3). abundance, cell volume and carbon stock maximum abundances of the small nanoplankters (1.3´10 6 cells l –1 and 1.1´10 6 cells l –1 ) were registered in june and august with a sharp decrease in july, which coincided with the increase of oligotrich ciliate density (results not shown) and could be explained as a acta bot. croat. 68 (1), 2009 37 phytoplankton in stella maris lagoon c a rb o n st o c k ( g c l ) m – 1 0 50 100 150 200 250 300 350 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se p 2004 2005 sampling days fig. 5. seasonal changes in phytoplankton carbon stock c e ll v o lu m e (m m l ) 3 – 1 0 1 2 3 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se p 2004 2005 sampling days fig. 4. seasonal changes in phytoplankton cell volume u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:34 color profile: disabled composite 150 lpi at 45 degrees consequence of the high microzooplankton grazing pressure. all the other taxonomic groups reached the maximum abundances in late spring and summer, with the exception of prymnesiophytes which had their peak in late winter. seasonal dynamics of phytoplankton volume and carbon showed a quite different pattern. in may, an explosive growth of large-sized dinoflagellates occurred and despite their low number (up to 5´10 4 cells l –1 ), they provoked a marked increase in phytoplankton volume, rising up to 2.7 mm 3 l –1 (fig. 4) and consequently in carbon stock, reaching its maximum of 347 mg c l –1 (fig. 5). in other months the carbon content never exceeded 100 mg l –1 . thus, in terms of biovolume and carbon stock, the dinoflagellates were the most prominent group, contributing up to 90% of total carbon stock in may, between 20% and 80% in other months (fig. 6), and with an average annual contribution of 73% (fig. 7). discussion in winter season the global solar irradiance of the area is 15 kj cm–2, while in summer it reaches a fivefold value, of 80 kj cm–2 (fanuko 1986). in the phytoplankton assemblages of the adjacent open sea an inverse fivefold increase in cell chlorophyll content was ob38 acta bot. croat. 68 (1), 2009 fanuko n., val^i] m. 0 10 20 30 40 50 60 70 80 90 100 % o f c a rb o n st o c k others minute nanoplankton dinoflagellates pennatae centricae 0 8 se p 2 5 n o v 1 0 d e c 1 5 m a r 1 9 a p r 1 5 m a y 2 7 m a y 2 ju n 1 5 ju n 1 7 ju l 3 0 ju l 2 a u g 1 3 a u g 1 3 se p 2004 2005 sampling days fig. 6. annual phytoplankton composition given as % of carbon stock centricae 7% pennatae 17% dinoflagellates 74% others 2% fig. 7. average annual contribution of different taxonomic groups to the carbon stock u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:34 color profile: disabled composite 150 lpi at 45 degrees served in winter months. while in july the average monthly concentration of chlorophyll a per cell was 1.1 pg per cell–1, in december it rose to 5.5 pg per cell–1 (fanuko 1986). more numerous and larger chloroplasts in the winter pennate diatoms of the lagoon of stella maris could be the adaptation of this group to the reduced light conditions in winter. the same phenomenon was never observed in other taxonomic groups. the number of phytoplankton species found in the lagoon of stella maris appeared to be high compared with other mediterranean lagoons (tab. 2). a higher number of species was found only in the southern part of the lagoon of venice (tolomio and bullo 2001), in a pool approximately 70 times larger than the stella maris lagoon and within 506 samples taken daily throughout the year. among the 198 taxa found in the adjacent open sea of the gulf of trieste (fanuko 1986), 151 (76%) were also registered in the stella maris lagoon. in two shallow bays of the gulf of trieste a total of 100 species of armoured dinoflagellates were registered (france and mozeti^ 2006) while 212 phytoplankton taxa were reported off-shore in the gulf of venice (bernardi aubry et al. 2006). in the more southern area off rovinj 689 phytoplankton species were found (relevante 1986) whereas 888 species were registered for the whole adriatic sea (vili^i] et al. 2002). in an in situ enrichment experiment, exhibited in the nearby lagoon of strunjan (fanuko 1984), the phytoplankton was significantly altered in terms of reduction of species diversity, cell density and chlorophyll biomass in the experimental lagoon which received settled municipal sewage during the period of two years. similarly, the dystrophic acta bot. croat. 68 (1), 2009 39 phytoplankton in stella maris lagoon tab. 2. phytoplankton diversity in several mediterranean lagoons. 1 this study; 2 fanuko 1980; 3 sarno et al. 1993; 4 andreoli et al. 1989; 5 tolomio et al. 1990; 6 tolomio and bullo 2001; 7 andreoli and tolomio 1988 taxonomic group lagoon s te la m a r is 1 s tr u n ja n 2 s tr u n ja n e x p .2 f u sa r o 3 v a ll e p o z z a ti n i4 v a r a n o 5 v e n ic e la g o o n c h io g g ia a r e a 6 v e n ic e la g o o n v a ll e d o g à 7 cyanobacteria 5 0 0 2 2 0 0 0 cryptophyceae 1 0 0 3 0 0 0 0 chrysophyceae 4 1 0 9 0 1 2 0 prymnesiophyceae 10 9 6 2 2 12 6 0 bacillariophyceae (centrales) 27 28 16 38 10 15 35 7 bacillariophyceae (pennales) 55 47 43 18 37 50 148 96 euglenophyceae 2 1 1 3 1 1 3 2 dinophyceae 43 29 20 31 9 20 42 13 chlorophyceae 4 0 0 4 1 1 0 1 total number of species 151 115 86 110 62 100 236 119 u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:34 color profile: disabled composite 150 lpi at 45 degrees status of the shallow santa giusta lagoon in sardinia (sechi et al. 2001) remained unchanged even after waste water diversion, with blooms of the toxic cochlodinium polykrikoides and chattonella marina, as well as the nitrophile macrobenthic alga ulva rigida, whose massive proliferation in other eutrophic mediterranean lagoons is attributed to industrial, agriculture and domestic wastes introduced in shallow lagoon waters (acri et al. 1995, socal et al. 1999, sfriso et al. 2003, facca et al. 2003). none of these algae has ever been observed in the lagoon of stella maris. the coccolithophorids, predominantly oceanic in distribution (heimdal 1993), are completely absent in some mediterranean lagoons (sarno et al. 1993), but they are common in the stella maris lagoon. during the investigation period, 9 different species of coccolithophorids were found, among which acanthoica aculeata predominated. tychopelagic pennate diatoms, found in the stella maris lagoon, are also a representative and perennial group in the mediterranean (andreoli and tolomio 1988, andreoli et al. 1989, sarno et al. 1993, socal et al. 2006) and some other lagoons worldwide (conde et al.1999, macedo et al. 2001) due to their capacity to support large and highly frequent changes in the physical conditions of the environment (bonilla et al. 2005). nevertheless, as shown in our study as well, other groups are responsible for the phytoplankton peaks: the small-sized picoand nanoplankton cells (vaquer et al. 1996) as primary producers in the microbial loop, centric diatoms, especially chain-forming chaetoceros spp. and skeletonema sp. (socal et al. 1999, bec et al. 2005, socal et al. 2006) and finally dinoflagellates (carrada et al. 1991) which, due to their possible toxicity or other palatability issues, may be subject to relatively low grazing pressure (badylak and phlips 2004). in the northern adriatic, skeletonema has been recently identified as s. marinoi (sarno et al. 2005). the phytoplankton abundance of the stella maris lagoon was comparable to the oligotrophic mar chiquita lagoon in argentina (de marco et al. 2005). compared to the western adriatic lagoons (socal et al. 1999, 2006) and the eutrophic thau lagoon in france (vaquer et al. 1996, bec et al. 2005), values in the stella maris lagoon were lower by two or three orders of magnitude. even when the unialgal blooms occurred (for example skeletonema sp.), their abundances in the stella maris lagoon never resulted in brown tides, as was the case in the industrial area of the lagoon of venice (socal et al. 1999). the seasonal pattern of cell abundance is similar to that of other lagoons of the temperate zone, showing low winter values and summer peaks (facca and de casabianca 2003, facca et al. 2004). the specific volume of diatom cells in the stella maris lagoon was higher than those found in the artificially fertilized fish ponds of the po estuary (andreoli et al. 1989), whereas the volumes of dinoflagellate species were in the same range (tab. 3). neglecting the possible inaccurate microscopy measurements (montagnes et al. 1996) and great variations in cell size (vili^i] 1985), cell volume may give better phytoplankton quantification than abundance. great differences in diatom cell size could be explained by different diatom division rates in different environments. probably the diatoms in the nutrient-rich pond multiplied more rapidly, with more frequent reduction in cell size. as far as the total phytoplankton volumes and the estimated carbon content are concerned, both parameters were still lower in the stella maris lagoon than in mediterranean (sarno et al.1993, andreoli et al. 1989) and atlantic (badylak and phlips 2004, bonilla et al. 2005) lagoons. 40 acta bot. croat. 68 (1), 2009 fanuko n., val^i] m. u:\acta botanica\acta-botan 1-09\fanuko.vp 16. travanj 2009 8:49:34 color profile: disabled composite 150 lpi at 45 degrees presuming that nutrients are not limiting in such a shallow environment and considering the high tidal dynamics, additionally enhanced by winds, low phytoplankton abundances might be the result of low residence time of the water inside the lagoon and its rapid export in the adjacent sea. acknowledgements thanks to giuliano orel and romina zamboni for their assistance in the project and for making laboratory facilities available. the research was supported by the ministry of science, education and sport of croatia, grant no. 0236001. references acri, f., alberighi, l., bastianini, m., bianchi, f., boldrin, a., cavalloni, b., cioce, f., comaschi, a., rabiti, s., socal, t., turchetto, m. m., 1995: variazioni ad alta frequenza dei parametri idrobiologici nella laguna di venezia. atti della società italiana di ecologia 16, 31–34. anderson, a., rudehäll, å., 1993: proportion of plankton biomass in particulate organic carbon in the northern baltic sea. marine ecology progress series 95, 133–139. andreoli, c., tolomio, c., 1988: ciclo annuale del fitoplancton in una valle da pesca della laguna di venezia valle dogà. archivio di oceanografia e limnologia 21, 95–115. andreoli, c., casellato, s. et al. 1989: densità e biomassa del fitoplancton e del macrobenthos in bacini fertilizzati di una valle da pesca del delta del po. atti della società italiana di ecologia 7, 89–99. babi], k., 1911: aspects on biological and bionomical relationships in the adriatic sea (in croatian). tisak dioni~ke tiskare, zagreb. badylak, s., phlips, e. j., 2004: spatial and temporal patterns of phytoplankton composition in a subtropical coastal lagoon, the indian river lagoon, florida, usa. journal of plankton research 2610, 1229–1247. acta bot. croat. 68 (1), 2009 41 phytoplankton in stella maris lagoon tab. 3. average cell volume (in mm 3 ) of some phytoplankton species in two adriatic lagoons. 1 andreoli et al. 1989; 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elementary school, music school (piano) and high school (pharmacy technician). in 1992 she finished the graduate course of pharmacy at the faculty of pharmacy and biochemistry, university of zagreb. she gained her master's degree from the postgraduate school of natural sciences (field: biology – molecular and cell biology) at the faculty of science, university of zagreb in 1997. in 2003, she gained her doctoral degree from the faculty of science, university of zagreb. from 1992 to 2004 she worked as an assistant at the department of pharmaceutical botany at the faculty of pharmacy and biochemistry, university of zagreb, after which she was appointed senior research assistant (2004), assistant professor (2007), associate professor (2011) and full professor (2017) at the same department. she participated in teaching the graduate courses pharmaceutical botany (1992–2017) and cell biology with genetics (1992–1998, 2013) at the faculty of pharmacy and biochemistry, university of zagreb, croatia, and furthermore, the graduate course pharmaceutical botany at the faculty of chemistry and technology, university of split, croatia (2004–2006) and the faculty of pharmacy, university of mostar, bosnia and herzegovina (2012). finally, she taught the graduate course medicinal plants at the faculty of forestry, university of zagreb, croatia (2007–2017). she was author and co-author of two chapters in a university textbook, author of two pharmaceutical botany handbooks and a mentor of 85 graduate students and one doctoral student. kroata’s scientific interests included plant population genetics, molecular phylogeny and systematics, functional genomics, plant biochemistry, morpho-anatomical characteristics of plants, secondary plant metabolites and their biological effects. she was particularly interested in species from the genus hypericum l. during her master’s studies, through intensive field work, under the supervision of professor ivan šugar from the department of pharmaceutical botany, faculty of pharmacy and biochemistry, university of zagreb, she gained a vast knowledge of botany and phytocoenology and expanded her knowledge of morpho-anatomical plant characteristics, plant physiology, secondary metabolites and their biological effects. she received additional scientific training at the department of plant ecology, university of bordeaux i, talence, france during 1995 and 1996, where she perfected her botanical knowledge, application of isoenzymes as molecular markers and methods of plant population genetics. she had further training in plant biology, molecular phylogeny and plant systematics at the division of molecular biology of the ruđer bošković institute in zagreb, where she conducted research for her doctoral thesis. the latter was largely financed by funds from her project for young researchers, funded by the ministry of science, education and sports of the republic of croatia. afterwards, she continued her scientific development in functional genomics and plant biochemistry, working on a project of professor hrvoje fulgosi at the laboratory for electron microscopy of the ruđer bošković institute. she herself was also the head of five projects funded by the university of zagreb and one project funded by the foundation of the croatian academy of sciences and arts, and was an associate on five projects funded by the ministry of science, education and sports of the republic of croatia, as well as on the croatianfrench scientific program of integral actions called cogito. she published 36 scientific papers and participated with oral and/or poster presentations in about 40 scientific meetings and was a member of the organisation committee of the 1st croatian congress on molecular life sciences. she was also a co-author of one professional book and author and coauthor of several professional papers and popular science articles. moreover, she gave many professional lectures and workshops and was the creator of two interactive exhibitions that popularize science. she was a member (1992–2017) and a vice president (2001–2008) of the croatian society of plant biology, as well as a member of the federation of european societies of plant biology (1992–2017), croatian genetic society (1995– 2017), iris croatica society (2004–2017) and croatian botanical society (2009–2017). she was also a member of the recognition of foreign professional qualifications commission at the croatian chamber of pharmacists (2015–2017). at the faculty of pharmacy and biochemistry she held the following positions: member of the education committee s4 acta bot. croat. 77 (1), 2018 (2010–2014), president of the student papers and awards committee (2015–2016), member of the awards and honours committee (2016) and member of the student papers and awards committee (2016–2017). having almost 25 years of teaching experience, kroata was one of the most beloved teachers at the faculty of pharmacy and biochemistry, valued and respected by her students, with whom she nurtured a caring relationship. most of the students knew her as an excellent professor, full of love and enthusiasm for the plant world, one who enjoyed teaching and knew how to spark an interest in those who were not plant lovers in the beginning. in teaching, she tended to be always positive and full of understanding and was very respectful to all living beings; plants, animals and people alike. the recognition and appreciation of her work as an educator by students and colleagues were also confirmed through the awards she received: the highest grade for an assistant in the academic year 2005/2006 (according to the university student survey), and the award of the faculty of pharmacy and biochemistry, university of zagreb for exceptional achievements in student education in the academic year 2013/2014. apart from genuinely enjoying working with students, kroata had a great curiosity for science as well. however, she tended to be more cautious and realistic in her research. her colleagues knew her as a hard-working, persistent, responsible and conscientious person, who was also unpretentious and modest. she was a patient and encouraging mentor, always ready to help and give useful advice, not just in terms of work and experimental planning, but also in terms of human relations and life in general. i thought of her as a truly warm, motivating, creative and honest person. i am so grateful that, as my phd mentor, she was open to new ideas and was always ready to encourage my independence in work, while at the same time being very supportive in all my activities, including going to education events, experiencing international stays, applying for projects or competing for awards. more importantly, she was a loving wife and caring mother of two children, of whose achievements, abilities and sense of responsibility she was very proud. i would also like to mention and appreciate some of the small things that made her happy, like baking cakes, gardening, traveling, sailing, ballooning, dancing, enjoying pleasant aromas and finding pleasure in small aesthetic details. we spent a lot of time together, frequently collaborating on a daily basis and occasionally presenting research at congresses or engaging in fieldwork. i had the privilege of learning from her as both her graduate and postgraduate student, through talking and discussing, as well as through sharing new ideas for the future. we were both looking forward to the continuation of our co-operation, both in science and in class, and planning new projects for the years to come. although she passed away so suddenly, with years of her greatest scientific achievements, creations and personal challenges still before her, leaving us all in pain and disbelief, she has undoubtedly left an indelible mark on all who knew her. while her scientific work will certainly be continued, her contributions to the education of thousands of students and to community in general encourage us to be better people and the best possible in everything we do, until the day we see her again, picking her favourite flowers in the eternal fields. maja friščić, phd department of pharmaceutical botany, faculty of pharmacy and biochemistry, university of zagreb, schrottova 39, 10 000 zagreb, croatia prof. kroata hazler pilepić (right) and maja friščić, phd (left), during fieldwork in 2012. the photo was taken by kroata’s husband, associate professor viktor pilepić from the department of physical chemistry, faculty of pharmacy and biochemistry, university of zagreb. acta bot. croat. 77 (1), 2018 s5 list of publications šugar, i., brkić, d., hazler, k., 1994: medvednica i njezin biljni svijet. ekološki glasnik 4, 9–10. vladimir-knežević, s., kalođera, z., pepeljnjak, s., blažević, n., hazler, k., 1994: activity of essential oil and ethanolic extract isolated from micromeria thymifolia (scop.) fritsch against dermatophytes. periodicum biologorum 96, 383–385. hazler, k., 1997: genetička struktura bukovih populacija (fagus sylvatica l.) s područja jugoistočne europe. prirodoslovnomatematički fakultet sveučilišta u zagrebu, zagreb, master’s thesis. hazler, k., comps, b., šugar, i., melovski, l., tashev, a., gračan, j., 1997: genetic structure of fagus silvatica l.: populations in southeastern europe. silvae genetica 46, 229–236. hazler pilepić, k., comps, b., šugar, i., šolić, e., 1999: genetic variability of the croatian beechwoods. periodicum biologorum 101, 165–170. maleš, ž., plazibat, m., hazler pilepić, k., cetina čižmek, b., 2001: istraživanje aminokiselinskog sastava drače (paliurus spinachristi mill.). farmaceutski glasnik 57, 257–265. šugar, i., brkić, d., ružanović, z., partl, a., hazler pilepić, k., 2001: stručna podloga za izradu pravilnika o korištenju i zaštiti samoniklih farmaceutski i prehrambeno iskoristivih biljnih vrsta. ministarstvo zaštite okoliša i prostornog uređenja, expertise. hazler pilepić, k., 2002: analysis of chloroplast dna variability among some hypericum species in croatia. periodicum biologorum 104, 457–462. maleš, ž., plazibat, m., hazler pilepić, k., bilušić, v., 2002: kvalitativna analiza aminokiselina mekolisne majčine dušice – thymus bracteosus vis. ex benth. farmaceutski glasnik 58, 155–160. šugar, i., gostl, i., hazler pilepić, k., 2002: hrvatsko biljno nazivlje: analiza hrvatskog biljnog nazivlja u djelu liber de simplicibus benedicti rinij, hrvatska sveučilišna naklada, zagreb. hazler pilepić, k., 2003: populacijsko-genetička struktura vrste hypericum perforatum l. (guttiferae) s područja hrvatske. prirodoslovno-matematički fakultet sveučilišta u zagrebu, zagreb, phd thesis. maleš, ž., plazibat, m., bilušić vundać, v., žuntar, i., hazler pilepić, k., 2004: thin-layer chromatographic analysis of flavonoids, phenolic acids and amino acids in some croatian hypericum taxa. journal of planar chromatography – modern tlc 17, 280–285. jurić, s., lepeduš, h., hazler-pilepić, k., jeličić, b., fulgosi, h., 2006: trol integrates chloroplast redox signalling, 46–47. slovenian society for plant physiology, ljubljana. maleš, ž., brantner, a., sović, k., hazler pilepić, k., plazibat, m., 2006: comparative phytochemical and antimicrobial investigations of hypericum perforatum l. subsp. perforatum and h. perforatum subsp. angustifolium (dc.) gaudin. acta pharmaceutica 56, 359–367. fulgosi, h., hazler pilepić, k., 2007: izdvajanje kloroplastne dna ekstrakcijom fenolom. in: ambriović ristov, a., brozović, a., bruvo mađarić, b., ćetković, h., herak bosnar, m., hranilović, d., katušić hećimović, s., meštrović radan, n., mihaljević, s., slade, n., vujaklija, d. (eds.), metode u molekularnoj biologiji, 161–163. institut ruđer bošković, zagreb. hazler pilepić, k., 2007: izdvajanje genomske dna iz biljnog tkiva. in: ambriović ristov, a., brozović, a., bruvo mađarić, b., ćetković, h., herak bosnar, m., hranilović, d., katušić hećimović, s., meštrović radan, n., mihaljević, s., slade, n., vujaklija, d. (eds.), metode u molekularnoj biologiji, 163– 165. institut ruđer bošković, zagreb. fulgosi, h., jurić, s., lepeduš, h., hazler-pilepić, k., prebeg, t., ljubešić, n., 2008: thylakoid system disassembly during bleaching of aurea mutant of maple acer negundo hassk. var. odessanum. croatica chemica acta 81, 89–95. hazler pilepić, k., maleš, ž., plazibat, m., 2008: genetic structure in hypericum perforatum l. population. periodicum biologorum 110, 361–365. maleš, ž., babac, m., hazler pilepić, k., zovko, m., 2008: kvantitativna analiza polifenola u nekim vrstama roda teucrium l. farmaceutski glasnik 64, 169–177. jurić, s., hazler-pilepić, k., tomašić, a., lepeduš, h., jeličić, b., puthiyaveetil, s., bionda, t., vojta, l., allen, j. f., schleiff, e., fulgosi, h., 2009: tethering of ferredoxin:nadp+ oxidoreductase to thylakoid membranes is mediated by novel chloroplast protein trol. plant journal 60, 783–794. maleš, ž., hazler pilepić, k., bojić, m., jebrini, s., 2009: određivanje količine 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human glioblastoma a1235 and breast cancer mda mb-231 cells. journal of environmental science and health, part a 51, 1157–1163. hazler pilepić, k., friščić, m., 2017: skriveni svijet ljekovitih biljaka, izložba. farmaceutski glasnik 73, 465–467. friščić, m., maslo, s., garić, r., maleš, ž., hazler pilepić, k., 2018: comparative analysis of specialized metabolites and antioxidant capacity in vitro of different natural populations of globularia spp. acta botanica croatica 77, 1–9. 172 acta bot. croat. 77 (2), 2018 acta bot. croat. 77 (2), 172–180, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0019 issn 0365-0588 eissn 1847-8476 genetic variability and distance between lactuca serriola l. populations from sweden and slovenia assessed by ssr and aflp markers michaela jemelková1, miloslav kitner1, eva křístková1, ivana doležalová2, aleš lebeda1* 1 palacký university in olomouc, faculty of science, department of botany, šlechtitelů 27, 783 71 olomouc, czech republic 2 department of genetic resources for vegetables, medicinal, and special plants of crop research institute in olomouc, šlechtitelů 29, 783 71 olomouc, czech republic abstract – the study involved 121 samples of the common weed, lactuca serriola l. (prickly lettuce), representing 53 populations from sweden and slovenia. the seed materials, originating from different habitats, were regenerated and taxonomically validated at the department of botany, palacký university in olomouc, czech republic. the morphological characterizations of the collected plant materials classified all 121 samples as l. serriola f. serriola; one sample was heterogeneous, and also present was l. serriola f. integrifolia. differences in the amount and distribution of the genetic variations between the two regions were analyzed using 257 amplified fragment length polymorphism (aflp) and 7 microsatellite (ssrs) markers. bayesian clustering and neighbor-network were used for visualization of the differences among the samples by country. under the bayesian approach, the best partitioning (according to the most frequent signals) was resolved into three groups. while the absence of an admixture or low admixture was detected in the slovenian samples, and the majority of the swedish samples, a significant admixture was detected in the profiles of five swedish samples collected near malmö, which bore unique morphological features of their rosette leaves. the neighbor-network analysis divided the samples into 6 groups, each consisting of samples coming from a particular country. reflection of morphology and eco-geographical conditions in genetic variation are also discussed. key words: biogeography, dinaric alps and the pannonian plain, dna polymorphism, ecology, habitats, morphological variation, prickly lettuce, scandinavia * corresponding author, e-mail: ales.lebeda@upol.cz introduction prickly lettuce (lactuca serriola l., asteraceae) is the most common species in the genus lactuca l. (feráková 1977), and has a circumglobal distribution (lebeda et al. 2004). it is an annual or winter-annual therophyte (feráková 1977), and an ‘r’strategist (tilman 1988). its evolution has trended towards a short life cycle, strong self-fertilization ability, good adaptation for wind dispersal, and quick germination (frietema de vries 1992, lebeda et al. 2001). l. serriola is a drought-tolerant species (werk and ehleringer 1986), mainly growing in sunny microhabitats within anthropogenic habitats such as roadsides, railways, dumps, and urban areas (feráková 1977, lebeda et al. 2001, 2004); it is considered a good colonist of a wide spectrum of different habitats with different degrees of invasivity. prickly lettuce is of euro-asian origin, also being native in north africa (feráková 1977). it has primarily spread in the mediterranean and the near east (de vries 1997, lebeda et al. 2007a), and is considered an archaeophyte dependent on a culture from the northern part of central europe (meusel and jäger 1992). the species belongs to a group of mediterranean ruderal plants that have enlarged their distribution area during the last few centuries (landolt 2001). the northern boundary of the european distribution area runs near latitude 65 °n through finland, and 55 °n through great britain (feráková 1977). the expanding distribution of this species is accomplished by the transport of reproductive propagules, achenes. the ripened achenes with attached pappus are primarily dispersed by the wind, probgenetic variability of lactuca serriola populations acta bot. croat. 77 (2), 2018 173 ably also by water (weaver and downs 2003). the spread of this species is also closely related with human activities, which primarily produce an increase in their transport (lebeda et al. 2001). prickly lettuce has drastically increased its geographical range, invading many european, (north-) american, and australian regions during the last 50-60 years (de vries 1996, lebeda et al. 2001, 2004); recently l. serriola has spread as an invasive weed throughout europe (lebeda et al. 2004, 2007b, d’andrea et al. 2009), including scandinavia (rydberg 2013). its synanthropic distribution has also been recorded from australia, including tasmania and new zealand (burbinge and gray 1970, webb et al. 1988), as well as taiwan (wang and chen 2010), north america, southern africa, and argentina (strausbaugh and core 1978, zohary 1991, zuloaga and morrone 1999). the study by alexander (2010) supported a genetic basis for the differences in the elevation limits of l. serriola populations between two parts of its native and introduced ranges. two primary morphological forms are recognized within l. serriola l. based on cauline leaf-shape variability; the pinnatifid-leaved form l. serriola l. f. serriola, and the unlobed-leaved form l. serriola l. f. integrifolia (s.f. gray) s.d. prince et r. n. carter. the serriola form is recorded as the most frequent species, occurring at a very high density in europe; the form integrifolia is not so common, and has been recorded in e.g., switzerland, italy, france, western germany, the netherlands, and is prevalent in the uk (lebeda et al. 2001, 2004, 2007a, b). lactuca serriola is the best known wild species of the genus lactuca, the geographic distribution, morphological, and phenological variations of which have been intensively studied (lebeda et al. 2004, 2007a, alexander 2010). l. serriola is also an important genetic resource for new resistance to diseases and pests (lebeda et al. 2014), abiotic factors, as well as for genes responsible for physiological and quality characters (lebeda et al. 2007a). prickly lettuce has been used in commercial lettuce breeding for more than 80 years (lebeda et al. 2007a), especially as a source of race-specific resistance genes against lettuce downy mildew (bremia lactucae regel) (parra et al. 2016). it has also been used over the last decade in various molecular studies to characterize genetic variation and diversity in both germplasm collections and natural populations (e.g. koopman et al. 2001, kitner et al. 2008, 2015). the most commonly used methods for the analysis of dna polymorphism include amplified fragment length polymorphism (aflp; vos et al. 1995), and microsatellites (simple sequence repeats, ssrs); simko (2009) contributed significantly to the development of these for the genus lactuca, and in particular for l. serriola riar et al. (2011). these markers have been successfully applied in lactuca research, addressing e.g., the distribution of the genetic variation of prickly lettuce across europe (lebeda et al. 2009a), distribution of genetic variation in natural populations of l. serriola, l. saligna, and l. aculeata in israel (kitner et al. 2015), or analyses of gene flow from crops to their wild relatives (uwimana et al. 2012). southern / central sweden is the northern limit of l. serriola distribution in europe; slovenia represents an area between the central european and mediterranean / balkan distributions (feráková 1977). the two areas differ in their climatic, ecogeographic, and ecologic conditions. in slovenia, prickly lettuce is distributed throughout the entire territory, from the lowlands to the mountain regions (martinčič and sušnik 1984), and it most often grows in association with stellarietea mediae – annual weed communities species (šilc and košir 2006). in sweden, l. serriola populations are found in southeastern areas, and mostly grow on surfaces and among stones in dry and sunny exposures (doležalová et al. 2001). the genetic structure of populations represented by prickly lettuce plants growing at a specific time in a particular site could emerge in at least four different ways: i) achenes can survive in a soil seed bank for 1 to 3 years (marks and prince 1982); at the moment of soil disturbance, the seeds can germinate, and these plants bear/represent “old” genotypes for a given population; ii) plants can grow from achenes newly transported to a particular locality by wind, humans, or other transport mechanisms, with such plants bearing “new” genotypes; iii) plants can grow on permanently disturbed soil from generation to generation, and such plants represent a “modified” genotype resulting from continuous evolution under local conditions; iv) “hybrid” plants may appear after natural hybridization between different plant species within the genus lactuca. the main purpose of this research was to describe the differences in genetic variability and population genetic structures between populations of prickly lettuce (lactuca serriola) coming from two different and distant biogeographic areas of the species’ distribution in europe. materials and methods plant materials a set of 121 samples of l. serriola l. plants, representing 53 populations, was collected by the authors in sweden (47 samples) and slovenia (74 samples) during 2000 (doležalová et al. 2001). the collected seed samples were regenerated in a greenhouse at the department of botany (palacký university in olomouc, czech republic). during regeneration, the plants were described morphologically according to doležalová et al. (2002), and the taxonomic status of each sample was verified (feráková 1977, doležalová et al. 2002). from each plant two mature leaves were used for dna extraction (i.e., 121 samples). data from the individual samples are provided in on-line suppl. tab. 1., with the geographic positions of the collection sites given in fig. 1. dna extraction, ssr, and aflp analyses total genomic dna was extracted from 100 mg of fresh leaf tissue using the ctab method (kump and javornik 1996), with minor modifications. after dna extraction, the jemelková m., kitner m., křístková e., doležalová i., lebeda a. 174 acta bot. croat. 77 (2), 2018 quality of the dna was inspected by 1.5% agarose gel electrophoresis, and the concentration measured on a nanodrop nd-1000 spectrophotometer (nanodrop technologies, delaware, usa). for microsatellite genotyping, seven ssr loci were used: sml-002, sml-019, sml-045, sml-055 (simko 2009), as well as wsuls-18, wsuls-75, and wsuls-163 (riar et al. 2011). the primer pairs were selected according to their high diversity indices in previously published papers (simko 2009, riar et al. 2011); however, randomly without any previous knowledge of their chromosome positions. amplification of the ssrs was performed according to jemelková et al. (2015). the length of the ssr alleles was scored based on their migration relative to the molecular weight size markers 30-330bp aflp® dna ladder (invitrogen, carlsbad, california, usa). the aflp analyses were carried out according to the protocol of vos et al. (1995), with modifications, and the aflp fragment detection according to kitner et al. (2008, 2012). five selective primer combinations, with two to three selective nucleotides, were chosen to generate the aflp profiles (tab. 2). the pcr products were separating on a 6%, 0.4 mm thick denaturating polyacrylamide gel using a t-rex sequencing gel electrophoresis apparatus (thermo scientific owl separation systems, rochester, ny, usa). tab. 1. microsatellite (ssr) loci used to assess genetic variability in lactuca sativa l. and l. serriola l.; na – number of alleles; pic – allelic polymorphic information content. marker reference na allele size (bp) pic (%) sml-002 simko (2009) 6 168-207 0.594 sml-019 simko (2009) 2 163-164 0.599 sml-045 simko (2009) 4 229-238 0.838 sml-055 simko (2009) 5 221-240 1.072 wsuls-18 riar et al. (2011) 4 208-235 0.494 wsuls-75 riar et al. (2011) 4 161-206 0.684 wsuls-163 riar et al. (2011) 7 183-197 1.052 fig. 1. collecting sites of the 121 samples lactuca serriola in sweden and slovenia. colors of spots correspond to the results of bayesian clustering presented in fig. 2. tab. 2. amplified fragment length polymorphism (aflp) primer sets for amplification reactions with the total number of scored and polymorphic fragments in the lactuca serriola samples; nf – total number of fragments; npol – number of polymorphic fragments; p(%) – percentage of polymorphic fragments. primer combination nf npol p(%) e agc, m ctg 45 37 82.2 e agc, m caac 49 36 73.5 e agc, m caat 72 54 75.0 e acc, m caac 43 35 81.4 e acc, m caat 48 30 62.5 total 257 192 mean 51.4 38.4 74.9 data scoring microsatellite profiles were scored based on the length of the pcr product. the allele frequencies, percentage of polymorphic loci (p%), number of private alleles (pa), observed and expected heterozygosity (ho and he) were all performed using genalex 6 software (peakall and smouse 2012). the mean number of alleles per locus (a) was calculated manually. the relative discriminatory value of each microsatellite locus was estimated by the polymorphic information content (pic), which measures the information content as a function of a marker system´s ability to distinguish between genotypes (powell et al. 1996). the number of different genotypes (ng), number of samples with a heterozygous constitution (nhet), and maximal number of heterozygous loci (nhetmax) were calculated manually. aflp profiles were checked visually, and only clear and unambiguous bands were scored for their presence (1) or absence (0) across all samples. for aflp data, the number of private bands (pa), the proportion of polymorphic loci (p%) and gene diversity (he) were calculated using genalex 6 software (peakall and smouse 2012). to evaluate the population genetic structure, a bayesian clustering approach was used as implemented in structure genetic variability of lactuca serriola populations acta bot. croat. 77 (2), 2018 175 2.3.4 (falush et al. 2007). structure attempts to assign individuals to clusters/groups/populations on the basis of their genotypes, while simultaneously estimating population allele frequencies. this allows one to compute the likelihood of a given genotype having originated in a predefined number (k) of clusters. in the simplest, ‘no-admixture’ model, it assumes that each individual belongs to a single cluster. in the more general ‘admixture model’ it estimates admixture proportions for each individual, allowing one to identify admixed individuals represented by a proportional mixture of two or more signals characteristic for the various clusters. in our analyses, ssr co-dominant data were transferred into binary data based on the presence/absence of a particular allele, and merged with the aflp binary data; the samples were then ordered according to the increasing latitude of the sampling site within a particular country. an admixture model was used, with correlated allele frequencies. k was set at 1–10, and the highest k value was identified as the run with the highest likelihood value, as recommended by pritchard et al. (2000). in addition, k values were averaged across 10 replicate runs for each k (100 000 burn-in iteration followed by 1 000 000 mcmc iterations). for the graphical interpretation of clustering for the appropriate k, structure harvester (earl and von holdt 2012), clumpp (jacobsson and rosenberg 2007), and distruct (rosenberg 2004) software packages were used. the optimal k value was selected according to evanno et al. (2005), who suggested the use of the ∆k value for identifying the correct number of clusters. to visualize the genetic relationships within and among the analyzed samples, a neighbor-network based on dice´s similarity coefficient (d) was constructed in splitstree 4 (huson and bryant 2006). the nexus input file for splitstree 4 was exported from genalex. also, for this purpose, the ssr data were transformed into a binary matrix and merged with the aflp binary data. the reliability and robustness of the network were tested by bootstrap analysis with 1.000 bootstrap replicates. results taxonomic verification of l. serriola for all 121 plants, the taxonomic status of lactuca serriola f. serriola according to feráková (1977) was confirmed. moreover, in one sample (no. 205_00, bostahusen, sweden) the plants were morphologically heterogeneous; with divided stem leaves belonging to l. serriola f. serriola, plants with entire stem leaves that ranged toward l. serriola f. integrifolia. in our analyses, this sample was split into two subsamples 205_00a (f. serriola) and 205_00b (f. integrifolia) and treated (analyzed) separately. genetic polymorphism the seven polymorphic ssr loci produced a total of 32 alleles across the 121 individual l. serriola plants. the number of alleles per locus ranged from 2 to 7, with an average of 4.57 alleles per locus (tab. 1). the allele sizes varied from 161 to 240 bp. the mean pic per ssr polymorphic allele was 0.762, within a range of 0.494 to 1.072. null alleles only appeared in two accessions from slovenia (13_00 and 22_00) at the locus sml-055. private alleles (pa) were present within both sampled regions (tab. 3). the l. serriola samples from sweden possessed 5 unique alleles: 193 bp, 204 bp, and 207 bp for locus sml-002, 221 bp for locus sml-055 (i.e., 221 bpsml-055), and 188 bpwsuls-163. the samples from slovenia possessed eight unique alleles: 172 bp, 198 bp for locus sml-002, 238 bpsml-045, 228 bpsml-055; 217 bp and 235 bp for locus wsuls-18, and lastly 183 bp and 195 bp for locus wsuls-163. the observed and expected heterozygosity (ho and he) ranged from 0.036 to 0.054 (mean 0.045), and from 0.341 to 0.432 (mean 0.387), respectively. the proportion of polymorphic loci (p%) was higher in the slovenian (84.4%) than in the swedish samples (75%). based on ssr data, in all, 51 different genotypes (ng) were recognized (sweden = 17; slovenia = 34) (on-line suppl. tabs. 2,3). genotype g3 was the most common in the samples from sweden (36.2%), while genotype g29 represented 32.4% of the slovenian samples (on-line suppl. tabs. 2,3). we recorded 17 slovenian samples that had at least one heterozygous locus (nhet = 17), in contrast to eight samples from sweden (on-line suppl. tabs. 2,3). three samples from slovenia and one sample from sweden bore the maximum number of heterozygous loci (nhetmax = 3) observed from among all analyzed samples. in total, five primer combinations, with two to three selective bases, were applied for aflp genotyping (tab. 2), resulting in 257 unambiguously scored fragments. detailed overall statistics calculated for each primer combination used are presented in table 2. the number of private bands (pa) ranged from 19 (slovenian samples) to 20 (swedish samples). the expected heterozygosity (he) ranged from 0.130 to 0.149 (mean he = 0.140) (tab. 3), and the proportion of polymorphic loci (p%) in the l. serriola samples ranged from 44.8% (swedish population) to 52.9% (slovenian population). the genetic variability indices for all populations are summarized in table 3. cluster analysis of molecular data based on seven microsatellite and 257 aflp markers, bayesian clustering and construction of a neighbor-network were used for visualization of the putative relationships among the analyzed individuals. under the bayesian approach implemented in structure, the best partition into three clusters (k = 3, fig. 2) was resolved (∆k = 214.73; st. dev. lnp(k) = 6.07); they are represented by the green (gcluster), red (r-cluster), and blue (b-cluster) color signals in figure 2. in general, a relatively low admixture was detected in the slovenian samples, which were clearly identified as genotypes from the gor b-cluster. while the b-cluster can be considered as characteristic for l. serriola genotypes jemelková m., kitner m., křístková e., doležalová i., lebeda a. 176 acta bot. croat. 77 (2), 2018 from the southern part of central europe and the northern balkans (representing ca. 1/3 of the slovenian samples), the g-cluster represents the genotype largely dispersed across europe, contributing significantly to the genotypic composition of the swedish populations. the signal characteristic for genotypes from the r-cluster was nearly absent in the slovenian samples, but was recorded in each sample from sweden; and 48.9% of the swedish samples fell into the rcluster with no admixture signal (fig. 2). for 19 samples, the signal from the r-cluster contributes up to 30% of a particular genotype, and is accompanied with an admixture of the g signal, which prevails in the slovenian samples (fig. 2). further, we observed a nearly equal admixture of signals from all three clusters in five samples collected in southern sweden near malmö. the neighbor-network analysis divided the analyzed samples into 6 groups (a-f; fig. 3), each consisting of samples coming from a separate country. the results fit the results of the bayesian clustering in terms of assigning individuals from a separate country to the revealed clusters (r-, g-, b-). the samples from sweden were placed into the a, c, and d groups. while individuals placed in group c represent the genotype from the r-cluster, group d is formed by samples with the g-cluster prevailing. finally, group a is formed by five samples 215_00, 217_00, 218_00, 219_00, and 220_00, having a strong admixture signal from all three structure clusters. these samples represent populations no. 16 and 17 from collecting sites close to malmö (on-line suppl. tab. 1). the samples from slovenia were split into three groups: a majority of the samples fell in groups b and e, both representing the g-cluster in fig. 2. samples originating from slovenian localities below 46°14'34" lat. fell into a separate group f, which represents genotypes from a unique b-cluster (fig. 2). it is interesting, that all three fig. 2. results of bayesian clustering based on the microsatellite (ssr) and amplified fragment length polymorphism (aflp) data of 121 lactuca serriola samples from sweden (swe) and slovenia (slo), ordered according to the increasing latitude of the sampling site within a specific country. each individual is represented by a horizontal line partitioned into segments of different color, the lengths of which indicate the posterior probability of membership in each group as identified by structure. fig. 3. neighbor-network cluster analysis of 121 samples lactuca serriola from sweden and slovenia, based on ssr and aflp analysis. resulting groups are highlighted by coloring that corresponds to the results of bayesian clustering presented in fig. 2. genetic variability of lactuca serriola populations acta bot. croat. 77 (2), 2018 177 “g-cluster” groups from both countries are in the center of the neighbor-network, which resemble their characteristics closely. on the other hand, group c (swe, r-cluster) and group f (slo, b-cluster) are placed on opposite sides of the network. discussion verification of the taxonomic status of the plants showed that lactuca serriola f. serriola is predominant in both countries. in the entire territory of slovenia only l. serriola f. serriola was recorded, which is in agreement with previous observations in central europe (lebeda et al. 2001, 2004, 2007b). within one sample from southern sweden (bostahusen, sample 205_00), apart from l. serriola f. serriola plants, there were plants identified as l. serriola f. integrifolia. all remaining samples from sweden were represented only by l. serriola f. serriola. it is evident that both populations are very taxonomically homogeneous on the subspecific level. the very rare occurrence of l. serriola f. integrifolia in southern sweden could be caused by the repeated introduction (e.g., through truck or ship transportation) of this form from the netherlands or uk, where it is prevalent (lebeda et al. 2007a, b). however, from our previous results (doležalová et al. 2001) it is evident, that this variety is not spreading into northern scandinavia, where the northern limit of the european distribution for this species is (feráková 1977). these conclusions are supported by recent observations in sweden made by rydberg (2013). also, in norway only l. serriola f. serriola has been recorded (lebeda 2013, unpubl. results). the leaf shape (i.e., the division of the leaf blade), can be interpreted as an ecological adaptation of the plant to different factors, including a means of leaf thermoregulation in arid or hot environments, or in reaction to hydraulic constraints (nicotra et al. 2011). doležalová et al. (2009) also confirmed the differences in the morphology of rosette and cauline leaves of swedish and slovenian l. serriola samples. the cauline leaves of swedish l. serriola plants were longer and wider; plants from slovenia had longer and narrower rosette leaves (divided) (doležalová et al. 2009). the width and length of cauline leaves (divided) correlate with the latitude, which could be explained as adaptations of the plants to drought. drier areas of lower latitudes are increasingly represented by plants with smaller leaves. regarding altitude, a negative correlation with the length and width of the leaves was found (doležalová et al. 2009), which could mean they are adapting to ecologically worse conditions at higher elevations. the occurrence of l. serriola f. integrifolia in temperate areas without a dry season (but with a warm summer) in the uk, western part of germany, benelux, and france (peel et al. 2007) supports the theory of the ecological adaptation of leaves presented by nicotra et al. (2011). the areas in sweden where lettuce samples were collected belong to the cold climate type, without a dry season or warm summer (peel et al. 2007). similarly significant differences in morphological parameters of achenes of l. serriola from slovenia and sweden were found between populations within countries and between samples within population (křístková et al. 2014). the higher phenotypic and genetic variability of the slovenian samples can be explained by the more favorable climatic and ecological conditions in the country (see peel et al. 2007). l. serriola is distributed throughout the entire country, and movement of diaspores among the surrounding countries is feasible (lebeda et al. 2004). this is in opposition to sweden, where the distribution is limited to the southern part (doležalová et al. 2001), with very limited migration from the surrounding countries. in general, plant species occurring almost in and/or near the center of their diversity, with suitable environmental and ecological conditions, display more genetic/phenotypic variability. conversely, at the edge of the distribution area, where less favorable conditions exist, the selection prioritizes stable and well-adapted genotypes. our results on genetic variability are in relationship to the general principles of diversity and allele distribution formulated by vavilov (1950). kuang et al. (2008) suggested that eastern turkey and armenia, along with the surrounding regions, might be the center of diversity of l. serriola (and possibly its center of origin). l. serriola might have spread from its center of origin first to the mediterranean basin and then to central and western europe after the glaciers retreated in the upper-pleistocene / holocene period (kuang et al. 2008). recent climatic changes and anthropogenic disturbances contributed substantially to the rapid spread of l. serriola into new areas (d´andrea et al. 2009, rydberg 2013), as well as increasing the genetic diversity of their populations in the central parts of their natural distribution areas (lebeda et al. 2009a, van de wiel et al. 2010, kitner et al. 2015). this phenomenon tab. 3. summary data based on 7 ssr and 257 amplified fragment length polymorphism (aflp) loci of 121 lactuca serriola samples from sweden and slovenia in recent study: n – sample size; passr – private microsatellite alleles; paaflp – private aflp bands; a – mean number of alleles per locus; p(%) – percentage of polymorphic loci; observed ho and expected he heterozygosity; se – standard error. country n microsatellite (ssr) data aflp data passr a p(%) ho he ± se paaflp p(%) he ±se sweden 47 5 3.42 75.0 0.036 0.341 ± 0.065 20 44.8 0.130 ± 0.011 slovenia 74 8 3.86 84.4 0.054 0.432 ± 0.049 19 52.9 0.149 ± 0.011 jemelková m., kitner m., křístková e., doležalová i., lebeda a. 178 acta bot. croat. 77 (2), 2018 was also clearly demonstrated in the genetic diversity of the central european population of l. serriola (van de wiel et al. 2010), as well as the resistance of the same population to bremia lactucae. while the czech republic has the greatest diversity of resistance phenotypes, the lowest was recorded in the uk (lebeda et al. 2008, petrželová and lebeda 2011). the results of our study on genetic variability are in good agreement with the different climatic conditions in sweden and slovenia. from the viewpoint of genetic variation, the results have proven the existence of l. serriola genotypes characteristic for each country. these clearly differ from one another, as is evident from bayesian clustering and neighbor-network analysis, where the r-cluster characteristic for the swedish samples (group c), and the b-cluster (group f) unique for slovenian samples were distinguished (figs. 2, 3). a number of samples from both countries were characterized by genotypes characteristic for the g-cluster, which might represent a common genotype resulting from the rapid spread of l. serriola in central europe (lebeda et al. 2001, 2007b, d´andrea et al. 2009). we have not recorded a prevailing microsatellite genotype for the samples representing this g-cluster, and no linkage to the latitude or altitude of the sampled sites. the same phenomenon was described by lebeda et al. (2009a), demonstrating that some l. serriola populations (e.g., scandinavian, british, some mediterranean) are quite isolated genetically from the heterogeneous central and west european populations. genetic analysis (pcr-rflp and ssr markers) on 101 populations of l. serriola from seventeen countries of western and central europe made by d´andrea et al. (2017) revealed a strong genetic differentiation between populations, and high inbreeding coefficients within populations. a clear geographical pattern of isolation by increasing distance was found; however, only a weak pattern of correlation between genetic diversity and geographical distance was found on the continental scale. the greatest amount of genetic diversity was characterized in central europe, while populations from the western mediterranean (spain and portugal), southern italy, great britain, the alps, and southern scandinavia generally possessed lower gene diversities (d´andrea et al. 2017). discrepancies were present in scandinavia with some polymorphic populations, and a monomorphic one. further, in a recent study, higher genetic variability in the slovenian samples was observed in terms of the recorded genetic variability indices (tab. 3) and the higher number of ssr genotypes (sweng = 17/slong = 34) (on-line suppl. tabs. 2,3). the level of genetic variation within and between populations can also result from intraspecific crossing. although autogamy is the predominant breeding system within the genus lactuca l., especially in the marginal parts of the distribution area (feráková 1977); in the center of the distribution, a higher occurrence of allogamy was estimated (stebbins 1957). lindquist (1960) proved experimentally that all species belonging to the “serriola” group were self-fertile. l. serriola is primarily a self-pollinated species; however, not only intermediate plants between the two l. serriola forms, but also interspecific hybrids of l. serriola can be detected in natural populations (zohary 1990, křístková et al. 2012). the main differences between the samples from sweden and slovenia can be characterized by the presence of genotypes characteristic for the ror b-cluster, determined by bayesian clustering (fig. 2), each unique (with a few exceptions) to a given country. the signal from the r-cluster was present in all swedish samples and prevails in 48.9% of them. these samples formed group c on the neighbornetwork (fig. 3), 65.2% of them represent the ssr genotype g3, with a completely homozygous character at all loci, and originating from localities at a higher latitude (on-line suppl. tab. 2). a rather interesting characteristic of five l. serriola samples was found in a group of plants collected near malmö. these samples forming group a on the neighbor-network, are represented by a significant admixture signal on the bayesian diagram, and also bore unique morphological features of their rosette leaves. the apical parts of the rosette leaves in samples 215_00, 217_00, 218_00, 219_00, and 220_00 were not divided, forming a long apex; the remaining two-thirds of the leaves were slightly divided (pinnately lobed). surprisingly, specific dna patterns fit better to specific phenotypes of the rosette leaves than to phenotypes of the cauline leaves. this is in contrast to the generally accepted view that morphological traits of the cauline leaves have a more significant taxonomic value than do the rosette leaves. the city of malmö is an international harbor in the region, and it is possible to explain the exceptional phenotypic characteristics of these samples by the human-moderated introduction of non-indigenous genotypes into the southern parts of northern europe, with subsequent natural hybridization with indigenous l. serriola genotypes. the bcluster in slovenian samples showed, with a few exceptions, a continuity with samples from a lower latitude; 96% of these samples are represented by the completely homozygous microsatellite genotype g29 (on-line suppl. tab. 3). this study provides interesting insights into the genetic variability of l. serriola populations originating from completely different eco-geographical areas. specifically those from slovenia, near the mediterranean, a world diversity hotspot (myers et al. 2000), the center of the greatest diversity of the genus lactuca (lebeda et al. 2009b); additionally, those from sweden, a region at the northern border of l. serriola european distribution (feráková 1977, lebeda et al. 2004). this study showed that l. serriola populations originating from various eco-geographical conditions differ significantly in their genetic background, which is also reflected in the geographic patterns of their phenotypic features. to obtain more comprehensive information on the genetic structure and variations of this species, it would be interesting to analyze: i) more populations with more individuals from sweden, and for a comparative study ii) additional samples originating from areas with greater contrasting ecological conditions. genetic variability of lactuca serriola populations acta bot. croat. 77 (2), 2018 179 acknowledgments this study was supported projects msm 6198959215 (ministry of education, youth and sports) and qh 71254 (czech ministry of agriculture), and by the internal grant agency of palacký university in olomouc (iga_ prf_2015_001, iga_prf_2016-001, iga_prf_2017_001, iga_prf_2018_01). references alexander, j. m., 2010: genetic differences in the elevational limits of native and introduced lactuca serriola populations. journal of biogeography 37, 1951–1961. burbinge, n. t., gray, m., 1970: flora of the australian capital territory. australian national university press, canberra, australia. d’andrea, l., broennimann, o., kozlowski, g., guisan, a., morin, x., keller-senften, j., felber, f., 2009: climate change, anthropogenic disturbance and the northward range expansion of lactuca serriola (asteraceae). journal of biogeography 36, 1573–1587. d’andrea, l., meirmans, p., van de wiel, c., guadagnuolo, r., van treuren, r., kozlowski, g., den nijs, h., felber, f., 2017: molecular biogeography of prickly lettuce (lactuca serriola l.) shows traces of recent range expansion. journal of heredity 108, 194–206. de vries, i. m., 1996: characterization and identification of lactuca sativa cultivars and wild relatives with sds-electrophoresis (lactuca sect. lactuca, compositae). genetic resources and crop evolution 43, 193–202. de vries, i. m., 1997: origin and domestication of lactuca sativa l. genetic resources and crop evolution 44, 165–174. doležalová, i., křístková, e., lebeda, a., vinter, v., 2002: description of morphological characters of wild lactuca l. spp. genetic resources (english-czech version). horticultural science 29, 56–83. doležalová, i., lebeda, a., křístková, e., 2001: prickly lettuce (lactuca serriola l.) germplasm collecting and distribution study in slovenia and sweden. plant genetics resources newsletter 128, 41–44. doležalová, i., lebeda, a., špoková, m., křístková, e., 2009: comparison of phenotypic variability of lactuca serriola genetic resources from slovenia and sweden. in: meglic, v., bastar, m.t. 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f. o., morrone, o., 1999: catálogo de las plantas vasculares de la república argentina. ii. acanthaceae-euphorbiaceae (dicotyledoneae). monographs in systematic botany from the missouri botanical garden 74 (in spanish). 208 acta bot. croat. 80 (2), 2021 acta bot. croat. 80 (2), 208–214, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-025 issn 0365-0588 eissn 1847-8476 habrosia (caryophyllaceae) a monotypic genus endemic to western asia: morphological and molecular remarks duilio iamonico university of rome sapienza, department of planning, design and technology of architecture, via flaminia 72, 00196 rome, italy abstract – habrosia (sagineae, caryophyllaceae) is a genus that includes only h. spinuliflora, a species occurring in iran, iraq, syria, lebanon, and turkey (irano-turanian floristic chorological element). based on the available molecular data published in 2011, habrosia appears to be nested in a minuartia-clade, which includes taxa currently recognized under the genus sabulina. consequently, habrosia should be treated as a genus to be included in sabulina. however, the molecular tree published in 2011 considered only 9 sabulina members whereas, according to the current concept, sabulina is a genus comprising about 65 species. unfortunately, the molecular phylogeny including a larger sabulina sample published in 2014 did not include h. spinuliflora and the taxonomic position of habrosia remains, therefore, uncertain. with the aim of verifying the correct position of habrosia in the tribe sagineae with respect to its relationship to sabulina, a comprehensive molecular investigation based on its sequences, linked to detailed morphological data, is presented. the results obtained revealed that habrosia is not part of sabulina. a detailed description of h. spinuliflora, its ecological preference, and a distribution map are provided. eventually, the name arenaria spinulifolia (basionym of h. spinuliflora) is lectotypified on a specimen preserved at g (barcode g00212963). keywords: arenaria spinulifolia, iraq, its, lebanon, syria, turkey, typification. introduction the family caryophyllaceae juss. comprises ca. 100 genera and 3000 species, occurring mainly in the northern hemisphere (hernández-ledesma et al. 2015). caryophyllaceae is monophyletic as circumscribed by bittrich (1993), but the traditional recognition of three subfamilies (alsinoideae fenzl, caryophylloideae arnott, and paronychioideae meisner; see e.g., bittrich 1993) based on features of stipules, petals, sepals, and fruits does not provide monophyletic groups and should be replaced with the tribe-based scheme as reported by harbaugh et al. (2010) and confirmed by subsequent studies (e.g. greenberg and donoghue 2011). at genus rank, several studies have been carried out on arenaria l., minuartia l., dianthus l., gypsophila l., polycarpon l., silene l., etc. (see e.g., kool et al. 2007, iamonico 2013, 2014, 2015, 2016, 2018, dillenberger and kadereit 2014, iamonico and domina 2015, iamonico et al. 2015, sadeghian et al. 2015, dillenberger and rabeler 2018, madhani et al. 2018), but various questions are still open. as part of the ongoing studies on caryophyllaceae (e.g., iamonico 2013, 2014, 2015, 2018, 2019, 2020, iamonico and domina, 2015), i here present a note about the monotypic genus habrosia fenzl [including the species h. spinuliflora (ser. ex dc.) fenzl], since some issues about its position in the tribe sagineae j.presl still need clarification. the aims of the research are: 1) to verify the correct position of habrosia in the tribe sagineae with special regards to its relationship to sabulina, 2) to consider the morphology of h. spinuliflora in comparison with its position in the molecular tree, 3) to clarify the identity of the name of arenaria spinuliflora ser. ex dc. (basionym of h. spinuliflora). materials and methods the present research is based on both the analysis of the relevant literature and the examination of the specimens preserved at bag, g, mo, p, sav, and w (codes according to thiers 2021-onward). the its sequences, used for the alignment and phylogenetic reconstruction, were publicly available in genbank (see smissen et al. 2003) and refer to 65 members of sabulina rchb., colobanthus bartl., drypis l., facchinia rchb., habrosia, minuartia, mcneillia dillenb. & kadereit, * corresponding author e-mail: d.iamonico@yahoo.it morphological and molecular remarks on habrosia acta bot. croat. 80 (2), 2021 209 sagina l., and (outgroups) bufonia tenuifolia l. and cherleria garckeana (asch. and sint. ex boiss.) a.j. moore and dillenb. raxml v8.2.12 (stamatakis 2014) was run under the gtrgamma model (bootstrapping was stopped automatically) for phylogenetic reconstruction. the distribution map was prepared using google earth pro (2021). data derive from both herbarium specimens and literature. the articles cited throughout the text follow the international code of nomenclature for algae, fungi, and plants (turland et al. 2018, hereafter as icn). results literature data arenaria spinuliflora ser. ex dc., after its original description in the 1st volume of candolle’s prodromus ( candolle 1824: 406), was treated under the genus arenaria up until 1833, when fenzl (1833: 57) proposed transferring this taxon to the genus alsine l. (note that neither morphological information nor a general reason about this nomenclatural choice was given by fenzl 1833). no subsequent papers or monographs, in which fenzl’s al. spinuliflora was accepted, have been traced. ten year later, fenzl (1843: 323–324) validly described the new genus habrosia to accomodate candolle’s species, correcting his previous choice under alsine. a detailed generic description was provided, as well as a diagnosis and description of his h. spinuliflora (ser. ex dc.) fenzl. the genus habrosia has been accepted until today (see e.g., hernández-ledesma et al. 2015, rabeler 2020). at suprageneric rank, habrosia spinuliflora is currently to be included in the tribe sagineae, together with sagina, colobanthus, sabulina, and bufonia sauvage. fenzl (1843: 326) proposed to treat his new genus habrosia as belonging to a new subtribe (named “habrosieae”) of the tribe sclerantheae link. in the same year, endlicher (1843), proposed transferring fenzl’s subtribe into tribe rank [habrosieae (fenzl) endl.]. more recently, novosel (1982) published “habrosieae novosel, tribus nov.” which would be morphologically similar to the tribe pollichieae dc. (including the genus pollichia aiton) from which it would differ by the indehiscent fruit, absence of sterile branches, occurrence of petals in flowers, and 1-4 ovules [see the diagnostic key (steps 1-3) provided by novosel 1982: 222]. according to art. 6.3 (note 2) of icn, novosel’s name “habrosieae” is an isonym (and therefore invalid) being based on the same type (h. spinuliflora) of endlicher’s name. the first authors to have included habrosia in a molecular analysis were smissen et al. (2003) in their study on the genus scleranthus l. where fenzl’s genus resulted to be sister of drypis. a more detailed study was carried out by greenberg and donoghue (2011) who investigated many samples of caryophyllaceae members (630 accessions) and revealed that drypis (a monotypic genus with drypis spinosa l.) was basal to a well-supported clade (bootstrap value: 91) including species of sagina, colobanthus, minuartia, habrosia, and bufonia, plus arenaria fontinalis (short and r.peter) shinners. this clade corresponds to the tribe sagineae. dillenberger and kadereit (2014), who investigated in detail the genus minuartia, did not consider the genus habrosia in their analysis. however, the species of minuartia, included in the tribe sagineae by greenberg and donoghue (2011), were treated by dillenberger and kadereit (2014) as belonging to the resurrected genus sabulina rchb. in contrast to habrosia, dillenberger and kadereit (2014) included arenaria fontinalis in their analysis, which was also investigated by greenberg and donoghue (2011), and confirmed that it is to be treated as a member of sabulina and, in fact, a new combination, s. fontinalis (short and r. peter) dillenb. and kadereit, was proposed. based on greenberg and donoghue (2011), habrosia should be a genus to be included in sabulina. molecular data greenberg and donoghue (2011: 1642, fig. 2) considered nine members of sabulina (sub minuartia spp.). however, according to the current concept, sabulina is a genus comprising ca. 65 species (hernández-ledesma et al. 2015, rabeler 2020). as a consequence, the position of habrosia in the its tree (clade sagineae) published by greenberg and donoghue (2011) cannot be considered as conclusive. as discussed above (see paragraph “literature data”), dillenberger and kadereit (2014) studied the majority of the minuartia s.l. taxa, and included in their analyses the sabulina clade by greenberg and donoghue (2011, sub minuartia), reaching the conclusion that these taxa are to be transferred to a different genus, i.e. the resurrected sabulina. however, habrosia was not included in the study by dillenberger and kadereit (2014), and an indirect inclusion of habrosia into sabulina would represent a risk from the taxonomical point of view. all things considered, i decided to merge the molecular data of dillenberger and kadereit (2014) and greenberg and donoghue (2011) in a single matrix and run a new comprehensive tree to verify if habrosia is actually nested in the sabulina clade or not. the results obtained (fig. 1) reveal that habrosia is not nested in the clade comprising the members belonging to sabulina, but is in an unresolved position outside of sabulina. morphological data starting from important works by mcneill (1962, 1967), the genus minuartia (at that time morphologically related to arenaria), was later accepted by most authors until the molecular studies by dillenberger and kadereit (2014). these latter authors demonstrated that minuartia is highly polyphyletic, and 11 different genera were recognized and later accepted by many authors (e.g., iamonico 2014, legler and dillenberger 2017, moore and dillenberger 2017, dillenberger and rabeler 2018). iamonico d. 210 acta bot. croat. 80 (2), 2021 among the resurrected genera, there is sabulina which comprises mcneill’s sects. acutif lorae (fenzl) hayek, alsinanthe (fenzl) graebn., greniera (gay) mattf., sabulina (rchb.) graebn., sclerophylla mattf., and tryphane (fenzl) hayek, as well as stellaria fontinalis short & r.peter (dillenberger and kadereit 2014, hernández-ledesma et al. 2015: 330). sabulina can be morphologically distinguished in having the following characters (see dillenberger and kadereit 2014: 80-81): stems neither spiny nor quadrangular, leaves linear to subulate, flowers white without episepalous staminoids, nectary glands not cup-shaped, calyx not hardened at the base, sepals acute and 1–3-veined, petals usually not exceeding the sepals, styles 3 and free, fruit dehiscent (capsule) 3-toothed. according to my examination of herbarium specimens, habrosia differs from sabulina by the following characters, which have a high taxonomic value in caryophyllaceae (see e.g., dillenberger and kadereit 2014, hernández-ledesma et al. 2015): apex of the sepals [awned (awns about 2/3 as long as the membranous part of the sepals) in habrosia vs. not awned in sabulina], number of styles (2 vs. 3), and fruit [indehiscent (utricule) in habrosia vs. dehiscent (capsule) in sabulina]. fig. 1. maximum likelihood phylogeny based on its sequences obtained with raxml under the gtrgamma model. bootstraps values are shown. arrow indicates the position of habrosia spinuliflora (ser. ex dc.) fenzl. morphological and molecular remarks on habrosia acta bot. croat. 80 (2), 2021 211 typification of arenaria spinuliflora arenaria spinuliflora ser. ex dc. [basionym of habrosia spinuliflora (ser. ex dc.) fenzl] was validly published by candolle (1824: 406) who provided a short diagnosis and the provenance (“in oriente”) and cited a syntype (“v. s. [ vidi sicco] comm. à cl. rosseau”). candolle (1824) reported “ser. mss.” just after the binomial, so referring to an unpublished seringe’s manuscript. tropicos (2021) does not list the name arenaria spinuliflora, erroneously reporting “habrosia spinuliflora fenzl” and citing, as syntypes, a specimen at mo (barcode mo256214, available at http://legacy.tropicos.org/image/59784) collected by t. kotschy (no. 120) in aleppo ( syria) in april 20, 1841. however, not only did fenzl (1843: 323–324) not propose a new species (rather a new combination of candolle’s name), but the cited specimen of kotschy (mo256214) cannot be regarded as syntype (or included in the original material) since it was not cited by candolle (1824: 406) but only by fenzl (1843). also the plant list (2013) and ipni (2021-onward) accepted the citation “habrosia spinuliflora fenzl” [note that the plant list (2013) reported, as synonym of “habrosia spinuliflora fenzl”, the name arenaria spinuliflora which is therefore (and wrongly) considered as heterotypic synonym]. among the main online database of plant names, only powo (2021-onward) correctly cited the name by fenzl (1843) which is considered a new combination of candolle’s name (basionym). i traced one sheet at g (barcode g00212963) bearing two plants which are clearly part of the same gathering. in fact, both the plants were collected by m. rousseau in “orient” in 1818. g00212963 is part of the original material for arenaria spinuliflora, matches candolle’s diagnosis (1824: 406), and it is here designated as the lectotype (arts. 9.3 and 9.4 of icn). in addition, an original label by seringe (“a. [arenaria] spinuliflora ser.”) occurs on the right-corner of g00212963, supporting the choice of this specimen as lectotype. taxonomic treatment habrosia fenzl., bot. zeitung (berlin) 1: 323. 1843. original type: habrosia spinuliflora (ser. ex dc.) fenzl. habrosia spinuliflora (ser. ex dc.) fenzl, bot. zeitung (berlin) 1: 323-324. 1843 (as “habrosias-pinuliflora” which is a typographic error) ≡ arenaria spinuliflora ser. ex dc., prodr. [dc.], 1: 406 (1824) ≡ alsine spinuliflora (ser. ex dc.) fenzl, vers. darstell. alsin.: 57 (1833), fig. 2). lectotype (designated here) – unknown country, orient, 1818, m. rousseau s.n. (g00212963!). description – annual herb, erect, 4–15 cm tall. stems filiform, dichotomously branched, slightly purple in colour, glabrous to sparsely pubescent [trichomes short, uniseriate, multicellular, eglandular, see chandra et al. 2019]. leaves opposite, setaceous, 0.5–2.0(–2.5) cm long, up to 0.5 mm width, connate at the base, glabrous to sparsely pubescent, sessile, margins entire, apex obtuse. stipules adnate to the margins at the base of the leaf, with membranous borders. inflorescences 4–10-flowered, terminal, lax; bracts leaf-like, shorter than leaves (ca. 3 mm long). flowers peryginous, on pedicels up to 6 mm long; sepals 5, glabrous, ovate-sublanceolate, 1.5–2.5(–3.0) mm long, 1(–3)-veined, membranous at the margins, apex awned, 2/3–1 as long as the membranous part of the sepals; petals 5, ovate-rounded, 1.0–1.5 mm long, shorter than sepals, white, entire; stamens 5, alternate to the sepals, with distinct glands attached to the adaxial surface extending in front of petal bases; styles 2, free, very short; ovary small, up to 1 mm long, subsessile, each ovary including 2 ovules. fruit indehiscent nutlet, ovoid, ca. 1 mm long; seed 1 per fruit, globose. pollen grain spheroidal, diameter about 27 μm, polyporate with 12–13 pores, each pore circular, in diameter about 4 μm, granulate; interpolar distance 5–8 μm. exine ca. 3 μm thick, tectate. sexine ca. 2.5 μm thick, punctulate. tegillum < 0.5 μm thick with minute spines. bacula broader at the apex than the base (chanda 1962: 73–74, and pl. 3, figs. 10–12). etymology – from the greek habros (χάβρος) which means “delicate, graceful”, presumably referring to the thinness of the plant, especially of stem and leaves. fig. 3. distribution map of habrosia spinuliflora (ser. ex dc.) fenzl. scale bar: 200 km. fig. 2. habrosia spinuliflora (ser. ex dc.) fenzl from mardin, se-turkey. a – habit, b – detailed of the inflorescence (photo by musa geçit, @musageçit). iamonico d. 212 acta bot. croat. 80 (2), 2021 vernacular names – çöl kumotu (turkish; ekim 2012), western asian sandwort (english common name, here proposed). habitat and phenology – rocky limestone slopes, gullies, scrubs, calcareous steppe, arable field, open banks, 300–1500 m a.s.l. flowering and fruiting times march to june. distribution and chorology – ne iran (see also rechinger et al. 1988), n iraq (see also blakelock 1957, ghazanfar and edmandson 2016), cand nw syria (see also boissier 1867, post 1932), ne lebanon (see also musselman 2011), se turkey (see also davis 1988, kaya and ketenoğlu 2010, bakis et al. 2011, ekim 2012) plus mt taurus (fig. 3). according to takhtajan (1986), the distribution area of habrosia spinuliflora is included in the irano-turanian floristic region, western asiatic subregion, mesopotamian province. additional specimens – iran: kurdistania persica: montes supra pagum režab dit. kasr-i-širin, in lapidosis, 05 may 1910, f. nábělek 4114 (sav0005141!). iraq: in montis kuh-sefin reg. infer. supra pagum schaklava (ditionis erbil), 1050 m, 15 may 1893, j. f. n. bormüller 949 (p05380908!); ibidem (p05380911!); mindan, 20 april 1947, bradburne 44 (bag); khormal, sulaimaniya liwa, 900 m, 21 april 1947, rawi 8857 (bag); jabal sinjar north of the town, mosul liwa, 900 m, coppiced quercus aegilops forest on limestone, localy abundant, 26 may 1948, c. c. gillet 11091 (bag); acra, mosul liwa, 30 may 1948, rawi 11305 (bag); bikhair mt., 900 m, 03 july 1957, rawi 23144 (bag); jabal maqlub, 550-750 m, 17 april 1958, shahwani 25204 (bag); serkupkan village c. 7 km nw of rania, on hillside, rawi 28533 (bag); dokan, near water, hillside, 15 may 1971, omar and karin 38029 (bag); 20 km to dahuk, clay soil wheat field, 310 m, 15 april 1980, al kaisi 52008 (bag); jebel sinjan, racky clay mountain, 16 april 1980, al kaisi 52034 (bag); bidem, 18 april 1980, al kaisi 52224 (bag). lebanon: ad antilibani radices occidentales, in declibvitatibus supra baalbek, 1150-1300 m, 20 may 1910, j. bornmüller 11506 (p05380909!); baalbek, 11 may 1933, m. r. gombault 2233 (p05110463!); ibidem (p05110464!). syria: in collibus lapidosis pr. aleppum, 20 april 1841, t. kotschy 120 (p05049676!, as “habrosyne spinufiflora”); ibidem (p00712781!); ibidem (p00712782!); ibidem (p00712783!); ibidem (mo256214!); aleppo, in graminis, 1330 ped. (= feet), 24 march 1865, c. haussknecht s.n. (p05380913!, the nineteen plants on the botton-half of the sheet); syria borealis, aleppo, 1865, a. de bunge s.n. (p00712788!); dans djebel belas, 28 may 1895, coll. illeg. 2964 (p05380913!, the seven plants of the top-half of the sheet); alep., 1834, a. montbret s.n. (p00712786!); ibidem (p00712787!); alep., s.d., s.coll. 590 (p00712785!); alep., s.d., a. montbret s.n. (p05380910!). turkey: mont taurus, 1837, m. aucher-eloy 590 (p00712784!); birecjik: djebel taken, 30 april 1888, o. stapf 461 (p05380912!); ibidem (p05380915!). discussion the available molecular data for habrosia placed this genus as sister of drypis (smissen et al. 2003) or minuartia s.l. (tribe sagineae; greenberg and donoghue 2011). more recently, dillenberger and kadereit (2014) resurrected the genus sabulina in which to place the minuartia species included in the tribe sagineae by greenberg and donoghue (2011). although dillenberger and kadereit (2014) did not consider the genus habrosia in their analysis, habrosia should be indirectly treated as a genus to be included in sabulina based on published data. on the contrary, the phylogenetic tree obtained in the present study, which derives from a single matrix including the sequences by both dillenberger and kadereit (2014) and greenberg and donoghue (2011), shows that habrosia cannot be merged with sabulina and it should be left separate. this result highlights the relevance, in molecular analisys, of considering all the taxa involved in the investigated genus and the related ones, especially in taxonomically critical groups as minuartia s.l. (specifically sabulina in this case). the morphological study confirms that habrosia clearly differs from sabulina in some characters (apex of the sepals, number of styles, and fruit dehiscence/indehiscence) which have a high taxonomic value in caryophyllaceae (see e.g., mcneill 1962, tutin et al. 1993, rabeler and hartman 2005, greenberg and donoghue 2011, dillenberger and kadereit 2014, pignatti 2017) and that it has to be recognized at generic rank. acknowledgements many thanks are due to m. dillenberger (johannes gutenberg-universität mainz, germany) for the useful discussion about molecular results. i also thank to m. geçit (mardin, mardin province, turkey) for the permission to reproduce his photo of habrosia from mardin and thanks to r. gül (datça, muğla province, turkey) who helped me getting in contact m. geçit. references bakis, y., babac, m.t., uslu, e., 2011: updates and improvements of turkish plants data service (tübi̇ves). in: allmer, j. 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(eds.), 1993: flora europaea, vol. 1, ed. 2. cambridge university press, cambridge. opce-str.vp acta bot. croat. 68 (2), 179–182, 2009 coden: abcra 25 issn 0365–0588 preface it is with a sense of great satisfaction that i introduce this issue of acta botanica croatica that contains twenty-two papers presented at 20th international diatom symposium (ids), held from 7–13 september 2008 at the grand hotel park in dubrovnik, croatia. as editor of this issue, i first must extend my sincere thanks to the authors who provided the contributions that formed the essential content of the symposium and to the referees who applied their professional expertise to review these contributions in such a short time. the results of this joint effort are contained in the present publication. it was in may 2006 that i first discussed the idea of hosting the 20th international diatom symposium with my colleagues at the institute. this quickly coalesced into a concrete proposal that was submitted to our rectorate. after review, i was very pleased to receive the full support of our rector, professor mateo milkovi}, and of our scientific board. i also solicited and received the support of the algology group of the university of zagreb’s faculty of science and of the croatian botanical society. organizing the 20th ids symposium in dubrovnik thus became our common task. in september 2006 i introduced my country, hometown, university, and institute at the general meeting of the international society for diatom research (isdr) in lystvianka, baikal, russia. i submitted our proposal to host isdr’s 20th anniversary symposium, which the council accepted in december 2007. the 20th international diatom symposium thus would be convened in dubrovnik in september 2008. when september finally arrived, i had the pleasure, as symposium chair, of delivering the opening remarks on behalf of the institute for marine and coastal research and the university of dubrovnik. professor vjekoslav dami}, pro-rector of the university, then welcomed the assembled participants and professor dra`en viki} topi}, state secretary of the ministry of science, education, and sports of the republic of croatia, officially declared the symposium’s opening. delegates were received afterwards by local officials at sponza palace, one of the landmarks of dubrovnik’s historic center. a total of 204 participants (exclusive of accompanying persons) representing 41 countries attended the 20th international diatom symposium (fig. 1). of these, 103 were isdr members and one-fourth students. the break-down was: usa (22), uk (16), poland (15), russia (15), croatia (14), japan (13), italy (12), germany (11), france (8), belgium (7), canada (6), the netherlands (6), spain (5), czech republic (4), australia (3), hungary (3), india (3), iran (3), republic of macedonia (3), serbia (3), south africa (3), ukraine (3), argentina (2), austria (2), estonia (2), grand-duchy of luxembourg (2), israel (2), portugal (2), sweden (2), bolivia (1), bosnia and herzegovina (1), brazil (1), greece (1), kuwait (1), mexico (1), new zealand (1), pr china (1), republic of belarus (1), republic of kosovo (1), taiwan (1), and turkey (1). acta bot. croat. 68 (2), 2009 179 u:\acta botanica\acta-botan 2-09\jasprica.vp 5. listopad 2009 12:51:48 color profile: disabled composite 150 lpi at 45 degrees 180 acta bot. croat. 68 (2), 2009 jasprica n. f ig . 1 . p ar ti ci pa nt s of th e 20 th in te rn at io na l d ia to m s ym po si um . u:\acta botanica\acta-botan 2-09\jasprica.vp 9. listopad 2009 12:58:23 color profile: disabled composite 150 lpi at 45 degrees oral presentations were assigned to one of the following categories: paleoecology and biostratigraphy (freshwater and marine); methodology, biotechnology, and astrobiology; ecology of marine and estuarine diatoms; ecology of freshwater diatoms; taxonomy, morphology and ultrastructure of diatoms; biodiversity and biogeography; genetic variability, phylogeny, molecular data, and evolution; eu diatomics project; application of diatoms in water quality assessment. while it is customary to have no more than 65 oral presentations over the four working days, we received more than 80 requests. the croatian organizing committee worked to accommodate as many of these as possible, given the time constraints. in the end, we succeeded in having 72 oral presentations, of which ten were made by students. some of the remaining requests had to be transferred to poster sessions. the plenary address, »revealing the molecular secrets of diatoms through the whole genome«, was delivered by dr. chris bowler of the département de biologie, école normale supérieure, paris. one hundred and forty posters, of which 40 were prepared by students, were presented in the following sessions: paleoecology and sediment record; ecology of marine and estuarine diatoms; biodiversity and biogeography of marine diatoms; diatoms of terrestrial ecosystems; ecology and water-quality assessment; biodiversity and biogeography of terrestrial water bodies; taxonomy, morphology, and ultrastructure; genetics, phylogeny and evolution; and methodology and collections. the symposium also included two workshops: »use of diatoms in forensic science« and »dna bar-coding«. the former, moderated by stefan uitdehaag of the netherlands forensic institute (the hague), and dr. herman van dam, consultancy for water and nature (amsterdam), highlighted the successful application of diatoms studies, particularly systematics and taxonomy, in forensic investigations. professor david g. mann of the royal botanic garden, edinburgh, moderated the bar-coding workshop and also presented »the tree of life website project« (http://www. tolweb.org/tree/), a multi-author encyclopedia of biodiversity that aims to provide a complete guide to the world’s species and their interrelationships. a roundtable, »functional morphology and diatom nanotechnology«, was moderated by professor mario de stefano of the environmental science department of the 2nd university of naples; and a meeting for nordic and baltic phycologists, »nordic network for benthic algae in freshwater (norbaf)«, was convened by dr. maria kahlert of the department of environmental assessment of the swedish university of agricultural sciences, uppsala. i was especially pleased to see students play such an active role in the symposium. this is very encouraging for the future of diatom research. the best student presentations were recognized at the closing banquet. the committee chose lourenço ribeiro of the faculty of sciences, university of lisbon, for the best student oral presentation: »benthic diatom communities in contrasting sandy and muddy shores: a spatiotemporal study in two intertidal flats of the tagus estuary, portugal«. elizabeth c. ruck, department of botany, university of texas, received the award for the best student poster: »estimating the phylogeny of the order surirellales with morphological and multi-gene sequence data«. acta bot. croat. 68 (2), 2009 181 preface u:\acta botanica\acta-botan 2-09\jasprica.vp 12. listopad 2009 11:34:49 color profile: disabled composite 150 lpi at 45 degrees i express my sincere thanks to all our sponsors: the croatian ministry of science, education, and sports; the city of dubrovnik; the croatian environmental protection and energy efficiency fund; dubrovnik airport ltd.; koeltz scientific books, koenigstein, germany; and fluid imaging technologies, yarmouth, maine (usa); and, of course, the institute for marine and coastal research of the university of dubrovnik. i owe special thanks to the then-mayor of dubrovnik, mme. dubravka [uica, who hosted our opening reception in sponza palace. members of her staff, especially mmes. nike sudarevi} and marija crn~evi}, also made valuable contributions to the organization of the symposium. i thank mr. ranko filipovi} of atlas travel agency, the official partner of the symposium, for making his very important contribution to the symposium with the highest professional competence. thanks to mr. @eljko schnattinger, who designed the attractive symposium logo, the cover of the abstract book, and other official materials. my thanks also go to dr. nick staresinic, who offered his unselfish support and advice throughout. i reserve special thanks for professor andrzej witkowski of the university of szczecin, poland. dr. witkowski was president of the international society for diatom research during the preparation of the dubrovnik symposium and was extremely generous in offering his kind help and very useful advice. i must, of course, warmly thank all of my colleagues at the institute for marine and coastal research in dubrovnik, and division of biology, university of zagreb. they are very well aware of my genuine gratitude for their exceptional support and hard work, without which we never could have realized this symposium. finally, it is my sincere hope that the dubrovnik symposium was as intellectually stimulating and scientifically valuable for the participants as it was for the organizers. i very much look forward to the 21st international diatom symposium to be held in the usa. i am confident that it will provide a venue that continues to assemble students and experienced researchers in an open and productive scientific climate. nenad jasprica chair, 20th international diatom symposium the guest editor of acta botanica croatica 68(2) 2009 dubrovnik, october 2009 182 acta bot. croat. 68 (2), 2009 jasprica n. u:\acta botanica\acta-botan 2-09\jasprica.vp 12. listopad 2009 11:34:49 color profile: disabled composite 150 lpi at 45 degrees 140 acta bot. croat. 80 (2), 2021 acta bot. croat. 80 (2), 140–145, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-014 issn 0365-0588 eissn 1847-8476 emergence of a new salt-tolerant alien grass along roadsides? occurrence of diplachne fusca subsp. fascicularis (poaceae) in hungary kristóf süveges1, attila molnár v.*1, attila mesterházy2, júlia tüdősné budai3, réka fekete1 1 university of debrecen, department of botany, egyetem tér 1, h-4032 debrecen, hungary 2 directorate of hortobágy national park, sumen u. 2, h-4024 debrecen, hungary 3 university of debrecen, research institute of karcag of the centre for agricultural and applied economic sciences, kisújszállási u. 166, h-5300 karcag, hungary abstract – this paper reports the occurrence of a north american salt-tolerant taxon, diplachne fusca subsp. fascicularis (lam.) p.m.peterson et n.snow in hungary (central-europe). two earlier hungarian observations of d. fusca were known from 1915, near győr (west transdanubia), later the taxon was collected by pénzes in 1958, in downtown budatétény (central hungary). both observations seem to be occasional. recently, the taxon has started spreading in europe, mainly on rice paddy fields, with a serious invasion potential. in north america its appearance on ruderal habitats, as well as along roads and other linear infrastructures is a well known phenomenon. the hungarian population was found near cegléd (central hungary) on the roadside of the e40 primary main road in september 2018. in july 2019 more than one thousand (mostly vegetative) individuals were detected. the salt content of the habitat shows remarkable temporal and spatial variability. at one meter distance from the edge of the paved road soil salt content was higher in spring (after the winter de-icing regime), than in autumn. salt concentration was highest in the vicinity of the road, and decreased with increasing distance from it. germination tests revealed a significant negative effect of nacl concentration on germination rates, but germination occurred even on extremely saline substrates with 1.5% nacl concentration. considering its biology and reproduction strategy, the further spread of diplachne fusca is highly presumable. keywords: central europe, de-icing salt, plant-invasion, roadside verges, salt-tolerance, seed dispersal introduction biological invasion is one of the recent global challenges (drake et al. 1989, perrings et al. 2002). increase of global cargo and passenger transport, as well as the growth of global road networks, now 64 million kilometres long in total(van der ree et al. 2015) is remarkable even from a biological point of view, especially because roads play an important role in the spread of invasive species worldwide (forman 2000, gelbard and belnap 2003, kalwij et al. 2008). long-distance dispersal of native and alien plant species by vehicles is frequent, but appears to be more common among alien species (von der lippe and kowarik 2007). the construction and maintenance of roads are usually associated with anthropogenic disturbances, including chemical de-icing, use of herbicides, mowing verges, trampling, introduction of a range of pollutants (e.g. petroleum products) and modified soils used for construction, the last of which may contain propagules of alien species (šerá 2008, van der ree et al. 2015). these factors act in synergy, favouring the spread of alien plant species, since native species are usually less able to adapt to the altered, anthropogenic conditions at roadsides (greenberg et al. 1997). in the case of plant species producing lightweight seeds, dispersal may be facilitated by the air turbulence caused by cars (von der lippe et al. 2013), but seeds may also travel long distances in mud attached to cars (clifford 1959, ross 1986, schmidt 1989, zwaenepoel et al. 2006, ansong and pickering 2013). moreover, machines used for mowing verges have also been shown to transport seeds, potentially aiding dispersal in some taxa at least (strykstra et al. 1997, vitalos and karrer 2009). * corresponding author e-mail: mva@science.unideb.hu diplachne fusca subsp. fascicularis in hungary acta bot. croat. 80 (2), 2021 141 winter de-icing (using mostly nacl, with a small proportion of cacl2, rarely mgcl2; houska 2007) became widespread in europe during the second half of the 20th century. de-icing salts have complex effects on the roadside environment (amrhein et al. 1992). the resulting increased soil salt content in the vicinity of roads can facilitate the spread of halotolerant, or even of halophytic plant species (davison 1971). for instance, the eastand northward spread of a mediterranean coastal halophyte species, plantago coronopus l., was detected in hungary between 2013 and 2016 (schmidt et al. 2016). similarly, the spread of the atlantic coastal halophyte, cochlearia danica l. was also documented in continental europe (fekete et al. 2018). during systematic surveys of the roadside vegetation along hungarian paved roads, on 25 september, 2018, we found diplachne fusca subsp. fascicularis (lam.) p.m. peterson et n. snow (syn: d. fascicularis (lam.) p. beauv., festuca fascicularis lam., leptochloa fusca subsp. fascicularis (lam.) n. snow) on the roadside of the e40 primary main road near cegléd (central hungary). the taxon was identified according to snow et al. 2018. this species has a native distribution range restricted to the americas, spanning the area from southern canada to argentina. it has been previously reported from disturbed habitats of several countries in europe: belgium (lambinon 1957, verloove and vandenberghe 1999), the netherlands (van der meijden 1975), ukraine (dubyna et al. 2003), portugal (valdés and scholz 2009) and the czech republic (pyšek et al. 2012). it is considered an invasive weed of rice paddy fields in italy (romani and tabacchi 2000), spain (osca 2013), turkey (altop et al. 2015) and bulgaria (vladimirov and delcheva 2016). here we document the first european appearance of the species on roadside verges, which raises the possibility of further spread or even invasion of the taxon along european roads. occupancy of this habitat could help the species to conquer new habitats (possibly even arable fields) and possibly reach new geographic regions. the central aims of this paper were: (i) to document the circumstances of occurrence of diplachne fascicularis along hungarian roadsides; (ii) to examine soil salt content of this newly found habitat and to test the effect of salt content on seed germination of the taxon in an in vitro germination experiment. materials and methods the geographic coordinates and the elevation of the locality were determined using a garmin etrex legend handheld gps device and recorded in wgs84 format. the number of diplachne fusca subsp. fascicularis individuals was estimated on 14th december 2018 and 22nd july 2019. the nomenclature of vascular plant taxa follows király (2009). soil samples were collected from root depth (1.5–6.5 cm) before and after the de-icing season (on 18th september 2018 and 11st march 2019, respectively). they were collected at six different distances from the paved road. these distances were 1, 2, 3 m in september 2018 and 0.1, 0.5 and 1 m in march 2019. soil total soluble salt content was quantified by measuring electric conductivity of a saturated paste of soil and water using a conductivity meter (tetra con 325) (hungarian technical standard msz-08-0213:1978 2.2). soil analyses were carried out by the accredited laboratory of the research institute of karcag of the centre for agricultural and applied economic sciences of the university of debrecen. in order to test the ability of diplachne fusca subsp. fascicularis seeds to germinate at different nacl concentrations we conducted an in vitro germination test. seeds were collected on 18th september 2018 and were stored in paper bags at room temperature until germination tests were initiated. we tested germination on a 1% agar substrate in petri dishes with the following 13 nacl concentrations (m/m% = mass percent): 0 (control), 0.15%, 0.30%, 0.45%, 0.60%, 0.75%, 0.90%, 1.05%, 1.20%, 1.35%, 1.50%, 2% and 2.50%. these concentrations were used to determine whether the species requires salt for germination and to assess the maximum concentration of nacl at which it is able to germinate. in one petri dish 25 seeds were placed on the agaragar medium. each concentration of medium was repeated three times. thus, at one given concentration, 75 seeds were germinated in three petri dishes with 25-25 seeds per petri dish. thus, a total of 975 seeds were tested at 13 different concentrations. petri dishes were stored at room temperature under natural light conditions (i.e. near a window in a laboratory) and germination was followed from 16th october 2018 to 6th november 2018. seedlings were counted on the 21st day of the experiment. data analyses were carried out in the r statistical environment (r core team 2018). to test the effect of substrate salt content on germinability, we performed a binomial generalized linear mixed model (glmm), where germination status (0/1) was used as dependent variable and nacl concentration was included as the sole explanatory variable in form of a second-degree orthogonal polynomial. the i. d. of the petri dish was included as a random factor in the model. prediction intervals were calculated using ’predictinterval’ function from mermod objects (knowles and frederick 2016). results were visualized using a sunflower plot. results diplachne fusca subsp. fascicularis (lam.) p.m.peterson et n.snow was found on 25th september 2018, near cegléd, on the roadside of the e40 primary main road (coordinates 47.19151 n, 19.91483 e, elevation 89 m a.s.l.), in the immediate vicinity of road reconstruction and overpass construction works. in total, 117 individuals were found, distributed along a 250 meter-long section of road, in a narrow lane (at 100–380 cm distance from the paved road margin). on 22nd july 2019 the latter site was revisited and altogether 1082 (mainly vegetative) individuals were recorded, along a 200 m-long section (at 5–330 cm distance from the paved road). illustration of the taxon (fig. 1) was made based on herbarium specimens and photographs. voucher specimens were süveges k., molnár v. a., mesterházy a., budai t. j., fekete r. 142 acta bot. croat. 80 (2), 2021 deposited in the herbaria of the university of debrecen (de), hungarian natural history museum (bp) and eszterházy károly university (egr). morphological description of the taxon, based on vladimirov and delcheva (2016) and on our own observations can be summarized as follows: annual plant. generative stems 20–40(–100) cm tall. leaf sheaths rolled up, 2–4(–5) mm wide, pointed, scabrid to subglabrous; ligules 2–8 mm, membranous, becoming lacerate at maturity; the uppermost leaf exceeds the panicle. the panicle is generally narrow, elongated. panicle partly enclosed in the uppermost leaf sheath (even at maturity), 10–60 cm long, with 8–25 branches, with (1)2–7(8) spikelets per branch; branches (2)3–12 cm long, erecto-patent to suberect (fig. 1). spikelets subsessile (peduncle 0.4–0.6 mm), (4)5–11 mm long, 5–11-flowered; lower glume 2–2.5 mm, fig. 1. diplachne fusca subsp. fascicularis (lam.) p.m. peterson et n. snow. a – habit, b – one branch of the panicle, c – spikelet, d, e – diaspore (caryopsis with lemmas) (drawn by jana táborská). diplachne fusca subsp. fascicularis in hungary acta bot. croat. 80 (2), 2021 143 lanceolate, upper glume ca. 4 mm, elliptic; lemmas lanceolate, 3-veined, with silky hairs at base and along the margin in the lower half, bifid at apex, with 0.5–2.5 mm long apical awn arising from the notch, midrib keeled, usually scabrid. diplachne fusca subsp. fascicularis was found to co-occur with the following taxa (halophytes are marked with asterisks, other grass species also spreading characteristically along roads are marked with hashtags): achillea collina, ambrosia artemisiifolia, artemisia santonicum*, atriplex prostrata*, a. tatarica*, bromus inermis, chenopodium album, cichorium intybus, cirsium arvense, cynodon dactylon, daucus carota, elymus elongatus#, e. repens, festuca pratensis, f. pseudovina*, inula britannica, limonium gmelinii*, phragmites australis, plantago lanceolata, podospermum canum*, polygonum aviculare, populus × euramericana (seed ling), populus alba (seedling), portulaca oleracea, puccinellia distans*, setaria glauca, sorghum halepense#, taraxacum sect. ruderalia, tragus racemosus#. the total soluble salt content of the habitat showed a remarkable temporal and spatial variability. at a one meter distance from the edge of the paved road soil salt content was remarkably higher (0.58%) in spring (after the winter de-icing), than in autumn (0.07%). in both cases the highest salt concentration (0.07% in autumn and 1% in spring) was detected in the sampling point closest to the road margin (1 m and 0.1 m). altogether, 486 seeds (51.4%) germinated from the 975 seeds tested. binomial generalized linear mixed model (glmm) showed a significant negative quadratic effect (p < 0.001) of substrate nacl concentration on germination (fig. 2). the maximum concentration of nacl where germination was detected was 1.5%. in the latter case, only 2.7% of the seeds germinated (fig. 2). the highest germination rate (97%) was detected with the 0.3% nacl concentration. discussion the species diplachne fusca has a secondary cosmopolitan range. it can be divided into four subspecies (snow et al. 2018). the native range of subspecies fascicularis extends from southern canada to argentina. in these areas it occurs in different marshy habitats, on wet, muddy surfaces and in ditches (broyles 1987, bartgis and hutton 1988), but it also appears on ruderal sites, such as places where cars are parked, or on roadsides (stevens 1917), being documented in both southern florida (atlas of florida plants) and wisconsin (virtual flora of wisconsin 2020). the occurrence of d. fusca (without it being recognised as a subspecies) in hungary was published by sándor polgár (polgár 1918), in győr (nw hungary) from the area of a vegetable oil factory. this record was also mentioned by jávorka (1924–1925). no recent herbarium voucher specimen was found in the following hungarian natural history collections (on january 9, 2020): natural history museum, budapest (bp); herbarium of the university of eötvös (bpu, nótári et al. 2017); university of debrecen (de, takács et al. 2014) and eszterházy university, eger (egr, e. vojtkó et al. 2014). the only hungarian specimen of diplachne fusca available in herbarium carpato-pannonicum (bp-380842) of the hungarian natural history museum was collected in 1958 by antal pénzes in a private garden in budatétény (central hungary), but our revision has shown that this specimen surely represents another subspecies and not the one the presence of which in hungary is described above. in southern europe, diplachne fusca subsp. fascicularis is considered as a quickly spreading, potentially invasive alien plant species (weber and gut 2005). it is considered a problematic weed in california, as well as in europe, interfering with rice production (driver et al. 2019, romani and tabacchi 2000, osca 2013). the newly found occurrence is located 60 km west from the closest rice paddy fields near kisújszállás. the number of individuals near cegléd (central hungary) increased almost 10 times from 2018 to 2019. this suggests that d. fusca subsp. fascicularis is able to reproduce under the habitat and climatic circumstances present at roadsides. however, we note that the species was able to colonize only otherwise open, gravel surfaces in the close vicinity of the road, but not the roadside verge, where dense, perennial grassland vegetation was dominant. d. fusca shows some important biological and physiological characteristics, which raise the strong possibility that it will continue to spread. with the help of its symbiotic bacteria d. fusca subsp. fascicularis is able to fix n2 (zafar et al. 1986, reinhold-hurek et al. 1993), furthermore it is a plant species of the c4 photosynthetic pathway (snow et al. 2018). these characteristics can help the species to adapt even to extreme habitat conditions. moreover, its ability to build a persistent seedbank (mcintyre et al. 1989), its cleistogamy and anemochory (jurado et al. 1991) can enhance its reproductive success and dispersal ability, as shown by the case of two sporobolus species, s. neglectus and s. vaginiflorus ( jogan 2017). finally, its high salt tolerance (myers and morgan 1989, and this study) facilitates its spread along roads. fig. 2. sunflower plot illustrating the relationship between germination rate and germination substrate supplemented with nacl. black dots show average germination rate in each petri dish and each petal of sunflowers represents a single observation (germination of individual seeds). süveges k., molnár v. a., mesterházy a., budai t. j., fekete r. 144 acta bot. croat. 80 (2), 2021 acknowledgments the authors are grateful to tünde abonyi for her assistance during germination test, and to zoltán barina and dániel pif kó for their help in herbarium work, to jana táborská for the drawing of the species, and to orsolya vincze for her linguistic corrections. this research was funded by national research, development and innovation office of hungary (grant number nkfi-otka k132573). kristóf süveges was funded by the new national excellence programme of the hungarian ministry for innovation and technology (únkp-19-3i-de-238). references altop, e.k., mennan, h., phillippo, c.j., zandstra, b.h., 2015: effect of the burial depth andenvironmental factors on the seasonal germination of bearded sprangletop (leptochloafusca [l.] kunth ssp. fascicularis [lam.] n.snow). weed biology and management 15, 147–158. amrhein, c., strong, j.e., mosher, p.a., 1992: effect of deicing salts on metal and organic matter mobilization in roadside soils. environmental science&technology 26, 703–709. ansong, m., pickering c., 2013: are weeds hitchhiking a ride on your car? a systematic review of seed dispersal on cars. 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survey of weeds that are increasingly spreading in europe. agronomy for sustainable development 25, 109–121. zafar, y., ashraf, m., malik, k.a., 1986: nitrogen fixation associated with roots of kallar grass leptochloa fusca (l.) kunth. plant and soil 90, 93–105. zwaenepoel, a., roovers, p., hermy, m., 2006: motor vehicles as vectors of plant species from road verges in a suburban environment. basic and applied ecology 7, 83–93. 214 acta bot. croat. 77 (2), 2018 acta bot. croat. 77 (2), 214–217, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0013 issn 0365-0588 eissn 1847-8476 short communication resurrection of a regionally extinct taxon in croatia – the case of ammophila arenaria (l.) link (poaceae) sandro bogdanović1,2*, vedran šegota3, antun alegro3 1 university of zagreb, faculty of agriculture, department of agricultural botany, svetošimunska 25, 10000 zagreb 2 centre of excellence for biodiversity and molecular plant breeding, svetošimunska 25, 10000 zagreb 3 university of zagreb, faculty of science, department of biology, division of botany, herbarium croaticum, marulićev trg 20/2, 10000 zagreb, croatia abstract – a regionally extinct taxon, ammophila arenaria (l.) link subsp. arundinacea h. lindb., has been rediscovered in the croatian flora after 78 years. previously it was known only from two coastal sand dune sites in northern dalmatia. the habitat at the locality of crnika near lopar on the northern adriatic island of rab is destroyed and a. arenaria subsp. arundinacea does not grow there anymore. at the second locality, on the sand dunes of kraljičina plaža in the vicinity of the town of nin, a. arenaria subsp. arundinacea was rediscovered and confirmed after 174 years. this is the only population of this taxon in croatia, counting 48 mature individuals where the psammophylous habitat of kraljičina plaža is under strong anthropogenic influence. this taxon is now classified as critically endangered (cr) and merits adequate active protection and conservation of its psammophylous habitat. key words: ammophila, conservation, extinction, nin, psammophytes, rare species * corresponding author, e-mail: sbogdanovic@agr.hr introduction the species european beachgrass, ammophila arenaria (l.) link, belongs to the family poaceae and it is a typical psammophylous grass that inhabits coastal sand dunes from the eastern atlantic coasts of europe to the mediterranean area (tutin 1980, valdés and scholz 2009). the species is represented by two subspecies along the distribution area: the typical a. arenaria (l.) link subsp. arenaria is distributed along the atlantic sandy coasts of north and west europe, southwards to the french basque country, while the second one, a. arenaria subsp. arundinacea h. lindb is distributed on the coasts of south europe, from romania to portugal, with the northern limit in the french basque county (tutin 1980, biurrun et al. 2012, marcenὸ and jiménez-alfaro 2017). in the croatian flora the taxon a. arenaria subsp. arundinacea was mentioned for the first time by alschinger (1832: 27) as psamma arenaria beauv. for the area of aenona (the town of nin) in the vicinity of the town of zadar in northern dalmatia. subsequently, visiani (1842: 79) cited the same locality “circa nona” in his flora dalmatica, as did schlosser and vukotinović (1869: 1232) in flora croatica. afterwards, it was recorded by morton (1915: 249) as ammophila pallida (c. presl) fritsch var. australis mabille and by horvatić (1939: 25), both for the same locality (crnika near the village of lopar) on the island of rab. to conclude, these were the only two known localities of a. arenaria subsp. arundinacea in the croatian flora for more than eight last decades. during the re-evaluation of the national red list of vascular flora of croatia, ilijanić (2005) evaluated the taxon a. arenaria subsp. arundinacea as a regionally extinct (re) with a note “extinct in the wild, with a very small possibility of re-finding due to habitat loss“. the author underlined various intensive touristic activities on sandy beaches as a main reason for the extinction of this taxon. materials and methods field investigation was carried out on kraljičina plaža (44°15'0.89" n, 15°10'37.28" e) in the vicinity of the town of nin in northern dalmatia, and in crnika near the village of lopar (44°49'25" n, 14°44'26"e) on the island of rab during the vegetation season of may, june and july of 2016 (fig. 1). additionally, the flora of the entire south-eastern coast resurrection of extinct ammophila arenaria acta bot. croat. 77 (2), 2018 215 around lopar, consisting of dozens of sandy beaches was carefully checked. plant material of psammophylous flora was collected and deposited in herbaria za and zagr. abbreviations are according to thiers (2016). for the purpose of documentation and for available public access to herbaria, specimens of a. arenaria subsp. arundinacea were geo-coded, digitalized and inserted in flora croatica database (nikolić 2016) and in the publicly accessible virtual herbarium zagr (bogdanović et al. 2016). vegetation relevés were made using the expanded braun banquet scale (braun-blanquet 1964, barkman et al. 1964, dierschke 1994). category 2 was subdivided into 2m, 2a and 2b. in total, three phytosociological relevés with plot sizes of 5 x 5 m were made in june of 2016. the species nomenclature follows the flora croatica database (nikolić 2016) and euro+med (2017). for the assessment of population status and conservation issues of a. arenaria subsp. arundinacea the iucn criteria and categories were adopted according to guidelines of iucn (2014). results and discussion our finding of a. arenaria subsp. arundinacea on the sand dunes around the town of nin in northern dalmatia raised the question of its extinction and its conservation status in croatia. the resurrection of a regionally extinct plant taxon in croatian flora after the last red list evaluations is uncommon. in eleven years, only botrychium matricariifolium w.d.j.koch has been rediscovered (but at a different locality), thirty years after its disappearance (borovečki-voska et al. 2011). more recently franjić et al. (2016) confirmed the existence of hippophae rhamnoides l. in the croatian flora, and both species were consequently removed from the list of extinct taxa. the case of a. arenaria subsp. arundinacea is the third one, and here we provide detailed information about the morphology, distribution, habitat, phytosociology and conservation status of this taxon, which is one of the rarest plant species in the croatian flora: morphology – a. arenaria subsp. arundinacea is a perennial rhizomatous grass with erect stems up to 170 cm. leaves are convolute, rigid, strongly ribbed, ca 5 mm wide, with bifid, up to 30 mm long ligule. inflorescence is erect, a dense cylindrical panicle up to 30 cm long (fig. 2). spikelets are 12–14 mm long, with 1 floret. glumes are linear-lanceolate, about equal to the lemma. lemma has 4–5 mm long hairs at the base, while in a. arenaria subsp. arenaria the glumes distinctly exceed the lemma, and the lemma has hairs ca 2 mm long at the base. ovary is glabrous (tutin 1980). distribution in croatia – the only site of this taxon is in northern dalmatia on the sand dunes of kraljičina plaža in the vicinity of the town of nin. the habitat of the historical record on crnika in lopar from the island of rab is devastated and destroyed by touristic development (fig. 1), as foreseen by ilijanić (1987). a field survey of all the sandy beaches on north-eastern part of the island of rab revealed no new findings. specimina visa – croatia: otok rab, pješčane sipine uz dragu crniku u loparskoj dolini, asoc. agropyretum mediterraneum, 31.05.1935, s. horvatić s.n. (za 7197); dalmacija, nin, ninsko blato, kraljičina plaža, obalna pješčana dina, 44°15'0,89" n, 15°10'37,28" e, 07.05.2016, s. bogdanović & v. šegota s.n. (zagr 41250). fig. 1. map of the investigated area in the vicinity of the town of nin (kraljičina plaža) and the island of rab (lopar). symbols indicate presence at (○) and absence from a previous known locality (x) of ammophila arenaria (l.) link subsp. arundinacea h. lindb. fig. 2. inflorescence of ammophila arenaria (l.) link subsp. arundinacea h. lindb. (photo by s. bogdanović). habitat and phytosociology – a. arenaria subsp. arundinacea is a characteristic species of the order ammophiletalia br.-bl. et tx. ex westhoff et al. 1946, growing on primary embryonic mobile sand dunes (biondi and galdenzi 2014). the species was found to be in a good state, with average height and at the time of our visit majority of the plants were in bloom. at the locality kraljičina plaža it grows in typical psammophytic vegetation (fig. 3) with some other very rare psammophylous taxa of the croatian flora: cyperus capitatus vand., phleum arenarium l., stachys maritima gouan, elytrigia juncea (l.) nevski subsp. juncea, armeria canescens (host) boiss. in dc. subsp. dalmatica (beck) trinajstić and calystegia soldanella (l). r. br. sandy coastal habitats in bogdanović s., šegota v., alegro a. 216 acta bot. croat. 77 (2), 2018 croatia are very rare and psammophytic vegetation of the order ammophiletalia is extremely endangered (alegro et al. 2004). due to the very small and localized areas of these habitats, as well as by the quoted psammophytic species they are also colonized by many species of neighbouring ruderalized habitats as can be seen from those three phytosociological relevés (tab. 1). therefore, the researched community can be considered an impoverished wet variant of the association euphorbio paraliae-agropyretum junceiformis tüxen in br.-bl. et tüxen 1952 corr. darimont, duvigneaud et lambinon 1962 (ammophilion br.-bl. 1921, ammophiletalia br.-bl. et tüxen ex westhoff et al. 1946) as defined by šilc et al. (2016). conservation status – in june 2016, we observed and counted 48 mature individuals along the sand dune at only one location. according to iucn (2014) for the evaluation of a. arenaria subsp. arundinacea the criterion d (very small or restricted population) could be applied. the area of occupancy (aoo) and the extent of occurrence (eoo) are both less than 10 km2. the number of locations is one, continued decline is observed in eoo and aoo, in habitat quality and number of locations. adopting the iucn (2014) criteria a. arenaria subsp. arundinacea should be classified as critically endangered (cr) based on following criteria: b1a(i,ii,iii,iv) + b2b(i,ii,iii,iv) and d. the sand dunes of kraljičina plaža are under strong anthropogenic influence, and the major threat to this taxon and to other critically endangered plant taxa of this area (cyperus capitatus and calystegia soldanella) is the human impact due to various touristic activities, e.g. removing the plant cover and exploitation and transportation of the sand for enlargement of sandy beaches, construction of infrastructure for local beach bars, etc. conclusions according to the flora croatica database (nikolić 2016), the croatian flora has eight regionally extinct taxa: caldesia parnassifolia (l.) parl., cuscuta epilinum weihe, fig. 3. habitat of ammophila arenaria (l.) link subsp. arundinacea h. lindb. in kraljičina plaža near the town of nin (photo by v. šegota). tab. 1. phytosociological relevés collected from kraljičina plaža near the town of nin. coordinates of relevés: rel. 1: 44°15'14" n, 15°10'15" e, rel. 2: 44°15'10" n, 15°10'22" e, rel. 3: 44°15'06" n, 15°10'30" e. relevés no. 1 2 3 surface (m2) 25 25 25 vascular plant cover (%) 80 80 80 ammophila arenaria (l.) link subsp. arundinacea h. lindb 1 2b 2b elytrigia juncea (l.) nevski subsp. juncea 1 2a 1 armeria canescens (host) boiss. in dc. subsp. dalmatica (g. beck) trinajstić . 2m 2m euphorbia paralias l. 1 . . vulpia ciliata dumort. . 1 + phleum arenarium l. . . 1 stachys maritima gouan + . . cyperus capitatus vand. 2a . . schoenus nigricans l. . 2a . juncus acutus l. . 1 2a limonium virgatum (willd.) fourr. . 2m 2m cynodon dactylon (l.) pers. 1 1 2a papaver rhoeas l. + 2m 1 silene vulgaris (moench) garcke + 1 + anthericum liliago l. + . . scolymus hispanicus l. + + . clematis flammula l. + + r allium sphaerocephalon l. . + + dactylis glomerata l. . + . scirpus holoschoenus l. . 1 1 melica ciliata l. . 1 + rubus ulmifolius schott . + . chrysopogon gryllus (l.) trin. . + . elymus pycnanthus (godr.) melderis . + . tamarix sp. (planted) . + + elymus repens (l.) gould . . 1 anagallis arvensis l. . . + arenaria leptoclados (rchb.) guss. . . + cakile maritima scop. . . 1 catapodium rigidum (l.) c. e. hubb. . . + herniaria glabra l. . . + lagurus ovatus l. . . 2m petrorhagia saxifraga (l.) link . . + reichardia picroides (l.) roth . . + sedum acre l. . . + seseli montanum l. subsp. tommasinii (rchb. f.) arcang. . . + trifolium scabrum l. . . + valantia muralis l. . . + helichrysum italicum (roth) g. don . . 2a cyperus glaber l., drosera anglica huds., d. intermedia hayne, eriophorum gracile roth, eryngium planum l. and ammophila arenaria (l.) link subsp. arundinacea h. lindb. due to our finding, the last one has to be removed now from that list and treated as critically endangered; however urgent protection actions are needed for conservation of this taxon rediscovered in the wild. resurrection of extinct ammophila arenaria acta bot. croat. 77 (2), 2018 217 acknowledgements the authors would like to thank the president of the croatian botanical society (hbod) for logistic and financial support during this research and to our colleagues nikola koletić for providing the distribution map and igor boršić phd for valuable comments on conservation issues. references alegro, a., biljaković, m., bogdanović, s., boršić, i., 2004: psammo-halophytic vegetation on the largest sand area on the croatian coast: the island of mljet, southern adriatic. biologia, bratislava 59, 435–445. alschinger, a., 1832: flora jadrensis. typ. battara, zadar (jaderae). barkman, j. j., doing, h., segal. s., 1964: kritische bemerkungen und vorschläge zur quantitativen vegetationsanalyse. acta botanica neerlandica 13, 394–419. biondi, e., galdenzi, d., 2014: syntaxonomic considerations of the mediterranean vegetation dominated by perennial psammophilous graminaceous plants. plant sociology 51, 25–32. biurrun, i., garcía-mijangos, i., campos, j.a., herrera, m., loidi, j., 2012: vegetation-plot database of the university of the basque country (bioveg). biodiversity and ecology 4, 328. bogdanović, s., britvec, m., dujmović purgar, d., ljubičić, i., vitasović kosić, i., 2016: herbarium zagr of the faculty of agriculture (zagreb, croatia). agriculturae conspectus scientificus 81, 1–5. borovečki-voska, lj., čičmir, r., šincek, d., 2011: new finding of the species botrychium matricarifolium (retz.) a. br. ex koch (ophioglossaceae) in croatia. natura croatica 20, 229–233. braun-blanquet, j., 1964: pflanzensoziologie. grundzüge der vegetationskunde. 3. aufl. springer verl., wien, new york. dierschke, h., 1994: pflanzensoziologie. grundlagen und methoden. eugen ulmer verl., stuttgart. euro+med, 2017: euro+med plantbase – the information resource for euro-mediterranean plant diversity. retrieved may 16, 2017 from http://ww2.bgbm.org/europlusmed/. franjić, j., horvat, g., krstonošić, d., 2016: new localities and syntaxonomic characteristics of sea buckthorn (hippophaë rhamnoides l., elaeagnaceae) in croatia. šumarski list 3–4, 111–116. horvatić, s., 1939: overview of the vegetation of the island of rab in terms of phytosociology. prirodoslovna istraživanja kraljevine jugoslavije 22, 1–96 (in croatian). ilijanić, lj., 1987: vegetational and biogeographic characteristics of the island of rab. in: mohorovičić, a. (ed.), rapski zbornik, 83–97. jazu and skupština općine rab (in croatian). ilijanić, lj., 2005: ammophila arenaria (l.) link ssp. arundinacea h. lindb. in: nikolić, t., topić, j. (eds.), red book of vascular flora of croatia, 82–83. ministarstvo kulture, državni zavod za zaštitu prirode, zagreb (in croatian). iucn, 2014: iucn standards and petitions subcommittee. guidelines for using the iucn red list categories and criteria. version 11.1. prepared by the standards and petitions subcommittee. retrieved december 21, 2016 from http://www. iuc-nredlist.org/documents/redlistguidelines.pdf marcenò, c., jiménez-alfaro, b., 2017: the mediterranean ammophiletea database: a comprehensive dataset of coastal dune vegetation. phytocoenologia 47, 95–105. morton, f., 1915: pflanzengeographische monographie der inselgruppe arbe, umfassend die inseln arbe, dolin, s. gregorio, goli und pervicchio sammt den umliegenden scoglien. botanische jahrbücher für systematik, pflanzengeschichte und pflanzengeographie 116, 67–273. nikolić, t., (ed.) 2016: flora croatica database. university of zagreb, faculty of science, department of botany. retrieved december 27, 2016 from http://hirc.botanic.hr/fcd/. schlosser, j. c., vukotinović, lj. f., 1869: flora croatica: exhibens stirpes phanerogamas et vasculares cryptogamas quae in croatia, slavonia et dalmatia sponte crescunt nec non illas quae frequentissime coluntur. zagrabiae. šilc, u., mullaj, a., alegro, a., ibraliu, a., dajić stevanović, z., luković, m., stešević, d., 2016: sand dune vegetation along the eastern adriatic coast. phytocoenologia 46, 339–355. thiers, b., 2016: index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden's virtual herbarium. retrieved december 27, 2016 from: http://sweetgum.nybg.org/ih/. tutin, t. g. 1980: ammophila (l.) link. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea vol. 5, 236, cambridge university press, cambridge. valdés, b., scholz, h., 2009: poaceae (pro parte majore). euro+med plantbase the information resource for euromediterranean plant diversity. retrieved december 27, 2016 from: http://ww2.bgbm.org/europlusmed/query.asp visiani, r., 1842: flora dalmatica 1. apud fridericum hofmeister, lipsiae. acta bot. croat. 79 (1), 2020 1 preface from the new editor-in-chief in 2019, i accepted the role of editor-in-chief for acta botanica croatica after my 10-year term within the editorial board as the subject editor in phycology and vegetation ecology. i want to thank professor emeritus damir viličić, editor-in chief from 1998–2013, for recruiting me to the editorial board. it was a pleasure to be able to work with such a dedicated and talented group of section editors. it went by all too quickly. in 2020, acta botanica croatica has reached volume 79. this issue of acta botanica croatica is the first to appear under my editorship. the previous editor (2014–2019), dr. branka salopek sondi, the ruđer bošković institute, zagreb, has helped to develop acta botanica croatica into a prominent international journal for botanical research. papers published in acta botanica croatica are more and more cited in highly competent journals, increasing the impact factor (woscc, 2014–2018) from 0.516 to 0.985 (5-year impact factor: 1.006; the third quartile, q3, in plant science) over the time window examined. in 2020, acta botanica croatica also celebrates its 95th anniversary. its longevity continues to be a testament to the vitality of the field of botany, and i hope that acta botanica croatica continues to be a review resource for different field botanists. high quality standards and modern scientific content should be a permanent editorial goal in order for the success of acta botanica croatica to be maintained. finally, on behalf of myself and the editorial board, i want to convey our general thanks to the authors and reviewers. it is they who are primarily to be credited with the actual content and the success of acta botanica croatica. nenad jasprica opce-str.vp acta bot. croat. 69 (2), 229–235, 2010 coden: abcra 25 issn 0365–0588 wall protuberance formation and function in secreting salt glands of tamarix aphylla l. artemios m. bosabalidis* department of botany, school of biology, aristotle university, thessaloniki 54124, greece salt glands of tamarix aphylla consist of three pairs of secretory cells arranged one upon the other. at the stage of secretion, the upper and middle pair of secretory cells develop in their walls an internal system of anastomosed rods, the protuberances. in the formation of the wall protuberances, golgi vesicles and microtubules appear to participate. the stage of salt secretion is also characterized by the presence of numerous mitochondria and microvacuoles. microvacuoles contain the secreted solution and accumulate in the region of the wall protuberances. the interaction between microvacuoles and wall protuberances as well as the genesis of wall protuberances constitute new findings on the subject. keywords: tamarix aphylla, salt glands, wall protuberances, microscopy, sem, tem introduction wall protuberances are rod-like projections of the cell wall towards the cytoplasm. they may be single or branched and may develop in the vicinity of the wall or extend deep into the cell interior. cells bearing wall protuberances are considered to participate in short-distance transportation of solutes (gunning and pate 1974). wall protuberances have been observed in angiosperms (marinos 1970), ferns (gunning and pate 1969), bryophytes (duckett et al. 1977), algae (franceschi and lucas 1980), etc. more specifically, they have been localized in the stem (pate et al. 1970), the root (kramer et al. 1977), the leaf (evert 1980), the flower (peterson et al. 1979), the fruit (cochrane and duffus 1980), the seed (newcomb 1978), the root nodule (newcomb et al. 1977), the mycorrhiza (hadley et al. 1971), etc. as concerns the type of tissue, wall protuberances have been identified in the epidermal tissue (birch 1974), the ground tissue (butterfield et al. 1981), the secretory tissue (schnepf and pross 1976, rozema et al. 1977) and particularly, the conductive tissue (bowes 1973, peterson and young 1975). the bulk of publications dealing with wall protuberance-bearing cells appeared in the seventies and many issues became resolved, and yet questions referring to their formation and development remain, to our knowledge, open. acta bot. croat. 69 (2), 2010 229 * corresponding author, e-mail: artbos@bio.auth.gr u:\acta botanica\acta-botan 2-10\319 bosabalidis.vp 11. listopad 2010 12:48:57 color profile: disabled composite 150 lpi at 45 degrees the present work provides ultrastructural evidence for the genesis of wall protuberances and their functional association with the secreted microvacuoles in the salt glands of tamarix aphylla. materials and methods for light microscopy (lm) and transmission electron microscopy (tem), small segments of tamarix aphylla l. leaves were initially fixed for 4 h with 3% glutaraldehyde in 0.05 m phosphate buffer, ph 7.2. after washing in buffer, the segments were postfixed for 3 h with 2% osmium tetroxide (similarly buffered) and then dehydrated in an ethanol series. dehydration was followed by infiltration and embedment in spurr’s resin. semithin sections for lm were obtained in a reichert om u2 ultramicrotome, stained with toluidine blue o and photographed in a zeiss iii photomicroscope. ultrathin sections for tem were cut in a reichert-jung ultracut e ultramicrotome, stained with uranyl acetate and lead citrate and examined in a jem 2000 fxii transmission electron microscope. for scanning electron microscopy (sem), leaf segments, after fixation and dehydration, were critical point dried in a balzers cpd 030 device and then coated with carbon in a jee-4x vacuum evaporator. observations were made in a jsm 840-a scanning electron microscope. results the salt glands of tamarix aphylla are epidermal structures located on both surfaces of the leaf (figs. 1 a, b). under the scanning electron microscope, they appear as local dome-like projections which are clearly distinguished from the surrounding typical epidermal cells (fig. 1 a, asterisk). in leaf cross-sections, a developed salt gland seems to consist of three pairs of secretory cells arranged one upon the other (fig. 1 b). the cells increase in size towards the top of the gland and their nuclei are large and centrally located. light microscopical examination of secreting salt glands reveals that the prominent characteristics associated with secretion are the raising of the cuticle from the apical walls (fig. 1 b, arrow) and the presence in these walls of internal protrusions (fig. 1 b, arrowheads). electron microscopical observations show that in the outer pair of secretory cells, the walls (particularly the apical walls) locally extend towards the cytoplasm forming a system of rod-like structures, the protuberances (fig. 1 c). protuberances may penetrate the cytoplasm as single rods, or may branch and anastomose, forming an elaborate interconnected system. in the region of the protuberances, numerous mitochondria occur. high magnification of the protuberances reveals that they have a fine granular constitution and their structure is more compact than that of the normal wall (fig. 1 d). the protuberance rods were measured to have an average thickness of 120 nm. protuberances do not exist until divisions of secretory cells in the salt glands are completed. when a gland becomes fully formed and salt secretion initiates, protuberances start to develop in the secretory cells, an indication that their presence is directly associated with the secretory process. in the middle pair of secretory cells, wall protuberances are not restricted to the periphery of the cells but often extend deep into the innermost cytoplasmic region (fig. 2 a). 230 acta bot. croat. 69 (2), 2010 bosabalidis a. m. u:\acta botanica\acta-botan 2-10\319 bosabalidis.vp 11. listopad 2010 12:48:57 color profile: disabled composite 150 lpi at 45 degrees the initial stages of protuberance formation have been identified and followed. thus, at the wall point where a protuberance is formed, an accumulation of vesicles and short elements of the rough endoplasmic reticulum takes place (fig. 2 b)., microtubules appear to acta bot. croat. 69 (2), 2010 231 wall protuberances in salt glands of tamarix aphylla fig. 1. light, sem and tem micrographs illustrating salt glands. a – sem micrograph illustrating a salt gland (asterisk) on the leaf surface; b – lm micrograph of a secreting salt gland. the cuticle (arrow) is detached from the apical cell walls, which bear a system of internal protuberances (arrowheads); c – tem micrograph taken at the upper part of a salt gland. the cuticle (cu) is raised forming a subcuticular space (ss). the apical walls (cw) bear towards the cytoplasm many anastomosed protuberances (pr) m=mitochondrion; d – higher magnification of the protuberances. they exhibit a fine granular substructure, denser than that of the typical wall. bars: 12 mm (a), 10 mm (b), 1 mm (c), 0.4 mm (d). u:\acta botanica\acta-botan 2-10\319 bosabalidis.vp 11. listopad 2010 12:49:00 color profile: disabled composite 150 lpi at 45 degrees 232 acta bot. croat. 69 (2), 2010 bosabalidis a. m. fig. 2. tem micrographs showing the development and function of wall protuberances in a secreting salt gland. a – wall protuberances (pr) extending deep into the cytoplasm of the middle pair of secretory cells; b – early stage of a protuberance formation. at the region of the protuberance (pr), many vesicles (vs) and converging microtubules (mt) appear; c – active dictyosomes (ds) during the stage of protuberance formation; d – endoplasmic reticulum elements (er) and mitochondria (m) in the vicinity or in contact with wall protuberances; e – numerous microvacuoles (mv) accumulated along the apical wall protuberances in the upper pair of secretory cells. note the presence of many mitochondria; f – a lysed secretory cell of a salt gland. the cytoplasmic components are disorganized, whereas the wall protuberances (pr) are well retained. bar = 0.5 mm. bars: 0.4 mm (a), 0.5 mm (b, c, d, f), 0.8 mm (e). u:\acta botanica\acta-botan 2-10\319 bosabalidis.vp 11. listopad 2010 12:49:03 color profile: disabled composite 150 lpi at 45 degrees converge on this wall point. vesicles most probably originate from the golgi apparatus, since during this phase numerous active dictyosomes exist (fig. 2 c). in the progress of protuberance development, mitochondria and endoplasmic reticulum elements approach the protuberances and occasionally come into contact with them (fig. 2 d). apart from wall protuberances, the secretion stage is characterized by the presence of high numbers of microvacuoles and mitochondria (fig. 2 e). microvacuoles move in a great population towards the periphery of the secretory cells, where they become attached to the wall protuberances (fig. 2 e). after secretion becomes completed, the secretory cells start to disorganize until they finally become fully lysed. at this stage, no organelles or other cytoplasmic components can be discerned and the whole cytoplasm has a dark foamy appearance. the only cellular element which retains its entity is the wall protuberance network (fig. 2 f). discussion when cell divisions in the salt glands of tamarix aphylla are completed, the glands enter the secretory phase. the principal structural feature characterizing the stage of early secretion is the formation in the walls of the outer and middle pair of secretory cells of ingrowths penetrating the cytoplasm. these ingrowths (protuberances) are highly developed in the apical wall of the outer pair of cells, less so in the middle pair, whereas the inner pair of cells devoids of protuberances. wall protuberances constitute a typical trait of a type of cells called »transfer cells«, which are considered to be involved in the short distance flux of solutes (gunning and pate 1974). the dual feature »wall protuberances-lining plasmalemma« is probably associated with both fast fluxing of solutes and fluxing of large amounts of solutes. these two parameters are quite important in: 1) emergence of leaves. in spring, leaves emerge in a rather short time, and so large amounts of water and nutrients are immediately needed. these needs are covered with the formation and function of transfer cells in the conductive tissue. 2) germination of seeds. the growth of the seed embryo into a seedling is relatively rapid, and a quick mobilization of nutrients from the endosperm/cotyledons takes place via transfer cells (gori 1977). 3) survival of halophytes. in the halophytes, the excess of the uptaking salty solution should be fast removed in order to prevent cell hyperosmosis. salt removal is performed through salt glands, the secretory cells of which bear wall protuberances (rozema et al. 1977, faraday and thomson 1986). 4) pollination of flowers. when a flower opens, the process of pollination becomes immediately activated and floral nectaries start producing insect-attracting nectar. in order to facilitate secretion of large amounts of nectar within a short time (as long as flower opening lasts), nectaries bear numerous protuberances in their cell walls (fahn 1979). at the wall point where a protuberance starts to form, an accumulation of golgi vesicles as well as converging microtubules were observed. this figure resembles that of the formation of the cell plate where golgi vesicles are moved on phragmoplast microtubules via acta bot. croat. 69 (2), 2010 233 wall protuberances in salt glands of tamarix aphylla u:\acta botanica\acta-botan 2-10\319 bosabalidis.vp 11. listopad 2010 12:49:03 color profile: disabled composite 150 lpi at 45 degrees motor proteins to the concrete region of cell wall formation (evert 2006). the complement of protuberance formation is followed by a great increase in the number of mitochondria and microvacuoles. microvacuoles appear only in the upper pair of secretory cells and move towards the apical walls where protuberances are highly developed. there, they come into contact with the protuberance plasmalemma and by exocytosis release their content into the cell wall (and then into the subcuticular space). the content of the microvacuoles has been cytochemically identified as corresponding to the secreted salty solution (thomson et al. 1969). the fact that the salt is secreted by microvacuoles and not in the form of free cytoplasmic molecules contradicts the generally received opinion that the increase of the plasmalemma surface at the protuberance region is related to an eccrine secretion (via molecules passing across the plasmalemma, durkee et al. 1981, nepi et al. 1996). in that case, what is the advisability of such an increase of the plasmalemma surface? to this point the interpretation could be expressed that the high plasmalemma surface probably aims at creating a great number of disposable positions for the numerous microvacuoles, facilitating thus their fusion with the plasmalemma, a fact which would not be possible if a great number of microvacuoles became thickly crowded in a small plasmalemma surface. the network of wall protuberances is well retained even after the secretory cells become lysed during salt gland collapse. in other cell types under lysis, the cell walls appear disintegrated or refolded (bosabalidis and tsekos 1986). this event stresses the important role the wall protuberances play in the process of salt secretion. references birch, w. r., 1974: the unusual epidermis of the marine angiosperm halophila thou. flora 163, 410–414. bosabalidis, a. m., tsekos, i., 1986: ultrastructure of the essential oil secretion in the oil glands of the fruit peel of mandarin (citrus deliciosa 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(ed.), progress in essential oil research, 449–459. walter de gruyter, berlin. bowes, b. g., 1973: observations on xylem transfer cells in the leaf traces of linum usitatissimum l. (flax). zeitschrift für plfanzenphysiologie 68, 319–322. butterfield, b. g., maylan, b. a., exley, r. r.,1981: intercellular protuberances in the ground tissue of cocos nucifera l. protoplasma 107, 69–78. cochrane, m. p., duffus, c. m., 1980 : the nucellar projection and modified aleurone in the crease region of developing caryopses of barley (hordeum vulgare l. var. distichum). protoplasma 103, 361–375. duckett, j. g., prasad, a. k. s. k., davies, d. a., walker, s., 1977: a cytological analysis of the nostoc bryophyte relationship. new phytologist 79, 349–362. durkee, l. t., gaal, d. j., reisner, w. h., 1981: the floral and extrafloral nectaries of passiflora. i. the floral nectary. american journal of botany 68, 453–462. evert, r. f., 1980: vascular anatomy of angiospermous leaves with special consideration of the maize leaf. berichte der deutscher botanischer gesellschaft 93, 43–55. evert, r. f., 2006: esau’s plant anatomy. wiley, hoboken, new jersey. 234 acta bot. croat. 69 (2), 2010 bosabalidis a. m. u:\acta botanica\acta-botan 2-10\319 bosabalidis.vp 11. listopad 2010 12:49:04 color profile: disabled composite 150 lpi at 45 degrees fahn, a., 1979: ultrastructure of nectaries in relation to nectar secretion. american journal of botany 66, 977–985. faraday, ch., thomson, w. w., 1986: morphometric analysis of limonium salt glands in relation to ion efflux. journal of experimental botany 37, 471–481. franceschi, v. r., lucas, w. j., 1980: structure and possible functions of charasomes; complex plasmalemma-cell wall elaborations present in some characean species. protoplasma 104, 253–271. gori, p., 1977: wall ingrowths in the embryosac of euphorbia helioscopia. israel journal of botany 26, 202–208. gunning, b. e. s., pate, j. s., 1969: cells with wall ingrowths (transfer cells) in the placenta of ferns. planta 87, 271–274. gunning, b. e. s., pate, j. s., 1974: transfer cells. in: robards, a.w. (ed.), dynamic aspects of plant ultrastructure, 441–480. mcgraw hill, london, new york. hadley, g., johnson, r. p. c., john, d. a., 1971: fine structure of the host-fungus interface in orchid mycorrhiza. planta 100, 191–199. kramer, d., lauchli, a., yeo, a. r., 1977: transfer cells in roots of phaseolus coccineus: ultrastructure and possible function in exclusion of sodium from the shoot. annals of botany 41, 1031–1040. marinos, n. g., 1970: embryogenesis of the pea (pisum sativum), 1. the cytological environment of the developing embryo. protoplasma 70, 261–279. nepi, m., ciampolini, f., pacini, e., 1996 : development and ultrastructure of cucurbita pepo nectaries of male flowers. annals of botany 78, 95–104. newcomb, w., syono, k., torrey, j. g., 1977: development of an ineffective pea root nodule: morphogenesis, fine structure and cytokinin biosynthesis. canadian journal of botany 55, 1891–1907. newcomb, w., 1978: the development of cells in the coenocytic endosperm of the african blood lily haemanthus katherinae. canadian journal of botany 56, 483–501. pate, j. s., gunning, b. e. s., milliken, f. f., 1970: function of transfer cells in the nodal regions of stems particularly in relation to the nutrition of young seedlings. protoplasma 71, 313–334. peterson, r. l., yeung, e. c., 1975: ontogeny of phloem transfer cells in hieracium floribundum. canadian journal of botany 53, 2745–2758. peterson, r. l., scott, m. g., miller, s. l., 1979: some aspects of carpel structure in caltha palustris l. (ranunculaceae). american journal of botany 66, 334–342. rozema, j., riphagen, i., sminia, t., 1977: a light and electron microscopical study on the structure and function of the salt gland of glaux maritima l. new phytologist 79, 665–671. schnepf, e., pross, e., 1976: differentiation and re-differentiation of a transfer cell: development of septal nectaries of aloe and gasteria. protoplasma 89, 105–115. thomson, w. w., berry, w. l., liu, l. l., 1969: localization and secretion of salt by the salt glands of tamarix aphylla. proceedings of the national academy of sciences (usa) 63, 310–317. acta bot. croat. 69 (2), 2010 235 wall protuberances in salt glands of tamarix aphylla u:\acta botanica\acta-botan 2-10\319 bosabalidis.vp 11. listopad 2010 12:49:04 color profile: disabled composite 150 lpi at 45 degrees in memoriam professor ana zlata štefanac (1934-2019) the sad news of the demise of professor ana zlata štefanac reached me on a busy monday morning on march 18, 2019. she passed away some time during the preceding weekend in her zagreb home. prof. ana zlata štefanac, phd was a croatian scientist and a full professor at the department of biology, faculty of science, university of zagreb, who retired in 2004. professor štefanac was born ana zlata uđbinac on december 6, 1934 in zagreb, where she completed all her levels of education. she finished her undergraduate studies in biology in 1959, received her msc degree in 1964 and phd in 1967 under the mentorship of professor and academician davor miličić (1915-1993). she worked as a teaching assistant at the department of biology from 1960 and later on advanced to the ranks of assistant professor (1972), associate professor (1978) and full professor (1984). from her early days at the university of zagreb, she was involved in teaching practical courses in botany and plant anatomy. as she advanced in her career, she lectured more and gave fewer practical instructions. nevertheless, she always relied on the power of practical examples in teaching. her students remember her insisting on meticulous preparation of slides containing plant anatomical structures, correctly depicting and describing them in their own hand. no discrepancy between the objects seen under the microscope and student notebook descriptions could escape her attention. this was part of her strategy for motivating students to gain deeper insight into plants structures and functions. her teaching methods were classical and today would be called old fashioned. however, they were efficient and produced more than a passing impression in the student’s mind. although professsor štefanac was a devoted teacher, her real passion was research. when prof. miličić started gathering a team of young collaborators for his pioneering work on plant viruses, she was the first to join in as early as in 1959. this was the time of establishing the laboratory and building capacities needed for the research. being very practical and energetic, prof. štefanac was closely involved in procuring the laboratory equipment, organizing the building of an additional experimental greenhouse and establishing the unit for producing plant virus antisera in rabbits. those were infrastructural prerequisites for the research projects resulting in many scientific papers, as well as in undergraduate, master and doctoral theses for students all over yugoslavia. prof. štefanac’s msc research focused on the turnip yellow mosaic virus and her phd enlarged the scope of the research to include other viruses of cruciferous hosts, thus directing her future interests towards plant virus infections and their cytopathological effects. in the late 1960s and early 1970s, during the time in which postdoctoral specializations were neither required nor easily available for scientists in croatia, prof. štefanac did two of them in renowned virological laboratories. the first one was at the scottish crop research institute (invergowrie, dundee) where she stayed for 13 months thanks toa british council scholarship. the second one was at the department of plant pathology of the university of california in davis where she was a fulbright scholar for 11 months. working with eminent experts in the field, and mastering new methods at these prestigious institutions enabled her to advance and successfully disseminate her research. one of the most important papers she co-authored investigated the role of mitochondria in the establishment of inclusions formed by the tobacco rattle virus in the cells of experimental plant nicotiana clevelandii (harrison et al., 1970). the paper on cell inclusions of holmes’ ribgrass virus (miličić et al., 1969) falls into the same category of papers drawing very much international attention as one of the early studies describing pathogenic effects of plant viruses at the cellular level. in addition, prof. štefanac left her mark in the field describing and characterizing, biologically and molecularly, numerous plant virus species and strains. her distinctive handwriting is easily discerned in the lists of plant virus isolates collected over the years as a part of our laboratory’s collection. this bears witness to the amount of biological assays she performed with plant viruses during her career. professor štefanac was a member of the society for general microbiology, the association of applied biologists and the international society for horticultural science. she attended numerous international and national conferences, published papers in the most important plant pathology and virology international journals like the journal of general virology, virology, annals of applied biology, phytopathologische zeitschrift (now journal of phytopathology) and protoplasma. most of them are still highly relevant in these fields. nonetheless, she, as well as her colleagues from the same laboratory, did not refrain from publishing in journals that had a more local character like mikrobiologija, agronomski glasnik and, at that time, acta botanica croatica. she considered her papers published in the latter highly acta bot. croat. 78 (2), 2019 s5 in memoriam relevant and often said that her most important papers were those published in acta. she and her peers were very good researchers and prolific writers, contributing significantly to the international status and quality of this journal. professor a. z. štefanac was not only an author published in but also served on the editorial board of acta botanica croatica from 1982 until 2008. i crossed paths with prof. štefanac in 1992 as an msc student in the laboratory. even though she was not one of my lecturers during previous studies or my mentor in the following years, she was a highly appreciated senior colleague, a practical guide to the inner workings of the laboratory as well as somebody on whose help and expertise i could always rely. she performed her duties with efficiency, thoroughness and pride. her honesty, dedication and fairness were as much a part of her moral fibre as of her research and teaching. students described her as tough but just. as coworkers, we saw other aspects of her personality too. she was an excellent baker, cook, interested in gardening and fruit cultivation. not a birthday or a birth of a baby in the group passed without her baking an old-fashioned full-flavoured cake. as a member of the post-world war ii generation, she did not let anything go to waste. she practiced recycling before it became a modern concept for a sustainable way of life. grounded and direct as she was, i am sure she would have described it as common sense. professor ana zlata štefanac’s name stays recorded in many chapters of plant virology books as one of the researchers whose results contributed to the basic knowledge on viruses we almost take for granted nowadays. she will be remembered as a scientist for her integrity, industriousness and enthusiasm but also as a person who had the best interest of the people around her at heart. prof. dijana škorić, phd department of biology, faculty of science, university of zagreb marulićev trg 9a, zagreb, croatia list of publications miličić, d., uđbinac, z., 1960: die eiweisskristalle von capsicum annuum sind viruskörper. protoplasma 52, 446–456. miličić, d., uđbinac, z., 1961: virus-eiweissspindeln der kakteen in lokalläsionen von chenopodium. protoplasma 53, 584–596. štefanac-uđbinac, z., 1962: kristali virusa mozaičke bolesti duhana iz plodova paprike. acta botanica croatica 20–21, 261–263. miličić, d., štefanac-uđbinac, z., mamula, đ., 1963: rasprostranjenost nekih virusa krucifera u jugoslaviji. agronomski glasnik 13, 92–100. štefanac-uđbinac, z., miličić, d., zeljko, m., 1963: virus mozaika postrne repe (turnip mosaic virus) u jugoslaviji. acta botanica croatica 22, 107–117. miličić, d., štefanac-uđbinac, z., mamula, đ., 1963: rasprostranjenost nekih virusa krucifera u jugoslaviji. agronomski glasnik 13, 92–100. štefanac, z., 1964: prilog poznavanju virusa mozaika postrne repe (marmor brassicae holmes). magistarski rad. prirodoslovnomatematički fakultet, zagreb. štefanac, z., miličić, d., 1965: zelleinschlüsse des kohlrubenmosaikvirus. phytopathologische zeitschrift 52, 349–362. štefanac, z., 1967: neka svojstva kukuruznog soja virusa mozaika šećerne trske iz jugoslavije. agronomski glasnik 17, 673–684. štefanac, z., 1967: prilozi poznavanju svojstava virusa krucifera. disertacija miličić, d., štefanac, z., 1967: plastidenverändreungen unter dem einfluss des wasserrübengelbmosaik-virus (turnip yellow mosaic virus). phytopathologische zeitschrift 58, 285–296. miličić, d., štefanac, z., juretić, n., wrischer, m., 1968: cell inclusions of holme’s ribgrass virus. virology 35, 356–362. harrison, b. d., štefanac, z., roberts, i. m., 1970: role of mitochondria in the formation of x-bodies in cells of nicotiana clevelandii infected with tobacco rattle virus. journal of general virology 6, 127–140. miličić, d., štefanac, z., 1971: cell inclusions of the cucumber green mottle mosaic virus and the odontoglossum ringspot virus. acta botanica croatica 30, 33–40. štefanac, z., mamula, đ., 1971: a strain of radish mosaic virus occurring in turnip in yugoslavia. annals of applied biology 69, 229–234. štefanac, z., ljubešić, n., 1971: inclusion bodies in cells infected with radish mosaic virus. journal of general virology 13: 51–57. cvjetković, b., pleše, n., štefanac, z., miličić, d., 1972: nalaz virusa nekrotične prstenaste pjegavosti trešnje na ruži u jugoslaviji. acta botanica croatica 31, 15–20. štefanac, z., 1972: the occurrence of narcissus mosaic virus in yugoslavia. acta botanica croatica 31, 37–40. štefanac, z., borović, n., 1973: the relative concentration of radish mosaic virus in turnip. acta botanica croatica 32, 37–42. štefanac, z., 1974: belladonna mottle virus in yugoslavia. acta botanica croatica 33, 17–21. štefanac, z., ljubešić, n., 1974: the spindle-shaped inclusion bodies of narcissus mosaic virus. phytopathologische zeitschrift 80: 148–152. štefanac, z., 1977: onion yellow dwarf virus in yugoslavia. acta botanica croatica 36, 39–45. štefanac, z., 1978: investigation of viruses and virus diseases of spinach in croatia. acta botanica croatica 37, 39–46. štefanac, z., 1980: cucumber mosaic virus in garlic. acta botanica croatica 39, 21–26. štefanac, z., grbelja, j., erić, ž., 1981: a cucumovirus isolated from pea (pisum sativum l.). acta botanica croatica 40, 35–41. štefanac, z., plazibat, m., 1981: biological, serological and immunoelectrophoretic studies of tomato aspermy virus from chrysanthemums in yugoslavia. acta botanica croatica 40, 43–49. bezić, n., štefanac, z., miličić, d., 1983: rasprostranjenost virusa karanfila u jugoslaviji. agronomski glasnik 45, 187–196. štefanac, z., wrischer, m., 1983: spinach latent virus: some properties and comparison of two isolates. acta botanica croatica 42, 1–9. bezić, n., štefanac, z., miličić, d., wrischer, m., 1983: occurrence of carnation vein mottle and cucumber mosaic viruses on carnations in yugoslavia. acta botanica croatica 42, 21–27. s6 acta bot. croat. 78 (2), 2019 in memoriam bezić, n., krajačić, m., štefanac, z., miličić, d., wrischer, m., 1984: occurence of carnation necrotic fleck virus in yugoslavia. acta botanica croatica 43, 7–12. šarić, a., štefanac, z., wrischer, m., 1984: two rare types of particle aggregates in infections caused by a yugoslavian isolate of broad bean wilt virus. phytopathologia mediterranea 23, 88–90. erić, ž., štefanac, z., plavšić, b., 1986: characteristics of the tombusvirus from spinach (spinacia oleracea). acta botanica croatica 45, 7–19. rana, g. z., krajačić, m., štefanac, z., pleše, n., rubino, l., miličić, d., 1987: properties of a new strain of tobacco streak virus from clematis vitalba (ranunculaceae). annals of applied biology 111: 153–160. štefanac, z., bezić, n., miličić, d., 1988: some new data on robinia mosaic cucumovirus. acta botanica croatica 47, 1–5. šarić, a., štefanac, z., 1988: the incidence and variation of cucumber mosaic virus in four vegetable species in croatia. acta botanica croatica 47, 7–13. mamula, đ., štefanac, z., thaler, i., gailhofer, m., 1988: detection of a variant of henbane mosaic virus in physalis alkekengi l. acta botanica croatica 47, 15–19. štefanac, z., wrischer, m., 1989: ultrastructural peculiarities of turnip mosaic virus (massive) inclusions in two host species. acta botanica croatica 48, 11–14. krajačić, m., štefanac, z., 1990: standardizing the conditions for performance of immunoelectrophoretic experiments with tobacco streak ilarvirus particles. acta botanica croatica 49, 1–5. štefanac, z., krajačić, m., 1991: glycerol efficiently lessens tobacco streak ilarvirus particles aggregation during formaldehyde fixation. acta botanica croatica 50, 13–17. bezić, n., štefanac, z., 1992: light microscopy of robinia mosaic cucumovirus crystalline inclusion. acta botanica croatica 51, 7–11. štefanac, z., miličić, d., 1992: observations on infection of garlic (allium sativum l.) with cucumber mosaic virus. acta botanica croatica 51, 1–5. štefanac, z., gailhofer, m., thaler, i., mamula, đ., 1993: some characteristics of crystalline inclusions associated with henbane mosaic potyvirus. acta botanica croatica 52, 1–4. štefanac, z., pleše, n., 1994: academician davor miličić (1915– 1993) – in memoriam. acta botanica croatica 53, 177–179. krajačić, m., štefanac, z., 1999: some physicochemical characteristics of native and formaldehyde treated tobacco streak ilarvirus particles. acta botanica croatica 58, 5–14. acta bot. croat. 78 (2), 2019 s7 opce-str.vp acta bot. croat. 69 (1), 7–18, 2010 coden: abcra 25 issn 0365–0588 russian wheat aphid causes greater reduction in phloem transport capacity of barley leaves than bird cherry-oat aphid. sefiu a. saheed1,2,3*, lisbeth m. v. jonsson2, christiaan e. j. botha1 1 department of botany, rhodes university, grahamstown 6140, south africa 2 school of life sciences, södertörn university college, s-141 89 huddinge, sweden 3 current address: department of botany, faculty of science, obafemi awolowo university, ile-ife 22005, nigeria the effects of feeding by the russian wheat aphid (rwa), diuraphis noxia mordvilko and the bird cherry-oat aphid (bca), rhopalosiphum padi l on the transport capacity of barley hordeum vulgare l leaves were investigated and compared with a view to relating these effects to the visible symptoms shown by the respective infested plants. rwa causes extensive chlorosis and necrosis on an infested plant whereas bca causes no observable symptoms. our results using the xenobiotic, phloem mobile fluorophore, 5, 6 carboxyfluorescein diacetate (5, 6-cfda) revealed striking differences in damage to the transport of assimilates through the phloem by these two aphids. the result clearly suggests that short-term feeding by rwa causes a reduction in transport of assimilates and a more severe reduction or perhaps even permanent cessation of transport during long-term feeding. in contrast, feeding by bca does not lead to a marked decrease in transport during short-term feeding period, however, a reduction in the transport was recorded during long-term feeding activities. these results perhaps suggest that damage to transport capacities of the barley leaves appears to be partly responsible for the observed symptoms in rwa-infested plants and the lack of them during bca infestations, symptoms such as reduction or cessation in transport of assimilates to growing tissues may lead to such observable symptoms. keywords: aphid, feeding, hordeum vulgare, phloem transport, diuraphis noxia, rhopalosiphum padi abbreviations: bca – bird cherry-oat aphid, rwa – russian wheat aphid, cfda – carboxyfluorescein diacetate, cf – carboxyfluorescein. introduction transport of synthesised assimilates by the phloem in the vascular bundles of monocots has been extensively studied (see evert et al. 1977, 1978, 1988; fritz et al. 1989; botha and van bel 1992). barley leaves, like other typical monocots, contain three different vein acta bot. croat. 69 (1), 2010 7 * corresponding author: saheed@oauife.edu.ng u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:39:33 color profile: disabled composite 150 lpi at 45 degrees orders of vascular bundles, which are interconnected by cross veins (evert et al. 1996, botha and cross 1997). these vascular bundles in the leaf blades are known to serve either as loading bundles (in cross, small and intermediate veins) or in the longitudinal transport of assimilates (in the large bundle) across different plant tissues (lush 1976, altus and canny 1982, fritz et al. 1989, botha and cross 1997). the major constituent of the transported assimilate is sucrose, which in barley leaves is compartmentalised into transport (mesophyll and vascular tissues) and vacuolar pools (farrar and farrar 1986). phloem-feeding aphids derive their nutrient supply from these pools, and they aggregate on the parts of the plant where food is of high quality (kennedy and booth 1951). because phloem sap is low in protein, aphids need to ingest large quantities of the sap in order acquire enough amino acids, necessary for their survival, while excess water and sugars are later excreted as 'honeydew' (douglas 1993). aphids feed specifically from the sieve tubes of the vascular bundles and preferentially from the thin-walled sieve tubes (matsiliza and botha 2002). this shows that they select their feeding sites according to the quality and quantity of the food that is yielded by the feeding site. it is obvious therefore, that aphid feeding activities will adversely affect the transport capacity of the infested plants. a number of authors have reported that aphids become strong secondary sinks, due to the diversion of assimilates meant for other growing plant tissues. assimilate diversion may therefore result in morphological symptoms in infested plants (cagampang et al. 1974, hicks et al. 1984, nielsen et al. 1990, botha and matsiliza 2004). nevertheless, an interesting case occurs when two phloem-feeding aphids – russian wheat aphid, diuraphis noxia mordvilko (rwa) and bird cherry-oat aphid, rhopalosiphum padi l (bca) infest barley leaves. in this case, rwa-infested leaves express feeding symptoms with a much lower feeding populations over a two week period while bca-infested leaves do not, despite a much higher bca feeding population (saheed et al. 2007a). we have recently focused our attention on the study of the feeding mechanisms of aphids during the infestation of plants (see saheed et al. 2007a, b; 2009). this feeding mechanism has been further investigated with respect to the effects that rwa and bca feeding have on the transport capacity of the phloem in barley leaves. we used the phloem mobile fluorophore, 5, 6 carboxyfluorescein (5, 6-cf), to investigate the potential differences in the damage to the phloem transport as caused by bca and rwa. the diacetate 5, 6-cfda and some other fluorescein compounds used in situ to study phloem transport in plants provide reliable information (turgeon and beebee 1991, farrar et al. 1992, botha et al. 2000). 5, 6-cfda is non-polar compound, and when applied to damaged cells, it is taken up by plant cells and then moved across cell walls and membranes. once in physiologically intact tissues, the diacetate is cleaved, resulting in polar free 5, 6-cf which is non-permeable to cell membranes. it fluoresces while moving symplasmically within the contiguous cell of the phloem. 5, 6-cf has been used to study phloem transport during rwa infestation in wheat (botha and matsiliza 2004), and during infestation of wheat cultivars by sitobion yakini (de wet and botha 2007). these two studies provide background information concerning the reduction of phloem transport capacity during aphid feeding. however, literature on the effect of feeding by bca on the transport capacity of the phloem is non-existent or rare. the experiment reported in this paper was designed to examine the effects the rwa and bca had on the transport capacity of the phloem in barley leaf and to possibly relate it to 8 acta bot. croat. 69 (1), 2010 saheed s. a., jonsson l. m. v., botha c. e. j. u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:39:33 color profile: disabled composite 150 lpi at 45 degrees the diverse morphological symptoms expressed by infested plants (saheed et al. 2007a). we hypothesized that rwa causes a greater reduction in phloem transport capacities of the infested plants, leading to the visible symptoms observed and that a much lesser reduction (if any) is caused by the bca, perhaps, which is why there are no morphological symptoms in bca-infested plants. these results, we hope, will further elucidate the effects of aphid feeding on the transport capacities of the phloem as it relates it to visible symptoms shown by infested plants. materials and methods plant material, aphid colony maintenance and treatments barley (hordeum vulgare l. cv clipper) seeds were pre-germinated in petri dishes and sown in plastic pots containing 60:40; peat: vermiculite mixture potting soil. they were watered twice a week with long-aston nutrient solution (hewitt 1966), and grown under controlled environment (conviron s10h controlled environments limited, winnipeg, manitoba, canada) at 24 °c, 66% rh day and 22 °c, 60% rh night, 14 h photoperiod. illumination in the cabinets was provided using a combination of fluorescent tubes (f48t12. cw/vho1500, sylvania, danvers, ma) and frosted incandescent 60w bulbs (philips, eindhoven, the netherlands) and the irradiation level was 250 mmol m–2 s–1. the colonies of the rwa and bca were from the arc-small grain institute, bethlehem, south africa. the raising and maintenance of the aphid colonies was on young barley plants over at least three successive generations, to avoid any effects from previous hosts (shufran et al. 1992). insect cages were used to cover the aphids in separate growth cabinets. the maintenance of the colonies was at 18 °c, 66% rh day and 15.5 °c, 66% rh night, 14 h photoperiod. in all cases, five adult aphids were confined using clipcages, to feed on either sink or source leaves for 72 h (short-term) and 14 days (long-term). the leaves used in these experiments were about 12 cm long, while the clipcages, placed at the mid-region of the fully expanded leaves, were about 5 cm in length. source and sink leaves were used during short-term feeding experiments while only source leaves were used for long-term experiments, with 20 replicate plants established for each treatments. leaf material treatment intact plants were used for all the treatments. immediately after the infestation period, the leaves were gently abraded (to allow access to the fluorophore) on the abaxial surface with a sterilized needle. source leaves were abraded on the part above the clipcage; while the part below the clipcage was used in sink leaves. this is taking into consideration the classical pattern of assimilates movement, which is known to be basipetal (lamina tip to base) in source leaves and acropetal (lamina base to tip) in sink leaves (turgeon 1989). the abraded portion was rinsed with distilled water before treatment with 100 ml of 5,6-cfda (carboxyfluorescein diacetate, c-195 molecular probes, eugen, oregon usa, 217 mm in distilled water, kept foil-wrapped at –5 °c until needed) was added and covered with transparent polythene film (housebrand, brackenfell, south africa) to prevent evaporation. the 5, 6-cfda was allowed to be taken up through the abrasion and transported for 3h. at the end of the loading and transportation time, the leaves were detached, placed on a glass slide and covered with silicone oil to prevent 5, 6-cfda from leaking from the leaf. acta bot. croat. 69 (1), 2010 9 effects of aphids feeding on phloem transport u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:39:33 color profile: disabled composite 150 lpi at 45 degrees the fluorescence front, amount (intensity) as well as the distribution was observed under uv light in both control and aphid-infested leaves. this was done with the use of an olympus bx61 wide-field fluorescence microscope with a u-yfp filter set (10c/topaz 41028, chroma technologies, battlebro usa) with excitation of 513 nm and an emission of 527 nm. images were saved in a database using the program analysis (soft imaging system gmhb, germany), and imported as bitmaps to corel draw 12 (corel corporation ottawa, canada 2003) for presentation. the rate of transport (measured by the distance moved by the cleaved 5, 6-cf from the point of application to the fluorochrome front in three hours) was recorded and statistical analysis (anova) was used to compare the differences in the treatments. the results presented are based on acropetal and basipetal movements of the cleaved 5, 6-cf along the assimilate stream in the longitudinal bundles. results transport of 5, 6-cf in control barley leaves the movement of the dissociation product of 5,6-cfda (5, 6-cf) occurs from the site of application in the control, source, as well as sink leaves and it shows that the fluorochrome moved acropetally in sink leaves and basipetally in source leaves. repeated experiments revealed that the 5, 6-cf uptake starts with the mesophyll, then moves through the bundle sheath before loading into the vascular bundles (fig. 1a). loading was seen to start with small and intermediate bundles after which the fluorochrome moves towards the large exporting bundles. the transport of 5, 6-cf appears continuous and undisturbed (figs. 1b and c) up to the fluorochrome front (fig. 1d). the unloading sequence of the fluoro10 acta bot. croat. 69 (1), 2010 saheed s. a., jonsson l. m. v., botha c. e. j. fig. 1. transport of cleaved 5, 6-cf in control leaves of barley, a – the point of application of the fluorochrome (arrowheads), where the 5, 6-cf is been taken up and transported in the intermediate vein. b, c – undisrupted transport of the cleaved 5, 6-cf along intermediate veins, where the transport is smooth, even and continuous. d – the fluorochrome front (arrowheads) in an intermediate vein. scale bars: a = 100 mm; b, c = 200 mm; d = 150 mm. u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:39:35 color profile: disabled composite 150 lpi at 45 degrees chrome as expected shows a movement from the sieve tubes into the mesophyll tissue via the vascular parenchyma and bundle sheath (data not included). all these movements are purely symplasmic. three hours after the application of the 5, 6-cfda, the fluorochrome had moved approximately 5 cm from the point of application. transport of 5, 6-cf in bca-infested leaves after 72h (short-term) of feeding by bca, distribution of the 5, 6-cf was patchy (fig. 2a). despite this patchiness, there are no apparent reductions in the amount (intensity) or the distance moved by 5, 6-cf from the point of application when compared to the control tissue. however, after 14d (long-term) of continuous feeding, there is an apparent reduction in the intensity and distance moved by the fluorochrome from the point of application in the infested leaves when compared to the control leaves. we present the observed gradual reduction in the intensity of the transported 5, 6-cf (figs. 2 c–e), and the points of stylet penetrations and an increased fluorochrome concentration (figs. 2 c–e). interestingly, traces of ingested 5, 6-cf can be seen in the honeydew excreted by the aphid (fig. 2b). transport of 5, 6-cf in rwa-infested leaves transport of 5, 6-cf in rwa-infested barley leaves shows a dramatic reduction in the distance moved as well as the intensity of the fluorochrome after short-term feeding (72h). the intensity of the color of the fluorochrome 5, 6-cf before the point of aphid feeding (arrows) is much higher than after 72h of feeding point (arrowheads)(figs. 2f–g). prolonged feeding (long-term) by rwa results in a greater reduction in the intensity and also the distance moved by 5, 6-cf and in most cases, cessation of the transport ensues. we present transport of 5, 6-cf after 14d (long-term) feeding (figs. 2 h–j), and apparent leakages of the cleaved 5, 6-cf through many points of stylet penetrations (arrowheads; fig. 2h). we observed fluorochrome in the abdomen of an aphid (fig. 2i), and the total cessation in the movement of 5, 6-cf which occurred on many occasions (fig. 2j). comparison of the distance moved by 5, 6-cf in control and infested leaves analysis of variance (anova) of the difference of means of the distance moved by 5, 6-cf in control, bca and rwa infested leaves from 20 replicate samples was calculated and confirmed with tukey’s post hoc test (95%). the short-term feeding experiments showed no significant difference (p-values <0.0001) between the distance moved by 5, 6-cf in control and bca of the infested leaves (fig. 3). a significant difference was however found between the distances moved by 5, 6-cf in control and bca infested leaves on one hand and the rwa infested leaves on the other (fig. 3). in this case, rwa infestation led to a significant reduction in the distance moved by the fluorochrome after 72 h of feeding when compared to the distance moved in both control and bca infested tissue. however, after 14 d of feeding by the aphids (long-term), the result shows that there is significant difference (p < 0.0001) between the distances moved by the 5, 6-cf in the control, bca and rwa infested leaves. the movement of 5, 6-cf in the leaves infested with bca was reduced when compared to what was observed in the control leaves (fig. 4). on the acta bot. croat. 69 (1), 2010 11 effects of aphids feeding on phloem transport u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:39:35 color profile: disabled composite 150 lpi at 45 degrees 12 acta bot. croat. 69 (1), 2010 saheed s. a., jonsson l. m. v., botha c. e. j. fig. 2. transport of cleaved 5, 6-cf in bca and rwa-infested barley leaves. a–e – the pattern of transport and distribution of 5, 6-cf in bca-infested barley leaves. a – patchy transport pattern of the fluorochrome during short-term feeding by the aphids in intermediate vein (iv). b – the fluorochrome in the honeydew (arrowheads) ejected by the aphid. c–e – a gradual reduction in the transported 5, 6-cf during long-term feeding by bca. stylet points (arrowheads) show a higher concentration of the fluorochrome. f–j – the pattern of transport and distribution of 5, 6-cf in rwa-infested barley leaves. f – reduction in the transported 5, 6-cf after short-term feeding (72h) by rwa. note the feeding points (arrows) and reduced transport after the points (arrowheads). g – feeding in an intermediate vein, reduced transport of 5, 6-cf was observed after the point of aphid feeding. h – several stylet points (arrowheads) after long-term feeding. i – cleaved 5, 6-cf in the abdomen of an aphid. j – complete cessation of further transport of the fluorochrome after severe, long-term feeding by the aphids. note the leakages of the 5, 6-cf through many stylet tracks (arrows) out of the intermediate vein and stoppage of further movement (arrowheads) of the 5, 6-cf from the feeding points. scale bars: a = 100 mm; b = 150 mm; c = 100 mm; d = 150 mm; e = 200 mm; f = 150 mm; g = 200 mm; h = 50 mm; i = 150 mm; j = 150 mm. u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:39:59 color profile: disabled composite 150 lpi at 45 degrees other hand, transport of 5, 6-cf in rwa-infested leaves is further reduced (fig. 4) when compared to the movement in control and bca infested leaves, and on many occasions no transport or total cessation of transport occurs. discussion the results presented here only relate to longitudinal phloem transport as visualised by 5, 6-cf transport and its fluorescence. transport is generally acropetal in sink leaves and basipetal in source leaves. longitudinal transport of assimilates (visualised by transport of 5, 6-cf) in control barley leaves shows that the rate (the distance moved from the point of application of 5, 6-cfda to the 5, 6-cf front in 3 h) of phloem transport is at an average of 5 cm over a 3h period in all classes of veins. this observation is true in both source and sink acta bot. croat. 69 (1), 2010 13 effects of aphids feeding on phloem transport fig. 3. comparison of the means of distance moved by 5, 6-cf from point of application in control, bcaand rwa-infested leaves after short-term (72h) feeding by the aphids (+ standard deviation); anova: f2, 57 = 300.98; p < 0.0001 fig. 4. comparison of the means of distance moved by 5, 6-cf from point of application in control, bcaand rwa-infested leaves after long-term (14d) feeding by the aphids (+ standard deviation); anova: f2, 57 = 621.94; p < 0.0001 u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:40:00 color profile: disabled composite 150 lpi at 45 degrees leaves. the movement of the phloem-mobile 5, 6-cf follows classical source-sink transport patterns. the data presented here thus lend strong support to an earlier report that demonstrated similar movement of the fluorochrome in wheat leaves (botha and matsiliza 2004). once applied, the symplasm of the mesophyll tissues takes up the fluorochrome (fig. 1a) and subsequently loads it into the phloem through many symplasmic connections that exist between bundle sheath-vascular parenchyma and companion cells-sieve tube complexes (evert et al. 1996, botha and cross 2001, botha 2005) and then moved longitudinally. this transport is unrestricted and confined within the transporting veins of the control tissues (figs. 1 b–c). however, this movement pattern ends up in severe disruption upon feeding by the aphids. the focus of this work was on the impact on the longitudinal transport of assimilates when rwa and bca feed on barley leaves. the observed differences in the damage caused by the two aphid species to the leaf ultrastructures (saheed et al. 2007a) and corresponding deposition of wound callous (saheed et al. 2009) are further confirmed with the current phloem transport results. in short-term (72 h) feeding experiments, infestation by bca does not result in obvious reduction in the capacity of the phloem to transport assimilate (fig. 2a), as there was no significant difference in the transport of 5, 6-cf in the assimilate stream of control and bca-infested leaves (fig. 3). by contrast, rwa infestation led to a significant reduction in the 5, 6-cf transported in the assimilate stream and by implication, the phloem transport capacity within 72 h of feeding (figs. 2f-g, 3). however, reduction in the 5, 6-cf transported which resulted in a significant reduction in the phloem transport capacity was found when bca fed for 14 d (long-term) in comparison to the control plant (figs. 2c–e, 4). obviously, rwa infestation results in a pronounced reduction in the 5, 6-cf transported, and in most cases complete cessation of transport ensues (fig. 2j). this infestation causes a greater reduction in phloem transport capacity (fig. 4) when compared to observations in both control and bca infested leaves. interestingly, the ingested 5, 6-cf in theassimilates were visible in the egested honeydew of bca (fig. 2b) and also in the abdomen of rwa (fig. 2i). the data presented here show that feeding by rwa resulted in patchiness in 5, 6-cf distribution (figs. 2h, j) to a much greater extent than observed in bca infested leaves (figs. 2c–e). these results clearly support the position that aphids redirected assimilates that are normally transported in the veins of the leaves, thereby depressing the sink strength in growing regions of the plant. diversion and disruption of transported assimilates have been reported in different species of phloem feeding insects. nielsen et al. (1990) have reported the disruption of assimilate transport when the potato leafhopper (empoasca fabae) feeds on medicago sativa (alfalfa). feeding by the planthopper (nilaparvata lugens) on rice has also been shown to result in removal of assimilates and reduction in photosynthesis (watanabe and kitagawa 2000), this feeding eventually causeing a reduction in rice growth and yield. hill (1962) had earlier shown that if the redirection of assimilates by the phloem feeding insects is strong and localized, the plant will react to it in some respect, as if a bud were involved. this work revealed that diversion and disruption of the assimilate transport pathway in barley leaves is more apparent in response to rwa than to bca feeding. this suggests that the damage inflicted by rwa is greater than that caused by bca. therefore, we can say that rwa is a more aggressive feeder than bca. rwa disrupts the phloem system thereby 14 acta bot. croat. 69 (1), 2010 saheed s. a., jonsson l. m. v., botha c. e. j. u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:40:00 color profile: disabled composite 150 lpi at 45 degrees reducing its transport capacity; this is evident with the reduction in the transport of the 5, 6-cf – a phloem-mobile xenophore. the rwa apparently redirects more assimilate to itself than the bca and this position finds support in our earlier papers where we have shown that damage to leaf ultrastructures was more severe and callous formation (wounding effects) was more intense when rwa infested plants than when bca infested them (saheed et al. 2007a, 2009). in terms of yield losses, we suggest that the greater disruption and diversion of assimilates in rwa infested barley leaves appears to be responsible for the higher yield losses (30% to 60%) reported during rwa-infestation (du toit and walters 1984). on the other hand a reduced disruption and diversion of assimilates results in a reduced (21%) yield loss that occurs during bca-infestation (riedell et al. 1999). this suggestion finds supports in earlier observations in rice, where disruption and diversion of assimilates causes yield losses in infested plants (watanabe and kitagawa 2000). the results thus presented further elucidate the mechanism behind aphid feeding and responses of the infested plants, by revealing more reasons why rwa-infested leaves show symptoms of chlorosis, necrosis and leaf roll and leaves infested by bca do not. in conclusion, the symptoms shown by rwa infested plants, which eventually lead to the subsequent losses in yield greater than in bca-infested plants, are suggested to be partly due to the aphid’s ability to inflict severe damage on the phloem transport of the infested plant. this damage leads to noticeable reduction in the transport of assimilates within 72h of rwa infestation and a significant reduction during prolonged feeding which, in most cases, results in total cessation of phloem transport. bca feeding, conversely, does not appear to cause as much serious damage to the transport capacities of the phloem, with only reduced assimilate transport occurring during a prolonged infestation. acknowledgements the authors thank dr vicky tolmay of the arc bethlehem, south africa for the supply of aphids used in this study, the national research foundation, pretoria south africa for supporting cejb research programme and for grant-holder bursary given to ssa (2006–7). we also thank the swedish foundation for international cooperation in research and higher learning and the swedish international development co-operation agency for their grants to cejb and lmvj and the swedish foundation for strategic environmental research (mistra) for support to the plantcommistra program. references altus, d. p., canny, m. j., 1982: loading of assimilates in wheat leaves. the specialization of vein types for separate activities. australian journal of plant physiology 9, 571–581. botha, c. e. j., van bel, a. j. e., 1992: quantification of symplastic continuity as visualized by plasmodesmograms: diagnostic value for phloem-loading pathways. planta 187, 359–366. acta bot. croat. 69 (1), 2010 15 effects of aphids feeding on phloem transport u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:40:00 color profile: disabled composite 150 lpi at 45 degrees botha, c. e. j., cross, r. h. m., 1997: plasmodesmatal frequency in relation to short-distance transport and phloem loading in leaves of barley (hordeum vulgare). phloem is not loaded directly from the symplast. physiologia plantarum 99, 355–362. botha, c. e. j., cross, r. h. m., van bel, a. j. e., peter, c. i., 2000: phloem loading in the sucrose-export-defective (sxd-1) mutant maize is limited by callose deposition at plasmodesmata in bundle sheath-vascular parenchyma interface. protoplasma 214, 65–72. botha, c. e. j., cross, r. h. m., 2001: regulation within the supracellular highway-palsmodesmata are the key. south african journal of botany 67, 1–9. botha, c. e. j., matsiliza, b., 2004: reduction in transport in wheat (triticum aestivum) is caused by sustained phloem feeding by russian wheat aphid (diuraphis noxia). south african journal of botany 70, 249–254. botha, c. e. j., 2005: interaction of phloem and xylem during phloem loading – functional symplasmic roles for thinand thick-walled sieve tubes in monocotyledons? in: holbrook, n. m., zweiniecki, m. a. (eds.), vascular transport in plants, 115–130. elsevier, london. cagampang, g. b., pathak, m. d., juliano, o. b., 1974: metabolic changes in the rice plant during infestation by brown planthopper, nilaparvata lugens stål. (hemiptera: delphacidae). applied entomology and zoology 9, 174–184. de wet, l. r., botha, c. e. j., 2007: resistance or tolerance: an examination of aphid (sitobion yakini) phloem feeding on betta and betta-dn wheat (triticum aestivum l.). south african journal of botany 71, 35–39. douglas a. e., 1993: the nutritional quality of phloem sap utilized by natural aphid populations. ecological entomology 18, 31–38. du toit, f., walters, m. c., 1984: damage assessment and economic threshold values for chemical control of the russian wheat aphid diuraphis noxia (mordvilko) on winter wheat. in: walters m. c. (ed.), progress in the russian wheat aphid (diuraphis noxia (mordw.) research in the republic of south africa. technical communication 191, 58–62. department of agriculture, republic of south africa. evert, r. f., eschrich, w., heyser, w., 1977: distribution and structure of the plasmodesmata in mesophyll and bundle-sheath cells of zea mays l. planta 136, 77–89. evert, r. f., eschrich, w., heyser, w., 1978: leaf structure in relation to solute transport and phloem loading in zea mays l. planta 138, 279–294. evert, r. f., mierzwa, r. j., eschrich, w., 1988: cytochemical localization of phosphatase activity in vascular bundles and contiguous tissues of the leaf of zea mays l. planta 159, 193–206. evert, r. f., russin, w. a., botha c. e. j., 1996: distribution and frequency of plasmodesmata in relation to photoassimilate pathways and phloem loading in the barley leaf. planta 198, 572–579. farrar, s. c., farrar, j. f., 1986: compartmentation and fluxes of sucrose in intact leaf blades of barley. new phytologist 103, 645–657. 16 acta bot. croat. 69 (1), 2010 saheed s. a., jonsson l. m. v., botha c. e. j. u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:40:00 color profile: disabled composite 150 lpi at 45 degrees farrar, j., van der schoot, c., drent, p., van bel, a. j. e., 1992: symplastic transport of lucifer yellow in mature leaf blades of barley: potential mesophyll-to-sieve-tube transfer. new phytologist 120, 191–196. fritz, e., evert, r. f., nasse, h., 1989: loading and transport of assimilates in different maize leaf bundles. digital image analysis of 14c-microautoradiographs. planta 178, 1–9. hewitt, e. j., 1966: sand and water culture methods used in the study of plant nutrition. technical communications 22. commonwealth agricultural bureau, farnham, england. hicks, p. m., mitchell, p. l., dunigan, e. p., newsom, l. d., bollich, d., 1984: effect of three-cornered alfalfa hopper (homoptera: membracidae) feeding on translocation and nitrogen fixation in soybeans. journal of economic entomology 77, 1275–1277. hill, g. p., 1962: exudation from aphid stylets during the period of dormancy to bud break in tili americana (l). journal of experimental botany 13, 144–151. kennedy, j. s., booth, c. o., 1951: host alternation in aphis fabae scoop. i. feeding preferences and fecundity in relation to the age and kind of leaves. annals of applied biology 38, 25–64. lush, w. m., 1976: leaf structure and translocation of dry matter in a c3 and c4 grass. planta 130, 234–244. matsiliza, b., botha, c. e. j., 2002: aphid (sitobion yakini, eastop) investigation shows thin-walled sieve tubes to be more functional than thick-walled sieve tubes. physiologia plantarum 115, 137–143. nielsen, g. r., lamp, w. o., stutte, g. w., 1990: potato leafhopper (homoptera: cicadellidae) feeding disruption of phloem translocation in alfalfa. journal of economic entomology 83, 807–813. riedell, w. e., kieckhefer, r. w. haley, s. d., langham, m. a. c., evenson, p. d., 1999: winter wheat responses to bird cherry-oat aphids and barley yellow dwarf. crop science 39, 158–163. saheed, s. a., botha,c. e. j., liu, l., jonsson, l., 2007a: comparison of structural damage caused by russian wheat aphid (diuraphis noxia) and bird cherry-oat aphid (rhopalosiphum padi) in a susceptible barley cultivar, hordeum vulgare cv. clipper. physiologia plantarum 129, 429–435. saheed, s. a., liu, l., jonsson, l., botha, c. e. j., 2007b: xylem – as well as phloem – sustains severe damage due to feeding by russian wheat aphid. south african journal of botany 73, 593–599. saheed, s. a., cierlik, i., larsson, k., delp, g., bradley, g., jonsson, l. m. v., botha, c. e. j., 2009: stronger induction of callose deposition in barley by russian wheat aphid than bird cherry-oat aphid is not associated with differences in callose synthase or b-1,3-glucanase transcript abundance. physiologia plantarum 135, 150–161. acta bot. croat. 69 (1), 2010 17 effects of aphids feeding on phloem transport u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:40:01 color profile: disabled composite 150 lpi at 45 degrees shufran, k. a., margolies, d. c., black, i. v. w. c., 1992: variations between biotype e clones of schizaphis graminum (homoptera: aphididae). bulletin of entomological research 92, 407–416. turgeon, r., 1989: the sink-source transition in leaves. annual review of plant physiology 40, 119–138. turgeon, r., beebe, d. u., 1991: the evidence for symplastic phloem loading. plant physiology 96, 349–345. watanabe, t., kitagawa, h., 2000: photosynthesis and translocation of assimilates in rice plants following phloem feeding by planthopper nilaparvata lugens (homoptera: delphacidae). journal of economic entomology 93, 1192–1198. 18 acta bot. croat. 69 (1), 2010 saheed s. a., jonsson l. m. v., botha c. e. j. u:\acta botanica\acta-botan 1-10\saheed.vp 9. travanj 2010 11:40:01 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 69 (2), 259–274, 2010 coden: abcra 25 issn 0365–0588 palynological classification of onosma l. (boraginaceae) species from east mediterranean region in turkey riza bi·nzet1*, i · rfan kandemi·r2, nermi·n orcan3 1 adiyaman university, faculty of science and art, department of biology, 02040 adiyaman, turkey 2 ankara university, faculty of science, department of biology, ankara, turkey 3 mersin university, faculty of science and art, department of biology, mersin, turkey twenty-five onosma (boraginaceae) taxa belonging to two subsections haplotricha and asterotricha from the east mediterranean region in turkey were studied by palynological analysis and numerical taxonomy. application of discriminant function analysis to raw data obtained from the acetolysis and wodehouse methods resulted in very good allocation of species to their original groups. however the results obtained from acetolysis (99%) resulted in better discrimination than the wodehouse (97%) method. a similar outcome was reached in principal component analysis and upgma. higher percentage of phenetic variation was explained by the acetolysis method. the utility of palynological data in taxonomic classification with the use of using numerical methods is discussed. keywords: onosma, pollen, numerical taxonomy, classification, turkey abbreviations: dfa – discriminant function analysis, upgma – unweighted pair group method with arithmetic mean introduction recent studies and revisions have increased the number of species in the genus onosma l. to over 230 species (boissier 1897, dinsmor 1932, hayek and markgraf 1970, tutin et al. 1972, shishkin 1974, riedl 1978, meikle 1985, teppner 1991, ge-ling et al. 1995). this genus has biennial and perennial members, and is generally suffruticose. onosma species are recognized on the basis of indumentum characteristics along with flowers in terminal cymes, calyx accrescence, stamens inserted at the middle of the corolla. the genus onosma (boraginaceae) is represented by about 102 taxa (97 species) in turkey and the endemism among native species is higher than 50 % (riedl 1978, yildirimli 2000, riedl et al. 2005, bi·nzet and orcan 2007). in the flora of turkey, the general classification of onosma species was based only on indumentum characteristics, and palynological acta bot. croat. 69 (2), 2010 259 * corresponding author, e-mail : rbinzet@gmail.com u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:45 color profile: disabled composite 150 lpi at 45 degrees data were not utilized. in addition, maggi et al. (2008) mentioned the lack of palynological data on the genus onosma. only a limited number of studies can be found in the literature; the first palynological study in the boraginaceae was that of erdtman (1952). the genera of lithospermae comprising 45 species of onosma were palynologically studied with light microscopy (johnston 1954). the pollen morphology of the family boraginaceae have been studied by erdtman (1952), marticorena (1968), huynh (1971, 1972), nowicke and ridgway (1973), nowicke and skvarla (1974), diez (1984), qureshi and qaiser (1987) and perveen et al. (1995). in recent years several onosma species from turkey have been examined for pollen morphology by bi·nzet and orcan (2003a, b). although palynological features can provide a wealth of taxonomic characters that have been important in inferring phylogenetic relationships and future classifications, there was not enough palynological data for the genus onosma. in the present study we investigated the pollen morphology of the 25 taxa belonging to onosma which is a difficult genus from systematic and taxonomic point of view. in order to resolve these difficulties and make use of palynological data in the genus onosma we applied the numerical taxonomic approach. this method has had a wide application to different plant taxa in turkey (togan et al. 1983, kence 1988, doðan et al. 1992, doðan and tosunoðlu 1992, doðan 1997, tütel et al. 2005). thus, we have attempted to classify 25 onosma taxa on the basis of palynological characters obtained by two different methodologies (acetolysis and wodehouse) and tried to find a future methodology to be used in palynological data collection in the future. materials and methods pollen grains obtained from herbarium and fresh materials were prepared using the methods described by wodehouse (wodehouse 1935) and acetolysis by erdtman (erdtman 1952). the wodehouse and acetolysis methods were followed by light microscopic (lm) study. polar and equatorial axis, pollen shape, length of pores (pori) and colpus (colpi), width of pores (pori) and colpus (colpi), exine thickness, intine thickness and length of polar triangular edge were measured with an olympus bx 40 microscope (x10; x 100). the terminology used is in accordance with erdtman (1952) and faegri and iversen (1964). at least 30 pollen grains were measured from each of 25 onosma taxa. the raw data obtained from both acetolysis and wodehouse were analysed with discriminant function analysis (dfa) using the statistical package spss (2004) separately in order to find out the palynological relationships between 25 taxa (tab. 1). the second sets of onosma data matrix were formed (the averages of the species used in dfa) from 9 palynological characters for acetolysis and 10 for wodehouse methods (tabs. 2, 3). the matrices obtained from the two different methods were standardised and the new character distribution has mean 0 and standard deviation 1. principal component analysis was applied to the standardized data sets using correlation matrices and finally upgma dendrograms were constructed based on the average taxonomic distance using ntsys-pc (rohlf 2004). statistical analyses were performed using statistical package (2004). 260 acta bot. croat. 69 (2), 2010 bi·nzet r., kandemi·r i · ., orcan n. u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:45 color profile: disabled composite 150 lpi at 45 degrees results the acetolysis and wodehose average values are listed in tables 2 and 3. principal component analysis and upgma were applied to acetolysis and wodehouse data in order to visualize the palynological relationships of onosma genus in the eastern mediterranean region. discriminant function analysis of acetolysis and wodehouse data all characters used both in acetolysis and wodehouse were significantly different among onosma taxa (p<0.01) based on univariate anova. a scatterplot of 25 onosma acta bot. croat. 69 (2), 2010 261 palynological classification of onosma tab. 1. onosma species and collection localities along with grid square information species locality (grid square) onosma albo-rosea fisch. et mey. kahramanmaraþ, kayseri: b6; mersin: c4 o. angustissima hausskn. et bornm.* antalya: c3; mersin: c4; adana: c5 o. armena dc.* mersin, karaman: c4 o. aucherana dc. mersin: c4 o. auriculata aucher ex dc. mersin: c4 o. bornmuelleri hausskn.* mersin: c5 o. bracteosa hausskn. et bornm.* karaman, mersin: c4; mersin: c5 o. caerulescens boiss. kahramanmaraþ: c6 o. cassia boiss. hatay: c5 o. frutescens lam. antalya: c3; mersin: c4 o. gigantea lam. adana, kahramanmaraº: b6; mersin: c4; mersin: c5; osmaniye, adana, osmaniye, kahramanmaraº: c6 o. inexspectata teppner * osmaniye: c6 o. isaurica boiss. et heldr.,* mersin, karaman: c4; kilis: c6 o. lycaonica hub.-mor.* mersin: c4 o. mersinana h. riedl, binzet et orcan * mersin: c5 o. mutabilis boiss.* kahramanmaraþ: b6; mersin, adana: c5 o. papillosa h. riedl * adana: b6 o. rascheyana boiss. kahramanmaraþ: c6 o. riedliana binzet et orcan * mersin: c5 o. roussaei dc. mersin: c4 o. rutila hub.-mor.* mersin: c4 o. sericea willd. hatay, kilis, gaziantep, kahramanmaraþ, osmaniye: c6; adana: b6 o. sieheana hayek * karaman, mersin: c4 o. stenoloba hausskn. ex h. riedl * kahramanmaraþ: b6; mersin: c5 o. taurica pallas ex willd. mersin: c4; hatay: c6 *endemic species u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:45 color profile: disabled composite 150 lpi at 45 degrees 262 a c t a b o t .c r o a t .69 (2),2010 b i ·n z e t r .,k a n d e m i ·r i ·.,o r c a n n . tab. 2. palynological characters of the onosma species for wodehouse (measurements in mm). species a p e plg plt clg clt ex i ect/end t o. albo-rosea m 17.56 15.47 3.54 3.98 13.21 2.75 0.72 0.78 0.25 6.67 sd 0.46 0.52 0.47 0.58 0.49 0.24 0.12 0.07 0.3 min–max 16–18.5 14–16 2.5–4.5 3–5 12.5–13.5 2.5–3 0.5–0.9 0.6–1 6–7 o. angustissima m 14.99 12.72 3.01 3.63 12.38 3.36 0.37 0.68 0.66 6.4 sd 0.57 0.71 0.25 0.37 0.43 0.33 0.03 0.08 0.25 min–max 13.5–16 11.5–13.5 2.4–4.2 3–4.2 11.5–14 3–4 0.3–0.5 0.5–0.8 6–7 o. armena m 15.83 13.28 3.15 3.6 12.31 2.45 0.55 0.8 1.5 6.53 sd 0.51 0.71 0.46 0.43 0.5 0.28 0.07 0.07 0.29 min–max 14.5–16.5 12–14 2–4 2.5–4.5 11–13 1.5–3 0.4–0.7 0.6–1 6–7 o. aucherana m 14.63 11.32 2.65 3.08 10.62 2.87 0.44 0.62 0.5 5.7 sd 0.58 0.43 0.43 0.4 0.56 0.27 0.04 0.06 0.27 min–max 13.5–16 10–12 1.5–3.5 2–4 9–12 2.4–3 0.3–0.5 0.5–0.8 5–6 o. auriculata m 17.92 13.86 3.72 4.1 14.37 3.49 0.6 0.8 0.33 7.46 sd 0.47 0.46 0.46 0.5 0.64 0.31 0.08 0.06 0.32 min–max 16.5–18.5 12.5–14.5 2.5–4.5 3–5 13–15 3–4 0.4–0.8 0.6–1 6.5–8 o. bornmuelleri m 16.46 12.9 3.91 4.35 12.24 3.68 0.46 0.77 0.33 6.32 sd 0.49 0.56 0.54 0.51 0.53 0.3 0.06 0.08 0.38 min–max 15–17 11.5–13.5 3–4.5 3.5–5 11–14 3–4 0.3–0.7 0.5–1 4–7 o. bracteosa m 15.69 13.26 3.53 3.6 12.61 2.85 0.75 1.03 3 6.57 sd 0.58 0.62 0.53 0.64 0.71 0.23 0.04 0.12 0.32 min–max 14.2–16.5 12–14 3–3.9 2.5–4.5 11–13 2.5–3.2 0.5–0.8 0.8–1.2 6–7 o. caerulescens m 17.03 14.01 3.28 3.73 13.09 3.02 0.41 0.63 0.66 5.86 sd 0.62 0.24 0.22 0.3 0.61 0.28 0.07 0.07 0.29 min–max 15.5–18 13–14.5 2.8–3.6 3.2–4.2 12.5–13.5 2.5–3.5 0.3–0.5 0.5–0.8 5–6.5 o. cassia m 14.84 10.37 2.75 3.66 11.31 3.19 0.39 0.71 0.6 5.1 sd 0.5 0.47 0.57 0.61 0.52 0.32 0.05 0.09 0.29 min–max 13.5–15.5 9.5–11.5 1.5–4 2.5–5 10–12 2.5–3.5 0.3–0.5 0.6–0.8 4–6 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 8 2 b i n z e t . v p 1 1 . l i s t o p a d 2 0 1 0 1 3 : 5 1 : 4 6 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s a c t a b o t .c r o a t .69 (2),2010 263 p a l y n o l o g ic a l c l a s s if ic a t io n o f o n o s m a tab. 2. – continued species a p e plg plt clg clt ex i ect/end t o. frutescens m 14.82 11.44 3.25 3.13 12.01 2.72 0.6 0.73 0.66 6.49 sd 0.51 0.55 0.59 0.52 0.57 0.25 0.05 0.08 0.35 min–max 13–15.5 10–12.5 2–4.5 2–4 10–13 2–3 0.4–0.8 0.6–0.9 5.5–7 o. gigantea m 17.56 13.64 3.76 3.76 14.21 2.83 1 0.41 1.5 7.04 sd 0.64 0.48 0.55 0.55 0.48 0.25 0.14 0.08 0.33 min–max 15–20 11–16 2.5–4.5 2.5–4.5 13–14.5 2.5–3.5 0.8–1.1 0.3–0.5 6–8 o. inexspectata m 15.81 12.75 2.78 3.07 11.77 2.83 0.36 0.75 0.5 5.9 sd 0.51 0.4 0.23 0.21 0.43 0.27 0.07 0.09 0.26 min–max 14.5–16.5 11.5–13.5 2.2–3.4 2.6–3.4 11.5–13 2.5–3 02–0.5 0.6–0.9 5–6.5 o. isaurica m 17.88 15.13 3.35 3.79 14.05 3.11 0.44 0.67 0.5 6.66 sd 0.65 0.5 0.32 0.28 0.51 0.3 0.06 0.07 0.37 min–max 16–19 13.5–16 2.8–4 3.2–4.4 12–15 2–3.5 0.3–0.6 0.5–0.8 5–8 o. lycaonica m 16.78 14.14 3.84 4.34 13.6 3.46 0.46 0.82 1.5 7.03 sd 0.81 0.65 0.61 0.5 0.62 0.31 0.06 0.08 0.4 min–max 15–18 13–15 2.5–5 3–5.5 12.5–14 3–4 03–0.6 0.7–1 4.5–8 o. mersinana m 16.47 11.7 2.82 3.49 11.87 2.81 0.36 0.72 0.5 7.59 sd 0.57 0.44 0.44 0.58 0.38 0.22 0.03 0.08 0.37 min–max 15–17 10.5–12.5 2–3.5 2.4–4.5 11–12.5 2–3.5 0.3–0.4 0.8–0.9 7–8 o. mutabilis m 16.03 14.28 4.18 4.15 13.06 3.25 0.52 0.89 2.5 8.05 sd 0.56 0.64 0.48 0.57 0.87 0.23 0.07 0.07 0.43 min–max 14.5–17 12.5–14.5 3.5–4.7 3–5 12–14 2.5–4 0.3–0.6 0.7–0.9 7–9 o. papillosa m 16.05 11.46 3.04 3.5 12.1 2.67 0.4 0.84 0.66 6.29 sd 0.63 0.52 0.47 0.67 0.54 0.26 0.05 0.08 0.33 min–max 14.5–17 10–12 2–4 2.5–4.5 10–13 2–3 0.3–0.5 0.7–0.9 5–8 o. rascheyana m 15.73 12.68 3.07 3.18 12.16 2.74 0.62 0.77 0.5 4.86 sd 0.43 0.48 0.19 0.25 0.65 0.23 0.04 0.05 0.22 min–max 14.5–16.5 11.5–13.5 2.6–3.4 2.6–3.6 11–13 2–3 0.4–0.8 0.6–1 4–6 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 8 2 b i n z e t . v p 1 1 . l i s t o p a d 2 0 1 0 1 3 : 5 1 : 4 6 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s 264 a c t a b o t .c r o a t .69 (2),2010 b i ·n z e t r .,k a n d e m i ·r i ·.,o r c a n n . tab. 2. – continued species a p e plg plt clg clt ex i ect/end t o. riedliana m 16.32 12.72 2.79 3.59 12.61 3.26 0.41 0.65 0.66 7.75 sd 0.55 0.39 0.44 0.43 0.73 0.26 0.05 0.07 0.33 min–max 15–17 12–13.5 2–3.5 2.5–4.5 11.5–13.5 2.5–3.5 0.3–0.5 0.5–0.8 7–8 o. roussaei m 14.84 10.5 2.79 2.82 10.71 2.45 1 0.51 2 5.96 sd 0.51 0.44 0.27 0.27 0.33 0.2 0.16 0.07 0.24 min–max 13–16 9–11.5 2–3 2–3 10–11 2–3 0.8–1.2 0.4–0.6 5.5–6 o. rutila m 14.88 10.68 2.66 2.78 12.98 2.3 0.43 0.67 0.33 5.68 sd 0.58 0.45 0.18 0.27 0.43 0.28 0.07 0.08 0.27 min–max 13.5–15.5 9.5–11 2.2–3 2.2–3.2 12–13.5 1.5–3 0.2–0.6 0.4–0.9 4–7 o. sericea m 17.43 14.7 3.43 4.13 13.9 3.62 0.29 0.71 0.5 6.49 sd 0.58 0.58 0.29 0.27 0.56 0.28 0.06 0.07 0.32 min–max 16–18 13–16 2.8–4 3.4–4.6 13–15 3–4 0.2–0.4 0.5–0.9 3–8 o. sieheana m 15.63 13.85 3.25 3.72 12.71 3.37 0.47 0.8 0.66 6.2 sd 0.72 0.5 0.62 0.55 0.63 0.31 0.08 0.06 0.3 min–max 14–16.5 12.5–14.5 2–4.5 2.5–5 11–14 2.5 4 0.3–0.7 0.7–0.9 5–7 o. stenoloba m 15.78 12.36 3.13 3.59 10.94 3.11 0.41 0.71 0.66 6.78 sd 0.45 0.48 0.43 0.44 0.38 0.29 0.03 0.08 0.28 min–max 14.5–16.5 11–13 2–4 2.5–4.5 9–12 2.5–3.5 0.3–0.6 0.6–1 6–7 o. taurica m 16.32 15.28 3.64 4.12 13.16 4.03 0.64 0.93 0.66 6.79 sd 0.55 0.54 0.47 0.47 0.5 0.3 0.1 0.1 0.3 min–max 14.5–17 14–16 2.5–4.5 3–5 12–13.5 3.5–4.5 0.5–0.8 0.7–1.1 6–8 m – mean, sd – standard deviation, min–max – minimum and maximum values), p – length of polar axis, e –width of equatorial axis, plg – length of pores (pori), plt – width of pores (pori), clg – length of colpus (colpi), clt – width of colpus (colpi), ex – exine thickness, i – intine thickness (only for wodehouse method), ect./end – ectexine to endexine ratio, t – length of polar triangular edge u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 8 2 b i n z e t . v p 1 1 . l i s t o p a d 2 0 1 0 1 3 : 5 1 : 4 6 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s a c t a b o t .c r o a t .69 (2),2010 265 p a l y n o l o g ic a l c l a s s if ic a t io n o f o n o s m a tab. 3. palynological characters of the onosma species for acetolysis (measurements in mm). species a p e plg plt clg clt ex ect/end t o. albo–rosea m 22.51 18.82 2.33 10.82 15.3 2.8 0.99 0.25 9.12 sd 0.92 1.08 0.27 0.81 0.8 0.24 0.1 0.55 min–max 20–24 16–21 1.8–2.8 9–12 14–16 1.5–2.5 0.8–1.1 8.5–10 o. angustissima m 15.2 13.21 1.29 7.07 11.06 2.82 0.89 0.33 5.4 sd 0.81 0.76 0.26 0.79 0.64 0.43 0.08 0.38 min–max 13.0–16.5 11.5–15.0 0.8–1.8 5.5 –9 10–12 2.4–3.2 0.7–1 5–6 o. armena m 16.51 14.18 1.76 6.39 13.26 1.94 0.98 1.5 6.4 sd 0.89 0.65 0.26 0.66 0.77 0.18 0.09 0.47 min–max 14.5–18 12.5–15 1–2.2 5–7 12–14 1.5–2.5 0.8–1.2 6–7 o. aucherana m 19.59 14.54 2.13 7.51 13.42 2.27 0.98 0.5 6.56 sd 0.99 0.95 0.21 0.8 0.75 0.25 0.12 0.32 min–max 17–21 13–16 1.6–2.6 6–9 12–14 1.8–2.5 0.8–1.1 6–7 o. auriculata m 27.13 20.83 2.59 10.78 17.65 2.49 1.24 0.33 9.97 sd 1.76 1.71 0.28 1.49 1.2 0.21 0.15 0.76 min–max 22–29 16–23 2–3.2 8–14 13–21 1.5–3.5 1–1.6 8–11 o. bornmuelleri m 20.82 16.12 2.45 9.39 14.83 2.58 1.11 0.33 7.7 sd 1.01 0.71 0.33 0.5 0.8 0.26 0.09 0.68 min–max 18–23 14.5–17 1.8–3.2 7.5–11 13–15 2–3 1–1.2 6.5–9 o. bracteosa m 15.94 13.39 0.79 7.07 13.09 3.28 0.66 0.33 7.56 sd 0.67 0.45 0.21 0.66 0.58 0.25 0.07 0.41 min–max 14.5–17 12–14 0.4–1.2 5.5–8.5 9–15 2.5–4.5 0.5–0.9 6–8 o. caerulescens m 15.55 13.2 1.59 7.07 10.36 2.64 0.76 0.66 5.68 sd 0.91 0.88 0.18 0.95 0.4 0.32 0.08 0.48 min–max 13–17 12–15 1–2 5–9 9.5–11 2–3 0.6–1 5–6.5 o. cassia m 17.3 4 13.19 1.62 6.63 14.79 4.78 0.84 0.66 7.14 sd 0.95 0.54 0.18 0.63 0.76 0.43 0.08 0.48 min–max 15–19 11.5–14 1.2–2 5–8 13–16 4–5.5 0.6–1 6–8 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 8 2 b i n z e t . v p 1 1 . l i s t o p a d 2 0 1 0 1 3 : 5 1 : 4 6 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s 266 a c t a b o t .c r o a t .69 (2),2010 b i ·n z e t r .,k a n d e m i ·r i ·.,o r c a n n . tab. 3. – continued species a p e plg plt clg clt ex ect/end t o. frutescens m 18.27 14.07 1.08 8.89 14.79 3.43 0.89 0.33 9.28 sd 1.18 0.57 0.3 0.69 0.7 0.34 0.09 0.76 min–max 15–21 12.5–15 0.6–1.8 7–10 13–16 3–4 0.7–1 8–10 o. gigantea m 19.33 15.91 1.98 9.11 17.51 2.41 0.77 0.33 8.9 sd 0.82 0.7 0.29 0.87 0.97 0.2 0.11 0.57 min–max 17.5–20 14–17 1.4–2.4 7–10.5 16–18 2–3 0.6–1 8–10 o. inexspectata m 14.46 11.89 1.3 6.91 10.2 2.49 0.7 0.5 5.22 sd 0.47 0.45 0.25 0.71 0.57 0.23 0.06 0.52 min–max 13.5–15 11–12.5 0.8–1.8 5.5–8 9.5–10.5 2–3 0.5–1 4.5–6 o. isaurica m 16.39 14.21 1.66 7.04 15.5 2.07 0.99 0.5 11.83 sd 0.74 0.72 0.34 1.13 0.92 0.33 0.1 0.67 min–max 14.5–17.5 12.5–15 1–2.2 5–9 14–16 1.8–2.4 0.8–1.2 10–13 o. lycaonica m 20.31 16.8 2.26 9.01 15.75 3.12 1.07 0.25 8.76 sd 1.09 0.65 0.32 0.77 0.8 0.31 0.13 0.54 min–max 17–22 15–18 1.6 –3 7.5–10.5 15–16 2.5–4 0.9–1.2 7–10 o. mersinana m 16.1 13.14 1.02 7.01 14.23 3.06 0.77 0.5 7.07 sd 0.78 0.72 0.2 0.79 0.87 0.3 0.08 0.5 min–max 14–17 11.5–14 0.6–1.4 5.5–8.5 13–14.5 2–4 0.6–1 6.5–7.5 o. mutabilis m 15.83 13.7 1.14 7.78 13.76 2.99 0.65 0.33 8.83 sd 0.54 0.45 0.26 0.99 0.73 0.2 0.06 0.73 min–max 14.5–16 12 –14 5 0.8–1.4 6.5–8.5 12.5–14.5 2–4 0.5–0.8 8–10 o. papillosa m 18.41 13.3 2.09 6.93 12.98 2.24 1.19 0.33 6.63 sd 0.83 0.63 0.48 0.74 0.62 0.32 0.9 0.39 min–max 15–20 11–14.5 1–3 4.5–9 12–14 2–2.5 0.8–1.2 5–8 o. rascheyana m 15.55 13.21 1.33 6.85 10.3 2.29 0.71 0.33 5.19 sd 0.62 0.47 0.26 0.66 0.6 0.25 0.08 0.38 min–max 14–16.5 12–14 0.8–2 5.5–8 9–11 1.5–3 0.6–0.9 4.5–5.5 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 8 2 b i n z e t . v p 1 1 . l i s t o p a d 2 0 1 0 1 3 : 5 1 : 4 7 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s a c t a b o t .c r o a t .69 (2),2010 267 p a l y n o l o g ic a l c l a s s if ic a t io n o f o n o s m a tab. 3. – continued species a p e plg plt clg clt ex ect/end t o. riedliana m 19.38 14.05 2.08 6.92 14.99 3.61 0.95 0.66 7.84 sd 1.52 0.86 0.46 0.98 0.87 0.4 0.11 0.5 min–max 17–21 13–16.5 1–3 6–9 12–15.5 3–4 0.8–1 7–9 o. roussaei m 14.8 12.13 1.75 6.9 12.34 3.46 0.69 0.5 7.09 sd 0.87 0.71 0.68 0.25 0.75 0.33 0.08 0.57 min–max 13–16.5 10.5–14 1.2–2.2 5.5–8 11.5–13 2.5–4 0.6–0.8 6–8 o. rutila m 20.45 14.88 2.38 6.57 13.89 2.5 1 0.33 7 sd 1.35 0.65 0.5 0.97 0.8 0.24 0.13 0.45 min–max 18–23 13–16 1.5–3.5 5–8.5 12.5–15 2–3 0.8–1.2 6–8 o. sericea m 16.25 13.73 1.56 7.12 11.37 2.52 0.87 0.5 6.7 sd 0.86 0.62 0.3 0.3 0.58 0.22 0.09 0.52 min–max 15–18.5 12.5–15 1–2 5.5–8.5 10.5–14 2.2–3 0.7–1 5–7 o. sieheana m 19.25 16.41 2.05 9.33 14 2.73 0.98 0.66 7.69 sd 0.95 0.93 0.21 0.72 0.76 0.2 0.1 0.61 min–max 17–21 14–18 1.6–2.6 7.5–10.5 13–16 2–3.5 0.8–1.1 7–8 o. stenoloba m 16.34 13.57 1.41 7.11 14.32 3.64 0.9 0.66 8.03 sd 0.54 0.62 0.27 1.12 0.67 0.3 0.09 0.5 min–max 14.5–17 12–14.5 0.8–2 5–10 13–16 2.5–5 0.6–1.2 6.5–9 o. taurica m 22.85 19.09 2.64 9.43 19.53 1.61 1.02 0.66 9.55 sd 1.24 0.7 0.29 1.2 1.3 0.15 0.09 0.72 min–max 20–25 17–20 2–3.2 7–12 15–21 1–2 0.8–1.2 9–10 m – mean, sd – standard deviation, min–max – minimum and maximum, p – length of polar axis, e – width of equatorial axis, plg – length of pores (pori), plt – width of pores (pori), clg – length of colpus (colpi), clt – width of colpus (colpi), ex – exine thickness, i – intine thickness (only for wodehouse method), ect./end – ectexine to endexine ratio, t – length of polar triangular edge u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 8 2 b i n z e t . v p 1 1 . l i s t o p a d 2 0 1 0 1 3 : 5 1 : 4 7 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s taxa analyzed with the two preparation methods, provided similar groupings. the correct classifications obtained by acetolysis and wodehouse were 99.0% and 96.9 % respectively. the data collected after acetolysis resulted in a better resolution in the genus onosma. that is why only the scatterplot obtained from acetolysis data is shown in figure 1. the first three canonical variates explained 82.7% of the variation in acetolysis and 78.5% of the variation in the wodehouse pollen preparation method (tab. 4). thus the data collected after acetolysis resulted in higher explanation of variation than the wodehouse method. such a difference was also seen in character correlations to the vectors in both methods. the characters p, e and plt were highly correlated with the first axis in the data collected after acetolysis and p, e and clg were highly correlated with the first axis in the data collected after wodehouse. in the second axis, however, totally different sets of characters were correlated with this axis: t, clg and plg in acetolysis and p, ex, and i in wodehouse. similarly to the second axis, totally different character sets were highly correlated in two different data 268 acta bot. croat. 69 (2), 2010 bi·nzet r., kandemi·r i · ., orcan n. fig. 1. the dfa scatterplot obtained from the analysis of acetolysis data. tab. 4. eigenvalues, % of variance and cumulative % comparison between acetolysis and wodehouse. function acetolysis wodehouse eigenvalue % of variance cumulative % eigenvalue % of variance cumulative % 1 53.809 48.5 48.5 32.801 43.2 43.2 2 23.659 21.3 69.8 16.144 21.3 64.5 3 14.241 12.8 82.7 10.679 14.1 78.5 u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:47 color profile: disabled composite 150 lpi at 45 degrees collection methods; plg, plt and clt were highly correlated with the third axis in acetolysis data whereas p, t and ex were highly correlated with the third axis in wodehouse data. principal component analysis (pca) and upgma phenogram: acetolysis and wodehouse data two different data sets obtained from acetolysis and wodehouse were subjected to pca and upgma clustering using ntsys-pc. the results were more or less similar to the results of dfa. although the numbers obtained for % variation explained the eigenvalues, which were close to each other, the scatterplot drawn after pca shows differences. similarly the character correlations to pc vectors show differences. the first three axes explained 82.1% of total variation in acetolysis data and 76.3% in wodehouse, which is close to the dfa result in which acetolysis resolves variation better than wodehouse. p, e and plt characters showed high correlations to the first axis in acetolysis, whereas e, plt and plg characters were highly correlated with the first axis in wodeouse data. on the second axis t, clt and ect/end in acetolysis and ex, clg and ect/end in wodehouse data have high correlations. the characters t, clg and ect/end in acetolysis data and p, i and ex in wodehouse data were highly correlated with the third axis. the pca scatterplot obtained for both type of methods showed different groupings and affinities between species. the scatterplot obtained from acetolysis data after pca did not form any kind of strong groupings in the genus onosma; however the formation of weak clusters did not correspond to any close affinity between species or subsections: haplotricha and asterotricha (fig. 2). also, the presence of weak affinities between species did acta bot. croat. 69 (2), 2010 269 palynological classification of onosma fig. 2. pca scatterplot of acetolysis data after standardization u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:48 color profile: disabled composite 150 lpi at 45 degrees not correspond to any relationships in onosma key. the scatterplot obtained from wodehouse data after pca resulted in a very similar outcome, that the relationship between species could not be seen in the species key to the genus onosma (fig. 3). 270 acta bot. croat. 69 (2), 2010 bi·nzet r., kandemi·r i · ., orcan n. fig. 3. pca scatterplot of wodehouse data after standardization fig. 4. upgma constructed from acetolysis data based on the general distance u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:53 color profile: disabled composite 150 lpi at 45 degrees acetolysis and wodehouse data are visualized by pca and also by upgma dendrograms (figs. 4, 5). the upgma dendrograms constructed do not match each other or the morphological relationships in the species of the genus onosma. discussion in this study, twenty-five onosma (boraginaceae) taxa belonging to 2 subsections (haplotricha (boiss.) gürke. and asterotricha (boiss.) gürke.) from the east mediterranean region in turkey were studied by palynological data approached by numerical taxonomic methods. it seems that none of the palynological methods (acetolysis and wodehouse) actually resulted in a good classification, similar to that given in the flora of turkey, for the species belonging to the genus onosma. numerical taxonomy uses a number of characters to construct a classification of onosma. the boraginaceae is a eurypalynous family (clarke 1977, diez 1984) in which a large number of species can be recognized by their pollen characters (diez and valdes 1991). pollen morphology may be a useful diagnostic tool in onosma taxonomy. for example o. stenoloba hausskn.ex riedl and o. mersinana riedl, binzet et orcan are very close to each other and pollen shape can help to distinguish these two species. interestingly these two species grouped together in all ordination methods (dfa, pca) except in upgma. however in cluster analysis (upgma) these two species were not very close to each other but were found in the same large group. the main outcome of this study is that the acetolysis methods resulted in a better explanation of palynological relationship within the genus onosma than the wodehouse method. this is due to some of the characters being not correctly measured in wodehouse method. acta bot. croat. 69 (2), 2010 271 palynological classification of onosma fig. 5. upgma constructed from wodehouse data based on the general distance u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:55 color profile: disabled composite 150 lpi at 45 degrees however in acetolysis, the fossilization of the pollen grains resulted in a better visualization of the edge of the characters and resulted in a better measurement. on the other hand some of the characters like p, e, plt, clg and t have a fairly good positive correlations between acetolysis and wodehouse, whereas, plg and ex had negative correlations and clt has a small correlation within two methods (data not shown). these characteristics resulted in differences in numerical taxonomic outcomes and the better results obtained by acetolysis method. thus as clearly shown in the present study, acetolysis methods would be more useful in further palynological, phylogenetical and numerical taxonomic studies. also data from karyology and molecular markers should be included for a more complete visualization of the relationships within the genus onosma. acknowledgements this study was supported by mersin university research fund (project no. bap-fbe bb (rb) 2004-1 dr). references bi·nzet, r., orcan, n., 2003a: morphological, anatomical and palynological study of onosma bracteosum hausskn. et bornm. and onosma mutabile boiss. 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(ed.), numerical taxonomy, pp. 552–556. nato asi series, springer verlag, berlin. tütel, b., kandemir, i., kuª s., kence, a., 2005: classification of turkish plantago l. species using numerical taxonomy. turkish journal of botany 29, 51–61. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a., 1972: flora europaea, 3. cambridge university press, cambridge. wodehouse, r.p., 1935: pollen grains. mcgraf-hill, new york. yildirimli, ª., 2000: the chorology of the turkish species of boraginaceae family. the herb journal of systematic botany 7, 257–272. 274 acta bot. croat. 69 (2), 2010 bi·nzet r., kandemi·r i · ., orcan n. u:\acta botanica\acta-botan 2-10\282 binzet.vp 11. listopad 2010 13:51:55 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 80 (1), 2021 99 acta bot. croat. 80 (1), 99–105, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-001 issn 0365-0588 eissn 1847-8476 ecophysiological study of some coastal dune species of zemmouri el bahri (algeria) sadjia rabhi*, réda djebbar, aicha belkebir university of science and technology houari boumediene, faculty of biological sciences, laboratory of biology and physiology of organisms, bp 32, el alia, bab ezzouar 16111, algiers, algeria abstract – species of coastal dunes are subjected to several environmental stress factors such as drought, high temperature, light intensity as well as salinity, which led to development of specific morphological, physiological and biochemical adaptation mechanisms. studying these strategies allows explaining the survival of these species in such hostile and stressful conditions. in this study we compared some parameters (morphological, physiological and biochemical) of two brassicaceae species matthiola tricuspidata (l.) w.t.aiton, cakile maritima scop., and two fabaceae species lotus creticus l. and ononis variegata l., harvested from their natural environment in coastal dunes of zemmouri el bahri (algeria), with the aim to understand their mechanisms of adaptation. the results revealed that c. maritime showed the highest relative water content, leaf area, leaf mass area, and succulence index. based on the highest levels of total phenols, flavonoids and anthocyanins as well as high contents of photosynthetic pigments, m. tricuspidata can be classified as “homoiochlorophyllous” plant. the dry mass content varied considerably among species, with the highest values observed in l. creticus and the lowest in c. maritima. as both fabaceae species l. creticus and o. variegata showed high chlorophylls, carotenoids, total phenols and flavonoids could also be classified as “homoiochlorophyllous” species. the relatively high levels of total phenols, total flavonoids, anthocyanins and carotenoids implies a biochemical adaptation that allows these plants to maintain necessary photosynthetic activity under a water deficiency condition. keywords: adaptation, anthocyanins, flavonoids, photosynthetic pigments, relative water content, total phenols * corresponding author e-mail: sadjiarabhi@gmail.com introduction coastal dunes are characterized by high ecological diversity, environmental heterogeneity, and variability of species (martinez and psuty 2005), with close interaction between biotic and abiotic factors (rucco et al. 2014). vegetation in the sand dunes is also influenced by a strong seasonality with two distinct periods being rainy cold winters and dry hot summers, the latter characterized by high irradiance and little or no precipitation. consequently, plants experience intense stress during summer due to drought, large evaporative demand and high irradiance at high temperatures (antunes et al. 2018). the ecological conditions of coasts are often stressful or even critical, their flora grows under unfavourable environmental conditions including low capillarity, salt spray, and dryness, water holding capacity, high light intensity, extreme temperature, sand burial, deficiency of nutrients and negative action of violent wind (gratani et al. 2009). these abiotic stress factors generate secondary osmotic and oxidative stresses, which then negatively influence and induce changes in the normal growth, development, and metabolism of the plant (kròl et al. 2014). in spite of all these constraints, these environmental conditions promote the development and evolution of some particular species belonging to different botanical families. many plant species have adapted on genetic, morphological and physiological level, which help them partially or completely withstand these stress conditions (gad et al. 2002). these established strategies are an important step that could explain the preservation and survival of these species in such extreme and hostile environmental conditions. to explore these strategies and in order to value plant resources native to saline biotopes, we have chosen four species belonging to two important families brassicaceae and fabaceae. rabhi s, djebbar r., belkebir a. 100 acta bot. croat. 80 (1), 2021 the genus matthiola w.t.aiton (brassicaceae) contains 50 species of annual and biannual herbs and perennial subshrubs, characterized by a silicle that features horns or swellings in its apical end (sánchez et al. 2006). matthiola tricuspidata (l.) w.t.aiton is a small annual plant characterized by a sturdy stem softly drooping, sinuous leaves, large flowers and a potential oilseed (demiral 2003); this species is widespread on the sandy and rocks of the coast (quezel and santa 1962). cakile maritima scop. is an annual, succulent and halophyte plant (ksouri et al. 2007), growing in sandy habitats along the north atlantic ocean, the mediterranean sea coasts, the canary islands and in southwestern asia (clausing et al. 2000). it is usually positioned in front of the pioneer dune and is fixed by its developed roots, it has fleshy and pinnatifid leaves (parisod and baudière 2006). this plant has an ecological value as its roots bind dunes, potential economical use as its seeds contain oil (40% of dry weight), and therapeutic value (ciccarelli et al. 2010). among the fabaceae two species have also been selected: ononis variegata l. and lotus creticus l. the genus ononis l. comprises about 75 species occurring in europe, central asia, and africa (wollenweber et al. 2003), being generally perennial herbs and shrubs (wdowiak-wróbel et al. 2017). many species of ononis genus carry glandular trichomes that are more or less viscent, and contain flavonoids in several cases (wollenweber et al. 2003). the genus lotus l. comprises between 120 to 130 species of perennial and annual native plants, distributed mainly in the mediterranean region (sandral et al. 2010). in this genus, l. creticus is considered an important perennial species due to its ability to stabilize dunes (sandral et al. 2010). plants living under hostile conditions synthesize higher amounts of protecting specialized metabolites such as phenolic compounds with free-radical quenching activity which can be primarily related to the substitutions on the aromatic ring and the structure of the side chain ( abdeen et al. 2015). these compounds are involved in various plants physiological and developmental processes such as growth regulation, pigmentation, reproduction, and resistance to pathogens. they protect plants against adverse factors including drought, uv radiations, infection and physical damage (slatnar et al. 2016). various biotic and abiotic factors generate oxidative stress that can be very harmful to cells (xiao et al. 2014). in such situations, antioxidant molecules intervene and decrease the deleterious effect of free radicals (apel and hirt 2004). thus, we evaluated the contents of total phenols, total flavonoids (as one of the largest groups of plant phenols) and anthocyanins (a sub class of flavonoids) in the leaves of the four selected plants growing on the coastal dunes of zemmouri el bahri, algeria. moreover, major photosynthetic pigments and certain morphological parameters were also determined as they are usually affected by drought and other environmental conditions as well. in this paper, we compared morphological and physiological traits of matthiola tricuspidata, cakile maritima, lotus creticus and ononis variegata with the aim to explore their possible adaptive mechanisms to environmental stress. materials and methods study site and plants materials the study is conducted in the coastal dunes of east-algiers (zemmouri el bahri), located between 3°32’, 3°38’ e and 36°48’, 36°50’ n geographical coordinates. the area is characterized by a mediterranean climate, with an annual rainfall mean of 579 mm. the air temperature mean of the coldest months reaches 9.3 °c whereas the mean maximum air temperature of the warmest month goes up to 28.9 °c, with a drought period spreading over five months, a sub­ humid bioclimatic zone and a variant hot winter (hanifi et al. 2007). the species m. tricuspidata, c. maritima, l. creticus and o. variegata were collected at the floral stage during april and may from the study station (sand dunes of zemmouri el bahri). the leaves were freshly harvested, washed, dried, finely ground and then stored in a dry dark place. morphological parameters of the leaf the morphological leaf traits were determined by the method described by ciccarelli et al. (2010). the leaves from three individual plants per each selected species were collected, and weighed to determine the fresh weight (fw). leaf projected area (la) was obtained with a canon scan using a meserium software area and expressed in cm². plant material was dried at 70 °c to constant weight (dry mass, dm). leaf dry matter content (ldmc) was calculated as the ratio of leaf dry mass (dm) to fresh mass (fm), expressed in mg g–1 (li et al. 2005): ldmc = dm fm leaf mass per area (lma) was expressed as the ratio of dry mass (dm) to leaf area (la), expressed in mg cm–2 (ogaya and penuelas 2007): lma= dm la succulence index(si) is the ratio of the difference between the dry mass (dm) and fresh mass (fm) to the leaf area (la), expressed in mg cm–2 (gratani et al. 2009): si= dm-fm la where dm corresponds to: dry mass, fm: fresh mass, la: leaf area. physiological parameters leaf relative water content (rwc) was evaluated using the equation introduced by ladigues (1975): rwc(%)= fw dw tw dw( ) / ( )� ��� ���100 where fw corresponds to: fresh weight, dw to: dry weight and tw to: full turgor weight, measured after soakecophysiology of algerian costal dune species acta bot. croat. 80 (1), 2021 101 ing the leaves in water for 24 hours at room temperature and in the presence of light. the dry weight was estimated after drying the leaves at 60 °c for 48 hours (clarck and mac-caig 1982). to extract the photosynthetic pigments chlorophyll a (chl a) chlorophyll b (chl b), total chlorophylls (chl a+b) and total carotenoids (car), 0.05 g of fresh leaf samples were ground in 5 ml of acetone solution (80%) . the homogenate was centrifuged at 3000×g for 10 min at 4 °c. the supernatant was recuperated and the absorbance was measured at 647 nm, 663 nm and 470 nm for chl a, chl b and car, respectively. the amounts of pigments present in the extract were calculated using the equation of lichtenthaler (1987): chl a aa � � � �12 25 663 2 79 647. . chl a ab � � � �21 5 647 5 1 663. . chl a aa b� � � � �7 15 663 18 71 647. . cx c� � � � � � �1000 470 1 82 85 02 198 a chla chlb. , biochemical parameters sample extract was obtained by stirring 1g of dry leaf powder with 7 ml of pure methanol and 3 ml deionized water for 30 min. the extracts were then kept for 48 hours at 4 °c, filtered through n°1 watman filter paper, evaporated under vacuum to dryness and then stored at 4°c until analysis (ksouri et al. 2007). total phenolic contents were determined using the folin-ciocalteu reagent, according to the method of singleton and rossi (1965). a volume of 0.025 ml of sample extract was added to 3.975 ml of distilled water before adding 0.25 ml of the folin-ciocalteu reagent. the mixture had the ability to react for 3 minutes before an amount of 0.75 ml of 20% na2co3 was added. after incubation for 40 minutes at 40 °c, the absorbance was measured at 760 nm. there has been a sequence of three assays, and the phenolic contents was expressed as gallic acid equivalent in milligrams per gram of dry weight (mg gae g–1 dw). total flavonoids were measured using a colorimetric aluminum chloride (alcl3) assay (lamaison and carnet 1990, chang et al. 2002). volume of 1.5 ml of each extract were mixed with 1.5 ml of 2% alcl3 6h2o. the preparation was shaken and the absorbance was read at 440 nm after 10 minutes at room temperature. the assay was repeated three times and the total flavonoid contents were expressed as rutin equivalent in milligrams per gram of dry weight (mg eru g–1 dw). the anthocyanins content was determined according to the procedure described by gould et al (2002) as follows; anthocyanins were extracted by adding 2 ml of acidified methanol 1% hcl (v/v) to 50 mg of dry leaves. the supernatant aliquot was retrieved and the absorbance was measured at 530 nm. total anthocyanin was calculated by endoresing the following formula: t . ( anthocyanin absorbance dilution factor weight � � � � 449 2 29600 gg) the anthocyanin content was expressed in mg of cyanidin-3-glucoside equivalent per gram of dry weight. statistical analysis the statistical study was performed by excel and statistica v.8 (statsoft, inc., tulsa, ok). results were analysed by one-way anova, followed by tukey test to compare means. the differences were held significant if p < 0.05. all experiments were carried in triplicates and expressed ± sd (n = 3). results plant morphological study there were significant differences between species for all morphological traits studied. leaf succulence index (si) shows different values in the four species (tab. 1). cakile maritima had the largest leaf area and the highest succulence index (12.51 ± 0.03 cm–2, 131.37 ± 0.83 mg cm–2, respectively), though both species of brassicaceae showed high leaf area and a high succulence index. fabaceae family species have narrow leaves, while l. creticus leaves exhibit the lowest leaf succulence (22.86 ± 0.56 mg cm–2). leaf mass area (lma) was also evaluated, this parameter varied from 6.55 ± 0.45 mg cm–2 (l. creticus) to 8.52 ± 0.74 mg cm–2 (c. maritima). leaf dry matter content (ldmc) indicates that fabaceae species have the highest values with 219.51 mg g–1, 147.64 mg g–1 for l. creticus and o. variegata, respectively. among the brassicaceae family species, c. maritima leaves show the lowest ldmc (58.52 ± 0.53 mg g–1). leaf area (la) of c. maritima was significantly higher, but the lowest ldmc was found there. on the other hand, the leaves of l. creticus, having the smallest foliar surfaces but highest dry matter accumulation (tab. 1). tab. 1. leaf area (la), leaf dry matter content (ldmc), leaf mass area (lma), and succulence index (si) of matthiola tricuspidata, cakile maritima, lotus creticus and ononis variegata. numbers present average of three replicates ± strandard deviation. different letters within each column mean significant differences at p < 0.05 (tukey’s hsd test). species la (cm²) ldmc (mg g–1) lma (mg cm–2) si (mg cm–2) matthiola tricuspidata 10.68 ± 0.95a 91.15 ± 0.33a 7.39 ± 0.29a 57.39 ± 0.78 a cakile maritima 12.50 ± 0.02b 58.52 ± 0.53b 8.52 ± 0.74a 131.37 ± 0.83b lotus creticus 0.57 ± 0.02c 219.51 ± 0.54c 6.55 ± 0.45b 22.86 ± 0.56 c ononis variegata 0.54 ± 0.01c 147.64 ± 0.90d 6.84 ± 0.29b 39.14 ± 0.74d rabhi s, djebbar r., belkebir a. 102 acta bot. croat. 80 (1), 2021 plant physiological responses the relative water content (rwc) was significantly higher in c. maritima (77.14%) than other species in which that parameter ranged from 63.10 to 65.15% (fig. 1). phylls (1.39 ± 0.1 mg g–1 fw and 1.34 ± 0 .12 mg g–1 fw, respectively) and carotenoids content (0.41 ± 0.06 mg g–1 fw, 0.47 ± 0.08 mg g–1 fw), while the lowest content of chlorophyll a, total chlorophylls and carotenoids were obtained for c. maritima. the content of chl b was similar among all species and the chl a/b ratio varied from 2.82 (m. tricuspidata) to 2.33 (l. creticus). the chlorophyll/carotenoid ratio was in the range of 3.42 to 2.78 (fig. 2b). plant biochemical responses the results of total phenolic compounds and total flavonoids in plant extracts are presented in tab. 2. the results obtained show that the content of total phenolic compounds varied between different species; m. tricuspidata had the highest total phenolic compounds with 72.72 ± 0.45 mg gae g–1 dw, and the lowest were found in c. maritima (24.17 ± 0.25 mg gae g–¹ dw). the quantities of total phenolic were 40.73 ± 0.77 mg gae g–1 dw and 50.01 ± 0.37 mg gae g–1 dw for l. creticus and o. variegata, respectively. flavonoid content also varied between species; m. tricuspidata showed the highest content (7.36 ± 0.09 mg eru g–1 dw), and the lowest were found in c. maritima (1.93 ± 0.02 mg eru g–1 dw) which is 4 times lower than that of m. tricuspidata. the values recorded for fabaceae species were 5.86 ± 0.21 mg eru g–1 dw and 3.7 ± 0.08 mg eru g–1 dw for l. creticus and o. variegata, respectively. the results of anthocyanin contents of species present some notable differences (fig. 3). leaves of m.tricuspidata displayed the highest levels of anthocyanins with 0.54 ± 0.06 fig. 1. relative water content (%) in leaves of matthiola tricuspidata, cakile maritima, lotus creticus and ononis variegata. data are mean values of three replicates ± standard deviation. different letters are significantly different at p < 0.05 (tukey hsd test). fig. 2. chlorophyll a (chl a), chlorophyll b (chl b), total chlorophylls (chl a+b), total carotenoids (car) contents (a), and ratio of chlorophyll a to chlorophyll b (chl a/b) and total chlorophyll to carotenoid ratio (b) in leaves of matthiola tricuspidata, cakile maritima, lotus creticus and ononis variegata. data are mean values of three replicates ± standard deviation. different letters are significantly different at p < 0.05 (tukey hsd test). tab. 2. phenolic contents (total phenols, total flavonoids) in leaf methanolic extracts of matthiola tricuspidata, cakile maritima, lotus creticus and ononis variegate. numbers present average of three replicates ± standard deviation. different letters within each column are significantly different at p < 0.05 (tukey’s hsd test). species total phenols (mg eag g-1 dw) total flavonoids (mg eru g-1dw) matthiola tricuspidata 72.72 ± 0.45a 7.36 ± 0.09a cakile maritima 24.17 ± 0.25b 1.93 ± 0.02b lotus creticus 40.73 ± 0.77c 5.86 ± 0.21c ononis variegata 50.01 ± 0.37d 3.70 ± 0.08d the variations of photosynthetic pigments were shown in fig. 2a. among the four species, m. tricuspidata and l. creticus showed the highest levels of chlorophyll a (1.02 ± 0.04 mg g–1 fw and 1.11 ± 0.09 mg g–1 fw), total chloroecophysiology of algerian costal dune species acta bot. croat. 80 (1), 2021 103 mg g–1 dw, compared to other species. the values of those compounds were 0.36 ± 0.05 mg g–1 dw for o. variegata, 0.29 ± 0.05 mg g–1 dw for l. creticus and 0.25 ± 0.02 mg g–1 dw for c. maritima. discussion the results of the study show that matthiola tricuspidata, cakile maritima, lotus creticus and ononis variegata, living on the sand dunes have different adaptive strategies for morphological, physiological and biochemical to survive in hostile conditions of the coastal dunes. drought, salinity and uv ray’s intensity, affect the photosynthesis, growth and development of species (shah et al. 2017). relative water content is probably the most appropriate measure of plant water status in terms of the physiological consequence of cellular water deficit (tátrai et al. 2016). cakile maritima showed the highest relative water content (rwc), a large leaf area (la), leaf mass area (lma) and succulence index (is), but its chl a, chl b and total chlorophyll contents were the lowest. the high lma represents a morphological character of mediterranean species that plays a protecting role for plants exposed to drought stress (gratani et al. 2009). the values of leaf mass area (lma) and index of succulence (is) are similar to those reported in some species of the mediterranean coastal dunes (gratani et al. 2009). cakile maritima is a halophyte and succulent plant, which is naturally tolerant under salinity and drought conditions (ksouri et al. 2007), with taproots very developed allowing it maximum water absorption, and fleshy leaves, ensuring the accumulation of large tissue water reserves (parisod and baudière 2006). the survival of plants in circumstances of water deficiency is dependent on their osmotic adjustment (baroowa and gogoi 2015). different studies have indicated that the drought stress decreases chl a, chl b and the total chlorophyll content of the various crops (hailemichael et al. 2016). in this regard, c. maritima could be classified as a “poikilochlorophyllous” species (challabathula et al. 2018), which reduce their chlorophyll concentration to absorb less light energy and thus have less excess electrochemical energy, and therefore their needs for antioxidant defence are less important. content of phenolic compounds in c. maritima is close to that reported by meot-duros et al. (2008): 22.24 mg eag g–1 dw and ksouri et al. (2007): 42.84 ± 7.67 mg eag g–¹ dw. the total content of flavonoids in plants from the brassicaceae family ranged from 0.72 ec.g ¹ dw to 15.38 ec.g–¹ dw (grigore and oprica 2015), which are close to those reported in this study. the flavonoid content found in lepidium crassifolium (2.37 ec.g–1 dw) was similar to that in c. maritima (1.93 mg eru g–1 dw). ivan and oprica (2013) also reported total flavonoids levels comparable to those in our results. cakile maritima adopts a “drought avoidance” strategy to resist drought, which is defined as the ability of the plant to maintain a relatively higher water content in tissues despite reduced water content in the soil (omprakash et al. 2017). in comparison with c. maritima, m. tricuspidata had the lower relative water content and higher content of chl a and car. the levels of total phenols, total flavonoids, and anthocyanins were the highest with respect to other species. based on those results, m. tricuspidata can be classified as one of the “homoiochlorophyllous” species, which maintain their chlorophyll pool relatively high under high salinity and light intensity (challabathula el al. 2018). as a large part of the light energy captured and converted into electrochemical energy at the reaction centers would then be in excess, these species need to have efficient protective mechanisms against the free radicals formed. the carotenoids (xanthophylls) are necessary for photoprotection of photosynthesis and they play an important role as a precursor in the signaling during plant growth under abiotic/biotic stress (ola and abd el-maboud 2013). this could explain the high levels of antioxidant molecules such as carotenoids, total flavonoids and anthocyanins that we have recorded in m. tricuspidata. the chlorophyll content results reported in this study were similar to those found in plants growing in coastal dunes (frosi et al. 2017). many studies have shown that the concentrations of specialized metabolites in plant exposed to drought are higher than in those grown under well-watered conditions (al-gabbiesh et al. 2015). the results of total phenol contents of species are in accordance with studies on several halophytes (ksouri et al. 2007, ivan and opricã 2013). the increase of content in phenolic compounds in the different tissues under the effect of salinity has been recorded in a certain number of plants (ksouri et al. 2007, abideen et al. 2015). plants react to various environmental factors by increasing the production of polyphenols, particularly flavonoids (anket et al. 2019). these molecules have important properties such as the protection of tissues against the harmful effects of uv radiations and are an important antioxidant activity (xiao et al. 2014). the two species of fabaceae family have different morphological strategies, l. creticus and o. variegata have smaller leaf areas (la), lower leaf mass area (la), and succulence index (si). the two species show a combination of fig. 3. anthocyanins contents in leaves of matthiola tricuspidata, cakile maritima, lotus creticus and ononis variegata. data are mean values of three replicates ± standard deviation. different letters are significantly different at p < 0.05 (tukey hsd test). rabhi s, djebbar r., belkebir a. 104 acta bot. croat. 80 (1), 2021 high leaf dry matter content (ldmc) and low leaf mass area (lma). the species with high ldmc and low lma show a slow growth rate, with long leaf lifespan and more efficient conservation of nutrients (saura-mas and lloret 2007). the reduced leaf area is a strategy to conserve water and control excessive transpiration (melo júnior and boeger 2016). the species with low lma (i.e. high specific area, sla), is characterized by high photosynthetic capacity per unit mass (gratani et al. 2009). since both l. creticus and o. variegata displayed high chlorophylls, carotenoids, total phenols and flavonoids contents, they could also be classified as “homoiochlorophyllous” species. the results of total phenolics and flavonoids contents were similar to those found by sayari et al. (2016). thus, m. tricuspidata, l. creticus and o.variegata use the “drought tolerance” strategy to resist water deficit, which involves maintaining cell turgor by osmotic adjustment, the elevation of protoplasmic resistance (supratim et al. 2016), the extension of antioxidant capacity, and development of desiccation tolerance (osmolovskaya et al. 2018). conclusion this paper shown the various strategies adopted by the four species under unfavorable environmental conditions present in coastal dunes. on the morphological and physiological level, c. maritima has a high water potential (high rwc) with a succulent character, which is an adaptive trait that comprises a large leaf area and water reserve. in comparison, m. tricuspidata, l. creticus and o. variegata show lower relative water content, have relatively high chlorophyll contents and fight the consequent higher oxidative damage by greater accumulation of antioxidant molecules such as phenolic compounds and carotenoids. in addition, l. creticus and o. variegata showed reduced leaf area and a high leaf dry matter content, i.e. leaf morphological adaptations enabling protection against water deficit and limitation of water losses. references abideen, z., qasim, m., rasheed, a., adnan, m.y., gul, b., ajmal khan, m., 2015: antioxidant activity and polyphenolic content of phragmites karka under saline conditions. pakistan journal of botany 47(3), 813–818. al­gabbiesh, a., kleinwächter, m., dirk, s., 2015: influencing the contents of secondary metabolites in spice and medicinal plants by deliberately applying drought stress during their cultivation. jordan journal of biological sciences review 8(1), 1–10. anket, s., babar, s., rehman, a., bhardwaj, r., landi, m., zheng, b., 2019: review response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. molecules 24, 2452. antunes, c., pereira, a.j., fernandes, p., ramos, m., ascensão, l., correia, o., máguas, c., 2018: understanding plant drought resistance in a mediterranean coastal sand dune ecosystem: differences between native and exotic invasive species. journal of plant ecology 11(1), 26–38. apel, k., hirt, h., 2004: reactive oxygen species: metabolism, oxidative stress, and signal transduction. the annual review of plant biology 55, 373–399. baroowa, b., gogoi, n., 2015: changes in plant water status, biochemical attributes and seed quality of black gram and green gram genotypes under drought. international letters of natural sciences 42, 1–12. challabathula, d., zhang, q., bartels, d., 2018: protection of photosynthesis in desiccation-tolerant resurrection plants. journal plant physiology 227, 84–92. chang, c., yang, m., wen, h., chern, j., 2002: estimation of total flavonoids contents in propolis by two complementary colorimetric methods. journal of food and drug analysis 10, 178–182. ciccarelli, d., balestri, m., pagni., a.m., forino, l.m.c., 2010: morpho-functional adaptations in cakile maritima scop. subsp. maritima: comparation of two different morphological types. caryologia 63, 411–421. clarck, j.m., mac-caig, t.n., 1982: excised leaf water relation capability as an indicator of drought resistance of triticum genotypes. canadian journal plant science 62, 571–576. clausing, g., vickers, k., kaderiet, j.w., 2000: historical biogeography in a linear system: genetic variation of sea rocket (cakile maritima) and sea holly (eryngium maritimum) along european coasts. molecular ecology 9, 1823–1833. demiral, m.a., 2003: determination of salt tolerance of stock (matthiola tricuspidata) as a potential oil crop. turkis journal of agriculture and forestry 27, 229–235. frosi, g., harand, w., teixeira de oliveira, m., pereira, s., cabral, p.s., montenegro, a.a., santos, m.g., 2017: different physiological responses under drought stress result in different recovery abilities of two tropical woody evergreen species. acta botanica brasilica 31, 153–160. gad, m.r.m., el-hadidy, m.e.a., el-nabarawy, a.a.a., 2002: comparative study on the adaptation of some natural plants grown under macronutrients limitation at north sinai sand dunes (egypt). annals of agricultural science 57, 81–90. gould, k.s., neill, s.o., vogelmann, t.c.a., 2002: a unified explanation for anthocyanins in leaves? in: gould, k.s, lee, d.w. 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2019 acta bot. croat. 78 (2), 142–146, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0016 issn 0365-0588 eissn 1847-8476 a new variety of plocama calabrica (rubiaceae) from denizli (turkey) confirmed by morphological and molecular issr markers ramazan süleyman göktürk1, olcay düşen2*, ergun kaya3, betül gürcan2, uygar sarpkaya2 1 akdeniz university, faculty of science, department of biology, 07058 antalya, turkey. 2 pamukkale university, faculty of arts and science, department of biology, 20160, kinikli, denizli, turkey. 3 muğla sıtkı koçman university, faculty of science, molecular biology and genetic department, 48000, kötekli, muğla, turkey. abstract – plocama calabrica (l.f.) m.backlund & thulin var. alba göktürk, o.d.düşen, b.gürcan & u. sarpkaya variety nova is described from south-west anatolia. the new variety grows on limestone slopes between akpınar and yaylapınar villages in the çameli district in denizli. it is closely related to p. calabrica var. calabrica, and can be readily distinguished by morphological and molecular characters from the related variety. taxonomic comments such as descriptive and diagnostic characters, distribution and ecology, phenology and proposed conservation status for this new variety are given in the current study. morphological affinities and the inter-simple-sequence repeat (issr)-pcr based phylogenetic relationships between the new and the related variety are also discussed keywords: issr-pcr, molecular marker, new variety, plocama, systematics, turkey * corresponding author e-mail: odusen@pau.edu.tr introduction the family rubiaceae comprises 650 genera and ca. 11000 species, distributed throughout almost all the regions of the world, although they are found mainly in tropical and subtropical regions (ortiz et al. 2000). putoria is a monotypic genus which belongs to rubiacaeae in turkey, and was revised by ehrendorfer in the flora of turkey and the east aegean islands (ehrendorfer 1982). the genus of putoria was treated as a synonym of plocama genus by backlund et al. 2007. plocama was represented by 34 species and 1 subspecies until 2009 (backlund et al. 2007, ipni 2015). since then, one new species has been described from uzbekistan (khassanov et al. 2014). with the description here of p. calabrica var. alba there are now two taxa in turkey and worldwide there are in all 37 plocama taxa. in the flora of turkey, the genus plocama is represented only by p. calabrica (l. fil) dc. p. calabrica is known from spain, italy (incl. sicily), malta, montenegro, bosnia and herzegovina, croatia, albania, greece (incl. crete), cyprus, turkey, israel, lebanon, iraq, syria, morocco, algeria, tunisia, and libya (backlund and thulin 2007). true and rapid determination of degrees of genetic relationship and genetic diversity levels are required for conservation and/or utilization of plant breeding programs. during the last twenty years, usage of dna-based molecular marker systems has increased faster than chemical or other morphological characterization systems (collard et al. 2005, bernardo 2008). formerly, these markers were quite expensive and laborious, but technological improvements have made them cheaper, faster and relatively easy (yang et al. 2015). the inter-simple-sequence repeat (issr) marker system is based on amplification of dna fragments using a single microsatellite sequence primer designed with six trinucleotide or eight dinucleotide repeats and one more anchor nucleotide (zietkiewicz et al. 1994). this marker technique has been verified as a simple, fast and low-cost way to determine genetic diversity (sarla et al. 2003), to investigate relationships between cultivars (martins et al. 2003, kaya 2015), to use evolutionary studies such as gene flow (wolfe et al. 1998), and to detect genetic stability (kaya et al. 2017). the practicality and utility of issr primers has been evalua new variety of plocama calabrica (rubiaceae) acta bot. croat. 78 (2), 2019 143 ated in this work for identifying the differences between two taxa of plocama calabrica. materials and methods morphological study in june 2017, during a project named “biodiversity and monitoring studies of terrestrial and inland water ecosystems in denizli province”, the authors collected some interesting plocama specimens (fig. 1, 2). while the normal color of p. calabrica var. calabrica individuals is pink, individuals of this new variety were interesting because of their white flowers. in total, 10 herbarium specimens of the new variety were collected from the type locality. plocama specimens were dried for morphological and molecular phylogenetic studies according to standard herbarium techniques and preserved in the pamukkale university herbarium (pamuh). after drying process, these specimens were checked using the basic floras, flora of turkey (ehrendorfer 1982), flora europaea (ball 1976), flora italiana (tanfani 1887) and related papers (backlund et al. 2007, backlund and thulin 2007, karabacak 2012) and also confirmed by comparison with the e-herbarium samples in the b, bgbm, c, br, e, m, p, sav, w and wu herbaria. after detailed morphological and molecular phylogenetic studies, we decided that p. calabrica var. alba was a variety new to science. ucts were separated on 1.5% agarose gel and visualized under uv light after being stained with ethidium bromide. pcr band profiles were recorded as 1 (present) or 0 (absent) and cluster analysis was performed to construct dendrograms, with the unweighted pair-group method by arithmetic averages (upgma) from the similarity data matrices using jaccard’s coefficient (d-upgma, 2002). results taxonomy plocama calabrica (l.f.) m. backlund & thulin var. alba göktürk, o. d. düşen, b. gürcan & u. sarpkaya var. nov. (figs. 1, 3) fig. 1. a general view of plocama calabrica var. alba in nature (a) with detail of flowers (b). molecular phylogenetic study genomic dna was isolated from three individual plants of two different characteristic taxa belonging to dry p. calabrica herbarium samples using a modified protocol developed by ferdous et al. (2012). the method based on cetyltrimethylammonium bromide (ctab) extraction included one more step, the use of chloroform:isoamyl alcohol:phenol (24:1:5, v/v). polymerase chain reactions were carried out using 40 ng µl–1 dna template, 1×pcr buffer, 2.5 mm mgcl2, 0.4 mm dntp, 1 unit taq dna polymerase and ten issr primers (tab. 1) in a 25 µl reaction mix (martins-lopes et al. 2009, ozudogru et al. 2011, smykal et al. 2011, düşen et al. 2018). dna band profiles were amplified at 95 °c for 3 min initial denaturation followed by 35 reaction cycles (95 °c 15 s; 55 °c 30 s; 72 °c 3 min) and finally extended with 72 °c 10 min. pcr prodtab. 1. issr primer sequences and genbank accessions numbers (martins-lopes et al. 2009, smykal et al. 2011) used in molecular phylogenetic analysis of plocama calabrica var. calabrica and p. calabrica var. alba. primer sequence genbank accesion number issr1 (ag)8t ubc 807 issr2 (ag)8g ubc 809 issr3 (ga)8t ubc 810 issr4 (ga)8c ubc 811 issr5 (ca)8a ubc 817 issr6 (tc)8c ubc 823 fig. 2. a general view of plocama calabrica var. calabrica in nature (a) with detail of flowers (b). holotype:—turkey. c2 denizli: çameli, between akpınar and yaylapınar villages, limestone slopes, 1166 m, 21 june 2017, o. d. düşen (2584) & r. s. göktürk (holotype pamuh!, isotypes akdeniz univ. herb.!). procumbent shrubs. stem much branched, woody at base, 5–25 cm, forming mats up to 1.5 m in diameter, puberulent. leaves opposite, 10–15(–20) × 2–3.5(–4.5) mm, lanceolate to oblong, obtuse, narrowed into a short petiole, revolute, slightly scabrid on margins and midrib, somewhat leathery-succulent, blackening when dry; stipules small, linear-oblong, ± fused. flowers grouped in laxly contracted shortly pedicellate cymes, hermaphrodite, 4-merous. calyx greenish, tubular, persistent and inflated in fruit; teeth 4, greenish, unequal triangular. corolla infundibular with a long tube, 8–15 (–20) mm, white, glabrous outside, hairy göktürk r.s, düşen o., kaya e., gürcan b., sarpkaya u. 144 acta bot. croat. 78 (2), 2019 inside; lobe 4, valvate. stamen 4, inserted at corolla throat; anther white, dorso-basifixed, exserted; filament white. style white, filiform, with very short bifid stigma. ovary bilocular, each cell with one basal ovule. fruit drupe, 4–6 mm, oblong, with two pyrenes, glossy. plocama calabrica includes 2 varieties and the diagnostic keys are presented below; calyx teeth reddish; corolla pink; anther and filament pink ..................................................................var. calabrica calyx teeth greenish; corolla white; anther and filament white ........................................................................ var. alba the new variety is different from p. calabrica var. calabrica (figs. 1, 2) and the two are compared on the basis of morphological characters in tab. 2. ed that this new variety should be placed under the iucn threat category “critically endangered (cr)” (iucn 2012), because the estimated area of occupancy is less than 10 km2 (criterion b2) and it is known only from one locality (criterion b2a). the population size of the new species is estimated to be less than 50 mature individuals (criterion c2-ai). in addition, the distribution area of the new taxon may be destroyed by anthropogenic effects such as road construction or grazing in the near future. etymology: the specific epithet is derived from corolla, anther and filament color. specimens examined of plocama calabrica var. calabrica: turkey. c3 antalya: çakırlar, south of çakırlar, roadside, 25 m, 13 august 1993, r. s. göktürk (3080) (akdeniz univ. herb.!). c3 antalya: saklıkent, pinus clearings, 1600 m, 8 july 1995, o.dinç (1087) (akdeniz univ. herb.!)). c2 denizli: kocabaş, limestone slopes, 604 m, 21 may 2017, o.d.düşen (1871) & r. s. göktürk (pamuh!). c2 denizli: çakıroluk, pinus clearings, 1172 m, 20 june 2017, o.d.düşen (2391) & r. s. göktürk (pamuh!). c2 denizli: çameli, between akpınar and yaylapınar villages, limestone slopes, 1166 m, 21 june 2017, o.d.düşen (2583) & r. s. göktürk (pamuh!). c2 denizli: çamlık, roadside, 815 m, 8 june 2018, o.d.düşen (5170) & r. s. göktürk (pamuh!). molecular phylogenetic data leaf samples belonging to six individuals from p. calabrica var. calabrica and p. calabrica var. alba (three individuals from each) were analyzed using six issr primers to prove that they are separate varieties. the total 35 reproducible bands ranging 565 to 2010 bp were obtained from pcr reactions using issr1, 5 and 6 primers (tab. 3). the polymorphism rate was calculated as 68.6% between the two taxa and the genetic differences were able clearly to be seen from stained band profiles on agarose gel (fig. 5). the similarity matrix values, ranging up to 0.462, generated by jaccard’s coefficient method showed considerable distinction between two taxa and dendogram analyses divided them into two main clusters. the results obtained by molecular fingerprinting and by morphological analyses were tab. 2. morphological comparison of plocama calabrica var. calabrica and p. calabrica var. alba flowers. characters plocama calabrica var. calabrica plocama calabrica var. alba cymes in flowering densely contracted laxly contracted calyx teeth reddish greenish corolla pink white anther pink white filament pink white style pink white distribution and ecology this variety is endemic to south-west anatolia, turkey (fig. 3). it grows on limestone slopes at an elevation of 1166 m (fig. 4). it is associated with some plants such as plocama calabrica (l.f.) m. backlund & thulin var. calabrica (fig. 2), dianthus zonatus fenzl var. zonatus, pinus brutia ten var. brutia, tussilago farfara l. and glaucosciadium cordifolium (boiss.) b. l. burtt & p. h. davis. phenology: flowering time is may to june. fruiting time is july to august. proposed conservation status: plocama calabrica var. alba is known only from one restricted locality. it is suggestfig. 3. distribution of plocama calabrica var. calabrica (blue dots) and p. calabrica var. alba (red dot) in turkey. a new variety of plocama calabrica (rubiaceae) acta bot. croat. 78 (2), 2019 145 complementary and strongly confirmed the classification of p. calabrica var. calabrica and p. calabrica var. alba taxa into two different varieties. discussion systematic botany is based on the plant morphological characteristics that provide the major information for identification between taxa and these characteristics are obtained from morphological, physiological and anatomical features of plant tissues, organs, seeds, embryos and pollens. on the other hand, use of these features as taxonomic information source these days has been overtaken by the use of molecular markers to obtain dna-based data, because these data provide a universal standard for the taxonomical comparison of all organisms. dna-based systematic information has been provided by many kind of molecular marker systems such as pcr based approaches (badr 2008). in this study, two p. calabrica taxa that have morphological differences such as different colour flowers and especially different colour of characteristic petal margins were verified using the pcr based marker system, issr, to classify into a new variety. these morphological differences were strongly supported by the issr marker system for two p. calabrica taxa. according to the results of pcr reaction analyses, it is highly probable that these two taxa belonging to p. calabrica may be different varieties. there are many studies carried out using a combination of both morphological and molecular characteristics on different plant species. for example, prasad (2014) obtained a polymorphism rate of 71.2% between nine different variety of hibiscus rosa-sinensis linn. using random amplified polymorphic dna (rapd) analyses. another, similar, study was performed to determine the polymorphism rate between secale cereale l. subspecies using rapd and amplified fragment length polymorphisms (aflp) analyses by ćwiklińska and his colleagues (2010) and they obtained an up to 79% polymorphism rate. results of previous studies based on molecular analyses, similarly to our study, have demonstrated that polymorphism rate of taxa belonging to different plant species are consistent. for example, the polymorphism rate can be up to 90% or more for species, up to 80% for subspecies and up to 72% for varieties. fourteen species belonging to the coffea genus were compared using issr primers and 96.5% polymorphism rate was obtained between them (ruas et al. 2003). in another study, microsatellite markers were used for the evaluation of genetic diversity in oryza sativa l. subspecies and an approximately 79% polymorphism rate was obtained (junjian et al. 2002). ko and his colleagues (1994) used rapd primers for determination of oryza sativa l. varieties and they obtained 67% polymorphism. like previous studies, the current study also showed that molecular markers strongly supported the morphological differences between two different taxa of p. calabrica. the data obtained from analysis of band profiles derived from pcr reactions have been enough for it to be classified into a new variety of p. calabrica. tab. 3. analysis of band profiles obtained from pcr reactions with six samples belonging to three individuals of plocama calabrica var. calabrica and p. calabrica var. alba using three productive primers. primer total bands the biggest band size (bp) the smallest band size (bp) the total polymorphic bands the total monomorphic bands polymorphism (%) issr 1 13 1950 570 9 4 69.2 issr 5 11 1700 565 7 4 63.6 issr 6 11 2010 565 8 3 72.7 total 35 2010 565 24 11 68.6 fig. 4. habitat of plocama calabrica var. calabrica (red cycle) and p. calabrica var. alba (yellow cycle) in limestone slopes. fig. 5. pcr band profiles produced from six samples belonging to three individuals of plocama calabrica var. calabrica (pcc1-3) and p. calabrica var. alba (pca1-3) using issr5 and issr6 primers. m – marker lambda dna/hindiii. arrows indicate polymorphic band profiles. göktürk r.s, düşen o., kaya e., gürcan b., sarpkaya u. 146 acta bot. croat. 78 (2), 2019 acknowledgement this work was supported by the ministry of forestry and water affairs and the general directorate of nature conservation and national parks (the project “biodiversity and monitoring studies of terrestrial and inland water ecosystems in denizli province”). references backlund, m., bremer, b., thulin, m., 2007: paraphyly of paederieae, recognition of putorieae and expansion of plocama (rubiaceaerubioideae). taxon 56, 315–328. backlund, m., thulin, m., 2007: revision of the mediterranean species of plocama (rubiaceae). taxon 56, 516–520. badr, a., 2008: molecular approaches in plant systematics and evolution. taeckholmia 28, 127–167. ball, p.w., 1976: putoria pers. in: tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. 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(eds), current technologies in plant molecular breeding, 15–50. springer, dordrecht. zietkiewicz, e., rafalski, a., labuda, d., 1994: genome finger printing by simple sequence repeat (ssr) anchored polymerase chain reaction amplification. genomics 20, 176–183. opce-str.vp acta bot. croat. 69 (2), 237–247, 2010 coden: abcra 25 issn 0365–0588 a morphometric study on scorzonera l. taxa (asteraceae) from northeast anatolia serdar makbul1*, zafer turkmen2, kamil coskuncelebi3, osman beyazoglu3 1 rize university, faculty of arts and sciences, department of biology, 53100 rize, turkey 2 giresun university, faculty of arts and sciences, department of biology, giresun, turkey 3 karadeniz technical university, faculty of arts and sciences, department of biology, trabzon, turkey phenetic traits of 39 populations belonging to 19 taxa of scorzonera l. (asteraceae) from north anatolia were analyzed with the use of numerical methods. principal component analysis (pca) showed that pubescence and length of achenes, the shape of outer phyllaries, and average length of flowering capitula, plant pubescence, root shape and state of the plant stem are the best variables to distinguish the examined taxa. in addition, it was also found that binary are more important than quantitative characters in discriminating the examined scorzonera taxa. numerical results based on 25 morphological characters were discussed and compared with traditional taxonomic treatments. key words: phenetics, scorzonera, systematics, turkey introduction the genus scorzonera l. (asteraceae) numbers about 160 species, ancient mediterranean by origin, belonging to the subtribe scorzonerinae dumort. (lactuceae cass., cichorioideae) is widely spread in the arid regions of eurasia and africa (nazarova 1997, bremer and anderberg 1994). the genus includes up to 47 species in turkey, some of which are endemic (duran 2008). this genus appears simple at first sight. however, it is a taxonomically difficult genus with several complexes of closely related species (chamberlain 1975). the difficulties of identifying scorzonera derive from the genus not having been sufficiently well investigated by taxonomists, many questions thus remaining controversial (nazarova 1997). in recent years scorzonera has been the subject of caryological (nazarova 1997, guardia and blanca 1987), ethnobotanical (rivera et al. 2006, ertuð 2000), chemical (zidorn et al. 2003, magiatis et al. 2001) and phenetic (mavrodiev et al. 2004) studies that have improved our understanding of the systematics of this genus. acta bot. croat. 69 (2), 2010 237 * corresponding author, e-mail: smakbul@hotmail.com u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:38 color profile: disabled composite 150 lpi at 45 degrees nevertheless, there are still systematic problems. to date, no comprehensive numerical taxonomic study has been conducted on scorzonera species, which are often difficult to distinguish from each other and whose infrageneric classification is only preliminary. the numerical approach is indeed a powerful tool in resolving the relationships and taxonomy of closely related species with complex variation patterns. the purpose of this study is to evaluate the extent of the variations in morphological characters and determine the taxonomic value of phenetic traits in classifying scorzonera taxa as represented by 39 populations distributed in ne anatolia. materials and methods thirty-nine populations of nineteen scorzonera taxa were collected from ne anatolia and used as operational taxonomic units (otus) in this study. locality information and a distribution map of the otus are given in table 1 and figure 1. vouchers are deposited in the herbarium of karadeniz technical university, department of biology (ktub). twenty-five morphological characters were assessed: 11 related to vegetative structure, and 14 to floral structures (tab. 2). trait selection was based on floras and our own observations. a total of 234 individuals with flowers were scored for all these characters and the averages of all the individuals from each species were combined to yield the basic data matrix. the raw data matrix of 25 variables as columns and 39 objects (populations) as rows were used for numerical analysis (tab. 3). identifications were made according to flora of turkey (chamberlain 1975), flora of europe (chater 1976), flora iranica (rechinger 1977) and flora of ussr (lipschiz 1964) and assigned to nineteen taxa as follows: scorzonera cana var. jacquiniana , s. cana 238 acta bot. croat. 69 (2), 2010 makbul s., turkmen z., coskuncelebi k., beyazoglu o. fig. 1. distribution map of the investigated taxa in ne anatolia. population numbers are specified in table 1. u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:40 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (2), 2010 239 morphometric study on scorzonera tab. 1. collection data of the examined specimens. no taxa locality altitude (m) collection numbers 1 scorzonera cana (c.a. mey.) hoffm. var. jacquiniana (w. koch) chamb. trabzon: araklý-daðbaºý 1850 makbul 054 2 rize: ovit 2400 makbul 028 3 giresun: alucra 1490 makbul 078 4 s. cana (c.a. meyer) hoffm. var. cana rize: cimil 2300 makbul 057 5 rize: ovit 2400 makbul 030 6 giresun: eðribel geçidi 2350 makbul 095 7 erzurum: ýspir-moryayla 2450 makbul 093 8 s. cana (c.a. meyer) hoffm. var. alpina (boiss.) chamb. rize: ovit 2400 makbul 029 9 artvin: ardanuç 640 makbul 076 10 s. eriophora dc. gümüºhane: tersun daðý 2040 makbul 050 11 gümüºhne: mogoldas daðý 1650 makbul 044 12 s. armeniaca (boiss. et huet.) boiss. bayburt: bayburt kalesi 1650 makbul 059 13 erzurum: aºkale 2000 makbul 048 14 artvin: yusufeli-ýspir road 40. km 815 makbul 073 15 s. parviflora jacq. erzurum: aºkale 1630 makbul 088 16 s. mollis bieb. ssp. mollis gümüºhane: tersun daðý 2040 makbul 051 17 giresun: findikbeli geçidi 1730 makbul 080 18 s. insica dc. bayburt: kop daðý 2150 makbul 085 19 s. laciniata l. ssp. laciniata bayburt: kop daðý 2100 makbul 045 20 bayburt: çerçi köyü 1700 makbul 070 21 artvin: yusufeli-yokuºlu köyü 815 makbul 074 22 s. inaequiscapa boiss. bayburt 1500 makbul 068 23 giresun: alucra-ªiran, 15. km 1670 makbul 079 24 s. sericea dc. bayburt: kop daðý 2450 makbul 089 25 s. tomentosa l. bayburt: kop daðý 2160 makbul 010 26 giresun: alucra 1400 makbul 012 27 s. mollis bieb. ssp. szowitzii (dc) chamb. gümüºhane: kelkit 1635 makbul 039 28 gümüºhane: tersun daðý 2000 makbul 064 29 erzurum. aºkale 1640 makbul 090 30 s. sosnowskyi lipschitz. erzurum: ýspir-moryayla 2400 makbul 091 31 bayburt: kop daðý 2150 makbul 086 32 s. suberosa c. koch erzincan: erzincan-kelkit 20. km 1750 makbul 041 33 bayburt: çerçi köyü 1700 makbul 069 34 s. cinerea boiss. bayburt: kop daðý 2150 makbul 087 35 s. pseudolanata grossh. bayburt 1500 makbul 072 36 bayburt: köse 1650 makbul 040 37 s. seidlitzii boiss. artvin: ªavºat, sahara mezrasý 2150 makbul 022 38 artvin: yalnizçam daðlarý 2200 makbul 025 39 s. latifolia (fish. et mey.) dc. bayburt: kop daðý 2160 makbul 094 u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:40 color profile: disabled composite 150 lpi at 45 degrees var. cana, s. cana var. alpina, s. eriophora, s. armeniaca, s. parviflora, s. mollis ssp. mollis, s. insica, s. laciniata ssp. laciniata, s. inaequiscapa, s. sericea, s. tomentosa, s. mollis ssp. szowitzii, s. sosnowskyi, s. suberosa, s. cinerea, s. pseudolanata, s. latifolia, s. seidlitzii. two multivariate analyses were performed using syn-tax pc 5.0 (podani, 1993): cluster analysis (ca) and principal components analysis (pca). for the ca, a pair-wise matrix of resemblance values was calculated from the standardized data matrix, using gower’s coefficient as the coefficient of resemblance that is designed for mixed data sets (sneath and sokal 1973). a dendrogram was generated by the unweighted pair-group method using arithmetic averages (upgma). for pca, the standardized data were used to create a covariance matrix, and three eigenvectors were extracted. 240 acta bot. croat. 69 (2), 2010 makbul s., turkmen z., coskuncelebi k., beyazoglu o. tab. 2. list of characters used in this study. symbol characters x1 total plant high (cm) x2 state of the plant stem; 0: scape, 1: caulescent x3 root; 0: cylindrical, 1: tuberose x4 residue leaves at the base of the stem; 0: absent, 1: present x5 stem; 0: not branched, 1: branched x6 leaf; 0: entire, 1: pinnatisect or pinnatifit x7 width of leaf (mm) x8 length of leaf (mm) x9 lamina; 0: linear, lanceolat, 1: ovat, eliptic x10 leaf margin; 0: smooth, 1: undulate x11 plant; 0: densely hairy (tomentose, pallose, villous), 1: slightly hairy (pubescent and sericea) x12 number of capitula x13 average length of flowering capitula (mm) x14 average length of outer phyllaries (mm) x15 average length of inner phyllaries (mm) x16 shape of outer phyllaries; 0: linear, lanceolat, 1: ovat, eliptic x17 ligulae color; 0: yellow, 1: purple x18 length of achenes (mm) x19 pappus color; 0: white or cream, black or brownish x20 pappus; 0: completely plumose or barbellat, 1: plumose together with barbellat x21 lower surface of inner phyllaries; 0: hairless, 1: hairy x22 average row number of phyllaries x23 apex of outer phyllaries; 0: unforked, 1: forked x24 surface of achenes; 0: smooth, 1: rough x25 achenes; 0: glabrous, 1: hairy u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:40 color profile: disabled composite 150 lpi at 45 degrees results dendrogram of 25 phenetic variables corresponds to 19 taxa (fig. 2). all taxa fall into two main clusters. the first group is labeled »a« and consists of 30 populations belonging to 15 taxa. the second cluster is labeled »b« and consists of the rest of the examined populations belonging to 4 taxa (scorzonera mollis subsp. mollis, s. inaequiscapa, s. mollis subsp. szowitzii, s. suberosa). the cluster »a« is divided into small subclusters labeled »c« and »d«. pca results using 25 characters are given in figures 3 and 4, showing the distribution of otus and variables on the first two components (axis). the results of principal component analysis show eigenvalues as percentages of explained variance, and cumulative percentages (tab. 4). pc-1 and pc-2 had the highest score for the eighteen of the twenty five traits (tab. 4). these variables are x3, x10, x13, x14, x15, x16, x17, x18, x25 for pc-1 and x4, x6, x9, x11, x12, x19, x20, x23, x24 for pc-2. pc-3 received the highest score only for the remaining seven variables used in this study. eleven of the twenty-five traits analyzed in the present study explained more than 50% of the total variation. the highest variation rates observed among the twenty-five characters and accounting for the first three acta bot. croat. 69 (2), 2010 241 morphometric study on scorzonera fig. 2. cluster analysis – upgma. the clusters discussed in the text are marked with letters. population numbers are given in table 1. u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:41 color profile: disabled composite 150 lpi at 45 degrees 242 a c t a b o t .c r o a t .69 (2),2010 m a k b u l s .,t u r k m e n z .,c o s k u n c e l e b ik .,b e y a z o g l u o . tab. 3. raw data matrix using numerical analysis (raw: character, column: taxa). 25.83 0 0 1 0 1 3.13 10.51 0 0 1 3.33 21.60 7.50 20.16 0 1 9.33 0 0 1 1 3 1 0 25 0 0 1 1 1 2.98 12.16 0 0 1 5.16 22.50 8 22.16 0 1 10 0 0 1 1 3 1 0 24.83 0 0 1 0 1 2.42 13.33 0 0 1 1.33 24.66 6 17 0 1 9.66 0 0 1 1 3 0 0 20.83 0 0 1 0 1 1.88 8.83 0 0 1 3.16 22.16 6.83 19.16 0 1 8.50 0 0 1 0 3 1 0 11.33 0 0 1 0 1 1.80 11.33 0 0 1 2.83 22.50 5.58 16.66 0 1 9.66 0 0 1 1 3 1 0 18.66 0 0 1 0 1 2.61 12.50 0 0 1 3.83 22.16 8 23.83 0 1 11.60 0 0 1 1 3 1 0 18.66 0 0 1 1 1 2.15 10 0 0 1 6 19.16 6 18.16 0 1 9.50 0 0 1 1 3 0 0 6 0 0 1 0 1 1.10 7.25 0 0 1 1.66 16.25 5.83 14.16 0 1 8.16 0 0 1 1 3 1 0 9.33 0 0 1 0 1 1.80 7 0 0 1 3.50 13.83 6.50 18 0 1 9 0 0 1 1 3 1 0 24 1 0 0 0 0 1.33 16.60 0 0 0 3.83 22.66 13 24 0 1 11 1 1 1 1 2 0 0 33.16 1 0 0 1 0 1.85 15.33 0 0 0 2.50 26.50 12.50 31.50 0 1 6.33 1 1 1 1 2 0 0 20 1 0 1 1 1 2.56 15.66 0 0 1 5.16 15.83 4.83 16.83 0 1 8.66 1 0 1 1 3 1 0 34.16 1 0 1 1 1 3.18 13.83 0 0 1 3.16 15.66 5 21.83 0 1 10.33 1 0 0 1 3 1 0 31.16 1 1 1 1 1 2.60 13.83 0 0 1 8 18.83 4.83 16.66 0 1 8.50 1 0 1 1 3 1 0 54.66 1 0 0 0 0 11.16 27.66 0 0 1 1 24.66 7.83 23.33 1 1 7.66 0 0 1 0 3 1 0 27.33 1 1 1 1 0 0.66 13.33 0 1 1 2.66 32.16 10.16 29.50 1 1 16.66 0 0 0 0 2 0 1 21.50 1 1 1 1 0 0.45 13.66 0 1 1 4 32.50 11.16 30.66 1 1 14 0 0 1 0 2 0 1 33.50 1 0 1 0 0 3.45 17.66 1 0 1 3.16 31.33 9.83 28.33 1 1 20.16 0 0 1 0 3 0 1 51.66 1 0 1 1 1 4.50 20 0 0 1 3.33 18.16 5.16 22.33 0 1 9.16 0 0 0 1 3 1 0 48.83 1 0 1 1 1 3.50 19.16 0 0 1 3 13.50 5.33 21.16 0 1 11.33 0 0 0 1 3 0 0 49 1 0 0 1 1 3.73 10.50 0 0 1 3.66 22.33 6.16 21.16 0 1 11.33 0 0 0 0 3 1 0 12 0 1 1 0 0 0.90 8.83 0 1 1 1 26.66 7.50 27.83 1 1 15.50 0 1 1 1 3 0 1 13.33 0 1 1 0 0 0.96 9 0 1 1 1 26.66 8.83 34.50 1 1 16 0 1 1 1 3 0 1 4 0 0 1 0 0 2.66 3 0 0 1 1 14.66 5 10.83 0 1 7.33 0 0 1 1 3 0 0 49 1 0 0 1 0 2.13 8.25 1 1 0 12.66 19.66 9 16.50 0 1 10.33 0 1 0 1 2 0 0 32.16 1 0 0 1 0 1.83 8.83 1 1 0 6.16 16.83 7.16 17.16 0 1 8.66 0 1 0 1 2 0 0 13.83 0 1 1 0 0 0.48 10.50 0 1 1 3 27.66 8.83 26.33 1 1 15.83 0 1 1 1 2 0 1 12.16 0 1 1 0 0 2.41 11.50 0 1 1 1.66 24.16 9.83 24.33 1 1 16.50 0 1 1 1 2 0 1 23.66 0 1 1 0 0 2.33 13.66 0 1 1 6.50 27.50 8.16 29.33 1 1 14.16 0 1 1 1 2 0 1 66 1 0 1 1 0 2.18 10.33 1 0 1 10.66 16.83 5.16 15.66 0 1 11 1 0 1 1 2 0 0 65.33 1 0 1 1 0 1.96 11.83 1 1 1 18 15 5.83 16.83 0 1 11.16 1 0 1 1 2 0 0 10.16 0 1 1 0 0 0.58 8.66 0 1 1 1.66 22 7.50 17.16 1 0 15.66 0 1 0 1 3 0 1 8.83 0 1 1 0 0 0.96 8.33 0 1 1 2.50 25 9 20.33 1 0 16 0 1 0 0 3 0 1 52.16 1 0 0 1 0 1.41 11.83 1 0 1 5.33 19.16 6.16 13 0 1 8.66 0 0 1 1 3 0 0 8.16 0 1 0 0 0 0.81 7.25 0 1 0 3.16 12.66 4.50 11.83 0 1 6.33 1 0 1 1 2 0 0 7.50 0 1 0 0 0 0.86 6.83 0 1 0 3.33 13.33 3.50 12 0 1 5.41 1 0 1 1 2 0 0 11.83 0 0 0 0 0 2.08 7.83 0 0 0 1.83 17.66 8 14.83 0 1 5.91 0 1 1 1 2 0 0 10.16 0 0 0 0 0 1.33 5.50 0 0 0 2.50 14.16 5.50 14.16 0 1 5.66 0 1 1 1 2 0 0 75 1 0 0 1 0 2.18 11.83 1 0 0 44.16 18.16 7.50 15.50 0 1 9.50 1 1 1 1 3 0 0 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 0 0 m a k b u l . v p 1 1 . l i s t o p a d 2 0 1 0 1 3 : 2 8 : 4 1 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s acta bot. croat. 69 (2), 2010 243 morphometric study on scorzonera fig. 3. principal component analysis of 39 populations projected onto the first two axes. for population number explanations see table 1. fig. 4. principal component analysis of 25 variables projected onto the first two axes. for variables number explanations see table 2. u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:44 color profile: disabled composite 150 lpi at 45 degrees components are 92.25% (x25), 83.705% (x16) and 69.471% (x2). these characters are among the binary traits examined in this study. only the first three components were taken into account because of their eigenvalues. the three components account together for 60.55% variation (tab.4). the first component accounts for 27.23% variation. the second component accounts for 17.75%, the third component accounts for 15.57% and together they account for 60.55 %. discussion there are many clustering methods and similarity coefficients in the literature as can be seen in sneath and sokal (1973). in this study, upgma along with gower’s coefficient has been used for clustering the 39 otus (fig. 2). in order to determine how well this dendrogram represents the underlying matrix of resemblances, the cophenetic correlation 244 acta bot. croat. 69 (2), 2010 makbul s., turkmen z., coskuncelebi k., beyazoglu o. tab. 4. percentage of variance of variables accounted for by first three components. names of variables pc-1 pc-2 pc-3 x1 15.981 7.052 61.736 x2 7.373 7.770 69.471 x3 57.119 0.730 4.527 x4 8.108 45.162 0.147 x5 13.995 4.502 34.571 x6 26.887 51.816 0.339 x7 10.890 11.774 25.331 x8 0.385 6.451 59.081 x9 3.948 30.556 17.282 x10 49.736 14.570 1.507 x11 3.927 65.503 3.842 x12 8.451 24.685 9.668 x13 56.878 3.881 16.841 x14 36.768 3.774 12.378 x15 43.064 3.743 21.436 x16 83.705 1.424 4.333 x17 17.493 0.209 2.170 x18 64.405 3.017 10.716 x19 14.161 20.058 0.687 x20 25.361 30.846 1.906 x21 0.655 0.023 8.316 x22 11.527 7.026 21.506 x23 5.888 51.826 0.362 x24 21.179 47.062 0.389 x25 92.789 0.340 0.715 percentage of variance explained 27.23 17.75 15.57 cumulative percentages of variance explained 27.23 44.98 60.55 u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:44 color profile: disabled composite 150 lpi at 45 degrees coefficient (rcs) was also calculated. it has generally been found to vary from 0.6 to 0.95, depending on the methods producing the dendrogram and the nature of the differences among the specimens classified (sneath and sokal 1973). our dendrogram had a cophenetic correlation of 0.72. this means that the dendrogram provides a fairly accurate representation of the resemblances among otus. all the examined otus fall into two major clusters at 88.2% dissimilarity levels (fig. 2). one, labeled »a«, consists of populations that belong to scapigerous and caulescent taxa, the other, labeled »b«, includes all the remaining otus representing entirely scapigerous taxa. when the dendrogram is carefully examined, it can be seen that cluster »a« consisting of most of the examined otus belongs to fifteen taxa, but cluster »b« consisting only of nine otus belongs to four taxa. there are no significant differences in the anatomical peculiarities among the taxa in group »b« (lipschiz 1964, kamelin and tagev 1986). cluster »a« splits into two small clusters (c, d). cluster »c« consists of otus corresponding to scapigerous, subscapigerous and caulescent taxa. they are morphologically and anatomically related to each other. all otus in this group have a typically cylindrical root system, but the representatives of s. parviflora (otu 15) and s. insica (otu 18) form a small group because of the unforked stem and the pubescence. the representatives of s. armeniaca (otus 12–14) and s. laciniata ssp. laciniata (otus 19–21) are close to each other because of the similar morphological traits as indicated by chamberlain (1975). these views are correlated with our results obtained from upgma. additionally, our findings also support the results based on caryological data as indicated by nazarova (1997). although otu 24 (s. sericea) display some different morphologic properties and grow in different habitats from the other examined populations in cluster »c«, it occurs in cluster »c«, together with the other otus because of the shared characters such as cylindrical root, pinnatisect leaf, similar achene and flower peculiarities. although all representatives of s. cana (otus 1–7) are uniform at the specific level, they are not separated at the subspecific level. s. cana is said to be close to s. laciniata ssp. laciniata (chamberlain 1975), but our results from upgma do not support this view and show that s. cana is more closely related to s. armeniaca (fig. 2). this situation might be explained as follows; s. laciniata prefers dry habitats and is characterized by an entire leaf and s. cana prefers humid habitats and is characterized by a compound leaf. cluster »d« includes otus characterized by densely hairy, generally caulescent and a few lanate scapigerous taxa (s. pseudolanata, s. seidlitzii). although the representatives of s. pseudolanata (otus 35–36) and s. seidlitzii (otus 37–38) are morphologically similar to the representatives of cluster »b«, these scapigerous otus that are densely lanate and hairy occur in cluster »d«. the major group of the cluster »d« includes only caulescent tomentose taxa. but s. latifolia (otu 39) characterized by numerous capitula (more than 20) and s. cinerea (otu 34) characterized by globrous achenes form a small group in cluster »d«. s. tomentosa is closed to s. latifolia (chamberlain 1975). our results do not support this idea; those from upgma show that s. latifolia is more closely related to s. cinerea and s. sosnowskyi than s. tomentosa. otus characterized by scapigerous or semi-caulescent, tuberose-root, glabrous or pubescent and entire linear lamina with undulate margins were clustered in group »b«. it seems that the representative of s. mollis ssp. mollis forms a small sub-group in cluster »b«. acta bot. croat. 69 (2), 2010 245 morphometric study on scorzonera u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:44 color profile: disabled composite 150 lpi at 45 degrees the reason for this can be explained with the different phenetic traits such as the forked and semi-caulescent stem. the other otus in cluster »b« have very similar morphological characters. the representatives of s. suberosa (otus 32–33) with lilac to purple flowers are separated from the other otus (fig. 2). this delimitation is coincided with the caryological and its results of mavrodiev (2004). the ranks of subgenus and section have been used in scorzonera by several scientists (lipschiz 1964, kamelin and tagev 1986, nazarova 1997). scorzonera cana, s. armeniaca and s. laciniata ssp. laciniata treated in subg. podospermum (dc.) lipschiz, s. suberosa in subg. pseudupodospermum (lipschiz et krash) lipschiz, s. seidlitzii in subgen. scorzonera sect. pulvinares lipschiz and s. latifolia in subg. scorzonera sect. nervosae lipschiz (lipschiz 1964). all the otus of these listed taxa are grouped as indicated by lipschiz (1964) and our results from upgma support the use of such ranks within the genus (fig. 2). the position of the 39 examined populations on the first two components of the pca (fig. 3) shows that all populations examined in this study are split into three distinct clusters. these clusters are almost associated with the results from upgma except for the representatives of s. insica. this situation could be explained by the differences in the root systems and the shapes of lamina, which are the most important characters, explaining most of the variation among the examined taxa. this might be caused by the insufficient specimens or population examined in this study. there is no other contradiction between pca and upgma with respect to the distribution of otus. the first two extracted components explain 44.98% of the total variation among the examined populations (fig. 3). the three components account together for 60.55% of the variation among populations (tab. 4). the first component emphasizes pubescence and length of achenes, shape of outer phyllaries, average length of flowering capitula, root shape and leaf margin and the second component with apex of outer phyllaries, hair types of plant and shape of lamina. the third component emphasizes length of the leaf, total plant height and state of the plant stem. however, weights are all under 50% and almost equal. in summary, the state of residue leaves at the base of the stem, pubescence and length of achenes, shape of outer phyllaries, average length of flowering capitula, root shape and leaf margin are the most important characters separating the scorzonera species. these characters are the basic ones used in most floras for separating these species (chater 1976, chamberlain 1975). the present study is a preliminary step in the analysis of morphological characters by means of numerical analysis. the results basically agree with the traditional taxonomic treatments of scorzonera. although qualitative variables such as shape of outer phyllaries, achenes and root shape explain most of the total variation, binary characters seem to be more important than quantitative ones in separating scorzonera taxa. acknowledgements the authors would like to express their thanks to dr. ahmet duran from the education faculty, selçuk university (konya-turkey) for kindly helping on the identification of samples. thanks to the scientific and technological research council of turkey for financial support. 246 acta bot. croat. 69 (2), 2010 makbul s., turkmen z., coskuncelebi k., beyazoglu o. u:\acta botanica\acta-botan 2-10\200 makbul.vp 13. listopad 2010 12:02:49 color profile: disabled composite 150 lpi at 45 degrees references bremer, k., anderberg, a. a., 1994: asteraceae: cladistics and classification. timbers press, portland. chamberlain, d. f., 1975: scorzonera l. in: davis, p. h. (ed), flora of turkey and the east aegean islands. edinburgh university press, edinburgh. chater, a. o., 1976: scorzonera. in: tutin, t. g, heywood, v. h, burges, n. a, valentine, d. h, walters, s. m, webb, d. a. (eds.), flora europaea. cambridge university press, cambridge. duran, a., 2008: rediscovery of the poorly known scorzonera argyria and its relationships in turkey. biologia 63, 1078–1084. ertuð, f., 2000: an ethnobotanical study of central anatolia (turkey). economic botany 54, 155–182. guardia, c. d., blanca, g., 1987: karyology of the scorzonera (compositae) species from the iberian peninsula. plant systematic and evolution 156, 29–42. kamelin, r. v., tagev, i. u., 1986: survey of the species of the genus scorzonera (asteraceae). botanical journal 71, 1972–1982. lipschiz, s. j., 1964: scorzonera. in: shishin, b. k. (ed.), flora of the ussr, 29 (in russian). academy of science of the ussr, moscow, leningrad. magiatis, p., mitaku, s., skaltsounis, a., tillequin, f., 2001: 1-oxo-2-hydroxy-1,2-dihydroacronycine: a useful synthon in the acronycine series for the introduction of amino substituents at 6-position and for the conversion into isopropylfuroacridones. chemical and pharmological bulletin 49, 1304–1307. mavrodiev, e. v., edwards, c. e., albach, d. c., gitzendanner, a., soltis, p. s., soltis, d. e., 2004: phylogenetic relationships in subtribe scorzonerinae (asteraceae: cichorioideae: cichorieae) based on its seguence data. taxon 53, 699–712. nazarova, e. a., 1997: karyosystematic investigation of the genus scorzonera l. s.l. (lactuceae, asteraceae). caryologia 50, 239–261. sneath, p. h. a., sokal, r. r., 1973: numerical taxonomy: the principles and practice of numerical classification. w. h. freeman and company, san francisco. podani, j., 1993: multivariate data analysis in ecology and systematic, a methodological guide to syn-tax 5.0 package. spb academic publishing, netherlands. rechinger, k. h., 1977: scorzonera l. in: rechinger, k. h. (ed.), flora iranica, 122, 16–79. graz akademische druck und verlagsanstalt. rivera, d., obón, c., heinrich, m., inocencio, c., verde, a., fajardo, j., 2006: gathered mediterranean food plants–ethnobotanical investigations and historical development. forum of nutrition 59, 18–74. zidorn, c., ellmerer, e. p., sturm, s. stuppner, h., 2003: trylobibenzyls e and f from scorzonera humilis and distribution of caffeic acid derivatives, lignans and tyrolobibenzyls in european taxa of the subtribe scorzonerinae (lactuceae, asteraceae). phytochemistry 63, 61–67. acta bot. croat. 69 (2), 2010 247 morphometric study on scorzonera u:\acta botanica\acta-botan 2-10\200 makbul.vp 11. listopad 2010 13:28:44 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 69 (2), 173–182, 2010 coden: abcra 25 issn 0365–0588 enhancement of betanin yield in transformed cells of sugar beet (beta vulgaris l.) bojana kri@nik, dubravko pavokovi]* department of molecular biology, division of biology, faculty of science, university of zagreb, horvatovac 102a, hr-10000 zagreb, croatia betanin belongs to a large family of betacyanin pigments, betalains, and in the food industry it is established as a powerful antioxidant and natural colorant. the main source of betanin is a red beet (beta vulgaris l.), the yield depending on abiotic and biotic factors in the field. sugar beet cells, transformed by a wild strain b6s3 of agrobacterium tumefaciens, strongly produce betanin and could be a stable production source. with the aim of enhancing the yield, cell suspensions were initiated from friable calli. biomass accumulation, betanin content and yield were monitored over 21 days in relation to changes of sucrose concentration, modifications of minerals in nutrient medium, and the usage of elicitors. results showed that elevating sucrose levels from 3% to 4%, 5%, or 6% (w/v) in the original medium strongly induced biomass accumulation followed by an increase in betanin yield of up to 250%. modification of minerals in the medium additionally increased betanin yield up to 20% in a few instances: 40 mg l–1 was recorded at day 10 with 5% and 6% of sucrose. the highest yield at 53 mg l–1 was reached at day 21 on 4% suc, again with the modified medium. high sucrose concentrations positively affected betanin accumulation only during lag phase of the cell suspension, but afterwards the trend reversed. calcium and yeast extract were used as abiotic and biotic elicitor, respectively, in the early exponential phase of subculture (day 7). calcium ions (at 10 fold higher concentration than in the control) failed to increase betanin yield as well as yeast extract at 0.25% (w/v). yeast extract at 1.25% provoked excretion of betanin at day 4, and cell necrosis at day 7 after elicitation. taken together, in our system, sucrose affected betanin yield more strongly than medium modifications or elicitors. yeast extract could be used for reverse-sequestration of betanin where the cells can be used over an extended period. key words: elicitors, plant tissue culture, pigment, antioxydant, betanin, beta vulgaris abbreviations: dopa – 3,4-dihydroxyphenylalanine, hb – high betacyanin, ye – yeast extract, suc – sucrose introduction betalains are a class of water soluble nitrogenous plant pigments characteristic of the order caryophyllales (girod and zryd 1991, tanaka et al. 2008). they are classified into red (crimson) betacyanins and yellow betaxanthins. betalain biosynthesis starts with acta bot. croat. 69 (2), 2010 173 * corresponding author, e-mail: dubravko@zg.biol.pmf.hr u:\acta botanica\acta-botan 2-10\304 kriznik.vp 13. listopad 2010 11:36:55 color profile: disabled composite 150 lpi at 45 degrees amino acid tyrosine and continues through two independent pathways from dopa; the betalamic acid biosynthetic pathway and the cdopa synthetic pathway. betalamic acid is the chromophore molecule of both betacyanins and betaxanthins, and cdopa and its derivatives are essential to betacyanin production (strack et al. 2003). the addition of amines or amino acids such as betalamic acid conjugates produces numerous betaxanthins. however, in comparison to flavonoids and carotenoids, biosynthetic pathways of betalains, enzymes and genes involved in the pathway are much less understood (tanaka et al. 2008). betalains have aroused a huge interest in the food industry as a natural food colorant as well as in biopharmaceuticals. their advantages as food colorants are that the color does not depend on the ph, and their stability (tanaka et al. 2008). additionally, betalains are up to twice fold better free radical scavenger than some anthocyanins (lee et al. 2005, gliszczyñska-œwi£go et al. 2006). moreover, they have desirable bioactive properties: they have an anti-inflammatory effect, they activate phase ii detoxifying enzymes, or they can protect low density lipoproteins from oxidation (tesoriere et al. 2004, lee et al. 2005, gliszczyñska-œwi£go et al. 2006). the most important commercial source of betalains is red beetroot (beta vulgaris l.), which mostly contains betanin, a violet-red betacyanin. the yield and quality of betanin obtained from red beet root depends on field conditions and can be significantly lowered by lack of minerals, drought, salinity, pathogen attacks or even by an unfavorable political climate (ramachandra rao and ravishankar 2002). plant cell and tissue cultures are attractive alternative sources of bioactive plant substances, including betalain pigments. such a type of production offers several advantages over field cultivation, notably optimal and stable growth conditions, possibility for voluntary modulation of growth parameters and constant quality control (moreno et al. 2008). in vitro systems for producing betanin include callus cultures, cell suspensions of red beet root and hairy roots transformants of red beet root, but the yield of betanin is still lower than in field-raised red beet (georgiev et al. 2008). new approaches are being tested to enhance betanin production in in vitro systems. this involves optimization of nutrient medium, elicitors – molecules of biological or non biological origin which effectively induce secondary metabolite production, or reverse sequestration using a »permeabilization, release and recovery« approach with limited oxygen supply, sonication or the use of detergents (akita et al. 2002, thimmaraju et al. 2003, uozumi 2004, savitha et al. 2006). we transformed sugar beet cells by infecting leaf fragments with wild strain b6s3 of agrobacterium tumefaciens. subsequently a cell line characterized by high betanin production was established (pavokovi] et al. 2009). this tumor tissue stably maintained its phenotype during more than 10 years of subculturing. the betanin production was light-dependent. when placed in darkness, the tumor stopped producing betanin and became colorless after few subcultures. a switch from the red-violet to white phenotype was reversible (pavokovi] et al. 2009). the aim of present study was to optimize the betanin yield of the transformed cells by modifying the composition of nutrient media and by using elicitors. sucrose is the best carbohydrate source as it gives the highest betalain production and yield (monroy et al. 1994, bhagyalakshmi et al. 2004). yeast extract is frequently used as a widely available and successful biotic elicitor in a number of systems, including red beet hairy root transformants 174 acta bot. croat. 69 (2), 2010 kri@nik b., pavokovi] d. u:\acta botanica\acta-botan 2-10\304 kriznik.vp 11. listopad 2010 11:31:31 color profile: disabled composite 150 lpi at 45 degrees (sánchez-sampedro et al. 2005, savitha et al. 2006, shinde et al. 2009) while calcium is the most successful among abiotic elicitors in elevating betalain productivity, as was recently shown (savitha et al. 2006). material and methods sugar beet tumor tissue was obtained by transformation of leaf fragments by a. tumefaciens wild strain b6s3 (pavokovi] et al. 2009). tissue was cultivated on a hormone-free pg0 medium supplemented with 3% (w/v) suc and solidified by 0.9% (w/v) agar (negrutiu et al. 1975). the light (photon density of 42 mmol m–2 s–1) / dark photoperiod was 16 / 8 hours. cell suspensions were initiated by transferring 2 g of very friable callus to 50 ml of pg0 medium supplemented with 3% suc. suspensions were shaken on reciprocal shaker at 100 rpm under the same light/dark conditions as cultures on solid medium. to initiate the experiment, two-week-old cell suspensions (10 ml of packed cells volume) were added to the flask containing 35 ml of the pg0 or improved linsmeier-skoog medium (hb, tab. 1) (akita et al. 2002). control suspensions were supplemented with 3% suc, while treatments were with 4%, 5% or 6% (w/v) suc. cells were collected at days 4, 7, 10, 14 and 21 of subculture and were weighed, lyophilized and stored until usage. yeast extract (sigma, germany) as elicitor was dissolved in hb medium with 5% suc at 0.25 and 1.25% (w/v) concentration. calcium, as cacl2, was dissolved at 10-fold higher concentration than in hb medium. after seven days of subculture in hb with 5% suc, old acta bot. croat. 69 (2), 2010 175 betanin production in transformed sugar beet cells tab. 1. concentrations of inorganic salts in pg0, ls and hb media, expressed in mmol l–1. component pg0 ls medium hb medium macroelements nah2po4 ´ h2o kcl nh4no3 (nh4)2so4 kno3 nano3 mgso4·7h2o cacl2 kh2po4 na2fe-edta 1.81 8.05 – 3.03 19.20 – 2.03 2.04 0.1 – – 20.6 – 18.8 – 1.5 3.0 1.25 0.1 – – 2.0 – 18.8 7.2 1.5 3.0 1.25 0.1 microelements h3bo3 mnso4·4h2o znso4·7h2o ki na2moo4·2h2o cuso4·5h2o cocl2·6h2o 0.01 0.01 0.03 0.09 0.0001 0.00001 0.00001 0.1 0.1 0.03 0.005 0.001 0.0001 0.0001 0.1 0.1 0.0003 0.005 0.001 – – u:\acta botanica\acta-botan 2-10\304 kriznik.vp 11. listopad 2010 11:31:31 color profile: disabled composite 150 lpi at 45 degrees medium was removed from cell suspensions and 50 ml of medium with elicitors was added to the cells (savitha et al. 2006). cell suspensions were returned to the shaker and samples were collected at days 4, 7, 10 and 14 after elicitation, and processed as mentioned above. for betanin quantification, 20 mg of the lyophilized sugar beet tumor cells were pulverized in a bead mill homogenizer (retsch mm 200, retsch gmbh, germany) and resuspended in 1 ml of 80% methanol (v/v) supplemented with 50 mm ascorbic acid. homogenates were clarified by centrifugation for 20 minutes at 15000 ´ g. pigments were reextracted twice with additional 2 ml of the same buffer. extracts were joined and the absorbance was measured spectrophotometrically at 534 nm (ati/unicam uv4-100, cambridge, uk). betanin was quantified using molar absorption coefficient of betanin e = 60 000 l cm–1 mol–1. 176 acta bot. croat. 69 (2), 2010 kri@nik b., pavokovi] d. fig. 1. biomass accumulation of sugar beet tumor line grown using 0.8 g inoculum in 50 ml of a) pg0 or b) hb liquid medium with sucrose concentrations ranging from 3% to 6% (w/v). each data point is a result from two independent experiments with 4 replicates ±sd. u:\acta botanica\acta-botan 2-10\304 kriznik.vp 11. listopad 2010 11:31:37 color profile: disabled composite 150 lpi at 45 degrees results a sugar beet tumor line producing betanin was subcultured in liquid pg0 medium with 3% (w/v) suc. in this control medium, exponential growth of the cells began after day 4 and continued until day 14 when the growth ceased (fig. 1a). elevation of suc concentration in the pg0 from 3% to 4%, 5% or 6% resulted in the increased dry biomass (fig. 1a). maximum biomass at these concentrations was produced at day 14, except on 6% suc where the highest biomass was accumulated at day 21. changing nutrient composition from pg0 to hb induced slightly higher accumulation of dry biomass (tab. 1, fig. 1b). the positive influence of suc on dry biomass was apparent after 10 days of subculture in both media, but only between 3% and the higher concentrations of suc. an increase in suc concentration increased betanin content in both media at days 4 (fig. 2). at day 7 differences in betanin content were observed between 4% and 3% suc on pg0 and between 6% and 3% on hb, and at day 10 only between 6% and 3% suc on hb. afterwards, the trend reversed, and a higher suc concentration negatively affected betanin content. compared to pg0, hb medium increased the pigment content in only a few instances: at day 4 with 4% suc and at day 21 with 4 and 6% suc. the resulting effect of increase of suc concentration was visible as a higher total betanin yield at days 7 and 10 of the subculture on pg0, and at days 4, 7, and 10 of the sub culture on hb (fig. 3). when compared to pg0, hb medium produced higher pigment acta bot. croat. 69 (2), 2010 177 betanin production in transformed sugar beet cells fig. 2. betanin content of sugar beet tumor line subcultured during 21 days in either pg0 or hb medium in relation to 3 – 6% (w/v) sucrose. each data point is a result from two independent experiments with 4 replicates ±sd. u:\acta botanica\acta-botan 2-10\304 kriznik.vp 13. listopad 2010 11:50:27 color profile: disabled composite 150 lpi at 45 degrees yield at day 4 with 4% and 5% suc, at day 7 and 14 with 6% of suc. the highest total betanin yield was obtained in hb medium and was 40 mg l–1 at day 10 and 14 at maximum suc concentration and 50 mg l–1 at day 21 with 4% suc. as elicitors of betanin biosynthesis, calcium at 10-fold of the normal concentration, and ye at 0.25 and 1.25% (w/v) were used for cells grown in the hb medium supplemented with 5% suc (fig. 4). ye at 0.25% and ca did not significantly improve betanin yield. ye at 1.25% provoked expulsion of betanin in liquid medium which decreased betanin yield. cell necrosis followed 7 days after elicitation with higher concentration of ye. discussion previously, it was shown that a sugar beet cell line transformed with a. tumefaciens wild type b6s3 produced betanin (pavokovi] et al. 2009). no phenotype changes were observed during more than ten years of subculturing of the tumor tissue. suc was a better source of carbohydrates for high betanin yield than monosaccharides glucose, fructose or their combination (pavokovi] et al. 2009). in order to optimize betanin content and yield, cell suspensions from the transformed line were initiated and cells were incubated in increasing concentrations of suc, in different composition of mineral nutrients and using biotic and abiotic elicitors. 178 acta bot. croat. 69 (2), 2010 kri@nik b., pavokovi] d. fig. 3. total betanin yield of sugar beet tumor line subcultured during 21 days in either pg0 or hb medium in relation to 3 – 6% (w/v) sucrose. each data point is a result from two independent experiments with 4 replicates ±sd. u:\acta botanica\acta-botan 2-10\304 kriznik.vp 11. listopad 2010 11:31:51 color profile: disabled composite 150 lpi at 45 degrees increase of suc concentration from 3% to 4%, 5% or 6% had a positive effect on biomass accumulation and total betanin yield during subculture (figs. 1, 3). suc concentrations higher than 6% did not additionally increase betanin yield and were not used (akita et al. 2002). when linsmeier-skoog medium optimized for betanin production was used instead of pg0, a slight increase in biomass and betanin yield was observed on few instances (tab. 1, figs. 1, 3). these improvements were most likely due to the modification of ratio of ammonium to nitrate ions (1:14), reduction of zinc ions and complete removal of cobalt and copper ions from the medium (akita et al. 2002). the effect of increasing suc concentrations on betanin content was complex. during the first days of subculture, a higher suc concentration increased betanin content, but this trend quickly changed and after 10 days high suc negatively affected betanin content (fig. 2). the initial and positive effect of high suc on betanin yield could be a result of the high osmolarity of the medium which caused an osmotic stress on the cells. it is known that this type of stress can stimulate production of secondary metabolites such as anthocyanins in vitis vinifera l. cv (grape) cells, saponins in panax notoginseng cells or alkaloids in catharanthus roseus cells (do and cormier 1990, zhang et al. 1995, godoy-hernández et al. 2000). on the other hand, the inhibitory effect of suc on betanin accumulation after 10 days was also reported in cell suspensions of beta vulgaris, phytolacca americana and chenopodium rubrum (berlin et al. 1986, sakuta et al. 1987, akita et al. 2002). a possible explanation lies in the positive correlation of betacyanin accumulation and cell division (hirose et al. 1990, sakuta et al. 1994), where betacyanins are produced during intensive cell division. when a number of cell divisions was reduced, as in the stationary phase, betanin accumulation was also decreased (fig. 3). this suggests that the main factor contributing to increased betanin yield with high suc concentrations could be the increase in cell number, rather than increased accumulation of betanin per cell. production of secondary metabolites can be enhanced even further using abiotic or biotic elicitors. calcium ions, at 10 fold higher concentration than found in hb medium, were used as they displayed the highest potential as abiotic elicitors of betanin production in hairy roots of red beet (savitha et al. 2006). in our system, however, calcium failed to acta bot. croat. 69 (2), 2010 179 betanin production in transformed sugar beet cells fig. 4. total betanin yield of sugar beet tumor line grown in hb medium with 5% sucrose for 7 days and treated with either: a) calcium at 10 fold concentration than in hb medium. b) 0.25 % (w/v) yeast extract. c) 1.25 % yeast extract. each data point is a result from two independent u:\acta botanica\acta-botan 2-10\304 kriznik.vp 11. listopad 2010 11:31:57 color profile: disabled composite 150 lpi at 45 degrees provoke similar cellular response (fig. 4). calcium acts as the secondary messenger in signaling the responses following elicitation (nishi 1994) but is also a messenger in a number of other signaling processes involving cellular growth, development, redox homeostasis, or light signal transduction (demidchik and maathuis 2007, tuteja and mahajan 2007, yang 2008), which might impede yield of betanin. yeast extract at 0.25 and 1.25% failed to increase and decreased yield of betanin, respectively (fig. 3). the same concentrations of ye were only marginally successful in increasing betanin yield in red beet hairy roots (savitha et al. 2006). however, the higher concentration of ye used was toxic. it caused extraction of the pigment from vacuole to extracellular space and after 7 days of elicitation it induced cell necrosis. although toxic, this effect of ye could be used for repeated sequestration of betanin. in such a process, cells are briefly introduced to a tightly controlled stressor, such as abrupt changes of ph, sonication, temperature shock or hypoxia (thimmaraju et al. 2003). this causes non-lethal expulsion of pigments from the cells to medium which can be harvested. then the conditions of cultivation are returned to the original state and pigments can be harvested again. taken together, betanin yield was successfully induced in sugar beet tumor cells mostly by increasing carbohydrate concentration and to a lesser extent by modifying composition of nutrient medium. while our results are comparable with in vitro red beet production systems (akita et al. 2002, pavlov et al. 2005), they are lower than red beet hairy root transformants (savitha et al. 2006) suggesting further testing, involving a broader number of elicitors or betanin precursor feeding should be performed to additionally increase the betanin content and yield of the tumor cells. acknowledgements the financial support of this work was provided by the ministry of science, education and sports of republic of croatia within the project no. 119-1191196-1200. the authors thank professor marijana krsnik-rasol for transforming sugar beet cells and establishing the transformed tissue lines. references akita, t., hina, y., nishi, t., 2002: new medium composition for high betacyanin production by a cell suspension culture of table beet (beta vulgaris l.). bioscience biotechnology and biochemistry 66, 902–905. berlin, j., sieg, s., strack, d., bokern, m., harms, h., 1986: production of betalains by suspension cultures of chenopodium rubrum l. plant cell tissue and organ culture 5, 163–174. bhagyalakshmi, n., thimmaraju, r., narayan, m. s., 2004: various hexoses and di-hexoses differently influence growth, morphology and pigment synthesis in transformed root cultures of red beet (beta vulgaris). plant cell tissue and organ culture 78, 183–195. demidchik, v., maathuis, f. j. m., 2007: physiological roles of nonselective cation channels in plants: from salt stress to signalling 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in the era of global agri-food science, technology and nutritional health. phytochemistry reviews 7, 261–280. negrutiu, i., beeftink, f., jacobs, m., 1975: arabidopsis thaliana as a model system in somatic cell genetics i. cell and tissue culture. plant science letters 5, 293–304. nishi, a.,1994: effect of elicitors on the production of secondary metabolites. in: ryu, d. d. y., furusaki, s. (eds.), advances in plant biotechnology, 135–51. amsterdam, elsevier science. pavlov, a., georgiev, v., ilieva, m., 2005: betalain biosynthesis by red beet (beta vulgaris l.) hairy root culture. process biochemistry 40, 1531–1533. pavokovi], d., rusak, g., besendorfer, v., krsnik-rasol, m., 2009: light-dependent betanin production by transformed cells of sugar beet. food technology and biotechnology 47, 153–158. ramachandra rao, s., ravishankar, g. a., 2002: plant cell cultures: chemical factories of secondary metabolites. biotechnology advances 20, 101–153. sakuta, m., takagi, t., komamine, a., 1987: effects of sucrose on betacyanin accumulation and growth in suspension-cultures of phytolacca americana. physiologia plantarum 71, 455–458. sakuta, m., hirano, h., kakegawa, k., suda, j., hirose, m., joy, r. w., sugiyama. m., komamine. a., 1994: regulatory mechanisms of biosynthesis of betacyanin and acta bot. croat. 69 (2), 2010 181 betanin production in transformed sugar beet cells u:\acta botanica\acta-botan 2-10\304 kriznik.vp 11. listopad 2010 11:31:57 color profile: disabled composite 150 lpi at 45 degrees anthocyanin in relation to cell division activity in suspension cultures. plant cell tissue and organ culture 38, 167–169. sánchez-sampedro, m. a., fernández-tárrago, j., corchete, p., 2005: yeast extract and methyl jasmonate-induced silymarin production in cell cultures of silybum marianum (l.) gaertn. journal of biotechnology 119, 60–69. savitha, b. c., thimmaraju, r., bhagyalakshmi, n., ravishankar, g. a., 2006: different biotic and abiotic elicitors influence betalain production in hairy root cultures of beta vulgaris in shake-flask and bioreactor. process biochemistry 41, 50–60. shinde, a. n., malpathak, n., fulzele, d.p., 2009: optimized production of isoflavones in cell cultures of psoralea corylifolia l. using elicitation and precursor feeding. biotechnology and bioprocess engineering 4, 612–618. strack, d., vogt, t., schliemann, w., 2003: recent advances in betalain research. phytochemistry 62, 247–269. tanaka, y., sasaki, n., ohmiya, a., 2008: biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. the plant journal 54, 733–749. tesoriere, l., allegra, m., butera, d., livrea, m. a., 2004: absorption, excretion, and distribution of dietary antioxidant betalains in ldls: potential health effects of betalains in humans. american journal of clinical nutrition 80, 941–945. thimmaraju, r., bhagyalakshmi, n., narayan, m. s., ravishankar, g. a., 2003: kinetics of pigment release from hairy root cultures of beta vulgaris under the influence of ph, sonication, temperature and oxygen stress. process biochemistry 38, 1069–1076. tuteja, n., mahajan, s., 2007: calcium signaling network in plants: an overview. plant signaling and behavior 2, 79–85. uozumi, n., 2004: large-scale production of hairy root. advances in biochemical engineering biotechnology 91, 75–103. yang, z., 2008. cell polarity signaling in arabidopsis. annual review of cell and developmental biology 24, 551–575. zhang, y. h., zhong, j. j., yu, j.t., 1995: effect of osmotic pressure on cell growth and production of ginseng saponin and polysaccharide in suspension cultures of panax notoginseng. biotechnology letters 17, 1347–1350. 182 acta bot. croat. 69 (2), 2010 kri@nik b., pavokovi] d. u:\acta botanica\acta-botan 2-10\304 kriznik.vp 11. listopad 2010 11:31:57 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 69 (2), 163–171, 2010 coden: abcra 25 issn 0365–0588 cryoseston in stara planina (balkan) mountains, bulgaria jaromír lukavský*, vladislav cepák academy of sciences of czech republic, institute of botany, department of plant ecology, centre for bioindication and revitalisation, dukelská 135, cz-379 82 tøeboò, czech republic. numerous snow fields persisted in the stara planina (central balkan) mountains, some with a very light tint, but with dirty surfaces, at the end of may 2009. the cryoseston community was quite different in individual snow fields. we found chlorophyta: zygospores of chloromonas nivalis and chloromonas rostafiñski, chromophyta: chromulina elegans, bacillariophyceae: aulacoseira granulata var. angustissima, aulacoseira italica, cyclotella menenghiniana, navicula nivalis, hantzschia amphioxys, fungi: chionaster nivalis and selenotila nivalis. alpine species such as chlamydomonas nivalis were not found in the snow fields studied, even though they were located in the alpine zone. key words: cryoseston, stara planina (balkan), bulgaria introduction a luminary of cryoseston research, kol (1968) described 78 taxa classified as cryoseston, 63 of which were chlorophyta, 6 cyanobacteria, 1 xanthophyceae, 1 dinophyceae and 7 fungi. many of the described taxa were synonymous, representing different stages of a single life cycle, or were observed incompletely, and they have been progressively eliminated through cultivation and molecular biology (hoham 1973, 1979; hoham and mullet 1977; hoham and blin 1979; komárek and nedbalová 2007). the last authors listed 47 chlorophyta in the cryoseston. in bulgaria, cryoseston was recorded for the first time by kol (1956) in the rila and the pirin mts. these reports were confirmed by wodenitscharoff (1962) in the rila mts., and a new locality was found by lukavský et al. (2009) in the vitosha mountains. although the stara planina (balkan) mountains have the same geographic attributes that enable the occurrence of cryoseston in other mountains in bulgaria, snow algae have not previously been recorded there. acta bot. croat. 69 (2), 2010 163 * corresponding author, e-mail: lukavsky@botany.cas.cz in memory of stefan petkov 1866–1951, luminary of bulgarian phycology stefan petkov, bulgarian botanist, born on 5th of june 1866 in lovech, ottoman empire; died on 8th of december 1951 in sofia, bulgaria. he was a lecturer in sofia university »st. kliment ohridski«, the author of the first bibliography of the bulgarian flora. from 1907, he was a member of bulgarian literary society, today the bulgarian academy of sciences. u:\acta botanica\acta-botan 2-10\300 lukavsky.vp 11. listopad 2010 11:28:35 color profile: disabled composite 150 lpi at 45 degrees recently, extreme habitats (e.g. snow, polar soils, thermal springs) have received increasing attention as a potential source of algae with unusual physiological characteristics and biochemical pathways and products (øezanka et al. 2007, 2008). the isolation of new algal strains with such characteristics is the goal of this project. material and methods localities snow fields were sampled in the central stara planina (balkan) mts., between the mazalat chalet to the dermenka chalet, during the last week of may 2009. the snow fields were tens of square meters in size. gps positions and altitudes of the sites are given in table 1. sampling and sample processing snow samples were collected in plastic bags of volume ca 50 ml and transported in thermos bottles to the laboratory of the institute of plant physiology in sofia. samples were melted and centrifuged 20 min. at 3000 g, and living material was examined using a nu2 microscope (c. zeiss, jena, d) with 63/0.8 and hi 100/1.35 objectives, and photographed with a dp10 digital camera (olympus, japan). the samples were also inoculated into z nutrient solution, according to zehnder in staub (1961), to be isolated into unialgal culture. all data in this paper were derived only from samples collected in the field. results and discussion in snow samples from the stara planina mts., we found and determined the following organisms, (figs. 1–55): chlorophyta, chlorophyceae chloromonas nivalis (chodat) hoham and mullet 1978 (syn. scotiella nivalis, chloromonas cryophila), figs. 1–4, 10, 11. cysts ellipsoid, 26–31 ´ 12–15 mm, a smooth, thick cell wall, with 6 longitudinal, twisted ribs on surface, joined into prominent thickenings at each pole (fig. 2). cell volume 164 acta bot. croat. 69 (2), 2010 lukavský, j., cepák, v. tab. 1. localities studied in central stara planina (balkan) mountains at end of may 2009. no location gps altitude, m a.s.l. 1 near chalet taza n 42° 44' 19.6" e 025° 04' 16.3" 1994 2 main chain n 42° 44' 02.8" e 025° 03' 77.4" 2037 3 main chain n 42° 44' 04.6" e 025° 03' 17.5" 1993 4 main chain n 42° 44' 06.4" e 024° 59' 02.1" 1882 5 main chain n 42° 43' 64.3" e 024° 56' 35.1" 2133 6 under peak botev n 42° 43' 02.0" e 024° 56' 30.4" 2201 7 peak botev n 42° 43' 02.8" e 024° 55' 05.3" 2376 8 botev shelter n 42° 43' 21.0" e 024° 54' 32.8" 2057 9 near peak zhaltets n 42° 43' 45.4" e 024° 53' 02.3" 2108 u:\acta botanica\acta-botan 2-10\300 lukavsky.vp 11. listopad 2010 11:28:35 color profile: disabled composite 150 lpi at 45 degrees filled with amorphous green chloroplast, grains, and yellow-red oil drops; green cells were seen rarely. dominated in fields 5 and 8. flagellates not observed. the characteristic big oil drops at each cell pole (lukavsky et al. 2009) were lacking but the size range and external ornamentations of the cells confirm our identification. resting stages of several snow algae in the genus chloromonas were formerly described as vegetative cells, and given the genus name scotiella fritsch. the life history of chloromonas nivalis was described in detail by hoham and mullet (1977, 1978) and kawecka (1981). a characteristic feature of the cysts of some biflagellate volvocalean algae, which allows identification to species level, is the number and shape of ribs, or shape and length of spines. komáromy (1982) with respect to cyst morphology distinguished five groups, where chloromonas nivalis stands apart. this alga dominated in spring as well as in summer snow collected in the vitosha mts. (lukavský et al. 2009). it is recognised as a characteristic organism of middle altitude, open localities (hoham and mullet 1977, komárek and nedbalová 2007). chloromonas rostafiñski (starmach et kawecka) gerloff et ettl (nom. dubium chlamydomonas flavo-virens, syn. chlamydomonas rostafiñski), figs. 5–9, 35, 36, 41–55 cysts ellipsoidal, 15 ´ 25 mm, cell wall thick, uniformly covered with blunt protrusions or spines (figs. 52, 53), cell content green – orange (fig. 43), chloroplast without pyrenoid. flagellates egg-shaped 12 ´ 8 mm, papilla lacking, chloroplast cup-shaped without pyrenoid (figs. 5, 36), flagella not observed. dominant in fields 5 and 6. chloromonas rostafiñski was discovered by rostafiñski and published as chlamydomonas flavo-virens, but without the diagnosis and description required by the botanical code. a valid description of this organism (but from a different locality) was provided by starmach and kawecka (1965) as chlamydomonas rostafiñski, from yellowish-green snow in the alpine zone of the polish tatra mts. it has been recorded in the high tatra mts. (kawecka et al. 1979, kawecka 1984, lukavský 1994) and vitosha mts. (lukavský et al. 2009). the cell wall of resting cells is covered by numerous longitudinal ribs which show indentations of different length, and hence the papillae-like structures visible by light microscopy are formed (figs. 45, 49, 52, 53, 55). these non-motile cells are believed to be formed from vegetative cells through gradual development of cell wall ornamentation (kawecka and eloranta 1986). sexual reproduction was described by kawecka (1984) as isogamy. a small joined cell, and probably the sporangium of a parasitic chytrid attached to a big spore can be seen in figure 51. more research on this species in the stara planina mts. is clearly necessary. chromophyta, chromophyceae chromulina elegans doflein cells spherical (rarely with pointed posterior), diameter 6–8 mm, 1 flagellum 6–8 mm in length. one cup-shaped chromatophore, yellow-orange, without pyrenoid or stigma, a few small drops or grains in protoplasm. present in field 9. this alga was described by doflein from a peat-bog in germany. novis (2002) tentatively identified it in cryoseston collected on mt. philistine, arthur´s pass national park, acta bot. croat. 69 (2), 2010 165 cryoseston in stara planina (balkan) mts., bulgaria u:\acta botanica\acta-botan 2-10\300 lukavsky.vp 11. listopad 2010 11:28:35 color profile: disabled composite 150 lpi at 45 degrees new zealand, at 1600–1700 m. a.s.l. lukavský et al. (2009) reported the species from snowfields in the vitosha mountains. it is similar, also to chromulina ettlii hindák, which was described from brownish snow in the high tatra mts (hindák 1969). the last had cells spherical, 7–9 mm in diameter, flagellum of 12–15 mm long, chromatophore one, cup-shaped, yellow-brown, lacking pyrenoid, contaractile vacuoles 1–2. another similar species, chromulina chionophila stein was reported from ny usa, in coniferous canopy as yellow-green snow (hoham 1975), but this species has 2–3 chromatophores per cell and it is flattened in cross section. some species ascribed to the genus chromulina, originally defined as uniflagellate, have 166 acta bot. croat. 69 (2), 2010 lukavský, j., cepák, v. figs. 1–27. cryoseston of snow fields in the stara planina mountains, collected in may 2009. 1–4, 10, 11 – zygospores of chloromonas nivalis; 5 – zoospore, 6–9 – cysts of chloromonas rostafiñski; 12–21 – chionaster nivalis, variability of spores, 14, 15 – cf. c. bicornis; 22–27 – selenotila nivalis, variability of thalli. origins of samples: snow field no. 1. (12, 22–24, 27); no. 2. (13, 21); no. 4. (1–4); no. 5. (25, 26); no. 6. (7–11); no.7. (5, 6). u:\acta botanica\acta-botan 2-10\300 lukavsky.vp 11. listopad 2010 11:28:42 color profile: disabled composite 150 lpi at 45 degrees since been shown to possess a second smaller flagellum not visible by lm (novis 2002). ultrastructural studies would be required to determine this material. bacillariophyceae the centric diatoms cyclotella menenghiniana (fig. 28), aulacoseira italica and a. granulata var. angustissima (figs. 33, 34) were found in fields 1 and 8 (tab. 2), but only as empty frustules. similarly, the pennate diatom cf. navicula nivalis (fig. 29, cf. also achnanthes coarctata) was also only observed as empty frustules. the only living diatom species was hantzschia amphioxys (figs. 30–32) from snow fields 2 and 3. acta bot. croat. 69 (2), 2010 167 cryoseston in stara planina (balkan) mts., bulgaria figs. 28–55. cryoseston of snow fields in the stara planina mountains, collected in may 2009. 28 – cyclotella meneghiniana; 29 – cf. navicula nivalis; 30–32 – hantzchia amphioxys; 33 – aulacoseira granulata var. angustissima; 34 – aulacoseira italica; 35, 36 – zoospores of chloromonas rostafiñski; 37–40 – zoospores »small«; 41–44, 46–48, 50, 51, 54, 55 – cysts of chloromonas rostafiñski; 49, 52, 53 – empty cell walls, 51 – sporangium of a chytridiomycet?. scale bar = 10 mm. origins of samples: snow field no. 1. 49–55; no. 2. 29, 30; no. 3. 31, 32, 37–40; no. 5. 41–48; no. 6. 33, 34; no. 8. 28, 35, 36. u:\acta botanica\acta-botan 2-10\300 lukavsky.vp 11. listopad 2010 11:28:46 color profile: disabled composite 150 lpi at 45 degrees navicula nivalis ehr. 1854 (syn. navicula mutica var. nivalis (ehr.) hustedt 1911 is a cosmopolitan of high mountains from aerial biotopes such as moss mats, wet rocks (krammer and lange-bertalot 1986). the other species, from our samples can be ubiquists transported by wind. samples were not sufficient for the creation of permanent preparations, and further studies and determinations are necessary to distinguish the genera navicula and achnanthes (the last has raphae only on one valve). surprisingly no bacillariophyceae were listed by kol (1968), but later 33 taxa were determined in antarctic cryoseston (zidarova 2008). fungi chionaster nivalis (bohl.) wille, figs. 12–21. cells colourless, 2–3 (rarely more) projections up to 25 mm long arise from a triangular centre (diameter 12–14 mm; e.g. fig. 17) filled with numerous small spheres. rarely, there were only 2 projections (figs. 14–15); this form has been described as chionaster bicornis kol. the variability in morphology and the number of projections is very wide in our material; the specimen closest to typical chionaster (sensu kol) is fig. 17. reproduction was not observed. dominant in field 2. notes: this species was already recorded in bulgaria, in the pirin mts. by kol (1956), the vitosha mts. by lukavský et al. (2009), and the rila and pirin by lukavský (unpublished). according to the list of genera of fungi (kirk et al. 2001), chionaster wille (1903) 168 acta bot. croat. 69 (2), 2010 lukavský, j., cepák, v. tab. 2. cryoseston of central stara planina (balkan) collected at end of may 2009. species distributions in individual snow fields. gps positions and altitudes see tab. 1. d = dominate, x = present, r = rare, zsp = zoospores. species no of locality 1 2 3 4 5 6 8 9 chromophyceae chromulina elegans x bacillariophyceae, centrales x cyclotella meneghiniana x aulacoseira granulata var. angustissima x aulacoseira italica x bacillariophyceae, pennales hantzschia amphioxys x x cf. navicula nivalis x chlorophyceae chloromonas nivalis, x x x d chloromonas rostafiñski d d x zspchl. rostafinski x d zsp »small«, diameter 8–10 mm x x x x fungi chionaster nivalis x d x x selenotila nivalis x r x x x x x u:\acta botanica\acta-botan 2-10\300 lukavsky.vp 11. listopad 2010 11:28:46 color profile: disabled composite 150 lpi at 45 degrees is a »nomen dubium«, and its reproduction was never observed. it is probably a spore of some yet unidentified fungus from hyphomycetes. selenotila nivalis lagerh. (syn. selenozyma), figs. 22–27. cells cylindrical-clavate, 30 ´ 3–5 mm, arranged into irregular clusters, colourless, filled by small globes, reproducing through budding. dominant in fields 7 and 9, present in all sampled snow fields. there is a wide variability in morphology of thalli. notes: selenotila nivalis is an invalid taxon according to kirk et al. (2001). sometimes it is included in the genus selenozyma (arx et al. 1977). similarly to chionaster nivalis, this species often occurs with snow algal populations. species richness and composition we have determined 13 taxa together, including fungi. the species composition and general distribution pattern of snow algae in the stara planina mts. was similar to that in the bohemian forest (lukavský 1993). only low–moderate altitudes species (chloromonas nivalis and c. rostafiñski) were found, despite the large areas of snow habitats with open exposure. in contrast, the cryosestons of the rila and pirin mts. were dominated by giant cells of cf. chlamydomonas nivalis, accompanied by only a few other species, e.g., koliella viretii (chod.) hind. (kol 1956, wodenitscharoff 1962, lukavský unpublished data). the vitosha and the giant mts, however, contained a combination of ecotypes: chlamydomonas nivalis was present but not dominant (kociánová et al. 1989, lukavský et al. 2009). in the stara planina we found a single »giant spore« resembling chlamydomonas nivalis (fig. 7), but it was brown rather than the typical brick-red and we believe that this is a cell of chloromonas rostafiñski. surprisingly, some common genera such as koliella, raphidonema, stichococcus (komárek et al. 1973, hoham 1973, hoham and duval 2001, novis 2002, lukavský et al. 2009) were absent from our samples. all our snow samples were heavily contaminated by dust and pollen grains, so nutrients should be present in excess. perhaps the reason for the absence of the species is that stara planina mts. are high and long, but narrow and chain-like mountains, not huge and compact complexes as the rila, the pirin or the vitosha mts. the former are accordingly more influenced by their surrounding, and the snow fields melted too early. bacillariophyceae were present in a few fields (tab. 2.) but usually as dead valves. living cells of hantzschia amphioxys (figs. 31–32) were found only in fields 3 and 4. living diatoms are known as components of the cryoseston, for example on livingstone island, antarctica, where zidarova (2008) determined 33 taxa of bacillariophyceae, 2 ochrophyceae (chromophyta) and 4 taxa of chlorophyta. we can conclude that the stara planina mts. provide suitable localities for cryoseston, particularly volvocales, and fungi, but so far not in such density as to prominently colour the snow. acknowledgements this work was supported by a grants of the ministry of education, youth and sports of the czech republic (msmt 1m0571), and of av0z60050516 of academy of sciences. we thank p. novis for valuable comments on the manuscript, staff members of the institute of plant physiology and genetics of bas sofia, g. gaceva and v. titlová for technical assistance. acta bot. croat. 69 (2), 2010 169 cryoseston in stara planina (balkan) mts., bulgaria u:\acta botanica\acta-botan 2-10\300 lukavsky.vp 11. listopad 2010 11:28:46 color profile: disabled composite 150 lpi at 45 degrees references arx, j. a., miranda, r., smith, m. t., yarrow, d., 1977: the genera of yeasts and the yeast-like fungi. studies in mycology 14, 12–78. hindák, f., 1969: brownish snow in the high tatras. biologia (bratislava) 24, 80–85. hoham, r. w., 1973: pleiomorphism in the snow alga, raphidonema nivale lagerh. 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vegetation in urban gardens mahmoud omar hassan, howida yacoup mohamed*, ayman hassan aboellil beni-suef university, faculty of science, department of botany and microbiology, 62511 beni-suef, egypt abstract – pruning ficus trees in urban green spaces may lead to the accumulation and spread of their leaf litter on the understory vegetation. this study was conducted to evaluate the allelopathic effect of ficus retusa l. leaf litter on the understory species in urban gardens. a field study showed that the plant cover and species richness of litter-affected plots were lower than those of litter-free areas. the litter-affected soils had substantially lower ph and higher electrical conductivity. in a greenhouse experiment, litter-affected soil significantly inhibited the emergence and growth of understory species selected for the purpose of this study: melilotus indicus (l.) all., trifolium resupinatum l. and amaranthus viridis l. osmotic potentials equivalent to those of the litter-affected soils did not affect emergence or growth of these species. a spectrophotometric analysis indicated that the litter-affected soils contained larger amounts of phenolics and flavonoids. an hplc analysis revealed that the litter-affected soils contained higher concentrations of free phenolic and flavonoid allelochemicals. these results demonstrate that f. retusa leaf litter may reduce plant cover and species richness. the significant inhibition in both field and greenhouse experiments could be attributed to phenolic and flavonoid allelochemicals released from the tree litter, as the osmotic potential of the litter had no effect on the understory species. the allelopathic potential of f. retusa leaf litter plays at least a partial role in reducing urban vegetation. keywords: allelopathy, ficus retusa, leaf litter, understory species, urban gardens introduction allelopathy is a process that occurs when a donor plant releases chemical compounds into the environment that exert an adverse or positive effect on associated species (rice 1984). these compounds can leach from leaf litter, be exuded from roots, or arise from the decomposition of plant residues. their release is modulated by environmental factors such as temperature, soil moisture, microorganisms and nutrients (el-khawas and shehata 2005). depending on their concentrations, these substances can hinder the germination and growth of plant species, and their effect varies according to species (quddus et al. 2014). leaf litter can be found in abundance under the canopies of some trees. in urban ecosystems, leaf litter may arise naturally from deciduous trees (hassan 2018) or be generated when evergreen trees undergo pruning. several toxic compounds may be released from this leaf litter into the surrounding environment, adversely affecting the cover, diversity, species richness and species composition of the understory species (chou 1999, hassan 2018). for example, toxic compounds released from the leaf litter of eucalyptus globulus labill. and acacia melanoxylon r.br. have been shown to suppress some understory species (souto et al. 1994). under precipitation conditions, acacia dealbata link canopy released phytotoxic compounds that inhibited the net photosynthetic rate and consequently affected the distribution of understory species (lorenzo et al. 2011). in this article, we will highlight such phenomena. soil is a complex medium that influences the availability of phytotoxic compounds released from plant residues, thereby affecting plant growth (el-khatib et al. 2004). when these compounds are released into the soil, retention, transformation and transport processes may occur. these processes can be influenced by soil properties, chemical compound nature, and the physical environment (cheng 1992, kobayashi 2004). substantial amounts of toxic substances that leach from litter or are released by its decay accumulate on the soil surface and interfere with plant growth (facelli and pickett 1991). in general, allelochemicals released from * corresponding author e-mail: howidayacoup71@yahoo.com hassan m. o., mohamed h. y., hassan aboellil a. 132 acta bot. croat. 80 (2), 2021 plant residues can influence the germination and growth of tested species when they are present in sufficient concentrations in the associated soil (inderjit et al. 1996, el-khatib 2000, el-khatib et al. 2004). therefore to assess the allelopathic effect of a particular plant, it is necessary to study its associated soil. several studies have been undertaken to assess the potential release of allelochemicals and their effects on the understory vegetation (molina et al. 1991, espinosagarcia et al. 2008). however, knowledge of the allelopathic potential of the leaf litter of some trees is still lacking. ficus is a genus of about 750 woody species belonging to the moraceae family (semwal et al. 2013). ficus species are highly distributed all over the world, widely used in medicinal purposes and are native to india, southwest china and nepal (rawat et al. 2012). ficus retusa is an evergreen tree that can grow to a height of 15 m with a wide-spread canopy ( semwal et al. 2013, khan 2017). it was introduced to egypt for ornamental purposes. trees dominate the urban ecosystem of the new city of beni-suef governorate, and most of them are f. retusa trees. they are widely distributed in gardens, streets and along roadsides. previous studies revealed the presence of a variety of phenolic compounds and flavonoids in the stem and leaves of f. retusa (takahashi et al. 2002, khan et al. 2011, rawat et al. 2012, aly et al. 2013, singhal et al. 2017). additionally, several compounds such as luteolin, ß-sitosterol acetate, ß-amyrin, friedelenol and new polyphenolic compounds called retusaphenol and (+)-retusa afzelechin were first isolated from the aerial parts of f. retusa (sarg et al. 2011). many of these compounds were shown to be allelochemicals (rice 1984). nevertheless, the allelopathic activity of f. retusa leaves has not been investigated. the goal of this study was to fill this gap in scientific knowledge. the annual pruning of ornamental trees, such as f. retusa, and the residents’ lack of interest in removing the falling leaves cause the accumulation of substantial amounts of leaf litter underneath their canopies. there is a lower cover of some species under these canopies than in adjacent regions. in the light of litter effects, two main hypotheses were tested. the first hypothesis was that potential allelochemicals can be released from ficus litter, accumulate in the soil, and affect the cover, richness and diversity of the understory vegetation. to test this hypothesis, a field study was conducted to measure the cover, richness and diversity indices of species under f. retusa canopies and compared them with those in neighboring areas away from the canopies. additional testing in a greenhouse assessed the effects of litteraffected soils on the germination and growth of selected cooccurring species detected in the field study by comparing them with the effects of soils unaffected by litter. to confirm the allelopathic effect of field soil, hplc analysis was used to investigate the putative allelochemicals (phenolics and flavonoids) present in litter-affected and unaffected soils. this analysis reflected the presence of allelochemicals released from leaf litter into the soil under natural conditions. the second hypothesis stated that the litter altered the chemical properties of the soil in a way that led to adverse effects on the cover and diversity of understory plants. in other words, the litter may interfere with soil ph, electrical conductivity (ec), organic matter (om) and nutrient availability. to test this hypothesis, soil samples collected from under and outside the tree canopy were analyzed to study the potential changes in these properties. since changes in the ec of field soil may exert an osmotic effect on the understory vegetation, a polyethylene glycol (peg) 6000-based bioassay was used to assess this possibility. the primary goal of the present study was to assess the allelopathic effects of f. retusa leaf litter on the cover, composition and floristic diversity of the understory vegetation. material and methods study area this study was conducted in the second district of the city new beni-suef (29°01.50′ to 29°02.50′ n, 31°06′ to 31°07.30′ e). this region is considered one of the common new urban areas in egypt. it is found about 124 km south of cairo and located east of the nile valley at an elevation of 32 to 42 m above sea level. it has been completely built since 1990 and has an area of about 1.42 km2. the climate of this city is characterized by mild winters with low precipitation and hot, dry summers (hassan and hassan 2019). the average annual precipitation, which falls from november through april, is about 11.98 mm. the texture of the soil in this region is sandy, sandy loam or sandy clay loam. other properties of the soil in the gardens of this area, such as ph, ec, organic matter and some available nutrients, were measured by hassan (2018). many tree species have been introduced into this area for ornamental purposes. f. retusa trees are among the most common trees in this territory. field study to assess the effect of f. retusa tree litter on the understory vegetation, a total of 62 plots, each of 3 × 0.1 m2, were set randomly under and just outside the tree canopies. thirty-one plots located under tree canopies were designated as treatment areas and the remaining plots outside those canopies (about 2 m away) were set as controls. these plots were selected in different seasons (mid-winter and mid-summer of 2018) to include the total species richness present in this area through the entire year. in each plot, the vegetative parameters measured in this study were the cover of each species, total plant cover of all species, species richness, bare length (measured by the area not occupied by plant cover), relative cover of species [(cover of species i/cover of all species) × 100] and diversity indices (simpson’s index (d), shannon-weaver index (h’) and evenness index (e)) as shown by hassan (2018). the species detected were identified using boulos (2000, 2002, 2005): ′ − ×h = pi ln pi i=1 s s( ) e = pi ln pi s i=1 s − ×  s( ) / ln d =1/ pi i=1 s 2s where pi is the relative cover of species i. allelopathic potential of ficus retusa l. leaf litter acta bot. croat. 80 (2), 2021 133 soil samples taken from 0-20 cm depth of each plot were air-dried, passed through a 2 mm sieve and stored in plastic bags prior to analysis. the measured parameters in soil samples were ph, ec, available nutrients (n, p, k, cu and zn) and organic carbon. soil ph was measured in a soil-water extract (1:2.5, w/v) using a ph meter (ad 3000), while soil ec was measured in a soil-water extract (1:5, w/v) using a conductivity meter (jenway 3305). available nitrogen was determined as described by allen (1989). briefly, 5 g of soil was added to 50 ml of 2n kcl, shaken for 30 min and then filtered. ammonium nitrogen and nitrate nitrogen in the filtrate were measured with a technician auto analyzer. total available nitrogen is expressed as the sum of ammonium and nitrate nitrogen. available p, k, cu and zn were determined as described by soltanpour (1991). briefly, 20 g of soil sample was added to 40 ml of a solution containing diethylenetriaminepentaacetic acid (dtpa, 97%) and ammonium bicarbonate at ph 7.6 and then mixed well. after 15 minutes of shaking, the extract was filtered and the filtrate used for further analyses. p, cu and zn were measured using inductively coupled plasma (icp) spectrometry (ultima 2 jy plasma), while k was measured using a flame photometer. soil organic carbon was determined using the method described by walkley and black (1934). allelopathic potential of litter-affected soils under ficus canopy to assess the allelopathic effect of f. retusa leaf litter, its residual toxicity in field soil collected under tree canopies was determined using selected understory species, melilotus indicus (l.) all., trifolium resupinatum l. and amaranthus viridis l. these species showed appreciably suppressed cover under canopies in field conditions. litter-affected soil samples were collected under f. retusa canopies and mixed well to form a composite treatment soil, while litter-free soil samples collected outside those canopies were used as a control. these samples were used for germination and growth of the three tested species. specifically, the soil samples were shade-dried, passed through a 2-mm sieve, and then put in plastic pots (11 cm diameter × 11 cm deep) that received about 0.5 kg soil each. in each pot, ten seeds of each tested species were sown at a depth of 0.2 cm. regular irrigation was carried out by spraying when needed. this experiment was kept with four replicates in a protected area for 30 days under prevailing environmental conditions (12 h light and 12 h dark photoperiod, 24 to 34 °c daytime temperature, 14 to 22 °c nighttime temperature, and 27 to 28% relative humidity). the germination rate (in %) was calculated after the emergence of the tested species ceased. three growth criteria were measured at harvest: shoot length, root length and biomass. individuals from each target species were dried in an oven at 70 °c for 72 h then weighed to determine the dry mass. to assess the osmotic potential of the litter-affected soil and separate its osmotic effect from its allelopathic effect, another experiment was conducted using peg 6000. in this experiment, solutions with osmotic potentials equivalent to those of the field soils (litter-affected and litter-free) were prepared. from soil analysis, ec values of the field soil were converted to osmotic potentials (osm·kg–1 h2o) by using the following equation (gomaa et al. 2014): mpa = osm · kg–1 h2o / 0.407 the osmotic potentials of litter-affected and unaffected soil were -0.0097 and -0.0039 osm·kg–1 h2o, respectively. to obtain a peg solution with osmotic potential equal to that of litter-affected soil, 32.3 g of peg 6000 was dissolved in 1 liter of h2o at room temperature (28 °c) (michel and kaufmann 1973). by diluting the previous peg solution, we prepared a solution of osmotic potential value equivalent to that of the unaffected soil. to show the effect of the osmotic potential of field soil on the target species, a germination test was performed using the prepared peg solutions. due to the hard seed coat and dormancy characteristics of the legume and amaranthus species, scarification treatments were done (ates 2011, assad et al. 2017). seeds of the legume species were soaked in concentrated h2so4 (98%, v/v) for 10 min while amaranthus seeds were soaked for 2 min. after soaking, the seeds were washed with distilled h2o, sterilized with 5% (w/v) sodium hypochlorite for 2 min, and rinsed three times with distilled h2o. twenty-five seeds of each tested species were put on filter paper in sterile petri dishes (9 cm diameter) and then supplied with 5 ml of the prepared peg solutions. all the experimental samples were kept in a dark chamber at 23 ± 2 °c in a completely randomized design with four replicates. after seven days, germination (in %) was assessed by counting the number of germinated seeds. root lengths, shoot lengths and seedling biomasses were determined seven days after seeding by measuring similar seedlings in each dish. root and shoot lengths were estimated as described by hussain et al. (2011). determination of phenols and flavonoids in litter-affected and unaffected soils polyphenols were extracted from the field soil samples with aqueous methanol (80%) in a 250 ml erlenmeyer flask using an ultrasound-assisted method (kim and lee 2002). briefly, aqueous methanol (80%, 100 ml) was added to soil (10 g) and the mixture was subjected to continuous sonication for 60 min. after filtration, the filtrate was evaporated in a vacuum evaporator at 40 °c. the residue was dissolved in 50 ml methanol then diluted to a final volume of 100 ml with distilled h2o. the resulting solution was centrifuged for 15 min at 12,000 rpm and stored at −20 °c until analysis. the total phenolic content was determined according to kim et al. (2003) protocol. one ml of the stock extract, 9 ml of distilled h2o and then 1 ml of folin-ciocalteu phenol reagent were added to a 25-ml volumetric flask and mixed well. after 5 min, 10 ml of 7% na2co3 solution were added to the mixture with shaking. finally, distilled h2o hassan m. o., mohamed h. y., hassan aboellil a. 134 acta bot. croat. 80 (2), 2021 was added to reach 25 ml and then the solution was left standing for 90 min. at this point, the absorbance of the solution at 750 nm was measured versus a prepared blank. gallic acid was used to prepare a calibration curve. total phenolic content was expressed as mg gallic acid equivalent (gae) per gram of soil sample (mg·g–1 soil sample). the total flavonoid content was performed using the method described by chun et al. (2003). one ml of the stock extract and 4 ml of distilled h2o were added to a 10ml volumetric flask. after 5 min, 300 μl of 5% nano2 followed by 300 μl 10% alcl3 were added to the mixture. this mixture was allowed to stand for 6 min, and then 2 ml of 1 m naoh was added and the final volume was adjusted to 10 ml using distilled h2o. the absorbance at 510 nm was measured versus a prepared blank. rutin was used to prepare a calibration curve. total flavonoids in the soil sample were expressed as mg of rutin equivalents (re) g–1 soil (mg·g–1 soil sample). hplc analysis phenolics and flavonoids were analyzed according to hassan (2018). this analysis was carried out using a shimadzu hplc system equipped with an lc 1110 pump, a kromasil c8 column (4.6 mm × 250 mm; particle size, 4.6 μm; pore size, 100 å) and a diode-array uv detector and run using wincrome chromatography software (version 1.3). phenolic compounds were analyzed using a solvent gradient consisting of acetonitrile: 0.05% h3po4 (99:1; solvent a) and water: h3po4 (99:1; solvent b) and a flow rate of 1 ml·min–1. the elution program consisted of 90% a from 0 to 30 min, a linear decline to 50% a from 30.01–40 min, and a further decline to 0% a from 40.01 to 55 min. flavonoid compounds were analyzed using a solvent gradient composed of methanol:h3po4 (99:1; solvent a) and water:h3po4 (99:1; solvent b) and a flow rate of 1 ml·min–1. flavonoid compounds were detected using uv (250–400 nm) absorbance. phenolic and flavonoid compounds were identified by comparison of their retention time with those of phenolic standards including trans-cinnamic, vanillic, p-coumaric acids, catechol, sinapic, protocatechuic, syringic, caffeic, p-hydroxybenzoic acids, vanillin and resorcinol; and flavonoid standards including hesperidin, luteolin, rutin, apigenin, catechin, kaempferol and quercetin. the concentrations, expressed as mg g–1, were estimated according to knowledge of the heights and areas under peaks of detected compounds in soil samples. values reported are the average of three replicates. statistical analysis kolmogorov–smirnov and levene’s tests were used to check the normality and homogeneity, respectively, of the data obtained from field, greenhouse and laboratory experiments. when the data were normally distributed, they were analyzed using the independent samples t test. data showing abnormality and heterogeneity were analyzed using the nonparametric mann-whitney u test. all data in this study were analyzed with the use of the spss statistics software package, version 20.0 (ibm corporation, usa) at probability levels *p < 0.05 and **p < 0.01. results field study a total of 12 species belonging to 11 genera and five families were detected throughout the study area (tab. 1). poaceae had the highest number of species (five) followed by fabaceae (three), plantaginaceae (two), amaranthaceae and euphorbiaceae (in each one). nine species were detected as annuals and three species were detected as perennials (tab. 1). sites heavily covered with f. retusa leaf litter attained lower cover of some understory species. among the annuals, the covers of amaranthus viridis l., medicago polymorpha l., melilotus indicus (l.) all. and trifolium resupinatum l. tab. 1. plant cover of each plant species detected in plots with and without ficus retusa leaf litter. values are given as means ± standard error from 31 replicates. an asterisk means there is a significant difference between litter-unaffected plots and litter-affected plots by independent samples t test (*p < 0.05, **p < 0.01). species/family life span litter-unaffected plots litter-affected plots amaranthus viridis l. (amaranthaceae) annual 1.40 ± 0.32 0.13 ± 0.13** cenchrus echinatus l. (poaceae) annual 1.27 ± 0.62 0.37 ± 0.15 cynodon dactylon (l.) pers. (poaceae) perennial 1.35 ± 0.17 0.86 ± 0.09* digitaria sanguinalis (l.) scop. (poaceae) annual 1.00 ± 0.17 0.70 ± 0.20 eragrostis pilosa (l.) p.beauv. (poaceae) annual 0.84 ± 0.34 0.30 ± 0.04 euphorbia peplus l. (euphorbiaceae) annual 0.16 ± 0.11 0.42 ± 0.20 medicago polymorpha l. (fabaceae) annual 0.55 ± 0.13 0.10 ± 0.05* melilotus indicus (l.) all. (fabaceae) annual 1.20 ± 0.22 0.07 ± 0.04** paspalum dilatatum poir. (poaceae) perennial 0.67 ± 0.31 0.27 ± 0.13 plantago amplexicaulis cav. (plantaginaceae) annual 0.15 ± 0.03 0.00 ± 0.00** plantago major l. (plantaginaceae) perennial 0.74 ± 0.22 0.16 ± 0.08 trifolium resupinatum l. (fabaceae) annual 1.66 ± 0.29 0.05 ± 0.05** allelopathic potential of ficus retusa l. leaf litter acta bot. croat. 80 (2), 2021 135 under tree canopies were significantly lower than those outside the canopies. in addition, plantago amplexicaulis cav. was completely absent from the infested sites (tab. 1). for perennials, only the cover of cynodon dactylon (l.) pers. was significantly reduced at the litter-affected sites, whereas the remaining perennial species were not affected. significant reductions in vegetation cover, and species richness were also recorded in the plots affected by litter, compared with those free from litter (tab. 2). also, there was a significant increase in bare length in litter affected plots. however diversity, as measured using shannon-weaver, evenness and simpson’s indices, was not influenced (tab. 2). most of the soil analysis criteria were unaffected by the presence of litter. however, the litter-affected soils exhibited lower ph and higher ec values (tab. 3). greenhouse experiment generally, litter-affected soil collected from under f. retusa canopies drastically reduced germination and some of the growth variables of studied species. litter-affected soils significantly decreased seed germination of the selected target species (p < 0.01) (fig. 1a). litter-affected soil significantly decreased the root lengths of a. viridis and t. resupinatum (fig. 1b), while the shoot length was significantly reduced tab. 2. vegetation cover, bare length, species richness, and diversity indices (mean ± standard error; n = 31) in the stands with and without ficus retusa leaf litter. an asterisk means there is a significant difference between plots without litter and plots with litter by mann-whitney u test (*p < 0.05, **p < 0.01). parameters plots without litter plots with litter vegetation cover (%) 83.33 ± 4.63 36.58 ± 2.81** bare length (%) 16.67 ± 4.63 63.42 ± 2.81** species richness (s) 2.68 ± 0.23 1.9 ± 0.19* shannon-weaver index (h’) 0.60 ± 0.077 0.41 ± 0.075 evenness index (e) 0.55 ± 0.06 0.45 ± 0.07 simpson’s index (d) 1.81 ± 0.14 1.51 ± 0.12 tab. 3. soil properties in stands with and without ficus retusa litter. values are given as means ± standard error from 31 replicates. an asterisk means there is a significant difference in the parameters between stands without litter and stands with litter by independent samples t test (*p < 0.05, **p < 0.01). soil parameter stand without litter stand with litter ph 7.9 ± 0.03 7.63 ± 0.06** electrical conductivity (µs cm–1) 263.75 ± 36.85 663.8 ± 146.55* organic carbon (%) 2.18 ± 0.58 2.21 ± 0.4 available nutrient concentrations n (mg kg–1) 73.75 ± 13.24 85.50 ± 3.52 p (mg kg–1) 6.29 ± 1.82 9.8 ± 2.52 k (mg kg–1) 102.82 ± 31.08 182.64 ± 31.74 cu (mg kg–1) 0.91 ± 0.16 1.85 ± 0.54 zn (mg kg–1) 3.59 ± 0.60 5.35 ± 0.78 fig. 1. effect of the residual toxicity of ficus retusa leaf litter in soil on selected understory species melilotus indicus, trifolium resupinatum and amaranthus viridis (n = 4). a – germination percent (%), b – root length (cm), c – shoot length (cm), d – biomass (g). test refers to litter-affected soil and control refers to unaffected soil. the bars on each column represent the standard error. significant difference were at *p < 0.05 and **p < 0.01 (independent samples t test). hassan m. o., mohamed h. y., hassan aboellil a. 136 acta bot. croat. 80 (2), 2021 for the later (fig. 1c). moreover, the biomass of both species was significantly suppressed (fig. 1d). the peg solution with an osmotic potential equivalent to that of the litter-affected soil did not significantly affect the germination (fig. 2a) or growth parameters (fig. 2b-d) of the tested species, compared with the control. biochemical analysis the concentrations of the phenolic and flavonoid compounds detected in litter-affected and unaffected soils are summarized in tab. 4. among the free compounds, quercetin and resorcinol were completely absent in control soils (tab. 4). litter-affected soils also contained significantly more quercetin, resorcinol, caffeic acid, coumaric acid and ellagic acid than unaffected soils. furthermore, the total phenolics and f lavonoids were significantly higher (by 65.84% and 47.54%, respectively) in litter-affected than in control soils (tab. 4). discussion the results of this study clearly demonstrate that f. retusa leaf litter has an inhibitory effect on the selected understory species. significant reductions in the cover of many species, total plant cover and species richness were observed in plots under tree canopies. these observations are similar to those of previous studies, which illustrated considerable inhibition of plant cover beneath the tree canopy, compared with areas outside the canopy, due to the presence of leaf litter (ahmed et al. 2008, souza et al. 2010, hassan 2018). many tree species have been shown to negatively affect the cover, diversity and composition of some understory herbaceous species (barbier et al. 2008). moreover, loydi et al. (2013) indicated that a high amount of oak tree litter reduced the cover, composition, species richness and biomass of some associated fig. 2. effects of polyethylene glycol (peg) 6000 solutions with osmotic potentials equivalent to those of field soil under and outside the tree canopy on selected target understory species melilotus indicus, trifolium resupinatum and amaranthus viridis (n = 4). a – germination (%), b – root length (cm), c – shoot length (cm), d – biomass (g). the bars on each column represent standard error. data were analyzed using the independent samples t test. tab. 4. free and total concentrations of phenolic and flavonoid compounds (mean ± standard error, mg·g-1 soil, n = 4) detected in the field soil affected and unaffected by ficus retusa leaf litter using hplc and spectrophotometric analyses, respectively. significant difference at *p < 0.05 and **p < 0.01 according to independent samples t test, –: not detected. compounds litter-unaffected soil litter-affected soil phenolics caffeic acid 1.28 ± 0.15 2.10 ± 0.07** coumaric acid 0.40 ± 0.23 2.01 ± 0.00** ellagic acid 1.10 ± 0.07 1.66 ± 0.03** gallic acid 2.52 ± 0.10 2.56 ± 0.04 resorcinol – 1.39 ± 0.05** total phenolics 16.42 ± 0.72 48.07 ± 3.28** flavonoids quercetin – 1.56 ± 0.02** total flavonoids 22.79 ± 3.07 43.44 ± 6.54* allelopathic potential of ficus retusa l. leaf litter acta bot. croat. 80 (2), 2021 137 species. this effect may be due to toxic compounds leaching from litter residues (batish et al. 2007). the soil analysis revealed that the differences in organic matter and available nutrients between litter-affected and unaffected soils were not significant. this result suggests that the litter does not interfere with these soil criteria. it also indicates that ficus trees do not have a competitive effect on associated weed species. therefore, the reduction in plant cover and species richness could not be attributed to decreased soil organic matter or nutrient availability. these results are similar to those obtained by hassan (2018). in contrast, there was a considerable reduction in ph and a significant increase in the ec of litter-affected soils. these results may be due to phenolic compounds and minerals liberated from litter residue, as mentioned by hassan et al. (2014). in this study, the ph value decreased from 7.9 to 7.63. these values seem to be in the normal range for the germination and growth of plants (xuan et al. 2005). on the other hand, the ec value of litter-affected soil in this study was 0.664 ms·cm–1. xuan et al. (2005) reported that ec values < 1 ms·cm–1 did not affect the growth of tested species. similarly, hassan et al. (2014) confirmed that ec had a significant role in the inhibition of target species growth when it was >1.5 ms·cm–1. therefore, since the ec value observed in this study was within a range appropriate for plants, the decline in vegetation cover and richness is not attributable to a high ec value in the field soil. under greenhouse conditions, the litter-affected soil collected under the canopies of f. retusa trees substantially reduced the emergence and growth of the tested species. moreover, hplc and spectroscopic analyses showed that the litter-affected soil contained greater amounts of phenols and flavonoids. these findings suggest that the litter-affected soil collected under the ficus canopy had an inhibitory effect on the understory species. this inhibition could be due to the presence of phenolic and flavonoid compounds released into the soil during ficus leaf litter decay or leached from ficus leaf litter during irrigation or rainfall. among different classes of secondary metabolites, phenolic compounds are the major group that inhibits plant growth ( appel 1993). the compounds detected by hplc are potent allelochemicals that frequently inhibit the seed germination, growth and productivity of some species (qasem and foy 2001). for example, golisz et al. (2007) indicated that some phenolics, such as ferulic acid, gallic acid and chlorogenic acid, reduced the germination and seedling growth of lettuce to differing degrees. likewise, quercetin, apigenin, rutin and kaempferol are considered inhibitory substances that suppress the emergence and performance of many species (basile et al. 2000, sadeghi and bazdar 2018). these compounds harmfully affect cell division, the ability to absorb nutrients, membrane permeability, protein creation and the activities of enzymes, eventually reducing the growth of target species (li et al. 2010). additional studies have demonstrated that the cover and diversity of plant species could be changed by allelopathic compounds such as those detected during the hplc analysis conducted in this study (chou 1999, hassan 2018, hassan and mohamed 2020). the inhibitions observed under field conditions and in greenhouse experiments may be equivalent. in this study, litter-affected soils attained substantially higher values of ec than those of litter-free soils. although the values observed were normal for plant growth, the effect of the osmotic potential of soil solutes on seed germination and seedling growth had to be assessed to exclude potential osmotic interference with the allelopathic effect in soil. the results of our work showed that peg solutions with osmotic potentials equivalent to those of litter-affected soils did not significantly reduce the germination and growth of the target species. therefore, osmotic potential did not play a significant role in the inhibition of target species under greenhouse conditions or in the understory vegetation. accordingly, the inhibitory impact of field soil collected under the tree canopy on the germination and growth of tested species under greenhouse conditions was due to the allelochemicals present in the field soil. conclusion the field study indicated that the cover of selected understory species and species richness were reduced under the canopy of f. retusa trees. the greenhouse experiments showed that litter-affected soil suppressed the germination and growth parameters of the selected species. the biochemical analysis of the litter-affected soils confirmed the presence of elevated levels of many phenolic and flavonoid compounds, as compared with unaffected soils. these compounds have been shown to be phytotoxins that negatively affect the germination and growth of some understory species. the results of the soil analysis indicated that there were no statistically significant differences between soils obtained underneath and outside the ficus tree canopy; 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(rutaceae). acta botanica brasilica 24, 169– 174. takahashi, s., tamashiro, a., sakihama, y., yamamoto, y., kawamitsu, y., yamasaki, h., 2002: high-susceptibility of photosynthesis to photoinhibition in the tropical plant ficus microcarpa l. f. cv. golden leaves. bmc plant biology 2, 2. walkley, a., black, i.a., 1934: an examination of the degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. soil science 37, 29–38. xuan, t.d., tawata, s., khanh, t.d., chung, i.m., 2005: cropping and forage systems/crop ecology/organic farming. journal of agronomy and crop science 191, 162–171. acta bot. croat. 80 (1), 2021 1 acta bot. croat. 80 (1), 1–11, 2021 coden: abcra 25 doi: 10.37427/botcro-2020-029 issn 0365-0588 eissn 1847-8476 silver nanoparticles affect germination and photosynthesis in tobacco seedlings renata biba1, mirta tkalec1, petra cvjetko1, petra peharec štefanić1, sandra šikić2, dubravko pavoković1, biljana balen1* 1 university of zagreb, department of biology, faculty of science, horvatovac 102a, 10000 zagreb, croatia 2 dr. andrija štampar institute of public health, department of ecology, mirogojska cesta 16, 10000 zagreb, croatia abstract – extensive commercialization of silver nanoparticles (agnps) raises the risk of their accumulation in the soil-plant system. once released into the environment, agnps are prone to chemical transformations, which make it hard to determine whether their phytotoxic effects are purely np-related or a consequence of released ag+ ions. in this study the effects of 25, 50, 75, 100 and 150 μm agnps and agno3 on seed germination and early growth of tobacco (nicotiana tabacum l.) seedlings were compared. additionally, the effects on photosynthetic performance and pigment content were investigated. germination rate and index values indicated delayed and slower germination in some agnp treatments. lower agnp concentrations stimulated root growth, but induced a prominent reduction in fresh weight. by contrast, all agno3 concentrations inhibited root growth but only the higher ones decreased fresh weight. obtained results imply that the observed agnp toxicity could be ascribed to np form and can be correlated with high agnp stability in the solid medium. on the other hand, the majority of agnp and agno3 treatments induced an increase in chlorophyll content that was accompanied by significantly lower values of relative electron transport rate and coefficient of photochemical quenching, implying an inhibition of the electron transport chain. a similar impact of agnps and agno3 on photosynthesis can be correlated with lower stability of agnps in a liquid medium, resulting in agnp aggregation and dissolution of ag+ ions. keywords: chlorophyll fluorescence, germination, nicotiana tabacum, photosynthetic pigments, silver ions, silver nanoparticles introduction the rapid development and extensive commercialization of engineered nanomaterials (enms) have expanded their application in industry and daily life, raising serious concerns about their impact on the environment and human health (scheringer 2008, wiesner et al. 2009). among the various types of enms, silver nanoparticles (agnps) are involved in nearly 25% of consumer products (vance et al. 2015). special physicochemical properties of ag nanomaterials play a crucial role in their antibacterial activity, which is being utilized in the environmental, biomedical and industrial sectors (deshmukh et al. 2019). increased use of agnps raises the risk of their discharge into the environment and accumulation in the soil-plant system (yang et al. 2017, lv et al. 2019). through plants agnps could be transported and accumulated in high trophic-level consumers, including humans (rico et al. 2011, mckee and filser 2016). previous studies have shown both positive and negative impacts of agnps on plant metabolisms, mainly depending on their concentration, size, shape and coating (pallavi et al. 2016, jasim et al. 2017, cvjetko et al. 2017, 2018, peharec štefanić et al. 2019). experimental methodology (growth medium, exposure method, exposure time) as well as plant system used (species and developmental stage) also significantly affect agnp phytotoxicity (yan and chen 2019). agnp suspensions are prone to chemical transformations (oxidation, dissolution, aggregation and agglomeration) (levard et al. 2012, gorham et al. 2014) making it * corresponding author e-mail: bbalen@biol.pmf.hr biba r., tkalec m., cvjetko p., peharec štefanić p., šikić s., pavoković d., balen b. 2 acta bot. croat. 80 (1), 2021 dissolved in ultrapure water (ion-free milli-q water, 18.2 mω.cm resistivity, merck millipore, usa) and used as a 100 mm stock solution. prior to treatments, the concentration of ag in the agnp and agno3 stock solution was determined by elan drc-e inductively coupled plasma mass spectrometry (icp-ms) (perkin elmer, usa). for preparation of treatment solutions of agnps, the concentration of silver was considered in calculations. plant material and culture treatments in this study we used solidified half strength murashige and skoog (ms) nutrient medium (0.2% (w/v) phytagel, 1.5% (w/v) sucrose; both sigma aldrich, usa) (murashige and skoog 1962) to study the effects of agnps and agno3 on the germination and early growth of tobacco (nicotiana tabacum l. cv. burley). nutrient medium was sterilised and supplemented with agnp or agno3 stock solutions to obtain 25, 50, 75, 100 and 150 µm concentrations and subsequently poured into petri dishes (90 mm diameter) and left to solidify. tobacco seeds were surface sterilised for 15 min using 50% (v/v) naocl (kemika, croatia), and then rinsed three times with sterile deionised h2o before being placed on the half strength ms medium. control seeds were germinated on a medium devoid of agnps and agno3. two days before the beginning of the experiment, petri dishes with sown seeds were placed in cold stratification (+4 °c) to promote and synchronise seed germination (kucera et al. 2005). germination was monitored for 5 consecutive days, starting from the 3rd day after seed stratification. seedlings were harvested after three weeks and used for measurements of root length as well as fresh and dry weight. all experiments were conducted two times. in each experiment, 100 seeds were sown for every treatment with either agnps or agno3 (200 seeds in total per treatment). for analysis of effects of agnps and agno3 on photosynthesis and photosynthetic pigments and for measurement of ag content, sterilized and stratified seeds were placed in sterile 100 ml erlenmeyer flasks filled with 5 ml of liquid half strength ms medium and left to germinate on a shaker. germinated seeds were grown for three weeks in the same erlenmeyer flask, which was periodically supplemented with fresh sterile nutrient medium. after three weeks, the nutrient medium was replaced with fresh sterile liquid half strength ms medium supplemented with either agnps or agno3 in the above-mentioned concentrations. seedlings were treated for 7 days. during the experiments, all plant material was kept in the culture room at 24 ± 1 °c with 16/8 h light/dark cycles and 90 µmol m–2 s–1 light intensity. agnp stability in solid and liquid culture medium agnps stability in the solid nutrient medium was analysed as previously reported (peharec štefanić et al. 2018). briefly, one ml of half strength ms medium, solidified with phytagel (agar substitute) and supplemented with agnp stock solution to obtain a 150 µm concentration (the highharder to determine whether the effects of agnps are purely nanoparticle-related or a consequence of silver ion release from the nanoparticles. so far, research has provided evidence for both ag+ ionand agnp-specific toxicity (tkalec et al. 2019). ag+ ions released from agnps can induce oxidative stress through excessive reactive oxygen species (ros) production (park et al. 2009), disturb cell function by binding to cell components and modifying their activities (montes et al. 2017) and affect photosynthesis through competitive substitution of cu+ ions in plastocyanin (sujak 2005, jansson and hansson 2008). however, in some cases agnps proved to be more toxic than ag+ ions at the same concentrations, due to the inhibition of apoplastic trafficking caused by the clogging of pores and barriers in the cell wall or plasmodesmata (tripathi et al. 2017, ruotolo et al. 2018). high sensitivity to agnps caused by inhibition of photosynthetic processes was shown in several algae and plant species (navarro et al. 2008, oukarroum et al. 2012, jiang et al. 2017, dewez et al. 2018). reduced photosynthetic activity and decreased atp and nadph synthesis can disturb the biochemical and physiological processes needed for cell growth, which in the end affects plant development (gerst et al. 1994, stirbet and govindjee 2012). measurement of chlorophyll a fluorescence together with the content of photosynthetic pigments can provide valuable insight into the mechanism of agnp phytotoxicity, and coupled with germination percentage, root elongation and mass measurements, fast and reliable toxicity indicators, gives a more comprehensive view of the effects of agnps on plants (wang et al. 2001, dewez et al. 2018). to elucidate the nature of agnp-phytotoxicity, the effects of citrate-coated agnps and ionic ag in the form of silver nitrate (agno3), both applied in concentrations of 25, 50, 75, 100 and 150 μm, on seed germination, early growth as well as on photosystem ii (psii) performance and the photosynthetic pigment content of tobacco (nicotiana tabacum l.) seedlings, were compared. the significance of the work we are reporting on derives from interactions that have received little attention to date; the difference of possible phytotoxic effects of silver nanoparticles and ionic silver on germination and early growth as well as on photosynthesis. as object of our study we chose tobacco, not only an economically interesting and important plant but also a frequently used model organism in abiotic stress research (gichner et al. 2004, peharec štefanić et al. 2012, tkalec et al. 2014). materials and methods agnps and agno3 suspensions all experiments were performed with commercial agnps with citrate coating (50 nm citrate biopure silver nanospheres, nanocomposix, san diego, ca, zeta potential of –47.8 mv). the concentration of agnp stock solution was 9.27 mm. agno3 (sigma aldrich st. lois, mo, usa) was agnps affect tobacco germination and photosynthesis acta bot. croat. 80 (1), 2021 3 est applied agnp concentration in this study), was prepared in a 1 cm quartz cuvette for spectrophotometric absorbance measurements. the cuvette was sealed with parafilm m to prevent medium from drying and was kept in the same conditions as plant material during 5 days. measurements of spectrophotometric absorbance were performed using the uv-visible spectrophotometer (ati unicam, cambridge, uk) in the wavelength range of 300–800 nm. for instrument zeroing, solid half strength ms medium devoid of agnps was applied. stability of agnps was monitored regularly during the period of 5 days. for measurements of agnp stability in the liquid half strength ms medium a similar procedure for spectrophotometric measurements was applied. the differences were that a liquid half strength ms medium was used instead of a solid and that the cuvette was kept for 7 days in the same conditions as the plant material. measurements of spectrophotometric absorbance were performed in the abovementioned wavelength range. for instrument zeroing, liquid half strength ms medium devoid of agnps was applied. stability of agnps was monitored regularly during the period of 7 days. to confirm the data obtained by spectrophotometric analysis, the size and charge of nanoparticles in 150 µm agnp solution in liquid half strength ms medium were measured with the dynamic light scattering (dls) technique using a zetasizer nano zs (malvern, uk) equipped with green laser (532 nm). intensity of scattered light was detected at the angle of 173°. all measurements were conducted at 25 °c. the data processing was done with the use of the zetasizer software 6.32 (malvern instruments). measurements were performed at 0 and 15 min as well as 1, 5 and 24 h after the solution of agnps in nutrient medium was prepared. results are reported as an average value of 10 measurements and the size distributions are reported as volume distributions. the charge of agnps was evaluated by measuring electrophoretic mobility of agnps and results are reported as an average value of 5 measurements. in addition, agnps were visualized in nutrient media of all tested agnp concentrations after exposure of tobacco seedlings in a liquid medium using a fei morgagni 268d electron microscope. tem samples were prepared by depositing a drop of the sample suspension on a formvar®/carbon copper grid. samples were air-dried at room temperature. germination parameters seed germination was monitored for 5 days, each day at the same time. seeds were considered germinated when the radicle emerged from the seed. germination percentage was calculated using the formula: germination percentage (%) = final number of germinated seeds/total number of seeds × 100. to calculate germination index (gi) and germination rate (t50), daily counts of germinated seeds were used by employing the following formulas (farooq et al. 2005): gi = ∑ nt (number of germinated seeds on the t day)/dt (germination days) t50 = ti+{[(n/2)-ni]*(ti-tj)/ni-nj} where n is the final number of germinated seeds, ni and nj are cumulative number of seeds germinated by consecutive counts at times ti and tj, considering that ni<n/2<nj. growth parameters three weeks after exposure to agnps and agno3 on the solid half strength ms medium, seedlings were harvested and measured. the length of the main root was measured using a ruler and root length reduction percentage (rlpr) was calculated using the formula by zafar et al. (2015): rlpr % = 100 × [1 – (root length stress/root length control)]. seedlings fresh weight was measured and the fresh weight reduction percentage (fwpr) was calculated by employing the following formula (zafar et al. 2015): fwpr % = 100 × [1 – (fresh weight stress/fresh weight control)]. for dry weight measurements, seedlings were oven dried at 60 °c for 24 hours, and dry matter content (dmc) percentage was calculated using the formula: dmc % = 100 × [dry weight/(fresh weight + dry weight)] chlorophyll fluorescence chlorophyll a fluorescence measurement was carried out with a fluorpen fp100 (photon systems instruments, brno, czech republic). first the minimal fluorescence (f0) was determined in dark-adapted leaflets of three week old tobacco seedlings, followed by a short pulse of saturating light (3.000 µmol photons m–2 s–1) to induce maximum fluorescence (fm). then the leaflets were illuminated with actinic light (100 µmol photons m–2 s–1) to measure steadystate fluorescence (f) and maximum fluorescence (f’m) in light-adapted state. maximum photochemical quantum efficiency of psii (fv/fm), effective quantum efficiency of psii (φpsii), nonphotochemical quenching (npq) and coefficient of photochemical quenching (qp) were calculated according to maxwell and johnson (2000). the relative electron transport rate (retr) was calculated from the φpsii and photosynthetic photon flux density (genty et al. 1989) and shown in the results section. photosynthetic pigment analysis pigments were extracted from freeze dried leaf samples by homogenization in 96% ice-cold acetone with 0.3 mg ml–1 caco3. a high performance liquid chromatography (hplc, perkin elmer, usa) with the diode array detector and nonendcapped zorbax ods column (4.6 × 250 mm, 5 µm particle size) preceded by an ods guard column, both from agilent, was used for separation. the pigments were eluted using a method described by thayer and bjorkman (1990) biba r., tkalec m., cvjetko p., peharec štefanić p., šikić s., pavoković d., balen b. 4 acta bot. croat. 80 (1), 2021 with a slight modification: 100% solvent a (acetonitrile: metha nol:water 84:12:4) for the first 2 min followed by a 14 min linear gradient to 100% solvent b (methanol:ethyl acetate 68:32) which continued isocratically for the next 9 min. the column was re-equilibrated in solvent a for 10 min prior to the next run. the flow rate was 1 ml min–1. the pigments neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, lutein, β-carotene, chlorophyll a and chlorophyll b were detected by their absorbance at 440 nm and quantified against known amounts of standards (dhi water and environment, denmark). determination of ag content for determination of ag content, whole three week old seedlings exposed to 25, 50, 75, 100 and 150 µm concentrations of agnps and agno3 in a liquid half strength ms medium were taken. firstly, they were washed with 0.01 m hno3 to remove agnps potentially adsorbed on the root surface, and subsequently rinsed with ultrapure water. washed plant material was dried at 80 °c for 24 h and prepared for analysis as previously reported (cvjetko et al. 2017, peharec štefanić et al. 2018). determination of the total ag concentration was done using an elan drc-e icp-ms (perkin elmer, usa). to calculate the ag concentration, a calibration curve obtained with a set of standards of known concentrations was applied. detection limit and limit of quantification (loq) were 0.05 µg g–1 and 0.1 µg g–1, respectively. spike recovery tests were 96.6% and 96.8% for seedlings exposed to agnps and agno3, respectively. statistical analysis statistical analysis was performed using the statistica 13.0 (tibco) software package. the data were analysed by one-way anova followed by the least significant difference (lsd) test. differences between means were considered statistically significant at p ≤ 0.05. results agnps stability in culture media the results of the stability analyses obtained by spectrophotometric absorbance measurements of 150 µm agnps in solid and liquid half strength ms media are presented in fig. 1. in the solid medium used for germination and early growth of tobacco seeds, a reduction of the absorption maximum (absorption of 0.8) and a small peak shift towards lower wavelengths (peak maximum at 422 nm) were recorded immediately after medium solidification (zero minute) compared to the absorption maximum obtained for the 150 µm agnp solution in ultrapure water (absorption of 1.3 and peak maximum at 425 nm) (fig. 1a). the intensity of the surface plasmon resonance (spr) peak and its position remained relatively constant over 5 days suggesting little agglomeration of agnps had occurred. obtained results indicate that in the solid medium agnps were encapsulated, which prevented their aggregation and/or the dissociation of ag+ ions. agnp stability measurements in the liquid half strength ms medium, used for seedling growth for photosynthesis and photosynthetic pigment analyses, revealed stronger reduction of the absorption maximum compared to solid medium at zero minute (absorption of 0.5) and a peak shift towards lower wavelengths (peak maximum at 419 nm) compared to the absorption maximum obtained for the 150 µm agnp solution in ultrapure water (absorption of 1.3 and peak maximum at 425 nm) (fig. 1b). moreover, an exponential decrease of the spr intensity was observed over a period of 5 h, thus suggesting rapid agglomeration of agnps, although the position of the spr peak remained relatively constant. after a 5 h period, no spr peak could be detected, which indicates that agnps were completely aggregated. additional dls and tem analysis of 150 µm agnps in the liquid half strength ms medium corroborated these findings (on-line suppl. figs. 1, 2). dls analysis config. 1. stability analyses of agnps by uv-vis absorption spectra in: a – solid half strength ms medium supplemented with 150 µm agnps recorded over a period of five days, b – liquid half strength ms medium supplemented with 150 µm agnps recorded over a period of seven days. arrows indicate wavelengths of peak maximums. agnps affect tobacco germination and photosynthesis acta bot. croat. 80 (1), 2021 5 firmed the strong aggregation of agnps, which started almost immediately after the 150 µm agnp solution in the liquid half strength ms medium was prepared; the appearance of nanoparticles with sizes larger than 100 nm was observed after 15 min and it continued progressively until 24 h when nanoparticles below 200 nm were no longer present (on-line suppl. fig. 1). the charge of agnps remained negative with a slight shift towards less negative values (on-line suppl. fig. 1). tem analysis exhibited evident agnp aggregation in exposure solutions of all concentrations, which was the most prominent in 150 µm agnps solution (online suppl. fig. 2). effects on germination and early growth none of the applied concentrations of either agnps or agno3 had a significant influence on germination percentage compared to control. moreover, no significant difference was observed in the treatments with agnps and agno3 of the corresponding concentrations (fig. 2a). germination rate was increased after exposure to all agnp treatments in comparison to control, although significantly only at 50 µm concentration, while exposure to agno3 had no significant effect. comparison of treatments with corresponding concentrations of agnps and agno3 revealed significant difference only for the 50 µm concentration (fig. 2b). germination index decreased after treatments with agnps compared to control, although significantly different values were obtained only at 50 and 100 µm concentrations (fig. 2c). by contrast, no significant difference was obtained after exposure to agno3. corresponding concentrations of agnps and agno3 were not significantly different (fig. 2c). root growth was significantly enhanced after exposure to lower agnp concentrations (25 and 50 µm) compared to fig. 2. germination parameters of tobacco seeds after 5 days of germination on a solid half strength ms medium supplemented with 25, 50, 75, 100 or 150 μm agnps or agno3. a – germination percentage, b – germination rate (t50), c – germination index. values are the means ± standard error of three different experiments with 100 seeds per experiment (n = 300). if values are marked with different letters, the treatments are significantly different at p ≤ 0.05 (one-way anova followed by lsd post-hoc test); capital letters mark the differences among different concentrations of the agnp treatment as well as control, small letters mark the differences among different concentrations of the agno3 as well as control, while asterisks mark the differences among different treatments of the same concentration. fig. 3. growth parameters of three week old tobacco seedlings on a solid half strength ms medium supplemented with 25, 50, 75, 100 or 150 μm agnps or agno3. a – root length reduction percentage, values are the means ± standard error of three different experiments with 20 seedlings per experiment (n = 60), b – fresh weight reduction percentage, c – dry matter content. for fresh and dry weight, values are the means ± standard errors of three different experiments with 3 replicas per experiment (n = 9). if values are marked with different letters, the treatments are significantly different at p ≤ 0.05 (lsd test); capital letters mark the differences among different concentrations of the agnp treatment as well as control, small letters mark the differences among different concentrations of the agno3 as well as control, while asterisks mark the differences among different treatments of the same concentration. biba r., tkalec m., cvjetko p., peharec štefanić p., šikić s., pavoković d., balen b. 6 acta bot. croat. 80 (1), 2021 control, while treatments with 75, 100 and 150 µm agnps significantly reduced root elongation in a dose-dependent manner (fig. 3a, fig. 4). on the contrary, exposure to all agno3 concentrations induced a significant reduction in root growth in comparison to control (fig. 4). comparison of treatments with agnps and agno3 of the corresponding concentrations revealed a significant difference between them at 25 and 50 µm concentration (fig. 3a). all agnp concentrations applied induced a significant reduction in fresh weight compared to control, unlike agno3-treatments, among which only higher applied concentration (75, 100 and 150 µm) significantly decreased fresh weight content in comparison to control (fig. 3b). a significant difference between the corresponding agnpand agno3-treatments was obtained only for the 25 µm concentration, at which agnps induced a more prominent reduction in fresh weight than ionic ag (fig. 3b). compared to control, dry matter content increased after the majority of the treatments, being significantly different after exposure to 75 and 100 µm agnps and 50, 75, 100 and 150 µm agno3. no significant difference was noticed between the corresponding treatments with agnps and agno3 (fig. 3c). effects on photosynthesis and photosynthetic pigments measurements of chlorophyll a fluorescence showed that none of the applied concentrations of either agnps or agno3 had a significant influence on fv/fm compared to control (tab. 1). on the other hand, relative electron transport rate and qp decreased markedly after exposure to all agnp and agno3 treatments, except for the lowest agnp concentration (25 µm), where the observed decrease was not statistically different from control value (tab. 1). moreover, agno3 applied in all concentrations, except the lowest one, had a more negative effect on both parameters than the corresponding agnp treatments, leading to a reduction of over 50% (tab. 1). non-photochemical quenching was also significantly lower after exposure to all agnp and agno3 treatments, except for the 25 µm agnps, where the observed decrease was not statistically different from the control value (tab. 1). there was no difference between agnp and agno3 treatments of corresponding concentrations (tab. 1). the majority of the applied concentrations of both agnps and agno3 increased the content of total chlorophylls compared to control (tab. 2). the only exceptions were the highest concentration of agnps and the lowest concentration of agno3, where the values were similar to those of control. in both types of treatments, the highest concentration of total chlorophylls was measured after exposure to 50 µm concentration, although the value was higher in seedlings exposed to agnps compared to agno3 (tab. 2). on the other hand, higher concentrations of agnps (100 and 150 µm) resulted in lower pigment content than the corresponding concentrations of agno3. compared to control, chlorophyll a/b ratio was decreased after treatments with lower agnp concentrations (25, 50 and 75 µm) as well as with the 50 µm agno3 (tab. 2). regarding total carotenoids, exposure to 25, 50 and 75 µm agnps significantly increased their content compared to control, while among treatments with agno3, the 50, 75 and 100 µm concentrations resulted in significant augmentation (tab. 2). comparison of corresponding agnp and agno3 concentrations revealed that significantly lower values were measured in seedlings exposed to 100 and 150 µm agnps than in the corresponding concentrations of agno3, tab. 1. contents of ag in dry weight (dw) and chlorophyll fluorescence parameters (expressed as % of control) in tobacco seedlings after 7 days of exposure to 0, 25, 50, 75, 100 and 150 µm agnps and agno3. fv/fm – maximum fluorescence in dark-adapted state, retr – relative electron transport rate, qp – coefficient of photochemical quenching, npq – non-photochemical quenching. values are the means ± standard error of three different experiments, each with three replicas. if values are marked with different letters, the treatments are significantly different at p ≤ 0.05 (lsd test); capital letters mark the differences among agnp concentrations as well as control, small letters mark the differences among agno3 concentrations as well as control, while asterisks mark the differences among different treatments of the same concentration. # – ag content was below the limit of quantification (< 0.1 µg g–1). ns – no significant difference. conc (µm) ag (µg g–1 dw) fv/fm retr qp npq control 0 0d,# 100.00 ± 0.82 100.00 ± 4.46a,a 100.0 ± 5.17a,a 100.0 ± 4.33a,a agnp 25 180.03 ± 28.67c 98.57 ± 0.64ns 83.04 ± 7.44ab 89.30 ± 6.68ab 86.84 ± 8.9ab 50 214.85 ± 30.88bc 98.33 ± 0.46ns 60.76 ± 5.85b 64.78 ± 6.99bc 77.62 ± 5.21b 75 270.26 ± 18.93b 98.13 ± 0.73ns 57.38 ± 3.37b 58.91 ± 3.49c 66.67 ± 6.33b 100 358.60 ± 28.02a 98.94 ± 0.73ns 60.76 ± 5.06b 61.46 ± 5.47bc 62.03 ± 5.80b 150 404.98 ± 19.20a 100.15 ± 0.97ns 65.82 ± 9.69b 63.04 ± 9.39bc 68.45 ± 7.41b agno3 25 76.92 ± 16.52cd,* 99.26 ± 0.67ns 76.66 ± 4.46b 75.81 ± 4.78b 75.17 ± 7.31b 50 152.19 ± 23.33bc,* 98.43 ± 1.24ns 46.72 ± 7.44c,* 44.84 ± 4.91c,* 64.02 ± 6.58b 75 204.72 ± 15.11b,* 99.17 ± 0.84ns 42.14 ± 5.85c,* 41.56 ± 4.28c,* 63.33 ± 4.03b 100 230.87 ± 27.13ab,* 99.64 ± 1.32ns 38.75 ± 3.37c,* 39.69 ± 7.93c,* 57.34 ± 5.29b 150 296.03 ± 15.24a,* 99.17 ± 0.98ns 36.89 ± 5.06c,* 35.17 ± 6.12c,* 62.92 ± 3.95b agnps affect tobacco germination and photosynthesis acta bot. croat. 80 (1), 2021 7 while the opposite result was observed for treatments with 25 and 75 µm agnps and agno3 (tab. 2). similar results as those of total carotenoids were obtained for xanthophylls involved in the xanthophyll cycle (violaxanthin, zeaxanthin and antheraxanthin, vza), although the differences among the agnp and agno3 treatments were significant only for 100 and 150 µm concentrations (tab. 2). other xanthophylls, neoxanthin and lutein, followed a similar trend; namely, exposure to lower agnp concentrations (25, 50 and 75 µm) significantly increased their content compared to control, while treatments with agno3 resulted in a significant increase with all tested concentrations, except the lowest one (25 µm) (tab. 2). comparison of corresponding agnp and agno3 treatments revealed that the two highest agnp concentrations resulted in significanlty lower pigment content than treatments with 100 and 150 µm agno3. in contrast, β caroten content was increased only after the treatment with 75 µm agnps, while the two highest concentrations significanly lowered its value. among all tested treatments only exposure to 100 µm agnps resulted in a lower carotenoid/chlorophyll ratio in comparison to the control (tab. 2). moreover, seedlings exposed to higher agnp concentrations (100 and 150 µm) had reduced carotenoid/chlorophyll ratio compared to seedlings treated with corresponding concentrations of agno3. vza/ chlorophyll ratio was significantly decreased after all agnp treatments compared to the control, especially with the highest concentrations of agnp (100 and 150 µm), which also provoked a significantly lower vza/chlorophyll ratio than the corresponding agno3 concentration (tab. 2). by contrast, only treatment with 50 µm agno3 induced a significantly low vza/chlorophyll ratio compared to the control (tab. 2). ag content ag content in tobacco seedlings exponentially increased with the increasing concentrations of both agnps and agno3 treatments, being significantly the highest after exposure to 150 µm agnps and agno3 compared to control (tab. 1). comparison of agnp and agno3 treatments of the corresponding concentrations revealed that in general ag uptake in seedlings was higher after exposure to agnps compared to ionic ag. in control seedlings, ag content was below the instrument detection limit loq (<0.1 µg g–1). discussion germination rate and index values indicated that germination of tobacco seeds was delayed and slower only in some agnp-treatments than in the control, while agno3 had no significant effect. previous experiments performed with nanoparticles on germination of different plant species showed negative (geisler-lee et al. 2014, tripathi et al. 2017), positive (parveen and rao 2015, almutairi and alharbi 2015) as well as neutral effects (el-temsah and joner 2012, yin et al. 2012), depending on physico-chemical char-ta b. 2 . c on te nt s of p ho to sy nt he tic p ig m en ts a nd th ei r ra tio s ex pr es se d as % o f c on tr ol in to ba cc o se ed lin gs a ft er 7 d ay s of e xp os ur e to 0 , 2 5, 5 0, 7 5, 1 00 a nd 1 50 µ m a gn ps a nd a gn o 3 in a li qu id h al f st re ng th m s m ed iu m . v z a v io la xa nt hi n, z ea xa nt hi n an d an th er ax an tin ( xa nt ho ph yl l c yc le p oo l) , c hl a /b c hl or op hy ll a/ b, c ar /c hl c ar ot en oi ds /c hl or op hy ll, v z a /c hl v z a / c hl or op hy ll. v al ue s ar e th e m ea ns ± st an da rd e rr or o f t hr ee d iff er en t e xp er im en ts , e ac h w ith th re e re pl ic as . i f v al ue s a re m ar ke d w ith d iff er en t l et te rs , t he tr ea tm en ts a re si gn ifi ca nt ly d iff er en t a t p ≤ 0 .0 5 (l sd te st ); ca pi ta l l et te rs m ar k th e di ffe re nc es a m on g a gn p co nc en tr at io ns a s w el l a s c on tr ol , s m al l l et te rs m ar k th e di ffe re nc es a m on g a gn o 3 c on ce nt ra tio ns a s w el l a s c on tr ol , w hi le a st er is ks m ar k th e di ffe re nc es a m on g di ffe ren t t re at m en ts o f t he sa m e co nc en tr at io n. c on c (µ m ) to ta l c ar ot en oi ds to ta l c hl or op hy ll v z a n eo -x an th in lu te in βca ro te ne c hl a /b c ar /c hl v z a /c hl co nt ro l 0 10 0 ± 3. 29 b, c 10 0 ± 2. 82 d ,d 10 0 ± 3. 74 b c , b 10 0 ± 3. 90 b, d 10 0 ± 4. 13 b, d 10 0 ± 4. 46 b 10 0 ± 1. 30 a ,a 10 0 ± 2. 79 a 10 0 ± 3. 27 a ,a a gn p 25 12 7. 86 ± 3 .9 6a 13 0. 77 ± 2 .2 5b 11 5. 41 ± 8 .9 6a b 14 8. 87 ± 2 .1 3a 13 9. 52 ± 4 .2 9a 11 3. 64 ± 5 .5 5a b 94 .4 6 ± 1. 32 b c 97 .6 7± 2 .3 9a 90 .2 7± 3 .3 0b 50 13 0. 51 ± 2 .5 2a 14 6. 02 ± 2 .9 7a 12 1. 27 ± 2 .0 8a 15 5. 66 ± 1 .8 4a 14 7. 83 ± 2 .8 6a 10 8. 34 ± 3 .6 9a b 93 .1 7 ± 0. 93 c 89 .3 2 ± 3. 75 a b 84 .3 3 ± 1. 87 b c 75 13 2. 54 ± 3 .9 3a 13 4. 14 ± 3 .1 5b 11 4. 79 ± 5 .8 7a b 15 2. 00 ± 4 .3 3a 14 4. 28 ± 5 .3 5a 12 1. 28 ± 2 .7 7a 96 .1 4 ± 1. 76 b c 95 .7 4 ± 2. 46 a 88 .4 4 ± 3. 42 b c 10 0 94 .0 3 ± 2. 40 b 11 6. 10 ± 1 .2 4c 82 .5 7 ± 2. 02 c 11 1. 80 ± 3 .4 7b 10 8. 89 ± 2 .4 3b 78 .7 9 ± 1. 68 c 97 .5 1 ± 4. 15 a b 80 .9 4 ± 1. 58 b 71 .0 4 ± 3. 12 d 15 0 90 .6 9 ± 1. 88 b 10 2. 97 ± 1 .6 3d 80 .7 1 ± 2. 37 c 10 8. 58 ± 5 .7 6b 10 1. 66 ± 1 .9 6b 79 .9 2 ± 2. 19 c 10 0. 65 ± 0 .9 8a 88 .0 1 ± 2. 75 a b 78 .3 0 ± 5. 20 c d a gn o 3 25 99 .9 6 ± 4. 35 c, * 10 1. 31 ± 1 .9 7d ; * 95 .5 0 ± 2. 93 b 10 3. 87 ± 2 .5 3d , * 10 6. 55 ± 1 .7 9d , * 94 .7 6 ± 4. 02 * 95 .7 0 ± 4. 64 ab 98 .6 6 ± 4. 57 94 .2 2 ± 2. 38 ab 50 12 2. 92 ± 5 .1 5a 13 2. 78 ± 2 .1 1a , * 11 6. 49 ± 2 .7 4a 15 1. 46 ± 2 .2 9a 15 3. 7 ± 3. 99 a 10 3. 60 ± 9 .9 0 92 .4 7 ± 1. 78 b 96 .3 0 ± 3. 11 87 .6 8 ± 2. 22 b 75 11 9. 16 ± 2 .9 1a b, * 12 3. 74 ± 1 .9 1b 11 4. 63 ± 2 .5 9a 13 5. 78 ± 3 .8 7b c, * 14 1. 49 ± 3 .3 1a b 97 .0 7 ± 5. 07 97 .1 2 ± 3. 72 ab 96 .5 2 ± 1. 90 91 .7 9 ± 1. 52 ab 10 0 12 0. 31 ± 2 .1 6a b, * 12 1. 78 ± 1 .8 7b , * 11 5. 49 ± 2 .8 5a , * 14 0. 95 ± 2 .6 7a b, * 13 4. 21 ± 2 .8 1b c, * 11 0. 13 ± 1 .7 3* 95 .4 6 ± 1. 35 ab 10 0. 44 ± 3 .3 6* 94 .8 5 ± 2. 77 ab , * 15 0 11 1. 79 ± 3 .2 3b c, * 11 3. 61 ± 1 .1 3c , * 10 3. 31 ± 3 .3 5a b, * 12 5. 54 ± 3 .4 2c , * 12 0. 97 ± 1 .3 8c , * 10 5. 85 ± 2 .7 3* 96 .9 0 ± 1. 42 ab 98 .4 2 ± 1. 70 * 90 .9 9 ± 3. 85 ab , * biba r., tkalec m., cvjetko p., peharec štefanić p., šikić s., pavoković d., balen b. 8 acta bot. croat. 80 (1), 2021 acteristics of the particles, plant species and the applied concentration of agnps. root growth was significantly enhanced after exposure to lower agnp concentrations (25 and 50 µm), but significantly reduced with higher concentrations in a dose-dependent manner, which is in accordance with the effects of agnps on corn and common bean plantlets (salama 2012) as well as on wheat seedlings (asanova et al. 2019). moreover, higher concentrations of agnps had effects on root growth of tobacco seedlings that were as retarding as agno3, which inhibited root growth from the lowest applied concentration. prominent toxic effects of agno3 on root elongation have already been reported for mustard (vishwakarma et al. 2017), barley seedlings (fayez et al. 2017) and he castor bean (yasur and rani 2013). however, in this study the lowest agnp concentration, which stimulated tobacco root growth, induced prominent reduction in fresh weight compared to corresponding concentration of agno3, where no reduction was recorded. our results indicate that effects induced by agnps and agno3 are similar but not identical implying that the observed agnp toxicity could be ascribed to nanoparticle form of ag and not ag+ ions released from nanoparticles. agnp stability analyses in the solid half strength ms medium showed high stability of the applied agnps, with minor aggregation and/or dissociation of ag+ ions. as previously reported by peharec štefanić et al. (2018), this is probably a result of agnps stabilization with phytagel, a natural polymer used for solidification of nutrient media. it is known that some polymer ligands can control access of molecular oxygen to the nanoparticle surface, which would decrease the oxidative dissolution rate of agnps (gunsolus et al. 2015). therefore, in our treatments with agnps added in the solid nutrient medium for germination and growth experiment, ag was accessible to seeds and developing seedlings in the form of nanoparticles, although the presence of ag+ ions due to release from the agnp surface cannot be completely excluded. the mechanisms explaining the impact of agnps on germination and plant biomass are still not completely elucidated. positive effects can be ascribed to agnps ability to generate new pores on the seed coat during penetration, which may help in the influx of nutrients inside the seed or else agnps may carry the nutrients along with them, leading to accelerated germination and increased growth rate (tkalec et al. 2019). on the other hand, zuverza-mena et al. (2016) demonstrated that agnp-treatment of radish sprouts induced a decrease in water content in a dose-dependent manner, which is in accordance with increased dry matter content after agnp-exposure of tobacco seedlings recorded in our study. these results together with findings that agnps affect the balance of water by changing the transcription of aquaporin genes (qian et al. 2013), imply that agnps can negatively affect plant growth by reducing water uptake. chlorophyll fluorescence measurement showed that exposure of tobacco seedlings to agnps resulted in reduced values of φpsii and qp, implying an inhibition of the electron transport chain. significantly reduced qp was previously reported in mustard (vishwakarma et al. 2017) and pea seedlings (tripathi et al. 2017) after exposure to agnps. furthermore, a severe disruption of photosynthesis and co2 assimilation efficiency as well as decrease in photosynthesis rate was found in tomato seedlings treated with agnps (das et al. 2018). moreover, in these studies a decline in chlorophyll fluorescence parameters was accompanied with a decrease in chlorophyll content, suggesting severe negative effects of agnps on photosynthesis. to the contrary, in our study, a significant increase of total chlorophyll was obtained at almost all tested concentrations of agnps. an increase in chlorophyll content along with significantly lower values of φpsii and qp was also recorded in tobacco seedling after almost all agno3-treatments. a similar impact of agnps and agno3 on photosynthesis can be correlated with lower stability of agnps in the liquid medium used for plant growth, resulting in agnp aggregation as well as dissolution of ag+ ions. several studies suggested that conditions present in the culture medium such as high ionic strength and neutral ph can cause agnp dissolution (sharma et al. 2014, domazet jurašin et al. 2016). therefore, our results suggest that tobacco seedlings exposed to either agnps or agno3 increased synthesis of chlorophyll pigments to overcome the impaired electron transfer imposed by ag+ ions. this is because ag+ ions can competitively replace cu+ ions and bind to plastocyanin, which results in disturbance or inactivation of the photosynthetic electron transport (sujak 2005, jansson and hansson 2008). moreover, previous proteomic analysis of tobacco seedlings (peharec štefanić et al. 2018) revealed up-regulation of proteins involved in the photosynthesis after agnp and agno3 treatments. however, the reduction of φpsii and qp in tobacco seedlings was significantly more pronounced after exposure to agno3 compared to corresponding agnp treatments, although total ag content was much higher in seedlings exposed to agnps. this could be correlated with prominent oxidative stress found in agno3-treated seedlings (peharec štefanić et al. 2018), which could additionally damage photosynthetic apparatus. in the study of pardha-saradhi et al. (2018) it was shown that exposure of wheat fig. 4. three week old tobacco seedlings after growing on a solid half strength ms medium supplemented with 25, 50, 75, 100 or 150 μm agnps or agno3. c – control (solid half strength ms). agnps affect tobacco germination and photosynthesis acta bot. croat. 80 (1), 2021 9 seedlings to agnps had a less toxic effect on light harnessing photosynthetic machinery than agno3, which corroborates our results. contents of analysed carotenoids were also elevated in tobacco seedlings exposed to lower concentrations of agnps and most concentrations of agno3. increased total carotenoids and xanthophylls might play a role in the regulation of light harvesting and energy flow of chlorophyll-excited states. also, carotenoids are important antioxidant molecules which may represent a defence against elevated ros (he et al. 2011). a strong increase of carotenoid content has already been found in plants after agnp exposure, suggesting that carotenoids are implicated in antioxidant defence responses of plants to agnps (mirzajani et al. 2013, yan and chen 2019). it has been suggested that xanthophylls, especially zeaxanthin, are involved in the npq of excess energy in the antenna of psii (jahns and holzwarth 2012). however, in our study npq was decreased in almost all agnp and agno3 treatments. still, the vza/chlorophyll ratio was mostly lower in agnp treated tobacco seedlings, so it is possible that agnps in interaction with chlorophylls acted as quencher removing the excited electron from the chlorophyll (queiroz et al. 2016). different effects induced by agnps and agno3 on germination and growth compared to effects of photosynthesis can be related to the stability of the agnps in the media used in the experiments. since the observed effects of agnps compared to agno3 were not identical, some of the toxic effects can be attributed to the nanoparticle form of ag. acknowledgements this research has received funding from the croatian 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dataset (2404 relevés) was divided according to different locations (arable land, semi-natural land, settlement) used as surrogates for disturbance types differing in areal extent, regularity, predictability and frequency. using multivariate methods we detected main gradients in species composition and correlated them to plant traits. plants in arable land are mainly annuals, therophytes and alien species. species comprising ruderal habitats are richer in perennials and c strategists. the latter habitats were further divided into semi-natural sites and settlements. phanerophytes and c strategists are more common in semi-natural vegetation and r strategists and archeophytes in settlements. differences in village and town vegetation are observed in alien species that are more frequent in towns and on sites that are warmer and lighter. keywords: vegetation, ruderal, segetal, disturbance, slovenia introduction anthropogeneous vegetation is by definition a product of human influence. anthropogenic pressure can be understood as a disturbance that differs according to areal extent, magnitude (intensity), frequency, predictability and turnover rate (sousa 1984). disturbance alters the site and recolonization and succession of open space will appear. human activities are reflected in different vegetation types that thrive in man-made habitats. synanthropic vegetation can be generally divided into two broad types: weed and ruderal vegetation (mucina et al. 1993, lososová et al. 2006). weed vegetation is found on arable land and ruderal vegetation is found in settlements, waste deposits, along transportation routes, but also in semi-natural landscape (according to westhoff 1983) comprising disturbed river shores and woodland fringes. major differences in species composition are the product of different disturbance regimes and the life histories of plant species that occupy these disturbed habitats. with greater disturbance (especially human activity) a landscape becomes a mosaic of ecosystem patches with sharper boundaries (forman and godron 1981) and sharper differences in ecological conditions and floristic composition. acta bot. croat. 69 (2), 2010 215 * corresponding author, e-mail: urban@zrc-sazu.si u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:01 color profile: disabled composite 150 lpi at 45 degrees arable land is regularly disturbed at least once a year. agrotechnical measures (plowing, tilling, harvesting) are highly predictable and of the intensive disturbance type (fig. 1). the areal extent of disturbance is large and ecological conditions within large fields are uniform. semi-natural habitats are small scale and the magnitude of human activity is rather low. they are less frequently disturbed (once in a few years) and predictability is low. settlements are a synonym for human activity, although there are differences between village and town environments. the sites of these plant communities are generally small. intensity of disturbance depends on the level of urbanization, where human density could be used as a surrogate. usually it is higher in towns. frequency of disturbance is high (again higher in urban settlements), but predictability is uncertain. these land use types that are the product of mostly human activities show differences in species composition since plant traits are filtered by different environmental conditions (zobel 1997, knapp et al. 2008). the aim of our study was to analyze a large dataset of synanthropic vegetation and detect differences in species composition and plant traits of different types of vegetation of man-made habitats and to correlate these differences to different disturbance regimes. materials we have collected a dataset of 2404 vegetation relevés of anthropogeneous vegetation from slovenia stored in slovenian phytosociological database ([ilc 2006). relevés were selected according to the original author’s assignment to the synanthropic vegetation classes (polygono-poetea, stellarietea medie, artemisietea, galio-urticetea) (fig. 2, tab. 1). using outlier analysis in the pc-ord 5 software (mccune and grace 2002) we have eliminated one plot deviating more than 3sd in floristic composition. to avoid oversampling we divided the territory of slovenia into grid squares (1.25 longitudinal and 0.75 latitudinal minutes) and performed stratified re-sampling. if more than one relevé of the same syntaxon and date were recorded in the same grid square we randomly selected only one. this procedure yielded a matrix of 1537 relevés x 461 species that was used in further analyses. 216 acta bot. croat. 69 (2), 2010 [ilc u. fig. 1. schematic representation of dichotomous division of synanthropic vegetation according to disturbance type. u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:02 color profile: disabled composite 150 lpi at 45 degrees explanatory variables for each vegetation plot a set of variables that are supposed to affect species composition was compiled. mean annual temperature, mean annual amount of precipitation, and altitude were collected from plot locations and climatic maps (mekinda-majaron 1995, zupan^i^ 1995) as layers in the arc gis 9.2 programme. each plot was also categorized according to the location (given by the author) where the relevé was made: arable fields (two subsets – root crops and cereals), settlement (two subsets – town and village; 1000 inhabitants was the limit) and semi-natural. location is a surrogate of disturbance type and further in the text we use the term disturbance type. plant traits plant traits from the biolflor (klotz et al. 2002) database were used to characterize species. life span, life form, life strategy and residence time of alien species were used. to evaluate habitat characteristics ecological indicator values (ellenberg et al. 1992) were used. acta bot. croat. 69 (2), 2010 217 synanthropic vegetation fig. 2. distribution of relevés of synanthropic vegetation in slovenia with indicated phytogeographical regions. tab. 1. classification of relevés used in the analysis into vegetation classes according to the location (disturbance) type and original author’s assignment. no. of relevés settlement arable land semi-natural polygono-poetea 135 135 0 0 stellarietea mediae 670 233 435 2 artemisietea 306 268 16 22 galio-urticetea 426 321 0 105 u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:06 color profile: disabled composite 150 lpi at 45 degrees subsets we have divided the dataset into three subsets according to the location where vegetation plots were recorded and the type of disturbance that corresponds to that location. first analyses were made with the whole dataset. then we divided the dataset into segetal and ruderal. additionally, the ruderal subset was subdivided into semi-natural and settlement subsets. the latter was partitioned into town and village subsets. subsets were analyzed with the same statistical procedure as described for the whole dataset. methods the general pattern of variation in synanthropic vegetation was detected by using detrended correspondence analysis (dca) in canoco 4.5 software package (ter braak and [milauer 2002). as a long gradient (5.198 sd) was stated, we decided to use unimodal methods in further analyses (dca, cca) (lep[ and [milauer 2003). to evaluate the importance of explanatory variables on species composition canonical correspondence analysis (cca) was used with the monte carlo test with 999 permutations. dca analysis was used to visualize the variation in samples and explanatory variables that are passively projected. partial detrended correspondence analysis (pdca) was performed to present species variation and passively projected locations (presenting disturbance type) of vegetation plots. 218 acta bot. croat. 69 (2), 2010 [ilc u. fig. 3. detrended correspondence analysis (dca) ordination of samples (dots) with passively projected explanatory variables. triangles represent nominal variables. u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:10 color profile: disabled composite 150 lpi at 45 degrees partial canonical correspondence analysis (pcca) was used to obtain species scores according to the disturbance type, while the influence of other explanatory variables was partialled out. location was used as an only explanatory variable, while other variables were used as covariables in the analysis. this procedure was used on the entire dataset (segetal vs. ruderal) and further on the subsets (semi-natural vs. settlement and town vs. village). a method combining the net effects of variables from pcca and logistic regression (lososová et al. 2004, 2006) was used to identify the correlation between plant traits and disturbance type of vegetation plot. species scores on the first axis from pcca of disturbance type as a single explanatory variable, and plant traits were used in logistic regression. species scores were the independent and plant traits (as presence/absence of a single trait) acta bot. croat. 69 (2), 2010 219 synanthropic vegetation -1 5 0 6 malvneg planmaj poa ann6 urtidio chenalb polyper polyare echicru galipar lamimac rumeobt dactglo erigann taraoff loliper dauccar ranurep setapum achimil aegopod capsbur convarv stelmed veroper digisan elytrep lamipur trifrep calysep poa tri rubucae artevul galiapa cirsarv anthsyl silelat polyavi violarv arable semi-natural settlement fig. 4. partial detrended correspondence analysis (pdca) of species and passively projected disturbance types. other variables were used as covariables. species abbreviations: achimil-achilea millefolium, aegpod-aegopodium podagraria, anthsyl-anthriscus sylvestris, artevul-artemisia vulgaris, calysep-calystegia sepium, capsbur-capsella bursa pastoris, chenalb-chenopodium album, cirsarv-cirsium arvensis, dactglo-dactylis glomerata, dauccar-daucus carota, digisan-digitaria sanguinalis, echicru-echinochloa crus-galli, elytrep-elytrigia repens, erigann-erigeron annuus, galiapa-galium aparine, galipar-galinsoga parviflora, lamimac-lamium maculatum, lamipur-lamium purpureum, loliper-lolium perenne, malneg-malva neglecta, polyper-polygonum persicaria, planmaj-plantago major, poaann-poa annua, polyare-polygonum arenastrum, polyavi-polygonum aviculare, ranurep-ranunculus repens, rubucae-rubus caesius, rumeobt-rumex obtusifolius, silelat-silene latifolia, stelmed-stellaria media, taraoff-taraxacum officinale, trifrep-trifolium perenne, urtidio-urtica dioica, veroper-veronica persica, violarv-viola arvensis. u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:10 color profile: disabled composite 150 lpi at 45 degrees dependent variable in analysis. for ellenberg indicator values for particular species and their scores on the first pcca axis we performed linear least-square regression. regression analyses were performed by statistica 8.0 (statsoft 2007). results dca analysis with passively projected explanatory variables shows their importance for species composition (fig. 3). disturbance type has less effect on species composition than climatic variables and year of sampling. disturbance type correlates to the first axis. the eigenvalue of axis 1 is 0.607 and of axis 2 0.363, respectively. 220 acta bot. croat. 69 (2), 2010 [ilc u. tab. 2. relationships between life history plant traits and ellenberg indicator values and different habitat types. the negative sign is correlated with the first variable. *p < 0.01, ** p < 0.05. segetal vs. ruderal seminatural vs. settlement town vs. village estimate p estimate p estimate p life span annuals –1.283 ** 0.702 ** –0.767 ** biannuals 0.152 0.524 * 0.220 perennials 1.124 ** –0.562 ** 0.608 ** alien status neophytes –0.360 * –0.210 –0.892 ** archaeophytes –0.855 ** 1.542 ** –0.527 * life form t –0.080 ** 0.637 ** –0.748 ** g 0.056 –0.282 0.595 * h 0.232 * 0.322 * 0.439 ** c 1.585 ** –0.382 –0.326 ** p 2.336 ** –0.610 * 0.147 strategy c 0.957 ** –0.331 * 0.399 ** cr –0.554 ** 0.563 ** –0.418 * cs 1.094 ** –0.664 ** 0.444 csr 0.337 * 0.444 * 0.298 r –0.828 ** 0.906 ** –0.748 ** sr –0.551 * 1.080 –1.692 s not enough variables –1.138 –0.197 ellenberg indicator values light 0.009 –0.128 * 0.196 * temperature 0.268 ** –0.084 0.313 ** continentality –0.016 0.021 0.143 moisture –0.178 ** 0.355 ** –0.301 ** reaction –0.264 ** 0.076 0.082 nutrients –0.091 –0.056 0.050 u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:10 color profile: disabled composite 150 lpi at 45 degrees the results of pcca revealed that the most important factor in the whole dataset is the location of the vegetation plot, i.e. the disturbance type. the importance of other variables is lower, they follow in the following order: precipitation, temperature, year of sampling and altitude. all variables are significant at p<0.001. the influence of disturbance type (segetal vs. ruderal) after partialling out the effects of other explanatory variables is shown on the pdca graph (fig. 4). plant life history traits show significant differences between segetal and ruderal vegetation (tab. 2). weed vegetation is composed of annuals, therophytes and r-strategists (and correlated cr and sr strategy). notably, segetal vegetation is richer in archeophytes and neophytes. species are thermophilous. in ruderal vegetation perennial species are more common, mostly phanerophytes, chamephytes and hemicriptophytes. they have c-strategy and related cs and csr strategy. they tend to thrive on moist sites and are basiphilous. species characteristic of segetal and ruderal habitats are presented in table 3. there is an evident disproportion in favor of species typical of arable land and especially cereal fields. fifty species were determined with highest fit and species scores on the first axis of pcca with disturbance type of the vegetation plot as the only explanatory variable (tab. 3). differences between semi-natural and settlement vegetation in life history traits show that in settlements there are annuals and biennials, archeophytes, therophytes and hemicryptophytes. their strategy is ruderal (r-strategy and also cr and csr). plants in settlements are heliophilous. on the other hand, perennials and phanerophytes are more common in semi-natural vegetation and have a c-strategy. habitats in semi-natural land are more humid. if we compare village and town, vegetation in towns is composed of annual species; therophytes and chamephytes are more common. alien species (archeophytes and neophytes) are more frequent, and the strategy is ruderal (r and cr). species of vegetation in towns are more heliophilous and thermophilous whereas village vegetation has more perennials, hemicryptophytes and geophytes, which are c-strategists. the habitat is wetter. discussion our aim was to detect differences in species composition and life history traits in anthropogenous vegetation related to disturbance. according to our previous knowledge we made a hypothesis that the primary division is between arable and ruderal habitats. the latter have different disturbance regimes and can be further divided into semi-natural land and habitats in settlements. according to the number of inhabitants and related urbanization we can further divide this type of vegetation into village and town habitats. differentiation of weed and ruderal vegetation in the narrower sense is obvious. disturbance in segetal vegetation is regular and uniform in large areas (lososová et al. 2006), while in ruderal habitats it varies in all characteristics mentioned above (sousa 1984) thus resulting in patchy vegetation. the mosaic of vegetation types is composed of temporally and spatially differentiated patches. acta bot. croat. 69 (2), 2010 221 synanthropic vegetation u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:10 color profile: disabled composite 150 lpi at 45 degrees 222 a c t a b o t .c r o a t .69 (2),2010 [ il c u . tab. 3. fifty species with highest fit and species scores on the first axis of pcca with disturbance type of the vegetation plot as the only explanatory variable. segetal vs. ruderal ax1 score fit semi-natural vs. settlement ax1 score fit town vs. village ax1 score fit valerianella locusta 2.67 0.071 carex acutiformis 2.66 0.021 viola odorata 2.14 0.011 buglossoides arvensis 2.40 0.043 populus alba 2.64 0.016 veronica cymbalaria 1.94 0.008 sherardia arvensis 2.09 0.058 galega officinalis 2.53 0.023 euphorbia peplus 1.81 0.010 legousia speculum-veneris 2.03 0.130 iberis amara 2.52 0.018 catapodium rigidum 1.78 0.009 ranunculus arvensis 2.01 0.056 populus nigra 2.44 0.027 eragrostis minor 1.73 0.020 centaurea cyanus 1.93 0.076 antirrhinum majus 2.42 0.033 trifolium hybridum 1.72 0.008 anthemis arvensis 1.86 0.135 impatiens glandulifera 2.17 0.125 euphorbia lathyris 1.69 0.011 aphanes arvensis 1.83 0.195 verbascum austriacum 2.12 0.055 euphorbia maculata 1.61 0.007 vicia angustifolia 1.82 0.078 rudbeckia laciniata 2.01 0.081 verbena officinalis 1.55 0.018 vicia hirsuta 1.75 0.076 echinops exaltatus 1.93 0.097 chenopodium hybridum 1.53 0.010 apera spica-venti 1.68 0.078 salix purpurea 1.91 0.055 solidago canadensis 1.53 0.022 veronica hederifolia 1.65 0.053 cerastium sylvaticum 1.85 0.030 conyza bonariensis 1.31 0.007 myosotis scorpioides 1.65 0.217 erucastrum gallicum 1.91 0.025 carduus acanthoides 1.31 0.018 raphanus raphanistrum 1.64 0.069 euphorbia stricta 1.77 0.016 hordeum leporinum 1.24 0.008 papaver rhoeas 1.64 0.180 helianthus tuberosus 1.83 0.057 galinsoga ciliata 1.26 0.015 viola arvensis 1.54 0.251 barbarea vulgaris 1.64 0.038 portulaca oleracea 1.23 0.009 mentha arvensis 1.43 0.107 galeopsis speciosa 1.52 0.072 panicum capillare 1.20 0.009 euphorbia helioscopia 1.41 0.111 epilobium hirsutum 1.38 0.031 raphanus raphanistrum 1.18 0.009 arenaria serpyllifolia 1.41 0.071 scrophularia canina 1.45 0.031 verbascum nigrum 1.18 0.011 polygonum lapathifolium 1.35 0.097 peucedanum verticillare 1.37 0.020 mercurialis annua 1.15 0.010 lamium purpureum 1.26 0.135 phalaris arundinacea 1.36 0.048 oenothera biennis 1.08 0.012 stachys palustris 1.24 0.064 senecio ovatus 1.32 0.019 tripleurospermum inodorum 0.92 0.014 amaranthus hybridus agg. 1.23 0.044 saponaria officinalis 1.21 0.050 microrrhinum minus 0.79 0.012 chenopodium polyspermum 1.23 0.106 myosoton aquaticum 1.25 0.135 saponaria officinalis 0.84 0.010 matricaria chamomilla 1.22 0.072 solidago gigantea 1.17 0.054 hedera helix 0.76 0.009 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 3 1 8 u r b a n . v p 1 1 . l i s t o p a d 2 0 1 0 1 2 : 4 5 : 1 0 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s a c t a b o t .c r o a t .69 (2),2010 223 s y n a n t h r o p ic v e g e t a t io n tab. 3. – continued segetal vs. ruderal ax1 score fit semi-natural vs. settlement ax1 score fit town vs. village ax1 score fit veronica arvensis 1.21 0.081 fallopia dumetorum 1.16 0.032 polygonum lapathifolium 0.71 0.010 echinochloa crus-galli 1.19 0.101 poa palustris 1.07 0.028 galeopsis tetrahit 0.68 0.008 amaranthus retroflexus 1.17 0.081 petasites hybridus 1.00 0.054 diplotaxis tenuifolia 0.59 0.016 cerastium glomeratum 1.16 0.097 humulus lupulus 0.99 0.054 setaria viridis 0.57 0.010 oxalis fontana 1.08 0.083 filipendula ulmaria 0.92 0.024 hordeum murinum 0.56 0.008 anagallis arvensis 1.06 0.067 deschampsia cespitosa 0.85 0.019 lactuca serriola 0.50 0.016 veronica persica 1.06 0.131 lysimachia nummularia 0.88 0.017 oxalis fontana 0.52 0.008 setaria pumila 1.00 0.075 chaerophyllum hirsutum 0.79 0.018 conyza canadensis 0.46 0.018 galinsoga parviflora 0.99 0.060 cuscuta europaea 0.75 0.028 sonchus oleraceus 0.41 0.012 stellaria media 0.99 0.127 angelica sylvestris 0.76 0.040 picris hieracioides 0.41 0.016 fallopia convolvulus 0.94 0.077 symphytum officinale 0.71 0.032 artemisia vulgaris 0.28 0.020 chenopodium album agg. 0.81 0.118 cirsium oleraceum 0.58 0.032 medicago lupulina 0.25 0.010 convolvulus arvensis 0.81 0.113 eupatorium cannabinum 0.68 0.045 erigeron annuus 0.27 0.019 polygonum persicaria 0.80 0.066 polygala comosa 0.55 0.016 dactylis glomerata agg. –0.12 0.009 cirsium arvense 0.69 0.086 vicia cracca 0.46 0.031 poa trivialis –0.14 0.008 capsella bursa-pastoris 0.63 0.082 rubus caesius 0.33 0.021 urtica dioica –0.14 0.012 urtica dioica –0.68 0.092 calystegia sepium 0.32 0.029 galium aparine –0.20 0.008 arrhenatherum elatius –0.69 0.042 taraxacum officinale agg. –0.22 0.030 lamium maculatum –0.21 0.014 dactylis glomerata agg. –0.71 0.117 plantago major –0.30 0.019 veronica chamaedrys –0.21 0.010 heracleum sphondylium –0.73 0.054 lolium perenne –0.31 0.019 galium album –0.22 0.012 veronica chamaedrys –0.74 0.045 cichorium intybus –0.32 0.019 stachys sylvatica –0.34 0.009 artemisia vulgaris –0.76 0.067 trifolium repens –0.33 0.020 scrophularia nodosa –0.36 0.010 lamium maculatum –0.77 0.074 capsella bursa-pastoris –0.34 0.024 pimpinella major –0.45 0.010 rubus caesius –0.79 0.047 convolvulus arvensis –0.35 0.016 sorghum halepense –0.98 0.007 galium album –0.82 0.059 poa annua –0.36 0.021 petasites paradoxus –1.71 0.009 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 3 1 8 u r b a n . v p 1 1 . l i s t o p a d 2 0 1 0 1 2 : 4 5 : 1 0 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s life span vegetation of arable fields harbors mainly annual species (sutherland 2004), as they are best adapted to regular disturbance (plowing, harvesting). other ruderal types have a higher proportion of perennials, as there are habitats that are less frequently disturbed and are succesionally more developed. this is particularly evident for semi-natural vegetation and vegetation of villages. the occurrence of more annuals in towns could be explained by intensive and irregular disturbances (knapp et al. 2008) but also by ecological conditions. drought in sealed urbanized soils could be as severe as disturbance is for plant species (hill et al. 2002). life form therophytes are most common in arable land as they take advantage of disturbance, i.e. disturbance of the soil (mcintyre and lavorel 1995). chamaephytes and phanerophytes are less adapted to regular disturbance because they cannot complete their life cycle. in semi-natural land and settlement this division is not so clear except for therophytes, as in urban areas disturbance is more intense. phanerophytes are more common in semi-natural vegetation as they thrive in fringe vegetation and on shores of water bodies with less frequent disturbance. therefore successionally more developed communities are able to evolve. anthropogeneous communities in semi-natural landscape are usually in contact with natural communities that are a source of perennials. more therophytes in towns than villages have already been reported (sukopp and werner 1983, py[ek and py[ek 1990). the high proportion of chamaephytes (for example, sagina procumbens, silene vulgaris, chenopodium ambrosioides) in towns is rather surprising, but they can also be found in trampled communities with low soil disturbance. invasion status the high proportion of alien species (archaeophytes and neophytes) in disturbed habitats is consistent with several papers (kowarik 1995, hill et al. 2002, lososová et al 2006). but in other studies the pattern is different: more archaeophytes in arable land and more neophytes in settlements (lososová et al 2006). in our case we found a higher proportion of both types of alien species in the arable land. our dataset consists of all types of ruderal vegetation (annual and perennial) and this could be the reason for differences with the reported results. representation of archaeophytes and neophytes may vary considerably between different habitats (lososová et al. 2006), and the mosaic structure of some landscapes shows varying human impact with differently disturbed areas (kowarik 1995). comparing semi-natural vegetation and settlements, archaeophytes are more common in the latter, while proportions of alien species are higher in towns. strategy ruderal strategy is most common in arable land, while other ruderal communities in this dichotomous division are richer in competitors. disturbance is frequent and regular and resources are abundant in arable fields. ruderal vegetation is less intensively or infrequently disturbed and is invaded by species from natural vegetation. these species are mostly perennial herbs that exhibit csr strategy with the widest range of strategies (grime 2002). 224 acta bot. croat. 69 (2), 2010 [ilc u. u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:10 color profile: disabled composite 150 lpi at 45 degrees the strategy of plants in a semi-natural habitat (c and cs) shows that the habitat is relatively undisturbed (compared to other anthropogeneous habitats) and it is mainly competition that influences species composition. the vegetation of settlements is composed of ruderal strategists, indicating that disturbance is the main gradient. intensity and frequency of human disturbance decrease along this coenocline from trampled habitats towards ruderals in the narrower sense (mucina 1989). differences in disturbance between town and village flora again favor ruderal strategists in towns. ecological conditions and permanent disturbances in urban landscapes are factors to which r strategists are best adapted (sukopp and werner 1983, benvenuti 2004, lososová et. al. 2006). habitat preferences and species ruderal habitats are more shaded and humid than arable land, but only when we compare the whole ruderal dataset. generally, moist habitats are found in semi-natural landscapes, while urban habitats are drier and warmer (lososová et al. 2006). in this study, differences between cereals and root crops were not tested, as this has been done in previous studies ([ilc 2008, [ilc et al. 2009). but segetal species with the highest fit (table 2) are mostly from cereal fields indicating that weeds of root crops are less specialized and are found also in other ruderal habitats. this could be explained by similar disturbance types in root crops and in settlements. other reasons for floristic similarity could be differences in seasonal aspect (pinke et al. 2009) as ruderal communities and root crops are generally best developed in summer and autumn. species in towns are more lightand warmth-demanding, while in villages they thrive in more humid habitats. we found more c4 plants in towns (e.g. eragrostis minor, euphorbia maculata) that are indicators of an environment more suitable to thermophilous species (hügin 1999). we confirmed the heat island effect in urban settlements (sukopp and werner 1983), although it was not found in a similar study by lososová et al. (2006). however, their dataset consisted only of annual anthropogeneous vegetation. acknowledgements i would like to thank a. ^arni for comments in previous versions of the manuscript. the english was revised by a. mcconnell-duff. this work was supported by a grant from the slovenian research agency arrs p1-0236. references benvenuti, s., 2004: weed dynamics in the mediterranean urban ecosystem: ecology, biodiversity and management. weed research 44, 341–354. ellenberg, h., weber, h. e., düll, r., wirth, v., werner, w., pauliszen, d., 1992: zeigerwerte von pflanzen in mitteleuropa. erich goltze, göttingen. forman, r. t. t., godron, m., 1981: patches and structural components for a landscape ecology. bioscience 31, 733–740. grime, j. p., 2002: plant strategies, vegetation processes and ecosystem properties. j. wiley and sons, chichester. acta bot. croat. 69 (2), 2010 225 synanthropic vegetation u:\acta botanica\acta-botan 2-10\318 urban.vp 11. listopad 2010 12:45:10 color profile: disabled composite 150 lpi at 45 degrees hill, m. o., roy, d. b., thompson, k., 2002: hemeroby, urbanity and ruderality: bioindicators of disturbance and human impact. journal of applied ecology 39, 708–720. hügin, g., 1999: was sind wärmezeiger? untersuchungen zum wärmbedürfnis von ruderalund segetalpflanzen in mitteleuropa. tuexenia 19, 425–446. klotz, s., kuhn, i., durka, w., 2002: biolflor-eine datenbank mit biologisch-oekologischen merkmalen zur flora von deutschland. schriftenreihe fuer vegetationskunde 38, 1–334. knapp, s., kühn, i., wittig, r., ozinga, w. a., poschlod, p., klotz, s., 2008: urbanization causes shifts in species’ traits state frequencies. preslia 80, 375–388. kowarik, i., 1995: on the role of alien species in urban flora. in: py[ek, p., prach, k., rejmanek, m., wade, m. 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(ed.), man’s impact on vegetation, 7–24. w. junk publishers, the hague. zobel, m., 1997: the relative role of species pools in determining plant species richness: an alternative explanation of species coexistence? trends in ecology and evolution 12, 266–269. zupan^i^, b., 1995: climatography of slovenia. precipitation: period 1961–1990 (in slovenian). hidrometeorolo{ki zavod republike slovenije, ljubljana. acta bot. croat. 69 (2), 2010 227 synanthropic vegetation u:\acta botanica\acta-botan 2-10\318 silc.vp 13. listopad 2010 11:53:45 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 78 (1), 2019 17 acta bot. croat. 78 (1), 17–24, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0002 issn 0365-0588 eissn 1847-8476 morpho-chemical divergence and fatty acid profile of shea tree seeds (vitellaria paradoxa) collected from different locations in kwara state, nigeria david adedayo animasaun1*, stephen oyedeji1, kehinde stephen olorunmaiye1, musibau a. azeez2*, idowu abdulfatah tijani3, joseph akintade morakinyo1 1 department of plant biology, faculty of life sciences, university of ilorin, ilorin, kwara state, nigeria 2 department of pure and applied biology, ladoke akintola university of technology, ogbomoso, oyo state, nigeria 3 department of chemical engineering, university of ilorin, ilorin, kwara state, nigeria abstract – the present study characterizes seed-related traits, phytochemical, physiochemical parameters and fatty acid profile of shea (vitellaria paradoxa) seeds collected from the kosubosu, fufu and sare areas of kwara state, nigeria to determine the effects of microclimate on seed morphology, biochemical and oil constituents. seed morphological data were analyzed for variability. seed oil was extracted for phytochemical constituents, physicochemical properties, and fatty acid profiling by gas chromatography equipped with mass spectrometry (gc/ms). results showed intra and inter-locational variations in seed characters. most fruits had 1–2 seeds. seeds were predominantly brown and very few were dark brown. phytochemicals and physicochemical parameters of the seed oil varied with place of collection. alkaloid, saponin, tannin and phytate contents ranged between 0.79–0.84, 1.20–1.26, 1.48–1.56 and 0.15–0.18 mg g–1 respectively. the density of the oil was less than that of water, acid value ranged from 10.58–13.56 mg koh g–1 and iodine values were between 36.63 to 40.32 g i2 (100 g)–1. saponification values lie between 160.39 and 184.14 mg koh g–1; and free fatty acid was within 5.32–6.81 %. peroxide, ɑ-tocopherol, total phenol and oxalate values as well as viscosity of the oil also varied; however, refractive index was similar. ethyl oleate and octadecanoic acids were present and most abundance in all the locations, while glycidol stearate was only found in fufu samples with three other fatty acids. five fatty acids were present in kosubosu, while sare had only two. the results obtained in the present study indicate that shea oil could be used for medicinal, nutritional and industrial purposes. since seed characters, phytochemical, physicochemical and fatty acid compositions varied with the microclimate, environmental and micro-ecological conditions should be considered when collecting seeds for oil utilization. keywords: fatty acids, phytochemical analysis, physicochemical parameters, shea butter, vitellaria paradoxa * corresponding authors: animasaun.ad@unilorin.edu.ng, musibau_azeez@yahoo.com introduction vitellaria paradoxa gaertn. f., commonly known as the shea tree is a tree of the sapotaceae family indigenous to sub-sahara africa where it grows in the wild and has huge economic and ecological potentials (ademola et al. 2012). the tree occurs in west african and is popular among the local dwellers for its numerous social and economic utilizations (kobomoje et al. 2013). the shea tree grows wild across a wide belt of savanna including west african countries of senegal, mali, cote d’ ivoire, burkina faso, togo, ghana, benin, nigeria, niger and cameroon. the tree is also found further to the east, in uganda, sudan and ethiopia (maranz et al. 2004, goreja 2004, masters et al. 2010). among these countries, ghana and burkina faso are the main shea net exporters (walter et al. 2003). in nigeria, the shea tree is known as “emi” among the yoruba people of the western part. the shea grows up to 9–12 m in height with profuse branches. commercial fruiting quantity usually commences at approximately 20 years and the tree can continuously produce shea fruit for many more years (alander 2004). late maturity of the tree has been the bane of its commercial plantation and shea butter related industries depend on nuts collected from scattered, naturally growing shea trees. in nigeria, the animasaun d. a., oyedeji s., olorunmaiye k. s., azeez m. a., tijani i. a., morakinyo j. a. 18 acta bot. croat. 78 (1), 2019 shea is found in the guinea savanna with high tree concentrations in niger, kwara, kebbi and kaduna states (warra 2011). shea trees blossom between february to march, and the fruit matures and falls in may-june. the fruit is ellipitic, yellow green or light green, about 5–8 cm in circumference and similar to the fig (chalfin 2004). the fruit has a butterlike, mucous pericarp covering an oval brown or light brown seed surrounded by a fragile shining shell with a large hilum on a broad base (olaniyan et al. 2007). usually, a shea fruit contains one seed but occasionally has two to three oil-rich nuts (alander 2004). the shea tree has the potential to improve nutrition, boost healthcare, reduce rural poverty and support sustainable land care (moore, 2008). the fruit pulpy pericarp can be eaten while the nut is discarded or crushed for shea butter extraction. the fat obtained from the shea nut known as shea butter (ori: yoruba; nigeria), which contains 40–60% oil, is the most valued product from the shea tree (warra and komo, 2014). shea butter is used for local domestic purposes such as cooking, lightning, soap making, skin moisturizer and cosmetics as well as traditional medicine among the local population where the tree grows in abundance in nigeria. also, it is used to treat wounds, suppress inflammation, relieve rheumatic and joint pains and other health conditions (kraft and lynde 2005, soro et al. 2011, warra and komo 2014). the potential of shea oil to protect the skin from damaging ultra violet rays was demonstrated by velasco et al. (2008). on a global scale, the importance of the shea tree is related to the usefulness of its seed fat in food and cosmetic industries. decolourised shea butter could be used as a cheaper alternative to cocoa butter due to their similar physical and chemical properties (lipp et al. 2001, alander 2004). the high levels of unsaponifiable matter in shea nut oil compared to other vegetable fats could be exploited for the development of more shea butter products (rogers and lenick 2009). shea oil finds applications in traditional and social rituals such as marriages, funerals, coronations and rainmaking (ferris et al. 2004, gwali et al. 2011, hall et al. 1996, moore 2008). also, high grade charcoal and furniture are made from the wood while the latex yields a considerable amount of glue (lovett and haq 2000). consequently, the shea tree could generate significant incomes for the rural households. however, despite the enormous economic and healthcare potentials, shea butter and its products are underutilised in nigeria and only little information is available on the seed variability. in particular, not much work has been done on locational variation of seed characters in relation to the nut phytochemicals and fatty acid characterization of seeds from different locations. the present work was carried out to characterize seed related traits, quantify phytochemical constituents of the nut and profile the fatty acids of the shea seed collected from different locations in kwara state, nigeria. this study hopes to identify existing variations in the shea seeds from different locations using seed and oil characteristics to throw more light on how microclimate affects seed morphology and chemical constituents in shea tree. materials and methods fruit and seed collection vitellaria paradoxa fruits were collected from three locations in kwara state, nigeria, because of the abundance of shea trees, the folkloric processing and utilization of shea butter for socio-economic purposes. kwara state is located in the southern guinea savanna belt of nigeria, which is dominated by grasses interspersed mainly with tree species such as daniellia oliveri, vitellaria paradoxa, prosopis africana and parkia biglobosa. the state is within the coordinates 8°30'0.00"n and 4°32'32"e and at an elevation of 303 m above sea level (ngia, 2016). the names and coordinates of the three locations from where shea fruits and seeds are collected are shown in table 1. the soil was mainly sandy, lateritic or ferralitic, mean annual rainfall is 640–1350 mm. usually, the rainy season lasts from may to october and dry season from november to april. relative humidity is 75–80% in the wet season and about 65% during the dry season (ajadi et al. 2011). the average monthly temperature is 31.4–33.5 °c, but fluctuates between 33 and 34 °c from november to january and 34–35 °c (february to april) (ilorin atlas, 1982; nimet, 2016). tab. 1. names, local division, coordinates and elevations of the locations from where shea fruits and seeds were collected for the study in kwara state of nigeria. location name local government area coordinates altitude (m) kosubosu baruten 9°35'n, 3°15'e 273-364 fufu ilorin west 8°30'n, 4°32'e 300-310 sare ifelodun 9°40'n, 3°22'e 370-418 fruit and seed morphological studies ten mature fruit bearing trees were selected at each location (kosubosu, fufu and sare) from may to july, 2016. each location has five sites from which two fruiting trees were selected. thirty ripe fruits were randomly plucked from each tree for characterization. epicarp thickness and seed diameter were determined using an electronic vernier calliper (atd-8656). number of seed per fruit, seed dimension and seed coat colour were also recorded; the mean values of ten readings were used for the seed characters. shea nuts oil extraction the pulpy pericarp of the fruits were removed, the seeds cleaned and sun dried for two weeks. the dried seeds were deshelled, the kernels were ground and kept in airtight containers prior to oil extraction. oil extraction was carried out in soxhlet apparatus using n-hexane as solvent. 50 g of the ground material was transferred to a 30 mm × 200 mm cellulose thimble placed in the extraction chamber of a 250 ml soxhlet apparatus fitted with a condenser on a 500-ml distillation flask containing 250 ml of n-hexane solvent. shea nut oil was extracted under reflux with n-hexane following variation in shea seeds from different locations acta bot. croat. 78 (1), 2019 19 the procedure described by ali et al. (2015). after extraction, hexane was removed by using a heated rotary evaporator (stuart, england), under vacuum conditions to recover the oil. extractions of oil from seeds of different locations were performed in triplicate, and the mean values were reported. oil yield was expressed as percentage weight of oil obtained relative to the weight of shea butter used for extraction according to warra et al. (2011). phytochemical and physicochemical properties determination methanolic extracts of the shea nut were screened for presence or otherwise of bioactive compounds using standard procedures (sofowora 1993, kokate 1999, kumaran and karunakaran 2006). the amount of tannin was estimated using the method described by hagerman et al. (2000), saponin, and alkaloid present in the shea nut extract were determined according to procedure of obadoni and ochuko (2001). total phenol, oxalate and phytate contents in the extract were quantified according to the methods of yadav et al. (2011) and falana et al. (2016), while the amount of α-tocopherol was determined by the standard procedure of he association of official analytical chemists (2010). iodine values (iv) of the shea oil were estimated using the method of gafar et al. (2012). the value was calculated from fatty acid methyl ester compositions of oil. saponification value was calculated from fatty acid methyl ester compositions of oil using the equation of kalayasiri et al. (1996). determination of the acid value, which is the amount of carboxylic acid groups in the oil, was carried out based on a titration method using the aoac (2010) procedure. viscosity of the shea butter oil was recorded using an ostwald viscometer (dv-iii ultra, uk). the free fatty acid of the oil was estimated by placing 0.2 g of oil into 250 cm3 erlenmeyer flask, 100 cm3 of ethanol was added and followed by 2 cm3 of phenolphthalein indicator. the mixture was titrated against 0.1 m naoh till endpoint (slight pink colour that persists for 30seconds), the free fatty acid was calculated according to chopra and kanwar (1991). to determine the peroxide value of the oil, 2.0 g of oil was added to 22 cm3 of a solution containing 12 cm3 chloroform and 10 cm3 acetic acid. 0.5 cm3 saturated potassium iodide was added and allowed to stay with intermittent agitation for 1 minute before 30 cm3 distilled water was added. the mixture was titrated against 0.1 m of na2s2o3 in the presence of starch indicator until the blue colour had just disappeared. the peroxide value determination was carried out in accordance with neilson (2003). refractive index of the oil with reference to air was measured with the abbe refractometer (mettler-toledo, usa). the specific gravity of the oil relative to water was measured by hydrometer. fatty acid profiling fatty acid composition of the oil was determined at the chemical engineering laboratory, university of ilorin, nigeria by a gas chromatograph equipped with a mass spectrometer (gc/ms; agilent technologies; model: 7890a) with hp column, 30 cm long × 0.32 mm id × 0.25 μm thickness film (sge, australia). the gc is fitted with hewlett packard (usa) flame ionization detector (fid) and 99.9% helium was used as carrier gas. the oil triacylglycerides (tg) was converted to fatty acid methyl ester (fame) to decrease the boiling point. 0.2 ml of biodiesel was added to a 1 ml mixture consisting of hexane and 2-propanol (4/5 volume ratio). before charging the oil, the samples were purified by filtration using 0.2 μm polytetrafluoroethylene syringe micro filter, then 1 μl of the sample was injected into the gc/ms using a 5 μl micro syringe (sge, australia). the gas chromatography condition was continuous flow mode. the inlet temperature was 250 °c while the oven temperature was 250 °c. the injection volume was 1 µl and the split ratio 1:50. the procedure was run for 20 minutes with an average velocity of 37.789 cm s–1 at 17.04 psi pressure. on the other hand, the mass spectrometry was run under transfer line temperature of 240 °c, source temperature at 220 °c. the solvent delay was for 2 minutes while the scan mass range was 45–500. data analysis was conducted with gc/msd chemstation integrator software. results the fruits and seeds of the shea tree collected from three selected local government areas of kwara state, nigeria varied in number of seed/fruit, pericarp thickness, seed coat colour and seed dimension as well as phytochemical and fatty acid constituents. among the 300 shea fruits (30/tree) collected from kosubosu, baruten local government area, 265 (88.33%) had 1 seed/fruit, 34 were 2-seeded and only one fruit had 3 seeds (tab. 2). the average ratios of seeds to fruit were 26.5, 3.4 and 0.1 for 1-seed, 2-seed and 3-seed fruits respectively. in addition, 164 of the seeds had brown seed coats, 112 were light brown while 24 were dark brown. the fruits collected from fufu (ilorin west local govt area) are predominantly one seeded. 254 (84.66%) of the fruits were 1-seed, 42 had 2 seeds per fruit and 4 fruits were found with 3 seeds each (tab. 2). the seed colour distribution showed 129 light brown seeds, 121 brown and 50 dark brown seed coats. similarly, 1-seed fruits occurred most among the fruits collected from sare (ifelodun local govt area). 252 out of the 300 sampled fruits had 1 seed, 2 seeds per fruit were found in 44 and 3 seeds occurred in 5 fruits. brown seeds were the most frequent (160) followed by light brown (119) and the few (21) dark brown seeds were from sare samples. the mean epicarp thickness ranged from 2.77–3.48 mm in fruits collected from kosubosu (fig. 1a). mean seed diameter and circumference ranged between 2.41 to 3.95 cm and 6.44 to 7.46 cm, respectively. similarly, seed lengths among the kosubosu seed population were alike. considering fruit and seed characters, fufu fruits had mean pericarp thickness between 2.88–3.36 mm (fig. 1b). the seed diameter and circumference were smaller than those of kosubosu and ranged between 2.41 and 87 cm and 5.66 and 7.10 cm respectively. the greatest seed length was 4.96 cm while the least was 3.88 cm. seed diamanimasaun d. a., oyedeji s., olorunmaiye k. s., azeez m. a., tijani i. a., morakinyo j. a. 20 acta bot. croat. 78 (1), 2019 nol and alkaloids were found in nuts from kosubosu which invariably had the least amount of ɑ-tocopherol. the order of the amount of ɑ-tocopherol in the samples was sare>fufu>kosubosu while saponin content was highest (1.26 mg g–1) in samples from sare, followed by kosubosu (1.21 mg g–1) and the least (1.20 mg g–1) in fufu. in contrast, fufu samples produced the highest amount of oxalate (0.82 mg g–1) as against 0.80 mg g–1 from kosubosu and 0.78 mg g–1 obtained from sare samples. eter and circumference were similar for the fruits collected from 10 different trees in sare, but pericarp thickness and seed length varied (fig. 1c). the fruits and seeds are smaller than those of kosubosu and fufu areas. the average pericarp thickness, seed diameter and length were between 2.61 and 3.37 mm, 2.47 and 2.66 cm, 5.02 and 5.82 cm respectively, however, seed circumference varied from 7.02–7.70 cm. the phytochemical compositions of the shea nut are presented in table 3. highest phytate, tannin, total phefig. 1. morphological seed related characters of shea tree seeds collected from different locations in kwara state, nigeria: (a) kosubosu, baruten, (b) fufu, ilorin west, (c) sare, ifelodun. tab. 2. morphological characteristics of shea tree seeds collected from selected locations in three local government areas of kwara state, nigeria. tn – number of tree, nfs – number of seeds collected, dbdark brown, lblight brown, b – brown. tn nfs kosubosu fufu sare no of seed/fruit seed colour no of seed/fruit seed colour no of seed/fruit seed colour 1 2 3 db lb b 1 2 3 db lb b 1 2 3 db lb b 1 30 29 1 0 13 17 22 7 1 6 13 11 13 16 2 1 13 16 2 30 24 6 0 7 11 12 26 3 1 0 22 8 29 1 0 3 10 17 3 30 30 0 0 3 3 24 28 2 0 0 14 16 28 2 0 3 7 20 4 30 30 0 0 4 9 17 30 0 0 25 4 1 29 0 1 4 9 17 5 30 30 0 0 0 15 15 23 7 0 3 10 17 25 5 0 0 14 16 6 30 14 15 1 10 5 15 26 4 0 1 4 25 28 2 0 3 8 19 7 30 29 1 0 0 21 9 28 2 0 0 19 11 21 8 1 1 21 8 8 30 28 2 0 0 19 11 24 5 1 4 18 8 26 3 1 2 18 10 9 30 22 8 0 0 3 27 28 2 0 7 14 9 23 7 0 0 8 22 10 30 29 1 0 0 13 17 19 10 1 4 11 15 30 0 0 4 11 15 ʃ 300 265 34 1 24 112 164 254 42 4 50 129 121 252 44 5 21 119 160 mean 30 26.5 3.4 0.1 2.4 11.2 16.4 25.4 4.2 0.4 5.0 12.9 12.1 25.2 4.4 0.5 2.1 11.9 16.0 variation in shea seeds from different locations acta bot. croat. 78 (1), 2019 21 percentage oil yield of the shea nut was highest (53.06%) for kosubosu, followed by sare (49.26%) and the least in fufu (47.31%) (tab. 4). the specific gravity and refractive index of the oil from the different locations were similar, but the least acid value, iodine value and free fatty acid were recorded for kosubosu samples. saponification value was higher (184.14 mg koh g–1) in the sare sample than in the kosubosu (182.15 mg koh g–1) but a much lower value was obtained for fufu (160.39 mg koh g–1) which invariably had the highest viscosity. furthermore, the order of peroxide values in the shea oil was kosubosu>sare>fufu (tab. 4). table 5 shows the retention time, types and percentage composition of the fatty acids present in shea nut oils. five fatty acids were found in kosubosu samples, four in fufu and only two in sare. 2-hydroxyl-1(hydroxymethyl) ethyl ester with percentage compositions of 38.89 and 47.43% was the most abundant fatty acid in kosubosu and fufu samples respectively; it was however not found in sare samples. ethyl oleate constituted 59.96% of the total fatty acids in the sare sample and octadecanoic acid accounted for 40.04%. while ethyl oleate and octadecanoic acids were common to all the locations, hexadecanoic acid was present in a trace amount (1.36%) only in kosubosu. discussion analysis of seed/fruit revealed that fruits from different locations are predominantly one-seeded, a few had 2 seeds per fruit, and 3 seeds per fruit are rare. although, shea fruit is a berry, occurrence of more than two seeds in a fruit is a rare phenomenon. the higher frequency of one-seeded fruit in kosubosu than in other locations may be due to environmental and/or edaphic variations. this observation aligned with the seed number variation among fruits from the same tree as reported by abbiw (1990) and djekota et al. (2014) who claimed that 2–3 seeds were found in some shea tab. 3. phytochemical composition of shea butter from different locations in kwara state, nigeria. phytochemical constituent location kosubosu fufu sare phytate (mg g–1) 0.18 0.15 0.16 tannins (mg g–1) 1.56 1.50 1.48 oxalate (mg g–1) 0.80 0.82 0.78 total phenol (mg g–1) 0.52 0.50 0.49 α-tocopherol (mg g–1) 26.38 28.60 34.42 alkaloid (mg g–1) 0.84 0.79 0.80 saponin (mg g–1) 1.21 1.20 1.26 tab. 4. oil yield and physicochemical parameters of extracted shea butter oil from different locations of kwara state, nigeria. physicochemical property location kosubosu fufu sare oil yield (%) 54.60±2.71 46.35±2.18 49.25±2.63 specific gravity (g cm-3) 0.90±0.01 0.90±0.01 0.90±0.01 acid value (mg koh g-1) 10.58±0.70 13.56±0.94 13.16±0.91 iodine value (g i2 (100 g)-1) 36.63±2.67 40.32±3.52 39.28±2.91 free fatty acid (% oleic acid) 5.32±0.49 6.81±0.53 6.60±0.56 reflective index 1.46±0.09 1.46±0.09 1.46±0.09 saponification value (mg koh g-1) 182.15±3.18 160.39±2.90 184.14±3.14 peroxide value (meq kg-1) 5.20±0.07 4.10±0.08 4.82±0.04 viscosity 2.60±0.02 2.70±0.01 2.61±0.02 tab. 5. chemical composition of the extracted shea nut oil from different locations in kwara state, nigeria. location compound name retention time (min) % composition kosubosu hexadecanoic acid, ethyl ester 33.175 1.36 ethyl oleate 36.267 20.89 octadecanoic acid, ethyl ester 36.768 15.78 2-hydroxyl-1(hydroxymethyl) ethyl ester 41.262 38.80 2-hydroxyl-1,3-propanediyl ester 41.685 23.18 fufu ethyl oleate 36.267 7.21 octadecanoic acid ethyl ester 36.768 4.25 2-hydroxyl-1(hydroxymethyl) ethyl ester 44.306 47.43 glycidol stearate 41.729 41.13 sare ethyl oleate 36.278 59.96 octadecanoic acid ethyl ester 36.796 40.04 animasaun d. a., oyedeji s., olorunmaiye k. s., azeez m. a., tijani i. a., morakinyo j. a. 22 acta bot. croat. 78 (1), 2019 fruits collected from different locations of mondoul in the chad republic. fruit and seed morphological parameters such as pericarp thickness, petiole length, seed dimensions were used to partition populations of shea tree (djekota et al. 2014). seed coat colours were mainly in three categories (dark brown, light brown and brown), but while light brown and brown seeds occurred frequently, only very few seeds had dark brown coat for fruits obtained from same tree in a location. variation in seed colour observed in the present study matched previous findings on shea nut seed coat (nafan et al. 2007, lamien et al. 2007). since seed formation involves meiosis, coat colouration in this case is most likely genetic and can be caused by mutation or recombination of genes. also, these variations can be explained by natural and/ or human selection and gene flow mediated from genetic drift (tremblay et al. 2010, abasse et al. 2011). differences recorded in quantitative seed characters intra and inter-locational in the present study concurred with the findings on tamarindus indica (soloviev et al. 2004). furthermore, significant intra and inter-locality variations have been demonstrated in shea tree and seed characters (mbaiguinam et al. 2007, gwali et al. 2011). the phytochemical constituents varied with locations, the variations could be accounted for by environmental stress, rainfall regimes and soil characteristics as opined by sanou et al. (2005). amount of phytochemicals were less than those obtained by falana et al. (2016) who worked on v. paradoxa collected from onipako village, ilorin and this could be due to locational differences. the presence of similar phytochemicals has been reported in other tropical plants in nigeria and some of them exhibit varying biological activities (sofowora 1993, onwuliri 2004). the oil yield varied with locations, and the higher amount of oil obtained from the kosubosu seeds could be explained by the same factors that account for the morphological and biochemical compositions. the yield of the oil from the study locations were in accordance to the values earlier reported (warra and komo 2014). the present study revealed that in terms of oil yield, kosubosu samples are the best. physicochemical parameters are of great importance in determination of oil quality. pure oils have marked ranges of specific gravity and refractive index; thus the degree of variation of typical oil from its true values may indicate its relative purity. the specific gravity of the extracted shea nut oil was less than 1.0 g cm–3, which implies the oil is less dense than water. the specific gravity of 0.902 g cm–3 obtained in this study correlates with the 0.90 g cm–3 documented by raimi et al. (2014) and is congruent with those of persea macrophylla (0.89 g cm–3) and persea gratesima (0.90 g cm–3) (akubugwo et al. 2008). furthermore, the refractive index (1.468), which was the same for the locations, was also the same as the value reported by raimi et al. (2014) and similar to anacandium occidentlis oil (1.458) (akinhanmi et al. 2008) and walnut kernel oil (1.534) (ozcan et al. 2010). the iodine value of the oil suggests it is a typical nondrying oil containing saturated and a low level of unsaturated fatty acid. hence, the oil may be utilized for vegetable oil-based ice cream manufacturing, but, the non-drying nature of the oil makes it not applicable for paint and varnish production. shea oil from fufu has low saponification value compared with the other two locations. the range of saponification indicates that the oil may be useful in making soap and shampoo (ugbogu et al. 2013). low acid value of the shea nut oil although higher than value reported by raimi et al. (2014) was far less than of livistona. chinensis (nwosu et al. 2012) and this signify it could be used as an edible oil. the free fatty acid which is also an indicator of oil edibility was low and close to the range reported by ugbogu et al. (2013). this suggests a low level of hydrolytic and lipolytic activities in the oil, thus, the extracted oil could be used as raw materials for industries (obasi et al. 2012). analysis of the fatty acid profile of shea nut oil revealed the presence of 6 fatty acids in varying quantities. it is of interest to note that while five fatty acids were found in kosubosu samples, four were present in fufu and only two in sare samples. ethyl oleate and octadecanoic acids were present in all the samples irrespective of their locations. this result buttresses an earlier report that variation exists in fatty acid composition of same seed oil from different locations (wara, 2015). ethyl oleate acid which is the most abundant in the extracted shea nut oil is an unsaturated fatty acid, naturally present in most seed oil and can be used for lotion and pharmaceutical solvents (pubchem, 2014). the variation in chemical constituents and fatty acid composition of the studied samples may be accounted for by environmental variations (sanou et al. 2005, mbaiguinam et al. 2007). this explains why 2-hydroxyl,3-propanediyl ester, was only present in kosubosu samples while glycidol stearate occurred only in fufu samples. hexadecanoic acid ethylester (palmitic acid), used mainly for soaps, cosmetics and release agent production, was present only in kosubosu shea nut oil. thus, considering the fatty acid composition of the shea nut oil, its industrial and domestic application potentials, locational variation should be taken into consideration in collecting seeds for oil production. the present study revealed that shea tree seeds from kosubosu had a higher yield in term of quantity and quality of shea nut oil than the other two locations. conclusion the present study showed that morphological variations exist within and among locations in the seed characters of v. paradoxa from different areas of kwara state in central nigeria. also, from the results, shea nut oil phytochemical constituents and physicochemical parameters varied with locations of seed collection. the fatty acid also varied with locations and the presence of vital fatty acids in a large percentage in the oil enhances the potential of shea oil as a candidate oil for industrial revolution and rural economy development. however, in sourcing for shea seed for butter and oil production, locational variations should be taken into consideration as important factors that could affect oil quantity and quality. the present study can be extended to other regions where the variation in shea seeds from different locations acta bot. croat. 78 (1), 2019 23 trees are endemic and the molecular approach could be used for a better understanding of the existing variability among the shea tree and nut populations. references abasse, t., weber, j., katkore, b., boureima, m., larwanou, m., kalinganire, a., 2011: morphological variation in balanites aegyptiaca fruits and seeds within and among parkland agroforests in eastern niger. 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ecology, university of £ódÿ, banacha 12/16, 90-237 £ódÿ, poland 2 centre for medical biology polish academy of sciences, lodowa 106, 93-232 £ódÿ, poland 3 university of £ódÿ, department of cytogenetics and plant molecular biology, banacha 12/16, 90-237 £ódÿ, poland nine taxa of potentilla species from poland representing p. sect. terminales (dõll.) gren et godr. and p. sect. aureae (wolf) juz. were analyzed via a series of random amplified polymorphic dna (rapd) analyses to test (1) the hypothesis that the six species representing p. subsect. collinae juz. of the p. terminales sect., i.e. p. collina wibel, p. thyrsiflora zimmeter, p. wimannania günther et schummel, p. leucopolitana p. j. müll., p. ´gabarae kolodziejek, p. koernickei zimmeter, are genetically differentiated enough to be considered as separate taxa, and (2) the position of populations of p. thyrsiflora and p. collina with respect to the terminales sect. (p. argentea l) and the aureae sect. (p. tabernaemontani ascherson and p. incana p. gaertner, b. meyer et scherb.). rapd-based genetic similarity values using the upgma method and corresponding dendrogram exhibited incomplete accordance between rapd and morphological variations. according to 'overgenomic' associations based on a series of genomic loci selected at random, p. thyrsiflora and p. collina are closely related and similarly related to the species: p. argentea, p. tabernaemontani and p. incana hypothesized as being 'parental'. within terminales sect., our dendrogram shows p. argentea to be relatively isolated from the other members of the section analysed, p. thyrsiflora and p. collina. key words: average similarity, multilocus diversity, potentilla, rapd, rosaceae, taxonomy abbreviations: rapd – random amplified polymorphic dna; upgma – unweighted pairs group method using mathematical averages introduction the genus potentilla l. comprises approximately 490 species distributed mainly in the northern hemisphere (eriksson et al. 1998, soják 2005). within potentilla seven subgenacta bot. croat. 69 (1), 2010 71 * corresponding author, e-mail: kolo@biol.uni.lodz.pl u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees era have been distinguished, i.e. schistophyllidium juz., micropogon bge., fragariastrum ser., closterostyles torr. et gr., hypargyrium fourr., dynamidium fourr. and argentina (lam.) jepson (juzepczuk 1941, soják 2005). this study included seven species of the p. sect. terminales (dõll.) gren. et godr. (p. argentea and six species of p. subsect. collinae) and two species of the p. sect. aureae (wolf) juz. (p. tabernaemontani ascherson and p. incana p. gaertner, b. meyer et scherb.) within p. subgen. hypargyrium. in europe, a total of 25 taxa have been described in the p. subsect. collinae, most of them being not very well defined (as separate species, subspecies or varietas). this divergence in taxonomic viewpoints is accompanied by problems in distinguishing them and mistakes in the use of synonyms. species identification strictly on the basis of morphological features is however difficult, because all species are closely related and morphologically similar. the high degree of polymorphism is partly caused by apomictic reproduction (pseudogamy) in conjunction with polyploidy and hybridization, as first pointed out by müntzing (l928, 1931). in poland, there are 12 taxa of p. subsect. collinae, i.e. p. collina wibel, p. thyrsiflora zimmeter, p. silesiaca r. uechtr., p. wimannania günther et schummel, p. leucopolitana p. j. müll., p. microdons schur, p. schultzii f. w. schultz, p. koernickei zimmeter, p. leucopolitanoides b³ocki, p. scholziana callier, p. tynieckii b³ocki and p. ´gabarae ko³odziejek (p. leucopolitana p. j. müll. ´ p. incana p. gertner, b. meyer et scherb) (ko£odziejek unpublished). since it is widely accepted (ascherson and graebner 1904, wolf 1908, asker and frõst 1970, kurtto et al. 2004) that the species of p. subsect. collinae has been derived from natural hybridization between different potentilla species, we attempted to use rapd analysis to deduce the putative parents of this species. in morphology, the taxa of p. subsect. collinae shows traits both from the terminales (p. argentea) and the aurea sections (p. tabernaemontani and p. incana). the characteristic features of the p. subsect. collinae are primarily style conical shape with a few papillae at base, slightly clavate at apex; leaflets 5–7 oblong-obovate or oblanceolate, variously toothed, sparsely pubescent to white-tomentose or sericeous beneath with mixture of four types of hairs, i.e. crispate, straight, curved and imperfectely stellate. in contrast, p. argentea has a subcylindrical style, a dense crispate indumentum on the lower side of leaves and leaves with inrolled margins. the p. subsect. collinae differs from the p. tabernaemontani and p. incana by its mixed (straight, curved, flexuous and incomplete stellate) hairs on the upper and lower side of the leaves. p. tabernaemontani have only straight hairs. from p. incana it differs by the lack of the stellate hairs. apart from by the different indumentum, the members of the collinae subsect. can easily be separated from species of the aureae sect. (p. tabernaemontani and p. incana) by their style shape. in species of the aureae sect., the styles towards the apex are widened. the members of the collinae subsect. occurs in isolated sites in the plains of poland and have slightly different habitats. the species prefer open habitats, mostly on dry sandy soils and calcareous rendzinas. potentilla argentea and p. incana can be found growing abundantly on dry mineral soils over the whole territory, while p. tabernaemontani occurs locally on dry soils in west, central and south poland. however, in a few localities, for instance on the wy¿yna czêstochowska upland the seven taxa, i.e. p. collina, p. thyrsiflora, p. wimannania, p. leucopolitana, p. tabernaemontani, p. incana and p. argentea occurred side by side on gravelly or sandy, not or only slightly calcareous ground (ko£odziejek 2004). 72 acta bot. croat. 69 (1), 2010 ko£odziejek j., cieœlikowski t., sakowicz t. u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees in the present paper we explore the value of a detailed, molecular (rapds) approach for unravelling complex variation at low taxonomic level, in order to use this approach to (1) test the hypotheses whether the morphologically defined species of the collinae subsect. represent genetically distinct units. (2) secondly, we consider the position of p. thyrsiflora and p. collina with respect to the terminales (p. argentea) and aureae sections (p. tabernaemontani and p. incana), in order to obtain a better taxonomic classification of potentilla. material and methods plant material and genomic dna isolation in this study, we investigated six species of p. subsect. collinae, i.e. p. collina, p. thyrsiflora, p. wimannania, p. leucopolitana, p. koernickei, p. ´gabarae and a group representing the terminales and aureae sections, i.e. p. argentea, p. tabernaemontani and p. incana considered (ascherson and graebner 1904, wolf 1908, asker and frõst 1970) as the potential parental species for the group. the geographic localities and information on the population samples are given in table 1. some of the plants investigated were preserved in the form of herbarium specimens and deposited in the department of geobotany and acta bot. croat. 69 (1), 2010 73 multilocus genomic associations of potentilla tab. 1. collection data from potentilla material sampled from cytogenetic analyses. the chromosome counts after ilnicki and kolodziejek (2008). population code taxon 2n sample collection site a p. collina 28,35 silesia prov., k³obuck 50°57’n/18°59’e b p. collina 28,35 silesia prov., rzêdkowice 50°35’n/19°27’e c p. collina 28,35 silesia prov., mirów 50°37’n/19°28’e d p. thyrsiflora 21,28,35 silesia prov., cisowa near pilica 50°26’n/19°42’e e p. thyrsiflora – silesia prov., ostra góra near siewierz 50°27’n/19°09’e f p. wimannania 35 silesia prov., mirów 50°37’n/19°28’e g p. leucopolitana 35 pomerania prov., cha³upy 54°43’n/18°23’e h p. ´gabarae – silesia prov., jaroszów village near ¯arki 50°39’n/19°21’e i p. koernickei 28,35,42 pomerania prov., czarna woda near czersk 53°51’n/18°08’e j p. argentea 35 silesia prov., cisowa near pilica 50°26’n/19°42’e k p. argentea 35 £ódÿ prov., bolimów landscape park 52°05’n/20°10’e l p. tabernaemontani – ma³opolska prov., szaflary 49°25’n/19°33’e m p. tabernaemontani – silesia prov., jaroszów village near ¯arki 50°39’n/19°21’e n p. incana 28 ojców nationale park 50°14’n/19°50’e o p. incana 28 sl¹sk prov., kusiêta village nar olsztyn 50°45’n/19°16’e u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees plant ecology of the university of lodz. others were transplanted into the experimental garden for further studies. the selected species of collinae are the most common taxa of the subsection occurring in poland. two of them, i.e. p. collina and p. thyrsiflora are encountered in relatively the greatest number of localities and their populations are the richest, consisting of more than fifty individuals. the relative abundance and incidence of the accessions observed for these two species suggests more frequent genetic material exchange between them and the species of the terminales and aureae sections. the studied plant material was collected during field trips in poland. for rapd analysis we used five plants representing each population. each plant selected was separated from another by at least 0.5 m. all of them were dug in may – july. leaves were dried in fine-grained silica gel. the samples of the plant genomic dna were extracted from approximately 0.25 g of fresh and young leaves from field-collected plants. the frozen material, prior to the cell-lysis, was disrupted by grinding in liquid nitrogen, then digested by rnase-a in a lysis buffer at 65°c. the extraction itself, performed using dneasy plant mini kit (qiagen), was followed by the spectrophotometrical quantification at 260 nm. the uv-spectrophotometer hitachi u-2000, japan was used for determination of dna purity and concentration. similar to the other experimental work (e.g. román et al. 2003, sakowicz and cieœlikowski 2006), ours followed the template-mixing strategy where equal amounts of working solution dnas from each group of individuals of the same species were pooled as the 'species-template dna' prior to the pcr reaction. dna was extracted from single plants and each population was represented by the bulk of a variable number of individuals, depending on the availability of samples after collection. that was to increase the relative contribution of the common target sequences and consequently to highlight the species-specific features (amplicons) at the step of amplification, before starting numerical analysis. rapd amplification the random polymorphic dna amplification was performed according to the subtle modified (temperature profile) procedure of williams at al. (1990). the reaction mixture included 10 mm tris-hcl ph 8.3, 50 mm kcl, 2 mm mgcl2 (finnzyme, finland), 0.2 mm dntp (promega, usa). the aliquot of 25 ml contained: the reaction mixture, 30 ng of primer (sigma-ark, germany), 5 ng of genomic dna and 2 units of termostable polymerase dynazymeä(finnzyme, finland) and milli-q water (millipore, austria). the temperature profile was as follows: initial denaturation at 95 °c for 1 min., followed by 35 cycles (denaturation at 94 °c for 1 min., annealing for 1 min. and extension at 72 °c for 2 min.) with a final extension on the step at 72 °c for 10 min. the annealing temperature was primer-specific and was each time 5°c below its melting temperature supplied by the manufacturer – sigma-ark (tabs. 2, 3). the amplification was performed in thin-wall vials (mj research, usa) with a thermocycler uno ii (biometra, germany). the amplification products were separated against molecular-weights marker (100 bp dna ladder, mbi fermentas) in 1.5% agarose gel and tae buffer (40 mm tris-acetate, 1 mm edta, ph 7.8) at 80 v in mgu 602t electrophoresis unit (cbs scientific, usa). the agarose gel stained with ethidium bromide was visualized under ultra-violet light and documented using computer image system (vilber lourmat, france, agarose, tae, etbr from serva – 74 acta bot. croat. 69 (1), 2010 ko£odziejek j., cieœlikowski t., sakowicz t. u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees germany). the gel stained with ethidium bromide was visualized under ultra-violet light and documented using computer image system (vilber lourmat, france). numerical analysis of dna fingerprinting electrophoretic patterns the computer assisted analysis was carried out with the aid of bionumerics programme (applied maths, kortijk, belgium), and the unweighted pairs group method using mathematical averages – upgma was used for the data cluster analysis. numbers at branches are bootstrap values (%) generated after 1000 replications. according to specificity of the rapd method, the electrophoretic patterns were compared on the basis of the whole densitometric curve shape and the pearson’s product-moment correlation coefficient has been applied. the final dendrograms describing averaged phenetic similarities were calculated according to upgma algorithm and euclidean distances (statistica 5.1, statsoft). the bootstrap method (felsenstein 1985) employed to evaluate the reliability of tree topology was evaluated after 1000 samples. the calculations were performed with the mega 4.0. software (tamura et al. 2007). results rapd-based genetic diversity among the six species of p. subsect. collinae rapds generated a total of 110 bands using 13 decamer primers (an average of 8.5 bands per assay) of which 39.1% were polymorphic. the size of amplification products ranged between 300–800 bp (tab. 2). the dendrograms formed (not shown) as a result of 13 experiments were diverse in their shape and global homology level. for seven out of the thirteen rapd tests, p. leucopolitana and p. koernickei were rather similar and their electrophoretic patterns acta bot. croat. 69 (1), 2010 75 multilocus genomic associations of potentilla tab. 2. names, percentage of polymorphic bands and melting temperatures (tm) of different primers used to rapd analysis for six species of p. subsect. collinae. code sequence 5’ to 3’ percentage of polymorphic bands tm (°c) j-01 tgggtccctc 42.9 34 j-02 cggcggacta 40.0 38 j-03 ggcggatagc 50.0 34 j-04 gagtcagcag 36.4 32 j-05 gtcagggca 37.5 32 j-06 gtcagggcaa 33.3 32 j-07 tcacgtccac 40.0 32 j-08 caggggtgga 41.7 34 j-09 caggggtgga 40.0 34 j-10 gggccgttta 28.6 32 j-11 gccctcggat 44.4 32 j-12 gtgcgcgacc 37.5 29 j-13 tgagggtccc 36.4 34 39.1 (average) u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees formed common subcluster. these two species were weakly associated with p. ´gabarae. in 8 experiments, three populations of p. collina were clustered altogether, while in 3 rapd tests they were clustered together with p. wimannania. in an analysis of the total rapd data set, three very distinct clusters of rapd phenotypes (fig. 1) appeared. the first distinct minor cluster consisted of p. leucopolitana and p. koernickei. these species showed a similarity coefficient of 0.43 with the cluster supporting by a bootstrap value of 82%. the middle cluster included p. thyrsiflora (population d). the third cluster was further divided into two, somewhat less distinct, subclusters of which the left included population of p. collina located in mirów (population c), p. wimannania, p. ´gabarae, and while the right subcluster was formed by two populations (a, c) of p. collina and p. thyrsiflora (population e). in the present study, significant divergence was found between two p. thyrsiflora populations, and the genetic distance values between populations was 0.48. populations from different sites did not cluster together. one rapd phenotype of p. thyrsiflora observed in plants from the cisowa population clustered at a high level (0.49) with the p. leucopolitana and p. koernickei cluster, and the other phenotype of p. thyrsiflora observed in the ostra góra population from clustered well within the p. collina subcluster. the associations determined with rapd markers among p. subsect. collinae showed genetic similarity coefficients ranging from 0.22 (population c of p. collina and p. wimannania) to 0.55 (p. leucopolitana, p. koernickei and p. thyrsiflora) revealing medium levels of genetic variation among the species studied. rapd-based genetic diversity among p. collina, p. thyrsiflora, and the putative parents using 19 decamer primers, a total of 168 rapd marker loci were scored (an average of 8.8 bands per primer) and 43.4% were polymorphic over all accessions (tab. 3). for most of the rapd tests the electrophoretic patterns of p. collina and p. thyrsiflora formed common cluster (15/19 rapds) or were weakly connected in a stair shape manner (4/19 rapds), while the species representing aureae sect. remained separated (not 76 acta bot. croat. 69 (1), 2010 ko£odziejek j., cieœlikowski t., sakowicz t. 0,45 0,40 0,35 0,30 0,25 0,20 0,15 0,10 4 (g) 5 (i) 2 (d) 1 (c) 3 (f) 6 (j) 1(a) 1(c) 2 (e) a v e ra g e d g e n o m ic s im il a ri ty fig. 1. taxonomic similarity among the potentilla subsect. collinae juz. species – the final dendrogram. the averaged similarity between nine populations found on the basis of thirteen rapd analyses. u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees shown). the internal homology level of p. collina and p. thyrsiflora ranged between 75% and 30% (in stair-shaped dendrograms). the separation of p. collina and p. thyrsiflora was distinct in two tests. these two species were occasionally included into the aureae sect. in 11 experiments, p. tabernaemontani and p. incana of the aureae sect. were gathered together, while p. argentea clustered out of this section, or was weakly associated with it. cluster analysis, presented in the form of a final dendrogram grouped all species into two groups (fig. 2). the first group is represented by two species, p. collina and p. thyrsiflora with the high genetic similarity of 0.88. the second, mixed cluster is formed by three species, i.e. p. argentea, p. tabernaemontani and p. incana. (supported by a 64% bs). out of these species, p. tabernaemontani and p. incana are joined as a distinct minor subcluster with a low genetic similarity (0.35), supported by a bootstrap value of 89%. interestingly, the analysis of species similarity showed that rapd markers placed p. argentea (terminales sect.) closer to p. tabernaemontani and p. incana (aureae sect.) than to p. collina and p. thyrsiflora, both from the terminales section (fig. 2). the association of p. argentea and the subcluster containing p. tabernaemontani and p. incana was supported by a moderate bootstrap (bs) value of 64%. p. tabernaemontani and p. incana (classified in the same section) showed a similarity coefficient of 0.35 with the cluster supported by a bootstrap value of 81%. acta bot. croat. 69 (1), 2010 77 multilocus genomic associations of potentilla tab. 3. names, percentage of polymorphic bands and melting temperatures (tm) of different primers used to rapd analysis for potentilla collina and p. thyrsiflora, and the putative parents, i.e. p. argentea, p. tabernaemontani and p. incana. code sequence 5’ to 3’ percentage of polymorphic bands tm (°c) s-01 gtcagggcaa 37.5 32 s-02 gggctcgtga 54.5 34 s-03 gagtcagcag 44.4 32 s-04 tgggggtccc 33.3 36 s-05 ggcggatagc 50.0 34 s-06 caggggtgga 28.6 34 s-07 cctgggccac 44.5 36 s-08 cccgcctccc 50.0 38 s-09 gagcactagc 45.5 34 s-10 gagcacggga 44.4 34 s-11 atctgcgagc 50.0 32 s-12 tccgatgctg 42.9 32 s-13 accgtcggca 37.5 34 s-14 gcttgcaccg 45.5 35 s-15 ctaccgtggc 58.3 36 s-16 aggggcggtc 33.3 36 s-17 gtccacacgg 42.6 32 s-18 gtgtgagaga 37.5 31 s-19 ccagtgcatg 44.4 31 43.4 (average) u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees discussion in the investigation presented here, 39.1% of the rapd fragments were polymorphic. compared to other plant species, the members of collinae subsect., therefore, showed a medium level of genetic variability. in a study of the widespread tanacetum vulgare, 85% polymorphic bands were observed (), in the rare vicia pisiformis 7.2% () and in apomictic species of rosa sect. caninae only 3% (). these differences are not astonishing, since the level of genetic variability strongly depends on the plant’s life history traits (). the species of the subsection are perennial, facultatively apomictic plants with a narrow ecological amplitude and these biological characteristics all contribute to create and maintain the observed medium level of genetic variability. within terminales sect., our dendrogram shows p. argentea to be relatively isolated from the other members of the section analysed, p. collina and p. thyrsiflora (fig. 2). previous morphological studies have also found the same differentiation between p. argentea and other members of the section (leht 1997). in earlier studies (ko£odziejek 2007, ko£odziejek and gabara 2007, 2008), based on morphological analyses in p. subsect. collinae material, i.e. p. collina, p. thyrsiflora, p. wimannania, p. leucopolitana, p. ´gabarae and p. koernickei, was identified at the species level. however, these conclusions do not fit our observations based on rapd markers. the clustering obtained with morphological characters after research based largely upon herbarium material (ko£odziejek data not shown) was not altogether compatible with the dendrogram based on rapd marker. the absence of a relationship between the morphological and genetic similarities was also found for wild populations of other plants (greene et al. 2004, steiner and santos 2001). several reasons may account for the discordance between the morphological traits and rapd marker. first, molecular markers represent a sample of the plant genome, and even so, are used to make an inference concerning the whole genome. second, morphological variation is strongly associated with environmental 78 acta bot. croat. 69 (1), 2010 ko£odziejek j., cieœlikowski t., sakowicz t. 1,00 0,95 0,90 0,85 0,80 0,75 0,70 0,65 0,60 0,55 0,50 1 (a+b+c) 2 (d+e) 3 (l+m) 4 (n+o) 5 (j+k) a v e ra g e d g e n o m ic s im il a ri ty fig. 2. taxonomic similarity among potentilla collina, p. thyrsiflora, and the putative parents, i.e. p. argentea, p. thyrsiflora and p. incana – the final dendrogram. the averaged similarity among five species found on the basis of nineteen rapd analyses. u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees variation; the morphological similarities observed may be due to different combinations of alleles producing similar phenotypes that might result in morphological similarities or differences that are not proportional to the underlying genetic differences. third, the p. subsect. collinae is a very variable species complex in some respects appearing intermediate between p. argentea, p. tabernaemontani, p. incana and regarded by some authors as derived from hybridisation between them, the occurrence of polyploids and ability to reproduce both sexually and apomictically (müntzing 1928, asker and frõst 1970, holm and ghatnekar 1996, gregor at al. 2002). apparently, taxa of the collinae subsect. occasionally hybridise with each other and with their parents (kurtto et al. 2004). the similarity between p. leucopolitana and p. koernickei has been a matter of debate. these two species have been traditionally considered to be closely related, and p. koernickei have even been treated as forms within p. leucopolitana by some authors (e.g. wolf 1908). in contrast, other authors have classified both taxa as separate species (zimmeter 1887). however, recent morfological studies (data not shown), clearly support the separation of p. leucopolitana from p. koernickei. our results also support these findings since the dendrogram separated both species. the rapd variation in four other species of p. subsect. collinae, i.e. p. praecox, p. alsatica, p. leucopolitana and p. alpicola and the putative parents, i.e. p. argentea, p. incana and p. tabernaemomtani has been investigated earlier gregor et al. 2002). according to their research, p. argentea is probably a parental species of p. praecox, p. alsatica, p. leucopolitana and p. alpicola, and p. tabernaemontan might be the parent species of p. lindackeri. based on chemotaxonomical studies, asker and frõst (1970) showed a close relationship between taxa from p. subsect. collinae and p. argentea and p. tabernaemontani. according to theim, the taxa of p. subsect. collinae could have emerged from crosses between p. argentea of the terminales sect. on one side, and p. tabernaemontani or p. incana of the aureae sect. on the other side. even other taxa from the aureae sect., such as p. tommasiniana f. w. schultz and p. gaudinii gremli (= p. pusilla host), can participate in the emergence of new species belonging to p. collina (gustafsson 1947). the molecular data thus are consistent with the supposed intermediacy in morphological features, which has been cited to support the hybrid origin of the taxa of p. subsect. collinae (ascherson and graebner 1904, wolf 1908, ball et al. 1968). as has been noted to be the case for many hybrids and hybrid derivatives (rieseberg 1995), the morphology of the taxa of p. subsect. collinae is not strictly intermediate between its putative parental species, but rather consists of a mixture of qualititative characters that match one or the other parental species as well as intermediacy in quantitative characters. conclusion morfological differences and traditional taxonomy practice seem to justify recognition of the six taxa p. collina, p. thyrsiflora, p. wimannania, p. leucopolitana, p. ´gabarae and p. koernickei as a separate taxa at species level. however, the grouping of nine populations representing the six species within collinae subsect. in the rapd trees, as partitioned in traditional taxonomic treatments, was not altogether compatible with the well defined species. the authors state (asker and frõst 1970, gregor et al. 2002). that molecular data agree with the intermediacy of species of collinae subsect.between p. argentea (termiacta bot. croat. 69 (1), 2010 79 multilocus genomic associations of potentilla u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees nales sect.), p. tabernaemontani and p. incana (aureae sect.), but our own results do not agree with this. further analyses are needed to determine the correct infrageneric taxonomic treatment of the collinae subsect. as it is outside the terminales sect. in our and leht (1997) analyses. to obtain a deeper insight into the position of this species, it will be necessary to increase both the number of potentilla populations and the number of specimens analysed. acknowledgements part of this work was supported by grant no. 2 p04f 040 26 from the ministry of science and information society technologies (kbn). references ascherson, p., graebner, p., 1904: potentilla l. in: ascherson, p., graebner, p. (eds), synopsis der mitteleuropäischen flora 6, 664–872. wilhelm engelmann, leipzig. asker, s., frõst, s., 1970: the »potentilla collina problem« – a chemotaxonomic approach. hereditas 66, 49–70. ball, p. w., paw£owski, b., walters, s. m., 1968: potentilla l. in: tutin, t. g., heywood, v. h., borges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. 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(eds), flora urss 10, 78–223 (in russian). academy of science urss, moskow. 80 acta bot. croat. 69 (1), 2010 ko£odziejek j., cieœlikowski t., sakowicz t. u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees keskitalo, m., lindén, a., valkonen, j. p. t., 1998: genetic and morphological diversity of finnish tansy (tanacetum vulgare l., asteraceae). theoretical and applied genetics 96, 1141–1150. ko£odziejek, j., 2004: distribution of the potentilla species in the northern part of the landscape park »orle gniazda« (silesia-cracow upland) (in polish). fragmenta floristica et geobotanica polonica 11, 263–270. ko£odziejek, j., 2007: morphological analysis of carpel styles of polish members of the potentilla collina group (rosaceae). genus (supplement) 14, 35–39. ko£odziejek, j., gabara, b., 2007: characteristics of achenes in potentilla collina group (rosaceae). acta societatis botanicorum poloniae 76, 35–42. ko£odziejek, j., gabara, b., 2008: palynological study of polish taxa of potentilla subsect. collinae (rosaceae). acta botanica croatica 67, 139–146. kurtto, a., lampinen, r., junikka, l., (eds) 2004: atlas florae europaeae. distribution of vascular plants in europe. rosaceae (spiraea to fragaria, excl. rubus) 13, 195–203. the committee for mapping the flora of europe and societas biologica fennica vanamo, helsinki. leht, m., 1997: the genus potentilla l. in estonia, latvia and lithuania: distribution, morphology and taxonomy. dissertationes biologicae universitatis tartuensis 27, 1–186. müntzing, a., 1928: pseudogamie in der gattung potentilla. hereditas 11, 267–283. müntzing, a., 1931: note on the cytology of some apomictic potentilla-species. hereditas 15, 166–178. nybom, h., bartish, i. v., 2000: effects of life history traits and sampling strategies on genetic diversity estimates obtained with rapd markers in plants. perspectives in plant ecology, evolution and systematics 3, 93–114. román, b., alfaro, c., torres, a. m., moreno, m. t., [atovi], z., pujadas, a., rubiales, d., 2003: genetic relationships among orobanche species as revealed by rapd analysis. annals of botany 91, 637–642. rieseberg, l. h., 1995: the role of hybridization in evolution: old wine in new skins. american journal of botany 82, 944–953. sakowicz, t., cieœlikowski, t., 2006: phylogenetic analyses within three sects of the genus vicia. cellular molecular biology letters. 11, 594–615. soják, j., 2005: potentilla l. s.l. (rosaceae) in flora europae orientalis (notes on potentilla, 18). candollea 60, 59–78. steiner, j. j., los santos, g. g., 2001: adaptive ecology of lotus corniculatus l. genotypes: i plant morphology and rapd maker characterizations. crop science 41, 552–563. tamura, k., dudley, j., nei, m., kumar, s., 2007: mega4: molecular evolutionary genetics analysis (mega) software version 4.0. molecular biology and evolution 24, 1596–1599. werlemark, g., nybom, h., 2001: skewed distribution of morphological character scores and molecular markers in three interspecific crosses in rosa sect. caninae. hereditas 134, 1–13. acta bot. croat. 69 (1), 2010 81 multilocus genomic associations of potentilla u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees williams, j. g. k, kubelik, a. r., livak, k. j., rafalski, j. a., tingey, s. v., 1990: dna polymorphisms amplified by arbitrary primers are useful as genetic markers. nucleic acids research 18, 6531–6535. wolf, t., 1908: monographie der gattung potentilla l. bibliotheca botanica 71, 1–714. zimmeter, a., 1887: schlüssel zur bestinmung der deutschen, österreichischungarischen und schweizer arten der gattung potentilla. botaniker-kalender, 66–83. 82 acta bot. croat. 69 (1), 2010 ko£odziejek j., cieœlikowski t., sakowicz t. u:\acta botanica\acta-botan 1-10\kolodziejek2.vp 9. travanj 2010 12:43:21 color profile: disabled composite 150 lpi at 45 degrees 68 acta bot. croat. 79 (1), 2020 acta bot. croat. 79 (1), 68–77, 2020 coden: abcra 25 doi: 10.37427/botcro-2020-007 issn 0365-0588 eissn 1847-8476 the impact of drying on bioactive compounds of blue honeysuckle berries (lonicera caerulea var. edulis turcz. ex herder) mateja senica1*, franci stampar1, sezai ercisli2, barbara sladonja3, danijela poljuha3, maja mikulic-petkovsek1 1 university of ljubljana, biotechnical faculty, department of agronomy, chair for fruit growing, viticulture and vegetable growing, jamnikarjeva 101, si-1000, ljubljana, slovenia 2 ataturk university, agricultural faculty, department of horticulture, 25240 erzurum, turkey 3 institute of agriculture and tourism, karla huguesa 8, hr-52440 poreč, croatia abstract – drying fruit is one of the simplest ways to extend the shelf-life of fruit, especially berries. both higher temperature and time of heating significantly change the contents of some primary and secondary metabolites in honeysuckle fruit. differences in their contents arising from different heat treatments were determined with the aid of high-performance liquid chromatography (hplc) coupled with mass spectrophotometry (ms). the content of sugars showed a small change with drying, while organic acid contents decreased with a longer drying time. ascorbic acid was totally degraded, regardless of the time or heating temperature. different phenolic groups responded differently to heat intensity and time of drying. flavanols were more sensitive to higher temperature than to duration of heating and they decreased by more than 70% at 75 °c. in contrast, the content of hydroxycinnamic acids, increased with drying by more than 75%, regardless of the time and temperature. keywords: heating, lonicera, phenolics, time of drying *corresponding author e-mail: mateja.senica@bf.uni-lj.si introduction small fruit berries, such as blueberries, strawberries, blackberries and raspberries, are widely consumed all over the world. they are a rich source of polyphenolics, especially flavonols and anthocyanins, which have health benefits (sablani et al. 2010, mikulic-petkovsek et al. 2012). minor fruits, such as quince, rose hip, hawthorn, saskatoon, chokeberries and honeysuckles, are easier to grow and hardy in nature, producing a crop even under adverse soil and climatic conditions (gündüz and özbay 2018). their fruits have a unique aroma and taste and play a vital role in nutrition and as a source of livelihood, in particular providing employment and income generation for rural and tribal groups (vijayan et al. 2008, mikulic-petkovsek et al. 2012, ercisli et al. 2012, cuce and sokmen 2017). the blue honeysuckle berry (lonicera caerulea var. edulis turcz. ex herder) (wfo 2019) from the lonicera genus and the plants can be organically grown. their natural growth areas are wetland spaces along rivers, marshes or forest clearings in northeastern asia and america (thompson 2008, miyashita et al. 2009). the berries are similar in color to blueberries, dark purple with a waxy coating, but with an obvious difference in the berry shape. blue honeysuckle berries have a more elongated or cylindrical shape than the blueberry (thompson 2008, hummer et al. 2012). the taste is bitter to tart-sweet, a mixture of known berry flavors (hummer et al. 2012). the berries from blue honeysuckle have become popular because of their health-promoting properties. they contain nutraceutical compounds, such as vitamins, minerals, polyphenolics, iridoids and saponins (jurikova et al. 2009, 2012, becker et al. 2017, oszmiański and kucharska 2018). additionally, they contain a low sugar content compared to some other fruits, which could make them a good source of nutrition for people with diabetic troubles (palíková et al. 2009). the berries have a short shelf-life and it is important to keep the product fresh to maintain its nutritional value as far as possible. most storage techniques require low temperatures, which are difficult to maintain throughout a distribution chain (sagar and kumar 2010). in addition to freezing, drying is the simplest drying of blue honeysuckle berries acta bot. croat. 79 (1), 2020 69 procedure for preserving fruit (senica et al. 2016). not only does it extend the shelf-life of fruit, but it retains the characteristics of natural products, reduces the costs of packing, storage and transportation, due to the reduced weight and volume of the product, and additionally inhibits the growth of micro-organisms (wang and xu 2007, chauhan and srivastava 2009, sagar and kumar 2010, mundada et al. 2010). another advantage is the higher prices of commodities and accessibility of food in the season when fresh berries are not available. disadvantages of drying are an alternation of color, in terms of browning, lipid oxidation, degradation of enzymes and change of aroma, flavour and taste. the texture of dried products is influenced by their moisture content, composition, ph, and product maturity. during drying, the collapse of cell structures causes, reduction in size through shrinkage of the berries (sagar and kumar 2010). freshly harvested blue honeysuckle berries have a short shelf-life. in europe, fresh blue honeysuckle berries can be purchased from mid-may to the end of june. there are many food preservation methods for prolonging the presence of fruit in the market and to ensure product nutritional and health quality. the most suitable fruits for drying are apples, pears, plums, grapes, apricots, figs, persimmons and peaches. they can be purchased dried on the market or prepared at home. they are principally dried on average for less than 30 hours (depending on the fruit) at 60 degrees (gardenrobinson 2012, fao 2019). there have been a several studies investigating anthocyanin contents during different preservation processes in blue honeysuckle berries (khattab et al. 2016, oszmiański et al. 2016) and some other fruit species, such as blueberries (brownmiller et al. 2008, sablani et al. 2010), persimmon (karaman et al. 2014, senica et al. 2016), gooseberry (kucner et al. 2014) and strawberry (wojdyło et al. 2009). mineral content changes were observed in strawberries stored at different temperatures (çavuşoğlu 2018). the aim of this study was to identify appropriate drying conditions for preparing dried blue honeysuckle berries while achieving a high quality. we evaluated the influence of a combination of different temperatures (40, 50, 60, 65 and 75 °c) and times of heating (240, 200, 66, 30 and 20 hours) on the thermal stability of ascorbic acid, sugars, organic acids and phenolic content of blue honeysuckle berries. materials and methods plant material the blue honeysuckle berries were from the cultivar 'aurora', grown organically in slovenia. the berries were handharvested at the fully ripe stage on 11 june 2017, at the location šmartno pri litiji (46°2'38.7ʺ n 14°50'47ʺ e, 250 m a.s.l.). fresh berries were immediately used for control treatment. other berries were dried in a drying oven at different durations and temperatures. in addition to the control, we applied five treatments: at 40 °c for 240 hours (10 days), at 50 °c for 200 hours, at 60 °c at 66 hours, at 65 °c for 30 hours and at 75 °c drying for 20 hours. fifty grams of blue honeysuckle berries were used per repetition (10 repetitions). drying process the drying process was carried out using a hot-air drying oven (suša 6, wood dryer, splošno mizarstvo, slovenia) at 40, 50, 60, 65 and 75 °c. drying was continued for up to 30 h or the time needed to reach 15% of moisture or lower. determination of ascorbic acid in dried honeysuckle berries for control 5 g of berries mashed and extracted with 10 ml of 2% meta-phosphoric acid was used. half of one gram of dried berries from each drying treatment was extracted with 5 ml of 2% meta-phosphoric acid. the control and all dried mixtures were then left at room temperature for 1 hour on a shaker (grant-bio pos-300, grant instruments, shepreth, england) for ascorbic acid extraction. the samples were afterward centrifuged at 4 °c at 10 000 rpm for 7 min and filtered through a chromafil a-20/25 mixed ester filter (macherey-nagel, düren, germany) into vials and left to wait until hplc analysis. determination of ascorbic acid was carried out with the thermo finnigan surveyor hplc system (thermo scientific, san jose, ca). conditions were previously described in mikulic-petkovsek et al. (2016) study. contents were expressed in mg of ascorbic acid per 100g of dry weight. determination of sugars and organic acids in dried honeysuckle berries sugar and organic acid contents among various dried honeysuckle berries were estimated according to the method of mikulic-petkovsek et al. (2016). for control 5 g of fresh honeysuckle berries was mixed with an ultra-turrax t-25 macerator and extracted with 25 ml double-distilled water. half of one gram of dried honeysuckle berries was extracted with 10 ml of double-distilled water. each sample was then left at room temperature for 1 hour on a shaker. samples were afterwards centrifuged at 4 °c at 10 000 rpm for 7 min and filtered through a chromafil a-20/25 mixed ester filter into vials and allowed to wait until hplc analysis. determination of individual sugars and organic acids was carried out with the thermo finnigan surveyor hplc system, as previously described in mikulic-petkovsek et al. (2016) study. contents were expressed in mg per g of dry weight. determination of individual phenolics the extraction of phenolic compounds for 5 different honeysuckle berry products was carried out as described by senica et al. (2016) with some modifications. for control honeysuckle berries were homogenized with an ultra-turrax t-25 and 5 g of fruit paste was extracted in 30 ml-centrifuge tubes with 15 ml methanol containing 3% formic acid. for other treatments, one gram of dried honeysuckle berries was extracted with 10 ml of methanol containing 3% formic acid. all samples were then placed in a cool ultrasonic bath for 1 hour. the mixtures were then centrifuged for 10 min at 12 000 rpm. each supernatant was filtered through a chromafil ao-20/25 polyamide filter (macherey-nagel, düren, germany) and transferred into vials until hplc and ms senica m, stampar f, ercisli s, sladonja b, poljuha d, mikulic-petkovsek m 70 acta bot. croat. 79 (1), 2020 analysis. separation of phenolic compounds was performed on a mass spectrometer (lcq deca xp max, thermo scientific) with electrospray ionization (esi) operated in negative and positive ion modes. the esi parameters were described by senica et al. (2016). analyses were carried out using the accela hplc system (thermo scientific, san jose, ca), equipped with a diode array detector (dad), controlled by cromquest 4.0 chromatography workstation software, with technical characteristics as described by senica et al. (2016) with the mobile phase gradient according to wang et al. (2002). individual phenolic compounds were identified by fragmentation with hplc-ms, comparison of retention times with standards and monitoring uv-vis spectra from 200–550 nm. calibration curves were prepared from all standards and the individual compounds were identified and quantified by comparison with pure standards. chemicals quinic acid, shikimic acid, 5-caffeoylquinic acid, neochlorogenic acid 3-caffeoylqinic acid, cyanidin-3-glucoside, ellagic acid, naringenin and luteolin-3-rutinoside and ascorbic acid standards as well as meta-phosphoric acid and methanol were obtained from sigma aldrich chemie (steinheim, germany). we obtained fructose, glucose, sucrose, citric, malic, fumaric and tartatic acid, standards for sugars and organic acids; additionally epicatechin, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-rutinoside, p-coumaric acid, procyanidin b2, luteolin-3-glucoside, genistein and kaempferol-3-glucoside for standards of phenolics from fluka chemie (buchs, switzerland). phenolic standards catechin, p-coumaric acid and caffeic acid were obtained from roth (karlsruhe, germany); quercetin-3-xyloside, quercetin-3-arabinofuranoside from apin chemicals (abingdon, uk) and isorhamnetin-3-rutinoside, loganin, petunidinand peonidin-3-glucoside from extrasynthese (genay, frence). ultrapure water used to prepare all water extractions and the mobile phases was obtained from the milli-q system (millipore, bedford, ma, usa). for phenolics where standards were lacking, they were tentatively identified based on their fragmentation pattern obtained from ms2/ms3 analysis and by comparison with data from the literature. their contents were calculated using chemically similar phenolic compounds. thus quercetin glycosides were quantified in equivalents of quercetin-3-galactoside, isorhamnetin glycosides in equivalents of isorhamnetin-3-rutinoside, luteolin derivatives in equivalents of luteolin-3-glucoside, genistein derivate in equivalents of genistein, kaempferol glycosides in equivalents of kaempferol-3-glucoside, procyanidins in equivalents of procyanidin b2, pelargonidinand peonidin derivatives on pelargonidin or peonidin-3-glucoside and ellagic acid derivatives in equivalents of ellagic acid. statistical analysis the results were analyzed statistically using a one way analysis of variance (anova) with the statistical program r commander. duncan’s mean separation tests were done for comparisons of the contents of the primary and secondary metabolites studied. statistically significant differences were accepted at p < 0.05. results all compounds were expressed on a dry weight basis to ensure reliable comparison among different thermal treatments. at lower temperatures, they needed a longer time to dry and for water to be removed from the fruit. the contents of ascorbic and organic acids as well as of sugars in dried honeysuckle berries are shown in tab. 1. in tab. 2, the identification of individual phenolic compounds is given, while their contents are presented in tab. 3. sugars, ascorbic and organic acid levels contents of sugars, glucose, fructose and sucrose in blue honeysuckle berry fruit were determined. fructose content ranged from 51 to 58%, glucose from 41 to 47% and sucrose from 1–5% of total sugar content (tab. 1). determined fructose contents were from 15.5–22.6 g 100 g–1 dw, glucose 11.5 to 17.7 g 100 g–1 dw and sucrose 0.47 to 1.8 g 100 g–1 dw of honeysuckle berries. in general, sugar contents in dried berries dropped one third less than non-treated berries. the highest total sugar content was measured in honeysuckle tab. 1. the content of sugars and organic acids (g 100 g–1 dw) in fresh (control) and blue honeysuckle berries dried at different temperatures and times. means ± standard deviation are presented. different letters (a-d) in rows denote statistically significant differences in some primary metabolites among fresh and dried blue honeysuckle berries by duncan multiple range test (p < 0.05); n = 10. parameters control 40 °c (240 h) 50 °c (200 h) 60 °c (66 h) 65 °c (30 h) 75 °c (20 h) fructose 22.57±0.51a 15.58±1.37c 17.5±0.87b 17.21±1.13b 17.83±0.41b 15.96±0.63c glucose 17.75±0.28a 14.23±0.98c 15.29±0.72b 12.82±0.99d 13.24± 0.45d 11.50±0.54e sucrose 0.86±0.21c 0.65±0.22c 0.47±0.31cd 1.40 ±0.52b 1.78±0.22a 0.18±0.04d citric acid 18.10±1.30a 6.28±0.54f 9.74±1.16d 8.44±1.07e 15.14±0.50b 11.67±0.30c fumaric acid 0.002±0.00d 0.009±0.002c 0.010±0.001bc 0.003±0.001d 0.013±0.001a 0.012±0.001b malic acid 5.91±0.28b 3.71±0.40c 5.37±0.66b 2.63±0.46d 7.79±0.53a 7.82±0.28a shikimic acid 0.002±0.001c 0.022±0.007b 0.038±0.008a 0.004±0.001c 0.019±0.002b 0.023±0.001b tartaric acid 1.23±0.12d 2.49±0.52c 3.54±0.61b 1.38±0.05d 4.61±0.38a 4.37±0.10a quinic acid 6.62±0.26a 4.76±0.42b 6.10±0.78ab 3.16±0.33c 6.56±0.46a 7.44±0.92a drying of blue honeysuckle berries acta bot. croat. 79 (1), 2020 71 berries dried at 50 °c for 200 hours and at 65 °c for 30 hours, after which sugar contents were 20% lower than before drying (fig. 1). the lowest total sugar content was measured in honeysuckle berries dried at 75 °c for 20 hours, with their contents 33% lower than in the control. the drying process caused a decrease of ascorbic acid as well. fresh berries contained 154.89 mg 100 g–1 dw of ascorbic acid. after the heat treatment there was no detected vitamin c content in any dried blue honeysuckle berries. five organic acids were identified in the blue honeysuckle berries. the most abundant was citric acid (36 to 57% of total organic acids), followed by malic and quinic acids (19 to 28%), tartaric acid (4 to 14%), while fumaric and shikimic acids contributed under 1% of total organic acids (tab. 1). in general, the contents of organic acids significantly varied according to the different heat treatments (fig. 1), but all organic acids slightly increased (tab. 1) with heating above 65 °c (fig. 2). phenolic compounds composition forty different individual phenolics were quantified by hplc-ms in the honeysuckle berries (tab. 2). we detected and identified some less known phenolics, such as loganinpentoside, an iridoid determined according to its molecular ion at m/z 521 [m-h]– and its corresponding fragment ions m/z 389, 227. an isoflavone genistein hydroxyhexoside (tab. 2) was confirmed according to the fragmentation pattern; from molecular ion at m/z 449 we got fragment ion tab. 2. identification of phenolic compounds in blue honeysuckle fruits in positive and negative ions with hplc-ms, ms2 and ms3. phenolic group [m]+ or [m-h]–(m/z) ms2 (m/z) ms3 (m/z) phenolic compound hydroxycinnamic acid 353 191, 179, 135 neochlorogenic acid (3-caffeoylquinic acid) 353 173, 179, 191 cryptochlorogenic acid (4-caffeoylquinic acid) 353 191, 179, 173, 135 chlorogenic acid (5-caffeoylquinic acid) 337 191, 173, 163 5-coumaroylquinic acid 325 163, 119 p-coumaric acid hexoside 515 353 191, 179, 173 dicaffeoylquinic acid hydroxybenzoic acids 463 301 257, 229 ellagic acid hexoside flavanols 289 245 catechin 289 245 epicatechin 577 425, 407, 289 procyanidin dimer 865 577, 451, 425, 407, 289 procyanidin trimer flavones 447 285 luteolin hexoside 593 447 285 luteolin-3-rutinoside isoflavones 449 269 genistein hydroxyhexoside flavonols 519 315 isorhamnetin acetyhexoside 665 315 isorhamnetin acetyl rhamnosylhexoside 609 315 isorhamnetin hexosylpentoside 623 315 isorhamnetin-3-rutinoside 489 285 kaempferol acetylhexoside 579 285 kaempferol hexosylpentoside 593 285 kaempferol-3-rutinoside 447 285 kaempferol-3-glucoside 505 301 quercetin-3-acetylhexoside 433 301 quercetin-3-arabinofuranoside 463 301 quercetin-3-galactoside 463 301 quercetin-3-glucoside 463 301 quercetin hexoside 595 301 quercetin hexoside pentoside 609 301 quercetin-3-rutinoside 595 301 quercetin-3-vicianoside 433 301 quercetin-3-xyloside flavanones 433 271 naringenin hexoside iridoid 521 389, 227 loganin-7-pentoside anthocyanins 611 449/287 cyanidin-3,5-diglucoside 449 287 cyanidin-3-glucoside 595 449/287 cyanidin-3-rutinoside 595 433/271 pelargonidin dihexoside 433 271 pelargonidin-3-glucoside 625 463/301 peonidin dihexoside 463 301 peonidin-3-glucoside senica m, stampar f, ercisli s, sladonja b, poljuha d, mikulic-petkovsek m 72 acta bot. croat. 79 (1), 2020 m/z 269. ellagic acid hexoside was identified according to its molecular ion at m/z 463 [m-h]– and its corresponding fragment ions m/z 301, 229 and 257. all phenolic compounds were divided into 9 groups (tab. 2). the groups in our study had different tolerances to temperature and time of heating (fig. 2). the group of hydroxycinnamic acids (hca) represented only 3% of total phenolics in fresh blue honeysuckle berries. the main contributors to hca in fresh berries were neochlorogenic (3-caffeoylquinic acid) and dicaffeoylquinic acids, while in dried blue honeysuckle berries there were neochlorogenic and p-coumaric acids (tab. 3). in our study their content was higher after the drying than in fresh berries. the highest content of total hca derivatives was measured in honeysuckle berries dried at 65 °c for 30 h (355.94 mg 100 g–1) (fig. 2). flavanols (catechin, epicatechin and procyanidins) contribute approximately 50% of total analyzed phenolics in fresh berries. their contents decreased with heating. the highest level, which was still only half that of fresh berries, was determined in berries dried at 60 °c for 66 h (374.13 mg 100 g–1). berries dried at 50 °c for 200 h (190.45 mg 100 g–1 dw) had the lowest flavanol content (tab. 3). the group of flavonols (quercetin, kaempferol and isorhamnetin glycosides), with up to 18% of total analyzed phenolics, did not show a clear trend of increase or decrease with heating. at temperatures of 60 °c and 75 °c, the content of flavonols was higher than in the control, while their contents in other treatments slightly decreased (fig. 2). honeysuckle berries dried at 75 °c for 20 h had the highest flavonol content (326.60 mg 100 g–1), while berries dried at 65 °c for 30 h had the lowest flavonol content (248.61 mg 100 g–1) (tab. 3). flavanones and flavones contributed less than 1% of total phenolics. naringenin hexoside of the flavanone group showed a decrease in content with longer heating time and an increase at higher temperatures. flavones in our study decreased with all heat treatments, but a higher decrease occurred after longer drying than at higher temperature (tab. 3). in our study, anthocyanins comprising 26% of the total phenolics, had the lowest content among phenolics in all heat treatments (fig. 2). the most abundant of total anthocyanins was cyanidin-3-glucoside and the least was pelargonidin-dihexoside. cyanidin-3-glucoside represented from 45 to 65% of total anthocyanins in the various drying treatments. their level decreased by 64% with drying at 40 °c for 240 hours, by 80% at 50 °c for 200 hours, by 57% at 60 °c fig. 1. the content of total sugars and organic acids during the different heating treatments expressed per 100 g dw. different letters (a-d) mean significant differences among different heat treatments (p < 0.05) by duncan’s multiple range test. fig. 2. the content of different phenolic groups during the different heating treatments expressed per 100 g dw. different letters (a-d) mean significant differences among different heat treatments (p < 0.05) by duncan’s multiple range test. drying of blue honeysuckle berries acta bot. croat. 79 (1), 2020 73 tab. 3. individual phenolics content (mg 100 g–1 dw) in six different blue honeysuckle berry products. means ± standard deviation are presented. different letters (a-f ) in rows denote statistically significant differences in individual phenolic levels among blue honeysuckle berry products by duncan’s multiple range test (p < 0.05); n = 10. control 40 °c (240 h) 50 °c (200 h) 60 °c (66 h) 65 °c (30 h) 75 °c (20 h) hydroxycinamic acids neochlorogenic acid (3-cqa) 19.69±0.34 e 154.56±8.97b 86.70±3.56d 161.58±20.94b 198.99±15.14a 119.10±23.42c cryptochlorogenic acid (4-cqa) 0.57±0.01 c 56.22±5.72a 32.78±3.54b 57.30±8.80a 54.35±3.61a 40.34±8.92b chlorogenic acid (5-cqa) 0.26±0.01a 0.04±0.00d 0.02±0.00e 0.05±0.00c 0.07±0.00b 0.01±0.00f coumaroylquinic acid 1.84±0.01e 5.87±0.55d 9.21±0.46c 34.56±0.70a 19.75±1.10b 6.72±0.75d p-coumaric acid hexoside 2.99±0.27d 68.05±5.65a 30.26±3.78bc 48.90±4.65ab 69.99±2.81a 14.96±1.84cd dicaffeoylquinic acid 14.13±0.39a 10.51±0.73c 7.48±0.40d 14.38±0.56a 12.79±1.34b 11.77±1.71bc hhydroxybenzoic acids ellagic acid hexoside 16.09±1.75b 1.6±0.27e 7.12±0.41d 9.40±1.03c 10.73±0.72c 23.44±1.28a flavanols (+)catechin 20.04±2.76a 16.03±1.31bc 7.93±0.63e 17.21±1.36b 14.59±1.11c 10.05±0.79d (-)epicatechin 232.83±23.50a 89.40±8.11c 81.62±14.64c 142.13±18.31b 91.77±6.70c 75.25±2.55c procyanidin dimer 539.20±40.48 a 184.93±18.17c 100.73±14.99d 214.37±11.93b 169.72±6.68c 122.70±10.15d procyanidin trimer 0.55±0.05b 0.49±0.03bc 0.16±0.02d 0.42±0.03c 0.21±0.03d 1.13±0.15a flavones luteolin hexoside 3.01±0.15a 1.55±0.81cd 1.13±0.09d 1.83±0.11bc 1.49±0.12cd 2.09±0.31b luteolin-3-rutinoside 4.85±0.21a 3.84±0.65b 4.56±0.47a 4.81±0.48a 2.05±0.15c 4.56±0.51a isoflavone genistein hydroxyhexoside 1.91±0.07a 1.05±0.01d 1.05±0.01d 1.30±0.10c 2.07±0.19a 1.53±0.07b flavonols isorhamnetin acetylhexoside 1.47±0.16a 0.03±0.00b 0.01±0.00b 0.02±0.00b 0.01±0.00b 0.02±0.00b isorhamnetin acetyl rhamnosyl hexoside 10.91±0.61 ab 10.47±1.31ab 6.31±0.38c 11.78±0.56a 10.26±1.06b 9.69±1.40b isorhamnetin hexosylpentoside 1.95±0.11 d 4.49±0.86c 5.55±0.53b 6.20±0.29a 5.28±0.44b 6.35±0.10a isorhamnetin-3-rutinoside 14.08±0.92a 9.21±0.97c 9.90±1.64c 12.99±0.44ab 9.32±1.17c 11.98±1.27b kaempferol acetylhexoside 0.51±0.00d 1.30±0.16a 0.47±0.03d 0.97±0.03b 0.74±0.05c 0.89±0.09b kaempferol hexosylpentoside 3.55±0.08 a 1.23±1.72b 0.58±0.05b 0.70±0.03b 0.75±0.05b 1.01±0.16b kaempferol-3-rutinoside 2.20±0.25e 2.53±0.29de 3.03±0.37cd 3.56±0.34bc 3.88±0.58b 5.47±0.86a kaempferol-3-glucoside 0.06±0.00a 0.10±0.01a 0.15±0.02a 0.12±0.01a 0.09±0.01a 0.09±0.01a quercetin-3-acetylhexoside 1.95±0.12e 3.09±0.06b 2.54±0.51d 3.53±0.22a 3.04±0.28bc 2.64±0.44cd quercetin-3arabinofuranoside 18.90±0.96 a 7.68±0.99c 4.88±0.28d 9.46±0.38b 7.82±0.63c 7.15±0.70c quercetin-3-galactoside 30.73±1.90 d 30.43±2.66d 23.90±3.05e 43.93±2.68b 35.98±2.97c 54.52±2.97a quercetin-3-glucoside 4.29±0.12d 37.74±8.85a 30.38±4.40bc 34.06±3.37ab 24.20±1.92c 37.95±4.90a quercetin-hexoside 2.33±0.19a 0.57±0.08d 0.15±0.03e 0.76±0.09c 2.37±0.14a 1.92±0.24b q hexoside-pentoside 6.60±0.05a 3.11±0.82d 3.89±0.65c 3.88±0.31c 3.49±0.32cd 4.85±0.18b quercetin-3-rutinoside 173.44±15.19a 163.47±16.10a 157.39±17.29a 165.13±11.90a 131.02±12.15b 167.08±14.52a quercetin-3-vicianoside 16.24±0.55a 11.89±0.81cd 12.45±2.42bc 14.11±1.72ab 10.02±0.91d 14.56±2.09ab quercetin-3-xyloside 0.40±0.03ab 0.32±0.03c 0.36±0.06bc 0.45±0.05a 0.34±0.04c 0.44±0.05a flavanones naringenin hexoside 1.36±0.04c 0.76±0.13e 0.98±0.09de 1.72±0.11b 1.19±0.14cd 2.27±0.37a iridoid loganin-7-pentoside 3.28±0.38b 4.23±0.75a 1.81±0.16c 2.06±0.10c 1.80±0.31c 0.99±0.03d anthocyanins cyanidin-3,5-diglucoside 29.53±0.36a 0.51±0.09d 0.16±0.01e 2.82±0.17b 2.11±0.10c 0.44±0.08d cyanidin-3-glucoside 268.46±17.30a 83.41±8.49c 42.90±9.15e 105.21±5.19b 107.60±11.54b 67.53±1.81d cyanidin-3-rutinoside 44.19±2.52b 30.61±2.49d 18.61±4.91e 30.27±1.28d 93.03±3.20a 37.31±5.17c pelargonidin dihexoside 7.21±0.45a 2.89±0.33d 1.46±0.11e 5.74±0.52b 3.85±0.21c 2.87±0.22d pelargonidin-3-glucoside 10.76±0.43a 3.50±0.16e 3.94±0.15d 3.63±0.46de 6.03±0.21c 6.42±0.11b peonidin dihexoside 12.39±0.90bc 14.75±0.81a 6.79±0.60d 11.46±1.14c 13.38±0.38b 11.79±0.55c peonidin-3-glucoside 40.61±1.52a 13.07±0.74c 6.58±0.59d 16.70±1.79b 15.72±0.35b 12.17±0.21c total 1565.37±42.52a 1035.56±61.54c 715.94±57.15e 1199.52±74.99b 1141.90±59.62b 902.06±49.25d senica m, stampar f, ercisli s, sladonja b, poljuha d, mikulic-petkovsek m 74 acta bot. croat. 79 (1), 2020 for 66 hours, by only 40% with berries dried at 65 °c for 30 hours and by 66% at 75 °c for 20 hours (fig. 2). discussion oszmiański et al. (2016) reported that honeysuckle berries mainly consist of water and soluble solids. selected bioactive compounds start to alter soon after harvest. particularly, after harvesting, different fruit ingredients are subject to various enzymatic and non-enzymatic reactions. enzymatic reactions involve some enzymes, which react in the presence of individual compounds and oxygen, resulting in a change in their contents and composition. other, non-enzymatic processes do not require enzymatic catalysis, but they include three main reaction pathways: maillard reaction, caramelization and ascorbic acid oxidation (sanz et al. 2001). the drying process additionally changes the contents of selective primary and secondary metabolites. water solubility and heat sensitivity are the main two factors that alter the content of selected compounds in dried fruits (karaman et al. 2014). honeysuckle berries have extremely firm skins, which impeded water evaporation in our study. we thus needed much more time to dry the berries to an acceptable dryness (85%) than is needed for other often-dried fruits. fao (2019) reported the desirable final moisture content in dried fruits to be 15%. in general, heat treatment means water conversion into vapor, which passes in a gaseous state across a disrupted cell and consequently changes the concentration of solid components and the evaporation of some volatile compounds (karathanos 1999, yadav and singh 2014, karaman et al. 2014). at lower temperatures, the transformation process lasts longer than at higher temperatures, in which water passes from the fruit faster. enough moisture (85%) must be removed for the product to be considered dried, otherwise mold starts grow on the berries in a few days (fao 2019). singh et al. (2006) found that a low temperature of drying caused minimum damage to dried material, retaining more nutrients in the fruits than other drying methods. sugars in blue honeysuckle berries in general decrease with drying. our sugar contents were in accordance with previous published studies (oszmiański et al. 2016, auzanneau et al. 2018). one of the reasons for the decrease in the sugar content was the maillard reaction. this is a complex series of reactions between amines, amino acids and proteins with sugars and it is the major cause of fruit browning during heating processes. the result of that reaction is reduced sugar content and the formation of brown pigments (yilmaz and toledo 2005). the maillard reaction was not the only reason for sugar reduction. caramelization also occurs, taking place above the melting point of sugar, which darkens to a brown color and decreases the sugar content (sanz et al. 2001). furthermore, heating of the berries caused water evaporation and partial decomposition of sugars i.e. transformation to volatiles, such as water vapor and carbon dioxide, or other types of carbon-containing volatiles (karathanos 1999). the other elements of sugars were modified into crystallized structures (senica et al. 2016), which only diffuse from the berry with difficulty. all heat treatment caused some extent of water evaporation and the formation of soluble sugars, but a longer duration at higher temperatures can destroy sugar molecules, which are prone to chemical transformation at elevated temperatures (karaman et al. 2014). high ascorbic acid content in fresh blue honeysuckle berries is also found in the study of jurikova et al. (2009). ascorbic acid content can be affected by many factors like heat intensity, drying time, final moisture content and air velocity (santos and silva 2008). ascorbic acid is a water-soluble compound (khattab et al. 2017) and it is lost from the berries with vapor. khattab et al. (2017) reported the reduction of vitamin c by 90% at a temperature of 60 °c following 24 h drying. our results are in agreement with khattab et al. (2017) as ascorbic acid was lost from berries after less than 24 hours of drying at 75 °c. degradation of ascorbic acid in dried fruit starts with the first-order reaction, followed by the effect of the reduction of the moisture content which, as the process of drying proceeds with the temperature effect, becomes predominant (santos and silva 2008, goula and adamopoulos 2006, qiu et al. 2018; di scala and crapiste 2008). the first reactions may depend on water activity, ph and the presence of degraded enzymes. with an increase of the water content, the aqueous phase becomes less viscous, which enhances diffusion in the media. this facilitates the reaction of oxidation and, consequently, the degradation of certain compounds (santos and silva 2008). the same authors also reported that both heat intensity and time of drying have a major effect on ascorbic acid degradation. a longer drying time results in a lower retention of ascorbic acid. in addition, higher temperatures (> 65 °c) and the consequent increase in relative humidity resulted in a lower retention of ascorbic acid. during the drying process, moisture and temperature altered with drying time and their combination caused degradation, and the formation of organic acids. tartaric, fumaric and shikimic acids increased with both higher temperature and duration of heating. quinic, citric and malic acids, the most abundant of the organic acids in blue honeysuckle berries seem to be sensitive to drying time (more than 30 hours), but their contents increased with temperatures above 60 °c. it seems that organic acids tolerate higher temperatures better than a long time of heating. regardless of the temperature, dry matter was similar among treatments. higher temperatures mean faster water transfer from the berries, which is linked to a higher loss of low-molecular weight components (kucner et al. 2014, zorenc et al. 2017), the same as with sugars. on the other hand, organic acid content seems to be more sensitive to a long duration of heating. lower temperatures (40 and 50 °c) imply low porosity of the epidermal layer of various berries, which is reflected in slower mass transfer, and the destruction of molecules (kucner et al. 2014). chen et al. (2012) reported that organic acids, including citric acid, degraded via the gamdrying of blue honeysuckle berries acta bot. croat. 79 (1), 2020 75 ma-aminobutyrate shunt pathway following a longer time of heating at lower temperatures. blue honeysuckle berries are rich in phenolic compounds and their presence as established here is in accordance with some other studies (senica et al. 2018a, b; oszmiański et al. 2016). the last mentioned study put special emphasis on iridoids with high health properties. in our study, significant changes occurred in their contents during the thermal treatment at different heating times and heat intensities. total phenolic content has been reported to diminish as a result of food processing (može bornšek et al. 2015). the main reason for phenolic content decrease is the transfer of phenolics to the hypertonic solution. heat treatment causes cellular disruption and the exposure of phenolics to oxidative and hydrolytic enzymes (wojdyło et al. 2009, karaman et al. 2014). the most important enzymes are polyphenol oxidases (ppo), which catalyze the oxidation of colorless phenolic compounds into o-quinones, which are red to brown in color. heating and poor handling of fruits or vegetables cause greater ppo activation and, accordingly, a decrease in the content of some phenolic compounds (sanz et al. 2001, yilmaz and toledo 2005). additionally, with the drying process, a longer time or a higher heat intensity increases the presence of ppo (kucner et al. 2014). additionally, wojdyło et al. (2009) reported that drying temperatures between 55 and 85 °c reduced the content of phenolics. they suggested that an irreversible oxidative process and prolonged exposure to thermal degradation may be the cause of altered levels of phenolic compounds. unlike other groups of phenolics, the content of hydroxycinnamic acids (hca) increased with both heating time and temperature. that is in agreement with several reports (brownmiller et al. 2008, wojdyło et al. 2009, oszmiański et al. 2016). zorić et al. (2014) also noted the high heat stability of hca. kaneko et al. (2016) reported that selected hca have extremely high thermomechanical performance. the thermal degradation temperature for hca was around 300 °c (kaneko et al. 2016), which was four times higher than our highest studied drying temperature. flavanols, flavonols, flavanones, flavones and isoflavones made up the group of flavonoids. their heat sensitivity caused by drying depends on the structural stability of the selected compound. in particular, compounds with a double bond in the structure need more energy in order to be degraded (chaaban et al. 2017). wang et al. (2000) reported that compounds from the flavanol group, especially epicatechin, are highly sensitive to oxidation processes. in our study, their contents greatly decreased regardless of the temperature and time of heating. flavonols have two more double bonds in their basic molecular structures than the previously described flavanols. they are therefore more stable during heat treatment. the structure of flavonones resulted in greater stability with respect to the intensity but not with respect to the duration of drying. degradation of these contents occurred, but more slowly than with some more thermolabile compounds, such as anthocyanins and flavanols (zorić et al. 2014). anthocyanins, with high beneficial properties for human health, are mostly concentrated in the skin of the blue honeysuckle berry (oszmiański et al. 2016). accordingly they are more exposed to degradation, including from heating. total anthocyanins showed significant differences among the various heat treatments (figure 2). berries dried at 65 °c for 30 h (241.72 mg 100 g–1) had the highest anthocyanin content, but still only approximately half that of fresh berries (413.15 mg 100 g–1). berries dried at 50 °c for 200 h had the lowest anthocyanin content. khattab et al. (2016) also found a positive correlation between drying temperature and anthocyanin degradation. those results are also in accordance with zorić et al. (2014) and zorenc et al. (2017), who reported that degradation of anthocyanins is significantly greater at higher temperatures. a higher stability of anthocyanins during heating was achieved by using a lower temperature and shorter duration of heating during processing (wang and xu 2007, zorić et al. 2014). our study showed that dried berries had the same losses at 40 °c as at 75 °c, which confirms that heat treatment in itself negatively affects the cyanidin content. additionally, loss of anthocyanins can be attributed to various factors, such as residual enzyme activity or condensation reactions with other phenolics (brownmiller et al. 2008). some anthocyanins are more vulnerable than other phenols from that group, because of their different chemical structure (srivastava et al. 2007), mainly due to different sugar and hydroxyl moieties. cyanidin glycosides are considered to be less stable in relation to heating, which is in accordance with our study, in which cyanidin-3-glucoside and cyanidin 3.5-diglucoside was 70 to 98% lower than in fresh berries. the results showed that peonidin and pelargonidin are more stable in relation to heat, which is in agreement with previous studies (srivastava et al. 2007, khattab et al. 2016). dried blue honeysuckle berries are a durable and convenient product available throughout the year. it is important to recognize that drying has many advantages; apart from cost reduction, it also accelerates water loss and consequently prevents the growth of bacteria, fungi, and other microorganisms. the chief disadvantage is the loss of some important nutritional compounds. sagar and kumar (2010) reported that an optimal drying system for the preservation of fruits should be cost effective, with a short drying time and minimum damage to the food product. the studied contents of some primary and secondary metabolites in our study responded differently to drying conditions. sugars, flavonols and hca appeared to be more thermostable substances, especially hca, which increased by more than 75% with drying, regardless of the drying time and temperature. on the other hand, anthocyanins and flavanols were highly thermolabile substances, their contents decreasing with an increase of both drying time and temperature. additionally, ascorbic acid totally degraded with heat treatment. what is more, organic acids seem to be more sensitive to long exposure to drying, than to higher temperature of heating, while iridoids are more sensitive to higher heating temperatures. senica m, stampar f, ercisli s, sladonja b, poljuha d, mikulic-petkovsek m 76 acta bot. croat. 79 (1), 2020 in conclusion, we found that the optimal treatment was drying at 60 °c for 33 h, which is in agreement with garba and kaur (2014). understanding the structural stability of selected active compounds in blue honeysuckle berries will help their processors to provide high quality dried berry products with rich nutritional properties. acknowledgement the research is part of the program horticulture no. p4-0013-0481, which is funded by the slovenian research agency (arrs). the authors would like to thank haskap d.o.o. for contributting the plant material. references auzanneau, n., weber, p., kosińska-cagnazzo, a., andlauer, w., 2018: bioactive compounds and antioxidant capacity of lonicera caerulea berries: comparison of seven cultivars over three harvesting years. journal of food composition and analysis 66, 8189. becker, r., pączkowski, c., szakiel, a., 2017: triterpenoid profile of fruit and leaf cuticular waxes of edible honeysuckle lonicera caerulea var. kamtschatica. acta societatis botanicorum poloniae 86, 3539–3548. brownmiller, c., howard, l.r., prior, r.l., 2008: processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blueberry products. journal of food science 73, h72–h79. chaaban, h., ioannou, i., chebil, l., slimane, m., gérardin, c., paris, c., charbonnel c., chekir, l., ghoul, m., 2017: effect of heat processing on thermal stability and antioxidant activity of six flavonoids. journal of food processing and preservation 41, e13203. chauhan, a.k.s., srivastava, a.k., 2009: optimazing drying conditions for vacuum-assisted microwave drying of green peas (pisum sativum l.). drying technology 27, 761–769. chen, m., jiang, q., yin, x.-r., lin, q., chen, j.-y., allan, a.c., xu, c.-j., chen, k.s., 2012: effect of hot air treatment on organic acidand sugarmetabolism in ponkan (citrus retuculata) fruit. scientia horticulturae 147, 118–125. cuce, m., sokmen, a., 2017: in vitro production protocol of vaccinium uliginosum l. 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review. journal of food science and technology 51, 1654–1673. yilmaz, y., toledo, r., 2005: antioxidant activity of water-soluble maillard reaction products. food chemistry 93, 273–278. zorenc, z., veberic, r., stampar, f., koron, d., mikulic-petkovsek, m., 2017: thermal stability of primary and secondary metabolites in highbush blueberry (vaccinium corymbosum l.) purees. lwt-food science and technology 76, 79–86. zorić, z., dragović-uzelac, v., pedisić, s., kurtanjek, ž., garofolić, i.e., 2014: kinetics of the degradation of anthocyanins, phenolic acids and flavonols during heat treatments of freezedried sour cherry marasca paste. food technology and biotechnology 52, 101–108. opce-str.vp acta bot. croat. 70 (1), 41–51, 2011 coden: abcra 25 issn 0365–0588 effects of selected groundwater chemical traits on a salt marsh community mahmut kilinç1,*hamdý güray kutbay1, erkan yalçin1, alý býlgýn2, kenan avci3, solmaz gencoglu topaloglu3 1 university of ondokuz, mayis faculty of arts – sciences, department of biology, 55139 kurupelit-samsun, turkey 2 rize university, faculty of arts – sciences, department of biology, 53100 rize, turkey 3 ministry of agriculture and rural affairs, soil and water resources research institute, samsun, turkey abstract – electrical conductivity, exchangeable sodium ratio and water depth have negative impacts, whereas soil organic matter concentration has a positive impact on black sea salt marsh vegetation. the most saline soils were characterized by salicornia prostrata vegetation and associated with exchangeable sodium ratio. alhagi pseudalhagi and tamarix smrynensis populations were associated with water depth, while juncus littoralis, ammophila arenaria and e. paralias were associated with soil organic matter. euphorbia paralias, ammophila arenaria and iris orientalis were associated with acidity. key words: black sea, groundwater, salt marsh, vegetation, black sea introduction salt marsh ecosystems are known to be a highly structured environment providing a gradient of environmental conditions from extremely inundated and saline to relatively mesic (zhang 1996, cantero et al. 1998a). coastal salt marshes vegetated by herbs, grasses, and low shrubs bordering saline water bodies are unique ecosystems universally recognized for their exceptional ecological value (mitsch and gosselink 1993). they comprise areas of land bordering the sea largely covered with vegetation and subject to periodic tidal inundation (kennish 2001). a small number of halophytic species that are spatially segregated in pronounced vegetation zones dominate these ecosystems (bertness et al. 1992, asri and ghorbanli 1997, abd el-ghani 2000a, apaydin et al. 2009). the use of halophytes as indicators of groundwater and soil chemical traits could be an effective and useful method for scientists to inform extension agents, and end users about the state of the environment (li wei-quiang et al. 2008, shaltout and al-sodany 2008). acta bot. croat. 70 (1), 2011 41 * corresponding author, e-mail: hguray@omu.edu.tr copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\kilinc.vp 28. o ujak 2011 15:57:58 color profile: disabled composite 150 lpi at 45 degrees it has been reported that vegetation zonation in salt marshes is primarily related to the salinity of the groundwater (chapman 1974, abdel-razik and ismail 1990, pennings and callaway 1992, jongman et al. 1995, cantero et al. 1998a, kutbay and demir 2001) and groundwater chemical traits are often considered to determine the possibility of restoring or rehabilitating a salt marsh. this study is aimed to determine the role of chemical traits of groundwater on plant communities in a salt marsh located in the north of turkey using numerical methods. for this reason, selected groundwater and soil chemical traits were used and the roles of these traits on plant communities in the study area were evaluated. study area the study area is situated on the east bank of the kýzýlýrmak river in the northern and northeastern parts of bafra town (41°43'10.09'' n, 35° 59' 26.32'' e), north turkey, in the central black sea region (fig. 1). the area is characterised by a semi-humid mediterranean climate with the highest potential evaporation rate (140 mm) occurring during june (engin and korkmaz 1990, apaydin et al. 2009). the study area is located at kýzýlýrmak river delta. the kýzýlýrmak river delta is an exception in that coastal erosion is an environmental threat that increases inundation, thus leading to land loss. coastal retreat in the study area is between 2.5–5.0 m per year. the sediments supplied by the kýzýlýrmak river feed the coastal barriers in the study area under the influence of the longshore flows with an average speed of 40–50 cm s–1. the soils of the study area are formed from alluvial materials. the sediments in the area consist of upper pleistocene and holocene alluviums and vary from fine sand, silt and clay in varying thickness and extents. the thickness of quaternary deposits increases northwards. the soils are 42 acta bot. croat. 70 (1), 2011 kilinç m., güray kutbay h., yalçin e., býlgýn a., avci k., gencoglu topaloglu s. fig. 1. map of the study area u:\acta botanica\acta-botan 1-11\kilinc.vp 31. o ujak 2011 13:25:49 color profile: disabled composite 150 lpi at 45 degrees fine-textured with moderate hydraulic conductivity. sublayers of these soils are massive in structure (alpar 2009, demir et al. 2009). soil ph and electrical conductivity are rather high and soils are alkaline (arslan et al. 2007). additionally, groundwater in the study area has high electrical conductivity (demir et al. 2009). coastal dunes occur along the shoreline and they extend about 30–40 m from the shoreline and coastal dunes are characterised by ammophila arenaria subsp. arundinacea, euphorbia paralias and tamarix smrynensis communities. coastal sand dunes are replaced by salt pans and these salt pans are characterised by salicornia prostrata communities. at the edge of these communities alhagi pseudalhagi communities occur. about 200 m from the shoreline juncus littoralis c. a. meyer and juncus acutus communities occur. about 270 m from the shoreline inland dunes are characterised by iris orientalis miller communities (fig. 2). we use taxonomic nomenclature according to davis (1965–1985) and davis et al. (1988) and brummitt and powell (1992). materials and methods the research transect was 350 m long from sand dunes in the north-east to the water level below the salt marsh in the south-west (fig. 1). ground water samples were taken along the transect populated by the ammophila arenaria subsp. arundinacea, euphorbia paralias and tamarix smrynensis communities (fig. 2). the cover of species (in %) was estimated in 4 square meters (2 m � 2 m) for each community according to the braun–blanquet scale, as proposed by van der maarel (1979), using standard relevé methods (mueller-dumbois and ellenberg, 1974). groundwater samples were collected during july 2000 with a sample bottle after soil cores were with the acta bot. croat. 70 (1), 2011 43 soil characteristics and salt marsh community fig. 2. the transect showing plant zonation in the study area. 1. ammophila arenaria, 2. euphorbia paralias, 3. tamarix smrynensis, 4. salicornia prostrata subsp. prostrata, 5. alhagi pseudalhagi, 6. juncus littoralis, 7. juncus acutus, 8. iris orientalis u:\acta botanica\acta-botan 1-11\kilinc.vp 31. o ujak 2011 13:25:49 color profile: disabled composite 150 lpi at 45 degrees use of a 7 cm diameter soil auger to a depth of 80 cm because mean root depth was about 80 cm for the studied species and four water samples were taken per community. the groundwater level was determined at the sampling time by sinking a hole and allowing the interstitial water to refill it. groundwater samples were taken by boreholes which, as specified by soil survey staff guidelines, were sunk at each sampling point (faulkner et al. 1989, sánchez et al. 1998, álvarez-rogel et al. 2007, bornman et al. 2008). all materials that might come in contact with the groundwater were rigorously cleaned with high purity reagents (hcl and hno3) and pure water (creasey and flegal 1999). soil samples were taken from 4 m � 4 m (16 m2) plots with a soil auger and soil samples were air-dried, crushed and sieved using a 2 mm mesh. the groundwater samples were analysed for: 1) electrical conductivity using a jenway analyser, 2) acidity using a beckman ph meter (black 1968), 3) k+, ca2+ and mg2+ using a perkin elmer atomic absorption spectrophotometer (hanlon 1998), 4) so42– and cl– (meq l–1) concentrations were determined by turbidimetric and gravimetric methods, respectively (allen et al. 1986, kilinc et al. 2006), 5) exchangeable sodium ratio and sodium adsorption ratio were calculated according to hussein and rabenhorst (2001). 6) hco3– concentration was determined by titration with sulphuric acid (álvarez-rogel et al. 2006). 7) organic matter was determined according to the walkley-black method (kilinc et al. 2006). to examine the relationships between plant communities and groundwater variables, canonical correspondence analysis (cca) was applied (jongman et al. 1995) using ecom version 1.33 (henderson and seaby 2001). the cover-abundance symbols of the braun-blanquet scale (r, +, 1, 2, 3, 4 and 5) were replaced by 1, 2, 3, 4, 5, 7, 8 values according to van der maarel (1979) and focht and pillar (2003). results chloride concentrations of groundwater and soil were rather high and sodium adsorption ratio and exchangeable sodium percentage values were also found to be high (tab. 1). 44 acta bot. croat. 70 (1), 2011 kilinç m., güray kutbay h., yalçin e., býlgýn a., avci k., gencoglu topaloglu s. t a b . 1 . m ea n ± st an da rd de vi at io n va lu es of st ud ie d gr ou nd w at er (g w ) an d so il tr ai ts . t ra it ph e s p e c s a r k + c a2 + m g2 + h c o 3– c l– s o 42 – o m g w 7. 44 ± 0. 25 – 7. 71 ± 5. 41 35 .4 4± 10 .3 0 1. 90 ± 1. 63 25 .1 3± 17 .8 6 61 .4 1± 21 .2 2 24 .3 1± 10 .3 5 16 7. 94 ± 34 .0 8 2. 02 ± 1. 52 – s oi l 7. 46 ± 0. 33 23 .6 9± 8. 10 8. 32 ± 3. 40 – 1. 40 ± 0. 40 15 .3 8± 3. 95 56 .3 2± 10 .2 5 22 .9 1± 1. 69 46 .3 6± 4. 35 1. 70 ± 0. 53 2. 22 ± 0. 70 e c – el ec tr ic al co nd uc ti vi ty ; e s p – ex ch an ge ab le so di um pe rc en ta ge ; s a r – so di um ab so rp ti on ra ti o; o m – or ga ni c m at te r. u:\acta botanica\acta-botan 1-11\kilinc.vp 31. o ujak 2011 13:25:49 color profile: disabled composite 150 lpi at 45 degrees pearson correlations between species and environment scores in canonical axis 1 and 2 were highly significant and explained 76 % and 77 % of the cumulative variance, respectively (tab. 2). a monte carlo permutation test (999 permutations) confirmed the significance of the first two axes (p< 0.001). from the intra-set correlations of the soil factors with the first two axes of the cca, electrical conductivity, exchangeable sodium ratio, water depth and organic matter concentration were the most significant parameters in axis 1 and all of these parameters were negatively correlated except for the soil organic matter concentration which was positively correlated. along axis 2, only soil ph was negatively correlated and none of the other parameters were significant (tab. 3). according to the cca analysis s. prostrata was associated with exchangeable sodium ratio, while a. pseudalhagi and t. smrynensis were associated with water depth. j. littoralis, a. arenaria and e. paralias were associated with soil organic matter. along axis 2 e. paralias, a. arenaria and i. orientalis were associated with soil ph (fig. 3). the results from the detrended correspondence analysis (dca) were similar to the results of the cca analysis in that the species were arranged according to groundwater salinity along axis 1. species grouped along a groundwater salinity gradient with s. prostrata grouping separately on the right of axis 1 followed by j. acutus and then rest of the species along the left of axis 1. dca axis 2 represents the distance from the sea and sand-dune species like e. paralias, a. arenaria and j. littoralis grouped along the bottom of axis 2 (fig. 4). acta bot. croat. 70 (1), 2011 45 soil characteristics and salt marsh community tab. 2. eigen values and species-groundwater chemical traits correlation coefficients. canonical axis 1 canonical axis 2 canonical eigenvalue 0.422 0.379 % variance explained 5.877 5.284 cumulative % variance 5.877 11.16 pearson correlation species/environment scores 0.756 0.774 tab. 3. intraset correlation coefficients of soil and groundwater. statistically significant correlations (p<0.05) are marked in bold. can. axis 1 can. axis 2 ph 0.345 –0.523 ec –0.817 0.131 esr –0.644 –0.145 om 0.677 –0.011 wd –0.579 –0.394 sar –0.151 0.039 k+ –0.069 0.210 ca2+ –0.005 0.424 mg2+ 0.254 0.375 hco3 – 0.055 –0.282 cl– 0.308 0.236 so4 2– 0.001 0.245 u:\acta botanica\acta-botan 1-11\kilinc.vp 31. o ujak 2011 13:25:49 color profile: disabled composite 150 lpi at 45 degrees discussion correlations between species and environmental scores indicated a strong association between communities and measured soil parameters, as has been already reported (jongman et al. 1995, abd el-ghani and amer 2003). salinity (electrical conductivity and ex46 acta bot. croat. 70 (1), 2011 kilinç m., güray kutbay h., yalçin e., býlgýn a., avci k., gencoglu topaloglu s. fig. 3. the relationship among soil and groundwater traits and species by cca. ä salicornia prostrata,�tamarix smrynensis,�juncus acutus,� iris orientalis, + alhagi pseudoalhagi,�juncus littoralis, � euphorbia paralias, � ammophila arenaria fig. 4. the relationship among soil and groundwater traits and species by detrended correspondence analysis (dca). ä salicornia prostrata, � tamarix smrynensis, � juncus acutus, � iris orientalis, + alhagi pseudoalhagi, � juncus littoralis, � euphorbia paralias, � ammophila arenaria. u:\acta botanica\acta-botan 1-11\kilinc.vp 31. o ujak 2011 13:25:50 color profile: disabled composite 150 lpi at 45 degrees changeable sodium ratio), water depth and organic matter concentrations were found to be most significant environmental variables affecting salt marsh zonation. groundwater salinity and depth (cantero et al. 1998b, mashaly 2001, bornman et al. 2002) and soil salinity (ji et al. 2009) have been identified as the most important factors in shaping vegetation patterns in other salt marsh ecosystems. sodic soils are widespread in the study area because sodium adsorption ratio values >13 (amezketa and de lersundi 2008) salicornia prostrata was associated with the most saline soils in the studied coastal salt marsh. this species forms monospecific stands along coastal salt marshes and inhabits salt-pan areas. these salt-pan areas are characterised by extreme conditions and salicornia (chenopodiaceae) species are regarded as fugitive species of these hypersaline bare patches and salt pans, because of their inability to compete with the dominant perennials (bertness et al. 1992). it has been found that communities of halophytes were definitely better indicators of soil salinity than individual species and salicornia occurred on extremely saline soils (piernik 2003). it is known that groundwater salinity is determined by tidal influences (sánchez et al. 1998, hussein and rabenhorst 2001, apaydin et al. 2009, salama and bokhari 2009). flooding has been classified as a determinant factor of vegetation patterns in coastal estuaries (ji et al. 2009). salicornia prostrata subsp. prostrata is widely subject to flooding and closely associated with exchangeable sodium ratio and may therefore be used reliably to evaluate the impact of flooding in salt marshes. alhagi pseudalhagi and tamarix smrynensis was closely associated with water depth and both species have been identified as groundwater indicating plants (abd el-ghani 2000b; el-bana and al-mathnani 2009). soil organic matter is an important factor that regulates the distribution of euphorbia paralias, juncus littoralis and ammophila arenaria (abd el-ghani and amer 2003, omer 2004, zahran and willis 2008). luxuriant growth of j.littoralis is associated with soil organic matter concentrations making individual clumpings, reaching a height of 100 cm (abd el-ghani 2000a, el-sheikh and abbadi 2004). juncus littoralis was also associated with ph. according to the dca diagram the two different gradients are interpreted as salinity and distance from the sea. salicornia prostrata occurred in the right of the dca diagram and indicated the most saline soils. burchill and kenkel (1991) stated that the most saline areas in salt marshes are generally dominated by succulent annual species like salicornia. salicornia species are subject to regions characterized by downward flow, more frequent tidal recharge, and thus hypersaline soil conditions. these conditions permit the long-term persistence of fugitive species like salicornia by maintaining a physically harsh hypersaline environment that is intolerable to neighbouring communities (thibodeau et al. 1998). juncus acutus and j. littoralis occurred in the middle and left of the diagram. juncus l. species are classified as slow-growing plants with extensive below-ground reserves, and hence tend to respond slowly to changes in soil factors (pennings et al. 2005, apaydin et al. 2009). salinity is decreased to the left of the diagram. coastal dune species (ammophila arenaria, euphorbia paralias) inhabit the lowest part, while iris orientalis inhabits the upper part of the dca diagram along the axis 2. i. orientalis occurred on inland dunes. these species usually adapted to less saline conditions as compared to salicornia species (ihm et al. 2007). acta bot. croat. 70 (1), 2011 47 soil characteristics and salt marsh community u:\acta botanica\acta-botan 1-11\kilinc.vp 31. o ujak 2011 13:25:50 color profile: disabled composite 150 lpi at 45 degrees the importance of particular factors is likely to vary geographically in salt marshes. in particular, salinity stress probably plays a much more important role in mediating plant zonation patterns at lower latitudes (pennings et al. 2005). álvarez-rogel et al. (2007) stated that salinity is more effective on zonation of communities than the vegetation and distance to the shoreline in a dune coastal salt marsh ecosystem. in the present study both factors (salinity and distance from the sea) were effective on plant zonation. the similarity between cca and dca suggests that there might be no other environmental variables missed in sampling (abd el-ghani and amer 2003). cisneros et al. 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of italy valeria tomaselli, massimo terzi* institute of biosciences and bioresources, cnr, via amendola 165/a, bari, italy abstract – in the south-east of italy, rocky coasts are almost entirely concentrated in the apulia region. several phytosociological papers have already dealt with the rocky coastal vegetation of the class crithmo-staticetea in some parts of the apulian coast. however, there is still no overall revision carried out by using modern statistical treatments of phytosociological data. this paper aims to revise the syntaxonomy and nomenclature of the class crithmo-staticetea in the south-east of italy. the revision is based on a data set of 225 relevés consisting of new and original phytosociological relevés (66) and others already published. the data matrix was classified with the use of flexible beta clustering. indicator species analysis was employed to identify the indicator species of the main clusters of relevés. results were interpreted from a syntaxonomic point of view. non-metric multidimensional scaling ordination was performed in order to visualize the floristic relationships among associations. rocky coastal vegetation of the crithmo-staticetea class in the south-east of italy is represented by two orders, crithmo maritimi-staticetalia and helichrysetalia italici. the first one includes two alliances, crithmo-staticion and limonion anfracti-cancellati, with four associations and one, respectively. however, since they rely on very few character species, the floristic and syntaxonomic relationships between these two alliances need to be deepened by further investigations, involving a larger data set and investigation area. the second order, helichrysetalia italici, includes two associations, well differentiated by their ecology, structure and floristic composition. for this reason, they were classified within two different alliances, anthyllidion barbae-jovis and helichrysion litorei. the helichrysion litorei is here validated. keywords: adriatic, coastal vegetation, helichrysion litorei, limestone coasts, mediterranean basin, syntaxonomy, crithmo-staticetea * corresponding author, e-mail: massimo.terzi@ibbr.cnr.it introduction coastal environments have an important ecological value, providing ecosystem services essential to people and the environment (e.g. food provision, erosion control, water control, and habitats for many threatened and endangered species) and are worthy of attention and conservation. coastal areas are one of the most threatened environments, both in the mediterranean region and worldwide. in fact, especially in the last decades, they are undergoing rapid anthropogenic development. increasing human pressure (e.g. urbanization, exploitation of natural resources, plant invasion) is causing coastal areas to diminish, in concert with the degradation and isolation of their habitats (médail and quézel 1997, biondi 1999, gibbs 2000, van der maarel 2003, underwood et al. 2009). the need to preserve these habitats was recognized by european policies so that they were included in annex i of directive 92/43/eec, the ‘habitat directive’. cliffs and rocky coasts, identified by the habitat type code 1240, ‘vegetated sea cliffs of the mediterranean coasts with endemic limonium spp.’, are currently represented within many protected areas along the north mediterranean basin. vegetation of rocky coasts is composed of a typical flora, with halophilous chasmophytes and comophytes directly exposed to the action of marine aerosol, wind and waves, tolerating a high concentration of sodium chloride in the substrate and large temperature ranges. a large part of this vegetation belongs to the class crithmo-staticetea, which includes halophytic and halotolerant plant communities structurally formed by hemicryptophytes, chamaephytes and nanophanerophytes (rivas-martinez et al. 2002, fanelli et al. 2004, biondi et al. 2014). among the character species, many the crithmo-staticetea in the south-east of italy acta bot. croat. 78 (1), 2019 47 limonium species play a pivotal role, being represented by numerous endemics with a very restricted distribution area, and often including groups of vicariant species (dolcher and pignatti 1971, diana 1992). due to the presence of such endemics, most of the associations of the crithmo-staticion have endemic closely regional distribution. along the coasts of the mediterranean basin, and up to the black sea, the class is traditionally represented by only one order (crithmo-staticetalia), whilst another two have been described for the european atlantic coasts (crithmo-armerietalia maritimae, habitat code 1230), the canary island, the azores and morocco (frankenio-astydamietalia, habitat code 1250). a fourth order, helichrysetalia italici, was described to represent the sub-aerohaline coastal dwarf scrub vegetation on the inland edges of the mediterranean coasts (mucina et al. 2016). the coastline of the italian peninsula extends over nearly 8000 km. the apulia region is characterized by nearly 900 km of coast and represents the region of the italian peninsula with the longest coastline (giandonato 2003). many phytosociological surveys have been carried out along the apulian coast, on both sandy and rocky shores (e.g., cristofolini et al. 1967, curti and lorenzoni 1968, corbetta 1970, caniglia et al. 1984, géhu et al. 1984, corbetta et al. 1989, bartolo et al. 1992, brullo and de marco 1989, mariotti et al. 1992, beccarisi et al. 2003, biondi et al. 2006, corbetta et al. 2006, biondi and casavecchia 2010, tomaselli et al. 2011, pirone 2014, sciandrello and tomaselli 2014). the vegetation of the rocky coasts was classified in several associations of the orders crithmo-staticetalia, helichrysetalia italici and senecionetalia cinerariae. since the rocky coasts of the south-east of italy are almost entirely concentrated in the apulian region (biondi, 1999), these associations are representative of the class crithmo-staticetea in the south-east of italy. this paper aims to revise the syntaxonomy and nomenclature of the class crithmo-staticetea in the south-east of italy and to establish the floristic, coenological, syntaxonomic and synchorological relationships among its associations. several phytosociological papers have already dealt with this vegetation type in some parts of the apulian coast (e.g. cristofolini et al. 1967, curti and lorenzoni 1968, bartolo et al. 1992, biondi et al. 2006); however, there is still no overall revision carried out by using modern statistical treatments of phytosociological data. to this end, new original phytosociological data, sampled from the gargano promontory to the tip of the salento peninsula, and already published data were analysed together. moreover, since different syntaxonomic schemes of the crithmo-staticetea have been proposed for this area, the results have been discussed in the light of these different interpretations. materials and methods this revision is based on a data set consisting of 225 relevés from the apulia region (fig. 1). some of these relevés (123) were taken from phytosociological literature (tab. 1), selecting those that have already been classified in the crithmo-staticetea class. another 33 relevés published by biondi et al. (2006) and describing two communities dominated by arthrocaulon macrostachyum (= arthrocnemum macrostachyum), namely ‘arthrocnemum macrostachyum community’ and ‘limonio virgati-arthrocnemetum macrostachyi’, were added to the data set in order to define the syntaxonomic and floristic relationships with the crithmo-staticetea class, and clarify the typical zonation of the apulian rocky coast vegetation. in fact, these communities were originally classified in the arthrocnemion glauci alliance of the class salicornietea fruticosae but show floristic and habitat similarities with vegetation of the crithmo-staticetea growing in a close catenal relationship. for analogous reasons, we also added to the data set another three relevés assigned to the salicornietea fruticosae by mariotti et al. (1992). the data set includes also 66 new relevés carried out in rocky coastal habitats, along the distribution area of four apulian endemic limonium species (fig. 1, on-line suppl. tab. 1). three of them, l. apulum, l. diomedei and l. japygicum, were frequently recorded in the relevés whereas the fourth, l. peucetium, was not found. this latter was originally identified by pignatti (1982) from old plant material collected in the 19th century along the rocky coast near bari where, however, it is no longer observed (wagensommer et al. 2014, bartolucci et al. 2018). the new relevés were collected by using the braun-blanquet approach (westhoff and van der maarel 1980). for each sample, geographical coordinates, slope, exposure, distance from the shoreline, total vegetation cover and plot size, the latter ranging from 10 to 50 m2, were detected. species abundance–dominance values were estimated in the field by using the braun-blanquet scale. considering the entire set of relevés, the average plot size turned out to be 43.5 m2, with a median of 20 m2 and minimum and maximum values of 5 and 500 m2, respectively. fig. 1. map of the study area (apulia region, south-east of italy) with indications of the relevés’ locations. empty circles indicate the new relevés; full circles indicate relevés taken from phytosociological literature (see tab. 1). tomaselli v., terzi m. 48 acta bot. croat. 78 (1), 2019 relevés sampled on extremely large (> 100 m2) and extremely small (<10 m2) plots were excluded from the data analysis, since they could have affected the results of statistical analyses (see otýpková and chytrý 2006). taxa recorded only at the genus level as well as lichens and bryophytes were omitted from the data set. taxon scores originally recorded according to the braun-blanquet scale, were replaced with the ordinal scale as proposed by van der maarel (1979). the resulting data matrix consisted of 205 relevés sharing 115 taxa. relevés were hierarchically clustered by using flexible beta linkage, with the bray–curtis coefficient. beta was set at −0.25 so that flexible beta clustering became a space-conserving method (mccune and grace 2002). the dendrogram was pruned at the level yielding the highest number of indicator species (indsp). to this end, indicator species analysis (isa, dufrêne and legendre 1997) was run for the first 20 partitioning levels of the resulting dendrogram, further divisions dealing with minor variations. a taxon was considered as the indsp of a cluster for a given partition if its indicator value (indval, dufrêne and legendre 1997) turned out to be higher for that cluster than for the others of the same partition. the statistical significance (p < 0.01) of indval was assessed by means of a monte carlo test with 10000 permutations. in order to enhance the interpretability of the results, each indsp was assigned to only one cluster of relevés along the hierarchical descending typologies of the dendrogram, that is to the cluster for which the indval of that taxon first reached its maximum value (dufrêne and legendre 1997, see also terzi 2015). two taxa yielded the highest value for the first trivial partition with all the relevés in one cluster (fig. 2, cluster c1), and they were considered as diagnostic for the crithmo-staticetea class. the clusters of relevés were interpreted from a syntaxonomical viewpoint, on the basis of the occurrence of nomenclatural type-relevés within each of them. diagnostic species of syntaxa were selected from the indsp of the relevant cluster or of its subdivisions. the relevés were also ordinated by means of nonmetric multi-dimensional scaling (nmds), using the bray–curtis coefficient as a dissimilarity measure. the whole of the statistical analysis described above was carried out by using pc-ord software, version 6.22 (mccune and mefford, 2011). for nmds, the ‘slow and thorough’ option of the auto-pilot mode provided in pc-ord was used. taxonomic nomenclature follows bartolucci et al. (2018), except for plantago holosteum scop. subsp. grovesii (beg.) brullo. in fact, the taxonomic status of this taxon is controversial and requires further in-depth studies, being considered as a species by conti et al. (2005, ‘plantago grovesii’), as a subspecies of plantago holosteum by brullo (1988) and as a synonym of plantago subulata by hassemer et al. (2017). syntaxonomic nomenclature follows mucina et al. (2016), except where indicated. the nomenclatural decisions were taken according to the 3rd edition of the international code of phytosociological nomenclature (icpn, weber et al. 2000), whose articles (art) are cited within brackets. tab. 1. data sources of relevés of the crithmo-staticetea class and limonio-arthrocnemetum (arthrocnemion glauci) from apulia region, south-east of italy, taken from phytosociological literature and included in the data set. bibliographic source, number of relevés (no.), and original classification (at association/alliance level) are reported. source no. association and alliance as reported in the original paper bartolo et al. (1992: tab. 9, rel. 1–6) 6 ‘crithmo-limonietum apuli’, ‘crithmo-limonion’ bartolo et al. (1992: tab. 10, rel. 1–9) 9 limonietum japygici,‘crithmo-limonion’ bartolo et al. (1992: tab. 11, rel. 1–20) 20 crithmo-limonietum diomedei, ‘crithmo-limonion’ bartolo et al. (1992: tab. 12, rel. 1–8) 8 limonio virgati-plantaginetum grovesii, ‘crithmo-limonion’ bartolo et al. (1992: tab. 15, rel. 1–16) 16 agropyro-helycrisetum italici, ‘plantagini-thymelaeion hirsutae’ biondi et al. (2006: tab. 8, rel. 1–18) 18 ‘arthrocnemum macrostachyum comm.’, ‘arthrocnemion macrostachyi’ biondi et al. (2006: tab. 9, rel. 1–11) 11 limonio virgati-arthrocnemetum macrostachyi, ‘arthrocnemion macrostachyi’ biondi et al. (2006: tab. 9, rel. 12–15) 4 limonio virgati-arthrocnemetum macrostachyi subass. crithmetosum maritimi, ‘arthrocnemion macrostachyi’ biondi et al. (2006: tab. 10, rel. 1–7) 7 limonietum japygici, ‘crithmo-limonion’ biondi et al. (2006: tab. 11, rel. 1–3) 3 limonietum japygici subass. capparidetosum spinosae, ‘crithmo-limonion’ biondi et al. (2006: tab. 13, rel. 1–12) 12 crithmo maritimi-inuletum crithmoidis, ‘crithmo-limonion’ brullo and de marco (1989: tab. 3, rel. 34–42) 9 anthyllido-centaureetum diomedeae, ‘anthyllidion barbae-jovis’ caniglia et al. (1984: tab. 16, rel. 1–9) 9 ‘limonietum japygici subass. a salicornia e inula’, ‘crithmo-staticion s.l.’ cristofolini et al. (1967: tab. 1, rel. 1–6) 6 ‘crithmo-staticetalia’ curti and lorenzoni (1968: tab. 2, rel. 6–9) 4 limonietum japygici typicum, ‘crithmo-staticion’ curti and lorenzoni (1968: tab. 2, rel. 10–17) 8 ‘limonietum japygici subass. a salicornia fruticosa e inula crithmoides’, ‘crithmostaticion’ mariotti et al. (1992: tab. 5, rel. 29–31) 3 ‘aggr. a limonium virgatum e sarcocornia fruticosa’, ‘limonion galloprovincialis’ mariotti et al. (1992: tab. 10, rel. 53–54) 2 ‘frankenio laevis-limonietum cancellati’, ‘crithmo-limonion’ mariotti et al. (1992: tab. 10, rel. 55–58) 4 ‘frankenio laevis-limonietum cancellati’ ‘subass. sarcocornietosum fruticosae’, ‘crithmo-limonion’ the crithmo-staticetea in the south-east of italy acta bot. croat. 78 (1), 2019 49 results the dendrogram was pruned to give 16 clusters of relevés (fig. 2). few clusters turned out to be clearly differentiated from the others by having a high number of indsp (e.g., clusters 5b, 7b); most have no or few indsp (fig. 3). therefore, only 9 out of these 16 clusters can be considered as representative of associations, the others representing only minor variations without syntaxonomic relevance. cluster 2a includes the relevés dominated by arthrocaulon macrostachyum. further subdivision of this cluster does not show a clear distinction between the ‘arthrocnemum macrostachyum community’ and ‘limonio virgate-arthrocnemetum macrostachyi’, the relevés of these two community types being mixed together in clusters 16a and 16b. the relevés of these clusters are characterized by very low species richness (1-6 taxa for relevés), with four indsp: arthrocaulon macrostachyum, halimione portulacoides, limonium narbonense and juncus maritimus (fig. 3). the relevés with limonium apulum are grouped in clusters 9b and 10a. few indsp characterize these clusters and, with the exception of l. apulum, all of them have a low indval and are usually considered diagnostic for other vegetation types (e.g., suaeda vera for the salt-marsh shrub vegetation of the salicornietea fruticosae, parapholis incurva for the therophytic vegetation of the saginetea maritimae). cluster 9a has no indsp and includes relevés dominated by limbarda crithmoides subsp. longifolia and without limonium apulum. fig. 2. flexible beta clustering of relevés of the crithmo-staticetea class and limonio-arthrocnemetum (arthrocnemion glauci) from apulia region, south-east of italy. each cluster of the first 16 partitions of the dendrogram, is identified by a two digit code: the number refers to the partitioning level, while the letters (‘a’ or ‘b’) identify the two clusters originating at that partition. the first trivial partition, with all the relevés in only one group, is indicated as ‘c1’. ab – anthyllido barbae-jovis-centaureetum diomedeae; ah – agropyro pungentis-helichrysetum italici; am1/2 – limonio virgati-arthrocnemetum macrostachyi; ci: crithmo maritimi-inuletum crithmoidis; la1/2 – crithmo maritimi-limonietum apuli; ld: crithmo maritimi-limonietum diomedei; lj – limonietum japygici typicum; pg – limonio virgati-plantaginetum grovesii; sf – relevés with salicornia fruticosa. fig. 3. results of the indicator species analysis, according to dufrêne and legendre (1997), for the first 16 partitioning levels of the dendrogram (see fig. 2). indicator species associated to each cluster are reported together with the corresponding indicator values (within brackets). each cluster of the dendrogram is identified by a two digit code: the number refers to the partitioning level, while the letters (‘a’ or ‘b’) identify the two clusters originating at that partition. the first trivial partition, with all the relevés in only one group, is indicated as ‘c1’. tomaselli v., terzi m. 50 acta bot. croat. 78 (1), 2019 this cluster represents the crithmo-inuletum. cluster 10b includes the relevés of the limonio-plantaginetum grovesii that is a typical aspect of coastal vegetation, developing in a restricted area on marls near otranto. this cluster is characterized by several indsp, such as plantago holosteum subsp. grovesii, pl. macrorhiza and pl. crassifolia. cluster 4b and its subdivisions represent the limonietum japygici. few indsp are associated with these clusters and they do not support the identification of more than one subassociation, the typical one. a second subassociation is represented by cluster 6b, which includes relevés already classified in the subassociation ‘a salicornia e inula’ by curti and lorenzoni (1968) and caniglia et al. (1984) together with others previously assigned to the ‘frankenio laevis-limonietum cancellati subass. sarcocornietosum fruticosae’ by mariotti et al. (1992). the relevés from the gargano and tremiti coasts are grouped in cluster 3b. the three main subdivisions of this cluster represent the agropyro-helichrysetum italici (cluster 5b), anthyllido-centaureetum diomedeae (7b) and crithmolimonietum diomedei (7a), respectively. the first two associations turned out to be differentiated by many indsp, some of them having a high indval (fig. 3). the crithmo-limonietum diomedei (7a) is recognizable for the occurrence of the sole limonium diomedei, and its two sub clusters (13a and 13b) are not associated to any indsp. the nmds ordination resulted in a three-axis solution, with a final stress of 14.7. the three axes accounted for 71.3% of the variance in the bray–curtis dissimilarity matrix (first axis 47.8%, second axis 11.7% and third axis 11.9%). in the nmds diagram (fig. 4), axis 1 clearly separates the communities dominated by arthrocaulon macrostachyum (am) on one side, and agropyro-helichrysetum italici (ah), anthyllido-centaureetum diomedeae (ab) and crithmo-limonietum diomedei (ld) on the other. in the middle, there are the other vegetation types without clear separation among them (a similar situation is observed on the diagram for axis 1 and 2 – data not shown). axis 3 roughly separates the relevés of the limonietum japygici (filled circular marks in fig. 4: lj), concentrated in the lower part of the diagram, from those of the crithmo-limonietum apuli (la), limonio-plantaginetum grovesii (pg) and crithmo-inuletum (ci), which are in the upper part. results of the nmds ordination approximately confirm the general syntaxonomic pattern highlighted by the cluster analysis. a synoptic table (tab. 2) summarizes the nine groups identified, with the percentage frequency for each species in each plant community. discussion the syntaxonomic classification of the rocky coastal vegetation of the italian peninsula has been subjected to numerous changes and revisions in the last decades. in a thorough survey on the crithmo-staticetea class, bartolo et al. (1992) reported two alliances of the order crithmo-staticetalia for the italian peninsula, crithmo-staticion and plantagini-thymelaeion hirsutae, with the first one including the halophilous pioneer communities next to the coastline, and the second one including halotolerant shrub vegetation, located towards the hinterland and in catenal contact with the crithmo-staticion. the plantagini-thymelaeion hirsutae was, however, originally invalidly published (art. 5, 8 and 3g icpn). subsequently mayer (1995: 101) explicitly listed the diagnostic taxa of the alliance – as required by article 8 of the icpn. however, the alliance remained invalid because it is unclear from what plantago species the alliance name is formed (art 3g) and because it still lacks the nomenclatural type (art. 5). brullo and de marco (1989) described another alliance, anthyllidion barbae-jovis, for the halotolerant nanophanerophytic vegetation of vertical high cliffs next to the coast, including this syntaxon in the order crithmo-staticetalia. subsequently, the anthyllidion barbae-jovis and plantagini-thymelaeion hirsutae were moved to the helichrysetalia italici, of the class helichryso-crucianelletea maritimae (biondi et al. 1997, biondi 1999). in a subsequent contribution, biondi (2007) separated the chasmophytic halophilous pioneer vegetation of the crithmo-staticetalia from the chomophytic and halotolerant vegetation dominated by chamaephytes as well as nanophanerophytes, clearly distinct from both an ecological and a physiognomic-structural point of view (see fanelli et al. 2004), and described a new order, senecionetalia cinerariae, for these last communities. the alliance anthyllidion barbae-jovis was thus attributed to this new order, whilst halotolerant dwarf shrub communities were included in the helichrysion litorei, of the helichrysetalia italici (biondi 2007; biondi et al. 2014). as pointed out by biondi et al. (2013), the helichrysion litorei was originally fig. 4. nonmetric multi-dimensional scaling ordination of relevés of the crithmo-staticetea class and limonio-arthrocnemetum (arthrocnemion glauci) from the apulia region, south-east of italy. ab ‒ anthyllido barbae-jovis-centaureetum diomedeae; ah ‒ agropyro pungentis-helichrysetum italici; am ‒ limonio virgati-arthrocnemetum macrostachyi; ci ‒ crithmo maritimi-inuletum crithmoidis; la ‒ crithmo maritimi-limonietum apuli; ld ‒ crithmo maritimilimonietum diomedei; lj ‒ limonietum japygici typicum; pg ‒ limonio virgati-plantaginetum grovesii; sf ‒ relevés with salicornia fruticosa. the crithmo-staticetea in the south-east of italy acta bot. croat. 78 (1), 2019 51 invalidly described because the sole association of its original diagnosis, the senecioni-helichrysetum litorei barbagallo, brullo et signorello 1983, had been in turn invalidly published, it being unclear from what senecio species the association name was formed (art. 3g). biondi (in: biondi et al. 2013) described the new association ‘senecioni bicoloris-helichrysetum litorei’ (cf art. 6) but failed to validate the alliance helichrysion litorei. in fact, biondi (in: biondi et al. 2013) designated as nomenclatural type of the alliance the invalid ‘senecioni-helichrysetum litorei barbagallo, brullo et signorello 1983’ instead of the new and valid senecioni bicolorishelichrysetum litorei biondi in biondi et al. 2013. more recently, mucina et al. (2016) proposed a new classification scheme. according to this contribution, two orders of the crithmo-staticetea can be recognized for the italian peninsula: the crithmo-staticetalia for the rupicolous vegetation of salt-sprayed cliffs, and the helichrysetalia italici for the sub-aerohaline coastal dwarf scrub on the inland edges of salt-sprayed cliffs of the seaboards. within the crithmo-staticetalia, two alliances are recognized: crithmo-staticion for the rupicolous dwarf-herb vegetation of salt-sprayed limestone cliffs of the tyrrhenian and ligurian coasts, and limonion anfracti-cancellati for the rupicolous herb-rich vegetation of salt-sprayed rocky cliffs of the adriatic coasts. the tab. 2. abridged synoptic table of the crithmo maritimi-staticetea class in the south-east of italy (taxon scores represent the taxa percentage frequencies). columns: i – arthrocaulon macrostachyum group (limonio virgati-arthrocnemetum macrostachyi); ii – limbarda crithmoides group (crithmo maritimi-inuletum crithmoidis); iii – limonium apulum group (crithmo-limonietum apuli); iv – plantago holosteum subsp. grovesii group (limonio virgati-plantaginetum grovesii); v – salicornia fruticosa group (limonietum japygici salicornietosum fruticosae); vi – limonium japygicum group (limonietum japygici); vii – limonium diomedeum group (chrithmo-limonietum diomedei); viii – anthyllis barba-jovis group (anthyllido-centauretum diomedeae); ix – helichrysum italicum group (agropyro-helichrysetum italici). group i ii iii iv v vi vii viii ix no. of relevès 42 23 25 6 12 43 29 9 16 char. and diff. species of associations limonium apulum 0 0 90 0 25 0 0 0 0 plantago holosteumsubsp. grovesii 0 0 0 67 0 0 0 0 0 limonium japygicum 21 12 0 0 50 98 0 0 0 centaurea diomedea 0 0 0 0 0 0 0 100 0 asperula staliana subsp. diomedea 0 0 0 0 0 0 0 89 0 aurinia leucadea 0 0 0 0 0 0 0 44 0 all. limonion anfracti-cancellati limonium diomedeum 2 0 0 0 0 0 100 44 13 cl. crithmo-staticetea, ord. crithmo-staticetalia, all. crithmo-staticion crithmum maritimum 31 100 97 100 33 86 100 100 94 limonium virgatum 43 65 84 100 100 51 41 0 19 plantago macrorrhiza 0 0 0 67 0 12 0 0 0 lotus cytisoides 0 29 58 50 17 56 86 78 94 frankenia hirsuta 12 24 32 0 8 33 7 0 0 allium commutatum 0 6 32 33 0 5 45 56 69 daucus carota subsp. hispanicus 0 6 3 50 0 7 62 89 94 frankenia laevis subsp. laevis 14 6 6 0 67 2 0 0 0 plantago holosteum subsp. scopulorum 0 0 0 0 0 0 17 56 19 senecio leucanthemifolius subsp. leucanthemifolius 0 0 0 0 0 0 3 0 0 reichardia picroides 0 29 29 67 0 37 62 78 75 ord. helichrysetalia italici, all. anthyllidion barbae-jovis and all. helichrysion litorei anthyllis barba-jovis 0 0 3 0 0 0 0 89 0 matthiola incana subsp. incana 0 0 0 0 0 0 0 67 0 helichrysum italicum subsp. pseudolitoreum 0 0 0 0 0 0 17 56 0 helichrysum italicum subsp. italicum (d) 0 0 0 0 0 0 0 0 100 thymelaea hirsuta 0 0 0 0 0 0 0 0 62 cl. salicornietea fruticosae, ord. salicornietalia fruticosae, all. arthrocnemion glauci arthrocaulon macrostachyum 100 54 58 0 8 37 48 0 0 limbarda crithmoides subsp. longifolia 19 100 19 33 67 19 10 0 0 suaeda vera 7 54 13 0 0 2 28 0 13 halimione portulacoides 24 24 0 0 17 7 0 0 0 salicornia fruticosa 7 0 0 0 92 0 0 0 0 limonium narbonense 21 0 0 0 8 0 0 0 0 puccinellia festuciformis subsp. festuciformis 2 0 0 0 0 0 0 0 0 tomaselli v., terzi m. 52 acta bot. croat. 78 (1), 2019 plantagini-thymelaeion hirsutae and helichrysion litorei are dealt with as syntaxonomic synonyms of the anthyllidion barbae-jovis, within the helichrysetalia italici. according to the syntaxonomic proposal of mucina et al. (2016), the associations limonio virgati-plantaginetum grovesii, crithmolimonietum apuli, crithmo maritimi-inuletum crithmoidis and limonietum japygici should be classified in the crithmostaticion, whilst the crithmo-limonietum diomedei has to be ascribed to the adriatic alliance limonion anfracti-cancellati. on the other hand, the anthyllido-centauretum diomedeae and agropyro-helichrysetum italici should be classified in the anthyllidion barbae-jovis which encompasses both the nanophanerophytic vegetation of the high cliffs (anthyllido-centauretum diomedeae) and the coastal dwarf scrub communities (agropyro-helichrysetum italici). however, these two vegetation types are characterized by different structures and ecological requirements, and in the end by a different floristic composition. the clusters representing these two associations turned out to be clearly distinguished, being separated both in the ordination diagram (fig. 4) and in the dendrogram (fig. 2). moreover, each of them was characterized by many indsp, indicating their floristic autonomy (fig. 3). therefore, in our opinion, these two associations should be kept at least in separate alliances, as already proposed by other authors (biondi 2007; pirone 2014; biondi et al. 2014). as a consequence, the helichrysion litorei is here validated (see below) and the agropyro-helichrysetum italici is classified in this alliance. as regards the spatial distribution and vegetation zonation of the rocky coasts, several belts can be described (fig. 5 and 6). in a thorough analysis of the coastal vegetation of the salento peninsula (south apulia), biondi et al. (2006) described a first belt, subject to continuous salt-water spraying and characterized by plant communities formed by succulent chamaephytes or nanophanerophytes such as arthrocaulon macrostachyum (arthrocaulon macrostachyum communities and limonio virgati-arthrocnemetum macrostachyi) or by limbarda crithmoides (crithmo maritimi-inuletum crithmoidis), depending on the geological nature of the substrate (cluster 16a-16b and 8a in fig. 2). although these communities with arthrocaulon macrostachyum develop on rocky substrates and in close catenal contact with communities of the crithmo-staticetea class, they were originally classified in the arthrocnemion glauci, of the salicornietea fruticosae class (biondi et al. 2006). in our analyses, this vegetation type turned out to be well differentiated from all the other types (fig. 2) and characterized by few indsp, most of them of the salicornietea fruticosae class. in two previous contributions about the vegetation of some coastal sites of southern apulia, curti and lorenzoni (1968) described a subassociation of the limonietum japygici with ‘salicornia fruticosa e inula crithmoides’ whereas mariotti et al. (1992) described the subassociation sarcocornietosum fruticosae of the frankenio laevis-limonietum cancellati (here considered as a synonym of the crithmo-limonietum apuli). in our numerical analyses, these relevés segregate into a group (6b) characterized by salicornia fruticosa and including both subassociations. the first subassociation could be considered as a halophilous aspect of the limonietum japygici, subject to periodic submersion with stagnation of sea water, due to the low elevation above sea level (see below). however, as regards the subassociation ‘sarcocornietosum fruticosaei’ of the frankenio laevis-limonietum cancellati, described by mariotti et al (1992) in the site of torre guaceto, it is necessary to point out that, during our surveys in torre guaceto and other coastal sites of the same area, in the same habitat types we have found arthrocaulon macrostachyum instead of salicornia fruticosa. therefore, we suppose that the frankenio laevis-limonietum cancellati salicocornietosum fruticosae could actually refer to a transition between the crithmo-limonietum apuli and limonio virgati-arthrocnemetum macrostachyi. following the zonation, the belt immediately towards the inland is occupied by plant communities characterized by the presence of limonium sp.pl. (figs. 5 and 6). in the apulian region, four vicariant and endemic associations have been described. the limonietum japygici is distributed along the south-western part of the apulian coast and is characterized by limonium japygicum, endemic to the salento peninsula, from taranto to otranto (le) (pignatti 1971, pignatti et al. 2014). this association was invalidly published by curti and lorenzoni (1968) because its name-giving taxon, limonium japygicum, had not been validly published. this taxon has recently been validated (pignatti et al. 2014), and consequently the limonietum japygici and its two subassociations (typicum and sarcocornietosum fruticosae) are here validated. based on the results of our revision, these two subassociations are sufficient to describe the floristic variability of this association. the crithmo-limonietum apuli, extendfig. 5. schematic outline of the distribution of plant communities on rocky coasts of central-southern apulia: a) arthrocnemion glauci (limonio virgati-arthrocnemetum macrostachyi)/ crithmo maritimi-staticion (crithmo-inuletum crithmoidis); b) crithmo maritimi-staticion (limonietum japygici in southern apulia / crithmo-limonietum apuli in central apulia); c) juniperion turbinatae or oleo-ceratonion. the crithmo-staticetea in the south-east of italy acta bot. croat. 78 (1), 2019 53 ing along the adriatic coast of apulia from otranto to the gargano peninsula, is characterized by limonium apulum, a taxon belonging to the l. cancellatum group and endemic to the adriatic coast of the apulia region (brullo et al. 1990, bogdanović et al. 2012). this association was effectively published in 1992, although the year 1989 is reported on the first page of the article by bartolo et al. (1992). in the same year, another association, frankenio laevis-limonietum cancellati, was described for torre guaceto for a similar habitat type (mariotti et al. 1992). according to the results of our analysis, these two associations should be united. in fact, the nomenclatural types of the crithmo-limonietum apuli and frankenio laevis-limonietum cancellati were grouped in the same cluster (fig. 2: cluster la2). having no information about the month of publication of the two volumes where these associations were validly published (vol. 47 of candollea and vol. 19 of colloques phytosociologiques, both published in 1992), we choose to retain the name crithmo-limonietum apuli. in fact, limonium cancellatum, one of the name-giving taxa of the other association, was excluded from the flora of italy (wagensommer et al. 2012) so that frankenio laevis-limonietum cancellati turns out to be incorrect. the limonio virgati-plantaginetum grovesii is a restricted endemic association that develops on marl substrates near the alimini lakes (otranto), within the distribution range of the crithmo-limonietum apuli. it is characterized by plantago holosteum subsp. grovesii and differentiated by the higher frequency and cover of plantago macrorhiza and pl. crassifolia. the limonio virgati-plantaginetum grovesii could be considered a geographic vicariant of the plantagini holostei-staticetum cancellatae (i.e. the lectotype of the limonion anfracti-cancellati, cf. mucina et al. 2016) that has been recorded in numerous localities along the croatian coasts (horvatić 1934, 1939, pandža et al. 2007, stančić et al. 2008). the crithmo-limonietum diomedei is characterized by limonium diomedeum which is endemic to the tremiti islands and gargano peninsula (perrino and wagensommer 2011) and is considered as a vicariant of l. vestitum which is endemic to the kamik islet of the vis archipelago in central dalmatia (brullo 1988, bogdanović and brullo 2015). following the syntaxonomic scheme proposed by mucina et al. (2016), this association has been here classified in the limonion anfracti-cancellati. nevertheless, this last attribution relies on the presence of one only diagnostic species of the alliance, that is, limonium diomedeum. more generally, the floristic differences between this alliance and the crithmo-staticion rely on few character/differential stenoendemic taxa. for this reason, in our opinion, further investigations are required in order to confirm the autonomy of the limonion anfracti-cancellati as a distinct alliance. a third zone, sheltered from the direct action of marine waters, is characterized by halotolerant shrub communities, with different ecologies and structures (fig. 6). the agropyrohelichrysetum italici garrigues are discontinuously distributed along the adriatic coasts of apulia, in some localities near brindisi and along the gargano and tremiti coasts. this association makes catenal contact with the crithmo-limonietum apuli, and with the crithmo-limonietum diomedei, depending on the geographic zone. the anthyllido-centaureetum diomedeae brullo et de marco 1990, on the high cliffs of the gargano peninsula and tremiti islands, makes catenal contact with the crithmo-limonietum diomedei communities. fig. 6. schematic outline of the distribution of plant communities on rocky coasts of northern apulia (gargano and tremiti islands): a) limonion anfracti-cancellati (crithmo maritimi-limonietum diomedei); b) helichrysion litorei (agropyro pungentis-helichrysetum italici); c) anthyllidion barbae-jovis (anthyllido barbae-jovis-centaureetum diomedeae); d) juniperion turbinatae or oleo-ceratonion. tomaselli v., terzi m. 54 acta bot. croat. 78 (1), 2019 syntaxonomic scheme cl. crithmo maritimi-staticetea br.-bl. in br.-bl. et al. 1952* ord. crithmo maritimi-staticetalia molinier 1934* all. crithmo maritimi-staticion molinier 1934* limonietum japygici curti et lorenzoni ex tomaselli et terzi ass. nov hoc loco [holotypus: see below the limonietum japygici typicum; synonym: limonietum japygici curti et lorenzoni 1968 (art. 3l)] limonietum japygici typicum subass. nov. hoc loco [holotypus rel. 49 of the on-line supplement tab. s1, 15 may 2014, locality ponte ciolo (le), italy: allium commutatum +, capparis spinosa s.l. +, catapodium balearicum +, crithmum maritimum 3, dittrichia viscosa subsp. viscosa +, elymus acutus 1, limonium japygicum 1, lotus cytisoides 1, silene sedoides subsp. sedoides +, silene vulgaris subsp. tenoreana 1]; limonietum japygici salicornietosum fruticosae subass. nov. hoc loco [holotypus rel. 14, tab. 2, in curti and lorenzoni 1968: 887]; crithmo maritimi-limonietum apuli bartolo, brullo et signorello 1992 [syn. frankenio laevis-limonietum cancellati mariotti et al. 1992] crithmo maritimi-inuletum crithmoidis biondi, casavecchia et guerra 2006 limonio virgati-plantaginetum grovesii bartolo, brullo et signorello 1992 all. limonion anfracti-cancellati (horvatić 1934) mucina in mucina et al. 2016 crithmo maritimi-limonietum diomedei bartolo, brullo et signorello 1992 ord. helichrysetalia italici biondi et géhu in géhu et biondi 1994 all. anthyllidion barbae-jovis brullo et de marco 1989 anthyllido barbae-jovis-centaureetum diomedeae brullo et de marco 1989 all. helichrysion litorei biondi ex tomaselli et terzi all. nov. hoc loco [holotypus: senecioni bicoloris-helichrysetum litorei biondi in biondi, allegrezza, casavecchia, galdenzi, gigante et pesaresi 2013 (p. 189); diagnostic taxa (cf. biondi 2007): helichrysum litoreum, helichrysum italicum subsp. pseudolitoreum, helichrysum italicum subsp. italicum (diff.), jacobaea maritima subsp. bicolor, thymelaea hirsuta; synonyms: plantagini-thymelaeion hirsutae bartolo et brullo in bartolo, brullo et signorello 1992 nom. inval. (art. 3g, 5, 8); plantagini-thymelaeion hirsutae bartolo et brullo ex mayer 1995 (art. 3g, 5); helichrysion litorei biondi 2007 nom. inval. (art. 5); helichrysion litorei biondi in biondi, allegrezza, casavecchia, galdenzi, gigante et pesaresi 2013 nom. inval. (art. 5)] agropyro pungentis-helichrysetum italici bartolo, brullo et signorello 1992 cl. salicornietea fruticosae br.-bl. et tx. ex a. de bolos y vayreda et o. de bolos in a. de bolos y vayreda 1950 ord. salicornietalia fruticosae br.-bl. 1933 all. arthrocnemion glauci rivas-mart. et costa m. 1984 limonio virgati-arthrocnemetum macrostachyi biondi, casavecchia et guerra 2006 *as already pointed out by mucina et al. 2016, the presence of more than one species of the genus limonium in the original diagnoses of the crithmo-staticetea, crithmo-staticetalia and crithmo-staticion, do not allow the addition of any epithet to the original forms of the names. however, since only one species of the genus crithmum occurs (i.e. cr. maritimum), the epithet has been added. acknowledgements we thank jean-paul theurillat and laco mucina for their help in resolving a nomenclatural issue. we are also grateful to two anonymous referees for their comments and suggestions. references barbagallo, c., 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(ed.), classification of plant communities, 2nd edition, 287–399. junk, the hague, nl. acta bot. croat. 79 (2), 2020 105 acta bot. croat. 79 (2), 105–113, 2020 coden: abcra 25 doi: 10.37427/botcro-2020-019 issn 0365-0588 eissn 1847-8476 validation of variants using cost effective highresolution melting (hrm) analysis predicted from target re-sequencing in eucalyptus abdul bari muneera parveen, divya lakshmanan, modhumita ghosh dasgupta* institute of forest genetics and tree breeding, r.s. puram, coimbatore–641002, tamil nadu, india abstract – the advent of next-generation sequencing has facilitated large-scale discovery and mapping of genomic variants for high-throughput genotyping. several research groups working in tree species are presently employing next generation sequencing (ngs) platforms for marker discovery, since it is a cost effective and time saving strategy. however, most trees lack a chromosome level genome map and validation of variants for downstream application becomes obligatory. the cost associated with identifying potential variants from the enormous amount of sequence data is a major limitation. in the present study, high resolution melting (hrm) analysis was optimized for rapid validation of single nucleotide polymorphisms (snps), insertions or deletions (indels) and simple sequence repeats (ssrs) predicted from exome sequencing of parents and hybrids of eucalyptus tereticornis sm. × eucalyptus grandis hill ex maiden generated from controlled hybridization. the cost per data point was less than 0.5 usd, providing great flexibility in terms of cost and sensitivity, when compared to other validation methods. the sensitivity of this technology in variant detection can be extended to other applications including bar-hrm for species authentication and tilling for detection of mutants. key words: exome sequencing, genotyping, high resolution melting analysis, validation, variants * corresponding author e-mail: gmodhumita@gmail.com introduction high resolution melting (hrm) for dna analysis is a closed-tube analysis system and post real-time pcr analytical technique based on the principle that the melting curves of dna fragments vary depending on base composition (montgomery et al. 2007). the melting kinetics from the amplification profile of hrm-designed primers facilitates scanning and cataloging of single nucleotide polymorphisms (snps), mutations, and methylation in the genomic signature of individual species (wojdacz and dobrovic 2007, toi and dwyer 2008, wittwer 2009). the advantages of hrm analysis include reduced risk of cross contamination and decreased analytical time. it is considered an accurate and sensitive pcr-based genotyping technology for cost-effective screening of genetic markers without a priori knowledge of the variation in template dna sequence (wittwer et al. 2003). this fluorescence-based melting analysis allows greater discrimination between homozygote and sensitive detection of heterozygote (montgomery et al. 2007). hrm based genotyping has been used for snp (han et al. 2012, marshall et al. 2015, gomes et al. 2018), insertion/ deletion (indel) (zhou et al. 2015) and simple sequence repeat (ssr) genotyping (mackay et al. 2008, distefano et al. 2012) in several plant species like vitis vinifera l., podocarpus l'hér. ex pers. and medicago sativa l. further, the applications of hrm has also been extended to analysis of candidate dna barcode marker as reported in leguminous forage and pasture species (ganopoulos et al. 2012), crocus sativus l. (jiang et al. 2014), sideritis l. (kalivas et al. 2014), phyllanthus amarus schumach. et thonn. (buddhachat et al. 2015), calendula officinalis l. (schmiderer et al. 2015), artemisia l. (song et al. 2016) and senna alexandrina mill. (mishra et al. 2018), where hrm was reported as an efficient molecular tool to authenticate species of traded medicinal plants. targeting induced local lesions in genomes (tilling) is a high-throughput technique and reverse genetic strategy to identify induced mutants. it involves steps such as endonuclease digestion, cloning and electrophoresis, which are critical and time-consuming. in addition, lack of complete genome sequence information for many plant species has limited the development of suitable tilling targets. taheri et al. (2017) reported that tilling-hrm is a mumuneera parveen a b, lakshmanan d, ghosh dasgupta m 106 acta bot. croat. 79 (2), 2020 tant detecting method that is accurate, sensitive, fast and cost-effective when compared to conventional methods. in plants, tilling-hrm is reported in wheat for the detection of ems induced mutation (botticella et al. 2011), solanum lycopersicum l. (gady et al. 2009) and brassica rapa l. (lochlainn et al. 2011) for detection of point mutation, and oryza sativa l. (shan et al. 2018) for screening γ rayinduced mutations. however, in woody perennials, use of this technology is limited to gene based snp marker assay in sweet cherry (ganopoulos et al. 2013), validation of sex-linked markers in pistacia vera l. (kafkas et al. 2015) and identification of genetically distinct species in podocarpus sp. (marshall et al. 2015). the genus eucalyptus l'hér. is a widely planted hardwood species because of its superior growth, adaptability and wood properties and occupies 19.61 million hectares globally. this genus is targeted world-wide for genetic improvement programs due to its high commercial value as raw material for the paper and pulp industries. in eucalypts, application of ngs technology has led to the identification of genetic markers for linkage map construction (grattapaglia et al. 2011, neves et al. 2011, bartholomé et al. 2015), genetic diversity analyses (novaes et al. 2008, dillon et al. 2014), genome wide association studies (cappa et al. 2013, silva-junior et al. 2015) and marker-assisted selection (resende et al. 2012). however, marker prediction using deep sequencing has been predominantly validated using sanger sequencing (novaes et al. 2008) and goldengate assay (grattapaglia et al. 2011). to our knowledge, no reports on the use of hrm analysis for confirmation of variants (snps/ indels/ ssrs) are reported in forest tree species, including eucalyptus. in our previous study, we reported the use of target capture and deep sequencing of xylogenesis-related genes for identification of high-throughput genetic markers in three eucalyptus species for family-based qtl and association analysis (dasgupta et al. 2015). the study was further extended and 7 genes were selected for sequencing in parents, eucalyptus tereticornis sm. (clone et86) and eucalyptus grandis hill ex maiden (clone eg9) and their hybrids generated from controlled hybridization for variant identification. the present study was undertaken to demonstrate the use of hrm-based genotyping for rapid validation of predicted variants (snps/indels/ssrs) in both heterozygous and homozygous conditions in parents and hybrids. the use of hrm as a rapid, homogenous, highly specific, sensitive and cost effective methodology for high-throughput validation of markers in eucalyptus is reported. materials and methods plant material and dna isolation a bi-parental mapping population of eucalyptus tereticornis (clone et86) × eucalyptus grandis (clone eg9) was developed for construction of linkage map and qtl tagging for wood property traits (rajasugunasekar et al. 2015). the leaf tissues from the two parents and eight hybrids (h13, h48, h137, h160, h190, h218, h265, and h275) were harvested and immediately frozen at −80 °c. genomic dna was isolated from the leaf tissues using the arboreasy dna isolation kit (institute of forest genetics and tree breeding, india) and quantified using a nanodrop nd1000 spectrophotometer (thermo scientific, usa). target capture and deep sequencing seven genes presumed to be involved in wood formation (on-line suppl. tab. 1) were mined from literature (dasgupta et al. 2015) for in-solution target capture and deep sequencing in parents and hybrids. the regions targeted for capture included the exon, 3’ and 5’ utrs and 500-1000 bp upstream regions. approximately 200 ng of genomic dna from each sample was made up to 50 µl with tris edta buffer and sonicated for ~210 s to fragment dna into a size range of 200 to 400 bp. the size distribution was checked on the agilent high sensitivity tape station. subsequently, libraries for each sample were constructed using sureselectxt target enrichment system for illumina paired-end sequencing (agilent technologies, santa clara, california, usa) using the manufacturer’s protocol. the library was hybridized at 65 °c for 65 hours and the captured library was isolated using magnetic streptavidin-coated beads (thermo fisher scientific, usa). the captured library was pcr amplified (12 cycles) and purified using the highprep pcr clean-up system (magbio genomics, md, usa). the enriched amplified library was quantified using qubit fluorometer (thermo scientific, usa) and 2 × 150 bp paired end sequencing was conducted using nextseq 550 system (illumina inc., san diego, ca, usa). raw reads were quality checked using fastqc (andrews, 2010). the quality passed raw reads were processed using trim galore (krueger, 2015) for adapter clipping and low quality base trimming with filtering criteria of minimum read length 20 bp and minimum base quality q30. the processed reads were aligned to the reference e. grandis genome database hosted by the phytozome portal (goodstein et al. 2012) using bowtie 2–2.0.5 (langmead and salzberg 2012) allowing one mismatch in a seed length of 22. the consensus sequences of all ten files were generated to identify the variants with samtools v.1.23 (li 2011) and varscan v. 2.34 (koboldt et al. 2009). the genes were sequenced with an average read depth of 268.62 x to 561.72 x. the regions that were monomorphic between the parent were filtered out and positions that had polymorphic alleles between the parents were further explored in all the eight progenies and polymorphic variants (snps and indels) were cataloged. the presence of polymorphic ssrs across parents and hybrids were mined from the consensus sequences using misa computational tool (beier et al. 2017). in the present study, hrm analysis was conducted in the parents (et86 and eg9) and eight hybrids (h13, h48, h137, h160, h190, h218, h265, and h275) (tab. 1). validation of variant using hrm analysis acta bot. croat. 79 (2), 2020 107 synthesis of lignin, cellulose, xyloglucan, cell wall formation and phytohormone signaling were selected (on-line suppl. tab. 1) based on our earlier study (dasgupta et al. 2015). the length of the gene, position of the variants and their biological functions are presented in on-line suppl. tab. 1. the presence of polymorphic snps, indels and ssrs across parents and eight hybrids was predicted using different computational pipelines (tab. 1). the raw sequence data was deposited in ncbi short read archive with the accession number srp152786 including eucalyptus tereticornis (srx4367138), e. grandis (srx4367137), eucalyptus hybrids h13 (srx4367139), h48 (srx4367141), h137 (srx4367144), h160 (srx4367136), h190 (srx4367130), h218 (srx4367129), h265 (srx4367132) and h275 (srx4367131). dna isolation and primer design hrm analysis was conducted for two eucalyptus species (e. tereticornis and e. grandis) and eight hybrids generated by controlled hybridization. total dna isolated from leaves of two parents and hybrids was quantified and the concentration ranged from 173.5 ng μl–1 to 868.8 ng μl–1 with od 260/280 = 1.69 to 1.98 across all samples. seven primer pairs amplifying fragments less than 160 bp were chosen for the study. hrm analysis for variant determination hrm assay targeted four snp regions of four different genes to determine if the hrm curves were in concordance with the exome sequencing results as shown in tab. 1. distinct hrm melting curves were observed for each snp genotype in the parents and hybrids, each of which is represented ïn different color (tab. 1, figs. 1–4, on-line suppl. figs.1–4). in all samples evaluated, the replicates resulted in similar curves and were assigned to the same group, indicating the consistency and reproducibility of the hrm assay. all the four snp markers produced polymorphic melting curves among the parents and hybrids investigated through hrm analysis. all the three snps (bp, paapa and xth) showed polymorphic melting curves differentiating between the homoprimer design and hrm analysis primer pairs targeting specific snps, indels and ssrs were designed using beacon designer version 7.90 (on-line suppl. tab. 2). primers were designed following the criteria: predicted annealing temperature (tm) of 58–61 °c, limited self-complementarity and amplicon lengths of 80–160 bp. pcr optimization was done to ascertain amplification of single product. after testing of different combinations of primers and template dna concentrations, the reaction was optimized at 30 ng of dna, 100 nmol l–1 of each primer, 0.8 mg ml–1 bsa, 2.5 mm mgcl2 and 5 µl of kappa fast hrm master mix (sigma, st. louis, mo, usa) in a total volume of 10 µl. as suggested by the manufacturer, the amplification cycle was initially performed in two steps and the pcr program was as follows: 95 °c for 10 minutes; 40 cycles of 95 °c for 15 seconds, 55–60 °c for 1 minute (on-line suppl. tab. 2) and a dissociation cycle of 95 °c for 10 seconds, 60 °c for 1 minute, and 95 °c for 15 seconds (ramp rate, 0.3%) was used. quantitative real time pcr was performed in the abi prism 7500 step one plus sequence detection system and hrm curves were analyzed using hrm software version 2.3 (applied biosystems, usa). pcr amplification was analyzed through the assessment of the ct value, end point fluorescence level, and the amplification efficiency for data qc. data from low quality amplification including runs with ct value of over 30; outliers having end point fluorescence less than 50% of average fluorescence were removed from the analysis. hrm curve for each genotype was visually scored and variants were identified by examining normalized, difference and derivative melt plots. melting temperatures of parents and hybrids for the genes used in hrm analysis are presented in on-line suppl. tab. 3. hrm assays for all samples and variants were conducted in duplicate and in independent replicates to ascertain the consistency of the results. results selection of candidate genes for in-solution target capture in the present study, seven xylogenesis-related genes involved in different stages of wood formation including biotab. 1. genotypes of parents (eucalyptus tereticornis clone et86 and eucalyptus grandis clone eg9) and eight hybrids (h13, h48, h137, h160, h190, h218, h265, and h275) of eucalyptus mapping population obtained from target capture and exome sequencing data. snp – single nucleotide polymorphism, indel – insertion deletion, ssr – simple sequence repeat. gene name eg9 et86 h13 h 48 h 137 h 160 h 190 h 218 h 265 h 275 snps bp c/c t/t c/t c/t c/t c/t c/t c/t c/t c/t xth c/c t/t c/t c/t c/t c/t c/t c/t c/t c/t paapa t/t c/c c/t c/t c/t c/t c/t c/t c/t c/t lim1 c/t t/t c/t c/t t/t t/t t/t c/t t/t c/t indel cesa4 g/ac g/-ac g/-ac g/-ac g/-ac g/-ac g/-ac g/-ac g/-ac g/-ac ssrs mur3 (tc)7 (tc)5 (tc)7 (tc)7 (tc)7 (tc)7 (tc)7 (tc)7 (tc)7 (tc)7 arf4 (tgg)6 (tgg)4 (tgg)6 (tgg)6 (tgg)4 (tgg)4 (tgg)6 (tgg)4 (tgg)3 (tgg)6 muneera parveen a b, lakshmanan d, ghosh dasgupta m 108 acta bot. croat. 79 (2), 2020 tide change in the pcr product or presence of secondary structures, resulting in inconsistent melting of pcr product. the presence of rare or unknown snps near the region selected for study may also interfere with the analysis and interpretation. targeted exome sequencing data for cesa4 predicted the presence of deletion in et86 (g/-ac) and insertion in eg9 (g/g) with the amplicon size differing by 2 bp between the two parents (tab. 1). all the eight hybrids selected for hrm analysis had the presence of indel and so they grouped with the et86 (red), where as eg9 had a distinct melting profile (green) in the analysis (tab. 1, fig. 5, on-line suppl. fig. 5). the hrm curve results were consistent with sequencing results (tab. 1). two ssr markers chosen for this study produced polymorphic melting curves differentiating the parents and hybrids when investigated using hrm analysis. for the ssr loci of mur3, the parents eg9 had the (tc) 7 repeat while et86 had the (tc) 5 repeat and all the hybrids sequenced had the (tc) 7 repeat, similar to eg9. the derivative melting curve and difference plot of ssr loci of mur3 showed a narrow curve for eg9 (tc) 7 and a broader curve for et86 (tc) 5 (tab. 1, fig. 6, on-line suppl. fig.6). all the hybrids grouped with parent eg9 but a slight deviation in the shape of the melt curves for h218, h275 and h265 (green) was observed. et86 was identified as different variant (red) (fig. 6, on-line suppl. fig.6). zygous parents and heterozygous hybrids (tab. 1, figs. 1–3, on-line suppl. figs. 1–3). in xth, the melting curve of hybrids (blue) was above that of the parents (red) (fig. 3, online suppl. fig. 3) since the melting temperatures of hybrids were higher than those of the parents (fig. 3, on-line suppl. fig. 3). in bp (fig. 1, on-line suppl. fig. 1) and paapa (fig. 2, on-line suppl. fig. 2) the melting curves of the hybrids were intermediate between the parents. the exome sequencing data for the parent et86 showed a presence of nucleotide change in the position (718 bp) around the target snp for the hrm analysis (fig. 3). the change in melting curve profile of xth in the parents when compared to bp and paapa could be attributed to the nucleotide change in the parent (et86) at this locus. melting curve of lim1 indicated a heterozygous (c/t) and homozygous (t/t) allelic composition at the target locus for eg9 (green) and et86 (red) respectively (fig. 4, online suppl. fig. 4). the melting curve of the four hybrid genotypes (h13, h48, h218, and h275) clustered in the same group as eg9 (green), indicating similar allelic status. the remaining hybrids (h137, h160, h190 and h265) (red) grouped with et86 and represented a homozygous condition. however, in h13 and h275, the melting curves did not show a decrease in fluorescence to the same baseline as other hybrids (h48 and h218) (fig. 4, on-line suppl. fig. 4). the lack of decrease in fluorescence to the baseline in the two hybrids may indicate the presence of a second nucleofig. 1. melting curve analysis of the single nucleotide polymorphic (snp) marker in bp. polymorphic high resolution melting (hrm) curves differentiated the genotypes into three groups. a – normalized melting plot, b – difference melting curve. the curves with different color represent different genotypes. c – sequence alignment of the bp gene sequence for the parents. snps are shown and highlighted in the red box. fig. 2. melting curve analysis of the single nucleotide polymorphic (snp) marker in paapa. polymorphic high resolution melting (hrm) curves differentiated the genotypes into three groups. a – normalized melting plot, b – difference melting curve. the curves with different color represent different genotypes. c – sequence alignment of the paapa gene sequence for the parents. snps are shown and highlighted in the red box. validation of variant using hrm analysis acta bot. croat. 79 (2), 2020 109 a lig ne d f lu or es ce nc e (% ) 0 20 40 60 80 100 64 67 70 73 76 79 eg 9 : c/c et 86 : t/t 8 hybrids : c/t a -9 -6 -3 0 3 6 64 67 70 73 76 79 d iff er en ce b fig. 3 temperature (oc) temperature (oc) c h13 h275 0 20 40 60 80 100 a lig ne d f lu or es ce nc e (% ) 65 70 75 80 85 90 95 a eg 9, h13, h48, h218, h275 : c/t et 86, h137, h160, h190, h265 : t/t -5 0 5 10 15 20 25 65 70 75 80 85 90 95 d iff er en ce b h13 h275 fig. 4 temperature (oc) temperature (oc) c fig. 3. melting curve analysis of the single nucleotide polymorphic (snp) marker in xth. polymorphic high resolution melting (hrm) curves differentiated the genotypes into three groups. a – normalized melting plot, b – difference melting curve. the curves with different color represent different genotypes. c – sequence alignment of the xth gene sequence for the parents. snps are shown and highlighted in the red box. the change in the second nucleotide is highlighted with orange box. fig. 4. melting curve analysis of the single nucleotide polymorphic (snp) marker in lim1. polymorphic high resolution melting (hrm) curves differentiated the genotypes into two groups. a – normalized melting plot, b – difference melting curve. the curves with different color represent different genotypes. c – sequence alignment of the lim1 gene sequence for the parents. snps are shown and highlighted in the red box. -10 -5 0 5 10 15 fig. 5 67 70 73 76 79 82 a lig ne d f lu or es ce nc e (% ) 0 20 40 60 80 100 eg 9 : g/ac et 86 and 8 hybrids : g/-ac a 67 70 d iff er en ce b 73 76 79 82 temperature (oc) temperature (oc) c fig. 6 74 77 80 83 86 h218, h275 h265 a lig ne d f lu or es ce nc e (% ) 0 20 40 60 80 100 eg 9 and 8 hybrids : (tc)7 et 86 : (tc)5 a -30 -20 -10 0 10 20 30 h218, h275 h265 d iff er en ce 74 77 80 83 86 b temperature (oc) temperature (oc) c fig. 5. melting curve analysis of the insertion or deletion (indel) marker in cesa4. polymorphic high resolution melting (hrm) curves differentiated the genotypes into two groups. a – normalized melting plot, b – difference melting curve. the curves with different color represent different genotypes. c – sequence alignment of the cesa4 gene sequence for the parents. position of indel is shown and highlighted in the red box. fig. 6. melting curve analysis of the simple sequence repeat (ssr) marker in mur3. polymorphic high resolution melting (hrm) differentiated the genotypes into two groups. a – normalized melting plot, b – difference melting curve. the curves with different color represent different genotypes. c – sequence alignment of the mur3 gene sequence for the parents. position of ssr is shown and highlighted in the red box. muneera parveen a b, lakshmanan d, ghosh dasgupta m 110 acta bot. croat. 79 (2), 2020 the sequencing results for the ssr loci in arf4 showed the (tgg) 6 repeat in eg9 and (tgg) 4 in et86. three distinct variants were identified in the hrm analysis with melt curve difference between parents (et86 and eg9). the hybrids (h13, h48, h190 and h275) grouped with eg9 (green) and hybrids (h137, h160 and h218) clustered with et86 (red). the hybrid h265 showed distinct melting curve (blue) with repeat ssr motif of (tgg) 3. the sensitivity of hrm analysis allowed the detection of a complex situation for arf4, which is the presence of (tgg)6 repeat for parent eg9, h13, h48, h190, h275, (tgg)3 repeat for h265 and (tgg) 4 repeat in et86, h137, h160 and h218 (tab. 1). the variants identified using the assay is consistent with the sequencing results, confirming the discriminating power of the analysis (fig. 7, on-line suppl. fig. 7). -10 -5 0 5 10 15 20 25 fig. 7 78 80 82 84 86 88 a lig ne d f lu or es ce nc e (% ) 0 20 40 60 80 100 eg 9, h13, h48, h190, h275 : (tgg)6 et 86, h137, h160, h218 : (tgg)4 h265 : (tgg)3 d iff er en ce 78 80 82 84 86 88 b a temperature (oc) temperature (oc) c fig. 7. melting curve analysis of the simple sequence repeat (ssr) marker in arf4. polymorphic high resolution melting (hrm) differentiated the genotypes into three groups. a – normalized melting plot, b – difference melting curve. the curves with different color represent different genotypes. c – sequence alignment of the arf4 gene sequence for the parents. position of ssr is shown and highlighted in the red box. discussion next generation sequencing has revolutionized genome research since it enables cost effective sequencing of millions of loci in a single run and has replaced the conventional approaches for genotyping like polyacrylamide gel electrophoresis system and denaturing capillary electrophoresis (wenz et al. 1998, wang et al. 2003). it is presently an indispensable approach for identifying and cataloging the full spectrum of genetic variation across the genome at a scale unattainable by traditional techniques such as sanger sequencing (ku et al. 2012). this technology has facilitated and accelerated large-scale discovery, validation and assessment of genetic markers for high-throughput genotyping (harismendy et al. 2009, paszkiewicz and studholme 2012). however, data generated using ngs platforms may suffer from high error rates mainly due to base-calling and alignment errors, limiting accurate variant prediction. determining the rate of false positives and false negatives is one of the critical areas in genome analysis (shigemizu et al. 2013), since it drastically influences downstream analyses including the identification of rare mutation and estimation of allele frequencies (nielsen et al. 2011). variants predicted using ngs platforms are validated with secondary, orthogonal methods such as sanger sequencing, kompetitive allele specific pcr (kasp), taqman system (applied biosystems, foster city,ca) and illumina goldengate assay (myakishev et al. 2001, fan et al. 2003, de la vega et al. 2005, mu et al. 2016). while the cost of marker discovery using ngs platforms has drastically reduced over the past decade, inclusion of additional confirmation of variants has added additional costs to genetic testing. hence, the need for alternative assays that are rapid, simple, homogenous, highly specific, sensitive and cost effective has increased for high throughput validation of markers. hrm analysis has been reported as a convenient and cost effective pcr based method for genotyping snps, ssrs and indels in plant species (simko 2016). it was used in gene scanning, species identification (słomka et al. 2017), as an alternate to snapshot analysis (mehta et al. 2017), barcoding (song et al. 2016, mishra et al. 2018) and mutant screening (taheri et al. 2017). in the present study hrm was used to predict variants (snps/indels/ssrs) in both heterozygous and homozygous conditions in eucalyptus parents and hybrids. the sensitivity and utility of this technology for snp genotyping was demonstrated in tetraploid medicago sativa l., where parental genotypes and segregating progenies were characterized (han et al. 2012). mutation scanning for homozygous or heterozygous substitution and genotyping with the use of hrm assays was reported in olea europaea l. germplasm (muleo et al. 2009). furthermore, hrm assay was used for species discrimination and phylogenetic analysis in podocarpus sp. (marshall et al. 2015), varietal discrimination in vitis vinifera l. (gomes et al. 2018), cultivar discrimination in sweet cherry (ganopoulos et al. 2012) and validation of sex linked markers in pistacia vera l (kafkas et al. 2015). the discriminating power of hrm melt curves was proven in cesa4, where the analysis could differentiate parents with and without the predicted indel. this is in agreement with the earlier report in barley, where indel markers were scored using electrophoresis and the hrm method (zhou et al. 2015). they reported the discriminating ability of the hrm technique to distinctly differentiate samples with 2 bp difference. the two ssr markers (mur3 and arf4) targeted for this study produced polymorphic melting curves differentiating https://www.ncbi.nlm.nih.gov/pubmed/?term=de la vega fm%5bauthor%5d&cauthor=true&cauthor_uid=15829242 https://www.ncbi.nlm.nih.gov/pubmed/?term=de la vega fm%5bauthor%5d&cauthor=true&cauthor_uid=15829242 validation of variant using hrm analysis acta bot. croat. 79 (2), 2020 111 the parents and hybrids when investigated through hrm analysis. however, mild changes in the shape of the melt curves for the same variants could be probably due to the minor systematic errors like pipetting of reagents. there are a number of potential problems and challenges that could affect the sensitivity of hrm assays. the quality and quantity of the dna, the presence of salt from dna isolation procedure, presence of primer dimer or other agents that interfere with pcr amplification can affect the sensitivity and reproducibility of hrm analysis, leading to incorrect genotyping or variant calling (twist et al. 2013). results on ssr genotyping using hrm assay in trees is limited to grapevine (mackay et al. 2008), citrus (distefano et al. 2012) and sweet cherry (ganopoulos et al. 2013). the studies have reported hrm as a flexible, cost-effective and closed-tube microsatellite genotyping method well suited for varietal certification and as an effective alternate to gel based platforms. in the present study, for the first time hrm analysis was optimized for the validation of variants predicted using deep seqencing methods in eucalyptus. it could detect the presence of variants with high resolution and grouped genotypes based on their predicted allelic composition. this enabled rapid scanning for the presence of snps/indels/ ssrs among heterozygous mapping population. the use of small amplicons (shorter than 150 bp) for genotyping allowed differentiation between homozygous genotypes and easy identification of heterozygous genotypes. a cost per data point analysis revealed that hrm assay per reaction was less than 0.5 usd and hence provided a greater flexibility for marker validation in terms of cost than sanger sequencing and other genotyping platforms. acknowledgements the authors acknowledge department of biotechnology, government of india for funding the research work (project no. bt/pr10539/pbd/16/1064/2013). the funding support as research fellowship was provided to amp by department of biotechnology, government of india. the technical support extended by dr. neeta s. madan, senior scientist, reliance research and development centre, mumbai, india in conducting hrm assay is gratefully acknowledged. references andrews, s., 2010: fastqc: a quality control tool for high throughput sequence data, available online. retrieved may 17, 2018 from http://www.bioinformatics.babraham.ac.uk/ projects/fastqc bartholomé, j., mandrou, e., mabiala, a., jenkins, j., nabihoudine, i., klopp, c., schmutz, j., plomion, c., gion, j.m., 2015: highresolution genetic maps of eucalyptus improve eucalyptus grandis genome assembly. new phytologist 206, 1283–1296. beier, s., thiel, t., münch, t., scholz, u., mascher, m., 2017: misa-web: a web server for microsatellite prediction. bioinformatics 33, 2583–2585. botticella, e., sestili, f., hernandez-lopez, a., phillips, a., lafiandra, d., 2011: high resolution melting analysis for the detection of ems induced mutations in wheat sbeiia genes. bmc 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dubey2, sahu keshavkant1* 1 school of studies in biotechnology, pt. ravishankar shukla university, raipur 492 010, india 2 central laboratory facility, chhattisgarh council of science and technology, raipur 492 010, india abstract – arsenic (as)-toxicity is a major constraint for crop production. the present study was intended to examine the comparative ameliorative effects of diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) and proline (pro) on as-stress in glycine max l. seeds of glycine max l. were subjected to as (100 µm) singly, and together with dpi (10 µm), ebl (0.5 µm) or pro (10 mm), for five days, and were then analyzed. experimental results showed that as treatment caused a substantial fall in growth traits like germination percentage, radicle length and dry mass, which was accompanied by as accumulation. additionally, as application also revealed reduced viability, total protein content and activities of antioxidative enzymes (superoxide dismutase, catalase and ascorbate peroxidase), while it increased the levels of total sugar, proline and oxidative stress markers such as electrolyte leakage, reactive oxygen species, lipid oxidized products, protein carbonyls and hydroperoxides, amadori and maillard reaction products, malondialdehyde-/4-hydroxy-2-nonenal-protein adducts, protease and proteasome. isozymes of antioxidative enzymes were also observed to be altered considerably under as-stress. impressively, dpi, ebl and pro played their role as protective agents, hence caused enhanced growth and reduced as accumulation. these protective chemicals also improved the viability, accruals of total protein, total sugar and endogenous proline, and activities of antioxidants, while they reduced the levels of oxidative stress markers. our findings demonstrated the involvement of dpi, ebl and pro in as-stress tolerance in glycine max l. further, pro appears to be superior to dpi and ebl, in alleviating as-induced responses in glycine max l. keywords: arsenic, diphenylene iodonium, 24-epibrassinolide, oxidative stress, proline, protein metabolism, reactive oxygen species * corresponding author, e-mail: sneza.dragicevic@t-com.me introduction arsenic (as) is a hazardous metalloid, which ranks 20th in the earth’s crust and is ubiquitously present in the natural environment. its concentration above the permissible limit (10 µg l–1, who) hampers the normal growth, development and overall metabolic functioning of plants, resulting in toxicity symptoms. the symptoms of as-stress in plants include reduced growth and biomass accumulation, leaf gas exchange, chlorophyll synthesis and thereby photosynthesis, nutrient supply, cellular water potential, protein turnover, and enzymic dysfunction (chandrakar et al. 2016a). a plant’s root serves as the foremost and most susceptible site for the perception of abiotic stress responses including astoxicity. after entering into the plant’s body, as readily binds with sulfhydryl groups of both proteins and enzymes, thereby perturbing the cellular metabolism and inhibiting enzymatic activities (farooq et al. 2015). a well-known consequence of as-toxicity is over-production of reactive oxygen species (ros) such as superoxide (o2˙ˉ), hydroxyl radical (˙oh) and hydrogen peroxide (h2o2), affecting the oxidative condition inside the plants (siddiqui et al. 2015, chandrakar et al. 2016b). this over-produced ros are largely shown to attack cellular macromolecules such as lipids, proteins, nucleic acids, etc. (chandrakar et al. 2017a). the polyunsaturated fatty acid (pufa) fractions of membrane lipids are the prime targets of ros attack (chandrakar et al. 2016b). accruals of malondialdehyde (mda) and 4-hydroxy-2-nonenal (hne), chief products and biomarkers of lipid peroxidation reaction in stressed cells, are linked directly with the disturbed integrity or leakiness of the membranes (yadu et al. 2016). accumulation of ros has also been shown to cause reduced fluidity of cellular membranes and chandrakar v., dubey a., keshavkant s. 52 acta bot. croat. 77 (1), 2018 thereby increased leakage of electrolytes, damage to membrane proteins, loss of tissue viability and inactivation of both enzymes and receptors of it (kaur et al. 2012). exposure to ros is shown to modify the structure of proteins, oxidation of side chain groups, cross-linking, generation of new reactive groups and fragmentation of backbone (chandrakar et al. 2016b). moreover, direct oxidation of a number of amino acids, particularly his, lys, arg, pro and thr, gives rise to protein carbonyls (pco), which are quite susceptible to proteolysis (parkhey et al. 2014a). in addition, protein hydroperoxides (proohs), the most abundant and reactive molecules, are also produced as a derivative of ros-mediated oxidation of proteins, and are shown to contribute to pco turnover (mallick et al. 2013). moreover, in stressed cells, proteins are also shown to combine readily with lipid peroxidized products such as malondialdehyde (mda) and 4-hydroxy-2-nonenal (hne), thus forming cytotoxic compounds mda-/hne-protein adducts, which are genotoxic in nature and are resistant to proteases (parkhey et al. 2014a, chandrakar et al. 2017b). proteasomes are shown to be involved actively in elimination of degraded, aggregated and/ or misfolded proteins (karmous et al. 2014). in addition to oxidation, proteins also undergo non-enzymatic modification following amadori and maillard reaction pathways (murthy and sun 2000). in the amadori reaction, reducing sugars attack on amino groups of proteins, while the maillard reaction involves subsequent interactions and/ or rearrangements of amadori products to form brown colored polymeric cytotoxic products (murthy and sun 2000). these glycated products lead to alterations in metabolic processes, damage to dna, failure of repairing system and loss of enzymatic function as well, consequently, of loss of cell viability (parkhey et al. 2014a). literature pertaining to glycation of protein in any of the plant species subjected to heavy metal/ metalloid stress is absolutely lacking. therefore, the biochemical mechanisms governing as-induced protein metabolism need to be elucidated empirically. fortunately, plants have an intricate and impressive array of ros processing systems comprising superoxide dismutase (sod), catalase (cat), ascorbate peroxidase (apx), etc., which often impart abiotic stress tolerance to plants (nikolic et al. 2014). however, this defensive system is often insufficient to withstand severe stress conditions. hence, efforts have been invested in finding exogenous additives to counter ros-induced injury symptoms and enhance stress tolerance in plants and their parts. diphenyleneiodonium (dpi) is one of the potential ros scavengers in plants, providing tolerance against drought-induced oxidative condition (jiang and zhang 2002). it lessens the ros level by inhibiting nadph oxidase and nitric oxide synthase, enzymes of o2˙ˉ biosynthetic pathway (ye et al. 2012). although, having the potential to scavenge ros, exogenous application of dpi against any metal/ metalloidinduced stresses, still remains to be revealed completely in plants. thence, exogenous use of dpi to compensate for asinduced injuries in plants is a matter of scientific interest. therefore, dpi was used in the current study to provide tolerance against oxidative stress by lowering the accumulation of as-induced ros in glycine max l. similarly, 24-epibrassinolide (ebl) has been shown to regulate various metabolic processes such as seed germination, cell division and elongation, ions uptake, membrane integrity, nucleic acid and protein syntheses, photosynthesis, etc. in plants (fariduddin et al. 2015). exogenous addition of ebl enhances antioxidant enzyme expression, thus protecting the plants from oxidative injury. 24-epibrassinolide is well known for the synthesis of phytochelatins, a short polypeptide that binds to metals/ metalloids and sequesters them inside the cell. studies have confirmed the efficacy of ebl in increasing plant tolerance to a number of abiotic stresses such as mn-toxicity (fariduddin et al. 2015), chilling stress (wu et al. 2015), etc., but its role in as-toxicity conditions remains to be resolved fully. further, under abiotic stress conditions, plants mostly accumulate osmolytes such as proline (pro), which serves as osmoprotectant, membrane plasticizer and direct scavenger of free radicals (yadu et al. 2016). additionally, it also acts as a momentary source of both carbon and nitrogen that helps in the recovery of plants in stressful conditions. it also prevents enzymes from denaturation caused under stressful conditions, hence can be used against as, which readily binds with sulfhydryl groups and inactivates enzymes and proteins. thus, pro may play an imperative role in enhancing plant stress tolerance to various abiotic stresses (hayat et al. 2012, agami 2014). our previous study has shown that oxidative stress is a major component in the expression of as-toxicity in glycine max l. (chandrakar et al. 2016a). therefore, in order to explore the possibility of whether the ros scavengers viz dpi, ebl or pro, could be used to alleviate as-induced oxidative injury, the present study was aimed at examining the comparative effects of dpi, ebl and pro on growth and development, as accumulation, tissue viability, oxidative stress markers (electrolyte leakage, ros, mda, hne, endogenous pro and total sugar), total protein and its oxidized products (pco, prooh, amadori and maillard reaction products and mda-/hne-protein adducts), activities of protease and proteasome, and responses of antioxidant enzymes in asstressed glycine max l. materials and methods seed collection, treatments and germination assessment fresh seeds of glycine max l. were collected from the farmers of kabirdham (n22°01', e81°23', 353 msl), 110 km to the south of raipur. healthy seeds were sorted out and stored in plastic trays in laboratory conditions (26±2 °c, relative humidity 52±2%) for experimental usage. assorted seeds were surface sterilized applying 1% (v/v) sodium hypochlorite solution for 5 min and then washed thoroughly (5 times) with milliq water (mw) (millipore, gradient a-10, usa). subsequently, seeds (n=80) were kept for germination on sterile boxes (30×15×15 cm) and over filter paper towels soaked with 100 µm of as (naaso2 was used as source). in addition, the same number of seeds were also germinated over paper towels moistened with dpi (10 µm), ebl (0.5 arsenic toxicity and its amelioration in glycine max l. acta bot. croat. 77 (1), 2018 53 µm) and pro (10 mm) separately, and in their combination with as (100 µm). the dose of as chosen was the concentration at which glycine max l. seeds revealed toxicity symptoms in the form of 26% germination only. moreover, the doses of dpi, ebl and pro selected were those in which 58%, 70% and 78% respectively germination percentage against as-stress (100 µm) in glycine max l. were noted. all the seeds were allowed to grow in darkness at 26±2 °c, for 5 days. the solution (10 ml each) was supplied to the growing seeds after every 24 h until day 5. all the analyses were performed in five replicates, and repeated twice. only seeds having radicles 5 mm long were considered germinated, and were scored (%) on fifth day of incubation. radicle length and biomass to assess change in length (rl) and dry mass (dm), the radicles were removed precisely from the seeds, blotted gently over a filter paper, and changes in length and dm were monitored (chandrakar et al. 2016a). estimation of as content to determine as content, shade dried tissues (100 mg) were digested in a mixture of conc. hno3, h2o2 and h2o (3/2/10, v/v/v) at 80 °c, until a clear solution was obtained, and made up to 15 ml with mw (chandrakar et al. 2016a). arsenic content was determined using an atomic absorption spectrometer coupled to a hydride generation system (agilent, aa240, usa). the standard reference materials of metals (1.19773.0500, merck, darmstadt, germany) were used for calibration and quality assurance for analysis. reactive oxygen species for o2•ˉ estimation, 0.2 g radicles were homogenized with cold (4 °c) sodium phosphate buffer (0.2 m, ph 7.2) comprising diethyldithiocarbamate (10–3 m) to inhibit active sod (sangeetha et al. 1990). the homogenate was centrifuged (10000 rpm, 10 min, 4 °c) and in the collected supernatant, o2•ˉ was estimated by its capacity to reduce nitro blue tetrazolium (nbt, 2.5 × 10–4 m) at 540 nm using an uv-vis spectrophotometer (lambda-25, perkin elmer, usa), and expressed as µmol o2•ˉ min–1 g–1 fresh mass (fm). to measure •oh, radicles (0.2 g) were homogenized in 2 ml of phosphate buffer (10 mm, ph 7.4) consisting of 15 mm 2-deoxyribose and incubated at 37 °c for 2 h. after centrifugation (12000 rpm, 15 min), 0.7 ml of supernatant was mixed with 3 ml of 0.5% (w/v) thiobarbituric acid (tba, prepared in 5 mm naoh) and 1 ml of glacial acetic acid (kaur et al. 2012). this reaction mixture was heated at 100 °c for 30 min and then cooled immediately (4 °c, 10 min). absorbance was recorded at 532 nm and corrected for nonspecific absorbance at 600 nm. content of oh was calculated using an extinction coefficient of 0.155 mol–1 cm–1 and expressed as nmol g–1 fm. to assess h2o2 content, 0.2 g of radicles were extracted with 2 ml of 0.1% (w/v) trichloroacetic acid (tca) and centrifuged at 12000 rpm for 15 min (velikova et al. 2000). in an aliquot (0.75 ml) of supernatant, equal volumes of both phosphate buffer (10 mm, ph 7) and potassium iodide (1 m) were added and absorbance of the sample was read at 390 nm. content of h2o2 was calculated using an extinction coefficient 0.28 µmol–1 cm–1 and was expressed as µmol g–1 fm. viability assessment ten randomly selected radicles were immersed in the 0.5% (w/v) 2,3,5-triphenyl tetrazolium chloride (ttc) solution, and incubated in the dark for 24 h at room temperature (lakon 1949). afterwards, seedlings were homogenized in 2 ml of ethanol and centrifuged (5000 rpm, 10 min). in the collected supernatant, reduced ttc was assayed at 520 nm using ethanol as blank, and values were expressed as a520 g–1 fm. leakage of electrolytes rate of electrolyte leakage was measured following the protocol of blum and ebercon (1981). 0.2 g of radicles was immersed in 20 ml of mw and kept on an orbital shaker (50 rpm, 24 h). thereafter, electrical conductance of the solution was measured (c0) using a ec-tds analyzer (cm183, elico, india). then, the radicles were boiled for 20 min and electrical conductance was once again determined (c1). results were expressed as percentages of electrolyte leakage = c0/ c1 × 100. lipid peroxidized products for the estimation of mda, 0.1 g radicles was crushed with 20% (w/v) tca consisting 0.5% (w/v) tba (velikova et al. 2000). homogenate was boiled for 30 min, cooled and centrifuged (11000 rpm, 10 min). absorbance of the supernatant was recorded at 532 nm and mda was estimated following the extinction coefficient of 157 mmol–1 cm–1. data was expressed as nmol g–1 fm. to monitor hne, 0.1 g radicles was homogenized in borate buffer (0.2 m, ph 7.4) and tca (10%, w/v). the homogenate was centrifuged (11000 rpm, 20 min, 4 °c), and the supernatant was mixed with 2,4-dinitrophenylhydrazine (dnph, 1%, w/v) prepared in hcl (0.5 m), and incubated at room temperature for 2 h. it was then extracted with hexane, and dried under liquid nitrogen. residue was dissolved in methanol and absorbance was recorded at 350 nm (ray et al. 2007). an extinction coefficient of 13750 mol–1 cm–1 was used to calculate hne content and data was expressed as mmol g–1 fm. protein extraction and quantification 0.2 g radicles were extracted with 2 ml of 10 mm phosphate buffer (ph 7.2) containing 1 mm edta, 2 mm dithiothreitol and 0.2% (v/v) triton x-100, and centrifuged (1000 rpm, 20 min, 4 °c). supernatant thus obtained was used for estimations of protein content and antioxidant enzymes. protein content was assessed following bradford (1976). bochandrakar v., dubey a., keshavkant s. 54 acta bot. croat. 77 (1), 2018 vine serum albumin was used to prepare a standard curve. content of protein was expressed as mg g–1 fm. estimation of protein carbonyl carbonyl groups were monitored, following their reactivity with dnph to form hydrazones (levine et al. 1994). the extracted protein (500 µl) was incubated with 200 µl each of 0.03% (v/v) triton x-100 and 1% (w/v) streptomycin sulphate for 20 min. after centrifugation (11000 rpm, 10 min, 4 ºc), supernatants were mixed with 10 mm dnph, prepared in 2 m hcl. the blank was incubated with 2 m hcl only. after 1 h of incubation, proteins were precipitated with 10% (w/v) tca. the pellets were finally dissolved in 6 m guanidine hydrochloride, prepared in 20 mm potassium phosphate buffer (ph 2.3), and absorbance was recorded at 370 nm. content of pco was calculated using extinction coefficient 22000 mol–1 cm–1 and values were expressed in terms of mol mg–1 protein. determination of protein hydroperoxide to measure prooh content, isolated protein was initially dissolved in 25 mm sulphuric acid. to it, 5 mm each of ferrous ammonium sulphate, and xylenol orange prepared in 25 mm sulphuric acid were added. this mixture was incubated for 1 h in the dark, and then centrifuged (12000 rpm, 20 min) (gay et al. 1999). absorbance of the supernatant was recorded at 560 nm, and content of prooh was calculated using the extinction coefficient of 35.5 mmol–1 cm–1. amount of prooh was referred as mmol mg–1 protein. measurements of mda-/hne-protein adducts an isolated protein pellet (5 mg ml–1) was re-dissolved in sodium phosphate buffer (0.2 m, ph 7.4). fluorescence for mda-protein adducts (395 nm excitation/460 nm emission) and hne-protein adducts (356 nm excitation/460 nm emission) was determined using a fluorescence spectrophotometer (ls-45, perkin elmer, usa) (chairpotto et al. 1997). values were expressed as unit fluorescence mg–1 protein. monitoring of total sugar for total sugar estimation, 0.2 g radicles were extracted with sodium phosphate buffer (50 mm, ph 7.2) and centrifuged (12000 rpm, 15 min, 4 °c) (spiro 1966). the supernatant thus obtained was mixed with anthrone (0.2%, w/v), and allowed to stand for 10 min in a water bath (100 °c). the sample was cooled and the absorbance was read at 620 nm, and denoted as mg g–1 fm. determinations of amadori and maillard reaction products radicles (0.2 g) were macerated with sodium phosphate buffer (50 mm, ph 7.2), consisting of 10% (w/v) streptomycin sulphate, dissolved in 50 mm hepes buffer (ph 7.2) (murthy and sun 2000). the homogenate was centrifuged (11000 rpm, 15 min) and streptomycin sulphate (200 µl) was once again added to it and then re-centrifuged. thereafter, proteins were precipitated with ammonium sulphate (0.55 g ml–1) and were re-dissolved in sodium phosphate buffer (50 mm, ph 7.2). it was then used for amadori and maillard reaction products measurement. to measure the amadori reaction product, the isolated protein was mixed with nbt (0.5 mm in 100 mm sodium carbonate, ph 10.3) and incubated in a water bath (40 °c). absorbance was recorded at 550 nm, after 10 and 20 min of incubation. absorbance (∆od) value was considered as the measure of the amadori reaction product. to monitor the maillard reaction product, isolated proteins were scanned at excitation wavelength 375 nm and emission wavelength 440 nm. content was denoted in terms of protein fluorescence. assay of protease for protease assay, 0.2 g radicles was homogenized with borate buffer (0.2 m, ph 7.4) and centrifuged (11000 rpm, 15 min). the supernatant was acetone-precipitated and resuspended in sodium phosphate buffer (0.02 m, ph 6.4), and 1 ml of it was incubated with an equal volume of casein (1%, w/v) at 37 °c for 3 h (merheb et al. 2007). then, 10% (w/v) tca was added to it to terminate the reaction, which was allowed to stand for 30 min. the mixture was centrifuged at 11000 rpm for 10 min, and the collected supernatant was mixed with folin ciocalteu reagent (1 n) and sodium hydroxide (0.2 n), and incubated for 30 min. absorbance was read at 570 nm, and activity was referred as units g–1 protein. estimation of proteasome activity for proteasome assay, 0.2 g radicles was extracted with potassium phosphate buffer (50 mm ph 7) comprising 2 mm mgcl2, 5% (v/v) glycerol, 5 mm 2-mercaptoethanol and 10 mm atp, and then centrifuged (11000 rpm, 15 min, 4 °c) (yang et al. 2004). supernatant obtained was incubated with 50 m succinyl-leu-leu-val-tyr-7-amido-4-methylcoumarin (suc-llvy-amc) prepared in potassium phosphate buffer (80 mm, ph 7.0), at 37 °c for 20 min. then, sodium acetate buffer (80 mm, ph 4.3) was added to it, to inhibit the reaction. the amc released was measured at 380 nm excitation and 440 nm emission and expressed in terms of units mg–1 protein. assay of antioxidant enzymes the sod (ec 1.15.1.1) was assayed by monitoring percent inhibition of pyrogallol auto-oxidation by the enzyme at 420 nm (marklund and marklund 1974). 2.74 ml of trishcl buffer (50 mm, ph 8.2) comprising diethylenetriaminepentaacetic acid and edta, 1 mm of each, was mixed with 0.2 ml of extracted enzyme. to initiate the reaction, 60 µl of pyrogallol (0.2 mm prepared in 10 mm hcl) was added to it, and change in absorbance was read at 420 nm. activity of sod was denoted as units min–1 mg–1 protein. to monitor cat (ec 1.11.1.6) activity, decomposition of h2o2 was measured by recording the fall in absorbance at 240 nm (chance and maehly 1955). in a test tube, 2.74 ml arsenic toxicity and its amelioration in glycine max l. acta bot. croat. 77 (1), 2018 55 of potassium phosphate buffer (37.5 mm, ph 6.8) and 60 µl of enzyme were taken, and then a reaction was triggered by adding 200 µl of h2o2 (60 mm). the change in absorbance was recorded for 5 min at an interval of 15 sec. its activity was calculated using an extinction coefficient of 0.039 mol–1 cm–1 and denoted as nmol min–1 mg–1 protein. apx (ec 1.11.1.11) was estimated by monitoring the rate of ascorbate oxidation at 290 nm (nakano and asada 1981). in a test tube, 2.3 ml of 0.025 m potassium phosphate buffer (ph 7.0), 500 µl of 0.0025 m ascorbic acid, 190 µl of 0.001 m edta and 10 µl of enzyme extract were added. just after the addition of 10 µl of 0.1 m h2o2 into the assay mixture, initial absorbance, and after 20 min of incubation, final absorbance was measured, at 290 nm. apx activity was derived using extinction coefficient of 0.0028 mol–1 cm–1 and expressed as mmol min–1 mg–1 protein. isozyme analysis electrophoretic analyses of sod, cat and apx were performed on native-polyacrylamide gels (10%) using trisglycine buffer (5 mm, ph 8.3) (in case of apx, running buffer consists of 4 mm ascorbate), at 4 °c for 2 h with a constant current of 20 ma, using mini-protean tetra cell (biorad, usa) (chandrakar et al. 2016a). after a complete run, gels were imaged and analyzed using a gel-doc system (biorad, usa). to visualize the sod, gels were incubated in the dark for 20 min in the 2.45 mm nbt solution, and were then immersed in dipotassium hydrogen phosphate (36 mm, ph 7.8) comprising riboflavin and temed, 28 µm of each, until the gel turned blue except the region showing sod activity (chandrakar et al. 2016a). to stain cat isozymes, the gels were incubated in 0.03% (v/v) h2o2 solution for 10 min. the gels were rinsed quickly in mw and stained in a solution consisting 1% (w/v) each of potassium ferric chloride and ferricyanide. soon after the green color appeared, the gels were washed with mw. similarly, to detect apx, the gels were equilibrated with sodium phosphate buffer (50 mm, ph 7) and ascorbate (2 mm) for 30 min. then, the gels were incubated with sodium phosphate buffer (50 mm, ph 7) comprising 4 mm of each of ascorbate and h2o2 for 20 min. finally, the gels were rinsed twice with 50 mm sodium phosphate buffer (ph 7) and stained in sodium phosphate buffer (50 mm, ph 7.8) consisting temed (28 mm) and nbt (2.45 mm). the reaction was continued for 10–15 min and stopped by a brief wash with mw. proline content to measure proline, 0.5 g radicles was extracted with 10 ml of sulfo-salicylic acid (3%, w/v) and centrifuged (6000 rpm, 15 min) (bates et al. 1973). the supernatant (2 ml) was mixed with 2 ml each of ninhydrin reagent and glacial acetic acid. the mixture was incubated at 100 °c for 60 min, and cooled at room temperature. then, 4 ml toluene was added and the chromophore containing toluene was aspirated out and its absorbance was recorded at 520 nm taking toluene as a blank. its amount was expressed as mg g–1 fm. statistical analysis all the investigations were performed twice with five separate replications. the data obtained were subjected to oneway anova, and the mean differences were compared by duncun’s multiple range tests using spss software (ver. 16.0). results the results demonstrated that the tested growth traits were significantly decreased (germination: 74%, radicle length: 86%, dm: 83%) with as addition in glycine max l., as compared to control, indicating a negative relationship between growth attributes and as (tab. 1). however, the addition of dpi, ebl or pro with as favored the growth traits by reducing (up to 86%) the inhibitory impacts of stress in glycine max l. (tab. 1). accumulated data revealed the growth-promoting nature of potential compounds. additionally, amongst the tested treatments, pro was found to be more effective in as-stress amelioration than dpi and ebl. radicles of glycine max l. subjected to as (100 µm) for five days accrued 31.97 µg as g−1 dm (tab. 1). on the other hand, as accumulation in the radicles of glycine max l. grown in dpi-, eblor pro-amended as-solution measured tab. 1. comparative effects of diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) and proline (pro) in alleviation of arsenic (as)induced changes in germination percentage, radicle length, dry mass, arsenic content, electrolytes leakage and tissue viability in glycine max l. values presented are mean ± se of ten (for physiological parameters) or five (viability and arsenic content) different observations. different letters indicate significant difference among treatments at p < 0.05. germination % radicle length (mm) dry mass (g) as content (µg g–1 dm) electrolyte leakage (%) viability (a520 g–1 fm) control 100a±0 65c±2 0.11a±0.01 – 0.19c±0.05 1.28c±0.008 dpi 100a±0 70b±2 0.12a±0.005 – 0.18c±0.002 1.31b±0.004 ebl 100a±0 68bc±2 0.12a±0.005 – 0.17c±0.002 1.34a±0.005 pro 100a±0 76a±2 0.13a±0.02 – 0.16d±0.004 1.35a±0.017 as 26e±3 9f±3 0.02c±0.01 31.97±0.06 2.91a±0.04 0.19f±0.006 as+dpi 58d±3 22e±2 0.03bc±0.01 26.25±0.04 1.54b±0.05 0.22e±0.001 as+ebl 70c±4 22e±2 0.03b±0.01 23.02±0.03 1.51b±0.004 0.24e±0.005 as+pro 78b±2 28d±2 0.04b±0.005 21.87±0.04 1.48b±0.007 0.29d±0.008 chandrakar v., dubey a., keshavkant s. 56 acta bot. croat. 77 (1), 2018 only 26.25, 23.02 and 21.87 µg as g−1 dm respectively, which indicates that the above treatments permitted only a limited uptake of as in glycine max l. the results revealed that pro was the most effective treatment, allowing less as-uptake than the other two. exposure of glycine max l. to as for five days caused enhanced accumulation of all the three ros (o2˙ˉ: 1340%, ˙oh: 239%, h2o2: 244%), as compared to control (tab. 2). however, exogenous application of dpi, ebl and pro, separately, with as significantly lowered the amounts of ros (tab. 2). gathered data suggested that pro played a more prominent role than dpi and ebl in countering the deleterious impacts of as in glycine max l. a remarkable fall in the tetrazolium staining capacity/ viability of glycine max l. exposed to as for five days was observed, as compared to control, revealing an inverse association between the two (tab. 1). however, exogenous application of dpi, ebl and pro, separately, with as was seen to maintain tissue viability to some extent (77–82%), as compared to as-alone treated samples. however, pro performed better than dpi and ebl in the amelioration of as-induced loss in tissue viability. compared to control, a significantly high rate of electrolyte leakage from radicles of glycine max l. subjected to as for five days was discernible (tab. 1). in contrast, exogenous addition of dpi, ebl or pro with as resulted in statistically lower, 721, 737 and 753% respectively, release of electrolytes. however, pro was found to maintain membrane integrity better and in consequence allow lower leakage than dpi and ebl. arsenic-induced peroxidation of membrane lipid was measured in terms of both mda and hne, and found to be increased by 2674 and 2272% respectively as compared to controls (tab. 2). however, in dpi-, ebland pro-supplied as-subjected radicles, significantly low levels of mda and hne were measured (tab. 2). in general, pro exhibited a leading role in prevention of as-prompted lipid peroxidation in glycine max l., greater than that of dpi and ebl. exposure of glycine max l. to as for five days manifested a massive loss (91%) in protein turnover, as compared to control. however, a considerably greater amount of it was measured in those subjected to as-solution amended with dpi, ebl or pro (tab. 2). the results suggested that pro served as more effective treatment in compensation of protein level, than the dpi and ebl. activity measurements of both protease and proteasome in as-grown glycine max l. revealed increases of 1390 and 204%, respectively, as compared to their controls (tab. 3). however, activities of both the enzymes were significantly less (protease: 994–1088%, proteasome: 104–152%) in those treated with dpi, ebl or pro amended as solution, for five days. moreover, pro was seen to allow lower activities of these two enzymes than dpi and ebl in as-stressed glycine max l. arsenic-impelled oxidation of protein was measured in terms of pco, prooh and mda-/hne-protein adducts, which were found to be raised considerably i.e. 381%, 200%, 204% and 34% respectively, in as-treated five-day-old radicles as compared to control (tabs. 3 and 4). in contrast, fewer accruals of these products were discernible in those samples grown in as solution amended with dpi, ebl or pro (tabs. 3 and 4). in general, pro exhibited a leading role in the prevention of as-prompted protein oxidation in glycine max l., more so than dpi and ebl. arsenic application manifested enhanced accumulation of both pro (561%) and total sugar (277%) in glycine max l., as compared to control (tab. 3). the results witnessed that exogenous addition of dpi, ebl or pro with as, caused further improvements in their accumulations. maximum of both pro (2.86 mg g–1 fm) and total sugar (15.74 mg g–1 fm) were measured in pro-supplied as-grown radicles, proving it a better preventer of as-injury than dpi or ebl (tab. 3). approximately 89 and 37% more accumulations of both amadori and maillard reaction products were registered in the as-grown radicles than in the control (tab. 4). however, their levels were comparatively less in those samples subjected tab. 2. variations in the levels of superoxide anion, hydroxyl radical, hydrogen peroxide, malondialdehyde and 4-hydroxy-2-nonenal in glycine max l. exposed to arsenic (as) alone or in combination with diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) or proline (pro). given values are mean ± se of five separate observations. different letters indicate significant difference at p < 0.05. superoxide anion (µmol min–1 g–1 fm) hydroxyl radical (nmol g–1 fm) hydrogen peroxide (µmol g–1 fm) malondialdehyde (nmol g–1 fm) 4-hydroxy-2-nonenal (mmol g–1 fm) control 1.68c±0.79 8.46e±0.07 0.36e±0.007 1.64e±0.10 0.11e±0.007 dpi 1.36c±0.48 4.34f±0.52 0.35e±0.062 1.62e±0.43 0.09e±0.005 ebl 1.10c±0.48 3.91g±0.14 0.27f±0.026 1.56e±0.53 0.08e±0.002 pro 0.63c±0.31 3.67g±0.17 0.11g±0.007 1.57e±0.32 0.07e±0.001 as 24.20a±0.48 28.71a±0.05 1.24a±0.009 45.50a±0.61 2.61a±0.03 as+dpi 21.67a±1.27 17.52b±0.07 1.06b±0.011 37.80b±0.10 1.12b±0.008 as+ebl 19.88a±6.5 13.10c±0.03 1.00c±0.038 35.22c±0.04 1.05c±0.001 as+pro 10.31b±3.37 10.62d±0.12 0.94d±0.004 34.11d±0.05 1.00d±0.001 arsenic toxicity and its amelioration in glycine max l. acta bot. croat. 77 (1), 2018 57 to dpi-, eblor pro-supplied as solutions. in this study, pro exhibited a leading role in compensating as-prompted protein glycation in glycine max l., more so than dpi and ebl. treatment of as significantly decreased the activities of sod (48%), cat (75%) and apx (69%) in the five-day-old radicles, but exogenous addition of dpi, ebl or pro alleviated this decline by up to 67%, under as-stress (figs. 1–3). however, activities of these were higher (63, 51 and 36% respectively) in pro added as-subjected radicles, than the dpi or ebl added as-grown (figs. 1–3). isozyme analysis of sod unveiled a total of six isoforms in response to as and/ or dpi, ebl and pro. exposure to as revealed only one band but isozymes-ii-iv were restored when dpi, ebl or pro was added with as (fig. 1). additionally, intensities of isoforms-ii-iv were higher in presence of pro as compared to that of the dpi and ebl. in gel activity analysis of cat in glycine max l. unveiled presence of at least three isoforms in control, dpi, ebl or pro-treated samples (fig. 2). on the other hand, reductions in both intensities and number of bands were observed in as-treated samples (fig. 2). band intensity was higher in pro-supplied as-grown samples than in dpior ebl added as-subjected samples. staining of apx identified four isoforms of it in as and/ or dpi-, ebland pro-subjected samples (fig. 3). out of these, isoform-i is common in all the treatments. additiontab. 3. levels of proteins, protein hydroperoxides, protease, proteasome, sugar and proline in glycine max l. exposed to arsenic (as) alone or in combination with diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) or proline (pro). data presented are mean ± se of five individual replicates. different letters indicate significant difference among treatments at p < 0.05. proteins (mg g–1 fm) protein hydroperoxides (mmol mg–1 protein) protease (units g–1 protein) proteasome (units mg–1 protein) sugar (mg g–1 fm) proline (mg g–1 fm) control 8.57d±0.77 1.21c±0.04 0.053e±0.002 0.21e±0.03 3.1g±0.27 0.31d±0.006 dpi 17.66c±0.98 0.89d±0.06 0.049ef±0.007 0.17f±0.008 4.04f±0.09 0.28d±0.01 ebl 20.38b±0.59 0.77e±0.01 0.047ef±0.002 0.16f±0.01 4.91e±0.11 0.29d±0.005 pro 27.17a±0.32 0.73e±0.02 0.042f±0.001 0.15f±0.004 5.47d±0.11 0.29d±0.005 as 0.69h±0.23 4.23a±0.07 0.79a±0.007 0.64a±0.02 11.71c±0.72 2.05c±0.19 as+dpi 3.22g±0.02 1.85b±0.12 0.63b±0.002 0.53b±0.01 12.03c±0.04 2.65b±0.11 as+ebl 6.42f±0.07 1.43c±0.006 0.60c±0.003 0.47c±0.01 15.13b±0.07 2.72ab±0.04 as+pro 8.01e±0.15 1.35c±0.02 0.58d±0.007 0.43d±0.01 15.74a±0.06 2.86a±0.09 tab. 4. amendments in the levels of amadori and maillard reaction products, protein carbonyls and malondialdehyde (mda)-/ 4-hydroxy2-nonenal (hne)-protein adducts in glycine max l. subjected to arsenic (as) alone or in combination with diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) or proline (pro) for five days. observations are mean ± se of five independent replications. different letters indicate significant difference among treatments at p < 0.05. amadori products (∆od) maillard products (protein fluorescence) protein carbonyls (µmol mg–1 protein) mda-protein adduct (uf mg–1 protein) hne-protein adduct (uf mg–1 protein) control 0.011e±0.001 110.86e±0.56 54.20e±0.17 155.83e±0.53 379.28e±0.73 dpi 0.008f±0.00001 102.13f±0.75 52.69fg±0.68 153.74e±1.11 271.12f±1.45 ebl 0.007f±0.00001 97.42g±0.34 50.63ef±0.39 151.22e±0.38 251.96g±0.32 pro 0.004g±0.0005 94.43h±0.29 49.21g±0.47 136.38g±0.06 240.24h±0.57 as 0.100a±0.002 152.11a±0.62 261.24a±1.40 474.10a±2.47 510.24a±0.90 as+dpi 0.082b±0.0007 147.25b±0.35 145.49b±4.56 344.55b±0.72 475.39b±1.23 as+ebl 0.075c±0.0001 142.70c±0.46 98.11c±0.83 341.26c±1.13 462.62c±1.21 as+pro 0.062d±0.0004 137.38d±0.43 90.20d±0.67 246.45d±1.48 391.47d±0.65 fig. 1. spectrophotometric activity (upper part) and isoenzymic pattern (lower part) of superoxide dismutase in glycine max l. grown in various experimental solutions of arsenic (as), diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) or proline (pro) for five days. bars presented in the graphs are mean ± se of five separate observations. different letters indicate significant differences among treatments at p < 0.05. chandrakar v., dubey a., keshavkant s. 58 acta bot. croat. 77 (1), 2018 discussion arsenic is a non-essential metalloid, ubiquitously present in the natural environment. the presence of it in the soil above the permissible limit adversely affects plant growth, development and productivity. a recent report of ours revealed that as-treatment leads to oxidative stress in glycine max l. (chandrakar et al. 2016a). however, the current study was aimedat evaluating the comparative efficacy of dpi, ebl and pro in the amelioration of as-promoted oxidative injuries in glycine max l. the results showed that the exogenous application of dpi, ebl and pro enabled glycine max l. to cope with as-induced oxidative stress, although in differing rates, consequentially improved growth traits (germination percentage, rl and dm), viability, levels of protein, sugar, pro and antioxidant enzymes, and reduced electrolyte leakage, ros, mda, hne, pco, prooh, amadori and maillard reaction products, mda-/ hne-protein adducts, and activities of both protease and proteasome. however, pro was found to be more effective than dpi and ebl in improving as-stress tolerance in glycine max l. experimental results proved that glycine max l. subjected to as for five days exhibited considerable reductions in germination percentage (74%), rl (86%) and dm (83%), as compared to controls (tab. 1). this reduced germination and inhibited growth in response to as might be related with the lower mitotic activity that decreased the rate of cell division, expansion and elongation of newly formed cells (chandrakar et al. 2016a). additionally, inside the cell, as causes disruption in the cellular energy flow by replacing the phosphate of the atp molecule. hence, reduced germination and growth responses are possibly due to the detrimental effects of as on the cellular functioning of the plants, where most of the available energy is consumed in the formation of stresslinked essentials like phytochelatins, anti-oxides, etc. (farooq et al. 2015). similarly, reduced dm is possibly an outcome of as-promoted increase in membrane permeability and thereby loss of electrolytes and other important constituents of the cell, essentially required for growth and development processes. these results are consistent with the findings of anjum et al. (2016) and begum et al. (2016) in as-exposed zea mays l. and oryza sativa l. respectively. however, exogenous addition of dpi, ebl or pro, particularly pro, with as, remarkably improved the growth traits of glycine max l., probably by protecting the structures of plasma membranes and cell walls, under stress conditions. dpi, ebl and pro are have been recorded in publications as enhancing seed germination and root/ shoot growth in different plants under various abiotic stresses (hayat et al. 2012, ye et al. 2012, agami 2014, chandrakar et al. 2017a). inhibited growth responses coincided well with enhanced accumulation of as in the glycine max l. radicles. accrual of as inside the cells might be the result of damaged cell membranes, alterations in the cell wall structures and/ or rapid influx of it through transporters of the exposed cells (singh et al. 2015). a similar result was recently recorded by chandrakar et al. (2017b). on the other hand, exogenous application of dpi, ebl or pro with fig. 2. activity of catalase, spectrophotometric assay (upper part) and isoenzymic pattern (lower part) of glycine max l. exposed to various treatments of arsenic (as), diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) or proline (pro). each bar is mean ± se of five distinct observations. different letters indicate significant differences at p < 0.05. fig. 3. quantitative estimation (upper part) and isoenzymic pattern (lower part) of ascorbate peroxidase in glycine max l. subjected to different experimental solutions of arsenic (as), diphenyleneiodonium (dpi), 24-epibrassinolide (ebl) or proline (pro). each bar is mean ± se of five distinct observations. different letters indicate significant differences at p < 0.05. ally, two new isoforms (ii and iii) were induced exclusively in samples treated with ebl and pro only. more bands appeared in pro + as-treated samples than in the dpior eblamended as-grown samples. arsenic toxicity and its amelioration in glycine max l. acta bot. croat. 77 (1), 2018 59 as revealed lesser accumulation of as, possibly by preventing entry of it via the maintenance of the structures of the plasma membranes and cell walls and the modification of as-transporters. inside the cell, as readily binds with sulfhydryl groups of both proteins and enzymes, consequently inhibiting cellular metabolism, which finally results in cell death (farooq et al. 2015). results of ttc test revealed a notable fall in the vitality of as-stressed glycine max l. tissue, which was in agreement with the report of kaur et al. (2012) in phaseolus aureus l. on the other hand, in protective treatments, exogenous dpi, ebl or pro, more precisely pro, maintained the vitality of glycine max l. radicles, up to a large extent, even under as-stress. dpi, ebl and pro decreased as accrual, which resulted in a better antioxidant system, hence lesser oxidative stress and increased viability. this condition might help glycine max l. to survive under as-stress. exogenous ebl and pro were shown to increase the viability of the roots in lycopersicon esculentum mill. and cicer arietinum l. exposed to extreme heat (kaushal et al. 2011, khan et al. 2014). exposure to as raised accumulation of all the three ros (o2˙ˉ: 1340%, ˙oh: 239%, h2o2: 244%) in glycine max l., and was accompanied with an enhanced rate of electrolyte leakage (tab. 1). in regard, singh et al. (2015) demonstrated that interaction between as and intracellular components may result in overproduction of ros in stressed cells. further, being an oxidizing agent, ros disturbs membrane integrity drastically, consequentially enhancing the leakage of cellular constituents. a similar change in free radical generation and related electrolyte leakage under as-stress has been observed in oryza sativa l. (begum et al. 2016) and zea mays l. (anjum et al. 2016). the results of the present study indicate that the as-induced oxidative damage in glycine max l. was compensated for remarkably by exogenous dpi, ebl or pro, by limiting ros production and electrolyte release. it has been suggested that dpi directly scavenges ros or decelerates the ros generation process via inhibiting o2˙ˉ synthase enzyme (ye et al. 2012). similarly, ebl and pro provide tolerance to oxidative stress either by accumulating endogenous osmolytes or activating antioxidant defense mechanism or both in the stressed tissues (agami 2014, shu et al. 2015). in this manner, dpi, ebl and pro, particularly pro, lower the ros in as-stressed tissues. this is in accordance with the reports of ye et al. (2012), shu et al. (2015), singh et al. (2015) and chandrakar et al. (2017a) under different abiotic stresses. arsenic-impelled alterations in lipid moieties of glycine max l. were analyzed by measuring contents of both mda and hne. accumulated data showed that the contents of mda and hne, the chief indicators in the monitorin gof oxidative injury, rose considerably in the as-exposed glycine max l. (tab. 2), which might be attributed to the enhanced accumulation of ros, known to cause membrane lipid oxidation (parkhey et al. 2014b). increased mda was also measured in as-exposed oryza tenuiflorum l. and zea mays l. by siddiqui et al. (2015) and anjum et al. (2016) respectively. the detrimental impacts of as in glycine max l., however, was attenuated substantially after the addition of dpi, ebl or pro; moreover, among the three treatments, pro was recognized to be the most effective against as-stress. since pro acts as a free radical scavenger and membrane stabilizer, it protects the lipid fractions from being oxidized, and thereby there is less accumulation of lipid peroxidized products (siddiqui et al. 2015, chandrakar et al. 2017b). arsenic-prompted increase in ros accumulation led oxidation of proteins and was analyzed by measuring protein and its oxidized products (pco and prooh)/ aldehydic adducts (mdaand hne-protein adducts). the results revealed that on exposure to as, there was a fall in protein content with concomitant increase in both pco and prooh, compared to the control. decreased protein with increased pco was recently reported in as-exposed seedlings of zea mays l. by mallick et al. (2013). however, there is nothing in the literature concerning the measurement of prooh, and mdaand hne-protein adducts in response to any metal/metalloid-toxicity in plants. the detrimental impacts of as in glycine max l. were attenuated substantially after the exogenous addition of dpi, ebl and pro, hence protein content was increased substantially and the levels of pco, prooh, and mdaand hne-protein adducts were lowered (tabs. 3 and 4). however, pro was recognized to be more effective than dpi and ebl against as-stress; it is well known to stabilize protein structures and conformations (wu et al. 2015). similarly, a massive rise in both amadori and maillard reaction products was observed in as-subjected glycine max l. in this regard, mostofa (2015) demonstrated that increased accumulation of glycation end products led to oxidative injury in oryza sativa l. upon cd exposure. however, treatment with dpi, ebl or pro decreased these products to a certain degree, and proved their potentialities in as-stress alleviation (tab. 4). these compounds decrease ros accumulation and stabilize protein structure, and thereby enhances as-stress resistance in glycine max l. compatible osmolytes such as pro, sugar, etc., regulate the osmotic potential of cells exposed to abiotic stresses (yadu et al. 2016, chandrakar et al. 2017a). the results revealed that in response to as, accumulation of endogenous pro and sugar were increased as compared with their respective controls. our data are in agreement with those of jha and dubey (2005) and anjum et al. (2016). however, accumulations of these osmolytes were further increased by the application of dpi, ebl or pro with as. at an increased level, endogenous pro directly scavenges ˙oh radicals and lowers as accumulation, hence providing protection against as-toxicity. likewise, enhanced sugar accrual may contribute to the osmotic balance in the cell, and provide sufficient storage reserves for faster recovery of plants under stressful conditions (jha and dubey 2005). exposure to as not only leads to protein oxidation, but also to accrual of damaged/oxidized proteins. if not eliminated readily, such proteins may form large aggregates due to covalent cross-linking, that may lead to cell death (karmous et al. 2014). both protease and proteasome are involved in the removal of such aggregated and damaged proteins from the cell (jin et al. 2016). the results revealed that chandrakar v., dubey a., keshavkant s. 60 acta bot. croat. 77 (1), 2018 activities of both protease and proteasome increased in the presence of as more than in their respective controls, and this is well supported by the report of jin et al. (2016). improved protease and proteasome could be the consequence of an increased need to remove oxidatively damaged proteins from the cell (karmous et al. 2014). data from the current study showed that upon supplementation of dpi, ebl or pro along with as protease and proteasome activity decreased, as they lowered the formation of protein oxidized products. to maintain ros homeostasis, plants possess an enzymatic defense system, which includes sod, cat, apx, etc. (yadu et al. 2017). exposure to as caused a decline in the activities of the above enzymes in glycine max l., which is consistent with the results of kaur et al. (2012). hence, antioxidant system might not be properly stimulated to attain the desired regulation over ros accumulation, which in turn contributes to greater oxidative stress in as-exposed glycine max l. further, dpi, ebl or pro supplementation to as activates the antioxidant system, hence confirms their protective roles in mitigating as-induced deleterious impacts in glycine max l. similar change in the activities of antioxidant enzymes in relation to exogenous dpi, ebl or pro into different abiotically stressed plants has previously been documented by jiang and zhang (2002), agami (2014) and fariduddin et al. (2015). exogenous application of dpi, ebl or pro was effective in alleviating the detrimental effects to glycine max l. of asstress. the beneficial effects of dpi, ebl and pro may be attributed to improvement in germination, growth, viability and antioxidant defense system to alleviate the ros-induced membrane damage and increased lipid and protein oxidized products. thus, exogenous application of dpi, ebl and pro provides tolerance to as-stress in glycine max l. moreover, pro performed better in reducing as-induced oxidative injury than dpi or ebl. the current approach could be useful in unravelling the mechanism(s) behind amelioration of as-induced oxidative injury in plants. acknowledgements the authors would like to thank the department of science & technology, new delhi, for awarding an inspire fellowship (no. dst/inspire fellowship/2013/791, dated 23.01.2013) to vibhuti chandrakar. the authors are also grateful to the department of science & technology, new delhi and the 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abscisic acid in rice seeds. journal of experimental botany 63, 1809–1822. acta bot. croat. 80 (2), 2021 191 acta bot. croat. 80 (2), 191–198, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-022 issn 0365-0588 eissn 1847-8476 response of bacillus cereus on zea mays under different doses of zinc sulphate asim shahzad1,2*, mingzhou qin1, mudassar nazir2, abdul shakoor3,4,, motsim billah5, gul zaib6 1 henan university, the college of geography and environment science, jinming ave, kaifeng, china 2 mohi-ud-din islamic university nerian sharif, department of botany, azad jammu & kashmir pakistan 3 henan university, college of environment and planning, jinming ave, kaifeng, china 4 henan university, laboratory of geospatial technology for the middle and lower yellow river regions, ministry of education kaifeng 475004, henan, china 5 university of haripur, faculty of applied sciences, khyber pakhtunkhwa, pakistan 6 institute of epigenetics and epigenomics, yangzhou university, 48 east wenhui road, yangzhou, jiangsu, 225009, china abstract – anthropogenic activities have added a large amount of heavy metals to the environment. heavy metal contaminants affect the physiological and biological properties of soil and plant health. zinc (zn) is an essential micronutrient and it promotes plant growth and development but a higher concentration of the metal causes reduction in plant growth. the present study was aimed to evaluate the response of bacillus cereus on maize plants at different concentrations of znso4 (20, 40 and 60 mg kg–1) amended in the soil under pot experiment conditions. the experiment was conducted by using complete randomized design (crd) with three replications. higher doses of znso4 inhibited maize growth and nutrient uptake. however, inoculation of maize seeds with bacillus cereus at 20 mg kg–1 concentration of znso4 increased seed germination about 39% and plant height by 15%. moreover, 17% increase in leaf length and a 7% increase in leaf number were observed as compared to control at 20 mg kg–1 concentrations of znso4. reductions in all growth parameters were observed with 60 mg kg–1 concentration of znso4. the zn uptake was 75% higher in treatment t8 (uninoculated seeds with 60 mg kg–1 concentration of znso4) as compared to treatments which were inoculated and grown under different zinc concentrations. the results suggest that bacillus cereus has good potential to remediate zn from soil as well as to reduce the phyto-availibility and phytotoxicity of zinc. keywords: maize, nutrient availability, phytoextraction, phytotoxicity, plant microbial interaction, zinc introduction soil is a mixture of different components, organic matter (5%), minerals (45%), gases (25%), and liquids (25%). besides this, several organisms which support and maintain life on earth are also found in the soil. soil provides several ecosystem services by maintaining biogeochemical cycles and protection against erosion (tahat et al. 2020). healthy soil acts as a dynamic living system that delivers multiple ecosystem services, such as plant productivity, sustaining water quality and controlling soil nutrient recycling decomposition. soil health is closely associated with sustainable agriculture, because soil microorganism diversity and activity are the main components of the soil health (tahat et al. 2020). anthropogenic and industrial activities, facilities and products such as mining of natural resources, electroplating, smelting, fertilizers, pesticides, tanneries add a large number of contaminants to our natural environment. these contaminants affect the physiological and biological properties of soil (arivalagan et al. 2014). among different contaminants, heavy metals such arsenic (as), cadmium (cd), lead (pb), copper (cu), chromium (cr), nickel (ni), zinc (zn), aluminum (al) and manganese (mn) are the most common (ullah et al. 2015). heavy metals have shown negative impacts on soil fertility and soil microbial community (chu 2018). they are not soluble in water and are not read* corresponding author e-mail: agron12@yahoo.com shahzad a., qin m., nazir m., shakoor a., billah m., zaib g. 192 acta bot. croat. 80 (2), 2021 ily degradable; therefore, they remain insoluble in the soil. later on, they are assimilated by plant roots. in this way, heavy metals are transferred to different trophic levels of the ecosystem and contaminate the food chain (tchounwou et al. 2012). zinc is an essential micronutrient which promotes plant growth and development (hefferon 2019); however, higher concentration of zn causes root blunt, cell wall thickening, reduction in cell division, cell elongation, and disruption in cell organelles and an increase in the number of the nucleoli (glińska et al. 2016, taghizadeh et al. 2018). plants have different defense mechanisms or strategies for tolerance or detoxification, whenever they are exposed to heavy metal stress condition. in addition, microorganisms from soil assist plants to degrade a huge variety of contaminants. in contaminated soil, microorganisms can modify and adopt various mechanisms that convert toxic substances into a less toxic form (jaiswal et al. 2017). these mechanisms include biosorption, bioaccumulation, biotransformation, and biomineralization. pseudomonas, alcaligenes, sphingomonas, rhodococcus and mycobacterium have the potential to detoxify contaminants in nature (vergani et al. 2017). some bacterial species such as bacillus and pseudomonas are generally used for the remediation of heavy metals from contaminated water and soil, as these bacterial strains have more metal binding affinities (arivalagan et al. 2014, ullah et al. 2015). plant growth-promoting bacteria increase plant growth and development by increasing accessibility of nutrients (bulgarelli et al. 2013), which contributes to plant health and increases their biomass and absorbing capacity to overcome stress (weyens et al. 2013). rhizobia have the potential to remove organic and metal contamination by degrading organic contaminants (teng et al. 2015). besides, bacteria have the ability to produce phytohormones such as auxins, cytokinins and gibberellins, which control all aspects of plant growth and development (glick 2010). plant growth-promoting bacteria enhance plant growth in stress conditions through various mechanisms including nitrogen (n) and phosphorus (p) solubilization, siderophore production and 1-aminocyclopropane-1-carboxylic acid (acc) deaminase production (fatima et al. 2017). increased concentration of heavy metals inhibits normal plant functioning by disturbing protein structure; this alteration hampers metabolic processes (hall 2002). the change in in protein structure can cause mutation in various physiological processes in plants such as photosynthesis, respiration, and enzymatic activities (hossain et al. 2012). plants have different defense mechanisms or strategies for tolerance or detoxification whenever they are exposed to elevated concentration of heavy metals. the first step in metal tolerance is inhibition or restriction of heavy metal entry into plant root (viehweger 2014). failure of the first step activates the mechanism of phytosequestration which permits heavy metals to enter in intracellular compartments (patra et al. 2004), which produce different substances like phytochelatins (pcs), organic acid, polysaccharides and metallothionein (mts), which are the two best-characterized metalbinding ligands in plant cells that help a plant to detoxify heavy metals (dalvi and bhalerao 2013). if all these strategies fail, plants activate their antioxidant defense mechanism which can help the plant by inducing reactive oxygen species (ros), which inhibit most cellular processes at various levels of metabolism. ros, being highly unstable, could play dual role (1) damaging cellular components and (2) act as an important secondary messenger for inducing plant defense system. cells are equipped with enzymatic and nonenzymatic defense mechanisms to counteract this. (sytar et al. 2013, manara 2012). any plant used for phytoremediation should not be metal-tolerant, and must be fast growing with the potential to produce high biomass. maize is mainly used in phytoextraction due to the fact that it is a common and rapidly growing crop, which possesses an extensive fibrous root system and can tolerate stress conditions (farooq et al. 2015). therefore, maize plants are suitable for the extraction of heavy metals from contaminated soil (lu et al. 2015). there is insufficient information about the fate of bacterial inoculation in maize edible and non-edible parts to enhance maize tolerance in heavy metal soils (shahzad et al. 2016). henceforth, present investigation is aimed to evaluate the role of bacillus cereus strain in enhancing the phytoextraction ability of maize plant under zn contaminated soil conditions. materials and methods bacterial strain bacillus cereus (accession no. kr232400) was selected on the basis of its plant growth promoting potential (shahzad et al. 2020). the strain was obtained from phytohormone laboratory at quaid-i-azam university, pakistan. bacterial strain was previously identified by 16s rrna gene sequencing (shahzad et al. 2016). preparation of heavy metal solution three different concentrations of zn solution (20, 40 and 60 mg kg–1) for maize plant treatments were prepared in pure distilled water by dissolving zinc sulfate (znso4). we selected these three concentrations on the basis of previous scientific data (long et al. 2003, yang et al. 2005). pure distilled water was used as the control for the experiment. 200 ml of each solution was added in 1 kg of potted soil. preparation of inoculum for the preparation of inoculum, five ml of nutrient broth medium (oxoid, uk) was autoclaved in test tubes for cultivation of bacterial strain. bacteria were incubated in a shaker incubator (excella e24 germany) at 37 °c and 150 rpm for 48–72 h. thereafter, the culture was centrifuged at 3000 rpm for 10 min. the pellet was re-suspended in autoclaved distilled water and the optical density (od) was adjusted to 0.100 at 660 nm with uv-vis spectrophotometer (uv-vis double beam spectrophotometer, model no: maize response against zinc acta bot. croat. 80 (2), 2021 193 ae-s90-2d a & e lab (uk) co., ltd.). bacterial density was 106 cells ml–1 at optical density of 0.10 at 660 nm. the optical density was adjusted by adding about 200 ml sterile water to pure bacterial pellets. preparation of treatment applications and seed inoculation kashmir gold variety maize (zea mays l) seeds were collected from the national agricultural research centre, islamabad, pakistan. the seeds were surface sterilized by washing with 95% ethanol, following by soaking in 10% clorox for 2–3 minutes. the seeds were washed successively 2–3 times in autoclaved distilled water. for experiment preparation, seeds were soaked in the b. cereus inoculum for 2–3 hrs, after that about 10 seeds were sown in each pot. different concentrations of zn solutions (100, 200 and 300 mg l–1) and distilled water was added to each pot. eight different treatments with three replicates were made. detailed explanation of treatments is given in table 1. parameter measured the percentage germination was noted after seven day of sowing. maize seedlings were uprooted after 21 days of sowing, after which root length (cm), plant height (cm), leaf length (cm), number of leaves and fresh plant weight parameters were recorded. each treatment contained three replicates and 5 plants from each replicate were measured. soil nutrient analyses determination of macroand micro-nutrients (na, ca, mg, k, p, n, fe, cu, cr, co, zn, and mn) was carried out by following the ammonium bicarbonate and diethylenetriamine pentaacetic acid (dtpa) method as described by soltanpour and schwab (1977). the 0.005 m ammonium bicarbonate and (dtpa) solution was prepared by adding 1.97 g of dtpa to 800 ml of distilled water. about 2 ml of ammonium hydroxide (nh4oh) was added to facilitate dilution and to prevent effervescence due to ammonium bicarbonate addition. when dtpa was dissolved completely, 79.06 g of ammonium bicarbonate (nh4hco3) was added. the ph of the solution was adjusted to 7.6 by adding 2 ml of ammonium hydroxide. final volume was adjusted to 1 l with the addition of distilled water. for the analysis of different nutrient, 10 g of soil sample was mixed with 15 ml of distilled water. the suspension was stirred for 30 minutes on a magnetic stirrer. after stirring step, suspension was filtered. two ml of filtrate was removed and 18 ml of distilled water was added for nutrient analysis. plant nutrient analysis the perchloric-acid digestion scheme was used to determine the content of various nutrients in maize plants (allen et al. 1974). a solution of nitric acid, sulfuric acid, and perchloric acid in the ratio 5:1:0.1 (6.5 ml) was added to 0.25 g of plant material in a 50 ml flask and the mixture was heated on the hot plate in the fume hood to complete the digestion process. the formation of white fumes in the mixture confirmed the completion of digestion of the plant extract. thereafter, distilled water (few drops) was poured into the flask and the mixture was allowed to cool. the digested plant extract samples were transferred to the volumetric flasks (50 ml) and the volume was raised up to 50 ml by the addition of distilled water. the samples were filtered with whatman no.42 filter paper and were stored for further element analysis. the concentration of different elements/nutrients present in the plant samples were measured by atomic absorption spectrophotometer (shimadzu aa-670 germany). statistical analysis the experiment was conducted in a completely randomized design (crd) by using statistic 8.1.1. (https://statistix. informer.com/8.1/). the results are the compare means and standard error of means of three replicates of a treatment. five plants from each replicate were measured. results effects on seed germination percentage germination of maize seeds has shown variations among inoculated and uninoculated seeds (fig. 1a). maximum seed germination (%) was observed in b. cereusinoculated seeds in combination with 20 mg kg–1 znso4 (treatment t3), which was significantly higher than control (39%) and the majority of the treatments. high germination percentage was also recorded in b. cereus-inoculated seeds in combination with 40 mg kg–1 znso4 (treatment t4), which was significantly higher than the treatment of uninoculated seeds with 60 mg kg–1 znso4 (treatment t8), where the lowest values were obtained (germination reduction of 9% as compared to control). effects on root length, leaf length and number of leaves the root length was only mildly affected by investigated treatments (fig. 1b). the maximum increase in the root length (15%) was observed in b. cereus inoculated seeds in tab. 1. kashmir gold variety zea mays seeds were inoculated (+) or not (–) with bacillus cereus, germinated and grown in soil supplemented with 20, 40 or 60 mg kg–1 znso4. eight treatments (t) were applied. treatments bacillus cereus inoculation znso4 (mg kg–1) t1 – 0 t2 + 0 t3 + 20 t4 + 40 t5 + 60 t6 – 20 t7 – 40 t8 – 60 shahzad a., qin m., nazir m., shakoor a., billah m., zaib g. 194 acta bot. croat. 80 (2), 2021 combination with 20 mg kg–1 znso4 (treatment t3), although it was not significant compared to control. similar increase in root length (14%) was also recorded for b. cereusinoculated seed (treatment t2 treatments). on the contrary, the 19 % reduction in root length was observed in the treatment of uninoculated seeds exposed to60 mg kg–1 znso4 (treatment t8), which was not significant compared to control, but was significantly reduced in comparison to treatments t2 and t3. other treatments exhibited values similar to those of control maximum increase in leaf length (17%) was observed in b. cereus-inoculated seeds in combination with 20 mg kg–1 znso4 (treatment t3), although it was not significant as compared to control and other treatments of inoculated seeds; however, it was significantly higher in comparison to result obtained for uninoculated seeds (fig. 1c). namely, all treatments of uninoculated seeds resulted in reduction in leaf length, which was particularly after exposure to 20 mg kg–1 znso4 (treatment t6). maximum number of leaves (1% increase compared to control) was observed in b. cereus-inoculated seeds in combination with 40 mg kg–1 znso4 (treatment t4), although this value was not significantly different as compared to the majority of the treatments; the only exception was the treatment of uninoculated seeds exposed to 20 mg kg–1 znso4 (treatment t6), which exhibited the lowest value (7% decrease compared to control) (fig. 1d). effects on plant height, fresh weight and zinc uptake the inoculation of maize seeds with b. cereus significantly increased the fresh weight and height of maize plants (fig. 2). a significantly higher increase in fresh weight was observed upon exposure of inoculated seeds to 20 mg kg–1 znso4 (treatment t3, 80% increase) and 40 mg kg–1 znso4 (treatment t4, 62% increased) in comparison to control and the treatments of uninoculated seeds. on the contrary, a significant reduction in plant fresh weight as compared to control was observed in all treatments of uninoculated seeds, with the maximum reduction of 27% recorded upon exposure to 60 mg kg–1 znso4 (treatment t8) (fig. 2b). fig. 1. effect of zinc on kashmir gold variety zea mays seed germination % (a), root length (b), leaf length (c) and number of leaves (d). plants were measured at 21 day after sowing. results, expressed as means ± standard error (se) of 3 replicates and each replica contains 10 plants. results were compared by using compare means with completely randomized design (crd). treatments sharing a common letter (a>b>c) are similar, otherwise differ significantly at p < 0.05. t1 – control, t2 – bacillus cereus-inoculated seeds, t3 – b. cereus-inoculated seed + 20 mg kg-1 znso4, t4 – b. cereus-inoculated seeds + 40 mg kg-1 znso4, t5 – b. cereusinoculated seeds + 60 mg kg-1 znso4, t6 – uninoculated seeds + 20 mg kg-1 zn so4, t7 – uninoculated seeds + 40 mg kg-1 znso4, t8 – uninoculated seeds + 60 mg kg-1 znso4. fig. 2. effect of zinc on kashmir gold variety zea mays plant height (a), plant fresh weight (b) zinc uptake (c). plants were measured at 21 day after sowing. results, expressed as means ± standard error (se) of 3 replicates and each replica contains 10 plants. results were compared by using compare means with completely randomized design (crd). different letters (a> b> c) indicate treatments sharing a common letter are similar but otherwise differ significantly at p < 0.05. t1 – control, t2 – b. cereus-inoculated seeds, t3 – b. cereus-inoculated seed + 20 mg kg-1 znso4, t4 – b. cereus-inoculated seeds + 40 mg kg-1 znso4, t5 – b. cereus-inoculated seeds + 60 mg kg-1 znso4, t6 – uninoculated seeds + 20 mg kg-1 zn, t7 – uninoculated seeds + 40 mg kg-1 znso4, t8 – uninoculated seeds + 60 mg kg-1 znso4. maize response against zinc acta bot. croat. 80 (2), 2021 195 exposure of inoculated seeds to znso4 slightly improved the plant height (treatments t3, t4 and t5) as compared to control; the maximum value (12% increase as compared to control) was obtained upon exposure to 20 mg kg–1 zn (treatment t3). on the other hand, exposure of uninoculated seeds to the same zn concertation (20 mg kg–1) resulted in a reduction in plant height up to 19% (fig. 2a). zinc uptake was reduced in the majority of the treatments with inoculated seeds as compared to control; significantly for treatments t2 and t3 (exposure to 100 mg l–1 znso4), and notably for treatment t4 (exposure to 49 mg kg–1); reduced zn uptake was 49%, 44% and 22% in t2, t3 and t4 respectively (fig. 2c). however, the exposure of inoculated seeds to the highest tested zn concentration (treatment t5) resulted in a significantly higher zn uptake as compared to control. among treatments with uninoculated seeds, exposure to 20 mg kg–1 (treatment t6) resulted in a value similar to that of control, while upon treatment with 40 and 60 mg kg–1 zn (treatments t7 and t8, respectively) zn uptake was significantly higher; the maximum increase (75% as compared to control) was observed in treatment t8 (fig. 2c). nutrients status of soil and accumulation of nutrients by maize plant different nutrients including ca, mg, k, na, fe, cd, cu, cr, zn, co and ni were detected in soil samples (tab. 2). the plant nutrient status (tab. 3) showed that the cu content decreased in majority of the treatments as compared to control; the only exceptions were treatments t3 and t8. the mn content was significantly elevated in the treatment of plants from inoculated seeds exposed to 20 mg kg–1 znso4 (treatment t3), while the exposure of plants from uninoculated seeds to 40 mg kg–1 znso4 (treatment t7) resulted tab. 2. analysis of soil micro and macro nutrients before addition of znso4 and sowing of zea mays measured in mg kg–1. results are expressed as means ± standard error (se) of three soil samples. nutrient soil content (mg kg–1) ca 19.6 ± 1.11 mg 2.46 ± 0.23 k 1.67 ± 0.21 na 22.39 ± 0.66 fe 3.18 ± 0.12 cd 4.39 ± 0.19 cu 11.20 ± 0.44 cr 10.5 ± 0.12 zn 8.9 ± 0.11 co 0.19 ± 0.02 ni 2.23 ± 0.15 mn 6.22 ± 0.27 ta b. 3 . a cc um ul at io n of m ic ro a nd m ac ro n ut ri en ts b y k as hm ir g ol d va ri et y z ea m ay s p la nt s. pl an ts w er e m ea su re d at 2 1 da y af te r so w in g. r es ul ts , e xp re ss ed a s m ea ns ± s ta nd ar d er ro r (s e) o f th re e re pl ic at es a nd e ac h re pl ic a co nt ai ns 5 p la nt s. r es ul ts w er e co m pa re d by u si ng c om pa re m ea ns w ith c om pl et el y ra nd om iz ed d es ig n (c r d ). tr ea tm en ts s ha ri ng a c om m on le tt er (a >b >c ) a re si m ila r, ot he rw is e di ffe r s ig ni fic an tly a t p < 0. 05 . t 1 – co nt ro l, t 2 – ba ci llu s c er eu sin oc ul at ed se ed s, t 3 – b. ce re us -i no cu la te d se ed + 2 0 m g kg –1 z ns o 4, t 4 – b. ce re us -i no cu la te d se ed s + 4 0 m g kg –1 z ns o 4, t 5 – b. c er eu sin oc ul at ed se ed s + 6 0 m g kg –1 z ns o 4, t 6 – un in oc ul at ed se ed s + 2 0 m g kg –1 z n, t 7 – un in oc ul at ed se ed s + 4 0 m g kg –1 z ns o 4, t 8 – un in oc ul at ed se ed s + 6 0 m g kg –1 z ns o 4. n ut ri en t n ut ri en t c on ce nt ra tio n (m g kg –1 ) t 1 t 2 t 3 t 4 t 5 t 6 t 7 t 8 c u 0. 33 ± 0 .0 1 a b 0. 15 ± 0 .0 01 d 0. 38 ± 0 .0 2 a 0. 20 ± 0 .0 01 d 0. 30 ± 0 .0 2 b c 0. 21 ± 0 .0 2 c d 0. 16 ± 0 .0 2 d 0. 2± 0. 01 d m n 0. 12 ± 0 .0 1 b c 0. 14 ± 0 .0 1 b 0. 2 ± 0. 02 a 0. 07 ± 0 .0 03 c d 0. 09 ± 0 .0 1 b c d 0. 06 ± 0 .0 1 c d 0. 05 ± 0 .0 1 d 0. 09 ± 0. 01 c d n i 0. 02 ± 0 .0 01 b 0. 00 8 ± 0. 00 02 d 0. 03 ± 0 .0 02 a 0. 01 3 ± 3. 33 b c 0. 00 7 ± 0. 00 1 d 0. 00 7 ± 0. 00 04 d 0. 00 9 ±0 .0 02 c d 0. 00 9 ± 0. 00 03 c d c o 0. 10 ± 0 .0 01 b 0. 11 ± 0 .0 02 a 0. 03 ± 0 .0 01 f 0. 05 ± 0 .0 02 d e 0. 03 ± 0 .0 00 1 f 0. 04 ± 3 .8 5 ef 0. 05 ± 0 .0 05 c d 0. 06 ± 0 .0 01 c n a 11 .3 ± 0 .3 4 e 16 .1 ± 0 .7 c d e 13 .3 ± 1 .0 d e 29 .1 9 ± 0. 5 a b 33 .8 ± 0 .5 2 a 35 .9 ± 0 .9 a 23 .6 ± 3 .6 b c 19 .1 ± 0 .2 c d c r 0. 20 ± 0 .0 1 b c 0. 3 ± 0. 02 a b 0. 4 ± 0. 02 a 0. 32 ± 0 .0 5 a 0. 20 ± 0 .0 14 b c 0. 12 ± 0 .0 03 c 0. 13 ± 0 .0 2 c 0. 1 ± 0. 03 c fe 30 .3 ± 0 .1 c 34 .1 ± 0 .8 b c 43 .9 ± 0 .3 a b c 47 .0 1 ± 0. 5 a b 49 .9 ± 2 .0 3 a 28 .1 ± 8 .1 7 a b c 11 .8 ± 1 .5 d 10 .1 ± 0 .3 1 d c a 13 .1 ± 0 .2 e 31 .4 ± 0 .6 c 39 .8 2 ± 0. 4 b 49 .6 0 ± 1. 2 a 37 .4 ± 0 .7 b 18 .7 ± 0 .1 d 17 .8 ± 0 .9 d 14 .0 7 ± 0. 3 e m g 7. 10 ± 0 .6 e 29 .3 ± 0 .4 b c 35 .2 3 ± 0. 9 a 30 .9 ± 0 .3 a b c 32 .5 ± 1 .2 a b 25 .7 ± 1 .2 c 14 .5 ± 1 .5 d 13 .4 ± 0 .4 d k 34 .7 ± 0 .5 b 58 .3 ± 4 .1 a 61 .3 4 ± 2. 01 a 56 .2 0 ± 1. 1 a 47 .3 ± 0 .8 a b 30 .0 7 ± 4. 7 b 10 .4 ± 4 .3 c 6. 7± 0. 7 c shahzad a., qin m., nazir m., shakoor a., billah m., zaib g. 196 acta bot. croat. 80 (2), 2021 in significantly lower mn content as compared to control. reduced ni and co contents were recorded in the majority of treatments as compared to control. seed inoculation with b. cereus in general decreased the na content as compared to values obtained in uninoculated plants, since the higher values were obtained in treatments of plants from uninoculated seeds (treatments t6, t7 and t8) as compared to treatments of plants from inoculated seeds to the corresponding zn concentrations. seed inoculation with b. cereus increased cr content in plants as compared to control as well as to the treatments of plants from uninoculated seeds upon exposure to 20 and 40 mg kg–1 znso4 (treatments t3 and t4) as compared to treatments t6 and t7, respectively). fe content was significantly elevated in plants from inoculated seeds upon exposure to 40 and 60 mg kg–1 znso4 compared to control as well as to the exposure of plants from uninoculated seeds to the same zn concentration. seed inoculation with b. cereus in general increased the ca and mg uptake in all investigated treatments as compared to control, with higher values obtained in plants from inoculated seeds compared to the uninoculated ones. k content was significantly higher in plants from inoculated seeds as compared to the control as well as to plants from uninoculated seeds exposed to the corresponding zn concentrations. discussion zinc is a trace element that performs a vital role in plant growth and development. however, at higher concentrations, it reduces seed germination, length of seedlings and stem as well as contents of chlorophyll, carotenoid, sugar, and amino acids (todeschini et al. 2011, glińska et al. 2016). in this study, the effects of zinc toxicity on plant height, leaf length, number of leaves, root length, fresh weight, and nutrient content was observed. our results indicated that high zn concentrations inhibited seed germination as compared to the control. previous studies found a negative impact of zn on plant growth parameters. current findings are in agreement with previous studies (islam et al. 2014, glińska et al. 2016, boi et al. 2020), in these studies, they reported that increased heavy metal concentration causes decrease in plant biomass and growth. nonetheless, maize inoculated with b. cereus showed increases in seed germination, plant height and fresh weight as well as nutrient uptake as compared to control and uninoculated plants. enhanced root growth results due to inoculation of b. cereus under higher concentrations of zinc and thus a positive impact of b. cereus on root growth was observed, which may be due to the stress tolerance capacity of b. cereus (hassan 2018). improvement in plant root development under zinc concentration resulted in an increase in nutrient uptake, which increased seed germination, plant height, leaf length, number of leaves, root length and plant fresh weight. inoculated seeds showed an increase in leaf growth and number of leaves as compared to un-inoculated plants grown in the soil supplemented with zinc. we argue that gibberellins play a vital role in plant growth development. scientific data suggests that b. cereus helps the plant by producing different plant promoting hormones i.e gibberellic acid (ga). therefore, the increase in some plant growth parameters due to inoculation of b. cereus might be because of production of certain plant promoting enzymes, which can stimulate seed germination by enhancing growth ability (finch-savage and leubner-metzger 2006, tuan et al. 2018). gibberellins increase leaf area by suppressing peroxidase secretion (de souza and macadam 2001), and induce cell mitotic division (takatsuka and umeda 2014, fonouni-farde et al. 2019). moreover, some researchers suggested that b. cereus induces production of indole acetic acid (iaa), which may help to increase root length, plant height, and leaf area (shafi et al. 2017). henceforth, phytohormones produced by b. cereus might have played vital role in maize growth. zn uptake decreased in b. cereus-inoculated maize plants. common logic suggests that microbes including bacillus species have ability to convert zn into smaller, inaccessible and immoveable form in the soil (saravanan et al. 2004, li et al. 2016). another explanation for reduced zn uptake is production of metal binding proteins in the rhizosphere. bacillus cereus produces metal binding proteins to counter heavy metal toxicity and these proteins have ability to restrict phytoavailability of heavy metals (islam et al. 2014). we also demonstrated the accumulation of macro and micronutrients in maize plant owing to b. cereus. bacillus cereus showed increase uptake of cu, mn, ni, na, cr, fe, ca, and mg in maize plant. tiwari et al. 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d.v., 2005: uptake and accumulation of cadmium and zinc by sedum alfredii hance at different cd/zn supply levels. journal of plant nutrition 27, 1963–1977. acta bot. croat. 80 (1), 2021 29 acta bot. croat. 80 (1), 29–34, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-005 issn 0365-0588 eissn 1847-8476 plant development, gas exchanges and pigments of mesosphaerum suaveolens submitted to osmoconditioning and saline stress francisco romário andrade figueiredo1*, jackson silva nóbrega2, reynaldo teodoro de fátima3, toshik iarley da silva4, rodrigo garcia da silva nascimento2, maria de fátima de queiroz lopes2, thiago jardelino dias2, riselane de lucena alcântara bruno2 1 federal rural university of the semi-arid region, department of agronomic and forestry sciences, mossoró, brazil 2 federal university of paraíba, department of plant science and environmental sciences, areia, brazil 3 academic unit of agricultural engineering, federal university of campina grande, campina grande, brazil 4 federal university of viçosa, department of plant science, viçosa, brazil abstract – salinity is one of the main plant abiotic stresses affecting the establishment and development of crops. it is thus a matter of prime importance to search for technologies that minimize the damage caused by salinity. the aim of the present work was to evaluate the effect of salinity stress and osmotic conditioning of seeds on the biomass, gas exchanges and chlorophyll pigments in mesosphaerum suaveolens (l.) kuntze. the statistical design adopted was a randomized block design, combined according to the central composite design, referring to electrical conductivities of irrigation water and osmotic potentials, with minimum (–α) and maximum (α) values of 0.5 and 10.0 ds m–1 and 0.0 and –1.0 mpa, respectively, totaling nine combinations. the characteristics of dry biomass, gas exchange and chlorophyll indices were evaluated at 45 days after the beginning of irrigation with saline water. the salinity of irrigation water severely affected the dry biomass and the gas exchanges of m. suaveolens. irrigation water of electrical conductivity above 3.2 ds m–1 caused reductions in chlorophyll a, b and total contents in m. suaveolens plants. seed osmoconditioning did not attenuate the negative effects of saline stress on m. suaveolens plants. keywords: bamburral, chlorophyll, plant growth, photosynthesis, salinity introduction mesosphaerum suaveolens (l.) kuntze, also known as “bamburral” in the brazilian northeast, is a species belonging to the botanical family lamiaceae, which presents subshrub growth habit and leaves with high aromatic potential (sabóia et al. 2018). the species, besides being widely used by folk medicine in several regions of brazil, is also rich in secondary compounds (alkaloids, flavonoids, tannins and others), which are used in the pharmaceutical and cosmetics industries (alves et al. 2017, bezerra et al. 2017). the chemical quality of irrigation water is one of the factors that directly affect plants’ metabolic processes, especially in semi-arid regions, where water is scarce and, in many cases, saline (guimarães et al. 2019). in the semi-arid region of brazil, salinity is among the abiotic stresses that most hinder the growth and production of crops (sales et al. 2015). salinity can affect plants in two ways: by reducing the soil osmotic potential, thus reducing water and nutrient uptake, and by accumulating specific ions (na+ and cl–), leading to toxicity and nutritional imbalance (taiz et al. 2017). osmotic and ionic effects on crops can cause changes in the plants’ physiological and biochemical functions (ouhaddach et al. 2018). therefore, it is necessary to search for technologies that might attenuate these salinity stress negative effects on irrigated agricultural systems. in this sense, seed osmoconditioning is a practice that has been adopted with the purpose of reducing the germination time, increasing the germinability, uniformity and * corresponding author e-mail: romarioagroecologia@yahoo.com.br figueiredo f. r. a., nóbrega j. s., de fátima r. t., da silva t. y., nascimento r. g. s., lopes m. f. q., dias t. j., bruno r. l. a. 30 acta bot. croat. 80 (1), 2021 performed: ph = 7.8; organic matter (g kg–1) = 22.2; p (mg kg–3) = 85.3; k+ (mg kg–3) = 693.6; ca+2 (cmolc dm–3) = 2.9; mg+2 (cmolc dm–3) = 1.59; na+ (cmolc dm–3) = 0.23; h++al+3 (cmolc dm–3) = 0.0; al+3 (cmolc dm–3) = 0.0; sum of bases (cmolc dm–3) = 6.5; cation exchange capacity (cmolc dm–3) = 6.5. the evaluations were carried out at 45 days after the irrigation with saline water started. to determine root and shoot dry mass, these plant parts were packed in kraft paper bags and dried in a forced air circulation oven at 65 °c until reaching constant weight. subsequently, the material was weighed, with results expressed in g plant–1; the total dry mass was obtained from the sum of the dry masses of the root and shoot, with the results in g plant–1. using the dry mass data, the shoot: root ratio was estimated (shoot dry mass/root dry mass). the gas exchange evaluations were performed on the fourth leaf, from apex to base, between 9:00 and 10:00 a.m., using the portable infrared gas analyzer irga (model li6400xt, li-cor®, nebraska, usa) with 300 ml min–1 airflow, 400 µmol co2 m–2 s–1 and a coupled light source of 1200 μmol m–2 s–1. the variables assessed were: net co2 assimilation rate (a) (μmol co2 m–2 s–1), stomatal conductance (gs) (mol h2o m–2 s–1), co2 concentration in intercellular spaces (ci) (μmol co2 mol air–1), transpiration rate (e) (mmol h2o m–2 s–1) and foliar temperature (°c). from these variables, the water use efficiency (wue: a/e), intrinsic water use efficiency (iwue: a/gs) and instantaneous carboxylation efficiency (ice: a/ci) were calculated. the contents of chlorophyll a, b and total, as well as the chlorophyll a/b ratio, were also determined on the fourth leaf, from apex to base, between 9:00 and 10:00 am, by a non-destructive method using a portable chlorophyllometer (clorofilog®, model cfl 1030, porto alegre, rs). the values were expressed as the falker chlorophyll index (fci). the data were submitted to analysis of variance by the f-test (p < 0.05), and in the case of a significant effect, the data were submitted to regression analysis, using the statistical program sas university (cody 2015). results there was no significant interaction between the studied factors, seed osmoconditioning and salinity levels of irrigation water. for the shoot (sdm), root (rdm) and total (tdm) dry mass, a decreasing effect is observed, with reductions of 80.7%, 89.4% and 86.8%, respectively, at 10.00 ds m–1 salinity level compared to the control treatment (fig. 1a-c). however, the sdm/rdm ratio presented an inverse behavior to the above-mentioned variables, increasing linearly as a function of increases in salinity level of the irrigation water (fig. 1d). regarding the gas exchange variables, a decreasing linear effect was observed for a. plants irrigated with water at 10.00 ds m–1 salinity level presented a reduction of 54.9% in a when compared to the control treatment (fig. 2a). howvigor of the seedlings, which is advantageous in plants subjected to salt stress conditions (cardoso et al. 2012). during physiological conditioning, seed hydration occurs slowly, which gives more time for the repair or reorganization of plasma membranes, allowing the formation of tissues in a more orderly manner, reducing the risk of damage to the embryonic axis (windauer et al. 2007). polyethylene glycol (peg) has been widely used for the formulation of solutions with different osmotic potentials, simulating drought stress, which could induce secondary dormancy (souza et al. 2011). thus, the aim of the present work was to evaluate the effect of salinity stress and osmotic conditioning of seeds on the biomass accumulation, gas exchanges and chlorophyll pigments in m. suaveolens plants. materials and methods the research was carried out in a greenhouse belonging to the department of plant science and environmental sciences at the federal university of paraíba, located in the city of areia, paraíba, brazil. the statistical design adopted was a randomized block design, factorially combined according to the central composite matrix of box, referring to electrical conductivities of irrigation water (ecw) and osmotic potentials, with minimum (–α) and maximum (α) values of 0.5 and 10.0 ds m–1 and 0.0 and –1.0 mpa, respectively, with four replicates consisting of two plants, totaling nine combinations (mateus et al. 2001). saline water was prepared by adding sodium chloride (nacl) to the water of the supply system (ecw = 0.5 ds m–1) in the proportions required. the salinity level of the waters was measured using the instrutherm® (model cd – 860) micro-processed portable conductivity meter. irrigation was performed daily, with the application of saline water 10 days after sowing. the irrigation volume applied was established through drainage lysimetry, from the difference between the amount applied and drained. osmoconditioning was performed by soaking seeds in peg 6000 (dinâmica®, brazil) solutions. peg 6000 was diluted in 200 ml of distilled water in the proportions required for each osmotic potential. the seeds were soaked for 8 hours at 25 °c, in containers covered with aluminum foil, being the amounts of peg 6000 established according to villela et al. (1991). subsequently, the seeds were washed with distilled water. the seeds used in the experiment were obtained from native plants found at the novo horizonte settlement, municipality of várzea, paraíba, brazil. the seedlings were produced in 1.2 l capacity polyethylene bags, 10 seeds being sown per container. after seedling emergence, thinning was performed, keeping only the most vigorous plant in each container. the polyethylene bags were filled with a substrate composed of soil (latosol type), washed sand and tanned cattle manure in a 3:1:1 ratio. a substrate chemical analyse was ecophysiology of m. suaveolens under saline stress acta bot. croat. 80 (1), 2021 31 ever, ci presented an increasing linear effect as a function of the increase in salinity level, with an increase of 11.3% (fig. 2b). for ice there was a 58.7% reduction in ecw at 10.0 ds m–1 salinity level when compared to the control treatment (fig. 2c). it was observed that increasing salinity levels in the irrigation water provided a linear reduction in the transpiration rate (e), decreasing by 33.8% at 10.0 ds m–1 when compared to the control (fig. 2d). the plants tended to use water less efficiently with increasing salinity levels, decreasing by 36.6% (fig. 2e). it can be observed that only from 3.2 ds m–1 salinity level was there a reduction in the indexes of chlorophyll a, b and total, with decreases of 25.8%, 42.3% and 29.4%, respectively, at the highest salinity level when compared to the control treatment (fig. 3a-c). regarding the chlorophyll a/b ratio, the data show that there were increases in this variable from the ecw of 3.2 ds m–1 (fig. 3d). seed osmoconditioning with peg did not provide significant differences for chlorophyll a. however, total chlorophyll and chlorophyll b were negatively affected by the increase in osmotic potential reduction of the solution, with decreases of 27.2 and 18.2%, respectively, when comparing the highest osmotic potential (–1.00 mpa) with the control (fig. 4a,b). the reduction of the osmotic potential of the solution provided increases in the chlorophyll a/b ratio, with a 16.2% increase at the potential of –1.00 mpa (fig. 4c). discussion the results for the dry mass may be related to the excessive absorption of ions such as na+ and cl–, causing reductions in the accumulation of photoassimilates due to a lower photosynthetic rate. also, there is an increase in energy expenditure by the plant due to the reduction of the osmotic potential, which makes it difficult to absorb water through the roots (araújo et al. 2016). similar results were verified by bione et al. (2014) in salt-stressed basil plants (ocimum basilicum l.) cultured in a hydroponic nft system and oliveira et al. (2016) in maize (zea mays l.) submitted to saline stress and biostimulant treatment. these authors verified that dry mass production was reduced with the increase of salinity. the reduction in a was possibly due to the damage caused by salts to the chloroplast, thus reducing co2 fixation by rubisco. high concentrations of na+ and cl– cause toxicity at different levels and may alter several biochemical and physiological processes, among them the fixation of carbon dioxide during the photosynthesis process (rouphael et al. 2012). the increase in ci can be directly related to the photosynthetic rate reduction, which results in a lower co2 consumption during the carboxylation process. reductions in the photosynthetic rate lower the atp and nadph availability as well as the substrate for rubisco, which is associated with the reduction of intrinsic carboxylation effifig. 1. shoot dry mass (a), root dry mass (b), total dry mass (c) and shoot/root dry mass ratio (d) of mesosphaerum suaveolens subjected to electrical conductivities of irrigation water (0.50, 1.45, 5.00, 8.55 and 10.00 ds m–1). figueiredo f. r. a., nóbrega j. s., de fátima r. t., da silva t. y., nascimento r. g. s., lopes m. f. q., dias t. j., bruno r. l. a. 32 acta bot. croat. 80 (1), 2021 ciency as a function of the increase in salinity (silva et al. 2015). this decrease in e can usually be an adaptation mechanism in response to abiotic stresses, specifically saline and hydric, in order to reduce water loss. several authors have observed reductions in net co2 assimilation rate, transpiration rate and carboxylation efficiency as a function of saline stress, for example huang et al. (2015) in boehmeria nivea l., yarami and sepaskhah (2015) in crocus sativus l., and rouphael et al. (2016) in cucurbita pepo l. this reduction in chlorophyll indices may be related to several factors, such as: decreased chlorophyll a biosynthesis, increased chlorophyllase activity and instability of protein complexes caused by saline stress effects (houimli et al. 2010). melo et al. (2017), studying the effect of irrigation with saline water in bell pepper plants, obtained similar results, stating that chlorophyll a was the most sensitive variable to salinity. however, the increase in the chlorophyll a/b ratio, possibly, can be attributed to the greater reduction observed in chlorophyll b (42.3%) than in chlorophyll a (25.8%). corroborating these results, shimoda et al. (2012) report that the first step in the degradation of chlorophyll b is its conversion to chlorophyll a. these decreases in total chlorophyll and chlorophyll b can reflect a biochemical adaptation of the plant in response to the osmotic conditioning and the action of oxidative degfig. 2. net co2 assimilation rate – a (a), internal co2 concentration – ci (b), instantaneous carboxylation efficiency – ice (c), transpiration rate – e (d) and water use efficiency – wue – a/e (e) of mesosphaerum suaveolens subjected to electrical conductivities of irrigation water (0.50, 1.45, 5.00, 8.55 and 10.00 ds m–1). ecophysiology of m. suaveolens under saline stress acta bot. croat. 80 (1), 2021 33 fig 3. chlorophyll a (a), chlorophyll b (b), total chlorophyll (c) and chlorophyll a/b ratio (d) of mesosphaerum suaveolens subjected to electrical conductivities of irrigation water (0.50, 1.45, 5.00, 8.55 and 10.00 ds m–1). fig. 4. chlorophyll b (a), total chlorophyll (b) and chlorophyll a/b ratio (c) of mesosphaerum suaveolens subjected to osmoconditioning with peg (–1.00, –0.85, –0.50, –0.15 and 0.00 mpa). figueiredo f. r. a., nóbrega j. s., de fátima r. t., da silva t. y., nascimento r. g. s., lopes m. f. q., dias t. j., bruno r. l. a. 34 acta bot. croat. 80 (1), 2021 radation agents (cardoso et al. 2012). this increase in chlorophyll a/b ratio was possibly due to the reduction in chlorophyll b and unchanged chlorophyll a indices. however, in basil plants santos et al. (2012) observed divergent results, where plants cultivated without water deficit showed a higher chlorophyll a/b ratio, attributing this behavior to the fact that chlorophyll a degradation by oxidative damage occurs faster when compared to chlorophyll b. conclusions seed osmoconditioning did not attenuate the negative effects of saline stress on m. suaveolens plants. the salinity of irrigation water severely affected the dry biomass and the gas exchanges of m. suaveolens. irrigation water with electrical conductivity above 3.2 ds m–1 caused reductions in chlorophyll a, b and total contents in m. suaveolens plants. acknowledgements the authors thank the coordination for the improvement of higher education personnel (capes) and the brazilian national council for scientific and technological development (cnpq) for granting the scholarships to the postgraduate students involved in this research. references alves, j.j.l., resende, o., oliveira, d.e.c., branquinho, n.a.a., 2017: drying 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polyethilene glycol 6000 concentration and temperature. pesquisa agropecuária brasileira 26, 1957–1968 (in portuguese). windauer, l., altuna, a., benech-arnold, r., 2007: hydrotime analysis of lesquerella fendleri seed germination responses to priming treatments. industrial crops and products 25, 70– 74. yarami, n., sepaskhah, a.r., 2015: physiological growth and gas exchange response of saffron (crocus sativus l.) to irrigation water salinity, manure application and planting method. agricultural water management 154, 43–51. pyropia yezoensis mutant with high nitrogen availability acta bot. croat. 79 (2), 2020 1 on-line suppl. tab. 1. sequences of primer sets used for rt-qpcr to validate results of transcriptome analysis. contig name primer sequence py2k_unigene_122397 f 5´-gaagattaccaagaagagcggc-3´ r 5´-actttgtctcgtccttccactc-3´ py2k_unigene_173936 f 5´-gctacaagatcaagccattcgt-3´ r 5´-tcagaaccaccaacaaactcac-3´ py2k_unigene_43753 f 5´-caagacggaccataatcgatacg-3´ r 5´-aaggcacccaagtacgatgaat-3´ pywt_unigene_402 f 5´-aacagccatcaattaccgtcac-3´ r 5´-acagtcttccaggtttcagtct-3´ py2k_unigene_145511 f 5´-gccatggtcgagttcctcta-3´ r 5´-gatgacaccacccgagtagaa-3´ py2k_unigene_90598 f 5´-gccataggattagtagtagttgca-3´ r 5´-cttgcaactcaccaacttttcg-3´ py2k_unigene_146357 f 5´-tctgaagcctatgttgagagca-3´ r 5´-tcttgaactcataccactccgt-3´ py2k_unigene_159094 f 5´-attttatgccgtggttagtggg-3´ r 5´-agggaatttatcaaacacgcca-3´ py2k_unigene_115858 f 5´-ccattccgtcactgtacaacac-3´ r 5´-cccattcttctgtaggtcttgc-3´ py2k_unigene_159070 f 5´-ggtgtggacgagcgacaatacag-3´ r 5´-cggacggggggtaagcaaaagaag-3´ on-line suppl. tab. 2. summary statistics of raw and clean reads in pyropia yezoensis wild type (pywt) and mutant (py2k). sample raw reads clean reads reads (no.) bases (total) avg. length (bp) gc (%) reads (no.) % of raw data gc (%) pywt wt1 75,146,820 11,347,169,820 151 45.2 49,337,074 65.65 45.08 wt2 69,260,700 10,458,365,700 151 44.41 50,952,456 73.57 44.03 wt3 64,414,678 9,726,616,378 151 44.41 45,643,162 70.86 43.27 py2k 2k1 65,639,484 9,911,562,084 151 44.59 54,125,262 82.46 44.31 2k2 70,666,770 10,670,682,270 151 43.16 56,377,874 79.78 42.85 2k3 79,066,906 11,939,102,806 151 44.02 53,554,388 67.73 44.56 on-line suppl. tab. 3. list of upregulated genes in mutant pyropia yezoensis (py2k) annotated as ‘antioxidant’. trinity name log2-fold description identity pfam description trinity_py2k_dn131974_c0_g1 7.97 chloroplastic putative glutathione peroxidase 7 [arabidopsis thaliana] 52.17% glutathione peroxidase trinity_py2k_dn254273_c0_g1 4.28 glutathione peroxidase-like peroxiredoxin gpx1 [schizosaccharomyces pombe] 57.14% glutathione peroxidase trinity_py2k_dn63293_c0_g1 6.42 glutathione s-transferase [aplysia californica] 32.94% glutathione s-transferase, n-terminal domain trinity_py2k_dn59285_c0_g4 2.53 1-cys peroxiredoxin [dictyostelium discoideum] 49.28% c-terminal domain of 1-cys peroxiredoxin trinity_py2k_dn64707_c0_g1 7.55 glutathione synthetase [bordetella pertussis] 66.12% glutathione synthetase, atp-grasp domain trinity_py2k_dn59959_c0_g1 3.74 chloroplastic 2-cys peroxiredoxin bas1 [hordeum vulgare] 38.39% c-terminal domain of 1-cys peroxiredoxin trinity_py2k_dn51886_c0_g1 4.60 catalase [pyropia yezoensis] 91.20% catalase trinity_py2k_dn66210_c7_g3 2.71 thioredoxin [porphyra purpurea] 40.00% thioredoxin trinity_py2k_dn65307_c1_g2 4.79 thioredoxin [ictalurus punctatus] 46.79% thioredoxin trinity_py2k_dn59105_c0_g1 4.97 mitochondrial aldehyde dehydrogenase [pongo abelii] 55.51% aldehyde dehydrogenase family park s, choi j 2 acta bot. croat. 79 (2), 2020 on-line suppl. tab. 4. list of differentially expressed genes in pyropia yezoensis mutant (py2k) related to the shikimate pathway. trinity name log2-fold description identity pfam description trinity_py2k_dn61445_c1_g1 2.76 chorismate synthase [ruegeria pomeroyi] 83.33% chorismate_synt trinity_py2k_dn61820_c0_g2 4.87 3-dehydroquinate synthase [thioalkalivibrio sulfidiphilus] 65.69% 3-dehydroquinate synthase trinity_py2k_dn61821_c0_g1 4.02 shikimate kinase [pseudomonas aeruginosa] 60.00% shikimate kinase trinity_py2k_dn63681_c0_g1 6.94 shikimate dehydrogenase [methylobacillus flagellatus] 60.44% shikimate dehydrogenase substrate binding domain on-line suppl. tab. 5. list of differentially expressed genes in pyropia yezoensis mutant (py2k) related to nitrogen assimilation and reassimilation, ribosome biogenesis, and amino acid biosynthesis. trinity name log2fold description identity pfam description nitrogen assimilation trinity_py2k_dn59400_c0_g2 4.22 high affinity nitrate transporter 2.5 [pyropia yezoensis] 99.20% major facilitator superfamily trinity_pywt_dn101823_c0_g1 2.76 chloroplastic ferredoxin-nitrite reductase [arabidopsis thaliana] 28.94% nitrite and sulfite reductase 4fe-4s domain trinity_py2k_dn61283_c1_g2 2.87 glutamine synthetase [cryptococcus neoformans var. neoformans serotype d] 56.34% glutamine synthetase, beta-grasp domain ribosome biogenesis trinity_py2k_dn141935_c0_g1 7.05 casein kinase ii subunit beta [arabidopsis thaliana] 61.28% casein kinase ii regulatory subunit trinity_py2k_dn278700_c0_g1 6.53 serine/threonine-protein kinase rio1 [capsaspora owczarzaki atcc 30864] 92.40% calcineurin-like phosphoesterase trinity_py2k_dn56816_c1_g1 6.97 gtp-binding nuclear protein ran [sphaeroforma arctica jp610] 74.50% adp-ribosylation factor family trinity_py2k_dn303240_c0_g1 6.68 13 kda ribonucleoprotein-associated protein [nicotiana tomentosiformis] 76.00% ribosomal protein l7ae/ l30e/s12e/gadd45 family trinity_py2k_dn63805_c1_g4 5.18 oligoribonuclease [pseudomonas aeruginosa] 67.98% exonuclease trinity_py2k_dn19438_c0_g1 6.26 ntf2-related export protein [caenorhabditis elegans] 28.57% nuclear transport factor 2 (ntf2) domain trinity_py2k_dn56108_c0_g1 6.42 guanine nucleotide-binding protein-like 3 homolog [dictyostelium discoideum] 45.45% gnl3l/grn1 putative gtpase trinity_py2k_dn292600_c0_g1 7.81 nucleolar protein 56 [schizosaccharomyces pombe] 50.62% nop5nt (nuc127) domain trinity_py2k_dn30246_c0_g1 5.84 ribosomal rna small subunit methyltransferase nep1 [dictyostelium discoideum] 61.22% emg1/nep1 methyltransferase purine ring catabolism trinity_py2k_dn50780_c0_g1 6.37 probable allantoinase 1 [dictyostelium discoideum] 40.86% ohcu decarboxylase trinity_py2k_dn63532_c0_g1 4.69 probable xanthine dehydrogenase subunit a [bacillus subtilis] 27.03% xdhc and coxi family biosynthesis of amino acids trinity_py2k_dn203287_c0_g1 2.61 d-amino-acid n-acetyltransferase [saccharomyces cerevisiae] 37.93% acetyltransferase (gnat) family trinity_py2k_dn251932_c0_g1 3.69 cystathionine gamma-lyase [dictyostelium discoideum] 57.89% cys/met metabolism plp-dependent enzyme trinity_py2k_dn33143_c0_g2 6.30 argininosuccinate synthase [achlya hypogyna] 58.80% arginosuccinate synthase pyropia yezoensis mutant with high nitrogen availability acta bot. croat. 79 (2), 2020 3 on-line suppl. tab. 6. list of differentially expressed genes in pyropia yezoensis mutant (py2k) related to the electron transport chain. trinity name log2fold description identity pfam description trinity_py2k_dn188135_c0_g1 2.41 nadh dehydrogenase ubiquinone ironsulfur protein 8-b [arabidopsis thaliana] 61.19% 4fe-4s binding domain trinity_py2k_dn63881_c0_g1 3.02 succinate dehydrogenase assembly factor 2 [dictyostelium discoideum] 39.77% flavinator of succinate dehydrogenase trinity_py2k_dn63855_c0_g2 2.50 mitochondrial succinate dehydrogenase ubiquinone flavoprotein subunit [oryza sativa subsp. japonica] 74.75% fumarate reductase flavoprotein c-term trinity_py2k_dn65270_c1_g1 5.93 alternative oxidase [mangifera indica] 59.60% alternative oxidase trinity_py2k_dn38046_c0_g1 6.50 cytochrome b-c1 complex subunit rieske-1 [arabidopsis thaliana] 65.22% ubiquinol cytochrome reductase transmembrane region trinity_py2k_dn65488_c2_g5 8.24 ubiquinol-cytochrome c reductase ironsulfur subunit [allochromatium vinosum] 56.68% ubiquitinol-cytochrome c reductase fe-s subunit tat signal trinity_py2k_dn28456_c0_g1 2.46 cytochrome c [pectinaria gouldii] 71.96% cytochrome c trinity_py2k_dn63840_c0_g2 2.11 cytochrome c oxidase subunit 1 [marchantia polymorpha] 76.81% cytochrome c and quinol oxidase polypeptide trinity_py2k_dn3622_c0_g1 2.29 mitochondrial atp synthase subunit beta [fistulifera solaris] 84.10% atp synthase alpha/beta family, beta-barrel domain trinity_py2k_dn61343_c0_g1 5.44 mitochondrial isocitrate dehydrogenase [candida tropicalis] 67.74% isocitrate/isopropylmalate dehydrogenase trinity_py2k_dn129036_c0_g1 5.79 putative cytochrome b5 [neurospora crassa] 45.45% cytochrome b5-like heme/ steroid binding domain trinity_py2k_dn62822_c0_g1 2.55 mitochondrial manganese-dependent superoxide dismutase [dictyostelium discoideum] 58.26% iron/manganese superoxide dismutases, alpha-hairpin domain on-line suppl. fig. 1. random amplified polymorphic dna (rapd) analysis of pyropia yezoensis wild-type (pywt) and mutant (py2k). rapd analysis was conducted to compare pywt and py2k using 20 rapd primer sets (opa 1-20 primer sets). representative amplifications with opa 4, 7, 11, 12, 15 17 and 20 are shown. arrows indicate the bands that were different between pywt and py2k. wt, wild-type; mt, mutant. park s, choi j 4 acta bot. croat. 79 (2), 2020 on-line suppl. fig. 2. relationship among samples based on log2-transformed count data between pyropia yezoensis wild-type (pywt) and mutant (py2k) with hierarchically clustered heatmap using pearson’s correlation coefficients. on-line suppl. fig. 3. reverse transcription quantitative pcr (rt-qpcr) results for verification of transcriptome analysis results. all experiments were performed in triplicate and error bar indicated standard deviation. pyropia yezoensis mutant with high nitrogen availability acta bot. croat. 79 (2), 2020 5 on-line suppl. fig. 4. description of differentially expressed genes (degs) in the shikimate biosynthesis pathway related to phenol biosynthesis in py2k. the shikimic acid pathway was described by santos-sánchez et al. (2019). the green arrows indicate upregulated genes in py2k. pep – phosphoenolpyruvic acid, d-erythores-4-p – d-erythores-4-phosphate, dahp – 3-deoxy-d-arabion-heptulosonic acid 7-phosphate, dhq – 3-dehydroquinic acid, dhs – 3-dehydroshikimic acid, s3p – shikimic acid 3-phosphate, epsp – 5-enolpyruvylshikimic acid 3-phosphate, dahps – 3-deoxy-d-arabion-heptulosonic acid 7-phosphate synthase, dhqs – 3-dehydroquinic acid synthase, sdh – shikimate dehydrogenase, sk – shikimate kinase, epsps – 5-enolpyruvylshikimic acid 3-phosphate synthase, cs – chorismite synthase. 74 acta bot. croat. 80 (1), 2021 acta bot. croat. 80 (1), 74–81, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-004 issn 0365-0588 eissn 1847-8476 an analysis of research into urban flora and vegetation in southeast europe slobodan jovanović1*, milan glišić1,2 1 university of belgrade, faculty of biology, institute of botany and botanical garden, belgrade 11000, serbia 2 academy of applied studies šabac, šabac 15000, serbia abstract – in the last two decades, the number of research articles focused on urban ecosystems in europe has increased significantly. however, the cities investigated are very unevenly distributed, and most of the studies are focused on central europe. the aim of this analysis was to provide a realistic insight into the results of previous research into the urban flora and vegetation of southeast europe. the analysis covers a total of 149 articles, classified according to the topic and concept of research. the rates of exploration of urban flora and vegetation vary considerably across the countries of southeast europe. the floristic approach was the most common. although some countries of southeast europe have a significant number of floristic studies (e.g. serbia and croatia with more than 20 each), their urban flora is still insufficiently explored compared to other european regions. also, the use of different methodologies makes it impossible to compare results in an adequate way and draw relevant conclusions. unlike the studies in most of europe, with a broader spatial framework and uniform methodology, in southeast europe they usually referred to individual cities, specific habitats or certain parts of the cities. hence, including southeast europe in large-scale studies would be beneficial. keywords: balkan peninsula, current state of research, invasive plants, literature review, ruderal flora, ruderal vegetation introduction human settlements present a specific environment for living beings with unique conditions that significantly influence the diversity of plant species (mckinney 2006). urban areas are subject to intensive and frequent habitat disturbance, above all by trampling, and are characterized as well by herbicide use (knapp et al. 2012) and nutrient enrichment leading to habitat eutrophication (lososová et al. 2012a). due to the presence of buildings in close proximity to each other, areas with a hard surface (asphalted, concreted or paved), mowed lawns, and pollution (heat and air), urban areas have climatic conditions that are different from those in the surrounding natural environment. called the urban heat island effect, the phenomenon of higher temperatures in urban areas is very prominent in big cities (gaston et al. 2010) and exerts strong influence on the species composition of urban plant communities (wittig 2002). from the viewpoint of island biogeography, cities can be regarded as a type of ecological island isolated by its surroundings from other urban ecological islands (mcgregorfors et al. 2011). urban flora is mainly distinguished by high species richness and consists of native and alien species from different parts of the world (lososová et al. 2012a). the heterogeneity of urban habitats (kühn et al. 2004) and the high influx of propagules, factors that are in positive correlation with the level of urbanization and size of the city (pyšek 1998), result in high species diversity in the total urban flora (kühn et al. 2004). the total number of plant species increases with size of the city (pyšek 1998), but city size also influences species richness in particular habitats (čeplová et al. 2017). urbanization is often considered as the greatest threat to native species diversity (mckinney 2004). it affects the variety of living beings and their functional characteristics (lososová et al. 2006). although richer than the flora in rural areas * corresponding author e-mail: sjov@bio.bg.ac.rs urban flora and vegetation in se europe acta bot. croat. 80 (1), 2021 75 tigation in these countries and point out gaps in the research. an additional goal was to affirm the importance and necessity of adopting a systematic methodological approach to research on this regioń s urban flora and vegetation in the future. materials and methods in order to find relevant references, we searched google scholar, scopus, web of science, researchgate and other online databases.a search of these databases was conducted using the following keywords: urban flora, urban vegetation, urban plants, urban plant species, urban plant communities, urban forests, ruderal flora, ruderal vegetation, wall flora and wall vegetation, in combination with names of the countries (albania, bosnia and herzegovina, bulgaria, croatia, greece, montenegro, north macedonia, romania, slovenia, serbia and kosovo) and cities (at least 15 cities with the largest population of each country) of southeast europe. among the 13,700 offered and examined titles, 174 were found relating to urban flora and vegetation (on-line suppl. tabs. 1, 2). all studies that partially or completely treat characteristics of recent spontaneous flora and vegetation of urban habitats were included in the analysis. in addition to studies employing the floristic and phytosociological approaches, studies based on the application of remote sensing technology in research on urban vegetation from the aspect of landscape ecology were also taken into consideration. studies of individual species or a small number of species in cities were only taken into account if they dealt with the spatial distribution, abundance, population dynamics and habitat preferences of the species in question, whereas those treating the specieś morpho-anatomy, ecophysiology and phenology were not considered. in addition, the analysis did not include studies of planted trees in parks, explorations of heavy metal content, radioactivity research, studies of the effects of pollution on plants, articles considering the importance (economic, social and psychological) of plants to humans or palynological and paleobotanical studies. only results published in the period from 1989 to 2018 with an abstract in english were subjected to statistical analysis. available articles were classified according to the following criteria: topic, geographical location and the year of publication. with respect to topics, all the studies were grouped into one or more categories: 1) floristic studies, including not only those which consider just the total flora of cities, but also ones treating the flora of separate city parts or certain habitats, as well as those dealing with specific plant groups (e.g., woody plants, medicinal herbs, alien plants, allergenic plants, invasive plants, etc.); 2) phytosociological studies, including ones employing the classical approach with phytosociological tables of certain plant communities, as well studies based on the syntaxonomic approach on different levels; 3) landscape ecological studies of urban vegetation, i.e., habitat studies, using the methods of remote detection; 4) studies that refer to plant species protection, management of habitats, vegetation and plant re(kühn et al. 2004), the urban flora is distinguished by lower functional variety. the largest number of plants in central european cities are human-dispersed species with big demands for nutrients and a preference for drier to mesic soil conditions (kalusová et al. 2016). urban areas are especially interesting in the context of biological invasions. to be specific, human activities in cities result in a high input of propagules, resulting in a large proportion of foreign species (pyšek 1998). alien plant species make up about 40% of the total flora in central european cities (pyšek 1998), with similar participation in individual urban habitats (lososová et al. 2012a). the number of alien species in the cities of central europe is still increasing (pyšek et al. 2004), and urban areas can represent centres for the dissemination of alien introduced species into surrounding areas (hulme et al. 2008), where some of them can become naturalized or even invasive. according to lososová et al. (2018), new alien species in central european cities will increase the risk of invasion in the future. this can not only induce a decrease of native species diversity but can result in biotic homogenization, i.e., to an increase in the similarity of different areas with respect to species composition (olden et al. 2004). however, the influence of alien species on biodiversity depends on the time of their introduction (rejmánek 2000). in cities, the presence of archaeophytes (species introduced before 1500 ad) generally led to increasing floristic homogenization, while the presence of neophytes (species introduced after 1500 ad) usually had the opposite effect (lososová et al. 2012b). although urban habitats are characterized by the presence of allochthonous plant species, some preserved natural or semi-natural habitats in cities may represent a hiding place for certain specialized species, including even rare and endangered ones (kühn et al. 2004). urban flora is often surprisingly rich in species and presents a combination of plants with the most different habitat preferences and distribution types. also, urban vegetation contributes to ecosystem services and affects the citizens’ well-being (bratman et al. 2012). because of that and since the human population of europe mostly lives in urban areas, it is of great importance to know and understand the processes that form the composition of species in spontaneous urban vegetation. urban ecology is a young ecological discipline. interest in exploring urban ecosystems emerged in the 1960s and 1970s (wittig 2002), and the number of studies with a focus on urban ecosystems in europe has significantly increased in the last two decades (celesti-grapow and blasi 1998, kühn et al. 2004, knapp et al. 2012, nobis et al. 2009, lososová et al. 2011, 2012a, b, 2016a, b, 2018, čeplová et al. 2017, kalusová et al. 2016). however, the investigated cities are very unevenly distributed, mainly in central europe, with results that can hardly be considered applicable to the whole of europe. the aims were to provide an analysis of research on urban flora and vegetation in the countries of southeast europe during the last three decades, consider trends of invesjovanović s., glišić m. 76 acta bot. croat. 80 (1), 2021 sources or planning of green surfaces in cities; 5) studies of urban and suburban forests; 6) studies of individual or several species in cities in relation to their abundance, distribution or habitat preference; 7) studies of invasive and alien species, with different approaches and types of research, including floristic, phytosociological, population-ecological, cartographic, etc. also included in the analysis were review articles summarizing previous research in this field, which are classified into appropriate categories. the articles were classified on the basis of geographical location in order to provide insight into the extent to which the countries of southeast europe and their cities have been investigated. some studies refer to wide regions, others treat a few cities and compare them, while some pertain to one or more countries of southeast europe. in order to discern the trend of production of articles in this field, special attention was paid to the period of their publication. in this connection, articles were grouped into one of the following three categories, of articles published: 1) from 1989 to 1998; 2) from 1999 to 2008; and 3) from 2009 to 2018. results in total, 149 articles dealing with urban flora and vegetation of southeast europe were found by online search (online suppl. tab. 1). the number increased significantly during the last three decades (fig. 1). in the period from 1989-1998, only 12 articles were published (8% or 1.2 per year), while a total of 40 articles (26% or 4 per year) were published between 1999 and 2008. in the last decade, i.e., from 2009 to 2018, 97 articles were published (65% or 9.7 per year). the publication of the most articles in one year (17) occurred in 2016, while the average number of published references per year was 5 during the whole analysed period. most of the analysed studies related to the urban flora (103 titles, i.e., 69% of all analysed articles). in contrast to floristic investigations, the phytosociological approach was represented with only 12 articles (i.e., about 8%), whereas articles employing the landscape ecological approach to investigation of urban vegetation are becoming more and more interesting (27 titles, i.e., 18%). a total of nine articles (6%) partially or completely dealt with plant protection, management of urban habitats and planning of green surfaces. floristic and spatial studies of urban and suburban forests were separated as a special category that includes 16 analysed articles (11%). there were nine articles treating the presence, abundance, distribution or habitat preference of a particular species or a small group of species in cities (6%). special attention was paid to invasive and alien species, and 19 articles (13%) in titles or abstracts contained the words “invasive”, “alien”, “adventive”, “neophytes” or “neophytic” (fig. 2). the rates of exploration of urban flora and vegetation varied considerably throughout the countries of southeast europe, with the following production and distribution of articles: serbia – 31 (21%), croatia – 27 (18%), romania – 27 (18%), slovenia – 24 (16%), greece – 18 (12%), bosnia and herzegovina – 17 (11%), bulgaria – 14 (9%), montenegro – 10 (7%), albania – 7 (5%), north macedonia – 5 (3%) and kosovo – 3 (2%), with several papers (9, i.e., 6%) relating to cities from more than one country (figs. 3, 4). the cities of southeast europe have been unevenly investigated in terms of urban flora and vegetation. the majority of analysed articles dealt (partially or completely) with the flora and vegetation of the following cities: belgrade (16 articles), ljubljana (13), maribor (8), bucharest (7), mostar (6), podgorica and sofia (5), etc. (fig. 5). among the analysed articles, 125 (i.e., 84%) were published in scientific journals, 17 (11%) were published in proceedings from scientific conferences, while the rest or 5% relate to books or monographs. the analysed articles were published in 74 different journals and in proceedings from 16 scientific conferences. journals with the largest number of articles in this field are natura croatica and acta herbofig. 1. distribution and trend of published articles about urban flora and vegetation of southeast europe per year (1989-2018). urban flora and vegetation in se europe acta bot. croat. 80 (1), 2021 77 logica, with 11 and 10 published articles, respectively (14% of all articles). the largest number of scientific conferences were organized in serbia (5). discussion analysing articles from the field of urban flora and vegetation of southeast europe during the last 30 years, we note that significant disparities are evident regarding the chronological and territorial distribution of investigations conducted (fig. 3), as well as with respect to the distribution of topics of published results (fig. 4). chronological aspect of conducted investigations the fewest articles were published in the period from 1989 to 1998, with only 8% of articles treating topics in urban flora and vegetation. although most of the articles were conceived on the classical floristic principle, which mainly gave a survey and analysis of ruderal flora in certain cities, several articles employing the phytosociological approach relating to belgrade (jovanović 1994a, b) were published in that period, in addition to one such article referring to some smaller cities in croatia and slovenia (čarni 1996). one of the articles from that period was devoted to the distribution and ecology of ailanthus altissima (mill.) swingle on the territory of belgrade (jovanović et al. 1997), indicating that even then the problem of alien and invasive species in the cities of this region was recognized. during the period between 1999 and 2008, significantly more articles were published (27%). most of them were concerned with floristic research, special attention being paid to alien and invasive species in cities such as thessaloniki (krigas and kokkini 2004), šibenik and knin (milović 2001) and banja luka (topalić-trivunović and šumatić 2004). also during this period, urban and suburban forests were investigated in cities like ljubljana (pirnat fig. 3. number of published articles about urban flora and vegetation in southeast europe per country and research period. fig. 2. number of published articles about urban flora and vegetation in southeast europe per research theme and period. jovanović s., glišić m. 78 acta bot. croat. 80 (1), 2021 2000) and podgorica (stešević 2002), as well as in numerous cities in greece (christopoulou et al. 2007). phytosociological articles were still less present, and the only cities whose urban plant communities were the subject of phytosociological research in this period were timisoara (coste and arsene 2003) and kranj (šilc and košir 2006). about 65% of all the articles analysed were published after 2009. the floristic approach is still prevalent, but the last decade was distinguished by significantly more articles relating to alien and invasive species, as well as to landscape aspects of urban vegetation. this period was also one when investigations of urban flora and vegetation beyond the borders of certain countries and parallel analysis of the flora in different cities of southeast europe were conducted (šilc et al. 2012, jasprica et al. 2017, salvati et al. 2017, krajter ostoić et al. 2018, lososová et al. 2018, etc). thematic aspect of conducted investigations apart from the fact that floristic investigations were generally the most numerous (69%), the largest increase in their number was noticed especially in the last decade. however, floristic studies of southeast european cities were mostly restricted to a certain group of plants, particular habitats or specific city parts (e.g., parks, walls, old fortresses, roads and railways, lawns, cemeteries, etc). some floristic articles consider only a specific group of plants, such as woody plants, weeds, allergenic plants and endangered or endemic species, but articles dealing with alien and invasive species were the most numerous and were additionally categorized in a separate group (see later). phytosociological investigations were less numerous (8%) than studies employing the floristic approach, with more than one third pertaining to cities in slovenia. some vegetation studies referred only to particular ruderal communities, e.g., chenopodio rubrii-amaranthetum adscendentis s. jovanović et d. lakušić 1990 in belgrade (jovanović and lakušić 1990) and sambucetum ebuli felföldy 1942 in pale (petronić and bratić 2016), whereas some articles treated other syntaxa of ruderal vegetation, e.g., the alliance euphorbion prostratae rivas-mart. 1976 in the region of istria (čarni 1996). there were articles that yielded more comprehensive phytosociological information in some cities such as belgrade (jovanović 1994b), timisoara (coste and arsene 2003), kranj (šilc and košir 2006), ljubljana (the city ś cemetery) (šilc 2009), žabljak (jovanović et al. 2013) and koper (the town harbour) (šilc et al. 2014). however, the most extensive research of synanthropic vegetation from the classes polygono-poetea annuae rivas-mart. 1975, papaveretea rhoeadis s. brullo et al. 2001, artemisietea vulgaris fig. 4. distribution of published articles about urban flora and vegetation of southeast europe per country and research field. urban flora and vegetation in se europe acta bot. croat. 80 (1), 2021 79 lohmeyer et al. in tx. ex von rochow 1951 and epilobietea angustifolii tx. et preising ex von rochow 1951 was conducted by šilc (2010) in slovenia using 2404 phytocoenological relevés. studies dealing with the landscape ecology of urban areas have recently become more and more of interest (18%). they usually treated the spatial patterns of urban vegetation and their dynamics, and for the most part involved the use of remote sensing methods. half of these articles referred to the cities of romania (rosu and oiste 2013, petrişor 2015, anastasiu et al. 2017). there were similar studies of wider areas, e.g., southeastern romania (vlad and brătăşanu 2011), or even studies covering a large number of cities in the eu, including ones in slovenia, greece, romania and bulgaria (salvati et al. 2017). studies of conservation, management and urban planning accounted for 6% of the articles analysed. for example, grand park in tirana (mesiti et al. 2015), urban forests of western macedonia (tsitsoni and samara 2002), forest fragments and corridors of the suburban area of ljubljana (pirnat 2000), etc., have been investigated from this point of view. investigations of urban and suburban forests from the floristic or spatial aspects were separated as a special category that included 11% of the analysed articles (pirnat 2000, lacan and mcbride 2009, karlo and sajna 2017, etc.). similar investigations, but on larger spatial scales, have been carried out for cities in western macedonia (tsitsoni and samara 2002), the whole of greece (christopoulou et al. 2007) and the mediterranean region, including southeast european countries (krajter ostoić et al. 2018). investigations on the presence, abundance, distribution or habitat preference of a particular species in the cities of southeast europe (6%) mainly include articles that treat a particular invasive species, such as ambrosia artemisiifolia l. (memišević-hodžić et al. 2015), reynoutria japonica houtt. (topalić-trivunović and šumatić 2004), helianthus spp. (filep et al. 2010), aster lanceolatus (l.) kuntze (obratov-petković et al. 2016), etc. additionally, there were several contributions on the subject of endangered species in urban conditions, e.g., scirpus supinus l. (prlić 2017). the largest number of articles devoted to alien and invasive species in cities of southeast europe came from serbia. however, certain cities of slovenia were included in extensive and significant investigations of urban flora in the whole area of central europe, while some articles based on these investigations paid special attention to alien species and their effect on the biotic homogenization of urban floras (lososová et al. 2012a, b, 2016a). in addition, šilc et al. (2012) analysed the presence of alien species in anthropogenic communities of numerous cities in the countries of ex – yugoslavia. comparative aspect and proposal to future research the urban flora and vegetation of southeast europe has been less explored in comparison with the cities of other european regions, especially in central europe. at the same time, current data from southern europe show that plant species diversity in urban ecosystems is higher there than in climatically different parts of central europe (celesti grapow and blasi 1998). moreover, the methods used by different research teams vary greatly, and only few of them compare the cities of larger regions using a standardized research protocol (lososová et al. 2011, 2012a, b, 2016a, b, 2018, kalusová et al. 2016, čeplová et al. 2017, etc.). thus, it is still impossible to draw conclusions about wider regions of southern and especially southeast europe. the low proportion of articles published in sci journals for the region of southeast europe was also noted. a large number of contributions (25) were available only in the form of abstracts from different scientific conferences and symposia (on-line supplementary tab. 2), whereas some of the studies were only available in the local language. in addition, the methods used by researchers from different countries differed, making it impossible to adequately compare results. at the same time, studies in other parts of europe usually had a broader spatial framework and uniform methodology, while studies in southeast europe often referred to individual cities, to specific habitats within the city or to certain parts of the city. although is widely known that there are no universal standard methods that can be applied to every type of research, we can make some suggestions as to what should be taken care of during planning of field research of urban flora and vegetation regarding different spatial scales: community level: a) sample plot size for ruderal vegetation should be limited to max. 10 m2, and urban forests to max. 100 m2; b) number and arrangement of samples should be adequate for the area of the cities/towns; c) duration of survey should cover the whole vegetation season; and d) each sample plot should be georeferenced. habitat level: a) species composition of all vascular plants except those deliberately planted should be recorded in seven 1-ha plots in each city, each plot representing one habitat type (according lososová et al. 2011). typical reprefig. 5. geographical location of investigated cities in southeast europe (size of the circle represents the number of publications per city). jovanović s., glišić m. 80 acta bot. croat. 80 (1), 2021 sentative plots should be selected before from aerial photographs. b) because such research aims to obtain comparable samples with limited phenological variation, the duration of research should be limited to the period from midjune to lateaugust (lososová et al 2011); c) if the research aims to collect floristic data about ephemeral plant species, then multiple fieldwork campaigns through the whole vegetation season are needed; and d) each sample plot should be georeferenced. it would be beneficial if the region of southeast europe were included with other european countries in studies on a large scale. moreover, conducting studies in the entire region of southeast europe, using the standardized habitat level model applied in central europe by lososová et al. (2011), should also be considered. collaboration of researchers and scientific institutions from different countries of the region should be promoted through international meetings, as well as by means of national and international projects. this would make possible the realization of more effective comparative research on urban flora and vegetation in southeast europe. in addition, it would enable us to prepare a comprehensive synthesis on different levels of knowledge. acknowledgements the ministry of education, science and technological development of the republic of serbia supported this research through grant 451-03-68/2020-14/ 200178. we are grateful to dr. ksenija jakovljević for helpful comments on the manuscript. we would also like to thank mr. raymond dooley for linguistic editing, as well as reviewers and editorin-chief for useful comments towards a better manuscript. references anastasiu, p., comănescu, c.p., nagodă, e., liţescu, s., negrean, g., 2017: nature reclaiming its territory in urban areas. case study: văcăreşti nature park, bucharest, romania. acta horti botanici bucurestiensis 44, 71–99. bratman, g.n., hamilton, j.p., daily, g.c., 2012: the impacts of nature experience on human cognitive function and mental health. annals of the new york academy of sciences 1249, 118–136. celesti grapow, l., blasi, c., 1998: a comparison of the urban flora of different phytoclimatic regions in italy. global ecology and biogeography 7, 367–378. christopoulou, o., polyzos, s., minetos, d., 2007: peri-urban and urban forests in greece: obstacle or advantage to urban development? management of environmental quality: an international journal 18, 382–395. coste, i., arsene, g-g., 2003: notes on anthropohilous flora and vegetation in the city of timisoara. proceedings 7 international symposium interdisciplinary regional research, hundeora, 211–216. čarni, a., 1996: thermophilous vegetation of trampled habitats in istria (croatia and slovenia). biologia 51, 405–409. čeplová, n., kalusová, v., lososová, z., 2017: does the size of settlement matter? effects of urban heat island, settlement size and habitat type on urban plant biodiversity. landscape and urban planning 159, 15–22. filep, r., balogh, l., csergő, a-m., 2010: perennial helianthus taxa in târgu-mureş city and its surroundings. journal of plant development 17, 69–74. gaston, k.j., davies, z.g., edmondson, j.l., 2010: urban environments and ecosystem functions. in: gaston, k.j. 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an invasive species in ruderal flora of banja luka. acta herbologica 13, 13–18. tsitsoni, t., samara, t., 2002: the existing situation and management of urban forests and trees in western macedonia. proceedings 10 panhellenic forest science conference, tripoli, 136–147. vlad, m.i., brătăşanu, d., 2011: quality of life assessment based on spatial and temporal analysis of the vegetation area derived from satellite images. romanian review of regional studies 7, 111–120. wittig, r., 2002: siedlungsvegetation. stuttgart, ulmer. acta bot. croat. 79 (2), 2020 s social news zlatko pavletić (1920-1981) – on the 100th anniversary of his birthday professor zlatko pavletić (april 4, 1920 – march 19, 1981) was a milestone person for biology in croatia in the second half of 20th century. he was one of the first native bryologists, founder of microbiology teaching at the faculty of science, university of zagreb, prominent limnologist and a tireless populariser of science (fig. 1). he was born in rijeka. during the turbulent years of world war ii, he lost a leg, but persisted in the struggle against fascism with his commitment to the education of the dalmatian refugees in the el shatt camp in egypt. having started to study biology in zagreb before the war, he remained dedicated to biological research and teaching to the end of his life. in 1950 he became an assistant, in 1964 an associate and in 1969 a full professor at the botanical department of the faculty of science, university of zagreb. one of the first tasks confided to him by his supervisor, professor vale vouk, was to start filling the gap in bryological research into croatia and the whole of yugoslavia. the result was a masterpiece, the prodromus flore briofita jugoslavije [prodromus of yugoslav bryophyte flora] published in 1955. it is remarkable work and from the present point of view, it is impressive how he succeeded in compiling all the literature referring to the bryophyte flora of yugoslavia without any text or spreadsheet processor, or databases. on 578 pages, he quotes all the bryophyte species known for the yugoslav flora, including the synonymy and comprehensive lists of localities. this work will stay the keystone for all other species catalogues and distributional atlases for the whole territory at issue. it was the basis and main source of data for many other checklists (e.g. martinčič 1968, düll et al. 1999, sabovljević 2000, 2003, 2006, sabovljević et al. 1999, 2006, 2008). the second step in filling the bryological gap was the preparation of identification keys. this task was finished in 1968 with the handbook flora mahovina jugoslavije [the bryophyte flora of yugoslavia] containing identification keys for families, genera and species provided by short morphological descriptions, basic data on species habitats, ecology, phenology and chromosome numbers. this is not only the first identification key for bryophytes published in croatian, fig. 1. professor zlatko pavletić with his colleagues and associates before the fieldtrip (second half of 1970-ies). from the left to the wright: ivančica krulik, božidar stilinović, nada mrkša, paula durbešić, ivo matoničkin, biserka primc, ivan habdija and zlatko pavletić. photo by courtesy of professor biserka primc. s1 acta bot. croat. 79 (2), 2020 but the first in the south slavic languages (the rare exceptions are the identification keys for the bryophytes of mt medvednica published by a. heinz in 1887 and 1888). with this work pavletić became the first native croatian bryologist (however, there are several earlier sporadic bryological papers published by croatian authors). for although croatia has a long tradition of bryological research dating back to the first decades of the 19th century, all of these researchers came mainly from other parts of the austrian-hungarian empire, of which croatia was a part. simultaneously with his work on these monographic publications, pavletić started with his own field researches. at the focus of his scientific interest was the bryophyte vegetation of tufa barriers in karst rivers. in 1954 he started with comprehensive researches into the krka river and over the years he expanded them to take in almost all karst waters in croatia including plitvice lakes, and several in adjacent areas. the results of these researches were published in a series of papers that are fluently written and with a feeling for language, informed by many detailed observations and additional data. they reveal a passionate naturalist and analytical scientist with an ability to synthesize collected data in well-worked-out descriptions and theories on the structure and functioning of the biocoenoses in karst rivers. during the 1960s he worked with his colleague and friend, zoologist and professor ivo matoničkin, with whom he studied algae, bryophytes and macrozoobenthos, the main constituents of the biocoenoses of karst rivers. it is important to emphasize that pavletić’s approach was not only descriptive, for from the very beginning he included in his research measurements of physicochemical parameters as environmental drivers crucial for the structure and development of river biocoenoses. his papers remain an invaluable and not-to-be-missed source of information for any further study of karst river ecosystems, providing the grounds for studies of changes that have occurred in the last sixty years. unfortunately, pavletić did not have a direct successor in bryological research, and after him we had again a gap of several decades. his second field of interest, microbiology of ecosystems, was more fortunate in this respect. with his assistant, later professor, božidar stilinović he started to research into the microbiology of soils, rivers and the sea, mostly in terms of pollution, water quality classification and saprobiology. for the purpose of these researches, he established the first microbiological laboratory at the faculty of science. this laboratory is continuously working and is very successful in research into the bacteriology of polluted waters. the results of his researches were the basis for many feasibility studies and action plans on nature protection, water management and the development of methods for estimation of ecological status of rivers. besides his scientific work, pavletić was inexhaustible populariser of science. he wrote dozens of popular science articles, over 50 in the journal priroda, where he was the main editor for 10 years. with his friend ivo matoničkin he wrote the book život naših rijeka [the life of our rivers] published in 1971, which is still an excellent introduction to river biology and conservation. it is written fluently and understandably in a clear style, summarizing several decades of their research. another popular book životopis života [biography of life], published posthumously in 1984, is thematically very accurate, despite the huge amount of new knowledge on the topic, and the growing amount of pseudo-scientific and creationist literature that was penetrating the educational system. he is remembered as a professor dedicated to his students, an excellent lecturer and patient supervisor of many msc and phd theses. moreover, he always tried to help his former students to find positions in the profession. a more detailed biography with a list of scientific and professional publications was published by miličić (19791980) and miličić and stilinović (1981). professor antun alegro division of botany, department of biology, faculty of science, zagreb references düll, r., martinčič, a., pavletić, z., 1999: a contribution to the yugoslavian bryoflora – checklist of the yugoslavian bryophytes. bryologische beiträge 11(1), 1–94. heinz, a., 1887: briofiti zagrebačke okolice, dio i. pravi mahovi. glasnik hrvatskoga naravoslovnoga društva 2(1), 217–266. heinz, a., 1888: briofiti zagrebačke okolice, dio ii. jetrenjače. glasnik hrvatskoga naravoslovnoga društva 3(1), 57–86. martinčič, a., 1968: catalogus florae jugoslaviae ii/1. bryophyta – musci. academia scientarum et artium slovenica, ljubljana. matoničkin, i., pavletić, z., 1971: život naših rijeka. školska knjiga, zagreb. miličić, d., 1979-1980: u povodu 60. godišnjice života prof. dra zlatka pavletića i 10. godišnjice uređivanja časopisa “priroda”. priroda 68 (5-6), 149-151. miličić, d., stilinović, b., 1981: prof. dr. zlatko pavletić (1920– 1981). acta botanica croatica 40, 269–277. pavletić, z., 1954: istraživanja briofita na travertinskim slapovima rijeke krke. ljetopis jugoslavenske akademije znanosti i umjetnosti. 61(1), 331–351. pavletić, z., 1955: prodronomus flore briofita jugoslavije. jugoslavenska akademija znanosti i umjetnosti, zagreb. pavletić, z., 1968: flora mahovina jugoslavije. institut za botaniku sveučilišta u zagrebu, zagreb. pavletić, z., 1984: životopis života. školske novine, zagreb. sabovljević, m., 2000: checklist of hepatics of the federal republic of yugoslavia. lindbergia 25, 37–42. sabovljević, m., 2003: the hepatic check list of croatia. archives of biological science belgrade 55(1-2), 59–66. sabovljević, m., 2006: checklist of mosses of croatia. archives of biological science belgrade 58(1), 45–53. sabovljević, m., natcheva, r., 2006: a check-list of the liverworts and hornworts of southeast europe. phytologia balcanica 12(2), 169–180. sabovljević, m., natcheva, r., dihoru tsakiri, e., dragićević, s., erdag, a., papp, b., 2008: check-list of mosses of se europe. phytologia balcanica 14(2), 207–244. sabovljević, m., stevanović, v., 1999: moss conspectus of federal republic of yugoslavia. flora mediterranea 9, 65–95. acta bot. croat. 80 (1), 2021 s3 in memoriam in memoriam zorana sedlar (1980-2020) a year bad in so many ways, 2020 is especially bitter for the small botanical community in croatia due to the premature and sudden loss of zorana sedlar (fig. 1). she was not only a member of the community, she was also its cheerful, smiling and optimistic side, always ready to help and support her colleagues and friends. zorana was born in zagreb, but she was strongly connected with the adriatic and the island of molat, where her mother’s family comes from. she completed her primary and secondary education in zagreb and in 1998 started the study of biology at the faculty of science. she graduated in 2004 with a thesis in plant physiology, and in the same year she started working at the botanical division of the same faculty as a research assistant. she was involved in projects related to research into vegetation ecology and the biogeography of flora and vegetation of croatia. she was also involved in teaching as an assistant in practical classes in plant ecology, geobotany, vegetation ecology and the related fieldwork. her excellent knowledge of the italian language enabled her to visit the university of camerino, where she acquired specialist knowledge in vegetation dynamics and established permanent cooperation with colleagues there. the acquired knowledge and lasting love for the island of her ancestors prompted her to dedicate her doctoral thesis to research into changes in the vegetation cover of the island of molat during the last hundred years caused mainly by depopulation. she also started to study the flora of the island, and with her friend and colleagues she began research into the island’s entomofauna and its connection with the vegetation. at the same time, she was involved in floristic research into the island of mljet, the krka national park and mt žumberak. in the krka national park, in addition to floristic research, she also researched vegetation dynamics, the impact of grazing and controlled fires on the restoration of grassland vegetation. in 2016, zorana moved from the position of senior assistant at the faculty to the position of curator at the croatian museum of natural history. she became involved in the work on the herbarium collection, especially in its digitization, but she also worked on a number of other museum projects. she collected material for exhibitions, was engaged in education, participated in botanical field research with colleagues from the museum, but at the same time went on with research and cooperation with colleagues from the faculty. she published the results of her research in a number of scientific and professional articles and studies, as well as conference papers. since zorana’s career was abruptly interrupted by a serious illness during the first half of it, it would be inappropriate here to give any summary of her work. so much more was planned, started, conceived, desired and imagined. she coped bravely with her illness, not showing any signs that something might be wrong, so that even her closest associates had no idea how severe her illness was. as i said at the beginning, she belonged to the bright and smiling side, persisting bravely and modestly until the very end. for all of us, zorana will remain a person with a smile, someone who never had a single bad word to say. professor antun alegro university of zagreb faculty of science department of biology fig. 1 zorana in field research near barać caves in spring 2020. foto by sara essert. opce-str.vp acta bot. croat. 70 (2), 269–288, 2011 coden: abcra 25 issn 0365–0588 phytoplankton composition and abundance assessment in the nador lagoon (mediterranean coast of morocco) faid el madani1*, abderrahim chiaar1, abdelhafid chafi2 1 institut national de recherche halieutique – centre régional de nador, 13 boulevard zerktouni, nador, morocco 2 unité de formation et de recherche techniques de gestion de l’environnement, département de biologie, faculté des sciences, oujda, morocco abstract – we evaluated phytoplankton abundance, composition and trophic state of the nador lagoon (morocco) on the basis of data taken in the period november 2007 to august 2008. sampling was performed at 11 stations (bottle samples at 0.5 m depth and horizontal plankton net tows with mesh size of 20 mm). among seven identified phytoplankton classes, diatoms and dinoflagellates dominated with 133 and 169 species, respectively. frequent phytoplankton blooms were contributed by one to three species in the lagoon. abundance and seasonality of phytoplankton characterized the nador lagoon as a highly eutrophicated environment. keywords: mediterranean sea, nador lagoon, phytoplankton, diversity, eutrophication, introduction taxonomic composition and size structure of phytoplankton is regulated by eutrophication in coastal lagoons (pérez-ruzafa et al 2002). eutrophication can induce massive blooms (coloured water) of phytoplankton species and the reduction in diversity. in our study, both the qualitative and quantitative aspects were considered in order to determine (a) the actual phytoplankton community structure of the nador lagoon, (b) the spatial distribution and (c) the temporal succession of the dominant species. description of the study area the moroccan nador lagoon is a semi-enclosed coastal ecosystem with a surface area of about 115 km2 (fig. 1). the lagoon is isolated from the mediterranean sea by a 25 km long sand bar (le lido), crossed by one channel (boccana). it is located in a region characterised by a mediterranean climate with, often, a low and irregular rainfall – (annual mean acta bot. croat. 70 (2), 2011 269 * corresponding author, e-mail: elmadanifaid@hotmail.com copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:44 color profile: disabled composite 150 lpi at 45 degrees 116–430 mm). during the year, there is a distinction between the rainy season from november to march and the dry season from april to october (fig. 2). the average depth of the lagoon is 4–7 m (fig. 1). the depths increase from edges to the middle part of the lagoon. the salinity range is 32.7–40.2 in the confined extremity of the lagoon where the mixing of the water column is very low (benbrahim 2009). the water temperature variation closely follows the temperature of the air (lakhdar et al. 2005) and generally ranges from 11 °c in january to 30 °c in august. stratification of the water column is weak. the nador lagoon is one of the largest costal lagoons on the mediterranean coast that suffer from a substantial anthropogenic wastewater overload in organic matter, nitrogen and phosphorus in addition to other various kinds of chemicals including heavy metals and 270 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. fig. 1. the bathymetric map of nador lagoon and its geographic position. 0 10 20 30 40 50 60 jan feb mar apr may jun jul aug sep oct nov dec months 0 5 10 15 20 25 30 t e m p e ra tu re (° c ) months jan feb mar apr may jun jul aug sep oct nov dec precipitation (mm) 45.8 48.4 36.7 27.7 17.5 10.4 1.4 3 9.1 22.1 32.2 32.5 temperature (°c) 12.2 13.1 14.3 15.7 18.2 21.65 24.3 25.1 23.1 19.3 16.15 13.6 p re c ip it a ti o n (m m ) fig. 2. monthly mean rainfall and temperature in nador city, in the period 1977–1996. u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:45 color profile: disabled composite 150 lpi at 45 degrees hydrocarbons (bloundi 2005). this organic and inorganic complex may cause dysfunctions in the food web that might lead a total ecosystem imbalance, especially because of the low water exchange rate with the open sea. the turnover time of the water in the lagoon was estimated to be 80 days (hilmi 2005). however, despite the narrowness of the channel, the hydrological balance of this system shows a quasi-permanent predominance by sea water. in 1993 the channel (boccana) was dredged and widened after a progressive accumulation of the sand that since 1987 had almost completely isolated the lagoon from the open sea; this was done in order to re-establish normal water circulation. the macrophytes recorded in the lagoon, during the present study, belong to three groups (i) climax phanerogams cyamodocea nodosa, and nanozostera nolti and the chlorobiontes caulerpa prolifera, (ii) the opportunist algae: ulva spp, enteromorpha spp, chaetomorpha linum and (iii) the drifting rhodophyceae species gracilaria gracilis and alscidium corlinum. generally, the invasive macroalgae, caulerpa prolifera, cover most of the bottom, except the central part that is devoid of macrophytes, restricting the seaweed cymodocea nodosa to small bands in the shallowest areas surrounding the lagoon. diatoms are the dominant planktonic algae in the lagoon (el madani et al 2001). several watercourses drain into the lagoon but most of them become functional only during episodic flood periods (mahjoubi 2003). some of them, such as wadi caballo, wadi afelioune and wadi akhandouk, became true open wastewater collectors that may contribute to increase the organic matter and various kinds of chemical element input. materials and methods surface phytoplankton sampling was conducted, five times, roughly each two months, at 11 stations along nador lagoon, (fig. 3). for qualitative study, samples were collected using a standard plankton net (20 mm mesh size), in a horizontal tow for about 2 minutes at acta bot. croat. 70 (2), 2011 271 phytoplankton in the nador lagoon (marocco) fig. 3. the sampling plan at stations: phytoplankton (�); temperature and salinity (�). u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:47 color profile: disabled composite 150 lpi at 45 degrees each station. for quantitative plankton sampling a bottle was used. once collected, samples were fixed immediately with neutral formalin. for identification and enumeration of phytoplankton an inverted microscope leica dm-irb was used. phytoplankton identification was performed according to dodge (1982), balech et al. (1984), ricard (1987), balech (1988), larsen and moestrup (1989), delgado and fortuño (1991), hallegraeff (1991), hallegraeff et al. (1991), paulmier (1992, 1994), nezan (1996), nezan and piclet (1996), tomas (1997), faust et al. (1999), matsuoka and fukuyo (2000), hansen et al. (2001), sar et al. (2002), kashima (2002), koening and lira (2004). the salinity and temperature measurements were made in situ (20 stations) with wtw cond-197i model conductivity-salinometer (fig. 3). results water temperature and salinity salinity and temperature increase from january to august (fig. 4). the salinity minimum is due to the rainfall maximum. if we consider spring the reference season, because of its moderate temperature and low rainfall, the normal salinity in the nador lagoon is around 36.7 with small variations. the range between minimum and maximum water temperature is about 13 °c, and photoperiod seems to be the main limiting factors for the development of the phytoplankton community. taxonomic composition a total of 311 phytoplankton species belonging to seven groups were identified during the period of study; 133 diatom species, 169 dinoflagellates, 2 cyanophyceae, 2 dyctiochophyceae, 2 euglenophyceae, 1 chlorophyceae, 1 coccolithophorid species and 1 raphydophyceae (tab. 1). among phytoplankton species, the most dominant were diatoms, such as: chaetoceros spp, pseudonitzschia spp, nitzschia longissima, skeletonema sp, neocera272 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. 0 5 10 15 20 25 30 35 nov. 2007 jan. 2008 apr. 2008 june 2008 aug. 2008 months t e m p e ra tu re (° c ) 33,5 34 34,5 35 35,5 36 36,5 37 37,5 38 38,5 39 s a li n it y (p su ) temperature salinity fig. 4. temporal variation of the means of the temperature and salinity of the lagoon waters during the study period. u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:47 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 273 phytoplankton in the nador lagoon (marocco) tab. 1. the spatial distribution of phytoplankton taxa (result from five samplings during 2007 and 2008). stations 1 2 3 4 5 6 7 8 9 10 11 dictyochophyceae 1 hermesinum adriaticum o. zacharias – + + + + + – + + + + 2 dictyocha fibula ehrenberg – – + – – – – – – + + cyanophyceae 3 oscillatoria sp. + + + + + + + – + – + 4 spirulina sp. + + – – – – – – – – – chlorophyceae 5 scenedesmus rastro-spinosus chodat – – – + + – – – – – – 6 eutreptia sp. + – + + + + – + – – – 7 euglena sp. – – – + – – – – – – – coccolithophorids 8 calciosolenia murrayi gran – + + + – – – – + + + bacillariophyceae 1 achnanthes brevipes agadh + + + – – – – – – – + 2 achnanthes catenata bily et marvan – + + – – – – – – – + 3 actinoptechus sp. – – – – – – – – – – + 4 actinoptychus senarius (ehrenberg) ehrenberg – – – – – – – – – – + 5 actinoptychus splendens (shadbolt) ralfs ex pritchard – – – – – – – – – – + 6 amphipleura pellucida(kütz.) kütz. + + + + + + + + + + + 7 amphiprora angustata hendey + – – – – – – – – – – 8 amphiprora sp. – – – – – – – – – – – 9 amphora egregia ehrenberg – – – – – – – – – – + 10 amphora hyalina kützing – – – – – – – – – – + 11 amphora laevis gregory – – + – – – + + + – – 12 amphora recta grunow – – – – – – – – – – – 13 amphora sp. + + + + – – + + – – + 14 amphora ventricosa w. gregory – – – – – – – – – – + 15 asterionella japonica cleve et moller + + – + – – – – – – + 16 auliscus sculptus (w. smith) ralfs ex pritchard – – – – – – – – – – + 17 auliscus sp. – – – – – – – – – – + 18 bacillaria paxillifera (müller) hendey + + + + + + + + + + + 19 biddulphia edwardsii febiger ex grunow – – – – – – – – – + – 20 biddulphia pulchella s.f. gray – – – – – – – – – – + 21 biddulphia rhombus (ehrenberg) w. smith – – + – – – – – – – – 22 biddulphia sp. – – – – – – – – – – + 23 biddulphia tuomeyi (bailey) roper – – – – – – – – – – + u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:47 color profile: disabled composite 150 lpi at 45 degrees 274 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. stations 1 2 3 4 5 6 7 8 9 10 11 24 campylodiscus decorus de brébisson – – – – – – – – – – + 25 cerataulina pelagica (cleve) hendey + + + – + – + + + + + 26 chaetoceros affinis var. willei (gran) hustedt – – + – – – – + – – – 27 chaetoceros brevis shütt + – – – – – – – – – – 28 chaetoceros curvisetus cleve + + + + – – – – – + – 29 chaetoceros danicus cleve – – + – – + – + – – – 30 chaetoceros decipiens cleve – + + + + + + + + + + 31 chaetoceros didymus ehrenberg – + – – – – – – – – – 32 chaetoceros laciniosus schütt – – – – – – – + – – – 33 chaetoceros laevis leuduger–fortmorel – + – – – – – – – – – 34 chaetoceros lauderi ralfs – – – – – + – – – – – 35 chaetoceros mitra (j.w. bailey) cleve + + + + – + + + + + + 36 chaetoceros peruvianus brightwell – + + + + – – + + + + 37 chaetoceros pseudocurvisetus mangin – + – – – – – – – – – 38 chaetoceros simplex ostenfeld – – – + – – – – – – – 39 chaetoceros spp. + + + + + + + + + + + 40 chaetoceros teres cleve – + – + – + + – – – + 41 chrysanthemodiscus floriatus mann – – – – – – – – – – + 42 climaconeis sp. – – – – – – + – – – – 43 cocconeis scutellum ehrenberg – + – – – + – + – – + 44 cocconeis sp. + – + – – – – – + – + 45 corethron criophilum castracane – + – – – – – – – – – 46 coscinodiscus sp. – – + – – – – – – – + 47 dactyliosolen fragilissimus (bergon) g. r. hasle – – – + – – – – – + – 48 diatoma mesodon (ehrenberg) kützing – – – – – – – – – – + 49 diploneis chersonensis (grunow) cleve – – – – – – – – – – + 50 diploneis sp. – – – – – – – – + – – 51 ditylum brightwellii (t. west) grunow + – + – – – – – – + – 52 entomoneis alata (ehrenberg) ehrenberg + – + – + + + – + – – 53 entomoneis sp. + + – – – – – – – – – 54 epithemia turgida (ehrenberg) kütz – – – – – – – – – – + 55 goniothecium odontella ehrenberg – – – – – – – – – – + 56 grammatophora angulosa ehrenberg – – – – – – – – – – + 57 grammatophora marina (lyngbye) kützing – + + – – – – – – – – 58 guinardia delicatula (cleve) hasle – – – – – + + – – – + 59 guinardia flaccida (castracane) h. peragallo + – – – – – – – – – + 60 guinardia striata (stoltherfoth) hasle – + + – + + + + + + + tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 26. rujan 2011 14:34:20 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 275 phytoplankton in the nador lagoon (marocco) stations 1 2 3 4 5 6 7 8 9 10 11 61 gyrosigma fasciola (ehrenberg) j.w.griffith et henfrey + + – + + – + – – + – 62 gyrosigma scalproide (rabenhorst) cleve – + – + – – – – + – + 63 gyrosigma sp. – – – + – + – – – – – 64 gyrosigma wansbeckii (donkin) cleve – – – – + – – – – – – 65 hantzschia amphioxus (ehrenberg) grunow – – – – – – – – – – + 66 haslea wawrikae (husedt) simonsen – + + + – + – – – – + 67 hemialus hauckii grunow ex van heurck + + + + + + + – + + + 68 hemiaulus sinensis greville – – – – + – – – – – – 69 lauderia annulata cleve – – – – – – – – – – + 70 lauderia sp. – – – – – – – – + – – 71 leptocylindrus danicus cleve – + + + – + – + + + + 72 leptocylindrus minimus gran – + + + + + + + + + + 73 licmophora flabellata (greville) agardh + + + + + + + + + + + 74 licmophora gracilis (ehrenberg) grunow + + + + + + – – – + + 75 licmophora sp. + + – – – – – – – + + 76 lioloma pacificum (cupp) hasle – – – + – + – – – + – 77 lioloma sp. – + – – – – – – – – – 78 melosira nummuloides c.a. agardh – + – – – – – – – – – 79 melosira sp. – – – – – – – – – + + 80 meuniera membranacea (cleve) p.c. silva in hasle et syvertsen + – – – – – – – – – + 81 navicula cf. carinifera grun. grunow in schmidt – + – – – – – – – – – 82 navicula forcipata greville – – – – – – – – – – + 83 navicula hasta pantocsek – – – – – – – – – – + 84 navicula hennedyi w.smith – – – – – – – – – – + 85 navicula humerosa brébisson ex w. smith – – – – – – – – – – + 86 navicula lanceolata (c. agardh) kützing – – – – – – – – – – + 87 navicula menaiana hendey – – – – – – – – – – + 88 navicula smithii navicula smithii (agardh) van heurck – – – – – – – – – – + 89 navicula spp. – + + – + + – – + – + 90 navicula tuscula (ehrenberg) grunow – + – – – – – – – – – 91 cylindrotheca closterium (ehrenberg) reimann et j.c.lewin kingston – + + + + + + – + + + 92 nitzschia levidensis (w. smith) grunow in cleve et grunow – – – – – – – – – – + 93 nitzschia longissima (brébisson) ralfs + + + + + + + + + + + tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:47 color profile: disabled composite 150 lpi at 45 degrees 276 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. stations 1 2 3 4 5 6 7 8 9 10 11 94 nitzschia panduriformis w. gregory – – – – – – – – – – + 95 nitzschia sigma (kützing) w. smith – + – – – + – – – – + 96 nitzschia sp. – + + + + + + + + + + 97 nitzschia ventricosa kitton + + – – + + + + + + + 98 oestrupia musca (gregory) hustedt – – – – – – – – – – + 99 plagiotropis lepidoptera (greg.) reimer navarro – – – – – – + – – – – 100 planctoniella sol (wallich) schutt – – – – – – – – – – + 101 pleurosigma elangatum w. smith + + + – + + + + + + + 102 pleurosigma itium ricard – + + + + + + – + + + 103 pleurosigma spp. + – + + – – – – – – + 104 podocystis adriatica (kützing) ralfs – + – – – – – – – – + 105 proboscia alata (brightwell) sundström – – – – – – – – – – + 106 pseudoguinardia recta von stosch – – – – – – – – – + + 107 pseudo-nitzschia spp. + + + + + + + + + + + 108 rhizosolenia alata brightwell – – – – – – – – – – + 109 rgizosolena setigera brightwell – + – + + – – + – + – 110 rhizosolenea sp. – – – + – – – – – – – 111 rhizosolenia alata forma indica (h. peragallo) gran – – – – – – – – + – + 112 rhizosolenia setigera brightwell + + + + + + – + + + + 113 rhizosolenia sp. – – – – – – – – – – + 114 rhizosolenia styliformis brightwell – – – – + + – – – + + 115 rhoicosigma sp. – – – – – – – – – – + 116 skeletonema sp. – + + + + + + + + + + 117 stauroneis amphioxys gregory – – – – – – – – – – + 118 stenopterobia intermedia (lewis) brébisson ex van heurck – – – – – – – – – – + 119 helicotheca tamesis ricard – – – + – – – – – – – 120 striatella unipunctata (lyngbye) c. agardh + + + + – – + – – – + 121 surirella amoricana h. peragallo – – – – – – – – – – + 122 surirella fastuosa ehrenberg – – – + + + – – – + + 123 surirella gemma (ehrenberg) kützing – + + + – + + – – – – 124 surirella sp. – – – – – – – – – – + 125 synedra sp. + – – – – – – – – – – 126 thalassionema nitzschioides (grunow) mereschkowsky + + + + + + + + + + + 127 thalassiosira hyalina (grunow) gran – – – – – – – – – – + 128 thalassiosira sp. + + – – + – – – – + + tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:47 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 277 phytoplankton in the nador lagoon (marocco) stations 1 2 3 4 5 6 7 8 9 10 11 129 thalassiothrix froenfeldeii grunow + + + – + – + – + – + 130 thalassiothrix spp. + + + + + + + + + + + 131 toxonidea sp. – – – – – – – – – – + 132 triceratium alternans j. w. bailey – – – – – – – – + – + 133 trichotoxon reinboldii (van heurck) reid et round – + – – – – – – – + – dinoflagellates 1 achradina pulchra lohmann + + – – – + + – – – – 2 alexandrium catenella (whedon et kofoid) balech – – + + – – – – – – + 3 alexandrium margalefi balech – – – – – – – – – + + 4 alexandrium minutum halim + + + + + + + + + + + 5 alexandrium pseudogonyaulax (biecheler) horiguchi ex yuki et fukuyo – + + – – + – + + + + 6 alexandrium sp. – – – – – – – – + – + 7 alexandrium tamarense (lebour) e.balech – – – – – – + – – – – 8 amphidinium sp. – – – + + – – – – – – 9 amphidoma caudata halldal + + + + + + + + + + + 10 amylax sp. – – – + – – – – – – – 11 archaeperidinium sp. – – – – – – – – – – + 12 coolia monotis meunier + + + – – – + + – + + 13 cyst alexandrium minutum – – – + + + – + + + – 14 cyst of dinoflagellates + + + – – + + + + + + 15 dinophysis caudata saville–kent – + – – – – + + + + + 16 dinophysis contracta (kofoid et skogsberg) balech – – – – – – – – – – + 17 dinophysis diegensis kofoid – – – – – – – – – – + 18 dinophysis exigua kofoid and skogsberg – – + – – – – – + + + 19 dinophysis rapa (stein) balech – – – – – – – – – – + 20 dinophysis rotundata claparède et lachmann – – – – – – – – – – + 21 dinophysis sacculus stein + + + + + + + + + + + 22 dinophysis sp. – – – – – – – – – – + 23 diplopelta asymetrica (mangin) lindermann – – – – + – – + – – – 24 diplopelta steinii (t. h. abé) e. balech – – – – – – – – – – + 25 diplopeltopsis minor pavillard – + – – – – – – – – + 26 diplopeltopsis sp. – + – – – – – – – – – 27 diplopsalis sp. + + + – + + + + + + + 28 diplopsalopsis bomba (stein ex jörgensen) j. d. dodge et s. toriumi – – – – – – – – + – – tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 26. rujan 2011 14:05:26 color profile: disabled composite 150 lpi at 45 degrees 278 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. stations 1 2 3 4 5 6 7 8 9 10 11 29 diplopsalopsis sp. – – – – – – – – – + – 30 ensciculifera sp. + + + + – + + + + – + 31 ensiculifera angulata e. balech – – – – – – – – – + – 32 ensiculifera angulata e. balech – – – – – – – – – – + 33 gambierdiscus toxicus adachi and fukuyo + – – – – – – – – – – 34 goniodoma polyedricum (pouchet) jörgensen – – + – – – – – – + + 35 gonyaulax dicantha (meunier) schiller – – + + + + + + + – + 36 gonyaulax digitale (pouchet) kofoid + – – + – + + + – + + 37 gonyaulax grindleyi reinecke – – – – – – – + – – + 38 gonyaulax polygramma stein – – – – – + – – + + + 39 gonyaulax sousae balech + + + + + + + + + + + 40 gonyaulax spinifera (claparède et lachmann) diesing + + + + + + + + + + + 41 gonyaulax striata mangin – – – – – – – – – – + 42 gonyaulax turbynei murray et whitting – – – – – – – – – + + 43 gonyaulax unicornis lebour + + – + + – + + + – + 44 gonyaulax veriore sournia – – – – – – – + + – – 45 gotoius mutsuensis abé – – – – – – – – – + – 46 gymnodinium catenatum graham – + – + – – – – – – – 47 gymnodinium sanguineum k. hirasaka + + + + + + + + + + + 48 gymnodinium sp. – + – – – – + + – + + 49 gyrodinium sp. – – + + – – – + – – + 50 gyrodinium spirale (bergh) kofoid et swezy – + + + – + + + + + – 51 heterocapsa circularisquama horiguchi – – – – – – – – – + – 52 heterocapsa niei (loeblich) morrill et loeblich iii – – – + – – – + + + – 53 heterocapsa rotundata (lohmann) g. hansen – – – – – – – + + + – 54 heterocapsa triquetra (ehrenberg) f. stein – – + – – – – – – – – 55 katodinium sp. – + – – – + + – – + + 56 lingulodinium polyedrum (stein) dodge + + + + + + + + + + + 57 neoceratium azoricum (cleve) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 58 neoceratium candelabrum (ehrenberg) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 59 neoceratium contrarium (gourret) f. gómez, d. moreira et p. lópez-garcía – + – – – – – – – + + 60 neoceratium declinatum f. declinatum (sournia) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – + – tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 26. rujan 2011 14:05:26 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 279 phytoplankton in the nador lagoon (marocco) stations 1 2 3 4 5 6 7 8 9 10 11 61 neoceratium declinatum f. majus (jörgensen) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – + – 62 neoceratium deflexum (kofoid) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 63 neoceratium extensum (gourret) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 64 neoceratium furca (ehrenberg) f. gómez, d. moreira et p. lópez-garcía + + + + + + + + + + + 65 neoceratium fusus (ehrenberg) f. gómez, d. moreira et p. lópez-garcía – + + + – – – – + + + 66 neoceratium karstenii (pavillard) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 67 neoceratium lineatum (ehrenberg) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 68 neoceratium longipes (bailey) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 69 neoceratium macroceros (ehrenberg) f. gómez, d. moreira et p. lópez-garcía – + – – – – – – – + – 70 neoceratium massiliens (gourret) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – + + 71 neoceratium massiliens armatum (karsten) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 72 neoceratium pentagonum var. tenerum (jörgensen) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 73 neoceratium teres (kofoid) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – + – – 74 neoceratium trichoceros (ehrenberg) f. gómez, d. moreira et p. lópez-garcía – + + – – – – + – + + 75 neoceratium tripos (o. f. müller) f. gómez, d. moreira et p. lópez-garcía – – + – – – – – + – – 76 neoceratium tripos f. tripodoides (jörgensen) f. gómez, d. moreira et p. lópez-garcía – – – – – – – – – – + 77 noctiluca scintillans (macartney) kofoid et swezy – + + + + + – + + + + 78 oblea baculifera balech ex loeblich jr. et loeblich iii + – + + + + + + + + + 79 ornithocercus magnificus stein – – – – – – – – – – + 80 ostreopsis ovata fukuyo – – + – – – – – – – + 81 palaeophalacroma unicinctum schiller – – – – – – – – – – + 82 pentapharsodinium sp. – – + – – – – – – + – tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 26. rujan 2011 14:05:26 color profile: disabled composite 150 lpi at 45 degrees 280 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. stations 1 2 3 4 5 6 7 8 9 10 11 83 peridiniella sp. + + + + – – – – – – – 84 periperidinium sp. – – + – + + – – – + – 85 podolampas palmipes stein – – – – – – – – – + – 86 polykrikos schwarzii bütschli – + – – + + + + + + + 87 prorocentrum balticum (lohmann, 1908) loeblich – – – – – – – – – – + 88 prorocentrum compressum (bailey) abé ex dodge – – – – – – – – – – + 89 prorocentrum lima (ehrenberg) dodge + – – – – – – – – + + 90 prorocentrum mexicanum tafall + + + + + + + + – + + 91 prorocentrum micans ehrenberg + + + + + + + + + + + 92 prorocentrum minimum (pavillard) schiller + + – + + + – + + + – 93 prorocentrum ruetzlerianum faust + – – – – – – – – – – 94 prorocentrum sigmoide bohm + + – + + + + – + + – 95 prorocentrum sp. – – – – – – – – + – + 96 prorocentrum triestinum schiller + + + + + + + + – + + 97 protoceratium reticulatum (claparède et lachmann) butschli – – – – – – – + – – + 98 protoceratium sp. – – – – – – – – – + + 99 protoperidinium acanthophorum (balech) balech + – – – – – – – – – – 100 protoperidinium bipes (paulsen) balech + + + + + + + + + + + 101 protoperidinium bispinum (schiller)balech + + + + + + + + + + + 102 protoperidinium brevipes (paulsen) balech + + – – + + – – + – + 103 protoperidinium capurroi (balech) balech + – + – – – – – – – – 104 protoperidinium cf. avellana (meunier) balech + – – – – – – – – – – 105 protoperidinium cf. capurroi subpellucidum (e. balech) e. balech – – – – – – – – – – + 106 protoperidinium cf. nanum (balech) balech – – – – – – – – – – + 107 protoperidinium cf. obtusum (karsten) parke et dodge – – – – – – – – – + – 108 protoperidinium claudicans (paulsen) balech – – – – – – – – – + + 109 protoperidinium conicoides (paulsen) balech + + – – – – + – + + + 110 protoperidinium conicum (gran) balech – + – + – – – + + + + 111 protoperidinium conicum (gran) balech avr. conicum in balech – – + + – – – – – – – 112 protoperidinium conicum (gran) balech var. concavum matzenauer – – – – – – – – – – + 113 protoperidinium cruciferum (balech) balech – – – – – – – – + – – 114 protoperidinium curtipes (jörgensen) balech – – – – – – – + + – – tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:48 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 281 phytoplankton in the nador lagoon (marocco) stations 1 2 3 4 5 6 7 8 9 10 11 115 protoperidinium decipiens (jörgensen) parke et dodge (jörgensen) parke et dodge – – + – – – – – – – – 116 protoperidinium depressum (bailey) balech – + – – – – – – – – + 117 protoperidinium diabolus (karsten) balech – + + + + + + + + + + 118 protoperidinium divaricatum (meunier) balech – – – – – – + – + – – 119 protoperidinium divergens (ehrenberg) balech (ehrenberg) balech + + – – – + + + + + + 120 protoperidinium excentricum (paulsen) balech – – – – – – – – – – + 121 protoperidinium fartum balech – – – – – – – – – – + 122 protoperidinium gibbosum (matzenauer) balech – – – – – + – – – – – 123 protoperidinium granii (ostenfield) balech (ostenfield) balech – – – – – + – – – – – 124 protoperidinium hirobis abè + + + + + + – + + + + 125 protoperidinium hirobis abè – – – – – – – – – + – 126 protoperidinium incognitum (balech) balech – – – – – – – + – – – 127 protoperidinium latispinum (mangin) balech – – – – – – – – – – + 128 protoperidinium leonis (pavillard) balech – – – – – – + + + – + 129 protoperidinium mastophorum (balech) balech – – + + – – – + – + + 130 protoperidinium metananum (balech) balech – – + + – – – + – – – 131 protoperidinium minutum (kofoid) loeblich iii – – – – + – – – – – + 132 protoperidinium mite (pavillard) balech – + + – + – – + – + + 133 protoperidinium nanum (balech) balech – – + – – – – – – – – 134 protoperidinium nudum (meunier) balech – – – – – – – + – + – 135 protoperidinium oblongum (aurivillius) parke et dodge + + + + + + + + + + + 136 protoperidinium obtusum (karsten) parke et dodge – – – – – – – + + – – 137 protoperidinium ovatum subsp. asymmetricum pouchet + + + – – – – – + – – 138 protoperidinium oviforme (dangeard) balech – + – – – – – – – – + 139 protoperidinium ovum (schiller) balech – – – – – – – + – – + 140 protoperidinium pallidum (ostenfeld) balech – – + – – – – – – – – 141 protoperidinium parapyriforme (hermosilla) balech – – – + + – – – – + + 142 protoperidinium parcum (balech) balech – – – – – – – – + – – 143 protoperidinium parviventer balech – – – – – – – – + – – 144 protoperidinium paulseni pavillard – – – – – – – – + – – 145 protoperidinium punctulatum (paulsen) balech + + + + + + + + + + + tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:48 color profile: disabled composite 150 lpi at 45 degrees tium furca, gonyaulax souseae, alexandrium minutum, scrippsiella trochoidea, prorocentrum triestinum and achradina pulchra. a maximum of 201 taxa were observed at station 11, the minimum of 72 species at station 7 (fig. 5). abundance the maximum phytoplankton abundance was found in august 2008 (fig. 6), due to the bloom of nitzschia longissima (1.7 ´ 107 cells l–1 at station 1, located in the n-w beninsar area, and the bloom of skeletonema (7.4 ´ 106 cells l–1) observed at station 4. the minimum abundance was recorded in november 2007. 282 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. stations 1 2 3 4 5 6 7 8 9 10 11 146 protoperidinium pyriforme var. pyriforme (paulsen) balech – – + – + – – – – – + 147 protoperidinium quarnerense (schröder) balech – – – – – – – – – – + 148 protoperidinium quinquecorne (abé) balech – – – + – – – – – – – 149 protoperidinium simulum (paulsen) balech – – – – – – – + – – – 150 protoperidinium sp. + + + – – – – + – + + 151 protoperidinium sphaeroideum (mangin) balech – – – – – – – – + – + 152 protoperidinium spinulosum schiller – – + + + + + – + – + 153 protoperidinium steidingerae balech – – – – – – – – – – – 154 protoperidinium steinii (jørgensen) balech + – – – – – – – – + + 155 protoperidinium subcrassipes e. balech – – – – – – – – – – + 156 protoperidinium subpyriforme (dangeard) balech – – – – – – + – – – – 157 protoperidinium subsphaericum (broch) balech + – – – – – – – – – – 158 protoperidinium thorianum (paulsen) balech + – – + – – – – – – – 159 protoperidinium ventricum (abé) balech – – – + – – – – – – – 160 protoperidinium vulgare balech – – + – – – – – – – – 161 pyrocystis noctiluca murray ex haeckel murray ex haeckel – – – + – – – – – + – 162 pyrophacus horologicum stein + + + + + + + + + + + 163 pyrophacus sp. – – – – – – – – + – – 164 pyrophacus steinii (schiller) wall et dale – – – – – – – – – – + 165 scrippsiella precaria montressor et zingone – – – – – – – + – – – 166 scrippsiella spinifera g. honsell et m. cabrini – + + + + + – + + + + 167 scrippsiella sweeneyae balech ex loeblich iii + – + + – + – + + + + 168 scrippsiella trochoidea (stein) loeblich iii + + + + + + + + + + + 169 spirilax jollifei (murray et whitting) kofoid + + + – – – – + – – – tab. 1. – continued u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:48 color profile: disabled composite 150 lpi at 45 degrees the abundances in january and june differ due to the bloom of chaetoceros observed in january at station 10, with abundance exceeding 2 ´ 106 cells l–1. november 2007 and january 2008: the community was dominated by the diatom chaetoceros which contributed in 96.14% and 99.06% respectively in november and january. in november, the maximum phytoplankton abundance was recorded at station 2, with 4.4 ´ 104 cells l–1 (fig. 7). the dominance of chaetoceros was observed all over of the lagoon with the exception of stations 5 and 6 where neoceratium furca dominated. acta bot. croat. 70 (2), 2011 283 phytoplankton in the nador lagoon (marocco) fig. 5. spatial distribution of the phytoplankton taxa numbers. 0 40 80 120 160 200 nov. 2007 jan. 2008 apr. 2008 jun. 2008 aug. 2008 samling period g e o m e tr ic m e a n o f p h y to p la n k to n a b u n d a n c e (c e ll s m l ) – 1 fig. 6. temporal variation of the geometric mean of phytoplanktonic abundance of the 11 stations. u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:49 color profile: disabled composite 150 lpi at 45 degrees in january 2008, beside the dominance of chaetoceros, dinoflagellates such as gonyaulax souseae, alexandrium minutum, scrippsiella trochoidea and prorocentrum triestinum co-dominated at stations 1 and 2. april 2008: high abundances were recorded at the station 10, with about 9.5 ´ 105 cells l–1 of pseudo-nitzschia, which contributed 72.19% of the community abundance. jun 2008: the maximum abundance was recorded at the station 1, with 1.7 ´ 107 cells l–1 which was mostly contributed by nitzschia longissima (99.9% of the community abundance). august 2008: the maximum abundance was recorded at station 5, with 8.5 ´ 105 cells l–1 of unidentified rounded cells, 17 to 20 mm in diameter (dinoflagellates or rhaphidophycean flagellatres), contributing 95.7% of the community abundance. we also found abundant 284 acta bot. croat. 70 (2), 2011 el madani f., chiaar a., chafi a. june 2008 0 3000 6000 9000 12000 15000 18000 1 4 6 8 10 3 2 7 5 11 9 january 2008 0 500 1000 1500 2000 2500 10 4 11 5 8 6 3 7 2 1 9 november 2007 0 5 10 15 20 25 30 35 40 45 50 2 8 10 4 1 3 5 11 7 9 6 a b u n d a n c e (c e ll s m l ) – 1 april 2008 0 200 400 600 800 1000 10 8 9 7 1 4 11 5 6 3 2 stations a b u n d a n c e (c e ll s m l ) – 1 august 2008 0 200 400 600 800 1000 5 8 3 9 10 1 2 7 11 6 4 stations a b u n d a n c e (c e ll s m l ) – 1 fig. 7. spatial variation of phytoplankton abundance during the investigated period. u:\acta botanica\acta-botan 2-11\el madani.vp 9. rujan 2011 13:04:49 color profile: disabled composite 150 lpi at 45 degrees dinoflagellate achradina pulchra (5.9 ´ 105 cells l–1) at station 8, and nitzschia longissima (4 ´ 105 cells l–1) at station 3. the dominant species (with abundance greater than 104 cells l–1) for each sampling cruise are summarized in table 2. generally, diatoms dominated the phytoplankton community, with the occasional dominance of unidentified flagellates and dinoflagellates in august 2008. discussion in the present study, diatoms were generally dominant. the maximum number of taxa was observed at station 11, situated in boccana channel, with a greater exchange of water between the lagoon and the open sea. the minimum number of taxa was found at station 7 located in the confined kariat arekman area as found in the period 1982–1993 (lefebvre et al 1996). the difference in diversity between these two areas shows that more than half of the phytoplankton population comes from the open sea. the highly eutrophic conditions in the lagoon cannot maintain populations. in addition, gilabert (2001a) explains the low diversity in the lagoon by strong physical perturbations. anyway, more than 114 species are adapted to the lagoon’s ecological conditions. about 44 phytoplankton species are common in most sampling stations (those found in at least 6 sampling stations). the number of diatoms and dinoflagellates identified in different parts of the mediterranean sea mostly varies between 107 and 183 of diatoms, and 107 to 205 dinoflagellates (vili^i] et al. 2002). in the nador lagoon, we found 133 diatoms and 169 dinoflagellates. acta bot. croat. 70 (2), 2011 285 phytoplankton in the nador lagoon (marocco) tab. 2. seasonality of dominant and accompanying species in the nador lagoon. sampling campaign dominants species accompanying species (density > 104) november 2007 chaetoceros spp. neoceratium furca (ehrenberg) f. gómez, d. moreira et p. lópez-garcía, pseudo-nitzschia spp. january 2008 chaetoceros spp. prorocentrum triestinum schiller small dinoflagellates (*) skeletonema sp. pseudo-nitzschia spp. divers bacillariophyceae pennates nitzschia longissima (brébisson) ralfs april 2008 pseudonitzschia spp. leptocylindrus minimus gran thalassionema nitzschioides (grunow) mereschkowsky nitzscha longissima, (brébisson) ralfs small dinoflagellates (*) chaetoceros spp. eutreptia sp. prorocentrum triestinum schiller (*): alexandrium minutum, gonyaulax verrior, g. sousea, g. dicantha scrippsiella spp, protoperidinium bipes, p. quinquecorne, p. hirobis, and others similar forms. u:\acta botanica\acta-botan 2-11\el madani.vp 26. rujan 2011 14:07:11 color profile: disabled composite 150 lpi at 45 degrees in the nador lagoon we recorded a high abundance of nitzschia longissima (1.7 ´ 107 cells l–1) in june at station 1. this station is situated in the confined area in the north-west part of the mar chica that receives wastewater from beninsar city. an increasing abundance of phytoplankton, especially diatoms to 5 ´ 106 cells l–1, usually reveals an area of anthropogenic influence (vili^i] 1989). nador lagoon is slightly eutrophicated in october, with an tendency to increase with the approach of summer, due to the increasing temperature, light intensity and input of nutrients into the lagoon (fig. 4, 7). the eutrophication of the lagoon is manifested by the reduction of the water clarity, which is the direct result of the phytoplankton blooms, corresponding to the diatoms chaetoceros, pseudo-nitzschia, nitzschia longissima etc. in summer, chlorophyll concentrations range from below 5 mg l–1 to 20 mg l–1, and oxygen decrease to anoxic conditions at stations 1 and 4. strong eutrophic conditions were observed in the last decade. the blooms cause a green, brown or yellowish-brown discoloration of water. the bloom degradation results in increasing oxygen demand and degradation of water quality. seasonal variations of primary production are the result of external physical chemical environmental variables and the shallowness of the lagoon (gilabert 2001a, b). during several years, we have observed that summer phytoplankton blooms are accompanied by appearance of abundant jellyfish rhyzostoma pulmo. occurrences were reported in the mar menor, where these organisms may play an important role in controlling eutrophication by feeding on diatoms and zooplankton (pérez-ruzafa et al. (2002). the qualitative and quantitative results lead us to conclude that, in general, the nador lagoon seem to be highly affected by eutrophication. a new channel (300 m wide) is going to be opened to increase the lagoon water exchange with the open sea, with an expected reduction of the residence time of water in the lagoon. this action is expected to have a great impact on the water quality by reducing the present eutrophication level, which will be accompanied by the modification of current phytoplankton population structure. acknowledgements the authors thank saida motia and najib el ouamari for their comments on the first version, and professor amany ismael for the review of the last version of the paper; siham el madani for language verification. references balech, e., 1988: los dinoflagelados del atlántico sudoccidental. publicaciones especiales del instituto español de oceanografía 1, 1–310. balech, e., akselman, r., benavides, h. r., negri, r. m., 1984: suplemento a los 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doi: 10.2478/botcro-2019-0005 issn 0365-0588 eissn 1847-8476 climatic drivers of dry grassland phylogenetic diversity in the republic of macedonia marco antonio batalha1*, renata ćušterevska2, vlado matevski2 1 department of botany, federal university of são carlos, po box 676, 13565-905, são carlos, brazil 2 institute of biology, faculty of natural sciences and mathematics, ss. cyril and methodius university, arhimedova 3, 1000, skopje, macedonia abstract – climatic gradients can be used to predict the extent to which climate drives biodiversity and to which biodiversity may be affected by global climate changes. climate and evolutionary history are linked by the ecological adaptations of species and the history of earth’s climate. if so, phylogenetic diversity may be a good metric to estimate biodiversity. we aimed to test whether the phylogenetic diversity of macedonian dry grasslands was related to climatic variables. we sampled 575 plots, identifying the species and building a phylogenetic tree for them. we calculated two metrics of phylogenetic diversity and regressed them against climatic variables. we also tested whether there were nodes in the tree responsible for the main observed spatial patterns of phylogenetic diversity. we found a strong signature of evolutionary history in species sorting across a gradient driven by climate in macedonian dry grasslands. first, the amount of evolutionary history decreased towards drier and more seasonal climates, suggesting a phylogenetic niche conservatism. second, there was an air temperature filter and a temperature seasonality filter, acting in opposite directions and leading to phylogenetic clustering. third, there were few nodes in the phylogenetic tree with high degrees of allopatry, associated with clades that differed not only in their geographic distribution, but also in their climatic preferences. macedonian dry grassland communities developed over centuries of traditional land use but are threatened nowadays by human activities. the use of phylogenetic approaches may lead to more effective conservation policies and help us preserve this highly diverse vegetation. keywords: balkans, climate, evolutionary history, macedonian flora, phylogeny, plant diversity * corresponding author, e-mail: marcobat@fastmail.fm introduction considering the current rates at which natural habitats are being destroyed (wwf 2016), understanding which factors influence community structure is critical to managing, preserving, and restoring biodiversity (cavender-bares et al. 2009). biodiversity provides multiple environmental services and, thus, contributes to human welfare (naeem et al. 1999). species richness has been used as the primary method for mapping biodiversity (marchese 2015), but, when only the number of species is taken into account as a biodiversity metric, all species are considered equally distinct (chao et al. 2015). community structure, however, depends not only on the number of species, but also on their ecological differences (mcgill et al. 2006). hence, the quantification of biodiversity has shifted lately from species counting to more integrative approaches, which use information about functional traits or evolutionary history or both (marchese 2015). as a matter of fact, there has been a growing interest in including phylogenetic information into community ecology studies (lean and maclaurin 2016), since phylogeny has been recognised as a major source of biological variation (jombart et al. 2010). as long as a phylogenetic tree depicts the evolutionary relations among species, it can be used to calculate the amount of evolutionary history in a community or the “phylogenetic diversity” (lean and maclaurin 2016). the less related the species in a community, the higher the phylogenetic diversity of that community (vellend et al. 2011). by taking into account the evolutionary relations, phylogenetic diversity provides additional information to guide conservation decisions (marchese 2015). for instance, assessing the loss of phylogenetic diversity may be a better indicator of community vulnerability than simply batalha m. a., ćušterevska r., matevski v. 26 acta bot. croat. 78 (1), 2019 assessing the loss of species (marchese 2015). information on functional traits is often lacking, especially in species-rich communities (webb 2000). however, if traits are conserved on the phylogeny (ackerly 2003), closely related species are expected to play similar functional roles and tend to be limited by their similarity, and phylogenetic diversity may be used to predict community functioning (lean and maclaurin 2016). phylogenetic diversity, thus, offers information on evolutionary history and potentially on functional similarity, which can shed light on the drivers of community structure and the responses of communities to environmental change (marchese 2015). it is possible to integrate phylogenetic diversity and species distributions in site-based approaches, which quantify spatial variation in the relatedness of co-occurring species using some metric of community phylogenetic structure (borregaard et al. 2014). even though there are many phylogenetic diversity metrics, all of them can be grouped into three conceptual dimensions (pavoine et al. 2009, tucker et al. 2017): richness, the sum of accumulated phylogenetic differences among taxa in a community; divergence, the mean phylogenetic distance among taxa in a community; and regularity, the variance in distances among taxa in a community. helmus et al. (2007), for example, developed a metric of phylogenetic richness, “phylogenetic species richness”, which combines species richness and phylogenetic relatedness, and a metric of phylogenetic divergence, “phylogenetic species variability”, which is independent of species richness and quantifies how, on average, species in a community are related to each other. since ecological questions consider how accumulated differences among species are related to biodiversity patterns, these dimensions of phylogenetic diversity can be an outcome of processes under study (tucker et al. 2017). the fact that biodiversity varies from place to place has caught the attention not only of ecologists but of anyone who observes the natural world (begon et al. 2006). knowing the spatial distribution of biodiversity and what drives it are preconditions for prioritising conservation efforts on local, regional, and global scales (begon et al. 2006). climate, especially temperature and precipitation, has been considered the main predictor of biodiversity on a large scale (andersen et al. 2015). the relationship between biodiversity and latitude, a surrogate for temperature, is a well-established biogeographical pattern (kerkhoff et al. 2014) and assumed to be “ecology's oldest pattern” (hawkins 2001). current climatic conditions can be successfully used to predict biodiversity, especially in plants (kerkhoff et al. 2014). plant diversity usually peaks in warm, wet, and stable climatic conditions, declining towards cold, dry, and seasonal ones (francis and currie 2003). climatic gradients can be used to predict the extent to which climate drives biodiversity and to which biodiversity may be affected by global climate changes (andersen et al. 2015). climatic gradients can also be used to identify sensitive zones, with high rates of compositional changes (williams et al. 1995). when trying to relate biodiversity and climate, species richness is generally used, but phylogenetic diversity has been increasingly taken into account (qian et al. 2013). since the loss of a more distinct species implies a greater loss of biodiversity than the loss of a non-distinct species with many close relatives (purvis et al. 2000), species without close relatives may be granted special protection, so that overall evolutionary history can be maximised (begon et al. 2006). although climate and evolutionary history are usually considered competing explanations for biodiversity patterns, they are linked by the environmental adaptations of species and the history of earth’s climate (kerkhoff et al. 2014). because seed plants have been evolving since the late devonian, about 400 million years ago (rothwell and erwin 1987), but temperate habitats have expanded over tropical ones only since the oligocene, about 35 million years ago, most of the temperate clades have diversified recently (kerkhoff et al. 2014). as a consequence, two predictions can be made: first, communities in colder, drier, and more seasonal climates will be phylogenetically less diverse and, second, clades in colder, drier, and more seasonal climates will be younger (kerkhoff et al. 2014). in this sense, when trying to integrate phylogenetic diversity and species distributions, site-based approaches may be combined with clade-based approaches, which quantify the spatial overlap between sister clades (borregaard et al. 2014). the simultaneous use of both approaches can be helpful to test whether environmental gradients are relevant in shaping the phylogenetic structure of local communities (parra et al. 2010). borregaard et al. (2014), for instance, developed a metric that uses both approaches, the “specific overrepresentation score”, which goes through each node of the phylogenetic tree and compares the species richness of sister clades in each community to what is expected by chance. these scores can be summarised for all communities to produce the “geographic node divergence”, which quantifies the spatial divergence between the two sister clades of a given node and identifies which nodes are mainly responsible for the observed spatial patterns of phylogenetic diversity (borregaard et al. 2014). the use of such a metric can be a powerful strategy to detect community patterns more thoroughly and unravel the interplay of processes behind them (graham et al. 2016). the republic of north macedonia, with an area of 25,713 km2, is located in the southern portion of the balkan peninsula, bound by albania to the west, greece to the south, bulgaria to the east, and serbia and kosovo to the north. macedonia is a mountainous country, cut by valleys, canyons, and plateaus, going from 60 m to 2,764 m above sea level. from the geomorphological point of view, the country can be divided into two main regions: the western part with carbonaceous rocks and the eastern part with siliceous rocks (čarni and matevski 2015). macedonia is at the top of the list of “european hotspots” due to its great biodiversity, a result of its long historical development (gaston and david 1994). the differentiation of indigenous species and the migration of species from other areas played an important role in its genesis, so that it is not possible to understand its current biodiversity patterns without looking at its evolutionary history (čarni and matevski 2015). for instance, grassland phylogenetic diversity in macedonia acta bot. croat. 78 (1), 2019 27 related species (helmus et al. 2007). if so, this community would have lower phylogenetic diversity (lean and maclaurin 2016). by sampling dry grassland plant communities in the republic of macedonia, southern balkans, calculating their phylogenetic diversity, and relating it to climatic variables, we aimed to answer three questions: (1) does evolutionary history decrease towards colder, drier, and more seasonal climatic conditions? (2) is environmental filtering more important in colder, drier, and more seasonal climatic conditions? (3) are there nodes on the phylogenetic tree that identify evolutionary divergences associated with spatial segregation? materials and methods we used a database consisting of 575 plots, 259 on carbonaceous rock and 316 on siliceous rock, placed on dry grasslands all over the country (fig. 1). each plot had 100 m2, because of the relatively homogenous vegetation at this scale. plots were located within targeted patches of dry grasslands in such a way that edge effects were excluded (matevski et al. 2015) and sampled according to braun-blanquet (1964), in late spring, the optimal phenological period of the year. data have already been partially analysed, but with different aims (matevski et al. 2007, 2008, 2011, 2015, ćušterevska et al. 2012, ćušterevska 2016). in each plot, we recorded the presence of seed plants, identifying them to species level and lodging vouchers at the mknh herbarium, ss. cyril and methodius university, skopje, macedonia. we used plantminer (carvalho et al. 2010) to search for families and synonyms concerning our sampled species. we built a phylogenetic tree for them, using bell et al. (2010) as the backbone and improving resolution by consulting references on specific clades, as suggested by beaulieu et al. (2012). when only the topology of the clade was available, we placed the glaciations during the pleistocene in the balkans had a significant impact on the heterogeneity of macedonian biodiversity (grunewald and scheithauer 2010). consequently, the country has a high level of endemics and relicts (chemonics international 2001). although macedonia is small, the heterogeneity of its climatic conditions is very high (bergant 2006). the country lies in a transitional zone, where climate goes from mediterranean to continental, generally with warm summers and moderately cold winters, and annual precipitation going from 500 m in the eastern region to 1,700 mm in the western region (filipovski et al. 1996). southern mountains prevent hot air masses to move from south to north, whereas the šar mountains, located in the northwestern portion, block cold northern winds (bergant 2006). in the highest and coldest parts, winters are long and snowy and summers are short and cold, whereas, in the warmest parts, summer temperatures reach over 40 °c (bergant 2006). based on the annual cycle of mean daily air temperature and precipitation, four climatic types may be recognised in macedonia (filipovski et al. 1996): mediterranean in the southeastern part, mediterranean-continental in the central part, continental in the southwestern part, and alpine in the northwestern part. regions with a continental climate are expected to have the weakest response to large‐scale climate change and regions with an alpine climate the strongest (bergant 2006). four phytogeographic regions can be distinguished, characterised by their different climates, altitudes, flora, and fauna (chemonics international 2001): (1) submediterranean region, covering about 40% of the country, up to 600 m above sea level, characterised by the presence of ostrya carpinifolia and carpinus orientalis; (2) sub-continental region, covering about 37% of the country, between 600 and 1,200 m above sea level, characterised by the presence of quercus frainetto; (3) sub-humid region, covering about 22% of the country, with a lower belt, between 900 and 1,250 m above sea level, characterised by beech forest, and a higher belt, between 1,250 m to 1,700 m, with mountainous beech forest and mixed beech and fir forest; and (4) sub-alpine region, covering about 1% of the country, above 1,700 m, on the highest mountains. in the first three phytogeographic regions, dry grasslands may be found (matevski et al. 2015). dry grasslands in europe are mostly seminatural habitats, but they harbour some of the most diverse plant communities in the world on small scales (janišová et al. 2011). despite that diversity, they are one of the most endangered vegetation types in europe due to urbanisation and afforestation (janišová et al. 2011). conservation is only possible if we understand what drives community structure and dynamics in both ecological and evolutionary terms, for which phylogenetic diversity is a suitable metric (lean and maclaurin 2016) and climate is a potential driver (andersen et al. 2015). understanding what drives community structure and dynamics can also give us insights about assembly rules (cavender-bares et al. 2009). as long as closely related species might have similar tolerances, when the climate becomes harsher, they are more likely to occur within the same community than with less fig. 1. altitudinal map with the location of the examined plots (blue dots) in the republic of north macedonia. batalha m. a., ćušterevska r., matevski v. 28 acta bot. croat. 78 (1), 2019 undated nodes in the tree evenly between the dated nodes (webb et al. 2008). plots were placed in different altitudes, from 60 m to 1,500 m above sea level, under different climatic conditions, from mediterranean to continental. in each plot, we recorded the coordinates with a global positioning system and, based on them, extracted altitude (google maps 2016) and the 19 climatic variables available on worldclim (on-line suppl. tab. 1; hijmans et al. 2005). we applied spearman correlation analyses to test whether altitude and the climatic variables were correlated, excluding from further analyses variables highly correlated to others (ρ > |0.7|). thus, for the remaining analyses, we kept annual mean temperature (bio 1), isothermality (bio 3), mean temperature of wettest quarter (bio 8), mean temperature of driest quarter (bio 9), annual precipitation (bio 12), and precipitation seasonality (bio 15). these six climatic variables were related to minimum temperature, maximum temperature, precipitation, or a combination of them (on-line suppl. tab. 1) and brought additional information when used as explanatory variables. we standardised these six climatic variables by the range and applied a principal component analysis (jongman et al. 1995), using the first two principal components as a means to summarise the climate in the studied region. to answer the first question, we calculated phylogenetic species ichness (helmus et al. 2007) per plot, using the community matrix and the phylogenetic tree. this metric is directly comparable to the traditional metric of species richness but includes phylogenetic relatedness (helmus et al. 2007) and, thus, sums up the quantity of phylogenetic differences present in a community (tucker et al. 2017). since our data was non-normally distributed, heteroscedastic, and with outliers, instead of applying ordinary least square regression, we applied robust regression (huber and ronchetti 2009), with phylogenetic species richness as response variable, the six climatic variables as explanatory ones, and the bisquare algorithm as the weighting function. to answer the second question, we calculated phylogenetic species variability (helmus et al. 2007) per plot. this metric quantifies how phylogenetic relatedness decreases the variance of some unspecified neutral trait shared by all species in the sample (helmus et al. 2007) and, thus, represents the mean phylogenetic difference among taxa in a community (tucker et al. 2017). we also applied robust regression following the previous procedure, but with phylogenetic species variability as response variable. to answer the third question, we calculated the specific overrepresentation scores for all nodes in the phylogenetic tree as a measure of clade overrepresentation (borregaard et al. 2014), with 999 randomisations. we summarised these values to calculate the geographic node divergence scores (borregaard et al. 2014), mapping them across the plots. we used a cutoff value of 0.55 to identify which nodes were mainly responsible for the observed spatial patterns of phylogenetic diversity, also mapping them across the plots. to test whether phylogenetic spatial patterns could be explained by climate, we applied robust regressions, with the specific overrepresentation scores of the main nodes as response variables and the six climatic variables as explanatory ones. we carried out all analyses in r (r development core team 2016), using the “ape” (paradis et al. 2004), “foreign” (r development core team 2015), “geiger” (harmon et al. 2008), “ggmap” (kahle and wickham 2013), “mass” (venables and ripley 2002), “nodiv” (borregaard et al. 2014), “picante” (kembel et al. 2008), “raster” (hijmans 2015), “rgdal” (bivand et al. 2015), and “vegan” (oksanen et al. 2013) packages. results in the 575 plots, we found 767 species, belonging to 59 families, for which we built a phylogenetic tree (on-line suppl. fig. 1). the richest families were asteraceae (113 species), fabaceae (100), and poaceae (80), which, together, accounted for 38% of the total number of species. the first principal component of the ordination analysis explained 44% of the climatic variation, being negatively related to mean temperature of the wettest quarter and annual mean temperature and positively related especially to annual precipitation, precipitation seasonality, and isothermality (fig. 2). according to that component, there was a climatic gradient from northeast to southwest, in which plots in the former were warmer and plots in the latter were wetter, more seasonal with respect to precipitation, and less seasonal with respect to temperature (fig. 3a). the second principal component explained an additional 33% of the climatic variation, being negatively related especially to mean temperature of the driest quarter and positively related especially to isothermality and mean temperature of the wettest quarter (fig. 2). according to it, there was a warmer region during the driest quarter in the central part, along the vardar river basin, from skopje, through veles and negotino, to gevgelija (fig. 3b). tab. 1. phylogenetic species richness (psr) and phylogenetic species variability (psv) of macedonian dry grassland communities as a function of annual mean temperature (bio 1), isothermality (bio 3), mean temperature of wettest quarter (bio 8), mean temperature of driest quarter (bio 9), annual precipitation (bio 12), and precipitation seasonality (bio 15). response variable explanatory variable coefficient p psr bio 1 1.44 × 10–2 0.610 bio 3 1.92 × 10 <0.001 bio 8 2.39 × 10–3 0.716 bio 9 –1.01 × 10–2 0.005 bio 12 1.51 × 10–2 0.023 bio 15 –8.08 × 10–1 <0.001 psv bio 1 –1.68 × 10–4 <0.001 bio 3 –2.04 × 10–3 <0.001 bio 8 –2.42 × 10–5 <0.001 bio 9 –1.65 × 10–5 <0.001 bio 12 –1.22 × 10–5 0.059 bio 15 3.94 × 10–4 0.062 grassland phylogenetic diversity in macedonia acta bot. croat. 78 (1), 2019 29 phylogenetic species richness varied from 3.9 to 37.1, with a mean of 18.7 and a standard deviation of 5.2. we found the lowest values in the central part of macedonia, between veles, štip, and negotino, and the highest values in kozjak mountain near prilep (fig. 4a). when regressing phylogenetic species richness against the climatic variables, we found positive relationships with isothermality and annual precipitation and negative relationships with mean temperature of the driest quarter and precipitation seasonality (tab. 1). overall, phylogenetic species richness decreased towards warmer in the driest period, drier, and more seasonal climates. phylogenetic species variability varied from 0.33 to 0.43, with a mean of 0.35 and a standard deviation of 0.01. we found low values in almost all plots, but high values in the jakupica and labinica mountain ranges (fig. 4b). when regressing phylogenetic species variability against the climatic variables, we found negative relationships with annual mean temperature, isothermality, mean temperature of the wettest quarter, and mean temperature of the driest quarter (tab. 1). overall, phylogenetic species variability decreased towards warmer climates with less seasonal temperatures. fig. 2. principal component analysis biplot of climatic data in the republic of north macedonia. bio 1 ‒ mean annual temperature, bio 3 ‒ isothermality, bio 8 ‒ mean temperature of wettest quarter, bio 9 ‒ mean temperature of driest quarter, bio 12 ‒ annual precipitation, bio 15 ‒ precipitation seasonality. fig. 3. climate of the plots based on the first two principal components. (a) first component. the more yellow the point, the more negative the scores (higher annual mean temperature and mean temperature of wettest quarter); the more blue, the more positive the scores (higher annual precipitation, precipitation seasonality, and isothermality). (b) second component. the more yellow the point, the more negative the scores (higher mean temperature of driest quarter); the more blue, the more positive the scores (higher isothermality and mean temperature of wettest quarter). climatic data extracted from worldclim (hijmans et al. 2005). fig. 4. phylogenetic species richness (a) and phylogenetic species variability (b) of macedonian dry grasslands. the more blue the point, the higher its value; the more yellow the point, the lower its value. batalha m. a., ćušterevska r., matevski v. 30 acta bot. croat. 78 (1), 2019 most of the nodes in the phylogenetic tree presented low geographic node divergence scores (fig. 5). only four nodes presented scores higher than 0.55, corresponding to major distributional divergences in macedonian dry grassland communities (fig. 5). the specific overrepresentation scores for these four nodes were related to some of the climatic variables, from two to five depending on the node (tab. 2). the first node separated caryophyllales from asterids, that is, santanales, ericales, campanuliidae, and lamiidae (fig. 5). the occurrence of asterids, with lower scores, was related to warmer climates (figs. 6a; tab. 2). the second node separated, within the fabids, malpighiales, with higher scores, related to colder climates with more seasonal temperatures, from fabales, fagales, and rosales (figs. 5 and 6b; tab. 2). the third node separated, within fabaceae, loteae, with higher scores, related to colder climates with more seasonal precipitation, from hedysareae, galegeae, vicieae, and trifolieae (figs. 5 and 6c; tab. 2). the fourth node separated trifolium, with higher scores, related to drier, more isothermal climates, from other genera of trifolieae (figs. 5 and 6d; tab. 2). tab. 2. specific overrepresentation scores (sos) of the four nodes with the highest degree of allopatry (see fig. 5) as a function of annual mean temperature (bio 1), isothermality (bio 3), mean temperature of wettest quarter (bio 8), mean temperature of driest quarter (bio 9), annual precipitation (bio 12), and precipitation seasonality (bio 15) for macedonian dry grassland communities. response variable explanatory variable coefficient p sosa bio 1 –1.64 × 10–2 0.021 bio 3 5.68 × 10–1 <0.001 bio 8 1.24 × 10–3 0.451 bio 9 –3.27 × 10–3 0.002 bio 12 –3.95 × 10–3 0.018 bio 15 –5.01 × 10–2 0.354 sosb bio 1 –2.39 × 10–2 <0.001 bio 3 –8.44 × 10–1 <0.001 bio 8 –6.69 × 10–3 <0.001 bio 9 4.51 × 10–4 0.578 bio 12 1.27 × 10–3 0.401 bio 15 5.89 × 10–3 0.904 sosc bio 1 –6.02 × 10–2 <0.001 bio 3 –4.69 × 10–1 <0.001 bio 8 –1.04 × 10–2 <0.001 bio 9 –9.28 × 10–4 0.210 bio 12 –7.74 × 10–3 <0.001 bio 15 3.24 × 10-1 <0.001 sosd bio 1 –1.57 × 10–2 0.117 bio 3 8.70 × 10–1 <0.001 bio 8 5.78 × 10–4 0.788 bio 9 –7.07 × 10–5 0.945 bio 12 –7.21 × 10–3 0.003 bio 15 8.07 × 10–2 0.284 fig. 5. phylogenetic tree of the species sampled in macedonian dry grasslands, showing the four nodes with the highest geographic node divergence (gnd) scores. node a separates caryophyllales from asterids; node b separates, within fabids, malpighiales from fabales, fagales, and rosales; node c separates, within fabaceae, loteae from hedysareae, galegeae, vicieae, and trifolieae; node d separates trifolium from other trifolieae. in the detail, a histogram of all scores, showing that few nodes have high scores. fig. 6. specific overrepresentation scores of the four nodes with the highest degree of allopatry (see fig. 5). (a) node a, which separates caryophyllales, with higher scores, from asterids, with lower scores; (b) node b, which separates malpighiales, with higher scores, from fabales, fagales, and rosales, with lower scores; (c) node c, which separates loteae, with higher scores, from other tribes of fabaceae, with lower scores; (d) node d, which separates trifolium, with lower scores, from other genera of trifolieae, with higher scores. color gradation from blue to red presents the lowest to the highest scores. grassland phylogenetic diversity in macedonia acta bot. croat. 78 (1), 2019 31 discussion in only 5.75 hectares, we sampled 767 species, almost one fourth of the whole macedonian seed plant flora, which contains about 3,200 species (matevski et al. 2003). this high number of species highlighted how diverse dry grasslands are on small scales, more diverse than wet grasslands or even tropical rainforests (janišová et al. 2011). the main climatic pattern we found was the transition from the mediterranean, in the eastern part of the country, to the continental climate, in the western part. under mediterranean influence, the climate is warmer and with more seasonal temperatures, but, as the continental influence increases, it becomes progressively colder, wetter, and with more seasonal precipitation (bergant 2006). the high mountains in the western and southern parts prevent the influence of the mediterranean climate from going deep into the country despite its proximity to the aegean and adriatic seas (čarni and matevski 2015). the influence of the aegean sea is restricted to the vardar river basin (čarni and matevski 2015), where heat waves coming from the sea make the climate warmer during the dry summer (baldi et al. 2006), which was highlighted by the second climatic pattern we found. climatic variables are strong predictors for plant phylogenetic diversity (weigelt et al. 2015). as a matter of fact, the amount of evolutionary history in macedonian dry grassland communities, as measured by phylogenetic species richness, decreased towards drier and more seasonal climatic conditions. we found the lowest values in central macedonia, where, during summer rains, warm and dry winds cause the water to evaporate and salt-rich sediments lead to salinised soils (matevski et al. 2008). in this region, there is a steppe-like vegetation, with an impoverished flora able to stand the harsh conditions (matevski et al. 2008). as climatic conditions became milder, the amount of evolutionary history increased, peaking in the kozjak mountain, in highland pastures, with numerous balkan endemics (stevanović et al. 2009, palpurina et al. 2015). this pattern may be partially due to phylogenetic niche conservatism, that is, the idea that related species tend to occur in the same type of environment (harvey and pagel 1991). if many extant lineages began to diversify under tropical climatic conditions and if these lineages colonised temperate climates only recently, regions with wetter and more stable climates should be phylogenetically more diverse (donoghue 2008). the pattern of phylogenetic clustering, indicated by the low values of phylogenetic species variability in almost all plots, may be a signal of environmental filtering: only a few lineages of closely related species with conserved traits occurred under given climatic conditions (hoiss et al. 2012). in macedonian dry grasslands, temperature seems to be the main environmental filter. if phylogenetic niche conservatism holds true, we may expect decreased phylogenetic clustering when temperatures become higher and less seasonal. however, in the studied region, the warmer plots in the northeastern part were also more seasonal, whereas the colder plots in the southwestern part were also less seasonal. there appear, thus, to be two main environmental filters, acting in opposite directions: an air temperature filter, whose importance decreases from northeast to southwest, and a temperature seasonality filter, whose importance decreases from southwest to northeast. we found high values of phylogenetic species variability only in the jakupica and labinica mountain ranges, where the not so cold and not so seasonal climate may have led to competition as the main ecological force and, thus, to phylogenetic overdispersion (webb 2002). as in new world tyrant birds (borregaard et al. 2014), the current pattern of species distributions in macedonian dry grasslands was the outcome of major divergences at a few nodes. the most basal main node separated asterids, more frequent in the steppe-like vegetation. because of their derived phylogenetic position, asterids may be considered a relatively young group that diversified during the cretaceous (bremer et al. 2004) and, in macedonia, are one of the few clades able to persist in the steppe. the other three main nodes occurred along the lineage leading to presentday fabids, one of the few seed plant lineages in which temperate species are strongly clustered (kerkhoff et al. 2014). even malpighiales, typically tropical but related to colder climates in macedonia, include some speciose genera, such as euphorbia and linum, that are well-known in northern temperate regions (davis et al. 2005). these four nodes were key locations within the phylogeny in which clades differed not only in their geographic distribution, but also in their climatic preferences. these geographical shifts accompanied by climatic shifts might indicate that adaptation to new environments allows clades to colonise new areas (borregaard et al. 2014). in the last 40 years, macedonia has experienced a slight increase in temperature and a decrease in precipitation (čarni and matevski 2015). climate change projections for the country in the 21st century predict a more intensive increase in temperature in summer than in winter, almost no change in winter precipitation, and a strong decrease in summer precipitation (bergant 2006, karanfilovski 2012). moreover, changes are expected to be more pronounced in a mediterranean than in a continental climate (bergant 2006). biodiversity is subject to the impact of global changes and reacts to these changes with its own adaptive capacity or migrating to other areas with more suitable conditions (čarni and matevski 2015). in this sense, we may predict: (1) a decrease in phylogenetic diversity of dry grasslands, especially in the eastern part of the country, under a mediterranean climate; (2) an increased pattern of phylogenetic clustering due to the higher summer temperatures and lower summer precipitation; (3) major geographical shifts of the clades associated with the nodes with a high degree of allopatry; (4) an increase in phylogenetic diversity in lowland areas due to transhumance, which may have brought species with different origins and evolutionary history (chang 1993). under this scenario, phylogenetic diversity should be also considered when selecting priority conservation areas (zhang et al. 2015). the increasing availability of phylogenetic data and computational tools has stimulated the inclusion of phylogenetic batalha m. a., ćušterevska r., matevski v. 32 acta bot. croat. 78 (1), 2019 information in community ecology (cavender-bares et al. 2009). we found a strong signature of evolutionary history in species sorting across a gradient driven by climate in macedonian dry grasslands. first, the amount of evolutionary history decreased towards drier and more seasonal climates, suggesting a phylogenetic niche conservatism. second, there appear to be an air temperature filter and a temperature seasonality filter, acting in opposite directions and leading to phylogenetic clustering. third, there were few nodes in the phylogenetic tree with a high degree of allopatry, associated with clades that differed not only in their geographic distribution, but also in their climatic preferences. most european dry grassland communities, including those in macedonia, are 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(eds.), the impact of climate change on ecosystems and species: terrestrial ecosystems, 39–65. iucn, gland. wwf, 2016: living planet report. wwf international, gland. zhang, j., nielsen, s. e., stolar, j., chen, y., thuiller, w., 2015: gains and losses of plant species and phylogenetic diversity for a northern high-latitude region. diversity and distributions 21, 1441-1454. acta bot. croat. 81 (2), 2022 221 acta bot. croat. 81 (2), 221–232, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-020 issn 0365-0588 eissn 1847-8476 the relationship between biodiversity and the biomass of grasslands in the zagreb area (nw croatia) marta justić1, sven d. jelaska2* 1 geonatura ltd., fallerovo šetalište 22, 10000 zagreb, croatia 2 university of zagreb, faculty of science, department of biology, division of botany, marulićev trg 20/ii, 10000, zagreb, croatia. abstract – research into the floristic and ecological characteristics of grasslands in the zagreb area was conducted in june and july 2020. eight localities were processed, each containing two 1 m2 plots. the floristic composition of each quadrat was analysed and the presence of two vegetation communities, festuco-brometea and molinio-arrhenatheretea, was determined. although indigenous taxa dominate, two invasive plants: echinocystis lobata (michx.) torr. et a. gray and erigeron annuus (l.) desf. have been recorded. based on the functional traits of taxa, the functional diversity of plots was calculated and compared with the number of taxa and the weight of plant biomass collected within the plots. functional diversity values ranged from 1.121 to 2.060 and dry biomass from 251.4 to 586.8 g m-2 per quadrat. correlation analyses linked a decrease in the number of taxa and functional diversity with an increase in biomass and specific leaf area. functional diversity is positively correlated with the number of taxa, plant dry matter content and leaf dry matter content. it is necessary to conduct this type of research in other grassland communities in croatia to obtain more detailed insights into the relations of these parameters. keywords: ecological indicator values, functional diversity, functional traits, life-forms, origin introduction the biological diversity of an ecosystem is defined by the total number and composition of taxa, genotypes, functional groups and landscape units present (diaz and cabido 2001). favourable relationships among the taxa that make up the ecosystem improve its stability and ability to recover from disturbances. changes in environmental conditions such as negative anthropogenic impacts (excessive mowing, over-fertilization, deforestation, urbanization, etc.), global warming and succession all lead to biodiversity loss ( perrings et al. 1995, alhamad 2006). to conserve biodiversity, it is necessary to understand natural processes and sources of stability and the resilience of the communities to these disturbances (alhamad 2006). one of the problems that arises is the multidimensionality of biodiversity because diversity among taxa can be functional, phylogenetic, genetic and taxonomic. biodiversity is determined not only by the number of taxa but also by other factors such as species uniformity, diversity and rarity; macroclimate; amount of water and nutrients in the soil; functional diversity and human activity (willig 2011, grace et al. 2016). the most studied pattern is the relationship between biodiversity and plant biomass because these are key factors in predicting the impact of species loss on ecosystem functions and services. these relationships are not yet completely clear and vary depending on the spatial scale of the research, the number of woody plants, the competition among taxa, etc. (guo and berry 1998). the model that claims that biodiversity of herbaceous plant taxa can be predicted based on plant biomass is called the “hump-backed model” (hbm) and it predicts maximum biodiversity at the mean values of biomass. according to this model, the data are presented by quadratic regression ( fraser et al. 2014). at low biomass, the number of taxa is limited by disturbances and stress (extremes in temperature, water or nutrient concentrations in the soil), conditions that only some taxa can tolerate. at high biomass, the number of taxa is limited by mutual competition so only a small number of highly competitive taxa will survive (fraser et al. 2015). * corresponding author e-mail: sven.jelaska@biol.pmf.hr justić m., jelaska s. d. 222 acta bot. croat. 81 (2), 2022 previously, there have been only few studies on biomass and functional diversity in european temperate grasslands. fraser et al. (2015) recorded the biomass of natural grasslands worldwide, showing that it varies from 1 up to 5711 g m-2. hector et al. (1999) recorded biomass for experimental grasslands in several european countries where average biomass values vary from 336.5 g m-2 in greece to 802.2 g m-2 in germany. schumacher and roscher (2009) measured biomass in arrhenatheretalia type grasslands in germany (thuringia) ranging from 182 to 592 g m-2. grace et al. (2016) studied the relationship between biomass and functional diversity at a global scale but did not provide biomass values. on experimental savannah-grasslands, tilman et al. (1997) recorded biomass ranging from approximately 20 to 180 g m-2. in a study conducted by grace et al. (2016) the relationship between dry biomass and the number of taxa is negatively correlated, whereas in a study by fraser et al. (2015) the number of taxa increases until a biomass of about 100 g m-2 is reached, after which, in most cases, it decreases with any further increase in biomass. in contrast, on experimental grasslands (tilman et al. 1997, hector et al. 1999) the number of taxa and dry biomass are positively correlated. recently, research into the effect of biomass and the number of taxa on biodiversity is less frequent in published papers, whereas a growing importance is attributed to the dominant taxa and the composition of functional traits and functional diversity in determining species richness, community productivity and processes of ecosystem (diaz and cabido 2001, tredennick et al. 2016). some scientists claim that the value and range of functional traits define the interactions of taxa and the functioning of an ecosystem better than the actual richness of species (tilman et al. 1997, lepš et al. 2006, cadotte et al. 2009, van’t veen et al. 2020). functional traits are the morphological, physiological and phenological attributes of plants measured at the level of cells, organs, or organism (violle et al. 2007). they develop as an adaptation to environmental conditions, and, because of their ability to absorb the resources provided by the ecosystem, enable successful survival and reproduction of plants (lepš et al. 2006). functional traits of individual taxa are similar, but vary depending on environmental conditions, which makes them good indicators of the state of the ecosystem (lepš et al. 2006). functional diversity is defined by the range and value of functional traits. it is one of the components of biodiversity and as such affects the balance of nutrients in the soil, vegetation resistance and dynamics, stability and productivity of the ecosystem. in cases of f luctuations and disturbances greater functional diversity increases ecosystem stability because more numerous taxa provide a greater range of functional traits, which vary in their responses to different environmental conditions, thus making resource use more efficient (diaz and cabido 2001, tilman 2001). the relationship between species richness and functional diversity of natural grasslands is still quite unexplored. the assumption is that the more favourable the habitat conditions for plant growth, the more plants will be present because they will differ in adaptations for absorbing available resources, and these adaptations are quantified by functional trait values. therefore, greater diversity of functional traits generally implies a larger number of taxa in the community and vice versa (diaz and cabido 2001). grassland productivity/biomass research is scarce for croatia, and has mostly been conducted from the agricultural perspectives and so focused only on yield, i.e., biomass. the aim of this research was to determine the relationship between plant biomass, functional diversity and taxonomic diversity using quantitative analysis and floristic data. material and methods the research area was the city of zagreb, which is in the northwestern part of croatia. although surrounded by hills, most of the area of the city is located in the valley of the sava river where the soil is dominated by sand, clay and loam (anonymous 2008). the climate of the zagreb area belongs to the cfa climate type, according to köppen’s climate classification. it is a moderately warm humid climate with relatively mild winters, warm summers and no extremely dry periods. maximum precipitation occurs at the beginning of the warm part of the year and during late autumn (šegota and filipčić 2003). the average annual temperature is 11.4 °c and the average annual precipitation is 844 mm/m2 (nikolić and kovačić 2008). the climazonal vegetation of the entire northwestern part of croatia is forest, but, due to vegetation clearing to obtain pastures and arable land, forest now makes up only a third of the area. forests now predominate in elevated areas where agricultural development was not possible due to steep terrain (stančić 2000). grassland communities occupy a third of the area of northwestern croatia and are used for mowing and grazing. most grasslands at low and midaltitudes, along watercourses and on fertile, deep and mostly moist soils developed into communities of the molinioarrhenatheretea tx. 1937 class. a smaller number of grasslands consist of dry grasslands of the festuco-brometea br.-bl. et tx. ex soo 1947 class which develop only on dry upland basophilic ground (stančić 2000, nikolić 2020). the research was conducted at the peak of the vegetation season in june and july 2020 at eight different sites within the city of zagreb (tab. 1, fig. 1). criteria for locality selection were presence of grassland vegetation, homogenous vegetation cover on an area between 25 and 100 m2 and peak biomass of vegetation. methodology used was adapted according to alhamad (2006) and schumacher and roscher (2009). on each locality two 1 m2 plots were randomly selected and divided into four equal smaller squares of 0.25 m2. biomass was collected within two diagonally placed smaller squares, therefore, on a total of 0.5 m2 area due to the small capacity of the dryer in the laboratory. we collected the biomass by cutting the above ground vegetabiodiversity and biomass of grasslands in nw croatia acta bot. croat. 81 (2), 2022 223 tion and storing it in paper bags. biomass was weighed before and after drying at 70 °c for 72 hours. all vascular plant taxa present at the locality within the plots and outside the plots were recorded. the number of taxa within a 1 m2 plot is expressed as the number of taxa per plot. the number of taxa within both plots of one locality and outside the plots, but within the existing grassland vegetation, is expressed as the number of taxa per locality. some of the taxa were identified in the field, and some were collected, pressed and dried in order to be identified or additionally confirmed. to avoid reduction in the final measure of biomass, subsequently identified individuals were collected either within the plot from smaller squares on which biomass was not collected or from the nearby area surrounding the plot. for plant taxa identification excursion flora by nikolić (2019) was used, sometimes supplemented with additional identification keys and iconographies (jávorka and csapody 1991, domac 2002, eggenberg and möhl 2007, rothmaler and jäger 2007). nomenclature was given according to flora croatica database (nikolić tab. 1. list of localities with corresponding markings, coordinates (htrs96 / tm coordinate system), dates of fieldwork, altitudes and brief description. locality mark coordinates date altitude [m] locality description x coordinate y coordinate 1 466562 5084815 25. 6. 365 on a slope, s exposure 2 463938 5069645 2. 6. 120 northern side of the sava embankment, near savica lakes, on a slope, n exposure 3 457669 5071654 4. 6. 120 south side of the sava embankment, on level ground 4 456580 5070719 4. 6. 120 south side of the sava embankment, on a slope, n exposure 5 451870 5073306 25. 6. 120 industrial zone, on level ground 6 467447 5085184 25. 6. 280 surrounded by forest, on a slope, e exposure 7 463409 5078138 2. 7. 148 on level ground, by the štefanovec stream 8 464622 5078994 2. 7. 153 on level ground, abandoned agricultural land, by the trnava stream fig. 1. map of the city of zagreb with marked localities. justić m., jelaska s. d. 224 acta bot. croat. 81 (2), 2022 2020). the vegetation type was determined according to stančić (2000) and nikolić (2020). taxa determined at the genus level are not included in the analyses due to the impossibility of associating accompanying floristic data. life-form, origin, ellenberg ecological indicator values and functional trait values were attributed to each species of the checklist. data used in the preparation of the life-form spectrum were given according to the leda database (kleyer et al. 2008) and flora indicativa (landolt et al. 2010), and for doubtful taxa according to the flora croatica database (nikolić 2020). species origin was standardized according to biolflor (klotz et al. 2002) database and flora indicativa (landolt et al. 2010), while the invasiveness status in croatia was given according to flora croatica database (nikolić 2020). origin data were divided into the following four categories: au – autochthonous taxa, ar – archeophytes, ne – neophytes, and in – invasive alien taxa. data on ecological indicator values were obtained from ellenberg et al. (1992), borhidi (1995), pignatti et al. (2005) and landolt et al. (2010). for each locality and plot the most frequent values were calculated for the following environmental variables: l – light, m – moisture, n – nutrients, r – reaction, s – salinity and t – temperature. functional trait values were given according to the leda database (kleyer et al. 2008) for seed mass, leaf dry matter content (ldmc), specific leaf area (sla) and plant height. the functional trait values distribution test was performed in the statistica 13.3.0 (tibco software inc. 2017). in the case of the non-normal distribution, a logarithmic transformation was performed followed by another distribution test. using normally distributed functional traits the functional diversity index was calculated in the program canoco 5 (šmilauer and lepš 2014), defined as (lepš et al. 2006): 1 1 s s ij i ji j fd d p p = = = ∑ ∑ where s is the number of species in the community, pi and pj are the relative abundance of species i and j, and dij describes the functional dissimilarity between species i and j. the weighed values of fresh and dried biomass were doubled to represent the biomass per 1 m2 in order to enable a straightforward comparison with the literature, in which it is usually expressed per 1 m2. using weighed values, the percentage of dry biomass in the individual plots was calculated as well. the linear correlation between the measured variables was tested using the pearson correlation coefficient (with the significance level p < 0.05). correlation analysis was performed and graphically represented in statistica 13.3.0 (tibco software inc. 2017). results altogether 100 taxa (seven identified to the genus level, 92 species and one subspecies) were recorded in the flora of researched area (tab. 2). based on the recorded taxa, two classes of grassland vegetation were identified. taxonomic composition at locality 1 indicates the presence of bromion erecti koch 1926 alliance belonging to the festuco-brometea class, while at all other localities’ vegetation belongs to the arrhenatheretum elatioris association belonging to the molinio-arrhenatheretea class. regarding life-form spectrum, hemicryptophytes dominate on every locality, while other life-forms vary depending on the locality and plot. at locality 5, only hemicryptophytes were recorded. an increased share of chamaephytes was recorded on the plots of site 1, and of therophytes on the plots of site 3. phanerophytes are represented by only two species, both present with a small number of individuals (fig. 2). vegetation consists largely of autochthonous plants. among the 13 allochthonous taxa recorded, he most frequent are archaeophytes. neophytes are represented by two species: anthyllis vulneraria l. and lolium multiflorum lam. with l. multiflorum lam. comprising a significant part of the vegetation of both plots on the site 3. invasive alien taxa are also represented by two species: erigeron annuus (l.) desf. which was recorded on both plots of sites 3, 6 and 8 and on one plot of site 4 and echinocystis lobata fig. 2. life form spectrum for localities (h – hemicryptophytes, ch – chamaephytes, t – therophytes, g – geophytes, p – phanerophytes). numbers from 1-8 represents the localities were the research was conducted. biodiversity and biomass of grasslands in nw croatia acta bot. croat. 81 (2), 2022 225 tab. 2. list of recorded taxa with associated localities and plots. family taxa localities and plots amaryllidaceae allium sp. 8 apiaceae daucus carota l. 4, 4.2, 6, 6.2, 8 pastinaca sativa l. 2, 2.2, 5, 5.1 asteraceae achillea millefolium l. 1, 1.1, 2, 2.2, 3, 4, 4.1, 4.2, 8, 8.2 artemisia vulgaris l. 3, 3.2 bellis perennis l. 4, 4.2 buphthalmum salicifolium l. 1, 1.1, 1.2 centaurea jacea l. 2, 2.1 centaurea macroptilon borbás 2, 2.2, 5, 5.2, 8, 8.2 centaurea nigrescens willd. 5, 5.1, 6, 7 centaurea scabiosa l. 1, 1.2 cirsium arvense (l.) scop. 2, 2.2,7, 7.1, 8 erigeron annuus (l.) desf. 1, 3, 3.1, 3.2, 4, 4.2, 6, 6.1, 6.2, 7, 8, 8.1, 8.2 inula salicina l. 1 leucanthemum vulgare lam. 1, 1.2 tanacetum vulgare l. 3 boraginaceae symphytum officinale l. 2, 4 brassicaceae arabis hirsuta (l.) scop. 1, 1.1, 1.2 caryophyllaceae cerastium brachypetalum pers. 2, 2.2, 4, 4.2,7, 7.1 silene vulgaris (moench) garcke 1, 2, 3, 4, 4.2 cichoriaceae crepis biennis l. 2, 2.1, 3, 3.1, 5, 5.1, 5.2, 6 chondrilla juncea l. 3, 3.1, 3.2 picris hieracioides l. 6, 7, 7.2 tragopogon pratensis l. ssp. orientalis (l.) čelak. 3 clusiaceae hypericum perforatum l. 4, 4.1 convolvulaceae calystegia sepium (l.) r. br. 2, 2.1 convolvulus arvensis l. 2, 3, 3.2, 6, 6.2, 7, 7.1, 7.2, 8, 8.1, 8.2 cucurbitaceae echinocystis lobata (michx.) torr. et a. gray 2 cyperaceae carex distans l. 1, 1.2 carex spicata huds. 7, 7.2, 8 dipsacaceae knautia drymeia heuff. 2 knautia arvensis (l.) coult. 2, 2.2, 4 scabiosa triandra l. 1, 1.2 equisetaceae equisetum arvense l. 2, 2.1, 2.2, 4, 4.1, 4.2, 8, 8.2 euphorbiaceae euphorbia esula l. 6, 6.2 fabaceae anthyllis vulneraria l. 1, 1.1, 1.2, 4 coronilla varia l. 1, 1.1, 3, 3.2, 4, 4.1, 6 dorycnium herbaceum vill. 1, 1.1, 1.2 lathyrus hirsutus l. 4, 4.2 lathyrus nissolia l. 8, 8.1, 8.2 lathyrus pratensis l. 1, 1.1, 6, 6.1 lembotropis nigricans (l.) griseb. 1 lotus corniculatus l. 2, 4, 4.1, 6, 6.2, 7, 7.2, 8 medicago lupulina l. 1, 1.1, 1.2, 3, 3.1, 6, 6.2 medicago sativa l. 3, 5, 5.1, 7, 7.1 trifolium campestre schreb. 3, 3.1, 3.2 trifolium pratense l. 2, 3, 3.2, 4, 4.1, 5, 5.1, 5.2, 7, 7.1 trifolium repens l. 3, 3.1, 3.2, 5, 5.2, 6, 6.1, 6.2 vicia angustifolia l. 3, 3.1, 3.2 vicia cracca l. 1, 2, 2.1, 2.2, 4, 4.2, 6, 7, 7.1, 8, 8.1, 8.2 vicia hirsuta (l.) gray 8, 8.1, 8.2 lamiaceae clinopodium vulgare l. 2, 2.1, 2.2, 4, 6, 6.1, 6.2 glechoma hederacea l. 5, 5.1, 7, 7.1, 8, 8.1, 8.2 salvia pratensis l. 2, 2.2, 3, 4, 4.1, 4.2, 6, 6.1 salvia verticillata l. 1 teucrium chamaedrys l. 1, 1.1, 4 thymus pulegioides l. 1, 1.1, 1.2, 4, 4.1 justić m., jelaska s. d. 226 acta bot. croat. 81 (2), 2022 linaceae linum catharticum l. 1, 1.2 orchidaceae anacamptis pyramidalis (l.) rich. (nt) 1 plantaginaceae plantago lanceolata l. 1, 1.1, 2, 2.1, 2.2, 3, 3.1, 3.2, 5, 5.1, 5.2, 6, 6.1 plantago major l. 5, 5.1 plantago media l. 1, 1.1, 3, 3.1, 4, 4.1, 4.2, 6, 6.1, 6.2 poaceae arrhenatherum elatius (l.) j. presl et c. presl 2, 2.1, 2.2, 3, 3.1, 6, 6.1, 6.2, 8 brachypodium pinnatum (l.) p. beauv. 1, 1.1, 1.2, 2, 2.1, 4, 4.1 briza media l. 1, 1.1, 1.2, 2, 4, 4.1, 7, 7.1 bromus erectus huds. 1, 1.1, 1.2, 2, 2.2, 4, 4.1, 4.2, 6, 6.1 bromus racemosus l. 3, 3.1, 3.2, 4, 4.2 calamagrostis epigejos (l.) roth 7, 7.1, 7.2, 8 dactylis glomerata l. 1, 1.2, 2, 2.1, 2.2, 3, 3.2, 4, 4.2, 6, 6.2, 7, 7.1, 7.2, 8, 8.1, 8.2 elymus hispidus (opiz) melderis 1 elymus repens (l.) gould 3, 8, 8.1 festuca pratensis huds. 2, 2.1, 3, 4, 4.1, 4.2, 7, 7.1, 7.2, 8, 8.1, 8.2 festuca rubra l. 4, 4.1, 4.2, 6, 6.1, 7, 7.1, 7.2 holcus lanatus l. 2, 2.1, 2.2, 7, 7.1, 7.2, 8, 8.1, 8.2 hordeum murinum l. 3, 3.1 lolium multiflorum lam. 3, 3.1, 3.2 lolium perenne l. 5, 5.1 lolium sp. 3, 3.1, 5, 5.1, 5.2 poa pratensis l. 3, 3.1, 3.2, 4, 4.1, 6, 6.1, 6.2, 7, 7.1, 7.2, 8, 8.1 trisetum flavescens (l.) p. beauv. 2, 4, 4.1, 4.2, 6, 6.1 polygonaceae rumex crispus l. 8 rumex obtusifolius l. 3 primulaceae lysimachia nummularia l. 2, 2.1, 8, 8.2 primula vulgaris huds. 2, 2.1, 2.2 ranunculaceae clematis vitalba l. 3, 3.1, 7, 7.1 ranunculus acris l. 2, 2.1, 2.2 ranunculus bulbosus l. 8, 8.2 ranunculus sp. 1, 1.2, 4, 4.1, 4.2 rosaceae agrimonia eupatoria l. 1, 1.2, 4, 4.1, 4.2, 6, 8, 8.1, 8.2 potentilla reptans l. 2, 2.1, 2.2, 5, 5.1, 5.2, 8, 8.1, 8.2 potentilla sp. 3, 3.2, 6, 6.2 rubus sp. 2, 2.1, 7, 7.2, 8, 8.1 sanguisorba minor scop. 1, 1.1, 1.2, 4, 4.1, 4.2 rubiaceae galium lucidum all. 2, 2.1, 2.2 galium mollugo l. 4, 4.1, 4.2, 5, 5.1, 5.2, 6, 6.1, 7, 7.2, 8, 8.1, 8.2 galium verum l. 1, 1.1, 6, 6.1, 6.2, 7, 7.1, 7.2 scrophulariaceae melampyrum barbatum willd. 1, 1.2 veronica chamaedrys l. 6, 6.1, 6.2, 7, 7.1, 7.2, 8, 8.2 veronica sp. 4, 4.1 violaceae viola sp. 1, 1.2, 2, 2.1, 2.2, 4, 4.2 fig. 3. percentages of autochthonous (au) and allochthonous (ar – archaeophytes, ne – neophytes, in – invasive alien) taxa for each plot (decimal numbers). tab. 2. continued biodiversity and biomass of grasslands in nw croatia acta bot. croat. 81 (2), 2022 227 (michx.) torr. et a. gray, which was recorded only outside the plots at site 2 (fig. 3). according to the analysis of ellenberg ecological indicator values most of the studied area is dominated by half-light plants. locality 1 differs from others given the lower indicator values for moisture and nutrients and higher values for reaction. other localities have higher values for moisture or nutrients, and the reaction is lower, corresponding to the more acidic soil in the sava valley (tab. 3). the highest functional diversity index was calculated for localities 1, 2 and 8 and for plots 7.1, 8.1 and 8.2; while the lowest functional diversity was recorded for locality 5 and its associated plot 5.2 (tab. 4). the highest values of fresh biomass were recorded on localities 3 and 5, and of dry biomass on locality 6. fresh biomass is the lowest on plots of localities 1 and 4, while the minimum of dry biomass was recorded on plots of locality 4. the largest dry biomass share belongs to the vegetation of locality 1, whereas the lowest share is in the vegetation of locality 3. the taxonomically richest grasslands were on localities 2 and 4, while the poorest was recorded on locality 5 (tab. 4). our results show that fresh biomass and the number of taxa are negatively correlated, while the correlations of dry biomass and percentages of dry biomass with the number of taxa are not shown as they are not statistically significant. a statistically significant positive correlation between the number of taxa and functional diversity was recorded. therefore, the greater the number of taxa, the greater the functional diversity. functional diversity is negatively correlated with fresh biomass and positively with the percentage of dry biomass, but it is not correlated with dry biomass. although neither dry nor fresh biomass correlated with the mean values of functional traits, the calculation proved a statistically significant positive correlation between the percentage of dry biomass and ldmc. also, there is a positive correlation between the mean values of ldmc and the number of taxa (r = 0.5617, p = 0.024) and a negative correlation between the mean values of sla and the number of taxa (fig. 4). tab. 3. the most frequent ellenberg ecological indicator values for light (l), moisture (m), nutrients (n), reaction (r), salinity (s) and temperature (t) for each locality (single number) and plot (decimal numbers). localities and plots most frequent values l m n r s t 1 7 4 3 7 0 5 1.1 7 4 3 8 0 5 1.2 7 4 3 8 0 5 2 7 6 5 6 0 5 2.1 7 5 4 6 0 5 2.2 7 4 5 6 0 5 3 7 4 5 7 0 5 3.1 7 4 5 6 0 5 3.2 7 4 5 6 0 6 4 7 4 3 7 0 6 4.1 7 4 3 6 0 5 4.2 7 5 4 7 0 5 5 8 4 5 6 0 6 5.1 8 4 5 6 0 6 5.2 7 5 5 6 0 5 6 7 4 4 7 0 5 6.1 7 5 6 7 0 5 6.2 7 4 6 7 0 5 7 7 5 6 6 0 5 7.1 7 5 5 7 0 5 7.2 7 5 6 7 0 5 8 7 5 4 7 0 5 8.1 7 6 6 6 0 6 8.2 7 6 5 7 0 6 tab. 4. functional diversity index (fd(rao)), plant biomass before and after drying, percentages of dry biomass and number of taxa for localities (single numbers) and plots (decimal numbers). localities and plots fd (rao) fresh biomass (g m-2) dry biomass (g m-2) % of dry biomass number of taxa 1 2.073 35 1.1 1.840 944.8 391.0 54.0 17 1.2 1.893 958.4 414.4 55.3 20 2 2.047 36 2.1 1.851 1241.0 350.0 40.6 19 2.2 1.905 1031.4 315.8 44.5 20 3 1.728 31 3.1 1.667 1615.2 387.4 34.5 16 3.2 1.392 1597 381.0 34.5 15 4 1.785 36 4.1 1.802 925.4 268.2 44.5 21 4.2 1.732 964.4 251.4 41.7 22 5 1.509 14 5.1 1.509 1695.6 361.2 31.7 12 5.2 1.121 1196.2 309.4 39.1 8 6 1.810 28 6.1 1.694 1374.6 561.8 50.2 15 6.2 1.579 1457.4 585.6 49.2 15 7 1.818 24 7.1 2.060 1023.8 432.0 53.9 17 7.2 1.562 1426.0 586.8 50.2 14 8 1.906 29 8.1 1.965 995.4 435.2 55.3 15 8.2 1.997 1224.2 511.2 51.9 18 justić m., jelaska s. d. 228 acta bot. croat. 81 (2), 2022 discussion plant biomass is associated to grassland vegetation type. grasslands of the molinio-arrhenatheretea class develop at low to medium altitudes, and near watercourses, which is why they are the most productive grasslands of temperate europe. for this reason, they are used as fodder for cattle, and, to additionally increase their biomass, are often fertilized. the plant biomass of dry grassland of the festucobrometea class at locality 1 is lower due to more extreme fig. 4. relationships between biomass, number of taxa, functional diversity and mean values of functional traits measured at the plot level. (a) a decrease in fresh biomass is followed by an increase in the number of taxa per plot (p = 0.026). (b) an increase in functional diversity follows an increase in the number of taxa per plot (p = 0.002). (c) fresh biomass increases as the functional diversity of the plots decreases (p = 0.022). (d) an increase in the percentage of dry biomass is followed by an increase of functional diversity (p = 0.027). (e) as the ldmc mean value of the leaves increases, so does the percentage of dry biomass (p = 0.006). (f) the number of taxa on a plot is higher when the sla mean value is lower (p = 0.005) biodiversity and biomass of grasslands in nw croatia acta bot. croat. 81 (2), 2022 229 conditions such as nutrient low substrate and lower temperatures. the dominance of hemicryptophytes in the vegetation cover was expected given the temperate climate of the study area (horvat 1949). the main cause for the high share of chamaephytes at locality 1 is the vegetation type (festucobrometea) since low shrubs, which better tolerate cold, drought and a small amount of nutrients in the soil thrive better in such communities (horvat 1949). the presence of phanerophytes is most likely due to dispersion from the nearby forests. it is expected that locality 3, which is closest to the city centre, is under the greatest anthropogenic influence. anthropogenic activity is a cause of disturbances and in such conditions annual plants with high seed productivity, such as therophytes, thrive best. the high share of autochthonous taxa in the study area indicates a still relatively weak anthropogenic influence and habitat stability. even though they make up a relatively small share in the composition of taxa, the presence of neophytes lolium multiflorum lam. and the invasive erigeron annuus (l.) desf. should be emphasized because, in addition to being quite numerous they are robust plants and therefore make up a significant part of the plant biomass. in addition, due to their exceptional ability of colonization they represent a potential threat to future ecosystem stability. ecological indicator values for light indicate most plants recorded at the localities grow in habitats frequently exposed to sunlight, for the researched area consists of open habitats such as grasslands. other values such as moisture, nutrients and reaction vary according to different management type, soil type, geographical position, slope, proximity to water and so on. therefore, the observed variations in plant indicator values reflect the environmental conditions of the different types of grasslands. at locality 1, the ecological indicator values of the belonging taxa indicate the presence of dry and basophilic grassland of the festucobrometea class. values calculated for other localities are characteristic for grasslands of molinio-arrhenatheretea class which grow at medium to low altitudes on more acidic soils near watercourses. plant taxa were recorded only once, in the fieldwork before the vegetation was cut, thus those taxa that appear earlier or later in the growing season are not included in the research. this resulted in a smaller number than the expected approximate amount of 37 taxa per locality as found by stančić (2000), who used the relevé method, which can include multiple field visits during the season. in this research, a statistically significant negative correlation between biomass and the number of taxa coincided with the results of fraser et al. (2015) and grace et al. (2016) but was obtained only with fresh biomass. the reason for this is the much larger range of fresh biomass values compared to the values of dry biomass, while the small range of dry biomass values is a consequence of one dominant grassland type with uniform life forms. for the results to coincide with those of fraser et al. (2015) and grace et al. (2016), the research should be further extended to different types of grasslands, which would increase the range of values of both species and dry biomass. a positive correlation between the number of taxa and dry biomass on experimental grasslands in a study conducted by tilman et al. (1997) is most probably a consequence of the low biomass which, according to the “hump-backed” model by fraser et al. (2014), at these values, increases with increasing number of taxa. a positive correlation was also obtained in a research by hector et al. (1999) where the values of biomass are higher. a possible explanation for such results is a different composition of vegetation on experimental grasslands as it was created by random selection of taxa while the impact on them is minimal because environmental factors are uniform. that means that the vegetation composition on experimental grasslands is not conditioned by long-term impact of abiotic and biotic factors. such research should be re-conducted in the context of natural grassland ecosystems so, for each community, the characteristic environmental factors influencing the vegetation and the properties of the vegetation influencing the environmental factors could be considered. in this research, the number of taxa is generally lower on localities with higher biomass. this may be due to disturbances such as human activities because anthropogenic habitats usually have more fertile soils which enable faster growth and higher habitus of plants. larger plants overshadow the smaller ones and thus prevent their germination resulting in dominance of several larger taxa (grace et al. 2016). the plots of locality 5 are dominated by few large plant taxa that overshadow the smaller plants, resulting in a reduction of the number of taxa to only 8 and 12 per plot. furthermore, the invasive taxon erigeron annuus (l.) desf. on localities 3, 6 and 8 is a possible cause of high biomass and a small number of taxa due to its large habitus and the ability successfully to colonize disturbed habitats. habitats exposed to constant disturbance are less stable and more susceptible to colonization by invasive species which also modify habitat conditions by increasing the amount of nutrients in the soil (wu et al. 2018). the highest biomass recorded on the plots of locality 3 could be a possible consequence of the large number of legumes (fabaceae) which enrich the soil with nutrients by fixing atmospheric nitrogen, which then increases the growth of all plants on a locality. the positive correlation between the number of taxa and the functional diversity coincides with the theory that in favourable conditions plant species differ more from each other according to their functional traits that allow them to make better use of the available resources. therefore, a greater diversity of functional traits, in most cases, including grasslands, means a greater number of taxa in the community. the relationship found here between fresh biomass and functional diversity is expected given the previously presented results because the number of taxa decreases as biojustić m., jelaska s. d. 230 acta bot. croat. 81 (2), 2022 mass increases and is positively correlated with functional diversity. thus, functional diversity also decreases as biomass increases. the reason is that biomass is generally higher in habitats with a few dominant taxa possessing a greater number of similar functional traits. however, the results of other research do not coincide with our results. tilman et al. (1997) and zhang et al. (2016) investigated dry grasslands, savannah, and mongolian steppes, respectively. drought conditions are better tolerated by grassland plants with a greater number of similar functional trait values (e. g. lower sla reduces transpiration, lower plant height due to insufficient water), which means that the functional diversity on dry grasslands is lower than on mesic and wet grasslands. also, dry grasslands have a lower biomass, so, given the “hump-backed” model, it is to be expected that the increase in biomass will be accompanied by an increase in the number of taxa, and accordingly by an increase in the value of functional diversity. a study on european grasslands conducted by petchey et al. (2004) is not comparable because it took as its subject an experimental grassland with a manipulated composition of taxa. this means that the vegetation composition was not a result of natural processes and that the functional traits of the associated taxa did not develop as a result of long-term effects of biotic and abiotic factors on the habitat. positive correlations of the percentage of dry biomass with functional diversity and ldmc are associated, given that both the percentage of dry biomass and ldmc express the ratio of the mass of dry and fresh (water-saturated), plant material. the percentage of dry biomass expresses the average density of plant tissue which indicates higher soil fertility, i. e. high concentrations of nitrogen in the soil (hodgson et al. 2011). therefore, the more favourable the habitat conditions, the greater the functional and taxonomic diversity. the reciprocal function of ldmc is the sla (hodgson et al. 2011). thus, as the number of taxa increases with increasing ldmc then it is to be expected that the number of taxa will decrease with increasing sla. the reason for this is that leaves with a large surface area overshadow the plants growing beneath them and in this way prevent their growth and germination. such a situation was recorded at locality 5, where the highest value of sla and the lowest number of taxa were recorded. the most stable habitat in this research is locality 2 because it has one of the highest functional diversity indices and the highest taxonomic diversity, composed of exclusively autochthonous taxa. also, the ecological indicator values reflect the environmental conditions of the area and do not show any extremes. localities 1 and 7 also emerge as some of the more stable grasslands whose composition of life-forms and ecological indicator values describe well the characteristics of the belonging vegetation communities. th high functional and taxonomic diversity at both localities confirm that these are undisturbed habitats without the dominance of a few aggressive taxa. the most unstable habitat is locality 3, which, despite its high taxonomic diversity, has one of the lowest values of functional diversity. it also has the smallest share of autochthonous taxa and the maximum share of allochthonous taxa with invasive alien taxa on both plots, which, together with a large share of therophytes indicate a ruderal habitat. unlike locality 3, locality 5 is yet unstable because of its low both taxonomic and functional diversity. even though it does not contain any invasive alien taxa, the anthropogenic impact on the habitat is expressed in the larger proportion of thermophilic plants, which is due to the proximity of the city and the increased anthropogenic impact. such an impact resulted in the dominance of several competitive taxa (centaurea macroptilon borbás, lolium perenne l., medicago sativa l., trifolium pratense l.), which caused the biomass of plot 5 to be one of the highest measured. the 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oikos 116, 882–892. willig, m.r., 2011: biodiversity and productivity. science 333, 1709–1710. wu, a., liu, j., he, f., wang, y., zhang, x., duan, x., liu, y., qian, z., 2018: negative relationship between diversity and productivity under plant invasion. ecological research 33, 949–957. zhang, q., buyantev, a., yonghong li, f., jiang, l., niu, j., ding, y., kang, s., ma, w., 2016: functional dominance rather than taxonomic diversity and functional diversity mainly affects community aboveground biomass in the inner mongolia grassland. ecology and evolution 7, 1605–1615. opce-str.vp acta bot. croat. 69 (2), 183–197, 2010 coden: abcra 25 issn 0365–0588 transgenerational stress memory in arabidopsis thaliana (l.) heynh.: antioxidative enzymes and hsp70 katarina ]uk1, marko gogala2, mirta tkalec3, @eljka vidakovi]-cifrek3* 1 molecular epidemiology group, german cancer research center (dkfz), im neuenheimer feld 581, d-69120 heidelberg, germany; molecular biology of breast cancer group, university women's clinic, voßstraße 9, d-69115 heidelberg, germany 2 gene center munich and center for integrated protein science, department of chemistry and biochemistry, ludwig-maximilians-universität münchen, feodor-lynen-strasse 25, d-81377 munich, germany. 3 university of zagreb, faculty of science, division of biology, department of botany, rooseveltov trg 6, hr-10000 zagreb, croatia transgenerational transmission of information about stress exposure is manifested as an increase in the somatic homologous recombination frequency in plants. our aim was to investigate whether information about changes of antioxidative enzyme activities and protein hsp70 induction are also transmitted in response to stress caused by uv-c irradiation. these stress indicators were investigated in arabidopsis thaliana plants exposed to uv-c irradiation (6 and 600 j m–2) and its non-irradiated progeny. the activity of catalase was significantly decreased in the irradiated plants in comparison to the non-irradiated control plants, while the activity of guaiacol peroxidase was increased. the ascorbate peroxidase activity was not significantly changed. in irradiated plants there was an induction of a new hsp70 protein isoform. in the non-irradiated progeny of irradiated plants, a significant decrease in catalase and ascorbate peroxidase activity was noticed in comparison to plants whose parents were not irradiated. there was no significant change in guaiacol peroxidase activity or induction of hsp70 isoforms in the progeny. the obtained results indicate that, besides the already known increase in frequency of somatic homologous recombination, transmission of information about stress exposure can also include changes in activities of antioxidative enzymes catalase and ascorbate peroxidase. the explanations for the observed changes and the mechanism by which they occur have to be established in further research. keywords: arabidopsis thaliana, uv-c, stress memory, antioxidative enzyme, catalase, ascorbate peroxidise, guaiacol peroxidise, hsp70 abbreviations: apx – ascorbate peroxidase, cat – catalase, pod – peroxidase, hsp – heat shock protein, page – polyacrylamide gel electrophoresis, ros – reactive oxygen species, shr – somatic homologous recombination acta bot. croat. 69 (2), 2010 183 * corresponding author, e-mail: zcifrek@zg.biol.pmf.hr u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:20 color profile: disabled composite 150 lpi at 45 degrees introduction as sessile organisms, plants have to cope with various environmental stresses such as drought, soil salinity, low or high temperature, excess light, toxic substances and pathogen attack. plant response to stress conditions includes not only changes in physiological processes, regulation of cellular metabolism and gene expression but also alterations of the dynamics of the genome. in the last decade several studies demonstrated an increased rate of somatic homologous recombination (shr) in response to various stress factors, e. g. uv irradiation, herbicides, osmotic stress and high temperature (lebel et al. 1993; ries et al. 2000a, b; lucht et al. 2002; molinier et al. 2006; pecinka et al. 2009). uv irradiation affects plants at several levels. dna damage causes heritable mutations, while disturbances of physiological processes (stapleton 1992) reduce growth, development, photosynthesis, flowering, pollination and transpiration (rozema et al. 1997, jansen et al. 1998). in order to minimize the effects of uv irradiation plants activate different physiological responses, such as the biosynthesis of protecting compounds (stapleton 1992), the reactive oxygen species (ros) scavenging system (xu et al. 2008) and dna repair mechanisms (molinier et al. 2005). one of the most important dna repair mechanism is shr (ries et al. 2000b). molinier et al. (2006) noticed an elevation of its frequency in arabidopsis thaliana (ecotype columbia) not only in plants exposed to uv-c irradiation and flagellin (an elicitor of defence mechanisms to pathogen attack) but also in four generations of their progeny, which were not exposed to irradiation themselves. for this transgeneration »stress memory« the authors suggested an epigenetic mechanism. epigenetic changes are mitotically and/or meiotically heritable alterations in the chromatin structure, which occur without a change in the dna sequence. they are mediated by changes in dna methylation, histone modifications and by non-coding rnas, which can cause changes in the chromatin architecture or dna methylation patterns (bond and finnegan 2007). some physiological effects related to epigenetic regulation in plants have already been investigated and described, like the process of vernalization (burn et al. 1993, bond and finnegan 2007, schmitz and amasino 2007). most of the stress-induced epigenetic modifications are reset to the basal level once the stress is relieved, while some of them may be stable, that is, may be inherited as »stress memory« across mitotic and even meiotic cell divisions (chinnusamy and zhu 2009). oxidative stress in plant cells, as a consequence of the accumulation of reactive oxygen species (ros) such as superoxide radical, hydroxyl radical, hydrogen peroxide, singlet oxygen and organic hydroperoxides is one of the earliest responses to most, if not all, biotic and abiotic environmental stress conditions, among them uv irradiation. if not scavenged, ros can damage plants by oxidizing the proteins, nucleic acids, photosynthetic pigments and membrane lipids. however, ros can also act as signalling molecules and trigger a range of cellular responses important for stress tolerance. tight control of ros levels in cells is achieved by the antioxidative defence system including various antioxidants (mittler 2002). superoxide dismutase catalyses the dismutation of superoxide into h2o2 and molecular oxygen. catalases (cat; e.c.1.11. 1.6.), the main h2o2-scavenging enzymes in plants, convert h2o2 to water and molecular oxygen, but have low substrate affinity. in addition to having a role in lignin biosynthesis and indole-3-acetic acid (iaa) degradation, 184 acta bot. croat. 69 (2), 2010 ]uk k., gogala m., tkalec m., vidakovi]-cifrek @. u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:20 color profile: disabled composite 150 lpi at 45 degrees peroxidases (pod; e.c.1.11.1.7) convert h2o2 to water using different organic cellular substrates. under in vitro conditions they can use a wide range of artificial electron donors, e.g. guaiacol or pyrogallol, so they are usually referred as guaiacol peroxidases. ascorbate peroxidase (apx; e.c.1.11.1.11.) is the key enzyme of the ascorbate cycle, as it eliminates h2o2 by converting ascorbic acid to monodehydroascorbate. in addition to antioxidative enzymes, the antioxidative system also includes non-enzymatic antioxidants such as ascorbic acid, glutathione, a-tocopherol and carotenoids. there is much evidence showing that the activities of antioxidative enzymes, their isoenzyme patterns and amounts of antioxidants are changed by various stress conditions (gaspar et al. 1991, alscher et al. 1997, dat et al. 2000, arora et al. 2002, mittler 2002). all organisms respond to excess heat by inducing the synthesis of a group of evolutionarily conserved proteins known as the heat shock proteins (hsps). most hsps are expressed constitutively, but in plants their expression can also be additionally induced and/or enhanced by various environmental stress conditions. the correlations between hsp synthesis and stress response led to the assumption that these proteins protect cells from the destructive effects of stress conditions. they act as molecular chaperones and are involved in protein folding, unfolding, assembly and disassembly (vierling 1991). as already mentioned, the mechanism of the transfer of epigenetic information between generations could provide a »memory« of environmental stresses experienced in earlier generations and has an important ecological role in preparing the progeny for potentially harmful conditions (molinier et al. 2006). therefore, we wanted to investigate whether there is a possibility that the transmission of information about exposure to uv-c stress from irradiated plants to their non-irradiated first generation progeny includes changes in antioxidative enzyme activities (cat, apx and pod) and the induction of the heat shock protein hsp70. for this purpose arabidopsis thaliana plants were exposed to uv-c irradiation doses of 6 and 600 j m–2 and the antioxidative enzyme activities and hsp70 protein induction were investigated in treated plants as well as in their non-irradiated progeny. materials and methods plant material, growth conditions and uv-c treatments arabidopsis thaliana seeds (ecotype columbia) were germinated in the soil and incubated in a growth chamber at 24 ± 2 °c with a photoperiod of 16 h light/8 h darkness under fluorescent white light (35–40 mmol m–2 s–1) for a period of five weeks. prior to entering the flowering phase the plants were divided into three groups. the first one was the non-irradiated control group, while the second and third were irradiated with uv-c doses of 6 j m–2 and 600 j m–2, respectively. the plants were irradiated with uv-c lamps (osram, germany), l = 254 nm, for time intervals previously determined with an uv radiometer (vlx-3w, france) to achieve irradiation doses of 6 and 600 j m–2. half of the plants of each group were allowed to continue growth, pass the flowering phase and produce seeds. the rest of the plants of each group were used for the preparation of protein extracts. plants exposed to a 600 j m–2 dose were immediately taken for protein extraction, while the plants exposed to a 6 j m–2 dose were first kept in the dark for 30 minutes before protein extraction to elicit a response, since the uv-c treatment itself was very short. acta bot. croat. 69 (2), 2010 185 antioxidative enzymes and hsp70 in stress memory u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:20 color profile: disabled composite 150 lpi at 45 degrees seeds produced by the first, second and third group were collected and planted. five week old progeny of irradiated as well as control plants were used for protein extraction, without ever being irradiated themselves. enzyme assays and isoenzyme analysis plant extracts were prepared for the determination of ascorbate peroxidase (apx), guaiacol peroxidase (pod) and catalase (cat) activities as well as isoenzyme analyses by gel electrophoresis. the plants (100 mg) were homogenized in 1 ml of a cold 50 mm potassium phosphate buffer (ph 7.0), containing 1 mm ascorbate and 5% (w/v) polyvinylpyrrolidone (pvp). the homogenate was centrifuged at 20,000 g for 30 minutes at 4 °c and the supernatant was used for enzyme assays. apx activity was determined by the decrease in absorbance at l = 290 nm (e = 2.8 mm–1 cm–1), as described by nakano and asada (1981) and expressed as mmol of oxidised ascorbate per minute per gram of total soluble proteins. the reaction mixture consisted of a 50 mm potassium phosphate buffer (ph 7.0), 0.5 mm ascorbate, 5 mm h2o2 and 120 ml of an enzyme extract. cat activity was assayed by measuring the decrease in absorbance at l = 240 nm (e = 36 mm–1 cm–1) according to aebi (1984) and expressed as mmol of decomposed h2o2 per minute per gram of total soluble proteins. the reaction mixture consisted of a 50 mm potassium phosphate buffer (ph 7.0), 20 mm h2o2 and 50 ml of an enzyme extract. pod activity was determined by the increase in absorbance at l = 470 nm (e = 26.6 mm–1 cm–1) according to chance and maehly (1955) and expressed as mmol of oxidised guaiacol per minute per gram of total soluble proteins. the reaction mixture consisted of a 50 mm potassium phosphate buffer (ph 7.0), 10 mm h2o2, 18 mm guaiacol and 20 ml of an enzyme extract. the protein content in the enzyme extracts was determined by a dye-binding technique (bradford 1976) using bovine serum albumin as a protein standard. for isoenzyme analysis, vertical page according to laemmli (1970) without sodium dodecylsulfate (sds) was performed on 12% (w/v) polyacrylamide gels at 4 °c. approximately equal amounts of proteins, 30–35 mg per well, were loaded. for apx, the gel was pre-run for 30 min before the samples were loaded and ascorbate was added to the electrode buffer. for apx detection the gels were stained by the protocol according to mittler and zilinskas (1993), for cat detection according to woodbury et al. (1971) and for pod according to chance and maehly (1955). protein extraction and hsp70 expression the plants (50 mg) were homogenized in 1 ml of a cold 0.1 m tris–hcl buffer (ph 8.0) (staples and stahmann 1964) containing 5% (w/v) pvp and centrifuged at 20,000 g for 30 minutes, at 4 °c. the soluble protein content was determined according to bradford (1976). protein extracts were mixed with an equal volume of sample loading buffer (laemmli 1970) and separated by 10% (w/v) sds-page. after electrophoresis, one gel was electro-transferred to a nitrocellulose membrane in a mini trans-blot cell (biorad, germany). the other one was stained with coomassie blue and served as an internal control of equal loading. after overnight incubation with primary antibodies (1:1000) against pea hsp70 (raised in rabbits), the protein blots were probed 186 acta bot. croat. 69 (2), 2010 ]uk k., gogala m., tkalec m., vidakovi]-cifrek @. u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:20 color profile: disabled composite 150 lpi at 45 degrees with alkaline phosphatase-conjugated anti-rabbit igg antibodies (1:2000) and detected with bromo-4-chloro-3-indolyl phosphate/nitrotetrazolium blue chloride (bcip/nbt) as a substrate. statistics the results were calculated as the mean value of at least six replicates ± standard error. statistical analysis was performed using the statistica 7.1 (statsoft, inc., usa) software package. significant differences between mean values were established by one-way anova followed by duncan’s multiple range test. differences were considered to be significant at p < 0.05. enzyme activities staining after page and immunoblotting after sds-page were repeated at least two times. results antioxidative enzyme activities and hsp70 induction were determined in plants irradiated with uv-c doses of 6 and 600 j m–2 and corresponding non-irradiated control plants (p generation), as well as in their non-irradiated progeny (f1 generation). besides the changes in antioxidative enzyme activities and the occurrence of a new hsp70 isoform, irradiated plants showed symptoms of tissue damage and stunted growth, which were more prominent in plants irradiated with a higher uv-c dose (600 j m–2). in the progeny of plants irradiated with a 6 j m–2 dose we observed an earlier progression to the flowering phase than in the progeny of plants exposed to an irradiation dose of 600 j m–2 and the non-irradiated (control) plants. antioxidative enzymes when cat activity was monitored, a statistically significant decrease of enzyme activity in the plants exposed to uv-c irradiation was observed. cat activity of the parental plants exposed to an uv-c dose of 6 j m–2 was 3.5 times lower, while plants exposed to an uv-c dose of 600 j m–2 showed a 5.5 times lower activity in comparison to the control plants. the progeny of the control plants did not show a significantly decreased catalase activity in comparison to the parental generation, while the progeny of plants exposed to an uv-c dose of 6 j m–2 showed a significantly decreased catalase activity in comparison to the progeny of the control plants. exposure of plants to a dose of 600 j m–2 did not cause a significant change in catalase activity in their progeny (figure 1a). results obtained from the detection of the catalase activity in gel after separation of proteins by gel electrophoresis in native conditions were in accordance with the activities measured spectrophotometrically: one catalase isoenzyme (marked k1) was present in extracts of both irradiated and non-irradiated parental plants. it is also evident that its activity was strongly decreased in plants irradiated with uv-c doses of 6 and 600 j m–2. the same band of particularly similar activity as in the corresponding control plants was detected in plant extracts of the progeny of plants exposed to both investigated irradiation doses (fig. 1b). although apx activities of parental plants exposed to uv-c irradiation doses of 6 and 600 j m–2 were not significantly decreased in comparison to control plants, their progeny acta bot. croat. 69 (2), 2010 187 antioxidative enzymes and hsp70 in stress memory u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:21 color profile: disabled composite 150 lpi at 45 degrees showed significantly decreased activities. this decrease was more evident in the progeny of plants exposed to a uv-c dose of 6 j m–2 (fig. 2a). in plant extracts of non-irradiated and irradiated parental plants two distinct ascorbate peroxidase isoenzymes (a1 and a2) were detected. the isoenzyme a1 was present in extracts of both, non-irradiated and irradiated plants, whereas a2 was seen only in non-irradiated plants and plants irradiated with a lower uv-c dose (6 j m–2). furthermore, the a2 isoenzyme band was evidently weaker in irradiated plants. the progeny of non-irradiated (control) and irradiated plants also showed two ascorbate peroxidase isoenzymes (a1 and a2). the progeny of the exposed plants showed activity of the isoenzyme a1 and a2 less prominent than in the extracts from the corresponding control plants (fig. 2b). the pod activity was significantly increased in the parental plants exposed to uv-c irradiation in comparison to the non-irradiated control plants. the plants exposed to a dose of 6 j m–2 showed a 1.8 times higher and the ones exposed to a 600 j m–2 dose a 2.4 times higher activity. in the progeny of irradiated plants, the pod activity did not differ significantly from the activity of the corresponding control plants (fig. 3a). by gel-electrophoresis two guaiacol peroxidase isoenzymes (g1 and g2) were resolved. the isoenzyme g1 was detected in both the extracts of non-irradiated (control) and irradiated plants, its band being more prominent in irradiated plants, especially in those exposed to a higher uv-c 188 acta bot. croat. 69 (2), 2010 ]uk k., gogala m., tkalec m., vidakovi]-cifrek @. a b b a b ab 0 0.05 0.1 0.15 0.2 control +uv –uv 6 j m +uv –uv 600 j m c a ta la se a c ti v it y ( m o l d e c o m p o se d m h o m in m g p ro te in ) 2 2 – 1 – 1 p generation f1 generation –2 –2 fig. 1. activity of cat (a) and isoenzyme pattern (b) in parental plants (p generation) exposed to uv-c radiation and their non-irradiated progeny (f1 generation). letters denote a statistically significant difference (p < 0.05) between p generation (uppercase letters) and f1 generation (lowercase letters). 1 – nonirradiated (control) plants (p-generation) 2 – parental plants exposed to 6 j m–2 uv-c 3 – parental plants exposed to 600 j m–2 uv-c 4 – progeny (f1 generation) of control plants 5 – nonirradiated progeny (f1 generation) of plants exposed to 6 j m–2 uv-c 6 – nonirradiated progeny (f1 generation) of plants exposed to 600 j m–2 uv-c a) b) u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:23 color profile: disabled composite 150 lpi at 45 degrees dose (600 j m–2). the isoenzyme g2 was found only in extracts of irradiated plants. in the progeny plants only one pod isoenzyme (g1) was observed in both plant groups – the progeny of non-irradiated (control) and the progeny of irradiated plants. the progeny of control plants had an isoenzyme band slightly weaker than the parental generation, whereas the progeny of exposed plants had an isoenzyme g1 activity decreased even more than the extracts of corresponding parental plants (fig. 3b). hsp70 expression we detected two isoforms of the protein hsp70 (marked h1 and h2) in the extracts of parental plants. the h1 isoform was present in both the non-irradiated (control) and the irradiated plants, whereas we only observed h2 in plants exposed to uv-c irradiation doses of 6 and 600 j m–2. in plant extracts of the progeny we observed only the isoform h1. it was present in both the progeny of the control and the irradiated plants (fig. 4). discussion since rearrangements between homologous dna sequences in somatic cells are strongly stimulated by dna damage (ries et al. 2000a), the elevated frequency of shr found in arabidopsis thaliana plants exposed to uv-c irradiation in research done by molinier et al. (2006) was not a surprise. nevertheless, it was surprising that the information about stress exposure was transmitted to four generations of their non-irradiated progacta bot. croat. 69 (2), 2010 189 antioxidative enzymes and hsp70 in stress memory fig. 2. activity of apx (a) and isoenzyme pattern (b) in parental plants (p generation) exposed to uv-c radiation and their non-irradiated progeny (f1 generation). letters denote a statistically significant difference (p < 0.05) between p generation (uppercase letters) and f1 generation (lowercase letters). samples 1–6 are the same as in figure 1b. a a aa b b 0 0.2 0.4 0.6 0.8 a sc o rb a te p e ro x id a se a c ti v it y p generation f1 generation control +uv –uv 6 j m +uv –uv 600 j m–2 –2 ( m o l p ro d u c t m in m g p ro te in ) m – 1 – 1 a) b) u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:25 color profile: disabled composite 150 lpi at 45 degrees eny plants. the authors suggested the basis for this transgeneration »memory« to be epigenetic. pecinka et al. (2009) tested the effect of ten abiotic stress factors, among them uv-c irradiation, on parental and two non-exposed subsequent generations of arabidopsis thaliana plants. results confirmed the enhanced frequency of shr in the treated generation, while the two subsequent generations showed a low and stochastic increase in shr. since the subject of our investigation was antioxidative enzymes, an interesting result for our work was the effect of the herbicide paraquat, a well-known ros producer. this treatment, as a rare exception, showed a significantly increased frequency of shr not only in the treated parental plants, but also in the first non-treated progeny generation. the mechanism of the transgenerational stress memory is not fully elucidated yet, so our aim was to investigate whether this process could also include other stress responses, not only genetic material repair mechanisms. for this purpose we investigated (1) the activities of antioxidative enzymes (catalase, ascorbate peroxidase and guaiacol peroxidase), which have an important role in the process of plant adaptation to stress and (2) the induction of the heat shock protein hsp70, which catalyzes the correct folding of proteins denatured as a consequence of stress conditions. symptoms of tissue damage and stunted growth were observed in the plants irradiated with a higher uv-c dose (600 j m–2), while the plants exposed to the lower irradiation dose (6 j m–2) showed milder symptoms. that was expected, because the severity of uv-c irradiation symptoms increases with the irradiation dose, since a higher dose represents a more pronounced stress for the plant. in previous reports it was shown that an uv-c irradiation 190 acta bot. croat. 69 (2), 2010 ]uk k., gogala m., tkalec m., vidakovi]-cifrek @. fig. 3. activity of pod (a) and isoenzyme pattern (b) in parental plants (p generation) exposed to uv-c radiation and their non-irradiated progeny (f1 generation). letters denote a statistically significant difference (p < 0.05) between p generation (uppercase letters) and f1 generation (lowercase letters). samples 1–6 are the same as in figure 1b. a b b a a a 0 1 2 3 4 5 g u a ia c o l p e ro x id a se a c ti v it y p generation f1 generation control +uv –uv 6 j m +uv –uv 600 j m–2 –2 ( m o l p ro d u c t m in m g p ro te in ) m – 1 – 1 b) a) u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:26 color profile: disabled composite 150 lpi at 45 degrees dose of 500-2000 j m–2 can trigger a stress response in arabidopsis thaliana plants (martínez et al. 2004) and that a dose of 10 kj m–2 caused severe symptoms of damage at the level of the whole plant (danon et al. 2004). for even higher uv-c doses (10–50 kj m–2) dna fragmentation was confirmed (danon and gallois 1998). the exposure to stress conditions which cause oxidative stress usually increases antioxidative enzyme activities (dat et al. 2000, arora et al. 2002, mittler 2002). nevertheless, in our research a decreased activity of cat in parental plants exposed to uv-c irradiation in comparison to non-irradiated plants was observed (fig. 1a). the decrease was slightly more pronounced in the plants exposed to an uv-c dose of 600 j m–2 than in the plants exposed to a dose of 6 j m–2. the obtained results are in accordance with the results of willekens et al. (1994), which confirmed the decrease of some cat isoenzyme activities as a consequence of stress induced by uv-b irradiation. also, cho and seo (2005) have shown that other forms of abiotic stress (e.g. exposure to cadmium) decreased cat activity. apx activity in the extracts of plants exposed to uv-c irradiation did not differ significantly from the activity in the non-irradiated (control) plant extracts (fig. 2a). willekens et al. (1994) and yannarelli et al. (2006) monitored the apx transcripts in plants after exposure to uv-b irradiation and observed only insignificant differences. similarly, shigeoka et al. (2002) and davletova et al. (2005) showed that the levels of the apx isoenzyme transcripts increased in the cytosol under conditions of high light intensities, but did not increase in the chloroplasts. such results support the hypothesis that the free radical detoxifying system in chloroplasts is strong enough to monitor ros production even in stressful conditions and does not need to be induced additionally (davletova et al. 2005). in contrast to cat and apx, the activity of pod was significantly increased in parental plants exposed to uv-c irradiation, especially those exposed to a higher irradiation dose, 600 j m–2 (fig. 3a). an explanation for the higher pod activity, when compared to the cat and apx activities, is their different intracellular function. although all the three enzymes use h2o2 as a substrate, the regulation of its intracellular levels is primarily the function of apx, but not of all the other classes of peroxidases (mittler 2002, shigeoka et al. 2002). yannarelli et al. (2006) studied the effect of uv-b irradiation on the isoforms and activities of peroxidases in sunflower cotyledons. they concluded that some types of peroxidases act as ros scavengers in the acclimation to uv-b but others rather play a role either in the metabolism of polyphenols by increasing the antioxidative capacity of the cell or in the cross-linking of uv-absorbing phenolic compounds. furthermore, pod in apoplastic space participates in the generation of ros (kawano 2003). zacchini and de agazio (2004) reported that the activities of both apx and pod were enhanced 24 hours after acta bot. croat. 69 (2), 2010 191 antioxidative enzymes and hsp70 in stress memory fig. 4. immunoblot probed with antibody against hsp70. isoforms of hsp70 are marked h1 and h2. m – molecular mass markers. samples 1–6 are the same as in figure 1b. u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:27 color profile: disabled composite 150 lpi at 45 degrees uv-c irradiation (50 kj mol–1) of tobacco calluses, whereas the activity of cat remained unchanged. research done by willekens et al. (1994) showed that uv-b irradiation also causes increased pod activity. molinier et al. (2005) studied the expression and regulation of genes involved in abiotic and biotic stress response. immediately after uv-c exposure (6 j m–2), a rapid but transient increase of enzymes responsible for the production of ros (nadph oxidase, peroxidase) was detected, while a few hours later genes encoding ros scavengers (including apx) as well as other elements involved in the stress response (e.g. resistance proteins and proteins involved in signalling pathways) were up-regulated. there was thus reason to expect the detection of a higher apx activity, but this was not the case with the irradiated parental plants in our experiment. this could be explained by the difference in time period between treatment and sampling. in our work plant extracts were made 30 minutes after exposure to 6 j m–2, while molinier et al. (2005) took plants few hours after treatment. the isoenzyme pattern of cat, apx and pod after page in native conditions is in line with the enzyme activity levels determined spectrophotometrically for the individual enzymes. uv-c irradiation induced the g2 isoenzyme of pod in the parental generation which explains the increased guaiacol peroxidase activity seen in the extracts of irradiated plants. concerning the reaction of progeny of irradiated plants, our first observation was the earlier progression to flowering phase. a variety of stressful conditions, e.g. a pathogen infection, extreme temperatures and even uv-c irradiation, can promote flowering (martínez et al. 2004). in our experiment the plants were irradiated prior to entering the flowering phase to avoid the possibility of exposing the gametophyte and the germ cells from which progeny plants will develop, to uv-c irradiation. in other words, if irradiation had affected the gametophyte itself, there would have been a possibility of direct effect on the progeny, which would be an explanation for the observed changes in the progeny of irradiated plants. an earlier progression to the flowering phase was observed in the progeny of plants exposed to an uv-c dose of 6 j m–2, while the progeny of plants exposed to a higher uv-c dose (600 j m–2) did not show this effect. this phenomenon could potentially have an important role for plants in their native environment, since it increases the chance of survival in periods of unfavourable conditions (troyer and brown 1976, martínez et al. 2004). it is known that salicylic acid is a stress signal molecule involved in non-photoperiodic flowering mechanisms (endo et al. 2009) and could cause earlier flowering. furthermore, salicylic acid binds to and inactivates cat (chen et al. 1993, conrath et al. 1995), resulting in increased levels of ros, including h2o2. in our work significantly lower cat activity was seen in the progeny of those plants that were exposed to an irradiation dose of 6 j m–2 in comparison to the corresponding control plants (progeny of non-irradiated plants) (fig. 1a). vranová et al. (2002) showed that transgenic plants with constitutively increased levels of h2o2 posses a better pathogen resistance and produce more salicylic acid. therefore, the decrease of cat activity observed in the progeny of irradiated plants may be explained in the context of the role of h2o2 as a signalling molecule. the progeny of irradiated plants also showed a significant reduction in apx activity (fig. 1b) when compared to the corresponding control group (progeny of non-irradiated plants). as for cat, this would result in increased level of ros molecules, which could then be involved in signalling processes. as described earlier, salicylic acid and nitrogen oxide, both 192 acta bot. croat. 69 (2), 2010 ]uk k., gogala m., tkalec m., vidakovi]-cifrek @. u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:27 color profile: disabled composite 150 lpi at 45 degrees synthesized in stress conditions, can lead to reduction of cat and apx activities (willekens et al. 1994), whereby cat is repressed on a transcriptional and apx on a post-transcriptional level (mittler 2002). reduced activities of both enzymes in the progeny demonstrate that the information about stress exposure could be obtained from the parental generation in order to achieve better response to stress conditions. however, it remains unclear how the information about the need for increased h2o2 levels and thus decreased activity of antioxidative enzymes was transmitted to the progeny. the progeny of stressed plants does not show a significantly different pod activity when compared to corresponding control plants (the progeny of non-stressed plants). furthermore, the pod activity in the progeny of all groups was similar to the activity observed in the parental generation of control plants (fig. 3a). it could be concluded that the information about increased pod activity in the parental generation was not transferred to the progeny. an explanation could be a more important role of pod in polyphenol metabolism than in ros scavenging. in irradiated plants immunodetection revealed an induction of an additional isoform of hsp70, which was not observed in the control plants. since hsp70 protein family members play a role in the correct re-folding or controlled degradation of denatured proteins, the induction of a new isoform of hsp70 in plants exposed to uv-c irradiation probably contributes to the minimization of the harmful effects of irradiation damage. the hsp70 family in a. thaliana has 14 members, three of which are constitutively expressed at low levels while the rest of them are induced during thermal stress, viral infections, etc. (sung et al. 2001, aparicio et al. 2005). furthermore, as mentioned earlier, the elevated levels of h2o2 enhance the induction of hsps during light stress in arabidopsis plants deficient in apx (pnueli et al. 2003). the decreased cat activity observed in our plants exposed to uv-c could also cause higher amounts of h2o2 in the cell and be the reason for the induction of an additional hsp70 isoform. the lack of difference with respect to the number of the hsp70 isoforms in the progeny generations of stressed and non-stressed plants suggests that there was no transmission of the hsp70 induction from the parental generation to the progeny and that elevated levels of h2o2 are not the only signal for hsp induction. the absence of a difference in the pod activities and hsp70 isoform induction between the progeny of irradiated plants and their corresponding control means that no information about the changes in their activity and pattern, respectively, was transmitted to the progeny. since the activities of cat and apx, the enzymes responsible for h2o2 scavenging, were reduced it seems that only the information about the need for increased amounts of ros was transmitted to the progeny. the basis of the described transmission of information about the response to an uv-c induced stress across generations is probably of an epigenetic nature because the entire population of plants changes its behaviour in a relatively homogeneous manner. by contrast, when comparing the effect of ten abiotic stress factors on the dna repair process by shr, pecinka et al. (2009) concluded that two subsequent non-treated generations showed a low and stochastic increase in shr frequency and that transcripts coding for shr enzymes returned to the pre-treatment levels in the progeny. in other words, an increased frequency of shr as a transgenerational stress effect was not a general response to abiotic stress in arabidopsis thaliana plants. the authors explained this stochastic response as part of an evolutionarily successful adaptation mechanism in irregularly changing environmental conditions. acta bot. croat. 69 (2), 2010 193 antioxidative enzymes and hsp70 in stress memory u:\acta botanica\acta-botan 2-10\336 cuk.vp 11. listopad 2010 11:36:27 color profile: disabled composite 150 lpi at 45 degrees our results showed that, besides the already known increase in frequency of somatic homologous recombination, transmission of information about stress exposure to progeny can also include changes in activities of antioxidative enzymes catalase and ascorbate peroxidase while changes in guaiacol peroxidase activity and hsp70 isoforms were not confirmed. in both cases – the increased frequency of shr as a part of a dna repair system and the antioxidative stress response as a ros scavenging system – the exact nature of the transmission of information about exposure to stress conditions from the parental plants to their progeny remains to be elucidated. acknowledgements this work was supported by the croatian ministry of science, education and sports, project no. 119-1191196-1202. special thanks to prof. gordana rusak and sa{a liki} for arabidopsis thaliana (ecotype columbia) seeds, dr. davor zahradka for help with and advice about uv exposure and prof. hrvoje fulgosi for the primary antibodies against pea hsp70. references aebi, h., 1984: catalase in vitro. methods in enzymology 105, 121–126. alscher, r. g., donahue, j. l., cramer, c. l., 1997: reactive oxygen species and antioxidants: relationships in green cells. physiologia plantarum 100, 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81 (2), 2022 149 acta bot. croat. 81 (2), 149–158, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-012 issn 0365-0588 eissn 1847-8476 seed and pollen morphology and numerical analysis of tephrosia pers. (fabaceae) and their taxonomic significance ahmed elkordy1*, ahmed k. osman2, mohamed o. badry2 1 sohag university, faculty of science, botany and microbiology department, 82524, sohag, egypt 2 south valley university, faculty of science, botany and microbiology department, qena 83523, egypt abstract – the seed and pollen grain morphology of the genus tephrosia pers. in egypt was studied using light and scanning electron microscopy. qualitative and quantitative characters of seeds and pollen grains are presented. the data suggest that several seed and pollen morphological characters can be used to distinguish the taxa of tephrosia. based on upgma clustering analysis and pca, three main clades were recognized: clade a comprising t. kassasii, clade b comprising t. apollinea, t. purpurea, t. quartiniana, and t. uniflora, and clade c comprising t. nubica. the seed and pollen morphological data obtained in this study provide additional characters that assist in the classification of the genus. dichotomous artificial keys based on seed and pollen data of the investigated taxa are presented. keywords: pollen, principal component analysis, scanning electron microscopy, seeds, taxonomy, tephrosia introduction tephrosia pers. is one of the larger genera of the tribe millettieae, subfamily papilionoideae, of fabaceae. the genus comprises ca. 350 species and infraspecific taxa with a pantropical distribution, concentrated in africa-madagascar (170 taxa), asia (40), central and tropical n. america (45), and australia (90) (lewis et al. 2005, zhi and pedley 2010, powo 2021). taxonomically, the genus tephrosia is a very problematic complex as a distinction between some taxa of the genus is still subject to discussion and needs much further investigation (schrire 2005, lakshmi et al. 2008). many taxonomists classified the genus tephrosia into subgenera or sections based mainly on morphological traits. de candolle (1825) suggested classifying the genus tephrosia into four sections: brissonia, craccoides, mundulea, and reineria. bentham (1862) classified tephrosia into two sections: sect. brissonia (neck.) dc. and sect. recueria benth. later, three subgenera of tephrosia were proposed by bentham (1865) and baker (1876) as macronyx, which comprises t. tenuis, brissonia, which comprises t. candida, and reineria, which comprises the remaining species of tephrosia. the most recent infrageneric taxonomy was suggested by brummitt (1980), who classified tephrosia into two subgenera: t. subgenus tephrosia and t. subgenus barbistyla based on the presence or absence of trichomes on styles and stigmas (de queiroz et al. 2015). although this ranking has been largely agreed upon, there is some overlap in diagnostic characteristics among the taxa of the two suggested subgenera (lakshmi et al. 2008). based on a few analyzed taxa, tephrosia was considered monophyletic (hu et al. 2002), but the subgenera of brummitt (1980) were not supported by the genetic and other molecular studies conducted on the genus (hu et al. 2002, acharya et al. 2004, lakshmi et al. 2008). instead, three species complexes in t. subgenus tephrosia, represented by t. purpurea, t. cinerea, and t. adunca were distinguished based on density and coloration of the indumentum, flower location, and size (de queiroz 2012). the seed surface micromorphology of angiosperms and gymnosperms can exhibit significant structural details (barthlott 1981). seed micromorphology has proven to be a good taxonomic tool, giving several characters significant for classifying some genera of faboideae (papilionoideae), particularly for the genus tephrosia (rao and rao 2008, al-ghamdi and al-zahrani 2010, de queiroz et al. 2013). the seed surface of tephrosia subgenus tephrosia has a simple reticulate pattern, while in tephrosia subgenus * corresponding author e-mail: aelkordy@science.sohag.edu.eg elkordy a., osman a. k., badry m. o. 150 acta bot. croat. 81 (2), 2022 barbistyla, the surface has a crested ornamentation pattern (de queiroz et al. 2013). moreover, the variations in the morphology of pollen grains are significant for the identification delimitation of taxa in tephrosia (buril et al. 2011, antonio-domingues et al. 2019). combining the macro and microstructure of seeds and pollen grains is important for the taxonomy of many angiosperm taxa (abdelkhalik et al. 2008). in egypt, the genus tephrosia is represented by six species and two subspecies, mainly distributed in gebel elba, desert wadis, the nile valley, and oases (täckholm 1974, boulos 1999). the current study aims to investigate the macro-and micromorphology of seeds and pollens of the genus tephrosia in egypt using light microscope and scanning electron microscope (sem) to assess their taxonomic significance. provision of identification keys to the studied tephrosia species based on seed and pollen macroand micromorphological characters is aimed at further applications in other taxonomic studies of the genus tephrosia. materials and methods plant material the current study was mainly based on specimens kept in the following egyptian herbaria: cai herbarium at cairo and south valley university herbarium at qena ( herbarium acronyms follow thiers (2020) (on-line suppl. tab. 1). the nomenclature of the studied taxa was updated according to powo (2021). samples and techniques to study seeds’ macro and micro-morphological characters, 15-30 seeds per species taken from five healthy and fully mature plants and seed specimens were studied by a conventional method using a stereomicroscope (sm) and scanning electron microscope (sem). seed samples were examined with an olympus szx7 stereo dissecting microscope. for sem, dried seed specimens were mounted on metallic stubs utilizing double adhesive tape and coated with gold for 4-6 minutes (150-200 angstrom) in a sputtering chamber employing a jeol jfc1100e ion-sputtering device. all quantitative measurements of seed characters from the sm and sem images were obtained utilizing the program imagej v1.45 (schneider et al. 2012). mean quantitative measurements were obtained from 35 readings from each specimen. pollen grains of the studied taxa were extracted from 5 to 10 flowers per species, taken from five healthy plant specimens, to study macro and micro-morphological characters of tephrosia pollen grains. examination of pollen grains using light microscopy (lm) was achieved employing an olympus bh-2 research microscope. the pollen grain samples were acetolyzed and mounted on a metallic stub in some drops of ethanol based on the methods summarized in moore et al. (1991). the mean quantitative measurements of pollen grains were conducted based on a minimum of 40 pollen grains per specimen. pollen grain specimens were studied employing scanning electron microscopy (jeol jsm 5400 lv), under an accelerated voltage of 15 kv (150200 angstrom). all photomicrographs were captured at the unit of electron microscope, university of assiut, egypt. all quantitative measurements were compiled using the imagej v1.45 program (schneider et al. 2012). the main morphological characters, terminology, and concepts of pollen grains and seeds used here follow previous studies (huysmans et al. 2003, punt et al. 2007, de queiroz et al. 2013, lawrence 2017) with some modifications by the authors. data analyses aspects of plant, seed, and pollen micro-and macromorphology based on 62 characters were recorded and scored for the otus (operational taxonomic units) in a data matrix: 30 qualitative (nine binary and 21 multi-state characters) and 32 quantitative continuous characters. two analyses were performed with past (paleontological statistics software, ver. 4.03) (hammer et al. 2001). a hierarchical cluster analysis (hca) based on euclidean distance similarity index and using the unweighted pair group method using the arithmetic averages (upgma) clustering method (sokal 1958, seberg et al. 1991) was generated to classify the studied taxa into clusters based on overall character similarity. then, a principal component analysis (pca) was conducted to reveal whether the analyzed characters could cluster ta xa a nd to recog ni z e t he most d ist i nc t ive morphological character(s) for the studied taxa, as in other studies (e.g., coutinho et al. 2011, lopes et al. 2013, badry et al. 2020). eigenvalues were plotted in a two-dimensional scatter plot along the first two principal component axes (pca1, pca2), accounting for the highest character variation. results seed seed shape among the studied taxa showed large variation, from reniform, elliptical to oblong, oblong reniform, oval, rectangular, and quadrate (tab. 1). however, seed shape is reniform in t. apollinea (fig. 1a), elliptical to oblong in t. nubica (fig. 1d), oblong reniform in t. purpurea (fig. 1g), oval in t. quartiniana (fig. 1j), rectangular in t. uniflora (fig. 1m) and quadrate in t. kassasii (fig. 1p). the seed size of the investigated taxa ranged from 1.96 × 1.34 mm in t. quartiniana to 4.35 × 2.77 mm in t. apollinea. the rest of the taxa have intermediate-sized seeds. however, it is approximately 4.00 × 2.45 mm in t. nubica, 3.34 × 2.30 mm in t. purpurea, 2.54 × 1.34 mm in t. uniflora, and 2.09 × 1.80 mm in t. kassasii (tab. 1). seeds with the lowest length-width ratio occur in t. quartiniana (1.12) and the highest in t. uniflora (1.90) (tab. 1). seed and pollen morphology of tephrosia acta bot. croat. 81 (2), 2022 151 three different patterns of testa texture have been reported: reticulated, multi reticulated, and multi crested. however, it is reticulated in t. apollinea and t. purpurea (fig. 1b, h) respectively; multi reticulated in t. nubica, t. quartiniana, and t. kassasii (fig. 1e, k, q) respectively; multi crested in t. uniflora (fig. 1n). fig. 1. scanning electron microscope micrographs of tephrosia seeds. side view of whole seed (a, d, g, j, m, p), testa ornamentation (b, e, h, k, n, q) and hilum shape (c, f, i, l, o, r). t. apollinea (a–c), t. nubica (d–f), t. purpurea (g–i), t. quartiniana (j–l), t. uniflora (m–o), t. kassasii (p–r). elkordy a., osman a. k., badry m. o. 152 acta bot. croat. 81 (2), 2022 the cellular shapes can be of considerable diagnostic and systematic value among the studied taxa. they vary from polygonal; 5, 6 gonals; 4, 5, 6 gonals. however, it is polygonal in t. quartiniana (fig. 1k); 5, 6 gonals in t. apollinea and t. purpurea (fig. 1b, h) respectively; 4, 5, 6 gonals in t. nubica, and t. kassasii (fig. 1e, q) respectively, while it was crested in t. uniflora (fig. 1n). the area of the epidermal cells shows a highly significant variation among the studied taxa. however, it varies from 2.45 µm2 in t. kassasii to 19.02 µm2 in t. apollinea (tab. 1, fig. 1q, b) respectively, while the rest of the studied taxa are characterized by intermediate epidermal cell areas: 5.38 µm2 in t. quartiniana, 7.73 µm2 in t. nubica, 8.67 µm2 in t. purpurea (fig. 1k, e, h) respectively. the form of the anticlinal boundaries is of less taxonomic and systematic value among the studied taxa. however, all investigated taxa are characterized by raised and straight anticlinal boundaries. the surface of the anticlinal boundaries of the studied taxa is smooth to fine folded in t. kassasii (fig. 1q), coarse folded in t. purpurea (fig. 1h), wavy in t. uniflora (fig. 1n), and fine folded in the rest of the taxa. the thickness of the anticlinal boundaries varies from 2.34 µm in t. quartiniana to 3.92 µm in t. nubica (tab. 2). the form of the periclinal cell wall is concave in t. quartiniana (fig. 1k), convex in t. uniflora (fig. 1n), and flat in the rest of the studied taxa. there are three different shapes for the surface of the periclinal cell wall: rugulate in t. apollinea (fig. 1b), favulariate in t. nubica and t. purpurea (fig. 1e, h) and smooth in t. quartiniana, t. uniflora and t. kassasii (fig. 1k, n, q). three different shapes of hilum have been recognized among the studied taxa: ovate in t. apollinea, t. nubica, t. tab. 1. morphological characteristics of the studied tephrosia taxa. min – minimum, max – maximum, l – length, w – width. the number of measurements for each taxon n = 35. taxon shape seed dimensions testa texture epidermal cell l (mm) (min–max) w (mm) (min–max) l/w (min–max) shape area (µm2) (min–max) tephrosia apollinea (delile) dc. reniform 4.35 (4.27–4.43) 2.77 (2.71–2.93) 1.57 (1.58–1.76) reticulate 5,6 gonals 19.02 (6.79–32.03) tephrosia nubica (boiss.) baker elliptical to oblong 4.00 (3.91–4.29) 2.45 (2.23–2.66) 1.63 (1.68–1.74) multi reticulate 4,5,6 gonals 7.73 (2.14–10.19) tephrosia purpurea (l.) pers. oblong reniform 3.34 (2.97–3.91) 2.30 (2.01–2.62) 1.45 (1.43–1.58) reticulate 5,6 gonals 8.67 (3.41–15.68) tephrosia quartiniana cufod. oval 1.96 (1.82–2.29) 1.75 (1.71–1.89) 1.12 (1.06–1.27) multi reticulate polygonal 5.38 (2.89–8.24) tephrosia uniflora pers. rectangular 2.54 (2.43–2.84) 1.34 (1.28–1.49) 1.90 (1.44–1.97) multi crested polygonal 2.45 (1.19–6.41) tephrosia kassasii boulos quadrate 2.09 (2.05–2.32) 1.80 (1.54–2.16) 1.16 (1.33–1.03) multi reticulate 4,5,6 gonals 3.80 (0.29–5.51) tab. 2. morphological characteristics of the studied tephrosia taxa. min – minimum, max – maximum, l – length, w – width. the number of measurements for each taxon n = 35. taxon anticlinal boundaries periclinal cell wall hilum form thickness (µm) (min–max) surface form surface shape position l (mm) w (mm) area (mm2) t. apollinea raised, straight 3.24 (2.68–3.61) fine folded flat rugulate ovate central 0.44 0.33 0.10 t. nubica raised, straight 3.92 (3.27–4.46) fine folded flat favulariate ovate sub–central 0.50 0.42 0.15 t. purpurea raised, straight 3.55 (2.47–4.00) fine folded flat favulariate ovate to elliptic central 0.43 0.32 0.10 t. quartiniana raised, straight 2.34 (1.14–3.20) course folds concave smooth ovate sub–central 0.24 0.20 0.030 t. uniflora raised, wavey 3.96 (3.29–4.48) smooth convex smooth circular sub–central 0.33 0.25 0.06 t. kassasii raised, straight 3.30 (2.67–3.78) smooth to fine folded flat smooth ovate sub–central 0.25 0.20 0.04 seed and pollen morphology of tephrosia acta bot. croat. 81 (2), 2022 153 quartiniana, and t. kassasii (fig. 1c, f, l, r) respectively, ovate to elliptic in t. purpurea (fig. 1i) and circular in t. uniflora (fig. 1o). the position of the hilum is either central in t. apollinea and t. purpurea (fig. 1c, i), or subcentral in the rest of the investigated taxa. the diameter of the hilum varies from 0.25 × 0.20 mm in t. kassasii to 0.50 × 0.42 mm in t. nubica (tab. 2, fig. 1r, f), respectively. the hilum area varies from 0.03 mm2 in t. quartiniana to 0.15 mm2 in t. nubica (tab. 2, fig. 1l, f) respectively. fig. 2. scanning electron microscope micrographs of tephrosia pollen grains. polar view (a, d, g, j, m, p), equatorial view (b, e, h, k, n, q) and exine ornamentation (c, f, i, l, o, r). t. apollinea (a–c), t. nubica (d–f), t. purpurea (g–i), t. quartiniana (j–l), t. uniflora (m–o), t. kassasii (p–r). elkordy a., osman a. k., badry m. o. 154 acta bot. croat. 81 (2), 2022 artificial key to taxa of tephrosia based on the seed morphological characteristics: 1.a. seed testa texture multicrested ........................ t. uniflora 1.b. seed testa texture reticulate to multireticulate ............ 2 2.a. seed epidermal cell shape polygonal ..... t. quartiniana 2.b. seed epidermal cell shape 4,5,6 gonals or 5,6 gonals .. 3 3.a. seed width 1.8 (1.54-2.16) mm ....................... t. kassasii 3.b. seed width 2.00-3.00 ........................................................ 4 4.a. seed length 3.34 (2.97-3.91) .......................... t. purpurea 4.b. seed length 3.90-4.5 mm ................................................. 5 5.a. seed epidermal cell area 19.02 (6.79-32.03) mm ............................................................................ t. apollinea 5.b. seed epidermal cell area 7.73 (2.14-10.19) mm ................................................................................ t. nubica pollen pollen dispersed as monads, isopolar, radially symmetric, 3-zonocolporate. colpi with pointed apices, with granulate, scabrate to granulate–scabrate membrane; endoaperture lolongate with endexine protrusions (main characteristics of the analyzed pollen grains of the studied taxa are summarized in detail in (fig. 2, tabs. 3-5). the shape in the equatorial view varies from the prolate to the subprolate (tab. 5), while they are circular, either fossaperturate (fig. 2a, p) or planaperturate (fig. 2d) or triangular planaperturate (fig. 2g), in polar view. pollen grains of the studied taxa are relatively small to medium size, the polar axis length (p) exceeds 30.0 μm in only one species (t. nubica) at 31.11 μm (fig. 2e). the average polar axis (p) ranges from 25.02 μm in t. purpurea to 31.11 μm in t. nubica, while the average equatorial axis length (e) ranges from 18.43 μm in t. apollinea to 26.62 in t. nubica. all the investigated taxa have tectate exine with either punctuate ectexine with small, rounded granules in the luminae (fig. 2c, o) or foveolate to microreticulate in the rest of the studied taxa (fig. 2c-r). artificial key to taxa of tephrosia based on the palynological results of this study 1a. pollen grains have punctuated ectexine, small, rounded granules in the luminae .................................................... 2 1b. pollen grains have foveolate to microreticulate ectexine ................................................................................................. 3 2a. colpus length ranges from 10.68 to 19.67 µm ................................................................................ t. uniflora 2b. colpus length ranges from 20.25 to 26.78 µm .............................................................................. t. apollinea 3a. pollen grains prolate-spheroidal .................... t. purpurea 3b. pollen grains prolate or sub prolate ................................ 4 4a. pollen grains sub prolate with microreticulate ectexine .................................................................................. t. nubica 4b. pollen grains prolate with rugulate to rugulate-reticulate ectexine ......................................................................... 5 5a. polar axis ranges from 23.81 to 26.75 µm ....... t. kassasii 5b. polar axis ranges from 27.00 to 33.92 µm .. t. quartiniana statistical analyses the phenetic relationships of the studied taxa of tephrosia reflected in the morphological diversity of the seeds and pollens are presented through two statistical analyses. the dendrogram of all otus studied, clustered by the upgma method, is shown in (fig. 3). the cophenetic correlation of the distance matrix and tree matrix was 0.9883, indicating a good fit of the dendrogram to the distance matrix (see rohlf 1990). three main clades were recognized: clade a comprising t. kassasii, clade b comprising t. apollinea, t. purpurea, t. quartiniana, and t. uniflora, and clade c comprising t. nubica. generally, clade b is divided into two subgroups: subgroup (i) comprising t. apollinea, t. purpurea, and subgroup (ii) comprising t. quartiniana and t. uniflora. the scatter plot of six otus on the first two principal component axes is shown in (on-line suppl. fig. 1), interpreting 99.81% of the total observed variation. on the first component axis, 74.81% of the total variation, segregation is veritab. 3. morphological characteristics of the studied tephrosia taxa. min – minimum, max – maximum, l – length, w – width, p – polar axis, e – equatorial diameter. the number of measurements for each taxon n = 40. taxon p e (p/e) (min–max)axis (µm) (min–max) distance (µm) (min–max) view area (µm2) (min–max) diameter (µm) (min–max) view area (µm2) (min–max) t. apollinea 28.31 (26.29–30.14) 14.22 (13.54–14.60) 302.71 (231.64–332.03) 18.43 (16.59–19.83) 420.98 (336.06–448.09) 1.54 (1.46–1.69) t. nubica 31.11 (27.89–32.54) 15.56 (13.94–16.27) 604.24 (555.73–624.13) 26.62 (26.00–27.08) 639.13 (583.99–669.25) 1.18 (1.06–1.23) t. purpurea 25.02 (24.98–25.06) 20.04 (12.49–25.20) 397.06 (360.16–437.04) 24.22 (23.95–24.96) 433.35 (430.56–435.54) 1.02 (1.00–1.04) t. quartiniana 30.28 (27.00–33.92) 15.14 (13.50–16.96) 355.15 (351.26–358.30) 19.85 (17.49–22.64) 470.57 (420.22–521.70) 1.56 (1.22–1.92) t. uniflora 29.23 (23.64–30.96) 14.61 (11.82–15.48) 258.55 (238.31–286.20) 18.80 (18.35–19.37) 428.68 (340.64–481.55) 1.55 (1.29–1.69) t. kassasii 25.52 (23.81–26.75) 12.76 (11.91–13.37) 240.99 (230.74–255.84) 18.65 (16.99–20.21) 380.33 (343.92–409.88) 1.37 (1.20–1.57) seed and pollen morphology of tephrosia acta bot. croat. 81 (2), 2022 155 fied between two groups: 1) t. kassasii, 2) t. apollinea, t. purpurea, t. quartiniana, and t. uniflora. the main characters explaining this segregation (characters with factor loading ≥ ± 0.6) (on-line suppl. tab. 2) were stem type, leaflet shape, abaxial leaflet surface, pod indumentum, epidermal cell shape, intercolpium area, mesocolpium diameter, equatorial view surface sculpture, and endoaperture. on the second component axis, 24.13% of the total variation in (on-line suppl. fig. 1) segregation is verified between two groups: 1) t. nubica, 2) t. apollinea, t. purpurea, t. quartiniana, and t. uniflora. the main characters explaining this segregation (characters with factor loading ≥ ± 0.6) were stem type, stem indumentum, stem branching, pod length, number of seeds per pod, hilum width, hilum area, polar view area, equatorial diameter, equatorial view area, apocolpium diameter, apocolpium index, apocolpium field, colpus width, and endoaperture. generally, the results show unity between the principal component analysis (pca) and upgma clustering, suggesting three groups. discussion the sculpture and structure of seed coats are conservative and have stable characters, which have been used effectively in the taxonomy and phylogeny of different plant groups (el-naggar 2005, gabr 2018). seed size and shape among the studied taxa of tephrosia are quite consistent. still, some characters are diagnostic, e.g., seeds in t. nubica and t. apollinea equal or exceed 4.0 mm in length (fig. 1d, a), respectively, while seeds in t. apollinea, t. nubica, and t. purpurea exceed 2.0 mm in width. the seed shape of the studied taxa is of a high taxonomic significance, and it could be used as a tool to distinguish taxa tab. 4. morphological characteristics of the studied tephrosia taxa. min – minimum, max – maximum, l – colpus length, w – colpus width. the number of measurements for each taxon n = 40. taxon lumen murus thickness (µm) (min–max) colpus apocolpium mesocolpium diameter (µm) (min–max) diameter (µm) (min–max) area (µm2) (min–max) l (µm) (min–max) w (µm) (min–max) area (µm2) (min–max) diameter (µm) (min–max) index (min–max) t. apollinea 0.81 (0.32–1.29) 0.17 (0.03–0.37) 0.49 (0.16–0.85) 24.04 (20.25–26.78) 4.48 (3.58–5.55) 87.58 (72.07–99.70) 5.95 (4.97–6.55) 0.49 (0.45–0.53) 13.98 (12.37–15.25) t. nubica 0.56 (0.28–1.06) 0.16 (0.06–0.25) 0.58 (0.36–0.84) 19.26 (16.17–21.23) 6.12 (4.60–8.13) 99.30 (66.03– 119.80) 12.86 (12.59–13.68) 0.86 (0.80–0.94) 20.35 (19.21–21.15) t. purpurea 0.52 (0.26–0.98) 0.12 (0.03–0.32) 0.31 (0.16–0.55) 23.88 (20.90–24.90) 3.97 (2.21–8.15) 61.37 (43.68–86.95) 7.88 (3.79–10.92) 0.40 (0.33–0.43) 19.32 (19.31–19.33) t. quartiniana 0.26 (0.11–0.62) 0.03 (0.01–0.10) 0.52 (0.32–0.82) 15.78 (10.68–19.67) 4.08 (2.22–5.96) 56.84 (35.59–76.60) 6.37 (3.99–7.67) 0.52 (0.46–0.59) 14.29 (13.82–14.52) t. uniflora 0.50 (0.17–0.81) 0.12 (0.03–0.26) 0.45 (0.15–0.89) 15.78 (10.68–19.67) 4.25 (2.90–5.12) 74.96 (48.95–93.96) 6.44 (4.80–8.44) 0.60 (0.56–0.67) 12.11 (10.61–13.88) t. kassasii 0.48 (0.10–1.05) 0.13 (0.03–0.72) 0.40 (0.18–0.62) 15.78 (10.68–19.67) 4.67 (2.58–6.13) 47.07 (38.85–52.46) 6.22 (5.14–7.69) 0.58 (0.54–0.64) 23.48 (22.91–24.07) tab. 5. morphological characteristics of the studied tephrosia taxa. taxon pollen shape sculpture type endoaperture lumina shapesurface aperture ectoaperture equatorial view polar view t. apollinea prolate punctuate psilate psilate granulate lalongate regular, with small, rounded granules t. nubica subprolate foveolate to microreticulate scabrate perforate scabrate granulate lolongate irregular t. purpurea prolatespheroidal foveolate to microreticulate psilate psilate-scabrate granulate lalongate regular t. quartiniana prolate foveolate to microreticulate scabrateperforate perforate granulate scabrate lalongate irregular t. uniflora prolate punctuate psilate perforate psilate scabrate lalongate regular, with small, rounded granules t. kassasii prolate foveolate to microreticulate scabrate perforate scabrate granulate scabrate lolongate irregular 156 acta bot. croat. 81 (2), 2022 elkordy a., osman a. k., badry m. o. at the interspecific level. these results align with those of al-ghamdi and al-zahrani (2010). the epidermal cell area is polygonal in t. uniflora, as the testa has multi crested texture. in tephrosia, the epidermal cell shape is a less diagnostic character at the interspecific level. however, it could aid in the classification at the infrageneric level. the epidermal cell area is highly taxonomic, particularly in distinguishing some studied taxa, with the largest area in t. apollina. in contrast, the smallest is found in t. kassasii. the form, thickness, and surface of anticlinal boundaries of the studied taxa are of less taxonomic significance, with the form of anticlinal boundaries being raised and straight in all studied taxa (fig. 1b, e, h, k, q) excluding t. uniflora, in which it is raised and wavy (fig. 1n). moreover, the surface of anticlinal boundaries is less of a diagnostic feature among the studied taxa (tab. 2, fig. 1b-q). periclinal cell wall form is useful in distinguishing t. quartiniana from other studied taxa, being concave in t. quartiniana (fig. 1k). at the same time, it is convex in t. uniflora (fig. 1n) and flat in the rest of the studied taxa (fig. 1b, e, h, q). the surface of the periclinal cell wall is a good taxonomic character in segregation of the studied taxa. moreover, the hilum shape, position, size, and area are of high taxonomic value among the studied taxa. these results agree with the findings of al-ghamdi and al-zahrani (2010). the morphology of pollen grains was an important taxonomic tool in tephrosia as formerly observed in other palynological studies (perveen and qaiser 1998, buril et al. 2011). the genus tephrosia is commonly stenopalynous. the pollen morphology is more or less similar, especially the size and surface sculpture (antonio-domingues et al. 2019). although pollen grains of the studied taxa of tephrosia are very similar morphologically, the analyses executed here highlight the importance of both quantitative and qualitative characters in differentiating the studied taxa. the most common shape of pollen is prolate. the pollen grain size of the examined taxa is of less taxonomic significance. according to measurements of the polar axis, the largest pollen grains are those of t. nubica 31.11 (27.89-32.54) μm, and the smallest ones are those of t. purpurea 25.02 (24.98-25.06) μm. the equatorial diameter measurements showed that the largest pollen grains are t. nubica 26.62 (26.00-27.08) μm, and the smallest ones are t. apollinea 18.43 (16.59-19.83) μm. venkateswarlu and kameswara rao (1967) performed only size measurements on tephrosia purpurea pollen grains, and their data agree with the speci men analyzed in our study. moreover, the length of ectocolpus and apocolpium can also distinguish the studied taxa. the longest colpus is found in t. apollinea, and the shortest in t. quartiniana, t. uniflora, and t. kassasii. the longest apocolpium is found in t. nubica and the shortest in t. apollinea. the ectexine of the studied taxa showed a high taxonomic value for segregation of closely related species. it is punctuated with small, rounded granules in the luminae in t. apollinea and t. uniflora, while it is foveolate to microreticulate in the rest of the studied taxa. these results agree with those of buril et al. (2011) and, antonio-domingues et al. (2019) but disagree with the findings of perveen and qaiser (1998). generally, the seed and pollen morphological characters observed in this study along with the upgma and pca analyses confirm the subgenera classification suggested by bentham (1865), baker (1876), and de queiroz et al. (2013), but did not prove the two subgenera proposed by brummitt (1980). conclusion the seed and pollen grain morphologies were significant differentiating tools for the studied taxa of tephrosia. this work contributes to the understanding of the diversity of seeds and pollen grains in tephrosia and the considerable variation through a range of morphological characters. some characters, which include seed shape and size, testa texture, epidermal cell shape and area, the sculpture of ectexine and ectoaperture membranes, length of the ectocolpi, apocolpium diameter, p/e ratio, and lumina area, bring new taxonomic clarification to the genus tephrosia. these results are congruent with other seed and pollen studies on different genera within the tribe milletieae. the quantitafig. 3. cluster analysis of the studied tephrosia taxa based on 62 measured variables using the unweighted pair group method with arithmetic averages (upgma) and euclidean similarity index showing three clades: a – subgenus macronyx, b – subgenus reineria, c – subgenus brissonia. seed and pollen morphology of tephrosia acta bot. croat. 81 (2), 2022 157 tive and qualitative seed and pollen characters offer a helpful taxonomic key for segregating closely related species. these data support morphological knowledge and can be used in future phylogenetic approaches to help develop the systematic and morphological study of the fabaceae-papilionoideae subfamily. acknowledgments we are grateful to the directors and curators of cairo university herbarium (cai) for the loan of specimens. sincere thanks and deepest appreciation to dr. john gaskin, botanist/research leader pmru, usda ars nparl. sidney, usa, for going through the manuscript and making valuable suggestions that always inspired us to learn something new. references abdel khalik, k., abd el-ghani, m., elkordy, a., 2008: fruit and seed morphology in galium l.(rubiaceae) and its importance for taxonomic identification. acta botanica croatica 67, 1–20. acharya, l., mukherjee, a.k., panda, p.c., 2004: genome relationship among nine species of millettieae (leguminosae: papilionoideae) based on random amplified polymorphic dna (rapd). zeitschrift für naturforschung c 59, 868–873. al-ghamdi, f.a., al-zahrani, r.m., 2010: seed morphology of some species of tephrosia pers. 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(ed.), flora of china, 190–193. missouri botanical garden press, st. louis. acta bot. croat. 81 (2), 2022 s11 social news the thirty-ninth meeting of the eastern alpine and dinaric society for vegetation ecology (eadsve) was held in dubrovnik, croatia (may 4-7, 2022) at the end of the 1950s vegetation scientists erwin aichinger from austria, sandro pignatti from italy and maks wraber from slovenia met and decided to found a society to stimulate cross-border cooperation in studies of the botanically interesting area of the eastern alpine and dinaric region. in 1960, the first meeting was held in klagenfurt. the eastern alpine and dinaric society for vegetation ecology (eadsve) was established in 1960. the society organises meetings every second year. the meeting is open not only to members, but also to other researchers interested in the flora and vegetation of the region. the society cultivates contacts among scientists of central and south-eastern europe and it gives young scientists in particular the possibility to present themselves and their work with presentations and posters. invitations for discussions and field trips are also organised as additional activities. thirty-eight meetings have been organised up to the present. in the last ten years they were in pörtschach, austria (2009), camerino, italy (2011), ohrid, north macedonia (2013), osijek, croatia (2015), prizren, kosovo (2017) and colfiorito, italy (2019). the 39th eadsve meeting was held in dubrovnik, croatia, from 4 to 7 may 2022. the meeting was organised by the university of dubrovnik in dubrovnik led by nenad jasprica and željko škvorc. dubrovnik, the pearl of the adriatic, was inscribed on the unesco world heritage list in 1979. the city has always been keen on preserving its identity in all aspects, which is also true with respect to higher education in dubrovnik. the collegium ragusinum established by the jesuits in 1624 was the forerunner of the university of dubrovnik, i.e., modern higher education in dubrovnik. dubrovnik is also the place where lujo adamović (1864-1935), fig. 1. participants of the 39th meeting of the eastern alpine and dinaric society for vegetation ecology (eadsve), dubrovnik, croatia, may 4-7, 2022 during the botanical field excursion on the pelješac peninsula (photo: i. brautović). s12 acta bot. croat. 81 (2), 2022 social news a world-famous croatian botanist, spent his youth and later life. scientific sessions included both poster and oral presentations during the first day of meeting held at the student centre dormitory of the university of dubrovnik. the meeting program included 15 oral and 12 poster presentations. in all there were 99 authors from 17 countries. dr massimo terzi from the institute of bioscience and bioresources, italian national council of research, bari (italy) was the invited plenary speaker. other contributions included vegetation mapping, description of syntaxa new for science, plant recolonization, notes on flora of islets, vegetation of grasslands and forests, assessment of the impact of climate change on the potential natural vegetation, phytosociology and ecology of some plant taxa, monitoring and management analyses in natura 2000 sites and habitats, and ethnobotanical research. the book of a group of authors “interpretative manual of european riparian forests and shrublands“ was also presented. abstracts of all presentations are included in the book of abstracts published by the university of dubrovnik and edited by nenad jasprica, željko škvorc and daniel krstonošić (http://www.eadsve.org/). finally, during the closing remarks, the focus was on excellence, research, training, skills and mobility. during the general assembly, an updated bylaw was adopted. new honorary members of the society were admitted – harald niklfeld (university of vienna, austria) and paul heiselmayer ( university of salzburg, austria). a minute of silence was held in honour all members – jozo franjić, ferat rexhepi, zorana sedlar and ljerka marković – who deceased since the last meeting in colfiorito, italy, in 2019. the next meeting will be held in the city of banja luka, bosnia and herzegovina, in 2024. all participants took part in two one-day botanical excursions – to the pelješac peninsula and the konavle region (fig. 1). nenad jasprica željko škvorc daniel krstonošić eastern alpine and dinaric society for vegetation ecology http://www.eadsve.org/ acta bot. croat. 80 (2), 2021 125 acta bot. croat. 80 (2), 125–130, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-012 issn 0365-0588 eissn 1847-8476 morphological and genetic diversity of senecio vulgaris l. (asteraceae) in iran mostafa ebadi1*, rosa eftekharian2 1 azarbaijan shahid madani university, faculty of science, department of biology, 53714-161, tabriz, iran 2 shahid beheshti university, faculty of life sciences and biotechnology, tehran, iran abstract – senecio vulgaris l., an annual herb belonging to the asteraceae, is widely distributed in different regions of the world. there is no information on the intraspecific variations of the morphological and molecular features of this species. in the present investigation, we studied the morphological and genetic diversity of 81 accessions of s. vulgaris collected from 10 geographical populations. eleven inter simple sequence repeat (issr) primers were used for the examination of genetic variations among the populations. analysis of molecular variance (amova) and gst analyses revealed significant differences among the investigated populations. a significant correlation between genetic distance and geographical distance was revealed by the mantel test. however, reticulation analysis indicated the occurrence of gene flow among most of the populations studied. principal component analysis (pca) plot showed that the number of capitula, length of the cauline leaf and plant height were the most variable morphological characters. principal coordinates analysis (pcoa) plot revealed two groups of populations, according to molecular and morphological data. the results suggested the existence of possible intraspecific taxonomic ranks within this species. keywords: gene flow, groundsel, morphology, principal component analysis introduction the genus senecio l. (asteraceae) includes ca. 1250 species and is one of the largest genera of flowering plants (bremer 1994, calvo et al. 2015). in iran, some species of senecio have a widespread distribution in the different geographical regions, such as s. vulgaris l., s. glaucus l., and s. leucanthemifolius subsp. vernalis (waldst. et kit.) greuter. senecio vulgaris, commonly called groundsel or oldman-in-the-spring, is an annual herb that grows in a variety of habitats from open grassland to woodland. it is usually regarded as a temperate weed and although it is almost cosmopolitan it appears to be more localized in temperate regions of america and asia, mediterranean areas and south africa (chater and walters 1976). senecio vulgaris has a long history of herbal use and is widely used as an anthelmintic, antiscorbutic, diaphoretic, diuretic, emmenagogue and purgative (hatfield 1977, launert 1981). a homeopathic remedy is made from aerial parts of the plant. the therapeutic activity of groundsel is related to a variety of biologically active substances such as phenols and flavonoids, essential oils, polysaccharides, triterpenes, amines, saponins, tannins and mucilage (uzun et al. 2004, conforti et al. 2006). a population genetics survey plays the main role in the planning of genetic and breeding programs. it provides data about genetic diversity, gene migration, allelic drift, genetic fragmentation, genetic bottleneck and any other evolutionary forces acting on population divergence (sheidai et al. 2013). intraspecific taxonomic entities can be identified after a detailed population-based investigation within plant species. in general, extensive morphological and genetic divergences among populations result in speciation events. intraspecific variation occurs within both wild and cultivated crop plants (maxted and hunter 2011). in iran, s. vulgaris grows in different areas and forms numerous local geographical populations. there is no information about its genetic diversity and adaptation against population divergence. population genetic studies not only can provide information on these aspects but can also unravel the speciation process of senecio in general. one method for the study of genetic diversity employs morphological * corresponding author e-mail: ebadi2023@yahoo.com ebadi m., eftekharian r. 126 acta bot. croat. 80 (2), 2021 material and methods plant specimens eighty-one plant specimens were collected from ten geographical regions (on-line suppl. fig. 1). details of localities and voucher specimens are showed in tab. 1. morphological characteristics of s. vulgaris accessions were measured by a binocular microscope (olympus bh2 ×400). dna extraction and pcr amplification for the molecular study, a sub-sample of 0.5 g leaves from each population was taken and genomic dna was extracted by the ctab method (sheidai et al. 2013). eleven issr primers were tested for pcr amplification; (agc) 5gg, (agc) 5gt, (ca) 7gt, (ca) 7at, (ga) 9c, ubc807, ubc810, ubc811, ubc823, ubc834, and ubc847. pcr reactions were done in a total volume of 25 μl containing 1 μl primer (12.5 pmol), 0.5 μl dntps mix (10mm), 20 ng template dna, 1.5 μl reaction buffer (10×), 1.2 μl mgcl2, 1.5 u taq dna polymerase and 18.5 μl deionized water. the pcr reaction was performed with a thermal cycler (techne, germany) with the following program: 94 °c for 5 min, 30 cycles at 94 °c for 30 sec, 55 °c for 1 min, 72 °c for 2 min and a final extension at 72 °c for 10 min. the pcr products were electrophoresed at 85 v on 1.5% agarose gels and the resulting bands were observed under a uv transilluminator. the dna fragment sizes were estimated after comparison with a 1-kb dna ladder. polymorphic issr markers were manually scored as binary data with presence as “1” and absence as “0”. multivariate analysis the one-way analysis of variance (anova) was used to indicate a significant difference of morphological characters among the studied populations. principal component analysis (pca) was used to recognize important variable morphological characters in the studied populations. principal coordinate analysis (pcoa) was carried out based on morphological and molecular data with euclidean distance as a characteristics, but molecular markers are the potential tools to study genetic relations, phylogeny, population dynamics or geneand genome mapping. . the advent of molecular markers resulted in an improved ability to track evolution through a good understanding of genetic diversity among populations and new phylogenetic viewpoints (müller-schärer and fischer 2001, stuessy et al. 2014). among molecular markers, inter simple sequence repeat (issr) deserves special attention as a tool for analyzing diversity. issr is easy to use, quick, simple and highly reproducible (azizi et al. 2014, eftekharian et al. 2016). issr markers usually show high polymorphism and have the very important advantage that no prior information about the genomic sequence is required (kojima et al. 1998, bornet and branchard 2001). this study aimed to investigate the genetic variation between s. vulgaris populations collected from different geographic regions by using issr and morphological characters. also, gene flow and correlation of genetic and geographical distances were estimated. fig. 1. principal component analysis (pca) among studied population based on quantitative morphological characters. component 1 and component 2 refer to the first and second principal components, respectively. arrows represent the correlations between the independent variables and the two principal components represented. each point represents an individual sample. tab. 1. geographical information concerning the studied populations of senecio vulgaris. population locality habitat type altitude (m a.s.l.) longitude (e) latitude (n) voucher no. pop1 fars, kazerun waste ground 904 29.64 51.64 95109 pop2 khuzestan, andimeshk roadsides 185 32.44 48.34 95105 pop3 khuzestan, masjed soleiman roadsides 150 31.13 49.20 95104 pop4 fars, nurabad roadsides 985 30.11 51.54 95110 pop5 tehran, velenjak mountain meadows 1758 35.48 51.23 95101 pop6 lorestan, khorramabad roadsides 1215 33.29 48.22 95103 pop7 alborz, karaj mountain meadows 1327 35.38 50.92 95107 pop8 ilam, ivan waste ground 1144 33.82 46.30 95106 pop9 tehran, lavasan mountain meadows 1720 35.75 51.60 95102 pop10 khuzestan, ahwaz roadsides 15 31.31 48.67 95108 intraspecific diversity of senecio vulgaris l. acta bot. croat. 80 (2), 2021 127 similarity index. statistical analysis was performed using the program past v. 2.17c. (hammer et al. 2001). genetic diversity and population variation parameters were performed by popgene software v. 1.32 (yeh et al. 1999). mantel permutation test was used to check the correlation between genetic distances and corresponding geographical distances of the studied populations by genalex software v. 6.5. (peakall and smouse 2006). analysis of molecular variance (amova) test was used to determine significant genetic differences among the studied populations with the use of genalex v. 6.5. genetic differentiation was estimated by d,st = jost measure of differentiation (jost 2008) and g,st = standardized measure of genetic differentiation (hedrick 2005). the occurrence of allele flow among populations was estimated by reticulation analysis (legendre and makarenkov 2002). results morphological variability twenty-three qualitative and quantitative characters were selected for morphological evaluation of s. vulgaris populations (tab. 2). anova test showed that there are significant differences (p ≤ 0.01) among the quantitative morphological characters of studied populations. the most important morphological characters among the studied populations were identified by pca-biplot. the biplot of the pca based on quantitative morphological characters showed that the number of capitula, length of the cauline leaf and plant height are important markers in the distribution of the populations studied (fig. 1). it showed two factors (pc1 and pc2) which together explained 82% of the total variance. the pc1 (51% of variance) had positive correlations with the number of capitula, and a negative significant correlation with length of the cauline leaf. pc2 (variance 31%) had positive correlations with plant height (tab. 3). the pcoa plot of both quantitative and qualitative characters divided the studied populations into two groups (groups i and ii) based on morphological characters (fig. 2). populations no. 5, 7 and 9 were grouped into i and the rest of the studied populations were placed in group ii. the capitula numbers of populations of the first group ranged between 6 to 9 and between 4 to 7 in populations of group ii. the means of the length of cauline leaf were 6 cm tab. 2. quantitative and qualitative morphological characters of senecio vulgaris accessions (length measured values are in cm). sd – standard deviation; n – number of analyzed samples. quantitative characters mean ± sd (n) plant height 21.45 ± 8.71 (81) length of basal leaf 3.96 ± 1.08 (176) width of basal leaf 1.35 ± 0.53 (176) thickness in base of stem 3.21 ± 0.91 (81) length of cauline leaf 4.54 ± 1.16 (214) width of cauline leaf 1.97 ± 0.62 (214) number of capitula 6.13 ± 1.42 (81) length of peduncle 0.21 ± 0.08 (295) length of bract on a peduncle 0.37 ± 0.08 (205) number of involucral bracts 21.19 ± 0.59 (81) length of involucral bracts 0.64 ± 0.05 (232) number of calyculus bracts 10.1 ± 2.49 (81) length of corolla 0.67 ± 0.05 (217) length of corolla tube 0.40 ± 0.05 (217) length of corolla lamina 0.28 ± 0.04 (217) length of anther in disc florets 0.18 ± 0.01 (110) length of style in disc florets 0.09 ± 0.01 (192) length of papus 0.6 ± 0.08 (305) length of nutlet 0.31 ± 0.03 (273) qualitative characters type of stem branchedunbranched stem indumentum yes no blackness of calyculus bracts less than 1mm – more than 1mm blackness of involucral bracts less than 1mm – more than 1mm tab. 3. correlation of morphological characteristics of the studied senecio vulgaris populations with two components of principal component analysis (pca). character component pc1 pc2 number of capitula 0.908 0.132 length of the cauline leaf –0.895 0.115 length of peduncle 0.531 0.043 plant height 0.144 0.951 fig. 2. two dimensional plot of principal coordinate analysis (pcoa) of the studied senecio vulgaris populations based on morphological characters. different colors indicate the plant specimens from each geographical population. group 1: the populations in north iran (populations 5, 7 and 9); group 2: the populations in west and south west iran (populations 1, 2, 3, 4, 6, 8 and 10). ebadi m., eftekharian r. 128 acta bot. croat. 80 (2), 2021 and 4 cm in group i and ii populations, respectively. also, the mean of plant height of group i populations was 25 cm whereas it was 18 cm in group ii populations. moreover, the stem type of group i populations was often branched and the black tips of the bracts were more than 1 mm long whereas populations of another group mostly have an unbranched stem and the black tips of the bracts were less than 1 mm long (fig. 3). issr marker in this study, 121 reproducible issr fragments (200-800 bp) were obtained utilizing the pcr amplification. anova analysis revealed significant genetic differences among the studied populations (phipt = 0.83, p ≤ 0.01). it also showed that most of the genetic variation (60%) occurred within populations, while 40% was due to inter-population genetic differences. these results revealed the presence of a high level of genetic differentiation among s. vulgaris populations. this result was also supported by the gst analysis and hickory test. moreover, population differentiation parameters determined among the studied populations produced high values for hedrick, standardized fixation index after 999 permutation (g’st = 0.886, p ≤ 0.001) and jost, differentiation index (d-est = 0.275, p ≤ 0.001). various parameters of genetic diversity calculated in 10 geographical populations of s. vulgaris are shown in online suppl. tab. 1. the results obtained from the common genetic diversity indices are similar to the unbiased gene diversity parameter (which is free from the sampling size). the polymorphic loci percentage in each population varied from 10.54% to 55.61% (on-line suppl. tab. 1). kazerun population (pop1) showed the highest percentage (55.61%) of polymorphic loci of all the populations while the velenjak population (pop 5) exhibited the lowest amount of polymorphism (10.54%). the values of shannon’s information index (i) varied from 0.062 to 0.298 in all the populations. the gene diversity (he) for all loci in each of the population ranged from 0.039 to 0.189. pcoa analysis showed that there were genetic differences among studied populations of s. vulgaris based on issr data. populations 5, 7 and 9 were categorized into separate groups while the rest of the studied populations were distanced from them (fig. 4). the mantel test showed that there is a significant correlation (r = 0.5, p < 0.01) between genetic and geographic distance (on-line suppl. fig. 2). this result demonstrated that gene exchange occurred between populations that were close to each other. a reticulogram revealed some degree of shared alleles among most of the populations. (fig. 5) these shared alleles might be due to ancestral and or ongoing limited gene flow among the studied populations. discussion population genetic investigations are useful in understanding genetic variability, allele flow, inbreeding against fig. 3. morphological characters of two different types of senecio vulgaris. a – unbranched stem and bract black tip less than 1 mm long, b – branched stem and bract black tip more than 1 mm long (adapted and modified from klinkenberg 2019). fig. 5. reticulogram of the studied senecio vulgaris populations based on a neighbour joining tree of issr data. populations are marked with numbers from 1-10 according to tab. 1. dashed lines indicate gene flow. reticulation analysis revealed that limited amount of shared alleles or gene. fig. 4. two dimensional plot of principal coordinate analysis (pcoa) of the studied senecio vulgaris populations based on issr data. different colors indicate the plant specimens from each geographical population. group 1: the populations in north iran (populations 5, 7 and 9); group 2: the populations in west and south west iran (popu;atopms 1, 2, 3, 4, 6, 8 and 10). intraspecific diversity of senecio vulgaris l. acta bot. croat. 80 (2), 2021 129 outbreeding and effective population size. this information is valuable in choosing effective management in conservative plans and also throws light on the presence of intraspecific taxonomic forms and suggests an appropriate hybridization strategy (freeland et al. 2011, sheidai et al. 2013, 2014). genetic diversity of s. vulgaris was studied by müllerschärer and fischer (2001). according to their investigation different geographical areas include different habitats and populations representing different habitats varied in their sizes. in our recent study, the genetic structure of s. glaucus showed that population fragmentation, restricted gene flow, genetic drift, and local adaptation have played a role in the genetic divergence of s. glaucus populations in iran (eftekharian et al. 2016). in the present investigation, s. vulgaris populations presented a high degree of genetic variability. species that are dispersed over a wide area face different environmental conditions and therefore can possess a wide genetic and morphological variability to manage ecological challenges (freeland et al. 2011, el-amier et al. 2014, eftekharian et al. 2015, 2016). in this study, population fragmentation in s. vulgaris is revealed by pcoa. however, some degree of gene flow and genetic admixture occurred among populations as shown by a reticulogram. the probable loss of genetic variation within these populations can be prevented by this limited gene flow due to the action of genetic drift (habibollahi et al. 2015). according to the mantel test, which presented a pattern of isolation-by-distance in the studied s. vulgaris populations, adjacent populations have more chance for gene exchange than more distant populations, which is why more genetic similarities have been shown in closely situated populations. comparison of the two datasets indicated that in some cases, the grouping of populations based on the morphological method was consistent with molecular groupings. therefore, the pcoa plot based on molecular and morphological data showed two groups of populations. genetic and morphological differences between the two groups may be related to different ecological conditions. ecological and environmental factors (e.g., temperature, humidity, and soil factors) can be significantly effective in shaping genetic diversity patterns (huang et al. 2016). according to müller-schärer and fischer (2001) different habitats can affect the genetic structure of s. vulgaris populations. tang et al. (2015) showed that there was a strong relationship between the soil factors (especially salinity and soil texture) and plant diversity. in our study, the habitat of studied populations varied from mountain meadows (populations 5, 7 and 9) to roadsides (populations 2, 3, 4, 6 and 10) and waste grounds (populations 1 and 8). the mountain meadow habitats had gravelly soils while loamy soils were predominant in the waste ground and roadside habitats. however, the effects of some factors in plant population genetic diversity remain unclear and more investigations have to be done. conclusion morphological characters revealed that two groups of the studied geographical populations differed from each other in two qualitative morphological features (black tips of calyculus bracts and type of stem), as evidenced in fig. 3 and three quantitative characters (capitula number, length of the cauline leaf and plant height). therefore, in these populations, morphological changes accompanied genetic differences and this pattern of arrangement has confirmed the existence of possible intraspecific taxonomic ranks within this species. references azizi, n., sheidai, m., mozafarian, v., noormohammadi, z., 2014: genetic, cytogenetic and morphological diversity in helicrysum leucocephalum (asteraceae) populations. biologia 6, 1–8. bornet, b., branchard, m., 2001: nonanchored inter simple sequence repeat (issr) markers: reproducible and specific tools for genome fingerprinting. plant molecular biology reporter 19, 209–215. bremer, k., 1994: asteraceae: cladistics and classification. timber press, portland. calvo, j., alvarez, i., aedo, c., 2015: systematics of senecio section crociseris (compositae, senecioneae). phytotaxa 211, 1–105. chater, a.o., walters, s.m. 1976: senecio l. in: tutin, t.g., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. 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(asteraceae: senecioneae). nucleus 60, 43–49. el-amier, y.a., el-halawany, e.f., abed zaid, a., 2014: ecological study on senecio glaucus l. in the deltaic mediterranean coastal land of egypt. journal of environmental science 43, 597–614. freeland, j.r., kirk, s., petersen, d., 2011: molecular ecology. 2nd ed. willy, london. habibollahi, h., noormohammadi, z., sheidai, m., farahani, f., 2015: genetic structure of cultivated flax (linum usitatissimum l.) based on retrotransposon-based markers. genetika 47, 1111–1122. hammer, ø., harper, d.a.t., ryan, p.d., 2001: past: paleontological statistics software package for education and data analysis. palaeontologia electronica 4, 9. hatfield, a.w., 1977: how to enjoy your weeds. frederick muller ltd, london. hedrick, p.w., 2005: a standardized genetic differentiation measure. evolution 59, 1633–1638. huang, w., zhao, x., zhao, x., li, y., lian, j., 2016: effects of environmental factors on genetic diversity of caragana microphylla in horqin sandy land, northeast china. ecology and evolution 6, 8256–8266. ebadi m., eftekharian r. 130 acta bot. croat. 80 (2), 2021 jost, l., 2008: gst and its relatives do not measure differentiation. molecular ecology 17, 4015–4026. klinkenberg, b., 2019: e-flora bc: electronic atlas of the plants of british columbia. retrieved july 28, 2020 from https:// ibis.geog.ubc.ca/biodiversity/eflora/ kojima, t., nagaoka, t., noda, n., ogihara, y., 1998: genetic linkage map of issr and rapd markers in einkorn wheat in relation to that of rflp markers. theoretical and applied genetics 96, 37–45. launert, e., 1981: edible and medicinal plants. hamlyn, london. legendre, p., makarenkov, v., 2002: reconstruction of biogeographic and evolutionary networks using reticulograms. systematic biology 51, 199–216. maxted, n., hunter, v., 2011: crop wild relatives: a manual of in situ conservation. experimental agriculture 47, 735. müller-schärer, h., fischer, m., 2001: genetic structure of the annual weed senecio vulgaris in relation to habitat type and population size. molecular ecology 10, 17–28. peakall, r., smouse, p.e., 2006: genalex 6: genetic analysis in excel. population genetic software for teaching and research. molecular ecology notes 6, 288–295. sheidai, m., afshar, f., keshavarzi, m., talebi, s.m., noormohammadi, z., shafaf, t., 2014: genetic diversity and genome size variability in linum austriacum (linaceae) populations. biochemical systematic and ecology 57, 20–26. sheidai, m., zanganeh, s., haji-ramezanali, r., nouroozi, m., noormohammadi, z., ghasemzadeh-baraki, s., 2013: genetic diversity and population structure in four cirsium (asteraceae) species. biologia 68, 384–97. stuessy, t.f., takayama, k., lopez-sepulveda, p., crawford, d.j., 2014: interpretation of patterns of genetic variation in endemic plant species of oceanic islands. botanical journal of the linnean society 174, 276–288. tang, l., dong, s., liu, s., wang, x., li, y., su, x., zhang, y., wu, x., zhao, h., 2015: the relationship between soil physical properties and alpine plant diversity on qinghai-tibet plateau. eurasian journal of soil science 4, 88–93. uzun, e., sariyar, g., adsersen, a., karakoc, b., otuk, g., oktayoglu, e., pirildar, s., 2004: traditional medicine in sakarya province (turkey) and antimicrobial activities of selected species. journal of ethnopharmacology 95, 287–296. yeh, f.c., yang, r.c., boyle, t.j., ye, z.h., mao, j.x., 1999: popgene ver. 1.32, the user-friendly shareware for population genetic analysis. molecular biology and biotechnology centre, university of alberta, edmonton. opce-str.vp acta bot. croat. 69 (2), 291–297, 2010 coden: abcra 25 issn 0365–0588 growth and yield response of cowpea (vigna unguiculata l. walp.) to soils from different fallow physiognomies in the rainforest zone of nigeria samson o. oke*, damilola l. eyitayo department of botany, obfemi awolowo university, ile-ife, nigeria a study was conducted to evaluate the response of cowpea (vigna unguiculata) seedlings to soil collected from four fallows of different physiognomy. seedlings of cowpea were grown from seeds on soil samples collected from the four different fallow statuses (panicum maximum-dominated fallow soil, chromolaena odorata-dominated fallow soil, tithonia spp.-dominated fallow soil and bush fallow soil that contains many herbaceous plant species) in plastic containers each having fifteen replicates. among the growth characteristics assessed the number of nodules and stem dry weight being the most influenced. panicum maximum-dominated fallow soil with greater organic matter content produced the most desirable growth characteristics during the growing period and at harvest. the results were used to deduce the best type of fallow soil for cowpea cultivation. key words: growth, yield, fallow soil, vigna unguiculata, physiognomy, rainforest, nigeria introduction cowpea (vigna unguiculata l. walp.) is a legume grown under rain-fed conditions in the tropics (sangakkara 1998). although it occupies a smaller proportion of the crop area than cereals, it contributes significantly to household food security in west and central africa. compared with many other crops, the cowpea has received little attention from plant breeders and a large efforts need to be made to break the yield barriers. if cowpea production is to keep pace with other crops, especially cereals, its yield potential must be improved. in nigeria, 80% of the cowpea production is mainly from the savanna zone of the country (fao 1999). a wide range of seed yields have been recorded for cowpeas but they are generally low. among the factors responsible for the low yields are low soil fertility, as most tropical soils are deficient in essential nutrients particularly nitrogen and phosphorus (jones and wild 1975). traditionally, soil fertility in west africa has been maintained acta bot. croat. 69 (2), 2010 291 * corresponding author: soke@oauife.edu.ng u:\acta botanica\acta-botan 2-10\279 oke.vp 13. listopad 2010 12:28:07 color profile: disabled composite 150 lpi at 45 degrees through fallow. in nigeria intensive cropping is gradually replacing the traditional shifting cultivation that is associated with long fallow and low crop yields are among the results of this practice. the steady decline in food production due to reduced length of fallow on land has prompted farmers to improve soils with different materials (organic and inorganic) in order to enhance plant growth and increase yield. there is limited documentation on the effect of status of soil of different fallow physiognomies on cowpea growth and yield in nigeria. such information would be useful and important in agricultural extension delivery to farmers for increased crop productivity and poverty alleviation. an attempt to find out the growth and yield responses of cowpea to soils from different fallow status in the rainforest zone inform this study. since fallows not only improve subsequent crop performance but also restore soil fertility and organic matter content over the long term, this experiment seeks to evaluate the effects of four fallow soils of different physiognomies on the growth characteristics and yield of the cowpea (vigna unguiculata var. ife brown) in the ile-ife area of southwestern nigeria. materials and methods four fallow plots of different physiognomies were selected within the same area at obafemi awolowo university, ile-ife, southwestern nigeria. these sites had to be in close proximity to one another to satisfy the conditions of the chronosequence approach of the study. topsoil from the four fallow plots of different physiognomies (a panicum maximum -dominated fallow plot, a chromolaena odorata -dominated fallow plot, a fallow plot that contains many herbaceous plant species (mixed) and a tithonia spp -dominated fallow plot) that are common in ile-ife area of southwestern nigeria were collected. the soil was air dried and loosened before being put in 20cm wide plastic containers with holes at the bottom to facilitate effective drainage. the four soil types were analyzed for ph; particle size distribution, organic matter content, sodium, potassium, calcium, magnesium and nitrogen contents. the soil particle size distribution was determined by bouyoucos hydrometer and the ph electrometrically. organic matter was determined by the chromic acid digestion method (black 1965), the exchangeable cations and nitrogen by the semi-micro kjeldahl method (tel and rao 1982). pure lines of cowpea seeds (ife brown variety) were collected from the international institute for tropical agriculture (i.i.t.a) ibadan. the seeds were planted in each soil type in may 2008 and seedlings were established in each soil type at densities of 1, 2, and 3 per 5 litre pot. each density had fifteen replicates, watering was with tap water every two days in the morning and in the evening to field capacity to give the best soil moistures regime for growth, thus there was no competition for water. when the seedlings were nursed to full and equal establishment in the pots, measurement of some growth parameters such as leaf length, leaf breadth, leaf area, leaf number, shoot height were taken weekly. at harvest in august 2008 ten weeks after planting, the plants were removed and seedling mean dry matter yield (leaves, stems, roots, total), pod numbers and nodule numbers were measured after oven drying at 80 °c. the effects of soils from different fallow on the seedlings growth characteristics were determined using analysis of variance (anova) with the aid of the software sas release 8.1. significant means were separated by duncan’s multiple range test at the 5% probability level. 292 acta bot. croat. 69 (2), 2010 oke s. o., eyitayo d. l. u:\acta botanica\acta-botan 2-10\279 oke.vp 11. listopad 2010 15:44:20 color profile: disabled composite 150 lpi at 45 degrees results physical and chemical properties of soil a chemical and mechanical analysis of the soils prior to cropping with cowpea is shown in table 1. the panicum maximum -dominated fallow soil was found to have higher organic matter, organic carbon, ph and greater percentage of exchangeable cations than other fallow soils. the tithonia spp -dominated fallow soil had higher nitrogen and phosphorus content, while chromolaena odorata -dominated fallow soil had intermediate values for most of the soil properties determined. growth characteristics the cowpea seedlings in panicum maximum -dominated fallow soil had better growth characteristics such as greener leaves, higher number of leaves, bigger leaves, bigger stems, leaf area, shoot height and greater canopy than seedlings from other fallow soils. the cowpea seedlings growing in the chromolaena odorata -dominated fallow soil were observed to have the slowest growth rate, the smallest number of leaves, the lowest leaf area, shoot height and the least canopy while the seedlings from the other two fallow soils had intermediate values (figs. 1, 2, 3). the analysis of variance for the seedling growth parameters that were measured during the growing period revealed that the soils from the different fallows had a significant influence on leaf area (p<0.05). vigna unguiculata seedlings had the highest mean stem dry weight per plant in panicum maximum -dominated fallow soil, the lowest in chromolaena odorata -dominated fallow soil and intermediate values in the other two fallow soils (tab. 2). the fallow soil types significantly affected the mean stem dry weight (p £ 0.05). vigna unguiculata seedlings had the highest mean root dry weight in mixed fallow soil, the least in chromolaena odorata -dominated fallow soil and intermediate values in panicum maximum and tithonia spp fallow soils. the fallow soil types had no significant effects on the mean root dry weight.vigna unguiculata seedlings had the highest mean leaf dry weight in panicum maximum -dominated fallow soil, the least in chromolaena odorata -dominated fallow soil and intermediate values in the other two fallow soils. the fallow soils had significant effects on the mean leaf dry weight (p £ 0.05). vigna unguiculata seedlings had the highest mean dry weight of pods in tithonia spp fallow soil, the lowest in chromolaena odorata -dominated fallow soil and intermediate values in the other two fallow soils. the fallow soils had significant effects on the mean dry weight of pods (p £ 0.05). vigna unguiculata seedlings had the highest mean number of pods in panicum maximum -dominated fallow soil, the lowest in chromolaena odorata -dominated fallow soil and intermediate values in the other two fallow soils. the fallow soil types had significant effects on the mean number of pods (p £ 0.05). vigna unguiculata seedlings had the highest mean total biomass in tithonia spp fallow soil, the lowest in chromolaena odorata fallow soil and intermediate values in panicum maximum and mixed fallow soils. the fallow soil types had a significant effect on the mean total biomass (p £ 0.05).vigna unguiculata seedlings had the highest mean number of nodules in panicum maximum -dominated fallow soil, the lowest in chomoleana odorata -dominated fallow soil and intermediate values in the two other fallow soils. the fallow acta bot. croat. 69 (2), 2010 293 growth and yield of vigna unguiculata in the rainforest u:\acta botanica\acta-botan 2-10\279 oke.vp 11. listopad 2010 15:44:20 color profile: disabled composite 150 lpi at 45 degrees 294 a c t a b o t .c r o a t .69 (2),2010 o k e s .o .,e y it a y o d .l . tab. 1. the chemical and mechanical analyses of the fallow soils prior to cropping with cowpea. fallow soil sample % sand % silt % clay % o.c % o.m phosphorus mg kg–1 % k % ca % na total n ph electrical conductivity panicum 54 18 28 4.24 7.3 8.47 0.55 7.0 3.17 1.5 7.8 h2o 7.5 caci2 0.37 chromokena 62 14 24 2.21 3.8 6.64 0.21 3.4 2.26 0.8 6.6 h2o 6.2 caci2 0.27 mixed 36 26 38 3.66 6.3 7.13 1.03 6.2 3.04 1.3 6.9 h2o 6.6 caci2 0.55 tithonia 72 14 14 2.04 3.5 9.86 0.36 5.1 2.48 2.48 8.1 h2o 7.6 caci2 0.30 tab. 2. some growth characteristics and yield of seedlings of the cowpea (vigna unguiculata) grown in the four different fallow soil types at harvest. fallow soil types stem dry weight root dry weight leaf dry weight pod dry weight pod number total dry weight no of root nodules leaf number leaf area panicum dominated 1.41a 0.70ab 3.95a 2.81b 4.62a 8.26ab 15.24a 24.01a 35.49a tithonia dominated 1.31a 0.81ab 3.52b 3.67a 4.17ab 9.31a 15.12a 16.86b 31.85a chromolaena dominated 0.62b 0.59b 1.30c 1.48c 2.92b 4.04c 10.33b 17.23b 23.75b mixed 1.00c 1.10a 2.53b 2.13bc 3.51ab 6.77b 12.37b 18.47b 28.83ab mean within the same column and with the same letter are not significantly different according to dmrt u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 0 \ 2 7 9 o k e . v p 1 1 . l i s t o p a d 2 0 1 0 1 5 : 4 4 : 2 0 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s acta bot. croat. 69 (2), 2010 295 growth and yield of vigna unguiculata in the rainforest 0 5 10 15 20 25 30 35 40 45 50 panicum chromolaena mixed tithonia fallow types l e a f n u m b e r wap1 wap2 wap3 wap4 wap5 wap6 wap7 wap8 wap9 wap10 fig. 1. response of cowpea leaf number to various fallow soil types (wap1–wap10) during the growing period 0 5 10 15 20 25 30 35 40 panicum chromolaena mixed tithonia fallow types m e a n l e a f a re a (c m ) 2 wap1 wap2 wap3 wap4 wap5 wap6 wap7 wap8 wap9 wap10 fig. 2. response of cowpea leaf area to various fallow soil types (wap1–wap10) during the growing period. 0 10 20 30 40 50 60 70 panicum chromolaena mixed tithonia fallow types m e a n s h o o t h e ig h t (c m ) wap1 wap2 wap3 wap4 wap5 wap6 wap7 wap8 wap9 wap10 fig. 3. response of cowpea shoot height to various fallow soil types (wap1–wap10) during the growing period. u:\acta botanica\acta-botan 2-10\279 oke.vp 11. listopad 2010 15:44:20 color profile: disabled composite 150 lpi at 45 degrees soil type had significant effects on the mean number of nodules (p £ 0.05). vigna unguiculata seedlings had the highest mean number of leaves in panicum maximum fallow soil, the lowest in chromolaena odorata fallow soil and intermediate values in mixed and tithonia spp. fallow soils. the fallow soil types had significant effects on the mean number of leaves (p £ 0.05). discussion the study set out to highlight the response of the growth characteristics of cowpea (vigna unguiculata) seedlings to soils from different fallow physiognomy. the analysis carried out on the performance of cowpea in relation to number of leaves, shoot height, leaf area and total biomass at harvest, showed a significantly higher value in panicum maximum -dominated fallow soil than in the other fallow soils. the result showed clearly that fallow soil types are important factors in the cultivation of the cowpea. the rapid growth rate observed in panicum maximum fallow soil may be due to its richness in organic matter content, organic carbon content and in exchangeable cations. the binding influence of panicum maximum roots on the soil is considered to be responsible for the ability of the grass to effectively check erosion (amusan and oke 2000). as a result the enriched soil will be able to retain its nutrients due to its better ability to check soil erosion. enriched soils through proper management of organic amendments such as crop residues and manure can increase the yield of cowpea. cowpea seedlings (vigna unguiculata) growing in panicum maximum -dominated fallow soil were observed to have the highest number of leaves and leaf area and so when considering growing cowpea as a fodder or vegetable, panicum maximum fallow soil is highly recommended. comparison of the biomass of different parts of cowpea seedlings indicated that leaves had the highest yield. this could be attributable to the fact that the leaf is the site of food manufacture through photosynthesis and hence is expected to have a greater biomass accumulation compared to the stem and root. heuvelink (1995) while monitoring the growth, development and yield of a tomato crop found that in all experiments the ratio between leaf and stem dry weight was 7:3 and that this ratio did not change with crop development. the highest yield of pod biomass obtained at harvest in the tithonia spp -dominated fallow soil which was due to its high phosphorus content and is in agreement with the observations of israel (1987) that cowpea has a high phosphorus requirement. the highest number of pods produced was observed in the panicum maximum fallow soil because it had the highest number of root nodules which can fix atmosphere nitrogen and hence contribute significantly, towards the nitrogen nutrition of cowpea. similar results were obtained by awonaike (1991). in this study, it was observed that soils from different fallows had significant effects on the leaf number, leaf area, shoot height, pod number, stem dry weight, leaf dry weight, root dry weight and pod dry weight of cowpea. it was interesting to note that panicum maximum fallow soil had the best growth characteristics and highest yield of biomass. there were significant lower values for almost all the growth parameters examined in chromolaena odorata -dominated fallow soil. it is often suggested that, as a short fallow species, chromolaena odorata has serious adverse effects on agricultural productivity and on the weed composition and subsequent food crops (mcfayden and skarratt 1996). 296 acta bot. croat. 69 (2), 2010 oke s. o., eyitayo d. l. u:\acta botanica\acta-botan 2-10\279 oke.vp 11. listopad 2010 15:44:20 color profile: disabled composite 150 lpi at 45 degrees in conclusion, we suggest that farmers who are interested in cultivating cowpea, fallow lands dominated by chromolaena odorata should as much as possible be avoided, as cowpea growth is adversely affected on such fallow soil, while fallow lands dominated by panicum maximum is highly recommended for farmers who are interested in vegetative growth as such fallow soils tend to have greater organic matter content, organic carbon content and exchangeable cations, which favour cowpea (vigna unguiculata) seedling growth. farmers who are interested in yield of pods are recommended to grow their cowpea in tithonia spp -dominated fallow soil. references amusan, a. a., oke, s. o., 2000: effect of different fallow plant species on regeneration of degraded ultisols in southwestern nigeria. annals of agricultural sciences 2, 53–59. awonaike, k. o., 1991: nitrogen assimilation and distribution in field grown cowpea at various growth stages. agronomy journal 55, 81–85. black, c. a., 1965: methods of soil analysis i. a. s. a. monogram, madison. fao, 1999: production yearbook, fao, rome. heuvelink, e., 1995: growth, development and yield of a tomato crop. scientia horticulturae 61, 77–79. israel, d. w., 1987: investigation of the role of phosphorus in symbiotic dinitrogen fixation. plant physiology 84, 835–840. jones, m. i., wild a., 1975: soils of west african savanna. the maintenance and improvement of their fertility. commonwealth bureau of the soils technical communication of the harpenden, u.k. commonwealth agricultural bureau (cab), farnham royal, uk. 55, 246. mcfayden, r. c., skarratt b., 1996: potential distribution of chromolaena odorata (siam weed) in australia, africa and oceania. agriculture ecosystem and environment 59, 89–96. sangakkara,u. r., 1998: growth and yields of cowpea (vigna unguiculata (l.) walp) as influenced by seed characters, soil moisture and season of planting. journal of agronomy and crop science 180, 137–142. tel, d. a., rao p. v., 1982: automated and semi-automated methods of soil and plant analysis. manual series 7. international institute for tropical agriculture, ibadan, nigeria. acta bot. croat. 69 (2), 2010 297 growth and yield of vigna unguiculata in the rainforest u:\acta botanica\acta-botan 2-10\279 oke.vp 11. listopad 2010 15:44:20 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (1), 53–64, 2011 coden: abcra 25 issn 0365–0588 cytochemical and ultrastructural observations of anthers and pollen grains in lathyrus undulatus boiss. fýlýz vardar*, meral ünal marmara university, science and art faculty, department of biology, göztepe, 34722, ýstanbul, turkey. abstract – in lathyrus undulatus boiss. (fabaceae), the young microspore stage of anther development was characterized by the enlarged secretory tapetal cells, which presented an intense reaction with regard to protein, insoluble polysaccharides and lipids. at bicellular pollen stage, the middle layer and the tapetum degenerated. after degradation of the tapetum, epidermis and single row u-shaped endothecium existed in the mature anther wall, and pollen grains remained in the locus. young microspores had a spherical and centrally located nucleus with one or two nucleoli, many spherical lipid bodies and starchy plastids. a mature pollen grain contains insoluble polysaccharides, proteins, lipids and calcium. the mature pollen had the following morphological characteristics: 3-zonocolporate, prolate, tectate (imperforate) type of exine and perforate type of structure. the intine formed an important constituent portion of the wall, and consisted two sublayers: an outer intine (exintine) and an inner intine (endintine). the well-defined exine was made up of lipoidal substances and protein, but the intine composed of insoluble polysaccharides and protein. the bicellular state of the pollen grains persisted to anthesis. keywords: anther, cytochemistry, fabaceae, lathyrus undulatus, microsporogenesis, pollen, sem, tem, tapetum. abbreviations: pmc – pollen mother cell, pas – periodic acid-schiff introduction microsporogenesis and pollen grain development have been a focus of interest since generative reproduction in plants depends on pollen structure and function. disturbances decrease the chance of effective pollination and fertilization of maternal plants (bohdanowicz et al. 2005). male sterility is mostly due to defects in the development of the microspore or the surrounding nutritive layer, the tapetum. during microsporogenesis, the tapetum performs a secretory role, providing essential nutrients to the developing sporogenous tissue, and later degenerates to facilitate the release of the mature pollen (pacini 2000). the mature pollen grain is a nutrient storage site necessary for the germination and growth of the pollen tube (rodriguez-garcia et al. 2003). acta bot. croat. 70 (1), 2011 53 * corresponding author: e-mail: filiz.vardar@gmail.com copyright ® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:01:58 color profile: disabled composite 150 lpi at 45 degrees for structural and molecular analysis of anther tapetum, many authors divide the tapetum life span into stages correlated with microspore/pollen grain development (platt et al. 1998, koltunow et al. 1990). this paper reports observations on the development and cytochemistry of anthers in lathyrus undulatus boiss. (fabaceae), which belongs to the papilionoideae subfamily, endemic to northwestern turkey. the genus lathyrus l. (fabaceae) comprises approximately 200 species, represented by 77 lathyrus taxa, 23 of which are endemic to turkey (davis 1970, 1988; güneª and çirpici 2008). pollen morphological characters have been established to elucidate the phylogenetic relationship of lathyrus genus within the papilionoideae (evren et al. 1994, mantar et al. 2003, tosheva and tonkov 2005). taxonomy within the genus cannot be solved by morphological analysis (mantar et al. 2003). embryological characters and the study of the male reproductive organ within the lathyrus genus, mainly relating to meiotic chromosomes of pollen mother cells (latter 1925). a few studies described the female gametophyte development in lathyrus (rembert 1969, davies and williams 1985), but no attention has been given to the structural and cytological aspects of the male gametophyte. the present paper provides research on anther and pollen grain development in lathyrus undulatus, by the application of light, fluorescence and electron microscopy. material and methods flower buds of lathyrus undulatus boiss. (fabaceae) growing in natural habitats in the vicinity of beykoz-ýstanbul (turkey) were collected in march-april. one anther from the each flower bud was gently dissected and squashed in dapi (concentration 1 µg ml–1) for estimation of the development stage (schweizer 1976). flower buds were fixed in 3% glutaraldehyde in 0.05 m cacodylate buffer at ph 7.4 for 6 h at 4 °c and post-fixed in 1% osmium tetroxide in the same buffer for 4 h at 4 °c. the samples were dehydrated in ethanol series, and embedded in epoxy resin using propylene oxide. ultrathin sections (~70 nm) contrasted with uranyl acetate and lead citrate, and examined with a jeol jem 1011 transmission electron microscope (tem). for cytochemical observations, the osmication step was omitted from the fixation. semi-thin sections (1 µm) were stained with periodic acid-schiff (pas) (feder and o’brien 1968) for insoluble polysaccharides, coomassie brilliant blue (fisher 1968) for proteins, sudan black b for lipids (pearse 1961), alizarin red s (mcgee-russel 1958) for intracellular ca2+ and auramine o (heslop-harrison and shivanna 1977) for sporopollenin and exine. results the anther development of lathyrus undulatus boiss. was separated into 5 stages: premeiotic stage, tetrad stage, young microspore stage, vacuolated pollen stage and bicellular pollen grain stage. the undifferentiated anthers of l. undulatus were ovoid and consisted of meristematic cells encircled by an epidermal layer. concurrent with development, the anther turned out 54 acta bot. croat. 70 (1), 2011 vardar f., ünal m. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:01:58 color profile: disabled composite 150 lpi at 45 degrees to be tetrasporangiate. in each anther lobe 2–4 hypodermal cells differentiated into archesporial cells, going in a parallel plane to the outer wall of the anther (periclinal divisions), cutting off parietal cells toward the epidermis and primary sporogenous cells toward the interior of the anther. the cells of the parietal layer formed concentric layers of wall endothecial, middle and tapetal layer (dicotyledonous type) by a series of periclinal and anticlinal divisions. meanwhile the sporogenous cells enlarged and underwent mitotic divisions once generating pollen mother cells (pmcs). the enlargement of anthers was in progress during meiotic division of pmcs. pollen sacs combined by tissue fusion were subsequent to the pollen maturation. mature pollen grains scattered to the environment concurrent with dehiscence of stomium. in lathyrus undulatus an anther locule containing the developing microspores was bordered by four different layers: the tapetum, the middle layer, the endothecium and the epidermis at the early development stages. the secretory tapetal cells with emphatic nuclei and large volume resembled pmcs (fig. 1a). cytochemical reactions revealed that anther wall cells presented a weak reaction with regard to protein, insoluble polysaccharides, lipids and intracellular ca+2 as carried out by coomassie brilliant blue, pas, sudan black b and alizarin red s, respectively (figs. 1a, 2a, 3a, 4a). at the tetrad stage, the organic compounds and intracellular ca+2 started to accumulate in the tapetal cells in comparison to the other anther wall cells. the tapetal cell was polarized, and most of the cytoplasm was located on the locular side. the vacuole of tapetum grew progressively, and took up almost the whole cellular volume (figs. 1b, 2b, 3b). at young microspore stage, uninucleated tapetal cells enlarged and the cytoplasm became denser. insoluble polysaccharides, proacta bot. croat. 70 (1), 2011 55 anther development in lathyrus undulatus boiss. fig. 1. semi-thin sections of lathyrus undulatus anthers at different developmental stages stained with coomassie brilliant blue. a – pmcs at premeiotic stage and anther wall layers, epidermis (ep), endothecium (en), middle layer (ml) and tapetum(ta). b – enlarged tapetal cells at tetrad stage with large central vacuole. c – young microspore stage, tapetal cells with denser cytoplasm. d, e – vacuolated microspore stage, degenerating tapetum (arrow in e) and pollen-tapetum connection (arrow in d). f – bicellular pollen stage, disintegrated tapetal cells. bar denotes 10 µm in a–f. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:00 color profile: disabled composite 150 lpi at 45 degrees teins and lipids were indicated in the cytoplasm (figs. 1c, 2c, 3c). auramine o dye indicated cytoplasmic lipoidal globules containing sporopollenin precursors which were denser in the locular side of tapetum (fig. 3d). as well as the organic compounds, intracellular ca+2 was increased in the tapetal cytoplasm and nucleus (figs. 4b, c). at vacuolated pollen stage, the tapetum underwent important modifications. during this period, the volume of tapetal cell was reduced, the central vacuole collapsed, and the cytoplasm dispersed in the locule. the degenerating tapetal cytoplasm was abundant in protein, insoluble polysaccharides and calcium (figs. 1d, e; 2 d, e; 4d) although it was poor in lipoidal substances (figs. 3e, f). microspore mother cells of l. undulatus showed a regular meiotic division, and the processes of pollen development progressed normally within each individual anther as well as in all anthers of the same flower. meiotic division was followed by simultaneous cytokinesis. callose accumulation started at the corners of pmcs in the early stages of prophase i. microspore tetrads mostly showed a tetrahedral arrangement, and four microspores were separated from each other by a callose wall (fig. 5) as tapetal degeneration progresses, cytochemical tests showed that tapetal remnants consisted of dense protein and polysaccharides were in contact with the pollen grains. the contact between tapetal remnants and pollen exine were visible ultrastructurally (figs. 6b, c). endothecial wall thickenings initiated at the mentioned stage and reacted pas-positive (figs. 2e, f). at bicellular pollen stage, the middle layer and the tapetum degenerated completely. after degradation of the tapetum, epidermis and single row u-shaped endothecium existed in the mature anther wall, and pollen grains remained in the loculus (fig. 1f) 56 acta bot. croat. 70 (1), 2011 vardar f., ünal m. fig. 2. semi-thin sections of lathyrus undulatus anthers at different developmental stages stained with pas. a – pmcs at premeiotic stage and anther wall cells. b – enlarged tapetal cells at tetrad stage with large central vacuole. c – young microspore stage, tapetal cells with denser cytoplasm. d, e – vacuolated microspore stage, degenerating tapetum and pollen-tapetum connection (arrow). f – bicellular pollen stage. arrow denotes pas positive endothecial thickening. bar denotes 10 µm in a–f. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:03 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (1), 2011 57 anther development in lathyrus undulatus boiss. fig. 3. semi-thin sections of lathyrus undulatus anthers at different developmental stages stained with sudan black b (a-c, e) and auramine o (d, f). a – pmcs at premeiotic stage and anther wall cells. b – tetrad stage. c – young microspore stage, tapetal cells with denser cytoplasm. d, e – vacuolated microspore stage, cytoplasmic lipophilic globules containing sporopollenin precursors (arrow). f – pollen grains with exine. bar denotes 10 µm in a–f. fig. 4. lathyrus undulatus anthers at different developmental stages stained with alizarine red s. a – pmcs at premeiotic stage and anther wall cells. b – tetrad stage. c – young microspore stage, tapetal cells with intracellular ca2+ deposits. d – vacuolated microspore stage, degenerating tapetum (arrow). bar denotes 10 µm in a–d. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:07 color profile: disabled composite 150 lpi at 45 degrees ultrastructural observations confirmed that a pmc resembled a meristematic cell with spherical nucleus, dense cytoplasm and thin cell wall at premeiotic stage. at the end of meiosis, the callose wall surrounding tetrads underwent progressive lysis, and the young microspores were liberated into the locule cavity. the free microspores which had spherical and centrally located nucleus with one or two nucleoli started to round up (fig. 6a). many spherical lipid bodies and starchy plastids were distributed throughout the cytoplasm of the young microspore. then the vacuole expanded concomitantly with the increase in microspore volume, and the nucleus displaced to a peripheral position in the thin layer of cytoplasm (figs. 6b, c). division of the pollen took place in the vacuolated stage, and resulted in a large vegetative and generative cell (fig. 6d). at shedding time the pollen grains were 2-celled with a generative cell and a vegetative cell. the vegetative nucleus was spherical and usually had one nucleolus. the elliptical generative nucleus became elongated in further. the generative cell was bounded by a plasma membrane (fig. 6e). the mature pollen had the following morphological characteristics: 3-zonocolporate, prolate, tectate (imperforate) type of exine and perforate type of structure. the pollen wall 58 acta bot. croat. 70 (1), 2011 vardar f., ünal m. fig. 5. microsporogenesis in lathyrus undulatus anthers. a – pmc, b – prophase i., c – metaphase i., d – anaphase i., e – telophase i., f – metaphase ii., g – anaphase ii., h – telophase ii., i – tetrads., j – small young microspore, k – enlarged young microspores, l – mature pollen. bar denotes 10 µm. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:17 color profile: disabled composite 150 lpi at 45 degrees composed of exine and intine. the exine was thin in respect to the intine, initially. ultrastructural studies revealed that tectate was thick and composed of a darkly stained globular material. therefore the nexine was considerably thinner than the sexine. the intine formed an important constituent portion of the wall and consisted of two sublayers: an outer intine (exintine), presenting a thick zone, and an inner intine (endintine), which consisted of a reticular network. the endintine was thickened at the germ pore region. the pollen grain surface and anther dehishence is shown by scanning electron micrographs (figs. 7a, b). the bicellular state of the pollen grains of l. undulatus persisted to anthesis (figs. 6f, 7b). cytochemical experiments indicated that the cytoplasm of a mature pollen grain was filled with insoluble polysaccharides, proteins, lipids and calcium. it was detected that a well-defined exine was made up of lipid-like substances and proteins, but intine was composed of insoluble polysaccharides and proteins (fig. 8). acta bot. croat. 70 (1), 2011 59 anther development in lathyrus undulatus boiss. fig. 6. ultrastructure of pollen grains, from young microspore to bicellular polen stage. a – young microspore with central nucleus. b, c – vacuolization in microspore, degenerating tapetum and pollen connection (arrows). d – mature pollen grain with vegetative (vn) and generative nucleus (gn). e – mature pollen grain with elongated generative nucleus (arrow). f – exine and two sublayers of intine; exintine (ex) and endintine (en). bar denotes 5 µm (a-e) and 1 µm (f). u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:18 color profile: disabled composite 150 lpi at 45 degrees discussion beside chromosome number (güneª and çirpici 2008) and pollen morphology (güneª and aytuð 2010), the male gametophyte development was not investigated in lathyrus. the anther wall development in papilionoideae was reported as dicotyledonous type (davis 1966), including trifolium (hindmarsh 1964), pisum and lens (biddle 1979, indigofera (ashrafunnisa and pullaiah 1995), lotus glaber (galati et al. 2006). the 60 acta bot. croat. 70 (1), 2011 vardar f., ünal m. fig. 7. sem micrograph of mature pollen (a), and (h) anther dehiscence (b) in lathyrus undulatus. fig. 8. cytochemistry of lathyrus undulatus pollen grains. a – protein depositions stained with coomassie brilliant blue. b – insoluble polysaccharide depositions stained with pas. c – lipid depositions stained with sudan black b. d – intracellular calcium depositions stained with alizarin red s. bar denotes 10 µm. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:20 color profile: disabled composite 150 lpi at 45 degrees presented results confirmed that anther walls of l. undulatus also belonged to dicotyledonous type. the undifferentiated anther of dumasia miaoliensis (papilionoideae) was ovoid-shaped and tetrasporangiated (liu and huang 2003), as the observations were showed the similar evidences in lathyrus undulatus. furthermore periclinal divisions of sporogenous cells resulted in the formation of pollen mother cells in d. miaoliensis. the researchers stated that at early developmental stages the epidermal, endothecial and middle layer cells had prominent nuclei, large vacuoles, numerous rough endoplasmic reticula and mitochondria. besides, the microspore mother cells structurally resembled to the tapetal cells. analysis of glycine max has shown that the endothecial thickenings started to accumulate in inner tangential and radial wall at vacuolated pollen stage (albertsen and palmer 1979), the same in lotus glaber (galati et al. 2006). the middle layer degenerated at young microspore stage. they found similar timing of developmental alterations as we found in lathyrus undulatus. the uninucleate tapetal cells were commonly observed in members of mimosoideae and papilionoideae (buss and lersten 1975, albertsen and palmer 1979). the cells of secretory tapetum maintain their position, and eventually undergo degeneration in situ towards the end of pollen development (pacini et al. 1985). although, in recent years, it has been known that tapetal cells undergo programmed cell death during degeneration (papini et al. 1999, wu and cheung 2000), the time of the degeneration varies greatly from species to species. in l. undulatus, the uninucleated tapetal cells developed at young microspore stage, underwent degeneration at vacuolated pollen stage and degenerated completely at bicellular pollen stage. the tapetum has been considered to be the nutritive tissue for the developing pollen. in the secretory tapetum metabolites in the form of soluble carbohydrates, amino acids and peptides are released into the loculus from which they are taken up by the developing pollen (pacini 1994). the proteins are located in exine cavities (tectate grain) as pollenkitt/tryphine. it has been reported that (shivanna 2003) orbicules originate in the cytoplasm of the tapetal cells as lipoidal pro-orbicular bodies that accumulate below the membrane and eventually extrude to the cell surface (facing the locule) where they provide sporopollenin precursors for exine formation. according to our results, the tapetal cells of l. undulatus accumulate protein, carbohydrate and lipoidal substances during development, and after breakdown these derivatives contribute to pollen coat formation. the results confirmed that tapetal derivatives were in contact with pollen grains, and had a possible role in pollen maturation. several researchers reported that polysaccharides, proteins and lipids existing in pollen cytoplasm had important metabolic roles in pollen germination and pollen tube formation (bedinger 1992, hess 1993, li et al. 1995). calcium plays an important role in plant growth and development. it is implicated in the movement of cellular organelles such as the spindle apparatus and secretory vesicles, and may play a key role in integrating plant cell metabolism (manivannan et al. 2007). it has been shown that ca2+ is one of the key elements of apoptotic pathway and alteration of the intracellular ca2+ concentration can lead to cell death (olofsson et al. 2008). intracellular ca2+ increase in tapetal cells of l. undulatus as evidenced by the alizarin red s may be related to cell death. acta bot. croat. 70 (1), 2011 61 anther development in lathyrus undulatus boiss. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:20 color profile: disabled composite 150 lpi at 45 degrees cytochemical results of lathyrus undulatus anthers showed that the cells of epidermis, endothecium and middle lamella gave weak reaction for protein, polysaccharides, lipids and intracellular ca2+. however pas positive reaction showed that endothecial wall thickenings consisted of polysaccharide. in chenopodium rubrum (de fossard 1969) and triticale (bhandari and khosla 1982) endothecial thickenings were described as a kind of polysaccharide, a-cellulose. lathyrus undulatus underwent simultaneous cytokinesis in the pollen mother cells. isobilateral microspore tetrads in l. undulatus were shown to be comparable to vicia galileae (papilionoideae), as found by dane and merýç (1999). the morphological characters of pollen obtained from tem and sem studies were compatible with the other lathyrus species; however the intine structure was first indicated in the genus lathyrus. acknowledgements this work was supported by the research foundation of marmara university (bapko no. fen-dkr 151105-0227). references albertsen, m. c., palmer, r. g., 1979: a comparative light and electron microscopic study of microsporogenesis in male sterile and male fertile soy beans (glycine max l. merr.). american journal of botany 66, 253–265. ashrafunnisa, pullaiah, t., 1995: embryology of indigofera (fabaceae). taiwania 40, 391–402. bedinger, p. a., 1992: the remarkable biology of pollen. plant cell 4, 879–887. bhandari, n. n., khosla, r. 1982: development and histochemistry of anther in triticale cv. tri-1. i. some new aspects in early ontogeny. phytomorphology 32, 18–27. biddle, j. a., 1979: anther and pollen development in garden pea and cultivated lentil. canadian journal of botany 57, 1883–1900. 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(eds.), genetic control of self-incompatibility and reproductive development in flowering plants, 289–308. kluwer academic publishers, dordrecht. pacini, e., 2000: from anther and pollen ripening to pollen presentation. plant systems and evolution 222, 19–43. papini, a., mosti, s., brighigna, l., 1999: programmed cell death events during tapetum development of angiosperms. protoplasma 207, 213–221 pearse, a. g. e., 1961: histochemistry, theoretical and applied. j. a. churchill ltd., london. platt, k. a., huang, a. h. c, thomson, w. w., 1998: ultrastructural study of lipid accumulation in tapetal cells of brassica napus l. cv. westar during microsporogenesis. international journal of plant science 159, 724–737. rembert, d. h., jr., 1969: comparative megasporogenesis in papilionaceae. american journal of botany 56, 584–591. rodriguez-garcia, m. i., m’rani-alaoui, m., fernández, m. c., 2003: behavior of storage lipids during development and germination of olive (olea europaea l.) pollen. protoplasma 221, 237–244. schweizer, d., 1976: reverse fluorescent chromosome banding with chromomycin and dapi. chromosoma 58, 307–324. shivanna, k. r., 2003: pollen biology and biotechnology, 22. science publishers inc., new hampshire. tosheva, a., tonkov, s., 2005: pollen morphology of bulgarian species from the section orobus (l.) gren. et godr. (genus lathyrus, fabaceae). acta botanica croatica 64, 275–287 wu, h. m., cheung, a. y., 2000: programmed cell death in plant reproduction. development 44, 267–281. 64 acta bot. croat. 70 (1), 2011 vardar f., ünal m. u:\acta botanica\acta-botan 1-11\vardar.vp 28. o ujak 2011 16:02:20 color profile: disabled composite 150 lpi at 45 degrees 158 acta bot. croat. 80 (2), 2021 acta bot. croat. 80 (2), 158–168, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-016 issn 0365-0588 eissn 1847-8476 biodiversity and seasonal distribution of benthic diatom assemblages as an indicator of water quality of small karstic river in bosnia and herzegovina anita dedić1*, dubravka hafner2, ana antunović1, jasmina kamberović3, svjetlana stanić-koštroman1, martyn g. kelly4 1 university of mostar, faculty of science and education, department of biology, matice hrvatske bb, bih-88000 mostar, bosnia and herzegovina 2 bartulovići 4, hr-20357 blace, croatia 3 university of tuzla, faculty of natural sciences and mathematics, department of biology, urfeta vejzagića 4, bih75000, tuzla, bosnia and herzegovina 4 bowburn consultancy, 11 monteigne drive, bowburn, durham dh6 5qb, uk abstract – the aims of this paper were to describe seasonal changes in the qualitative and quantitative composition of diatom taxa and the potential application of benthic diatoms for ecological status evaluation. diatom indices (ips and ti) were calculated from data from three different locations along a longitudinal profile of the bunica, a small karstic river in bosnia and herzegovina. a total of 147 taxa were recorded in 12 samples. the most common taxa were meridion circulare (greville) c.agardh and ulnaria ulna (nitzsch) compère. physical and chemical analyses showed low concentrations of nutrients, good oxygenation, typical ph for carbonate bed/origin and generally oligotrophic conditions and high ecological status. all sites had similar physico-chemical conditions and there were only few seasonal differences. ordination of the diatom data showed that samples showed neither longitudinal nor seasonal patterns. median value for ips (16.8) and for ti (7.3) can be possible ‘‘expected’’ values for ecological status assessment for small karstic rivers in the mediterranean region. we propose the use of the phytobenthos intercalibration common metric (picm – an index that combines the ips and ti) as a national metric for countries developing wfd diatom methods at a late stage. one situation is described, and a solution, which is potentially transferable to other locations in bosnia and herzegovina and also to other countries facing similar challenges. keywords: bacillariophyceae, diatom indices, ecological status, mediterranean river, phytobenthos, picm introduction the water framework directive (directive 2000) led to the adjustment of existing assessment systems for water quality and encouraged the design of new classification systems for the ecological status of rivers. the new systems included new approaches to the definition of reference conditions (pardo et al. 2011, 2012, 2018), the establishment of national and european river typologies, the definition of ecological status class boundaries, and the mandatory intercalibration of boundaries between european countries (van de bund 2009). ecological status assessment has to be type-specific as streams have different biological communities and reference conditions due to their physical and morphological attributes, such as stream size, altitude, catchment geology, etc. benthic diatoms are one group of organisms widely used as ecological indicators in water quality and ecological status assessment and monitoring and are widely used as proxies for “phytobenthos” in wfd assessments. many indices based on species-specific sensitivities and tolerances have been developed to infer the environmental conditions in streams and rivers. the wfd requires that methods are expressed as ecological quality ratios (eqrs), defined as the “observed” (o) value of a metric divided by the value “expected” (e) at reference condition (o/e). in this study two * corresponding author e-mail: anita.dedic@fpmoz.sum.ba benthic diatoms of small karstic river acta bot. croat. 80 (2), 2021 159 diatom indices; ips (indice de polluo-sensibilite, cemagref 1982), and ti (trophic index, rott et al. 1999) were used to estimate ecological status in a small karstic river in bosnia and herzegovina (bh). it presents the relationship between measured water quality variables in the bunica river and values of diatom indices. two diatom metrics (ips and ti) were compared and then combined into the phytobenthos intercalibration common metric (picm, kelly et al. 2009). also, various diversity indices are used to determine the distribution of benthic diatoms. diversity index is a statistical method which is planned to evaluate the variety of a data group consisting of different types of components. number of existing species (richness), distribution of individuals equally (evenness) and total number of existing individuals underlie the basis of diversity indices. three diversity indices (the shannon diversity index, simpson diversity index, margalef diversity index) and one evenness index (pielou’s evenness index) were used in this paper. the purposes of this paper are: to present the list of benthic diatom assemblages; to assess the seasonal variations and ecological status of the river; to test the use of two diatom indices as tools for estimating the ecological status of small karstic rivers; and to explore the potential for using the picm as a national metric for countries such as bh which are developing wfd-compatible phytobenthos methods at a relatively late stage. material and methods study area the bunica river is a limestone karstic river in the south of bosnia and herzegovina and is a tributary of the buna river, itself part of the neretva river basin (fig. 1). in terms of geology and geomorphology the bunica river is part of the dinaric karst or dinaric carbonate platform – external dinarides (milanović 2006). thick layers of carbonateformed long carbide precipitation characterize this area. the dominant clasts of the sediment are cobbles and sand. the siphon spring of the bunica river is the underground extension of the flow of the zalomka river (fig. 1). the siphon is 73 m deep and ranks among the largest sources of the dinaric karst (milanović 2006). the minimum and maximum discharges registered for the bunica spring were 0.72 and 207 m3 s–1 (milanović 2006). it is located in an area with a mediterranean climate, characterized by long, hot summers and rainy winters with rare occurences of snow (galić 2011). the average annual air temperature is 14.6 °c. the highest daily average temperature is 23.2 °c in july and the lowest below 5.8 °c in january (data for the city of mostar, for the period 1971–2000, recorded by meteorological and hydrological service of bosnia and herzegovina). the total annual precipitation is about 1515 mm. this area has approximately 2291 hours of sunshine per year. average relative humidity is 69. so far, diatoms were studied only in the spring area of the bunica river, revealing high diversity of taxa on artificial (glass slides) and natural substrates (dedić 2015, dedić et al. 2015). sampling and analyses the ecological river typology in bosnia and herzegovina was developed according to system b of the wfd annex ii, which allows any natural environmental parameter-influencing communities to be included. a total of 216 water bodies of surface waters have been identified in the adriatic sea river basin district in the federation of bh, specifically 211 running waters, four (4) stagnant waters and one (1) coastal water body (mrđen et al. 2018). within this typology the bunica river belongs to type 12, subundertype 12a, small and medium lowland rivers on limestone sediment. for this study, a total of 12 benthic diatom samples (three sampling locations: the source of the river, the middle course, and the lower course, each each once per season: spring – may, summer – july, autumn – october in 2013, and winter in january 2014) were collected by scraping the biofilm from rock surface, applying the the standard procedure of scraping sludgy material from the rock surface while making sure that the total surface of all rocks is about 500 cm2 (cen 2003). the upper surface of each rock was scratched with a scalpel and carefully scrubbed with a toothbrush. the samples were fixed with 4% formaldehyde and stored in appropriately labelled bottles. in the laboratory, samples were treated with concentrated sulphuric acid, potassium-permanganate and oxalic acid (krammer and lange-bertalot 1986). the cleaned suspension was used to make permanent diatom microscope slide preparations. the composition and relative abundance of diatoms were estimated at 1000× magnification, using a carl zeiss jena light microscope. at least 400 valves were counted and identified. identification and nomenclature were based on the relevant scientific literature and keys: krammer and lange-bertalot (1986–1991), langebertalot (2001), krammer (2000–2003), krammer and lange-bertalot (2004). the nomenclature of taxa follows algaebase (guiry and guiry 2020). physico-chemical parameters (ph, electric conductivity (ec), dissolved oxygen (do), oxygen saturation (sa) and fig. 1. sampling sites marked with numbers 1, 2 and 3 along the studied bunica river, tributary of the river buna in bosnia and herzegovina. dedić a., hafner d., antunović a., kamberović j., stanić-koštroman s., kelly m. g. 160 acta bot. croat. 80 (2), 2021 temperature of water (t)) were measured at the same time that diatom samples were collected using a wtw probe (wissenschaftliche-technische wersättem gmbh & co. kg-weilheim). water samples were also taken and analysed by standard spectrophotometric methods in in the laboratory of public health mostar to current norms. the following chemical parameters were analyzed: dissolved oxygen (do), chemical oxygen demand (cod), nitrate (no3–), ammonium (nh4+), total nitrogen (tn), total phosphorus (tp), orthophosphate (po43–) and silica (sio2). diatom indices were calculated using omnidia gb 5.3 software ( lecointe et al. 1993). indices taken into account for the assessment of water quality are those with the highest proportion of species used in the calculation. values of indices in omnidia software range from 1 to 20, indicating the quality range from polluted to clean water. the following indices were used: ips (cemagref 1982) and ti (rott et al. 1999). we estimated diatom diversity using shannoń s (h´), simpsoń s (1-lambda), margalef ś (d) diversity indices and pielou ś evenness index (j). indices were calculated with the past software (hammer et al. 2001). chemical data were processed using principal component analysis (pca) to obtain different water quality groups. the variables used in these analyses were ph, electrical conductivity, dissolved oxygen, oxygen saturation, water temperature, chemical oxygen demand, ammonium, nitrate, total nitrogen, total phosphorus, orthophosphate and silica. diatom data were analyzed using primer v.6 (clarke and gorley, 2006): hierachical group average clustering, simprof test, principal coordinates analysis (pco), to represent the distribution of the communities; simper analysis (similarity percentage) to estimate the degree of similarity within and among different groups (i.e., reference vs non-reference), and to estimate the individual contribution and the importance of each taxon to the global similarity among groups. these analyses were based on the braycurtis similarity matrix which combines information on the relative abundance of species and faithfulness of the occurrence of species abundances in particular groups. the phytobenthos intercalibration common metric (picm) was conceived by kelly et al. (2009) as a means of converting national assessment metrics to a common scale. it is calculated as the average of eqrs (ecological quality ratios) computed using the indice de polluosensibilité specifique (ips, cemagref 1982) and the trophic index (ti: rott et al. 1999). the picm is defined as the average of two eqrs: icm = (eqrips + eqr ti )/2. the picm metric responds along a long gradient of inorganic and organic enrichment (kelly et al. 2009, 2012). for this study we adopted the “expected” values of 16.8 (ips) and 7.3 (ti) used for streams similar to the karstic streams in this study during the mediterranean phytobenthos intercalibration exercise (almeida et al. 2014). we also used the average position of the high/good and good/moderate ecological status class boundaries (0.9 and 0.69) established during this study as a starting point for understanding the ecological condition of these streams in comparison to others in the mediterranean basin. both metrics were calculated using omnidia version 5.3 (lecointe et al. 1993) and converted to eqrs by dividing the observed value by the ‘expected’ value. the picm was then transformed against national metrics and national eqrs for high/good and good/moderate status boundaries converted to equivalent values of picm. results physico-chemical and chemical variables per seasons the main abiotic variables measured in the bunica river are shown in tab. 1. nutrient concentrations are low, the water is well-oxygenated and ph is typical for water of tab. 1. main abiotic variables in the bunica river measured in three sites along a longitudinal profile of the bunica river once per season (spring, summer, autumn and winter in the period from 5th may 2013 to 9th january 2014). min. – minimum value, max. – maximum value, avg. – average value, sd – standard deviation, t – temperature, ec –conductivity, do – dissolved oxygen, sa – oxygen saturation, cod – chemical oxygen demand, nh4+ – ammonium, no3– – nitrate, tn – total nitrogen, sio2 – silica, tp – total phosphorus, po43– – orthophosphate. parameter/ station bunica 1 (n=4) bunica 2 (n=4) bunica 3 (n=4) min. max. avg. sd min. max. avg. sd min. max. avg. sd t (°c) 11 14.2 12.8 1.4 11.4 15.5 13.7 1.7 11.4 17 14.6 2.3 ph 7.6 8 7.8 0.17 7.7 7.9 7.8 0.05 7.8 7.9 7.8 0.04 ec (µs cm–1) 313 403 372.5 40.4 314 406 371.5 39.8 312 406 369.2 40.3 do (mg l–1) 9.65 12.25 11.28 1.16 11.2 12.7 11.88 0.66 11.43 13.3 12.2 0.84 sa (%) 90.1 119.3 106.9 13.4 90.1 127.9 107.2 15.6 103.4 132 120.8 12.3 cod (mg l–1) 0.77 1.28 1.02 0.23 1.02 3.58 2.07 1.17 0.89 2.43 1.47 0.66 nh4+ (mg l–1) 0 0 0 0 0 0.003 0.0007 0.0015 0 0.003 0.0007 0.001 no3– (mg l–1) 0.18 0.45 0.35 0.12 0.29 0.49 0.37 0.08 0.27 0.43 0.36 0.067 tn (mg l–1) 0.30 0.61 0.47 0.13 0.37 0.55 0.47 0.07 0.37 0.52 0.45 0.062 sio2 (mg l–1) 2.68 4.8 3.63 0.97 3.10 4.71 4.04 0.68 3.14 4.20 3.86 0.48 tp (mg l–1) 0.006 0.011 0.008 0.002 0.004 0.01 0.007 0.0009 0.006 0.008 0.0066 0.0009 po43– (mg l–1) 0.002 0.005 0.0035 0.00 0.002 0.003 0.0026 0.0007 0 0.005 0.0018 5.13 benthic diatoms of small karstic river acta bot. croat. 80 (2), 2021 161 carbonate bed/origin and generally oligotrophic conditions. water quality corresponds to natural status without anthropogenic influence, following anonymous (2014) and should support high ecological status. differences between sites along the bunica river, and fluctuations among the seasons were not pronounced. water temperature ranged from 11–17 °c (tab. 1) and showed relatively low values for all the seasons. the lowest seasonal fluctuations were recorded at the spring. dissolved oxygen concentration (9.65–13.3 mg l–1) and saturation (90.1–132%) (tab. 1) were high at all sites and in all seasons. conductivity ranged from 312 to 406 µs cm–1 (tab. 1) with the lowest values in spring, and the highest in winter. all sites had slightly alkaline ph values (7.6 – 8) (tab. 1). the most significant positive correlation was achieved among total nitrogen and silica (r = 0.9, p < 0.01), and negative correlation was found for total nitrogen and oxygen saturation (r = 0.677, p < 0.01). multivariate analysis of the main chemical variables (pca) in three dimensions accounted for 74.2% of the variance. the first three axes (pc1, pc2 and pc3) accounted for 45.4%, 18.9%, and 9.9% of the variance, respectively (tab. 2). the most important parameters for the pca axis 1 were tn and no3–, with the coefficients in the linear combinations of variables of 0.400 for tn, and 0.374 for no3–. the variables that weighted most for ordination for the pca axis 2 were the cod and po43– (coefficients in the linear combinations of variables: –0.477 and 0.432, respectively). the first axis differentiated spring and summer samples with low scores on pc1 from autumn and winter samples with high pc1 scores (fig. 2). analysis of benthic diatoms in total, 147 diatom taxa from 43 genera were identified in the 12 samples from the bunica river (tab. 3). the genera with the highest number of taxa were gomphonema (23 taxa), navicula (18), cymbella (10), nitzschia (10) and cocconeis (7). meridion circulare (greville) c.agardh and ulnaria ulna (nitzsch) p.comparé, recorded in all samples, followed by cocconeis placentula ehrenberg, c. euglypta (ehrenberg) grunow, c. lineata ehrenberg, diatoma vulgaris bory de saint-vincent, d. vulgaris var. capitulata grunow, d. vulgaris var. producta grunow, encyonema ventricosum (c.agardh) grunow, e. silesiacum (bleisch) d.g. mann, gomphonema olivaceum (hornemann) brébisson, melosira varians c.agardh, navicula tripunctata (o.f. müller) bory de saint-vincent, rhoicosphenia abbreviata (c. agardh) lange-bertalot and navicula cryptocephala kützing. all sampling stations had a similar species richness in all seasons. the number of taxa at bunica 1 (spring area) ranged from 30 (in winter and summer) to 43 (in autumn), from 33 (spring) to 53 (autumn) for bunica 2 (middle part of the river) and 40 (summer) to 49 (autumn) for bunica 3 (the mouth of river). values of diversity and evenness indices per samples are shown in tab. 4. shannon’s diversity index ranged from 1.80 to 2.75, simpson’s diversity index from 0.69 to 0.90, margalef ’s diversity index from 4.50 to 8.42 and pielou’s evenness index from 0.20 to 0.41. the higest values of indices were recorded for the middle part of river (bunica 2), mostly in summer (tab. 4). the lowest values were in spring but for different locations (tab. 4). simper analyses showed high disimilarity among the seasons. the dissimilarities among the samples in different seasons ranged from 60.83% (spring-summer) to 70.24% (autumn-winter). the spring group showed 36.8% similarity, and was characterized by melosira varians (mvar), diatoma vulgaris var. capitulata (dvca), meridion circulare (mcir) and gomphonema olivaceum (goli). the summer group showed 37.77% similarity, characterized by encyonema ventricosum (even), navicula tripunctata (ntpt), and cocconeis placentula (cpla). autumn samples showed the lowest similarity, 35%, with c. euglypta (cple), achnanthes sp. (acsp) and m. circulare (mcir) as the most typical species. winter samples had 35.9% similarity and were characterized by m. circulare (mcir), ellerbeckia arenaria (eare) and c. placentula (cpla). similar results were obtained for the analysis of longitudinal gradient/locations with dissimilarities ranging from 64.79% between the first and second sites to 68.6%, between the second and third locations. fig. 2. principal component analysis (pca) ordination diagram performed on the environmental parameters for all sampling stations (1, 2, 3) in the bunica river during four periods (a-spring, b-summer, c-autumn, d-winter) and overlapped with pearson correlation vectors with pc1 and pc2 axes (r > 0.4). abbreviations: ec – conductivity, t – temperature, nh4+ – ammonium, no3– nitrate, tn – total nitrogen, sio2 – silica, po43– orthophosphate, tp – total phosphorus, cod – chemical oxygen demand, do – dissolved oxygen, sat. – oxygen saturation. tab. 2. characteristic values of all five axes of principal component analysis (pca) with percentage variance for a total of 12 investigated parameters. pca 1 2 3 4 5 eigenvalues 5.44 2.27 1.19 1.04 0.872 % variation 45.4 18.9 9.9 8.7 7.3 cumulative % variation 45.4 64.3 74.2 82.9 90.1 dedić a., hafner d., antunović a., kamberović j., stanić-koštroman s., kelly m. g. 162 acta bot. croat. 80 (2), 2021 tab. 3. identified diatom taxa with their maximum abundances (%) in a total of 12 samples in the bunica river. taxon max (%) achnanthes inflata (kützing) grunow 8.33 achnanthes sp. 83.33 achnanthidium minutissimum (kützing) czarnecki 16.67 achnanthidium pyrenaicum (hustedt) h.kobayasi 41.67 amphipleura pellucida (kützing) kützing 8.33 amphora ovalis (kützing) kützing 41.67 amphora pediculus (kützing) grunow 58.33 amphora sp. 8.33 aneumastus sp. 8.33 aneumastus tuscula (ehrenberg) d.g.mann et a.j.stickle 25 aulacoseira italica (ehrenberg) simonsen 41.67 brachysira microcephala (grunow) compère 16.67 brebissonia lanceolata (c.agardh) mahoney et reimer 16.67 caloneis bacillum (grunow) cleve 8.33 caloneis placentula ehrenberg 8.33 caloneis silicula (ehrenberg) cleve 8.33 caloneis ventricosa var. truncatula (grunow) meister 33.33 campylodiscus noricus ehrenberg ex kützing 8.33 cocconeis euglypta ehrenberg 91.67 cocconeis lineata ehrenberg 91.67 cocconeis pediculus ehrenberg 66.67 cocconeis placentula ehrenberg 83.33 cocconeis placentula var. klinoraphis geitler 66.67 cocconeis pseudolineata (geitler) lange-bertalot 8.33 cocconeis robusta a. jurilj 25 cyclotella meneghiniana kützing 8.33 cyclotella sp. 75 cymatopleura apiculata w. smith 8.33 cymatopleura elliptica (brébisson) w. smith 25 cymbella affinis kützing 33.33 cymbella excisa kützing 8.33 cymbella excisiformis krammer 8.33 cymbella hungarica (grunow) pantocsek 8.33 cymbella hustedtii var. crassipunctata lange-bertalot et krammer 8.33 cymbella laevis nägeli 25 cymbella parva (w.smith) kirchner 16.67 cymbella parvula krasske 8.33 cymbella sp. 16.67 cymbella subleptoceros krammer 16.67 denticula elegans kützing 8.33 denticula tenuis kützing 33.33 denticula tenuis var. crassula (nägeli) hustedt 41.67 diatoma vulgaris bory 91.67 diatoma vulgaris var. capitulata grunow 83.33 diatoma vulgaris var. producta grunow 8.33 diploneis oblongella (nägeli ex kützing) a. cleve 25 ellerbeckia arenaria (moore ex ralfs) r.m.crawford 66.67 encyonema leibleinii (c. agardh) w.j.silva, r.jahn, t.a.veiga ludwig et m.menezes 25 encyonema minutum (hilse) d.g.mann 8.33 taxon max (%) encyonema silesiacum (bleisch) d.g.mann 66.67 encyonema ventricosum (c. agardh) grunow 91.67 encyonopsis microcephala (grunow) krammer 8.33 epithemia adnata var. saxonica (kützing) r.m.patrick 8.33 epithemia muelleri fricke 25 epithemia sp. 8.33 epithemia turgida (ehrenberg) kützing 16.67 eunotia arcus ehrenberg 16.67 eunotia valida hustedt 8.33 fragilaria vaucheriae (kützing) j.b.petersen 41.67 fragilaria recapitellata lange-bertalot et metzeltin 8.33 fragilaria sp. 41.67 gomphonema acuminatum ehrenberg 33.33 gomphonema angustum c. agardh 33.33 gomphonema apicatum ehrenberg 8.33 gomphonema augur ehrenberg 41.67 gomphonema auritum a.braun ex kützing 8.33 gomphonema calcareum cleve 25 gomphonema capitatum ehrenberg 8.33 gomphonema constrictum ehrenberg 8.33 gomphonema gracile ehrenberg 8.33 gomphonema grunowii r.m.patrick et reimer 8.33 gomphonema micropus kützing 8.33 gomphonema minutum (c.agardh) c.agardh 8.33 gomphonema montanum (j. schumann) grunow 8.33 gomphonema olivaceum (hornemann) brébisson 91.67 gomphonema parvulum (kützing) kützing 16.67 gomphonema productum (grunow) lange-bertalot et reichardt 8.33 gomphonema pseudoaugur lange-bertalot 8.33 gomphonema pumilum (grunow) e. reichardt et lange-bertalot 8.33 gomphonema sp. 50 gomphonema subclavatum (grunow) grunow 25 gomphonema tergestinum (grunow) fricke 25 gomphonema truncatum ehrenberg 33.33 gyrosigma acuminatum (kützing) rabenhorst 41.67 gyrosigma attenuatum (kützing) rabenhorst 8.33 gyrosigma sciotoense (w.s.sullivant) cleve 8.33 gyrosigma sp. 16.67 halamphora veneta (kützing) levkov 8.33 hantzschia amphioxys (ehrenberg) grunow 25 iconella helvetica (brun) ruck et nakov 33.33 iconella linearis (w.smith) ruck et nakov 25 iconella spiralis (kützing) e.c.ruck et t.nakov 8.33 melosira sp. 33.33 melosira varians c.agardh 91.67 meridion circulare (greville) c.agardh 100 navicula amphibola cleve 8.33 navicula capitatoradiata h.germain ex gasse 8.33 navicula cari ehrenberg 16.67 benthic diatoms of small karstic river acta bot. croat. 80 (2), 2021 163 however, similarity in species compostion was much higher between sampling periods at the first location of bunica river (40.1%) than at the second (29%) or third (25.7%) locations. the most abundant taxa at bunica 1 were: e. ventricosum, e. silesiacum and planothidium lanceolatum; at bunica 2 halamphora veneta, c. euglypta and c. placentula and at bunica 3 g. olivaceum, achnanthes sp. and c. placentula var. klinoraphis. hierarchical group average clustering and the simprof test followed by simper analysis identified two groups and three ungrouped samples. figure 3 shows the results of pco analysis conducted using a bray curis similarity matrix and overlain with hierarchical clustering with taxon max (%) navicula cincta (ehrenberg) ralfs 16.67 navicula cryptocephala kützing 75 navicula cryptonella lange-bertalot 16.67 navicula jakovljevicii hustedt 16.67 navicula lanceolata ehrenberg 8.33 navicula microdigitoradiata lange-bertalot 8.33 navicula oblonga (kützing) kützing 25 navicula radiosa kützing 50 navicula reinhardtii (grunow) grunow 50 navicula tripunctata (o.f.müller) bory de saintvincent 91.67 navicula trivialis lange-bertalot 25 navicula veneta kützing 8.33 navicula viridula (kützing) ehenberg 8.33 navicymbula pusilla (grunow) k.krammer 8.33 navigeia decussis (østrup) bukhtiyarova 8.33 neidium dubium (ehenberg) cleve 8.33 nitzschia acicularis (kützing) w.smith 16.67 nitzschia capitellata hustedt 8.33 nitzschia dissipata (kützing) rabenhorst 8.33 nitzschia linearis w. smith 41.67 nitzschia palea (kützing) w.smith 16.67 nitzschia pusilla grunow 8.33 nitzschia recta hantzsch ex rabenhorst 8.33 nitzschia sigmoidea (nitzsch) w.smith 33.33 nitzschia sp. 41.67 nitzschia sublinearis hustedt 41.67 odontidium mesodon (kützing) kützing 33.33 pinnularia microstauron (ehrenberg) cleve 8.33 pinnularia sudetica hilse 16.67 pinnularia sp. 8.33 placoneis gastrum (ehrenberg) mereschkovsky 8.3 planothidium hauckianum (grunow) round et bukhtiyarova 8.33 planothidium lanceolatum (brébisson ex kützing) lange-bertalot 25 reimeria sinuata (w. gregory) kociolek et stoermer 16.67 rhoicosphenia abbreviata (c. agardh) lange-bertalot 91.67 sellaphora bacillum (ehrenberg) d.g.mann 41.67 sellaphora gregoryana (cleve et grunow) metzeltin et lange-bertalot 8.33 staurosira construens ehrenberg 8.33 staurosira venter (ehrenberg) cleve et moeller 16.67 staurosirella lapponica (grunow) d.m.williams et round 8.33 surirella angustata kützing 41.67 surirella grunowii kulikovskiy, lange-bertalot et witkovski 16.67 surirella librile (ehrenberg) ehrenberg 33.33 surirella minuta brébisson ex kützing 8.33 tryblionella angustata w.smith 8.33 ulnaria acus (kützing) aboal 8.33 ulnaria danica (kützing) compère et bukhtiyarova 41.67 ulnaria oxyrhynchus (kützing) m.aboal 25 ulnaria ulna (nitzsch) compère 100 fig. 3. principal coordinates analysis (pco) diagram comparing the community composition between samples in different seasons. pco plot coded by the species vectors indicates taxa (a) and environmental vectors (b) positively correlated to pc1 and pc2 axes (r > 0.5; r > 0.3, respectively), and overlapped with the cluster groups based the 35% resemblance level of the group average clustering. abbreviations: acsp – achnanthes sp., cped – cocconeis pediculus, cpll – c. lineata, cplk – c. placentula var. klinoraphis, cple – c. euglypta, mvar – melosira varians, even – encyonema ventricosum, esil – e. silesiacum, goli – gomphonema olivaceum, mcir – meridion circulare, nrad – navicula radiosa, ntri – n. tridentula, uuln – ulnaria ulna; t – temperature, nh4+ – ammonium, sio2 – silica, ec –conductivity, po43– orthophosphate. tab. 3. continued dedić a., hafner d., antunović a., kamberović j., stanić-koštroman s., kelly m. g. 164 acta bot. croat. 80 (2), 2021 vectors given by the pearson correlation coefficient with environmental data and most significant taxa. the first group on the plot included all samples from location 1, except winter samples, summer and winter samples from location 2, and spring and winter samples from location 3. this group was characterized by g. olivaceum (goli), e. ventricosum (even), and m. circulare (mcir) up to 35% cumulative contribution and is associated with lower values of po43–, consumption of chemical oxygen demand (cod) and electrical conductivity (ec). the second group is composed of autumn samples from the second and third location and was characterized by c. euglypta (cple) and c. placentula var. klinoraphis (cplk) with up to 50% cumulative contribution; these samples are associated with higher values of sio2 and no3–. the ungrouped samples were 1d (dominated by p. lanceolata (alan) and c. placentula (cpla), 2a with high abundance of ulnaria oxyrhynchus (uoxy) and h. veneta (aven) and 3b, dominated by cymbella laevis (clae) and nitzschia pusilla (nipu). diatom indices the data were used to calculate values of ips and ti for all samples. ips calculations used 95–100% of diatoms per sample whilst ti calculations used 65–95%. the values of indices for ips ranged from 8.9 to 18.5 and for ti from 4.9 to 12.6. the two indices were not significantly correlated with each other or with measured physical and chemical parameters (p > 0.05). the picm values ranged from 0.21 (2a) to 1 (2c) (tab. 5), with mean values suggesting good status at bunica 3 but not at the other two sites (based on an intercalibrated boundary value of 0.69). the concentrations of physical and chemical parameters should mean that the bunica river can support high ecological status (tab. 5), so the reason for this mismatch needs to be explored. biotic index classes assigned by omnidia showed variations in results from high to low ecological status. ips values mostly in autumn season on all sites referred to the first category, while ti values mostly varied from fourth to fifth category, tab. 4. values of diversity and evenness indices in 12 samples at three stations on the bunica river. h´ – shannon’s diversity index, 1-lambda – simpson’s diversity index, d – margalef ’s diversity index, j – pielou’s evenness index. station season sample code h´ 1-lambda j d bu n ic a 1 spring 1a 1.94 0.71 0.24 4.50 summer 1b 2.51 0.88 0.41 4.82 autumn 1c 2.17 0.80 0.20 6.94 winter 1d 1.88 0.69 0.21 4.82 bu n ic a 2 spring 2a 1.85 0.69 0.20 5.40 summer 2b 2.75 0.91 0.41 6.15 autumn 2c 2.72 0.88 0.29 8.42 winter 2d 2.64 0.87 0.35 6.48 bu n ic a 3 spring 3a 2.44 0.85 0.25 7.27 summer 3b 2.26 0.81 0.25 6.13 autumn 3c 2.40 0.81 0.22 8.09 winter 3d 2.11 0.77 0.20 6.45 tab. 5. values of phytobenthos intercalibration common metric (picm) and water quality classes according to diatom indices (ips and ti) and chemical conditions at three stations on the bunica river. ips – specific pollution-sensitivity index (cemagref 1982), ti –trophic index (rott et al. 1999), nh4+ – ammonium, no3-– nitrate, tn – total nitrogen, tp – total phosphorus. station season sample code ips ti picm nh4+ no3tn tp bu n ic a 1 spring 1a ii v 0.57 i i i i summer 1b i v 0.64 i i i i autumn 1c i v 0.77 i i i i winter 1d ii v 0.47 i i i i bu n ic a 2 spring 2a iv v 0.21 i i i i summer 2b ii v 0.61 i i i i autumn 2c i iii 1 i i i i winter 2d ii v 0.61 i i i i bu n ic a 3 spring 3a i v 0.63 i i i i summer 3b ii iv 0.74 i i i i autumn 3c i iv 0.76 i i i i winter 3d i v 0.70 i i i i benthic diatoms of small karstic river acta bot. croat. 80 (2), 2021 165 indicating extremely high trophic status (tab. 5). however, these classes are over-simplistic and do not give nuanced insights into conditions in any particular river. the percentage of pollution tolerant taxa for site 1 were 0.3% in spring, 1.8% in summer, while for autumn and winter they were not registered. for site 2 there were 39.7 pollution tolerant taxa registered and for site 3 those taxa were not recorded. discussion in this paper the benthic diatom assemblages in karstic river in bh were studied in order to estimate ecological status and to test the future applicability of diatom indices. benthic diatoms have been recommended in recent decades as appropriate tools for pollution assessment in rivers (e.g., coste et al. 1991, whitton and kelly 1995), and have been used worldwide (porter et al. 2008, mangadze et al. 2015). although our results indicate that the nutrient concentrations of the water do not determine the composition and structure of the benthic diatom assemblage in the bunica river, this is likely to be due to the short gradient encountered during this study. all sites in all seasons had low nutrient concentrations and good oxygenation. the high oxygen saturation and water transparency, as well as low nutrient concentrations, indicate the oligotrophic character of the river. for the investigated stations in different seasons, no major seasonal fluctuations and differences in physical and chemical parameters were identified, which is probably due to the proximity and impact of springs along this short river (just 5.8 km in length). water temperature (11–17 °c) showed relatively low values for all seasons. the smallest fluctuations were closest to the spring due to the temperature stability of spring water. high values of dissolved oxygen (9.65–13.3 mg l–1) and saturation (90.1–132%) are due to lower water temperature, porosity of the rock aquifer and contact between groundwater and the atmosphere (cantonati 1998). higher conductivity values indicate higher mineralization and our results suggest that the bunica river has moderate mineralization. physical and chemical values in this study are in accordance with values for limestone substrate (mogna et al. 2015). the analysis of the diatom assemblages revealed high species diversity. the investigated sites in the bunica river were characterized by similar numbers of diatom taxa, and high species diversity indicates that there were favorable conditions for development. a similar number of diatom taxa (117) was recorded in the mura river (krivogradklemenčić and balabanič 2010), while in the bunica spring 104 (dedić et al. 2015) and 87 (hafner and jasprica 2010) taxa were recorded. although this is not universally applicable, similar correspondence between low levels of pollution and high species diversity were also observed by noga et al. (2014). the bunica river has a low level of organic pollution and it is not impacted by many anthropogenic stressors (high density of population, livestock farming, developed industry, etc.). this was also supported by diversity and evenness indices. although values of indices varied for longitudinal and temporal patterns they indicated that the structure of habitats were stabile and balanced. their variations reflect the heterogeneity of the diatom population. those genera that were well represented in this study (gomphonema, navicula and nitzchia) were also well represented in other studies of karstic rivers (wojtal 2009, dedić et al. 2015). all three genera are found across the whole range of the bunica river, with particular species favouring different levels of ions and nutrients. meridion circulare and ulnaria ulna were recorded in all samples. meridion circulare is a widespread, mesosaprobous and oligo to eutraphentic alkaliphilous diatom (van dam et al. 1994). ulnaria ulna occurs in oligoto polytrophic and oligo-saprobic to α-mesosaprobic waters (hofmann et al. 2013) and is also classified as alkaliphilous (van dam et al. 1994). the occurence of large numbers halamphora veneta in a spring at the middle location is, however, very unusual. it occurs in fresh and slightly brackish water. according to van dam et al. (1994) it is an alpha/polisaprobic and eutrophic taxa. taxa of the genera amphora, cocconeis and diatoma, frequently found in this research, have a high indicator values for ph, organic nitrogen, oxygen, saprobity and trophic state (van dam et al. 1994). diatoms are influenced by many factors including those that are site-specific on various temporal and species scales (denicola et al. 2004) as well as those that reflect human interventions in the environment. those factors include chemical properties of the water (potapova 1996), nutrient load (rott et al. 1997) and also flow velocity and substratum type (rimet 2009). the results of this study showed that physical and chemical variables and seasons did not have a notable effect on the diversity of benthic diatoms in the bunica river. it is possible that hydromorphological and other water conditions such as flow velocity, type of substrates, discharge of water, marginal vegetation in karstic river also influenced the structure and composition of benthic diatom assemblages more than physical and chemical variables. ips and ti did not always give consistent results along the entire length of the bunica river. one important reason is that they were developed for different purposes – ips measures “general degradation” whilst ti was developed, specifically, to measure the impact of inorganic nutrients. ips index showed better water quality along the entire length of the bunica river (mostly moderate, ii and iii class) compared with the ti trophic index, which indicated a poor or bad ecological status of the river (iv-v class). this variation has also been noted by others, kitner and poulícková (2003), poulíčková et al. (2004), and stenger-kovács et al. (2007), szczepocka and szulc (2009), kalyoncu and şerbetci (2013). because the indices were developed for different purposes, naive division of the scale into equal-sized segments, though an easy option (e.g., eloranta and soininen 2002), is unlikely to give meaningful results or consistency between indices. others have noted that indices developed in certain regions of europe were not effective in others (pipp 2002, rott et al. 2003). dedić a., hafner d., antunović a., kamberović j., stanić-koštroman s., kelly m. g. 166 acta bot. croat. 80 (2), 2021 our study also noted a mismatch between status assessment using the average picm boundary set during the mediterranean intercalibration exercise (almeida et al. 2014) and observations in this study. the nature of the bunica river is such that we should estimate high or good status based on diatom assemblages, whereas our results indicate less than good status at two of the sites. kamberović et al. (2019 a) used ti index in a study of springs on konjuh mountain (bh) and reached similar conclusions about the suitability of existing indices, recommending some modifications to the ti. our study has also identified potential mismatches between ecological status boundaries used elsewhere in the mediterranean basin and those used in bh, suggesting a need to recalibrate the picm, if it is to be used successfully here. based on this study, we recommend “expected” values for picm calculations of 16.8 and 7.3 for ips and ti, respectively. diatom indices have been shown to be one of the most effective tools for evaluating ecological status in european rivers (eloranta and soininen 2002, kelly et al. 2008); however, further testing of ips and ti diatom indices was needed to ensure their utility in small karstic rivers in bh. the suitability of indices depends, to some extent, on the percentage of taxa used for index calculation. in this study 95– 100% of taxa were used for the ips calculation, suggesting that it is highly suitable. for the ti, a smaller percentage was used (60–90%). in the study of epiphytic diatoms in lake modrac in northeastern bh, kamberović et al. (2019b) also concluded that the ips and ti were most suitable. however, consideration should also be given to how sensitivity values are assigned to taxa and, in this respect, the ips does not have a transparent process for determining such scores whilst the ti has never been updated since first published (rott et al. 1999). on the other hand, both are widely used around europe (poikane et al. 2016) and, therefore provide a means by which results from bh can be compared with data from other countries. rather than develop a new index, therefore, we have adopted the ‘‘phytobenthos intercalibration common metric’’ (picm, kelly et al. 2009), based on these two metrics, as an interim tool for evaluating ecological status using phytobenthos in bh. these values are similar to those proposed for mediterranean streams by kelly et al. (2012), and higher similarities were recorded in ips than in ti, but have the benefit for being more closely tuned to local conditions within bh. conclusions the bunica river has a high diversity of benthic diatom taxa (147). physical and chemical analyses indicated oligotrophic conditions in all locations and high ecological status. measured physical and chemical parameters and seasons did not have a notable effect on the biological diversity of benthic diatoms. it is possible that hydromorphological and other water conditions such as flow velocity, type of substrates, discharge of water, marginal vegetation had a greater influence on structure and composition of benthic diatom taxa than physical and chemical variables. the diatom indices ips and ti were used in our study and we concluded that combining them into the “phytobenthos intercalibration common metric’’ (picm) gave us more results that were more easily compared with those from elsewhere in europe than would have been the case if only a single index had been used. we therefore recommend picm as a good starting point for ecological status assessment in bh and propose it as a potential national metric for phytobenthos assessment in the future. however, testing on a larger number of sites and along a longer gradient of ecological quality is necessary before this can be confirmed. references almeida, s.f.p., elias, c., ferreira, j., tornés, e., puccinelli, c., delmas, f., dőrflinger, g., urbanič, g., marcheggiani, s., rosebery, j., mancini, l., sabater, s., 2013: water quality assessment of river using diatom metrics across mediterranean europe: a methods intercalibration exercise. science of the total environmental 476–477, 768–776. anonymous, 2014: decision on surface and groundwater characterization, reference conditions and parameters for water status assessment and water monitoring. official gazzete of federation of bosnia and herzegovina 01/14, 61–119 (in bosnian/croatian/serbian). cantonati, m., 1998: diatom communities of springs in the southern alps. diatom research 13, 201–220. cemagref, 1982: etude des méthods biologiques quantitatives d’appréciation de la qualité des eaux. rapport division qualite des eaux lyon – agence financière de bassin rhône – méditerranée – corse, pierre-bénite. cen, 2003: the european standard. water quality. guidance standard for the routine sampling and pre-treatment of benthic diatoms from rivers. en 13946. european committee for standardization, brussels. clarke, k.r., gorley, r.n., 2006: primer v.6. primer-e, plymouth. coste, m., boca, c., dauta, a., 1991: use of algae for monitoring rivers in france. in: whitton, b.a., rott, e., friedrich, g. 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10.37427/botcro-2021-026 issn 0365-0588 eissn 1847-8476 analysis of hypericum accessions by dna fingerprinting and flow cytometry anca butiuc-keul1,2,3, ana coste4,*, holger budahn1, frank dunemann1, anca farkas2,3, dragoș postolache5, evelyn klocke1 1 institute for breeding research on horticultural crops, julius kühn institute, federal research centre for cultivated plants, erwin-baur-str. 27, 06484 quedlinburg, germany 2 babeş-bolyai university, faculty of biology and geology, department of molecular biology and biotechnology, m. kogalniceanu st. 1, 400084 cluj-napoca, romania 3 babeş-bolyai university, centre for systems biology, biodiversity and bioresources, clinicilor st. 5-7 400006, cluj-napoca, romania 4 institute of biological research cluj-napoca, branch of national institute of research and development for biological sciences, republicii st. 48, 400015 cluj-napoca, romania 5 national institute for research and development in forestry “marin drăcea”, eroilor boulevard 128, voluntari, 077190 ilfov county, romania abstract – hypericum perforatum, h. umbellatum, h. maculatum, and h. hircinum accessions originating from botanical gardens across europe were examined by flow cytometry and molecular markers. 2c dna content of 17 hypericum perforatum accessions (hp) and the h. perforatum cultivar topaz amounted to between 1.56 pg and 1.62 pg. in four hp accessions some individual plants were found with a dna content corresponding to 6cx (2.34 2.39 pg). all plants of accession hp8 showed a dna content of 6cx (2.41 pg). in root tips of hp plants with an average dna amount of 1.58 pg, 32 chromosomes were detected, corresponding to 2n = 4x. this is the first ploidy and/or dna content report for h. umbellatum, h. maculatum and h. hircinum. h. umbellatum and h. maculatum, each contained 0.76 pg dna and 16 chromosomes were counted. the 2c dna content of h. hircinum was 1.00 pg with the best metaphase plate revealing 32 chromosomes. additionally, a combined marker analysis, based on inter-simple sequence repeats (issr) and sequence related amplified polymorphism (srap), was conducted to gain a better understanding of diversity especially within the accessions of h. perforatum. a total of 27 (11 issr and 16 srap) primer combinations were screened, showing 699 bands, of which 661 were polymorphic. upgma clustering revealed that accessions from the same geographic area tended to be more closely related, while h. maculatum was grouped separately from all h. perforatum accessions. both methods have shown similar sensitivities in detecting the genetic diversity of the analyzed genotypes. our results may be useful for hypericum breeding programs and the development of effective conservation strategies. keywords: chromosome number, dna, genetic diversity, molecular markers, st. john’s wort introduction hypericum l. (hypericaceae) is a species-rich genus that colonized the temperate regions of the northern hemisphere and underwent rapid radiation during the pleistocene ( scheriau et al. 2017). the genus consists of almost 500 species of shrubs, herbs and a few trees (nürk and blattner 2010), which were grouped into 36 sections (robson 1981). among hypericum species only h. perforatum is widely used in medicine. so far, a limited number of species within the genus was studied, and the chemical compounds of approximately three quarters of hypericum species have not been surveyed yet (karioti and bilia 2010). hypericum perforatum extracts have multiple effects as an antidepressant, antiviral, antimicrobial and anti-inflammatory drug due to the main constituents, such as naphthodianthrones (hypericin and pseudohypericin), phloroglucinol derivatives (hyperforin), and flavonoids (quercetin, quercitrin, hyperoside and rutin). * corresponding author e-mail: ana.coste@icbcluj.ro accepted, ahead of print version of the manuscript 81(1), 1st april 2022 please cite this article as: butiuc-keul a, coste a, budahn h, dunemann f, farkas a, postolache d, klocke e: analysis of hypericum accessions by dna fingerprinting and flow cytometry. acta botanica croatica, doi: 10.37427/botcro-2021-026 running title: analysis of hypericum accessions butiuc-keul a., coste a., budahn h., dunemann f., farkas a., postolache d., klocke e. 2 acta bot. croat. 81 (1), 2022 because the substances are present only in small amounts but of great commercial interest, there have been strong efforts to enhance their production by biotechnological methods (coste et al. 2011, franklin et al. 2016). for this purpose, molecular characterization of native plants and/or accessions is necessary in order to identify the genotypes and to develop molecular markers associated with valuable traits. in addition, the knowledge of the full range of ploidy variation is valuable for a proper management of genetic resources of hypericum ssp. extensive cytological investigations of hypericum perforatum have made this species a model for examining aposporous apomixis (barcaccia et al. 2007, mártonfi and mártonfiová 2011). this species has a basic chromosome number of 8, but mostly it is tetraploid (2n = 4x = 32), although diploids (2n = 2x = 16) and hexaploids (2n = 6x = 48) occur in natural populations (matzk et al. 2001). apomictic (polyploid) individuals can facultatively produce both sexual and variable apomictic seeds (matzk et al. 2001, 2003, barcaccia et al. 2007, galla et al. 2011). despite comprehensive studies about its modes of reproduction in relation to different ploidies, there is little knowledge about the distribution of different genome sizes and ploidies of individual plants within accessions, this distribution being an important feature especially for management of genetic resources. furthermore, aneuploid individuals have been identified in australian populations (2n-1 = 31) (mayo and langridge 2003). h. perforatum is hypothesized to hybridize easily with its sister h. maculatum, with mainly diploid populations (brutovská et al. 2000; barcaccia et al. 2007). up to date, there have been reports of two diploid subspecies for h. maculatum (subsp. maculatum and subsp. immaculatum) and one tetraploid subspecies h. maculatum subsp. obtusiusculum (koch et al. 2013). regarding h. hircinum, this species was first reported as a possible pentaploid cytotype, with chromosome number varying between 40 (loon and jong 1978, robson 1981) and 32 (matzk et al. 2003, castro and rosselló 2006). to our knowledge, no data regarding h. umbellatum ploidy level and dna content have been reported. in the present paper, flow cytometry (fcm) was conducted to estimate the 2c dna content of single plants of 17 accessions of h. perforatum (hp) from botanical gardens all over europe and the cultivar topaz as well as romanian accessions of wild species h. maculatum crantz (hm), h.  umbellatum a. kern. (hu) and h. hircinum l. (hh). the occurrence of various ploidies within the accessions was noted. for confirmation of the ploidy, chromosome counting was performed. molecular characterization of hypericum germplasm was accomplished by issr and srap markers. materials and methods plant material within this study, different accessions (populations) belonging to the genus hypericum and covering four species namely hypericum perforatum (hp), hypericum maculatum (hm), h. umbellatum (hu) and h. hircinum (hh) from different botanical gardens of europe were analyzed. in most of the accessions, seeds were from plants cultivated in botanical gardens (hp1-8, hp11-13, hp15, hm23, hh24) but some of them were collected from plants from natural populations (hp9-10, hp14, hp16-17, hu21, hm22) according to tab. 1. seeds were germinated in soil and plants grown in the greenhouse (at least 30 seeds/accession). flow cytometry analysis estimation of dna content and ploidy by fcm was performed using fresh leaf material from young plants. small amounts of leaf tissue of a sample and the internal standard were chopped together in 0.5 ml of galbraith’s buffer ( galbraith et al. 1983), supplemented with 0.5 ml partec cystain propidium iodide solution containing dnase-free rnase following the manufacturer’s instructions (partec) and filtered through a cell-strainer cap (bd falcontm) with 35 µm pore size. analyses were performed with a flow cytometer bd facs calibur (usa). for each sample, no fewer than 5000 particles were registered by 488 nm laser beam. for estimation of the nuclear dna content, raphanus sativus l. was used as an internal standard (2c = 1.11 pg; doležel et al. 1992). analyses were carried out for at least tab. 1. accessions of hypericum spp. studied and their origin (species code are given in parenthesis, * – seeds collected from plants cultivated in a botanical garden, ** – seeds collected from natural populations). accession name origin seeds / dna source h. perforatum (hp) 1 germany botanical garden ulm hp2* germany botanical garden frankfurt hp3* germany botanical garden regensburg hp4* germany humboldt university berlin hp5* germany botanical garden constance hp6* germany botanical garden hamburg hp7* switzerland botanical garden st. gallen hp8* austria botanical garden salzburg hp9** austria botanical garden graz hp10** france botanical garden nancy hp11* france botanical garden ville de renne hp12* france botanical garden talence hp13* poland botanical garden wrocław hp14** norway botanical garden oslo hp15* italy botanical garden trento hp16** italy botanical garden siena hp17** estonia läänemaa, hort bot tartu university hp cultivar ’topaz’ germany seed provider, fürstenwalde h. umbellatum (hu21) ** romania gilău h. maculatum (hm22) ** romania piatra craiului h. maculatum (hm23) * romania botanical garden cluj-napoca h. hircinum (hh24) * romania botanical garden cluj-napoca analysis of hypericum accessions acta bot. croat. 81 (1), 2022 3 five randomly selected individuals per accession and three repetitions for each individual. for accessions with differing cytotypes up to 30 individual plants were tested (tab. 2). data evaluation was accomplished with bd software cellquesttmpro. nuclear dna content was calculated using the linear relationship between the ratio of the 2c peak positions of the target species/internal standard on the area histogram of fluorescence intensities. the statistics were carried out using the real statistics resource pack version 4.9 for microsoft excel. squash preparations of root tip cells root tips were harvested from greenhouse plants and pretreated with 8-hydroxyquinoline for 2.5 hours at room temperature and fixed overnight in ethanol: glacial acetic acid (3:1 v/v) at 4 °c. the fixed root tips were transferred to 70% ethanol and stored at 4 °c. for further investigation fixed root tips were washed in distilled water for 10 min and then incubated at 37 °c for 45 min in an enzyme mix consisting of 4% celullase, 1% pectolyase and 45% acetic acid, ph 4.0. after being washed with distilled water, the tips were squashed on the slide in 45% acetic acid. if under a bright microscope in phase contrast metaphase chromosomes were observed, the slide was frozen for at least 15 min at -80 °c and the cover slip was blown off. after an air drying for 10 min or longer 15 µl dapi vectashieldò antifade mounting medium with dapi (4’, 6-diamidino-2-phenylindol, vector laboratories) was added. the chromosomes were detected and photographed in fluorescent light (microscope axioimager z1 with ccd-camera axiocam, zeiss). the image analysis was carried out with the software program isis (metasystems, germany). issr and srap markers analysis marker analysis was conducted with 90 individuals (5 individuals/available accession), a representative sub-set of accessions and individuals screened by flow cytometry. genomic dna was isolated from young leaves of plants grown tab. 2. dna content of accessions of hypericum perforatum (hp), h. umbellatum (hu), h. maculatum (hm), h. hircinum (hh) assessed with internal standard raphanus sativus (2c = 1.11 pg). -1, -2: plants belonging to the same accession but with different dna content. accession № of plants investigated № of measurements mean dna content (pg) standard deviation ploidy 1cx content (pg) hp1 5 15 1.58 0.03 4x 0.40 hp2 15 35 1.57 0.04 4x 0.39 hp3-1 15 33 1.58 0.03 4x 0.40 hp3-2 1 3 1.99 0.02 ? ? hp4 5 15 1.56 0.01 4x 0.39 hp5 5 15 1.59 0.02 4x 0.40 hp6-1 24 30 1.57 0.03 4x 0.39 hp6-2 2 7 2.38 0.05 6x 0.40 hp7-1 30 38 1.56 0.02 4x 0.39 hp7-2 5 10 2.34 0.02 6x 0.39 hp8 5 12 2.41 0.07 6x 0.40 hp9-1 25 33 1.58 0.03 4x 0.40 hp9-2 1 3 2.37 0.03 6x 0.40 hp10-1 23 44 1.57 0.02 4x 0.39 hp10-2 2 5 2.39 0.05 6x 0.40 hp11 5 15 1.59 0.03 4x 0.40 hp12 5 14 1.62 0.01 4x 0.40 hp13 5 15 1.58 0.01 4x 0.40 hp14 5 15 1.57 0.02 4x 0.39 hp15 5 15 1.59 0.02 4x 0.40 hp16 5 15 1.59 0.01 4x 0.40 hp17 5 15 1.59 0.02 4x 0.40 hp ’topaz’ 6 7 1.58 0.01 4x 0.40 hp (average for 4x) 188 369 1.58 0.01 0.40 hp (average for 6x) 15 37 2.38 0.02 0.40 other species hu21 5 13 0.76 0.02 2x 0.38 hm22 10 14 0.76 0.03 2x 0.38 hm23 5 14 0.76 0.01 2x 0.38 hh24 11 14 1.00 0.04 4x 0.25 butiuc-keul a., coste a., budahn h., dunemann f., farkas a., postolache d., klocke e. 4 acta bot. croat. 81 (1), 2022 under standard greenhouse conditions, by using the ctab method described by doyle and doyle (1987). dna concentration was estimated using a uv-vis spectral photometer nanodrop 8000. issr (inter simple sequence repeats) amplification was performed with 11 primers (rostami -ahmadvandi et al. 2013). for srap (sequence related amplified polymorphism) analysis, sixteen primer combinations (li and quiros 2001) were used. primer sequences are given in tab. 2. pcr amplification was performed in a 0.2 ml tube containing 12.5 μl 2x dreamtaq green pcr master mix (thermo fisher scientific, usa), 10.25 μl nuclease-free water (lonza, switzerland), 25 pmol of each primer (eurogentec, belgium) and 5 ng of genomic dna in a final volume of 25 μl. for issr analysis the geneamp pcr system 9700 (applied biosystems, forster city, usa) was programmed as follows: 94 °c for 4 min, 35 cycles of 94 °c for 30 sec, 46 °c for 30 sec and 72 °c for 55 sec followed by a final elongation step at 72 °c for 5 min. for srap analysis, the following program was used: 94 °c for 4 min, 5 cycles of 94 °c for 30 sec, 35 °c for 30 sec, and 72 °c for 55 sec followed by 30 cycles of 94 °c for 30 sec, 50 °c for 30 sec, 72 °c for 55 sec, and a final elongation at 72 °c for 5 min. amplification products were separated in 1.5% w/v agarose (cleaver scientific, united kingdom) gel in 1×tbe buffer (lonza, switzerland) and stained with 0.5 μg/ml ethidium bromide (thermo fisher scientific, usa). issr and srap patterns were assessed as dominant markers. band patterns for both marker systems were recorded in 1/0 matrices to determine the level of genetic similarity between the different accessions on the basis of jaccard’s coefficient (jaccard 1908). the resulting matrix of similarity was analyzed by the unweighted pairgroup method with arithmetic mean (upgma) and the dendrogram was obtained using multivariate statistical package 3.21 (kovach 2007). shannon’s information index (i) (shannon and weaver 1949) and expected heterozygosity (he) were calculated, using genalex software version 6.5 (peakall and smouse 2012). the polymorphism information content (pic) value of each individual locus was calculated according to sehgal et al. (2009) as: picj = 1–ʃpi2n=1 where i is the ith allele of the jth marker, n is the number of alleles at the jth marker and p is the allele frequency. resolving power (rp) for the individual marker system was bases on the distribution of detected bands within the sampled clones and was calculated based on the formula described by prevost and wilkinson (1999): rp = σib where ib (informativeness) is 1[2 x |0.5-p|] and p is the ratio of present bands among the analyzed accessions. results estimation of nuclear dna content and determination of ploidy the flow cytometric measurements consistently showed a high reproducibility in repeated measurements of the same plant despite the presence of numerous secondary metabofig. 1. flow cytometric histograms (1: 2c peak of internal standard raphanus sativus, 1.11 pg). a – hypericum perforatum hp7-1: 2: 2c = 4cx peak, 1.55 pg, b – hp7-2: 2: 2c = 6cx peak, 2.34 pg, c – h. maculatum hm23: 2: 2c = 2cx peak, 0.76 pg. analysis of hypericum accessions acta bot. croat. 81 (1), 2022 5 lites, which often influence the peak performance. the cv (coefficient of variation) of histogram peaks was below 5.0 in each case. raphanus sativus served as an internal standard for genome size estimation. both 2c peaks from internal standard and hypericum sample were well separated from one another (fig. 1). the genome size of 17 hp accessions and the cultivar topaz of the tetraploid genotypes amounted to between 1.56 pg and 1.62 pg (tab. 2, fig. 1a) with an average genome size of 2c = 4cx = 1.58 pg. the hypericum accessions were obtained from various european botanical gardens and from romanian regions (tab. 1). the accession hp8 from the salzburg botanical garden, austria, revealed for five tested plants a hexaploid cytotype with a dna content of 6cx = 2.41 pg (tab. 2). besides tetraploid plants we found in hp6, hp7, hp9 and hp10 at least one plant which showed a genome size corresponding to the hexaploid chromosome level (fig. 1a, b), taking it into account that the 1cx dna content for all hp accessions amounted to 0.40 pg. however, one exception was noticed. in the hp3 from the botanical garden in regensburg, germany, one plant with a genome size of 1.99 pg was observed. only speculation could be offered about the nature of this cytotype. for both romanian h. maculatum accessions as well as for h. umbellatum a small genome size with 0.76 pg was estimated (fig. 1c). the genome size of h. hircinum was 1.00 pg (tab. 2). thus, the 1cx value of h. hircinum differed considerably from the others and was only 0.25 pg. chromosome number for correct assignment of genome size to ploidy, the numbers of metaphase chromosomes were counted in stained root tips. the chromosomes of hypericum are very small and morphologically similar, making chromosome counting difficult. in addition, the plant tissue digestion posed problems with regard to achieving well-spread chromosome plates. the unsatisfactory quality in connection tab. 3. srap (sequence related amplified polymorphism) and issr (inter-simple sequence repeats) primers used for amplification with their respective codes and nucleotide sequences. primer sequence primer sequence srap issr srp2 fw 5'-tgagtccaaaccggagc-3' rv 5'-gactgcgtacgaattaat-3' a (gaca)3rt srp5 fw 5'-tgagtccaaaccggaag-3' rv 5'-gactgcgtacgaattaat-3' c (gacac)2 srp6 fw 5'-tgagtccaaaccggata-3' rv 5'-gactgcgtacgaatttgc-3' ubc808 (ag)8c srp11 fw 5'-tgagtccaaaccggata-3' rv 5'-gactgcgtacgaattgac-3' ubc809 (ag)8g srp12 fw 5'-tgagtccaaaccggagc-3' rv 5'-gactgcgtacgaattgac-3' ubc811 (ga)8c srp13 fw 5'-tgagtccaaaccggaat-3' rv 5'-gactgcgtacgaattgac-3' ubc112 (gaca)4 srp14 fw 5'-tgagtccaaaccggacc-3' rv 5'-gactgcgtacgaattgac-3' ubc818 (ca)8g srp15 fw 5'-tgagtccaaaccggaag-3' rv 5'-gactgcgtacgaattgac-3' ubc855 (ac)8yt srp17 fw 5'-tgagtccaaaccggagc-3' rv 5'-gactgcgtacgaatttga-3' ubc856 (acac)4yg srp20 fw 5'-tgagtccaaaccggaag-3' rv 5'-gactgcgtacgaatttga-3' ubc857 (ac)8t srp25 fw 5'-tgagtccaaaccggaag-3' rv 5'-gactgcgtacgaattaac-3' ubc873 (atg)6 srp26 fw 5'-tgagtccaaaccggata-3' rv 5'-gactgcgtacgaattgca-3' srp27 fw 5'-tgagtccaaaccggagc-3' rv 5'-gactgcgtacgaattgca-3' srp28 fw 5'-tgagtccaaaccggaat-3' rv 5'-gactgcgtacgaattgca-3' srp29 fw 5'-tgagtccaaaccggacc-3' rv 5'-gactgcgtacgaattgca-3' srp30 fw 5'-tgagtccaaaccggaag-3' rv 5'-gactgcgtacgaattgca-3' butiuc-keul a., coste a., budahn h., dunemann f., farkas a., postolache d., klocke e. 6 acta bot. croat. 81 (1), 2022 with low mitotic index resulted in a fluctuating number of chromosomes being counted. in the accessions hp3, hp4, hp7, and hp9 we noticed 30 – 34 chromosomes, mainly 32 (fig. 2a, tab. 4). in h. umbellatum and h. maculatum 16 chromosomes were detected (fig. 2b, tab. 4). in squash preparations of h. hircinum 30 – 40 chromosomes were found, in the best preparations 32 chromosomes (fig. 2c, tab. 4). especially in h. hircinum, chromosomes often so stuck together that even at different focal levels an unambiguous assessment of the chromosomes was problematic. issr and srap marker analysis in all, 699 amplified dna bands were scored using 11 issr primers (298 markers) and 16 srap primers (401 markers) for the 17 h. perforatum accessions and one h. maculatum accession (hm22). the overall number of detected individual bands per primer ranged from 9 (srp17) to 53 (srp30), with an average of 27.1/25.1 (issr/srap) (tab. 5). the combined analysis of issr and srap markers revealed a total of 661 (94.6%) polymorphic bands. the marker performance was estimated by two parameters: pic value and resolving power (rp). issr primers and srap primer combinations showed the same mean pic value (0.38). the highest pic value was determined for primers ubc808, ubc809, ubc857, ubc873 and srp26 (0.49). the resolving power (rp) of primers tested varied between 0.44 (ubc818/ srp29) and 1.69 (ubc112). the mean values for shannon’s information index (i) and expected heterozygosity (he) were 0.39/0.25 for issr primers, and 0.44/0.29 for srap markers (tab. 5). combined issr-srap upgma revealed two major clusters (fig. 3): cluster i comprising all h. perforatum accessions and cluster ii represented by h. maculatum (hm22) as a clearly separated outgroup. within cluster i we observed 6 subclusters: hp1 and hp2 from germany; hp7 (switzerland) and hp9 (austria); hp13 (poland) and hp14 (norway); hp5 and hp6 from germany. french hp10 and hp11 and italian hp16 and hp15 grouped with hp17 from estonia. four h. perforatum accessions, hp3 and hp4 from germany as well as hp8 from austria and hp12 from france were separated in individual branches from the other genotypes. h. maculatum is clearly distinguished from all h. perforatum accessions. discussion nuclear dna content and ploidy the key to a successful breeding program is a better understanding of the extent and nature of genetic diversity present in wild, conserved and/or actively utilized germplasm of various species. there is a broad interest in gaining a better understanding of diversity within hypericum species, especially in h. perforatum, due to its pharmaceutical importance but also for its remarkable evolutionary and adaptive capacities. therefore, we employed fcm for analysis of ploidy and genome size as well as two types of molecular markers (issr and srap) to reveal the genetic diversity and relationships among several hypericum accessions. fcm is a powerful tool for genome size estimation and ploidy determination. with all the advantages and possifig. 2. chromosomes in root tips stained with dapi. a – hypericum perforatum hp7, 32 chromosomes, b – h. maculatum hm22, 16 chromosomes, c – h. hircinum hh24, 32 chromosomes. scale bar: 5 µm. tab. 4. chromosome number in root tips of different hypericum accessions (hp h. perforatum, hu – h. umbellatum, hm – h. maculatum, hh – h. hircinum). accession № of chromosome counts counted chromosomes hp3 3 31 32 hp4 2 30 31 hp7 23 28 34, of which 10 times 32 chromosomes hp9 1 32 34 hu21 11 14 16 hm22 9 13 16 hh24 13 30 40, of which 5 times 32 chromosomes analysis of hypericum accessions acta bot. croat. 81 (1), 2022 7 bilities of fcm, however, it must be taken into account that the results are always expressed in relation to a known standard. for genome size estimation (pg value) a plant with an already defined dna content as internal standard should be measured together with the sample plant in the staining solution. for ploidy estimation, plants with a cytologically verified chromosome number within the same species serve as standard, providing that different ploidy degrees are in linear dependency. for 17 h. perforatum accessions from different botanical gardens across europe and the cultivar topaz, the genome size was on average 1.58 pg ranging from 1.56 to 1.62 tab. 5. estimation of the genetic diversity of 18 hypericum accessions. tnb – total number of bands, npb – number of polymorphic bands, rp – resolving power-average, pic – polymorphic information content, i – shannon’s information index, he – expected heterozygosity, issr – inter-simple sequence repeats, srap – sequence related amplified polymorphism. primer detected amplification products % of polymorphic loci rp pic i he tnb npb issr a 23 21 91.3 0.99 0.45 0.43 0.28 c 13 13 100.0 0.50 0.25 0.33 0.20 ubc808 24 20 83.3 1.38 0.49 0.49 0.33 ubc809 21 18 85.7 1.25 0.49 0.44 0.29 ubc811 46 46 100.0 0.58 0.27 0.41 0.25 ubc112 20 8 40.0 1.69 0.35 0.23 0.16 ubc818 31 31 100.0 0.44 0.21 0.33 0.20 ubc855 42 42 100.0 0.51 0.25 0.33 0.20 ubc856 24 22 91.7 0.98 0.45 0.42 0.27 ubc857 23 21 91.3 1.21 0.49 0.47 0.31 ubc873 31 27 87.1 1.27 0.49 0.45 0.30 total 298 269 90.3 average 27.1 24.5 0.98 0.38 0.39 0.25 srap srp2 22 22 100.0 0.92 0.42 0.47 0.31 srp5 22 22 100.0 0.76 0.35 0.45 0.29 srp6 25 23 92.0 0.81 0.39 0.42 0.27 srp11 21 21 100.0 0.78 0.37 0.43 0.28 srp12 18 18 100.0 0.97 0.42 0.54 0.37 srp13 26 26 100.0 0.88 0.41 0.46 0.30 srp14 26 26 100.0 0.90 0.41 0.47 0.31 srp15 16 16 100.0 1.08 0.45 0.55 0.38 srp17 9 9 100.0 0.68 0.36 0.31 0.19 srp20 21 20 95.2 0.89 0.41 0.47 0.31 srp25 25 25 100.0 1.09 0.46 0.52 0.35 srp26 22 18 81.8 1.37 0.49 0.48 0.33 srp27 30 29 96.7 1.01 0.44 0.52 0.35 srp28 30 30 100.0 0.46 0.22 0.33 0.20 srp29 35 35 100.0 0.44 0.21 0.32 0.19 srp30 53 52 98.1 0.58 0.29 0.34 0.21 total 401 392 97.8 average 25.1 24.5 0.85 0.38 0.44 0.29 issr + srap total 699 661 94.6 average 25.9 24.5 0.90 0.38 0.42 0.28 butiuc-keul a., coste a., budahn h., dunemann f., farkas a., postolache d., klocke e. 8 acta bot. croat. 81 (1), 2022 pg in the different tetraploid accessions. chromosome counting in root tips of different hypericum plants with the corresponding genome size of 1.58 pg revealed 2n = 4x = 32 chromosomes. these results are in agreement with temsch et al. (2010) (2c = 1.59 pg), and alan et al. (2015) (2c = 1.50 pg). the slight differences result from employment of different standards, extraction / staining buffers and flow cytometers (doležel and bartoš 2005) but the use of various accessions and the physiological state of the plant material also have an influence on the measurement. matzk et al. (2003) developed the flow cytometric seed screen (fcss), boosted the investigation of apomixes of hypericum and broaden the knowledge about apomictic pathways in general. however, reports about the germination capacity of the differently developed seeds and their contribution to plant populations are very limited. thought, such information would have an impact on germplasm management and preservation strategies of rare natural populations. mixed cytotypes in populations could provide novel traits for crop development and therefore the seed samples in gene banks should be adapted to that situation. hypericum perforatum is predominately tetraploid (matzk et al. 2003, galla et al. 2011). barcaccia et al. (2007) describe wild populations of h. perforatum in more detail as populations with diploid and polyploid (mainly tetraploid) plants. savaş tuna et al. (2017) analyzed three seedlings from 39 hp accessions each from different regions of turkey and revealed a nuclear dna content between 0.8 – 2.57 pg. of the 39 accessions, one was diploid, 5 hexaploid and 33 tetraploid but no ploidy variation was noticed inside the accessions. perhaps the test of only three seedlings per accession is not sufficient to provide reliable findings. here we present flow cytometric dna size determination of plants of 17 belonging to hp accessions and the cultivar topaz. tetraploidy was revealed with only one exception: all plants from hp8 from salzburg have shown a dna size of 2.41 pg, corresponding to the hexaploid level. this uniformity could be explained by the origin of seeds in plants cultivated in botanical garden, most probably the collection being not very diverse. in four accessions (hp6-7 and hp9-10), there were mixed cytotypes (4x + 6x). this is an interesting situation, as hp 6-7 seeds were collected from plants cultivated in botanical gardens and hp 9-10 belong to plants from natural populations. similar results were found in three h. perforatum accessions (qu et al. 2010). despite the diverse embryo and endosperm ratios observed in seeds, qu et al. (2010) found only tetraploids and hexaploids in seedling populations with tetraploids constituting 87 97% but a complete hexaploid population was not detected. among the accessions presented here the accession hp8 shows only hexaploid plants indicating that such accessions at least with a high proportion of hexaploid plants could exist and depend on the seed source. moreover, in accession hp3 a single plant was found with 1.99 pg. since it does not fit the 1c content of 0.40 pg, one can assume that it is an aneuploid plant reflecting additionally the high plasticity of the h. perforatum genome, even if hp3 comes from plants that are cultivated in a botanical garden. to our knowledge we report for the first time the genome size of h. maculatum (two accessions, one from a natural habitat and one cultivated in a botanical garden), h. umbellatum (from natural habitat) (0.76 pg each) and h. fig. 3. upgma dendrogram generated by jaccard’s similarity coefficients showing relationships among 18 hypericum accessions based on combined data from issr-srap (inter-simple sequence repeats analysis – sequence related amplified polymorphism). the samples are labeled with the codes listed in tab. 1. analysis of hypericum accessions acta bot. croat. 81 (1), 2022 9 hircinum (from botanical garden) (1.00 pg). the first three accessions are native to romania. hence, the dna size of h. hircinum of 1.0 pg is between the estimated dna size for the diploid species h. maculatum und h. umbellatum (0.76 pg) and the tetraploid h. perforatum (1.58 pg). the genome size for h. maculatum and h. umbellatum corresponds to the chromosome number 2n = 2x = 16. for h. hircinum loon and jong (1978) published a chromosome number 2n = 40. they explained the high chromosome number of 40 as a possible pentaploid cytotype. castro and rosselló (2006) found 2n = 32 in h. hircinum subsp. cambessedesii, an endemic plant from the balearic islands. the h. hircinium accession investigated in the present paper was provided by the alexandru borza botanical garden from cluj-napoca, romania. in this accession, we observed 30 40 chromosomes; in the best metaphase plates 32, which is in agreement with the findings of castro and rosselló (2006). the result was surprising because h. hircinum has a much lower dna size than h. perforatum (1.58 pg) although it also has 32 chromosomes. this fact underlines the high variability of the genus hypericum. genetic polymorphism characterization of hypericum species by different molecular marker types has been performed over the years (corral et al. 2011, he and wang 2013). issr and srap markers were chosen due their advantages: cost efficiency, informativeness, versatility and reproducibility. we employed a set of 11 issr primers and 16 srap primer combinations to assess the genetic diversity among one hm and 17 hp accessions. issr and srap markers have proved to be highly polymorphic. the average number of polymorphic bands per primer was the same (24) for both types of markers. several studies report that when several marker types are used, they can be complementary tools for genetic diversity analysis, because they can be used to amplify different regions of the genome (chen et al. 2013). our study revealed a significantly higher rate of polymorphism in the analyzed hypericum germplasm for both srap (97.8%) and issr (90.2%) markers than previously reported (he and wang 2013). this might be explained by a larger sampling area with very different hp accessions from botanical gardens in europe and native wild accessions from romania. the two marker systems used in our study revealed close degrees of resolution (tab. 5). species cluster analyses based on combined issr-srap data (fig. 3) indicate that h. maculatum is closely related to some h. perforatum accessions. these taxa, belonging to section hypericum “core hypericum” (nürk 2011), were proven to be in close contact and apparently hybridize frequently, which might explain the sympatric occurrence of morphologically similar taxa (robson 2003, koch et al. 2013). thus, according to brutovská et al. (2000), h. perforatum probably originates from autopolyploidization of an ancestor closely related to diploid h. maculatum, while robson (2003) regards h. perforatum as an allopolyploid, derived from a cross between h. maculatum subsp. immaculatum and h. attenuatum. this close relationship between the two species was also supported by later studies based on different marker types, such as rflp and cpdna markers, as well as phylogenetic studies using nuclear internal transcribed spacer (its) sequences (pilepić et al. 2011). however, it was recently implied that h. perforatum is not of hybrid origin (koch et al. 2013). the authors suggest that h. perforatum has a single evolutionary origin arising from independent and recurrent polyploidization of two different ancestral gene pools along with occurrence of substantial gene flow within and between h. perforatum and h. maculatum. regarding the cluster analysis of h. perforatum accessions, we have noticed a partially regional-based relationship. some accessions from the same regions shared similar genotypes and ploidy levels (hp1 and hp2 from germany; hp15 and hp16 from italy), and the same was noticed for accessions from different geographical locations (hp9 from austria and hp7 from switzerland; hp13 from poland and hp14 from norway) (tab. 1, 3; fig. 3). moreover, mixed ploidy accessions (4x and 6x) (hp5 and hp6; hp9 and hp7; hp10 and hp11) shared similar genotypes, while the complete hexaploid accession hp8 from austria is clearly of different genetic origin from that of the tetraploid hp9 accession from the same country (tab. 1, 3; fig. 3). this endorses the high plasticity in ploidy and reproductive system of h. perforatum, regardless of geographic origin (koch et al. 2013). differences among h. perforatum genotypes were reported in different h. perforatum wild populations and landraces as confirmed by molecular, morphometric and cytogenetic analyses (he and wang 2013, morshedloo et al. 2014). the high genetic diversity exhibited by our analysis might be explained by the diverse mating systems of h. perforatum from sexual cross to apomixis. conclusions we report the 2c dna content of 17 h. perforatum accessions from different botanical gardens in europe. 2c dna content of h. perforatum found in our study was 1.58 pg. the tetraploid degree of the plants was confirmed by chromosome counting. fcm is a fast and reliable method for screening the variability inside a hypericum accession concerning ploidy distribution. besides the tetraploids, few hexaploid plants and one putative aneuploid plant were found in the h. perforatum accessions, independent of the origin of the seed. mixed cytotypes (4x+6x) were identified in accessions from natural populations and cultivated in botanical gardens as well. one h. perforatum accession was characterized as hexaploid. the genome size of h. maculatum, h. umbellatum and h. hircinum was not previously reported and is 0.76 pg dna for h. maculatum and for h. umbellatum, whereas h. hircinum has 1.00 pg dna. this study demonstrated that both issr and srap markers were highly polymorphic in hypericum, showing the prevalence of a wide range of diversity among the studied accessions. the relative performance of issr and srap butiuc-keul a., coste a., budahn h., dunemann f., farkas a., postolache d., klocke e. 10 acta bot. croat. 81 (1), 2022 markers was quite close, indicating that these markers are suitable for the determination of genetic diversity with high resolution among the hypericum genotypes tested. overall, marker analysis ensures information for potential applications of the srap and issr marker systems in molecular breeding of hypericum species. complementary analysis of ploidy level and molecular markers of different accessions of hypericum species could provide information for the selection of valuable accessions producing high level of natural compounds useful for biotechnological applications. acknowledgments this work was supported by daad scholarship (research stays for university academics and scientists, 2016, id-57210259), bioserv 25n/2019 (core program pn20192022 biodivers 3) and 22pfe/2018. the authors acknowledge to dr. alexandra șuteu from alexandru borza botanical garden, cluj-napoca, romania for providing the seeds and information about the origin of the seeds, simone abel for technical assistance of fcm analysis, anika kunze for technical assistance of molecular analysis and kerstin maier for root tip preparations. references alan, a.r., murch, s.j., saxena, p.k., 2015: evaluation of ploidy variations in hypericum perforatum l. 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imehregan@srbiau.ac.ir introduction plants are an important component of traditional food, but are also central to healthy diets of the modern urban population (benjak et al. 2005, ercisli 2009, ercisli et al. 2010, rop et al. 2014, canan et al. 2016, zorenc et al. 2016). essential oils obtained from plants have applications in food, chemistry, pharmacy, medicine and perfumery (hay and waterman 1993, mehregan and ghannadi 2013). members of the family apiaceae (umbelliferae) are well known for the production of essential oils with high pharmaceutical and economic value (olivier and van wyk 2013). based on the most recent researches, the plant family apiaceae with about 430 genera and 3800 species has a worldwide distribution, especially in the northern hemisphere (stevens 2012). apiaceae has about 110 genera and 400 species distributed in iran (mozaffarian 2007). the genus leutea pimenov belongs to the “ferula group” including ferula l., dorema l. and leutea (kurzyna-młynik et al. 2008). the genus ferula includes more than 170 species distributed in central and southwestern asia, and the mediterranean region including northern africa (pimenov and leonov 1993, kurzynamłynik et al. 2008). it has about 30 aromatic species in iran (mozaffarian 2007). the genus dorema has seven species in iran (mozaffarian 2007). the taxonomic status of leutea is more complex. the genus leutea with its few species is limited to sw asia (pimenov 1987). the taxonomy of the group has changed considerably, especially since publication of flora iranica, volume 162 (pimenov 1987). the type of the genus, i.e. leutea petiolaris was first effectively published as part of the genus ferula as f. petiolaris dc. (de candolle 1830). boissier (1872) integrated l. petiolaris with other species of the genus peucedanum l. sect. juncea. he described two other new species, which are today known to be part of leutea. pimenov (1987) transferred all six known species of the group to the new genus named leutea pimenov. spalik and downie integrated the whole leutea species into the genus ferula (see kurzyana-mlynik et al. 2008). recent works of panahi et al. (2015) suggested that species of leutea should be re-established from ferula again. leutea elbursensis mozaff. (syn.: ferula elbursensis (mozaff.) spalik et. s. r. downie), an endemic to northern iran, emami-tabatabaei s.-s., larijani k., mehregan i. 120 acta bot. croat. 77 (2), 2018 is a glabrous perennial tough herb, 1.5 to 3 m high, with compound pinnate leaves with tubular lobes. it has compound umbels consisting of yellow 5-merous flowers. the fruits are schizocarp, compressed, elliptic and up to 10 × 4 mm in size (mozaffarian 2007, kanani et al. 2013). l. elbursensis can be distinguished from the other species by its narrow pedicels, ovate-elliptic fruits, greenish-yellow petals, and large and strong habits (mozaffarian 2007). despite the recent publication date of its name, many populations of l. elbursensis have been known for a long time. material of l. elbursensis was first considered part of l. cupularis (pimenov 1987), and was later segregated from it by mozaffarian in 2003 (kurzyna-młynik et al. 2008). mozafarian (2007) identifield two different species in the north of iran (tehran province) i.e. l. petiolaris and l. elbursensis. these taxonomic complexities have resulted in ambiguity attaching to those works already performed on three species l. elbursensis, l. cupularis and l. petiolaris (masoudi et al. 2004, kurzynamłynik et al. 2008, alipour et al. 2015). members of the family apiaceae in iran are widely studied for their essential oil contents (olivier et al. 2013, safaeian et al. 2015). most of those studies are single records based on the material collected from a single locality. chemical composition of the essential oils depends on many factors such as collection time, geographical locality and genetic structure (munoz-bertomeu et al. 2007). therefore, a single report of essential oil composition for a given species could not be generalized for all of its populations. looking for their chemotaxonomic value, kanani et al. (2011) studied chemical composition of the essential oils for 18 ferula species from iran. since its introduction in 1995, amplified fragment length polymorphism (aflp) has become a popular research tool for detecting genetic structure of populations as well as differences at intra-species level (vos et al. 1995). in this paper we try to find out if different populations growing wild in the north of iran (tehran province) belong to the same species. material from the western part of the province was previously identified as l. elbursensis by mozaffarian (2007). we used analysis of its (internal transcribed spacer) of the nuclear genome to find the phylogenetic placement of those plants. in addition, aflp fingerprinting techniques are used for distinguishing the genetic structures of different populations. we also aim to study the possible correlation between the genetic structure and the essential oil profile of l. elbursensis populations collected from different localities in northern iran (tehran province), using gc/ms and aflp fingerprinting techniques. finally, we will try to clarify the taxonomic position of l. elbursensis. materials and methods between august and september 2013, plants from three populations of l. elbursensis and one population of l. petiolaris were collected from northern iran, the province of tehran (tab. 1). plant materials were identified by authors after mozaffarian (2007) and vouchers were deposited in the herbarium of islamic azad university, science and research branch, tehran, iran (iauh). fresh leaf fragments from at least six individuals from each population (24 individuals in total) were taken and gradually dried in silica-gel pearls. total dna was extracted for each individual sampled using nucleospin® plant ii kit after the manufacturer’s manual (machery-nagel, dueren, germany). the complete internal transcribed spacer (its) region of the dna was amplified using the primer pair ab101 (5ʹacg aat tca tgg tcc ggt gaa gtg ttc g – 3ʹ) and ab102 (5ʹ-tag aat tcc ccg gtt cgc tcg ccg tta c – 3ʹ) (douzery et al. 1999), in a pcr reaction under the following conditions: a pretreatment of 5 minutes at 95 °c, 35 cycles of 30 seconds at 95 °c, 30 seconds at 50 °c, and 1 minute 30 seconds at 72 °c, and a final extension of 7 minutes at 72 °c. the complete its region was sequenced on an abi 3730 sequencer machine (applied biosystems, waltham, massachusetts, usa). sequences were visually checked and edited with sequencher 4 (gene codes corporation, ann arbor, mi usa), and then aligned using macclade 4.08 (maddison and maddison 2000), alongside additional sequences taken from the genebank. ferula violacea korovin and f. olivacea (diels) h. wolff. were chosen as outgroup taxa after panahi et al. (2015). maximum parsimony (mp) analysis of the its dataset was performed with paup* (swofford 2002). bayesian analysis (ba) of the its dataset was performed using mrbayes v3.1.2 (huelsenbeck and ronquist 2001). the aflp™ procedure for this work followed vos et al. (1995), and scalone et albach (2012) with the following modifications: three primer combinations used for selective pcr were e38-hex labelled (5ʹ – gac tgc gta cca att cac t – 3ʹ) combined with m57 (5ʹgat gag tcc tga gta acg g – 3ʹ), e45-fam labelled (5ʹ-gac tgc gta cca att cat g – 3ʹ) combined with m54 (5ʹ – gat gag tcc tga gta acc t – 3ʹ), and e40-ned labelled (5ʹgac tgc gta cca att cag c – 3ʹ) with m55 (5ʹ-gat gag tcc tga gta acg a – 3ʹ). pcr products of each sample were combined equally, and 2 μl of this multiplex product was run with 7.75 μl hidi formamide (applied biosystems) and 0.25 μl internal size standard genescan rox (applied biosystems) on an abi 3730 automated capillary sequencer. fragments were analyzed and scored using genemarker 2.4.1 (softgenetics). structure 2.3.4 (pritchard et al. 2000) was used for analyzing the genetic structure of populations. an analysis of molecular variance (amova) test was performed using genalex 6.5 (peakall and smouse 2012). 100 g of the ripened and dried fruits of the plant for each population were chopped in distilled water and a hydro-distilled fraction of it was isolated by hydrodistillation for 3 hours. we used a hewlett packard 5972a mass selective detector coupled with a hewlett packard 6890 gas chromatograph, equipped with a cross-linked 5% ph me siloxane hp5ms capillary column (50 m × 0.25 mm, film thickness 0.32 μm) for gas chromatography-mass spectrometry (gc-ms) analysis. the following gc operating conditions were used: carrier gas, helium with a flow rate of 1 ml min–1; column molecular identification of leutea elbursensis acta bot. croat. 77 (2), 2018 121 temperature, 60 °c with 7 °c temperature increase per minute, up to 230 °c; injector and detector temperatures, 280 °c; volume injected, 0.1 μl of the oil; split ratio, 1:25. in addition, the following ms operating parameters were used: ionization potential, 70 ev; resolution, 1000; ion source temperature, 200 °c. components in the oil were identified based on gc retention indices relative to n-alkanes and computer matching with the wiley 275.l library, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature (adams 1995, masoudi et al. 2004, alipour et al. 2015). spss v. 20 (ibm corporation) was used to perform cluster analyses based on the chemical components of essential oils with ward’s method (ward 1963). results figure 1 shows the phylogeny of different species of leutea based on the bayesian analysis (ba) of the its region. two species of ferula i.e. f. violacea and f. olivacea, were included in the tree as outgroups. posterior probabilities (pp) are indicated by numbers above each clade. bootstrap supports (bs) for those clades also retrieved in the maximum parsimony (mp) analysis are indicated by the numbers below each clade (fig. 1). as seen on the phylogenetic tree, species of the genus leutea are mainly grouped into two clades: clade a) a firmly supported clade including all samples of l. elbursensis with posterior probability (pp) = 1.00 and bootstrap support (bs) = 100%, and clade b) another well supported clade including a polythomy of eight species with pp = 1.00 and bs = 85%. leutea elbursensis is sister to rest of species (fig. 1). all our three accessions of l. elbursensis together with another two samples taken from the genbank (both from nw of tehran) formed a monophyletic clade. all four samples are taken from a relatively small locality in n iran (central elburz) with distances of less than 100 km among them. the monophyletic clade b presents a polythomy of different species. these include three samples of l. petiolaris, two samples of l. cupularis, and one sample of each species l. galucopruinosa, l. rechingeri, l. polyscias, l. avicennae, l. gracillima and l. nematoloba (fig. 1). two samples of l. cupularis collected from the dena mountain ranges (sw iran) formed a monophyletic clade with strong support (pp = 1, bs = 85%). different samples of l. petiolaris did not form a monophyletic clade. the results showed that our samples from the area clearly belong to two different species. phylogenetic relationships within this clade are not essentially resolved. this clade consists of samples collected from a larger region involving distances of hundreds of kilometres. in the selective pcr of aflp analysis, the e38-m57 primer combination yielded 62 bands, the e45-m54 primer combination 36 bands, and the e40-m55 primer combinatab. 1. populations of leutea elbursensis from northern iran included in the molecular and phytochemical analysis with voucher information and genbank accession numbers. no. species locality herbarium number genbank number 1 leutea elbursensis mozaff. iran: tehran, emamzadeh-davoud,35.898, 51.288, 2400 m emami 14391 (iauh) kp793684 2 leutea elbursensis mozaff. iran: tehran, morad-abad, 35.796, 51.322, 1800 m emami 14393 (iauh) kp793683 3 leutea elbursensis mozaff. iran: tehran, hesarak35.796, 51.305, 1700 m emami 14394 (iauh) kp793686 4 leutea petiolaris (dc.) pimenov iran: tehran, dizin,36.043, 51.441, 3400 m emami 14392 (iauh) kp793685 5 leutea elbursensis mozaff. iran: tehran, karaj, 35.933, 51.067, 1600 m valiejo-roman et al., 526 (mw) ay941276 ay941304 6 leutea elbursensis mozaff. iran: nw of tehran, souleghan mozaffarian & jamzad 33570 (tari) kj660832 7 leutea glaucopruinosa (rech. fil.) akhani & salimian iran: mazandaran termeh & zargari 040483-e (w 0023608) kj660833 8 leutea petiolaris (dc.) pimenov iran: azarbayejan mozaffarian & massoumi 78152 (tari) kj660836 9 leutea petiolaris (dc.) pimenov iran: tehran,35.767, 51.950, 2400 m valiejo-roman et al. 63 (mw) ay941278 ay941306 10 leutea rechingeri (leute) pimenov iraq: suleymanieh, mt. algurd rechinger 11416 (w 05825) kj660838 11 leutea cupularis (boiss.) pimenov iran: sw, dena mts., 2900-3100 m valiejo-roman et al.235 (mw) ay941277 ay941305 12 leutea cupularis (boiss.) pimenov iran: sw, dena mts., 3500-3900 m assadi & mozaffarian. 31236 (tari) kj660831 13 leutea polyscias pimenov iran: manjil, 1650 m mozaffarian 64227 (tari) kj660837 14 leutea gracillima pimenov iran: ne, golestan park. akhani 12060 (w 1999-03655) kj660834 15 leutea nematoloba (rech.f.) pimenov iran: n, chalus valley. rechinger & rechinger 6668 (w 02835) kj660835 16 ferula violaceae korovin sw asia valiejo-roman et al., 119899 (mw) af077891 17 ferula olivacea (diels) h. wolff. china chamberlain, ming, yuan & al. 229 (e) ef560691 emami-tabatabaei s.-s., larijani k., mehregan i. 122 acta bot. croat. 77 (2), 2018 tion 36 bands. the amova test results showed that of 100% total variation, 34% was among regions, 6% was among populations and 60% was within populations (tab. 2). analysis of the populations using structure 2.3.4 software showed that all individuals belonged to two different clusters showed here with different colours (fig. 2). as seen in figure 2, individuals of l. petiolaris from “dizin” population are homogenous in their genetic structure (cluster “a”). all six individuals collected from “dizin” (l. petiolaris) had genetic structures 90–100 % consisting of cluster “a” alleles. genetic profiles of three populations of l. elbursensis mainly consist of a different cluster (cluster b). genetic structures of all six individuals from the “emamzadeh-davoud” population consisted 100 % of alleles from cluster b. except for one individual from the “hessarak” population and another one individual from the “morad-abad” population, all other individuals from those two populations were 97-100% made by alleles from cluster “b”. aflp profiles of two species are clearly different. this clearly shows that two different species are distributed in the area n and nw of tehran. this is the first report of chemical composition of essential oils extracted from the ripened fruits of l. elbursensis. the essential oils obtained from dried fruits were clear, pale yellow liquids. the essential oil content of l. elbursensis was between 0.5% (w/w; in the emamzadeh-davoud population) – 0.6% (w/w; in hessarak and moradabad populations). between 15 to 29 natural compounds were identified, accounting for 98.7 – 100% of the oils (tab. 3). ripened fruits of l. petiolaris collected for two successive years did not yield fig. 1. phylogenetic tree obtained from the bayesian analysis of the internal transcribed spacer region of different species of leutea alongside two ferula species as outgroup (total characters = 442; constants = 299; parsimony informatives = 60. model of evolution = sym +g; a-c constitution rate = 1.7930; a-g constitution rate = 2.2561; a-t constitution rate = 2.0384; c-g constitution rate = 0.4189; c-t constitution rate = 5.9481; g-t constitution rate = 1.0000. gamma distribution rate = 0.6465). numbers above clades are posterior probabilities. numbers below clades are the bootstrap supports (100 replicates) for those clades retrieved in the maximum parsimony analyses (bootstrap supports less than 50% are not shown). tab. 2. amova test results showing the variations of leutea elbursensis from northern iran: within populations, among populations (pops) and among regions. df – degrees of freedom; sssum of the squares; ms – mean squares; est. var. – estimation of variance. source df ss ms est. var. % among regions 1 124.972 124.972 10.602 34 among pops 2 59.111 29.556 1.798 6 within pops 20 375.333 18.767 18.767 60 total 23 559.417 173.295 31.167 100 fig. 2. amplified fragment length polymorphism finger printing results showing the genetic profile of 24 individuals collected from northern iran (leutea elbursensis from hessarak, morad-abad and emamzadeh (e-) davoud; l. petiolaris from dizin). molecular identification of leutea elbursensis acta bot. croat. 77 (2), 2018 123 any traceable essential oil. the highest number of components was identified in the moradabad population (29 components). we also identified 20 components for the “hessarak” population. the smallest number of components was 15 and was identified in the “emamzade-davoud” population. the main components identified in our three populations were α –pinene (33.18 – 43.22%) and β-pinene (32.4 – 40.9%). there were few other important but less abundant components (ca. 5% or more) i.e. myrtenol (5.6%), transverbenol (5.6%) and isobornyl acetat (4.95%) which were identified only in the hesarak population. discussion different populations of l. elbursensis had identical its sequences and relatively similar genetic structures. its segregation from l. petiolaris was supported by its and aflp data. this segregation is also supported by morphological and geographical evidence. leutea elbursensis has unique tall tab. 3. composition of the essential oils of different populations of leutea elbursensis from northern iran. ki – kovats index. number component name ki percentage of components in each population morad-abad hessarak emamzadeh-davoud 1 α-thujene 930 1.38 0.54 1.2 2 α-pinene 939 39.77 33.18 43.22 3 camphene 954 1.32 0.57 1.24 4 thuja-2,4 (10)-diene 960 0.14 0 0 5 sabinene 975 2.05 0.71 1.9 6 β-pinene 979 36.1 32.4 40.9 7 myrcene 990 0 1 0 8 р-cymene 1024 0.35 0 0 9 limonene 1029 3.14 1.9 3.5 10 β-phelandrene 1029 0.91 0.64 0 11 cineol 1031 0.78 0 0.8 12 z-β-ocimene 1037 0.18 0 0 13 γ-terpinene 1059 0.19 0 0 14 α-campholenal 1126 0.41 0.53 0 15 trans-pinocarveol 1139 3.5 0 1.3 16 transverbenol 1144 0.16 5.62 0 17 pinocarvone 1164 0.9 1.52 0.8 18 α-terpineol 1188 0.33 0.78 0.46 19 myrtenol 1195 1.34 5.6 1.2 20 verbenone 1205 0.19 0 0 21 endo-fenchyl acetate 1220 1.32 3.02 0.8 22 isobornyl acetate 1285 1.36 4.95 1.73 23 cis-pinocarvyl acetate 1312 0.25 0.75 0 24 myrtenyl acetate 1326 0.57 2.12 0.45 25 neryl acetate 1361 0.26 2.01 0.5 26 daucene 1381 0.52 0.81 0 27 α-trans bergamotene 1434 0.1 0 0 28 z-β-farnesene 1442 0.15 0 0 29 pentadecane 1500 0.2 0 0 30 trans-β-guaiene 1502 0.2 0 0 31 α-copaen-11-01 1539 0 0.8 0 total 98.07 99.45 100 flowering stems 1.5–3 m high, while other species are clearly shorter (up to 1.5 m high). leutea species have an allopatric distribution. leutea elbursensis is endemic to n and nw tehran, especially the rocky slopes of souleghan valley (mozaffarian 2007). despite the similarities in morphology, its sequence and genetic structure, the essential oil contents of our three populations were highly variable. the results showed that leutea plants distributed in the region n and nw of tehran do not belong to a single species. pimenov (1987) mistakenly identified all herbarium material collected from the area under l. cupularis. we showed here that they belong to two different species. our results are in agreement with mozaffarian (2007), who identified the material from “central elburz” under two different species i.e. l. elbursensis and l. petiolaris. these results indicate that f. petiolaris and f. elbursensis are two different entities. regardless of the disputable taxonomic position of the genus leutea, our results clearly showed that l. elbursensis is a distinct species sister to the rest of leutea species (fig. 1). emami-tabatabaei s.-s., larijani k., mehregan i. 124 acta bot. croat. 77 (2), 2018 the genus ferula s. l. (including leutea) in iran is widely studied chemically. we compared our results to one of the most comprehensive studies, that performed by kanani et al. (2011) on 18 different populations belonging to different species of ferula (fig. 3). our analysis showed that the essential oil profile of our three samples was most similar to that of a sample from f. stenocarpa boiss. & hausskn. all these four samples formed a distinct group “rich” and “balanced” of α – pinene (33.18 – 48.8%) and β-pinene (30.1 – 40.9%), and this group is also related to another group consisted of f. gummosa boiss. and f. galbaniflua boiss. & buhse with a higher amount of β-pinene (26.8 – 69.2%) and a lower amount of α– pinene (1.4–33.9%) (ghannadi and amree 2002, ghasemi et al. 2005, jahansouz et al. 2008, talebi kouyakhi et al. 2008, kanani et al. 2011). the chemical composition of essential oils of leutea glaucopruinosa rech.f. yassa et al. (2003) showed no close similarity to our samples (“lg” in figure 3). the chemical composition of dorema glabrum fisch. & c. a. mey. delnavazi et al. (2015) is similar to that of some ferula species. comparing the dendrogram shown in figure 3 with the leutea phylogeny (fig. 1) and the phylogeny obtained by kurzyna-mlynik et al. (2008), it becomes clear that similarities in chemical composition of the essential oils of the “ferula group” does not reflect the phylogenies based on the molecular data. regarding their uniform its sequences and similar aflp profiles, the essential oil profiles of different populations of l. elbursensis were very variable, most probably as the result of different ecological conditions. the chemical composition of essential oils in the family apiaceae can be very variable, especially according to the plant parts the oils are extracted from (kanani et al. 2011). alipour et al. (2015) reported different chemical compositions for the essential oils extracted from different parts of l. cupularis. they found following major components in different parts of the plant: ð-2-carene (15.81%) and dl-limonene (25.04%) in flowers; β-pinene (13.87%), β-ocimene (9.05%), bornyl angelate (6.55%) and allo-ocimene (6.08%) in leaves; and ð-3-carene (8.38%), α-terpinyl isobutyrate (8.69%) and bornyl angelate (7.45%) in stems. in addition, the chemical composition of essential oil can be highly affected by ecological conditions and genetic structure (munoz-bertomeu et al. 2007). therefore, we here suggest that a standard method should be used for analyzing and interpreting the data obtained from essential oils. as mentioned above, different parts of the plants might yield essential oils with different contents. ripened fruits of the members of the family apiaceae could be an appropriate references adams, r. p., 1995: identification of essential oil components by gas chromatography/mass spectrometry. allured publishing corporation, illinois. alipour, z., taheri, p., samadi, n., 2015: chemical composition and antibacterial activity of the essential oils from flower, leaf and stem of ferula cupularis growing wild in iran. pharmaceutical biology 53, 483–487. fig. 3. dendrogram showing classification of different populations of ferula spp. based on the similarities in essential oil composition. amount of components for leutea populations are shown in table 3 and for other populations are presented in kanani et al. 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acta bot. croat. 80 (2), 2021 book review ii dubravka hafner and anita dedić – diatoms of the adriatic sea basin in bosnia and herzegovina diatoms – do we know them well? we know that diatoms are the only organisms on our planet whose cell walls are made of transparent opaline silica, that they convert solar energy into sugars, produce 50% of the air we breathe, feed the oceans, lakes and rivers, are the most diverse protists on earth, and inform us about the health of aquatic systems. to date, there are numerous applications of diatoms in various scientific fields, be it nanotechnology, biotechnology, environmental science, biophysics or biochemistry, bringing all facets of diatom biology under one roof. there is a particular focus on biosilication, biomineralization and the use of diatoms as “nanomaterials”, vehicles for drug delivery, optical and immunological biosensors, philters, immunodiagnostics, aquaculture feeds, lab-on-a-chip, metabolites and biofuels. for these reasons, it is extremely important to have a good knowledge of the ecology of diatoms, their spatial and temporal distribution, especially in our immediate environment. the monograph diatoms of the adriatic sea basin in bosnia and herzegovina, in bilingual form (croatian and english), writen by dubravka hafner and anita dedić, published in july 2020 (publisher: university of mostar, bosnia and herzegovina, 216 pp.), does not provide a definitive account of the diatoms of bosnia and herzegovina, but is a starting point that provides insight into the richness of their natural heritage. it was written as a result of the scientific work of professor dubravka hafner, who has devoted her whole life and work to the study of cyanobacteria and algae, with a special interest in diatoms. the book is a useful resource for anyone interested in the ecology and conservation of the nation’s freshwaters, and for anyone studying algae in school, at university, or for their own enjoyment. compiling a definitive list of diatoms from bosnia and herzegovina involves considerable scientific effort, and i am sure that both authors will acknowledge that knowledge of freshwater habitats is still incomplete and there is much to learn. this book describes the state of the art in 2020 and provides a challenge for future researchers. it also provides a basis for the development of ecological assessment protocols that will be necessary if bosnia and herzegovina is to meet the requirements of the european union’s water framework directive. the book begins with an introduction describing the biology of diatoms and the history of research on these organisms in bosnia and herzegovina, before providing an overview of aspects of the landscape that influence diatom distribution. the authors provide a list of all sites, with enough information to locate these sites in the future, before proceeding with a list of all diatom species recorded in bosnia and herzegovina. this list includes synonyms (important, as many diatom names have changed in the last 30 years) and information on their ecology and conservation status. finally, there is a comprehensive reference list. professor anđelka plenković-moraj university of zagreb, faculty of sicence, department of biology opce-str.vp acta bot. croat. 70 (2), 121–132, 2011 coden: abcra 25 issn 0365–0588 physiology and biochemistry of leaf bleaching in prematurely aging maple (acer saccharinum l.) trees: i. hydrogen peroxide level, antioxidative responses and photosynthetic pigments zvonimir u@arevi]1, ivna [tolfa2, nada para\ikovi]3, vera cesar2*, hrvoje lepedu[4 1 faculty of education, university of j. j. strossmayer in osijek, l. jägera 9, hr-31000 osijek, croatia 2 department of biology, university of j. j. strossmayer in osijek, trg lj. gaja 6, hr-31000 osijek, croatia 3 faculty of agriculture, university of j. j. strossmayer in osijek, trg sv. trojstva 3, hr-31000 osijek, croatia 4 agricultural institute osijek, ju`no predgra|e 17, hr-31000 osijek, croatia abstract – essential signaling processes such as changes in calcium mobilization, protein phosphorylation and gene expression are known to be modulated by hydrogen peroxide (h2o2). a lot of silver maple trees in the city of osijek (croatia) were noticed to have bleached leaves by early summer as well as during the whole vegetation season. in this study we aimed to investigate the processes that regulate h2o2 levels in healthy (green) and prematurely aged (bleached) leaves. for that purpose, photosynthetic performance and antioxidative response of green and bleached silver maple leaves were studied. bleached leaves had higher hydrogen peroxide level, a three-fold level of total soluble proteins as well as a lower level of ascorbic acid. concentrations of chlorophyll a, chlorophyll b, total chlorophylls and total carotenoids as well as maximum quantum yield of photosystem ii were lower in bleached leaves. this indicated their impaired photosynthetic performance. further more, bleached leaves were characterized by lower specific activities of the main antioxidative enzymes, which influenced their reactive oxygen species scavenging capability. the higher level of h2o2 content in bleached leaves as the consequence of reduced antioxidative enzyme specific activities as well as ascorbic acid level could be the reason for the down-regulation of photosynthetic performance and premature aging of those leaves. keywords: antioxidative enzymes, reactive oxygen species, photosynthesis, premature aging, acer saccharinum acta bot. croat. 70 (2), 2011 121 * corresponding author, e-mail: vcesarus@yahoo.com copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:41 color profile: disabled composite 150 lpi at 45 degrees abbreviations: aa – ascorbic acid, apx – ascorbate peroxidase, bsa – bovine serum albumin, car – total carotenoids, cat – catalase, chl a – chlorophyll a, chl b – chlorophyll b, chl a+b – total chlorophylls, dm – dry mass, edta – ethylenediamine tetraacetic acid, fm – fresh mass, f v /f m – maximum quantum yield of photosystem ii, gpod – guaiacol peroxidase, nbt – nitroblue tetrazolium, psii – photosystem ii, pvp – polyvinylpyrrolidone, ros – reactive oxygen species, sod – superoxide dismutase, tba – thiobarbituric acid, tbars – thiobarbituric acid reactive substances introduction hydrogen peroxide (h2o2) is reactive oxygen species (ros), continually generated from various sources during normal metabolism and also as a result of oxidative stress (neill et al. 2002a, neill et al. 2002b, yan et al. 2010). h2o2 is mostly generated via superoxide during electron transport processes in chloroplasts and mitochondria (dat et al. 2000, arora et al. 2002). also, h2o2 generation is induced in plants following exposure to a wide variety of abiotic and biotic stimuli. these include extremes of temperatures, drought, salinity, uv-b irradiation, excess excitation energy, ozone exposure, wounding, pathogens and herbivores (lamb and dixon 1997, costa et al. 2002, karpinski et al. 2003, œlesak et al. 2007). oxidative stress arises from an imbalance in the metabolism of ros, with more ros being produced than are catabolized (neill et al. 2002b). the high rates of h2o2 production are normally balanced by very efficient antioxidant mechanisms, both enzymatic and non-enzymatic antioxidants, by which it is removed from the cell (noctor and foyer 1998, chen and gallie 2006, œlesak et al. 2007). the balance between superoxide dismutase (sod) and ascorbate peroxidase (apx) or catalase (cat) activities in cells is crucial for determining the steady-state level of hydrogen peroxide and superoxide radicals. these defence processes are not restricted to the intracellular compartments, but are also found in the apoplast to a limited extent (arora et al. 2002). abiotic and biotic stresses can disturb this balance, by increased h2o2 initiating signaling responses that include enzyme activation, gene expression, programmed cell death and cellular damage (neill et al. 2002a, hung et al. 2005, van dorn 2008). well established deleterious effects of ros include damage to nucleic acids, protein oxidation, enzyme inhibition, and membrane lipid peroxidation (mittler 2002). silver maple (acer saccharinum l.) is a relatively fast-growing tree that is also highly adaptable, although it has higher sunlight requirements than other maples. this species is highly tolerant to a wide range of soils, drought, seasonal flooding and urban conditions; therefore it is frequently planted next to streets. also, it is widely used as ornamental tree in parks especially because of its ease of propagation and transplanting (day et al. 2000, hardin et al. 2001). however, a lot of silver maple trees in the city of osijek (croatia) were noticed to have bleached leaves by early summer as well as during the whole vegetation season. in this study we aimed to investigate the processes that regulate h2o2 levels in healthy (green) and prematurely aged (bleached) leaves. for that purpose, hydrogen peroxide level, antioxidative responses and photosynthetic pigment content in green and bleached silver maple leaves were studied. 122 acta bot. croat. 70 (2), 2011 u@arevi] z., [tolfa i., para\ikovi] n., cesar v., lepedu[ h. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:41 color profile: disabled composite 150 lpi at 45 degrees material and methods plant material and sampling the materials for study were healthy (green) and prematurely aged (bleached) silver maple (acer saccharinum l.) leaves (fig. 1). leaves were sampled from trees grown in the city of osijek (croatia). the sampling was done in july 2006. branches from ten trees of each type were sampled from the lower part of the crown, put in a plastic bag and delivered to the laboratory within one hour. for all extractions, leaves were cut into small pieces without main veins and ground with liquid nitrogen in a mortar in order to obtain fine tissue powder. determination of photosynthetic parameters leaf pigments were extracted from about 0.1 g of liquid-nitrogen-powdered leaves with absolute ice-cold acetone in the presence of magnesium hydroxide carbonate. the contents of chlorophyll a (chl a), chlorophyll b (chl b), total chlorophylls (chl a+b) and total carotenoids (car) per dry mass of tissue (dm) were determined spectrophotometrically (specord 40 analytic jena) at 470, 644.8 and 661.6 nm, respectively (lichtenthaler 1987). fluorescence measurements were done on dark-adapted (for 30 minutes) plant material. maximum quantum yield of photosystem ii (psii) (fv/fm) was measured by the saturating pulse method (mini-pam, waltz) according to schreiber et al. (1994). enzyme assays activities of ascorbate peroxidase (apx; ec 1.11.1.11), guaiacol peroxidase (gpod; ec 1.11.1.7), catalase (cat; ec 1.11.1.6) and superoxide dismutase (sod; ec 1.15.1.1) were assayed in leaf tissue extracts. the extraction of proteins for apx activity measurements was done in ice-cold 100 mm potassium phosphate buffer (ph 7.0) with 5 mm sodium ascorbate and 1 mm ethylenediamine tetraacetic acid (edta), with the addition of polyvinylpyrrolidone (pvp). the extractions for gpod, cat and sod activity determiacta bot. croat. 70 (2), 2011 123 hydrogen peroxide, antioxidative responses and pigments in acer fig. 1. silver maple (acer saccharinum l.) leaves: (a) – healthy (green), (b) – prematurely aged (bleached). bar = 2 cm. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:42 color profile: disabled composite 150 lpi at 45 degrees nation were done in ice-cold 100 mm potassium phosphate buffer (ph 7.5) with the addition of pvp. the apx activity was measured spectrophotometrically by monitoring the decrease in absorbance at 290 nm, after nakano and asada (1981). the reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0) with 0.1 mm edta, 50 mm ascorbic acid (aa) and enzyme extract. the reaction was started with the addition 12 mm h2o2. the gpod activity was measured at 470 nm as described by siegel and galston (1967). the reaction mixture consisted of 5 mm guaiacol and 5 mm h2o2 in 200 mm phosphate buffer (ph 5.8) and enzyme extract. the cat activity was determined in the presence of 50 mm potassium phosphate buffer (ph 7.5), 10 mm h2o2 and enzyme extract according to aebi (1984). the decrease in absorbance at 240 nm was monitored. the activity of sod was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (nbt) as described by giannopolitis and ries (1977). the reaction mixture consisted of 50 mm potassium phosphate buffer (ph 7.5), 13 mm methionine, 75 mm nbt, 0.1 mm edta, 2 mm riboflavin and enzyme extract. the reaction mixture that was not exposed to light did not develop color, and served as control. the absorbance was measured at 560 nm. one unit of sod activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of nbt reduction at 560 nm. hydrogen peroxide and ascorbic acid determination concentrations of hydrogen peroxide (h2o2) and ascorbic acid (aa) were determined by method described in mukherjee and choudhuri (1983). for the determination of h2o2 level, the absorbance was measured at 415 nm and concentration was calculated using an extinction coefficient of 1.878 nm–1 cm–1. for the measurement of aa level the absorbance was measured at 530 nm and concentration was calculated using an extinction coefficient of 226.2 mm–1 cm–1. thiobarbituric acid reactive substances determination lipid peroxidation was measured as the amount of thiobarbituric acid reactive substances (tbars) determined by the thiobarbituric acid (tba) reaction as described by verma and dubey (2003). the absorbance was measured at 532 nm. the value for non-specific absorption at 600 nm was subtracted. the concentration of tbars was calculated using an extinction coefficient of 155 mm–1 cm–1. soluble proteins, organic nitrogen and tissue dry weight determination the soluble proteins were assayed as described by bradford (1976) using bovine serum albumin (bsa) as a standard. organic nitrogen was determined by standard kjeldahl method. the amount of the tissue dry mass (dm) was determined by drying on 105 °c for 24 hours. statistical analysis obtained data were analyzed using student’s t-test modified for small samples (n = 10, p < 0.05) (petz 1997). 124 acta bot. croat. 70 (2), 2011 u@arevi] z., [tolfa i., para\ikovi] n., cesar v., lepedu[ h. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:42 color profile: disabled composite 150 lpi at 45 degrees results hydrogen peroxide level in green leaves was 39.94 ± 2.96 nmol g–1 dm, while in bleached leaves it was 45.99 ± 6.42 nmol g–1 dm (fig. 2). that is 15.16% higher in respect to green leaves. oppositely, the level of ascorbic acid in bleached leaves was 1.11 ± 0.15 mmol g–1 dm and in green ones 1.69 ± 0.15 mmol g–1 dm (fig. 2). so, the amount of aa in bleached leaves was 65.69% of the value in green leaves. tbars level in green leaves was 19.01 ± 4.80 nmol g–1 dm and in bleached ones it was 20.42 ± 6.93 nmol g–1 dm (fig. 2) showing no difference between green and bleached leaves. the mean values of antioxidative enzyme specific activities, ascorbate peroxidase, guaiacol peroxidase, catalase and superoxide dismutase in green and bleached maple leaves are shown on figure 3. the specific activities of apx were 1.82 ± 0.45 da min–1 mg–1 proteins in green leaves and 0.78 ± 0.21 da min–1 mg–1 proteins in bleached leaves. gpod specific activity was 1.84 ± 0.50 da min–1 mg–1 proteins in green leaves towards 0.63 ± 0.16 da min–1 mg–1 proteins in bleached ones. in green leaves the specific activity of cat was 1.18 ± 0.13 da min–1 mg–1 proteins while in bleached leaves it was 0.52 ± 0.11 da min–1 mg–1 proteins. relative activity of sod was 15.62 ± 2.32 u mg–1 proteins in green and 8.79 ± 2.19 u mg–1 proteins in bleached leaves. the enzyme specific activities of apx, gpod, cat and sod in green leaves were almost twice those in bleached leaves. the mean values of chlorophyll a, chlorophyll b, total chlorophyll and total carotenoid concentrations in bleached leaves were significantly lower than in green leaves (tab. 1). concentration of chlorophyll a in bleached leaves was 33.69% of that in green leaves. chlorophyll b concentration in bleached leaves was even lower, only 23.32% of the concentration in green leaves. consequently, the concentration of total chlorophylls in bleached leaves was 29.13% of that in green leaves. concentration of total carotenoids in bleached leaves was 59.45% of that for green leaves. chlorophyll a to chlorophyll b ratio was 47.95% higher in bleached leavesthan green leaves). the total chlorophylls to total acta bot. croat. 70 (2), 2011 125 hydrogen peroxide, antioxidative responses and pigments in acer fig. 2. the levels of hydrogen peroxide (h2o2), thiobarbituric acid reactive substances (tbars) and ascorbic acid (aa) in silver maple leaves (green leaves – grey columns, bleached leaves – white columns). collumn lengths indicate mean values expressed as 100% in green leaves giving relative levels in white leaves in comparison to green ones. vertical bars indicate ± s.d., n = 10, p(t) < 0.05, * – significant difference, ns – not significant difference. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:43 color profile: disabled composite 150 lpi at 45 degrees carotenoids (chl a+b/car) ratio was 52.50% lower in bleached than in green leaves. the mean value of maximum quantum yield of photosystem ii (fv/fm) in bleached leaves was also significantly lower, 78.59% of the value in green leaves. in green leaves the amount of organic nitrogen was 2.72 ± 0.14 m/m% while in bleached leaves it was 3.26 ± 0.38 m/m%, or 19.85% more (fig. 4). the amount of soluble proteins was almost 3 times higher in bleached leaves (12.21 ± 1.61 mg g–1 fm) than in green leaves (3.97 ± 0.90 mg g–1 fm). the amount of dry mass was lower in bleached leaves (0.29 ± 0.02 g g–1 fm) than in green leaves (0.34 ± 0.02 g g–1 fm). discussion hydrogen peroxide level in bleached leaves was 15.16% higher in respect to green leaves (fig. 2). higher level of h2o2 content in bleached leaves could be due to decline in its antioxidant response. ascorbic acid level in bleached leaves was lower (65.69%) than 126 acta bot. croat. 70 (2), 2011 u@arevi] z., [tolfa i., para\ikovi] n., cesar v., lepedu[ h. fig. 3. the antioxidative enzyme specific activity in silver maple leaves (green leaves – grey columns, bleached leaves – white columns): (a) – ascorbate peroxidase (apx), guaiacol peroxidase (gpod) and catalase (cat), (b) – superoxide dismutase (sod). vertical bars indicate ± s.d., n = 10, p(t) < 0.05, * – significant difference. tab. 1. mean values (± standard deviation) of photosynthetic parameters in green (g) and bleached (b) leaves of silver maple (acer saccharinum). standard deviation (± s.d) is given in parentheses, n = 10. chlorophyll a – chl a, chlorophyll b – chl b, total chlorophylls – chl a+b, total carotenoids – car, maximum quantum yield of psii – fv/fm, p(t) – percent of similarity, dm – dry mass. parameter g b p (t) chl a (mg g–1 dm) 5.18 (± 0.62) 1.75 (± 0.31) < 0.05 chl b (mg g–1 dm) 4.07 (± 0.78) 0.95 (± 0.38) < 0.05 chl a+b (mg g–1 dm) 9.25 (± 0.93) 2.69 (± 0.63) < 0.05 car (mg g–1 dm) 1.17 (± 0.26) 0.69 (± 0.09) < 0.05 chl a/chl b 1.32 (± 0.34) 1.96 (± 0.41) < 0.05 chl a+b/car 8.26 (± 1.85) 3.92 (± 0.97) < 0.05 fv/fm 0.81 (± 0.01) 0.64 (± 0.08) < 0.05 u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:43 color profile: disabled composite 150 lpi at 45 degrees the value in green leaves (fig. 2). aa has multiple functions in photosynthesis and photoprotection, and it plays an important role in the antioxidant defense system in plants (noctor and foyer 1998, davey et al. 2000). as the aa is involved in the detoxification of ros including h2o2 (chen and gallie 2006) reduced aa level in bleached leaves could contribute to an increased level of h2o2 content in those leaves. also, the correlation between increase in ros and suppressing dehydroascorbate reductase expression resulting in less efficient aa recycling was shown (chen and gallie 2006). the apx, gpod, cat and sod enzyme specific activities were lower in bleached than in green leaves: 43.05, 34.08, 43.66 and 56.27% of the values in green, respectively. bleached leaves characterized by lower specific activities of the antioxidative enzymes showed declined antioxidative response and ros scavenging capability. free radicals and antioxidants play a significant role during the natural senescence process (arora et al. 2002). prochazkova et al. (2001) have reported in the case of maize that early senescence was due to enhanced h2o2 production and lipid peroxidation, and lower sod, apx and cat activity towards aging. similar results were reported for ginkgo, and birch leaves were h2o2 production increased and cat activity decreased in the early phase of leaf senescence (kukavica and veljovic-jovanovic 2004). decline in antioxidant enzymes activity has been reported as the possible cause for leaf senescence in plants (ye et al. 2000). the mean values of concentrations of chlorophyll a, chlorophyll b, total chlorophylls and total carotenoids in bleached leaves were lower: 33.69, 23.32, 29.13 and 59.45% of the values in green leaves, respectively (tab. 1). in chloroplasts, the chlorophyll pigments associated with the electron transport system are the primary source for production and scavenging of ros in leaf cells (arora et al. 2002). the lower pigment contents in maize leaves were described previously as the consequence of increased hydrogen peroxide content (prochazkova et al. 2001). chlorophyll a to chlorophyll b ratio was 47.95% higher in bleached than in green leaves (tab. 1). such increased chl a/chl b ratio showed that chlorophyll b was degraded faster in bleached leaves. total chlorophylls to total carotenoids (chl a+b/car) ratio was lower in bleached (for 52.50%) than in green leaves (tab. 1), due to faster chlorophyll degradation. lower chlorophyll content in bleached leaves reflected on its maximum quantum yield of psii (fv/fm). the mean value of fv/fm in bleached leaves was significantly lower, 78.59% of the value in green leaves (tab. 1). the fv/fm in acta bot. croat. 70 (2), 2011 127 hydrogen peroxide, antioxidative responses and pigments in acer fig. 4. organic nitrogen (n), soluble proteins (sp) and dry mass (dm) in silver maple leaves (green leaves – grey columns, bleached leaves – white columns). column lengths present mean values expressed as 100% in green leaves giving relative levels in white leaves in comparison to green ones. vertical bars indicate ± s.d., n = 10, p(t) < 0.05, * – significant difference. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:43 color profile: disabled composite 150 lpi at 45 degrees bleached leaves was below 0.75, which is considered the boundary value for fully functional psii (bolhár-nordenkampf et al. 1989). a lower fv/fm value in bleached leaves indicates a stress situation as well as declined photosynthesis. the lowering of photosynthetic parameters was previously reported in leaves of maize, ginkgo and birch that also contained higher level of h2o2 (foyer et al. 2002, kukavica and veljovic-jovanovic 2004, ougham et al. 2008). accumulation of thiobarbituric acid-reactive substances is often used as an indicator of lipid peroxidation. in the present study, tbars level did not show a difference between green and bleached leaves (fig. 2). because of the unchanged tbars level, increased h2o2 level seems to be not very harmful for bleached leaves. generally, a coordinated regulation of the free radical scavenging system comprising cat, sod, apx, ascorbate and glutathione is essential (zimmermann and zentgraf 2005). the promoted ability in scavenging does not always prevent the increase in h2o2 content (lu et al. 2009). also, among the different ros, only h2o2 is relatively stable and able to penetrate the plasma membrane as an uncharged molecule. in addition to being a toxicant h2o2 has been regarded as a signaling molecule acting in initiating transduction pathway towards plant cell death (chakrabarty et al. 2009). the increased radical levels displayed during senescence are not only caused by the elevated production of radicals but also by a loss in antioxidant capacity. the degradation of chlorophylls and the membranes causes an increase in the production of free radicals. in addition, the amount of reduced oxygen, e. g. h2o2, increases greatly during senescence. lipid peroxidation, measured as tbars level leads to the generation of free radicals which in turn initiates an increase in ethylene formation leading to the promotion of senescence (zimmermann and zentgraf 2005, yan et al. 2010). although our investigations showed an increase of h2o2 and no significant increase of tbars level, we could speculate that the increase of 15% in h2o2 and increase of 7% in tbars together with the lowering of cat, gpod, apx and sod activity leads to a certain loss in antioxidant capacity and the slow promotion of senescence. accelerated leaf senescence is one of the harmful effects of elevated troposferic ozone concentrations on plants (gielen 2007, yan et al. 2010). moreover, according to the data on the photosynthesis performance and biochemistry this could indicate that bleaching was not a simple symptom of premature aging but also an adaptation to high-light induced oxidative stress during summer (lepedu[ et al. 2011). the relative levels of organic nitrogen (for 19.85%) and soluble proteins (for 208.05%), were higher in bleached leaves while dry mass was lower than in green ones (85.49% of the value in green leaves) (fig. 4). perry and hickman (2001) have reported that mean leaf nitrogen concentrations for silver maple healthy leaves was 2.52%. data revealed that the nitrogen level in bleached leaves (3.26%) was higher than that mean value (perry and hickman 2001) and higher than the value in green leaves (2.72%). natural senescence is characterized by protein degradation and mobilization of released nitrogen to the developing parts of the tree or to the storage tissues (valjakka et al. 1999, vanacker et al. 2006). in our study, bleached leaves do have more nitrogen and especially more soluble proteins, although antioxidative enzymes specific activities were reduced showing that some other proteins were having a more intensive biosynthesis. this is rather the indication of some type of stress situation leading to premature aging of bleached leaves, than of the natural senescence process. 128 acta bot. croat. 70 (2), 2011 u@arevi] z., [tolfa i., para\ikovi] n., cesar v., lepedu[ h. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:43 color profile: disabled composite 150 lpi at 45 degrees it can be concluded that decreased chloroplast pigments concentrations and reduced maximum quantum yield psii (fv/fm) in bleached leaves indicated their impaired photosynthetic performance. bleached leaves were also characterized by lower specific activities of the antioxidative enzymes showing declined antioxidative response and ros scavenging capability. increased level of hydrogen peroxide content in bleached leaves as the consequence of deficient antioxidative response could be the reason for the down-regulation of photosynthetic performance as well as premature aging of those leaves. judging from unchanged the tbars level, the increased h2o2 level was not deleterious to bleached leaves. it can be concluded that the down-regulation of photosynthetic performance was sufficient to enable the bleached leaves to keep their functionality in the beginning of summer. extended investigations will comprise detailed analysis of psii photochemistry and abundance of key photosynthetic proteins in order to investigate mechanisms of photosynthetic down regulation in bleached leaves. acknowledgments this work was supported by the scientific research grants to h. l. (research agreement 073-0731674-1673), v.c. (research agreement 073-0731674-0841) and d.[. 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e., 1999: expression of photosynthesisand senescence-related genes during leaf development and senescence in silver birch (betula pendula) seedlings. physiologia plantarum 106, 302–310. vanacker, h., sandalio, l. m., jiménez, a., palma, j. m., corpas, f. j., meseguer, v., gómez, m., sevilla, f., leterrier, m., foyer, c. h., del rio, l. a., 2006: roles for redox regulation in leaf senescence of pea plants grown on different sources of nitrogen nutrition. journal of experimental botany 57, 1735–1745. verma, s., dubey, r. s., 2003: leads toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants. plant science 164, 645–655. yan, k., chen, w., he, x., zhang, g., xu s., wang, l., 2010: responses of photosynthesis, lipid peroxidation and antioxidant system in leaves of quercus mongolica to elevated o3. environmental and experimental botany 69, 198–204. acta bot. croat. 70 (2), 2011 131 hydrogen peroxide, antioxidative responses and pigments in acer u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 26. rujan 2011 13:49:34 color profile: disabled composite 150 lpi at 45 degrees ye, z., rodriguez, r., tran, a., hoang, h., de los santos, d., brown, s., vellanoweth, r. l., 2000: the developmental transition to flowering represses ascorbate peroxidase activity and induces enzymatic lipid peroxidation in leaf tissue in arabidopsis thaliana. plant science 158, 115–127. zimmermann, p., zentggraf, u., 2005: the correlation between oxidative stress and leaf senescence during plant development. cellular and molecular biology letters 10, 515–534. 132 acta bot. croat. 70 (2), 2011 u@arevi] z., [tolfa i., para\ikovi] n., cesar v., lepedu[ h. u:\acta botanica\acta-botan 2-11\uzarevic et al.vp 8. rujan 2011 15:46:43 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (1), 9–21, 2011 coden: abcra 25 issn 0365–0588 early post-fire changes in pinus brutia forests (amanos mountains, turkey) necattin türkmen*, atabay düzenli department of biology. faculty of science and letters. çukurova university, 01330 adana, turkey abstract – we studied the species composition and soil nutrients in a pinus brutia forest after a fire that occurred in 1989. four permanent plots were created in the burnt and not burnt areas in the amanos mountains of turkey. the floristic richness, biological spectra, above ground phytomass and soil features in the study areas were assessed during the first three years after the fire. after the fire, we found a reduced amount of organic matter (14.3%), total nitrogen (22%) and soil water saturation (13.1%), but an increased amount of available phosphorus (71%), acidity (3.6%), cation exchange capacity (9.9%), exchangeable sodium (20.8%) and exchangeable potassium (37.1%). the aboveground phytomass in the burned area reached 5284 kg ha–1, the third year after the fire. forty-six pre-fire species were renewed in the first three years after the fire. juniperus oxycedrus could not renew within three years after the fire. pine phytomass has increased five times within three years after the fire. key words: forest, fire, pinus brutia, succession, soil introduction fire is one of the most important ecological factors in mediterranean-type ecosystems. most mediterranean-type vegetation is composed of woodland in various degradation stages created by the long history of human activities (le houreou 1974, naveh 1990, trabaud 2000, bilgili and saðlam 2003). depending on their post-fire reaction, plant species are classified as »seeders«, »sprouters«, or both types. pine forest communities are very flammable during drought periods as they contain rich resins, essential oils, litter and understory shrub layers. on the other hand, these are resistant to fire in the absence of human pressures such as grazing, cutting and cultivation (mazzoleni and esposito 1993, ne’eman et al. 2004). acta bot. croat. 70 (1), 2011 9 * corresponding author, e-mail: nturkmen@cu.edu.tr copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:50:24 color profile: disabled composite 150 lpi at 45 degrees pinus brutia ten. covers extensive areas in the eastern mediterranean: mainly turkey, greece, cyprus, w. syria, lebanon and italy; scantly n. iraq, w. caucasus and crimea (gezer 1986, fady et al. 2003) (fig. 1). the total p. brutia forest cover is estimated to be over 4 million hectares, 3.8 million hectares of which are in turkey, accounting for 20.2% of turkey’s total forest area. of this, 2.2 million hectares are productive while 1.6 million hectares are degraded. economically, it is turkey’s most important forest tree species (davis 1965–1985, fady et al. 2003, boydak 2004). fires are started for reasons like acquiring new grazing land, clearing for new farmland, smoking, arson, camping, glass waste and various accidents with 99% of the forest fires in turkey being caused by humans; with only ca 1% of the recorded forest fires being started by lightning. the origin of about half the human-caused fires is shown to be negligence (25%) and deliberate fire-setting (26%). it is assumed that most of the fires with unknown origins are intentionally set fires, including arson (serez 1995). forty-eight percent (9 732 840 ha) of the forests in turkey are susceptible to fire due to the mediterranean climate (türkmen and düzenlý 2005), and the flammable and combustible vegetation. an average of 23 127 ha y–1 of natural vegetation was burnt in turkey between 1937–2003 with 72 316 fires occurring and 1 549 506 ha of forest being burnt. the post-fire succession in pine forests has not previously been studied in turkey, although some studies on this subject were conducted in other countries with mediterranean type ecosystems such as france (trabaud 2000), greece (arianoutsou-faraggitaki 1984, thanos et al. 1989), italy (mazzoleni and esposito 1993), spain (faraco et al. 1993), the united states (hanes 1970, keeley 1987) and israel (naveh 1975). the aim of the present study is therefore to examine the effects of fire on the soil properties and the floristic diversity of pinus brutia forests in turkey, from the immediate post-fire period up to three years after the fire. 10 acta bot. croat. 70 (1), 2011 türkmen n., düzenli a. fig. 1. study area and distribution of pinus brutia woods (•). u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:15:56 color profile: disabled composite 150 lpi at 45 degrees study area the study area is located on a hillside near erzin town (36° 57' n. 36° 16' e), hatay province, turkey, ca. 550 m above sea level and 14 km from the ýskenderun gulf (fig. 1). climate the mediterranean climate in the study area is characterized by long summer droughts and mild and rainy winters. the mean annual precipitation is about 1019.3 mm, while the monthly precipitation is approximately 22.4 mm in july and 130.3 mm in january. the mean maximum temperatures range from 15.2 °c in january to 32.2 °c in august and the mean minimum temperatures from 6.8 °c in january to 23.8 °c in august. the bioclimatic diagram prepared for the study area shows the months with dry and rainy periods (fig. 2). geology the geological structure of the area is of mesozoic and cretaceous limestone, upper cretaceous ultra-basic rocks (gabro and serpentine) and tertiary marls. the common soil formation distinguished in the area is brown forest soils (türkmen and düzenli 1998). vegetation the native woody vegetation of the study area is mainly composed of pinus brutia ten., quercus cerris l., styrax officinalis l.. fraxinus ornus l.. cotinus coggyrea scop.. pistacia terebinthus l.. cistus salviifolius l.. cercis siliquastrum l.. erica manipuliflora salisb.. sorbus torminalis (l.) crantz. genista lydia boiss. and smilax aspera l.. a part of the pinus brutia vegetation in the amanos mountains under the responsibility of the dörtyol forestry administration of hatay province in turkey was intensely burnt at the end of february 1989 as a result of negligence by a shepherd. acta bot. croat. 70 (1), 2011 11 post-fire changes in the pinus brutia forests fig. 2. ombrothermic diagram of the study area. the dry period in july – august, the rainy period (>100 mm / month in december – april, and transitional period in may and november. u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:15:57 color profile: disabled composite 150 lpi at 45 degrees materials and methods in order to analyze the changes in species composition, soil elements, above ground phytomass and physiognomy of the plant community in this area, four permanent plots were established in the burned area and four in the adjacent unburned area. the unburned plots are the control plots. each plot was 100 m2 (10 m � 10 m). both burned and unburned plots were evaluated as single plots. all floristic records were made monthly from february 1989 to february 1992. in each of the burned plots, four sub-plots (2 m � 2 m) were randomly created for post-fire plant biomass determination. all the vascular plants in subplots were cut with hand equipments (with bucksaw, hatchet and prune scissors) as close to the soil surface as possible. to determine phytomass, the harvested material was air dried in an oven at 105 °c. five robust suckers from each woody species were cut, dried in the oven and weighted. in total, 24 soil samples from the study plots were taken (24 of these samples from burned plots. and 4 from unburned plots). each soil sample was created by mixing the upper soil layer (0–15 cm as depth) in the center of the corner of each plot. these soil samples were dried in the open air, and after being sieved in a fine sieve in the laboratory were analyzed using relevant methods. air-dry sieved soil samples were analyzed in the department of soil sciences, çukurova university. the soil analysis was performed according to bayrakli (1987). lime (caco3) was determined with a scheibler calcimeter measuring the carbon dioxide pressure. organic carbon was determined by walkley-black wet oxidation method, total nitrate by the semi-micro kjeldahl method. available phosphorus (p2o5 kg ha–1) was determined according to olsen et al. (1954) (using phosphorus solubility in sodium bicarbonate). exchangeable sodium (expressed as cmolc kg–1, i.e. centimoles of charge per kilogram of dry soil) was determined by 1n ammonium acetate, exchangeable potassium (cmolc kg–1) by 1n ammonium acetate. total salt was determined by electrical conductivity within the 100 g water-saturated soil. for cation exchange capacity (cmolc kg–1) 1n sodium acetate-soil mixture was washed with 2n ammonium acetate. acidity (ph) was determined using distilled water-saturated soil samples, after waiting 24 hours, using a beckman ph meter connected to a glass-calomel electrode pair. soil water saturation (%) was determined by measuring the amount of saturated pure water of 100 g air-dried soil. sorensen’s similarity index (si) was used to compare the species diversity of the burned and unburned areas: si = 2 w / (a + b) where a is the total number of species in sample 1, b is the number of species in sample 2 and w is the number of species common to both samples. the collected plant material was numbered and kept as samples for botanical identification. taxonomical determination was performed according to davis (1965–1985). a voucher specimens of each species was kept in the herbarium of çukurova university, faculty of science and letters, department of biology. 12 acta bot. croat. 70 (1), 2011 türkmen n., düzenli a. u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:15:57 color profile: disabled composite 150 lpi at 45 degrees results the flora of the unburned area (400 m2) consisted of 47 resident species and floristically it did not change during the three years after the fire. in the burned area (400 m2), the presence of plant species that changed throughout the observation period is as follows: 26 species (24 autochthonous specific for the brutia pine community, and 2 allochthonous or exotic for the brutia pine community in the first year; 55 species (39 autochthonous and 16 allochthonous) in the second year; and 66 species (46 autochthonous and 20 allochthonous) in the third year (tab. 1). ninety-eight percent of the pre-fire species (i.e. 46 species) occurred in the first three years after the fire, while the remaining 2% (i.e. 1 species) did not occur during this time. the floristic richness of the burnt community showed a tendency that resembles the stabile balance that existed before the fire (fig. 3, tab. 2). the phytomass above ground of the burnt vegetation reached 5283.8 kg ha–1 (herbaceous phytomass 2845.9 kg ha–1, woody phytomass 2437.9 kg ha–1) showing rapid growth at the end of third year. phytomass of the pine seedlings were much lower (9.9 g per 5 seedlings) than a woody resprouter species (e.g., phytomass of oak species is 401 times greater than that of pine species) due to both the pine species being an obligate seeder and woody resprouter species being capable of vigorous re-growth with spare nutrients (tabs. 3, 4). the first three years, root growth of the pine was faster than stem growth (the root length was 18.3 cm in second year and 29.6 cm in third year while the stem length was 7.1 cm and 19.8 cm respectively). the soil analysis in the study area (fig. 4) indicated that immediately after the fire organic c (%)decreased from 13.37 to 11.46, later was reduced to 5.14. total n (%) immediately after the fire decreased from 0.391 to 0.305, and then later was reduced to 0.217. soil water saturation immediately after the fire decreased from 103.2 to 89.7, later was reduced acta bot. croat. 70 (1), 2011 13 post-fire changes in the pinus brutia forests fig. 3. development and floristic composition after the fire. u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:15:58 color profile: disabled composite 150 lpi at 45 degrees 14 acta bot. croat. 70 (1), 2011 türkmen n., düzenli a. tab. 1. occurrence of the plant species in the study area. lf denotes life form: th – terophyte, ge – geophytes, h – hemicryptophyte, ph – phanerophyte, ch – chamaeophyte. l denotes life span: a – annual, b – biannual, p – perennial. rs denotes reproductive strategy: g – generative, v – vegetative, vg – both generative and vegetative. presence of species in terms of time after fire: m – first three mounts, 1 – first year, 2 – second year, 3 – third year. – denotes absence, + denotes presence. species family lf l rs m 1 2 3 autochthonous species alyssum strigosum banks et sol. ssp. strigosum t. r. dudley brassicaceae th a g – – – + asparagus acutifolius l. liliaceae ge p g – – + + asperula cymulosa (post) post rubiaceae ch p vg – – + + asperula stricta boiss. ssp. stricta ehrend. et schönb.-tem. rubiaceae ch p vg – – + + asplenium adiantum-nigrum l. aspleniaceae h p g – – – + brachypodium pinnatum (l.) p. beauv. poaceae h p v + + + + centaurea ptosimopappa hayek asteraceae h p g + + + + cercis siliquastrum l. ssp. hebacarpa (bornm.) yalt. fabaceae ph p v + + + + chrysopogon gryllus (l.) trin. poaceae h p v + + + + cistus salviifolius l. cistaceae ch p g + + + + clinopodium vulgare l. ssp. arundanum (boiss.) nyman lamiaceae ch p vg – – + + cotinus coggyrea scop. fabaceae ph p v + + + + crepis reuterana boiss. asteraceae h p g + + + + cytisopsis dorycniifolia jaub. et spach ssp. dorycniifolia d.f.chamb. fabaceae ch p g – – + dactylis glomerata l. ssp. hispanica (roth) nyman poaceae h p v + + + + dorycnium graecum (l.) ser. fabaceae ch p vg – – + + epipactis helleborina (l.) crantz orchidaceae ge p vg + + + + erica manipuliflora salisb. ericaceae ph p vg + + + + eryngium falcatum delar apiaceae h p vg + + + + euphorbia apios l. var. lamprocarpa boiss. euphorbiaceae ge p v – – + + euphorbia macrostegia boiss. euphorbiaceae h p g – + + + ferulago cassia boiss. apiaceae h p g – – – + fraxinus ornus l. ssp. cilicica (lingelsh.) yalt. oleaceae ph p v + + + + genista lydia boiss. var. antiochia (boiss.) p. gibbs. fabaceae ch p vg + + + + gladiolus italicus miller iridaceae ge p vg – – + + hedera helix l. araliaceae ch p v – – – + iris unguicularis poiret iridaceae ge p vg – – + + juniperus oxycedrus l. ssp. oxycedrus coode et cullen cupressaceae ph p g – – – – lesquereuxia syriaca boiss. et reut. scrophullariaceae h p vg – – + + melica minuta l. poaceae h p v + + + + muscari tenuiflorum tausch liliaceae ge p vg – – + + origanum laevigatum boiss. lamiaceae ch p vg – + + + u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:15:59 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (1), 2011 15 post-fire changes in the pinus brutia forests species family lf l rs m 1 2 3 osyris alba l. santalaceae ch p g – – + + pinus brutia ten. pinaceae ph p g – – + + pistacia terebinthus l. ssp. palaestina (boiss.) engl. anacardiaceae ph p v + + + + potentilla micrantha ramond ex dc. rosaceae h p g – – + + quercus cerris l. var. cerris hedge et yalt. fagaceae ph p v + + + + quercus infectoria oliv. ssp. boissieri (reut.) o.shwarz fagaceae ph p v – – – + rubus canescens dc. var. glabratus (godr.) davis et meikle rosaceae ch p v + + + + ruscus aculeatus l. var. angustifolius boiss. liliaceae ch p v – + + + saccharum strictum(host) sprengel poaceae h p vg + + + + salvia tomentosa l. lamiaceae h p g – – + + smilax aspera l. liliaceae ph p vg + + + + sorbus torminalis (l.) crantz var. torminalis gabrieljan rosaceae ph p vg + + + + stachys diversifolia boiss. lamiaceae ch p vg – – + + lparstyrax officinalis l. styracaceae ph p v + + + + viola alba besser ssp. dehnhardtii (ten.) becker violaceae h p g – – – + allochthonous species carex flacca schreber ssp. serrulata (biv.) greuter cyperaceae h p g – – – + cephalaria taurica szabo scrophullariaceae h p g – – + + chenopodium album l. ssp. album chenopodiaceae th a g – – – + conyza bonariensis(l.) cronquist asteraceae th a g – – + + dorycnium penthaphyllum scop. ssp. haussknetchii (boiss.) gams fabaceae ch p g – – + + hypericum montbretii spach hypericaceae th a g – – – + inula vulgaris(lam.) trevis. asteraceae h b g – – + + lactuca serriola l. asteraceae h b g – – + + lathyrus spathulatus cel. fabaceae h p g – – + + lotus peregrinus l. var. peregrinus heyn fabaceae th a g – – – + michauxia campanuloides l’her. ex aiton campanulaceae h b g – – – + orobanche crenata forssk. orobanchaceae th a g – – + + pterdium aquilinum (l.) kuhn hypolepidaceae ge p g + + + + rhus coriaria l. anacardiaceae ph p g – – – + senecio vernalis waldst. et kit. asteraceae th a g – – + + sideritis perfoliata l. var. condensata boiss. lamiaceae h p g – + + – silene confertifolia chowdh. caryophyllaceae h p g – – + + sonchus oleraecus l. asteraceae th a g – – + – thesium bergeri zucc. santalaceae ch p g – – + + thlaspi annuum koch brassicaceae th a g – – + + torilis arvensis (huds.) link ssp. arvensis cullen brassicaceae th a g – – + + trifolium campestre schreb. fabaceae th a g – – + – verbascum galileum boiss. scrophulariaceae h b g – – – + tab. 1. – continued u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:15:59 color profile: disabled composite 150 lpi at 45 degrees 16 acta bot. croat. 70 (1), 2011 türkmen n., düzenli a. tab. 2. relative contribution of autochthonous and allochthonous species at the burnt site, and the species similarity between burned and unburned areas after fire. time afterthe fire number of autochthonous species (and relative contribution) number of allochthonous species (and relative contribution) sorensen similarity index (%) 1 year 24 (51.1) 2 (8.7) 67.5 2 years 15 (31.9) 14 (60.9) 76.4 3 years 7 (14.9) 7 (30.4) 81.4 total 47 (97.9) 23 (100) – tab. 3. characteristics of the plant species appearing in the first three years after fire (pine seedlings did not occur within the first year; to avoid damage to the vegetation. the biomass of the second year was not determined). characteristics first year second year third year pine seedlings: height (cm) – 7.1 19.8 root lenght (cm) – 18.3 29.6 stem phytomass (g per 5 suckers) – 1.1 5.2 needle phytomass (g per 5 suckers) – 0.7 3.1 root phytomass (g per 5 suckers) – 0.3 1.2 total pine phytomass (g per 5 suckers) – 2.2 9.9 vegetation biomass (kg per ha): 162 – 5283.2 tab. 4. height and phytomass values of dominant understory woody species of the pine forest in the third year. species woody (g of 5 suckers) herbaceous (g of 5 suckers) total (g of 5 suckers) max. heights (cm) quercus cerris 2455 849 3304 391 styrax officinalis 1636 272 1908 305 pistacia terebinthus 923 311 1235 171 fraxinus ornus 947 106 1052 226 cercis siliquastrum 619 250 869 230 cotinus coggyrea 525 210 736 198 cistus salviifolius 213 76 289 82 sorbus torminalis 94 57 151 30 erica manipuliflora 44 19 63 38 u:\acta botanica\acta-botan 1-11\turkmen.vp 31. o ujak 2011 13:18:46 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (1), 2011 17 post-fire changes in the pinus brutia forests fig. 4. changes of soil ph, organic carbon content, water saturation, contribution of coluble salt, lime (caco3), cation exchange capacity (cec), exchangeable na, exchangeable k, total nitrogen, c/n ratio, and available phosphorus (p2o5) after the fire. u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:16:03 color profile: disabled composite 150 lpi at 45 degrees to 77.9. c/n ratio immediately after the fire increased from 34.83 to 38.27, later was reduced to 23.55. available phosphorus (p2o5 kg ha–1) immediately after the fire increased from 0.390 to 0.667, later was reduced to 0.250; ph value (in soggy soil) immediately after the fire increased from 6.88 to 7.13, later was reduced to 7.04 cmolc kg–1; exchangeable k. immediately after the fire increased from 0.70 to 0.96, later was reduced to 0.56 cmolc kg–1; exchangeable na immediately after the fire increased from 0.24 to 0.29, later was reduced to 0.22 cmolc kg–1; cation exchange capacity immediately after the fire increased from 39.86 to 43.80, later was reduced to 36.45 cmolc kg–1; lime (caco3) immediately after the fire increased from 3.0 to 3.1, later was reduced to 2.8 %; total salt immediately after the fire increased from 0.665 to 0.091, later was reduced to 0.072 %. similar trends were found in the earlier studies, elsewhere (ahlgren and ahlgren 1960, raison 1979, kutiel and shaviv 1989). discussion all the existing species before the fire (species at the control site), except for juniperus oxycedrus l., emerged again within the first three years after the fire. juniperus oxycedrus did not resprout from the lignotubers or burls. the new habitat conditions created by fire led to the emergence of allochthonous opportunistic species such as sonchus olereacus, sideritis perfoliata, senecio vernalis, trifolium campestre, conyza bonariensis, lactuca serriola and verbascum galileum. these species caused an increase in the floristic richness of the area in the first three years (the richness includes both native and non-native species) but an elimination started by the dominance of the autochthonous sprouting phanerophytes. the most typical sprouter phanerophytes were quercus cerris. fraxinus ornus. cotinus coggyrea. pistacia terebinthus and erica manipuliflora which can vigorously regenerate by root sprouting. the woody species that reproduce by seedlings only were pinus brutia and cistus salviifolius. these two woody species are known pyrophytes because of better germination and growth show in burned areas. the re-sprouting species remained alive in the fire, stored food reserves are developed by sprouting from vegetative organs. many herbaceous re-sprouters having subterranean root organs (rhizomes or tubers) such as saccharum strictum, brachypodium pinnatum, dactylis glomerata and epipactis helleborina regenerated easily in the first year after the fire. these results agreed with those presented by the other authors (daubenmire 1968, trabaud 1973, verroius and georgiadis 2002, türkmen and düzenli 2005). the allochthonous species (species not seen in the control site occurred only from seeds in the burned areas. in the presence of autochthonous species, their seeds and / or vegetative organs were able to continue. subterranean organs are protected from fire by the soil, which is a good insulator and conducts little heat produced by the burning vegetation (aston and gill 1976). mooney and dunn (1970) found that nearly 50% of the small woody shrubs in california and chile sprouted after a fire. naveh (1975) in israel, trabaud and lepart (1980) in southern france, thanos et al. (1989) in samos island of greece and türkmen and düzenli (2005) in the turkish mediterranean region found that nearly all the woody species of these regions sprouted after a fire within 3-5 years. the emergence of the original species was 51.1% in the first year, 31.9% in the second year and 14.7% in the third year. it is known that some mediterranean ecosystems evolve 18 acta bot. croat. 70 (1), 2011 türkmen n., düzenli a. u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:16:03 color profile: disabled composite 150 lpi at 45 degrees with fire and that most plant species have developed adaptive mechanisms (trabaud 1987). these mechanisms could be associated with the strategies of persistence after a fire (e.g. species that regenerate well or disseminate numerous seeds after a fire). among the allochthonous species, the most prolific families were asteraceae (5 species) and fabaceae (4 species). the fabaceae are of special interest due to their waterproof hard-coated seeds and ability to fix atmospheric nitrogen. these characteristics increased the germination abilities of their seeds in the soil seed bank after the fire. the diminished nitrogen in the burnt soils can be slightly resolved through the quick nitrogen fixation of the leguminous species. the dispersal abilities of the many species that belong to the asteraceae are usually more than those in other families (verroius and georgiadis 2002). most of these plants occur usually in open areas such as forest roadsides, forest-adjacent agricultural areas and abandoned fields and have small, light seeds with pappus-like structures. therefore, it was observed that these families were more abundant during the study period than before the fire. the pre-fire species were detected in early post-fire vegetation and species richness increased during the three years after the fire due to the high abundance of short-lived herbaceous plants, which benefit from enriched supply with nutrients and light, as has been explained before (buhk et al. 2006). acknowledgements we wish to thank the soil laboratory personnel of çukurova university for their valuable help in making the soil analysis, the çukurova university research fund (fbe-89-e 29) for their financial assistance, and the anonymous referees for their helpful comments. references ahlgren. i., ahlgren, c. e., 1960: ecological effects of forest fires. the botanical review 26, 48–533. arianoutsou-faraggitaki, m., 1984: post-fire successional recovery of a phryganic (east mediterranean) ecosystem. acta oecologica 5, 387–394. aston, a. r., gill, a. m., 1976: coupled soil moisture, heat and water vapor transfer under simulated fire conditions. australian journal of soil research 14, 55–56. bayrakli, f., 1987: soil and plant analyses (in turkish). ondokuz mayýs university, samsun. university of ondokuz mayis, faculty of agriculture publications, samsun. bilgili, e., saglam, b., 2003: fire behavior in maquis fuels in turkey. forest ecology and management 184, 201–207. boydak, m., 2004: silvicultural characteristics and natural regeneration of pinus brutia ten. – a review. plant ecology 171, 153–163. buhk, c., götzenberger, l., wesche, k., sànchez gómez, p., hensen, i., 2006: post-fire regeneration in a mediterranean pine forest with historically low fire frequency. acta oecologica 30, 288–298. daubenmire, r., 1968: ecology of fire in grasslands. advances in ecological research 5, 209–266. acta bot. croat. 70 (1), 2011 19 post-fire changes in the pinus brutia forests u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:16:03 color profile: disabled composite 150 lpi at 45 degrees davis, p. h. (ed.), 1965–1985: flora of turkey and the east aegean islands. 1–9. edinburgh university press, edinburgh. fady, b., semerci, h., vendramin, g. g., 2003: euforgen technical guidelines for genetic conservation and use for aleppo pine (pinus halepensis) and brutia pine (pinus brutia). international plant genetic resources institute, rome. faraco, a. m., fernandez, f., moreno, j. m., 1993: post-fire vegetation dynamics of pine woodlands and shrublands in the sierra de gredos, spain. in: trabaud, l., prodon, r. (eds.), fire in mediterranean ecosystems, 101–112. commission of the european community, brussels. gezer, a., 1986: the sylviculture of pinus brutia in turkey. options méditerranéennes ciheam 86, 55–66. hanes, t. l., 1970: succession after fire in the chaparral of southern california. ecological monograph 41, 27–50. keeley, j. e., 1987: role of fire in seed germination on woody taxa in california chaparral. ecology 68, 434–443. kutiel, p., shaviv, a., 1989: effect of simulated forest fire on the availability n and p in mediterranean soils. plant soil 120, 57–63. le houreou, h. n., 1974: fire and vegetation in the mediterranean basin. proceedings 13 annual tall timbers fire ecology conference, florida, 237–277. mazzoleni, s., esposito, a., 1993: vegetative regrowth after fire and cutting of mediterranean macchia species. in: trabaud, l., prodon, r. (eds.), fire in mediterranean ecosystems, 87–99. commission of the european communities, brussels. mooney, h. a., dunn, e. l., 1970: convergent evolution of mediterranean climate evergreen sclerophyll shrubs. evolution 24, 292–303. naveh, z., 1975: the evolutionary significance of fire in mediterranean region. vegetatio 29, 199–208. naveh, z., 1990: fire in the mediterranean: a landscape ecological perspective. in: goldammer, j. g., jenkins, m. j. (eds.), fire in ecosystem dynamic: mediterranean and northern perspectives, 1–20. spb academic publishing, the hague. ne’eman, g., goubitz, s., nathan, r., 2004: reproductive traits of pinus halepensis in the light of fire – a critical review. plant ecology 171, 69–79. olsen, s. r., cole, c. v., watanabe, f. s., dean, l. a., 1954: estimation of available phosphorus in soils by extraction with sodium bicarbonate. united states department of agriculture circular, washington, d.c. raison, r. j., 1979: modifications of the soil environment by vegetation fires. with particular reference to nitrogen transformations: a review. plant soil 51, 73–108. serez, m., 1995: status of forest fires and fire management in turkey. international forest fire news 12, 13–16. thanos, c. a., marcou, s., christodoulakis, d., yannitsaros, a., 1989: early post-fire regeneration in pinus brutia forest ecosystems of samos island (greece). acta oecologica 10, 79–94. trabaud, l., 2000: post-fire regeneration of pinus halepensis forests in the west mediterranean. in: ne’eman, g., trabaud, l. (eds.), ecology, biogeography and management 20 acta bot. croat. 70 (1), 2011 türkmen n., düzenli a. u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:16:03 color profile: disabled composite 150 lpi at 45 degrees of pinus halepensis and p. brutia ecosystems in the mediterranean basin, 257–268. backhuys publishers. leiden. trabaud, l., 1973: experimental study on the effects of prescribed burning on a quercus coccifera l. garrigue: early results. proceedings 13 annual tall timbers fire ecology conference, florida, 97–129. trabaud, l., 1987: dynamics after fire of sclerophyllous plant communities in the mediterranean basin. ecologia mediterranea 13, 25–37. trabaud, l., lepart, j., 1980: diversity and stability in garrigue ecosystems after fire. vegetatio 43, 49–57. türkmen, n., düzenli, a., 1998: the flora of dörtyol and erzin districts of hatay province of turkey. turkish journal of botany 22, 121–141. türkmen, n., düzenli, a., 2005: changes in floristic composition of quercus coccifera macchia after fire in the çukurova region (turkey). annales botanici fennici 42, 453–460. verroius, g., georgiadis, t., 2002: post-fire vegetation succession: the case of aleppo pine (pinus halepensis miller) forests of northern achaia (greece). fresenius environmental bulletin 11, 186–193. acta bot. croat. 70 (1), 2011 21 post-fire changes in the pinus brutia forests u:\acta botanica\acta-botan 1-11\turkmen.vp 28. o ujak 2011 15:16:03 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (1), 91–97, 2011 coden: abcra 25 issn 0365–0588 comparative effects of 4-cl-iaa and kinetin on photosynthesis, nitrogen metabolism and yield of black cumin (nigella sativa l.) shoukat h. shah* plant physiology division, department of botany, aligarh muslim university, aligarh 202002, india abstract – the leaves of 40-day old plants of black cumin (nigella sativa l.) were sprayed with 10–7, 10–6, 10–5 m 4-cl-iaa, and 10–6, 10–5 and 10–4 m kinetin. both the hormones improved vegetative growth, photosynthetic efficiency and seed yield of the test plants as compared to deionized water (control). however, 10–6 m 4-cl-iaa was most prominent in its effect, generating 42, 30, 40, 41 and 51% higher values for carbonic anhydrase, nitrate reductase, net photosynthetic rate, leaf protein content and dry mass respectively, as compared to the control in 70-day old plants. similarly, capsule number and seed yield per plant were elevated by 41 and 43% over the untreated control at harvest (130 days after sowing) following the same treatment. overall, the auxin showed a higher efficiency than kinetin in all treatment concentrations. key words: 4-chloroindole-3-acetic acid, photosynthesis, nitrate reductase, carbonic anhydrase, protein, kinetin, yield, nigella sativa. abbreviations; ca – carbonic anhydrase, 4-cl-iaa – 4-chloroindole-3-acetic acid, nr – nitrate reductase, par – photosynthetic active radiation, p n net photosynthetic rate, rubpco – ribulose-1, 5-bisphosphate carboxylase/oxygenase introduction auxins are a class of naturally occurring plant growth substances found throughout the plant kingdom. they are known actively to influence a number of developmental processes such as stem elongation, ethylene biosynthesis and tissue vascularization (nordstrom et al. 1991). the first known ubiquitously occurring auxin is indole-3-acetic acid (iaa), but various other analogues such as 4-cl-iaa have also been identified in certain species. this halogenated compound is characterized by very high auxin-like activity (reinecke et al. 1995), demonstrated in the growth of excised tissue (katekar and geissler 1982), rooting acta bot. croat. 70 (1), 2011 91 * corresponding address, e-mail: shoukathusseine@gmail.com copyright ® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\shah.vp 28. o ujak 2011 16:28:05 color profile: disabled composite 150 lpi at 45 degrees in mung bean (stenlid et al. 1987), parthenocarpy and epinasty in tomato (sell et al. 1953) and the synthesis/induction of specific enzymes in detached pea cotyledons (hirasawa 1989, ahmad and hayat 1999, ahmad et al. 2001a). in addition, acceleration of the rate of photosynthesis resulting in enhancement of the economic yield has also been reported (ahmad et al. 2001b). closely associated in function with auxins are another group of endogenous plant growth substances, the cytokinins. these hormones have an equally potent effect on plant physiology and environmental responses, and are intimately involved in the regulation of cell division, apical dominance, chloroplast development, anthocyanin production and maintenance of the source-sink relationship (hutchkinson and kieber 2002). also, cytokinins are regarded as the most important senescence-retarding hormones and their exogenous application has been demonstrated to prevent the degradation of chlorophyll and photosynthetic proteins (wingler et al. 1998), as well as reverse leaf and fruit abscission (pospí[ilová et al. 2000). owing to the interlinked nature of the action of these hormones and because of their potential benefits in relation to plant performance and productivity, the present study was designed to compare the effect of auxin (4-cl-iaa) and cytokinin (kinetin) supplementation on growth, nitrogen metabolism and yield of black cumin (nigella sativa l.). though natively middle eastern, this herb is valued highly in domestic and international markets owing to its characteristic medicinal and aromatic properties. it is also a significant source of essential fatty acids, carbohydrates, vitamins (a, d, b2 and niacin) and minerals (calcium, potassium, iron, magnesium, selenium and zinc) (saeed et al. 1996). materials and methods seeds of black cumin (nigella sativa l.) were obtained from the regional research institute of unani medicine, aligarh, and were surface sterilized by soaking in 0.01% mercuric chloride solution for 3 minutes, followed by repeated washing with double distilled water. the seeds were then sown in earthen pots (25 cm diameter), filled with 10 kg of a mixture of sandy loam soil and farmyard manure in a ratio of 9:1 (w/w). a uniform basal dose (45, 300 and 78 mg) of n, p and k, in the form of urea, single superphosphate and potassium chloride, was applied at the time of sowing to each pot. the pots were lined in the department’s net house, according to simple randomized block design, with three replications. 5 uniform plants were maintained per pot. 4-chloroindole-3-acetic acid (4-cl-iaa) and kinetin were obtained from sigma chemicals co., st. louis, usa. the plants of 40-days age (vegetative stage) were sprayed with 10–7, 10–6 or 10–5 m aqueous solution of 4-cl-iaa or 10–6, 10–5 or 10–4 m that of kinetin at the rate of 5 cm3 per plant. control plants were sprayed with double distilled water only. the stage of spraying was determined through an earlier experiment (shah 2007). five plants from each replicate were randomly selected and carbonic anhydrase activity (ca), dry mass, nitrate reductase activity, net photosynthetic rate (pn), and protein content were assessed at 50 and 70 days after sowing, corresponding to vegetative and flowering stage respectively. the ca activity in the leaves was measured following the procedure described by dwivedi and randhawa (1974). nitrate reductase (nr) activity was determined in fresh leaf samples, followed the method of jaworski (1971). the net photosynthetic rate was measured using a li-6200 portable photosynthetic system (li-cor 6200, lincoln, ne, 92 acta bot. croat. 70 (1), 2011 shah s. h. u:\acta botanica\acta-botan 1-11\shah.vp 28. o ujak 2011 16:28:05 color profile: disabled composite 150 lpi at 45 degrees usa) on fully expanded uppermost leaves, at atmospheric conditions (between 1100–1200 h): photosynthetic active radiation (par) about 990 mmol m–2 s–1, relative humidity 63%, temperature 23 °c. protein content of the leaves was estimated by the method of lowry et al. (1951). shoot dry mass was determined after dehydrating the plants at 80 °c for 24 h. capsule number and seed yield per plant were estimated at harvest (130 days after sowing). treatment means were compared by analysis of variance using the statistical package spss (spss 7.5.1 for windows, standard version 1996). least significant difference was estimated at 0.05 level of probability. results foliar treatment of either hormone was found appreciably to enhance all the studied parameters; however, a higher degree of stimulation was noted following the application of 4-cl-iaa, especially at the 10–6 m concentration. among different concentrations of kinetin, 10–5 m was found to elicit the maximal response (tabs. 1, 2). the activities of enzymes ca and nr were significantly elevated in the hormone-treated test plants as compared to the water sprayed control. a maximum enhancement of 35 and 42% was noted in the activity of ca at the 50 and 70 days after sowing sampling stages respectively, following treatment with 10–6 m 4-cl-iaa. on the same sampling days, treatment with kinetin was found to cause an enhancement of 28 and 37%, respectively (tab. 1). concurrently, the activity of nr was optimized by 30 and 39% following the auxin treatment as noted at 50 and 70 days after sowing, respectively. on the other hand, corresponding enhancements of 24 and 32 % in enzyme activity were achieved at the respective samplings from treatment with kinetin. in correspondence with the increase in nr activity, protein content was also found to be enhanced in the leaves of the test plants as compared to the control; there was a maximum enhancement of 27 and 38% with the auxin treatment in the samplings, respectively. analogous enhancements of 21 and 29% were recorded subsequent to treatment with kinetin. pn of the treated plants was also appreciably elevated as compared to the control, registering a maximum increase of 26 and 38% at the 50 and 70 days after sowing stages following 4-cl-iaa spray. comparatively, an enhancement of 20 and 30% was brought about by the kinetin treatment at respective samplings. consequent to the increased pn, the test plants also exhibited an increased dry mass production, the maximum enhancement being by 35 and 50% with 4-cl-iaa, whereas that with kinetin was 26 and 42% over the control at respective sampling stages. finally, at harvest, the hormone treated plants showed an appreciable improvement of the yield parameters. number of capsules and seed yield plant–1 were increased by 39 and 41% following the auxin treatment. correspondingly, treatment with kinetin achieved an increase of 31 and 32%, respectively in the mentioned parameters as compared to the control (tab. 2). discussion test plants receiving either of the hormone treatments exhibited an increase in net photosynthetic rate (pn). this effect could be a consequence of the direct stimulation of the process itself by the hormones, as well as of the optimization of other parameters to faciliacta bot. croat. 70 (1), 2011 93 effects of 4-cl-iaa and kinetin on nigella sativa u:\acta botanica\acta-botan 1-11\shah.vp 28. o ujak 2011 16:28:05 color profile: disabled composite 150 lpi at 45 degrees 94 acta bot. croat. 70 (1), 2011 shah s. h. tab 1. carbonic anhydrase (ca) activity [mol (co2) kg –1 s–1], nitrate reductase (nr) activity [nmol (no2) g –1 min–1], net photosynthetic rate (pn) [mmol (co2) m –2 s–1], protein content [% (dm)] and dry mass (dm) [g plant–1], in nigella sativa leaves, sprayed with water (control), 4-cl-iaa or kinetin (kin) at 40-d after sowing and sampled at 50 (vegetative stage) and 70 das (flowering stage). each value is mean ± s.e, (n = 3) lsd for p = 0.05. parameter treatment [m] 50 das % increase over control 70 das % increase over control ca control 2.17±0.20 – 2.64±0.18 – kin 10–6 2.51±0.22 16 3.19±0.23 21 10–5 2.79±0.17 28 3.61±0.24 37 10–4 2.73±0.22 26 3.56±0.21 35 4-cl-iaa 10–7 2.58±0.15 19 3.30±0.20 25 10–6 2.93±0.25 35 3.74±0.25 42 10–5 2.88±0.22 33 3.70±0.23 40 lsd 0.068 0.057 nr control 7.11±0.65 – 7.56±0.81 – kin 10–6 7.97±0.55 12 8.93±0.54 18 10–5 8.82±0.65 24 9.97±0.75 32 10–4 8.89±0.81 25 9.90±0.81 31 4-cl-iaa 10–7 8.32±0.64 17 9.30±0.71 23 10–6 9.25±0.61 30 10.50±0.71 39 10–5 9.17±0.71 29 10.36±0.81 37 lsd 0.17 0.18 pn control 15.01±1.3 – 16.05±1.6 – kin 10–6 16.66±1.6 11 18.65±1.9 16 10–5 18.01±1.7 20 20.88±1.4 30 10–4 17.71±1.5 18 20.38±1.7 27 4-cl-iaa 10–7 17.10±1.6 14 19.75±1.4 23 10–6 18.95±1.9 26 22.15±1.6 38 10–5 18.61±1.7 24 21.66±1.7 35 lsd 0.48 0.58 protein control 11.35±1.1 – 12.75±1.1 kin 10–6 12.83±1.2 13 14.65±1.4 15 10–5 13.74±1.4 21 16.46±1.3 29 10–4 13.63±1.3 20 16.34±1.2 28 4-cl-iaa 10–7 13.30±1.5 17 15.31±1.5 20 10–6 14.42±1.6 27 17.62±1.8 38 10–5 14.21±1.1 25 17.48±1.5 37 lsd 0.32 0.78 dm control 1.15±0.11 1.85±0.17 kin 10–6 1.29±0.12 12 2.18±0.18 18 10–5 1.45±0.13 26 2.63±0.16 42 10–4 1.44±0.10 25 2.61±0.19 41 4-cl-iaa 10–7 1.35±0.09 17 2.31±0.16 24 10–6 1.55±0.16 35 2.79±0.20 50 10–5 1.53±0.15 33 2.74±0.21 48 lsd 0.06 0.08 u:\acta botanica\acta-botan 1-11\shah.vp 28. o ujak 2011 16:28:05 color profile: disabled composite 150 lpi at 45 degrees tate efficient photosynthetic activity. the elevation of the activities of enzymes ca and nr can be said to have been of significant consequence. after the substrate for carbon fixation, co2, diffuses into the stomata and is transported into the chloroplast, it is reduced by rubpco, the main c fixing enzyme. however, this supply of co2 to rubpco depends on the activity of ca, which catalyses the reversible hydration of hco3 to co2 in close proximity to rubpco. this entire mechanism could have been augmented by the enhancement in ca activity, as achieved in the present study through hormone supplementation. in context of the 4-cl-iaa treatment, such results can be ascribed to the action of auxins in de-repressing certain genes and in activating the process of translation and transcription (key 1969, hopkins 1995). similarly, the enhancement effect of kinetin on ca activity can be said to have arisen at the level of transcription and / or stabilization of the transcripts while they increased the content of ca-mrna through translation (sugiharto et al. 1992). our hypothesis that enhanced ca activity brought about an optimization of co2 availability, consequently to affect pn, is strongly supported by the positive correlation observed between ca and pn of the hormone treated plants (r = 0.875). another level of influence for the hormones on pn was the protein content in leaves of the test plants, which was enhanced following an increase of nr activity (tab. 1). nr regulates the major rate limiting stage in the reduction of nitrate to ammonium, which is then incorporated in amino acids (hopkins 1995).the hormone treatment resulted in an increase in nr activity and thus elevated the useable form of nitrogen (ammonia) to produce a larger pool of amino acids/amides, in addition to stimulating their incorporation to polypeptide synthesis (by activating transcription and/or translation) and optimizing protein formation. this conjecture is supported by the correlation obtained between nr activity and the level of protein in leaves of the test plants (r = 0.821) (singh and singh 1985). in the context of nr, similar enhancements have been reported following auxin and kinetin treatment, although not from the modulation of the activity of the existing enzyme, but by induction of its de novo synthesis (premabatidevi 1998). hence in the present study, the influence of auxins and cytokinins on translation/transcription mechanisms, as aforementioned, can be referred to in the context of increased nr activity and protein content of the test plants. acta bot. croat. 70 (1), 2011 95 effects of 4-cl-iaa and kinetin on nigella sativa tab 2. number of capsules and seed yield plant–1 in nigella sativa l. plants, sprayed with water (control), 4-cl-iaa or kinetin (kin) at 40-d after sowing and sampled at harvest (130 das), lsd for p = 0.05. each value is mean ± s.e, (n=3). treatment [m] number of capsules [plant –1 ] % increase over control seed yield [g plant –1 ] % increase over control control 16.15±1.4 – 1.20±0.17 – kin 10–6 18.50±1.7 14 1.35±0.20 13 10–5 21.28±1.8 31 1.58±0.21 32 10–4 20.98±1.6 30 1.57±0.19 31 4-cl-iaa 10–7 19.57±1.9 21 1.43±0.17 19 10–6 22.46±2.2 39 1.69±0.20 41 10–5 22.15±1.8 37 1.65±0.11 38 lsd 0.65 0.05 u:\acta botanica\acta-botan 1-11\shah.vp 28. o ujak 2011 16:28:05 color profile: disabled composite 150 lpi at 45 degrees in the present study, leaves of plants receiving either of the hormone treatments not only photosynthesized at a faster rate, but also possessed an extended period of metabolic activity, because of the delayed senescence caused by the phytohormones (davies 1995). as a result, these test plants accumulated a large quantity of metabolites, which is evident from the increase observed in their dry mass (tab. 1). a similar positive relation between pn and dry mass production has been observed by khan (1994). the sufficient availability of nutrient facilitated ample vegetative growth in the treated plants, thus resulting in an increase in the number and size of reproductive sink (number of capsules per plant) (tab. 2). this, when coupled with the 4-cl-iaa-enhanced mobilization of photoassimilates to the developing capsules (davies 1995), may have further stimulated their growth and hence caused an increase in seed yield, as observed herein (tab. 2). likewise, the effect of kinetin on economic yield, though relatively mild, may be ascribed to the increased opportunity for formation of new buds (bruinsma 1977), and successful reversal of flower and fruit abscission (nagel et al. 2001). conclusively, the efficacy of auxin (4-cl-iaa) was found to be superior to that of kinetin with regard to performance and productivity of nigella sativa. the relatively subdued effect of kinetin may have been because endogenous cytokinin is seldom 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1847-8476 influence of dehydration on cryopreservation of musa spp. germplasm ergun kaya1*, fernanda vidigal duarte souza2, janay almeida dos santos-serejo2, selin galatali1 1 mugla sitki kocman university, faculty of science, molecular biology and genetics department, 48000 kotekli, mugla, turkey 2 embrapa cassava and fruits, caixa postal 007, cruz das almas, ba 44380-000, brazil abstract – cryopreservation is an important technique for the long-term storage of economically important plant germplasm. in this study, an efficient protocol was developed for the long-term conservation of seven economically important musa taxa: m. acuminata colla ssp. burmannica n.w. simmonds, m. acuminata colla ssp. zebrina (van houtte) r.e. nasution, m. balbisiana colla, m. basjoo sieb., m. ornata w. roxburgh (st. lavender), m. velutina h. wendl. et drude (velvet pink banana), and m. acuminata × balbisiana. the seeds were dehydrated in a sterile laminar flow cabinet for different exposure times and then they were directly immersed in liquid nitrogen. the critical point was to support the initial germination of cryopreserved seeds and this was achieved by the excision of zygotic embryos after liquid nitrogen treatment that allowed the seed germination. the best moisture content for tolerance to cryopreservation ranged from 15.8% (m. acuminata ssp. zebrina) to 17.1% (m. ornata) and the maximum post-cryopreservation germination rates varied from 86.4% (m. velutina) to 55.0% (m. ornata). all seedlings derived from seeds germinated after cryopreservation were easily rooted and acclimated to greenhouse conditions. keywords: banana, long-term conservation, moisture content, seed desiccation * corresponding author e-mail: ergunkaya@mu.edu.tr introduction conservation strategies can be divided into two main categories, ex situ and in situ, and include many procedures for the preservation of plant biodiversity. ex situ strategies consist of the conservation of biodiversity in an area different from that in which plants have their natural habitat. in situ strategies, on the other hand, mean the maintenance of biodiversity as the domestication and/or cultivation of species, in developed artificial media (engelmann 2012). conservation of agrobiodiversity and plant biodiversity is important for food security and the sustainability of plant genetic resources. genetic diversity increases the options for improvement through crop selection and breeding, including higher yields and greater resistance to adverse environmental conditions (rao 2004). the banana is the one of most important nutrition sources in the world and many efforts have been made to support the conservation of musa germplasm (fao 2010, langhe et al. 2018). banana and plantain germplasm has conventionally been preserved in field genebanks, but there are many other different preservation strategies and methods with specific advantages and disadvantages for short-, medium and longterm conservation, depending on different situations, such as plant material and the method to be applied (sipen et al. 2011). the basic procedure for seed conservation in seed banks consists of dehydration and storage of seeds at low temperatures (below 0 °c). however, active collections are stored above 0 °c. additionally, different types of genebank, such as in vitro and field collections, complement each other for musa spp. germplasm conservation and seed storage, and clonal collections need further complementary conservation procedures (chin 1996). the embryo metabolism of orthodox seeds is suspended as a result of maturation drying and seedswith low moisture contents (~10–15%) can not germinate until their water content becomes fully restored, when they are also not dormant (bewley and nonogaki 2003). the long-term storage of the seeds of orthodox species in genebanks requires certain conditions: a temperature between –15 °c and –20 °c and a kaya e, souza fvd, almeida dos santos-serejo j, galatali s 100 acta bot. croat. 79 (2), 2020 moisture content between 3% and 7%. orthodox seeds can maintain germination and viability under these conditions for long periods of time, a hundred years or more (hintum and menting 2003). musa spp. seeds are classified as orthodox and can be dehydrated and conserved for long periods of time at low temperatures (vineesh et al. 2015, kaya 2016) and they are also known as very dormant. after harvesting, these seeds can easily germinate, however, they usually stay dormant when they lose their moisture content (chin 1996). the hard and thick seed coat of musa spp. seeds can prevent the water and oxygen uptake that are essential for germination under natural conditions (puteh et al. 2011). the very small embryo of the seed is under a seed lid named an operculum which blocks water uptake for dried seeds (ellis et al. 1985, graven et al. 1996). because only the wild musa and plantain species produce large numbers of seeds, the storage of seeds of commercial varieties has limited applicability. while orthodox seeds can be dehydrated and stored for long periods at low temperatures, seed dormancy is frequently a problem after drying and cold treatment processes. for development of an efficient way to start the initial germination, further work on understanding the mechanisms of germination is required. additionally, more information is needed about the optimization of storage conditions, effective germination and the handling of these seeds for succesful achievement of longterm storage of musa spp. seeds (kaya 2016). cryopreservation has a significant role in global programs to preserve plant genetic resources (reed 2008). seeds stored in seed banks are divided into three main groups according to dehydration responses: orthodox (dehydrationtolerant); intermediate (relatively dehydration-tolerant); and recalcitrant (dehydration-sensitive). orthodox seeds accumulate sugars and proteins in their cytoplasm to achieve vitrification at positive temperatures and to resist damage to their cells and membranes by desiccation at low temperature (kaya et al. 2017). for this reason, such seeds are cryopreserved easily by being directly plunged into liquid nitrogen after desiccation, without any substantial reduction of seed viabilityor germination rate upon thawing (gakhova et al. 2006). furthermore, musa seed cryopreservation may have applications beyond the conservation of germplasm and can be a valuable tool for breeding programs. some crosses generate a large number of seeds and the rescue of embryos becomes long and labor-intensive. for the transfer of considerable properties to exclusive varieties, the fertile and viable seeds of musa spp. are important germplasms for breeding programs. this provides an advantage for sterile vegetatively propagated clones which is available as trading varieties. the possibility of cryopreserving these seeds allows the work to be done within the capacity of the laboratory. the aim of this study was to develop a protocol for the long-term conservation of musa seeds using dehydration/direct immersion in liquid nitrogen. materials and methods plant material m. acuminata colla ssp. burmannica n.w. simmonds, m. acuminata colla ssp. zebrina (van houtte) r.e. nasution, m. balbisiana colla, m. basjoo sieb., m. ornata w. roxburgh (st. lavender), m. velutina h. wendl. et drude (velvet pink banana) and m. acuminata × balbisiana (hybrid) seeds were provided by the genebank of the berlin-dahlem botanical garden. decontamination of musa spp. seeds musa spp. seeds belonging to the seven taxa were surface sterilized by treatment for 5 minutes in 70% ethanol, 5 minutes in 10% h2o2 and two times 10 minutes in 20% concentrated commercial bleach (domestos®). after each step, the seeds were rinsed in sterile distilled water (kaya 2016). embryo germination media and culture conditions zygotic embryos obtained from seeds of the seven musa spp taxa were extracted for in vitro germination from all of control (before and after dehydration of seeds) and liquid nitrogen groups (after cryopreservation of seeds) under a microscope (fig. 1a, b) before being transferred to semisolid ms (murashige and skoog 1962) germination medium supplemented with 20 g l–1 of sucrose as carbon source, 1.5 g l–1 of phytagel and 3.5 g l–1 of agar as solidifying agents, and 0.1 µm of gibberellic acid (13 mg l–1) as dormancy breaker (fig. 1c). until the start of germination, the zygotic embryos were kept in the dark at 27 ± 2 °c and then, they were transferred to the standard culture conditions (16/8 h photoperiod, with cool daylight fluorescent lamps rated at 50 μmol−1 m−2 s−1). determination of moisture content and evaluation of germination rate after the musa spp. seeds were weighed (initial weight), they were placed in sterile petri dishes in a laminar flow cabinet at room temperature. additionally, to test the initial germination rate, ten zygotic embryos removed from non-dehydrated seeds were directly transferred to germination medium, while the other seeds were kept in the laminar flow cabinet (up to 9 hours) to measure the seed weight every hour for determination of the water loss rate. the environmental conditions (temperature and relative humidity) of the laminar flow cabinet were recorded during the dehydration procedure and ten seeds were also transferred to germination medium to test the effect of dehydration on germination after each hour. in all, 200 seeds (20 seeds for each hour of dehydration time from 0 to 9 hours, ten for moisture content measurement and the others for germination testing) were used. each test was repeated at least three times. seed moisture content was calculated using the formula of pixton (1966): % moisture content (mc) = [(initial weight of seeds – dry wight of seeds) / initial weight of seeds] × 100. cryopreservation of musa germplasm acta bot. croat. 79 (2), 2020 101 long-term storage via cryopreservation after calculation of seeds’ mc in the dehydration test, the dehydrated seeds (from each dehydration time in hours) were placed in 2 ml cryovials, five seeds per vial, and were directly plunged into a storage tank containing liquid nitrogen. the thawing process was performed by placing the seeds in a sterile laminar flow cabinet at room temperature for 5 minutes after storage of 24 hours at −196 °c. the zygotic embryos removed from the cryopreserved and thawed seeds were transferred to germination medium and kept in the dark at 27 ± 2 °c, and then transferred to culture media under the conditions stated above. evaluation of data and statistical analyses zygotic embryo germination of the control and liquid nitrogen group was evaluated with respect to dormancy for three and four weeks (for control group) and six and eight weeks (for liquid nitrogen group) after being transferred to germination medium. there were the germination time differences between control group and liquid nitrogen group. germination of the liquid nitrogen group took longer than the control group. zygotic embryos of both groups producing at least one morphologically normal seedling were accepted as successful germinations. thirty musa spp. seeds were used for each treatment step for mc determination. the treatments of all control and liquid nitrogen groups were repeated at least three times. embryo germination porcentage was compared by multiple x2 test by the spss program (ibm spss statistics 21.0) and statistical analysis was also performed with anova, followed by the lsd test at p ≤ 0.05 (marascuilo and mcsweeney 1977). results relation between seed moisture content and embryo germination percentage after treatment of successful surface sterilization (which obtained approximately 90% sterile material), the seeds presented normal morphology. initial moisture content of all seven musa taxa ranged between 45.1% (m. ornata) and 58.5% (m. acuminata × balbisiana) and embryo germination percentage ranged between 80% (m. acuminata ssp. zebrina) and 100% (m. balbisiana, m. basjoo and m. acuminata × balbisiana). the moisture content of seeds was reduced by dehydration in the laminar flow cabinet, to values between 9.2% fig. 1. in vitro germination of zygotic embryos excised from musa spp. after cryopreservation: seeds of m. velutina (a), embryo of m. ornata (arrow indicates embryo in seed) (b), embryo germination of cryopreserved seed of m. acuminata ssp. burmannica in semi-solid germination medium after four weeks incubation (c). seedlings having well-formed shoots and strong roots derived from embryos of cryopreserved seeds of m. acuminata ssp. burmanica (d), m. acuminata ssp. zebrina (e), m. balbisiana (f), m. basjoo (g), m. ornata (h), m. velutina (i), m. acuminata’ balbisiana (j), all musa spp. cryopreserved seeds were very easily acclimated to greenhouse conditions (k). scale bars: 1 cm. kaya e, souza fvd, almeida dos santos-serejo j, galatali s 102 acta bot. croat. 79 (2), 2020 (m. acuminata ssp. zebrina) and 15.9% (m. velutina) after nine hours. the germination percentage decreased as the water content declined. the seeds most sensitive to dehydration belonged to m. ornata, because germination decreased to 55.2% when the moisture content declined to 15.8% after nine hours of dehydration. on the other hand, seeds that had almost the same moisture content (between 15.8% and 16.2% comparing four taxa of musa), such as 16.0% (m. balbisiana, 6 h), 15.9% (m. velutina, 9 h), 16.2% (m. acuminata × balbisiana, 8 h) and 15.8% (m. acuminata ssp. zebrina, 6 h) had good germination rates, of 100%, 86.2%, 84.7% and 70.3%, respectively (tab. 1, fig. 2). these dehydration time differences were caused by the different shapes and sizes of the different musa taxa seeds used in the study. cryopreservation of seeds the best cryopreservation results, ranging from germination of 55% (m. ornata) to 86.4% (m. velutina) were obtained from seeds that had moisture content reduced to between 15.9% (m. acuminata ssp. zebrina) and 17.1% (m. ornata) (tab. 1, fig. 2). there was a positive correlation between moisture content and germination percentage in the control group. on the other hand, there was a negative correlation after cryostorage. another significant result was that most of the musa spp. seeds with a moisture content of 20% or more did not germinate, the exceptions being m. acuminata subsp. burmanica and m. ornata, with 21.3% and 20.8% moisture content respectively, although the germination percentage were very low (4.2% for m. acuminata ssp. burmannica and 10% for m. ornata). furthermore, the blocking effect of liquid nitrogen delayed the in vitro germination compared to control groups. for example, the zygotic embryos removed from cryopreserved seeds started to germinate after approximately six or eight weeks for all the musa spp., while the germination times of embryos removed from control group seeds were shorter (maximum four weeks) (fig. 1c). finally, all seedlings obtained from zygotic embryos of cryopreserved seeds had well-formed shoots and strong roots (fig. 1d-j) and they were very easily acclimated to greenhouse conditions (fig. 1k). discussion although musa spp. seeds are classified as orthodox, they have been found to be dormant after preservation, depending on the different morphological, physical and physiological characteristics among species (chin 1996, burgoshernández et al. 2014). their hard and strict seed coat causes physical dormancy by reducing water intake, which is crucial for germination, thus decreasing or completely preventing germination (baskin et al. 2000). musa spp. seed endosperms are surrounded by a tegmen and testa. the first one is a weak inner integument and the other is a multilayer outer integument. the musa seed zygotic embryos are very soft, smooth and small, and are located under a seed lid called the operculum (graven et al. 1996) and in some seeds, fig. 2. effect of varying moisture content on musa spp. seed germination: a – m. acuminata ssp. burmannica, b – m. acuminata ssp. zebrina, c – m. balbisiana, d – m. basjoo, e – m. ornata, f – m. velutina, g – m. acuminata × balbisiana (enviromental conditions during seed dehydration: temperature 23.04 ± 0.06 °c; relative humudity, 46 ± 2.65%). cryopreservation of musa germplasm acta bot. croat. 79 (2), 2020 103 the cells surrounding them can inhibit water uptake during germination (finch-savage and leubner-metzger 2006). in this study, to overcome this problem, the zygotic embryos were removed from all the dehydrated seeds before transfer to the germination medium. in this way, they germinated easily (germination start of the control seeds took three to four weeks, while for the liquid nitrogen group it took six to eight weeks). many treatments aimed at breaking the dormancy of musa seeds have been described, such as mechanical scratching, treatment with gibberellic acid, removal of zygotic embryos, chemical exposure (treatment with sulfuric acid with different exposure times), but only removal of zygotic embryos, as in this study, and/or chemical exposure have shown successful germination (puteh et al. 2011, uma et al. 2011, burgos-hernández et al. 2014). the capability of orthodox seeds to survive significant dehydration tends to be obtained during seed maturation at which seeds reach highest dry weight, or earlier, based upon the environment conditions to which they have been subjected (hong and ellis 1992). this capability can be lost during seed germination. the dried seeds turn out to be sensitive to dehydration if it is permitted to develop too far (koster and leopold 1988). loss of dehydration tolerance tends to overlap with radicle emergence (senaratna and mckersie 1983). dehydration sensitivity of intermediate and recalcitrant seeds have also been shown to be decreased during the development, but not to the same extent as in orthodox seeds (ellis et al. 1985). cryopreservation (storage at ultra-low temperatures) of different types of plant materials (seeds, meristems, buds, embryos, calli) is used for long-term conservation of plant genetic resources (reed 2008). the main aim of this study was to optimize a protocol for long-term storage of seven different musa taxa using a one-step freezing technique (dehydration and direct immersion in liquid nitrogen). the results were successful, with germination percentage of embryos from cryopreserved seeds between 55% and 86.4%. the other critical result was that optimum germination was obtained with seed moisture contents between 15.9% and 17.1% (tab. 1, fig. 2). it can be deduced that for long-term cryopreservation of musa spp seeds in liquid nitrogen, the moisture content should be less than 17% for optimal viability and germination. panis (2009) cryopreserved zygotic embryos of m. acuminata and m. balbisiana and obtained an average germination rate of 53% from zygotic embryos having 14% moisture content. in another study, kaya (2016) used two different musa species (m. velutina and m. acuminata) for cryopreservation and obtained an optimum germination rate (up to 84.3%) from seeds with moisture content of less than 17%. our results corroborate these earlier findings. however, some orthodox seeds have critical problems such as dormancy as seen in the current study. they are still very suitable material for successful cryopreservation, because they have strong sugar reserves and their zygotic embryos have high sucrose/raffinose contents, preventing sucrose crystallization at high concentrations. these properties have a protective role during seed desiccation (steadman et al. 1996). one explanation for this supposition is that sugars cause the vitrification of cell cytoplasm. thus, the intracellular matrix becomes concentrated via dehydration and does not form ice crystals (buitink and leprince 2004). another possible mechanism is that oligosaccharides change with water bound to membrane phospholipids via hydrogen bonding (crowe et al. 1988). tab. 1. germination percentages of control group and cryopreserved seeds belonging to seven musa taxa: 1: m. acuminata colla ssp. burmannica n.w.simmonds; 2: m. acuminata colla ssp. zebrina (van houtte) r.e. nasution; 3: m. balbisiana colla; 4: m. basjoo sieb.; 5: m. ornata w. roxburgh (st. lavender); 6: m. velutina h. wendl. et drude (velvet pink banana; 7: m. acuminata × balbisiana (cont. – control group, ln+ – liquid nitrogen group). values in bold indicated the best germination percentages obtained from cryopreserved seeds. germination percentage ± standard error values were statistically analyzed by a nonparametric test, the post hoc multiple comparisons test (marascuilo and mcsweeney 1977). statistical analysis performed by anova, followed by lsd test at p ≤ 0.05. different letters at apex of values indicate statistically significant differences between the values obtained from treatment results, p ≤ 0.05. musa taxon dehidration time 1st h 2nd h 3rd h 4th h 5th h 6th h 7th h 8th h 9th h 1 cont. 89.5 ± 0.1c 90.0 ± 0.6c 83.0 ± 1.1de 79.3 ± 1.8e 78.9 ± 0.9e 70.7 ± 1.1g 64.8 ± 0.7h 60.0 ± 0.7i 59.3 ± 0.9i ln+ 0.0 0.0 4.2 ± 0.3r 23.3 ± 0.5p 68.3 ± 0.4h 48.5 ± 0.5k 40.0 ± 1.3m 40.4 ± 0.4m 32.7 ± 0.6no 2 cont. 71.0 ± 0.0g 70.6 ± 0.2g 73.1 ± 1.3f 69.5 ± 0.6g 70.5 ± 0.6g 70.3 ± 0.5g 50.3 ± 0.3k 42.5 ± 0.7lm 48.0 ± 0.3k ln+ 0.0 0.0 0.0 6.3 ± 0.3r 48.4 ± 0.3k 60.9 ± 0.7i 42.3 ± 0.3lm 0.0 10.5 ± 0.3q 3 cont. 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 99.0 ± 0.9a 95.5 ± 0.1b 100 ± 0.0a 89.6 ± 0.1c 81.1 ± 1.1e 73.3 ± 0.1f ln+ 0.0 0.0 0.0 10.2± 0.9q 70.4 ± 0.3g 79.6 ± 0.1e 48.4 ± 0.37k 38.3 ± 0.3m 41.7 ± 1.3m 4 cont. 100 ± 0.0a 98.3 ± 0.1a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 89.6 ± 0.1c 78.3 ± 1.1e 73.3 ± 0.1f 75.4 ± 0.1f ln+ 0.0 0.0 0.0 0.0 30.9 ± 0.4o 56.0 ± 0.4j 71.5 ± 0.1g 48.4 ± 0.3k 0.0 5 cont. 79.3 ± 0.5e 73.0 ± 0.3f 73.3 ± 0.2f 65.1 ± 0.2h 69.5 ± 0.4g 66.7 ± 0.5h 68.3 ± 0.3h 60.0 ± 0.5i 55.2 ± 0.3j ln+ 0.0 0.0 0.0 0.0 10.0 ± 0.3q 48.0 ± 0.7k 55.0 ± 0.4j 43.3 ± 0.7l 40.7 ± 0.1m 6 cont. 100 ± 0.0a 100 ± 0.0a 96.7 ± 0.1b 96.0 ± 0.1b 91.0 ± 1.1c 89.3 ± 0.4c 91.5 ± 0.1c 89.4 ± 0.2c 86.2 ± 0.1d ln+ 0.0 0.0 0.0 0.0 0.0 34.0 ± 0.2n 48.2 ± 0.3k 86.4 ± 0.1d 83.3 ± 0.3d 7 cont. 98 ± 0.0a 100 ± 0.0a 96.7 ± 0.1b 99.7 ± 0.1a 100 ± 0.0a 93.3 ± 0.3b 90.7 ± 0.1c 90.3 ± 0.1c 92.1 ± 0.1c ln+ 0.0 0.0 0.0 0.0 51.8± 1.1k 64.0 ± 0.2h 77.2 ± 0. 8ef 84.7 ± 0.1d 75.0 ± 0.3f kaya e, souza fvd, almeida dos santos-serejo j, galatali s 104 acta bot. croat. 79 (2), 2020 references baskin, j.m., baskin, c.c., li, x., 2000: taxonomy, anatomy and evolution of physical dormancy in seeds. plant species biology 34, 139–152. bewley, j.d., nonogaki, h., 2003: seed maturation and germination. in: akkerman, a.j.j., andré, j.p. 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(eds.), non-parametric and distribution-free methods for the social sciences, 141–147. brooks/cole publications, pacific grove, ca, usa. murashige, t., skoog, f., 1962: a revised mediumfor rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum 15, 473–497. panis, b., 2009: cryopreservation of musa germplasm. in: engelmann, f., benson, e. (eds.), technical guidelines, 27–36. bioversity international, montpellier. pixton, s.w., 1966: moisture content-its significance and measurement in stored products. journal of stored products research 3, 35–47. puteh, a.b., aris, e.m., sinniah, u.r., rahman, m.m., mohamad, r.b., abdullah, n.a.p., 2011: seed anatomy, moisture content and scarifi cation infl uence on imbibition in wild banana (musa acuminata colla) ecotypes. african journal of biotechnology 10(65), 14373–14379. rao, n.k., 2004: plant genetic resources: advancing conservation and use through biotechnology. african journal of biotechnology 3, 136–145. reed, b.m., 2008: plant cryopreservation: a practical guide. springer, new york. senaratna, t., mckersie, b.d., 1983: dehydration injury in germinating soybean (glycine max l. merr.) seeds. plant physiology, 72, 620–624. sipen, p., chubo, j. k., king, p.j.h., ong, k.h., davey, m.r., 2011: genetic improvement of banana using conventional and in vitro technologies. journal of crop improvement 25(6), 697–727. steadman, k., pritchard, h.w., dey, p.m., 1996: tissue-specific soluble sugars in seeds as indicators of storage category. annals of botany 77(6), 667–674. uma, s., lakshmi, s., saraswathi, m.s., akbar, a., mustaffa, m.m., 2011: embryo rescue and plant regeneration in banana (musa spp. l.). plant cell tissue and organ culture 105, 105–111. vineesh, p.s., skaria, r., mukunthakumar, s., padmesh, p., decruse, s.w., 2015: seed germination and cryostorage of musa acuminata ssp. burmannica from western ghats. south african journal of botany 100, 158–163. this study showed a variation in the response of the different musa spp. seeds to dehydration and cryopreservation. however, despite this, the presented technique can be applied to the cryopreservation of musa orthodox seeds. as for the problem of identified dormancy, it can be overcome by the excitation of the embryo and its cultivation in culture medium. acknowledgements the study was supported by mugla sitki kocman university, scientific research projects coordination unit (mugla, turkey, msku-bap, project number: 17–135). opce-str.vp acta bot. croat. 68 (1), 79–92, 2009 coden: abcra 25 issn 0365–0588 diversity in common bean landraces from south brazil tamara pereira1, cileide m. m. coelho1*, amauri bogo1, altamir f. guidolin1, david j. miquelluti2 1 departamento de agronomia, universidade do estado de santa catarina, avenida camões 2090, bairro conta dinheiro cep 88520-000, lages sc, brasil 2 departamento de solos e recursos naturais, universidade do estado de santa catarina (udesc). avenida camões, 2090, bairro conta dinheiro, cep 88520-000, lages, sc, brasil phaseolin is the major protein in legume seeds and has provided evidence for protein diversity studies, particularly for subdividing phaseolus vulgaris in two major gene pools: the central american (s-type) and the andean (t-type) groups. in the work reported here, a total of 73 representative landrace genotypes from santa catarina state, brazil, were evaluated according to their phaseolin patterns using sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). the seed traits analyzed were: a) the 100-seed weight (p100); b) seed shape degree and seed flattening as determined by j and h coefficients; c) soluble and total protein contents; and d) seed colours. the data indicated that landrace genotypes of common bean collected in southern brazil were from both gene pools (central america and andes) with both »s« (53.42%) and »t« (42.46%) phaseolin types. the p100 was the main character that grouped these gene pools. the landrace genotypes of the common bean showed a wide range of seed size associated with seed colour. the grouping used by comparison of means, allowed efficiently identify promising genotypes to compose valuable source of genetic diversity that would be highly useful for future studies of representative genotypes from each group. the accession numbers 11, 25, 26, 46, 48 and 74 are of interest for breeding purpose, for they showed higher productivity associated with high protein content. keywords: bean, phaseolus, genetic variability, phaseolin, sds-page, protein, polymorphism introduction the common bean (phaseolus vulgaris l.) is one of the most important legumes in the world. brazil is the major producer of this crop with approximately 3 million ton/year and an annual per capita consumption average of around 17.5 kg (broughton et al. 2003). in southern brazil, mainly santa catarina state, in the year of 2006/2007 there was a production of around 217.000 tons, which constituted 5.8% of brazilian production, which is characterized by low-input and medium-input sustainable farming systems (ibge 2007). acta bot. croat. 68 (1), 2009 79 * corresponding author, email: a2cmm@cav.udesc.br u:\acta botanica\acta-botan 1-09\pereira.vp 22. travanj 2009 12:18:46 color profile: disabled composite 150 lpi at 45 degrees beans provide essential proteins (20 to 25%) in the human diet, complementing other food sources like maize and rice (broughton et al. 2003). phaseolin is the major protein in the bean seed and has provided evidence for studies into protein diversity. common bean cultivars are products from multiple domestications in the american continent (van schoonhoven and voysest 1991). archaeological, morphological, biochemical and molecular evidence has suggested two well-defined andean and mesoamerican centers of common bean origins, which were confirmed through morphological markers (gepts et al. 1986, singh et al. 1991, chacón et al. 2005, de la cruz et al. 2005), isozyme markers (koenig and gepts 1989, santalla et al. 2004, chacón et al. 2005), phaseolin types by eletrophoretic profiles (gepts et al. 1986, pereira and souza 1992, maciel et al. 1999, solano 2005), and molecular markers of rflp (chacón et al. 2005), aflp (maciel et al. 2003), rapd (beebe et al. 2000) and issr (de la cruz et al. 2005). the primary center of the central american type is characterized by cultivars with predominantly the »s« phaseolin type and smaller seeds (< 25 g/100 seeds). the other primary center, the andean, is characterized by cultivars with the »t« phaseolin type and larger seeds (> 40 g/100 seeds) (singh et al. 1991). the expression of the phaseolin phenotype is not influenced by environment (brown et al. 1981), or by human selection, or any other type of selection (gepts et al. 1986). in addition, the heritability of seed may reach values of up to 0.8–0.9 (van schoonhoven and voysest 1991), showing that the phaseolin type can be used as a potential tool to indicate genetic diversity and gene flow between wild and domesticated beans. in a previous study with 192 landrace beans (pereira and souza 1992) from the cnpaf (centro nacional de pesquisa de arroz e feijão – embrapa, brazil) germplasm bank 80.6% and 19.4% were established for the »s« and »t« phaseolin types, respectively. amongst all 192 genotypes only 1 was related with the »t« type from santa catarina state. on the other hand, gepts et al. (1988) found predominantly the »t« type only in the minas gerais and santa catarina regions from the 72 brazilian genotypes studied. but these studies did not include a representative number of landraces from santa catarina state. the genetic diversity available for the common bean in brazil has been reduced drastically by rigorous consumer preference by seed size, shape and colour (conafe 2005). this reduction has limited the source of genetic variability for breeding and conservation programs. particularly in santa catarina state currently in use are landraces of phaseolus vulgaris that display a wide range of seed and colour patterns, maintained for generations by farmers, contributing to the genetic resource (nass and paterniani 2000). the preservation and study of landraces are a challenge for the future, as landraces represent a source of variation that can be useful in crop science or breeding programs to increase seed quality. moreover, these genotypes can contribute important characteristics like resistance to biotic and abiotic stresses, diseases, and seed technology quality, to reduce the vulnerability of improved genotypes (gepts et al. 1986). the traditional bean cropping in santa catarina state is based on small to medium properties. this activity is basically an income earner for the farmers. however, it has not been sufficient for the maintenance of the families, leading the farmer to look for new economic alternatives, like the grain differential yield. moreover, governmental incentives and scientific support concerning the qualities of the landraces that they are producing exist. based on the above considerations, the objective of the present study was to characterise the di80 acta bot. croat. 68 (1), 2009 pereira t., coelho c. m. m., bogo a., guidolin a. f., miquelluti d. j. u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:09 color profile: disabled composite 150 lpi at 45 degrees versity among landraces of the common bean from southern brazil through phaseolin pattern and other traits such seed shape, colour patterns and soluble and total protein, to provide information to the farmer about the cultivation and preservation of these landraces. materials and methods plant material a collection of 73 common bean genotypes were used in the study for comparison against 5 controls of phaseolin type (active bank common beanbafs: 113-»s«, 116-»h«, 117-»t«, 118-»b« and 119-»c«) that were supplied by cnpaf (embrapa, centro nacional de pesquisa de arroz e feijão, brazil). the 5 commercial cultivars (bafs: 110, 111, 112, 115 and 121) and 63 landraces of common bean were randomly chosen from the different regions of the state of santa catarina, and the seeds were obtained from the active bank bean (baf), from santa catarina state university (udesc, brazil). the landraces are defined as locally adapted or domesticated unimproved. a field experiment was conducted in the 2005/06 season in a randomized complete block design, with three replications, located at lages, santa catarina state, south-brazil (27 ° 52’ south, 50 ° 18’ east, 930 m above sea level), characterized by mild summers and regularly distributed rains (epagri 2006). the objective of the experiment was to obtain seeds from the same field at the same time. the unit plot consisted of 4 rows, each 4 m long, with 0.5 m between rows; 1 m between plots and 208 plants per plot. the soil fertility and moisture conditions were adequate for bean cultures in southern brazil as described by chemical and fertility committee for santa catarina and rio grande do sul states, brazil (cqfs-rs/sc 2004). insect and pest control were implemented as needed. at harvest, seeds from the central plants, from each plot from each genotype, was bulked and dried in a forced-air oven (30 °c) until the moisture content of the seeds reached 12%, which was monitored with moisture-testing equipment (dole model 500) (gehak). seed traits assessed after harvest, the moisture content of the seeds was standardized at 12%, and samples were stored at 10 �c and 40% relative humidity. the characteristics of the seeds were evaluated in 100-seed weight (p100), seed shape degree and seed flattening (determined by j coefficient and h coefficient), soluble and total protein content, and seed colour. the phaseolin data were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). the j coefficient was estimated by the ratio between the seed length and width and the h by the ratio between the seed thickness and width (ipgri 2001). the seeds were grouped in market classes: manteigão, white, mulatinho, black, coloureds, yellow, red and carioca (ipgri 2001). acta bot. croat. 68 (1), 2009 81 diversity in bean landraces u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:09 color profile: disabled composite 150 lpi at 45 degrees total protein quantification the total protein was determined by total n content of samples, where % of protein is equal to n content (%) ´ 6.25, according to kjeldahl (aoac 1995). soluble protein extraction and quantification the dry seeds without coat were ground with pestle and mortar and the fine powder produced (100 mg) was added to 1 ml of 0.5 m nacl buffer (ph 2.4), for 30 minutes with constant stirring at room temperature on a rotator shaker. the samples were centrifuged at 10.000 g for 20 minutes to clarify the supernatant and finally stored at –20 o c. the supernatant was used for quantification and electrophoretic profiles. the colorimetric protein assay was used according to bradford. protein solution was added to 5 ml of the bradford reagent and the absorbance was measured at 595 nm. a bovine serum albumin (bsa) dilution curve was used as standard (bradford 1976). phaseolin analysis by electrophoretic profiles (sds-page) the supernatant (10 mg) obtained by extraction was mixed with an equal volume of cracking buffer containing 0.25 m nacl, ph 2.4; 0.625 m tris-hcl, ph 6.8; 0.5 mm edta, ph 6.8; 1% (w/v) sds; 8% glycerol (v/v); 0.5% b-mercaptoethanol (w/v); 0.01% bromophenol blue marker dye (gepts et al. 1986). the mixture was heat-treated at 100 o c for 5 min and centrifuged at 10000 g and used for electrophoresis. electrophoretic profiles of the polypeptides were obtained by separation using 13% polyacrilamide gels in the presence of sds under alkaline conditions (laemmli 1970). after the quantification of soluble protein, 10 mg was applied in the gel. a high molecular weight calibration kit (45–200 kda, bio rad) provided standard of known molecular weight for the polypeptides profiles. page was performed in the presence of the running buffer (50 mm tris-hcl, 0.384 m glicina, 5mm edta and 0.25% sds) at a constant 35 ma/gel at 8 °c until the bromophenol blue dye reached the bottom of the gels. the gels were rinsed three times with water, ultrapure, (millipore system) for 5 min each and fixed overnight in 400 ml l –1 methanol and 100 ml l –1 acetic acid. subsequently they were silver stained according to blum (blum et al. 1987). after staining, the phaseolin type from each genotype was scored by comparing the patterns to those of reference genotypes (table 1, bafs: 113, 116, 117, 118 and 119). the phaseolin polypeptides were identified from the other seed proteins by their molecular weight (45–51 kda) (brown et al. 1981). data analysis the univariate analysis of variance to design model and multivariate analysis were performed on the quantitative characters (weight of the seeds, total protein, soluble protein, seed size). the multivariate analysis was conducted by using canonical variable technique (rao 1952). the contribution of the different character to the total variation was determined by using singh’s methodology (singh 1981). the scott and knott (1974) and the tukey (steel and torrie 1980) methods were used in the mean separation procedure. the statistical analysis was performed using the genes program (cruz and carneiro 2003). 82 acta bot. croat. 68 (1), 2009 pereira t., coelho c. m. m., bogo a., guidolin a. f., miquelluti d. j. u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:09 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 83 diversity in bean landraces tab. 1. identification, origin of collection, phaseolin type, seed weight, total and soluble protein of common bean genotypes from santa catarina, brazil. baf origin of collection phaseolin type weight 100 seeds total protein (%) soluble protein (mg ml –1 ) 01 ponte serrada s 36.64 25.70 8406.49 03 palmitos t 29.14 28.98 8947.77 04 lages t 40.81 29.53 9839.25 07 lages s 17.61 29.53 8847.53 10 palmitos s 19.80 26.80 7482.43 11 lages t 39.93 26.80 8367.97 13 caxambú do sul s 22.23 30.63 9186.15 17 palmitos s 17.36 24.61 8228.26 19 palmitos s 20.97 27.89 9863.99 20 palmitos t 21.34 27.89 9149.19 21 palmitos s 22.99 27.89 9690.78 22 palmitos s 20.20 24.06 8514.25 23 chapecó s 22.80 25.16 9011.99 24 são josé do cerrito s 42.73 25.16 9044.88 25 palmitos t 19.66 27.90 9801.67 26 palmitos s 21.95 26.80 9472.45 28 saudades t 27.94 23.52 9089.36 29 ituporanga t 36.37 26.25 8942.77 32 coronel freitas t 34.02 24.61 9667.28 33 concórdia t 42.82 26.25 10237.07 36 são josé do cerrito s 25.98 28.98 8636.42 39 bom jardim da serra t 37.43 25.70 9946.38 40 capão alto s 20.56 26.80 8601.02 41 bom jardim da serra s 21.40 28.98 10431.27 42 capão alto s 17.32 26.81 9273.23 43 capão alto t 43.71 24.61 7781.27 44 capão alto s 19.78 26.25 8983.80 45 capão alto s 23.50 24.06 8863.82 46 lages t 39.19 21.88 9849.60 47 piratuba s 38.85 27.90 8068.19 48 capão alto t 42.57 27.90 9204.00 49 oeste de sc t 33.91 32.02 8272.43 50 lebon régis s 20.45 24.61 9094.99 51 cunha porá t 35.25 25.16 9270.72 52 cunha porá s 17.96 25.70 7965.45 53 cunha porá t 47.73 30.62 8242.98 54 cunha porá t 33.59 26.80 8994.76 55 cunha porá s 18.43 25.16 8154.02 56 cunha porá s 18.89 26.25 7939.14 u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:09 color profile: disabled composite 150 lpi at 45 degrees 84 acta bot. croat. 68 (1), 2009 pereira t., coelho c. m. m., bogo a., guidolin a. f., miquelluti d. j. baf origin of collection phaseolin type weight 100 seeds total protein (%) soluble protein (mg ml –1 ) 57 cunha porá t 36.01 20.23 8328.50 58 cunha porá t 36.93 27.34 8893.27 59 bom jardim da serra s 15.86 26.25 8193.49 60 lebon régis s 18.78 31.02 9403.54 61 painel t 35.50 23.51 8497.33 62 são joaquim s 20.55 28.44 8679.64 63 são joaquim t 36.59 19.14 8851.61 64 campo belo do sul t 33.00 24.06 8320.98 65 lebon régis s 22.85 26.25 8641.11 69 bocaína do sul s 51.51 26.80 9437.68 74 irineópolis t 18.01 29.53 8772.67 78 lebon régis s 17.32 25.70 9255.37 80 fraiburgo s 19.58 26.80 8409.63 81 lebon régis s 17.04 25.16 9146.99 87 fraiburgo s 50.12 26.25 8249.25 88 curitibanos t 41.45 21.87 7479.30 89 curitibanos t 47.75 23.52 8834.69 90 lebon régis t 31.05 21.87 8244.86 91 fraiburgo t 33.00 25.16 8893.89 92 fraiburgo s 16.21 26.25 8004.92 93 bom retiro t 39.21 28.98 8663.66 110 lages s 25.25 27.89 12927.80 111 lages t 21.07 30.81 9849.28 112 lages s 21.90 27.90 9183.33 113 goiáis–cnf5057 s a 19.50 31.00 10512.72 114 capão alto s 19.56 24.61 8217.09 115 lages s 15.21 29.53 9612.47 116 goiáis–cnf5493 h b 17.03 26.80 9520.37 117 goiáis–cnf6551 t c 27.67 26.80 10218.27 118 goiáis–cnf11513 b d 27.47 26.80 11880.32 119 goiáis–cnf1446 c e 50.85 30.62 9521.01 120 lages t 64.68 23.52 7986.75 121 lages s 24.72 28.99 8544.09 sh80 lages s 25.50 22.97 8207.90 means – – 28.94 26.60 8996.89 cv (%) – – 5.85 8.10 6.68 baf = number of collection bank of germplasm from udesc, santa catarina, brazil. s a = sanilac, h b = pampa, t c = tendergreen, b d = boyaca and c e = contender. bafs: 110 = guará, 111 = pérola, 112 = uirapuru, 115 = valente, 121 = iapar 81. tab. 1. – continued u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:09 color profile: disabled composite 150 lpi at 45 degrees results the phaseolin type profiles of the seed proteins from 73 common bean genotypes were analyzed by sds polyacrylamide gels, showing protein subunit variations. two different patterns of phaseolin were found among the 73 genotypes analyzed (tab. 1). the most common was the »s« type, found in 39 genotypes (53.42%), and »t« type was the second, found in 31 genotypes (42.46%). however, the types h, c and b, contributed only 4.12% (controls), because these types were not found in the landraces from santa catarina state. in additional, the commercial cultivars (bafs: 110, 111, 112, 115 and 121) at the moment used in south-brazil exhibited a typical »s« phaseolin type (tab. 1). the diversity was evidenced by the weight of 100 seeds, where genotypes that presented the »t« phaseolin type showed a higher mean seed weight (36 g/100 seeds), while the »s« type, a low mean seed weight (23 g/100 seeds) (tab. 1). in terms of total and soluble protein, the values ranged from 19 (baf 63) to 32% (baf 49) and 7.5 (baf 10) to 10.4 mg/ml (baf 41) respectively (tab. 1). the quantitative traits (p100, total and soluble protein) showed significant difference (p <0.05), which indicated a contribution from each character to genetic diversity (tab. 2). the p100 had higher relative contribution to genetic divergence (91.67%) in overall genotypes, while other traits (total and soluble protein content) showed a smaller contribution (~ 8%) (tab. 2). the grouping based in p100, showed a separation in eleven groups within germplasm bank samples from southern brazil (fig. 1, tab. 3). in the association between p100 and phaseolin types two major groups were found (fig. 1). the first group (a, b, c, d, e, f, g and h) showed p100 higher than the second group (mean of 39 g), with most of the landraces classified as »t« type phaseolin (77%). the second group (i, j and k) was classified as »s« type (87%), and had p100 value lower than the first group (~19 g). the two groups analyzed showed diversity also with respect to seed colour as well as in the degree of seed flattening. in relation to seed colour, in the andean group we found seeds of the market classes yellow (11.7%), manteigão (17.6%), mulatinho (2.9%), coloured (29.5%), red (23.54%), black (14.7%); and in the mesoamerican group, we encountered the types carioca (15.4%), white (7.6%), red (15.4%), black (48.7%) and coloured (12.9%) (tab. 3, fig. 1). the degree of seed flattening assessed by the j and h coefficient proved to be similar to the group of the p100. the andean group showed seeds with the j coefficient ranging from 1.41–2.25, and the h coefficient from 0.7–0.9. in the mesoamerican group the j coefficient acta bot. croat. 68 (1), 2009 85 diversity in bean landraces tab. 2. relative importance of characters analyzed in 73 genotypes of bean. s.j. – contribution of each character to total distances between the genotypes according to singh (1981). variables s.j. value in % weight of 100 seeds 237791.996 91.67 soluble protein 11628.383 4.48 total protein 9976.991 3.85 u:\acta botanica\acta-botan 1-09\pereira.vp 22. travanj 2009 12:18:46 color profile: disabled composite 150 lpi at 45 degrees ranged from 1.52–1.91 and h coefficient from 0.74–0.76 (tab. 3). indeed, significant differences were observed between phaseolin types with regard to seed length, height, width, and h, and j coefficients. multiple comparisons of the means (p <0.05) revealed that the andean group had a higher size seed (1.25 cm length; 0.74 cm height; 0.58 cm width) than the mesoamerican group (tab. 4). 86 acta bot. croat. 68 (1), 2009 pereira t., coelho c. m. m., bogo a., guidolin a. f., miquelluti d. j. fig. 1. representative bean market classes included in each group (a, b, c, d, e, f, g, h, i, j, k) from santa catarina state/brazil. u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:10 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 87 diversity in bean landraces tab. 3. groups from common bean genotypes based on 100 seeds weight, market classes, phaseolin type, j and h coefficient groups genotypes (baf) variation of 100 seeds weight (g) phaseolin type market classes *j coefficient *h coefficient a 120 64.68 t manteigão 2.25 0.74 b 69, 119, 87 51.51–50.12 s,c manteigão, red 2.17 0.82 c 89, 53 47.75–47.73 t red, yellow 1.98 0.79 d 43, 33, 24, 48, 88 43.71–41.75 s,t coloured, black 1.60 0.76 e 4, 11, 93, 46, 47 40.81–38.85 t,s red, manteigão, yellow, black 1.64 0.70 f 39, 58, 01, 63, 29, 57, 61,51 37.43–35.25 t, s manteigão, black, red, coloured 1.46 0.76 g 32, 49, 54, 91, 64,90 34.02–31.05 t manteigão, coloured, yellow, mulatinho 1.41 0.90 h 3, 28, 117, 118,36 29.14–25.98 t,s,b coloured, red, black 1.74 0.73 i sh80, 110, 121, 45, 21, 65, 23, 13 25.50–22.23 s carioca, red, coloured, black, 1.91 0.74 j 26, 112, 41, 20, 111, 19, 40, 62, 50, 22, 10, 44, 25, 80, 114, 113, 56, 60 21.90–18.80 s,t black, red, carioca, coloured, white 1.60 0.76 k 55, 74, 52, 7, 17, 42, 78, 81, 116, 92, 59,115 18.40–15.20 s,t,h white, black, coloured 1.52 0.76 * the results were based in 20 seeds average each sample and grouped by scoot knott (p <0.05). tab. 4. seed size differences of bean genotypes and relationships with groups andean and mesoamerican. groups mean seed sizes (cm)* length (cm) height (cm) width (cm) j coefficient h coefficient andean 1.25 a 0.74 a 0.58 a 1.69 a 0.79 a mesoamerican 1.04 b 0.63 b 0.47 b 1.58 b 0.75 b cv (%) 15.35 7.18 11.41 13.87 9.8 * the values in the column followed by same letter are not significantly different, tukey (p <0.05). u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:10 color profile: disabled composite 150 lpi at 45 degrees discussion the estimation of the relationship among landraces of the common bean is of great interest to the management of the germplasm bank. the active bank bean from santa catarina showed the existence of two patterns of phaseolin, 53.42% »s« type and 42.46% »t« (tab. 1). in a study executed by gepts et al. (1988) with 72 brazilian genotypes, the »t« type was predominant in minas gerais and santa catarina states. therefore, in 192 landrace beans from the cnpaf (centro nacional de pesquisa de arroz e feijão – embrapa, brazil) germplasm bank 80.6% and 19.4% were found for the »s« and »t« phaseolin types respectively. from 20 genotypes of santa catarina state, only one was related to the »t« type (pereira and souza 1992), although this study did not include a representative number of genotypes from santa catarina state. the landraces of the common bean analyzed from rio grande do sul state showed t, s, h, a, and c phaseolin types, with the »t« type accounting for 77.27% (maciel et al. 1999). in contrast, our results showed only »t« and »s« types, both around 50%. most of the data corroborate the hypothesis that in brazil there are two origins, from the andean region and the mesoamerican (gepts et al. 1988). regarding the concentration of total protein in the grains found in table 1, a variation from 19–31% was encountered, which was similar to the values obtained by santalla (santalla et al. 2004) in common bean species, with values of 26.5–31%. in brazilian commercial cultivars values around 20–27% of total protein were found (antunes et al. 1995, santalla et al. 1999, dalla corte et al. 2003). particularly, the landraces baf 11, 25, 26, 42, 46, 48, and 74 showed high values of total protein (27%), and the soluble protein was around 9000 mg/ml (tab. 1). the genotypes mentioned above were associated with high productivity (3500–4000 kg ha –1 ) and also with other important agronomic characters like determinate growth (bafs: 48 and 74) and indeterminate growth (bafs: 11, 25, 26 and 46), number of seeds per pod (~4,2 in the bafs 11, 46 and 48; and 5,7 in the bafs 25, 26 and 74), number of pods per plant (~13.5 in the bafs 11, 25, 46, 48, 74 and 19.5 for genotype baf 26). averages of p100 and phaseolin type classes were very similar to those already observed in the andes and central america, with »t« phaseolin type with large seeds (>40 g per100 seeds) and cultivars with predominantly »s« phaseolin type, with smaller seeds (<25 g per 100 seeds) (singh et al. 1991). these results are in agreement with those of other authors who studied different gene pools (maciel et al. 1999, de la cruz et al. 2005, logozzo et al. 2006). the singh methodology showed p100 had a high relative importance for genetic divergence (tab. 2). this higher contribution found with p100 was observed by other works (benin et al. 2002, chiorato et al. 2005, coelho et al. 2007a). there is a wide diversity of color of the tegument (tab. 3 e, fig. 1). in the mesomerican group, the black seed colour was predominant and in the andean both the coloured and the red seeds. in the mesoamerican group, our results were in agreement with another study (logozzo et al. 2006). this work indicated that the proportion observed in the black colour seeds (47.2%) was mostly similar with the darker colour seeds in the american »s« phaseolin type. 88 acta bot. croat. 68 (1), 2009 pereira t., coelho c. m. m., bogo a., guidolin a. f., miquelluti d. j. u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:11 color profile: disabled composite 150 lpi at 45 degrees those variations show a wide range of seed size associated with seed colour, which might be an important alternative for the consumer of southern brazil, where their preference is for black beans. the commercial cultivars showed a high similarity with the »s« phaseolin type, of mesoamerican origin. traits like seed size, weight and colour found in the landraces studied represent a source of variation that can useful in the crop science or breeding programs, particularly to reduce the vulnerability of improved genotypes. these ranges of variation of seed coefficient (j and h) estimated in the present study, found in the andean group were similar in cultivars representing andean dry bean and snap bean marked classes (santalla et al. 2004). this similarity was observed by gepts et al. (1986) where »t« phaseolin (andes) showed 1.36 cm (length), 0.82 cm (height) and 0.63 cm (width). means of seed size by j (1.69) and h (0.79) coefficients (andean) were similar to those obtained by santalla et al. (2004), which were 1.9 and 0.76 in andean dry bean and snap bean marked classes respectively. the data proved to be similar to the brazilian commercial cultivars, in which values around 1.68 for j coefficient and 0.79 for h coefficient were encountered (dalla corte et al. 2003). this work indicate that landraces of the common bean collected in southern brazil came from two gene pools, that of central america (small seeds, »s« type phaseolin) and andean (higher size seeds and phaseolin type »t«) gene pool, which had 53.42 and 42.46% respectively. the p100 was the principal character that grouped these two gene pools. the grouping used by comparison of means allowed for the efficient identification of promising genotypes to compose a valuable source of genetic diversity highly useful for future breeding programs and in crop science. the genotypes suggested are the bafs 11, 25, 26, 46, 48 and 74, which had a higher productivity level and high protein content. it would be very interesting to further investigate the genetic diversity of protein and micronutrient concentrations associated with phytate concentration in dry bean and other legumes. regarding the nutrient level in grain, there appears to be little research at the molecular and biochemical level. recent studies have identified in rice grain significant correlation between phytate and inorganic phosphorus, fe, zn, cu, and mn, but those compounds were not located on the same chromosomal regions, suggesting that they were genetically different (stangoulis et al. 2007). additionally, the understanding the genetic and control mechanisms of phytate accumulation in seeds is interesting for breeding programs and to improve phosphorus nutrition in bean-eating populations. results have been published for common bean seed development with evidence that phytate synthesis has regulation points with mips (myo-inositol-3-phosphate synthase) enzyme. it is also attractive to investigate further the genetic variability of mips activity and gene expression associated with phytate concentration during seed development in the common bean (coelho et al. 2005, coelho et al. 2007b). acknowledgments the authors would like to thank the capes – prodoc (coordenação de aperfeiçoamento de pessoal de nível superior) and udesc for support. we thank dr heloisa torres da silva (embrapa-cnpaf, brazil) that provided bean seed samples used as controls of phaseolin type. acta bot. croat. 68 (1), 2009 89 diversity in bean landraces u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:11 color profile: disabled composite 150 lpi at 45 degrees references antunes, p. l., bilhalva, a. b., elias, m. c., soares, g. j. d., 1995: valor nutricional do feijão (phaseolus vulgaris l.) cultivares rico 23, carioca, piratã-1 e rosinha-g2. revista brasileira agrociência 1, 12–18. aoac, 1995: official methods of analysis. association of official analytical chemists, washington. beebe, s., skroch, p. w., duque, m. c., pedraza, f., 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(http://www1.ibge.gov.br/ home/estatistica/indicadores/agropecuaria/). ipgri, 2001: descritores para phaseolus vulgaris. international plant genetic resources institute, rome. koenig, r., gepts, p., 1989: segregation and linkage of genes for seed proteins, isozymes, and morphological traits in common bean (phaseolus vulgaris). journal of heredity 80, 455–459. laemmli, u. k., 1970: cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227, 680–685. logozzo, g., donnoli, r., macaluso, l., papa, r., knupffer, h., zeuli, p. s., 2006: analysis of the contribution of mesoamerican and andean gene pools to european common bean (phaseolus vulgaris l.) germplasm and strategies to establish a care collection. genetic resources and crop evoution 54, 1763–1779. maciel, f. l., echeverrigaray, s., gerald, l. t. s., grazziotin, f. g., 2003: genetic relationships and diversity among brazilian cultivars and landraces of common beans (phaseolus vulgaris l.) revealed by aflp markers. genetic resources and crop evoution 50, 887–893. maciel, f. l., gerald, l. t. s., echeverrigaray, s., 1999: variation of phaseolin and other soluble proteins among cultivars and landraces of common beans of south-brazil. journal of genetics and breeding 53, 149–154. nass, l. l., paterniani, e., 2000: pre-breding: a link between genetic resources and maize breeding. scientia agricola 57, 581–587. pereira, p. a. a., souza, c. r. b., 1992: tipos de faseolina em raças crioulas de feijão no brasil. pesquisa agropecuaria brasileira 27, 1219–1221. acta bot. croat. 68 (1), 2009 91 diversity in bean landraces u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:11 color profile: disabled composite 150 lpi at 45 degrees rao, r. c., 1952: advanced statistical methods in biometrics research. jonh wiley and sons, new york. santalla, m., rodiño, a. p., ron, a. m. d., 1999: breeding for culinary and nutritional quality of common bean (phaseolus vulgaris l.) in intercropping systems with maize (zea mays l.). biotechnology agronomy society and environment 3, 225–229. santalla, m., sevillano, m. c. m., monteagudo, a. b., de ron, a. m., 2004: genetic diversity of argentinean common bean and its evolution during domestication. euphytica 135, 75–87. scott, a. j., knott, m., 1974: a cluster analysis methods for grouping means in the analysis of variants. biometrics 30, 507–512. singh, d., 1981: the relative importance of characters affecting genetic divergence. indian journal of genetics and plant breeding 41, 237–245. singh, s. p., gepts, p., debouck, d. g., 1991: races of common bean (phaseolus vulgaris, fabaceae). economic botany 45, 379–396. solano, j. p. l., 2005: patterns of phaseolins and rapd analysis in domesticated species of phaseolus. revista fitotecnia mexicana 28, 195–202. stangoulis, j. c. r., huynh, b. l., welch, r. m., choi, e. y., graham, r. d., 2007: quantitative trait loci for phytate in rice grain and their relationship with grain micronutrient content. euphytica 154, 289–294. steel, r. g. d., torrie, j. h., 1980: principles and procedures of statistics–a biometrical approach. megraic hill, new york. van schoonhoven, a., voysest, o., 1991: common beans: research for crop improvement. ciat, cali, colômbia. 92 acta bot. croat. 68 (1), 2009 pereira t., coelho c. m. m., bogo a., guidolin a. f., miquelluti d. j. u:\acta botanica\acta-botan 1-09\pereira.vp 17. travanj 2009 10:21:11 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 81 (1), 2022 89 acta bot. croat. 81 (1), 89–100, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-006 issn 0365-0588 eissn 1847-8476 physiological responses of resistant and susceptible pepper plants to exogenous proline application under phytophthora capsici stress esra koç ankara university, faculty of science, department of biology, ankara, turkey abstract – phytophthora capsici leon. is the main pathogen that limits the production of peppers. in this study, the effects of 1 and 10 mm proline (pro), prior to exposure of resistant (cm-334) and susceptible (sd-8) pepper seedlings to p. capsici, on some physiological parameters were investigated. a lower pro concentration (1 mm) was found to be more effective than 10 mm pro in increasing the stress tolerance of the cm-334 cultivar. namely, in cm-334 cultivar, the highest chlorophyll a, chlorophyll b, carotenoid, glucose and fructose content and 1,1-diphenyl-2-picrylhydrazyl (dpph) scavenging activity percentage were detected on the seventh day after application of 1 mm pro + p. capsici, while the lowest malondialdehyde (mda) amount was measured on the third day in the same treatment. the highest ferric reducing antioxidant power (frap) increase was determined on the seventh day in the 10 mm pro + p. capsici application. the effects of the same pro treatments on the sd-8 cultivar somewhat differed; the highest amounts of chlorophyll a, chlorophyll b, anthocyanins, fructose, total protein and endogenous pro were detected on the seventh day in the 1 mm pro + p. capsici application, while the lowest mda amount was measured on the third day after the 10 mm pro + p. capsici application, the highest dpph % and frap values were detected on the seventh day with 10 mm pro + p. capsici application. although some differences were detected between the cultivars, pro application against the p. capsici stress in general resulted in a positive effect on photosynthetic pigments, soluble carbohydrates and antioxidant capacity in pepper. the exogenous application of pro helped the non-resistant cultivar to overcome the stress. keywords: antioxidant capacity, lipid peroxidation, pepper, photosynthetic pigments, soluble carbohydrate introduction pepper (capsicum annuum l.) is a vegetable that belongs to the solanaceae family. it has both economic and high nutritional value. according to food and agriculture organization (fao) data for 2017, pepper was among the ten most cultivated vegetables in the world with a production of approximately 34 million tons. however, various diseases that threaten production of the pepper worldwide are a limiting factor. phytophthora capsici leon. is a widespread, destructive and invasive soil-borne oomycete pathogen that causes decomposition of root and root collar in pepper and therefore poses a serious threat to its production (siddique et al. 2019). increasing the plant’s tolerance to p. capsici-imposed stress is vital in agriculture and horticulture. stress tolerance is a complex trait that is controlled by multiple genes and includes different physiological and biochemical mechanisms (zhang and shi 2013, sharma and prasad 2017, rabuma et al. 2021). it is essential to develop economically viable strategies to increase and improve plants’ stress tolerance under adverse environmental conditions. therefore, in the fight against p. capsici, various approaches have been developed to increase the genetic resistance of the host, as well as crop rotation, soil solarizations, application of fungicides, fumigation and cultural methods (hausbeck and lamour 2004, jin et al. 2016, xu et al. 2016, kim et al. 2017, rabuma et al. 2021). application of amino acids such as proline (pro), which is also synthesized by a wide variety of plants during abiotic and biotic stress, can be a promising alternative strategy for the management of root rot disease. pro has many beneficial traits in the plant organism; it acts as an osmolyte, which accumulates in plant tissues exposed to stress; it is an antioxidant compound (hoque et al. 2008) a source of carbon and nitrogen, both of which are essential for plant growth; it stabilizes protein structure and protects biological membranes and macromolecules from denatur* corresponding author e-mail: ekoc@science.ankara.edu.tr koç, e. 90 acta bot. croat. 81 (1), 2022 ation (trovato et al. 2008). the effect of pro depends on its concentration since an excessive amount of free pro has adverse effects on cell growth and protein functions (nanjo et al. 2003), application time, plant species and plant growth stage (ashraf and foolad 2007, elewa et al. 2017). thus, it is essential to determine optimal concentrations of exogenously applied pro which has positive effects on plants exposed to stress. there are many studies which reported the successful application of exogenous pro for increase of stress tolerance in plants (nounjan and theerakulpisut 2012, medeiros et al. 2015, abdelaal et al. 2020, hayat et al. 2021). while these studies mostly focused on abiotic stress, there is little information on the effects of exogenous application of pro to plants exposed to biotic stress. the aim of this study was to determine the extent to which exogenous application of pro could change important physiological parameters in pepper cultivars with different tolerances to p. capsici. the strain cm-334 is a hot pepper cultivar originating from southern mexico, which shows consistently high resistance to various pathogens, including p. capsici, pepper mottle virus and root-knot nematodes (ortega et al. 1991; kim et al. 2014). cm-334 is a genotype with very high resistance to multiple p. capsici strains (foster and hausbeck 2010). on the other hand, sd-8 is a sweet pepper cultivar commercially grown in turkey and susceptible to p. capsici (göçmen 2006). to establish the possible positive effect of exogenously applied pro on pepper plants exposed to p. capsici, the content of soluble carbohydrates and starch, photosynthetic pigments, anthocyanins and total flavonoids as well as endogenous pro and total protein were examined along with the 1,1-diphenyl-2picrylhydrazyl (dpph) scavenging activity, reducing antioxidant power and lipid peroxidation in pepper leaves on the 3rd, 5th and 7th day post inoculation. in the framework of the findings obtained, the relationship of pro with the investigated metabolic pathways is discussed. according a survey of the literature, there is no record of the effect of exogenous pro pre-applications on the investigated parameters in peppers exposed to p. capsici. materials and methods plant material in this study, two pepper (capsicum annuum l.) cultivars, criollo de morelos 334 (cm-334; resistant to p. capsici) and sera demre-8 (sd-8; susceptible to p. capsici), have been used. after germination, pepper seedlings were grown in plastic pots containing a steam-sterilized soil/fertilizer/sand mix (1/1/1, v/v/v) in a growth chamber (digitech glopg42) under controlled environmental conditions (25±2 °c, 16-h light/8-h dark photoperiods and 60% humidity). at the end of the two months period, when seedlings reached the six-leaf stage, they were collected and the leaves were separated, frozen in liquid nitrogen, and stored at -80 °c until analysis. preparation of p. capsici zoospore suspension phytophthora capsici strain 22 (p. capsici-22) was obtained from the fungal culture collection of ankara university, faculty of agriculture, ankara, turkey. zoospore production and spore concentrations were determined as described previously (jones et al. 1974). p. capsici-22 was grown on v8 agar (200 ml v8 juice, 20 g agar, 3 g caco3 and tap water to 1 l) plates at 25 °c in the dark. zoospores were produced from mycelia. drops of mycelial suspension was placed onto the surface of water-agar plates using a sterile syringe and the cultures were incubated for an additional 3 days at 25 °c under fluorescent lights (40 w daylight). the release of zoospores was induced by incubating the culture plates in sterile water at 4 °c at room temperature for 1 h. the zoospores were collected and filtered through a whatman no. 54 filter to remove sporangial cases. the concentration of 104 zoospores ml-1 was the desired inoculum concentration and the optimal zoospore concentration for inducing disease in pepper (koç et al. 2011). proline application and plant inoculation pro application and plant inoculation were performed according to koç (2017). for each pepper cultivar four applications were used: 1 control (no p. capsici or pro), 2 p. capsici alone, 3 1 mm pro + p. capsici and 4 10 mm pro + p. capsici. for both cultivars, each application was repeated three times. in all, 30 seedlings were used for each repetition of each application. the roots of the seedlings were washed with tap water and disinfected with 0.75% (v/v) sodium hypochlorite for 1-2 min and then washed with sterile distilled water several times. the seedlings were placed into a sterile glass bottle containing 400 ml of liquid hoagland medium. pro was applied to the plants once before the p. capsici inoculation by spraying the leaf surface of pepper seedlings. for the seedlings in the control group, sterile distilled water was used instead of pro application and then control group and pro-applied pepper seedlings were transferred back to the growth chamber and incubated for 3 days at 25±2 ºc, 16-h light/8-h dark photoperiods and 60% humidity. inoculation of p. capsici zoospores was performed 72 hours after the pro application. 100 ml of zoospore suspension (104 zoospores ml-1) was placed into 250 ml beakers in which seedling roots were dipped for 1 h. afterwards, seedlings were placed into sterile glass bottles containing 400 ml of hoagland solution and kept in the growth chamber. for control seedlings, sterile water was used instead of p. capsici suspension. samples were taken on the 3rd, 5th and 7th day post inoculation (dpi) according to the random blocks design model. the leaves were harvested and homogenized in liquid nitrogen and stored at -70 ºc until the analysis. all chemicals and reagents used in the analyses were of analytical grade. distilled water was used throughout the study. determination of pigments and flavonoid content for chlorophyll extraction, 0.1 g of fresh leaf sample was ground with 8 ml of 80% acetone with a mortar and pestle. exogenous proline modulates physiological responses acta bot. croat. 81 (1), 2022 91 the mixture was centrifuged at 5000 rpm for 10 minutes. the absorbance of the resulting supernatant was recorded at 664 and 647 nm using an uv-visible spectrophotometer (cecil 5000, cecil instruments, milton, uk) with 80% acetone as blank. chlorophyll a and b amounts were calculated using the equations given by porra et al. (1989). the extraction of carotenoids (xanthophyll + β-carotene) was done with 0.1 g fresh leaf material using 1 ml of 100% acetone. the mixture was centrifuged at 3500 rpm for 10 minutes. the absorbance of the resulting supernatant was recorded at 470 nm using an uv-visible spectrophotometer. the carotenoids amount was calculated using the equations given by lichtenthaler (1987). anthocyanin content was determined using the method described by mancinelli et al. (1975). fresh leaf sample (0.1 g) was extracted with 1 ml of a solution containing 79% (v/v) methanol, 20% distilled water and 1% (v/v) hcl. absorbances were measured at 530 and 657 nm wavelengths. the amount of anthocyanin was expressed as mg ml-1. flavonoid content was determined with the method of mirecki and teramura (1984). absorbance was measured at 300 nm. flavonoid content was expressed as the percentage of content of control plants. values obtained in the control plants at 3rd dpi were set as 100%, and all other values were calculated in reference to this value. determination of soluble carbohydrates, starch and protein content glucose and fructose content were determined according to halhoul and kleinberg (1972). fresh leaf sample (0.1 g) was extracted with 2 ml of 80% (v/v) ethyl alcohol and the supernatant was transferred into an erlenmeyer flask and filled to 100 ml with distilled water after the alcohol had evaporated. glucose and fructose contents were analyzed by mixing 1 ml of the extract with 2 ml of anthrone solution. for measurement of glucose content, the mixtures were placed water bath at 95 ºc for 15 minutes, while for measurement of fructose content, mixtures were placed in a water bath at 40 ºc for 30 min and the reaction was finished in an ice bath. five minutes later, absorbance was measured at 620 nm and calculated as ppm g-1 fresh weight (fw) against the glucose (merck k13654437) and fructose ( merck k04317907) standards. starch content was determined using the method described by mccready et al. (1950). fresh leaf sample (0.1 g) was extracted with 1.6 ml of 52% (v/v) perchloric acid and 1 ml of extract was mixed with 2 ml of anthrone solution. the reaction mixture was incubated in a boiling water bath for 5 minutes and the reaction was finished in an ice bath. absorbance was measured at 520 nm and starch content was calculated as ppm g-1 fw against the glucose standard. total soluble proteins extraction was done according to kurkela et al. (1988). fresh leaf sample (0.1 g) was extracted with 1.5 ml of 50 mm tris hcl buffer ph 6.8 containing 1% (v/v) 2-β mercaptoethanol (sigma-aldrich m6250) and 50 mg l-1 phenylmethylsulfonyl fluoride (pmsf) (sigmaaldrich p7626). protein contents were measured according to bradford method (bradford 1976) using the bovine serum albumin (bsa) (sigma-aldrich b4287) as a standard protein. determination of proline and mda content free pro extraction and determination were made according to bates et al. (1973). fresh leaf sample (0.1 g) was extracted with 3 ml of 3% (w/v) sulfosalicylic acid. two ml of extract was mixed with 2 ml of ninhydrin solution and 2 ml of glacial acetic acid. the reaction mixture was incubated in a boiling water bath for 1 h and the reaction was finished in an ice bath. four ml of toluene was added to the reaction mixture and the absorbance of the toluene phase was measured at 520 nm in a uv-visible spectrophotometer and calculated as µg g-1 fw against the proline (sigma aldrich p5607) standard. to determine the level of lipid peroxidation, the malondialdehyde (mda) method was used. after 0.1 g of fresh leaf sample was homogenized with 1.5 ml of 1% (w/v) trichloroacetic acid (tca), a solution containing 20% (w/v) tca and 0.5% (w/v) thiobarbituric acid (tba) was added to the extract obtained. subsequently, the solution was incubated in a water bath at 95 °c and absorbance was measured in a spectrophotometer at 532 and 600 nm. mda content was measured according to thiobarbituric acid reaction and content was calculated using extinction coefficient of 155 mm-1 cm-1 (devasagayam et al. 2003). evaluation of antioxidant capacity the measurement of the 1,1-diphenyl-2picrylhydrazyl (dpph) radical scavenging activity was performed according to a methodology described by blois (1958). the percentage of antioxidant activity of methanol extracts of pepper cultivars was assessed by dpph free radical assay. a fresh leaf sample (0.1 g) was incubated overnight at room temperature in 2 ml methanol, 1.5 ml of 0.1 mm dpph (sigma-aldrich d9132) was mixed with 100 μl of extract and the samples were incubated in a water bath for 30 min at 24 °c. the reduction of dpph radicals was determined by measuring the absorption at 517 nm. percent inhibition was calculated using the formula: dpph scavenging activity (% inhibition) = [(ac – as)/ ac ] × 100, where “ac” is the absorbance of the control reaction (absorbance of the dpph solution), while “as” is the absorbance of the extracts. ferric reducing antioxidant power (frap) of extracts was determined by the method of vijayalakshmi and ruckmani (2016). to determine reducing power, 100 mg of fresh leaves was extracted with 1.5 ml of methanol. the reaction mixture, which consisted of 250 μl of extract, 1.25 ml of 0.2 m phosphate buffer ph 6.6 and 1.25 ml of 1% (m/v) potassium hexacyanoferrate (k3fe(cn)6) (sigma-aldrich p8131), was incubated at 50 °c for 20 min. the reaction was stopped by the addition of 1.25 ml of 10% (m/v) tca and centrifuged at 3000 g for 10 min. 1.25 ml of the supernatant upper layer was mixed with 1.25 ml of distilled water and koç, e. 92 acta bot. croat. 81 (1), 2022 250 μl of 0.1% (m/v) of ferric chloride (fecl3) and incubated at 24 °c for 10 min. the absorbance was measured at 700 nm. a higher absorbance value of the reaction mixture indicated greater reducing power. the frap value of the extracts was calculated and expressed as ascorbic acid equivalent (aae) (µg aae g-1 fw) through the calibration curve of ascorbic acid (sigma-aldrich a4544) (10-100 µg/ml). statistical analysis variance analysis (anova-factorial design: cultivar* day*application) was conducted using a test arrangement in which data analysis was completely random. the trials were arranged to create an experimental design with three repetitions in randomized blocks. variance analyses were conducted using spss 24 software package. a 5% significance level was used in the tukey test and in the interpretation of the results. the statistical significance is indicated by appropriate letters within the tables. results as a result of variance analysis for chlorophyll a, chlorophyll b, carotenoid, flavonoid, fructose, starch, pro, mda, dpph and frap content, cultivar*day*application triple interaction was found to be statistically significant (p < 0.01). as a result of variance analysis for glucose and protein content, cultivar*day*application triple interaction was found to be statistically significant (p < 0.05). pigments and flavonoid content chlorophyll a, b and carotenoid contents of the leaves of control seedlings of resistant cm-334 cultivar on the 3rd, 5th and 7th dpi were found to be higher than in the control leaves of susceptible sd-8 cultivar (p < 0.05). chlorophyll a and b content mostly decreased due to p. capsici-imposed stress in both cultivars compared to control (tab. 1). in the cm334 cultivar, the highest values of chlorophyll a and b as well as of carotenoids were determined on the 5th dpi upon 1 mm pro + p. capsici application. although p. capsici application generally induced an increase in the amount of anthocyanin and flavonoids in the cm-334 cultivar compared to the control group, the highest values were obtained upon applications of both pro concentrations + p. capsici (p < 0.05) (tab. 1). when the cultivars were compared, the highest amounts of chlorophyll a and b were found in the sd-8 cultivar upon 1 mm pro + p. capsici application on the 7th dpi (p < 0.05). in the sd-8 cultivar, the highest amounts of chlorophyll a and b as well as of anthocyanin were detected after exposures to both pro concentrations + p. capsici on the 7th dpi, while the highest carotenoid amount was detected upon 1 mm pro + p. capsici application on the 3rd dpi. in the sd-8 cultivar, the highest chlorophyll a and b increases were determined respectively as 247% and 170% after exposure to 1 mm pro + p. capsici on the 7th dpi (p < 0.05) as compared to exposure to p. capsici alone. a significantly higher flavonoid level was detected in cm-334 cultivar than in all corresponding controls and treatments of the sd-8 cultivar. the highest flavonoid increases were determined in cm-334 cultivar upon exposures to both pro concentrations + p. capsici (p < 0.05) when compared to control and treatment with p. capsici alone (tab. 1). soluble carbohydrates, starch and protein content glucose content in the leaves of sd-8 cultivar control seedlings was significantly higher than in the cm-334 cultivar (p < 0.05) at all dpi (tab. 2). in the sd-8 cultivar exposed to p. capsici, glucose amount was similar to the control value on the 3rd and 5th dpi, but significant reduction was recorded on the 7th dpi; however, upon exposure to the combined treatments with both pro concentrations, the values were significantly elevated and even exceeded the control value. moreover, the highest increase in glucose content was approximately 63.7% and 57.8% on the 7th dpi with 1 and 10 mm pro + p. capsici applications respectively (p < 0.05) when compared to the values obtained after exposure to p. capsici alone. in the cm-334 cultivar, the highest glucose increase of 7.7% was recorded on the 5th dpi upon 1 mm pro + p. capsici application (p < 0.05) when compared with p. capsici application alone. fructose content in the leaves of sd-8 cultivar control seedlings was significantly higher at the 3rd and 5th dpi (p < 0.05) than in the cm-334 cultivar (tab. 2). the greatest, significant changes in fructose content were recorded in sd-8 cultivar on the 7th dpi between control and all treatments. in the cm-334 cultivar, the highest fructose increase of 40% was detected on the 7th dpi upon exposure to 1 mm pro + p. capsici (p < 0.05) when compared with p. capsici application alone (tab. 2). the starch content was found to be higher in the leaves of the control seedlings of the sd-8 cultivar than in the cm334 cultivar at all dpi (p < 0.05) (tab. 2), while the highest value was detected in the sd-8 cultivar upon infection with of p. capsici on the 3th dpi (p < 0.05). in the cm-334 cultivar, the starch content increased at all dpi after infection with p. capsici alone compared to the control, while combined treatments with both pro concentrations resulted in values that were not significantly different from control values, with the exception of the combined treatment with 10 mm pro + p. capisici (tab. 2). in the sd-8 cultivar, the most prominent increase in starch content was detected upon p. capsici application on the 3rd dpi (p < 0.05). combined treatments with both pro concentrations and p. capisici failed to increase the starch content to the control value, which was particularly pronounced on the 5th and the 7th dpi (tab. 2). total protein levels increased in leaves of both cultivars on the 7th dpi in control and p. capsici-exposed plants (p < 0.05) (tab. 2). compared to the values obtained in treatment with p. capsici alone, the highest total protein increase was detected after exposure to 1 mm pro + p. capsici on 7th dpi with approximately 23% in cm-334 cultivar (p < 0.05). likewise, an 3.5% increase was detected in the 1 mm pro + exogenous proline modulates physiological responses acta bot. croat. 81 (1), 2022 93 ta b. 1 . t he c on te nt o f p ig m en ts a nd fl av on oi ds in le av es o f c m -3 34 a nd s d -8 p ep pe r cu lti va rs e xp os ed to p ro lin e (p ro ) an d ph yt op ht ho ra c ap si ci . t yp e of a pp lic at io ns ( a pp ): 1 co nt ro l ( no p ro , n o p. ca ps ic i) , 2 p. c ap si ci a lo ne , 3 1 m m p ro + p . c ap si ci , 4 10 m m p ro + p . c ap si ci . m ea n ± st an da rd d ev ia tio n of 3 r ep lic at es is p re se nt ed . f la vo no id v al ue s ob ta in ed in th e co nt ro l p la nt s at 3 rd d ay p os t i noc ul at io n (d pi ) w er e se t a s a 10 0% , a nd a ll fla vo no id c on te nt s w er e ca lc ul at ed in r ef er en ce to th is v al ue . d iff er en t l et te rs in di ca te s ig ni fic an t d iff er en ce s at p < 0 .0 5 by th e tu ke y te st . c ap ita l l et te rs r ep re se nt d iff er en ce s i n ap pl ic at io ns fo r th e sa m e cu lti va r an d th e sa m e da y. l ow er c as e le tt er s r ep re se nt d iff er en ce s i n da ys fo r th e sa m e cu lti va r an d th e sa m e ap pl ic at io n. s up er sc ri pt lo w er c as e le tt er s r ep re se nt d iff er en ce s i n cu lti va rs fo r th e sa m e da y an d th e sa m e ap pl ic at io n. f w – fr es h w ei gh t. c ul ti va r dp i a pp c hl a (m g g1 fw ) c hl b ( m g g1 fw ) c ar ot en oi d (m g g1 fw ) a nt ho cy an in s (m g m l1 ) fl av on oi d (% o f c on tr ol ) c m -3 34 3 1 1. 15 7 ± 0. 05 0 a aa 0. 79 1 ± 0. 03 1 a aa 0. 92 4 ± 0. 17 9 a aa 0. 04 3 ± 0. 00 2 b ca 10 0. 0 ± 0. 00 d ca 2 0. 92 5 ± 0. 26 6 ba a 0. 65 3 ± 0. 15 6 a aa 0. 90 1 ± 0. 04 8 a aa 0. 04 2 ± 0. 00 2 b cb 11 5. 3 ± 4. 12 c ca 3 1. 04 1 ± 0. 20 0 a ba 0. 73 5 ± 0. 15 1 a bb 0. 82 0 ± 0. 04 6 a bb 0. 08 5 ± 0. 00 1 b ca 26 3. 6 ± 5. 57 a aa 4 0. 79 5 ± 0. 02 6 b cb 0. 56 8 ± 0. 26 4 a bb 0. 90 0 ± 0. 07 7 a aa 0. 21 3 ± 0. 02 7 a aa 18 3. 8 ± 0. 38 b ba 5 1 1. 20 6 ± 0. 50 0 b c aa 0. 90 5 ± 0. 09 9 a ba a 0. 87 0 ± 0. 02 9 ba a 0. 05 1 ± 0. 00 1 c ab 12 3. 5 ± 5. 54 c ba 2 0. 97 2 ± 0. 07 2 c aa 0. 73 5 ± 0. 19 4 ba a 0. 88 2 ± 0. 02 6 ba a 0. 08 9 ± 0. 00 0 ba a 20 7. 9 ± 3. 62 b aa 3 1. 65 7 ± 0. 16 4 a aa 1. 13 4 ± 0. 08 6 a aa 0. 96 5 ± 0. 02 6 a aa 0. 11 2 ± 0. 00 0 a ba 23 4. 3 ± 9. 28 a aa 4 1. 34 6 ± 0. 02 3 ba a 0. 97 4 ± 0. 10 9 a ba ba 0. 44 9 ± 0. 03 2 c cb 0. 10 9 ± 0. 01 0 a ba 23 7. 8 ± 3. 49 a aa 7 1 1. 15 5 ± 0. 12 3 a aa 0. 82 5 ± 0. 04 6 ba a 0. 76 7 ± 0. 03 3 a aa 0. 04 8 ± 0. 01 0 d bb 13 3. 1 ± 0. 94 c aa 2 1. 12 0 ± 0. 05 1 a aa 0. 77 0 ± 0. 05 9 ba a 0. 77 0 ± 0. 05 3 a bb 0. 08 2 ± 0. 00 0 c ba 19 6. 1 ± 1. 89 b ba 3 1. 12 6 ± 0. 09 4 a bb 0. 79 3 ± 0. 03 9 bb b 0. 69 0 ± 0. 02 2 a ca 0. 12 0 ± 0. 00 0 ba b 22 7. 4 ± 24 .7 1a ba a 4 1. 27 1 ± 0. 00 2 a bb 1. 03 4 ± 0. 03 4 a ab 0. 72 0 ± 0. 01 7 a ba 0. 15 2 ± 0. 00 0 a aa 23 1. 9 ± 6. 51 0a aa sd -8 3 1 0. 89 3 ± 0. 28 1 ba b 0. 76 5 ± 0. 03 3 b c aa 0. 85 0 ± 0. 01 2 bb b 0. 04 0 ± 0. 01 0 d ca 10 0. 0 ± 0. 00 a aa 2 0. 66 3 ± 0. 13 5 b cb 0. 65 2 ± 0. 01 4 c aa 0. 79 7 ± 0. 00 4 c cb 0. 05 0 ± 0. 00 2 c ba 91 .4 9 ± 10 .0 9a bb 3 0. 75 9 ± 0. 19 2 b cb 0. 92 0 ± 0. 04 2 a ba 0. 97 0 ± 0. 00 4 a aa 0. 07 4 ± 0. 00 0 a bb 10 3. 1 ± 2. 15 a ab 4 1. 04 3 ± 0. 37 8 a ba 0. 79 5 ± 0. 01 2 bb a 0. 84 0 ± 0. 00 1 ba a 0. 06 2 ± 0. 00 6 b cb 98 .8 7 ± 8. 35 a ab 5 1 0. 92 1 ± 0. 17 5 ba b 0. 68 5 ± 0. 03 5 bb b 0. 88 2 ± 0. 02 1 a aa 0. 06 2 ± 0. 00 5 bb a 98 .0 6 ± 4. 30 a ab 2 1. 00 3 ± 0. 05 3 a aa 0. 62 3 ± 0. 11 4 ba a 0. 87 1 ± 0. 00 2 a aa 0. 06 8 ± 0. 01 4 bb b 10 7. 2 ± 3. 57 a ab b 3 1. 33 0 ± 0. 10 2 a bb 1. 11 0 ± 0. 09 5 a ba 0. 56 5 ± 0. 02 7 b cb 0. 06 0 ± 0. 00 4 b cb 99 .9 8 ± 6. 98 a ab 4 1. 62 6 ± 0. 49 5 a aa 1. 10 6 ± 0. 11 1 a ab a 0. 58 8 ± 0. 03 1 b ca 0. 11 1 ± 0. 00 5 a ba 10 6. 2 ± 0. 85 a ab 7 1 0. 83 3 ± 0. 02 9 c ab 0. 70 5 ± 0. 03 4 c ab b 0. 80 4 ± 0. 00 5 a bc a 0. 07 5 ± 0. 00 0 ba a 10 2. 3 ± 0. 10 b ab 2 0. 82 1 ± 0. 04 1 c bb 0. 66 7 ± 0. 01 3 c aa 0. 82 7 ± 0. 00 3 a ba 0. 11 2 ± 0. 02 7 a aa 11 1. 2 ± 3. 31 a ab 3 2. 84 9 ± 0. 22 3 a aa 1. 80 3 ± 0. 12 1 a aa 0. 73 0 ± 0. 01 3 bb a 0. 15 1 ± 0. 01 0 a aa 10 8. 8 ± 2. 77 a ba b 4 2. 08 1 ± 0. 37 1 ba a 1. 35 9 ± 0. 19 5 ba a 0. 73 9 ± 0. 06 0 bb a 0. 13 9 ± 0. 01 6 a aa 10 4. 9 ± 3. 02 a ba b koç, e. 94 acta bot. croat. 81 (1), 2022 ta b. 2 . s ol ub le c ar bo hy dr at e, st ar ch a nd to ta l p ro te in a m ou nt in le av es o f c m -3 34 a nd s d -8 p ep pe r c ul tiv ar s e xp os ed to p ro lin e (p ro ) a nd p hy to ph th or a ca ps ic i. ty pe o f a pp lic at io ns (a pp ): 1 c on tr ol (n o pr o, n o p. c ap si ci ), 2 p . c ap si ci a lo ne , 3 1 m m p ro + p . c ap si ci , 4 10 m m p ro + p . c ap si ci . m ea n ± st an da rd d ev ia tio n of 3 re pl ic at es is p re se nt ed . d iff er en t l et te rs in di ca te si gn ifi ca nt d iff er en ce s a t p < 0 .0 5 by t uk ey te st . c ap ita l l et te rs r ep re se nt d iff er en ce s i n ap pl ic at io ns fo r th e sa m e cu lti va r an d th e sa m e da y. l ow er c as e le tt er s r ep re se nt d iff er en ce s i n da ys fo r th e sa m e cu lti va r an d th e sa m e ap pl ic at io n. s up er sc ri pt lo w er c as e le tt er s r ep re se nt d iff er en ce s i n cu lti va rs fo r t he s am e da y an d th e sa m e ap pl ic at io n. f w – fr es h w ei gh t, dp i – da y po st in oc ul at io n. c ul ti va r dp i a pp g lu co se (p pm g -1 f w ) f ru ct os e (p pm g -1 f w ) st ar ch (p pm g -1 f w ) to ta l p ro te in (m g g1 f w ) c m -3 34 3 1 12 .6 07 ± 0 .1 58 a ab 22 .1 78 ± 1 .2 44 a ab 8. 65 8 ± 0. 11 3 c bb 36 .3 35 ± 1 .6 08 a aa 2 11 .6 59 ± 0 .2 36 a ab 24 .3 73 ± 0 .5 72 a aa 12 .7 44 ± 0 .5 96 a cb 38 .3 33 ± 0 .6 44 a aa 3 15 .8 97 ± 2 .6 01 a aa 24 .0 71 ± 3 .1 63 a aa 10 .1 92 ± 0 .3 61 b bb 37 .7 66 ± 1 .1 54 a ca 4 14 .3 91 ± 3 .1 91 a ab 20 .7 65 ± 0 .6 53 a bb 9. 43 5 ± 0. 37 8 b c bb 36 .3 85 ± 0 .5 15 a ab 5 1 12 .2 38 ± 1 .0 20 b ab 19 .4 99 ± 0 .7 00 c ab 8. 76 7 ± 1. 19 2 b bb 36 .1 70 ± 1 .1 15 b ab 2 12 .5 05 ± 0 .7 76 b ab 21 .0 13 ± 0 .7 86 b bb 17 .8 65 ± 0 .3 50 a aa 39 .6 23 ± 1 .1 45 a ba a 3 17 .2 26 ± 2 .7 31 a aa 29 .4 04 ± 2 .8 00 a aa 9. 16 6 ± 0. 64 2 b bb 42 .6 71 ± 0 .6 54 a ba 4 11 .4 03 ± 0 .8 55 b ab 25 .8 03 ± 3 .7 34 a ba ba 10 .8 45 ± 0 .9 84 b ba 38 .9 00 ± 3 .0 78 a ba b 7 1 12 .3 69 ± 0 .2 77 a ab 23 .0 85 ± 2 .6 21 b aa 11 .1 19 ± 0 .9 41 b ab 41 .7 60 ± 3 .3 26 b aa 2 12 .3 18 ± 0 .6 69 a ab 24 .9 09 ± 1 .3 28 a ba b 14 .4 97 ± 0 .9 46 a bb a 42 .6 85 ± 1 0. 49 b ab 3 13 .2 55 ± 1 .0 09 a ab b 29 .8 92 ± 3 .2 42 a ab 12 .1 96 ± 0 .9 93 b ab 52 .5 28 ± 1 .4 07 a aa 4 10 .0 01 ± 0 .5 84 b ab 27 .9 09 ± 1 .4 72 a ba b 17 .6 50 ± 2 .7 26 a aa 40 .9 56 ± 2 .1 14 b ab sd -8 3 1 15 .4 76 ± 1 .3 01 b ba 25 .3 98 ± 1 .2 30 a aa 20 .6 90 ± 0 .5 39 b aa 34 .5 28 ± 0 .1 43 a ba 2 15 .1 36 ± 0 .6 53 b ba 24 .5 68 ± 0 .0 57 a ba 31 .5 48 ± 2 .3 92 a aa 39 .6 71 ± 3 .7 11 a ba 3 15 .5 62 ± 0 .5 40 b ca 24 .7 71 ± 0 .7 90 a ba 19 .8 10 ± 0 .2 20 b aa 38 .0 52 ± 3 .9 28 a ba 4 20 .9 18 ± 3 .4 31 a aa 25 .8 58 ± 1 .1 80 a ba 18 .5 93 ± 0 .5 08 b aa 41 .8 13 ± 2 .3 38 a aa 5 1 17 .9 48 ± 1 .2 61 a ab a 24 .9 55 ± 0 .1 11 a aa 18 .6 93 ± 0 .5 39 a ba 44 .0 99 ± 0 .7 56 a aa 2 17 .9 19 ± 0 .1 97 a aa 24 .6 89 ± 0 .7 43 a ba 12 .2 43 ± 0 .7 26 b cb 37 .7 63 ± 4 .1 45 b ba 3 18 .5 95 ± 0 .5 55 a ba 25 .0 66 ± 1 .1 28 a bb 11 .1 69 ± 0 .1 24 b c ca 44 .5 75 ± 0 .2 97 a ba 4 22 .7 05 ± 5 .5 09 a aa 26 .3 77 ± 0 .5 41 a ba 10 .2 12 ± 0 .5 79 c ca 43 .3 85 ± 2 .3 51 a ba a 7 1 20 .6 12 ± 2 .1 55 b aa 24 .3 85 ± 1 .4 80 b aa 17 .4 51 ± 0 .1 21 a ba 42 .3 85 ± 2 .5 71 b aa 2 15 .2 08 ± 0 .1 19 c ba 32 .6 37 ± 1 .6 19 a aa 16 .2 91 ± 0 .9 55 a bb a 53 .4 80 ± 1 .5 01 a aa 3 24 .9 06 ± 0 .5 67 a aa 34 .6 73 ± 2 .6 32 a aa 14 .8 66 ± 0 .4 46 b ba 55 .3 55 ± 3 .6 61 a aa 4 24 .0 03 ± 2 .2 03 a ba a 33 .1 07 ± 2 .7 29 a aa 13 .6 01 ± 0 .6 02 c bb 45 .6 71 ± 0 .8 68 b aa exogenous proline modulates physiological responses acta bot. croat. 81 (1), 2022 95 ta b. 3 . p ro lin e an d m al on di al de hy de ( m d a ) co nt en t, 1, 1di ph en yl -2 pi cr yl hy dr az yl ( d pp h ) ra di ca l s ca ve ng in g ac tiv ity ( % ) an d fe rr ic r ed uc in g an tio xi da nt p ow er ( fr a p) i n le av es o f c m -3 34 a nd sd -8 p ep pe r cu lti va rs e xp os ed to p ro lin e (p ro ) an d ph yt op ht ho ra c ap si ci . t yp e of a pp lic at io ns ( a pp ): 1 co nt ro l ( no p ro , n o p. c ap si ci ), 2 p. c ap si ci a lo ne , 3 1 m m p ro + p . c ap si ci , 4 1 0 m m p ro + p . ca ps ic i) . m ea n ± st an da rd d ev ia tio n of 3 r ep lic at es is p re se nt ed . d iff er en t l et te rs in di ca te s ig ni fic an t d iff er en ce s at p < 0 .0 5 by t uk ey te st . c ap ita l l et te rs r ep re se nt d iff er en ce s in a pp lic at io ns fo r th e sa m e cu lti va r an d th e sa m e da y. l ow er c as e le tt er s re pr es en t d iff er en ce s in d ay s fo r th e sa m e cu lti va r an d th e sa m e ap pl ic at io n. s up er sc ri pt lo w er c as e le tt er s re pr es en t d iff er en ce s in c ul tiv ar s fo r th e sa m e da y an d th e sa m e ap pl ic at io n. a a e – as co rb ic a ci d eq ui va le nt , d pi – d ay p os t i no cu la tio n, f w – fr es h w ei gh t. c ul ti va r dp i a pp p ro lin e ( µg g -1 f w ) m d a ( nm ol g -1 f w ) d p p h s ca ve ng in g a ct iv it y (% ) f r a p ( µg a a e g1 f w ) c m -3 34 3 1 28 8. 37 9 ± 3. 89 11 b bb 77 .7 98 ± 1 .3 16 b aa 31 .3 46 ± 1 .4 52 c aa 63 8. 55 ± 2 1. 30 9 bb a 2 35 9. 40 7 ± 12 .0 53 b cb 78 .0 80 ± 3 .4 32 b ca 33 .0 98 ± 0 .8 86 c aa 65 7. 30 ± 1 0. 22 9 ba a 3 97 9. 03 3 ± 11 9. 23 a ab 76 .9 40 ± 0 .4 95 b ca 43 .0 21 ± 0 .8 33 b bb 99 1. 08 ± 4 5. 15 9 a aa 4 26 0. 34 2 ± 2 6. 52 1b bb 90 .1 00 ± 2 .9 72 a ca 51 .7 23 ± 2 .9 10 a aa 92 2. 16 ± 5 9. 62 6 a bb 5 1 32 6. 69 7 ± 42 .9 78 b ab b 77 .7 98 ± 0 .9 90 c aa 32 .3 20 ± 2 .2 21 d aa 72 0. 27 ± 8 .6 32 6 b c ab a 2 10 13 .9 2 ± 21 5. 88 a ab 89 .2 33 ± 2 .6 24 b bb 34 .4 28 ± 1 .2 64 c aa 69 6. 19 ± 3 6. 59 2 c ab 3 33 3. 11 5 ± 5. 10 65 b bb 89 .2 41 ± 3 .4 32 b ba 43 .0 75 ± 1 .8 15 b bb 78 6. 62 ± 5 3. 69 7 bb b 4 36 6. 88 4 ± 9. 45 41 b ab 15 0. 45 6 ± 4. 31 9 a aa 54 .7 61 ± 1 .6 96 a aa 10 20 .2 7 ± 15 .7 8 a aa 7 1 36 4. 08 0 ± 6. 56 42 b ab 79 .7 99 ± 2 .5 74 d aa 32 .3 78 ± 0 .3 70 d aa 77 8. 60 ± 5 6. 94 5 ba a 2 74 6. 94 6 ± 31 .7 24 a bb 12 2. 41 7 ± 1. 78 6 ba b 33 .9 35 ± 0 .9 71 c aa 67 7. 21 ± 5 .5 53 0 ba b 3 30 6. 75 9 ± 12 .0 17 c bb 11 2. 71 3 ± 1. 98 1 c aa 62 .5 33 ± 1 .3 55 a aa 73 4. 62 ± 5 .6 13 5 bb b 4 25 4. 42 3 ± 24 .7 68 c bb 12 8. 99 5 ± 1. 31 0 a ba 46 .0 69 ± 0 .6 63 b bb 10 61 .4 7 ± 94 .7 1 a ab sd -8 3 1 69 0. 87 2 ± 23 .3 70 c aa 73 .7 90 ± 3 .9 34 a ba 27 .7 27 ± 1 .7 08 b ab 66 4. 25 ± 2 5. 80 8 c ba 2 11 88 .7 25 ± 5 2. 07 b ba 76 .9 40 ± 1 .3 10 a ba 26 .4 74 ± 1 .4 55 b cb 66 3. 74 ± 2 3. 24 1 c ca 3 11 73 .1 50 ± 3 12 .4 9 bb a 5. 72 0 ± 0. 49 5 b cb 54 .8 82 ± 3 .4 95 a ba 76 3. 88 ± 1 6. 22 3 bb b 4 17 80 .8 42 ± 2 7. 06 a ba 3. 43 20 ± 1 .4 86 b cb 54 .7 42 ± 2 .4 64 a ba 11 86 .0 1 ± 8. 37 1 a aa 5 1 67 8. 09 9 ± 37 .4 80 b aa 78 .3 70 ± 2 .7 58 b ab a 28 .0 33 ± 1 .3 44 c ab 67 9. 53 ± 6 .5 62 2 c ab b 2 15 04 .3 02 ± 2 16 .6 a ab a 19 1. 24 0 ± 4. 72 6 a aa 37 .0 29 ± 2 .0 98 b aa 86 1. 47 ± 2 6. 29 3 bb a 3 13 74 .9 30 ± 2 77 .3 a ab a 21 .1 62 ± 1 .3 10 d bb 58 .9 67 ± 1 .4 62 a aa 11 71 .6 2 ± 27 .8 1 a aa 4 12 69 .3 76 ± 9 .0 47 a ca 41 .4 71 ± 2. 6 21 c bb 39 .0 47 ± 0 .9 87 a b cb 76 1. 79 ± 5 8. 92 7 c bb 7 1 65 9. 40 7 ± 27 5. 75 c aa 86 .6 91 ± 5 .5 73 c aa 28 .2 95 ± 1 .7 62 d ab 72 1. 65 ± 2 4. 68 6 c aa 2 15 92 .4 29 ± 9 .3 45 b aa 18 7. 89 6 ± 0. 82 8 a aa 35 .3 94 ± 2 .4 91 c ba 10 33 .2 3 ± 27 .4 45 ba a 3 20 16 .0 25 ± 1 16 .8 a aa 37 .4 68 ± 5 .1 72 d ab 43 .7 94 ± 1 .1 49 b cb 11 00 .9 1 ± 39 .2 45 ba a 4 19 99 .5 94 ± 1 10 .3 a ba a 11 9. 13 9 ± 7. 53 9 ba a 64 .5 71 ± 3 .2 95 a aa 13 77 .2 1 ± 13 3. 63 a aa koç, e. 96 acta bot. croat. 81 (1), 2022 p. capsici application on the 7th dpi when compared with p. capsici in sd-8 cultivar, but the difference was not found to be significant. when the two cultivars were compared, the highest total protein level was detected in the sd-8 cultivar after application of 1 mm pro + p. capsici on the 7th dpi with 55.385 ± 3.661 mg g-1 fw, but the difference was not found to be significant (tab. 2). proline and mda content the content of endogenous pro in all controls and pro treatments of sd-8 cultivar was higher than in the cm-334 cultivar (p < 0.05) (tab. 3). although exposure to p. capsici alone caused an increase in the amount of endogenous pro in both cultivars on all dpi, pro application before inoculation increased the endogenous pro accumulation ability only in the sd-8 cultivar (p < 0.05). in the cm-334 cultivar, the application of 1 mm pro + p. capsici on the 3rd dpi resulted in the highest pro increase of 239% when compared with the control group and 172.2% compared to exposure to p. capsici alone (p < 0.05) (tab. 3). in the sd-8 cultivar, the highest pro increase of 205% was determined on the 7th dpi upon exposure to 1 mm pro + p. capsici (p < 0.05) when compared with the control group. in addition, a significant increase of approx 49.8% in pro amount was detected on the 3rd dpi after exposure to 10 mm pro + p. capsici compared to treatment with p. capsici alone (p < 0.05) (tab. 3). mda content increased due to p. capsici-imposed stress in both cultivars on all dpi compared to the control group, although the difference was found to be statistically significant on the 5th and 7th dpi (p < 0.05) (tab. 3). when the cultivars were compared, the lowest mda amounts were found in the sd-8 cultivar in both pro + p. capsici applications (p < 0.05). compared to exposure to p. capsici alone in the cm334 cultivar, the application of 1 mm pro before inoculation was more effective against lipid peroxidation than 10 mm pro (p<0.05). in the sd-8 cultivar, both pro applications had a significant effect on the mda amount when compared with treatment with p. capsici alone, while the highest mda decrease of 95.5% was determined on the 3th dpi in the 10 mm pro + p. capsici application (p < 0.05) (tab. 3). antioxidant capacity dpph radical scavenging activity (%) increased in both cultivars on all dpi upon exposure to both combined treatments with pro + p. capsici compared to the control group and treatment with p. capsici alone (p < 0.05) (tab. 3). in cm-334 cultivar, the highest increase in dpph radical scavenging activity of approximately 84% was recorded after the treatment with 1 mm pro + p. capsici on the 7th dpi in comparison to p. capsici application alone. in sd-8 cultivar, the highest increase in dpph radical scavenging activity of approximately 82% was observed upon exposure to 10 mm pro + p. capsici on the 7th dpi (p < 0.05) in comparison to p. capsici application alone (tab. 3). frap values in control groups at all dpi were of similar values in the cultivars. in the cm-334 cultivar, antioxidant power increased on all dpi upon exposure to both combined treatments with pro + p. capsici compared to control and treatment with p. capsici alone (tab. 3). similar results were obtained in the sd-8 cultivar as well with the exception of the treatment with 10 mm pro + p. capisici on the 5th dpi. when the two cultivars were compared, the highest frap value was detected on the 7th dpi in the 10 mm pro + p.  capsici application in the sd-8 cultivar (p < 0.05), while the highest frap increase of 56.6% was determined in the treatment with 10 mm pro + p. capsici in comparison to p. capsici applied group in cm-334 cultivar (p < 0.05) (tab. 3). discussion in this study the possible beneficial effects of exogenously applied pro on physiological parameters in pepper plants exposed to infection with ph. capisici were investigated. pro is considered an important indicator of environmental stress caused by either abiotic or biotic factors (claussen 2005, hayat et al. 2012, liang et al. 2013). verslues and sharma (2010) reported evidence that pro plays a role in programmed cell death and development in plant-pathogen interactions. penetration of p. capsici usually takes place in the host cell wall, a passive barrier that limits the access of pathogens to plant cells. pro is an important source of cell wall matrix (hydroxyproline and proline-rich proteins), which indicates that it is necessary in the first line of defence against fungal pathogens (lehmann et al. 2010, kavi kishor et al. 2015). moreover, biotic stress results in increased production of reactive oxygen species (ros) molecules, which play important roles in protecting plants against harmful pathogens. however, nucleic acid damage, oxidation of proteins and lipids and degeneration of chlorophyll pigments may occur due to excessive ros accumulation, which increases depending on the severity and duration of the stress (huang et al. 2019). proline plays an important role in protection against oxidative damage as a powerful ros scavenger as well as in stabilising the 3d structure of membranes and proteins (hossain et al. 2014), protecting organelles such as mitochondria and chloroplasts (ashraf and foolad 2007) and by induction of stress-sensitive genes (kahraman et al. 2019). in the current study, p. capsici infection caused a decrease in chlorophyll a and b content, which was accompanied by an increase in leaf starch content on all days following the application in the resistant cm-334 cultivar. the increase in starch accumulation in infected leaves may have led to a decrease in the photosynthesis rate, which in turn caused the observed decrease in the amount of chlorophyll a and b in the leaves. the main reason for the decrease in chlorophyll content in plants exposed to stress may be disorganisation of thylakoid membranes due to the formation of proteolytic enzymes such as chlorophyllase, rather than chlorophyll synthesis (sharma et al. 2019). the present results corroborate the findings of mandal et al. (2009) and llave (2016). namely, llave (2016) reported that viral infection caused an increase in starch accumulation in leaves and exogenous proline modulates physiological responses acta bot. croat. 81 (1), 2022 97 a decrease in photosynthesis, while mandal et al. (2009) found that downy mildew infection caused an increase in the amount of starch, a decrease in the amount of soluble sugars and in the rate of photosynthesis in leaves of the plantago ovata forsk. both studies reported that the increase in the amount of starch in the infected leaves may be the reason for the decrease in photosynthesis. moreover, decrease in the amount of chlorophyll content was accompanied with the decrease in the amount of glucose in infected leaves of sd-8 cultivar as well as with the increase in the amount of pro. proline’s multiple roles as an osmolyte, ros scavenger, signalling molecule and energy source are similar to glucose’s multiple roles acting as a carbon and energy source (trovato et al. 2008). dawood et al. (2014) found that pro application caused significant increases in photosynthetic pigments in faba bean plants under seawater stress, while kaushal et al. (2011) reported that exogenous pro protected the chlorophyll content and activity of rubisco and antioxidant enzymes against heat stress in chickpea. in my previous study, the pre-application of pro in pepper exposed to p. capsici caused an increase in the activities of antioxidant enzymes peroxidase (pox) and catalase (cat) and a decrease in the amount of hydrogen peroxide (h2o2) (koç 2017). therefore, the increase in the chlorophyll a and b content in peppers exposed to p. capsici stress can be attributed to the stimulation of chlorophyll biosynthesis or inhibition of its degradation and the more efficient removal of stress-induced increased ros by pro. moreover, application of 1 mm pro significantly increased carotenoid concentration in sd-8 pepper leaves in the early days (3rd dpi) after infection. ramel et al. (2012) reported that carotenoids play a role in the protection of the photosynthetic apparatus by directly deactivating singlet oxygen, which causes photoinhibition damage. this study, with the detection of an increase in the amount of anthocyanin and flavonoids in parallel with the increase in the amount of fructose due to p. capsici stress, also supports the view of landi et al. (2013) that fructose is necessary for the biosynthesis of many defence compounds such as anthocyanin and phenolic compounds. in the sd-8 cultivar, pro, exogenously applied under p. capsici stress, increased both endogenous pro and soluble sugar content, and these results support moustakas et al. (2011)’s conclusion that the pro signalling pathway interacts with the soluble sugar signalling pathway. the increase in soluble sugar content in pro + p. capsici applications revealed the positive effect of pro on photosynthetic activity. in addition, soluble sugars participate in the defence by their capacity to directly or indirectly scavenge ros in chloroplasts by stimulating antioxidative defence systems, as well as acting as an energy source (van den ende and valluru 2009). however, exposure to 10 mm pro + p. capsici caused a decrease in glucose content in the cm-334 cultivar. this result indicates that the high concentration of exogenously applied pro may have negatively affected rubisco activity. the same application caused an increase in glucose content only on the 7th day in sd-8 cultivar. there is information that the exogenously applied pro can cause damage in some plants and have a stimulating effect on the defence (hayat et al. 2012). the reason for this difference can be explained by the fact that genotypes react differently to the applied pro concentration. the peroxidation of lipids in biological membranes is the most obvious symptom of oxidative stress and mda is a marker of stress-induced oxidative lipid damage. in this study, an increase in the amount of endogenous pro was determined in parallel with the increase in the amount of mda under p. capsici stress in both cultivars, but it seems that this endogenous pro accumulation was not sufficiently effective in reducing the lipid peroxidation damage caused by p. capsici stress. the exogenous 1 mm pro pre-application was determined as the most effective joint application in stabilising the protein and membrane structure by causing an increase in the amount of endogenous pro and total protein and a significant decrease in the amount of mda in both cultivars, although there was no decrease in mda on the 5th dpi for cm-334. the increased level of endogenous pro in pepper exposed to exogenous pro can be attributed to the ros scavenging function of pro. roychoudhury and chakraborty (2013) reported that low concentrations of exogenous pro may activate cytosolic pro biosynthesis from glutamate and induce pro catabolism in mitochondria. despite the positive effects of applied pro in inducing plant stress tolerance, there are some reports on the inhibitory effect of pro (ashraf and foolad 2007). in this study, the high concentration of 10 mm pro application caused a decrease in the endogenous pro and total protein accumulation and an increase in the mda in the cm-334 cultivar when compared with p. capsici infected plants without previous pro application, thus supporting the results of ashraf and foolad (2007). in addition, pro degradation can provide carbon, nitrogen, and energy sources. perhaps, despite the negative effect of 10 mm pro application, endogenous pro oxidation may also have been used as an energy source for repair of stress-induced damage. the same application caused an increase in the amount of proline and total protein in general and a significant decrease in the amount of mda in the sensitive sd-8 cultivar. these different defence responses are attributed to the different genotypes. hao et al. (2016) reported that the resistance and susceptibility levels of genotypes to p. capsici may be due to their different genetic structures. dpph is a free radical that easily damages the cell membrane. the high radical scavenging activity of the organism is directly proportional to its protective effect against oxidative damage. it has been reported that the antioxidant activity in plants is mostly due to phenolic compounds ( subramanian et al. 2013). phenolic compounds are effective hydrogen donors, which makes them good antioxidants. krishnan et al. (2015) reported that besides phenolic compounds, flavonoid compounds that tend to accumulate under stress conditions also contribute to total antioxidant activity. in my study, the effect of pro applied through leaf on cm-334 and sd-8 cultivars under p. capsici stress was different based on cultivars. dpph scavenging activity in the koç, e. 98 acta bot. croat. 81 (1), 2022 leaves of sd-8 cultivars reached the highest level in 1 and 10 mm pro applications, and in 1 mm pro application in cm-334 cultivar. findings show that the dpph scavenging activity is positively associated with the amount of flavonoids and the exogenous pro application directly contributes to the antioxidant activity. in this study, p. capsici stress applied alone caused an increase in the amount of flavonoid content, especially in the cm-334 cultivar. when pro was applied before inoculation, it was observed that it caused further increases in flavonoid levels in the cm-334 cultivar. the results are consistent with the findings of zhang et al. (2014), which stated that pro application stimulates flavonoid synthesis. the rapid increase in the synthesis of anthocyanins (3rd day), a class of flavonoids, which are secondary metabolites after infection, indicated that it is among the earliest defence responses against the pathogen. anthocyanins are synthesized through the phenylpropanoid pathway. phenylalanine is the precursor of this synthesis, and the conversion from phenylalanine to anthocyanins occurs as a result of reactions catalyzed by enzymes. a previously conducted study determined that pro application before inoculation significantly increased phenylalanine ammonia lyase (pal) enzyme activity in cm-334 cultivar (koç 2017). findings from this study show that this increase in anthocyanin is associated with an increase in pal enzyme activity. in this context, the results corroborate the findings of elewa et al. (2017) and zhang et al. (2014), who reported that flavonoids and anthocyanins tend to accumulate under stress conditions. in addition, pro contributed to hydrogen bonding removal of the dpph radical as a hydrogen donor (zou et al. 2016) and demonstrated its antioxidant property by acting as a free radical inhibitor or scavenger. the reducing capacity of a compound is considered an important indicator of its potential antioxidant activity. antioxidant compounds can donate electrons to reactive radicals, reducing them to more stable and non-reactive species (santos-sánchez et al. 2019). a higher absorbance indicates a higher ferric reducing power. in this study, although pro + p. capsici applications showed different effects in the periods following the inoculations in both cultivars, the fact that there was an overall increase in frap indicates that exogenous pro may have stimulated the production of metabolites responsible (flavanoids, anthocyanins, pro etc.) for the reductive mechanisms of plants under the p. capsici stress. 1 and 10 mm pro pre-applications caused an increase in the amount of f lavonoid and anthocyanin in cm-334, and endogenous pro in sd-8 cultivar. also, the findings show that the cm-334 cultivar responds earlier (3rd dpi) to the p.  capsici infection in some parameters such as flavonoid, anthocyanins, dpph radical scavenging activity than the sd-8 cultivar. comparing the two pro concentrations used, in terms of plant activity performance, in cm-334, application of 1 mm of pro was more effective than 10 mm pro in alleviating the damage caused by p. capsici. the low concentration of 1 mm pro applied exogenously increased stress tolerance in the cm-334 cultivar. in the sd-8 cultivar, 1 mm pro + p. capsici application on the 5th dpi and 10 mm pro + p. capsici on the 7th dpi were more effective in parameters such as dpph radical scavenging activity and frap value. in this study and in previous studies, it was seen that different plants have diverse responses to different 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e0151401. zhang, y., butelli, e., martin, c., 2014: engineering anthocyanin biosynthesis in plants. current opinion in plant biology 19, 81–90. zhang, j.l., shi, h., 2013: physiological and molecular mechanisms of plant salt tolerance. photosynthesis research 115(1), 1–22. zou, t.b., he, t.p., li, h.b., tan, h.w., xia, e.q., 2016: the structure-activity relationship of the antioxidant peptides from natural proteins. molecules 21(1), 72. opce-str.vp acta bot. croat. 70 (1), 81–90, 2011 coden: abcra 25 issn 0365–0588 biological activity of the red alga laurencia brandenii aseer manilal1, sugathan sujith1, balu sabarathnam1, george s. kiran1, joseph selvin1*, chippu shakir1, aaron p. lipton2 1 department of microbiology, bharathidasan university, tiruchirappalli 620 024, india 2 central marine fisheries research institute, vizhinjam 695 521, thiruvananthapuram, india abstract – the marine red alga laurencia brandenii collected from the southwest coast of india (indian ocean) was extracted and fractioned using column chromatography. the individual fractions were evaluated in vitro via antimicrobial activity against six species of microbial type culture collection and three species of clinical human pathogens, antipest activity on sitophilus oryzae, maggoticidal activity against 2nd instar larvae of sarcophaga sp. and termiticidal activity against microtermes obesi. it was found that the fraction eluted using petroleum ether:chloroform (6:4) exhibited broader biological activities. the phyco-constituents of the active fraction were identified by gas chromatography-mass spectrometry (gc-ms) analysis. the gc-ms profile of the active fraction revealed that the main constituent was octadecadienoic acid (49.75%) followed by n-hexadecanoic acid (14.24%), which might have a functional role in the biological activities. the overall activity profile envisages that these bioactive compounds from l. brandenii could be utilized as a renewable natural resource for the development of novel environmental-compatible formulations for the control of human pathogens, pests, termites and maggots. keywords: alga, laurencia brandenii, antimicrobial activity, antipest activity, maggoticidal activity, termiticidal activity, larva, sarcophaga, octadecadienoic acid, n-hexadecanoic acid introduction natural marine products are in great demand due to their prolific biological activities and serve as source for the discovery of novel bioactive compounds (blunden 2001). during the past few decades, over 14,000 novel natural products from marine organisms have been isolated and described (proksch 2002). such biogenic compounds exhibit unique structural and functional properties that are not found in terrestrial natural products (fenical 1982). among the diverse group of marine flora, seaweeds are the most primitive vegetation, surviving over a large range of environmental conditions and being continuacta bot. croat. 70 (1), 2011 81 * corresponding author, e-mail: selvinj@rediffmail.com copyright ® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:51 color profile: disabled composite 150 lpi at 45 degrees ously exposed to high density of harmful micro and macrofauna. the survival of this sedentary vegetation has primarily relied on efficient defence mechanism against diverse invaders. seaweeds from varied locales have been evaluated for a wide range of biological activities, e.g., antibacterial (tuney et al. 2006), antiviral (serkedjieva 2004), antifungal (tang et al. 2002) and antialgal activities (hellio et al. 2002). over 2,400 secondary metabolites have been isolated and described from the divisions rhodophyta, phaeophyta, and chlorophyta, many of which have been reported to have excellent biological activity (faulkner 2001 and references therein). of the three groups of seaweeds, the class rhodophyta produces a plethora of structurally diversified novel halogenated natural products, which in fact symbolize the extraordinary wealth of biogenic compounds principally for pharmaceutical leads (faulkner 2001). among the red algae, the genus laurencia is known to produce the largest number and diversity of secondary metabolites, ultimately making it the world’s most chemically complex seaweed genus (pereira et al. 2003). moreover, the anticandidal, antibacterial (manilal et al. 2009a, shanmughapriya et al. 2008), nematicidal and mosquito larvicidal activity, as well as the ichthyotoxicity and brine shrimp cytotoxicity (manilal et al. 2009b) of crude extracts of different red algal species from the indian coast has already been reported. therefore, the present study was aimed to evaluate the biological activity of l. brandenii against human pathogens, pests, maggots and termites and to detect the bioactive compounds from l. brandenii that could be useful for the development of novel environmental compatible formulations for the control of human pathogens, pests, termites and maggots. to this day, information regarding biological activities of flora and fauna from the southwest coastline of india (kollam coast) remains scanty. materials and methods study area and collection of samples the study area was located along the 45 km long southwest coast of india (indian ocean), between latitude 08°54' n and longitude 76°38' e. seaweed specimens were collected from the intertidal and subtidal habitat of kollam prefecture (thirumullavaram) located on the southwest coast. the area is rich in flora and fauna (manilal et al. 2009a). samples were collected during december 2007 to february 2008 when red algal diversity remains dominant. cleaned plant materials were shade-dried under a stream of air for one week to prevent photolysis and thermal degradation. the completely dried material was weighed and ground coarsely in a mechanical grinder. in the present study we evaluate the biological potencies of a column-purified fraction of the red alga laurencia brandenii, on the following bioassays: 1) antimicrobial activity on nine species of human pathogens; 2) antipest activity on sitophilus oryzae; 3) maggoticidal activity on 2nd instar larvae of sarcophaga sp. and 4) termiticidal activity against microtermes obesi. extraction of seaweed bioactives the shade-dried samples (5 kg) were extracted using methanol and purified in column chromatography to yield eleven fractions (f1-f11). the individual fractions were evaluat82 acta bot. croat. 70 (1), 2011 manilal a., sujith s., sabarathnam b., kiran g. s., selvin j., shakir c., lipton a. p. u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:51 color profile: disabled composite 150 lpi at 45 degrees ed for biological activities (data not shown). the fraction eluted using petroleum ether: chloroform (6:4), fraction f8, showed a wide spectrum of activity in all the bioassays. for the extraction of bioactive compounds, 100 g of the dried seaweed powder was weighed and immersed in a flask containing 1000 ml of methanol (purity grade 99%) and placed at 35 °c in a shaker at 120 rpm for 7 days for the extraction of active ingredients. the algal material was re-extracted with methanol in a 1 l capacity round bottom flask in a water bath at 60 °c for 3 h. the individual crude mixture was pooled and filtered using paper filter fitted with a buchner funnel using suction pressure followed by centrifugation (eppendorf) at 6000 � g for 5 min at 20 °c. the supernatant was collected in a round-bottomed flask and the remaining solvent was concentrated up to 15–20 ml in a rotary vacuum evaporator (yamato). the residue collected was evaporated to dry completely in a vacuum desiccator and stored in the refrigerator. chromatography of laurencia brandenii the methanolic extract of laurencia brandenii (200 g) was applied in a silica gel (60–120 mesh) column developed with petroleum ether and eluted with petroleum ether and chloroform (9:1 to 1:9 and 100% chloroform) followed by chloroform and methanol (9:1 to 1:9 and 100% methanol) and yielded eleven fractions. the individual fractions were screened for biological activity (data not shown). the fraction that was eluted using petroleum ether: chloroform (6:4), which exhibited activity was used for hewlett packard gas chromatography-mass spectrometry (gc-ms) analysis. peak identification was carried out by comparison of the mass spectra with those available in the nist version 2 (2005). test microorganisms to characterize the antimicrobial activity, the algal fractions were evaluated against a battery of human pathogenic bacteria. antimicrobial activity (inhibition area) was determined against six species of type cultures from microbial type culture collection (mtcc) of human gram-positive pathogens, such as staphylococcus aureus (mtcc 2940), bacillus subtilis (mtcc 1306), micrococcus luteus (mtcc 106), rhodococcus rhodochrous (mtcc 265), and the gram-negative bacteria escherichia coli (mtcc 739) and pseudomonas aeruginosa (mtcc 2453), as well as three species of clinical isolates, vibrio cholerae, salmonella typhi, and streptococcus pneumoniae. the resistant patterns of these human pathogenic isolates were confirmed using selective antibiotics: streptomycin, oxytetracycline, ampicillin and erythromycin, in a preliminary experiment. antimicrobial assay the antibacterial assay was carried out following the methodology of selvin and lipton (2004). anti-pest activity pesticidal activity of the algal fraction was evaluated by a modified area preference test (mcdonald et al. 1970) using the common rice weevil sitophilus oryzae (l.). the organism was collected from infested rice obtained from a local market and reared in glass bottles under standard insectaria conditions at ambient temperature (28 � 3 °c) and the relative humidity 70 � 5%. a uniformly saturated filter paper disc of different concentrations of albioactivity of laurencia brandenii 83 acta bot. croat. 70 (1), 2011 u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:51 color profile: disabled composite 150 lpi at 45 degrees gal extracts (2, 4, 6, 8 and 10 mg cm–2) was pasted in the bottom of each petri dish. ten unknown-sex insects of same age were released in the petri dish and kept in a dark room. mortality was observed after 24 h of exposure. all the experiments performed in the present study were repeated six times to validate the findings statistically. the mortality percentage was converted into probit scale to determine the ld50 values. maggoticidal activity for the determination of maggoticidal activity, 2nd instar maggots of the flesh fly, sarcophaga sp. (miegen) were used as test organism. a stock colony of the insect was established using mature flies collected from local slaughter house. a couple of adults were released into a wide-mouth glass chamber covered with a copper mesh screen and a piece of chicken meat was placed for the oviposition. the whole setup was left for 72 h with constant monitoring. ten successfully hatched larvae were picked using a blend end probe and placed in a 40 mm disposable petri dish filled with boiled egg yolk uniformly mixed seaweed extracts (20, 40, 60, 80 mg g–1). petri dishes containing no seaweed extracts and methanol were used as positive and negative control. since this was the active feeding stage of the maggots, they could consume the appropriate quantity of the algal fraction mixed with boiled egg yolk, so the effect of the algal extracts could be evaluated effectively. mortality rate of treated maggots was recorded after 24 and 48 h of exposure. based on the percent mortality, the ld50 value was determined using probit scale (wardlaw 1985). termiticidal activity the test termite microtermes obesi (holmgren) worker was collected from the thickets of bharathidasan university. the collected organisms were maintained for three days on a degraded log in a glass chamber at 30 °c ± 2 °c, 75 ± 5% relative humidity and a 12:12 h photoperiod. ten worker termites were transferred to a 30 mm petri dish uniformly lined with different concentration of algal fractions which dried overnight to remove the solvent completely. the petri dishes were covered and kept in a dark cabinet maintained at 25 °c for 24 h. the survivors were counted under mild light and the percentage of survivors was calculated. all the experiments were repeated six times to validate the findings statistically. results in antimicrobial assay, the algal fraction exhibited a growth inhibition range of 213 mm2 to 87 mm2 against the nine species of microbial type culture collection and clinical isolates (fig.1). the results of the antipest activity indicated that the algal fraction was toxic to sitophilus oryzae which produce an ld50 value of 3.7 mg cm–2 (fig. 2). maggoticidal assay showed the algal fraction was active against the 2nd instar maggots of sarcophaga with an ld50 of 43 mg g–1 (fig. 3). concerning the termiticidal assay, the active fraction of l. brandenii was found to be highly lethal at 2.6 mg ml–1 causing 50% mortality at a 6 h exposure (fig. 4). the fraction f8 exhibited biological activity and proceeded to gc-ms analysis, to show the relative percentage of identified active compounds in laurencia brandenii (tab. 1). the main phycoconstituents of the active fraction were octadecadienoic acid (49.75%) followed by n-hexadecanoic acid (14.24%). 84 acta bot. croat. 70 (1), 2011 manilal a., sujith s., sabarathnam b., kiran g. s., selvin j., shakir c., lipton a. p. u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:51 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (1), 2011 85 bioactivity of laurencia brandenii 0 50 100 150 200 250 sa bs ml rr ec pa vc st sp test organisms a re a o f in h ib it io n (m m ) 2 fig. 1. antibacterial potentials of laurencia brandenii against microbial type culture collection and clinical isolates. bs – bavillus subtilis, ec – escherichia coli, ml – micrococcus luteus, rr – rhodococcus rhodochrous, pa – pseudomonas aeruginosa, sa – staphylococcus aureus, sp – streptococcus pneumoniae, st – salmonella typhi, vc – vibrio cholerae. 0 20 40 60 80 100 2 4 6 8 concentration (mg cm )–2 m o rt a li ty (% ) ld 3.7 mg cm50 –2 fig. 2. antipest activity of laurencia brandenii against sitopzhilus oryzae 0 20 40 60 80 100 20 40 60 80 concentration (mg g )–1 m o rt a li ty (% ) ld 43 mg g50 –1 fig. 3. maggoticidal activity of laurencia brandenii against sarcophaga albiceps larvae u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:51 color profile: disabled composite 150 lpi at 45 degrees 86 acta bot. croat. 70 (1), 2011 manilal a., sujith s., sabarathnam b., kiran g. s., selvin j., shakir c., lipton a. p. concentration (mg ml )–1 m o rt a li ty (% ) 0 20 40 60 80 100 1 2 3 4 ld 2.6 mg50 ml –1 fig. 4. termiticidal activity of laurencia brandenii against microtermes obesi tab. 1. gc-ms data of active fraction of laurencia brandenii no rt name of the compound molecular formula molecular weight peak area % 1 5.01 cyclohexasiloxane – dodecomethyl c12h36o6si6 444 1.07 2 8.20 trisiloxane 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethyl silyl)oxy]c12h36o4si5 384 0.89 3 9.36 3,5-dimethyl-5-hexan-3-ol c18h6o 128 0.11 4 11.64 3,5-dimethyl-5-hexan-3-ol c18h6o 128 0.11 5 13.59 4-dodecanol c12h26o 186 0.08 6 16.04 2-decanone. 5,9-dimethylc12h24o 184 0.13 7 18.65 n-hexadecanoic acid c16h32o2 256 14.24 8 20.90 9-dodecanoic acid, methyl ester,(e)c13h24o2 212 0.48 9 21.11 oxalic acid, allyl hexadecyl ester c21h38o4 354 0.40 10 21.85 9,12-octadecadienoic acid (z,z)c18h32o2 280 49.75 11 24.33 cyclohexanecarboxylic acid, decyl ester c17h32o2 268 2.70 12 25.96 9,12-octadecadienoyl chloride (z,z)c18h31clo 298 3.50 13 28.26 oxalic acid, allyl pentadecyl ester c20h36o4 340 1.23 14 28.75 cis9,10-epoxyoctadecan-1-ol c18h36o2 284 0.61 15 29.79 heptanoic acid, 9-decen-1-yl ester c17h32o2 268 0.65 16 30.63 9-octadecenal c18h34o 266 5.91 17 34.00 9,12-octadecadienoic acid (z,z)-, phenylmethyl ester c25h38o2 370 0.44 18 34.51 oxalic acid, allyl hexadecyl ester c21h38o4 354 1.11 19 37.64 oxirane, tetradecylc16h32o 240 3.49 20 38.61 1,2-15,16-diepoxyhexadecane c16h30o2 254 3.01 21 40.00 7,11-hexadecadienal c16h28o 236 5.99 22 40.40 thunbergol c20h34o 290 4.11 u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:51 color profile: disabled composite 150 lpi at 45 degrees discussion data obtained from the antimicrobial activity of the column purified fraction (f8) of laurencia brandenii exhibited considerable activity against all the tested microbial type culture collection and clinical isolates. the magnitude of inhibition against organisms was on the decreasing order of bacillus subtilis (213 mm2) > micrococcus luteus (194 mm2) > staphylococcus aureus (158 mm2) > rhodococcus rhodochrous (132 mm2) > pseudomonas aeruginosa (118 mm2) > escherichia coli (103 mm2). a high activity range was extended against one of the microbial type culture collection human pathogens, b. subtilis with 213 mm2, whereas the activity was moderate against e. coli 103 mm2. regarding the clinical isolates, salmonella typhi was found to be the most resistant bacteria, producing an inhibition area of 64 mm2 whereas the fraction was moderately able to inhibit the growth of streptococcus pneumoniae (87 mm2). the bactericidal potency of the algal fraction was high against the clinical pathogen, vibrio cholerae to the extent of 113 mm2 at 37 °c. in the present study, algal fraction of l. brandenii showed maximum antibacterial activity against the gram positive bacteria, whereas the activity was found to be moderate against gram negative bacteria. the resistant mechanism of gram negative bacteria could be due to the permeability provided by the cell wall or to the membrane accumulation tactics (adwan and abu-hasan 1998). antimicrobial potentials of red algae have already been reported from different locales, but their efficacy against clinical and human pathogens have scarcely been investigated. concerning the prevalence of microbial resistance to conventional antibiotics, our study provides a new insight into the development of novel phytotherapeutics for the management of infectious diseases. data pertaining to the susceptibility of gram positive strains to algal extracts were reported by many authors (pesando and caram 1984, rosett and srivastava 1987). our result is in accordance with the antibacterial activity of laurencia spp. reported from various geographic regions (de nys et al. 1996, hellio et al. 2001, taskin et al. 2007) against marine, human and fish bacterial pathogens (vairappan et al. 2001, 2003; bansemir et al. 2006). based on the present findings l. brandenii could be utilized as renewable bioresource for the development of potential antimicrobials. in the present study, the algal fraction of laurencia brandenii exhibited considerable effects on the rice pest, sitophilus oryzae showing an ld50 value of 3.7 mg after 24 h, significantly higher than that of solvent control, clearly demonstrating the direct contact toxicity as the rationale. the mode of action of this seaweed might be due to the shutdown of different biosynthetic routes of the pest’s metabolic pathways. these insecticidal compounds can inhibit the feeding behaviour and growth regulators, disrupting the endocrinological balance of the insects (balandrin et al. 1985). marine flora and fauna with their broad chemical diversity are still an untapped resource for the development of new agro-chemical agents (crombie 1990) and insecticides (saxena 1987). the findings of the present study indicate that red alga laurencia could be employed as a biological grain protectant to combat invading pests. the maggoticidal activity of laurencia brandenii was studied by feeding the maggots on an artificial diet containing different concentrations of the active fraction of the seaweed. it showed only a moderate toxicity effect on maggots. response varied with different concentrations of the algal fraction i.e., the toxicity of the extracts varies with an increase in concentration. maximum mortality (100%) was recorded at 60 mg g–1, whereas at acta bot. croat. 70 (1), 2011 87 bioactivity of laurencia brandenii u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:52 color profile: disabled composite 150 lpi at 45 degrees 20 mg g–1 extract showed a decrease in activity. the results indicate that the l. brandenii fraction contains compounds that are toxic to maggots since the ld50 value is moderately low. the literature proved that the present study would be the first report on the maggoticidal property of seaweeds. the mortalities of the larvae were 100% and 50% after 24 h of exposure to 60 and 43 mg g–1 of algal extract respectively. at higher concentrations, the seaweed fraction was an annoyance to maggots, which showed feed aversion, avoidance, and repellence. the present findings proved that a direct feeding trial assay would be useful to prove the maggoticidal property of seaweed. according to this finding, l. brandenii could be used for developing a novel natural veterinary grade maggoticides, since these natural products would likely be biodegradable, thus posing no threat to the environment. the inexorable use of synthetic termiticides is a major threat to the natural environment, and as a result there is an urgent need to explore and utilize naturally occurring products for combating harmful termites (carter 1976). historically, plants are well-known for their various bioactivities. the toxicity of plants in termites has been reported since 1947 (wolcott 1947). the bioactive compounds have evolved in plants for defense against phytophagous insects. the sesquiterpenes from cryptomeria japonica reported to possesses termiticidal activity (arihara et al. 2004). similarly, essential oils from melaleuca species can suppress the growth of termites (sakasegawa et al. 2003). the termiticidal assay represents a rapid, inexpensive and simple bioassay for testing seaweed bioactivity. in the present study, a fatal effect was observed when 3 mg ml–1 concentration of algal fraction of l. brandenii was applied to the petri dish, causing 90% mortality at a 6 h exposure. the mortality rate for the positive control experiment was much lower (<10) while that of negative control gave a mortality of 15%. the study showed that the termite population declined drastically relative to the concentration and time of exposure. the termiticidal potential of l. brandenii has not been investigated or reported in the literature. the tested seaweed fraction may be a valuable source of biorational compounds and could be employed for the development of novel natural termiticide. spectral data by gc-ms showed that a mixture of fatty acids characterised the chemistry of the active fraction. in all, 22 peaks with different retention times were observed. it is possible that bioactive compounds primarily consisting of 9, 12-octadecadienoic acid (z, z) – (49.75 %) may be involved in the biological activity. the fatty acid composition of the active fraction revealed that the main acid was octadecadienoic acid (49.75 %) followed by n-hexadecanoic acid (14.24 %). seaweeds exhibit a high level of fatty acid diversity and many of them possess potential bioactivity. therefore in the present study, the biological activity of l. brandenii might be attributed due to the presence of the fatty acid octadecadienoic acid (49.75 %) in a high percentage. acknowledgements a. m. is grateful to the council of scientific and industrial research, new delhi for the award of senior research fellowship. thanks are extending to professors m. jayakumari and c. k. thankachi, faculty of zoology, sree narayana college, kollam, kerala for identification of insects. this work was funded by the department of biotechnology, government of india (bt/pr8064/ aaq/03/290/2006). 88 acta bot. croat. 70 (1), 2011 manilal a., sujith s., sabarathnam b., kiran g. s., selvin j., shakir c., lipton a. p. u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:52 color profile: disabled composite 150 lpi at 45 degrees references adwan, k., abu-hasan, n., 1998: gentamicin resistance in clinical strains of enterobacteriaceae associated with reduced gentamicin uptake. folia microbiologica 43, 438–440. arihara, s., umeyama, a., bando, s., imoto, s., ono, m., yoshikawa, k., 2004: three new sesquiterpenes from the black heartwood of cryptomeria japonica. chemical and pharmaceutical bulletin 52, 463–465. balandrin, m. f., klocke, j. 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coast of india tested against shrimp, human and phytopathogens. annals of microbiology 59, 207–219. mcdonald, l. l., guy, r. h., speirs, r. d., 1970: preliminary evaluation of new candidate materials as toxicant, repellents and attractants against stored product pests i. marketing research report 882. agricultural research service, u.s. department of agriculture, washington dc, 1–8. pereira, r. c., da gama, b. a. p., teixeira, v. l., yoneshigue-valentin, y., 2003: ecological roles of natural products of the brazilian red seaweed laurencia obtusa. brazilian journal of biology 63, 665–672. acta bot. croat. 70 (1), 2011 89 bioactivity of laurencia brandenii u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:52 color profile: disabled composite 150 lpi at 45 degrees pesando, d., caram, b., 1984: screening of marine algae from the french mediterranean coast for antibacterial and antifungal activity. botanica marina 27, 381–386. proksch, p., edrada, r. a., ebel, r., 2002: 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annals of microbiology 58, 535–541. tang, h. f., yi, y. h., yao, x. s., xu, q. z., zhang, s. y., lin, h. w., 2002: bioactive steroids from the brown algae sargassum carpophyllum. journal of asian natural product research 4, 95–105. taskin, e., ozturk, m., taskin, e., kurt, o., 2007: antibacterial activities of some marine algae from the aegean sea (turkey). african journal of biotechnology 6, 2746–2751. tuney, i., cadirci, b. h., unal, d., sukatar, a., 2006: antimicrobial activities of the extracts of marine algae from the coast of urla (izmir, turkey). turkish journal of biology 30, 171–175. vairappan, c. s., 2003: potent antibacterial activity of halogenated metabolites from malaysian red algae, laurencia majuscule (rhodomelaceae, ceramiales). biomolecular engineering 20, 255–259. vairappan, c. s., suzuki, m., abe, t., masua, m., 2001: halogenated metabolites with antibacterial activity from the okinawan laurencia species. phytochemistry 58, 517–523. wardlaw, a. c., 1985: practical statistics for experimental biologists. john wiley and sons, chichester. wolcott, g. n., 1947: permanence of termite repellents. journal of economic entomology 40, 124–129. 90 acta bot. croat. 70 (1), 2011 manilal a., sujith s., sabarathnam b., kiran g. s., selvin j., shakir c., lipton a. p. u:\acta botanica\acta-botan 1-11\manilal.vp 28. o ujak 2011 16:13:52 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 79 (1), 2020 3 acta bot. croat. 79 (1), 3–14, 2020 coden: abcra 25 doi: 10.37427/botcro-2020-001 issn 0365-0588 eissn 1847-8476 the effect of salinity gradient and heavy metal pollution on arbuscular mycorrhizal fungal community structure in some algerian wetlands warda sidhoum1,2*, kheira bahi2,3, zohra fortas1 1 laboratory of microorganisms biology and biotechnology, university of oran 1 ahmed ben bella, oran, algeria 2 abdelhamid ibn badis university, mostaganem, algeria 3 department of biology, faculty of natural science and life, university of oran 1 ahmed ben bella, oran, algeria abstract – algerian natural wetlands suffer from anthropogenic disturbances due to industrial development and urbanization. this study was designed to draw attention to arbuscular mycorrhizal fungi (amf) distribution and community assemblages following heavy metal and salinity concentrations in two wetlands subjected to domestic and industrial effluents. rhizospheric soil and roots of 18 plant species were collected in two wetlands along a decreasing salinity gradient. the results showed that 72.72% of plant species exhibit an association within arbuscular mycorrhizas (am), and 36.36% a dual association between am and dark septate endophytes (dse). a total of 33 amf morphospecies were distinguished on the basis of morphological criteria dominated by taxa belonging to glomeraceae and acaulosporaceae. soil contamination was investigated by determining metallic trace elements (mte) (cd, cu, ni, pb, cr and zn) using an atomic absorption spectrophotometer. values of the pollution index revealed wetlands that were particularly polluted by lead. two-way anova showed significant variations in metal content among sampling locations and transects. principal component analysis showed that species richness, and mycorrhizal frequency were slightly affected by mte. this opens possibilities for their utilization in polluted soil remediation. keywords: dark septate endophytes, metallic trace elements, mycorrhizal association, saline wetlands, soil pollution * corresponding author e-mail: sidhoumwarda@yahoo.fr introduction oran is located in the north-west of algeria, and constitutes a wetland complex of eight zones, four of which are classified as of international importance. great sebkha and macta (since 2001), telamine lake (lt) and les salines d’arzew (since 2004), while others, though not be classified as ramsar, have nonetheless received attention from the ramsar convention (chenchouni and si bachir 2010). these wetlands are ecologically important ecosystems, providing important winter grounds for several world populations of endangered bird species. in particular, species belonging to the anas and tadorna orders, overwinter in significant numbers in these areas (boucheker et al. 2011, samraoui et al. 2015). the wetland soils are mostly solonchak types, containing large amounts of exchangeable sodium and soluble salts (benziane 2013). these habitats are characterized by the presence of flooded or water saturated soils for at least part of the growing season. these natural hydrosystems have halophylic plants, such as amaranthaceae (ghodbani and amokrane 2013, megharbi et al. 2016). however, these ecosystems can be modified by various factors, among them aridity causing changes in soil properties and trace element pollution. this is due to human activities, including, urbanization processes, domestic sewage discharges, livestock wastewater and industrial effluent (bouldjedri et al. 2011, domínguez-beisiegel et al. 2016). several studies have shown that arbuscular mycorrhizal fungi (amf) exist in the roots of wetland plants and woody species grown on flooded soils (d’souza and rodrigues 2013), in aquatic macrophytes, freshwater wetland plant communities and salt marshes (xu et al. 2016). it has also been shown that am fungal diversity in wetlands is comhttps://www.scirp.org/journal/articles.aspx?searchcode=laboratory+of+biology+of+development+and+differanciation%2c+department+of+biology%2c+faculty+of+natural+science+and+life%2c+university+of+oran+1+(ahmed+benbella)%2c+oran%2c+algeria&searchfield=affs&page=1&skid=0 sidhoum w, bahi k, fortas z 4 acta bot. croat. 79 (1), 2020 parable to that of most terrestrial ecosystems and for the growth and development of wetland plant species, and thus am fungi are functionally essential (tuheteru et al. 2015). the primary abiotic factors: soil flooding, nutrient, oxygen availability, salinity, and high levels of heavy metals in soil strongly affect the abundance and distribution of am fungi in aquatic ecosystems (millar and bennett 2016). as previously reported, the intraradical and extraradical mycelia of metallic stress adapted amf isolates are capable of sequestering heavy metals and alleviating metal toxicity to plants (cabral et al. 2015). this indicates that these fungi have evolved a tolerance to metallic trace elements (mte), and play a role in the phytoremediation of metal polluted sites, even in polluted aquatic and semi-aquatic habitats (wężowicz et al. 2015). a number of surveys on amf associated with wetland plants have been performed in order to investigate their diversity and colonization potential (d’souza and rodrigues 2013, kumar and muthukumar 2014) along a soil hydrological gradient (de marins et al. 2009, miller and bever 1999, turner et al. 2000), salinity (roda et al. 2008, saint-etienne et al. 2006, yang et al. 2010), or nutrient content (cornwell et al. 2001, jayachandran and shetty 2003). there has been a small amount of research focused on amf communities in mte polluted wetlands (carrasco et al. 2006, ban et al. 2017), but there has been no report of the simultaneous effect of salinity and heavy metals on amf distribution in wetland habitats. the present study, therefore, was aimed at evaluating the am fungal diversity in heavy metals polluted saline wetlands and at adding to knowledge on these populated areas with heterogeneous plant species that have rarely been considered mycorrhizal, as well as at exploring the impact of soil salinity gradient and trace element pollution on amf community structures. materials and methods study area this survey was carried out in two wetlands located in oran city (western region of algeria), namely telamine lake (lt) (35°42’50”n 0°23’30’’w) located in the district of gdyel (eastern oran) at 7 km from hassi amer industrial zone ii. dayet morsli (dm) (35°39’58”n 0°36’27”w) located in essénia district in the south of oran at a distance of 2 km north of the industrial zone i of es-sénia. the altitude ranged between 50 and 87 m a.s.l. the regional climate is of the semiarid mediterranean type characterized by a cold and rainy winter followed by a hot dry summer spread over 4 to 6 consecutive months where the average temperature varies between 14.1 °c and 22.5 °c and precipitation varies between 250 and 400 mm per year. sampling the investigations were conducted at each site (lt and dm) along four transects 200 m long and 10 m wide. starting from the wetland water edge to the periphery. each transect was divided into three plots according to the salinity gradient and plant distribution: from 0 m (where no vegetation was present) to 30 m, and electrical conductivity (ec) varying between 6 to 9.5 dsm–1, and 30 m to 60 m (2 dsm–1 <ec<6 dsm–1), and more than 60 m (ec<2 dsm–1). a total of 24 soil samples representing 12 plots in each wetland were selected. from each plot, one soil sample was used for chemical analysis. also, five replicates per plant species of rhizospheric soil and roots of dominant plant species were sampled as well. in order to establish am fungal diversity, about 500 g of rhizospheric soil was collected from topsoil (10 to 30 cm depth), put into plastic bags, air-dried and stored at room temperature at the laboratory until use. plant species encountered were recorded, with their relative abundances estimated visually and rated on a scale of i (very rare) to v (very abundant) (bradai et al. 2015). the determination of herbarium specimens for floristic inventory was carried out using flore de l’afrique du nord (maire 1958–1976), and a previous study used as a reference on saline wetlands vegetation of oran region (quézel and simonneau 1960). plant nomenclature was brought up to date according to the synonymic index proposed by dobignard and chatelain (2010-2013). chemical analyses of soil soil samples were dried at room temperature and sieved in a 2 mm mesh size sieve. dried soil samples were analyzed for ph and electrical conductivity on 1:2.5 (soil: distilled water suspension ratio) (mathieu and pieltain 2003). trace elements were extracted according to the aqua regia method iso 11466: 1995. about 0.5 g of dried soil at 105 °c was digested in 10 ml of freshly prepared aqua regia solution (1/3 of hno3 and hcl, v/v) on a hotplate for 2 h at 100 °c. after evaporation to near dryness, the extracts were cooled to room temperature before being filtered. the filtrates were further transferred to 25 ml volumetric flasks and brought to volume with distilled water, then stored at 4 °c until the spectrophotometric measurement using the aas shimadzu aa–7000 atomic absorption flame emission spectrophotometer. soil pollution index multiple contaminations by metallic trace elements increase soil toxicity. soil pollution index is the criterion that allows us to evaluate soil toxicity, to classify the soil contamination and to assess potential ecological risk (müller 1979, belabed et al. 2014). this index is calculated by the ratio of heavy metal content in the soil (ppm) based on the corresponding values, according to the following formula proposed by kloke (1979): pi cd cu pb zn cr ni = + + + + + 2 100 100 300 150 50 6 where pi>1indicates that the soil is polluted. amf diversity in wetland stresses acta bot. croat. 79 (1), 2020 5 assessment of amf and dse colonization young roots (with root tips) were washed in tap water to remove soil particles, and then fixed in faa formalin, glacial acetic acid, and ethanol (1:1:18, v/v/v). roots were washed several times in tap water, cleared in 10% (w/v) koh while heating to approximately 90 °c for 1h, acidified in 10% lactic acid, and stained with 0.1% trypan blue in lactophenol at 90 °c for 1h, according to a modified method of phillips and hayman (1970). for each root system, amf colonization was estimated by optical microscopy (olympus cx22) from 50 root fragments of approximately 1 cm in length. mycorrhizal development was evaluated according to the method of trouvelot et al. (1986), and expressed as mycorrhizal frequency (f%), intensity of colonization (m%), mycorrhizal intensity of colonized root fragments (m%), arbuscules abundance (a%) and arbuscules abundance of colonized root fragments (a%). in the case of dse colonization, microsclerotia and hyphae were scored collectively, and the frequency of dse occurrence in roots (dse%) was calculated as the ratio between colonized root fragments by dse and the total number of examined root fragments. isolation and taxonomic identification of am fungal spores the plants were carefully dug out from the soil and the majority of bulk soil was manually removed from the roots. only the soil closely attached to the root system was analyzed. am fungal spores were isolated via wet sieving (gerdemann and nicolson 1963) followed by soil centrifugation (1398 × g during 10 minutes at room temperature) in 50% sucrose solution and filtration (mesh size 40 µm) (brundrett et al. 1996). intact, noncollapsed spores, with cytoplasmic content were separated into groups according to general morphological similarities recorded under a stereomicroscope (leica ez4hd), and the diameter of spores was measured. also, permanent slides of all spores were prepared with the use of a drop of polyvinyl alcohol/lactic acid/glycerol (pvlg) mixed with melzer’s reagent (1:1, v/v) on a slide. species identification was based on the examination of spore morphological and subcellular characteristics. obtained results were compared to the descriptions of oehl et al. (2011), and redecker et al. (2013); other available descriptions are found on the sites www.agro.ar.szczecin.pl/~jblaszkowski/ and https://invam.wvu.edu/, while the nomenclature employed follows that used by the mycobank (www.mycobank.org). statistical analysis ecological measures of diversity used to describe the structure of amf communities included spore density (number of spores in 100 g of soil), species richness (number of identified amf species per soil sample), relative abundance (ra), isolation frequency (if), shannon’s diversity index (h’), evenness (e), simpson’s index of dominance (d), and sorensen’s coefficient (cs) (simpson 1949). the relative abundance refers to how common or rare a species is relative to other, and it was calculated from the formula: ra = spore number of a species genus total number of identifie ( ) dd spore samples ×10 isolation frequency was calculated following the formula: if = number of soil samples where a species genus total number ( ) of soil samples the shannon diversity index is a mathematical measure of species diversity in a community. h’ value was calculated according to the formula: h’ = -∑pi ln pi, where pi is the relative abundance of each identified species per sampling site and calculated by the following formula pi = ni/n, where ni is the number of spores of a species and n is the total number of all identified spores. the evenness also called “equitability”, refers to the homogeneity of the species, 0<e<1, e = 1 means that all species have the same frequency. it was calculated according to the formula e= h h max ’ ’ , where h’ max is the maximal h’ that can be determined by the following formula: h’ max = ln s, where s is the total number of identified species per sampling site. simpson’s index of dominance (d) measuring the probability that two randomly selected individuals in a community belong to the same species (0<d<1) was calculated from the formula: d = ∑[ni(ni – 1)/n(n – 1)]. sorensen’s coefficient was used to compare the similarity of two sites, and was calculated following the formula: cs = 2j/ (a+b), where a or b is the total number of identified species per sampling site and j is the number of identified species common to both sites. we determined the dominant amf species with respect to the if > 50% and the ra > 5%. all statistical analyses were performed using the spss software package (version 23.0). the data were analyzed by two-way anova with site (geographical location of the wetlands) and plots (the position in the transect) as main factors. multiple mean comparisons were performed using tukey’s hsd post hoc test at the 0.05 level of probability. a principal component analysis (pca) was performed in order to verify the environmental variables that best explain amf community structure. because the variables were measured in different units, a correlation test was used (all variables were normalized using division by their standard deviations). results species composition and abundance the floristic survey on sites reveals the presence of a variable phytodiversity comprising 46 vascular plant species (34 in dm, and 28 in lt) belonged to 44 genera and 21 families, thus indicating that dayet morsli was the most diversified site (tab. 1). moreover, flora was characterized by the dominance of taxa belonging to the families amaranthaceae (26.47% in dm and 25% in lt) and asteraceae (23.52% in dm and 17.85% lt), followed in dm by poaceae (17.64%), solanaceae (8.82%), malvaceae, liliaceae (5.88%), while 2.94% of the remaining species belonged to other families. https://invam.wvu.edu/ https://invam.wvu.edu/ sidhoum w, bahi k, fortas z 6 acta bot. croat. 79 (1), 2020 on the other hand, in lt the asteraceae were followed by poaceae and apiaceae (each represented with 7.14% of the species), and the rest of surveyed plant families (each represented with 3.57% of the species). soil chemical properties results of soil physicochemical analyzes (tab. 2) revealed an average ec value 4.72 dsm–1 in lt (2.07–7.98 dsm–1) and 4.42 in dm (0.2–9.5 dsm–1), in addition to a slightly alkaline ph varying from 7.3 to 8.5 in dm and from 7.33 to 8.31 in lt. overall, the results showed higher levels of heavy metals in dm topsoils than those found in lt. these values are greater than the maximum concentration foreseen in the environmental soil quality guidelines afnor nfu 44–041. a high level of pb was found at both sites, while cd and cr were present in dm only. the pollution index in dm is considerably greater with values exceeding 1, indicating that dm is a moderately polluted site. two-way anova showed significant differences between the investigated sites along transects in salinity degree (ec) (p < 0.001), cr (p < 0.001), as well as zn (p < 0.05) and cu concentrations (p < 0.01), which significantly and steadily decreased from sea to inland regions. also, significant differences were noticed between wetland sites for all mte concentrations (except zn), pollution index, and salinity (ni, cr, cu and pi at p < 0.001, pb (p < 0.01), cd and ec (p < 0.05)). no significant effect was observed in the interaction (site*plot) on zn and ni. the obtained results highlight the effect of distance from water which has a very important role in soil mte concentration. tab. 1. abundances of plant species in the wetlands dayet morsli and telamine lake, algeria, and their accession numbers at the plant ecology laboratory herbarium of the university oran 1 ahmed ben bella, oran, algeria. abundance classes: i – very rare, ii – rare, iii – occasional, iv – frequent, v – abundant (bradai et al. 2015). / = no accession number in the herbarium. family plant species herbarium accession no. dayet morsli telamine lake aizoaceae mesembryanthemum crystallinum l. 00790 ii i amaranthaceae chenopodium album l. 00739 ii i amaranthaceae arthrocnemum macrostachyum (monic.) k.koch 00745 iii iii amaranthaceae atriplex halimus l. 00727 iv iii amaranthaceae atriplex canescens (pursh) nutt. / . iii amaranthaceae sarcocornia perennis (mill.) a.j.scott 00747 iii iii amaranthaceae salicornia patula duval-jouve 00746 ii iii amaranthaceae suaeda vera forssk. ex j.f.gmel. 00767 iii iv amaranthaceae atriplex prostrata dc. 00720 i . amaranthaceae beta macrocarpa gus. 00732 i . apiaceae daucus carota subsp. carota (l.) thell. 01924 . iii apiaceae smyrnium olusatrum l. 01993 . ii arecaceae chamaerops humilis l. 00449 i i asteraceae scolymus maculatus l. 03033 i i asteraceae calendula stellata cav. 02824 i ii asteraceae centaurea pullata l. 02966 i ii asteraceae glebionis coronaria (l.) spach 02878 i . asteraceae limbarda crithmoides subsp. longifolia (arcang.) greuter 02764 . i asteraceae dittrichia viscosa (l.) greuter. 02763 i . asteraceae silybum marianum (l.) gaertn. 02941 ii . asteraceae taraxacum erythrospermum andrz. ex besser 03096 . ii brassicaceae sinapis arvensis l. subsp. arvensis 01146 i i cucurbitaceae ecballium elaterium (l.) a.rich. 02682 i . cynomoriaceae cynomorium coccineum  (l.) subsp. coccineum 01873 . i fabaceae trifolium tomentosum l. 01446 i . joncaceae juncus maritimus lamk. 00461 . i lamiaceae marrubium vulgare l. 02346 i ii liliaceae asphodelus tenuifolius cav. 00496 i . liliaceae asphodelus ramosus l. 00493 . ii malvaceae malva sylvestris l. 01819 ii i malvaceae lavatera multiflora (cav.) soldano et al. 01826 i . oxalidaceae oxalis pes-caprae l. 01683 . ii plantaginaceae plantago lagopus l. 02583 i . poaceae avena sterilis l. 00252 i . poaceae hordeum murinum l. subsp. glaucum (steud.) tzvelev 00386 ii ii poaceae anisantha madritensis (l.) nevski 00345 ii . poaceae hordeum maritimum with. subsp. maritimum / i poaceae phalaris canariensis l. 00143 ii . poaceae phragmites australis subsp. altissima (benth.) 00227 i . primulaceae lysimachia arvensis (l.) u. manns & andreb. 02112 i . rhamnaceae ziziphus lotus (l.) desf. 01802 . ii solanaceae datura stramonium l. 02437 i . solanaceae nicotiana glauca graham 02426 i . solanaceae solanum indicum l. / i . tamaricaceae tamarix sp. / . i urticaceae urtica pilulifera l. 00698 i i http://www.tela-botanica.org/bdtfx-nn-49948-synthese?referentiel=bdtfx&niveau=2&module=fiche&action=fiche&num_nom=101080&type_nom=nom_scientifique&nom=plantaginaceae amf diversity in wetland stresses acta bot. croat. 79 (1), 2020 7 fungal root colonization arbuscular mycorrhizae were observed in 72.72% of plant species since the dual association between am fungi and dse was found in 36.36% of plant species. one species (asphodelus tenuifolius) showed am, dse, and ectomycorrhizae (tab. 3). no am was noted in roots of some species: mesembryanthemum crystallinum, arthrocnemum macrostachyum, salicornia patula and tamarix gallica. the mean am frequency (f%) varied with particular species, ranging from 10% (beta macrocarpa and sarcocornia perennis) to 100% (calendula stellata and marrubium vulgare). am colonization intensity (m%) varied from 0.69% in sarcocornia perennis to 67.62% in centaurea pullata. the mean arbuscule richness (a%) was also diverse and being lowest in all amaranthaceae, in particular, atriplex canescens (0.93%) and highest in asteraceae, i.e. in centaurea pullata (60.61%) (tab. 3). arum type morphology was present in 76.47% of mycorrhized plants, 11.76% in paris type morphology and the rest were not identified as am morphology, their association being generally characterized by hyphal proliferation with few arbuscules. dse was found in 54.54% of collected plants, although the dse occurrence frequency in roots was high in the case of several taxa (tab. 3). several forms of mycelia were encountered in both the outer cortex and the rhizoderm. here, brownish, thick mycelium (4-5 μm) was developed into microsclerotia around the central cylinder or thin mycelium (2 μm) stained with trypan blue-lacking dark pigmentations. they subsequently formed hyphal complexes in cortical cells or aggregates to form a puzzle or brain-like microsclerotium and thick-walled mycelium for plate lobed and unlobed hyaline mycelium. other fungal endophytes were noted: the remaining olpidium spp. (chytridiomycota) spores found in asphodelus tenuifolius, beta macrocarpa, and atriplex halimus rhizodermis and outer cortex. chlamydospore-like structures in beta macrocarpa roots and fine hyphae with fanshaped branches and glomus tenue swellings (glomeromycota) were discovered in a. halimus roots. amf community composition and diversity on the basis of morphological criteria, 33 morphospecies were distinguished from analyzed site samples (tab. 4). am community was characterized by the presence of several taxa belonging to: glomeraceae (16 taxa) represented by five genera, glomus (5 spp.), rhizoglomus (4 spp.), funneliformis (3 spp.), sclerocystis (2 spp.) and septoglomus (1 sp.), acaulosporaceae (acaulospora 10 spp.), diversisporaceae (diversispora 2 spp. and tricispora 1 sp.), claroideoglomeraceae (claroideoglomus 2 spp.), archaeosporaceae (archaeospora 1 sp.), paraglomeraceae (paraglomus 1 sp.), and ambisporaceae (ambispora 1 sp.). however, fourteen morphospecies remained unidentified since their morphological characteristics did not fit with any description of known species. species richness of the sampled plant species varied in the rhizospheric soils, with maximum levels in dm (28 species) followed by lt (20 species). fourteen species are classified as general and were found at all sites. a total of 12 species were classified as exclusive. there were ten only in dm (acaulospora cf. collicosa, a. alpina, a. rehmii, diversispora sp. 1, divercispora tortuosa, tricispora nevadensis, glomus macrocarpum, g. diaphanum, sclerocystis rubiformis and rhizoglomus sp. 3) and 2 amf species that were restricted to lt (acaulospora sp. 3 and g. lamellosum). the highest number of spores in both sites belonged to acaulosporaceae (tab. 4), followed by archaeosporaceae and glomeraceae. in dm the glomeraceae were dominated by rhizoglomus (6.74%), glo-ta b. 2 . v ar ia tio n in tr ac e el em en t c on ce nt ra tio ns (p pm ) a nd so il pa ra m et er s m ea su re d in th e w et la nd s d ay et m or sl i a nd t el am in e la ke a t d iff er en t d is ta nc es fr om th e w at er ’s ed ge . e c – e le ct ri ca l c on du ct iv ity . c sq – c ri te ri a in so il qu al ity a cc or di ng to q ua lit y n or m es a fn o r n fu 4 404 1. d at a ar e m ea n va lu es ± s d , n = 4 . m ea ns in th e sa m e co lu m n w ith d iff er en t l et te rs a re si gn ifi ca nt ly d iff er en t fr om e ac h ot he r ( p < 0. 05 ) a cc or di ng to th e tu ke y– te st (n s = n ot si gn ifi ca nt , * p < 0 .0 5, * * p < 0. 01 , * ** p < 0 .0 01 ). t he n um be rs fo r s ite , p lo t, an d si te *p lo t o n th e ta bl e bo tt om a re th e va lu es o f t w ow ay a n o va f -s ta tis tic s. pl ot s – th e di st an ce fr om sa m pl in g ar ea a nd th e w at er e dg e (0 -3 0 m , 3 060 m , a nd > 60 m ). si te pl ot s ph ec (d sm –1 ) pb n i c d c r c u z n po llu tio n in de x d ay et m or sl i 030 8. 24 ±0 .2 8a 7. 36 ±0 .3 8a 72 1. 97 ±0 .0 0a 3 2. 07 ±0 .0 0a 5 .6 1± 0. 00 a 50 8. 87 ±0 .0 0a 1 18 .0 4± 0. 00 a 1 88 .0 ±0 .0 0a 2 .6 3± 0. 00 ab 30 -6 0 7. 79 ±0 .4 2a 5. 12 ±0 .6 5b 82 0. 01 ±4 5. 31 a 3 8. 02 ±3 .1 7a 8 .0 1± 1. 25 ab 29 8. 60 ±1 7. 55 b 1 18 .5 5± 6. 57 a 18 2. 00 ±7 .4 6a 2. 79 ±0 .2 0a >6 0 8. 00 ±0 .2 7a 0. 42 ±0 .1 7c 27 0. 32 ±4 39 .3 a 2 8. 96 ±1 0. 81 a 1 .8 4± 3. 59 b 9 7. 14 ±9 0. 44 c 42 .0 4± 46 .9 2b 9 8. 07 ±9 2. 30 a 0. 95 ±1 .2 2b te la m in e la ke 030 7. 45 ±0 .2 4a 7. 29 ±0 .7 5a 6 5. 91 ±5 4. 59 a 1 5. 60 ±5 .3 5a 1 .7 9± 3. 87 a 4 4. 65 ±2 5. 66 a 11 .3 3± 6. 29 a 14 9. 13 ±3 9. 85 a 0. 45 ±0 .3 0a 30 -6 0 7. 80 ±0 .3 1a b 2. 98 ±0 .9 3b 1 01 .4 ±1 63 .0 5a 1 5. 22 ±4 .2 7a 0 .4 2± 0. 48 a 2 4. 41 ±6 .8 3a 14 .1 5± 6. 63 a 16 6. 91 ±5 0. 26 a 0. 40 ±0 .2 5a >6 0 8. 01 ±0 .0 6b 0. 76 ±0 .4 6c 27 8. 23 ±3 91 .9 7a 7 .9 9± 8. 45 a 3 .1 7± 3. 45 a 1 5. 01 ±3 .5 4a 5 .6 ±1 .5 5a 5 3. 75 ±7 6. 01 b 0. 90 ±0 .2 8a c sq 10 0 50 2 15 0 10 0 30 0 1 si te 4. 21 n s 5 .1 5 * 4. 22 * * 45 .0 9 ** * 7. 7 * 16 1. 65 ** * 57 .0 8* ** 1. 59 n s 27 .4 0 ** * pl ot 0. 94 n s 19 2. 5 ** * 0. 88 n s 2. 40 n s 0. 65 n s 33 .9 7 ** * 5. 96 * * 5. 56 * 1. 92 n s si te *p lo t 4. 87 * 8 .0 4 ** 3. 91 * 0. 42 n s 4. 32 * 25 .3 2 ** * 4. 09 * 0. 12 n s 5. 91 * * sidhoum w, bahi k, fortas z 8 acta bot. croat. 79 (1), 2020 mus (1.66%), funnelifomis (1.21%), septoglomus (0.71%), and sclerocystis (0.34 %), while funnelifomis (5.98%), septoglomus (3.45%), glomus (0.37%), rhizoglomus (1.12%), and sclerocystis (0.45%) dominated in lt. the other families were not equally present in the studied sites. in dm, claroideoglomeraceae (2.29%) were more dominant than ambisporaceae (1.94%) and diversisporaceae (1.6%), but in lt, we found paraglomaceae (1.02%) then ambisporaceae (0.6%). spore density is higher in lt (1077.09/100 g of soil) than that seen in dm (640/100 g of soil). based on relative abundance and isolation frequency, six species were dominant in dm (acaulospora thomii, a. cf. morrowae, a. rehmii, a. leavis, a. melea and archaeospora undulata), whereas only three dominated in lt: a. thomii, a. leavis and ar. undulata (tab. 4). on the other hand, a few amf species had low relative abundances but high isolation frequency, such as the species in dm: a. leavis (6.96% ra, 100% if), a. melea (6.18% ra 100% and 80% if), and tricispora nevadensis (1.23% ra and 71.42% if). ra of claroideoglomus claroideum is 2.29 % in dm and 2.75 in lt, while if is 85.71% in dm and 60% lt. in addition, ra and if of some other species in lt are: f. mosseae (0.75%, 60%), se. constrictum (3.45%, 60%), and a. cf. morrowae (4.34%, 60%). two-way anova showed (table 4) the existence of significant negative effects in am frequency (p < 0.001), species richness (p < 0.01), and spore density (p < 0.01) under increasing gradient salinity (transect from water to inland), while dse was affected positively (p < 0.001). moreover, only spore density was significantly different (p < 0.01) among wetland sites. no significant effect was noted of the interaction (site*plot) on species richness. in general, the mean values of the shannon index showed that dm was slightly more diversified (1.69 ± 0.33) than lt (1.32 ± 0.29). calculation of the am fungal community evenness indicates a more equitable distribution among am fungal communities found in dm (0.73 ± 0.13) than that found in lt (0.60 ± 0.12). simpson’s index was higher in lt (0.24 ± 0.09) than dm (0.19 ± 0.06) reflecting a slight am fungal species dominance among am fungal communities. sorensen’s coefficient indicated 58% of similarity in amf community composition between wetland sites. no significant differences were noted in diversity indices affected by sites or the (site*plot) interaction except for dominance index (f = 4.25, p < 0.05) which was significantly increased with salinity gradient (tab.5). multivariate analysis eigenvalues of the two first principal components explained a large proportion (component 1 43.58% and component 2 22.53%) of the total variance (fig. 1). component 1 was strongly correlated with heavy metals values and dse frequency, which showed positive correlation coefficient values (0.52 to 0.96). these metal concentrations were highly correlated with each other except for zn (0.66 ≤ r ≤ 0.96; p < 0.001). the component 2 was more correlated with ph (0.54), f% (0.67), ec (0.77), spore density (–0.54), species richness (–0.75), and zn (–0.63). tab. 3. fungal root endophyte colonization of plant species in the wetlands dayet morsli (dm) and telamine lake (lt). arbuscular mycorrhizal types: p – paris-type, a – arum-type, -: not determined, nm – non mycorrhizal, e – ectomycorrhizal, mycorrhizal frequency f% – the ratio between colonized root fragments by arbuscular mycorrhizal fungi and the total number of examined root fragments, m% – mycorrhizal intensity of colonized root fragments, m% – the colonization intensity in all root system, a% – arbuscule abundance of colonized root fragments, a% – arbuscule abundance of all root system, dse% – the ratio between colonized root fragments by dark septate endophytes and the total number of examined root fragments. family plant species location am type mycorrhizal frequency f% m% m% a% a% dse % aizoaceae mesembryanthemum crystallinum dm nm 0 0 0 0 0 70 amaranthaceae arthrocnemum macrostachyum dm nm 0 0 0 0 0 0 atriplex canescens lt a 48 7.14 14.88 13.07 0.93 8 atriplex halimus lt a 58.49 18.74 32.03 11.54 2.16 13.2 atriplex halimus dm a 57.25 21.63 38.44 10 2 12 beta macrocarpa dm – 10 3.4 5.63 5.76 0.2 72 salicornia patula dm nm 0 0 0 0 0 0 sarcocornia perennis lt – 1.82 0.02 1 0 0 7.27 sarcocornia perennis dm – 10 0.69 1.35 0 0 28 suaeda vera lt a 36 8.16 22.67 21.69 1.77 4 suaeda vera dm a 76.19 28.9 39.16 41.26 11.93 0 asteraceae silybum marianum dm a 93.55 8.53 8.93 34.9 2.92 0 calendula stellata dm p 100 61.27 61.27 85.26 52.25 0 centaurea pullata lt a 98 67.62 69 89.64 60.61 46 dittrichia viscosa dm p 89.66 56.12 62.6 81.8 45.91 limbarda crithmoides subsp. longifolia lt a 87.88 31.03 35.31 93.38 28.98 12 scolymus maculates dm a 45 13 28.89 64.67 8.41 0 juncaceae juncus maritimus lt a 60 9.06 15.1 3.89 0.35 0 lamiaceae marrubium vulgare lt a 98.08 48.67 49.3 88.14 42.8 0 liliaceae marrubium vulgare dm a 100 52.21 52.21 70.76 36.94 0 asphodelus tenuifolius dm e a 10 2.5 0 0 0 0 27.5 tamaricaceae tamarix sp. lt nm 0 0 0 0 0 30 amf diversity in wetland stresses acta bot. croat. 79 (1), 2020 9 tab. 4. the relative abundance of arbuscular mycorrhizal fungi (amf) isolates in the wetlands dayet morsli (dm) and telamine lake (lt). values are expressed as mean values ± standard deviation, n = 12. ra – relative abundance (%), if – isolation frequency (%), sr – species richness, average sr – the mean of species number in all plant rhizospheres for each site. sd – spore density. amf species ra dm (%) if (%) ra lt (%) if (%) acaulosporaceae 48.11 – 31.86 acaulospora cf. collicosa 0.29±0.77 14.28 – – acaulospora cf. alpina 0.09±0.24 14.28 – – acaulospora thomii 12.23±5.51 100.00 15.94±3.96 100.00 acaulospora leavis 6.96±4.20 100.00 6.09±5.74 60.00 acaulospora melea 6.18±5.47 100.00 4.55±5.39 80.00 acaulospora tortuosa 0.05±0.12 14.28 – – acaulospora aff. morrowae 11.58±7.58 100.00 4.34±3.89 60.00 acaulospora rehmii 10.88±9.04 85.71 – – acaulospora sp. 1 – – 0.6±0.89 40.00 acaulospora elegans – – 0.32±0.71 20.00 ambisporaceae 1.94 – 0.6 ambispora reticulata 1.94±4.59 28.57 0.6±1.34 20.00 archaeosporaceae 15.67 – 18.39 archaeospora undulata 15.67±15.62 71.42 18.39±15.63 80.00 diversisporaceae 1.6 – – – diversispora sp. 0.09±0.24 14.28 – – diversispora tortuosa 0.28±0.65 28.57 – – tricispora nevadensis 1.23±1.45 71.42 – – claroideoglomeraceae 2.29 – 2.75 – claroideoglomus claroideum 2.29±2.07 85.71 2.1±3.12 60.00 claroideoglomus lamellosum – – 0.65±0.99 40.00 glomeraceae 10.53 – 12.43 funneliformis caledonium 0.38±1.00 14.28 1.36±2.31 40.00 funneliformis geosporum 0.03±0.07 14.28 3.56±6.43 40.00 funneliformis mosseae 0.80±1.24 42.85 1.06±1.46 60.00 glomus achrum – – – – glomus nanolumen 0.62±0.82 42.85 0.37±0.51 40.00 glomus macrocarpum 0.64±1.70 14.28 – – glomus diaphanum 0.25±0.67 14.28 – – glomus sp. 1 0.15±0.40 14.28 – – rhizoglomus microagregatum 0.23±0.42 28.57 0.2±0.27 40.00 rhizoglomus sp. 1 2.91±7.27 28.57 0.42±0.93 20.00 rhizoglomus sp. 2 3.42±9.07 14.28 0.50±1.13 20.00 rhizoglomus sp. 3 0.18±0.49 14.28 – – sclerocystis rubiformis 0.03±0.07 14.28 – – sclerocystis sp.1 0.11±0.30 14.28 0.45±0.63 40.00 septoglomus constrictum 0.71±1.49 28.57 3.45±5.30 60.00 paraglomeraceae – – 1.02 – paraglomus sp. – – 1.02±1.43 40.00 unidentified 16.93±23.45 – 33.81±31.81 – total 100 100 sr 28 20 average sr 10.43±2.37 10.2±4.6 sd (100 g soil) 640.3±269.9 1077.9±364.16 it is worth noting that along the component 1, the variables were clustered in three main groups. the etm formed a distinct group within dm site and the nearest plots to the water (0–30 and 30–60). this group was negatively correlated to amf spore density which recorded the highest level near to the water (plots 0–30 and 30–60) in telamine lake site. the third group gathering species richness and mycorrhizal frequency in both sites and in plot (> 60) was strongly reduced by both salinity and zinc concentration. discussion in this study, we present a detailed report on fungal root endophyte occurrence in eighteen plant species. heavy metal contents in soil and their effect on the fungal composition and root colonization was also investigated. the most represented families of the two study sites are amaranthaceae and asteraceae, whose dominance in wetlands was reported by several studies (megharbi et al. 2016, neffar et al. 2013). halophytic species of amaranthaceae and asteraceae are known to be naturally better adapted to survive environmental stresses, compared to salt-sensitive crop plants commonly chosen for phytoremediation purposes as described by manousaki and kalogerakis (2011). research findings suggest them as ideal candidates for phytoremediation of both saline and non-saline, heavy metal polluted soils, such as atriplex halimus (mesnoua et al. 2016), mesembryanthemum crystallinum (lutts and lefèvre sidhoum w, bahi k, fortas z 10 acta bot. croat. 79 (1), 2020 2015), atriplex canescens, suaeda vera (ayyappan and ravindran, 2014), calendula officinalis l. (hristozkova et al. 2016), marrubium vulgare (belabed et al. 2014), limbarda crithmoides subsp. longifolia and dittrichia viscosa (turnau et al. 2010). the floristic survey showed that dm site has more diversified plant species than lt (34 plant species in dm, and 28 in lt), while the flora has not been affected, despite the high pb, cr and cd concentrations in the soil, due to the presence of different native heavy metal accumulator plants. soil salinity values were heterogeneous, and interestingly, salinity is the critical factor controlling the distribution of plant communities within the study sites, corroborating those of several algerian wetland studies (aliat et al. 2016, chenchouni 2017). the current study shows that the levels of pb (270–820 ppm) in dm and (101–278 ppm) in lt), cd (1.84–8 ppm in dm and 1.79–3.17 in lt), cu (118 ppm in dm) and cr (97.14–508.87 ppm in dm) recorded exceeded afnor nfu 44–041 reference values (100, 2 and 150 ppm, respectively), and are higher than those reported by several authors of wetland soil studies (teuchies et al. 2013, belabed et al. 2017, esmaeilzadeh et al. 2017). in addition, nickel and zinc concentrations in dm and lt soils in the present study are homogeneous and vary only slightly. they do not exceed the reference values, but the highest values for ni and cu are recorded in dm and the highest zn value is found in lt. similar values were reported in algerian wetlands near to industries, including elhadjar iron and steel complex (belabed et al. 2017, louhi et al. 2012). on the other hand, tab. 5. changes in fungal colonisation, fungal spore density, species richness and diversity indexes along transects at the dayet morsli and telamine lake wetland sites. results are expressed as mean values ± standard deviation, n = 4. means in the same column with different letters are significantly different from each other (p < 0.05) according to the tukey–test (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001). plots – the distance from sampling area and the water edge (0-30 m, 30-60 m, and >60 m), dse% – dark septate endophytes frequency, f% – mycorrhizal frequency, sr – species richness, sd – spore density, h’ – shannon index, d – dominance index, e – evenness. the numbers for site, plot, and site*plot on the table bottom are f-statistics values of two-way anova analysis of variance. sites: telamine lake and dayet morsli. site plots dse (%) f (%) sr sd h’ d e dayet morsli 0-30 70.00±0.00a 0.00±0.00a 9.00±0.00a 125.00±0.00a 1.50±0.30a 0.25±0.03a 0.69±0.13a 30-60 12.00±0.00b 47.08±17.39b 10.66±3.05ab 580.66±298.57a 1.45±0.18a 0.22±0.09a 0.61±0.04a >60 2.58±6.01c 96.63±4.79c 13.42±1.61b 542.85±240.17a 1.72±0.33a 0.18±0.05a 0.76±0.14a telamine lake 0-30 11.86±11.63a 50.49±17.40a 10.00±0.63a 1209.38±519.85a 1.50±0.28a 0.28±0.10a 0.72±0.07a 30-60 5.83±6.88a 62.02±18.22a 9.00±2.44a 1156.66±368.16a 1.41±0.43a 0.18±0.05a 0.57±0.14a >60 36.00±50.9a 54.00±62.22a 12.00±0.00a 480.00±28.284a 1.64±0.41a 0.14±0.02a 0.70±0.13a site 3.27ns 0.85ns 0.84ns 12.77** 0.07ns 0.17ns 0.23ns plot 11.96*** 12.20*** 6.65** 6.65** 1.00ns 4.25* 2.72ns site*plot 21.19*** 10.27** 1.33ns 4.73* 0.02ns 0.56ns 0.29ns fig. 1. principal component analysis (pca) ordination biplot of 24 soil samples in the wetlands telamine lake (lt) and dayet morsli (dm), algeria, and the following variables: spore density (sd), f (mycorrhizal frequency), dse (dark septate endophytes frequency), species richness (sr), ph, electrical conductivity (ec), pollution index (pi), and trace elements cr, cd, cu, ni, pb, zn. amf diversity in wetland stresses acta bot. croat. 79 (1), 2020 11 pca test showed a good correlation between the different total metal contents except for zn, suggesting a common origin as confirmed by carrasco et al. (2006). the presence of these metals in stations located close to industrial areas confirms their anthropogenic origin. according to vardanyan et al. (2008), very high local concentrations of metals often occur as a result of a strong reducing environment coupled with industrial and municipal discharges. the variation of mte between sites was significant at p < 0.001. however, their variation between plots was not significant, except for cr, cu, and zn which decreased inversely with salinity, indicating that they might arise from flood water. elevated metal (pb, zn, and cr) contents and toxic phosphate levels in dm are likely to impose toxic effects for plant establishment in addition to other constraints, but it is difficult to prove mte toxicity because it depends on their soil availability which is generally reduced. our data also showed higher mycorrhizal frequency in asteraceae varied with particular species, that confirms several previous studies, showing high total colonization in centaurea stoebe (gucwa-przepiora and blaszkowski 2007) and calendula officinalis (hristozkova et al. 2016). these results may explain the considerable effectiveness of calendula officinalis and m. vulgare extensively mycorrhized in the phytoremediation of heavy metals (hristozkova et al. 2016). as the genus asphodelus is known to form am (cavagnaro et al. 2001), we detected in our study the presence of am, ectomycorrhizae and dse in a. tenuifolius roots. the microsclerotes (7.5%) and sporangia (27.5%) observed are reported here for the first time. this seems to be a kind of plant species adaptation to environmental stress caused by salinity, inundation and metal pollution. in this study, tamarix sp. was not mycorrhized but bencherif et al. (2015) reported 16 to 65% of am frequency in tamarix articulata vahll in algerian saline soils. no mycorrhization was found in salicornia patula and arthrocnemum macrostachyum roots which confirm the results of landwehr et al. (2002), in saline, sodic and gypsum soils. arbuscule rates in amaranthaceae were very low (a<12%), like those reported by becerra et al. (2016). plenchette and duponnois (2005) hypothesized about the existence of a third am morphological type with no arbuscules in the amaranthaceae family. the high incidence (>50%) of dse in the present study is in accordance with several reports on their great abundance in highly stressed ecosystems (schmidt et al. 2008), as in wetlands (kumar and muthukumar 2014) and in heavily polluted soils (čevnik et al. 2000). additionally, the findings showed that dse colonization is positively correlated with cr and cu concentration in soil, indicating that these fungal endophytes infect plant root systems occupying highly cr and cu stressed areas, or that plants might utilize them as an adaptation mechanism to resist abiotic stress in cr and cu polluted soil, due to their role in the accumulation of some trace elements, and the amelioration of the growth of some plants (wang et al. 2016). dse correlated negatively with mycorrhizal frequency in the investigated plant root systems, indicating the important competitive and antagonistic interaction between the two fungal endophytes, like those found by (kandalepas et al. 2010), confirming our result. according to the same authors, dse could be more resistant to adverse environmental conditions than mycorrhizal fungi by being either more competitive in disturbed or moderately polluted soils or better equipped to survive. the distribution of am fungal species of the current study showed a great amf species richness, recorded in both dm and lt (28 and 20, respectively). these results are comparable to those of wang et al. (2011) (23 phylotypes) and de marins et al. (2009) (27 morphotypes). the dominance of glomeraceae and acaulosporaceae was also reported by previous studies of wetlands (sun et al. 2016). this dominance is owing to their ability to propagate via mycelial fragments, mycorrhizal root fragments and spore germination (yang et al. 2015), and adaptation to stressful conditions of soil contamination with heavy metals compared with other amf species metals (lopes leal et al. 2016). septoglomus constrictum, funneliformis mosseae and funneliformis geosporum isolated from study sites were also isolated in algerian saline areas (bencherif et al. 2015). these fungi and others encountered in our investigation (cl. claroideum, a. mellea and ar. undulata) were also recorded in saline or heavy metal-polluted areas (hammer et al. 2011). am frequency cannot be affected by spore density, unlike species richness, and hence the infective amf are not automatically those producing a greater proportion of spores, but an increase in fungal colonization results from a greater amf species diversity in root systems. these results are in line with those of de marins et al. (2009) done in wetlands and hence they confirmed that spores occur both in the rhizosphere of macrophytes whose roots were internally colonized by amf and in non-colonized macrophytes. however, mycorrhizal roots and extraradical mycelia in the soil also could be involved in plant root infection. on the other hand, salinity strongly affects am plant status and species richness, indicating that mycorrhizal colonization and diversity are indeed reduced with increasing salt levels, as with those previously found by füzy et al. (2008). no correlation is observed between spore density and salinity, because they are halotolerant and more adapted to soil salt levels in these areas. this result suggest that the soil salt concentration is not sufficiently high to affect negatively the sporulation in these amf species and that probably they develop their own ability to survive and naturally occur in saline environments (hammer et al. 2011). inversely there are reports of low amf spore density in saline areas (barrow et al. 1997) while aliasgharzad et al. (2001) suggest that higher fungal spore density in saline soils may be due to the fact that sporulation is stimulated under salt stress, and this means the possibility of producing spores by amf under low root-colonization levels in severe saline conditions. sidhoum w, bahi k, fortas z 12 acta bot. croat. 79 (1), 2020 in addition, heavy metal pollution rates including pb, ni, cu, and cr concentrations disturb spore density but not mycorrhizal status, except for cr which may inhibit root hyphal colonization. these results are in agreement with those of vogel-mikuš et al. (2005), indicating that the elevated concentrations of as, pb, zn, cd, and cu exerted harmful effects on amf spore numbers in polluted plots. heavy metals have been reported to reduce, delay, or even eliminate amf colonization and spore density at heavy metal polluted sites (wei et al. 2015). salinity seems to affect amf diversity and infectivity, but not sporulation, which is more influenced by heavy metal pollution. mte may play a major role in the distribution of spore density. in sum, this is the first report on algerian am diversity in saline and contaminated wetlands with heavy metals. it revealed a specific plant community and environmental stress, including high salinity as well as trace element pollution. the salinity gradient and etm pollution were found to be the dominant factors affecting am community structures. both amf diversity and infectivity decreased with the increase in soil salinity, while am fungal sporulation rates were attenuated by mte augmentation. this native 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the land-use pattern is based on extensive agricultural systems and only 10% is covered by forests that are mainly composed of oak woods. it is the region in italy showing the highest number of oak species, among which four putative species of the quercus pubescens group, have been reported in floras and checklists with uncertain taxonomic value because of the overlapping of diagnostic characters. in this paper, we carried out a molecular analysis on natural populations of q. pubescens s.l. distributed throughout the apulia region. individuals from 24 pubescent oak populations were sampled and each tree was genotyped at 11 polymorphic microsatellite markers. overall, the average expected heterozygosity (he) was 0.629, and the allelic richness (ar) ranged between 2.130 and 7.187. no differentiation was observed among the populations investigated, and the genetic differentiation coefficient (fst) was 0.036. gene flow among populations was found to be relatively high (nm = 6.664). from a taxonomic point of view, the possibility of the coexistence of more than one species among the apulian pubescent oaks reported in the taxonomic and syntaxonomic literature is not supported by the results of this molecular analysis. key words: genetic diversity; italy; population structure; quercus pubescens; ssr; taxonomy * corresponding author e-mail: fortini@unimol.it introduction the genus quercus l. subgenus quercus has a wide distribution in the northern hemisphere, especially in central and southern europe, where quercus species form important forest communities in the temperate and the mediterranean bioclimatic regions (nixon 1993, govaerts and frodin 1998). italy is the country showing the highest oak species diversity in europe, although there is still incomplete agreement on the exact number of oak taxa occurring in the territory. most of the taxonomic uncertainty regarding the italian (and european) oaks concerns the so-called white oaks and in particular the pubescent oaks (quercus pubescens s.l. subgen. quercus sect. quercus). the most recent italian flora and checklists (pignatti et al. 2017, bartolucci et al. 2018) report various pubescent oaks in southern italy (q. amplifolia guss., q. apennina lam., q. congesta c. presl, q. dalechampii ten., q. leptobalana guss., q. ichnusae mossa, bacch. et brullo, q. humilis mill., q. virgiliana ten.) which are all considered steno-mediterranean vicariant species of q. pubescens willd. apulia is the easternmost italian administrative region and all italian oak species, both evergreen and deciduous, occur in this region with the exception of q. petraea (matt.) liebl. despite this richness in oak species and a wide potential range for the oak woodlands, only 10% of the apulian territory is covered by forests. the q. pubescens s.l. forests are the most widespread and can be found in the flooded depressions of the plain, in the limestone plateaus and on the sub-montane rocky slopes. molecular marker studies have not yet been published for the genus quercus in the apulian region. however, the di pietro r, di marzio p, antonecchia g, conte al, fortini p 16 acta bot. croat. 79 (1), 2020 evaluation of genetic variation can be of great importance for the conservation and management of forest ecosystems, especially in areas particularly vulnerable to climatic change (peñuelas et al. 2017) and in forests whose original extent has been significantly reduced. this is the case of the oak forests of the apulia region. in fact, the dry mediterranean climate that characterizes this region places it at risk of desertification if the climate should experience a further increase in aridity. moreover, the widespread practice of extensive agriculture (wheat, olive and vine) during the last century led to the destruction of most of the natural forest resources (biondi et al. 2010). several reports on the population genetics of the white oaks q. robur l., q. petraea, q. pubescens have been published for south-eastern europe in the last two decades (franjić et al. 2006, curtu et al. 2007a, jerše and batič 2007, slade et al. 2008, curtu et al. 2009, ballian et al. 2010, enescu et al. 2013, gailing et al. 2013). in italy, only few data are available. fineschi et al. (2002) and fineschi and vendramin (2004) presented a study on the genetic diversity of the italian white oaks analyzing chloroplast dna. subsequently, several studies that compared leaf morphology and molecular data on some white oaks populations of central italy have been published (fortini et al. 2009, 2013, 2015). in the most recent papers published on the apulia forests (biondi et al. 2004, di pietro and misano 2009) the following pubescent oaks were reported in the phytosociological tables: q. pubescens, q. virgiliana, q. amplifolia and q. dalechampii. a recent study (di pietro et al. 2016) statistically analyzed 25 morphological characters of leaves and fruits within 24 pubescent oak populations in the apulia region, in order to identify possible diagnostic traits useful for taxonomic differentiation. the results provided no evidence for differentiation among tree individuals based on comparisons of morphological traits. in the present study plant material from the same pubescent oak populations was used to analyze their genetic diversity, structure and gene flow. the aims of this study are twofold. first, to assess whether any group of genetic structure could be identified among oak individuals and/or populations, notwithstanding the morphological and taxonomical uniformity highlighted in the previous morphological work (di pietro et al. 2016). second, to investigate the pattern of genetic diversity in the apulian pubescent oaks in order to provide useful information for further studies. material and methods study area the apulia region is located in the south-eastern part of the italian peninsula where it is largely open to the adriatic and ionian seas. it is slightly sloping, with more than 60% of the territory occurring below 200 m a.s.l. five physiographic units can be distinguished in the apulia region and these are the daunian sub-apennine (1150 m), the gargano promontory (1080 m), the murgian plateau (680 m), the salento peninsula and the large plain tavoliere delle puglie. the bedrock of the mountainous systems is mainly composed of cretaceous limestone and paleocenic calcarenites. marls occur in the lower parts of the daunian sub-apennine and in the tavoliere plain together with conglomerates and pleistocenic and holocenic sand deposits. the bioclimate is thermo-mediterranean and meso-mediterranean with the temperate region occurring only in the sub-and lower-montane belts. the rainiest areas are the gargano, the daunian sub-apennine and the south-eastern part of the salento peninsula (slightly over 800 mm year–1). annual precipitation values on average of less than 500 mm year–1 are recorded in the western side to the murgian plateau and in the tavoliere plateau whereas the remaining portion of the territory exhibits an average annual rainfall of between 500 and 700 mm year–1 (blasi and michetti 2007, cotecchia et al. 2014). as far as the climax vegetation is concerned almost the entire apulian territory would potentially be covered by oak forests except for some scattered coastal and subcoastal areas dynamically linked to the pinus halepensis mill. woods and maquis, and the inner part of the gargano promontory where the wide “foresta umbra” beech wood occurs (biondi et al. 2004, 2010). population sample information individual mature oak trees were randomly selected in each population trying to maintain a distance of at least 20 meters from each other. leaves were randomly collected from 379 individuals within 24 populations (subdivided in squares 50 × 50 m), which covered approximately uniformly the physiographic units of the apulia region (fig. 1, tab. 1, on-line suppl. tab. 1). both the individuals and the populations they belong to are the same on which the morphological analysis of leaves and acorns was performed in the paper by di pietro et al. (2016). the collected oak specimens were dried on silica gel, and stored at room temperature until analysis. voucher specimens for each individual sampled were stored at the herbarium of the university of molise. dna extraction, est-ssr amplification total genomic dna was extracted from 0.5 g of dried leaves from all samples using spin columns of “invisorb® spin plant mini kit” and following the protocol of the manufacturer. eleven microsatellite loci or simple sequence repeats (est-ssrs), developed by durand et al. (2010) (pie020, pie102, pie152, pie215, pie223, pie227, pie239, pie242, pie243, pie267, pie271) and polymorphic in white oaks (guichoux et al. 2011, antonecchia et al. 2015) were selected. polymerase chain reaction (pcr) amplification was performed in a single multiplex reaction in a dna engine tetrad (mj research bio-rad) thermocycler and pcr products were run on an abi 3730xl capillary sequencer (applied biosystems) using genescan 600 liz internal size standard (applied biosystems). alleles were scored using str and software version 2.3.106 (toonen and hughes 2001) and alquercus pubescens complex in southern italy acta bot. croat. 79 (1), 2020 17 fig. 1. locations of the 24 oak collection stands. populations. est-ssr amplification was unsuccessful for 41 of the 379 individuals sampled. two populations (pop23 and pop24) were not included in the statistical analyses because of the low number of amplified individuals. the basic statistics for each locus: number of alleles (na), number of individuals (n), observed heterozygosity (ho), expected heterozygosity (he), and number of private alleles (pa) were computed using the statistical program genalex version 6.503 (peakall and smouse 2012). lele binning was performed as described in guichoux et al. (2011). microchecker (van oosterhout et al. 2004) was used to check for the presence of null alleles and to check for potential problems related to allele dropout with 1000 randomizations and a 95% confidence interval. data analysis polymorphic est-ssr markers were used to analyze the genetic diversity of 338 q. pubescens s.l. individuals in 24 tab. 1. quercus species composition of the 24 oak populations (according to biondi et al. 2004, biondi and guerra 2008, di pietro and misano 2009) with number of genotyped individuals and coordinates. population code analyzed individuals genotyped individuals latitude (n) longitude (e) quercus species pop01 16 16 4524805 650840 q. pubescens pop02 16 16 4557220 526723 q. pubescens pop03 16 16 4624669 567854 q. pubescens pop04 16 16 4624043 561581 q. virgiliana, q. dalechampii, q. pubescens pop05 16 15 4562416 534533 q. virgiliana, q. dalechampii, q. pubescens pop06 16 15 4564231 529921 q. pubescens pop07 16 16 4558496 530175 q. pubescens pop08 16 16 4512469 618357 q. pubescens pop09 18 16 4497035 647716 q. pubescens pop10 16 16 4582747 553553 q. pubescens, q. dalechampii, q. virgiliana pop11 16 16 4551101 622237 q. virgiliana, q. dalechampii, q. amplifolia pop12 15 15 4504016 656599 q. pubescens pop13 12 12 4491270 658881 q. pubescens, q. dalechampii, q. virgiliana pop14 16 16 4446957 278106 q. virgiliana, q. amplifolia, q. dalechampii pop15 15 15 4514201 647317 q. pubescens pop16 16 11 4502657 686693 q. pubescens pop17 16 13 4534971 632507 q. pubescens, q. dalechampii, q. virgiliana pop18 16 14 4523011 649110 q. dalechampii, q. virgiliana, q. pubescens pop19 16 14 4499728 654902 q. pubescens pop20 17 14 4635503 582284 q. pubescens, q. dalechampii, q. virgiliana pop21 16 16 4504890 744270 q. virgiliana, q. dalechampii, q. pubescens pop22 14 11 4518821 650238 q. virgiliana, q. dalechampii, q. pubescens pop23 16 6 4540867 597285 q. pubescens pop24 16 7 4635677 587298 q. pubescens di pietro r, di marzio p, antonecchia g, conte al, fortini p 18 acta bot. croat. 79 (1), 2020 value were done to test the consistency of the results. each (k) value was tested from k = 1 to k = 10. the optimum k value was predicted using the web-based software structure harvester web version 0.6.94 which implements the evanno method (earl and vonholdt 2012). individuals with probabilities above 0.80 (q ≥ 0.800) were assigned as putative purebred while all the other individuals were assigned as “putative hybrids” although the term “hybrid” could be a little misleading since no plant material of possible parental species was collected. for this reason we have opted for using the much more neutral term “off-type” in the rest of the text. bottleneck software version 1.2 (cornuet and luikart 1996) was used to detect the likelihood of a bottleneck effect. heterozygosity excesses were displayed, identified based on the estimates of multilocus genotypes calculated by using the wilcoxon signed rank tests and evaluating departures from the ratio 1:1 deficiency/excess (cornuet and luikart 1996, luikart and cornuet 1998, piry et al. 1999). a two-phase mutation model (tpm) and a stepwise mutation model (smm) for wilcoxon signed-rank tests were used considering a 90% smm proportion and tpm with a variance of 10, and 1000 repeats. results ssr polymorphism and genetic diversity all the loci were polymorphic (pj = q ≤ 0.99). the total number of alleles identified was 100 out for the 325 individuals analyzed. the allele fragment sizes (tab. 2) matched with the relative ssr reference size, as described in guichoux et al. (2011). the number of alleles per locus (k) ranged between 4 and 17, with an average of 11 alleles. the pic values varied from 0.233 (pie227) to 0.879 (pie152). according to (hildebrand et al. 1992) eight est-ssr loci were found to be informative (pic ≥ 0.5), whereas two were found to be slightly informative (pic < 0.4). the observed heterozygospolymorphic information content (pic), deviation from hardy-weinberg equilibrium (hw), and null allele frequency (f(null)) were computed for each locus using cervus software version 3.0.7 (kalinowski et al. 2007). the basic statistics for each population: number of different alleles, number of effective alleles, allelic richness, observed heterozygosity, expected heterozygosity, index of fixation (fis) calculated as f = 1 – (ho / he) and number of private alleles, were computed using arlequin software version 3.5.2.2 (excoffier and lischer 2010). allelic richness per locus and population were computed using fstat version 2.9.4 (goudet 2003). analysis of molecular variance (amova) was performed using genalex in order to partition the total microsatellite diversity, among and within populations, and within the individuals of each q. pubescens s.l. population, based on 11 est-ssr markers loci. the variance components were statistically tested by non-parametric randomization tests of significance based on 1000 bootstraps. gene flow among populations was estimated using the indirect method based on the number of migrants per generation (nm) using the formula, nm = 0.25 (1 − fst) / fst (slatkin and barton 1989). to investigate population differentiations, pairwise fst between all pairs of populations was computed, fst was calculated according to weir and cockerham (1984) in program fstat 2.9.4. principal coordinate analysis (pcoa) with data standardization based on nei’s genetic distance was performed using genalex. bayesian clustering analysis, using structure software version 2.3.4 (pritchard et al. 2000, 2010), with population ids as sampling location information, has been carried out. we opted for the admixture with correlated allele frequencies model between populations. we used a burn-in period of 25,000 and a number of mcmc of 100,000 cycles. ten runs for each k (k = number of possible clusters) tab. 2. allelic diversity of eleven microsatellite loci scored in 325 individuals of quercus pubescens s.l.; s – size range fragments (bp); na – number of alleles at the locus, ar – allelic richness per locus, n – number of individuals typed at the locus, ho – observed heterozygosity, he – expected heterozygosity, pic – polymorphic information content, hw – significance of deviation from hardyweinberg equilibrium (hardy-weinberg equilibrium test chi-square value, p value and significance with bonferroni correction: ns = not significant, * = p < 0.05; ** = p < 0.01; *** = p < 0.001), f(null) – null allele frequency estimate, fis – fixation index (* = p < 0.05). locus s na ar n ho he pic hw f(null) fis pie020 97–109 7 3.386 312 0.583 0.585 0.537 35.434*** –0.026 –0.067 pie102 139–167 13 5.367 321 0.523 0.750 0.726 87.243 *** 0.187 0.279* pie152 228–260 17 7.187 314 0.834 0.890 0.879 3.756 ns 0.032 0.043* pie215 185–218 12 6.295 281 0.384 0.707 0.673 193.668*** 0.311 0.417* pie223 197–234 11 5.980 318 0.811 0.822 0.803 0.525 ns 0.006 –0.002 pie227 156–165 4 3.387 323 0.223 0.251 0.233 3.776 ns 0.060 0.095* pie239 69–93 9 4.689 302 0.248 0.435 0.420 56.089*** 0.274 0.399* pie242 102–132 14 5.924 318 0.748 0.841 0.821 16.677ns 0.058 0.093* pie243 204–224 10 4.392 282 0.688 0.683 0.649 37.002*** –0.029 –0.038 pie267 84–106 12 4.029 313 0.425 0.667 0.622 82.591*** 0.227 0.334* pie271 182–204 12 2.130 311 0.949 0.843 0.823 37.040*** –0.063 –0.153 mean – 11 – 308.64 0.583 0.679 0.653 – 0.094 – standard error – 1.053 – 4.386 0.074 0.058 0.059 – 0.040 – quercus pubescens complex in southern italy acta bot. croat. 79 (1), 2020 19 ity (ho) for microsatellite loci ranged from 0.223 (pie227) to 0.949 (pie271), and the average was 0.583. a high level of ho was detected in seven est-ssr loci ranging from 0.523 to 0.949, whereas four loci detected low ho with values ranging from 0.223 to 0.425. the expected heterozygosity (he) ranged from 0.251 to 0.890 with an average value of 0.679 and was higher than ho for all the microsatellite loci, except for pie243 and pie271. the hardy-weinberg exact test for all populations (hw) revealed that seven loci (pie020, pie102, pie 215, pie239, pie243, pie267, pie271) exhibited significant deviation from hardy–weinberg equilibrium (p<0.001) whereas four loci (pie152, pie223, pie227, pie242) did not show significant departures from hardyweinberg equilibrium. the indexes of genetic diversity of the 22 q. pubescens s.l. populations are summarized in tab. 3. the number of different observed alleles (na) and effective alleles (ne) averaged across all loci ranged from 4.727 (pop22) to 7.182 (pop09) and 2.559 (pop22) to 4.363 (pop20), respectively. nine private alleles (pa) was found and these are distributed within ten individuals in turn distributed in eight oak populations (two other private alleles were found in pop24; on-line suppl. tab. 2). the allelic richness (ar), ranged between 3.865 (pop3) and 5.171 (pop20). the average observed heterozygosity (aho) ranged from 0.487 (pop03) to 0.665 (pop11) with mean value of 0.583. the average expected heterozygosity (ahe) ranged from 0.510 (pop03) to 0.709 (pop09) with mean value of 0.629. overall, the observed heterozygosity showed mostly slightly lower values than the expected heterozygosity. the mean fixation index (fis) values are close to zero for all populations (ranging from 0.058 in pop11 to 0.188 in pop21) that are the values expected under a random mating. population genetic structure and gene flow the overall population differentiation degree is reported in tab. 4. amova analysis showed that 3.6% (p < 0.001) of the genetic variations was among populations while 96.4% was within populations. the majority of molecular variance was partitioned within individuals (78.5%, p < 0.01) while 17.9% (p < 0.01) of variance was found to occur among individuals. the pairwise fst matrix between populations is shown in on-line suppl. tab. 3; eleven population pairs exhibited negative values but all of them showed no significant p values (all largely > 0.05). the differentiation coefficient between population pairs having significant p values showed that pop11 (incoronata-tavoliere) and pop15 (palmariggisalento) was the smallest (fst = 0.0025; p ≤ 0.001); the differentiation coefficient between pop02 (deliceto-daunian sub-apennine) and pop22 (vico del gargano-east gargano) was the largest (fst = 0.1053; p < 0.01). nei’s genetic distance among the populations belonging to the five units are graphically illustrated in fig. 2, constructed on the basis of principal coordinate analysis (pcoa with the physiographic units superimposed). the first two axes of the pcoa explained 42.26% of the variation. the scattergram did not show an evident correlation between the physiographic units and the genetic similarities of the populations. in fact, populations belonging to four different physiographic units are closely associated along the first axis in the left part of the diagram. the mantel test comparing the matrices of geographic and genetic distances among populations did not evidence any correlation between these two variables. tab. 4. analysis of molecular variance among populations, within populations, and within individuals of quercus pubescens s.l. populations; df – degree of freedom, ss – sum of squares, ms – mean squares, p – probability based on standard permutation across the full data set, nm –average estimate of gene flow among populations. source of variation df ss ms estimated variation percentage variation % f-statistics p-value gene flow among populations 21 180.496 8.595 0.141 3.6 fst = 0.036 0.001 nm = 6.664 among individuals 303 1346.241 4.443 0.695 17.9 fis = 0.185 within individuals 325 992.500 3.054 3.054 78.5 fit = 0.215 total 649 2519.237 3.889 100 tab. 3. genetic diversity parameters for the quercus pubescens s.l. populations (pop) analyzed through eleven microsatellite loci. n – number of individuals; na – average number of different alleles; ne – average number of effective alleles; ar – allelic richness; ahe – average expected heterozygosity; aho – average observed heterozygosity; fis – average of fixation index tested by 1023 permutation of gene copies between individuals within population (* = p < 0.05); pa – number of private alleles. pop n na ne ar aho ahe fis pa pop01 16 6.182 3.641 4.642 0.581 0.604 0.035 0 pop02 16 5.091 3.353 4.064 0.498 0.584 0.125* 0 pop03 16 4.818 2.968 3.865 0.487 0.510 0.029 0 pop04 16 6.364 3.684 4.605 0.572 0.638 0.094* 0 pop05 15 5.182 3.097 4.192 0.604 0.610 -0.036 1 pop06 15 5.182 3.055 3.940 0.529 0.590 0.037 0 pop07 16 5.636 3.213 4.156 0.557 0.544 -0.059 0 pop08 16 6.455 3.888 4.775 0.577 0.632 0.086* 0 pop09 16 7.182 4.166 4.903 0.605 0.709 0.155* 0 pop10 16 6.455 3.979 4.601 0.587 0.656 0.093* 0 pop11 16 6.000 3.369 4.313 0.665 0.614 -0.112 0 pop12 15 5.909 3.790 4.420 0.628 0.655 0.025 1 pop13 12 5.545 3.883 4.760 0.595 0.705 0.092 2 pop14 16 6.091 3.749 4.487 0.655 0.648 -0.018 1 pop15 15 5.727 3.503 4.583 0.596 0.634 0.066 0 pop16 11 5.273 3.400 4.606 0.627 0.647 0.006 1 pop17 13 6.091 3.700 4.560 0.580 0.639 0.062 0 pop18 14 5.909 3.688 4.651 0.605 0.642 0.005 1 pop19 14 6.545 3.955 4.841 0.583 0.669 0.064 0 pop20 14 7.091 4.363 5.171 0.617 0.692 0.120* 1 pop21 16 6.000 3.753 4.510 0.557 0.684 0.116* 1 pop22 11 4.727 2.559 3.941 0.527 0.540 -0.129 0 mean 14.8 5.884 3.580 4.481 0.583 0.629 0.039 di pietro r, di marzio p, antonecchia g, conte al, fortini p 20 acta bot. croat. 79 (1), 2020 when structure was run using population ids as sampling location information, a maximum value of the rate of change in the log probability of data was revealed at k = 2, using evanno's method (evanno et al. 2005) (on-line suppl. fig. 1). the individual membership proportion determined by bayesian clustering analysis suggested a subdivision of the whole data-set of oak individuals (325) into two genetic clusters: a first cluster including 305 pure individuals (94.15%) and a second one composed of 19 off-type individuals (5.85%) (fig. 3a). the distribution of the 19 off-types involved 13 populations out of the 22 investigated (fig. 3b, on-line suppl. tab. 4). population 13, where the sampled individuals were selected from a mixed wood with q. pubescens and q. robur that developed in a humid area, is the population showing the highest number (4) of off-types. populations 2, 9, and 10 count two off-types each and relate to q. pubescens s.l. woods with the occurrence of q. frainetto. the wilcoxon sign-rank test showed no significant results as regards bottleneck effect in all the populations analyzed, using both tpm and smm models. the observed number of loci with heterozygosity excess (obs lhexc) was found to be always lower than the expected number of loci with heterozygosity excess (exp lhexc) except for population 13 where “obs lhexc” is about 1.5 times greater than “exp lhexc”. however, the wilcoxon sign-rank test was not significant (tab. 5). discussion the data-set exhibited the following levels of genetic diversity at the microsatellite loci examined: mean expected heterozygosity (he) = 0.679, mean observed heterozygosity (ho) = 0.583, average number of alleles per locus (k) = 11.0 (from 4 to 17 alleles). four (pie102, pie215, pie239, pie267), out of the eleven loci investigated, showed a positive fis (0.279-0417) which turned out to be statistically significant (tab. 2). it is known that the fixation of alleles could be favored by directional selection (andolfatto 2001), or be the results of genetic drift. molecular studies regarding othfig. 3. genetic assignment obtained with structure clustering analysis. oak individuals are distributed in progressive order along the horizontal axis while the vertical axis expresses the group membership percentage degree per single individual per k=2. clustering of all the data-set oak individuals into two different genetic clusters with decreasing membership degrees for group 2 and increasing degrees for group 1 (a). clustering of all the data-set oak individuals ordered per populations; the latter separated by white lines (b). fig. 2. principal coordinate analysis (pcoa) via distance matrix (nei genetic distance) with data standardization. daunian sub-apennine populations: pop02, 06, 07, 08; w-gargano populations: 04, 05; e-gargano populations: 21, 22; murgia plateau populations: 01, 03, 09, 10, 12, 13, 14, 16, 17, 18, 19, 20; salento population: 15; tavoliere population: 11. quercus pubescens complex in southern italy acta bot. croat. 79 (1), 2020 21 tab. 5. results of tests for genetic bottlenecks using the two-phase mutation model (tpm) and a stepwise mutation model (smm), and the wilcoxon sign-rank tests for heterozygosity excess; the number of polymorphic loci for all populations (pop) is 11; obs lhexc – observed number of loci with heterozygosity excess; exp lhexc – expected number of loci with heterozygosity excess; p – probability of no significant heterozygosity excess. tpm smm pop obs lhexc exp lhexc p obs lhexc exp lhexc p pop01 4 6.46 0.9492 2 6.31 0.9954 pop02 6 6.44 0.7114 4 6.43 0.8398 pop03 4 6.43 0.9385 4 6.43 0.9585 pop04 4 6.37 0.9126 4 6.37 0.9585 pop05 3 6.29 0.8608 3 6.29 0.9263 pop06 5 6.39 0.7676 4 6.35 0.8799 pop07 4 6.36 0.9585 4 6.38 0.9731 pop08 5 6.49 0.9492 4 6.58 0.9663 pop09 3 6.55 0.9492 2 6.50 0.9976 pop10 5 6.27 0.7676 3 6.36 0.8970 pop11 2 6.39 0.9895 2 6.49 0.9966 pop12 5 6.49 0.5845 5 6.57 0.7114 pop13 8 6.52 0.1602 8 6.61 0.2598 pop14 4 6.59 0.8608 4 6.58 0.9585 pop15 6 6.58 0.7676 5 6.51 0.8608 pop16 4 6.59 0.9263 4 6.55 0.9663 pop17 5 6.46 0.8799 2 6.51 0.9919 pop18 3 6.47 0.9126 3 6.50 0.9126 pop19 4 6.39 0.9126 4 6.50 0.9492 pop20 4 6.48 0.8799 3 6.59 0.9919 pop21 6 6.56 0.6812 4 6.57 0.8970 pop22 3 6.56 0.9663 2 6.53 0.9954 er oak species stated that the genetic variation removed by genetic drift would affect the genome rather uniformly (alberto et al. 2010). this would not seem to be the case of the apulian pubescent oak populations, since the other est-ssr loci examined showed only slightly positive values of fis, or even negative ones. due to the relatively low number of sampled individuals per population, it cannot be completely excluded that these results could be addressed, at least in part, to the sampling effect. however, there is no uniformity of views at present on the minimum number of individuals per population that should be sampled to detect “reliable” results for genetic differentiation. hale et al. (2012) stated that the number of individuals per population should be established using a “case by case” approach. on the other hand the genetic literature on this issue shows a wide range of opinions and experiences from which it emerges that the number of individuals sampled can be highly variable. in many cases it was considered as acceptable to take a minimum of 10 to 30 individuals. for some pinus sylvestris l. populations in the baltic area, the number of 20-25 individuals was found to be large enough to detect all the alleles (danusevičius et al. 2016) whereas other studies have even considered significantly lower numbers (kitamura et al. 2017, rinaldi et al. 2019). in contrast, in kalinowski (2005) it was shown particularly clearly that when fst is less than 0.01 it can be useful to sample up to 100 individuals. as far as we are concerned, we believe that these results, although deriving from the analysis of populations composed of a seemingly limited number of samples, can still provide interesting information that should not be completely ignored. the highest allelic variability was found in locus pie152 with 17 alleles and pic = 0.879. a similar variability was found in other pubescent oak populations investigated in southern italy and central italy (antonecchia 2015) where locus pie152 exhibited the highest polymorphic degree and in particular the highest values of allelic richness for q. pubescens. the still insufficient knowledge about the q. pubescens genome do not allow it to be established if the high variability in locus pie152 might be associated with adaptive traits. more in-depth investigations would be needed to clarify this point. the level of genetic variability within the pubescent oak populations of the apulia region was here found to be lower than that reported for q. pubescens in other countries. curtu et al. (2007b) reported ahe = 0.891, ar = 17.8 on a study based on 6 microsatellite loci for 73 q. pubescens individuals while enescu et al. (2013) reported ahe=0.847, k = 22 and ar = 14.22 in a 7 microsatellite loci study in a geographically related area. the heterozygosity values reported in these studies, however, would not be directly comparable with those obtained by us due to the use of different type of markers (genomic ssrs vs. est-ssrs). some authors (durand et al. 2010, parthiban et al. 2018) showed that the use of est-ssrs highlights the values of polymorphism slightly lower than those obtained from the use of genomic ssrs. however, there are some studies on the quercus genus in which both these types of markers were tested. curtu et al. (2011) investigated the genetic variability of 65 individuals of q. pubescens using 7 microsatellite loci, of which 5 gssr and 2 est-ssrs. the overall average values for the genetic parameters in issue were the following: ahe = 0.859, k = 17, ar = 16.5. calculating the values of these parameters solely for the two est-ssrs markers we obtained values (ahe = 0.819, k = 17, ar = 16.8) that are lower than those obtained averaging both kind of markers but still significantly higher than those we obtained from our apulian pubescent oak data-set. the only available analyses of genetic differentiation within italian white oak populations including q. pubescens s.l. carried out using est-ssrs markers are those reported for the mixed q. petraea, q. frainetto, q. pubescens woods of the mount vairano range in southern italy (antonecchia et al. 2015). comparison between these analyses and our data revealed that the genetic variability of the apulian populations is significantly lower than that recorded in mount vairano (ahe = 0.72, k = 9.46 ar = 9.2) as regards ahe and ar, and slightly higher as regards k. also the level of genetic variability among the q. pubescens s.l. populations of the apulia region (fst = 0.036) was found to be lower than the fst values found for other oak species in other parts of the world, e.g. q. rubra l. (fst = 0.080) (sullivan et al. 2013), quercus mongolica fisch. ex ladeb. (fst = 0.077) (ueno and tsumura 2008), q. variabilis bl. (fst = 0.063) (shi et al. 2017). di pietro r, di marzio p, antonecchia g, conte al, fortini p 22 acta bot. croat. 79 (1), 2020 as regards gene flow, the present study showed that the apulian q. pubescens s.l. populations are characterized by a relatively high values (nm = 6.664). although q. pubescens is a typical wind-pollinated and outcrossing species such high values are quite surprising considering the progressive contraction suffered by the apulian oak woods in the last millennium. the value of gene flow is determined by many factors, such as the biological means of spreading via pollen and seeds, the occurrence of physical barriers among populations, population dimension and so forth; in addition, it can be facilitated by physical proximity of the populations. it is probable that the short distance separating some of the apulian pubescent oak populations and the lack of significant mountain barriers have contributed to keep the average gene flow value high. it is not possible, at present, to establish whether the above gene-flow values are stable over time, or are the result of a contraction due to over-exploitation of forests. in studies on other oak species (shi et al. 2017) it was established that gene-flow values can decrease significantly in a relatively short time (ten years) following the transition from primary forest to natural secondary forest. in the apulia region the millennial exploitation of the woodlands led to the disappearance of primeval forests as early as roman times. however, the secondary forest communities that have since replaced the primary ones show a high degree of naturalness and a rather stable floristic composition. it is conceivable therefore that the gene flow rate will remain stable over time and that the only parameter capable of negatively acting on the geneflow rate is the spatial reduction of the population size. the apparent relatively low level of intraspecific diversity observed for the apulian q. pubescens populations could be addressed to their marginal geographical position not only in the italian peninsula but also in the central mediterranean context. in terms of biological conservation of deciduous oak forests the apulia region is particularly exposed to environmental risks due to climate change drying effects which overlap a bioclimatic pattern dominated by the thermo-mediterranean thermotype the latter having in the mediterranean maquis the main type of potential vegetation (rivas-martínez et al. 2004, ladisa et al. 2012). threats to pubescent oak woods are further increasing in many localities that are experiencing greater aridity and water deficiency due to the overexploitation of water reserves resulting from the ever increasing consumption for agricultural purposes and by tourist settlements. in the long term, the combined effect of these two factors could lead to a change in the floristic composition of the upper structural layers of the thermophilous deciduous oak forests with a progressive replacement of the pubescent oak individuals by evergreen woody species, such as quercus ilex l. phillyrea latifolia l., rhamnus alaternus l., arbutus unedo l. and pistacia lentiscus l. conversely, the high gene flow values observed in the study area seem to move in the opposite direction and might positively contribute to preventing possible activations of genetic drift. yet the lack of bottleneck effects in all investigated populations implies a certain degree of resilience despite the relatively small areas covered by the single oak populations. as regards possible taxonomic implications arising from this study, the results of structure bayesian clustering showed that the populations of apulian pubescent oaks could not be divided into groups. none of the populations investigated displayed significant differences in their genetic composition. however, the highest percentage of the possible “putative hybrids” that we simply named “off-types”, was found in pop13, which also showed the highest percentage of private alleles. pop13 is located within a flat sub-humid area in the western side of the murgian plateau in the municipality of laterza, and lies in spatial contact with a small stand of q. robur wood (the only known site of q. robur currently recorded for the apulia region). it is conceivable that in this area q. robur was much more abundant in the past and that both climate change and the millennial work of deforestation carried out by the local populations, has led to its current extreme impoverishment. it is very probable that during the climatic oscillations that took place during the quaternary, environmental situations favorable to the development of mixed forests of q. robur and q. pubescens s.l. were created several times with consequent hybridization or introgression between these two species. in fact, a possible key of interpretation of genetic structure of the q. pubescens apulian populations cannot fail to consider some physiographic and geographical features of this region such as the lack of sizeable mountain systems and its deep wedges jutting into the adriatic sea. these features led to the apulian peninsula experiencing the effects of quaternary glaciations only mildly and allowed some parts of it to work as glacial refuges for the thermophilous oak forests during the quaternary cold periods, when most of the italian peninsula was covered by steppic grasslands (follieri et al. 1988). the occurrence of restricted areas where the climate forced the various species of white oaks to coexist for long (cold) periods may have favored the generation of hybrids. the innumerable past hybridization or introgression events, which led to the current high morphological variability of q. pubescens s.l., may also have played a role in enlarging the gene flow of the apulian populations. white oaks commonly live in sympatry, making them particularly suitable for gene exchange and production of hybrid individuals (rushton 1993, williams et al. 2001, lepais and gerber 2011). according to burger (1975), applying the biological concept of species to the genus quercus would cause an upheaval of the nomenclature, since the binomial species would then not correspond to the biological one. this makes the systematics of this genus extremely susceptible to disagreement among botanists and the hope of arriving at the proposal of a largely shared and possibly unambiguous taxonomic framework (in particular for the south-european white oaks) is something not soon to be realized. the problematic classification of pubescent oaks in the apulian peninsula and in the entire italian peninsula is perfectly evidenced by the discrepancy that emerges when comparing the taxonomy of the genus quercus as reported in latest versions of flora d’italia (pignatti et al. 2017) and in the checklist of the italian vascular flora (bartolucci et al. 2018). although both works are worthy of the utmost respect and quercus pubescens complex in southern italy acta bot. croat. 79 (1), 2020 23 consideration, they show enormous differences in both the numbers and the names of the taxa considered good species for the same taxonomic group (white oaks). actually, based on what emerges from our work, the high taxonomic differentiation among the italian pubescent oaks as reported in most of the italian floras and checklists does not seem to have much foundation (see also di pietro et al. 2012) or at least does not seem to have it with regard to the pubescent oaks of apulian peninsula. in fact, our results established that it was not possible to identify genetic clusters among the pubescent oak populations of the apulian region basing on 11 highly polymorphic markers as it was not possible to identify morphological clusters basing in a previous biometric study on the morphological traits of leaves and fruits on the same set of specimens (di pietro et al. 2016). summarizing, the presence of more than one taxon at species rank belonging to q. pubescens s.l. (e.g., q. virgiliana, q. dalechampii, q. amplifolia, etc.) is not confirmed (at least at present) for the apulia region. at the same time, the phytosociological frameworks that are currently supposed to classify different types of pubescent oak forests (phytosociological associations), dominated by different pubescent oak species and including up to five different species of pubescent oaks in the dominant tree layer, need at the very least to be reconsidered. obviously we do not consider this work to be a solution to all the problems concerning the taxonomy of white pubescent oaks, not even to some of them of topical relevance in south-eastern europe, such as the eternal debate on the dualism between q. pubescens and q. virgiliana (cf. borazan and babaç 2003, škvorc et al. 2005, franjić et al. 2006, jerše and batič 2007, trinajstić 2007, enescu et al. 2013). we simply believe that this work represents a small step forward, a substantial contribution, albeit on a regional scale, to being able to arrive in the near future at a possible solution of the problem on a global scale. finally, it is worth emphasizing, that all the taxonomical and phytosociological considerations mentioned in this paragraph should not be viewed as useful only for updating floras, checklists or vegetation maps. in fact, they have direct implications for the application of european policies for nature conservation. it is only necessary to recall the 92/43/eec european directive where the identification of some of the forest habitats listed in the manual of interpretation (e.g., 91h0* “pannonian woods with quercus pubescens”, 91aa* “eastern white oak woods”, 91m0 “pannonian-balkanic turkey oak-sessile oak forests”, 91y0 “dacian oak and hornbeam forests”), is also based on the occurrence of species such as q. virgiliana and q. dalechampii. acknowledgments the authors wish to acknowledge g. vendramin for his critical discussion of the preliminary results of the paper and p. medagli, g. misano, g. silletti, v. viscosi, r.p. wagensommer for their help in collecting oak populations. references alberto, f., niort, j., derory, j., lepais, o., vitalis, r., galop, d., kremer, a., 2010: population differentiation of sessile oak at the altitudinal front of migration in the french pyrenees. molecular ecology 19, 2626–2639. andolfatto, p., 2001: adaptive hitchhiking effects on genome variability. current opinion in genetics and development 11, 635–641. antonecchia, g., 2015: analisi della variabilità genetica del sottogenere quercus oerst. in italia centro-meridionale. phd thesis, department of bioscience and territory, university of molise, pesche (is). retrieved april 10, 2016 from http://hdl. handle.net/2192/296. antonecchia, g., fortini, p., lepais, o., gerber, s., léger, p., scippa, g. s., viscosi, v., 2015: genetic structure of a natural oak community in central italy, evidence of gene flow between three sympatric white oak species (quercus, fagaceae). annals of forest research 58, 205–216. ballian, d., ivanković, m., gračan, j., perić, s., marjanović, h., bobinac, m., slade, d., 2010: analysis of pubescent oak (quercus pubescens willd.) in the western part of the balkan peninsula. acta societatis botanicorum poloniae 79, 189–195. bartolucci, f., peruzzi, l., galasso, g., albano, a., alessandrini, a., ardenghi, n.m.g., astuti, g., bacchetta, g., ballelli, s., banfi, e., barberis, g., bernardo, l., bouvet, d., bovio, m., cecchi, l., di pietro, r., domina, g., fascetti, s., fenu, g., festi, f., foggi, b., gallo, l., gottschlich, g., gubellini, l., iamonico, d., iberite, m., jiménez‐mejías, p., lattanzi, e., marchetti, d., martinetto, e., masin, r.r., medagli, p., passalacqua, n.g., peccenini, s., pennesi, r., pierini, b., poldini, l., prosser, f., raimondo, f.m., roma‐marzio, f., rosati, l., santangelo, a., scoppola, a., scortegagna, s., selvaggi, a., selvi, f., soldano, a., stinca, a., wagensommer, r.p., wilhalm, t., conti, f., 2018: an updated checklist of the vascular flora native to italy. plant biosystems 152, 179–303. biondi, e., casavecchia, s., beccarisi, l., marchiori, s., medagli, p., zuccarello, v., 2010: le serie di vegetazione della regione puglia. in: blasi, c. 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10.2478/botcro-2019-0019 issn 0365-0588 eissn 1847-8476 nectar and pollen production of helianthus tuberosus l. – an exotic plant with invasiveness potential bożena denisow, karolina tymoszuk, marta dmitruk* department of botany and plant physiology, subdepartment of plant biology, university of life sciences in lublin, 15 akademicka st., 20-950 lublin, poland abstract – in central europe, helianthus tuberosus l. is a late summer/autumn bloomer (august/november). the disc florets produce both nectar and pollen. floral reward is available in male-phase flowers (pollen and nectar) and in female-phase flowers (nectar). the floral reward is attractive to a variety of insect visitors (honey bees, wasps, flies and butterflies). the season of blooming as well as the total sugar yield (25.4 – 47.4 kg ha–1) and pollen yield (57.8 – 212.7 kg ha–1) indicate that h. tuberosus is important in the enhancement of food resources for pollinators. the generative reproduction in h. tuberosus is impaired (the species does not set seeds/ fruits). however, due to its attractiveness for a variety of pollinators in both rural and urban areas, the spread of h. tuberosus should be monitored. moreover, its propagation needs to be attended with restrictions. keywords: alien plant, apis mellifera, bombus spp., insect visitors, nectar, pollen *corresponding author e-mail: marta.dmitruk@up.lublin.pl introduction alien plant species can be introduced by animals and/or accidentally or intentionally by humans (tokarska-guzik et al. 2010). such species can spread at a high rate in both urban and rural landscapes due to their biological properties, e.g. anthropochorous and anemochorous dispersal modes and long-term or transient seed banks, and/or recruitment of reproductive offspring (lockwood et al. 2007, wrzesień et al. 2016a, denisow et al. 2017). over recent decades, the acceleration in the rate of biological invasions has been noted worldwide possibly due to globalization of trade and transport, and climate change (tokarska-guzik et al. 2010, denisow and malinowski 2016). many negative impacts of invasive species on native species richness, the composition of local biocoenoses and the functioning of local ecosystems have been recorded (weber 2003, tokarska-guzik et al. 2010). in particular, the arrival of novel entomophilous plant species in new areas is considered to be harmful for local biocoenoses as it can potentially interrupt plant-insect interactions (aizen et al. 2008, stout and morales 2009). therefore, there is a growing body of interest in invasive entomophilous species, which can be expected to be visited and pollinated by native insect pollinators (denisow et al. 2016a). however, the impacts of alien species on pollinators vary according to the floral traits of the plant species (stout and tiedeken 2017). in many areas alien plant species are responsible for the reduction of food resources and initiate gaps in food availability (goulson et al. 2015, jachuła et al. 2018a, wrzesień et al. 2016b). on the other hand, several alien species (e.g. solidago spp., impatiens grandulifera royle) due to their prolific production of nectar sugars per unit area have attracted considerable attention from beekeepers (guzikowa and maycock 1986, brodschneider and crailsheim 2010). helianthus tuberosus l. (asteraceae), the jerusalem artichoke or topinambour, is an entomophilous, perennial plant native to eastern north america. the species is attractive to pollinators due to its floral traits (impressive inflorescences, late summer blooming) and the reward offered (cabi 2018). it was intentionally introduced into europe in the 17th century as an ornamental plant, attractive for its showy, eye-catching inflorescences (cabi 2018). nowadays, the species is widely cultivated across the temperate and tropical climate regions (yang et al. 2015). there is an increasing interest in commercial-scale cultivation of h. tuberosus, due to needs of the food, cosmetic and pharmaceutical industry (seiler denisow b., tymoszuk k., dmitruk m. 136 acta bot. croat. 78 (2), 2019 and campbell 2006). in particular, the inulin gathered in h. tuberosus tubers is evidenced to have therapeutic properties being a dietary fiber and known to have prebiotic effects, and enhances the immune system in humans (ma et al. 2011). the plant is also grown as an energetic plant or good quality feedstuff for animals (yang et al. 2015). however, the likelihood that it will escape from cultivation and its invasiveness is documented in many regions, in europe, asia, new zealand and south america (cabi 2018; weber 2003). further expansion into new areas is expected due to the globalization of transport and climate change (lockwood et al. 2007, tokarska-guzik et al. 2010). in plant-pollinator interactions, nectar and pollen are the main floral rewards important in establishing a relationship (antoń and denisow 2014, rodríguez-riaño et al. 2014). nectar predominately contains sugars and is regarded as a cost-effective (easy to digest and absorb) major energetic floral reward for pollinators (nicolson and thornburg 2007). the nectar traits (volume, sugar concentration) vary among species (chalcoff et al. 2006, denisow et al. 2016b, strzałkowska-abramek et al. 2016a, b, jachuła et al. 2018c). nectar production and the sugar concentration are known to be considerably influenced by environmental conditions (nicolson and thornburg 2007, strzałkowska-abramek et al. 2018). pollen also attracts pollinators as it is a major source of proteins and other nutrients (vitamins, lipids, hormones) crucial for a well-composed pollinator diet essential for insect growth and development as well as immunocompetence (filipiak et al. 2017, jachuła et al. 2018c). nectar and pollen traits are recognized to be important for the foraging behavior of pollinators (pacini and hesse 2005, denisow 2011, antoń and denisow 2018). helianthus tuberosus is a tall perennial plant, 1–2.3 m in height. the plant forms head inflorescences (ca. 3–5 cm in diameter). the heads are composed of outer sterile, bright-yellow ligulate florets and inner bisexual golden-yellow disc florets (swanton et al. 1992). h. tuberosus inhabits areas on moist, nutrient-rich, sandy or loamy soils, especially along rivers, fallows, railway embankments, abandoned fields (kays and nottingham 2007, tokarska-guzik et al. 2010, eppo 2014). the objective of the present study were: (i) to assess the phenology of blooming, (ii) to evaluate nectar and pollen quantity that can be used as food by insects, (iii) monitor the spectrum of insect visitors, and (iv) check the pollination requirements of helianthus tuberosus, an exotic plant in europe with high invasiveness potential. we also checked if the plant traits and insect visitor composition differ among plant populations grown in an urban and a nearby rural area. materials and methods study area the study was made in the years 2014–2015 in two populations of helianthus tuberosus localized in the lublin upland, se poland (51°08’–51°18’n, 21°27’–21°41’e; elevation: 170–220 m a.s.l.). the first population was grown in an urban area, the second in a rural area in the vicinity. the urban site was localized in lublin, the largest city of south-eastern poland, with flora characteristic specific for cities with a so-called atmospheric urban heat island (uhi) (rysiak and czarnecka 2018). the average annual air temperature was lower in the rural than in the urban area – in 2014 by 0.8 °c and in 2015 by 1.3 °c. the experimental plots of the urban site (approx. 10 m2 each; n = 3) were established on ruderal spaces located close to built-up areas. the rural plots (approx. 10 m2 each; n = 3) were localized in a zone adjacent to the urban area. the rural area is characterized by ongoing residential development, however agricultural production is still evolving. the distance between the experimental plots localized in both the urban and the rural area was approximately 10 km. flowering observations the flowering phenology was monitored at each study site. we visited the study populations every 3–7 days and recorded the onset (= beginning), peak (= full bloom), and the end (= termination) of blooming (denisow et al. 2014). the beginning of blooming was defined by 10% of inflorescences per individuals in bloom, full bloom was indicated if ca. 50% of the inflorescences were in bloom, and the end of blooming was identified when 80% of inflorescences on individuals had completed flowering. in each population, 10 individuals were randomly selected for phenological observations. the duration of the male and female phases, life-span of disc florets (n = 20 disc florets per population, per year) and individual inflorescences (n = 10 heads) were assessed. the observations were made at 1-hour intervals. the male phase was recognized from the beginning of pollen presentation to the beginning of opening of stigma lobes. the female phase was defined as a period between opening of stigma lobes and corolla wilting. the duration of inflorescence life-span was recorded by marking the inflorescences with just opened disc florets. these inflorescences were followed until the end of last floret blooming. the disc floret life-span was defined as the period from the time when the flower bud was opened to its ending when the corolla was shed. we established the density of shoots in each population. the shoots were counted in 6 randomly selected areas of 1 m2 (frame method, denisow 2011). the total number of florets per unit area was calculated by multiplying the number of heads per shoot (n = 20–30 per year per population) with the average number of disc florets per head (n = 30 per year per population) and with the number of individual shoots. nectar and pollen production nectar production was assessed using capillary pipettes (stpiczyńska et al. 2014). prior to nectar collection, we excluded insect visitors from entire head inflorescences (n = 10–20) using tulle isolators. sampling of nectar was attempted at 4 different time points of the blooming period (between 20th august and 1st october). at each time 3–9 samples floral reward in helianthus tuberosus acta bot. croat. 78 (2), 2019 137 tukey’s multiple comparisons test. for statistical evaluation of the results, the statistica software ver. 6 (statsoft, poland, 2001) was used. results flowering and floral morphology helinathus tuberosus bloomed in august-november with a full bloom in late august (2015) and early-mid september (2014). each year the start of blooming was noted earlier (9–14 days) in the urban (site a) than in the rural landscape (site b). in a single head, the flowering starts from the outer disc florets. anthesis of the ray florets occurred in the morning (7.00–9.00 gmt+2 h). furthermore, the diurnal opening of disc florets was documented and the process was most intensive in the early afternoon (15.00–18.00 gmt+2 h). the inflorescence life-span ranged from 5.6 to 9.3 days (mean = 7.4 days). the length of corolla tube in disc florets ranged between 3.57 and 4.88 mm. neither population nor the year affected the disc florets’ length. disk florets are protandrous, therefore three stages of floret development can be distinguished from the edge of the inflorescence towards the inner part, i.e. (i) male disc florets presenting pollen and offering nectar, (ii) female disc florets with nectar available, and (iii) immature buds. the study populations differed in the number of developed disc florets per inflorescence and heads per stem (tab. 1). individuals of h. tuberosus in the urban population produced fewer (ca. 12%) flower heads, bearing fewer disc florets (ca. 6%) than the individuals in the rural population. interpopulation variation in plant density was noted. the density of the stems of h. tuberosus varied over the study period only in the urban site. nectar and pollen rewards nectar production began in 1-day disc florets and lasted 3–5 days in individual flowers of h. tuberosus. the nectar was available till the end of anthesis. the amount of secreted nectar was significantly higher in disc florets of the rural population than in those of the urban population (f1,15 = 8.589, p = 0.011) (tab. 2). year-to-year disparities (from 20–30 disc florets) were collected. nectar was collected in previously weighed micro-capillary pipettes. the nectar mass was assessed reweighting the pipettes with collected nectar (wps-36 analytical balance; radwag, poland). sugar concentration was established with abbe refractometer (rl-4 pzo, warsaw, poland). then the total sugars mass was calculated for florets (in mg), head inflorescences (mg), and for unit area (kg ha–1). pollen production was evaluated using the ether-ethanol method (denisow 2011). we extracted the buds of disc florets from head inflorescences. unopened anthers from buds were collected in weighed glass containers (250 anthers per trial × 4 replications). next, the glass containers with anthers were placed into a dryer (elcon cl 65) at ca. 33 °c. the pollen was rinsed from anthers once with pure ether (1–2 ml) and then 4–6 times with 70% ethanol (10–20 ml). the mass of produced pollen was calculated per disc floret (in mg), per head inflorescence (in mg), per stem (in g), and per 1 ha (in kg). pollination requirements and insect visitors we checked the fruit/seed set using different pollination treatments, i.e. (i) open pollination – with free access of insect visitors to flowers, (ii) self-pollination – with exclusion of insect visitors by bagging flowers with tulle isolators, (iii) artificial cross-pollination – flowers open-pollinated and additionally hand-pollinated with pollen collected from flowers of other individuals. simultaneously with the blooming observations, we recorded the pattern and intensity of insect visits. the observations were made at the same time and were conducted for two days at one week intervals (6 days of observations for each site, in total). insect foraging at 8.00, 12.00 and 18.00 h (gmt + 2.00 h) was noted. during each census of observation, the total number of visiting insects was recorded. the following categories were determined: 1. apis mellifera, 2. bombus species, 3. other hymenoptera (solitary bees), 4. vespula species 5. diptera, 6. syrphidae, 7. lepidoptera, 8. coleoptera. data analysis means of data measured at urban and rural sites and in different years were compared with one-way anova with tab. 1. dates of flowering, duration of blooming stages and blooming abundance of helianthus tuberosus in 2014–2015 in urban and rural sites, lublin upland, se poland. means ± sd (standard deviation) are presented. means followed by the same small letters are not significantly different between years and means followed by the same capital letters are not significantly different between study sites, at α = 0.05 based on tukey’s test. site year flowering period duration of flowering (days) number of disc florets per head number of heads per stem number of disc florets per stem (thous.) number of stems per m2 urban 2014 10 august – 2 november 85 118.4±27.5a 23.2±9.3a 2.7±0.8a 42.8±15.6b 2015 20 july – 20 november 124 120.2±31.6a 29.4±2.7b 3.5±0.5b 24.5±7.8a mean 104.5 119.3±14.9a 26.3±6.8a 3.1±0.8a 29.7±10.5a rural 2014 19 august – 12 november 86 129.8±11.6a 29.8±1.8a 3.8±0.5a 39.7±0.5a 2015 04 august – 03 november 92 123.6±13.5a 28.5±2.0a 3.5±0.6a 39.8±0.4a mean 89.0 126.7±12.8b 29.2±2.0b 3.7±0.6b 39.8±0.4b denisow b., tymoszuk k., dmitruk m. 138 acta bot. croat. 78 (2), 2019 in the amount of produced nectar were also recorded (f1,15 = 7.64, p = 0.004). on average, sugar nectar concentration amounted to 29.7% at the rural site, while nectar in the urban site was more concentrated. the nectar sugar mass varied among populations (f1,15 = 13.619, p = 0.004) and years of study (f1,15 = 3.752, p = 0.037). the total mass of sugar per disc floret was 0.029 mg, on average. the head inflorescences of rural population produced 35% more sugars than the urban population. in h. tuberosus, the disc florets are protandrous. during sunny days, the dehiscence of anthers started just after opening of the floret and pollen was available to insects for 1–2 days, if the air temperature was > 20 °c. the pollen presentation lasted 3–4 days, if the air temperature was < 15 °c. a significant population effect (f1,15 = 4.067, p = 0.041; tab. 3) and a year effect (f1,15 = 11.808, p = 0.048) were found for the amount of pollen produced per disc floret. the average mass of pollen produced was 0.11 mg per disc floret in 2014 and 0.07 mg per disc floret in 2015. the mass of pollen produced in disc florets of the urban population was 60% lower than in rural population florets. the total sugar and pollen yield varied in the urban and rural populations (fig. 1). helianthus tuberosus produced 47.3 kg ha–1 of nectar sugars in the rural and 25.4 kg ha–1 in the urban area. the pollen productivity was 212.7 kg ha–1 of pollen in rural and 57.8 kg ha–1 in urban environments, on average. no set of seeds/fruits was observed in any of the treatments applied. in h. tuberosus, head inflorescence is a unit of attraction for insect foragers. numerous insects foraged the florets. the spectrum of insect visitors was similar in urban and rural habitats (fig. 2). however, the proportion of insect visitors differed. a higher proportion of syrphids was observed fortab. 2. nectar production, sugar concentration and sugar mass in helianthus tuberosus in 2014–2015 in urban and rural sites, lublin upland, se poland. means ± standard deviation are presented. means with the same small letter do not differ significantly between study seasons within sites, whereas means with the same capital letter do not differ significantly between study sites at α = 0.05, based on tukey's test. site year nectar amount per disc floret (mg) sugar concentration (% w/w) sugar amount per disc floret (mg) sugar amount per head (mg) sugar amount per stem (g) urban 2014 0.053±0.012a 39.0±8.9a 0.021±0.011a 2.49±1.58a 0.057±0.022a 2015 0.062±0.015b 48.8±6.9b 0.030±0.016b 3.61±2.65b 0.105±0.034b mean 0.058±0.021a 43.9±10.3b 0.026±0.013a 3.05±2.15a 0.081±0.051a rural 2014 0.079±0.017a 32.5±3.6b 0.026±0.012a 3.37±2.95a 0.099±0.027a 2015 0.145±0.012b 26.8±7.2a 0.039±0.145b 4.82±3.58b 0.137±0.065a mean 0.112±0.015b 29.7±5.8a 0.033±0.089b 4.10±3.74b 0.118±0.054b fig. 1. sugar (a) and pollen (b) yield of helianthus tuberosus in 2014–2015 in urban and rural sites, lublin upland, se poland. means ± standard deviation are presented. means with the same small letter do not differ significantly between study seasons within sites, whereas means with the same capital letter do not differ significantly between study sites at α = 0.05, based on tukey's test. fig. 2. spectrum of insect visitors in helianthus tuberosus in 2014– 2015 in urban and rural sites, lublin upland, se poland. percentage relation of each group of insects to the total number of insect visitors (n) noted is shown. floral reward in helianthus tuberosus acta bot. croat. 78 (2), 2019 139 between populations. we assume that the uhi environmental conditions (higher temperature, lower humidity) impacted on plant species phenotypic traits and impaired the number of developed disc florets and inflorescences. the flowers of h. tuberosus are arranged in head inflorescence, which is a characteristic trait of asteraceae (wist and davis 2006, czarnecka and denisow 2014). it is suggested that outer ray florets attract pollinators visually, while the inner disc florets’ function is to reward the pollinators with food (wild et al. 2003). in disc flowers of h. tuberosus, floral reward is available in male-phase flowers (pollen and nectar) and in female-phase flowers (nectar). such a pattern of insect visitor-rewarding is commonly found in asteraceae plants (hadisoesilo and furgala 1986, wist and davis 2006, czarnecka and denisow 2014). protandry is a trait characteristic for the asteraceae family (howell et al. 1993, ladd 1994) and is reported to be an adaptation to cross-pollination. although the disc florets were willingly visited by diverse groups of pollinators, seeds/ fruits were obtained neither in open-pollination nor in the self-pollination treatments applied. in its natural range, h. tuberosus is recognized as highly self-incompatible species that requires cross-pollination for seed production (kays and nottingham 2007). however, poor seed set has been reported, i.e. fewer than 5 per head inflorescence (swanton et al. 1992). this strategy seems to be reasonable for a plant species that is propagated vegetatively and impaired generative reproduction in h. tuberosus does not restrict its effective spread to natural habitats. interpopulational differences in nectar production and nectar sugar concentrations were found. moreover, in each population differences in nectar traits were observed between growing seasons. the variability in the nectar amount produced in flowers is quite common and has been reported among plant species, plant populations or even among individual flowers (nicolson and thornburg 2007, denisow et al. 2014). nectar production is a complex physiological process dependent on variable environmental factors, i.e. temperature, relative humidity, light, co2 concentration, or physico-chemical soil properties (petanidou and smets 1996). habitat/environment conditions were found to have an impact on nectar/sugar production in many plant species, e.g. in linaria vulgaris (jachuła et al. 2018b), lamium maculatum and ajuga reptans (mačukanović-jocić et al. 2004, jarić et al. 2010) or allium ursinum l. ssp. ucrainicum (farkas et. al. 2012). nectar production can also be affected by soil nutrient availability or fertilizer application (denisow et al. 2016a). in our study, approximately 2-fold more nectar was produced in disc flowers of h. tuberosus grown in the rural than in the urban habitat. the rather diluted nectar in flowers at the rural site can be explained by the more humid microclimate conditions associated with rural habitat (located closed to the woodland). on the contrary, the drier microclimate at the urban site may be a background for higher sugar concentration in the nectar. aging on h. tuberosus in the urban habitat. honey bees were more frequently noted in rural plants than in urban. approximately 3-fold more insect visitors were noted in the urban habitat. honey bees foraged for nectar and pollen, while wasps, flies, and butterflies foraged for nectar. discussion in se poland, helianthus tuberosus is a late summer/autumn bloomer. a similar blooming season has been reported from north america and other european countries (cabi 2018, yang et al. 2015). our short–term study indicated that the blooming season of h. tuberosus differed in urban and rural areas. in both study years, the species started to bloom earlier in the urban than in the nearby rural zone. the acceleration of flowering in urban compared to rural areas was noted in diverse european regions for a variety of species (kasprzyk 2016, stępalska et al. 2016). the prolongation of the flowering season within cities was also documented (e.g., in the largest cities in britain), as compared to rural surroundings (dallimer et al. 2016). it is accepted that phenological processes are affected by diverse weather factors (e.g., air temperature, humidity, insolation) (mckinney 2008, masierowska 2012). in compact built-up areas of many cities higher temperatures than in neighboring areas are evidenced, a phenomenon referred to as an urban heat island (uhi) (taha 1997). the uhi phenomenon is evidenced in lublin city and is well-known to have an impact on the vegetation (rysiak and czarnecka 2018). however, in addition to microclimatic factors, plant phenology can be modified in relatively close areas by disease/pest occurrence, soil nutrients, or soil water availability (menzel et al. 2008). we did not observe plants affected by disease/pests in our study plots, and therefore we assume that the difference in the flowering of h. tuberosus in the study plots was determined by weather-related factors. in particular, a higher average air temperature was noted in urban areas. in our study, disparities in flowering abundance (i.e., the number of disc florets per head, head inflorescence per shoot) and quantitative traits of floral reward were evidenced tab. 3. the mass of pollen produced per disc floret, per head, and per stem of helianthus tuberosus in the years 2014–2015 in urban and rural sites, lublin upland, se poland. means ± standard deviation are presented. means followed by the same small letters are not significantly different between years and values followed by the same capital letters are not significantly different between study sites, at α = 0.05 based on tukey’s test. site year mass of pollen per disc floret (mg) mass of pollen per head (mg) mass of pollen per stem (g) urban 2014 0.07±0.02b 8.29±3.24b 0.19±0.08a 2015 0.04±0.01a 4.81±1.61a 0.14±0.03a mean 0.06±0.01a 6.55±2.89a 0.17±0.04a rural 2014 0.14±0.04a 18.17±3.62a 0.54±0.35a 2015 0.10±0.07a 18.54±7.31a 0.53±0.18a mean 0.12±0.06b 18.36±8.48b 0.54±0.22b denisow b., tymoszuk k., dmitruk m. 140 acta bot. croat. 78 (2), 2019 in our study, the pollen production per flower, per head and per unit area differed between populations (i.e., between rural and urban sites). disc florets of the urban population produced ca. twice as little pollen as those of the rural habitat. pollen potential of rural plants was almost 3-fold higher. the disparity in pollen production between plant populations may be related to differences in environmental conditions. in particular, a water deficit and air temperatures that exceed the norm is known to impair the mass of pollen produced in anthers (hedhly et al. 2009, denisow 2011). such environmental conditions are probably associated with an uhi (rysiak and czarnecka 2018). the estimated sugar yield 25.4 – 47.4 kg ha–1 and pollen yield 57.8 – 212.7 kg ha–1, indicates that h. tuberosus may be considered a good forage-yielding plant. the floral reward in h. tuberosus was attractive to insect visitors. the availability of food resources in h. tuberosus flowers is important to enhance food for pollinators during late summer time, i.e. in a period of poor resources for pollinators. in particular, the pollen available in h. tuberosus is very important in the late season and can enhance the overwintering of bee colonies and their strength in spring (di pasquale et al. 2016). the pollen produced in h. tuberosus was attractive to a variety of pollinators and not just apis mellifera. the fact underlines the importance of the plants in the maintenance of general insect biodiversity. in conclusion, the season of blooming (late summer/ autumn), high nectar and pollen production as well as attractiveness of the reward for insect visitors indicate that h. tuberosus is important to enhance food resources for pollinators. however, due to the invasiveness potential of h. tuberosus, the species should be propagated with caution. acknowledgements the research was supported financially by the ministry of science and higher education of poland as a part of 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the marine littoral of the central mediterranean gabrielle zammit1,2*, sarah schembri1,2, mark fenech1,2 1 laboratory of applied phycology, centre for molecular medicine and biobanking, university of malta, msida, malta 2 microbiology lab, department of biology, faculty of science, university of malta abstract – phototrophic biofilm and microbial mat communities grow along the rocky coastline of the maltese islands. this research involved studying phototrophs from the mediolittoral and supralittoral zones over a two-year period and seasonal changes were observed. attachment of pioneer microorganisms to the porous eroded limestone bedrock was facilitated via a gelatinous matrix composed of exopolymeric substances (eps). in submerged areas, such as undisturbed rock pools, these progressively formed green or brown compact biofilms, some of which thickened over the spring to form microbial mats via the production of more extensive eps layers. microbial mats gradually attained a lighter colouration due to the presence of ultraviolet (uv) screening pigments. in full summer, they were observed to shrink, detach from the exposed substrate, harden and progressively calcify. biofilm microorganisms survived the harsh summer months in sheltered areas. the major biofilm formers were filamentous non-heterocytous cyanobacteria belonging to the leptolyngbyaceae, pseudanabaenaceae and oscillatoriaceae. their sheaths were thick, lamellated and often confluent. a higher biodiversity of phototrophs was observed in late autumn and winter, when tufts of heterocytous calothrix sp. grew on thin compact biofilms of nodosilinea sp., toxifilum sp. and phormidesmis spp., while lyngbya spp. trichomes were surrounded by thick brown sheaths. germlings of green and brown macroalgal species belonging to ulva, cladophora and sphacelaria were embedded in biofilms and microbial mats and gradually grew to form extensive macroalgal covers submerged in rock pools. erythrotrichia sp. filaments colonised the mediolittoral zone and were confined to areas that were exposed to wave action and submerged intermittently. over the summer, macroalgal coverage diminished and microalgal biofilms and microbial mats prevailed in rock pools. keywords: biofilm, cyanobacteria, marine littoral, microalgae, microbial mat, mediterranean introduction phototrophic biofilms and microbial mats grow on coastal rocky shores around the maltese islands. they constitute under-explored communities as there is an overall lack of research about the biotic assemblages of maltese rocky shores (schembri et al. 2005) and of the mediterranean area in general, as evidenced bythe lack of scientific literature published on the subject. a biofilm can be defined as a self-sustaining community of microorganisms associated with a substrate that is several millimetres thick (dang and lovell 2015). on the other hand, microbial mats (also referred to as biomats) are stratified microbial communities, commonly associated with aquatic habitats, which for the purpose of this study were over one centimetre in thickness. many of the microbial associations within biofilms and microbial mats are symbiotic, which confers a selective advantage on the community as a whole. in phototrophic communities, a significant proportion of the microorganisms are photosynthetic and highly dependent on the presence of light. research interest in these communities is related to the discovery of new taxa that produce novel enzymes with high biotechnological potential in green environmental solutions and biomedical applications (prieto-barajas et al. 2018). this study consists of a structural exploration of phototrophic biofilms and microbial mats occurring on the rocky shores of the maltese islands and a morphological descrip* corresponding author e-mail: gabrielle.zammit@gmail.com marine biofilms and biomats in the central mediterranean acta bot. croat. 80 (1), 2021 113 tion of the cyanobacterial and algal biodiversity comprising these communities. the main objectives were to identify any seasonal variations and to attempt to further knowledge about the strategies adopted by these communities that enable them to form on hostile surfaces and thus persist throughout different seasons. areas of the maltese coastline colonised by phototrophic communities were visually observed and documented over a two-year period, and representative biofilms and microbial mats were then seasonally sampled and directly observed by light and electron microscopy. to our knowledge, this is the first such study of marine phototrophic biofilms and microbial mats growing on rocky shores in the central mediterranean region. materials and methods phototrophic microbial communities growing in the mediolittoral and supralittoral zones of different coastal locations around the maltese islands were studied over a twoyear period starting in autumn 2017 and seasonal changes were visually observed and documented. representative biofilms and microbial mats were then sampled from the rocky shoreline around st julian’s tower in sliema (35°55’05.6” n, 14°29’57.8” e) and from the coastline of the village of baħar iċ-ċagħaq (35°55’06.4” n, 14°29’58.0” e) in malta during autumn 2017, spring and autumn 2018 and again during spring 2019. sampling was carried out during autumn and spring, as these were the seasons in which visible changes in biofilm and biomat structure and extent of growth were recorded. sampling took place during november 2017, may and december 2018 and again during april 2019. since the growth of endoliths was not expected, sampling of the phototrophic communities was carried out using techniques that were non-invasive to the underlying rock. the biofilms and microbial mats were transferred to enriched sea water (provasoli 1968) in petri dishes and incubated at 20 °c with a photoperiod of 10 hours. the sampled phototrophic communities and the same phototrophic communities growing in culture were observed by means of light microscopy using an olympus bx 51 microscope equipped with an olympus dp-71 digital camera. for transmission electron microscopy (tem), biofilm microsamples were fixed in 2.5% glutaraldehyde, postfixed in a 1% osmium tetroxide solution, dehydrated in a graded ethanol series, and embedded in epoxy resin (epoxy 812 resin kit, multilab supplies, newcastle upon tyne, uk). thin sections were collected on copper grids, stained with uranyl acetate and lead citrate (reynolds 1963) and were observed using an h-7100 tem (hitachi, tokyo, japan) operating at 100 kv. identification of phototrophic biofilm and biomat-forming organisms was carried out using morphological keys for cyanobacteria (komárek and anagnostidis 1999, 2005, komárek 2013), chlorophyta (ettl and gärtner 2013, cormaci et al. 2014, lange-bertalot et al. 2017, škaloud et al. 2018), phaeophyceae (cormaci et al. 2012) and rhodophyta (cormaci et al. 2017). results and discussion seasonal changes in biofilm and biomat structure different phototrophic communities grew as biofilms and microbial mats in the marine littoral zone (fig. 1). in submerged areas, such as undisturbed rock pools or salt pans, microorganisms formed green or brown coloured compact biofilms (fig. 1a). during spring and summer, similar phototrophic communities prevailed. these were characterised by an abundance of microbial mats located in rock pools found in the mediolittoral and supralittoral zones. towards the end of spring, the warm weather and restricted wave action led to biofilms becoming restricted to rock pools in the mediolittoral zone. seasonality was a crucial factor affecting ecological succession. in fact, some microbial communities thickened over spring to form microbial mats via the production of more extensive eps layers. these gradually attained a lighter colouration, probably due to the presence of eps and uv screening pigments such as carotenoids and scytonemins in the upper layers. in full summer, both biofilms and microbial mats survived only close to the shore, submerged in rock pools. on the other hand, exposed biofilms appeared shrivelled, fragmented and progressively detached from the rock surface (fig. 1b). the mediolittoral zone became reduced during summer due to high average temperatures above 28 °c, a uv index above 9 and low average wind speeds of 2.9 km/h. this led to biofilms being restricted to the area of the mediolittoral zone closest to the shore. biofilm and microbial mat organisms survived only in rock crevices and shaded areas (fig. 1c). dry biomats hardened and became progressively calcified. during autumn, succession occurred, when the rock surfaces previously conditioned by biofilm growth, became colonised by macroalgae. composition of the phototrophic communities microscopic observations showed that these phototrophic biofilms and biomats were highly diverse communities composed of both phototrophic and heterotrophic microorganisms. each phototrophic community had a distinctive morphology and species composition which depended on the respective microhabitat. the predominant microorganisms in communities that were submerged from autumn to spring were filamentous cyanobacteria including fine leptolyngbyaceae filaments belonging to leptolyngbya sp., nodosilinea sp., pseudanabaenaceae of toxifilum sp. and non-heterocytous oscillatoria spp., phormidium spp. and lyngbya spp. strains. dense networks of these filamentous cyanobacteria provided structural strength to the biofilms and microbial mats due to their ability to grow intertwined in sheets or bundles of filaments (fig. 2), in which other organisms, such zammit g., schembri s., fenech m. 114 acta bot. croat. 80 (1), 2021 fig. 1. dark biofilms grow submerged in man-made salt pans or natural rock pools along the maltese coastline (a), dry biofilms shrink and completely detach from dry horizontal rock surfaces in full summer (b), biofilm microorganisms survive in crevices and shaded areas on vertical rock faces (c). fig. 2. biofilm and biomat forming cyanobacteria lyngbya sp. trichomes surrounded by dark brown sheaths (a), calothrix sp. filaments with hyaline hair (b), arrows indicate profuse trichome fragmentation and formation of hormogonia (c), release of spherical propagules from the open ends of sheaths (d). tem micrographs of biofilm-forming pseudanabaenaceae and leptolyngbyaceae. a biofilm consisting exclusively of fine filaments of toxifilum sp., arranged both longitudinally and horizontally, each surrounded by thick polysaccharide sheaths (e). a different biofilm composed of a more diverse community of nodosilinea sp. filaments with heterotrophic bacteria embedded in a dense eps matrix (f ). scale bars: 25 µm (a, b), 20 µm (c), 10 µm (d), 5 µm (e, f ). as the filamentous heterocytous cyanobacteria nunduva sp. and calothrix sp. (fig. 2b) and diverse coccal microalgae became embedded. coccal cyanobacteria included species of aphanocapsa sp. and chroococcus sp., while microalgal strains belonged to species of chlamydomonas, chlorella, coelastrella and navicula. a higher biodiversity of phototrophs was observed in autumn and winter, mainly due to the occurrence of macroalgal germlings in addition to the microbial taxa. the included the filamentous macroalgae ulva croatica, ulva flexuosa, cladophora ruchingeri and sphacelaria sp. filaments of erythrotrichia sp. colonised the mediolittoral zone and were mostly confined to areas that were exposed to wave action and submerged intermittently in autumn and winter. streptomycetes, micronematodes, ciliates and microcrustaceans were also observed living and feeding within the biofilm and biomat communities. adaptation and survival strategies these biofilms and microbial mats formed in response to the prevailing stressful environmental conditions related to temperature, solar irradiation, dehydration and salinity prevalent in the maltese islands. the layered 3-d structure gave better overall protection than that of constituent microorganisms, such as for instance, the uv protection conferred individually by the cyanobacterium lyngbya and the thermotolerant microalga coelastrella. in such microorganmarine biofilms and biomats in the central mediterranean acta bot. croat. 80 (1), 2021 115 isms, salt and light stress also accelerate carotenoid pigment and oil production (hu et al. 2013). lyngbya aestuarii was common in microbial mat structure. trichomes in the upper layers of microbial mats were surrounded by a thick brown lamellated sheath (fig. 2a), which probably contributed to protection against uv radiation (280-400 nm). the filaments formed several separation discs and frequently fragmented into short filaments, which have the potential to grow under favourable conditions (rath and adhikary 2007). the species is known to grow within microbial mats present in the marine intertidal zone, in which it performs roles vital to the community and connected to photosynthesis, protection, and hydrogen production, which is considered a key metabolite (kothari et al. 2013). tufts of heterocytous calothrix and nunduva spp. filaments provided for nitrogen (n) fixation. calothrix filaments were heteropolar, with a heterocyte at the basal part of the filament that provided for n fixation and the end of the trichome narrowed into a long hyaline hair to facilitate the uptake of phosphorus (p) (fig. 2b). each of these survival strategies provided a competitive advantage which contributed to the success of the biofilm or microbial mat community as a whole. tem revealed that the attachment of pioneer microorganisms to the porous eroded limestone bedrock was facilitated via a gelatinous polymeric matrix, in which the whole community ultimately became embedded (fig. 2e, f ). individual trichomes and cells were surrounded by thick gelatinous sheaths that were often confluent (fig. 2) and that enabled cells and trichomes to glide in a protected microenvironment that retained the moisture around them (fig 2f). the production and subsequent release of propagules were frequent (fig. 2c, d). the microorganisms were also observed to alter their morphology as a survival strategy to adapt to fluctuating seasonal environmental conditions. one such change was fragmentation observed in lyngbya sp., in which filaments became fragmented into several smaller pieces allowing faster adaptation. other filaments, especially those belonging to the leptolyngbyaceae and oscillatoriaceae were observed to coil and compress, allowing the organism to selfshade within biofilm or biomat structure (wu et al. 2005). microbial mats provided an adequate survival strategy capable of withstanding the harsh environmental conditions prevailing towards the end of spring and summer. in fact, the top protective eps layer became thicker, allowing microorganisms living in the lower layers to survive. the biomats formed to counteract water shortage, since the larger volume of eps retains more water via hydrogen bonding, facilitating water absorption and preventing its quick loss, thus allowing the communities to survive in the supralittoral zone, where they are only hydrated by sea spray. the eps also acted as a buffer preventing rapid micro-environmental changes from occurring within the microbial mat. lyngbya aestuarii was often found in the top layers of microbial mats, where it provided uv protection due to its thick lamellated pigmented sheaths that are known to contain scytonemin (rath and adhikary 2007). other microorganisms which required uv protection, such as leptolyngbyaceae and rivulariaceae filaments, were found in the bottom layers closer to the substrate. in fact, another survival strategy involved the migration of filaments, where microorganisms such as leptolyngbyaceae were able to glide and migrate to the bottom layer of the microbial mat, especially during spring and summer. this strategy has also been recorded and studied in other genera, for instance, synechococcus isolates from octopus spring, yellowstone national park were observed to migrate away from a strong light source via cell gliding (ramsing et al. 1997). the biofilms and microbial mats of the marine littoral constitute unexplored microbial communities in the central mediterranean region. in fact, they often contain understudied taxa such as those belonging to the genera toxifilum sp. and nunduva sp. that were identified also in this study and have only been recently described (zimba et al. 2017, gonzález-resendiz et al. 2018). these microorganisms are currently being investigated via a polyphasic approach, involving the application of molecular genetics and bioinformatics tools to identify relevant species. an improved understanding of the biodiversity and survival adaptations of such phototrophic biofilms and biomats would contribute greatly 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(cyanobacteria, cyanophyceae). journal of phycology 53, 188–197. acta bot. croat. 77 (2), 2018 141 acta bot. croat. 77 (2), 141–151, 2018 coden: abcra 25 doi: 10.2478/botcro-2018-0016 issn 0365-0588 eissn 1847-8476 antibacterial, cytotoxic and trypanocidal activities of marine-derived fungi isolated from philippine macroalgae and seagrasses kin israel notarte1,2,3*, takashi yaguchi4, keisuke suganuma5, thomas edison dela cruz1,2,3* 1 the graduate school, 2 college of science and 3 fungal biodiversity, ecogenomics and systematics group, research center for the natural and applied sciences, university of santo tomas, españa 1008, manila, philippines 4 medical mycology research center, chiba university, 1-8-1 inohana, chuo-ku, chiba 260-8673, japan 5 national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, inada, obihiro, hokkaido 080-8555, japan abstract – the occurrence and bioactivities of marine-derived fungi are evaluated in this paper. a total of 16 morphospecies of marine-derived fungi (mdf) were isolated from four host macroalgae and two seagrasses and identified as belonging to the genera aspergillus, fusarium, paecilomyces, penicillium, sclerotinia, thamnidium and trichoderma, including five mycelia sterilia. among these host organisms, the rhodophyte laurencia intermedia harboured the highest number of isolated mdf. selected mdf were then assayed and showed to inhibit pseudomonas aeruginosa (8-19 mm zone of inhibition) and staphylococcus aureus (6-19 mm zone of inhibition), and were cytotoxic against the brine shrimp artemia salina nauplii (ld50: 201.56-948.37 µg ml-1). the screening led to the selection of five of the most bioactive morphospecies, all belonging to the genus aspergillus. these marine aspergilli were subjected to β-tubulin gene sequence analysis for species identification, and to mass production in different culture media with or without marine salts, and screening of the crude culture extracts for their cytotoxic and trypanocidal activities. aspergillus tubingensis cultivated in potato dextrose broth with marine salt proved to be the most cytotoxic against p388 (ic50: 1028 ng ml-1) and hela (ic50: 1301 ng ml-1) cancer cells. on the other hand, a. fumigatus cultivated in malt extract broth without marine salt was shown to be the most potent against trypanosoma congolense (ic50: 298.18 ng ml-1). our study therefore showed that salinity may influence the bioactivities of some species of mdf. key words: bioactivity, fungal natural products, marine fungi, philippines abbreviations: asw – artificial seawater; dmso – dimethyl sulfoxide; mdf – marine-derived fungi; mea – malt extract agar; meas – malt extract agar with marine salt; meb – malt extract broth; mebs – malt extract broth with marine salt; pdb – potato dextrose broth; pdbs – potato dextrose broth with marine salt; zoi – zone of inhibition * corresponding authors, e-mails: kinotarte@gmail.com, tedelacruz@ust.edu.ph introduction the ocean has vast biological resources, accounting for more than eighty percent of total world biodiversity, which makes it a reservoir for many kinds of organisms, including marine-derived fungi (bugni and ireland 2004). these marine-derived fungi (mdf) grow and possibly sporulate in the marine ecosystem (kohlmeyer and kohlmeyer 1979). interestingly, many of these marine-derived fungi produce structurally diverse and bioactive compounds (dela cruz et al. 2006a, schulz et al. 2008, silber et al. 2016), utilized varied substrata (dela cruz et al. 2006b), and may play an important ecological role in marine habitats (solis et al. 2010). in spite of their economic and ecological importance, some of the less explored mdf thrive inside healthy tissues of macroalgae, e.g. in sargassum thunbergii (miao et al. 2012), and seagrasses, such as cymodocea serrulata and halophila ovalis (supaphon et al. 2013). notarte k. i. r., yaguchi t., suganuma k., dela cruz t. e. 142 acta bot. croat. 77 (2), 2018 various biochemical properties with anti-infective and anti-tumor activities have also been documented in algae and seagrass-derived marine fungi (greve et al. 2008, supaphon et al. 2013, 2014). for instance, aspergillus flavus isolated from the green alga enteromorpha tubulosa synthesized two new 5-hydroxy-2-pyrone derivatives that could induce production of camp on gpr12-transfected cells (lin et al. 2008). penicillium chrysogenum isolated from the red alga laurencia produced the novel antifungal and cytotoxic penicisteroids a and b (gao et al. 2011). despite the promising chemicals that these fungi produce, there are no drugs from these fungi that have been commercially approved (gerwick and moore 2012). obviously, this necessitates the need to do more research on these organisms in order to find bioactive and potentially novel chemicals for drug discovery and development. thus, this recent study isolated marine-derived fungi in philippine macroalgae and seagrasses, and tested their biological activities for potential chemotherapeutic uses. materials and methods screening of marine-derived fungi for biological activities collection of macroalgae and seagrasses four species of algae, belonging to phaeophyta [sargassum piluliferum (turner) c. agardh], chlorophyta [caulerpa racemosa (forsskål) j. agardh], and rhodophyta [laurencia intermedia yamada and portieria hornemannii (lyngbye) p. c. silva], and two species of seagrasses, enhalus acoroides (linnaeus) royle and syringodium isoetifolium (ascherson) dandy, were collected from the intertidal (9°19'15.82"n, 123°18'45.56"e) and subtidal (9°19'15.82"n, 123°18'46.53"e) zones of piapi beach, dumaguete city, negros oriental, philippines. the collected specimens were washed three times with filtered seawater (fsw) to get rid of adhering soil and epiphytes. these were placed in clean ziplock bags containing an ample amount of fsw. the samples were kept on ice and processed in the laboratory within 48 h. identification of the host algae and seagrasses was done following detailed morphological characterization of voucher specimens. isolation and identification of marine-derived fungi the thalli, roots, fronds and leaves of the collected algae or seagrasses were initially washed with sterile artificial seawater (asw, 33 g marine salt dissolved in 1l distilled water). with a sterile razor blade, the specimens were cut into 3-5 mm explants and immersed in 70% ethanol (etoh) for 60 s to remove surface-associated microorganisms. the explants were then washed three times with sterile asw for 3 min and blotted dry with sterile cotton cloth. afterwards, the explants were transferred onto petri plates pre-filled with ½-strength malt extract agar containing 33 g l–1 marine salt (meas). to prevent growth of contaminating bacteria, the culture media were supplemented with streptomycin (450 µg ml-1). six explants were placed on each culture plate. in all, five plates containing a total of 30 explants were used for each algal or seagrass species collected. tissue printing was performed by carefully placing some of the explants over the surface of meas plates using sterilized tweezers to evaluate the effectivity of the surface-sterilization treatment. for sterility control, two uninoculated plates were exposed to the working environment to rule out air contamination. the culture plates were incubated for 1 week at room temperature and were monitored daily for fungal growth. fungal colonies that grew out of the edges of the explants were subcultured on freshly prepared full-strength meas and purified by spore touch technique. identification of isolates was achieved by comparing their colony, hyphal, and spore morphologies with the published literature, i.e. klinch (2002), raper and fennell (1977), and quimio (1988). to test if the isolated fungi were adapted to the marine environment, the colony extension rate (cer) for the isolates grown in mea with or without salt was computed using the formula: [mean colony radial growth (day 7) — mean colony radial growth (day 3)] / number of days of incubation (4 days). a paired ttest was computed for the cer on mea and meas to determine if the presence or absence of marine salt in the medium significantly affected the colony extension rate of the mdf. mass production and extraction of metabolites twenty-one mdf were mass produced by transferring an agar block cut from the margin of a 7-day old fungal colony onto erlenmeyer flasks containing 100 ml malt extract broth supplemented with 33 g l–1 marine salt (mebs). a total of five culture flasks were prepared for each fungal isolate and were maintained in stationary condition at room temperature for 4 weeks. following incubation, the contents of the culture flasks coming from the same fungal isolate were pooled and extraction of secondary metabolites was performed by initially separating the broth and mycelia. the culture broth was extracted with ethyl acetate in 1:1 (v/v) proportion, while the harvested mycelia were homogenized and then soaked in methanol. the ethyl acetate and methanol extracts were concentrated in vacuo and stored in preweighed vials. screening for antibacterial activities the test bacteria (staphylococcus aureus atcc 25923, escherichia coli atcc 25922, and pseudomonas aeruginosa atcc 27853) were acquired from the university of santo tomas collection of microbial strains (ustcms), manila, philippines and maintained on tryptic soy broth (tsb). following the protocol of quinto and santos (2005), the 24-h old bacterial cultures were transferred to tubes containing 5 ml of 0.9% normal saline solution (nss) and standardized to 0.5 mcfarland. then, a sterile swab was dipped on the cell suspension and streaked onto mueller hinton agar (mha) plates. the crude extracts from the mycelia and culture broth were weighed and dissolved in dmso to achieve a concentration of 50 mg ml–1. the sterile whatman paper disks (6 mm in diameter) were then impregnated with 20 µl of crude extracts and air-dried aseptically, giving a final concentration of 1 mg per disk of extract. the treated paper bioactive marine-derived fungi associated with macroalgae and seagrasses acta bot. croat. 77 (2), 2018 143 disks were placed on top of the mha plate seeded with the test bacteria. the control antibiotic was streptomycin (10 μg) while the negative control was dmso. all culture plates (in triplicates) were incubated at 37 °c for 24 h and the zone of inhibition (zoi) was measured in mm. the observed zone of inhibition was then compared with the controls and the reference antibiotics listed in eucast ver. 7 (2017). screening for brine shrimp cytotoxicity dried cysts of artemia salina were hatched in asw (1 g cyst per liter) at room temperature with continuous illumination. asw was prepared by dissolving 38 g of commercially available marine salt in distilled water. after hatching, the nauplii were collected with a pipette and concentrated in a vial. the testing of fungal culture extracts was conducted in 6-well microplates. the stock solution was prepared by dissolving the extracts in dmso at 5 mg ml–1. aliquots of 0.10, 0.20, 0.40, 0.60, 0.80, and 1 ml of stock solution were diluted with 4.90, 4.80, 4.60, 4.40, 4.20, and 4 ml of brine water to make up a final concentration for the extracts of 100, 200, 400, 600, 800, and 1000 µg ml–1, respectively. the negative control was dmso. ten brine shrimps were then carefully transferred to each well with a pipette and these were incubated with the fungal culture extracts for 24 h. the treatments and the control were tested in triplicate, and the number of nauplii that survived was counted for ld50 determination using probit regression analysis at 95% confidence interval. effects of salinity and culture media on the bioactivities of marine aspergilli selection and molecular characterization of marine aspergilli the marine aspergilli were selected based on the results of the disk diffusion and the brine shrimp cytotoxicity assays. identification of these strains was done via molecular methods. the dna of the five marine aspergilli was extracted using the gentorukun® (takara bio inc., ltd., otsu, japan) from approximately 100 µl volume of fungal mass cultured at 25 °c for 5 days on potato dextrose agar (pda) slants. the β-tubulin gene was sequenced directly from pcr products using bt2a (5’-ggtaaccaaatcggtgctgctttc-3’) and bt2b (5’-accctcagttgagtgacccttggc-3’) primer pair (glass and donaldson 1995). the pcr conditions consisted of denaturation at 95 °c for 10 min followed by 35 cycles of 95 °c for 1 min, 55 °c for 1 min, and 72 °c for 1 min, with a final extension at 72 °c for 10 min. the pcr products were then sequenced using the bigdye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, ca, usa) on an abi prism®3130abi genetic analyzer (applied biosystems) and the dna sequences were edited using atgc ver. 4 sequence assembly software (genetyx co., tokyo, japan). using the blast algorithm, the β-tubulin gene sequence was used to search the genbank database at the ncbi website (http:// blast.ncbi.nlm.nih.gov/blast.cgi). the phylogenetic tree was then constructed using the consensus sequence based on paup maximum parsimony analysis. mass production and extraction of metabolites from marine aspergilli five selected species of marine aspergilli were mass produced in solid rice medium and the liquid media, malt extract broth (meb/mebs) and potato dextrose broth (pdb/ pdbs) with and without marine salt. a total of five erlenmeyer flasks each containing 100 ml of culture media were prepared for each marine aspergillus. these were maintained under stationary conditions at room temperature for 4 weeks. for the marine aspergilli cultivated in the liquid media, the culture broth and the fungal mycelia were separated following incubation. the culture broth was extracted with ethyl acetate in 1:1 (v/v) proportion while the mycelia were homogenized and soaked in methanol. for the marine aspergilli cultivated in solid rice medium, only ethyl acetate extraction was performed. the ethyl acetate and methanol extracts were concentrated in vacuo and stored in preweighed vials. methyl thiol tetrazolium (mtt) cytotoxicity assay following the protocol of takada et al. (2012), hela cervical cancer cells were cultured in dulbecco's modified eagle medium (wako pure chemical industries, osaka, japan), containing 10% fetal bovine serum, 2 mg ml–1 gentamycin, and 10 mg ml–1 antibiotics adjusted to ph 7.0–7.4 by 1 m hcl, while p388 murine leukemia cancer cells were cultivated in roswell park memorial institute medium-1640 (wako pure chemical industries), containing 10% fetal bovine serum, 100 mg ml–1 kanamycin, and 10 mm 2-hydroxyethyldisulfide. the cell lines were incubated at 37 °c under an atmosphere of 5% co2. afterwards, an aliquot of 200 µl tumor cell suspension (1 × 104 cells ml–1) was dispensed unto each well of the 96-well microplate and pre-incubated for 24 h. following pre-incubation, 1 µl of marine aspergilli extracts dissolved in dmso concentrated at 1 mg ml–1 was added to the cell suspension to prepare a screening concentration of 5 µg ml–1. the reference chemotherapeutic drug was adriamycin. after treatment, the cells were further incubated for 72 h and to each well 50 µl of 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2h-tetrazolium bromide (mtt) saline solution (1 mg ml–1) was added, followed by incubation for 3 h under the same condition to stain live cells. finally, the culture medium was withdrawn and 150 µl of dmso was added to dissolve cells. using the fusiontm α microplate reader (packard bioscience company, ct, usa), the absorbance of each well was read. the ic50 for the most bioactive aspergilli extracts was calculated by plotting a dose-dependent curve in graphpad prism 5 software (graphpad software inc., ca, usa). atp-based luciferase viability assay for trypanosoma congolense following the protocol of suganuma et al. (2014), the blood stream form of trypanosoma congolense il 3000 was cultivated at 33 °c in air using iscove's modified dulbecco's medium (sigma-aldrich, tokyo, japan), containing 20% heat inactivated-fetal bovine serum, 60 mm hepes, notarte k. i. r., yaguchi t., suganuma k., dela cruz t. e. 144 acta bot. croat. 77 (2), 2018 1 mm pyruvic acid sodium salt, 0.1 mm bathocuproine, 1 mm hypoxanthine, 16 μm thymidine, 10 μg l–1 insulin, 5.5 μg l–1 transferrin, 6.7 ng l–1 sodium selenite, 0.0001% 2-β-mercaptoethanol, 0.4 g l–1 bsa, and 2 mm l-cysteine. the screening concentration for the fungal extracts was set at 2.5 µg ml–1. this was prepared by initially diluting 0.5 µl of fungal crude extract (1 mg ml–1) with 100 µl hmi9. from this solution, a 50-µl aliquot was dispensed to the microplate and added to each well containing 50 µl suspension of t. congolense (2 × 105 cells ml–1). the reference antiprotozoal drug was pentamidine. the microplates were incubated for 72 h, and subsequently, 50 µl of celltiter-glo® luminescent cell viability assay reagent (promega japan, tokyo, japan) was added to each well to evaluate atp concentration. following the incubation of the microplates for another 10 min at room temperature, luminescence was read using a glomax®-multi detection system plate reader (promega japan, tokyo, japan). the ic50 values of the most bioactive aspergilli extracts were computed by plotting a dosedependent curve in graphpad prism 5 software (graphpad software inc.). moreover, the selectivity index (si) of the bioactive extracts was also calculated following the formula of koch et al. (2005): ic50 hela or p388 / ic50 trypanosoma congolense. results occurrence of marine-derived fungi in macroalgae and seagrasses a total of 40 mdf were isolated from algae and seagrasses collected from piapi beach, philippines. these mdf belonged to 16 morphospecies from the genera aspergillus, fusarium, paecilomyces, penicillium, sclerotinia, thamnidium, and trichoderma (tab. 1). among these genera, aspergillus was isolated in the highest frequency. five of the mdf could not be identified as they remained sterile even after prolonged incubation, hence were simply designated as mycelia sterilia. among the host macroalgae and seagrasses, the red alga l. intermedia harbored the highest number of isolates, followed by the seagrass e. acoroides, then the brown alga s. piluliferum, and the seagrass s. isoetifolium. intriguingly, only one mdf was isolated from the red alga p. hornemannii and from the green alga c. racemosa. the identified fungal genera were known to be of terrestrial origin. hence, to assess the adaptability of the isolates in the marine environment, the mdf were cultured in mea with or without marine salt. of the 21 mdf tested, 17 grew much better in meas than in mea (fig. 1). statistical analysis by one-tailed paired t-test confirmed that the mean colony extension rates in meas were greater than mea and that the difference was highly significant (p-value 0.000349 < α = 0.01). antibacterial and cytotoxic activities of marine-derived fungi several mdf showed antibacterial properties and brine shrimp cytotoxicity (tab. 2). of the 21 mdf tested, five fungal isolates belonging to the genus aspergillus and one mycelia sterilia inhibited p. aeruginosa and/or s. aureus with zoi greater than 10 mm. the most bioactive crude extracts were the mycelial extract of a. fumigatus against p. aeruginosa (19 mm zoi) and the broth extract of a. tubingensis against s. aureus (19 mm zoi). the zoi of the most bioactive culture extracts were comparable to the control antibiotic streptomycin (18 – 21 mm zoi) and with some reference antibiotics listed in eucast ver. 7 (2017). interestingly, no antagonism to e. coli treated with the mdf culture extracts was observed. meanwhile, of the 21 mdf tested for cytotoxicity, seven mdf, belonging to the genera aspergillus, fusarium, paecilomyces, penicillium and trichoderma, proved to be cytotoxic to a. salina nauplii with ld50 ranging from 201.56948.37 µg ml–1. the most cytotoxic was a. tubingensis. thus, members of the genus aspergillus were noted to be generally bioactive in this study. furthermore, there was higher record of bioactivities with the ethyl acetate extracts derived from the fungal culture broth as opposed to the methanolic extracts derived from the fungal mycelia. molecular characterization of marine aspergilli the marine aspergilli that showed promising bioactivities in the previous screening were subjected to molecular characterization to validate their identity. comparison of their β-tubulin gene sequences by blast searching showed fig. 1. colony extension rates of marine-derived fungi grown on malt extract agar with (meas) or without (mea) marine salt (n=3). mean standard deviation is expressed in error bar. bioactive marine-derived fungi associated with macroalgae and seagrasses acta bot. croat. 77 (2), 2018 145 100% similarities between the sequences of the isolated marine aspergilli and those deposited at genbank. this homology was further supported by their phylogenetic tree constructed according to maximum parsimony analysis with bootstrap values 90% and above (fig. 2). molecular analysis confirmed the marine aspergilli isolates as a. fumigatus (af-st-02), a. flavus (af-sl-02), a. niger (af-c-01), a. terreus (af-l-12) and a. tubingensis (sf-el-06). all the dna sequences were deposited at the dna data bank of japan (ddbj) with the accession number lc176429 for a. terreus, lc176430 for a. niger, lc176431 for a. flavus, lc176432 for a. fumigatus, and lc168474 for a. tubingensis. tab. 1. marine-derived fungi (mdf) isolated from macroalgae and seagrasses collected from piapi beach, dumaguete city, philippines. anames in bold grew better in malt extract agar with salt. names with asterisks were isolated in more than one host alga/seagrass. host alga/seagrass substrata marine-derived fungia total no. of isolated mdf laurencia intermedia (rhodophyte) thallus aspergillus sp. 1* aspergillus sp. 2 aspergillus flavus* aspergillus terreus fusarium sp.* thamnidium sp. trichoderma sp. 1* trichoderma sp. 2* mycelia sterilia 1 mycelia sterilia 2 13 portieria hornemannii (rhodophyte) thallus mycelia sterilia 3 1 sargassum piluliferum (phaeophyte) frond aspergillus sp. 1* aspergillus flavus* 4 thallus aspergillus fumigatus fusarium sp.* trichoderma sp. 2* 4 caulerpa racemosa (chlorophyte) thallus aspergillus niger 2 enhalus acoroides (seagrass) root paecilomyces sp.* trichoderma sp. 1* 4 leaf aspergillus sp. 1* aspergillus sp. 3 aspergillus tubingensis mycelia sterilia 4 mycelia sterilia 5* 6 syringodium isoetifolium (seagrass) leaf aspergillus sp. 4 paecilomyces sp. * penicillium sp. sclerotinia sp. mycelia sterilia 5* 6 notarte k. i. r., yaguchi t., suganuma k., dela cruz t. e. 146 acta bot. croat. 77 (2), 2018 effects of culture media to the cytotoxicity of marine aspergilli a total of 45 culture extracts were obtained from the marine aspergilli cultivated in five different media with and without marine salt. of the marine aspergilli tested at 5 µg ml–1 screening concentration, a. tubingensis and a. fumigatus were shown to be the most cytotoxic against cancer cells (fig. 3). the ethyl acetate crude extracts of a. tubingensis derived from the culture media supplemented with salt (mebs and pdbs) had a percent inhibition ranging from 98–99% against p388 and from 70–76% against hela. the bioactivity of these extracts against p388 was comparable with the chemotherapeutic drug adriamycin, which completely inhibited the cancer cell lines. interestingly, the crude extracts derived from the same fungus cultivated in media without salt (meb, pdb and solid rice) provided less inhibition, which ranged from 21–76% for both cancer cell lines. for a. fumigatus, the ethyl acetate crude extract derived from meb was the most active with 62.81 and 68.55% inhibition for hela and p388, respectively. for the other marine aspergilli, a. niger, a. terreus, and a. flavus showed an inhibitory activity of less than 40% for both cancer cells regardless of whether the fungi were cultivated in media with or without marine salt and of whether the extracting solvent was methanol or ethyl acetate. based on the initial screening of 45 extracts, four candidate extracts derived from a. fumigatus and a. tubingensis were selected for ic50 determination (tab. 3). in this study, only the crude culture extracts with more than 50% inhibition were considered for ic50 determination. the most bioactive was the ethyl acetate extract of a. tubingensis mass produced in pdbs with an ic50 at 1028 ng ml–1 for p388 and 1301 ng ml–1 for hela. effects of culture media to the trypanocidal activity of marine aspergilli consistent with the results of the cytotoxicity assay, a. tubingensis and a. fumigatus showed the highest trypanocidal activity at 2.5 µg ml–1 screening concentration (fig. 4). the crude ethyl acetate extracts of a. tubingensis cultivated in media with salt (mebs and pdbs) demonstrated an inhibitory activity of 99% comparable with the antiprotozoal drug pentamidine. in contrary, the crude extracts derived from the same fungus cultivated in media without salt (meb, pdb and solid rice) showed lesser or no inhibitory activity that ranged from 0.53–87.75%. on the other hand, only one extract from a. fumigatus showed promising activity. the crude ethyl acetate extract of the same fungus in meb exhibited an inhibitory activity of 99%, while the remaining extracts from other media provided an inhibitory activity of less than 55%. a. niger, a. flavus, and a. terreus gave an inhibitory activity of less than 52% regardless of culture fig. 2. phylogenetic analysis of marine aspergilli. the consensus sequence was downloaded from genbank and can be retrieved through the following accession numbers: a. flavus kacc46454 (kt354303.1), a. flavus kacc46449 (kt354304.1), a. flavus sousse c12 (kj136090.1), a. terreus nrrl1913 (ef669518.1), a. terreus uoa/hcpf3355 (gq376127.1), a. terreus ibt24859 (fj491707.1), a. niger cm4213 (fj828913.1), a. niger sousse c2 (kj136065.1), a. niger dr02 (kc311845.1), a. tubingensis sousse c41 (kj136086.1), a. tubingensis cm4000 (kj136086.1), a. tubingensis cm4899 (kj136086.1), a. fumigatus af117 (kf410677.1), a. fumigatus af23 (kf410682.1) and a. fumigatus sousse c8 (kj136109.1). bioactive marine-derived fungi associated with macroalgae and seagrasses acta bot. croat. 77 (2), 2018 147 media and addition of marine salts during mass production. furthermore, all the methanolic extracts from marine aspergilli showed weak inhibition towards t. congolense (less than 40%). the ic50 of the most bioactive crude culture extracts was determined (tab. 3) and showed that the ethyl acetate extract of a. fumigatus cultivated in meb was the most potent (ic50: 298.18 ng ml–1) with selectivity index ranging from 7.21–15.50. discussion marine-derived fungi are known colonizers of various organic substrates, including corals, echinoderms, seagrasses, algae, and vertebrates, and can act as endophytes, pathogens, saprobes, and parasites in the marine communities (loque et al. 2010). in this study, 16 morphospecies of marine-derived fungi belonging to the genera aspergillus, fusarium, paecilomyces, penicillium, sclerotinia, thamnidium, and trichoderma including five mycelia sterilia were isolated from macroalgae and seagrasses (tab. 1). among the mdf, the genus aspergillus was isolated in highest frequency and in wide distribution in brown, red and green marine algae, and seagrasses. it is well-reported that aspergillus is not only isolated from terrestrial substrata, but is also widely distributed in the marine environment (kato et al. 2007, cui et al. 2010, li et al. 2011). in this study, all the isolated mdf genera were observed to be terrestrial in origin. however, it is possible for terrestrial fungi to be introduced into the marine ecosystem tab. 2. results of the disk diffusion and brine shrimp cytotoxicity assays of the crude ethyl acetate (etoac) and methanolic (meoh) extracts from marine-derived fungi (n=3). azoi assessment: inactive < 10 mm; partially active 10–13 mm; active 14–19 mm; very active > 19 mm (quinto and santos 2005). bld50 assessment: cytotoxic < 1000 µg ml–1; non-cytotoxic > 1000 µg ml–1 (meyer et al. 1982). marine-derived fungi test bacteria zone of inhibition – zoi (mm)a brine shrimp assay ld50 (µg ml–1)bpseudomonas aeruginosa staphylococcus aureus escherichia coli etoac meoh etoac meoh etoac meoh etoac meoh laurencia intermedia (rhodophyte) aspergillus sp. 1 0 0 0 0 0 0 >1000 >1000 aspergillus sp. 2 0 0 0 0 0 0 >1000 >1000 aspergillus flavus 13 0 10 0 0 0 836.24 948.37 aspergillus terreus 8 0 10 0 0 0 824.71 857.75 thamnidium sp. 0 0 0 0 0 0 >1000 >1000 trichoderma sp. 1 0 0 0 0 0 0 834.14 >1000 mycelia sterilia 1 0 0 0 0 0 0 >1000 >1000 mycelia sterilia 2 13 0 11 0 0 0 >1000 >1000 portieria hornemannii (rhodophyte) mycelia sterilia 3 0 0 0 0 0 0 >1000 >1000 sargassum piluliferum (phaeophyte) aspergillus fumigatus 15 19 14 15 0 0 555.22 807.23 fusarium sp. 9 0 6 0 0 0 834.14 863.29 trichoderma sp. 2 11 0 9 0 0 0 >1000 >1000 caulerpa racemosa (chlorophyte) aspergillus niger 13 0 11 0 0 0 706.76 930.12 enhalus acoroides (seagrass) aspergillus sp. 3 9 0 7 0 0 0 >1000 >1000 aspergillus tubingensis 17 0 19 0 0 0 201.56 333.27 mycelia sterilia 4 0 0 0 0 0 0 >1000 >1000 mycelia sterilia 5 9 0 7 0 0 0 >1000 >1000 syringodium isoetifolium (seagrass) aspergillus sp. 4 0 0 0 0 0 0 >1000 >1000 paecilomyces sp. 0 0 0 0 0 0 >1000 875.01 penicillium sp. 8 0 0 0 0 0 >1000 873.90 sclerotinia sp. 9 0 9 0 0 0 >1000 >1000 dmso (– control) 0 0 0 0 0 0 >1000 >1000 streptomycin (+ control) 21 21 20 20 18 18 – – notarte k. i. r., yaguchi t., suganuma k., dela cruz t. e. 148 acta bot. croat. 77 (2), 2018 and to evolve as a result of selective pressure from this new habitat (jones 1994). as shown in fig. 1, the colony extension rates of the isolated marine-derived fungi were greater in meas as opposed to mea. this shows the adaptability of the mdf to the salinity in the marine environment. following kohlmeyer and kohlmeyer (1979), the isolates were regarded as facultative marine fungi that might have originated from freshwater or terrestrial environment and undergone physifig. 3. cytotoxicity profile of ethyl acetate (e) and methanolic (m) crude extracts (5 µg ml–1) from marine aspergilli against p388 murine leukemia cancer cell line (a) and hela cervical cancer cell line (b). marine aspergilli were previously cultivated in malt extract broth with and without marine salt (meb/mebs), potato dextrose broth with and without marine salt (pdb/pdbs), and solid rice. data are average values ± sd (n=3). tab. 3. median inhibitory concentration (ic50) of ethyl acetate crude extracts from selected marine aspergilli. aassessment of cytotoxicity: ic50 < 30 µg ml–1 signifies anticancer activity (suffness and pezzuto 1990). bassessment of antiprotozoal activity: ic50 < 10 µg ml–1 signifies trypanocidal activity (koch et al. 2005). cassessment of si value for trypanocidal activity: si value > 2 signifies selectivity (koch et al. 2005). marine aspergilli culture medium test organism/cell line (ic50 ng ml–1) selectivityc index (si)p388a helaa trypanosomab congolense aspergillus fumigatus meb 2150 4623 298.18 7.21-15.50 aspergillus tubingensis mebs 2247 1168 381.16 3.06-5.90 aspergillus tubingensis meb 3462 1857 2010.04 0.92-1.72 aspergillus tubingensis pdbs 1301 1028 485.58 2.12-2.68 adriamycin + control 92.85 72.96 – – pentamidine + control – – 100.45 – ological adaptations for their survival in the marine environment. this is further supported by the ability of the mdf to grow in mea with or without marine salt. similar growth characteristic was observed in other marine fungi, as in the case of dendryphiella (dela cruz et al. 2006b). the continuing prevalence of infectious diseases and the challenge to chemotherapy brought about by drug resistance call for the development of a new pipeline of drugs. owing bioactive marine-derived fungi associated with macroalgae and seagrasses acta bot. croat. 77 (2), 2018 149 to their taxonomic and metabolic diversity, marine organisms including marine fungi are now considered ideal sources of new secondary metabolites (schulz et al. 2008). among the tested mdf, a. tubingensis and a. fumigatus conferred the greatest antibacterial and cytotoxic properties (tab. 2). comparing the zones of inhibition of these crude culture extracts with reference antibiotics specified by eucast ver. 7 (2017), the antibacterial property of the mycelial extract of a. fumigatus was more potent than netilmicin while the broth extract of a. tubingensis showed greater inhibition than ampicillin and benzylpenicillin. this is not surprising as the observed bioactivities of the members of the genus aspergillus are well reported (lee et al. 2013). in fact, many novel bioactive compounds have been isolated from marine aspergilli, including waikialoid a (wang et al. 2012) and pseudodeflectusin (ogawa et al. 2004). however, the other isolated mdf (fusarium, paecilomyces, penicillium, sclerotinia, thamnidium, and trichoderma) showed either no bioactivities or fewer than marine aspergilli, which further supports the idea that marine aspergilli are worthy target microorganisms for drug discovery. interestingly, a higher record of bioactivity was observed in the ethyl acetate extracts derived from the culture broth as opposed to the methanolic extracts derived from the mycelia. this means that the bioactive metabolites must have been extracellularly produced by the mdf. thrane et al. (2007) explained that most of the secondary metabolites, organic acids, enzymes, and other bioactive proteins are extracellularly produced by the fungi to their environment. based on the initial screening, the marine aspergilli a. tubingensis, a. fumigatus, a. niger, a. terreus, and a. flavus were selected for further testing against cancer cells and the protozoan t. congolense. consistent with the previous results, a. fumigatus and a. tubingensis showed the greatest anticancer and trypanocidal activities (tab. 3). the study reported for the first time the bioactivity of a marine-derived a. fumigatus against t. congolense. previous studies reported the isolation of a. fumigatus from macroalgae, sea sediment, and holothurians, and showed to produce several cytotoxic compounds such as n-acetyltyramine (zhao et al. 2010) and the apoptosis-inducing metabolite fumigaclavine c (li et al. 2013). moreover, furtado et al. (2005) also reported that a. fumigatus derived from soil samples produced metabolites that are bioactive against t. cruzi, which causes chagas disease. another promising bioactive mdf is a. tubingensis, which was found to be cytotoxic against hela and p388. the isolation and the possible mode of action of its bioactive compound is fully discussed in a separate paper (notarte et al. 2017). nonetheless, the cytotoxicity of a. tubingensis is supported by zhan et al. (2007) when a terrestrial strain derived from the rhizosphere of festuca paradoxa produced metabolites that were active against several cancer cells. the ‘osmac (one strain-many compounds) approach’, which involves manipulation of cultivation parameters, such as the media composition, is an efficient strategy for enhancing the chemical diversity of compounds that may be of interest as lead structures in conducting high-throughput biological screening (bode et al. 2002). wang et al. (2014) demonstrated the potential application of the osmac approach that induced the mdf ascotricha sp. to synthesize three new caryophyllene derivatives when cultured in an oligotrophic rather than the typical eutrophic medium. in the current research, there was no general pattern on the influence of marine salt on the bioactivities of marine aspergilli, although some species were directly influenced by salinity in the production of bioactive metabolites (figs. 3 and 4). interestingly, cultivation in solid rice led to a weaker or loss fig. 4. trypanocidal activity of ethyl acetate (e) and methanolic (m) crude extracts (2.5 µg ml-1) from marine aspergilli cultivated in malt extract broth with and without marine salt (meb/mebs), potato dextrose broth with and without marine salts(pdb/pdbs), and solid rice. data are average values ± sd (n=2). notarte k. i. r., yaguchi t., suganuma k., dela cruz t. e. 150 acta bot. croat. 77 (2), 2018 of bioactivity for the marine aspergilli. indeed, it is worth noting that a. tubingensis proved to have elicited better bioactivities against cancer cells and t. congolense when cultivated in the presence of marine salt regardless of the culture medium used for cultivation. this indicates that salt plays an important role in the synthesis of biologically active components present in the crude extracts of a. tubingensis. the crucial role of salt in the production of compounds was best demonstrated by wang et al. (2011) when cultivation of the marine-derived fungus spicaria elegans in 10% salinity led to the production of novel and chlorinated compounds that were not synthesized when the same fungus was cultured in 3% saline condition. another interesting observation is that of a. fumigatus, which showed promising cytotoxic and trypanocidal activities only when cultivated in meb without marine salt. calvo et al. (2002) showed that the type of carbon and nitrogen source used as precursor molecules during anabolic chemical reactions affects the synthesis of certain compounds. as in the case of meb, this culture medium contains malt extract and peptone, which served as the carbon and nitrogen sources for the production of various metabolites by a. fumigatus. moreover, the production of bioactive chemicals from a. fumigatus without marine salt may suggest that the presence of salt could block the expression of certain biosynthetic pathways essential for the synthesis of its bioactive components. as described by yu and keller (2005), salinity is one factor that can regulate production of secondary metabolites. acknowledgements the authors would like to extend their modest gratitude to dr. paciente a. cordero, jr. for the identification of marine algae and seagrasses, to dr. irineo j. dogma, jr. for aid in the morphological identification of fungi, and to the research center for natural and applied sciences of the university of santo tomas. kir notarte acknowledges the department of science and technology – science education institute for the graduate scholarship and research grant. references bugni, t. s., ireland, c. m., 2004: marine-derived fungi: a chemically and biologically diverse group of microorganisms review. natural product reports 16, 143–163. bode, h. b., bethe, b., höfs, r., zeeck, a., 2002: big effects from small changes: possible ways to explore nature's chemical diversity. chembiochem 3, 619–627. calvo, a. m., wilson, r. a., bok, j. w., keller, n. p., 2002: relationship between secondary metabolism and fungal development. microbiology and molecular biology reviews 66, 447–459. cui, c. m., li, x. m., meng, l., li, c. s., huang, c. g., wang, b. g., 2010: 7-norergosterolide, a pentalactone-containing norsteroid and related steroids from the marine-derived endophytic aspergillus ochraceus en-31. journal of natural products 73, 1780–1784. dela cruz, t. e. e., wagner, 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q., zhu, w. m., 2010: three new dioxopiperazine metabolites from a marine-derived fungus aspergillus fumigatus fres. natural product research 24, 953–957. 146 acta bot. croat. 80 (2), 2021 acta bot. croat. 80 (2), 146–157, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-015 issn 0365-0588 eissn 1847-8476 seed micromorphology and anatomy of 36 muscari (asparagaceae) taxa from turkey with notes on their systematic importance hüseyin eroğlu1, mehmet cengiz karaismailoğlu2*, süleyman mesut pinar3, mehmet fidan2 1 yüzüncü yıl university, faculty of sciences, department of biology, 65090 tuşba-van, turkey 2 siirt university, faculty of arts and sciences, department of biology, 56100 kezer-siirt, turkey 3 yüzüncü yıl university, van school of health, 65090 tuşba-van, turkey abstract – this study presents the first in-depth evaluation of the morphological and anatomical characters, as well as their taxonomic importance, of the seeds of 36 taxa in subgenera muscari, leopoldia, pseudomuscari and botryanthus of the genus muscari in turkey, where 24 of the taxa are endemic. the results indicate that the taxa generally differ from each other in terms of seed shape and dimension. seed dimensions vary between 1.66 mm and 3.21 mm in length, and between 1.12 mm and 2.63 mm in width. the seed surface ornamentation is grouped into nine forms: ruminate, reticulate, reticulate-areolate, reticulate-foveate, alveolate, scalariform, rugose, verrucate and areolate. the most common type is ruminate, while areolate, reticulate-foveate and scalariform ornamentation forms were found to be taxon-specific. testa structures of the taxa examined consist in general of two different layers: the epidermis and the subepidermis in scleranchymatous or parenchymatous structures. the subepidermis may be absent in some of taxa. the structure and thickness of the epidermis and the subepidermis are very important characteristics that disclose interspecific relations among the examined taxa. we also provide a key for the identification of the studied taxa based on seed features. keywords: crystal, morphology, muscari, scanning electron microscopy, taxonomy, testa, turkey introduction the genus muscari mill. is found across the european continent, mediterranean region and northwest asia (jafari and maassoumi 2011). according to the latest checklist study, the genus is represented by 51 species worldwide ( govaerts 2019). according to other recent studies, muscari includes 40 species belonging to four subgenera as muscari, leopoldia and botryanthus and pseudomuscari with controversial status in turkey, 26 of which are endemic (dizkırıcı et al. 2019, eker 2019a,b, demirci-kayıran et al. 2019). the genus is characterized by its bulbs, basal leaves, inflorescences, pedicels, flower form and colour, filament placement relative to the tube, and capsule shape (davis and stuart 1984). major taxonomic problems of the genus include the many synonyms among taxa, the fact that type specimens are often cultivated material of unknown origin, that widespread taxa show a lot of variation and the color difference between fresh and dried f lowers (davis and stuart 1984). moreover, reliable classification is impossible in the genus because morphological characteristics and karyological information are not complete or consistent enough to make uncontroversial taxonomic judgements (dizkırıcı et al. 2019). the genus was placed in hyacinthaceae until the angiosperm phylogeny group (apg) re-evaluated its taxonomic position as a result of subsequent studies, and placed it within the family asparagaceae (reveal and chase 2011, guner et al. 2012, demirci and özhatay 2017). several morphological, anatomical, cytological, palynological and ecological studies on taxa belonging to various genera of asparagaceae have been performed previously (shoub and halevy 1971, bentzer et al. 1974, küçüker 1990, uysal 1999, herrmann et al. 2006, lynch et al. 2006, uysal et al. 2007, gürsoy and şık 2010, kahraman et al. 2010, doğu and bağcı 2009, doğu et al. 2011, sezer et al. 2013). however, the morphological and anatomical features of * corresponding author e-mail: cengiz.karaismailoglu@siirt.edu.tr seed structures of muscari from turkey acta bot. croat. 80 (2), 2021 147 seeds have been largely ignored in the systematics of taxa in the family, except in a few new species descriptions (yıldırım 2015, 2016, doğu and uysal 2019). the purpose of this study is to: (i) examine the morphological and anatomical characteristics of seeds of 36 taxa in subgenera muscari, leopoldia, pseudomuscari and botryanthus of the genus muscari in turkey, and (ii) debate the taxonomic use of these characters. the study will also serve as a guide to further related studies on various genera in the family. material and methods the plant specimens were collected from various phytogeographical regions of turkey during the fruiting season and were deposited at vanf (van yüzüncü yıl university herbarium). details are provided in tab. 1. macromorphological features of the seeds including colour, shape and size were documented for 100 seeds of 10 individuals per species utilizing a leica ez4 binocular microscope with a hd camera (on-line suppl. fig. 1, tab. 2). for the micromorphological features of surface ornamentation, anticlinal and periclinal cell walls, and the form of epidermal cells, the samples were studied with a scanning electron microscope (on-line suppl. fig. 2, tab. 3). seeds were first placed on the stub with silver epoxy and coated with gold, then examined with a zeiss leo 440 sem. a survey of seed anatomical characters was done with dry herbarium materials. cross-sections were taken from the middle of the seed with a fully automatic microtome (thermo shonda met finesse, thermo). they were brought through a series of alcohol and xylene, dyed with hematoxylin and eosin-y in a staining device (asc 720 medite) and mounted using entellan (on-line suppl. fig. 3, tab. 4) (karaismailoğlu 2015, karaismailoğlu and erol 2018, karaismailoğlu and güner 2019). anatomical characteristics were examined with an olympus cx31 light microscope and kameram imaging software (kameram12 ccd, argenit micro system ltd., turkey). the terminology used for seed morphological and anatomical characteristics is compatible with stearn (1985). grouping of taxa was performed using the clustering analysis method (upgma) in multivariate statistical package (mvsp) in accordance with the 44 characters in tables 2-4 (fig. 1). characters used in statistical analysis were: seed colour (1); shape: orbicular (2), ovate (3), oblong (4), elliptic (5), lanceolate (6); sizes: length (7), width (8), l/w (9); surface ornamentation: reticulate (10), alveolate (11), areolate (12), verrucate (13), ruminate (14), foveate (15), rugose (16), scalariform (17); anticlinal cell walls: sunken (18), raised (19), unclear (20); periclinal cell walls: convex (21), concave (22), unclear (23); epidermal cell structure: polygonal (24), alveolar (25), rectangular (26), flat (27), unclear (28); anatomical structure of the epidermis: flat (29), rectangular (30), crushed (31), polygonal (32), scleranchymatous cells (33), parenchymatous cells (34); anatomical structure of the subepidermis: crushed (35), flat (36), orbicular (37), square (38), polygonal (39), rectangular (40), scleranchymatous cells (41), parenchymatous cells (42); testa thickness (43); presence of crystals (44). the dissimilarity matrix of the studied taxa was created with mvsp (kovach 2007) (on-line suppl. tab. 1). a dendrogram was created. also, the cophenetic correlation coefficient is designed to explain the relation between the dendrogram and similarity matrix (on-line suppl. tab. 1, fig. 1). results this work assesses macromorphologically the seed features of the studied taxa, including colour, shape and dimensions. all of the taxa examined have the same seed color (black) but the shape and size of seeds vary considerably. seeds examined can be divided into 7 shapes; orbicular, ovate-orbicular, ovate, oblong-ovate, oblong-elliptic, elliptic fig. 1. cluster analysis of the studied taxa. eroğlu h., karaismailoğlu m. c., pinar s. m., fidan m. 148 acta bot. croat. 80 (2), 2021 and elliptic-lanceolate. orbicular is the most common type (found in 20 taxa). however, oblong-ovate, oblong-elliptic and elliptic-lanceolate are characteristic types for muscari mirum, m. longipes and m. macbeathianum, respectively. seed dimensions range from 1.66 mm to 3.21 mm in length, and from 1.12 mm to 2.63 mm in width. while m. erdalii and m. racemosum have the largest seeds, m. macbeathianum has the smallest seeds (tab. 2, on-line suppl. fig. 1). the surface ornamentation, anticlinal and periclinal cell walls, and epidermal cell structures of the seeds have been micromorphologically evaluated in this study. seed surface ornamentation is grouped into nine types: ruminate, reticulate, reticulate-areolate, reticulate-foveate, alveolate, scalariform, rugose, verrucate and areolate. the most common form is ruminate, while areolate, reticulate-foveate and scalariform ornamentation forms were found to be taxon-specific (tab. 3, on-line suppl. fig. 2). the reticulate-foveate (in m. elmasii), areolate (m. neglectum), and scalariform (m. azureum) ornamentation types are each displayed by only one taxon. the anticlinal cell walls in the studied taxa are raised, sunken or unclear. while sunken cell walls are widely seen in the alveolate, verrucate, areolate, reticulate-areolate and scalariform ornamentation types, the reticulate and reticulate-foveate ornamentation types are found where epidermal cells are enclosed by raised walls. rugose and ruminate types are associated with unclear form (tab. 3). no clear relationship exists between convex or concave periclinal cell walls and surface ornamentation types; however, ruminate and rugose types are found only with unclear periclinal cells. the shape of epidermal cells on the seed surface has also showed diversity and may be grouped into polygonal, alveolar, rectangular and unclear categories. the most common cell type is unclear, while rectangular and alveolar are fairly rare (tab. 3). the results of the examination of the anatomical structures of the seeds are indicated in on-line suppl. fig. 3 and tab. 4. testa structures of the seeds of the examined taxa generally consisted of 2 main layers, the epidermis and the subepidermis, formed in either the scleranchymatous or parenchymatous tissue. the epidermis layer displays important variations in cell form, consisting of f lat, rectangular, crushed, or polygonal cells, in 1-3 layers, and has undulated or straight wall structure. the most frequent form is flat, while the rarest ones are the rectangular and polygonal types (tab. 4, on-line suppl. fig. 3). the subepidermis layer consists of crushed, orbicular, rectangular, square, flat or polygonal cells in 1-10 layers. the most commonly seen types are crushed and polygonal, whereas the rarest ones are the orbicular and square types. the subepidermis layer is not found in some of the examined taxa (m. discolor, m. inconstrictum, m. parviflorum, m. botryoides and m. turcicum) (tab. 4). the thickness of the epidermis layers varies between 16.64 μm (in m. turcicum) and 128.46 μm (in m. longipes). raphide crystals are seen in the epidermis or subepidermis layers of seeds in m. comosum, m. tenuiflorum, m. babachii, m. discolor and m. vuralii (tab. 4, on-line suppl. fig. 3). a dendrogram indicating differences and similarities among the studied taxa was created by numerical analyses of the seed morphological and anatomical characters, based on the variation of 44 characteristics in 36 taxa. the cophenetic correlation between the similarity matrix and dendrogram has been computed as 0.59, representing a good match. cluster a2 includes the highest number of taxa when compared to other clusters. muscari sandrasicum forms a clade separate from these clusters in the dendrogram (fig. 1). m. discolor and m. parviflorum are the most closely related taxa (with a dissimilarity coefficient of 1.01), the most distantly related taxa recorded are m. sandrasicum and m. turcicum (with a dissimilarity coefficient of 136.31) (on-line suppl. tab. 1). discussion the morphological features of seeds offer valuable information about evolutionary relationships among flowering plants (corner 1976, karaismailoğlu and erol 2018). however, seed morphological and anatomical features have so far not been extensively used to elucidate inter-species relationships within genera of the family asparagaceae. this is the first study to reveal the morphological and anatomical features of the seeds of a genus in the family, and it will be a model for subsequent studies on various genera. the macromorphological characters of seeds display variation among the examined muscari taxa, with the exception of seed colour, which is consistently black. the general appearance among populations, including floristic characters and capsule structures, of m. macrocarpum and m. racemosum in subgenus muscari, m. caucasicum and m. weissii in subgenus leopoldia, m. aucheri and m. armeniacum in subgenus botryanthus are very similar, but they can be easily distinguished using seed shape and size. comparison of the surface micromorphological structure of seeds is of taxonomical importance (karaismailoğlu and erol 2018). heywood (1971) discusses the significance and efficiency of scanning electron microscopy in elucidating taxonomic problems and distinguishing taxa. however, there are few studies on the importance of seed micromorphology in the family asparagaceae (yıldırım 2015, 2016). this study on 36 muscari taxa shows that seed microstructures are useful characteristics in separating the taxa within the family. almost all of the studied taxa have been examined in this way for the first time, with the exceptions of m. elmasii (smooth) and m. atillae (smooth) (yıldırım 2015, 2016). we recorded nine seed surface ornamentation types in this study. in the genus, the most common seed ornamentation types are ruminate and reticulate. in contrast to this study, reticulate and reticulate-areolate types have been commonly seen among taxa from various angiosperm fami lies (ta ntaw y et a l. 20 04, ka ra isma i loğ lu 2015, karaismailoğlu and erol 2018). two closely related taxa in the subgenus muscari, m. macrocarpum and m. racemosum, have the same reticulate surface ornamentation type; however, m. macrocarpum has different secondary cuticular seed structures of muscari from turkey acta bot. croat. 80 (2), 2021 149 ta b. 1 . t he e xa m in ed ta xa a nd th ei r l oc at io ns (* =e nd em ic ta xo n) . n o su bg en us ta xa lo ca tio n v ou ch er 1 m us ca ri m us ca ri m ac ro ca rp um s w ee t c 1 m uğ la ; b et w ee n m ar m ar is a nd e m ec ik , a ft er b al ık p as s, ro ck y va lle y, 3 6° 4 6’ 2 7” n , 2 7° 5 9’ 3 6” e , 3 24 m , 01 .0 3. 20 16 h . e ro ğl u 12 15 2 *m . r ac em os um m ill . c 2 d en iz li; ç am el i, d en iz life th iy e ro ad , 5 k m to a liv er en v ill ag e, p in us y ar ds , s er pe nt in e fie ld s, 37 ° 1 3’ 3 9” n , 2 9° 26 ’ 5 2” e , 1 26 4 m , 0 4. 05 .2 01 7. h . e ro ğl u 13 17 3 le op ol di a m . c au ca si cu m (g ri se b. ) b ak er b9 v an ; e re k m ou nt ai n, so ut h of s ar m aç v ill ag e, st ep pe , 3 8° 2 9’ 1 6” n , 4 3° 2 9’ 2 6” e , 2 20 0 m , 2 4. 05 .2 01 6. h . e ro ğl u 12 81 4 m . w ei ss ii fr ey n a nt al ya : s er ik , k um kö y, p in us p in ea fo re st n ea r th e se a, d un es u nd er th e w oo od , 3 6° 5 2’ 0 7’ ’ n , 3 0° 5 6’ 3 6’ ’ e , 3 m , 02 .0 4. 20 16 . h . e ro ğl u 12 20 5 m . c om os um (l .) m ill . c 2 m uğ la , m ar m ar is , b et w ee n m ar m ar is a nd d at ça , h is ar ön ü ba y, r oa ds id e, 3 6° 4 7’ 5 9” n , 2 8° 0 5’ 3 1” e , 7 0 m , 16 .0 4. 20 17 . h . e ro ğl u 13 01 6 m . t en ui flo ru m t au sc h b6 a da na ; f ek e, e se nd er e c an yo n, p in us y ar ds , 3 7° 4 5’ 4 4” n , 3 5° 5 5’ 0 3” e , 6 51 m , 1 5. 06 .2 01 6. h . e ro ğl u 12 88 7 *m . b ab ac hi i e ke r & k oy un cu c 6 h at ay , a nt ak ya , k is ec ik v ill ag e, r ad ar r oa d, sc ru b ya rd s, 36 ° 1 8’ 1 5” n , 3 6° 0 2’ 5 9” e , 1 43 0 m , 1 2. 06 .2 01 6. h . e ro ğl u 12 86 8 *m . e rd al ii n .ö zh at ay & s .d em ir ci c 4 i̇ç el ; m ut , s ou th o f i̇ br ah im li v ill ag e, sc ru b ya rd s, 36 ° 4 0’ 5 5” n , 3 3° 3 9’ 2 3” e , 9 00 m , 0 2. 05 .2 01 6. h . e ro ğl u 12 55 9 m . l on gi pe s b oi ss . b6 s iv as ; h afi k, w es t o f d ur ul m uş v ill ag e, m ar ly h ill s, 39 ° 5 0’ 0 8” n , 3 7° 1 8’ 2 0” e , 1 31 2 m , 3 0. 05 .2 01 7. h . e ro ğl u 13 27 10 *m . m as sa ya nu m c .g ru ne rt c 5 a da na ; p oz an tı, u pw ar ds o f h am id iy e v ill ag e, se rp en tin e sl op es , 3 7° 3 2’ 2 7” n , 3 5° 0 0’ 5 1” e , 1 35 7 m , 0 1. 05 .2 01 6. h . e ro ğl u 12 53 11 *m . m ir um s pe ta c 2 d en iz li; ç am el i, d en iz life th iy e ro ad , 4 k m to a liv er en v ill ag e, se rp en tin e sl op es , 3 7° 1 2’ 4 1” n , 2 9° 2 6’ 1 7” e , 14 75 m , 0 4. 05 .2 01 6. h . e ro ğl u 12 59 12 *m . e lm as ii yı ld ır ım c 2 m uğ la ; d al am an , a bo ve g ür le yi k v ill ag e, ç al m ou nt ai n, p in us y ar ds , 3 6° 5 2’ 4 9” n , 2 9° 0 7’ 1 0” e , 1 27 1 m , 14 .0 5. 20 16 . h . e ro ğl u 12 70 13 *m . u fu ki i e .k ay a & d em ir ci b9 v an ; ç at ak , b et w ee n ç at ak -b ilg i v ill ag e, st ep pe , 3 8° 0 3’ 4 8” n , 4 3° 1 1’ 4 9” e , 1 67 0 m , 1 7. 07 .2 01 7. h . e ro ğl u 13 41 14 ps eu do m us ca ri *m . c oe le st e fo m in b9 v an ; e re k m ou nt ai n, si de o f k eş iş l ak e, h um id m ea do w s, 38 ° 2 7’ 4 3” n , 4 3° 3 4’ 5 1” e , 2 56 4 m , 1 8. 05 .2 01 7. h . e ro ğl u 13 19 15 *m . a zu re um f en zl c 5 n iğ de ; u lu kı şl a, k ar ag öl , h um id m ea do w s, 37 ° 2 4’ 1 6” n , 3 4° 3 3’ 3 8” e , 2 59 9 m , 0 1. 05 .2 01 6. h . e ro ğl u 12 51 16 bo tr ya nt hu s *m . a uc he ri (b oi ss .) ba ke r a 9 k ar s; sa rı ka m ış to h an de re 5 . k m , m ea do w s, 40 ° 1 8’ 3 3’ ’ n , 4 2° 3 0’ 4 3’ ’ e , 2 19 6 m , 0 8. 06 .2 01 6. h . e ro ğl u 12 85 17 m . a rm en ia cu m l ei ch tli n ex b ak er c 4 k ar am an ; s ar ıv el ile r, a tm ey da nı p la ce , s te pp e, 3 6° 4 1’ 4 4” n , 3 2° 3 1’ 0 0” e , 1 66 5 m , 0 1. 05 .2 01 7 h . e ro ğl u 13 06 eroğlu h., karaismailoğlu m. c., pinar s. m., fidan m. 150 acta bot. croat. 80 (2), 2021 18 *m . s iv ri hi sa rd ag hl ar en sis y ıld . & b .s el vi b3 e sk iş eh ir ; s iv ri hi sa r, be tw ee n k uz uö re n an d k ar ac aö re n vi lla ge s, st on yro ck y st re am si de , 3 9° 1 8’ 5 2” n , 3 1° 4 2’ 42 ” e, 1 41 6 m , 0 2. 05 .2 01 7. h . e ro ğl u 13 09 19 m . n eg le ct um g us s. ex t en . b3 e sk iş eh ir ; s iv ri hi sa r, g ün yü zü c ro ss , s te pp e, 3 9° 2 9’ 4 2” n , 3 1° 3 6’ 4 1” e , 1 00 9 m , 1 7. 04 .2 01 6. h . e ro ğl u 12 42 20 *m . a na to lic um c ow le y & ö zh at ay c 5 i̇ç el ; t or os la r, a rs la nk öy , a bo ve d üm be le k g eç id i r oc ky sl op es , 3 7° 0 3’ 5 6” n , 3 4° 1 7’ 5 3” e , 2 21 2 m , 1 1. 05 .2 01 8. h . e ro ğl u 13 82 21 *m . t uz go lu en si s y ıld . b4 a ks ar ay , e sk il, 1 k m to w ar s t uz gö lü fr om e sk il, st ep pe , 3 8° 2 4’ 4 3” n , 3 3° 2 7’ 2 0” e , 9 22 m , 1 3. 04 .2 01 6. h . e ro ğl u 12 33 22 *m . d is co lo r b oi ss . & h au ss kn . e x b oi ss . c 8 m ar di n; a rt uk lu , m ar di nd iy ar ba kı r ro ad , a kr es ta p as s, st on y st re am si de , 3 7° 2 2’ 5 7” n , 4 0° 3 9’ 0 9” e , 1 13 8 m , 07 .0 4. 20 17 . h . e ro ğl u 12 97 23 m . i nc on st ri ct um r ec h. f. c 6 k ili s; so ut h of k oc ab ey li v ill ag e, st on yro ck y fie ld s, 36 ° 4 8’ 0 7” n , 3 6° 5 4’ 5 9” e , 4 50 m , 2 8. 02 .2 01 6. h . e ro ğl u 12 12 24 *m . l at ifo liu m j. k ir k b2 ç an ak ka le ; b ay ra m iç , a ya zm a pr om en ad e, u nd er th e fo re st , h um id a re as , 3 9° 4 4’ 4 5” n , 2 6° 5 0’ 4 7” e , 4 76 m , 03 .0 5. 20 17 . h . e ro ğl u 13 13 25 *m . a di lii m .b .g ün er & h .d um an a 3 a nk ar a, b ey pa za rı , a bo ve h ır ka te pe v ill ag e, a ro un ds o f k oç ah m et f ou nt ai n, m ar ly v al le ys , 4 0° 1 1’ 4 3” n , 3 1° 4 6’ 39 ” e, 1 00 0 m , 0 2. 05 .2 01 7. h . e ro ğl u 13 08 26 *m . b ou rg ae i b ak er c 4 k ar am an ; s ar ıv el ile r, a tm ey da nı p la ce , m ea do w s, st re am si de , 3 6° 4 1’ 2 5” n , 3 2° 3 2’ 4 1” e , 1 60 3 m , 0 1. 05 .2 01 7. h . e ro ğl u 13 04 27 *m . s an dr as ic um k ar lé n c 2 m uğ la ; k öy ce ği z, s an dr as m ou nt ai n, s an dr as h ig hl an d, d eğ ir m en bo zu ğu p la ce , s to ny st re am si de , 3 7° 0 5’ 3 6” n , 28 ° 5 3’ 2 3” e , 1 35 6 m , 1 1. 04 .2 01 6. h . e ro ğl u 12 26 28 m . m ic ro st om um p .h .d av is & d .c .s tu ar t b5 k ay se ri ; b ün ya n, b et w ee n bü ny an a nd p ın ar ba şı 4 . k m , h um id m ea do w s, 38 ° 4 9’ 4 0” n , 3 5° 5 4’ 2 4” e , 1 38 9 m , 19 .0 5. 20 16 . h . e ro ğl u 12 77 29 *m . m ac be at hi an um k it ta n b6 a da na ; t uf an be yl i, 2 km fr om g üz el im v ill ag e to t uf an be yl i, du ne u nd er p in us , 3 8° 0 9’ 2 4” n , 3 6° 1 0’ 4 5” e , 14 42 m , 0 9. 05 .2 01 8. h . e ro ğl u 13 74 30 *m . v ur al ii ba ğc ı & d oğ u c 4 k ar am an ; s ar ıv el ile r, a tm ey da nı p la ce , m ea do w s, 36 ° 4 1’ 2 5” n , 3 2° 3 2’ 0 1” e , 1 60 3 m , 1 4. 04 .2 01 6. h . e ro ğl u 12 34 31 m . p ar vi flo ru m d es f. c 5 i̇ç el ; y en iş eh ir, b et w ee n em ir le r an d tu ru nç lu v ill ag es , g ar de n ed ge s, 36 ° 5 0’ 1 0” n , 3 4° 2 8’ 4 2” e , 2 88 m , 28 .0 9. 20 16 . h . e ro ğl u 12 91 32 *m . s er pe nt in ic um y ıld ır ım , a ltı oğ lu & pi rh an c 2 m uğ la ; k öy ce ği z, s an dr as m ou nt ai n, s an dr as h ig hl an d, d eğ ir m en bo zu ğu p la ce , s to ny st re am si de , 3 7° 0 5’ 3 6” n , 28 ° 5 3’ 2 3” e , 1 35 6 m , 1 1. 04 .2 01 6. h . e ro ğl u 12 24 33 m . b ot ry oi de s ( l. ) m ill . b9 a ğr ı; tu ta k, b et w ee n a şa ğı kö şk a nd d oğ an üs tü n vi lla ge s, m ea do w s, 39 ° 2 4’ 2 1” n , 4 2° 4 5’ 3 6” e , 1 66 9 m , 10 .0 5. 20 16 . h . e ro ğl u 12 62 34 *m . a rt vi ne ns e d em ir ci & e .k ay a a 9 a rt vi n; m ur gu l, ab ov e k or uc ul ar v ill ag e, m ea do w s, 41 ° 1 8’ 0 0” n , 4 1° 3 8’ 5 8” e , 7 62 m , 1 3. 05 .2 01 6. h . e ro ğl u 12 66 35 *m . a til la e yı ld ır ım b7 m al at ya , a kç ad ağ , l ev en t c an yo n, m ar ly -m ov em en t s lo pe s, 38 ° 2 6’ 0 3” n , 3 7° 5 5’ 5 6” e , 1 19 7 m , 0 7. 04 .2 01 7. h . e ro ğl u 12 96 36 *m . t ur ci cu m u ys al , e rt ug ru l & d ur al c 4 k on ya ; b oz kı r, ab ov e a vd an h ig hl an d, sn ow pa tc he s, st ep pe , 3 7° 0 1’ 1 5” n , 3 2° 1 0’ 4 1” e , 1 97 8 m , 1 1. 05 .2 01 8. h . e ro ğl u 13 79 seed structures of muscari from turkey acta bot. croat. 80 (2), 2021 151 protrusions. seed surface ornamentation is a useful character in distinguishing the taxa of the subgenus leopoldia, which exhibits five ornamentation types in 11 taxa. in the subgenus pseudomuscari, m. coeleste and m. azureum taxa are very similar in terms of population appearance, flowers and fruit capsule characteristics; however, they are distinctly different in terms of seed ornamentation types: ruminate and scalariform, respectively. in the subgenus botryanthus, ornamentation types are diverse (seven types), and the distinct surface ornamentation in nearly identical taxa, such as m. armeniacum-m. aucheri, m. armeniacum-m. bourgaei, m. armeniacum-m. microstomum is proof of the taxonomi cal significance of this characteristic in the subgenus. earlier seed surface studies have indicated that the views and structures of anticlinal and periclinal cell walls are good diagnostic characters in the establishment of inter-species relationships (barthlott 1981, karaismailoğlu 2015, 2016). the types of anticlinal and periclinal cell walls, and epidermal cell structures of the examined taxa vary among the taxa, except for those of the subgenus muscari. tab. 2. macromorphological characters of the seeds of the studied taxa (mean values ± standard deviation, l=length, w=width). subgenus taxa shape seed dimensions l (mm) w (mm) l/w muscari muscari macrocarpum orbicular 2.98 ± 0.32 2.61 ± 0.29 1.14 m. racemosum broadly ovate-orbicular 3.16 ± 0.22 2.63 ± 0.30 1.20 leopoldia m. caucasicum broadly ovate 2.34 ± 1.18 1.85 ± 0.11 1.26 m. weissii orbicular 2.08 ± 0.14 1.76 ± 0.12 1.18 m. comosum orbicular 2.23 ± 0.13 2.03 ± 0.11 1.10 m. tenuiflorum broadly ovate-orbicular 2.45 ± 1.15 2.17 ± 0.14 1.13 m. babachii broadly ovate 2.70 ± 0.19 2.22 ± 0.15 1.22 m. erdalii broadly ovate 3.21 ± 0.25 2.43 ± 0.15 1.32 m. longipes oblong-elliptic 2.42 ± 0.24 2.01 ± 0.12 1.20 m. massayanum broadly ovate 3.13 ± 0.23 2.56 ± 0.18 1.22 m. mirum oblong-ovate 2.82 ± 0.22 2.34 ± 0.17 1.20 m. elmasii broadly ovate 2.81 ± 0.25 2.25 ± 0.20 1.24 m. ufukii broadly ovate 3.06 ± 0.17 2.61 ± 0.13 1.17 pseudomuscari m. coeleste orbicular 2.18 ± 0.16 1.52 ± 0.09 1.43 m. azureum orbicular 2.06 ± 0.10 1.35 ± 0.09 1.52 botryanthus m. aucheri orbicular 2.11 ± 0.13 1.37 ± 0.08 1.54 m. armeniacum broadly ovate-orbicular 1.96 ± 0.16 1.71 ± 0.11 1.14   m. sivrihisardaghlarensis broadly ovate-orbicular 2.18 ± 0.17 1.69 ± 0.12 1.28 m. neglectum broadly elliptic 2.06 ± 0.15 1.64 ± 0.10 1.25 m. anatolicum orbicular 2.09 ± 0.15 1.72 ± 0.19 1.21 m. tuzgoluensis orbicular 1.94 ± 0.13 1.65 ± 0.13 1.17 m. discolor orbicular 2.23 ± 0.26 1.73 ± 0.11 1.28 m. inconstrictum orbicular 1.99 ± 0.12 1.80 ± 0.12 1.10 m. latifolium ovate-orbicular 2.39 ± 0.20 1.98 ± 0.13 1.20 m. adilii orbicular 2.45 ± 0.16 2.20 ± 0.17 1.11 m. bourgaei orbicular 1.82 ± 0.09 1.42 ± 0.11 1.28 m. sandrasicum orbicular 1.94 ± 0.17 1.53 ± 0.16 1.26 m. microstomum orbicular 1.87 ± 0.13 1.55 ± 0.16 1.20 m. macbeathianum broadly elliptic-lanceolate 1.66 ± 0.13 1.12 ± 0.09 1.48 m. vuralii orbicular 2.15 ± 0.14 1.60 ± 0.12 1.34 m. parviflorum orbicular 1.90 ± 0.16 1.62 ± 0.17 1.17 m. serpentinicum orbicular 1.72 ± 0.11 1.51 ± 0.10 1.13 m. botryoides broadly ovate 1.80 ± 0.14 1.36 ± 0.11 1.32 m. artvinense orbicular 1.70 ± 0.12 1.41 ± 0.10 1.21 m. atillae orbicular 2.25 ± 0.10 1.87 ± 0.11 1.20   m. turcicum orbicular 1.76 ± 0.13 1.34 ± 0.09 1.31 eroğlu h., karaismailoğlu m. c., pinar s. m., fidan m. 152 acta bot. croat. 80 (2), 2021 revisions of the anatomy of the testa of the various angiosperm families are influential in solving systematic problems (vaughan et al. 1976, karaismailoğlu and erol 2018). koul et al. (2000) have shown that testa structures may be utilized as a valuable characteristic in the separation of the taxa and the clarification of their phylogenetic relationships. the seed anatomical characters are frequently as useful as morphological characters for plant taxonomy, and they are valuable in the discrimination of closely correlated taxa in various families and genera (karamian et al. 2012, karaismailoğlu and erol 2018, karaismailoğlu et al. 2018). a detailed review of the literature has not found a previous study aiming at the exploration of phylogenetic relationships among the taxa with a comparative investigation of anatomical structures of the testa in members of the family asparagaceae. this work is the first such study for the family and is the precursor to subsequent investigations. in this study, we found that the testae mostly consist of two layers, tab. 3. micromorphological characters of the seeds of the studied taxa. subgenus taxa seed surface anticlinal periclinal epidermal ornamentation cell wall cell wall cell structure muscari muscari macrocarpum reticulate raised concave polygonal cells m. racemosum reticulate raised concave polygonal cells leopoldia m. caucasicum alveolate sunken concave alveolar cells m. weissii alveolate sunken concave alveolar cells m. comosum verrucate sunken convex unclear m. tenuiflorum reticulate-areolate sunken convex polygonal cells m. babachii reticulate-areolate sunken convex polygonal cells m. erdalii ruminate unclear unclear unclear m. longipes ruminate unclear unclear unclear m. massayanum ruminate unclear unclear unclear m. mirum reticulate-areolate sunken convex polygonal cells m. elmasii reticulate-foveate raised convex polygonal and alveolar cells m. ufukii ruminate unclear unclear unclear pseudomuscari m. coeleste ruminate unclear unclear unclear m. azureum scalariform sunken convex rectangular and polygonal cells botryanthus m. aucheri ruminate unclear unclear unclear m. armeniacum reticulate-areolate sunken concave polygonal cells   m. sivrihisardaghlarensis rugose unclear unclear unclear m. neglectum areolate sunken concave polygonal cells m. anatolicum rugose unclear unclear unclear m. tuzgoluensis ruminate unclear unclear unclear m. discolor ruminate unclear unclear unclear m. inconstrictum slightly reticulate raised concave polygonal cells m. latifolium ruminate unclear unclear unclear m. adilii ruminate unclear unclear unclear m. bourgaei ruminate unclear unclear unclear m. sandrasicum ruminate unclear unclear unclear m. microstomum verrucate sunken convex unclear m. macbeathianum ruminate unclear unclear unclear m. vuralii ruminate unclear unclear unclear m. parviflorum ruminate unclear unclear unclear m. serpentinicum verrucate sunken convex unclear m. botryoides rugose unclear unclear unclear m. artvinense rugose unclear unclear unclear m. atillae ruminate unclear unclear unclear   m. turcicum ruminate unclear unclear unclear seed structures of muscari from turkey acta bot. croat. 80 (2), 2021 153 ta b. 4 . t es ta a na to m ic al fe at ur es o f t he st ud ie d ta xa (m ea n va lu es ± st an da rd d ev ia tio n, + = pr es en ce , = ab se nc e) . su bg en us ta xa e pi de rm is la ye rs pr es en ce / ab se nc e of c ry st al s ep id er m is st ru ct ur es su be pi de rm is st ru ct ur es t hi ck ne ss (μ m ) m us ca ri m . m ac ro ca rp um 1 la ye r, sc le ra nc hy m at ic fl at c el ls 67 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 59 .7 5 ± 2. 48 – m . r ac em os um 1 la ye r, sc le ra nc hy m at ic r ec ta ng ul ar c el ls 34 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 54 .2 3 ± 3. 09 – le op ol di a m . c au ca si cu m 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 34 la ye rs , p ar en ch ym at ic o rb ic ul ar o r fla t c el ls 10 5. 44 ± 2 .3 7 – m . w ei ss ii 1 la ye r, sc le ra nc hy m at ic r ec ta ng ul ar c el ls 3 la ye rs , s cl er an ch ym at ic la rg e fla t c el ls 46 .7 1 ± 1. 82 – m . c om os um 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 1 la ye r, pa re nc hy m at ic r ec ta ng ul ar o r sq ua re c el ls 38 .4 5 ± 3. 63 + m . t en ui flo ru m 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 23 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 11 6. 59 ± 3 .8 8 + m . b ab ac hi i 1 la ye r, sc le ra nc hy m at ic fl at c el ls 23 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 12 1. 10 ± 5 .6 4 + m . e rd al ii 1 la ye r, sc le ra nc hy m at ic fl at c el ls 34 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 40 .3 7 ± 4. 21 – m . l on gi pe s 1 la ye r, sc le ra nc hy m at ic fl at c el ls 57 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 12 8. 46 ± 4 .2 3 – m . m as sa ya nu m 1 la ye r, sc le ra nc hy m at ic fl at c el ls 2 la ye rs , s cl er an ch ym at ic fl at o r cr us he d ce lls 68 .8 3 ± 3. 47 – m . m ir um 12 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 56 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 10 8. 54 ± 2 .8 8 – m . e lm as ii 1 la ye r, sc le ra nc hy m at ic r ec ta ng ul ar c el ls 45 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 44 .1 6 ± 2. 72 – m . u fu ki i 23 la ye rs , s cl er an ch ym at ic la rg e fla t c el ls 1 la ye r, sc le ra nc hy m at ic fl at c el ls 85 .3 5 ± 2. 41 – ps eu do m us ca ri m . c oe le st e 1 la ye r, sc le ra nc hy m at ic r ec ta ng ul ar c el ls 23 la ye rs , s cl er an ch ym at ic fl at c el ls 33 .6 2 ± 3. 13 – m . a zu re um 1 la ye r, sc le ra nc hy m at ic fl at c el ls 23 la ye rs , p ar en ch ym at ic fl at o r po ly go na l c el ls 39 .7 7 ± 2. 54 – bo tr ya nt hu s m . a uc he ri 2 la ye rs , s cl er an ch ym at ic la rg e fla t c el ls 1 la ye r, pa re nc hy m at ic p ol yg on al c el ls 38 .7 6 ± 1. 85 – m . a rm en ia cu m 12 la ye rs , s cl er an ch ym at ic fl at c el ls 2 la ye rs , p ar en ch ym at ic fl at o r po ly go na l c el ls 71 .1 9 ± 4. 06 – m . s iv ri hi sa rd ag hl ar en si s 1 la ye r, sc le ra nc hy m at ic fl at c el ls 34 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 37 .8 4 ± 3. 71 – eroğlu h., karaismailoğlu m. c., pinar s. m., fidan m. 154 acta bot. croat. 80 (2), 2021 su bg en us ta xa e pi de rm is la ye rs pr es en ce / ab se nc e of c ry st al s ep id er m is st ru ct ur es su be pi de rm is st ru ct ur es t hi ck ne ss (μ m ) m . n eg le ct um 1 la ye r, sc le ra nc hy m at ic fl at c el ls 2 la ye rs , p ar en ch ym at ic fl at c el ls 41 .6 7 ± 3. 24 – m . a na to lic um 2 la ye rs , s cl er an ch ym at ic fl at c el ls 23 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 23 .0 8 ± 3. 92 – m . t uz go lu en si s 2 la ye rs , s cl er an ch ym at ic fl at c el ls 1 la ye r, sc le ra nc hy m at ic p ol yg on al c el ls 48 .3 3 ± 2. 18 – m . d is co lo r 2 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 18 .4 1 ± 2. 38 + m . i nc on st ri ct um 23 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 22 .0 5 ± 1. 14 – m . l at ifo liu m 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 23 la ye rs , p ar en ch ym at ic fl at c el ls 66 .1 5 ± 3. 52 – m . a di lii 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 23 la ye rs , s cl er an ch ym at ic c ru sh ed c el ls 31 .1 7 ± 1. 84 – m . b ou rg ae i 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 2 la ye rs , s cl er an ch ym at ic fl at c el ls 64 .2 6 ± 2. 29 – m . s an dr as ic um 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 23 la ye rs , p ar en ch ym at ic fl at c el ls 71 .2 2 ± 2. 31 – m . m ic ro st om um 1 la ye r, sc le ra nc hy m at ic fl at o r po ly go na l c el ls 23 la ye rs , s cl er an ch ym at ic p ol yg on al c el ls 69 .9 8 ± 3. 53 – m . m ac be at hi an um 2 la ye r, sc le ra nc hy m at ic la rg e fla t o r re ct an gu la r ce lls 1 la ye r, sc le ra nc hy m at ic p ol yg on al c el ls 41 .1 3 ± 2. 36 – m . v ur al ii 1 la ye r, sc le ra nc hy m at ic fl at o r po ly go na l c el ls 23 la ye rs , s cl er an ch ym at ic p ol yg on al c el ls 44 .8 6 ± 1. 71 + m . p ar vi flo ru m 1 la ye r, sc le ra nc hy m at ic la rg e fla t c el ls 18 .0 8 ± 0. 86 – m . s er pe nt in ic um 1 la ye r, sc le ra nc hy m at ic la rg e re ct an gu la r ce lls 1 la ye r, sc le ra nc hy m at ic r ec ta ng ul ar c el ls 39 .7 3 ± 3. 15 – m . b ot ry oi de s 23 la ye rs , s cl er an ch ym at ic fl at c el ls 25 .8 1 ± 2. 03 – m . a rt vi ne ns e 1 la ye r, sc le ra nc hy m at ic p ol yg on al c el ls 810 la ye rs , s cl er an ch ym at ic c ru sh ed a nd o rb ic ul ar ce lls 11 7. 46 ± 3 .5 5 – m . a til la e 3 la ye rs , s cl er an ch ym at ic fl at c el ls 23 la ye rs , s cl er an ch ym at ic p ol yg on al c el ls 82 .1 9 ± 2. 68 – m . t ur ci cu m 1 la ye r, sc le ra nc hy m at ic c ru sh ed c el ls 16 .6 4 ± 3. 22 – seed structures of muscari from turkey acta bot. croat. 80 (2), 2021 155 the epidermis and the subepidermis, in the sclerotic or parenchymatous structure. the epidermis type differs among the taxa. this 1-3 layered epidermis may consist of flat, rectangular, crushed, or polygonal cells. the most frequent form is flat, while the rarest are the rectangular and polygonal types. the structure of the subepidermis layer, which is mostly a compressed tissue under the epidermis layers, also displays significant differences among the taxa. the subepidermis layer consists of crushed, orbicular, rectangular, square, flat or polygonal cells in 1-10 layers, except for m. discolor, m. inconstrictum, m. parviflorum, m. botryoides and m. turcicum, which do not have a subepidermis layer. testa characters such as the structures of the epidermis and subepidermis, thickness of the testa, and the presence or absence of crystals are fairly effective and beneficial in discriminating almost all of the studied taxa, especially in the pairs of closely correlated taxa m. macrocarpum-m. racemosum, m. caucasicum-m. weissii, m. coeleste-m. azureum, and m.  aucheri-m. armeniacum. this can be interpreted as follows: the anatomy of the testa is a useful additional character in the muscari, and it can aid in the classification of this huge genus. the results obtained are also in agreement with similar previous studies performed on seed structure of some taxa of the genera crocus l. and romulea maratti in the closely related family iridaceae, in terms of the differences observed at interspecific level in testa anatomical structures such as epidermis cell types and thickness of the testa (grilli caiola et al. 2010, karaismailoğlu 2015, karaismailoğlu et al. 2018). the dendrogram showing two main clusters largely agree with the results of davis and stuart (1984). the seed morphological and anatomical variations have been observed at the species level and subgenus level, especially in shapes, ornamentation types, dimensions, and thicknesses and structures of epidermis and subepidermis layers. the proximity between taxa belonging to subgenera muscari and pseudomuscari has been preserved; however, there are taxon transitions between leopoldia and botryanthus subgenera. while m. atillae, m. latifolium, m. microstomum and m. armeniacum taxa are among the taxa belonging to botryanthus subgenus, m. mirum and m. caucasicum taxa are located between leopoldia taxa. in conclusion, the study of morphological and anatomical seed characteristics of the studied muscari taxa offers important insights into the systematics of taxa within the genus. key to studied muscari taxa, based on seed characteristics 1. seed shape is orbicular . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1. seed shape is ovate, ovate-orbicular, oblong-elliptic, oblong-ovate, elliptic, elliptic-lanceolate . . . . . . . . . . 21 2. seed ornamentation is reticulate . . . . . . . . . . . . . . . . . 3 2. seed ornamentation is alveolate, verrucate, ruminate, scalariform or rugose . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3. outer epidermis of testa consists of crushed cells, with 2-3 layers . . . . . . . . . . . . . . . . . . . . . . . . m. inconstrictum 3. outer epidermis of testa consists of flat cells, with 1 layers . . . . . . . . . . . . . . . . . . . . . . . . . . m. macrocarpum 4. seed ornamentation is alveolate or scalariform . . . . . 5 4. seed ornamentation is verrucate, ruminate or rugose . .6 5. seed ornamentation is alveolate . . . . . . . . . . m. weissii 5. scalariform . . . . . . . . . . . . . . . . . . . . . . . . . . m. azureum 6. seed ornamentation is verrucate or rugose . . . . . . . . . 7 6. ruminate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 7. anticlinal cell walls are sunken . . . . . . . . . . . . . . . . . . 8 7. anticlinal cell walls are unclear . . . . . . . . . . . . . . . . . 10 8. outer epidermis of testa consists of flat or polygonal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 8. outer epidermis of testa consists of rectangular cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. serpentinicum 9. subepidermis of testa consists of rectangular cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. comosum 9. subepidermis of testa consists of polygonal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. microstomum 10. outer epidermis of testa consists of flat cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. anatolicum 10. outer epidermis of testa consists of polygonal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. artvinense 11. outer epidermis is 1 layer . . . . . . . . . . . . . . . . . . . . . . 12 11. outer epidermis is 2 or 3 layers . . . . . . . . . . . . . . . . . . 19 12. outer epidermis of testa consists of crushed or rectangular cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 12. outer epidermis of testa consists of flat or polygonal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 13. outer epidermis of testa consists of crushed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. turcicum 13. rectangular . . . . . . . . . . . . . . . . . . . . . . . . . . m. coeleste 14. subepidermis layer is absent . . . . . . . . m. parviflorum 14. subepidermis layer is present . . . . . . . . . . . . . . . . . . . 15 15. subepidermis is in parenchymatous structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. sandrasicum 15. subepidermis is in scleranchymatous structure . . . 16 16. subepidermis consists of crushed cells . . . . . m. adilii 16. subepidermis consists of flat or polygonal cells . . . . 17 17. subepidermis consists of flat . . . . . . . . . . . m. bourgaei 17. polygonal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 18. crystals are present in the epidermis or subepidermis layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. vuralii 18. crystals are absent . . . . . . . . . . . . . . . . m. microstomum 19. outer epidermis is 3 layers . . . . . . . . . . . . . . . .m. atillae 19. 2 layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 20. subepidermis is in parenchymatous structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. aucheri 20. subepidermis is in scleranchymatous structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. tuzgoluensis 21. seed shape is elliptic or oblong . . . . . . . . . . . . . . . . . . 22 eroğlu h., karaismailoğlu m. c., pinar s. m., fidan m. 156 acta bot. croat. 80 (2), 2021 21. seed shape is ovate or ovate-orbicular . . . . . . . . . . . . 25 22. seed shape is elliptic . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 22. oblong . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 23. seed shape is elliptic-lanceolate . m. macbeathianum 23. broadly elliptic . . . . . . . . . . . . . . . . . . . . . . m. neglectum 24. seed shape is oblong-ovate . . . . . . . . . . . . . . . m. mirum 24. oblong-elliptic . . . . . . . . . . . . . . . . . . . . . . . . m. longipes 25. seed shape is ovate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 25. seed shape is ovate-orbicular . . . . . . . . . . . . . . . . . . . 32 26. seed surface ornamentation is alveolate or reticulate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 26. seed surface ornamentation is ruminate or rugose . 29 27. seed surface ornamentation is reticulate . . . . . . . . . 28 27. alveolate . . . . . . . . . . . . . . . . . . . . . . . . . m. caucasicum 28. seed surface ornamentation is reticulate-foveate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. elmasii 28. reticulate-areolate . . . . . . . . . . . . . . . . . . . . .m. babachii 29. seed surface ornamentation is rugose . . m. botryoides 29. ruminate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 30. outer epidermis of testa is 2-3 layers . . . . . . . m. ufukii 30. outer epidermis of testa is 1 layer . . . . . . . . . . . . . . . . 31 31. subepidermis is 3-4 layers . . . . . . . . . . . . . . . m. erdalii 31. subepidermis is 2 layers . . . . . . . . . . . m. massayanum 32. seed ornamentation is ruminate or rugose . . . . . . . . 33 32. seed ornamentation is reticulate or reticulate-areolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 33. seed surface ornamentation is ruminate . . m. latifolium 33. rugose . . . . . . . . . . . . . . . . . . . m. sivrihisardaghlarensis 34. seed surface ornamentation is reticulate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m. racemosum 34. reticulate-areolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 35. crystals are present in the epidermis or subepidermis layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .m. tenuiflorum 35. crystals are absent . . . . . . . . . . . . . . . . . m. armeniacum acknowledgments this article is adapted from the first author’s doctoral thesis. the authors thank yyu-bapb for supporting this study financially (project number: fdk-2017-5960). references barthlott, w., 1981: epidermal and seed surface characters of plants: systematic applicability and some evolutionary aspects. nordic journal of botany 1, 345–355. bentzer, b., bothmer, r., wendelbo, p., 1974: cytology and morphology of the genus hyacinthus l. s. str. 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(eds.), the biology and chemistry of the cruciferae, 119–144. academic press, london. yıldırım, h., 2015: muscari atillae (asparagaceae): a new species from eastern anatolia, turkey. phytotaxa 213, 291–295. yıldırım, h., 2016: muscari elmasii sp. nova (asparagaceae): a new species from western anatolia, turkey. turkish journal of botany 40, 380–387. opce-str.vp acta bot. croat. 70 (2), 147–155, 2011 coden: abcra 25 issn 0365-0588 ecological conditions, flora and vegetation of a large doline in the mecsek mountains (south hungary) zoltán bátori*, róbert gallé, lászló erdõs, lászló körmöczi department of ecology, university of szeged, 6726 szeged, közép fasor 52, hungary abstract – vegetation-environment relationships were investigated in a large doline of the mecsek mts (south hungary). to reveal the vegetation pattern, we collected vegetation data and environmental variables along a 243 m long transect. a total of 144 vascular plant species and 4 vegetation types were identified in the doline. we found that both the species composition and the vegetation pattern are significantly influenced by air temperature, air humidity, soil moisture and altitude. our results confirm the putative temperature and vegetation inversion in the doline. keywords: direct gradient analysis, doline, flora, vegetation types, vegetation inversion, redundancy analysis, species composition, mecsek mountains, hungary introduction karst surfaces and environments are extremely vulnerable to degradation and pollution (caló and parise 2006, breg 2007), thus they are currently in the focus of research and conservation efforts. dolines are funneland bowl-shaped closed depressions formed by water infiltration, which range from a few meters to a few hundred meters in diameter and depth (veress 2004). at night, cold-air lakes develop in the depressions (bárány-kevei 1999), resulting in low air temperatures and high air humidities, which in turn strongly influences the composition of local flora and vegetation (beck-mannagetta 1906, morton 1936, özkan et al. 2010). many karst depressions around the world have developed into excellent refuge areas for relict, mountain (horvat 1953) and endemic species (egli et al. 1990, brullo and giusso del galdo 2001), which play a central role in the knowledge about vegetation history. the study was carried out in the karst area of 30 km2 in the mecsek mountains (south hungary), near the city of pécs. sub-mediterranean type, middle-aged oak-hornbeam (asperulo taurinae-carpinetum soó et borhidi in soó 1962) and beech forests (helleboro odori-fagetum soó et borhidi in soó 1960) dominate the present vegetation of the plateaus acta bot. croat. 70 (2), 2011 147 *corresponding author, e-mail: zbatory@gmail.com copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:15 color profile: disabled composite 150 lpi at 45 degrees and slopes of the study site. the bottom of larger valleys is covered by beech forests or ravine forests (scutellario altissimae-aceretum (horvát 1958) soó et borhidi in soó 1962). this latter vegetation type is also found in the deeper and larger dolines of the mecsek mountains (cf. bátori et al. 2009). the purpose of this study was to analyse the vegetation pattern of the herb layer in a doline of the mecsek mountains. the following questions were addressed: how many vegetation types and vascular plant species can be identified in the doline? what environmental variables influence the vegetation pattern on the slopes? 148 acta bot. croat. 70 (2), 2011 bátori z., gallé r., erdõs l., körmöczi l. u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:21 color profile: disabled composite 150 lpi at 45 degrees material and methods our surveys were conducted in a large (diameter > 200 m) and deep (depth > 25 m) doline of the mecsek mountains. the herb layer was sampled along a 2 m wide and 243 m long transect consisting of 1 m × 2 m contiguous plots (figs. 1, 2). the transect was established in a north-south direction traversing the deepest point of the depression. each plot was surveyed twice: in summer 2008 and spring 2009. in addition, a complete plant species list was compiled for the total area of doline. the total area included the area of the slopes (where the slope angle was over 10°) and the area of the edge (an approximately 15–20 m wide stripe around the doline where the slope angle was less than 10°). acta bot. croat. 70 (2), 2011 149 flora and vegetation of a large doline fig. 1. species occurrences and relief profile along the doline transect. numbers on the right indicate plant species as follows: 1 – fraxinus excelsior l., 2 – cardamine bulbifera (l.) crantz, 3 – allium ursinum l., 4 – tilia tomentosa moench, 5 – arum maculatum l., 6 – asarum europaeum l., 7 – hedera helix l., 8 – alliaria petiolata (m. b.) cavara et grande, 9 – galium aparine l., 10 – veronica hederifolia l., 11 – helleborus odorus w. et k., 12 – viola reichenbachiana jord., 13 – acer platanoides l., 14 – acer campestre l., 15 – fraxinus ornus l., 16 – stellaria holostea l., 17 – carex pilosa scop., 18 – melica uniflora retz., 19 – dactylis polygama horvátovszky, 20 – polygonatum multiflorum (l.) all., 21 – quercus petraea (mattuschka) lieblein, 22 – euphorbia amygdaloides l., 23 – ligustrum vulgare l., 24 – rosa arvensis huds., 25 – lathyrus vernus (l.) bernh., 26 – galium schultesii vest, 27 – hepatica nobilis mill., 28 – crataegus laevigata (poir.) dc., 29 – bromus ramosus huds. agg., 30 – fallopia dumetorum (l.) holub, 31 – quercus cerris l., 32 – fagus sylvatica l., 33 – viola alba bess., 34 – carpinus betulus l., 35 – festuca drymeja m. et k., 36 – lathyrus venetus (mill.) wohlf, 37 – campanula rapunculoides l., 38 – symphytum tuberosum l. subsp. nodosum (schur), 39 – convallaria majalis l., 40 – iris graminea l., 41 – glechoma hirsuta w. et k., 42 – ajuga reptans l., 43 – clinopodium vulgare l., 44 – taraxacum officinale weber ex wiggers, 45 – sorbus torminalis (l.) cr., 46 – luzula luzuloides (lam.) dandy et wilm., 47 – luzula forsteri (sm.) dc., 48 – primula vulgaris huds., 49 – hordelymus europaeus (l.) c. o. harz, 50 – mycelis muralis (l.) dum., 51 – tamus communis l., 52 – milium effusum l., 53 – geum urbanum l., 54 – stachys alpina l., 55 – euonymus europaeus l., 56 – galium odoratum (l.) scop., 57 – arabis turrita l., 58 – geranium robertianum l., 59 – moehringia trinervia (l.) clairv., 60 – cardamine impatiens l., 61 – isopyrum thalictroides l., 62 – galeobdolon luteum huds. s.l., 63 – ruscus hypoglossum l., 64 – galanthus nivalis l., 65 – cardamine enneaphyllos (l.) crantz, 66 – anemone ranunculoides l., 67 – mercurialis perennis l., 68 – pulmonaria officinalis l., 69 – fragaria vesca l., 70 – carex divulsa stokes, 71 – pyrus pyraster (l.) burgsdorf, 72 – hypericum hirsutum l., 73 – veronica chamaedrys l., 74 – erigeron annuus (l.) pers., 75 – cornus sanguinea l., 76 – rumex sanguineus l., 77 – dryopteris affinis (löwe) fras.-jenk., 78 – rubus hirtus w. et k. agg., 79 – clematis vitalba l., 80 – scrophularia nodosa l., 81 – solanum dulcamara l., 82 – paris quadrifolia l., 83 – dryopteris carthusiana 8vill.) h. p., 84 – rubus fruticosus l. agg., 85 – veronica montana l., 86 – aconitum vulparia rchb., 87 – sambucus nigra l., 88 – brachypodium sylvaticum (huds.) roem. et schult., 89 – carex sylvatica huds., 90 – ranunculus ficaria l., 91 – polystichum setiferum (forskål) woynar, 92 – corydalis cava (l.) schw. et koerte, 93 – epilobium montanum l., 94 – gagea lutea (l.) ker-gawl., 95 – polystichum aculeatum (l.) roth, 96 – stachys sylvatica l., 97 – eupatorium cannabinum l., 98 – dryopteris filix-mas (l.) schott, 99 – atropa bella-donna l., 100 – athyrium filix-femina (l.) roth, 101 – acer pseudoplatanus l., 102 – circaea lutetiana l., 103 – urtica dioica l. � u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:21 color profile: disabled composite 150 lpi at 45 degrees presence-absence data were analysed using hierarchical classification (hc; complete link, sorensen index) to identify the vegetation types on the slopes. however, field observations were also performed to specify the result of the classification. cluster analysis was done with the program package syn-tax 2000 (podani 2001). air temperature (°c) and humidity (%) were measured for 24 hours by sn21140ca sensors 25 cm above the ground surface in 50 sampling plots at 5 m intervals. spectrum – fieldscout tdr-300 was used to detect soil moisture values (%; in 12 cm depth) at the same 50 plots. all measures were carried out in summer, after a dry period, under clear weather conditions. slope angles were also measured along the transect, from which altitude values were calculated for each plot. we performed constrained ordinations using the vegan r package (oksanen et al. 2009, r development core team 2009) and syn-tax 2000 to determine the main environmental parameters affecting the distribution pattern of plant species along the transect. we 150 acta bot. croat. 70 (2), 2011 bátori z., gallé r., erdõs l., körmöczi l. fig. 2. cover of the 12 most abundant plant species along the transect. u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:23 color profile: disabled composite 150 lpi at 45 degrees used a series of the linear ordination method, redundancy analysis (rao 1964) to identify parameters that explain significant variation of species. we calculated the marginal and conditional effect of each term and assessed their significance using monte-carlo permutation tests with 5000 permutations (e.g. muff et al. 2009, gallé and torma 2009). the following data were used in the analysis: presence-absence data of species, mean air temperature, mean air humidity, soil moisture and altitude values (fig. 3). for interpretability, data of the summer aspect were used only in the redundancy analysis. figures were prepared with microsoft excel and adobe photoshop cs2. plant community names were used according to borhidi (2003), while the names of plant taxa follow simon (2000). results a total of 144 vascular plant species were detected in the doline (fig. 1, tab. 1). we found a strong correlation between species composition and doline morphology. some species occur in every part of the doline (e.g. asarum europaeum, cardamine bulbifera, fraxinus excelsior), while others occur only on the south-facing slope (e.g. clinopodium vulgare, festuca drymeja, lathyrus venetus), the north-facing slope (e.g. arabis turrita, isopyrum thalictroides, milium effusum) or in the doline bottom (e.g. dryopteris affinis, polystichum aculeatum, solanum dulcamara). from a floristic point of view, the doline acta bot. croat. 70 (2), 2011 151 flora and vegetation of a large doline fig. 3. redundancy analysis ordination diagram with 50 plots (�: plots of the south-facing edge, �: plots of the south-facing slope, �: plots of the north-facing edge, �: plots of the north-facing slope, �: plots of the doline bottom), environmental variables (arrows) and the most frequent plant species (italic numbers 1, 8, 15, 17–19, 62, 67, 103, according to fig. 1) of the plot groups. u:\acta botanica\acta-botan 2-11\batori.vp 26. rujan 2011 13:54:59 color profile: disabled composite 150 lpi at 45 degrees bottom is the most important, because it preserves the highest number of mountain species (e.g. aconitum vulparia, actaea spicata, asplenium scolopendrium, dryopteris affinis and polystichum aculeatum). the glacial relict plant stachys alpina was found only on the upper part of the north-facing slope. species cover is also strongly correlated with slope position and exposition along the transect (fig. 2). for example, festuca drymeja is abundant only on the south-facing slope, allium ursinum and cardamine enneaphyllos on the north-facing slope and in the doline bottom, while galeobdolon luteum s.l. is abundant only in the doline bottom. four vegetation types were detected along the transect. a turkey oak-sessile oak forest (potentillo micranthae-quercetum dalechampii horvát a. o. 1981) and an oak-hornbeam forest transition occurs on the south-facing edge (0–43 m), an oak-hornbeam forest on the south-facing slope and on the north-facing edge (44–71 m and 218–243 m), a beech forest on the lower part of the south-facing slope and on the north-facing slope (72–90 m and 146–217 m), and a ravine forest fragment in the bottom of the doline (91–145 m). summary statistics for redundancy analysis are shown in table 2. the subsequent monte-carlo permutation test indicates the significance of both the marginal and the conditional effect of the studied parameters (p < 0.001). the redundancy analysis triplot shows that doline vegetation is arranged along a moisture and temperature gradient (fig. 3). the south-facing edge and south-facing slope are the driest and warmest, while the doline bottom is the moistest and coldest. plots of the north-facing edge and north-facing slope occupy a transitional area in the ordination space. the maximum mean diurnal air temperature was detected in a plot of the south-facing edge (19.87 °c) and the minimum in a plot of the doline bottom (17.56 °c). in contrast, the maximum (57.9%) and minimum (7.3%) soil moisture, and the maximum (93.86%) and minimum (78.58%) mean diurnal air humidity values showed a reverse distribution. 152 acta bot. croat. 70 (2), 2011 bátori z., gallé r., erdõs l., körmöczi l. tab. 1. list of plant species outside the doline transect. south-facing edge and slope 104: allium oleraceum l., 105: astragalus glycyphyllos l., 106: buglossoides purpureo-coerulea (l.) i. m. johnst., 107: campanula persicifolia l., 108: chaerophyllum temulum l., 109: cornus mas l., 110: crataegus monogyna jacq., 111: cruciata glabra (l.) ehrend., 112: euonymus verrucosus scop., 113: euphorbia cyparissias l., 114: festuca heterophylla lam., 115: galium lucidum all., 116: lamium maculatum l., 117: lapsana communis l., 118: lathyrus niger (l.) bernh., 119: lilium martagon l., 120: lysimachia nummularia l., 121: lysimachia punctata l., 122: melittis carpatica klok., 123: poa nemoralis l., 124: potentilla micrantha ram., 125: scutellaria altissima l., 126: silene viridiflora l., 127: vicia sepium l., 128: viola odorata l. north-facing edge 129: asperula taurina l. doline bottom 130: aethusa cynapium l., 131: actaea spicata l., 132: arctium minus (hill) bernh, 133: asplenium scolopendrium l., 134: calamagrostis epigeios (l.) roth, 135: carex pendula huds., 136: cirsium arvense (l.) scop., 137: festuca gigantea (l.) vill., 138: galeopsis speciosa mill., 139: heracleum sphondylium l., 140: knautia drymeia heuff., 141: phytolacca americana l., 142: staphylea pinnata l., 143: tilia cordata mill., 144: tilia platyphyllos scop. u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:26 color profile: disabled composite 150 lpi at 45 degrees discussion our study revealed the close relationships between the variation of geomorphological and vegetation pattern in a karst depression at the spatial scale of the study. the most important finding of this study is that the vegetation pattern is strongly correlated with slope exposition and the depth of the doline. a former study by bátori et al. (2009) showed that plant associations of the karst of mecsek mountains are arranged along a moisture and nutrient gradient. however, despite being the first detailed description of the doline vegetation and its surroundings of this area, that study only addressed the large-scale vegetation pattern (braun-blanquet scale) of the bottom of the dolines, and used exclusively ecological-indicator values to reveal the habitat conditions in the depressions. in contrast, our present study reveals the fine-scale vegetation pattern along a doline transect and shows that there are remarkable differences among the species composition of the south-facing slope, the north-facing slope and the bottom of the doline. this is a consequence of the special microclimate that often determines both abiotic and biotic parameters of karst depressions (geiger 1950, whiteman et al. 2004, gargano et al. 2010, ozimec et al. 2010, vrbek et al. 2010). in hungary, south-facing slopes receive higher solar radiation (jakucs 1971), which affects temperature, air humidity and soil moisture, and ultimately, affects the vegetation. in the case of the investigated doline, mixed-oak forests cover the major part of the south-facing slope. in contrast, the north-facing slope receives lower solar radiation, which results in lower mean temperatures and lower evapotranspiration. due to the cooler climate, the north-facing slope and the lower part of the south-facing slope are covered by beech forests. therefore, a vegetation inversion is formed, which is a common phenomenon in karst depressions (beck-mannagetta 1906, horvat 1953, lausi 1964, favretto and poldini 1985). the vegetation pattern also changes in the bottom of the doline, where the special ecological conditions have led to the development of a ravine forest. our results are in good agreement with those of hoyk (1999), who pointed out that dolines of the mecsek mts play an important role in the landscape due to their characteristic shape and valuable flora. considering the special microclimatic conditions, geomorphological features, vegetation pattern and species composition, dolines are especially important and valuable for scientific research and nature conservation. acta bot. croat. 70 (2), 2011 153 flora and vegetation of a large doline tab. 2. summary statistics for redundancy analysis with the four environmental variables. axes 1 2 3 4 eigenvalues 0.110 0.044 0.038 0.029 species-environment correlations 0.9305 0.8834 0.8298 0.8047 cumulative % variance of species data 11.0 15.4 19.2 22.1 of species-environment relationship 50 69.7 86.8 100 sum of all eigenvalues: 1 sum of all canonical eigenvalues: 0.22 u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:26 color profile: disabled composite 150 lpi at 45 degrees acknowledgement we would like to thank andrás bíró, sándor csete, miklós maróti, tamás morschhauser, csaba németh and jános podani for the useful comments and suggestions. this research was supported by the támop-4.2.2/08/1/2008-0008 program of the hungarian national development agency. references bárány-kevei, i., 1999: microclimate of karstic dolines. acta climatologica universitatis szegediensis 32–33, 19–27. bátori, z., csiky, j., erdõs, l., morschhauser, t., török, p., körmöczi, l., 2009: vegetation of the dolines in mecsek mountains (south hungary) in relation to the local plant communities. acta carsologica 38, 237–252. beck-mannagetta, g., 1906: die umkehrung der pflanzenregionen in den dolinen des karstes. sitzungsberichte der kaiserliche akademie der wissenschaften in wien 65, 3–4. borhidi, a., 2003: plant associations of hungary (in hungarian). akadémiai kiadó, budapest. breg, m., 2007: degradation of dolines on loga{ko polje (slovenia). acta carsologica 36, 223–231. brullo, s., giusso del galdo, g., 2001: astracantha dolinicola (fabaceae): a new species from crete. nordic journal of botany 21, 475–480. calò, f., parise, m., 2006: evaluating the human disturbance to karst environments in southern italy. acta carsologica 35, 47–56. egli, b., gerstberger, p., greuter, w., risse, h., 1990: horstrissea dolinicola, a new genus and species of umbels (umbelliferae, apiaceae) from kriti (greece). willdenowia 19, 389–399. favretto, d., poldini, l., 1985: the vegetation in the dolinas of the karst region near trieste (italy). studia geobototanica 5, 5–18. gallé, r., torma, a., 2009: epigeic spider (araneae) assemblages of natural forest edges in the kiskunság (hungary). community ecology 10, 146–151. gargano, d., vecchio, g., bernardo, l., 2010: plant-soil relationships in fragments of mediterranean snow-beds: ecological and conservation implications. plant ecology 207, 175–189. geiger, r., 1950: das klima der bodennahen luftschicht. ein lehrbuch der mikroklimatologie. die wissenschaft, 4. verlag f. vieweg and sohn, braunschweig. horvat, i., 1953: vegetation of sinkholes (in croatian). geografski glasnik 14/15, 1–25. hoyk, e., 1999: investigations of the vegetation and soil in the dolinas of mecsek mountains, south hungary. acta carsologica 28, 105–113. jakucs, l., 1971: morphogenetics of karsts (in hungarian). akadémiai kiadó, budapest. lausi, d., 1964: vorläufiger überblick über die vegetation der triester karstdolinen. acta botanica croatica 4, 65–71. 154 acta bot. croat. 70 (2), 2011 bátori z., gallé r., erdõs l., körmöczi l. u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:26 color profile: disabled composite 150 lpi at 45 degrees morton, f., 1936: relazione sulla vegetazione delle doline del carso triestino. i. communicazione. alpi giulie 37, 57–70. muff, p., kropf, c., frick, h., nentwig, w., schmidt-entling, m., 2009: co-existence of divergent communities at natural boundaries: spider (arachnida: araneae) diversity across an alpine timberline. insect conservation and diversity 2, 36–44. oksanen, j., kindt, r., legendre, p., o’hara, b., simpson, g. l., solymos, p., stevens, m. h. m., wagner, h., 2009: vegan: community ecology package. r package version 1.15-4. retrieved february 6, 2011, from http://cran.r-project.org/package=vegan ozimec, s., bo[kovi], i., florijan^i], t., jelki], d., opa^ak, a., pu[kadija, z., labak, i., 2010: the lichen flora of risnjak national park (croatia). acta botanica croatica 69, 19–29. özkan, k., gulsoy, s., mert, a., ozturk, m., muys, b., 2010: plant distribution-altitude and landform relationships in karstic sinkholes of mediterranean region of turkey. journal of environmental biology 31, 51–61. podani, j., 2001: syn-tax 2000 computer programs for data analysis in ecology and systematics. scientia publishing, budapest. r development core team, 2009: r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. retrieved february 6, 2011, from http://www.r-project.org rao, c. d., 1964: the use and interpretation of principal components analysis in applied research. sankhya a 26, 329–358. simon, t., 2000: vascular flora of hungary (in hungarian). nemzeti tankönyvkiadó, budapest. veress, m., 2004: the karst. bdf természetföldrajzi tanszék, szombathely. vrbek, m., buzjak, n., buzjak, s., vrbek, b., 2010: floristic, microclimatic, pedological and geomorphological features of the balinovac doline on north velebit (croatia). proceedings 19 world congress of soil science, brisbane, 9–11. whiteman, c. d., haiden, t., pospichal, b., eisenbach, s., steinacker r., 2004: minimum temperatures, diurnal temperature ranges, and temperature inversion in limestone sinkholes of different sizes and shapes. journal of applied meteorology 43, 1224– 1236. acta bot. croat. 70 (2), 2011 155 flora and vegetation of a large doline u:\acta botanica\acta-botan 2-11\batori.vp 8. rujan 2011 15:57:26 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (2), 259–267, 2011 coden: abcra 25 issn 0365-0588 shift of the western boundary of the distribution area of micromeria cristata (hampe) griseb. and steptorhamphus tuberosus (jacq.) grossh. danka petrovi]*, danijela ste[evi] biology deptartment, faculty of natural sciences and mathematics, university of montenegro, d`ord`a va{ingtona bb, 81 000 podgorica, montenegro abstract – during field investigations of mt rumija, two new taxa for the flora of montenegro were recorded: micromeria cristata (hampe) griseb. and steptorhamphus tuberosus (jacq.) grossh. from the phytogeographic point of view these data indicate a change in the distribution area of both taxa, which have shifted to the west. a short overview of the taxonomic treatment of both genera is given. key words: flora, montenegro, mt rumija, micromeria cristata, steptorhamphus tuberosus introduction rumija is the southernmost part of the dinaric alps, the adriatic coastal mountain chain, extending in a nw-se direction, situated between skadar lake and the adriatic sea. part of the mountain lies in albanian territory, and can be considered as both a geographic and a phytogeographic link between the albanian, macedonian and greek mountains. several taxa reach the limits of their distribution area on mt rumija: valeriana dioscoridis sibth. et sm. and ramondia serbica pan~. are the easternmost limit (hayek 1931, stevanovi] and buli] 1992), cionura erecta (l.) griseb. and gymnospermium altaicum ssp. scipetarum (e. mayer et pulevi}) kit tan et mullaj are the westernmost (ble^i] and pulevi] 1979, tan and mullaj 2001) and centaurea incompta vis. is the southwestern (hayek 1931). mt rumija, accordingly, can be characterised as a kind of a floristic crossroad between the western and eastern balkans. on mt rumija the following parts are clearly distinguished: a) a longitudinal ridge with its high summitsthe summit of rumija, (1593 m),vrsuta (1184 m) and lonac (1179 m); b) numerous cliffs, ravines, defiles, karst valleys, sharp peaksin this area the hill called vladimir (486 m) is situated; c) the mountain of lisinj (1351 m), which rises in the south-west. southern slopes up to 300–400 m are exposed to the mediterranean climate, acta bot. croat. 70 (2), 2011 259 * corresponding author, email: danka.petrovic@t-com.me copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:17 color profile: disabled composite 150 lpi at 45 degrees while the northern and higher altitude southern slopes are affected by a modified mediterranean or sub mediterranean per humid variant of climate. the geological substrate is polymorphic: flysch is dominant on southern and chalk on northern slopes (milanovi] 1964). such specific geographic position, climate and geology are reflected in the development of a very interesting plant-life that has attracted attention of botanists since the second half of the 18th century. although numerous publications (e.g. ebel 1844; baldacci 1891, 1892, 1894, 1900, 1901; pan^i] 1875; adamovi] 1913; rohlena 1942; ^ernjavski et al. 1949; pulevi] 2005) have provided data about the flora of this area, it is still incompletely known. a summary of all the literature data (only the most important contributions are listed in the paragraph above) and of our own field investigations, which began in early spring 2000, the checklist of vascular flora of mt rumija consists of more than 1500 taxa (excluding the psamophilic and halophilic vegetation of the coastal part of the mountain). during the field investigations several species that are new for the montenegrin flora were recorded (petrovi] 2005). in this paper we will present micromeria cristata (lamiaceae) and steptorhamphus tuberosus (cichoriaceae the westernmost limits of the distribution area of which are on mt rumija. in addition we will give a short overview of the taxonomic treatment of both genera. material and methods material was collected during field investigations of mt rumija, conducted in mid-summer 2006 (micromeria cristata (hampe) griseb) and late spring 2007 (steptorhamphus tuberosus (jacq.) grossh.). it is preserved in the herbarium collection of the faculty of natural sciences in podgorica, voucher numbers: 968/06 and 642/07. identification of steptorhamphus was conducted according to ferákova (1976, 1977) and davis (1982), while nomenclature follows ferákova (1977). identification of micromeria was conducted according to dikli] (1974), [ili] (1979), ball and getliffe (1972) and nomenclature according to bräuchler et al. (2008). material is also compared with herbarium specimens preserved in the herbarium collection of the institute of plant sciences in graz. due to the diverse taxonomic treatments of micromeria and steptorhamphus genera a short taxonomic overview is given (ball and getliffe 1972; chater and guinea 1972; [ili] 1979; davis 1982; greuter et al. 1986; wagstaff et al. 1995; prather et al. 2002; trusty et al. 2004; bräuchler et al. 2005; koopman et al. 1998; stebbins 1937; tuisl 1968; ferákova 1977; kilijan 2001; lebeda et al. 2001, 2004). in order to avoid confusion with similar plants in our flora, some important morphometric features are specified, such as length of the flower and verticillaster peduncle (micromeria) and the presence of an outer minute pappus row (steptorhamphus). iucn characterization is in accordance with the most recent guidelines for using the iucn red list categories and criteria (anonymous 2010), while the regional justification of categories follows the guidelines for application of iucn criteria at regional levels (anonymous 2003). sources of threats and recommendation measures are defined due to the authority files, prepared by iucn, major threats authority file and conservation actions authority file (http://www.iucn.org). from the time of the first record, the species was monitored up to september 2010 at a frequency of several times a year. only mature individuals were counted. 260 acta bot. croat. 70 (2), 2011 petrovi] d., ste[evi] d. u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:17 color profile: disabled composite 150 lpi at 45 degrees results during the field investigations of mt rumija two new taxa for the flora of montenegro were recorded: micromeria cristata (hampe) griseb. subsp. cristata and steptorhamphus tuberosus (jacq.) grossh. the first taxon was collected in seoca village, northern slopes, n 42° 13' 83'' e 19° 08' 27'', altitude ca 250 m, in rock crevices in the stipo-salvietum officinalis h-i} (1956) 1958 association, developed as a degradation stage of white hornbeam shrubland (rusco-carpinetum orientalis ble~i} et laku{i} 1966.) and representing the most common type of rocky pastures in the southern part of the country. the population has fewer than 50 individuals. they share a habitat with the related micromeria juliana (l.) bentham. although very similar in habitus and ecology, these species can be clearly distinguished by the length of the flower and verticillaster peduncle (fig 1). micromeria cristata flowers are distinctly pedicellate and verticillasters are pedunculate, in distinction to m. juliana, which has sessile flowers and subsessile verticillaster. however, we suspected there was a possibility of confusion as result of which in previous surveys m. cristata might have been overlooked in rich populations of m. juliana. after the first record in 2006, no new populations of this species have been found in similar habitat types in the sub-mediterranean part of montenegro. the first records of steptorhamphus tuberosus (fig. 2) date from 2007. the species was collected in the vicinity of the village of godinje (northern slopes of rumija mountain, n 42° 13' 52'', e 19° 06' 32'', altitude 86 m), in the plant community carpinetum orientalis punicosum o. greb 1949., one of the degradation stages of white hornbeam forests and acta bot. croat. 70 (2), 2011 261 distribution area of micromeria and steptorhamphus fig 1. micromeria cristata, verticillasters (photo by d. petrovi}) fig. 2. steptorhamphus tuberosus, a) inflorescens, b) root, and c) pappus (photo by d. petrovi}) u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:22 color profile: disabled composite 150 lpi at 45 degrees shrublands. the population consisted of approximately 30 individuals and it has remained stable. another population of s. tuberosus has been found on the southern slopes of rumija mountain (spilica, n 42° 05' 50'', e 19° 08' 37'', altitude 195 m). it inhabits a site similar to that of the previous populationa fringe of rusco-carpinetum orientalis ble~i} et laku{i} 1966., a degradation stage of white hornbeam shrubland. population size is much smaller (fewer than 10 individuals) and has remained stable. discussion taxonomic position of micromeria cristata (hampe) griseb. according to some taxonomists, the genus micromeria benth. is considered a part of the »satureja complex«, which is, due to morphological diversity, divided into several genera: satureja l., calamintha mill., clinopodium l., acinos mill., micromeria benth. (ball et getliffe 1972, davis 1982, [ili] 1979). according to others, it is included in the genus satureja l. (greuter et al. 1986). recently conducted studies of the phylogeny and generic status of satureja s.l. (wagstaff et al. 1995, prather et al. 2002, trusty et al. 2004, bräuchler et al. 2005) clearly show that the genus is not monophyletic. the genus micromeria includes about 54 accepted species with 32 subspecies and 13 varieties bräuchler et al. (2008). following this taxonomic approach and revising rohlena (1942), [ili] (1979) and pulevi] (2005) we found that in the montenegrin flora micromeria is represented by 5 species: micromeria graeca (l.) benth. ex rchnb., micromeria croatica (pers.) schott., m. juliana (l.) benth. ex rchnb., m. longipedunculata bräuchler and m. kerneri murb. during field investigations of mt rumija an additional micromeria species was recorded – m. cristata, which is according to bräuchler et al. (2008) subdivided into 6 subspecies: m. cristata subsp. carminea (p. h. davis) p. h. davis, m. cristata (hampe) griseb. subsp. cristata, m. cristata subsp. kosaninii ([ili}) bräuchler et govaerts, m. cristata subsp. orientalis p. h. davis, m. cristata subsp. phrygia p. h. davis, m. cristata subsp. xylorrhiza (boiss. et heldr. ex benth.) p. h. davis. data on general distribution (greuter et al. 1986, davis 1982, hand 2006 and jamzad 2009) show that four subspecies are endemic to anatolia, while the type species and m. cristata subsp. kosaninii are distributed in the balkan peninsula. in the flora of montenegro only the subspecies cristata exists. geographic distribution of micromeria cristata the main part of the distribution area occurs on the balkan peninsula, in albania, bulgaria, greece, serbia, kosovo, macedonia ([ili] 1979) (fig. 3), but the species is also reported for anatolia (davis 1982) and iran (jamzad 2009). thus the new finding of micromeria cristata on mt rumija means a shift of the limit of its distribution area towards the west. endangered status and protection during the field survey we estimated that the population of micromeria cristata in montenegro consists of fewer than 50 mature individuals. due to this fact, and according to criterion d1, the iucn threat category is cr (critically endangered) (anonymous 2010). but, taking into account the possibility of immigration from the neighbouring area (albania in the first place), the regional red list category (anonymous 2003) should be 262 acta bot. croat. 70 (2), 2011 petrovi] d., ste[evi] d. u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:22 color profile: disabled composite 150 lpi at 45 degrees changed to en (endangered). monitoring conducted in the period 2006–2010 showed that the population is stable. however, even a small environmental change could seriously endanger this species. taxonomic position of steptorhamphus tuberosus (jacq.) grossh. similar to that of micromeria, the taxonomic position of the genus lactuca s. l. is not simple. there are 3 basic concept of the genus given by stebbins, tuisl and feráková (koopman et al., 1998). according to stebbins (1937), the genus lactuca s. l. also includes the following genera mulgedium cass., lactucopsis schultz-bip. ex vis. et panc., phaenixopus cass, mycelis cass. and part of cicerbita. tuisl (1968) gave a stricter concept of the genus and singled out mulgedium, scariola fw. schmidt (= phaenixopus), cicerbita, cephalorrhynchus boiss. and steptorhamphus. clasification by ferákova (1976, 1977) is intermediate so the genera mulgedium, lactucopsis and phaenixopus/scariola are included in genus lactuca, while mycelis, cicerbita, cephalorrhynchus and steptorhamphus are not. the presence of an outer minute pappus row (fig. 2) was the main feature used by ferákova to keep this genus separate from lactuca. acta bot. croat. 70 (2), 2011 263 distribution area of micromeria and steptorhamphus fig. 3. utm map of montenegro (source: geokarta's utm basemap) with the record of micormeria cristata on mt rumija, and general distribution map of m. cristata (with permission from atlas florae europaeae). u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:22 color profile: disabled composite 150 lpi at 45 degrees flora europaea follows ferakova's concept where lactuca cretica desf. contains two unequal rows of hairs, and is transferred to the genus steptorhamphus (s. tuberosus (jacq.) grossh.). molecular analysis based on its-1 dna sequences (koopman et al. 1998), has not supported a distinction between lactuca and steptorhamphus. nevertheless, these investigations were not sufficient for a revised delimitation of lactuca and related genera (kilijan 2001). despite this, greuter et al. (2008) included the genus steptorhamphus into lactuca. in the recent publications on the distribution and ecology of lactuca species in europe (lebeda et al. 2001, lebeda et al., 2004), the taxon l. tuberosa jacq. is not treated within this genera. in our paper we decided to follow the concept of the monographer (ferakova 1977), arguing that molecular analysis still has not resolved the »lacucasteptorhamphus« taxonomical problem. geographic distribution of steptorhamphus tuberosus only a part of the steptorhamphus tuberosus distribution area occurs in the southern and eastern part of the balkan peninsula (fig. 4). an interesting detail is that in spite of a 264 acta bot. croat. 70 (2), 2011 petrovi] d., ste[evi] d. fig. 4. utm map of montenegro (orig., map, source: geokarta's utm basemap) with the record of streptorhaphus tuberosus on mt rumija, and general distribution map of s. tuberosus (with permission from atlas florae europaeae). u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:22 color profile: disabled composite 150 lpi at 45 degrees previous record of this taxon in macedonia (so[ka 1939, bornmüller 1926), flora europaea did not include ex-yugoslavia in its geographic distribution. just as in the case of micromeria cristata, a new record of the species on mt rumija (montenegro) means a moving of the limit of its distribution area towards the west. endangered status and protection during the field survey we estimated that the population of steptorhamphus tuberosus in the village of godinje consists of approximately 30 mature individuals, while on the southern slopes only a few (less than 10) have been counted. due to this fact, and according to criteria d1, the iucn category of threat is cr (critically endangered) (anonymous 2010). since the species is not recorded in neighbouring countries, the regional red list category need not to be changed. monitoring of steptorhamphus tuberosus population during the period 2007–2010 showed that the population is stable. acknowledgements we are grateful to our colleague bojan zlatkovi} for valuable help with the literature and the editors of atlas florae europaeae arto kurtto et al. for permission to use the copyrighted afe basemap. references adamovi], 1913: contribution to the knowledge of flora of the kingdom of montenegro (in serbian). rad jazu 195 1–96. anonymous, 2003: guidelines for application of iucn red list criteria at regional levels: version 3.0. iucn species survival commission. iucn, gland, switzerland and cambridge, uk. anonymous, 2010: iucn standards and petitions subcommittee. 2010. guidelines for using the iucn red list categories and criteria, version 8.1. prepared by the standards and petitions subcommittee in march 2010. downloadable from http://intranet.iucn.org/ webfiles/doc/ssc/redlist/redlistguidelines.pdf. ball, p. w., getliffe, f. m., 1972: satureja l., acinos miller, clinopodium l., calamintha miller. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a., (eds.), flora europaea 3, 163–167. cambridge university press, cambridge. baldacci, a., 1891: cenni ad appunti intorno alla flora del montenegro iv. malpighia (genova) 5, 61–81. baldacci, a., 1892: altra notizie intorno alla flora del montenegro. malpighia (genova) 6, 1–123. baldacci, a., 1894: contributo alla conoscenza della flora dalmata, montenegrina, albanesa, epirota e greca. nuovo giornale botanico italiano (nuova serie) 1, 90–103. acta bot. croat. 70 (2), 2011 265 distribution area of micromeria and steptorhamphus u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:22 color profile: disabled composite 150 lpi at 45 degrees baldacci, a., 1900: contributo alla conoscenza della flora del confine montenegro-albanese. memorie della reale accademia delle scienze dell'istituto di bologna, 1–43. baldacci, a., 1901: rivista della collezione botanica fatta nell 1897 nell'albania settentrionale. memorie della reale accademia delle scienze dell'istituto di bologna 9, 513–553. ble^i], v., pulevi], v., 1979: new data for the flora of montenegro (in serbian). glasnik republi~kog zavoda za za{titu prirode i prirodnja~kog muzeja u titogradu 12, 189– 193. bornmüller, j., 1926: beiträge zur flora mazedoniens ii. botanische jahrbücher für systematik, pflanzengeschichte und pflanzengeographie 40, 1–125. bräuchler, c., meimberg, h., abele, t., heubl, g., 2005: polyphyly of the genus micromeria benth. 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(eds.), med-checklist notulae. wildenowia 31, 320. tuisl, g., 1968: der verwandtschaftskreis der gattung lactuca l. im iranischen hochland und seinen randgebieten. annalen des naturhistorischen museums in wien 72, 587–638. trusty, j. l., olmstead, r. g., bogler, d. j., santos-guerra, a., francisco-ortega, j., 2004: using molecular data to test a biogeographic connection of the macaronesian genus bystropogon (lamiaceae) to the new world: a case of conflicting phylogenies. systematic botany 29, 702–715. wagstaff, s. j., olmstead, r. g., cantino, p. d., 1995: parsimony analysis of cpdna restriction site variation in subfamily nepetoideae (labiatae). american journal of botany 82, 886–892. acta bot. croat. 70 (2), 2011 267 distribution area of micromeria and steptorhamphus u:\acta botanica\acta-botan 2-11\petrovic.vp 9. rujan 2011 13:02:22 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 78 (1), 2019 1 acta bot. croat. 78 (1), 1–8, 2019 coden: abcra 25 doi: 10.2478/botcro-2019-0008 issn 0365-0588 eissn 1847-8476 expression of small gtpases in the roots and nodules of medicago truncatula cv. jemalong abdul razaque memon1*, christiane katja schwager2, karsten niehaus3 1 department of molecular biology and genetics, faculty of science and arts, usak university, usak, turkey 2 department of genome research, faculty of biology, bielefeld university, bielefeld, germany 3 department of proteome and metabolome research, faculty of biology, bielefeld university, bielefeld, germany abstract – in this study we used medicago truncatula, to identify and analyze the expression of small gtp-binding proteins (arf1, arl1, sar1, rabs, rop/rac) and their interacting partners in the infection process in the roots and nodules. a real-time polymerase chain reaction analysis was carried out and our results showed that arf1 (atarfb1c-like), mtsar1, atraba1e-like, atrabc1-like, msrab11-like and atrop7-like genes were highly expressed in the nodules of rhizobium inoculated plants compared to the non-inoculated ones. on the contrary, atraba3 like, atrab5c and msrac1-like genes were highly expressed in non-infected nitrogen supplied roots of m. truncatula. other rab genes (atraba4a, atraba4c and atrabg3a-like genes) were nearly equally expressed in both treatments. interestingly, rbohb (a respiratory burst nadph oxidase homologue) was more highly expressed in rhizobium infected than in non-infected roots and nodules. our data show a differential expression pattern of small gtp-binding proteins in roots and nodules of the plants. this study demonstrates an important role of small gtp-binding proteins in symbiosome biogenesis and root nodule development in legumes. keywords: gene expression, medicago truncatula, root nodules, small gtp-binding proteins * corresponding author, e-mail: armemon@usak.edu.tr, abdulrezzak.memon@gmail.com introduction intracellular membrane trafficking plays a major role in plant growth and development including root hair formation, rhizobium infection process and nodule formation in legumes (oldroyd and downie 2008). it is evident from the literature that small gtp-binding proteins perform a critical role in different aspects of root nodule development (blanco et al. 2009). for example several research groups have shown the importance of rab1, rab7 and rab11f in peribacteroid membrane formation in different legume species like glycine max, vigna aconitifolia, medicago sativa and medicago truncatula (cheon et al. 1993, son et al. 2003, schiene et al. 2004, limpens et al. 2009). during rhizobium infection, raba2 is reported to participate in the polar growth of root hairs of phaseolus vulgaris (dalla via et al. 2017). these authors showed that raba2 is localized to mobile vesicles around the infection thread (it) and is required to maintain the integrity of the membrane during it progression. rab7 is reported to be required in vesicular fusion during symbiosome membrane transport in soybean (cheon et al. 1993). furthermore, a total of 33 genes of small gtp-binding proteins were identified from a lotus japonicus genome and among them, some of the rabs like rab1, rab2, rab5, rab7, and rac were predominantly expressed in root nodules of these plants (borg et al. 1997). likewise, schiene et al. (2004) have shown that rabf1 is preponderantly expressed in the nodules and possibly involved in membrane vesicular trafficking and symbiosome formation in m. sativa and m. truncatula. interestingly rab7 has been shown to be recruited by symbiosome as an endosomal marker when they stop dividing and possibly to regulate the symbiosome maturation process (clarke et al. 2015). rab gtpases are the largest member of gtpase family in most of the eukaryotes. they work in combination with rab effector proteins and regulate nearly all steps of membrane traffic from vesicle budding to vesicle fusion (pfeffer 2017, biver et al. 2011). arf and sar1 are known to be the key gtp-binding proteins in the regulation of membrane trafficking between er and ga in plant, yeast and mammalian cells (pimpl et al. 2000, jumemon a. r., schwager c. k., niehaus k. 2 acta bot. croat. 78 (1), 2019 rgens 2004, memon 2004, memon 2012). several observations in a. thaliana and tobacco cells indicate that arf1 not only functions in retrograde protein transport from golgi to er but also in some way affects the anterograde pathway (memon, 2004). several isoforms of arf1 have been identified in the m. truncatula genome (vernoud et al 2003, yuksel and memon 2008) but their exact function in root and/or nodules has not been defined. it will be interesting to examine the possible involvement of these proteins in intracellular vesicle trafficking in the early steps of symbiotic interactions. accumulating evidence shows that rop/rac acts as a predominant switch in signal transduction in plants and is reported to regulate several developmental processes in plants, such as polarized cell growth, cell morphogenesis, hormone signalling, defence and responses to oxygen deprivation (craddock et al. 2012). in root hairs (foreman et al. 2003, ke et al. 2016, lei et al. 2015) and in pollen tubes (potocky et al. 2007), these gtp-binding proteins activate nadph oxidase (wong et al. 2007) and are responsible for the apical accumulation of reactive oxygen species (ros). ros is shown to be essential for cell elongation and to play a vital role in root hair deformation and curling during rhizobial infection (damiani et al. 2016a, damiani et al. 2016b). recently it has been shown that rop activates the nadph oxidase homologue of plants termed rboh (for respiratory burst oxidase) and generates ros during early microbial infection (kiirika et al. 2012). membrane protein targeting in the symbiosome varies depending on the mosaic nature of the symbiosome which includes the properties of both plasmalemma and tonoplast. this indicates that the participation of several small gtpbinding proteins in symbiosome membrane biogenesis is more complex and this process could be studied in detail. to elucidate the mechanism of membrane transport in root nodules, we made an attempt to retrieve the est databases of two model legume plants, medicago and lotus and searched for the nodule-specific expressed transcripts of small gtpases (yuksel and memon 2008). this analysis showed a group of 10 small gtpases which are likely to be expressed in root nodules and among them, seven belonged to the rab cladistic group and two belonged to the arf subfamily group (yuksel and memon 2008). in this study, we made a detailed expression analysis of these gtp-binding proteins in rhizobium inoculated and non-inoculated roots and nodules of m. truncatula with the intention of exploring the role of these proteins in rhizobium infection process and symbiosome formation in m. truncatula roots. materials and methods plant cultivation medicago truncatula cv. jemalong seeds were surface sterilized in concentrated hcl (37%) for 10 min. after several washings with sterile water, seeds were spread on agar plates with plant minimal medium adjusted to ph 5.7 (rolfe et al. 1980) and germinated for two days in the dark at room temperature. for nodulation, each seedling was inoculated on agar plates with 0.1 ml sinorhizobium meliloti 2011 grown to an optical density of 0.6 in yep medium (5 g l–1 yeast extract, 10 g l–1 peptone, 5 g l–1 nacl, ph 7.0) supplemented with 600 mg l–1 streptomycin. mature roots and nodules were harvested four weeks after infection. plants were grown under the following conditions: 25 °c, 65% humidity, 200 µe m–2 s–1 and 16 h light/8 h dark. growth conditions for inoculated and non-inoculated plants were the same except that non-inoculated plants were supplied with nh4no3 as the nitrogen source. nodules and roots were separated and were used for rna isolation, cdna synthesis, and quantitative real-time pcr (qpcr). real-time qpcr total rna was isolated from the roots and nodules of m. truncatula grown with and without rhizobium with the use of the rneasy plant mini kit (qiagen, hilden, germany) following the manufacturer’s instructions. rna was quantified using a nanodrop analyser 1000 (thermo scientific, wilmington, de, usa) and cdna was synthesized from 2 µg of extracted rna as previously described by (mehrtens et al. 2005). forward and reverse primers of the targeted genes were designed using primer 3 software (rozen and skaletsky 2000) and are listed in table 1. real-time qpcr (rt-qpcr) was performed using the platinum sybr green qpcr supermix udg kit (invitrogen, karlsruhe, germany) following the manufacturer’s instructions on a rotor-gene 6000 (corbett research; http://www.corbettlifescience.com/) cycler. in our preliminary experiments ubiquitin, actin and ribosomal 18s genes were used for internal control. our data showed that ubiquitin (tc102473) was the most suitable for use as an internal control for rt-qpcr under our experimental conditions. therefore, in our further experiments we used ubiquitin as an internal control in data evaluation. raw data were analyzed with the camper software tool implementing an algorithm based on a four parameter logistic model (fplm) (tichopad et al. 2003) of the fluorescence curve. the fplm algorithm was used to calculate efficiency-corrected crossing points (ccps) that were subsequently analyzed using the rest (relative expression software tool) software (tichopad et al. 2003, bucciarelli et al. 2006). each experiment was replicated 3 times and each sample was replicated 3 times for real-time qpcr analysis. statistical analysis all the analyses were performed in triplicate and the results were recorded as mean ± standard deviation (sd). visualizations were created using graphpad prism 7.5 for windows (graphpad software, san diego usa). analysis of variance (anova) and post-hoc test (tukey) was carried out by using ibm spss statistics 23 software. tukey test was used to evaluate the significance of different treatment groups. small gtp-binding proteins in the root nodules acta bot. croat. 78 (1), 2019 3 results to obtain a global view of the gene expression of small gtp-binding proteins in roots and nodules of m. truncatula, rt-qpcr experiments were carried out with plants inoculated with s. meliloti for 4 weeks. the efficiency of cdna synthesized was assessed by endpoint rt-qpcr using constitutive gene-specific primers encoding ubiquitin as a reference gene. expression patterns of mtrab, mtarf, mtarl, mtsar, mtrop7, mtrac1, mtrbohb and mtcop in nodules and rhizobia inoculated and non-inoculated roots of m. truncatula are shown in figs. 1, 2, and 3. the rt-qpcr experiments with these genes showed that atraba1e and atrabc1 homologues and msrab11 were highly expressed in the nodules compared to the roots of non-inoculated plants (see figs. 1a, f, h). on the other hand, the expression pattern of homologues of atraba3, atraba4a, atraba5c and atrabg3a was significantly higher in the roots of noninoculated plants compared to the nodules and roots of inoculated plants (see figs. 1b, c, e, g). in contrast, homologues of atraba4c was predominantly expressed both in the roots of inoculated and non-inoculated plants (fig. 1d). inoculation of m. truncatula roots with s. meliloti caused a strong induction of the mtsar1 transcript (see fig. 2c). we found that mtsar1 expression was induced around 6 times more in inoculated than in the roots of non-inoculated nodules (fig. 2). post-hoc test showed that mtsar1 expression in nodules was statistically highly significant (p < 0.05) as compared to the expression in control roots and roots treated with s. meliloti. interestingly arfb1c transcript accumulation was significantly reduced in the roots of non-inoculattab. 1. primer pairs used for quantitative real-time pcr. primer3 software was used to design the primers of the each gene. gene forward primer sequence (5’–3’) reverse primer sequence (5’–3’) atraba1e cgaatcttcagttgaaaccgctc gctggtcaagaaaggtaccgag atraba3 accctccacattcacaccag gaggtgcattaggagcaatgc atraba4a ggccaagaacgatatagagcag tgcatcctctgtgggtactg atraba4c gtgcgtattaccgtggagctg gagcatcttcagtaggtactgcc atraba5c gctggacaagaaaggtttagagc cccaccaacattctcgcaac atrabc1 gttcagggcagcaagaattcga tcccaaatggcaagcttaagc atrabg3a cacttgataactggcacgaggag ggaatgcagcatcaacattgagg msrab11 gtgctgattggtgattctgg cttgatcgcctcgttatgtc atarfb1c catcgctcctctcaggtcttg gaggacactctggaggcac atarla1d gaggacatgattcctacagtggg cactcaatgatggcttgctcag mtsar1 cgctcaggtggatgctgtagtc catgaacacctccaaaggacg mtarf1 ctggatgtagtggcgctgt ccaagacaagattcgtccactg mt-z-cop-2 ctccttccaaactcgagttg gctccagattctgtactattac msrac1 gcttgaccttcgggatgataag gacaactcttatggctgcgtc mtrbohb gctcgctctgctcttattgc tgcgcttgtagacactacgc athrop7 ctcttcaccctgggcagtag ggccattgagctatagaggagc ubiquitin gcagatagacacgctggga aactcttgggcaggcaataa fig. 1. expression levels of rab group gtp binding proteins (at mrna level) in the nodules and roots (roots-sm) of medicago truncatula inoculated with rhizobium sinorhizobium meliloti. ubiquitin was used as internal control. control roots (control n+) were harvested from un-inoculated plants which were supplied with nitrogen fertilizer. bars represent standard deviation of three replicates. different letters show significant differences between groups (p < 0.05) based on tukey test. memon a. r., schwager c. k., niehaus k. 4 acta bot. croat. 78 (1), 2019 ed plants (fig. 2a). this clearly suggests that arfb1c is possibly nodule specific and expressed in host plant roots after rhizobium infection, indicating its importance in nodule development. intriguingly, another mtarf1(mtrgb57486383) transcript was equally expressed in both roots treated with s. meliloti and nodules of inoculated plants (fig. 2d) but its expression was increased in non-inoculated roots suggesting its general role in vesicular trafficking. our data show that one of the interacting partners of arf1, a z-subunit of mtcop2 (a component of cop1 vesicles) was predominantly expressed in rhizobia inoculated roots (fig. 2e) and this expression pattern was quite different than that of arf1 expression in roots and nodules. fig. 2b shows that atarla1d was highly expressed in both nodules and roots of non-inoculated plants and this expression pattern of at arla1d was different than that of the expression pattern of the other genes in the arf/sar1 group (see figs. 2 a, b, c, d). to assess the role of mtrop in the infection process, mtrop gene expression, and their relative transcriptional levels were analyzed in nodules and roots of inoculated and non-inoculated plants. the rt-qpcr experiments showed that indeed our previously identified transcript (homologues to a. thaliana rop7 and a8ik57_medtrrop 8) is predominantly expressed in root nodules (fig. 3c), indicating its role in nodule biogenesis. there are few data available related to the participation of nadph oxidases in rhizobium and symbiotic interactions in legumes. this prompted us to investigate the rac and rbohs role during the initial stages of nodulation (figs. 3a, b). our results showed that the expression of mtrbohb transcript was significantly increased in roots and nodules of inoculated as compared with non-inoculated plants (see fig. 3b). discussion the cdna array analysis performed with m. truncatula has identified the genes involved in nitrogen and carbon metabolism (tesfaye et al. 2006). however, very little is known about the genes and proteins involved in vesicular trafficking which is reported to increase in root cells during the infection process (navazio et al. 2007, yuksel and memon 2008). the symbiotic interaction between rhizobia and roots of legumes results in the formation of an infection thread followed by the formation of a bacteria containing compartment, the symbiosome, which continuously fuses with numerous vesicles and finally gives rise to plant cells colonized by a huge number of bacteroids within mature nitrogen-fixing nodules (wang et al. 2010, ivanov et al. 2010). since a huge amount of membrane material is needed to be transported from er to the ga for symbiosome formation (oldroyd and downie 2008), it is assumed that the transport of the symbiotic membranes should be dramatically enhanced due to rhizobium infection. consequently, we carried out a differential expression analysis, which provided a comprehensive view of the rate of fig. 2. expression levels of arf/sar group gtpbinding proteins (at mrna level) in nodules and roots (roots sm) of medicago truncatula inoculated with rhizobium sinorhizobium meliloti. ubiquitin was used as internal control. control roots (control n+) were harvested from un-inoculated plants which were supplied with nitrogen fertilizer. bars represent standard deviation of three replicates. different letters show significant differences between groups (p < 0.05) based on tukey test. fig. 3. expression level of rop/rac group gtp binding proteins and mt rboh gene in nodules and roots (roots sm) of medicago truncatula inoculated with rhizobium sinorhizobium meliloti. ubiquitin was used as internal control. control roots (control n+) were harvested from un-inoculated plants which were supplied with nitrogen fertilizer. bars represent standard deviation of three replicates. different letters show significant differences between groups (p < 0.05) based on tukey test. small gtp-binding proteins in the root nodules acta bot. croat. 78 (1), 2019 5 transcription of small gtp-binding proteins during nodule development in m. truncatula. our previous research conducted on databases of two model legume plants (medicago and lotus) containing ests of genes expressed in legume-rhizobium infection process have enabled the identification of 14–15 genes of gtp-binding proteins possibly involved in root hair formation and nodule development in m. truncatula (yuksel and memon 2008). analyses of sequence similarities and of the presence of specific family and subfamily conserved motifs in their primary sequence allowed the identification of closest homologues from arabidopsis and the assignment of around 8 medicago and lotus sequences to the rab family, 4 to arf/arl/sar family and 2 to rop/rac family involved in root hair formation and nodule development (yuksel and memon 2008). rab proteins have been functionally linked to the development of nodules in legumes (limpens et al. 2009). for example, rab1 and rab7 proteins are known to be part of symbiosome membrane biogenesis (cheon et al. 1993). within the medicago rab family, the most abundant subclass is the raba subclade and this is consistent with the significant expansion of the raba group reported in arabidopsis (woollard and moore 2008). two rab gene homologues to atraba1e and atrabc1 and one gene homologue to msrab11 were shown to be dominantly expressed in nodules of rhizobium inoculated plants (figs. 1a, f, h). although other rabs (raba3, raba4a, raba4c, raba5c rabg3a) were also expressed in nodules and roots of inoculated plants their expression level was not significantly increased compared to the roots of un-inoculated plants (figs. 1 b,c,d,e,g). the precise role of these rabs at different stages of infection process cannot be clarified at this stage; however, our gene expression data clearly indicates that these proteins play an important role in symbiosome formation. in arabidopsis most of the genes in the raba4 group were spatially expressed in root tissues except atraba4d which was specifically expressed in stamen and pollen (yuksel and memon 2008, szumlanski and nielsen 2009). a subclade of rab gtpase is reported to be involved in golgi to plasma membrane trafficking in both plant and mammalian cells (woolward and moore 2008), therefore it could fulfil the complementary role in different steps of the exocytic pathway in root cells. evidence from different sources supports the involvement of raba gtpases in post golgi trafficking (lycett 2008) and could play a substantial role in nodule development. another rabg3 class (rabg3a) is related to rab7 (mazel et al. 2004) and has been highly expressed in roots and nodules. this rab gtpase has been reported an endosomal marker and acquired by symbiosomes when they stop dividing and that rab7 regulates the maturation of the symbiosomes into nitrogen fixing organelles in m. truncatula (son et al. 2003, limpens et al. 2009). the level of arfb1c expression in the nodules of inoculated plants was significantly higher than in the roots of noninoculated ones. indeed, very little arfb1c transcript accumulation has been observed in the roots of non-inoculated plants (fig. 2a). this indicates that in medicago truncatula, this arf is possibly a nodule specific and expressed in the host plant roots after rhizobium infection. intriguingly another mtarf (mtrgb57486383) transcript was expressed both in roots and nodules of inoculated and noninoculated plants suggesting its general role in vesicular trafficking in plant cells (fig 2d). in our previous experiment with arf1 protein expression, we observed a significant increase in arf1 protein expression with rhizobial infection and the major pool of the arf1 in both nodules and roots is possibly present in active form (memon 2012). it is interesting to note that sar1, which is required in vesicular trafficking in early anterograde trafficking from er to cis-golgi, has shown a very high expression in nodules as compared to roots in rhizobium inoculated plants (fig. 2c). these observations indicate that the genes of these proteins involved in early secretory pathway may play a central role in nodule development and biogenesis (memon 2012, oldroyd and downie 2008). the expression of atarl a1d was more or less similar in both nodules and non-inoculated roots (fig. 2b). this suggests the dual role of this arl in both the early and late secretory pathway. interestingly the expression pattern of mtzcop-2 was shown to be different than that of atarfb1c (figs. 2a, e). these differences in expression could be due to the fact that arf1is not only required in the retrograde pathway from ga to er transport but also involved in late secretory transport from trans-golgi to endosomes and to the plasma membrane (memon 2004, matheson et al. 2007, park and jurgens 2012). rop gtp-binding proteins work as signalling molecules to control the polar growth of root hairs, and the formation of the interdigitated pattern and leaf pavement and pollen tube growth in arabidopsis (fu et al. 2005, gu et al. 2005). recent data show that the spatiotemporal dynamics of rop proteins, the cytoskeleton, endocytosis, and exocytosis are intertwined (yalovsky et al. 2008). the expression of some rops in roots of m. truncatula is altered in response to rhizobia infection (liu et al. 2010). previously we identified a single rop gene ljatc 11919 from lotus which was predominantly expressed in nodules (yuksel and memon 2008) and is closely associated with a gene from m. truncatula (mtr imgacc140545-16.1). this gene is homologous to atrop7 and mtrrop8. our realtime qpcr results showed about 3 times more expression of athrop7 transcript in the nodules of inoculated plants than in those of non-inoculated one (fig. 3c), indicating its role in nodule biogenesis. some reports have shown the increased expression of mtrop5 and mtrop6 after rhizobium inoculation (riely et al. 2011). it is assumed that these genes could be involved in cytoskeleton reorganization of the host cell during rhizobium infection process. there is increasing evidence that rop/rac proteins are involved in the generation of ros during the early stages of microbial infection via the activation of respiratory burst nadph oxidase homologues of plants (rboh) (ke et al. 2016). the ros are reported to be involved in signalling in response to rhizobia–legume interaction (pauly et al. 2006, jamet et al. 2007) and it appears that ros production is essential for optimal symbiosis formation. rac and rbohs seem to play an important role in ros production memon a. r., schwager c. k., niehaus k. 6 acta bot. croat. 78 (1), 2019 during symbiotic interaction (marino et al. 2011, montiel et al. 2012, arthikala et al. 2014). interestingly the downregulation of rbohb through rnai in phaseolus vulgaris roots reduced ros production and lateral root density and greatly impaired nodulation (montiel et al. 2012). on the contrary, over-expression of rbohb in p. vulgaris significantly enhanced the ros production, infection thread formation and increased the density of symbiosomes in nodules, and the density and size of bacteroids in symbiosomes (arthikala et al. 2014). our data in fig. 3b showed significantly high expression of mtrbohb in rhizobium inoculated roots compared to non-inoculated ones. these observations illustrate the importance of the nadph oxidases and their interacting gtpases (rops) in rhizobium infection process and show that is possibly plays an important role in the early infection process. rac1 is an orthologue to small gtpase mtrop9 in m. truncatula (kiirika et al. 2012) and our data show that it was expressed in both roots and nodules but its expression was significantly higher in the roots of non-inoculated plants (fig 3a). this implies that rac1 is not only involved in ros-mediated early infection process as reported earlier (kiirika et al 2012) but also involved in the activation of plant defence response. the early defence response of rac/rop-like gtpases in plants has been previously reported (wong et al. 2007). for example rac1 in rice activates rboh mediated ros signalling and is reported to be a positive regulator in disease resistance. this work provides important experimental evidence for the involvement of small gtp-binding proteins and the respiratory burst oxidase homologue (rboh) in rhizobium infection process and nodule development in the roots of m. truncatula. conclusion in this communication, we described the role of small gtp-binding proteins (arf1, arl1, sar1, rabs, rop/rac) and some of their interacting components in the biogenesis of the symbiosome membranes in the root nodules of m. truncatula. our gene expression data showed that several rabs, rop, arf and sar proteins are involved in early and late secretory pathways during rhizobium-legume interaction and nodule development. this study gives an important clue that in addition to the proteins involved in the late secretory pathway (like rab7, rab11), the gtp-binding proteins of the early secretory pathway (sar1, arfb1c raba1e, rabc1) are also more highly expressed in rhizobium inoculated roots and nodules than in non-inoculated roots. this suggests that the activation of multiple gtp-binding proteins (both in early and late secretory pathway) is required for symbiosome membrane biogenesis and root nodule development. further work would identify the additional components of protein trafficking in the early and late secretory pathway in legume roots and nodules and elucidate the mechanism of transport through the golgi complex to the symbiosome compartment in legumes. acknowledgments we appreciate the support and laboratory facilities given by cebi-tec, bielefeld university, bielefeld, germany. this work was supported by bap 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(cistaceae) along an elevational gradient in turkey sevda turkis, tugba ozbucak* department of biology, faculty of arts and sciences, ordu university, 52750 ordu, turkey foliar nitrogen and phosphorus dynamics, leaf resorption efficiency, proficiency, changing of chlorophyll a/b proportions in leaves of cistus creticus l. (cistaceae) along an elevational gradient (sea level-30 m, middle-670 m, high-880 m) were investigated. statistically significant differences were found in foliar nitrogen and phosphosrus content in terms of growth periods, while no significant differences were found according to elevations. nitrogen and phosphorus resorption efficiency and proficiency values were high as compared to the other evergreen species. cistus creticus effectively used nitrogen and phosphorus. no statistically significant differences were found among elevations in terms of specific leaf area. however, statistically significant differences were found in terms of growth periods. there were significant differences in chlorophyll a/b proportion according to both growth periods and elevations. besides, the chlorophyll a/b proportion increased along senescence period. key words: leaf, nitrogen, phosphorus, resorption, chlorophyll, cistus creticus, elevation introduction the cistaceae family includes 8 genera with 175 species distributed in the temperate zone of the northern hemisphere, especially in mediterranean climates. five cistus l. species are found in turkey (davis 1965). cystus creticus is an evergreen shrub and distributed all along the coastal belt of the turkish mediterranean phytogeographical region, as well as in some enclaves along the black sea coast. these species adorn habitats with their purple flowers from late march till june, extending from sea level up to an altitude of 1000 m (davis 1965, baslar et al. 2001). the leaves of these species exude a fragrant, sticky gum called ladanum used in perfumery and folk medicine (baytop 1994). c. creticus is one of the pioneer plants of secondary succession and it succeeds pine after fire. therefore, sites where ladanum communities are distributed are evidence of the existence of pine forest communities before a fire in the environs (turkmen and duzenli 2005). acta bot. croat. 69 (2), 2010 275 * corresponding author, e-mail: tsiozbucak@hotmail.com concentrations of nutrients in mature leaves can indicate the nutritional status of a plant. for this reason, foliar analysis is a classic tool for diagnosing nutrient efficiencies and has long been applied to forests (mayor and roda 1992). but leaf nutrient concentrations vary with species, age of the tissue, climate, soil and other factors (schlesinger 1997, teklay 2004). forest trees, shrubs and herbs retranslocate sizeable proportions of the nutrient content of leaves before leaf abscission (mayor and roda 1992). one of the most important methods to measure of nutrient use efficiency in plants is to determine foliar resorption, the process of nutrient translocation from the leaves into storage tissues during senescence (killingbeck 1988, luken 1988). in particular, seasonal changes in leaf nutrients occur in response to resorption or retranslocation before senescence (chapin 1980). the rate of nutrient resorption from senescing leaves may also vary with the availability of nutrients for resorption. the duration of retention of leaf nutrients in a plant is largely a function of leaf resorption (escudero et al. 1992). especially, n and p are largely withdrawn from senescing leaves before abscission, and used for new growth or stored in vegetative tissue until the next growing season (van heerwaarden et al. 2003). this process plays an important role in nutrient conversation (chapin 1980). obviously, species and seasonal pattern of nutrients strongly influence nutrient resorption (teklay 2004, aerts 1996, chapin and kedrowski 1983, killingbeck 1996). it has been reported that individuals growing in less fertile sites may use nutrients more efficiently than those growing in more fertile sites. however, some stress factors such as low soil moisture may reduce resorption, especially of nitrogen. several studies examining foliar nutrient resorption among temperate deciduous stands support these hypotheses for n and p (boerner 1984, escudero et al. 1992, minoletti and boerner 1994). the nutrients resorbed from the trees during senescence are directly available for further plant growth, which makes a species less dependent on current nutrient uptake. nutrients which are not resorbed, however, will be circulated through litterfall in the longer term. all of this has important implications for element cycling at the ecosystem level (aerts and chapin 2000, martíáez-sánchez 2005). resorption can be expressed in two ways: as resorption efficiency and resorption proficiency. resorption efficiency is most accurately calculated for any nutrient as area-specific mass in green leaves minus area-specific mass in senesced leaves divided by area-specific mass in green leaves, and the quantity multiplied by 100. a new measure of resorption was introduced by killingbeck (1996) as resorption proficency. proficiency is simply the amount of a nutrient that remains in fully senesced leaves (killingbeck 2004). from a biological perspective, an important advantage of measuring resorption as proficiency rather than efficiency is that proficiency is a more unequivocal measure of the degree to which selection has acted to minimize nutrient loss in ephemeral leaves (killingbeck 2004). the chlorophylls (chl a and chl b) are the most important photosynthetic pigments, and thus virtually essential for the oxygenic conversion of light energy to the stored chemical energy that powers the biosphere. from an applied perspective, leaf pigmentation is important to both land managers and ecophysiologists (richardson et al. 2002, lin et al. 2005). the amount of solar radiation absorbed by a leaf is largely a function of the concentrations of leaf photosynthetic pigments and, therefore, low concentrations of chlorophyll can directly limit photosynthetic potential and hence primary production (curran et al. 1990, filella et al. 1995) much of the leaf nitrogen is incorporated in chlorophyll, so 276 acta bot. croat. 69 (2), 2010 turkis s., ozbucak t. quantifying chlorophyll content gives an indirect measure of nutrient status (filella et al. 1995, moran et al. 2000). pigmentation can be directly related to stress physiology, as concentrations of chlorophylls generally decrease under stress and during senescence (penuelas and filella 1998). the relative concentrations of pigments are known to change with abiotic factors such as light, so quantifying these proportions can provide important information about relationship between plants and their environment. the seasonal changes of leaf ecophysiological and ecomorphological characters depend on both internal and external factors. particularly, seasonal changes are informative for evergreens, because the leaf character of a plant changes according to age of plant, growth of leaf and vegetative reproductive phases (nunez-olivera et al. 1996). the present study addresses three main objectives : (1) the seasonal variation of n and p contents, efficiency and proficiency, and (2) to find whether n and p resorption efficiency and proficiency is changed along an elevational gradient or not, (3) determining chl a/b in leaves of the evergreen c. creticus l. along an elevational gradient. material and methods study area this study was conducted in natural cystus creticus populations at samsun (41°17’ n; 36°20’ e) and amasya (40° 39’ n; 35°51’ e) counties in 2004–2006. samsun and amasya are situated on the north, black sea region of turkey (a6 square based on the grid system of davis) (fig.1). mean annual temperature and precipitation in samsun (30 m a.s.l. 670 m a.s.l.) and amasya (880 m) are 12.16 °c, 65.7 mm and 13.37 °c, 450.3 mm, respectively acta bot. croat. 69 (2), 2010 277 foliar resorption and chlorophyll content of cistus creticus l. fig. 1. the map of study area. 1 – kurupelit (30 m), 2 – kavak (670 m), 3 – yenice (880 m). (tab. 1). a western mediterranean type precipitation regime is present in samsun. a western black sea region 2nd type oceanic precipitation regime is seen in amasya (akman 1990). cistus creticus occurs on loamy and strongly alkaline soils (tab. 1). sampling plant samples were collected from along an elevational gradient from 30 to 880 m. five (25 m ´ 25 m) plots were chosen in homogeneous places at altitudes of 30 m a.s.l., 670 m a.s.l. and 880 m a.s.l. in homogeneous places. in each plot, at least five individuals were randomly selected and flagged. individuals were selected ³2.5 m from the stems of neighboring canopy trees to avoid potential microsite variation (boerner and koslowsky 1989) leaf samples from throughout the midcrown per individual were taken to avoid effects of crown position of the canopy and subcanopy species and consisted of leaves with no evidence of insect attack. chemical analyses leaf samples were dried at 60 °c until constant weight, ground, and sieved and digested in a mixture of nitric and perchloric acids with the exception of samples for nitrogen (n) analysis. nitrogen was determined by the micro kjeldahl method with a kjeltec auto 1030 analyser (tecator, sweden) after the samples were digested in concentrated h2so4 with a selenium catalyst. phosphorus (p) was determined with the stannous chloride method with the use of a jenway spectrophotometer (allen et al. 1986). concentrations of chlorophyll a and b were determined according to standard methods (odabas 1981). leaf samples were scanned and leaf area was calculated with the use of a spss 10.0 for windows (anonymous 1999). specific leaf area (sla) was calculated according to (cornelissen et al. 1997, kutbay 2001): sla = s la (cm2) / s ldw (mg) n contents = s ldw (mg) ´ crude n concentration/ sla = mg cm–2 p contents = s ldw (mg) ´ crude p concentration/ sla = mg dm–2 la – leaf area (cm2) ldw – leaf dry weight (mg) 278 acta bot. croat. 69 (2), 2010 turkis s., ozbucak t. tab. 1. general characters of the study areas. (see fig.1) locality mean annual temperature (°c) mean annual precipitation (mm) soil texture ph composition of the study area samsun 12.16 65.70 loamy 8.35 pinus pinea dominates, quercus ilex, cistus salviifolius. amasya 13.37 45.03 loamy 8.20 pinus pinea dominates, cistus salviifolius. acta bot. croat. 69 (2), 2010 279 foliar resorption and chlorophyll content of cistus creticus l. fig. 2. concentration of n and p (µg cm–2), percentage of n resorption efficiency (nre), p resorption efficiency (pre), n resorption proficiency (nrp) and p resorption proficiency (prp). seasonal n concentrations (a), n concentrations along the elevational gradient (b), seasonal p concentrations (c), p concentrations along the elevational gradient (d), nre (%) along the elevational gradient (e), pre (%) along the elevational gradient (f), nrp concentration along the evational gradient, prp concentration along the evational gradient (g) (standard errors are indicated. means followed by the same letter are not significantly different at the 0.05 level using tukey’s hsd test). resorption efficiency was calculated as the percentage of n, p and recovered from senescing leaves (orgeas et al. 2002, rejmankova 2005): [(nutrient in live leaves – nutrient in senescent leaves)/ nutrient in live leaves] ´ 100 statistical analyses one and two-way analysis of variance (anova) tests and multivariate general linear models procedure were carried out with the use of the programme spss 10.0. the dependent and independent variables were foliar nutrient concentrations and foliar resorption and, growth period and localities, respectively. tukey’s hsd test was used to rank means following analysis of variance with the use of spss 10.0. pearson correlation coefficients were also calculated with spss 10.0 version (anonymous 1999). results nitrogen and phosphorus dynamics and specific leaf area nitrogen and phosphorus concentrations of c. creticus changed according to months and altitudes (figs. 2a, 2b, 2c, 2d). there were statistically significant differences in terms of n and specific leaf area (sla) (p<0.01) but there were no significant differences in p concentration (tabs. 2, 3). the highest n concentration was observed in august (55.5 mg cm–2). in the beginning of senescence (september), the n concentration of leaves decreased (25.3 mg cm–2). the highest p concentration was observed in august (0.49 mg cm–2) while the lowest p concentration was found in june and november (0.21 mg/cm–2, 0.25 g cm–2) while the flower were in bloom (figs. 2a, 2b, 2c, 2d). 280 acta bot. croat. 69 (2), 2010 turkis s., ozbucak t. tab. 2. the comparison of the monthly changes in n, p, specific leaf area (sla) and leaf mass area (lma) in c.creticus by using one-way anova. parameter sum of square df mean square f sig. n between groups 8100.518 8 1012.565 8.307 0.000** within groups 18648.795 153 121.888 total 26749.313 161 p between groups 0.756 8 9.448e-02 1.784 0.084 ns within groups 8.103 153 5.296e-02 total 8.858 161 sla between groups 0.747 8 9.338e-02 6.961 0.01** within groups 0.966 72 1.342e-02 total 1.713 80 lma between groups 59.338 8 7.417 6.535 0.01** within groups 81.725 72 1.135 total 141.063 80 *p< 0.05 **p< 0.01 ns: not significant nitrogen and phosphorus concentrations of c. creticus changed with respect to study period and along the elevation gradient. n concentration was higher in july (28 mg cm–2) and at 670 m (44 mg cm–2) (figs. 2a, 2b). the lowest n concentration was found in september (25.3 mg cm–2) and at 30 m (38 mg cm–2). the highest p concentration (37.5 mg cm–2) was determined at 880 m, while the lowest p concentration (34 mg cm–2) was found at 670 m (figs. 2c, 2d). in the present study, it was found that mature leaf nutrients was higher than in senescent leaf. there were significant differences during the study period (p<0.01**), whereas there were no significant differences along the elevational gradient (tabs. 2. 4). acta bot. croat. 69 (2), 2010 281 foliar resorption and chlorophyll content of cistus creticus l. tab. 3. the comparison of the monthly changes in n, p and specific leaf area (sla) in c.creticus by using tukey’s hsd test. months n p sla march 4.76bc 0.20 ab 0.34 bcd april 7.16bc 0.66 a 0.38 abc may 15.03bc 0.92 a 0.38 abc june1 6.78ab 0.18 ab 0.38 abc july 28.00a 0.24 ab 0.33 bcd august 15.93bc 0.14 ab 0.26 cd september 10.39 bc 0.26 ab 0.19 d october 9.80 bc 0.26 ab 0.44 ab november 3.94 c 0.22 ab 0.54 a f-value 8.307 1.784 6.961 std. error 0.368 0.767 0.546 *p< 0.05 **p< 0.01 tab. 4. the comparison of the elevation gradient in n, p, specific leaf area (sla) and leaf mass area (lma) in c.creticus by using one-way anova. parameter sum of square df mean square f sig. n between groups 441.810 2 220.905 1.320 0.270ns within groups 26601.618 159 167.306 total 27043.428 161 p between groups 0.318 1 0.318 5.661 0.020ns within groups 4.442 79 0.056 total 4.760 80 sla between groups 0.035 1 0.035 1.486 0.226ns within groups 1.844 79 0.023 total 1.878 80 lma between groups 0.502 1 0.502 0.282 0.597ns within groups 140.561 79 1.779 total 141.063 80 *p< 0.05 **p< 0.01 ns: not significant significant correlations were found among n, p and n/p (p<0.01**, p<0.05*) (tab. 5). strong positive correlations were reported between green-leaf n concentration and n/p and n contents, respectively for c. creticus. however, significant differences were found along the elevational gradient and during the growth period in respect to the n/p ratio. negative correlations were found between green-leaf p concentrations and the n/p ratio (tab. 5). n/p ratios of c. creticus were > 16 at only at 30 m, but < 14 at 670 and 880 m, respectively. except for march, june and july the n/p ratio was < 14 during the study period (tab. 6). nitrogen and phosphorus resorption efficiency and resorption proficiency the highest n resorption efficiency (85.60) was found at 30 m, while the lowest n resorption efficiency value (80.36) was found at 880 m (fig. 2e). the highest and lowest n resorption proficiency (11.18, 4.85) were found at 670 and 30 m, respectively (figs. 2g). however, the highest p resorption efficiency (78) was found at 670 m while the lowest p resorption efficiency value (53.48) was found at 30 m (figs. 2f). the highest and lowest p resorption proficiency (1.95, 0.79) were found at 30 and 880 m, respectively (fig. 2h). chlorophyls (chl a, chl b, chl a+ b, chl a/ b) it was found that chlorophyll content of c. creticus changed both seasonally and along the elevational gradient (tab. 7, figs. 3a, b, c, d). in c. creticus, the highest chl a content was found in october (38 mg cm–2) and at 880 m (28 mg cm–2), while the lowest chl a content was found in november (13 mg cm–2) and at 670 m (17 mg cm–2) (figs. 3a, b). the highest chl b content of c. creticus was observed in june (70 mg cm–2) and at 880 m (9.7 mg cm–2) while the lowest chl b was found in august (17.5 mg cm–2) and at the 670 m (8.2 mg cm–2) (figs. 3c, d). chl a and chl b values exhibited significant changes seasonally and along the elevational gradient. chl a+ b content was higher in june (73 mg cm–2) and at 670 m (37 mg cm–2) (figs. 3e, f), while the lowest chl a/b content was found in july (0.49 mg cm–2) and at 880 m (1.08 mg cm–2), respectively (figs. 3g, h). seasonal chl a, chl b, chl a+ b and chl a/b of c. creticus are compared in table 8 and 9, using one way anova and tukey’s hsd. according to statistical analysis there were significant differences in terms of seasonal variation (p<0.01**, p<0.05*) but no significant differences along the elevational gradient (tabs. 8, 9). 282 acta bot. croat. 69 (2), 2010 turkis s., ozbucak t. tab. 5. pearson correlations among n, p content and n/p in c. creticus (**p<0.01; *p<0.05) n p n/p n 1.000 0.059 0.199* p 0.059 1.000 –0.289** n/p 0.199* –0.289** 1.000 tab. 6. n/p ratio in c. creticus along the elevational gradient n/p locality 30m 18.89 >16 670m 13.65 <14 880m 10.07 <14 months march 18.47 >16 april 13.72 <14 may 15.92<14 june 16.51 >16 july 20.34 >16 august 12.16 <14 september 9.73 <14 october 5.15 <14 november 2.87<14 discussion it has been found that foliar nutrient contents of deciduous species in the early growing season are high. these values are stable from mid-growing season to the beginning of senescence, but low in the beginning of abscission. similar results were reported for some evergreen species. however, foliar nutrient concentrations for some evergreen species increase in the abscission phases (kutbay and kilinç 1994, hevia et al. 1999). in deciduous species the most mature phases of leaf are mid-summer (diaz and cabido 1997). however, in evergreen species fully-expanded leaves are found in the middle of spring but this phase may change according to climatic factors (hevia et al., 1999). there were notable seasonal acta bot. croat. 69 (2), 2010 283 foliar resorption and chlorophyll content of cistus creticus l. tab. 7. the comparison of the monthly changes in chl a, chl b, chl a+b, chl a/b in c.creticus by using one-way anova. parameter sum of square df mean square f sig. chl a betweengroups 1478.563 5 295.713 10.528 0.000** within groups 1348.237 48 28.088 total 2826.800 53 chl b between groups 21111.373 5 4222.275 64.882 0.000** within groups 3123.641 48 65.076 total 24235.015 53 chl a+ b between groups 15462.828 5 3092.566 21.764 0.000** within groups 4262.787 30 142.093 total 19725.615 35 chl a/b between groups 13.720 5 2.744 6.029 0.001* within groups 13.653 30 0.455 total 27.373 35 *p< 0.05 **p< 0.01 ns: not significant tab. 8. the comparison of the monthly changes in chl a, chl b, chl a+b, chl a/b in c.creticus by using tukey’s hsd test. months chl a chl b chl a+b chl a/b june 23.40 a 45.57 a 70.07 a 0.65 b july 23.53 a 55.00 a 62.26 a 0.47 b august 12.90 b 7.00 b 21.20 b 1.89 a september 11.16 b 4.63 b 21.49 b 2.15 a october 15.51 b 11.79 b 26.06 b 1.71 ab november 11.16 b 13.35 b 21.64 b 1.60 ab f-value 10.528 64.882 21.764 6.029 std. error 2.49 3.80 6.88 0.38 *p< 0.05 **p< 0.01 variations in n and p concentrations in c. creticus. the n and p dynamics of c. creticus are a bit different from similar species (i.e. cistus laurifolius) and n concentration peaked in july, while p concentration peaked in august. in other words, a summer peak was observed in both n and p concentrations. peak concentrations of n and p of similar species in mediterranean region were may and march for n and p, respectively, on an area basis and this may be due to differences in phenological patterns. however, the overall pattern for p concentrations was quite similar to that of c. laurifolius (milla et al. 2004). foliar n and p concentrations in the present study were low in the early growing season as compared to the mid-growing season. although p concentrations declined, n concentrations increased in the senescence period (figs. 2a, c). hobbie and gough (2002) stated that species with short leaf lifespans (deciduous trees, sedges, and forbs) have higher foliar nutrient concentrations than evergreen species. however, n and p resorption efficiency values in the present study were found to be higher than that of other evergreen species (fig. 2e). a longer leaf life span is regarded as a mechanism for conserving nutrients since it reduces the loss of minerals during leaf abscission (lima et al. 2006). the greater the resorption efficiency, the more nitrogen is reused by the plant (corte et al. 2009). n resorption efficiency was higher at 30 m in c. creticus and decreased along the elevational gradient. however, p resorption efficiency was higher at 670 m. c. creticus individuals effectively used nitrogen at low elevations, whilst phosphorus was effectively used at high elevations. it has been hypothesized that n and p resorption efficiency usually decreased as nutrient concentrations in green leaves increased (ratnam et al. 2008). n and p resorption efficiency usually and green leaf n and p concentrations in c. creticus leaves supported that hypothesis. 284 acta bot. croat. 69 (2), 2010 turkis s., ozbucak t. tab. 9. the comparison of the elevation gradient in chl a, chl b, chl a+b, chl a/b in c.creticus by using one-way anova. parameter sum of square df mean square f sig. chl a betweengroups 398.7 5 199.3 4.187 0.201 ns within groups 2428.1 48 47.6 total 2826.8 53 chl b between groups 890.5 2 445.3 0.973 0.385 ns within groups 23344.5 51 457.7 total 24235.0 53 chl a+ b between groups 609.1 2 304.5 0.526 0.596 ns within groups 19116.5 33 579 total 19725.6 35 chl a/b between groups 0.2 2 0.1 0.137 0.873 ns within groups 27.1 33 0.8 total 27.4 35 *p< 0.05 **p< 0.01 ns: not significant acta bot. croat. 69 (2), 2010 285 foliar resorption and chlorophyll content of cistus creticus l. fig. 3. pigment content (µg cm–2). seasonal chlorophyll a content (a), along the evational gradient (b), seasonal chlorophyll b content (c), chlorophyll b content along the elevational gradient (d), seasonal chlorophyll a+b content (e), chlorophyll a+b content along the elevational gradient (f), seasonal chlorophyll a/b content, chlorophyll a/b content along the elevational gradient (standard errors are indicated. means followed by the same letter are not significantly different at the 0.05 level using tukey’s hsd test). in some evergreen species, n resorption efficiency values were found to range from 25.7% – 75.1% (killingbeck and costigan 1988, hevia et al. 1999, mediavilla and escudero 2003, escudero et al. 1992, kutbay et al. 2003, özbucak et al. 2008). woody evergreens show a higher resorption than deciduous species because mature evergreens have lower nutrient concentrations than deciduous leaves (killingbeck 1996). in some deciduous species like quercus suber, populus nigra and frangula alnus n resorption efficiency was found to be 47.9, 62.6 and 61.6, respectively (escudero et al. 1992). in the present study, n and p resorption efficiency values of evergreen c. creticus were found that are higher than those of other evergreen species (killingbeck and costigan 1988, escudero et al. 1992). the n and p resorption processes are more efficient due to the higher n and p concentrations before senescence (milla et al. 2004). low n and p concentrations were found in some deciduous species at high elevations. however, it was found that n and p concentrations in evergreen species increased along an elevation gradient (hevia et al. 1999). similar results were observed in the present study for the evergreen c. creticus (figs. 2b, 2d). this may probably be due to the decrease of soil moisture along the elevational gradient (kutbay and ok 2003). resorption proficiency is considered a more stable indicator of the plant capacity to reuse nutrients than resorption efficiency (killingbeck 1996, lima et al. 2006). according to killingbeck (1996) n and p resorption proficiencies are high when ey are below 50 g cm–2 and 3 g cm–2, respectively. based on these threshold values n and p resorption proficiencies are biochemically complete in c. creticus (figs. 2 g, h). n/p ratios are more important than n and p concentrations in terms of mineral nutrition (gusewell 2005). if n/p <14, n-limitation is present. however, if n/p>16, p-limitation is present (koerselman and meuleman 1996). in present study, the n/p ratio of c. creticus was found to be below 14 at 30, 670 and 880 m, whilst the n/p ratio was found to be > 16 at 30 m (tab. 6). as a result of this, p-limitation is present at low elevations, while the n-limitation is present at high elevations. n and p resorption efficiency in c. creticus were quite high as compared to the other evergreen species, whichmay probably be due to nand p-limitation along the elevational gradient (tab. 6). chlorophyll a is located only in the reaction centres of the photosystems, while chl b is located both in the reaction centres and the light harvesting complexes (lin et al. 2005). a change in the chl a/b ratio reflects an adaptation mechanism to balance the amount of light captured by the leaf and its utilization for photochemical processses (lin et al. 2005). filella and peòuelas (1999) found that chlorophyll concentrations were not significantly changed along the elevational gradient. however, covington (1975) and richardson and berlyn (2002) found chlorophyll concentrations changed significantly along the elevational gradient, which the present study confirms. these results are further evidence of the usefulness of reflectance measures for the rapid and noninvasive detection of plant stress (richardson and berlyn 2002). chlorophyll a and b contents were curvilinear in style. similar results were reported for the chlorophyll a and b contents in phyllostachys pubescens (lin et al. 2005). chl a, b content and chl a+b content of mature leaves was higher in summer than in autumn with respect to leaf growth phases (p<0.01) (figs. 3a, c, e). chlorophyll a content was higher at 30 m and 880 m (fig. 3b), while chlorophyll b was higher at 880 m (fig. 3d). chl a+b content 286 acta bot. croat. 69 (2), 2010 turkis s., ozbucak t. was similar in all the elevations (figs. 3e, f). chl a/b ratio of mature leaves was higher in autumn than summer with respect to leaf growth phases (p<0.01) (figs. 3g, h). in particular, chl b content declined more than that of chl a (figs. 3a, b, c, d). similar results were found in some studies (lin et al. 2005). scheuman et al. (1999) observed that the chl a/b ratio of barley seedling increased during senescence from 2.9 at day 0 to 5 at day 8, and suggested that either degradation of chl b was faster than that of chl a or that chl b was transformed into chl a. gossauer and engel (1996) proposed that the conversion of chl b to chl a should precede chlorophyll degradation in higher plants. lin et al. (2005) reported a chlorophyll content decrease but a chl a/b ratio increase during leaf senescence. in the present study the chl a/b ratio increased in august, september and october(fig. 3g). chlorophyll a and b contents were decreased after senescence due to vulnerability to winter stress and the chl a/b ratio was compatible to the shade acclimation hypothesis, which indicates the increasing shade characteristics of leaves (zeliou et al. 2009). the resorbed nitrogen may be used for the synthesis of chlorophyll a (jain and gadre 2004). leaf ecological and physiological traits are important factors to define plant photosynthetic rates. these traits also play an important role in determining the nutrient cycle. references aerts, r., 1996: nutrient resorption from senescing leaves of perennials. are there general patterns? journal of ecology 84, 597–608. aerts, r., chapin, f. s., 2000: the mineral nutrition of wild plants revisited. a re-evaluation of processes and patterns. advances 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in green and senesced leaves of three agroforestry species in southern ethiopia. plant and soil 267, 297–307. turkmen, n., duzenli, a., 2005: changes in floristic composition of quercus coccifera macchia after fire in the çukurova region (turkey). annales botanici fennici 42, 453– 460. van heerwarden, l. m., toet, s. aerts, r., 2003: current measures of nutrient resorption efficiency lead to a substantial underestimation of real resorption efficiency: facts and solutions. oikos 101, 664–669. zeliou, k., manetas y., petropoulou y., 2009: transient winter leaf reddening in cistus creticus characterizes weak (stress-sensitive) individuals, yet anthocyanins cannot alleviate the adverse effects on photosynthesis. journal of experimental botany 60, 1–12. 290 acta bot. croat. 69 (2), 2010 turkis s., ozbucak t. colonization of bacteria and diatoms in a marine lake acta bot. croat. 79 (2), 2020 1 on-line suppl. tab. 1. schedule of sampling physico-chemical data, bacteria and diatoms in lake mrtvo more in 2016. t – temperature, s – salinity, no3– – nitrate, no2– – nitrite, nh4+ – ammonium, tin – total inorganic nitrogen, po43– – phosphate, sio44– – silicate, chl a – chlorophyll a concentrations, o2/o2′ – oxygen saturation. se as on m on th date physico-chemical parameter bacteria samples diatom samples t s no3– no2– nh4+ tin po43– sio44– chl a o2/o2' sp ri ng a pr il 19-apr-2016 1 1 1 1 1 1 1 1 1 1 . . 26-apr-2016 1 1 1 1 1 1 1 1 1 1 . . m ay 4-may-2016 1 1 1 1 1 1 1 1 1 1 . . 10-may-2016 1 1 1 1 1 1 1 1 1 1 1 1 20-may-2016 1 1 1 1 1 1 1 1 1 1 . 1 25-may-2016 1 1 1 1 1 1 1 1 1 1 . 1 31-may-2016 1 1 1 1 1 1 1 1 1 1 . 1 su m m er ju ne 7-jun-2016 1 1 1 1 1 1 1 1 1 1 1 1 18-jun-2016 1 1 1 1 1 1 1 1 1 1 1 1 24-jun-2016 1 1 1 1 1 1 1 1 1 1 1 1 29-jun-2016 1 1 1 1 1 1 1 1 1 1 1 1 ju ly 7-jul-2016 1 1 1 1 1 1 1 1 1 1 . 1 13-jul-2016 1 1 1 1 1 1 1 1 1 1 1 1 20-jul-2016 1 1 1 1 1 1 1 1 . 1 1 1 28-jul-2016 1 1 1 1 1 1 1 1 1 1 1 1 a ug us t 11-aug-2016 1 1 1 1 1 1 1 1 1 1 1 1 17-aug-2016 1 1 1 1 1 1 1 1 1 1 . 1 24-aug-2016 1 1 1 1 1 1 1 1 1 1 1 1 a ut um n se pt em be r 2-sep-2016 1 1 1 1 1 1 1 1 1 1 1 1 6-sep-2016 1 1 1 1 1 1 1 1 1 1 1 1 14-sep-2016 1 1 1 1 1 1 1 1 1 1 . 1 21-sep-2016 1 1 1 1 1 1 1 1 1 1 . 1 27-sep-2016 1 1 1 1 1 1 1 1 1 1 . . o ct ob er 3-oct-2016 1 1 1 1 1 1 1 1 1 1 . 1 12-oct-2016 1 1 1 1 1 1 1 1 1 1 . 1 number of samples: 25 25 25 25 25 25 25 25 24 25 12 21 on-line suppl. tab. 2. results of anosim test performed on physico-chemical data. physico-chemical parameters varied significantly (anosim, p < 0.05) among: seasons (spring, summer, autumn), months (april-october), between the significantly different clusters of samples for analysis physico-chemical parameters collected before the 18th june (group 1) and afterwards (group 2, and group 3 containing only sample from 12th october), and between the significantly different clusters of diatom assemblages. av.abund. – average abundance. season month environmental simprof groups 1 & 2 & 3 av. abund. simprof groups 1 & 2 & 3 av. abund. simprof subgroups 1a & 1b & 2a & 2b & 3 p 0.001 0.001 0.001 0.001 > 0.05 global r 0.545 0.466 0.891 0.386 0.381 car a, hafner d, ljubimir s, dupčić radić i, bobanović-ćolić s, jasprica n 2 acta bot. croat. 79 (2), 2020 on-line suppl. tab. 4. list of diatoms taxa and their percentage contribution to total diatom community composition (taxa with relative abundances ≥ 3.5%, ra, are only shown) on artificial substrat (glass) in lake mrtvo more in 2016. trix index was computed in order to identify the trophic level of the research area. groups and sub-groups were established upon the cluster analysis with similarity profiles (simprof) performed to determine significant levels of similarity between diatom samples. only relative abundances > 25% are framed with black rectangle. t r ix o lig ot ro ph ic m es ot ro ph ic ex tr em e eu tr op hi c eu tr op hi c m es ot ro ph ic eu tr op hi c ex tr em e eu tr op hi c eu tr op hi c m es ot ro ph ic o lig ot ro ph ic group 1 2 3 subgroup 1a 1b 2a 2b month may june july august september october taxa / date 10 -m ay 20 -m ay 25 -m ay 31 -m ay 07 -j un 18 -j un 24 -j un 29 -j un 07 -j ul 13 -j ul 20 -j ul 28 -j ul 11 -a ug 17 -a ug 24 -a ug 02 -s ep 06 -s ep 14 -s ep 21 -s ep 03 -o ct 12 -o ct achnanthes kuwaitensis 11 .1 4 8. 75 amphora sp. 10 .0 0 3. 50 cocconeis costata 6. 75 3. 50 7. 25 6. 00 4. 73 8. 50 10 .0 0 6. 25 6. 75 4. 09 cocconeis dirupta var. flexella 22 .5 0 14 .7 5 20 .7 5 19 .7 5 5. 50 29 .8 5 51 .0 0 37 .2 5 65 .0 0 28 .8 9 47 .0 0 27 .5 0 24 .0 0 15 .5 0 11 .8 8 23 .7 5 15 .5 0 10 .0 0 8. 25 6. 27 cocconeis dirupta 4. 02 cocconeis pseudomarginata 3. 75 7. 47 cocconeis scutellum var. scutellum 53 .2 5 73 .0 0 73 .0 0 70 .7 5 89 .2 5 47 .2 6 39 .0 0 55 .5 0 7. 25 17 .0 9 14 .7 5 10 .0 0 4. 25 7. 00 6. 00 9. 75 4. 09 cocconeis woodii 4. 50 on-line suppl. tab. 3. results of anosim test performed on diatom species (s) and growth form (gf) relative abundance data. diatom assemblages differed significantly (anosim, p < 0.05) among months, between the significantly different clusters of diatom samples collected up to the middle of july (group 1) and afterwards (group 2, and group 3 containing only sample from 12th october), and between the significantly different sub-clusters of diatom samples. season month simprof groups 1 & 2 & 3 simprof subgroups 1a & 1b & 2a & 2b & 3 s gf s gf s gf s gf p > 0.05 > 0.05 0.001 0.001 0.001 0.001 0.001 0.001 global r 0.268 0.274 0.650 0.591 0.903 0.668 0.968 0.774 colonization of bacteria and diatoms in a marine lake acta bot. croat. 79 (2), 2020 3 t r ix o lig ot ro ph ic m es ot ro ph ic ex tr em e eu tr op hi c eu tr op hi c m es ot ro ph ic eu tr op hi c ex tr em e eu tr op hi c eu tr op hi c m es ot ro ph ic o lig ot ro ph ic group 1 2 3 subgroup 1a 1b 2a 2b month may june july august september october diploneis crabro 6. 50 fragilaria sp.2 13 .7 5 6. 50 halamphora coffeiformis 3. 50 3. 75 7. 93 13 .5 0 3. 75 halamphora hyalina 9. 50 11 .2 5 14 .3 6 6. 50 halamphora subangularis 3. 50 5. 72 licmophora flabellata 13 .5 0 8. 29 3. 75 12 .2 6 licmophora paradoxa 7. 50 3. 75 3. 50 16 .8 9 43 .6 7 mastogloia cuneata 5. 47 navicula directa 3. 75 navicula flagellifera 4. 50 6. 00 navicula salinicola 3. 50 5. 25 13 .2 5 6. 94 10 .0 0 13 .2 5 3. 81 navicula sp.1 7. 67 nitzschia compressa var. compressa 4 .0 2 nitzschia frustulum 5. 03 on-line suppl. tab. 4. continued car a, hafner d, ljubimir s, dupčić radić i, bobanović-ćolić s, jasprica n 4 acta bot. croat. 79 (2), 2020 t r ix o lig ot ro ph ic m es ot ro ph ic ex tr em e eu tr op hi c eu tr op hi c m es ot ro ph ic eu tr op hi c ex tr em e eu tr op hi c eu tr op hi c m es ot ro ph ic o lig ot ro ph ic group 1 2 3 subgroup 1a 1b 2a 2b month may june july august september october nitzschia laevis 5. 25 nitzschia sp.2 3. 73 opephora mutabilis 4. 00 4. 00 7. 67 6. 00 10 .0 0 6. 25 5. 50 22 .3 4 pinnularia quadratarea var. cuneata 5 .0 0 pinnularia sp. 6. 50 placoneis flabellate 3. 50 psammodictyon rudum 8. 29 6. 25 rhabdonema adriaticum 5. 50 7. 00 seminavis sp. 3. 75 striatella unipunctata 5. 18 trachyneis aspera 19 .0 0 tryblionella coarctata 5. 50 4. 28 on-line suppl. tab. 4. continued colonization of bacteria and diatoms in a marine lake acta bot. croat. 79 (2), 2020 5 on-line suppl. fig. 1. a – lake mrtvo more on 7th july 2016, red dot indicates the sampling site position, b – plate with microscopic slides submerged in lake mrtvo more at the depth of 1 m on 19th of april 2016, c-e – hauling up the plate with microscopic slides so one glass could be removed for diatom analyses (c, d – 7th june 2016, e – 14th september 2016). on-line suppl. fig. 2. the average monthly precipitation (mm) in dubrovnik for the period 1961-2017 and during 2016 (data for dubrovnik meteorological station, croatian meteorological and hydrological service). car a, hafner d, ljubimir s, dupčić radić i, bobanović-ćolić s, jasprica n 6 acta bot. croat. 79 (2), 2020 on-line suppl. fig. 3. cluster analysis (a) and non-metric multidimensional scaling (nmds) ordination (b) based on the data of physicochemical parameters (temperature, salinity, tin, po43–, sio44–, chlorophyll a concentrations, oxygen saturation, no3–, no2–, nh4+) in 25 sampling dates (lake mrtvo more, the island of lokrum, april-october 2016). euclidean distance as a similarity measure was used. n = 25. opce-str.vp social news u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:14 color profile: disabled composite 150 lpi at 45 degrees u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:14 color profile: disabled composite 150 lpi at 45 degrees professor nikola ljube{i} – on the occasion of his seventieth birthday professor nikola ljube{i} was born on may 18, 1940, in komarevo near sisak. he attended elementary school in cepeli{te and petrinja and completed high school in sisak. he enrolled in the study of biology at the faculty of science of university of zagreb and obtained a bsc degree in 1964. at the same faculty, he enrolled in the postgraduate biology programme in the field of experimental biology. in 1966, he earned his msc degree and, in 1971, he defended his phd thesis. in 1964, professor ljube{i} joined the laboratory for electron microscopy at the ru|er bo{kovi} institute (rbi) where he spent his entire carrier. in 1974, he acquired the title of research associate and continued his career path by becoming higher research associate in 1980, followed by the title of senior scientist in 2001. over the years, he carried out a number of duties at rbi: he was the head of the laboratory for electron microscopy, and served, in several mandates, as the head of the division of organic chemistry and biochemistry, and afterwards of the division of molecular genetics. also, for a number of years he was appointed a member of the rbi’s executive board. in 1973, and again from 1986 till 1988, professor ljube{i} continued his scientific specialization in the laboratory of prof. eberhardt schnepf at the department for cellular biology (lehrstuhl für zellenlehre), university of heidelberg, germany. for his professional achievements and endeavors in the popularization of science professor ljube{i} received several awards. in 1986 he was awarded the »narodna tehnika« republic’s prize. for his dedicated work on the investigation of plastids, he won in 1998, together with professor mercedes wrischer, the croatian academy of sciences and arts (hazu) prize. in 2005, professor ljube{i} received the annual state prize for popularization and promotion of science (in the field of natural sciences). in 2006, he was elected an associate member of croatian academy of sciences and arts. professor nikola ljube{i} is the author or co-author of 112 scientific papers, and the co-author of one university textbook. during his scientific career, his interest was primarily focused on the structure and function of plastids, plant organelles with versatile morphology and functions. in his research, he placed special emphasis on electron-microscopic investigations of the conversions between various plastid types. he initiated his career with investigations on ultrastructural changes occurring in plastids during yellowing (senescence) and subsequent regreening of tobacco leaves, in which he particularly followed the acta bot. croat. 69 (2), 2010 301 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees processes of thylakoid degradation and reconstitution as well as morphology of plastoglobules. these investigations became the main part of his msc thesis entitled »a contribution to understanding the submicroscopical structure of chloroplasts«. later on, in his doctoral thesis entitled »transformations of plastids in the subepidermis of the fruits in the genus cucurbita«, he described the processes of plastid conversion during growth, ripening and decay of pumpkin fruits. these investigations revealed immense ultrastructural variability in chromoplasts found in fruits of varieties of cucurbita pepo and cucurbita maxima. especially intriguing was the discovery of the regreening of mature fruits, more specifically, the possibility of re-building the thylakoid system in photosynthetically inactive chromoplasts. his fascination with chromoplasts in pumpkin fruits led to further extensive research on chromoplasts found in flowers and fruits of different species. especially interesting were chromoplasts in the flowers of hypericum perforatum, thunbergia alata and liriodendron tulipifera, as well as in the fruit and sepals of physalis alkekengi and the fruit of solanum capsicastrum. these studies not only demonstrated the amazing morphological diversity of chromoplasts in different plant species, but also showed that ultrastructurally distinct chromoplasts can be found in different tissues of the same part of the plant, as exemplified by the flowers of thunbergia alata. his studies of chromoplasts showed not only their fascinating morphology, but also their complex biogenesis. in his efforts to elucidate the mechanisms underlying formation of different chromoplast substructures, especially important were his studies on different herbicides influencing carotenoid biosynthesis. these studies showed a strong connection between the amount and composition of carotenoids and the formation of the particular carotenoid-containing structure. apart from chromoplasts, a significant part of the scientific interest of professor ljube{i} was dedicated to chloroplasts found in different 'aurea' plant varieties (such as zelkova serrata 'aurea' and euonymus japonicus 'aureomarginatus'), in which leaves exposed to high-light intensities turn golden-yellow. these investigations led to the detailed descriptions of the processes involved in the disassembly of the photosynthetic apparatus during leaf yellowing, as well as the processes of thylakoid system reassembly, which can occur if such yellowed leaves are exposed to low light conditions. in more than 45 years of dedicated scientific investigation using electron microscopy led to professor ljube{i} becoming widely known as an expert in the field of plant cell ultrastructure. moreover, with his exceptional knowledge and expertise in microscopy, he has been a collaborater in solving many scientific problems of his fellow biologists, as well as other scientists, notably biomedical researchers and chemists with whom he established long and fruitful collaborations. he has also restlessly catalyzed the connectivity of electron microscopists from different fields and institutions, thus immensely contributing to the development of electron microscopy in croatia. professor ljube{i} contributed significantly to publishing of the most significant croatian scientific biological journals, »periodicum biologorum« and »acta botanica croatica«, either by his regular submissions of scientific papers, or by being a member of the editorial boards for a number of years. in »priroda«, the oldest croatian journal for the popularization of science, he was a member of the editorial board for more than ten years, and also acted as the editor in chief. along with his work in science, for a number of years he was involved in education, enthusiastically sharing his broad knowledge and experience in microscopy and cell biology 302 acta bot. croat. 69 (2), 2010 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees with generations of students. he participated in lecturing within the scope of graduate and post-graduate studies at the faculty of science (at which he taught as a full professor since 1997), faculty of pharmacy and biochemistry, faculty of medicine, and academy of fine arts of university of zagreb, and at the faculty of education of j. j. strossmayer university of osijek. he was also a supervisor of a number of bsc, msc and phd thesis. professor nikola ljube{i} was, and still is, exceptionally active in a number of professional societies. in the croatian natural sciences society (hpd) he performed many duties, while in one mandate he was its president. he was one of the founders of the croatian society for plant biology and of the croatian biological society (hbd), in the latter being awarded the »zdravo lorkovi}« plaque for exceptional contribution to the work of the society. professor ljube{i} was also one of the founders of the section for electron microscopy within the hpd which later developed into the croatian society for electron microscopy and finally croatian microscopy society. he has also greatly contributed to the work of »matica hrvatska«, in which he was the founder of a division for technical culture and division for natural sciences and mathematics. professor ljube{i} was also the president of the 7th croatian biological congress, one of the organizers of a symposium dedicated to zdravko lorkovi}, as well as two international scientific meetings, numerous professional and scientific conventions, and a number of professional excursions. in more than 45 years of fruitful work by professor ljube{i}, it is hard to distinguish scientific and professional achievements from his exceptional talent in the popularization of the natural sciences. in this respect, he was very fond of working with younger generations. already after completion of his studies, he became a member of the committee for biology within the movement »nauka mladima« that later developed into the association of young natural scientists – »znanost mladima«. he was the leader of many summer schools for young biologists, with those organized on the velebit mountain being particularly successful. without exaggeration, today we can say that he was, and still is, the »spirit« of this movement, and that he is well known as such even in the most remote corners of croatia. with the same passion and joy that he showed in the laboratory studying microscopic structures, professor ljube{i} also discovered nature in its »macroscopic« dimensions – as a passionate mountain climber conquering mountain tops, and also as an admirer of the stars in the night sky. he is a member of the croatian climbing society, croatian astronomical society, and was even the director of zagreb’s observatory. therefore, although professor ljube{i} is by vocation a biologist, it would be more correct to describe him as versatile natural scientist. along with his many interests and duties, too many to mention, professor ljube{i} has always found time to patiently share his wide knowledge and experience with younger colleagues but also to help and support them, as a mentor and as a colleague. he has always been able to bring the spirit of tolerance and optimism in the working environment, and his enthusiasm and friendliness has always been the source of a pleasant and constructive atmosphere. all of his co-workers have unforgettable memories of excursions that he has organized, and that have always been a very welcom break from the laboratory work. on behalf of all his colleagues, we wish professor nikola ljube{i}, on the occasion of his 70th birthday, that he may continue to solve the »microscopically tiny« puzzles of nature acta bot. croat. 69 (2), 2010 303 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees with the same curiosity and enthusiasm. also, it is our special wish that he may enjoy many more beautiful moments discovering the splendors of nature by hiking on mountain trails and catching magnificent sights from mountain tops. tatjana prebeg, hrvoje fulgosi, mercedes wrischer laboratory for electron microscopy, division of molecular biology, ru|er bo{kovi} institute, bijeni~ka 54, hr-10000 zagreb, croatia scientific papers: ljube[i], n., 1968: feinbau der chloroplasten während der vergilbung und wiederergrünung der blätter. protoplasma 66, 369–379. ljube[i], n., 1969: plastoglobuli kod vrste chlorophytum comosum (thumb.) baker. acta botanica croatica 28, 227–231. oberman, b., ljube[i], n., 1969: elektronsko-mikroskopska istra`ivanja melanoti~nog i amelanoti~nog melanoma hr~ka. lije~ni~ki vjesnik 91, 947–953. hirtzler, r., oberman, b., kuli[, m., ljube[i], n., 1969: acinuszelladenocarcinome der speicheldrüsen. archiv für klinische und experimentelle ohren-, nasenund kehlenheilkunde 195, 68–80. ljube[i], n., 1970: fine structure of developing chromoplasts in outer yellow fruit parts of cucurbita pepo cv. pyriformis. acta botanica croatica 29, 51–56. ljube[i], n., 1970: osmiophile substanz in blattzellen der brombeere (rubus fruticosus l. s.l.). protoplasma 69, 49–59. oberman, b., ljube[i], n., 1970: elektronska mikroskopija adenoma jetre. radovi medicinskog fakulteta u zagrebu 18, 515–521. [tefanac, z., ljube[i], n., 1971: inclusion bodies in cells infected with radish mosaic virus. journal of general virology 13, 51–57. oberman, b., ljube[i], n., 1971: elektronsko mikroskopska istra`ivanja lejomioma ezofagusa. lije~ni~ki vjesnik 93, 761–766. devidé, z., ljube[i], n., 1972: plastid transformations in pumpkin fruits. naturwissenschaften 59, 39–40. ljube[i], n., 1972: ultrastructural changes of plastids during the yellowing of the fruit of cucurbita pepo var. pyriformis. acta botanica croatica 31, 47–53. ljube[i], n., 1973: transformation of plastids in white pumpkin fruit. acta botanica croatica 32, 59–62. devidé, z., ljube[i], n., 1974: the reversion of chromoplasts to chloroplasts in pumpkin fruits. zeitschrift für pflanzenphysiologie 73, 296–306. buturac, i., [ari], a., ljube[i], n., 1974: nalaz virusa mozaika celera u jugoslaviji. acta botanica croatica 33, 37–44. [tefanac, z., ljube[i], n., 1974: the spindle-shaped inclusion bodies of narcissus mosaic virus. phytopathologische zeitschrift 80, 148–152. 304 acta bot. croat. 69 (2), 2010 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees wrischer, m., ljube[i], n., devidé, z., 1975: transformation of plastids in the leaves of acer negundo l. var. odessanum (h. rothe). journal of cell science 18, 509–518. grbelja, j., ljube[i], n., 1975: clover phyllody disease in jugoslavia. acta botanica croatica 34, 25–31. wrischer, m., ljube[i], n., devidé, z., 1975: ultrastructural studies of plastids in leaves of fraxinus excelsior l. var. aurea (willd.). journal de microscopie biologie cellulaire 23, 105–112. mamula, \., ljube[i], n., 1975: identification of turnip mosaic virus in tropaeolum majus l. acta botanica croatica 34, 33–42. mili^i], d., [tefanac, z., ljube[i], n., 1975: two plant viruses isolated from soil in croatia. rad jugoslavenske akademije znanosti i umjetnosti 371, 161–170. wrischer, m., ljube[i], n., devidé, z., 1976: ultrastructural and functional characteristics of plastids in the leaves of ligustrum ovalifolium hassk. var. aureum. acta botanica croatica 35, 57–64. horvath, j., jureti], n., ljube[i], n., besada, w. h., 1976: natural occurrence of celery mosaic virus in hungary. acta phytopathologica academiae scientiarum hungaricae 11, 17–24. ljube[i], n., 1976: phytoferritin in plastids of blackberry leaves. acta botanica croatica 35, 51–56. schnepf, e., deichgräber, g., ljube[i], n., 1976: the effects of colchicine, ethionine, and deuterium oxide on microtubules in young sphagnum leaflets. a quantitative study. cytobiologie 13, 341–353. vlatkovi], g., belicza, m., suboti], r., uhlik, z., batini], d., ljube[i], n., 1976: alpertov sindrom u dje~joj dobi. elektronsko-mikroskopska studija dva bolesnika. acta medica iugoslavica 30, 109–123. ljube[i], n., 1977: the formation of chromoplasts in fruits of cucurbita maxima duch. 'turbaniformis'. botanical gazette 138, 286–290. ljube[i]. n., 1977: chromoplasts of forsythia suspensa (thunb.) vahl. i. ultrastructure and pigment composition. acta botanica croatica 38, 23–28. ljube[i], n., radi], m., 1979: chromoplasts of forsythia suspensa (thunb.) vahl. ii. the effect of isopropyl n-phenylcarbamate. acta botanica croatica 38, 29–34. ljube[i], n., 1979: chromoplasts in the petals of liriodendron tulipifera l. zeitschrift für pflanzenphysiologie 91, 49–52. musi], s., ljube[i], n., 1980: electron microscopy of v2o5 microcrystals in the v2o5nh3-h2o colloid system. coloid and polymer science 258, 194–195. [erman, d., ljube[i], n., 1980: novije spoznaje o stanici. medicinar 29, 3–71. ljube[i], n., 1981: the regreening of tubulous chromoplasts in fruits of cucurbita maxima duch. cv. turbaniformis. acta botanica croatica 40, 61–66. ljube[i], n., 1982: phytoferritin accumulation in chromoplasts of sorbus aucuparia l. fruits. acta botanica croatica 41, 29–32. wrischer, m., ljube[i], n., 1984: plastid differentiation in calceolaria petals. acta botanica croatica 43, 19–24. acta bot. croat. 69 (2), 2010 305 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees ljube[i], n., 1984: structural and functional changes of plastids during yellowing and regreening of lemon fruits. acta botanica croatica 43, 25–30. hlou[ek, a., ljube[i], n., 1985: the effect of san 9789 on tulip tree chromoplasts. acta botanica croatica 44, 15–18. wrischer, m., hlou[ek-radoj^i], a., kunst, lj., ljube[i], n., 1986: differentiation of chloroplasts in leaves of aurea plants. in: akoyounoglou, g., senger, h. (eds.), regulation of chloroplast differentiation, 685–690. a. r. liss. inc., new york. wrischer, m., ljube[i], n., mar^enko, e., kunst, lj., hlou[ek-radoj^i], a., 1986: fine structural studies of plastids during their differentiation and dedifferentiation. acta botanica croatica 45, 43–54. hlou[ek-radoj^i], a., ljube[i], n., 1988: the development of daffodil chromoplasts in the presence of herbicides san 9789 and san 9785. zeitschrift für naturforschung 43c, 418–422. modru[an, z., ljube[i], n., wrischer, m., 1989: study of nucleoides in bean chloroplasts by fluorescent and electron microscopy. acta botanica croatica 48, 19–25. ljube[i], n., quader, h., schnepf, e., 1989: correlation between protonema morphogenesis and the development of the microtubule system in funaria spore germination under normal conditions and at high auxin concentrations: an immunofluorescence study. canadian journal of botany 67, 2227–2234. devidé, z., ljube[i], n., 1989: plastid transformation in greening scales of the onion bulb (allium cepa, alliaceae). plant systematics and evolution 165, 85–89. muraja, j., ljube[i], n., wrischer, m., 1990: seasonal changes in the chloroplasts of cherry-laurel leaves. acta botanica croatica 49: 23–28. [erman, d., ljube[i], n., 1990: molekularna biologija stanice, medicinski fakultet, zagreb, 1990. ljube[i], n., wrischer, m., devidé, z., 1991: chromoplasts – the last stages in plastid development. international journal of developmental biology 35, 251–258. wrischer, m., ljube[i], n., modru[an, z., 1991: development of calceolaria chromoplasts in the presence of herbicides affecting carotenoid biosynthesis. acta botanica croatica 50, 25–30. wrischer, m., ljube[i], n., devidé, z., 1992: ultrastructural studies of degradational processes in amitrole-damaged photosynthetic membranes. journal of structural biology 108, 1–5. ljube[i], n., wrischer, m., 1992: different illumination dependent behaviour of chloroplast ultrastructure in the gall and leaf tissues of zelkova serrata 'aurea'. biochemie und physiologie der pflanzen 188, 97–103. fulgosi, h., ljube[i], n., 1992: dynamics of plastid nucleoids changes in wildand aurea-type leaves of ligustrum ovalifolium hassk. var. aureum. acta botanica croatica 51, 21–26. ljube[i], n., matijevi], d., 1992: the effect of amitrole on the pigment composition and ultrastructure of chromoplasts of tulip tree flowers. acta botanica croatica 51, 13–19. wrischer, m., ljube[i], n., 1993: the effect of light and norflurazon on the bleaching processes in chloroplasts. periodicum biologorum 95, 267–268. 306 acta bot. croat. 69 (2), 2010 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees salopek, b., ljube[i], n., 1994: the fine structure of pepper chromoplasts: the effect of bleaching herbicides. acta botanica croatica 53, 7–13. goti], m., popovi], s., ljube[i], n., musi], s., 1994: structural properties of precipitates formed by hydrolysis of fe3+ ions in aqueous solutions containing no3– and cl– ions. journal of materials science 29, 2474–2480. pavlica, m., lorkovi], z., ljube[i], n., pape[, d., 1994: cytogenetical, immuno-fluorescence, ultrastructural and biochemical investigations of pesticide genotoxicity in plants. periodicum biologorum 96, 410–412. musi], s., goti], m., ljube[i], n., 1995: influence of sodium polyanethol sulphonate on the morphology of ß-feooh particles obtained from the hydrolysis of a fecl3 solution. materials letters 25, 69–74. ljube[i], n., wrischer, m., devidé, z., 1995: development of chromoplast tubules in hypericum flowers. periodicum biologorum 97, 333–336. ljube[i], n., wrischer, m., devidé, z., 1996: chromoplast structures in thunbergia flowers. protoplasma 193, 174–180. selakovi], d., ljube[i], n., prebeg, t., 1996: the influence of carotenoid composition on chromoplast structures. journal of computer-assisted microscopy 8, 275–276. wrischer, m., ljube[i], n., salopek, b., 1996: fibrillar and tubular structures in chromoplasts. journal of computer-assisted microscopy 8, 213–214. musi], s., czakó-nagy, i., salaj-obeli], i., ljube[i], n., 1997: formation of a-fe2o3 particles in aqueous medium and their properties. materials letters 32, 301–305. gale[i], m., te@ak, \., tudja, m., ljube[i], n., babi]-ivan^i], v., 1997: characterization of lyotropic liquid crystalline phases by transmission-, and scanning electron microscopy. fizika a 6, 15–22. ljube[i], n., dugonji], b., fulgosi, h., 1997: gamma-ray-induced changes in the chloroplasts of hamatocactus setispinus. periodicum biologorum 99, 61–66. prebeg, t., ljube[i], n., selakovi], d., 1997: the effect of light intensity on the chloroplasts of tomato aurea mutant. periodicum biologorum 99, 409–414. salopek, b., ljube[i], n., wrischer, m., magnus, v., 1998: greening of non-transformed and agrobacterium rhizogenes transformed adventitious potato roots. biologia (bratislava) 53, 127–132. [ari], a., nomura, k., popovi], s., ljube[i], n., musi], s., 1998: effects of urotropin on the chemical and microstructural properties of fe-oxide powders prepared by the hydrolysis of aqueous fecl3 solutions. materials chemistry and physics 52, 214–220. te@ak, \., jal[enjak, n., ljube[i], n., 1998: formation of the lamellar phase of alkyl-benzenesulphonates from surfactant/water/electrolyte solutions. progress in colloid and polymer science 110, 204–207. wrischer, m., ljube[i], n., salopek, b., 1998: the role of carotenoids in the structural and functional stability of thylakoids in plastids of dark-grown spruce seedlings. journal of plant physiology 153, 46–52. muraja-ljubi^i], j., wrischer, m., ljube[i], n., 1998: formation of the photosynthetic apparatus in plastids during greening of potato microtubers. plant physiology and biochemistry 36, 747–752. acta bot. croat. 69 (2), 2010 307 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees wrischer, m., ljube[i], n., devidé, z., 1998: the influence of norflurazon on the formation of chromoplast tubules in hypericum perforatum flowers. acta botanica croatica 57, 11–18. wrischer, m., ljube[i], n., prebeg, t., magnus, v., 1999: the succession of chromoplast structures in impatiens noli tangere flowers. phyton 39, 49–59. muraja-ljubi^i], j., wrischer, m., ljube[i], n., 1999: influence of herbicides amitrole and norflurazon on greening of illuminated potato microtubers. zeitschrift für naturforschung 54c, 333–336. prebeg, t., ljube[i], n., wrischer, m., 1999: structural and physiological characteristics of the coloured spots on the leucojum perigone. phyton 39, 75–78. fulgosi, h., ljube[i], n., 1999: molecular structure of spinach chloroplast nucleoids. acta botanica croatica 58, 15–25. ljube[i], n., prebeg, t., devidé, z., 1999: chromoplasts in the sepals of physalis alkekengi: the effect of norflurazon on chromoplast differentiation. acta botanica croatica 58, 79–86. salopek-sondi, b., kova^, m., ljube[i], n., magnus, v., 2000: fruit initiation in helleborus niger l. triggers chloroplast formation and photosynthesis in the perianth. journal of plant physiology 157, 357–364. franjevi], d., krajna, a., kalafati], m., ljube[i], n., 2000: toxic effects of copper upon the planarian polycelis felina (daly.). periodicum biologorum 102, 283–287. franjevi], d., krajna, a., kalafati], m., ljube[i], n., 2000: the effects of zinc upon survival and regeneration of planaria polycelis felina. biologia, bratislava 55, 689– 694. wrischer, m., ljube[i], n., magnus, v., devidé, z., 2000: structural and functional characteristics of overwintering blackberry leaves. acta botanica croatica 59, 5–16. [egota, s., te@ak, \., ljube[i], n., 2001: formation of vesicles in diluted aqueous solution of surfactant investigated by direct analysis of light scattering. advances in colloid and interface science 89–90, 283–291. britvec, m., reichenauer, t., soja, g., ljube[i], n., eid, m., pecina, m., 2001: ultrastructure changes in grapevine chloroplasts caused by increased tropospheric ozone concentrations. biologia, bratislava 56, 417–424. lepedu[, h., cesar, v., ljube[i], n., 2001: chloroplast ultrastructure and chlorophyll levels in vegetative buds and needels of norway spruce (picea abies l. karst.). periodicum biologorum 103, 61–65. kalafati], m., kova^evi], g., ljube[i], n., [unji], h., 2001: effects of ciprofloxacin on green hydra and endosymbiotic alga. periodicum biologorum 103, 267–272. kova^evi], g., kalafati], m., ljube[i], n., [unji], h., 2001: the effect of chloramphenicol on the symbiosis between alga and hydra. biologia, bratislava 56, 605–610. ljube[i], n., wrischer, m., prebeg, t., brki], d., 2001: carotenoid-bearing structures in fruit chromoplasts of solanum capsicastrum link. acta botanica croatica 60, 131–139. wrischer, m., prebeg, t., magnus, v., ljube[i], n., 2001: ultrastructural study of chromoplast components rich in glycolipids. acta botanica croatica 60, 141–147. 308 acta bot. croat. 69 (2), 2010 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:18 color profile: disabled composite 150 lpi at 45 degrees lepedu[, h., ljube[i], n., cesar, v., 2001: the effect of 2,4-d on the photosynthetic apparatus in cotyledons of spruce (picea abies l. karst.) seedlings grown in the dark. acta botanica croatica 60, 211–218. fulgosi, h., ester, l., ljube[i], n., 2002: essential role of peptidyl-propyl isomerase sll0408 in synechocystis sp. pcc 6803 development. periodicum biologorum 104, 413–419. poljuha, d., balen, b., bauer, a., ljube[i], n., krsnik-rasol, m., 2003: morphology and ultrastructure of mammillaria gracilis (cactaceae) in in vitro culture. plant cell, tissue and organ culture 75, 117–123. lepedu[. h., cesar,v., ljube[i], n., has-schön, e., 2003: photosynthetic pigments, chloroplast distribution and fine structure in vegetative buds of two spruce species. biologia, bratislava 58, 867–873. lepedu[, h., cesar, v., ljube[i], n., 2003: the appearance of vacuolar polyphenols in vegetative buds and developing needles of norway spruce (picea abies l. karst.). periodicum biologorum 105, 295–300. cesar, v., lepedu[, h., ljube[i], n., 2004: histochemical observations on the needles of norway spruce (picea abies l.) trees affected by cement dust pollution. phyton annales rei botanicae 44, 205–217. ba^i], t., ljube[i], n., u@arevi], z., grgi], lj., ro[a, j., 2004: tem investigation of tannins and chloroplast structure in needles of damaged silver fir trees (abies alba mill.). acta biologica cracoviensia series botanica 46, 145–149. lepedu[, h., viljevac, m., cesar, v., ljube[i], n., 2005: functioning of the photosynthetic apparatus under low and high light conditions in chlorotic spruce needles as evaluated by in vivo chlorophyll fluorescence. russian journal of plant physiology 52, 165–170. ljube[i], n., wrischer, m., prebeg, t., devidé, z., 2005: structural changes of the lamellar cells in the leaves of the moss polytrichum formosum hedw. during winter freezing and thawing processes. acta botanica croatica 64, 219–226. kova^evi], g., kalafati], m., ljube[i], n., 2005: endosymbiotic alga from green hydra under the influence of cinoxacin. folia microbiologica 50, 205–208. fulgosi, h., lepedu[, h., cesar, v., ljube[i], n., 2005: differential accumulation of plastid preprotein translocon components during spruce (picea abies l. karst.) needle development. biological chemistry. 386, 777–783. lepedu[, h., cesar, v. ljube[i], n., 2005: photosystem ii efficiency, chloroplast pigments and fine structure in previous-season needles of norway spruce (picea abies l. karst.) affected by urban pollution. periodicum biologorum 107, 329–333. ivankovi], s., musi], s., goti], m., ljube[i], n., 2006: cytotoxicity of nanosize v2o5 particles to selected fibroblast and tumor cells. toxicology in vitro 20, 286–294. ljube[i], n., britvec, m., 2006: tropospheric ozone-induced structural changes in leaf mesophyll cell walls in grapevine plants. biologia 61, 85–90. prebeg, t., ljube[i], n., wrischer, m.: differentiation of chromoplasts in cucumis sativus petals. international journal of plant sciences 167, 437–445. acta bot. croat. 69 (2), 2010 309 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:19 color profile: disabled composite 150 lpi at 45 degrees prebeg, t., ljube[i], n., wrischer, m., 2006: chloroplast biogenesis in chelidonium majus petals. acta societatis botanicorum poloniae 75, 107–112. kova^evi], g., kalafati], m., ljube[i], n., 2007: new observations on green hydra symbiosis. folia biologica (kraków) 55, 77–79. wrischer, m., prebeg, t., magnus, v., ljube[i], n., 2007: crystals and fibrils in chromoplast plastoglobules of solanum capsicastrum fruit. acta botanica croatica 66, 81–87. [ijakovi]-vuji^i], n., ljube[i], n., @ini], m., 2007: transcription of gel assemblies of bola type bis(oxalamide)-dicarboxylic acid and -diester gelators into silica nanotubes and ribbons under catalyzed and non-catalysed conditions. croatica chemica acta 80, 591–598. dunki], v., bezi], n., ljube[i], n., bo^ina, i., 2007: glandular hair ultrastructure and essential oils in satureja subspicata vis. ssp. liburnica [ili}: acta biologica cracoviensia – series botanica 49, 45–52. prebeg, t., wrischer, m., fulgosi, h., ljube[i], n., 2008: ultrastructural characterization of chloroplasts in cucumber fruit. journal of plant biology 51: 122–131. tkalec, m., prebeg, t., roje, v., pevalek-kozlina, b., ljube[i], n., 2008: cadmium-induced responses in duckweed lemna minor l. acta physiologiae plantarum 30, 881–890. fulgosi, h., juri], s., lepedu[, h., hazler-pilepi], k., prebeg, t., ljube[i], n., 2008: thylakoid system disassembly during bleaching of aurea mutants of acer negundo hassk. var. odessanum. croatica chemica acta 81, 89–95. buri], z., vili^i], d., caput mihali], k., cari], m., kralj, k., ljube[i], n., 2008: pseudo-nitzschia blooms in the zrmanja river estuary (eastern adriatic sea). diatom research 23, 51–63. kova^evi], g., kalafati], m., ljube[i], n., 2009: effects of norflurazon on green and brown hydra. folia biologica (kraków) 57, 91–96. wrischer, m., prebeg, t., magnus, v., ljube[i], n., 2009: unusual thylakoid structures appearing during degradation in the photosynthetic apparatus in chloroplasts. acta botanica croatica 68, 1–9. bo^ina, i., ljube[i], n., saraga-babi], m., 2010: cilia-like structures anchor the amphioxus notochord to its sheat. acta histochemica, in press. doi:10.1016/j.acthis.2009. 08.002 310 acta bot. croat. 69 (2), 2010 u:\acta botanica\acta-botan 2-10\social news.vp 11. listopad 2010 15:52:19 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp in memoriam u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:36 color profile: disabled composite 150 lpi at 45 degrees u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:36 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (1), 125–133, 2010 coden: abcra 25 issn 0365–0588 in memoriam dr. volker magnus dr. volker magnus, an outstanding croatian plant physiologist and member of the editorial board of this journal, passed away on july 30, 2009. volker was born march 22, 1942 in eckernförde, germany. he came to zagreb as deutscher akademischer austauschdienst (daad) fellow to study experimental biology at the faculty of natural sciences and mathematics, university of zagreb, where he obtained a b.s. degree in 1967. volker decided to continue his education in zagreb, and as a graduate student began to carry out research in the tracer laboratory, department of organic chemistry and biochemistry at ru|er bo{kovi} institute, under the supervision of drs. dina keglevi}, sonja iskri} and sergije kveder, scientists interested in indole metabolism in biological systems. volker’s fascination with plants led him to choose indole metabolism in plants as his research topic. his msc thesis, completed in 1971, dealt with the isolation of indole-3-methanol from pea seedlings, a metabolite of indole -3-acetic acid (iaa) (magnus et al. 1971). his thesis marked the beginning of his life-long interest in plant hormone auxins. in his doctoral study, volker isolated and synthesized a variety of tryptophol glycosides (magnus et al. 1973, magnus 1979), and obtained a ph.d. in the field of plant biochemistry in 1976. as one of his mentors and a long time colleague, i want to stress the many outstanding qualities of volker magnus: he was a very intelligent and gifted man, with a broad knowledge about plants and had a very good dexterity in organic synthesis. he was modest and self-effacing. early in his career he became an independent scientist, proving himself in planning and running experiments and in writing papers. with time he became adept in helping colleagues around him with advice and in correction of their papers, making use of his excellent mastery of english, german and even croatian. he left us much too soon. sonja iskri} acta bot. croat. 69 (1), 2010 125 u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:37 color profile: disabled composite 150 lpi at 45 degrees volker moved for postdoctoral training to prof. robert s. bandurski’s laboratory, michigan state university, (michigan, usa) in 1977. he worked on the development of a gas chromatography-mass spectrometry based isotope dilution assay for the plant hormone iaa. combining his expertise in plant indoles with extensive knowledge of synthetic organic chemistry, he produced two stable deuterated forms of iaa (fig. 1), thus enriching the auxin field with its first quantitative gc-ms isotope-dilution based assay (magnus et al. 1980). his work provided the foundation stone for the development of quantitative measurements of auxin in plants. upon completing his work on the first heavy isotope labeled iaa, he returned to the ru|er bo{kovi} institute where he remained for the duration of his career. beginning in 1979, volker and his first ph.d. student, goran la}an, continued doing research on volker’s »first love«: tryptophol and its glycosides. they synthesized a number of conjugate standards and identified endogenous glycosides in 120 different samples ranging from bacteria and fungi, to algae and plants (la]an et al. 1984, la]an et al. 1985). for that scientific achievement they obtained, in 1986, the »award for outstanding research in plant physiology«, from the gli{i} foundation, university of belgrade. in addition to auxin conjugates, volker worked on alkyl and halogen auxins derivatives with his ph.d. students, neboj{a ili} and eduard dolu{i}, who later continued their careers abroad. i met volker in the late 1960s at the ru|er bo{kovi} institute’s laboratory of stereochemistry and natural products, where he worked part-time as a technician, and i was an organic chemistry novice in my junior year at the university of zagreb. at a time when thousands of its citizens were emigrating to germany in search of work, a soft-spoken german gastarbeiter in yugoslavia was an oddity. all of us who knew him then quickly recognized his keen sense of a language that was not native to him, as well as his meticulous approach to scientific endeavors. the willingness to learn from and selflessly share his knowledge with others was a lifelong trait. volker was an excellent synthetic organic chemist for whom synthesis was not just a tool for probing plant physiology’s unanswered questions, but also a challenge to which he responded with dedication and passion. as his first graduate student, i enjoyed his friendship and was given the chance to learn critical thinking and rigorous scrutiny crucial for scientific research. volker’s professional and personal legacy will always remind me of my dear friend, »the best plant physiologist among chemists and the best organic chemist among plant physiologists.« goran la}an 126 acta bot. croat. 69 (1), 2010 iskri] s., jelaska s., koji]-prodi] b. et al. fig. 1. deuterated iaa with the labels on the ring (magnus et al. 1980). u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:38 color profile: disabled composite 150 lpi at 45 degrees volker always maintained excellent contacts with colleagues abroad. he returned to michigan state university in 1982, working for three years with norman good and roger hangarter on iaa-conjugates and their application in tissue cultures (magnus et al. 1992a, b). in the period between 1991 and 1996 he took a scientific leave to work first in the sandberg laboratory in umea, sweden, until 1993 (jakas et al. 1993, ili] et al. 1997), and then moved to mark brenner’s laboratory at the university of minnesota, minneapolis, mn, to work on 4-cl-iaa in pea with jocelyn ozga and dennis reinecke (reinecke et al. 1995, magnus et al. 1997). upon returning to the ru|er bo{kovi} institute, in 2003, volker founded the laboratory of chemical biology, at the department of molecular biology. volker was a scientist with interdisciplinary interests. as an excellent chemist and a passionate plant biologist, he collaborated with colleagues from divergent scientific fields. at his home institute, he collaborated for more than twenty years with dr. koji}-prodi} and her group (biljana nigovi}, sanja tomi}, snje`ana antoli}-steiner and branimir berto{a) on the structure/activity relationship of auxins (fig. 2). more than 50 indolic compounds were characterized structurally (most notably through use of x-ray diffraction, nmr and ir spectroscopy), and classified as active auxins, antagonists, or inactive compounds, based on different bioassays and modeling approaches. the collaboration resulted in more than 30 scientific papers, among which there were some representative ones on iaa amino acid conjugates (duddeck at al. 1989; koji]-prodi] et al. 1991a, 1993, 1999), n-alkyl-substituted iaas (ili] et al.1991; koji]-prodi] et al. 1991b; nigovi] et al. 1996, 2000; antoli] et al. 2003), and halogen-substituted iaas (antoli] et al. 1996, 1999).these papers represent in the open literature the most comprehensive study of the 3-dimensional characterization of auxin and auxin-like molecules. volker was also deeply involved in discussions on auxin binding protein (abp1) and its role in the auxin signaling pathway (fig. 3) (berto[a et al. 2008.). volker was a silent and unobtrusive person, very analytical in thinking, and sometimes indecisive and cautious in reaching conclusions. through the years, we successfully worked on many international projects funded by the us national science foundation, united states department of agriculture, the european community, and in bilateral collaborative projects, producing more than 30 scientific papers, exhibiting with mutual respect and tolerance. collaboration with volker was fruitful, exhilarating, sometimes followed by surprises, an inseparable part of his personality. biserka koji}-prodi} acta bot. croat. 69 (1), 2010 127 in memoriam dr. volker magnus fig. 2. 3d structure of indole-3-acetic acid (iaa) (koji]-prodi] et al. 1999). u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:39 color profile: disabled composite 150 lpi at 45 degrees more recently, volker’s interests became focused on advanced methods for auxin localization, in the production of a novel class of antibodies based on linking ring-aminoalkyl substituted iaa to proteins (dolu[i] et al. 2001, ili] et al. 2005, [o[ki] and magnus 2007, toma[i] et al. 2007, [o[ki] and magnus, 2009), and the use of carbon nanotube microelectrodes to record the auxin flux (mancuso et al. 2005). as a plant biologist, and that was how he primarily identified himself, he was interested in plant physiology and hormonal regulation of plant growth and developmental processes. he collaborated with prof. sibila jelaska, biology department, faculty of science, university of zagreb, in testing indole-3-ethanol and its sugar conjugates in the embryonic cultures of cucurbita pepo (jelaska et al. 1985). for more than twenty years they taught a joint course entitled: »mechanisms of plant development«, at the doctoral study of biology at the faculty of science. collaborating with his close friend and colleague from student days, dr. nikola ljube{i}, and later with dr. mercedes wrischer, volker was involved in research into plastids in different plant species. in a fruitful collaboration with the laboratory of electron microscopy, he was involved in studies of the effect of growth substances on the greening process of non-green plastids (salopek et al. 1998, 2000, 2002). recently, he also took part in a collaborative study of ultrastructure and function of different types of plastids (wrischer et al. 1999, 2000, 2001, 2007, 2009). working with his ph.d student branka salopek-sondi during the last decade, he became actively involved in research on the role of plant hormones in the regulation of postanthesis development of the christmas rose (helleborus niger l.) flower. in this un128 acta bot. croat. 69 (1), 2010 iskri] s., jelaska s., koji]-prodi] b. et al. fig. 3. hypothesis on the mechanism of the auxin binding protein, abp1, and its role in the auxin signaling pathway (berto[a et al. 2008). u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:40 color profile: disabled composite 150 lpi at 45 degrees usual plant, attractive flowers, white at anthesis, become intensively green upon fruit development, representing an excellent model for understanding cellular control of plastid development (fig. 3) (salopek-sondi et al. 2000, 2002; salopek-sondi and magnus 2007). in collaboration with colleagues from abroad, the hormones cytokinins, gibberellins, and auxins have been identified in this plant model and their role was discussed (tarkowski et al. 2006, ayele et al. 2009, pen^ik et al. 2009). volker was my mentor and teacher since 1993, when i started my scientific career at the ru|er bo{kovi} institute. the enjoyable moments of our collaboration were field trips to the forests of gorski kotar where we collected the flowers of the christmas rose (helleborus niger l.) for our research (fig. 4). during our trips through the forest volker was able to identify almost every plant along our way and had little stories about them, from either the biological or culinary aspects. even more fascinating, he used to recognize birds hidden in the tree bushes based on their songs. volker was a passionate naturalist, a very good botanist and a true plant lover. in the last few years, we shared an office and spent many afternoons in discussions over a cup of coffee. i will miss him. branka salopek-sondi auxin conjugates attracted volker’s attention during his entire scientific career. in the last few years, in collaboration with prof. jutta ludwig-müller from the technical university of dresden (germany), he participated in research into the structure, function, and regulation of auxin amidohydrolases, enzymes that hydrolyze amino acid conjugates of auxin, releasing free active forms (fig. 5.) (campanella et al. 2004, savi] et al. 2009). volker was a member of the croatian society of plant biology (cspb) for a long time, and its president from 1997 until 2001. he was the creator of the most recent statutes of the society and of the society’s representative emblem: the flower of aquilegia kitaibelii, an acta bot. croat. 69 (1), 2010 129 in memoriam dr. volker magnus fig. 4. the christmas rose (helleborus niger l.) flowers: white at anthesis (a), becoming green during fruit development (b). u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:42 color profile: disabled composite 150 lpi at 45 degrees endemic croatian plant residing in the mountains of velebit and plje{ivica (fig. 5). it will always remind us of volker, as well as volker’s famous bean soup, the one he used to cook for his friends and colleagues (the recipe for volker’s bean soup is posted on the cspb web site: http://www.hdbb.hr). he was member of the editorial board of acta botanica croatica from 1998 and was responsible for plant physiology and biochemistry. volker was entirely dedicated to science until the very end of his life. although retired since 2008, he was in the laboratory daily, continuing his research, writing, and discussing problems with young colleagues. volker magnus came to croatia and joined our scientific community silently; he lived and worked among us for more than 40 years, mostly unno130 acta bot. croat. 69 (1), 2010 iskri] s., jelaska s., koji]-prodi] b. et al. fig. 5. comparative model of auxin amidohydrolase from brassica rapa l. with highlighted mn2+ as a metal cofactor (violet spheres) (savi] et al. 2009). fig. 6. the flower of the croatian endemic plant (aquilegia kitaibelii schot) (left) inspired volker to create the emblem of the croatian society of plant biology (right). photo of aquilegia flowers is provided by professor toni nikoli} from the department of botany, division of biology, faculty of science, university of zagreb. u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:45 color profile: disabled composite 150 lpi at 45 degrees ticed and silent, and left us, unexpectedly, in the same way. his work and scientific contributions to our and international science was anything but silent and unnoticed; it will remain priceless. references antoli], a., koji]-prodi], b., tomi], s., nigovi], b., magnus, v., cohen, j. d., 1996: structural studies on monofluorinated derivatives of the phytohormone indole-3-acetic acid (auxin). acta crystallographica b 52, 651–661. antoli], s., salopek, b., koji]-prodi], b., magnus, v., cohen, j. d., 1999: structural characterization and auxin properties of dichlorinated indole-3-acetic acids. plant growth regulation 27, 21–31. antoli], s., dolu[i], e., ko@i], e. k., koji]-prodi], b., magnus, v., ramek, m., tomi], s., 2003: auxin activity and molecular structure of 2-alkylindole-3-acetic acids. plant growth regulation 39, 235–252. ayele, b. t., magnus, v., mihaljevi], s., prebeg, t., ^o@-rakovac, r., ozga, j. a., reinecke, d. m., mander, l. n., kamiya, y., yamaguchi, s., salopek-sondi, b., 2009: endogenous gibberellin profile during christmas rose (helleborus niger l.) flower and fruit development. journal of plant growth regulation doi: 10.1007/ s00344-009-9124-5. berto[a, b., koji]-prodi], b., wade, r. c., tomi], s., 2008: mechanism of auxin interaction with auxin binding protein (abp1): a molecular dynamics simulation study. biophysical journal 94, 27–37. campanella, j. j., olajide, a. f., magnus, v., ludwig-müller, j., 2004: a novel auxin conjugate hydrolase from wheat with substrate specificity for longer side-chain auxin amide conjugates. plant physiology 135, 2230–2240. dolu[i], e., kowalczyk, m., magnus, v., sandberg, g., normanly, j., 2001: biotinylated indoles as probes for indole-binding proteins. bioconjugate chemistry 12, 152– 162. duddeck, h., hiegemann, m., simeonov, m. f., koji]-prodi], b., nigovi], b., magnus, v., 1989: conformational study of some amino acid conjugates of indol-3-yl-acetic acid (iaa) by 1h-noe-difference spectroscopy. structure/auxin activity relationships. zeitschrift für naturforschung c 44, 543–554. ili], n., klai], b., magnus, v., viki]-topi], d., gács-baitz, e., 1991: synthesis of 5-alkylindole-3-acetic acids for use as plant hormone analogues. croatica chemica acta 64, 79–88. ili], n., magnus, v., ostin, a., sandberg, g., 1997: stable-isotope labeled metabolites of the phytohormone, indole-3-acetic acid. journal of labelled compounds and radiopharmaceuticals 39, 433–440. ili], n., habu[, i., barkawi, l. s., park, s., [efani], z., koji]-prodi], b., cohen, j. d., magnus, v., 2005: aminoethyl-substituted indole-3-acetic acids for the preparation of tagged and carrier-linked auxin. bioorganic and medicinal chemistry 13, 3229–3240. jakas, a., magnus, v., horvat, [., sandberg, g., 1993: synthesis of the -d-glucosyl ester of [carbonyl-13c]-indole-3-acetic acid. journal of labelled compounds and radiopharmaceuticals 33, 933–939. acta bot. croat. 69 (1), 2010 131 in memoriam dr. volker magnus u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:45 color profile: disabled composite 150 lpi at 45 degrees jelaska, s., magnus, v., seretin, m., la]an, g., 1985: induction of embryogenic callus in cucurbita pepo hypocotyl explants by indole-3-ethanol and its sugar conjugates. physiologia plantarum 64, 237–242. koji]-prodi], b., nigovi], b., horvati], d., ru@i]-toro[, @., magnus, v., duax, w. l., stezowski, j. j., bresciani-pahor, n., 1991a: comparison of the structures of the plant growth hormone, indole-3-acetic acid, and six of its amino acid conjugates. acta crystallographica b 47, 107–115. koji]-prodi], b., nigovi], b., tomi], s., ili], n., magnus, v., giba, z., konjevi], r., duax, w. l., 1991b: structural studies on 5-(n-alkyl)-substituted derivatives of the plant hormone, indole-3-acetic acid. acta crystallographica b 47, 1010–1019. koji]-prodi], b., nigovi], b., puntarec, v., tomi], s., magnus, v., 1993: structural comparison of biologically active and inactive conjugates of a-amino acids and the plant growth hormone (auxin) indole-3-acetic acid. acta crystallographica b 49, 367–374. koji]-prodi], b., magnus, v., antoli], s., tomi], s., salopek-sondi, b., 1999: structure/activity correlations for auxins. acta botanica croatica 58, 27–37. la]an, g., magnus, v., jeri^evi], b., kunst, lj., iskri], s., 1984: formation of tryptophol galactoside and an unknown tryptophol ester in euglena gracilis. plant physiology 76, 889–893. la]an, g., magnus, v., [imaga, [., iskri], s., hall, p. j., 1985: metabolism of tryptophol in higher and lower plants. plant physiology 78, 447–454. magnus, v., iskri], s., kveder, s., 1971: indole-3-methanol – a metabolite of indole-3-acetic acid in pea seedlings. planta 97, 116–125. magnus, v., iskri], s., kveder, s., 1973: the formation of tryptophol glucoside in the tryptamine metabolism of pea seedlings. planta 110, 57–62. magnus, v., 1979: tryptophyl b-d-glucopyranoside: chemical synthesis, metabolism, and growth promoting activity. carbohydrate research 76, 261–264. magnus, v., bandurski, r. s., schulze, a., 1980: synthesis of 4,5,6,7 and 2,4,5,6,7 deuterium-labeled indole-3-acetic acid for use in mass spectrometric assays. plant physiology 66, 775–781. magnus, v., hangarter, r. p., good, n. e., 1992a: interaction of free indole-3-acetic acid and its amino acid conjugates in tomato hypocotyl cultures. journal of plant growth regulation 11, 67–75. magnus, v., nigovi],, b., hangarter, r. p., good, n. e., 1992b: n -(indol-3-ylacetyl) amino acids as sources of auxin in plant tissue culture. journal of plant growth regulation 11, 19–28. magnus, v., ozga, j. a., reinecke, d. m., pierson, g. l., larue, t. a., cohen, j. d., brenner, m. l., 1997: 4-chloroindole-3-acetic and indole-3-acetic acids in pisum sativum. phytochemistry 46, 675–681. mancuso, s., marras, a. m., magnus, v., balu[ka, f., 2005: noninvasive and continuous recordings of auxin fluxes in intact root apex with a carbon nanotube-modified and self-referencing microelectrode. analytical biochemistry 341, 344–351. nigovi], b., koji]-prodi], b., antoli], s., tomi], s., puntarec, v., cohen, j. d., 1996: structural studies on monohalogenated derivatives of the phytohormone indole-3-acetic acid (auxin). acta crystallographica b 52, 332–343. 132 acta bot. croat. 69 (1), 2010 iskri] s., jelaska s., koji]-prodi] b. et al. u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:45 color profile: disabled composite 150 lpi at 45 degrees nigovi], b., antoli], s., koji]-prodi], b., kiralj, r., magnus, v., salopek-sondi, b., 2000: correlation of structural and physico-chemical parameters with the bioactivity of alkylated derivatives of indole-3-acetic acid, a phytohormone (auxin). acta crystallographica b 56, 94–111. pen^ik, a., rol^ik, j., novak, o., magnus, v., bartak, p., buchtik, r., salopek-sondi, b., strnad, m., 2009: isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction. talanta 80, 651–655. reinecke, d. m., ozga, j. a., magnus, v., 1995: effect of halogen substitution of indole-3-acetic acid on biological activity in pea fruit. phytochemistry 40, 1361–1366. salopek, b., wrischer, m., ljube[i], n., magnus, v., 1998: greening of non-transformed and agrobacterium rhizogenes transformed adventitious potato roots. biologia 53, 127–132. salopek-sondi, b., kova^, m., ljube[i], n., magnus, v., 2000: fruit initiation in helleborus niger l. triggers chloroplast formation and photosynthesis in the perianth. journal of plant physiology 157, 357–364. salopek-sondi, b., kova^, m., prebeg, t., magnus, v., 2002: developing fruit direct post-floral morphogenesis in helleborus niger l. journal of experimental botany 53, 1949–1957. salopek-sondi, b., magnus, v., 2007: developmental studies in the christmas rose (helleborus niger l.). international journal of plant developmental biology 1, 151–159. savi], b., tomi], s., magnus, v., gruden, k., barle, k., grenkovi], r., ludwig-müller, j., salopek-sondi, b., 2009: auxin amidohydrolases from brassica rapa cleave the alanine conjugate of indolepropionic acid as a preferable substrate: a biochemical and modeling approach. plant and cell physiology 50, 1577–1589. [o[ki], m., magnus, v., 2007: binding of ring-substituted indole-3-acetic acids to human serum albumin. bioorganic and medicinal chemistry 15, 4595–4600. [o[ki], m., magnus, v., 2009: interaction of indole derivatives with immobilized 3, 5-dinitrobenzoyl-phenylglycine. qsar & combinatorial science 28, 1276–1283. tarkowski, p., tarkowska, d., novak, o., mihaljevi], s., magnus, v., strnad, m., salopek-sondi, b., 2006: cytokinins in the perianth, carpels and developing fruit of helleborus niger l. journal of experimental botany 10, 2237–2247. toma[i], a., berto[a, b., tomi], s., [o[ki], m., magnus, v., 2007: binding behavior of amino acid conjugates of indole-3-acetic acid to immobilized human serum albumin. journal of chromatography a 1154, 240–249. wrischer, m., ljube[i], n., prebeg, t., magnus, v., 1999: the succession of chromoplast structures in impatiens noli-tangere flowers. phyton 39, 49–59. wrischer, m., ljube[i], n., magnus, v., devidé, z., 2000: structural and functional characteristics of overwintering blackberry leaves. acta botanica croatica 59, 5–16. wrischer, m., prebeg, t., magnus, v., ljube[i], n., 2001: ultrastructural study of chromoplast components rich in glycolipids. acta botanica croatica 60, 141–147. wrischer, m., prebeg, t., magnus, v., ljube[i], n., 2007: crystals and fibrils in chromoplast plastoglobules of solanum capsicastrum fruit. acta botanica croatica 66, 81–87. acta bot. croat. 69 (1), 2010 133 in memoriam dr. volker magnus u:\acta botanica\acta-botan 1-10\magnus.vp 9. travanj 2010 13:35:45 color profile: disabled composite 150 lpi at 45 degrees wrischer, m., prebeg, t., magnus, v., ljube[i], n., 2009: unusual thylakoid structures appearing during degradation of the photosynthetic apparatus in chloroplasts. acta botanica croatica 68, 1–9. sonja iskri}, sibila jelaska, biserka koji}-prodi}, goran la}an, nikola ljube{i}, mercedes wrischer, branka salopek-sondi ru|er bo{kovi} institute, zagreb faculty of science, university of zagreb 134 acta bot. croat. 69 (1), 2010 iskri] s., jelaska s., koji]-prodi] b. et al. u:\acta botanica\acta-botan 1-10\magnus.vp 13. travanj 2010 9:15:45 color profile: disabled composite 150 lpi at 45 degrees 56 acta bot. croat. 80 (1), 2021 acta bot. croat. 80 (1), 56–62, 2021 coden: abcra 25 doi: 10.37427/botcro-2021-003 issn 0365-0588 eissn 1847-8476 ornithogalum sibthorpii greuter (asparagaceae), a species overlooked in croatia milica rat1, sandro bogdanović2,3* 1 university of novi sad, faculty of sciences, department of biology and ecology, trg dositeja obradovića 2, 21000 novi sad, serbia 2 university of zagreb, faculty of agriculture, department of agricultural botany, svetošimunska cesta 25, 10000 zagreb, croatia 3 centre of excellence for biodiversity and molecular plant breeding, svetošimunska cesta 25, 10000 zagreb, croatia abstract – ornithogalum sibthorpii (asparagaceae) is an early flowering species, with populations scattered across the balkan peninsula and turkey. it inhabits rocky places and clearings, open habitats, parks and marginal parts of wetlands. based on the known distribution, habitat preferences and literature records for the balkan peninsula, it was hypothesised that this species might be distributed in croatia as well. to confirm this, herbarium material was revised, and field investigations were organized. the first report confirmed that o. sibthorpii is widespread along the eastern adriatic coast, reaching the inland dinaric region too. to present the currently known localities in croatia, a distribution map is provided. detailed morphological and leaf anatomy descriptions are given. morphological affinities with similar species, o. excapum and o. refractum, are also briefly discussed, and an identification key is given. all croatian populations of o. sibthorpii proved to be diploids with chromosome number 2n = 18. keywords: anatomy, geophytes, karyology, morphology, ornithogalum introduction the genus ornithogalum l. is characterized by a wide ecological tolerance and pronounced morphological variability. according to stevens (2001) it is one of the largest genera in the asparagaceae family, with approximately 160 species (300 taxa). it is distributed in africa, europe and southwest asia (speta 1998, martínez-azorin et al. 2013). global databases record nearly 120 species with distributions in the mediterranean region and in the rest of europe (govaerts 2019, euro+med 2006–2019). according to nikolić (2019a, b) in the croatian flora there are 19 orni­ thogalum species, three stenoendemic, and 10 species belonging to the subgenus ornithogalum. several discrete european ornithogalum groups are differentiated based on flowering time, that is, species flowering in late winter/early spring, spring, and in late spring/ early summer. one of the species that flower in late winter/ early spring is ornithogalum sibthorpii greuter. it is a rare and scattered species, distributed in the western part of turkey and in the balkan peninsula, in the north reaching dobrugea in romania (sibthorp and smith 1809, baker 1873a, b, markgraf 1932, radenkova 1964, zahariadi 1966, 1977, 1980, diklić 1975, landström 1989, speta 1990, rat and barina 2017, rat 2019). it prefers karst, rocky ground, clearings and open habitats (zahariadi 1980), and belongs to the group of ornithogalum species that have underground scape, bearing from one to many flowers on the shortened inflorescence. taxonomically significant characters compared with morphologically similar species in the investigated region (o. exscapum ten. and o. refractum kit. ex schltdl.) are the more or less pronounced pulvinus, refracted pedicels at anthesis and bulbs without bulbils (zahariadi 1980, landström 1989). there is one literature reference indicating that o. sib­ thorpii is distributed in croatia. ascherson and graebner (1905–1907: 249) stated that ornithogalum nanum smith in sibth. et smith (today synonym of o. sibthorpii) is recorded * corresponding author e-mail: sbogdanovic@agr.hr ornithogalum sibthorpii in croatia acta bot. croat. 80 (1), 2021 57 1997). image analysis and measurements of chromosomes were completed using karyotype 2 software (altinordu et al. 2016). results ornithogalum sibthorpii greuter, boissiera 13: 160 (1967) (fig. 1) type – “in arcadia, et prope abydum, martio florens”, smith loc. cit. (oxf!; file name: sib-0793). synonyms – ornithogalum nanum smith in sibth. et smith, fl. graec. prodr. 1: 230 (1809); fl. graec. (sibthorp). 4: 28, t. 333 (1823); non o. nanum (burm. f.) thunb., prodr. fl. cap. 62 (1794). morphological description (based on croatian material, fig. 1) ‒ bulb hypogeal, ovoid, 12 – 15 × 0.8 – 20 mm, within ljubuški and stolac in bosnia and herzegovina, near to the croatian border. furthermore, for general distribution they cited the adriatic region which covers an important part of the croatian coast. for that reason, herbarium revisions and field trips were organized, with the aim of providing new data about the potential distribution of the species in the western part of the balkan peninsula i.e. croatia, and to complement it with biological and ecological descriptions. materials and methods field trips were organized during season in 2015 and 2016. plant material was collected for morphological, anatomical and karyological analysis (tab. 1). voucher specimens are kept in the herbarium collections zagr and buns. herbarium materials of o. sibthorpii were revised in beo, beou, bp, buns, k, mknh, som, sara, w, wu, za, zagr and zaho. virtual collections g, gzu and oxf were accessed as well. herbaria acronyms follow thiers (2019). a distribution map of o. sibthorpii in croatia was produced in qgis software ver. 3.10. observations and morphological analyses included qualitative and quantitative description of bulb, leaves, scape and inflorescence with flowers. ten fresh plants were collected and pressed for analysis. all observations were performed on fresh material using a leica m205c binocular stereomicroscope, while measurements were carried out using digimizer image analysis software ver. 4.2.6.0. anatomical investigations included a description of leaf structure. cross sections were cut from the middle part of the leaf blade, using leica cm 1850 cryostat, at –20 ºc with a cutting interval of 60 ηm. observations and measurements were performed using a zeiss light microscope axiovision a2, equipped with a progres speed xtcore5 camera and capturepro v.2.8.8 image analysis software. for chromosome counting and analyses, 10 bulbs were planted in pots, and young root tips were collected, treated with 0.5% colchicine solution for 1 h at room temperature, and then fixed with a fresh solution of ethanol and glacial acid (3:1) for storage. to visualize metaphase chromosomes, root tips were hydrolysed with 1m hcl at room temperature, and then stained with schiff ’s reagent. standard squash technique was used for preparation of slides (jong tab. 1. list of examined plant materials of ornithogalum sibthorpii in croatia. locality latitude / longitude collecting date collector(s) herbarium voucher number croatia, brgat (south dalmatia) 42°38’45.06” 18°09’30.63” 28.03.2015 m. rat buns-2-1151 croatia, nin (north dalmatia) 44°14’48.58” 15°10’38.10” 23.04.2016 s. bogdanović, v. šegota, z. ljubešić zagr-41135 croatia, donji lapac (lika) 44°31’36.82” 15°58’44.48” 06.05.2016 s. bogdanović zagr-55520 zagr-55521 croatia, sukošan (north dalmatia) 44°02’59.10” 15°24’17.05” 05.04.2018 m. pandža, m. milović zagr-46301 fig. 1. ornithogalum sibthorpii (croatia, material from brgat locality): a – ascapose form with prominent pulvinus (bulb without outer tunics), b – scapose form with small pulvinus, c – flower, d – gynoecium, e – seed micromorphology. scale bar: a-d – 1 mm, e – 200 μm. rat m., bogdanović s. 58 acta bot. croat. 80 (1), 2021 out bulbils; outer tunics light brown to brown. leaves numerous, glabrous, longer than inflorescence, up to 3.5 mm wide, canaliculate, with median white stripe on the adaxial side. scape short, mostly hypogeal, 1 – 5 cm long. inflorescence up to 20 mm long, corymbose, with 1 – 8 (12) flowers. the overall length (scape + inflorescence) is ca 5 cm. the lower pedicels refracted, 10 – 25 mm long, with the pulvinus at the base. bracts shorter than or equal to pedicels. outer tepals 16 – 20 × 4 – 7 mm, with abaxial green stripe 4 – 6 mm wide. inner tepals 15 – 21 × 5 – 8 mm, with abaxial green stripe 3 – 6 mm wide. filaments 7 – 11 mm long; anthers 2.5 – 5 mm long. ovary elongated to rounded, 5 – 6 × 3.5 – 5.5 mm, with 6 prominent ribs; style 4 – 6 mm long, longer than or equal to ovary. seeds globose, 1 – 2 mm in diameter, black, luminous, with reticulate testa. phenology – flowering and fruiting time from march to june. leaf anatomy – leaf has typical “umbellatum-type”, that is “u” shape on transverse cross section. leaf blade is canaliculate, on abaxial side with 3 – 6 ribs. longitudinal white stripe visible on the adaxial side is a consequence of noncontinuous palisade tissue in the central part. visible on the cross section are 1-layered epidermis, 1-layered palisade tissue and mesophyll. vascular bundles are arranged in two lines: larger bundles are in the centre of mesophyll, and smaller ones along palisade tissue of abaxial side. other than vascular bundles, mesophyll contains large and small cavities, later usually with raffid crystals (fig. 2). karyology – for karyological studies, bulbs were available from localities brgat, donji lapac and nin. all investigated individuals were diploid, 2n=2x=18 (fig. 3a). combined chromosome formula is 2n=2x=6m + 12sm, and stebbins karyotype asymmetry degree is 2a. two chromosome pairs are long, three of medium length and four pairs are small chromosomes. karyotype is characterized with chromosomes that have more or less gradual transition in size (fig. 3b). two pairs are metacentric, while others are submetacentric, with total haploid chromosome length 59.01±0.89 µm (tab. 2). satellited chromosomes were not detected. fig. 2. leaf “u” cross section of ornithogalum sibthorpii (croatia, material from brgat locality). scale bar: 50 μm. tab. 2. chromosome parameters for ornithogalum sibthorpii in croatia. ten individuals are analyzed (2n=2x=18): four from donji brgat, three each from nin and donji lapac. number of analyzed metaphase plates is 40 in total (four per individua). abbreviations: l – long arm length, s – short arm length, tal – total absolute length, trl – total relative length, m – metacentric, sm – submetacentric. chromosome pairs l (µm) s (µm) tal (µm) trl (%) type i 6.20±0.72 5.13±1.03 11.32±1.39 10.51+8.68=19.19 m ii 5.96±0.48 3.12±0.65 9.08±0.84 10.10+5.29=15.38 m iii 5.41±0.55 2.08±0.51 7.49±0.97 9.17+3.52=12.69 sm iv 4.52±0.99 2.18±0.50 6.71±1.30 7.66+3.70=11.36 sm v 4.17±0.71 2.01±0.54 6.18±0.98 7.07+3.41=10.47 sm vi 3.80±1.25 1.67±0.41 5.47±1.27 6.44+2.83=9.27 sm vii 3.04±0.51 1.87±0.58 4.92±0.99 5.15+3.18=8.33 sm viii 2.82±0.58 1.54±0.29 4.36±0.62 4.78+2.61=7.39 sm ix 2.10±0.82 1.39±0.51 3.49±1.18 3.55+2.36=5.91 sm fig. 3. mitotic metaphase chromosome plate of ornithogalum sibthorpii (croatia, from brgat locality): a – metaphase plate; b – karyogram (2n=2x=18). scale bar: 10 μm. ornithogalum sibthorpii in croatia acta bot. croat. 80 (1), 2021 59 distribution and habitat – first herbarium specimens that confirmed presence of o. sibthorpii in croatia were found in the collection of ivo horvat (zaho), and date back to the first half of the 20th century. since then this species was omitted in collections, most probably due to the short vegetation period and early spring flowering time. however, herbarium revision of collections in gzu, w and wu revealed that the species was recorded earlier, but sparse data led to its neglect in croatia. recent field trips expanded the known distribution range of o. sibthorpii in croatia. it is spread from the southeast of the eastern adriatic cost to the central dalmatian region in the west, and in the north the central dinaric region, which is the only continental location in croatia. according to all the data gathered, a distribution map was created (fig. 4). o. sibthorpii inhabits dry, clear, open habitats, dry hillsides as well as anthropogenic areas and bare surfaces. the estimated altitude in croatia ranges from sea level up to 1000 m. in other regions of the balkan peninsula habitats and altitude are similar (radenkova 1964, rat et al. 2014, rat and barina 2017, rat 2019). examined specimens (specimina visa) – croatia: lika, krbava – donji lapac, bare, u nižem vlažnijem dijelu polja, 07.06.1958, i. horvat s.n. (zaho-41129, zaho-41130); makarska (dalmat.), 17.04.1931, m. salzmann s.n. (gzu057466); orebić (dalmatien), 16.04.; 19.04.1930, m. salzmann s.n. (gzu 057466); süddalmatien, halbinsel pelješac: zw. orebić u. sattel östl. des, mte. vipera, 16.04.1930, j. eggler s.n. (gzu 103693); süddalmatien, halbinsel peljesac: kräuterfluren u. macchie westl. von orebic., 14.04.1930, j. eggler s.n. (gzu 103694); dalmatien, gravosa [dubrovnik], 10.04.1933, k. ronniger s.n. [sub ornithogalum exscapum ten.] (w, herbarium karl ronniger 5580); dalmatia, in vineis pr. spalato [split], 04.1870, pichler s.n. [sub ornithoga­ lum umbellatum l. (vis.)] (wu, herbarium kerner). identification key for o. sibthorpii and morphologically similar species in croatia 1. bulb with numerous bulbils (>10) outside of tunics; bracts equal to or longer than pedicels ..... o. refractum – bulb without bulbils; bracts equal to or shorter than pedicels .............................................................................. 2 2. scape hypogeous, 20–50 mm long; inflorescence 1–8 (15)-flowered; lower pedicels refracted at anthesis, up to 30 mm long; pulvinus prominent; bracts subequal the pedicels ................................................... o. sibthorpii – scape only partly hypogeous, 10–30 mm long; inflorescence 5–10-flowered; pedicels erect to divergent (basal pedicel), 35–42 mm long; pulvinus absent; bracts shorter than pedicels ....................... o. exscapum discussion this species has been known since the pre-linnean period. buxbaum (1728) described it for the first time from the region around istanbul (constantinopolium) in turkey and he also provided the first iconography of the species. it was overlooked by linne, but later botanists who investigated greece and the eastern mediterranean, sibthorp and smith, described ornithogalum nanum as new species. ornitho­ galum nanum smith in sibthorp and smith (1809) was fig. 4. distribution map of ornithogalum sibthorpii in croatia. rat m., bogdanović s. 60 acta bot. croat. 80 (1), 2021 described as: „o. nanum, corymbo simplici paucifloro glabro scapo longiore, bracteis ventricosis scariosis, foliis linearibus numerosis“. the locality is cited as “in arcadia, et prope abydum, martio florens”, with reference to buxbaum (1728). abydum is ancient name for the region close to today’s cannakalle in turkey, and arcadia is an ancient region in peloponnese, but also in antalya (turkey) (stearn 1967). because of this, the precise locus classicus is not known. a detailed description of the species was published later (sibthorp and smith 1823). herbarium specimen marked as type material is deposited in oxf herbarium, under the file number sib-0793. since o. nanum is a later homonym of o. nanum (burm. f.) thunb., replacement name o. sibthorpii was published by greuter (greuter and rechinger 1967), in honour of john sibthorp (1758–1796) who collected plant material (stearn 1984). although in most of the relevant databases (e.g. theplantlist, euro+med checklist) o. sibthorpii is synonymised with o. sigmoideum freyn et sint. we retain viewpoint of landström (1989), later supported by the results of speta (1990), that o. sibthorpii is a good species which should not be underestimated without systematic investigations. the results of field trips organized in 2015 and 2016 confirmed our assumptions that o. sibthorpii is distributed in croatia, and these data were presented by rat and bogdanović (2016) at the 5th croatian botanical symposium. based on this report and deposited herbarium specimens in zagr herbarium (zagr-55520, 55521), nikolić (2019a, b) included it in the national list of vascular flora. historical data about distribution in italy (parlatore 1857) however have to be revised, since this record was included in distribution data for the species o. mutabile de not., which is in meantime synonymised with o. exscapum by peruzzi and passalacqua (2002) and garbari et al. (2003). landström (1989) summarized differential morphological characters of o. sibthorpii and o. exscapum, describing o. sibthorpii with shorter pedicels, larger flowers, longer styles and anthers. an analysis of the material from croatia shows that the additional differential qualitative morphological characters for these two species are: pedicel shape (in o. sib­ thorpii refracted at anthesis, in o. exscapum ascending to deflexed), visible pulvinus in o. sibthorpii, and absence of pulvinus in o. exscapum. another informative parameter is bract/pedicel length ratio; in o. sibthorpii bract and pedicel are almost of the same length, while in o. exscapum bract is about half the length of the pedicel. comparative morpho-anatomical and cytotaxonomical studies of ornithogalum species that belong to the group of small plants (overall length up to 10 cm) with hypogeal scape, including o. sibthorpii, have been done to clearly describe morphologically similar species in the area of turkey and the balkan peninsula (zahariadi 1962, 1965, 1977, speta 1990, 2000). both authors recognized three separated species: o. sintenisii freyn, o. sigmoideum and o. sibthorpii. in addition, speta (1990) described two new species, o. sag­ inatum speta and o. plutullum speta, first from romania and moldavia, and later from the balkan peninsula. moreover, landström (1989) recognized two ecotypes (montane and lowland) of o. sibthorpii and compare them to the intermediate type, localised for the region of i̇stanbul (turkey). these ecotypes are confirmed in croatia as well, since for plants collected at higher altitudes in the continental region (donji lapac, lika, dinaric area, 900 m a.s.l.) the flowering time is in june. in croatia, two distinct morphotypes of o. sibthorpii are observed: ascapose and scapose. the ascapose form (fig. 1a) is characterized by a short scape, and the inflorescence is more or less sessile on the ground, with numerous flowers; pulvinus is strong and prominent. the scapose form (fig. 1b) is differentiated with a scape up to 5 cm long, and an inflorescence with only a few (1 to 2) flowers (fig. 1b). comparing our results with those of speta (1990) who investigated o. sibthorpii and related species in the eastern part of the balkan peninsula, we can confirm that both morphotypes are mostly present in the same population. going further, he indicated that it is not unusual for two or more species from this group, that are morphologically indivisible, to coexist in one locality. in the balkan peninsula, o. sibthorpii could be easily misidentified with o. refractum. the most informative discriminate characteristic is a bulb without bulbils in o. sib­ thorpii (landström 1989, rat et al. 2014). it is not unusual to find both species in close localities, when in addition to bulb parameters flower characteristics can contribute to species identification (rat et al. 2014). furthermore, according to rat et al. (2017) seed testa micromorphology can be used as a taxonomically important character for the differentiation of o. sibthorpii from other ornithogalum species. globose seeds are typical only for o. sibthorpii (fig. 1e) and o. refractum, while, former is differentiated as species with rather small seeds. cytological review by cullen and ratter (1967), with doubtful discussion on plant material, reported three main cytotypes for o. sibthorpii ‒ o. sigmoideum group, in the region from caucasus to italy, with numerous aloploid series reported from different sources (2n = 12, 16, 17, 18, 19, 20, 28). they did not undertake any morphological measurements, and it was impossible for them to compare morphotypes and cytotypes or to discuss in detail obtained results. nonetheless, they recognized only two species, o. sigmoideum and o. sintenisii, while for them o. sibthorpii was a synonym of o. sigmoideum. an opposite view was presented by speta (2000), who included both morpho anatomical and cytological reports for 18 species that belong to this group. he recognized all species based on taxonomical characters, including the karyotypes described. it is clear from his results that o. sibthorpii is separated from all other species, with 2n = 14, 28 chromosome complements, while in o. sigmoideum they are 2n = 20, 24. chromosome arrangement 2n = 18 was documented also by speta (1990) in an unresolved discussion in short notes. in addition to these species, for o. sintenisii chromosome number 2n = 12 is recorded. diverse chromosome numbers (2n = 14, 16, 18, 24, 28) for o. sibthorpii were recorded and documented from the balkan peninsula, by other authors ornithogalum sibthorpii in croatia acta bot. croat. 80 (1), 2021 61 as well. for bulgaria, markova (1972) reported three different chromosome numbers (2n = 14, 16, 28), while lungeanu (1971) reported 2n = 18, 24 for romania. following evolutionary patterns in different ornithoga­ lum groups, it can be recognized that in almost all subgenera there are several groups or complexes that are characterized with “high morphological variability” and extensive chromosome numbers among samples. by now, for most of them, systematic studies have been undertaken and it has been confirmed that every complex (i.e. o. tenuifolium in africa and o. umbellatum in europe) includes “good species” and “transitional forms” (cytotypes and morphotypes) that exist in nature thanks to vegetative reproduction (stedje and nordal 1984, 1987, van raamsdonk 1985, van raamsdonk and heringa 1987, andrić et al. 2015). stedje and nordal (1984) concluded that rapid evolutionary processes in ornithogalum, visible as cytotype differentiation, do not express their changes in morphological characters, but do indicate the evolutionary progress in the taxon. this statement evidently can be accepted for the o. sibthorpii related group that was studied in this research, taking into consideration that many similarities were observed. acknowledgements we would like to thank zrinka ljubešić ex-president of the croatian botanical society (hbod) for financial support during the field trips to nin and to donji lapac. we are grateful to our colleagues milenko milović and marija pandža who provide a new locality of o. sibthorpii from sukošan. the research work of milica rat for her phd thesis was funded by the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-68/2020-14/200125). references altinordu, f., peruzzi, l., yu, y., he, x., 2016: a tool for the analysis of chromosomes: karyotype. taxon 65, 586–592. andrić, a., kočiš tubić, n., rat, m., obreht vidaković, d., 2015: diversity and genetic structure of ornithogalum l. 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(ed.), flora of socialistic republic of romania, 317–349. academiei republicii socialiste romania, bukurest (in romanian). zahariadi, c., 1977: notes on the intrageneric classification of the genus ornithogalum l. (liliaceae). botaničeskij žurnal 62, 1624–1639 (in russian). zahariadi, c., 1980: ornithogalum l. in: tutin, t., heywood, v.h., burges, n.a., moore, d.m., valentine, d.h., walters, s.m., webb, d.a. (eds.), flora europaea vol. 5, 35–40. cambridge university press, cambridge. 12 acta bot. croat. 81 (1), 2022 acta bot. croat. 81 (1), 12–22, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-027 issn 0365-0588 eissn 1847-8476 an asphodelus ramosus dominated plant community in montenegro: fringe or grassland? milica stanišić-vujačić¹*, danijela stešević¹, sead hadžiablahović², danka caković¹, urban šilc3 1 university of montenegro, faculty of natural sciences and mathematics, džordža vašingtona bb, 81000 podgorica, montenegro 2 environmental protection agency of montenegro, iv proleterske 19, 81000 podgorica, montenegro 3 zrc sazu, institute of biology, novi trg 2, 1000 ljubljana, slovenia abstract – our phytosociological study in montenegro (ćemovsko polje) deals with the syntaxonomy of arid grasslands in the adriatic region and, in particular, different interpretations of plant communities dominated by asphodelus ramosus. the main aims of this study were to contribute to knowledge of the composition of dry grasslands dominated by asphodelus ramosus in montenegro and to compare instances of asphodelus ramosus dominated vegetation along the adriatic. our vegetation dataset included 82 phytosociological relevés: 17 from our recent field work and 72 relevés of south european asphodelus ramosus communities. ordination analysis (nmds) was used for comparison of asphodelus ramosus dominated communities in the adriatic region. the asphodelus ramosus community from montenegro was classified into bromo erecti-chrysopogonetum grylli. the analysis revealed two distinct vegetation groups: grassland communities of the vegetation class festuco-brometea from montenegro, croatia and albania, and edge vegetation of the new class charybdido pancratii-asphodeletea ramosi from italy. comparison with similar vegetation types shows high similarity with associations on the eastern adriatic coast, where they are treated as grassland communities belonging to the alliance chrysopogono grylli-koelerion splendentis, order scorzoneretalia villosae, class festuco-brometea. keywords: asphodelus ramosus, bromo erecti-chrysopogonetum grylli, dry grasslands, festuco-brometea, montenegro introduction secondary dry grassland communities in the mediterranean have a zoo-anthropogenic origin; they have developed over centuries or even millennia of traditional land use, featuring practices such as mowing, grazing, temporary abandonment of arable fields, and/or other disturbance regimes (apostolova et al. 2014, valkó et al. 2018). mediterranean and sub-mediterranean dry grasslands are considered to be among the floristically richest vegetation types (apostolova et al. 2014) and, at the same time, a very important habitat for endangered, rare and endemic species, so they are included in the list of habitats of european interest (council directive 92/43/eec on the conservation of natural habitats and of wild fauna and flora, 1992), as well as the european red list of habitats (janssen et al. 2016). compared to other parts of europe, dry grasslands of the balkan peninsula are still well preserved (apostolova et al. 2014). the balkan peninsula is well known for its rich flora and well-preserved vegetation, because it was a glacial refuge for animal and plant species (griffiths et al. 2004). its biodiversity is considered to be among the highest in europe (apostolova et al. 2014). the peninsula is characterized by the presence of a broad spectrum of dry grasslands (e.g., horvat et al. 1974, jovanović et al. 1986, apostolova et al. 2014, terzi 2015, aćić et al. 2015, matevski et al. 2018). in some balkan countries (slovenia, serbia, bulgaria, croatia, north macedonia), dry grassland vegetation has been intensively researched (apostolova et al. 2014, matevski et al. 2018). in contrast to these countries, research into this type of vegetation in montenegro has considerable discontinuity, and comprenhensive studies, especially in the submediterranean and mediterranean region, are rare (pulević and bulić 2012). * corresponding author e-mail: milicas@ucg.ac.me asphodelus ramosus dominated plant community acta bot. croat. 81 (1), 2022 13 in our study, we focused on research into asphodelus ramosus-dominated vegetation. asphodelus ramosus is a species native to the mediterranean and macaronesian regions: southern europe, northern africa, the middle east, mediterranean islands and canary islands. it can be found in forest edges and grasslands, generally on basic soils of a certain depth, forming very dense populations in grazed areas, from the coast up to 1000 m, occasionally reaching 2150 m in the mountain systems of north africa (diazlifante and valdés 1996). it is unpalatable for grazing animals. research into plant communities dominated by asphodelus spp. (and asphodelus ramosus in particular) has become very intensive in recent years, especially in the western and central mediterranean (allegrezza et al. 2015, biondi et al. 2016, 2017). in the balkan peninsula, similar vegetation has been described from albania (fanelli et al. 2015) and croatia (horvatić 1934, 1939, 1963, šegulja 1969, 1970, hećimović 1984, jasprica and ruščić 2013, jasprica et al. 2016). today, there are different opinions about the syntaxonomical classification of asphodelus ramosus dominated vegetation in europe and its position in the landscape (as grasslands or fringe (saum) communities). in the eastern adriatic, these communities have been classified within the grassland class festuco-brometea, while in the central and western adriatic, they have been classified as heliophilous edge vegetation of trifolio-geranietea sanguinei (biondi et al. 2014, allegrezza et al. 2015) or recently into a new class charybdido pancratii-asphodeletea ramosi biondi et al. 2016 (biondi et al. 2016, 2017). the aim of this study was to (i) contribute to knowledge of the composition of dry grasslands dominated by asphodelus ramosus in montenegro and (ii) compare examples of asphodelus ramosus dominated vegetation along the adriatic and its syntaxonomical classification and classification into natura 2000 habitat types. materials and methods study area ćemovsko polje is a part of zetska ravnica plain in montenegro, between the rivers morača, cijevna and ribnica. it covers 165 km2 and extends from podgorica, the capital of montenegro, to skadar lake. the altitude of the investigated area ranges from 12 to 30 m a.s.l. during the pleistocene period, moraine material eroded from the mountains was carried along the rivers morača and cijevna into the area of ćemovsko polje (radojičić 2015). the dominant types of soil are eutric cambisol and rendzina, which are formed on fluvio-glacial deposits, and consequently are often shallow and skeletoid (burić et al. 2017). the area of ćemovsko polje has a mediterranean climate with hot summers – csa (burić and micev 2008). a grassland ecosystem is dominant in this area. the studied asphodelus ramosus – dominated vegetation occurs in pastures used for grazing by cattle and sheep. a significant area of ćemovsko polje is occupied by vineyards and plantations of peach and other kinds of fruit (radojičić 2015). the area investigated has also recently been heavily impacted by urbanization (burić et al. 2017). although many floristic studies have been performed in the area of ćemovsko polje (černjavski et al. 1949, hadžiablahović 2010, 2018, stešević et al. 2014), the vegetation has remained poorly studied (černjavski et al. 1949, hadžiablahović 2018). potential natural vegetation of the wider area of podgorica and lake skadar is apulian-southeast adriatic meso-supra-mediterranean quercus trojana forests with pistacia species (bohn et al. 2000-2003) or precisely macedonian oak forest ‘quercetum trojanae montenegrinum blečić et lakušić 1975’ (recte: quercetum trojanae em 1958). illyrian sub-mediterranean rocky grasslands on shallow calcareous soils of the alliance chrysopogono grylli-koelerion splendentis horvatić 1973 prevail in the area of ćemovsko polje (hadžiablahović 2010). sampling and data analysis from april to june 2019, we sampled asphodelus ramosus-dominated plant communities on ćemovsko polje according to the braun-blanquet method (braun-blanquet 1964). we made 17 relevés and the size of plots was 25 m2. the minimum distance between plots was 100 m. the minimum coverage value of asphodelus ramosus for it to be considered dominant was 25%. each plot was visited in april and again in june. cover values of species and total vegetation cover are based on the summer aspect (tab. 1, appendix 1). gps coordinates were recorded for each plot (appendix 2). all relevés made during fieldwork and relevés from the literature were entered into the turboveg (hennekens and schaminée 2001) database. relevés from the literature were used for comparison with vegetation data from our fieldwork (appendix 3). we obtained characteristic species of the association bromo erecti-chrysopogonetum grylli according to horvatić (1963). diagnostic taxa of alliance and order were assigned according to terzi (2015), while the diagnostic taxa of classes were determined according to terzi (2015) for the class festuco-brometea, mucina et al. (2014) for other classes and biondi et al. (2016) for the class charybdido pancratii asphodeletea ramosi. non-metric multidimensional scaling (nmds, kruskal 1964) was used to examine the overall variation in the species composition in the whole relevé dataset. hellinger transformation of percentage cover values (5=87.5 %, 4=62.5 %, 3= 37.5 %, 2=12.5 %, 1=2.5 %, +=0.5 %, r=0.1 %) was used and bray-curtis as a measure of dissimilarity. nmds was performed using the r package ‘vegan’ (oksanen et al. 2017). for ecological interpretation of vegetation patterns, ecological indicator values (pignatti et al. 2005) were passively projected onto the nmds graph. weighted by species cover, mean indicator values were calculated for each relevé using juice software (tichý 2002). stanišić-vujačić m., stešević d., hadžiablahović s., caković d., šilc u. 14 acta bot. croat. 81 (1), 2022 the nomenclature of taxa is in accordance with euro+med (2006) and the nomenclature of higher syntaxa according to mucina et al. (2014). the taxonomy of asphodelus ramosus species was sometimes ambiguous. previously, the species asphodelus microcarpus viv. was accepted as valid by many authors but, after a taxonomic revision of the genus asphodelus in the western mediterranean (diaz-lifante and valdés 1996), it has been considered a synonym of asphodelus ramosus l. (euro+med 2006). results the asphodelus ramosus plant community from ćemovsko polje (montenegro) is represented by 17 relevés in the phytosociological table (tab. 1). the dominant species of the association are asphodelus ramosus and chrysopogon gryllus, while the most frequent species are asphodelus ramosus, sanguisorba minor, poa bulbosa, teucrium capitatum, sideritis romana subsp. purpurea, and anacamptis papilionacea. asphodelus ramosus is also considered to be the only characteristic species of the association. the community develops in two clear phenological aspects. in the spring aspect (fig. 2a), the dominant species is asphodelus ramosus, while in summer (fig. 2b), dominance is taken by the tall grass chrysopogon gryllus. the spring aspect is also characterised by high frequency and coverage of anemone hortensis, poa bulbosa and sanguisorba minor, and the summer one by bupleurum veronense and teucrium capitatum. in addition to the species characteristic for the class festuco -brometea, there are several species of annual and ephemeral grasslands of the classes stipo-trachynietea distachyae and helianthemetea guttate, and seasonal perennial and ephemeroid pastures of the class poetea bulbosae, all indicating the mediterranean and grassland character of the studied plant community. the impact of grazing is evident from the many ruderal species: avena barbata, scandix pecten-veneris, daucus guttatus, scolymus hispanicus, bromus squarrosus, euphorbia exigua, sonchus oleraceus (tab. 1). the studied stands constitute an open grassland community, which develops on stony soil and is used for grazing (cattle and sheep). total vegetation coverage is 60 – 80% in the summer aspect. stones and pebbles have dimensions of up to 20 cm and their cover is 5 – 40%. if the stones are removed and these areas are used for mowing, the vegetation changes into dry grasslands of the alliance vulpio -lotion (hadžiablahović 2018). using various numerical classifications, we tried to classify the association bromo erecti-chrysopogonetum grylli into subassociations. this kind of vegetation is fairly uniform in the study area, and there were no ecologically evident/logical subunits. a comparison of the studied asphodelus community from montenegro with asphodelus ramosus-dominated communities from around the adriatic (fig. 1) revealed two distinct vegetation groups (tab. 2, fig. 3). communities from albania, croatia and montenegro form one group, fig. 2. asphodelus ramosus dominated community on ćemovsko polje, vicinity of podgorica, montenegro. a – spring aspect, b – summer aspect. fig. 1. localities of data used of asphodelus ramosus-dominated communities. see tab. 2 for community abbreviations. al – albania, ba – bosnia and herzegovina, hr – croatia, it – italy, me – montenegro, rs – serbia, si – slovenia. asphodelus ramosus dominated plant community acta bot. croat. 81 (1), 2022 15 table 1. phytosociological table of the association bromo erecti-chrysopogonetum grylli in ćemovsko polje (montenegro). relevé number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 % plot size (m2) 25 vegetation cover (%) 80 60 80 70 75 60 60 75 60 65 70 70 60 60 60 60 60 bromo erecti-chrysopogonetum grylli asphodelus ramosus 4 3 4 3 4 3 3 3 3 4 3 3 3 3 4 3 4 17 chrysopogono grylli-koelerion splendentis bupleurum veronense + + + . . + . + . . + . + + + . . 9 carduus nutans subsp. micropterus . + . + . . . + . . . . . + . . . 4 scorzoneretalia villosae chrysopogon gryllus 1 2 2 3 2 2 1 2 . 1 3 2 2 2 2 1 . 15 eryngium amethystinum + + 1 . 1 + 1 + + . 1 1 + 1 1 1 1 15 koeleria lobata + + + . + 1 . . + . 1 . + 1 1 + 1 12 seseli montanum subsp. tommasinii 1 . . . + . + . + . . 1 . . + 1 . 7 satureja subspicata . . . + . . . . + 2 . . . 2 2 . + 6 festuca stricta subsp. sulcata . . . + . + . 1 2 . . + . . . . . 5 medicago prostrata + . . . . . . . . . 1 + . + . . . 4 plantago holosteum . + + + . . . . . . . + . . . . . 4 bunium alpinum subsp. montanum . . + . . . . . + + . . . . + . . 4 scorzonera villosa . . 1 + . . . . + + . . . . . . . 4 festuco-brometea sanguisorba minor 3 2 3 2 2 + 1 2 + 1 2 2 2 1 1 2 1 17 poa bulbosa 1 1 1 2 1 2 2 1 + + 1 + 2 1 1 2 1 17 teucrium capitatum + 1 1 + + 1 1 1 2 + 1 2 + 2 2 1 1 17 hypericum perforatum + + + + + + . 1 + . + + + + + 1 + 15 hippocrepis ciliata + . 1 . + 1 + . . 1 + + + + + + + 13 centaurea deusta + + . . + + + 3 1 + 1 1 1 1 . 3 . 13 leontodon crispus . . + + + . . . 1 1 1 1 1 . 1 + + 11 ranunculus millefoliatus . + + + . . . + + . . + 1 + + . + 10 convolvulus cantabrica + . . . 2 1 2 . . . 1 1 1 + . 1 . 9 petrorhagia saxifraga . . . + + + + + . . + + . . + + . 9 linum tenuifolium . + + . . + . . 1 . . + + + . . + 8 anthyllis vulneraria subsp. polyphylla . . + . + + . . + + + + + . . . . 8 carex caryophyllea . . . 1 . . . . . . 1 + + 1 1 1 . 7 podospermum laciniatum + + . + + . . . . . . . . . . . + 5 bothriochloa ischaemum . . . . . . . + 1 + 1 . . . 1 . . 5 anacamptis morio . + . + + . . . . . + . . . . . . 4 trifolium campestre . + . . . . + . . + . . . . . + . 4 ophrys sphegodes . . . . . . + . + . . . . . + . . 3 cuscuta epithymum subsp. epithymum . . . . + . . . . . + + . . . . . 3 thymus striatus . . . . . . . . + . . . . + + . . 3 leopoldia comosa . + . . . . . . + . . . . . . . + 3 chenopodietea avena barbata + + + + 1 1 1 . . 2 . . 1 . . + + 11 valantia muralis . . 1 . + . . . . 1 . . . . + . + 5 scandix pecten-veneris . . . . . . . + + . . + . . . . . 3 stipo-trachynietea distachyae sideritis romana subsp. purpurea + + 1 + + 1 + + + + + + + + + + 1 17 crepis sancta 1 1 + + 1 1 1 1 + 1 + + . . . 1 + 14 arenaria leptoclados 1 . + 1 + + + . . . . . + + + + . 10 polygala monspeliaca . + + . + + + . . + + . . + . 1 + 10 ornithogalum collinum + . . . + + + 1 + . 1 1 2 . . . . 9 tordylium apulum . . . + . + + + . . + . . . . . + 6 stanišić-vujačić m., stešević d., hadžiablahović s., caković d., šilc u. 16 acta bot. croat. 81 (1), 2022 relevé number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 % plot size (m2) 25 vegetation cover (%) 80 60 80 70 75 60 60 75 60 65 70 70 60 60 60 60 60 petrorhagia dubia  . . + + . . . + . . . . + . . . . 4 filago germanica + . . . . + . . . . . + . . . . + 4 ononis reclinata + . . . + + . . . . . . . . . + . 4 trachynia distachya . . + . + . . . . . . 1 . . . . . 3 neatostema apulum . . . . . . . . . . . . 1 + + . . 3 helianthemetea guttati helianthemum salicifolium 1 + 1 + 1 1 2 2 + 1 1 1 1 . 1 1 . 15 crupina vulgaris + + + . + + 1 . 1 + + + + + . . . 12 asterolinon linum-stellatum + 1 + + + 1 1 . . . . . + + 1 + . 11 galium divaricatum 1 1 + + . + . + . . . . . + . + + 9 plantago bellardii + . . . . . + + . + . . 1 + + . . 7 linaria pelisseriana . + . + . . . + + . . . . . . . + 5 filago gallica . + . . . + . . . . . . + . + . . 4 aira elegantissima . . . + . . . . . . . . . + . + + 4 trifolium stellatum + . + . + . . . . . . . . . . . . 3 poetea bulbosae anthoxanthum odoratum + 2 . 1 + + + . + + . . + + . 1 + 12 trifolium subterraneum + 1 . 1 + . + + . . 1 . + . . 1 1 10 plantago lanceolata . + + 1 . . . + + + . . . . . + + 8 leontodon tuberosus . . + + + . + 1 + . . . . . . . . 6 herniaria glabra . . . . . . . . . . . . + + + + . 4 prospero autumnale . . + . . . . . . . . . + . . + . 3 trifolium nigrescens . + . . . . . . . 1 . . . . . . + 3 sedo-scleranthetea cerastium pumilum subsp. glutinosum + + . 1 1 1 . + . . . . 1 + + 1 + 11 viola kitaibeliana 1 1 + + + + . + . + . . + + . . . 10 clinopodium acinos . . + . + . 1 . . . + + + + + . . 8 draba verna + . . + . . . . . . . . + + + + . 6 artemisietea vulgaris daucus guttatus + + + + + 1 1 . . 2 + . + . . + + 12 scolymus hispanicus . + + 1 . + 1 1 + + + . . . + 1 . 11 tyrimnus leucographus 1 . . + . + + 1 . + . . . . . + + 8 potentilla recta . . . + 1 . + 1 . . . . . + . . . 5 sisymbrietea geranium columbinum + + . + + + + + + + + . + + . + + 14 erodium cicutarium + . . 1 . . . 1 . . + + 1 + + . 1 9 bromus squarrosus 1 . . . . . 1 + . . 1 . + . + . . 6 papaveretea rhoedis euphorbia exigua + . . . + 1 1 . + + + + 1 1 + + + 13 sherardia arvensis + + . . + + 1 . + . . . + . . + + 9 anagallis arvensis . . + + + + 1 . + 1 . . . . . + . 8 sonchus oleraceus + + . . . + + . . + + . + . . . . 7 euphorbia helioscopia . . . . . . . . + . . + + . . + . 4 euphorbia taurinensis . . . . . . . . . . . . + + + . . 3 molinio-arrhenatheretea serapias vomeracea . . + . . . . . . . + + . + . + . 5 hypochaeris radicata . + . + . . . . . . . . . . + + 1 5 ononido-rosmarinetea carlina corymbosa . . + . 1 + 1 . . + + 1 1 + . . 1 10 asphodelus ramosus dominated plant community acta bot. croat. 81 (1), 2022 17 relevé number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 % plot size (m2) 25 vegetation cover (%) 80 60 80 70 75 60 60 75 60 65 70 70 60 60 60 60 60 lygeo sparti-stipetea tenacissimae anacamptis papilionacea + + + + + . + + + 1 1 1 1 1 + 1 + 16 anemone hortensis 2 2 2 2 2 1 2 1 + 1 1 . . . . 2 . 12 other species cynodon dactylon . + + + + + + . + 1 + + + + + + + 15 allium guttatum subsp. sardoum + 1 . 1 + . + + . . + . + . + + + 11 cerastium ligusticum subsp. trichogynum 2 1 + 1 1 . . . + . . 1 . 1 1 . . 9 silene italica . + + + + + . . . . + 1 . 1 . . . 8 alkanna tinctoria . . . . . + . . . . 1 1 1 1 1 . . 6 hainardia cylindrica . . . . . . 1 . . . . . + + . . . 3 trifolium scabrum . . . . . . . . . + . + + . . . . 3 table 2. shortened synoptic table of plant communities with asphodelus ramosus. the original classification indicated by authors is used. communities: 1. bromo erecti-chrysopogonetum grylli (montenegro, new relevés from ćemovsko polje), 2. asphodelo-chrysopogonetum grylli (albania, fanelli et al. 2015), 3. bromo-chrysopogonetum grylli subass. asphodeletosum microcarpi (croatia, horvatić 1934), 4. narcisso tazzettae-asphodeletum microcarpi (croatia, šegulja 1970), 5. narcisso tazzettae-asphodeletum microcarpi (croatia, hećimović 1984), 6. narcisso tazzettae-asphodeletum microcarpi (croatia, jasprica et al. 2016), 7. asphodelus ramosus community (albania, fanelli et al. 2015), 8. charybdido pancratii-asphodeletum ramosi (italy, biondi et al. 2016), 9. alkanno tinctoriae-asphodeletum ramosi (italy, biondi et al. 2016), 10. euphorbio characiae-thapsietum garganicae (italy, biondi et al. 2017), 11. asphodelo ramosi-feruletum communis (italy, biondi et al. 2016), 12. asphodelino luteae-feruletum communis (italy, biondi et al. 2016). plant community 1 2 3 4 5 6 7 8 9 10 11 12 number of relevés 17 4 12 8 10 3 3 14 3 5 2 7 bromo erecti-chrysopogonetum grylli asphodelus ramosus 100 25 100 100 100 100 100 100 100 60 100 100 cytisus spinescens . . 50 . . . . . . . . . narcisso tazzettae-asphodeletum microcarpi narcissus tazetta . 25 . 100 70 100 . 7 . . . . anacamptis papilionacea 94 . . 75 . . . 21 . 40 . 29 chrysopogono grylli-koelerion splendentis carduus nutans subsp. micropterus 24 25 50 50 50 . . . . 80 . 43 bupleurum veronense 53 . 42 . 90 . 67 . . . . . centaurea cristata . . . 13 . . . . . . . . salvia officinalis . 50 8 . . . . . . . . . scorzoneretalia villosae chrysopogon gryllus 88 100 100 88 . 67 33 . . . . . koeleria lobata 71 25 67 88 40 67 . . . . . . plantago holosteum 24 25 33 88 . . . . . . . . eryngium amethystinum 88 50 58 88 . . 33 7 . 60 . 57 salvia pratensis . 25 33 88 . . . . . . . . medicago prostrata 24 . 25 . . . . . . . . . festuca valesiaca . . 83 13 . . . . . . . . seseli montanum subsp. tommasinii 41 . . . . . . . . . . . satureja subspicata 35 . . . . . . . . . . . potentilla heptaphylla subsp. australis . . 8 . . . . . . . . . dorycnium pentaphyllum subsp. germanicum . . 17 . . . . . . . . . festuco-brometea sanguisorba minor 100 25 17 88 . . . 29 . 20 . . bothriochloa ischaemum 29 25 . 50 100 . 33 . . . . . bromopsis erecta . 25 100 75 . 33 . . . . . . hippocrepis comosa . . 17 25 . . . . . . . . stanišić-vujačić m., stešević d., hadžiablahović s., caković d., šilc u. 18 acta bot. croat. 81 (1), 2022 plant community 1 2 3 4 5 6 7 8 9 10 11 12 number of relevés 17 4 12 8 10 3 3 14 3 5 2 7 linum tenuifolium 47 25 8 63 . . . . . . . . ruta graveolens . 25 17 75 . . . 36 . . . . leopoldia comosa 18 . . . 70 . . . . . . 43 carex caryophyllea 41 25 . 50 . . . . . . . . satureja montana . 25 8 . . . 33 . . . . . ranunculus millefoliatus 59 . . . . . . . . 20 . 14 charybdido pancratii-asphodeletum ramosi carlina corymbosa 76 50 100 63 . . 33 50 . 80 100 29 asparagus acutifolius 6 25 50 100 50 . . 79 100 60 100 86 drimia pancration . . . . . . . 100 67 20 100 57 anemone hortensis 71 25 . 63 70 . . 64 . 80 50 57 asphodeline lutea . . . . . . . 7 . 60 . 100 thapsia garganica . . . . . . . 57 . 100 . 100 ferula communis . . . . . . . . . 20 100 100 hypochaeris radicata 29 . . . . . . 29 33 . . . asphodeline liburnica . . . . . . . 7 . . . . other dactylis glomerata subsp. hispanica . 25 100 88 50 100 . 93 67 . . . plantago lanceolata 47 25 33 100 90 . . 29 . . . 71 reichardia picroides . 25 50 75 100 67 . 43 . 40 . 43 catapodium rigidum 6 50 75 88 100 . 100 . . . . . crepis sancta 82 . . . . . . 43 67 . 100 43 avena barbata 65 . 33 50 . . 67 . 33 . 100 29 trifolium scabrum 18 25 25 25 50 . 67 . . . . . helichrysum italicum . 25 75 50 90 67 33 7 . . . . paliurus spina-christi 25 63 . . . . . 43 . . 100 . olea europaea 17 . . . . . . 43 . . 100 . pinus halepensis . . . . . . . . 33 20 . . fig. 3. nmds ordination of asphodelus ramosus-dominated communities from the adriatic region with passively projected ecological indicator values. symbols indicate classification into the classes: – festuco-brometea, – artemisietea vularis, – charybdido pancratii-asphodeletea ramosi. numbers indicate centroids of relevés of particular communities (see tab. 2). asphodelus ramosus dominated plant community acta bot. croat. 81 (1), 2022 19 with abundant grassland species of the class festuco brometea, which are not present in the second group of edge communities from italy. two dimensional solution of the nmds ordination attained a minimum stress of 0.21. the nmds ordination clearly distinguishes the two groups along gradients of nutrients, light and soil reaction. edge communities from italy thrive on more nutrient-rich, shaded sites (fig. 3). based on floristic composition and all the comparative analyses, we decided to classify the studied stands dominated by asphodelus ramosus into the already described grassland association bromo erecti-chrysopogonetum grylli. discussion in recent years, several studies on the distribution and dynamics of plant communities with asphodelus ramosus have been performed, especially in the central and western mediterranean (biondi et al. 2016, biondi et al. 2017). in contrast, this type of vegetation has been poorly studied in montenegro. the association bromo erecti-chrysopogonetum grylli was described for the first time in croatia (island of pag, horvatić 1934), with two subassociations (bromochrysopogonetum grylli dorycnietosum herbacei and bromo-chrysopogonetum grylli asphodeletosum microcarpi), and classified in the alliance ‘chrysopogoneto-satureion subspicatae horvat i horvatić 1934’ (recte: chrysopogono grylli-koelerion splendentis), order ‘brometalia erecti br. bl.’ subsequently, bromo-chrysopogonetum grylli asphodeletosum microcarpi was raised by horvatić (1963) to the rank of association, with the name asphodelo microcarpi-chrysopogonetum grylli, and included in the order ‘scorzonero-chrysopogonetalia h-ić et ht (1956) 1958’ (recte: scorzoneretalia villosae). the association has so far been reported in montenegro and albania (černjavski et al. 1949, fanelli et al. 2015, hadžiablahović 2018). in his nomenclatural revision of the order ‘scorzonero villosae-chrysopogonetalia grylli horvatić et horvat in horvatić 1963’, terzi (2011) considered the associations asphodelo-chrysopogonetum grylli and bromo -chrysopogonetum grylli to be valid, while terzi (2015) later united them and retained the earlier valid name, bromo -chrysopogonetum grylli (cf. theurillat et al. 2021). in montenegro, bromo erecti-chrysopogonetum grylli was reported by blečić and lakušić (1976) in the coastal part of montenegro, as well as in the vicinity of podgorica, and by černjavski et al. (1949) and hadžiablahović (2018) in the area of skadar lake. these publications were not supported with phytosociological studies and it was not possible to make a comparison with our results from ćemovsko polje. according to černjavski et al. (1949), bromo-chrysopogonetum grylli is developed on stony hills with thin soils, while our researched stands are found in the lowlands of ćemovsko polje on deeper alluvial soils. according to černjavski et al. (1949) bromo-chrysopogonetum grylli is characterized by many species of shrub vegetation (paliurus spina-christi, salvia officinalis, rubus ulmifolius, euphorbia veneta, helichrysum italicum, ruscus aculeatus, cyclamen neapolitanum, arum italicum, phlomis fruticosa, cistus villosus, cistus salviaefolius, nephrodium filix-mas, pteridium aqiulinum, etc.) and chasmophytic vegetation (asplenium trichomanes, edraianthus tenuifolius, cardamine glauca, silene quadridentata, ceterach officinarum, sedum album, moltkea petraea, etc.) that were absent from our stands from ćemovsko polje. further investigation of the association bromo-chrysopogonetum grylli reported by černjavski (1949) is needed to determine whether there are differences in relation to our community from ćemovsko polje. fanelli et al. (2015) reported asphodelo-chrysopogonetum grylli in albania in the buna river protected landscape and it shows high similarity with associations from montenegro and croatia. the species cytisus spinescens, which is a charac teristic species of the association (horvatić 1963) is not present in stands from montenegro and albania. stands from montenegro are also characterized by an absence of species characteristic of dry grasslands of the classes festuco-brometea – salvia officinalis and bromopsis erecta. according to horvatić (1934, 1939), the development and distribution of bromo-chrysopogonetum grylli asphodeletosum microcarpi is conditioned by agro-pastoral activities, i.e., moderate grazing. in the case of ćemovsko polje, it is grazed particularly by sheep and rarely by cattle. intensive and permanent grazing leads to degradation of this community, which is especially evident from the absence of asphodelus ramosus from relevés on rab island (horvatić 1939). intensive grazing leads to a higher abundance of shrubs ( helichrysum italicum, cytisus spinescens, euphorbia spinosa, salvia officinalis) (horvatić 1934), or ruderal and subruderal species (fanelli et al. 2015). overgrazed stands can be included in the order carthametalia lanatae ( artemisietea vulgaris) because of abundant therophytes: carthamus lanatus, dasypyrum villosum, catapodium rigidum, nigella arvensis, etc. (fanelli et al. 2015). on the other hand, stands are abandoned, after which succession leads to rhamno-paliuretum trinajstić 1996, or are turned into mowed grasslands of the vulpio-lotion horvatić 1963 alliance (hadžiablahović 2018). in several localities in croatia along the adriatic coast, similar vegetation types with asphodelus ramosus have been reported. narcisso tazettae-asphodeletum microcarpi was described in istria (šegulja 1970). the same association was later reported on the islands of bobara and mrkan (hećimović 1984), supetar (jasprica and ruščić 2013) and olib (jasprica et al. 2016). originally, it was classified into scorzonerion villosae, but was later moved to chrysopogono grylli-koelerion splendentis. ecological conditions are different to those of asphodelo ramosi-chrysopogonetum grylli and it occurs on deep skeletoid soils and under the influence of salt spray (šegulja 1969). according to jasprica et al. (2016), the association is considered to be the most thermophilous grassland along the eastern adriatic coast. the characteristic species of the association are asphodelus ramosus, narcissus tezzeta and orchis papilionacea. thermophilous grassland communities with domination of stanišić-vujačić m., stešević d., hadžiablahović s., caković d., šilc u. 20 acta bot. croat. 81 (1), 2022 asphodelus ramosus have a distribution along the eastern adriatic, influenced by the mediterranean climate. there is a different situation along the western adriatic coast, where asphodelus ramosus-dominated communities are considered to be heliophilous fringe and tall-herb vegetation, which develops after the abandonment of agro-pastoral activities (tesei et al. 2020). comprehensive studies on heliophilous edge vegetation have been performed in italy (gargano peninsula, central part of the apennines, northern part of sardinia) and southern spain (biondi et al. 2016, 2017). as a result, a new class of edge vegetation charybdido pancratii-asphodeletea ramosi was described, focused on areas with a mediterranean macrobioclimate (biondi et al. 2016, 2017). the proposal to update the eurovegchecklist (mucina et al. 2014) by adding this new class was provisionally rejected by the european vegetation classification committee of the european vegetation survey working group of the international association of vegetation science, due to the lack of evidence concerning a clear floristic delimitation of charybdido pancratii-asphodeletea ramosi from lygeo sparti stipetea tenacissimae and trifolio-geranietea sanguinei ( biurrun and willner 2020). nonetheless, we can accept the existence of a new fringe class replacing trifolio-geranietea in the mediterranean, but we are of the opinion that bromo erecti-chrysopogonetum grylli clearly represents grassland vegetation erroneously classified as fringe vegetation by biondi et al. (2016). this is clearly indicated by the numerous species of festuco-brometea present in stands (tab. 2) from eastern adriatic asphodelus-dominated communities. fringe communities with asphodelus ramosus from italy, due to overgrazing, are dominated by monocotyledons, many of them toxic to animals, while in montenegro these areas represent pastures. another difference is the presence of shrub species with higher cover in stands of charybdidoasphodeletea ramosi (paliurus spina-christi, olea europaea, pinus halepensis), missing in grasslands from the eastern adriatic. according to biondi et al. (2016) the diagnostic species of the newly described class are asphodelus ramosus subsp. ramosus, a. fistlosus, a. tenuifolius, a. ayardii, charybdis pancration, c. maritima, c. glaucophylla, c. aphylla, c. hesperia, thapsia garganica, asparagus acutifolius, ornithogalum etruscum subsp. umbratille, anemone hortensis, carlina corymbosa, hypochoeris radicata, iris planifolia, i. bicapitata, asphodeline liburnica, a. lutea, ferula communis, f. communis subsp. cardonae, f. glauca, f. arrigonii and hermodactylis tuberosus. the diagnostic species group of charybdido-asphodeletea should also be revised, since many of them are attributed to other vegetation classes (festuco -brometea, lygeo sparti-stipetea tenacissimae, ononido rosmarinetea etc.) according to mucina et al. (2014). asphodelus ramosus, anemone hortensis, carlina corymbosa and hypochoeris radicata, which are considered to be character species by biondi et al. (2016), are also very frequent in grassland vegetation of bromo erecti-chrysopogonetum grylli. in terms of natura 2000 habitat types (council directive 92/43/eec on the conservation of natural habitats and of wild fauna and flora, 1992) there are differences between grassland and fringe communities dominated by asphodelus ramosus. heliophilous asphodelus spp. edge communities in the western and central mediterranean do not represent any habitats of european community interest and a progressive increase in asphodelus spp. cover in grasslands can result in the disappearance of grassland habitat types ( tesei et al. 2020). according to a report of habitat types of montenegro important for the european union (petrović et al. 2019), bromo erecti-chrysopogonetum grylli is classified within the eastern sub-mediterranean dry grasslands (scorzoneretalia villosae) habitat type (code 62a0). the same situation applies to narcisso tazettae-asphodeletum microcarpi in croatia (jasprica et al. 2016). syntaxonomical scheme based on the analyses performed, the following syntaxonomical scheme is proposed for asphodelus ramosus-dominated communities in the adriatic region. festuco-brometea br.-bl. et tx. ex soó 1947 scorzoneretalia villosae kovačević 1959 chrysopogono grylli-koelerion splendentis horvatić 1973 bromo erecti-chrysopogonetum grylli horvatić 1934 narcisso tazettae-asphodeletum microcarpi šegu lja 1969 artemisietea vulgaris lohmeyer et al. ex von rochow asphodelus ramosus community charybdido pancratii-asphodeletea ramosi biondi in biondi et al. 2016 asphodeletalia ramosi biondi in biondi et al. 2016 charybdido pancratii-asphodelion ramosi biondi et al. 2016 charybdido pancratii-asphodeletum ramosi biondi et al. 2016 alkanno tinctoriae-asphodeletum ramosi biondi et al. 2016 euphorbio characiae-thapsietum garganicae biondi et al. 2017 asphodelo ramosi-ferulion communis biondi et al. 2016 asphodelo ramosi-feruletum communis biondi et al. 2016 asphodelino luteae-feruletum communis biondi et al. 2016 acknowledgments we thank editor massimo terzi and two anonymous reviewers for their work that substantial improved our manuscript. martin cregeen kindly checked our english. the research was partly financed by the slovenian research agency (arrs) through a research program (p1-0236) and bilateral project with montenegro (bi-me/16-17-018). asphodelus ramosus dominated plant community acta bot. croat. 81 (1), 2022 21 references aćić, s., šilc, u., petrović, m., tomović, g., dajić stevanović z., 2015: classification, ecology and biodiversity of central balkan dry grasslands. tuexenia 35, 329–353. allegrezza, m., biondi, e., ballelli, s., tesei, g., ottaviani, c., 2015: the edge communities of asphodelus macrocarpus subsp. macrocarpus: the different ecological aspects and a new case study in the central apennines. plant sociology 52, 19–40. apostolova, i. dengler, j., di pietro r., gavilán, r.g., tsiripidis, i., 2014: dry grasslands of southern europe: 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labadessa, r., loos, j., 2018: the challenge of abandonment for the sustainable management of palaearctic natural and semi-natural grasslands. hacquetia 17, 5–16. appendix 1: species present in only 1 or 2 relevés from ćemovsko polje in table 1. asparagus acutifolius 17: +; catapodium rigidum 6: +; geranium molle 2: +; trifolium angustifolium 3: +; romulea bulbocodium 2: +; sonchus asper 10: +; pyrus amygdaliformis 3: 1; carthamus lanatus 6: +; salvia verbenaca 16: +; teucrium chamaedrys 17: +; geranium purpureum 7: +; alyssum minus 10: 1; ajuga chamaepitys 12: +, 16: +; vicia angustifolia 9: +; crepis foetida 2: +, 6: +; tragopogon porrifolius 9: +, 11: 1; bellis perennis 4: +; parentucellia latifolia 14: +, 15: +; aphanes arvensis 8: +, 17: +; valerianella rimosa 6: +, 8: +; knautia integrifolia 9: +, 10: +; stipa pulcherrima 3: +, 4: 1; cardamine hirsuta 4: +, 17: +; linum bienne 5: +, 7: +; trifolium cherleri 3: +, 5: +; bromus madritensis 7: +, 16: +; trifolium resupinatum 5: +; vulpia myuros 10: +; trigonella gladiata 1: +; veronica arvensis 17: +; reseda phyteuma 9: +; bromus sterilis 11: +; calepina irregularis 6: +; bromus hordeaceus 3: +; orchis ustulata 11: +; orlaya grandiflora 9: +; medicago rigidula 5: +; poa annua 10: +; hyacinthella dalmatica 5: +; matthiola incana 15: 1; lactuca viminea 3: +; aegilops neglecta 11: +; alyssum alyssoides 7: +; alyssum campestre 11: 1; astragalus illyricus 11: 2, 14: +; matricaria chamomilla 13: +, 14: +; onosma echioides 9: 1, 12: +. appendix 2: relevé dates (year/month/day) and coordinates (wgs84 reference system, in decimal degrees). 1. 2019/04/07, 42.3968090, 19.3021110; 2. 2019/04/07, 42.3964610, 19.3030050; 3. 2019/04/07, 42.3960000, 19.3040000; 4. 2019/04/07, 42.3978055, 19.3048888; 5. 2019/04/07, 42.3985277, 19.3019638; 6. 2019/04/07, 42.4001488, 19.3031944; 7. 2019/04/07, 42.4006410, 19.3023280; 8. 2019/04/07, 42.4021890, 19.3049660; 9. 2019/04/07, 42.3880040, 19.2856690; 10. 2019/04/07, 42.3803160, 19.2743840; 11. 2019/04/09, 42.3708888, 19.2303056; 12. 2019/04/09, 42.3728888, 19.2316944; 13. 2019/04/09, 42.3697222, 19.2339166; 14. 2019/04/09, 42.3705580, 19.2360490; 15. 2019/04/09, 42.3709350, 19.2383990; 16. 2019/04/10, 42.4021370, 19.3210555; 17. 2019/04/10, 42.3885277, 19.3110277. appendix 3: relevés from literature sources (name of the plant community, authors, table and number of relevés from original source paper). 1. bromo-chrysopogonetum grylli subass. asphodeletosum microcarpi (horvatić 1934), tab. 23, rels. 11-16, 18-23; 2. narcisso tazettae-asphodeletum microcarpi (šegulja 1970), tab. 2, rels. 1–8; 3. narcisso tazettae-asphodeletum microcarpi subass. sisymbrietosum officinalis (hećimović 1984), tab. 5, rels. 1–10; 4. narcisso tazettae-asphodeletum microcarpi (jasprica and ruščić 2013), rel. on the page 128; 5. asphodelus ramosus community (fanelli et al. 2015), tab. 41, rels. 15, 70, 492; 6. asphodelo-chrysopogonetum grylli (fanelli et al. 2015), tab. 25, rels. 31–33; 7. narcisso tazettaeasphodeletum microcarpi (jasprica et al. 2016), tab. 11, rels. 1–3; 8. charybdido pancratii-asphodeletum ramosi ( biondi et al. 2016, tab. 1, rels. 1–14; 9. alkanno tinctoriae asphodeletum ramosi (biondi et al. 2016), tab. 2, rels. 1–3; 10. asphodelo ramosi-feruletum communis (biondi et al. 2016), tab. 3, rels. 1–2; 11. asphodelino luteae-feruletum communis (biondi et al. 2016), tab. 4, rels. 1–7; 12. euphorbio characiae-thapsietum garganicae (biondi et al. 2017), tab. 6, rels. 1–5. acta bot. croat. 81 (1), 2022 s1 in memoriam in memoriam sister edita, marija šolić, phd (1946 – 2021) marija šolić was born on august 26, 1946, in brštanovo, klis municipality, now in split dalmatia county, croatia. her parents, petar and luca, had nine children. two of them did not survive the second world war when their parents lived in slavonia. after the war, the parents returned to brštanovo, where the youngest daughter, baptized marija, was born. marija completed primary school in her birthplace, and in 1963 joined the order of the school sisters of st. francis, in 1965 she took her vows as a nun, choosing religious name edita. she was to use this name, along with the baptized name of marija, until the end of her life. in 1966 sister edita was sent to the franciscan convent in makarska, where she was employed at the malacology museum. soon she became the associate of jure radić ofm, phd, the founder of the museum in makarska, working with him on the establishment of the mountain and the sea institute and forming scientific collections of marine mollusk shells (malacological collection), collections of fossil mollusks and other invertebrates, as well as live and herbaria plant collections. the arrival of sister edita in makarska was a watershed moment for her. there she became jure radić’s active associate, his diligent pupil, which opened up to her new horizons in the sphere of nature in general, especially of mt. biokovo with makarska at its foot, and the sea on the coast of which makarska lies. craving for knowledge and radić’s initiative led her to further education. the most obvious way was to take studies in biology. however, as sister edita did not have a certificate of secondary education, she needed to take the entrance exam for university. she passed this exam on 26 september 1972 at the department of biology, faculty of science, university of sarajevo. in the academic year 1972/73 she was enrolled as a part–time student in the year one of the biology department studies, in the group environment conservation. this also enabled her to become a permanent employee at the malacology museum in makarska as an assistant curator. as an employee of the museum, she was requested to come to sarajevo from time to time and attend lectures and practical lessons organized for part–time students. that was not an easy part of sister edita’s life, but with great efforts and her great wish for knowledge, she successfully graduated on 24 august 1980. she completed her diploma thesis “significant biokovo plants with special emphasis on their protection” under the supervision of professor radomir lakušić at the department of biology, and defended it with the best grade (10). she earned the title bsc in biology – environmental conservation. in the academic year, 1981/82 sister edita continued her education at the department of biology of the faculty of science in sarajevo, within the two years of graduate studies in ecology. her graduate studies were approved by the postgraduate council, based on previously completed pre– graduate studies and published works. she completed all the exams of the postgraduate curriculum with an average grade of 9.2. her master’s thesis was titled “chorologic-ecological and phenologic-morphological differentiation of biokovo’s endemic centaureas from subgenus acrolophus (cass.) dobr.-kov.”. the thesis was written under the mentorship of radomir lakušić. having successfully defended the master’s thesis on 30 may 1986, she was awarded the degree of msc in biology – field of ecology. while still pursuing her education, marija edita šolić worked with jure radić in the malacology museum and the mountain and the sea institute. she helped to take care of the existing, and to create new mollusk collections, as the museum received more and more new material through exfig. 1. marija edita šolić (1946 – 2021). in memoriam s2 acta bot. croat. 81 (1), 2022 change and donations. that helped her get knowledge in malacology and museology, so she advanced to the position of museum curator. she was constantly engaged in the presentation of the already renowned museum displays to numerous visitors, domestic and foreign, as she was fluent in scientific english, italian, and german. a great number of pupils and students visited the museum as a place of knowledge and a source of education, and sister edita played a special role during those visits. she had to the gift of conveying knowledge and made great use of it with these groups of visitors. in the institute she worked on the acquisition and processing of literature, exchange of publications, on keeping extensive correspondence with associates of the institute, and also worked with the herbarium. along with work in the institute, sister edita did extensive field research on mt. biokovo. on these occasions she would replace her habit with a mountaineering outfit and gear, always wearing a big backpack in which she would bring live plant material for the experiments in the institute’s small botanic garden. she would also bring carefully collected specimens for the institute’s herbarium as well as for her own research. her backpack would often contain material collected for the scientific research of her colleagues from universities and institutes in croatia and neighboring countries, and further afield. she processed collected plant material, classified it according to scientific criteria, and stored it in the herbarium. she became the curator of the herbarium, which was established in 1963, and which after several years was recognized and registered as the herbarium of the biokovo area (index herbarium id: 124939, makar). the herbarium contains carefully prepared vascular plants and algae, ca. 25,000 exsiccations. plant material in the herbarium is mostly from mt. biokovo, but there are also specimens from all over dalmatia. as sister edita gained a deeper and deeper knowledge of nature in general, especially of the plant kingdom, which was of her utmost interest, education at a higher level and the attainment of a doctorate became her next goal. she acquired her doctoral degree in natural sciences – field of biology at the faculty of science of the university of zagreb on july 14, 1993. the title of her doctoral thesis was “floristic-ecological features of the coastal slopes of mt biokovo “. in the thesis committee were ernest mayer (slovenian academy of science and arts, ljubljana), and ljudevit ilijanić and milan meštrov (faculty of science, university of zagreb). with the rescript issued by the ministry of science, technology and informatics of the republic of croatia of 17 july 1991 sister edita was entered in the register of researchers as an associate researcher in a scientific field of biology. she attained the rank of scientific assistant in the field of biology on 17 december 1992, and upon the decision of the ministry of science of the republic of croatia was entered in the register of researchers on 11 february 1993. in parallel to her progress in scientific research work in the field of biology, she also advanced in the field of museology. she began her museology career as an assistant curator, continued as curator (1993), to become a senior curator in 1999. marija edita šolić continuously worked on obtaining new knowledge and skills, occasionally with academician ernest mayer (slovenia), at the faculty of science in sarajevo (bosnia and herzegovina), at the faculty of science and the croatian natural history museum in zagreb, and with čedomil šilić, phd, at the national museum of bosnia and herzegovina in sarajevo. doctor šolić also consulted numerous scientists from various scientific institutions home and abroad. there is a long list of plant genera in which she did researche on one or more species. here are some of them: abies, allium, centaurea, edraianthus, echinops, fagus, iris, juniperus, lilium, narcissus, olea, salvia, satureja, stachys, sternbergia, teucrium, thymus, etc. marija edita šolić had various fields of interest in her scientific work in biology: morphology, systematics, chorology, autecology, synecology, and others, connected with the kingdom of plants in the first place. interesting enough, and not frequent among botanists, is the fact that she also studied and was highly knowledgeable about malacofauna of marine mollusks, especially of the adriatic sea. thus she was fully competent to work on the conservation of marine and terrestrial ecosystems. the scientific problems dr šolić was working on were numerous and various. a great number of her papers were about mt. biokovo floristic endemism, and about the vegetation of this mountain. she studied the chorology of numerous species, their morphological and physiological characteristics, systematic-taxonomical problems, as well as the possibilities for and necessities of their conservation. she joined the team of professor sonja šiljak-yakovlev (faculté des sciences d’orsay, universié paris-saclay, france), and professor milka maksimović (department of chemistry, faculty of science, sarajevo), which allowed her to gain new and modern knowledge in the fields of cytogenetic, evolutionary systematics, and biochemical characteristics of plants, namely the microscopic level of species she already knew very well on a macroscopic level. together they went very often to the field to collect material from biokovo and wider area for further laboratory research. marija šolić materialised her rich knowledge in scientific and professional papers. she published alone or in co– authorship in different domestic and foreign periodicals, as well as in presentations and reports at symposia, congresses, and other conferences. it could be generally concluded that her scientific and professional opus was comprehensive. over the period from 1978, when as a student she published her first paper, to 2021 when she died, she published dozens of papers. as m. e. šolić possessed very good organizational skills, she was trusted with membership in the organizational committees of the following scientific conferences: the second (1983), third (1987), and fourth (1992) scientific conference on the nature of biokovo held in makarska; congress natural history researches of the biokovo area, held in memoriam acta bot. croat. 81 (1), 2022 s3 in makarska in 1993 (where she also was the congress secretary); 14th international symposium on biospeleology (makarska, 1999). most of the proceedings from the mentioned conferences were published in the journal acta biocovica, whose editor-in-chief was jure radić, with sister edita šolić being his first assistant. she was a member of the editorial board over the period 1983-2001. after radić’s death in 1990 nikola tvrtković, phd, became editor-in-chief. from 1997 to 2001 edita šolić was the editor of the journal. in february 2002 she signed an employment contract with the institute of oceanography and fishery in split, croatia, where she was employed as associate for maintaining and upgrading floristic and faunistic collections. the contract was mutually terminated on 31 december 2010, when she retired. she also was active in the popularization of biological science and ecology. she was invited to give lectures to audiences of different ages and educational levels, to children in kindergarten, pupils in primary and secondary schools, university students, and to all those who had an interest in nature and its conservation. the lectures showed her great knowledge of the biokovo nature, with a sound knowledge of ecology supporting it. she always drew on her great knowledge and acted as activist for nature conservation using scientific arguments. frequently, she was invited as a guest in radio and television programs, when the topic of discussion was nature conservation for the benefit of future generations. thus she strongly and recognizably acted in the local community and further afield. her life was one of sustained care for a healthy environment, for natural resources, both marine and terrestrial, for cohabitation with nature. she was an uncompromising supporter of the nature conservation of mt. biokovo (one of the centers of endemism), and makarska with the surroundings as the nucleus of natural and social resources. in those efforts, she did not always meet with the understanding and support of some fellow citizens from the local authorities. lack of understanding was often related to the forced development of tourism, frequently at the expense of natural resources. “marija sister edita šolić, phd, remained faithful to the profession and conclusions of the first scientific conference on the nature of biokovo area, which already then clearly formulated problems that makarska would face within the years to come. upon the proclamation of the nature park to continue with the preparation of the terrain for a higher level of conservation of mt. biokovo, the preservation of its flora garden in a natural ambiance, to conduct research into water and the sea bed, preserve forests, downsize to a minimum any polluting industrial development, with urgent solutions for wastewaters in the entire makarska area…” (in memoriam: dr. sc. marija s. edita šolić, dobitnica nagrade grada makarske, www.makarska.hr, 3.11.2021.). although marija edita šolić in her active struggle for leaving nature to the future generations in the best possible state often experienced lack of understanding on the part of and disappointments from the local community, she also received understanding and acknowledgments for that struggle. she received the silver plaque for the exceptional achievements and merits in the promotion of the croatian economy in 1992 (awarded by the croatian chamber of commerce – regional chamber split), award of the biokovo croatian mountaineering association in 1996, the annual award and recognition of the achievements in the fields of environment conservation and biological and environmental diversity (1997). at the formal assembly of the city council, on the occasion of the day of makarska in 1998, she was awarded the city prize for merits in scientific research into the flora and fauna of mt. biokovo, the sea, and the coast, and conservation and protection of nature. along with scientific, professional, and educational work in the field of nature, sister edita lived her religious life, for which she decided in the early days and which lasted for 57 years. even before becoming an educated biologist, sister edita was a nun and remained so until the end of her life. in makarska, she lived in the nunnery of the school sisters of st. francis at the franciscan convent. the closing of the nunnery in 1999 was a critical moment in her life, as continuation of her life in makarska was came into question, as did her work at the mountain and the sea institute, the herbarium, the malacology museum, the natural botanical garden in kotišina, the biokovo nature park. the only thing that remained was to address the provincial board in split and the general board in rome with a request that she be allowed to stay in makarska. the request was granted and she was permitted to live outside the convent community and stay in makarska, living and working at the mountain and the sea institute. the overview of sister marija edita šolić’s life and work tells undoubtedly that she was an exceptional person in mafig. 2. marija edita šolić during the fieldwork on mt komovi, montenegro, 26 may 2007 (photo: d. šoljan). in memoriam s4 acta bot. croat. 81 (1), 2022 ny ways. in the first place, she was a nun, devoted to the faith in god she received in the religious catholic family, deciding in her early days on a religious life. yet, that calling did not prevent her from understanding and practising, within permitted and strict limits, the life of people who are not in a religious community. she knew how to balance between those two ways of living, being freely active on both tracks, in their fullness and clarity. she helped her friends, acquaintances, and all those who needed help in spiritual and material spheres as much as she could. i had the honor of being her friend for over 50 years. our friendship started the day when we first met in sarajevo, on the occasion of her preparations for the entrance exam at the biology department of the faculty of science. those were precious moments of her preparing for the exams and happiness for its successful pass and her eligibility for studying biology. our cooperation and friendship continued during her further education until she earned the master’s degree. the war period in the 1990s made the work on her doctoral thesis in sarajevo impossible. many events in human lives cannot be foreseen. so, it was unforeseen that mentor of my doctoral thesis, professor radomir lakušić, would give me, as the subject of a thesis, research in the endemic taxa of the genus edraianthus from mt biokovo. my task, among other things, was to conduct extensive field research in a mountain i was not at all familiar with. i needed help, and i unconditionally received it from my longtime friend, sister edita. every time i went to the field over the years, in different seasons, i went with her. often with us was jure radić, who had an excellent knowledge of the mountain, from the foot to the peak, from the littoral to the inland side. he also was an excellent connoisseur of the biokovo flora and the sites where i could collect interesting and necessary floristic material, as well as information on habitats. our fieldwork frequently started before sunrise, ending after sundown, at twilight. i felt very thankful to both of them for the engaged effort, energy, time, but also for the opportunity to become familiar with them in the moments when they, with prayers and liturgical chants, celebrated god and his creation seen in the beauty of the sky, rocks, sea, watched from the heights of the mountain, of flora and fauna. these are unforgettable events in my life. sister edita did not accept boundaries among people, she connected with people of different educations, religious and ideological beliefs, not caring about their origins or possessions. most important for her was their wish and intention to become acquainted with nature and protect it, and she wanted to help them with that. thus she made a wide range of acquaintances and a great number of friends. she met all of them with a wide smile and open arms. because of all this, a great emptiness will remain after her departure, on october 31 of this year, and the sincere if sad memories of all those who knew and were close to her. professor emeritus dubravka šoljan university of sarajevo, faculty of science, sarajevo, bosnia and herzegovina selected bibliography book chapters šolić, m.e., 1983: poznavanje flore biokova od visianija do danas. in: pavletić, z., matković, p., grubšić, s. (eds.), zbornik roberta visianija šibenčanina, 349–364. muzej grada šibenika, šibenik. šolić, m.e., 1999: prirodna baština nezaobilazani temelj odgoja i obrazovanja. in: macan, t. (ed.), hrvatska i održivi razvitak. humane i odgojne vrednote. ministarstvo razvitka i obnove, zagreb. pustahija, f., šolić, e.m., siljak-yakovlev, s., 2017: karyological study of some mediterranean species from bosnia and herzegovina, croatia and lebanon. in: kamari, g., blanché, c., siljak-yakovlev, s. (eds.), mediterranean chromosome data – 27. flora mediterranea 27, 295–301. original scientific papers šolić, m.e., 1981: rod edraianthus dc. na biokovu. acta biokovica l, 161–168. lakušić, r., kutleša, l., šolić, m.e., 1983: horološko-ekološka i morfološko-anatomska diferencijacija sekcije oxycedrus sp. roda juniperus l. na biokovu. acta biokovica 2, 309–316. šoljan, d., šolić, m.e., 1986/87: prilog poznavanju jele na biokovu. glasnik zemaljskog muzeja bosne i hercegovine prirodne nauke 25/26, 53–69. lakušić, r., kutleša, l., šoljan, d., šolić, m.e., 1987: prirodni sistem populacija i vrsta roda juniperus na biokovu. acta biokovica 4, 47–54. šolić, m.e., 1990: in memoriam dr. fra jure radić (1920-1990). acta botanica croatica 49, 161–166. puizina, j., šolić, m.e., papeš, d.,1994: studies of the karyotype and sexual mechanism of propagation in allium neapolitanum cyr. periodicum biologorum 96, 367–371. puizina, j., šolić, m.e., papeš, d., 1995: mediterranean chromosome number reports 524-527. flora mediterranea 5, 337– 340. besendorfer, v., samardžija, m., bušic, m., šolić, m.e., papeš, d., 1997: distribution of heterochromatin and location of nucleolar organizing region in allium commutatum guss. periodicum biologorum 99, 415–421. puizina, j., jelisavac, m., šolić, m.e., papeš, d., 1997: b-chromosomes in the population of allium pallens ssp. tenuiflorum from the region of makarska. periodicum biologorum 99, 129–134. hazler-pilepic, k., comps, b., sugar, i., solic, m.e., 1999: isozyme polymorphism of croatian beech populations (fagus sylvatica l.). periodicum biologorum 101, 165–170. pevalek-kozlina, b., pavlica, m., šolić, m.e., 1999: shoot and root regeneration from callus tissue of allium commutatum guss. acta botanica croatica 58, 57–64. šilić, č., šolić, m.e., 1999: contribution to the knowledge of the neophytic flora in the biokovo area (dalmatia, croatia). natura croatica 8, 109–116. šilić, č., šolić, m.e.,1999: sternbergia colchiciflora waldst. & kit. var. dalmatica reichenb. on the biokovo massif (croatia). natura croatica 8, 155–160. šilić, č., šolić, m.e., 2001: new ferns (filicopsida) in the flora of the massif of the biokovo mt. (dalmatia, croatia). natura croatica 10, 97–101. besendorfer, v., šop, k., šolić, m.e., papeš, d., 2001: karyotypes of allium senescens l. ssp. montanum (fries) holub populations from the mt. biokovo region. acta botanica croatica 60, 177–186. in memoriam acta bot. croat. 81 (1), 2022 s5 besendorfer, v., samardžija, m., zoldoš, v., šolić, m.e., papeš, d., 2002: chromosomal organization of ribosomal genes and nor–associated heterochromatin, and nor activity in some populations of allium commutatum guss. (alliaceae). botanical journal of the linnean society 139, 99–108. šilić, č., šolić, m.e., 2002: addition to the vascular flora in the region of biokovo (dalmatia, croatia). natura croatica 11, 341–363. šilić, č., šolić, m.e., 2002: the taxonomy, chorology and ecology of stachys menthifolia vis. 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(caryophyllaceae) in croatia dario kremer1, iztok jože košir2, tanja potočnik2, nikolina rogulj1, karla načinović1, marko randić3, siniša srečec4, renata jurišić grubešić1* 1 university of zagreb, faculty of pharmacy and biochemistry, a. kovačića 1, hr-10000 zagreb, croatia 2 slovenian institute of hop research and brewing, cesta žalskega tabora 2, si-3310 žalec, slovenia 3 public institution “priroda”, grivica 4, hr-51000 rijeka, croatia 4 križevci college of agriculture, m. demerca 1, hr-48260 križevci, croatia abstract – as a contribution to chemotaxonomic relations, a quantitative analysis of bioactive phenolic compounds was carried out for the first time in drypis spinosa l. subsp. spinosa and d. spinosa subsp. jacquiniana murb. et wettst. ex murb. in croatia. total polyphenols (tp), tannins (t) and total flavonoids (tf) were determined in samples of leaves, stems, and roots using uv-vis spectrophotometric methods. for the subsp. spinosa, the highest content of tf was in leaves (0.09%), as was the highest amount of tp (2.36%) and t (1.12%). in the subsp. jacquiniana, the highest contents of tf (0.10%), tp (1.96%), and t (0.88%) were measured in stems. coumaric, ferulic and rosmarinic acid were identified and quantified by hplc analysis in both subspecies. quercetin and sinapic acid were identified only in subsp. spinosa, while rutin and naringenin were found only in subsp. jacquiniana. among them, ferulic acid was identified only in flowers of both subspecies. the results of this study represent a useful basis for further research of phytochemical and eventually phytotherapeutic potential of d. spinosa. keywords: drypis, eastern adriatic, flavonoids, polyphenols, subspecies, tannins introduction the balkan peninsula is known to be very rich in endemic plant species. this is the meeting point of two phytogeographic regions: the alpine – high nordic regions and the euro siberian – north american (redžić et al. 2011). the genus drypis l. includes one (wielgorskaya 1995, erhardt et al. 2014) or two (euro+med 2006–2019) species. drypis spinosa l. subsp. spinosa (syn. d. spinosa, d. spinosa subsp. linneana murb. et wettst.) is distributed in italy, slovenia, croatia, montenegro, albania, north macedonia and greece, while d. spinosa subsp. jacquiniana murb. (syn. d. jacquiniana murb. et wettst.) is known from italy, slovenia and croatia (šilić 1990, stešević and caković 2013, euro+med 2006–2019). subspecies spinosa is a xerothermic, lithophytic species that grows on open, stony or gravelly habitats between mountain and subalpine vegetation belts (domac 1964, šilić 1990, stešević and caković 2013). on the other hand, subsp. jacquiniana is distributed along the eastern adriatic coast from the close to sea level to 800 m a.s.l. and it grows predominantly on coastal rocks and screes (domac 1964, šilić 1990, jasprica 2015). drypis spinosa is a typical representative of endemic plants with horticultural and possible medicinal potential. investigation of biologically active compounds in d. spinosa is part of our continuous work on balkan plant species. among biologically active compounds, phenolic compounds have attracted a great deal of scientific and public interest due to their health-promoting effects as antimicrobials, antivirals and antioxidants. the content of biologically active compounds varies among species, including closely related species, making them useful chemotaxonomic markers. the plants of the caryophyllaceae family contain significant amounts of polyphenols, including flavonoids. ethnopharmacologica l studies indicate that caryophyllaceae possess anticancer, antibacterial, antifun* corresponding author e-mail: rjurisic@pharma.hr kremer d., košir i. j., potočnik t., rogulj n., načinović k., randić m., srečec s., jurišić grubešić r. 44 acta bot. croat. 80 (1), 2021 spectrophotometric determination of tp and t at 720 nm. tannic acid was used as the standard substance. the content of total flavonoids (tf; colorimetric assay with alcl3) was obtained using spectrophotometric method prevalidated by jurišić grubešić et al. (2007). this procedure encloses hydrolysis of glycosides, extraction (with ethyl acetate) of tf aglycones and complex formation with alcl3 at 425 nm. the yield of tf was calculated as quercetin according to the following expression: tf (%) = a × 0.772 / b in this expression, a denotes measured absorbance, 0.772 is the conversion factor related to the specific absorbance of quercetin at 425 nm, while b is mass (g) of air-dried herbal material. tp, t and tf contents were performed in triplicate (technical replicates) and the results were expressed as mean ± sd. hplc analysis the ultrasonic extraction was performed on finely powdered air-dried herbal material (500 mg) with 20 ml of methanol at 25 °c for 60 min, then 20 ml of ethyl-acetate at 25 °c for 60 min, and finally with 20 ml of distilled water at 25 °c and 45 °c for 60 min. after that, samples were filtered (qualitative analytical filter paper) and diluted with the same solvent mixture to a volume of 25.0 ml. an aliquot of 5 μl of each sample solution was injected into the hplc system for chromatographic analysis. prior to injection samples were filtered through 0.45 μm ptfe 25 mm filter (restek co., usa). the stock solutions of standard compounds: chrysin, quercetin, quercitrin, rutin, chicoric acid, coumaric acid, ferulic acid, protocatechuic acid, rosmarinic acid, syringic acid and tannic acid were prepared according to čeh et al. (2007) and kremer et al. (2013). at first, the stock solutions of standards were diluted separately in the mixture of water and methanol (1:1, v/v) at a concentration of 1.0 mg ml–1. after that, the working standard solutions (at a concentration of 0.01 mg ml–1) were made by the dilution of each stock solution with the same mixture of water and methanol. the mixture of standards was made by the dilution of each stock standard solution with the same solvent mixture at final concentration of each standard of 0.01 mg ml–1. hplc analysis was performed according to čeh et al. (2007) and kremer et al. (2013). a c18 reversed-phase packing column was used for separation at the temperature of 30 ºc. a gradient elution type of chromatographic systems was used. the mobile phase for gradient elution was composed of phase a (water, ph = 2.50 adjusted with acetic acid), and phase b (acetonitrile), which was applied according to the following elution scheme: starting with 15% b and 85% a, followed with gradient from 15% b to 22.5% b in 15 min, then from 22.5% b to 40% b in 10 min and kept constant with 40% b and 60% a for another 5 min. in the addition 5 min, the initial conditions were set up. the injecgal, antiviral, antioxidant, and anti-inflammatory properties (chandra and rawat 2015). this is the reason why the investigations of phenolic compounds were conducted with various species from the caryophyllaceae family. the aim of this study is to gain an insight into the content of phenolic compounds in d. spinosa subsp. spinosa and d. spinosa subsp. jacquiniana growing in croatia. to the best of our knowledge this is the first report on the chemical properties of d. spinosa. materials and methods sampling, chemicals and apparatus the samples of the investigated subspecies were collected during the blooming period in july 2017. plant material of drypis spinosa subsp. spinosa was collected on mt velebit at 1200 m a.s.l., while the samples of d. spinosa subsp. jacquiniana were collected in the town of rijeka at 70 m a.s.l. above-ground parts of several randomly selected plants were harvested on a dry day and mixed to obtain a randomly selected sample. the stems, leaves and flowers were collected separately. herbal material was air-dried in a wellventilated room at 60% relative humidity and room temperature (22 ºc) for three weeks. the plant material was protected from direct sunlight and single-layered during drying. after drying, the plant samples were placed into double paper bags and stored in a dry place at room temperature (22 ºc, 60% of humidity), protected from the light until analysis. voucher specimens of herbal material were deposited in the fran kušan herbarium of the faculty of pharmacy and biochemistry, university of zagreb, croatia. folin-ciocalteu reagent (fcr), chrysin, quercetin, quercitrin, rutin, chicoric acid, coumaric acid, ferulic acid, protocatechuic acid, rosmarinic acid, syringic acid, tannic acid, linoleic acid, and gallic acid (sigma-aldrich chemical co., usa); methanol, ethanol, and hno3 (kemika, croatia) were used. all chemicals and reagents were of analytical grade. double distilled water was used throughout the study. agilent 8453 e uv/vis spectrophotometer (hewlett packard, germany) equipped with the pc-hp 845x uvvisible system (hewlett packard, germany) and 1 cm quartz cells were used for absorbance measurements. hplc analysis was performed using agilent 1100 series hplc system and a c18 reversed-phase packing column (zorbax eclipse xdb-c18, 150 mm × 4.6 mm, 5 μm; agilent, usa). phenolic compounds analyses the quantitative analysis of biologically active phenolic compounds was carried out using spectrophotometric methods, following adequate extractions. spectrophotometric determination of total polyphenols (tp) and tannins (t) was performed with the use of fcr. comprehensive prevalidation of this method was carried out by jurišić grubešić et al. (2005). this method is based on a reaction with fcr and, after precipitation with casein, phenolic compounds in drypis spinosa acta bot. croat. 80 (1), 2021 45 tion volume was 5 μl, while flow rate was 1.0 ml min–1. detection was performed by diode array detector at wavelengths 280 nm (chrysin, chicoric acid, syringic acid, protocatechuic acid, tannic acid), 320 nm (ferulic acid, rosmarinic acid, coumaric acid) and 370 nm (quercetin, quercitrin and rutin). particular compounds were identified by comparing the retention times of standards and unknown peaks in the samples. the method of standard addition was applied in order to avoid misinterpretation of results. quantification was conducted using the external standards. method was previously validated to confirm the linear range, reproducibility (rsd, between 3.8% and 8.6%) and accuracy (r, 76% to 87%) for particular compounds (detailed data not shown here). limit of quantification (loq) was determined at the concentration of 0.001%. data analysis significance of differences between analytical data was checked by the least significant difference (lsd) test, using the statistica software (hill and lewicki 2006). results total polyphenols (tp), tannins (t) and flavonoids (tf) content uv-vis spectrophotometric methods were used for a quantitative analysis of tp, t and tf in d. spinosa subsp. spinosa and d. spinosa subsp. jacquiniana. the results of the determinations of tp, t and tf are presented in tab. 1. for d. spinosa subsp. spinosa, the highest content of tp (2.36%) was in leaves, as was the highest amount of t (1.12%) and tf (0.09%). in d. spinosa subsp. jacquiniana, the highest contents of tp (1.96%), t (0.88%) and tf (0.10%) were measured in stems. it is evident that there was no dependence among plant parts and the quantity of investigated phenolic substances. the largest statistically significant differences in the content of all analyzed polyphenolic substances were observed for leaves of subsp. spinosa and subsp. jacquiniana, while the least significant difference in the content of polyphenols was noticed among flowers of the investigated plant subspecies (tab. 1). hplc analysis seven phenolic compounds were identified and quantified by hplc analysis in d. spinosa subsp. spinosa and d. spinosa subsp. jacquiniana and their concentrations in leaves, flowers and stems are shown in tab. 2. among standard compounds only caffeic acid was not identified in the investigated subspecies. content of phenolic compounds varied between 0.002% (coumaric acid) and 0.839% (naringenin). among them, quercetin and sinapic acid were identified only in subsp. spinosa, while rutin and naringenin were identified only in subsp. jacquiniana. on the other hand, coumaric acid, ferulic acid and rosmarinic acid were found in both subspecies. ferulic acid was identified only in flowers of both subspecies. discussion quantitative analysis of tp, t and tf was performed with the leaves, flowers and stems of drypis spinosa subsp. spinosa and d. spinosa subsp. jacquiniana. obtained results showed that there was no dependence among plant parts and the quantity of the phenolic substances investigated. tab. 1. contents (mean ± sd) of total polyphenols (tp), tannins (t) and flavonoids (tf) in drypis spinosa subsp. spinosa and d. spinosa subsp. jacquiniana obtained by uv-vis spectrophotometric methods. the asterisk (*) in superscript indicates a significant difference obtained by lsd test for content of tp, t and tf between different plant parts (leaf, flower, stem) of subsp. spinosa and subsp. jacquiniana at p < 0.05. n = 3. phenolic compund (%) leaf flower stem subsp. spinosa subsp. jacquiniana subsp. spinosa subsp. jacquiniana subsp. spinosa subsp. jacquiniana tp 2.357 ± 0.094* 1.773 ± 0.032 1.810 ± 0.035 1.808 ± 0.016 1.698 ± 0.137 1.955 ± 0.040 t 1.119 ± 0.081* 0.350 ± 0.113 0.557 ± 0.021 0.468 ± 0.113 0.503 ± 0.144* 0.895 ± 0.050 tf 0.092 ± 0.006* 0.057 ± 0.003 0.073 ± 0.002* 0.056 ± 0.002 0.063 ± 0.005* 0.097 ± 0.006 tab. 2. contents of phenolic compounds (%) in dried plant material of drypis spinosa subsp. spinosa and d. spinosa subsp. jacquiniana obtained by hplc analysis. – : not detected, n = 1. phenolic subsp. spinosa subsp. jacquiniana compound (%) leaf flower stem leaf flower stem rutin – – – – – 0.047 quercetin – – 0.008 – – – naringenin – – – – 0.839 – sinapic acid 0.017 – 0.004 – – – coumaric acid – – 0.004 0.002 0.004 – ferulic acid – 0.060 – – 0.066 – rosmarinic acid 0.041 0.043 0.041 – 0.030 – kremer d., košir i. j., potočnik t., rogulj n., načinović k., randić m., srečec s., jurišić grubešić r. 46 acta bot. croat. 80 (1), 2021 this is in accordance with some other investigations. for example, satureja montana l. and s. subspicata vis. (lamiaceae) had the highest tp content in leaves (dunkić et al. 2012), while moltkia petraea (tratt.) griseb. (boraginaceae) had the highest tp content in flowers (vuković rodríguez et al. 2016). additionally, investigations of tp content in ten moltkia petraea populations obtained by kremer et al. (2016) showed that tp content was the highest in leaves in six populations. but in another four populations the tp content was the highest in flowers. both subspecies of d. spinosa contain relatively small amounts of tp, which ranged from 1.70% in the stems to 2.36% in the leaves of d. spinosa subsp. spinosa. for comparison, tp content ranged from 4.43% in stem to 12.41% in leaves of satureja montana, and from 5.96% in stem to 20.04% in leaves of s. subspicata (dunkić et al. 2012). additionally, tp content in moltkia petraea ranged from 3.26% in stem to 6.61% in leaves (kremer et al. 2016). a similar independence among plant parts and the quantity of investigated phenolic substances was obtained for t content in drypis spinosa. the highest t content was obtained for the leaves and flowers of d. spinosa subsp. spinosa and d. spinosa subsp. jacquiniana, respectively. according to dunkić et al. (2012), leaves of satureja montana and s. subspicata generally contained higher concentrations of t than stems and flowers. content of t varied between 0.08% in stem and 4.90% in leaves of s. montana, and between 0.21% in stem and 6.95% in leaves of s. subspicata. the lowest t content (0.76%) was also measured in stems and the highest (7.33%) in leaves of teucrium arduinii l. (kremer et al. 2013). on the other hand, the highest t content was determined in the flowers in most of the ten investigated populations of moltkia petraea (kremer et al. 2016). the content of catechins in stellaria media (l.) vill. (caryophyllaceae), a herb obtained from five ukrainian populations ranged from 0.67% to 0.90% (vodoslavskyi 2017). the content of tf was the lowest among three investigated groups of phenolic compounds. dunkić et al. (2012) found that the lowest tf content was in the stem of satureja montana (0.06%), and the highest in leaves (0.40%). that investigation also showed that samples of leaves and flowers of s. montana have a comparable quantity of tf. the lowest tf content was measured in s. subspicata stem (0.07%), and the highest in the leaves (0.57%). additionally, the highest tf content was identified in leaves in all moltkia petraea populations (kremer et al. 2016). although some exceptions exist, the literature data show that stems contain the lowest tf content. the content of tf was also analyzed in five ukrainian populations of stellaria media herb, ranging from 1.22% to 1.40% (vodoslavskyi 2017). the most intensive investigations of the caryophyllaceae family have been carried out on species with medical properties. these investigations showed that caryophyllaceae contain a great number of bioactive compounds belonging to several chemical groups. seven phenolic compounds were identified in d. spinosa subsp. spinosa and d. spinosa subsp. jacquiniana using hplc analysis. the same phenolic compounds were also identified in some other caryophyllaceae species. quercetin was identified in silene littorea brot (richardson 1978) and arenaria serpyllifolia l. (zhou and tu 2013), while naringenin was detected in dianthus caryophyllus l. (spribille and forkmann 1982). teğin at al. (2018) identified naringenin and rutin in spergularia rubra (l.) j. presl et c. presl. rutin, ferulic acid, caffeic acid and p-coumaric acid were detected in lychnis floscuculi l. (ferry and darbour 1979, costea et al. 2017). ferulic acid was also identified in stellaria media (kitanov 1992), while p-coumaric acid was detected in gypsophila paniculata l. (chou et al. 2008). conclusion the content of different phenolic compounds was determined in leaves, flowers and stems of drypis spinosa subsp. spinosa and d. spinosa subsp. jacquiniana growing in croatia. there was no dependence among plant parts and the quantity of investigated phenolic substances. seven phenolic compounds were identified and quantified by hplc analysis and their content ranged between 0.002% (coumaric acid) and 0.839% (naringenin). quercetin and sinapic acid were identified only in subsp. spinosa, while rutin and naringenin were detected only in subsp. jacquiniana. further investigations will show if these phenolic compounds really are specific for one or another subspecies and if it is possible to use them as valuable chemotaxonomic markers. acknowledgements this work was supported by the ministry of science, education and sports of the republic of croatia (project no. 177–1191192–0830). authors would like to thank to valentina papić bogadi for helpful comments on the manuscript and for correcting the english style. references chandra, s., rawat, d.s., 2015: medicinal plants of the family caryophyllaceae: a review of ethno-medicinal uses and pharmacological properties. integrative medicine research 4, 123–131. chou, s.c., everngam, m.c., beck, j.j., 2008: allelochemical phenolic acids from gypsophila paniculata. journal of undergraduate chemistry research 7, 40–42. costea, t., nencu, i., gîrd, c.e., popescu, m.l., 2017: ragged robin (lychnis flos-cuculi l.) aerial parts – botanical characterization, phytochemical screening and antioxidant activity. studia universitatis vasile goldiş arad, seria ştiinţele vieţii 27, 231–238. čeh, b., kač, m., košir, i.j., abram, v., 2007: relationships between xanthohumol and polyphenol content in hop leaves and hop cones with regard to water supply and cultivar. international journal of molecular science 8, 989–1000. domac, r., 1964: drypis l. in: tutin, t.g., heywood, v.h., burges, n.a., valentine, d.h., walters, s.m., webb, d.a. (eds.), flora europaea, vol. 1, lycopodiaceae to platanaceae, 181. cambridge university press, cambridge. phenolic compounds in drypis spinosa acta bot. croat. 80 (1), 2021 47 dunkić, v., kremer, d., dragojević müler, i., stabentheiner, e., kuzmić, s., jurišić grubešić, r., vujić, l., kosalec, i., randić, m., srečec, s., bezić, n., 2012: chemotaxonomic and micromorphological traits of satureja montana l. and s. subspicata vis. (lamiaceae). chemistry & biodiversity 9, 2825– 2842. erhardt, w., götz, e., bödeker, n., seybold, s., 2014: zander – handwörterbuch der pflanzennamen. 19. aufl., eugen ulmer gmbh & co., stuttgart. euro+med, 2006–2019: euro+med plantbase – the information resource for euro-mediterranean plant diversity. retrieved november 15, 2019 from http://w w2.bgbm.org/europlusmed/ ferry, s., darbour, n., 1979: phenolic acids of three species of lychnis: lychnis alba mill., lychnis flos-cuculi l., lychnis githago scop. plantes medicinales et phytotherapie 13, 192– 198. hill, t., lewicki, p., 2006: statistics methods and applications. statsoft, inc., tulsa. jasprica, n., 2015: drypis spinosa l. ssp. jacquiniana murb. et wettst. in: nikolić, t., milović, m., bogdanović, s., jasprica, n. (eds.), endemic species of the croatian flora, 235–237. alfa d.d., zagreb (in croatian). jurišić grubešić, r., vuković, j., kremer, d., vladimir-knežević, s., 2005: spectrophotometric method for polyphenols analysis: prevalidation and application on plantago l. species. journal of pharmaceutical and biomedical analysis 39, 837– 842. jurišić grubešić, r., vuković, j., kremer, d., vladimir-knežević, s., 2007: flavonoid content assay: prevalidation and application on plantago l. species. acta chimica slovenica 54, 397– 406. kitanov, g., 1992: phenolic acids and flavonoids from stellaria media (l.) vill. (caryophyllaceae). pharmazie 47, 470–471. kremer, d., košir, i.j., kosalec, i., zovko končić, m., potočnik, t., čerenak, a., bezić, n., srečec, s., dunkić, v., 2013: investigation of chemical compounds, antioxidant and antimicrobial properties of teucrium arduini l. (lamiaceae). current drug targets 14, 1006–1014. kremer, d., jurišić grubešić, r., ballian, d., stešević, d., kosalec, i., vuković rodríguez, j., vukobratović, m., srečec, s., 2016: influence of soil traits on polyphenols level in moltkia petraea (tratt.) griseb. (boraginaceae). acta botanica croatica 75, 266–271. redžić, s., barudanović, s., trakić, s., kulijer, d., 2011: vascular plant biodiversity richness and endemo-relictness of the karst mountains prenj, čvrsnica and čabulja in bosnia and herzegovina (w. balkan). acta carsologica 40, 527–555. richardson, m., 1978: flavonols and c-glycosylflavonoids of the caryophyllales. biochemical systematics and ecology 6, 283–286. spribille, r., forkmann, g., 1982: chalcone synthesis and hydroxylation of flavonoids in 3’-position with enzyme preparations from flowers of dianthus caryophyllus l. (carnation). planta 155, 176–182. stešević, d., caković, d., 2013: catalogue of vascular plants of montenegro. montenegrin academy of sciences and art, vol. 1, 161–162 (in montenegrin). šilić, č., 1990: endemic plants. sp svjetlost, sarajevo, beograd (in bosnian). teğin, i̇., canpolat, g., fidan, m., 2018: the antioxidant capacity, total phenolic content and phenolic compounds of spergularia rubra (l.) j. presl & c. presl in naturally distributed five different saline areas in siirt province. in: polat, r., gören, k. (eds.), proceedings of the second international symposium on multidisciplinary studies and innovative technologies (ismsit), 450–454. institute of electrical and electronics engineers, ankara. vodoslavskyi, v.m., 2017: the quantitative content of the phenolic compounds in the stellaria media herb. the pharma innovation journal 6, 174–175. vuković rodríguez, j., jurišić grubešić, r., kremer, d., kokot, v., 2016: quality assessment of two spectrophotometric procedures for polyphenol determination and application in moltkia petraea (tratt.) griseb. journal of the chinese chemical society 63, 677–687. wielgorskaya, t., 1995: dictionary of generic names of seed plants. columbia university press, new york. zhou, g., tu, p., 2013: flavanoids and xanthones isolated from arenaria serpyllifolia l. journal of chinese pharmaceutical sciences 22, 192–198. opce-str.vp acta bot. croat. 70 (1), 109–114, 2011 coden: abcra 25 issn 0365–0588 achene slime content in some taxa of matricaria l. (asteraceae) huseyin inceer* karadeniz technical university, faculty of science, department of biology, 61080 trabzon, turkey abstract – the achenes of matricaria aurea and two varieties of m. chamomilla (var. chamomilla and var. recutita) have slime cells on the surface and they are characterized by slime envelope formation during hydration. the slime in these taxa is composed of pectins and cellulose. the slime could play important role in the distribution and colonisation of new habitats in matricaria taxa. key words: achene, slime, cellulose, pectin, matricaria introduction matricaria l. is a small genus of the tribe anthemideae with 6 species and mostly distributed in europe, northern africa, macaronesia, western, south-western and central asia, western north america (oberprieler et al. 2007). the wide range of geographical distributions and the diversity of habitats such as disturbed meadows, vacant lots, areas along roads and railroads, waste and dry areas in which matricaria occurs may result in different adaptations to diverse environments. the taxonomy of matricaria is controversial and very confused. depending on the authors, several species are classified in either matricaria or tripleurospermum. therefore, they have been confused with each other, both taxonomically and nomenclaturally (jeffrey 1979, xifreda 1985, kerguélen et al. 1987, pobedimova 1995, applequist 2002). achene morphology has been demonstrated to be paramount importance for the taxonomy of these genera (kyno^lova 1970). matricaria is characterized by achenes that are obovoid-oblong, circular to sligthly dorsiventrally flattened in the cross-section, with 3–5 adaxially arranged thin ribs that are sometimes furnished with longitudinal resin canals and are covered with myxogenic (slime, mucilage producing) cells mainly on their abaxial suracta bot. croat. 70 (1), 2011 109 * corresponding address, e-mail: inceer@ktu.edu.tr copyright ® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\inceer.vp 28. o ujak 2011 16:41:08 color profile: disabled composite 150 lpi at 45 degrees face and on the adaxial ribs (oberprieler et al. 2007). however, the slime content of the achenes in the genus has not been studied so far. the aim of this study has been to examine the slime characteristics of the achenes. materials and methods plant material the mature achenes of matricaria aurea, m. chamomilla var. chamomilla and m. chamomilla var. recutita were collected from a native environment (tab. 1). plant vouchers have been deposited in the herbarium of karedeniz technical university biology (ktub) or h. inceer collections. slime identification by chemical reactions the behaviour of the fruit after wetting was observed and tests on the ability of its coat to hydrate were performed. wetting experiments, with tap water, were performed at room temperature for 1–5 min, which was sufficient for the achenes to hydrate (kreitschitz et al. 2009). methylene blue and safranine staining were carried out to identify the slime type. the images were taken using leica dm 4000 microscope and a leica dfc 490 digital camera. results the wetting experiments show that slime cells on the surface of the pericarp produce mucilage after which the achenes are surrounded by a slime envelope. the slime is a distinct gel-like envelope. it belongs to a cellulosic type representing a heterogenous system. staining with methylene blue and safranine dyes demonstrate that the matricaria slime consists of a pectinous matrix and a cellulosic skeleton (fig. 1). methylene blue and safranine staining showed a characteristic pattern. methylene blue revealed a very faint blue envelope around the achene while an orange-red coloration of the slime was obtained with safranine (fig. 1). staining produced almost the same colour in both pectin and cellulose. cellulosic threads or fibrils forming a characteristic radical skeleton around the achene were clearly visible, whereas the pectin color was spread homogenously within the envelope. 110 acta bot. croat. 70 (1), 2011 inceer h. tab. 1. the origin of the materials studied taxon locality voucher matricaria aurea (loefl.) sch. bip. c6 gaziantep/sanlýurfa: between nizip and birecik, dutlu, roadsides, near cultivated area, 440 m a.s.l., 08.v. 2007. inceer 322 m. chamomilla l.var. chamomilla c1 muðla: marmaris, between marmaris and köyceðiz, roadsides, 20 m a.s.l., 18. iv. 2007. inceer 305 m. chamomilla l. var. recutita (l.) fiori c1 muðla: marmaris, between kizilkaya and fethiye, roadsides, 24 m a.s.l., 18. iv. 2007. inceer 307 u:\acta botanica\acta-botan 1-11\inceer.vp 28. o ujak 2011 16:41:09 color profile: disabled composite 150 lpi at 45 degrees discussion the results obtained from micro-staining reactions demonstrated that the matricaria slime is of the cellulosic type, consisting of two components i.e., pectin and cellulose. this is the first report on the slime structure of matricaria. the staining results correspond to already published data in artemisia and eragrostis (broda 1971, gerlach 1972, braune et al. 1975, o' brien and mccully 1981, kreitschitz and vallès 2007, kreitschitz et. al. 2009, tab. 2). in the literature, the presence of slime has been reported for many other angiosperm genera, e.g. in brassica, salvia, plantago, linum, anthemis, artemisia and matricaria (muhlethaler 1950, young and evans 1973, grubert 1974, grubert 1982, kreitschitz and vallès 2007). the cells producing slime could be arranged in isolated rows on the fruit surface, as in anthemis, artemisia and matricaria (grubert 1974). in addition, grubert (1982) emphasized that proper epidermal cells of the pericarp have produced the slime in m. chamomilla. the present results confirm that slime cells on the surface of the pericarp produce the slime. acta bot. croat. 70 (1), 2011 111 achene slime content in matricaria fig. 1. slime envelope in the achenes of matricaria. a-b – m. aurea (methylene blue). c – m. aurea (safranine). d-f – m. chamomilla var. chamomilla (disc, methylene blue). g – m. chamomilla var. chamomilla (disc, safranine). h – m. chamomilla var. chamomilla (ray, methylene blue). i-k – m. chamomilla var. recutita (methylene blue). l – m. chamomilla var. recutita (safranine). arrow with a line indicates slime cell, arrow with two lines indicates pappus, arrow with three lines indicates cellulose threads. u:\acta botanica\acta-botan 1-11\inceer.vp 28. o ujak 2011 16:41:12 color profile: disabled composite 150 lpi at 45 degrees the relative amount of slime is different in the studied taxa (fig. 1). differences in slime production can result from habitat diversity of the taxa. such a relationship was reported in lamiaceae (mosquero et al. 2004). the achene of m. aurea growing in particularly dry habitats has higher amounts of slime in the envelope than the other taxa. high slime production in the achene may be an advantageous adaptive feature faciliating germination. the functional significance of the slime in seeds and/or fruits has been reported in many works. it plays an important role in the control of germination, mostly in plants that grow in conditions of water deficiency in arid and semiarid environments, thus facilitating intake and maintenance of the water (kreitschitz and vallès 2007). furthermore, it can delay germination due to impeded penetration by oxygen. slime helps in fruit or seed dispersal and in the defence against pathogens (fahn and werker 1972, korobkov 1973, young and evans 1973, young and martens 1991, huang and gutterman 1999, huang et al. 2000). matricaria chamomilla var. chamomilla and var. recutita, which are the most widespread taxa of the genus, occur in diverse habitats such as dry and wet environments, roadsides, field margins and ruderal places as a weed. matricaria aurea also grows on limestone deposits, sand, saline land, flood-plain meadows with stony soil, sometimes as a weed. it is assumed that the presence of slime cells on the surface of the achenes could play an important role in the dispersal and competitive ability of these taxa. in some particular cases, production of slime on the fruit and or seed surface may also be an adaptation to ruderal, disturbed environments (young and evans 1973, kreitschitz and vallès 2007). the presence of a slime envelope is associated with a short life cycle and facilitates the quick colonization of such places (kreitschitz and vallès 2007). this study confirmed this kind of adaptation in matricaria, which is an annual. similar adaptive mechanisms are present in many common annual weeds colonizing ruderal habitats, e.g. lepidium flavum, l. nitidum, plantago lanceolata, cardaria draba (young and evans 1973) arabidopsis thaliana (western et al. 2000) and artemisia annua, a. biennis and neopallasia pectinata (kreitschitz and vallès 2007). acknowledgements the author thanks dr. faik ahmet ayaz, dr. melahat ozcan and murat bal for collecting plant materials, nursen aksu for technical assistance, the scientific and technical research 112 acta bot. croat. 70 (1), 2011 inceer h. tab. 2. slime staining in artemisia and eragrostis species staining target obtained color literature data references methylene blue pectin cellulose blue violet-blue blue violet, blue gerlach (1972) broda (1971 in kreitschitz and vallès 2007) safranine cellulose pectin orange orange-red orange-red red, orange-red orange braune et al. (1975), o’brien and mccully (1981), kreitschitz et al. (2009) kreitschitz and vallès (2007) kreitschitz et al. (2009) u:\acta botanica\acta-botan 1-11\inceer.vp 28. o ujak 2011 16:41:12 color profile: disabled composite 150 lpi at 45 degrees council of turkey (tubitak, tbag project no. 106t162) for financial support. we thank the two anonymous referees for providing helpful suggestions to improve the manuscript. references applequist, w. l., 2002: a reassessment of the nomenclature of matricaria l. and tripleurospermum sch. bip. (asteraceae). taxon 51, 757–761. braune, w., leman, a., taubert, h., 1975: practicum of plant anatomy (in polish). pwn, warszawa. broda, b., 1971: methods of plant histochemistry (in polish). pwzl, warszawa. fahn, a., werker, e., 1972: anatomical mechanisms of seed dispersal. in: kozlowski, t. t. (ed), seed biology, i. importance development and germination, 151–221. academic press, new york. gerlach, d., 1972: basics of botanical microtechnique (in polish). pwril, warszawa. grubert, m., 1974: studies on the distribution of myxospermy among seeds and fruits of angiospermae and its ecological importance. acta biologica venezuelica 8, 315–551. grubert, m., 1982: studies on the mucilage content of myxospermatic diaspores from various angiosperm families. plant systematics and evolution 141, 7–21. huang, z., gutterman, y., 1999: water absorption by mucilaginous achenes of artemisia monosperma: floating and germination as affected by salt concentrations. israel journal of plant sciences 47, 27–34. huang, z., gutterman, y., hu, z., 2000: structure and function of mucilaginous achenes of artemisia monosperma inhabiting the negev desert of israel. israel journal of plant sciences 48, 255–266. jeffrey, c., 1979: note on the lectotypification of the names cacalia l., matricaria l. and gnaphalium l. taxon 28, 349–351. kerguélen, m., bosc, c., lambýnon, j., 1987: données taxonomiques nomenclaturales et chrologiques pour une révision de la flore de france. lejeunia 120, 1–264. kreitschitz, a., vallès, j., 2007: achene morphology and slime structure in some taxa of artemisia l. and neopallasia l. (asteraceae). flora 202, 570–580. kreitschitz, a., tadele, z., gola, e. m., 2009: slime cells on the surface of eragrostis seeds maintain a level of moisture around the grain to enhance germination. seed science research 19, 27–35. korobkov, a. a., 1973: morpho-anatomical peculiarities of achene of artemisia ssp. from north-east of the ussr. botanicheskii zhurnal 58, 1302–1315. kyno^lova, m., 1970: comparative morphology of achenes of the tribe anthemideae cass. (family asteraceae) and its taxonomic significance. preslia, 42, 33–53. mosquero, m. a., juan, r., pastor, j., 2004: observaciones micromorfológicas y anatómicas en núculas de prunella l. y cleonia l. (lamiaceae) del suroeste de españa. acta botanica malacitana 29, 203–214. muhlethaler, k., 1950: the structure of plant slimes. experimental cell research 1, 341–350. o' brien, t. p., mccully, m. e., 1981: the study of plant structure principles and selected methods. termarcarphi pty. ltd., melbourne, australia. acta bot. croat. 70 (1), 2011 113 achene slime content in matricaria u:\acta botanica\acta-botan 1-11\inceer.vp 28. o ujak 2011 16:41:12 color profile: disabled composite 150 lpi at 45 degrees oberprieler, c., vogt, r., watson, l. e., 2007: tribe anthemideae cass. in: kadereýt, j.w., jeffrey, c. (eds.), the families and genera of vascular plants, 8, flowering plants, eucots, asterales, 342–374, springer-verlag, berlin. pobedimova, e. g., 1995: tripleurospermum sch. bip. in: shiskin, b. k., bobrov, e. g. (eds), flora ussr, 26, 181–213, bishen singh mahendra pal singh, dehra dun, india/koeltz scientific books, koenigsten, germany. western, t. l., debra, j. s., haughn, g. w., 2000: differentiation of mucilage secretory cells of the arabidopsis seed coat. plant physiology 122, 345–355. xifreda, c. c., 1985: sonre el nombre cientifica correcta de la manzanilla (matricaria recutita l., asteraceae). darwiniana 26, 373–375. young, j. a., evans, r. a., 1973: mucilaginous seed coats. weed science 21, 2–54. young, j. a., martens, e., 1991: importance of hypocotyl hairs in germination of artemisia seeds. journal of range management 44, 438–442. 114 acta bot. croat. 70 (1), 2011 inceer h. u:\acta botanica\acta-botan 1-11\inceer.vp 28. o ujak 2011 16:41:12 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (2), 157–166, 2011 coden: abcra 25 issn 0365-0588 fire risk in austrian pine (pinus nigra) plantations under various temperature and wind conditions imre cseresnyés*, orsolya szécsy, péter csontos research institute for soil science and agricultural chemistry of the hungarian academy of sciences, herman o. út 15, h-1022 budapest, hungary. abstract – the austrian pine (pinus nigra), an introduced conifer in hungary, forms a highly flammable vegetation type. the fire risk of such stands was examined using mcarthur’s empirical forest fire danger model. our study focused on the effects of temperature and wind speed on fire behaviour. by keeping the input parameters of the model constant while changing temperature and wind speed within a specified interval the resulting fire danger index (fdi) and fire behaviour were examined. the applied fixed parameters were: 30 °c temperature, 30% relative humidity, 30 km h–1 wind speed, 30 degree of slope and drought factor value 10. the annual trends of the byram-keetch drought index (bkdi) and the drought factor were also calculated. our results show that increasing temperature and wind speed raises the fdi, flame height, rate of fire spread (ros) and spotting distance. the amount of fuel does not influence the fdi, but increasing the amount promotes the ros and raises the flame height. wind speed was the most important factor in the ros. a serious fire risk of these plantations was determined. the reliability of mcarthur’s model was proved by comparison of our results with experimental laboratory data based on literature. key words: drought factor, fire danger index, flame height, mcarthur’s model, pinus nigra, rate of spread, plantation, wind abbreviations: bkdi – byram-keetch drought index, fdi – fire danger index, ros – rate of fire spread introduction in hungary, austrian pine (pinus nigra arn.) plantations were first established for soil preservation and landscape protection purposes. the species was also used in afforestation in the mediterranean areas of europe to prevent soil erosion (bar^i] et al. 2006, topi] et al. 2008). in hungary, the primary goal of the afforestation shifted towards wood production. today, this alien species is responsible for some serious nature conservation problems. owing to the accumulated litter fuels and strong shading by the pine canopy, the botaniacta bot. croat. 70 (2), 2011 157 * corresponding author, e-mail: cseresnyes.imre@rissac.hu copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:26 color profile: disabled composite 150 lpi at 45 degrees cally valuable floras of dolomite and limestone rock grasslands have been extensively impoverished or made locally extinct (csontos et al. 1996, tamás 2003, cseresnyés et al. 2006). consequently, monodominant pinus nigra stands are occupying habitats that were previously occupied by native grasslands. however, the most serious damage is caused by repeated forest fire events. that the frequency of forest fires has been increasing in recent decades was demonstrated world-wide (niklasson and granström 2000, hartley 2002, palik et al. 2002, muzy et al. 2008). this trend in increasing forest fires can be traced to human negligence or intentional burning (johnson and larsen 1991, granström 1993, viegas et al. 1999, burrows 2008). the risk of fire initiation in a forest depends not only on current meteorological and topographical factors, but also on the amount of accumulated flammable vegetation (millán et al. 1998, viegas 1998). as a consequence of its slow decomposition, needle litter tends to accumulate in large quantities under austrian pine stands (cseresnyés et al. 2006, csontos et al. 2007, küçük et al. 2007). for these reasons exotic pine plantations of hungary may be more vulnerable to damaging fires than grasslands (which once occupied these sites) and the native broad-leaved forests of the country. the dryness of the litter and the vegetation has a considerable influence on the scale of fire risk, such that the frequency and scale of forest fires increase during dry years (viegas et al. 1990, 1992, granström 1993, swetnam 1993). with the application of an adequate fire danger model the potential degree of fire risk of the hungarian pinus nigra plantations was studied. we also aimed to determine the effects of air temperature and wind speed on the characteristics of fire. materials and methods hungary is situated in the warm temperate zone, which is ranked third among climatic regions regarding fire risk, after the mediterranean and the wet subtropical areas. in hungary, the yearly average of forest area damaged by fire ranges between 600 ha and 800 ha, but in extremely dry years the burnt area may exceed 2000 ha (zambó 1995). modelling objects were austrian pine forests planted on dolomite hills of pilis and budai mountains. data on accurate localities and age of the stands (which ranged from 21 to 108 years) were known from local forest inventories. each stand was larger (considerably larger in most cases) than 5 ha. fire danger modelling was carried out by applying mcarthur’s empirical model (noble et al. 1980). this model was developed in australia, but later it was successfully used under different climatic and fuel conditions in north america and europe (pastor et al. 2003, snyder et al. 2006). the model requires the following input data for the prediction of fire risk and fire behaviour: air temperature, relative humidity, wind speed, slope degree, amount of fuel and drought factor. determination of the actual value of drought factor was based on the amount of last precipitation, the number of days since last rain and the byram-keetch drought index (bkdi). given this knowledge, the fire danger index (fdi), flame height, rate of spread (ros) and spotting distance can be calculated by forest fire danger calculator software (fig. 1). fire danger index means the probability of combustion, which has the following classes: 0–4: low; 5–11: middle; 12–23: high; 24–49: very high; above 50: extreme. bkdi 158 acta bot. croat. 70 (2), 2011 cseresnyés i., szécsy o., csontos p. u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:26 color profile: disabled composite 150 lpi at 45 degrees is an index characterizing the cumulative moisture deficiency in the 200 mm stratum of litter and upper soil layer expressed in mm-equivalent (keetch and byram 1968). it also indicates water deficit in living plants and hence their flammability (xanthopoulos et al. 2006). daily bkdi has been calculated from 1985 to 2009 using daily maximum temperature and rainfall data from the budapest-lörinc meteorological station (n 47°25'45"; e 19°10'56"; 138 m a.s.l.). by employing drought factor calculator software, daily drought factor values were calculated for the 1985–2009 period. these daily drought factors were averaged for every ten days, then the average of the 25 years was illustrated together with the wettest and the driest years. to study the effect of changing temperature or wind speed on fire-risk relations, one of these two factors was changed within a specified interval (leaving the other five input parameters constant) and the resulting four outputs of the model (see fig. 1) were examined. the following constant values and intervals of change were used: 1) temperature: its fixed value was 30 °c. this temperature is common in summer (kakas 1960). when temperature was the selected variable parameter, its value was changed from 0 to 40 °c. the daily maximum temperature of 40 °c is rare but not negligible, since there were 10 consecutive days above 40 °c in hungary in the year 2008. 2) relative humidity: 30%. similar or lower air humidity values occur frequently in hungary between july and september (bacsó et al. 1953). 3) wind speed: 30 km h–1 (gale force 5 in the beaufort-köppen scale). such strong winds are frequent in each season of the year. intervals of the step-wise testing ranged between 0 and 70 km h–1. the windiest months are june and july in hungary (bacsó et al. 1953). commonly, the highest wind speeds are usually recorded between 12 p. m. and 3 p. m., though they can vary to a great extent depending on the local weather conditions. steep slopes and sharp ridges are typical of dolomite hills, thus when examining these areas we have to reckon with strong or stormy squalls even on relatively calm days. acta bot. croat. 70 (2), 2011 159 fire risk in pinus nigra plantations fig. 1. schematic diagram of mcarthur’s forest fire danger model. u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:30 color profile: disabled composite 150 lpi at 45 degrees 4) degree of slope: 30°. this value is typical of dolomite hills. besides enhancing the flame height, increase in the degree of slope is also often a driving factor for crown fire initiations (santoni and balbi 1998). dolomite areas abound in steep slopes, consequently this topographical effect contributes significantly to fire risk conditions. 5) amount of fuel: according to mcarthur’s model the fire behaviour depends on the amount of litter only with a diameter of less than 6 mm (noble et al. 1980). from previous studies in austrian pine plantations, the litter fuel less than 6 mm is 10,574 kg ha–1 in the age class of 20–35 years, 14,024 kg ha–1 in the age class of 35–60 years, 18,564 kg ha–1 in the age class of 60–80 years and 13,056 kg ha–1 in stands above 80 years (cseresnyés et al. 2006). since the amount of fuel reaches a maximum mass in the 60–80 year stand age class, forest fires are expected to be most severe in this age class. for the purpose of this study, the stand age class above 80 years was ignored, firstly because such old monodominant austrian pine forests are relatively rare and secondly, because according to our former statistical analysis, the amount of accumulated fuel in this age class corresponds essentially to the fuel load in the stand age of 35–60 years. although the probability of combustion (namely the fdi) is independent of the amount of fuel, higher amounts of litter fuel increase the flame height, the ros and the spotting distance. some experiments of morvan and dupuy (2001) verified linear rate connection between the ros and the fuel mass until about 20,000 kg ha–1, as above that value they became independent of each other. 6) drought factor: this indicates the dryness of vegetation, litter and upper soil layer. its value is an integer number between 1 and 10. based on our previous investigations (cseresnyés and csontos 2006), we chose the highest figure (10) as the constant value. detailed analyses proved that periods characterized by the highest drought conditions can prevail not only in extreme dry years but also in years with average weather conditions. only the length and frequency of drought periods are of importance. results based on bkdi values and daily amounts of rainfall the annual drought factor trend was calculated. drought factor changes are evident in the period 1985 to 2009, in the wettest and the driest years, as well as in the 25 year average (fig. 2). from january to june the 25 year mean value of the drought factor was about five. in june it began to increase, reaching its maximum (above value 7) between the middle of august and september. until the end of november it was decreasing continuously, when it stabilized around value five. thus, august and september are generally the driest months of the year. in the driest year (2000) the drought factor reached the highest possible value (10), maintaining this for four ten-day periods. in 2000, the extreme arid conditions lasted 99 days, from 15th june to 4th november. the longest unbroken period, when the drought factor was at value 10, was continuously observed for 29 days (between 7th october and 4th november). fdi is influenced by temperature and wind speed (figs. 3a, b). fdi is independent of the amount of fuel, thus also of the stand age, age classes being not distinguished. fdi increased by the rising air temperature (fig. 3a). fdi was claimed to be »high« at 10 °c temperature and »very high« at 30 °c. increasing wind speed causes an exponential increase in the fdi (fig. 3b). under the chosen constant parameters the fire risk is »high« even if the weather is entirely calm, but at a wind speed of 60 km h–1 fdi becomes »extreme«. 160 acta bot. croat. 70 (2), 2011 cseresnyés i., szécsy o., csontos p. u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:30 color profile: disabled composite 150 lpi at 45 degrees flame height was not only increased by air temperature (fig. 3c) and wind speed (fig. 3d), but also by the amount of fuel. therefore the lowest flame height was observable in the stand age class 20–35 years, while the highest flame height was predicted in the age class of 60–80 years. expected flame heights are 4.22 m in stand age class 20–35 years, 6.70 m in the age class of 35–60 years and 9.19 m in the age class of 60–80 years. in the stand age class of 60–80 years, in case of a 70 km h–1 wind speed, flame height approximates to 20 m, but even in the youngest stands it exceeds 10 m. spotting distance also rose with the amount of fuel. consequently, we have to expect the minimum spotting distance in the youngest stands, while the highest values are observable in the age class of 60–80 years. spotting distance was also enhanced by air temperature (fig. 3e), and its value may be notable even at relatively low temperature. in the stand age class 60–80 years it can exceed 1.5 km if temperature reaches 30 °c. spotting distance was principally influenced by wind speed (fig. 3f). in the highly inflammable stand age class the spotting distance is nearly 0.6 km in calm weather, but a squall with 70 km h–1 wind speed can transport the glowing embers to a distance of 4.5 km. rate of fire spread (ros), like flame height and spotting distance, increases with the amount of fuel. the ros is much larger upslope than downslope or on flat terrain. on a 30 degree slope, the ros of an ascending fire is eight fold higher than that of a descending fire. our result graphs (fig. 3g and 3h) show only the upslope spreading rates. a rising temperature increases the ros (fig. 3g). in stand age class 60–80 years the predicted ros can approach 5 km h–1 on a hot summer day, when the temperature reaches 35 °c. naturally, the first factor enhancing the ros is wind speed (fig. 3h). based on our described constant parameters the ros is 4.19 km h–1 upslope in the age class of 60–80 years. the ros of a descending fire does not exceed 1.35 km h–1 at any of the used parameter combination. acta bot. croat. 70 (2), 2011 161 fire risk in pinus nigra plantations fig. 2. annual changes of the mean value of the drought factor as the average of years between 1985 and 2009, as well as in the wettest year (2005) and in the driest year (2000) of that period. u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:36 color profile: disabled composite 150 lpi at 45 degrees discussion the drought factor trend clearly shows that august and september are the most crucial months from the point of view of fire events. we obtained similar results during the calculation of bkdi in a former study (cseresnyés and csontos 2006). snyder et al. (2006) also demonstrated the close connection between bkdi and drought factor values. the importance of these two drought indicators is proved by one of the most serious forest fires of budai mountains, which devastated more than 100 ha of pine stand on 15th august 1993, near village nagykovácsi, 15 km west from budapest. on this day the calculated bkdi was rather high, 80.25 mm-equivalent, and the drought factor reached value 9 (cseresnyés and csontos 2006). these data precisely show the drought conditions prevailing in the mentioned day, and furthermore the daily maximum temperature was 33 °c, thus further increasing the fire risk. the fire danger index as well as fire behaviour are both affected by air temperature and wind speed. in general, wind speed has a stronger influence on fire risk than temperature. 162 acta bot. croat. 70 (2), 2011 cseresnyés i., szécsy o., csontos p. fig. 3. changes of the fire danger index (fdi), flame height, spotting distance and rate of fire spread (ros) with the temperature and wind speed in pinus nigra stands. (since the fdi is independent of the amount of fuel, age classes are not distinguished in fig. 3a and 3b.) u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:41 color profile: disabled composite 150 lpi at 45 degrees fdi can reach the »extreme« class only in strong winds, and the flame height also rises more vigorously with wind speed (increasing the probability of crown fires). spotting distance is also principally influenced by wind conditions; therefore windy weather makes fire fighting considerably difficult by forming local fire-seats far away from the main fire front. a large ros also contributes to this problem. in the highly inflammable stand age class (60–80 years) the ros is more than 5 km h–1 with a 40 km h–1 wind speed, but at 70 km h–1 wind speed it exceeds 10 km h–1. experimental field works in turkish pinus nigra stands and shrublands, provided evidence that wind speed, air temperature and the moisture content of litter, greatly contributed to ros (küçük et al. 2007, 2008b; bilgili and saglam 2003). moreover, the amount of flammable biomass has considerable importance in respect of fire behaviour and fire management (küçük et al. 2008a, saglam et al. 2008), thus in hungary the 60–80 years stand age class of austrian pine plantations is the most vulnerable to devastating fires. in order to test the correspondence between the results of our study and reality, we compared the outputs of the model with experimental laboratory results known from the scientific literature. on an experimental basis, viegas (1998) proved that a fire front propagating upslope has a rate of advance much greater than on horizontal terrain, and its value increases with terrain inclination. the ros of a descending fire is very similar to that on a flat terrain and besides, it is independent of terrain inclination. if the degree of slope reaches 30, the measurable ros will be 4–6 fold higher than on flat ground (viegas et al. 1994), so this difference is smaller than predicted by mcarthur’s model. in accordance with morandini et al. (2001), on a 30 degree slope an ascending fire has an 8–10 fold higher ros than a fire on horizontal terrain. their experiments were carried out in the laboratory, by burning a pinus pinaster aiton fuel bed. the 7.8–8 fold higher difference between upslope and downslope ros predicted by mcarthur’s model is practically equivalent to these laboratory results. in other experimental circumstances less (5–7 fold higher) difference was observed between the upslope and downslope ros, but the variances depended on the moisture content of the litter (santoni and balbi 1998, gonzalez et al. 2008). influence of wind speed on the ros was examined under similar conditions (simeoni et al. 2001). ros showed more increase with wind speed than predicted by mcarthur’s model; consequently this model might underestimate the effect of wind speed on the ros. morvan and dupuy (2001) measured ros with changes to the amount of fuel. in the present study, when the fuel mass was changed from 10,574 kg ha–1 (stand age class 20–35 years) to 18,564 kg ha–1 (stand age class 60–80 years), an increase of about 75%, it caused a 1.8 fold higher ros. morvan and dupuy (2001) noticed only 1.5–1.6 fold higher differences by using of similar range of fuel quantity, whereas glitzenstein et al. (2006) observed much larger (2.1–2.5 fold) differences among spreading rates. these examples imply that mcarthur’s model properly estimates the influence of fuel mass on ros. summarizing these comparative results, we can conclude that the outputs of mcarthur’s model generally correspond to the experimental data, but in some cases they differ to a certain degree. more detailed comparison with literature data was not possible, because in many publications relative humidity, air temperature and in some cases even the moisture content and the amount of fuel were not described. acta bot. croat. 70 (2), 2011 163 fire risk in pinus nigra plantations u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:41 color profile: disabled composite 150 lpi at 45 degrees the results of our studies show considerable fire risk in the austrian pine stands of hungary, more or less similar to that of mediterranean areas. consequently we have to pay more attention to these plantations not only during dry and warm, but also during windy periods of the year to prevent forest fires and to avoid serious natural and economic losses. acknowledgements the authors thank erika bózsing for helping with the field-work and foresters gábor bakon, istván apatóczky and viktor farkas for supplying data concerning austrian pine stands. we are grateful to the reviewers for their useful comments and for language copy editing. this research was supported by the hungarian national science foundation (otka t037732). references bacsó, n., kakas, j., takács, l., 1953: climate of hungary (in hungarian). országos meteorológiai intézet, budapest. bar^i], d., hr[ak, v., [panjol, @., 2006: the ameliorative effects of pine cultures on forest sites on the island of rab in southwest croatia. forest ecology and management 237, 39–46. bilgili, e., saglam, b., 2003: fire behavior in maquis fuels in turkey. forest ecology and management 184, 201–207. burrows, n. d., 2008: linking fire ecology and fire management in south-west australian forest landscapes. forest ecology and management 255, 2394–2406. cseresnyés, i., csontos, p., 2006: change in drought conditions of pinus nigra stands in hungary (in hungarian). tájökológiai lapok, 255–268. cseresnyés, i., csontos, p., bózsing, e., 2006: stand age influence on litter mass of pinus nigra plantations on dolomite hills in hungary. canadian journal of botany 84, 363–370. csontos, p., horánszky, a., kalapos, t., lokös, l., 1996: seed bank of pinus nigra plantations in dolomite rock grassland habitats, and its implications for restoring grassland vegetation. annales historico-naturales musei nationalis hungarici 88, 69–77. csontos, p., rocchini, d., bacaro, g., 2007: modelling factors affecting litter mass components of pine stands. community ecology 8, 247–255. glitzenstein, j. s., streng, d. r., achtemeier, g. l., naeher, l. p., wade, d. d., 2006: fuels and fire behaviour in chipped and unchipped plots: implications for land management near the wildland/urban interface. forest ecology and management 236, 18–29. gonzalez, j. r., del barrio, g., duguy, b., 2008: assessing functional landscape connectivity for disturbance propagation on regional scales – a cost-surface model approach applied to surface fire spread. ecological modeling 211, 121–141. granström, a., 1993: spatial and temporal variation in lightning ignitions in sweden. journal of vegetation science 4, 737–744. hartley, m. j., 2002: rationale and methods for conserving biodiversity in plantation forest. forest ecology and management 155, 81–95. 164 acta bot. croat. 70 (2), 2011 cseresnyés i., szécsy o., csontos p. u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:42 color profile: disabled composite 150 lpi at 45 degrees johnson, e. a., larsen, c. p. s., 1991: climatically induced change in fire frequency in the southern rockies. ecology 72, 194–201. kakas, j., 1960 (ed.): climatic atlas of hungary (in hungarian). akadémiai kiadó, budapest. keetch, j. j., byram, g. m., 1968: a drought index for forest fire control. u.s.d.a. forest service research paper se-38. southeastern forest experiment station, asheville, nc. küçük, ö., bilgili, e., baysal, i., 2007: fire development from a point source in surface fuels of a mature anatolian black pine stand. turkish journal of agriculture and forestry 31, 263–273. küçük, ö., bilgili, e., saglam, b., 2008a: estimating crown fuel loading for calabrian pine and anatolian black pine. international journal of wildland fire 17, 147–154. küçük, ö., bilgili, e., saglam, b., baskaya, s., dinç durmaz, b., 2008b: some parameters affecting fire behavior in anatolian black pine slash. turkish journal of agriculture and forestry 32, 121–129. millán, m. m., estrela, m. j., badenas, c., 1998: synoptic analysis of meteorological processes relevant to forest fire dynamics on the spanish mediterranean coast. in: moreno, j. m. (ed.), large forest fires, 1–30. backhuys publishers, leiden. morandini, f., santoni, p. a., balbi, j. h., 2001: the contribution of radiant heat transfer to laboratory-scale fire spread under the influences of wind and slope. fire safety journal 36, 519–543. morvan, d., dupuy, j. l., 2001: modelling of fire spread through a forest fuel bed using a multiphase formulation. combustion and flame 127, 1981–1994. muzy, a., nutaro, j. j., zeigler, b. p., coquillard, p., 2008: modeling and simulation of fire spreadig through the activity tracking paradigm. ecological modelling 219, 212– 225. niklasson, m., granström, a., 2000: numbers and sizes of fires: long-term spatially explicit fire history in a swedish boreal landscape. ecology 81, 1484–1499. noble, i. r., bary, g. a. v., gill, a. m., 1980: mcarthur’s fire-danger meters expressed as equations. australian journal of ecology 5, 201–203. palik, b. j., mitchell, r. j., hiers, j. k., 2002: modelling silviculture after natural disturbance to sustain biodiversity in the longleaf pine (pinus palustris) ecosystem: balancing complexity and implementation. forest ecology and management 155, 347–356. pastor, e., zárate, l., planas, e., arnaldos, j., 2003: mathematical models and calculation system for the study of wildland fire behaviour. progress in energy and combustion science 29, 139–153. saglam, b., küçük, ö., bilgili, e., dinç durmaz, b., baysal, i., 2008: estimating fuel biomass of some shrub species (maquis) in turkey. turkish journal of agriculture and forestry 32, 349–356. santoni, p. a., balbi, j. h., 1998: modelling of two-dimensional flame spread across a sloping fuel bed. fire safety journal 31, 201–225. acta bot. croat. 70 (2), 2011 165 fire risk in pinus nigra plantations u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:42 color profile: disabled composite 150 lpi at 45 degrees simeoni, a., santoni, p. a., larini, m., balbi, j. h., 2001: on the wind advection influence on the fire spread across a fuel bed: modelling by a semi-physical approach and testing with experiments. fire safety journal 36, 491–513. snyder, r. l., spano, d., duce, p., baldocchi, d., xu, l., paw, u. k. t., 2006: a fuel dryness index for grassland fire-danger assessment. agricultural and forest meteorology 139, 1–11. swetnam, t. w., 1993: fire history and climate change in giant sequoia groves. science 262, 885–889. tamás, j., 2003: the history of austrian pine plantations in hungary. acta botanica croatica 62, 147–158. topi], v., ani], i., butorac, l., 2008: effects of stands of black pine (pinus nigra arn.) and aleppo pine (pinus halepensis mill.) on the protection of soil from erosion. ekológia bratislava 27, 287–299. xanthopoulos, g., maheras, g., gouma, v., gouvas, m., 2006: is the keetch-byram drought index (kbdi) directly related to plant water stress? forest ecology and management 234 (supplement), 27. viegas, d. x., 1998: weather, fuel status and fire occurrence: predicting large fires. in: moreno, j. m. (ed.), large forest fires, 31–48. backhuys publishers, leiden. viegas, d. x., bovio, g., ferreira, a. d., nosenzo, a., sol, b., 1999: comparative study of various methods of fire danger evaluation in southern europe. international journal of wildland fire 9, 235–246. viegas, d. x., varela, v. g. m., borges, c. p., 1994: on the evolution of a linear fire front in a slope. proceedings 2 international conference on forest fire research, coimbra, portugal, 301–318. viegas, d. x., viegas, m. t., ferreira, a. d., 1990: characteristics of some forest fuels and their relation to the occurence of fires. proceedings 1 international conference on forest fire research, coimbra, portugal, 13. viegas, d. x., viegas, m. t., ferreira, a. d., 1992: moisture content of fine forest fuels and fire occurence in central portugal. international journal of wildland fire 2, 69–86. zambó, p., 1995: forest fires in years 1993 and 1994 on the territory of pilis forest inventory, degree of losses and efforts for restoration (in hungarian). erdészeti lapok 130, 152. 166 acta bot. croat. 70 (2), 2011 cseresnyés i., szécsy o., csontos p. u:\acta botanica\acta-botan 2-11\cseresnyes et al.vp 8. rujan 2011 15:59:42 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp book reviews u:\acta botanica\acta-botan 1-09\book review.vp 17. travanj 2009 13:24:22 color profile: disabled composite 150 lpi at 45 degrees u:\acta botanica\acta-botan 1-09\book review.vp 17. travanj 2009 13:24:22 color profile: disabled composite 150 lpi at 45 degrees book review the concise geologic time scale james g. ogg, gabi ogg, felix m. gradstein cambridge university press, new york, 2008, isbn 978-0-521-89849-2. 184 pp (vii + 177), 276 references (217: further reading + 59: on line), 69 color figs (65 + 4), 1 bw halftones, 16 tables, size 253 x 192 mm, hardback, weight 0.7 kg, price £ 20.00 contents 1. introduction 2. planetary time scale (kenneth l. tanaka and william k. hartmann) 3. precambrian (martin j. van kranendonk, james gehling and graham shields) 4. cambrian period (shanchi peng and loren babcock) 5. ordovician period 6. silurian period 7. devonian period 8. carboniferous period (philip h. heckel) 9. permian period 10. triassic period 11. jurassic period 12. cretaceous period 13. paleogene period 14. neogene period 15. quaternary period (philip gibbard, kim cohen, and james ogg) appendix 1. standard colors of international divisions of geologic time appendix 2. ratified gssfs for geologic stages index acta bot. croat. 68 (1), 2009 165 u:\acta botanica\acta-botan 1-09\book review.vp 17. travanj 2009 13:24:23 color profile: disabled composite 150 lpi at 45 degrees »this concise handbook presents a summary of earth’s history over the past 4.5 billion years as well as a brief overview of contemporaneous events on the moon, mars and venus«. this first sentence of the preface (p. iii) and the colored plate »geologic time scale« before (left) the title page (p. iv) define and summarize in the best way the rich, highly interesting and important contents of this excellent book. the geologic time scale is especially impressive and instructive, representing in fact the whole »calendar« of earth’s history from the very beginning up to the present day. therefore it is worth looking first carefully at that time scale beginning in the right lower corner of the page: the deeply colored purple field shows, at its left border (see fig. 1 in this text), a duration from more than 4600 to 4000 ma (million years)*, thus more than about 600 ma, which is more than that of the whole eon phanerozoic (542 ma). the wavy line at the bottom symbolizes the uncertainty in time determination. due to the absence of any reliable data the hadean is left out of the eon – era … system. on the left side of the same column, one sees that, after the hadean, the eons archean (4000–2500 ma) and proterozoic (2500–542 ma) follow which, together with the hadean, represent the enormously long time interval commonly called precambrian (4600–542 ma). the time interval from 542–0 ma is the eon phanerozoic, the already well known part of earth’s history, which has been, for a century, an important chapter of the natural history teaching in grammar-schools. it should explicitly be pointed out that the precambrian was about 7.5 (7.487) times longer than the whole phanerozoic. the eons are subdivided into eras, eras into periods, periods into epochs, epochs into age/stage units. to facilitate the reading of age-values (ma) the age scale is doubled. the scale on the left side is continuous, while the scale on the right border marks only the exact values of the boundaries between the divided and the subdivided parts of the eon-era-period-epoch-age/stage system (see fig. 1). in the introduction (p. 1), the book the concise geologic time scale is characterized as the framework for deciphering the history of our planet earth, and, in this sense, this book is a summary of the status of that scale and is intended to be a handbook. the readers who desire more background and details will find the necessary references at the end of each chapter and especially in the book by gradstein, f.m., ogg, j.g., smith, a.g. et al.: a geologic time scale 2004, cambridgeuniversity press, cambridge. in the introduction, the following topics are also explained and discussed: international divisions of geologic time and their global boundaries; biologic, chemical, sea-level, geomagnetic and other events or zones; methods for assigning numerical ages; time scale creator database and chart-making package; a geologic time scale 2010. in the second chapter the formal stratigraphic systems, developed for the surfaces of earth’s moon, mars, mercury and venus, using the results of contemporaneous interdisciplinary space research, are presented and discussed. some of the results are visible from the developed time scales. 166 acta bot. croat. 68 (1), 2009 * in the book, the following time units are used: ma megaannuum = l myrs: l million years = 106 years; ga gigaannuum = 1 billion years = 109 years. u:\acta botanica\acta-botan 1-09\book review.vp 22. travanj 2009 13:16:37 color profile: disabled composite 150 lpi at 45 degrees according to the data in the book (p. 18–19) the following time intervals are proposed, on the: ¿ moon of the earth: the pre-nectarian (4600–4200), the nectarian (4200–3800), the imbrian (3800–3200), the eratosthenian (3200–2200,? (2200–1250) and the copernican period (1250–0) ma. ¿ mars: the pre-noachian (4600–4200), the noachian (4200–3600), the hesperian (3600– 2600) and the amazonian period (2600–0) ma. ¿ venus/mercury: the pre-tolstojan (4600–4000), the tolstojan(4000–3800), the calorian (3800–3200), the mansurian (3200–1700) and the kuiperian period (1700–0) ma. about other bodies in the solar system there is not much known as yet. the third chapter treats the precambrian, which is not a stratigraphic unit at all. the period just refers to all rocks originating from the beginning of the earth to the onset of the cambrian (the first period of the phanerozoic), i.e. during the time interval from 4600–542 (= 4058) ma (see fig. 2). before the formation of the oldest preserved rock, the earth already passed the first phase of its history in a time interval of more than 537 ma, called mostly the hadean, which was nearly as long as the whole eon phanerozoic. the hadean is followed by the eons archean (4000–2500 ma) and proterozoic (2500–542 ma). the whole precambrian (hadean + archean + proterozoic) lasted thus, as already mentioned, 4058 ma, i.e. 7.5 (7.487) times longer than the whole phanerozoic, which is the only subject of the following 12 chapters (4 th –15 th , pp. 37–158). the eon archean comprises 4 eras: the eoarchean (4000–3600), the paleoarchean (3600–3200), the mesoarchean (3200–2800) and the neoarchean (2800–2500) ma (see fig. 2). the archean-proterozoic transition is generally considered significant and useful, although it is not yet scientifically well defined. the eon proterozoic is subdivided into 3 eras: paleoproterozoic (2500–1600), mesoproterozoic (1600–1000), and neoproterozoic (1000–542) ma (see fig. 2). the paleoproterozoic has four periods: the siderian (2500–2300), the rhyacian (2300– 2050), the orosirian (2050–1800) and the statherian (1800–1600) ma. the mesoproterozoic has 3 periods: the calymmian (1600–1400), the ectasian (1400–1200), and the stenian (1200– 1000) ma. the neoproterozoic era has three periods: the tonian (1000–850), the cryogenian (850–650), and the ediacaran (650–542) ma. the eras archean and proterozoic are still under investigation by the chronostratigraphs, while the cryogenian and the ediacaran already appear to be better explored, to some extent. in the north pole dome area of the pilbara craton (craton is a relatively rigid and immobile region of the earth’s crust), northwestern australia, in the dresser formation, there is some evidence of the earliest life on earth in the form of fossil stromatolites and highly fractionated d 13 c values of kerogen indicating a methan consuming life. this sedimentary unit of 3.49–3.48 ga is conformably bound by well preserved pillow basalts of the warrawoona group. many scientists believe that the most significant change in earth’s history was the development of an oxygenated atmosphere, as this allowed for the evolution of complex life acta bot. croat. 68 (1), 2009 167 u:\acta botanica\acta-botan 1-09\book review.vp 22. travanj 2009 13:16:37 color profile: disabled composite 150 lpi at 45 degrees on earth, including even primates and the genus homo. oxygenation is considered to be the result of the processes of gas exchange in cyanobacteria which use co2 and sunlight to produce energy-rich carbon compounds releasing free oxygen as a waste product. some geological changes followed this change in atmospheric composition. so e.g. detrital uraninite and pyrite disappeared in sandstones, and redbets and mn-rich sedimentary rocks appeared. many redox-sensitive chemical tracers could be identified, including mo-isotopes, ce, fe, re-os data, sulfur isotopes, and platinum group element concentration. as these products have various sensitivities to redox conditions, their ages could be determined according to various stages of the increase of oxygen levels. the archean-proterozoic boundary only approximately indicates the rise of cyanobacteria and oxygenation of the atmosphere. there is some strong evidence that cyanobacteria arose considerably earlier (2.7 ga) than the onset of oxygenation of the atmosphere. a critical step in the oxygenation of the atmosphere occurred at ~2.32 ga with the disappearance of the mass-independently fractionated isotope signature (s-mif), which indicates the development of an ozone layer requiring a partial pressure of oxygen of ~10 –5 of the present atmospheric level. this signal disappeared within the period of global, low-latitude paleoproterozoic glaciations and near the onset of the lomagundi-jatuli positive d 13 c isotopic excursion. the predicted drop in partial pressure of co2 in the atmosphere across the archean-proterozoid boundary should produce a change from predominantly chemical weathering to dominantly physico-mechanical weathering. this change in weathering manner is the consequence of decreasing concentration of carbonic acid (h2co3 formed by the binding of h2o and co2 being under higher partial pressure, increasing in this way the acidity (lowering the ph) of the water in oceans, rivers and rain (taken from pp. 28 and 29). beginning with chapter 3 (precambrian period) there is, at the top of the first paragraph, a paleogeographic map showing the geographic distribution and position of the developing continents during the treated period. these instructive and attractive paleogeographic maps were provided by christopher scotese. it should be mentioned that some positions of continents are, however, still uncertain. in chapter 3 (precambrian period), the paleogeographic map shows the situation at the cryogenian period (650 ma), thus, just before the beginning of the cambrian period. on the paleogeographic map of chapter 9 (permian period) in the complex of later continents and continental parts one can recognize the future: australia, antarctica, india, africa (with its whole southern part), south america, parts of north america (with alaska), siberia, kazahstan, north and south china, and malaya (see fig. 3). each of the chapters 4 to 15 treats one period of the phanerozoic eon (see contents) and contains, according to the schedule mentioned in the introduction, the following sections: history and base of the treated period; international subdivisions of the period; selected aspects of the period’s stratigraphy; numerical time scale (current status / gts 04, corrections –or:/current status and future developments; acknowledgments – further reading -selected on-line references. after the last chapter (15) the book finishes with appendix 1 (standard colors of international divisions of geologic time), appendix 2 (ratified global boundary sections and points for geologic stages) and the index. 168 acta bot. croat. 68 (1), 2009 u:\acta botanica\acta-botan 1-09\book review.vp 22. travanj 2009 13:16:37 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 169 fig. 1. fig. 2. fig. 3. fig. 4. figs. 1–4. illustrations in the book. fig. 1 – geologic time scale (p. ii). fig. 2 – current international stratigraphic chart for the precambrian… (p. 25). fig. 3 – paleogeographic map showing the distribution of the continents during the permian period (p. 85). fig. 4 – quaternary time scale (p. 155). u:\acta botanica\acta-botan 1-09\book review.vp 23. travanj 2009 13:12:17 color profile: disabled composite 150 lpi at 45 degrees the text of the book is perfect in any regard, being accompanied by many illustrations of the highest quality: drawings, photographs, in the chapters 4 to 15 predominantly photographs and profiles of gssp-localities as well as time scales containing also regional subdivisions, and in the last columns, details about biostratigraphy, magnetic stratigraphy, cycleand stableisotope stratigraphy, eustatic stratigraphy (sea level changes) etc. (see fig. 4). in this book, it is repeatedly pointed out that this is an essential reference book for all geoscientists, including researchers, students and mining professionals. the present book review, written by a non-geoscientist, has the explicit intention to draw the attention of the reader to the fact that biologists (especially paleobiologists, paleobotanists, plant phylogeneticists, plant taxonomists, plant physiologists, paleozoologists, evolutionists, cell biologists, molecular geneticists etc. as well as general biologists) are also highly interested in the concise geologic time scale 2008, which is doubtlessly a fascinating, attractive and in any regard most important book. if we remember that about a half of century ago we did not know any fact which would allow us to make even some of the simplest reasonable speculations about the beginning of the early history of our planet earth, then we must now confess that one can indeed hardly find words to express one’s admiration of the magnificent progress which the geoscientists-chronostratigraphs and their multidisciplinary coworkers accomplished in this part of geosciences. in this sense, it is even not easy to praise enough the high value of the concise geologic time scale 2008. therefore there is no doubt that this handbook will soon be on the bookshelf of any natural scientists in the world. acknowledgment the author of this book review wants to express his best thanks to the cambridge university press for giving him the permission to publish at reduced size the front cover and the pages ii, 25, 85, and 155 of the book the concise geological time scale, by james g. ogg, gabi ogg and felix m. gradstein, cambridge university press, cambridge, 2008. zvonimir devidé 170 acta bot. croat. 68 (1), 2009 u:\acta botanica\acta-botan 1-09\book review.vp 17. travanj 2009 13:24:32 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 81 (2), 2022 213 acta bot. croat. 81 (2), 213–220, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-019 issn 0365-0588 eissn 1847-8476 exogenous arginine treatment additively enhances growth and tolerance of salicornia europaea seedlings under salinity fereshteh shakhsi-dastgahian1, jafar valizadeh1, monireh cheniany2, alireza einali1* 1 university of sistan and baluchestan, faculty of science, department of biology, zahedan, iran 2 ferdowsi university of mashhad, faculty of science, department of biology, mashhad, iran abstract – the effects of foliar application of 5 mm arginine (arg) on the growth and control of salinity-induced osmotic and oxidative stresses (0, 200, 400 and 600 mm nacl) in salicornia europaea seedlings were investigated. despite higher levels of lipid peroxidation, lower membrane stability index (msi), decreased pigment content and phenolic compounds, and reduced activity of antioxidant enzymes observed under salinity, seedling growth indices, including plant height and biomass, increased significantly, and some protective and antioxidant molecules such as proline and flavonoids accumulated. soluble protein level increased at the low salt concentration (200 mm) but decreased at other doses. exogenous arg treatment alone had less or no effect on plant biomass and other metabolites, but in combination with salt, further enhanced growth parameters, msi and accumulation of soluble protein, phenolic compounds and proline. arg-induced changes under salinity were associated with decreased lipid peroxidation, flavonoids content and antioxidant enzymes activity. these results show that s. europaea seedlings are well tolerant to applied salt doses. the treatment with exogenous arg alone affects plant growth slightly, but in combination with salt, synergistically increases growth and salt tolerance of these plants by enhancing the accumulation of proline and antioxidant molecules instead of enzymatic antioxidant. keywords: arginine, lipid peroxidation, salicornia europea, salinity, proline introduction in arid and semi-arid regions, lack of rainfall and high temperatures have increased soil salinity. because most plants are sensitive to high salt contents, their growth and yield are affected by the osmotic and oxidative stresses induced by saline soils (li et al. 2011, wu et al. 2012). production of reactive oxygen species (ros) due to oxidative stress is an inevitable process in plants exposed to salt and other environmental stresses (allen 1995, garg and manchanda 2009). increased ros levels under salinity stress damage plant metabolism and destroy cell membrane lipids and proteins as well as other biomacromolecules (bor et al. 2003, gill and tuteja, 2010), which subsequently alters selective membrane permeability and causes the material to leak out of the cell (weckx and clijsters 1997). in saline areas, one of the best ways to reduce the harmful effects of salinity and increase productivity is to cultivate salt-resistant plants (oba et al. 2001). many halophyte plants can grow in saline soils due to changes in their energy metabolism (winicov and bastola 1997). the salinity tolerance mechanisms of halophytes are not well understood but may be due in part to the synthesis of proline and other osmolytes, ion homeostasis, and the activity of the antioxidant defense system to scavenge ros (hasegawa et al. 2000). l-arginine (arg) is one of the proteinogenic amino acids of plant cells, which is a precursor to the synthesis of proline, polyamines, and nitric oxide (liu et al. 2006). arg treatment improved chlorophyll synthesis and photosynthesis and prevented chlorophyll degradation and ageing (zeid 2009, mostafa et al. 2010). exogenously applying arg in plants exposed to environmental stresses upregulated antioxidant enzymes (barand et al. 2015) and induced the accumulation of compatible solutes (ramadan et al. 2019). salicornia europea l. is a halophyte plant that is widely distributed in coastal areas and salt marshes (davy et al. 2001). because it can survive in salt concentrations that are toxic to most plants, salicornia is known as a salt-tolerant plant species (flowers and colmer 2008, patel 2016). it is taken not only as a model species for salinity research, but * corresponding author e-mail: aeinali@science.usb.ac.ir mailto:aeinali@science.usb.ac.ir shakhsi-dastgahian f., valizadeh j., cheniany m., einali a. 214 acta bot. croat. 81 (2), 2022 it has also recently been considered as a potential food and pharmaceutical plant due to the accumulation of suitable nutrients such as polyphenols, fibres, and flavonoids (patel 2016). despite some studies on salt stress and the role of arg in its tolerance (nejadalimoradi et al. 2014, ramadan et al. 2019), there is no report on the possible effects of this amino acid on salt stress tolerance in salicornia. therefore, the present work aimed to evaluate the impact of exogenous arg on the growth and control of osmotic and oxidative stresses caused by salinity in s. europea. materials and methods plant materials and experimental design uniform seeds of salicornia europaea l. were obtained from the pakan bazr company at esfahan. seeds soaked in tap water for 12 h were planted in trays filled with wet cocopeat and placed at 28 ± 1 °c to germinate. the 4 to 5 cm seedlings were transferred to plastic pots (two seedlings per pot) containing 1 kg of well-watered cocopeat-perlite (2:1) in a phytotron at 25 °c, a relative humidity of 60%, and a photoperiod of 16 h light/ 8 h dark. the seedlings were fed with 1/2 hoagland’s nutrient solution at three-day intervals until their length reached 20 to 23 cm. three days before salinity treatment, the seedlings were divided into two groups of 20 pots containing 40 seedlings. one group was treated with foliar application of 5 mm arg, and the other group was left untreated and just received distilled water. both groups were exposed to four levels of nacl (0, 200, 400, and 600 mm) by the addition of salt to the 1/2 hoagland’s nutrient solution. salt and arg treatments were applied to seedlings at three-day and six-day intervals for up to 30 days, respectively. seedlings were then harvested and evaluated for morphological and biochemical responses to salinity and arg treatments. all studies were independently repeated using separate seedlings at least three times for each morphological and biochemical analysis. assessment of morphological traits and membrane stability index (msi) the measured weight characteristics included fresh weight (fw) and dry weight (dw) of both shoot and root, and longitudinal traits included shoot and root length. msi was determined according to sairam and saxena (2000). two samples of fresh shoot tissue (0.1 g) were placed separately in a test tube containing 10 ml of distilled water. one sample was heated in a water bath at 40 °c for 30 min, and the other sample was boiled at 100 °c for 10 min. after determination of the electrical conductivity (ec) of the samples, msi was calculated by the following formula: msi=1-(ec40/ec100) × 100 determination of photosynthetic pigments chlorophyll (chl) and carotenoids (car) were extracted from 0.1 g of fresh shoot tissue with 2 ml of 95% (v/v) ethanol. the extracts were filtered through a whatman no.1 filter paper, and their absorbance was read at 664, 648, and 470 nm to measure chl a, chl b, and car, respectively ( lichtenthaler and buschmann 2001). total chl was obtained from the sum of chl a and chl b. pigment contents were expressed in mg per g fresh weight. enzyme extraction and assay fresh shoot tissue (0.1 g) was ground in liquid nitrogen and homogenized with 1 ml of enzyme extraction buffer containing 100 mm cold potassium phosphate buffer (ph 7.4), 1 mm edta, 5 mm ascorbate, 50 mm 2-mercaptoethanol, and 1% (w/v) polyvinylpolypyrrolidone (pvpp). the homogenate was filtered through three layers of cheesecloth and centrifuged at 12,000 g for 10 min at 4 ºc. decoloration of the extract was done by adding 10 mg of charcoal before centrifugation. the 12,000 g supernatant was used for enzyme assay and soluble protein determination. soluble protein content was determined by bradford’s method (1976) using bovine serum albumin (bsa) as a standard. ascorbate peroxidase (apx) activity was determined spectrophotometrically by measuring the generation rate of dehydroascorbate in a reaction mixture (1 ml) containing 50 mm potassium phosphate buffer (ph 7.0), 1 mm h2o2, 0.5 mm ascorbic acid, and enzyme extract at 290 nm, assuming an extinction coefficient of 2.8 mm-1 cm-1 (chen and asada 1992). peroxidase (pox) activity was determined by evaluating the rate of guaiacol oxidation in an assay mixture (1 ml) containing 50 mm potassium phosphate buffer (ph 7.0), 5 mm guaiacol, 1 mm h2o2, and enzyme extract at 470 nm, using an extinction coefficient of 26.6 mm-1 cm-1 (srinivas et al. 1999). the assay of polyphenol oxidase (ppo) was done according to the rate of purpurogallin production in 1 ml of a reaction mixture containing 50 mm potassium phosphate buffer (ph 7.0), 40 mm pyrogallol, and enzyme extract. the assay mixture was monitored at 430 nm, and the activity of the enzyme was calculated using the extinction coefficient of 2.47 mm-1 cm-1 (nakano and asada 1981). determination of total phenolic and flavonoid contents phenol compounds were determined by the method of singleton et al. (1999) with some modifications (einali et al. 2018) using gallic acid as the standard. total flavonoids were measured according to krizek et al. (1998). fresh shoot tissue (0.1 g) was extracted with 1 ml of acidified ethanol (ethanol: acetic acid, 99:1, v/v). the extract was centrifuged at 5,000 g for 10 min, and the absorbance of the resultant supernatant was recorded at 300 nm after heating at 80 °c for 10 min. total flavonoid content was expressed as absorbance per mg of shoot tissue fw. other analytical methods proline content was estimated according to bates et al. (1973) using proline as a standard. the level of lipid peroxidation was determined by measuring malondialdehyde (mda) content as described by heath and packer (1968), assuming an extinction coefficient of 155 mm-1cm-1. arginine enhances tolerance of salicornia europaea acta bot. croat. 81 (2), 2022 215 statistical analysis all data were expressed as the mean ± standard error (se) of triple analyses. normality and equal variance were also tested. statistically significant differences were determined in a factorial design by a two-way analysis of variance (anova) at p < 0.05 using the duncan method. results effect of exogenous arg on plant growth and membrane stability during salt treatment salt and arg treatments alone or in combination increased the apparent growth of s. europaea seedlings (tab. 1, on-line suppl. fig. 1). both shoot and root lengths were tab. 1. exogenous effect of 5 mm arginine (arg) on plant longitudinal characteristics, plant biomass and membrane stability index (msi) of salicornia europaea seedlings under different concentrations of salinity. each value is the mean ± se of three independent measurement from three separate seedlings and the different letters in each column indicate significant differences among the various treatments at p < 0.05 according to the duncan test. msi (%)dry weight (g)fresh weight (g)root length (cm) shoot length (cm) arginine treatment salt treatment (mm) rootshootrootshoot 48.30±4.10a0.018±0.001e0.01±0.002g0.23±0.03c1.23±0.09f9.97±0.61d13.70±0.87c-arg0 51.10±6.30a0.029±0.002d0.04±0.003c0.25±0.07c2.76±0.28c13.00±0.93c16.90±2.24c+arg 43.70±1.30b0.021±0.004e0.02±0.003f0.57±0.06b1.88±0.33e11.97±1.05c16.00±2.00c-arg200 46.80±2.60ab0.035±0.003d0.04±0.002bc0.29±0.06c3.63±0.23b15.60±0.80b23.30±1.31b+arg 42.10±3.30b0.032±0.005d0.03±0.003d0.59±0.10b2.22±0.16e14.43±1.62b21.57±2.79b-arg400 45.20±1.20b0.060±0.002a0.05±0.003a0.26±0.03c4.80±0.24a17.77±0.38a27.43±1.67a+arg 18.80±2.10c0.042±0.003c0.03±0.003e0.96±0.10a2.68±0.19d14.03±1.04b19.80±1.91b-arg600 44.70±4.40b0.053±0.004b0.04±0.004ab0.22±0.08c3.39±0.25b15.77±0.94b22.70±2.35b+arg fig. 1. changes in the content of the photosynthetic pigments of salicornia europaea seedlings under salinity treated with or without 5 mm arg. (a) chl a, (b) chl b, (c) total chl, (d) chl a/b, (e) total car, and (f) chl/car ratio. data are mean ± se of three independent experiments from three separate seedlings, and the different letters indicate significant differences among the various treatments at p < 0.05 according to the duncan test. shakhsi-dastgahian f., valizadeh j., cheniany m., einali a. 216 acta bot. croat. 81 (2), 2022 positively affected by salinity in a concentration-dependent manner. the highest increase in length was found in seedlings treated with a concentration of 400 mm nacl. arg treatment showed a significant effect on improving plant height either alone or in combination with salinity. however, shoot and root height remained statistically unchanged in arg-treated seedlings grown at 600 mm nacl compared to untreated controls (tab. 1). the fresh and dry weight of seedlings increased in response to salt treatment in shoot and root. plant biomass and shoot fresh weight were positively changed by arg treatment alone or in combination with salinity. however, fresh weight of root in arg-treated seedlings remained unchanged or was drastically decreased compared to untreated controls (tab. 1). salt treatment alone reduced msi at all concentrations, especially in seedlings treated with 600 mm nacl. arg treatment alone or in combination with salinity up to 400 mm did not affect msi but greatly improved this index in seedlings treated with 600 mm nacl (tab. 1). effect of exogenous arg on plant pigments during salt treatment salt treatment at all concentrations significantly reduced the photosynthetic pigments of s. europaea seedlings (fig. 1). arg treatment alone did not change chl a content, but its effect in combination with salinity depended on the salt concentration (fig. 1a). in contrast, arg treatment alone significantly reduced chl b levels but had no effect when applied in combination with salinity (fig. 1b). total chl content was positively affected by arg treatment only in seedlings grown at 200 mm nacl (fig. 1c). the chl a/b ratio increased in response to both salinity and arg treatments (fig. 1d). however, this ratio decreased in arg-treated seedlings grown in 600 mm nacl. arg treatment enhanced carotenoid content in 200 mm nacl-grown seedlings but did not change it when applied alone or in combination with other salinities (fig. 1e). the chl / car ratio was affected by 200 mm nacl but not by other salt concentrations. arg treatment alone or in combination with 200 mm nacl reduced this ratio but was not effective at other salt doses (fig. 1f). effect of exogenous arg on plant phenolics during salt treatment salt treatment at all concentrations caused a significant reduction in the total phenol concentration of s. europaea seedlings (fig. 2a). arg treatment alone decreased phenol content, while it was effective in a salt-dependent manner when applied to seedlings grown at different salinity concentrations. in contrast, the flavonoid content of seedlings grown at 400 and 600 mm nacl enhanced compared to the control (fig. 2b). while arg treatment reduced the level of seedling flavonoids exposed to the mentioned salt doses, it was ineffective when used alone or with 200 mm nacl (fig. 2b). effect of exogenous arg on proline, soluble protein and lipid peroxidation during salt treatment the proline content in seedlings grown at salinity up to 400 mm remained statistically unchanged but increased significantly in response to 600 mm nacl (fig. 3a). arg fig. 2. changes in total phenol (a) and total flavonoid (b) contents of salicornia europaea seedlings under salinity treated with or without 5 mm arginine (arg). values are mean ± se of three independent experiments from three separate seedlings. the different letters show significant differences among the various treatments at p < 0.05 according to the duncan test. fig. 3. changes in proline (a), soluble protein (b), and malondialdehyde (mda) (c) contents of salicornia europaea seedlings under salinity treated with or without 5 mm arginine (arg). data are mean ± se of three independent experiments from three separate seedlings. the different letters indicate significant differences among the various treatments at p < 0.05 according to the duncan test. arginine enhances tolerance of salicornia europaea acta bot. croat. 81 (2), 2022 217 treatment alone did not change proline levels, whereas this metabolite was highly accumulated when combined with salt. arg-treated seedlings grown in 600 mm nacl showed more than 4-fold proline accumulation than untreated controls (fig. 3a). soluble protein concentration increased in 200 mm nacl-grown seedlings but decreased in seedlings fed higher doses of salt. protein content was not affected by arg treatment alone or with salt up to 400 mm, whereas when combined with 600 mm nacl, it changed positively (fig. 3b). lipid peroxidation was increased by salinity in a dosedependent manner. arg treatment increased the mda content in seedlings nourished with salt-free medium but decreased it in plants grown in combination with salt (fig. 3c). changes in enzyme activities in arg-treated seedlings during salt treatment the activities of apx, pox and ppo in s. europaea seedlings were negatively changed by salt treatments (fig. 4). exogenous arg treatment highly decreased the apx activity of salt-treated or salt-untreated seedlings (fig. 4a). however, pox activity of seedlings treated with arg alone or in combination with salinity up to 400 mm remained roughly unchanged but decreased abruptly in seedlings growing at 600 mm nacl compared to untreated controls (fig. 4b). in contrast, ppo activity was positively affected by arg treatment alone or in combination with salt (fig. 4c). discussion in contrast to the destructive effects of salinity on plant growth and biomass in most plants (qiu et al. 2014, ahmad et al. 2018, ghanem et al. 2021), our results showed that salinity could have a positive effect on height, fresh weight, and biomass of s. europaea seedlings. similarly, the maximum growth of another salicornia species (s. dolichostachya) was observed at 300 mm nacl (katschnig et al. 2013), which is consistent with our results. arg treatment alone or with salt has an additive effect on plant growth and biomass. this agrees with most studies showing that foliar spraying of mung bean (qados 2010), wheat (mostafa et al. 2010), and sunflower (nejadalimoradi et al. 2014, ramadan et al. 2019) with arg increased the growth of these plants under salinity stress. the ameliorative effect of arg on plant growth under salinity has been attributed to its role as a precursor of polyamines (mostafa et al. 2010) or as a source of nitric oxide (no) (ramadan et al. 2019). however, the negative effect of arg on root fw during salinity was noticeable. in contrast to the synergic effects of arg and salinity on plant growth, root fw showed an antagonistic relationship between arg and salinity treatments. due to the enhancement effect of arg and salt treatments on root biomass, this diminishing effect may refer to the potential for water maintenance in root tissues of salt-treated seedlings in the presence of arg. reduction of photosynthetic pigments of s. europaea occurred under salt treatment, which is consistent with other studies (zeid 2009, ramadan et al. 2019, ghanem et al. 2021). the decrease in pigment content under salinity can be attributed to their degradation by free radicals generated during stress (ma et al. 2020) or activation of chlorophyll catabolic processes along with inhibition of biosynthetic enzymes (rady et al. 2015). unlike other studies (zeid 2009, ramadan et al. 2019), arg treatment alone did not affect or decrease the pigment content of s. europaea seedlings. the positive effect of arg on pigment content was observed only with 200 mm nacl. this implies that arg does not change photosynthetic pigments under high salinities. thus, the improvement in the growth of arg-treated seedlings under salinity is not directly related to photosynthetic pigments but may be due to the increased photosynthetic capacity through maintaining chloroplast structure. the reduction of lipid peroxidation during salinity due to arg treatment associated with an increase in msi could further support this suggestion. one of the effects of salinity is lipid peroxidation, which occurs in plants both sensitive to and tolerant of salinity, due to oxidative damage, although its severity is higher in salt-sensitive plants (kumar et al. 2020). accumulation of mda, as an indicator of lipid peroxidation, in s. europaea seedlings under salinity was associated with a decrease in msi, which indicates the destructive effect of salt. however, fig. 4. changes in the activity of ascorbate peroxidase (apx) (a), peroxidase (pox) (b), and polyphenol oxidase (ppo) (c) of salicornia europaea seedlings under salinity treated with or without 5 mm arginine (arg). results are mean ± se of three independent experiments from three separate seedlings. the different letters show significant differences among the various treatments at p < 0.05 according to the duncan test. shakhsi-dastgahian f., valizadeh j., cheniany m., einali a. 218 acta bot. croat. 81 (2), 2022 these seedlings had a higher growth in the presence of salt, showing that they can tolerate these salinity concentrations well. arg treatment in combination with salt but not alone, caused less lipid peroxidation and increased msi. similar results were reported in canola seedlings treated with arg under salinity (nasibi et al. 2014). this indicates that arg enhances salt tolerance in s. europaea. to counteract the oxidative damage caused by abiotic stress, plants use defense mechanisms, including the accumulation of osmo protectants and non-enzymatic compounds along with antioxidant enzymes (ali et al. 2017, polash et al. 2019). proline is a compatible solute that acts as an osmo protectant, stabilizer and protector of membranes, enzymes, and proteins, and scavenger of free radical (ashraf and foolad 2007, kumar et al. 2020). as recently demonstrated (ghanem et al. 2021), the increase in proline during salt treatments of s. europaea seedlings indicates the strategy of these plants in salt tolerance. accumulation of proline in seedlings treated with arg under salinity can be related to the positive effects of this amino acid on the production of proline, no and polyamines (liu et al. 2006). thus, a more than fourfold increase in the proline content of arg-treated seedlings exposed to 600 mm nacl could explain a decrease in lipid peroxidation versus an increase in msi in these plants. polyphenols and flavonoids, as antioxidant molecules, play an important role in scavenging free radicals by themselves and inducing antioxidant enzymes (kofroňová et al. 2020). our results showed that the content of total phenols decreases, but the flavonoids increase during salinity. however, increases in both polyphenols and flavonoids have been reported in plants under salt stress (lim et al. 2012, sarker et al. 2019). the phenolics changes during salinity were associated with a decrease in ppo activity, which can be related to salt tolerance. this is consistent with studies showing that decreased ppo activity is associated with stress tolerance (thipyapong et al. 2004, sofo et al. 2005). it indicates that among phenolic compounds, flavonoids may have a role in s. europaea tolerance to salinity. in contrast, arg treatment combined with salinity had an additive effect on total phenols but decreased flavonoids content along with an increase in ppo activity. considering the role of arg in enhancing the growth of s. europaea seedlings under salinity, arg treatment probably induces the production of phenolic compounds other than flavonoids to scavenge free radicals. it means that arg with salt can change the pattern of phenolic production with ppo activity to induce high salt tolerance. this result in conflict with ppo activity points to the unclear role for the enzyme in stress tolerance, as reported previously (boeckx et al. 2015). the soluble protein content of s. europaea increased at 200 mm nacl but decreased under more salt concentrations (400 and 600 mm). this is consistent with previous reports of pisum sativum (velitcukova and fedina 1998) and tomato (manan et al. 2016) under salt stress, indicating a decrease in soluble protein levels due to inhibition of protein biosynthesis or increased protein degradation (mersie and singh 1993). arg treatment increased the protein levels of seedlings fed with 600 mm nacl. the positive effect of arg on the increase of soluble proteins, which has also been reported in lupinus termis under salinity ( akladious and hanafy 2018), refers to the synthesis of specific proteins that are involved in the salinity tolerance of plants (qados, 2010). in contrast to the findings of most studies (ali et al. 2017, polash et al. 2019, kumar et al. 2020), in this research the activity of antioxidant enzymes, such as apx and pox as h2o2-decomposing enzymes in s. europaea seedlings decreased or remained unchanged in response to different salt concentrations. similarly, arg treatment decreased apx activity at all salinities but reduced pox at only 600 mm nacl. since arg is involved in the production of proline, no and polyamines (liu et al. 2006), its protective effect, increasing salt tolerance in s. europaea seedlings, can be attributed to the subsequent metabolism of this amino acid. there are two lines of evidence that support this suggestion. first, the positive effect of arg on proline accumulation, especially in 600 mm nacl, confirms the role of arg in inducing proline biosynthesis. the second line of evidence comes from a study showing that the protective effect of arg on sunflower seedlings under salt stress may be due to the release of no from arg (nejadalimoradi et al. 2014). however, in contrast to this report, our results show that arg treatment decreased the activity of apx and pox in seedlings under salinity. therefore, the role of arg in enhancing the salt tolerance of this plant may be related to proline accumulation. in the current study, salinity per se played a positive role in the growth and biomass of root and shoot in s. europaea seedlings, demonstrating that they tolerate these salt doses. salt treatment was also associated with increased lipid peroxidation and decreased msi, pigment content and phenolic compounds. soluble protein content increased at low salinity but decreased under other salt concentrations. while the activity of antioxidant enzymes decreases, proline and flavonoids accumulate during salinity. arg treatment alone exhibited a slight or no change in seedling growth and metabolite content, but in combination with salt showed a synergistic effect on enhancing growth parameters, msi and accumulation of soluble protein, phenolic compounds and proline. it was associated with decreased lipid 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(leguminosae, loteae) based on nuclear ribosomal its sequences miguel a. faria1, d. james harris2,3,*, tatiana visnevschi-necrasov2, manuel tavares de sousa4, eugénia nunes2 1 requimte, laboratório de bromatologia e hidrologia, faculdade de farmácia da universidade do porto, rua de aníbal cunha, 164, 4099-030 porto, portugal 2 cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, 4485-661 vairão, portugal. 3 departamento de biologia, faculdade de ciências da universidade do porto, 4099-002 porto, portugal. 4 estação nacional de melhoramento de plantas, estrada de gil vaz, 7350-951 elvas, portugal abstract – the nrdna its sequence determined in lotus conimbricensis in a previous phylogenetic study was unusual, in that it was almost identical to those retrieved from the morphologically distinct species l. subbiflorus. in the present study we sequenced new specimens of both species to reassess the phylogenetic position of l. conimbricensis. we conclude that the its sequence of l. conimbricensis used in the earlier analyses was most likely erroneous, and in fact l. conimbricensis is not closely related to l. subbiflorus. critical reexamination of previously published data indicates that several other similar errors may exist for other lotus species, and these should be checked before taxonomic conclusions are made. key words: lotus conimbricensis, lotus subbiflorus, its, phylogeny, taxonomy, nrdna introduction lotus is the largest genus within the tribe loteae, with approximately 130 species. historically there has been little agreement in the taxonomic literature regarding the generic limits of lotus and its infrageneric subdivision (degtjareva et al. 2006). however, this has changed considerably with the advent of phylogenetic studies based on nrits sequences. these have clearly shown that the new world species of lotus are not closely related to the acta bot. croat. 71 (1), 2012 87 * corresponding author, e-mail: james@mail.icav.up.pt copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\481 faria.vp 26. o ujak 2012 11:00:29 color profile: disabled composite 150 lpi at 45 degrees old world species (allan and porter 2000), and in particular degtjareva et al. (2006) revised sectional classifications proposed by kramina and sokoloff (2003) and sokoloff (1999a, b). some sections appeared as non-monophyletic, including the section lotus, which was resolved as paraphyletic since lotus conimbricensis brot. (lotus sect. erythrolotus brand) had an its sequence type identical to those found in lotus subbiflorus lag. (lotus sect. lotus). most lotus species are distinct from each other in their its sequences, so it is surprising that two morphologically dissimilar species such as l. subbiflorus and l. conimbricensis should be identical in this dna region. errors in genbank are well known (harris 2003), and so unusual results that may have important taxonomic implications deserve careful investigation. furthermore, the its region is known to display intra-individual variation in some taxonomic groups, that can confound phylogenetic studies (harris and crandall, 2000). degtjareva et al. (2006) combined new data with previously published sequences and noted that in the case of lotus creticus they obtained a very different sequence and phylogenetic placement when compared to the sequence by allan et al. (2003, 2004). the aim of this study was to sequence several individuals of both l. subbiflorus and l. conimbricensis to resolve their phylogenetic relationship, and to look for some other possible examples of discordance in previous studies by examination of sequence data available for different lotus species. materials and methods specimens were obtained from the estação nacional de melhoramento de plantas (elvas, portugal). dna was isolated from leaf tissue using standard methodologies (sambrook et al. 1989). the entire its1 and 1ts2 region was amplified using universal primers (white et al. 1990). amplifications were performed in a biometra t3 thermalcycler (biometra, goettingen, germany) in 20 ml reactions consisting of approximately 10 ng dna template, 1 mm of each primer, 200 mm of each dntp, 0.5 u ecotaq dna polymerase (ecogen, barcelona, spain), 2 ml of 10x pcr buffer and 1.5 mm mgcl2, using the following amplification protocol: initial denaturation at 95 °c for 2 min followed by 30 cycles of 95 °c for 30s, 53 °c for 30s and 72 °c for 1 min. a final extension step at 72 °c for 7 minutes was performed. pcr products were purified using the jetquick (genomed, löhne, germany) micro spin kit based on a surface modified silica membrane and sequenced using the same primers on an abi 3730 dna sequencer (applied biosystems, foster city, usa) using the kit bigdyeterminator v3.1 from the same supplier. six specimens of l. conimbricensis, and two l. subbiflorus were sequenced. 101 sequences of lotus were taken from genbank, as well as the three closest outgroups, following degtjareva et al. (2006) – cytisopsis pseudocytisus, hammatolobium lotoides and anthyllis tetraphylla. sequences were aligned using clustalw with default parameters (thompson et al. 1997). phylogenetic analysis was run using mrbayes v3.1 (huelsenbeck and ronquist 2001), using the gtr+i+g model, as selected by modeltest (posada and crandall 1998). parameters were estimated as part of the analysis, with four markov chains. the analysis was run for 106 generations, saving one tree every 100 generations. 88 acta bot. croat. 71 (1), 2012 faria m. a., harris d. j., visnevschi-necrasov t., tavares de sousa m., nunes e. u:\acta botanica\acta-botan 1-12\481 faria.vp 26. o ujak 2012 11:00:29 color profile: disabled composite 150 lpi at 45 degrees the log-likelihood values of the sample point were plotted against the generation time and trees obtained prior to reaching stationary (25%) were discarded. remaining trees were combined in a 50% majority consensus tree . two independent runs were made to check for convergence. results in total 106 taxa were analysed, including the three outgroup taxa. aligned sequences were 646 nucleotides long (383 constant sites, 208 informative sites). as expected the two species sequenced as part of this study showed limited intraspecific variation – both samples of l. subbiflorus were identical, while of the six samples of l. conimbricensis five were identical and one differed by a single c-g mutation. therefore in the analysis only one sequence per species was included. all new sequence data have been deposited in genbank (accession numbers jq655098 to jq655105). the phylogram estimated from the bayesian analysis is shown in figure 1. the its sequences of l. subbiflorus generated here are identical to several other l. subbiflorus sequences from genbank. one l. subbiflorus, however, has a very different sequence (af450160), being identical to that from two specimens of lotus arenarius (af450193 and af218528). the its sequence of l. conimbricensis previously published (af450186) is almost identical to l. subbiflorus as sequenced in this and other studies. however, sequences from our six samples of l. conimbricensis are very different from the previously reported sequence, and appeared in a different part of the tree (fig. 1). the closest relative of l. conimbricensis would be a sample of lotus halophilus (af450208), which differs by just two or three mutations. however, another sample of lotus halophilus (dq160283) is extremely distinct and part of another clade. discussion the use of nrits sequences has revolutionized lotus systematics. nevertheless, some conclusions based on poor data or uncritical data treatment can be premature or even incorrect. an example is the single specimen of lotus conimbricensis, previously sequenced and found to be almost identical to lotus subbiflorus. later studies included the same sequence from genbank and reached the same conclusions (e.g. degtjareva et al. 2006). however, it is now clear that several published sequences in genbank are anomalous, with extremely divergent sequence types recovered by different authors from the same species. there may be various explanations for it. one is that in some groups intraindividual variation is very common (harris and crandall 2000). in these cases divergent copies obtained from the same individual can appear in different phylogenetic positions. this seems unlikely in the case of lotus however, since when this happens many heterozygous positions are usually observed, unless the initial pcr products are cloned. also, when multiple individuals are sequenced, variation would be expected. although we sequenced six l. conimbricensis, only a single nucleotide difference was found. another explanation may be that considerable variation exists within species. for instance, degtjareva et al. (2006) identified an its sequence in l. creticus, which was very different from those reported by allan et al. (2003), and suggested that further studies are needed to assess intraspecific variation within this species. again, however, we think that this explanation is unlikely in most cases acta bot. croat. 71 (1), 2012 89 the phylogenetic position of lotus conimbricensis u:\acta botanica\acta-botan 1-12\481 faria.vp 26. o ujak 2012 11:00:29 color profile: disabled composite 150 lpi at 45 degrees 90 acta bot. croat. 71 (1), 2012 faria m. a., harris d. j., visnevschi-necrasov t., tavares de sousa m., nunes e. u:\acta botanica\acta-botan 1-12\481 faria.vp 26. o ujak 2012 11:00:31 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .71 (1),2012 91 t h e p h y l o g e n e t ic p o s it io n o f l o t u s c o n im b r ic e n s is fig 1. phylogram of relationships of available lotus species estimated using a bayesian approach. * indicate nodes with >95% bayesian posterior probability support. arrows indicate newly sequenced haplotypes. u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 8 1 f a r i a . v p 2 6 . o u j a k 2 0 1 2 1 1 : 0 0 : 3 2 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s of lotus. as in this study, when multiple individuals are sequenced, limited variation is generally found. this can also be seen in figure 1. when haplotypes are different, intraspecific variation can be expected to be much greater than variation found between sister species. however, in the cases of l. creticus, l. subbiflorus and l. conimbricensis, where different haplotypes were found, they were placed in completely different lineages within lotus. more importantly, the unexpected haplotypes are identical, or nearly so, to those of other species. thus one l. subbiflorus is identical to l. arenarius, and one l. conimbricensis is almost identical to the more typical l. subbiflorus. in these cases it seems that errors have been made either in identifying the sample sequenced, or during the pcr and sequencing procedures. the example of lotus creticus has recently been reassessed using additional samples (sandral et al. 2010), confirming that the sample l. creticus 2 reflects its actual relationships. unfortunately, this seems to be quite common in lotus, as it is clearly illustrated here for l. conimbricensis. various published sequences appear unusual in being very different from the other of the same species, but similar or identical to sequences of a different species. for example, lotus corniculatus and l. subbiflorus show a similar pattern to this. another example is the highly divergent sequences of lotus glinoides. two samples (dq166220 and dq166282, from dagtjareva et al. 2006) cluster with lotus schimperi, while another (af450189, from allan et al. 2003) clusters unexpectedly as part of a clade including l. corniculatus. this latter sequence could be another possible error. regarding l. conimbricensis our results unambiguously indicate that it is not closely related to l. subbiflorus as previously reported. in addition, degtjareva et al. (2008) also sequenced another individual of l. conimbricensis, and found it was not closely related to l. subbiflorus. our newly sequenced accessions of l. subbiflorus are identical to some of those previously published (degtjareva et al. 2006), so there can be no doubt regarding the identification of this species. rather our six new sequences of l. conimbricensis are quite distinct from other species of lotus, except for one sample of lotus halophilus. unfortunately, this is another critical species, with two specimens sequenced being very different from each other. in the analysis of allan et al. (2003) the relationships of these taxa are unresolved in the strict consensus trees. however, given the highly divergent and unrelated sequences, we suspect that this l. halophilus sample is another error. although many phylogenetic relationships established in earlier studies of lotus are maintained in our analysis, we recommend extreme caution in making taxonomic changes based on single specimens sequenced for this marker. not only are there general articles regarding possible errors in genbank, but various studies have found similar problems in other plant families. for example, kristiansen et al. (2005) recently highlighted how errors in genbank were responsible for the incorrect phylogenetic placement of the genus oxychloe (juncaceae) in an analysis based on rbcl sequences. by sequencing multiple individuals from the same species, the probabilities of errors are greatly reduced. at the same time true levels of intraspecific variation can be assessed in different species and sections. as has been stressed by others (e.g. hodkinson et al. 2007), dna databanks require integration with herbaria and seed banks so that cross-referencing can maximize the utilization and value of the dna collections. finally, users of the published sequences of lotus in particular should be aware that many apparent misidentifications or errors exist, and should be especially wary of basing taxonomic decisions on single specimens. 92 acta bot. croat. 71 (1), 2012 faria m. a., harris d. j., visnevschi-necrasov t., tavares de sousa m., nunes e. u:\acta botanica\acta-botan 1-12\481 faria.vp 26. o ujak 2012 11:00:32 color profile: disabled composite 150 lpi at 45 degrees acknowledgements this work was partially funded by the fct (fundação para a ciência e tecnologia) project pocti/agr/55696/2004. m. a. faria gratefully acknowledges the fct „ciência e inovação 2010”, for the attribution of grant bpd/20725/2004. thanks to the two anonymous reviewers for their helpful comments on an earlier version of the manuscript. references allan, g. j., porter, j. m., 2000: tribal delimitation and phylogenetic relationships of loteae and coronilleae (faboideae: fabaceae) with special reference to lotus: evidence from nuclear ribosomal its sequences. american journal of botany 87, 1871–1881. allan, g. j., francisco-ortega, j., santos-guerra, a., boerner, e., zimmerf, e. a., 2004: molecular phylogenetic evidence for the geographic origin and classification of canary island lotus (fabaceae: loteae). molecular phylogenetics and evolution 32, 123–138. allan, g. j., zimmer, e. a., wagner, sokoloff, w. l., 2003: molecular phylogenetic analysis of tribe loteae (leguminosae): implications for classification and biogeography. in: klitgaard, b. b., bruneao, a. (eds.), advances in legume systematics, 371–393. royal botanic garden, kew. degtjareva, g. v., kramina, t. e., sokoloff, d. d., samigullin, t. h., valiejo-roman, c. m., antonov, a. s., 2006: phylogeny of the genus lotus (leguminosae, loteae): evidence from nrits sequences and morphology. canadian journal of botany 84, 813–830. degtjareva, g. v., kramina, t. e., sokoloff, d. d., samigullin, t. h., sandral, g., valiejo-roman, c. m., 2008: new data on nrits phylogeny of lotus (leguminosae, loteae). wulfenia 15, 35–49. harris, d. j., crandall, k. a., 2000: intragenomic variation within its1 and its2 of crayfish (decapoda, cambaridae): implications for phylogenetic and microsatellite studies. molecular biology and evolution 17, 284–291. harris, d. j., 2003: can you bank on genbank? trends in ecology and evolution 18, 317–319. hodkinson, t. r., waldren, s., parnell, j. a. n., kelleher, c. t., salamin, k., salamin, n., 2007: dna banking for plant breeding, biotechnology and biodiversity evaluation. journal of plant research 120, 17–19. huelsenbeck, j. p., ronquist, f., 2001: mrbayes: bayesian inference of phylogeny. bioinformatics 17, 754–755. kramina, t. e., sokoloff, d. d., 2003: on lotus sect. erythrolotus and related taxa (leguminosae). bulletin of the moscow society of naturalists biological series 108, 59–62. kristiansen, k. a.; cilieborg, m., drábková, l., jørgensen, t., petersen g., seberg o., 2005: dna taxonomy – the riddle of oxychloë (juncaceae). systematic botany 30, 284–286. acta bot. croat. 71 (1), 2012 93 the phylogenetic position of lotus conimbricensis u:\acta botanica\acta-botan 1-12\481 faria.vp 26. o ujak 2012 11:00:32 color profile: disabled composite 150 lpi at 45 degrees posada, d., crandall, k. a., 1998: modeltest: testing the model of dna substitution. bioinformatics 14, 817–818. sandral, g., degtjareva, g. v., kramina, t. e., sokoloff, d. d., samigullin, t. h., hughes, s., valiejo-roman, c. m., 2010: are lotus creticus and lotus cytisoides (leguminosae) closely related species? evidence from nuclear ribosomal its sequence data. genetic resources and crop evolution 57, 501–514. sambrook, j., fritsch e.f., maniatis,t., 1989: molecular cloning: a laboratory manual. cold spring harbor press, new york. sokoloff, d. d., 1999a: ottleya, a new genus of papilionaceae – loteae from north america. feddes repertorium 110, 89–97. sokoloff, d. d., 1999b: what is tetragonolobus wiedmannii boiss. (fabaceae)? novitales systematicae plantarum vascularium 31, 139–142. thompson, j. d., higgins, d. g., gibson, t. j., 1994: clustal w: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position-specific gap penalties and weight matrix choice. nucleic acids research 22, 4673– 4680. white, t. j., bruns, t. d., lee, s. b., taylor, j. w., 1990: amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis, n., gelfand, d., sninsky, j., white, t. (eds.), pcr – protocols and applications – a laboratory manual, 315–322. academic press, new york. 94 acta bot. croat. 71 (1), 2012 faria m. a., harris d. j., visnevschi-necrasov t., tavares de sousa m., nunes e. u:\acta botanica\acta-botan 1-12\481 faria.vp 26. o ujak 2012 11:00:32 color profile: disabled composite 150 lpi at 45 degrees s6 acta bot. croat. 81 (1), 2022 in memoriam jozo franjić (1966–2021) croatia’s small botanical community mourns the premature and sudden loss of our professor, colleague and friend jozo franjić, who passed away on 7 november 2021. jozo franjić (fig. 1) was a croatian scientist and a full professor at the department of forestry genetics, dendrology and botany, faculty of forestry and wood technology, university of zagreb. professor franjić was born on 7 january 1966 in musić (near the town of đakovo), croatia. he completed his undergraduate studies in forestry at the faculty of forestry in 1990 and received his msc degree in 1993 and phd in biology under the mentorship of professor emeritus ivo trinajstić at the faculty of sciences, university of zagreb, in 1996. he spent his whole working life at the faculty of forestry and wood technology teaching botany and related courses. he was a devoted professor, especially emphasizing practical courses and fieldwork, trying not only to transfer knowledge about plants to students but also to arouse their interest in and love for the natural world. he always tried to arouse students’ interest in ongoing research into the complex relationships among the different parts of ecosystems. as a passionate hunter, he went around and got to know many diverse natural areas, so that he had a good feeling for how to organize very interesting fieldwork across the whole of the country, in both the lowlands and the mountains, as well as in the coastal region. many generations of foresters have happy memories of fieldwork in botany with professor franjić. furthermore, he took on many responsibilities in the management of the faculty, especially during the bolognainduced reform of study programmes. he was head of the department of research in forestry (2003–2004), head of the department of forestry genetics, dendrology and botany (2004–2008), vice dean of the department of forestry (2004–2006) and dean (2006–2008). while he was dean, the faculty moved into new buildings, equipped with laboratories and student practical work classrooms. although professor franjić always had many duties and interests, his real passion was observing and exploring the natural world around him. he was a real old-fashioned field botanist. from an early age, he was distinguished by his curiosity, refined feeling for nature and a great desire to explore and understand it, especially plants and forests. he published over 100 scientific and professional papers. he began his scientific career studying morphological variability of common oak populations in croatia. plant taxonomy and the morphological and genetic variability of various plant taxa remained the focus of his interest in the following years. his persistence and enormous energy led to the establishment and equipping of the molecular biology laboratory at the faculty of forestry and wood technology. he also researched into the distribution of alien plant species in croatia and made a great contribution to many phytosociological research in his many papers dealing with the distribution, syntaxonomy and structure of plant communities in croatia and southeast europe. while working on various scientific and professional projects, he conducted floristic and phytosociological research in almost the whole of the republic of croatia (especially in the areas of baranja and spačva, on mountains such as dilj, papuk, krndija, kalnik, žumberak, samoborsko gorje, velebit, biokovo, and also on the island of mljet) as well as in neighbouring countries. in addition, he authored numerous articles in which he popularized the flora of croatia and other parts of the world, trying to bring plants closer to foresters and to the general public. he also published four university textbooks with surveys of forest flora and woody species in the republic of croatia. fig. 1. jozo franjić (1966–2021) in memoriam acta bot. croat. 81 (1), 2022 s7 working through many scientific and professional associations, professor franjić promoted botany and nature conservation trying to connect professionals working in different disciplines (foresters, biologists, ecologists, agronomists, etc.). he always tried to apply complementary knowledge from these different scientific disciplines in nature conservation as well as in sustainable use of nature for human needs. professor franjić was a member of the academy of forestry sciences, croatian chamber of forestry and wood processing engineers, croatian forestry society, croatian botanical society, croatian biological society, eastern alpine and dinaric society for vegetation ecology, and the international association for vegetation science. unfortunately, the rich and meaningful career of professor franjić was abruptly interrupted by a short but serious illness. he had planned, started, conceived and imagined so much more. however, professor jozo franjić’s name will go down in the history of croatian botany and of forestry science as one of those productive researchers who contributed to a better knowledge and understanding of croatian flora and plant communities. to all of us he will remain a dear colleague and friend, an enthusiastic researcher and nature lover who lived his life to the fullest. željko škvorc and daniel krstonošić university of zagreb, faculty of forestry and wood technology, zagreb selected bibliography books vidaković, m., franjić, j., 2004: golosjemenjače. sveučilište u zagrebu, šumarski fakultet. zagreb. franjić, j., škvorc, ž., 2010: šumsko drveće i grmlje hrvatske. sveučilište u zagrebu, šumarski fakultet, zagreb. franjić, j., škvorc, ž., 2014: šumsko zeljasto bilje hrvatske. sveučilište u zagrebu, šumarski fakultet, zagreb. franjić, j., škvorc, ž., 2021: croatian forest plants. sveučilište u zagrebu, fakultet šumarstva i drvne tehnologije, zagreb. book chapters vidaković, m., franjić, j., 2003: razmnožavanje obične bukve. in: matić, s. (ed.), obična bukva (fagus sylvatica l.) u hrvatskoj, 264–271. akademija šumarskih znanosti, zagreb. trinajstić, i., franjić, j., škvorc, ž., 2005: flora poplavnih i močvarnih šuma. in: vukelić, j. (ed.), poplavne šume u hrvatskoj, 93–99. akademija šumarskih znanosti, zagreb. franjić, j., škvorc, ž., 2010: biljni svijet park–šuma grada zagreba. in: matić, s., anić, i., (eds.), park–šume grada zagreba, 45–50. akademija šumarskih znanosti, zagreb. trinajstić, i., franjić, j., idžojtić, m., škvorc, ž., 2011: taksonomska problematika i rasprostranjenost glavnih vrsta drveća. in: matić, s. (ed.), šume hrvatskoga sredozemlja, 162–171. akademija šumarskih znanosti, zagreb. franjić, j., temunović, m., 2022: taksonomski status poljskog jasena (fraxinus angustifolia vahl, oleaceae). in: anić, i. (ed.), poljski jasen (fraxinus angustifolia vahl) u hrvatskoj. akademija šumarskih znanosti, zagreb. peer–reviewed journal papers franjić, j., 1991: rasprostranjenost vrste cornus hungarica kárpati u hrvatskoj. šumarski list 115, 461–466. trinajstić, i., franjić, j., kajba, d., samardžić, j., 1991: današnje stanje rasprostranjenosti vrste reynoutria japonica houtt. (polygonaceae) u hrvatskoj. fragmenta herbologica 20, 63– 67. franjić, j., 1992: nova nalazišta vrste eranthis hyemalis (l.) salisb. (ranunculaceae) u hrvatskoj. acta botanica croatica 51, 131–134. franjić, j., 1992: some morphological differences between species cornus australis c.a. meyer and cornus hungarica kárpati (cornaceae). natura croatica 1, 13–18. trinajstić, i., pavletić, zi., franjić, j., liber, z., 1992: prilog poznavanju korovne f lore makarskoga područja (dalmacija, hrvatska). fragmenta phytomedica et herbologica 21, 57– 62. franjić, j., 1993: veličina žira kao pokazatelj individualne varijabilnosti hrasta lužnjaka (quercus robur l.). glasnik za šumske pokuse, posebno izdanje 4, 195–205. franjić, j., 1993: nova nalazišta vrste datura inoxia miller ( solanaceae) u hrvatskoj. acta botanica croatica 52, 97–100. trinajstić, i., franjić, j., kajba, d., 1993: dosadašnje stanje rasprostranjenosti vrste galinsoga quadriradiata ruiz et pavon (asteraceae) u hrvatskoj. fragmenta phytomedica et herbologica 1, 117–121. britvec, m., dubravec, k., žutić, i., franjić, j., 1993: anatomska građa sjemene ljuske domaćih kultura i populacija graha (phaseolus vulgaris l.). poljoprivredna znanstvena smotra 58, 215–232. franjić, j., 1994: morphometric leaf analysis as an indicator of common oak (quercus robur l.) variability in croatia. annales forestales 19, 1–32. trinajstić, i., franjić, j., 1994: ass. salicetum elaeagno–daphnoides (br.-bl. et volk 1940) m. moor 1958 (salicion elaeagni) in the vegetation of croatia. natura croatica 3, 253–256. trinajstić, i., franjić, j., 1994: prilog poznavanju rasprostranjenosti vrste impatiens glandulifera royle (balsaminaceae) u hrvatskoj. fragmenta phytomedica et herbologica 22, 21–24. trinajstić, i., franjić, j., kajba, d., 1994: prilog poznavanju rasprostranjenosti vrste reynoutria japonica houtt. ( polygonaceae) u hrvatskoj. acta botanica croatica 53, 145–149. franjić, j., 1995: present distribution of the pubescent birch ( betula pubescens ehrh., betulaceae) in croatia. acta botanica croatica 54, 125–129. franjić, j., 1995: dosadašnje stanje rasprostranjenosti vrste cornus hungarica kárpati u hrvatskoj. šumarski list 119, 119–123. franjić, j., trinajstić, i., 1995: morphologische merkmale kroatischer populationen der taxa aristolochia lutea desf. und a. pallida willd. (aristolochiaceae). annali del museo civico di rovereto, sezione archeologia, storia e scienze naturali 11, suppl. 2, 231–241. trinajstić, i., franjić, j., 1995: as. impatienti-solidaginetum m. moor 1958 (calistegion sepium) u vegetaciji republike hrvatske. fragmenta phytomedica et herbologica 23, 25–30. krčmar, s., durbešić, p., franjić, j., 1995: uzimanje krvnog obroka nekih vrsta obada (diptera, tabanidae) na konjima (equus cabalus). stočarstvo 49, 15–21. gračan, j., trinajstić, i., orešković, ž., perić, z., franjić, j., 1995: growth of common oak (quercus robur l.) provenances in croatia. radovi šumarskog instituta jastrebarsko 30, 109– 123. in memoriam s8 acta bot. croat. 81 (1), 2022 franjić, j., pandža, m., 1995: morfometrijska analiza ploda kao pokazatelj varijabilnosti hrasta crnike (quercus ilex l.). ekološke monografije 4, 299–308. franjić, j., 1996: multivariate analysis of leaf properties in the common oak (quercus robur l., fagaceae) populations of posavina and podravina in croatia. annales forestales 21, 23–60. franjić, j., 1996: morfometrijska analiza varijabilnosti lista posavskih i podravskih populacija hrasta lužnjaka (quercus robur l., fagaceae) u hrvatskoj. glasnik za šumske pokuse 33, 153–214. franjić, j., trinajstić, i., 1996: sadašnje stanje rasprostranjenosti vrste datura inoxia miller (solanaceae) u hrvatskoj. fragmenta phytomedica et herbologica 24, 5–9. trinajstić, i., franjić, j., 1996: listovi kratkoga plodnoga izbojka, osnova za morfometrijsku analizu lista hrasta lužnjaka (quercus robur l., fagaceae). in: matić, s., gračan, j., (eds.), skrb za hrvatske šume od 1846. do 1996. unapređenje proizvodnje biomase šumskih ekosustava 1, 169–178. hrvatsko šumarsko društvo, zagreb. trinajstić, i., franjić, j., samardžić, i., samardžić j., 1996: fitocenološke značajke šuma sladuna i cera (as. quercetum frainetto-cerris rudsk i 1949) u slavoniji (hr vatska). šumarski list 120, 305–321. krstinić, a., trinajstić, i., gračan, j., franjić, j., kajba, d., britvec, m., 1996: genetska izdiferenciranost lokalnih populacija hrasta lužnjaka (quercus robur l.) u hrvatskoj. in: matić, s., gračan, j., (eds.), skrb za hrvatske šume od 1846. do 1996. zaštita šuma i pridobivanje drva 2, 159–168. hrvatsko šumarsko društvo, zagreb. franjić, j., 1997: current state of distribution of the species eranthis hyemalis (l.) salisb., (ranunculaceae) in croatia. natura croatica 6, 125–130. franjić, j., škvorc, ž., 1997: zimotrenost nekih vrsta hrastova (quercus l., fagaceae) na krndiji i dilju (slavonija, hrvatska). šumarski list 121, 599–607. franjić, j., trinajstić, i., škvorc, ž., 1998: prilog poznavanju širenja nekih neofita u hrvatskoj. fragmenta phytomedica et herbologica 26, 5–17. trinajstić, i., franjić, j., 1999: waterplant and swamp vegetations of velika and mala čambina in podravina (croatia). periodicum biologorum 101, 237–243. franjić, j., trinajstić, i., 1999: interspecific and intraspecific variability of aristolochia lutea desf. and a. pallida willd. ( aristolochiaceae) in croatia. periodicum biologorum 101, 259–264. franjić, j., trinajstić, i., škvorc, ž., presečan, m., samardžić, i., 1999: a contribution to the knowledge of the distribution of equisetum hyemale l. (equisetaceae) in croatia. natura croatica 8, 95–100. franjić, i., gračan, j., kajba, d., škvorc, ž., dalbelo–bašić, b., 2000: multivariate analysis of leaf shape of the common oak (quercus robur l.) in the ”gajno” provenance test (croatia). glasnik za šumske pokuse 37, 469–479. trinajstić, j., franjić, j., škvorc, ž., 2000: a new locality for the ass. acoro-glycerietum maximae slavnić 1956 (phragmition) in croatia. natura croatica 9, 163–167. franjić, j., škvorc, ž., krčmar, s., 2001: da li je naglo širenje vrste cornus hungarica kárpati u slavoniji i baranji posljedica promjenjenih stanišnih uvjeta? osječki zbornik 24/25, 181– 184. franjić, j., škvorc, ž., čarni, a., 2001: numerička analiza fitocenoloških snimaka u bukovo-jelovim šumama (abietifagetum s.l.) u hrvatskoj. šumarski list 125, 19–26. franjić, j., dalbelo-bašić, b., škvorc, ž., 2001: acorn form variability in the common oak (quercus robur l.) in croatia. sauteria 11, 383–394. trinajstić, i., franjić, j., škvorc, ž., 2001: water plant and swamp vegetation of virovi in posavina (croatia). natura croatica 10, 305–313. trinajstić, i., franjić, j., škvorc, ž., 2001: floristička analiza as. impatienti-solidaginetum m. moor 1958 (calystegion sepium) u međimurju. agronomski glasnik 6, 305–313. pandža, m., franjić, j., trinajstić, i., škvorc, ž., stančić, z., 2001: the most recent state of affairs in the distribution of some neophytes in croatia. natura croatica 10, 259–275. franjić, j., škvorc, ž., 2001: dosadašnji rezultati istraživanja varijabilnosti hrasta lužnjaka (quercus robur l., fagaceae) u hrvatskoj. in: matić, s., krpan, a.p.b., gračan, j., (eds.), znanost u potrajnom gospodarenju hrvatskim šumama, 53– 59. šumarski fakultet zagreb, šumarski institut jastrebarsko, hrvatske šume zagreb škvorc, ž., franjić, j., 2001: morfološka diferencijacija hrastova medunca (quercus pubescens willd.) i duba (q. virgiliana / ten./ ten.) u hrvatskoj. in: matić, s., krpan, a.p.b., gračan, j., (eds.), znanost u potrajnom gospodarenju hrvatskim šumama, 133–140. šumarski fakultet zagreb, šumarski institut jastrebarsko, hrvatske šume zagreb. trinajstić, i., franjić, j., škvorc, ž., 2002: fitocenološke značajke as. impatienti-solidaginetum m. moor 1958 (calystegion sepium) u hrvatskoj. fragmenta phytomedica et herbologica 27, 25–30. čarni, a., franjić, j., škvorc, ž., 2002: vegetacija grmastih šumskih rubova u slavoniji. šumarski list 126, 1–10. franjić, j., škvorc, ž., pandža, m., kekelić, b., 2002: šumska vegetacija poluotoka oštrica (dalmacija, hrvatska). šumarski list 126, 11–18. pandža, m., franjić, j., škvorc, ž., 2002: actual status of the spread of ass. oleo-euphorbietum dendroidis trinajstić 1973 (oleo-ceratonion) in croatia. acta botanica hungarica 44, 357–369. pandža, m., franjić, j., škvorc, ž., 2002: the flora of some uninhabited šibenik archipelago islands (dalmatia, croatia). natura croatica 11, 367–385. trinajstić, i., franjić, j., škvorc, ž., 2003: sintaksonomska analiza bukovih šuma međimurja (hrvatska). šumarski list 127, 3–9. franjić, j., pandža, m., škvorc, ž., 2003: rasprostranjenost vrste anthyllis barba-jovis l. (fabaceae) u hrvatskoj. šumarski list 127, 45–49. čarni, a., franjić, j., škvorc, ž., 2004: crataegus nigra waldst. et kit. dominated community in the flooded danube river area in croatia. hacquetia 3, 81–90. pandža, m., franjić, j., škvorc, ž., trinajstić, i., pavletić, zi., 2004: šumska vegetacija otoka murtera. radovi šumarskog instituta jastrebarsko 39, 131–162. škvorc, ž., franjić, j., idžojtić, m., 2005: population structure of quercus pubescens willd. (fagaceae) in croatia according to morphology of leaves. acta botanica hungarica 47, 193–206. ballian, d., škvorc, ž., franjić, j., kajba, d., bogdan s., bogunić, f., 2005: procjena nekih morfoloških značajki munike (pinus heldreichii christ.) u dijelu areala. šumarski list 129, 475– 480. pandža, m., franjić, j., škvorc, ž., 2005: weed and ruderal vegetation (stellarietea mediae r. tx. et al. ex von rochow 1951) in the central part of the east adriatic coast. periodicum biologorum 107, 361–372. in memoriam acta bot. croat. 81 (1), 2022 s9 franjić, j., škvorc, ž., filipović, k., vitasović-kosić, i., 2005: phytosociological characteristics of quercus cerris l. forests in east slavonia (croatia). hacquetia 4, 27–35. čarni, a., franjić, j., šilc, u., škvorc, ž., 2005: floristical, ecological and structural diversity of vegetation of forest fringes of the northern croatia along a climatic gradient. phyton 45, 287–303. idžojtić, m., kogelnik, m., franjić, j., škvorc, ž., 2006: hosts and distribution of viscum album l. ssp. album in croatia and slovenia. plant biosystems 140, 50–55. franjić, j., škvorc, ž., čarni, a., 2006: rasprostranjenost panonskoga crnog gloga (crataegus nigra waldst. et kit.) u hrvatskoj i njegov značaj u formiranju vegetacije šumskih rubova. šumarski list 130, 3–8. franjić, j., liber, z., škvorc, ž., idžojtić, m., šoštarić, r., stančić, z., 2006: morphological and molecular differentiation of the croatian populations of quercus pubescens willd. 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10000 zagreb, croatia 2 university of split, faculty of philosophy, teslina 12, 21000 split, croatia abstract – a new member of the croatian flora, pimpinella tragium vill. subsp. lithophila (schischk.) tutin (apiaceae), was found on the island of vis in the central adriatic. it grows on the calcareous rocky coast near the sea and is a member of a halophilous community within the crithmo-limonion br.-bl. et molinier 1934 alliance. morphological similarities within the pimpinella tragium group are briefly discussed. a determination key is given for all croatian pimpinella taxa. keywords: pimpinella, taxonomy, flora, island of vis, adriatic, croatia introduction the genus pimpinella l. (apiaceae) is represented by approximately 150 species in europe, asia and africa. these are annual, biennial and perennial species, usually growing on dry rocky places rocky crevices, fields, meadows, mountain pastures and grasslands (hartvig 1968, tutin 1968 a, pignatti 1982, velayos 2003). in the croatian flora according to hirc (1908), degen (1937), domac (1994: 235), luka^ (1997:113) and flora croatica database (nikoli] 2010) the genus pimpinella is represented by seven taxa. these are p. alpina host, p. anisum l., p. major (l.) huds., p. peregrina l., p. saxifraga l., p. tragium vill. and p. tragium vill. subsp. polyclada (boiss. et heldr.) tutin. among them is p. tragium vill. which is distributed in the mediterranean area from spain to lebanon, north-west africa, asia minor, the caucasus, and in the south of the east european plain (yurtseva and tikhomirov 1998). in the countries and territories around croatia and the adriatic basin, p. tragium has been noted for italy (pignatti 1982, conti et al. 2005), serbia (sari] and dikli] 1986), macedonia (matevski 2005), bosnia and herzegovina (beck-mannagetta 1927), montenegro (rohlena 1942), albania (qosja et al. 1992) and greece (hartvig 1968). acta bot. croat. 70 (1), 2011 115 * corresponding author, e-mail: sandro@botanic.hr copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\bogdanovic.vp 28. o ujak 2011 16:54:32 color profile: disabled composite 150 lpi at 45 degrees pimpinella tragium represents a variable group of taxa with great morphological variability and confused taxonomy (tutin 1968 a, b; yurtseva and tikhomirov 1998). some authors (tutin 1968 a, b; matthews 1972; engstrand 1987; brullo and brullo 2009) distinguish several subspecies within this group: p. tragium subsp. lithophila (schischk.) tutin distributed in southern europe, crimea, turkey and iran, p. tragium subsp. depressa (sieber ex spreng.) tutin from crete, p. tragium subsp. pseudotragium (dc.) matthews from turkey, iran, armenia and iraq, p. tragium subsp. titanophila (woronow) tutin from east europe, p. tragium subsp. polyclada (boiss. et heldr.) tutin from the balkans and asia minor and p. tragium subsp. glauca (c. presl) c. brullo et brullo from sicily. materials and methods field research on the central adriatic island of vis was undertaken from 2007 to 2010. for determination of plant species we used relevant taxonomic literature (hartvig 1968; tutin 1968 a, b; pignatti 1982; velayos 2003; matevski 2005). all the localities from herbaria, literature data and field observations were geocoded in the flora croatica database (nikoli] 2010). the positions of the localities of pimpinella tragium subsp. lithophila were determined by gps garmin etrex vista and a distribution map of the newly recorded taxon was produced by combined usage of esri gis arcmap 9.2 tool and the flora croatica database (nikoli] 2010). voucher specimens of p. tragium subsp. lithophila are deposited in cnhm and za herbarium. results a new taxon, pimpinella tragium subsp. lithophila (fig. 1 a) was found within halophytic vegetation on the northern side of the island of vis, in the vicinity of the village oklju~ina and of the town of vis. description pimpinella tragium vill. subsp. lithophila (schischk.) tutin in feddes repert. 79 (1–2): 62 (1968). synonyms: pimpinella lithophila schischk., not. syst. (leningrad) 12: 206 (1950), pimpinella tragium var. typica halácsy, concept. fl. graec. 1: 682 (1901). plant perennial, up to 60(–100) cm high, pubescent or crisped-tomentose, rarely glabrescent or glabrous. stems erect, basal parts form rhizomes covered by numerous scale-like remains of petioles. rosette leaves numerous, basal leaves 5–25 cm long, long petiolate, oblong, ovate in outline, pinnatisect or bipinnatisect, with 3–7 pairs of segments, segments cuneate, ovate or orbiculate, crenate to deeply dentate. upper cauline leaves vaginate with small sessile reduced lamina, usually reduced to a sheath. umbels with 5–15(–20) unequal rays, densely or sparsely pubescent. bracts and bracteoles absent or 1–2. umbellula with 10–20 white flowers, sometimes pinkish; petals to 1 mm long, hairy on dorsal surface. fruit 2–3 mm long, ovoid, densely tomentose, hairs short (fig. 1 b). stylopodium cushion-shaped, hemispherical, stylodium 1–2.5 mm long. flowering time from jun to august. chromosome number: 2n = 20. life form: chamaephyte. 116 acta bot. croat. 70 (1), 2011 bogdanovi] s., ru[^i] m. u:\acta botanica\acta-botan 1-11\bogdanovic.vp 28. o ujak 2011 16:54:32 color profile: disabled composite 150 lpi at 45 degrees distribution in croatia: central adriatic, dalmatia, island of vis (fig. 2). specimina visa: croatia: island of vis, tiha inlet, slatina inlet rocky shore, may 18, 2008, ru{~i} s.n. (za); island vis, tiha inlet rocky shore near oklju~ina, june 15, 2008, bogdanovi} and ru{~i}, s.n. (cnhm, za); island of vis, dobra inlet shore, june 15, 2008, bogdanovi} and ru{~i}, s.n. (cnhm, za); island of vis, mala travna inlet rocky shore near oklju~ine, june 12, 2010, bogdanovi} and bor{i} s.n. (za). acta bot. croat. 70 (1), 2011 117 new pimpinella in croatia fig. 1. pimpinella tragium subsp. lithophila. a – habit on the island of vis, b – tomentose ovaries (young fruits) (photo by s. bogdanovi}). fig. 2. distribution of pimpinella tragium subsp. lithophila in croatia. u:\acta botanica\acta-botan 1-11\bogdanovic.vp 28. o ujak 2011 16:54:35 color profile: disabled composite 150 lpi at 45 degrees discussion for the croatian flora, the species pimpinella tragium at a specific level was cited by visiani (1842:34) in his flora dalmatica he wrote »hab. ad rupes maritimas insulae lissa«. afterwards, hirc (1908) and domac (1955) reported this finding for the island of vis, but without any precise locality. trinajsti] (1998) also cited visiani’s (1842) and petter’s (1852) localities for the island of vis. it is clearly evident that nether domac nor trinajsti} had found p. tragium in the flora of the island of vis. for the island of svetac, p. tragium has been recorded by pavleti] (1978). according to hirc (1908:69), potential localities of the species p. tragium could be found on the island of krk and around rijeka. on these sites p. tragium was never confirmed and remain dubious. in za and zaho herbaria there were no herbarium specimens of this taxon. pimpinella tragium subsp. lithophila in croatia was found along the northern coast of the island of vis, near the village of oklju~ina, in the bays of tiha, slatina, mala travna and dobra near the town of vis. these are all littoral localities influenced by the sea spray, where p. tragium subsp. lithophila was found on the calcareous rocky places near the sea. a new taxon grows in the narrow belt along the coast, as a member of halophilous vegetation within crithmo-limonion br.-bl. et molinier 1934 alliance. on those sites p. tragium subsp. lithophila grows together with lotus cytisoides l., crithmum maritimum l., limonium subanfractum trinajsti}, reichardia picroides (l.) roth, parapholis incurva (l.) c. e. hubb., anthyllis vulneraria (l.) subsp. praepropera (a. kern.) bornm., centaurium erythrea rafn, desmazeria rigida (l.) tutin, d. marina (l.) druce, blackstonia perfoliata (l.) huds., valantia muralis l., silene sedoides poir., scorpiurus muricatus l., trifolium angustifolium l., centaurea issaea lovri}, brassica incana ten., allium commutatum guss., helichrysum italicum (roth) g. don and others. those rocky littorals of the bays tiha, slatina, mala travna and dobra are the only know localities of this taxon in croatia. recently, the island of vis has become an important plant area (ipa) where those sites are included (bogdanovi] and vukovi] 2010). morphological variability and density of leaf indumentum, as well as leaf incision and geographical pattern of those morphological characters along the whole geographic area of the p. tragium, make the taxonomy of the p. tragium group difficult. based on anatomical and morphological study of the p. tragium group, two types of plants differing mainly in the hair length on leaves and on the lower part of the stem can be distinguished. p. tragium with hairs 0.05–0.2 mm long and p. polyclada 0.3–1 mm long (yurtseva and tikhomirov 1998). although the taxonomy of the p. tragium group has been studied by yurtseva and tikhomirov (1998), it still remains unclear and unresolved. yurtseva and tikhomirov (1998) suggest including p. tragium subsp. lithophila and some other subspecific taxa (p. tragium subsp. depressa and p. tragium subsp. titanophila) of this group under the name of p. tragium, while suggesting treating p. tragium subsp. polyclada at the specific level as p. polyclada boiss. et heldr. from a morphological point of view, p. tragium subsp. lithophila has smaller leaf lobes that are 5–10(12) mm long with a deeply serrate margin, while p. tragium subsp. polyclada has bigger leaf lobes that are 10–15(20) mm long and a margin that is not deeply serrate (hartvig 1968; tutin 1968 a, b; velayos 2003; matevski 2005). here we adopt the opinion of tutin (1968 a, b), velayos (2003) and matevski (2005) and treat this new taxon for the croatian flora at the subspecific level. 118 acta bot. croat. 70 (1), 2011 bogdanovi] s., ru[^i] m. u:\acta botanica\acta-botan 1-11\bogdanovic.vp 28. o ujak 2011 16:54:36 color profile: disabled composite 150 lpi at 45 degrees determination key for croatian pimpinella taxa morphological characters and description of the taxon pimpinella tragium subsp. lithophila were compared with those of p. tragium subsp. polyclada, since it is the only known subspecies of this group in croatian flora. here we give determination key for the croatian taxa of the genus pimpinella: 1 ovary and fruit glabrous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 – ovary and fruit pubescent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 stem usually sharply angled, hollow; ridges of fruit prominent, whitish . . . . p. major – stem usually cylindrical, nearly or quite solid; ridges of fruit inconspicuous . . . . . 3 3 petals on abaxial side pubescent, stem cylindrical, glabrous, hairy only in basal part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. saxifraga – petals on abaxial side glabrous, stem slightly angled, completely glabrous, shiny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. alpina 4 plants perennial, stem ligneous at base; with fibres and scale-like leaf bases p. tragium 5 – plants annual or biannual; without fibres or scale-like leaf bases . . . . . . . . . . . 6 5 plant slender; leaf lobes 5–10(12) mm long, margin incise-serrate . . subsp. lithophila – plant stout; leaf lobes 10–15(20) mm long, margin not deeply serrate subsp. polyclada 6 fruit with long patent hairs; umbel with more than 15 rays . . . . . . . . p. peregrina – fruits with short appressed hairs; umbel with 2–12 rays . . . . . . . . . . . p. anisum acknowledgements field investigations were supported by the project »conservation and sustainable use of biodiversity in the dalmatian coast through greening coastal development – coast«. we extend our thanks to our colleague igor bor{i}, for providing distribution map and comments on the manuscript. references beck-mannagetta, g., 1927: flora bosnae, hercegovinae et regiones novipazar, 3. choripetalae. glasnik zemaljskog muzeja u bosni i hercegovini 63, 1–487. bogdanovi], s., vukovi], n., 2010: vis. in: nikoli], t., topi], j., vukovi], n. (eds.), important plant areas of croatia (in croatian), 454–459. prirodoslovno-matemati~ki fakultet i [kolska knjiga, zagreb. brullo, c., brullo, s., 2009: considerazioni su alcune specie critiche della flora sicula. in: peccenini, s., domina, g. (eds.), gruppi critici della flora d’italia, 43–44. società botanica italiana, firenze. conti, f., abbate, g., alessandrini, a., blasi, c. (eds.), 2005: an annotated checklist of the italian vascular flora. palombi editori, roma. degen, a., 1937: flora velebitica. verlag der ungar, akademie der wissenschaften, budapest. domac, r., 1994: flora of croatia – handbook for plant identification (in croatian). [kolska knjiga, zagreb. acta bot. croat. 70 (1), 2011 119 new pimpinella in croatia u:\acta botanica\acta-botan 1-11\bogdanovic.vp 28. o ujak 2011 16:54:36 color profile: disabled composite 150 lpi at 45 degrees domac, r., 1955: flora of the island of vis (in croatian). acta pharmaceutica jugoslavica 5, 3–42. engstrand, l., 1987: pimpinella l. in: rechinger, k. h. (ed.), flora iranica 126, 324–325. graz. hartvig, p., 1986: pimpinella l. in: strid, a. mountain flora of greece 1, 677–680. cambridge university press, cambridge. hirc, d., 1908: revision of croatian flora (in croatian). rad jugoslavenske akademije znanosti i umjetnosti 173, 38–136. luka^, g., 1997: pimpinella l. in: nikoli], t. (ed.), flora croatica. index florae croaticae 2. natura croatica 6, suppl. 1, 113. matevski, v., 2005: pimpinella l. in: micevski, k. (ed.), the flora of the republic of macedonia 1(6), 1564–1567. macedonian academy of science and arts, skopje. matthews, v. a., 1972: pimpinella l. in: davis, p. h. (ed.), flora of turkey and the east aegean islands 4, 360–362. edinburgh university press, edinburgh. nikoli], t. (ed.), 2010: flora croatica database. department of botany and botanical garden, faculty of science, university of zagreb. http://hirc.botanic.hr/fcd, accessed february 2010. pavleti], zi., 1978: vascular flora of the island of svetac (in croatian). acta botanica croatica 37, 215–224. petter, f., 1852: insel–flora von dalmatien. oesterreichisches botanisches wochenblatt 2, 34. pignatti, s., 1982: flora d’italia 2. edagricole, bologna. qosja, x., paparisto, k., demiri, m., vangeli, j. (eds), 1992: flora of albania (in albanian). academie des science de la république d’albanie. le centre de researches biologiques, tirana. rohlena, j., 1942: conspectus florae montenegrinae. preslia 20/21, 1–506. sari], m. r., dikli], n., 1986: flora of serbia (in serbian), 10, suppl. 2. srpska akademija nauka i umetnosti, beograd. trinajsti]. i., 1998: vascular flora of the island of vis (in croatian). hrvatska zora, glasilo matice hrvatske, vis 21, 10. tutin, t. g., 1968a: pimpinella l. in: tutin, t. g. et al. (eds.), flora europaea 2, 331–333. cambridge university press, cambridge. tutin, t. g., 1968b: pimpinella tragium vill. feddes repertorium 79, 62. velayos, m., 2003: pimpinella l. in: castroviejo, s., nieto feliner, g., jury, s. l., herrero, a. (eds.), flora iberica 10, 181–191. real jardín botánico, madrid. visiani, r., 1852: flora dalmatica 3. lipisiae. yurtseva, o. v., tikhomirov, v. n., 1998: morphological diversity and taxonomy of the pimpinella tragium vill. group (umbelliferae – apioideae) in the mediterranean. feddes repertorium 109, 479–500. 120 acta bot. croat. 70 (1), 2011 bogdanovi] s., ru[^i] m. u:\acta botanica\acta-botan 1-11\bogdanovic.vp 28. o ujak 2011 16:54:36 color profile: disabled composite 150 lpi at 45 degrees s1 acta bot. croat. 79 (1), 2020 social news the spiridion brusina medal for 2019 has been awarded to german plant biologist professor jutta ludwig-müller since 1997 the croatian society of natural sciences has acknowledged foreign scientists who have made substantial contributions to the croatian natural science field by awarding the spiridion brusina medal. spiridion brusina, a croatian biologist and zoologist, was the founder of marine biology in croatia and the first professor in the topic at the university of zagreb. renowned scientists from many countries and different life science fields over the world have been awarded this medal. awardee for 2019 is prof. dr. jutta ludwig-müller from the faculty of biology at technische universität dresden, germany, who is honored for her accomplishments in the field of plant biology and the promotion of croatian science for the last 20 years. jutta ludwig-müller graduated in 1986 and obtained a phd in 1990 in plant science at goethe university, frankfurt, germany. she was habilitated in the field of botany in 1996 at the same university. since 1999 she has been a professor at the technische universität dresden, and since 2001 director of the dresden botany institute. she is the editor-in-chief of the journal of plant growth regulation and is a member of the editorial boards of the journal of botany, plant signaling & behavior, mutation research. she is a member of numerous scientific and professional societies, and leader of numerous projects in the field of plant biology and biotechnology. professor ludwig-müller has extensive experience and expertise in plant physiology, molecular plant biology, as well as protein biochemistry and hormone determination. her main research interest is the function and regulation of the activity of the plant hormone auxin by studying the synthesis and hydrolysis of conjugates, as well as the interaction with other plant hormones during plant growth and development. in addition, she investigates the role of auxin in the interactions of plants with pathogenic and beneficial microorganisms. professor ludwig-müller intensively studies the disease of cabbages caused by the pathogen plasmodiophora brassicae, which causes extensive damage within the brassicaceae family. working at physiological, biochemical and molecular levels, her aim is to discover possible defense mechanisms. furthermore, her work addresses the possibility of the biocontrol of this important disease with endophytes. the scientific interests of professor ludwig-müller are also focused on the response of plants to factors of abiotic stress with particular interest in the role of plant bioactive compounds, in particular the plant hormone auxin and specialized metabolites. for the last twenty years professor jutta ludwig-müller has been actively involved in cooperation with croatian scientists from several institutions in croatia: faculty of science, university of zagreb, ruđer bošković institute in zagreb, and josip juraj strossmayer university in osijek. ludwig-müller is active as the head of numerous croatiangerman bilateral projects with colleagues from the faculty of science, as a manager of an alexander von humboldt foundation project that was conducted with colleagues from the ruđer bošković institute in zagreb, and a collaborator on projects of the croatian science foundation. under the ages of these collaborative ventures, 18 scientific papers were published in renowned journals and, at her initiative, an in memoriam dedicated to a long-time associate from croatia, dr. volker magnus. within the framework of these projects, numerous associates from croatia have advanced as phd students, postdoctoral fellows and visiting scientists in the group of professor ludwig-müller learning new modern methods in the field of plant molecular bifig. 1. professor jutta ludwig-müller presented her twenty years of collaboration with croatian scientists by giving a lecture on “control of the plant hormone auxin in different plant species and during the interaction of plants with their environment” at a mini-symposium of the croatian society of plant biologists, held in zagreb on november 29 2019.. acta bot. croat. 79 (1), 2020 s2 ology and analytics. within the alexander von humboldt project, valuable equipment was also purchased and used at the rudjer bošković institute. she has been a member of the ruđer bošković institute's international scientific council since 2011. she was the evaluator of the phd thesis of ivana šola from faculty of science zagreb: molecular mechanisms of transport and biological function of phenolic derivatives in arabidopsis thaliana (l.) heynh. in 2013. she is an active peer reviewer of the journal acta botanica croatica. she was a plenary lecturer at the croatian botanical congress, zagreb (2007) and an invited lecturer organized by the croatian society of plant biologists and the phytobracro project of the croatian science foundation (2018). in 2014 and 2015, she was the coordinator of the erasmus biology program at technische universität dresden for universities in zagreb and split. thus, professor jutta ludwig-müller has made a significant contribution to the development of science in plant biology in croatia, because through these collaborative efforts new topics have been opened up in croatia in the field of research into specialized metabolites and the role of the plant hormone auxin in abiotic and biotic stresses. particularly useful are the training sessions of young scientists, doctoral students and postdoctoral students from croatia in her group, which enable them access to modern analytical instrumentation and work in an international environment. professor jutta ludwig-müller supports the career development of young scientists from croatia with letters of support when they are applying for scholarships and research projects. in view of the above, the members of the croatian society for plant biologists warmly recommended professor jutta ludwig-müller for the spiridion brusina medal award. the award ceremony took place on 29th november 2019 in zagreb at a mini-symposium of the croatian society of plant biologists. the medal was presented by the president of the croatian society of natural sciences professor zrinka kovarik. branka salopek sondi, phd ruđer bošković institute, zagreb fig. 2. professor zrinka kovarik, the president of the croatian society of natural sciences, presenting the spiridion brusina medal (a), and awarding the medal to professor jutta ludwig-müller (b). b joint scientific publications by professor jutte ludwigmüller and croatian scientists: 1. smolko, a., ludwig-müller, j., salopek-sondi, b., 2018: auxin amidohydrolases – from structure to function: revisited. croatica chemica acta 91(2), 17. 2. karačić, z., vukelić, b., ho, g.h., jozić, i., sučec, i., salopek-sondi, b., kozlović, m., brenner, s.c., ludwig-müller, j., abramić, m., 2017: a novel plant enzyme with dual activity: an atypical nudix hydrolase and a dipeptidyl peptidase iii. biological chemistry 398, 101–112. 3. smolko, a., šupljika, f., martinčić, j, jajčanin-jozić, n., grabar-branilović, m., tomić, s., ludwig-müller, j., piantanida, i., salopek-sondi, b., 2016: the role of conserved cys residues in brassica rapa auxin amidohydrolase: the cys139 is crucial for the enzyme activity and the cys320 regulates enzyme stability. physical chemistry chemical physic 18, 8890–8900. 4. lovelock, d.a., šola, i., marschollek, s., donald, c. e., rusak, g., van pee, k.-h. ludwig-müller, j., cahill, d.m., 2016: analysis of salicylic acid-dependent pathways in arabidopsis thaliana following infection with plasmodiophora brassicae and the influence of salicylic acid on disease. molecular plant pathology 17, 1237–1251 5. salopek-sondi, b., pollmann, s., gruden, k., oelmüller, r., ludwig-müller, j., 2015: improvement of root architecture under abiotic stress through control of auxin homeostasis in arabidopsis and brassica crops. journal of endocytobiosis and cell research 26, 100–111. a s3 acta bot. croat. 79 (1), 2020 6. mittag, j., šola, i., rusak, g., ludwig-müller, j., 2015: physcomitrella patens auxin conjugate synthetase (gh3) double knockout mutants are more resistant to pythium infection than wild type. journal of plant physiology 183, 75–83. 7. ludwig-müller, j., jülke, s., geiß, k., richter, f., mithöfer, a., šola, i., rusak, g., keenan, s., bulman, s., 2015: a novel methyltransferase from the intracellular pathogen plasmodiophora brassicae methylates salicylic acid. molecular plant pathology 16, 349–364. 8. likić, s., šola, i., ludwig-müller, j., rusak, g., 2014: involvement of kaempferol in the defence response of virus infected arabidopsis thaliana. european journal of plant pathology 138(2), 257–271. 9. salopek-sondi, b., šamec, d, mihaljević, s., smolko, a., pavlović, i., janković, i., ludwig-müller, j., 2013: influence of stress hormones on auxin homeostasis in brassica rapa seedlings. plant cell reports 32, 1031– 1042. 10. leljak-levanić, d., ježić, m., cesar, v., ludwig -müller, j., lepeduš, h., mladinić, m., katić, m., ćurković-perica, m., 2010: biochemical and epigenetic changes in phytoplasma-recovered periwinkle after indole-3-butyric acid treatment. journal of applied microbiology 109, 2069–2078. 11. rusak, g., cerni, s., stupin polancec, d., ludwig -müller, j., 2010: the responsiveness of the iaa2 promoter to iaa and iba in arabidopsis thaliana is differentially affected in roots and shoots by flavonoids. biologia plantarum 54, 403–414. 12. rusak, g., piantanida, i., bretschneider, s., ludwig -müller, j., 2009: complex formation of quercetin with lanthanum enhances binding to plant viral satellite double stranded rna. journal of inorganic biochemistry 103, 1597–1601. 13. savić, b., tomić, s., magnus, v., gruden, k., barle, k., grenković, r., ludwig-müller, j., salopek-sondi, b., 2009: auxin amidohydrolases from brassica rapa cleave the alanine conjugate of indolepropionic acid as a preferable substrate: a biochemical and modeling approach. plant and cell physiology 50, 1577–1589. 14. walz, a., seidel, c., rusak, g., park, s., cohen, j.d., ludwig-müller, j., 2008: heterologous expression of iap1, a seed protein from bean modified by indole3-acetic acid, in arabidopsis thaliana and medicago truncatula. planta 227, 1047–1061. 15. gutzeit, h.o., tokalov, s.v., ludwig-müller, j., rusak, g., 2005: monitoring flavonoid metabolism in human cells by exploiting fluorescence elicited upon quercetin/protein interactions. croatica chemica acta 78, 337–342. 16. rusak, g., gutzeit, h., ludwig-müller, j., 2005: structurally related flavonoids with antioxidative properties differentially affect cell cycle progression and apoptosis of human acute leukemia cells. nutrition research 25, 143–155. 17. campanella, j. j., olajide, a. f., magnus, v., ludwig-müller, j., 2004: a novel auxin conjugate hydrolase from wheat with substrate specificity for longer side-chain auxin amide conjugates. plant physiology 135(4), 2230–2240. 18. rusak, g., gutzeit, h., ludwig-müller, j., 2002: effects of structurally related flavonoids on hsp gene expression in human promyeloid leukaemia cells. food technology and biotechnology 40, 267–273. 19. bandurski, s.r., baraldi, r., cohen, d.j., ilić, n., ludwig-müller, j., michalczuk, l., salopek-sondi, b., 2009: in memory of dr. volker magnus, plant biologist. journal of plant growth regulation 28(4), 305– 308. opce-str.vp in memoriam u:\acta botanica\acta-botan 2-10\in memoriam.vp 11. listopad 2010 15:58:18 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees u:\acta botanica\acta-botan 2-10\in memoriam.vp 11. listopad 2010 15:58:18 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 69 (2), 313–316, 2010 coden: abcra 25 issn 0365–0588 in memoriam prof dr. ernest mayer academician ernest mayer was born in zgornji tuhinj near kamnik (slovenia) on november 10, 1920, into a family of teachers and died on march 17, 2009, in ljubljana. from 1939 to 1942, he studied biology at the faculty of arts in ljubljana. he then continued his studies in vienna, until september 1944, when he returned home and joined the partisans. he was demobilised in december 1945 and became a temporary assistant in the botanical institute of the faculty of arts in ljubljana. in the same year, he left again for vienna, where he took his doctorate under the mentorship of prof. erwin janchen, with a dissertation »floristic articulation of the high mountain zone in the south-eastern alps and its position in the eastern alps«. after returning home, he devoted himself to a university career, which took him from assistant to full professor at the biotechnical faculty in ljubljana. he lectured in general and systematic botany. he was an excellent teacher and educator. after 32 years of teaching, at his own wish he left the university in 1978. he was employed in the same year at the jovan had`i institute of biology zrc sazu as a scientific adviser, right up until his retirement in 1991. he received numerous awards and recognitions for his work, including election as an extraordinary member (1974) and full member (1983) of the slovenian academy of sciences and arts, a member of the european academy of sciences and arts in salzburg (1993), the award of the boris kidri~ fund (1975), the kidri~ award for lifetime achievement (1986) and the order of the republic with silver wreath (1981) and many other distinctions. his scientific work was primarily directed at the extensive morphological, taxonomic and phytogeographic questions of the flora of the central balkan peninsular, with a particular stress on its polymorphism and endemism. he was critical in his research, which gave rise to numerous valuable results that were difficult to dispute. he undertook planned reacta bot. croat. 69 (2), 2010 313 u:\acta botanica\acta-botan 2-10\in memoriam.vp 11. listopad 2010 15:58:19 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees search of all the important phytogeographic regions of the then yugoslavia, alpine, dinaric, steppe, mediterrannean or areas of psamophytic or ofiolithic flora. academician mayer was the top expert in the flora and a leading researcher into plant taxonomy, floristics and phytogeography of slovenia and the former yugoslavia. this enabled him to write scientific papers in which he critically reviewed claims to date and supplemented them with his own findings, and he reinterpreted some taxa and reviewed and described various new findings. for the area of slovenia and the central balkan peninsula, either alone or with associates, he treated more than 50 taxa of various ranks. he cooperated with many domestic and foreign botanists, notably academician s. horvati}, dr. j. radi}, prof. dr. lj. ilijani} and prof. dr. r. domac from croatia, dr. @. bjel~i} from bosnia, academician v. ble~i}, dr. n. dikli} and dr. v. nikoli} from serbia and academician k. micevski from macedonia. mayer’s scientific oeuvre covers more than 100 units, among which the monograph «list of ferns and flowering plants in slovenia«, which »flora europaea« counts among the 75 standard works on flora from individual parts of europe, deserves special mention. professor mayer was mentor to 18 doctoral students and many master students and undergraduates at home and abroad. academician mayer was one of the best specialist in botany. we will remember him with gratitude as a good colleague and sincere friend. academician mitja zupan~i~ slovenian academy of sciences and arts novi trg 3, si-1000 ljubljana i would like to write something of my memories of professor dr. ernest mayer, first as his student, then technical associate and later as his colleague at the department of biology of the university of ljubljana. the educational work of prof. dr. ernest mayer for many years played an important role in the education of botanists at the university of ljubljana. students listened to his lectures with great interest. in the early years, when there were no lecturers for individual fields of botany, he familiarised students not only with systematics, which was his research field, but also with the cytology, anatomy and morphology of plants. he did not then have modern aids at his disposal, such as a computer and a projector for showing illustrative material. however, with the coloured chalk with which he drew on a blackboard he conjured up the material with which he was dealing very clearly. even today, i am of the opinion that such a method, whereby the professor creates a story and the content of the theme on a blackboard in front of you, sticks better in the memory than quickly presented pictures, sketches, formulae, metabolic pathways through a computer, such as we use today. he often held spring and autumn exams in the premises in the botanical gardens, where students could answer the questions set alongside living plant material. when, after 1960, it was possible to employ a number of assistants, prof. mayer directed each of them to a specific field of botany, thus performing a great service. in addition to an assistant for systematics, he directed one into cytology, another into physiology and later yet another into ecology. after having completed their doctorates and post-doctoral degrees, they each took over their own field of botany, including both research and pedagogic work. 314 acta bot. croat. 69 (2), 2010 zupan^i^ m., gogala n. u:\acta botanica\acta-botan 2-10\in memoriam.vp 11. listopad 2010 15:58:19 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees as a demonstrator and later for two years a technical associate, during my time of study i took part in major expeditions with the professor throughout all the republics of former yugoslavia. because he had contact with all the current botanists in this region, he often organised these excursions with them and their assistants and also took biology students with him. a journey through the adriatic islands that prof. mayer organised in 1960 together with prof. radovan domac from zagreb, was unforgettable for me. we became familiar with endemic flora on the islands of vis, bi{evo, svetac, jabuka and palagru`a. the outer islands, such as jabuka and palagru`a, were visited by very few biologists, let alone tourists, especially in the sixties. profesor mayer was a great connoisseur of the flora in the wider region of the central balkans. our excursions took us to velebit, biokovo and [ar planina, to enumerate only those that remained among our the finest memories. when i became assistant for plant physiology, i no longer took part in excursions with prof. mayer. after 1978, when the professor became a member of the jovan had`i institute zrc sazu and left the department of biology of the university of ljubljana, his lectures were taken over by his associates. i would like to mention at the end his great service in mentoring doctoral, master and graduation theses. candidates came from the university of ljubljana as well as other republics of former yugoslavia. he thus left an indelible trace as an educator in botanical research and educational activities, among both those who are sadly no longer with us and those of us who still think of him and his work. prof. emeritus dr. nada gogala pot na ti~nico 6 1351 brezovica pri ljubljani e-mail: nada.gogala@siol.net acta bot. croat. 69 (2), 2010 315 in memoriam prof dr. ernest mayer prof. dr. ernest mayer and dr. nada gogala during fieldwork in 1959 u:\acta botanica\acta-botan 2-10\in memoriam.vp 11. listopad 2010 15:58:19 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees selected bibliography of prof dr. ernest mayer mayer, e., 1951: kriti~ni prispevki k flori slovenskega ozemlja. razprave 4. razreda sazu 1, 25–80 + 17 zemljevidnih prilog. mayer, e., 1952: seznam praprotnic in cvetnic slovenskega ozemlja. sazu, dela 4. razreda 5, in{titut za biologijo, 3, 427. str. mayer, e., 1953: raziskovanje flore slovenskega ozemlja. godi{njak biolo{kog instituta u sarajevu 5 (spomenica karlu malý-u), 311–314. mayer, e., 1956: genusa aretia l. in androsace l. v jugovzhodnih apneni{kih alpah. biol. vestnik 5, 18–31. mayer, e., 1958: doprinos k poznavanju flore zahodnih julijskih alp. razprave 4. razreda sazu, 4, 5–37. mayer, e., 1960: chrysanthemum atratum jacg. subsp. lithopolitanicum e. mayer, subsp. nov., eine neue endemische sippe aus den südöstlichsten kalkalpen. acta bot. croat. 18–19, 69–77. mayer, e., 1960: endemi~ne cvetnice obmo~ja jugovzhodnih apneni{kih alp, njihovega predgorja in ilirskega prehodnega ozemlja. ad annum horti botanici labacensis solemnem, 25–48. mayer, e., 1961: südöstliches alpenvorland – ein pflanzengeographisches prachtgebiet. jahrbuch des vereins zum schutze der alpenpflanzen und -tiere 25, 136–144. mayer, e., 1963: pedicularis julica e. mayer spec. nov., eine bisher verkannte art der südöstlichsten kalkalpen. phyton 9, 299–305. mayer, e., 1963: pregled pteridofitov jugoslavije. razprave 4. razreda sazu 7, 45–73. mayer, e., 1963: die floristische und taxonomische tätigkeit in jugoslawien von 1945– 1961. webbia 18, 347–365. mayer, e., horvati], s., 1967: pteridophyta (papratnja~e). v: s. horvati}, analiti~ka flora jugoslavije 1, 1, 81–155. ble^i], v., mayer, e., 1969: zur taxonomie und chorologie von edraianthus sectio uniflori. phyton 13, 241–247. mayer, e., micevski, k., 1970: zur taxonomie und chorologie von tulipa scardica bornm. feddes repert. 80, 591–598. mayer, e., 1970: zur bewertung der einblütigen sippen in edraianthus graminifoliuskomplex. fragm. fl. geobot. 16, 109–113. mayer, e., 1972: pedicularis. l. in: t. g. tutin & al. (red.), flora europaea 3, 269–276. mayer, e., 1982: primitiae florae montenegrinae. glasnik republi~kog zavoda za za{~itu prirode i prirodnja~kog muzeja u titogradu 15, 27–48. mayer, e., 1983: beitrag zur flora des gebriges biokovo. acta biokovica 2, 403–411. mayer, e., 1983: compositae subfam. asteroideae trib. cardueae cass. v: flora bosnae et hercegovinae iv sympetalae 4, 90–115. mayer, e., 1983: visianijeva »flora dalmatica« s stali{~a sodobnih botani~nih dose`kov. zbornik roberta visianija [iben~anina, povremena izdanja muzeja grada [ibenika 10, 49–55. nikoli], v., mayer, e., 1988: über den plymorphismus der gattung corydalis vent. in südost-serbien. bull. acad. serb. sci. arts, cl. sci. math. nat., sci. nat. 28, 13–21. micevski, k., mayer, e., 2002: dianthus myrtinervius griseb. subsp. zupancicii k. micevski et e. mayer subsp. nov. in der flora von makedonien. razprave 4. razreda sazu 43, 3, 399–415. 316 acta bot. croat. 69 (2), 2010 zupan^i^ m., gogala n. u:\acta botanica\acta-botan 2-10\in memoriam.vp 11. listopad 2010 15:58:19 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 404 not found opce-str.vp acta bot. croat. 70 (2), 191–208, 2011 coden: abcra 25 issn 0365–0588 cyanobacteria of the thermal spring at pancharevo, sofia, bulgaria jaromír lukavský*1, sevdalina furnadzhieva2, plamen pilarski2 1 academy of sciences of the czech republic, institute of botany, department of plant ecology, centre for bioindication and revitalisation, dukelská 135, cz-37982 tøeboò, the czech republic 2 academy of sciences of bulgaria, institute of plant physiology and genetics, academician bontschev street 21, 3311 sofia, bulgaria abstract – eight taxa of cyanobacteria were identified in the thermal spring at pancharevo (in the sofia basin, bulgaria). as well as the widespread lyngbya thermalis, phormidesmis molle (syn. phormidium molle), phormidium papyraceum, phormidium corium and mastigocladus laminosus, four species were identified for the first time in bulgaria: calothrix thermalis, gloeocapsa gelatinosa, leibleinia epiphytica and symploca thermalis. key words: bulgaria, cyanobacteria, pancharevo, thermal spring introduction thermophilic cyanobacteria are interesting study organisms for basic as well as for applied research. their ancestors are possibly the oldest primary producer organisms common in the distant past, and they perhaps used thermal springs as refugia (gold 1992, 1999; plescia et al. 2001; adhikary 2006; izagiurre et al. 2006; hindák 2008). miller et al. (2006, 2007) have recently compared 37 strains of mastigocladus laminosus isolated from sites throughout the world, analyzed 839 nucleotides of the 16s rrna gene, and reconstructed phylogenies for the nitrogen metabolism genes. they concluded that, although the species per se is cosmopolitan, its populations are genetically differentiated on local geographic scales and genetically isolated by distance. a common ancestor may have been located in the yellowstone area (usa). hexadecenoic acids have a possibly important role in the adaptation of cyanobacteria to high temperatures and the ratio between saturated and unsaturated fatty acids (s/u ratio) decreases with decreasing temperature of a thermophilic strain of synechococcus (maslova et al. 2004). in mesophilic strains cultivated at 25–32 oc the s/u ratio was increased. a change in the s/u ratio, the mechanism of adaptation of algae to high temperatures, was acta bot. croat. 70 (2), 2011 191 * corresponding author, e-mail: lukavsky@botany.cas.cz copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:31 color profile: disabled composite 150 lpi at 45 degrees documented from a quite opposite spectrum of conditions. cryoseston species of chloromonas, isolated from antarctica, had a higher proportion of unsaturated acids, enabling the functioning of cells in temperatures just above zero (bidigare et al. 1993). thermal springs represent pools of new strains possessing attractive biochemical pathways and unusual metabolic products for biotechnological applications. for example, the thermotolerant phormidium sp. produced an anti-microbial material against g–, g+ bacteria, candida albicans and cladosporidium resinae (fish and codd 1994). another phormidium sp., immobilized in calcium alginate, was used for treatment of dye-rich wastewater, (ertugrul et al. 2007). cancer drugs were produced from thermophilic cyanobacteria by javor (1999). some unusual fe-proteins, siderophores, were identified in some thermophiles (øezanka and lukavský, not published). ferredioxins from mastigocladus laminosus, however, act as toxins and restrict public use exploitation of a spring in saudi arabia (mohamed 2008). thermophilic synechococcus sp. is a potential producer of poly-b-hydroxybutyrate, which is the basis of biologically degradable plastics (miyake et al. 1996). production of hydrogen by some cyanobacteria is a promising source of energy for the future (mitsui 1987). the exploitation of natural hot water with a large content of co2 is highly profitable for algal biotechnology, e.g. production of spirulina (fournadzhieva et al. 2002) and potentially for production of thermal species. precipitation of travertine, using cultures of thermal cyanobacteria, is a promising method for capturing and sinking co2 of anthropogenic origin (hayashi et al. 1994, ono and cuello 2007). thermophilic cyanobacteria also have the potential to remove nutrients from thermal effluents (weismann et al. 1998). cultivation of thermophilic algae is easy, due to the rapid growth and resistance to common »weedy« species, under extreme temperature. in bulgaria there are more than 850 thermal springs and boreholes (pentcheva et al. 1997). they have been exploited since antiquity; the majority of springs were in semi-natural conditions and populated with cyanobacteria and algae. presently, many of the springs are being exploited (captured belowground), and thus the original algal flora is disappearing. in the period, 1898–2001, over 200 taxa of algae and cyanobacteria comprising 67 genera were identified from hot springs in bulgaria (stoyneva 2003). in algal flora of bulgaria, vodenicharov et al. (1971) listed 29 species of cyanobacteria and 4 species of thermophilic chlorophyta. later, 26 genera (75 taxa) of chlorophyta were identified in thermal springs and published, together with a comprehensive review of historical data and literature (stoyneva 2003, stoyneva and gärtner 2004). recently, 26 springs were investigated in the sofia and sandanski basins (furnadzhieva et al. 2006). the sofia basin is famous for mineral hot springs, five of them are generally known, three are of the nitrogen type, two of co2 type (pentcheva et al. 1997). the aims of our project were to contribute to the check-list of bulgarian flora and to identify and isolate strains promising for biotechnology. all organisms were carefully documented, including their morphological variability, because of continuous changes in the taxonomy of cyanobacteria. material and methods the study site the study site has been already characterized by pentcheva et al. (1997). measurement of temperature was focused on the actual value at the sampling site. live samples were col192 acta bot. croat. 70 (2), 2011 lukavský j., furnadzhieva s., pilarski p. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:31 color profile: disabled composite 150 lpi at 45 degrees lected into eppendorf tubes, of 2 ml volumes, replicates were preserved with formaldehyde to a final concentration of 2%. live cells were inoculated into wells of immunological plates (9 � 13 cm, with 96 wells a 0.25 ml), filled with 0.1 ml per well of nutrient solution d after castenholz (1969), solidified with 2% of agar, and sealed with parafilm foil with a lid. live samples were stored in the dark at room temperature during transport (castenholz 1969). minicultures in wells were later inoculated into e-flasks with liquid d medium and exposed to standard environmental conditions (46 °c, irradiated by fluorescent tubes (day type, phar = 30 w.m–2), co2 was not added). pancharevo, region of sofia, (42° 36' 07.57'' n, 23° 24' 13.12'' e) spring is near a bath house. type is dolomite + limestone, pipes with water 49.5 °c, 10 l sec–1, ph 7.3, conductivity 84.7 ms cm–1, composition in mg l–1: o2 – 1.2, hco3 – 315000, co3 2– – 410, no2 – 20, no3 – 2000, po4 3– – 7, k – 1860, ca – 54300, hs < 50, s2o3 2– < 300, so3 2– < 210, so4 2– – 38500, fe – 35. detailed data are available in pentcheva et al. (1997). outlet of water is open to the atmosphere and the flow is oscillating, is diverted into a reservoir, and is used for public laundry purposes (plate 4, fig. 3). growth on concrete walls was green-blue to green-red, in different places (plate 4, fig. 2). microscopic observations microscopic observations were conducted with nu 2 and amplival microscopes (carl zeiss, jena) equipped with objective lens hi 100/1.3, and microphotographs were taken with an olympus bx50, with hi 100/1.32 and equipped with a digital camera dp10. determination cyanobacteria were determined using monographs and keys of anagnostidis (1961), starmach (1966), anagnostidis and komárek (1990), komárek and anagnostidis (1998, 2005), and other literature cited in references. results and discussion we have determined eight taxa in the hot spring of pancharevo (plates 1–3, 5). the mats of the cyanobacteria differ according to their position with respect to the outlet of the hot water, i.e. splashing and temperature. the species marked with asterisk (*) are new records for bulgaria, as compared to vodenicharov et al. (1971). *gloeocapsa gelatinosa (meneghini) kützing 1843 plate 1, fig. 5; plate 2, figs. 8–11; plate 5, figs. 8, 17–19. description: cells rounded, 3 mm in diameter, daughter cells together in 2more colonies in transparent, not laminated mucilage of a mother cell. small granules scattered in cell, colour bright green-blue. division of daughter cells in 3 perpendicular planes, colonies two-dimensional and grow up into a great irregular mass (plate 2, fig. 18). it grows as a black growth on the concrete walls of the spring (plate 4, fig. 4, arrow). notes: g. gelatinosa has been observed in central and south-east europe, israel, africa, asia and usa. similar to gloeocapsa gelatinosa lemmermann 1905, with respect to morphology of colonies and also ecology, but the latter has smaller cells, only 1–1.6 µm in diacta bot. croat. 70 (2), 2011 193 thermal spring cyanobacteria u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:31 color profile: disabled composite 150 lpi at 45 degrees 194 acta bot. croat. 70 (2), 2011 lukavský j., furnadzhieva s., pilarski p. plate 1. cyanobacteria in thermal spring in pancharevo, leg. 10.vii.2005 and 15.vii.2006. 1–3 – phormidesmis molle 4 – symploca thermalis. 5 – gloeocapsa gelatinosa. 6, 8 – phormidium corrium, with epiphytic lyngbya epiphytica. 7 – phormidium papyraceum. 9–17 – mastigocladus laminosus. 9 – lateral branch, 10, 11 – m.l. fa. oscillarioides, 12–14, 16, 17 – m.l. fa. typica. 15 – m.l. with elongated heterocyte. scale = 10 mm. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:32 color profile: disabled composite 150 lpi at 45 degrees ameter. it was described from hawaii (hot spring near volcano, mauna kea), later recorded in the hot springs of europe (greece, france, hungary) mainly as atmophytic (komárek and anagnostidis 1998). the alga was studied as a producer of capsular polysaccharides and a potential bioadsorber of pb from waste (raungsomboon et al. 2006). the black colour is for protection against uv irradiation (lewin 2006). *leibleinia epiphytica (hieronymus) compère 1985 (syn. schizothrix calcicola (c. agardh) gomont, syn. lyngbya epiphytica hieronymus in kirschner 1898. incl.) plate 1, figs. 6, 8. description: cells 1.7 � 5 mm, cylindrical, longer than wide, terminal cells rounded, content bright-blue, with very fine structure, sheath not visible. filaments twisted around phormidium corium, attached mainly by central part, both ends of filaments grow upwards. notes: our species agrees with the description in morphology and size of cells, thin or invisible sheath. in the original description, whole filaments are attached by their entire length to host filaments. it has been recorded in freshwater, and also in thermal springs of lower temperature. in thermal springs, an epiphytic cyanobacterium was observed also in yellowstone national park, on calothrix and described as leibleinia calotrichicola (komárek and anagnostidis 2005). *phormidesmis molle (gomont) turichia et al. 2009 (syn. phormidium molle gomont 1892, lyngbya mollis (gomont) compère 1974), was described by anagnostidis 1961 and turicchia et al. (2009). plate 1, figs. 1–3; plate 2, figs. 6, 12–16, 19–20, 23, 24. description: cells cylindrical, barrel-shape, little or more constricted at terminal cells, 2–2.3 � 3 mm, sheath fine, colourless. terminal cells rounded to little conical, colour of protoplast green – green-red, dominate, with lyngbya thermalis, on concrete walls. notes: our species agrees with the morphological description and size of cells and sheath. it has been recorded occasionally in thermal springs in europe, mostly on margins and walls (komárek and anagnostidis 2005, komárek et al. 2009). p. molle is a pantropical species; it occurs in waters with abundant water vegetation, growing in clusters and mats. morphologically, it is very similar to phormidesmis pristley (fritsch) (turicchia et al. 2009), which was collected from antarctica, in rapidly streaming water, attached on stones as reddish-brown mats. the similarity of the clade of p. molle and p. pristley to typical phormidium, is 91%. the genus phormidesmis is, however, based on the typical tropical type species of p. molle. the species was shown to produce bioactive substances such as microcystins and compounds with anti-tumor activities (teneva et al. 2005). the strain lukavsky 2008/31 is deposited in the culture collection of ccala tøeboò. sp.n. for bulgaria. phormidium corium gomont 1892 (lynbya corium (agard) ex hansgirg 1892, lynbya paulistana senna 1983, phormidium corium f. woronichiana elenkin 1949, phormidium corium fa. sensu anagnostidis 1961). plate 1, figs. 6, 8. description: cells cylindrical, wide 4 – 4.5 � 7 – 12 mm, contents of cells fine, dirty blue-green, terminal cells longer, rounded – little conical, sheath fine, can be empty at ends, filaments straight. on concrete walls splashed with hot water, subdominate with mixture with symploca thermalis. acta bot. croat. 70 (2), 2011 195 thermal spring cyanobacteria u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:32 color profile: disabled composite 150 lpi at 45 degrees notes: our species agrees in morphology and size of cells, and also in little conical terminal cells. nevertheless, after komárek and anagnostidis (2005), occurrence in thermal springs is problematic, including a record of p. corium sensu anagnostidis (1961). nevertheless, the drawings of anagnostidis (1961, tab. x, figs. 59–60) are identical with our species. p. corium, which is recognised as a marine species (bhandari and sharma 2006, bhandari et al. 2007). it has been investigated with respect to photosynthesis, fatty acids, dna, and its reaction to uv-b radiation. 196 acta bot. croat. 70 (2), 2011 lukavský j., furnadzhieva s., pilarski p. plate 2. cyanobacteria in pancharevo, leg. 10 july 2005 and 15 july 2006. 1–3, 7, 17 – symploca thermalis. 4–6, 12–16, 19–20, 23, 24 – phormidesmis molle. 8–11, 18 – gloeocapsa gelatinosa. 21, 22, 25–30 – lyngbya thermalis. 7, 27, 30 – precipitation of travertine by cyanobacteria. bars indicate 10 mm. scale in right up is valid only for figs. 8–11,18. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:40 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 197 thermal spring cyanobacteria plate 3. morphological variability of mastigocladus laminosus. pancharevo, leg. 10 july 2005. 1, 5, 9, 10, 15, 16, 19, 21, 22, 23, 24, 25 – m.l. fa. typica. 6, 17 – m.l. fa. phormidioides. 2, 3 – terminal heterocyte. 1, 6, 11, 20 – intercalary heterocytes. 14, 18 – rudimentary heterocytes. 7, 17 – lateral branches. 8 –hormogonium. bar denotes 10 mm. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:53 color profile: disabled composite 150 lpi at 45 degrees *symploca thermalis gomont ex gomont 1892 plate 2, figs. 1–3, 7, 17; plate 5, figs. 6, 7, 10, 21–22. description: cells cylindrical, 0.8–1.5 � 5–7 mm, slightly constricted by cross walls, green-blue colour, cell content fine with two distinct granules by both cross walls, filaments straight or little curved, in dense mats, sheaths hyaline, very fine or invisible, with no empty ends, terminal cells rounded not different from filament ones. notes: the species dominated in some samples, trichomes were joined in dense clusters, and precipitating crystals of calcium carbonate were inside (plate 2, fig. 7). it developed, together with phormidium molle, a dense growth, coloured blue-green to green-red, on concrete walls. the trichomes are fine, cells 2–3 � 7 mm long, cell content fine with 2 granules at both poles of every cell, sheath fine or invisible. the species is known from hot springs all over europe as well as kamchatka peninsula, iceland, israel, algeria, canada, usa, etc. it grows on walls and rocks up to 50 °c. symploca differs from phormidium only by growth form in fascicles (komárek and anagnostidis 2005). it is easy to cultivate and its biomass contains interesting siderophores, proteins including fe (øezanka and lukavský, not published). the strain lukavsky 2008/29 is deposited in culture collection of ccala tøeboò. sp.nov. for bulgaria. lyngbya thermalis kûtzing ex gomont 1892 (lyngbya martensiana sensu anagnostidis 1961). plate 2, figs. 21, 22, 25–30; plate 5, figs. 1–5. description: cells 11 � 5 – 8 mm, inside cells fine and greater spheres, not constricted at poles terminal cells rounded, sheath fine, colourless, by aging become thick and lamellated, filaments later ca 15 mm in diameter, with calcium carbonate crystals on surface. necroidal cells brightly blue, they separate the filament into short parts, which move inside the sheath. it dominates in growth on walls, colour is blue-green to reddish. notes: our species was morphologically identical with the drawing of palik (1949). anagnostidis (1961) has an identical organism and determined it as lyngbya martensiana in his drawing (tab. x., fig. 53a). the species occurred on the margins of thermal springs of europe (komárek and anagnostidis 2005). in the list of thermal algae of vodenicharov et al. (1971) it is listed as lyngbya martensiana in plovdiv and tynsko. *calothrix thermalis (schwabe) hansgirg (calothrix parietina f. thermalis g. s. west, mastichonema thermale schwabe). plate 5, figs. 11–16. description: cells width 4 mm, filaments width 5 mm, including yellow coloured mucillage sheath, bearing heterocysts, basal, transparent. the end of filament rounded, width 3 mm, emerging from sheath. notes: c. thermalis, together with gloeocapsa gelatinosa, dominated a yellow-red mat on the concrete wall. this species proved to produce the inhibitor of acetylcholinesterase, and is a potential drug for curing alzheimer´s disease (becher et al. 2009). it is a new species for bulgaria. mastigocladus laminosus cohn ex kirschner 1898 plate 1, figs. 9–17; plate 3, figs. 1–25. description: cells of basal filaments rounded, 3 – 8 mm in diameter, branch cells cylindrical, 4 � 8 mm, bright blue-green, distinct granules scattered in cytoplasm. heterocytes 198 acta bot. croat. 70 (2), 2011 lukavský j., furnadzhieva s., pilarski p. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:53 color profile: disabled composite 150 lpi at 45 degrees colourless, rounded, 6 mm diameter, rarely cylindrical 5 � 8 mm, intercalar, rarely apical (plate 3, figs. 2, 3). basal filaments are branched, reverse y branching according anagnostidis and komárek 1990, (plate 1, figs. 16; plate 3, fig. 15), saddle-shape (plate 3, fig. 9) or branches emerge upright from basal filament (plate 3, fig. 24). notes: mastigocladus laminosus was described from karlovy vary (bohemia) by cohn (1862), later it was approved by ka[tovský (2001) and ka{tovský and komárek (2001). it is considered in many textbooks as a typical thermal cyanobacteria, growing in temperatures < 60 oc, ph > 7.5 and low salinity. its taxonomic position, however, is complicated, because of its extreme morphological variability. it has been described as having different morphotypes: status, or forma »anabaenoides, nostocoides, oscillarioides, tolypotrichoides, scytonematoides, plectonematoides, chlorogloeopsis«, also as subforma »normalis, subrecta, spiroides« etc. ka[tovský and johansen (2008) concluded that the name mastigocladus laminosus should be reserved for populations with true branching filaments, growing only in thermal springs (f. laminosus). their conclusions were based on comparison of the morphology of four of their own strains as well as comparison of analyses of 16s rrna of 256 heterocytous cyanobacteria. m. laminosus f. nostocoides frèmy is the second thermal taxon, but with unbranched filaments, and with life cycle steps morphologically similar to another taxon, m. laminosus f. oscillarioides frèmy. it forms nostocacean-like filaments, ensheated filaments, also filaments with chlorogloeopsis-like branching, producing akinetes, heterocytes, and hormogonia. homogenity in dgge banding profiles of the populations of a hot spring at ranong, thailand, proved that 16s rdna gene distributions change along the thermal gradient 40–60 °c, regardless of seasons (udomluk et al. 2006). also, miller et al. (2009) found sympatric diversification along a temperature gradient (39–54 °c) as a potent source of evolution, in white creek, yellowstone n.p. geographic isolation may be an important aspect of cyanophycean evolution, including mastigocladus, studied in costa rica, (finsinger et al. 2008). castenholz (1972) forecasted the existence of the two genetic types in mastigocladus in his paper about surtsey island. they differ not only in morphology, but also in growth optima, since the branched form grew in <53 °c, whereas the unbranched form grew in 60 °c. in our materials collected from other localities in bulgaria, some of these forms were in coexistence, also with transient forms as well as together with the typical branched »fo. typica«. characteristic colour, and granules in cytoplasma (plate 1, fig. 3) nevertheless, speak for an identical taxon. the validity of a gap between mastigocladus and unbranched chlorogloeopsis (komárek and hauer 2010) was proved also by 16s rrna analysis (wilmotte et al. 1993); they also proposed that the ability to produce heterocytes can be lost by mutation. also, chlorogloeopsis fritschii is not so homogenous a species, since two different types of germination of akinetes were observed (hindák 2008). heterocytes: population of typical m. laminosus in pancharevo generally has heterocytes and a content of ntot was 0.7 mg l–1 (pentcheva et al. 1997). this concurs with the results of miller et al. (2006), who reported that m. laminosus from white creek (ntot <0.1 mg l–1) produced heterocytes, whereas m. laminosus in boiling water where nitrogen was more rich (ntot = 0.15 mg l–1) did not. also acetylene reduction activity fits with the acta bot. croat. 70 (2), 2011 199 thermal spring cyanobacteria u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:23:53 color profile: disabled composite 150 lpi at 45 degrees presence of heterocytes for the white creek population 151.5 and hot water culture 1.5 mg n l–1. unfortunately, they are no available data about n content in springs on surtsey island (castenholz p.c.), where heterocytes were also present. n-fixation, growth rate, pigment contents, etc. were compared in a group of 10 strains of stigonematales, and m. laminosus proved to be not the best biomass producer, but a good n-fixer (singh et al. 2007). nitrogen fixing domain showed high frequency (18%) among the genera mastigocladus, nostoc, anabaena etc. (lakshmi et al. 2009). mastigocladus laminosus was indentified from other hot springs: kaziczene, ravno pole, zheleznitza, in sofia basin, strelcza near , gradeshnitza, in the sandanski basin (lukavský et al., not published). mastigocladus laminosus was found in 2000–2001 at rupite, near sandanski also (zidarova 2001). vodenicharov et al. (1971) did not list mastigocladus laminosus, but they mentioned hapalosiphon fontinalis in the thermal waters of the pirin and the rila ranges. maybe mastigocladus was not recognised, since there are no drawings of the species. 200 acta bot. croat. 70 (2), 2011 lukavský j., furnadzhieva s., pilarski p. plate 4. thermal spring in pancharevo, sofia. 1 – growth inside and on outer wall of the pipe, temperature of water was 48.5 °c, flowing periodically, dominated by mastigocladus laminosus. 2 – stratified growth on concrete wall, with respect to different temperature and splashing, a – 43 °c, dominated by lyngbya thermalis. b – 21 °c, dominated by symploca thermalis and gloeocapsa gelatinosa. c – 16 °c, dry, wetted only by vapour, was settled with phormidium molle, calothrix thermalis, gloeocapsa gelatinosa and symploca thermalis. 3 – the outlet of the thermal spring at pancharevo is used as a public laundry. 4 – black growth on concrete wall by pipe, temperature 22 °c, dominated by lyngbya thermalis and phormidesmis molle. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:24:02 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 201 thermal spring cyanobacteria plate 5. microscopy picture of mat, on concrete wall, from different places in pancharevo, leg. december 16, 2008, see plate 4, fig. 2, a – 1–6, b – 7–8, c – 11–21. 1–5 – lyngbya thermalis, 6 – symploca thermalis, (a, 46 °c, blue mat). 7 – symploca thermalis. 8 – gloeocapsa gelatinosa 9 – mastigocladus laminosus (b, 21 °c, green mat). 10 – symploca thermalis (21 °c, green mat in another place). 11–16 – calothrix thermalis, 17–20 –gloeocapsa gelatinosa. 21–22 – symploca thermalis (c, 16 °c, red mat). bar denotes 10 mm. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:24:08 color profile: disabled composite 150 lpi at 45 degrees sulphur concentration: mastigocladus proved to be sensitive to soluble sulphur. mastigocladus laminosus f. typica tolerates max. of 5–7 mg l–1. mastigocladus laminosus f. nostocoides was sensitive to concentrations of s < 0.15–0.25 mg l–1 (castenholz 1976). the content of total s was 12.9 mg l–1 according to pentcheva et al. (1997). the other conditions in pancharevo, temperature of 49.5 oc and ph 7.3, agree with limits cited in the literature. biotechnology prospects: m. laminosus has become a »hot model« organism studied in laboratories, also with a potential for biotechnological applications e.g. gliding of its hormogonia through agar (robinson et al. 2007), and production of capsular polysaccharides and their anti-cancer activity (gloaguen et al. 1999, 2007). it is also interesting that its ferredoxin activity was optimal at 65 °c (fish et al. 2005), but the alga was shown to grow as a branched form at <53 °c, and unbranched form in 60 °c (castenholz 1972). m. laminosus has been demonstrated as a pioneer species for a laboratory model of cyanobacterial mat (bryanskaya et al. 2008). the problem of mastigocladus laminosus obviously needs further observation in the field, but also followed with cultivation experiments and sequencing in the lab. the strain lukavsky 2008/33 is deposited in the culture collection ccala tøeboò. spatial variability of mat the cyanobacterial mat changed its composition and colour with respect to its position with respect to the source of hot water (plate 1, fig. 4), i.e. the intensities of splashing and the resulting temperatures. mastigocladus laminosus dominated at the temperature of 48.5 °c, e.g. at the mouths of the pipes (plate 4, fig. 1). a deep blue-green growth, washed direct with water of 43 °c (plate 4, fig. 3-a,) was colonized with lyngbya thermalis. a green mat, splashed only with a small amount of water of 21 °c (plate 4, fig.3-b), was dominated by symploca thermalis and gloeocapsa gelatinosa. a green mat in another place, splashed intensively with water, even under an identical temperature 21 °c, was colonized with m. laminosus and symploca thermalis (plate 5, fig.10). a red growth (plate 4, fig. 3-c), with only a small increase over air temperature, 16 °c, dry, wetted only by vapour, was colonized with phormidium molle, calothrix thermalis, gloeocapsa gelatinosa and symploca thermalis. the last species showed a greater spectrum of adaptability to temperature and conditions. bblack spots on the wall, near the outlets of hot water of 22 °c (plate 1, fig. 4) were dominated by lyngbya thermalis and phormidesmis molle. the data concur with castenholz (1969, 1973), who stressed that dramatic changes of temperature occurred along the gradient from the mouth of the spring, with concomitant changes in species composition. species richness seven species belonging to five genera of cyanobacteria, from a spring at pancharevo is comparable with the published literature, e.g. 13 springs studied in central africa where mpawenayo et al. (2005) determined a total of 92 taxa of algae including cyanobacteria and 80 taxa were bacillariophyceae. in 14 springs in sklené teplice, slovakia, 29 genera and 30 species of cyanobacteria were identified (hindák and hindáková 2007). in three maturing concrete basins of pie{ any spa, slovakia (temperature 45–60 °c), 19 genera with 15 species were identified (hindák and hindáková 2006). 202 acta bot. croat. 70 (2), 2011 lukavský j., furnadzhieva s., pilarski p. u:\acta botanica\acta-botan 2-11\lukavsky et al colori.vp 9. rujan 2011 9:24:09 color profile: disabled composite 150 lpi at 45 degrees precipitation of limestone the pancharevo spring is of a dolomite and limestone type, and it has over 3 g l–1 of co3 2– and co3 –. grains of travertine precipitated on sheaths of phormidium papyraceum (plate 2, figs. 27, 28, 30), phormidium mollum (plate 3, figs. 6, 14), and also in clusters of filaments of symploca thermalis (plate 2, figs.4, 6, 7), mastigocladus laminosus (plate 3, fig. 13) and lynbya thermalis (plate 5, fig.3). this precipitation is caused by photosynthesis, which intensively consumes co2, and also by cooling the water outside the spring (cohn 1862, ferrari et al. 2002). because travertine is a quite stable and environment-friendly substance, these species are potential candidates for capturing, mitigation and as a sink for co2 from point sources of the gas (hsueh et al. 2007, ono and cuello 2007, obst et al. 2009). the advantages of thermophilic species are their capacity to grow in both a high temperature, and a high concentration of co2, which are common in point sources of co2. future experiments are necessary to evaluate the potency of such cultures. conservation of the locality pancharevo is very attractive locality, having a small spa and public swimming pool with thermal water, and with marginal capital investment should result in a great economic exploitation for the locality. the outlet of hot water is still semi-natural, exposed to light and it is a suitable place for growth of thermophilic cyanobacteria. it would be important to protect this important ecological habitat and to conserve this refugium of interesting and valuable genotypes. four new species were recorded for bulgaria. it is essential to set up some protective measures to prevent the deterioration of the site in the same way as at karlovy vary, czech rep., where »almost all springs were closed and changed into a hot water supply system, and remain completely without phototrophic microvegetation«, in spite of the fact that it is classic algological locality (ka[tovský 2001). acknowledgements we thank grant no. 1m 0571 of the ministry of education of the czech republic, avo z60050516 of acad. sci. the czech republic, grant »new microalgal strains – potential producers of economical and medical important products«, do 02-299/08.12.2008 funded by the national science fund, and »study of cyanoprokaryotes and algae from extreme habitats« of bulgarian academy of sciences for financial support; 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the largest group consists of pure insect pollination (43%), followed by both insect and self-pollination (27%), pure wind pollination (22%), insect and wind pollination (2.6%), and so on. overall, 54% of plant species useful to european honey bees were found, 51% of which provide pollen and 47% nectar. these results suggest that a. mellifera could be a potential pollinator for about half of the flora. analysis shows significant differences in pollination patterns among habitat types and that most entomophilous plant taxa are found in grassland, forest and ruderal sites, indicating that these habitats are most important for pollinators. other characteristics of plant species, such as flowering time, plant family, life form and origin, were also analysed to determine a possible relationship with pollination. keywords: european honey bee, insect pollinators, life forms, plant families introduction pollination is one of the key ecosystem services, enabling the reproduction of wild and cultivated plant species, i.e. the production of seeds and fruits. in europe, in the area of temperate continental climate, various insects are pollinators. most numerous are the hymenopterans (hymenoptera), butterflies (lepidoptera), flies (diptera) and beetles (coleoptera) (kevan and baker 1983, ollerton 2021). in addition to wild insects, european honey bees (apis mellifera l.) play a very important role in pollination. beekeeping is also used for the production of honey, pollen, propolis, royal jelly, bee venom, wax, queens and bee communities, as well as in apitherapy and apitourism. scientific studies have shown a declining trend in pollinator numbers (potts et al. 2010, goulson et al. 2015, sánchez-bayo and wyckhuys 2019), mostly relating to habitat degradation and loss, urbanisation, agricultural intensification, pesticide and fertiliser use, pollution, pathogens, climate change, alien species and synergistic action of several factors. the most common declines involve specialists or species closely associated with a particular plant species or habitat, while a small number of generalists are increasing in number (klein et al. 2007, sánchez-bayo and wyckhuys 2019). however, some generalists are also declining, including the european honey bee. there are also other problems, e.g. competition between european honey bees and wild pollinators for forage (goulson et al. 2015), a large knowledge gap about wild pollinators, etc. along with the decline in pollinators, a decline in wild plant species pollinated by insects has been observed in some parts of the world (e.g., the uk) (biesmeijer et al. 2006, potts et al. 2010). ollerton et al. (2011) indicate that, in temperate regions of the world, about 78% of wild plant species are pollinated by animals, while klein et al. (2007) have found that, of 107 leading crops worldwide, 91 species (85%) depend to varying degrees on animal pollination. according to potts et al. (2010) pollination by insects, primarily bees, is necessary for 75% of all crops. however, there is relatively little literature on this topic. in croatia, there are studies that deal with pollination from different aspects. one study refers to different taxonomic groups and species of insect pollinators in different habitats in north-eastern croatia (kovacic et al. 2016). a few papers present the results of melissopalynological analysis of honey samples from different areas of continental croatia (sabo et al. 2011, štefanić et al. 2012, * corresponding author e-mail: zvstan@gfv.hr pollination patterns in northern croatia acta bot. croat. 82 (1), 2023 35 špoljarić maronić et al. 2017, rašić et al. 2018), where the botanical origin (plant species used by european honey bees as nectar and pollen sources) was determined on the basis of pollen grains. nevertheless, due to the economic importance of beekeeping in croatia, several books and lists of plant species useful for a. mellifera have been published (e.g., umeljić 2004, 2018, bačić and sabo 2007, zima 2007, bučar 2008, 2018, zima and štefanić 2018). there are several botanical studies that include an analysis of plant species useful for pollinators, especially european honey bees, according to specific habitat types (martinis and lovašeneberhart 1986, dujmović purgar and hulina 2007, britvec et al. 2013, dujmović purgar et al. 2015, ljubičić et al. 2017, štefanić et al. 2020). franić (2019) provides an overview of the interaction between forestry and beekeeping in croatia. however, none of the above papers includes an analysis of the proportion of insect-pollinated plant species and those useful to a. mellifera in the entire flora and all habitat types. given the lack of data on the proportion of plant species pollinated by insects in the total flora and in all habitat types, at both regional and global level, this paper presents such an analysis in croatia for the first time. given the aforementioned decline in pollinators and insect-pollinated plant species, such scientific research data is of the utmost importance, as it can help in determining best practices for ecosystem management. the objectives of this study were therefore (i) to determine the pollination patterns of the flora and vegetation in the continental part of croatia, (ii) to determine the proportions of plant species useful to a. mellifera in the flora and by habitat type, and (iii) to analyse how pollination is related to by various characteristics of plant species, including flowering time, plant family, origin and life form. materials and methods study area the study of flora and habitats was carried out in the area of the settlement bedekovčina, with about 3400 inhabitants, in northern croatia (on-line suppl. fig. 1). the study area is located partly in the valley of the river krapina and partly in a hilly area at an altitude of 148 to 237 m a.s.l., over an area of about 30 km2. the landscape consists of a builtup area, arable land with annual crops, traditional gardens, vineyards, orchards, forest, a small number of mown meadows, abandoned arable land and meadows in various stages of succession. aquatic ecosystems include the river krapina, numerous streams and canals, and five artificial lakes that have an area of about 11.2 ha. the area is characterized by a temperate continental climate, belonging to the cfwbx type according to the köppen classification, and to the humid climate according to the thornthwaite classification, with an average annual air temperature between 10 and 11 °c and an average annual precipitation from 900 to 1000 mm (zaninović et al. 2008). data collection the field research into the flora and habitats was carried out in the period from 1992 to 2021. plant species were identified using the flora europaea (tutin et al. 1964-1980, 1993) and exkursionsflora von österreich (adler et al. 1994). the nomenclature of the plant taxa and their taxonomic positions follows euro+med plantbase (2006-2021). for some taxa only, flora croatica database (hereafter: fcd) (nikolić, 2021) and pladias (2021) were used, because these taxa could not be found in the euro+med plantbase (2006-2021). these include aggregate species, subspecies of the genus leontodon, genus corydalis and medicago x varia martyn. species were classified into 11 habitat groups according to their affiliation to plant communities: (i) forest unaffected by flooding (ii) scrubland unaffected by flooding, (iii) floodplain forest and scrubland, (iv) forest-edge vegetation, (v) wet and mesic grassland, (vi) dry grassland, (vii) aquatic freshwater vegetation, (viii) marsh vegetation, (ix) ruderal vegetation, (x) weed vegetation and (xi) vegetation of walls. for each habitat group, the corresponding habitat types according to the national habitat classification of the republic of croatia (anonymous, 2018) and vegetation classes according to the classification system for european vegetation (euroveg checklist, mucina et al. 2016) were added (see on-line suppl. tab. 1). data on the mode of pollination (autogamy, entomophily, anemophily, hydrophily), flowering time, origin of taxa and life forms were taken from fcd (nikolić 2021) and pladias (2021). plant species useful to a. mellifera have been divided into the following categories depending on the food source they offer: nectar, pollen, honeydew and propolis. the data were taken from maurizio and grafl (1969), bačić and sabo (2007), and bučar (2008, 2018). all collected data are presented in on-line suppl. mat. data analysis the data were treated statistically using excel and statistica v7. contingency tables, displaying the multivariate frequency distribution of the variables, were constructed using excel, while pearson chi-squares (χ2) were calculated using statistica v7 software. results flora in the bedekovčina area, a total of 507 plant taxa (on-line suppl. mat.) were identified, belonging to 95 plant families (on-line suppl. tab. 2), of which compositae are the most numerous (54 taxa), followed by poaceae (51), fabaceae (28), lamiaceae (26), cyperaceae (23), etc. according to the affiliation to higher taxonomic groups, the class magnoliopsida prevailed (496 taxa), followed by polypodiopsida (10) and pinopsida (1). relatively few threatened species were found: one endangered (en), seven vulnerable (vu) and five near-threatened species (nt) (on-line suppl. mat.). stančić z., fiket ž. 36 acta bot. croat. 82 (1), 2023 habitat types regarding habitat types, most plant taxa were recorded in ruderal vegetation (30%), followed by wet and mesic grassland (28%), forest unaffected by flooding (28%), weed vegetation (12%), marsh vegetation (9%), floodplain forest and scrubland (5%), scrubland unaffected by flooding (5%), forest-edge vegetation (4%), dry grassland (2%), freshwater aquatic vegetation (2%) and vegetation of walls (0.2%). some plant species occur in two or more habitat types. pollination patterns among the pollination modes, expressed in absolute percentages in relation to the total number of plant species, insect pollination (entomophily) is the most widespread, with 73.6%, followed by self-pollination (autogamy) with 30%, wind pollination (anemophily) with 25%, and water pollination (hydrophily) with 0.6% (fig. 1a). there are also ferns whose fertilisation requires water (2%). the sum of the percentages exceeds 100% because some plant species have more than one mode of pollination. pollination in the largest proportion of species is done exclusively by insects (43%) (fig. 1b). both insect and selfpollination occur in 27% of plant species, followed by wind pollination (22%), insect and wind pollination (2.6%), etc. (fig. 1b). the values are expressed in relative percentages. certain modes of pollination are associated with specific plant families. among the families with the largest number of species, compositae, fabaceae, lamiaceae, apiaceae, rosaceae, caryophyllaceae and plantaginaceae are predominantly insect-pollinated and to a lesser extent self-pollinated, while poaceae and cyperaceae are wind pollinated (on-line suppl. fig. 2). insect pollination is prevalent in all habitat types, and is shown in absolute percentages (fig. 2a), with the highest proportion in ruderal (24%), forest (22%) and grassland habitats (20%). as can be seen from tab. 1, for the grassland, forest and ruderal habitats, the calculated chi-square (χ2 = 14.5, p < 0.05) indicates their statistically significant difference, with insect pollination as the dominant mode. the proportion of windand self-pollinated plant species varies by habitat group (fig. 2a). the largest proportion of windpollinated plant species (9%) is found in open habitats, such as grassland. no wind-pollinated species were found in forest-edge vegetation, probably because these habitats are sheltered from the wind. self-pollinated plant taxa make up a significant proportion in ruderal (11%) and weed habitats (6%), because there are many annual species with a short life cycle, thus ensuring survival. pollination by water is represented only in aquatic vegetation. representation of pollination modes by habitat type in relative percentages and with an overlap of pollination modes (fig. 2b) shows that pollination patterns vary considerably among habitat types (χ2 = 39.8, p < 0.001). obtained variability of pollination modes (fig. 2b): insect pollination in the range of 26–60%, both insect and self-pollination ranging between 6 and 45%, wind pollination ranging from 0 to 38%, self-pollination ranging from 0 to 9%, and both insect and wind pollination fig. 1. contributions of the different modes of pollination in the flora studied in the northern croatia: a) representation of individual modes of pollination in absolute percentages (where the sum exceeds 100% because some plant species have more than one mode of pollination), b) contribution and overlap of specific modes of pollination in relative percentages. tab. 1. contingency table showing number of plant species in certain habitat type in relation to pollination modes. * for denoted habitats, there is a statistically significant difference (χ2: 14.5; p < 0.05). habitat type insect pollination insect and self-pollination wind pollination other modes of pollination total grass veg.* 48 22 37 3 110 forest veg.* 62 36 23 11 132 ruderal veg.* 54 37 22 8 121 other habitats types 55 44 29 16 144 total 219 139 111 38 507 pollination patterns in northern croatia acta bot. croat. 82 (1), 2023 37 ranging between 0 and 6%. pure insect pollination is most prevalent in forest-edge vegetation, followed by forest, grassland and ruderal vegetation. both insectand self-pollination are best repesented in weed, scrub, forest-edge and ruderal vegetation. pure wind pollination is most prevalent in marsh and grassland vegetation. plant species useful for apis mellifera the european honey bee plays a very important role in the pollination of plant species. in this study, a total of 54% of plant taxa useful to a. mellifera were identified: 47% as a nectar source, 51% as a pollen source, 4% as a honeydew source, and 1% as a propolis source (on-line suppl. tab. 3). of the plant species that depend only on insect pollination (43% of total species), 67% (29% of total species) can be used by european honey bees as a nectar source and 63% (27% of total species) as a pollen source (fig. 3). of the plant species with both insect and self-pollination (27% of total species), european honey bees can potentially use 63% (17% of total species) each as a nectar and/or pollen source. of the wind-pollinated plant species (22% of total species), european honey bees can use 18% (4% of total species) as a pollen source. the distribution of plant species useful to a. mellifera per habitat type is shown in fig. 4. as can be seen from the figure, most plant species providing nectar to a. mellifera were found in ruderal (16%), grassland (15%) and forest habitats (14%), while there were fewer in other habitat types. a similar trend was observed for plant species serving as a source of pollen: the highest numbers were found in ruderal (17%), forest (16%) and grassland habitats (16%). relatively few species are known to be a source of honeydew (up to 2%) and propolis (< 1%), and they grow in forest and scrub vegetation. fig. 2. percentages of different pollination modes in different habitat groups: a) representation of individual modes of pollination by habitat in absolute percentages (sum exceeding 100% because some plant species have more than one mode of pollination), b) representation of the proportion and overlap of specific modes of pollination by habitat type in relative percentages (where the habitat groups differ significantly with respect to pollination mode: χ2 = 39.8, p < 0.001). forest veg. – forest vegetation unaffected by flooding, scrub veg. – scrub vegetation unaffected by flooding, flood f&s veg. – floodplain forest and scrub vegetation, f-edge veg. – forest-edge vegetation, grass veg. – wet and mesic grassland vegetation, dry grass veg. – dry grassland vegetation, aqu. veg. – aquatic freshwater vegetation, marsh veg. – marsh vegetation, ruderal veg. – ruderal vegetation, weed veg. – weed vegetation, wall veg. – wall vegetation. fig. 3. percentages of plant species useful for apis mellifera (as a source of nectar, pollen and honeydew) by pollination mode. stančić z., fiket ž. 38 acta bot. croat. 82 (1), 2023 flowering time most plant species flower in june (66%), and fewest in december (0.6%) and january (0.8%). during the ten month flowering period, from february to november, pollinators and a. mellifera can use nectar and pollen (fig. 5). life forms with regard to life forms in the flora, herbaceous perennials or hemicryptophytes predominate (53%), followed by annual plant species or therophytes (21%), geophytes (17%), woody plants or phanerophytes (11%), hydrophytes and chamaephytes (4% each), with some species associated with two life forms. by habitat type, hemicryptophytes predominate in grassland, ruderal and forest habitats; therophytes have a high proportion in ruderal and weed habitats; geophytes are most numerous in forest habitats; phanerophytes in forest and scrub vegetation; chamaephytes in forest, and hydrophytes in marsh and aquatic vegetation (on-line suppl. fig. 3). insect pollination prevails in all life forms (tab. 2, online suppl. fig. 4), while wind and self-pollination are less well represented. theorophytes also have a considerable amount of self-pollination, whereas aquatic pollination occurs only in hydrophytes (on-line suppl. fig. 4). origin of plant species by origin, indigenous or native plant species are most abundant (79.1%), followed by archaeophytes (11.8%), neophytes (8.5%) and three taxa (0.6%) of uncertain origin. indigenous plant species dominate in all habitat types except weed vegetation, where archaeophytes have a higher proportion (on-line suppl. fig. 5). furthermore, ruderal and weed vegetation contains a considerable proportion of archaeophytes and neophytes. analysis of pollination modes by origin of plant species shows that, in all three groups (indigenous plant species, archaeophytes and neophytes), plant species pollinated by insects dominate, while wind pollination and self-pollination are less well represented (on-line suppl. fig. 6). only among the archaeophytes are there slightly more plant species with self-pollination than with wind pollination. the importance of insect pollination for plants of different origins can also be seen in tab. 3, which shows that this mode of pollination is particularly prevalent in native plant species and neophytes (χ2 = 19.6, p < 0.01). discussion the flora studied depends mostly on insect pollination (73.6%). our results are in agreement with ollerton et al. (2011) and potts et al. (2010), who state that about 78–80% of wild plant species in temperate zones are pollinated by insects. a similar percentage was obtained in a study by fig. 4. percentage contribution of plant species that are a source of nectar, pollen and honeydew for apis mellifera by habitat group. (for habitat abbreviations see caption of fig. 2). fig. 5. percentage contribution of plant species in bedekovčina flora according to flowering time. tab. 2. contingency table showing number of plant species by life form in relation to pollination modes. life form abbreviations: h – hemicryptophytes, t – therophytes, g – geophytes, ch – chamaephytes, p – phanerophytes, hy – hydrophytes. life forms insect pollination insect and self pollination wind pollination other forms of pollination total h 112 53 48 9 222 t 27 38 17 9 91 g 28 11 16 10 65 p 25 12 14 2 53 ch 6 7 1 14 hy 5 2 6 13 combinations 16 18 14 1 49 total 219 139 111 38 507 pollination patterns in northern croatia acta bot. croat. 82 (1), 2023 39 štefanić et al. (2020) in ne croatia, with the finding that 72.6% of plant species on field margins are beneficial to pollinators, although not all habitat types were included. for the flora of the czech republic, chytrý et al. (2021) show only maps with the proportions of pollination modes inf luenced by relief and climate. melendo et al. (2003) indicate, for the endemic flora in the south of the iberian peninsula with a mediterranean climate, that 91% of the plant species are biotically pollinated, mainly by insects. according to the data collected, about two thirds of plant species depend on only one mode of pollination, while about one third of plant species have two or, less frequently, several pollination modes. durka (2002) determined exactly the same proportion of insect pollination (43%) for the flora of germany as in n croatia, slightly less for both insect and self-pollination (21%), much more for self-pollination (22%), less for wind pollination (18.5%), and almost the same for water pollination (0.5%). the data are not fully comparable, as durka (2002) used, for plant species with several pollination modes, only the dominant one. somewhat later, kühn et al. (2006) mapped the distribution of pollination modes across the whole of germany, with the help of modelling. altitude and wind speed were strongly correlated with the proportions of pollination modes. remarkable spatial differences were obtained: insect pollination in the range of 41.9– 63.1%, wind pollination in the range between 15.5–32.7%, and self-pollination in the range of 16.1–29.9%. a coarse spatial resolution was used with a cell size of about 130 km2 and a different method for calculating the proportion of pollination modes than in this paper. to our knowledge, an approach combining multiple pollination modes of the whole flora and all habitat types, as used in this study, is not to be found in the available literature, so further comparison is not possible. the proportion of certain pollination modes in a given area is influenced by ecology and evolution. the dominance of insect-pollinated plant species on the global level is explained by the high rate of diversification during evolution (givnish 2010). wind pollination of angiosperms probably evolved from insect pollination in response to unfavourable weather conditions in some areas (strong wind, heavy rain and low temperatures) and the associated lack of insect pollinators (culley et al. 2002, friedman and barrett 2008). in some plant species, a transitional stage between wind and insect pollination i.e. ambophily is still present (culley et al. 2002). in the flora studied, plant species that use both wind and insect pollination are relatively rare. self-pollination is a typical feature of annual species (lloyd 1992) or therophytes. such plant species are not dependent on the availability of pollinators, weather conditions and pollen transmitters (animals, wind and water), which is particularly important when a species is rare in its habitat (lloyd 1992). according to pyšek et al. (2011), self-pollination is a crucial feature for the alien plant species invasion process. in the flora studied, there are very few plant species that are only self-pollinated, but a considerable proportion that are both insectand self-pollinated. to ensure their survival, some plant species exhibit multiple pollination modes. on a broad spatial scale, according to givnish (2010), 202 out of 379 plant families are animal-pollinated, and only 39 are windor water-pollinated. the same trend, with the largest number of insect-pollinated plant families, has been found in n croatia, and a small number are wind pollinated. most wind-pollinated species belong to herbaceous families of open habitats such as marsh and grassland vegetation (poaceae, cyperaceae, juncaceae) and woody species (betulaceae, corylaceae, fagaceae, moraceae) which are tall and exposed to the wind and flower before they form leaves. the results of this study revealed that insect pollination is the predominant mode of pollination for most life forms as well as for plant species of different origins. however, the analyses showed that the distribution pattern of life forms and plant species by origin is more influenced by habitat types rather than pollination modes. in fact, it has been found that habitat types, and then affiliation to plant families, have the greatest influence on the distribution of pollination modes. different plant species have different flowering times, thus occupying different temporal niches and providing food for different species of pollinators during the vegetation season (fenster et al. 2004). depending on the species, the duration of the flowering period varies. there are also rare species that bloom all year, and even in december and january, but due to low temperatures, short daylight and lack of dormant insects, it is hard to speak of pollination. from february, the number of flowering species and active pollinators increases until june, and then the number decreases until november. recently, the phenology of plant species has been significantly affected by climate change (tylianakis et al. 2008, tab. 3. contingency table showing number of plant species by origin in relation to pollination modes. a – archaeophytes; i – indigenous; n – neophytes. all types of analysed plant species were found to be different with respect to existing pollination modes (χ2: 19.6; p < 0.01). origin of plant species insect pollination insect and self pollination wind pollination other modes of pollination total i 172 104 91 34 401 a 19 28 10 3 60 n 27 7 8 1 43 total 218 139 109 38 504 stančić z., fiket ž. 40 acta bot. croat. 82 (1), 2023 gordo and sanz 2009). that is, climate change is causing plant species to begin flowering much earlier than usual, which can affect the temporal matching of pollinators and plant species (tylianakis et al. 2008). among pollinators, a. mellifera could be a potential pollinator for about half of the flora, according to the research results of this study. the actual number is probably even higher, because there are no data for each wild plant species on whether it is visited by european honey bees. as already mentioned, for bees the most important group is that of insect-pollinated plant species, and somewhat less the group of insectand self-pollinated plant species. in these groups, about two thirds of the plant species can be used by a. mellifera as a source of nectar and pollen. in addition, bees use less than one fifth of wind-pollinated plant species as a pollen source. comparison with the literature is not possible, as no comparable data are available, which underlines the need for further studies in this field. potts et al. (2010) also highlight the fact that the contribution of european honey bees to the pollination of wild plant species is not well supported by empirical data. for example, regarding a. mellifera, the entomophilous plant species are relatively well known. they all produce pollen in greater or lesser amounts, and most nectar, but not all (nectarless species: chelidonium majus l., clematis vitalba l., papaver rhoeas l., rosa canina l., and others) (maurizio and grafl 1969). anemophilous plant species produce large amounts of pollen through wind pollination, which is a very important food for many insect pollinators and the european honey bee. these include many widespread tree species (e.g., alnus glutinosa (l.) gaertn., betula pendula roth, corylus avellana l., fagus sylvatica l., populus tremula l., quercus petraea (matt.) liebl., q. robur l., etc.), and also common herbaceous plant species (e.g. plantago lanceolata l., p. major l., rumex spp., etc.) (maurizio and grafl 1969). of the other anemophilous plant species, a. mellifera is known to use plant taxa from poaceae (total annual pollen yield may be as high as 1–10%), cyperaceae (maurizio and grafl 1969), and probably many others. however, it is not completely known which species are involved. thus, the number of anemophilous species used by a. mellifera is probably much higher than presented in this paper. it is known that bees use the most suitable species among those available (maurizio and grafl 1969). which plant species are used by european honey bees can be determined by melissopalynological analysis. several such studies have been published for the continental part of croatia (sabo et al. 2011, štefanić et al. 2012, špoljarić maronić et al. 2017, rašić et al. 2018). in the papers cited, pollen grains from 4 to 33 plant taxes were found in honey samples. however, the final number of plant species visited by the bees is certainly much higher, since in the cited works not all honey samples were analysed during the vegetation season, and pollen samples collected separately by the bees were not analysed at all. as a. mellifera is the best-studied insect pollinator, many findings from this study can be applied to wild pollinators, especially from the hymenoptera group, which have similar foraging behaviour. which pollinators are associated with particular plant species can be found, in part, in the cryptra database (ellis and ellis-adam 1993), whose analysis shows that relationships are not characterised by specialisation. in the plant pollination system, johnson and steiner (2000) point out that, in europe, generalists among pollinators prevail over specialists. the study area is characterised by a diverse relief and a mosaic landscape. the great diversity of habitats is enhanced by the very small areas of land individually owned characteristic of this part of n croatia. as some plant species only grow in certain habitats, habitat diversity is a prerequisite for flora biodiversity. the results show that habitat types differ significantly in terms of pollination patterns. in this study, three groups of habitats were identified where most insect-pollinated plant species occur, and which are also useful for a. mellifera. these habitats include grassland, forest and ruderal sites. grassland habitats belong mostly to the wet and mesic meadows of the class molinio-arrhenatheretea tx. 1937. these are still very species-rich habitats, although much of the former meadows have been abandoned and are in various stages of succession. the reason for this is the change in the way of life of the local residents in the last 30 years. people have abandoned traditional agriculture and livestock breeding (mainly cows). significantly reduced grassland areas result in a reduced food source for pollinators. the importance of such habitats for a. mellifera in the continental part of croatia is highlighted by ljubičić et al. (2017), and in the mediterranean part of croatia by britvec et al. (2013). comprehensive research in several european countries has also shown that semi-natural habitats (grassland) are very rich in bee pollinators (hymenoptera: apiformes) (westphal et al. 2008). restoration of grassland habitats is possible and involves the reintroduction of traditional extensive management, e.g. mowing two to three times a year. forest habitats belong mainly to beech (fagus sylvatica l.) communities of the class carpino-fagetea sylvaticae jakucs ex passarge 1968. they are located in the hills, outside the influence of flood waters. other types of woody vegetation (scrubland unaffected by flooding, floodplain forest and scrubland) cover relatively small areas. compared to other habitat types, forest is the least changed. however, it is highly fragmented which negatively affects insect pollination (kolb and diekmann 2005), mostly privately owned, and affected by frequent and unplanned logging. wind-pollinated plant species predominate among woody species. herbaceous plant species develop in the ground layer and usually flower in the spring before tree leaves form. in ruderal habitats there is a very heterogeneous group of plant communities in phytosociological terms (mucina et al. 2016). in the study area, these are places alongside pollination patterns in northern croatia acta bot. croat. 82 (1), 2023 41 buildings, roads, railway lines and ditches, on construction sites, yards, landfills, composting sites, and filled and trampled areas. in general, these are habitats where humans prevent the development of natural vegetation through various disturbances. in addition to typical ruderal species, those of weed, grassland and, to a lesser extent, other habitat types grow in these stands. a large part of these habitats is mown and forms replacement habitat for grassland species, namely those that are resistant to frequent mowing. for pollinating insects, such habitats can be a food source, but only if mowing is not too frequent and if the plants have enough time to form flowers. the results of other studies (dujmović purgar and hulina 2007, dujmović purgar et al. 2015) in the continental part of croatia show the importance of ruderal habitats for a. mellifera. studies in urban areas in the uk have also confirmed the importance of such habitats for flower-visiting insects (baldock et al. 2015). the entire study area in n croatia is under significant anthropogenic influence. this is evident not only from the large areas covered with ruderal and weed vegetation, but also from a significant proportion of archaeophytes and neophytes in the composition of the flora, as well as from a small number of threatened species. although neophytes pose a threat to native plant species and habitat diversity, some neophy tes (robinia pseudoacacia l., amorpha fruticosa l., solidago gigantea aiton, etc.) can also serve as an additional nectar and pollen source for a. mellifera ( zima and štefanić 2018). even a common invasive alien species that is allergenic to humans, ambrosia artemisiifolia l., serves as a pollen source for european honey bees (špoljarić maronić et al. 2017). similarly, entomophilous neophytes serve as a food source for many wild pollinators (suni et al. 2022). visitation of alien plant species by entomofauna demonstrates their integration into the network of native pollinators, but there are controversial views on whether this is a positive or negative phenomenon (potts et al. 2010). on the positive side, alien plant species, including many ornamental plants, provide food for pollinators; and, on the negative side, native plant species may be deprived of pollinators (tylianakis et al. 2008). suni et al. (2022) have shown that pollinators in urban areas prefer invasive alien plant species over native ones. various anthropogenic activities are known to cause declines in biodiversity at all levels of biological organization, including declines in insect pollinators (potts et al. 2010, goulson et al. 2015, sánchez-bayo and wyckhuys 2019), which can lead to declines in plant species (biesmeijer et al. 2006), and vice versa. of all pollination modes, only insect pollination is threatened. to preserve the biodiversity of pollinators, it is necessary to preserve the biodiversity of flora and natural and seminatural habitats. dennis et al. (2003, 2007), garibaldi et al. (2014), goulson et al. (2015) and bretagnolle and gaba (2015) suggest implementing various practices: providing nesting opportunities for pollinators, increasing heterogeneity of agricultural land (smaller fields), leaving or restoring areas of natural or semi-natural vegetation between or near crops, leaving weeds between crops (which can reduce crop yields but promote pollinator biodiversity), sustainable and/or organic agriculture, reducing the use of pesticides and machinery, no-tillage farming, seeding (wild) flower strips between and along crops and roads, planting hedgerows, seeding flowering crops, managing plant phenology (sowing plants that flower at different times), introducing pollinator monitoring, preventing the introduction of nonnative bees, prohibiting the keeping of european honey bees in some natural areas to stimulate wild pollinators, enforcing effective quarantine measures for the movements of european honey bees to prevent the spread of pathogens and parasites, etc. some scientists point to the importance of cultivated plant species in maintaining wild pollinator biodiversity and providing food for a. mellifera (garbuzov and ratnieks 2014a, b, salisbury et al. 2015). however, cultivated plant species can only be considered an additional food source when a particular crop is sown or planted and for only a certain period of year. it is unlikely that a diversity of cultivated plant species in a given area will provide food for pollinators throughout the vegetation season. from the midtwentieth century to the present, various pesticides used in crop production have had lethal or sublethal effects on pollinators (goulson et al. 2015), which is difficult to reconcile with pollinator stimulation. in addition, studies of insect foraging show that some commonly planted non-native ornamental species are unused or rarely used by pollinators (garbuzov and ratnieks 2014b, lowenstein et al. 2019). in croatia, the food source for insect pollinators is still dominated by wild plant species. in wild plant and insect species, there is an evolutionary specialization of individual functional groups of insect pollinators to specific plant functional groups, which are linked in so-called pollination syndromes (fenster et al. 2004). conclusions the pollination pattern of the flora studied shows that insect pollination predominates, followed by self-, wind and water pollination. about two-thirds of the plant species depend on only one mode of pollination (mostly insect and wind pollination), while about one-third of the plant species depend on two (mostly both insect and self-pollination) and less frequently on several modes of pollination. the distribution of pollination patterns is mainly influenced by habitat types. detailed studies on this topic are needed in the future. most insect pollinated plant species are found in grassland, forest, and ruderal habitats, highlighting their importance to pollinators. among habitats, semi-natural grassland is most threatened because of the cessation of mowing. in addition to habitat types, plant family affiliation also has a considerable influence on the distribution of pollination modes. the european honey bee can potentially participate in the pollination of about half of the flora. stančić z., fiket ž. 42 acta bot. croat. 82 (1), 2023 given the predominance of wild plant species in n croatia as a food source for pollinators in terms of the number of species, the area they cover, and their various temporal niches, it is crucial to preserve the biodiversity of wild flora and associated habitats. the results of this work, with minor variations, can most likely be generalized to most of inland croatia and to other temperate regions with similar relief, climatic conditions and habitats. acknowledgment this work is dedicated to zvjezdana’s dear friend janko (24/01/2022). references adler, w., oswald, k., fischer, r. 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(eds.), 91–95. agroecology, ecological agriculture and environmental protection, proceedings 53rd croatian & 13th international symposium on agriculture, faculty of agriculture, university of j. j. strossmayer in osijek, 91-95. osijek (in croatian). 544 vizintin et al.vp acta bot. croat. 71 (2), 207–213, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 changes of photosynthesis and carbon metabolism in typha angustifolia l grown in conditions of nitrate nitrogen overload vladimir i. chikov1, elvira v. isaeva1, anna a. ratushnyak2, oleg yu tarasov2, kseniya i. abramova2, maxim v. trushin1,2,3* 1 kazan institute of biochemistry and biophysics, russian academy of sciences, lobachevskii st. 2/31, 420111 kazan, russia 2 state budgetary establishment research institute for problems of ecology and mineral wealth use, tatarstan academy of sciences, daurskaya 28, 420089 kazan, russia 3 kazan (volga region) federal university, faculty of biology and soil sciences, kremlyovskaya 18, 420008 kazan, russia abstract – nitrates may induce alterations in no-signaling system and change photosynthesis in plants. significant reduction of 14co2 fixation was noted at concentration of 3.96 mm nano3 in an aquatic macrophyte (typha angustifolia l.). assimilation of 14co2 seven days after the introduction of nitrates did not differ between control and experimental samples. there were changes in distribution of 14c among products of 4co2 fixation 4 h after nano3 addition, resulting in increased sugar radioactivity in experimental plants. it was suggested that the observed changes may have regulatory importance. keywords: aquatic macrophyte, carbon metabolism, nitrate, photosynthesis introduction the role of c / n proportion in the regulation of plant metabolism has been actively studied in recent years. it is known that during interaction between carbon and nitrogen metabolisms the start of trigger systems occurs; this may have an effect on the physiological and biochemical processes in plants (corruzzi and bush 2001). the functional role of the nitrate ion for the regulation of c / n status in plants has already been demonstrated (krapp and stitt 1995). with respect to of flax plants (linum usitatissimum ) it has been shown that introduction of nitrate solution (50 mm) to the apoplast of plant bine may result in the inhibition of the acta bot. croat. 71 (2), 2012 207 * corresponding author, e-mail: mtrushin@mail.ru copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. outflow of assimilates from leaves after 30 min as well as accumulation of 14csucrose in donor leaves and an increase of the ascending transport of photosynthetic products (batasheva et al. 2007). the blocking of 14csucrose export from leaves was found at the level of the transition of phloem terminals to vessel fascicles; vacuolization of cells-satellites in phloem terminals was noted (abdrakhimov et al. 2008). introduction of sodium nitroprusside, a generator of no, (in concentrations of 2 orders lower) into plant apoplast induced similar changes (batasheva et al. 2010). this suggests the participation of no-signaling in the rearrangement of physiological and biochemical changes in plants with increasing nitrate concentrations. then, it was proposed that the entry of nitrate into plants results in the formation of nitrogen oxide by enzymatic or nonenzymatic ways from the nitrate ion; no may activate no-signaling system and trigger genetically determined alterations of plant metabolism (khamidullina et al. 2011). probably, the ability of no to increase the content of callose (b-1,3glucan) in a leaf (parís et al. 2007) influences the blocking of sucrose transport at the level of footstalk (and, likely, root) phloem. the data on the ability of salicylic acid to induce a synthesis of no (zottini et al. 2007) and to inhibit callose destruction confirms indirectly the proposed mechanism (serova et al. 2006). it may also mean that inhibition of callose destruction may be mediated by the action of no. plants with both symplast and apoplast type of phloem sugar loading have their carbon metabolism changed due to nodifications of assimilate transport and leaf cell ultrastructure (khamidullina et al. 2011). the importance of no-signaling systems in animals was convincingly presented (glyan’ko et al. 2009). this allows the conclusion that the action of nitrates on metabolism via no-signaling system may have a universal character. in this connection, it was important to test the reaction of an aquatic macrophyte (typha angustifolia l.) to increased concentration of nano3 in the environment. the choice of the plant was governed by the fact that it is the important provider of exometabolites participating in the regulation of structure and functions of aquatic organisms (ratushnyak and abramova 2011). the hypothesis of the present study was to check whether nitrogen overload may result in both morphological and physiological alterations in aquatic macrophyte. materials and methods to detect the intensity of photosynthesis and to evaluate the distribution of 14c in leaf cells in vivo, we used the radioactive indicators method. plants of typha angustifolia l. were taken from sredniy kaban lake (55°44'29" n.l., 49°09'37" e.l.) and placed into experimental tanks (v = 30 l) according to the recommendations of (tsirtsis and karydis 1997, xu et al. 1999): each tank contained 10 plants that were cultivated in natural lighting at 24–26 °c. the plants were transferred to experimental tanks with sediments and natural water. data from this experimental design may be extrapolated to natural ecosystems (tsirtsis and karydis 1997, xu et al. 1999). to assess quantitatively the content of exometabolites of t. angustifolia in its natural environment, water samples were taken from environment a month after the beginning of the experiment. the liquid was evaporated gradually (2 ml) at 30 °c, and then the pellet was supplied with scintillation liquid zhs-1 to assess radioactivity (ratushnyak and abramova 2011). biomass of the macro208 acta bot. croat. 71 (2), 2012 chikov v. i., isaeva e. v., ratushnyak a. a., tarasov o. y., abramova k. i., trushin m. v. phyte was presented as g of dried substance per 1 m2. the number of bines and the average plant height were detected, then the plants were segregated into underground and above ground parts, and their dried and wet mass was registered. detection of nitrate ions was performed using photometric assay with salicylic acid. the assay is based on interaction between nitrate ion with salicylic acid with formation of a yellow complex compound with 410 nm absorption spectrum. the drange of detected concentrations was 0.1 to 100 mg dm–3. the grade dependence was stated according to a set of standard solutions with concentrations of 0.1 to 10 mg dm–3. to detect concentration of nitrate ions in water samples, the tested solution (10 ml) was mixed with 2 ml of salicylic acid, and then dried. after cooling, 2 ml of sulphuric acid was added to the mixture and stored for 10 min. after that, 10–15 ml of distilled water and 15 ml of a complex solution (naoh with rochelle salt) were added. then, this solution was used for photometric analysis at 410 nm. two weeks later (a period of adaptation), a solution of sodium nitrate (final concentration 0.39 and 3.96 mm) was added to the plant root area. unexposed plants served as controls. four hours and seven days later, the medial part of a leaf from the medial plant tier (1.5–2.0 cm2) was exposed to photosynthetic chamber with 14co2 (0.03%) and 3 and 4 min at natural illumination. then, this part of the leaf was fixed with boiling ethanol (80%), the fixed leaves were ground with ethanol (60%), and the content of 14c was detected in the homogenized samples. for each sample, total volume was measured and radioactivity was analyzed in 0.1 ml volume. radioactivity was measured using a delta-300 counter (tracor analytic, usa). spirit-water fraction was analyzed using 2d paper (fn-3) chromatogram and autoradiography on agfa radiographic film. start dots of chromatogram were coated with sample concentrate corresponding to 100,000 pulses per min. a liquid scintillation detector tri-carb b2810tr (perkin elmer, usa) was used to detect the amount of 14c in the samples. all data are presented in 5-fold replication, mean and standard errors are indicated in tables and figures. the level p<0.05 was considered to indicate significance. results it was revealed that t. angustifolia plants showed an activation of production processes under conditions of nitrate overload. we detected an increase of total biomass, number of bines and average heights (p<0.05, tab. 1). in parallel with morphological changes, we detected little change in the intensity of 14co2 fixation 4 h after the introduction of nano3 acta bot. croat. 71 (2), 2012 209 photosynthesis and carbon metabolism in typha angustifolia tab. 1. dried biomass of underground and above ground parts of t. angustifolia (per 1 m2) in conditions of nitrate overload variants number of bines average height (cm) total biomass of dried matter (g) overground biomass underground biomass dried matter (g) % of total biomass dried matter (g) % of total biomass control 14 140±7 246±11 79±4 32±4 166±8 67±4 n0.396 17 150±7 346±14 122±6 35±4 224±11 64±4 n3.96 22 162±8 459±22 246±10 53±3 213±9 46±3 (0.39 mm) to the root area of t. angustifolia (p<0.05, fig. 1). a significant reduction of 14co2 fixation was noted at 3.96 mm nano3 concentration (p<0.05). assimilation of 14co2 seven days after introduction of nitrates did not differ between control and experimental samples (fig. 2). there were changes in distribution of 14c among products of 4co2 fixation 4 h after nano3 addition; this resulted in increased sugar radioactivity in experimental plants (p<0.05, fig. 3). discussion our study indicated that nitrogen overload may induce morphological and physiological changes in the aquatic macrophyte t. angustifolia. significant reduction of 14co2 fixation was noted at a concentration of 3.96 mm. since in symplast plants the rate of assimilate efflux is lower than in apoplast plants (khamidullina et al. 2011), it is possible to suggest that the influence of nitrates was not distributed far from the root zone of the exper210 acta bot. croat. 71 (2), 2012 chikov v. i., isaeva e. v., ratushnyak a. a., tarasov o. y., abramova k. i., trushin m. v. fig. 1. radioactivity of 14co2 in leaves of t. angustifolia l. measured after nitrate nitrogen addition (4 h before the experiment). fig. 2. radioactivity of 14co2 in leaves of t. angustifolia l. measured after nitrate nitrogen addition (7 days before the experiment). fig. 3. the action of nitrate nitrogen 4 h after its introduction on the distribution of 14c among products of 3-min exposition. radioactivity corresponds to ethanol-watersoluble fraction. imental plants since the height of the plants was 1.5–1.7 m and the experimental part of the leaf functioned normally. probably, the 7 day period favored the utilization of nitrates by aquatic hydrobionts, and carbon dioxide metabolism was at a stationary level. this was supported by hydrochemical data (ratushnyak and abramova 2011). it was previously reported (ratushnyak 2002) that the excretory activity of macrophytes was raised after the addition of nitrogen to the environment. an increase in plant excretion results in enhancement (primarily at the periphytic area) of bacterial biomass for 3 days; these are basic destroyers of polluting compounds; these microbes may be competitors for biogenic elements of hydrobionts of other taxonomic groups, firstly phytoplankton (ratushnyak et al. 2008, 2009). also reported was the predominance of saprophytes (15.5-fold domination in open biotopes), denitrite bacteria (10–1000-fold domination), nitrite bacteria (38–100-fold domination) after the addition of nitrate nitrogen (3.96 mm); these bacteria participate actively in processes of organic destruction that facilitate their action in the hydroecosystem (ratushnyak and abramova 2011). interestingly, increase of nitrate (a basic substrate of inorganic nitrogen for plants) concentration did not result in elevated synthesis of amino acids: previous studies showed that nitrate increases the amount of 14c within amino acids in experiments with terrestrial (batasheva et al. 2007, khamidullina et al. 2011) and aquatic plants (ratushnyak et al. 2010). most likely this difference is connected with different exposition times and concentrations of nitrate ion used. at the same time, signal systems may inhibit enzyme synthesis in calvin’s cycle. this was confirmed by a significant reduction of 14c content at the starting spot at chromatogram where mainly spirit-water soluble proteins are concentrated (primarily, ribulosodiphosphatcarboxylase) (andreeva 1982). increase of 14c in sucrose and maltose (transport products of photosynthesis in the symplast plant, t. angustifolia) (weise et al. 2004, chia et al. 2004, lu and sharkey 2004) may be considered as difficulties with sugar export from the experimental part of a leaf. very likely the induction of callose synthesis (under the action of nitrate entering the underground organs of the plants) reduced the carrying capacity of phloem. as a result, the sieve tube located above started to overfill with sugars. these data allow the suggestion that the action of nitrates has a trigger mechanism. a sudden increase of nitrate concentration in roots opposes the stationary abilities of enzyme systems to reduce nitrates to amino groups. as a result, no, nitric oxide may be formed as a product of the incomplete reduction of nitrate; no may trigger no-signal system. the triggering of the no-signal system may occur during endogenous increase of the nitrate concentration in leaves or organs. there are many ways for nitric oxide to be generated from nitrite, both enzymatic and nonenzymatic (neill et al. 2003). the detected changes are characteristic of both concentrations of nitrate (fig 3). the increased radioactivity of citrate suggests the activation of respiration activity in connection with the forthcoming metabolic changes. it should be noted here that the used concentration of nano3 was 25 times lower than those for terrestrial plants (batasheva et al. 2007, abdrakhimov et al. 2008) and 2.5 times lower than the nitrate concentration used usually in nutrient solutions for hydroponic plant cultivation. the detected effects may have a regulatory character; the interaction between downward flow of sugars from leaves and the upward flow of nitrates with transpiration water may occur at different distances from the zone at which nitrate is introduced to the plant. synthesis of callose is the first reaction to the appearance of no (paris et al. 2007); this acta bot. croat. 71 (2), 2012 211 photosynthesis and carbon metabolism in typha angustifolia compound may occlude pores in cribriform lamellas of phloem. this contradicts to the transport of saccharose from leaves to roots and may result in repletion of phloem vessels with sugars (including those labeled with 14c). as a result, an increased radioactivity of saccharose and maltose was detected in the experimental part of a leaf that had received labeled 14c. thus, it was revealed in this study that nitrogen overload may result both in morphological and physiological alterations. this compound induced growth of the aquatic macrophyte and altered assimilation of carbon dioxide and transport of 14c-products. although no measurement was not performed, we discuss its possible formation since sodium nitroprusside and nitrates may have similar effects, according to literature data (wildt et al. 1997, khamidullina et al. 2011). references abdrakhimov, f. a., batasheva, s. n., bakirova, g. g., chikov, v. i, 2008: dynamics of ultrastructure changes in sheet plate 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5927. xu, f-liu, jørgensen, s. e., tao, s., 1999: ecological indicators for assessing freshwater ecosystem health. ecological modelling 116, 77–106. zottini, m., costa, a., de michele, r., ruzzene, m., carimi f., lo schiavo, f., 2007: salicylic acid activates nitric oxide synthesis in arabidopsis. journal of experimental botany 58, 1397–1405. acta bot. croat. 71 (2), 2012 213 photosynthesis and carbon metabolism in typha angustifolia opce-str.vp acta bot. croat. 70 (2), 133–146, 2011 coden: abcra 25 issn 0365–0588 physiology and biochemistry of leaf bleaching in prematurely aging maple (acer saccharinum l.) trees. ii. functional and molecular adjustment of psii. hrvoje lepedu[1, lidija begovi]2, selma mlinari]2, domagoj [imi]1, ivna [tolfa2, nada para\ikovi]3, zvonimir u@arevi]4, vlatka jurkovi]1, vera cesar2* 1 agricultural institute osijek, ju`no predgra|e 17, hr-31000 osijek, croatia 2 department of biology, university of j. j. strossmayer in osijek, trg lj. gaja 6, hr-31000 osijek, croatia 3 faculty of agriculture, university of j. j. strossmayer in osijek, trg sv. trojstva 3, hr-31000 osijek, croatia 4 faculty of education, university of j. j. strossmayer in osijek, l. jägera 9, hr-31000 osijek, croatia abstract – in the present study we aimed to investigate physiological and molecular mechanisms of photosynthetic performance decline in prematurely aged bleached leaves of silver maple (acer saccharinum l.) trees. we used in vivo chlorophyll a fluorescence measurement to analyze changes in psii photochemistry, relative abundance of photosynthetic proteins (d1, lhcii, cyt f and rubisco lsu), relations between chlorophylls and their precursor protochlorophyllide as well as elemental composition of the leaves. decreases in al, cr, cu, fe, k, zn and an increase in s concentrations were found in bleached leaves in comparison to healthy green ones. the bleached leaves were visually expressing symptoms characteristic of fe deficiency. further, they had considerably decreased chlorophyll contents and protochlorophyllide contents, overall photosynthetic activity and relative abundance of major photosynthetic proteins. all the results indicate that modifications in the molecular organization of photosynthetic electron-transport chain components in bleached leaves led to functional adaptation of the psii achieved by modifications of some reaction centres (rcs), turning them from active to dissipative. this provided efficient adaptation of bleached leaves to high-light induced oxidative damage during summer. key words: acer saccharinum, bleached leaves, chlorophyll, photosynthesis, premature aging acta bot. croat. 70 (2), 2011 133 * corresponding author, e-mail: vcesarus@yahoo.com copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees abbreviations: bcip – 5-bromo-4-chloro-3-indolyl phosphate, bsa – bovine serum albumin, chl a+b – total chlorophylls, cyt f – cytochrome f, dtt – dithiothreitol, df/f' m – effective quantum yield of the photosystem ii, f 0 – minimal fluorescence yield, f m – maximal fluorescence yield, f v /f m – maximum quantum yield of the photosystem ii, fm – mass of fresh tissue, icp-oes – inductively-coupled plasma optical emission spectroscopy, lhcii – light harvesting complex of photosystem ii, nbt – nitroblue tetrazolium, ojip test – chlorophyll a fluorescence transient measurement, pchl – protochlorophyllide, ppfd – photosynthetically active photon flux density, psii – photosystem ii, rcs – reaction centres, rubisco lsu – large subunit of rubisco, sds – sodium dodecyl sulfate introduction silver maple (acer saccharinum l.) is a widely distributed tree species. it constitutes sustainable resource for the production of maple syrup, wood and lately has been seen as good quality source for the production of biofuel (shapouri and duffield 1993). because of its marked tolerance to different kind of stresses, silver maple trees also have horticultural value and are often used for ornamentation of urban parks and landscapes (day et al., 2000, hardin et al., 2001). however, a considerable number of chlorotic silver maple trees were noticed in the city park of osijek (croatia) growing besides healthy silver maple trees with green leaves. leaves in these trees of decreased vitality became bleached as early as late spring (may) and kept such status during the whole vegetation season. such symptoms observed in trees of decreased vitality correlate with symptoms of premature aging, sometimes called decline, which is characterized by chlorosis of foliage (bleaching), crown thinning and tree dieback (houston 1999). decline could be caused by various abiotic, biotic and unknown factors which are the source of stress to a tree. silver maple is sensitive to nutrient deficiency, e.g. iron deficiency will cause a distinctive pattern on the leaves – cells close to the veins are green and the rest of the leaf area is bleached (sinclair et al. 1987). such a pattern can be seen in the maple leaves we investigated here (u@arevi] et al. 2011). our previous investigation on the regulation of hydrogen peroxide level in silver maple bleached leaves revealed reduced antioxidative enzyme specific activity levels as well as decreased ascorbic acid content (u@arevi] et al. 2011). also, bleached leaves had lower levels of chlorophyll a, chlorophyll b, total carotenoids and fv/fm value (maximum quantum yield of photosystem ii) than green leaves. premature aging involves various mechanisms of adaptation in plants including changes in cell structure, metabolism, degradation of macromolecules and changes in gene expression (jing et. al. 2003). psii, which functions as water-plastoquinone oxidoreductase, is one of major regulatory components in photosynthetic apparatus (barber et al. 1997). accordingly, we aimed to investigate changes in psii photochemistry, relative abundance of major photosynthetic proteins (d1, lhcii, cytf and rubisco lsu), relations between chlorophyll and its precursor protochlorophyllide as well as leaf elemental composition in order to understand functional and molecular adjustments of psii in bleached leaves. materials and methods plant materials branches from the lower parts of the crowns of ten different trees (five green and five bleached) of silver maple (acer saccharinum) were sampled, put in a plastic bag and deliv134 acta bot. croat. 70 (2), 2011 lepedu[ h., begovi] l., mlinari] s., [imi] d., [tolfa i., para\ikovi] n. et al. u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees ered to the laboratory within one hour. sampling was done in may 2008, in the city park in osijek (croatia) every day at 10.30 am. for all extractions, leaves were cut into small pieces without main veins and ground with liquid nitrogen in a mortar in order to obtain fine tissue powder. determination of photosynthetic pigment content for determination of photosynthetic pigment content combined samples of 5 replicates for every leaf type were used. fine tissue powder was extracted in absolute ice-cold acetone for 15 minutes at +4 °c and centrifuged for 10 minutes at 3000 rpm. this procedure was repeated until the leaf material was completely uncoloured. the concentrations of total chlorophylls (chl a+b) and protochlorophyllide (pchl) per mass of fresh tissue (fm) were determined spectrophotometrically (specord 40, analytic jena, germany) according to lichtenthaler (1987). protein extraction, sds-page and immunodetection of d1, lhcii and rubisco lsu fresh leaf tissue was ground in liquid nitrogen and 0.5 g of powder was used for extraction. soluble and membrane proteins were extracted by adding solution containing 0.013m tris ph=8, 4.6% sds, 1.54% dtt and 15% glycerol pre-warmed at 80 °c. after 10 minutes of extraction at 80 °c, samples were centrifuged at +4 °c for 10 minutes at 18000g. the concentration of total protein content was determined spectrophotometrically according to bradford (1976) using bovine serum albumin (bsa) as a standard. aliquots containing 30 mg proteins per lane were mixed with laemmli sample buffer (0.065 m tris-hcl buffer containing 6% sds, 6% b-mercaptoethanol, 30% glycerol and 0.01 % bromphenol blue), boiled for 5 minutes and loaded on the gel. the samples were separated by sdspage (mini-gel twin, biometra, germany) using 12% polyacrylamide gels and blotted onto a nitrocellulose membrane (fastblot b43, biometra, germany). the membranes were blocked with 5% non-fat powdered milk solution in pbs buffer (58 mm na2hpo4, 17 mm nah2po4, 68 mm nacl) ph = 7.4 containing 1% tween 20 over night at 4 °c and incubated with rabbit monoclonal antibodies raised against the pea rubisco lsu, d1, lhcii and cytochrome f for 2 hours at room temperature. after washing, membranes were probed with an anti-rabbit alkaline – phosphatase conjugated secondary igg antibody diluted 1:30 000 in pbst. protein bands were visualized using bcip/nbt (5-bromo-4-chloro3-indolyl phosphate and nitroblue tetrazolium and images were analyzed using kodak 1d 3.6 software (lepedu[ et al. 2005, 2008). measurement of chlorophyll a fluorescence in vivo chlorophyll a fluorescence measurements were performed using a pulse-amplitude-modulated photosynthesis yield analyser (mini-pam, waltz). the plant material (10 leaves (n=10) taken from 5 different trees) was dark-adapted for approximately 30 minutes before measurement. minimal (f0) and maximal (fm) fluorescence yields were measured in the dark-adapted leaves. the same parameters (f) and (f’m) were measured upon light applications (photosynthetic photon flux density (ppfd) of 100, 500 and 1100 mmol m–2 s–1). the radiation was maintained until both, f and f’m were stable. the maximum quantum yield of the photosystem ii (fv/fm) as well as the effective quantum yield of the photosystem ii (df/f’m) were calculated according to schreiber et al. (1994). acta bot. croat. 70 (2), 2011 135 functional and molecular adjustment of psii in acer saccharinum u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees the chlorophyll a fluorescence transient measurement (ojip test) was performed with the use of a plant efficiency analyser (pea, hansatech). plant material (25 leaves (n=25) taken from 5 different trees) was dark-adapted for about 30 minutes before measurements. chlorophyll fluorescence transients were induced by applying the pulse of saturating red light (peak at 650 nm, 3000 mmol m–2 s–1). changes in fluorescence were measured for 1s, starting from 50 ms after onset of illumination. during the first 2 ms changes were recorded every 10 ms and every 1 ms afterward. the obtained data were used in ojip test (strasser et al. 2004) in order to calculate several parameters of psii photochemistry (tab. 1). oxygen evolution oxygen evolution was measured using the gas-phase clark-type oxygen electrode (hansatech). five bleached and five green leaves were analyzed, each taken from a different tree. the leaf discs (2.5 cm2) were placed in the reaction chamber and the oxygen evolution was measured at three different light levels 100, 500 and 1100 mmol m–2 s–1. the temperature inside the reaction chamber was 25 °c. 136 acta bot. croat. 70 (2), 2011 lepedu[ h., begovi] l., mlinari] s., [imi] d., [tolfa i., para\ikovi] n. et al. tab. 1. ojip test parameters and expressions. ojip test data and parameters f0 – fluorescence intensity at 50 ms (o step) f300 – fluorescence intensity at 300 ms fj – fluorescence intensity at 2 ms (j step) fi – fluorescence intensity at 30 ms (i step) fm – maximal fluorescence intensity (p step) fv – maximal variable fluorescence; fv = fm – f0 vj – variable fluorescence at j step; vj = (fj – f0)/(fm – f0) vi – variable fluorescence at i step; vi = (fi – f0)/(fm – f0) sm – normalized total complementary area above ojip transient; sm = area/(fm – f0) n – turnover number; n = sm � [(dv/dt)0] / vj rc/cs0 – density of reaction centers; rc/cs0 = fv/fm � (vj/m0) � abs/cs0 abs/rc – absorption per active reaction center; abs/rc = m0 � (1/vj) � [1/(fv/fm)] tr0/rc – trapping per active reaction center; tr0/rc = m0 � (1/vj) et0/rc – electron transport per active reaction center: et0/rc = m0 � (1/vj) � (1-vj) di0/rc – dissipation per active reaction center; di0/rc = (abs/rc) – (tr0/rc) piabs – performance index; pi = (rc/abs) � (tr0/di0) � [et0/(tr0/et0)] rc/abs – density of reaction centers on chlorophyll basis; rc/abs = (rc/tr0) � (tr0/abs) = [(fj – f0)/4(f300 – f0)] � (fv/fm) tr0/di0 – flux ratio trapping per dissipation; tr0/di0 = fv/f0 et0/(tr0/et0) – electron transport beyond qa¯; et0/(tr0/et0) = (fm – fj)/(fj – f0) u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees inductively-coupled plasma optical emission spectroscopy (icp-oes) analysis for mineral concentrations in leaves for determination of mineral concentration content combined samples of 3 replicates for every leaf type were used. leaves were dried at 105 °c for 72 hours and ground into a powder. mineral concentrations in leaves were determined by inductively-coupled plasma optical emission spectroscopy (icp-oes) after microwave digestion. leaves were digested in 65% nitric acid (hno3) + 30% hydrogen-peroxide (h2o2) with a milestone mls 1200 microwave (zarcinas et al. 1987). the analyses were performed with a jobin-yvon ultrace 238 icp-oes spectrometer. statistical analysis data on chl a+b, pchl, elemental analysis and ojip parameters were analyzed by t-test. data on the maximum quantum yield of the photosystem ii (fv/fm), effective quantum yield of the photosystem ii (df/f’m) and oxygen evolution were analyzed by one-way analysis of variance (anova) and fischer lsd (least significant difference) for post hoc analysis. differences were considered significant at p � 0.05. all statistical analyses were done with statistica 7.1. software (statsoft, inc. 2005). results total chlorophyll (chl a+b) concentration, protochlorophyllide (pchl) concentration and total chlorophyll to protochlorophyllide ratio in green leaves were significantly higher (72 %) than in bleached leaves (tab. 2). bleached leaves had lower al, cr, cu, fe, k, zn and higher s concentrations than green leaves (tab. 3). in comparison to green, bleached leaves had 34.6, 35.7, 38 and 27.7 % reduced abundances of d1, lhcii, cytochrome f and rubisco lsu, respectively (fig. 1). down-regulation of psii photochemical efficiency was found (fig. 2a). maximum quantum yield of psii (fv/fm) was 0.81 in green leaves and 0.72 in bleached leaves. effective quantum yields of psii (df/f’m) were also decreased in bleached leaves. the values of df/f’m at ppfd of 100, 500, 1000 mmol m–2 s–1 were 0.72, 0.52 and 0.37, respectively, in green leaves. in bleached leaves the values of df/f’m at the same light intensities were acta bot. croat. 70 (2), 2011 137 functional and molecular adjustment of psii in acer saccharinum tab. 2. mean values (± standard deviation) of total chlorophyll (chl a+b) concentration, protochlorophyllide (pchl) concentration and total chlorophylls to protochlorophyllide ratio in green (g) and bleached (b) leaves of silver maple (acer saccharinum). p(t) – percent of similarity. parameters g b t p(t) chl a+b (mg g–1 fm) 2.57±0.09 0.72±0.05 60.514 <0.1% pchl (mg g–1 fm) 0.33±0.06 0.19±0.03 6.502 <0.1% chl a+b / pchl 8.07±1.71 3.84±0.31 7.696 <0.1% u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees 0.59, 0.35 and 0.24, respectively. relative electron transport rates (rel. etr), shown in figure 2b, at higher light intensities were lower in bleached than in green leaves. at 100, 500, 1000 mmol m–2 s–1 measured relative electron transport rates were 35.86, 130.80 and 202.62 138 acta bot. croat. 70 (2), 2011 lepedu[ h., begovi] l., mlinari] s., [imi] d., [tolfa i., para\ikovi] n. et al. tab. 3. mean values (± standard deviation) of element concentrations (mg kg-1 dry mass) in green (g) and bleached (b) leaves of silver maple (acer saccharinum). p(t) – percent of similarity, ns – not significant. elements g b t p(t) al 70.97±0.68 45.67±2.94 11.390 <5% b 117.19±8.32 10.78±3.29 3.068 ns ba 2.70±0.51 2.70±0.04 2.393 ns ca 10617.46±676.84 10617.46±96.18 6.594 ns cr 1.88±0.30 0.50±0.03 8.549 <5% cu 12.21±0.32 5.61±0.05 37.933 <5% fe 116.77±4.71 57.99±2.17 19.835 <5% k 11187.92±895.99 14770.64±88.62 7.514 <5% mg 3783.79±95.05 3889.90±60.19 1.578 ns mn 33.55±1.58 27.59±0.16 7.082 ns mo 0.19±0.07 0.18±0.01 0.323 ns na 11.81±4.10 7.97±1.47 1.585 ns 0ni 0.85±0.32 0.32±0.09 2.859 ns p 3574.91±214.73 3255.21±52.24 2.671 ns sr 8.42±1.19 8.96±0.09 0.856 ns zn 36.44±0.22 21.01±0.35 54.242 <5% s 2131.34±55.53 2419.62±26.37 8.178 <5% fig. 1. relative abundance of d1, rubisco lsu, cytochrome f and lhcii proteins on imunoblots in green (g) and bleached (b) leaves of silver maple (acer saccharinum l.). the abundance of each protein in g leaves was taken as 100 %. horizontal bars indicate relative standard deviation. u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees in green leaves, respectively while measured relative electron transport rates in bleached leaves were 29.53, 87.95 and 131.96, respectively. in figure 2c capacity for oxygen production is shown. at ppfd of 500 and 1100 mmol m–2 s–1 it was lower in bleached than in green leaves, while no difference was observed at low light level (100 mmol m–2 s–1) acta bot. croat. 70 (2), 2011 139 functional and molecular adjustment of psii in acer saccharinum fig. 2. the arithmetic mean values and standard deviations of photosystem ii efficiency (given as maximum quantum yield (f v /f m ) at 0 ppfd and effective quantum yields (df/f' m ) (a), relative electron-transport rate (rel. etr) (b) and psii capacity for oxygen evolution (c) in green (grey circles) and bleached (white circles) leaves of silver maple (acer saccharinum l.). significant differences between g, yg and y leaves were designated by different letters (a, b, c) placed near the circles. ppfd (in µmol m–2 s–1) – photosynthetically active photon flux density; vertical bars indicate standard deviation. u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees the chlorophyll a fluorescence transient, revealed a usual ojip curve shape in both leaf types (fig. 3a). o-p normalized curves revealed the increase of fluorescence in j and i steps in bleached leaves (fig. 3b). chlorophyll a fluorescence parameters revealed that the f0 value corresponded in green and bleached leaves, but maximal fluorescence intensity, fm values were lower in bleached leaves (tab. 4). also, the turnover number (n), sm value and density of active reaction centres (rc/cs0) significantly decreased in bleached leaves. specific fluxes or specific activities per active reaction centre were calculated (tab. 5) to show functioning of psii reaction centres. parameters describing absorption (abs/rc) and trapping (tr0/rc) of light energy per active reaction centre were higher in bleached leaves than in green leaves, as was dissipation of excess energy (di0/rc). the capability of bleached leaves for electron transport beyond qa – (et0/rc) was significantly lower. values of performance index (piabs) and its components in green and bleached leaves revealed that green leaves had higher piabs, as well as density of reaction centres on chlorophyll basis (rc/abs), ratio of trapping and dissipation fluxes (tr0/di0) and efficiency of the conversion of excitation energy to electron transport (et0/(tr0-et0) (tab. 6). 140 acta bot. croat. 70 (2), 2011 lepedu[ h., begovi] l., mlinari] s., [imi] d., [tolfa i., para\ikovi] n. et al. fig. 3. ojip chlorophyll a fluorescence transients without normalization (a) and o-p normalized (b) in green (grey circles) and bleached (white circles) leaves of silver maple (acer saccharinum l). u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:15 color profile: disabled composite 150 lpi at 45 degrees discussion numbers of reports have revealed that changes in chlorophylls content and photosynthetic performance can be considered as very sensitive indicators for different biotic and abiotic stresses as well as developmental processes in higher plants (munne-bosch 2008, takahashi and murata 2008, ]urkovi]-perica et al. 2007, fulgosi et al. 2005, lepedu[ et al. 2005, pfannschmidt 2003, mittler 2002). bleaching is often caused by disturbance in mineral nutrition. our investigation of maple leaf premature aging (u@arevi] et al. 2011) acta bot. croat. 70 (2), 2011 141 functional and molecular adjustment of psii in acer saccharinum tab. 4. the mean values (± standard deviation) of chlorophyll a fluorescence parameters in green (g) and bleached (b) leaves of silver maple (acer saccharinum). all parameters are expressed in relative units. p(t) – percent of similarity, ns – not significant. parameters g b t p(t) f0 565.36±92.09 650.92±206.35 12.940 ns fm 3133.56±308.72 1908.12±359.03 1.893 <0.1% vj 0.383±0.02 0.588±0.05 17.896 <0.1% vi 0.820±0.03 0.883±0.03 7.322 <0.1% sm 17.82±2.99 12.45±2.59 6.785 <0.1% n 44,09±5.61 37,06±7.78 3.662 <1% rc/cs0 184.95±18.45 141.57±36.43 5.312 <0.1% tab. 5. the mean values (± standard deviation) of specific fluxes or specific activities per active reaction centre in green (g) and bleached (b) leaves of silver maple (acer saccharinum). all parameters are expressed in relative units. p(t) – percent of similarity. parameters g b t p(t) abs/rc 3.044±0.24 4.558±0.54 12.870 <0.1% tr0/rc 2.493±0.16 2.981±0.16 11.574 <0.1% et0/rc 1.539±0.10 1.228±0.16 8.173 <0.1% di0/rc 0.550±0.09 1.577±0.52 9.788 <0.1% tab. 6. the mean values (± standard deviation) of performance index (piabs) and its components in green (g) and bleached (b) leaves of silver maple (acer saccharinum). all parameters are expressed in relative units. p(t) – percent of similarity. parameters g b t p(t) piabs 2.475±0.44 0.324±0.11 23.812 <0.1% rc/abs 0.330±0.03 0.222±0.02 15.475 <0.1% tr0/di0 4.602±0.48 2.064±0.58 17.085 <0.1% et0/(tr0-et0) 1.619±0.12 0.716±0.16 22.687 <0.1% u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:16 color profile: disabled composite 150 lpi at 45 degrees revealed increased levels of organic nitrogen (for 19.85%) and soluble proteins (for 208.05%). in this study we analyzed the abundance of some other elements in leaves (tab. 3). differences in elemental concentrations were found between green and bleached leaves. bleached leaves had decreased al, cr, cu, fe, k, zn and increased s concentrations in comparison to healthy green leaves. it can be speculated that increased s content might account for the increase in the synthesis of glutathione (mendoza-cózatl et al. 2005, rausch and wachter 2005) and in this way enhance the protection against potential oxidative damage. surprisingly, mg and mn content did not differ between green and bleached leaves, indicated that the bleaching symptom was not due to any deficiency of these elements. chlorophyll degradation is an enzymatically regulated process (rodoni et al. 1998, 1997, matile et al. 1996,) accompanied by breakdown of pigment-protein complexes in photosynthetic membranes (tang et al. 2005). the concentration of protochlorophyllide, a precursor in chlorophyll biosynthesis (suzuki et al. 1997), was decreased in bleached leaves to 57% of that in green leaves. the decreased total chlorophyll to protochlorophyllide ratio in bleached leaves indicated that lowering of total chlorophyll content was not brought about merely by degradation but also due to decreased biosynthetic capability of bleached leaves. chlorophyll biosynthesis is to a large degree dependent on iron, which was shown to be deficient in bleached leaves (tab. 3). iron is known to be a co-factor of many enzymes of the chlorophyll biosynthetic pathway and also an important part of the electron-transport chain in chloroplasts. a deficiency is usually accompanied by bleaching symptoms and decrease in photosynthetic performance (iturbe-ormaexte et al. 1995). decreased photosynthesis was due not only to decreased pigments content but also to changes in chloroplast proteome (briat et al. 2007). relative abundance of major photosynthetic proteins investigated here (fig. 1) showed that in comparison to green leaves, bleached leaves had 34.6, 35.7, 38 and 27.7 % reduced abundances of d1, lhcii, cytochrome f and rubisco lsu, respectively. andaluz et al. (2006) reported reduced abundance of chloroplast electron-transport proteins and an increased amount of carbon assimilation proteins, in response to fe deficiency. since our results revealed that the abundance of rubisco lsu was also decreased in bleached leaves (fig. 1) it can be speculated that premature bleaching of the investigated maple leaves was influenced not by fe deficiency alone, rather, in combination with some other ecological and/or physiological factors such as high light, drought and elevated temperatures. this also implies that the observed bleaching is most likely a pleiotropic effect influenced by many cellular energetic and metabolic processes. in this study down-regulation of psii photochemical efficiency was shown (fig. 2a). maximum quantum yield of psii (fv/fm) in bleached leaves (0.72) was significantly decreased in comparison to green leaves (0.81). since the value of 0.75 for fv/fm has been considered to be a boundary value for fully functional psii (bolhàr-nordenkampf et al. 1989) the values measured in bleached leaves indicated impaired psii photochemistry. effective quantum yields of psii (df/f’m) were also significantly decreased in bleached leaves (fig. 2a). since df/f’m corresponds to the proportion of the light absorbed by chlorophylls associated with psii (maxwell and johnson 2000), decrease in df/f’m (fig. 2a) was in accordance with the observed chlorophylls content decrease (tab. 2) in bleached leaves. down-regulation of df/f’m in bleached leaves is reflected in psii-driven electron transport (fig. 2b). decrease in relative electron transport rates (rel. etr) at higher light intensities might be related to a reduced abundance of rubisco enzyme, since a 142 acta bot. croat. 70 (2), 2011 lepedu[ h., begovi] l., mlinari] s., [imi] d., [tolfa i., para\ikovi] n. et al. u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:16 color profile: disabled composite 150 lpi at 45 degrees clear relationship between co2 fixation and psii driven electron transport was established (krall et al. 1992). in addition to marked down-regulation of photochemical efficiency, reduced capacity for oxygen production at 500 and 1100 mmol m–2 s–1 was also found in bleached leaves (fig. 2c). oxygen is produced as the by-product of photosynthesis during water oxidation at oxygen evolving complex (oec) which is constitutive part of psii (iwata and barber 2004, raymond and blankenship 2008). it has been postulated that s state of oec plays an important role in chlorophyll fluorescence rise at j-step of ojip curve (strasser et al. 2004). the chlorophyll a fluorescence transient revealed usual ojip curve shape in both leaf types (fig. 3a). the f0 did not differ between green and bleached leaves, but fm values appeared to be much lower in bleached leaves (tab. 4). since fm is obtained when all qa molecules are reduced (schreiber et al. 1994), changes in fm level usually reflect heat dissipation events in psii (maxwell and johnson 2000). o-p normalized curves revealed the increase of fluorescence in j and i steps (fig. 3b). rise in the o – j step in bleached leaves, seen as an increase in variable fluorescence at 2 ms (vj), can be explained by the accumulation of reduced primary plastoquinone (qa –) because of their inability to transfer electrons efficiently further than qa –. parameters that describe the accumulation of qa – very well are the turnover number (n) representing the number of qa reduction events between f0 and fm and normalized total complementary area above oijp transient (sm), which corresponds to the energy needed to close all reaction centres (strasser et al. 2000). both parameters were significantly lower in bleached (tab. 4) than in green leaves, indicating the reduced transport from qa – to qb. also, there was a rise in the j – i step that was evident as increase in variable fluorescence at 30 ms (vi) (tab. 3). while the o – j step represents the full reduction of qa (photochemical phase), the j – i – p steps represent thermal phases associated with reduction of secondary plastoquinone (non-photochemical phase) (strasser et al. 1995). the latest steps reflect the psii heterogeneity due to the presence of fast and slow reducing plastoquinone (pq) reaction centres (stirbet et al 1998). in order to analyze the functioning of psii reaction centres specific fluxes or specific activities per active reaction centre were calculated (tab. 5). parameters describing absorption (abs/rc) and trapping (tr0/rc) of light energy per active (qa reducing) reaction centre were higher in bleached leaves than in green leaves. the increase in average functional antenna size (abs/rc) (tab. 5) together with decreased density of active reaction centres (rc/cs0) (tab. 4) indicate the presence of non-qa reducing reaction centres in bleached leaves. such non-qa reducing reaction centres are also called silent reaction centres and act as heat sinks (strasser et al. 2004). according to strasser et al. (2004) the fraction of remained active reaction centres in bleached leaves might be calculated using the expression: rccontrol (green)/rcstressed (bleached) = (abs/rc)stressed (bleached) / (abs/rc)control (green) and it was 66.78 % of that in green leaves. further, the capability of bleached leaves for electron transport beyond qa – (et0/rc) was significantly impaired (tab. 5) due to reduced transport from qa – to qb. this resulted in an about three-fold increased dissipation of excess energy (di0/rc) (tab. 5) in order to avoid oxidative damage of psii. described differences in the shape of chlorophyll a fluorescence transient curves (fig. 3) between green and bleached leaves were reflected on values of performance index (piabs) (tab. 6). this parameter was established by strasser et al. (2000) and describes overall photosynthetic performance. it combines several parameters that describe three main functional characteristics of psii reaction centre, namely rc/abs (density of reaction centres on chlorophyll basis), tr0/di0 (ratio of trapping and dissipation fluxes) and et0/(tr0-et0) (efficiency of the acta bot. croat. 70 (2), 2011 143 functional and molecular adjustment of psii in acer saccharinum u:\acta botanica\acta-botan 2-11\lepedus et al.vp 8. rujan 2011 15:53:16 color profile: disabled composite 150 lpi at 45 degrees conversion of excitation energy to electron transport). green leaves had about two-fold higher piabs than bleached, with all of its components decreased in bleached leaves (tab. 6). from the presented results, it can be concluded that maple leaves of lower vitality revealed bleaching caused by disturbed mineral nutrition with marked fe deficiency but with mg and mn content comparable with green healthy leaves. data on the photosynthesis performance and biochemistry indicated that bleaching was not a simple symptom of premature aging but rather an adaptation to high-light induced oxidative stress during summer. in that manner, down-regulation of total chlorophylls and major photosynthetic proteins content (d1, lhcii, cytochrome f and rubisco lsu) occurred. these biochemical processes were accompanied with marked changes in psii photochemistry. the most important feature of psii was the down-regulation of its efficiency, which was achieved by modifications of a certain fraction of rcs, turning them from active to dissipative. such dissipative rcs were characterised by the presence of non-qa and non-qb reducing rcs which resulted by in reduced overall photosynthetic performance, seen as decrease in oxygen evolution and piabs value. acknowledgments this work was supported by the scientific research grants to h. l. (research agreement 073-0731674-1673), v.c. (research agreement 073-0731674-0841) and d.[. 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(cyperaceae) for the flora of the balkans, with emphasis in albania santiago martín-bravo1*, carmen benítez-benítez1, mónica míguez1, marjol meco2, pedro jiménez-mejías3,4 1 universidad pablo de olavide, department of molecular biology and biochemical engineering, botany area, ctra. de utrera km 1, 41013 seville, spain 2 university of tirana, faculty of natural sciences, department of biology, bulevard zogu i parë, no. 25/1, 1001 tirana, albania 3 autonomous university of madrid, department of biology (botany), campus cantoblanco, 28049 madrid, spain 4 autonomous university of madrid, center for research on biodiversity and global change (cibc-uam), 28049 madrid, spain abstract – relevant chorological notes of genus carex l. (cyperaceae) for the flora of the balkans are provided, with an emphasis on albania and adjacent countries (north macedonia and montenegro). our findings include new national species records and/or confirmations for albania, montenegro and north macedonia (c. agastachys, c. atrata, c. curvula, c. demissa, c. hispida, c. parviflora), as well as other interesting records of rare and/or endangered carex species in albania (c. castroviejoi, c. myosuroides). eventually, we provide relevant comments in order to clarify the taxonomy, distribution and/or ecology of carex sections rhynchocystis (c. agastachys, c. pendula) and aulocystis (c. ferruginea, c. kitaibeliana, c. lazarei) in the balkans. keywords: albania, balkans, carex, chorology, conservation, endangered flora introduction the balkan peninsula is one of the three large peninsulas in south europe projecting into the mediterranean sea. it harbors a high plant species diversity and endemism as compared to the rest of europe, with part of it included in the mediterranean basin, one of the world ś hotspots of biodiversity (myers et al. 2000, mittermeier et al. 2011). some of the countries belonging to the balkan peninsula, like albania, croatia or bulgaria, span territories included in both the mediterranean and the eurosiberian floristic regions, which considerably increases their plant richness. croatia and greece, for example, also include areas that are considered to have been climatic refugia for plants during historical climatic oscillations like the miocene cooling or the pleistocene glaciations. these refugia are reservoirs of unique genetic diversity and evolutionary potential due to the long-term persistence of species, and therefore have high conservation priority (médail and quézel 1999, médail and diadema 2009). carex l. (cyperaceae) is a megadiverse plant genus with an almost cosmopolitan distribution although with higher species diversity in the temperate and cold areas of both hemispheres. with ca. 2000 species it ranks among the three largest angiosperm genera in the world (powo 2020), but it is only the 17th in the euro-mediterranean territory (ca. 232 species; raab-straube, pers. comm.). in the balkan peninsula (considered here to include albania, bosnia and herzegovina, bulgaria, croatia, european turkey, greece, kosovo, north macedonia, montenegro, serbia, and slovenia) there are 92 native carex species according to the most up-to-date official checklist (govaerts et al. 2021). however, a big issue is that this checklist does not consider the current political borders in the balkans resulting from the breakup of the former yugoslavia in the 1990s, which complicates the elucidation of the real number of carex species in the balkan countries formerly belonging to yugoslavia. therefore, floristic studies are needed to solve this problem for carex and many other genera. * corresponding author e-mail: smarbra@upo.es martín-bravo s., benítez-benítez c., míguez m., meco m., jiménez-mejías p. 102 acta bot. croat. 81 (1), 2022 in albania, 58 carex species have been recognized by govaerts et al. (2021). the flora of this country has been intensively studied in recent years, and new floras and checklists have been recently published (vangjeli 2015, pils 2016, barina et al. 2017, 2018). materials and methods the sampling of carex species was performed during a field campaign in albania during july 2019. fieldwork was complemented by the study of carex specimens housed at tirana university herbarium, albania (tir). vouchers collected in the field were deposited at pablo de olavide university herbarium, seville, spain (upos) and tir. they were carefully studied and identified with the help of a nikon smz645 stereoscopic microscope. a special effort was made to examine the most important diagnostic characters in the genus, using carex sections as a gateway for taxonomic identification (e.g., jiménez-mejías et al. 2016). identified species were checked against relevant global ( govaerts et al. 2021), european (e.g., jiménez-mejías and luceño 2011), and albanian (e.g., vangjeli 2015, pils 2016, barina et al. 2017, 2018) floras and checklists. finally, we performed a preliminary assessment of the conservation status of c. atrata and c. parviflora at the national level in albania following criteria, categories, and guidelines from iucn (2012a, b). results our fieldwork trips resulted in 134 carex herbarium vouchers belonging to 44 different species. an additional species (c. agastachys) was studied from a collection housed at tir. the finding of 13 of these species was considered relevant for the knowledge of the flora of the balkans. as a result, we herein report new national records for two species: (i) c. atrata for albania and north macedonia, (ii) c. parviflora for albania, montenegro and north macedonia. we confirmed the occurrence in albania of four species whose presence was considered unproven or their status disputed: c. agastachys, c. demissa, c. curvula and c. hispida. we also provide additional records for two rare and/or endemic species to the balkans (c. castroviejoi, c. myosuroides). the studied collections also shed light on the taxonomy, distribution and/ or ecology in the balkans of two problematic carex groups (sections rhynchocystis and aulocystis). discussion carex atrata l. subsp. atrata (fig. 1a) this species is an arctic-alpine element distributed in the high latitudes and mountains of the western palearctic, reaching marginally greenland. in the balkans, govaerts et al. (2021) indicate its presence in greece and the former yugoslavia. jiménez-mejías and luceño (2011) attributed these records to bosnia, croatia, slovenia and serbia, to which they added bulgaria. carex atrata has recently been reported for albania by pils (2016), barina (2017a; also implicitly indicating north macedonia, montenegro and kosovo) and barina et al. (2018), based on a single known population from mt. korab, the highest peak (2764 m a.s.l.) of both albania and north macedonia. we visited this albanian-north macedonian population, and noticed it is nowadays composed of very few individuals (probably not more than a hundred) and confined to the very summit of mt. korab. this is a relatively large and conspicuous species, which is relatively easy to spot in the alpine meadows where it is found, so we believe individuals previously collected from lower altitudes collected at the beginning of 20th century (as low as 1800 m a.s.l.; j. andrasovszky in 1917, see barina 2017a) could have disappeared perhaps due to climatic warming and/or habitat degradation. if so, the only known albanian-north macedonian population of this species could be facing extinction, since individuals were only observed above 2650 m a.s.l. in addition, we observed flocks of sheep very close to the summit that were intensively grazing and nitrifying the alpine meadows where the species is found. our preliminary assessment of the national conservation status of this species in albania (and possibly also in north macedonia) indicates that it probably fulfills the criteria to be considered critically endangered [cr b1ab(iii,v)+2ab(iii,v)]. other populations located at lower altitudes from the balkans (montenegro, kosovo; cf. barina 2017a) and other european countries could be also endangered or have already disappeared (gebauer, pers. comm). while barina (2017a) cited the albanian specimens as belonging to c. aterrima hoppe (as c. atrata subsp. aterrima (hoppe) hartm.), we confirm that the korab population is c. atrata subsp. atrata. both species can be distinguished by the utricle color, light-colored (yellow or greenish) above in c. atrata, thus contrasting with the blackish glumes, while they are dark-colored (blackish to almost black) in c. aterrima, thus not contrasting with the glumes (gebauer and jiménez-mejías, unpubl. data). albania. dibër county, radomirë, mt. korab, alpine meadows at the summit, 41°47’23.62”n 20°32’49.81”e, 2764 m a.s.l., 18 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 174cbb19, upos-13414, tir-8961 . north macedonia. polog region, mavrovo and rostusa, mt. korab, alpine meadows near the summit, 41°47’13”n 20°32’50”e, 2662 m a.s.l., 18 jul 2019, c. benítezbenítez, m. míguez, s. martín-bravo and p. jiménez-mejías 172cbb19, upos-13412, tir-8960. carex castroviejoi luceño and jim.mejías (fig. 1b) a member of the c. flava l. species complex (sect. ceratocystis dumort.), a group characterized by its difficult taxonomy, due to faint morphological boundaries, frequent hybridization processes (jiménez-mejías et al. 2012a, 2014), and even morphological convergence (jiménez-mejías et al. 2017). carex castroviejoi has been recently described from the pindos range in northern greece, where it grows in mountain bogs on ophiolitic rocks (jiménez-mejías and new records of carex in albania acta bot. croat. 81 (1), 2022 103 luceño 2009). it was later reported from serpentines in central-southern albania (jiménez-mejías et al. 2012b), where it was apparently overlooked by barina et al. (2018). the population cited here constitutes the new northernmost limit of this restricted balkan endemic, hitherto only known from albania and northern greece (jiménez-mejías and luceño 2011, govaerts et al. 2021). it was found in the typical habitat reported for this species, boggy ground on serpentine rocks, in the fushë-lurë mountain range (ne albania). other balkan reports for species belonging to the c. flava species complex have to be carefully examined (e.g., pils 2016, barina et al. 2018). albania. dibër county, fushë lurë, path to lakes from lura hotel, wet meadows in pinus heldreichii forest clearings, on serpentines, 41°48’27.05”n 20°13’11.28”e 1125 m a.s.l., 10 jul 2019, c. benítez-benítez, m. míguez, s. martínbravo and p. jiménez-mejías 6cbb19, upos-13246, tir8949; ibidem, lura lakes, close to liqeni i madh, boggy fig. 1. representative photos of some carex species from albania considered in this paper. a – c. atrata l. subsp. atrata, b – c. castroviejoi luceño and jim.mejías, c – c. curvula all. subsp. curvula, d – c. ferruginea scop., e – c. kitaibeliana degen ex bech., f – c. parviflora host, g – c. lazarei jac.koopman, niketic, wieclaw and govaerts. scale bar: 1 cm, except in e: 30 centimeters. martín-bravo s., benítez-benítez c., míguez m., meco m., jiménez-mejías p. 104 acta bot. croat. 81 (1), 2022 meadows, on serpentines, 41°47’13.41”n 20°11’40.56”e, 1735 m a.s.l., 10 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 20cbb19, upos-13260, tir-8950. carex curvula all. subsp. curvula (fig. 1c) it is distributed in the mountains of central and southern europe. in the balkans, it has been accepted for albania, bosnia-hercegovina, bulgaria and slovenia (jiménez-mejías et al. 2011, govaerts et al. 2021) but excluded from the flora of greece (dimopoulos et al. 2020). the presence of this species in albania has been recently considered unproven by barina et al. (2018), although it is recorded by jiménez-mejías and luceño (2011), pils (2016) and govaerts et al. (2021). this was due to the lack of verified vouchers with specific localities (barina et al. 2018). we here provide a voucher to confirm that this species should be included in the checklist of the flora of albania. albania. dibër county, radomirë, mt. korab, psicroxerophylous stony and blown pastures (fellfields) with vaccinium uliginosum, 41°47’23.62”n 20°32’49.81”e, 2488 m a.s.l., 18 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 169cbb19, upos-13409, tir-8963. carex demissa hornem. like c. castroviejoi (see above), c. demissa is another member of the c. flava group (sect. ceratocystis). this amphi-atlantic species is mostly distributed in eastern north america and western europe, and is absent from most of eastern europe (govaerts et al. 2021). nonetheless, in the balkans, it has been reported from the former yugoslavia (north macedonia; jiménez-mejías and luceño 2011) and greece (govaerts et al. 2021). however, its presence in albania has been considered doubtful (barina et al. 2018, govaerts et al. 2021). carex demissa is often confounded with the superficially similar c. oederi retz., which is widely distributed across the northern hemisphere (europe, asia and north america), and has been confirmed for albania (jiménez-mejías and luceño, 2011, barina et al. 2018 – sub c. serotina mérat –, govaerts et al. 2021; also confirmed from our own collections). we here confirm the presence in albania of specimens that clearly fall within the morphological variation of c. demissa (see jiménez-mejías et al. 2014). interestingly, in ne albania (fushë-lurë range), it co-occurs with the closely related c. castroviejoi, with which it seems to form hybrids with intermediate morphology. albania. dibër county, fushë lurë, path to lakes from lura hotel, wet meadows in pinus heldreichii forest clearings, on serpentines, 41°48’27.05”n 20°13’11.28”e, 1125 m a.s.l., 10 jul 2019, c. benítez-benítez, m. míguez, s. martínbravo and p. jiménez-mejías 11cbb19, upos-13251, tir8971; ibidem, lura lakes, close to liqeni i madh, boggy meadows, on serpentines, 41°47’13.41”n 20°11’40.56”e, 1735 m a.s.l., 10 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 33cbb19, upos-13273, tir-8972; borders of streams in beech forest, 42°30’21”n 19°58’23”e, 1495 m a.s.l., 14 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 69cbb19, upos-13309, tir-8973; kukës county, between novosej and shistavec, boggy meadows, 41°59’15”n 20°36’5”e, 1375 m a.s.l., 16 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 129cbb19, upos-13309, tir-8974 (typical, not-introgressed individuals); dibër county, fushë lurë, path to lakes from lura hotel, wet meadows in pinus heldreichii forest clearings, on serpentines, 41°48’27.05”n 20°13’11.28”e, 1125 m a.s.l., 10 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 14cbb19, upos-13254, tir-8975 (deviant specimens, probably introgressed with c. castroviejoi). carex hispida willd. ex schkuhr a circum-mediterranean species whose distribution in the balkans is in need of clarification. it is only reported for greece by jiménez-mejías and luceño (2011), to which govaerts et al. (2021) added albania, since it had been included in several floristic accounts (meyer 2011, vangjeli 2015). however, it has subsequently been excluded from the albanian flora since the source voucher was misidentified and corresponded to the close relative c. flacca schreb. (pils 2016, barina 2017b, barina et al. 2018). we have collected and identified a voucher of c. hispida from northern albania (bajram curri), easily recognized and distinguished from c. flacca by its wider leaves and rhizomes and longer male spikes and utricles, which confirms the presence of c. hispida in albania. albania. kukës county, road from bajram curri to kukës (sh23), borders of stream, on serpentines, 42°21’45”n 20°7’47”e, 500 m a.s.l., 15 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 106cbb19, upos-13346, tir-8959. carex myosuroides vill. an artic-alpine species (formerly known as kobresia myosuroides (vill.) fiori) with a wide distribution in the northern hemisphere and shared by many of the main european mountain ranges (pyrenees, alps, apennines, carpathians and balkans). in the balkans, it has been reported for bulgaria and the former yugoslavia (govaerts et al. 2021), where it has been indicated from bosnia, croatia, montenegro, kosovo, north macedonia, serbia and slovenia (cf. stevanović et al. 2009, jiménez-mejías and luceño 2011). it was not cited for albania until barina et al. (2011, sub kobresia myosuroides; see also pils 2016) indicated a single small population in the north (albanian alps, mt. maja e shënikut). another previous report from southeastern albania was discarded (barina et al. 2011, 2018). we herein cite the second confirmed known population in albania, also from the albanian alps, close to the previously known one. it is reported here due to the apparent rarity of the species in the country, although it has been indicated from several bordering areas (stevanović et al. 2009). albania. kukës county, prokletije (albanian alps), valbonë valley, path from valbonë valley to kolata peak, new records of carex in albania acta bot. croat. 81 (1), 2022 105 dry rocky grassland on limestone, shelves among rocky crests, 42°28’39.98”n 19°48’37.15”e, c. 1900 m a.s.l., 12 jul 2019, c. benítez-benítez, s. martín-bravo and p. jiménezmejías 52cbb19, upos-13292, tir-8952. carex parviflora host (fig. 1f) this orophilous species is disjunctly distributed in the mountains of central and southern europe. in the balkans, it has been reported from bulgaria and the former yugoslavia (govaerts et al. 2021), which corresponds to slovenian populations (jiménez-mejías and luceño 2011). to our knowledge, these are the first national reports of this species for albania, north macedonia and montenegro. two distant populations were found during our field campaign in border areas of two mountain ranges, one in the valbonë valley (albanian alps, n albania) and the second one on mt. korab (ne albania), in both cases with individuals across the albania-montenegro and albania-north macedonia border, respectively. this implies a considerable increase in the species’ range in southeastern europe, with a disjunction of more than 230 km to the closest previously known populations in sw bulgaria ( pirin national park) and about 500 km to those in slovenia. due to these biogeographical peculiarities, and since the number of observed individuals was very small in both populations, especially on mt. korab (probably less than a hundred), they should be granted legal protection in the corresponding countries, and further searches for more populations and/or individuals would be desirable. a tentative assessment of the national conservation status suggests that this species should be considered at least as vulnerable (vu d2) in albania, but further data may indicate that the actual conservation category could be more critical (en or cr). albania. kukës county, prokletije (albanian alps), valbonë valley, path from valbonë valley to kolata peak, suba lpi ne me soph i lou s g r a s sla nd on l i mestone , 42°29’3.70”n 19°53’8.80”e, ca. 1900 m a.s.l., 12 jul 2019, c. benítez-benítez, s. martín-bravo and p. jiménez-mejías 50cbb19, upos-13290, tir-8951; dibër county, radomirë, korab mt., stony grassland in stream borders, 41°47’13.08”n 20°32’29.42”e, 2350-2400 m a.s.l., 18 jul 2019, c. benítezbenítez, m. míguez, s. martín-bravo and p. jiménez-mejías 171cbb19, upos-13411; ibidem, alpine meadows in the summit, 41°47’23.62”n 20°32’49.81”e, 2764 m a.s.l., 18 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 175cbb19, upos-13415, tir-8962. north macedonia. polog region, mavrovo and rostusa, mt. korab, alpine meadows near the summit, 41°47’13”n 20°32’50”e, 2662 m a.s.l., 18 jul 2019, c. benítezbenítez, m. míguez, s. martín-bravo and p. jiménez-mejías 173cbb19, upos-13413. montenegro. kukës county, prokletije (albanian alps), valbonë valley, path from valbonë valley to kolata peak, international border at saddle, subalpine mesophilous grassland on limestone, 42°29’3.70”n 19°53’8.80”e, ca. 1950 m a.s.l., 12 jul 2019, c. benítez-benítez, s. martín-bravo and p. jiménez-mejías 51cbb19, upos-13291. carex sect. rhynchocystis dumort. (c. agastachys l.f. and c. pendula huds.) only two species of this section were traditionally recognized to be present in the western palearctic until recently: the central mediterranean endemic c. microcarpa bertol. ex moris (míguez et al. 2021a) and the widely distributed c. pendula (e.g. jiménez-mejías and luceño 2011), a wellknown large sedge commonly inhabiting riparian habitats and frequently used as an ornamental in european gardens. however, the detailed study of the systematics of section rhynchocystis, based on morphological and molecular data, has initiated a revolution in the classification of this group (míguez et al. 2017, 2018, 2021b). two different species have been distinguished across the former c. pendula palearctic mainland range (plus two additional macaronesian endemic species; míguez et al. 2021b): c. pendula s.s., from europe and the mediterranean basin, and c. agastachys l.f., from c, se europe and sw asia (míguez et al. 2018). the range of both species overlaps from c to se europe, including part of the balkans. although this contact area is still poorly known and in need of detailed delimitation, recent studies are making progress with this issue (meierott 2019, bergmeier 2020, christians et al. 2020, dimopoulos et al. 2020, sutorý and repka 2020). in the balkans, c. agastachys has been confirmed from bulgaria, greece, european turkey, serbia and slovenia, and c. pendula from croatia, greece, montenegro and slovenia (míguez et al. 2018). govaerts et al. (2021) added albania for c. agastachys but did not clarify the distribution in the former yugoslavian countries. finally, recent albanian checklists and floras include c. pendula s.l. without taking into account the potential presence of c. agastachys (vangjeli 2015, pils 2016, barina et al. 2018). as a result of our field campaigns and study of tir herbarium specimens, we can confirm the presence of both species in albania. carex pendula: albania. lezhë county, sh 34 road, between rrëshen and perlat, just before perlat, degraded quercus cerris forest, puddly roadsides, 41°44’3.3”n 19° 58’21.1”e, ca. 285 m a.s.l., 9 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 1cbb19, upos-13241, tir-8948; vlorë county, vlorë, depressions under pinus halepensis coastal forest, 1-2 m, 40°27’34.70”n 19°27’57.78”e, 30 may 2021, m. meco s.n., tir-8964; tirana county, tirana, parku i madh kodrat e liqenit, wet soils in mixed broadleaf forest, 41°18’35.64”n 19°49’39.84”e, ca. 135 m a.s.l., 29 jun 2021, m. meco s.n., tir-8965. carex agastachys: albania. tirana county, tirana, 17 may 1958, xh. qosja s.n, tir-00706, 00707, 00708. carex sect. aulocystis dumort. (c. ferruginea scop., c. kitaibeliana degen ex bech., c. lazarei jac.koopman, niketic, wieclaw and govaerts) (fig. 1d,e,g) within sect. aulocystis, the c. sempervirens and c. ferruginea aggregates or complexes are two groups of closely related taxa of complex taxonomy, disjunctly distributed in the mountains of southern and central europe, with balkan martín-bravo s., benítez-benítez c., míguez m., meco m., jiménez-mejías p. 106 acta bot. croat. 81 (1), 2022 mountains as the centre of diversity of both groups. during our field work in albania, we observed that c. ferruginea, c. kitaibeliana and c. lazarei are sometimes sympatric, but there is apparently a certain degree of habitat differentiation and frequent ecological turnover between them. the three species may be easily distinguished using the identification key provided by flora europaea (chater 1980). in the balkans, the distribution and ecology of c. ferruginea are obscure and debated. while it has been reported from all the countries except for montenegro and north macedonia (jiménez-mejías et al. 2011, govaerts et al. 2021), barina et al. (2015) newly reported it for albania and considered it a very rare species in the balkans only known from albania, bosnia-hercegovina, kosovo, montenegro and serbia. most albanian records have been attributed to misidentifications, especially involving the similar c. kitaibeliana (barina et al. 2015, pils 2016). in albania it is known only from two populations in dibër county (mali i bardhë and mali i dejës in ne albania), where it grows along mountain streams on serpentine and evaporitic substrates (barina et al. 2015). we found additional populations of this rare species on mt. korab (ne albania) and in the albanian alps (çerem, n albania), also along flushes. carex kitaibeliana is widely distributed from se europe to turkey (govaerts et al. 2021) and has been reported for all balkan countries (jiménez-mejías and luceño 2011). in albania, the species is widespread in mountain areas throughout the country (barina 2017b), and we have found numerous populations, mostly growing in fissures of limestone rocks. finally, c. lazarei (formerly known as c. bulgarica (domin) lazare) is a balkan endemic (jiménez-mejías and luceño 2011, govaerts et al. 2021) reported from mountain acid soils (koopman et al. 2019). in albania, it has frequently been subsumed under the highly variable c. sempervirens vill. (pils 2016, barina 2017b, barina et al. 2018). we have found populations of c. lazarei in mountain grasslands of two different ranges in n and ne albania (çerem and mt. korab, respectively). c. ferruginea scop. albania. kukës county, çerem, qafa e kunji i armeves pass, borders of streams on serpentines, 42°28’49.26”n 20°0’10.22”e, ca. 1920 m a.s.l., 14 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 93cbb19, upos-13333, tir-8958. ibidem, qafa e kunji i armeves pass, borders of stream, 42°30’11.44”n 19°59’48.08”e, ca. 1660 m a.s.l., 14 jul 2019, c. benítezbenítez, m. míguez, s. martín-bravo and p. jiménez-mejías 89cbb19, upos-13329, tir-8966; dibër county, radomirë, korab mt., stream borders in pinus nigra forest, 41°48’43”n 20°29’48”e, ca. 1450 m a.s.l., 18 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 161cbb19, upos-13401, tir-8967. c. lazarei jac.koopman, niketic, wieclaw and govaerts albania. kukës county, çerem, qafa e kunji i armeves pass, grasslands on serpentines, 42°28’49.26”n 20°0’10.22”e, c. 1920 m, 14 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 94cbb19, upos-13334, tir-8957; ibidem, subalpine grasslands with vaccinium uliginosum, 42°29’12”n 19°59’56”e, 2010 m a.s.l., 14 jul 2019, c. benítez-benítez, m. míguez, s. martínbravo and p. jiménez-mejías 75cbb19, upos-13315, tir8956; dibër county, radomirë, mt. korab, stony grasslands, c. 1800 m, 18 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 166cbb19, upos-13406, tir-8968. c. kitaibeliana degen ex bech. albania. dibër county, fushë lurë, lura lakes, clearings in pinus peuce forest, 41°47’13,41”n 20°11’40,56”e, 1735 m a.s.l., 10 jul 2019, c. benítez-benítez, m. míguez, s. martín-bravo and p. jiménez-mejías 34cbb19, upos-13274, tir-8969; dibër county, radomirë, korab mt., stream borders in pinus nigra forest, 41°48’43”n 20°29’48”e, ca. 1450 m a.s.l., 18 jul 2019, c. benítez-benítez, m. míguez, s. martínbravo and p. jiménez-mejías 162cbb19, upos-13402, tir-8970. author contributions smb, cbb, mm (mónica míguez) and pjm conducted the main field campaign in albania. mm (mariol meco) performed additional collections. all authors contributed to the study of herbarium specimens. smb drafted the manuscript and all authors contributed to the writing of the final version. acknowledgements the authors thank professor a. mullaj (university of tirana) for essential help for the organization of the field campaign and facilitating work at tir herbarium. we are particularly indebted to g. pils and z. barina for their very useful advice on fieldwork in albania. c. barciela from upos herbarium provided technical help with processing of specimens. thanks are also due to e. von raab-straube, s. gebauer, and m. luceño for providing critical comments or relevant information. projects cgl2016-77401-p and pid2020-113897gb-i00 from the spanish ministries of economy and competitiveness, and science and innovation, respectively, are acknowledged for funding. references barina, z., 2017a: number 46 (carex atrata subsp. aterrima). in: csiky, j., kovács, d., deme, j., takács, a., óvári, m., molnár v., malatinszky, á., nagy, j., barina, z. 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gametophyte; sporophyte; arbuscular mycorrhiza; septate endophytic fungi introduction the life-cycle of ferns is unique as it alternates between two free-living and fundamentally different generations, the haploid gametophyte and the diploid sporophytes. sporophytic roots of ferns are often associated with a wide range of fungi, the relationship varying from parasitism to mutualism (johnson et al. 1997, neuhauser and fargione 2004). investigations on the root fungal association in fern sporophytes indicates the widespread occurrence of arbuscular mycorrhizal (am) association (muthukumar and udaiyan 2000, wang and qiu 2006). in addition, roots of fern sporophytes are associated with other fungal endophytes like the septate endophytic fungi (cooper 1976, berch and kendrick acta bot. croat. 71 (1), 2012 139 * corresponding author, e-mail: tmkum@yahoo.com copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:35:58 color profile: disabled composite 150 lpi at 45 degrees 1982). generally, reports on fungal associations in the gametophyte of pteridophytes are limited. the achlorophyllous subterranean gametophytes of taxa in ophioglossaceae (ophioglossales), lycopodiaceae (lycopodiales) and psilotaceae (psilotales) (winther and friedman 2007, 2008, 2009) are invariably colonized by endophytic fungi (read et al. 2000). in contrast, photosynthetic green gametophytes of higher ferns are considered free from fungal colonization (read et al. 2000). however, studies do indicate the occasional occurrence of aseptate endophytic fungi in gametophytes of marattiaceae (marattiales), osmundaceae (osmundales), gleicheniaceae (gleicheniales) and schizaeaceae (schizaeales) (campbell 1908, bower 1923, nayar and kaur 1971, boullard 1979, schmid and oberwinkler 1995). turnau et al. (2005) reported the association of am fungal endophytes glomus tenue and glomus intraradices with gametophytes and sporophytes of pellaea viridis. more recently reyes-jaramillo et al. (2008) described mycorrhizal-like interaction in chlorophyllous gametophytes and young sporophytes of dryopteris muenchii raised under laboratory conditions. polypodiales is the largest order among extant ferns, encompassing about 15 families. however, there is no information on the occurrence of fungal endophytes in gametophytes or young sporophytes of polypodiales. nephrolepis exaltata (l.) schott., is an epiphytic or epilithic fern belonging to the family lomariopsidaceae of the order polypodiales (smith et al. 2006). it is native to tropical regions throughout the world, and in india, this fern is distributed in the eastern himalayas, and in south and central india. n. exaltata is one of the most popular and widely cultivated plants in home gardens. medicinal uses of this fern include its use in menstrual disorders and as a birth-aid in parturition (cambie and ash 1994). though n. exaltata is not a hyperaccumulator of heavy metals, it can still accumulate 200 mg kg–1 dry weight of arsenic (srivastava et al. 2005). spores of this fern occur in irrigation water causing the most serious weed problem in the cultivation of orchids (ko et al. 2005). nephrolepis exaltata is one of the pioneer species involved in recolonization of disturbed sites (turrill 1935). information on the root fungal association in n. exaltata is limited. muthukumar and udaiyan (2000) reported the presence of am fungal association in sporophytes of n. exaltata growing in the western ghats of southern india. a few studies also indicate a growth response of in vitro raised n. exaltata to am inoculation (ponton et al. 1990, wang et al. 1993). as, there is no information on the endophytic fungal associations in the naturally occurring chlorophyllous gametophytes and young sporophytes of taxa belonging to polypodiales, we examined the gametophytes and young sporophytes of n. exaltata for endophytic fungal associations. materials and methods a total of 219 chlorophyllous gametophytes and 150 young sporophytes growing naturally on different substrates (soil, brick, and coir) were collected during may 2010 from a home garden at coimbatore, tamil nadu, india (tab. 1, figs. 1a–c). the gametophytes and sporophytes were growing on brick pieces (1–2 cm in diameter) and coir that were used as mulches to cover the soil surface of potted plants. the gametophytes and young sporophytes were confirmed to those of n. exaltata as there were no other fern taxa within a radius of 100m. care was taken to retrieve the entire gametophytes and sporophytes along with their rhizoids and roots. the soil (1 cm depth) on which the gametophytes and young sporophytes were growing were collected for assessing the presence of am fungal spores. 140 acta bot. croat. 71 (1), 2012 muthukumar t., prabha k. u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:35:59 color profile: disabled composite 150 lpi at 45 degrees the gametophytes and young sporophytes were cleared in 2.5% koh, acidified with 5 n hcl and stained with trypan blue (0.05%) in lactoglycerol (koske and gemma 1989). the stained specimens were mounted on glass slides and examined under bx 51 olympus trinocular compound microscope for the presence of am fungal structures. aseptate linear or coiled hyphae with arbuscules or arbusculate coils were considered am. hyaline or brown, regularly septate hyphae accompanied with moniliform cells or microsclerotia characterized septate fungal endophytes. ten grams of the air-dried soil were subjected to modified wet-sieving and decanting technique (muthukumar et al. 1996) to assess the presence of am fungal spores. results the gametophytes of n. exaltata were cordate, 0.25–0.55 cm, dark green with an apical notch, one cell thick with the region posterior to the notch six to eight cells thick. unicellular hairs occurred all over the surface of the gametophyte. mature gametophytes were made up of polygonal cells containing chloroplasts, hermaphrodite, with archegonia distributed below the notch on the ventral surface and numerous hyaline to light brown rhizoids with interspersed antheridia at the pointed end (fig. 1d, e). no am fungal structure was found in gametophytes growing on different substrates (tab. 1). in contrast, 62–90% of the gametophytes from different substrates had septate fungal structures. septate fungal colonization was frequent in gametophytes growing on brick followed by coir and soil. the endophytic fungal hyphae were mostly intracellular, regularly septate, hyaline or brown, 2–6 mm in diameter with moniliform cells or microsclerotia (figs. 2 f, g). we found mycorrhization in roots of 47% and 94% of the young sporophytes collected from brick and soil respectively (tab. 1). similarly, 34–55% of the young mycorrhizal sporophytes growing on different substrates also had the septate fungal association. am fungal entry into roots was characterized by the presence of an appressorium on the root cell surface (fig. 2a). am fungal colonization in young sporophytic roots was characterized by aseptate hyphal coils and arbusculate coils with occasional intercellular hyphae (fig. 2b–e). we did not observe any vesicles in the mycorrhizal roots of the young sporophytes. although the roots of young sporophytes growing on coir lacked am fungal colonization, roots of more than half (55%) of the young sporophytes examined had septate fungal colonization. the septate fungal colonization in young sporophytes was similar to acta bot. croat. 71 (1), 2012 141 fungal associations in nephrolepis exaltata tab. 1. incidence of root fungal association in gametophytes and young sporophytes of nephrolepis exaltata. n – total number of gametophytes/sporophytes examined; m – number of gametophytes/sporophytes with arbuscular mycorrhizal association; d – number of gametophytes/ sporophytes with septate fungal association. given in parenthesis are the percentage of gametophytes/sporophytes with arbuscular mycorrhizal/ septate fungal association. substratum gametophytes young sporophytes m/n d/n m/n d/n brick 0/40 (0%) 36/40 (90%) 28/60 (47%) 21/60 (35%) coir 0/110 (0%) 81/110 (74%) 0/20 (0%) 11/20 (55%) soil 0/69 (0%) 43/69 (62%) 66/70 (94%) 24/70 (34%) u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:35:59 color profile: disabled composite 150 lpi at 45 degrees those of the gametophytes except for slightly broader hyphae of 2–8 mm in diameter. the proportion of sporophytes possessing septate endophytic fungi was low compared to the gametophytes (tab. 1). discussion the morphology of gametophytes of n. exaltata examined in the present study is similar to those reported by javalgekar and mahabale (1959). however, we did not observe any abnormal branched gametophytes bearing antheridiophore-like structures as observed by javalgekar and mahabale (1959). observations of the present study disagree with the general view that the photosynthetic green gametophytes of ferns lack fungal association. although the gametophytes of n. exaltata growing on different substrata lacked colonization by am fungi, a large percentage (62–90%) of these gametophytes was colonized by septate endophytic fungi. to our knowledge, the presence of septate endophytic fungal colonization in green fern gametophytes is being reported for the second time. swatzell et al. (1996) isolated a septate fungus from the gametophytes of schizaea pusilla. however, the fungus failed to exhibit any specific recognition and rendering 10.3% of the host non-via142 acta bot. croat. 71 (1), 2012 muthukumar t., prabha k. fig. 1. gametophytes and young sporophytes of nephrolepis exaltata. gametophytes (arrow head) and young sporophytes (double arrow head) growing on coir (a), brick (b) and soil (c). rhizoids (rh) and archegonia (ar) on ventral side of the gametophyte (d). antheridium (an) on the ventral surface of the gametophyte (e). scale bars denote 0.50 cm (a–c), 200 mm (d) and 100 mm (e). u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:36:03 color profile: disabled composite 150 lpi at 45 degrees ble. the presence of septate endophytic fungal structures like moniliform hyphae and microsclerotia indicates that the fern gametophytes may also act as niches for endophytic fungal reproduction. the lack of am fungal colonization in the gametophytes of n. exaltata is in contrast to those studies (campbell 1908, bower 1923, boullard 1979, schmid and oberwinkler 1995, turnau et al. 2005, reyes-jaramillo et al. 2008) where such an observation has acta bot. croat. 71 (1), 2012 143 fungal associations in nephrolepis exaltata fig. 2. root fungal association in nephrolepis exaltata. a – appressorium (ap) and arbusculate coil (ac) in root of young sporophyte. b – hyphal coil (hc) within root cortical cell. c – cell to cell spread (double arrow head) of intracellular hyphal coils (hc) in young sporophyte root. d – arbusculate coil (ac) in root cortical cell. e – intercellular aseptate hyphae (arrow heads) in roots of young saprophyte. f – moniliform cells (mh) in gametophyte growing on coir. g – microsclerotia (ms) in gametophytic cells growing on coir. scale bars denote 50 mm. u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:36:09 color profile: disabled composite 150 lpi at 45 degrees been reported. lack of am propagules cannot be cited as a cause for the absence of am association in these gametophytes as the primary roots of the young sporophytes growing on some of these substrates exhibited colonization by am fungi. the presence of am colonization in young roots of n. exaltata is in accordance with schmid and oberwinkler (1995), turnau et al. (2005) and reyes-jaramillo et al. (2008) where such an observation was made in young sporophytic roots of different fern species. am morphology in primary roots of n. exaltata conform to an intermediate type characterized mostly by intracellular hyphal coils or arbusculate coils with limited intercellular hyphae as frequently observed in the roots of mature fern sporophytes (dickson et al. 2007). the presence of am fungal colonization in the roots of young sporophytes without any retrievable spores indicates the involvement of propagule other than spores in mycorrhizal formation. experimental evidence does indicate that the extramatrical hyphae arising from mycorrhizal roots can initiate mycorrhization (smith and read 2008). dual occurrence of am and septate endophytic fungi in n. exaltata young sporophyte root is similar to their presence in roots of mature fern sporophytes. septate endophytic fungal colonization tends to dominate under circumstances not conducive to am formation (haselwandter and read 1982). though few studies (cooper 1976, turnau et al. 2005, ponton et al. 1990, wang et al. 1993) do indicate the role of am fungi on growth of ferns, there is no information on the role of the septate endophytic fungi on fern development and growth. we suspect a greater role of septate endophytic fungi in gametophyte and sporophyte development in ferns as spores of ferns like n. exaltata often develop on substrates other than soil where am inoculum is rare or absent. further investigations on the role of septate endophytic fungi would draw a clear picture of the relationship among these fungi and fern gametophytes. references berch, m., kendrick, b., 1982: vesicular arbuscular mycorrhizae of southern ontario ferns and fern-allies. mycologia 74, 769–776. boullard, b., 1979: consideration sur la symbiosefongique chez les pteridophytes. syllogeus 19, 1–59. bower, f. o., 1923: the ferns, 1. cambridge university press, cambridge. cambie, r. c., ash, j., 1994: fijian medicinal plants. csiro, canberra, australia. campbell, d. h., 1908: symbiosis in fern prothallia. the american naturalist 42, 154–165. cooper, k. m., 1976: a field survey of mycorrhizas in new zealand ferns. new zealand journal of botany 14, 169–181. dickson, s., smith, f. a., smith, s. e, 2007: structural differences in arbuscular mycorrhizal symbiosis. more than 100 years after gallaud, where next? mycorrhiza 17, 375–393. haselwandter, k., read, d. j., 1982: the significance of a root-fungus association in two carex species of high-alpine plant communities. oecologia 53, 352–354. javalgekar, s. r., mahabale, t. s., 1959: germination of spores and prothalli in two species of nephrolepis, n. exaltata schott., and n. acuta presl. proceedings of the national institute of science india 25, 333–338. 144 acta bot. croat. 71 (1), 2012 muthukumar t., prabha k. u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:36:09 color profile: disabled composite 150 lpi at 45 degrees johnson, n. c., graham, j. h., smith, f. a., 1997: functioning of mycorrhizal associations along the mutualism-parasitism continuum. new phytologist 135, 575–585. ko, w-h., ko, s. s., chen, m., 2005: origin and control of fern weeds in orchid production in greenhouse in hawaii. crop protection 24, 487–490. koske, r. e., gemma, j. n., 1989: a modified procedure for staining roots to detect va-mycorrhizas. mycological research 92, 486–488. muthukumar, t., udaiyan, k., manian, s., 1996: vesicular – arbuscular mycorrhizae in tropical sedges of southern india. biology and fertility of soils 22, 96–100. muthukumar, t., udaiyan, k., 2000: vesicular arbuscular mycorrhizae in pteridophytes of western ghats, southern india. phytomorphology 50,132–142. nayar, b. k., kaur, s., 1971: gametophytes of homosporus ferns. botanical review 37, 295–396. neuhauser, c., faragione, j. e., 2004: a mutualism-parasitism continuum model and its application to plant-mycorrhizae interactions. ecological modelling 177, 331–352. ponton, f., piche, y., parent, s., caron, m., 1990: use of vesicular-arbuscular mycorrhizae in boston fern production. ii. evaluation of four inocula. horticultural science 25, 416–419. read, d. j., duckett, j. g., francis, r., ligrone, r., russell, a., 2000: symbiotic fungal associations in lower land plants. philosophical transactions of the royal society of london b-biological sciences 355, 815–831. reyes-jaramillo, i., camargo-ricalde, s. l., aquiahuatl-ramos, m. a., 2008: mycorrhizal-like interaction between gametophytes and young sporophytes of the fern dryopteris muenchii (filicales) and its fungal endophyte. revista de biologia tropical 56, 1101–1107. schmid, e., oberwinkler, f., 1995: a lightand electron-microscopic study on a vesicular-arbuscular host-fungus interaction in gametophytes and young sporophytes of the gleicheniaceae (filicales). new phytologist 129, 317–324. smith, a. r., pryer, k. m., schuttplez, e., korall, p., schneider, h., wolf, p. g., 2006: a classification of extant ferns. taxon 55, 705–731. smith, s. e., read, d. j., 2008: mycorrhizal symbiosis. academic press inc, san diego. srivastava, m., ma, l. q., singh, n., singh, s., 2005: antioxidant responses of hyper accumulator and sensitive fern species to arsenic. journal of experimental botany 56, 1335–1342. swatzell, l. j., powell, m. j., kiss, j. z., 1996: the relationship of endophytic fungi to the gametophyte of the fern schizaea pusilla. international journal of plant sciences 157, 53–62. turnau, k., anielska, t., jurkiewicz, a., 2005: mycothallic/mycorrhizal symbiosis of chlorophyllous gametophytes and sporophytes of a fern, pellaea viridis (forsk.) prantl (pellaeaceae, pteridales). mycorrhiza 15, 121–128. turrill, w. b., 1935: krakatau and its problem. new phytologist 34, 442. wang, b., qiu, l., 2006: phylogenetic distribution and evolution of mycorrhizas in land plants. mycorrhiza 16, 299–363. acta bot. croat. 71 (1), 2012 145 fungal associations in nephrolepis exaltata u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:36:09 color profile: disabled composite 150 lpi at 45 degrees wang, h., parent, s., gosselin, a., desjardins, y., 1993: vesicular-arbuscular mycorrhizal peat-based substrates enhance symbiosis establishment and growth of three micropropagated species. journal of the american society for horticultural science 118, 896–901. winther, j. l., friedman. w. e., 2007: arbuscular mycorrhizal symbionts in botrychium (ophioglossaceae). american journal of botany 94, 1248–1255. winther, j. l., friedman. w. e., 2008: arbuscular mycorrhizal symbionts in lycopodiaceae. new phytologist 177, 790–801. winther, j. l., friedman. w. e., 2009: phylogenetic affinity of arbuscular mycorrhizal symbionts in psilotum nudum. journal of plant research 12, 485–496. 146 acta bot. croat. 71 (1), 2012 muthukumar t., prabha k. u:\acta botanica\acta-botan 1-12\498 muthukumar.vp 26. o ujak 2012 13:36:09 color profile: disabled composite 150 lpi at 45 degrees 200 acta bot. croat. 76 (2), 2017 acta bot. croat. 76 (2), 200–224, 2017 coden: abcra 25 doi: 10.1515/botcro-2017-0014 issn 0365-0588 eissn 1847-8476 vegetation of croatia: phytosociological classification of the high-rank syntaxa željko škvorc1*, nenad jasprica2, antun alegro3, sanja kovačić4, jozo franjić1, daniel krstonošić1, ana vraneša5, andraž čarni6 1 university of zagreb, faculty of forestry, svetošimunska 25, hr-10000 zagreb, croatia. 2 university of dubrovnik, institute for marine and coastal research, kneza damjana jude 12, hr-20000 dubrovnik, croatia. 3 university of zagreb, faculty of science, department of biology, division of botany, marulićev trg 20/ii, hr-10000 zagreb, croatia. 4 university of zagreb, faculty of science, department of biology, botanical garden, marulićev trg 9a, hr-10000 zagreb, croatia. 5 ministry of culture of the republic of croatia, runjaninova 2, hr-10000 zagreb, croatia. 6 scientific research center of the slovenian academy of sciences and arts, institute of biology, novi trg 2, si-1000 ljubljana, slovenia. abstract – croatia is among the most ecologically diverse and floristically rich countries in europe, with a great variety of communities. the vegetation elaboration according to the standard central european method was initiated in croatia at the beginning of the 20th century. in previous overviews of croatian vegetation, the number of classes and alliances was underrepresented in relation to the country’s floristic richness. furthermore, the level of knowledge and the amount of available data varied greatly among the various types of vegetation. the aims of this paper are mainly to compile a stabile syntaxonomic list of classes, orders and alliances dominated by vascular plants in croatia and to adjust croatian vegetation to the new european syntaxonomic system (eurovegchecklist). it introduces a consistent description of high-rank syntaxa in croatian. in conclusion, the vegetation of croatia comprises 66 classes, 121 orders and 201 alliances. the number of syntaxa shows vegetation diversity that is rather high compared to most other european countries; this is related to the high floristic richness and endemism. the list points out the obvious problems and gaps in our knowledge of vegetation in croatia and can serve as a baseline for the future vegetation studies. keywords: braun-blanquet approach, phytosociology, syntaxonomy, vegetation * corresponding author, e-mail: zskvorc@sumfak.hr introduction croatia, extending from the adriatic sea over the dinaric alps toward the pannonian plain, is among the floristically richest countries in europe and one of the hotspots of european biodiversity (nikolić 2001, nikolić et al. 2014). this floristic and ecological richness, resulting in a great variety of communities, has always attracted research. beck-mannagetta (1901) and adamović (1909) were the first botanists systematically to study the vegetation, and published extensive monographs based on the physiognomic-ecological approach. the vegetation elaboration according to the standard central european method was initiated at the beginning of the 20th century (braun-blanquet 1921). shortly thereafter research according to this method began in croatia as well (pevalek 1924, horvat 1925, horvatić 1927) and the new approoach was widely accepted among botanists. since then, many researchers have elaborated the vegetation of croatia, providing the extensive results that enabled the preparation of this overview. the level of knowledge and the amount of available data varies greatly among the various types of vegetation. for example, forest vegetation was investigated and documented to a greater extent. the first reviews of forest vegetation appeared in the works of horvat (1937, 1938), and since then many comprehensive reviews have been published (e.g. rauš et al. 1992, vukelić et al. 2008, vukelić 2012). additionally, some of the most important vegetation research results in croatia were published by horvatić (1963), marković-gospodarić (1966), ilijanić (1968), šugar (1973), šegulja (1976), topić (1984), trinajstić (1998, 2008) and hulina (2002). vegetation of croatia acta bot. croat. 76 (2), 2017 201 to gain a comprehensive view of vegetation, botanists arrange complex vegetation patterns obtained from the field, in conceptually manageable and functionally logical units, called plant communities or vegetation types (mucina et al. 2016). in this way, vegetation description and classification provides suitable objects for ecosystem research and inventory on a different scale. these objects are also used for the planning and managing of various conservation programs and the use of natural resources. due to the enormous complexity involved, there are many different approaches to vegetation classification (peet and roberts 2013). the most widely applied approach is based on total floristic composition of stands reflecting the environmental heterogeneity and biogeographic processes (the braun-blanquet approach), and it is developed within the framework of the scientific discipline called phytosociology. phytosociology provides standardized protocols for vegetation sampling, description and delimitation of abstract vegetation types (syntaxa), and their hierarchical ordering into a practical and efficient framework (syntaxonomy) (braun-blanquet 1964, westhoff and van der maarel 1978, dengler et al. 2008). the international code of phytosociological nomenclature is a formal framework for the naming and organization of syntaxa (weber et al. 2000). initial attempts to prepare an overview of syntaxa on a larger scale were made in the first half of the 20th century by braun-blanquet (1933) and tüxen (1950, 1966), but the results were inadequate due to the lack of data. as vegetation scientists have been following this approach for decades, an enormous amount of phytosociological data has accumulated. in the last two decades, new syntaxonomic overviews have appeared for almost all european countries (jiménezalfaro et al. 2014) and many efforts have been made to unify the classification system of european vegetation (mucina 1997, rodwell et al. 2002). recently, finally, the first comprehensive and consistent syntaxonomic system of alliances, orders and classes for vascular plants, bryophytes and lichens, as well as for the algal communities of europe has been established (mucina et al. 2016). the first overview of croatian vegetation was published by horvat (1942), followed by that of trinajstić and šugar (1976). in addition, the vegetation of croatia has also been included within the review of ex-yugoslav (zupančič et al. 1986) or southeast-european vegetation (horvat et al. 1974). the latest and most comprehensive overview of croatian vegetation was given by trinajstić (2008), who reported 407 associations, 121 alliances, 66 orders and 42 classes. however, according to jiménez-alfaro et al. (2014), the number of classes and alliances in trinajstić (2008) was underrepresented when compared with the country’s actual floristic richness. the vegetation of croatia has been intensively studied during the last decade and many new syntaxonomical contributions have been published. therefore, the aims of this paper are: 1) to compile a stabile syntaxonomic list of classes, orders and alliances dominated by vascular plants in croatia, 2) to adjust the croatian vegetation survey to the new european syntaxonomic system (mucina et al. 2016), 3) to introduce a consistent description of classes, orders and alliances in croatian, and 4) to point out the obvious problems and gaps in our knowledge of the vegetation of croatia. methods the syntaxonomical scheme and nomenclature of all syntaxa follows the eurovegchecklist (mucina et al. 2016). classes are grouped into broad informal groups according to mucina (2013) and mucina et al. (2016). the baselines for the preparation of this paper were data originating from trinajstić (2008) and vukelić (2012) and references therein. in addition, the paper includes vegetation types reported in different sources, as well as those occurring in croatia according to our own knowledge and experience. vegetation types indicated by an asterisk (*) pro bably do occur in croatia, but to confirm their presence and distribution further research is needed. additional comments are included in the descriptions of vegetation types in the cases of: (1) different opinions on their syntaxonomy, (2) significant differences compared to the previous syntaxonomic treatment in croatian phytosociological literature. the eurovegchecklist provides brief text descriptions for all included syntaxa which contain the physiognomy of the vegetation classified within the given unit, their unifying ecological context, and their distribution (mucina et al. 2016). these original descriptions have been retained in the list with short descriptions in croatian added. only synonyms that have been frequently used in the croatian literature are listed. each alliance was associated by the eunis habitat code, according to schaminée et al. (2012), adjusted for croatian territory. this paper does not provide a compilation of all the phytosociological literature references in croatia, because they have been already listed in trinajstić (2008) and vukelić (2012). therefore, only references directly used during the preparation of this paper are listed. results and discussion according to our estimations, the vegetation of croatia comprises 66 classes, 121 orders and 201 alliances (see appendix). the number of syntaxa shows that the vegetation diversity of croatia is high compared to most european countries (jiménez-alfaro et al. 2014), which is related to the high floristic richness and endemism (nikolić 2001, nikolić et al. 2014), in common with other mediterranean countries (e.g. spain, italy and france, jiménez-alfaro et al. 2014). the total floristic composition reflects the biogeography, environmental heterogeneity and, consequently, vegetation diversity of the region. european countries with two biogeographical regions (eurosiberian and mediterranean) have the greatest vegetation richness in relation to their size (izco and amigo 2011). this paper lists 24 classes, 55 orders and 80 alliances more than were previously noted by trinajstić (2008). this discrepancy can be explained at least partly by the insufficient elaboration of certain vegetation types and/or regions škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 202 acta bot. croat. 76 (2), 2017 of croatia, since vegetation research was based mostly on the preference of particular researchers, institutions or projects. for example, for robinietea, sedo-scleranthetea, montio-cardaminetea and hypno cupressiformi-polypodietalia vulgaris there were few or no literature data. therefore, this paper may serve as a guideline for further studies, with the aim of better elaborating less known vegetation types in croatia. the other reason for the discrepancy in number of previously published syntaxa can be found in the different nomenclatural concepts applied. for example, the syntaxonomy of tall-herb vegetation (mulgedio-aconitetea) and zonal mediterranean forests and scrubs differs greatly from that given in trinajstić (2008) and vukelić (2012). furthermore, intrazonal mediterranean grasslands and herblands have been classified in a larger number of syntaxa than in horvatić (1963) and trinajstić (2008). from our own experience, the 17 alliances marked with an asterisk (*) probably are present in croatia, but due to the lack of relevant vegetation data their occurrence is unconfirmed. therefore, further research is needed. likewise, it would be necessary better to define the syntaxonomy of several other vegetation types with different opinions on their syntaxonomical treatment (e.g. alno glutinosae-populetea albae). the need for further comprehensive vegetation studies is evident also from the treatment of ruderal vegetation which is here represented by 34 alliances, in contrast to 22 alliances in trinajstić (2008). these vegetation types have been intensively studied in the past (marković-gospodarić 1966, marković 1984, 1992, topić 1984, hulina 2002, pandža et al. 2005), but such discrepancies in the number of alliances indicates the need for new field research and synthesis. in sum, this paper harmonizes the classification of croatian vegetation with the eurovegchecklist (mucina et al. 2016) in order to meet the common european standards. nevertheless, the classification of vegetation could have been done by using a different approach. with this in mind, the applied syntaxonomic concept represents just one view on this subject. however, it is a baseline for future syntaxonomic analyses that will result in a complete elaboration of croatian vegetation based on relevé-databases and numerical techniques. acknowledgements the authors thank steve latham (uk) for improving the english. references adamović, l., 1909: die vegetationsverhältnisse der balkanländer. in: engler, a., drude, o. 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(ed.), anthropogene vegetation, 75–82. dr w junk, den haag. trinajstić, i., 1998: plantgeographycal division of climazonal forest vegetation of croatia. šumarski list 122, 407–421 (in croatian). trinajstić, i., 2008: plant communities of croatia. akademija šumarskih znanosti, zagreb (in croatian). trinajstić, i., šugar, i., 1976: prodromus of plant communities of croatia. institut za botaniku sveučilišta u zagrebu (in croatian). vukelić, j., 2012: forest vegetation of croatia. šumarski fakultet sveučilišta u zagrebu, državni zavod za zaštitu prirode, zagreb (in croatian). vukelić, j., mikac, s., baričević, d., bakšić, d., rosavec, r., 2008: forest sites and forest communities in croatia. national ecological network. državni zavod za zaštitu prirode rh, zagreb. weber, h.e., moravec, j., theurillat, j.-p., 2000: international code of phytosociological nomenclature. 3rd edition. journal of vegetation science 11, 739–768. westhoff, v., van der maarel, e., 1978: the braun-blanquet approach. in: whittaker, r.h. (ed.) classification of vegetation, 287–399. dr w. junk, the hague, nl. zupančič, m., jovanović, b., lakušić, r., rizovski, r., trinajstić, i. (eds.) 1986: prodromus phytocoenosum jugoslaviae. ad mappam vegetationis m 1:200 000. bribir-ilok. appendix. vegetation list remarks to the syntaxa descriptions (in croatian) napomene uz opise sintaksona na hrvatskom jeziku svakom ovdje navedenom sintaksonu uz izvorne opise na engleskom jeziku dodijelili smo i hrvatske nazive, pri čemu smo u najvećoj mjeri nastojali preuzeti postojeću domaću terminologiju koja je tijekom prošlog stoljeća ko rištena pri imenovanju vegetacijskih tipova. u slu ča jevima kada se unutar višeg sintaksona nalazi samo jedan niži sintakson, hrvatski naziv nižeg sintaksona jednak je nazivu višeg. kada se u nazivu navodi fitogeografski položaj sintaksona koristili smo pojmove „sredozemni” i „kontinentalni”. pri tome se „sredozemni” odnosi na cijelu mediteransku vegetacijsku regiju unutar koje smo razlikovali submediteransko i primorsko područje. korištena je i uobi ča jena neformalna botanička podjela zeljastih biljka na jed nogodišnje, dvogodišnje i trajnice te na zeleni i trave, pri čemu su „zeleni” zeljaste biljke širokih listova (biljke koje nemaju graminoidne listove). 1. zonal and intrazonal vegetation zonalna i intrazonalna vegetacija 1.1. vegetation of the boreal zone vegetacija borealne zone 1.1.1. zonal boreal and hemiboreal forests zonalne borealne i hemiborealne šume pic vaccinio-piceetea br.-bl. in br.-bl. et al. 1939 gorske i pretplaninske šume smreke i jele holarctic coniferous and boreo-subarctic birch forests on oligotrophic and leached soils in the boreal zone and at high-altitudes of mountains in the nemoral zone of eurasia pic-01 piceetalia excelsae pawłowski et al. 1928 (syn. vaccinio-piceetalia excelsae br.-bl. in br.-bl. et al. 1939) acidofilne gorske i pretplaninske šume smreke i jele na siromašnim tlima european boreo-montane and subalpine spruce and pine forests on nutrient-poor soils pic-01a piceion excelsae pawłowski et al. 1928 (syn. vaccinio-piceion excelsae br.-bl. in br.-bl. et al. 1939) – eunis g3.1 škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 204 acta bot. croat. 76 (2), 2017 acidofilne gorske i pretplaninske šume smreke i jele na siromašnim tlima european boreo-montane spruce forests and subalpine open pine woods on nutrient poor podzolic soils pic-06 athyrio filicis-feminae-piceetalia hadač in hadač et al. 1969 gorske i pretplaninske šume smreke i jele na bogatim tlima european boreo-montane spruce, fir and pine forests on nutrient-rich soils pic-06a chrysanthemo rotundifolii-piceion (kra jina 1933) březina et hadač in hadač 1962 – eunis g3.1 pretplaninske šume smreke na bogatim tlima mesic herb-rich spruce forests of the central and northern european mountains pic-06b abieti-piceion (br.-bl. in br.-bl. et al. 1939) soó 1964 – eunis g3.1 gorske šume smreke na bogatim tlima mesophilous fir forests on brown forest soils of the central and southwestern european mountains pic-06c calamagrostio-abietion horvat 1962 nom. invers. propos. – eunis g3.1 šume jele na vapnenačkim stijenama i blokovima mesic herb-rich fir forests on limestone and dolomite boulder screes in the montane and subalpine belts of the western balkans bra brachypodio pinnati-betuletea pendulae ermakov et al. 1991 šume breze hemiboreal pine and birch-pine herb-rich open forests on fertile soils of the southern urals and southern siberia, and relict birchpoplar forests of europe bra-02 fragario vescae-populetalia tremulae willner et mucina in willner et al. 2016 nom. inval. šume breze relict extrazonal temperate deciduous birch-poplar woods on mineral soils of europe comment: this order comprises natural pioneer and secondary birch-poplar woods (willner et al. 2016). in croatian literature this communities were elaborated with in quercetalia roboris tx. 1931 (trinajstić 2008, vu kelić et al. 2012). bra-02a fragario vescae-populion tremulae will ner et mucina ined. – eunis g1.9 šume breze relict extrazonal temperate deciduous birch-poplar woods on mineral soils of europe 1.2. vegetation of the nemoral forest zone vegetacija šumske zone umjerenih područja 1.2.1. zonal temperate broad-leaved forests zonalne listopadne šume umjerenih područja fag carpino-fagetea sylvaticae jakucs ex passarge 1968 (syn. querco-fagetea sylvaticae br.-bl. et vlieger in vlieger 1937) mezofilne listopadne i mješovite šume mesic deciduous and mixed forests of temperate europe, anatolia, the caucasus and southern siberia fag-01 luzulo-fagetalia sylvaticae scamoni et passarge 1959 acidofilne šume bukve acidophilous beech and mixed fir-beech forests on nutrient-poor soils in the nemoral zone of temperate europe and as relicts at high altitides of corsica fag-01a luzulo-fagion sylvaticae lohmeyer et tx. in tx. 1954 – eunis g1.6 acidofilne šume bukve acidophilous beech and mixed fir-beech forests of central europe fag-02 fagetalia sylvaticae pawłowski 1928 neutrofilne i bazofilne šume bukve i šume bukve i jele basiphilous beech and mixed fir-beech forests in the nemoral zone and in the montane belt of the submediterranean regions of temperate europe fag-02a aremonio-fagion (horvat 1950) bor hi di in török et al. 1989 – eunis g1.6 ilirske neutrofilne i bazofilne šume bukve i šume bukve i jele refugial basiphilous beech and mixed fir-beech forests of the northwestern balkans and the eastern alps fag-02b fagion sylvaticae luquet 1926 – eunis g1.6 srednjoeuropske neutrofilne i bazofilne šume bukve partly refugial post-glacial basiphilous beech and mixed fir-beech forests of the temperate europe fag-03 carpinetalia betuli p. fukarek 1968 šume kitnjaka i običnog graba oak-hornbeam and mesic oak forests on deep nutrient-rich soils of the temperate europe fag-03a carpinion betuli issler 1931 – eunis g1.a srednjoeuropske šume kitnjaka i običnog graba oak-hornbeam forests on deep nutrient-rich soils of central and eastern europe fag-03c erythronio-carpinion (horvat 1958) marinček in wallnöfer et al. 1993 – eunis g1.a ilirske šume kitnjaka i običnog graba oak-hornbeam forests on deep nutrient-rich soils of the balkans and northern italy fag-05 aceretalia pseudoplatani moor 1976 nom. conserv. propos. šume plemenitih listača scree and ravine maple-lime forests of the nemoral zone of the temperate europe *fag-05a tilio-acerion klika 1955 – eunis g1.a srednjoeuropske mezofilne šume plemenitih listača sycamore maple forests in the montane belt and cool ravines of the central european mountain ranges *fag-05b melico-tilion platyphylli passarge et g. hofmann 1968 – eunis g1.a srednjoeuropske termofilne šume plemenitih listača thermophilous lime forests on scree slopes at low altitudes of the southern regions of central europe fag-05d fraxino excelsioris-acerion pseudoplatani p. fukarek 1969 – eunis g1.a ilirske mezofilne šume plemenitih listača submediterranean mesophilous broad-leaved ash-maple scree and ravine forests of the balkan peninsula fag-05e ostryo carpinifoliae-tilion platyphylli (košir et al. 2008) čarni in willner et al. 2016 – eunis g1.a ilirske kserotermofilne šume plemenitih listača submediterranean xero-thermophilous broad-leaved scree and ravine forests of the balkan peninsula vegetation of croatia acta bot. croat. 76 (2), 2017 205 pub quercetea pubescentis doing-kraft ex scamoni et passarge 1959 termofilne šume listopadnih hrastova oak, mixed deciduous and conifer open forests of warm regions in the cool-temperate nemoral zone of central and southern europe and in the supramediterranean belt of the mediterranean, asia minor and middle east pub-01 quercetalia pubescenti-petraeae klika 1933 termofilne šume listopadnih hrastova oak forests of the warm cool-temperate regions in the nemoral zone of central and southern europe and relic supramediterranean fir-pine and oak forests of the mediterranean pub-01a quercion petraeae issler 1931 – eunis g1.7, g1.8 srednjoeuropske acidotermofilne šume listopadnih hrastova thermophilous central european acidophilous oak forests pub-01b quercion pubescenti-petraeae br.-bl. 1932 nom. mut. – eunis g1.7 srednjoeuropske termofilne kalcifilne šume listopadnih hrastova thermophilous central european calciphilous oak forests pub-01c aceri tatarici-quercion zólyomi 1957 – eunis g1.7 termofilne panonske šume hrastova na lesu thermophilous oak forests on deep soils in the foreststeppe zone of the pontic-pannonian region pub-01f fraxino orni-ostryion tomažič 1940 – eunis g1.7 šume crnog graba i medunca amphiadriatic mesic calcareous submediterranean (sub)montane and inland oak and hop-hornbeam forests on shallow soils comment: in croatian literature all thermophilous pubescent oak forests were elaborated within ostryocarpinion orientalis horvat 1959 (trinajstić 2008, vukelić et al. 2012). in this paper two alliances differ within this complex – fraxino orni-ostryion tomažič 1940 and carpinion orientalis horvat 1958 (čarni et. al. 2009, mucina et al. 2016), wherein ostryo-carpinion orientalis horvat 1959 is a synonim of fraxino orni-ostryion tomažič 1940 pub-01g carpinion orientalis horvat 1958 – eunis g1.7, f5.3 submediteranske šume medunca i bijelog graba amphiadriatic low-altitude calcareous thermophilous oak and oriental hornbeam forests pub-01n quercion confertae horvat 1958 – eunis g1.7 termofilne šume sladuna thermophilous deciduous oak forests on slightly acid ic deep soils of the central balkans *pub-01o quercion petraeo-cerridis lakušić et b. jovanović in b. jovanović et al. ex čarni et mucina 2015 – eunis g1.7 termofilne šume cera i kitnjaka thermophilous montane oak forests of the central balkans. que quercetea robori-petraeae br.-bl. et tx. ex oberd. 1957 acidofilne šume kitnjaka i pitomog kestena acidophilous oak and oak-birch forests on nutrient-poor soils of europe que-01 quercetalia roboris tx. 1931 (syn. quercetalia robori-sessiliflorae tx. 1937) acidofilne šume kitnjaka i pitomog kestena acidophilous oak forests on nutrient-poor soils of europe que-01c agrostio-quercion petraeae scamoni et passarge 1959 (syn. genisto germanicae-quercion neuhäusl et neuhäuslová-novotná 1967) – eunis g1.8 acidofilne šume kitnjaka temperate acidophilous oak forests on nutrient-poor soils of central and eastern europe comment: in croatian literature these forests were elaborated within quercion roboris malcuit 1929 (quercion robori-sessiliflorae br.-bl. 1932; tri naj stić 2008, vukelić 2012). however, this alliance comprises temperate atlantic and subatlantic acidophilous oak forests on nutrient-poor soils of western europe (mucina et al. 2016). que-01e castaneo-quercion petraeae soó 1964 – eunis g1.8 acidofilne šume kitnjaka i pitomog kestena acidophilous chestnut-oak forests on nutrient-poor soils of the southeastern europe 1.2.2. intrazonal scrub of the nemoral zone intrazonalne šikare šumske zone umjerenih područja rha crataego-prunetea tx. 1962 nom. conserv. propos. (syn. rhamno-prunetea rivas goday et borja carbonell 1961) živice, šikare i šumski rubovi scrub and mantle vegetation seral or marginal to broad-leaved forests in the nemoral zone and the submediterranean regions of europe rha-01 prunetalia spinosae tx. 1952 kontinentalne živice, šikare i šumski rubovi scrub and mantel vegetation seral or marginal to broadleaved forests in the nemoral zone of europe rha-01a berberidion vulgaris br.-bl. ex tx. 1952 nom. conserv. propos. – eunis f3.1, f3.2 termofilne živice, šikare i šumski rubovi southern temperate and submediterranean thermophilous scrub of southern and central europe rha-01e astrantio-corylion avellanae passarge 1978 – eunis f3.1 šikare lijeske u brdskom i gorskom pojasu hazel scrub on nutrient-rich soils in the submontane and montane belts of western, central and southeastern europe rha-01f pruno-rubion radulae weber 1974 – eunis f3.1 mezofilne srednjoeuropske živice, šikare i šumski ru bovi bramble scrub on neutral and base-rich soils of western and central europe rha-01i brachypodio pinnati-juniperion commu nis mucina in mucina et al. 2016 – eunis f3.1, f3.2 termofilne šikare obične borovice na vapnenačkoj podlozi low-altitude thermophilous juniper scrub on calcarous substrates of western and central europe škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 206 acta bot. croat. 76 (2), 2017 rha-01j prunion fruticosae tx. 1952 – eunis f3.1 mezofilne panonske živice, šikare i šumski rubovi subcontinental and continental scrub in the foreststeppe and steppe zones of central and eastern europe rha-02 paliuretalia trinajstić 1978 submediteranske živice, šikare i šumski rubovi thermophilous mantle, pseudomaquis and šibljak fringing oak forests of the submediterranean regions of southeastern europe *rha-02c fraxino orni-cotinion soó 1960 – eunis f3.2 panonske kserotermofilne živice, šikare i šumski rubovi thermophilous mantle vegetation of the southern pannonian oak forests comment: this vegetation type is extrazonal submediterranean vegetation of the southern pannonian plain. rha-02e paliuro-petterion p. fukarek 1962 (syn. paliurion adriaticum trinajstić 1977, rhamno intermediae-paliurion spinae-christi trinajstić /1978/ 1996) – eunis f3.2 dračici submediterranean thermophilous šibljak of the eastern adriatic seaboards of the balkan peninsula rob robinietea jurko ex hadač et sofron 1980 šumske sječine i antropogene šikare seral forest-clearing and anthropogenic successional scrub and thickets on nutrient-rich soils of temperate europe rob-01 sambucetalia racemosae oberd. ex doing 1962 šumske sječine elder, willow and hazel scrub on nutrient-rich soils in forest clearings of temperate europe rob-01a sambuco-salicion capreae tx. et neumann ex oberd. 1957 – eunis f3.1, g5.2, g5.6, g5.8 šumske sječine elder, willow and hazel scrub on nutrient-rich soils in forest clearings of temperate europe rob-02 chelidonio-robinietalia pseudoacaciae jurko ex hadač et sofron 1980 antropogene šikare i šumarci subspontaneous anthropogenic scrub and low-grown forest groves rob-02a aegopodio podagrariae-sambucion nigrae chytrý 2013 – eunis f3.1 šikare bazge anthropogenic elder scrub in ruderal habitats of western and central europe rob-02b balloto nigrae-robinion pseudoacaciae hadač et sofron 1980 – eunis g5.2 kserofilne antropogene šikare i šumarci bagrema robinia groves with weedy understorey on loamy-sandy dry soils of central and eastern europe rob-02c chelidonio majoris-robinion pseudoacaciae hadač et sofron ex vítkováin chytrý 2013 – eunis g5.2 mezofilne antropogene šikare i šumarci bagrema robinia groves with weedy understorey on nutrientrich mesic soils of central and eastern europe *rob-02e chelidonio-acerion negundo l. ishbirdin et a. ishbirdin 1989 – eunis f3.1, g5.2 antropogene šikare i šumarci negundovca subspontaneous groves and scrub of acer negundo of eastern europe 1.2.3. intrazonal boreo-temperate grasslands and heath intrazonalni travnjaci i vrištine borealnih i umjerenih područja uli calluno-ulicetea br.-bl. et tx. ex klika et hadač 1944 vrištine i acidofilne šikare obične borovice heath on acidic nutrient-poor soils in the lowland to montane belts of the temperate and boreal zones of europe uli-02 vaccinio myrtilli-genistetalia pilosae schu bert ex passarge 1964 vrištine heath of cold-atlantic, subcontinental and subboreal and boreal regions of western, central and northeastern europe and scandinavia uli-02b calluno-genistion pilosae p. duvigneaud 1945 (syn. genistion pilosae böcher 1943) – eunis f4.2 vrištine low-altitude heath of the atlantic and subcontinental regions of temperate europe uli-03 vaccinio-juniperetalia communis passarge 1972 acidofilne šikare obične borovice low-altitude acidophilous juniper scrub of temperate subatlantic regions of europe uli-03a vaccinio-juniperion communis passarge in passarge et g. hofmann 1968 – eunis f3.1 acidofilne šikare obične borovice low-altitude acidophilous juniper scrub of temperate subatlantic regions of europe nar nardetea strictae rivas goday et borja carbonell in rivas goday et mayor lópez 1966 nom. conserv. propos. travnjaci tvrdače na siromašnim tlima secondary mat-grass swards on nutrient-poor soils at low and mid-altitudes of the temperate, boreal and subarctic regions of europe nar-01 nardetalia strictae preising 1950 travnjaci tvrdače na siromašnim tlima secondary mat-grass swards on nutrient-poor soils at low and mid-altitudes of temperate, boreal and subarctic regions of europe nar-01b violion caninae schwickerath 1944 – eunis e1.7, e5.3 travnjaci tvrdače u nizinskom pojasu meso-subxerophytic oligotrophic pastures in the lowland to submontane belts of western and central europe nar-01d nardo-agrostion tenuis sillinger 1933 – eunis e1.7 travnjaci tvrdače u pretplaninskom pojasu mat-grass dry pastures in the submontane to subalpine belts of the mountain ranges of central europe and the northern balkans nar-01h achilleo-arnicion horvat et pawłowski in horvat 1960 (syn. calluno-festucion capillatae horvat ex horvat et al. 1974) – eunis e1.7 travnjaci tvrdače u brdskom i gorskom pojasu oligotrophic pastures in the lowland to submontane belts of the western balkans cor koelerio-corynephoretea canescentis klika in klika et novák1941 panonski travnjaci na pješčanim tlima dry grasslands on sandy soils and on rocky outcrops of the temperate to boreal zones of europe, the north atlantic islands and greenland vegetation of croatia acta bot. croat. 76 (2), 2017 207 cor-02 festucetalia vaginatae soó 1957 panonski travnjaci na pješčanim tlima european (sub)continental fescue sandy steppes in the forest-steppe and steppe zones of europe cor-02a festucion vaginatae soó 1929 – eunis e1.1 panonski travnjaci na pješčanim tlima pannonian subcontinental fescue sandy steppes sed sedo-scleranthetea br.-bl. 1955 pionirska vegetacija na plitkim i kamenitim tlima pioneer vegetation on shallow soils on rocky siliceous outcrops on siliceous rocks of the temperateand boreal europe sed-03 thero-airetalia rivas goday 1964 pionirska vegetacija na plitkim i kamenitim silikatnim tlima pioneer vegetation on acidic shallow soils of the wintermild atlantic and subboreal regions of western europe, the northern iberian peninsula and madeira sed-03a thero-airion tx. ex oberd. 1957 – eunis e1.1, e1.9 pionirska vegetacija na plitkim i kamenitim silikatnim tlima pioneer vegetation on acidic shallow soils of the winter-mild atlantic and subboreal regions of western europe, the northern iberian peninsula and madeira sed-04 alysso-sedetalia moravec 1967 pionirska vegetacija na plitkim i kamenitim vapne nač kim tlima te bazičnim pijescima european temperate pioneer therophyte and stonecrop swards on calcareous shallow skeletal soils and base-rich sands sed-04a alysso alyssoidis-sedion oberd. et t. müller in t. müller 1961 – eunis e1.1, h3.6 pionirska vegetacija na plitkim i kamenitim vap ne nač­ kim tlima thermophilous stonecrop vegetation on weathered calcareous rocks of temperate europe *sed-04h bassio laniflorae-bromion tectorum borhidi 1996 nom. conserv. propos. – eunis e1.1 panonska pionirska vegetacija na bazičnim pijescima pannonian annual open swards on base-rich sandy substrates ger trifolio-geranietea sanguinei t. müller 1962 šumski rubovi s prevlašću visokih zeleni thermophilous forest fringe and tall-herb vegetation in nutrient-poor sites in the submediterranean to subboreal zones of europe and the macaronesia ger-01 origanetalia vulgaris t. müller 1962 mezofilni šumski rubovi s prevlašću visokih zeleni meso-subxerophytic fringe and tall-herb vegetation on nutrient-poor but base-rich soils of temperate and subboreal europe ger-01b trifolion medii t. müller 1962 – eunis e5.2 mezofilni šumski rubovi s prevlašću visokih zeleni meso-subxerophytic fringe vegetation on nutrient-poor but base-rich soils at lower altitudes of temperate western and central europe ger-02 antherico ramosi-geranietalia sanguinei julve ex dengler in dengler etal. 2003 termofilni šumski rubovi s prevlašću visokih zeleni xerophilous fringe and tall-herb vegetation on nutrientpoor and base-rich soils in the submediterranean, temperate and subboreal zones of europe ger-02a geranion sanguinei tx. in t. müller 1962 – eunis e5.2 srednjoeuropski termofilni šumski rubovi s prevlašću visokih zeleni xerophilous fringe and tall-herb vegetation of the subcontinental western and central europe ger-02c dictamno albi-ferulagion galbaniferae (van gils et al. 1975) de foucaultet al. ex čarni et dengler in mucina et al. 2009 – eunis e5.2 ilirski termofilni šumski rubovi s prevlašću visokih zeleni xerophilous fringe and tall-herb vegetation of the illyrian and dinaric regions of the balkan peninsula ger-05 melampyro-holcetalia mollis passarge in theurillat et al. 1995 acidofilni šumski rubovi s prevlašću visokih zeleni meso-xerophytic fringe and tall-herb on acidic soils in the submediterranean to subboreal zones of europe ger-05a melampyrion pratensis passarge 1979 – eunis e5.2 acidofilni šumski rubovi s prevlašću visokih zeleni meso-xerophytic forest-edge communities on acidic soils in semi-shady to sunny habitats of temperate and (sub)boreal europe mol molinio-arrhenatheretea tx. 1937 vegetacija travnjaka i visokih zeleni na dubokim tlima anthropogenic managed pastures, meadows and tall-herb meadow fringes on fertile deep soils at low and mid-altitudes (rarely also high altitudes) of europe mol-01 arrhenatheretalia elatioris tx. 1931 mezofilne livade i pašnjaci mown meadows and pastures on well-drained mineral soils at low and mid-altitudes of temperate and subboreal europe mol-01a arrhenatherion elatioris luquet 1926 – eunis e2.2, e2.7 mezofilne livade od nizinskog do brdskog pojasa mesic mown meadows on mineral-rich soils in the lowland to submontane belts of temperate europe mol-01b phyteumato-trisetion ellmauer et mucina 1993 – eunis e2.3 mezofilne livade u brdskom i gorskom pojasu mesic mown meadows on relatively mineral-poor soils in the submontane and montane belts of central europe mol-01c cynosurion cristati tx. 1947 – eunis e2.1, e2.6 mezofilni pašnjaci mesic pastures on well-drained mineral-rich soils at low to mid-altitudes of temperate europe mol-01d alchemillo-ranunculion repentis passarge 1979 – eunis e2.8 slabo gaženi mezofilni travnjaci slightly trampled herb-rich grasslands in shaded ha bit ats of the temperate and subboreal regions of europe mol-05 molinietalia caeruleae koch 1926 vlažne livade wet mown meadows on mineral and peaty soils in the temperate to subarctic zones of europe mol-05a molinion caeruleae koch 1926 – eunis e3.5 vlažne livade u nizinskom pojasu škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 208 acta bot. croat. 76 (2), 2017 mown meadows on temporarily wet soils at low altitudes of temperate western and central europe mol-05b calthion palustris tx. 1937 – eunis e3.4 vlažne livade s higrofilnim zelenima herb-rich temporarily wet mown meadows on mineral soils at low altitudes of suboceanic western and subcontinental central europe mol-05d deschampsion caespitosae horvatić 1930 (syn. alopecurion pratensis passarge 1964) – eunis e3.4 periodično vlažne nizinske livade na teškim tlima mown temporarily wet meadows on heavy soils on floodplains in the forest and forest-steppe zones of (sub)continental central and eastern europe mol-06 trifolio-hordeetalia horvatić 1963 vlažni djetelinski travnjaci amphiadriatic wet meadows on gleyic soils of the river floodplains and karstic poljes of the apennine and balkan peninsulas mol-06a molinio-hordeion secalini horvatić 1934 (syn. alopecurion utriculati zeidler 1954) – eunis e3.3 vlažni djetelinski travnjaci u krškim poljima vegetation of wet meadows of the submediterranean precipitation-rich regions of the balkans mol-06d trifolion pallidi ilijanić 1969 – eunis e3.3 kontinentalni vlažni djetelinski travnjaci vegetation of wet meadows of the subhumid continental regions of northern serbia mol-08 filipendulo ulmariae-lotetalia uliginosi passarge 1975 zajednice visokih zeleni uz rubove potoka i vlažnih trav­ njaka tall-herb wet meadow fringe vegetation on mineral soils of temperate europe mol-08a filipendulo-petasition br.-bl. ex duvigneaud 1949 – eunis e5.4 zajednice visokih zeleni uz rubove potoka i vlažnih travnjaka u brdskom i gorskom pojasu tall-herb fringe wet meadow vegetation on neutral and slightly basic mineral soils in the submontane and montane belts of western and central europe mol-08e mentho longifoliae-juncion inflexi t. müller et görs ex de foucault 2009 – eunis d5.3, e3.4 zajednice visokih zeleni uz rubove potoka i vlažnih travnjaka u nizinskom pojasu tall-herb temporarily flooded lightly-grazed nutrientrich meadow fringes in riparian and alluvial habitats of temperate europe mol-10 potentillo-polygonetalia avicularis tx. 1947 (syn. agrostietalia stoloniferae oberd. in oberd. et al. 1967) periodično plavljeni pašnjaci u nizinskom pojasu temporarily flooded and heavily grazed zoo-anthropogenic nutrient-rich meadows and pastures of the temperate and mediterranean regions of europe mol-10a potentillion anserinae tx. 1947 – eunis e3.4 periodično plavljeni pašnjaci u nizinskom pojasu temporarily flooded and heavily grazed nutrient-rich pastures experiencing variable wet-dry or brackishfresh alternating conditions of temperate europe comment: the name agropyro-rumicion crispi nordhagen 1940 has been used predominantly for inland communities of flooded pastures (e.g. tri najstić 2008). however, agropyro-rumicion crispi nordhagen 1940 as including very different communities of maritime strandline vegetation (mucina et al. 2016). 1.2.4. vegetation of the nemoral orosystems vegetacija orosustava u šumskoj zoni umjerenih područja sab junipero-pinetea sylvestris rivas-mart. 1965 nom. invers. propos. oromediteranske borove šume relict oromediterranean and submediterranean orotemperate dry pine forests, juniper woods and related scrub of the mediterranean sab-03 berberido creticae-juniperetalia excelsae mucina in mucina et al. 2016 oromediteranske borove šume relict submediterranean supramediterranean dry pine forests and juniper woods of the central and eastern mediterranean sab-03d berberido creticae-juniperion foetidissimae s. brullo et al. 2001 – eunis g3.5 oromediteranske borove šume silicicolous montane pine and juniper woods and related scrub of continental hellas, cyprus, anatolia and lebanon comment: according to brullo et al. (2001) these communities are spread not only on silicicolous but various supstrata. furthermore, sedlar et al. (2011) suggest the dalmatian pine forests to be included within berberido creticae-juniperion foetidissimae. eri erico-pinetea horvat 1959 bazofilne šume običnog i crnog bora relict pine forests and related scrub on calcareous and ultramafic substrates of the balkans, the alps, the carpathians and crimea eri-01 erico-pinetalia horvat 1959 nom. conserv. propos. bazofilne šume običnog i crnog bora relict pinus nigra forests on dolomite and ultramafic substrates of the dinarides eri-01f erico-fraxinion orni horvat 1959 nom. invers. propos. (syn. fraxino orni-ericion horvat 1959) – eunis g3.5 bazofilne šume običnog i crnog bora relict pinus nigra forests on dolomite and ultramafic substrates of the dinarides mug roso pendulinae-pinetea mugo theurillat in theurillat et al.1995 klekovina krivulja pine krummholz in the subalpine belts of the nemoral moun tain ranges of europe mug-01 junipero-pinetalia mugo boşcaiu 1971 klekovina krivulja pine krummholz in the subalpine belts of the nemoral mountain ranges of europe mug-01d lonicero borbasianae-pinion mugo čarni et mucina 2015 – eunis f2.4 vegetation of croatia acta bot. croat. 76 (2), 2017 209 klekovina krivulja subalpine calcicolous pine krummholz of the balkan peninsula rho rhododendro hirsuti-ericetea carneae schubert et al. 2001 pretplaninske sastojine niskog grmlja supramontane to subalpine low heath on calcareous skeletal soils, rocky outcrops, lapies (karren) and boulders of the alps, apennines and dinarides rho-01 rhododendro hirsuti-ericetalia carneae grabherr et al. 1993 pretplaninske sastojine niskog grmlja supramontane to subalpine low heath on calcareous skeletal soils, rocky outcrops, lapiés and boulders of the alps, the apennines and the dinarides rho-01b aquilegio nigricantis-rhododendrion hirsuti čarni et mucina 2015 – eunis f2.2 pretplaninske sastojine niskog grmlja u središnjim dinaridima subalpine heath on rocky calcareous soils of the central dinarides comment: the name ericion carneae rübel ex grabherr et al. 1993 has been used for this communities (e.g. surina 2013). however, ericion carneae rübel ex grabherr et al. 1993 is vicariant alliance of the alps, the apennines and the northern dinarides (mucina et al. 2016). *rho-01c daphno oleoidis-genistion radiatae n. ranđelović et rexhepi 1980 – eunis f2.2 pretplaninske sastojine niskog grmlja u južnim dinaridima relic supramontane to subalpine low heath on ultramafic and calcareous substrates of the southern dinarides vir betulo carpaticae-alnetea viridis rejmánek ex boeuf, theurillat,willner, mucina et simler in boeuf et al. 2014 gorska i pretplaninska vegetacija listopadnog grmlja subalpine and subarctic herb-rich alder and willow scrub and krummholz of the alps, the carpathians, the hercynicum, the balkans, the caucasus, northern europe and greenland vir-01 alnetalia viridis rübel ex karner et willner in willner et grabherr 2007 pretplaninske zajednice listopadnog grmlja subalpine herb-rich alder and willow scrub and krummholz of the alps, the balkans and the caucasus vir-01a alnion viridis schnyder 1930 – eunis f2.3 pretplaninske zajednice listopadnog grmlja subalpine green alder scrub on fertile soils of the alps and the balkans vir-02 rhamnetalia fallacis p. fukarek 1969 dinarske gorske i pretplaninske zajednice listopadnog grmlja relict deciduous scrub in the montane and subalpine belts of the southern alps, dinarides and apennines vir-02b lonicero-rhamnion fallacis p. fukarek 1969 – eunis f2.3 dinarske gorske i pretplaninske zajednice listopadnog grmlja relict deciduous scrub in the supramontane and subalpine belts of the dinarides and apennines mul mulgedio-aconitetea hadač et klika in klika et hadač 1944 brdska do pretplaninska vegetacija visokih zeleni tall-herb vegetation in nutrient-rich habitats moistened and fertilized by percolating water at high altitudes of europe, siberia and greenland mul-01 adenostyletalia alliariae br.-bl. 1930 gorske i pretplaninske zajednice visokih zeleni tall-herb vegetation on fertile soils at high altitudes of temperate and mediterranean europe mul-01a adenostylion alliariae br.-bl. 1926 nom. conserv. propos. – eunis e5.5 pretplaninske zajednice visokih zeleni na dubokim dekalcificiranim tlima tall-herb vegetation on siliceous substrates at high altitudes in the nemoral zone of europe mul-01c delphinion elati hadač in hadač et al. 1969 – eunis e5.5 gorske i pretplaninske zajednice visokih zeleni na vapnenačkim tlima submontane to subalpine calcicolous tall-herb vegetation of the carpathians mul-03 petasito-chaerophylletalia morariu 1967 brdske i gorske zajednice visokih zeleni tall-herb vegetation on nutrient-rich soils along mountain streams of central europe, the balkans and the apen nines mul-03a petasition officinalis sillinger 1933 – eunis e5.4 brdske i gorske zajednice visokih zeleni na aluvijalnim nanosima tall-herb vegetation on raw alluvia of streams in the upper colline to supramontane belts of the carpathians and the hercynicum mul-03b arunco-petasition albi br.-bl. et sutter 1977 – eunis e5.4 gorske zajednice visokih zeleni strmih padina na skeletnim tlima tall-herb vegetation on skeletal nutrient-rich soils on steep slopes in the montane and supramontane belts of the alps mul-04 senecioni rupestris-rumicetalia alpini mucina et karner in mucina et al. 2016 gorske i pretplaninske antropogene zajednice visokih zeleni tall-herb anthropogenic vegetation on nutrient-rich soils in the upper montane to alpine belts of the nemoral mountain ranges of europe *mul-04a rumicion alpini scharfetter 1938 – eunis e5.5 gorske i pretplaninske antropogene zajednice visokih zeleni tall-herb anthropogenic vegetation on nutrient-rich soils in the upper montane to alpine belts of the nemoral mountain ranges of europe ses elyno-seslerietea br.-bl. 1948 pretplaninske i planinske rudine na vapnenačkoj podlozi alpine and subalpine calcicolous swards of the nemoral mountain ranges of europe ses-01 seslerietalia caeruleae br.-bl. in br.-bl. et jenny 1926 srednjoeuropske pretplaninske i planinske rudine na vapnenačkoj podlozi alpine and subalpine calcicolous grasslands of the ne moral mountain ranges of central europe ses-01c caricion ferrugineae g. br.-bl. et br.-bl. in g. br.-bl. 1931 – eunis e4.4 škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 210 acta bot. croat. 76 (2), 2017 srednjoeuropske pretplaninske i planinske rudine na vapnenačkoj podlozi supramontane to alpine calcicolous meso-hygrophilous sedge swards of the alps and the carpathians ses-02 seslerietalia tenuifoliae horvat 1930 dinarske gorske do planinske rudine na vapnenačkoj podlozi montane to alpine calcicolous tussock grasslands of the northern balkans and the apennines ses-02a seslerion tenuifoliae horvat 1930 – eunis e4.4 dinarske gorske i pretplaninske rudine izložene vjetru montane and subalpine calcicolous blue-grass tussock grasslands of the illyrian region and the northern dinarides ses-02b seslerio juncifoliae-caricion firmae trinajstić 2005 – eunis e4.4 dinarske planinske rudine izložene vjetru alpine calcicolous sedge swards in wind-exposed habitats in the alpine belt of the illyrian region and the northern dinarides ses-02 festucion pungentis horvat 1930 (syn. festucion bosniacae horvat 1930) – eunis e4.4 dinarske pretplaninske rudine zaštićene od vjetra subalpine calcicolous tussock grasslands on steep terraced slopes of the northern dinarides 1.3. vegetation of the steppe zone vegetacija stepske zone 1.3.1. zonal steppe grasslands zonalni stepski travnjaci fes festuco-brometea br.-bl. et tx. ex soó 1947 suhi bazofilni travnjaci dry grassland and steppe vegetation of mostly baseand colloid-rich soils in the submediterranean, nemoral and hemiboreal zones of europe fes-01 brachypodietalia pinnati korneck 1974 nom. conserv. propos. (syn. brometalia erecti koch 1926 nom. ambig. rejic. propos., brometalia erecti br.-bl. 1936 nom. ambig. rejic. propos.) umjereno suhi brdski travnjaci na vapnenačkoj podlozi meso-xerophytic grasslands on deep calcareous soils of western and central europe fes-01a bromion erecti koch 1926 – eunis e1.2 umjereno suhi brdski travnjaci na vapnenačkoj podlozi pod utjecajem atlantske klime meso-xerophytic basiphilous grasslands of western europe and subatlantic central europe fes-01b cirsio-brachypodion pinnati hadač et klika in klika et hadač ex klika1951 – eunis e1.2 umjereno suhi brdski travnjaci na vapnenačkoj pod lozi pod utjecajem kontinentalne klime meso-xerophytic basiphilous grasslands of the subcontinental regions of central and southeastern europe fes-02 festucetalia valesiacae soó 1947 stepski travnjaci na dubokim vapnenačkim tlima steppes and rocky steppic grasslands on deep soils in the steppe and forest-steppe zones of europe and northwestern central asia fes-02a festucion valesiacae klika 1931 nom. conserv. propos. – eunis e1.2 stepski travnjaci na dubokim vapnenačkim tlima steppe fescue grasslands on deep calcareous soils of subcontinental central europe, romania, bulgaria and northwestern ukraine fes-05 stipo pulcherrimae-festucetalia pallentis pop 1968 nom. conserv. propos. kamenjarski stepski travnjaci na vapnenačkoj podlozi xerophilous open steppic grasslands on shallow rocky calcareous and siliceous substrates of central and southeastern europe *fes-05d chrysopogono-festucion dalmaticae borhidi 1996 – eunis e1.1. peripanonski kamenjarski stepski travnjaci na vapnenačkoj podlozi xerophilous rocky steppic grasslands on calcareous substrates of the southern fringes of the pannonian basin fes-05i diantho lumnitzeri-seslerion (soó 1971) chytrý et mucina in mucina et kolbek 1993 – eunis e1.1, e1.2 dealpinski reliktni kamenjarski stepski travnjaci na vapnenačkoj podlozi dealpine relict xerophilous steppic grasslands on calcareous substrates of southeastern central europe fes-09 scorzoneretalia villosae kovačević 1959 (syn. scorzonero villosae-chrysopogonetalia grylli horvatić et horvat in horvatić 1957) submediteranski suhi travnjaci na vapnenačkoj podlozi amphiadriatic dry steppic submediterranean pastures of the prealpine, illyrian and dinaric regions fes-09a chrysopogono grylli-koelerion splendentis horvatić 1973 (syn. chrysopogono-saturejion subspicatae horvat et horvatić 1934, festucion illyricae / horvat 1962/ trinajstić 2000) – eunis e1.2 submediteranski suhi travnjaci na plitkim tlima illyrian submediterranean rocky grasslands on shallow calcareous soils fes-09b saturejion subspicatae tomić-stanković 1970 – eunis e1.2 submediteransko-montani suhi kamenjarski trav njaci dinaric submediterranean montane calcareous rocky grasslands on shallow soils fes-09d scorzonerion villosae horvatić ex kovačević 1959 – eunis e1.2 submediteranski suhi travnjaci na dubokim tlima prealpic and illyrian meso-xerophytic submediterranean grasslands on deep and partly decalcified soils 1.3.2. intrazonal saline vegetation of the steppe zone intrazonalna halofitska vegetacija stepske zone fep festuco-puccinellietea soó ex vicherek 1973 stepski travnjaci na zaslanjenim tlima saline steppes and secondary saline steppic grasslands of the continental regions of europe fep-01 puccinellietalia soó 1947 stepski travnjaci na zaslanjenim tlima meso-xerophytic saline pastures in the subcontinental and submediterranean zones of the southern regions of central and southern europe fep-01c puccinellion limosae soó 1933 – eunis e6.2 stepski travnjaci na zaslanjenim tlima pannonian hypersaline open grasslands on solonetz soils vegetation of croatia acta bot. croat. 76 (2), 2017 211 cry crypsietea aculeatae vicherek 1973 pionirska vegetacija povremenih slanih močvara pioneer ephemeral dwarf-grass vegetation in periodically flooded saline habitats of submediterranean and (sub)continental eurasia cry-01 crypsietalia aculeatae vicherek 1973 pionirske zajednice povremenih slanih močvara pioneer ephemeral dwarf-grass vegetation in periodically flooded saline habitats of submediterranean and (sub)continental eurasia cry-01b heleochloion schoenioidis br.-bl. ex rivas goday 1956 – eunis e6.1, c3.5 pionirska zajednice povremenih slanih močvara pioneer ephemeral dwarf-grass vegetation in periodically flooded saline habitats in the (sub)mediterranean regions of southern europe and north africa 1.4. vegetation of the mediterranean zone vegetacija primorske zone 1.4.1. zonal mediterranean forests and scrub zonalne primorske šume i šikare qui quercetea ilicis br.-bl. ex a. bolós et o. de bolòs in a. bolòs y vayreda 1950 primorske vazdazelene šume i makije thermo-mesomediterranean pine and oak forests and associated macchia of the mediterranean qui-01 quercetalia ilicis br.-bl. ex molinier 1934 primorske vazdazelene šume i makije crnike evergreen and semi-deciduous thermoto supramediterranean oak and relict laurel forests of the central and western mediterranean qui-01d fraxino orni-quercion ilicis biondi, casavecchia et gigante in biondi et al. 2013 – eunis f5.2, g2.1 primorske vazdazelene šume i makije crnike evergreen and semideciduous calciphilous holm oak forests of the central mediterranean qui-03 pinetalia halepensis biondi, blasi, galdenzi, pesaresi et vagge in biondi et al. 2014 šume alepskog bora thermo-mesomediterranean pine forests of the central and eastern mediterranean qui-03a pistacio lentisci-pinion halepensis biondi, blasi, galdenzi, pesaresi et vagge in biondi et al. 2014 – eunis g3.7 šume alepskog bora thermo-mesomediterranean aleppo pine forests on calcareous substrates of the central mediterranean qui-04 pistacio lentisci-rhamnetalia alaterni rivasmart. 1975 kserotermne makije thermo-mesomediterranean low-grown matorral, macchia and garrigue of the mediterranean basin qui-04h oleo-ceratonion siliquae br.-bl. ex guinochet et drouineau 1944 – eunis b1.6, f5.2, f5.4, f5.5, g2.4 kserotermne makije thermomediterranean calcicolous macchia of the liguro-tyrrhenian seaboards ros ononido-rosmarinetea br.-bl. in a. bolòs y vayreda 1950 (syn. cisto-micromerietea julianae oberd. 1954, ericocistetea trinajstić 1985) primorski bušici mediterranean scrub (tomillar, espleguer, romeral, garrigue, phrygana, batha) on base-rich substrates ros-06 cisto-micromerietalia julianae oberd. 1954 (syn. cisto-ericetalia horvatić 1958) primorski bušici themo-mesomediterranean phrygana of the continental hellas and the adriatic and ionian seaboards ros-06a cisto cretici-ericion manipuliflorae horvatić 1958 – eunis b1.6, f6.3 primorski bušici thermomediterranean calcicolous garrigue of the dalmatian and istrian adriatic seaboards 1.4.2. intrazonal mediterranean scrub intrazonalne primorske šikare ner nerio-tamaricetea br.-bl. et o. de bolòs 1958 primorske šikare uz trajne i povremene vodotoke circummediterranean and macaronesian riparian scrub ner-01 tamaricetalia africanae br.-bl. et o. de bo lòs 1958 primorske šikare uz trajne i povremene vodotoke circummediterranean and macaronesian riparian scrub ner-01e tamaricion dalmaticae jasprica in jasprica et al. 2016 – eunis f9.3 primorske šikare uz trajne i povremene vodotoke thermo-mesomediterranean tamarisk scrub of the balkan adriatic seaboards cyt cytisetea scopario-striati rivas-mart. 1974 šikare zečjaka mediterranean and (sub)atlantic temperate broomy scrub (retamal, piornal, escobonal) seral to forests on acidic substrates cyt-03 spartio juncei-cytisetalia scoparii mucina in mucina et al. 2016 šikare zečjaka temperate (sub)atlantic broom heath of western europe and the southern european peninsulas *cyt-03a sarothamnion scoparii oberd. 1957 – eunis f3.1 šikare zečjaka acidophilous broom and gorse mantle on forest edges and in forest clearings of the (sub)atlanticregions of western europe comment: this type of vegetation is of anthropogenic origin and is spreading subspontaneously. 1.4.3. intrazonal mediterranean grasslands and herblands intrazonalni primorski travnjaci lyg lygeo sparti-stipetea tenacissimae rivas-mart. 1978 nom. conserv. propos. (thero-brachypodietea br.-bl. in br.bl. et al. 1947) primorski travnjaci s prevlašću trajnica na vapnenačkoj podlozi circummediterranean pseudosteppes on calcareous rocky substrates and relict edaphic steppes ondeep clayey soils lyg-01 cymbopogono-brachypodietalia ramosi hor vatić 1963 primorski travnjaci s prevlašću trajnica na vapnenačkoj podlozi circum-mediterranean thermoto supramediterranean pseudosteppes on sandy-loamy soils over calcareous bedrocks škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 212 acta bot. croat. 76 (2), 2017 lyg-01g cymbopogono-brachypodion ramosi horva tić 1963 – eunis e1.3 primorski travnjaci s prevlašću trajnica na vapne na­ čkoj podlozi thermo-mesomediterranean pseudosteppes on calcareous sandy soils of the eastern mediterranean bul poetea bulbosae rivas goday et rivas-mart. in rivasmart. 1978 primorski pašnjaci na dekalcificiranim tlima mediterranean and magrebinian seasonal perennial and ephemeroid pastures in the thermoto oromediterranean belts bul-01 poetalia bulbosae rivas goday et rivas-mart. in rivas goday et ladero 1970 primorski pašnjaci na dekalcificiranim tlima mediterranean and maghrebinian seasonal perennial and ephemeroid pastures in the thermoto oromediterranean belts bul-01f romulion oberd. 1954 – eunis e1.3 primorski pašnjaci na dekalcificiranim tlima macedonian seasonal perennial pastures on acidic substrates tub helianthemetea guttati rivas goday et rivas-mart. 1963 primorski travnjaci s prevlašću jednogodišnjih biljaka na dekalcificiranim tlima mediterranean and submediterranean-atlantic annual lowgrown ephemeral herband grass-rich vegetation on acidic substrates tub-02 vulpietalia pignatti 1953 primorski travnjaci s prevlašću jednogodišnjih biljaka na dekalcificiranim tlima mediterranean and ibero-atlantic ephemeral therophytic vegetation on coastal sand dunes under influence of salt spray tub-02d vulpio-lotion horvatić 1963 (syn. loto angustifoliae-vulpion ciliatae horvatić 1960 nom. invers. propos.) – eunis e1.a, b1.4 primorski travnjaci s prevlašću jednogodišnjih bi ljaka na dekalcificiranim tlima ephemeral therophytic vegetation on the terra rossa and decalcified soils of the illyrian-dinaric coastal regions tra stipo-trachynietea distachyae s. brullo in s. brullo et al. 2001 primorski travnjaci s prevlašću jednogodišnjih biljaka na vapnenačkoj podlozi mediterranean calciphilous annual and ephemeroid swards and grasslands tra-02 ptilostemono stellati-vulpietalia ciliatae mucina ined. primorski travnjaci s prevlašću jednogodišnjih biljaka na vapnenačkoj podlozi central and eastern mediterranean therophytic swards on shallow sandy and loamy soils over limestone and gypsum substrates tra-02a vulpio ciliatae-crepidion neglectae poldini 1989 – eunis e1.3 primorski travnjaci s prevlašću jednogodišnjih bi ljaka na vapnenačkoj podlozi therophytic swards on disturbed calcareous rubblerich shallow soils of the adriatic and ionian seaboards 2. azonal vegetation azonalna vegetacija 2.1. alluvial forests and scrub aluvijalne šume i šikare pop alno glutinosae-populetea albae p. fukarek et fabijanić 1968 galerijske i plavljene šume uz vodotoke riparian gallery forests of the eurosiberian and mediterranean regions pop-01 populetalia albae br.-bl. ex tchou 1949 nom. conserv. propos. sredozemne galerijske šume uz vodotoke mediterranean and submediterranean riparian gallery forests *pop-01f lauro nobilis-fraxinion angustifoliae i. kárpáti et v. kárpáti 1961 – eunis g1.3 sredozemne galerijske šume uz vodotoke riparian gallery forests with relict laurisilva elements of the eastern submediterranean regions of the apen nine and balkan peninsulas comment: there are only remnants of the white poplar stands in mediterranean croatia (krčić, župa dubrovačka, lower neretva). some authors do not separate this alliance from the west-mediterranean populion albae br.-bl. ex tchou 1949 (douda et al. 2015). pop-02 alno-fraxinetalia excelsioris passarge 1968 vlažne i periodično plavljene šume na aluvijalnim nanosima floodplain riparian forests on nutrient-rich alluvial soils of temperate and boreal europe pop-02a alnion incanae pawłowski et al. 1928 – eunis g1.2 šume crne i bijele johe uz vodotoke alder-ash and oak riparian floodplain forests on nutrient-rich alluvial soils in the nemoral zone of europe pop-02d alno-quercion roboris horvat 1950 – eunis g1.2 šume s prevlašću lužnjaka, poljskog jasena i brijesta u nizinskom pojasu alder-oak riparian floodplain forests on nutrient-rich alluvial soils of the temperate regions of the balkan peninsula comment: there are different approaches to syntaxonomy of lowland forests (vukelić et al. 2012). some authors do not separate alno-quercion roboris horvat 1950 from alnion incanae pawłowski et al. 1928 (douda et al. 2015). pur salicetea purpureae moor 1958 šume i šikare vrba uz vodotoke willow and tamarisk scrub and low open forests of riparian habitats in the temperate to arctic zones of europe pur-01 salicetalia purpureae moor 1958 šume i šikare vrba uz vodotoke willow scrub and low open forests of riparian habitats in the temperate to arctic zones of europe pur-01a salicion elaeagno-daphnoidis (moor 1958) grass 1993 – eunis f9.1 šikare vrba uz vodotoke u brdskom i gorskom pojasu willow scrub on the gravelly stream banks in the submontane to subalpine belts of the alps, the pyrenees and the carpathians pur-01b salicion albae soó 1951 – eunis g1.1 šume vrba i topola uz vodotoke od nizinskog do brdskog pojasa, te u submediteranu vegetation of croatia acta bot. croat. 76 (2), 2017 213 willow and poplar low open forests of lowland to submontane river alluvia in the nemoral zone of europe and at high altitudes of the mediterranean pur-01c salicion triandrae t. müller et görs 1958 – eunis f9.1 šikare vrba uz vodotoke od nizinskog do brdskog pojasa willow scrub on loamy-sandy sedimentary river banks in the lowland to submontane belts of the nemoral zone of europe 2.2. swamp forests and scrub močvarne šume i šikare aln alnetea glutinosae br.-bl. et tx. ex westhoff et al. 1946 močvarne i periodično plavljene šume s crnom johom european mesotrophic regularly flooded alder carr and birch wooded mires aln-01 alnetalia glutinosae tx. 1937 močvarne i periodično plavljene šume s crnom johom european mesotrophic regularly flooded alder carrs aln-01a alnion glutinosae malcuit 1929 – eunis g1.4 močvarne šume crne johe european mesotrophic regularly flooded alder carrs *aln-01b frangulo alni-fraxinion oxycarpae poldini, sburlino et venanzoni in biondi et al. 2015 – eunis g1.4 sredozemne močvarne šume amphiadriatic mesotrophic interdune and karstic ash carrs fra franguletea doing ex westhoff in westhoff et den held 1969 močvarne šikare willow carrs of western europe, fennoscandia and the subatlantic regions of central europe fra-01 salicetalia auritae doing 1962 močvarne šikare willow carrs of western europe, fennoscandia and the subatlantic regions of central europe fra-01a salicion cinereae t. müller et görs ex passarge 1961 – eunis f9.2 močvarne šikare willow carrs of western europe and the subatlantic regions of central europe 2.3. vegetation of coastal cliffs and dunes vegetacija priobalnih stijena i dina sag saginetea maritimae westhoff et al. 1962 vegetacija sredozemnih slanih utrina atlantic-mediterranean and macaronesian ephemeral winterannual vegetation in disturbed saline habitats and inland saline badlands sad-01saginetalia maritimae westhoff et al. 1962 zajednice sredozemnih slanih utrina atlantic-mediterranean ephemeral vegetation on aerohaline sandy soils of disturbed salt-marsh fringes sag-01c junco ranarii-plantaginion commutatae horvatić 1934 – eunis a2.5, b1.8 zajednice sredozemnih slanih utrina adriatic short-lived aerohaline vegetation of sandy flats of disturbed salt-marshes cri crithmo-staticetea br.-bl. in br.-bl. et al. 1952 vegetacija na stijenama u zoni prskanja mora rupicolous vegetation of salt-sprayed coastal cliffs of the atlantic and mediterranean seaboards of europe, north africa and middle east cri-01 crithmo-staticetalia molinier 1934 halofitske zajednice grebenjača rupicolous vegetation of salt-sprayed cliffs of the atlantic and mediterranean coasts of europe, north africa and middle east cri-01d limonion anfracti-cancellati (horvatić 1934) mucina in mucina et al. 2016 (staticion dalmaticum horvatić 1934) – eunis b3.3 halofitske zajednice grebenjača rupicolous herb-rich vegetation of salt-sprayed rocky cliffs of the adriatic coasts cri-02 helichrysetalia italici biondi et géhu in géhu et biondi 1994 zajednice polugrmova na stijenama u zoni prskanja mora sub-aerohaline coastal dwarf scrub on inland edges of saltsprayed cliffs of the mediterranean seaboards cri-02e anthyllidion barbae-jovis s. brullo et de marco 1989 – eunis b3.3 zajednice visokih polugrmova na stijenama u zoni prskanja mora subaerohaline coastal dwarf scrub on salt-sprayed cliffs of the eastern tyrrhenian sea *cri-02f crucianellion rupestris s. brullo et furnari 1990 – eunis b3.3 zajednice niskih polugrmova na stijenama u zoni prskanja mora subaerohaline dwarf scrub on salt-sprayed cliffs of the european and north african coasts of the lybian sea cak cakiletea maritimae tx. et preising in tx. ex oberd. 1952 pionirska halonitrofilna vegetacija na pješčanim i šljun ča nim obalama pioneer halo-nitrophilous short-lived vegetation in strandlines of sandy and shingle beaches of the coasts of the north atlantic and arctic oceans, the mediterranean and the black sea cak-03 thero-atriplicetalia pignatti 1953 (syn. euphorbietalia peplidis tx. 1950) pionirske halonitrofilne zajednice na pješčanim i šljun­ čanim obalama pioneer halo-nitrophilous strandline vegetation of the cantabro-atlantic, the mediterranean and the black sea coasts cak-03a euphorbion peplidis tx. ex oberd. 1952 – eunis b1.1, b1.2, b2.1, b2.2 pionirske halonitrofilne zajednice na pješčanim i šljunčanim obalama pioneer halo-nitrophilous strandline vegetation of the cantabro-atlantic and the mediterranean coasts amm ammophiletea br.-bl. et tx. ex westhoff et al. 1946 vegetacija trajnica na priobalnim pješčanim dinama tall-grass perennial swards on mobile coastal dunes of the seaboards of europe, north america, greenland, north africa, middle east and the caspian sea amm-01 ammophiletalia br.-bl. et tx. ex westhoff et al. 1946 zajednice trajnica na priobalnim pješčanim dinama tall-grass perennial swards on mobile white and embryonic škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 214 acta bot. croat. 76 (2), 2017 coastal dunes of the warm-temperate to boreo-atlantic coasts of the mediterranean and the black and caspian seas amm-01a ammophilion br.-bl. 1921 – eunis b1.3 zajednice trajnica na priobalnim pješčanim dinama tall-grass perennial swards on mobile white and embryonic coastal sand dunes of the mediterranean 2.4. vegetation of rock crevices and screes vegetacija pukotina stijena i sipara adi adiantetea br.-bl. et al. 1952 vegetacija sredozemnih nakapnica i plitkih polušpilja relict chomophytic and chasmophytic vegetation in the shaded and water-splashed habitats of the mediterranean, the atlantic islands, north africa and middle east adi-01 adiantetalia br.-bl. ex horvatić 1934 zajednice sredozemnih nakapnica i plitkih polušpilja relict chomophytic and chasmophytic vegetation in shaded and water-splashed habitats of the mediterranean, the atlantic islands, north africa and middle east adi-01a adiantion br.-bl. ex horvatić 1934 – eunis c2.1, h3.4 zajednice sredozemnih nakapnica i plitkih polušpilja relict fern-rich chasmophytic communities in shaded and water-splashed habitats of the mediterranean, the atlantic islands, north africa and middle east pod polypodietea jurko et peciar ex boşcaiu, gergely et codoreanu in raţiu et al. 1966 vegetacija s prevlašću mahovina i paprati na sjenovitim stijenama, panjevima i kori stabala chomophytic, chasmophytic and epiphytic vegetation of fern and moss-rich communities in crevices and on the surface of rocky cliffs of temperate and mediterranean europe pod-01 hypno cupressiformi-polypodietalia vulgaris jurko et peciar ex mucina et theurillat 2015 zajednice na sjenovitim silikatnim stijenama, panjevima i kori stabala fernand moss-rich chomophytic, chasmophytic and epiphytic vegetation of shaded rock faces and bark of old trees of cool-temperate europe pod-01a hypno-polypodion vulgaris mucina 1993 – eunis h3.1 zajednice na sjenovitim silikatnim stijenama, pa­ njevima i kori stabala fern-rich vegetation of siliceous shaded rock crevices in the colline and submontane belts of central and eastern europe pod-03 ctenidio-polypodietalia vulgaris jurko et peciar ex boşcaiu, gergely et codoreanu in raţiu et al. 1966 zajednice na sjenovitim vapnenačkim stijenama vegetation of shady calcareous rock faces and crevices at low altitudes of cool-temperate and submediterranean europe pod-03a ctenidio-polypodion vulgaris s. brullo et al. 2001 – eunis h3.2 zajednice s češljastom mahovinom na sjenovitim vap­ nenačkim stijenama vegetation of shady calcareous rock faces and crevices of the alps and the carpathians pod-03b moehringion muscosae horvat et horvatić ex boşcaiu, gergely et codoreanu in raţiu et al. 1966 – eunis h3.2 zajednice s mahovinastom merinkom na sjenovitim vlažnim vapnenačkim stijenama vegetation of shady calcareous rock faces and crevices of southeastern europe asp asplenietea trichomanis (br.-bl. in meier et br.-bl. 1934) oberd. 1977 vegetacija polusjenovitih i otvorenih stijena chasmophytic vegetation of crevices, rocky ledges and faces of rocky cliffs and walls of europe, north africa, middle east, the arctic archipelagos and greenland asp-01 geranio robertiani-asplenietalia trichomanis ferrez ex mucina ined. zajednice polusjenovitih i otvorenih stijena od nizin skog do gorskog pojasa chasmophytic vegetation of semi-shaded and sunny rock faces and crevices in the lowland to submontane belts of temperate europe asp-01a asplenio scolopendrii-geranion robertiani ferrez 2010 – eunis h3.1, h3.2 zajednice polusjenovitih i otvorenih stijena od nizinskog do gorskog pojasa chasmophytic vegetation of semi-shaded and sunny rock faces and crevices in the lowland to submontane belts of temperate europe asp-02 potentilletalia caulescentis br.-bl. in br.-bl. et jenny 1926 zajednice otvorenih vapnenačkih stijena u gorskom i pretplaninskom pojasu chasmophytic vegetation of sunny calcareous rock faces and crevices at high altitudes of the nemoral and boreal mountain ranges of europe asp-2a potentillion caulescentis br.-bl. in br.-bl. et jenny 1926 – eunis h3.2 zajednice otvorenih vapnenačkih stijena u gorskom i pretplaninskom pojasu gorskog kotara chasmophytic vegetation of calcareous rock faces and crevices in the subalpine and alpine belts of the central and eastern alps and the western carpathians asp-2l micromerion croaticae horvat in blečić 1959 – eunis h3.2 zajednica otvorenih vapnenačkih stijena u gorskom i pretplaninskom pojasu like i velebita chasmophytic vegetation of calcareous rock faces and crevices in the subalpine belt of the northwestern dinarides asp-03 moltkeetalia petraeae lakušić 1968 zajednice vapnenačkih stijena od brdskog do pretplaninskog pojasa središnjih i južnih dinarida chasmophytic vegetation of limestone crevices in the montane to alpine belts of the central and southern dinarides asp-03a edraianthion lakušić 1968 – eunis h3.2 zajednice vapnenačkih stijena od brdskog do pretplaninskog pojasa središnjih i južnih dinarida chasmophytic vegetation of limestone crevices in the montane and supramontane belts of the central and southern dinarides asp-05 centaureo dalmaticae-campanuletalia pyramidalis trinajstić ex terzi et di pietro 2016 zajednice priobalnih vapnenačkih stijena thermo-mesomediterranean chasmophytic vegetation of limestone cliffs of the northern and central adriatic coastal regions vegetation of croatia acta bot. croat. 76 (2), 2017 215 asp-05a centaureo dalmaticae-campanulion horvatić 1934 – eunis h3.2 zajednice priobalnih vapnenačkih stijena kvarnersko­ liburnijskog područja thermo-mesomediterranean chasmophytic vegetation of limestone crevices of the northern adriatic seaboards asp-05b centaureo cuspidatae-portenschlagiellion ramosissimae trinajstić ex terzi et di pietro 2016 – eunis h3.2 zajednice priobalnih vapnenačkih stijena dalmacije thermo-mesomediterranean chasmophytic vegetation of limestone crevices of the central and southern adriatic seaboards asp-10 asplenietalia septentrionalo-cuneifolii mucina et theurillat 2015 zajednice polusjenovitih i otvorenih silikatnih i serpentinskih stijena chasmophytic vegetation of siliceous and ultramafic rock crevices at low altitudes of temperateand boreal europe asp-10b asplenion septentrionalis gams ex oberd. 1938 – eunis h3.1 zajednice polusjenovitih i otvorenih silikatnih stijena fern-rich chasmophytic vegetation of siliceous sunny rock crevices and boulder fields of temperate and boreal europe asp-10c asplenion serpentini br.-bl. et tx. ex eggler 1955 – eunis h3.2 zajednice polusjenovitih i otvorenih serpentinskih stijena fern-rich chasmophytic vegetation of ultramafic rock crevices of central europe cym cymbalario-parietarietea diffusae oberd. 1969 (syn. parietarietea judaicae oberd. 1977) termofilna vegetacija u pukotinama zidova thermophilous chasmophytic vegetation of walls of the mediterranean and the winter-mild atlantic to subcontinental regions of temperate europe, middle east and north africa cym-01 tortulo-cymbalarietalia segal 1969 (syn. parietarietalia judaicae /rivas-mart. ex rivas goday 1964/ oberd. 1977) termofilna vegetacija u pukotinama zidova thermophilous chasmophytic vegetation of walls of the mediterranean and the winter-mild atlantic to subcontinental regions of temperate europe, middle east and north africa cym-01a cymbalario-asplenion segal 1969 – eunis e5.1 kontinentalna termofilna vegetacija u pukotinama zidova fern-rich chasmophytic vegetation of sunny walls of the atlantic to subcontinental regions of cool-temperate europe cym-01b galio valantiae-parietarion judaicae rivas-mart. ex o. de bolòs 1967 (syn. parietarion judaicae segal 1969, parietario-centranthion rubri rivas-mart. 1960) – eunis e5.1 primorska termofilna vegetacija u pukotinama zidova thermomediterranean chasmophytic vegetation of limestone walls of the iberian peninsula and the western tyrrhenian archipelago cym-01c artemisio arborescentis-capparidion spinosae biondi, blasi et galdenzi in biondi et al. 2014 – eunis e5.1 termofilna vegetacija u pukotinama zidova vanjskih dalmatinskih otoka i hridi thermomediterranean chasmophytic vegetation of limestone walls of the apennine peninsula, corsica, sardinia, sicily and malta thl thlaspietea rotundifolii br.-bl. 1948 vegetacija na siparima i šljunčanim obalama vodotoka vegetation of scree habitats and pebble alluvia of the temperate, boreal and oromediterranean europe and the arctic archipelagos thl-01 thlaspietalia rotundifolii br.-bl. in br.-bl. et jenny 1926 zajednice na vapnenačkim siparima vršnih dijelova dinarida alpine and subalpine calcareous scree vegetation of europe and greenland thl-01j saxifragion prenjae lakušić 1968 – eunis h2.4 reliktne zajednice na vapnenačkim siparima dânā dubokih ponikvi velebita i dinare subalpine chionophilous calcareous scree communities of the southern and central dinarides thl-01k bunion alpini lakušić 1968 – eunis h2.4 zajednice na vapnenačkim siparima vršnih dijelova dinarida subalpine chionophilous calcareous scree communities of the northern dinarides thl-02 arabidetalia caeruleae rübel ex nordhagen 1937 zajednice na snježištima i umirenim vapnenačkim siparima u pretplaninskom pojasu vegetation of snow-beds on stabilized calcareous screes of the arctic zone and the alpine and subnival belts of european mountains thl-02c arabidion caeruleae br.-bl. in br.-bl. et jenny 1926 (syn. salicion retusae horvat 1949) – eunis e4.1 zajednice na snježištima i umirenim vapnenačkim siparima u pretplaninskom pojasu vegetation of snow-beds on stabilized calcareous screes in the alpine and subnival belts of european mountains thl-04 arabido alpinae-petasitetalia paradoxi mucina et valachovič ined. zajednice na vlažnim vapnenačkim točilima i siparima u gorskom i pretplaninskom pojasu vegetation of humid calcareous screes and boulder fields in the montane to subalpine belts of the nemoral mountain ranges of europe thl-04a petasition paradoxi zollitsch ex lippert 1966 – eunis h2.4 zajednice na vlažnim vapnenačkim točilima i siparima u gorskom i pretplaninskom pojasu vegetation of humid calcareous fine-grained screes in the montane and subalpine belts of the alps thl-05 stipetalia calamagrostis oberd. et seibert in oberd. 1977 zajednice na termofilnim vapnenačkim i dolomitnim siparima u brežuljkastom do gorskom pojasu thermophilous calcareous scree vegetation in the colline to montane belts of central and western europe thl-05c stipion calamagrostis jenny-lips ex br. bl. 1950 – eunis h2.6 škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 216 acta bot. croat. 76 (2), 2017 zajednice na termofilnim vapnenačkim i dolomitnim siparima u brežuljkastom do gorskom pojasu vegetation of thermophilous low-altitude calcareous screes of central and western europe thl-08 epilobietalia fleischeri moor 1958 nom. conserv. propos. zajednice na šljunčanim obalama vodotoka vegetation of montane to subalpine riverine gravel terra ces of the nemoral and boreal european mountain ranges and the caucasus thl-08c epilobion fleischeri g. br.-bl. ex br.-bl. 1950 – eunis c3.5 zajednice na šljunčanim obalama vodotoka vegetation of the montane-subalpine riverine gravel terraces of the alps and the carpathians dry drypidetea spinosae quézel 1964 vegetacija na siparima primorskih padina dinarida vegetation of scree habitats and pebble alluvia in the submediterranean montane and supra-oromediterranean belts of the central and eastern mediterranean and the black sea seaboards dry-01 drypidetalia spinosae quézel 1964 zajednice na siparima primorskih padina dinarida montane submediterranean and oromediterranean scree vegetation of the balkans, crete and crimea dry-01a peltarion alliaceae horvatić in domac 1957 – eunis h2.6 zajednice na siparima primorskih padina dinarida u brdskom i gorskom pojasu limestone scree vegetation in the submontane and montane belts of the central balkans dry-01b silenion marginatae lakušić 1968 (syn. silenion prostratae trinajstić 2008) – eunis h2.6 zajednice na siparima primorskih padina dinarida u pretplaninskom pojasu limestone scree vegetation in the montane to subalpine belts of the southern dinarides 2.5. vegetation of saline and brackish waters and swamps vegetacija slanih i bočatih voda i močvara zos zosteretea pignatti 1953 morske livade svilinā i posidonije vegetation of sea-grass meadows on muddy and sandy submerged substrates of the temperate and subarctic seas surrounding europe zos-01 zosteretalia béguinot ex pignatti 1953 morske livade svilinā vegetation of sea-grass meadows of the sandy-muddy sublittoral of the temperate seas surrounding europe zos-01a zosterion marinae br.-bl. et tx. ex pignatti 1953 – eunis a2.6 morske livade morske sviline vegetation of perennial sea-grass meadows of the sandy-muddy sea sublittoral of the coldand cool-temperate seas surrounding europe zos-01b nanozosterion noltii den hartog ex mucina in mucina et al. 2016 – eunis a2.6 morske livade patuljaste sviline vegetation of short-lived sea grass meadows of the sandy-muddy sea sublittoral of the cold-temperate and cool-temperate seas surrounding europe zos-02 posidonietalia oceanicae den hartog ex mucina in mucina et al. 2016 morske livade posidonije vegetation of perennial sea-grass meadows of the sandyrocky sublittoral of the warm-temperate waters of the mediterranean sea zos-02a posidonion oceanicae br.-bl. ex molinier 1960 – eunis a2.6 morske livade posidonije vegetation of perennial sea-grass meadows of the sandy-rocky sublittoral of the warm-temperate waters of the mediterranean sea hal halodulo wrightii-thalassietea testudinum rivasmart. et al.1999 morske livade čvoraste morske rese vegetation of eel-grass swards on muddy and sandy substrates of subtropical and tropical seas fringing atlantic ocean hal-01 thalassio-syringodetalia filiformis knapp ex borhidi et al. 1979 morske livade čvoraste morske rese vegetation of eel-grass swards on muddy and sandy substrates of the sublittoral of subtropical and tropical seas fringing atlantic ocean cymodoceion nodosae den hartog ex mucina in mucina et al. 2016 – eunis a2.6 morske livade čvoraste morske rese vegetation of eel-grass swards on muddy and sandy substrates of the sublittoral of the subtropical atlantic ocean and the mediterranean sea rup ruppietea maritimae j. tx. ex den hartog et segal 1964 vegetacija bočatih voda s rupijom submerged rooted herbaceous vegetation of brackish watersof the world rup-01 ruppietalia j. tx. ex den hartog et segal 1964 nom. conserv. propos. zajednice rupije u bočatim vodama submerged rooted herbaceous vegetation of temperate brackish waters of europe rup-01a ruppion maritimae br.-bl. ex westhoff in bennema et al. 1943 – eunis a2.6 zajednice rupije u bočatim vodama submerged rooted herbaceous vegetation of temperate brackish waters of europe spa spartinetea maritimae beeftink 1962 pionirska vegetacija sa spartinom u zoni plime i oseke pioneer vegetation of perennial cord grasses on tidal flats of temperate seas of the world spa-01 spartinetalia glabrae conard 1935 pionirske zajednice sa spartinom u zoni plime i oseke pioneer vegetation of perennial cord grasses on tidal flats of temperate seas of the world spa-01a spartinion glabrae conard 1935 – eunis a2.5 pionirske zajednice sa spartinom u zoni plime i oseke pioneer vegetation of perennial cord grasses on tidal flats of temperate seas of europe and north america the therosalicornietea tx. in tx. et oberd. 1958 pionirska vegetacija slanjača s jednogodišnjim biljkama pioneer vegetation of annual succulent halophytes on tidal mud flats and edges of the irregularly flooded saline inland waters of eurasia vegetation of croatia acta bot. croat. 76 (2), 2017 217 the-01 therosalicornietalia pignatti 1952 pionirske zajednice slanjača s jednogodišnjim biljkama pioneer vegetation of annual succulent halophytes of tidal mud flats and edges of the irregularly flooded saline inland waters of the mediterranean, and temperate, boreal and subarctic europe the-01a therosalicornion br.-bl. 1933 – eunis a2.5 pionirske zajednice slanjača s jednogodišnjim bilj kama mediterranean and thermo-atlantic pioneer vegetation of annual succulent plants of tidal flats and irregularly flooded inland depressions jun juncetea maritimi br.-bl. in br.-bl. et al. 1952 primorske sitine i halonitrofilni travnjaci perennial grasslands and herb-rich vegetation of coastal and inland salt-marshes and sea-cliffs of the mediterranean sea and the atlantic and arctic oceans jun-01 juncetalia maritimi br.-bl. ex horvatić 1934 primorske sitine i zaslanjeni travnjaci mediterranean and thermo-atlantic tall-rush saline wetland vegetation jun-01a juncion maritimi br.-bl. ex horvatić 1934 – eunis a2.5 primorske sitine mediterranean and thermo-atlantic coastal saline rush marsh vegetation under a prolonged flooding regime jun-01e agropyro-plantaginion maritimi hor va tić 1934 – eunis a2.5 primorski zaslanjeni travnjaci central and eastern mediterranean saline swards of margins of lagoons and damp dune-slacks jun-02 agropyretalia pungentis géhu 1968 primorski halonitrofilni travnjaci halo-nitrophilous grasslands of salt-sprayed sandy-loamy shores of the winter-mild atlantic and mediterranean regions of europe *jun-02c agropyro-artemision coerulescentis pignatti 1953 – eunis a2.5 primorski halonitrofilni travnjaci tyrrhenian-adriatic (sub)halo-nitrophilous saltsprayed grassy scrub of the edges of coastal lagoons sal salicornietea fruticosae br.-bl. et tx. ex a. bolòs y vayreda et o. de bolòs in a. bolòs y vayreda 1950 vegetacija slanjača s polugrmovima mediterranean and thermo-atlantic perennial salt-marsh herblands and scrub sal-01 salicornietalia fruticosae br.-bl. 1933 zajednice slanjača s polugrmovima mediterranean and thermo-atlantic halophilous coastal tidal and inland temporarily flooded succulent chenopod scrub sal-01a salicornion fruticosae br.-bl. 1933 – eunis a2.5 zajednice slanjača s polugrmovima mediterranean and thermo-atlantic intertidal succulent dwarf chenopod scrub 2.6. freshwater aquatic vegetation slatkovodna vegetacija lem lemnetea o. de bolòs et masclans 1955 vegetacija plutajućih makrofita u mirnim vodama free-floating duckweed vegetation of still and relatively nutrient-rich freshwater bodies of the holarctic lem-01 lemnetalia minoris o. de bolòs et masclans 1955 zajednice plutajućih makrofita u mirnim vodama vegetation of free-floating vegetation of still and relatively nutrient-rich freshwater bodies of temperate europe lem-01a lemnion minoris o. de bolòs et masclans 1955 – eunis c1.2, c1.3 zajednice vodenih leća vegetation of free-floating duckweed vegetation of still and relatively nutrient-rich freshwater bodies of the temperate europe lem-01b utricularion vulgaris passarge 1964 – eunis c1.2 zajednice mješinki vegetation of free-floating bladderworts in mesotrophic and eutrophic waters of europe lem-01c stratiotion den hartog et segal 1964 (syn. hydrocharition morsus-ranae /passarge 1964/ westhoff et den held 1969) – eunis c1.2, c1.3 zajednice resca vegetation of free-floating macrophytes in fairly nutrient-rich shallow waters of europe pot potamogetonetea klika in klika et novák 1941 vegetacija ukorijenjenih plutajućih i submerznih makrofita vegetation of rooted floating or submerged macrophytes of stagnant mesotrophic, eutrophic and brackish freshwater bodies and slowly flowing shallow streams of eurasia pot-01 potamogetonetalia koch 1926 zajednice s prevlašću mrijesnjaka i biljaka s plutajućim listovima vegetation of rooted floating or submerged macrophytes of mesotrophic and eutrophic freshwater bodies of eurasia pot-01a potamogetonion libbert 1931 (syn. magnopotamion/vollmar 1947/ den hartog et segal 1964) – eunis c1.2, c1.3, c2.3 zajednice s prevlašću mrijesnjaka vegetation of rooted and floating macrophytes of freshwater bodies at low and mid-altitudes of temperate eurasia pot-01b nymphaeion albae oberd. 1957 – eunis c1.2, c1.3, c2.3 zajednice s prevlašću lopoča, lokvanja i biljaka s rozetama plutajućih listova vegetation of rooted floating-leaf macrophytes of sheltered nutrient-rich freshwaters of western and central europe pot-02 callitricho hamulatae-ranunculetalia aquatilis passarge ex theurillat in theurillat et al. 2015 (syn. callitricho-batrachietalia den hartog et segal ex passarge 1978) zajednice s prevlašću vodenih žabnjaka i žabovlatki vegetation of crosswort, crowfoot and milfoil rooted macrophytes in shallow and intermittent freshwater streams of europe pot-02a batrachion fluitantis neuhäusl 1959 (syn. ranunculion fluitantis neuhäusl 1959) – eunis c2.2, c2.3 zajednice s prevlašću vodenih žabnjaka u tekućim vodama vegetation of crowfoot and milfoil rooted macrophytes in shallow moving freshwaters of europe pot-02b ranunculion aquatilis passarge ex theurillat in theurillat et al. 2015 – eunis c1.2, c1.3, škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 218 acta bot. croat. 76 (2), 2017 c1.6, c2.3 zajednice s prevlašću vodenih žabnjaka ili ža bo vlatki u sporotekućim i stajaćim vodama vegetation of crosswort rooted macrophytes in shallow stagnant freshwaters of temperate europe pot-03 zannichellietalia pedicellatae schaminée, lanjouw et schipper ex mucina in theurillat et al. 2015 zajednice žabljaka u bočatim vodama vegetation of rooted macrophytes in meso-eutrophic brackish waters of western and central europe pot-03a zannichellion pedicellatae schaminée, lanjouw et schipper ex passarge 1996 – eunis c1.5 zajednice žabljaka u bočatim vodama vegetation of rooted macrophytes in meso-eutrophic brackish waters of western and central europe 2.7. vegetation of freshwater springs, shorelines and swamps slatkovodna vegetacija izvorišta, obala i močvara mon montio-cardaminetea br.-bl. et tx. ex klika et hadač 1944 vegetacija izvorišta i sedrenih barijera vegetation of water springs of europe, the european arctic archipelagos and greenland mon-01 cardamino-chrysosplenietalia hinterlang 1992 zajednice oko sjenovitih šumskih izvora meke vode vegetation of soft-water springs in shady forest habitats in the submontane and montane belts of the central european mountains mon-01a caricion remotae kästner 1941 – eunis c2.1 zajednice oko sjenovitih šumskih izvora meke vode vegetation of soft-water springs in shady forest habitats in the submontane and montane belts of central european mountains mon-02 montio-cardaminetalia pawłowski et al. 1928 zajednice s prevlašću mahovina hladnih oligotrofnih iz­ vorišta i sedrenih barijera vegetation of cold oligotrophic water-springs in the nemoral to arctic zones and in the oromediterranean belt of europe mon-02f cratoneurion commutati koch 1928 – eunis c2.1 zajednice s prevlašću mahovina hladnih oligotrofnih izvorišta i sedrenih barijera u gorskom pojasu vegetation of moss-rich calcareous water springs in the montane and subalpine belts of europe and green land *mon-02g lycopodo europaei-cratoneurion commutati hadač 1983 – eunis c2.1 zajednice s prevlašću mahovina hladnih oligotrofnih izvorišta i sedrenih barijera u brežuljkastom i brdskom pojasu vegetation of moss-rich calcareous water springs in the colline and submontane belts of central europe comment: syntaxonomic position of that alliance is not clear and there are opinions that should be reduced to synonymy with the cratoneurion commutati (mucina et al. 2016). these moss-rich communities are very uniform in croatia and probably belong to only one alliance. here are two alliances listed until their syntaxonomy and presence in croatia is not better investigated. iso isoëto-nanojuncetea br.-bl. et tx. in br.-bl. et al. 1952 pionirska vegetacija niskih šaševa periodično plavljenih staništa pioneer ephemeral dwarf-cyperaceous vegetation in periodically freshwater flooded habitats of eurasia iso-02 nanocyperetalia klika 1935 (syn. cyperetalia fusci pietsch 1963) pionirske zajednice niskih šaševa periodično plavljenih staništa pioneer ephemeral herband graminoid-rich late-season vegetation on periodically flooded soils of temperate europe iso-02a nanocyperion koch 1926 – eunis c3.5 pionirske zajednice niskih šaševa periodično plavljenih staništa pioneer dwarf cyperaceous vegetation on moist calcium rich substrates of the submediterranean and atlantic regions of europe iso-02e verbenion supinae slavnić 1951 (syn. fimbristylion dichotomae horvatić 1954) – eunis c3.5 pionirske zajednice niskih šaševa periodično plavljenih antropogenih staništa na tlima bogatima hra njivim tvarima pioneer ephemeral herb-rich vegetation in periodically flooded nutrient-rich habitats in the nemoral zone of central and southeastern europe phr phragmito-magnocaricetea klika in klika et no vák 1941 tršćaci, rogozici i šašici reed swamp, sedge bed and herbland vegetation of freshwater or brackish water bodies and streams of eurasia phr-01 phragmitetalia koch 1926 tršćaci i rogozici reed swamps, sedge beds and herblands of mesotrophic and eutrophic stagnating or slowly flowing freshwater or brackish water bodies of eurasia phr-01a phragmition communis koch 1926 – eunis c3.2, d5.1 tršćaci i rogozici reed swamp vegetation of mesotrophic and eutrophic standing freshwater bodies or gently moving streams of boreo-temperate eurasia phr-02 bolboschoenetalia maritimi hejný in holub et al. 1967 zajednica primorskog rančića meso-eutrophic brackish swamp reeds of european temperate coasts and the subcontinental inland regions of central and southern europe phr-02a scirpion maritimi dahl et hadač 1941 – eunis a2.5, c3.2 zajednica primorskog rančića meso-eutrophic brackish swamp reeds of european temperate coastal regions phr-04 magnocaricetalia pignatti 1953 šašici sedge-bed marsh vegetation of boreal and temperate eurasia phr-04a magnocaricion elatae koch 1926 – eunis d5.2 šašici na oligotrofnim do mezotrofnim sedimentima sedge-bed marsh vegetation on oligotrophic to mesotrophic organic sediments of temperate europe vegetation of croatia acta bot. croat. 76 (2), 2017 219 phr-04b magnocaricion gracilis géhu 1961 – eunis d5.2 šašici na eutrofnim sedimentima sedge-bed marsh vegetation on eutrophic clayey sediments in riverine habitats of temperate europe phr-04c carici-rumicion hydrolapathi passarge 1964 – eunis c3.1, c3.2 šašici na muljevitim organskim sedimentima herbland vegetation on non-stabilized organic substrates in mesotrophic waters of boreal and temperate eurasia phr-05 nasturtio-glycerietalia pignatti 1953 helofitske zajednice periodično plavljenih obala, stajaćica i plitkih vodotoka herblands and sedge-beds of well-oxygenated freshwater flowing streams of the temperate and mediterranean regions of europe and madeira phr-05a glycerio-sparganion br.-bl. et sissingh in boer 1942 – eunis c2.5, c3.1 helofitske zajednice s prevlašću zeleni u stajaćicama i plitkim vodotocima herbland vegetation of small freshwater streams and in shallow water bodies of temperate europe phr-05b phalaridion arundinaceae kopecký 1961 – eunis c3.2 helofitske zajednice periodično plavljenih obala s prevlašću trstastog blještaca ili buekovog šaša reed vegetation of freshwater flowing and seasonally fluctuating streams of temperate europe phr-06 oenanthetalia aquaticae hejný ex balátovátuláčková et al. 1993 helofitske zajednice plitkih močvara promjenjivog vodostaja vegetation of emergent helophytes in shallow waters with fluctuating water table of temperate and boreal eurasia phr-06a eleocharito palustris-sagittarion sagittifoliae passarge 1964 – eunis c3.2 helofitske zajednice plitkih močvara promjenjivog vodostaja vegetation of emergent helophytes on muddy soils of shallows streams and ponds with fluctuating water table of temperate and boreal eurasia 2.8. vegetation of bogs and fens vegetacija cretova sch scheuchzerio palustris-caricetea fuscae tx. 1937 niski i prijelazni cretovi sedge-moss vegetation of fens, transitional mires and bog hollows in the temperate, boreal and arctic zones of the northern hemisphere sch-01 caricetalia davallianae br.-bl. 1950 nom. conserv. propos. bazofilni niski cretovi sedge-moss vegetation of calcareous and extremely mineral rich brown-moss fens of eurasia sch-01a caricion davallianae klika 1934 – eunis d4.1 bazofilni niski cretovi sedge-moss calcareous mineral-rich fen vegetation of europe and western asia sch-02 sphagno warnstorfii-tomentypnetalia lap shina 2010 neutrofilni cretovi sedge and brown-moss nitrogen-limited fen vegetation of western siberia and the northeastern european lowlands sch-02a sphagno warnstorfii-tomentypnion nitentis dahl 1957 – eunis d4.1 neutrofilni cretovi moderately calcium-rich sedge-moss fens of the boreal zone and montainous regions in the nemoral zone of europe sch-03 caricetalia fuscae koch 1926 acidofilni prijelazni cretovi sedge-moss vegetation of slightly to strongly acidic minerotrophic moderately-rich or poor fens in the boreal and temperate zones of the northern hemisphere and in the supramediterranean belt of southern european mountains sch-03b caricion fuscae koch 1926 nom. conserv. propos. – eunis d2.2 umjereno acidofilni cretovi sa smeđim mahovinama sedge-moss vegetation moderately to low calcium-rich slightly acidic fens dominated by calcifuge brownmosses or nutrient-demanding peat-mosses of europe sch-03d sphagno-caricion canescentis passarge (1964) 1978 nom. conserv. propos. – eunis d2.2 acidofilni cretovi s mahovima tresetarima peat-moss acidic poor yet minerotrophic fens of the boreal and temperate zones of the northern hemisphere oxy oxycocco-sphagnetea br.-bl. et tx. ex westhoff et al. 1946 visoki cretovi dwarf-shrub, sedge and peat-moss vegetation of the holarctic ombrotrophic bogs and wet heath on extremely acidic soils oxy-02 sphagnetalia medii kästner et flössner 1933 visoki cretovi dwarf-shrub and peat-moss vegetation of the continental, subcontinental, boreo-continental and high-altitude raised bogs of the northern hemisphere oxy-02b sphagnion medii kästner et flössner 1933 – eunis d1.1 visoki cretovi dwarf-shrub and peat-moss vegetation of the subcontinental, temperate and mountain raised bogs of eurasia 3. anthropogenic vegetation antropogena vegetacija par papaveretea rhoeadis s. brullo et al. 2001 nom. conserv. propos. kontinentalna acidofilna jednogodišnja korovna vegetacija annual weed segetal vegetation of arable crops, gardens and vineyards in the cool-temperate and boreal zones of eurasia par-01 aperetalia spicae-venti j.tx. et tx. in malatobeliz et al. 1960 nom. conserv. propos. (syn. chenopodietalia albi /tx. 1937/ tx. et lohmeyer in tx. 1950; atriplici-chenopodietalia albi /tx. 1937/ nordhagen 1940 nom. ambig. rejic. propos.) kontinentalne acidofilne zajednice jednogodišnjih korova weed vegetation of cereal fields and gardens on acidic and nutrient-poor soils in the cool-temperate and boreal zones of eurasia par-01a scleranthion annui (kruseman et vlieger 1939) sissingh in westhoff et al. 1946 – eunis i1.1, i1.2, i1.3 škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 220 acta bot. croat. 76 (2), 2017 kontinentalne acidofilne zajednice jednogodišnjih korova u usjevima ozimih žitarica weed segetal vegetation of winter cereal crops on neutral to acidic loamy and sandy-loamy soils of the (sub) atlantic regions in the nemoral zone of europe par-01b oxalidion europeae passarge 1978 (syn. spergulo-oxalidion görs in oberd. et al. 1967) – eunis i1.1, i1.2, i1.3 kontinentalne acidofilne zajednice jednogodišnjih korova u okopavinskim kulturama weed segetal vegetation of gardens and root crop fields on acidic loamy and sandy-loamy soils of the subatlantic to subcontinental regions in the nemoral zone of europe par-02 papaveretalia rhoeadis hüppe et hofmeister ex theurillat et al. 1995 nom. conserv. propos. kontinentalna bazofilna jednogodišnja korovna vegetacija weed segetal vegetation of arable crops on base-rich soils in the forest, forest-steppe, steppe and subboreal zones of europe par-02a caucalidion tx. ex von rochow 1951 – eunis i1.1, i1.3 kontinentalna bazofilna jednogodišnja korovna ve­ getacija u usjevima žitarica weed segetal vegetation of cereal crops on the baserich soils of western, central and southeastern europe par-02c veronico-euphorbion sissingh in passarge 1964 – eunis i1.1, i1.3 kontinentalna bazofilna jednogodišnja korovna ve­ getacija u okopavinskim kulturama weed segetal vegetation of vineyards and gardens on the base-rich soils of central and western europe sis sisymbrietea gutte et hilbig 1975 kontinentalna jednogodišnja ruderalna vegetacija zoo-anthropogenic and modern anthropogenic vegetation of animal shelters and disturbed ruderal sites in cooland coldtemperate regions of eurasia sis-01 sisymbrietalia sophiae j. tx. ex görs 1966 nom. conserv. propos. (syn. sisymbrietalia officinalis j.tx. in lohmeyer et al. 1962) kontinentalne jednogodišnje ruderalne zajednice ruderal vegetation of annual nutrient-demanding herbs and grasses on disturbed soils in the nemoral and steppe zones of europe sis-01c malvion neglectae (gutte 1972) hejný 1978 – eunis e5.1 kontinentalne jednogodišnje ruderalne zajednice koje se razvijaju ljeti ruderal vegetation of low-grown short-lived summerannual herbs on nutrient-rich loamy and slightly trampled soils of temperate europe sis-01d sisymbrion officinalis tx. et al. ex von rochow 1951 – eunis e5.1 kontinentalne jednogodišnje ruderalne zajednice koje se razvijaju u proljeće ruderal vegetation of nutrient-demanding short-lived winter-annual grasses on sandy anthropogenic soils of temperate europe che chenopodietea br.-bl. in br.-bl. et al. 1952 sredozemna jednogodišnja ruderalna vegetacija winter-annual weed segetal and ruderal vegetation of manmade habitats of the mediterranean, the mild-winter atlantic seaboards and macaronesia che-01 brometalia rubenti-tectori (rivas goday et rivas-mart. 1973) rivas-mart. et izco 1977 nom. conserv. propos. sredozemne ruderalne zajednice s prevlašću jedno go diš­ njih trava winter-annual ruderal vegetation of summer-dry manmade habitats of the mediterranean, the mild-winter atlantic seaboards and macaronesia che-01f hordeion murini br.-bl. in br.-bl. et al. 1936 – eunis e5.1 sredozemne ruderalne zajednice s prevlašću jedno go­ dišnjih trava mediterranean ruderal winter-annual grasslands che-02 chenopodietalia br.-bl. in br.-bl. et al. 1936 sredozemne ruderalne zajednice s prevlašću jednogo­ dišnjih zeleni winter-annual ruderal herb-rich vegetation on nutrient-rich disturbed soils of the mediterranean and the macaronesia che-02a chenopodion muralis br.-bl. in br.-bl. et al. 1936 (syn. malvion parviflorae (rivas-mart. 1978) s. brullo in s. brullo et marcenò 1985) – eunis e5.1 sredozemne ruderalne zajednice s prevlašću jedno­ godišnjih zeleni mediterranean nutrient-demanding ruderal vegetation dominated by low-grown non-succulent herbs che-03 geranio purpureae-cardaminetalia hirsutae s. brullo in s. brullo et marcenò 1985 sredozemne ruderalne zajednice šumskih rubova s prevlašću jednogodišnjih zeleni winter-annual fringe vegetation in shaded mesic habitats of the mediterranean, winter-mild temperate (sub)atlantic and submediterranean regions of temperate europe and the macaronesia *che-03g cardaminion graecae biondi, pinzi et gubellini in biondi et al. 2013 – eunis e5.1 sredozemne ruderalne zajednice šumskih rubova s prevlašću jednogodišnjih zeleni apeninskog polu otoka mesic nitrophilous winter-annual fringe vegetation of the apennines *che-03h euphorbio taurinensis-geranion lucidi matevski et čarni in mucina et al. 2009 – eunis e5.1 sredozemne ruderalne zajednice šumskih rubova s prevlašću jednogodišnjih zeleni balkanskog poluotoka mesic nitrophilous winter-annual fringe vegetation of the submediterranean regions of the balkan peninsula dig digitario sanguinalis-eragrostietea minoris mucina, lososová et šilc in mucina et al. 2016 termofilna antropogena vegetacija na ljeti suhim, pješ ča nim staništima thermophilous grass-rich anthropogenic vegetation rich in summer-annual c4 species in the southern nemoral, mediterranean, steppe and semi-desert zones of europe dig-01 eragrostietalia j. tx. ex poli 1966 termofilne antropogene zajednice na ljeti suhim, pješ­ čanim staništima thermophilous grass-rich anthropogenous vegetation rich in c4 species on summer-dry sandy soils of southern and central europe dig-01a spergulo arvensis-erodion cicutariae j.tx. in passarge 1964 (syn. panico-setarion sissingh in westhoff et al. 1946) – eunis i1.1, i1.2, i1.3, i1.5 vegetation of croatia acta bot. croat. 76 (2), 2017 221 kontinentalne termofilne korovne zajednice ranog ljeta na pješčanim staništima subthermophilous summer-annual weed vegetation on sandy and sandy-loamy soils of the atlantic to subcontinental regions in the nemoral zone of europe dig-01b eragrostion tx. in oberd. 1954 – eunis e5.1, h5.6, i1.1, i1.3 kontinentalne termofilne korovne zajednice kasnog ljeta na pješčanim staništima thermophilous late-summer weed vegetation on sandy soils of southeastern central europe and the balkan peninsula dig-01d diplotaxidion erucoidis br.-bl. in br.-bl. et al. 1936 (syn. calendulo arvensis-heliotropion europaei trinajstić 2008) – eunis e5.1 sredozemne zajednice okopavinskih korova weed vegetation on neutral to basic soils in the thermo and mesomediterrannean belts of the central and western mediterranean dig-01f salsolion ruthenicae philippi ex oberd. 1983 – eunis e5.1 kontinentalne termofilne ruderalne zajednice na pješčanim i šljunčanim staništima ruderal vegetation on disturbed gravelly and sandy soils of the subcontinental regions of central europe dig-02 euphorbietalia prostratae vicedo et al. 1997 termofilne zajednice utrina na ljeti suhim, pješčanim staništima summer-dry trampled vegetation on sandy soils in the southern nemoral and mediterranean zones of europe dig-02b polycarpo-eleusinion indicae čarni et mucina 1998 – eunis e1.e, h5.6 sredozemne termofilne zajednice utrina na pješ ča nim staništima summer-dry vegetation of sandy trampled habitats of northern italy and the illyrian region dig-02c eragrostio-polygonion arenastri couderc et izco ex čarni et mucina 1998 – eunis e1.e, h5.6 kontinentalne termofilne zajednice utrina na pješ ča­ nim staništima summer-dry trampled vegetation on sandy soils of western and central europe pol polygono-poetea annuae rivas-mart. 1975 vegetacija utrina s prevlašću jednogodišnjih biljaka subcosmopolitan therophyte-rich dwarf-herb vegetation of trampled habitats pol-01 polygono arenastri-poetalia annuae tx. in géhu et al. 1972 corr. rivas-mart. et al. 1991 zajednice utrina s prevlašću jednogodišnjih biljaka subcosmopolitan therophyte-rich dwarf-herb vegetation of trampled habitats pol-01a polygono-coronopodion sissingh 1969 (syn. polygonion avicularis br.-bl. 1931; matricario matricarioidis-polygonion arenastri rivas-mart. 1975 corr. rivas-mart. et al. 1991) – eunis e1.e, h5.6 kontinentalne nitrofilne zajednice utrina herb-rich vegetation in trampled habitats in the temperate to boreal zones of europe pol-01b polycarpion tetraphylli rivas-mart. 1975 – eunis e1.e, h5.6 sredozemne zajednice utrina herb-rich vegetation in trampled sunny habitats of the mediterranean pol-01c saginion procumbentis tx. et ohba in géhu et al. 1972 – eunis e2.8, h5.6 kontinentalne zajednice zasjenjenih, intenzivno ga­ ženih utrina herb-rich vegetation in strongly trampled shady habitats of europe art artemisietea vulgaris lohmeyer et al. in tx. ex von rochow 1951 ruderalna vegetacija visokih zeleni na suhim staništima perennial (sub)xerophilous ruderal vegetation of the temperate and submediterranean regions of europe art-01 onopordetalia acanthii br.-bl. et tx. ex klika et hadač 1944 kontinentalne ruderalne zajednice s prevlašću kratko­ živućih trajnica na suhim staništima subxeric ruderal vegetation dominated by short-lived perennials of temperate europe art-01a onopordion acanthii br.-bl. et al. 1936 – eunis e5.1 ruderalne zajednice na suhim staništima istočnih kontinentalnih područja thistle-dominated xero-mesophytic ruderal vegetation of subcontinental central europe and the northern balkans art-01b dauco-melilotion görs ex rostański et gutte 1971 – eunis e5.1, i1.5 ruderalne zajednice na suhim staništima zapadnih kontinentalnih područja xero-mesophytic ruderal vegetation dominated by biennial plants of temperate and subboreal europe art-03 agropyretalia intermedio-repentis t. müller et görs 1969 kontinentalne korovne i ruderalne zajednice na zapuštenim površinama semiruderal grasslands and herblands and weed segetal vegetation of perennial crops in the nemoral, forest-steppe and subboreal zones of europe art-03a convolvulo arvensis-agropyrion repentis görs 1967 – eunis e5.1 kontinentalne korovne i ruderalne zajednice na zapuštenim površinama semiruderal grasslands and herblands in the nemoral and subboreal zones of europe art-04 carthametalia lanati s. brullo in s. brullo et marcenò 1985 sredozemne ruderalne zajednice s prevlašću visokih glavočika thistle-dominated ruderal vegetation on disturbed calcareous substrates of the submediterranean regions of southern europe art-04a silybo mariani-urticion piluliferae sissingh ex br.-bl. et o. de bolòs 1958 – eunis e5.1 primorske ruderalne zajednice s prevlašću visokih glavočika thistle-dominated ruderal vegetation of the central mediterranean art-04c onopordion illyrici oberd. 1954 – eunis e5.1 submediteranske ruderalne zajednice s prevlašću visokih glavočika thistle-dominated ruderal vegetation of the submediterranean regions of the balkans škvorc ž., jasprica n., alegro a., kovačić s., franjić j., krstonošić d., vraneša a., čarni a. 222 acta bot. croat. 76 (2), 2017 art-05 elytrigio repentis-dittrichietalia viscosae mucina ined. sredozemne ruderalne zajednice na zapuštenim povr šinama anthropogenic sub-ruderal and ruderal grasslands and herblands of submediterranean and mediterranean southern europe art-05a inulo viscosae-agropyrion repentis bi ondi et allegrezza 1996 – eunis e5.1 sredozemne ruderalne zajednice na zapuštenim površinama anthropogenic sub-ruderal and ruderal grasslands and herblands of the submediterranean regions of the apennine and balkan peninsulas epi epilobietea angustifolii tx. et preising ex von rochow 1951 (syn. galio-urticetea passarge ex kopecký 1969) mezofilna poluprirodna vegetacija visokih zeleni tall-herb semi-natural perennial vegetation on disturbed forest edges, nutrient-rich riparian fringes an in forest clearings in the temperate and boreal zones of eurasia epi-01 galeopsio-senecionetalia sylvatici passarge 1981 nom. conserv. propos. acidofilne poluprirodne zajednice visokih zeleni na šum­ skim rubovima i čistinama tall-herb perennial semi-natural vegetation on acidic soils on forest margins and clearings of the eurosiberian region epi-01a epilobion angustifolii oberd. 1957 – eunis e5.3, g5.8 acidofilne poluprirodne zajednice visokih zeleni na šumskim rubovima i čistinama tall-herb perennial semi-natural vegetation on acidic soils of forest margins and in forest clearings in the boreal and nemoral zones of europe epi-02 circaeo lutetianae-stachyetalia sylvaticae passarge 1967 nom. conserv. propos. (lamio albi-chenopodietalia boni-henrici kopecký 1969) poluprirodne zajednice zeleni na šumskim rubovima i čistinama ruderal and semi-natural fringe mesic tall-herb vegetation of tall-herbs on nutrientand base-rich soils of cool-temperate and submediterranean europe epi-02a fragarion vescae tx. ex von rochow 1951 nom. conserv. propos. (syn. atropion br.-bl. ex br.-bl. et al. 1952) – eunis g5.8 poluprirodne zajednice zeleni početnih sukcesijskih stadija na šumskim čistinama semi-ruderal herb-rich clearing vegetation on nutrientrich calcareous soils in the nemoral zone of central and western europe epi-02b impatienti noli-tangere-stachyion sylvaticae görs ex mucina 1993 – eunis e5.4 poluprirodne zajednice visokih zeleni na zasjenjenim šumskim rubovima i čistinama semi-ruderal tall-herb vegetation of shaded mesic forest margins and clearings on loamy soils in the colline and submontane belts of central europe epi-02c aegopodion podagrariae tx. 1967 nom. conserv. propos. – eunis e5.1, e5.4 poluprirodne nitrofilne zajednice zeleni na zasjenjenim šumskim rubovima i čistinama semi-ruderal herb-rich clearing vegetation on mesic margins and clearings of forests and scrub in the temperate and subboreal zones of europe epi-03 arctio lappae-artemisietalia vulgaris dengler 2002 ruderalne zajednice visokih zeleni na šumskim rubovima i čistinama ruderal vegetation dominated by short-lived perennials on mesic loamy soils of the low-altitude cool-temperate central europe and at high-altitudes of submediterranean europe epi-03a arction lappae tx. 1937 – eunis e5.1 ruderalne zajednice visokih zeleni na šumskim ru bovima i čistinama zapadnih kontinentalnih pod ručja ruderal vegetation of short-lived perennials on mesic loamy soils of cool-temperate europe epi-03b balloto-conion maculati s. brullo et marcenò 1985 – eunis e5.1 ruderalne zajednice visokih zeleni na antropogenim staništima istočnih kontinentalnih područja tall-herb perennial ruderal vegetation in mesic habitats in the submontane and montane belts of submediterranean europe epi-04 galio-alliarietalia oberd. in görs et t. müller 1969 nitrofilne termofilne ruderalne zajednice zeleni na šum­ skim rubovima i čistinama ruderal and semi-natural thermophilous fringe vegetation of short-lived herbs on nutrient-rich soils in the sub montane and montane belts of submediterranean europe epi-04a geo urbani-alliarion officinalis lohmeyer et oberd. in görs et t. müller 1969 (syn. galio-alliarion lohmeyer et oberd. in oberd. et al. 1967) – eunis e5.1 nitrofilne termofilne ruderalne zajednice zeleni na šumskim rubovima i čistinama ruderal and semi-natural fringe thermophilous vegetation of short-lived low herbs on nutrient-rich soils of temperate europe epi-05 convolvuletalia sepium tx. ex moor 1958 poluprirodne zajednice visokih zeleni na vlažnim sta ni­ štima i uz obale vodotoka semi-natural fringe vegetation on banks of rivers and other water bodies of temperate europe and the mediterranean epi-05a senecionion fluviatilis tx. ex moor 1958 (syn. convolvulion sepium oberd. 1949) – eunis e5.4 poluprirodne zajednice visokih zeleni na vlažnim staništima i uz obale vodotoka tall-herb fringe vegetation on nutrient-rich river banks and in ditches of central europe bid bidentetea tx. et al. ex von rochow 1951 pionirska vegetacija vlažnih eutrofnih staništa summer-annual pioneer vegetation of seasonally flooded nutrient-rich river alluvia, lacustrine banks and heavily nutrientloaded anthropogenic habitats of boreo-temperate europe and north africa bid-01 bidentetalia br.-bl. et tx. ex klika et hadač 1944 pionirske zajednice vlažnih eutrofnih staništa summer-annual pioneer vegetation of seasonally flooded nutrient-rich river alluvia, lacustrine banks and heavily nutrient-loaded anthropogenic habitats of boreo-temperate europe bid-01a bidention tripartitae nordhagen ex klika et hadač 1944 – eunis c3.5 pionirske zajednice vlažnih eutrofnih staništa summer-annual pioneer vegetation of periodically nutrient-rich river banks and drained muddy bottoms of eutrophic lakes of boreo-temperate europe vegetation of croatia acta bot. croat. 76 (2), 2017 223 references biondi, e., blasi, c., allegrezza, m., anzellotti, i., azzella, m. m., carli, e., casavecchia, s., copiz, r., del vico, e., facioni, l., galdenzi, d., gasparri, r., lasen, c., 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2017: classification of european beech forests: a gordian knot? applied vegetation science 20, 494–512. acta bot. croat. 81 (1), 2022 117 acta bot. croat. 81 (1), 117–120, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-004 issn 0365-0588 eissn 1847-8476 short communication new records of salicornia s.l. in montenegro and bosnia and herzegovina danijela stešević1*, đorđije milanović2, milica stanišić-vujačić1, urban šilc3 1 university of montenegro, faculty of natural sciences and mathematics, džordža vašingtona bb, 81000 podgorica, montenegro 2 university of banja luka, faculty of forestry, s. stepanovića 75a, 78000 banja luka, bosnia and herzegovina 3 research centre of the slovenian academy of sciences and arts zrc sazu, institute of biology, novi trg 2, 1000 ljubljana, slovenia abstract – floristic investigations on the eastern part of adriatic coast in montenegro and bosnia and herzegovina led to the discovery of three glasswort taxa new for the area: arthrocaulon macrostachyum (moric.) piirainen et g. kadereit and salicornia procumbens sm. subsp. procumbens and s. perennis mill. all three taxa were recorded in the abandoned basins of tivat saline in montenegro, while s. perennis was also found in the klek peninsula in bosnia and herzegovina. according to the iucn criteria, the status of the newly reported taxa was classified as “critically endangered” (cr) in both countries. keywords: arthrocaulon macrostachyum, new records, salicornia perennis, salicornia procumbens introduction due to coexistence in similar ecological environments, representatives of glassworts (salicornia s.l. (incl. sarcocornia a.j. scott) and arthrocnemum moq.) have developed characteristic morphological traits, such as succulent, articulated and apparently leafless stems. although they are easy to recognize, sometimes it is difficult to distinguish between species or even between the related genera. according to the literature sources (kutleša and lakušić 1964, kaligarič and škornik 2007, kaligarič et al. 2008, stešević and caković 2013, barina et al. 2018, nikolić 2020), the following glasswort species/aggregates have been reported along the eastern adriatic coast (svn – slovenia, hrv – croatia, bih – bosnia and hercegovina, mne – montenegro, alb – albania): arthrocaulon macrostachyum (svn, hrv, bih, alb), salicornia fruticosa (svn, hrv, bih, mne, alb), s. perennis (hrv, alb), salicornia europaea aggr. (svn, hrv, mne, alb) and s. procumbens aggr. (svn, hrv, alb). to date, arthrocaulon macrostachyum, salicornia perennis and s. procumbens have not been included in the checklist of vascular plants in montenegro (stešević and caković 2013), while salicornia perennis has not been recorded from bih (kutleša and lakušić 1964), so the current results represent the first records in this part of the adriatic coast. the aim of this paper is to present new records of the glassworts a. macrostachyum, s. perennis and s. procumbens on the eastern adriatic coast and to highlight their importance for nature conservation. material and methods field investigation was conducted along the eastern adriatic coast in montenegro and bosnia and herzegovina. the collected plant material was deposited in the herbarium collection of the university of montenegro (tgu) and in the herbarium of the faculty of forestry university of banja luka under the following voucher numbers: tgu 1570521 (arthrocaulon macrostachyum), tgu 1570524 (salicornia perennis), sfunibl 753/2017 (salicornia perennis) and tgu 1644085 (salicornia procumbens subsp. procumbens). specimens were identified according to kaligarič et al. (2008), kadereit et al. (2012) and piirainen et al. (2017). phytosociological relevés were recorded according to the braun-blanquet method (1964) and included in the vegeta* corresponding author e-mail: danijela.stesevic@ucg.ac.me stešević d., milanović đ., stanišić-vujačić m., šilc u. 118 acta bot. croat. 81 (1), 2022 tion database of montenegro (eu-me-001, http://www. givd.info /id /eu-me-001). the nomenclature of the taxa given in tab. 1, and in the text follows euro+med (2006), except for the arthrocaulon and salicornia (piirainen et al. 2017). results and discussion arthrocaulon macrostachyum (moric.) piirainen & g. kadereit (syn. salicornia macrostachya moric., arthrocnemum macrostachyum (moric.) k. koch, salicornia virginica forssk., salicornia glauca delile). the species is reported at several sites within tivat saline in montenegro, at the edge of the front basin and on one of its islets. at all its microlocalities a. macrostachyum grows in association with salicornia fruticosa l., but the cover of its individuals changes with the inundation period by the sea. table 1 shows the four ecological microsites with a. macrostachyum from the slightly elevated site, which has the shortest exposure to direct seawater impact, to the rapid alternation of dry and wet phases, where it grows in a monodominant community (tab. 1, rel. 1) to the longest flooded site, where it grows in the association puccinellio festucaeformis-sarcocornietum fruticosae (braun-blanquet 1928) géhu 1976 (tab. 1, rel. 4). according to the iucn (2012) criteria a. macrostachyum is considered critically endangered (cr) in montenegro (tab. 2). salicornia perennis mill. (syn. sarcocornia perennis (mill.) a. j. scott, arthrocnemum perenne (mill.) moss). the species is found in tivat saline in montenegro and in the klek peninsula in bosnia and herzegovina. salicornia perennis is more widespread in tivat saline than a. macrostachyum; it occurs not only in the slightly elevated and drier parts of the front basin, but also in the back basins, which are not exposed to the direct influence of seawater and are outside the tidal framework. table 1 shows the phytosociological relevés with s. perennis recorded at different sites in tivat saline, starting with the wettest site, where s. fruticosa dominates, while s. perennis occurs only sporadically (rel. 5), through the transitional and less humid and flooded variants (rels. 6, 7), to the monodominant stands of s. perennis in the back basins, which are completely dry and out of the tidal range from may onwards (rels. 8, 9). in bosnia and herzegovina, s. perennis is an extremely rare species that is found in a narrow zone of salt-sprayed rocky cliffs. it grows in flattened and sheltered parts of plantagini-limonietum cancellati horvatić (1934) 1939, together with limbarda crithmoides (l.) dumort., crithmum maritimum l., limonium cancellatum (bernh. ex bertol.) kuntze, juncus acutus l., sonchus maritimus l., reichardia picroides (l.) roth, etc. according to the iucn (2012) criteria, s. perennis is classified as critically endangered (cr) in both montenegro and bosnia and herzegovina (tab. 2). salicornia procumbens sm. subsp. procumbens (syn. s. emericii duval-jouve, s. herbacea var. stricta g. mey., s. procumbens var. stricta (g. mey.) j. duvign. & lambinon in lambinon & al., s. oliveri moss, s. dolichostachya moss, s. strictissima gram, s. fragilis p.w. ball & tutin, s. lutescens p.w. ball & tutin, s. ramosissima var. vicensis j. duvign., s. vicensis (j. duvign.) j. duvign., s. emericii var. peltii géhu, géhu-franck & caron, s. veneta pignatti & lausi, s. borysthenica tzvelev) was found in tivat saline, both in the front and in the back basins as well as along the channels of the seawater drainage system on mudflats exposed to a regular tidal regime and nutrient flux, forming mostly immersed pioneer communities that are monospecific (tab. 1, rel. 10) or of low species richness (tab. 1, rels. 11-13). all relevés tab. 1. phytosociological table of the vegetation with arthrocaulon macrostachyum (rels. 1-4), salicornia perennis (rels. 5-9) and salicornia procumbens subsp. procumebens (rels. 10-13) in the tivat saline, montenegro. relevé no. 1 2 3 4 5 6 7 8 9 10 11 12 13 plot size (m2) 25 25 25 25 25 25 25 25 25 25 25 4 25 vegetation cover (%) 60 40 70 90 100 80 40 80 95 30 70 80 60 arthrocaulon macrostachyum (moric.) piirainen & g. kadereit 4 3 3 1 . . . . . . . . . salicornia perennis mill. . . . . + 3 3 3 2 . . . + salicornia fruticosa (l.) l. 1 2 3 5 5 3 2 . . . . 1 + salicornia procumbens sm. subsp. procumbens . . . . . . . . . 3 4 4 4 limonium narbonense mill. . . . 1 1 2 . 2 3 . + 1 1 puccinellia festuciformis (host) parl. . . . 1 1 2 . 2 2 . + 1 . atriplex portulacoides l. . . . . . . + 3 4 . . 1 . hordeum marinum huds. . . . . . . . 1 + . . . . plantago coronopus l. . . . . . . . + . . . . . parapholis incurva (l.) c.e.hubb. . . . . . . . + . . . + . spergularia marina (l.) besser . . . . . . . + . . . . . triglochin marituma l. . . . . . . . . . . . 1 . new records of salicornia s.l. acta bot. croat. 81 (1), 2022 119 could be assigned to salicornietum emerici o. bolós ex brullo et furnari 1976. according to the iucn (2012) criteria, s. procumbens sm. subsp. procumbens is classified as critically endangered (cr) in montenegro (tab. 2). the morphological similarity of genera and species of certain halophytic succulents from the subfamily salicornioideae, compounded by the lack of studies dealing with the diversity of this group, could be considered the main reasons why it took so long to identify and report a. macrostachyum, s.  perennis and s. procumbens subsp. procumbens in the studied parts of the eastern adriatic coast. based on the experience gained in recent surveys at two studied sites in montenegro and bosnia and herzegovina, further work on glasswort chorology all along the eastern adriatic coast is recommended. indeed, s. perennis is confirmed at both localities where only s. fruticosa is previously reported (kutleša and lakušić 1964, janković and stevanović 1983), while s. procumbens subsp. procumbens is reported at tivat saline where this annual glasswort has been treated as s. herbacea s.l. (janković and stevanović 1983). inspection of a small number of existing digitised herbarium specimens from croatia (using the platform of the flora croatica database, nikolić 2020), shows that the glasswort is still frequently confused and misidentified by many botanists (e.g. arthrocaulon macrostachyum is frequently confused with salicornia fruticosa). recent studies of annual glassworts (kadereit et al. 2012, kaligarič et al. 2008, šajna et al. 2013) have shown that two annual species occur on the adriatic coast: the tetraploid s. procumbens and the diploid s. perennans willd. along the inundation gradient, tetraploid species usually alternate with diploid. the vegetation belts containing these species are clearly distinguishable in late autumn when s. procumbens turns red and s. perennans remains dully green (fanelli et al. 2015). during our field surveys we did not find s. perennans individuals, but since our transects did not cover the entire area of the saline, as well as the late autumn aspect, the reports of s. procumbens do not exclude the possibility that diploid s. perennans species also grow in the back basin of the saline. thus, further investigation is required. based on the iucn category assessment, we propose the newly reported glasswort species as candidates for the national list of protected plant species: arthrocaulon macrostachyum, salicornia procumbens subsp. procumbens and s. perennis in montenegro and s. perennis in bosnia and herzegovina. acknowledgements the authors would like to thank mitja kaligarič for confirming the identification of salicornia procumbens, željko škvorc for bibliographic help; and anonymous reviewers for valuable comments and suggestions that helped us to improve the manuscript considerably. the research was funded by jp morsko dobro (through the projects: monitoring of status/changes in the special reserve at tivat saline in 2019 and welcome), and by undp-gef (through the project “achieving biodiversity conservation through creation, effective management and spatial designation of protected areas and capacity building for nature conservation in bosnia and herzegovina”). uš was supported by programme p1-0236 (arrs) and a bilateral grant (bime/16-17-018) from arrs. tab. 2. assessment of the status of the arthrocaulon macrostachyum, salicornia perennis and salicornia procumbens subsp. procumbens in montenegro (mne) and bosnia and herzegovina (bih) according to the iucn criteria. taxon arthrocaulon macrostachyum salicornia perennis salicornia procumbens subsp. procumbens country mne mne bih mne category critically endangered (cr) critically endangered (cr) critically endangered (cr) critically endangered (cr) assessment criteria b2; b2a; b2biii b2; b2a; b2biii d b2; b2a; b2biii date of assessment september 2019 september 2019 october 2018 september 2019 population size ca. 500 mature individuals very large less that 30 individuals very large habitat and ecology coastal salt marshes in intertidal area with varying degrees of inundation. frequently occurs in saline sandy to clayey soils, mostly accompanied by salicornia fruticosa coastal salt marshes, on mudflats exposed to regular tide regime and rich nutrient flow sheltered salt-sprayed rocky cliffs coastal salt marshes, on mudflats exposed to regular tide regime and rich nutrient flow threats ecosystem modification, changes in shoreline morphology, changes in the tidal regime, accelerated sea-level rise due to climate changes ecosystem modification, changes in shoreline morphology, changes in the tidal regime, accelerated sea-level rise due to climate changes urbanization, water pollution, waste deposition ecosystem modification, changes in shoreline morphology, changes in the tidal regime, accelerated sea-level rise due to climate changes estimated by d. stešević d. stešević đ. milanović d. stešević stešević d., milanović đ., stanišić-vujačić m., šilc u. 120 acta bot. croat. 81 (1), 2022 references barina, z., somogyi, g., pif kó, d., rakaj, m., 2018: checklist of vascular plants of albania. phytotaxa 378, 1–339. braun-blanquet, j., 1964: pflanzensoziologie. grundzüge der vegetationskunde. springer verlag, wien. euro+med, 2006: euro+med plantbase the information resource for euromediterranean plant diversity. retreived march 16, 2020 from http://ww2.bgbm.org/europlusmed/ fanelli, g., de sanctis, m., gjeta, e., mullaj, a., attorre, f., 2015: the vegetation of the buna river protected andscape (albania). hacquetia 14, 129–174. iucn, 2012: iucn red list categories and criteria, ver. 3.1., 2nd ed. iucn species survival commission, gland. janković, m., stevanović, v., 1983: contribution to the knowledge of halophytic vegetation of the boka kotorska bay. in: pavletić, z., matković, p., grubišić, s. (eds.), zbornik roberta visianija šibenčanina, 377–396 (in serbian). muzeja grada šibenika, šibenik. kadereit, g., piirainen, m., lambinon, j., vanderpoorten, a., 2012: cryptic taxa should have names: reflections in the glasswort genus salicornia (amaranthaceae). taxon 61, 1227–1239. kaligarič, m., škornik, s., 2007: vegetation of tall rush saltmarshes (juncetea maritimae) and saltmarsh scrubs (arthrocnemetea fruticosae) on the slovenian seacoast. annales histoire sciences sociales 17, 47–58. kaligarič, m., bohanec, b., simonovik, b., šajna, n., 2008: genetic and morphologic variability of annual glassworts (salicornia l.) from the gulf of trieste (northern adriatic). aquatic botany 89, 275–282. kutleša, l., lakušić, r., 1964: flora and vegetation of the klek peninsula. godišnjak biološkog instituta univerziteta u sarajevu 17, 61–115 (in bosnian). nikolić, t. (ed.), 2020: flora croatica database. prirodoslovnomatematički fakultet, sveučilište u zagrebu. retreived march 16, 2021 from http://hirc.botanic.hr/fcd piirainen, m., liebisch, o., kadereit, g., 2017: phylogeny, biogeography, systematics and taxonomy of salicornioideae (amaranthaceae/chenopodiaceae) – a cosmopolitan, highly specialized hygrohalophyte lineage dating back to the oligocene. taxon 66, 109–132. stešević, s., caković, d., 2013: catalogue of vascular flora of montenegro, vol. 1. canu, podgorica (in montenegrin). šajna, n., regvar, m., kaligarič, s., škvorc, ž., kaligarič, m., 2013: germination characteristics of salicornia patula duval-jouve, s. emerici duval-jouve, and s. veneta pign. et lausi and their occurrence in croatia. acta botanica croatica 72, 347–358. appendix data and coordinates (wgs84) of the relevés (tab. 1). all relevés were collected at 0 m.a.s.l., except relevés 8 and 9, collected at 1 m.a.s.l. rel. 1 – 2019/09/20, 42.39742n 18.71352e; rel. 2 –2019/09/20, 42.3973n 18.71381e; rel. 3 – 2019/09/20, 42.375n 18.7303e; rel. 4 – 2019/09/20, 42.39553n 18.71223e; rel. 5 – 2019/08/15, 42.39092n 18.7185e; rel. 6 – 2019/09/20, 42.39156n 18.71871e; rel. 7 – 2019/09/20, 42.39203n 18.71695e; rel. 8 – 2019/07/06, 42.39829n 18.71825e; rel. 9 – 2019/08/15, 42.39791n 18.71889e; rel. 10 – 2019/09/02, 42.39525n 18.71759e; rel. 11 – 2019/09/02, 42.39376n 18.71843e; rel. 12 – 2019/05/03, 42.39226n 18.71006e; rel. 13 – 2019/09/20, 42.39488n 18.71912e. opce-str.vp acta bot. croat. 70 (2), 315–319, 2011 coden: abcra 25 issn 0365-0588 first national week of croatian botanical gardens and arboreta (may 30 – june 4, 2011) although it has only a few botanical gardens, croatia is a member of the european botanic gardens consortium, convened by the botanic gardens conservation international (bgci). croatian botanical gardens are recognized for their valuable collections of indigenous flora, the third richest in europe, while the few arboreta keep collections of ornamental trees and shrubs older than 300 years. most croatian botanical gardens are statutorily protected, and all operate as integrated units of different faculties, schools or similar, rather than as independent institutions. botanical garden employees (biologists, foresters, agronomists, horticulturists, teachers) gathered in 2009 to constitute a section of croatian botanical gardens and arboreta, within the croatian botanical society. that was the easiest way of fulfilling some of the elementary bgci-tasks: to share resources and expertise, get a stronger constituency and voice, and cope with a number of different matters. at the round table of the section, organized during third croatian botanical congress (september 2011), the idea of an »open day« of croatian botanical gardens was accepted. it was than decided that the »open day« should be extended to a week of croatian botanical gardens and arboreta, in order to gain more attention from the public and the authorities. the week would be accompanied by the first national symposium of the croatian botanical gardens and arboreta, as well as an exhibition on the same subject. six croatian ministries agreed that the event would take place under their auspices, as did the state institute for nature protection, the universities of zagreb, split and dubrovnik, and the cities of zagreb and dubrovnik. the symposium was held on may 30th in the exhibition pavilion in the botanical garden (fig. 1), gathering 33 participants, 15 representatives of the sponsors and donators and more than 50 listeners. twelve presentations on croatian botanical gardens, arboreta and several other plant-collections were held, as well as presentations by invited lecturers – ms suzanne sharrock, director of the bgci-global programmes, and dr jo`e bavcon, director of ljubljana botanic garden (slovenia). two opening lectures were presented by croatian representative in bgci, ms biserka jureti}, who is also the manager of zagreb botanical garden of the faculty of science, and dr antun alegro, president of the croatian botanical society. summarized presentations of all the participants were published in the booklet of abstracts (in croatian). acta bot. croat. 70 (2), 2011 315 copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\social news kovacic.vp 15. rujan 2011 11:53:23 color profile: disabled composite 150 lpi at 45 degrees during the week that followed, various events open daily to the public without charge were organized in 12 croatian botanical gardens, arboreta and other facilities with smaller plant collections. in dubrovnik, many students attended workshops and guided tours of the garden on the island of lokrum (botanical garden of the institute for marine and coastal research, university of dubrovnik), famous for its collection of australian plants. in the more mysterious parts of the nearby arboretum of the croatian academy of arts and science in trsteno, the oldest existing arboretum in the world (founded in 1498), story-tellings were organized for children and their parents, along with workshops, lectures and guided tours. in the marjan botanical garden of the faculty of science, university of split, many citizens and pupils attended different workshops and lectures, expressing their hope that this neglected university botanical garden would be soon restored to its former beauty. the nearby city of ka{tela is home to the ostrog elementary school botanical garden, long-known for its rich and beautiful exotic plants collection. pupils and their teachers prepared various workshops, guided tours and competitions in recognizing garden plants. this school was given the honour of organizing the official closing of the first national week of the botanical gardens and arboreta. at the event that took place on june 4, awards were given to the best young botanists. in the koti{ina visitors centre of the biokovo mountain botanical garden (biokovo nature park) near the city of makarska, lectures, tours and presentations were given daily, as well as a screening of the documentary film »endemics of the biokovo mountain«. because mt velebit botanical garden (northern velebit national park) is fairly remote, situated high in the mountains of zavi`an, the biologist of the national park service went on a »botanical tour« through the cities and communities in the valley: in gospi}, senj, oto~ac and krasno she organized various workshops and lectures in elementary schools and public libraries, in order to bring attention to the velebit botanical garden and its valuable collection of local endemic plant species. the live plants collection of the liburnian karst flora growing in the small botanical garden of the natural history museum in rijeka was presented to public, and various other events included the presentation of the qr-codes in the garden and the museum, and an evening cocktail-party 316 acta bot. croat. 70 (2), 2011 fig. 1. exhibition pavilion in the botanical garden of the faculty of science, university of zagreb. u:\acta botanica\acta-botan 2-11\social news kovacic.vp 15. rujan 2011 11:53:25 color profile: disabled composite 150 lpi at 45 degrees with non-alcoholic herbal beverages. the oldest statutorily protected monument of horticultural architecture in croatia, the opeka arboretum in vinica (near city of vara`din) suffers greatly from irregular maintenance, being treated today as just a 'park' with no full-time employees. the organisation of the week was thus carried out by the nearby arboretum opeka high school. pupils and their teachers presented to the public various school-projects, photo-exhibitions and workshops, all related to the arboretum situated literally across the street. the students of the antun gustav mato{ grammar school (zabok) started a lovely young arboretum of their own. they participated in the week with various projects and presentations, meant for their parents and co-citizens. on the slopes of the northeast-croatian papuk mountain, in the village of lisi~ine, a large arboretum established in the 1960s was badly damaged during the croatian war of independence. the arboretum is administered by the local forestry service, which organized guided tours for the students and citizens. both botanical gardens of zagreb university came up with various events. the fran ku{an pharmaceutical botanical garden (faculty of pharmacy and biochemistry) gave information to visitors on poisonous and curative plants. the botanical garden of the zagreb faculty of science offered 15 guided tours, 8 workshops, 8 lectures, 2 puppet-shows (fig. 2) and an exhibition on croatian botanical gardens and arboreta. the first national week of croatian botanical gardens and arboreta, organized at 12 locations, was visited by more than two thousand people. croatian radiotelevision broadcast daily from different gardens and collections, bringing various week events to a million viewers and listeners. gardens and events during the week were covered by a large number of newspapers and web sites. many scientific, cultural and popular institutions and societies advertised the programme, inviting the public to visit their local botanical collections. because of positive attention given to the first national week of croatian botanical gardens and arboreta by the public, the media, as well as local communities, national authorities and institutions, we conclude it was a success and propose it become an annual event. acta bot. croat. 70 (2), 2011 317 fig. 2. the youngest garden visitors taking their places for the puppet show. u:\acta botanica\acta-botan 2-11\social news kovacic.vp 15. rujan 2011 11:53:27 color profile: disabled composite 150 lpi at 45 degrees the next national week of croatian botanical gardens and arboreta will be organised from monday 14th to saturday 19th of may, 2012. sanja kova~i} and vanja stamenkovi}, department of botany, botanical garden, division of biology, faculty of science, university of zagreb maruli}ev trg 9a, hr – 10000 zagreb, croatia e-mail: sanja@botanic.hr 318 acta bot. croat. 70 (2), 2011 u:\acta botanica\acta-botan 2-11\social news kovacic.vp 15. rujan 2011 11:53:27 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp book reviews u:\acta botanica\acta-botan 1-10\book review.vp 9. travanj 2010 13:45:58 color profile: disabled composite 150 lpi at 45 degrees u:\acta botanica\acta-botan 1-10\book review.vp 9. travanj 2010 13:45:58 color profile: disabled composite 150 lpi at 45 degrees book review lehrbuch der botanik textbook of botany founded by e. strasburger, f. noll, h. schenck and a. f. w. schimper 36th edition, revised by andreas bresinsky, christian körner, joachim w. kadereit, gunther neuhaus and uwe sonnewald. spektrum akademischer verlag, heidelberg 2008 the founders of this textbook were botany docents at the university of bonn (germany). they constantly exchanged scientific insights and cooperated in their teaching activities. their objective in publishing the first edition of the strasburger textbook was to share their accumulated knowledge. eduard strasburger wrote the introduction and the chapter on morphology, fritz noll the physiology part, heinrich schenck the cryptogams, and andreas franz wilhelm schimper the phanerogams. although each author primarily was responsible for his part, there was still a strong cooperation provided by the ongoing contacts among the coauthors so that in spite of the plurality of authors the book with its four parts appeared as a genuine unity. according to the preface of the first edition (1894) this textbook was intended to be used by students at high schools to advance their scientific interest, knowledge, and perceptions. at the same time the book also considered the practical aspects of botanical studies, especially in medicine and pharmacy. for the past 116 years the importance of the strasburger textbook was mainly due to the well balanced and complete presentation of the diverse parts of botany as well as its currency and high scientific level. further advances were the excellent text, its differentiation between elementary and advanced knowledge of the reader (distinguished by the type of print, normal and petit), the excellent illustrations, and an acceptable price. until 1991 the contents of the textbook were a little behind the actual level of scientific progress, in agreement with the generally respected rule that most recent discoveries should not be included in a textbook before they definitely are accepted to be correct. acta bot. croat. 69 (1), 2010 149 u:\acta botanica\acta-botan 1-10\book review.vp 9. travanj 2010 13:46:05 color profile: disabled composite 150 lpi at 45 degrees in 1991 (33rd edition) the introduction and the morphology part were written by peter sitte, a new member of the team of authors. his presentation of the matter, the choice and quality of illustrations and the actuality of explanations, which considerably differed from the moderate and temperate classic style of the nearly 100 years old textbook, clearly showed that with this author the strasburger textbook was entering a new era. the new style of sitte’s introduction and morphology did not remain without influence on or resonance with the coauthors of the team, which became clear as soon as the 34th edition (1998) and especially in the 35th edition in which peter sitte and andreas bresinsky with three new coauthors (elmar w. weiler, joachim w. kadereit, and christian körner) succeeded in reaching the present level of a high quality textbook that at the same time met the requirements of handbook and reference book. during the past 116 years of its existence the strasburger textbook was translated into eight foreign languages: into english (9 editions, the last in 1975!), italian (9 editions), polish (5), spanish (9), serbo-croatian (4), russian (1), turkish (l) and portuguese (in preparation). in the recent 36th edition two new authors, gunther neuhaus and uwe sonnewald, thoroughly worked up the first and second part of the book. sitte left the team of the authors because of his age. the first part of the book, now »structure« (previously termed morphology) deals with the chemical principles of life, the fundamentals of cell biology, and spreads over to the morphology of higher plants. special attention is paid to the consequent use of the international scientific terminology in cell biology, as well as to the actualization of cell biological facts. the first part – structure – begins thus with the molecular principles, that is with the building components of the cells [chapter 1] explaining the structure and basic functions of nucleic acids (their building components; structure and replication of deoxyribonucleic acid dna; ribonucleic acids, viruses, phages, and viroids), the proteins (aminoacids as building components of proteins and protein complexes), polysaccharides (monosacharides as their building components, the formation of glycosides, further the reserve polysaccharides and the structural polysaccharides), and finally the lipids and the formation of lipid double layers in biomembranes. in the following chapter [2] the structure and ultrastructure of the cell, the fundamental facts of cell biology, the plant cell, the cell structure of prokaryotes, as well as the endosymbiont theory and the hydrogen hypothesis are well presented and explained. then the tissues of cormophytes are treated, both the formative tissues (meristems) and the permanent tissues [3]. the first part of the book is finished with a presentation of the morphology and anatomy of cormophytes [4], containing sections on shoot axis, of leaf organs (forms and metamorphoses), and of roots. in the second part of the book, the main aspects of plant physiology are presented: metabolism, growth and development, movements and allelophysiology. in the present edition the sections concerning the primary metabolism were rewritten, and all other parts brought up to date. this second part begins with the physiology of the metabolism [5] in this section all aspects of plant metabolism are presented and explained: energetics of metabolism, economy of minerals and water, photosynthesis (light reactions and the path of carbon, the assimila150 acta bot. croat. 69 (1), 2010 u:\acta botanica\acta-botan 1-10\book review.vp 9. travanj 2010 13:46:05 color profile: disabled composite 150 lpi at 45 degrees tion of nitrate and sulphate), the transport of assimilates, the energy earning by the degradation of carbohydrates, the formation of structural and reserve lipids and their mobilization, the formation of aminoacids, purines and pyrimidines, of tetrapyrols, and finally secondary metabolism, plant polymers and the excretion of plant substances. in the next chapter [6] the fundamental principles of the developmental physiology are described and interpreted, as there are genetic and cellular principles of development, the systemic control of the development, and interaction of cells during the processes of development by means of phytohormones and other factors. all this is followed by the description of the many diverse kinds of movement in plants [7]. the last chapter [8] (introduced seven years ago in the 35th edition by elmar weiler) concerns allelophysiology in which the peculiarities of the heterototrophic nutrition, of symbiosis and pathogens, herbivory and allelopathy are fundamentally explained and discussed. in the third part of the book, concerned with evolution and systematics, special attention is paid to the changes of our conception of the phylogeny of bacteria, fungi and plants, mainly by means of the new molecular-systematical statements. the history of vegetation has been thoroughly revised in this edition. in the chapter on evolution [9] the variation, the pattern and the natural variation of variability, the formation of species and the macroevolution are fundamentally presented and explained. the following chapter on systematics and phylogeny is the most extensive chapter of the book [10] – about 340 pages long, a third of the whole book. this is understandable as there are about 300,000 species of plants on earth. after a diligent treatment of the methods of systematics, the world of bacteria, fungi, and plants is described corresponding to the three domains (or regna) of the bacteria, archaea, and eucarya (or eucaryotes). the third regnum is divided in seven subdomains: acrasiobionta, myxobionta, mycobionta (fungi with chitin), glaucobionta, rhodobionta, heterokontobionta, and chlorobionta (»viridiplantae«, containing two divisions: chlorophyta and streptophyta), the flower plants are described under the term spermatophytina = seedplants, from p. 799 to p. 923. (the designation »vierte unterabteilung« [fourth subdivision, see also on p. 620!] apparently is an error). an overview of the systematics of angiosperms is given on pp. 847 to 919 in the form of 57 »basal orders« with the data of families, each with the number of genera, species and geobotanic remarks, all located inside light brown rectangular fields, and with short but very well illustrated presentations. the third part of the book finishes with the newly worked up history of vegetation. in the fourth part of the book, the ecology of plants is treated in relation to life conditions at diverse locations. plant reactions to the factors of climate and soil, the processes in the populations and species communities, as well as the great vegetation zones of earth are explained. in this 36th edition the global aspects of ecology are strongly pointed out. numerous illustrations have been newly chosen or even added (about 260) and are highly impressive and very instructive due to their excellent quality and perfect reproduction in spite of their small size (21 × 30 to 50 × 70 mm). in this way the fourth part of the book in just a few pages touches on the whole contents of voluminous contemporary textbooks. acta bot. croat. 69 (1), 2010 151 u:\acta botanica\acta-botan 1-10\book review.vp 9. travanj 2010 13:46:05 color profile: disabled composite 150 lpi at 45 degrees this fourth part of the book was introduced under the title »ecology« by christian körner in the 35th edition (2002), while the former titles of this part were »plant geography« (20th–29th edition, 1939–1970, by franz firbas), and »geobotany« (30th–34th edition, 1971–2002, by friedrich ehrendorfer). in the present edition, ecology comprehends four chapters [11–14]. it begins with the principles of ecology [11], which are represented by the explanation of the terms limitation, fitness and optimum, stress and adaptation, the time factor and non-linear reactions, biological variation, ecosystems and their structure, and finally the main directions of plant ecological research. the next chapter [12], »plants in their living space«, deals with the many different factors influencing growth and distribution of plants such as light, temperature, availability of water and minerals, and also biotic interactions. at last, human influences are well interpreted. in the »population and vegetation ecology« [13] plant regions and the ecology of vegetations are described and interpreted. the last chapter [14] deals with the vegetation of earth, especially with the vegetation of the temperate zone, and with the biomass of the earth. after the end of the text on p. 1120 follow a list of references (p. 1121–1124), an index (p. 1125–1171), a list of abbreviations (p. 1172–1173) and a guide to units and symbols (p. 1174–1175). maps of the global biodiversity of vascular plants and of the vegetation of earth are placed at the beginning and the end of the book (first and last page plus the inner side of the book’s wrapper). at the beginning of the book there are data on the book (prefaces of the present 36th edition, as well as of the first edition; table of contents; indices of boxes and of tables, and a chronological table of important discoveries (pp. i–xvi). the book is very well organised, and by means of the corresponding indexes it is possible to find quickly any desired part of the text and corresponding illustrations. the book is excellently printed. the beautiful colour photograph on the title cover shows the flower of strasburgeria robusta guill. (strasburgeriaceae), an endemic plant from new caledonia, called after the founder of this textbook. in the preface of the 36th edition the authors point out that the permanent challenges for updating as well as other circumstances have been carefully considered. among other things they ask themselves whether the title »strasburger-textbook of botany« does not imply too much of an antiquated tradition that is now no longer justified with regard to the new goals, methods, results and finally even to the position of the discipline itself. would not perhaps the term plant sciences (pflanzenwissenschaften) instead of botany correspond to a better understanding of the discipline? the decision to retain the old title was done finally by anchoring in the strasburger tradition which permits the treatment of bacteria and fungi together with plants. considering this dilemma of the strasburger authors we may perhaps try to help them with the proposal that the term botany should be a bit modified for the strasburger textbook by widening its sense in the way that botany should mean the science of growth (in german: lehre von den gewächsen), corresponding to the slavic languages in which the term for the plants (raslina, raslinje, ra{}e, rastlina, roslina, rastenie etc.) is derived from the verb rasti = to grow (in german: wachsen), from which there exists in german the well known and commonly used word »die gewächse«. it should also be pointed out that in the 152 acta bot. croat. 69 (1), 2010 u:\acta botanica\acta-botan 1-10\book review.vp 9. travanj 2010 13:46:05 color profile: disabled composite 150 lpi at 45 degrees strasburger textbook the first botanical text in our history by theophrastos ereisios (371– 286) is cited as die naturgeschichte der gewächse. if we accept this widening of the term botany, then we may define the term die gewächse as concerning organisms which manifest their life activities mainly by growth. from the term »animals« the term gewächse is well separated by the meaning of the word itself. according to the fact that in all our european languages the bacteria, archaea, cyanobacteria, all algae, fungi (mushrooms), lichens, and all plants grow somewhere on adequate substrates the idea, to unite them all under the same common term gewächse appears as a reasonable solution. accepting this solution we cause minimal trouble in the terminology by simply inconsiderably widening the sense of the term botany to get a proper scientific term for a conception already universally used during history in all languages and their vernaculars. however, regardless of these terminological problems, there can be no doubt that the recent 36th edition of the strasburger textbook has reached the level of an excellent textbook for advanced students as well as a handbook for all whose activities are connected with any field of botany. it would therefore be an irreparable fault not to continue with further editions of this unique text. an edition of an english version would doubtlessly be welcomed on the global level. such an excellent, highly informative and instructive textbook will well serve not only students, but also docents, as well as scientists and experts of all branches as a reliable reference book. in fact, presumably the 36th edition of the strasburger textbook of botany will soon be on the desks of many students of biology in the german speaking part of europe as well as abroad. zvonimir devidé acta bot. croat. 68 (1), 2009 153 u:\acta botanica\acta-botan 1-10\book review.vp 9. travanj 2010 13:46:05 color profile: disabled composite 150 lpi at 45 degrees 647 vukovic et al.vp acta bot. croat. 72 (1), 185–191, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 short communication the orchid ophrys speculum link (orchidaceae) in croatia nina vukovi]1*, annalisa tommasoni2, tancredi d'onofrio2 1 university of zagreb, faculty of science, division of biology, department of botany with botanical garden, maruli}ev trg 20/ii, hr-10000 zagreb, croatia 2 pendice scoglietto 5, it-34127 trieste, italy abstract – the orchid ophrys speculum link is widely distributed in the mediterranean. since its inclusion in the checklist of croatian flora, the species has not been confirmed in the field, but has recently been found on cape (rt) kamenjak, southern istria. a few plants were growing within garrigue vegetation of cisto-ericetalia. keywords: ophrys speculum, orchid, rt kamenjak, istria, croatia introduction in croatian flora the genus ophrys is present with 67 taxa, the largest genus in terms of number of taxa from the orchidaceae family (hr[ak 2000, bogdanovi] 2004, nikoli] 2012). the genus is distributed throughout the whole of croatia, but more taxa can be found in the mediterranean part, as the genus is principally mediteranean (delforge 2006). the ophrys speculum group consists of three closely related species: o. speculum link, o. vernixia brotero and o. regis-ferdinandii (achtaroff et kellerer ex renz) buttler (greuter 2004, delforge 2006). ophrys speculum is a widespread mediterranean species commonly found (sometimes even abundantly) in many mediterranean countries (e.g. spain, greece, north africa), but rare in the centre of the range (soó 1980, pignatti 1982, delforge 2006). for example, the species is noted for france and corsica, but it appears to be extremely rare on the mainland (lepage and wilcox 2003, delforge 2006). similarly, o. speculum is found only sporadically on the italian mainland, but is common on sicily and sardinia (pignatti 1982, romolini and biagioli 2002, delforge 2006). two variants can be distinguished across the distributional range of the species; the western variant o. speculum link ssp. speculum and the eastern variant with darker flowers, o. speculum link acta bot. croat. 72 (1), 2013 185 * corresponding author, e-mail: nina.vukovic@biol.pmf.hr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 647 vukovic et al.ps u:\acta botanica\acta-botan 1-13\647 vukovic et al.vp 14. o ujak 2013 12:56:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees ssp. orientalis (paulus) paulus et salkowski (baumann et al. 2006, delforge 2006, paulus and salkowski 2007). it flowers in the period of (february–) march–april (–may) and inhabits open habitats with full sunlight to mid shade, such as poor grassland and garrigue, appearing mostly in coastal areas up to 1200 m asl. (delforge 2006). the species is pollinated by the wasp dasyscolia ciliata (scoliidae, hymenoptera) and this relationship is highly species-specific (pouyanne 1917, ayasse et al. 2003, baumann et al. 2006, paulus 2007). it has been found that the western variant is pollinated by d. ciliata ssp. ciliata, while the eastern variant is pollinated by d. ciliata ssp. araratensis (delforge 2006, paulus and salkowski 2007). although o. speculum is listed in the checklist of the croatian flora (hr[ak 2000, nikoli] 2012) no specific locality is given, and furthermore, the origin of the data is unknown. relevant determination keys do not mention the eastern adriatic coast as a part of the species range (soó 1980, baumann et al. 2006, delforge 2006). the cape of the istrian peninsula (rt kamenjak, north adriatic) has been protected since 1996, nowadays as a part of the significant landscape »lower kamenjak and medulin archipelago«. more than 500 plant taxa have been recorded in previous floristic studies (tommasini 1873; freyn 1877; perko 1998; starmühler 1998, 2004, 2010; topi] and [egulja 2000; hr[ak et al. 2011), some of them rare, endangered and/or endemic; therefore rt kamenjak has recently been estimated as an important plant area (vukovi] 2010). a former study of orchids of the same area (vukovi] et al. 2011) did not provide any note on o. speculum. materials and methods the area of rt kamenjak was surveyed during may 2010 when a few plants were found in the field. we used the relevant determination key for european orchids (delforge 2006) for determination of the species. the same area was surveyed again in april 2012, with one fieldtrip in the first, and other in the second half of the month. on the second fieldtrip, the exact position of the locality was determined using gps garmin etrex vista, and the checklist of the adjacent flora was prepared. the obtained coordinates of the field observation were incorporated into the flora croatica database (nikoli] 2012) and a distribution map was prepared using esri gis arcmap 9.3 software. the nomenclature of taxa is given according to baumann et al. (2006), paulus and salkowski (2007) and flora croatica database (nikoli] 2012). in addition to fieldwork, two herbarium collections (za and zaho) were examined for specimens and some old literature was searched for localities of o. speculum (visiani 1842, 1847, 1852; schlosser and vukotinovi] 1869; pospichal 1897; mannagetta 1901; adamovi] 1909; rossi 1924, 1930; hayek 1933). furthermore, o. speculum was evaluated under the iucn criteria (gärdenfors et al. 2001, anonymous 2010) to obtain the threat status of the species on the national level. results three individuals of ophrys speculum ssp. speculum (fig. 1) were found growing along the macadam road on the way to cape [kara on south-eastern kamenjak (13°54'55" n, 186 acta bot. croat. 72 (1), 2013 vukovi] n., tommasoni a., d'onofrio t. 647 vukovic et al.ps u:\acta botanica\acta-botan 1-13\647 vukovic et al.vp 14. o ujak 2013 12:56:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 44°46'20" e) (fig. 2). the road passes through garrigue vegetation of the order cisto-ericetalia h-i}. 1958. in the first half of april only the sprouts were observed, while in the second half the plants were fully flowering. the plants were up to 10 cm high but generally in a good condition. however, the evaluation of the threat status on the national level has resulted in proposing the species as cr d (critically endangered according to criteria d). accompanying plant species typical of the area were recorded in the close vicinity, given alphabetically in the following list: cerastium pumilum curtis ssp. glutinosum (fries) jalas, acta bot. croat. 72 (1), 2013 187 ophrys speculum in croatia fig. 1. ophrys speculum link ssp. speculum. a – habitus, b – flower (photo by nina vukovi}). fig. 2. locality of ophrys speculum link ssp. speculum on rt kamenjak. gray area – »lower kamenjak and medulin archipelago«. 647 vukovic et al.ps u:\acta botanica\acta-botan 1-13\647 vukovic et al.vp 14. o ujak 2013 12:56:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees convolvulus arvensis l., dittrichia viscosa (l.) greuter, dorycnium hirsutum (l.) ser., helichrysum italicum (roth.) g. don, hypericum perforatum l., juniperus oxycedrus l., orchis morio l., plantago coronopus l., plantago lanceolata l., sanguisorba minor scop. ssp. muricata briq., sherardia arvensis l. and spartium junceum l. the search within the za and zaho herbaria did not reveal any specimens of o. speculum. none of the reviewed literature references provided any note on o. speculum in croatia. discussion the species ophrys speculum was found within its usual mediterranean range, growing in a typical habitat and our finding is in accordance with the steno-mediterranean distribution of this species. therefore an even wider occurrence of this species in croatia could be expected. however, it is evident that o. speculum is extremely rare in croatia. possible confusion with other croatian orchids due to morphological similarity is excluded because of the rather specific morphology of its flowers, such as large, shiny lip with dense, brown hairs (fig. 1), which makes it easily identifiable. the only similar plants are two related species from the same group that are rarer than o. speculum and locally distributed (o. vernixia in portugal and spain, o. regis-ferdinandii in greece and anatolia). what is more, they can be distinguished from o. speculum by the shape of the labellum and its lobes (baumann et al. 2006, delforge 2006). the rather early flowering period of the species and the fact that it can flower sporadically (delforge 2006) could be a part of the explanation. the scarce presence of this species in croatia, as well as in italy and france, is most probably connected with the distribution of a specific pollinator wasp dasyscolia ciliata. recent data (fallahzadeh and saghaei 2010, osten 2012) show that the wasp is absent from france, italy and eastern-adriatic coast, with a confirmed presence in the portuguese mainland, spanish mainland, balearic islands, greek mainland, dodecanese islands, malta, north africa and the near east. in addition, existing data show the presence of d. ciliata on corsica, sicily and sardinia (cocquempot and harmon 1995, osten 2000, pagliano 2012). in the key for scoliidae from osten (2000) there is an indication that the wasp can be found in croatia and/or nearby countries, stating that the wasp is present on the eastern adriatic coast, but this probably refers to the record of maidl (1922), who mentions d. ciliata (as scolia ciliata) for 'durazzo' (durrës, albania). to date there has been no record of the presence of this wasp in croatia. from the available distributional data for o. speculum and d. ciliata it can be concluded that the range of the orchid, where it is widespread and common, mainly corresponds to areas with the confirmed presence of the wasp. since the plant-pollinator relationship is highly species-specific in the case of ophrys orchids (paulus 2007), the absence or scarce presence of the wasp in the range of ophrys speculum could prevent or seriously reduce the ability of the orchid to reproduce and therefore close its life-cycle. consequently, its occurrence in croatia is unusual and it is likely to be found only accidentally and no stabile occurrence is to be expected. the localized distribution of this orchid in croatia (only one confirmed population of three individuals), the vicinity of the road (potential threat from passengers and/or vehicles) and exposure to natural succession certainly amplify the risk of disappearance from the 188 acta bot. croat. 72 (1), 2013 vukovi] n., tommasoni a., d'onofrio t. 647 vukovic et al.ps u:\acta botanica\acta-botan 1-13\647 vukovic et al.vp 14. o ujak 2013 12:56:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees area. given all these facts, we conclude that there is an extremely high risk of extinction of o. speculum from the croatian flora, and as a result recommend the species to be classified as critically endangered. aknowledgements the file containing the borders of croatian protected areas was provided by the state institute for the protection of nature. the authors would like to thank dr sandro bogdanovi} and igor bor{i} for useful comments on the manuscript, and also vedran [egota for help in the making of the digital distribution map. references adamovi], l., 1909: die vegetationsverhältnisse der balkanländer (moesische länder) umfassend serbien, altserbien, bulgarien, ostrumelien, nordtrakien und nordmazedonien. wilhelm engelmann, leipzig. anonymous, 2010: guidelines for using the iucn red list categories and criteria, 8.1. retrieved july 17, 2012 from http://intranet.iucn.org/webfiles/doc/ssc/redlist/redlist guidelines.pdf ayasse, m., schiestl, f. p., paulus, h. f., ibarra, f., francke, w., 2003: pollinator attraction in a sexually deceptive orchids by means of unconventional chemicals. 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(eds.), notulae ad indicem florae croaticae 4. natura croatica 13, 407–420. cocquempot, c., hamon, j., 1995: capture de dasyscolia ciliata (fabricius, 1787) en corse-du-sud (hymenoptera, scoliidae). bulletin de la société entomologique de france 100, 276. delforge, p., 2006: orchids of europe, north africa and the middle east. timber press, oregon. fallahzadeh, m., saghaei, n., 2010: a brief study on the scoliidae in iran (insecta: hymenoptera). munis entomology and zoology 5, 792–795. freyn, j., 1877: die flora von süd istrien. verhandlungen der zoologisch-botanischen gesellschaft in wien 27, 241–490. gärdenfors, u., hilton-taylor, c., mace, g. m., rodríguez, j. p., 2001: the application of iucn red list criteria at regional levels. conservation biology 15, 1206–1212. greuter, w., 2004: proposal to conserve the name ophrys speculum (orchidaceae) with a conserved type. taxon 53, 1070–1071. hayek, a., 1933: prodromus florae peninsulae balcanicae 3. repertorium specierum novarum regni vegetabilis 30, 1–472. hr[ak, v., 2000: orchidaceae. in: nikoli], t. 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(ed.), 1998: vorarbeiten zu einer »flora von istrien« 1. carinthia ii 188/108, 535–576. 190 acta bot. croat. 72 (1), 2013 vukovi] n., tommasoni a., d'onofrio t. 647 vukovic et al.ps u:\acta botanica\acta-botan 1-13\647 vukovic et al.vp 14. o ujak 2013 12:56:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees starmühler, w. (ed.), 2004: vorarbeiten zu einer »flora von istrien« 7. carinthia ii 194/ 114, 591–651. starmühler, w. (ed.), 2010: vorarbeiten zu einer »flora von istrien« 13. carinthia ii 200/ 120, 465–524. topi], j., [egulja, n., 2000: floristic and ecological characteristics of the southernmost part of istria (croatia). acta botanica croatica 59, 179–200. soó, r., 1980: ophrys l. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea 5, 344–349. cambridge university press, cambridge. tommasini, m. r., 1873: die flora des südlichsten theiles von istrien bei promontore und medolino. oesterreichische botanische zeitschrift 23, 169–177, 219–227, 257–260. visiani, r., 1842: flora dalmatica 1. apud fridericum hofmeister, lipsiae, 1–252. visiani, r., 1847: flora dalmatica 2. apud fridericum hofmeister, lipsiae, 1–268. visiani, r., 1852: flora dalmatica 3. apud fridericum hofmeister, lipsiae, 1–390. vukovi], n., 2010: rt kamenjak. in: nikoli], t., topi], j., vukovi], n. (eds.), important plant areas of croatia (in croatian), 374–378. prirodoslovno-matemati~ki fakultet, [kolska knjiga, zagreb. vukovi], n., brana, s., miti], b., 2011: orchid diversity of the cape of kamenjak (istria, croatia). acta botanica croatica 70, 23–40. acta bot. croat. 72 (1), 2013 191 ophrys speculum in croatia 647 vukovic et al.ps u:\acta botanica\acta-botan 1-13\647 vukovic et al.vp 14. o ujak 2013 12:56:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 647 vukovic et al.ps u:\acta botanica\acta-botan 1-13\647 vukovic et al.vp 14. o ujak 2013 12:56:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 73 (1), 267–273, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication chromosome number of elatine gussonei (sommier) brullo (elatinaceae) and its distribution on the maltese islands anna kalinka1*, stephen mifsud2, agnieszka popiela3, magdalena achrem1 1 chair of cell biology, university of szczecin, waska 13,71-415 szczecin, poland 2 environment protection directorate, malta environment planning authority, malta 3 department of botany and nature conservation, university of szczecin, felczaka 3c,71-412 szczecin, poland abstract – elatine gussonei (sommier) brullo is an endemic species, with a distribution restricted to the central part of the mediterranean basin (maltese islands, lampedusa, southern part of sicily). this hydrophyte grows in rainwater pools and cavities in karstic limestone. although the morphology has been well studied, no karyological study has been carried out, and hence this work brings the first chromosome data for the maltese-pelago endemic e. gussonei. we have found a diploid number of 54 chromosomes in e. gussonei, which differs from the chromosome number of most of elatine species (2n = 36). additionally, this account gives a recent distribution of the species on the maltese islands. key words: elatine gussonei, chromosome number, phytogeography introduction elatinaceae dum. is a small cosmopolitan family of herbaceous aquatic and semi-aquatic plants and terrestrial shrubs composed of two genera, i.e. elatine l., comprising about 15–20 taxa, and occurring in areas of moderate temperatures in both hemispheres, and bergia l., with about 25 species occurring mostly in tropical areas of the old world, primarily in africa, and also in australia (tucker 1986, leach 1989). with regards to the genus elatine, in europe 10 species are acknowledged: e. triandra schuhr., e. alsinastrum l.; e. hydropiper l.; e. orthosperma dueben; e. hungarica moesz.; e. macropoda guss.; e. campylosperma seub.; e. hexandra (lapierre) dc; e. brochonii clavaud and e. gussonei (sommier) brullo. based on the arrangement of the leaves along the stem, niedenzu (1925, after seubert 1845) proposed the subdivision of the genus elatine into lower subacta bot. croat. 73 (1), 2014 267 * corresponding author, e-mail: akali@op.pl copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 700 kalinka et al.ps u:\acta botanica\acta-botan 1-14\700 kalinka et al.vp 24. o ujak 2014 11:41:21 color profile: generic cmyk printer profile composite default screen genera: the subgenus potamopithys (adanson) seub. (whorled leaves) and the subgenus elatine (hydropiper moesz.) seub. (opposite leaves). the latter subgenus includes two sections: elatinella seub., where the number of stamens is twice that of petals, and triandra seub. (= crypta (nutt. seub.), characterised by an equal number of stamens and petals (tucker 1986). elatine gussonei is one of eight species of the section elatinella, one of which is found in euroasia, six are found in europe (three of them also in north africa) and one in north america (california). elatine gussonei was first found and described by sommier (1908) on the island of lampedusa, under the taxon of elatine hydropiper l. var. gussonei sommier, based on color of corolla, the ratio in sizes of the flower parts and general seed morphology. later, similar plants were also found in malta. both populations were reported to grow in rainwater pools contained in superficial small basins and cavities in karstic limestone (sommier and caruana gatto 1915), commonly known as rock pools. much later, detailed morphological studies were carried out by brullo et al. (1988) resulted in the promotion of the taxon to species rank: elatine gussonei (sommier) brullo, lanfranco, pavone and ronsisvale. however the first record for the maltese islands should be attributed to grech delicata (1853) who recorded elatine macropoda guss. from wied balluta, san �iljan. since all local elatine populations have been identified as e. gussonei, it is most probable that grech delicata (1853) also found e. gussonei – a species which is closely related to e. macropoda. recently e. gussonei was also found in three localities in the south-eastern part of sicily (molnár et al. 2013). the number of chromosomes in elatine gussonei was not previously reported, in contrast to the unequivocal results of chromosome number for other species of the elatine genus. the aim of this paper is to present results of the study on chromosome number of the taxon, as well as the new records of its distribution for the maltese islands. materials and methods the list of sites of elatine gussonei and the distribution map for the maltese are based on published (borg 1927) and unpublished records by one of the authors. specimens were collected from 5 different populations in the maltese islands and were immediately delivered to department of biology, university of szczecin for karyological analysis supervised by three of the authors. because it is a protected species, the necessary permits were obtained from the malta environment and planning authority. roots were treated with 0.05% colchicine (sigma-aldrich, st. luis, usa) solution for 3 hours, followed by a wash of 10 min with ice-cold distilled water. then they were fixed in ethanol : glacial acetic acid, 3 : 1 (v / v) for 24 hours at 4 °c. fixed roots were stored at 4 °c in fresh fixing solution. roots were washed in distilled water, and under stereoscopic microscope the root tips were dissected. each root tip was macerated directly on a microscope slide in a 10 ml mixture of 4% (w/v) pectinase (fluka, buchs, switzerland), 6% (w/v) hemicellulase (sigma-aldrich, st. luis, usa) and 4% (w/v) cellulase (sigma-aldrich, st. luis, usa) in 0.01 m citric buffer for 2 hours at 37 °c in a humidity chamber. root tips were carefully transferred with hypodermic needles to a drop of 45% acetic acid onto a new slide, and 10 ml of 2 % (w / v) aceto-orcein (sigma-aldrich, st. luis, usa) were added. each preparation was covered with a cover glass and stained at 47 °c for 1 hour. during this time aceto-orcein was added 3–5 times. the stain under a cover glass was 268 acta bot. croat. 73 (1), 2014 kalinka a., mifsud s., popiela a., achrem m. 700 kalinka et al.ps u:\acta botanica\acta-botan 1-14\700 kalinka et al.vp 24. o ujak 2014 11:41:21 color profile: generic cmyk printer profile composite default screen replaced with 45% acetic acid. root tips were squashed, and this was followed by gentle heating (5–10 min at 47 °c). preparations were analyzed under an eclipse e600 microscope (nikon, tokyo, japan). images were captured at 1000 × magnification with ccd ds-fi1c camera (nikon, tokyo, japan) and analyzed by means of nis-elements ar3.1 (nikon, tokyo, japan) software. results and discussion phytogeographic study elatine gussonei is in general well scattered throughout the rural areas of the maltese islands, and therefore is not considered a locally rare species (tab. 1, fig. 1). it is specifically acta bot. croat. 73 (1), 2014 269 chromosome number of elatine gussonei tab. 1. the list of localities of elatine gussonei (sommier) brullo in the maltase islands. locality references mainland malta: attard – wied incita sommier and caruana gatto 1915 bir ebbu�a – wied hal-saptan sm, 2006* east of g�ar hasan sm, 2012* g�arg�ur – in-nigret sm, 2006* mellie�a – qammie� sm, 2006* il-mi ieb sm, 2006* l-im�ieba� sm, 2006* coastal cliffs near mistra area sm, 2008* mosta – wied il-g�asel sommier and caruana gatto 1915 tal-wej sm, 2006* wied filep borg 1927 1 rabat – buskett gardens borg 1927 wied il-girgenti haslam and borg 1998 mta�leb sm, 2006* sliema – wied tal-balluta grech delicata 1853 san �iljan – tal-minsija sommier and caruana gatto 1915 �arq �ammiem stevens 1995 pembroke sm, 2006* san pawl il-ba�ar – rocky areas near salini sm, 2006* si��iewi – tal-g�olja borg 1927 san �or� and �ebel ciantar area, limits of fawwara sm, 2010* �urrieq – wied il-bassasa sm, 2011* gozo: munxar – xlendi sommier and caruana gatto 1915 ras il-fekruna sm, 2007* ta’ sannat – ta’ �en� sommier and caruana gatto 1915 wied m�arr ix-xini sm, 2006* * new records (= sites of e. gussonei not found in present literature) 1 demolished to make space for a stone quarry (from wikipedia.org, accessed jun 2012) 700 kalinka et al.ps u:\acta botanica\acta-botan 1-14\700 kalinka et al.vp 24. o ujak 2014 11:41:21 color profile: generic cmyk printer profile composite default screen and restrictedly found in rock pools scattered on coralline limestone mostly situated in rocky valley sides, borders of escarpments or coastal cliffs, and sometimes exposed, sloped rocky ground. most natural rock-basins are not suitable to hold water for long and hence provide the habitat for e. gussonei because water seeps out through cracks and as a result they become void after few days without rain. however, the species often dominates suitable rock pools, forming a dense population composed of thousands of specimens. among the species included in the section elatinella, elatine gussonei is the one most closely related to e. macropoda; a species mainly found in the western and central part of mediterranean basin (popiela and �ysko 2010). many of the morphological differences used since sommier (1908) to distinguish the two species appear to be somewhat unreliable. it turned out that the distinctive characters used by sommier (1908) and sommier et al.(1915) vary considerably according to environmental conditions and as a result they intersect between the species. it was proposed that more important distinguishing differences are the presence of a conspicuous semilunar membrane at the acute curvature of the seed and the shape and number of 'pits' in the reticulation of the seed testa (molnár et al. 2013). karyological analysis a total of 100 metaphase plates were analysed. only well-spread metaphases were included in the study as overlapping chromosomes are the main cause of errors in determining the chromosome number (fig. 2a). the chromosome number in e. gussonei amounts to 270 acta bot. croat. 73 (1), 2014 kalinka a., mifsud s., popiela a., achrem m. fig. 1. distribution of elatine gussonei in the maltese islands. (1 grid box = 1km). 700 kalinka et al.ps u:\acta botanica\acta-botan 1-14\700 kalinka et al.vp 24. o ujak 2014 11:41:23 color profile: generic cmyk printer profile composite default screen 2n = 54 (fig. 2b). this count was confirmed through the detailed analysis of 30 metaphase plates. chromosomes of elatine gussonei are very small in size and for this reason the analysis was much more difficult than expected. chromosomes of elatine gussonei contain appreciable amount of heterochromatin, which is perfectly visible in the nuclei (fig. 2c). however, the chromosomes are so small that it can hardly be stated if the condensed fraction of chromatin occupies the centromeric or telomeric part of chromosomes. it seems likely that at least in some chromosome pairs there are large blocks of heterochromatin in the subcentromeric region (fig. 2d). this is the first report on chromosome number for this species. it was proposed that the basic number for elatine genus is x = 9 (löve and löve 1974, goldblatt 1981, 1985). there are also several polyploid levels in different elatine spp. such as tetraploids, octoploids and dodecaploids (uotila 1974). however, there are contradictory reports for chromosome numbers in the literature. for instance, contandriopoulos et al. (1987) reported that there are 2n = 40 chromosomes in elatine macropoda guss. the same chromosome number of 2n = 40 was reported for elatine hydropiper l. (löve and löve 1974). however, subsequent studies showed that chromosome number in this taxon is actually 2n = 36 (krahulcová 1990). there is an indication that reports of 2n = 40 probably refer to 2n = 36 (uotila 1974) and the counting error arises from the fact that the chromosomes of elatine are very small and hence difficult to count precisely. in present paper the measurements of chromosomes from 30 metaphase spreads were made. the mean length of the chromosome was 0.69 mm, with a minimum value of 0.26 mm and maximum acta bot. croat. 73 (1), 2014 271 chromosome number of elatine gussonei fig. 2. chromosomes and interphase nuclei of elatine gussonei stained with aceto-orcein; a – metaphase spread with overlapping chromosomes, giving the false impression of 36 visible chromosomes; b – metaphase spreads with 54 chromosomes; c – interphase nuclei; dark spots correspond to condensed chromatin; d – metaphase spread; the condensed chromatin at centromeric / subcentromeric regions of some chromosomes are marked with arrows. scale bar = 1 mm. 700 kalinka et al.ps u:\acta botanica\acta-botan 1-14\700 kalinka et al.vp 24. o ujak 2014 11:41:34 color profile: generic cmyk printer profile composite default screen value of 1.12 mm (s = 0.17 mm). the same discrepancies appear in other species: elatine americana (pursh) arn. 2n = 70–72 (probatova and sokolovskaya 1986), elatine hexandra (lapíerre) dc 2n = 72, 108 (jankun 1989, pogan et al. 1990). the most common, as well as the lowest number of chromosomes in species of elatine genus is 2n = 36 as was found in elatine gratioloides a. cunn. (de lange et al. 2004) and elatine triandra subsp. americana (pursh) (löve and löve 1982). acknowledgements this work was partially supported by the national science centre, poland (project no: n n303 470638). references borg, j., 1927: descriptive flora of the maltese islands. government printing office, malta. brullo, s., lanfranco, e., pavone, p., ronsisvalle, g., 1988: taxonomical notes on the endemic flora of malta. giornale botanico italiano 122, 45. contandriopoulos, j., noguet, d., zevaco-schmitz c., 1987: contribution à l’étude dequelques espèces interessantes de corse: cytotaxonomie et comportement écologique. biologie-ecologie méditerranéenne 10, 259–271. goldblatt, p., 1981: index to plant chromosome numbers 1975–1978. in: goldblatt, p. (ed.), monographs in systematic botany from the missouri botanical garden, 5. missouri botanical garden press, st. louis. goldblatt, p., 1985: index to plant chromosome numbers 1982–1983. in: goldblatt, p. 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(elatinaceae). acta societatis botanicorum poloniae 79, 81–86. probatova, n. s., sokolovskaya, a. p., 1986: chromosome numbers of the vascular plants from the far east of the ussr. botanicheskii zhurnal sssr 71, 1572–1575. seubert, m., 1845: elatinarum monographia. academia caesarea leopoldino-carolina nova acta 21, 34–60. sommier, s., 1908: le isole pelagie lampedusa, linosa, lampione e la loro flora. stabilimento pellas, firenze. sommier, s., caruana gatto, a., 1915: flora melitensis nova. stabilimento pellas, firenze. tucker, g. c., 1986: the genera of elatinaceae in the southeastern united states. journal of the arnold arboretum 67, 471–483. uotila, p., 1974: elatine hydropiper l. aggr. in northern europe. memoranda societatis pro fauna et flora fennica 50, 113–123. acta bot. croat. 73 (1), 2014 273 chromosome number of elatine gussonei 700 kalinka et al.ps u:\acta botanica\acta-botan 1-14\700 kalinka et al.vp 24. o ujak 2014 11:41:34 color profile: generic cmyk printer profile composite default screen opce-str.vp acta bot. croat. 71 (1), 177–185, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 short communication where does sedum cepaea l. (crassulaceae) – one of the rarest species of croatian flora – really grow? irena [api]1, vedran [egota2, antun alegro3* 1 department of ecology and silviculture, faculty of forestry, university of zagreb, sveto{imunska 25, p.p. 422, 10002 zagreb, croatia 2 institute for research and development of sustainable ecosystems, jagodno 100a, 10415 novo ^i~e, velika gorica, croatia 3 department of botany, faculty of science, university of zagreb, maruli}ev trg 20/ii, 10000 zagreb, croatia abstract – the distribution of sedum cepaea in croatia is limited to sciophilous or slightly heliophilous forest habitats in the zone dominated by quercus petraea, developed on acidic types of soil above siliceous bedrock. we confirm a finding spot on moslava~ka gora and describe new localities on zrinska gora (central croatia). two s. cepaea populations on nikolino brdo [hill] in topusko disappeared a century ago. key words: sedum cepaea, flora, croatia introduction the genus sedum with 428 species is the largest genus of the stonecrop family (crassulaceae) (eggli 2003). it is almost exclusively confined to the subtropical and temperate regions of the northern hemisphere ('t hart and eggli 2003). in europe, about 55 species occur throughout the region, with the highest diversity in the mediterranean region (webb et al. 1993, 't hart and eggli 2003). in croatia 27 taxa are known (nikoli] 2010). originally an eastern mediterranean plant, sedum cepaea l. (syn. s. galioides all., s. calabrum ten., s. paniculatum lam., s. spathulatum waldst. et kit.) has extended its area of distribution to parts of central and southern europe ('t hart and eggli 2003). it reaches the iberian peninsula only as far as pyrenees and neighbouring areas (castroviejo and velayos 2001), while in france it is spread up to the atlantic (bonnier 1919–1920, fournier 1961). therefore, it is sometimes described as a mediterranean-atlantic plant. sedum cepaea is present in italy, including sicily and sardinia (zángheri 1976, pignatti acta bot. croat. 71 (1), 2012 177 * corresponding author, e-mail: antun.alegro@biol.pmf.hr copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\429 sapic.vp 26. o ujak 2012 14:45:15 color profile: disabled composite 150 lpi at 45 degrees 1982, arrigoni 2010) and corsica (jeanmonod and gamisans 2007). in central europe it is limited to southern switzerland (hess et al. 1970, lauber and wagner 1998), while in germany and austria it escaped from the culture, but has recently been thought to be extinct (lippert 1995, schmeil and fitschen 2003). it can be found in all countries of the balkan peninsula, including croatia (hr[ak 1997), bosnia and herzegovina (beck and mannagetta 1903), montenegro (rohlena 1942), albania (paparisto et al. 1989), serbia (josifovi] 1972, koji] 2007), macedonia (micevski 1998), romania (r�v�rut 1956), bulgaria (jordanov 1970) and greece (strid 2009). the eastern border of its distribution is in western turkey (asia minor) (r�v�rut 1956, eggli 2003, 't hart and eggli 2003,) and in the south it reaches northern africa – tunisia, algeria (bonnier 1919–1920, pottier-alapetite 1979) and libya (jalas et al. 1999). the occurrence of s. cepaea on crete was recently reported by deschâtres and greuter (2001). sedum cepaea requires the least amount of light among sedum species; therefore, it is most abundant in shady places such as the edges of forests and shrubberies, shadowed walls and rocks (lippert 1995). it prefers fresh, nutrient and base rich, lime-deficient, moderately neutral to acidic, humic, stony or sandy clay soil (oberdorfer 1994, lippert 1995). although it usually appears above limestone bedrock (eggli 2003, 't hart and eggli, 2003), it often prefers siliceous rocks (bonnier 1919–1920, fournier 1961, pignatti 1982, castroviejo and velayos 2001, arrigoni 2010). in central europe sedum cepaea grows mostly in lowlands within sciophilous and nitrophilous vegetation of alliarion oberd. (1957) 1962 em. siss 1973 alliance, while in the subalpine belt it is frequent in plant communities dominated by mosses and liverworts (lippert 1995). elsewhere it can be found in woods as well, for example, in serbia it is present in forest communities of the orders qurcetalia pubescentis br.-bl. 1932 (e.g. quercetum frainetto-cerris rudski 1940, quercetum montanum (jov. 1948) ^ernj. et jov. 1953) and fagetalia sylvaticae pawl. 1928 (e.g. fagetum montanum (rudski 1949) jov. 1967, fagetum submontanum mi{i} (1963) 1972) (josifovi] 1972, koji] et al. 1997, koji] 2007). in greece, it has been reported as a species constituting the rodopi beech forest communities (tsiripidis and athanasiadis 2003). there are three known cytotypes of s. cepaea, but they cannot be morphologically distinguished and do not differ in ecological preference or altitudinal range ('t hart 1991, eggli 2003, strid 2009). materials and methods in order to verify the localities where sedum cepaea was recorded in literature and herbaria, field research was performed in the period from 2009 to 2011. the research included three areas of central croatia – nikolino brdo in topusko and moslava~ka gora, as finding spots already known from literature and herbaria, and zrinska gora, as a newly discovered finding spot of s. cepaea. moslava~ka gora is a mountain 489 m a. s. l. in the western part of the croatian pannonian plain, built of igneous (granite) and metamorphic (gneiss) rocks giving rise to brown siliceous soils. forest vegetation covers a large part of the mountain. forests of sessile oak prevail, whereas beech is more common on northern slopes. nikolino brdo is a relatively small hill situated in the town of topusko in banovina region. at the end of the 19th and beginning of the 20th century some of the famous older 178 acta bot. croat. 71 (1), 2012 [api] i., [egota, v., alegro a. u:\acta botanica\acta-botan 1-12\429 sapic.vp 26. o ujak 2012 14:45:15 color profile: disabled composite 150 lpi at 45 degrees croatian botanists (rossi, schlosser and vukotinovi}) collected plant specimens there, which are deposited in the za herbarium. nikolino brdo enjoys preventive protection and has been proposed for categorization as forest park. zrinska gora is a mountain located at the meeting point of two large relief units: the southern edge of the pannonian plain and the northern part of the pre-dinaric space. geologically, it is built out of perm/carbonian schists, sandstones and conglomerates, which are in the lower range covered with younger tertiary deposits. it reaches a height of up to 616 m. most of the massif is covered with deciduous forests of oak, beech and chestnut, whereas the lower slopes feature pastures and arable land (bari^evi] et al. 2009, medak 2009). the climate of the wider area is, according to the croatian state hydro-meteorological office, temperate warm and rainy. the gauss krueger coordinates and altitudes of all finding spots were recorded on field using garmin etrex gps device. in order to describe the habitat preferences of s. cepaea in croatia, soil samples from all finding spots were taken and shadiness of the stands was estimated by the coverage of the tree layer. the soil was sampled at maximum depth of 10 cm. each finding spot was represented with three soil subsamples. soil acidity in distilled water and in solution of potassium chloride was measured using a ph 330i ph meter set. results during the seasons of 2009 and 2010, small populations of sedum cepaea consisting of only a few specimens were found at three localities on zrinska gora (one at bukova kosa and two at jezi~evac) (figs. 1, 2). the populations were noticed in shaded forest stands dominated by sessile oak. an old finding spot of s. cepaea on moslava~ka gora (st. benedict hill) was confirmed during growing season 2010. here, the species grew on more heliophilous habitats such as degraded forest of sessile oak or along forest roads near forest edges. in both cases the populations were more numerous than those found on zrinska gora. the occurrence of s. cepaea on nikolino brdo in topusko was not confirmed during the research in 2010. in addition, in 2011 two new finding spots of s. cepaea were recorded on zrinska gora, again in the same type of forest and with a low number of specimens. on moslav~ka gora, one new finding spot (kleti{te) was found also in 2011, on the edge of the forest, featuring again a high number of specimens. acta bot. croat. 71 (1), 2012 179 sedum cepaea in croatia fig. 1. sedum cepaea; a – inflorescence (zrinska gora), b – basal rosettes (moslava~ka gora), (photo by i. [api}) u:\acta botanica\acta-botan 1-12\429 sapic.vp 26. o ujak 2012 14:45:19 color profile: disabled composite 150 lpi at 45 degrees the gauss krueger coordinates and altitudes of the finding spots on zrinska and moslava~ka gora are summarized in table 1. in the same table, the mean ph values of soil for finding spots found in 2009 and 2010 are shown. 180 acta bot. croat. 71 (1), 2012 [api] i., [egota, v., alegro a. fig. 2. distribution of sedum cepaea in croatia. new and confirmed (�), and old unconfirmed (+) finding spots. localities and data sources quoted in appendix 1. tab. 1. mean ph values of soil from findspots of sedum cepaea in croatia. phh20 – ph measured in soil suspension with distillated water, phkcl – ph measured in suspension with 1m kcl. findspot (name of locality, habitat, gps coordinates, altitude) phh20 phkcl moslava~ka gora (st. benedict hill, degraded forest of sessile oak, 5634779, 505276, 320 m a.s.) 5.0 4.2 moslava~ka gora (st. benedict hill, along forest roads, 5634699, 5053488, 332 m a.s.) 5.3 4.4 zrinska gora (bukova kosa, acido-thermophilic forest of sessile oak, 5611517, 5012903, 403 m a.s.) 4.8 3.7 zrinska gora (jezi~evac, acido-thermophilic forest of sessile oak, 5608568, 5012489, 441 m a.s.;) 5.0 4.0 zrinska gora (jezi~evac, acido-thermophilic forest of sessile oak, 5608726, 5012478, 432 m a.s.) 4.7 3.7 u:\acta botanica\acta-botan 1-12\429 sapic.vp 26. o ujak 2012 14:45:19 color profile: disabled composite 150 lpi at 45 degrees discussion the recent findings of a few large populations of sedum cepaea on zrinska gora raised the issue of distribution of this rare species in croatia. therefore, a literature study, herbarium survey and verification of old, already known localities, were performed. literature study revealed only a few historical records of sedum cepaea in croatia: »morinje, lepetane et perzagno propre cattaro« (visiani 1852, schlosser and vukotinovi] 1869), »circa semling« (schlosser and vukotinovi] 1869) and moslava~ka gora (^abranjski drum, stara stra`a, pleterac) by hru[ka-dell’uomo (1974), the first two of them, however, being no longer within croatian territory. in flora croatica database (nikoli] 2010) beside record from moslava~ka gora, a record for morinje near [ibenik is present (milovi] 2002). in the herbarium of za barely five herbarium sheets with s. cepaea, collected on moslava~ka gora and in topusko (nikolino brdo), were found. first record on distribution of sedum cepaea in croatia was noted by visiani (1852) »in rupestribus umbrosis morinje, lepetane et perzagno propre cattaro, nec non circa much«, and is cited later in schlosser and vukotinovi] (1869). these finding spots refer to the towns of morinj, lepetane and pr~anj near the town of kotor, which were once within the borders of province of dalmatia. today, they are no longer within croatian territory, but in montenegro. the same is also with another finding spot cited by schlosser and vukotinovi] (1869) »circa semling« which refers to the town of zemun in today’s serbia. it took a long time for another quotation of this species in croatia. in 1971 it was found at only one locality on moslava~ka gora within sessile oak forest (ass. festuco drymeiae-quercetum petraeae jankovi} (1968) hru{ka 1974)) (hru[ka-dell’uomo 1974). the old visiani record of s. cepaea in the surroundings of town of [ibenik (at morinje) was not confirmed by milovi] (2002), who actually misinterpreted visiani’s toponym morinje (relating to morinj near kotor in montenegro) as morinje (near [ibenik in croatia) and his misinterpretation entered the flora croatica database (nikoli] 2010) where morinje near [ibenik is cited as finding spot of this species. in addition, a few herbarium specimens of s. cepaea, collected in the town of topusko (nikolino brdo) at the end of the 19th century and on moslava~ka gora at the beginning of the 20th century, are deposited in za herbarium. on nikolino brdo s. cepaea was collected three times: by schlosser in 1871, by vukotinovi} in 1882 and by rossi in 1888 (data from za). it is interesting that schlosser and vukotinovi] (1876) have not included s. cepaea in »bilinar – flora excursioria«. vukotinovi] in shedula (no. 3552) discussed a possible horticultural origin of this population: »in pede monticoli »nikolino brdo« in topusko, copiosum in herbidis inibi ad marginem viae (promenadae), quae a schlammbad ducit ad spiegelbad. sed per hortulanum ante pluros annos semine disjecto spontaneum modo copiosissimum.« some of the specimens collected by vukotinovi} were included in »flora exsiccata austro-hungarica« (no. 617) by kerner, who pointed out (also in shedula) that the population from nikolino brdo was not established through escape from gardens: »da dieses sedum als zierpflanze bei den gärtnern nie eine rolle gespielt hat, ist eine derartige künstliche ansiedelung und einbürgerung aber wenig wahrscheinlich. dagegen spricht der umstand, dass s. cepaea auch in anderen florengebieten gewönlich nur auf sehr beschränkten localitäten angetroffen wird, dafür, dass dasselbe eine in südlichen croatien indigene pflanze ist…«. but hirc (1910) could not find this species again even as soon as acta bot. croat. 71 (1), 2012 181 sedum cepaea in croatia u:\acta botanica\acta-botan 1-12\429 sapic.vp 26. o ujak 2012 14:45:20 color profile: disabled composite 150 lpi at 45 degrees in 1908. he made several excursions there during 1908 looking for s. cepaea and concluded that he was there »too early« in may, or »too late« in august. to summarize, until this research, the only known finding spots of s. cepaea in croatia based on literature and herbarium data were moslava~ka gora and nikolino brdo in topusko, with the existence of the latter being in doubt (appendix 1). since a detailed search for s. cepaea on nikolino brdo in 2010 did not reveal any results, it is obvious that this population has vanished, most probably a century ago. this is probably due to anthropogenic influence, since nikolino brdo has been used an entire century as a recreation place. the population on moslav~ka gora (st. benedict hill), first found during a forest vegetation survey in 1974, was rediscovered in 2010. this population represents the most numerous population of s. cepaea in croatia. the only other population existing currently in croatia is that found in 2009 on zrinska gora by first author. this is the first record of this rare species in zrinska gora and the banovina region. although the flora of the wider area of zrinska gora has been already investigated by [egulja et al. (1998), resulting in a total of 682 taxa, s. cepaea was not found during that research. here the population is much smaller, consisting of only a few specimens at three localities. on both finding spots (moslava~ka gora and zrinska gora, respectively), s. cepaea grows in vegetation belt dominated by sessile oak. on zrinska gora the species lives in the recently described association potentillo micranthae-quercetum petraeae vukeli}, bari~evi} et [api} 2010 (vukeli] et al. 2010). this is a floristically relatively poorer type of acido-thermophilic forest of sessile oak, predominantly developed on southern exposures, at heights between 300 and 500 m. a. s., on luvisol and dystric cambisol. the habitat of the population of s. cepaea on zrinska gora is rather shaded due to denser tree canopy, while on moslav~ka gora the species is present in degraded forest stands and forest edges, characterized by somewhat more light. the noticed habitats are in accordance with those known from the literature, supporting the fact that s. cepaea is one of the most sciophilous sedum species that can easily grow inside forest stands. all measurements of soil acidity indicate that s. cepaea prefers acidic substrata (phh2o from 4,7–5,3). these types of soil are developed above siliceous geological bedrock predominantly present on moslava~ka gora (hru[ka-dell’uomo 1974) and zrinska gora. in central europe the species occurs on acidic soil too (landolt, e. 2010) while, similarly, in serbia, it has been categorized in the transitional group between acidophylous and neutrophylous plants (koji] et al. 1997). taken together, the results of this research suggest that according to current knowledge s. cepaea is an extremely rare species in the croatian flora, represented by only a few small populations, restricted to two mountain areas of central croatia (moslava~ka gora and zrinska gora) (fig. 2). ecologically, it is limited to sciophilous or slightly heliophilous forest habitats in the zone dominated by quercus petraea (300–450 m a.s.), developed on acidic types of soil above siliceous geological bedrock. due to its rareness, there is a definite need for further investigation and protection of this species. moreover if we consider the fact that it is a decoratively interesting plant, it is potentially at risk from collectors. according to iucn criteria (anonymous 2010) s. cepaea could enter the category of near threatened (nt) species in croatia. this is due to there being fewer than ten known, fragmented, localities, as well as to its extinction in one locality. furthermore, the categorization is reevaluated according to the probability of propagule immigration from adjacent populations from the region. 182 acta bot. croat. 71 (1), 2012 [api] i., [egota, v., alegro a. u:\acta botanica\acta-botan 1-12\429 sapic.vp 26. o ujak 2012 14:45:20 color profile: disabled composite 150 lpi at 45 degrees references anonymus, 2010: guidelines for using the iucn red list categories and criteria. version 8.1. standards and petitions subcommittee of the iucn species survival commission. http://intranet.iucn.org/webfiles/doc/ssc/redlist/redlistguidelines.pdf arrigoni, p. v., 2010: flora dellâ isola di sardegna, 2. carlo delfino, roma. bari^evi], d., vukeli], j., [api], i., 2009: ass. polysticho setiferi-fagetum zupan~i~ et al. 2000 in forest vegetation of zrinska gora (croatia). hladnikia 23, 81–91. beck, v., mannagetta, g., 1923: the flora of bosnia, herzegovina and former region novi pazar cont (in serbo-croatian). glasnik zemaljskog muzeja bosni hercegovini 35. bonnier, g., 1919–1920: flore complète illustrée en couleurs de france, suisse et belgique 4. neuchatel, paris, brussels. bonnier, g., 1919–1920: flore complète illustrèe en couleurs de france suisse et belgique, 4. delachaux et niestlé, librarie générale de l’enseignement, j. lebègue, neuchatel. castroviejo, s., velayos, m., 2001: sedum. in: castroviejo, s. 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't hart, h., 1991: evolution and classification of the european sedum species (crassulaceae). flora mediterranea 1, 31–61. tsiripidis, i., athanasiadis, n., 2003: contribution to the knowledge of the vascular flora of ne greece: floristic composition of the beech (fagus sylvatica l.) forests in the greek rodopi. wildenovia 33, 273–297. visiani, r., 1852: flora dalmatica, lipsiae. vukeli], j., bari^evi], d., [api], i., 2010: nomenclatural-phytocoenological analysis of the association potentillo micranthae-quercetum petraeae ass. nova in croatia. haquetia 9, 5–17. webb, d. a., akeroyd, r., t’hart, h., 1993: sedum. in: tutin, t. g., burges, n. a., chater, a. o., edmonson, j. r., heywood, v. h., moore, d. m.,valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea 1, 429–436. cambridge university press, cambridge. zángheri, p., 1976: flora italica, 1 (pteridophyta – spermatophyta). cedam – casa editrice dott. antonio milani, padova. appendix 1. herbarium localities of sedum cepaea in croatia. specimina visa schlosser, j.: ad vias ad termas topuskensis, 1871 (za); vukotinovi}, j.: in pede monticule »nikolino brdo« in topusko, 1882 (za); vukotinovi}, j.: na podru~ju nikolino brdo u topuskom, 1882 (za); rossi, l.: ad pedem monticule ad topusko, 1888 (za); hirc, d.: moslavina, na {umskim ~istinama sv. benedikta (brdo) kod jelovske gornje, 1911 (za); [api}, i.: kleti{te, moslava~ka gora, 2011 (za) acta bot. croat. 71 (1), 2012 185 sedum cepaea in croatia u:\acta botanica\acta-botan 1-12\429 sapic.vp 26. o ujak 2012 14:45:20 color profile: disabled composite 150 lpi at 45 degrees 10 acta bot. croat. 82 (1), 2023 acta bot. croat. 82 (1), 10–19, 2023 coden: abcra 25 doi: 10.37427/botcro-2023-002 issn 0365-0588 eissn 1847-8476 the epiphytic bryophyte succession of buxus sempervirens forests in the fırtına valley, rize (north türkiye) tülay ezer1, mevlüt alataş2, nevzat batan3, hüseyin erata4* 1 niğde ömer halisdemir university, faculty of architecture, department of landscape architecture, 51100, niğde, türkiye 2 munzur university, tunceli vocational school, 62000, tunceli, türkiye 3 karadeniz technical university, faculty of science, department of molecular biology and genetics, 61080, trabzon, türkiye 4 gümüşhane university, kürtün vocational school, 29810, gümüşhane, türkiye abstract – in this study, the epiphytic bryophyte succession of the buxus sempervirens l. forests in fırtına valley (çamlıhemşin-rize, north türkiye), one of the nine biodiversity hotspots in türkiye, was investigated. for this purpose, a total of 60 sampling plots were taken from the live trunks of b. sempervirens trees of different ages. twenty-nine epiphytic bryophyte species were determined (24 mosses and 5 liverworts) within the sample plots. also, six different life form types and four different habitat affinity categories were determined. among them, the mat type life form is in first place with 34.4% and the cortico-saxicolous species are the most common with 51.7%. two-way indicator species analysis (twinspan) classified the epiphytic bryophyte communities on b. sempervirens trunks at the second level into two main clusters (a and b) and three sub-clusters (a1, b1 and, b2). detrended correspondence analysis (dca) axis 1 was interpreted as gradient along the height of the epiphytic habitat (from the lower base to the upper zone) on trunks and the dca axis 2 was interpreted as gradient of moisture (from mesic to xeric). exsertotheca crispa (hedw.) s. olsson, enroth & d. quandt was the species with the highest index of ecological significance (ies) value on the lower bases of the aged trees. species diversity and epiphytic cover in the upper zones were lower than in the basal and middle zones in the study area. while metzgeria furcata, (l.) corda, oxyrrhynchium hians (hedw.) loeske, plagiothecium nemorale (mitt.) a.jaeger, and radula lindenbergiana gottsche ex c.hartm were only found on old trees, ctenidium molluscum (hedw.) mitt. and pseudoleskeella nervosa (brid.) nyholm were only found on middle-aged trees. keywords: community, index of ecological significance, liverworts, mosses, ordination analyses introduction bryophytes, the pioneer plants of different substrate types, are one of the most important component of forest ecosystems (longton 1992, baldwin and bradfield 2005, ezer 2017, mellado-mansilla et al. 2017). bryophytes are poikilohydric organisms whose their moisture content rapidly equilibrating with environmental conditions (green and lange 1994). therefore, they are highly sensitive to environmental factors (schofield 2001). in particular, abiotic ecological factors such as humidity directly or indirectly affect the colonization and the distribution of bryophytes in the epiphytic habitat (mazimpaka and lara 1995, schofield 2001, mishler 2003, mazimpaka et al. 2009). in addition to environmental drought, phorophyte-type, physical and chemical properties of bark characteristics such as rugosity, water retention capacity, bark ph, and dust deposition are also important for the spatial distribution of bryophytes on epiphytic habitats (lara and mazimpaka 1998, mazimpaka et al. 2010, ezer 2017). some studies on the succession of epiphytic bryophyte communities have revealed that the succession gradient of epiphytes is highly complex due to changes in positive and negative interactions among species within epiphytic communities as trees age (mazimpaka et al. 2010, ódor et al. 2013, bargali et al. 2014, ezer 2017, ezer et al. 2019). although phytosociological studies on epiphytic bryophytes in türkiye have made progress in the last decade * corresponding author e-mail: huseyin__erata@hotmail.com succession of epiphytic bryophytes in türkiye acta bot. croat. 82 (1), 2023 11 (alataş et al. 2017, 2021, alataş 2018, can gözcü et al. 2018), few studies have been done on the spatial distributions and community composition of epiphytic bryophytes in successional stages (ezer and kara 2013, ezer 2017, ezer et al. 2019). the present study focuses on the vertical distribution patterns of epiphytic bryophytes and community composition in the successional stages on trunks of buxus sempervirens trees in fırtına valley. this study aimed to reveal the successional trends of bryophyte communities on the epiphytic habitats of boxwood trees and to contribute to bryo-ecological studies in türkiye. the study area the çamlıhemşin (rize, türkiye) district, in which the study area is located, is surrounded by pazar and ardeşen to the north, çayeli, hemşin and i̇kizdere districts to the west, and artvin province yusufeli district to the east. the distance of the district to rize city center is 62 km. the b. sempervirens forests, located in the fırtına valley and within the boundaries of the çamlıhemşin district, are located within the a4 square according to the henderson (1961) grid-square system and are in the colchic zone of the eurosiberian phytogeographic region (anşin 1983, fig. 1). the fırtına valley, which exhibits a unique phytological diversity, hosts many rare species due to the presence of all the main habitats of the region. buxus sempervirens, which has a very wide distribution in the euro-siberian phytogeographic region, forms remarkable communities in the valley (kurdoğlu et al. 2004). moreover, in 1999, the www (world wildlife fund) identified europe’s 100 forest areas valuable in terms of biodiversity and in urgent need of protection (myers et al. 2000). nine of these areas, which are called “hot spots of european forests”, are located in türkiye (satar and güneş 2014). one of these nine hot spots is the fırtına valley. only one study on bryophytes has been conducted in the valley so far (abay et al. 2006). fırtına valley, like the whole of the eastern black sea region, is an area very open to natural disasters such as landslides, rockfalls and avalanches due to the very steep slopes, rainy climatic conditions and, soil cover (tunçel 1990). lithologically, there are units from almost all geological times in the valley (bayrakdar 2006). in türkiye, not rich in current glaciers, kaçkar mountains (3932 m) located to the south of the fırtına valley are one of the regions where current glaciers are found (çiner 2003). the kaçkar mountains, the verçenik (üçdoruk) mountain, the bulut mountains and, the altınparmak mountains are mountain ranges that limit the study area in the sw-ne direction. in the valley, which is also very rich in terms of rivers, fırtına stream is divided into branches at various degrees and forms the smaller tunca, hala, palovit, haçivanak and hemşin creeks. these creeks also forked among themselves and surround the valley like a net system. fig. 1. the fırtına valley where epiphytic bryophyte species were investigated (a), its location in türkiye (b) and in the henderson’s grid system (1961) (c). ezer t., alataş m., batan n., erata h. 12 acta bot. croat. 82 (1), 2023 the forest formation, which starts at 200 m in the north of the study area, is very rich in terms of under-forest flora, depending on the oceanic and temperate climatic conditions in all seasons. this forest formation, which is dominated by broadleaf trees, loses its colchic feature with the increase in altitude and gives way to mixed forests and then to coniferous forests (özçağlar et al. 2006). the vegetation in the fırtına valley shows a distinct difference from other valleys, especially with its particular forest formations and flora richness. there are basically three types of forest formation such as alluvial forests, hardwood forests found in river floodplains and regularly flooded for a portion of the growing season; boxwood forests; and old growth forest formations in the fırtına valley (kurdoğlu et al. 2004). the b. sempervirens forests spread along the fırtına stream and its tributaries, between 200-1500 meters of the study area. although these forests are seen along the streams, they are also found in large groups on the valley slopes. these forests are widely found between 900-1300 meters along the çamlıhemşin-meydan road, in şimşirlik place, within the gito forests and palovit valley. the study area generally has a temperate oceanic climate. the annual average precipitation is 2192 mm and, the annual average temperature is 8.3 ºc in çamlıhemşin. the hottest month of the year is august and the coldest is january. the absence of a dry season indicates that the fırtına valley is under the influence of an oceanic climate (akman 2011). materials and methods field sampling procedures the epiphytic bryophyte specimens were collected from the living trunks of b. sempervirens in fırtına valley during field studies in 2020. the locality details are given in tab. 1. a total of 60 sampling plots were taken from the trunks of 20 boxwood trees of different ages in the valley. spatial distributions and community structures of epiphytic bryophytes in successional stages on boxwood trees were investigated according to tree diameter at breast height (dbh) and tree age. the trees were divided into three age classes using an indirect method as young (dbh 20-35 cm, 21 plots), middle-aged (dbh 40-50 cm, 18 plots), and aged trees (dbh 60-80 cm, 21 plots). each boxwood tree was divided into the lower base zone (0 40 cm from the ground), the middle zone (40 120 cm), and the upper zone (120 180 cm) as proposed by moe and botnen (2000). sampling plots from tree zones were defined by 20 × 20 cm2, determined according to species diversity on the living trunks of b. sempervirens. in the present study the branches of the boxwood trees did not provide sample plots, only the trunks. the percentage cover of the species within the each sample plot was visually estimated and ecological data such as humidity, aspect and light of the epiphytic habitat were recorded. the nomenclature of the epiphytic bryophyte species determined within the sample plots follows ros et al. (2013) and hodgetts et al. (2020) (tab. 2). habitat affinity types of bryophytes were established following mazimpaka and lara (1995) and draper et al. (2003). life forms of the taxa were determined according to mägdefrau (1982). voucher specimens were deposited in the herbarium of niğde ömer halisdemir university. data analysis the relative frequency of each taxon in the sample plots was determined by the index of ecological significance (ies) described by lara and mazimpaka (1998), albertos et al. (2001) and mazimpaka et al. (2009). the formula used to calculate the ies values for each taxon is as follows: ies = f (1 + c) c = σ ci / x where f is the relative frequency (100 x / n), and c is the cover of the taxon (σ ci / x), while x represents the number of sample plots containing the taxon, n is total number of sample plots, and ci is cover class assigned to the taxon in each sample plot. cover classes of taxa were established using the six-point lara and mazimpaka (1998) scale: [0.5 (< 1%), 1 (1-5%), 2 (625%), 3 (26-50%), 4 (51-75%) and, 5 (76-100%)]. in addition, the ies values were combined in the following abundance classes: very scarce (< 25), scarce (26-50), moderately abundant (51-100), abundant (101-200), and dominant (> 200). tab. 1. sampling localities from where epiphytic bryophyte specimens were collected and their characteristics. localities altitude (m) date gps coordinates 1 çat valley, doğa village 1275 17.05.2020 n 40º51’48.95” e 40º55’58.08” 2 between çatköy and meydanköy 1231 17.05.2020 n 40º51’59.44” e 40º55’46.26” 3 meydanköy exit 1176 17.05.2020 n 40º52’13.82” e 40º55’37.52” 4 meydanköy 1108 26.08.2020 n 40º52’48.29” e 40º55’43.37” 5 meydanköy entrance 1061 26.08.2020 n 40º53’20.49” e 40º55’51.11” 6 meydanköy, pul place 1026 26.08.2020 n 40º53’40.83” e 40º56’31.71” 7 gito plateausoutheast slopes 1018 09.10.2020 n 40º54’18.08” e 40º56’52.30” 8 between zilkale and meydanköy 1004 09.10.2020 n 40º54’27.30” e 40º56’53.71” 9 zilkale place 956 09.10.2020 n 40º54’47.61” e 40º56’52.38” succession of epiphytic bryophytes in türkiye acta bot. croat. 82 (1), 2023 13 study area (tab. 2). exsertotheca crispa (hedw.) s.olsson, enroth & d.quandt is the most common species in the b. sempervirens forests of fırtına valley. ecological results six different life forms were determined. among them, the mat life form prevailed (34.4%), followed by the weft life form (24.1%). the dendroid life form was negligible (3.4%) (tab. 2). in addition, four different habitat affinity categories as cortico-saxicolous, indifferent, customary epiphyte and preferentially not corticolous were determined belonging to the species. while cortico-saxicolous species were the most common with 51.7% within the sample plots, the preferentially not corticolous type habitat affinity is least common with one species (tab. 2). lower base zone the spatial distributions and community structures analyses showed that a total of 11 species, all of them are mosses, were found on the base zone of young boxwood trees (dbh 20-35 cm). among of the mosses e. crispa was the most here, twinspan and decorana (hill 1979, seaby and henderson 2007) were used to explore the community composition and spatial patterns of epiphytic bryophyte communities and their relationship with the associated environmental factors of the epiphytic habitat. in this context, twinspan and decorana were applied to the matrix of cover in 60 sample plots according to the computer program cap (community analysis package-5) of seaby and henderson (2007). results floristical results twenty-nine species belonging to 17 families and 25 genera were determined as a result of the identification of 362 specimens. among them 24 are mosses (21 pleurocarpous and 3 acrocarpous), and 5 are liverworts. neckeraceae (6 species, 20.6%) and brachytheciaceae (5 species, 17.2%), both pleurocarpous moss families, are the most species-rich families found in epiphytic habitats on boxwood trees in the tab. 2. list of epiphytic bryophyte species found on the buxus sempervirens trees, their families and life form types and affinity for epiphytic habitats (mägdefrau 1982, mazimpaka and lara 1995, draper et al. 2003). species family life form affinity for epiphytic habitats frequency (%) mosses alleniella besseri (lobarz.) s.olsson, enroth & d.quandt neckeraceae fan cortico-saxicolous 65 alleniella complanata (hedw.) s.olsson, enroth & d.quandt neckeraceae fan cortico-saxicolous 76.6 anomodon viticulosus (hedw.) hook. & taylor anomodontaceae tail cortico-saxicolous 3.3 brachythecium rutabulum (hedw.) schimp. brachytheciaceae weft preferantially not corticolous 5 ctenidium molluscum (hedw.) mitt. myuriaceae weft indifferent 3.3 exsertotheca crispa (hedw.) s.olsson, enroth & d.quandt neckeraceae fan cortico-saxicolous 93.3 fissidens serrulatus müll.hal. fissidentaceae fan indifferent 5 homalia trichomanoides (hedw.) brid. neckeraceae fan cortico-saxicolous 16.6 hypnum cupressiforme hedw. hypnaceae weft indifferent 20 isothecium alopecuroides (lam. ex dubois) isov. lembophyllaceae mat cortico-saxicolous 28.3 leucodon sciuroides (hedw.) schwägr. leucodontaceae tail cortico-saxicolous 33.3 orthotrichum pumilum sw. ex anon. orthotrichaceae cushion customary epiphyte 30 oxyrrhynchium hians (hedw.) loeske brachytheciaceae weft indifferent 1.6 palamocladium euchloron (müll.hal.) wijk & margad. brachytheciaceae tail cortico-saxicolous 15 plagiothecium nemorale (mitt.) a. jaeger plagiotheciaceae mat indifferent 1.6 pseudanomodon attenuatus (hedw.) ignatov & fedosov neckeraceae mat cortico-saxicolous 31.6 pseudoamblystegium subtile (hedw.) vanderp. & hedenäs amblystegiaceae weft cortico-saxicolous 6.6 pseudoleskeella nervosa (brid.) nyholm pseudoleskeellaceae mat cortico-saxicolous 1.6 pterigynandrum filiforme hedw. pterigynandraceae tail cortico-saxicolous 6.6 sciuro-hypnum flotowianum (sendtn.) ignatov & huttunen brachytheciaceae mat cortico-saxicolous 35 sciuro-hypnum populeum (hedw.) ignatov & huttunen brachytheciaceae weft indifferent 3.3 thamnobryum alopecurum (hedw.) gangulee neckeraceae dendroid indifferent 5 thuidium delicatulum (hedw.) schimp. thuidiaceae weft indifferent 6.6 ulota crispa (hedw.) brid. orthotrichaceae cushion customary epiphyte 31.6 liverworts frullania dilatata (l.) dumort frullaniaceae mat cortico-saxicolous 10 frullania tamarisci (l.) dumort. frullaniaceae mat cortico-saxicolous 8.3 metzgeria furcata (l.) dumort. metzgeriaceae mat indifferent 5 radula complanata (l.) dumort radulaceae mat customary epiphyte 51.6 radula lindenbergiana gottsche ex c. hartm. radulaceae mat customary epiphyte 1.6 ezer t., alataş m., batan n., erata h. 14 acta bot. croat. 82 (1), 2023 frequent and the most dominantm with the highest ies value (271). isothecium alopecuroides was co-dominant with 214 ies values. homalia trichomanoides and sciuro-hypnum flotowianum were abundant species with 171 and 143 ies values on the lower base of trunks of b. sempervirens. brachythecium rutabulum, s. populeum and thamnobryum alopecurum which have the lowest ies values (43) were scarce on the lower base (tab. 3). whereas the weft life form was the most dominant (36.3%) in the base zone, the life forms mat and fan were co-dominant (27.2%). also, the cortico-saxicolous type habitat affinity of the species was the most common with 54.5% on the base zones of young boxwood trees. fifteen mosses were collected from the lower base of the middle-aged b. sempervirens (dbh 40-50 cm). among them, e. crispa (266 ies value) and h. trichomanoides (216 ies value) were the two most dominant species. sciuro-hypnum flotowianum was the most abundant on the base zones of the middle-aged trees with the 183 ies value. anomodon viticulosus, pseudoleskeella nervosa and s. populeum were scarce with the lowest ies values (33). moreover, ctenidium molluscum and p. nervosa were only found on the lower bases of middle-aged trees (tab. 3). while the fan life form is the most dominant (33.3%), the life forms mat and weft were co-dominant (26.6%) on the lower base of middle-aged boxwood trees. cortico-saxicolous type affinity of epiphytic habitats (60%) were the most common on the basal zone of middle-aged trees. twelve species (11 mosses, one liverwort) were determined on the lower bases of aged boxwood trees (dbh 60-80 cm). exsertotheca crispa was still the most frequent and the most dominant with the highest ies value (371). this value is also the highest among all tree-size groups (tab. 3). while, hypnum cupressiforme, i. alopecuroides, pseudanomodon attenuates and, s. flotowianum were abundant, fissidens serrulatus, oxyrrhynchium hians and, plagiothecium tab. 3. index of ecological significance (ies) values in each tree-size groups according to tree diameter at breast height (dbh) and tree age at lower base zone, middle zone and upper zone. species young trees (dbh 20–35 cm) middle-aged trees (dbh 40–50 cm) aged trees (dbh 60–80 cm) lower base middle zone upper zone lower base middle zone upper zone lower base middle zone upper zone mosses alleniella besseri – 328 271 50 316 233 – 314 300 alleniella complanata 100 328 285 115 266 250 71 342 342 anomodon viticulosus – 43 – 33 – – – – – brachythecium rutabulum 43 – – 50 – – 57 – – ctenidium molluscum – – – 67 – – – – – exsertotheca crispa 271 314 357 266 216 366 371 314 328 fissidens serrulatus – – – 67 – – 28 – – homalia trichomanoides 171 – – 216 50 – 57 – – hypnum cupressiforme 71 71 – – – – 128 114 28 isothecium alopecuroides 214 43 – 130 33 33 171 85 – leucodon sciuroides – – 257 – 50 300 – – 300 orthotrichum pumilum – – 200 – – 100 – 28 200 oxyrrhynchium hians – – – – – – 28 – – palamacladium euchloron – 128 – – 150 – – 85 28 plagiothecium nemorale – – – – – – 28 – – pseudanomodon attenuatus 85 128 71 100 150 33 114 57 – pseudoamblystegium subtile – 28 – – 33 – – 43 28 pseudoleskeella nervosa – – – 33 – – – – – pterygynandrum filiforme – 71 – – – – – 57 – sciuro-hypnum flotowianum 143 86 28 183 116 33 128 28 – sciuro-hypnum populeum 43 – – 33 – – – – thamnobryum alopecurum 43 – – 83 – – – – – thuidium delicatulum 85 – – 100 – – – – – ulota crispa – – 200 – – 167 – – 200 liverworts frullania dilatata – – 57 – – 33 – 28 57 frullania tamarisci – – – – 33 – – 86 43 metzgeria furcata – – – – – – – 57 28 radula complanata – 200 114 – 133 100 71 171 143 radula lindenbergiana – – – – – – – 28 – succession of epiphytic bryophytes in türkiye acta bot. croat. 82 (1), 2023 15 nemorale usually not epiphytic, were scarce with the lowest ies values (28). the mat life form was the most dominant with 41.6% and cortico-saxicolous species (50%) were still the most common on base zones of aged b. sempervirens. middle zone twelve species (11 mosses, one liverwort) were determined on the middle zones of the young boxwood trees. alleniella besseri and a. complanata were the most frequent and the most dominant with the highest ies values (328). while e. crispa was co-dominant with 314 ies values, radula complanata (200), p. attenuatus (128) and, palamacladium euchloron (128) were abundant on the middle parts of the trunks of the young b. sempervirens (tab. 3). the mat life form with 33.3% and the cortico-saxicolous type habitat affinity with 83.3% were still most dominant on the middle parts of trunks of young boxwood trees. twelve species were collected from the middle zones of the middle-aged boxwood trees. alleniella besseri was the most dominant with the highest ies values (316). alleniella complanata and e. crispa were co-dominant, both with 266 ies values. frullania tamarisci, i. alopecuroides and, pseudoamblystegium subtile were scarce with the same ies values (33) on the middle zones of middle-aged trees (tab. 3). the life form mat was the most dominant with 41.6% and cortico-saxicolous species were conspicuously the most common with the rate of 91.6% on middle parts of the middle-aged b. sempervirens. a total of sixteen species, five of which were liverworts, were determined in the middle zones of the aged boxwood trees. all of the liverworts, which were determined in the epiphytic habitats of the boxwood forests, were found on the middle parts of the old trees. while a. complanata was the most frequent and the most dominant, with the highest ies value (342), e. crispa and a. besseri were co-dominant with the same ies values (314) (tab. 3). the mat life form (50%) and the cortico-saxicolous type affinity (68.75%) were the most dominant on the middle zones of aged trees. upper zone ten species were found on the upper zones of young trees. among them, eight were mosses and, two were liverworts. exsertotheca crispa was still the most frequent and the most dominant with the highest ies value (357). alleniella complanata, a. besseri and, leucodon sciuroides were co-dominant with the higher ies values (>200) on the upper zones of the young b. sempervirens. while orthotrichum pumilum (ies value 200), ulota crispa (200) and, r. complanata (114) were abundant on these zones, s. flotowianum (28) was scarce with the least ies value (tab. 3). the mat life form (40%) and the habitat affinity type cortico-saxicolous (70%) were the most common on the upper parts of young trees. eleven species (nine mosses, two liverworts) were collected from upper zones of the middle-aged boxwood trees. while e. crispa was still the most frequent and the most dominant with the highest ies value (366), a. besseri, a. complanata and, l. sciuroides were co-dominant (> 200). and also, u. crispa (167) was abundant on the upper zones of the middle-aged b. sempervirens (tab. 3). the mats (45.45%) and cortico-saxicolous species (72.72%) were the most dominant. a total of thirteen species were determined on the upper zones of the old b. sempervirens. among them, four were liverworts and nine were mosses. alleniella complanata was the most frequent and the most dominant with the highest ies value (342). while a. besseri (ies value 300), e. crispa (328) and, l. sciuroides (300) were co-dominant with the higher ies values, o. pumilum (200), r. complanata (143) and, u. crispa (200) were abundant on the upper parts of fig. 2. classification of twinspan based on the matrix of 29 epiphytic bryophyte species over 60 sample plots. ezer t., alataş m., batan n., erata h. 16 acta bot. croat. 82 (1), 2023 aged boxwood trees (tab. 3). the life form mat (30.7%) and the habitat affinity type cortico-saxicolous (53.8%) were the most common again on the upper parts of old trees. twinspan classification twinspan classified the epiphytic bryophyte communities on the trunks of b. sempervirens in the fırtına valley at the second level into two main clusters (a and b) and three sub-clusters (a1, b1 and, b2) (fig. 2). these main and sub-clusters were named according to the dominant, co-dominant and abundant species which were the distinctive species within the communities. the main cluster, a, occurred in lower-base communities and it was characterized by dominant species e. crispa and h. trichomanoides, co-dominant i. alopecuroides and abundant p. attenuatus and s. flotowianum. the second main cluster, b, occurred in middle and upper zone communities. cluster b was characterised by dominant species a. besseri and a. complanata, co-dominant e. crispa and l. sciuroides, abundant species o. pumilum, p. euchloron, r. complanata, and u. crispa. epiphytic bryophyte communities the a1 community was named exsertotheca crispa isothecium alopecuroides due to the frequency, constancy and, dominancy of these species within the lower-base community. both e. crispa and i. alopecuroides had the highest ies value on the lower bases of all tree-size groups (young, middle-aged and old boxwood trees) (tab. 3). the community was represented with 19 moss species in a total of 20 sample plots. moderately abundant r. complanata was the only liverwort in the lower-base community. exsertotheca crispa-isothecium alopecuroides community was co-dominated by h. trichomanoides. in this community, p. attenuatus and s. flotowianum were also abundant. while the dominant life forms within the community were weft and mat (31.5%), cortico-saxicolous species (47.3%) were dominant and indifferent type affinity was also co-dominant (42.1%). the b1 community was named alleniella complanataexsertotheca crispa according to its dominant and co-dominant species. it was represented by 19 species (15 mosses, 4 liverworts) in a total of 17 sample plots from the middle parts of the young, middle-aged and old boxwood trees. the liverwort r. complanata was still present in the middle parts of the trunks with relatively high ies values. also, p. euchloron was abundant in the community b1 on the middle zones, particularly of young and middle-aged trees particularly (tab. 3). while mats were the most dominant with the rate of 42.1% within the middle parts of community b1, corticosaxicolous species were the most common with 63.1%. the b2 community was named alleniella besseri leucodon sciuroides according to its co-dominant species. this community, consisting of 17 species (13 mosses and four liverworts), was found on the upper zones of boxwood trees. the community b2 was represented by a total of 23 sample plots. orthotrichum pumilum, r. complanata, and u. crispa were abundant species in the upper zones community. while mats were the most dominant with the rate of 41.1% within the middle parts of community b2, the cortico-saxicolous type habitat affinity was the most common with 70.5%. decorana ordination decorana grouped the sample plots on axis 1 and axis 2 according to the similarity and the environmental gradients (fig. 3). while the dca axis 1 was interpreted as fig. 3. the relationship between the three epiphytic bryophyte groups generated after the application of twinspan classification technique on 60 sample plots and the distribution of the groups along the environmental gradient on the first and second axes of decorana. a–1: exsertotheca crispa-isothecium alopecuroides, b–1: alleniella complanata-exsertotheca crispa, b–2: alleniella besseri-leucodon sciuroides, axis 1: the gradient of height of the epiphytic habitat, axis 2: the gradient of moisture. succession of epiphytic bryophytes in türkiye acta bot. croat. 82 (1), 2023 17 gradient along the height of the epiphytic habitat (from the lower base to the upper zone) on trunks, the dca axis 2 was interpreted as gradient of moisture (from mesic to xeric) (fig. 3). discussion when the epiphytic habitats on the boxwood trees in the fırtına valley were examined in terms of species diversity and species composition, the lower bases and the middleparts were the richest zones with equal numbers of species (19 species), while the upper zones contain 17 epiphytic species. large pleurocarp mosses such as e. crispa, h. trichomanoides, and i. alopecuroides were common on the basal parts of the trunks of b. sempervirens. these strong competitor members of pleurocarpous type mosses are more sensitive to drought, have a faster growth habit than acrocarpous mosses and spread horizontally in a carpet-like appearance on the substratum (schofield 2001, ezer 2017). the basal parts of the trunks are usually more humid and more nutrient rich than the other parts (middle and upper) due to soil proximity. therefore, basal zones allow early establishment and rapid colonization of bryophytes due to higher water retention capacity, higher soil humidity, low evapotranspiration rate and low insolation (lara and mazimpaka 1998, mazimpaka et al. 2009, ezer and kara 2013, ezer 2017). in this respect, the lower base parts of the trees can be considered an extension of the forest floor (groundlayer) environment. for this reason, species such as b. rutabulum, c. molluscum, o. hians, p. nemorale, p. nervosa, s. populeum, t. alpecurum, thuidium delicatulum, and f. serrulatus, which usually grows on the soil, were encountered only in this part. this caused the weft life form and indifferent type habitat affinity to co-dominate with the mats and cortico-saxicolous species were the strong competitor of robust pleurocarpous in basal parts of boxwood trees. alleniella besseri, a. complanata and e. crispa were most dominant on the middle zones of the boxwood trees. also, the mesophytic species p. euchloron, which was not present in the base zone, was abundant in the middle zone. therefore, p. euchloron can be considered a characteristic and distinctive species of the communities in the middle zones. the customary epiphyte xerophytic species o. pumilum, which in the present study was generally abundant in the upper zones, in the present study was found for the first time only in the middle zones of old trees. as the middle and upper parts of the trunks move away from the soil effect, they are periodically exposed to higher insolation and desiccation that makes colonization of species on epiphytic habitats difficult (moe and botnen 2000). therefore, as in the present study, small cushion-type mosses which have xerophytic characters such as orthotrichaceae members and photophilous or heliophilous species such as a. besseri, a. complanata, and l. sciuroides are most common species within the middle and upper zone communities. mat type life forms were predominant in the middle parts of old trees, due to the presence of cortico-saxicolous liverworts. although mesoscale climatic factors such as seasonal climatic variables are among the most important determinants of bryophyte species richness and species diversity, forest structure and habitat characteristics are also important for community compositions and spatial distributions of epiphytic bryophytes on epiphytic habitats (medina et al. 2014, ezer 2017). species diversity and epiphytic cover on the upper zones were found to be lower than in the base and middle zones. the xerophytic robust pleurocarpic species l. sciuroides was dominant on the upper parts of all tree-size groups. besides a. besseri, a. complanata and e. crispa there were other dominant pleurocarpic species in the upper zones of boxwood trees in the fırtına valley. therefore, weak competitor species such as liverworts frullania dilatata and metzgeria furcata and small cushion-type mosses (such as o. pumilum and u. crispa) remain under these large pleurocarpous species and decreased or disappeared from the epiphytic habitats in the valley. some studies on the succession of epiphytic bryophyte communities have demonstrated that variables of the epiphytic bryophyte composition in the successional stages are closely related to tree age, trunk height (basal, middle and upper zones) and bark characteristics (lara and mazimpaka 1998, mazimpaka et al. 2010, ódor et al 2013, bargali et al. 2014, ezer 2017). however, in the present study, trunk height rather than tree age and bark characteristics were effective in the variability of epiphytic bryophyte composition in the per successional stage. hygrophytic pleurocarpous species that usually grow on soil, such as b. rutabulum, c. molluscum, f. serrulatus, o. hians, p. nemorale p. nervosa, s. populeum, t. alpecurum, and t. delicatulum were particularly the pioneer colonizers in the early successional stages on the basal parts particularly of middle aged b. sempervirens. while the xerophytic small cushions o. pumilum and u. crispa were the pioneer colonizers in the early successional stages, the large pleurocarpous l. sciuroides was a secondary colonizer in the advanced stages on the upper parts of all tree-size groups. in addition, cortico-saxicolous species p. euchloron, a characteristic and distinctive species on the middle zones, was pioneer colonizer in the early successional stages on the middle parts of all tree-size groups. in this study, a. besseri, a. complanata, and e. crispa were other colonizers in the advanced successional stages on the middle zones. considering the morphological physiognomy of the bryophyte communities on trunks of b. sempervirens in the fırtına valley; all communities were dominated by large pleurocarpous mosses. e. crispa was in particular the most constant and the most dominant within all communities in the b. sempervirens forests. in sum, the succession of the epiphytic bryophyte communities of the boxwood forests in the fırtına valley has reached the climax. references abay, g., uyar, g., çetin, b., keçeli, t., 2006: the moss flora of the communities of buxus sempervirens l. in fırtına valley (çamlihemşin, rize). süleyman 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pisces conservation ltd., lymington, uk. schofield, w.b., 2001: introduction to bryology. the blackburn press, new jersey. tunçel, h., 1990: avalanches as a natural environmental problem and avalanche events in turkey. atatürk culture, language and history high institution, geography science and practice branch. coğrafya araştırmaları dergisi 1, 43–70 (in turkish). 625 pise and gaikwad.vp acta bot. croat. 72 (2), 197–209, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/v10184-012-0024-6 oxidative stress and antioxidant indices of the marine red alga porphyra vietnamensis navnath m. pise1,*, dattatry k. gaikwad2, tanaji g. jagtap1 1 biological oceanography division, national institute of oceanography, council of scientific and industrial research, dona paula, goa 403004, india 2 botany department, shivaji university, kolhapur, maharashtra 416004, india abstract – oxidative stress and antioxidant defence systems were assessed in a marine red alga porphyra vietnamensis tanaka et pham-hoang ho, from india. lipid peroxidation (lpx) and hydrogen peroxide (h2o2) were measured as oxidative stress markers. antioxidant defences were measured as catalase (cat), glutathione s-transferase (gst) and ascorbic acid (asa), in order to understand their dissimilarity with respect to environmental conditions (pollution levels) from selective locations along the central west coast of india. levels of lpx, h2o2, cat and gst were significantly higher in samples collected from dona paula than in samples from malvan and kunkeshwar, while a lower concentration of asa was found in samples from dona paula. heavy metals such as cd, pb and hg in higher concentrations in these areas than in other sites were also observed. variation of oxidative stress indices in response to the accumulation of heavy metals within p. vietnamensis could be used as molecular biomarkers for the assessment and monitoring of environmental quality in ecologically sensitive marine habitats. key words: porphyra vietnamensis, lipid peroxidation, antioxidant, biomarker, oxidative stress introduction natural antioxidants are found in some vegetables, fruits and a variety of other foods (moon and shibamoto 2009). many researchers reported the occurrence of various antioxidants in seaweeds, including polysaccharides, dietary fibers, minerals, proteins, amino acids, vitamins, polyphenols and carotenoids (burtin 2003). seaweeds produce antioxidants to counteract environmental stresses (lesser 2006). in the intertidal zones of rocky beaches around the world, marine algae are exposed to constantly fluctuating and extreme abiotic conditions in, for example, temperature, salinity, ph, and heavy metal pollution, acta bot. croat. 72 (2), 2013 197 * corresponding author, e-mail: pisenavnath@gmail.com copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. resulting in intermittent intracellular oxidative stress conditions developed by the accumulation of reactive oxygen species (ros) (davison and pearson 1996, mittler 2002, pinto et al. 2003). in biological systems, reactive oxygen species (ros) such as singlet oxygen (1o2), hydrogen peroxide (h2o2), superoxide anion radical (o2–) and hydroxyl radical (oh–) are produced in normal metabolic pathways, as well as from exposure of different xenobiotic substances (halliwell and gutteridge 2001). reactive oxygen species are produced directly by excitation of o2 and subsequent formation of singlet oxygen, or by the transfer of electrons to o2, which results in the formation of superoxide radicals, hydrogen peroxide (h2o2), or hydroxyl radicals, respectively (baker and orlandi 1995). marine algae are a fundamental part of marine food webs, since they are responsible for most of coastal primary production (lobban and harrison 1994). adverse effects on macroalgae caused by natural or anthropogenic phenomena can directly or indirectly affect organisms at higher trophic levels and, finally, the integrity of entire ecosystems (medina et al. 2005). marine algae protect themselves against such reactive oxygen, to some extent by developing anti-oxidant defence systems, which constitute both enzymatic and non-enzymatic biomolecules, such as superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), reduced glutathione (gsh), and ascorbic acid (asa) (halliwell and gutteridge 2001). marine algae or seaweeds have been found to be notable bioindicator species in environmental pollution studies (sawidis et al. 2001, conti and cecchetti 2003, moacir et al. 2008). brown algae, for instance, accumulate considerable quantities metals due to the presence of negatively charged polysaccharides (salgado et al. 2005). the aim of this study was to examine the various oxidative stress and antioxidant indices in porphyra vietnamensis, from the central west coast of india, arabian sea. this is the first known study applying oxidative stress indices to p. vietnamensis, a potential model for evaluating marine environmental health. materials and methods site description algal samples (p. vietnamensis) were collected from the central west coast of india (fig. 1). two sites were selected from the coast of maharashtra: malvan (16°03’47.5” n, 73°27’22.5”e) and kunkeshwar (16°20’ 02.4” n, 73°31.3”e), which is a cliff-locked beach. from goa, one site was selected: dona paula (15°51’n, 73°53’e) which receives various effluents from the zuari estuary. sample collection the red alga p. vietnamensis (family bangiaceae) is common in the intertidal zone of india. fresh fronds of p. vietnamensis were collected from the intertidal zones at the three sites during low tides in april 2011. whole fronds with holdfast were handpicked, washed in seawater and then in ice cold distilled water, and immediately frozen in liquid nitrogen. 198 acta bot. croat. 72 (2), 2013 pise n. m., gaikwad d. k., jagtap t. g. physico-chemical parameters physico-chemical parameters at each site were measured during the study period (tab. 1). surface water salinity was measured with a portable salinity refractometer (atago, s/mill, japan). the ph of the surface water was measured by a scan microprocessor-based pocket ph tester (yk-35425-10, oakton, malaysia). surface water temperature was noted using a mercury thermometer at the time of collection. metal analysis metal contents were analyzed by digesting 0.5 g of dry algal powder in 20 ml of concentrated hno3 in a beaker covered with a watch glass. after complete digestion, the samples were diluted to 100 ml and filtered through whatman no.1 filter paper (merck, mumbai, india). the acid digest was analyzed for heavy metals such as cadmium (cd), lead acta bot. croat. 72 (2), 2013 199 oxidative stress and antioxidant indices in porphyra vietnamensis fig. 1. sampling sites for porphyra vietnamensis collected from the central west coast of india, arabian sea. tab. 1. physico-chemical parameters of water bodies in different locations during sampling periods parameter malvan kunkeshwar dona paula water temp. (°c) 27.5 ± 0.86 26.3 ± 2.20 25.36 ± 0.60 ph 8.06 ± 0.67 7.96 ± 0.73 8.10 ± 0.37 salinity (psu) 20.6 ± 1.50 25.3 ± 3.21 22.3 ± 4.93 (pb) and mercury (hg) using atomic absorption spectrophotometry (analyst 300 perkin-elmer, waltham, ma, usa; aoac 2005). oxidative stress indices lipid peroxidation lipid peroxidation (lpx) was measured according to the malondialdehyde (mda) content, a product of lpx estimated by the thiobarbituric acid (tba) reaction (heath and packer 1968). a fresh algal sample (0.5 g) was homogenized in 5 ml of 10% (w/v) trichloro acetic acid (tca), and the homogenate was centrifuged at 7000 × g for 10 min. one ml of the supernatant was mixed with 2 ml of 0.5% tba solution (in 10% tca). then the mixture was heated at 95°c for 45 min and then cooled at room temperature. the supernatant was read at 532 nm after the removal of interfering substances by centrifuging at 4000 x g for 10 min. the amount of thiobarbituric acid reactive substances (tbars) formed was calculated by using an extinction coefficient of 1.56 × 105 m–1 cm–1 (wills 1969), and expressed as nmol tbars per g of wet tissue. hydrogen peroxide content the h2o2 content was determined according to sergiev et al. (1997). algal material (0.5 g) was homogenized with 5 ml of 10 % (w/v) tca in an ice bath. the homogenate was centrifuged at 7000 × g for 10 min, and the supernatant (0.5 ml) was added with 1.5 ml of 50 mm potassium phosphate buffer (ph 7.0) and 1 ml of 1 m potassium iodide (ki), and the absorbance was measured at 390 nm (uv 1800, shimadzu, japan). h2o2 was used as a standard and expressed as nmol h2o2 per g of wet tissue. preparation of enzyme extracts frozen algal samples (0.5 g) were homogenized in ice cold 50 mm potassium phosphate buffer (ph 7.0) containing 0.1% (v/v) triton x-100 and 1% (w/v) polyvinylpyrollidone (pvp). the homogenate was centrifuged at 10,000 × g for 15 min at 4 °c, and the supernatants were used for enzyme assays. catalase the activity of cat was measured by slightly modifying the method of aebi (1974). the assay mixture contained 2.9 ml of 15 mm h2o2 in 50 mm potassium phosphate buffer (ph 7.0) and 0.1 ml of enzyme extract. the decomposition of h2o2 was followed by a decline in absorbance at 240 nm. the enzyme activity was expressed as nkat per mg of protein (1 katal = 1 mol sec–1). glutathione s-transferase gst activity was measured by using 1-chloro-2, 4-dinitrobenzene (cdnb) as a substrate, according to the protocol by habig et al. (1974). the reaction rate was recorded at 340 nm, and enzyme activity was expressed as nmol cdnb conjugate formed/(min·mg protein), using a molar extinction coefficient of 9.6 mm–1 cm–1. protein content was estimated by the folin-phenol reaction as described by lowry et al. (1951). 200 acta bot. croat. 72 (2), 2013 pise n. m., gaikwad d. k., jagtap t. g. ascorbic acid content algal material was homogenized in an ice bath with 5 ml of 10 % (w/v) tca and centrifuged at 7000 × g for 10 minutes. the deproteinised supernatant was used as assay for ascorbic acid following the stoichiometric reduction of phosphomolybdate by ascorbic acid (mitusi and ohata 1961). asa was used as the standard, and results were expressed as µg asa per g of wet tissue. statistical analysis microsoft excel (excel-2007) was used for statistical analyses. anova analysis was used to compare sampling stations and a tukey test was applied for post-hoc comparisons (zar 1996). differences between means (n = 5) were considered significant when p < 0.05. pearson correlation analyses (p < 0.05) were tested between metal levels in porphyra vietnamensis and oxidative stress responses. results physico-chemical parameter during the present study, water temperature varied from 25.36 to 27.5 °c; the minimum temperature (25.36 °c) of water was recorded at dona paula, and the maximum (27.5 °c) at malvan. the overall differences in temperatures are consistent with normal variations. the ph varied from 7.96 to 8.10 indicating normal variation. the minimum ph value (7.96) was recorded at kunkeshwar and maximum (8.10) at dona paula during the sampling period (tab. 1). the salinity of water ranged from 22.3 to 25.3. kunkeshwar showed the maximum salinity (25.3) and minimum at malvan (20.6), during the monsoon sampling period. metal analysis in the present study heavy metal contents in porphyra vietnamensis from kunkeshwar, malvan and dona paula ranged from 0.47 to 1.98 µg g–1 dry weight and occurred in the following rank order of abundance: hg < cd < pb (tab. 2). the comparatively higher values of these metals from samples of dona paula and malvan could be attributed to higher levels than the kunkeshwar. maximum concentrations of metals in p. vietnamensis, from dona paula during the monsoon study period, could be due to the disturbances to the substratum, resulting from intensive river runoff and strong currents. acta bot. croat. 72 (2), 2013 201 oxidative stress and antioxidant indices in porphyra vietnamensis tab. 2. metal accumulation (mg g–1 dry weight) in tissues of porphyra vieatnamensis at different sampling sites metal malvan kunkeshwar dona paula pb (µg g–1) 1.77 ± 0.09 1.22 ± 0.09 1.98 ± 0.17 cd (µg g–1) 0.68 ± 0.06 0.67 ± 0.15 0.98 ± 0.18 hg (µg g–1) 0.58 ± 0.09 0.47 ± 0.015 0.99 ± 0.19 lipid peroxidation in the present study, lpx values were significantly higher in the samples from dona paula and malvan than in the samples from kunkeshwar (fig. 2a, p < 0.05). similarly, enhanced levels of h2o2 were also noticed in the samples from dona paula (fig. 2b). increased levels of lpx and h2o2 in p. vietnamensis from different localities were significantly correlated with the heavy metal concentrations (tab. 2, p < 0.001). 202 acta bot. croat. 72 (2), 2013 pise n. m., gaikwad d. k., jagtap t. g. fig. 2. biochemical measurements at different sites along the central west coast of india. a – lipid peroxidation (lpx), b – hydrogen peroxide (h2o2), c – catalase (cat), d – glutathion s-transferase (gst), e – ascorbic acid content (asa). the alphabetical letters (a, b, c) mark significant difference at p < 0.05. antioxidant defence systems in the present study, cat activity was significantly higher in the sample from dona paula and malvan, further indicating metal stress increasing the formation rate of h2o2 (fig. 2b). interestingly higher levels of h2o2 were measured in samples from dona paula than those from other sites (fig. 2b). the induction of cat activity in p. vietnamensis from dona paula could be attributed to the unfavorable environmental conditions. also observed were increased levels of gst activity in p. vietnamensis samples from malvan and dona paula, compared to those from kunkeshwar (fig. 2d). high concentrations of asa were also recorded in the samples from dona paula and malvan as compared to kunkehwar (fig. 2e) which suggest that asa synthesis might have been stimulated to protect against a metal-contaminated environment. this response appeared to be particularly associated with physicochemical parameters (pb, cd, hg, temperature, ph and salinity) and also a significant correlation was found (p < 0.001, tab. 3). discussion this study represents the most extensive work on oxidative stress induced by temperature, salinity, ph and metal concentration from selected sites along central west coast of india. heavy metals are implicated in oxidative injury involved in the formation of ros and their subsequent attack on proteins, lipids, and nucleic acids, leading to loss of enzyme functions, altered membrane fluidity, and genomic damage (dietz et al. 1999). the physical, chemical and biological features of the coastline and estuarine systems of goa are adapted to a seasonal rhythm, the monsoon cycle. increased precipitation and land runoff during the monsoon season causes dynamic changes from typically marine to brackish water conditions (qasim and sen gupta 1981) resulting in changes in temperature, salinity, ph and metal levels along outer coasts and in estuaries. the water in the goan estuaries remains well mixed during the pre-monsoon season and gets stratified during the monsoon (qasim and sen gupta 1981), while diurnal variations in physico-chemical conditions are governed by the tides (singbal 1976). the high precipitation and freshwater influx brings cooler water from the upper reaches of the river, resulting in lower water temperatures during monsoons. it also results in higher ph values due to dilution of acta bot. croat. 72 (2), 2013 203 oxidative stress and antioxidant indices in porphyra vietnamensis tab. 3. correlation coefficients (r) between physico-chemical and biochemical parameters phy.–chem. parameter biochemical parameters cat asa gst lpx h2o2 pb r =0.99*** r=0.82*** r=0.76** r=0.84*** r=0.94*** cd r =0.77** r=0.98*** r=0.06 r=0.97*** r=0.89*** hg r=0.65* r=0.93*** r=–0.11 r=0.91*** r=0.80** water temp.(°c) r=0.45 r=0.81*** r=–0.35 r=0.78** r=0.63* ph r=0.99*** r=0.84*** r=0.73* r=0.86*** r=0.95*** salinity (psu) r=–0.99*** r=–0.87*** r=–0.64* r=–0.89*** r=–0.96*** significance level: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) seawater by fresh water inflow (dehadrai and bhargava 1972). solar radiation during the monsoon is reduced because of heavy cloud cover that decreases temperature and the rate of evaporation, thus maintaining lower salinity. however, decreased salinity can be attributed predominantly to heavy precipitation and the influx of fresh water (dehadrai and bhargava 1972). metal analysis the concentration of heavy metals, in marine environments is mainly influenced by both natural and anthropogenic sources. in coastal waters, metals can occur at much higher concentrations, probably due to inputs from river systems (morillo et al. 2004). however, other metals, such as cd, hg, and pb, are toxic even at very low concentrations (wood 1974, nies 1999). marine algae are known to concentrate metals, and it is the increased concentration especially of non-essential trace metals such as cd, pb, hg that could induce oxidative stress in their tissues (kumar et al. 2010). it has been reported that these polluted areas (along the goa coastline) receive varieties of pollutants including metals, petrochemicals as well as sewage generated from various anthropogenic activities (cpcb 1996). lipid peroxidation metal levels in tissue induce a variety of cellular changes, such as damage to membrane integrity (smeets et al. 2005), reduction in photosynthesis and impairment of co2 assimilation (van assche and clijsters 1990, gouia et al. 2003), which may produce ros and resulting lpx. similar results were reported in brown seaweed padina tetrastromatica at polluted locations in karwar and colaba (maharana et al. 2010). this suggests that changes in oxidative stress and antioxidant levels might be to the contaminant metals or abiotic factors such as cadmium, mercury, lead, temperature, salinity and ph. induction of lpx levels were also reported in plants exposed to cd (liu et al. 2007) and pb (reddy et al. 2005, dazy et al. 2009). antioxidant defence systems ozone, salt stress, drought, heat, heavy metals, toxins and organic pollutants also induce the formation of reactive oxygen (pflugmacher 2004). in addition, photosynthetic organisms continuously produce reactive oxygen during photosynthesis and other metabolic processes (foyer and noctor 2000). the defence system against reactive oxygen in plants includes antioxidants such as ascorbate, glutathione, ßcarotene and atocopherol and reactive oxygen scavenging enzymes, such as catalase (ec 1.11.1.6), superoxide dismutase (sod, ec 1.15.1.1), glutathione reductase (gr, ec 1.6.4.2) and ascorbate peroxidase (apx, ec 1.11.1.11). higher levels of antioxidants, and increased activities of reactive oxygen scavenging enzymes, correlate with stress tolerance in seaweeds, e.g. brown algae of the genus fucus (collen and davison 1999 a, b). catalase is a porphyrin-containing enzyme which catalyses the decomposition of hydrogen peroxide to water and oxygen (aruoma 1998). a significant correlation was observed between cat and h2o2 scavenging action (r = 0.97, p < 0.001). this suggests either that catalase is the most important enzyme for the breakdown of h2o2. increased cat activity has been described in several aquatic species from impacted areas both by metals 204 acta bot. croat. 72 (2), 2013 pise n. m., gaikwad d. k., jagtap t. g. and organic contaminants (orbea et al. 2002, nimptsch et al. 2005). catalase activity increased with treatements with cd and pb (dazy et al. 2009). collen et al. (2003) also reported that the heavy metals also induced the activity of catalase in the g. tenuistipitata. nevertheless, cat induction in plants has only been reported under laboratory conditions (roy et al. 1995, rama devi and prasad 1998, collén et al. 2003). gst is also capable of detoxifying ros and its activity increases in cases of oxidative stress in plant cells (pflugmacher et al. 1999, thom et al. 2001). the observed increase in gst activity at dona paula site (tab. 3), and the positive correlation with pb (p < 0.01;) and ph (p < 0.05) suggest that higher metal stress might have induced gst levels to combat tissues from xenobiotic substances. similar findings were observed in padina terastromastica (maharana et al 2010). gst activity has also been observed in plants treated with cd (aravind and prasad 2005, mishra et al. 2009). although few studies have reported on asa levels in response to polluted environmental conditions, interestingly, a significant increase in asa content was observed in response to cd (maharana et al. 2010, aravind and prasad 2005). a significant correlation between asa and cd (r = 0.98, p < 0.001) levels in the algal tissue also supports this statement (tab. 3). asa and gsh are direct scavengers of ros, and an additional substrate for various antioxidant enzymes (halliwell and gutteridge 1999). it has been reported that theascorbateglutathione cycle (agc) plays an important role in detoxification of ros (kuzniak and maria 2001). important activities of cat, gst and asa consequent to increased lpx and h2o2 suggest that p. vietnamensis maintains sufficient antioxidant defences to withstand major environmental turbulence related to different stations and abiotic parameters. the evaluation of abiotic parameters and metal concentrations in p. vietnamensis appears to confirm such a relationship (tab. 3). conclusions porphyra vietnamensis displays different biochemical responses to environmental conditions. elevated levels of lpx and h2o2, indicate a state of oxidative stress possibly due to accumulation of metal ions. the present data revealed that oxidative stress markers such as lpx and h2o2, and nonenzymatic antioxidant such as asa and antioxidant enzymes (cat and gst) may be useful biomarkers, suggesting that this species might be a model species for the evaluation and monitoring of marine environments. acknowledgments the authors are thankful to the director, national institute of oceanography, goa for the facilities and encouragement. this work was carried under the funds from contingency funds from the csir –srf (rf-82609) fellowship. references aebi, h., 1974: catalase. in: bergmeyer, h. u. 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effects of supplemental ca2+ in salinised soil on germination and plant growth response of castor plant (ricinus communis l. var. avani-31, euphorbiaceae). nacl amounting to 390 g was thoroughly mixed with soil of seven lots, of 100 kg each, to give electrical conductivity of 4.1 ds m–1. further, ca(no3)2 × 4h20 to the quantity of 97.5, 195, 292.5, 390, 487.5, and 585 g was separately mixed with soil of six lots to give 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25, and 1:1.50 na+/ca2+ ratios, respectively. the soil of the seventh lot contained only nacl and its na+/ca2+ ratio was 1:0. soil without addition of nacl and ca (no3)2 × 4h20 served as control, with a 0:0 na+/ca2+ ratio. salinity significantly retarded seed germination and plant growth, but the deleterious effects of nacl on seed germination were ameliorated and plant growth was restored with ca2+ supply at the critical level (1:0.25 na+/ca2+ ratio) to salinised soil. supply of ca2+ above the critical level further retarded seed germination and plant growth due to the increased soil salinity. salt stress reduced n, p, k+ and ca2+ content in plant tissues, but these nutrients were restored by addition of ca2+ at the critical level to saline soil. in contrast, na+ content in plant tissues significantly increased in response to salinity, but significantly decreased with increasing ca2+ supply to saline soil. the results are discussed in terms of the beneficial effects of ca2+ supply on the plant growth of ricinus communis grown under saline conditions. keywords: na+/ca2+ ratio, ricinus communis, seedling growth, salt tolerance, salt stress introduction soil salinity is a major abiotic stress to plant growth and development (slater et al. 2003). a high salt content lowers the osmotic potential of soil solution that reduces the soil water potential. plants can absorb water only as long as the water potential of roots is lower (more negative) than that of the soil solution. in saline soils, plant cells have to decrease their water potential below that of the soil solution by lowering their solute potential acta bot. croat. 71 (1), 2012 13 * corresponding author, e-mail: anpandey2001@gmail.com copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:02 color profile: disabled composite 150 lpi at 45 degrees through accumulation of solutes. this osmotic adjustment causes water stress to plants. in addition, ionic toxicity and many nutrient interactions in salt-stressed plants can reduce plant growth or damage the plants (marschner 1995, taiz and zeiger 2006). salt tolerance of plants requires compartmentalization of potentially toxic ions in the vacuole and accumulation of compatible solutes, (organic solutes) in the cytosol where they function in osmotic adjustment and osmoprotection. the osmoprotectants that accumulate most commonly are proline and glycine betaine, although other molecules can accumulate to high concentrations in certain species (hasegawa et al. 2000) application of gypsum has long been considered a common practice in reclamation of saline-sodic and sodic soils (marschner 1995). the addition of ca2+ to the soil (as gypsum, lime or other soluble calcium salts) displaces na+ from clay particles. this prevents the clay from swelling and dispersing (sumner 1993) and also makes it possible for na+ to be leached deeper into the soil. thus exogenously supplied ca2+ not only improves soil structure, but also alters soil properties in various ways (shabala et al. 2003) that benefit the plant growth. moreover, an improved ca2+/na+ ratio in the soil solution enhances the capacity of roots to restrict na+ influx (marschner 1995). the importance of interaction between na+ and ca2+ was recognized after lahaye and epstein (1969) reported that exogenously supplied ca2+ may significantly alleviate detrimental effects of na+ on the physiological performance of hydroponically grown plants. since that time, many investigators have become interested in understanding the effects of divalent cations, specifically the effects of ca2+ on various physiological processes in plants (cramer et al. 1985; lauchli 1990; rengel 1992; shabala et al. 2003, 2006; chen et al. 2007; vaghela et al. 2009). the spectrum of na+/ca2+ interactions in plants seems to be extremely broad, ranging from those at the molecular level, such as reduced binding of na+ to cell wall or plasma membrane, to those manifested at the whole-plant level, such as effects on root and shoot elongation growth, increased uptake and transport of k+ or reduced na+ accumulation in plants (lauchli 1990; rengel 1992). despite the impressive bulk of literature, the interaction of na+ with ca2+ in plants still remains unclear. castor plant (ricinus communis l.), an oil yielding crop, is native to india, the south eastern mediterranean region and eastern africa. it is cultivated in tropical regions of india. moreover, it is extensively cultivated in the marginal saline area of kutch (north – west saline desert) of gujarat state of india. this plant is the source of castor beans (used in ornamentation) and castor oil, which is extracted from seeds. the seed cake, which is left over after pressing contains a protein toxin known as ricin. there is evidence that na+ induces ca2+ deficiency in plant tissues (cramer 1997; patel et al. 2010). consequently, it is assumed that ca2+ supply to saline soils may mitigate na+ toxicity to plants. an understanding of how and how far ca2+ supply modifies responses of plant species to salinity may be of practical significance. in the present investigation calcium nitrate ca(no3)2 × 4h2o, which is a nitrogenous fertilizer, was supplied to saline soil and the remedial effects of ca2+ on salt stressed plants of r. communis were determined by studying germination, growth, water status and acquisition of macro-nutrients. thus, the present study was designed to improve understanding of na+/ca2+ interactions at the whole plant level for this crop species, as such studies are lacking. 14 acta bot. croat. 71 (1), 2012 joshi s. v., patel n. t., pandey i. b., pandey a. n. u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:02 color profile: disabled composite 150 lpi at 45 degrees materials and methods study site the present study was carried out in a greenhouse of the botanical garden of saurashtra university at rajkot (22°18’ n latitude, 70°56’ e longitude) in gujarat. for seedling emergence and plant growth the top 15 cm of black-cotton soil, which is predominant in the saurashtra region of gujarat, was collected from an agricultural field. this soil is a clayey loam containing 19.6% sand, 20.3% silt and 60.1% clay. the available soil water between wilting coefficient and field capacity ranged from 18.3% to 35.0%, respectively. the total organic carbon content was 1.3% and ph was 7.2. the electrical conductivity of soil was 0.3 ds m–1. n, p, k, ca and na contents were 0.15%, 0.05%, 0.03%, 0.05%, and 0.002%, respectively. this soil is fertile and fit for intensive agriculture. physical and chemical properties of soil are given earlier (pandya et al. 2004). na + /ca 2+ ratios surface soil was collected, air dried and passed through a 2 mm mesh screen. eight lots of soil, of 100 kg each, were separately spread, about 50 mm thick, over polyethylene sheets. sodium chloride (nacl) amounting to 390 g was thoroughly mixed with soil of 7 lots to give electrical conductivity of 4.1 ds m–1. the soil was salinised to this level because this plant is cultivated on marginal saline lands in kutch. further, calcium nitrate (ca(no3)2 × 4h2o) in quantities of 97.5, 195, 292.5, 390, 487.5 and 585 g was separately mixed with soil of six lots to give 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 na+/ca2+ ratios, respectively, and then soil salinity for the corresponding lots was 4.3, 4.6, 4.9, 5.0, 5.1 and 5.2 ds m–1. the soil of seventh lot containing only nacl was considered saline soil and its na+/ca2+ ratio was 1:0. there was no addition of nacl and ca(no3)2 × 4h2o to the eighth lot of soil, which served as control with 0:0 na+/ca2+ ratio. the electrical conductivity of control soil was 0.3 ds m–1 and this value was approximately equal to 3.0 mm salinity. a total of eight grades of soil, defined according to their na+/ca2+ ratios, were used in this study. for the measurement of electrical conductivity a soil suspension was prepared in distilled water at a ratio of 1:2 in terms of weight. the suspension was shaken and allowed to stand overnight. thereafter, electrical conductivity was determined with a systronics conductivity meter 304, india. available ca 2+ , k + , na + and mg 2+ in soil for all grades of soil, ca2+, k+, na+ and mg2+ were extracted with 1n ch3coonh4 adjusted to ph 7.0 and measured by shimadzu double beam atomic absorption spectrophotometer aa-6800, japan following jones, jr. (2001). seedling emergence twenty polyethylene bags for each grade of soil were each filled with 5 kg of soil. tap water was added to each bag to bring the soils to field capacity and soils were allowed to dry for 7 days. soils were then raked using fingers and seeds were sown on 15 august 2008. seeds of r. communis var. avani-31 were collected from the saline desert of kutch. bags were kept in an uncontrolled greenhouse under natural temperature and light. ten seeds acta bot. croat. 71 (1), 2012 15 effect of supplemental ca2+ on nacl-stressed castor plants u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:02 color profile: disabled composite 150 lpi at 45 degrees were sown in each bag at a depth of 8–12 mm. immediately after sowing soils were watered (300 ml water was added to raise the soil moisture to field capacity) and thereafter about 100–150 ml water was added to soil (just to wet the surface soil) on alternate days. irrigation of soil with the required amount of water was taken as a measure to control the na+/ca2+ ratio. emergence of seedlings was recorded daily over a period of 30 days and data of cumulative emergence of seedlings were analysed by t-test (compared 0:0 and 1:0 na+/ca2+ treatments) and one-way anova (compared treatments ranging from 1:0 to 1:1.50 na+/ca2+). plant growth for the growth studies, the two seedlings that emerged first were left in each of the 20 bags for each grade of soil and the others were uprooted. seedlings grown in soils at 0:0 (control), 1:0 (saline), 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 na+/ca2+ ratios exhibited emergence of the second leaf after 7, 9, 8, 8, 8, 9, 9 and 9 days, respectively. emergence of the second leaf confirmed the establishment of seedlings. following the emergence of the second leaf, the more vigorous of the two seedlings was allowed to grow in each bag and the other was uprooted. thus twenty replicates for each of eight grades of soil (0:0, 1:0, 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 na+/ca2+ ratios) were prepared. this gave a total of 160 bags, which were arranged in twenty randomized blocks. plants were watered (to raise the soil moisture to field capacity) on alternate days and allowed to grow for 4 months. the experiment was terminated on 15 december 2008. the mean maximum temperature of the greenhouse increased from 31.7 ± 0.6 °c in august to 35.9 ± 0.8 °c in october and thereafter consistently decreased to 30.5 ± 0.6 °c in december 2008. plants contained in 20 bags at each grade of soil were washed to remove soil particles adhered to roots. morphological characteristics of each plant were recorded. shoot height and root length (tap root) were measured. leaf area was marked out on graph paper. fresh and dry weights of leaves, stems, tap roots and lateral roots were determined. water content (g g–1 dry weight) in plant tissues (leaves, stems, tap roots and lateral roots) was calculated using fresh and dry weight values. data recorded for morphological characteristics, dry weight and water content of tissues were analyzed by t-test to assess the effect of salinity on plant growth and by one-way anova to assess the effect of calcium nitrate treatment on the growth of salinised plants. determination of water potential and proline content ten additional plants grown in soil at each grade of soil were used for the measurement of water potential and proline determination in plant tissues. water potential of leaves, stems, tap roots and lateral root tissues was measured by dewpoint potential meter wp4 (decagon devices, inc.pullman, wa, usa) following patel et al. (2010). all the measurements were taken between 8 to 10.30 a.m. concentration of proline in plant tissues was determined following bates et al. (1973). extract of 0.5g fresh plant material with aqueous sulphosalicylic acid was prepared. the extracted proline was made to react with ninhydrin to form chromophore. toluene was added to terminate the reaction. optical density of chromophores was measured at 520 nm by a systronics uv-vis spectrophotometer 118, india. a stock solution of proline was used to prepare a standard curve for proline concentration and optical density. data were analyzed by t-test and one-way anova. 16 acta bot. croat. 71 (1), 2012 joshi s. v., patel n. t., pandey i. b., pandey a. n. u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:02 color profile: disabled composite 150 lpi at 45 degrees mineral analyses of plant materials mineral analyses were performed in triplicate on leaves, stems, tap roots and lateral root tissues of seedlings grown at each level of na+/ca2+ ratio. total nitrogen was determined by a micro-kjeldahl method and phosphorus content was estimated by the chlorostannous molybdophosphoric blue color method in sulphuric acid (piper 1944). concentrations of ca2+, mg2+, na+ and k+ were determined by shimadzu double beam atomic absorption spectrophotometer aa-6800, (shimadzu corporation, kyoto, japan) after triacid (hno3: h2so4: hclo4 in the ratio of 10: 1: 4) digestion. data were analyzed by t-test and one-way anova. results the concentration of available ca2+, k+, mg2+ and na+ in salinised soil increased with increasing calcium nitrate (ca(no3)2 × 4h2o) concentrations (fig. 1). salt stress significantly (p<0.01) reduced the percent emergence of seedlings (tab. 1). supply of external ca2+ to the salinity treatment significantly enhanced the germination percentage (p<0.01) and the process was stimulated. these effects were evident until na+/ca2+ ratio in soil increased to 1:0.25 and 1:0.50. seed germination again decreased with further supply of ca2+ to salinised soil. acta bot. croat. 71 (1), 2012 17 effect of supplemental ca2+ on nacl-stressed castor plants y = 47.080 + 0.006x (r=0.877, p<0.01) y = 2.960 + 0.0004x (r=0.963, p<0.01) y = 20.130 + 0.001x (r=0.971, p<0.01) y = 40.610 + 0.001x (r=0.956, p<0.01) 0 20 40 60 80 100 0 1000 2000 3000 4000 5000 6000 7000 ca 2+ added (mg kg ) e x tr a c ta b le c a 2 + , m g 2 + , k + a n d n a + (m g k g ) –1 – 1 fig. 1. concentrations of available ca2+ (�), mg2+ (�), k+ (�) and na+ (�)(mg kg–1) in salinised soil in relation to increasing supply of ca(no3) × 4h2o. valus are mean ±sem. the data points shown correspond to 1:0, 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 na+/ca2+ ratios respectively, on the x axis. u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:02 color profile: disabled composite 150 lpi at 45 degrees 18 a c t a b o t .c r o a t .71 (1),2012 jo s h i s .v .,p a t e l n .t .,p a n d e y i.b .,p a n d e y a .n . tab. 1. effect of salinity and ca2+ nutrition on leaf, stem, shoot and root characteristics of ricinus communis seedlings as indicated by mean ± sem. na+/ca2+ total seedling shoot root leaf leaf stem shoot dry weight tap root lateral root total root root/shoot ratio emergence height length area dry weight dry weight (leaf+stem) dry weight dry weight dry weight dry weight (%) (cm) (cm) (cm2) (mg) (mg) (mg) (mg) (mg) (mg) ratio 0:0 93 ± 2 42 ± 1 27 ± 1 198 ± 18 660 ± 64 758 ±39 1419 ± 92 132 ± 5 106 ± 12 238 ± 12 0.17 ± 0.1 1:0 84 ± 2 36 ± 1 19 ± 1 144 ± 3 546 ± 24 556 ± 36 1103 ± 51 97 ± 5 72 ± 6 170 ± 7 0.16 ± 0.1 1:0.25 88 ± 2 41 ± 1 25 ± 1 208 ± 9 651 ± 38 762 ± 21 1413 ± 43 134 ± 12 110± 11 244 ± 22 0.17 ± 0.1 1:0.50 92 ± 2 37 ± 0.4 18 ± 1 165 ± 8 616 ± 51 661 ± 43 1277 ± 72 127 ± 11 96 ± 11 223 ± 15 0.18 ± 0.1 1:0.75 80 ± 2 35 ± 1 16 ± 1 152 ± 5 564 ± 31 542 ± 27 1106 ± 29 112 ± 12 81 ± 12 193 ± 18 0.17 ± 0.1 1:1 77 ± 2 31 ± 1 14 ± 1 137 ± 4 520 ± 23 525 ± 35 1045 ± 52 96 ± 5 70 ± 8 167 ± 8 0.16 ± 0.1 1:1.25 67 ± 2 29 ± 1 13 ± 1 128 ± 3 498 ± 20 476 ± 35 974 ± 47 89 ±7 56 ± 6 146 ± 11 0.15 ± 0.1 1:1.50 52 ± 1 27 ± 1 11 ± 0.3 114 ± 4 402 ± 36 404 ± 29 806 ± 50 68± 8 46 ± 6 114 ± 14 0.14 ± 0.1 t – values 3.48** 4.59** 7.29** 3.07** 3.11** 4.06** 4.52** 3.54** 3.12** 4.07** ns f – values 29.82** 39.45** 36.89** 28.16** 5.05** 12.80** 13.17** 4.48** 5.67** 8.00** ns lsd0.05 8.3 3.2 2.0 16.0 106.4 91.5 151.8 30.9 26.2 44.9 ns results of 1:0 and 0:0 na+/ca2+ treatments were compared by t-test. results of treatments ranging from 1:0 to 1:1.50 were compared by f-test. ** values are significant at p<0.01, n.s. = non significant. u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 6 7 j o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 2 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s salinity significantly retarded (p<0.01) elongation of stems and roots (tab. 1). supply of ca2+ to salinity treatment reversed the negative effect of nacl. for example, stem height and root length of plants grown in soil at 1:0.25 na+/ca2+ ratio were almost equal to those of plants grown under control conditions. a further increase in supply of external ca2+ where na+/ca2+ exceeded the 1:0.25 ratio caused reduction in stem height and root length. in addition, salinity significantly reduced (p<0.01) the expansion of leaves. there was recovery in leaf expansion with increase of ca2+ supply to salinised soil until 1:0.25 na+/ca2+ ratio. following this na+/ca2+ ratio in soil, leaf expansion exhibited a decreasing trend. the dry weight of leaves, stems, shoots (leaves + stems), and roots significantly decreased (p<0.01) in response to salinity (tab. 1). when compared with the control, the reduction of dry matter caused by salinity was 17.2%, 26.6%, 26.3% and 31.4% for leaves, stems, tap roots and lateral roots, respectively. however, dry weight of tissues exhibited either a complete or a significant recovery (p<0.01) in the plants grown with 1:0.25 na+/ca2+ ratio. ca2+ supplies to the saline soil exceeding 1:0.25 na+/ca2+ ratio caused significant decreases in the dry weight of all tissues. root/shoot dry weight ratio of plants did not change with salinity and ca2+ treatments. salt stress significantly reduced (p<0.01) the water content in leaves, stems, tap roots and lateral roots (tab. 2). supply of ca2+ to salinity treatment resulted in a significant recovery (p<0.01) of water content in tissues. the results suggested that water content in the tissues of seedlings increased up to 1:0.25 na+/ca2+ ratio and was almost equal to that in control plant tissues. moreover, water content in tissues exhibited a decreasing trend when na+/ca2+ exceeded the 1:0.25 ratio. tissues according to their water content can be arranged in the decreasing order of lateral roots, tap roots, leaves and stems. water potential of leaves, stems, tap roots and lateral roots of plants grown in saline soil became significantly (p<0.05) more negative than that in tissues of control plants. it is evident that water potential of tissues of plants grown in soil at 1:0.25 na+/ca2+ ratio was significantly (p<0.01) restored. further increase in the supply of external ca2+ to salinity treatment again reduced water potential of tissues. according to their water potential (low to high negative values), tissues can be arranged in decreasing order of lateral roots, tap roots, leaves and stems. proline content significantly increased (p<0.05) in leaves, stems, tap roots and lateral root tissues in response to salinity (tab. 2). results suggested that proline content in tissues decreased to minimum level with 1:0.25 na+/ca2+ treatments, but it further increased as the external supply of ca2+ to saline soil increased. according to their proline content tissues can be arranged in decreasing order of leaves, stems, tap roots and lateral roots. na+ content in the leaf, stem and root tissues of plants significantly increased (p<0.05) in response to salinity (tab. 3), but increasing the ca2+ in saline soil significantly reduced (p<0.01) the na+ content in the tissues. salinity significantly reduced (p<0.05) k+ content in the tissues. there was a complete recovery in k+ content of plants grown under the 1:0.25 na+/ca2+ ratio. reduction in k+ content in tissues was again recorded when na+/ca2+ in soil exceeded the 1:0.25 ratio. the k+/na+ ratio of tissues significantly decreased (p<0.05) in response to salinity, but increasing supply of ca2+ to salinity treatment significantly increased (p<0.01) their k+/na+ ratio. concentrations of n, p and ca2+ in the tissue of plants significantly decreased (p<0.05) in response to salinity. it is evident that concentrations of these nutrients were completely restored in tissues of plants acta bot. croat. 71 (1), 2012 19 effect of supplemental ca2+ on nacl-stressed castor plants u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:02 color profile: disabled composite 150 lpi at 45 degrees 20 a c t a b o t .c r o a t .71 (1),2012 jo s h i s .v .,p a t e l n .t .,p a n d e y i.b .,p a n d e y a .n . tab. 2. effect of salinity and ca2+ nutrition on water content, water potential and proline content in tissues of ricinus communis seedlings as indicated by mean ± sem. na+/ca2+ water content (g g–1 dw) water potential (-mpa) proline content (µ mol g–1 fw) ratio leaves stems tap roots lateral roots leaves stems tap roots lateral roots leaves stems tap roots lateral roots 0:0 3.4 ± 0.1 2.6 ± 0.1 3.8 ± 0.1 4.3 ± 0.1 2.9 ± 0.3 4.1 ± 0.1 2.2 ± 0.1 1.6 ± 0.2 26 ± 2 26 ± 1 22 ± 1 18 ± 1 1:0 2.9 ± 0.1 2.0 ± 0.1 3.4 ± 0.0 3.9 ± 0.1 3.8 ± 0.2 4.7 ± 0.1 2.9 ± 0.1 2.4 ± 0.1 34 ± 1 29 ± 1 26 ± 1 22 ± 1 1:0.25 3.3 ± 0.1 2.6 ± 0.1 3.8 ± 0.1 4.1 ± 0.1 3.1 ± 0.1 4.5 ± 0.1 2.4 ± 0.1 1.9 ± 0.2 27 ± 2 25 ± 1 23 ± 1 19 ± 1 1:0.50 3.1 ± 0.1 2.3 ± 0.1 3.6 ± 0.1 3.9 ± 0.1 3.5 ± 0.1 4.6 ± 0.1 2.5 ± 0.1 2.1 ± 0.2 29 ± 1 26 ± 1 24 ± 1 20 ± 1 1:0.75 3.0 ± 0.1 2.1 ± 0.1 3.4 ± 0.1 3.8 ± 0.1 3.7 ± 0.1 4.6 ± 0.0 2.6 ± 0.0 2.3 ± 0.2 32 ± 1 28 ± 2 25 ± 1 21 ± 1 1:1 2.8 ± 0.0 2.0 ± 0.1 3.2 ± 0.1 3.7± 0.0 3.9 ± 0.2 4.7 ± 0.2 2.8 ± 0.3 2.4 ± 0.1 34 ± 0.4 29 ± 1 26 ± 1 21 ± 0 1:1.25 2.7 ± 0.0 1.9 ± 0.1 3.0 ± 0.1 3.5 ± 0.1 4.1 ± 0.1 4.9 ± 0.1 3.0 ± 0.1 2.7 ± 0.2 35 ± 1 30 ± 2 27± 1 22 ± 1 1:1.50 2.6 ± 0.0 1.7 ± 0.1 2.9 ± 0.0 3.3 ± 0.1 4.5 ± 0.1 5.3 ± 0.2 3.9 ± 0.2 3.1 ± 0.1 38 ± 0.3 31 ± 1 27 ± 1 22 ± 2 t values 4.311** 3.795** 6.165** 4.701** 6.584* 7.235* 6.913* 6.235* 6.547* 6.333* 7.216* 6.621* f values 9.572** 7.578** 22.605** 32.176** 8.473** 8.672** 10.752** 6.637** 12.060** 5.683** 6.265** 5.625** lsd 0.05 0.2 0.3 0.2 0.1 0.2 0.2 0.2 0.2 1.4 1.5 1.3 1.2 results of 1:0 and 0:0 na+/ca2+ treatments were compared by t-test. results of treatments ranging from 1:0 to 1:1.50 were compared by f-test. values are significant at p<0.01 (**) and p<0.05 (*). u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 6 7 j o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 2 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s a c t a b o t .c r o a t .71 (1),2012 21 e f f e c t o f s u p p l e m e n t a l c a 2+ o n n ac l-s t r e s s e d c a s t o r p l a n t s tab. 3. effect of salinity and ca2+ nutrition on nutrient content (mg g–1 dw) of tissues (leaf, stem, tap root and lateral root) of ricinus communis seedlings as indicated by mean ± sem. tissue na+/ca2+ n k+ p na+ ca2+ mg2+ k+/na+ ratio (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) ratio 0:0 23.0 ± 0.7 27.3 ± 0.7 2.4 ± 0.0 8.8 ± 0.1 12.6 ± 0.8 1.1 ± 0.3 3.1 ± 0.1 1:0 20.0 ± 1.2 23.4 ± 0.3 2.0 ± 0.1 10.0 ± 0.3 10.1 ± 0.4 0.9 ± 0.2 2.3 ± 0.1 1:0.25 23.0 ± 0.5 26.6± 0.4 2.5 ± 0.2 9.0 ± 0.3 13.6 ± 0.5 1.1 ± 0.3 3.0 ± 0.1 1:0.50 22.0 ± 0.3 26.1 ± 0.4 2.5 ± 0.2 8.6 ± 0.3 12.6 ± 0.5 1.1 ± 0.2 3.0 ± 0.1 1:0.75 21.0 ± 0.5 24.8 ± 0.8 2.4 ± 0.1 8.2 ± 0.3 12.1 ± 0.2 1.1 ± 0.1 3.0 ± 0.2 leaf 1:1 21.0± 0.6 24.0 ± 0.3 1.9 ± 0.2 7.4 ± 0.3 11.8 ± 0.4 1.1 ± 0.2 3.3 ± 0.1 1:1.25 20.0 ± 0.2 22.3 ± 0.2 1.7 ± 0.1 6.9 ± 0.2 11.2 ± 0.6 1.0 ± 0.2 3.2 ± 0.1 1:1.50 19.0 ± 0.6 21.9 ± 0.3 1.5 ± 0.2 6.3 ± 0.3 10.9 ± 0.7 1.0 ± 0.2 3.5 ± 0.2 t – values 5.002* 7.986* 5.292* 6.928* 5.339* ns 11.003* f – values 4.679** 18.810** 6.369** 19.805** 5.581** ns 6.650** lsd 0.05 0.8 0.5 0.2 0.4 0.6 ns 0.1 0:0 21.0 ± 1.0 21.6± 0.3 2.2 ± 0.1 9.1 ± 0.1 12.5 ± 0.7 1.0 ± 0.3 2.4 ± 0.1 1:0 19.0 ± 1.2 18.5 ± 0.3 1.9± 0.0 10.8 ± 0.3 10.6± 0.4 0.8 ± 0.2 1.7 ± 0.1 1:0.25 22.0 ± 0.6 21.2 ± 0.8 2.2 ± 0.1 9.4 ± 0.3 13.4 ± 0.4 1.0 ± 0.3 2.3 ± 0.1 1:0.50 21.0 ± 0.5 19.5 ± 0.5 2.1 ± 0.1 8.6 ± 0.2 12.4 ± 0.3 1.0 ± 0.4 2.3 ± 0.1 1:0.75 20.0 ± 0.7 18.5 ± 0.8 2.0 ± 0.1 8.2 ± 0.4 11.8 ± 0.4 1.0 ± 0.2 2.3 ± 0.1 stem 1:1 18.0 ± 0.7 16.3 ± 0.7 1.8 ± 0.1 7.2 ± 0.2 11.6 ± 0.3 0.9 ± 0.2 2.3 ± 0.1 1:1.25 18.0 ± 0.7 15.2 ± 0.7 1.7 ± 0.1 7.0 ± 0.4 11.1 ± 0.2 0.9 ± 0.1 2.2 ± 0.1 1:1.50 18.0 ± 0.4 14.1 ± 0.8 1.7 ± 0.1 6.7 ± 0.2 10.9 ± 0.7 0.9 ± 0.2 2.1 ± 0.1 t – values 5.774* 5.529* 5.196* 4.348* 7.208* ns 5.232* f – values 4.996** 13.976** 4.807** 24.982** 5.067** ns 5.677** lsd 0.05 0.9 0.8 0.1 0.4 0.5 ns 0.1 u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 6 7 j o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 3 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s 22 a c t a b o t .c r o a t .71 (1),2012 jo s h i s .v .,p a t e l n .t .,p a n d e y i.b .,p a n d e y a .n . tab. 3. – continued tissue na+/ca2+ n k+ p na+ ca2+ mg2+ k+/na+ ratio (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) (mg g–1 dw) ratio 0:0 20.0 ± 1.0 18.4 ± 0.6 2.0 ± 0.1 9.7± 0.6 11.8 ± 0.1 0.9 ± 0.3 1.9 ± 0.1 1:0 16.0 ± 1.0 13.9 ± 0.4 1.7 ± 0.1 10.8 ± 0.8 10.0 ± 0.3 0.7 ± 0.3 1.3 ± 0.1 1:0.25 20.0 ± 0.4 18.2 ± 0.5 1.9 ± 0.1 9.9 ± 0.7 12.1 ± 0.3 0.9 ± 0.2 1.9 ± 0.2 1:0.50 19.0 ± 0.6 16.9 ± 0.7 1.9 ± 0.1 8.6 ± 0.4 12.1 ± 0.3 0.9 ± 0.2 2.0 ± 0.2 1:0.75 18.0 ± 0.6 16.7 ± 0.3 1.8 ± 0.1 8.4 ± 0.4 11.9 ± 0.3 0.8 ± 0.2 2.0 ± 0.1 tap root 1:1 18.0 ± 0.5 15.2 ± 0.2 1.5 ± 0.1 7.6 ± 0.2 11.8 ± 0.5 0.8 ± 0.2 2.0 ± 0.0 1:1.25 17.0 ± 0.6 14.6 ± 0.6 1.4 ± 0.2 7.3 ± 0.2 11.7 ± 0.4 0.8 ± 0.2 2.0 ± 0.1 1:1.50 16.0 ± 0.9 13.9 ± 0.9 1.3 ± 0.2 7.0 ± 0.3 10.7 ± 0.3 0.8 ± 0.1 2.0 ± 0.1 t – values 4.703* 4.666* 4.645* 4.715* 4.754* ns 5.871* f – values 4.870** 8.897** 4.508** 8.520** 4.723** ns 4.798** lsd 0.05 0.8 0.7 0.1 0.6 0.5 ns 0.2 0:0 19.0 ± 1.3 13.2 ± 0.5 1.7 ± 0.0 10.2 ± 0.5 14.4 ± 0.6 0.8 ± 0.0 1.3 ± 0.1 1:0 14.0 ± 0.9 8.9 ± 0.4 1.4± 0.0 11.6 ± 0.5 12.6 ± 0.3 0.7 ± 0.1 0.8 ± 0.0 1:0.25 19.0 ± 0.5 13.6 ± 0.7 1.6 ± 0.1 10.5 ± 0.4 15 ± 0.5 1.0 ± 0.1 1.3 ± 0.1 1:0.50 19.0 ± 0.6 13.4 ± 0.5 1.6 ± 0.1 9.7 ± 0.3 14.8 ± 0.4 1.0 ± 0.2 1.4 ± 0.1 1:0.75 18.0 ± 1.0 13.1 ± 0.5 1.6 ± 0.1 8.8 ± 0.3 13.5 ± 0.7 1.0 ± 0.2 1.5 ± 0.0 lateral root 1:1 18.0 ± 0.6 12.5 ± 0.4 1.4 ± 0.1 7.9 ± 0.5 13.1 ± 0.5 0.9 ± 0.0 1.6 ± 0.1 1:1.25 17.0 ± 1.0 11.7 ± 0.5 1.2 ± 0.0 7.7 ± 0.5 12.8 ± 0.3 0.9 ± 0.3 1.5 ± 0.1 1:1.50 16.0 ± 0.9 11.3 ± 0.6 1.2 ± 0.1 7.3 ± 0.7 12.1 ± 0.1 0.9 ± 0.3 1.6 ± 0.1 t – values 5.879* 5.000* 5.090* 11.094* 6.079* ns 5.120* f – values 4.948** 10.296** 4.963** 10.778** 6.063** ns 8.830** lsd 0.05 1.0 0.6 0.1 0.6 0.5 ns 0.1 results of 1:0 and 0:0 na+/ca2+ treatments were compared by t-test. results of treatments ranging from 1:0 to 1:1.50 were compared by f-test. values are significant at p<0.01 (**), and p<0.05 (*), ns = non significant. u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 6 7 j o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 3 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s grown in soil with a 1:0.25 na+/ca2+ ratio. moreover, high ca2+ in saline soil reduced the concentration of these nutrients in the tissues. concentrations of mg2+ in plants were not significantly affected by na+ and / or ca2+ levels in the soil. discussion the deleterious effects of nacl on germination of r. communis were ameliorated by increase of ca2+ to a critical level (1:0.25 na+/ca2+ ratio) in the salinised soil. the detrimental effect of nacl salinity on germination is associated with an accumulation of toxic ions (mohammad and sen 1990), a decrease of available water to the seeds (pujol et al. 2000) or both. the beneficial effect of ca2+ did not persist when ca2+ supply exceeded the critical level. in the present study, the concentration of available na+ and soil salinity increased with increase in the external supply of ca2+ to the saline soil. secondly, the water uptake by the germinated seeds decreased with both salinity (20.2 ± 0.5%) and increased ca2+ levels (13.6 ± 0.6%). therefore, the beneficial effect of ca2+ on r. communis seed germination appears due to counteraction of the toxic effect of na+. an insufficient level of ca2+ in the germination medium could result in a general deterioration and loss of selectivity of the plasma membrane (whittington and smith 1992). this aggravates salt effects, probably by increasing membrane permeability and leads to a higher accumulation of toxic ions and/or leakage of solutes (cramer et al. 1987, lauchli 1990). a positive response to ca2+ application on germination rate under saline conditions has also been reported in phaseolus vulgaris (cachorro et al. 1994), in wimmera ryegrass (marcar 1986), in barley (bliss et al. 1986), in salvadora oleoides (vaghela et al. 2009). the detrimental effect of ca2+, above 1:0.50 na+/ca2+ ratio, on seed germination might be due to the decreased osmotic potential of soil solution because soil salinity increased with increase in ca2+ supply. a reduction in water content and water potential of leaves, stems, tap roots and lateral roots of plants grown in saline soil might have resulted in internal water deficit to plants, which in turn, reduced the elongation of stems and roots and dry matter accumulation in tissues. it is found that plants subjected to water stress show a general reduction in size and dry matter production (taiz and zeiger 2006). in general, salinity can reduce plant growth or damage to the plants through (i) osmotic effect (causing water deficit), (ii) toxic effect of ions and (iii) imbalance of the uptake of essential nutrients (ramoliya et al. 2004). these modes of action may operate on the cellular as well as on higher organizational levels and influence all the aspects of plant metabolism (kramer 1983, garg and gupta 1997). r. communis exhibited a reduction in leaf area (photosynthetic area) in response to salinity treatment. garg and gupta (1997) reported that salinity causes reduction in leaf area as well as in rate of photosynthesis, which together result in reduced crop growth and yield. also, a high concentration of salt tends to slow down or stop root elongation (kramer 1983) and causes reduction in root production (garg and gupta 1997). supply of ca2+ to the salinised soil ameliorated the harmful effects of nacl on r. communis and plant growth was restored at the 1:0.25 na+/ca2+ ratio. it has been reported that supplemental ca2+ in salinised growth media alleviated inhibition of barley root growth (shabala et al. 2003), shoot growth of phaseolus vulgaris (cachorro et al. 1994), shoot and root growth both in salvadora oleoides (vaghela et al. 2009). in maize plants grown with a high na+:ca2+ ratio, the hydraulic conductance was reduced; supplemental ca2+ (10 mm) improved growth by restoring hydraulic conductance back to that of the control plants (cramer 1992). acta bot. croat. 71 (1), 2012 23 effect of supplemental ca2+ on nacl-stressed castor plants u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:03 color profile: disabled composite 150 lpi at 45 degrees the inhibiting effect of salinity on plant growth was lowest in leaves and highest for stems, tap roots and lateral roots. consequently, leaves were more resistant and other tissues were sensitive to soil salinity. likewise, the recovery of dry matter at 1:0.25 na+/ca2+ ratio was 98.6%, 100.4%, 101.5% and 103.8% for leaves, stems, tap roots and lateral roots, respectively. results suggested that there was a resemblance in the shoot and root growth of plants and their root/shoot dry weight ratio did not change with salinity and ca2+ treatments. salt tolerance in plants is associated with the accumulation of organic solutes in cytoplasm to balance the osmotic pressure of ions in the vacuoles. proline accumulates in the cytoplasm without having any detrimental effects on cytosolic enzymes activities (stewart and lee 1974, hasegawa et al. 2000). in r. communis, osmotic adjustment was achieved by k+ (as evidenced by higher k+ than na+ content in tissues) and increase in the quantity of proline in tissues when water content decreased because of salinity. in addition to its conventional osmoprotective role, proline prevents nacl-induced k+ efflux from roots and may operate as ion channel regulators (cuin and shabala 2005) or reactive oxygen species (ros) scavengers (bohnert et al. 1995). such a regulatory role does not require significant amounts of proline to be accumulated and is, therefore, of low carbon cost to the plant. results further indicated that increase in water content and water potential of tissues with ca2+ treatment was related to decrease in proline content. in the present study, there was a significant decrease of ca2+ content in all the tissues with salinity treatment. as a result, na+ induced ca2+ deficiency in tissues. it is reported that uptake of ca2+ from the soil solution may decrease because of ion interaction, precipitation and increase in ionic strength that reduce the activity of ca2+ (janzen and chang 1987). it is found that salinity can alter ca2+ uptake and transport leading to ca2+ deficiency in plants (cramer et al. 1987). consequently, addition of ca2+ to salinised soil to the critical level resulted in recovery of shoot and root growth. supply of ca2+ exceeding the critical level again reduced the shoot and root growth. in the present study, increased nitrate content together with chloride content caused an increase in soil salinity with ca2+ treatment. the increased soil salinity, in other words, the decreased osmotic potential, might be responsible for retardation of growth at high supply of ca2+. k+ is a major osmoticum in plant cells (marschner 1995) and, therefore is essential for all extension growth. it is evidenced that in salt-stressed roots of cotton, na+ displaced membrane-associated ca2+, which was believed to be primarily located at the plasma membrane (cramer et al. 1985). in addition, nacl-salinity displaced membrane-associated ca2+ on protoplasts of corn (lynch and lauchli 1988) and barley (bittisnich et al. 1989), and on plasma membrane vesicles of melon (yermiyahu et al. 1994). one consequence of the displacement of membrane-associated ca2+ by na+ is the immediate increase of k+ efflux across the plasma membrane of salt-stressed cotton roots (cramer et al. 1985). this effect may be related to the rapid depolarization of the membrane potential upon salinisation (cramer 1997). in the present study, the increased efflux of k+ might be one of the reasons for the significant decrease of k+ content in tissues of r. communis in response to nacl salinity. however, recovery of k+ content in tissues with external ca2+ supply at the critical level (1:0.25 na+/ca2+ ratio) may be the result of repolarization of membrane. there is abundant evidence that salinity alters the ion transport and contents of plants (cramer 1997). in general, na+ uptake and concentrations increase and ca2+ uptake and 24 acta bot. croat. 71 (1), 2012 joshi s. v., patel n. t., pandey i. b., pandey a. n. u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:03 color profile: disabled composite 150 lpi at 45 degrees concentrations decrease in plant cells and tissues as the external na+ concentration increases (rengel 1992, cramer 1997). likewise, as external ca2+ concentrations increase na+ uptake and concentrations decrease and ca2+ uptake and concentrations increase. one consequence of these na+:ca2+ interactions is the reduction of k+ content in salinised plants, which can be prevented with supplemental ca2+. shabala et al. (2006) reported that supplemental ca2+ may prevent k+ efflux from the cell by blocking the depolarization – activated outward – rectifying k+ channels. in addition, salinity generates reactive oxygen species (slater et al. 2003) which activates non-selective cation channels (nscc) inducing further k+ leak (demidchik et al. 2002). this leak is additional to one caused by membrane depolarization (chen et al. 2007). as a result supplemental ca2+ may prevent such ros – induced nscc activation and associated k+ leak. however, increase in soil salinity with high ca2+ supply caused a decrease in k+ content in tissues and it can be accounted for low osmotic potential of soil solution. isosmotic concentrations of mannitol have similar effects as saline treatments with supplemental ca2+ (10 mm) indicating that k+ efflux is affected by osmotic factors in these solutions and not associated with na+-specific displacement of membrane-associated ca2+ (cramer et al. 1985). na+ content significantly increased in tissues of salt-stressed plants, but decreased with increase in ca2+ supply to saline soil. it is reported that uptake mechanisms of both k+ and na+ are similar (schroeder et al. 1994). na+ can not move through the plasma membrane lipid bilayer, but the ion is transported through both lowand highaffinity transport systems, which are necessary for k+ acquisition. as a consequence, na+ could enter the cell through high affinity k+ carriers or through the low affinity channels (nscc) that are strongly influenced by ca2+. these cation channels could allow entry of large amount of na+ from a highly saline soil if not adequately regulated (amtmann and sanders 1999). low affinity k+ uptake is not inhibited by na+ but the high affinity process is restricted (schroeder et al. 1994). similarly na+ toxicity in plants is correlated with two proposed na+ uptake pathways (niu et al. 1995). the k+ and na+ profiles of r. communis suggest that a similar mechanism might operate in this species. it has been shown that ca2+ is an efficient blocker of nscc, a major route for na+ uptake into the cell (demidchik and tester 2002, demidchik and maathuis 2007) and, thus, may directly reduce the amount of na+ accumulation in plants. for r. communis, external supply of ca2+ reduced na+ content on the whole plant level. further, the high k+ content and low na+ content in leaves, stems and tap roots tissues suggest that this plant has the characteristic for rapid transport of k+ to shoot tissues. intracellular k+/na+ homeostasis is a key component of salinity tolerance in plants (tester and davenport 2003). in general, salinity reduces n accumulation in plants (feigin 1985).this is due to the fact that an increase in chloride uptake and accumulation is mostly accompanied by a decrease in shoot nitrate concentration (torres and bingham 1973, garg and gupta 1997). the interaction between salinity and p is very complex and there is no clear cut mechanism for decreased, increased or unchanged p uptake in response to salinisation in different species (grattan and grieve 1992). however, it is known that p concentration is related to the rate of photosynthesis, but it decreases the conversion of fixed carbon into starch (overlach et al. 1993) and therefore decrease of p in leaves will reduce shoot growth. besides the role of mg2+ in chlorophyll structure and as an enzyme cofactor, another important role of mg2+ in plants is in the export of photosynthates (marschner acta bot. croat. 71 (1), 2012 25 effect of supplemental ca2+ on nacl-stressed castor plants u:\acta botanica\acta-botan 1-12\467 joshi.vp 26. o ujak 2012 10:17:03 color profile: disabled composite 150 lpi at 45 degrees 1995). external ca2+ supply reversed the effects of na+ and concentrations of n and p were restored in tissues of seedlings grown at 1:0.25 na+/ca2+ ratio. the high influx or low efflux of nutrients might be responsible for restoration or recovery of nutrients. the increased salinity (low osmotic potential) can be accounted for decrease of nutrients when ca2+ supply exceeded the critical level. in the present study, available ca2+ in salinised soil with supplemental ca2+ at the critical level (1:0.25 na+/ca2+ ratio) was two times higher than that in non-saline control soil. thus, it can be suggested that available ca2+ in saline soil should be maintained nearly two times higher than that in normal soil in order to ameliorate the injurious effects of nacl on seed germination and growth of ricinus communis. conclusions results of the present investigation show that germination and growth of r. communis plants were dependent upon external supply of ca2+ up to the critical level (1:0.25 na+/ca2+ ratio) to the salinised soil. our results are in accordance with the assumption that external ca2+ supply to the saline soil may alleviate na+ toxicity to castor plants. the beneficial effects of high ca2+ concentration are reflected in: (a) the almost complete recovery in germination percentage; (b) the negative effect of soil salinity on elongation of stems and roots, leaf area development and dry matter accumulation in tissues can be reduced by additional supply of ca2+; (c) water content and water potential of leaves, stems, tap roots and lateral root tissues increased with increase in ca2+ up to the critical level in salinised soil; (d) it seems that much of growth reduction associated with salinity is due to high na+ and low ca2+ levels in tissues, thus increasing ca2+ concentration reduces the uptake of na+ and increases ca2+ uptake, consequently decreasing na+ toxicity; (e) a decrease in the efflux of k+ and probably other mineral nutrients resulted in the restoration of nutrients. moreover, the beneficial effects of ca2+ did not persist when the external supply of this element exceeded the critical level because further ca2+ supply increased soil salinity. acknowledgement this study was supported with funds from departmental special assistance 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(elatinaceae) agnieszka popiela1*, andrzej £ysko2, attila molnár3 1 department of botany and nature conservation, university of szczecin, felczaka 3c, 71-412 szczecin, poland 2 department of environmental protection and management, western pomeranian university of technology, szczecin, poland 3 department of botany, university of debrecen, egyetem sq. 1, 4032 hungary abstract – the general distribution of the endangered euro-siberian sub-mediterranean species elatine alsinastrum l. is provided using literature, web-sources and herbaria dataset. the distribution pattern shows some regularities: occurrence of locations along river valleys, formation of concentrated site clusters in some lowlands, wide distances between locations or site clusters or single locations between their clusters. the distribution patterns in central europe seem to be rather well related to the history of the human migration in europe at least since the late holocene. the scattered locations on the eastern part of the distribution area are likely to be a consequence of missing information, rather than to the fragmentation of its distribution. keywords: elatine alsinastrum, ephemerophyte, distribution introduction the genus elatine l. (malpighiales mart., elatinaceae dumort.) includes approximately 15–25 species occurring in temperate areas of both hemispheres, mostly in the northern. besides elatine, the family includes the genus bergia l. (tucker 1986). species of the genus elatine are phytogeographically quite interesting, since they are found in all continents, except antarctica, mainly in the temperate zones, and reach their greatest spread in the northern hemisphere, both in terms of species-diversity and number of locations. despite this wide distribution, the species of elatine occur rarely, and their sites are usually quite distant from each other (often more than 100 km). they are semi-aquatic therophytes adapted to live in periodically flooded environments. the numbers of populations and acta bot. croat. 72 (2), 2013 375 * corresponding author, e-mail: popiela@univ.szczecin.pl copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. plants within a population usually show remarkable yearly fluctuations. the plants, usually characterized by an outstanding phenotypic variability and plasticity, have a very short reproduction period and may produce, when environmental conditions are favourable, many seeds, thus preserving the long lasting germination capacity (deil 2005; popiela and £ysko 2010, 2011; popiela et al. 2010, 2011), as kasahara et al. (1967) that has been demonstrated for elatine triandra schkuhr (more than 50 years). elatine alsinastrum l. is taxonomically isolated within the family elatinaceae, being the only taxon included in the subgenus potamopithys (adanson) seub., and clearly differing from other species of the genus by the presence of leaves arranged in whorls and most frequently a longer and sturdier stem. other species, with opposite leaves, are comprised in the subgenus elatine (= hydropiper moesz.) with the sections: triandra seub. (= crypta (nutt.) seub.] and elatinella seub. (tucker 1986). accordingly, e. alsinastrum is relatively easy to identify and both herbarium materials and literature data can be considered reliable. it includes upright amphibian herbs, with stem erect or ascending, mostly simple; leaves in whorls of (4)8–17 (submerged) and of 3 (aerial); flowers usually 3 at a node, mostly sessile or short-stalked; sepals 4, petals 4 and stamens 8. seeds cylindrical, 0.6–0.9 mm long, 0.2–0.3 mm width, almost straight to slightly bent (0–55°), pits conspicuous, (15–)17–21(–24) in each row. the species occurs in two forms: the first is terrestrial, and it can be found in wet depressions, while the second is aquatic and can be found in shallow stagnant waters (30–50 cm deep), in flooded rice fields and meadows, pond waters and on their verges, lake shores, in periodically dry puddles and water bodies, drainage ditches, in wet depressions in cultivated fields and on wet roadsides. it frequently occurs with other ephemeral wetland species, such as e. triandra schkurh., limosella aquatica l., lindernia procumbens (krock.) philcox, cyperus fuscus l., plantago major l. s.l., gnaphalium uliginosum l., eleocharis acicularis (l.) roem. et shult., peplis portula schrnk. (class isoëto-nanojuncetea br.-bl. et tüxen ex br.-bl. et all. 1952, associations: myosotido siculae-isoëtetum velatae pottier-alapetite 1952, eleocharito-limoselletum aquaticae philippi 1968, lindernio-eleocharitetum ovatae pietsch 1973, eleocharito acicularis-schoenoplectetum supine (horvati} 1931) sao et ubrizsy in ubrizsy 1951 (=elatini-lindernietum procumbentis ubrizsy 1961), elatini alsinastri-juncetum tenageiae libbert 1932, ranunculo lateriflori-limoselletum aquaticae pop 1968 (libbert 1932; oberdorfer 1957; pietsch 1973a, b; pottier-alapetite 1952; brullo et minissale 1998). elatine alsinastrum is an euro-siberian species, quite rare in the whole area of its distribution range (meusel et al. 1978, hultén and fries 1986, uotila 2009a). the aim of this work is to provide the current distribution of this endangered taxon. material and methods this paper is based on both analysis of floristic literature, distribution atlases, websources and examination of the specimens kept in 31 herbaria, 20 of them accessed by web. (tab. 1). a total of 993 floristic data (700 from the literature and herbaria, 293 from the web) are analyzed, priority being given to literature data over websources. the latter were used only if considered to be reliable (e.g. if they did not deviate from the range or had an author and exact location or were delivered by herbarium servers). in consequence, 392 web data were removed. 376 acta bot. croat. 72 (2), 2013 popiela a., £ysko a., molnár a. data were manually mapped using a satellite base image from google earth. calibration, rectification to utm grid and digitalization were done. the vector layers were prepared by conversion to wgs84 cartographic grid. electronic data containing geographic co-ordinates were exported to shp format. when geographic coordinates were missing, records were converted into text format. if detailed descriptions are missing and only cartographic grid locations are given, spatial queries connecting grid area and map were used. all analyses and maps were made using postgis database extension software and qgis (quantum gis) application software working in the linux environment. results elatine alsinastrum is a euro-siberian sub-mediterranean species (fig. 1). the analysis of the current distribution of e. alsinastrum allows us to infer that its distribution centre is in the lowland and upland areas of europe situated to the north and west of alps and carpathians. the pattern of its distribution has the form of smaller and larger site clusters which are frequently far away from each other, even many kilometers. they are mainly located along the valleys of the major rivers. the highest concentration of sites was found in acta bot. croat. 72 (2), 2013 377 the distribution of elatine alsinastrum tab. 1. literature, herbaria and web sources. herbarium acronyms follow index herbariorum (thiers 2012), but the herbarium full name is given when not listed there. area data asia, north and eastern europe (russia, kazakhstan, armenia, georgia, ukraine, moldova, finland, lithuania, belarus) adylov 1983, czerepanov 1995, gagnidze 2005, geideman 1986, grossheim 1962, korshinsky 1898, krylov 1939, kuusk et al. 1996, maevsky 1954, papczenkov 2006, pavlov 1963, peschkova 2006, prokudin 1987, 1999, shishkin and bobrov 1974, shishkin 1961, smallhausen 1895, takhtajyan 1966, tichomirova 1986, tzvelev 1996, uotila 2009b,vedenkov 1998, volobaiev 1991. specimens found in the following herbaria: k*, b central, southern and south-eastern europe (poland, czech republic, slovakia, austria, hungary, romania, the balkans, italy) barina and pifkó 2007, bergmeier & abrahamczyk 2008, ble^i] 1972, boros and vajda 1957, cesati et al. 1868–86, danin 2004, davis 1967, desfayes 2008, dimitrova 2009, dostál and ]ervenka 1991, dostál 1989, farkas 1999, fintha 1994, fiori and paleotti 1896, gori et al. 1998, grdini] et al. 2001, hayek 1927, hejný s. and slavík 1990, jakab 2005, juhász-nagy 1959, moesz 1908, molnár and gulyás 2001, molnár and pfeiffer 1999, nikoli] and topi] 2005, nikoli] 1994, peev 1984, petkovi] et al. 1998, pignatti 1982, pinke et al. 2006, popiela 2001, popiela et al. 2012a, popiela et al. 2010, proházka and køisa 1999, sarika-hatzinikolaou et al. 1994, 1997, sãvulescu 1955, soó and máthé 1938, stevanovi] 1999, trinajsti] 1975, vladimirov et al. 2010, specimens found in the following herbaria: k*, b, bp, de, samu, kazinczy ferenc museum, sátoraljaújhely (hungary); university of szeged, szeged (hungary); szent istván university, gödöllo (hungary); kazinczy ferenc museum, sátoraljaújhely (hungary). biodiversity occurrence data were provided by the university museums of norway. the french plains (armorican massif, paris, and aquitaine basin), in the central zones of the loire and seine river valleys, in the upper part of the rhine river valley, in the east german lowlands and in the western area of poland, in the oder and vistula river valleys (fig. 2). 378 acta bot. croat. 72 (2), 2013 popiela a., £ysko a., molnár a. area data western and south-western europe and northern africa (france, germany, switzerland, spain, portugal, morocco, algeria, tunisia) anonymus 2005, antonetti et al. 2006, benkert et al. 1996, bolomier and cartin 1999, boreau 1849, boudin et al. 2007, bugnon et al. 1998, cirujano and velayos 1993, coutinho 1939, diard 2005, fennane et al. 1998, fortune 2003, franco 1971, fukarek and henker 2006, gamisans and jeanmonod, 1993, grenier 1992, guinochet et vilmorin 1982, haeupler and schönfelder 1989, hammada et al. 2004, jahandiez and maire 1932, jeanmonod and gamisans 2007, le floch and boulos 2008, lunais et al. 1986, muller 2006, netien 1993, pottier-alapetite 1979, quezel and santa 1962–1963, saint-lager 1873, schotsman and osserdet 1967, tourlet 1908, wohlgemuth 1993. speciments were found in the following herbaria: k*, b. biodiversity occurrence data were provided by the following herbaria: abh; bc; cibio; cofc; e; fco; glm; hss; ibf; leiner-herbar konstanz; lux; ma; maf; mgc; ohn; sala; sant; sev; str; stu. occurrence data were provided by information system of the plants of spain, real jardín botánico, csic – fundación biodiversidad; retrieved november 20, 2011 from www.anthos.es. biodiversity occurrence data were provided by following database: aranzadi zientzi elkartea; botany (ups); bundesamt für naturschutz/ netzwerk phytodiversitaet deutschland; cartografía de vegetación a escala de detalle 1:10.000 de la masa forestal de andalucía; flora exsiccata bavarica; florabank1; inventaire national du patrimoine naturel (inpn); observations du conservatoire botanique national du bassin parisien.; phanerogamie; phytochorologie des départements français; sistema de información de la vegetación ibérica y macaronésica tab. 1. – continued fig. 1. distribution area of elatine alsinastrum l. smaller site clusters are in western and central europe, in the bohemian massif, in the danube and tisza river valleys, and in the pannonian basin. furthermore, it is found in scattered locations in southern europe and at the southern limit of its distribution range in single locations in morocco, algeria, tunisia and palestine. the distribution in eastern europe (the area of the vistula-eastern carpathian mountains) appears to be unclear (fig. 1), probably because there is a scarcity of data on detailed locations from this area. most of the russian floras have scarce or no information about the occurrence of the taxon at issue or the available information is rather scattered. the analysis of floras of the baltic countries and information from belarus show that this species either occurs very rarely there or is absent. in ukraine, it is found scattered over the whole territory, except the crimea. there are only scattered locations of e. alsinastrum in the european part of russia from the north and the south by the volga and the kama rivers. site clusters of this taxon are also observed in the stavropol krai and georgia and armenia where it reaches the southern limit of its range in eastern europe. in siberia, single locations are found in the valleys of left tributaries of the tobol river (the iset and tura rivers), in the irtysh river valley, and in kazakhstan. the largest number of observations in siberia has come from natural locations: swamps, flooded meadows, lake shores, flooded lowlands, river meanders and the shores of drying up water bodies. discussion on the basis of fossil data and molecular findings, davis et al. (2001) have raised a hypothesis that families of the order malpighiales originate in the northern areas of south america, with subsequent migration to north america and later, via the atlantic, to the old world. this would suggest that the genus elatine in the northern hemisphere has a history going back at least to the early tertiary and floras of that period (e.g. the mediterranean tertiary and the madrean tertiary), while the present range of the genus originated in the acta bot. croat. 72 (2), 2013 379 the distribution of elatine alsinastrum fig. 2. distribution area of elatine alsinastrum l. in central europe. holocene. e. alsinastrum is the species with the most isolated taxonomic position within the family elatinaceae; accordingly, a question arises about the formation of its range in the holocene, the refuge for this species during the pleistocene glaciations and the migration routes. the distribution area of e. alsinastrum l. shows a remarkable geographic disparity and certain regularities: the formation of concentrated site clusters in some lowlands, and large distances between locations or site clusters or single locations between their clusters. the situation in central europe appears to be well known, with smaller and larger site clusters (often along river valleys) which are frequently far away from each other, even many kilometers away. so, disjunctions inside the range seem to occur, indicating that the spreading of diaspores does not occur easily despite good habitat conditions. it is possible that the species is a relic of the former natural wetland flora, its sites thus able to remain permanent for a very long time (soil seed bank) and the distribution pattern is a reflection of history of colonization in europe at least since the late holocene. many locations have been changed by human beings and thus, it seems that the range of e. alsinastrum may be also extended anthropogenically, with human-created habitats; however, the species is also spread by birds. dispersal of diaspores by water and marsh birds (epiand/or endozoochoria) is possible, although documentation in the genus elatine is only known for one species (kerner 1895). in a discussion of methodological problems in mapping the range of e. alsinastrum differences in the quality of data and the methods being used for their preparation should be emphasized. in this study, the universal wgs 84 coordinate system was used, while the maps employed are made in local or regional coordinate systems. the best method is calibration of maps in the system, in which they were made, followed by their vectorisation and reduction to a uniform coordinate system. using this method, data do not lose quality when the map is being scaled up, while a point on it always preserves the specific scale (see also: popiela et al. 2012b). the comparison of the maps resulting from the present study with those published earlier (meusel et al. 1978, hultén and fries 1986) shows a larger amount of data (also with reference to those available in the last twenty five years) and a greater precision, which was allowed by the mapping method adopted. changes in the distribution of e. alsinastrum relate mainly to central spain, and southern france. the method adopted has also allowed verification of the dispersal character of the distribution of the species in central europe. acknowledgements the authors are very grateful to gábor sramkó for linguistic improvements. the work was provided to a. popiela by a ministry, department of scientific research in warsaw (poland) grant nr po4c03525. we wish to thank the keepers of the herbaria for access to the material and to anonymous reviewers for giving valuable comments. references adylov, t. a., 1983: conspectus florae asiae mediae, 7 (in russian). ussr academy of sciences/uzbek academy of sciences, moscow-tashkent. 380 acta bot. croat. 72 (2), 2013 popiela a., £ysko a., molnár a. anonymus, 2005: catalogue raisonné des plantes vasculaires de la gironde. mémoires de la société linnéenne de bordeaux 4, bordeaux. antonetti, p., brugel, e., kessle, f., 2006: atlas de la flore d’auvergne. conservatoire botanique du massif central, chavaniac-lafayette. barina, z, pifkó, d., 2007: botanical research in the visegrád mountains (in hungarian]. kitaibelia 12, 9–25. benkert, d., fukarek, f., korsch, h. 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(ed.), falz-fein biosphere reserve, 20–25. ascania nova, kherson. vladimirov, v., dane, f., stevanovi], v., tan, k., 2010: new floristic records in the balkans, 14. phytologia balcanica 16, 415–445. volobaev, p. a., 1991: genus elatine in siberia. sibirskij biologicheskij zhurnal 6, 59–64. wohlgemuth, t., 1993: der verbreitungsatlas der farnund blütenpflanzen der schweiz (welten und sutter 1982) auf edv. die artenzahlen und ihre abhängigkeit von verschiedenen faktoren. botanica helvetica 103, 55–71. 386 acta bot. croat. 72 (2), 2013 popiela a., £ysko a., molnár a. vukovic.vp acta bot. croat. 70 (1), 23–40, 2011 coden: abcra 25 issn 0365–0588 orchid diversity of the cape of kamenjak (istria, croatia) nina vukovi]1*, slavko brana2, bo@ena miti]1 1 universty of zagreb, faculty of science, division of biology, department of botany with botanical garden, maruli}ev trg 20, hr-10000 zagreb, croatia. 2 natura histrica, obala a. rismondo 2, hr-52210 rovinj, croatia. abstract – twentytwo taxa have been recorded in the south of istrian peninsula (north adriatic coast, croatia). the research was performed in the period 2003–2004. a great majority of taxa belong to euri-mediterranean (seven taxa, 41.18%) and steno-mediterranean (six taxa, 35.29%) floral elements. eurasiatic (two taxa, 11.76%), atlantic (one taxa, 5.88%) and endemic (one taxon, 5.88%) plants were also present. almost a half of recorded orchids are abundant or frequent. the most of taxa are endangered s.l.; nine vulnerable (vu) plants (52.94%), and one species endangered s.s. (en) (5.88%). there are also near threatened (nt) (two taxa, 11.76%), and data deficient (dd) (one taxon, 5.88%) plants, while others have no category assigned (four taxa, 23.53%). keywords: distribution, mapping, orchidaceae, istria, croatia introduction the orchidaceae family is regarded as a delicate group of plants in croatia – 58 taxa are included in different categories of the red book of vascular flora of croatia (nikoli] and topi] 2005), and therefore the entire family is strictly protected by croatian law (anonymous 2009). according to the most recent floristic data 151 orchidaceae taxa have been noted in croatia (nikoli] 2009). in the literature, the presence of orchids was mostly mentioned sporadically, or in floristic papers dealing with orchid flora of certain parts of croatia (e.g. perko 1998, hr[ak et al. 1999), until the first checklist of croatian orchids (with supplements) was completed (hr[ak 2000, bogdanovi] 2004). acta bot. croat. 70 (1), 2011 23 * corresponding author, e-mail: nvukovic@lipa.botanic.hr copyright ® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. materials and methods study area the researched area of rt (cape) kamenjak is on the southernmost tip of istria, the largest croatian peninsula, northern adriatic sea, croatia. rt kamenjak is officially known as punta or the peninsula of premantura (fig. 1). it includes a 4 km long piece of land, extending from the village of premantura towards the south. many small bays and pebble beaches produce a winding coast, and the width of the peninsula varies between 600 and 1500 m. the land is assembled of alternating valleys and hills (not higher than 50 m) and extends over an area of around 400 ha, with a centroid at 44°53'00'' n and 13°55'00'' e. rt kamenjak, together with the medulin archipelago, has been protected since 1996 due to its great floristic and landscape richness, today in the category of important landscape named lower kamenjak and medulin archipelago. several decades ago, before the protection, most of the population was engaged in agriculture, transforming the native vegetation fraxino orni-quercetum ilicis h-i} (1956) 1958 into its degradation states that nowadays prevail on rt kamenjak. therefore, the original forest vegetation has finally been replaced with maquis, garrigue and dry stony grassland. there are altogether several types of vegetation occurring on rt kamenjak with domination of dry, eu-mediterranean grassland, and vegetation of rt kamenjak would be euphorbietum nicaeensis h-i} (1956) 1958 grassland with its succession stage towards cisto-ericetum arboreae h-i} 1958 garrigue, cymbopogo-brachipodion ramosi h-i} (1956) 1958 and vulpio-lotion h-i} (1960) grassland, agricultural surfaces (arable land and pastures) and pinus halepensis mill. cultures ([ugar 1977, alegro 2002). sunny and open habitats dominate throughout the landscape, making rt kamenjak a suitable site for many heliophilous plants, such as various orchids. also, many other interesting plants rare in croatia are present in this small area, such as anthemis tomentosa l., convolvulus linneatus l., erodium acaule (l.) becherer et thell., festuca lapidosa (degen) markgr.-dann. (topi] and [egulja 2000), as well as cicendia filiformis (l.) delarbre and ophioglossum lusitanicum l. (freyn 1877). around premantura and its surroundings the prevalent soil is eutric cambisol on eolian deposits and relict terra rossa. further south towards the cape the typical and shallow calcocambisol prevails (vida^ek 1979, [kori] and bogunovi] 1987). unfortunately, there is no meteorological station on rt kamenjak. according to data obtained from pula meteorological station (about 10 km distant from premantura) the climate of the wider area of rt kamenjak is intermediate, between semiarid and semihumid, considering the 10-year period (1994–2004) prior to the research (bertovi] 1975), and average annual precipitation is 849.7 mm. nevertheless, our experience during many years spent in pula has shown that the climate of rt kamenjak differs from its nearest surroundings, since rainfall is often absent further south, especially during the summer months. in general, the climate of rt kamenjak is characterized by dry, hot summers and mild winters. rainfall is most abundant during the autumn and winter period (september-january), while summer months july and august are the driest, sometimes with no rainfall at all. temperatures are relatively high, with winter temperatures rarely below zero. winter is the coldest period (december-february), and the highest temperatures appear during july and august. rainfall data for rt kamenjak have been obtained for the period of 1997–1999 by topi] 24 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. acta bot. croat. 70 (1), 2011 25 orchids in istria (croatia) fig. 1. geographic position of the researched area. (a) croatia, (b) position of rt kamenjak (black dot) in gauss-krüger grid, (c) important landscape 'lower kamenjak and medulin archipelago' (grey) with gauss-krüger grid subdivided into 125 x 125 m fields. and [egulja (2000), in order to compare the climatic aridity of rt kamenjak with its surroundings, pula and medulin. those measurements have shown considerably less rainfall at rt kamenjak, particularly during summer when droughts appeared, and the authors define its climate as arid (topi] and [egulja 2000). twenty-three orchid taxa have been previously recorded on rt kamenjak (freyn 1877; perko 1998; starmühler 1998, 2003; topi] and [egulja 2000; hr[ak pers. comm.; per^i] pers. comm.). this study intends to give an updated insight of the orchid flora on »rt kamenjak«, and provide new information about the distribution of species throughout the area. fieldwork orchid diversity and distribution were investigated during several fieldtrips during the spring and autumn of 2003, and again in the spring of 2004, considering the usual phenology of orchids. an orthophotograph created by geodetic department of rijeka supplied with gauss-krüger 1 � 1 km grid was used as a cartographic basis for mapping. prior to fieldwork the existing grid was divided into smaller, 125 � 125 m, grid fields (altogether 244 grid fields studied). the 125 � 125 m grid seemed more suitable than the initially considered 100 � 100 m grid, for several reasons: fieldwork is simpler with fewer fields (fewer unit surfaces and border areas among fields), it is precise enough for a qualitative study of distribution, and it was easy to draw within the existing 1000 � 1000 m grid. the entire research area was thoroughly searched several times, and the presence of orchids was entered onto the map and into the corresponding fields. this data were later used for creating digital maps of orchid distribution. data analysis identification and nomenclature of orchids followed standard determination keys and some specific scientific papers (freyn 1877, fleischmann 1904, baumann and künkele 1986, perko 1998, delforge 1995, nikoli] 2009), except in the case of ophrys x sooi, for which no suitable determination literature was available at the given moment and we determined it according to its morphological resemblance to parental species. recorded species and subspecies are given alphabetically in the list, along with the following data: chorological type, iucn category when applicable (en – endangered, vu – vulnerable, nt – near threatened, dd – data deficient), relative frequency (a – abundant, f – frequent, r – rare, rr – extremely rare), number and percentage of fields containing specified taxa, flowering period (in roman numerals corresponding to months of a year), and preferred habitats, respectively. when appropriate, some comments are given. chorological data were taken from pignatti et al. (2005), and in the case of ophrys sphegodes ssp. incubacea, orchis coriophora ssp. fragrans and o. morio ssp. picta from delforge (2006). abbreviations of floral elements, as defined in pignatti (1982), are given as follows: 1. endemic (en) 2. steno-mediterranean (sm) 3. euri-mediterranean (em) 4. eurasiatic (ea) 5. atlantic (atl.) 26 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. iucn categories were taken from the red book of vascular flora of croatia (nikoli] and topi] 2005). considering that all recorded orchids are geophytes (delforge 2006), no life form was indicated. taxa recorded for the first time on rt kamenjak are marked with an exclamation mark (!). unconfirmed taxa are marked with an asterisk (*) and later discussed. most of the orchids recorded were photographed since no herbarium material was collected. hybrids are presented in a separate list, but were omitted from mapping and other analysis. data on the presence of orchids in different fields were used in the making of digital distribution maps of orchid taxa on rt kamenjak (figs. 2 – 18) using autocad r14 software (autodesk, inc., san rafael, califórnia, usa). results altogether 29 orchid taxa have been recorded on rt kamenjak. a total of 22 orchid taxa were noted in this survey, and previous citations mentioned seven more. recorded species and subspecies (tab. 1) belong to the genera aceras (1), anacamptis (1), ophrys (5), orchis (6), serapias (3) and spiranthes (1). altogether five hybrids have been recorded, belonging to the following genera: ophrys (3), orchis (1) and serapias (1). findings of four species (ophrys apifera, orchis simia, orchis tridentata and serapias parviflora) and two hybrids (ophrys x cosana and ophrys x sooi) on rt kamenjak are mentioned for the first time. list of orchid species and subspecies aceras anthropophorum (l.) r. br. (fig. 2) – atl. – dd – (r) – 6 (2.46%) – (iv/v). dry grassland on calcareous ground. this taxon has been previously recorded by perko (1998) anacamptis pyramidalis (l.) rich. (fig. 3) – em – nt – (rr) – 1 (0.41%) – (v/vi). open, dry grassland near the macadam road. extremely rare species on rt kamenjak, present as a single plant. this taxon was previously recorded by perko (1998) *limodorum abortivum (l.) sw. this taxon was previously recorded by per^i] (pers. comm.). !ophrys apifera huds. (fig. 4) – em – en – (r) – 1 (0.41%) – (v). dry grassland surrounded with shrubs. one population of about 15–20 individuals was discovered. *ophrys araneola rchb. this taxon was previously recorded by perko (1998). ophrys bertolonii moretti (fig. 5) – sm – vu – (a) – 76 (31.15%) – (iv/v). dry stony grassland and garrigue, pastures, sometimes edges of macadam and paths. this taxon was previously recorded by perko (1998) and topi] and [egulja (2000). ophrys bombyliflora link (fig. 6) – sm – vu – (a) – 101 (41.39%) – (iv/v). dense populations were found within dry grassland on calcareous ground. this taxon has been previously recorded by perko (1998). ophrys fuciflora (f. w. schmidt) moench (fig. 7) – em – vu – (r) – 10 (4.10%) – (iv/v). grassland and open surfaces within sparse garrigue. this taxon was previously recorded by perko (1998) (as o. holoserica) and topi] and [egulja (2000). acta bot. croat. 70 (1), 2011 27 orchids in istria (croatia) 28 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. figs. 2–5. distribution of aceras anthropophorum (2), anacamptis pyramidalis (3), ophrys apifera (4) and ophrys bertolonii (5) on »rt kamenjak«. ophrys sphegodes mill. ssp. atrata (lindl.) e. mayer (fig. 8) – sm – vu – (a) – 98 (40.16%) – (iv/v). distributed widely on grassland, garrigue, pastures and edges of macadam and paths. all records of this species belong with no exception to this subspecies. this taxon was previously recorded by perko (1998) (as o. incubacea), topi] and [egulja (2000) (as o. sphegodes) and starmuhler (2003) (as o. incubacea). *ophrys sphegodes mill. ssp. sphegodes. this taxon was previously recorded by perko (1998). orchis coriophora l. ssp. fragrans (pollini) sudre (fig. 9) – em – vu – (r) – 5 (2.05%) – (v/vi). grassy surfaces along macadam roads. this taxon was previously recorded by perko (1998). orchis morio l. ssp. picta (loisel.) k. richter (fig. 10) – sm – nt – (a) – 138 (56.56%) – (iv/v). very widespread taxon found almost everywhere, on dry grassland, garrigue, pastures, along macadam and paths, edges of abandoned agricultural land and olive groves. this taxon was previously recorded by perko (1998) and topi] and [egulja (2000) (as o. morio). orchis papilionacea l. (fig. 11) – em – vu – (a) – 141 (57.79%) – (iv/v). very abundant species found in most habitats: dry grassland, garrigue, pastures, edges of macadam and paths and sometimes on abandoned agricultural land and olive groves. this taxon was previously recorded by topi] and [egulja (2000). *orchis papilionacea l. ssp. rubra (jacquin) h. sund. this taxon was previously recorded by perko (1998). *orchis provincionalis balb. ssp. pauciflora (ten.) e. g. camus. this taxon was previously recorded by freyn (1877), and topi] and [egulja (2000) (unconfirmed). orchis purpurea huds. (fig. 12) – ea – vu – (rr) – 1 (0.41%) – (iv). edge of maquis along roadside. extremely rare species on rt kamenjak, presented with a single plant. this taxon was previously recorded by perko (1998). !orchis simia lam. (fig. 13) – em – vu – (rr) – 1 (0.41%) – (iv/v). grassy surface in the vicinity of garrigue. this species was presented with a small group of individuals in one field. !orchis tridentata scop. (fig. 14) – em – vu – (rr) – 3 (1.23%) – (iv/v). grassland and grassy surfaces surrounded by garrigue. this species has been found in several locations but only a single plant per field. *serapias cordigera l. this taxon was previously recorded by topi] and [egulja (2000) and hr[ak (pers. comm.). serapias istriaca perko (fig. 15) – en (istria) – (f) – 71 (29.10%) – (v/vi). dry, calcareous grassland. distributed on the southern part of the investigated area, occasionally found quite close to seashore. this taxon was previously recorded by perko (1998) and starmuhler (2003). serapias lingua l. (fig. 16) – sm – (a) – 163 (66.80%) – (iv/v). extremely abundant species, found in almost every habitat: dry and stony grassland, garrigue, pastures, edges of macadam and paths, abandoned olive groves, edges of agricultural surfaces. it is the second most widespread species on rt kamenjak. this taxon was previously recorded by perko (1998), topi] and [egulja (2000) and starmuhler (2003). acta bot. croat. 70 (1), 2011 29 orchids in istria (croatia) 30 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. figs. 6–9. distribution of ophrys bombyliflora (6), ophrys fuciflora (7), ophrys sphegodes ssp. atrata (8) and orchis coriophora ssp. fragrans (9) on »rt kamenjak«. acta bot. croat. 70 (1), 2011 31 orchids in istria (croatia) figs. 10–13. distribution of orchis morio ssp. picta (10), orchis papilionacea (11), orchis purpurea (12) and orchis simia (13) on »rt kamenjak«. 32 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. figs. 14–17. distribution of orchis tridentata (14), serapias istriaca (15), serapias lingua (16) and serapias parviflora (17) on »rt kamenjak«. !serapias parviflora parl. (fig. 17) – sm – (rr) – 2 (0.82%) – (v). dry grassland near the macadam. findings of this species included only two single plants recorded in different fields. *serapias vomeracea (burm.) briq. this taxon was previously recorded by topi] and [egulja (2000). spiranthes spiralis (l.) chevall. (fig. 18) – ea – (a) – 179 (73.36%) – (ix/x). extremely abundant species found on dry and stony grassland, garrigue, edges of macadam, paths and maquis, in olive groves, on pastures and abandoned agricultural land. the distribution maps show that this is the most widespread species on rt kamenjak, occasionally found very close to the seashore, sometimes even at the edge of vegetation towards the sea. this taxon was previously recorded by perko (1998), topi] and [egulja (2000) and starmuhler (1999). list of orchid hybrids !ophrys x cosana h. baumann et künkele (=ophrys sphegodes mill. ssp. atrata (lindl.) e. mayer x ophrys bombyliflora link). one individual plant was found on dry pasture near the macadam. high abundance of parental species was detected in the immediate vicinity. acta bot. croat. 70 (1), 2011 33 orchids in istria (croatia) fig. 18. distribution of spiranthes spiralis on »rt kamenjak«. ophrys x lyrata h. fleischm. (=ophrys sphegodes mill. ssp. atrata (lindl.) e. mayer x ophrys bertolonii moretti). this taxon was quite widely distributed in the investigated area, occurring on almost every site along with the parental species. this hybrid was previously recorded by perko (1998). !ophrys x sooi a. fuchs (=ophrys sphegodes mill. ssp. atrata (lindl.) e. mayer x ophrys fuciflora (f. w. schmidt) moench). several plants were found on a dry grassy surface along a macadam road. orchis x gennarii rchb. f. (=orchis morio l. ssp. picta (loisel.) k. richter x orchis papilionacea l.). the taxon is very widespread, occurring in most habitats along with its parental species. this hybrid was previously recorded by perko (1998). serapias x pulae perko (=serapias istriaca perko x serapias lingua l.). endemic taxon. only several plants were recorded. this hybrid was previously recorded by perko (1998). floristic analysis chorological spectrum of the orchids noticed in this research (tab. 1., fig. 19) shows that a total of 13 species and subspecies (76.47%), belong to the mediterranean group of plants, with euri-mediterranean (seven taxa, 41.18%) and steno-mediterranean (six taxa, 35.29%) geoelements. other geoelements were eurasiatic (two taxa, 11.76%), atlantic (one taxa, 5.88%), and endemic (serapias istriaca endemic for istria, 5.88%). 34 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. tab. 1. threat level, flowering period and floral element of orchidaceae species and subspecies recorded on »rt kamenjak«. taxa threat level flowering period floral element aceras anthropophorum dd iv–v atl. anacamptis pyramidalis nt v–vi em ophrys apifera en v em ophrys bertolonii vu iv–v sm ophrys bombyliflora vu iv–v sm ophrys fuciflora vu iv–v em ophrys sphegodes ssp. atrata vu iv–v sm orchis coriophora ssp. fragrans vu v–vi em orchis morio ssp. picta nt iv–v sm orchis papilionacea vu iv–v em orchis purpurea vu iv ea orchis simia vu iv–v em orchis tridentata vu iv–v em serapias istriaca – v–vi en (istria) serapias lingua – iv–v sm serapias parviflora – v sm spiranthes spiralis – ix–x ec analysis of endangered taxa (tab. 1, fig. 20) shows that one species ophrys apifera is endangered (en – 5.88%), nine (52.94%) are vulnerable (vu), two (11.76%) are near threatened (nt) and one (5.88%) is data deficient (dd), while four taxa (23.53%) have not been assigned an iucn category. relative frequency of orchid species and subspecies on rt kamenjak is shown on figure 21, and also in table 2, which shows that they can be grouped, almost equally, into two groups based on their abundance (1) abundant/frequent and (2) rare/extremely rare species. the distribution of all species and subspecies recorded in this survey is presented with distribution maps (figs. 2–18). the most widespread species is spiranthes spiralis reacta bot. croat. 70 (1), 2011 35 orchids in istria (croatia) fig. 19. chorological spectrum of orchid species and subspecies recorded on »rt kamenjak«. fig. 20. threat level of orchid species and subspecies recorded on »rt kamenjak«. fig. 21. relative frequency of orchid species and subspecies recorded on »rt kamenjak«. corded in 179 fields (fig. 18), followed by serapias lingua recorded in 163 fields (fig. 16). the extremely rare species are anacamptis pyramidalis (fig. 3) and orchis purpurea (fig. 12), each represented with a single individual. most of the orchids recorded in this study (16 taxa, 94.12%) flowered during spring, with ophrys bombyliflora and orchis morio ssp. picta in middle april as the earliest, and 36 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. tab. 2. abundance of orchidaceae species and subspecies recorded on »rt kamenjak« (total number of surveyed fields: 244). fields (no.) – number of fields containing the taxa, fields (%) – percentage of fields containing the taxa, rel. freq. – relative frequency of the taxa. taxa fields (no.) fields (%) rel. freq. spiranthes spiralis 179 73,36 a serapias lingua 163 66,80 a orchis papilionacea 141 57,79 a orchis morio ssp. picta 138 56,56 a ophrys bombyliflora 101 41,39 a ophrys sphegodes ssp. atrata 98 40,16 a ophrys bertolonii 76 31,15 f serapias istriaca 71 29,10 f ophrys fuciflora 10 4,10 r aceras anthropophorum 6 2,46 r orchis coriophora ssp. fragrans 5 2,05 r ophrys apifera 1 0,41 r orchis tridentata 3 1,23 rr serapias parviflora 2 0,82 rr orchis simia 1 0,41 rr anacamptis pyramidalis 1 0,41 rr orchis purpurea 1 0,41 rr fig. 22. number of orchid species and subspecies recorded on rt kamenjak flowering in different periods of the year. serapias istriaca in late may as the latest spring flowering orchid. only spiranthes spiralis flowered in autumn (september-october) (tab 1, fig. 22). discussion this research has failed to confirm previous findings of some orchid species and subspecies: serapias cordigera was previously recorded by topi] and [egulja (2000) and hr[ak (pers. comm.), who found only one small population of s. cordigera, on a single locality. perhaps due to its localized occurrence the species was not confirmed during our survey. ophrys araneola was previously noted only by perko (1998). orchis provincionalis ssp. pauciflora was first noted by freyn (1877), with topi] and [egulja (2000) quoting those findings in their paper without confirmation. in the case of ophrys sphegodes, our records belong without exception to the subspecies atrata, but the typical subspecies has been noted by perko (1998). records of o. sphegodes in topi] and [egulja (2000) probably refer to the subspecies atrata, since it has shown to be rather abundant on rt kamenjak. there is a possibility that those taxa (ophrys araneola, orchis provincionalis ssp. pauciflora and ophrys sphegodes ssp. sphegodes) are also present but rare on rt kamenjak, and their individual or scarce appearance is the reason for their being overlooked. on the other hand, there is a possibility that some species are extinct on rt kamenjak. for example, a few individuals of limodorum abortivum were noticed a few years prior to the research at a single locality (per^i] pers. comm.), but since then there has been no trace of those individuals. it should also be taken into consideration that seasonal and annual variations of the climatic conditions could be associated with annual changes in emergence and flowering of some species (tyler 2001, coates et al. 2006). thus, it is possible for a species to be observed one year and not the other, but still be present on the site. the single appearance of orchis purpurea could be explained with the absence of some habitats like woodland edges and open woodland (the only plant was found on a shady spot at the edge of dense maquis). however, there is no obvious reason for the limited appearance of some species, such as anacamptis pyramidalis, since dry, calcareous mediterranean grasslands that cover most of rt kamenjak are common habitats for this species. several small groups of plants recorded in a few fields were identified as ophrys fuciflora, known as a very variable and polymorphic species (delforge 1995) with many described subspecies, varieties and forms. the relations within o. fuciflora group are still rather unclear, as a result of various approaches to complex and different interpretations among different authors. thus, some authors state the possibility that the medio-european o. fuciflora does not occur south of the central massif and the alps, but it is replaced by a number of locally distributed endemic populations (delforge 2006), such as istrio-quarnerian ophrys untchjii (devillers and devillers-terschuren 2004). ophrys untchjii (sometimes referred to as o. fuciflora var. untchjii) has a locus classicus in istria and seems to be endemic for croatia (delforge 2006). serapias vomeracea was previously recorded by topi] and [egulja (2000). the species is similar to the endemic s. istriaca, and a certain possibility of confusion between the two species exists (perko 1998). given the fact that fieldwork by topi] and [egulja (2000) was carried out before s. istriaca was described, this record probably refers to s. istriaca. acta bot. croat. 70 (1), 2011 37 orchids in istria (croatia) previous notation of orchis papilionacea as subspecies rubra mentioned by perko (1998) was not confirmed in this research, probably because the plant identification, in this case, did not go further than the species level. records of ophrys apifera on rt kamenjak are given here for the first time, although there are some dubious earlier citations of this species. ophrys holoserica is listed in the orchid flora of rt kamenjak (perko 1998), but it remains unclear whether the name refers to o. apifera or o. fuciflora. our research has shown that o. apifera has a very limited area of distribution on rt kamenjak and o. fuciflora is somewhat more widely distributed, so it is more likely that the above-mentioned name refers to the latter. orchis simia was presented with a small group of individuals in one field, and findings of orchis tridentata and serapias parviflora included only a few plants recorded in different fields and thus they were probably overlooked by other authors, due to their extremely rare appearance on rt kamenjak. distribution of those, as well as other rare orchid taxa on rt kamenjak, should be investigated more closely. the two most widespread species on »rt kamenjak«, spiranthes spiralis and serapias lingua, and also some other very abundant taxa like orchis morio ssp. picta and orchis papilionacea, are affiliated with a wide range of habitats, which seems to be responsible for such a wide distribution. it was often difficult to determine whether a certain individual is a parental species or a hybrid, due to a great variability of parental plants and many intermediate forms towards hybrids. similarity to the parent serapias lingua is probably the reason why only a few individuals of the endemic serapias x pulae were recorded. several plants found on dry grassy surface along macadam road were named ophrys x sooi, due to their morphological resemblance to both parental species, and also to the fact that parents were detected on the same locality. however, those findings should be investigated more thoroughly and further confirmation of this record is needed. it is the same in the case of ophrys x cosana, since only one plant was found in the surveyed area (surrounded with abundant populations of both parental species). our study has shown that serapias lingua, s. istriaca and spiranthes spiralis often inhabit areas in the near vicinity of the seashore; therefore those species probably have greater tolerance to salinity. since the investigated area is situated on the very coast of the eastern adriatic, a high number of mediterranean species was expected (topi] and [egulja 2000), a hypothesis borne out by our data. the size of the 125 x 125 m basic grid unit proved to be appropriate for acquiring precise and authentic distribution maps and could be applied in the mapping of other flora on »rt kamenjak«. we can conclude that rt kamenjak can be regarded as a remarkable orchid site since it contains at least 22 out of 151 croatian orchid taxa, while its area of 4 km2 is rather small in comparison with the 56,600 km2 area of croatia. moreover, rt kamenjak is characterised with large populations of some widely distributed taxa, as well as with an established population of the endemic serapias istriaca. the rich and diverse orchid flora of rt kamenjak makes this area a significant orchid site in croatia. as many as 58.82% of the orchids of rt kamenjak have the status of vulnerable and endangered species; this increases the floristic 38 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. importance of this orchid resort. the high number of threatened and endemic taxa and also the fact that croatian law strictly protects the orchidaceae family indicate that greater efforts should be made to preserve this site, perhaps by assigning it a higher level of protection. in order to expand the knowledge about plant distribution and improve the conservation of this important landscape, we highly recommend mapping in high resolution for other plant species of rt kamenjak, especially for rare, endemic and endangered plants. acknowledgments the authors would like to thank mladen per~i} from the public institution natura histrica for providing transport and sharing his orientation ability during numerous fieldtrips. special thanks go to andrej stroj, igor bor{i} and maja hlastec bi{}an for their help in the making of digital maps. we are also grateful to our colleague sandro bogdanovi} for helpful suggestions and critical comments on the manuscript. references alegro, a. l., 2002: morphometric, ecological and phytosociological characteristics of species from the genus festuca l. 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(orchidaceae). berichte aus den arbeitskreisen heimische orchideen 15, 13–27. pignatti, s., 1982: flora d’italia 1, 3. edagricole, bologna. pignatti, s., menegoni, p., pietrosanti s. 2005: valori di bioindicazione delle piante vascolari della flora d’italia. bioindicator values of vascular plants of the flora of italy. braun-blanquetia 39, 3–95. starmühler, w., 1998: vorarbeiten zu einer »flora von istrien«, 2. carinthia 2 189/109, 431–466. starmühler, w., 2003: vorarbeiten zu einer »flora von istrien«, 6. carinthia 2 193/113, 579–658. [kori], a., bogunovi], m., 1987: geography of soils. in: [kori], a. (ed.), pedosphere of istria (with pedological map) (in croatian). projektni savjet pedolo{ke karte hrvatske, posebna izdanja, knjiga 2. zagreb. [ugar, i. (ed.), 1977: vegetation map of croatia, section pula (in croatian). institute for botany, university of zagreb. topi], j., [egulja, n., 2000: floristic and ecological characteristics of the southernmost part of istria (croatia). acta botanica croatica 59, 179–200. tyler, g., 2001: relationships between climate and flowering of eight herbs in a swedish deciduous forest. annals of botany 87, 623–630. vida^ek, @., 1979: basic pedological map of section pula 1, scale 1:50 000 (in croatian). projektni savjet za izradu pedolo{ke karte sr hrvatske, zagreb. 40 acta bot. croat. 70 (1), 2011 vukovi] n., brana s., miti] b. 544 vizintin et al.vp acta bot. croat. 71 (2), 371–374, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication a note on pteris vittata l. (pteridaceae) in montenegro walter gutermann* department of biogeography, faculty centre of biodiversity, university of vienna, rennweg 14, a-1030 vienna, austria abstract – pteris vittata, formerly doubtfully indicated for dalmatia, is recorded for montenegro based on a hitherto disregarded collection preserved in the vienna university herbarium. key words: pteris vittata, pteridophyta, fern, flora, montenegro introduction pteris vittata l. (pt. longifolia auct. non l. s. str. – cf. walker 1964, 1993; tryon and tryon 1982) is a subtropical fern of the old world, with disjunct outliers in the southern mediterranean. additionally, a few naturalizations in europe are known further north (cf. jalas and suominen 1972). in the literature of the 19th century it was indicated also for dalmatia, e.g. in an authoritative survey of european ferns (milde 1867), although without any source adduced. probably this author took the record from hooker (1858, 157; hooker and baker 1868: 154), who had mentioned a specimen of his herbarium from dalmatia (leg. alexander). this was also the opinion of luerssen (1889), who asked for a confirmation, however, since the species was missing in all the geographically pertinent literature (e.g. schlosser and vukotinovi] 1869). from that time this record, never localized in more detail, obviously was suppressed (hayek 1927 ff.) nor in the analiti~ka flora jugoslavije (mayer and horvati] 1967) was it found worthy of consideration. in consequence no record for the former yugoslavia is found in either flora europaea or in the med-checklist (walker 1964, 1993; greuter et al. 1984). in atlas florae europaeae, the distribution map for pteris vittata shows zakinthos as the northernmost dot in the eastern mediterranean (jalas and suominen 1972: 56, map no. 60). results and discussion in connection with floristic work on the ionian islands (greece), and examining vouchers from the island of zakinthos in the vienna university herbarium (wu), i unexpectedly acta bot. croat. 71 (2), 2012 371 * corresponding author, e-mail: walter.gutermann@univie.ac.at copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. came across a well preserved collection of pteris vittata from kotor bay (boka kotorska; fig. 1), montenegro. this affords a late corroboration of an old indication, and, thanks to the statements on the herbarium label, it allows an exact localisation of this occurrence, decidedly the northernmost in the mediterranean east of italy. the plants were collected on july, 28, very probably in 1916 [and not 1917 – see below], in (then) southern dalmatia: „ »bocche di cattaro: trockener, sonniger steinriegel bei teodo, ca. 10 m«, that is near tivat (grid 34tcn2 of atlas florae europaeae), today part of montenegro. the fern has not previously been recorded for this country (cf. rohlena 1942, pulevi] 2005). 372 acta bot. croat. 71 (2), 2012 gutermann w. fig. 1. voucher specimen of pteris vittata from boka kotorska (herbarium wu). the collection was incorporated into the wu herbarium in 1917, on june 22 (according to the acquisitions journal of the university herbarium), as the donation of dr. a. ginzberger, at that time (1903–1912) »adjunkt« at the botanical institute of the vienna university (schönbeck-temesy 1992). so far it remains uncertain, whether all collections of this entry (»24 pflanzen aus süd-dalmatien«) came from h. spunar, who collected the fern under discussion. – there is a discrepancy in relation to the collection date as written on the herbarium label (»28. vii. 1917«): presumably 1917 is an error for 1916, because the entries in the herbarium daybook were listed continuously by day. so far, any other possible connections of the collector, hermann spunar, to botany in vienna are unknown to us, as are further herbarium sheets traceable to him. according to information from heeresgeschichtliches museum (vienna), hermann spunar (born in 1889 at schota near leibnitz, styria) was first lieutenant of the fortress artillery, regiment no. 5, at that time stationed at kotor for defence of the »k. u. k. kriegshafen« (imperial naval port). acknowledgements investigations on the flora and vegetation of the ionian islands have been supported by fwf (fonds zur förderung der wissenschaftlichen forschung; projekt p10466-bio). the author is furthermore indebted to dr. wolfgang etschmann (heeresgeschichtliches museum) who kindly conveyed data on h. spunar, and to dr. heimo rainer (herbarium wu) who provided the herbarium image. references greuter, w., burdet, h. m., long, g. (eds.), 1984: med-checklist. a critical inventory of vascular plants of the circum-mediterranean countries, 1: pteridophyta 2, gymnospermae, dicotyledones (acanthaceae-cneoraceae). conservatoire et jardin botanique de la ville de genève, genève. hayek, a., 1927: prodromus florae peninsulae balcanicae. 1. band: pteridophyta, gymnospermae, dicotyledoneae (apetalae et choripetalae). repertorium specierum novarum regni vegetabilis 30, 1, 1–1193. hooker, w. j., 1858: species filicum, being descriptions of the known ferns, 2. w. pamplin, london. hooker, w. j., baker, j. g., 1868: synopsis filicum, a synopsis of all known ferns. r. hardwicke, london. jalas, j., suominen, j. (eds.) 1972: atlas florae europaeae. distribution of vascular plants in europe. 1. pteridophyta (psilotaceae to azollaceae). the commitee for mapping the flora of europe and societas biologica fennica vanamo, helsinki. luerssen, ch., 1889: die farnpflanzen oder gefässbündelkryptogamen (pteridophyta). [dr. l. rabenhorst’s kryptogamenflora von deutschland, oesterreich und der schweiz, 2, 3], e. kummer, leipzig. acta bot. croat. 71 (2), 2012 373 a note on pteris vittata l. (pteridaceae) in montenegro mayer, e., horvati], s., 1967: i. pteridophyta (ferns). in: horvati], s. (ed.), flora analytica iugoslaviae (in croatian), 1, 1, 81–155. institut za botaniku sveu~ili{ta u zagrebu, zagreb. milde, j., 1867: filices europae et atlantidis, asiae minoris et sibiriae. a. felix, lipsiae. pulevi], v., 2005: material for vascular flora of montenegro. a supplement to »conspectus florae montegrinae« (j. rohlena) (in montenegrin). republi~ki zavod za za{titu prirode crne gore, podgorica. rohlena, j., 1942: conspectus florae montenegrinae. preslia 20/21, 1–506. schlosser, j. c., vukotinovi], l., 1869: flora croatica exhibens stirpes phanerogamas et vasculares cryptogamas quae in croatia slavonia et dalmatia sponte crescunt nec non. f. @upan, zagrabiae. schönbeck-temesy, e., 1992: zur geschichte des herbars der wiener universität. in: morawetz, w., (ed.), die botanik am rennweg. das institut für botanik und der botanische garten der universität wien. festband zur eröffnung des neuen instituts. abhandlungen der zoologisch-botanischen gesellschaft in österreich 26, 69–95. tryon, r. m., tryon, a. t., 1982: ferns and allied plants. with special reference to tropical america. springer-verlag, new york, heidelberg, berlin. walker, t. g., 1964: 1. pteris l. in: tutin, t. g., heywood, v. h., burges, n. a., valentine, d. h., walters, s. m., webb, d. a. (eds) with the assistance of ball, p. w., chater, a. o.), flora europaea 1, 11. university press, cambridge. walker, t. g., 1993: pteris l. in: tutin, t. g., burges, n. a., chater, a. o., edmondson, j. r., heywood, v. h., moore, d. m., valentin, d. h., walters, s. m., webb, d. a. (eds., assisted by akeroyd, j. r., newton, m. e.), flora europaea 1, 14. university press, cambridge. 374 acta bot. croat. 71 (2), 2012 gutermann w. opce-str.vp acta bot. croat. 69 (1), 31–46, 2010 coden: abcra 25 issn 0365–0588 vegetation spatial heterogeneity in a hyper arid biosphere reserve area in north africa kamal h. shaltout1, mohammed g. sheded2*, ashraf i. salem3 1 botany department, faculty of science, tanta university, tanta, egypt 2 botany department, faculty of science at aswan, south valley university, aswan, egypt 3 national conservation sector, wadi allaqi biosphere reserve, eeaa, aswan, egypt ninety eight species of angiosperms belonging to 34 families were identified in the wadi allaqi biosphere reserve (s. e. egypt): 33.7 % annuals and 66.3 % perennials. the members of leguminosae contributed 19.4% of the total flora, considering the most dominant family in wadi allaqi. three herbaceous species were recorded for the first time in this region: iphiona scabra, chenopodium album and lotus deserti. eight vegetation clusters were obtained and categorized into 4 distinct groups according to soil composition and chemical characteristics (concentration of bicarbonates, calcium, magnesium and chlorides), and intensity of inundation by the water of lake nasser. key words: vegetation, tamarix, iphiona scabra, chenopodium album, lotus deserti, scrubland, life form, diversity, desert, wadi allaqi, egypt introduction wadis represent one of the most prominent desert landforms, which exhibit physiographic irregularities that lead to parallel variations in species distribution (kassas and girgis 1964). wadi vegetation in the eastern desert of egypt is distinguished into plant communities where the dominant perennial species give the permanent character to the plant cover in each habitat (kassass and imam 1954). this may be attributed to the scanty rainfall, which is not adequate for the appearance of many annuals. this desert lies in the saharo-sindian region and is bounded from the south by sudano-zambezian region; it is dominated by saharo-sindian species (ozenda 1958). the south eastern part belongs to the saharo-arabian phytogeographical region. in general, the vegetation is characterized by sparseness of plant cover and the preponderance of a limited number of plant species (springuel et al. 1997). the life form distribution is closely correlated with topography and landform (kassas and girgis 1965, zohary 1973, orshan 1986). the composition of life forms expresses a typical desert flora, the majority of species being therophytes and acta bot. croat. 69 (1), 2010 31 * corresponding author, e-mail: sheded1960@yahoo.com u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:40 color profile: disabled composite 150 lpi at 45 degrees chamaephytes. the south eastern desert of egypt is interesting ecologically because of its physiographic variations and environmental gradients. considerable efforts have been made towards the elucidation of vegetation – environmental relationships in its wadi ecosystems (kassas and imam 1954, 1959; batanouny 1979; el-sharkawi et al. 1987). recently, the multivariate approach (hill 1979a,b; digby and kempton 1987) has been used for the study of spatial distribution and classification of the desert vegetation of wadi allaqi (springuel and sheded 1991, sheded 2002). the present study aims at analyzing the vegetation of wadi allaqi, in the south eastern desert of egypt, in order to depict the main vegetation types and to assess the role of the soil factors and human interference that influence the vegetation. on the other hand, it is also a diagnostic study to evaluate the recent situation of wadi allaqi vegetation in comparison with the previous studies (e.g. springuel et al. 1991, ali et al. 1997). study area the wadi allaqi biosphere reserve is situated in the south-eastern desert of egypt (i.e. egyptian nubian desert), about 180 km south of aswan, on the eastern side of lake nasser (22° and 23° n to 33° and 35° e). it forms one of the most extensive drainage systems in egypt’s eastern desert. its upstream tributaries drain some of the mountains that divide the red sea coastal plain from the nile valley. the wadi extends for about 350 km, in a nwse direction. it has an average width of about 1 km being narrower upstream and considerably broader downstream as it approaches lake nasser (fig. 1). 32 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. fig. 1. map of wadi allaqi showing the 19 sampled locations (1–19). u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:45 color profile: disabled composite 150 lpi at 45 degrees the main course of wadi allaqi starts in the area of gabal iss. it has a length of 350 km and runs generally west, reaching the nile at kurisku. the lower part of the wadi is now inundated by the water of lake nasser. the upstream part is ca 500 m. a.s.l. however the surrounding mountains are high; one such mountain is gabal eigat in the upstream part, rising to 1400 m. a.s.l. (belal and springuel 1996). the upstream tributaries may receive occasional rainfall and their drainage may accumulate in the main channel of wadi allaqi forming torrents that are the main source of water in this wadi (kassas and girgis 1969). the middle part of wadi allaqi on the north side receives large influents. wadi abu murra is one of these influents and lays a more clearly defined channel bounded by high ground on both sides. its channel was once a part of the main camelcaravan. wadi umm raylan is another influent. it is smaller wadi, of about 30 km. with a few short effluent runnels (kassas and girgis 1969).wadi allaqi consists of the el quleib core area, an extreme arid ecosystem comprising a small number of plants and animals. vegetation is sparse with acacia ehrenbergiana and aerva javanica as the characteristic species. the el quleib core area is located in the downstream part of the reserve, and the eigat core area is located in the upstream part of wadi allaqi in a remote area that is difficult to reach. the impacts of the local community include charcoal formation, collection of medicinal plants, grazing and cutting trees. soil in wadi allaqi consists of wadi fill deposits and varies in depth, physical and chemical composition depending on the soil-forming materials, transport processes and depositions (moalla and pulford 1991). tamarix litter has an important effect on surface soils as it accumulates salts and increases soil salinity. the study area is characterized by a hyperarid environment with an aridity index of less than 0.05 (ayyad and ghabbour 1986). data from the wadi allaqi meteorological station between 1996 and 2005 shows the annual mean temperature is 25.8 °c. it can be as low as –2 °c in january. on the other hand, the mean maximum temperature of 41.8 °c has been recorded in july, which can often be as hote as 45 °c or higher, especially in august. the monthly mean relative humidity ranged between 14.0 and 38%, with an annual mean of 22.7%. the annual rainfall rarely exceeds 5 mm and is highly variable in both time and space. the wind speed at aswan ranged between 4 and 8 km h–1 between 1960 and 1980 with an annual mean of 5.9 km h–1. the annual fluctuation of water in lake nasser during a forty-year period (1964–2003), reached its first peak of 178 m. a.s.l. in 1978, but by 1988 the level had dropped to 154 m. a.s.l. and 175.6 in 2003 m. a.s.l. (lake nasser authority). materials and methods one hundred and twelve stands were analyzed at 19 locations within the wadi allaqi biosphere reserve (upstream, midstream and downstream parts, including the different wadi tributaries), in the period from november 2004 to may 2005. the locations and stands were selected to represent a wide range of physiographic and environmental variation in each tributary. in each location, sampling stands were situated randomly using the réléve method described by muller-dombois and ellenberg 1974). species were identified after tackholm 1974, boulos 1999, 2000 and 2002. species life forms were determined depending upon the location of the regenerative buds and the shed parts during the unfavorable season (raunkier 1934). acta bot. croat. 69 (1), 2010 33 desert vegetation spatial heterogeneity u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:45 color profile: disabled composite 150 lpi at 45 degrees soil samples were collected from each stand. sizes of soil particles were estimated using the pipette method (kilmer and alexander 1949. soil water extracts (1:5) were prepared for determination of ec and ph using conductivity and ph meters, chlorides by direct titration against silver nitrate using potassium chromate as an indicator, carbonates and bicarbonates by direct titration against hcl using phenolphthalein and methyl orange as indicators, calcium and magnesium by titration against edta (ethylenediamine dihydrogen tetraacetic acid) using ammonium purpurate and eriochrome black t as indicators (jackson 1977). two-way indicator species analysis (twinspan), as a classification technique and detrended correspondence analysis (dca) as an ordination technique, were applied to the presence estimates of 98 species in 112 vegetation stands according to the computer programs of hill (1979 a, b). the relationship between the vegetation and edaphic variables were assessed by calculating the simple linear correlation coefficient (r) between the dca axes (reflect the vegetation gradient) and the soil variables. results stand classification according to twinspan led to the identification of 8 clusters of stands similar in terms of their species composition (fig 2); they are named after the dominant species as follows: balanites aegyptiaca – acacia tortilis subsp. tortilis, fagonia indica – leptadenia pyrotechnica – acacia tortilis subsp. tortilis – solenostemma arghel, acacia ehrenbergiana – morettia philaeana, acacia ehrenbergiana – aerva javanica – 34 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. 112 +20-92 tamarix nilotica cynodon dactylon glinus lotoides acacia ehrenbergiana -23 +69 acacia tortilis subsp. tortilis balanites aegyptiaca solenostemma arghel leptadenia pyrotechnica calotropis procera aerva javanica acacia ehrenbergiana +13-7 hyoscyamus muticushyoscyamus muticus -3 +20 -4 +16 a st ra g a lu s v o g e li i s a lv a d o ra p e rs ic a s o le n o st e m m a a rg h e l c a y lu se a h e x a g y n a c le o m e d ro se ri fo li a fagonia indica leptadenia pyrotechnica solenostemma arghel calotropis procera -59 +10 astragalus vogelii indigofera argentea -12 +1 eragrostis aegyptiaca eragrostis aegyptiaca -6 +6 -5 +2 astragalus vogelii astragalus vogelii chenopodium murale +4-1+7-3 s a ls o la im b ri c a ta -9 +50 -35 +15 m o re tt ia p h il a e a n a c o tu la c in e re a f a g o n ia in d ic a a . to rt il is su b sp . ra d d ia n a p u li c a ri a c ri sp a l o to n o n is p la ty c a rp a h e li o tr o p iu m su p in u m t y p h a d o m in g e n si s b a la n it e s a e g y p ti a c a 13 viii 7 vii 10 vi 15 v 35 iv 9 iii 20 ii 3 i fig. 2. dendrogram indicating the eight vegetation clusters resulting from the twinspan classification of the 112 sampled vegetation stands in wadi allaqi. u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:46 color profile: disabled composite 150 lpi at 45 degrees fagonia indica, acacia ehrenbergiana – aerva javanica, acacia ehrenbergiana – indigofera argentea, hyoscyamus muticus – tamarix nilotica – morettia philaeanaglinus lotoides and tamarix nilotica – glinus lotoides – cynodon dactylon (tab. 1, fig. 2). ninety-eight species recorded in the present study belonging to 34 families and 74 genera (tab. 4). 33 species are annuals (33.7%) and 65 perennials (66.3%). members of leguminosae contribute 19.4% of the total flora and so considered the most dominant family, followed by gramineae (13.3%), zygophyllaceae (6.1%) and compositae (6.1%). three herbaceous species were recorded for the first time in this region: iphiona scabra and lotus deserti at the upstream part, and chenopodium album the downstream. acta bot. croat. 69 (1), 2010 35 desert vegetation spatial heterogeneity tab. 1. floristic composition of the vegetation clusters (i–viii) identified after the application of twnispan on the 112 sampled stands in wadi allaqi. * new recorded species in wadi allaqi. the figures represent presence value. cluster i ii iii iv v vi vii viii p% number of stands per cluster 3 20 9 35 15 10 7 13 species present in seven clusters acacia ehrenbergiana 0.0 10.0 88.9 100.0 100.0 100.0 14.3 7.7 64.3 species present in six clusters aerva javanica 0.0 0.0 66.7 94.3 100.0 20.0 57.1 46.2 58.9 species present in five clusters faidherbia albida 33.3 10.0 11.1 0.0 0.0 0.0 14.3 7.7 5.4 balanites aegyptiaca 100.0 40.0 11.1 0.0 13.3 0.0 14.3 0.0 13.4 fagonia indica 0.0 70.0 66.7 94.3 6.7 0.0 42.9 0.0 50.9 pulicaria crispa 0.0 0.0 22.2 2.9 40.0 0.0 42.9 15.4 12.5 species present in four clusters acacia tortilis subsp. raddiana 0.0 50.0 11.1 11.4 66.7 0.0 0.0 0.0 22.3 astragalus vogelii 0.0 0.0 11.1 0.0 0.0 40.0 28.6 7.7 7.1 salsola imbricata 0.0 5.0 0.0 0.0 13.3 30.0 42.9 0.0 8.0 species present in three clusters fagonia glutinosa 33.3 5.0 22.2 0.0 0.0 0.0 0.0 0.0 3.6 senna alexandrina 0.0 20.0 44.4 8.6 0.0 0.0 0.0 0.0 9.8 citrullus colocynthis 0.0 20.0 0.0 0.0 0.0 0.0 57.1 7.7 8.0 glinus lotoides 0.0 5.0 0.0 0.0 0.0 0.0 57.1 76.9 13.4 pulicaria incisa 0.0 5.0 33.3 0.0 0.0 0.0 14.3 0.0 4.5 species present in two clusters acacia tortilis subsp. tortilis 100.0 55.0 0.0 0.0 0.0 0.0 0.0 0.0 12.5 cleome chrysantha 33.3 10.0 0.0 0.0 0.0 0.0 0.0 0.0 2.7 chrozophora oblongifolia 33.3 5.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 crotalaria microphylla 33.3 10.0 0.0 0.0 0.0 0.0 0.0 0.0 2.7 senna italica 0.0 20.0 11.1 0.0 0.0 0.0 0.0 0.0 4.5 cotula cinerea 0.0 25.0 66.7 0.0 0.0 0.0 0.0 0.0 9.8 forsskalea tenacissima 0.0 10.0 22.2 0.0 0.0 0.0 0.0 0.0 3.6 u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:46 color profile: disabled composite 150 lpi at 45 degrees 36 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. cluster i ii iii iv v vi vii viii p% number of stands per cluster 3 20 9 35 15 10 7 13 stipagrostis plumosa 0.0 5.0 44.4 0.0 0.0 0.0 0.0 0.0 4.5 cynodon dactylon 0.0 0.0 0.0 0.0 0.0 0.0 28.6 61.5 8.9 tamarix nilotica 0.0 0.0 0.0 0.0 0.0 0.0 85.7 84.6 15.2 heliotropium supinum 0.0 0.0 0.0 0.0 0.0 0.0 14.3 46.2 6.3 eragrostis aegyptiaca 0.0 0.0 0.0 0.0 0.0 0.0 28.6 7.7 2.7 indigofera argentea 0.0 5.0 0.0 0.0 0.0 70.0 0.0 0.0 7.1 cleome droserifolia 0.0 25.0 0.0 2.9 0.0 0.0 0.0 0.0 5.4 reseda pruinosa 0.0 5.0 0.0 0.0 0.0 0.0 28.6 0.0 2.7 haplophyllum tuberculatum 0.0 5.0 0.0 0.0 0.0 0.0 14.3 0.0 1.8 psoralea plicata 0.0 0.0 11.1 0.0 0.0 0.0 14.3 0.0 1.8 solenostemma arghel 0.0 55.0 0.0 0.0 0.0 0.0 14.3 0.0 10.7 trichodesma africanum 0.0 5.0 0.0 0.0 0.0 0.0 14.3 0.0 1.8 astragualus eremophilus 0.0 0.0 11.1 0.0 0.0 0.0 14.3 0.0 1.8 morettia philaeana 0.0 0.0 88.9 0.0 0.0 0.0 57.1 0.0 10.7 lotus sp 0.0 0.0 0.0 0.0 6.7 0.0 14.3 0.0 1.8 species present in one clusters euphorbia forsskalii 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 tribulus pentandrus 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 ziziphus spina-christi 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 iphiona scabra* 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 euphorbia granulata 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 cleome paradoxa 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 lupinus digitatus 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 zilla spinosa 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 dipterygium glaucum 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 tribulus ochroleucus 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 typha domingensis 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 chrozophora tinctoria 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 aizoon canariense 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 anticharis glandulosa 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 aristida adscensionis 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 arnebia hispidissima 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 asphodelus fistulosus 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 calotropis procera 0.0 45.0 0.0 0.0 0.0 0.0 0.0 0.0 8.0 caylusea hexagyna 0.0 25.0 0.0 0.0 0.0 0.0 0.0 0.0 4.5 cistanche phelypaea 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 farsetia aegyptiaca 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 cymbopogon schoenanthus l. spreng. subsp proximus 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 heliotropium pterocarpum 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 tab. 1. – continued u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:46 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (1), 2010 37 desert vegetation spatial heterogeneity cluster i ii iii iv v vi vii viii p% number of stands per cluster 3 20 9 35 15 10 7 13 monsonia heliotropoides 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 zygophyllum simplex 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 dichanthium foevulatum 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 trianthema triquetra 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 leptadenia pyrotechnica 0.0 65.0 0.0 0.0 0.0 0.0 0.0 0.0 11.6 lotus deserti * 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 maerua crassifolia 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 ochradenus baccatus 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 panicum turgidum 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 pergularia tomentosa 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 salvadora presica 0.0 20.0 0.0 0.0 0.0 0.0 0.0 0.0 3.6 orobanche ramosa 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 cocculus pendulus 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 fagonia bruguieri 0.0 0.0 22.2 0.0 0.0 0.0 0.0 0.0 1.8 capparis decidua 0.0 0.0 11.1 0.0 0.0 0.0 0.0 0.0 0.9 heliotropium arbainense 0.0 0.0 22.2 0.0 0.0 0.0 0.0 0.0 1.8 crotalaria aegyptiaca 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 0.9 hyphaene thebaica 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 0.9 lotononis platycarpa 0.0 0.0 0.0 0.0 40.0 0.0 0.0 0.0 5.4 chenopodium murale 0.0 0.0 0.0 0.0 0.0 0.0 14.3 0.0 0.9 hyoscyamus muticus 0.0 0.0 0.0 0.0 0.0 0.0 100.0 0.0 6.3 rumex dentatus 0.0 0.0 0.0 0.0 0.0 0.0 42.9 0.0 2.7 tephrosia purpurea 0.0 0.0 0.0 0.0 0.0 0.0 28.6 0.0 1.8 cyperus laevigatus 0.0 0.0 0.0 0.0 0.0 0.0 28.6 0.0 1.8 phragmites australis 0.0 0.0 0.0 0.0 0.0 0.0 28.6 0.0 1.8 zea mays 0.0 0.0 0.0 0.0 0.0 0.0 14.3 0.0 0.9 polycarpaea repens 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 aristida mutabilis 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 sonchus oleraceus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 acacia nilotica 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 imperata cylindrica 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 chenopodium album * 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 sesbania sesban 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 abutilon pannosum 0.0 0.0 0.0 0.0 0.0 0.0 0.0 15.4 1.8 fimbristylis bis-umbellata 0.0 0.0 0.0 0.0 0.0 0.0 0.0 46.2 5.4 crypsis schoenoides 0.0 0.0 0.0 0.0 0.0 0.0 0.0 46.2 5.4 senecio flavus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 ricinus communis 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 tamarix aphylla 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.7 0.9 tab. 1. – continued u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:47 color profile: disabled composite 150 lpi at 45 degrees six life forms of species were recognized (tab. 4, fig. 3) phanerophytes (19 species), chamaephytes (22 species), hemicryptophytes (16 species), geophytes (4 species), parasites (2 species), helophytes (2 species) and therophytes (33 species). regarding the floristic categories, only one species is a mediterranean element (lupinus digitatus) and another one is saharo-sindian with extension to the mediterranean region (heliotropium supinum). on the other hand, four species are cosmopolitan taxa: chenopodium album, chenopodium murale, crypsis schoenoides and sonchus oleraceus (tab. 4, fig. 4), while 30 species are sudano-zambezian with extension to saharo-sindian and 18 species belong to the saharo-sindian with extension to sudano-zambezian region. three species belong to saharo-sindian with extension to irano-turanian (fagonia bruguieri, haplophyllum tuberculatum and hyoscyamus muticus). 38 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. 19 phanerophytes (19%) 22 chamaephytes (22%) hemicryptophytes (16%) 4 geophytes (4%) 2 parasites (2%) 33 therophytes (35%) 2 helophytes (2%) fig. 3. biological spectrum of wadi allaqi vegetation. 4% 1% 1% 2% 1% 4% 7% 7% 3%18% 7%2% 10% 1% 31% cosm . cultivated me me+ir-tr me+sa-si+ir-tr pal. pan. sa-si. sa-si+ir-tr. sa-si+s-z. sa-si+s-z+ir-tr+me. sa-s-z s-z s-z+me s-z+sa-si. fig. 4. floristic category spectrum of wadi allaqi vegetation. cosm – cosmopolitan, pal – palaeotropical, pan – pantropical, sa-si – saharo-sindian, s-z – sudano-zambezian, me – mediterranean and ir-tr – irano-turanian. u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:47 color profile: disabled composite 150 lpi at 45 degrees sand attains the highest value in cluster i (85.5%) and the lowest in cluster viii (87.4). clay has the highest value in cluster viii (8.1%) and the lowest in cluster iii (1.9), while silt has the highest in cluster viii (12.0) and the lowest in cluster ii (10.7). the minimum value of ph is attained in cluster ii and iv (7.4) and the maximum in clusters vi, vii and viii (7.6). ec has a maximum in cluster viii (413 ms–1) and a minimum in cluster i (83.7 ms–1). cl attains the highest concentration in cluster viii [16.0 mg (100 mg)–1] and the lowest in cluster iii [10.0 mg (100 mg)–1]. hco3 has the highest value in cluster ii [46 mg (100 mg)–1] and the lowest in cluster iv [29 mg (100 mg)–1]. ca has a maximum in cluster viii [20.7 mg (100 mg)–1] and its minimum in cluster iv [9.1 mg (100 mg)–1], while mg attains a maximum in cluster viii [14.6 mg (100 mg)–1] and a minimum in cluster iii [3.0 mg (100 mg)–1]. organic matter attains the highest in cluster vii (1.7 %) and the lowest in cluster i (0.8 %) (tab. 2). the first dca axis (ax1) correlated positively with electric conductivity (r = 0.81), calcium (r = 0.59), clay (r = 0.89) magnesium (r = 0.78) and organic matter (r = 0.76); and negatively with sand (r = –0.94). the third axis (ax3) correlated positively with silt (r = 0.76) (tab. 3). the similarity between the vegetation clusters, as revealed by dca analysis, shows a distinct pattern along the two-dimensional plane of axes 1 and 2 (using the mean vectors of centroids of each cluster): group of clusters i and ii; cluster vi; group of clusters iii, iv and v; and group of clusters vii and viii. discussion ninety-eight species were recorded in the present study compared with 127 species recorded by springuel et al. (1991) in the same study area. this may be due to the severe environmental conditions as the area has been completely rainless since 1995/1996. overexploitation of the plant resources (e.g. overgrazing and overcutting) may also responsible for the decrease of species diversity in this region (ali et al. 2000). the common perennial species are: acacia ehrenbergiana, aerva javanica, fagonia indica, acacia tortilis subsp. raddiana, solenostemma arghel, pulicaria crispa, acacia tortilis subsp. tortilis, calotropis procera, morettia philaeana and leptadenia pyrotechnica. the common annuals are astragalus vogelii, pulicaria incisa, glinus lotoides, indigofera argentea and cotula acta bot. croat. 69 (1), 2010 39 desert vegetation spatial heterogeneity tab. 2. means of soil characteristics of the eight vegetation clusters identified in wadi allaqi. cluster ph ec ms–1 physical characteristics chemical characteristics silt clay total sand organic matter hco3 cl ca mg % mg (100 g)–1 i 7.5 83.7 11.5 2.5 85.5 0.8 42.4 13.7 12.1 2.7 ii 7.4 105.3 10.7 2.4 85.1 1.1 46.3 11.9 11.4 4.1 iii 7.5 122.9 11.0 1.9 85.1 1.2 34.6 10.0 12.2 3.0 iv 7.4 129.9 10.8 2.7 85.1 1.0 29.3 12.9 9.1 7.1 v 7.6 117.5 11.4 3.1 83.6 1.1 38.9 12.8 10.6 5.2 vi 7.6 108.1 10.9 3.4 84.2 1.0 38.0 12.2 13.4 3.2 vii 7.6 166.7 11.9 5.2 80.8 1.7 35.3 11.3 11.1 6.3 viii 7.6 413.3 12.0 8.1 78.4 1.3 39.8 16.0 20.7 14.6 u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:47 color profile: disabled composite 150 lpi at 45 degrees 40 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. tab. 3. the species recorded in wadi allaqi and their families, floristic categories and life forms. cosm – cosmopolitan, pal – palaeotropical, pan – pantropical, tr – tropical, sa-si – saharosindian, s-z – sudano-zambezian, me – mediterranean, and ir-tr – irano-turanian. the life forms are ph – phanerophyte, ch – chamaephyte, h – hemicryptophyte, g – geophyte, he – helophyte, p – parasite, and th – therophyte. species family floristic category life form aerva javanica (burm.f) juss.ex schult. amaranthaceae sa-si+s-z. ch abutilon pannosum (g.forst. f.) schltdl. malvaceae s-z+sa-si. ch faidherbia albida (delile). leguminosae s-z ph acacia ehrenbergiana (hayne). leguminosae s-z+sa-si. ph acacia nilotica l. (delile) leguminosae s-z ph acacia tortilis subsp. raddiana savi leguminosae s-z+sa-si. ph acacia tortilis subsp. tortilis (forssk.) hayne leguminosae s-z+sa-si. ph aizoon canariense l. aizoaceae s-z+sa-si. th anticharis glandulosa (asch). scrophulariaceae sa-si+s-z. th aristida adscensionis l. gramineae pan th aristida mutabilis (trin. and rupr.). gramineae s-z+sa-si. th arnebia hispidissima (lehm.) dc boraginaceae s-z+sa-si. th asphodelus fistulosus cav. liliacaae me+sa-si+ir-tr th astragalus eremophilus bioss leguminosae sa-si+s-z. th astragalus vogelii (webb) bornm. leguminosae sa-si+s-z. th balanites aegyptiaca (l.) del. balanitaceae s-z+sa-si. ph calotropis procera (ait.) asclepiadaceae sa-si+s-z. ph capparis decidua (forssk.) edgew capparaceae s-z+sa-si. ph senna italica (mill.). leguminosae s-z+sa-si. ch senna alexandrina (mill.). leguminosae s-z+sa-si. ch caylusea hexagyna (forssk.) m. l. green resedaceae s-z+sa-si. th chenopodium album l. chenopodiaceae cosm. th chenopodium murale l. chenopodiaceae cosm. th chrozophora obongifolia (delile) spreng. euphorbiaceae s-z. ch chrozophora tinctoria l.raf. euphorbiaceae s-z+sa-si. ch cistanche phelypaea (l.) cout. orobanchaceae sa-si+s-z+ir-tr+me. p citrullus colocynthis (l.) schrad. cucurbitaceae sa-si+s-z+ir-tr+me. h cleome chrysantha.decne. cleomaceae sa-si. ch cleome droserifolia (forssk.) delile. cleomaceae sa-si+s-z. h cleome paradoxa dc. cleomaceae s-z. h cocculus pendulus (j. r. and g. forst.) diels menispermaceae s-z+sa-si. ph cotula cinerea del. compositae sa-si. th crotalaria aegyptiaca benth. leguminosae sa-si+s-z. h crotalaria microphylla vahl leguminosae s-z. th crypsis schoenoides (l)lam gramineae cosm. th cymbopogon schoenanthus l. spreng. subsp proximus a.rich gramineae sa-si. g cynodon dactylon (l.) pers gramineae pan. g u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:47 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 69 (1), 2010 41 desert vegetation spatial heterogeneity species family floristic category life form cyperus laevigatus l. cyperaceae pan. g dichanthium foevulatum delile. gramineae sa-si+s-z+ir-tr+me. h dipterygium glaucum decne. cruciferae s-z. ch eragrostis aegyptiaca (willd.) delile. gramineae s-z. th euphorbia forsskalii j. gay. euphorbiaceae s-z+sa-si. th euphorbia granulata forssk. euphorbiaceae s-z+sa-si. th fagonia bruguieri dc. zygophyllaceae sa-si+ir-tr. h fagonia glutinosa delile zygophyllaceae sa-si. h fagonia indica burm.f. zygophyllaceae sa-si+s-z. ch farsetia aegyptiaca cruciferae s-z+sa-si. ch fimbristylis bis-umbellata (forssk) bubani. cyperaceae pal. th forsskalea tenacissima l. urticaceae sa-si+s-z. h glinus lotoides l. molluginaceae pal. th haplophyllum tuberculatum (forssk.). juss rutaceae sa-si+ir-tr. ch heliotropium arbainense (fresen.) boraginaceae sa-tr+-s-z ch heliotropium pterocarpum hochst boraginaceae sa-tr+-s-z th heliotropium supinum (l.) boraginaceae s-z+me th hyoscyamus muticus (l.) solanaceae sa-si+ir-tr. ch hyphaene thebaica (l.) mart. palmae s-z. ph imperata cylindrica l. gramineae pan. h indigofera argentea (burm.) leguminosae s-z+sa-si. h iphiona scabra dc. compositae sa-si+s-z. ch leptadenia pyrotechnica (forssk.) decne asclepiadaceae s-z+sa-si. ph lotononis platycarpa (viv.) pic serm. leguminosae s-z+sa-si. th lotus sp. l. leguminosae s-z+sa-si. th lotus deserti tackh. et boulos leguminosae sa-si. h lupinus digitatus forssk.ssp orientalis sensu. täckh. leguminosae me. th maerua crassifolia (forssk.) capparaceae s-z. ph monsonia heliotropoides (cav.) boiss. geraniaceae sa-si+s-z. h morettia philaeana (del.) dc. cruciferae sa-si+s-z. h ochradenus baccatus del. resedaceae sa-si+s-z. ph orobanche ramosa l. orobanchaceae me+ir-tr p panicum turgidum (forssk.) gramineae sa-si+s-z+ir-tr+me g pergularia tomentosa l. asclepiadaceae s-z+sa-si. ch phragmites australis (cav.)trin ex steud gramineae pal. he polycarpaea repens (forssk.) asch. et schweinf. caryophyllaceae sa-si+s-z. h psoralea plicata delile leguminosae sa-si+s-z. ch pulicaria crispa (forssk.) oliv. compositae s-z+sa-si. ch pulicaria incisa (lam.) dc. compositae s-z+sa-si. h tab. 3. – continued u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:47 color profile: disabled composite 150 lpi at 45 degrees 42 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. species family floristic category life form reseda pruinosa (delile) resedaceae sa-si th ricinus communis l. euphorbiaceae pan. ch rumex dentatus l. polyganacaea me+ir-tr th salsola imbricata (forssk.) chenopodiaceae sa-si+s-z. ch salvadora persica l. salvadoraceae s-z+sa-si. ph senecio flavus (decene) sch. bip. compositae sa-si+s-z. th sesbania sesban (l) merr. leguminosae s-z. ph solenostemma argel (del.) hayne asclepiadaceae sa-si+s-z. ph sonchus oleraceus l. compositae cosm. th stipagrostis plumosa (l.) munro ex t. andersson. gramineae sa-si+s-z+ir-tr+me. h tamarix aphylla (l.) h. karst. tamaricaceae sa-si+s-z+ir-tr+me. ph tamarix nilotica (ehrenb.) bunge. tamaricaceae sa-si+s-z+ir-tr+me. ph tephrosia purpura (l.) pers. leguminosae s-z+sa-si. ch trianthema triquetra (forssk). aizoaceae pal. th tribulus ochroleucus (maire) zygophyllaceae pan. th tribulus pentandrus forssk. zygophyllaceae s-z+sa-si. th trichodesma africanum (l.) r. br. boraginaceae s-z+sa-si. ch typha domingensis (pers.) poir. ex. steud. typhaceae pan. he zea mays l. gramineae cultivated. th zilla spinosa l. cruciferae sa-si. ch ziziphus spina-christi (l.) desf. rhamnaceae s-z+sa-si. ph zygophyllum simplex l. zygophyllaceae s-z+sa-si. th tab. 3. – continued tab. 4. simple linear correlation coefficient (r) between the soil variables and dca axes. *: p < 0.05, **: p < 0.01. edaphic variable dca axis ph ec hco3 cl ca mg silt clay sand ax1 ax2 ax3 ms–1 mg 100 g–1 % ax1 1.00 ax2 –0.22 1.00 ax3 0.33 0.16 1.00 ph 0.65 0.06 0.37 1.00 ec ms–1 0.81** –0.10 0.40 0.34 1.00 hco3 m g 10 0 g– 1 –0.32 0.67 –0.02 –0.08 0.00 1.00 cl 0.32 0.09 0.67 0.04 0.69 0.24 1.00 ca 0.59** 0.24 0.37 0.42 0.88** 0.29 0.67 1.00 mg 0.78* –0.27 0.40 0.19 0.95** –0.11 0.74* 0.72* 1.00 silt % 0.70 0.23 0.76* 0.59 0.64 0.08 0.49 0.55 0.58 1.00 clay 0.89** 0.05 0.50 0.50 0.93** 0.03 0.67 0.80* 0.90** 0.77* 1.00 sand –0.94* 0.03 –0.45 –0.58 –0.90** 0.02 –0.53 –0.73* –0.86** –0.81* –0.98** 1 o.m 0.76* –0.14 –0.01 0.47 0.38 –0.24 –0.22 0.10 0.37 0.54 0.52 –0.67 u:\acta botanica\acta-botan 1-10\sheded.vp 13. travanj 2010 9:22:15 color profile: disabled composite 150 lpi at 45 degrees cinerea. these species were also recorded by (kassas and girgis 1965, sheded 1992) as dominant species in some wadis in the eastern desert of egypt including wadi allaqi. three species were recorded for the first time in wadi allaqi: iphiona scabra, chenopodium album and lotus deserti. however, sheded (1992) recorded iphiona scabra in the red sea region (never recorded by other authors, such as ali et al. 1997& 2000). the appearance of these species may be due to the increasing grazing pressure in wadi allaqi that extends to the sudanese borders, the cultivation of some crops along the shoreline of lake nasser, the effects of lake nasser itself on the migration of certain species, and the camel trade between sudan and egypt (ali et al. 2000). the life form spectrum reflects a typical desert flora, the majority of species being therophytes and chamaephytes (about 57 %). life forms of desert plants are also closely related with topography (kassas and girgis 1965, zohary 1973 and orshan 1986). according to hassib (1951), therophytes are the most common life form in the egyptian flora. the communities of wadi ecosystems of the basement complex country are demonstrated by kassas and girgis (1969) into four types: ephemeral, suffrutescent woody, suffrutescent succulent and scrubland types. in the present study, the ephemeral type is represented by morettia philaeana and fagonia indica in the downstream and midstream parts of wadi allaqi. the suffrutescent type is represented by the community of aerva javanica and senna alexandrina in wadi tributaries particularly at the middle part. the suffrutescent succulent type is represented by salsola imbricata and aerva javanica which clearly appear in the main channel of wadi allaqi and some of its tributaries. the scrubland type is represented by the following shrubs and trees (see also sheded 1992): 1tamarix nilotica community,common in the downstream part, 2salvadora persica community, which appears in small scale in the upstream part (associated with solenostemma arghel, leptadenia pyrotechnica, balanites aegyptiaca, acacia tortilis subsp. raddiana and acacia tortilis subsp. tortilis), 3balanites aegyptiaca community, well represented in the upstream and midstream parts (associated with salvadora persica, acacia tortilis subsp. raddiana, acacia tortilis subsp. tortilis and calotropis procera), 4leptadenia pyrotechnica community, well represented in the upstream part (mainly associated with calotropis procera, solenostemma arghel and maerua crassifolia), 5acacia ehrenbergiana, which community could be dominant in the downstream part and in some scattered small-sized acta bot. croat. 69 (1), 2010 43 desert vegetation spatial heterogeneity i ii iii iv v vi vii viii 0 50 100 150 200 250 300 350 0 100 200 300 400 500 600 700 dca axies 1 (x) d c a a x ie s 2 (y ) a b c d fig. 5. dca ordination of the eight vegetation clusters identified in wadi allaqi. u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:48 color profile: disabled composite 150 lpi at 45 degrees places. 6acacia tortilis subsp. tortilis community, well represented in the upstream part (associated with acacia tortilis subsp. raddiana and balanites aegyptiaca), 7acacia tortilis subsp. raddiana community well represented in all parts of the wadi (in the downstream and midstream parts it is associated with acacia ehrenbergiana but in the upstream part with acacia tortilis subsp. tortilis, leptadenia pyrotechnica, balanites aegyptiaca, solenostemma arghel and salvadora persica). soil analysis in the present study, indicated that some stands appear to be acidic (it is indicative that the ph of some stands in cluster ii was 6.7). this is may be due to the inundation of these stands by the water of lake nasser, as well as the effects of livestock excreta on the soil characteristics. the soils of wadi allaqi are generally not highly saline, salt being brought to the surface by evaporation following surface irrigation (pulford et al. 1992). however, the electric conductivity in some stands in sidinab area (wadi umm hambol is located in the downstream part of wadi allaqi) is abnormally high values (up to 2960 ms–1) where tamarisk woodland is common. tamarix has been identified as a major cause of salt accumulation on the soil surface (ali 1987, springuel and ali 1990). tamarix also is known to concentrate a high amount of sodium chloride in specialized glands in its leaves (bosabalidis 1992). in addition, there is a relationship between the amount of tamarix litter and the electric conductivity of soil (ali 1987, briggs et al. 1993 and ali et al. 2001). in the downstream part, soil characteristics support the vegetation clusters. this part was inundated by lake nasser water due to the rising of water levels and the soil was characterized by relatively high contents of clay and silt. in contrast, the soil of the clusters in the other parts was not affected by inundation processes, and consequently, the sand fraction was relatively high (see the study of sheded 1992). the sand fraction was relatively high in the stands of the upstream part (eigat core area) compared with the other stands. this may be due to the weathering process of granite components, which distinguished the upstream part of wadi allaqi. organic matter had a wide range of variation in relation to the different vegetation clusters, especially in the downstream part of wadi allaqi. livestock and birds play an important role, due to the deposition of their dung in the soil, dead fish, due to the activity of anglers in lake nasser, as well as the little microbial activity in the soil surface (volk and loeppert 1982). the eight vegetation clusters, identified after twinspan, are categorized along the dca axes 1 and 2 into 4 distinct groups (hill 1979 a, b). the stands belonging to group a (clusters i and ii) are mainly located in the upstream part (eigat core area) and their soil differs from the that of other areas by a higher concentration of bicarbonates, calcium, magnesium and chlorides, perhaps due to animal grazing, rainfall, floods and their effects on the parent rocks (pulford et al. 1992).the stands of group b (cluster vi) are located in the middle part, those of group c (clusters iii, iv and v) are located in the downstream and middle parts, while those of group d (clusters vii and viii) are located in the downstream part (some stands are sometimes inundated by the water of lake nasser, and their soils are sandy loam)(sheded 1992). references ali, m. m., 1987: studies on the shoreline vegetation of aswan high dam lake (lake nasser) and impacts of the lake on the desert. msc. thesis, assiut university, assiut. 44 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:48 color profile: disabled composite 150 lpi at 45 degrees ali, m. a., badri, m. a., moalla, s. n., pulford, i. d., 2001: cycling of metals in desert soils: effects of tamarix nilotica and inundation by lake water. environmental geochemistry and health 23, 373–382. ali, m. m., badri, m. a., hassan, l. m., springuel, i. v., 1997: effects of physiogeographical factors on desert vegetation, wadi allaqi biosphere reserve, egypt: multivariate analysis. ecologie 28, 119–128. ali, m. m., dickinson, g., murphy, k. j., 2000: predictors of plant diversity in a hyperarid desert wadi ecosystem. journal of arid environments 45, 215–230. ayyad, m. a., ghabbour, s. i., 1986: hot deserts of egypt and sudan. in: evenari, m., noy-meir, i., goodall, d. w. (eds.), ecosystems of the world, 12b, hot desert and arid shrublands, b, 149–202. elsevier, amsterdam. batanouny, k. h., 1979: the desert vegetation in egypt. cairo university african studies revue, special publication 1, 9–37. belal, a. e., springuel, i. v., 1996: economic value of plant diversity in arid environments. nature and resources 32, 33–39. bosabalidis, a. m., 1992: a morphological approach to the question of salt gland lifetime in leaves of tamarix aphylla l. israel journal of botany 41, 115–121. boulos, l., 1999: flora of egypt, 1 (azollaceae – oxalidaceae). al hadara publishing, cairo. boulos, l., 2000: flora of egypt, 2 (geraniaceae – boraginaceae). al hadara publishing, cairo. boulos, l., 2002: flora of egypt, 3 (verbenaceae – compositae). al hadara publishing, cairo. briggs, j., dickinson, g., murphy, k., pulford, i., belal, a. e., moalla, s., springuel, i., ghabbour, s. i., mekki, a. m., 1993: sustainable development and resource management in marginal environments: natural resources and their use in wadi allaqi region of egypt. applied geography 13, 259–284. digby, p. c. n., kempton, r. a., 1987: multivariate analysis of ecological communities. chapman and hall, london. el-sharkawi, h. m., salama, p. m., fayed, a. a., 1987: vegetation of inland desert wadis in egypt. 8: vegetation of wadi kharit. feddes repertorium 98, 543–547. hassib, m.,1951: distribution of plants in egypt. bulletin of the faculty of science fouad 1 university. 29, 59–261. hill, m. o., 1979a.: decorana: a fortran program for detrended correspondence analysis and reciprocal averaging. cornell university, ithaca, new york. hill, m. o., 1979b: twinspan: a fortran program for arranging multivariate data in an ordered two-way table by classification of the individuals and attributes. cornell university, ithaca, new york. jackson, m. l., 1977: soil chemical analysis. prentice-hall, new delhi. kassas, m., girgis, w. a., 1964: habitat and plant communities in the egyptian desert. v: the limestone plateau, journal of ecology 52, 107–119. acta bot. croat. 69 (1), 2010 45 desert vegetation spatial heterogeneity u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:48 color profile: disabled composite 150 lpi at 45 degrees kassas, m., girgis, w. a., 1965: the units of a desert ecosystem. journal of ecology 53, 715–728. kassas, m., girgis, w. a., 1969: plant life in the nubian desert east of the nile. bulletin de l’institute d,égypte 31, 47–71. kassas, m., imam, m., 1954: habitat and plant communities in the egyptian desert. iii. the wadi bed ecosystem. journal of ecology 42, 242–441. kassas, m., imam, m., 1959: habitat and plant communities in the egyptian desert. iv. the gravel desert. journal of ecology 47, 289–310. kilmer, v. j., alexander, l. t., 1949: methods of making mechanical analysis of soil. soil science 86, 15 – 24. moalla, s. m. n., pulford, i. d., 1991: survey of soil resources in wadi allaqi. allaqi project working paper 18. university of glasgow and faculty of science at aswan, assiut university. muller-dombois, d., ellenberg, h., 1974: aims and methods of vegetation ecology. john wiley and sons, new york. orshan, g., 1986: the desert of the middle east. in: evenari, m., noy-meir, i., goodall, d. w., (eds.), ecosystems of the world, 12 b, hot deserts and arid shrublands, 1–28. el-sevier, elsevier, amsterdam. ozenda, p., 1958: flore du sahara septentrional. c.n.r.s., paris. pulford, i. d., murphy, k. j., dickinson, g., briggs, j. a., springuel, i., 1992: ecological resources for conservation and development in wadi allaqi, egypt. botanical journal of the linnaean society 108, 131–141. raunkier, c., 1934: life forms of plants and statistical plant geography. the clarendon press, oxford. sheded, m. g., 1992: environment and vegetation in the south eastern desert, egypt. phd. thesis, faculty of science at aswan, assiut university. sheded, m. g., 2002: vegetation analysis in the south eastern desert of egypt. journal of biological science 2, 573–581. springuel, i., ali, m. m., 1990: impact of lake nasser on desert vegetation in desert development. proceedings 2 international desert development conference, cairo, 557–568. springuel, i., el-hadidi, m. n., sheded, m. g., 1991: plant communities in the southern part of the eastern desert (arabian desert) of egypt. journal of arid environments 21, 307–317. springuel, i., sheded, m. g., 1991: spatial analysis of the plant communities in southern part of the eastern desert of egypt. journal of arid environments 21, 319–325. springuel, i., sheded, m. g., murphy, j. k., 1997: the plant biodiversity of the wadi allaqi biosphere reserve (egypt): impacts on lake nasser on a desert wadi ecosystem. biodiversity and conservation 6, 1259–1275. tackholm, v., 1974: student’s flora of egypt. cairo university publication. zohary, m., 1973: geobotanical foundations of the middle east gus. fischer verlag, stuttgart. volk, b. g., loeppert, r. h., 1982: soil organic matter. in kilmer, v. j. (ed.), handbook of soils and climate in agricuture, 211–268. crc press, boca raton, florida. 46 acta bot. croat. 69 (1), 2010 shaltout k. h., sheded m. g., salem a. i. u:\acta botanica\acta-botan 1-10\sheded.vp 9. travanj 2010 12:09:48 color profile: disabled composite 150 lpi at 45 degrees 534 el-sheikh.vp acta bot. croat. 72 (1), 97–111, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 population structure of woody plants in the arid cloud forests of dhofar, southern oman mohamed a. el-sheikh*, ** department of botany, faculty of science, damanhour university, damanhour, egypt abstract – this study evaluates the size frequency distribution of 11 trees and shrubs in the cloud forest in wadi garziz, southern oman, in order to assess their current situation, which is affected by cutting, overgrazing and other constraints. a size class distribution and a vegetation structure analysis were applied in order to analyze the dynamics of this forest using census data from 51 plots selected across 5 transects covering the hill-slope and wadi-bed habitats. some of the trees inhabiting the hill-slopes (e.g. acacia senegal and commiphora spp.) were found to exhibit an inverse j-shaped distribution with constant regeneration, whilst in the wadi bed these same trees exhibited a j-shaped distribution (i.e. of declining populations). on the other hand, acacia etbaica inhabiting the hill-slopes exhibited a tendency towards a j-shaped distribution and an inverse j-shaped distribution in the wadi-bed. all the populations (i.e. inhabiting the hill-slopes and the beds) of anogeissus dhofarica had a j-shaped distribution whereas all the populations of blepharispermum hirtum had a more or less inverse j-shaped distribution. the ziziphus spina-christi and acacia tortilis populations, meanwhile, exhibited the bi-modal shape of size distribution. the shrubs inhabiting the hill-slopes (e.g. coroton confertus and ormocarpum dhofarense) exhibited a tendency towards a j-shaped distribution; such distribution characterizes a declining population with a limited regenerational capacity. overall, most of the examined populations, except those of acacia etbaica in wadi-bed and blepharispermum hirtum, seemed to be under stress from both environmental and human factors, particularly in the wadi bed. this type of study can provide a basis for the development of a management plan to support the conservation and sustainable use of forest vegetation in an arid region. key words: cloud forest, distribution of trees, ecosystem management, seed dispersal introduction oman is situated in the latitudinal belt of subtropical deserts of the northern hemisphere. water is generally considered to be the most important limiting factor for the acta bot. croat. 72 (1), 2013 97 * corresponding address, e-mail: el_sheikh_eg@yahoo.co.uk **present address: botany and microbiology department, college of science, king saud university, p. o. box 2455, riyadh 11451, saudi arabia copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:29 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees germination, growth and productivity of woody plants in desert ecosystems like those of oman (baxter et al. 2005, shaltout et al. 2009). in the forests of northern oman, for example, fisher and grander (1995) have identified variations in the population size and regeneration of juniper woodlands as being affected by the microclimate, topography and hydrology of the area. in addition to these environmental and climatological factors, however, human activities (primarily over-grazing by livestock and related disturbances) are one of the principal influences on woodland species (oliver 1980, agren and zachrisson 1990, skarpe 1990, stewart and rose 1990, welden et al. 1991, veblen 1992, sakio 1997, lykke 1998, sakio et al. 2002, tanaka et al. 2008). even within desert regions, there may be significant local variations in water limitation. in cloud forests, for example, cloud condensation in fog-affected escarpment mountain slopes and fog precipitation during the summer season create a niche for moist woodland vegetation within the surrounding desert. in a cloud forest, woodland vegetation is able to gain enough water for survival through enhanced capture (filtering) of cloud water by tree canopies or from horizontal precipitation. this is the case in parts of the dhofar mountains of oman, which are a remnant of a much more extensive moist vegetation belt, which once spread across the arabian peninsula (radcliffe-smith 1980). the cloud forest of the dhofar mountains is classified as a centre of plant diversity in the arabian peninsula, with approximately 750 species, of which some 60 are endemic (radcliffe-smith 1980, miller 1994). dhofar, like other areas of oman, is developing rapidly, and this development has the potential to put the ecosystem under stress through increased human activities, such as cutting for firewood, housing, road building, and also from overgrazing. one of the features of a cloud forest is that a large proportion of total precipitation is filtered by the very presence of vegetation. once vegetation is degraded, therefore, it becomes more difficult to re-establish due to the reduced level of filtered precipitation. over-use of the dhofar cloud forest has the potential, therefore, to seriously threaten this diverse ecosystem (miller and morris 1988, ghazanfar 1998). throughout dhofar an urgent need exists for conservation measures and the adoption of sustainable utilization methods for keystone tree species, so that further degradation of natural resources can be avoided. this is especially the case since trees and shrubs can be of high ecological, economic and medicinal importance in arid regions (miller and morris 1988, sheded et al. 2006, briggs et al. 2007, shaltout et al. 2009). efficient management plans, however, must be based on knowledge of the current situation and the trends towards change in that situation. the present work assessed the actual status of tree and shrub populations in the dhofar cloud forests on the basis of static vegetation data. for this, the size structure of 11 populations of trees and shrubs in wadi garziz, southern oman, were analyzed. based on this analysis, factors affecting the survival of individuals in each population were identified and the regeneration, stability and decline status of these populations were described. the paper uses size-class distribution to consider the nature of the population changes in severely disturbed sites in the cloud forest and seeks to identify causes of population stress in order to inform subsequent population management plans. the structure of a population of plants may be described in terms of the age and size of the population, and the form of its life stages. since the fecundity and survival of plants are much more closely related to size than age (harper and white 1974, harper 1977, weiner 1986), some authors, e.g. (werner and casswell 1977, casswell 1986, shaltout and 98 acta bot. croat. 72 (1), 2013 el-sheikh m. a. 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:29 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees ayyad 1988, shaltout et al. 2009) have argued that it is preferable to classify the life history of a plant by size rather than by age. size-class distribution of trees has traditionally been employed in studies of forests specifically as an indicator of changes in species composition (anonymous 1983, newbery and gartlan 1996, poorter et al. 1996, lykke 1998). a reverse j-shaped size-class distribution curve is usually characteristic of a species with good regeneration and continuous replacement, whereas distribution curves deviating from such a reverse-j shape tend to indicate a lack of recruitment and may indicate changes in species composition (hall and bawa 1993, lykke 1998). like all ecosystems, forests undergo changes brought about by a combination of endogenous and exogenous processes (richard and daniel 2004). the population size structure of a woody species reflects regeneration processes and, when compared with the spatial structure of the forest, can reveal important insights regarding forest dynamics (takahashi et al. 2001, hou et al. 2004). study area the study area is wadi garziz, 17° 07' 55" n, 54° 01' 59" e, which is a part of the dhofar region, southwest oman. along the southern coast, there is a range of limestone mountains covered by a woody vegetation type unique in arabia (miller and morris 1988). the mountains extend eastwards from the yemen border as a crescent-shaped escarpment with a length of about 290 km (fig. 1). the top of the range consists of a rolling plateau rising to over 2100 m, dissected by deep wadis (el-baz 2002). there are no permanent water-courses, but pools persist in the wadi beds and a series of spring-fed pools can be found along the foot of the mountains. from june to mid-september the south-facing escarpments acta bot. croat. 72 (1), 2013 99 arid cloud forests of dhofar fig. 1. map of oman showing the study area of wadi garziz (a), and a topographical map showing wadi garziz and a part of plain of dhofar with 5 transects (b). 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees are blanketed by moisture-laden clouds, fed by southwest monsoon weather patterns (fig. 2). due to this, the region hosts a unique seasonally water-limited cloud forest, sustained by water droplets from the foggy clouds that condense on the plant surfaces and thence supply water to the soil. during the dry season, the trees can take up the required water from the supplemental water reserves percolated down to the ground water (radcliffe-smith 1980, miller and morris 1988, hildebrandt and eltahir 2006). meteorological data for the region from the period 1942–1972 (sale 1980) indicate that mean temperatures at the coastal plain of salalah reach a high of 32 °c in may and june, and a low of 27 °c in january and february. the highest relative humidity (97%) is in july and august during the period of monsoon currents, and the lowest (40%) is in february. unfortunately no data from continuous measurements are available for the mountains. however, at 'qayrun heriti' village, near the mountain, the total rainfall during the monsoon season is more than 200 mm in june, july and august (fig. 3) (anonymous 2004). the soil of the wadi beds is alluvial with fine particles (el-baz 2002). 100 acta bot. croat. 72 (1), 2013 el-sheikh m. a. fig. 2. meteorological situation in dhofar during the monsoon, and a diagrammatic representation of a section through dhofar showing the main habitats from sea to desert: 0 – ocean, 1 – coastal, 2 – foothills, 3 – escarpment, 4 – dry plateau, 5 – north cliffs, 6 – desert (modified from hildebrandt et al. 2007). 0 20 40 60 80 100 120 j a n f e b m a r a p r m a y j u n j u l a u g s e p o c t n o v d e c r a in fa ll (m m ) salalah qayrun heriti fig. 3. mean rainfall (in mm) in salalah (elevation: 25 m) and the mountain village 'qayrun heriti' (elevation: 880 m a.s.l.). 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees materials and methods a field survey was carried out in the western slope (600–1200 m a.s.l.) and the wadi-bed (500–600 m a.s.l.) of wadi garziz. the study was carried out during winter in 2007 after the monsoon season. five w–e transects were established in the study area (fig. 1a and b) and 51 plots of 20 x 20 m size were selected (27 plots on the hill-slope and 24 on wadi-bed) with the variation in the number of plots depending on the habitat heterogeneity. in each plot eleven taxa of trees and shrubs of the cloud forest were sampled (tab. 1). these species were selected as they are the most common species, they are native and endemic woody species and constitute the main body of the forest, which is under severe anthropogenic disturbances. these species were identified according to miller and morris (1988) and include eight trees (acacia tortilis, acacia etbaica, acacia senegal, anogeissus dhofarica, commiphora habessinica, commiphora gileadensis, blepharispermum hirtum and ziziphus spina-christi) and three shrubs (croton confertus, ormocarpum dhofarense and sideroxylon mascatense). seed and fruit dispersal types were described according to the scheme of dansereau and lems (1957), which distinguishes dispersal types primarily according to the morphology of the diaspore. the seed dispersal types recognized in the present study are: ballochore: parent plant with a mechanism for diaspore expulsion; sarcochore: diaspore with juicy or fleshy outer layer; pterochore: diaspore with scarious winglike appendages; desmochore: diaspore with short, stiff or hooked appendages; and pogonochore: with long hair-like appendages. the species dispersal frequency was calculated in each plot; the ratio between the number of individuals of the species and number of individuals of all species of dispersal types in the habitat. impacts of human activities such as cutting, firing, grazing and resultant gaps were recorded in each plot. in the concerned plot, the height (h) and mean crown diameter (cd: based on three crown diameters depending on the uniformity of the measured individual) of each indiacta bot. croat. 72 (1), 2013 101 arid cloud forests of dhofar tab. 1. characteristics of the sampled species in wadi gerziz, oman showing their families and dispersal type. endemic species according to (miller and morris 1988) are in bold. species family dispersal type trees acacia tortilis (forssk.) hayne fabaceae fruit legume, seed 6 mm 'ballochore' acacia etbaica schweinf. fabaceae fruit legume, seed 6 mm 'ballochore' acacia senegal willd. fabaceae fruit legume, seed 6 mm 'ballochore' anogeissus dhofarica a. j. scott combretaceae two winged 'pterochore' blepharispermum hirtum oliv. asteraceae with pappus and bristle 'pogonochore' commiphora habessinica (berg.) engl. burseraceae drupe 9 mm 'sarcochore' commiphora gileadensis (l.) c. christ burseraceae drupe 8 mm 'sarcochore' ziziphus spina-christi (l.) willd. rhamnaceae drupe 1cm, seed 5 mm 'sarcochore' shrubs croton confertus baker euphorbiaceae capsule with hairs, seed 7 mm 'desmochore' ormocarpum dhofarense hillcoat et gillett fabaceae legume 'ballochore' sideroxylon mascatense (falc.) a. dc. sapotaceae berry 15 mm 'sarcochore' 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:30 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees vidual belonging a certain species were measured (cm). species size index was calculated as: h+cd)/2 (modified from crisp and lange 1976, by shaltout et al. 2003). the size index values of each species were used to determine the frequency distribution of the different age (size) cohorts. in order to evaluate the characteristics of each size class, the total density of the plant species per ha was also calculated. mean and coefficient of variation of density, height, crown diameter and size index of each species were calculated. results vegetation structure in the vegetation of wadi garziz, acacia species represented the greatest proportion of the dominant family fabaceae, followed by burseraceae species (tab. 1). concerning seed and fruit dispersals, pogonochore (e.g. blepharispermum hirtum) species, which depend on wind dispersal, attained the highest individual density in the study area, followed by sarcochorous species that usually depend on dispersal by animals (fig. 4). because of the human impact, the cloud forest is a disturbed environment, the wadi-bed more so than the hill-slope (fig. 5). 102 acta bot. croat. 72 (1), 2013 el-sheikh m. a. 0 10 20 30 40 50 60 ball saro pter desm pogo wadi-bed hill-slope dispersal type s p e c ie s fr e q u e n c y (% ) fig. 4. the relative species dispersal frequency in the two habitats of cloud forests. ball – ballochore, saro – sarochore, pter – pterochore, desm – desmochore, pogo – pogonochore. 0 20 40 60 80 100 cutting firing grazing gap no. total impact impact type s p e c ie s fr e q u e n c y (% ) wadi-bed hill-slope fig. 5. frequency of the human impact types (%) in the two habitats of cloud forests. 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acacia etbaica exhibited twice as many individuals per hectare in the wadi-bed area (105 individuals ha–1) as on the hill-slopes (53 individuals ha–1). on the other hand, the density of most other taxa was distinctly higher on the slope plots. sometimes, they were exclusively present on slope plots. the trees acacia tortilis and ziziphus spina-christi, however, exhibited almost the same density of individuals at the hill-slope and the wadi-bed sites (tab. 2). the highest density of occurrence (398 ind./ha) was found in the blepharispermum hirtum population, followed by that of commiphora gileadensis established on the hill-slope, while the population of ormocarpum dhofarense showed the lowest density (67 individuals ha–1), being found exclusively on the hill-slopes. the height of most tree species exceeded five metres. in most cases relationships between the individual crown diameter and the height of the 11 studied species are simple linear with r values > 0.7 except for »sideroxylon mascatense and ormocarpum dhofarense«. the mean height/crown diameter ratio is >1.0 for all species (tab. 2) population size classes frequency distribution the size class frequency distribution of some trees inhabiting the hill-slopes (e.g. acacia senegal and commiphora spp.) showed an inverse j-shaped distribution, while they attained a j-shaped distribution in the wadi-bed populations (fig. 6). on the other hand, acacia etbaica inhabiting the hill-slopes demonstrated a j-shaped distribution, while in the wadi-bed it showed an inverse j-shaped distribution. the entire populations (i.e. inhabiting both of the hill-slopes and wadi-beds) of anogeissus dhofarica attained a j-shaped distribution, whereas all the populations of blepharispermum hirtum had more or less an inverse j-shaped or positively skewed size frequency distribution. meanwhile, all the populations of ziziphus spina-christi and acacia tortilis exhibited a bi-modal shaped size frequency distribution. acta bot. croat. 72 (1), 2013 103 arid cloud forests of dhofar tab. 2. mean values of some demographic variables of the common shrubs and trees in wadi gerziz, oman. b: wadi-bed, s: wadi slope, h: height and cd: crown diameter. species density (ind ha –1 ) height (cm) crown diameter (cm) h:cd size class index (cm) r-value between h and cds b trees acacia tortilis 117 106 538 341 1.57 440 0.89 acacia etbaica 53 105 413 281 1.47 348 0.92 acacia senegal 303 116 522 376 1.38 450 0.86 anogeissus dhofarica 194 139 680 462 1.47 572 0.79 blepharispermum hirtum 398 170 296 163 1.81 230 0.88 commiphora habessinica 130 35 154 100 1.53 127 0.89 commiphora gileadensis 311 — 72 18 3.90 45 0.97 ziziphus spina-christi 147 133 568 396 1.43 482 0.88 shrubs croton confertus 258 — 592 393 1.50 493 0.82 ormocarpum dhofarense 67 — 210 179 1.17 195 0.69 sideroxylon mascatense 215 — 145 75 1.94 110 0.27 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 104 acta bot. croat. 72 (1), 2013 el-sheikh m. a. 0 20 40 60 f re q u e n c y % 1-trees 0 20 40 60 0 20 40 f re q u e n c y % 0 20 40 60 commiphora habessinica 0 20 40 60 80 commiphora gileadensis 0 20 40 60 f re q u e n c y % 0 10 20 30 40 acacia etbaica 0 20 40 f re q u e n c y % 0 20 40 60 80 0 20 40 60 f re q u e n c y % 0 20 40 60 anogeissus dhofarica 2-shrubs. hillslope 0 20 40 60 80 croton confertus 0 20 40 ormocarpum dhofarense 0 20 40 60 1 2 3 4 5 6 size index classes sideroxylon mascatense 0 10 20 30 f re q u e n c y % 0 10 20 30 ziziphus spina-christei blepharispermum hirtum hillslope bed acacia senegal 0 20 40 1 2 3 4 5 6 f re q u e n c y % 0 10 20 30 1 2 3 4 5 6 size index classes size index classes acacia tortilis 1-trees fig. 6. size-frequency distribution of the 11 common woody populations in the dhofar region of oman. the ranges of size index classes are as follows: for 8 trees are 1=<100, 2 =100–250, 3=250–400, 4= 400–550, 5=550–700 and 6=>700. for the 3 shrubs are 1=<50, 2 =50–100, 3=100–150, 4= 150–200, 5=200–250, and 6=>250 cm. 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees the shrubs inhabiting the hill-slopes (e.g. coroton confertus and ormocarpum dhofarense) have a tendency towards the j-shaped distribution while sideroxylon mascatense population showed a bell-shaped distribution. (fig. 5). discussion the size differences in plant populations revealed in this study may be caused directly or through differences in growth rates due to age differences, genetic variation, heterogeneity of resources, competition and herbivory (weiner 1985). as reported by gray (1975), the topography and relative abundance of plants with a dense crown, along with the frequency and abundance of rainfall and human impacts are among the important variables to be accounted for in an attempt to quantify the capacity of species for regeneration. human impact in forest environments frequently leads to vegetation changes that take place faster than natural vegetation transformations, as human impact-caused disturbances often occur in the form of continuous and widespread stress, e.g. cutting, frequent fires, grazing, and construction of roads and buildings. in environments where population changes are significant, the impact of population change on vegetation structure might override other demographic parameters (lykke 1998). this discussion will consider the significance of the population changes in the species surveyed in this study. populations of acacia senegal, commiphora spp. and blepharispermum hirtum showed a greater density with a size distribution positively skewed or an inverse j-shaped size distribution towards the smaller individuals on the hill-slope as compared to the wadi-bed. the pattern of distribution of these species may represent rapidly growing populations with high reproductive capacity. while such distributions would tend to suggest a high level of juvenile mortality (harper 1977, el-sheikh 2005), this does not contra-indicate long-term population stability, since in most stable populations one would expect excess juveniles over mature individuals (crisp and lange 1976, goldberg and turner 1986, shaltout and ayyad 1988, el-sheikh 2005). if human activities increase in the hill-slope areas, however, small individuals will inevitably die before maturity (lykke 1998). this is well demonstrated by the commiphora spp., for example, which occurred in greater densities on the hill-slope than in the wadi-bed. whereas the steeper and less accessible hill-slopes tended to exhibit fewer signs of human activity, commiphora spp. in the wadi-bed appeared subject to over-cutting by natives on account of its aromatic secretions, which are used in folk medicine (batanouny 1987, miller 1994). a further advantage provided by the steep hill-slopes was that the tree crowns were generally arranged in a step-like fashion and tended to be asymmetrical. this imbricate arrangement allowed more sunlight to penetrate from all sides and enabled individual densities to increase through creating different microhabitats and therefore greater heterogeneity in the substrate (weissenhofer 2005). turning now to the endemic shrub ormocarpum dhofarense, this species demonstrated a lower density and a j-shaped size distribution on the hill-slopes and disappeared totally from wadi-bed. the ormocarpum dhofarense shrubs were particularly vulnerable to human impact in the wadi-bed, e.g. through overgrazing due to the palatability of this plant, its acta bot. croat. 72 (1), 2013 105 arid cloud forests of dhofar 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees edible fruits, and cutting for folk medicine, firing and construction materials. moreover, on the hill-slopes, the presence of tall trees represented a limiting factor for the shrubs with a negative influence on individual density. therefore, overall, ormocarpum dhofarense was found to be suffering from poor regeneration and insufficient seed dispersal »ballochorous« (weissenhofer 2005). among the regional endemic species blepharispermum hirtum had the highest density in the study area with an inverse j-shaped distribution. this may be because it has minute pogonochores for seed dispersal that allow dispersal by wind distribution. consequently the seeds of the plant are able to invade large areas, including those less used by human inhabitants. this suggests that micro-environmental sites are suitable for their establishment, growth and survival (grime 1979; green and johnson 1996; wang et al. 1999, 2000; manabe et al. 2000; hou, et al. 2004; el-sheikh 2005). on the other hand, only acacia etbaica, had a wide range of occurrence positively skewed towards small individuals in the wadi-bed rather than the hill-slope. this plant has a higher water demand than other acacia spp. it tends to proliferate in open environments such as hillsides and the mouths of wadi openings (delta) with deep alluvial soils that receive seasonal flood waters (batanouny 1987, miller 1994, fetene 2003). wadi mouths store a significant amount of shallow groundwater where seepage of moisture and runoff collect from the hill-slopes (miller and morris 1988, hildebrandt and eltahir 2006). moreover, the ground surface of this habitat is covered with conglomerates and boulders which decrease evaporation and provide shelter for saplings. acacia etbaica also has a hard stem nature to encroacher overgrazing and has a capacity to recover these degraded sites following enclosures of a few years (fetene 2003). field observation confirmed that this plant is able to regenerate following significant disturbance, such as cutting and overgrazing – events which also lead to a clumped distribution of young trees around older mother trees, as well as re-sprouting from dormant stems. overall, acacia etbaica ability for vigorous regeneration is a strategy well adapted to the environmental disturbance which is common in the contemporary cloud forest. the entire population of acacia tortilis and ziziphus spina-christi had the same density and bi-modal bell shaped size distribution, which tends to indicate a comparable proportion of juvenile and mature individuals. if the current situation continues, however, the bimodal size distribution would tend to predict a reduction in the population size of this species in the future (shaltout and mady 1993, al-sodany 2003, el-sheikh 2005). this distribution might have resulted from an initially unimodal size distribution with a discontinuous variation in exponential growth rates among individuals. sources of discontinuous variation may be due to environmental heterogeneity, gaps, variations in pedo-topography, altitude, impact gradients, undulation, light and soil conditions. it is known that the size structure of some species decreases with increasing undulation and decreasing soil depth (takahashi 1994, pelissier and goreaud 2001, nishimura and kohyama 2002). in addition, fluctuation in the availability of water from the short, wet, monsoon season to the long dry season may cause a variable input of new seedlings to the population each year (shaltout and ayyad 1988). regarding the gaps, once vegetation is degraded, it becomes more difficult to re-establish due to a reduced level of filtered precipitation by trees, which creates an unbalance in the availability of water (huston 1986, ghazanfar 1998, masaki et al. 1992, 106 acta bot. croat. 72 (1), 2013 el-sheikh m. a. 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees tanaka et al. 2008). moreover, mature trees change the conditions within their own microsite, which affects the establishment of the same or other species (wilson and agnew 1992) and thus the distribution of saplings is spatially associated with canopy crowns (either conspecific or heterospecific). some sapling distributions overlap mature trees of the other species along complex environmental gradients (stohlgren et al. 1998). the dominance of acacia species (as opposed to open canopy species) in the dhofar forests is reflective of their ability to survive and succeed in such adverse conditions combining long dry periods and increasing human activities. in particular it is recognised that the acacia's ability to utilise dormancy as a means of avoiding climatic stress is of great importance in allowing the colonization of habitats unfavourable for growth at certain times of the year (mahmoud 1977). in addition, the performance and vigour of acacia trees, as indicated by the density and size of individuals, is known to differ according to the type of habitat, altitude, relative ground water content in dry periods, and human activities (mahmoud 1977, batanouny 1987, fetene 2003). moreover, acacias are able to increase as the main encroacher in overgrazed habitats due to their large shallow lateral roots. tolsma et al. (1987) and skarpe (1990) attribute the success of shallow-rooted acacias as compared to deep rooted species in encroaching overgrazed areas to their better access to nutrients, including nitrogen fixation by rhizobium. this is probably of particular significance in a nutrient poor environment, such as a wadi-bed, but access to water is likely to be at least as important. therefore, the increase of acacia spp. in the cloud forest may give a good indication of the progress of wider vegetation changes that occur in the disturbed sites of this forest, from a wet closed forest habitat to more open dry savanna conditions. this steady replacement of the common and endemic woody trees and shrubs typical of the wet cloud forest is well illustrated by the case of anogeissus dhofarica (a closed canopy species) which exhibited a j-shaped distribution towards larger individuals in all habitats of the wadi, tending to indicate a declining population with limited regeneration capacity or higher mortality of young individuals. (narukawa and yamamoto 2002, el-sheikh 2005, coomes and allen 2007, tanaka et al. 2008). in conclusion, most of the studied woody trees and shrubs in the cloud forest, especially in the wadi-bed site, have been facing many stresses from human activities in southern oman during the past 40 years, due to modernization and road construction inside this natural cloud forest. some of these studied populations are the endemic species anogeissus dhofarica, maytenus dhofariensis, ormocarpum dhofarense and it is these that are considered to be among the most threatened species in the cloud forest of dhofar region due to their widespread collection for fodder, medicine, buildings and firewood (miller and morris 1988, ghazanfar 1998, brinkmann et al. 2009). in general, different types of human impacts, mainly obvious, cause degradation in the cloud forest in wadi garziz. the size structure frequencies of these woody species give a good indication of the vegetation changes consequent to this human activity and further indicate that these are occurring especially in the wadi-bed, which seems to be experiencing a greater level of disturbance than the hill-slope areas, with a number of threatened species. this information on species distributions and threats is important for formulating management plans, and the size-class assessment method can be recommended for use in disturbed environments where changes are occurring quickly and where environmental management plans are therefore needed urgently. acta bot. croat. 72 (1), 2013 107 arid cloud forests of dhofar 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acknowledgements i would thank prof. loutfy boulos botany department, faculty of science, alexandria university, prof. k. h. shaltout, botany department, faculty of science, tanta university and prof. a. k. hegazy, botany and microbiology department, college of science, king saud university for valuable comments. i also thank distinguished scientist fellowship program of king saud university and mr karl woodgett for assistance in proof reading a draft of this article. references agren, j., zackrisson, o., 1990: age and size structure of pinus sylverstris populations on mires in central and northern sweden. journal of ecology 78, 1049–1062. al-sodany, y. m., 2003: size structure and dynamics of the common shrubs in omayed biosphere reserve in the western mediterranean coast of egypt. ecologia mediterranea 29, 39–48. anonymous, 1983: ecosystem forestiers tropicaux d'afrique. orstom-unesco, paris. anonymous, 2004: water resources in sultanate of oman. ministry of regional municipalities, environment and water resources, muscut, sultanate of oman. batanouny, k. h., 1987. current knowledge of plant ecology in the arab gulf countries. catena 14, 291–316. baxter, w. j., getz, m., wayne, p., 2005: a model-farmed evaluation of elephant effects on tree, fire dynamics in africa savannas. ecological application 154, 1331–1341. briggs, j. m., schaafsma, h. d., trenkov, d., 2007: woody vegetation expansion in a desert grassland: prehistoric human impact? 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structure and vegetation dynamics of four selected one hectare forest plots in the lowland rain forests of the piedras blancas national park, costa rica, with notes on the vegetation diversity of the golfo dulce region. ph.d. thesis, university of wienna. wilson, j. b., agnew, a. d. q., 1992: positive-feedback switches in plant communities. advances in ecological research 23, 263–336. acta bot. croat. 72 (1), 2013 111 arid cloud forests of dhofar 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 534 el-sheikh.ps u:\acta botanica\acta-botan 1-13\534 el-sheikh.vp 14. o ujak 2013 10:43:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 785 kovacic social news.vp social news 785 kovacic social news.ps u:\acta botanica\acta-botan 1-13\785 kovacic social news.vp 14. o ujak 2013 12:11:00 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 785 kovacic social news.ps u:\acta botanica\acta-botan 1-13\785 kovacic social news.vp 14. o ujak 2013 12:11:00 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), s1–s4, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 second week of croatian botanical gardens and arboreta (may 14–19, 2012) following the successful first week of croatian botanical gardens and arboreta, organized at 12 locations across croatia in late may–early june of 2011, the croatian botanical society with many associates organized the second week in the spring of 2012 (may 14–19, 2012). various free events open daily to the public were organized in 14 croatian botanical gardens, arboreta and several other facilities with smaller plant collections. about ten thousand visitors participated in 187 garden-events. the dates suggested to the garden-managements as reference points were may 15 (international day of families), may 18 (international plant conservation day and international museum day) and may 22 (international day for biological diversity). this year’s central event (opening ceremony) was organized in the oldest croatian statutorily protected monument of horticultural architecture (since 1947), opeka arboretum in vinica (near the city of vara`din), founded in the 17th century. as that arboretum – once upon a time a fairytale place – today has no full-time employees, the organisation of the events was carried out by the public institution for the management of protected natural areas in the vara`din county and the near-by high school, named after the arboretum opeka mar~an. at the opening ceremony, students and their teachers presented a lovely costumed role play about the noble family that established the arboretum (»the love story of countess fernandine bombelles«), and demonstrated various crafts and school-projects all across the park (making bird-cages and »hotels« for insects, stone-carving, florist and gardening workshops, etc.). another important week-event was organized in the fran ku{an pharmaceutical botanical garden (faculty of pharmacy and biochemistry of zagreb university): the re-opening of the herbarium with ca 36 000 plant specimens collected by the famous croatian botanist fran ku{an, which is kept in the garden. the second statutorily protected zagreb university botanical garden, of the faculty of science, this year – beside everything else – organized many events for pre-school children: the »amazing forest« exhibition, puppet-shows and story-telling features inside the large persian ironwood tree (parrotia persica). in dubrovnik, many pupils, citizens and tourists attended workshops and guided tours across the garden on the island of lokrum (botanical garden of the institute for marine and coastal research, university of dubrovnik), famous for its collection of australian plants. besides lectures, tours and presentations given daily in the koti{ina visitors centre of the biokovo mountain botanical garden (nature park biokovo, city of makarska), the garden staff organized several events with children – for children (»villa kamenjarka – legend of the mt biokovo villas«, »a looong journey of a short acta bot. croat. 72 (1), 2013 s1 copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 785 kovacic social news.ps u:\acta botanica\acta-botan 1-13\785 kovacic social news.vp 14. o ujak 2013 12:11:00 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees s2 acta bot. croat. 72 (1), 2013 fig. 1. in the crown of a large persian ironwood tree. »inside the magic tree«, the youngest visitors of the botanical garden of the faculty of science (university of zagreb) are listening to the well-known croatian tale of long-ago, »stribor’s forest«, written a century ago by the famous croatian children’s writer and nobel-prize nominee, ivana brli} ma`urani} (photo by vanja stamenkovi}) fig. 2. high-and-low garden collaboration. as the velebit botanical garden (np northern velebit) is in may still under snow, biologist tea [ili} brings the mountainous garden down to the coast: children in the natural history museum rijeka, owner of the liburnian karst botanical collection, learning about plant adaptations to the rugged high-mountain conditions (photo by anita hodak) 785 kovacic social news.ps u:\acta botanica\acta-botan 1-13\785 kovacic social news.vp 14. o ujak 2013 12:11:05 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees snail«, »the brave adventures of lapitch«). because the mt velebit botanical garden (northern velebit national park) is in mid-may still under a thick snow-cap, the resources manager of the national park service went on a tour, this year again, »carrying« the garden to the coastal cities of zadar and rijeka, where she organized workshops for children, lectures and exhibitions for adults. the coastal city of ka{tela is home to the ostrog elementary school botanical garden, long-known and statutorily protected for its rich exotic plants collection. pupils and their teachers prepared various workshops and guided tours, gladly attended by the local citizens and many tourists. three new gardens joined the week in 2012, and one of them was, also in the city of ka{tela, the biblical garden of the sanctuary of our lady of a hundred rosaries. the manager of the garden prepared the guided tours, photo-exhibition and lectures with a theme of biblical plants and herbs. the city of karlovac (central croatia) is famous for its gardens and parks: two of them joined the second week, preparing various events for their students and opening their doors to the public. these are the arboretums of karlovac grammar school and the forestry and carpentry high school. the only croatian arboretum to participate in the first, but not in the second week was lisi~ine arboretum on the slopes of mt papuk (nw croatia), and for a very good reason; that large arboretum, badly damaged during the croatian war of independence (1991 – 1995), had prepared an extensive project founded by european union, and was in 2012 subjected to a broad reconstruction. at the beginning of the forthcoming third national week of croatian botanical gardens and arboreta in 2013, that beautiful arboretum has been chosen to organize the opening ceremony, to show its new face to the public. the event in acta bot. croat. 72 (1), 2013 s3 fig. 3. island garden joy. dubrovnik elementary school pupils taking part in an art-workshop on lokrum island (botanical garden of the institute for marine and coastal research, university of dubrovnik): catching the essence of the australian plant collection far-away from its home (photo by nenad jasprica) 785 kovacic social news.ps u:\acta botanica\acta-botan 1-13\785 kovacic social news.vp 14. o ujak 2013 12:11:07 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees lisi~ine arboretum will be organized by croatian forests – forest administration branch na{ice. the third week of croatian botanical gardens and arboreta across the country will be organised from monday 13th to saturday 18th of may, 2013, under the auspices and general sponsorship of croatian ministry of the environment and nature protection. sanja kova~i} croatian botanical society – section of botanical gardens and arboreta, department of botany and botanical garden, division of biology, faculty of science, university of zagreb, maruli}ev trg 9a, hr – 10000 zagreb, croatia e-mail: sanja.kovacic@biol.pmf.hr s4 acta bot. croat. 72 (1), 2013 785 kovacic social news.ps u:\acta botanica\acta-botan 1-13\785 kovacic social news.vp 14. o ujak 2013 12:11:07 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 544 vizintin et al.vp coden: abcra 25 issn 0365–0588 eissn 1847-8476 floristic analysis of a high-speed railway embankment in a mediterranean landscape goffredo filibeck1*, paolo cornelini2**, paolo petrella3 1 department of agriculture, forests, nature and energy, university of tuscia, i-01100 viterbo, italy 2 istituto sperimentale ferrovie dello stato, roma, italy 3 via tuscolana 909, i-00174 roma, italy abstract – we analyzed the floristic composition of a 4.5 km-long segment of a high-speed railway in lazio, central italy, which travels on an artificial embankment through an intensively-farmed landscape. in total, 287 vascular plant species were recorded. the life-form distribution was found to be similar to that of the regional species pool, with high percentages of therophytes (38%) and phanerophytes (13%). in the chorological spectrum the mediterranean floristic element prevailed (44%), while alien species were 8% of the flora. the phytosociological spectrum showed a high diversity of characteristic species from the class stellarietea mediae or its subordinate syntaxa (26%), and in particular from the order thero-brometalia (mediterranean, sub-nitrophilous annual communities). species from forest syntaxa had a relatively high diversity (9%). these results suggest that the ecological filtering provided by the mediterranean regional climate controlled species assemblage even in a completely artificial habitat, preventing floristic homogenization: the flora of the studied railway section is only partially »ruderalized«, while it keeps strong links with the regional (semi-) natural plant communities. however, in contrast to what is observed in central and north europe, the railway sides studied in the present paper do not seem to represent a refugial habitat for rare species from grassland communities, mainly because in italy semi-natural dry grasslands are still widely represented. key words: anthropogenic, habitat, artificial soil, life-form, railway flora, lazio, italy introduction railways and railway verges are colonized by a high number of plant species, and have been the subject of botanical studies for more than 150 years (see references in: mühlenbach acta bot. croat. 71 (2), 2012 229 * corresponding author, e-mail: filibeck@unitus.it ** present address: via scandriglia 7, i-00199 roma, italy copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. acta bot. croat. 71 (2), 229–248, 2012 1979; brandes 1983, 1993, 2008; cornelini and petrella 1996; schoenenberger et al. 2002; nowi�ska and czarna 2008). some works have focused on the negative role of rail lines as dispersal pathways for alien species (schoenenberger et al. 2002, christen and matlack 2006, ranta 2008, tret’yakova 2010) – although due to the decrease in grain and livestock rail transportation in the last few decades, railways have nowadays a less rich alien flora than observed in the first half of the 20th century (brandes 1983: 38, cornelini and petrella 1996, tinner and schumacher 2004). on the other hand, several central and north european studies have underlined the role of road and railway verges and embankments in hosting a surprisingly high percentage of a country’s native floristic diversity, and have showed that they can even act as habitats for rare or endangered species (e.g. sargent 1984, brandes 1993, tikka et al. 2000, schaffers and sykora 2002, wittig 2002, tinner and schumacher 2004, ranta 2008). a floristic survey of the dutch railway network found a total of 1,026 species, i.e. 63% of the country’s flora (koster 1987), while in britain a total of 1,632 vascular plant species and subspecies was recorded from railway land (sargent 1984). in finland, 4% of the nationally endangered species regularly occur on road and rail verges (tikka et al. 2000). in natural landscapes, roads and railways mainly function as a source of fragmentation for habitats and populations (christen and matlack 2006, fischer and lindenmayer 2007). in highly transformed landscapes, on the other hand, rail and road sides – being a relatively undisturbed habitat compared to the surrounding areas (messenger 1968, brandes 1993) – may act as corridors for the survival and dispersal of plant populations from endangered natural or semi-natural communities (tikka et al. 2001, ranta 2008), especially if the railway verges are managed according to biodiversity-oriented criteria (parr and way 1988; cornelini et al. 1990; cornelini 1994a, b; tikka et al. 2000, 2001). however, in italy (and, to our knowledge, in most of mediterranean europe) botanical studies on rail lines are much scarcer than in central european countries (cf. brandes 1993: 441) and are focused on railway stations only (cacciato 1952; cornelini and petrella 1996, 1997) or on the classification of plant communities (brandes 1992; cornelini 1994a, b). in this study, we present the results of a complete floristic survey of a 4.5 km segment of a high-speed railway (i.e. a passenger rail transport system that operates above 200 km h–1), across an intensively farmed landscape within the mediterranean region of central italy. the rail line segment was built in 1975; the floristic survey took place in 1993, within a project carried out by one of the authors (p. c.) at the research institute of italian state railways, aimed at monitoring the plant-cover evolution of rail embankments without hydroseeding or other revegetation practices. the results of the floristic survey remained unpublished. we believe they are nowadays of interest, for many reasons. first, no other complete floristic studies have been performed, to our knowledge, on a long segment of an italian railway line beyond railway stations and urban areas; second, it is now very difficult to obtain permission from the railway authorities to perform botanical studies on high-speed lines, because of increased operational speeds and the stricter security policy; finally, recently enforced maintenance procedures now include much more frequent mowing of embankments than at the time of our survey, so that the data collected under a lower disturbance regime can be useful for further studies comparing the effects of different management practices. 230 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. materials and methods the surveyed railway segment is 4.5 km long and belongs to the high-speed florence-rome line. this line was the first high-speed railway opened in europe (it started operating in 1977). the studied section is located in the municipality of rome (lazio, central italy), and travels on an uninterrupted embankment, i.e. a raised earth and gravel structure (with the exception of a bridge over the river tiber), thus providing with its sides a large surface of a man-made substrate, raising above the cultivated land. the embankment begins at the end of a viaduct near settebagni (at the 18 km milepost), and ends at the 22,5 milepost (where a series of tunnels begins). it was built in 1975 and is made up of a mixture of coarse-grained soil and crushed rock that was compacted during the construction work so as to ensure mechanical resistance and low permeability. in contrast to the current usual practice in analogous earthworks, the slopes of the embankment were not sown with stabilizing grass mixtures during or after construction, so the colonizing process was completely spontaneous. ever since the line came into operation, herbicide spraying has been done on the track ballast only; the embankment slopes, at the time of the field study, were left mostly undisturbed. the dominant vegetation types on the embankment slopes and sides included: annual mediterranean grasslands, patches of perennial grasslands, hygrophilous herbaceous communities (along the drainage ditches), stands of shrubs (mostly dominated by spartium junceum) (cf. cornelini 1994b). the rail segment travels in the tiber plain, at approx. 20 m a.s.l., with a n-s orientation; the railway is more or less parallel to the riverbed, although due to the many meanders the distance between the rail line and the river banks varies between ca. 100–1000 m (except where the railway crosses the river). the plain is ca 3 km wide and is surrounded by a series of pyroclastic hills (max. altitude 150 m). the climate is mediterranean; namely, the area belongs to the meso-mediterranean, sub-humid belt, i.e. a climate type with an annual rainfall of approx. 800–900 mm, a marked summer drought from june to august and mild winter temperatures (blasi 1994). much of the floodplain surrounding the rail segment is intensively farmed: hedgerows and other residual habitats for wild plants are scarce. the river banks host poorly-preserved fragments of riparian vegetation, dominated by populus sp.pl. and salix sp. pl. the heavily-trafficked milan-rome motorway, too, travels in the floodplain parallel to the rail line, at a distance of a few hundred meters. the hill-slopes feature deciduous, xerophilous coppices dominated by quercus cerris (teucrio siculi-quercion cerridis alliance) (blasi et al. 2010, filibeck and scoppola 2011). the floristic survey was done in 1993. the whole length of the segment of the railway was walked, on both sides of the embankment, in both directions. the walk was repeated in different seasons. all vascular plant species observed on the embankment slopes, including the drainage ditches at the base, were recorded. the survey area defined in this way was ca 20 m wide on each side of the rail line. the surveyed area did not include the ballast, because on high-speed lines the permanent way is kept free of vascular plants through frequent herbicide spraying. the plant list of the first 1,000 m was recorded separately, in order to compare the species richness of a 1-km segment with that of the whole 4.5 km stretch. it was not possible (due to the inherent safety problems of working on a high-speed line) to divide the study site into statistically formalized sampling units (plots or transects) in order to obtain acta bot. croat. 71 (2), 2012 231 flora of a high-speed railway embankment quantitative data on species abundance. however, 12 phytosociological relevés were done on the embankment slopes at the beginning of the field study (and published as a synoptic table in cornelini 1994b). no significant differences were observed, during the phytosociological survey between the eastand west-facing slopes of the embankment: therefore, during the subsequent floristic survey no separate floristic lists were recorded for the two sides. identification of species and nomenclature followed pignatti (1982). each species was then assigned a life-form type and a geographic element according to pignatti (1982), in order to analyze the relationships with the regional species-pool. the subdivision of floristic zones and regions, and thus of geographic elements, used by pignatti (1982), stresses some patterns relevant for the italian peninsula (e.g. a distinction between »steno-mediterranean« and »euri-mediterranean« species is adopted) – thus it does not match the other systems used in central europe perfectly. a species was defined as »alien« if included in the check-list of italian alien taxa by celesti-grapow et al. (2010), and its status (casual, naturalized or invasive) was taken from the same source. alien »species« defined in this way include also taxa originating in cultivation. finally, each species was assigned to a phytosociological unit, in order to analyze the relationships between the floristic assemblage of the railway sides and the (semi-)natural vegetation types of the surrounding landscape. in order to do so in a standardized and repeatable way, this was done assigning to each phytosociological class or order only those plants that are considered »characteristic species« of that unit, following reference manuals (oberdorfer 1992, mucina et al. 1993) and syntaxonomical revisions (izco 1977, biondi et al. 1995, blasi et al. 2004, gigante and venanzoni 2007). results a total of 287 vascular plant species was recorded in the research area (tab.1); 207 species (72%) were found within the first 1,000 m. the richest family was found to be asteraceae (16.4%), followed by fabaceae and poaceae (ca. 12%). the life-form types with the highest diversity were therophytes and hemicryptophytes – accounting for 38% each (fig. 1). as for hemicryptophytes, many of the species found in the 232 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. fig. 1. life-form distribution in the flora of the railway segment. for abbreviations see table 1. acta bot. croat. 71 (2), 2012 233 flora of a high-speed railway embankment tab. 1. list of recorded species. legend of abbreviations – life forms: ch – chamaephytes, g – geophytes, h – hemicryptophytes, h(b) – biennial hemicryptophytes, i – hydrophytes, p – phanerophytes, t – therophytes. geographic elements: »circumboreal« includes also the euro-siberian element; »cosmopolitan« includes also the sub-cosmopolitan element; »eurasiatic« includes also the european element. origin of aliens: afr – africa, eur – europe, hort. – horticultural (taxa originated in cultivation), n amer – north america, s amer – south or central america. status of aliens in lazio: cas – casual, inv – invasive, nat – naturalized. vegetation units: av – artemisietea vulgaris, c-m – cisto-micromerietea, f-b – festuco-brometea, g-u – galio-urticetea, hg – helianthemetea guttati, hy – riparian and hygrophilous communities, m-a – molinio-arrhenateretea, qi – quercetea ilicis, q-f – querco-fagetea, r-p – rhamno-prunetea, st – stellarietea mediae (except thero-brometalia),t-brom – thero-brometalia, t-g – trifolio-geranietea. species life form geographic element origin of aliens status of aliens (lazio) vegetation units acer negundo l. p alien n amer cas agrimonia eupatoria l. h cosmopolitan t-g agropyron repens (l.) beauv. g circumboreal av ailanthus altissima (miller) swingle p alien asia inv alcea setosa (boiss.) alef. h alien eur nat alisma plantago-aquatica l. i cosmopolitan hy allium ampeloprasum l. g euri-mediterranean alopecurus myosuroides hudson t cosmopolitan st althaea cannabina l. h eurasiatic amaranthus retroflexus l. t alien n amer inv anagallis arvensis l. t euri-mediterranean st anchusa hybrida ten. h steno-mediterranean anchusa italica retz. h euri-mediterranean andryala integrifolia l. t euri-mediterranean st anthemis arvensis l. t steno-mediterranean st anthemis tinctoria l. h(b) eurasiatic av apium nodiflorum (l.) lag. h euri-mediterranean hy arenaria serpyllifolia l. t cosmopolitan hg artemisia verlotorum lamotte h alien asia inv artemisia vulgaris l. h circumboreal av arum italicum miller g steno-mediterranean g-u arundo donax l. g alien asia inv asparagus acutifolius l. p steno-mediterranean qi astragalus hamosus l. t medit.-turanic t-brom avena barbata potter t euri-mediterranean t-brom avena fatua l. t eurasiatic st avena sterilis l. t euri-mediterranean t-brom bellevalia romana (l.) sweet g euri-mediterranean bellis perennis l. h eurasiatic m-a beta vulgaris l. cv. h alien hort. cas blackstonia perfoliata (l.) hudson t euri-mediterranean hg borago officinalis l. t euri-mediterranean briza maxima l. t subtropical hg 234 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. species life form geographic element origin of aliens status of aliens (lazio) vegetation units bromus gussonei parl. t euri-mediterranean st bromus hordeaceus l. t cosmopolitan t-brom bromus madritensis l. t euri-mediterranean t-brom bromus rigidus roth t subtropical t-brom broussonetia papyrifera (l.) vent. p alien asia inv calamintha nepeta (l.) savi h montane-mediterranean calendula arvensis l. t euri-mediterranean st calystegia sepium (l.) r.br. h eurasiatic g-u campanula rapunculus l. h(b) eurasiatic capsella bursa pastoris (l.) h(b) cosmopolitan st cardamine hirsuta l. t cosmopolitan cardaria draba (l.) desv. g medit.-turanic carduus nutans l. h(b) atlantic carduus pycnocephalus l. h(b) medit.-turanic st carex caryophyllea la tourr. h eurasiatic f-b carex divisa hudson g atlantic carex divulsa stokes h euri-mediterranean carex flacca schreber g eurasiatic carex otrubae podp. h atlantic hy carlina corymbosa l. h steno-mediterranean hg celtis australis l. p euri-mediterranean centaurea solstitialis l. h(b) steno-mediterranean av centaurium erythraea rafn h(b) eurasiatic f-b cephalaria transsylvanica (l.) schrader t eurasiatic cerastium glomeratum thuill. t euri-mediterranean st cercis siliquastrum l. p eurasiatic q-f chenopodium album l. t cosmopolitan st chondrilla juncea l. h eurasiatic chrozophora tinctoria (l.) juss. t medit.-turanic cichorium intybus l. h eurasiatic cirsium arvense (l.) scop. g eurasiatic st cirsium vulgare (savi) ten. h(b) eurasiatic av clematis vitalba l. p eurasiatic q-f coleostephus myconis (l.) cass. t steno-mediterranean convolvulus arvensis l. g eurasiatic av convolvulus cantabrica l. h euri-mediterranean hg conyza bonariensis (l.) cronq. t alien s amer inv conyza canadensis (l.) cronq. t alien n amer inv cornus sanguinea l. p eurasiatic r-p crataegus monogyna jacq. p eurasiatic r-p crepis bursifolia l. h endem. crepis neglecta l. t euri-mediterranean hg crepis sancta (l.) babc. t medit.-turanic st tab. 1. – continued acta bot. croat. 71 (2), 2012 235 flora of a high-speed railway embankment species life form geographic element origin of aliens status of aliens (lazio) vegetation units crepis vesicaria l. t atlantic st cruciata laevipes opiz h eurasiatic g-u cyclamen hederifolium aiton g steno-mediterranean q-f cynara cardunculus l. h steno-mediterranean cynodon dactylon (l.) pers. g cosmopolitan cyperus longus l. g eurasiatic hy cyperus rotundus l. g cosmopolitan hy dactylis glomerata l. h eurasiatic m-a dasypirum villosum (l.) borbas t medit.-turanic t-brom daucus carota l. h(b) eurasiatic av diplotaxis tenuifolia (l.) dc. h atlantic dipsacus fullonum l. h(b) euri-mediterranean av dorycnium hirsutum (l.) ser. ch euri-mediterranean ecballium elaterium (l.) a.rich. g euri-mediterranean st echium italicum l. h(b) euri-mediterranean av echium plantagineum l. t euri-mediterranean t-brom echium vulgare l. h(b) eurasiatic f-b epilobium hirsutum l. h eurasiatic epilobium tetragonum l. h eurasiatic equisetum arvense l. g circumboreal hy equisetum ramosissimum desf. g circumboreal hy erodium malacoides (l.) l’her. t steno-mediterranean st eryngium campestre l. h euri-mediterranean f-b euonymus europaeus l. p eurasiatic r-p eupatorium cannabinum l. h eurasiatic hy euphorbia falcata l. t euri-mediterranean hg euphorbia helioscopia l. t cosmopolitan st euphorbia platyphyllos l. t euri-mediterranean euphorbia prostrata aiton t alien s amer inv euphorbia segetalis l. t steno-mediterranean fallopia dumetorum (l.) holub t circumboreal ferula communis l. h euri-mediterranean festuca arundinacea schreber h eurasiatic ficus carica l. p medit.-turanic foeniculum vulgare miller h euri-mediterranean t-brom fraxinus ornus l. p eurasiatic q-f fraxinus oxycarpa bieb. p eurasiatic hy fumaria capreolata l. t euri-mediterranean st fumaria officinalis l. t eurasiatic st galactites tomentosa moench h(b) steno-mediterranean t-brom galega officinalis l. h eurasiatic g-u galium album miller h eurasiatic f-b galium aparine l. t eurasiatic g-u tab. 1. – continued 236 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. species life form geographic element origin of aliens status of aliens (lazio) vegetation units gastridium ventricosum (gouan) sch. et th. t atlantic t-brom geranium dissectum l. t eurasiatic st geranium molle l. t eurasiatic st geranium robertianum l. t cosmopolitan st hedera helix l. p euri-mediterranean q-f hedysarum coronarium l. h steno-mediterranean heliotropium europaeum l. t euri-mediterranean st holcus lanatus l. h circumboreal m-a hypericum perforatum l. h eurasiatic t-g hypochoeris achyrophorus l. t steno-mediterranean hg hypochoeris glabra l. t euri-mediterranean hypochoeris radicata l. h eurasiatic f-b inula conyza dc. h(b) eurasiatic t-g inula viscosa (l.) aiton h euri-mediterranean iris germanica l. g alien hort. nat juglans regia l. p alien asia cas knautia integrifolia (l.) bertol. t euri-mediterranean st lactuca serriola l. h(b) eurasiatic lathyrus clymenum l. t steno-mediterranean st lathyrus pratensis l. h eurasiatic m-a lathyrus sylvestris l. h eurasiatic t-g laurus nobilis l. p steno-mediterranean qi lavatera punctata all. t steno-mediterranean lemna minor l. i cosmopolitan hy linaria vulgaris miller h eurasiatic av linum bienne miller h(b) euri-mediterranean f-b lophochloa cristata (l.) hyl. t cosmopolitan lotus corniculatus l. h eurasiatic t-g lotus ornithopodioides l. t steno-mediterranean t-brom lycopus europaeus l. h eurasiatic hy malus domestica borkh. p alien hort. cas malus sylvestris miller p eurasiatic q-f malva sylvestris l. h circumboreal av medicago lupulina l. t eurasiatic st medicago orbicularis (l.) bartal. t euri-mediterranean t-brom medicago sativa l. h eurasiatic st melica ciliata l. h euri-mediterranean f-b melilotus alba medicus t eurasiatic melilotus indica (l.) all. t medit.-turanic melilotus officinalis (l.) pallas h(b) eurasiatic melissa romana miller h steno-mediterranean mentha suaveolens ehrh. h euri-mediterranean mercurialis annua l. t eurasiatic st tab. 1. – continued acta bot. croat. 71 (2), 2012 237 flora of a high-speed railway embankment species life form geographic element origin of aliens status of aliens (lazio) vegetation units micromeria graeca (l.) bentham ch steno-mediterranean c-m myosotis arvensis (l.) hill t eurasiatic st narcissus tazetta l. g steno-mediterranean nigella damascena l. t euri-mediterranean hg odontites rubra (baumg.) opiz t eurasiatic onobrychis viciifolia scop. h montane-mediterranean f-b ononis spinosa l. ch euri-mediterranean f-b onopordum illyricum l. h(b) steno-mediterranean av ophrys apifera hudson g euri-mediterranean f-b origanum vulgare l. h eurasiatic t-g ornithogalum umbellatum l. g euri-mediterranean st oryzopsis miliacea (l.) asch. et schweinf. h steno-mediterranean oxalis corniculata l. h euri-mediterranean oxalis dillenii jacq. h alien n amer inv pallenis spinosa (l.) cass. t euri-mediterranean papaver dubium l. t medit.-turanic papaver rhoeas l. t medit.-turanic st parentucellia viscosa (l.) caruel t atlantic parietaria diffusa m. et k. h euri-mediterranean parthenocissus quinquefolia (l.) planchon p alien n amer nat petrorhagia prolifera (l.) ball et heyw. t euri-mediterranean phalaris bulbosa l. h steno-mediterranean phalaris coerulescens desf. h steno-mediterranean phalaris truncata guss. h euri-mediterranean phleum pratense l. h circumboreal f-b phragmites australis (cav.) trin. g cosmopolitan hy picris echioides l. t euri-mediterranean st picris hieracioides l. h circumboreal st pyrus pyraster burgsd. p eurasiatic q-f pisum sativum l. t steno-mediterranean plantago lanceolata l. h eurasiatic m-a poa annua l. t cosmopolitan st poa trivialis l. h eurasiatic m-a polypogon monspeliensis (l.) desf. t subtropical populus alba l. p eurasiatic hy populus nigra l. p eurasiatic hy portulaca oleracea l. t cosmopolitan st potentilla reptans l. h eurasiatic prunus armeniaca l. p alien hort. cas prunus persica (l.) batsch p alien hort. cas prunus cerasifera ehrh. p alien eur, asia cas prunus spinosa l. p eurasiatic r-p pteridium aquilinum (l.) kuhn g cosmopolitan tab. 1. – continued 238 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. species life form geographic element origin of aliens status of aliens (lazio) vegetation units pulicaria dysenterica (l.) bernh. h euri-mediterranean pyracantha coccinea roemer p steno-mediterranean r-p pyrus pyraster burgsd. p eurasiatic q-f quercus robur l. p eurasiatic hy ranunculus bulbosus l. h eurasiatic f-b raphanus raphanistrum l. t euri-mediterranean st rapistrum rugosum (l.) all. t euri-mediterranean reichardia picroides (l.) roth h steno-mediterranean t-brom reseda phyteuma l. t euri-mediterranean st robinia pseudoacacia l. p alien n amer inv rosa canina l. p eurasiatic r-p rosa sempervirens l. p steno-mediterranean qi intbl rubia peregrina l. p steno-mediterranean qi rubus ulmifolius schott p euri-mediterranean r-p rumex conglomeratus murray h eurasiatic rumex crispus l. h cosmopolitan rumex obtusifolius l. h eurasiatic g-u rumex pulcher l. h euri-mediterranean salix alba l. p eurasiatic hy salvia verbenaca l. h atlantic sambucus ebulus l. g euri-mediterranean g-u sanguisorba minor scop. h eurasiatic f-b saponaria officinalis l. h circumboreal hy scabiosa maritima l. h(b) steno-mediterranean f-b scorpiurus muricatus l. t euri-mediterranean hg securigera securidaca (l.) deg. et doerfl. t euri-mediterranean hg senecio vulgaris l. t euri-mediterranean st setaria verticillata (l.) beauv. t subtropical setaria viridis (l.) beauv. t cosmopolitan st sherardia arvensis l. t euri-mediterranean st silene alba (miller) krause h(b) eurasiatic av silene bellidifolia juss. t steno-mediterranean silene latifolia poiret h(b) steno-mediterranean g-u silene vulgaris (moench) garcke h eurasiatic sinapis arvensis l. t steno-mediterranean st solanum nigrum l. t cosmopolitan st sonchus asper (l.) hill t eurasiatic st sonchus oleraceus l. t eurasiatic st sonchus tenerrimus l. t steno-mediterranean st sorghum halepense (l.) pers. g alien afr, asia inv spartium junceum l. p euri-mediterranean r-p stachys ocymastrum (l.) briq. t steno-mediterranean stellaria media (l.) vill. t cosmopolitan st tab. 1. – continued acta bot. croat. 71 (2), 2012 239 flora of a high-speed railway embankment species life form geographic element origin of aliens status of aliens (lazio) vegetation units sylibum marianum (l.) gaertner h(b) medit.-turanic g-u taraxacum officinale weber h circumboreal m-a thymus vulgaris l. ch steno-mediterranean c-m tolpis virgata (desf.) bertol. h steno-mediterranean st torilis arvensis (hudson) link t cosmopolitan tragopogon porrifolius l. h(b) euri-mediterranean av trifolium angustifolium l. t euri-mediterranean t-brom trifolium arvense l. t eurasiatic trifolium campestre schreber t eurasiatic st trifolium fragiferum l. h eurasiatic trifolium ochroleucum hudson h eurasiatic f-b trifolium pratense l. h circumboreal m-a trifolium subterraneum l. t euri-mediterranean trisetaria panicea (lam.) maire t steno-mediterranean t-brom tussilago farfara l. g eurasiatic av typha latifolia l. g cosmopolitan hy ulmus minor miller p eurasiatic q-f urospermum dalechampii (l.) schmidt h euri-mediterranean t-brom urtica dioica l. h cosmopolitan g-u urtica membranacea poiret t steno-mediterranean st valerianella eriocarpa desv. t steno-mediterranean st verbascum blattaria l. h(b) eurasiatic av verbascum densiflorum bertol. h(b) euri-mediterranean av verbascum pulverulentum vill. h(b) eurasiatic av verbascum sinuatum l. h(b) euri-mediterranean av verbena officinalis l. h eurasiatic st veronica arvensis l. t eurasiatic st veronica cymbalaria bodard t euri-mediterranean veronica hederifolia l. t eurasiatic st veronica persica poiret t alien asia inv vicia bithynica (l.) l. t euri-mediterranean vicia hirsuta (l.) gray t eurasiatic vicia hybrida l. t euri-mediterranean vicia lutea l. t euri-mediterranean st vicia sativa l. t medit.-turanic st vicia villosa roth t euri-mediterranean t-brom vitis vinifera l. p alien hort. cas vulpia ciliata (danth.) link t euri-mediterranean t-brom vulpia ligustica (all.) link t steno-mediterranean t-brom zea mays l. t alien hort. cas tab. 1. – continued embankment communities are either biennial or rosulate plants: typical examples include daucus carota, malva sylvestris, plantago lanceolata, picris hieracioides, and four different verbascum species. phanerophytes featured 13% of the recorded flora (37 species). the geographic element with the highest number of species (fig. 2) was the eurasiatic (ca. 30%), followed by euri-mediterranean and steno-mediterranean elements – however, the sum of these latter (and of the mediterranean-turanic element) is ca 44% of the recorded flora. alien species (including taxa that originated as cultivated plants) accounted for 8.4%. alien species classified as »invasive« in lazio were 4% of the recorded flora. in the phytosociological spectrum (fig. 3), the largest element (75 species, i.e. 26%) was made up by species from stellarietea mediae, but 20 out of these 75 species belong to the thero-brometalia order. species from festuco-brometea are also represented in the embankment flora (16 species, 5.6%), while the molinio-arrhenateretea element is poorer (8 species). artemisietea vulgaris class is represented by 19 species (6.6%), and galio-urticetea features only 10 species (3.5 %). a total of 26 species (9.1%) belongs to the floristic assemblage of forest or forest-edge communities. finally, 6.3% of the flora is made up of species from hygrophilous syntaxa. discussion the total richness found (with more than 200 taxa recorded in the first 1,000 m) is considerably high compared to some north european studies on railways (cf. e.g. messenger 1968, niemi 1969, lejmbach et al. 1975), but is consistent with the high floristic richness of mediterranean landscapes (the lazio region has 3,228 taxa: conti et al. 2005) and with species-area patterns known for central italy (chiarucci et al. 2012). in the family spectrum, the richest families recorded from the embankment are the same as in the whole italian flora (abbate et al. 2007), however in the embankment flora there is an anomalous proportion of scrophulariaceae, lamiaceae and boraginaceae, due to the disturbance regime of the railway sides, which promotes the diversity of ruderal and/or biennial species 240 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. fig. 2. geographic elements in the flora of the railway segment. for explanations see table 1. – particularly well represented in these families. the most common annuals in the embankment flora included avena barbata, bromus madritensis, papaver rhoeas and sherardia arvensis (cornelini 1994b). the life-form distribution, despite the artificial and recent origin of the study site, keeps a strong similarity with the spectrum of the regional pool (fig. 4). annuals feature a higher proportion than in the flora of lazio (38% vs. 33%), as expected due to three co-occurring factors: the warm climate belt where the study area is located; the dry topsoil of the embankment; and the disturbance caused by mowing of the embankment slopes. the proportion of therophytes found here is much lower than that recorded in a railway station in rome by cornelini and petrella (1996), where 50% of a 266-species flora was made up of annuals – however, the two study sites are not comparable as the railway station includes large paved areas and/or highly disturbed habitats, and it is surrounded by a completely urbanized district. perennial herbs have a slightly higher proportion in the embankment assemblage than in the regional flora. the richness of biennial and rosulate hemicryptophytes is well known to be connected to the features of railway verges (tinner and schumacher 2004, nowi�ska and czarna 2008): traits such as buds very close to the ground, long taproots and flat rosettes allow survival in spite of frequent mechanical mowing. geophytes, although with a lower percentage than in the regional flora, are well-represented, since these plants are favorably selected by an intermediate intensity of mechanical disturbance – as already observed e.g. in the archaeological areas in downtown rome (celesti-grapow et al. 1989, celesti-grapow 1995). among the geophytes found in the embankment flora, acta bot. croat. 71 (2), 2012 241 flora of a high-speed railway embankment fig. 3. proportion of character species of different syntaxa in the flora of the railway segment. the most noteworthy include an orchid, ophrys apifera, and cyclamen hederifolium, a species usually bound to the shady understorey of sub-mediterranean woods. on the other hand, the high proportion of phanerophytes highlights the role played by the railway sides as a substitute habitat for woody species, within an intensively farmed landscape devoid of suitable microsites such as hedgerows or dry stone walls. in fact, among the phanerophytes recorded on the rail embankment, there are species from different vegetation series of the toposequence of the tiber valley, and from different seral stages of each vegetation series: populus alba and salix alba are trees from the river-side forest vegetation (ceschin and salerno 2008); fraxinus oxycarpa and quercus robur are trees which potentially dominate the alluvial, hygro-mesophytic plains (blasi et al. 2010); cornus sanguinea and euonymus europaeus are shrubby species from the wood edges of the alluvial plain forest (ceschin and salerno 2008); fraxinus ornus and pyrus pyraster are small trees of the thermophylous quercus cerris woods of the hill slopes (blasi et al. 2004); cercis siliquastrum, crataegus monogyna and pyracantha coccinea are found in the wood edges and shrubby seral stages of the q.cerris forests (blasi et al. 2004); celtis australis and ficus carica are often found on the pyroclastic cliffs and boulders (scoppola and filibeck 2008). as for the geographic element distribution (fig. 2), the mediterranean element (in a wide sense) prevails in the studied railway side flora (ca. 44%), suggesting that the mediterranean climate acts as a strong ecological filter, so that a marked link with the regional species pool is preserved even in a completely artificial habitat. the richness of alien species found in the study site (8.4%) is relatively low for an artificial, disturbed habitat, considering that in the chorological spectrum of the whole flora of the lazio region alien units reach 10.4% (conti et al. 2005). studies from central european railway floras found percentages of alien species up to 36% (fig. 5) (lejmbach et al. 1975, tinner and schumacher 2004, nowi�ska and czarna 2008, warcholi�ska and suwara-szmigielska 2009). however, these studies are not fully comparable with the present work, as they include (or are limited to) stations and/or urban habitats. alien therophytes are a group that in central and north europe is often reported to play a significant role in the flora of railway areas (e.g. niemi 1969, brandes 1983, tinner and schumacher 2004, galera et al. 2011). for instance, in a floristic study on railway lines and 242 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. fig. 4. comparison between the life-form distribution in the embankment flora (black columns) and in the flora of lazio region (grey columns). for legend of life forms, see table 1. stations of pomerania (lejmbach et al. 1975), 22.3% of the total railway flora was made up of alien therophytes (including alien biennial-hemicryptophytes). this group, by contrast, had a very low diversity in our survey: only 6 species (2.1%) of exotic annuals were found (amaranthus retroflexus, conyza bonariensis, conyza canadensis, euphorbia prostrata, veronica persica, zea mays). although our result could be influenced by the fact that in the present study the ballast was not surveyed, it has already been underlined that in the floras of mediterranean cities (e.g. celesti-grapow 1995, celesti-grapow et al. 1997) alien annuals are not as characteristic as they are in central european urban environments. this is because in the temperate biome artificial habitats perform as extrazonal, dry and warm micro-environments: the local species are less adapted to this niche, which is more competitively exploited by aliens from warmer countries. in the mediterranean biome, instead, xeric conditions are widespread and thus railway grounds do not perform as especially favorable sites for therophytes (brandes 1993: 441). in the phytosociological spectrum (fig. 3), the richest group (26%) is made up by species recognized as characteristic taxa of stellarietea mediae (or of its subordinate orders), i.e. the widely-distributed class of the annual, ruderal communities (according to grime et al. 1988), bound to highly disturbed or anthropogenic soils (e.g. mucina et al. 1993). however, a large share of the stellarietea species recorded in the embankment flora is accounted for by taxa belonging to the order thero-brometalia (=brometalia rubenti-tectorum) and in particular to the echio-galactition alliance, i.e. to the mediterranean, sub-nitrophilous and medium-sized annual vegetation communities of fallow land, minor-road sides and other edge haacta bot. croat. 71 (2), 2012 243 flora of a high-speed railway embankment fig. 5. proportion of alien species recorded in various railway floras: a) present study (8.4%); b) railway stations of ne-switzerland (19.6%) (tinner and schumacher 2004); c) powodowo station, w-poland (25.7%) (nowi�ska and czarna 2008); d) railway lines and stations of pomerania, n-poland (27.7%) (lejmbach et al. 1975); e) urban railways and stations in pabianice, c-poland (36.4%) (warcholi�ska and suwara-szmigielska 2009). bitats (izco 1977, gigante and venanzoni 2007). all these species (e.g. avena barbata, bromus hordeaceus, dasypyrum villosum, lotus ornithopodioides, medicago orbicularis, reichardia picroides), although with a partially ruderal character, have a decidedly xerophilous ecology and a strictly mediterranean range – so that many of them were until recently classified among the characteristic species of the natural and semi-natural mediterranean annual grasslands of helianthemetea guttati (=thero-brachypodietea) (e.g. rivas-martinez 1977, fanelli and lucchese 1998). further, even this latter class, interpreted in a narrower sense (i.e. without the above mentioned sub-nitrophilous species), is represented in our survey by a significant group of species (e.g. briza maxima, blackstonia perfoliata, hypochoeris achyrophorus, scorpiurus muricatus). on the other hand, the taxa from artemisietea vulgaris [i.e. the euro-siberian communities of perennial, nitrophilous to sub-nitrophilous herbs and grasses with an intermediate c-r behaviour (according to grime et al., 1988), usually typical of roadand railway-sides (mucina et al. 1993)] are not particularly numerous in the embankment (19 species, 6.6%), as their functional traits are scarcely adapted to the mediterranean climate. the same holds for the species from galio-urticetea (woodand road-fringe communities of nitrophilous, tall herbs with a c-r or c strategy) (mucina et al. 1993), featuring only 10 species (3.5 %) and from molinio-arrhenateretea (nitrogen-rich and mesophytic anthropogenic hay-meadows of the temperate region) (8 species). we defined as forest species (9.1%) the taxa known as characteristic species of the following classes (or subordinate syntaxa): quercetea ilicis (mediterranean evergreen woods and maquis: e.g. rubia peregrina, asparagus acutifolius); querco-fagetea (european deciduous forests – namely, in the surroundings of the study area, sub-mediterranean thermophilous deciduous forests of the order quercetalia pubescenti-petraeae: e.g. cyclamen hederifolium, cercis siliquastrum, fraxinus ornus); rhamno-prunetea (deciduous forest-edge shrublands: e.g. crataegus monogyna, prunus spinosa); trifolio-geranietea (communities of the partially shaded, herbaceous fringes at the edge of deciduous woodlands; e.g. agrimonia eupatoria, holcus lanatus, lathyrus sylvestris, origanum vulgare, sanguisorba minor). species from (woody or herbaceous) hygrophilous syntaxa (lemnetea, phragmito-magnocaricetea, salicetea purpureae, populetalia albae) are also found in a noteworthy proportion (6.3 %): these plants (e.g. alisma plantago-aquatica, carex otrubae, lemna minor, typha latifolia) are common within the surveyed railway sides because of the numerous drainage ditches at the foot of the embankment – and because the vicinity of the river tiber is a source of propagules (cf. ceschin and salerno 2008). thus, the spectrum of the original plant communities of the recorded species shows that the flora of the studied railway section is only partially »ruderalized«: on the contrary, it seems to keep a strong link with the floristic composition of the plant communities of its biogeographic context. this confirms the suggestion of a comparative study on urban floras of italy (celesti-grapow et al. 1997), where the floristic assemblage in cities belonging to the mediterranean sector of the country was found to have a high degree of similarity with the floristic composition of the surrounding (semi-)natural landscapes, while such a correlation was much weaker in cities of the temperate sector of italy. further, the different ecological elements that can be traced in the recorded flora underline the extremely high heterogeneity of micro-habitats within the railway sides, and hence their potential importance for biodiversity: the co-occurrence in the surveyed flora of, for 244 acta bot. croat. 71 (2), 2012 filibeck g., cornelini p., petrella p. instance, hygrophytes such as salix alba and xerophytes such as micromeria graeca highlights the very steep gradient taking place within the 20-m wide embankment. in contrast to many central and north european studies (ranta 2008 with references), no national/regional red list taxa or other particularly rare species were found. however, in temperate europe rail verges have a high percentage of rare/endangered species because they provide a much drier micro-habitat than the surrounding landscape (brandes 1993), and thus are capable of hosting extrazonal mediterranean taxa and other thermophilous plants (which include many of the red list species in central european countries). as for boreal europe, semi-natural grasslands (meadows and pastures which have originated from grazing and hay-making on natural grasslands and in woodlands) account for up to 25% of the endangered plant species, and are a particularly threatened habitat-type because of the cessation of grazing and mowing and their conversion to arable and afforested land (tikka et al. 2000, 2001): for these reasons, mowed roadand rail-verges are nowadays among the largest surviving areas of grassland habitats in north europe. this is not applicable in mediterranean europe, where dry habitats are the rule rather than the exception, and where grasslands are still widely represented (blasi et al. 2007, 2010). thus, in south europe road and rail verges are not necessarily valuable habitats in every landscape context, and probably do not play a significant role for endangered species. however, in the highly transformed landscape of the present study, the railway sides provided a decidedly less disturbed habitat (although on a completely artificial soil), containing a significant sample of the floristic assemblages of many different natural and semi-natural communities of a wider area – thus probably playing an important role, at a local scale, in maintaining diversity patterns and in providing corridors through an unfavorable landscape matrix. the present work was the first study carried out in italy – and probably in mediterranean europe – on the floristic composition of a long railway segment beyond rail stations or urban areas, and perhaps the first study ever performed in europe on a high-speed line. the floristic assemblage recorded in the studied rail embankment suggests that the ecological filter operated by the mediterranean macroclimate prevents both complete »ruderalization« and »floristic homogenization« in the flora of even a highly artificial habitat – by contrast to what is observed in temperate europe and north america, where floras of anthropogenic habitats from different countries were found to be more similar to each other than to the surrounding (semi-) natural habitats (wittig and becker 2010). further studies gathering a large amount of floristic data from railway grounds and other artificial habitats in south europe are needed to confirm what is suggested by the present work. this is relevant also for planning restoration practices of artificial substrata (embankments, road cuts, etc.), since it means that in the mediterranean region the climate prevails over the edaphic filter. thus, the surrounding natural communities can provide a good reference list for choosing the species to be used for revegetation practices, and, as tested by recent studies in mediterranean areas (bochet et al. 2007, mola et al. 2011), it is even possible to rely only on natural colonization processes. further, as already found in central and north europe (tikka et al. 2000; 2001, tinner and schumacher 2004, ranta 2008, lososova and lanikova 2010), in the mediterranean region too railway sides can act as a substitute habitat for species from natural and semi-natural communities. however, the ecological role of railway sides in the temperate acta bot. croat. 71 (2), 2012 245 flora of a high-speed railway embankment and boreal regions – where road and railway verges are among the few surviving examples of dry and mowed habitats – is quite different compared to the mediterranean countries. finally, it is to be stressed that the maintenance regime has a crucial role: at the time of our survey, rail-side management consisted only of infrequent mechanical cutting, which allowed the development of a complex pattern of many different seral stages (including shrub communities), thus promoting floristic diversity. references abbate, g., alessandrini, a., conti, f., 2007: vascular plants. in: blasi, c., boitani, l., la posta, s., manes, f., marchetti, m. 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of china 2 university of chinese academy of science, beijing 100049, people’s republic of china 3 am stegskreuz 3b, d-65719 hofheim, germany abstract – this paper describes three new cymbella species from high altitude lakes of hengduan mountains region, southwest china. cymbella heihainensis li et gong nov. spec. is similar to c. modicepunctata krammer, c. asiatica metzeltin, lange-bertalot et li, y., but differs from them in valve size, stigmata, raphe ending and also striae number. cymbella shudunensis li et metzeltin nov. spec. is related to c. terrafuegiana, but differs in wider valve size, larger central area and coarser puncta. c. shudunensis differs from c. proxima reimer in patrick et reimer group in reverse lateral raphes at the proximal endings, and distinguished from c.cistula (ehrenberg) o.kirchner group in the absent central area on dorsal side. cymbella xingyunnensis li and gong sp. nov. resembles the group around c. proxima, and the most similar taxon is c. sinensis metzeltn et krammer, which can be distinguished by its lack of stigmata. key words: cymbella heihainensis, cymbella shudunensis, cymbella xingyunnensis, diatoms, china introduction the global warming and nitrogen deposition in the last century have enriched lakes on the hemispheric scale, and resulted in a loss of biodiversity in natural habitats (aber et al. 1995). as is well known, high altitude lakes are an important part of natural ecosystems. southeast asia is becoming a hot spot because of its unique topographic character and the increasing anthropogenic disturbance (galloway and cowling 2002). diatom community is used as a main indicator of environmental and biodiversity change, both in limnology and palaeolimnology (smol and stoermer 2010). acta bot. croat. 72 (2), 2013 359 * corresponding author, e-mail: ylli@niglas.ac.cn copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. there is limited investigation of freshwater diatoms in se asia. among species of the genus cymbella, 49 were reported from northeastern china (fan et al. 1993, bao et al. 1992), 8 from xingjiang province, nw china (you et al. 2005, 2008), 44 from india (jena et al. 2006, nautiyal et al. 2004), and 9 from japan (hirano 1972). one new variety of cymbella was described from qinghai province, nw china (li et al. 2003b). in the himalayan region, 4 new species of cymbella from alpine lakes and springs have been reported from everest national park of nepal (jüttner et al. 2010, 2000). there were also studies on cymbelloid diatoms near mount everest in china (li et al. 2004, 2007b), showing that 44 species belong to cymbella, of them 16 new records, some of them unique to this region. one new species of cymbella has been found from northwest tibet (li et al. 2003a), and one from the far east (lee et al. 1993), respectively. also, a few studies have been carried out on the diatoms from the hengduan mountains region, sw china (skuja 1937, zhu and chen 1994, li et al. 2003c, li et al. 2007a). one cymbella species was found that is unique to this area (li et al. 2007a), and some taxa belonging to cymbella are found that have different morphological features compared with previously published descriptions (li et al. 2003c). in this paper we describe three cymbella species new to science from three high altitude lakes in the hengduan mountains region. one is related to c. modicepunctata krammer and similar species; one is similar to c. terrafuegiana krammer; and we also give a detailed morphological description of a new taxon similar to c. sinensis metzeltn et krammer. study area the hengduan mountains region (23–33°n, 97–103°e) comprises western sichuan province, western yunnan province and part of eastern tibet, southwest china. the area is about 0.5 million km2 (zhang 1997). this region is a transitional zone between lowland tropical and subtropical climate zones because of the uplift of tibetan plateau (yang 1983). the elevation ranges from more than 5000 m a.s.l. to less than 2000 m a.s.l. it is predominantly affected by the sw asian monsoon from india in summer (chen 1998). plentiful monsoon precipitation promotes the development of forest, and its topographic complexity. besides, the unique geologic history makes this region become one of the world’s biodiversity hotspots (fan et al. 2012, myers et al. 2000). thus there are rich flora and fauna in this area, including many relict diatom species (li et al. 2007a). the high altitude lakes located are important reservoirs of regional biodiversity. methods samples were taken from the surface sediments of heihai lake (tab. 1), shudu lake and xingyun lake with the use of a kajak gravity corer in the deepest part of the lakes. heihai lake is above the tree line, the catchment is characterized by alpine meadows; shudu lake is located within the alpine/sub-alpine ecotone with the catchment primarily consisting of picea/abies, while xingyun lake is situated within subtropical evergreen coniferous and broad-leaved mixed forest, dominated by pinus yunnanensis. diatom samples were kept under 4 °c in refrigerator before laboratory treatment. they were treated with hcl and h2o2 (yang et al. 2008). permanent slides were made from cleaned materials and mounted in naphrax® for observation with light microscopy (olym360 acta bot. croat. 72 (2), 2013 hu z., li y., metzeltin d. pus, bx-51, dic). cleaned materials were also investigated with a leo 1530 scanning electron microscope (sem). at least 500 valves were counted in each surface sediment sample. lake water ph and specific conductance were measured in the field using a ysi 650 multi–parameter display system (650 mds, ysi incorporated 1700/1725 brannum lane, yellow springs, oh 45387 usa) with a 600xl probe. water samples were taken from about 50 cm under the surface. total nitrogen (tn) and total phosphorus (tp) were measured by a shimadzu uv2450 ultraviolet-visible spectrophotometer at nanjing institute of geography and limnology, chinese academy of sciences, using alkaline potassium persulfate digestion-uv spectrophotometric method (nydahl 1978) and ammonium molybdate spectrophotometry method (ebina et al. 1983), respectively. results cymbella heihainensis li et gong nov. spec. figs. 1–6. fig. 1 is of the holotype. description: valves are strongly dorsiventral. dorsal margins strongly convex, ventral margins slightly to moderately concave and slightly tumid in the middle. ends are bluntly rounded. length 82–105 mm, breadth 19–22.5 mm, maximal length/breadth is about 4.7. raphe is at middle line of valve, strongly lateral, and becoming slightly reverse-lateral near the proximal ends. axial area is moderately broad. central area is orbicular to broadly elliptical, about 1/2 breadth of the valve. striae slightly radiate in the middle portion, and radiate more towards the end, coarsely punctuate. 7–10 stigmata surrounding the ventral side on the central area separated from the shortened ventral striae by a narrow hyaline area, sometimes there are several smaller stigmata between the main stigmata and the middle ventral striae (fig. 2), on dorsal side, 0–3 stigmata distance from the shortened striae (figs. 1, 3). striae 7–8/10 mm in the middle, and becoming 8–10/10 mm towards the distal, with 14–16 areolae in 10 mm. in sem external valve view: proximal and distal raphe endings deflected on ventral side, raphe slit is located on a rib and thickened part of the centre axial areas, expanded drop-like central pores at proximal raphe ends (figs. 4, 6); the distal raphe endings are question mark-shaped and dorsally deflected, terminating in a large apical pore acta bot. croat. 72 (2), 2013 361 three new species of cymbella (bacillariophyta) tab. 1. location and environmental characteristics of the three lakes. heihai lake shudu lake xingyun lake latitude (°n) 27°21' 27°54' 24°20' longitude (°e) 100°04' 99°57' 102°47' altitude (m) 4117 3630 1748 lake area (km2) 0.18 1.70 34.71 maximum depth (m) 42.4 7.8 8.3 ph 7.79 7.77 9.15 sd (m) 4.5 0.8 0.9 tn (mg l–1) 0.26 1.52 1.22 tp (mg l–1) 0.013 0.036 0.263 conductivity (ms cm–1) 57 31.6 410 with a big hyaline area on the ventral side of the raphe slit, and small, round pore foramina are arranged irregularly at the margin pole fields (fig. 5); 6–7 large main stigma foramina on the ventral side of the central nodule, and a second row of some roundish smaller pores is present between the stigmata and the median ventral striae (fig. 6); striae composed of slit-like areolae becoming shortened, y-, xor irregular round shaped towards centre and margin of the valve (figs. 5, 6). distribution: heihai lake, which is slightly alkaline and oligotrophic with low tn and tp concentration (tab. 1). typus: praep. heihai-1, nanjing institute of geography and limnology, chinese academy of sciences (niglas). locus typicus: heihai lake (27°21’19.3”n, 100°04’12.1”), 11th september, 2007, surface sediment, leg. dr. rong wang. cymbella shudunensis li et metzeltin nov. spec. figs. 7–13. fig. 7 is of the holotype. description: valves are strongly dorsiventral and triangle. dorsal margin is strongly arched. ventral margin is slightly concave with slightly tumid central part. valve ends broadly rounded, not protracted. length 55–87 mm and width 24–27 mm, the maximal 362 acta bot. croat. 72 (2), 2013 hu z., li y., metzeltin d. figs. 1–3. cymbella heihainensis. light microscopy, valve views showing size diminution series for the species. figure 1 is the holotype. scale bar denotes 10 mm. length/width ratio is about 3.3. axial area is narrow, nearly linear, and the two branches form an obtuse angle. central area is rounded, and dorsal side is smaller than ventral side. raphe is strongly ventrally displaced as the axial area, distinctly lateral, becoming filiform near the distal and slightly reverse-lateral near the proximal ends. central pores small, ventrally bent, terminal fissures dorsally deflected. striae slightly radiate and become more radiate towards the valve ends. some short striae arranged on the convex dorsal side in the middle of the valve. 2–4 stigmata appear ventrally from the central nodule, and distant from the middle ventral striae. striae 7–10/10 mm in the middle and becoming 10–12/10 mm towards the distal, with 17–19 areolae in 10 mm. in sem external valve view: striae composed of elliptical areolae extending to the mantle, and becoming more rounded towards the valve centre and the valve margin, the striae on the convex dorsal middle part are slightly undulating (figs. 11, 12); round central pores at proximal raphe ends (fig. 12); roundish stigma foramina, arranged distant from the foramina of the areolae at central area on ventral side (figs. 11, 12); scythe-shaped in an angle of 90° at terminal fissures (fig. 13). acta bot. croat. 72 (2), 2013 363 three new species of cymbella (bacillariophyta) figs. 4–6. cymbella heihainensis. sem, external valve views. fig. 4 – entire valve view showing the proximal and distal raphe endings ventrally deflected, raphe is located on a thickened part of the centre axial areas. scale bar denotes 10 mm. fig. 5 – view of the pole with question mark-shaped raphe ending, pore foramina are arranged irregularly at the margin pole fields. scale bar denotes 2mm. fig. 6 – central portion of the valve showing enlarged proximal raphe ends and six rounded bigger stigma openings with some smaller circular stigma near the shortened striae on ventral side, sometimes small round stigmata which are differ from areolae present on the dorsal side. striae composed of silt-like areolae and becoming xor y-shaped towards centre and margin of the valve, the yshaped areolae are illustrate with arrows and one xshaped areolae is pointed with dashed line arrow. scale bar denotes 2 mm. distribution: shudu lake, china. this lake is characterized by high total nitrogen, low total phosphorus concentration, and also low specific conductance (tab.1). typus: praep. shudu-1, nanjing institute of geography and limnology, chinese academy of sciences (niglas). locus typicus: shudu lake (27°54’36.9”n, 99°56’58.4”), 15th september, 2007, surface sediment, leg. dr. rong wang. cymbella xingyunnensis li et gong sp. nov. figs. 14–32. fig. 14 is of the holotype. description: valves are strongly dorsiventral, and broadly lanceolate. dorsal margin is strongly arched. ventral margin is straight or slightly concave with slightly tumid central part. valve ends are narrow, rounded, not protracted. length 25–41 mm, width 11.5–13.5 mm, the maximal length/width ratio is about 3.0. axial area is narrow, and nearly linear. central area is small and orbicular, developed more on the ventral side. raphe is nearly in the midline or slightly ventrally displaced, moderately lateral, becoming filiform near the distal and the proximal ends. central pores indistinct in lm, and terminal fissures dorsally 364 acta bot. croat. 72 (2), 2013 hu z., li y., metzeltin d. figs. 7–10. cymbella shudunensis. light microscopy. fig. 7 is the holotype. figs. 8, 9 – the same valve at different focus. scale bar denotes 10 mm. deflected with big pole area. striae radiate, distinctly punctuate-lineolate. one big stigma appears on the ventral side of the central nodule. striae 12–14/10 mm in the middle and becoming 14–16/10 mm towards the distal, with 22–25 areolae in 10 mm. in sem external valve view: elongated areolae combined the striae and extending onto the mantle, becoming round towards the central area (figs. 27); terminal fissures dorsally bent off in an angle of nearly 60°, and small roundish foramina arranged in lines around the pole (fig. 28); one large, long stigma foramina at central area, expanded drop-like central pores at proximal raphe ends (fig. 29). in sem internal valve view: striae with internal areolae openings, and no papillae are present on the areolae inside (fig. 30); the distal raphe endings dorsally curved, knob-like helictoglossae with parallel arranged pole field alveoli (fig. 31); central area formed a small hock in proximal raphe ends, one large stigma alveoli with irregular structures near the shorten striae on the ventral side (fig. 32). distribution: xingyun lake, china. it is an alkaline, eutrophic lake with relative high specific conductance (tab. 1). erhai lake, china. typus: praep. xingyun-2, nanjing institute of geography and limnology, chinese academy of sciences (niglas). acta bot. croat. 72 (2), 2013 365 three new species of cymbella (bacillariophyta) figs. 11–13. cymbella shudunensis. sem, external valve views. fig. 11 – whole valve view showing striae composed of elliptical areolae extending to the mantle, and becoming more rounded towards the valve centre and the valve margin. scale bar denotes 2 mm. fig. 12 – valve centre with drop-like proximal raphe ends and four roundish stigma opening. v 2 mm. fig. 13 – pole field with an angle of almost 90° scythe-shaped terminal raphe end. scale bar denotes 1 mm. locus typicus: lake xingyun in china, (24°21’35.9”n, 102°46’33.0”), 4th september, 2007, surface sediment, leg. dr. rong wang. 366 acta bot. croat. 72 (2), 2013 hu z., li y., metzeltin d. figs. 14–26. cymbella xingyunnensis. light microscopy, valve views showing size reduction. fig. 14 is the holotype. scale bar denotes 10 mm. discussion taxonomy cymbella heihainensis appears closely allied to c. modicepunctata and c. asiatica metzeltin, lange-bertalot et li, y. (krammer 2002, metzeltin et al. 2009) (tab. 2). the most similar taxon is c. modicepunctata: it has larger valve size, bigger central area, and more striae in 10 mm, distinguishing it from c. heihainensis. cymbella heihainensis has bigger stigmata near the central area and smaller ones near the middle striae ventrally and sometimes stigmata on dorsal side, but c. modicepunctata only has the same size stigmata on the ventral side. the same stigmata feature of c. heihainensis is also found in c. schimanskii and its variety var. excelsa (meister) krammer. in sem view, c. schimanskii acta bot. croat. 72 (2), 2013 367 three new species of cymbella (bacillariophyta) figs. 30–32. cymbella xingyunnensis. sem, internal valve views. fig. 30 – internal valve view shows striae are a composite of internal areolae openings without papillae. scale bar denotes 1mm. fig. 31 – valve ends with dorsally curved knob-like helictoglossae and parallel alveoli are arranged on the pore field. scale bar denotes 1 mm. fig. 32 – on the central area of the valve, proximal raphe ends form a small hook, and the margins of the stigma alveoli are surrounded with irregular structures. scale bar denotes 2 mm. figs. 27–29. cymbella xingyunnensis. sem, external valve views. fig. 27 – whole valve view showing the striae composed of elongated elliptical areolae extending onto the mantle. scale bar denotes 3 mm. fig. 28 – view of valve end with an angle of nearly 60° dorsally bent terminal raphe ending and small roundish areolae present in lines around the pole. scale bar denotes 1 mm. fig. 29 – central portion of the valve showing drop-like proximal raphe ends and long stigma opening. scale bar denotes 1 mm. has y-shaped areolae openings in the middle of the valve and elsewhere they are slit-like, which is similar to the case of c. heihainensis, and the raphe is also located on a rib and thickened part (pl. 79, figs. 1–3 in krammer 2002). however, they differ from c. heihainensis in the bigger valve size and smaller central area, also in the central pores (roundish shaped vs expanded dropas in c. heihainensis). c. sturii grunow has stigmata on both side, but the ventral stigmata are the same size. c. heihainensis can be distinguished from c. asiatica in valve size, shape of ends and stigmata numbers. cymbella asiatica varies between 50 and 105 mm in length and 14–18 mm in width, with broadly rounded ends, 4–6 stigmata on the ventral side and no stigmata on the dorsal side. c. heihainensis is also similar to c. arctissima metzeltin, which can be separated by dorsally deflected proximal raphe endings, thinner (17–18 mm vs. 19–22.5 mm) valve size and finer punctuate (18–21/ 10 mm vs. 14–16/10 mm). 368 acta bot. croat. 72 (2), 2013 hu z., li y., metzeltin d. tab. 2. morphological characters of cymbella heihainensis li et gong, c. modicepunctata krammer, c. asiatica metzeltin, lange-bertalot et li, y., c. schimanskii krammer and c. arctissima metzeltin. new species species for comparison species/ feature c. heihainensis li et gong c. modicepunctata krammer c. asiatica metzeltin, lange-bertalot et li, y. c. schimanskii krammer c. arctissima metzeltin valve length, mm 82–100 102–140 50–105 100–200 98–105 valve width, mm 19–22.5 21–22 14–18 29–36 17–18 length/width ratio 4.7 max 6.4 max 5.8 max 5 max 5.8 striae in 10 mm 7–10 5.5–6 6–7 7–12 7–10 areolae in 10 mm 14–16 14–16 18–22 10–16 18–21 areolae slit-like, y-, xshaped lineolate not known slit-like, y-shaped slit-like stigmata ventral side: 7–10 big and some smaller, dorsal side: 0–5 ventral side: 7–10 ventral side: 4–6 ventral side: 6–10 big and some smaller, dorsal side: sometimes some ventral side: 6–8 central area 1/2 width 2/3–3/4 width 1/2 width 1/3 width 2/3 width central raphe endings expanded drop-like, slightly ventrally deflected distinct, very slightly reverse-lateral distinct, abruptly reverse-lateral small, slightly reverse-lateral small, dorsally deflected reference this study krammer (2002) metzeltin et al. (2009) krammer (2002) krammer (2002) based on the morphological characters, c. shudunensis belongs to the group around c. cymbiformis agardh (e.g. indistinct central area on dorsal side, less than 20 puncta in 10 mm, and 1–6 stigma). cymbella shudunensis differs from c. proxima reimer in patrick et reimer group in reverse lateral raphes at the proximal endings, and can be distinguished from c.cistula (ehrenberg) o. kirchner group in the absent central area on dorsal side. the outline shape of this species is rare, as it is in all the similar species (tab. 3). the taxon most similar to c. shudunensis is c. terrafuegiana which belongs to c. cymbiformis group with strongly dorsiventral broadly semirhomboid. the outline of c. shudunensis is more convex than c. terrafuegiana on a wider valve size, larger central area and coarser puncta (tab. 3). the similar taxa c. proxima (pl. 112, figs. 1–6b in krammer 2002), c. baicalensis skvortzow et meyer and c. perfossilis krammer all have a less convex dorsal outline and central axial area. except the above differences, c. proxima differs from c. shudunensis in bigger central area, larger length/width ratio, different raphe type, the lineolae-like areolae in contrast to the elliptical shaped areolae, and also the arched terminal fissures against the acta bot. croat. 72 (2), 2013 369 three new species of cymbella (bacillariophyta) tab. 3. morphological characters of cymbella shudunensis li et metzeltin, c. terrafuegiana, c. proxima reimer in patrick et reimer, c. baicalensis skvortzow et meyer and c. perfossilis krammer. new species species for comparison species/ feature c. shudunensis li et metzeltin c. terrafuegiana krammer c.proxima reimer in patrick et reimer c. baicalensis skvortzow et meyer c. perfossilis krammer valve length, mm 55–87 64–74 38–120 112–195 48–121 valve width, mm 24–27 18–22 18–24 51–60 20–26 length/width ratio 3.3 max 3.6 max 5 3.3 3.7 striae in 10 mm 7–12 8–11 7–11 middle 6–7 7–12 areolae in 10 mm 17–19 19–21 12–18 about 8 13–18 areolae slit-like, y-, xshaped not know slit-like not know not known stigmata ventral side: 2–4 ventral side: 2–4 ventral side: 2–5 ventral side: 2–4 ventral side: 2–5 central area small and indistinct dorsal absent or small round orbicular, 1/3 width orbicular, 1/3–1/4 width irregularly round, 1/3 width central raphe endings round, ventrally bent small bulbous, somewhat dorsally bent large distinct, ventrally bent reference this study krammer (2002) krammer (2002) krammer (2002) krammer (2002) scythe-shaped; c. baicalensis is different in the larger size and the much coarser puncta. cymbella perfossilis can be distinguished by the outline and larger dorsal central area. cymbella xingyunnensis resembles to the group around c. proxima, but with smaller size than the species included in this group now. all the similar species compared with this new taxa (tab. 4). cymbella xingyunnensis is quite similar with c. sinensis, belonging to c. proxima group. but cymbella sinensis has larger valve size, coarser puncta, without the observed stigmata. in sem, c. sinensis has cshaped areolae, the foramina areolae in the pole are arranged in both side of the terminal fissures in external view, in internal view the foramina surround the helictoglossa contrary to the parallel arrangement in cymbella xingyunnensis. cymbella parva w. smith has similar sem view (pl. 12, figs. 8–10, krammer 2002), but this taxa can be separated from the lm view, with smaller width, less dense striae and finer puncta. cymbella xingyunnensis is also similar to c. compacta østrup and c. turgidula grunow. the former can be distinguished by the lack of apical pore field 370 acta bot. croat. 72 (2), 2013 hu z., li y., metzeltin d. tab. 4. morphological characters of cymbella xingyunensis li and gong, c. sinensis metzeltn et krammer, c. parva (w. smith) kirchner in cohn, c. compacta østrup and c. turgidula grunow new species species for comparison species/ feature c. xingyunensis li and gong c. sinensis metzeltn et krammer c. parva (w. smith) kirchner in cohn c. compacta østrup c. turgidula grunow valve length, mm 25–41 48–58 15–47 28–76 30–50 valve width, mm 11.3–13.5 17–18 7–10 11–15 11–14 length/width ratio 3 max 2.8 max 4.5 max 5.1 max 3.3 striae in 10 mm 13–16 12–13 9–13 10–14 8–14 areolae in 10 mm 22–25 15–16 28–30 18–24 22–25 areolae elongated, become round towards the centre c-shaped, become round towards the centre elongated, become round towards the centre silt-like not know stigmata ventral side: one big no ventral side: one big ventral side: 4–8 ventral side: 1–3 central area small and orbicular small, orbicular, 1/4–1/5 width absent or small absent small, rounded, more on doarsal central raphe endings indistinct indistinct distinct, rounded small, slightly ventral bent slightly rounded reference this study krammer (2002) krammer (2002) krammer (2002) krammer (2002) and 4–8 stigmata on the ventral side. the latter has subrostrate to rostrate valve ends, commonly two stigmata, sometimes one to three stigmata on ventral side. cymbella xingyunnensis can be distinguished from c. subcistula krammer in valve size, length/ breadth, striae numbers in 10 mm, stigmata numbers, and central area. cymbella subcistula varies between 33 and 85 mm in length and 13.4–18 mm in width, with maximal length/ breadth ratio 4.8, 7–12 striae in 10 mm and 2–5 small stigmata on ventral side. ecology so far, cymbella heihainensis has only been found in the surface sediment of heihai lake. heihai lake is an alpine lake, which is slightly alkaline and base-poor (tab.1). the associated taxa from the surface sediment on which cymbella heihainensis was found include cyclotella distinguenda var. unipunctata (hustedt) håkansson et j. r. carter (42%), cyclotella ocellata pantocsek (13%), achnanthes minutissima kützing (10%) and small fragilaria spp. (15%). the small cyclotella species indicate an oligotrophic and open water environment in the pelagic zone (wunsam et al. 1995, corella et al. 2011). achnanthes minutissima also shows high abundance in low nutrient and base poor condition (ponader and potapova 2007). liu et al. (2012) reported from great xing’an mountains in ne china morphologically similar species, i. e. cymbella cf. arctissima metzeltin which should be re-examined to establish its identity and relationship to c. heihainensis. cymbella shudunensis has only been found in shudu lake, which is a high altitude, shallow, oligo-mesotrophic lake with relative high tn but low tp concentration (tab. 1). in shudu lake, the diatom community from surface sediment is diverse, and mainly dominated by cyclotella stelligera (cleve et grunow) van heurck (17%), fragilaria pinnata ehrenberg (15%), achnanthes minutissima (9%), tabellaria flocculosa (roth) kützing (5%) and gomphonema minutum (c. agardh) c. agardh (4%). the high diversity of diatom species may reflect the shallow lake conditions with rich available habitats. cyclotella stelligera and tabellaria flocculosa are known as oligo-mesotrophic taxa (little et al. 2000, lilham 1971). cymbella shudunensis may prefer mesotrophic water condition. the new species cymbella xingyunnensis has been found in xingyun lake, base-rich, and meso-eutrophic (with relative high tn and tp concentration). the associate diatom assemblage of surface sediment in xingyun lake showed the dominance of cyclostephanos dubius (fricke) round (49%), fragilaria crotonensis kitton (28.5%) and aulacoseira granulata (ehrenberg) simonsen (10%). this new taxon was also found in erhai lake, another meso-eutrophic lake in yunnan province (with 0.592 mg l–1 tn and 0.045 mg l–1 tp), while erhai lake was characterized by fragillaria crotonensis (43%), aulacoseira ambigua (grunow) simonsen (28%), cyclostephanos dubius (11%) and cyclotella ocellata (10%). cyclostephanos dubius and fragillaria crotonensis are indicators of eutrophic (bradshaw and anderson 2003, lotter 1998). cymbella xingyunnensis may be tolerant to nutrient-rich water. the most resemble taxa of cymbella xingyunnensis is c. sinensis, which was considered unique to the yunnan plateau (li et al. 2007b). 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(ed.), compilation of report on survey of algal resources in southwestern china, 79–130. science press, beijing. 374 acta bot. croat. 72 (2), 2013 hu z., li y., metzeltin d. 108 acta bot. croat. 81 (1), 2022 acta bot. croat. 81 (1), 108–116, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-008 issn 0365-0588 eissn 1847-8476 fungal diversity and ex vitro symbiotic germination of serapias vomeracea (orchidaceae) yasemin özdener kömpe*, vildan akin mutlu, i̇brahim özkoç, sevim demiray, serhat bozkurt university of ondokuz mayıs, faculty of arts and sciences, department of biology, samsun, turkey abstract – conservation of orchids can be possible with effective seed germination and seedling growth methods. in this context, ex vitro symbiotic seed germination and seedling growth of orchid seeds may be convenient and advantageous. in this study, both the diversity of the root endophytic fungi in serapias vomeracea (burm.f.) briq. and the ex vitro effects of these fungi on seed germination, seedling development and tuber formation were revealed. the fungi were isolated monthly for two years from s. vomeracea roots and the isolates were identified based on morphological characters and internal transcribed spacer (its) region of nuclear ribosomal dna (rdna) sequences. all of the rhizoctonia-like isolates that joined the mycorrhizal association were closely related to tulasnella calospora (thirty isolates). non-rhizoctonia isolates are closely related to fusarium tricinctum (two isolates), aspergillus spelaeus (one isolate) and talaromyces pinophilus (pezizales) (one isolate). the viability rate of the seeds was 90.32%. the seed packs were placed in soils containing fungus and the germination process was followed. all isolates associated with tulasnella calospora promoted germination and seedling development. isolate svl 21 (tulasnella sp.) was found to have the highest germination rate (98%) but isolate svl 4 developed seedlings with advanced leaves (stage 4 (s4): seedlings with advanced leaves and/or rooted, 13.67%). all seedlings at s4 were transferred to the natural environment; the first tubers were observed seven months after. in this study, for the first time, a tuberous european orchid, s. vomeracea developed from seed to adult plant in a natural environment. keywords: endophytic fungi, orchid conservation, symbiotic cultivation, serapias vomeracea introduction orchids are of great importance as medicinal, food and ornamental plants in the world. they are under threat of extinction as they are over-collected from nature and their habitats are destroyed (rasmussen 2002). in addition, orchid tubers are collected too much for salep production and medical purposes in greece, iran and turkey (sezik 2002, ghorbani et al. 2014, kreziou et al. 2016). although they produce a large number of seeds it is not easy to propagate them in the natural environment by seed germination ( rasmussen 2002). however, seed germination is the most important factor for both medical, commercial and in situ/ ex situ conservation and reintroduction. orchids propagated from seeds are used successfully in reintroduction, especially in orchid protection projects (paul et al. 2012). previous research results indicate that more than one fungus joins the mycorrhizal association continuously or seasonally in the roots of orchids (tondello et al. 2012). inoculation with appropriate fungus is the most important factor for the orchid to adapt to the natural environment and survive because the population size of orchids and the number of individuals are associated with the presence and abundance of suitable fungi in the soil. the decrease in numbers of particularly rare and threatened species results from the loss of mycorrhizal fungi due to habitat change or destruction (rasmussen 2002). in this context, understanding the relationship between orchids and fungi is important for the protection of orchids, their reintroduction and production for agricultural purposes. most orchid mycorrhizal fungi belong to rhizoctonia-like fungi, a diverse polyphyletic group of pathogens, endophytes, saprophytes and mycorrhizal fungi (bayman and otero 2006). this group includes the anamorphic (asexual) genera, ceratorhiza, epulorhiza, moniliopsis, and rhizoctonia (moore 1988). the teleomorphs (sexual stages) of these genera are ceratobasidium, tulasnella, sebacina and thanatephorus, respectively ( bayman and otero 2006). tropical and temperate orchids can also be produced by in vitro asymbiotic methods. however, the procedure re* corresponding author e-mail: yasemino@omu.edu.tr germination of serapias vomeracea acta bot. croat. 81 (1), 2022 109 quires complete axenic conditions and expensive laboratory equipments. also, asymbiotic seedlings are difficult for in situ or ex situ adaptation (aewsakul et al. 2013). alternatively, successful results have recently been obtained from ex vitro symbiotic seed germination studies. however, there are very few studies in which this method has been applied. quay et al. (1995) and aewsakul et al. (2013) germinated some epiphytic orchids symbiotically in ex vitro conditions. there is not enough research on ex vitro symbiotic seed germination and seedling development of temperate orchids (especially euroasian tuberous orchids). due to the glucomannan content of their tubers, orchids have a considerable economic importance as an additive in ice cream, salep and other foods (sezik 2002). despite their economic importance, they have not yet been cultured and are under serious threat of extinction due to the collection of thousands of tubers every year. in order to prevent the destruction and extinction of temperate orchids, it is essential to produce them with a fast, easy and inexpensive method. due to anthropogenic effects and global climate change many orchids will be under a threath of extinction in the near future. there are five species included in the serapias genus in turkey, and serapias vomeracea (burm.f.) briq. is one of the most collected orchid species because they have 1-5 tubers and a high glucomannan content. a large number of studies have been conducted on in vitro asymbiotic and symbiotic germination of the seeds (ozkoc and dalcı 1993, acemi and ozen 2019). however, temporal fungal diversity in s. vomeracea roots and the effects of these fungi on symbiotic germination and seedling growth in ex vitro conditions have not been investigated. hence, the main objectives of this study are: (i) to establish the diversity of fungi participating in mycorrhizal association in the roots of s. vomeracea, (ii) germination of s. vomeracea seeds and seedling formation in the presence of root endophytic fungi in ex vitro conditions, and (iii) to determine whether an ex vitro symbiotic method is suitable for orchid production. it is also hoped that the data obtained from this study will be useful for further reproduction and conservation of this orchid species. materials and methods study site serapias vomeracea is distributed in the coastal area of the black sea and aegean regions of turkey. the research area of this study is ondokuz mayıs university campus area. the habitats of the species are the open spaces located next to oak forests at about 50 m a.s.l. the species of the genera hordeum, avena and melilotus are present in these habitats and over 100 s. vomeracea individuals are also represented in the research area. they bloom in may and the seeds maturate in july. in 2015, seasonal avarage temperatures and amount of precipitation were 12.2 °c and 176 mm in spring, 23.9 °c and 85 mm in summer, 16.8 °c and 134 mm in autumn. in 2016, they were: 13.8 °c and 171 mm in spring, 24.8 °c and 69 mm in summer, and 15.1 °c and 133 mm in autumn. physical and chemical properties of the soil for soil analyses, an about 2 kg sample was taken from 0-15 cm depth of 1 m2 area after removing leaves and other debris from the surface of soil in research area where s. vomeracea is commonly distributed. the soil was dried at room temperature in laboratory and used for all analyses. in the soil saturated with water, a direct ph reading was taken with a glass electrode ph meter (aciego pietri and brookes 2008). electrical conductivity was determined with an ec – meter in saturation extract (rhoades 1996), organic matter using the smith-weldon method (nelson and sommers 1982). total sand, silt, lime and clay were determined by the densimeter method (bowman and hutka 2002). exchangeable cations (calcium, magnesium, and potassium) were determined with the mehlich-3 method (mehlich 1984). available phosphorus was analysed using the molybdate blue method (murphy and ridley 1962). fungus isolation before root samples were collected, s. vomeracea phenological stages were followed for one year in their habitats (campus area of ondokuz mayis university). it was determined that the leaves formed in february to march and the roots completely dried in july. the roots for fungi isolation were taken through two years (2015-2016). every month, complete roots of a plant were collected. cross-sections were then taken from the roots and examined under a microscope for the presence of fungi. all the roots containing fungal coils were used for fungi isolation, performed according to clements et al. (1986). the roots were sterilized in 1.5% naocl solution for 5 minutes and washed in sterile distilled water. under aseptic conditions, root pieces (1-2 cm) were placed in petri dishes containing fungi isolation medium (fim) (clements et al. 1986) and incubated at 27 °c for 2 days in the dark. following stereo microscopy examination, any fungal colonies were transferred to fim and purified. pure fungus cultures were stored at 4 °c. morphological and molecular identification of the fungi isolates for preliminary identification were grown in fim plates and thin agar blocks of 48-h-old cultures were viewed under a microscope for the mycelial and branching characteristics of the isolates. the following distinct morphological characters were observed: mycelia branched at acute to right angles, constrictions at or near the point of branching and septum formation near the branching point. isolates showing rhizomorph and asexual spore (conidia) structure were defined as non–rhizoctonia. the isolates were grown in potato dextrose agar (pda) at 25 ± 2 °c for özdener kömpe y., akin mutlu v., özkoç i̇, demiray s., bozkurt s. 110 acta bot. croat. 81 (1), 2022 7-8 days to determine the color and texture of the colonies. the color of the colony was defined according to the color chart of the royal horticultural society of london. the hypha diameter of the fungi was measured under light microscope. to determine the number of nuclei in the cell, isolates were grown for three days at 25 °c in petri dishes containing water-agar (wa), stained with safranine-o solution and nuclei were counted under the microscope (bandoni 1979). dna isolation from fungal mycelia was performed using the ctab (cetyltrimethyl ammonium bromide) method described by pascual et al. (2000) with some modifications (e.g., 50 mg of mycelium was crushed in a sterile mortar in 1 ml extraction buffer and incubated for 30 min.) for each isolate, the internal transcribed spacer (its) region of the nuclear ribo somal rdna was amplified by pcr. its1 and its2 regions, including the ribosomal 5.8s rna gene, were amplified using the universal primers its-1 (5’-tccgtaggtgaacctgcgg-3’) and its-4 (5’ tcctccg cttattgatatgc 3’) (white et al. 1990). pcr amplification reactions were performed in a 50 μl reaction containing 1 μl genomic dna (1 ng μl–1), 1 μl (2.5 mm) dntp mix (sigma), 0.25 μl taq dna polymerase (5 u/μl) (promega, go-taqflexi dna polymerase), 1 μl each of primers (25 pmoles), 10 μl 5 × pcr buffer supplied by manufacturer (promega, go-taq green buffer) and 3 μl mgcl2 (1.5 mm) (sigma) and 32.75 μl sterile ddh20. pcr amplification was carried out as follows: an initial denaturation at 94 °c for 3 min followed by 30 cycles of 94 °c for 1 min, 49 °c for 2 min, 72 °c for 3 min and a final extension at 72 °c for 7 min (salazar et al 1999). pcr products of the rdna-its region were sequenced by macrogen (macrogen inc., seoul, republic of korea) by using abi 3730 xl dna sequencer with its1 and its4 primers. for each pcr product, sequences of both strands were combined to generate a consensus sequence by using bioedit version 7.2.5 software (hall 1999). the consensus sequences of the rdna-its region were compared with the sequence data in genbank (national center for biotecnology information) by using blastn tool. we used 97 to 100% sequence identity to delimit fungal species and genera. one sequence for each isolate was deposited in the genbank database. dna polymorphism was determined for the its gene sequences using dna sequence polymorphism software (dnasp), version 6.0 (rozas et al. 2017). haplotype groups were formed according to the nucleotide polymorphism of the sequences. the data set formed for tulasnella species including the sequence of 15 fungal isolates and the 24 reference sequences obtained from ncbi. genetic distances for data sets were calculated using the mega 6 software package (tamura et al. 2013). the tree (rhizoctonia-like) was constructed using maximum likelihood (ml) analysis with 1000 bootstrap replications using mega 6. collection of the seeds and seed viability test capsules produced by natural pollination were collected from the individuals grown in the meadows next to oak forests in 2015. the seeds were removed from the capsules, kept in room conditions for a few days in the laboratory to lose moisture, and then stored at 4 °c. pre-treatments for viability test and ttc (2,3,5 triphenyl tetrazolium chloride) test were conducted according to kömpe et al. (2020) and van waes and deberg (1986), respectively. approximately 100-150 seeds were placed in each pack. the seeds were incubated in moist cocopeat for 7 days so that the seed coat could crack. thus, ttc would be allowed to enter into the embryo. the seeds were incubated in ttc (1%) solution for 12 hours at 28 °c, followed by 12 h incubation in sterile distilled water. the viability experiments were performed with six repetitions. the seeds with red-pink embryos were evaluated as live. viability rates (%) = number of stained embryos /total seed number × 100. germination of the seeds and development of the seedlings under ex vitro condition the soil samples were taken from 3-5 cm depth and 30 cm around the adult orchids individuals. a mixture of 2:1 soil: perlite was prepared and sterilized in autoclave at 121°c for 20 min. the sterile soil mixture was filled into pots (20 x 31 x 13 cm) to a depth of 15 cm. the seeds were placed between sheets of water-resistant nylon mesh (45 µm pore size). approximately 400-500 seeds (10 mg) were placed in each pack. ten packs of seeds were placed in each pot. fifteen rhizoctonia-like (tulasnella) and four non-rhizoctonia isolates (fusarium, aspergillus, talaromyces) were used in germination tests. for each fungal isolate, two independent pots containing sterile soil mixture were prepared. fungi were grown on fim for 7 days and fungus discs (1-2 mm in diameter) obtained from then were placed in each corner of the pots. seed packs were placed in control pots not inoculated with fungus. ex vitro germination and growth experiments were performed with six repetitions. the pots were incubated at 25 ± 2 °c in the climate chamber with a 16/8h light/dark photoperiod and 33 par (photosynthetic active radiation). it was irrigated with sterile distilled water once a week. three months after the seeds were embedded in the pots, 3 seed packs were randomly selected from each pot to calculate germination and seedling growth rates and evaluated according to the scale of clements et al. (1986). the extent of seed germination and development was divided into stages: s0, s1, s2, s3 and s4. these stages are represented as follows: s0: no germination (seed); s1: protocorm; s2: leaf premordium; s3: the first photosynthetic leaf; s4: seedling with advanced leaves (and/or rooted). germination percentage was calculated as number of seeds in stages 1-4 divided by the total number of seeds ( clements et al. 1986). germination rates (%) = number of germinated seeds /total seed number x 100. microscopic observations of mycorrhizal associations for showing that mycorrhizal association had been established, transverse sections were taken from experimental protocorms produced ex vitro, from the roots of experimengermination of serapias vomeracea acta bot. croat. 81 (1), 2022 111 tal seedling and from the roots of adult plants in natural habitat, and 0.1% lactophenol (r al diagnostic) was dropped and incubated for 10 min at 25 °c. the sections were then washed with distilled water to remove residual staining. each section was examined under a microscope for presence of fungal coil and photographed. statistical analysis the viability tests and the effects of fungal isolates on ex vitro germination and growth were analyzed using one wayanova. results were compared using the sd (standard deviation) of means, and the post-hoc duncan’s multiple range test. statistical significance was set at p < 0.05. all analyses were performed in spss 15.0 (spss inc., chicago, usa). results the soil properties were as following: electrical conductivity (ec, ds m-1) 0.896, ph 6.88, organic matters 7%, clay 12.43%, silt 21.79, sand 65.78%, lime: 3.17, ca + mg (cmol kg-1) 25.04 + 18.40, p (ppm) 6.21, k (cmol kg-1) 1.76. the fungi of serapias vomeracea in the first year of the experiment, the phenological stages continued from march to july. in the second year they took place between february and june. fungal isolations were made from march to july in 2015 and from february to june in 2016. from the roots of s. vomeracaea 18 isolates (svl 1-18) were found in the first year and 16 in the second year (svl 19-34). applying the pre-identification procedure, two main isolate groups were determined: binucleate rhizoctonia-like group with 30 isolates and multinucleate non-rhizoctonia group with 4 isolates. rhizoctonia-like ( tulasnella sp.) isolates were found every month when fungal isolation was carried out. among non-rhizoctonia isolates, two isolates of fusarium sp. were isolated in march 2015, one isolate of aspergillus sp. in june 2015 and one isolate of talaromyces sp. in february 2016 (fig. 1). morphological features of the isolated fungi in both groups (hyphae diameter, number of nuclei, colony color, colony appearance) were determined. three different colony colors of rhizoctonia-like isolates were determined after growth on pda medium at 25 ± 2 °c in the dark for two weeks. the most frequently observed colony color was grayish yellow, while orangewhite and yellowish white were observed less frequently. colony appearance was frequently submerged and powdery. vegetative hyphae color of all the isolates was transparent. hyphae diameter varied between 2.5 and 4.4 µm. vegetative hyphae color of all isolates was transparent. colony color of the non-rhizoctonia isolates were determined as red and orange (svl 10, svl 11, respectively), white (svl 13), grayed yellow (svl 20), while the colony appearance was determined as submerged aerial hyphae and powdery. vegetative hyphae color of all isolates was transparent. hyphae diameter varied between 2.5 and 4.4 µm. the pcr products of the its regions of all the fungal isolates were sequenced and aligned. obtained consensus sequences were uploaded to genbank and compared with other sequences from that database. accession numbers for our fungal its regions and identity percentages with most closely related sequences from genbank are given in tab. 1. according to blastn results, 2 of the 4 non-rhizoctonia isolates were found to show 100% identity to fusarium tritinctum (svl 10 – svl 11) (genbank acession number fig. 1. numbers of endophytic root fungi of serapias vomeracea during the isolation months at 2015 and 2016. rhizoctonia-like (tulasnella sp.) isolates were obtained every month when fungal isolation was carried out. among non-rhizoctonia isolates, two isolates of fusarium sp. were isolated in march 2015, one isolate of aspergillus sp. in june 2015 and one isolate of talaromyces sp. in february 2016. özdener kömpe y., akin mutlu v., özkoç i̇, demiray s., bozkurt s. 112 acta bot. croat. 81 (1), 2022 mk250655, mk250515 respectively), while one isolate was found to show 99% identity to aspergillus spelaeus (svl 13) (genbank accession number mk2505615) and one isolate was found to show 99% identity to talaromyces pinophilus (svl 20) (genbank acession number mk255324). all of the rhizoctonia-like isolates had identity rates to uncultured tulasnella (eg. svl 1, svl 3, svl 4 etc.) of between 97-100% (genbank acession number mk249887, mk250064, mk250062, respectively) (tab. 1). a phylogenetic tree was constracted to reveal the phylogenetic relationship between tulasnella isolates of s. vomeracea and tulasnella sp. from other orchids (fig. 2). among the its sequence data set, 15 haplotypes were detected based on sequence analysis of 30 tulasnella isolates. its sequences exhibited a haplotype diversity of 87%. haplotype 5 exhibited the highest frequency within the its sequences, being generated from 12 sequences. haplotype 4 and haplotype 7 exhibited low frequencies within the its sequences, and were generated respectively from 4 and 2 sequences. other haplotypes exhibited the lowest frequencies among the its sequences, and were generated from only one sequence each. tulasnella its sequences from our study grouped with t. calospora clade. in the phylogenetic tree, tulasnella isolates (e.g. svl 3, svl4, svl5, svl9 etc.) isolated from s. vomeracea roots were therefore found to be closely associated with t. calospora. (genbank accession no: ay373298.1, fj613176.1, gu166421.1, hq889722.1 etc.) (fig. 2). the seed viability test the embryos of seeds incubated in cocopeat for 1, 2 and 3 days were not stained. the viability rates of the seeds incubated for 5 days and 7 days were found to be 40.00% ± 9.27 and 90.32% ± 1.30, respectively. ex vitro symbiotic germination and symbiotic association with the seeds ex vitro germination tests were evaluated for total germination and development stages after three months of incubation. developmental stages (s1-4) are shown in fig. 3a. fusarium tricinctum (svl 10, svl 11), aspergillus spelaeus (svl 13) and talaromyces (svl 20) isolates did not promote germination. there was no germination in the control pots without fungi (fig. 3b). all the tulasnella isolates on the phylogenetic tree promoted germination and growth at varying rates (tab. 2, fig. 3c-e). according to the counts performed at the end of three months, 98% of seeds germinated in the packages inoculated with the isolate svl 21 and the seeds inoculated with this isolate developed until to s3. the seeds placed in the pots inoculated with svl 4, svl 14 and svl 34 germinated at the rates of 93.2%, 94% and 90% respectively, and these isolates supported the development of the seedling up to the s4 (13.67%, 10.8%, 9.8%, respectively) (fig. 3d, e). the percentage of seedlings that reached the s4 of development (13.67%) inoculated with the isolate svl 4 was tab. 1. molecular identification of mycorrhizal fungi isolated from serapias vomeraceae roots based on the closest match in the genbank. blast results show the top hit matching the sequencing. two fusarium tritinctum (mk250655 – mk250515) one aspergillus spelaeus (mk250156) and talaromyces pinophilus (mk255324) isolates were found. the other isolates had identity rates to uncultured tulasnella (eg. mk250064, mk250062, mk250060 etc.). length per base pair of dna (bp), genbank accession number (an), close relatives (a unique identifier assigned to records in the ncbi databases), the highest percent identity for fungi sequences (% id) and references are given). isolate designation bp genbank (an) close relatives (an) %id reference svl 3 567 mk250064 uncultured tulasnellaceae (kc243935.1) 98 tĕšitelová et al. 2013 svl 4 547 mk250062 uncultured tulasnella (jf926504.1) 100 girlanda et al. 2011 svl 5 583 mk250060 uncultured tulasnellaceae (jx649082.1) 98 bailarote et al. 2012 svl 9 603 mk250058 uncultured tulasnellaceae (jx649082.1) 99 bailarote et al. 2012 svl 10 548 mk250655 fusarium tricinctum (jx045791.1) 100 unpublished svl 11 609 mk250515 fusarium tricinctum (hq703409.1) 100 unpublished svl 12 573 mk256219 uncultured tulasnellaceae (jx649082.1) 99 bailarote et al. 2012 svl 13 609 mk250156 aspergillus spelaeus (mg976863.1) 97 unpublished svl 14 603 mk250061 uncultured tulasnellaceae (jf926504.1) 99 girlanda et al. 2011 svl 15 608 mk250075 uncultured tulasnellaceae (jx649082.1) 99 bailarote et al. 2012 svl 18 6 10 mk250519 uncultured tulasnellaceae (jf926504.1) 99 girlanda et al. 2011 svl 19 510 mk250524 uncultured tulasnellaceae (jx649082.1) 99 bailarote et al. 2012 svl 20 499 mk255324 talaromyces pinophilus (lt558963.1) 100 guevara-suarez et al. 2016 svl 21 450 mk281614 uncultured tulasnellaceae (jf926504.1) 99 girlanda et al. 2011 svl 22 615 mk250526 unculturedntulasnellaceae (jx024734.1) 99 jacquemyn et al. 2012 svl 29 496 mk250522 tulasnella sp (kf537647.1) 99 ding et al. 2014 svl 30 310 mk250656 uncultured tulasnellaceae (jx649083.1) 99 bailarote et al. 2012 svl 31 406 mk250520 tulasnella sp. (jq713578.1) 99 unpublished svl 34 603 mk250530 uncultured tulasnellaceae (jf926504.1) 100 girlanda et al. 2011 germination of serapias vomeracea acta bot. croat. 81 (1), 2022 113 found to be statistically significantly high when compared with seedlings reaching the same stage of development inoculated with other fungi. thirty seedlings grown in pots (s4seedlings with advanced leaves (and/or rooted)) were planted in their natural habitats and the first real tubers occurred at 5 months after fig. 2. maximum-likelihood (ml) tree based on an alignment of its-5.8 sequences, showing relationship of fifteen tulasnella species. node tips show ncbi accession numbers followed by fungal species name, host species and country name. the tree was rooted with the sequence of xylaria polymorpha (accesion number eu272539.1) numbers above branches are maximum likelihood bootstrap probalities (>50%). fig. 3. from the seed to the seedlings of serapias vomeracea. a – developmental stages (s1-4). arrows and numbers show the developmental stages. b – control (no fungal isolate). seed coats ruptured but, no advanced development (s0). c – protocorms and leaf primordiums, d – leafy plantlet, e – the seedling in natural area, f – the first tuberous adult plant. scale bars: a – 10 mm, b – 0.3 mm, c – 0.2 mm, d – 10 mm, e – 10 mm, f – 10 mm. özdener kömpe y., akin mutlu v., özkoç i̇, demiray s., bozkurt s. 114 acta bot. croat. 81 (1), 2022 the seedlings were transferred to the soil. all of the seedlings transferred to soil developed tubers (fig. 3f). the presence of symbiotic association was shown in cross-sections from the roots of adult plants, the ex vitro experimental protocorms and the ex vitro experimental seedling roots (fig. 4a, b, c, respectively). discussion orchids are in danger of extinction due to severe destructive factors such as destruction of natural habitats, excessive tuber harvesting for medical or commercial purposes, and global climate change (ghorbani et al. 2014). symbiotic propagation of orchids with suitable root endophytic fungi is a very advantageous method for reintroduction to the habitats of endangered orchids and for agricultural cultivation (aewsakul et al. 2013). during the annual life cycle of the plant, various fungi can join the mycorrhizal association, and a fungus obtained during isolation from roots, especially in the flowering period, may not encourage the germination of the seeds of the same orchid (girlanda et al. 2011). for this reason, all fungi participating in the mycorrhizal association were obtained by culture-dependent isolation method performed monthly for two consecutive years. it was determined that all our rhizoctonia-like isolates are closely related to tulasnella calospora. girlanda et al. (2011) reported that members of ceratobasidium as well as tulasnella joined the mycorrhizal association of s. vomeracea. however, ceratobasidium was not isolated in our study. all the tulasnella isolates supported the development at various rates. the differences in development to s4 may be due to the test period being limited to 3 months. differences in the effects of the fungal isolates on germination and development indicate that isolates retab. 2. germination and development rates after three months from inoculation of serapias vomeracea seeds with rhizoctonia-like fungi in ex vitro conditions. development of the seedlings was divided into stages: s0, s1, s2, s3 and s4. s0: no germination (seed); s1 – protocorm, s2 – leaf primordium, s3 – the first photosynthetic leaf, s4 – seedling with advanced leaves (and/or rooted). the seed packs in control pots were not inoculated with fungal isolate. the effects of fungal isolates on ex vitro germination and developmental stages were analyzed using one way-anova. results were compared using the sd (±: standard deviation) of means, and the post-hoc duncan’s multiple range test. statistical significance was set at p < 0.05. there is no statistically significant difference between groups with the same letters, n = 6. fungi %germination s0 s1 s2 s3 s4 control 0.00 ± 0.00e 100 ± 0.00a 0.00 ± 0.00g 0.00 ± 0.00h 0.00 ± 0.00e 0.00 ± 0.00d svl 3 94.00 ± 2.54ab 6.00 ± 2.54de 4.40 ± 1.67fg 78.00 ± 3.16a 11.60 ± 2.50d 0.00 ± 0.00d svl 4 93.20 ± 2.38ab 6.80 ± 2.38de 11.97 ± 8.79e 39.27 ± 8.07ef 27.94 ± 7.88a 13.67 ± 5.35a svl 5 87.00 ± 3.39cd 13.00 ± 3.39bc 4.80 ± 0.83fg 59.40 ± 3.28b 17.80 ± 3.49bcd 5.20 ± 1.64c svl 9 94.00 ± 3.16ab 6.00 ± 3.16de 7.60 ± 1.14ef 65.80 ± 4.14b 15.20 ± 2.94cd 5.40 ± 3.20c svl 12 87.00 ± 4.06cd 13.00 ± 4.06bc 19.00 ± 2.23d 52.00 ± 4.63c 10.60 ± 2.60d 5.40 ± 2.07c svl 14 94.00 ± 3.74ab 6.00 ± 3.74de 0.60 ± 0.89g 63.60 ± 5.59b 19.00 ± 5.47bcd 10.80 ± 2.16b svl 15 82.40 ± 3.28d 9.60 ± 6.84cd 22.80 ± 3.56d 42.40 ± 0.64de 12.00 ± 1.87d 4.80 ± 1.64c svl 18 87.00 ± 4.06cd 13.00 ± 4.06 19.00 ± 2.23d 52.00 ± 4.63c 10.60 ± 2.60d 5.40 ± 2.07c svl 19 87.00 ± 3.08cd 13.00 ± 3.08bc 32.8 ± 6.76b 43.60 ± 7.95de 10.20 ± 3.27d 0.40 ± 0.54d svl 21 98.00 ± 2.00a 2.00 ± 2.00d 21.80 ± 10.40d 47.40 ± 8.04cd 28.8 ± 17.81a 0.00 ± 0.00d svl 22 90.00 ± 4.69bc 10.00 ± 4.69cd 25.00 ± 0.09cd 41.00 ± 6.40de 24.00 ± 5.65ab 0.00 ± 0. 00d svl 29 94.60 ± 3. 97ab 5.40 ± 3.97de 60.40 ± 5.12a 20.60 ± 3.28g 10.40 ± 1.32d 0.00 ± 0. 00d svl 30 93.20 ± 3.56ab 6.80 ± 3.56de 34.20 ± 6.64b 33.20 ± 3.49f 27.80 ± 3.16a 0.00 ± 0. 00d svl 31 83.60 ± 3.84d 16.40 ± 3.84b 36.80 ± 2.86b 24.00 ± 5.33g 22.80 ± 2.85abc 0.00 ± 0. 00d svl 34 90.00 ± 3.16bc 10.00 ± 3.16cd 30.40 ± 2.96bc 39.00 ± 2.23ef 12.80 ± 1.49bc 9.80 ± 2.281b fig. 4. fungal pelotons stained with lactophenol cotton blue. a – in the cortical cells of adult serapias vomeracea roots, b – in the ex vitro protocorm cells, c – in the cortical cells of the roots of ex vitro seedlings. the arrows indicate fungal coils. scale bars: 50 µm. germination of serapias vomeracea acta bot. croat. 81 (1), 2022 115 lated to tulasnella may be different at species level. therefore, their precise identification is needed to make using molecular techniques. it was shown by fracchia et al. (2014) and vujanovic et al. (2000) that some fusarium species promoted germination of tropical orchid seeds. none of the following species, fusarium tricinctum (svl 10 and svl 11), aspergillus spelaeus (svl 13) and talaromyces pinophilus (svl 20), all isolated in our study, stimulated germination of the seeds. in recent years, it has been indicated that the members of pezizales are also members of the mycorrhizal association (stark et al. 2009). also, it has been determined that fungi included in the order of pezizales also join the mycorrhizal association in the roots of dactylorhiza (kömpe and mutlu 2017), anacamptis and orchis species (mutlu and kömpe 2020). although non-rhizoctonia fungi in orchid roots join the mycorrhizal association, it has been reported that they do not establish a symbiotic relationship with seeds (stark et al. 2009). our study also indicates that nonrhizoctonia fungi do not establish a symbiotic relationship with s. vomeraceae seeds. orchid tubers are economically important in turkey, iran, and greece. therefore, large-scale cultures should be set up for their commercialization. ex vitro symbiotic propagation of orchids has certain advantages over in vitro asymbiotic propagation since the asymbiotic method requires complex laboratory equipment, expensive chemicals and sterile laboratory conditions. in addition, studies on germination and seedling growth in natural conditions (in situ, ex situ) are also not suitable. structure of the soil, other microorganisms, various insect larvae or nematodes make the efficiency of this method difficult (mccormick et al. 2013). otherwise, it is stated that ex vitro production is easy, cheap and the most suitable method for mass production (aewsakul et al. 2013). soil or a substrate is sterilized to eliminate biotic factors, while superficial, surface sterilization of seeds and other complex laboratory methods and materials are not required. there are very few studies on ex vitro germination and seedling development of orchid seeds to date and they are about epiphytic orchids (quay et al. 1995, aewsakul et al. 2013). hence, this research is the first study in which the seeds of s. vomeracea, a temperate tuberous orchid, were germinated in ex vitro conditions and the tuber formation in natural conditions was observed. this orchid is one of the most collected orchids because it is one of the orchids with highest glucomanan content (ozkoç and dalcı 1993, acemi and özen 2019). according to the results of this study, ex vitro seedlings of s. vomeracea under heavy destruction may establish new populations after being transferred to nature and this may contribute to the rehabilitation of destroyed areas. the most important indicator of the adaptation of tuberous temperate orchids to their natural conditions is the occurence of the first tuber, because it remains in the soil and develops as a new individualin the following year (sezik 2002). production of tubers by all seedlings transferred to soil indicates that the reintroduction to natural conditions after ex vitro symbiotic germination is succesful. thus new populations may be established by applying this method to the other orchids under threat of extinction. tetrazolium test showed that viability of the seeds of s. vomeracea (90.32% ± 1.30) was lower than that observed in germination test with svl21 (98% ± 2). several studies indicated that the tetrazolium test was not a reliable indicator for orchid seeds (vujanovic et al. 2000, kömpe et al. 2020). there may be several reasons for this, which suggests that the viability test is not a good indicator for the germination of orchid seeds. incubation period may not be sufficient for the breakage of all the seed coats. water may not reach equally to all seeds in a seed package, or seed coats did not break in a way that the dye can penetrate evenly. for this reason, viability tests must be supported by seed germination tests. while viability test is not always a reliable indicator of germination potential for orchid seeds, performing a viability test on seeds for which germination potential is not known may be useful to prevent loss of time and materials. conclusions rhizoctonia-like fungi isolated from s. vomeracea roots are closely related to tulasnella, therefore it can be said that the dominant fungus of the mycorrhizal association in s. vomeracea is generally tulasnella. in this research, the life cycle of s. vomeracea from seed to tuberous seedling stage was presented for the first time in ex vitro conditions. with our method, it is possible to produce orchids on a large scale and to prevent orchid destruction and help reintroduction to their natural habitats. acknowledgment this research is supported by the scientific and technical research council of turkey (project no: 114z218). references acemi, a., özen, f., 2019: optimization of in vitro asymbiotic seed germination protocol for serapias vomeracea. the eurobiotech journal 3, 143–151. aciego pietri, j. c., brookes, 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(eds.), pcr protocols: a guide to methods and applications, 315–322. academicpress, san diego. 80 acta bot. croat. 81 (1), 2022 acta bot. croat. 81 (1), 80–88, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-005 issn 0365-0588 eissn 1847-8476 determination of salt tolerance levels and genetic relationships of vicia sativa cultivars using gene targeted functional markers iskender tiryaki*, nuray isidogru canakkale onsekiz mart university, faculty of agriculture, department of agricultural biotechnology, terzioglu campus, 17000 canakkale, turkey abstract – the objectives of the present study were to determine salt tolerance levels of 12 different common vetch (vicia sativa l.) cultivars at germination stage in the presence of 250 mm nacl and to reveal genetic relationships based on gene targeted functional markers (gtfms) associated with salt tolerance. the results revealed the presence of a significant genetic variation among the cultivars although salt stress significantly reduced all germination parameters tested. the cultivar ozveren was the most salt tolerant with 20.1% reduction in final germination percentage compared to control seeds while cultivars alınoglu, ayaz and bakir did not germinate. the maximum delays in germination rate (g50 = 3.78 days) and synchrony (g10-90 = 3.45 days) were obtained from the cultivars urkmez and ozveren, respectively. the gtfms provided a total of 53.1% polymorphism. the primers of mtsos2 gene gave the highest numbers of alleles per primer pair while the highest polymorphism rate (77.8%) was obtained from the mtp5cs gene. the first three components of principal component analysis explained 57.63% of total variation. this study concluded that the cultivars determined to be salt tolerant and sensitive at germination stage distributed into three main clades determined by upgma analysis while the gtfms associated with salt tolerance successfully determined the genetic relationships of common vetch cultivars. keywords: common vetch, germination, gene, markers, salt tolerance introduction soil salinity is one of the most important abiotic stress factors directly limiting plant yield in agricultural production areas of all over the world (shokat and großkinsky 2019). plants respond to salts in soil or in irrigation water at different levels by tuning complex physiological and molecular mechanisms (mel et al. 2019). high salt prevents the water uptake and creates a physiological drought (khayamim et al. 2014) or ion toxicity to the embryo of seeds, which directly limits fast and even seed germination (farooq et al. 2017). salt tolerance level of a plant can vary based on plant developmental stage (bu et al. 2015). seed germination and seedling stages of plants are generally less tolerant than mature plants (hussain et al. 2010). therefore, determination of salt tolerance levels of cultivars at germination stage is one of the most important tasks to promote successful plant development for sustainable agricultural production. the common vetch (vicia sativa l.) is considered one of the most important annual, self-pollinated, diploid forage crop species and has plasticity not only for adaptability to different soil and climate conditions but also its diverse use as grain, straw, hay, silage, and green manure along with soil improvement ability with nitrogen fixation (sherasia et al. 2017). the common vetch also provides valuable protein and mineral sources for cattle and poultry in turkey, australia, new zealand, china and eastern europe ( firincioglu et al. 2007, sherasia et al. 2017). like many other legume crops, common vetch has, however, reduced growth and yield under saline condition and is considered more vulnerable to salt stress than some other major crop species such as cereals (hussain et al. 2010). to discriminate individuals from various breeding sources, pcr-based molecular markers are the best tool for genetic characterization and the estimation of genetic diversity in various organisms including plants (poczai et al. * corresponding author e-mail: tiryaki46@yahoo.com salt tolerance in vicia sativa acta bot. croat. 81 (1), 2022 81 2013a). molecular markers are also more reliable than pedigree data for parental selection to estimate genetic diversity in plants (babic et al. 2016). however, the power of a molecular marker is mainly determined by the level of polymorphism detected (wan et al. 2004). as a complex taxon, v. sativa represents a diverse phylogenetic relationship (kartal et al. 2020). various types of molecular markers have been previously used to resolve interand intra-specific diversity in common vetch by using srap and issr (cil and tiryaki 2016), ssrs (raveendar et al. 2015), est-ssr, (liu et al. 2014), scot (chai et al. 2017) and snps (de la rosa et al. 2020). the recent sequencing advances in plant biotechnology resulted in an avalanche of information in terms of dna sequences and functional gene determination in various plant species (chai et al. 2017). therefore, not only has the transferability of molecular markers among the species been studied (raveendar et al. 2015) but new alternative molecular marker techniques have also been developed in diverse plant species (poczai et al. 2013b). of those, the molecular markers which are generated based on untranslated regions of expressed sequence tags (ests) are called gene-targeted markers (gtms) (poczai et al. 2013b) while the gene markers involved in the variation of phenotypic traits due to their functional gene sequences are called gene-targeted functional markers (gtfms) (arnholdtschmitt 2005). this study aimed to determine the salt tolerance levels of 12 common vetch cultivars at germination stage by using seed germination parameters and to reveal genetic relationships based on salt tolerance-related gene primers. the aim of the study was also to reveal whether or not gtfms can also distinguish the salt tolerance and sensitivity levels of common vetch cultivars at germination stage in the presence of 250 mm nacl. materials and methods seed material all available 12 common vetch cultivars were kindly provided from the ankara field crops central research institute (alınoglu, ayaz, ankaramoru, bakir, farukbey, zemheri), izmir aegean agricultural research institute (alper, cumhuriyet, selcuk, urkmez) and adana eastern mediterranean agricultural research institute in turkey (ozveren, yucel). a range of nacl concentrations were tested in pre-experimental trials and 250 mm nacl was chosen to determine the salt tolerance levels of the 12 common vetch cultivars. germination experiment a single layer of 50 seeds of each cultivar was placed in covered petri dishes (80 × 15 mm) on double layers of filter papers saturated with 4 ml of 250 mm nacl. a temperature-controlled incubator kept at 20 ± 0.5 °c in darkness was used for germination test. four replications of 50 seeds were arranged in a completely randomized block design. seeds showing a radicle exceeding 2 mm in length emerging from the testa were recorded as germinated. the germinated seeds were daily removed from the petri dishes until the germination count was unchanged for 3 subsequent days (total 15 days). the final germination percentage (fgp), germination rate and span of germination parameters were determined as described previously (tiryaki and kaplan 2019). germination rate was used to estimate days to 50% of fgp (g50) and the span of germination (g10-90) was used to estimate the time from 10% to 90% of fgp by using methods described previously (tiryaki and kaplan 2019). the control seeds of each cultivar were treated with 4 ml of dh2o and were germinated under the same germination conditions used for the salt experiment as described above. dna extraction and quality control the seeds of each cultivar were planted in rows with 10 cm raw space in plastic boxes (10 × 35 × 45 cm) filled with peat with 3 cm planting depth and were incubated in a plant growth chamber with a 12 h light (350 µmol m-2 s-1)/dark cycle at 20 oc. the young leaves of five seedlings from each cultivar were bulked and were used for dna extraction by using a plant genomic dna extraction kit (favorgen, pingtung, taiwan) following the manufacturer’s instruction. the dna concentrations were estimated by comparing known concentration of λ dna on 1% agarose-gel electrophoresis and were used in pcr analysis after concentration equalization. gene targeted functional markers and pcr amplification in all, nine gene specific primer pairs (tab. 1) were used after pcr amplifications of each primer pair were optimized in a gradient pcr (thermo fisher scientific, inc., usa) to determine the best annealing temperature in common vetch genome. eight of nine gene specific primer pairs were either directly or indirectly associated with salt tolerance in plants while mtactin (medicago truncatula actin) gene was used to reveal the level of variation of mtactin gene in the common vetch genome. the reaction mixtures of pcr were set in a 20.5 μl reaction mixture containing 100 μm each of datp, dgtp, dctp, and dttp, 10 mm trishcl, ph 8.8, 20 mm (nh4)2so4, 2.0 mm mgcl2, 1 unit of taq dna polymerase (invitrogen), and 20 ng of genomic dna (kang et al. 2002). the pcr cycles were set as follows: 5 pre-cycles of 1 min at 95 °c for denaturing, 45 seconds at 35 °c for annealing and 30 seconds at 72 °c for extension. annealing temperature (ta) of each primer was raised to the temperature given in table 1 for 1 min for another 35 cycles along with 15 seconds at 95 °c for denaturing, 1 min at 72 °c for extension and 7 min at 72 °c for final cycle extension. amplicons of pcr products were separated on a 2% (w/v) agarose gel in 1×tbe buffer at constant 80 v for 3 h, stained with ethidium bromide (2 µl/100 ml) and visualized under uv light source. tiryaki i., isidogru n. 82 acta bot. croat. 81 (1), 2022 data analysis an angular transformation (arcsine√fgp) was applied to the final germination percentage (fgp) data and was used in statistical analysis (tiryaki and kaplan 2019). to eliminate differences among the cultivars that might be due to the differences in percentages of live seeds in the seed stocks, an adjustment was performed for the fgp values of each cultivar determined under salt stress germination conditions as described before (tiryaki and andrews 2001) using the following equation: adjusted fgp value of cultivar n = [(fgp value of control seeds of cultivar n / fgp of salt stress seeds of cultivar n)] × 100. these values were then used to calculate the fgp reduction percentage due to salt stress for each cultivar. analysis of variance was used to calculate statistically significant differences of germination rate, span of germination and transformed fgp data using sas software (sas 1997). the mean separation was done using fisher’s least significant difference (lsd) test if the f-test was significant at p < 0.05. the presence (1) or absence (0) of dna bands with high intensity was used for allele scoring. the polymorphism information content (pic) of each primer was calculated as described before (powell et al. 1996): pic= 1−∑(pij)2 where pij is the frequency of the ith band revealed by the jth primer, p(ij) is summed for all the bands of each primer. to determine the genetic similarity of the genotypes, numerical taxonomy multivariate analysis system (ntsyspc-2.1) was used by using the dice coefficient (dice, 1945). the unweighted pair-group with arithmetic mean (upgma) method was used to construct the dendrogram. the eigen and proj modules of ntsyspc-2.1 program were used for pca (the principal component analysis) analysis. tab. 1. salt tolerance related genes used as gene targeted functional markers in the study. the name of genes, references, forward (f) and reverse (r) primer sequences, annealing temperature (ta), the number of amplicons, the number of polymorphic amplicons, poly morphism rate (%) and polymorphism information content (pic) values. medicago truncatula salt overly sensitive gene 1 (mtsos1), m. truncatula salt overly sensitive gene 2 (mtsos2), m. truncatula salt overly sensitive gene 3 (mtsos3), arabidopsis thaliana dehydration responsive element binding factor 1b (atdreb1b), a. thaliana delta1 pyroline-5-carboxylate synthase (atp5cs), m. truncatula delta1 pyroline-5carboxy late synthase (mtp5cs), m. truncatula proline dehydrogenase (mtprodh), a. thaliana vacuolar na+/h+ exchanger gene 1 (atnhx1) and m. truncatula actin (mtactin). gene reference f/ r primer 5’-3’ annealing temperature (ta. °c) no. of total amplicons no. of polymorphic amplicons polymorphism rate (%) pic mtsos1 (liu et al., 2015) gctgactttcccgtatg 48 8 6 75.0 0.50 tggcacccagttctttc mtsos2 (liu et al., 2015) ccgtggtatcttctgtt 48 11 5 45.5 0.29 caagggttaggtgtatt mtsos3 (liu et al., 2015) tctgaggcaaacagggta 48 1 0 0.0 0.00 ctgggaaatgctaaggtaat atdreb1b (wang et al., 2006) ggatcctgatcaatgaactacattttc 50 4 3 75.0 0.44 agctcccattctaaaaaggaac atp5cs (yamada et al., 2005) tcagaggactacgtgttgga 55 8 6 75.0 0.32 atgagtactaagcagagagg mtp5cs (quan et al., 2016) gagagggaacggccaagtg 52 9 7 77.8 0.45 cagatccttgtgtgtata mtprodh (planchet et al., 2014) ccaacgtccacgctgataaga 57 3 1 33.3 0.19 acaggtcctatagccgttgca atnhx1 (yokoi et al., 2002) caacaccccaaaatccatac 52 7 5 71.4 0.25 atatccctttgttggaccaa mtactin (wang et al., 2017) acgagcgtttcagatg 50 4 1 25.0 0.23 acctccgatccagaca mean 6.1 4.3 53.1 0.30 total 55 34 – – salt tolerance in vicia sativa acta bot. croat. 81 (1), 2022 83 results effects of salt stress on germination performance of the cultivars salt stress significantly reduced the fgps of all cultivars while the same cultivars had high final germination percentages (fgps) in nonstress (control) conditions with a few exceptions (fig. 1). the cultivars ayaz, alınoglu and bakir had very low levels of fgps under salt (0.4%, 0.7% and 0.6% of fgps, respectively) stress conditions and the germination data of those cultivars were, therefore, excluded from further analysis. the seeds of cultivar ozveren had the highest fgp in the presence of 250 mm nacl and were determined to be the most salt tolerant cultivar at germination stage (fig. 1). in contrast, the cultivars zemheri and ankaramoru were statistically the most sensitive cultivars to salt stress since they had the lowest fgps (5.8% and 6.0%, respectively). the reduction rates in fgps due to salt stress were 94.0% and 93.5% for the cultivars zemheri and ankaramoru, respectively while the cultivar ozveren had the lowest reduction rate (20.1%) (fig. 1). salt stress significantly increased the time required for 50% of fgp in all cultivars (fig. 2). the germination rates ranged from 1.07 days (urkmez) to 2.21 days (zemheri) under no salt stress conditions and from 4.0 days (zemheri) to 5.62 days (ankaramoru) (fig. 2) at 250 mm nacl. due to salt stress, the highest delay in the time required for 50% of fgp was determined in the cultivar urkmez with 3.78 days fig. 1. the final germination percentage (fgp) of vicia sativa cultivars germinated in the presence of 250 mm nacl and no salt conditions (control) at 20 ± 0.5 °c in darkness, and reduction rate (%) in fgp due to salt tolerance. decline in fgp of each cultivar due to salt stress was presented as reduction percentage. the bars indicate standard deviation (n = 4). the means that differ significantly (alpha = 0.05) are indicated by different letters. fig. 2. the time to 50% of final germination percentage (g50) of vicia sativa cultivars germinated in the presence of 250 mm nacl and no salt conditions (control) at 20 ± 0.5 °c in darkness. the bars indicate standard deviation (n = 4). the means that differ significantly (alpha = 0.05) are indicated by different letters. tiryaki i., isidogru n. 84 acta bot. croat. 81 (1), 2022 in comparison to control seeds while the cultivar zemheri had the least delay (1.79 days) (fig. 2). the salt stress significantly extended the time required from 10% to 90% of fgp for all cultivars tested in comparison to control seeds (fig. 3). the span of germination ranged from 2.02 days (ankaramoru) to 4.45 days (selcuk) and from 0.6 days (ozveren) to 2.35 days (zemheri) for stress and no stress conditions, respectively. the cultivars zemheri and ankaramoru had about the same span of germination in both salt stress (g10-90 = 2.37 days, g10-90 = 2.02 days) and no stress conditions (g10-90 = 2.35 days, g10-90 = 1.94), respectively (fig. 3). because of salt stress, the highest delay (g10-90 = 3.45 days) in the germination synchrony was determined with the cultivar ozveren with 3.45 days (fig. 3). genetic diversity based on gtfms nine loci specific markers produced a total of 55 alleles 34 of which were polymorphic (tab. 1). the mtsos3 (m. truncatula salt overly sensitive gene 3) gene marker provided one monomorphic band only. the polymorphism rate of the markers used in the study ranged from 25.0% to 77.8% with an average of 53.1% per locus. the numbers of amplicons per marker changed from 1 to 11, an average of 6.1 alleles. the marker mtsos2 had the highest number of amplicons (11 bands) and 5 of them were polymorphic while the polymorphism rate of mtp5cs (m. truncatula delta1 pyroline-5-carboxylate synthase) gene marker produced the highest polymorphism rate (77.8%). the average pic values fig. 3. the time from 10% to 90% of final germination percentage (g10-90) of vicia sativa cultivars germinated in the presence of 250 mm nacl and no salt conditions (control) at 20 ± 0.5 °c in darkness. the bars indicate standard deviation (n = 4). the means that differ significantly (alpha = 0.05) are indicated by different letters. fig. 4. genetic similarity dendrogram based on nei (1972) for 12 vicia sativa cultivars created according to the upgma method using 9 salt tolerance-related gene targeted functional markers. asterisk (*), indicates the cultivars which did not germinate at 250 mm nacl. the different letters given in the parentheses indicate statistical mean differences (alpha = 0.05) of final germination percentage for the cultivars germinated at 250 mm naci. salt tolerance in vicia sativa acta bot. croat. 81 (1), 2022 85 of nine markers changed from none to 0.50 with an average of 0.30 while the marker mtsos1 (m. truncatula salt overly sensitive gene 1) had the highest (0.50) (tab. 1). twelve common vetch cultivars were divided into three main clades based on upgma analysis generated by using 55 alleles (fig. 4). the cluster analysis revealed that the cultivars alınoglu and farukbey were grouped in clade i and distinctly separated from the others (fig. 4). clades ii and iii shared the remaining cultivars equally. the eigen values of the c1, c2 c3 components of pca analysis explained 57.63% of total variation. the twoand three-dimensional plots of pca analysis also showed that the cultivars ayaz and yucel were cultivars the most distinct from each other (fig. 5). discussion the results of this study showed that final germination percentage, germination rate and span of germination parameters of 12 common vetch cultivars were significantly reduced at 250 mm nacl and that the adverse effects of salt stress varied according to the cultivar used. these results revealed the presence of genetic variation among the common vetch cultivars for salt tolerance level at germination stage. cultivar ozveren was the most salt tolerant with 20.1% reduction rate in fgp at 250 mm nacl while cultivars alınoglu, ayaz and bakir did not germinate (fig. 1). high salt concentration also significantly delayed the time required for 50% of fgp in all cultivars (fig. 2) while span of germination was not changed for cultivars zemheri and ankaramoru (fig. 3). these findings suggested that fgp and germination rate are better parameters to determine salt tolerance levels of the common vetch cultivars than span of germination. similarly, a greater salt susceptibility and a better detection of genotypic variability in germination rate under salt stress were also reported for other plant species including vicia faba l. (el-bok et al. 2015) and oryza sativa l. (ologundudu et al. 2014). the results of this study also showed that the salt concentration used in this study can successfully be used to determine salt tolerance levels of common vetch cultivars at germination stage. akhtar and hussain (2009) reported that germination percentage of common vetch was significantly reduced at 150 mm nacl which was used as the highest nacl concentration tested. however, our pre-experiment trials showed that lower concentrations of nacl were not able to discriminate the high salt tolerant common vetch cultivars from the moderate salt tolerant ones. therefore, the right salt concentration is one of the most important prerequisites for the determination of salt tolerance levels of cultivars. salt concentrations that are too high fully inhibit seed germination while relatively low salt concentrations are not able to distinguish salt tolerant genotypes from nontolerant or moderately tolerant. previous reports indicated that vicia sativa can tolerate moderate salt levels at germination stage (akhtar and hussain 2009) and the level of salt tolerance is less in comparison to pisum sativum l. (bilgili et al. 2011). however, this study revealed that the cultivars ozveren, alper, urkmez and cumhuriyet can be used as salt tolerant cultivars and should be compared with other important seed legume crops since they had relatively high fgps at 250 mm nacl which was generally considered a high salt concentration for legume plants at germination stage (farooq et al. 2017). adverse effects of salt stress at lower salt concentrations than in the current study were reported for other vetch species including vicia pannonica crantz. (ertekin et al. 2018), v. faba and v. villosa roth. (lee et al. 2014) at germination stages. we have used gene-specific primers as gtfms which were directly or indirectly related to salt tolerance in plants (tab. 1). all the primers of gtfms amplified in common vetch genome and provided a total of 53.1% polymorphism rate (tab. 1), suggesting a high degree of transferability for the primers of salt tolerance-related genes among distantly related species. the highest polymorphism rate was obtained from mtp5cs gene primers with 77.8% while mtsos2 gene primers had the highest numbers of alleles per primer pair (11 bands). the mtsos1 gene primers produced more polymorphic bands (75.0%) and gave the highest pic value (0.50), higher than mtsos2 (45.5%) and mtsos3 (single monomorphic band only) genes. the mtsos1 gene is known to be among the most important loci and provides a better salt tolerance to plants than other members of sos genes (shi et al. 2000). this study confirmed that mtsos1 gene has more allelic variation in common vetch plants than the other members of the gene family and may play a more critical role in the control of salt tolerance in this genus. as a most abundant member of the sodium/proton antiporter gene family, atnhx1 (arabidopsis thaliana (l.) heynh. vacuolar na+/h+ exchanger gene 1) mediates salt tolerance in plants due to regulation of na+ homeostasis in the cell (shi and zhu 2002). the atnhx1 gene specific primers used in this study provided a 71.4% polymorphism rate, indicating the presence of a high level of allelic variation for atnhx1 gene in common vetch. transcriptional factor mtdreb1b (arabidopsis thaliana dehydration responsive element binding factor 1b) gene encodes dehydration responsive element binding protein and is involved in plant responses to several abiotic stresses including salt tolerance (donde et al. 2019). up regulation of such transcription factors in plants increases proline content, which is one of the key molecules protecting plant cells in their responses to various abiotic stresses including salt (dar et al. 2016). the results of this study revealed that atdreb1b gene primers have a higher polymorphism rate (75%) along with the genes related to proline biosynthesis, namely delta1 pyroline5-carboxylate synthase genes atp5cs (75%) and mtp5cs (77.8%) than mtprodh (m. truncatula proline dehydrogenase) gene primers which had 33.3% polymorphism rate (bouazzi et al. 2019). on the other hand, mtactin gene is generally used as control in transcription analysis due to its lower level of variation than other constitutive genes under various stress conditions including salt (zhang et al. 2019). the results of this study showed that mtactin gene primers tiryaki i., isidogru n. 86 acta bot. croat. 81 (1), 2022 had a relatively low polymorphism rate (25%) in common vetch compared to the other gene specific primers tested, suggesting that a low level of variation of mtactin gene in common vetch should be considered when this gene is used as a housekeeping gene in rt-qpcr analysis. the first three components of pca analysis explained 57.63% of total variation and the cultivars alınoglu and farukbey separated from the rest of the cultivars while the cultivars ayaz and yucel were determined as the most distant cultivars (figs 4 and 5). the upgma analysis provided three main clades (figs 4 and 5). of those, clade iii included 5 cultivars and four of them (urkmez, alper, ozveren and yucel) were determined to be salt tolerant at germination stage while cultivar zemheri in the same clade was determined to be salt sensitive. clade ii also included both salt tolerant and sensitive cultivars while clade i contained 2 salt sensitive cultivars only (figs 4 and 5). it was previously hypothesized that alternative oxidase (aox) gene can be used as a functional marker under stress conditions (arnholdtschmitt et al. 2006). recent advances in current genome sequencing technologies and gene expression studies also resulted in the identification of a large number of functional genes involved in controlling agronomic traits including salt tolerance (arnholdt-schmitt 2005) and opened a new era in which those complete or partial gene sequences as molecular markers can be employed. as a result of these efforts, caat box derived polymorphism (cbdp) and start codon targeted (scot) polymorphism become important types of molecular markers in plant genome analysis ( sharma et al. 2019). in this study, we have used the primers for salt tolerance related genes as gtfms to reveal genetic relationship as well as to determine the possibility of those gene primers to evaluate salt tolerance levels of the common vetch cultivars. the results revealed that salt tolerance-related gtfms did not clearly distinguish the salt tolerant and sensitive common vetch cultivars defined in germination stage at 250 mm nacl although the same markers were successfully able to determine the genetic relationships among the cultivars (figs. 4, 5). one of the reasons for such discrepancies may be due to gene primers indirectly related to salt tolerance being used in the study such as dreb1b, prodh and p5cs genes along with mtactin gene. another reason may come from the trait of salt tolerance per se which is a polygenic trait influenced by other environmental stimuli including drought stress, and its signaling cross talk with other important functional genes in various stages of plant growth and development. these findings suggested that a better clustering in terms of salt tolerance levels of the cultivars might be obtained if more specific gene markers associated with salt tolerance are used. however, some other genes which have a cross talk in response to salt tolerance in plants should also be considered to have a better resolution in such analysis. overall, the results of this study demonstrate that gene specific primers related to salt tolerance in plants could be used to discriminate the salt tolerant and salt sensitive cultivars at germination stage as well as to determine intraspecific genetic diversity if a higher number of salt tolerance related genes are used as gtfms. however, further studies should be conducted to test the reliability and reproducibility of gtfms and their relation to not only salt tolerance but also to other abiotic stresses at various plant growth and development stages in various plant species. acknowledgements this research is funded by canakkale onsekiz mart university (comu, bap grant #: fyl-2017-1156). author contribution statements i.t. conceived the idea, granted financial support, planned the experiments, processed the experimental data, performed the analysis, performed all the numerical calculations, interpreted the data, designed the figures and table, drafted the manuscript. n.i. carried out the experiments. fig 5. two (a) and three-dimensional (b) pca plots of 12 vicia sativa cultivars analyzed by 9 salt tolerance related gene targeted functional markers. the eigen values of c1, c2 and 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facilitates symbiosome accommodation during nodulation in medicago truncatula. new phytologist 221, 1049–1059. acta bot. croat. 82 (1), 2023 71 acta bot. croat. 82 (1), 71–79, 2023 coden: abcra 25 doi: 10.37427/botcro-2023-004 issn 0365-0588 eissn 1847-8476 effects of exogenous no on the growth and photosynthetic fluorescence characteristics of ryegrass seedlings under b[a]p stress yue li*, junqiang ma, yu wang, sunan xu, lei jiang, lihong zhang, wei hou* school of environment, liaoning university, 66 chongshan middle road, huanggu district, shenyang 110036, china abstract –benzoapyrene (b[a]p) pollution poses a threat to the environment and the food chain and consequently to human health. however, the alleviation of the harmful effects of b[a]p pollution in perennial ryegrass (lolium perenne l.) by the application of exogenous nitric oxide (no) has been ignored. thus, in this paper the effects of exogenous sodium nitroprusside (snp, a no donor) on the growth, photosynthetic fluorescence characteristics, and antioxidant enzyme activity of ryegrass exposed to b[a]p stress are investigated. b[a]p stress induced the reduction of the aboveground and belowground dry weights, chlorophyll (a, b), the total chlorophyll contents, the carotenoid content, the net photosynthetic rate (pn), the intercellular carbon dioxide concentration (ci), the water use efficiency (wue), the photosystem ii (psii) potential activity (fv/f0), the maximum quantum yield of psii photochemistry (fv/fm), the steady-state fluorescence yield (fs), and the non-photochemical quenching (qn), while enhancement was recorded in response to the foliar spray of snp at 200 and 300 μmol l–1 under b[a]p stress. gray correlation and principal component analyses show that 200 μmol l–1 of snp more drastically alleviated the damage caused by b[a]p stress than 300 μmol l–1 of snp. the exogenous no-mediated alleviation of b[a]p toxicity in ryegrass was associated with preserved photosynthetic characteristics and activation of antioxidant enzymes. keywords: no, b[a]p stress, ryegrass, growth, photosynthesis, chlorophyll fluorescence parameters introduction benzo(a)pyrene (b[a]p), a typical polycyclic aromatic hydrocarbon (pah) organic compound commonly found in the natural environment, has been recognized as one of the three major carcinogens by the world health organization and is often used as a representative indicator for determining pahs (ye et al. 2019). b[a]p has a high octanolwater partition coefficient and high vapor pressure, so it is difficult to degrade in the natural environment and can easily accumulate in the atmosphere, water bodies, and soil and cause serious environmental pollution (ncube et al. 2017). b[a]p contaminated soil, which is primarily distributed in industrially contaminated sites, such as in northeast and north china, is a major concern in several regions in china, with the b[a]p content of the soils of industrial areas peaking at over 1500 μg kg–1. the average b[a]p content of the soil in the yangtze river delta region of china exceeds 200 μg kg-1 (fismes et al. 2002). nitric oxide (no), which is a reactive nitrogen species, is recognized to play a very important signaling role in plants and has been reported to be involved in plant growth processes (dai et al. 2020) and responses to various environmental stresses, including salinity (ali et al. 2017), uv light (yan et al. 2016), water deficit (silveira et al. 2016), heat (song et al. 2013), and heavy metals (he et al. 2014). reportedly, exogenous no application is involved in various physiological mechanisms that improve plant tolerance to various stresses, including metal toxicity, by increasing the activity of antioxidant enzymes and subsequently reducing the accumulation of reactive oxygen species (ros) (nagel et al. 2019). foliar spray of snp can enhance metal transporters and reduce as uptake while inducing new adventitious root formation and enhancing antioxidant and defense capacities (souri et al. 2020), indicating the positive role of exogenous no in as detoxification. no also plays a key role in regulating plant stomatal movement and maintaining chlorophyll content under environmental stress. the application of no synthesis promotes the production of plant carotenoids and enhances photosynthetic capacity by increasing the quantum production of photosystem ii (psii) in stressed plants (tiwari et al. 2019). however, very * corresponding author e-mail: yuanlinliyue@163.com, houwei@lnu.edu.cn mailto:yuanlinliyue@163.com li y., ma j., wang y., xu s., jiang l., zhang l., hou w. 72 acta bot. croat. 82 (1), 2023 few studies have focused on the influence of exogenous no on a series of physiological changes and growth abnormalities in plants under b[a]p toxicity. perennial ryegrass (lolium perenne l.) is a commonly used turfgrass species in the urban areas of central and western europe and china and has a very wide scientific value (ding et al. 2002). given its high biomass production rates, ryegrass has been selected as a ditch plant for domestic wastewater treatment and plays an important role in nitrogen removal from wastewater (duan et al. 2017). ryegrass is also used as a forage crop and exhibits easy germination (dąbrowski et al. 2015). recent research confirms ryegrass’ potential in phytoextraction for single metal cd pollution or combined cd and zn pollution (zhang et al. 2019). ryegrass can be used as a material for the remediation of contaminated soil and is widely used for phytoremediation (xie et al. 2021). however, little information is available regarding the alleviation of the adverse effects of b[a]p stress in ryegrass through seeding treatment with exogenous no. therefore, the present study aims to explore (1) whether exogenous sodium nitroprusside (snp, an exogenous no donor) treatment ameliorates b[a]p toxicity in ryegrass and (2) the mechanisms by which no improves b[a]p tolerance, by investigating the growth parameters, photosynthetic characteristics, chlorophyll fluorescence properties, and antioxidant enzyme activities. materials and methods plant material and treatment seeds of ryegrass were available from liaoning fuyou seed co. ltd. in shenyang, china. snp and b[a]p were purchased from sigma-aldrich. all reagents used in this study were of analytical grade. ryegrass seeds of uniform size were selected and surface sterilized with 5% h2o2 for 3 min. after soaking in distilled water for 12 h, the seeds of uniform size and shape were pregerminated on a double layer of moist filter paper for 48 h. the germinated seeds were transferred to plastic pots (22 cm in height, 20 cm in diameter) with 2 kg of nutrient soil. the measured soil parameters were as follows: soil ph 6.25, organic matter 20.27 g kg–1, and cation exchange capacity 20.54 cmol kg−1, total nitrogen and total phosphorus 3.66 and 3.79 g kg−1, respectively. the soil was sieved through a 3-mm sieve, and the uncontaminated soil was air-dried and mixed thoroughly with the base fertilizer. the soil ph, organic matter, alkaline soluble nitrogen, available phosphorus, and available potassium after mixing were 5.56, 25.3 g kg−1, 5.37 g kg−1, 6.22 g kg−1 and 5.93 g kg−1, respectively. the pots were placed in an experimental field in the ecological research center of liaoning university in a controlled environment, with an environmental temperature of 29 ± 5 °c/22 ± 3 °c (12 h day/ 12 h night), relative humidity of 62%~76%, and a photosynthetic photon flux density (ppfd) of 625 μmol m–2 s–1 provided by the led light source. appropriate soil moisture was maintained by watering every four days throughout the experimental duration. excess seedlings were removed, leaving thirty ryegrass seedlings for growth in each pot. pre-experiments were conducted to analyze the inhibition rate and growth of ryegrass seedlings at different concentrations of b[a]p applied foliarly, and 30 μmol l–1 was identified as the b[a]p stress concentration (data not shown). in this experiment, these treatments were applied via leaf spraying: the control was sprayed with deionized water; b[a] p 30 was sprayed with a solution with 30 μmol l–1 b[a]p; snp 100 + b[a]p 30, snp 200 + b[a]p 30, snp 300 + b[a]p 30, and snp 400 + b[a]p 30 were sprayed with solutions with 30 μmol l–1 b[a]p plus 100, 200, 300, and 400 μmol l–1 snp, respectively. when the ryegrass plants reached approximately 15 cm in height, 100 ml of the prepared treatment solution was sprayed uniformly per pot every other day by foliar spraying at 16:00. these treatments were arranged in a randomized complete block design with at least three pots per treatment. the investigated parameters were measured and analyzed when the plants reached approximately 22 cm in height after 14 days of snp treatments under b[a]p stress. growth measurements ten seedlings were harvested and divided into roots and leaves. root length and aboveground plant height of ryegrass seedlings (five plants of each treatment) were measured with a sliding caliper. then they were rinsed with tap water and distilled water three times, blotted with filter paper to dry the surface water, weighed immediately for belowground and aboveground fresh weight. the fresh sample materials were dried at 85 °c for 60 hours and weighed for aboveground dry weight and belowground dry weight. photosynthetic pigment content measurements photosynthetic pigments contents were determined using fresh leaves according to the method described by lichtenthaler (1987). ryegrass leaves were accurately weighed to 0.5 g, and soaked in extraction solution for 24 h in the dark. the extraction solution was composed of 10 ml of 80% acetone and 5 ml of 95% ethyl alcohol. the absorbance of the extract was recorded at 663, 645 and 470 nm, and the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid were obtained using the equations described by arnon (1949). photosynthetic parameters measurements five leaves of similar height and shape were selected from each pot to measure net photosynthetic rate (pn), stomatal conductance (gs), intercellular carbon dioxide concentration (ci), transpiration rate (tr) and water use efficiency (wue) measurements were made using a li-6400xt (licor, usa) portable photosynthesis meter in each application. measurements were performed from 9:00 to 12:30 in sunny weather conditions. to ensure that the measurements were carried out under approximately ideal photosynthetic effects of exogenous no on ryegrass under b[a]p stress acta bot. croat. 82 (1), 2023 73 conditions, the leaf surface temperature was controlled at 25 °c, the relative humidity was controlled at about 60%, the photosynthetically active radiation (par) was set to 1400 mol m–2 s–1, the anaerobic conditions were set to t = 27 ± 2 °c, and airborne co2 concentration was 430 ± 20 µmol mol–1. chlorophyll fluorescence measurements five leaves of similar height and shape were selected from each pot to measure chlorophyll fluorescence parameters using a li-6400 portable photo synthesizer equipped with a pulse-modulated-fluorescent leaf chamber (6400-30, li-cor inc., usa). before fluorometer measurements, plants were dark-adapted for 6 hours with leaf clips. the dark-adapted minimal fluorescence (f0) and maximal fluorescence (fm) were measured by applying a saturating actinic pulse of 8000 μmol m−2 s−1 for 1 s. the variable fluorescence (fv = fm − f0), psii potential activity (fv/f0) and maximum quantum yield of psii photochemistry (fv / fm) were calculated from fm, fv and f0. steady-state fluorescence yield (fs) was recorded in the light. a saturating actinic pulse of 8000 μmol m−2 s−1 for 1 s was applied to produce maximum fluorescence yield in the light-adapted state (f’m). the actual quantum yield of psii photochemistry (φ psii), minimum fluorescence value in the light (f’0) (murchie and lawson 2013), photochemical quenching (qp) and non-photochemical quenching (qn) were calculated as follows ( schreiber et al. 1995): fv/f0 = (fm – f0) / f0, fv/fm = (fm – f0) / fm, φ psii = (f’m – fs) / f’m, qp = (f’m – fs) / (f’m – f’0), qn = 1 – (f’m – f’0) / (fm – f0). determination of antioxidant enzyme activity leaf samples (0.6 g) were ground with 10 ml 50 mmol l–1 phosphate buffer (ph 7.0). then the homogenate was centrifuged (13,000 g, 20 min), and the supernatant was used to determine antioxidant enzyme activity. sod activity was defined by measuring the inhibition of nitro blue tetrazolium (nbt) photochemical reduction (tandy et al. 1989). pod activity was determined by monitoring guaiacol oxidation using the method described by pinhero et al. (1997). cat activity was assayed in a reaction mixture containing 50 mmol l–1 sodium phosphate buffer (ph 7.0), 0.2 μmol l–1 h2o2 and a suitable aliquot of enzyme extract (dai et al. 2020). data analysis all experimental data were expressed as mean ± sd of at least three replicates. all figures were plotted by using origin pro 8.5. statistical significance analysis was performed by duncan’s multiple range test at 0.05 probability level using computer software spss 24.0 (spss inc, chicago, il, usa). the value of p < 0.01 or p < 0.05 represented a very significant difference or remarkable variance, respectively. significant differences at the p < 0.05 level were indicated by different lower-case letters. as a systemic analysis method, gray correlation degree theory is often used to measure the correlation degree between each factor according to the similar degree or different degree of their development situation. the comprehensive evaluation of evaluated parameters of ryegrass under b[a]p stress after snp application was carried out according to the equations of grey correlation degree and entropy weight method using spss 24.0. principal component analysis (pca) using origin pro 8.5 and spss 24.0 was performed to further evaluate the responses of growth, photosynthetic characteristics and chlorophyll fluorescence parameters and antioxidant enzymes activities of ryegrass to different snp treatments under b[a] p stress. the pca allowed the ordination of the parameters to discover potential groupings within the parameters. plots were generated using principal components (pc) 1, 2 and 3 as axes. therefore, pca can be used to determine the most appropriate exogenous no concentration which can alleviate b[a]p stress of ryegrass. results effect of exogenous no on the growth characters of ryegrass under b[a]p stress b[a]p stress significantly reduced the underground dry weights (p < 0.05) of the ryegrass plants compared with the control (fig. 1). in contrast, the aboveground fresh weight and the belowground root length significantly increased by 14.16% and 26.17%, respectively, in the b[a]p treatment compared with the control. b[a]p stress did not affect the aboveground dry weight and plant height and the belowground fresh weight. the exogenous application of 200 μmol l–1 snp considerably enhanced the belowground dry and fresh weights, the root length, and the aboveground plant height by 78.67%, 86.07%, 7.78%, and 14.38% compared with those under b[a]p stress, respectively. the foliar application of 200 μmol l–1 snp showed more pronounced results than that of the other three concentrations of snp treatments on ryegrass plants under b[a]p stress. effect of exogenous no on photosynthetic pigment contents of ryegrass under b[a]p stress the chlorophyll a, chlorophyll b, and total chlorophyll contents of the ryegrass under 30 μmol l–1 b[a]p stress drastically decreased (p < 0.05) and the carotenoid content declined but not radically compared with the control (fig. 2). compared with the sample under 30 μmol l–1 b[a]p stress, the chlorophyll b contents of ryegrass under the 100, 200, 300, and 400 μmol l–1 snp treatments were notably boosted by 60.92%, 72.73%, 161.11%, and 110.16%, respectively. similarly, the carotenoid contents dramatically increased by 71.29%, 91.72%, 94.24%, and 71.16% compared with those under b[a]p stress. the total chlorophyll content also increased following the 200 μmol l–1 snp treatment. li y., ma j., wang y., xu s., jiang l., zhang l., hou w. 74 acta bot. croat. 82 (1), 2023 fig. 1. effect of different concentrations (100, 200, 300 and 400 μmol l–1) of sodium nitroprusside (snp), a no donor, on the biomass of ryegrass seedlings under benzoapyrene (b[a]p) stress. (a) aboveground dry weight, (b) belowground dry weight, (c) aboveground fresh weight, (d) belowground fresh weight, (e) aboveground plant height, (f) belowground root length. data are mean ± standard deviation, n = 3. different letters indicate significant differences at p < 0.05. fig. 2. effect of different concentrations (100, 200, 300 and 400 μmol l–1) of sodium nitroprusside (snp), a no donor, on the photosynthetic pigment content of ryegrass seedlings under benzoapyrene (b[a]p) stress. (a) chlorophyll a content, (b) chlorophyll b content, (c) carotenoid content, (d) total chlorophyll content. fw – fresh weight. data are mean ± standard deviation, n = 3. different letters indicate significant differences at p < 0.05. effects of exogenous no on ryegrass under b[a]p stress acta bot. croat. 82 (1), 2023 75 effect of exogenous no on photosynthetic gas exchange parameters in ryegrass under b[a]p stress the pn, ci, and tr in ryegrass radically decreased under 30 μmol l –1 b[a]p stress compared with the control, but wue increased considerably by 42.21% (fig. 3). the exogenous snp application mostly alleviated the photosynthetic inhibition caused by b[a]p stress. the pn significantly increased when the concentration of snp was 200 μmol l–1 compared with that in the b[a]p treatment. under 100 μmol l–1 snp treatment, the gs and the ci significantly increased with the b[a]p treatment, indicating that 100 μmol l–1 snp had the best effect in alleviating b[a]p stress on photosynthetic gas exchange parameters. effect of exogenous no on chlorophyll fluorescence parameters of ryegrass under b[a]p stress compared with the control, the fv/f0 and фpsii decreased considerably due to b[a]p stress, whereas the qp and qn increased in ryegrass leaves under b[a]p stress conditions (fig. 4). the fv/f0, fs, and qn further increased in various degrees under low snp concentrations compared with the b[a]p stress, and the most significant effects were observed in 200 μmol l–1 snp treatment. after the application of 200 μmol l–1 snp significantly improved the fs and qn of ryegrass plants under 30 μmol l–1 b[a]p stress, the fs and qn were at their highest, namely, 116.20% and 24.37%, respectively, higher than those under b[a]p treatment. fig. 3. effect of different concentrations (100, 200, 300 and 400 μmol l–1) of sodium nitroprusside (snp), a no donor, on photosynthetic gas exchange parameters of ryegrass seedlings under benzoapyrene (b[a]p) stress. (a) net photosynthetic rate (pn), (b) stomatal conductance (gs), (c) intercellular carbon dioxide concentration (ci), (d) transpiration rate (tr), (e) water use efficiency (wue). data are mean ± standard deviation, n = 3. different letters indicate significant differences at p < 0.05. li y., ma j., wang y., xu s., jiang l., zhang l., hou w. 76 acta bot. croat. 82 (1), 2023 effect of exogenous no on antioxidant enzyme activity of ryegrass under b[a]p stress the sod, pod and cat activities in the presence of 30 μmol l–1 b[a]p increased by 6.29%, 56.33% and 26.73%, respectively, compared with those in the untreated control plants (fig. 5). however, no significant differences were found in the sod between the 30 μmol l–1 b[a]p treatment and the control. compared with the 30 μmol l–1 b[a]p treatment alone, the 100, 200, 300, and 400 μmol l–1 snp treatments increased the sod, pod and cat activities. among them, the 200 μmol l –1 snp supplementation of the 30 μmol l–1 b[a]p stress boosted the sod, pod and cat activities by 17.85%, 14.74% and 53.58%, respectively. meanwhile, the application of 300 μmol l –1 snp under b[a]p stress further boosted the sod, pod and cat activities by 27.36%, 10.16% and 63.60%, respectively. gray correlation analysis and pca we analyzed the investigated parameters of ryegrass under b[a]p stress after snp application using the gray correlation degree (tab. 1). our results show drastic differences in the 24 parameters among the different snp treatments. the associative order of first six parameters is as follows: chlorophyll a content > belowground root length > aboveground dry weight > net photosynthetic rate > total chlorophyll content > qp. the pca revealed that the first three components with eigenvalues could explain more than 70.4% of the total variation (fig. 6). pc1, pc2, and pc3 respectively account for 46.2%, 24.2%, and 14.6% of the physiological indexes. the pca provided a simplified classification of the growth, photosynthesis, f luorescence, and antioxidant enzyme activities of ryegrass for different snp treatments under b[a]p stress. pc1 tended to separate the effects of b[a]p fig. 4. effect of different concentrations (100, 200, 300 and 400 μmol l–1) of sodium nitroprusside (snp), a no donor, on chlorophyll fluorescence parameters of ryegrass seedlings under benzoapyrene (b[a]p) stress. (a) psii potential activity (fv/f0), (b) psii maximum light energy conversion efficiency (fv/fm), (c) actual quantum yield of psii photochemistry (фpsii), (d) steady-state fluorescence (fs), (e) photochemical quenching coefficient (qp), (f) non-photochemical quenching coefficient (qn). data are mean ± standard deviation, n = 3. different letters indicate significant differences at p < 0.05. effects of exogenous no on ryegrass under b[a]p stress acta bot. croat. 82 (1), 2023 77 stress and different concentrations of snp, and pc2 further segregated the differences of snp. according to the results, the application of 200 μmol l–1 snp had a greater alleviative effect on b[a]p stress than the other three concentrations of snp. discussion b[a]p stress on plants causes reduced growth and photosynthesis, posing a threat to plant life due to metabolic fig. 5. effect of different concentrations (100, 200, 300 and 400 μmol l–1) of sodium nitroprusside (snp), a no donor, on antioxidant enzyme activity of ryegrass seedlings under benzoapyrene (b[a]p) stress. (a) superoxide dismutase (sod) activity. (b) catalase (cat) activity. (c) peroxidase (pod) activity. data are mean ± standard deviation, n = 3. u – unit of enzyme activity, fw – fresh weight. different letters indicate significant differences at p < 0.05. fig. 6. principal component analysis (pca) plots of growth, photosynthetic characteristics and chlorophyll fluorescence parameters and antioxidant enzyme activity of ryegrass exposed to different snp treatments (100, 200, 00 and 400 μmol l–1) under benzoapyrene b[a]p stress. tab. 1. gray correlation degree and correlation sequence of indexes investigated in ryegrass under benzoapyrene stress after applica tion of sodium nitroprusside (snp) as a no donor. index correlation associative order chlorophyll a content 0.981 1 belowground root length 0.971 2 aboveground dry weight 0.970 3 net photosynthetic rate (pn) 0.970 4 total chlorophyll content 0.969 5 photochemical quenching coefficient (qp) 0.965 6 belowground dry weight 0.954 7 actual quantum yield of psii photochemistry (φpsii) 0.953 8 peroxidase (pod) activity 0.953 9 chlorophyll b content 0.953 10 aboveground fresh weight 0.953 11 aboveground plant height 0.952 12 catalase (cat) activity 0.950 13 belowground fresh weight 0.949 14 psii maximum light energy conversion efficiency (fv/fm) 0.947 15 superoxide dismutase (sod) 0.945 16 intercellular carbon dioxide concentration (ci) 0.942 17 transpiration rate (tr) 0.931 18 psii potential activity (fv/f0) 0.928 19 stomatal conductance (gs) 0.927 20 water use efficiency (wue) 0.905 21 steady-state fluorescence (fs) 0.887 22 non-photochemical quenching coefficient (qn) 0.877 23 carotenoid content 0.675 24 li y., ma j., wang y., xu s., jiang l., zhang l., hou w. 78 acta bot. croat. 82 (1), 2023 disorders (he et al. 2014). no, which is a small ubiquitous signaling molecule, plays a vital role in the response to abiotic stress in many plants (gadelha et al. 2017, fancy et al. 2017, li et al. 2018, wei et al. 2020). in this study, compared with the control, the growth and photosynthesis of ryegrass was inhibited as shown by the decreased aboveground and underground dry weights, and the photosynthetic characteristics. the decrease in ryegrass’ growth caused by b[a]p pollution could be attributed to the decrease in the leaf photosynthetic pigment content and the photosynthetic gas exchange parameters. moreover, b[a]p stress caused a decrease in fv/f0, fv/fm and фpsii suggesting a decline in psii function, and indicating that photosynthetic units and membrane-bound electron transfer processes were disrupted under this stress. after snp application, the chlorophyll b, total chlorophyll, and carotenoid contents increased compared with those under b[a]p stress, implying that the release of no from snp increased the photosynthetic pigments of ryegrass and maintained a high photosynthetic rate. the protective effect of snp on photosynthesis had a positive effect on plant growth as shown by the increased plant height, underground dry weight, and fresh weight. this result is consistent with the work of ahanger et al. (2019) who reported that the exogenous application of no evidently contributed to the improved growth and photosynthetic parameters of salt-stressed vigna angularis. moreover, the exogenous application of no reportedly enhances the carotenoid synthesis, effectively channels additional energy, and increases the quantum production of psii in stressed plants, resulting in enhanced photosynthesis and growth (ahmad et al. 2021). in this study, 200 μmol l–1 snp treatment increased fv/f0, fv/fm, and fs, implying that snp alleviated the disruption of psii caused by 30 μmol l–1 b[a]p stress. the exogenous application of snp was able to increase the sod, pod and cat activities. ahmad et al. (2018) reported that the observed decline in lipid peroxidation and membrane leakage in no-treated tomato plants can be attributed to the upregulation of the antioxidant system, which rapidly eliminates ros, including h2o2. our results show that snp treatments increased activities of cat and pod thus alleviating oxidative stress and preventing damage of the photosynthetic apparatus. grey correlation analysis is one of the most commonly used multivariable statistical methods (xiao et al. 2021). the gray correlation analysis enabled the combined evaluation value of 24 measured indicators, thus avoiding the limitation of using individual trait indicators to describe the response of ryegrass. according to the comparison of gray correlation, the chlorophyll a content, the underground root length, and the above ground dry weight have the highest correlation with resistance to b[a]p stress in ryegrass, which can be used as the analysis index for reflecting the response effect of ryegrass to stress. the results show that the comprehensive evaluation of ryegrass indicators using gray system theory is practical and feasible. the pca showed that the application of 200 μmol l–1 snp had an obvious alleviating effect on ryegrass’ growth and physiological properties under 30 μmol l–1 b[a]p stress. no could exert protective effects by increasing the plant tolerance to stress conditions, but high concentrations of no may be toxic to plants due to their high reactivity (reda et al. 2018). references ahanger, m.a., aziz, u., alsahli, a.a., alyemeni, m.n., ahmad, p., 2019: influence of exogenous salicylic acid and nitric oxide on growth, photosynthesis, and ascorbate-glutathione cycle in salt stressed vigna angularis. biomolecules 10, 42. ahmad, p., ahanger, m.a., alyemeni, m.n., wijaya, l., alam, p., 2018: exogenous application of nitric oxide modulates osmolyte metabolism, antioxidants, enzymes of ascorbate-glutathione cycle and promotes growth under cadmium stress in tomato. 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(in chinese) xie, h., ma, y., wang, y., sun, f., liu, r., liu, x., xu, y., 2021: biological response and phytoremediation of perennial ryegrass to halogenated flame retardants and cd in contaminated soils. journal of environmental chemical engineering 9, 106526. yan, f., liu, y., sheng, h., wang, y., kang, h., zeng, j., 2016: salicylic acid and nitric oxide increase photosynthesis and antioxidant defense in wheat under uv-b stress. biologia plantarum 60, 686-694. ye, x., ma, j., wei, j., sun, k., xiong, q., 2019: comparison of the bioavailability of benzo[a]pyrene (b[a]p) in a b[a]p-contaminated soil using the different addition approaches. scientific reports 9, 1-9. zhang, j., yang, n.n., geng, y.n., zhou, j.h., lei, j., 2019: effects of the combined pollution of cadmium, lead and zinc on the phytoextraction efficiency of ryegrass (lolium perenne l.). rsc advances 9(36), 20603-20611. 600 grozeva and cvetanova.vp acta bot. croat. 72 (1), 49–69, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 karyological and morphological variations within the genus dysphania (chenopodiaceae) in bulgaria neli h. grozeva1, yanka g. cvetanova2 1 department of biology and aquaculture, agriculture faculty, trakia university, stara zagora, bulgaria 2 department of informatics and mathematics, faculty of economics, trakia university, stara zagora, bulgaria abstract – the karyological and morphological variability of species from the genus dysphania were studied. the results demonstrated that genus dysphania is represented in bulgaria by five species: dysphania ambrosioides, d. multifida, d. botrys, d. schraderiana and d. pumilio. the first two species are tetraploids with chromosome number 2n = 32 for d. ambrosioides and 2n = 36 for d. multifida. the remaining three species are diploids with 2n = 18. the results from statistical analysis demonstrated that the main source of phenotype variation in the species is the interpopulation variation. the specific characters which allowed their recognition are the morphological characteristics of the perianth lobes, the upper leaves and the seeds. the distinction between d. multifida, d. ambrosioides and d. schraderiana is based on differences in the quantitative traits, while in d. botrys and d. pumilio qualitative traits are also important. the basic evolutionary mechanisms are polyploidy and diploidy. a tendency towards reduction in the size of generative organs and the number of perianth lobes was found. key words: dysphania, distribution, ecology, karyology, morphology, bulgaria introduction traditionally, the genus dysphania comprises 6–10 species, endemic for australia (mosyakin 1993, clements and mosyakin 2003, zhu et al. 2003). over the past decades, the systemic definition and composition of the genus has frequently been a subject of discussion (mosyakin 1993; mosyakin and clements 1996, 2002, 2008). currently the genus dysphania comprises about 40 species that earlier constituted the subgenus ambrosia of the genus chenopodium or were referred to the genera: neobotritium moldenke, roubieva moquin-tandon, teloxys moquin-tandon. the representatives of the genus are mainly ruderal and weed plants, more common in the tropics, subtropics and warm-temperature acta bot. croat. 72 (1), 2013 49 * corresponding author, e-mail: grozeva@uni-sz.bg copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:46 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees zone. most of them entered europe through the import of wool and agricultural products from australia, south america, eastern asia, and africa. a number of researchers (aellen 1960, uotila and suominen 1976, dostalek 1985, kudhn 1993, uotila 2001) pointed out their great morphological variability, especially prominent in the leaf lamina. the similar, rarely exceeding 1 mm, dimensions of the generative organs very often make their distinction difficult. for the bulgarian flora so far five species have been reported – chenopodium ambrosioides l. (= dysphania ambrosioides (l.) mosyakin et clements), c. botrys l. (= dysphania botrys (l.) mosyakin et clements), c. multifidum l. (= dysphania multifida (l.) mosyakin et clements), c. pumilio r. br. (= dysphania pumilio (r. br.) mosyakin et clements) and c. schraderianum schult. (= dysphania schraderiana (schult.) mosyakin et clements) (markova 1966, assyov and petrova 2006, grozeva 2007). dysphania ambrosioides forms populations on open sandy terrain, in close proximity to water basins from sea level to 250 m above sea level. it is usually a dominant species in anthropophyte or ruderal communities. d. multifida forms populations on open lowland areas, more rarely on those facing west or south at altitudes from sea level to 700 m above sea level. it dominates or is an accompanying species in anthropophyte or ruderal communities dominated by cereal species. d. botrys is widely spread on open, sunlit areas and as a weed in cultivated crops at altitudes from sea level to 900 m above sea level. most often, it dominates or is an accompanying species in ruderal communities. d. schraderiana was established for the bulgarian flora by pertti uotila in 1993 while revising the materials from the genus chenopodium in the herbarium of the biological faculty at sofia university (so). the species has very limited distribution. so far, only two populations have been known located in vitosha and in sredna gora mountain. its ecological requirements are quite similar to these of d. botrys and specimens from both species were recorded in the population under study. d. pumilio occurs in single localities in the eastern balkan range, thracian lowland, northeast bulgaria, black sea coast and tundzha hilly country as an accompanying species in ruderal communities (grozeva 2007, vladimirov and petrova 2010, vladimirov 2011). the species forms populations in open sunlit places at an altitude from 168 to 850 m. the objective of this study was to investigate the patterns and levels of morphological variation, chromosome numbers and karyotype morphology as well as ecological conditions of the natural local bulgarian populations of genus dysphania, and to attempt to trace the relationships between the species and some evolutionary trends in the genus. materials and methods morphological and karyological analyses were carried out on 27 natural bulgarian populations of the genus dysphania, referred to five species: dysphania ambrosioides, d. botrys, d. multifida, d. pumilio, and d. schraderiana. thirty plants from each population were used in the overall research. chromosome numbers and karyotypes have been reported on lasting preparations of metaphase root apex plates of seeds germinated in laboratory conditions collected in the nat50 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:46 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees ural habitats of the species. the root tips were treated and squashed according to the accepted methods (grozeva 2007). the chromosomal type was determined after the centromere index i = s/s+l, according to the classification proposed by grif and agapova (1986). three metaphase plates have been measured from each population. the voucher specimens are kept in the herbarium of the bulgarian academy of sciences (som). twenty-four quantitative characters were included in the morphological analysis: 1. height of plant; 2. length of basal leaf; 3. width of basal leaf; 4. length/width ratio; 5. length of basal leaf petiole; 6. length of upper leaf; 7. width of upper leaf; 8. length/width ratio; 9. length of upper leaf petiole; 10. length of inflorescence; 11. length of flower petiole; 12. diameter of flower; 13. length of perianth lobes in bisexual flower; 14. width of perianth lobes in bisexual flower; 15. length of perianth lobes in female flower; 16. width of perianth lobes in female flower; 17. length of seed; 18. width of seed; 19. length/width ratio; 20. thickness of seed; 21. length of fruit; 22. width of fruit; 23. length/width ratio; 24. thickness of fruit. the following qualitative features were employed too: colour of stem; type of inflorescence; degree of perianth concrescence; presence of keeled perianth lobes; colour of perianth, seed and pericarp. the mean value and coefficient of variation were calculated for each character of every population. they were used in the comparative analyses on different levels. the relative contributions of intraand interpopulation variation to the overall variation of each characteristic of the studied species were evaluated by one-way anova. unweighted pair-group average (upga) hierarchical cluster analysis (hca) was applied to the matrix with the euclidean distances between the populations of the genus dysphania in order to study the morphological pair-wise similarities and the underlying hierarchical classification structure. the discriminant function analysis was conducted on morphological data to determine the most parsimonious way to distinguish between species. the stepwise discriminant analysis (sda) procedure was used as it allows recognition of the most effectively discriminating variables among a large set of morphological characters. statistica 6.0 (statsoft inc. 2001) was used for the statistical analysis of morphological data. results karyology as a result of karyological study, 3 chromosome numbers: 2n = 18, 32, 36 have been found in the genus dysphania (tab. 1). two types of chromosomes: metacentric and submetacentric have been established in the karyotypes. the number 2n = 18 has been counted in three species – dysphania botrys, d. pumilio and d. schraderiana. the last species was studied karyologically in bulgaria for the first time and for the studied population karyotype 2n = 14 m + 4 sm = 18 chromosomes has been established (plate 1a). the diploid chromosome number found for d. schraderiana confirms the data known from literature sources (kjellmark 1934, schwarzova 1978a). for all three populations of d. pumilio the karyotype of 2n = 8 m + 10 sm = 18 chromosomes has been recorded (plate 1b). the chromosome number 2n = 18 found for the bulgarian popuacta bot. croat. 72 (1), 2013 51 karyological and morphological variations in dysphania 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:46 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 52 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. tab. 1. description of studied populations of genus dysphania. * – data published by grozeva and stoeva (2006). ** – data published by grozeva (2007) section, species and population number 2 n locality sect. adenois (moq.) mosyakin et clements. dysphania ambrosioides (l.) mosyakin et clemants o 32** danubian plain, island milka near belene town, at 27 m, 43° 40’n, 25° 10’e, in sandy hair no32 32** thracian lowland, plovdiv town, near the river maritza, at 164 m, 42°09’n, 24°45’e, in ruderal places no104 32** thracian lowland, the locality ostrova near plovdiv town, at 164 m, 42° 09’n, 24° 45’e, in ruderal places dysphania multifida (l.) mosyakin et clemants no39 36 danubian plain, island goljama barzina near belene town, at 20 m, 43° 40’n, 25° 10’e, in outskirts of the island and on sandy hair no40 36 danubian plain, vidin town, at 345 m, 43° 59’n, 22° 52’e, in ruderal places near the bus station with trifolium album and other legumes. no105 36 central balkan range, kalofer town, at 666 m, 42° 37’n,24° 59’e, grasslands with lolium perenne l. of 50 m from post no106 36** western sredna gora mt., ikhtiman town, at 658 m, 42° 09’n, 24° 45’e, in ruderal places in the western parts of the city together with cereal species no107 36** eastern rhodope mts, ivaylovgrad town, at 104 m, 41° 32’n, 26° 08’e, in ruderal places together with bromus tectorum l. and setaria viridis no108 36 thracian lowland, purvomay town, at 134 m, 42° 06’n, 25° 13’e, grasslands in kvartal debar. no109 36** thracian lowland, asenovgrad town, at 104 m, 42° 01’n, 24° 52’e, in ruderal places together with setaria viridis sect. botryoides (c. a. mey) mosyakin et clemants dysphania botrys (l.) mosyakin et clemants no29 18 northern black sea coast, around varnensko ezero lake, at 79 m, 43° 11’n, 27° 50’e, periodically flooded coastal alluvial sands deposits no97 18 southern black sea coast, pomorie town, at 0 m, 42° 35’n, 27° 37’e, in ruderal places near monastery of saint george no100 18** north-eastern bulgaria, razgrad town, at 200 m, 43° 46’n, 26° 31’e, between the sidewalk and fences together with polygonum aviculare no35 18** danubian plain, belene town, at 35 m, 43° 39’n, 25° 07’e, in ruderal places near the fishing pier no102 18 easthern balkan range, natural park »sinite kamani«, at 290 m, 42° 42’n, 26° 21’e, in ruderal places near road from hotel ablanovo to the lake asenovets no99 18** central balkan range, trojan town, at 400 m, 43° 53’n, 24° 43’e, in ruderal places from 50 m to bus station 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:46 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees lations of d. pumilio corresponds to the data of javourkova-jaromirova (1992) and mesib̂ ek (1992) from the czech republic, schwarzová (1978b) from slovakia and rahimenijad (2004) from iran. with the third diploid species d. botrys differences have been recorded in the karyotype of the various populations. in population no. 29 from the varna lake and population no. 30 from plovdiv the karyotype consists of 2n = 4m + 14 sm = 18 chromosomes (plate 1c). in population no. 99 from troyan and population no. 45 from tselina satellites can be seen on one pair of submetacentric chromosomes (plate 1d). in the remaining eight populations (tab. 1), a karyotype of 18 submetacentric chromosomes has been established. in four of these populations (no. 97 from pomorie; no. 102 from »sinite kamani« natural park; no. 53 from gorno novo selo; no. 100 from razgrad) one of the chromosome pairs has satellites (plate 2a), while in the other four (no. 35 from belene; no. acta bot. croat. 72 (1), 2013 53 karyological and morphological variations in dysphania section, species and population number 2 n locality no98 18 sofia region, malo buchino village, at 748 m, 42° 41’n, 23° 10’e, along the sidewalk between the plates together with polygonum aviculare and cynodon dactylon (l.) pers. no101 18 struma valley, blagoevgrad town, at 360 m, 41° 45’n, 23° 25’e, weed in the cultural community no53 18 easthern sredna gora mt., gorno novo selo village, at 597 m, 42° 27’n, 25° 14’e, in ruderal places near the road for a hut kavakliika together with cynodon dactylon. no30 18 thracian lowland, plovdiv town, near the river maritza, at 164 m, 42°09’n, 24°45’e, in ruderal places no34 18 thracian lowland, malka vereja village, at 235 m, 42° 24’n, 25° 33’e, in ruderal places at the eastern edge of the village together with atriplex tatarica l. and setaria viridis (l.) beauv no45 18 thracian lowland, tselina village, at 193 m, 42° 07’n 25° 27’e, weed in the cultural community dysphania schraderiana (schult.) mosyakin et clemants no103 18 easthern sredna gora mt., near kavakliika hut, at 650 m, 42° 27’n, 25° 12’e, in ruderal places around the hut of forestry together with dysphania botrys sect. orthospora (r. br.) mosyakin et clemants dysphania pumilio (r. br.) mosyakin et clemants no214 18* eastern balkan range, sinite kamani natural park, at 850 m, 42° 42’n, 25° 20’e, in ruderal places by roadsides together with polygonum aviculare l. no217 18 thracian lowland, stara zagora town, at 195 m, 42° 25’n, 25° 38’e, in ruderal places together with dysphania botrys no221 18 thracian lowland, chirpan town, at 168 m, 42° 25’n, 25° 38’e, in ruderal places together with cereal plants tab. 1. – continued 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:46 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 54 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. plate 1. microphotographs of the metaphase plate of dysphania species: a – d. schraderiana (population no 103) – 2n = 18; b – d. pumilio (no 221) – 2n = 18; c – d. botrys (no 29) – 2n = 18; d – d. botrys (no 45) – 2n = 18. – scale bars = 10 mm. plate 2. microphotographs of the metaphase plate of dysphania species: a – d. botrys (population no 53) – 2n = 18; b – d. botrys (no 98) – 2n = 18; c – d. ambrosioides (no 102) – 2n = 32; d – d. ambrosioides (no 201) – 2n = 32. – scale bars = 10 mm. 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:47 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 98 from malo buchino; no. 34 from malka vereya; no. 101 from blagoevgrad) satellites have not been found (plate 2b). the chromosome number 2n = 18 registered for the bulgarian populations of d. botrys confirms the familiar reference data (mulligan 1961; mehra and malik 1963; keener 1970; basset and crompton 1971, 1982; uotila 1973; queiros 1975, schwarzová 1978b, 1980, 1993; dvorfák et al. 1980; khatoon and ali 1993; lomonosova et al. 2003). the tetraploid chromosome number 2n = 32 has been found for dysphania ambrosioides. the same result has been found in various regions in the geographical area of the species (lorz 1937; woroschilov 1942; kawatani and ohno 1950; raghavan and arora 1958; mehra and malik 1963; sharma and dey 1967; muribn and ferakova 1974; queiros 1975; schwarzová 1978b, 1986; silvestre 1984; dvorfák 1989; khatoon and ali 1993; al-turki et al. 1999, 2000). for the three studied populations, differences were recorded in the karyotype structure. in the two populations from the thracian lowland (no. 32 from plovdiv and no. 104 from an island on the maritsa river) the karyotype consists of 2n = 20 m + 12 sm = 32 chromosomes (plate 2c). for the population from the danubian plain (no. 201 from the island of milka) a karyotype of 2n = 4 m + 28 sm = 32 chromosomes has been established (plate 2d). the highest chromosome number 2n = 36 is registered in the populations of d. multifida (tab. 1). the same number was reported by markova (1968) for another bulgarian population of the species from the village of koshava in the danubian plain. the data from the other parts of its habitat are for 2n = 32 (kawatani and ohno 1956; giusti 1970; queiros 1975; granado et al. 1988). in four of the studied populations (no. 39 from the island of golyama barzina, no. 108 from parvomay, no. 105 from kalofer, and no. 109 from asenovgrad) the karyotype of 2n = 20 m + 16 sm = 36 chromosomes has been registered (plate 3a). in the other three populations (no. 40 from vidin, no. 106 from ihtiman, no. 107 from ivaylovgrad) the karyotype of 2n = 16 m + 20 sm = 36 chromosomes has been established (plate 3b). morphology the intrapopulation variation was estimated on the basis of the coefficient of variation (tabs. 2–4). the data show that in all populations vegetative have higher level of variability than generative traits. acta bot. croat. 72 (1), 2013 55 karyological and morphological variations in dysphania plate 3. microphotographs of the metaphase plate of dysphania species: a – d. multifida (population no 105) – 2n = 36; b – d. multifida (no 40) – 2n = 36. – scale bars = 10 mm. 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:48 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 56 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. tab. 2. mean (first line) and coefficient of variation in % (second line) of dysphania schraderiana (103), d. pumilio (214, 217, 221), d. ambrosioides (201, 32, 104) populations for each of the 24 observed characters populations 103 214 217 221 201 32 104 1 79.3 17.5 41.2 36.9 34.29 28.39 30.1 37.2 52.1 34.1 77.0 27.0 65.4 15.8 2 5.6 27.4 2.6 38.7 2.17 13.25 2.0 32.2 4.5 20.2 4.1 22.9 4.0 20.3 3 3.5 23.4 1.3 28.3 1.24 22.41 1.2 31.3 1.1 25.2 1.2 21.6 1.1 21.3 4 1.6 12.1 2.3 38.2 1.73 17.82 1.3 16.8 4.5 27.3 3.7 25.7 3.8 24.3 5 1.5 13.0 1.4 42.3 1.39 47.3 1.3 32.3 0.6 33.3 0.54 37.9 0.3 13.6 6 3.0 8.6 0.9 32.1 0.84 11.97 0.7 23.9 1.9 23.7 2.0 30.4 1.8 18.4 7 1.4 11.3 0.5 27.4 0.39 18.18 0.3 23.9 0.4 23.6 0.5 36.2 0.4 15.9 8 2.2 6.9 1.5 44.2 1.52 21.34 1.4 19.8 5.8 25.2 4.5 29.0 5.1 21.0 9 0.3 26.2 0.5 35.2 0.53 12.30 0.6 14.6 0.4 32.7 0.3 35.8 0.1 23.0 10 50.9 22.2 21.7 24.3 19.94 38.61 15.1 32.2 29.6 46.9 45.4 40.8 40.8 25.2 11 0.4 30.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 23.4 0.1 28.3 0.1 31.0 12 0.8 15.0 0.8 15.4 0.75 12.34 0.7 11.3 1.0 3.4 1.0 5.2 1.1 5.9 13 0.8 10.2 0.8 18.5 0.85 11.25 0.9 9.5 1.0 5.7 1.0 5.2 1.1 8.1 14 0.5 16.1 0.4 12.3 0.37 18.42 0.3 14.2 0.6 9.3 0.6 8.9 0.6 0.0 15 0.8 7.2 0.8 9.4 0.75 9.40 0.8 8.4 1.0 4.7 1.0 9.0 1.0 6.4 16 0.5 11.9 0.4 11.5 0.31 9.72 0.3 9.3 0.5 7.0 0.6 8.6 0.6 7.2 17 0.8 16.7 0.8 4.9 0.78 4.95 0.72 5.2 0.7 16.3 0.5 13.7 0.6 6.3 18 0.7 11.6 0.7 7.5 0.67 7.50 0.7 5.6 0.6 12.6 0.5 8.9 0.6 4.1 19 1.1 12.0 1.2 4.8 1.24 4.35 1.3 5.8 1.1 14.0 1.0 13.0 1.0 5.1 20 0.4 12.5 0.2 6.8 0.34 17.62 0.4 13.3 0.4 12.1 0.3 10.9 0.4 5.1 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:48 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 57 karyological and morphological variations in dysphania populations 103 214 217 221 201 32 104 21 0.9 13.3 0.8 7.5 0.84 7.5 0.9 8.4 0.8 12.8 0.6 10.9 0.7 5.0 22 0.9 13.3 0.8 8.4 0.78 8.44 0.8 5.5 0.7 11.6 0.6 8.0 0.6 0.0 23 1.0 10.9 1.3 7.3 1.36 7.36 1.1 6.8 1.2 8.7 1.0 13.0 1.2 5.1 24 0.6 9.8 41.2 36.9 34.29 28.39 30.1 37.2 0.4 8.4 0.4 12.1 0.4 8.4 tab. 2. – continued tab. 3. mean (first line) and coefficient of variation in % (second line) of dysphania botrys populations for each of the 24 observed characters populations 29 97 100 35 102 99 98 101 53 30 34 45 1 44.3 51.4 48.2 46.0 51.7 40.6 52.4 59.3 43.9 45.5 47.9 24.7 24.3 25.2 17.5 20.2 20.8 40.2 12.4 16.1 27.5 27.6 14.4 44.2 2 2.4 3.7 4.0 2.2 3.3 3.7 4.3 3.1 3.3 2.1 3.0 2.9 30.4 38.5 40.3 12.9 41.4 34.1 28.8 28.2 40.6 24.4 47.9 46.0 3 1.4 1.6 0.9 0.9 1.4 1.7 2.0 1.5 1.7 1.2 1.6 1.4 26.5 44.6 19.4 19.8 39.7 44.6 27.5 26.8 40.8 24.4 41.8 43.0 4 2.3 2.5 4.7 3.2 2.4 2.4 2.2 2.1 1.9 1.6 1.8 2.2 35.4 31.7 29.2 39.7 32.5 21.3 27.5 26.2 24.1 22.6 20.3 15.1 5 1.1 1.2 1.5 1.5 1.2 0.6 1.2 1.3 1.4 1.4 1.3 0.6 37.3 26.8 17.5 17.5 37.4 32.5 20.9 26.5 28.4 23.1 19.6 32.2 6 0.8 0.8 0.7 0.7 0.8 0.6 0.8 0.7 0.7 0.7 0.7 0.6 26.1 29.4 12.8 11.7 28.7 27.1 12.9 17.0 11.7 16.0 14.6 31.5 7 0.3 0.3 0.4 0.3 0.4 0.4 0.3 0.3 0.3 0.3 0.3 0.3 18.2 30.8 14.4 11.4 23.0 24.1 11.0 26.5 22.9 21.9 16.5 39.0 8 2.7 2.7 2.0 2.2 2.3 1.8 2.4 2.7 2.5 2.7 2.6 2.1 39.2 40.2 19.6 10.9 38.5 22.5 16.6 18.3 17.7 17.2 24.6 23.9 9 0.9 0.4 0.0 0.0 0.1 0.4 0.2 0.1 0.5 0.1 0.9 0.4 29.5 45.8 39.3 40.4 49.5 47.8 46.6 55.1 11.3 46.3 49.6 47.0 10 25.6 25.4 24.9 24.9 25.6 19.0 40.5 46.7 34.5 25.6 40.2 19.7 34.7 35.3 18.9 56.6 34.7 35.8 16.6 8.8 30.6 34.7 17.0 38.7 11 0.2 0.7 0.2 0.2 0.2 0.3 0.7 0.2 0.7 0.2 0.3 0.3 40.1 43.3 51.6 51.6 34.5 15.8 43.2 45.9 43.2 43.0 35.6 15.6 12 0.9 0.9 1.1 1.1 0.9 0.9 1.0 1.0 1.1 1.0 1.0 0.9 8.6 8.6 4.9 4.9 8.5 9.7 4.8 4.9 5.0 4.8 4.8 9.5 13 0.9 0.9 1.1 1.1 0.9 0.9 0.8 0.8 0.7 0.8 0.8 0.8 12.0 10.4 7.2 7.2 11.5 14.9 6.8 10.2 6.7 14.3 6.8 13.8 14 0.3 0.4 0.4 0.3 0.4 0.3 0.3 0.3 0.4 0.3 0.3 0.3 12.7 15.8 16.4 14.1 17.3 16.6 14.1 9.8 14.5 10.5 13.8 13.3 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:48 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 58 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. populations 29 97 100 35 102 99 98 101 53 30 34 45 15 0.9 0.9 1.0 1.0 0.9 0.6 0.7 0.7 0.7 0.7 0.7 0.6 8.3 12.1 6.4 6.4 12.0 8.0 6.2 11.0 8.7 11.1 6.3 7.8 16 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.3 0.3 8.3 8.4 12.0 12.0 8.3 0.0 12.0 9.8 14.1 9.7 11.3 0.0 17 0.7 0.7 0.8 0.8 0.7 0.7 0.7 0.6 0.6 0.6 0.7 0.7 9.6 9.8 6.4 6.4 9.6 10.5 6.4 11.2 7.6 11.6 6.2 10.4 18 0.7 0.7 0.7 0.7 0.7 0.7 0.6 0.8 0.6 0.8 0.6 0.7 8.8 89.0 2.6 2.6 8.8 11.0 8.7 7.7 11.3 7.6 8.2 10.8 19 1.0 1.0 1.1 1.1 1.0 1.0 1.2 0.8 1.1 0.8 1.2 1.0 9.0 10.0 7.3 7.3 10.0 11.9 5.1 9.1 7.0 7.0 5.1 10.8 20 0.2 0.2 0.4 0.4 0.4 0.3 0.5 0.4 0.4 0.3 0.5 0.3 24.4 20.0 10.5 10.5 14.6 14.2 11.2 14.6 10.8 16.5 10.9 14.1 21 0.8 0.8 0.9 0.9 0.8 1.0 0.8 0.7 0.8 0.7 0.8 1.0 8.4 8.5 8.6 8.6 8.4 7.7 5.5 14.5 7.0 14.3 5.4 7.8 22 0.8 0.8 0.8 0.8 0.8 0.9 0.7 0.7 0.7 0.7 0.7 0.9 7.7 7.8 8.4 8.4 7.7 7.8 7.5 9.5 9.9 9.9 7.0 7.7 23 1.0 1.0 1.2 1.2 1.0 1.1 1.2 1.1 1.1 1.1 1.2 1.1 8.0 9.0 14.5 14.5 8.0 6.7 4.4 12.8 10.6 7.0 4.4 6.7 24 0.3 0.3 0.5 0.5 0.5 0.5 0.6 0.5 0.5 0.4 0.6 0.5 14.4 13.4 9.0 9.0 11.4 10.0 9.2 11.4 9.3 12.8 9.0 10.1 tab. 3. – continued tab. 4. mean (first line) and coefficient of variation in % (second line) of dysphania multifida populations for each of the 24 observed characters populations 103 214 217 221 201 32 104 1 51.2 90.9 73.4 88.6 83.6 54.2 65.5 21.9 15.6 30.6 24.6 24.9 29.1 32.5 2 1.3 1.3 1.4 1.6 1.4 1.2 1.2 32.9 11.9 24.7 15.2 20.8 32.1 25.7 3 0.5 0.6 0.6 0.8 0.7 0.6 0.6 35.5 18.5 23.8 9.3 19.9 35.7 32.8 4 2.6 2.3 2.5 1.9 2.1 2.5 2.1 39.1 23.4 26.5 20.1 21.1 51.4 44.0 5 0.4 0.4 0.4 0.5 0.4 0.2 0.4 20.9 21.1 18.7 11.7 17.0 22.6 20.7 6 0.5 0.8 0.7 0.7 0.8 0.5 0.6 31.5 17.2 23.6 10.6 15.3 36.6 32.4 7 0.3 0.4 0.4 0.5 0.4 0.3 0.3 35.2 20.5 17.6 11.0 17.0 21.3 37.8 8 1.9 2.3 1.8 1.6 1.8 1.9 1.9 22.2 20.4 28.8 15.1 20.2 26.3 23.9 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:49 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees the results of the anova (tab. 5) demonstrate that in all species, interpopulation variability is dominant in total variability. the interpopulation variation was evaluated in dysphania botrys, d. pumilio, d. ambrosioides and d. multifida, as d. schraderiana was investigated in a single population. the values of euclidean distances (ed) varied within different limits in the individual taxa (tabs. 6–9) the upga cluster analysis based on the morphological pairwise similarities (euclidean distances between population centroids) enabled us to detect two main clusters a and b acta bot. croat. 72 (1), 2013 59 karyological and morphological variations in dysphania populations 103 214 217 221 201 32 104 9 0.2 0.2 0.3 0.3 0.3 0.2 0.3 19.3 20.3 19.1 20.7 22.1 29.9 22.9 10 36.9 73.1 49.3 64.6 63.3 39.6 43.9 28.8 19.8 38.8 15.8 22.9 33.5 33.0 11 0.1 0.3 0.2 0.3 0.2 0.1 0.1 54.1 43.7 47.3 42.6 41.7 40.1 44.7 12 1.2 1.2 1.1 1.0 1.1 1.2 1.2 4.9 16.4 14.3 10.3 10.2 8.8 9.6 13 1.0 1.0 1.0 1.1 1.1 1.0 1.0 6.6 12.6 10.6 8.4 7.8 8.9 11.4 14 0.4 0.4 0.4 0.5 0.4 0.4 0.4 13.8 9.3 16.9 12.2 7.8 13.4 16.9 15 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 17 0.8 0.9 0.8 0.8 0.8 0.8 0.8 7.2 4.3 7.8 8.1 7.8 5.9 7.3 18 0.8 0.8 0.8 0.9 0.8 0.8 0.8 8.2 5.4 6.6 6.0 5.6 8.9 7.7 19 1.1 1.1 1.1 0.9 1.0 1.1 1.0 8.5 7.6 6.5 11.1 11.0 5.2 8.7 20 0.3 0.3 0.3 0.4 0.3 0.3 0.3 18.9 0.0 16.0 14.7 14.6 20.9 13.2 21 0.9 1.0 0.9 1.0 1.0 0.9 0.9 6.4 5.3 7.1 6.5 4.6 5.8 6.4 22 0.9 0.9 0.9 0.9 0.9 0.9 0.9 7.2 0.0 8.1 8.9 8.1 7.6 8.3 23 1.1 1.0 1.1 1.1 1.1 1.0 1.0 7.5 8.0 5.7 8.9 10.0 9.6 6.7 24 0.4 0.4 0.4 0.4 0.4 0.4 0.4 7.8 0.0 7.8 0.0 0.0 15.6 5.2 tab. 4. – continued 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:49 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees (fig. 1). cluster a consists of two subclusters a1 and a2. the populations of dysphania multifida form the subcluster a1, while the populations of d. botrys, d. pumilio and d. schraderiana were grouped by their own species in the subcluster a2. cluster b contains the populations of d. ambrosioides. 60 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. tab. 5. percentage of the interpopulation variation in the overall morphological variation for each character in dysphania character no species d. botrys d. pumilio d. ambrosioides d. multifida 1 37.7 30.1 66.0 43.2 2 29.2 45.7 47.7 29.2 3 65.1 47.6 43.4 64.5 4 46.4 70.9 38.1 49.9 5 69.1 32.2 48.0 61.1 6 61.8 51.3 31.1 37.0 7 66.5 46.1 68.0 53.9 8 67.7 55.8 40.2 57.3 9 45.1 66.3 74.6 57.9 10 76.6 65.3 71.1 48.7 11 69.3 0.0 59.4 50.6 12 52.1 55.7 62.3 55.7 13 66.5 50.9 62.9 52.9 14 55.7 59.0 64.9 38.2 15 78.9 52.7 55.7 0.0 16 33.1 56.8 59.9 0.0 17 41.6 59.5 85.1 61.4 18 57.0 56.2 94.0 63.0 19 57.0 49.3 65.1 52.4 20 76.8 52.8 78.7 61.5 21 52.5 54.9 91.5 58.7 22 66.4 52.2 91.6 55.2 23 64.0 50.1 56.6 68.4 24 61.7 50.6 0.0 52.1 tab. 6. values of euclidean distances between the pairs of populations of d. pumilio based on 24 characters. number of population 214 217 217 54 221 111 57 tab. 7. values of euclidean distances between pairs of populations of d. ambrosioides based on 24 characters. number of population 32 104 104 125 201 295 194 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:49 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees the results from the sda are presented in table 10. the first three discriminant (canonical) functions account for over 98% of the total variance. overall, the discrimination between species is highly significant (wilks’ lambda = 0.00026, f = 567.01, p < 0.001). wilks’ lambda is the standard statistic that has been used to denote the statistical significance of the discriminatory power of the current model. the closer to 0 the wilks’ lambda, the greater is the contribution of the model to the overall discrimination. the partial wilks’ lambda values in the first column (tab. 10) present the unique contribution of each character in the model to the discrimination between species. the lower the value in this column, the greater is the unique discriminatory power of the respective character. all discriminant functions and variables in the model were highly significant (p < 0.001) the standardized discriminant function coefficients determine the unique contribution of each character to the discriminant functions d1–d3. the first discriminant function which provides the most overall discrimination between the five species is loaded most heavily by the width and the length of perianth lobes in the female flower, and the length of perianth lobes in the bisexual flower. the second discriminant function seems to be marked acta bot. croat. 72 (1), 2013 61 karyological and morphological variations in dysphania tab. 8. values of euclidean distances between the pairs of populations within dysphania multifida based on 24 characters number of population 39 40 105 106 107 108 40 537 105 254 296 106 465 122 216 107 418 89 173 51 108 40 497 215 425 378 109 158 388 96 310 266 121 tab. 9. values of euclidean distances between the pairs of populations of dysphania botrys based on 24 characters number of population 29 97 100 35 102 99 98 101 53 30 34 97 72 100 43 34 35 22 59 31 102 74 5 37 60 99 77 126 97 83 129 98 171 152 162 171 150 245 101 296 265 273 283 266 267 410 53 90 119 106 100 119 158 105 378 30 13 62 34 15 63 84 166 290 92 34 151 152 153 155 151 224 47 417 69 148 45 205 274 241 220 276 160 347 398 243 216 309 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:49 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 62 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. fig. 1. dendrogram of the cluster analysis of the genus dysphania: no 105–40 = d. multifida (m); no 217–221 = d. pumilio (p); no 97–101 = d. botrys (b); no 103 = d. schraderiana (s); no 201–104 = d. ambrosioides (a) tab. 10. the sequence of the ten most significant characters for discrimination between five species of genus dysphania obtained by sda and corresponding values of partial wilks’ lambda, f-values and standardized coefficients for discriminant functions d1–d3. the eigenvalues, cumulative percentage of the variance explained by the first three roots are presented in the bottom two rows. characters f-remove wilks’ lambda d1 d2 d3 x16. width of perianth lobes in female flower 307.0124 0.000674 –0.75486 –0.096454 –0.255158 x7. width of upper leaf 169.4663 0.000488 –0.03937 –0.384323 0.715588 x6. length of upper leaf 137.8743 0.000445 –0.12250 –0.666162 –0.372245 x15. length of perianth lobes in female flower 137.6188 0.000445 –0.71658 0.390183 0.069843 x13. length of perianth lobes in bisexual flower 72.0499 0.000356 0.47251 –0.214634 –0.386907 x24. thickness of fruit 54.0739 0.000332 –0.15462 –0.220721 0.206799 x11. length of flower petiole 85.6233 0.000374 –0.01660 –0.107996 0.471146 x5. length of basal leaf petiole 62.8143 0.000343 0.10170 0.156215 0.544039 x12. diameter of flower 45.4535 0.000320 0.28564 –0.306588 –0.234661 x9. llength of upper leaf petiole 37.9717 0.000310 0.10216 –0.273894 –0.373920 eigenvalues 41.98092 7.680871 4.657472 cumulative prop. of variance 0.76118 0.900440 0.984887 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:50 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees mostly by the length of the upper leaf, while the width of the upper leaf, length of flower petiole and length of basal leaf petiole contribute mostly to the third discriminant function. the populations of d. multifida, d. ambrossoides and d. schraderiana have been classified 100% correctly on the fourth step of the sda while the populations of the other two species d. botrys and d. pumilio were classified with 98.46% and 92.22% correctness, respectively, on the tenth step. the scatter plot of the canonical scores of the individuals from the five species on the first two discriminant dimensions (fig. 2) enabled us to determine the nature of the discrimination for the first two discriminant roots. the first discriminant function mostly discriminates between d. multifida and the four other species. clearly, the populations of d. multifida are plotted much further to the right of the central line. the second function seems to provide some discrimination between the species of d. ambrosioides, d. schraderiana and d. multifida (which mostly show negative values for the second canonical function) and the other two species d. botrys and d. pumilio (which have mostly positive values). discussion the results of the present complete population research of genus dysphania confirm the data from our previous research (grozeva and cvetanova 2008, 2011) on the species from family chenopodiaceae, namely that vegetative traits are more variable than generative ones (tabs. 2–4). the lamina size of the lower leaves, petiole length of upper or lower leaves and stem height are the most variable traits in almost all populations. the characteristics of the seed dimensions (i.e., length, width and ratio between them) are some of the most conacta bot. croat. 72 (1), 2013 63 karyological and morphological variations in dysphania fig. 2. scatter plot of scores of the first two canonical roots d1 and d2 for the individuals of the five dysphania species 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:51 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees servative traits. no variability has been recorded for the trait length of female flower perianth in two of the populations of d. botrys, no. 45 from tselina and no. 99 from troyan. the high variability of stem height and leaf characters of the species from chenopodium senso lato, and the lack of any considerable variability in seed characters have been acknowledged by researchers of the genus from various parts of the world (kowal 1953, engstrand and gustafsson 1972–1974, reynolds and crauford 1980, basset and crompton 1982, dostálek et al. 1990, al-turki and ghafoor 1996, uotila 2001). the main source of phenotype variation in all species is the interpopulation variation. the interpopulation differences were fewer and varied within much narrower limits in d. pumilio (tab. 6). that is associated with the small differences between population habitats (tab. 1) as well as with the lack of karyotype variability. low variability level has been found among the populations of d. ambrosioides (tab. 7), which relates to their similar habitats (sandy terrains in close proximity to a water basin) and similar altitudes (28–164 m). the recorded higher variability for the population from the danubian plain than in the two populations from the thracian lowland could be explained by either the differences in the soil and climatic conditions due to their geographic remoteness or the established karyotype differences. large interpopulation morphological variation was observed in d. multifida (tab. 8) and the greatest differences have been recorded among populations with different karyotypes. among populations with the same structure of the karyotypes, strong morphological similarity was recorded between populations with similar community components – no. 39 from the island of golyama barzina and no. 108 from parvomay (ed=40), where d. multifida dominates and cereal species are quite limited; no. 106 from ihtiman and no. 107 from ivaylovgrad (ed = 51), where cereal species are dominant. according to our observations, the established relationship is directly linked to the biological characteristics of the species. d. multifida is a perennial grass plant with a shallow root system and highly branched creeping stems. its normal vegetative development needs space and efficient amount of water and food supplies in the topsoil, and these depend on the composition of the population and the existing relationships within it. the largest interpopulation morphological variation was observed in dysphania botrys (tab. 9) which is in agreement with the diversity of ecological conditions, altitudes (0–748 m), ecosystems (tab. 1), as well as the constituted kariotype differences. the interpopulation differences within this species were the least between populations no. 97 from pomorie, no. 102 from »sinite kamani« natural park (ed = 5), and population no. 29 from the varna lake and population no. 30 from plovdiv (ed = 13), which have identical karyotype structures. high morphological similarity was found between population no. 35 from belene and population no. 30 from plovdiv (ed = 15), and population no. 29 from varna lake (ed = 22), which are with different karyotypes, although they have similar ecological conditions (altitudes up to 200 m, flat terrain, close to a water basin with moist and sandy soil). of all studied populations, the greatest differences are those from tselina (population no 45) and blagoevgrad (population no 101) where the species is a weed in a cultivated community. this is related to better soil conditions (i.e., food substances, moisture, and aeration), and weaker competition between the cultivated and weed plant than in the species’ natural habitat. regardless of the similar composition of communities, both populations differ significantly from each otehr, which relates to their appurtenance to two different and quite distant floristic regions and to the differences found in their karyotypes. 64 acta bot. croat. 72 (1), 2013 grozeva n. h., cvetanova y. g. 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:51 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees the interpopulation variability is dominant within the total variability in all species (tab. 5), which is due mainly to differences in ecological conditions and especially in the various plant communities to which they belong, as for the populations of d. ambrosioides, d. botrys and d. multifida karyological variability is also a factor. the less pronounced intrapopulation variability relates to the smaller number and area of the population, and the uniform ecological conditions within its borders. after comparing all populations of the species in the genus according to the entire morphological set of quantitative traits (fig. 1), the greatest similarity was found between the two diploid species d. botrys and d. pumilio. quite similar to these two is the third diploid species – d. schraderiana. the three species also have similar ecological preferences. a certain morphological similarity was registered between the tetraploid d. multifida and the three diploid species, as well as between the tetraploid d. ambrosioides and all other species in the genus. such morphological similarity among the five species was also noted by uotila (2001) in flora nordica. in spite of the high morphological similarity among the species, each one of them could be recognized correctly by certain characteristics (fig. 2). the top ten significant features for differentiation of species, found by sda were: width of perianth lobes in female flower, width of upper leaf, length of upper leaf, length of perianth lobes in female flower, length of perianth lobes in bisexual flower, thickness of fruit, length of flower petiole, length of basal leaf petiole, diameter of flower, length of upper leaf petiole (tab. 10). the distinction between d. multifida, d. ambrosioides and d. schraderiana is based on differences in the quantitative traits, while such discrimination between d. botrys and d. pumilio is not always feasible. due to the similarity in the greater part of the qualitative traits of these two species, qualitative characteristics should be used as well for their clear-cut distinction. d. pumilio could be distinguished easily from d. botrys as well as from the other species of the genus by its 4–5 pale green perianth leaves that harden and whiten at the fruit, as well as by reddish-brown to brown-black seeds with an imperceptible blunt edge. d. botrys was characterized by 5 yellowish-green perianth leaves that do not fully cover the fruit and do not harden and greyish-black seeds with an edge longitudinally split up in two. d. schraderiana differed by 4–5 perianth lobes with clearly defined serrated dorsal edge and black seeds with blunt edge. d. ambrosioides is easily recognizable by its five pale green perianth lobes with clearly defined non-serrated dorsal edge, connate up to 1/2 of their length and brown seeds with blunt edge. d. multifida differed to the greatest degree from the other species with its five heavily furrowed perianth lobes, with no dorsal edge, connate along their entire length, that are preserved at the fruit and form a false pod and dark brown round seeds. each species could also be identified by its typical indentation of the leaf lamina, however, that is not always a sure method since it is necessary to collect a greater number of whole plants in order to trace the lamina changeability of the lower, middle, and upper leaves. the data from the present study confirm that polyploidy and diploidy are basic evolutionary mechanisms in genus dysphania. in tracing the morphological variability, the tendency, known for chenopodium senso lato, to reduce the sizes of the generative organs and the number of perianth lobes was established. the reduced size of perianth lobes and the decrease in their number facilitate anemophilous pollination. the retention of the perianth lobes at the fruit and the presence of glands on their surface is an adaptation to exozoochory. the nuts stick to animal fur and are carried as they move around, while the reduced size of acta bot. croat. 72 (1), 2013 65 karyological and morphological variations in dysphania 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:51 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees the seeds favours their transportation at greater distances. perianth lobes in some species have additional features securing reliable attachment: rough surface in d. multifida, pointed dorsal edge in d. ambrosioides, serrated dorsal edge in d. schraderiana). the lack of a dorsal edge on the perianth lobes of d. botrys and d. pumilio is perfectly compensated for by there being more glands on the entire surface of the first species, and on the upper parts of the second. acknowledgements this study was supported by the project scientific research fund of trakya university, agriculture faculty (project 2p/08). the authors are grateful to the anonymous reviewers for the valuable comments and 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(eds), flora of china, 5. ulmaceae through basellaceae, 376–378. science press, beijing and missori botanical garden press, st. louis. acta bot. croat. 72 (1), 2013 69 karyological and morphological variations in dysphania 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:52 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 600 grozeva and cvetanova ispravljeni.ps u:\acta botanica\acta-botan 1-13\600 grozeva and cvetanova.vp 18. o ujak 2013 10:16:52 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (1), 67–77, 2009 coden: abcra 25 issn 0365–0588 cytology and palynology of the clematis l. species (ranunculaceae) in iran masoud sheidai*, meysam habibi, dina azizian, mahboobeh khatamsaz shahid beheshti university, g. c., faculty of biological sciences, tehran, iran cytological and palynological studies were performed on clematis l. species (runanculaceae) of iran indicating 2n = 2´ = 16 and 2n = 4´ = 32 in them. they formed only bivalents in metaphase of meiosis-i with some amount of chromosome stickiness and laggard formation in anaphase. the species possessed a symmetrical karyotype but differed in karyotypic formulae indicating the occurrence of structural changes in the chromosomes during species diversification. clematis species usually possessed tricolpate pollens but differed in details of pollen morphology, colpi length, colpi width and apocolium length. keywords: clematis, cytology, palynology, iran introduction the genus clematis l. (ranunculaceae) comprises about 250 species world wide. the species are well adapted to highland areas and are economically important for their chemical properties including the saponin glycosides available in their leaves and roots and also as ornamentals. clematis is a vigorous climbing, with attractive flowers. almost all species are found throughout the temperate regions of both hemispheres, and also in mountains in the tropics (tamura 1993). clematis marks a peak of evolutionary development on the one hand and a high degree of speciation on the other. it is almost if not quite the most widely geographically distributed genus in the family and is very close to being the second largest genus in the family with 250 species. although only about 40 of its 250 species have been studied, every one but two were simple diploids with n = 8. obviously, polyploidy as a factor in both evolution and speciation has a limited role in this remarkable genus (keener 1967, yano 1993). clematis is considered to be a monophyletic genus within ranunculaceae. the genus clematis has been divided into two subgenera of 1 – clematis including the sections clematis, cheiropsis, lasiantha, aspidanthera and naravelopsis, and 2 – flammula including the sections flammula, pterocarpa, viticella and fruticella (tamura 1987, tamura 1968, johansson 1995, christopher grey-wilson 2000, magnus johnson 2001) acta bot. croat. 68 (1), 2009 67 * corresponding author, e-mail: msheidai@yahoo.com u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:02 color profile: disabled composite 150 lpi at 45 degrees based on calyx arrangement and pubescence of the stamens, the genus clematis has been divided into 4 subgenera: 1 – subgen. campanella tamura including the sections campanella tamura, tubulosae decne, atragen (l.) dc., meclatis (spach) tamura, bebaenanthera edgew and pseudanemone prantl., 2 – subgen. viorna (reichb.) tamura including only the section viorna (reichb.) prantl., 3 – subgen. clematis including the sections clematis, cheirppsis dc., naraveliopsis hand.-mazz., aspidanthera spach., and lasiantha tamura, 4 – flammula (dc.) peterm., including the sections flammula dc., angustifoliae (tamura) serov., fruticella (moench) dc., viticella (moench) dc., and pterocarpa tamura. the section clematis has been divided into 5 subsections such as angustifoliae, rectae, crassifoliae, baoninianae including 73 species and 47 varieties (wang 2003a, b; 2006). biosystematic, phylogenetic and molecular studies have been performed to reveal the phylogenetic relationships of the clematis species (polinkova 1936, keener 1967, tamura 1968, townsend and evans 1980, ziman 1981, yano 1993, kimberly et al. 2005, miikeda 2006, wang 2006). similar studies are lacking in iran. the number of clematis species growing in iran varies according to different authors. parsa reports 8 clematis species while rechinger et al. reported the occurrence of 6 species in the country: 1 – c. viticella l. from the section viticella, 2 – c. orientalis l., 3 – c. flammula l., 4 – c. ispahanica boiss., 5 – c. songarica bunge., and 6 – c. asplenifolia schrenk., all from the section clematis (parsa 1986, rechinger et al. 1992). the present report is a part of biosystematic study of the genus clematis in iran, considering cytogenetic and palynological studies in three species of c. orientalis, c. flammula and c. ispahanica. materials and methods cytology cytological studies were performed on 9 populations of 3 species of c. orientalis l., c. flammula l. and c. ispahanica boiss. for meiotic studies, young flower buds were collected randomly from 10 plants of each species/ population and fixed in glacial acetic acid: ethanol (1:3) for 24 hrs. flower buds were washed and preserved in 70% ethanol at 4 oc until used. cytological preparations were made using the squash technique and 2% aceto-orcein as the stain (sheidai et al. 2006, 2007). fifty to one hundred pollen mother cells (pmcs) were analysed for chiasma frequency and distribution at diakinesis and metaphase stages while 500 pmcs were analysed for chromosome segregation during the anaphase and telophase stages. pollen satiability as a measure of fertility was determined by staining a minimum of 500 pollen grains with 2 % acetocarmine: 50 % glycerin (1:1) for about 30 min. complete pollens which were stained were taken as fertile, while incomplete, shrunken pollens with no stain were considered to be infertile (sheidai et al. 2006). in order to compare the meiotic characteristics among the species/ populations with different chromosome numbers, relative meiotic data were used (meiotic data/chromosome number). c2 test was performed to detect any significant difference in the relative chiasma frequency and distribution as well as chromosomes association as well as to detect any significant difference in meiotic abnormalities (sheidai et al. 2007). statistical analysis used spss ver. 9 (1998). 68 acta bot. croat. 68 (1), 2009 sheidai m., habibi m., azizian d., khatamsaz m. u:\acta botanica\acta-botan 1-09\sheidai.vp 22. travanj 2009 11:35:46 color profile: disabled composite 150 lpi at 45 degrees for karyotypic studies we collected freshly grown root tips from the seeds of at least ten randomly selected plants in each species. details of the pre-treatment, fixation and squash technique as well as karyotype analyses followed the earlier report of sheidai and rashid (2007). the chromosomes were identified according to levan et al. (1964), karyotype symmetry was determined according to stebbins (1971), while other karyotypic parameters like total form percentage (tf %) and coefficient of variation of the chromosome size were also determined (sheidai and bagheri-shabestarei 2007). pollen morphology fully matured anthers were removed from the specimens and prepared by the standard acetolysis method, after which they were mounted in glycerin jelly and sealed with paraffin wax for light microscopy (lm) (erdtman 1969). measurements and morphological observations of the pollen grains were performed with the use of a minimum of 20 pollen grains. for scanning electron microscopy (sem), the pollen grainss were attached to the aluminum stubs with double sided cellophane tape, air dried at room temperature and coated with gold. the specimens were examined with phylips xl 30, leo 440i at 15 kv and 22 kv and photographed. results cytology meiotic and mitotic karyotype analyses of the clematis species and populations studied revealed the presence of 2n = 2´ = 16 chromosome number in c. orientalis and 2n = 4´ = 32 chromosome number in c. ispahanica and c. flammula (tabs. 1, 2; figs. 1, 2). the populations of c. orientalis formed ring and rod bivalents with the highest value of ring bivalents (5.50) in the dizbad population and the lowest value of the same (4.00) in the roodbar 84 population. the highest value of total chiasmata occurred in the dizbad population (13.27) while the lowest value of occurred in the roodbar 84 population (11.29). two tetraploid species of c. ispahanica and c. flammula showed diplontic behavior and formed only bivalents in metaphase of meiosis-i. meiotic metaphase and anaphase chromosome stickiness occurred in the species and populations studied. the degree of chromosome stickiness ranged from stickiness among two or more chromosomes to the involvement of all metaphase chromosomes forming a complete clump (fig. 1, c). the species and populations differed significantly (p<0.05) in percentage of chromosome stickiness. the percentage of metaphase i cells showing stickiness varied from 2.50 in c. flammula to 7.50 in c. ispahanica. the percentage of metaphase i cells showing stickiness in different populations of c. orientalis ranged from 5.00% in the roodbar 84 population to 6.60% in the dizbad population. the percentage of metaphase i cells showing complete chromosome clumping varied from 2.50 in c. flammula to 20.00 in shahrestanak population of c. orientalis. one to a few anaphase-i laggard chromosomes occurred in c. flammula, c. ispahanica and the roodbar 85 population of c. orientalis. the species and populations studied differed significantly (p<0.05) in the mean number of laggard chromosomes too. the clematis species showed >97 % pollen fertility. acta bot. croat. 68 (1), 2009 69 cytology and palynology in clematis u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:02 color profile: disabled composite 150 lpi at 45 degrees 70 acta bot. croat. 68 (1), 2009 sheidai m., habibi m., azizian d., khatamsaz m. t a b . 1 . m e io ti c c h a ra c te ri st ic s in c le m a ti s sp e c ie s a n d p o p u la ti o n s st u d ie d . m e a n a n d st a n d a rd d e v ia ti o n s. s p e c is l o c a li ty 2 n x r b r o b ix t e x t o x ix n t x n t o x n r b n r o b n c le m a ti s o ri e n ta li s n e y sh a b o o r 1 6 2 ´ 5 .5 2 .5 4 5 .4 7 .8 6 1 3 .2 7 0 .6 8 0 .9 8 1 .6 6 0 .6 9 0 .3 2 ± 0 .3 3 3 3 /0 ± ± 0 .4 5 ± 0 .5 6 ± 0 .3 3 c . o ri e n ta li s s h a h re st a n a k 1 6 2 ´ 4 .8 1 3 .1 8 4 .8 1 8 .1 3 1 3 0 .6 1 .0 2 1 .6 3 0 .6 0 .4 k a ra j ± 0 .3 0 ± 0 .1 8 ± 0 .3 1 ± 0 .5 7 ± 0 .3 2 c . o ri e n ta li s r o o d b a r 8 4 1 6 2 ´ 4 4 4 .8 8 7 .1 1 1 1 .2 9 0 .6 1 0 .8 9 1 .4 1 0 .5 0 .5 ± 0 .4 1 ± 0 .4 1 ± 0 .4 0 ± 0 .7 6 ± 0 .7 5 c . o ri e n ta li s r o o d b a r 8 5 1 6 2 ´ 4 .2 5 3 .7 5 5 .1 7 .3 1 2 .4 0 .6 4 0 .9 1 1 .5 5 0 .5 3 0 .4 7 ± 0 .3 4 ± 0 .3 4 ± 0 .3 3 ± 0 .6 4 ± 0 .3 6 c . is p a h a n ic a n o o r 3 2 4 ´ 6 .5 4 9 .5 4 1 2 .3 6 1 0 .3 6 2 2 .7 2 0 .7 7 0 .6 5 1 .4 2 0 .4 1 0 .6 ± 0 .8 2 ± 0 .7 9 ± 0 .6 2 ± 1 .3 7 ± 0 .9 5 c . fl a m m u la r o o d b a r 3 2 4 ´ 7 .5 8 .5 1 1 .6 8 1 2 .1 8 2 3 .8 7 0 .7 3 0 .7 6 1 .4 9 0 .4 7 0 .0 3 ± 0 .5 3 ± 0 .5 3 ± 0 .4 9 ± 0 .9 5 ± 0 .5 9 a b b re v ia ti o n s: ´ = p lo id y le v e l, r b = m e a n n u m b e r o f ri n g b iv a le n ts , r o b = m e a n n u m b e r o f ro d b iv a le n ts , ix = m e a n n u m b e r o f in te rc a la ry c h ia sm a ta , t e x = m e a n n u m b e r o f te rm in a l c h ia sm a ta a n d t o x = m e a n n u m b e r o f to ta l c h ia sm a ta , r b n = m e a n n u m b e r o f ri n g b iv a le n ts p e r c e ll , r o b n = m e a n n u m b e r o f ro d b iv a le n ts p e r c e ll , ix n = m e a n n u m b e r o f in te rc a la ry c h ia sm a ta p e r b iv a le n t, t e x n = m e a n n u m b e r o f te rm in a l c h ia sm a ta p e r b iv a le n t, t o x n = m e a n n u m b e r o f to ta l c h ia sm a ta p e r b iv a le n t. u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:02 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 71 cytology and palynology in clematis fig. 1. meiotic cells in clematis species. a = meiocyte showing n = 8 in dizbad population of c. orientalis, b – meiocyte showing n = 8 in roodbar 84 population of c. orientalis, c – meiocyte showing complete clump in metaphase of meiosis-i in roodbar 84 population of c. orientalis, d – meiocyte showing n = 8 in sharestanak population of c. orientalis, e – meiocyte showing n = 8 in roodbar 85 population of c. orientalis, f = meiocyte showing n = 16 in c. flammula. scale bar = 10 mm. tab. 2. karyotypic details of clematis species. species locality 2n s l tl l/s x tf% cv kf clematis orientalis roodbar 16 5.43 11.34 64.96 2.08 8.12 32.90 13.17 4m+1sm + 1st + 2t c. orientalis noor 16 5.47 10.13 62.86 1.85 7.85 36.20 19.87 5m+1st + 2t c. ispahanica roodbar 32 3.69 11.36 116.91 3.07 7.30 35.20 14.65 10m+2st + 4t abbreviations: s = size of the shortest chromosome (mm), l = size of the longest chromosome (mm), tl = total length of haploid chromatin length (mm), l/s = ratio of the longest to shortest chromosome, x = the mean chromatin length, tf% = total form percentage, cv = coefficient of variation, kf = karyotype formulae, fig. 2. somatic metaphase cells in clematis species. a – roodbar 84 population of c. orientalis showing 2n = 16, b – yoosh population of c. orientalis showing 2n = 16, c – roodbar population of c. ispahanica showing 2n = 32. scale bar = 10 mm. u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:03 color profile: disabled composite 150 lpi at 45 degrees the somatic chromosome numbers obtained also show a tetraploid chromosome number (2n = 4´ = 32) for c. orientalis (tab. 2, figs. 2, 3). the size of chromosomes varied from 3.69 mm to 11.35 mm in c. ispahanica. the highest value of cv (36.20) for the size of 72 acta bot. croat. 68 (1), 2009 sheidai m., habibi m., azizian d., khatamsaz m. fig. 3. ideograms of clematis orientalia roodbar population (a), c. orientalia yoosh population (b) and c. ispanica (c). u:\acta botanica\acta-botan 1-09\sheidai.vp 22. travanj 2009 11:35:48 color profile: disabled composite 150 lpi at 45 degrees chromosomes occurs in the noor population of c. orientalis indicating the highest degree of size variation among its chromosomes. the clematis species studied differ in their karyotypic formulae (tab. 2), possess tf% of >32 and are placed in 2a and 2b classes of stebbins system (tab. 2). pollen morphology since c. orientalis populations showed morphological variations and were grouped in four clusters (data not given), representative populations were included in the palynological study (tab. 3, fig. 4). the general shape of pollen in the roodbar 85 (figs. 4, a–d) and dizbad (figs. 4, e–f) populations of c. orientalis is prolate spheroidal in equatorial view and is round in polar view (figs. 4, q–t). the pollen is tricolpate, isopolar and radiosymmetric, the furrows are long, relatively wide and the furrow margins unfolded. tectum is soft, smooth with microchinate projections. the pollen shape in the sharestanak population of c. orientalis is equiprolate in equatorial view and round in polar view (figs. 4, i–l), trizonocolpate, isopolar and radiosymmetric. the furrows are long, slender and their margins are folded inwards and thickened. the furrow membrane is granulate with microchinate projections. acta bot. croat. 68 (1), 2009 73 cytology and palynology in clematis tab. 3. pollen measurements in clematis species. (the rows of data are: minimum, mean and maximum respectively). species p e p/e colpi length colpi width apocolpium apo index o1 30.03 32.40 34.82 24.13 27.07 29.18 1.19 1.19 1.24 15.70 16.15 16.85 1.74 2.27 2.78 3.59 3.98 4.52 0.20 0.22 0.24 o2 28.76 22.92 36.03 23.77 27.25 29.50 1.20 1.20 1.22 16.59 19.29 21.40 1.63 2.54 3.43 2.18 4.98 6.10 0.16 0.23 0.31 o3 29.98 33.18 37.27 20.71 24.70 27.11 1.33 1.34 1.44 21.56 25.92 29.30 1.12 1.63 2.15 6.60 5.80 7.11 0.36 0.39 0.47 f 24.99 26.79 28.00 24.15 24.70 25.63 1.03 1.08 1.09 22.69 24.86 26.58 2.36 4.04 4.90 3.48 4.98 6.21 0.17 0.20 0.25 i 30.13 35.00 38.23 27.18 29.18 30.75 1.10 1.20 1.24 26.10 24.18 28.63 2.02 2.99 3.68 3.85 5.18 6.37 0.18 0.24 0.31 abbreviations: o1–o3 = clematis orientalis dizbad, roodbar and karaj populations respectively, f = c. flamula, i = c. ispahanica. apo index = apocolpium index, p = polar view, e = equatorial view. u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:04 color profile: disabled composite 150 lpi at 45 degrees in c. ispahanica (figs. 4, m–p), the pollen shape is equiprolate in equatorial view and round in polar view, trizonocolpate, isopolar and radiosymmetric. the furrows are syncolpate, almost long, folded and wide with regular, upright margins which are blunt at the end. the furrow membrane is granulate with microchinate projections. 74 acta bot. croat. 68 (1), 2009 sheidai m., habibi m., azizian d., khatamsaz m. fig. 4. pollen shape and measurements in clematis species. a–d – roodbar 85 population of c. orientalis., e–f – dizbad population of c. orientalis, i–l – sharestanak population of c. orientalis, m–p – c. ispahanica. q–t – c. flammula. scale bar = mm. u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:06 color profile: disabled composite 150 lpi at 45 degrees in c. flammula (figs. 4, q–t), the pollen shape is prolate spheroidal in equatorial view and almost triangular in polar view, trizonocolpate, isopolar and radiosymmetric. the furrows are long, slender, not folding; their margins are irregular, pointed at he tip. the groove membrane is granulate with microchinate projections. discussion the earlier cytological studies report 2n = 2´ = 16 and 32 for c. orientalis and 2n = 2´ = 16 for c. flammula and c. ispahanica (beskaravaynaja et al. 1979, bir et al. 1987, pastor et al. 1988, serov 1989), therefore a tetraploid chromosome number is new for two species of c. ispahanica and c. flammula. considering the diplontic behavior of the species of c. ispahanica and c. flammula we can not say for sure if it is due to the presence of a controlling mechanism for chiasma formation or due to the allopolyploid nature of the species studied. c 2 test showed no significant difference in the relative meiotic characteristics of the clematis species and populations studied, suggesting the close affinity of these species. variation in chiasma frequency and localization is genetically controlled. such a variation in the species and populations with the same chromosome number is considered a means for generating new forms of recombination influencing the variability within natural populations in an adaptive way (rees and dale 1974, quicke 1993). the significant difference for the percentage of chromosome stickiness and laggards observed among clematis species and populations may indicate their genomic differences. genetic and environmental factors as well as genomic-environmental interaction have been considered the reason for chromosome stickiness in different plant species (nirmala and rao 1996, baptista-giacomelli et al. 2000). the meiotic abnormalities observed may be the reason for the low pollen infertility observed in the species studied. variations observed in the karyotypic formulae among the clematis species may indicate the occurrence of structural changes in their chromosomes. these species are placed in relatively primitive classes of 2a and 2b of the stebbins system indicating the presence of a symmetrical karyotype in clematis species studied. therefore in general, the variations observed in both meiotic chromosome pairing as well as the details of karyotypes (although the study considers only 3 species) may indicate the role of cytological changes in clematis species diversification. the occurrence of a granulate membrane in furrows in c. ispahanica has already been reported; however, 4 and 6 colpate pollens were not previously recorded (serov and trasevich 1986). differences observed in palynological characteristics of the clematis species may be of use in the species identification and systematics. the populations of c. orientalis also showed some variation in their pollen characteristics. the section meclatis has been considered as c. orientalis sensu lato as the most variable complex within the genus clematis, possessing the widest geographical distribution, and thus divided into seven varieties. the previously cited author believes that c. orientalis var. orientalis grows in iran (grey-wilson 1989). the morphological, cytological and palynological findings support such a consideration; however, at present we are not attempting to make any infraspecific division for c. orientalis, as this would need further detailed investigation. acta bot. croat. 68 (1), 2009 75 cytology and palynology in clematis u:\acta botanica\acta-botan 1-09\sheidai.vp 22. travanj 2009 11:35:50 color profile: disabled composite 150 lpi at 45 degrees references baptista-giacomelli, f. r., pagliarini, m. s., almeida, j. l., 2000: meiotic behavior in several brazilian oat cultivars (avena sativa l.). cytologia 65, 371–378. beskaravaynaja, m. a., djakova, m. i., sakharova, t. p., 1979: cytological research of clematis l (in russian). bjulleten glavnogo botanicheskogo sada 113, 81–84. bir, s. s., thakur, h. k., chatha, g. s., 1987: chromosomal studies in certain members of ranunculaceae and mensipermaceae. proceedings of indian science congress association 74, 184–185. erdtman, g., 1969: handbook of palynology. hafner publishing company, new york. grey-wilson, c., 1989: clematis orientalis (ranunculaceae) and its allies. kew bulletin 44, 33–60. grey-wilson, c., 2000: clematis: a comprehensive guide for gardeners, horticulturists and botanists. timber press, portland. johansson, j. t., 1995: a revised chloroplast dna phylogeny of the ranunculaceae. plant systems and evolution supplimentary 9, 253–261. johnson, m., 2001: the genus clematis. magnus johnson, stockholm. kimberly, f., garey, j., frederick, b. 2005: actin intron sequences can determine the hybridization history within clematis. learning from plants. austin. keener, c. s., 1967: a biosystematic study of clematis subsection integrifoliae (ranunculaceae). journal of elisha mitchell science society 83, 1–41. levan, a., fredga, k., sandberg, a., 1964: nomenclature for centromeric position on chromosomes. hereditas 52, 201–220. miikeda, o., 2006: phylogenetic relationships of clematis (ranunculaceae) based on chloroplast and nuclear dna sequences. botanical journal of linnean society 152, 153–168. nirmala, a., rao, p. n., 1996: genetics of chromosome numerical mosaism in higher plants. the nucleus 39, 151–175. parsa, a., 1986: flora of iran. 2. ministry of culture and higher education, tehran. pastor, j., fernández, i., díez, m., 1988: números cromosómicos para la flora española. lagascalia 15, 124–129. polinkova, j., 1936: a report on meiosis in clematis jackmani. cytologia 7, 35–53. quicke, d. l. j., 1993: principles and techniques of contemporary taxonomy. blackie academic and professional, glasgow. rechinger, k. h., riedle, v. h., iranshahr, m., 1992: clematis. in: rechinger, k. h. (ed.), flora iranica, 171, 229–239. akademische druck und verlagsanstalt, graz. rees, h., dale, p. j., 1974: chiasma and variability in lolium and festuca populations. chromosoma 47, 335–351. serov, v. p., 1989: the study of karyotypes in representatives of the genera clematis and atragene (ranunculaceae). botanicheski zhurnal moscow 74, 967–972. 76 acta bot. croat. 68 (1), 2009 sheidai m., habibi m., azizian d., khatamsaz m. u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:06 color profile: disabled composite 150 lpi at 45 degrees serov, v. p., trasevich, v.f., 1986: the morphology and ultrastructure of pollen in the genera clematis and atragen (ranunculaceae) in connection with the systematics. botanicheski zhurnal moscow 71, 1491–1501. sheidai, m., noormohammadi, z., sotodeh, m., 2006: cytogenetic variability in several canola cultivars. caryologia 39, 267–276. sheidai, m., rashid, s., 2007: cytogenetic study of some hordeum l. species in iran. acta biologica szegedensis 51, 107–112. sheidai, m., kalhor-home, n., poorneydanei, a., 2007: cytogenetic study of some populations of foeniculum vulgare (umbelliferae) in iran. caryologia 60, 257–261. sheidai, m., bagheri-shabestarei, e. s., 2007: cytotaxonomy of some festuca species and populations in iran. acta botanica croatica 66, 143–151. stebbins, g. l., 1971: chromosomal evolution in higher plants. edward arnold, london. tamura, m., 1968: morphology, ecology and phylogeny of the ranunculaceae, 7. science reports of the osaka university 16, 21–43. tamura, m., 1987: a classification of the genus clematis. acta phytotaxonomica et geobotanica 38, 33–44. tamura, m., 1993: ranunculaceae. in: en kubitzki, k., rohwer, j. g., bittrich, v. (eds.), the families and genera of vascular plants, 2. flowering plants – dicotyledons, 563–583. springer-verlag, berlín. townsend, c. c., evans, g., 1980: clematis. in: townsend, c. c. (ed.), flora of iraq 4, 743–750. baghdad ministry of agriculture and agrarian reform, baghdad. wang, w. t., 2003a: a revision of clematis sect. clematis (ranunculaceae). acta phytotaxonomica sinica 41, 61. wang, w. t., 2003b: a revision of clematis sect. clematis (ranunculaceae). acta phytotaxonomica sinica 41, 97–172. wang, w. t., 2006: a revision of clematis sect. clematis (ranunculaceae). acta phytotaxonomica sinica 44, 401–436. yano, y., 1993: pollen grain morphology in clematis (ranunculaceae). clematis international 1993, 42–43. ziman, s. n., 1981: analysis of phylogenetic relations in subtribe clematidinae (ranunculaceae). ukrainian botanical zhurnal 38, 36–43. acta bot. croat. 68 (1), 2009 77 cytology and palynology in clematis u:\acta botanica\acta-botan 1-09\sheidai.vp 16. travanj 2009 11:40:06 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 71 (1), 95–113, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 foliar epidermis morphology in quercus (subgenus quercus, section quercus) in iran parisa panahi1, 2, ziba jamzad2*, mohammad r. pourmajidian1, asghar fallah1, mehdi pourhashemi3 1 department of forestry, sari agricultural sciences and natural resources university, p.o.box: 737, sari, iran 2 botany research division, research institute of forests and rangelands of iran, p.o.box: 013185-116, tehran, iran 3 forest research division, research institute of forests and rangelands of iran, p.o.box: 13185-116, tehran, iran abstract – the foliar morphology of trichomes, epicuticular waxes and stomata in quercus cedrorum, q. infectoria subsp. boissieri, q. komarovii, q. longipes, q. macranthera, q. petraea subsp. iberica and q. robur subsp. pedunculiflora were studied by scanning electron microscopy. the trichomes are mainly present on abaxial leaf surface in most species, but rarely they appear on adaxial surface. five trichome types are identified as simple uniseriate, bulbous, solitary, fasciculate and stellate. the stomata of all studied species are of the anomocytic type, raised on the epidermis. the stomata rim may or may not be covered with epicuticular. the epicuticular waxes are mostly of the crystalloid type but smooth layer wax is observed in q. robur subsp. pedunculiflora. statistical analysis revealed foliar micromorphological features as been diagnostic characters in quercus. keywords: quercus, trichome, stoma, epicuticular wax, sem introduction quercus is the most frequent genus of fagaceae in the forests of iran. different species of oak are distributed in vast areas of the zagros, arasbaran and hyrcanian forests. in these forests, there is a remarkable morphological variation in the genus quercus. all of iran’s native oaks belong to the subgenus quercus, sections quercus and cerris. species with dentate leaves belong to the section cerris and lobed-leaves species, which are the object of this research, belong to the section quercus. acta bot. croat. 71 (1), 2012 95 * corresponding author, e-mail: jamzad@rifr-ac.ir copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:24 color profile: disabled composite 150 lpi at 45 degrees the first and the most complete study of quercus in iran, and also including the most important herbaria in europe was made by djavanchir (1967), in his phd thesis. he identified 9 taxa of lobed-leaves oak species (quercus macranthera fisch. and c. a. mey.; q. iberica steven = q. petraea (matt.) liebl. subsp. iberica (steven) krassiln.; q. komarovii a.camus = q. longifolia k.koch; q. longipes steven; q. cedrorum kotschy; q. hass kotschy = q. robur l. subsp. pedunculiflora (k.koch) menitsky; q. infectoria oliv.; q. infectoria oliv. var. tenuicarpa djav.-khoie; q. infectoria oliv. var. pfaeffingeri kotschy = q. pfaeffingeri kotschy). four years later, another treatment of quercus was published by menitsky in flora iranica (1971). he recognized 4 taxa in this group (q. robur subsp. pedunculiflora; q. infectoria oliv. subsp. boissieri (reut.) o. schwarz; q. petraea subsp. iberica; q. macranthera). since 1971, there have been many local studies and new collections revealing high diversity among the species of sect. quercus, so a comprehensive study of the genus is necessary. in the new review, micromorphological characters should be considered as well as investigations into ecological conditions, populations and possible hybridizations. trichome, stomata and wax characters demonstrated by previous investigators for american and european quercus species, proved to be useful characters for species delimitation. the use of trichome character in quercus dates back to the taxonomic studies of dyal (1936). she recognized glandular vs. non-glandular as two basic types of trichomes in oaks, then subdivided the latter into those with branches spreading from one point and those arising above one another along the hair axis. lutz (1938) described four different types and camus (1936–1954) developed a much more precise classification of seven types. their widespread application to oak taxonomy has been increased through the use of scanning electron microscopy (sem) so that hardin (1976, 1979a, b) and jones (1986) were able to present a complete classification of oak trichomes and their terminology. later, some researchers used sem images for separation of quercus species based on trichome types (ba^i] 1981; uzunova and palamarev 1985, 1992a, b, 1993; bussotti and grossoni 1997; lou and zhou 2001; scareli-santos et al. 2007). epicuticular waxes have considerable micromorphological diversity. many of them are of great systematic significance (barthlott et al. 1998). the taxonomic significance of epicuticular waxes in quercus has been studied by some researchers (bussotti and grossoni 1997, uzunova et al. 1997, scareli-santos et al. 2007). the first comprehensive study of epicuticular waxes was made by de bary (1871). in the course of his light microscopic studies, he investigated about 60 species and classified epicuticular waxes into four categories. the most recent comprehensive overview of the classification of epicuticular waxes was given by barthlott et al. (1998). the stomata of oak leaves have certain structural features which make them mutually distinguishable (nikoli] et al. 2003). different characteristics of stomata such as density, dimension, location, subsidiary cell, guard cells and rim have been used for delimitation of oak species by some investigators (uzunova and palamarev 1993, bussotti and grossoni 1997, uzunova et al. 1997, lou and zhou 2001). the analysis of epidermal structures has proved to be of some help in taxonomy of oaks and provided a valuable set of analytical characters for species delimitation (thamson and 96 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:24 color profile: disabled composite 150 lpi at 45 degrees mohlenbrock 1979, ba^i] 1981, safou and saint-martin 1989, manos 1993, pénas merino et al. 1994, llamas et al. 1995, uzunova et al. 1997, fortini et al. 2009). the present study was initiated to determine if quantifiable characters of trichomes, stomata and waxes could be combined with other morphological characters to produce a valid set of characters for recognizing iranian oak species. in this research, we focused on the foliar epidermis structure of quercus species of sect. quercus. furthermore, we did a complete survey on habitats of quercus species, their distribution patterns, populations and ecological conditions. materials and methods the majority of specimens used in this study were obtained during several field trips to areas throughout the geographic range of species of the section quercus in iran. ten individual trees were sampled for each taxon and some voucher specimens were prepared for each studied taxa. materials and collecting data of examined taxa are listed in table 1.the voucher specimens are deposited in the herbarium of the research institute of forests and rangelands of iran (tari). furthermore, other previously collected specimens of tari and the herbarium of the natural resources faculty, university of tehran (nrf) were sampled. the descriptions that follow are based on the mature condition of adaxial and abaxial leaf surfaces. all measurements were taken from trichomes and stomata occurring on a sample of 10 mm2 of surface midway between the base and apex, midvein and margin of leaves. only fully expanded, undamaged leaves without signs of scarring or disease were examined. the leaf specimens were mounted onto aluminum stubs and sputter-coated with gold palladium for 5 minutes in a humer éé sputtering device. all leaf materials were preacta bot. croat. 71 (1), 2012 97 foliar epidermis in quercus tab. 1. list of species and the collection data taxon collection data quercus macranthera iran: mazandaran, siahbisheh, djavanchir, 2054c (nrf) iran: ardabil, khalkhal, andebil forest, 2100 m, panahi and pourhashemi 95102 (tari) iran: golestan, 40 km south of ali-abad, between gorgan and shah-passand, 1800 m, assadi 25591(tari) iran: east azerbaijan, kallibar, protected region from makidi to veinagh, 1700 m, wendelbo and assadi 17057 (tari) q. petraea subsp. iberica iran: east azerbaijan, kallibar, protected region from makidi to veinagh, wendelbo and assadi 17026 (tari) iran: mazandaran, chalous road, 2 km to siahbisheh, 2450 m, runemark and mozaffarian, 28082 (tari) iran: east azerbaijan, arasbaran, mountains south of vaighan, 1200–1500 m, assadi and masoumi, 20365(tari) iran: east azerbaijan, arasbaran, 1190 m, panahi and pourhashemi 95103 (tari) transcaucasia, georgia, adigeni, flora of ussr, menitsky (tari) u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:24 color profile: disabled composite 150 lpi at 45 degrees 98 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. taxon collection data quercus komarovii iran: east azerbaijan, kallibar, arasbaran, djavanchir 2052 (nrf) iran: east azerbaijan, kallibar, arasbaran, djavanchir 2054 (nrf) iran: east azerbaijan, 10 km from kallibar to khodaafarin, 1850 m, mozaffarian and mohammadi 37616 (tari) iran: east azerbaijan, arasbaran, 1700 m, panahi and pourhashemi 95099 (tari) q. longipes iran: east azerbaijan, oskou, 1492 m, panahi and pourhashemi 95104 (tari) iran: west azerbaijan, khoy, djavanchir 2054 (nrf) iran: east azerbaijan, tabriz, oskou, 1400 m, mozaffarian 72830 (tari) iran: east azerbaijan, oskou, gunbarf village, 2240 m, panahi and pourhashemi 95105 (tari) q. robur subsp. pedunculiflora iran: kurdistan, baneh-marivan, shipaneh-jow, pirsharif cemetery, 1598 m, panahi and pourhashemi 91401 (tari) iran: kurdistan, baneh-marivan, bayan-darreh village, sheikh musa cemetery, 1736 m, panahi and pourhashemi 91401 (tari) iran: west azerbaijan, urmia, bardehsou, silvana, sabeti 30992 (tari) transcaucasia, azerbaijania, dist keessary, flora of ussr, menitsky (tari) q. cedrorum iran: west azerbaijan, mirabad, molla allah cemetery, 1425 m, panahi and pourhashemi 95052 (tari) iran: west azerbaijan, sardasht, djavanchir, no number (nrf) iran: west azerbaijan, mirabad, khedr-abad village, 1290 m, panahi and pourhashemi 95098 (tari) q. infectoria subsp. boissieri var. boissieri iran: kurdistan, baneh, between armardeh and nirvan, 1523 m, panahi and pourhashemi 91433 (tari) iran: kurdistan, banehsardasht road, djavanchir 2046 (nrf) iran: west azerbaijan, mirabad, khedr-abad village, 1290 m, panahi and pourhashemi 95050 (tari) iran: kurdistan, 45 km on road from saqqez to baneh, 1900 m, runemark and mozaffarian 25929 (tari) q. infectoria subsp. boissieri var. tenuicarpa iran: kurdistan, baneh, belakeh, 1650 m, djavanchir 25 (nrf) iran: kurdistan, baneh, soleiman bak cemetery, 1489 m, panahi and pourhashemi 91427 (tari) iran: kurdistan, baneh, between armardeh and nirvan, 1523 m, panahi and pourhashemi 91435 (tari) q. infectoria subsp. boissieri var. pfaeffingeri iran: kurdistan, baneh, mirhesam, 14-rigeh cemetery, 1550 m, panahi and pourhashemi 95022 (tari) iran: west azerbaijan, aliabad, on road of piranshahr to sardasht, 1200m, zehzad and siami 30983 (tari) iran: kurdistan, baneh, between belakeh and baneh, djavanchir 2050h (nrf) iran: kurdistan, baneh, soleiman bak cemetery, 1489 m, panahi and pourhashemi 91421 (tari) tab. 1. continued u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:24 color profile: disabled composite 150 lpi at 45 degrees pared without pretreatment or critical point drying. the micromorphological characters of epidermal structures were observed and measured by a hitachi (s–4160) sem at an accelerating voltage of 15 kv. in total, 20 micromorphological characters were scored (tab. 2). the number of each type present on a standard area of 1 mm2 was counted. the trichome ray length was measured from the point of divergence of the ray and base of the trichome. terminology used for trichome types is based on hardin (1976, 1979a). identification of wax layer types has been done according to barthlott et al. (1998) and the freedom of rim was estimated following bruschi et al. (2000). stomatal density was then measured on a standard area of 1 mm2. normality distribution of characters was assessed for all the 20 variables using the chi-square test (p< 0.001) and anova with post-hoc studentnewmankeuls mean comparisons was used to define the discrimination values for each variable. furthermore, discriminant analysis was used to assess the degree of separation of the studied taxa by multivariate measurements. results trichomes the studied oak species have different trichome types. we have identified five different trichome types in the examined taxa as follows: acta bot. croat. 71 (1), 2012 99 foliar epidermis in quercus tab. 2. leaf micromorphological characters examined abbreviation and units description nst-abs nfa-abs nso-abs nsi-abs nbu-abs tot-abs nst-ads nfa-ads nso-ads tot-ads nstr-abs nfar-abs lstr-abs lfar-abs lsor-abs lst (ìm) wst (ìm) std (no. mm–2) fsr (ìm) sai number of stellate trichomes on abaxial surface number of fasciculate trichomes on abaxial surface number of solitary trichomes on abaxial surface number of simpleuniseriate trichomes on abaxial surface number of bulbose trichomes on abaxial surface total number of trichomes on abaxial surface number of stellate trichomes on adaxial surface number of fasciculate trichomes on adaxial surface number of solitary trichomes on adaxial surface total number of trichomes on adaxial surface number of stellate rays on abaxial surface number of fasciculate rays on abaxial surface length of stellate rays on abaxial surface length of fasciculate rays on abaxial surface length of solitary rays on abaxial surface length of stomata width of stomata stomatal density freedom of stomata rim stomatal area index u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:24 color profile: disabled composite 150 lpi at 45 degrees 1. simple-uniseriate: thin-walled, unicellular or multicellular and uniseriate with different length diameter, number of cells and shape of cells. this trichome has been observed in all studied taxa except quercus infectoria subsp. boissieri var. pfaeffingeri and q. robur subsp. pedunculiflora (figs. 1b, 1c, 1d). 2. bulbous: thin-walled, multicellular, uniseriate or irregularly multiseriate with enlarged, swollen cell or cells. this type has been observed only in q. longipes (fig. 1e). 3. solitary: single, long, usually straight and unicellular often thin-walled. there are two very distinct subtypes in this type. one has erect, long, unicellular hair on the lamina 100 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. fig. 1. sem micrographs of trichomes in quercus sect. quercus (a–l): abaxial surface (a–j). q. macranthera (a), q. petraea subsp. iberica (b), q. komarovii (c), q. longipes (d–e), q. robur subsp. pedunculiflora (f), q. cedrorum (g), q. infectoria subsp. boissieri var. boissieri (h), q. infectoria subsp. boissieri var. tenuicarpa (i), q. infectoria subsp. boissieri var. pfaeffingeri (j), adaxial surface (k–l). q. infectoria subsp. boissieri var. pfaeffingeri (k), q. macranthera (l). scale bar denotes 100 µm (b–d, f–j); 300 µm (a, k–l) and 30 µm (e). u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:24 color profile: disabled composite 150 lpi at 45 degrees and veins. the second subtype presents more or less appressed, long, often thinner-walled and sometimes wavy hair. they occur mostly on juvenile leaves and may persist along the petiole and midrib. we observed this type in all studied taxa (figs. 1f, 1l). 4. fasciculate: erect, thick-walled cells, clustered and fused at base. this may be sessile, stipitate, or pedestaled. we have found this trichome type on the lamina of q. macranthera and on the edge of the midrib in q. infectoria subsp. boissieri var. boissieri (figs. 1a, 4g). 5. stellate: usually thick-walled, with a single set of radiating, slender rays, projecting horizontally from a common center, sessile or stipitate. a few rays may be somewhat erect (e.g. q. komarovii, fig. 1c) but the general aspect is of an appressed trichome with the rays spreading in one plane roughly parallel to the epidermis (q. longipes, fig. 1d). the trichome types identified may be present on the adaxial or on both surfaces of leaves. the differences of trichome types on the abaxial and adaxial surfaces of different species are summarized in table 3. waxes two main types and three subtypes of epicuticular waxes were recognized as follows: é. crystalloids: distinct wax projections, usually with a characteristic shape, size and orientation towards the surface, protruding from the ubiquitous wax film (barthlott et al. 1998). two subtypes are identified within this type as follows: a. membranous platelets: flat, membranous interconnected crystalloids, protruding from the surface at an acute angle, often with threadlike extensions (fig. 2f). b. irregular platelets: flat crystalloids with irregular margin, protruding perpendicularly from the surface (figs. 2a–d, 2g–i). acta bot. croat. 71 (1), 2012 101 foliar epidermis in quercus tab. 3. trichome types of quercus species sect. quercus taxon trichome types simpleuniseriate bulbous solitary fasciculate stellate quercus macranthera –/+ –/– +/+ +/+ –/– q. petraea subsp. iberica –/+ –/– –/+ –/– –/+ q. komarovii –/+ –/– –/+ –/– –/+ q. longipes –/+ –/+ –/+ –/– –/+ q. robur subsp. pedunculiflora –/– –/– –/+ –/– –/+ q. cedrorum –/+ –/– –/+ –/– –/+ q. infectoria subsp. boissieri var. boissieri –/+ –/– –/+ –/+ –/+ q. infectoria subsp. boissieri var. tenuicarpa –/+ –/– –/+ –/+ –/+ q. infectoria subsp. boissieri var. pfaeffingeri –/– –/– –/+ –/+ +/+ adaxial surface/abaxial surface. – = absent, + = present u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:24 color profile: disabled composite 150 lpi at 45 degrees éé. layers and crusts: continuous coverings with a considerable thickness, ca.0.5–10 ìm. it should be noted that most crusts are continuous coverings from the beginning, but some are the product of the erosion of crystalloids. sometimes contaminated smooth layers may appear as crusts (barthlott et al. 1998). several subtypes have been identified within this type but we observed one subtype as follows: smooth layers: continuous covering usually less than 1 ìm thick without a prominent surface sculpturing (fig. 2e). stomata according to the arrangement of stomata subsidiary cells, we recognized only anomocytic type. this type of stomata has epidermal cells around the guard cells not distinguishable from other epidermal cells. we identified three stomata shapes as follows: elliptical (quercus cedrorum, fig. 3f), roundish (q. infectoria subsp. boissieri var. pfaeffingeri, fig. 3i) and rounded (q. infectoria subsp. boissieri var. tenuicarpa, fig. 3h). stomata were raised above the epidermal surface in all cases. the rims of the stomata were covered by 102 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. fig. 2. sem micrographs of wax in quercus sect. quercus: q. macranthera (a), q. petraea subsp. iberica (b), q. komarovii (c), q. longipes (d), q. robur subsp. pedunculiflora (e), q. cedrorum (f), q. infectoria subsp. boissieri var. boissieri (g), q. infectoria subsp. boissieri var. tenuicarpa (h), q. infectoria subsp. boissieri var. pfaeffingeri (i), scale bar in a–i denotes 3.75 µm. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:25 color profile: disabled composite 150 lpi at 45 degrees epicuticular waxes of crystalloids type in some examined taxa like q. longipes (fig. 3d), while the others had uncovered rims (e.g. q. petraea subsp. iberica, fig. 3b). in one case, the rim and pore were entirely covered by crystalloids, so that the pore was invisible (q. infectoria subsp. boissieri var. tenuicarpa, fig. 3h). univariate analysis there were significant differences among studied taxa (anova, p < 0.001) for all micromorphological characters (tab. 4). snk tests indicated that the number of fasciculate trichomes on the abaxial surface was responsible for providing three groups: q. macranthera with highest value, three varieties of q. infectoria subsp. boissieri with a low number of fasciculate trichomes, while q. komarovii, q. petraea subsp. iberica, q. cedrorum, q. longipes and q. robur subsp. pedunculiflora had no fasciculate trichomes. the presence of bulbous trichomes on the abaxial surface and the highest number of stellate trichomes on the abaxial surface distinguishes q. longipes from other examined taxa. quercus macranthera and q. infectoria subsp. boissieri var. pfaeffingeri had trichomes on the adaxial surfaces of fasciculate, stellate and solitary types. acta bot. croat. 71 (1), 2012 103 foliar epidermis in quercus fig. 3. sem micrographs of stomata in quercus sect. quercus: q. macranthera (a), q. petraea subsp. iberica (b), q. komarovii (c), q. longipes (d), q. robur subsp. pedunculiflora (e), q. cedrorum (f), q. infectoria subsp. boissieri var. boissieri (g), q. infectoria subsp. boissieri var. tenuicarpa (h), q. infectoria subsp. boissieri var. pfaeffingeri (i), scale bar in a–i denotes 15 µm. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:25 color profile: disabled composite 150 lpi at 45 degrees 104 a c t a b o t .c r o a t .71 (1),2012 p a n a h i p .,ja m z a d z .,p o u r m a jid ia n m .r .,f a l l a h a .,p o u r h a s h e m im . tab. 4. anova results of leaf micromorphological characters. variables and units are specified in table 2. variables df f descriptive q. macranthera q. petraea subsp. iberica q. komarovii q. longipes q. robur subsp. pedunculiflora q. cedrorum q. infectoria subsp. boissieri var. boissieri q. infectoria subsp. boissieri var. tenuicarpa q. infectoria subsp. boissieri var. pfaeffingeri mean sd mean±sd mean±sd mean±sd mean±sd mean±sd mean±sd mean±sd mean±sd mean±sd nst-abs 8 1398* 17 22 0 a 11±2 b 47±3 c 65±3 d 8±2 ef 8±2 e 4±1 g 5±1 efg 4±2 fg nfa-abs 8 714* 4 9 28±3 a 0 b 0 b 0 b 0 b 0 b 3±1 c 3±1 c 2±1 c nso-abs 8 62* 3 2 4±1 a 0 b 2±1 c 2±1 c 7±7 d 2±1 c 3±1 c 2±0 c 2±1 c nsi-abs 8 265* 15 12 32±3 a 33±3 a 16±3 bc 20±2 b 0 d 2±1 d 19±4 b 15±3 c 0 d nbu-abs 8 376* 1 2 0 a 0 a 0 a 6±1 b 0 a 0 a 0 a 0 a 0 a tot-abs 8 771* 40 28 65±4 a 44±4 b 65±4 a 93±3 c 14±3 d 12±2 de 29±5 f 25±3 f 8±2 e nst-ads 8 565* 1 4 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 12±2 b nfa-ads 8 52* 0 1 2±1 a 0 b 0 b 0 b 0 b 0 b 0 b 0 b 0 b nso-ads 8 312* 1 2 5±1 a 0 b 0 b 0 b 0 b 0 b 0 b 0 b 0 b tot-ads 8 401* 2 4 7±1 a 0 b 0 b 0 b 0 b 0 b 0 b 0 b 12±2 c nstr-abs 8 24* 3 2 0 a 3±1 b 4±1 bc 4±2 bc 3±1 b 3±1 b 3±1 b 5±1 cd 6±2 d nfar-abs 8 60* 2 2 5±2 a 0 b 0 b 0 b 0 b 0 b 3±1 c 3±1 c 3±1 c lstr-abs 8 52* 96 51 0 a 89±13 b 82±9 bc 52±10 c 98±18 bd 132±23 de 156±43 e 105±18 bd 150±28 e lfar-abs 8 111* 119 144 341±91 a 0 b 0 b 0 b 0 b 0 b 277±78 ac 236±29 c 218±44 c lsor-abs 8 47* 243 105 381±83 a 247±73 b 237±70 b 339±9 ac 93±11 d 152±10 d 307±27 c 305±34 bc 128±9 d lst 8 14* 24 3 22±2 ab 25±2 bcd 21±2 a 24±2 abc 23±2 abc 20±1 a 27±3 d 26±1 cd 25±2 bcd wst 8 36* 18 3 16±1 a 18±1 b 18±2 b 18±2 b 15±2 a 14±1 a 20±2 b 23±1 c 20±1 b std 8 54* 191 38 204±19 a 194±7 ab 177±5 bc 169±10 bc 207±19 a 163±13 c 277±9 d 174±32 bc 156±10 c fsr 8 250* 6 3 8±1 a 9±0 b 7±1 a 7±0 a 5±1 c 4±0 d 4±1 d 0 e 8±1 a sai 8 47* 4557 1275 4554±601 a 4777±429 a 3631±510 bc 4013±436 bc 4770±627 a 3346±283 b 7523±848 d 4475±781 ab 3925±210 abc *p < 0.001; means followed by the same letter in the same row are not significantly different at p < 0.001 according to nsk test u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 3 4 p a n a h i . v p 2 6 . o u j a k 2 0 1 2 1 1 : 1 5 : 2 5 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s on the other hand, a freedom of stomatal rim enables q. infectoria subsp. boissieri var. tenuicarpa (stomatal rim completely covered) to be distinguished from the other taxa (stomatal rim covered to varying degrees). furthermore, high value of stomatal density distinguishes q. infectoria subsp. boissieri var. boissieri from other studied taxa. multivariate analysis discriminant analysis revealed that the three functions represented 87.6 % of the total variation in the data set (tab. 5). the first discriminant function accounted for 49.7 % of the total variance. the standardized coefficients of function 1 were highest for the number of stellate trichomes on the abaxial surface, number of solitary trichomes on the adaxial surface and number of fasciculate trichomes on the abaxial surface. function 2 represented another 26.9 % of the total variance. this function was related to number of solitary trichomes on the adaxial surface, number of stellate trichomes on the abaxial surface and number of fasciculate trichomes on the abaxial surface, respectively. function 3 accounted for 11 % of the total variation and belonged to number of stellate trichomes on the adaxial surface, the number of bulbous trichomes on the abaxial surface and the number of fasciculate trichomes on the abaxial surface (fig. 4). overall misclassification was zero. description of taxa quercus macranthera fisch. and c.a.mey. deciduous large tree, up to 25 m high, with spreading crown, gray-brown thick bark, leaves with long persistent stipules. this species is characterized with dense trichomes of different types on leaf surfaces. both abaxial and adaxial surfaces are covered with trichomes, but the density and variability of trichomes is higher on abaxial surface. the most acta bot. croat. 71 (1), 2012 105 foliar epidermis in quercus tab. 5. standardized coefficients for the first three discriminant functions of means micromorphological characters variables function 1 function 2 function 3 lstr-abs –.042 –.201 .209 lsor-abs .397 .247 .021 lfar-abs .302 .129 –.025 nst-abs –.723 .494 .190 nfa-abs .662 .489 .279 nso-abs –.189 .031 –.199 nsi-abs –.023 .396 –.202 nbu-abs –.269 .345 .285 nst-ads .194 –.402 .839 nso-ads .669 .501 .253 wst .006 –.150 .061 std .183 .060 –.217 fsr –.133 .039 .221 u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:25 color profile: disabled composite 150 lpi at 45 degrees frequent trichomes on abaxial surface are pedestaled-fasciculate and stipitate-fasciculate. these trichomes have 2–8 very long rays (190–455 ìm). they are not spread on the surface of the leaf (fig. 1a). stipitate-fasciculate trichomes are more abundant than pedestaled-fasciculate. furthermore, simple-uniseriate hairs are abundant. on adaxial surface trichomes are restricted only to a few solitary and 2-rayed fasciculate forms, irregularly and sparsely scattered on epidermal surface (fig. 1l). on the main veins, the solitary, pedstaled-fasciculate and 2-rayed fasciculate trichomes were frequently found. midrib is covered completely with long solitary (more than 300 ìm) and fasciculate trichomes with often 2–3 long rays (fig. 5a). the waxes on the abaxial surface are irregular platelets (fig. 2a). they are not very densely and evenly distributed. the platelets are shorter in open spaces between stomata and are also eroded. the stomata are raised, elliptical and the rim is uncovered (fig. 3a). quercus petraea (matt.) liebl. subsp. iberica (steven) krassiln. deciduous medium size tree, up to 12 m high, in uplands, large tree up to 18 m height in lowlands, with spreading crown, bright and cracked bark, branches glabrous. leaves have short persistent stipules. simple-uniseriate multicellular hairs with 2–3 cells and stellate trichomes with 2–4 (often 4) symmetric short rays (75–120 ìm) are spread regularly on the 106 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. fig. 4. canonical discriminant functions of micromorphological characters of studied taxa: q. cedrorum (1), q. petraea subsp. iberica (2), q. longipes (3), q. komarovii (4), q. robur subsp. pedunculiflora (5), q. macranthera (6), q. infectoria subsp. boissieri var. boissieri (7), q. infectoria subsp. boissieri var. tenuicarpa (8), q. infectoria subsp. boissieri var. pfaeffingeri (9). black squares represent centered groups of closely related taxa. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees surface of the leaf (fig. 1b). there are only a few long solitary hairs (more than 200 ìm) on the midrib (fig. 5b). there is no trichome on the adaxial surface. the waxes on the abaxial surface are irregular platelets that do not necessarily cover the entire leaf surface, leaving empty spaces between individual platelets (fig. 2b). the stomata are elliptical, raised and covered by a crown of wax platelets; the rim is elliptical and uncovered (fig. 3b). quercus komarovii a. camus deciduous small tree or shrub, up to 5 m high, with spreading crown, brown-red young branches and leathery leaves. the abaxial surface is covered by stellate trichomes with 2–4 (often 4) short rays (60–95 ìm), completely symmetric (fig. 1c). usually the middle rays are erect and the others spread roughly parallel to the epidermis. this trichome type is denser near the midrib. on the midrib a few solitary hairs are observed (fig. 5c). there are a few simple-uniseriate trichomes on the abaxial surface. densely irregular wax platelets have been observed on the abaxial surface (fig. 2c), and flat layer type wax is also observed in some sections of surface and around some stomata. the stomata are elliptical and raised; the rim is uncovered (fig. 3c). acta bot. croat. 71 (1), 2012 107 foliar epidermis in quercus fig. 5. sem micrographs of midrib in quercus sect. quercus: q. macranthera (a), q. petraea subsp. iberica (b), q. komarovii (c), q. longipes (d), q. robur subsp. pedunculiflora (e), q. cedrorum (f), q. infectoria subsp. boissieri var. boissieri (g), q. infectoria subsp. boissieri var. tenuicarpa (h), q. infectoria subsp. boissieri var. pfaeffingeri (i), scale bar denotes 300 µm (b, c, f) and 150 µm (a, d–e, g–i). u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees quercus longipes steven deciduous large tree, 15–20 m high, with spreading crown, gray-brown branches and large leathery dark-green leaves. the abaxial surface has high trichome density. different types of trichomes are found on abaxial surface. stellate with 2–8 (mostly 4) symmetric short rays (35–70 ìm), appressed to the surface of the leaf are the most frequent type. simple-uniseriate hairs are unicellular or multicellular (fig. 1d). a few bulbous trichomes have also been seen (fig. 1e). there were no trichomes on the adaxial surface and on the midrib, but long solitary trichomes (more than 300 ìm) are distributed sparsely on the midrib edges (fig. 5d). the wax type on the abaxial surface is that of irregular platelets, very densely and evenly distributed over the epidermal surface (fig. 2d). the stomata are elliptical and raised; the rim is completely covered with wax so that only the pore is visible (fig. 3d). quercus robur l. subsp. pedunculiflora (k. koch) menitsky deciduous large tree, up to 18 m high, with spreading crown, thick and cracked bark. leaves are smooth, with short petioles. on abaxial surface, few stellate trichomes with symmetric 2–4 rays measuring 60–130 ìm, sparsely spread on the surface of the leaf intermixed with a few short solitary hairs (80–115 ìm) were observed (fig. 1f). a few solitary trichomes are present on midrib (fig. 5e). the waxes are of smooth layer type (fig. 2e). the stomata are elliptical and completely raised. the guard cells of stomata are covered with smooth layer waxes (fig. 3e). quercus cedrorum kotschy deciduous small tree, 6–9 m high, with spreading crown and long branches. a few sparsely distributed multicellular simple-uniseriate, solitary and stellate trichomes were observed only on the abaxial surface. the stellate trichomes have 2–4 (often 4) rays measuring 80–160 ìm, fairly asymmetric and completely spread on the surface of the leaf. the solitary hairs are 135–170 ìm long (fig. 1g). no trichomes have been observed on the midrib, but there are some solitary and stellate trichomes on the midrib edges (fig. 5f). crystalloid waxes in the shape of membraneous platelets cover the abaxial surface (fig. 2f). the stomata are markedly elliptical and raised; the rim is visible and without wax (fig. 3f). quercus infectoria oliv. subsp. boissieri (reut.) o. schwarz var. boissieri deciduous small tree, 6–9 m high, with spreading crown, gray, cracked bark and leathery leaves. two types of trichome were observed on the abaxial surface: sparse trichomes as stellate with 2–5 rays (90–210 ìm) and a few multicellular simple-uniseriate trichomes (fig. 1h). long solitary (270–350 ìm) and fasciculate trichomes with 2–4 erect long rays (215–400 ìm) were observed on the midrib (fig. 5g). the irregular platelets waxes are evenly but irregularly distributed on the abaxial surface (fig. 2g). the stomata are elliptical and raised; the rim is completely covered with wax but the pore is visible (fig. 3g). quercus infectoria oliv. subsp. boissieri (reut.) o.schwarz var. tenuicarpa (djav.khoie) jamzad et panahi deciduous small tree, up to 10 m high, with spreading crown, gray, cracked bark. leaves are oblong, indistinctly lobed. the trichomes are observed only on the abaxial surface; simple-uniseriate trichomes are mainly distributed on the surface but a few stellate trichomes with 4–8 short rays (85–150 ìm) are also observed (fig. 1i). there are no tri108 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees chomes on the midrib, but a few solitary and fasciculate trichomes have been observed on the midrib edges (fig. 5h). the waxes are of the irregular platelet type, platelets are large and evenly distributed (fig. 2h). the stomata are raised and rounded, the rim is entirely covered with wax, pore is invisible (fig. 3h). quercus infectoria oliv. subsp. boissieri (reut.) o. schwarz var. pfaeffingeri (kotschy ex tchihatcheff) jamzad et panahi deciduous small tree, up to 10 m high, with spreading crown, narrow and slightly hairy branches turning glabrous in mature specimens. leaves are elliptical, entire, with obtuse apex. a few sparse stellate trichomes with 4–8 rays (110–190 ìm), mostly completely twisted occur on both abaxial (fig. 1j) and adaxial surfaces (fig. 1k). solitary and fasciculate trichomes with 2–4 long erect rays (150–270 ìm) are observed upon the midrib (fig. 5i). on the midrib, a higher trichome density at the edges is observed. irregular platelets waxes are irregularly distributed on the abaxial surface (fig. 2i). the waxes have been eroded in some spaces especially around the stomata. the stomata are roundish and raised; the rim is uncovered (fig. 3i). discussion our observations confirm the taxonomical usefulness of micromorphological features of the abaxial and adaxial leaf surfaces within the studied taxa. a very clear differentiation was observed for the types and number of trichome present on both abaxial and adaxial surfaces, types of epicuticular wax, shape and density of stomata and their position with respect to the leaf surface. regarding morphological characters and controversial nomenclature used by different authors, three species groups can be recognized in the examined taxa. the first group is formed by quercus komarovii, q. petraea subsp. iberica and q. macranthera. quercus komarovii was recorded from iran by djavanchir (1967). he reported it in the list of lobed-leaf oak species of iran, distributed in arasbaran forests, located in northwest iran, whereas menitsky (1971) did not report it from iran. from an ecological point of view, q. komarovii is only present in populations mixed with q. macranthera in upland forests of arasbaran, over a range of altitudes between 1700 m to 2200 m. quercus petraea subsp. iberica grows throughout hyrcanian forests, located in the north of iran. it mixes with q. castaneifolia in lower altitudes and with q. macranthera in upper altitudes. however, the distribution pattern of this subspecies extends to arasbaran in nw iran where it appears in elevations lower than 1700 m but separate from q. komarovii. besides, it mixes with q. macranthera in some parts of the upper boundaries of its distribution area. the form of this subspecies changes to shrub in arasbaran because of human activities and ecological factors. quercus komarovii and q. petraea subsp. iberica exhibit some morphological similarities, but differences in leaf shape, color of branches and shape of cup scales are recognized. the tree form is also different by its presenting a low-growing spreading crown. these similarities may have been the reason for identifying all iranian specimens as q. petraea subsp. iberica by some authors. we did not have access to q. komarovii specimens from the caucasus to compare its epidermal features with the iranian specimens and with q. petraea subsp iberica, but with the data available we considered the specimens from high altitude of arasbaran forest to be q. komarovii. in our studies we recognized some acta bot. croat. 71 (1), 2012 109 foliar epidermis in quercus u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees epidermal features such as the number of stellate trichomes, trichome type and wax distribution on abaxial surface and around the stomata as diagnostic characters to define these species. also both q. komarovii and q. petraea subsp. iberica have 2–4 rayed trichomes with more or less the same size range but in the former the middle rays of trichomes are erect and the others are appressed on lamina, while in the later species all trichome rays are spread regularly on lamina. quercus macranthera completely differs from the other species in this group because of presence of trichomes on both abaxial and adaxial surfaces and denser fasciculate trichomes on abaxial surface. the second group is formed by quercus longipes and q. robur subsp. pedunculiflora, which are more or less similar regarding to their morphological characteristics. quercus longipes is distributed in small stands in piranshahr and khoy (west azerbaijan) and osku (east azerbaijan), located in the west of iran. individuals of q. robur subsp. pedunculiflora grow in osku and pessan valley of urmia, between baneh and marivan (kurdistan) and between khoy and tabriz. in general, this species has a wider distribution and the individual tree specimens are seen in the area of its distribution, but q. longipes appears in small size populations. our observations showed that these taxa have distinct differences regarding to some aspects of epidermal structures. quercus longipes has densely stellate trichomes with 2–8 short rays, while q. robur subsp. pedunculiflora has sparse stellate trichomes with 2–4 longer rays. the presence of a few bulbous trichomes in q. longipes is another diagnostic character which can be used for determination of this species. furthermore, types of epicuticular waxes completely differ in these taxa. quercus longipes has crystalloids type of wax, whereas q. robur subsp. pedunculiflora has smooth layer type of wax. last group is formed by quercus infectoria subsp. boissieri, q. infectoria subsp. boissieri var. tenuicarpa, q. infectoria subsp. boissieri var. pfaeffingeri and q. cedrorum. quercus infectoria subsp. boissieri has wider distribution area than the others in northern zagros forests. it is the main taxon of zagros forests. it is widely distributed in west azerbaijan, kurdistan, kermanshah and luristan provinces. small stands of specimens named quercus infectoria var. tenuicarpa djavanchir and q. pfaeffingeri kotschy are only distributed in baneh, kurdistan province, mixed with q. infectoria subsp. boissieri. quercus cedrorum is only found in a small forest area in west azerbaijan, and was reported for the first time from zagros forests of iran (djavanchir khoie 1967). this species is easily distinguished from the rest by having leaves with deep lobes, membraneous platelet waxes, markedly elliptical stomata and two types of trichomes: solitary and stellate. fasciculate trichomes are absent in this species. quercus infectoria var. tenuicarpa is different from q. infectoria subsp. boissieri var. boissieri by having 4–8 rayed stellate trichomes, rays shorter than the typical variety, and rounded stomata completely covered by wax, so that the pore is invisible. within this group, special attention should be devoted to q. pfaeffingeri, which was recognized as a synonym of q. infectoria subsp. boissieri (menitsky 1971). in q. pfaeffingeri, almost all leaves are entire in an individual tree (about 5–6% with indistinctly crenate to more or less undulate margins), but in q. infectoria subsp. boissieri, heterophyly can be observed and only a few entire leaves are present in an individual tree. in addition to morphological differences, our study on epidermal structures showed distinct and obvious differences among these taxa. the main epidermal characteristics of 110 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees quercus pfaeffingeri are the presence of twisted stellate trichomes on the adaxial surface and roundish stomata with the rim not covered by wax. considering the morphological characters such as entire leaves, trichome types and stomata shape and distribution pattern we recognize it as a variety of q. infectoria subsp. boissieri as stated below: quercus infectoria oliv. subsp. boissieri (reut.) o.schwarz var. pfaeffingeri (kotschy ex tchihatcheff) jamzad et panahi comb nov. (syn.: q. pfaeffingeri kotschy, op. cit. t. 23 (1860); q. infectoria oliv. var. pfaeffingeri kotschy ex tchihatcheff, etude veget. hautes montagnes asia mineure et arménia, 17, 1857). quercus infectoria oliv. var. tenuicarpa as a variety of subsp. boissieri as stated below: q. infectoria oliv. subsp. boissieri (reut.) o.schwarz var. tenuicarpa (djav.-khoie) jamzad et panahi comb. nov. (syn.: q. infectoria oliv. subsp. boissieri (reut.) o. schwarz, feddes repert. 33: 336 (1934); q. infectoria oliv. var. tenuicarpa djav.-khoie, chenes iran, 172, 1967). the results of multivariate analysis show the different positions of all examined taxa which is congruent with recognizing nine distinct taxa in quercus sect. quercus in iran. in conclusion, epidermal features are useful taxonomic characters that can help to delimit different taxa in lobed-leaf oaks of iran. based on foliar epidermal characters an identification key of species of lobed-leaf oaks of iran has been prepared (tab. 6). acta bot. croat. 71 (1), 2012 111 foliar epidermis in quercus tab. 6. key to the species of quercus sect. quercus based on leaf epidermis features 1. trichomes present on both abaxial and adaxial surfaces, adaxial surface with a few solitary and 2 – rayed fasciculate trichomes; abaxial surface with densely 2 – 8 long rays (190 – 455 ìm long) fasciculate trichomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q. macranthera – trichomes present only on abaxial surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. abaxial surface with dense and various types of trichomes: 2 – 8 (often 4) symmetric short rays (35 – 70 ìm long) stellate, simple-uniseriate and bulbous. the stomata rim completely covered with wax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q. longipes – abaxial surface with a few sparse trichomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. abaxial surface with smooth layers type waxes . . . . . . . . . . . . . . q. robur subsp. pedunculiflora – abaxial surface with platelets and plates type waxes, membraneous platelets or irregular platelets . 4 4. membraneous platelets waxes with very small scales . . . . . . . . . . . . . . . . . . . . . . . . q. cedrorum – irregular platelets waxes with large scales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5. stomata rim completely covered by wax, stellate trichomes with 2 – 5 rays (90–210 ìm long), . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q. infectoria subsp. boissieri var. boissieri – stomata rim uncovered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6. abaxial leaf surface with multicellular simple-uniseriate and 2 – 4 (often 4) symmetric short rayed (75 – 120 ìm) stellate trichomes, spread on regularly on the lamina, simple uniseriate trichomes are more abundant than stellate trichomes. waxes of irregular lax platelets type with empty spaces between platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q. petraea subsp. iberica – abaxial leaf surface with completely symmetric 2 – 4 (often 4) short rays (60 – 95 ìm) stellate trichomes, the middle rays are erect and others appressed, roughly parallel to the epidermis. a few solitary trichomes present. stellate trichomes are more frequent than simple uniseriate trichomes. waxes of irregular dense platelets type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q. komarovii u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees acknowledgements we would like to thank the authorities of herbarium of research institute of forests and rangelands and herbarium of natural recourses faculty, tehran university for permitting to use the herbarium specimens. thanks are due to mr. m. saghafi for his help in preparing sem micrographs. references ba^i], t., 1981: investigations of stomata of three oak species with light and scanning electron microscope. acta botanica croatia 40, 85–90. barthlott, w., neinhuis, c., cutler, d., ditsch, f., meusel, i., theisen, i., wilhelmi, h., 1998: classification and terminology of plant epicuticular waxes. botanical journal of the linnean society 126, 237–260. bruschi, p., venderamin, g. g., bussotti, f., grossoni, p., 2000: morphological and molecular differentiation between q. petraea (matt.) liebl. and q. pubescens willd. (fagaceae) in northern and central italy. annals of botany 85, 325–333. bussotti, f., grossoni, p., 1997: european and mediterranean oaks (quercus l.; fagaceae): sem characterization of the micromorphology of the abaxial leaf surface. botanical journal of the linnean society 124, 183–199. camus, a., 1936–1954: les chênes. monographie du genre quercus, 3. paul lechevalier, paris. de bary, a., 1871: ueber die wachsüberzüge der epidermis. oesterreichische botanische zeitschrift 29, 128–139, 145–154, 161–176, 566–571, 573–585, 605–619. djavanchir khoie, k., 1967: les chênes de l'iran. phd thesis, universite de montpellier. dyal, s. c., 1936: a key to the species of oaks of eastern north america based on foliage and twig characters. rhodora 38, 53–63. fortini, p., viscosi, v., maiuro, l., fineschi, s., vendramin, g. g., 2009: comparative leaf surface morphology and molecular data of five oaks of the subgenus quercus oerst (fagaceae). plant biosystems 143, 543–554. hardin, j.w., 1976: terminology and classification of quercus trichomes. journal of elisha mitchell science society 92, 151–161. hardin, j.w., 1979a: atlas of foliar surface features in woody plants, i. vestiture and trichome types of eastern north american quercus. bulletin of torrey botanical club 106, 313–325. hardin, j. w., 1979b: patterns of variation in foliar trichomes of eastern north american quercus. american journal of botany 66, 576–585. jones, j. h., 1986: evolution of the fagaceae: the implications of foliar features. annals of missouri botanical garden 73, 228–275. llamas, f., perez-morales, c., acedo, c., penas, a., 1995: foliar trichomes of the evergreen and semi-deciduous species of the genus quercus (fagaceae) in the iberian peninsula. botanical journal of linnean society 117, 47–57. 112 acta bot. croat. 71 (1), 2012 panahi p., jamzad z., pourmajidian m. r., fallah a., pourhashemi m. u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees lutz, a., 1938: beiträge zur pharmakognosie der salicales juglandales and fagales. anatomie des laubblattes. phd thesis, universitäte basel. manos, p. s., 1993: foliar trichome variation in quercus section protobalanus (fagaceae). sida 15, 391–403. menitsky, g. l. 1971: fagaceae. in: rechinger, k. h. (ed.), flora iranica 77, 1–20. akademische drukund verlagsanstalt, graz. nikoli], n. p., merkulov, l. s., krsti], b. d., orlovi], s. s., 2003. a comparative analysis of stomata and leaf trichome characteristics in quercus robur l. genotypes. proceedings for natural sciences, matica srpska, novi sad, 105, 51–59. pénas merino, a., llamas, f., perez morales, c., acedo, c., 1994: aportaciones al conocimiento del genero quercus en la cordillera cantabrica. i, tricomas foliares de las especies caducifolias. lagascalia 17, 311–324. safou, o., saint-martin, m., 1989: leaf trichomes of some perimediterranean quercus species. bulletin de la société botanique de. france 136, 291–304. scareli-santos, c., herrera-arroyo, m. l., sanchez-mondragon, m. l., gonzalez-rodriguez, a., bacon, j., oyama, k., 2007: comparative analysis of micromorphological characters in two distantly related mexican oaks, quercus conzattii and q. eduardii (fagaceae) and their hybrids. brittonia 59, 37–48. thamson, p. m., mohlenbrock, r. h., 1979: foliar trichomes of quercus subgenus quercus in the eastern united states. journal of the arnold arboretum 60, 350–366. uzunova, k., palamarev, e., 1985: the foliar epidermis studies of fagaceae dumort. from the balkan peninsula. ii. quercus [subgenera sclerophyllodrys schwarz and cerris (spach) oersted] (in bulgarian). fitologia 29, 3–20. uzunova, k., palamarev, e., 1992a: study of the leaf epidermis of the balkan representatives of the fagaceae dumort. iii. quercus l. [subgenus quercus, sect. roburoides (schwarz) schwarz and dascia kotschy]. fitologia 42, 22–48. uzunova, k., palamarev, e., 1992b: the foliar epidermis studies of fagaceae dumort. from the balkan peninsula. iv. quercus l. (subgenus quercus, sect. robur reichend.). fitologia 43, 3–30. uzunova, k., palamarev, e., 1993: an investigation of the leaf epidermis of the european (non-balkan) species of the genus quercus. fitologia 45, 3–15. uzunova, k., palamarev, e., ehrendorfer, f., 1997: anatomical changes and evolutionary trends in the foliar epidermis of extant and fossil euro-mediterranean oaks (fagaceae). plant systematics and evolution 204, 141–159. acta bot. croat. 71 (1), 2012 113 foliar epidermis in quercus u:\acta botanica\acta-botan 1-12\434 panahi.vp 26. o ujak 2012 11:15:26 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (1), 65–80, 2011 coden: abcra 25 issn 0365–0588 mericarp micromorphology and anatomy of salvia hedgeana dönmez, s. huberi hedge and s. rosifolia sm. (section salvia hedge, lamiaceae) hatýce n. büyükkartal1, ahmet kahraman2,*, hatýce çölgeçen3, musa doðan2, ersýn karabacak4 1 ankara university, department of biology, 06100 ankara, turkey. 2 middle east technical university, department of biological sciences, 06531 çankaya, ankara, turkey. 3 zonguldak karaelmas university, department of biology, 67100 zonguldak, turkey. 4 çanakkale onsekiz mart university, department of biology, 17020 çanakkale, turkey abstract – mericarp (nutlet) micromorphology and pericarp structure of three morphologically similar endemic salvia species; salvia hedgeana, s. huberi and s. rosifolia were investigated using lm, sem and tem. salvia hedgeana has larger mericarps and abscission scars than s. huberi and s. rosifolia. mericarp length to width ratio ranges from 1.11 in s. hedgeana to 1.60 in s. huberi. mericarp shape is mainly ovoid, rarely broadly ovoid in s. hedgeana, and oblong in s. huberi. the mericarp surface sculpturing pattern in all species is colliculate. however, exocarp cells are pentangular-hexangular in s. hedgeana, irregular in s. huberi and rounded and smaller in s. rosifolia. in salvia huberi anticlinal walls are undulate whereas in s. hedgeana and s. rosifolia anticlinal walls are straight. salvia hedgeana was distinguished from the others by the thickest pericarp (146–185 µm). the sclerenchymatous region significantly varied between the species. it was 84–99 µm in s. hedgeana, 56–82 µm in s. huberi and 27–61 µm in s. rosifolia. the mesocarp was also thicker in s. hedgeana. the wetted mericarps produced mucilage, but s. huberi differed from the others in having translucent-milky white opaque mucilage with fibres or radiating cordons. keywords: mericarp, micromorphology, anatomy, salvia hedgeana, salvia huberi, salvia rosifolia, lamiacaeae, sem, tem acta bot. croat. 70 (1), 2011 65 * corresponding author: e-mail: ahmetk@metu.edu.tr copyright ® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:15 color profile: disabled composite 150 lpi at 45 degrees introduction lamiaceae (the mint family) has a cosmopolitan distribution represented by nearly 7200 species belonging to 236 genera, including many well-known plants, herbs, shrubs and trees of horticultural, economic and medicinal significance (harley et al. 2004). it is the third largest family in turkey with 45 genera and 574 species, 256 of which are endemic. the rate of endemism is 44.5% in this family (davis 1965–1985, davis et al. 1988, güner et al. 2000). some mericarp (nutlet) characters can be used successfully at many taxonomic levels, depending on the characters chosen and the variation present (guerin 2005). studies on mericarp micromorphology and pericarp anatomy in the family have proved the usefulness of these characters for species and generic level in the family lamiaceae (wagner 1914; isley 1947; wojciechowska 1958, 1961, 1966, 1972; hedge and lamond 1968; husain et al. 1990; rejdali 1990; ryding 1992, 1995, 2001, 2010; marin et al. 1994; turner and delprete 1996; budantsev and lobova 1997; zhou et al. 1997; duleti]-lauševic and marin 1999; mosquero et al. 2002; guerin 2005; moon and hong 2006; kaya and dýrmencý 2008; salmaki et al. 2008). isley (1947) distinguished the tribes ajugaea, scutellarieae and stachyeae in usa based on mericarp characters. wojciechowska (1966) investigated mericarp morphology including in some genera of lamiaceae and pointed out that there were often important differences at both generic and species level. ryding (1995) showed that the groups of prunella and cleonia plus lepechinia and chaunostoma were distinguishable from other labiates by differences in pericarp structure. budantsev and lobova (1997) divided the subtribe nepetinae (as tribe nepeteae) into three informal generic groups based on differences in pericarp structure and other characters. wojciechowska (1966) and moon and hong (2006) pointed out that the genus lycopus had a pericarp anatomy and mericarp morphology unique in the shape of corky crests and corky ring and the distribution of glandular trichomes, which were well distinguishable from the other genera in the tribe mentheae. moon and hong (2006) also found that mericarp morphological and anatomical characters of lycopus were useful as diagnostic characters at the specific and interspecific levels. salvia l., the largest genus of the family lamiaceae, is represented by about 1000 species. it is distributed extensively in tropical and temperate areas of the old and new world, with three distinct regions of population: central and south america (500 spp.), western asia (200 spp.) and eastern asia (100 spp.) (walker and sytsma 2007). based on the most recent classification (harley et al. 2004), lamiaceae is divided into seven subfamilies and the large subfamily nepetoideae is divided into the three tribes elsholtzieae, mentheae and ocimeae. mentheae is divided into the three subtribes salviinae, menthinae and nepetinae. however, the genera melissa and heterolamium are unclassified at this rank. according to their classification, the genus salvia is classified into the subtribe salviinae. salvia species possess only two thecae on each stamen separated by an elongate connective while most mentheae have four stamens (walker et al. 2004). mericarp micromorphology and anatomy are poorly reported in salvia. in twenty salvia species from afghanistan, hedge (1970) reported that characters of myxocarpy (i.e. the production of mucilage when mericarps get in contact with water), thickness of the pericarp and the proportions of its individual layers as well as morphology of mericarps 66 acta bot. croat. 70 (1), 2011 büyükkartal h. n., kahraman a., çölgeçen h., doðan m., karabacak e. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:15 color profile: disabled composite 150 lpi at 45 degrees could be used in the systematics of the genus. mericarp micromorphological characters of different salvia species was investigated by marin et al. (1996) and oran (1997). they both pointed out that the variation in thickness of exocarp (=epicarp), mesocarp and endocarp morphology may be useful additional taxonomical markers. in turkey, there are 98 salvia species reported (hedge (1982; kahraman et al. in press), 52 endemic species (53%), divided into seven sections salvia hedge (syn. eusphace benth.), aethiopis benth., plethiosphace benth., drymosphace benth., horminum benth. and hemisphace benth. (boissier 1879, doðan et al. 2007). leaf (pinnatisect, trisect or simple), calyx (membranous or thick textured, upper lip 2-sulcate or not, concave or not), corolla (upper lip falcate or not, tube squamulate or not, annulate or not) and stamen (type a, b or c) characteristics are the most important means for distinguishing these sections. the section salvia has usually pinnatisect leaves, thick-texture calyces, straight upper corolla lips, annulate corolla tubes and type a stamens with shorter staminal connectives than filaments and larger upper theca than the lower theca. salvia hedgeana dönmez, s. huberi hedge and s. rosifolia sm. included in the section salvia are endemic to turkey. they show morphologically close similarities to each other (dönmez 2001), especially s. huberi and s. rosifolia (hedge 1982, personal observation). salvia hedgeana is also close to s. caespitosa montbret et aucher ex benth. in terms of its habit and distribution area. even though micromorphological and anatomical studies can be used as a powerful tool in delimitation of species, there have been no mericarp micromorphological and anatomical studies on the species examined. this study aims to examine micromorphological and anatomical characteristics of mericarps of the three salvia species which have not been studied to date; using light microscopy, scanning electron microscopy and transmission electron microscopy. materials and methods different populations of salvia, salvia hedgeana dönmez, s. huberi hedge and s. rosifolia sm. were investigated in localities presented in table 1. the herbarium specimens were deposited in department of biological sciences, middle east technical university, ankara, turkey and the herbarium of çanakkale onsekiz mart university. light microscopy (lm) mericarps were first examined using a stereomicroscope to ensure that they were of normal size and maturity. the ripe mericarps from each population were measured in order to determine the average mericarp size and abscission scar diameter. the pericarp structure of the mericarps was investigated from softened herbarium specimens. the mature mericarps were placed in distilled water for 24 hours, 3% glutaraldehyde and then %1 osmium tetraoxide. the materials were dehydrated through a graduated ethanol series, embedded in epon 812 (luft 1961), sectioned, stained with methylene blue and toluidine blue, and permanently mounted. these sections were examined for the pericarp features, and thickness of the pericarp and mesocarp, sclerenchymatous region, endocarp, size of crystals and mucilaginous cells were measured and photographed under lm. mericarp micromorphology and anatomy of salvia hedgeana acta bot. croat. 70 (1), 2011 67 u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:15 color profile: disabled composite 150 lpi at 45 degrees scanning electron microscopy (sem) mature mericarps were directly placed on aluminium stubs using double-sided adhesive tape, sputter coated with gold using a hummer vii gold-coating apparatus, then viewed with jeol-6060 sem and photographed. transmission electron microscopy (tem) ultrathin sections of the mericarps were stained with uranyl acetate (stempak and ward 1964) and lead citrate (sato 1967), then examined with jeoll cx-100 tem and photographed. each quantitative character was analyzed for its mean and median values, range, standard deviation and significance, using the statistica version 9 software. significance of differences was tested using one-way anova and tukey’s honestly significant difference 68 acta bot. croat. 70 (1), 2011 büyükkartal h. n., kahraman a., çölgeçen h., doðan m., karabacak e. tab. 1. list of the studied populations of salvia hedgeana, s. huberi and s. rosifolia. species collection data collection number s. hedgeana b7 sivas: divriði, karasar pass, uzunkaya to kayaburun, 1450 m, steppe, 25.vii.2008 a. kahraman 1589ca,b b7 sivas: divriði, karasar pass, near uzunkaya village, 1460 m, steppe, 9.vii.2006 a. kahraman 1255aa,c s. huberi a8 bayburt: 12 km from bayburt to maden, 1615 m, steppe and slopes, 12.vii.2006 e. karabacak 4979a a8 erzurum: 41 km from erzurum to tortum, 2121 m, roadside slopes, 14.vii.2006 a. kahraman 1474a,b,c a8 erzurum: tortum waterfall, 1010 m, scrubs, 22.vi.2008 e. karabacak 6137a a8 erzurum: tortum, above balikli village, 1342 m, stony slopes, 22.vi.2008 e. karabacak 6144a a8 erzurum: 10 km from tortum to yusufeli, 1367 m, calcareous slopes and screes, 9.vii.2007 e. karabacak 5611a s. rosifolia a8 bayburt: gümüþhane to bayburt, niþantaþý village, osluk (korgan) bridge environs, 1616 m, steppe and rocky slopes, 11.vii.2006 e. karabacak 4963a a9 kars: 22 km from kaðýzman to kars, 1581 m, roadside slopes, 13.vii.2007 a. kahraman 1470a,c a9 kars: 28 km from kaðýzman to kars, 1819 m, roadside slopes, 29.vii.2008 a. kahraman 1607a,b a9 kars: kaðýzman to kars, near kötek village, 1642 m, screes, 8.vii.2007 e. karabacak 5585a a9 erzurum: oltu, çamlýdere, 1443 m, slopes, 22.vi.2008 e. karabacak 6161a aspecimens used for morphological studies of mericarps by stereomicroscopy; bspecimens used for micromorphological studies of mericarps by sem; cspecimens used for anatomical studies of mericarps by lm and tem. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:15 color profile: disabled composite 150 lpi at 45 degrees (hsd) test. the terminology was adjusted for mericarp surface sculpturing (stearn 1992, salmaki et al. 2008) and for pericarp structure (hedge 1970, ryding 2010). to determine for the absence or presence of myxocarpy, we treated with distilled water ten mericarps from each species examined, following ryding (1992). mucilage characteristics regarding colour, consistency and general appearance were followed according to hedge (1970). results the significance of differences in basic parameters defines each species (fig. 1, tab. 2). three characters such as mericarp width, pericarp thickness and sclerenchymatous region thickness allow clear discrimination of the three species. mericarp length/width ratio helps to separate salvia huberi from s. hedgeana and s. rosifolia whereas size of exocarp cells distinguishes between s. rosifolia and s. hedgeana and s. huberi. mericarp length, abscission scars and mesocarp thickness contribute the distinction between s. hedgeana and the remaining taxa. nonetheless, some of these characters indicate to some degree overlapping values. among the studied qualitative characters of the species, shape of mericarp and exocarp cells and structure of anticlinal walls were found to be the most significant diagnostic characters (tab. 3). mericarp micromorphology size of mericarps measured from different populations of the species varied from 2.46 mm (salvia huberi) to 4.36 mm (s. hedgeana) in length and 1.83 mm (s. huberi) to 3.44 (s. hedgeana) in width. shape of the mericarps was mainly ovoid, rarely broadly ovoid as in s. hedgeana or oblong as in s. huberi (fig. 2). they ranged in length to width ratio from 1.11 (s. hedgeana) to 1.60 (s. huberi). when mericarps were not fully matured, their colour was greenish at first, and then turned to dark brown or blackish. transverse sections of the mericarps were also rounded-trigonous. abscission scars were almost spherical and their diameter ranged between 0.41 mm as in s. huberi and 0.83 mm as in s. hedgeana. in the species examined, the mericarps were glabrous and the sculpturing pattern was of the colliculate type characterized by small hill-like eminences, spaced, covering throughout the mericarp surface. the colliculate sculpturing could be subdivided based on shape of exocarp cells: pentangular or hexangular in s. hedgeana (fig. 3a), irregular in s. huberi (figs. 3c-d) and rounded in s. rosifolia (figs. 3e-f). size of these cells varied from 7.45 µm as in s. rosifolia to 27.79 µm as in s. huberi. striation partially occurred on their surface (fig. 3b). anticlinal walls were represented by straight channels in s. hedgeana and s. rosifolia or undulate channels in s. huberi. outer periclinal walls were convex, but also consisted of small holes in s. huberi (figs. 3c-d). mericarp anatomy the lm and tem investigation revealed a pericarp differentiated into four main regions: the exocarp (outer epidermis), mesocarp, sclerenchyma region and endocarp (inner epidermis) (figs. 4a, c, f). the outermost region was the exocarp, consisting of a single layer of parenchymatous cells. these cells were differentiated into two types: large oblong or oval mucilaginous cells in groups of one or more and narrow non-mucilaginous cells acta bot. croat. 70 (1), 2011 69 mericarp micromorphology and anatomy of salvia hedgeana u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:15 color profile: disabled composite 150 lpi at 45 degrees 70 acta bot. croat. 70 (1), 2011 büyükkartal h. n., kahraman a., çölgeçen h., doðan m., karabacak e. fig. 1. box plots of the main discriminant quantitative characters in salvia species examined. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:15 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .70 (1),2011 71 m e r ic a r p m ic r o m o r p h o l o g y a n d a n a t o m y o f s a l v ia h e d g e a n a tab. 2. overview of quantitative characteristics of the species accessions examined. numbers refer to means ± standard deviations [medians] and (ranges). n = 50, *p < 0.001, ns = not significant. character s. hedgeana s. huberi s. rosifolia p tukey’s hsd test mericarp length (mm) 3.66 ± 0.37 [3.70] (3.12–4.36) 2.90 ± 0.27 [2.86] (2.46–3.54) 2.96 ± 0.26 [2.90] (2.56–3.63) * s. hedgeana-*-s. huberi, s. rosifolia mericarp width (mm) 2.88 ± 0.33 [2.78] (2.26–3.44) 2.17 ± 0.20 [2.13] (1.83–2.86) 2.36 ± 0.23 [2.34] (1.91–2.87) * s. hedgeana-*-s. huberi, s. rosifolia; s. huberi-*-s. rosifolia mericarp length/width ratio 1.28 ± 0.06 [1.28] (1.11–1.38) 1.34 ± 0.10 [1.32] (1.20–1.60) 1.25 ± 0.05 [1.25] (1.17–1.36) * s. huberi-*-s. hedgeana, s. rosifolia abscission scar diameter (mm) 0.69 ± 0.08 [0.71] (0.55–0.83) 0.56 ± 0.08 [0.55] (0.41–0.75) 0.60 ± 0.09 [0.60] (0.46–0.79) * s. hedgeana-*-s. huberi, s. rosifolia size of exocarp cells (µm) 15.88 ± 3.84 [15.06] (10.06–24.04) 17.49 ± 4.00 [17.26] (11.38–27.79) 11.66 ± 2.11 [11.39] (7.45–16.43) * s. rosifolia-*s. hedgeana, s. huberi pericarp thickness (µm) 169.39 ± 10.23 [170.00] (145.53–185.30) 129.30 ± 12.01 [130.65] (100.83–151.90) 114.87 ± 13.83 [122.35] (89.79–133.22) * s. hedgeana-*-s. huberi, s. rosifolia; s. huberi-*-s. rosifolia mesocarp thickness (µm) 64.26 ± 6.31 [62.73] (55.64–75.91) 47.17 ± 8.53 [45.44] (34.58–61.91) 45.06 ± 7.33 [47.08] (31.18–56.20) * s. hedgeana-*-s. huberi, s. rosifolia crystal length/width ratio 0.50 ± 0.09 [0.48] (0.33–0.65) 0.47 ± 0.10 [0.51] (0.30–0.60) 0.44 ± 0.06 [0.43] (0.35–0.55) ns sclerenchymatous region thickness (µm) 91.01 ± 3.77 [91.30] (84.00–98.90) 69.42 ± 8.05 [70.25] (55.50–82.34) 46.38 ± 11.27 [47.15] (26.50–60.90) * s. hedgeana-*-s. huberi, s. rosifolia; s. huberi-*-s. rosifolia endocarp thickness (µm) 7.16 ± 1.93 [6.80] (4.50–10.98) 6.82 ± 1.60 [6.77] (4.08–10.62) 7.43 ± 1.25 [7.18] (5.13–10.56) ns u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 1 \ b u y u k k a r t a l . v p 2 8 . o u j a k 2 0 1 1 1 6 : 0 7 : 1 5 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s with thin cavities, located between the former cells (figs. 4a, c, d, f, g). the mucilaginous cells had thickened walls and their size was very variable within the same mericarp. hence, we measured the pericarp thickness without the mucilaginous cells. it ranged from 89.79 µm in s. rosifolia to 185.30 µm in s. hedgeana. the mucilage pocket encircled the central cytoplasmic column with darkly staining mucilage (figs. 5c, f). below the exocarp, there is the mesocarp region (31.18–75.91 µm thick) composed of a dark amorphous mass of several strongly compressed and almost indistinguishable cells. this region contained several groups of sclerenchymatic cells with the luminar cavity enlarged (figs. 4b, d). brachysclereids (=stone cells) were characterized by thick walls (figs. 4b, 5a, c, e). these cells accumulated tannins. the innermost layer of the mesocarp contained prismatic crystals (figs. 4a, c, e, f). they were 6.67–13.22 long µm and 15.14–32.52 µm wide. they varied in length to width ratio between 0.30 and 0.65. the mesocarp was followed by the sclerenchymatous region consisting of a layer of thick-walled macrosclereids, i.e. malpighian cells (figs. 4e, 5b, d) in which tannins accumulated (fig. 5b). the sclerenchymatous region thickness varied between 26.50 µm in s. rosifolia and 98.90 µm thick in s. hedgeana, with small and rounded luminar cavities at or near the centre (fig. 5). the thickness of the sclerenchymatous layer was nearly half of the total pericarp thickness. 72 acta bot. croat. 70 (1), 2011 büyükkartal h. n., kahraman a., çölgeçen h., doðan m., karabacak e. tab. 3. characteristics in salvia examined. character s. hedgeana s. huberi s. rosifolia mericarp shape ovoid to broadly ovoid ovoid to oblong ovoid mericarp transverse section rounded-trigonous rounded-trigonous rounded-trigonous mericarp colour mostly blackish mostly blackish mostly blackish abscission scar shape ±spherical ±spherical ±spherical surface sculpturing type colliculate colliculate colliculate shape of exocarp cells pentangular-hexangular irregular rounded anticlinal walls represented by straight channels represented by undulate channels represented by straight channels periclinal walls convex convex with small holes convex fig. 2. sem micrographs showing general appearance of mericarps in salvia species examined. a – s. hedgeana, b – s. huberi, c – s. rosifolia. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:16 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (1), 2011 73 mericarp micromorphology and anatomy of salvia hedgeana fig. 3. sem micrographs showing mericarp surface sculpturing in salvia species examined. a-b – s. hedgeana, c-d – s. huberi, e-f – s. rosifolia. fig. 4. lm micrographs showing pericarp structure of salvia hedgeana (a-b), s. huberi (c-e) and s. rosifolia (f-g). bs – brachysclerids, cr – crystals, ex – exocarp, en – endocarp, me – mesocarp, mc – mucilaginous cells, nc – non-mucilaginous cells, s – sclerenhymatic cells, sc – sclerenchymatous region, t – tannins. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:17 color profile: disabled composite 150 lpi at 45 degrees the endocarp, consisting of a single layer of transversely arranged cells, was 4.08– 10.98 µm thick (figs. 4a, c, f). a mucilage reaction (myxocarpy) was seen in the wetted mericarps of the studied species. in s. huberi and s. rosifolia mucilage was formed after the first half hour of wetting, but in s. hedgeana the time taken was longer than one hour. according to colour, consistency and degree of transparency, two mucilage types were observed: transparent and fibreless or having radiating cordons embraced by the mass of mucilage, and translucent-milky white opaque with fibres or radiating cordons embraced by the mass of mucilage. the former type was observed in s. hedgeana (fig. 6a) and s. rosifolia (fig. 6d) whereas the latter type was detected in s. huberi (fig. 6b-c). discussion the size, shape, length to width ratio, abscission scar diameter, size and shape of exocarp cells and structure of anticlinal walls varied significantly in the investigated species (tabs. 2, 3). salvia hedgeana was easily distinguished from s. huberi and s. rosifolia by large mericarps and abscission scars. their mericarps were ovoid, but s. huberi had also oblong mericarps. in addition, the mericarps ranged in their length to width ratio from 1.11 in s. hedgeana to 1.60 s. huberi. hedge (1982) reported that some species of the turkish salvia had brown or black mericarps and rounded, trigonous or rounded-trigonous 74 acta bot. croat. 70 (1), 2011 büyükkartal h. n., kahraman a., çölgeçen h., doðan m., karabacak e. fig. 5. tem micrographs showing cells in pericarps of s. hedgeana with brachysclereids and tannins in the mesocarp (a), s. hedgeana with macrosclereids and tannins in the sclerechymatous region (b), s. huberi with mucilaginous cells in the exocarp and with brachysclereids in the mesocarp (c), s. huberi with macrosclereids in the sclerenchymatous region (d), s. rosifolia with brachysclereids in the mesocarp (e), and s. rosifolia with mucilaginous cells in the exocarp (f). br – brachysclereids, ma – macrosclereids, mu – mucilaginous cells, t – tannins. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:17 color profile: disabled composite 150 lpi at 45 degrees mericarps (the most common) in transverse sections. however, the investigated species had mericarps similar in colour, transverse sections, as well as, abscission scar shape. the abscission scar and mericarp shape were invariable in the tribe saturejeae while these characters varied significantly in the westringieae (husain et al. 1990). the studied species showed a similar sculpturing pattern, but exocarp cells were pentangular-hexangular in s. hedgeana, irregular in s. huberi and rounded and smaller in s. rosifolia. while exocarp cells of s. huberi had undulate anticlinal walls, those of s. hedgeana and s. rosifolia had straight anticlinal walls. small holes were also present on the outer periclinal walls of s. huberi. though mericarp sculpturing was found to be useful for separating species within the sections in the genus stachys, it did not seem to be a helpful character at the infrageneric level (salmaki et al. 2008). mericarp shape, nature of the abscission scar, nature of surface sculpturing, exocarp cell shape and sculpturing, and nature of the indumentum were mainly useful for infrageneric delimitation in the genera hemigenia and microcorys (guerin 2005). according to the mericarp anatomical properties of the species examined, the thickest pericarp was found in s. hedgeana, whereas s. rosifolia had the thinnest pericarp. moreover, the thickness of the sclerenchymatous region significantly varied among the species. duleti]-lauševic and marin (1999) determined that the pericarp thickness in the tribe nepetoideae of lamiaceae was often correlated with dimensions of mericarps. however, they also indicated that the pericarps of mentha aquatica mericarps were very thick (approximately 95 µm), even though the species had very small mericarps (nearly 0.8 mm � 0.6 mm). in our work, s. hedgeana had the largest mericarps that were composed of the thickest pericarp structure. in the mesocarp and sclerenchmatous region, tannins were recognized. they were thought to protect the plant against dehydration, rotting and damage by predators such as animals and insects (fahn 1990). acta bot. croat. 70 (1), 2011 75 mericarp micromorphology and anatomy of salvia hedgeana fig. 6. stereomicroscopic micrographs showing the mucilaginous reaction (myxocarpy) on mericarps in salvia hedgeana (a), s. huberi (b-c), and s. rosifolia (d). u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:18 color profile: disabled composite 150 lpi at 45 degrees mericarp data are not only useful as a diagnostic character in delimitation of the species of salvia, but also they provide support for separating sections of the genus as indicated by some early researchers (wojciechowska 1966, marin et al. 1994, özkan et al. 2009). according to data presented in the flora of turkey (hedge 1982), members of the sections plethiosphace and hemisphace can be distinguished from those of the other sections by their smaller mericarps. mericarp size of the section plethiosphace is 1.5–2.5 � 1.0–2.0 mm and mericarp size of the section hemisphace is 2.2–2.5 � 1.3–1.5 mm. in marin et al. (1996), mericarps of s. officinalis l. (sect. salvia) and s. glutinosa l. (sect. drymosphace) have convex anticlinal walls and concave periclinal walls in the exocarp cells. however, those of s. officinalis exhibit the isodiametric hexagonal or pentagonal exocarp cells while the mericarps of s. glutinosa l. have smaller exocarp cells, which are not clearly angular. moreover, salvia coccinea juss. ex murr. and s. splendens ker-gawl. in subgenus calosphace are particularly distinguished from all other species by regular pentangular or hexangular exocarp cells with convex periclinal walls. sem images of s. coccinea presented by the authors seem to be very similar to those of s. hedgeana in our study. s. ballsiana and s. macrochlamys in the section salvia studied by kahraman et al. (2010a, b) have a surface sculpturing similar to that of s. hedgeana, but s. ballsiana (4.5–5.3 � 3.8–4.2 mm) and s. macrochlamys (4.7–5.2 � 3.8–4.1 mm) have larger mericaps. s. verticillata in the section hemisphace had an irreegular, reticulate surface pattern with exocarp cells of varying size (marin 1996). özkan et al. (2009) reported mericarp properties of 12 turkish salvia taxa belonging to different sections. they grouped the species into three types of sculpturing: foveate, reticulate and verrucate. the three types were observed in the section aethiopis among the examined taxa. while the studied taxa of the sections salvia and hymenosphace had the foveate sculpturing pattern, those of the sections hemisphace and plethiosphace had verrucate sculpturing pattern. in our study, the species belonging to sect. salvia showed the colliculate pattern of mericarp sculpturing. some discrepancies hamper the usefulness of direct comparisons with the findings of this previous study. in some cases, the sem images of several taxa do not seem to correspond with surface sculpturing types assigned to the species. in some cases, the magnification seems to be insufficient to show the detail of the sculpturing patterns of exocarp cells. variation in thickness of the pericarp and its individual layers among species have been already indicated (oran 1997). the pericarp thickness in the investigated species ranged from 48 µm, as in s. viscosa (sect. plethiosphace), to 275.5 µm, as in s. fruticosa (sect. salvia). in our study, the species have pericarps varying between 89.79 and 185.30 µm in thickness.) two-staminate salviinae (e.g. salvia and rosmarinus) could be distinguished from four-staminate salviinae (e.g. lepechinia and chaunostoma) and other mentheae by having large crystals in the innermost cell layer of the mesocarp (ryding 2010). except in a few of the species (e.g. salvia splendens and perovskia abrotanoides), crystals were observed in all the genera. the sclerenchymatous region varied between 32 µm (s. splendens and s. taraxacifolia) and 115 µm (s. officinalis). s. officinalis was also found to differ from the other studied species in having a very much thicker sclerenchymatous region. the salviinae with four stamens differed from the two-staminate (3–9 µm) and other mentheae (2–12 µm) in having a thicker endocarp (17–40 µm). our findings confirmed data presented in the previous study. prismatic crystals were detected in the mesocarp of the species studied. the thickness of the sclerenchymatous region was measured between 26.50 µm in s. rosifolia and 98.90 µm in s. hedgeana. the endocarp thickness ranged from 4.08 µm to 10.98 µm. 76 acta bot. croat. 70 (1), 2011 büyükkartal h. n., kahraman a., çölgeçen h., doðan m., karabacak e. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:18 color profile: disabled composite 150 lpi at 45 degrees myxocarpy, the production of mucilage by wetted mericarps, is widespread in the subfamily nepetoideae (wagner 1914; hedge 1970; swarbrick 1971; witztum 1978; ryding 1992, 2001, 2010; duleti]-lauševic and marin 1999; harley et al. 2004). morphologically allied species had similar mucilage properties (hedge 1970). in this study, we observed that the species did produce mucilage on their mericarp surfaces, but s. huberi seemed to differ from the others in colour, consistency and degree of transparency. in a total of over forty salvia species investigated in southwest asia, only three failed to produce mucilage on the mericarp surface (hedge 1970). all the salvia species known from afghanistan had mucilage formation, with one exception (hedge 1970). these mucilage-producing species were grouped into four basic types according to their mucilage characteristics: transparent, translucent, milky opaque and brownish opaque. the occurrence of mucilage was found in 14 salvia species in jordan which had not been previously examined (oran 1997). however, one species, s. napifolia (sect. hemisphace), only sometimes produced a very small amount or no visible mucilage. the greatest mucilage production was detected in s. viridis (sect. horminum). there was no mucilage on the mericarp surface of all the taxa studied in lycopus, but a moderately strong mucilaginous reaction was seen in elsholtzia blanda benth. in the tribe elsholtzieae (moon and hong 2006). mucilage plays a significant role in anchoring the mericarp to the soil. presence or absence of mucilage seems to be a very homoplastic character in nepetoideae; most of the species-rich genera and many of the genera with few species contained myxocarpic as well as non-myxocarpic species (ryding 1992). therefore, the amount of mucilage may evolve quickly in order to adapt the species to different biological conditions. even though the presence of mucilage may provide a considerable selective advantage under certain conditions, the production of mucilage may be costly for the plants, and it may be quickly lost where it has no function. plants growing in moist habitats more often had non-mucilaginous mericarps (ryding 1992, 2001). the whole of lycopus species grow in low wet places and have non-mucilaginous mericarps (moon and hong 2006). the species of salvia examined grow in dry habitats and have mucilaginous mericarps. mericarp micromorphological and anatomical characteristics might be helpful in the identification of the species studied. nevertheless, the value of these characteristics can be better appreciated by examining other species of salvia. acknowledgements the authors thank the scientific and technical research council of turkey for the financial assistance (tubýtak-tbag-104 t 450) and çanakkale onsekiz mart university research fund (2007/14). references boissier, e. p., 1879: flora orientalis. genevae et basileae. budantsev, a. v., lobova, t. a., 1997: fruit morphology, anatomy and taxonomy of tribe nepeteae (labiatae). edinburgh journal of botany 54, 183–216 acta bot. croat. 70 (1), 2011 77 mericarp micromorphology and anatomy of salvia hedgeana u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:18 color profile: disabled composite 150 lpi at 45 degrees davis, p. h., 1965–1985: flora of turkey and the east aegean islands, 1–9. edinburgh university press, edinburgh. davis, p. h., mill, r. r., tan, k., 1988: flora of turkey and the east aegean islands, 10 (first supplement). edinburg university press, edinburg. doðan, m., akaydin, g., celep, f., bagherpour, s., kahraman, a., karabacak, e., 2007: infrageneric delimitation of salvia l. 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(labiatae). botanical gazette 139, 430–435. wojciechowska, b., 1958: taxonomy, morphology and anatomy of seeds in the genus salvia l. (in polish). monographie botanicae 6, 1–56. wojciechowska, b., 1961: fruits in the middle european species of some genera of stachyoideae (fam. labiatae). monographie botanicae 12, 89–120. wojciechowska, b., 1966: morphology and anatomy of fruits and seeds in the family labiatae with particular respect to medicinal species. monographie botanicae 21, 119–243. wojciechowska, b., 1972: morphology and anatomy of fruits of scutellaria, chaiturus, galeobdolon and sideritis of the family labiatae. monographie botanicae 37, 137–168. zhou, s. l., pan, k. y., hong, d. y., 1997: pollen and nutlet morphology in mosla (labiatae) and their systematic value. israel journal of plant sciences 45, 343–350. 80 acta bot. croat. 70 (1), 2011 büyükkartal h. n., kahraman a., çölgeçen h., doðan m., karabacak e. u:\acta botanica\acta-botan 1-11\buyukkartal.vp 28. o ujak 2011 16:07:19 color profile: disabled composite 150 lpi at 45 degrees 579 iamonico and panitsa.vp acta bot. croat. 72 (1), 193–196, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 short communication lectotypification of the linnaean name bryonia cretica (cucurbitaceae) duilio iamonico1*, maria panitsa2 1 laboratory of phytogeography and applied geobotany, department data, section environment and landscape, university of rome sapienza, via flaminia 72, 00196 rome, italy 2 department of environmental and natural resources management, university of western greece, seferi 2, 30100 agrinio, greece abstract – the typification of the name bryonia cretica is investigated and discussed. a specimen from the clifford herbarium is designated as the lectotype. the morphology of the species, notes on its cytology and geographical distribution and ecological features are also treated. key words: bryonia l., eastern mediterranean, typification introduction bryonia l. (bryonieae dumort., cucurbitales juss. ex brecht et j. presl) is a genus of 10 species distributed in europe, africa and central asia (schaefer and renner 2011). carolus linnaeus published 8 names under bryonia (jarvis 2007), six of which refer to other genera on the basis of the current circumscription in cucurbitaceae juss. the others (b. alba and b. dioica) are included in the genus bryonia according to the current nomenclatural and taxonomic point of view, (schaefer and renner 2011, apg iii 2009) and appearsto be as yet untypified. the aim of this study is to investigate the typification of the name bryonia cretica (a species of the eastern mediterranean area) and to provide morphological, cytological, ecological and chorological notes about the taxon. materials and methods the investigation of the typification of the name b. cretica included all the available literature (linnaeus 1738, 1753; royen 1740; bauhin 1620, 1623, 1651) as well as research of the linnaean herbarium (linn) and the clifford herbarium in bm. for the acta bot. croat. 72 (1), 2013 193 * corresponding author, e-mail: d.iamonico@yahoo.it copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 579 iamonico and panitsa.ps u:\acta botanica\acta-botan 1-13\579 iamonico and panitsa.vp 14. o ujak 2013 12:58:20 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees morphology, geographical distribution and cytology, we followed tutin et al. (1968), davis et al. (1972), greuter et al. (1986) and volz and renner (2008, 2009). authors’ field data were used for the description of the plant communities in which b. cretica participates while for the habitat types, the codes and description of the european nature information system (eunis 2012) and those of annex i of the council directive 92/43/eec (eur25 2003) were also used. information concerning ecological preferences of the species with respect to different indicators such as light, humidity and soil, follows böhling et al. (2002). results and discussion typification linnaeus’ protologue (linnaeus 1753: 1013) consisted of a diagnosis (»6. bryonia foliis palmatis supra calloso-punctatis«), with five synonyms cited from linnaeus (1738: 453), royen (1740: 264) and bauhin (1620: 135, 1623: 297, 1651: 146) and the provenance (»habitat in creta«). none of these references include an illustration of b. cretica. in the linnaean herbarium (linn) there are no sheets of b. cretica and we have been unable to trace any further original material in any of the other linnaean and linnaean-linked herbaria (see jarvis 2007). in the clifford herbarium there is one sheet (original material coded as bm000647452) that bears a plant identifiable as b. cretica according to the linnaeus description, both in the shape of the leaves (palmate) and in the surface of the leaves (punctate). the clifford phrase (»bryonia cretica maculata«) refers to the descriptions by bauhin (1620, 1623). a label on this sheet reports »bryonia cretica l. lectotypus det. c. jeffrey 2.i.1979«. however, no typification of b. cretica appears in the manuscripts published post 1978 by jeffrey (1978, 1980, 1982). so this can be considered an informal typification and it is not effective. as a sheet from the clifford herbarium is the only extant original material, and it has long been considered representative of the species, it is here designated as the lectotype of b. cretica: bryonia cretica l., sp. pl. 2, 1013 (1753) – type (designated): herb. clifford: 453 bryonia 2 (lectotype bm). retrieved july 07, 2012 from http://www.nhm.ac.uk/resources/ research-curation/projects/clifford-herbarium/lgimages/bm000647452.jpg description perennial dioecious, up to 4 m. leaves 5–10 cm long, 5-lobed, each lobe entire or with few obtuse teeth, the central slightly longer than the lateral lobes; young leaves with irregular whitish markings. inflorescence usually eglandular; calyx campanulate, 5-dentate, corolla rotate, stigma hairy; stamens 3; fruit in berry, red coloured, 6–10 mm in diameter. b. cretica is a hexaploid species (2n = 6x = 60) and probably of hybrid origin between b. dioica jacq. and b. syriaca boiss. and/or b. multiflora boiss et heldr. (volz and renner 2008). geographical distribution bryonia cretica is an eastern mediterranean species. it is distributed in greece, on the east aegean islands, crete and karpathos, asiatic turkey, cyprus, egypt, israel and jordan, libya, lebanon and syria (greuter et al. 1986). recently, volz and renner (2009) gave new sites of b. cretica indicating dna number and chloroplast haplotype for indi194 acta bot. croat. 72 (1), 2013 iamonico d., panitsa m. 579 iamonico and panitsa.ps u:\acta botanica\acta-botan 1-13\579 iamonico and panitsa.vp 14. o ujak 2013 12:58:20 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees viduals from different areas of greece such as the peloponnesian peninsula, kythera island and crete. jeffrey (1969: 448) noted that b. cretica has several morphological variants, a typical one from southern greece, crete, and the aegean islands and another that occurs in cyprus, syria, turkey, and palestine. tutin et al. (1968) distinguished b. cretica subsp. cretica as an aegean endemic taxon based on the characteristic irregular whitish markings on leaves and young fruits, as well as on the eglandular -or very nearly somale inflorescence. this taxon is also mentioned for the south and the east aegean area by böhling et al. (2002) and panitsa and tzanoudakis (2010). ecology this taxon includes rhizomatose geophytes growing in bushes and hedges, between sea level and 550 m (davis et al. 1972). on the basis of the correspondence between the habitat codes of the european nature information system (eunis 2012) and those of annex i of the council directive 92/43/eec (eur25 2003), habitat types where b. cretica has mostly been registered as a native species are: coastal dune thickets (eunis code b1.61), east mediterranean phrygana (eunis code f7.3, annex i code 5420), thermo-mediterranean and pre-desert scrub (eunis code f5.5, annex i code 5330), olea europaea-ceratonia siliqua woodland (eunis code g2.4, annex i code 9320), southern riparian galleries and thickets (eunis code f9.3, annex i code 92d0). b. cretica mainly grows in association with the following taxa: pistacia lentiscus l., olea europaea l. subsp. oleaster (hoffm. et link) negodi, calicotome villosa (poir.) link, prasium majus l., juniperus phoenicea l. subsp. phoenicea, rhamnus lycioides l. subsp. oleoides (l.) jahand. et maire, quercus coccifera l., ceratonia siliqua l., osyris alba l., euphorbia dendroides l., juniperus oxycedrus l. subsp. macrocarpa (sibth. et sm.) neilr., clematis cirrhosa l., prunus webbii (spach) vierh. and rubia tenuifolia d’urv. it is found in communities mainly belonging to the class quercetea ilicis br.-bl. ex a. de bolos 1950 and the order pistacio lentisci-rhamnetalia alaterni rivas-mart. 1975 but also in plant communities of the class cisto-micromerietea julianae oberd. 1954 and the order cisto-micromerietalia oberd. 1954. according to böhling et al. (2002), b. cretica is a semi-shade to semi-light plant of fairly hot to hot sites with a centre of occurrence between 50 and 300 m a. s. l., characterized by a mean annual temperature of 18.5 °c. it is also a fresh-sites indicator, found at moderately developed, temporarily wet places, in poorly irrigated land. it prefers mostly basic soils (ph 7.2–7.6) and it is an indicator of sites more or less rich in nutrient, on soils with narrow c/n 8–11 and a mostly narrow c/p <1. references apg iii, 2009: an update of the angiosperm phylogeny group classification for the orders and families of flowering plants: apg iii. botanical journal of linnean society 161, 105–121. bauhin, c., 1620: prodromos theatri botanici. puli jacobi, francofurtiad moenum. bauhin, c., 1623: pinat theatri botanici. ludovici regis, basileae. bauhin, c., 1651: historia plantarum universalis 2. ebroduni. acta bot. croat. 72 (1), 2013 195 typification of the name bryonia cretica 579 iamonico and panitsa.ps u:\acta botanica\acta-botan 1-13\579 iamonico and panitsa.vp 14. o ujak 2013 12:58:20 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees böhling, n., greuter, w., raus, t., 2002: zeigerwerte der gefässpflanzen der südaegeis (griechenland). braun-blanquetia 32, 1–106. davis, p. h. (ed.), 1972: flora of turkey and the east aegean islands 4. edinburgh univ. press, edinburgh. eunis, 2012: european nature information system. retrieved july 07, 2012 from http:// eunis.eea.europa.eu/ eur25, 2003: interpretation manual of european union habitats. european commission dg environment. greuter, w., burdet, m. m., long, g., 1986: med-checklist 3. conservatoire et jardin botaniques de la ville de genève, geneve. jarvis, c., 2007: order out of chaos: linnaean plant names and their types. linnean society of london and the natural history museum, london. jeffrey, c., 1969: a review of the genus bryonia l. (cucurbitaceae). kew bulletin 23, 441–461. jeffrey, c., 1978: further notes on cucurbitaceae, 4. some new-world taxa. kew bulletin 33, 347–380. jeffrey, c., 1980: further notes on cucurbitaceae, 5. the cucurbitaceae of the indian subcontinet. kew bulletin 34, 789–809. jeffrey, c., 1982: further notes on cucurbitaceae, 5. the cucurbitaceae of the indian subcontinet: corrigenda and addenda. kew bulletin 36, 737–740. linnaeus, c., 1738: hortus cliffortianus. salomonem schouten, amstelaedami. linnaeus, c., 1753: species plantarum 2. laurentii salvii, stockholm. panitsa, m., tzanoudakis, d., 2010: floristic diversity on small islands and islets: leros islets’ group (east aegean area, greece). phytologia balcanica 16, 271–284. royen, a., 1740: florae leydensis prodromus. samuelem luchtmans, lugduni batavorum. schaefer, h., renner, s. s., 2011: phylogenetic relationship in the order cucurbitales and a new classification of the gourd family (cucurbitaceae). taxon 60, 122–138. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. e. (eds.), 1968: flora europaea 2. cambridge university press, cambridge. volz, s. m., renner, s. s., 2008: hybridization, polyploidy, and evolutionary transitions between monoecy and dioecy in bryonia (cucurbitaceae). american journal of botany 95, 1297–1306. volz, s. m., renner, s. s., 2009: phylogeography of the ancient eurasian medicinal plant genus bryonia (cucurbitaceae) inferred from nuclear and chloroplast sequences. taxon 58, 550–560. 196 acta bot. croat. 72 (1), 2013 iamonico d., panitsa m. 579 iamonico and panitsa.ps u:\acta botanica\acta-botan 1-13\579 iamonico and panitsa.vp 14. o ujak 2013 12:58:20 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 544 vizintin et al.vp acta bot. croat. 71 (2), 195–205, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 genetic characterization of genista sericea wulfen (cytiseae – fabaceae) as revealed by nuclear dna content and its nrdna region analysis liliana vi@intin1*, vera kosovel2, laura feoli chiapella2, borut bohanec1 1 university of ljubljana, biotechnical faculty, jamnikarjeva 101, 1111 ljubljana, slovenia 2 university of trieste, department of life sciences, via giorgieri 10, 34127 trieste, italy summary – genista sericea wulfen, a northern illyrian amphi-adriatic species, presents a certain morphological variability. to clarify whether the genetic variations support the morphological differences among accessions of different geographic origin, analysis of nuclear dna content and polymorphism of the internal transcribed spacer (its) dataset was studied. the variation in nuclear dna content of g. sericea var. sericea and var. rigida is minimal (2.09 and 2.08 pg/nucleus respectively) and is correlated with equal chromosome numbers in both varieties. intraspecific variability of the its region was studied on 13 accessions of g. sericea, 6 belonging to var. sericea and 7 to var. rigida. these accessions were analyzed in comparison to closely related species already studied. its sequences of g. sericea revealed large polymorphism and formed two main clusters. one cluster (6 accessions) comprehends var. sericea of northern italy, slovenia and northern croatia; the other cluster (7 accessions) includes five accessions of var. rigida from southern croatia and montenegro and two from the pollino massif (southern italy). the later two accessions considerably differed from other accessions of var. rigida. this genetic analysis supports the previous assumptions, which subdivided g. sericea into at least two taxa. on the basis of the results presented, it is here suggested that the subdivision of g. sericea into var. sericea and var. rigida should be maintained. key words: fabaceae, genista sericea, genome size, its, phylogenic analysis. abbreviations: dapi – 4, 6-diamidino-2-phenylindole, its – internal transcribed spacer, pi – propidium iodide. introduction genista sericea wulfen, a northern illyrian amphi-adriatic species, has a range from northeastern italy to albania, with a disjunct distribution on the pollino massif, calabria, italy (pampanini 1912, gibbs 1966, conti et al. 2005). acta bot. croat. 71 (2), 2012 195 * corresponding author, e-mail address: lili.vizintin@gmail.com copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. within the species, pampanini (1912) distinguished var. sericea and var. rigida. in the first taxon the non-flowering branches are elongated and flexuous; the leaves are lanceolate and acute with a glabrous upper surface. in the second taxon the branches are shorter, more strongly ribbed and rigid; the leaves are ovate, broader and thicker, especially the young, with a sparsely pubescent upper surface. whereas var. sericea is distributed in the northern part of the range (northeastern italy and northern part of the illyrian region), var. rigida is spread in the southern part of the illyrian region (dalmatia, bosnia-hercegovina, montenegro, albania) and on the pollino massif (italy). in recent literature, var. rigida is not considered (gibbs 1966, pignatti 1982, conti et al. 2005). in northern italy, var. sericea occurs along the southern slopes of the alps; it is frequent in the east (friuli venezia giulia, particularly in the karst area near trieste), while it becomes rarer towards the west, in southern veneto and southern trentino. in slovenia, var. sericea is present only in the south west. in croatia it occurs in the northwestern area, especially in kvarner (pampanini 1912, pignatti 1982, feoli chiapella and rizzi longo 1987, conti et al. 2005). genista sericea belongs to sect. spartioides spach. according to its morphological and phytochemical characters, it is known as a heterogeneous section that does not form a monophyletic group. because of the molecular data, the species of this section are included in separate groups intermingled with species of other sections and several intersectional hybrids are also present (pardo et al. 2004). this heterogeneity was confirmed also by karyological and palynological data (cantó et al. 1997; cusma velari et al. 2003, 2009; rizzi longo and feoli chiapella 2009). the species of this section occur mostly in the mediterranean region, with two main distribution centers: a western (southern spain and northwestern africa) and an eastern one (balkan peninsula and anatolia) (gibbs 1966, cantó et al. 1997). rizzi longo and feoli chiapella (1993) carried out a pollen morphology analysis of g. sericea by light microscope and sem, where var. sericea and var. rigida differ in some quantitative (grain dimensions) and qualitative (shape of amb and furrows) characters. cusma velari et al. (1996) have determined the chromosome number of g. sericea var. sericea and var. rigida: 2n=4x=48. the nuclear ribosomal dna (nrdna) internal transcribed spacer region (its) has proven a useful source of characters for phylogenetic studies in many angiosperm families including leguminosae (álvarez and wendel 2003). many genera of cytiseae (kaess and wink 1997b), lupinus (badr et al. 1994, kaess and wink 1997a), genista (de castro et al. 2002), genista and related genera as teline, echinospartum, retama, stauracanthus (pardo et al. 2004), cytisus and allied genera such as argyrocytisus, calicotome, cytisophyllum (cubas et al. 2002), adenocarpus, genista and teline (percy and cronk 2002) and ulex (ainouche et al. 2003), have already been analyzed. although molecular data are considered the most convincing data set, which can be used to identify phylogenetic relationships, a combination of different data sets, including genome size data, provides useful information on genetic similarity and taxonomic position (vi@intin et al. 2006). most reports (leitch and bennett 2002) confirmed that genome size is usually a value with limited variations within a single species and could be considered characteristic of the species. flow cytometry could be used for estimations of genome size in absolute units using intercalating fluorochrome pi stained nuclei (noirot et 196 acta bot. croat. 71 (2), 2012 vi@intin l., kosovel v., feoli chiapella l., bohanec b. al. 2002) or for an intraspecific comparison in relative units using dapi, an at base specific dye (buitendijk et al. 1997). genome size data concerning the taxa genista, cytisus, lembotropis and argyrocytisus are rather scarce. the aim of our study is to assess the intraspecific variability of g. sericea, highlighting the possible differences between var. sericea and var. rigida at a molecular level through the analyses of nrdna regions its1 and its2. furthermore, as genome size has not yet been determined for g. sericea, our aim is to provide data on genome size in both varieties. materials and methods plant material genome size determination was performed with fresh plant material. accessions used for the flow cytometry analyses were: g. sericea var. sericea, collected at opicina and monrupino (trieste – italy) and g. sericea var. rigida, collected on the pollino massif (calabria, italy) (tab. 1). the extraction of dna for molecular analyses was performed from herbarium plant material. different accessions of g. sericea were examined: six belonging to var. sericea and seven to var. rigida. the studied accessions, with their geographical origin and ncbi genbank number are listed in table 2 and figure 1. voucher specimens are deposited in the herbarium of the department of life sciences, university of trieste, italy (tsb). the classification of pampanini (1912) was followed. determination of nuclear dna content by flow cytometry the relative and absolute value of dna content was assessed by flow cytometry using trifolium pratense l. as standard. the genome size (0.85 pg) was previously estimated for this species using trifolium repens l. cv. milo as standard (vi@intin et al. 2006). for the determination of absolute dna content, tissues from green and young stems of the analyzed samples, together with leaf tissues of the standard species, were chopped with a razor blade in plastic petri dishes in cold lb01 buffer, according to a technique adapted acta bot. croat. 71 (2), 2012 197 genetic characterization of genista sericea tab. 1. propidium iodide and dapi flow cytometric genome size determination in the examined populations of genista sericea. staining method taxon accession no. plant average 2c (pg) ± sd pi staining g. sericea var. sericea opicina (trieste, italy) 90579 (tsb) 5 2.09 ± 0.03 g. sericea var. rigida m. pollino (cosenza, italy) 90582 (tsb) 5 2.08 ± 0.03 dapi staining g. sericea var. sericea opicina (trieste, italy) 90579 (tsb) 4 1.96 ± 0.02 monrupino (trieste, italy) 90580 (tsb) 4 1.95 ± 0.02 g. sericea var. rigida pollino (cosenza, italy) 90582 (tsb) 4 1.95 ± 0.03 from dole@el et al. (1989). the suspension was passed through a 30 mm nylon-mesh filter and nuclei were stained with 50 mg ml–1 of pi and 50 mg ml–1 ribonuclease. for the determination of relative dna content, the staining was performed with dapi and the procedure was modified according to otto (1988). nuclei of our sample and of the standard species were released in 0.1 m citric acid containing 0.5% tween 20. the suspension was filtered through a 30 mm nylon-mesh filter. a 4x volume of staining buffer containing 4 mg ml–1 dapi and 0.4 m disodium hydrogen phosphate was added. measurements were done on a partec pas flow cytometer using a linear scale. for pi staining, samples were analyzed using an argon laser tuned to 488 nm, with emissions measured through an rg 590 long-pass optical filter. for dapi staining, the uv spectrum ex198 acta bot. croat. 71 (2), 2012 vi@intin l., kosovel v., feoli chiapella l., bohanec b. tab. 2. accession data of the populations of genista sericea sampled for phylogenetic analyses in the its region of nrdna: origin, collector and ncbi genebank accession numbers. taxon sample origin collector genebank accession no. its1 its 2 g. sericea var. sericea gs17 vivaro, pordenone, italy, 15/10/2000, 84731 (tsb) l. feoli chiapella eu525894 eu525906 gs19 pinedo di claut, pordenone, italy, 23/5/1999, 84728 (tsb) c. coran eu525896 eu525908 gs18 mt. ^aven, nova gorica, slovenia, 10/5/1999, 84730 (tsb) l. feoli chiapella eu525895 eu525907 gs20 mt. nanos, nova gorica, slovenia, 29/5/1999, 84726 (tsb) l. feoli chiapella eu525893 eu525909 gs2 ba{~anska draga – punat, krk, croatia, 24/4/1966, 84138 (tsb) m. tarabocchia eu525897 eu525910 gs15 capo pax tecum – plomin, istria, croatia, 70 m, 26/4/1964, 84139 (tsb) t. mozenich eu525898 eu525911 g. sericea var. rigida gs12 biokovo, kozica, croatia, 600 m, 30/4/2001, 84457 (tsb) l. feoli chiapella eu525904 eu525917 gs9 ku~i, donji medun, podgorica, montenegro, 3/6/1998, 84143 (tsb) v. karaman eu525900 eu525913 gs10 kakari~ka gora, masline, podgorica, montenegro, 173 m, 3/6/1998, 84456 (tsb) v. karaman eu525901 eu525914 gs11 piperi, podgorica, montenegro, 300 m, 17/5/1998, 90581 (tsb) v. karaman eu525902 eu525915 gs13 cijevna reka, montenegro, 27/5/2000, 84451 (tsb) s. had`iablahovi~ eu525903 eu525916 gs4 timpone dolcetti, pollino, cosenza, italy, 9/6/1997, 84141 (tsb) d. puntillo eu525899 eu525912 gs16 morano calabro, pollino, cosenza, italy, 3/6/1997, 84140 (tsb) a. vaccaro eu525905 eu525918 cited with a hbo lamp was used and emissions were measured through a gg 435 long-pass filter. seven thousand nuclei per sample were measured and at least four repetitions of different nuclear isolations were performed for each species. flomax® software (partec, münster) was used to calculate the positions of g0/g1 peaks of standard and investigated accessions. the analysis was completed in a short period to minimize the seasonal variation effect. analysis of the its region of rdna genomic dna was extracted from herbarium plant material applying ctab mini dna extraction protocol according to compton et al. (1998) with slight modifications. the ribosomal its 1 and 5.8 s -its 2 regions were separately amplified using, respectively, its1-its2 and its3-its4 primers designated by white et al. (1990). amplification was carried out by means of a polymerase chain reaction in 50 ml reaction mixture containing 1 x taq polymerase buffer (promega, madison, wi, usa), 200 mm of each dntp, 0.5 mm of each primer, 100 ng dna and 2.5 units of taq polymerase (promega). optimized amplification was performed in a ptc-150-16e-25 m.j. research inc. thermal minicycler programmed as follows: (a) an initial 3 min denaturation at 95 °c; (b) 35 cycles of denaturation at 93 °c for 35 sec, annealing at 49 °c for 35 sec and polymerisation at 72 °c for 2 min; (c) final extension at 72 °c for 7 min. the amplified dna products were sequenced from both sides by macrogen inc. (seoul, korea) using the same primers on an abi 3730 xl dna analyser (applied biosystems, renton, usa). sequence results from each amplified fragment were visualized and edited to obtain an optimized consensus sequence using codoncode aligner ver. 1.4.6 (codoncode corporation, usa). nucleotide sequences were deposited in the genbank database with the accession numbers reported in table 2. acta bot. croat. 71 (2), 2012 199 genetic characterization of genista sericea fig. 1. geographycal origin of the examined accessions of genista sericea. � g. sericea var. sericea, � g. sericea var. rigida phylogenetic analysis of its sequences the initial data matrix was aligned using clustalx (ver. 1.83). a phylogenetic tree was constructed using the maximum likelihood method based on the tamura 3-parameter model (tamura 1992) on mega 5.0 software (tamura et al. 2011). bootstrapping was performed at one thousand replicates to assess the confidence values of the clusters formed. bootstrap data are shown next to the branches. a discrete gamma distribution was used to model evolutionary rate differences among sites. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. there were a total of 508 positions in the final dataset. an accession of cytisus was used as an outgroup. several sequences of closely related genista species from the section spartioides, already available in the genbank, have also been included in the its analyses to obtain a more comprehensive picture. accession numbers and sources of these sequences are indicated in table 3. results analysis of nuclear dna content genome size values and base composition were determined for five plants of both varieties of g. sericea (var. sericea and var. rigida) using pi staining and for four plants of each accession of the two varieties using dapi (tab. 1). determination using pi serves as an estimation of absolute nuclear dna content, while determination using dapi serves as a comparison for further intraspecific analysis. the absolute value of nuclear dna content for var. sericea is 2.09 pg dna per nucleus. the value obtained for var. rigida is 2.08 pg dna per nucleus. because lower cv values cause higher resolution, dapi staining was additionally used to test more accurately the intraspecific genome size variation: g. sericea var. sericea presents a relative nuclear dna content of 1.95 – 1.96 pg per nucleus and var. rigida 1.95 pg per nucleus. results obtained with dapi staining confirmed that the variability of genome size is rather small between the two varieties of g. sericea and is not useful for the characterization of the varieties. it should be noted that the values obtained by pi staining have given higher estimations of dna content, suggesting unequal at/gc content. 200 acta bot. croat. 71 (2), 2012 vi@intin l., kosovel v., feoli chiapella l., bohanec b. tab. 3. accession data and sequences of its region obtained from ncbi genbank. taxon references genebank accession no. genista cinerea subsp. speciosa rivas mart. et al. pardo et al. 2004 ay263637 genista cinerea subsp. ausetana o. bolo` s et vigo pardo et al. 2004 ay263638 genista cinerea (vill.) dc. subsp. cinerea pardo et al. 2004 ay263636 genista majorica canto et m.j. sanchez pardo et al. 2004 ay263652 genista ramosissima (desf.) poir. pardo et al. 2004 ay263662 genista valentina (sprengel) steud. pardo et al. 2004 ay263676 cytisus scoparius (l.) link subsp. scoparius cubas et al. 2002 af351120 polymorphism of the its region of rdna the internal transcribed spacer (its1 and its2) region of ribosomal dna was sequenced from 13 accessions of g. sericea (tab. 2). each examined sequence produced a slightly different sequence profile; their alignment resulted in a matrix of 508 characters. a high variability of its nucleotide sequences was discovered exhibiting 22 variable sites. the nucleotide sequences of amplified its regions were used to construct a phylogenetic tree (fig. 2) to assess the relationships among the analyzed accessions. the its tree reveals two major clades, bootstrap support was 74. one clade is formed by all the accessions of var. sericea, from friuli – venezia giulia (italy), slovenia and northern coast of croatia. the other clade includes the accessions of var. rigida from southern croatia coast and montenegro. the two accessions of var. rigida from pollino massif (southern italy) stand at the base, but the bootstrap support is low. discussion the accessions of g. sericea var. sericea and var. rigida present a very similar nuclear dna content (2.09 and 2.08 pg per nucleus); also the relative nuclear dna content is similar in the two varieties (1.95 – 1.96 and 1.95 pg per nucleus). the nuclear dna content in a wild population of a species of genista sericea is presented for the first time in this study, as the only data in literature regarding this genus are those of bellenot-kapusta et al. (2006), who studied some ornamental brooms (as g. tinctoria, g. lydia, g. pilosa, g. hispanica and g. aetnensis) the 2c dna content of which varies from 1.60 to 3.56, and of suda et al. acta bot. croat. 71 (2), 2012 201 genetic characterization of genista sericea fig. 2. phylogram of 13 populations of genista sericea and of 6 related species of the genus genista. cytisus scoparius subsp. scoparius was added as an outgroup. (2005) who determined the nuclear dna content value for g. benehoavensis as 4.60 pg. in particular the 2c dna content of g. pilosa, the only species previously examined of sect. spartioides, is 1.99 pg, while in g. tinctoria and g. lydia of the affine sect. genista is 1.73 and 1.74 pg. data concerning other genera of cytiseae, wild and cultivated plants of cytisus, lembotropis, argyrocytisus and laburnum, were published by bellenot-kapusta et al. (2006) and olszewska and osiecka (1983, 1984): the analyzed 2c dna content varies from 1.10 to 4.40. misset and gourret (1996) determined the genome size of ulex (7.7 pg/2c). obermayer et al. (1999) and naganowska et al. (2006) have studied many species of lupinus (2c content from 1.15 to 2.68), a genus distant from the others of cytiseae. because of the obtained genome size data, morphological differences between the two varieties of g. sericea are not correlated with significant differences in genome size and this led us to suppose that no major insertions or deletions of dna segments occurred in any of varieties. this result is correlated with chromosome studies which showed the same number in both varieties (2n=4x=48; cusma velari et al. 1996). both varieties of g. sericea formed clearly separated clades (74 bootstrap supports) which did not intermingle with other related genista species. although analysis of its region is mainly used for interspecific phylogeny, this study indicates that genetic variation of its region in g. sericea was in this case sufficient to resolve both subspecies. in g. sericea the data presented reflect the separation of two distinct groups of accessions, the first comprehending all the accessions of var. sericea from northern italy, slovenia and northern croatia, the second including all the accessions of var. rigida collected in southern croatia, montenegro and in southern italy. molecular data are in accordance with morphological characteristics and pollen differences (rizzi longo and feoli chiapella 1993), as well as seed dimensions (cusma velari and feoli chiapella, personal observations). our analysis supports the subdivision of g. sericea into two taxa (var. sericea and var. rigida). on the basis of molecular data, the accessions of the pollino massif (calabria, italy) attributed by pampanini (1912) to var. rigida are related to those of southern croatia and montenegro, but do show diversification within var. rigida that could be further studied. acknowledgements this research was supported by a grant from the ministry of foreign affairs of the italian republic and in part by a project (p4-0077) of the ministry of higher education, science and technology of the republic of slovenia. we would like to thank prof. l. bernardo (cosenza), dr. d. puntillo (cosenza), prof. a. vaccaro (acri), dr. d. and v. vincek (kola{in) for supplying us with some plants and dr. lucia feoli, for correcting the english version. references ainouche, a., bayer, r. j., cubas, p., misset, m. t., 2003: phylogenetic relationships within tribe genisteae (papilionoideae) with special reference to genus ulex. in: klitgaard, b. b., bruneau, a. 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(eds.), pcr protocols: a guide to methods and applications, 315–322. academic press, san diego. acta bot. croat. 71 (2), 2012 205 genetic characterization of genista sericea 625 pise and gaikwad.vp acta bot. croat. 72 (2), 257–268, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/botcro-2013-0012 cryoseston of the pirin mountains, bulgaria vladislav cepák*, jaromír lukavský academy of sciences of the czech republic, institute of botany, centre of algology, dukelská 135, cz-37982 trfebonf, czech republic abstract – in the cryoseston community of the pirin mountains, 18 species were determined. chlorophyta: chlamydomonas nivalis (predominant), chloromonas brevispina, chloromonas rostafinski, chlainomonas rubra (new genus and species for bulgaria, documented in europe for only the second time), cystococcus nivicolus and stichococcus nivalis. bacillariophyceae: aulacoseira granulata var. angustissima, hantzchia amphioxys, cf. surirella. fungi: cf. chytridium chlamydococcii. deuteromycetes: selenotila nivalis, chionaster nivalis, and saprophytic fungae cf. myzocytium, rhodosporidium toruloides, alternaria sp. and cf. cladosporium, in pollen grains. bacteria: leptothrix ochracea. ciliata: vorticella campanula. the composition of cryoseston in the pirin mountains is characteristic for high mountains where chlamydomonas nivalis predominates. key words: chlainomonas rubra, cryoseston, pirin mountains, snow algae, bulgaria introduction extremophilic algae are attracting attention as a prospective pool of organisms producing compounds of biotechnological interest (hoham and duval 2001, pulz and gross 2004, seckbach 2007, rfezanka et al. 2008). the cryosestonic alga chlamydomonas nivalis produces large amounts of the red pigment, astaxanthin, which is an effective uv screen and, as a free radical acceptor, protects cells against photoinhibition (remias et al. 2005, rfezanka et al. 2007). moreover c. nivalis is not a strict cryophile as photosynthetic activity continued up to 20 °c for about one hour (remias et al. 2005), as was the case with the mesophile haematococcus pluvialis (wang et al. 2003). recently c. nivalis was patented for use in cosmetics as a uv filter (brooks and franklin 2011, schürch et al. 2010). in addition, cryosestonic algae contain anti-freeze proteins (afp) that bind to small ice crystals in cells and permit the survival of cells at temperatures below zero (li et al. 2009, gwak et al. 2010). for space research (astrobiology), cryoseston is used as an analogy for e.g. martian life (wharton et al. 1989, rothschild 1990, kanik 2009). acta bot. croat. 72 (2), 2013 257 * corresponding author, e-mail: cepak@botany.cas.cz copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. the first step towards the biotechnological exploitation of cryophilic algae is sample collection, cell isolation and screening. a checklist of species is built up via such research. in bulgaria, cryoseston was recorded for the first time by kol (1956) in the rila mountains, and in the pirin mountains. this report was confirmed by wodenitscharoff (1962) in the rila mountains and new localities were found later by lukavský et al. (2009) in the vitosha mountains, and stara planina mountains (lukavský and cepák 2010). the aim of this work was to confirm the species diversity in the locality of the pirin mountains and to isolate strains for further research. material and methods localities snow fields were sampled in the pirin mountains at altitudes of 1996–2930 m a.s.l., in may, 2011 (tab. 1). 258 acta bot. croat. 72 (2), 2013 cepák v., lukavský j. tab. 1. gps positions, altitudes and species presence in individual localities. d = dominant species. no. a lt it ud e (m a. s. l. ) gps l e p to th ri x o c h ra c e a a u la c o se ir a g ra n u la ta va r. a n g u st is si m a h a n tz c h ia a m p h io x y s cf .s u ri re ll a c h la in o m o n a s ru b ra c h la m y d o m o n a s n iv a li s c h lo ro m o n a s b re v is p in a c h lo ro m o n a s ro st a fi n sk i c y st o c o c c u s n iv ic o lu s s ti c h o c o c c u s n iv a li s v o rt ic e ll a c a m p a n u la c h io n a st e r n iv a li s cf .c h y tr id iu m c h la m y d o c o c i s e le n o ti la n iv a li s cf . m y zo c y ti u m r h o d o sp o ri d iu m to ru lo id e s a lt e rn a ri a cf . c la d o sp o ri u m 47 1996 n41 52.672 e23 31.782 x x x x 44 2118 n41 45.641 e23 24.570 x x x x x x x 48 2379 n41 45.619 e23 24.346 x x d x x 46 2426 n41 45.588 e23 24.248 d x x 45 2483 n41 45.614 e23 24.110 x x x 49 2483 n41 45.608 e23 24.177 41 2549 n41 45.649 e23 23.984 x x x x x 43 2729 n41 45.826 e23 23.883 x 42 2930 n41 46.036 e23 23.942 x sampling and sample processing snow samples were collected into plastic bags, in volumes of ca 20 ml, transported in a thermos bottle to the laboratory, melted, centrifuged for 20 min. at 3000 g, at temperatures below 5 °c, examined with the use of an olympus dx 50 microscope and photographed using a dp10 digital camera. autofluorescence of chlorophyll was observed under the same microscope, equipped with a filter combination of 360–370 nm excitation, >515 nm barrier filter. samples were inoculated into medium z (staub 1961)-containing tubes, solidified with 2% agar, and held in a refrigerator at a temperature of less than 7 °c with continuous irradiation from fluorescent tubes (ca 20 w m–2 par). the selected strains are maintained in the collection of biotechnological exploitable microorganisms (cobiem) in trfebonf (czech republic). results and discussion in snow samples from the pirin mountains, we isolated and identified 18 microorganisms (pl. 1–4, figs. 1–56). chlorophyta chlainomonas rubra (j. r. stein et r. c. brooke) hoham, pl. 1, figs. 1–8. syn. sphaerellopsis rubra stein et brooke 1964 description: cells egg-shaped, about 30 × 50 mm, sometimes with a small, but prominent papilla, flagella not observed. cell wall smooth, outer and inner envelopes separated by a wide space. protoplast contents brown, filled by small grains, pyrenoid not observed. division into 2 autospores is predominantly unequal. distribution: chlainomonas rubra and c. kolii were identified only from snow. c. kolii was described as a snow alga by hoham (1974a), who re-arranged trachelomonas kolii (euglenales) into chlorophyta, as chlainomonas kolii. this same alga was described in snow samples from canada, in the nw pacific mountains of the usa, and in mt philistine, new zealand (novis 2002, novis et al 2008). chlainomonas rubra (hoham 1974b) was identified in snow in tirole, n. island of new zealand, the pacific northwest usa, wenatchee natl for., washington, and mt. seymour park, brit. columbia, canada (novis et al. 2008). it represents a new genus and species for the bulgarian algal database, and this is only the second documented record in europe. cf. chlainomonas was also identified in central svalbard (kvíderová 2012). notes: type of the genus is c. ovalis h. r. christen. the monophyly of the genus chlainomonas was proven by dna analysis. it is the only quadriflagellate chlorophycean unicell that shares a robust affinity to biflagellates. maybe chlainomonas was derived from the fusion of biflagellate cells or is a diploid component of a biflagellate phase (sexual reproduction was not seen). the flagellar basal apparatus was also organized into two distinct pairs of basal bodies that lack connections (novis et al. 2008). the alga is very sensitive to temperature, and can only live at 0–4 °c (hoham 1975). perhaps for this reason we have not yet been able to culture this organism. the colour of the alga found in the pirin mountains is very unusual. while cells of c. rubra and c. kolii are a pink-red colour, while our material was more brown (fig. 7). the pigment composition could be unique. acta bot. croat. 72 (2), 2013 259 cryoseston of the pirin mountains chlamydomonas nivalis (bauer) wille, figs. 9–14, 19–22. syn. uredo nivalis bauer, sphaerella nivalis (bauer) sommerfelt, protococcus nivalis agardh, haematococcus nivalis flotow description: only spherical spores/hypnoblasts, deep-red coloured, diameter 35–40 mm, some in an envelope of dust grains of different thicknesses. division has not yet been observed. in adult cells, chloroplasts are split into a few disc-shaped parts (figs. 11, 21), in agreement with remias et al. (2005). notes: the characteristically brick-red colour of protoplasts is caused by astaxanthin and its fatty acid esters. this compound protects the alga from damage by uv irradiation. the first screen is a cover made up of inorganic dust particles that stick to the mucilage layer 260 acta bot. croat. 72 (2), 2013 cepák v., lukavský j. plate 1. cryoseston of the pirin mountains. collected in may 2011. figs. 1–4 – chlainomonas rubra, protoplast filled with a mass of small grains; 5 – autofluorescence of chlorophylls under uv irradiation; 6 – division of protoplast into 2 unequal parts (note small, but prominent papilla); 7 – coloured snow at locality no. 44 (algae are near the surface); 8 – outer envelope (cell wall), showing distance from inner envelope (stained with lugol solution), see also figures 3, 4 and 6. all figures loc. 44. (remias et al. 2005). this dust envelope is of different thicknesses (figs. 19, 20, 22). fatty acids, together with haematochromes, also protect cells from freezing. c. nivalis is characteristic at higher altitudes, open stands and under strong irradiation (kawecka 1981, hoham et al. 1993). surprisingly, photosynthesis proceeds up to 20 °c (but only in short-term experiments of about one hour), proving that the alga is more cryotolerant than cryophilic. in acta bot. croat. 72 (2), 2013 261 cryoseston of the pirin mountains plate 2. cryoseston of the pirin mountains collected in may 2011. figs. 9–14, 19–22 – chlamydomonas nivalis; 11, 12, 21 – fluorescence of chlorophyll under uv irradiation; 13, 18 – cells infected by some chytrid, cf. chytridium chlamydococci; 14 – cells infected by some filamentous fungus, selenotila nivalis; 15–18 – chloromonas brevispina. loc. 41: figures 14–16, 19; loc. 43: figures 22; loc. 44: figures17, 18; loc. 46: figure 20; loc. 48: figures 9–13, 21. addition, in drying experiments, the hypnospore showed high viability after storage at room temperature for 6 months (remias et al. 2005). all the physiological characteristics demonstrate that this species has a potential for biotechnological exploitation. chloromonas brevispina (fritsch) hoham, roemer et mullet, figs. 15–18. syn. chodatella brevispina fritsch, cryocystis brevispina (fritsch) kol description: cysts ellipsoidal, 15 × 20 mm, cell wall thick, smooth (fig. 17) or covered with small protrusions, cell content green or orange (fig. 17). sometimes cells were covered with dust grains (figs. 15, 18), pyrenoid was not observed. notes: chloromonas brevispina and chloromonas rostafinski are very similar in morphology, cell structure, and reproduction, and both are snow algae. some differences in size and in spines/blunt protrusions can be the result of growth conditions, including nutrients (kawecka 1983/84, kawecka and eloranta 1986, hoham et al. 1979). chloromonas rostafinski (starmach et kawecka) gerloff et h.ettl, figs. 23–25, 28–29, 33. syn. chlamydomonas rostafinski starmach et kawecka description: cysts ellipsoid, 12 × 25 mm; cell wall uniformly covered with papillae-like structures; cell contents green or orange; chloroplast without a pyrenoid. flagellate stages (figs. 25, 29) observed. notes: chloromonas rostafinski was described by starmach and kawecka (1965) as chlamydomonas rostafinski, isolated from yellowish-green snow in the alpine zone of the high tatra mountains (kawecka 1983/84, lukavský 1994) and later in the stara planina mountains (lukavský et cepák 2010). its characteristic structure was revealed by sem (kawecka and eloranta 1986). it is covered by numerous longitudinal ribs that feature indentations of different lengths, forming papilla-like structures visible by light microscopy. these non-motile cells are believed to be formed (probably asexually) from vegetative cells through gradual development of cell wall ornamentation. we also observed similar cells with a smooth, or almost smooth cell wall, and pink oil droplets near both poles (fig. 17) resembling cryodactylon japonica kol. we suppose that these cells could be immature stages of chloromonas rostafinski or chloromonas brevispina resting cells. kawecka and eloranta (1986) recorded cells of chloromonas rostafinski with ornamentation of the cell wall developed to varying degrees, and stated that they were stages of growth of resting cells. further culture studies, under different conditions, are necessary. stichococcus nivalis chodat, fig. 32 description: cells rod-shaped, 5 × 10 mm, arranged in short or longer, but not firm filaments. notes: stichocccus nivalis is described mainly as individual cells or as very short filaments (kol 1968), but the alga described here was mainly in longer filaments. cystococcus nivicolus kol, fig. 38 description: cells spherical, 15 mm in diameter, cell wall smooth, chloroplast central, with many irregular lobes, and a prominent median pyrenoid. under the cell wall ring are 262 acta bot. croat. 72 (2), 2013 cepák v., lukavský j. acta bot. croat. 72 (2), 2013 263 cryoseston of the pirin mountains plate 3. cryoseston of the pirin mountains collected in may 2011. figs. 23–25, 28–30, 33 – chloromonas rostafinski; 25 – zoospore; 30 – zoospore or a colourless flagellate; 26, 31, 35, 43 – selenotila nivalis; 27 – hantzchia amphioxys; 32 – stichococcus nivalis; 34 – unknown organism; 36 – aulacoseira granulata var. angustissima; 37 – fe bacteria leptothrix ochracea; 38 – cystococcus nivicolus; 39 – mature sporangium of some chytridiomycet, cf chytridium chlamydococci, see also figures 13, 18 – sporangia with characteristic oil drops; 40, 41 – spores of alternaria (deuteromycetes); 42 – chionaster nivalis; 44 – spore of deuteromycete cf. cladosporium; 45 – a filamentous fungus; 46 – cf. surirella; 47–50 – vorticella campanula (47–49 cysts, 50 – mature organism). loc. 41: figures 26, 42; loc. 42: figures 31, 34; loc. 44: figures 23–25, 27–30, 33, 35; loc. 45: figures 32, 38, 39, 47–50; loc. 46: figures 28, 44; loc. 47: figures 36, 40, 41, 43; loc. 48: figures 37, 46, 47. numerous, peripheral small vacuoles, between the lobes of the chloroplast. reproduction was by 2, 4 (8) autospores. notes: kol (1956) determined the species in the rila mountains and placed it into chlorophyta (chlorococcales), proving the classification by observation of zoospores with 2 equal flagella. in our material, green as well as yellow-green cells and only autospores were seen. zoospores in figures 25 and 29 are probably the products of chloromonas rostafinski. for further study, isolation of unialgal culture will be necessary. bacillariophyceae aulacoseira granulata var. angustissima (o. f. müll.) simonsen, fig. 36 syn. melosira granulata var. angustissima (o. f. müll.) hust. this species was found only in the form of dead frustulae, but our identification of living cells of identical species in snow from a few other localities e.g. the stara planina mountains, bulgaria (lukavský and cepák 2010) and the sierra nevada mountains, spain (cepák and lukavský 2012) proved that they are not accidental, but are true cryobionts. hantzchia amphioxys (ehrenb.) grun., fig. 27 dead frustulae of the species were determined in a few localities, as with previous species. fungi cf. chytridium chlamydococci a. braun f. cryophila kol., figs. 13, 18, 39 description: sporangia globular 7–12 mm in diam. no papilla or intercellular components were observed. notes: a few individual cells of chlamydomonas nivalis were infected by some chytrid (figs. 13, 18). also figure 39 probably shows a cluster of zoospores, with characteristic oil droplets, after being released from the sporangium. for exact determination, we need details of intracellular components of the fungus (rhizoid single or branched, hausotrium etc.). chytridium chlamydococci was described as a parasite of chlamydococcus pluvialis. it was also described as rhizophydium acuforme (zopf) fischer by letcher and powell (2012). rhizophydium acuforme has been observed several times, as common parasites of moving chlamydomonas cells (letcher and powell 2012, lukavský 1970). in cryoseston, kol (1968) established that similar organisms such as chytridium chlamydococcii f. cryophila were common parasites of chlamydomonas nivalis. it has been found globally in europe, america etc. characteristic features of rhizophydium acuforme are few sporangia/host cell, adult sporangia have prominent papilla, rhizoid is branched into a few parts, zoospores with a prominent oil droplet and a long flagellum, thick-walled spores are rounded. relationships betweenthe two organisms are unknown and further study is necessary. chionaster nivalis (bohl.) wille, fig. 42 this fungus is a common species of cryoseston. in bulgaria, it was identified for the first time in the pirin mountains (kol 1956) and in the vitosha mountains (lukavský et al. 2009). it is recognized as »nomen dubium« (arx et al. 1977, kirk et al. 2001). 264 acta bot. croat. 72 (2), 2013 cepák v., lukavský j. cf. myzocytium, figs. 51–54, 56 description: thallus thick, tube-like, later as a chain of rounded elongated blue cells, reproduction not seen. saprophytic fungus, decaying pollen grains. notes: myzocytium is a common parasite of living algae, especially conjugatophyceae, e.g. micrasterias (batko 1975), and it was identified in snow for the first time. for a precise identification, it would be necessary to observe its reproduction. phycomycetes, particularly chytridiomycetes, are recognized as the principal organisms causing the degradation of pollen grains (goldstein 1960). in cryoseston, pollen grains were found, but their decomposition has not yet been studied thoroughly. acta bot. croat. 72 (2), 2013 265 cryoseston of the pirin mountains plate 4. cryoseston of the pirin mountains collected in may 2011. figs. 51–54, 56 – saprophytic chytrid cf. myzocytium in pollen grains of pinus peuce; 55 – fungus rhodosporidium toruloides. material was not stained with lugol solution. loc. 41: figure 55; loc. 44: figure 54; loc. 47: figures 51–53, 56. rhodosporidium toruloides, fig. 55 yeast, a saprophyte in pollen grains, was identified in snow for the first time. for a precise identification, further observations are necessary. fungi and bacteria grow in the mucilage surrounding some species, e.g. chlamydomonas nivalis, but knowledge about them is fragmentary (remias et al. 2005). ciliata vorticella campanula ehr., figs. 47–50 description: characteristic bell-shape cells, 30 × 40 mm, seated on a spiral, contractile long stalk. figures 47 and 48 probably show cystic forms that enable survival in unfavourable conditions. notes: ciliata have been occasionally described as members of cryoseston, but published reports are scarce (hoham et duval 2001). usually, vorticella are found in eutrophic-polytrophic waters. a characteristic feature of this species is a mass of highly refractile, small reserve grains. acknowledgements this work was supported as a project of the technology agency of czech republic te 01020080 and ta03011027 and by the institutional long-term research plan no. av0z60050516, funded by the academy of sciences of the czech republic. we also thank the exchange programme of the bulgarian academy of sciences, the institute of plant physiology and genetics, sofia, for assistance, h. brabcová who participated in technical affairs, and j.d. brooker who kindly corrected the english. references arx, j. a., miranda, r., smith, m. t., yarrow, d., 1977: the genera of yeasts and the yeast-like fungi. study in mycology 14, 12–78. batko, a., 1975: introduction to hydromycology (in polish). wydawnictwo naukowe pwn, warszawa. brooks, g. r., franklin, s., 2011: cosmetic composition comprising microalgal components. patent us 2011/0250178 a1. cepák, v., lukavský, j., 2012: cryoseston in sierra nevada mountains (spain). nova hedwigia 94, 163–173. goldstein, s., 1960: degradation of pollen by phycomycetes. ecology 41, 543–545. gwak, g., jung, w., kim, h. j., kang, s. h., jin, e. s., 2010: antifreeze protein in antarctic marine diatom, chaetoceros neogracile. marine biotechnology 12, 630–639. hoham, r. w., 1974a: chlainomonas kolii (hardy et curl) comb. nov. 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croat. 82 (1), 2023 acta bot. croat. 82 (1), 52–59, 2023 coden: abcra 25 doi: 10.37427/botcro-2023-001 issn 0365-0588 eissn 1847-8476 effect of excess boron on growth, membrane stability, and functional groups of tomato seedlings abeer a. radi1, hussein kh. salam2, afaf m. hamada1*, fatma a. farghaly1 1 botany and microbiology department, faculty of science, assiut university, assiut 71516, egypt 2 biology department, faculty of applied science, thamar university, dhamar, yemen abstract with the scarcity of good quality water, plants like tomatoes will be more susceptible to excess boron (eb) in mediterranean regions. the effects of eb on the growth, free, semi-bound, and bound boron (b) concentrations, and macromolecules of the solanum lycopersicum l. cultivar castle rock, were investigated in this study. seedlings were exposed to four levels of eb using boric acid. the results showed that eb inhibited tomato growth, total water content, and photosynthetic pigments. eb harmed membrane stability, as seen by increased potassium (k) leakage, uv absorbance metabolites, and electrolyte conductivity (ec) in leaf disc solution. eb raised concentrations of free, semi-bound, and bound forms of b in seedlings. fourier-transform infrared spectroscopy (ftir) data revealed that eb induced uneven wax deposition, altered the shape of cell walls, and lowered cellulose synthesis in seedlings. eb affected the amide i and amide ii proteins indicating damage to the protein pools. these results provide new insights into understanding the specific effects of eb on the functional groups of different macromolecules of tomato seedlings. keywords: excess boron, ftir analysis, membrane stability, photosynthetic pigments, plant growth, tomato introduction boron (b) performs vital tasks in plant life at ideal levels, whereas excess boron (eb) has negative consequences. the difference in b insufficiency and toxicity levels is minimal (fang et al. 2016). b-rich soils can be found all over the world and are prevalent in arid and semi-arid areas (ardıc et al. 2009). with reduced precipitation in the mediterranean area (cervilla et al. 2012) and irrigation water shortage due to new dams, demand for desalinated water for agriculture is expected to rise, potentially raising the level of b in irrigation water. moreover, rising sea levels (mediterranean sea) can pollute groundwater, resulting in higher b levels in irrigation water (princi et al. 2016). in egypt the cultivated area is suitable for intensive cultivation and this, along with anthropogenic activity, may lead to b contamination ( elbehiry et al. 2017). eb produces different physiological and morphological changes in plants, resulting in decreased plant growth, leaf chlorophyll, membrane stability ( el-shazoly et al. 2019), and ultimately reduced production (metwally et al. 2018). tomatoes are grown all over the mediterranean region, where there is a disturbance with eb in the soil. tomatoes are among the most important vegetable crops in egypt throughout the year, with a total production of 6,729,004 tons and a total cultivation area of 166,206 hectares (faostat 2017). eb has led to alterations in tomatoes, including biomass, membranes, photosynthetic pigments, phenolic compounds, and antioxidant enzymes (cervilla et al. 2012, farghaly et al. 2022b), leading to reduced yields (kaya et al. 2009). the advantage of fourier-transform infrared spectroscopy (ftir) is its ability to produce spectra on different samples such as powders and liquids with minimal sample preparation, which reduces analysis time (canteri et al. 2019). ftir is an appropriate analytical tool for biological macromolecules, assessing the composition of organic components (wu et al. 2017). absorption outlines show fixed peaks area that identifies modest modifications of metabolites related to physiological processes after infrared spectra (400–4000 cm–1) pass through plant samples (renuka et al. 2016). the peak areas, positions, and bandwidth values are critical to changes in plant macromolecules (renuka et al. 2016). however, to our knowledge, there are no reports on the use of this technique to assess physiological changes produced by excess boron on tomato seedlings. in this study, we aimed to focus on how eb treatments alter tomato macromolecules, assessing the composition of * corresponding author e-mail: hamada@aun.edu.eg, afafhamada@yahoo.com mailto:hamada@aun.edu.eg mailto:afafhamada@yahoo.com boron toxicity and tomato seedling growth acta bot. croat. 82 (1), 2023 53 organic components using ftir analysis. additionally, we measured the growth, photosynthetic pigments, membrane stability, and b forms concentration in tomato seedlings. the findings reveal a fresh understanding of the various structural responses of tomato seedlings when exposed to eb. materials and methods growth conditions vegetables department, faculty of agriculture, assiut university gave seeds of solanum lycopersicum l. (tomato), cultivar castle rock. under a laminar airflow hood, seeds were surface sterilized for 15 minutes with a 5% naclo solution and rinsed four times with sterile water. we wanted to achieve data without any extraneous influences and repeat the experiment under the same settings, so we did it in vitro. sterilized seeds were grown on sterile, half-strength murashige and skoog (ms) medium (murashige and skoog 1962). the medium was supplemented with 2.2 g l–1 ms, 3% sucrose, various concentrations (0, 2, 4, and 6 mm) of h3bo3, and 0.3% gelrite added after adjusting the ph to 5.7. the medium was sterilized for 15 min at 121 °c, pressed at 105 kpa, and allowed to cool to room temperature. seedlings were cultured in a growth chamber at 25 ± 1 °c, 65–70% relative humidity, and a photoperiod of 16/8 h with 30 μm m–2 s–1 illumination. after 20 days, some seedlings were separated into shoots and roots, weighed quickly to estimate the fresh weight (fw), and stored at –80 °c. other seedlings were ovendried at 60 °c to determine the dry weight (dw). the total water content (twc) of shoots and roots was determined using the following formula: twc = fw – dw photosynthetic pigments using a spectroscopic approach, photosynthetic pigments, including chlorophyll a (chl a), chlorophyll b (chl b), and carotenoids (cars), were determined (lichtenthaler 1987). 0.1 g of a fresh leaf was dropped in 5 ml of 95% ethanol at 60 °c until colorless, and the volume was then finished to 10 ml using 95% ethanol. using a spectrophotometer (unico uv-2100), the concentrations of carotenoids and chlorophylls were determined using formulas: chl a = (13.36 × a663) – (5.19 × a644) chl b = (27.49 × a644) – (8.12 × a663) cars = {(1000 × a452) – (2.13 × chl a) – (9.76 × chl b)}/209. results were expressed as mg g–1 fw. cell membrane stability different parameters were assessed, including electrical conductivity (ec%), potassium leakage (k leakage), and uv-absorbing metabolites (metabolite leakage) for determining the cell membrane stability. the percentage of injury (electrical conductivity; ec) was measured according to the premachandra et al. (1992) method. fresh leaf discs (2.1 cm) were soaked in 10 ml of distilled water for 24 h at 10 °c. after measuring the initial electrical conductivity (ec1) of all test tubes at 25 °c, the leaf discs were autoclaved for 15 min, cooled to 25 °c, and then the last ec2 was measured again. the cell membrane stability index was estimated using a percentage of damage: electrical conductivity (%) = (ec1/ec2) × 100 potassium leakage was measured using a flame photometer in the same conductivity solution before and after sterilization (williams and twine 1960). metabolite leakage was assessed using the navari-lzzo et al. (1989) method in the same solution of conductivity measurements. boron analysis boron forms were extracted, according to du et al. (2002) and li et al. (2017). 5 ml of distilled water was added to 0.2 g of powdered dry seedlings, shaken at 25 °c for 24 h, filtered, and the free b was measured. the residue was shaken at 25 °c for 24 h in a plastic tube with 1 m nacl, filtered, and then semi-bound b was quantified in the filtrate. finally, the residue was shaken at 25 °c for 24 hours in a plastic tube with hcl (1 m), filtered, and then the bound b was quantified in the filtrate. according to mohan and jones (2018), b concentration was quantified using the curcumin-acetic acid method and detected at 550 nm. the curcumin-acetic acid (1 ml) solution was added to 1 ml of filtrates and 0.25 ml of concentrated h2so4, shaken for 30 min, and diluted with 95% ethanol to 5 ml after 30 min read at 550 nm using h3bo3 as a reference. fourier-transform infrared spectroscopy (ftir) analysis to analyze macromolecular alteration, we employed fourier-transform infrared spectroscopy (nicolet is 10 ftir) in chemistry department. a translucent, homogeneous tablet was prepared by a tablet-making machine using a little amount of the finely powdered sample (approximately 100 μg) mixed with kbr (1: 100 p/p). the absorbance of spectra was measured (400–4000 cm–1) against an ordinary kbr pellet (blank), then the resolution was 4 cm–1. the functional groups of the sample were determined by comparison of the spectroscopic result with a reference. statistical analysis the studies (25 jars/each treatment) were repeated at least twice, with the findings being an average ± standard deviation (sd) of four biological replicates. charts were generated by origin 8.6 and microsoft excel 2010. using spss radi a.a., salam h.kh., hamada a.m., farghaly f.a. 54 acta bot. croat. 82 (1), 2023 statistical package 22.0, a one-way analysis of variance test was performed and followed by a tukey’s test for significant differences (p � 0.05) to compare the means. the correlation between the mean values of different parameters of tomatoes under eb treatments was determined using pearson’s correlation analysis. results growth eb affected all growth parameters of the tested tomato seedlings, including fw, dw, and twc of shoots and roots (figs. 1a-d, on-line suppl. fig. 1). compared to control, tomatoes treated with 2 mm b showed a slight or considerable increase in fw, dw, and twc of shoots and roots. in contrast, treatments with 4 and 6 mm b lowered fw, dw, and twc of shoots and roots. after exposure to 6 mm b, the highest dw reductions of 56.90% and 86.52% were recorded in shoots and roots, respectively. further, treatment with eb showed a significant negative association between shoots and roots, fw, dw, twc, and an increase in free, semi, and bound b contents. however, the shoot/root ratio was positively and strongly associated with free, semibound, and bound b concentrations in the shoots (0.907**, 0.922**, and 0.646*, respectively, on-line suppl. tab. 1 and tab. 2). although the correlations between bound b and growth parameters were significant, they were the weakest of all the growth criteria associations. photosynthetic pigments eb had varied effects on the contents of chl a, b, a+b, and cars pigments in leaves (fig. 2a). compared to control, eb at a low-level (2 mm) stimulated chl a content in leaves by 31.28%, but at a high-level (6 mm), it significantly reduced it by 48.34%. in the shoots, there were strong negative associations between chl a and free, semi-bound, and bound b content (–0.768**, –0.822**, and –0.812**, respectively, on-line suppl. tab. 1). low eb treatments promoted the synthesis of chl b in tomato leaves. compared to control, the rise in chlorophyll b content at low eb levels (2 and 4 mm) was considerable (129.45% and 66.23%, respectively), but there was no significant increase at a high eb level (6 mm). insignificant relationships between chl b and free, semi-bound, and bound b levels in shoots confirmed these findings (–0.224, –0.311, and –0.341, respectively, on-line suppl. tab. 1). 6 mm b reduced chl a + b concentration by 42.69% compared to control. the moderate treatment (4 mm b) showed a lower increase in the chl a + b content (0.63%) than exposure to 2 mm b, which resulted in a 40.66% rise compared to control. moreover, results revealed a strong negative association between chl a + b and free, semi-bound, and bound b content in shoots (–0.704*, –0.767**, and –0.675**, respectively, on-line suppl. tab. 1). regarding carotenoids, eb boosted cars content by 37.18% at a low-level (2 mm) but lowered it by 44.72% at a fig. 1. fresh (a), dry weight (b), total water content (c), and shoot/root ratio (d) in tomato seedlings grown under 0, 2, 4, and 6 mm boron for 20 days. the data are means ± sd (n = 4). different letters, capital for roots and small for shoots, indicate statistically significant differences (p ≤ 0.05). boron toxicity and tomato seedling growth acta bot. croat. 82 (1), 2023 55 high level (6 mm) compared to control. carotenoids and free, semi-bound, and bound b levels had negative associations (–0.655*, –0.593*, and –0.686*, respectively), like chlorophylls (on-line suppl. tab. 1). moreover, the data revealed a significant and positive relationship between chl a, a + b, cars, and shoot dw (0.669*, 0.734**, and 0.785**, respectively), except for chl b, which was not. boron concentrations the most important factor in measuring a plant’s tolerance to eb is the b concentration in its tissues. therefore, the b forms in tomatoes grown under various eb treatments were measured (fig. 2b). our results indicated that free b content was higher than the content of semi-bound and bound b content in seedlings. our results indicated that free b content was higher than semi-bound and bound b content in seedlings. with increasing eb concentrations, the accumulation of all b forms also increased. compared with optimal b concentration, eb at the low level (2 mm) increased free, semi-, and bound b by 25.41%, 37.40%, and 88.61%, while the high level (6 mm) increased it by 149.69%, 134.98%, and 367.93%, respectively. fig. 2. photosynthetic pigments (chl a; chl b; chl a + b; cars; a) and boron concentration (free, semi-bound; and bound b) in tomato seedlings grown under 0, 2, 4, and 6 mm boron for 20 days. the data are means ± sd (n = 4). different letters, capital for chl a, free b and small for chl b, semi-bound b, small1 for chl a + b, bound b, small2 for cars, indicate statistically significant differences (p ≤ 0.05). fig. 3. electrical conductivity (ec; a), potassium leakage (k leakage; b), and uv absorbing metabolites (metabolite leakage; c) in tomato seedlings grown under 0, 2, 4, and 6 mm boron for 20 days. the data are means ± sd (n = 4). different letters indicate statistically significant differences (p ≤ 0.05). radi a.a., salam h.kh., hamada a.m., farghaly f.a. 56 acta bot. croat. 82 (1), 2023 membrane stability to quantify the degree of membrane integrity under eb stress, the ec, k leakage, and metabolite leakages in seedlings undergoing various treatments were measured (figs. 3a-c and on-line suppl. tab. 1 and tab. 2). 6 mm b raised the ec and incidence of k and uv metabolites in leaves by 67.22%, 101.54%, and 91.99%, respectively. furthermore, ec (0.931**, 890**, and 0.724**, respectively), k (0.943**, 0.915**, and 0.828**, respectively), and metabolite leakages (0.971**, 0.937**, and 0.687*, respectively) were shown to be strongly connected with free, semi-bound, and bound b levels in shoots. ftir analysis we employed ftir analysis to assess the effect of eb on seedling ultrastructure (fig. 4 and tab. 1). eb did not induce extensive alterations within the four peaks at 3405.17 cm–1, 2927.25 cm–1, 1384.45 cm–1, and 825.42 cm–1. treatment with 4 mm b raised the peak intensity of 3405.17 cm–1, but exposure to 2 and 6 mm b lowered it compared to control. moreover, treatments with 4 and 6 mm b raised the peak intensity at 2927.25 cm–1 and 1384.45 cm–1, respectively, while exposure to 2 mm b lowered them compared to control. however, eb levels raised the peak intensity of 825.45 cm–1 relative to control. regarding the peak at 1653.21 cm–1 (control), 2 mm b treatment did not significantly alter its transmission, while 4 and 6 mm concentrations lowered it by –6.34 cm–1 and –12.48 cm–1, respectively. moreover, 4 mm b raised this peak intensity, although other b treatments did not. meanwhile, the peak at 1540.60 cm–1 (control) disappeared under b treatments (2, 4, and 6 mm). the peak recorded at 1058.10 cm–1 (control) was negatively shifted by –3.20 cm–1, –4.27 cm–1, and –4.18 cm–1 upon exposure to 2, 4, and 6 mm b, respectively. moreover, treatments with 2 mm and 4 mm b stimulated this peak intensity, but treatment with 6 mm eb reduced it. compared to the peak recorded at 617.04 cm–1 (control), eb treatments increased from the transmission area by 1.04 cm–1, 5.31 cm–1, and 3.75 cm–1 at 2, 4 and 6 mm concentrations, respectively. furthermore, low levels of eb (2 and 4 mm) increased the peak intensity, while the treatment with 6 mm b decreased it. the peaks recorded at 540.52 cm–1, 536.78 cm–1, and 537.63 cm–1 appeared under treatments with 2, 4, and 6 mm b, respectively. under the high levels of eb (4 and 6 mm), the 483.52 cm–1 and 459.94 cm–1 peaks disappeared, while the exposure to 2 mm b stimulated their values by 0.74 cm–1 and 4.26 cm–1, respectively. discussion the shoot/root ratio of seedlings was enhanced at the lowest eb concentration, indicating the higher growth (figs. 1a-d). moreover, lower growth at high levels of eb was fig. 4. fourier-transform infrared spectroscopy (ftir) spectra (range 4000–400 cm–1; a, and 0–500 cm–1 region expanded; b) of tomato seedlings grown under 0, 2, 4, and 6 mm boron for 20 days. tab. 1. fourier-transform infrared spectroscopy (ftir) spectra showing observed peaks in tomato seedlings grown under 0, 2, 4, and 6 mm boron for 20 days. frequency (cm–1) excess boron (mm) peak 0 2 4 6 1 3405.17 3404.98 3405.43 3406.01 2 2927.25 2927.25 2926.60 2926.75 3 1653.21 1656.79 1646.87 1640.73 4 1540.60 – – – 5 1384.45 1384.28 1384.34 1384.30 6 1058.10 1054.9 1053.83 1053.92 7 825.42 825.55 825.68 825.42 8 617.04 618.08 622.35 620.79 9 – 540.52 536.78 537.63 10 483.52 484.26 – – 11 459.94 464.20 – – 12 – 453.29 – 453.74 boron toxicity and tomato seedling growth acta bot. croat. 82 (1), 2023 57 linked to the concentration of b forms within the seedlings, demonstrating that b buildup was limiting growth. as demonstrated by the strong positive relationships between shoot/root ratio and b level within shoots, the negative b effect was more evident in tomato roots than in shoots. eb has a comparable negative effect on tomato growth, according to kaya et al. (2020). cell division (liu and yang 2000), cell expansion, cell numbers (choi et al. 2007), water content (metwally et al. 2018), and cell wall matrix stiffness (farghaly et al. 2022b) are all linked to decreased seedling growth. conversely, promoting growth at a low eb level may be associated with active b influx, which lowers intracellular b levels (reid et al. 2004, ardic et al. 2009). the primary organelles damaged by eb are the chloroplasts (landi et al. 2019). the deficiency of photosynthetic pigments found in this study (fig. 2a) could be due to a structural damage to thylakoids as a result of abnormal spongy parenchyma growth (papadakis et al. 2004), oxidation of chlorophyll and chloroplast membranes (aftab et al. 2012), and a reduction in three types of thylakoid-related proteins (sang et al. 2015). our results match the findings of wheat and tomato, which are vulnerable to eb (elshazoly et al. 2019, kaya et al. 2020). thus, eb has a variety of consequences on photosynthetic processes, including changes in photosynthetic pigment levels, lower co2 assimilation, impaired photosystem ii performance, and a decreased electron transport rate (landi et al. 2019). high content of photosynthetic pigments at a low eb level suggests seedling tolerance. furthermore, the chloroplasts were less vulnerable to eb since the dw was high at this level of 2 mm eb. additionally, strong positive relationships between pigment contents and shoot dw revealed that pigment preservation is necessary to stimulate seedling growth. accordingly, eraslan et al. (2007) found no significant changes in chl a and b concentration in carrot plants when exposed to eb. boron amount in plants is considered the main physiological feature utilized to examine tolerance to eb in an environment. our findings revealed that all b forms were significantly increased in eb-stressed seedlings, explaining the symptoms of increased eb (fig. 2b). likewise, during exposure to eb, an accumulation of b forms was previously observed in tomato calli (farghaly et al. 2021, 2022b). free b demonstrated the ability to cross cell membranes and showed promise as being immediately accessible for potential physiological roles in the cell, according to dannel et al. (1998). in this study, the content of semi-bound and bound b forms varied from about 6%–76% and 2%–119%, respectively. these data may reveal that a small amount of eb was attached to the cell walls in exchange for increased b availability, but this amount was too low to actively participate in eb detoxification, as indicated by increased free b ( dannel et al. 1998, farghaly et al. 2022b). ionic solutes and cellular metabolites are widely applied to assess membrane integrity (palta et al. 1977, navari-izzo et al. 1993). according to our findings presented in figs. 3a-c, the membrane damage was more severe as eb levels in the medium increased. these findings showed that eb had a significant impact on the permeability of tomato membranes, revealed by the ec and leakage of k and uv metabolites, which were all confirmed in a prior work with wheat ( metwally et al. 2012). ftir spectra revealed further information about the influence of eb on seedling macromolecules (fig. 4 and tab. 1). türker-kaya and huck (2017) correlate the first peak, recorded at 3405.17 cm–1 in control, with o-h and n-h related to alcohol, carbohydrates, phenols, and proteins. eb did not affect the wavenumber, indicating that the lack of alterations in cell wall components and the reduction in bound-b in seedlings may clarify these findings. furthermore, eb lowered peak intensity, suggesting that eb may change the pattern in binding between alcohols, carbohydrates, proteins, phenols, and components of walls. riaz et al. (2021) demonstrated that eb increased lignin and suberin levels in rice plants, perhaps leading to cell wall stiffness. otherwise, changes in peak intensity can be referred to as changes in cell wall shape (zuverza-mena et al. 2016). the peak found around 3000–2800 cm–1 was assigned the c-h stretching area of lipids, wax, and fats (legner et al. 2018). eb did not affect this peak’s value (3000–2800 cm–1), but the intensity of the peak increased at 4 and 6 mm eb. these data indicate that no changes were made to wall wax amount, while the shape of wall wax may only change under eb (morales et al. 2013). mesquita et al. (2016) found irregular wax deposition on the surface of citrus leaves under eb, which might support our findings. the peak in the region of 1700–1600 cm–1 is characteristic of the c=o of the amide i (proteins) (dumas and miller 2003). the amide ii peak in the region of 1480–1580 cm–1 is a mixture of n-h and c-n vibrations that aid in ionic reaction response, although it is less well understood (zhao and wang 2016). the amide i peak value was reduced by high levels of eb (4 and 6 mm), demonstrating that the protein’s structure is changed to chelate eb, and this explanation might be confirmed by the finding of farghaly et al. (2022a). according to riaz et al. (2021), eb significantly affected the amide protein, amide ii, and amide iii, indicating damage to the protein pools. the disappearance of the amide ii peak under eb treatments can disclose the binding of b ions to nitrogen amide to chelate the eb, and plants can use this claw to withstand b toxicity. dunbar et al. (2012) reported the disappearance of amide ii (around 1550 cm–1; bending of amide n-h) owing to iminol structural coordination between the amide ii and magnesium, nickel, and cobalt. wei et al. (2015) assigned the peaks between 1500–1000 cm–1 fingerprint regions of the amide iii, nucleic acid functional groups, and carbohydrates. our results revealed that eb treatments induce a change in the intensity of the peak recorded at 1384.45 cm–1. the increasing peak intensity might reveal that the additional b has changed the fingerprint region’s components and linked eb with proteins. radi a.a., salam h.kh., hamada a.m., farghaly f.a. 58 acta bot. croat. 82 (1), 2023 eb reduced the 1058.10 cm–1 peak, which was attributed to cellulose (wu et al. 2017), indicating a reduction in cellulose production in seedlings under eb. this peak intensity was lowered by eb at its maximum level, indicating a decrease in cellulose synthesis. similarly, farghaly et al. (2022b) discovered that eb decreased the cellulose content of tomato calli in their study. the peak recorded at 825.42 cm–1 in control, which was not affected by eb treatments, was assigned to the trisaccharide (d-(+)-raffinose pentahydrate) with α-glycosidic bonds (wiercigroch et al. 2017). the intensity of this peak was increased, indicating b binding to the pentahydrate. eb also boosted the 617.04 cm–1 peak, which was assigned to d(+)-glucose (wiercigroch et al. 2017), showing the degradation of cellulose or sucrose into simple monosaccharides. this explanation might confirm a decrease in the cellulose wavelengths. under eb, the appearance of additional peaks, recorded at 540.52 cm–1, 536.78 cm–1, and 537.63 cm–1, may also demonstrate glucose buildup (farghaly et al. 2022b). furthermore, at high eb levels, the disappearance of ribose peaks (484 cm–1 and 460 cm–1; wiercigroch et al. 2017) suggested eb binding to ribose, and this confirmed the ability of eb to stabilize ribose to create a nucleotide of a borate ester (grew et al. 2011, scorei 2012). in conclusion, eb treatments exhibited unfavorable influences on fw, dw, twc, and photosynthetic pigments of tomato seedlings. eb also caused a reduction in membrane integrity, as seen by higher ec, and k and uv-metabolite leakage. b absorption matched the b content in the nutritional medium, resulting in increased accumulation of various b forms in seedlings. eb inhibited cellulose synthesis in seedlings and altered wax deposition in cell walls. moreover, eb affected the amide i and amide ii indicating damage to the protein pools. finally, our results reveal that decreased tomato growth under eb might be related to alterations in photosynthetic pigments, membrane stability, and macromolecules. acknowledgments the authors thank assiut university for providing the laboratory equipment. references aftab, t., khan, m.m., naeem, m., idrees, m., moinuddin, a.s., teixeira da silva, j.a., ram, m., 2012: exogenous nitric 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(eds.), modern methods of plant analysis, vol 5, 3–5. springer, berlin. wu, x.w., muhammad, r., yan, l., du, c.q., liu, y.l., jiang, c.c., 2017: boron deficiency in trifoliate orange induces changes in pectin composition and architecture of components in root cell walls. frontiers of plant science 8, 1882. zhao, j., wang, j., 2016: uncovering the sensitivity of amide-ii vibration to peptide-ion interactions. the journal of physical chemistry b 120, 9590–9608. zuverza-mena, n., armendariz, r., peralta-videa, j.r., gardeatorresdey, j.l., 2016: effects of silver nanoparticles on radish sprouts: root growth reduction and modifications in the nutritional value. frontiers of plant science 7, 90. opce-str.vp social news u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:10 color profile: disabled composite 150 lpi at 45 degrees u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:10 color profile: disabled composite 150 lpi at 45 degrees celebrating the eightieth birthday of professor peter sitte peter sitte was born on december 8, 1929, in innsbruck (austria), where he grew up and after the war studied biology, chemistry, physics and philosophy, finishing his studies in 1954 with a phd degree. from 1959 to 1965 he was associate professor at the university of heidelberg (germany). from time to time he worked as visiting professor in sweden, in the united states of america and in austria. from 1966 to 1995 he was appointed full professor of cell biology at the university of freiburg (germany). the main fields of his research and theoretical interests were: 1. fine structure of plant cells (techniques and methods in light, polarizing, and electron microscopy; structure of cell walls, particularly those of spores and pollen grains and of suberinised cells; general ultrastructure of plant cells; development and fine structure of plastids, especially chromoplasts and gerontoplasts; studies on the molecular structure and function of intracellular membranes). 2. cytosymbiosis (general organisation of the eucytes as a consequence of endosymbiosis – cells in cells; in this connection, intense investigation of genomes and nucleic acids in cryptomonads; the significance of cytosymbiosis in cell evolution. for some years, sitte led a german research program on endocytobiotic systems). 3. bioesthetics (the beauty of organisms; symmetry in plants, phyllotaxis; symmetry in nature and art; esthetics as a basic value in education and philosophy). 4. philosophical and social aspects of natural sciences (the historical development of cell research and the golden age of cell biology; the evolution of evolution theory; natural sciences and humanities in the evolution of culture; the seeming but not real contradiction of religious belief and evolution theory; biology as a key science in modern society: biology as the science of the century). peter sitte is author, coauthor, editor and coeditor of many important books and journals, among them the books »bau und feinbau der pflanzenzelle« (structure and fine structure of the plant cell), 1965; together with hans mohr: »molekulare grundlagen der entwickiung« (molecular fundaments of development) 1971; with g. c. hirsch and h. ruska: »grundlagen der cytologie« (fundamental principles of cytology) 1973; with hans kleinig: »zellbiologie – ein lehrbuch« (cell biology – a textbook), three editions in 1984, 1986 and 1992; with three and four coauthors respectively: »strasburger – lehrbuch der botanik« (strasburger's textbook of botany), three editions, 1991, 1998, 2002 (35th edition). with this extraordinary work sitte earned a great reputation, not only as an author but also as the leading editor, being the primus inter pares among his colleagues. acta bot. croat. 69 (1), 2010 137 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees he also edited »horizonte der biologie« (horizons of biology) 1993; he was editor of the interesting book »jahrhundertwissenschaft biologie« (biology as the science of the century) 1999. from 1971 to 1983 he was editor in chief of the popular journal »biologie in unserer zeit« (biology in our time). for certain periods of time, he also acted as president of various german scientific societies (elektronenmikroskopie, zellbiologie; gesellschaft deutscher naturforscher und ärzte). sitte is a member of the deutsche akademie der naturforscher leopoldina, as well as of the academies in göttingen and vienna. for some years he was a member of the academic council of the humboldt society. he received some awards among them the schleiden-medaille, the miescher-ishida award, the oken medaille and the treviranus-medaille of the vbiol. he became doctor honoris causa of natural sciences of the university of salzburg, and he is honorary member of the german botanical society. professor sitte is well known as a very kind and helpful personality who always is willing to assist and to support his colleagues and students. in this regard the croatian botanists and biologists are very much in his debt, and wish to express their sincere thanks for the great help professor sitte has given them ever since 1955 by sending papers, books, journals (e.g. many volumes of the journal »biologie in unserer zeit«), giving them hospitality in his laboratories and helping them by direct advice and suggestions. on this important birthday, we wish him good health, much vital strength and in general all the best for him to continue in his enjoyment of life, a life that he has devoted thoroughly to the biology and natural sciences as well as to the arts and to the piano, on which he is an impassioned performer. in the name of all our croatian colleagues and the editorial board of the acta botanica croatica, zvonimir devidé journal papers, congress proceedings papers, books and book chapters sitte, p., 1953: untersuchungen zur submikroskopischen morphologie der pollenund sporenmembranen. mikroskopie 8, 290–299. sitte, p., 1954: untersuchungen zum feinbau verkorkter zellwände. rapport europaean congress toegepaste electronenmicroscopie, gent, 83–89. sitte, h., sitte, p., 1955: technik und methoden der elektronenmikroskopie, 1. prinzip und arbeitsweise des elektronenmikroskopes. desgl. 2. elektronenmikroskopische präparationsmethoden. pyramide 4, 131–142; 5, 7–15. sitte, p., 1955: der feinbau verkorkter zellwände. mikroskopie 10, 178–200. sitte, p., sitte-lürzer, e., 1956: was ist palynologie? pyramide 5, 94–101. sitte, p., 1957: bildungsabweichungen an wirteligen sprossen und ihre entwicklungsphysiologische und genetische bedeutung. österreichische botanische zeitung 104, 234–302. sitte, p., 1957: der feinbau der kork-zellwände. sowie: morphologie des cutins und des sporopollenins. in: treiber, e., hrsg. (ed.), die chemie der pflanzenzellwand, 421– 432; 439–445. springer-verlag, berlin. 138 acta bot. croat. 69 (1), 2010 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees sitte, p., 1958: die ultrastruktur von wurzelmeristemzellen der erbse (pisum sativum). protoplasma 49, 447–522. bachmann, l., sitte, p., 1959: dickenbestimmung nach tolansky an ultradünnschnitten. mikroskopie 13, 289–304. sitte, p., 1959: mischkörperdoppelbrechung der kork-zellwände. naturwissenschaften 46, 260–261. sitte, p., 1959: polarisationsoptische untersuchungen an sporodermen. zeitschrift für naturforschung 14b, 575–582. falk, h., sitte, p., 1960: untersuchungen am caspary-streifen. proceedings europaean regional conference electron-microscopy. ii, delft, 1063–1066. sitte, p., 1960: die probleme der pflanzenhaut. pyramide 8, 42–49. sitte, p., 1960: die optische anisotropie der sporodermen. grana palynologica 2, 16–38. sitte, p., 1960: zur blasenfreien plexiglas-einbettung von pflanzenmaterial. mikroskopie 15, 227–229. sitte, p., 1960: das eindringen von osmiumtetroxyd-, kaliumpermanganatund formaldehydlösungen in pflanzengewebe. histochemie 2, 76–86. sitte, p., 1961: imbibition mit gemischen von methylenjodid und methoxy-essigsäure-methylester für die polarisationsmikroskopie. mikroskopie 16, 18–23. sitte, p., 1961: zum bau der plastidenzentren in wurzelproplastiden. protoplasma 53, 438–442. sitte, p., falk, h., 1961: artefakte in pflanzlichen zellwänden durch methacrylat-einbettung. zeitschrift wissenschaftliche mikroskopie und technik 65, 26–30. sitte, p., 1961: die submikroskopische organisation der pflanzenzelle. berichte der deutschen botanischen gesellschaft 74, 177–200. sitte, p., 1961: zum feinbau der suberinschichten im flaschenkork. protoplasma 54, 555–559. sitte, p., 1961: polarisationsmikroskopie der mitose in vivo bei staminalhaarzellen von tradescantia. protoplasma 54, 560–572. sitte, p., 1962: feinbau und funktion der pflanzenzellwand, i und ii. umschau 62, 236–9, 273–276. sitte, p., 1962: einfaches verfahren zur stufenlosen gewebe-entwässerung für die elektronenmikroskopische präparation. naturwissenschaften 49, 402–403. sitte, p., 1962: zum chloroplasten-feinbau bei elodea. portugalia acta biologica series a 6, 269–278. sitte, p., 1963: hexagonale anordnung der globuli in moos-chloroplasten. protoplasma 56, 197–201. falk, h., sitte, p., 1963: zellfeinbau bei plasmolyse i. der feinbau der elodea-blattzellen. protoplasma 57, 290–303. sitte, p., 1963: desgl. ii. der feinbau der elodea-blattzellen bei zuckerund ionenplasmolyse. protoplasma 57, 304–333. sitte, p., rennier, r., 1963: untersuchungen an cuticularen zellwandschichten. planta 60, 19–40. acta bot. croat. 69 (1), 2010 139 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees sitte, p., 1963: zur kenntnis der zwischenschichte im sporoderm von pinus mugo. grana palynologica 4, 41–52. sitte, p., 1963: bau und bewegung der sporen-hapteren bei equisetum arvense. berichte des naturwissenschaftlich-medizinischen vereins innsbruck 53, 193–207. sitte, p., 1963: feinbau und bewegung der sporenhapteren bei equisetum. berichte der deutschen botanischen gesellschaft 76, 342–343. sitte, p., 1965: feinbau der zelle bei höheren organismen. fortschritte der botanik 27, 15–35. sitte, p., 1965: bau und feinbau der pflanzenzelle. veb g. fischer verlag, jena, und gustav fischer verlag, stuttgart. sitte, p., 1966: submikroskopische cytologie der eukaryotischen zelle. fortschritte der botanik 28, 14–28. sitte, p., 1966: allgemeine mikromorphologie der zelle. in: metzner h. (hrsg.), die zelle – struktur und funktion, 7–56. wissenschaftl. verlagsgesellschaft, stuttgart. neubearbeitungen für 2. aufl. 1971: 8–69, sowie für 3. aufl. 1981: 9–89. geitler, l., tschermak-woess, e., sitte, p., 1967: morphologie und entwicklungsgeschichte der zelle. fortschritte der botanik 29, 1–24. sitte, p., 1968: molekulararchitektur von biomembranen. schriften des vereins zur verbreitung naturwissenschaftlicher kenntnisse wien 108, 129–168. sitte, p., 1969: biologie – kritisches zu ihrem selbstverständnis und ihrer situation an den universitäten der bundesrepublik. freiburger universitäts-blätter 24, 21–36. (vgl. auch mitteilungen des verbandes deutscher biologen 150, 723–730.) sitte, p., 1969: biomembranen: struktur und funktion. berichte der deutschen botanischen gesellschaft 82, 329–383. sitte, p., 1969: submikroskopische und molekulare struktur der zelle. fortschritte der botanik 31, 18–44. brown, r. m. jr., franke, w. w., kleinig, h., falk, h., sitte, p., 1969: cellulosic wall components produced by the golgi apparatus of pleurochrysis scherffelii. science 166, 894–896. brown, r. m. jr., franke, w. w., kleinig, h., falk, h., sitte, p., 1970: scale formation in chrysophycean algae i. cellulosic and noncellulosic wall components made by the golgi apparatus. journal of cell biology 45, 246–271. sitte, p., 1970: biomembranen. in: sund, h. (hrsg.), große moleküle, 119–159. suhrkamp, frankfurt/m. mohr, h., sitte, p., 1971: molekulare grundlagen der entwicklung. blv, münchen sitte, p., ab 1971: zahlreiche, nur z.t. namentlich signierte kurzbeiträge in 'biologie in unserer zeit', verlag chemie, weinheim. sitte, p., 1972: submikroskopische und molekulare struktur der zelle. fortschritte der botanik 34, 1–41. sitte, p., 1973: methodengefüge und erkenntnisfortschritt. naturwissenschaften 60, 333–339. sitte, p., 1973: zellwand-feinbau bei flughaaren der clematis-frucht. berichte der deutschen botanischen gesellschaft 86, 551–561. 140 acta bot. croat. 69 (1), 2010 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees sitte, h., sitte, p., 1973: methoden der cytologischen forschung. in: hirsch, g. c., ruska, h., sitte, p. (hrsg.), grundlagen der cytologie', 35–80. veb g. fischer, jena, und gustav fischer verlag, stuttgart. (2., unveränd. aufl. 1974). sitte, p., 1973: molekulare morphologie der zelle. ibidem, 81–116. egger, k., sitte, p., 1973: plastiden und photosynthese. ibidem, 345–387. sitte, p., 1973: allgemeine morphologie der pflanzenzelle. ibidem, 391–412. liedvogel, b., sitte, p., 1974: lipids and proteins in lipid-rich chromoplast membranes. naturwissenschaften 61, 131. sitte, p., 1974: plastiden-metamorphose und chromoplasten bei chrysosplenium. zeitschrift für pflanzenphysiologie 73, 243–265. falk, h., liedvogel, b., sitte, p., 1974: circular dna in isolated chromoplasts. zeitschrift für naturforschung 29c, 541–544. sitte, p., 1974: methoden und erfolge der zellbiologie. in: todt d. (hrsg.), funkkolleg biologie, 12. studienbegleitbrief, 45–81. deutsches institut für fernstudien, beltz verlag, weinheim. (vgl. auch 'systeme des lebendigen', bd. 2. fischer taschenbuch verlag, stuttgart 1976. sitte, p., 1975: zelluläres und molekulares: biologie der minimal-organismen. mannheimer forum 75/76, 55–117. sitte, p., 1975: die bedeutung der molekularen lamellenbauweise von korkzellwänden. biochemie und physiologie der pflanzen 168, 287–297. sitte, p., 1975: elektronenmikroskopie und biologie – schicksal einer symbiose. mikroskopie 32, 145–190. winkenbach, f., falk, h., liedvogel, b., sitte, p., 1976: chromoplasts of tropaeolum majus l.: isolation and characterization of lipoprotein elements. planta 128, 23–28. liedvogel, b., sitte, p., falk, h., 1976: chromoplasts in the daffodil: fine structure and chemistry. cytobiologie 12, 155–174. sitte, p., 1976: forschung und humanität. kassenarzt 16, 2018–2035. (vgl. auch umschau 76, 574–580.) sitte, p., 1976: zur chemischen fixierung von lipidstrukturen. mikroskopie 32, 208–209. sitte, p., 1976: 4 kapitel in 'biologie. in: czihak, g., langer, h., ziegler, h. (hrsg.), ein lehrbuch für studenten der biologie, 5–24; 25–47; 61–82; 133–138. springer-verlag, berlin. bearbeitungen f. weitere auflagen, bis incl. 5. aufl., 1992, 7–52; 134–174. sitte, p., 1977: chromoplasten – bunte objekte der modernen zellbiologie. biologie in unserer zeit 7, 65–74. sitte, p., 1977: die lebende zelle als system, systemelement und übersystem. nova acta leopoldina nf 47/226, 195–216. sitte, p., 1977: functional organization of biomembranes. in: tevini, m., lichtenthaler, h. k., (eds.), lipids and lipid polymers in higher plants, 1–28. springer-verlag, berlin. sitte, p., 1979: biomembranes: high protein concentration. naturwissenschaften 66, 315–316. acta bot. croat. 69 (1), 2010 141 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees sitte, p., 1979: unterwegs zu einem weltbild der naturwissenschaften. naturwissenschaften 66, 273–278. (vgl. auch verhandlungen der gesellschaft deutsch naturforscher und ärzte 1978, 10–17.) sitte, p., 1979: general principles of cellular compartmentation. in: nover, l., lynen, f., mothes, k. (eds.), cell compartmentation and metabolic channeling, 17–32. veb g. fischer verlag, jena, und elsevier/north holland biomed. press, amsterdam. sitte, p., 1980: naturwissenschaftliches weltbild und gesellschaft. freiburger universitäts-blätter 67, 48–61. sitte, p., 1980: electron microscopy and the understanding of life. in 'electron microscopy 1980' (brederoo, p., de priester, w., eds.), vol. 2, 818–825. leiden. sitte, p., 1980: nematic liquid crystals of lipid pigments in chromoplasts. europaean journal of cell biology 22, 280. sitte, p., falk, h., liedvogel, b., 1980: chromoplasts. in: czygan, f.-c. (ed.), pigments in plants, 2, 117–148. g. fischer verlag, stuttgart. sitte, p., 1981: vom maximum zum optimum. umschau 81, 584–588. sitte, p., 1981: quantitative bestimmung der lipophilie von osmiumtetroxid. verhandlungen der anatomischen gesellschaft 75, 655–656. sitte, p., 1981: die endosymbionten-hypothese – eine kritische betrachtung zur zell-evolution. nova acta leopoldina 56, nf 251, 41–58. sitte, p., 1981: wesen und auftrag der naturwissenschaften. in: sitter, b., weber, r., (hrsg.), wissenschaft in frage gestellt, 69–86. paul haupt, bern. sitte, p., 1981: role of lipid self-assembly in subcellular morphogenesis. in: kiermayer, o. (ed.), cytomorphogenesis in plants, 401–421, springer-verlag, wien. sitte, p., 1982: mutiger anstoß. stellungnahme zu 'wo stünde die biologie ohne darwin?' die zeit 37/16, 61. hansmann, p., sitte, p., 1982: composition and molecular structure of chromoplast globules of viola tricolor. plant cell reports 1, 111–114. sitte, p., 1982: das weltbild der naturwissenschaften als aufgabe der forschung. universitas 37, 375–384. sitte, p., 1982: die entwicklung der zellforschung. berichte der deutschen botanischen gesellschaft 95, 561–580. sitte, p., 1983: general organization of the eucyte and its bearing on cytosymbiosis and cell evolution. in: schenk, h. e. a., schwemmler, w. (eds.), endocytobiology ii, 101–119. de gruyter, berlin. sitte, p., 1983: science today, and science tomorrow. in: staudinger, m. (ed.), evolution of responsibilities and values today, 207–220. deutsche unesco-kommission, bonn. hansmann, p., sitte, p., 1984: comparison of the polypeptide complement of different plastid types and mitochondria of narcissus pseudonarcissus. zeitschrift für naturforschung 39c, 758–766. sitte, p., 1984: symmetrie bei organismen. biologie in unserer zeit 14, 161–170. kleinig, h., sitte, p., 1984: zellbiologie. ein lehrbuch. xii + 488 s. gustav fischer verlag, stuttgart. 142 acta bot. croat. 69 (1), 2010 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees sitte, p., 1985: keimplasma-theorie und genom-konstanz. freiburger universitäts-blätter 87/88, 91–98. hansmann, p., falk, h., ronai, k., sitte, p., 1985: structure, composition, and distribution of plastid nucleoids in narcissus pseudonarcissus. planta 164, 459–472. hansmann, p., falk, h., sitte, p., 1985: dna in the nucleomorph of cryptomonas demonstrated by dapi fluorescence. zeitschrift für naturforschung 40c, 933–935. hansmann, p., falk, h., scheer, u., sitte, p., 1986: ultrastructural localization of dna in two cryptomonas species by use of a monoclonal dna antibody. europaean journal of cell biology 42, 152–160. knoth, r., hansmann, p., sitte, p. 1986: chromoplasts of palisota barteri, and the molecular structure of chromoplast tubules. planta 168, 167–174. kleinig, h., sitte, p., 1986: zellbiologie. ein lehrbuch. 2. aufl. gustav fischer verlag, stuttgart. sitte, p., hansmann, p., 1986: cytosymbiosis. progress in botany 48, 30–55. sitte, p., 1987: das elektronenmikroskop in biologie und medizin. (festvortrag zur nobelpreis-verleihung an ernst ruska.) futura 87/1, 7–11. sitte, p., 1986: die moleküle des lebens – einführung. in 'die moleküle des lebens', s. 7–9. verlag spektrum d. wissenschaft, heidelberg. sitte, p., 1986: bio-symmetrie. symmetrie in kunst, natur und wissenschaft. mathildenhöhe darmstadt. sitte, p., 1986: zellen in zellen: endocytobiose und die folgen. in: lüst, r., et al. (hrsg.), beobachtung, experiment und theorie in naturwissenschaft und medizin, 431–446. wiss. verlagsges., stuttgart. hansmann, p., junker, r., sauter, h., sitte, p., 1987: chromoplast development in daffodil coronae during anthesis. journal of plant physiology 131, 133–143. hansmann, p., maerz, m., sitte, p., 1987: investigations on genomes and nucleic acids in cryptomonads. endocytobiology and cell research 4, 289–295. sitte, p., 1987: development and division of chromoplasts in petals of forsythia. la cellule 74, 59–77. sitte, p., 1987: geleitwort zu 'elektronenmikroskopische methodik in der zellund molekularbiologie', plattner, h., zingsheim, h. g., sn. vii–viii. gustav fischer verlag, stuttgart. sitte, p., 1988: ästhetik als grundwert der bildung. in: böhm, w., lindauer, m. (hrsg.), 3. würzburger symposium, nicht vielwissen sättigt die seele, 323–348. klett, stuttgart. hansmann, p., maerz, m., sitte, p., 1989: cytosymbiosis. progress in botany 51, 21–47. sitte, p., 1990: phylogenetische aspekte der zellevolution. biologische rundschau 28, 1–18. sitte, p., 1990: editorial: biuz im 20. jahrgang. biologie in unserer zeit 20, 7. eschbach, s., speth, v., hansmann, p., sitte, p., 1990: freeze-fracture study of the single membrane between host cell and endocytobiont in the dinoflagellates glenodinium foliaceum and peridinium balticum. journal of phycology 26, 324–328. acta bot. croat. 69 (1), 2010 143 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees emter, o., falk, h., sitte, p., 1990: specific carotenoids and proteins as prerequisites for chromoplast tubule formation. protoplasma 157, 128–135. sitte, p., baltes, s., 1990: morphometric analysis of two cryptomonads: quantitative evaluation of fine-structural changes in an endocytobiotic system. in: nardon, p., et al. (eds.), endocytobiology iv, 229–233. inra, paris. sitte, p., 1991: die zelle in der evolution des lebens. biologie in unserer zeit 21, 85–92. (vgl. auch: jb. akad. wiss. göttingen 1989, 30–47.) sitte, p., 1991: biologie und humanitas. freiburger universitäts-blätter 111, 57–67. sitte, p., 1991: schon der keim ist mensch. die welt v. 10-6-91, nr. 132, s. 19. sitte, p., 1991: wege, einsichten und möglichkeiten der biologie. naturwissenschaftliche rundschau 44, 337–347. eschbach, s., wolters, j., sitte, p., 1991: primary and secondary structure of the nuclear small-subunit ribosomal rna of the cryptomonad pyrenomonas salina as inferred from the gene sequence: evolutionary implications. journal molecular evolution 32, 247–252. eschbach, s., hofmann, c. j. b., maier, u.-g., sitte, p., hansmann, p., 1991: a eukaryotic genome of 660 kb: electrophoretic karyotype of nucleomorph and cell nucleus of the cryptomonad alga, pyrenomonas salina. nucleic acids research 19, 1779–1781. maerz, m., sitte, p., 1991: isolation, physical map and gene map of mitochondrial dna from the cryptomonad pyrenomonas salina. plant molecular biology 16, 593–600. maerz, m., wolters, j., hofmann, c. j. b., sitte, p., maier, u.-g., 1991: plastid dna from pyrenomonas salina (cryptophyceae): physical map, genes, and evolutionary implications. current genetics 21, 73–81. sitte, p., 1991: umdenken – das tut niemand gern. in: m. oesterreicher-mollwo (hrsg.), was uns bewegt. naturwissenschaftler sprechen über sich und ihre welt, s. 222–238. beltz, weinheim u. basel. sitte, p., 1991: einleitung und 1. teil: morphologie, in strasburger, lehrbuch der botanik für hochschulen, 33. aufl., v + 238 sn. g. fischer verlag, stuttgart u. new york. sitte, p., 1991: strukturen und funktionen lebender systeme: erkennung und deutung. in: marx, w. (hrsg.), die strukturen lebendiger systeme – zu ihrer wissenschaftlichen und philo-sophischen bestimmung, 9–25. klostermann, frankfurt/m. sitte, p., 1992: phyllotaxis – paradigma einer organismischen musterbildung. nova acta leopoldina n.f. 67, 285–307. sitte, p., eschbach, s., 1992: cytosymbiosis and its significance in cell evolution. progress in botany 53, 29–43. sitte, p., 1992: german research program on endocytobiotic systems and their evolution. endocytobiology andcell research 8, 197–198. sitte, p., maier, u.-g., 1992: evolution of 'complex plastids' from eukaryotic endosymbionts. endocytobiology and cell research 8, 223–225. sitte, p., 1992: a modern concept of the 'cell theory'. international journal of plant science 153, 1–6. 144 acta bot. croat. 69 (1), 2010 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees kleinig, h., sitte, p., 1992: zellbiologie. ein lehrbuch. xii, 591 sn., 515 abbn., 82 tab. 3., neubearb. aufl. g. fischer verlag, stuttgart. sitte, p., eschbach, s., maerz, m., 1992: the role of symbiosis in algal evolution. in: reisser, w., (ed.), algae and symbioses, 711–733. biopress, bristol. sitte, p., 1993: subjektive und objektive grenzen für das erkennen der grenzen unseres wissens am beispiel der evolutionstheorie. ethik und sozialwissenschaften 4, 102– 109. sitte, p., 1993: symbiogenetic evolution of complex cells and complex plastids. europaean journal of protistology 29, 131–143. sitte, p., 1993: zu diesem buch (vorwort). in: sitte, p. 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(eds.), endocytobiology v, 169–177. tübingen, univ. press. sitte, p., 1993: 'intertaxonic combination': introducing a new term in symbiognesis. ibid. 557–558. maier, u.-g., sitte, p., 1994: cryptomonad evolution: insights into a 'eucyte within a eucyte'. endocytobiology and cell research 10, 129–135. sitte, p., 1994: wissen wir genug vom 'yang' der evolution? ethik und sozialwissenschaften 5, 253–255. sitte, p., 1994: zukunft der biologie – biologie der zukunft. versuch einer perspektive. biologen in unserer zeit 4/94, 49–55. sitte, p., 1994: vorlesung 2001 – versuch einer enthüllung. biologie in unserer zeit 24, 286–290. sitte, p., 1994: physo-analyse? biologie in unserer zeit 24, 5. sitte, p., 1994: natura nusquam magis est tota quam in minimis: die enthüllung des lebenden mikrokosmos. in: moltmann, u. g. (red.), 100 jahre strasburgers lehrbuch der botanik für hochschulen 1894–1994, 79–99. g. fischer verlag, stuttgart. sitte, p., 1994: die evolution von zellen: innovation durch symbiogenese. in: wieser, w. (hrsg.), die evolution der evolutionstheorie, 77–108. spektrum akadem. verl., heidelberg. sitte, p., 1995: fahrten meines lebens: erlebtes und erdachtes. freiburger universitätsblätter 128, 23–41. sitte, p., 1995: das goldene zeitalter der zellbiologie. in: braune, w., krause, e., sitte, p. (hrsg.), die evolution des zellbildes seit eduard strasburger. festschrift eduard strasburger 1844–1912, 39–62. univ.-verlag jena. maier, u.-g., hofmann, c. j. b., sitte, p., 1996: die evolution von zellen. naturwissenschaften 83, 103–112. sitte, p., 1996: biologie und kunst. die besondere ästhetik des lebendigen. biologie in unserer zeit 27, 151–160. acta bot. croat. 69 (1), 2010 145 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees sitte, p., 1997: facts and concepts in cell compartmentation (review). progress in botany 59, 3–45. springer-verlag, berlin. sitte, p., 1997: ästhetik des lebendigen. das schöne als kulturund lebensproblem. in: kulturzeit der wissenschaft (hörsaal holzen 3), 25–41. hochschule holzen, kandern-holzen. sitte, p., 1999: die ‚listenreiche' evolution. in: von senger, h. (hrsg.), täuschung bei tieren, pflanzen, bakterien und viren. die list, 475–496. suhrkamp verlag, frankfurt/m. sitte p., hrsg., 1999: jahrhundertwissenschaft biologie – die großen themen. darin vorwort, einleitungstexte, und beitrag »von der schönheit des lebendigen«, s. 405– 425. c. h. beck, münchen. sitte, p., 2001: symbiogenese in der zellund lebensevolution. in: storch,v., welsch, u., wink, m. (hrsg.), evolutionsbiologie, 196–208. springer-verlag, berlin. sitte, p., 2003: wesen, werden und wachsen der lebenswelt. heidelberger jahrbuch 47, 71–96. sitte, p., 2003: die biologie als schlüsselwissenschaft in der modernen gesellschaft. studien verlag, innsbruck. sitte, p., 2003: natur und kunst. die besondere ästhetik des lebendigen. in: kummer, c. (hrsg.), die andere seite der biologie, 46–65. christian kummer, münchen. sitte, p., 2005: schöpfung oder evolution? das hartnäckige mißverständnis. zur debatte (themen der katholischen akademie in bayern) 35/5, 38–41. sitte, p., 2007: naturwissenschaften und medizin in der kulturellen evolution, i. rückblick und einblicke; ii. ausblick vom heute ins morgen. festschrift der universität freiburg zu ihrem 550-jährigen bestehen, 19–47, 77–88. verlag karl alber, freiburg/ münchen. sitte, p., 2008: evolutionäre ästhetik und funktionelle schönheit. in: klose, j., oehler, j. (hrsg.), gott oder darwin? vernünftiges reden über schöpfung und evolution, 331–348. springer-verlag berlin. 146 acta bot. croat. 69 (1), 2010 u:\acta botanica\acta-botan 1-10\social news.vp 9. travanj 2010 13:39:12 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (2), 301–306, 2011 coden: abcra 25 issn 0365-0588 short communication ammi visnaga (l.) lam. (apiaceae), a new taxon in croatian flora mirko ru[^i]1, toni nikoli]2* 1 university of split, faculty of science, department of biology, teslina 12, hr-21000 split, croatia 2 university of zagreb, faculty of science, department of botany and botanical garden, maruli}ev trg 9a, hr-10000 zagreb, croatia abstract – during floristic research into the island of bra~ (dalmatia, croatia) in 2010, ammi visnaga (l.) lam. (apiaceae), a new neophyte for croatia was found in several localities and natural habitats. at the altitude of 380–460 m above sea level, mostly in habitats disturbed by humans, the located populations were composed of numerous and vital specimens in blooms and with fruits. the gradual and successful integration of this species into the natural vegetation was noticed, particularly in grasslands of the association brachypodio retuso-trifolietum stellati horvati} 1958 and macchia of the association fraxino orno-quercetum ilicis horvati} (1956) 1958. key words: ammi visnaga, flora, neophyte, island bra~, croatia introduction the genus ammi l. (apiaceae) contains about 25 species. this taxon of european origin is distributed mostly in the mediterranean region, north africa, and in south-west asia (tutin 1968). because of their particular and useful chemical compounds (in the first place 4, 9-dimethoxy-7-methylfuro [3, 2-g] chromen-5-one or khellin, but also other compounds) plants of the genus ammi have a long tradition of usage in ethno-medicine (batanouny et al. 1999: 207, chevallier 2001). several ammi species are cultivated in the area of natural distribution, but some taxa are introduced into the culture in other continents as well (i.e. north america), where naturalization occasionally occurs. in europe several taxa are indigenous: ammi crinitum guss., a. huntii h. c. watson (endemic for the azores, incl. a. seubertianum (h. c. watson) trelease), a. majus l. (incl. a. topalii beauverd), a. trifoliatum (h. c. watson) trelease (endemic for the azores), and a. visnaga (l.) lam. (tutin 1968, pignatti 1982, bueno et al. 2006). acta bot. croat. 70 (2), 2011 301 * corresponding author, e-mail: toni@botanic.hr copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\ruscic and nikolic.vp 9. rujan 2011 13:11:37 color profile: disabled composite 150 lpi at 45 degrees in the flora of croatia the genus was previously represented with one species – ammi majus l. (nikoli] 2011). material and methods floristic investigations of the island of bra~ were performed during 2006–2010. (ru[^i] 2010). specimens of the genus ammi l. were collected, but these plants did not match the description of the already known ammi majus (domac 1994). additional determinations were performed using tutin (1968) and pignatti (1982). all localities where plants were noticed were geo-coded by using a gps device with an accuracy that alternated horizontally on the field between ±5 up to ±50 m. the basic geo-reference propositions (ellipsoid, projection, date, zone) are in accordance with nikoli] (2006). the mtb 64 mapping square identification mark was added to all findings (nikoli] et al. 1998). a herbarium specimen (voucher) is placed in herbarium croaticum (za, specimen id. 27668). the data were recorded in the flora croatica database (nikoli] 2011). results and discussion of the five european taxa of the genus ammi (tutin 1968: 353), in the croatian flora only a. majus was known (nikoli] 2011). during floristic investigation of the island of bra~ (central dalmatia) a new taxon for croatian flora, ammi visnaga (l.) lam. (flore françoise 3: 462, 1778) (= daucus visnaga l., species plantarum 1: 242, 1753) was established (fig. 1). the key for distinction of the two ammi species in croatian flora is as follow: 1. rays patent or erecto-patent in flower; in fruit become erect, thickened and indurate; basal leaves with linear leaflets (0.3–0.5 x 3–6 mm), leaflets margin entire 302 acta bot. croat. 70 (2), 2011 ru[^i] m., nikoli] t. fig. 1. ammi visnaga (l.) lam. (apiaceae) from bra~ (dalmatia, croatia) (photo m. ru{~i}). u:\acta botanica\acta-botan 2-11\ruscic and nikolic.vp 9. rujan 2011 13:11:39 color profile: disabled composite 150 lpi at 45 degrees ammi visnaga 2. rays patent and slender in flower and fruit; basal leaves with lanceolate leaflets (1–3 x 3–6 mm), leaflets margin dentate ammi majus ammi visnaga is a robust annual or biennial plant, with height up to 100 cm. the root is fattened and looks like the root of the carrot. lower leaves are pinnate, others 2to 3-pinnate, all with narrowly linear or filiforme lobes. rays are numerous (30–50, up to 150), slender and patent or erecto-patent in the flower. in the fruit, rays become erect, thickened and indurate. bracts are 1to 2-pinnatisect, equalling or exceeding the rays. bracteoles are subulate. flowers are pentamerous, tetracyclic, with radial symmetry, with five stamens and inferior ovary composed from two united carpels. petal colour is amber. plants usually flower from may to september, and on the island of bra~ up to october. fruit is dry, 2–2.5 mm long, with two mericarpes, characteristic of the family (tutin 1968, pignatti 1982). in comparison, ammi majus have patent rays and are slender in flower and fruit. there are fewer rays (15–30, up to 60). basal leaves are compounded of lanceolate leaflets (1–3 x 3–6 mm), the leaflet margin being dentate. petal colour is white-amber. the fruits are smaller (1.5–2 mm). the ammi visnaga were recorded in three localities in the central part of bra~, along the road that connects the villages pra`nica and gornji humac (tab. 1, fig. 2). all localities are placed inside the mapping square mtb 2666.33. acta bot. croat. 70 (2), 2011 303 ammi visnaga in croatian flora fig. 2. distribution map of known localities of ammi visnaga (l.) lam. in croatia u:\acta botanica\acta-botan 2-11\ruscic and nikolic.vp 9. rujan 2011 13:11:39 color profile: disabled composite 150 lpi at 45 degrees ecologically, all localities are in the eu-mediterranean zone of evergreen vegetation of the union quercion ilicis br.-bl. (1931) 1936 (horvati] 1963, 1967, 1971) between 380 and 460 m above sea level. habitats where species prosper are primarily ruderal. there are pebble embankments alongside the road and semi-natural stony habitats with elements of the vegetation of the association scolymo-marrubietum incane horvati} et hodak 1956 (trinajsti] 2008). in all the more or less separated micro localities, the species is present with the bulk of specimens that successfully develop flowers and fruits. efficient expansion in natural vegetation is registered as well, mostly in stony grasslands of the association brachypodio retuso-trifolietum stellati horvati} 1958 and macchia of the association fraxino orno-quercetum ilicis horvati} (1956) 1958 (trinajsti] 2008). the inclusions in natural compositions are particularly efficient at locality no. 3 (tab. 1) where ammi visnaga comes together with taxa characteristic of this kind of habitat, i.e.: quercus ilex l., paliurus spina-christi mill., phillyrea media l., fraxinus ornus l., clematis vitalba l., juniperus oxycedrus l., centaurea spinosociliata seenus ssp. spinosociliata, brachypodium retusum (pers.) p. beauv., salvia officinalis l., etc. ammi visnaga is an indigenous plant of north africa, west asia, and a great part of the european mediterranean (tutin 1968, pignatti 1982, batanouny et al. 1999, chevallier 2001, bueno et al. 2006). as a naturalized species ammi visnaga appears in north america, argentina, chile, mexico, south-west asia and some atlantic islands (kenner and requena 2001). the origin of this species in the croatian flora is not clear. regarding documented usage of this plant in popular and official medicine (i.e. asthma relief due to its coronary blood vessel dilating effects, a smooth muscle relaxer in the bronchial tissue, vasodilator and helps to relieve spasms, etc.; kenner and requena 2001, chevallier 2001), and long tradition of appliances in many parts of the world, we can assume that this species came as a healing plant into the garden flora of the island bra~. it is very difficult to trace when this introduction happened. however, the relatively long presence in culture issuggested by the existence of a croatian vernacular name – »mrkva divja« (meaning »wild carrot«, because of its high similarity with daucus species) mentioned by [ulek (1879) and used also by [ugar (2008). consequently, this plant has certainly been cultivated in the coastal area at least longer than 120–150 years. how long the species has existed as a naturalized plant or when this species secondarily escaped from the garden flora is also not clear. this estimation is particularly difficult, be304 acta bot. croat. 70 (2), 2011 ru[^i] m., nikoli] t. tab. 1. new localities of the ammi visnaga (l.) lam. on island bra~ (dalmatia, croatia) no. locality a.s.l. (m) habitat coordinates x y 1 island bra~, west from village pra`nica 457 ruderal, pebble embankment along the road 5636352 4798362 2 island bra~, pra`nica 421 ruderal, pebble embankment, partially stony grassland 5637188 4798425 3 island bra~, east from village pra`nice toward village gornji humac 388 ruderal, along the road on pebble embankment; part of population inside vegetation of natural stony grassland and macchia 5638839 4797986 u:\acta botanica\acta-botan 2-11\ruscic and nikolic.vp 9. rujan 2011 13:11:39 color profile: disabled composite 150 lpi at 45 degrees cause the flora of the island bra~ has not been systematically explored for a long time. in any case, based on standardization of the indigenous plants of croatia (miti] et al. 2008), for the species a. visnaga we propose the following status: introduced plant (code 2.), noticed outside cultivation (code 2.1.) and locally naturalized (code 2.1.1). regarding the unquestionably observed trend in successful integration in the natural vegetation, there is no doubt that the appearance of this species outside cultivation is not merely occasional (code 2.1.2). the potential invasive characteristics of the species should be monitored in the coming years. references batanouny, k. h., abou table, e., shabana, m., soliman, f., 1999: wild medicinal plant in egypt. palm press, cairo. bueno, e., juan, a., crespo, m. b., 2006: lactotypification of three endemic taxa of ammi l. (apiaceae) from the archipelago of the azores. anales de jardin botánico de madrid 63, 31–33. chevallier, a., 2001: encyclopaedia of medicinal plants. dorling kindersley, london. domac, r., 1994: small flora of croatia (in croatian). [kolska knjiga, zagreb. horvati], s., 1963: phytogeographic placemant and analysis of our seashore in the light of modern phytosociological investigations (in croatian). acta botanica croatica 22, 27–81. horvati], s., 1967: phytogeographic characteristics and analysis of the yugoslavia (in croatian). in: horvati}, s. (ed.), analytical flora of the yugoslavia 1. institut for botany, university of zagreb, 23–61. horvati], s., 1971: basic vegetation units in the seashore karst and question about additional protection (in croatian). proceedings of the symposium on nature protection in croatian karst. jazu, zagreb, 109–144. kenner, d., requena, y., 2001: botanical medicine, a european professional perspective. paradigm publications, massachusetts. miti], b., bor[i], i., dujmovi], i., bogdanovi], s., milovi], m., cigi], p., re[etnik, i., nikoli], t., 2008: alien flora of croatia: proposals for standards in terminology, criteria and related database. natura croatica 17, 73–90. nikoli], t. (ed.), 2011: flora croatica database (in croatian). url: http://hirc.botanic.hr/fcd. faculty of science, university of zagreb. nikoli], t., 2006: flora. manual for inventory and monitoring (in croatian). state institute for nature conservation, zagreb. nikoli], t., bukovec, d., [opf, j., jelaska, s. d., 1998: mapping the flora of croatia: possibilities and standards (in croatian). natura croatica 7 (suppl. 1), 1–62. pignatti, s., 1982: flora d’italia 2. edagricole, bologna. ru[^i], m., 2010: the flora of the island bra~ (in croatian). phd thesis, university of zagreb. [ugar, i., 2008: nomenclator botanicus croaticus (in croatian). matica hrvatska, zagreb. [ulek, b., 1879: yugloslavian vernacular plant names (in croatian). jazu, zagreb. acta bot. croat. 70 (2), 2011 305 ammi visnaga in croatian flora u:\acta botanica\acta-botan 2-11\ruscic and nikolic.vp 9. rujan 2011 13:11:39 color profile: disabled composite 150 lpi at 45 degrees trinajsti], i., 2008: plant communities of the republic of croatia (in croatian). academia scientiarum forestariarum, zagreb tutin, t. g., 1968: ammi. in: tutin, t. g., heywood, u. h., burges, n. a., d. m., valentine, d. h., walters, s. m., webb, d. a. (eds.), flora europaea 2, 353. cambridge university press. 306 acta bot. croat. 70 (2), 2011 ru[^i] m., nikoli] t. u:\acta botanica\acta-botan 2-11\ruscic and nikolic.vp 9. rujan 2011 13:11:39 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 73 (1), 93–106, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 pollen studies in the genus viola (violaceae) from iran shahryar saeidi mehrvarz1*, narjes yousefi1, maryam mohammadi1, thomas marcussen2 1 department of biology, faculty of science, university of guilan, p. o. box 41335-1914, rasht, iran. 2 department of plant and environmental sciences, university of gothenburg, p. o. box 461, se 405 30 gothenburg, sweden. abstract – pollen morphology of 17 species of viola representing five sections, melanium, plagiostigma, 'spathulidium' ined., sclerosium, and viola, was studied using light and scanning electron microscope. pollen grains were usually symmetrical, tetrazonocolporate to pentazonocolporate in section melanium and trizonocolporate to tetrazonocolporate in the other four sections. pollen shape was circular to subtriangular, tetragonal or pentagonal in polar view and prolate to oblate, spheroidal or pyramidal in equatorial view. exine ornamentation was granulate, psilate and mostly perforate. the psilate type was only observed in v. modesta. we found heteromorphy in aperture number in v. caspia of section viola, v. occulta of section melanium and v. behboudiana of section sclerosium, which corroborates their higher ploidy than in related species (octoploid versus tetraploid). key words: exine, morphology, ornamentation, palynology, pollen, viola introduction viola l. is the largest genus of violaceae, with ca 600 species and is the only one widely distributed in the northern hemisphere (ballard et al. 1999). infrageneric classification has varied, but recent phylogenetic analysis indicates that the genus can be subdivided into two subgenera and 16 sections worldwide (yousefi et al. 2012). five sections belonging to subgenus viola are present in iran: section melanium ging., section plagiostigma godr., section viola, section sclerosium w. becker, and the undescribed section 'spathulidium' ined. the three first sections are widespread in the northern hemisphere and together comprise a few hundred species, while the two last sections are restricted to parts of southwestern asia with fewer than a dozen species. the sections are ancient allotetraploid except for section 'spathulidium' which is inferred to be allooctoploid (marcussen et al. 2011). recently, molecular phylogenetic analyses of sect. melanium (including 25 species) were performed using its sequences and issr markers (yockteng et al. 2003); they have shown acta bot. croat. 73 (1), 2014 93 * corresponding author, e-mail: saeidimz@guilan.ac.ir copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:34 color profile: generic cmyk printer profile composite default screen that sect. melanium forms a derived and monophyletic group. section melanium, the pansies, is represented in iran by five annual-ephemeral species, v. arvensis, v. kitaibeliana, v. modesta, v. occulta, and v. tricolor. the only species of section plagiostigma occurring in iran, v. somchetica, belongs to subsect. patellares (boiss.) rouy and foucaud. section viola is restricted to northernmost iran, where it occurs with six species, v. alba (subsp. alba), v. odorata, and v. sintenisii in subsect. viola and v. caspia, v. reichenbachiana, and v. rupestris in subsect. rostratae. section sclerosium is distributed in the south of iran with three putative species, v. behboudiana, v. cinerea and v. stocksii. section 'spathulidium’ ined. refers to a small group of three central asian species, in iran represented by v. spathulata and v. pachyrrhiza; v. maymanica occurs in northern afghanistan (schmidt 1992). 17 species of viola are distributed in different parts of iran, mainly in the north. of these, only v. spathulata is endemic to the territory (elbrus mountains), but another five species are endemic to the region. the delimitation of viola species within sections and subsections can be problematic in many regions of the world, owing partly to phenotypic plasticity, partly to hybridisation and polyploidy, and partly to the few morphological characters separating taxa. introgression appears, however, to be rare and restricted to certain groups (valentine 1962, krahulková et al. 1996, marcussen et al. 2001, hildebrandt et al. 2006). pollen morphology of violaceae in general has been examined by some authors (erdtman 1952, pettet 1964, walker and doyle 1975). a few additional studies have been published on pollen morphology in viola. gorb (1994) studied 12 ukrainian species of viola sect. melanium (including v. arvensis, v. kitaibeliana, and v. tricolor) using both light and scanning electron microscopy. perveen and qaiser (2009) examined five viola species (including v. stocksii) from pakistan. dinç (2009) studied two species of subsection viola (v. sandrasea, v. kizildaghensis) endemic to turkey. pollen heteromorphism is defined as the production by the same plant of pollen grains differing in aperture number (till-bottraud et al. 1995). this phenomenon occurs in over 30% of angiosperm species (mignot 1994). in viola pollen heteromorphism occurs in about a third of the studied species and appears to be associated with polyploidy (nadot et al. 2000). the main aim of this study is to provide a descriptive investigation of pollen grains in the genus viola and also of pollen heteromorphism in the species distributed in iran, using light and scanning electron microscopy. in light of the nomenclatural confusion and paucity of morphological characters for discrimination of viola taxa in iran (yousefi et al. 2012), pollen morphology and heteromorphism may provide new characters useful for the delimitation of these taxa. in this study, pollen morphology of 13 species of the genus viola with the exception of v. arvensis, v. kitaibeliana, v. tricolor and v. stocksii are described for the first time. material and methods pollen of 24 taxa representing the 17 species of viola occurring in iran was studied by means of light microscopy (lm) and scanning electron microscopy (sem). pollen samples were obtained from herbarium specimens collected the same year. in order to ensure the constancy of pollen characters among different populations of a certain species, 2–3 specimens per population were included in the analysis; otherwise each species was represented by only one population. the voucher specimens were deposited in guilan university her94 acta bot. croat. 73 (1), 2014 mehrvarz s. s., yousefi n., mohammadi m., marcussen t. 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:35 color profile: generic cmyk printer profile composite default screen barium. a list of the voucher specimens is given in table 1. pollen grains were stained with basic-fuchsine, then mounted in glycerin jelly and prepared for lm observation. some measurements i.e. polar axis (p), equatorial axis (e), colpus length and exine thickness were made by lm (nikon microscope model optiphot-z optic) for 30 pollen grains under a magnification of 1000x. for analysing pollen heteromorphism, the number of apertures were counted for 100 pollen grains, then the percentage of each aperture-class –three, four and five– were calculated and the percentage lower than 95 was reported as heteromorphic pollen (mignot 1994). for sem observations, pollen grains soaked in absolute ethanol were pipetted directly on to 12.5 mm diameter stubs and air-dried at room temperature; they were then coated in a sputter coater with approximately 25 nm of gold-palladium. the micrographs were obacta bot. croat. 73 (1), 2014 95 pollen studies in the genus viola (violaceae) from iran tab. 1. collection data of viola species examined in the present. species endemic to iran are indicated by an asterisk (*). species sect. / subsect. collection data v. arvensis murray melanium ging. iran: mazandaran, javaherdeh, jirkoh, may 1999, naderifar, guh-13682 v. kitaibeliana schult. iran: mazandaran, east slope of damavand, above malar, june 2000, moosavi & mozafarian, tari-33184 v. modesta fenzl iran: kohgiluye-boyer ahmad, nourabad to yasuj road, 30 km to yasuj, may 2009, yousefi, guh-13263-1 v. occulta lehm. iran: guilan, deylaman, may 2009, yousefi & saeidi, guh-13683 v. tricolor l. iran: guilan, rudbar, april 2009, ghahremani, guh-13663 v. somchetica c. koch plagiostigma godr./ estolonosae kupffer iran: ardebil, almas road, april 2009, yousefi & shahi, guh-13674 v. behboudiana rech. f. et esfand. sclerosium iran: baluchestan, hudar, ghasre ghand, 380 m, foroughi, tari-10755 v. cinerae boiss. iran: bushehr, 2–3 km of ne khormoj, 150 m, mozafarian, tari17141 v. stocksii boiss. iran: hormozgan, bandar-abas, 20–30 km of w rudan, 200 m,wendelbo & foroughi, tari-15630 v. spathulata willd.* «spathulidium« ined. iran: mazandaran, chalous-karaj road, duzdbon, april 1977, ghahreman & aghoostin, tuh-11252 v. pachyrrhiza boiss. et hohen. iran: lorestan, aligudarz road to shoolabad, 2400 m, runemark & lazari, tari-26228 v. caspia (rupr.) freyn 1 viola/rostratae (kupffer) w. becker iran, guilan,, lahijan, sheitan koh, march 2008, tabarestani, guh-13634 v. caspia (rupr.) freyn 2 iran: guilan, masouleh, yousefi, guh-4369 v. caspia (rupr.) freyn 3 iran: guilan, lahijan, sheitan koh, march 2007, yousefi, guh-13681 v. reichenbachiana jord. ex bor. iran, guilan, asalem to khalkhal road, march 2009, yousefi & asaadi, guh-13651 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:35 color profile: generic cmyk printer profile composite default screen tained from a leo1430 vp (england) at an accelerating voltage of 10–15 kv under magnifications of 2040× to 11190×. pollen terminology according to walker and doyle (1975), punt (2007) and halbritter et al. (2008) has been followed. results general pollen characters of the genus viola the pollen grains were shed in monads, symmetrical, tetrazonocolporate to pentazonocolporate in section melanium and trizonocolporate to tetrazonocolporate in the other four sections (figs. 1–2, tab. 2). the apertures were colporate, equatorially elongated and reached the poles of the pollen with rounded or acute ends. the smallest pollen grains belonged to v. spathulata (p = 29 mm, e = 26.08 mm) and the largest ones to v. arvensis (p = 69.09 mm, e = 68.23 mm). the p/e ratio varied between 0.94 and 1.18 (tab. 2). the exine thickness varied between 0.6 µm (v. spathulata) and 1 µm (v. occulta). exine ornamentation proved to be a valuable taxonomic character in viola. it varies from perforate, granulate, to psilate (tab. 2). among these patterns the simple psilate type represented by v. modesta is very characteristic. section melanium (figs. 1a–g, 3a–g) section melanium is a morphologically well-defined group of about 125 species. of these, five species are distributed in iran, mostly in the north. members of this section have 96 acta bot. croat. 73 (1), 2014 mehrvarz s. s., yousefi n., mohammadi m., marcussen t. species sect. / subsect. collection data v. rupestris f. w. schmidt iran: ardebil, almas road, april 2009, yousefi & asaadi, guh-13672 v. alba bess. subsp. alba 1 viola/viola iran: guilan, masouleh, march 2008, royan, guh-13266 v. alba bess. subsp. alba 2 iran: guilan, tootkabun, april 2009, yousefi & saeidi, guh-13684 v. alba bess. subsp. alba 3 iran: guilan, lahijan, sheitan koh, 2008, yousefi, guh13271 v. odorata l. 1 iran: guilan, mountains of masouleh, march 2008, yousefi & saeidi, guh-13665 v. odorata l. 2 iran: guilan, masouleh road, march 2007, yousefi & saeidi, guh-13481-2 v. sintenisii w. becker 1 iran: guilan, lahijan, sheitan koh, february 2008, yousefi, guh-13675 v. sintenisii w. becker2 iran: guilan, lakan, february 2008, yousefi, guh-13281 v. sintenisii w. becker 3 iran: guilan, lashtenesha, the rice field, guh-13296-2 tab. 1. – continued 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:35 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 97 pollen studies in the genus viola (violaceae) from iran fig. 1. micrographs of pollen grains in viola arvensis (a–b), v. kitaibeliana (c), v. modesta (d), v. occulta (e), v. tricolor (f–g), v. somchetica (h), v. behboudiana (i–j), v. cinerea (k), v. stocksii (l). scale bar: 10 µm. fig. 2. micrographs of pollen grains in viola pachyrrhiza (a), v. spathulata (b), v. caspia (c), v. reichenbachiana (d–e), v. rupestris (f), v. alba (g), v. odorata (h), v. sintenisii (i). scale bar: 10 µm. 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:41 color profile: generic cmyk printer profile composite default screen 98 a c t a b o t .c r o a t .73 (1),2014 m e h r v a r z s .s .,y o u s e f in .,m o h a m m a d im .,m a r c u s s e n t . tab. 2. characteristic features of the investigated pollen in species of viola. abbreviations : p– polar axis (µm); e– equatorial axis (µm); m– mean value; sd – standard deviation; eo – exine ornamentation; pe: perforate, gr: granulate, ps: psilate. species sect. / subsect. e (mm) min (m ± sd) max p (mm) min (m ± sd) max p eo v. arvensis melanium 60.0 (68.2 ± 5.1) 85.0 62.5 (69.1 ± 3.7) 77.5 1/01 pe-gr v. kitaibeliana 42.5 (45.6 ± 1.8) 47.5 30.0 (43.2 ± 7.9) 55.0 0/94 – v. modesta 27.5 (34.8 ± 4.8) 40.0 40.0 (41.1 ± 3.2) 47.5 1/18 ps v. occulta 52.5 (60.0 ± 4.7) 67.5 55.0 (63.3 ± 4.0) 70.0 1/05 pe-sub ps v. tricolor 50.0 (52.6 ± 2.1) 57.5 45.0 (54.9 ± 3.6) 62.5 1/04 gr v. somchetica plagiostigma 25.0 (29.0 ± 1.5) 32.5 27.5 (31.9 ± 2.0) 35.0 1/1 pesub ps v. behboudiana sclerosium 29.4 (30.8 ± 1.1) 32.5 25.9 (29.5 ± 1.6) 31.7 0/95 pe (fine) v. cinerae 32.5 (33.9 ± 1.0) 35.7 33.0 (38.2 ± 2.0) 40.0 1/14 pe (fine and large) v. stocksii 38.2 (41.1 ± 1.7) 42.0 42.5 (44.0 ± 1.2) 45.0 1/07 pe (fine) v. pachyrrhiza spathulidium 23.0 (26.1 ± 1.7) 31.0 26.0 (29.0 ± 1.9) 34.0 1/11 pelarge v. spathulata 22.5 (26.1 ± 1.7) 30.0 25.0 (29.0 ± 1.9) 32.5 1/11 gr v. caspia 1 viola.rostratae 32.5 (36.4 ± 1.7) 37.5 32.5 (36.5 ± 1.8) 40.0 1/00 (micro) gr v. caspia 2 27.5 (31.3 ± 2.2) 37.5 27.5 (33.5 ± 2.9) 37.5 1/07 gr v. caspia 3 40.0 (34.4 ± 3.6) 30.0 40.0 (35.5 ± 2.9) 32.5 1/03 gr v. reichenbachiana 27.5 (32.8 ± 3.7) 37.5 27.5 (32.6 ± 3.5) 37.5 0/9 pesub gr v. rupestris 22.5 (25.3 ± 1.5) 30.0 27.5 (29.1 ± 1.6) 32.5 1/14 (micro) gr v. alba subsp. alba 1 viola.viola 25.0 (28.1 ± 1.9) 32.5 30.0 (31.6 ± 1.4) 35.0 1/12 pe-sub ps v. alba subsp. alba 2 25.0 (30.3 ± 2.6) 35.0 27.5 (32.3 ± 3.0) 37.5 1/06 pegr v. alba subsp. alba 3 25.0 (24.5 ± 1.1) 27.5 25.0 (28.8 ± 3.6) 30.0 1/17 pe-sub ps v. odorata 1 25.0 (27.9 ± 2.6) 35.0 25.0 (29.5 ± 2.6) 35.0 1/05 gr v. odorata 2 25.0 (26.7 ± 1.3) 30.0 25.0 (31.6 ± 1.7) 35.0 1/18 (micro) pe-gr v. sintenisii 1 25.0 (26.4 ± 2.0) 32.5 25.0 (30.3 ± 2.4) 35.0 1/14 pe-gr v. sintenisii 2 22.5 (26.9 ± 3.7) 32.5 25.0 (30.6 ± 4.1) 37.5 1/13 pegr v. sintenisii 3 25.0 (29.4 ± 3.7) 35.0 30.0 (33.8 ± 3.7) 40.0 1/14 pegr 7 5 5 m e h r v a r z e t a l . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 7 5 5 m e h r v a r z e t a l . v p 1 9 . o u j a k 2 0 1 4 1 7 : 0 0 : 4 1 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n four to five apertures, while a more frequent aperture number in members of other sections is three. the pollen type of v. arvensis is perforate–granulate (figs. 3a–b). at species level, v. kitaibeliana with pyramidal shape in equatorial view (fig. 1c) can be distinguished from the other species, which are prolate to spheroidal in shape. exine ornamentation does not provide valuable characters in delimitation of section melanium from other sections, but it is useful for species recognition within this section. for instance, two morphologically close species, v. tricolor and v. modesta, have different types of exine sculpturing, i.e. granulate and psilate, respectively (figs. 3c, f–g). furthermore, v. arvensis, which exhibits a perforate to granulate exine, can be separated from its relative v. occulta, which has perforate to subpsilate exine (figs. 3d–e). section plagiostigma (figs. 1h, 3h–i) viola somchetica is the only iranian species of this section and its pollen grain in equatorial view and exine ornamentation is prolate–spherodial (fig. 1h) and perforate–subpsilate (figs. 3h–i), respectively. from a palynological point of view, this species does not show any obvious morphological apomorphies. section sclerosium (figs. 1i–l, 3j–l, 4a–c) viola behboudiana is easily separated from the other iranian species of the section by two palynological characters: the shape of pollen grains, which is oblate–spheroidal in v. behboudiana (figs. 1i–j) and prolate–spheroidal in v. cinerea and v. stocksii (figs. 1k–l), acta bot. croat. 73 (1), 2014 99 pollen studies in the genus viola (violaceae) from iran fig. 3. sem micrographs of pollen grains in viola arvensis (a–b), v. modesta (c), v. occulta (d–e), v. tricolor (f–g), v. somchetica (h–i), v. behboudiana (j–k), v. cinerea (l). 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:42 color profile: generic cmyk printer profile composite default screen and the presence of pollen heteromorphism in v. behboudiana. all of the three species have perforate to perforate–subpsilate exine ornamentation (figs. 3j–l, 4a–c). section 'spathulidium' ined. (figs. 2a–b, 4d–f) the two iranian species of section spathulidium are morphologically similar but can be easily separated using palynological characters: v. pachyrrhiza has perforate exine (fig. 4d), whilst v. spathulata has granulate exine ornamentation (figs. 4e–f). pollen morphology shows a basic similarity between section spathulidium and section plagiostigma; however these two sections can be differentiated according to exine ornamentation (tab. 2). section viola no palynological characters separate this section from the others. subsection rostratae (figs. 2c–f, 4g–l, 5a) this subsection has three species in iran, v. caspia, v. reichenbachiana and v. rupestris. the morphological recognition of the first two species is very problematic due to the variable morphological characters; however palynological studies provide useful characters for delimiting these taxa. pollen grains of v. caspia are prolate–spheroidal to subprolate in equatorial view (fig. 2c) with a granulate exine (figs. 4g–j), while v. reichenbachiana has oblate–spheroidal pollen grains (figs. 2d–e) with a perforate–subgranulate exine (fig. 4k). furthermore, v. caspia shows pollen heteromorphism, in which the dominant pollen shape has three apertures and 6 % of pollen grains have four apertures. the characteristics of pollen sculpturing of v. rupestris are similar to v. caspia (figs. 2f, 4l, 5a), but the p/e ratio of v. rupestris is larger than v. caspia (tab. 2). subsection viola (figs. 2g–i, 5b–i) this subsection has three species in iran, v. alba, v. sintenisii, and v. odorata. whereas the latter can be easily recognised using morphological characters, i.e. broad stipules and high number of leaf crenulae, the division between two first species is still problematic. the shape in equatorial view of three species is prolate–spheroidal to subprolate (figs. 2g–i). pollen characteristics are also variable between the taxa, particularly, the exine ornamentation shows that v. alba has perforate to subpsilate and perforate to granulate exine (figs. 5b–c), v. sintenisii has perforate–granulate (fig. 5h–i). viola odorata has perforate to subpsilate and perforate to granulate exine (figs. 5d–g). discussion the exine sculpturing of the pollen grains, their size, shape and aperture numbers can be of taxonomic value in the studied taxa. the pollen size variability is a diagnostic value between some of the taxa within the same section, for example, between v. arvensis and v. kitaibeliana (sect. melanium), v. modesta and v. occulta (sect. melanium), v. cinerea and v. stocksii (sect. sclerosium). 100 acta bot. croat. 73 (1), 2014 mehrvarz s. s., yousefi n., mohammadi m., marcussen t. 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:42 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 101 pollen studies in the genus viola (violaceae) from iran fig. 5. sem micrographs of pollen grains in viola rupestris (a), v. alba (b–c), v. odorata (d–g), v. sintenisii (h–i). fig. 4. sem micrographs of pollen grains in viola cinerea (a), v. stocksii (b–c), v. pachyrrhiza (d), v. spathulata (e–f), v. caspia (g–j), v. reichenbachiana (k), v. rupestris (l). 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:44 color profile: generic cmyk printer profile composite default screen 102 a c t a b o t .c r o a t .73 (1),2014 m e h r v a r z s .s .,y o u s e f in .,m o h a m m a d im .,m a r c u s s e n t . tab. 3. characteristic features of the investigated pollen in species of viola. m – mean value; sd – standard deviation species sect. / subsect. colpate (mm) min (m ± sd) max exin thickness (mm) shape (equatorial view) shape (polar view) v. arvensis melanium 52.5 (60.4 ± 4.8) 75.0 2.2 ± 0.2 prolate spheroidala pentagonal v. kitaibeliana 35.0 (40.0 ± 2.8) 42.5 2.1 ± 0.2 pyramidal tetragonal v. modesta 20.0 (24.5 ± 2.2) 27.5 2.3 ± 0.2 prolate spheroidala tetragonal v. occulta 47.5 (53.9 ± 4.2) 62.5 2.5 ± 0.1 prolate spheroidala tetragonal v. tricolor 42.5 (46.0 ± 1.7) 47.5 2.3 ± 0.2 prolate spheroidala tetragonal v. somchetica plagiostigma 20.0 (22.8 ± 1.6) 25.0 1.6 ± 0.2 prolate spheroidala circular-subtriangular v. behboudiana sclerosium 22.0 (23.3 + 1.4) 25.0 1.8 ± 0.1 oblate spheroidal circular-subtriangular v. cinerae 16.5 (17.6 + 0.8) 18.7 1.2 ± 0.2 prolate spheroidala circular-subtriangular v. stocksii 19.7 (19.8 + 0.1) 20.0 2.7 ± 0.2 prolate spheroidala circular-subtriangular v. pachyrrhiza spathulidium 20.0 (22.3 ± 1.6) 26.0 1.4 ± 0.1 prolate spheroidala circular-subtriangular v. spathulata 20.0 (22.3 ± 1.6) 25.0 1.4 ± 0.1 prolate spheroidala circular-subtriangular v. caspia 1 viola.rostratae 22.5 (27.9 ± 2.7) 32.5 1.8 ± 0.1 prolate spheroidala circular-subtriangular v. caspia 2 v. caspia 3 30.0 (31.5 ± 1.2) 33.8 30.0 (26.8 ± 2.7) 22.5 1.7 ± 0.1 2.0 ± 0.2 spheroidal prolate spheroidala circular-subtriangular circular-subtriangular v. reichenbachiana 22.5 (28.0 ± 3.7) 35.0 2.4 ± 0.2 oblate spheroidal circular-subtriangular v. rupestris 17.5 (21.6 ± 1.8) 25.0 2.2 ± 0.2 prolate spheroidala circular-subtriangular v. alba subsp. alba 1 viola.viola 17.5 (21.8 ± 0.2) 25.0 2.0 ± 0.2 prolate spheroidala circular-subtriangular v. alba subsp. alba 2 22.5 (24.6 ± 2.5) 30.0 2.2 ± 0.2 prolate spheroidala circular-subtriangular v. alba subsp. alba 3 v. odorata 1 20.0 (22.5 ± 1.8) 25.0 17.5 (20.8 ± 1.6) 22.5 2.0 ± 0.6 2.0 ± 0.2 prolate spheroidala prolate spheroidala circular-subtriangular circular-subtriangular v. odorata 2 20.0 (21.2 ± 1.2) 20.0 2.2 ± 0.2 subprolatea circular-subtriangular v. sintenisii 1 v. sintenisii 2 v. sintenisii 3 20.0 (21.0 ± 1.2) 22.5 22.5 (24.4 ± 2.1) 27.5 25.0 (27.5 ± 1.8) 30.0 1.8 ± 0.2 1.9 ± 0.2 1.5 ± 0.1 prolate spheroidala prolate spheroidala prolate spheroidala circular-subtriangular circular-subtriangular circular-subtriangular 7 5 5 m e h r v a r z e t a l . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 7 5 5 m e h r v a r z e t a l . v p 1 9 . o u j a k 2 0 1 4 1 7 : 0 0 : 4 4 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 103 p o l l e n s t u d ie s in t h e g e n u s v io l a (v io l a c e a e ) f r o m ir a n tab. 4. the number of aperture and pollen heteromorphism in the investigated species of viola. species sect. . subsect. number of apertures (3 + 4 + 5) percent of most abundant pollen morph (%) hetero-morphism (<95 %) predominant pollen morph v. arvensis melanium 0 + 2 + 98 98 – 5 zonocolporate v. kitaibeliana 0 + 97 + 3 97 – 4 zonocolporate v. modesta 0 + 96 + 4 96 – 4 zonocolporate v. occulta 0 + 85 + 15 85 + 4 zonocolporate v. tricolor 0 + 97 + 3 97 – 4 zonocolporate v. somchetica plagiostigma 100 + 0 + 0 100 – 3 zonocolporate v. behboudiana sclerosium 92 + 8 + 0 92 + 3 zonocolporate v. cinerae 99 + 0 + 0 100 – 3 zonocolporate v. stocksii 98 + 2 + 0 98 – 3 zonocolporate v. pachyrrhiza 98 + 2 + 0 98 – 3 zonocolporate v. spathulata spathulidium 98 + 2 + 0 98 – 3 zonocolporate v. caspia 1 viola/rostratae 94 + 6 + 0 94 + 3 zonocolporate v. caspia 2 97 + 3 + 0 97 – 3 zonocolporate v. reichenbachiana 99 + 1 + 0 99 – 3 zonocolporate v. rupestris 100 + 0 + 0 100 – 3 zonocolporate v. alba subsp. alba 1 viola/viola 100 + 0 + 0 100 – 3 zonocolporate v. alba subsp. alba 2 100 + 0 + 0 100 – 3 zonocolporate v. alba subsp. alba 3 v. odorata 1 100 + 0 + 0 100 + 0 + 0 100 100 – – 3 zonocolporate 3 zonocolporate v. odorata 2 100 + 0 + 0 100 – 3 zonocolporate v. sintenisii 1 v. sintenisii 2 v. sintenisii 3 100 + 0 + 0 100 + 0 + 0 100 + 0 + 0 100 100 100 – – – 3 zonocolporate 3 zonocolporate 3 zonocolporate 7 5 5 m e h r v a r z e t a l . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 7 5 5 m e h r v a r z e t a l . v p 1 9 . o u j a k 2 0 1 4 1 7 : 0 0 : 4 4 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n pollen grains in polar view are variable within the sect. melanium and the other sections (tab. 2). a tetragonal to pentagonal shape in polar view is found in v. arvensis, v. kitaibeliana, v. modesta, v. occulta and v. tricolor (sect. melanium), whilst circular-subtriangular is present in the other sections. the pollen shape of sect. melanium was circular to sub-triangular, tetragonal or pentagonal in polar view and prolate to oblate, spheroidal or pyramidal in equatorial view according to the ratio of polar-equatorial axis (walker and doyle 1975). according to the classification of pollen grains based on size (walker and doyle 1975), this section has large pollen grains (50–99 mm), while the other sections of viola distributed in iran have medium sized pollen grains (25–49 mm). pollen grains with six apertures were reported for v. arvensis from ukraine (gorb 1994). the figures given in erdtman (1952) show that v. arvensis has 4-colpate with a few 5-colpate grains appearing to be based on misidentification with v. tricolor since this type is found in v. tricolor and not v. arvensis. pettet (1964) noted that the two very variable and often confused species, v. tricolor and v. arvensis, are readily separable on the basis of pollen assemblage. earlier studies suggested that section plagiostigma is not closely related to section viola, as previously assumed (clausen 1929, marcussen et al. 2012). our palynological results for iranian v. stocksii agree with findings for this species in pakistan (perveen and qaiser 2009). previously these species, i.e.. v. pachyrrhiza and v. spathulata were included in section plagiostigma subsection patellares (as section nomimium grex adnatae; becker 1918). however, this lineage takes a completely different phylogenetic position and will require a section of its own (marcussen et al. 2010). yousefi et al. (2012) demonstrated that viola somchetica (section plagiostigma) differs from v. spathulata (section spathulidium) in the number of collenchyma layers at the corners of petiole cross sections (4 vs. 3) and the shape of peduncle cross sections (quadrangular vs. circular). morphological and phylogenetic evidence supports two subsections, subsection rostratae, with aerial floriferous stems and explosive capsules, and subsection viola, lacking aerial stems and with inexplosive capsules (becker 1925, marcussen and karlsson 2010). anatomically, v. caspia can be separated from v. reichenbachiana by the presence of a pith region in root cross-sections and by the number of vascular bundles in stem cross sections (yousefi et al. 2012). viola behboudiana (section sclerosium), v. caspia (section viola) and v. occulta (section melanium) showed structural polymorphism. the number of apertures in the two first ones was 3–4, and 4–5 in the last one. all three species are high-polyploids. viola behboudiana and v. caspia are both octoploids (marcussen and borgen 2011), and this is probably the case also for v. occulta (n = 10 versus n = 3, 4, 5, 7 in its close tetraploid relatives; yockteng et al. 2003). in the genus viola, pollen heteromorphism is a direct result of polyploidy (nadot et al. 2000). acknowledgements we thank to mr. a. khodayari (university of ardebil) for his assistance in electron microscopy. this research was supported by a grant 989–27 from the research council of the university of guilan. 104 acta bot. croat. 73 (1), 2014 mehrvarz s. s., yousefi n., mohammadi m., marcussen t. 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:44 color profile: generic cmyk printer profile composite default screen references ballard, h. e., sytsma, k. j., kowal, r. r., 1999: shrinking the violets: phylogenetic relationships of infrageneric groups in viola (violaceae) based on internal transcribed spacer dna sequences. systematic botany 23, 439–458. becker, w., 1918: violae asiaticae et australenses iii. beihefte zum botanischen centralblatt 36, 15–59. becker, w., 1925: viola l. in: engler, a. (ed.), die natürlichen pflanzenfamilien, 363– 376. parietales und opuntiales. wilhelm engelmann: leipzig. clausen, j., 1929: chromosome number and the relationship of some north american species of viola. annals of botany 63, 741–764. dinç, m., 2009: comparative morphological and palynological study on poorly known viola sandrasea and its closest relative v. kizildaghensis. biologia 64, 81–87. erdtman, g., 1952: pollen morphology and plant taxonomy: angiosperms. chronica botanica co., waltham, massachusettes. gorb, o. v., 1994: pollen morphology of section melanium ging. of genus viola l. of ukraine (in ukrainian). ukrainian botanical journal 51, 78–85. halbritter, h., weber, m., zetter, r., frosch-radivo, a., buchner, r., hesse, m., 2007: paldat– illustrated handbook on pollen terminology. society of the promotion of palynological research in austria, vienna. hildebrandt, u., hoef-emden, k., backhausen, s., bothe, h., bozbek, m., siuta, a., kuta, e., 2006: the rare, endemic zinc violets of central europe originate from viola lutea huds. plant systematics and evolution 257, 205–222. krahulková, a., krahulec, f., kirschner, j., 1996: introgressive hybridization between a native and an introduced species: viola lutea subsp. sudetica versus v. tricolor. folia geobotanica and phytotaxonomica 31, 319–344. marcussen, t., borgen, l., nordal, i., 2001: viola hirta (violaceae) and its relatives in norway. nordic journal of botany 21, 5–17. marcussen, t., karlsson, t., 2010: violaceae. in: karlsson, t. 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(ed.), flora iranica 169, 1–29. akademische druckund verlagsanstalt. till-bottraud, i., mignot, a., de paepe, r., dajoz, i., 1995: pollen heteromorphism in nicotiana tabacum (solanaceae). american journal of botany 82, 1040–1048. valentine, d. h., 1962: variation and evolution in the genus viola. preslia 34, 190–206. walker, j. w., doyle, j. a., 1975: the bases of angiosperm phylogeny: palynology. annals of the missouri botanical garden 62, 664–723. yockteng, r., ballard, h. e., mansion, g., dajoz, i., nadot, s., 2003: relationships among pansies (viola section melanium) investigated using its and issr markers. plant systematics and evolution 241, 153–170. yousefi, n., saeidi mehrvarz, s., marcussen, t., 2012: anatomical studies on selected species of viola (violaceae). nordic journal of botany 30, 461–469. 106 acta bot. croat. 73 (1), 2014 mehrvarz s. s., yousefi n., mohammadi m., marcussen t. 755 mehrvarz et al.ps u:\acta botanica\acta-botan 1-14\755 mehrvarz et al.vp 19. o ujak 2014 17:00:44 color profile: generic cmyk printer profile composite default screen 168 acta bot. croat. 81 (2), 2022 acta bot. croat. 81 (2), 168–176, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-014 issn 0365-0588 eissn 1847-8476 morpho-anatomical diversity of five species of the genus asparagus (asparagaceae) from algeria kenza boubetra1*, nabila amirouche2, rachid amirouche2 1 national institute of forest research, inrf, po box 37, cheraga 16014 algiers, algeria 2 university of sciences and technology houari boumediene, faculty of biological sciences, laboratory for biology and physiology of organisms, usthb, bab ezzouar, 16111, algiers, algeria abstract – five species of the genus asparagus are recognized in the flora of algeria: a. acutifolius l., a. albus l., a. horridus l., a. officinalis l., and the endemic a. altissimus munby. the chorology of each of these species is fairly well known. in this study, morphological variation and the anatomical features of the cladodes have been evaluated in respect to each taxonomic unit and ecogeographical distribution and they suggest distinct adaptive strategies. analyses have been performed on twenty-nine natural populations sampled along the east-west bioclimatic gradient of northern algeria. multivariate analysis based on the main diagnostic descriptors underlines the interspecific differentiation particularly with respect to the stigma type, bifid versus trifid, shape of flowers, color of berry, and the number of cladodes in a fascicle. for each species, the anatomy of the cladodes is unique, unlike that of stems and roots. interspecific differentiation was observed in the form of cross-sections of the cladode, thickness of the cuticle, shape of epidermal cells, number of vascular bundles and presence of raphides. morphological and anatomical traits of the cladode constitute important interspecific criteria within the genus asparagus. keywords: adaptation, algeria, anatomy, asparagus, cladodes, morphology, taxonomy introduction the genus asparagus (asparagaceae) includes more than 200 species distributed in the arid and subarid regions of africa, europe, asia and australia (chen and tamanian 2000). most species of asparagus have great economic value, particularly a. officinalis l., cultivated as a vegetable crop all over the world. some wild mediterranean species such as a. acutifolius l., a. horridus l., a. aphyllus l. and a. albus l., are traditionally collected and sold in local markets (pieroni 2005, boubetra et al. 2017a, mantovani et al. 2019). the genus is noteworthy for its diversity of herbaceous, shrubby and climbing forms. plants are provided with underground rhizomes from which the aerial shoots arise. all species are characterized by cladodes that correspond to green photosynthetic stems; the true leaves are reduced to small scales. in mediterranean forests, the lianascent species grow preferentially in shady and wet ecological niches (schnitzler and arnold 2010). in contrast, shrubby species are encountered in open habitats especially in steppic areas, exhibiting tolerance to high temperatures and aridity. among the asparagaceae (sensu angiosperm phylogeny group iii 2009), species may display different phyllocladodes from flattened or cylindrical forms (kubitzki and rudall 1998, fukuda et al. 2005). for instance, species of the genus ruscus, develop a leaf-like organs, termed precisely phylloclades. they differ from cladodes in consisting of a stem with multiple internodes (bell 2008). it is through the variation of these photosynthetic organs (cladodes leaves, stems, phyllocladodes) that species express adaptation and physiological responses to environmental changes. molecular phylogenetic studies on asparagus, indicated that cladodes evolve from a leaf-like (flattened) to a rod-like (cylindrical) form, suggesting a rapid radiation particularly in arid regions (fukuda et al. 2005, kubota et al. 2012). the genes involved in the evolutionary pathway of the transformation of the leaf-like form to axillary shoots were identified by nakayama et al. (2012). their cooption into a gene network is well documented in nakayama et al. (2013). although numerous studies have been devoted to the biochemical characteristics of some asparagus species, the morpho-anatomical variations in response to changes in * corresponding author e-mail: kenzaboubetra@yahoo.fr genus asparagus in algeria acta bot. croat. 81 (2), 2022 169 environmental conditions have been poorly documented. in addition, current researches are more focused on roots and rhizomes for the detection of bioactive molecules. in the flora of algeria, five species have been recorded: a. acutifolius l., a. albus l., a. horridus l., (= a. stipularis forsk.), a. officinalis l., and the endemic a. altissimus munby. all these species are diploids with 2n = 2x = 20, and grow in contrasting ecological conditions in northern parts of algeria, except the endemic a. altissimus which is hexaploid with 2n = 6x = 60 (boubetra et al. 2017b). the most widespread species, a. acutifolius, is encountered from humid to arid bioclimates, while a. horridus and a. albus, are less common and have a predilection for open habitats, dry, sandy and stony soils. a. altissimus, is a narrow endemic to nw algeria and morocco. asparagus officinalis is very rare and seems a remnant of ancient cultures. however, spears of wild asparagus (a. acutifolius, a. horridus, a. albus) are widely harvested for food by local people. therefore, wild species constitute a very interesting potential as a genetic resource for breeding programs. the aims of this study were to evaluate morphological, floral and anatomical variations among the natural populations of the five asparagus species occurring in algeria. anatomical examinations have been performed on cross sections of the cladodes in order to understand the ecological affinities and adaptive strategies of these species. material and methods examined specimens twenty-four locations were selected in northern algeria in humid, subhumid, semiarid and arid zones corresponding to the main vegetation types of forest, shrub formation, open habitat and steppic highlands. the taxonomic determination of the specimens was made according to the flora of north africa (maire 1958) and algeria (quézel and santa 1962). overall, twenty-nine populations representative of the five studied species, recognized in the flora of algeria, were sampled, some of them occurring in sympatry (fig. 1, on-line suppl. tab. 1). specimens for morphological and anatomical analysis were sampled in reproductive stage with their whole vegetative structures. seeds collected in the field were sown in the experimental station of the national institute of forest research (baraki, algiers) in order to start a living collection of the studied species. vouchers were deposited in the official herbarium of ensa (algiers, algeria). fig.1. locations of the sampled populations of the five species of the genus asparagus in northern algeria. tab. 1. morphological and floral characters used in the multivariate analyses of five species of the genus asparagus from algeria. abbreviations characters qualitative values and measurement units 1 cl shape of cladodes smooth spiny strongly spiny 0 1 2 2 lcl lenght of cladodes mm 3 ncl number of cladodes in a fascicle 4 cfl color of flower yellow white purplish 0 1 2 5 tfl type of flower dioecious hermaphrodite 0 1 6 ffl shape of flower campanulate stelate tubular 0 1 2 7 nfl number of flowers 8 sti type of stigma bifid trifid 0 1 9 lpe pedicel length mm 10 per perianth length mm 11 cba color of berry red black purple 0 1 2 12 ngr number of seed per berry 13 dig diameter of seed mm boubetra k., amirouche n., amirouche r. 170 acta bot. croat. 81 (2), 2022 analysis of the variation of the vegetative and floral characters the morphological evaluation was carried out on three or four individuals per population directly taken in their natural environment. a total of five quantitative and eight qualitative morphological and floral characters were selected following the diagnostic criteria for species delimitation, and self-observations (tab. 1). the global raw data matrix consisting of 120 individuals belonging to the five studied species, and 13 variables, was first subjected to principal component analysis. this pca was performed on the basis of the correlation matrix of the variables, generated after standardization of the raw matrix data. this analysis was used to estimate the relative contribution of each variable to the main principal components. to detect the phenetic grouping and relationships among the species studied, a upgma dendrogram was constructed using the euclidean distances of the mean values per population. in order to assess the impact of the bioclimatic conditions on the morphological variability, another pca was focused on the populations of a. acutifolius which have the largest biogeographical distribution. this analysis was based on the average values of each variable for each population and included the main parameters of the mediterranean bioclimate and the altitude (on-line suppl. tab. 1). for all the analyses we used statistica software (ver. 12.0). anatomical analyses anatomical examinations were performed on cross sections of cladodes freshly collected and conserved in ethanol 70°. the cross sections were made using a microtome. the technique consists of making thin paraffin sections (about 10 μm). several successive steps are required: fixation, inclusion in paraffin, microtome sections, staining, assembly and observation. the technique of double staining with successively methyl green (7’) and congo red (10’) was used. the methyl green stains the lignified walls and the congo red stains the cellulosic walls. cross sections were photographed with a zeiss axiostar-plus microscope equipped with a canon digital camera. results interspecific variation for morphological and floral traits in aim to assess the morphological characters involved in the variability and the interspecific relationships, a pca fig. 2. principal components analysis of 29 natural populations of five asparagus species from algeria based on 13 morphological and floral traits. a – loading of the 13 variables on the principal components (see table 1 for abbreviations), b – overall scatter plot of 120 individuals representative of all species. tab. 2. loading of the morphological and floral characters on the first three pca axes of five species of genus asparagus from algeria (see table 1 for abbreviations). pca loadings > 0.60 are indicated in bold. characters pc1 pc2 pc3 lcl 0.65 -0.47 0.05 ncl 0.02 0.78 0.36 cl -0.40 -0.85 0.14 tfl 0.74 0.54 0.26 cfl 0.79 -0.49 0.28 ffl 0.78 -0.20 -0.44 nfl 0.79 0.37 0.32 sti 0.97 0.08 -0.03 lpe 0.56 0.62 -0.16 per 0.22 0.05 0.84 dig 0.22 -0.45 0.29 cba 0.81 -0.49 0.04 ngr 0.18 0.08 -0.54 eigenvalue 5.08 3.16 1.72 total variance (%) 39.13 24.35 13.29 cumulative variance (%) 39.13 63.49 76.79 genus asparagus in algeria acta bot. croat. 81 (2), 2022 171 followed by upgma analysis, were applied to all populations in the overall pca, we first examined the relative contribution of each variable on the first two principal components, then the distribution of individuals (fig. 2). the loading of the variables, the eigenvalues and the cumulative variance to the principal components are given in tab. 2. together, the two first components describe 63.58% of the overall variances with 39.13% and 25.35% for pc1 and pc2 respectively. as shown in fig. 2a, eight variables displayed a high absolute contribution to pc1 in respect to their correlation: pedicel length (lpe), type of flower (tfl), number of flowers (nfl), type of stigma (sti), shape of flower (ffl), color of berries (cba), color of flowers (cfl) and length of cladode (lcl). the highest contribution being that of the type of stigma bifid versus trifid. compared to the second axis, only the number of cladodes (ncl) and the seed diameter (dig) are discriminative. the length of the perianth (per) and the number of seeds per berry (ngr) show no significance in either pc1 or pc2. the scatterplot of individuals on the two first pcs display three main well-separated groups (fig. 2b). compared to pc1, a first group (i) is isolated in the negative part and corresponds to a. acutifolius. by contrast, towards the positive values of axis 1, the next two groups (ii, iii) concern individuals of other species. these last two groups are distinguished from each other in respect to pc2. one corresponds to the individuals of a. horridus, the other to those of a. albus. the individuals belonging to a. altissimus and a. officinalis occupy a neighboring position to a. albus. this principal component analysis makes it possible to discriminate the relevant diagnostic criteria of the five studied species. for instance, the color of berries and type of flowers discriminate all the species. the berries are red in a. albus, a. officinalis and a. altissimus, black in a. acutifolius and purple in a. horridus. the flowers are campanulate in a. acutifolius, stellate in a. albus, a. horridus and a. altissimus, and tubular in a. officinalis. the last two species, a. altissimus and a. officinalis, show affinities in relation to their smooth cladodes. on the other hand, a. horridus and a. acutifolius are outstanding, by one strongly spiny cladode and by bifid stigmas, respectively. a hierarchical analysis using the matrix of the means values by population of the 13 morphological and floral traits, brings out the same grouping of populations as in the previous pca (fig. 3). at the distance d < 100, two main clades are clearly distinct from each other. the first clade brings together the populations belonging to a. albus and a. horridus which constitute small distinctive groups. although developing in open and semi-arid habitats, the populations of these two species show unexpected inter-population variability. the other two species a. officinalis and a. altissimus, show close relationships with a. albus due to the smooth cladodes. within a. acutifolius, two clusters are quite well separated (at the distance d < 55). the first one is mainly composed of coastal populations from tipaza, el aouana and fig. 3. dendrogram analysis showing relationships among 29 algerian populations of the genus asparagus. analysis was performed on the matrix of mean values of 13 morphological and floral traits. the dotted lines correspond to the distances allowing the main clusters to be distinguished. boubetra k., amirouche n., amirouche r. 172 acta bot. croat. 81 (2), 2022 zemmouri. the second cluster concerns populations from the inland collecting sites at higher altitudes such as senalba (southern slope of the saharan atlas) mezloug, ain smara (east of the tellian atlas), redjredj and keddara (central part of the tellian atlas). in order to estimate the impact of climatic factors on morphological variability, we performed another pca focused on the 16 populations of a. acutifolius. indeed, unlike a. albus and a. horridus, populations of a. acutifolius are encountered in very diverse habitats from wetlands to semiarid and arid areas. this pca takes into account the five main parameters of the mediterranean bioclimate (fig. 4). in contrast to the two species a. albus and a. horridus, populations of a. acutifolius are encountered in very diverse habitats of undergrowth and open areas from wetlands to semi-arid and arid zones. in this aim, a second pca was focused on the 16 populations of a. acutifolius taking into account the five main parameters of the mediterranean bioclimate (fig. 4). the pca has been carried out on the average of the values of each variable for each population as well as the annual precipitation (p), the altitude (alt.), the average of the maximum in summer (m °c) and the minimal temperatures in winter (m °c) (on-line suppl. tab. 1). the first two axes pc1 and pc2 describe 55.26% of the total information with 34.45% and 21.81%, respectively (fig. 4a). the length of the cladodes, lcl, and, to a lesser extent ngr, ncl and nfl, are located on the positive part of pc1 and highly correlated with the precipitation p. in contrast, the altitude (alt) is located on the negative part of this axis. pc2 shows, on one hand, a positive correlation between the diameter of the seeds (dig) and the maximum temperature in summer m °c and, on the other hand, a negative correlation with the minimum temperature in winter (m °c). two groups of populations are opposed with respect to pc1 (fig. 4b). the populations from continental, semi-arid to arid bioclimates and higher altitudes (senalba, redjredj, ain smara and from mezloug in the east to mansourah in the west area) constitute a first group. the second one concerns populations from northeast coastal stations as zemmouri and el aouana located in subhumid and humid bioclimate. all other populations are from subhumid bioclimate (keddara, bainem, bouchaoui, tipaza, souidania) and are distributed in an intermediate situation between the two previous groups within the biogeographical sector of algiers. anatomical analysis of the cladodes the cross sections of the cladodes of the five studied species show a different structure. interspecific variations have been observed relatively to the shape of section, the thickness of the cuticle, the shape of epidermal cells, the number of layers of palisade cells, the number of vascular bundles and the presence of raphides (fig. 5, on-line suppl. tab. 2). circular cross sections are found in three species, a. acutifolius, a. officinalis and a. altissimus (fig. 5a, i, k). the first one is characterized by uniseriate epidermis with isodiametric cells strongly cutinized (fig. 5b) with the presence of stomata along this epidermis (fig. 5c). just below the epidermis, the palisade parenchyma consists of two layers of elongated cells rich in chloroplasts. spongy parenchyma varies highly in shape, and the cells may be isodiametric or elongated. at this level, some cells, called idioblasts, contain raphides (calcium oxalate crystal) in the form of rods (fig. 5d). in the pith, the cells are strongly sclerified, and only two vascular bundles have been observed. in a. officinalis, the cuticle is thin and the epidermal cells are rounded and not of the same size with some larger cells (fig. 5j). stomata were observed all around the cross section. the palisade parenchyma is composed of two layfig. 4. principal components analysis focused on 16 populations of a. acutifolius showing the relationships between the morphological and floral traits and the main bioclimatic parameters. a – loading of the variables on the circle of correlations, b – scatter of the mean values of each population. genus asparagus in algeria acta bot. croat. 81 (2), 2022 173 ers of elongated cells. in the pith, two vascular bundles are observed and surrounded by a single layer of spongy parenchyma. in the endemic a. altissimus, the epidermis is made up of a single layer of square cells also covered by thick cuticle with numerous stomata. the palisade parenchyma is composed of two layers of elongated cells. the cells of spongy parenchyma are irregular and some of them contain raphides (fig. 5l). four vascular bundles are observed. in the two other species, i.e. a. albus and a. horridus, the cross section is triangular. (fig. 5e, g). the epidermal cells in a. albus, are rounded to irregular in shape with variable size. they are covered by a thin cuticle (fig. 5e). stomata were also observed. the palisade parenchyma contains elongated cells arranged in three layers whereas the spong y parenchyma consists of relatively large, thin-walled cells. in the pith, the cells are sclerified with four vascular bundles (fig. 5f). in a. horridus, the epidermal cells are rounded with a thick cuticle (fig. 5h). in the palisade parenchyma, the cells were rearranged in several layers with small intercellular spaces. the pith occupies the greatest part and contains numerous vascular bundles arranged in a ring. here, in each bundle, the xylem is directed toward the center of the cladode. discussion taxonomic relationships algeria occupies a central biogeographic position in north africa, characterized by an impressive east-west bioclimatic gradient from humid to arid lands. in this mediterranean region, strong topographic and soil heterogeneity explains the highly contrasting habitats and the floristic richness. in this ecogeographical context, wild species of the genus asparagus occur in various ecological conditions, under forest cover as well as in open and dry habitats of the steppe vegetation of the highlands, showing high tolerance to drought and high temperatures (boubetra et al. 2017a, b). asparagus acutifolius is widespread and quite common in moist and shady biotopes of woodlands and shrublands of humid and semiarid bioclimates. asparagus albus and a. horridus were less common in this research; both are linked to dry and stony soils mostly in arid and semiarid habitats of nw algeria. sometimes, they occur in sympatry in steppic highlands making high xerophytic shrubs. asparagus altissimus is endemic to northwestern algeria consisting of small populations of scattered individuals along hedges on saline and dry rather sandy soils. asparagus fig. 5. cross sections of cladodes of the five algerian asparagus species. a. acutifolius: a – circular section, b – epidermis and cuticle, c – stomata, d – raphides. a. albus: e – triangular section, f – vascular bundles. a. horridus: g – triangular section, h – epidermis and cuticle. a. officinalis: i – circular section, j – epidermis. a. altissimus: k – circular section, l – raphides. cu: cuticle, ep: epidermis, pp: palissadic parenchyma, st: stomata, gc: guard cell, sc: substomatal chamber, r: raphides, pi: pith, vb: vascular bundles, xy: xylem, ph: phloem. boubetra k., amirouche n., amirouche r. 174 acta bot. croat. 81 (2), 2022 officinalis is very rare and appears to have naturalized as isolated individuals on the edges of cultivated fields. despite the wide distribution of the species of the genus asparagus in the mediterranean region and their ecological and economic interest, few studies have been performed to elucidate the morphological variability, particularly for wild a. acutifolius, a. albus and a. horridus. current researches are focused mainly on the cultivated species a. officinalis for its economic importance. studies based on molecular markers, aim to assess genetic diversity among germplasm including the related wild species from europe and asia (mousavizadeh et al. 2021). other studies combine both morphological and molecular data (irshad et al. 2019, chen et al. 2020). morphological variability, growth and production of spears may be correlated to environmental factors (altunel 2021). concerning wild species, a multivariate analysis performed on indian populations of a. racemosus willd. (chithra and siril 2017), showed that significant characters were relative to plant height, the diameter and color of stem, length and number of cladodes in fascicle. in the iranian species, a. officinalis, a. persicus baker, a. verticillatus l., and a. breslerianus schult. & schult., the length of cladodes and seed number per fruit are the most discriminating characters (mousavizadeh et al. 2015). in these species, the number of seeds per berry varies from 1 to 6, unlike the algerian species where the number of seeds is 1-2, exceptionally 3 in an individual of a. officinalis. despite the morphological similarities among some species of the genus asparagus, chen et al. (2020) have shown that individual variations could be linked to environmental factors. the analysis of the genetic variation of italian populations of a. acutifolius, evaluated by the issr markers (sica et al. 2005), indicated an inter-population diversity according to their geographical origin. a study on the systematics and the chorology of a. acutifolius, a. albus and a. fig. 6. illustrations of some cross sections of stems and roots from algerian specimens of asparagus. stem of a. horridus: a – global view of the section, b – arrangement of cribro-vascular bundles. c – trichomes in the stem of a. acutifolius. d – stomata in the stem of a. altissimus. e – vascular bundle in stem of a. horridus. root of a. acutifolius: f – general view of the section, g – root hairs, h – raphides within cells of the cortex, i – general view of vascular cylinder. cu: cuticle, ep: epidermis, st: stomata, co: collenchyma, pi: pith, vb: vascular bundles, xy: xylem, ph: phloem, cr: cortex (parenchyma cells), rh: root hairs, r: raphides, pi: pith, ed: endodermis, pr: pericycle. genus asparagus in algeria acta bot. croat. 81 (2), 2022 175 horridus in sardinia, allowed urbani et al. (2007) to confirm the presence of these species and to exclude a. aphyllus l., from the flora of this island on the basis of anatomical criteria of cladodes. in iran, the wild asparagus polyploids (8x, 10x) are adapted to saline and dry lands (mousavizadeh et al. 2022). these results agree with the repartition of the endemic hexaploid (6x) a. altissimus which occurs preferentially on saline soils. anatomical diversity of the cladodes compared to the stems and roots (fig. 6), the morphology and anatomy of the cladode in the genus asparagus, show a striking diversity and distinctive interspecific features. undoubtedly the structure of the cladodes has a taxonomic significance. it would also be linked to the ecological conditions with regard to the chorology and geographic distribution of each species (boubetra et al. 2017a, b). the cross section is circular in all the species, except a. albus and a. horridus, which show a triangular shape. circular sections have also been observed in asparagus species from bulgaria such as a. tenuifolius lam., and a. acutifolius (raycheva and stojanov 2013). the triangular shape is rare, having been reported only in a. adscendens roxb., (kawale et al. 2014). various other shapes are specific to this genus such as the oval in a. brachyphyllus turcz., and a. schoberioides kunth (ito et al. 2006), irregular elliptic in a. lycicus p.h. davis and a. persicus (güvenç and koyuncu 2002). in a. officinalis, a. maritimus (l.) mill., and a. verticillatus, the shape varies from irregular elliptic to stellar with unclear angles (raycheva and stojanov 2013). samples of a. officinalis from algeria are remarkable for their circular shape. the interspecific variability of cross-section of the cladodes was also observed within other genera such as ruscus where the shape is rectangular in r. aculeatus l., and elliptical in r. colchicus yeo (güvenç et al. 2011). furthermore, the epidermal cells also exhibit a variability. for the algerian specimens of a. acutifolius and a. officinalis, the cells are respectively isodiametric and rounded, whereas they are rectangular in bulgarian populations (raycheva and stojanov 2013), barrel-shaped in a. brachyphyllus (bercu 2008) and longitudinal in a. racemosus (durai prabakaran et al. 2015). in addition, these cells are covered with a cuticle varying in thickness depending on the species. anatomical studies performed on a. asparagoides (l.) druce show that the cuticle thickness correlates with the shape of the section of the cladode (coles et al. 2006). according to tamanian (1982), this variability expresses adaptation to ecological conditions. under the epidermis of each species two or three layers of palisade cells with different lengths are situated. these results are consistent with those obtained on a. adscendens (kawale et al. 2014) and a. racemosus (durai prabakaran et al. 2015). the most numerous and the longest palisade cells were observed in a. verticillatus, with four rows (raycheva and stojanov 2013). in a. tenuifolius, a. maritimus and a. officinalis, the parenchyma is represented by two rows of slightly prolonged cells (raycheva and stojanov 2013). these results correlate with our studied species except for a. albus and a. horridus whose show three and several layers respectively. compared to species of the genus ruscus, the palisade and spongy parenchyma are replaced by layers of cells containing chloroplasts (güvenç et al. 2011). the vascular system is delimited by the assimilation parenchyma by one cell row. in our study, raphides are remarkable in this parenchyma, and are present only in cladodes of a. acutifolius and a. altissimus. their presence in some taxa may have a taxonomic significance as mentioned also by prychid and rudall (1999), also shown in the genus ruscus (güvenç et al. 2011). the cladodes of the algerian samples of a. acutifolius are distinguished by the constant presence of two vascular bundles while in turkish and bulgarian specimens, this number were four and five respectively (güvenç and koyuncu 2002). two vascular bundles have also been reported for a. brachyphyllus and a. officinalis (begum et al. 2017). in a. horridus from algeria, the vascular bundles are can number up to 20 and occupy all the pith, as in the case of a. aphyllus (güvenç and koyuncu 2002). as suggested by raycheva and stojanov (2013), the anatomical characters of the cladodes particularly the number of vascular bundles can be correlated with the ecological environments. conclusion this study constitutes the first report on morpho-anatomy for the genus asparagus in algeria. the results highlight the taxonomic importance of morphological and floral characters as well as the anatomical features of the cladodes, mainly the shape, the number of vascular bundles and the raphides. they also provide perspective for a better understanding of the diversity of species and populations in relation to environmental conditions, particularly for a. acutifolius, which is widespread in highly contrasting bioclimatic conditions. acknowledgements this work was carried out in the framework of the project “asparagales in algeria”, between the university of sciences and techniques houari boumediene (usthb, algiers) and the national institute of forest research (inrf, algiers). the authors wish to thank the two anonymous reviewers for their suggestions and comments that have improved our manuscript. we are also grateful to s. benhouhou, the manager of the official herbarium of ensa (algiers). references altunel, t.a., 2021: morphological and habitat characteristics of asparagus (asparagus officinalis l.) and socio-economic structure of producers. turkish journal of agriculture food science and technology 9, 1092–1099. angiosperm phylogeny group iii, 2009: an update of the angiosperm phylogeny group classification for the orders and boubetra k., amirouche n., amirouche r. 176 acta bot. croat. 81 (2), 2022 families of flowering plants: apg 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(asparagaceae) in sardinia (italy). bocconea 21, 267–271. 701 besendorfer and mlinarec.vp acta bot. croat. 72 (1), 1–12, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 retention of relict satellite dna sequences in anemone (ranunculaceae) vi[nja besendorfer, jelena mlinarec* faculty of science, university of zagreb, division of biology, department of molecular biology, horvatovac 102a, hr-10000 zagreb, croatia abstract – satellite dna is a genomic component present in virtually all eukaryotic organisms. the turnover of highly repetitive satellite dna is an important element in genome organization and evolution in plants. here we study the presence, physical distribution and abundance of the satellite dna family ahtr1 in anemone. twenty-two anemone accessions were analyzed by pcr to assess the presence of ahtr1, while fluorescence in situ hybridization and southern hybridization were used to determine the abundance and genomic distribution of ahtr1. the ahtr1 repeat unit was pcr-amplified only in eight phylogenetically related european anemone taxa of the anemone section. fish signal with ahtr1 probe was visible only in a. hortensis and a. pavonina, showing localization of ahtr1 in the regions of interstitial heterochromatin in both species. the absence of a fish signal in the six other taxa as well as weak signal after southern hybridization suggest that in these species ahtr1 family appears as relict sequences. thus, the data presented here support the »library hypothesis« for ahtr1 satellite evolution in anemone. similar species-specific satellite dna profiles in a. hortensis and a. pavonina support the treatment of a. hortensis and a. pavonina as one species, i.e. a. hortensis s.l. keywords: anemone, fish, library hypothesis, satellite dna abbreviations: fish – fluorescence in situ hybridization, satdna – satellite dna introduction the genus anemone s.str. consists of approximately 150 species (tamura 1995), mainly distributed in the northern hemisphere. the ancestry, phylogenetic differentiation and systematic classification of anemone have been debated for years. to advance the knowledge of the phylogenetic relationships within anemone, considerable efforts based on dna-analytical approach have been applied successfully and provided new insights into interspecific relationships (hoot et al. 1994, ehrendorfer and samuel 2001, schuettpelz et al. 2002, acta bot. croat. 72 (1), 2013 1 * corresponding author, e-mail: jelena@biol.pmf.hr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:22 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees ehrendorfer et al. 2009, meyer et al. 2010). hoot et al. (1994) and meyer et al. (2010) suggest that hepatica, knowltonia and pulsatilla as well as the south american genera oreithales and barneoudia should be subsumed within the anemone s. lat and propose a preliminary classification that recognizes two subgenera (anemonidium and anemone), seven sections, and 12 informal subsection groupings. the european anemone taxa belong to the anemone section, with only one exception, a. narcissifolia, which belongs to the anemonidium section. the coronaria group is the largest in the anemone section, comprising all the mediterranean and american tuberous anemone. anemone species were considered favourable plant material in cytogenetic studies because of chromosomal polymorphism, different ploidy levels, as well as variation in dna content among species (böchner 1945, heimburger 1959, rothfels et al. 1966, baumberger 1970, mlinarec et al. 2006, mlinarec et al. 2012a, mlinarec et al. 2012b). following the discovery of differential staining methods such as c-banding in the 1970s, anemone again became a subject of interest due to the high quantities of heterochromatin and variation in distribution among species (marks 1974, marks and schweizer 1974). the greatest variations in heterochromatin amount and distribution on chromosomes are found among the members of the coronaria group (mlinarec et al. 2012b). satellite dna (satdna) is highly repetitive, non-coding and organized into long arrays composed of thousands to millions of tandemly arranged units. these arrays form constitutive heterochromatin (ugarkovi] and plohl 2002). different satdna sequences can coexist in genomes, forming what has been defined as a library of satdnas (fry and salser 1977, me[trovi] et al. 1998). despite well-recognized roles of telomeric and centromeric satdnas in the stabilization of chromosome ends and in cell division, their overall biological significance remains unclear (csink and henikoff 1998). since satdna evolves through evolutionary processes as predicted by the molecular drive model (dover 1986), dna turnover usually leads to a high interspecific divergence and low intraspecific variation. the balance, persisting between satellite homogenization and persistence of satellite variants, could generate sufficient sequence divergence to cause reproductive isolation between intraspecific lineages, ultimately leading to speciation. the ahtr1 satdna monomer is an at-rich sequence of 560 bp; this satdna sequence constitutes about 0.14% of the a. hortensis genome, roughly about 3.05×104 copies (mlinarec et al. 2009). the same authors only found evidence of ahtr1 in a. hortensis; the absence of southern hybridization signals was found in other relatives tested. here we characterize the presence, genomic distribution and abundance of the ahtr1 in 22 species of anemone using pcr, fluorescence in situ hybridization (fish) and southern hybridization. our goals were to: i) evaluate the phylogenetic signal of satdna family in a genus and ii) gain an insight into the karyotype and genome evolution of anemone using satdna markers. materials and methods plant material information on all plant taxa used in this study is given in table 1. plants were grown in pots in the botanical garden of university of zagreb. all species were identified by morphological and karyological characteristics. for karyological studies, actively growing 2 acta bot. croat. 72 (1), 2013 besendorfer v., mlinarec j. 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:22 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees root-tip meristems were pretreated with 0.05% colchicine (sigma-aldrich chemie gmbh, taufkirchen, germany) for 4 h at rt, fixed in a solution of ethanol and acetic acid (3:1) for 24 h at –20 °c, and stored in 70% ethanol at –20 °c until use. for cloning as well as for southern hybridization, high quality genomic dna was isolated from young leaves using the qiagen mini kit (gmbh, hilden, germany) according to manufacturer's instructions. acta bot. croat. 72 (1), 2013 3 satellite-dna evolution in anemone tab. 1. species and accessions used in this study. the sectional classification is according to meyer et al. (2010). positive (+) and negative (–) pcr amplifications of the ahtr1 of the analysed accessions are also reported. taxon accession no. origin and/or source section pcr anemone apennina l. 1446b dizdarica (montenegro), anemone + anemone blanda schott et kotschy 12418 bg university of vienna (austria) anemone + anemone coronaria l. 1725h anecor, esdraelo plain, tel shiron, 25 km se of haifa (israel) anemone + anemone hortensis l. 8216r island of hvar (croatia) anemone + anemone palmata l. 12434 morgion, marseille (france) anemone + anemone pavonina lam. 12724 bogdanci, plajurci (macedonia) anemone + anemone ranunculoides l. 289 delibatska pe{~ara (serbia) anemone + anemone sylvestris l. 1451b ^u~erje, medvednica (croatia) anemone + anemone parviflora michx. 20081630 rbg edinburgh (uk) anemone – anemone obtusiloba d. don 19861079 rbg edinburgh (uk) homalocarpus – anemone demissa hook et thomson 19910632 upper mo chu dist. (bhutan), rbg edinburgh (uk) homalocarpus – anemone rivularis buch.-ham. 28070 himalaya (nepal), rbg kew (uk) rivularidium – anemone hupehensis lemoine 28069 sichuan, huangtuliang hills (china), rbg kew (uk) rivularidium – anemone tomentosa (maxim.) c.'pei 28071 rbg kew (uk) rivularidium – anemone canadensis l. 28068 south dakota (usa), rbg kew (uk) anemonidium – anemone narcissiflora l. 1864l baden-würtemburg, german alps near beuron (germany), bg university of zagreb (croatia) homalocarpus – anemone baldensis l. 698g vácrátót (hungary), anemone – anemone trifolia l. 562 botanical garden of the university of zagreb anemone – anemone cylindrica a. gray 6559b chemnitz (germany) anemone – anemone multifida poir. 1247 chemnitz (germany) anemone – anemone nemorosa l. 558 dubravkin put, zagreb (croatia) anemone – anemone virginiana l. 11838b quebec, country deux-montaignes (canada) anemone – 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:22 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees pcr amplification and cloning pcr amplifications of the satdna family ahtr1 were carried out in a 50 ml reaction mixture containing 10 ng template dna, 0.4 mm of each primer, 200 mm dntps, 2.5 u gotaq® dna polymerase and corresponding 1x (1.5 mm mgcl2) green reaction buffer (promega cor., madison, usa), using the primer pairs ahtr1-1 (5'gtgtgaggtataacacactgt 3') and ahtr1-2 (5' tagtgttgtggaatacacatc 3'). after an initial denaturing step at 94 °c for 3 min, the amplification was carried out in 30 cycles consisting of denaturation at 94 °c for 1 min, annealing at 54 °c for 10 sec and primer extension at 72 °c for 1 min with final extension at 72 °c for 20 min. cloning and transformation procedure were carried out using the instaclonetm pcr cloning kit (fermentas gmbh, germany) or pgem®-t easy vector system (promega, usa) according to the manufacturer's instructions. sequencing was carried out by macrogen inc. (seoul, korea). sequence alignment and phylogenetic analysis the number of ahtr1 clones by anemone taxa is indicated in table 2. ahtr1 clones of a. hortensis (eu769127-eu769132) were taken from mlinarec et al. (2009). all sequences obtained in this study are the result of cloning and are deposited in genebank under the accession numbers: a. ranunculoides ahtr1 (kc148493kc148496), a. apennina ahtr1 (kc148497-kc148500), a. sylvestris ahtr1 (kc148501-kc148502), a. coronaria ahtr1 (kc148503-kc148505), a. blanda ahtr1 (kc148506-kc148509), a. palmata ahtr1 (kc148510-kc148514), a. pavonina ahtr1 (kc148515-kc148520). sequences were aligned with clustal x v1.81 (thompson et al. 1997). the evolutionary history was inferred using the neighbor-joining method (saitou and nei 1987). the evolutionary distances were computed using the kimura 2-parameter model (kimura 1980) and are in the units of the number of base substitutions per site. the analysis involved 34 nucleotide sequences. all positions containing gaps and missing data were eliminated. the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. evolutionary analyses were conducted in mega5 (tamura et al. 2011). 4 acta bot. croat. 72 (1), 2013 besendorfer v., mlinarec j. tab. 2. features of ahtr1 sequences by anemone species. taxa sequence number sequence length at % polymorphic sites anemone apennina 4 490–494 66 104 a. blanda 4 455–458 67 41 a. coronaria 3 457–492 65 106 a. hortensis 6 489–485 68 163 a. palmata 5 385–495 67 100 a. pavonina 6 492–496 69 139 a. ranunculoides 4 488–495 66 84 a. sylvestris 2 496 67 67 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:22 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees chromosome preparation and fluorescence in situ hybridization chromosome preparations for fish are described in mlinarec et al. (2006). fish experiments were done according to mlinarec et al. (2012b). the probes apav2ahtr1 and abla3ahtr were directly labelled with cy3-dctp (amersham, ge healthcare, little chalfont, buckinghamshire, uk) by using a nick-translation kit according to the manufacturer's instructions (roche diagnostics gmbh, mannheim, germany). after overnight hybridization, slides were given a stringent wash in 1 x ssc. these stringency conditions allowed the target sequences of approx. 56% homology to remain hybridized (schwarzacher and heslop-harrison 2000). the preparations were mounted in dako fluorescent mounting medium (dako north america, inc., ca93013, usa) and stored at 4 °c. signals were visualized and photographs captured on an olympus bx51 microscope, equipped with a highly sensitive olympus dp70 digital camera. an average of 10 well-spread metaphases was analyzed for each individual. two individuals per taxa were analyzed. southern hybridization southern hybridization analyses were performed using gene images alkphos direct labelling and detection system (amersham, ge healthcare, uk). genomic dna (gdna) (1.6 mg) was digested over night with ecorv (new england biolabs, ipswich, usa). digested gdna was loaded per lane on a 1% (w/v) agarose gel and electrophoretically separated for several hours at 100 v. dna was blotted onto a positively charged nylon membrane (roche, basel, switzerland) for one hour by using the model 785 vacuum blotter (biorad, hercules, usa). crosslinking was performed for 3 min (0.24 j cm–2) on an uvlink cl508m crosslinker (uvitec, cambridge, uk). the membrane was prehybridized for half hour and hybridized over night at 55 °c using the probe apav2ahtr. subsequently, the membrane was washed at 55 °c and room temperature according to manufacturer's instructions. results pcr amplification of the ahtr1 satellite family in anemone to detect the presence of ahtr1 satdna family we performed pcr amplification in anemone. the ahtr1 repeat unit was amplified in 8 of 22 anemone taxa. all eight species in which the ahtr1 was amplified were phylogenetically related, belonging to the anemone section. six of the eight taxa were members of the mediterranean group coronaria (a. apennina, a. blanda, a. pavonina, a. palmata, a. hortensis and a. coronaria). surprisingly, ahtr1 was amplified in a. sylvestris and a. ranunculoides, members of the multifida and nemorosa groups, respectively, but not in their closest relatives. as expected, successful pcr amplification usually resulted in a ladder pattern of products (data not shown), suggesting that the ahtr1 family exhibits a tandem repeat organization in the genome. sequence analysis to investigate diversity of ahtr1 satdna family in anemone we have sequenced a total of 32 monomeric repeats of the ahtr1 family belonging to a total of eight species of the genus anemone. table 2 details for each species the number of repeat analyses, acta bot. croat. 72 (1), 2013 5 satellite-dna evolution in anemone 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:22 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees monomer length, base composition and polymorphism. the anemone ahtr1 was 385–496 bp and 65–69% in a+t content. the number of polymorphic sites ranged from 41–163, being the lowest in a. blanda and the highest in a. hortensis. percentage similarity across all 32 ahtr1 sequences ranges from 53–99%. previously we found that ahtr1 satellite monomer of a. hortensis contains a pentanucletide sequence (caaaa) that may have consequence for chromatin packing and sequence homogeneity (mlinarec et al. 2009). here we have found this motif in 26 out of 32 cloned ahtr1 sequences. phylogenetic analysis to evaluate the phylogenetic signal of satdna family in a genus we performed analyses with ahtr1 sequences as molecular markers. un-rooted neighbor-joining tree showed 6 acta bot. croat. 72 (1), 2013 besendorfer v., mlinarec j. clade i clade ii a p a la h t r -3 a p a v a h t r -4a sy la h t r -1 a al ta h t r -3 ap a v ah tr -2 a p al a ht r4 a r an a h t r3 a h tr -2 9 a c o ra h t r1 a c ora ht r-3 a pa va htr -3 a a pea ht r-2 a a p e a h t r -3 a p a v a h t r -6 a a p e a h t r -4 a p a la h t r -5 a p a v a h t r -5 a h t r -3 4 a h t r -1 7 a h tr -1 6 as yl ah tr -2 ah tr 3 3 a a p eah tr1 a p al a h tr -2 a p al a h tr -6 a p avah t r1 a rana ht r1 a ra n a h t r -4 a ra n a h t r -2a h t r -2 2 a b la a h t r 2 a b la a h t r -4 a c o ra h t r -2 a b la a h t r -1 ablaahtr -3 0 .0 2 fig. 1. phylogenetic relationships among cloned ahtr1 sequences of a. apennina (aapeahtr1), a. blanda (ablaahtr1), a. coronaria (acorahtr1), a. hortensis (ahtr1), a. palmata (apalahtr1), a. pavonina (apavahtr1), a. ranunculoides (aranahtr1) and a. sylvestris (asylahtr1). 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:23 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees separation of sequences into two slightly divergent clades: i and ii. the sequences originating from a. coronaria, a. sylvestris, a. apennina, a. palmata, a. hortensis, a. ranunculoides and a. pavonina fell within the two distinct clades i and ii, while those from a. blanda fell only within clade ii (fig. 1). in both clades the sequences are intermingled. the only exception is a. blanda which showed a clear separation within clade ii. interestingly, one clone of a. coronaria associated with those of a. blanda. for more detailed phylogenetic analyses, more clones should be isolated from each individual. fish in anemone clone apav2ahtr1 was used as a probe to determine the chromosomal position of ahtr1 satdna family in the anemone taxa in which ahtr1 was pcr-amplified. the only exception was a. blanda in which clone abla3ahtr was used as a probe as this species proved to have a distinctive ahtr1 family. the ahtr1 sequences were either completely lacking or predominantly localized at the intercalary dapi-positive heterochromatic regions of chromosomes. clear fish signals were visible only in a. hortensis and a. pavonina (fig. 2), while negative in situ results were obtained in the karyotypes of other six species in which the ahtr1 was pcr-amplified (data not shown). southern blot analysis to determine the abundance and organization of ahtr1 isolated from a. hortensis within different species of anemone, clone apav2ahtr1 was hybridized to ecorv-digested genomic dna of a. apennina, a. blanda, a. coronaria, a. hortensis, a. palmata, a. pavonina, a. ranunculoides and a. sylvestris (fig. 3). in accordance with the fish experiments, only a. hortensis and a. pavonina exhibited a strong ladder-like hybridization signal of similar intensity, suggesting that in these two species satdna family ahtr1 is highly abundant and represented by a similar copy number. in the lanes of the six other species, the hybridization signal was hardly visible suggesting that in these species ahtr1 is present only as minor repeats. acta bot. croat. 72 (1), 2013 7 satellite-dna evolution in anemone fig. 2. fish on mitotic chromosomes of anemone hortensis (a) and a. pavonina (b) with labelled apav2jme probe (in red). bar = 10 µm. 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:27 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees discussion distribution of satdna family ahtr1 is in agreement with phylogeny: the ahtr1 satdna family is pcr-amplified in the european members of the anemone section. by contrast, the phylogenetic tree of ahtr1 sequences is not congruent with the phylogeny of these species based on other markers. using atpb-rbcl spacer region and its data meyer et al. (2010) demonstrated that anemone section contains three major clades: a basal clade composed of the members of the baldensis group; a second clade composed of the members of the nemorosa and multifida groups; and a third clade including all tuberous mediterranean and american anemone of the coronaria group. in contrast, the tree based on ahtr1 sequences shows an intermixture of sequences originating from the members of the coronaria, nemorosa and multifida groups with slight divergence of sequences into two clades. the lack of species-specific clustering can be partly explained by divergence of sequences in the ancestors of the species analyzed. however, it is possible that deeper sampling of sequences, within each species, might produce sub-trees that are more representative of species relationships. in the ahtr1 tree, the only exception is a. blanda, which shows low intra-individual nucleotide diversity and clear separation from the other anemone taxa in which ahtr1 was amplified. the phylogenetic position of a. blanda has been debated for years. in the tree based on atpb-rbcl spacer region and its data a. blanda has a basal position to all anemones of the anemone section. thus, the position of a. blanda in the ahtr1 tree agrees with the position based on other molecular markers suggesting that although similar morphological characteristics and geographic distribution unite a. blanda with other mediterranean anemones in the coronaria group, this species underwent different evolutionary pathways than the other mediterranean tuberous anemone from the coronaria group. the dna library model proposed by fry and salser (1977) hypothesized that closely related species share a set of satellite dna families (satdna library) differing in copy number and sequence divergence (ugarkovi] and plohl 2002). as a consequence of the 8 acta bot. croat. 72 (1), 2013 besendorfer v., mlinarec j. fig. 3. genomic restriction digests (a) and southern blotting analyses (b) of anemone apennina (1), a. blanda (2), a. coronaria (3), a. hortensis (4), a. palmata (5), a. pavonina (6), a. ranunculoides (7) and a. sylvestris (8) with restriction endonuclease ecorv and probed with clone apav2ahtr1. m – marker, bp – base pairs. 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:29 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees amplification of a particular satdna family in a species, this satdna becomes highly abundant, while the others are present only as minor repeats. this leads to species-specific profiles (ugarkovi] and plohl 2002). in anemone we have found a support for this hypothesis. the presence of ahtr1 in all mediterranean anemones as well as in the representatives of the other two groups of the anemone section (the multifida and nemorosa groups) suggests that this family was present in the common ancestor of the anemone section. however, the highly dynamic nature of satdna turnover resulted in considerable fluctuation in satellite copy number. in a. hortensis and a. pavonina it became highly abundant, while in other members it stayed only as minor repeats, even in the close relative a. coronaria. furthermore, the abs1 satdna family constitutes the major fraction of a. blanda interstitial at-rich heterochromatin, about 2 % of its genome, while in sister species a. apennina the family is present as minor repeats (hagemann et al. 1993). these two satellite dna families, ahtr1 and abs1, are highly divergent (<35% similarity) suggesting that in a. hortensis and a. blanda different satdna families were amplified and became abundant. this further supports the »satdna library« hypothesis. besides, it is worth mentioning that in the six species where ahtr1 was present in lower numbers, a ladder pcr pattern was still recovered. the tandem arrangement might be important for maintaining the identity of these repeats via homogenization mechanisms. there are, to our knowledge, few empirical examples for the »satdna library« hypothesis in plants (koukalova et al. 2010, quesada del bosque et al. 2011). the mediterranean anemones are mostly diploids (except for a. palmata in which the autotetraploid 2n=4x=32 populations are reported, médail et al. 2002). the experimental hybrids between a. coronaria, a. palmata and a. hortensis exhibit meiotic asyndesis and are completely sterile (maïa and venard 1976). as repetitive sequences constitute the major fraction of the genome, it is reasonable to suggest that these are responsive to maintaining reproductive isolation between the mediterranean anemone taxa. on the other hand, experimental hybrids between a. hortensis and a. pavonina exhibit relatively normal meiosis and fertility (maïa and venard 1976). considering this and the existence of molecular and morphological similarities between a. hortensis and a. pavonina, ehrendorfer et al. (2009) proposed to treat a. hortensis and a. pavonina as one polymorphic species, i.e. a. hortensis s.l. the results of this study showed that a. hortensis and a. pavonina share similar species-specific satdna profile and thus clearly support the treatment of a. hortensis and a. pavonina as one species, i.e. a. hortensis s.l. in summary, the study of ahtr1 satellite dna in anemone reveals a complex evolutionary history where differential levels of satellite dna amplification in different lineages, at different evolutionary times, and in different chromosomal places gave rise to sequence variants persisting as »library« (me[trovi] et al. 1998). acknowledgements we thank darko mihelj for maintaining the plant material and avionan danin, mare lisi~kova, peter brownless from rbg edinburgh and mario vallejo-marin for assistance with the collection of plant material. this work was funded by the ministry of science, education and sport of the republic of croatia, grant no. 119-1191196-1201. acta bot. croat. 72 (1), 2013 9 satellite-dna evolution in anemone 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:29 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees references baumberger, h., 1970: chromosomenzahlbestimmungen und karyotypanalysen bei den gattungen anemone, hepatica und pulsatilla. berichte der schweizerischen botanischen gesellschaft 80, 17–95. böchner, t. w., 1945: meiosis in anemone apennina with special reference to chiasmata localization. hereditas 31, 221–231. csink, a., henikoff, s., 1998: something from nothing: the evolution and utility of satellite repeats. trends in genetics 14, 200–204. dover, g.a., 1986: molecular drive in multigenes families. how biological novelties arise, spread and are assimilated. trends in genetics 168, 159–165. ehrendorfer, f., samuel, r., 2001: contributions to a molecular phylogeny and systematics of anemone and related genera (ranunculaceae-anemoninae). acta phytotaxonomica sinica 39, 293–307. ehrendorfer, f., ziman, s. n., könig, c., keener, c. s., dutton, b. e., tsarenko, o. n., bulakh, e. v., boscaiu, m., médail, f., kästner a., 2009: taxonomic revision, phylogenetics and transcontinental distribution of anemone section anemone (ranunculaceae). botanical journal of the linnean society 160, 312–354. fry, k., salser, w., 1977: nucleotide sequence of hs-a satellite dna from kangaroo rat dipomys ordii and characterization of similar sequences in other rodents. the cell 12, 1069–1084. hagemann, s., scheer, b., schweizer, d., 1993: repetitive sequences in the genome of anemone blanda: identification of tandem arrays and dispersed repeats. chromosoma 102, 312–324. heimburger, m., 1959: cytotaxonomic studies in the genus anemone. canadian journal of botany 37, 587–612. hoot, s. b., reznicek, a. a., palmer, j. d., 1994: phylogenetic relationships in anemone (ranunculaceae) based on morphology and chloroplast dna. systematic botany 19, 169–200. kimura, m., 1980: a simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. journal of molecular evolution 16, 111–120. koukalova, b., moraes, a. p., renny-byfield, s., matyasek, r., leitch, a. r., kovarik, a., 2010: fall and rise of satellite repeats in allopolyploids of nicotiana over c. 5 million years. new phytologist 186, 148–160. maïa, n., venard, p., 1976: contribution a l'étude cytotaxonomique d'espèces méditerranéennes d'anemone et de leurs hybrides. canadian journal of genetic and cytology 18, 151–168. marks, g. e., 1974: giemsa banding of meiotic chromosomes in anemone blanda l. chromosoma 49, 113–119. marks, g. e., schweizer, d., 1974: giemsa banding: karyotype differences in some species of anemone and in hepatica nobilis. chromosoma 44, 405–416. 10 acta bot. croat. 72 (1), 2013 besendorfer v., mlinarec j. 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:29 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees médail, f., ziman, s., boscaiu, m., riera, j., lambrou, m., vela, e., dutton, b., ehrendorfer, f., 2002: comparative analysis of biological and ecological differentiation of anemone palmata l. (ranunculaceae) in the western mediterranean (france and spain): an assessment of rarity and population persistence. botanical journal of the linnean society 140, 95–114. me[trovi], n., plohl, m., mravinac, b., ugarkovi], \., 1998: evolution of satellite dnas from the genus palorus-experimental evidence for the »library« hypothesis. molecular biology and evolution 15, 1062–1068. meyer, k. m., hoot, s. b., arroyo, m. t. k., 2010: phylogenetic affinities of south american anemone (ranunculaceae) including the endemic segregate genera, barneoudia and oreithales. international journal of plant sciences 171, 323–331. mlinarec, j., chester, m., siljak-yakovlev, s., pape[, d., besendorfer, v., 2009: molecular structure and chromosome distribution of three repetitive dna families in anemone hortensis l. (ranunculaceae). chromosome research 17, 331–343. mlinarec, j., pape[, d., besendorfer, v., 2006: ribosomal, telomeric and heterochromatin sequences localization in the karyotype of anemone hortensis. botanical journal of the linnean society 150, 177–186. mlinarec, j., [atovi], z., malenica, n., ivan^i]-ba]e, i., besendorfer, v. 2012a: evolution of the tetraploid anemone multifida (2n = 32) and hexaploid a. baldensis (2n = 48) (ranunculaceae) was accompanied by rdna loci loss and intergenomic translocation: evidence for their common genome origin. annals of botany 110, 703–712. mlinarec, j., [atovi], z., mihelj, d., malenica, n., besendorfer, v., 2012b: cytogenetic and phylogenetic studies of diploid and polyploid members of tribe anemoninae (ranunculaceae). plant biology 14, 525–536. quesada del bosque, m. e., navajas-perez, r., panero, j. l., fernandez-gonzalez, a., garrido-ramos, 2011: a satellite dna evolutionary analysis in the north american endemic diocious plant rumex hastatulus (polygonaceae). genome 54, 253–260. rothfels, k., sexsmith, e., heimburger, m., krause, m. o., 1966: chromosome size and dna content of species of anemone and related genera (ranunculaceae). chromosoma 20, 54–74. saitou, n., nei, m., 1987: the neighbor-joining method: a new method for reconstructing phylogenetic trees. molecular biology and evolution 4, 406–425. schuettpelz, e., hoot, s. b., samuel, r., ehrendorfer, f., 2002: multiple origins of southern hemisphere anemone (ranunculaceae) based on plastid and nuclear sequence data. plant systematics and evolution 231, 143–151. schwarzacher, t., heslop-harrison, j. s., 2000: practical in situ hybridization. oxford: bios. tamura, k., peterson, d., peterson, n., stecher, g., nei, m., kumar, s., 2011: mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. molecular biology and evolution 28, 2731– 2739. acta bot. croat. 72 (1), 2013 11 satellite-dna evolution in anemone 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:29 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees tamura, m., 1995: angiospermae: ordnung ranunculales, fam. ranunculaceae, anemoneae. in: hiepko, p. (ed.), die natürlichen pflanzenfamilien 17a, 4, 324–349. duncker and homblot, berlin. thompson, j. d., gibson, t. j., plewniak, f., jeanmougin, f., higgins, d. g., 1997: clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids research 25, 4876–4882. ugarkovi], \., plohl, m., 2002: variation in satellite dna profiles-causes and effects. the embo journal 21, 5955–5959. 12 acta bot. croat. 72 (1), 2013 besendorfer v., mlinarec j. 701 besendorfer and mlinarec.ps u:\acta botanica\acta-botan 1-13\701 besendorfer and mlinarec.vp 14. o ujak 2013 10:27:29 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees opce-str.vp book review plant cell compartments – selected topics. b. schoefs (ed.) 2008. research signpost (kerala, india). 215 usd (paperback) 450 pp. isbn 978-81-308-0104-9 this book has 16 chapters closely or loosely related to each other, grouped into 6 parts. in this book review, each chapter has been treated individually in order to give an account of the content of the volume. the reviewer admits that his view and his emphases may be subjective. chapter 1 (»the nuclear pore complex: from higher eukaryotes to plants«, by s. m. paulillo and b. fahrenkrog) gives an up-to-date description of this structure and its molecular constituents. the authors avoid mentioning a transporter, rather they identify the central plug with a cargo in transit, an old idea re-proposed now on the basis of recent afm and em studies. even so, how the targeted transport (import/export) actually happens still remains to be elucidated. the next chapter (»cell wall changes during strawberry fruit ripening«, by g. a. martinez) describes an applied and rather specific study about the highly dynamic changes that plant cell walls can undergo during fruit maturation. the strawberry model is compared with other fruit types. semi-autonomous organelles are treated in the following six chapters from different points of view. k. rohacek et al. give a very comprehensive treaty under the title »chlorophyll fluorescence: a wonderful tool to study plant physiology and plant stress«. their review is a practical manual (or guidebook) including the basic principles, instrumental set-ups, experimental protocols and data analysis, closing with an overview of chlorophyll fluorescence imaging. this is followed by »update in nucleotide dependent processes in plant chloroplasts« (c. spetea and s. thuswaldner), which extends to three classes of nucleotide binding proteins in the chloroplast: 1. atp-binding transporters, 2. nucleotide diphosphate kinases, 3. gtp-binding proteins. these transporters play crucial roles in the regulation of photosynthesis. in the next chapter (»prolamellar body: a unique plastid compartment, which does not only occur in dark-grown leaves«) k. solymosi and b. schoefs primarily address the debated question of whether the greening of etioplasts can be considered a model of chloroplast development or whether it is an unnatural experimental system. they give numerous arguments about the occurrence of the etiolated state in certain stages of the development of different plant organs under totally natural conditions. this convincingly speaks in favour of the usefulness of the etiolated system. after this they thoroughly discuss the structure, the molecular composition and the development of prolamellar bodies. writing their review, these authors traced their way back to the original articles describing the original concepts instead of referring only to recent literature data. subsequently a brief review is added (»control of electron transfer reactions in photosynthetic reaction centers by dielectric permittivity: a quantitative description based on the marcus theory«) by c. s. chamorovsky et al. the correlation between the parameters mentioned in the title is interpreted by the distance dependence of the electron tunnelling rate. acta bot. croat. 71 (1), 2012 b1 u:\acta botanica\acta-botan 1-12\book review.vp 26. o ujak 2012 14:58:42 color profile: disabled composite 150 lpi at 45 degrees then the reader can turn to »the uncoupling proteins of plant mitochondria: from gene expression patterns to putative activities« (by c. hourton-cabassa and f. moreau), which is a pioneering assay on the plant homologues of thermogenic proteins in brown adipose tissue. mitochondria are treated from quite another point of view by i. foisser under the title »microfilaments, microtubules and mitochondria in characean internodal cells«. she points out that the two sets of these organelles (cortical and endoplasmic) move differently in elongating cells, and this seems to be influenced by the photosynthesis-based ph-bands in the cell. the forthcoming three chapters are grouped under the main title »impact of abiotic factors on organelles«. the first one deals with the »cytoplasmic accumulation of astaxanthin by the green alga haematococcus pluvialis« by y. lemoine et al., and reviews how environmental factors can trigger complete reorientation of the algal cell metabolism. the second one, entitled »plastids and metals« by i. poirier et al., compares the impacts of several heavy metals on the photosynthetic machinery. the third chapter, »betalains: vacuolar pigments« by h. el gharras presents a review on the biochemistry of betalain pigments. the next three chapters under the title »impact of biotic factors on organelles« deal with mycorrhiza. the first (»plastid reorganization in arbuscular mycorrhizal roots«, by t. fester) gives an account of the proliferation of plastids and their increased biosynthetic capacity related to this symbiosis. the second (»effect of mycorrhizal and biocontrol fungi on peroxidase activity of phoenix dactylifera palms«, by l. ben khaled et al.) focuses on a specific consequence of fungal inoculation, while the third one (»plasma membrane proteins in arbuscular mycorrhiza«, by g. recorbet et al.) is more general, including data obtained by classical methods, transcriptomics and proteomics on the profound modifications that the plasma membrane surrounding the arbuscules can undergo upon cell colonization by an arbuscular mycorrhizal fungus. the closing part (special topics) contains two chapters about organisms that do not belong to the plant kingdom, yet do have a compartment characteristic for plants, the vacuole (»vacuole-like structures in prokaryotes – a personal account«, by h. guo), or the plastid (apicoplast) in parasites (»structure, function and biogenesis of the secondary plastid of apicomplexan parasites«, by c. bisanz et al.). rather than offering a classical systematic coverage of plant cell compartments, this book includes chapters focused on emerging, debated, or new areas. in this way, the book is quite original and provides a lot of information not found in earlier books on plant cell biology. áron keresztes department of plant anatomy eötvös loránd university budapest, hungary keraron@freemail.hu b2 acta bot. croat. 71 (1), 2012 u:\acta botanica\acta-botan 1-12\book review.vp 26. o ujak 2012 14:58:42 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 81 (2), 2022 197 acta bot. croat. 81 (2), 197–205, 2022 coden: abcra 25 doi: 10.37427/botcro-2022-017 issn 0365-0588 eissn 1847-8476 a systematic study of thlaspi s.l. taxa in the sections nomisma, thlaspi and pterotropis from turkey based on fruit morphological and molecular data mehmet cengiz karaismailoğlu1*, behcet i̇nal2, osman erol3 1 bartın university, faculty of science, department of molecular biology and genetics, bartın, turkey 2 siirt university, agriculture faculty, department of agricultural biotechnology, siirt, turkey 3 i̇stanbul university, faculty of science, department of biology, i̇stanbul, turkey abstract – the classification of thlaspi s.l. is still problematic. earlier phylogenetic research of the genus focused on several small groups within thlaspi s.str. and lacked detailed morphological observations. the relationships among eurasian taxa and the value of fruit morphology in defining them have yet to be studied. the aim of this study was to analyze 22 taxa belonging to the nomisma, thlaspi and pterotropis sections of thlaspi s.l. from turkey using maximum likelihood (ml) analysis of internal transcribed spacer (its) sequences. we also analyzed the morphological features of the fruit. according to the results, the examined taxa fell into 2 main clades. moreover, clade ii showed 3 sub-clusters. thlaspi huetii and t. aghricum were the most distant taxa with a distance of 0.49%; however, t. ochroleucum and t. violascens were found to be 99% similar. according to its region data based on multiple populations of each taxon, t. arvense, t. huetii, t. perfoliatum, t. violascens, t. cataonicum, t. elegans, t. rosulare and t. aghricum were placed together in one cluster, which indicates that they are monophyletic. thlaspi elegans was found to be a polyploid complex based on bootstrap (bs) (a resampling technique that uses replacement sampling to estimate statistics in a population) values, which varied widely among the studied t. elegans taxa (98, 65 and 49%). fruit morphology also supported the inter-specific relationships based on molecular data, and relationships found by its region data were compatible with fruit type and geographic distribution. a diagnostic key based on fruit morphology is provided for the identification of the examined thlaspi taxa. keywords: diagnostic key, fruit, its region, taxonomy, thlaspi, turkey introduction the genus thlaspi sensu lato (s.l.) is a large and dynamic complex in the brassicaceae family, which is widespread in eurasia and north america and represented by more than 75 species (karaismailoğlu and erol 2018). in turkey, it is represented by 36 taxa belonging to 6 sections, 22 of which are endemic. the first comprehensive study of the nomisma, thlaspi and pterotropis sections was conducted by hedge (1965). sixteen taxa belonging to the sections specified in the study are included in the flora of turkey (hedge 1965). subsequent floristic studies have added t. leblebicii (gemici and görk 1995, yıldırımlı 2001), t. praecox subsp. praecox wulfen, t. cariense carlström, t. syriacum bornm., t. aghricum p.h. davis & kit tan and t. watsonii p.h. davis (davis et al. 1988) to the flora of turkey, and today the specified sections are represented by 22 taxa. thlaspi consists of annual, biennial or perennial herbaceous plants with simple leaves and hairy or papillose stems. sepals are bulging or non-saccate, the base of the sepals is often inclined and oblique with broad white membranaceous margins, while the oval petals are white, rose, lilac or yellowish. filaments are narrow, straight or slightly curved. nectar glands are present around the outer short stamens, but not on the long stamens in the middle. the ovary contains 2-16 ovules. the fruit is a dehiscent narrowly septate silicula or rarely a silique, strongly or weakly horizontally compressed, winged or not, with 1-8 seeds per loculus. the * corresponding author e-mail: mkaraismailoglu@bartin.edu.tr karai̇smai̇loğlu m. c., i̇nal b., erol o. 198 acta bot. croat. 81 (2), 2022 septa is often wavy. seeds may or may not contain mucilage. the embryonic rootlet is accumbent (resting on the edge of the cotyledons) (hedge 1965). the genus thlaspi is a highly variable complex in eurasia, which includes turkey, and several researchers have conducted different types of studies on many of its taxa. first, some important characters that improved the classification of this giant complex were found by meyer (1973, 1991), who revised thlaspi based on seed coat anatomy and embryology and transferred many of the taxa previously included in the genus to noccaea moench. meyer (1973, 1991) also divided the genus thlaspi s.l. into 12 genera (thlaspi l., neurotropis (dc.) f.k. meyer, microthlaspi f.k. meyer, thlaspiceras f.k. meyer, noccidium f.k. meyer, kotschyella f.k. meyer, callothlaspi f.k. meyer, raparia f.k. meyer, noccaea moench, atropatenia f.k. meyer. vania f.k. meyer, masmenia f.k. meyer). according to this classification, 6 species remain in thlaspi sensu stricto (s. str.) (t. arvense l., t. huetii boiss., t. watsonii p.h. davis, t. kurdicum hedge, t. alliaceum and t. ceratocarpon murray) (meyer 1973 and 1991). many scientists do not accept this classification (hedge 1965, al-shehbaz 1986, karaismailoğlu 2018, karaismailoğlu and erol 2018, 2019, 2020) but it has been accepted by others (greuter and burdet 1983). although most of meyer’s classification was accepted by greuter and burdet (1983), this classification and the resulting new taxa were not accepted in the flora of turkey. davis et al. (1988) rejected the genus fragmentation of meyer (1973, 1991) and evaluated thlaspi in the broad sense because they found the putative new taxa to be unanalyzable and, as a result, were unable to assess their proper placement. in addition, the latin descriptions of many taxa were found to be insufficient by davis et al. (1988). some more recent studies using molecular markers (rubisco, chloroplast dna, nuclear ribosomal dna) clearly show that the boundaries of some genera (callothlaspi, microthlaspi, noccaea, noccidium and vania) in meyer’s classification are unnatural (mummenhoff and koch 1994, zunk et al. 1996, mummenhoff et al. 1997, koch et al. 1998, koch and mummenhoff 2001). according to al-shehbaz (2014), the systematic structure of meyer (1973, 1991) is inherently incorrect and its inter-genus relations cannot be resolved. however, the study partially agreed with meyer’s classification at the genus level. according to al-shehbaz (2012), the genus noccaea, a well-known taxonomic complex, contains most of the species transferred from thlaspi s.l. although he accepts that neither the genus boundaries nor the boundaries of the taxa within it can be fully resolved, he estimates the species diversity of noccaea to be quite high with approximately 85 to 120 taxa (al-shehbaz 2012). family-wide molecular phylogenetic studies place the genus noccidium in the camelineae tribe and indicate that the 10 genera (atropatenia, callothlaspi, kotschyella, masmenia, microthlaspi, neurotropis, raparia, thlaspiceras, vania, noccaeopsis). meyer (1973, 1991) distinguished from thlaspi are synonyms of noccaea (khosravi et al. 2009). however, almost all of the taxa transferred are lacking adequate field studies, with work most often based on herbarium specimens missing important organs. these studies also include few plants from turkey, which is a center of diversity with many endemic thlaspi taxa. it is therefore necessary to re-evaluate morphological characters in light of the intra-genus classification of meyer (1973) and the transfers made in the following years (meyer 1973, 1991), using molecular phylogenetic studies based on extensive field work. thlaspi is a difficult genus to study from a botanical point of view, mainly because it requires individuals with both flowers and ripe fruit for diagnosis. many of the perennial species studied in the past, especially those with alpine distributions, were described from a few individuals, often with neither flowers nor mature fruit. this makes the taxonomic status of many species uncertain (hedge 1965, karaismailoğlu 2018). fruits contain many typical morphological characteristics distinguishing taxa from each other; for instance, shape, colour, size and microstructure (involving ultrastructure) often offer useful contributions to the taxonomy of angiosperms (barthlott 1981, karaismailoğlu 2017, 2019). fruit morphology and surface micromorphology have been described as some of the best identification characters at the species and infra-generic levels in brassicaceae (hedge 1965, davis et al. 1988, karaismailoğlu 2018, 2019). it is difficult to morphologically distinguish thlaspi from closely related genera without mature fruits. however, detailed studies of fruit morphology covering the genus as a whole have so far been lacking. dna sequencing has become one of the most important and widespread methods of investigating the phylogenetic status and taxonomic relationships among taxa in recent years. the its (internal transcribed spacer) region is one of the most commonly used genomic regions in plant systematics, the reasons for which include the presence of several types of polymerase chain reaction (pcr) primer sets that can be used in different taxonomic groups (white et al. 1990, gardes and bruns 1993, gültepe 2014, moorhousegann et al. 2018), as well as the size of the region, fewer than 700 base pairs, which makes it easy to replicate and sequence (gernandt et al. 2001). this region provides information useful in determining the phylogenetic relationships between taxa at the species and subspecies levels (baldwin and markos 1998, gültepe 2014). although a handful of studies have used different molecular markers to investigate several taxa, no comprehensive study of the its region has been conducted for thlaspi s.l. in this article, we present the most comprehensive sampling of nomisma, thlaspi and pterotropis sections of thlaspi s.l. from turkey to date. phylogenetic analyses based on nuclear ribosomal dna (nrdna) its sequence data using an extensive sample set are used to elucidate relationships between the taxa. we also support molecular data with fruit morphological character data in an attempt the better to explain the fruit variation of thlaspi s.l. species and to make a diagnostic key utilizing these taxonomically important characteristics. a systematic study of thlaspi from turkey acta bot. croat. 81 (2), 2022 199 materials and methods sampling in the current study, 22 species belonging to the nomisma, thlaspi and pterotropis sections of the thlaspi genus were collected as both flowering and ripe-fruited specimens from various phytogeographic regions in turkey in march-july between 2013 and 2016. specimens were numbered, pressed, arranged on herbarium sheets and deposited at istanbul university science faculty herbarium (istf) and siirt university flora and fauna center (sufaf) (on-line suppl. tab. 1.). macromorphological characteristics of the fruits such as shape, size, apical sinus, wing and septum structures were analyzed for 100 fruits, from 10 individuals of each species, using an olympus szx7 stereomicroscope and kameram imaging software. for micromorphological examination of fruit surface ornamentation, we used a jeol neoscope-5000 scanning electron microscope to examine samples fixed to stubs with silver epoxy and coated with platinum-gold mix (karaismailoğlu and erol 2018, karaismailoğlu and güner 2019). dna extractions, its region amplification and sequencing process leaf samples were acquired from the field and istf and sufaf herbaria, for a total of 33 accessions belonging to 22 species representing all three defined sections of thlaspi. aethionema speciosum boiss. & a.huet subsp. compactum hartvig & å.strid [=ae. compactum (hartvig & å.strid) yild.], which is closely related, was selected as the outgroup. total genomic dna was isolated according to the cetyltrimethylammonium bromide (ctab) method developed by karaca et al. (2005). ctab buffer is a mixture of extraction buffer (eb): (0.35 m sorbitol, 100 mm tris-hci (ph 7.5), 5 mm edta (ph 7.5), 2% tween, 1% triton-x, 1% bme) and lysis buffer (lb): (200 mm tris-hci (ph 8.0), 50 mm edta (ph 8.0), 2m naci, 2% ppvp, 2% ctab, 2% triton-x, 2% bme). dna quantitation was performed using a thermo nanodrop® spectrophotometer. the its2 region sequences obtained from the genomic dna was used as a template to amplify the its region with a miniamp plus thermal cycler device using the primer pairs uniplantf (5'-tgtgaattgcarr atycmg-3') and uniplantr (5'-cccghytgayytgrggtcdc-3') ( moorhouse-gann et al. 2018). pcr was prepared in 25 µl volumes using the following reaction elements: 3 µl template dna, 11.25 µl water, 2.5 µl 10x buffer, 1 µl each of primers (50 ng μl–1), 4 µl mgcl2 (2.5 mm), 1 µl dntp mix (0.25 mm), 0.25 µl taq dna polymerase and 1 µl bovine serum albumin (bsa). pcr thermal cycle conditions were as follows: pre-denaturation = 95 °c (1 min.), dna denaturation = 94 °c fig. 1. fruits of the thlaspi taxa (two images per taxon; 1 – fruit general appearance, 2 – number of seeds per locus): 1 – t. arvense, 2 – t. huetii, 3 – t. orbiculatum, 4 – t. kotschyanum, 5 – t. perfoliatum, 6 – t. microstylum, 7 – t. annuum, 8 – t. bulbosum, 9 – t. leblebicii, 10 – t. ochroleucum, 11 – t. praecox subsp. praecox, 12 – t. cariense, 13 – t. violascens, 14 – t. densiflorum, 15 – t. tatianae, 16 – t. cataonicum, 17 – t. syriacum, 18 – t. elegans, 19 – t. rosulare, 20 – t. lilacinum, 21 – t. aghricum, 22 – t. watsonii. scale bars: 2 mm. karai̇smai̇loğlu m. c., i̇nal b., erol o. 200 acta bot. croat. 81 (2), 2022 (1 min.), annealing = 55 °c, extension =72 °c (0.45 h), number of cycles =35 and final extension =72 °c (5 min.). purification and sequencing were outsourced to genoks (istanbul, turkey). bioinformatic analysis of sequences we used its region base sequences of 33 accessions in our analyses. sequences with poor quality reads throughout were sequenced again. afterward, the first and last 30 bases were removed due to poor quality using the bioedit program (hall 2011) and these sequences were not included in the main analysis. the obtained sequences were analyzed with the ncbi-blast algorithm to confirm they belong to the studied material. we then used mega x version 10.0.05 (kumar et al. 2018) to perform phylogenetic analyses. the sequences were first loaded and then aligned with the outgroup, ae. speciosum subsp. compactum, which included using the base sequence, and clustal w (larkin et al. 2007). after alignment, a phylogenetic tree was constructed to interpret the genetic similarities among taxa. all raw sequences were arranged in fasta format for bioinformatics analysis. after editing of the sequences, statistical analyses were performed in mega x, using the predictive model algorithm to determine the most appropriate method for our study. bootstrap values for 1000 replicates were obtained according to the maximum likelihood (ml) phylogenetic method. results structure of fruit in thlaspi all examined taxa have siliculae as fruit. eight fruit shapes were observed: oval-circular, elliptical, oval, obcortab. 1. fruit macroand micromorphological features of the thlaspi taxa (l: length, w: width). the measurements of these features were made on 100 fruits for each taxon. taxa fruit shape fruit sizes wing apical sinus stylus length (mm) septum sizes number of seeds in each locus surface ornamentationl (mm) w (mm) w (mm) tip veining structure l (mm) l (mm) w (mm) t. arvense oval circular 10–18 10–16 2–5 rotundate reticulate narrow 2–4 0.1–0.2 7–11 1–2 4–8 reticulate t. huetii circular 6–10 5–10 1–2 rotundate reticulate narrow 1.5–2 0.1–0.2 4–7 1.5–2 2–4 rugose t. orbiculatum obcordate 10–12 13–15 2–5 rotundate parallel narrow 1–3 0.4–0.5 8–11 1–1.5 4–6 areolate t. kotschyanum obcordate 7–14 8–15 2–4 rotundate reticulate narrow 2–3 0.1 6–10 2–3 6 reticulate t. perfoliatum obcordate 4–6 4–7 0.5–1.2 rotundate reticulate broad 1–3 0.2–0.4 4–6 1–3 3–5 favulariate t. microstylum obcordate 6–10 3–7 1–2.5 obtuse reticulate narrow 1–1.5 1.1–1.5 6–8 0.5–1.5 2–4 slightly reticulate t. annuum obcordate 5–8 3–6 1–2 rotundate reticulate broad 0.8–1.1 0.8–1 4.5–7 1–2 3–5 rugose t. bulbosum obcordate 8–11 6–10 1–3 rotundate reticulate broad 2–3 2–3 8–11 2–3 2 rugose t. leblebicii obcordate 6–12 4–7 1–2 rotundate reticulate narrow 1–2 0.2–0.5 6–8 1.5–2.5 2 ruminate t. ochroleucum cuneateobcordate 5–8 4–6 0.5–1 acute reticulate broad 0.5–1 2–3 5–6 1–2 4 straight t. praecox subsp. praecox obcordate 5–8 3–4 1–2 rotundate reticulate broad 0.5–1 0.5–1 5–7 1–2 2–4 straight t. cariense obcordate 9–12 4–6 1–2 rotundate reticulate broad 0.4–0.8 1–3 6–8 3 2–4 rugose t. violascens obcordatetriangular 8–10 4–6 1–2 rotundate reticulate narrow 1.5–2 1–2 6–7 1.5–2 4–5 ruminate t. densiflorum obcordatetriangular 8–9 3.5–4 1–2 acute reticulate narrow 1.5–2.2 1.5–2 7–8 1.5–2 4 ruminate t. tatianae cuneate 6–10 2.5–4 0.3–1 obtuse reticulate broad 1–1.8 0.3–0.5 6–7 0.8–1.2 5–6 lineolate t. cataonicum oval obcordate 10–12 3–4 0.5–1 acute reticulate broad 2–2.5 2–2.3 7–8 1.5–2.1 5–6 rugose t. syriacum obcordatetriangular 6–8 3–4 0.5–1 obtuse reticulate broad 0.5–0.7 1.5–2 5–6 1–1.5 2–4 rugose t. elegans rectangular 5–7 3–4.5 0.5–1.5 acute reticulate broad 0.8–1.1 1–1.5 5–6 1–2 2–4 ruminate t. rosulare oval 7–9 5–6 0.7–1.2 obtuse reticulate narrow 1–1.2 0.2–0.5 5–6 2.5–3 2 ruminate t. lilacinum elliptical 5–9 3–4.5 – – – – – 2–2.5 5–8 3–4 4–6 rugose t. aghricum obcordate 8–12 5–9 1–2.2 obtuse reticulate broad 0.5–0.7 1.5–2 5–6 1–1.5 2–4 ocellate t. watsonii oval 5–7 3–4 1–2 rotundate reticulate absent or narrow 0.1–0.2 3.5–4 5–6 2–2.5 2–6 ruminate a systematic study of thlaspi from turkey acta bot. croat. 81 (2), 2022 201 date, cuneate-obcordate, cuneate, obcordate-triangular and rectangular (fig. 1). the most common type is obcordate (in 10 taxa), while oval-circular (t. arvense), circular (t. huetii), cuneate-obcordate (t. ochroleucum), cuneate (t. tatianae), oval-obcordate (t. cataonicum) and rectangular (t. elegans) types are species specific (fig. 1). fruit sizes range from 4 mm (t. perfoliatum) to 18 mm (t. arvense) in length, from 2.5 mm (t. tatianae) to 16 mm (t. arvense) in width. t. perfoliatum has the smallest fruits and t. arvense has the largest. fruit wing characteristics vary in width, wingtip structure and venation among taxa, except for t. lilacinum, which is not winged. the width of the wings varies between 0.3 mm (t. tatianae) and 5 mm (t. arvense and t. orbiculatum). wing tips may be rotundate, obtuse or acute. the most common form is rotundate (12 taxa), while acute is the least (4 taxa). the wing tips are the same length in all taxa studied except for t. elegans, which has different sized wing tips. the wings have reticulate surface venation, except for t. orbiculatum, which has parallel venation (fig. 1, tab. 1). the apical sinus also differs in terms of structure and size in the taxa studied. it is absent in t. lilacinum. it may be broad or quite narrow, as in t. watsonii. apical sinus length varies between 0.1 mm (t. watsonii) and 4 mm (t. arvense). the length of the fruit stylus and its relationship to apical sinus length are quite diverse among these taxa. stylus length is between 0.1 mm (t. arvense, t. huetii and t. kotschyanum) and 4 mm (t. watsonii). septum sizes vary from 4 mm (t. huetii and t. perfoliatum) to 11 mm (t. arvense, t. orbiculatum and t. bulbosum) in length, and from 0.5 mm (t. microstylum) to 4 mm (t. lilacinum) in width. the number of seeds per locus ranges from 2 (11 taxa) to 8 (t. arvense) (fig. 1, tab. 1). fruit surface ornamentation is categorized into 8 types: reticulate, rugose, areolate, favulariate, ruminate, straight, lineolate and ocellate. the most common types are rugose and ruminate (13 taxa). areolate (t. orbiculatum), favulariate (t. perfoliatum), lineolate (t. tatianae) and ocellate (t. aghricum) ornamentation types are represented by one taxon each (on-line suppl. fig. 1, tab. 1). fig. 2. phylogenetic tree for representatives of the examined thlaspi taxa based on its region data. numbers at nodes show the bootstrap values. numbers in parentheses indicate accession numbers. see on-line suppl. tab. 1 for locality data. karai̇smai̇loğlu m. c., i̇nal b., erol o. 202 acta bot. croat. 81 (2), 2022 phylogenetic relation of thlaspi based on its region sequences the its gene sequences of the 22 thlaspi taxa studied were used in analyses, plus that of ae. speciosum subsp. compactum as the outgroup. the bootstrap values of the phylogenetic tree obtained using the maximum likelihood (ml) phylogenetic method are shown in fig. 2. the dendrogram obtained from its data shows two different clusters, or clades i and ii. clade ii has six sub-clusters: a, b, c1, c2, c3 and c4 (fig. 2). only t. huetii in the nomisma section was placed in clade i. the other taxon of that section, t. arvense, was found in cluster a of clade ii, meaning that the nomisma taxa preserve their existing systematic proximity. t. orbiculatum and t. kotschyanum, closely related in the thlaspi section in the flora of turkey, remain closely linked to each other in cluster b of clade ii. cluster c of clade ii included 18 studied species and was further divided into four clusters: c1, c2, c3 and c4. due to the aggregation of taxa in this cluster, it can be thought that other taxa originate from this cluster. thlaspi annuum and t. perfoliatum from thlaspi section are closely associated with each other in c1 cluster. taxa from the thlaspi and pterotropis sections (t. leblebicii, t. rosulare, t. lilacinum and t. watsonii) are in c2. all of these are narrow endemics in turkey. t. ochroleucum, t. cariense, t. densiflorum, t. violascens, t. tatianae, t. cataonicum and t. syriacum taxa are included in the c3 cluster. common features among taxa in this cluster include generally obcordate-shaped siliculae and horizontally suppressed fruits. cluster c4 consists of t. bulbosum and t. microstylum, belonging to thlaspi section, plus t. praecox subsp. praecox, t. elegans and t. aghricum of the pterotropis section. although t. bulbosum and t. praecox subsp. praecox taxa are widely found in the european flora, they have a very limited distribution in turkey. the dissimilarity matrix based on its region data is provided in on-line suppl. tab. 2. all examined taxa have differences ranging from 0.01 to 0.49%, except for the outgroup. thlaspi huetii and t. aghricum are the most distinct taxa with a distance of 0.49% between them. thlaspi violascens and t. ochroleucum-t. violascens had a 99% similarity to each other. the lengths of the nrdna-its region ranged from 260 to 378 bp, and % gc contents varied between 45.91 and 54.73 (on-line suppl. tab. 3). alignment of the its regions of all examined taxa resulted in a data set consisting of 324 base pairs (bp). we found that 95 of this aligned data consisted of parsimony (informative) nucleotides. discussion fruit morphological characters provide valuable information regarding the evolutionary relationships of flowering plants (corner 1976). in this study, fruit macroand micromorphology generally varies across thlaspi species. meyer (1973, 1991, 2001, 2006) discusses fruit similarities within the genus and argues that the relationships among taxa are masked in fruit-based classification. however, we found ripe fruits do not show this similarity. instead, mature fruit morphology ranged from broad to narrow, winged to wingless and rounded to acute to obtuse in the wing tips. in addition, the width and length of the apical sinus on the fruit, and the comparative length of the stylus are among characters that distinguish species. a thorough diagnostic key based on fruit characters is found at the end of the discussion section. the examined thlaspi taxa have overall very high similarity rates for the its region, which indicates that the genus has not fully completed the differentiation process. previous studies conducted with various genera have shown that the its region contains a considerable number of parsimony informative nucleotides and therefore clearly reveals the relationship between taxa (baldwin and markos 1998, alvarez and wendel 2003, hughes et al. 2006). we targeted this region specifically because of its parsimony informative nucleotides and used the data to generate a phylogenetic tree (fig. 2). many studies have shown that the its gene region offers remarkable solutions in explaining the relationships between taxa in some species-rich genera (soltis et al. 1993, soltis et al. 2008). the its region shows significant divergence between species but is often highly conserved within taxa, making it one of the most chosen genetic markers for species-level delimitation (cheng et al. 2016). moreover, the prospects of amplification from processed or aged plant materials are good due to the its region’s large copy number (balasubramani et al. 2010). its has been effectively used to differentiate taxa in diverse plant groups. according to our its data, the examined taxa are monophyletic and highly similar, which does not support previous taxa transfers to different genera, and all examined taxa should be evaluated under the same genus. a similar result was reported in a dendrogram created by evaluating 215 macromorphological, micromorphological and anatomical characters (karaismailoğlu 2018), as well as a cladogram based on the detailed examination of palynological characters of these taxa (karaismailoğlu and erol 2019). taxa belonging to the nomisma section are easily distinguished from others by their broad wings and oval, elliptical or circular fruits. this data supports distinguishing this section from others according to fruit shape, as done by schulz (1936) and hedge (1965) in flora of turkey, who established the section using fruit shape and supported it with other classifications. thlaspi orbiculatum and t. kotschyanum taxa are morphologically very similar in their flower structure and broadly obcordate-shaped fruit; however, a contrast between the parallel-veined fruit wings of t. orbiculatum and the reticulate venation of t. kotschyanum distinguishes the two. plant height, leaf size and seed characteristics, used in karaismailoğlu (2018), also clearly differentiate these taxa. clusters in this group are consistent with the key characters and descriptions in flora of turkey described by hedge (1965) and davis et al. (1988) (fig. 2). thlaspi annuum and t. perfoliatum are morphologically similar in that their petals are in two segments, inner and outer. in the subset a systematic study of thlaspi from turkey acta bot. croat. 81 (2), 2022 203 formed by t. perfoliatum taxa, the length of the petals in the outer segment is equal to those of the inner segment, while in t. annuum the petals in the outer segment are longer than in the inner segment (karaismailoğlu 2018). surprisingly, t. leblebicii and t. watsonii branch from the same place in the dendrogram because these taxa are quite different in appearance and distributed in different phytogeographic regions. the dendrogram positions of t. rosulare, t. lilacinum and t. watsonii are compatible with their positions in the flora of turkey. thlaspi ochroleucum, t. densiflorum and t. violascens, which are morphologically similar, are closely related but separated from each other by their bs values. thlaspi bulbosum and t. praecox subsp. praecox are similar to each other in that they have underground stem metamorphoses, namely rhizomes and tubers, obcordate-shaped silicula, narrow or wide fruit wings, rounded wing tips and fruit styluses that generally exceed the apical sinus, all of which are also used as key characters in the flora of turkey (hedge 1965, davis et al. 1988). on the other hand, t. bulbosum differs from t. praecox subsp. praecox in gc% (51.38 in t. bulbosum, 50.99 in t. praecox subsp. praecox) and its region length (323 bp in t. bulbosum, 300 bp in t. praecox subsp. praecox). the rankings and relationships of taxa in the dendrogram obtained by molecular phylogenetic data are also supported by taxa descriptions in the flora of turkey (fig. 2). on the other hand, we see no parallel between the sections created using morphological data and molecular data. based on its comparisons, the close proximity between the nomisma section taxa (t. arvense and t. huetii) is preserved. on the other hand, we see a gradual transition in taxa belonging to the thlaspi and pterotropis sections, similar to what has been observed in previous detailed studies on the genus (karaismailoğlu 2018, karaismailoğlu and erol 2018, 2019, 2020, karaismailoğlu and fidan 2021). this shows that distinguishing sections based on fruit morphology is artificial and not taxonomically beneficial. according to its data, t. elegans is a non-monophyletic polyploid complex. the bs values among the studied t. elegans taxa differ considerably (98%, 65%, 49%,). also, its data from more than one population of t. arvense, t. huetii, t. perfoliatum, t. violascens, t. cataonicum, t. elegans, t. rosulare and t. aghricum indicate that they are same-arm taxa, and thus monophyletic. thlaspi is a systematically problematic genus because of the presence of many morphologically similar species. thlaspi arvense-t. huetii, t. orbiculatum-t. kotschyanum, t. violascens-t. densiflorum and t. lilacinum-t. watsonii are taxon pairs that are very similar to each other in terms of macromorphology. however, they are easily separated using the bs values of our its-based phylogenetic tree, telling us that despite the high morphological similarity among species, they can be differentiated by molecular methods. this study, which includes a phylogenetic comparison of the its region, places the studied taxa into a natural systematic group due to high base sequence similarity. as in previous detailed morphological, anatomical, palynological a nd c y tolo g ic a l s t ud ie s (k a r a i sm a i lo ğ lu 2 018 , karaismailoğlu and erol 2018, 2019, 2020, karaismailoğlu and fidan 2021) on the same taxa, we found that these taxa should be treated as part of thlaspi s.l. a diagnostic key for taxa studied based on fruit morphological characteristics 1. fruit shapes are oval, circular, oval-circular, elliptical, cuneate or rectangular ...................................................... 2 2. oval, oval-circular, circular or elliptical ........................ 3 3. oval, oval-circular ............................................................. 4 4. oval-circular ....................................................... t. arvense 4. oval ...................................................................................... 5 5. stylus exceeds apical sinus .............................. t. watsonii 5. stylus does not exceed apical sinus ................. t. rosulare 3. elliptical ............................................................................... 6 6. apical sinus absent ......................................... t. lilacinum 6. apical sinus present .............................................. t. huetii 2. cuneate or rectangular ...................................................... 7 7. cuneate ............................................................... t. tatianae 7. rectangular .......................................................... t. elegans 1. fruit shapes are obcordate, cuneate-obcordate, oval-obcordate or obcordate triangular ....................................... 8 8. cuneate-obcordate, oval-obcordate or obcordate ........ 9 9. cuneate-obcordate or oval-obcordate ......................... 10 10. cuneate-obcordate ................................... t. ochroleucum 10. oval-obcordate ........................................... t. cataonicum 9. obcordate ........................................................................... 11 11. fruit wings with parallel veins ................ t. orbiculatum 11. fruit wings with reticulate veins ................................... 12 12. stylus exceeds apical sinus ............................................. 13 13. wing tips are obtuse; septum width is 1.5-2 mm ............................................................................. t. aghricum 13. wing tips are rotundate; septum width is 3 mm ................................................................................. t. cariense 12. stylus does not exceed apical sinus or is the same length ................................................................................................. 14 14. stylus length >1 mm ........................................................ 15 15. septum width is 0.5-1.5 mm; ornamentation type is reticulate ......................................................... t. microstylum 15. septum width is 2-3 mm; ornamentation type is rugose ............................................................................ t. bulbosum 14. stylus length ≤1 mm ........................................................ 16 16. the number of seeds at each locus is 2-6 ...................... 17 17. 6 seeds at each locus .................................. t. kotschyanum 17. 2-5 seeds at each locus ..................................................... 18 18. ornamentation type is favulariate or straight ........... 19 19. favulariate ................................................... t. perfoliatum 19. straight ........................................ t. praecox subsp. praecox karai̇smai̇loğlu m. c., i̇nal b., erol o. 204 acta bot. croat. 81 (2), 2022 18. ornamentation type is rugose or ruminate ................ 20 20. rugose ................................................................. t. annuum 20. ruminate ........................................................... t. leblebicii 8. obcordate-triangular ....................................................... 21 21. wing tips are rotundate or obtuse .................................. 22 22. ornamentation type is rugose ....................... t. syriacum 22. ornamentation type is ruminate ................ t. violascens 21. wing tips are acute ....................................... t. densiflorum acknowledgments this study was financially supported by i̇stanbul university-scientific research projects coordination unit (project number: 33567) and i̇stanbul university-oyp unit (project number 113.2014-dr-35/26.04). we would like to thank the curators of herbaria iste, isto, kato, oufe, 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(subtribe thlaspidinae, lepidieae) and allied genera based on chloroplast dna restriction-site variation. theoretical and applied genetics 92, 375–381. opce-str.vp acta bot. croat. 73 (1), 1–19, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 morphological variation of lactuca serriola l. achenes as a function of their geographic origin eva k�ístková1, ale[ lebeda1*, al@b�ta novotná1, ivana dole@alová1**, tomá[ berka2 1 palacký university in olomouc, faculty of science, department of botany, [lechtitel� 11, 783 71 olomouc-holice, czech republic 2 podlesí 5411, 760 01 zlín, czech republic abstract – the morphological characteristics of achenes of lactuca serriola represented by 34 local populations from slovenia and 12 local populations from sweden were studied in relation to their eco-geographical conditions. in total, eight quantitative morphological characters were evaluated: length and width of achene body; index length/width of achene body; number of ribs on achene body; length of beak; length of pappus bristles; pappus area and discus diameter. nested anova analysis indicated significant differences in length and width of achene body, length of pappus bristles, and pappus area between slovenian and swedish populations. achenes from slovenia were longer, wider and possessed longer pappus bristles than achenes from sweden. among geographical factors, latitude had the greatest impact on the morphological characters evaluated. significant differences in seven parameters were also found between populations within countries and between samples within populations. it is probable that this variation has a genetic basis with sufficient variation within populations to permit continued selection. keywords: achen, lactuca serriola, eco-geographical conditions, fruit, morphology, pappus, wind-dispersed diaspore introduction wind dispersal is a natural mode of spreading achenes common in many plant communities, comprising 10–30% on average, and up to 70%, of the flora in temperate plant communities, with obvious morphological adaptations of achenes (willson et al. 1990). various morphological attributes such as hairs, wings, achene shape and size facilitate their aerodynamic transport, essentially by lowering wingand/or plume-loading (the ratio of mass to surface area). adaptive structural designs on the surface of achene body increase the acta bot. croat. 73 (1), 2014 1 * corresponding author, e-mail: ales.lebeda@upol.cz ** present address: crop research institute praha-ruzyn�, department of genetic resources for vegetables, medicinal and special plants, [lechtitelu 11, 783 71 olomouc-holice, czech republic copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:23 color profile: generic cmyk printer profile composite default screen roughness and thereby increase the drag that the air exerts on the achene in flight and prolong its travel time through the air (nathan et al. 2001). many studies have focused on the morphology of wind-dispersed diaspores of herbaceous members of the asteraceae or trees of different families in relation to their fall speed (terminal velocity, setting velocity) (green 1980, augspurger 1986, matlack 1987, andersen 1993a, gravuer et al. 2003, chmielewski and strain 2007). the morphology of achenes has been studied in numerous plant species. the proportion between size, shape, and weight of achene body, beak length, length of pappus bristles, and the number of ribs play an important role in plant dispersal under different eco-geographical and climatic conditions (andersen 1992, 1993a, gravuer et al. 2003, riba et al. 2005, lacroix et al. 2007, katinas and crisci 2008, monty and mahy 2010). elaborate mathematical models of diaspore dispersal take into account not only the morphological and physical parameters of the diaspores, but also physical environmental parameters (schultz et al. 1991, bullock and clarke 2000), and examine different emission scenarios of dispersal and spread of wind-spread (andersen 1993b), climate-limited species, including l. serriola (bullock et al. 2012). tackenberg et al. (2003) applied simulation modeling of wind dispersed plant diaspores to 335 plant species, and revealed considerable variation in the wind dispersal potential of species classified as the same morphological type of diaspores. thus, the dispersal and survival ability with regard to morphological parameters and mass of diaspores must be assessed for individual species. the dispersal of achenes is influenced by the height and structure of surrounding plants, the location of plants within the population (andersen 1993b, lavorel et al. 1994, welham and setter 1998, blumenthal and jordan 2001, oester et al. 2009), and by the position of plant population in the centre of species distribution or on the margin, and degree of isolation of islands vary significantly among species with the same morphological type of diaspore (fresnillo and ehlers 2008). fresnillo and ehlers (2008) stated that for plants producing wind dispersed seeds, the height of mother plant, the spacing of branches and the position of fruits on the plant may directly affect the dispersal potential of released seeds or fruits to be carried away by wind. differences in dispersal rate may thus occur even if there are no apparent differences in the morphology of the diaspores themselves (donohue 1999). peroni (1994) described evolutionary change in dispersal ability between old and young populations, where young often show higher dispersal rates than older populations. the chance that achenes will be dispersed over long distances increases with decreased setting velocity (fresnillo and ehlers 2008). the ability to be dispersed long distances is attributed to diaspores’ lower achene mass and advantageous proportion of achene mass to pappus mass and/or area (chmielewski and strain 2007). our scientific interest focuses on different aspects of infraspecific variation on lactuca serriola l. (prickly lettuce, compass lettuce, fam. asteraceae) (lebeda and astley 1999, lebeda et al. 1999, 2009). however, detailed data about variability in achene morphology related to eco-geographical conditions are limited (novotná et al. 2011). prickly lettuce is considered one of the direct progenitors of cultivated lettuce lactuca sativa l. (de vries 1997). it belongs to the primary gene-pool of lettuce and plays an important role in modern lettuce breeding programs as a source of race-specific resistance genes against lettuce downy mildew (bremia lactucae regel) (lebeda et al. 2007b). recently, l. serriola has spread throughout europe as an invasive weed (jehlík 1998, lebeda et al. 2001, 2004, brant and holec 2004, hooftman et al. 2006, d’andrea et al. 2009). 2 acta bot. croat. 73 (1), 2014 k�ístková e., lebeda a., novotná a., dole@alová i., berka t. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:23 color profile: generic cmyk printer profile composite default screen originally a meridional-temperate, western euro-asian species, prickly lettuce is now distributed worldwide (lebeda et al. 2004). the northern limit of its distribution in europe is near 65° n in finland and 55° n in great britain. the northernmost localities in norway and sweden are at 60° n (feráková 1977). the greatest genetic diversity of this species in europe is around the mediterranean basin, including the near east (asia) and coastal northern africa (lebeda et al. 2004). lactuca serriola is an annual or winter-annual therophyte; it is a ruderal species, growing preferably on disturbed soil. from an ecological viewpoint, this species is an »r« strategist with a short life cycle (feráková 1977, d’andrea et al. 2009). it expands along roads, highways, railways and embankments (lebeda et al. 2001). the spread of this species is very closely related to human activities, mainly by transportation, building constructions, while anthropogenic climate changes are likely to accelerate this process (d’andrea et al. 2009). according to many previous studies (dole@alová et al. 2001, lebeda et al. 2001, hooftman et al. 2006, lebeda et al. 2007b), during the last 20 years prickly lettuce has shown an enormous range expansion with populations increasing in europe and in some parts of scandinavia (d’andrea et al. 2009). in the netherlands, the occurrence of l. serriola has significantly increased since 1940, indicating that the species currently occurs in a broader range of vegetation types. except for its original ruderal habitats, l. serriola currently occurs in more closed vegetation types, which are less continental and more humid (hooftman et al. 2006). within this highly polymorphic species there are two forms distinguished by the shape of their cauline leaves; f. serriola with divided leaves, and f. integrifolia with entire leaves (feráková 1977).the two forms are not equally distributed around the world. in europe, l. serriola f. serriola is distributed in the majority of european countries, but the distribution of f. integrifolia is restricted to the southern and western parts of europe (lebeda et al. 2007a, b), and is most prevalent in the united kingdom (prince and carter 1977, carter and prince 1982). the beginning of the flowering and the period between bolting and flowering are strongly influenced by local eco-geographical conditions (lebeda et al. 2007b). prickly lettuce can only reproduce sexually, producing plumed achenes (figs. 1a, 1b). the spread of l. serriola is facilitated by the pappus on the top of achene body acting as a parachute (cousens et al. 2008). the diaspores of the genus lactuca are monomorphic, in contrast to many other members of the asteraceae where dimorphism or polymorphism are typical (harper et al. 1970, baker and dowd 1982, imbert et al. 1996). achenes of l. serriola are relatively small. the length of the achene body is 3 mm and together with the beak is 6–8 mm (dostál 1989, grulich 2004). achenes are ±1 mm broad, oblong-ovate in shape, moderately flattened, and brown to grayish in color. the achene surface has 5 to 10 ribs with short fine bristles located in the apical part of the ribs and pointing towards the apex of achene. the beak is white, filiform, and as long as, or longer than the achene body. the monomorphic pappus consists of 2 rows of whitish hairs, which exceed the involucral bracts (feráková 1977). the pappus is 3–4.5 mm long, white, and deciduous (feráková 1977, dostál 1989, grulich 2004). pappus bristles of l. serriola are smooth, consisting of two or three vertical rows of cells (tuisl 1968). the first compilation of data on morphological parameters of l. serriola achenes was presented by novotná et al. (2011) where achene parameters from 50 populations of prickly lettuce from the czech republic, germany, the netherlands and the united kingdom acta bot. croat. 73 (1), 2014 3 morphology of lactuca serriola achenes from slovenia and sweden 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:23 color profile: generic cmyk printer profile composite default screen were analyzed. novotná et al. (2011) showed that the two leafy forms of l. serriola, f. serriola, and f. integrifolia, differ in achene morphology. achenes of f. serriola are shorter, thinner, with a shorter beak, lower length/width index, and have a higher number of ribs than those of the f. integrifolia. novotná et al. (2011) concluded that achene morphology was significantly correlated with longitude, latitude, and soil texture of original habitats of samples from the four countries mentioned above. achene length and width increased along an east-west transect. mean beak length had a similar trend with the exception of achenes from german populations. the number of ribs increased from east to west in continental europe, whereas the lowest number of ribs was recorded in achenes collected in the czech republic and the united kingdom. novotná et al. (2011) also showed that with increasing latitude, i.e. from south to north, l. serriola achenes were longer, narrower, with fewer ribs, and longer beaks. to support the general conclusions based on the principles of morphological variation of achenes mentioned above, results need to be complemented by data on achene morphology from plants originating from additional regions with contrasting eco-geographical conditions. to do this, we analysed l. serriola achenes from slovenia and sweden. these countries differ not only in their geographical position, but also in environmental conditions. the mediterranean region including slovenia is characterized by the highest diversity of european lactuca spp. and is considered the probable area of lettuce domestication (de vries 1997, lebeda et al. 2004, 2007b). slovenian populations of prickly lettuce are older than scandinavian populations. the northernmost european localities of l. serriola in norway and sweden are located at 60° n (feráková 1977) and intense spread of prickly lettuce in some parts of scandinavia was observed during past 20 years only (d’andrea et al. 2009). 4 acta bot. croat. 73 (1), 2014 k�ístková e., lebeda a., novotná a., dole@alová i., berka t. fig. 1. lactuca serriola: a) position of freshly mature achenes with pappus (in the morning), b) final position of mature achenes (at noon), c) schematic drawing of achene with beak and pappus, and measured morphological parameters: lbr – length of bristles, lb – length of beak, lab – length of achene body, wab – width of achene body. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:27 color profile: generic cmyk printer profile composite default screen the main objective of the current study was to provide answers to the following questions: 1) are there differences in the morphological parameters of l. serriola achenes between populations in the northern european limits of the species’ distribution (sweden) and populations in southern europe (slovenia)? 2) which morphological parameters of achenes are significantly influenced by the original eco-geographical conditions? material and methods plant material initial experimental material was collected during explorations to slovenia and sweden in 2000 (dole@alová et al. 2001). the set of lactuca serriola samples used in the study consisted of 72 achene samples from 34 local populations in slovenia (svn) (fig. 2a) and 47 achene samples from 12 local populations in sweden (swe) (fig. 2b). description of the l. serriola local populations in their original habitats and collection sites in slovenia and sweden was given by dole@alová et al. (2001). one to six samples per collection site were collected, depending on the number of plants in the local population. samples from slovenia were collected from latitude 45°46’31’’ n to 46°40’50’’ n, and from longitude 14°12’51’’ e to 16°11’35’’ e (fig. 2a). collection locations were situated at an altitude of 187 to 594 m a.s.l. the mean annual temperature in ljubljana, the capital of slovenia, for the period of 1991–2000 was 10.9 °c (statistical office of the republic of slovenia 2011). collection sites in sweden were located at latitude of 55°36’11’’ n to 59°58’16’’ n, and between longitude of 12°49’48’’ e, and 18°03’52’’ e (fig. 2b). achenes were collected acta bot. croat. 73 (1), 2014 5 morphology of lactuca serriola achenes from slovenia and sweden fig. 2a. geographic locations of lactuca serriola local populations sampled in slovenia. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:29 color profile: generic cmyk printer profile composite default screen at an elevation of 3 to 158 m a.s.l. the mean annual temperature in stockholm is 6.6 °c (anonymous, 2011). detail passport data related to the achene samples are available from the authors. the achene samples were regenerated in the greenhouse in 2006 at the department of botany (faculty of science, palacký university in olomouc, czech republic) located at the 49°34’29,61’’ n, 17°16’54,29’’ e, and 208 m a.s.l. each sample was represented by 16 plants. plants were cultivated in plastic pots with garden soil under a standard protocol for regeneration of plant genetic resources of lactuca spp. (boukema et al. 1990, lebeda et al. 2007b). during vegetative growth, the taxonomic status of samples was verified and the infraspecific classification was determined. five plants per sample were used for morphological assessment of achenes. around 60 mature achenes were collected from 3 to 4 heads from single plants at the beginning of fruit maturity. achenes from each single plant were kept separate and measured. the samples are maintained at the department of botany (faculty of science, palacký university in olomouc, czech republic). 6 acta bot. croat. 73 (1), 2014 k�ístková e., lebeda a., novotná a., dole@alová i., berka t. fig. 2b. geographic locations of lactuca serriola local populations sampled in sweden. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:31 color profile: generic cmyk printer profile composite default screen morphological evaluation and measurements of achenes fifty achenes per plant were randomly chosen to determine their shape. eight morphological characters were studied: length and width of the achene body (lab and wab respectively); length/width index of achene body (lab/wab index); length of beak (lb); number of ribs (nr); length of pappus bristles (lbr); pappus area (paparea); and diameter of discus (discus). achene morphological features evaluated are schematically illustrated in fig. 1c. achenes were measured using the imagej computer program (imagej 1.32j, wayne rasband. national institutes of health, usa) after being scanned as jpgs at a resolution of 400 dpi. calibration was determined by the program on a scanned ruler. measurements of distance were converted into mm with an accuracy of 0.01 mm. length and width of achene were measured at the longest and widest point of the achene body. the shape of the achene body was determined from the lab/wab index. the length of the beak (lb) was the distance from the apical point of the achene body to the discus. length of the pappus bristles (lbr) was measured from the point of attachment of the bristle on the discus to the apex of the bristle. the area of the pappus (paparea) was calculated as the surface of the circle, where the radius is given by the length of the bristle. for this purpose five bristles per achene were measured. ten achenes of each plant were randomly chosen to determine the number of ribs (nr). a magnifying glass was used to count the number of ribs on each side of the achene and the numbers totaled. statistical analysis statistical analysis of morphological characters of lactuca serriola achenes were performed using ncss software, version 2007 (hintze 2001). morphological differences between achenes were evaluated using the nested anova general linear models (glm) in ncss. three geographical parameters, latitude, longitude, and altitude were analyzed. associations between morphological characters of the achenes and geographical parameters of the plant populations/collection sites were determined using canoco for windows 4.5 and canodraw for windows 4.5 (ter braak and [milauer 2002). according to the gradient length from detrended correspondence analysis (dca, the highest value 0.914) direct gradient analysis (rda, redundancy analysis) was used. rda relates evaluated morphological characters of l. serriola achenes to habitat factors. the significance of geographical factors was determined using the method 'forward selection'. monte-carlo permutation test with 999 permutations was used to test the significance of the environmental factors used in rda. results character of lactuca serriola populations according to the shape of cauline leaves, all plants from slovenia were determined to be l. serriola f. serriola. within samples from sweden, l. serriola f. serriola was the dominant form, while the occurrence of l. serriola f. integrifolia was determined in only three plants within one sample from population 45 (fig. 2b, tab. 1). acta bot. croat. 73 (1), 2014 7 morphology of lactuca serriola achenes from slovenia and sweden 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:31 color profile: generic cmyk printer profile composite default screen data for the morphological characters of l. serriola achenes from slovenia were obtained from 304 plants, representing 72 achene samples from 34 collection sites. corresponding data for achenes from sweden were obtained by measuring achenes from 186 plants, representing 47 achene samples from 12 collection sites. sample 192/00-3 (local population 41) was not analyzed for four achene body parameters, lab, wab, lb, and lab/wab index because achenes were damaged during measurements. comparison of morphological characters of achenes from slovenia and sweden achenes from slovenia were longer and wider than those from sweden (tab. 2, figs. 3a, 3b). there were significant differences in mean l. serriola achene length and width from slovenia and sweden (tab. 3). significant differences in the length and width of achenes were observed among local populations and within samples in both countries. no significant differences in the length/width index or the length of the beak were observed for 8 acta bot. croat. 73 (1), 2014 k�ístková e., lebeda a., novotná a., dole@alová i., berka t. tab. 1. lactuca serriola form serriola and integrifolia for populations collected in slovenia and sweden. l. serriola number of entities analysed slovenia sweden total populations 34 12 46 achene samples 72 47 119 plants f. integrifolia (frequency) 0 (0%) 3 (1.61%) 3 (0.61%) plants f. serriola (frequency) 304 (100%) 183 (98.39%) 487 (99.39%) tab. 2. descriptive statistics of morphological characteristics of l. serriola achenes from slovenia and sweden. morphological charactera parameters of l. serriola achenes slovenia sweden n no. of plants mean sd min max n no. of plants mean sd min max lab 15178 304 3.06 0.26 2.12 4.01 9236 185 2.89 0.24 1.93 3.82 wab 15178 304 1.04 0.13 0.48 1.61 9236 185 0.96 0.12 0.54 3.03 lab/wab index 15178 304 2.99 0.39 1.72 5.77 9236 185 3.04 0.39 0.97 5.19 lb 15178 304 4.20 0.53 1.73 6.52 9236 185 4.35 0.51 1.89 5.99 lbr 3040 304 5.07 0.38 3.18 6.24 1858 186 4.83 0.37 2.64 5.81 paparea 3040 304 81.08 11.99 31.71 122.34 1858 186 73.7 10.9 21.88 105.85 discus 3040 304 0.27 0.04 0.15 0.44 1858 186 0.27 0.04 0.15 0.41 nr 3040 304 11.71 1.36 8.00 16.00 1858 186 11.7 1.25 7.00 16.00 alab – length of achene body (mm). wab – width of achene body (mm). lab/wab index – index length/width of achene body. lb – length of beak (mm). lbr – length of bristles (mm). paparea – pappus area (mm2). discus – diameter of discus (mm). nr – number of ribs. n – number of l. serriola achenes measured. sd – standard deviation. min, max – minimum, maximum values. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:31 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 9 morphology of lactuca serriola achenes from slovenia and sweden fig. 3. morphology characteristics of achene bodies of lactuca serriola from slovenia (svn) and sweden (swe): a) length of achene body (lab), b) width of achene body (wab), c) index of length/width of achene body (lab/wab index), d) length of beak (lb). tab. 3. morphological characteristics of l. serriola achenes from119 samples and 46 local populations from slovenia and sweden analysed by nested anova. morphological characteristicsa source of variation df ss f-ratio lab country 1 167.34 28.97*** local populations within countries 44 254.14 2.27*** samples within countries 73 185.83 56.04*** wab country 1 31.15 29.42*** local populations within countries 44 46.59 3.8*** samples within countries 73 20.36 21.28*** lab/wab index country 1 13.73 1.3 ns local populations within countries 44 463.52 5.43*** samples within countries 73 141.64 15.41*** lb country 1 138.75 2.56 ns local populations within countries 44 2388.87 5.04*** samples within countries 73 786.58 75.61*** alab – length of achene body. wab – width of achene body. lab/wab index – index length of achene body/width of achene body. lb – length of beak. df – degree of freedom. ss – sum of squares. ***p < 0.05. ns – not significant. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:36 color profile: generic cmyk printer profile composite default screen slovenia and swedish samples. means are shown in table 2 and in figures 3c and 3d. however, significant differencesin lb and the lab/wab index were detected among local populations and within samples in both countries (p < 0.05) (tab. 3). comparison of mean length of pappus bristles (lbr) and pappus area (paparea) showed significant differences between countries, local populations within countries, and achene samples within countries (p < 0.05) (tab. 4). slovenia local populations had higher values for these morphological characters than the swedish local populations (tab. 2, figs. 4a, 4b). no significant differences in the diameter of discus (discus) and the number of ribs (nr) were found between countries (tab. 4). values of means are shown in table 2 and figures 4c and 4d. on the other hand, comparison of local populations and achene samples within countries showed significant differences (p < 0.05) (tab. 4). associations of achene morphology with geographical factors associations between morphological parameters of l. serriola achenes and geographical factors of collection sites were evaluated by rda (figs. 5, 6). latitude had a significant influence on the morphological characters of l. serriola achene body (f = 665.66, p < 0.001), and also on morphological characters of achene ribs and pappus (f = 240.47, p = 0.002) (tab. 5). as the latitude increases, the length/width index of the achene body (lab/wab index) and length of beak (lb) increased, conversely the length (lab) and width (wab) of the 10 acta bot. croat. 73 (1), 2014 k�ístková e., lebeda a., novotná a., dole@alová i., berka t. fig. 4. morphology characteristics of the pappus and achenes of lactuca serriola from slovenia (svn) and sweden (swe): a) length of bristles (lbr), b) pappus area (paparea), c) diameter of discus (discus), d) number of ribs (nr). 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:41 color profile: generic cmyk printer profile composite default screen achene body were decreased (fig. 5). length of the pappus bristles (lbr) and pappus area (paparea) had lower values at higher latitudes. for discus diameter (discus) and number of ribs (nr), no relationship with geographical location of collection sites was found (fig. 6). acta bot. croat. 73 (1), 2014 11 morphology of lactuca serriola achenes from slovenia and sweden tab. 4. morphological characteristics of achene ribs and pappus of l. serriola from 119 samples and 46 local populations from slovenia and sweden analysed by nested anova. morphological characteristics source of variation df ss f-ratio lbr country 1 64.32 18.31*** local populations within countries 44 154.57 2.85*** samples within countries 73 89.92 13.25*** paparea country 1 62664.8 18.11*** local populations within countries 44 152252 3.00*** samples within countries 73 84141.5 13.08*** discus country 1 0 0 ns local populations within countries 44 0.45 2.15*** samples within countries 73 0.35 3.87*** nr country 1 0.34 0.01 ns local populations within countries 44 1302.32 3.22*** samples within countries 73 671.89 6.75*** lbr – length of bristles. paparea – area of pappus. discus – diameter of discus. nr – number of ribs. df – degree of freedom. ss – sum of squares. ***p < 0.05. ns – not significant. fig. 5. correlation of morphological characteristics of achene body of lactuca serriola from slovenia and sweden with geographical factors selected by forward selection using redundancy analysis. axis 1 and axis 2 account for 4.5% of the variability in species data. lab – length of achene body, wab – width of achene body, lb – length of beak, lab/wab index – index length/width of achene body, lat – latitude, long – longitude, alt (m) – altitude. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:43 color profile: generic cmyk printer profile composite default screen longitude and altitude had a lower impact on morphological parameters of l. serriola achenes (tab. 5). with decreasing longitude, achenes were characterized by shorter, but wider bodies, longer beaks and lower length/width index (fig. 5). with increasing longitude, all pappus characters became longer (fig. 6). in places of higher elevation, longer and wider l. serriola achenes with shorter beaks, longer pappus, wider discus and higher number of ribs occurred (figs. 5, 6). discussion length and width of achenes samples of lactuca serriola from slovenia differed significantly from those from sweden in length and width of achene body. generally, achenes of prickly lettuce were longer and wider in slovenia than in sweden. this is supported by rda analysis showing shorter length and width of achene body as one goes north. mean values for l. serriola f. serriola achenes from the czech republic which is geographically located between slovenia and sweden reported by novotná et al. (2011) are: lab 2.95 mm, wab 0.93 mm, lab/wab 3.21. 12 acta bot. croat. 73 (1), 2014 k�ístková e., lebeda a., novotná a., dole@alová i., berka t. fig. 6. correlation of morphological characteristics of achene ribs and pappus of lactuca serriola achenes from slovenia and sweden with geographical factors selected by forward selection using redundancy analysis. axis 1 and axis 2 acount for 6.6% of the variability in species data. lbr – length of bristles, paparea – area of pappus, discus – diameter of discus, nr – number of ribs. lat – latitude, long – longitude, alt (m) – altitude. tab. 5. impact of geographical factors affecting morphological characteristics of l. serriola achenes using the forward selection method of redundancy analysis. variables achene bodya achene ribs and pappusb f-ratio p f-ratio p latitude 665.66 < 0.001 240.47 0.002 longitude 314.87 < 0.001 39.64 0.002 altitude 150.70 < 0.001 11.22 0.002 alab – length of achene body. wab – width of achene body. lab/wab index – index length of achene body/width of achene body. lb – length of beak. blbr – length of bristles. paparea – area of pappus. discus – diameter of discus. nr – number of ribs. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:44 color profile: generic cmyk printer profile composite default screen while the width of czech achenes reach the lowest value, the length of achene body for czech populations confirmed the trend of a decreasing value from the south to the north. the differences in the length/width index of the achene body between the slovenian and swedish populations of prickly lettuce are not statistically significant; however the lab/ wab achene index from these two countries increased toward the north. toward the north, achenes are shorter and narrower. this parameter must be considered with regard to the beak length and pappus area. achene ribs the number of ribs between populations from slovenia and sweden was not significantly different. in both countries, the mean value was 11.7 ribs, more than the 5–10 ribs described for this species by feráková (1977), and more than reported by novotná et al. (2011) for achenes from the czech republic, germany, the netherlands, and united kingdom. however, novotná et al. (2011) stressed that the achenes from central european populations (from the czech republic and germany) created a distinct group, significantly different based on the number of ribs from the group of dutch and english populations. nathan et al. (2011) paid particular attention to various morphological features of diaspores, including those which increase the roughness of the seed surface, thereby increasing the drag force on the seed in flight, both of which increase travel time through the air. our study shows that there is a variation in number of ribs. we did not measure the bristles on achene ribs, however we noticed differences in their quantity and quality among samples. the architecture of bristles and ribs with regard to species dispersion and evolution needs to be studied in more detail. beak the difference in the length of the beak between slovenian and swedish populations was not statistically significant. however, the beak of achenes from slovenia was shorter than the beak of achenes from sweden. similar phenomenon was observed by novotná et al. (2011) on lactuca serriola populations from czech republic, germany, the netherlands and great britain, where toward the west and the north the beak became longer. andersen (1993a) studied the morphology and settling velocity of some wind-dispersed species of the asteraceae, including l. serriola. he found that diaspores with beaked achenes have significantly lower setting velocities than diaspores with unbeaked achenes, even though beaked and unbeaked achenes do not differ in plume loading, i.e. the ratio of diaspore weight/plume area (matlack 1987). lower setting velocity is one of the prerequisites of better dispersal ability which is attributed to younger populations (chmielewski and strain 2007). so, toward the north, achenes of younger populations of prickly lettuces are favored by longer beaks that reach lower setting velocity compared to older southern populations. discus and pappus no significant difference was found for discus diameter between slovenia and swedish populations. the mean value of that trait from both countries was 0.27 mm. according to correlation analysis, a weak correlation was found with latitude and longitude. acta bot. croat. 73 (1), 2014 13 morphology of lactuca serriola achenes from slovenia and sweden 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:44 color profile: generic cmyk printer profile composite default screen our recent study showed significant differences in the length of pappus bristles and the area of pappus of l. serriola from slovenia and sweden. when we compared achene size to pappus area, slovenian achenes, with a significantly larger achene body, also possessed significantly longer pappus bristles and a larger pappus area than the smaller-sized swedish achenes. dispersal ability is positively related not only to lower mass of achene, but also by advantageous proportion between achene mass and pappus. this scenario was demonstrated empirically on several wind dispersed plant species from the asteraceae by cody and overton (1996). very detailed studies of the pappus characters, especially of the pappus bristles of weedy and non-weedy aster species were made by chmielewski and strain (2007). they concluded that the achenes of weedier aster species have the potential to remain in the air longer and thus can be dispersed further on average than achenes of non-weedy aster species. the greater dispersal ability of weedy species is due to comparatively lower values of terminal velocity resulting from the lower achene mass. sheldom and burrows (1973) found a negative curvilinear relationship between terminal velocity and the ratio of pappus diameter/achene diameter for 17 species. in our study we did not measure the diameter of achenes, and we substituted achene width for this parameter. the ratio of pappus diameter/achene width is for the slovenian achenes 10.01, and 10.34 for swedish achenes. from the curve constructed by sheldom and burrows (1973), the extrapolated terminal velocity for slovenian achenes is 0.8 m/s, and 0.6 to 0.4 m/s for swedish achenes. these data are in agreement with scenarios on wind dispersibility of achenes in old and young populations proposed by fresnillo and ehlers (2008). they stated that the terminal velocity of achenes in old populations is higher, so a subsequent reduction in dispersal ability as the population ages is expected due to the risk of long dispersal seeds landing in unsuitable habitats. in old populations, this would favor genotypes with a lower dispersal rate. populations in newly colonized areas are expected to have high dispersal potential, because they were established from seed with parameters enabling long-distance flight. however, diaspores with high dispersal ability may be blown off newly reached areas, and this should select for a reduction in dispersal ability in future generations originating from individuals that produce diaspores with low dispersal ability (cody and overton 1996). fresnillo and ehlers (2008) compared the dispersal ability of three wind dispersed plant species (cirsium arvense, epilobium angustifolium and e. hirsutum) from populations on a mainland and three islands. they found that the achenes of c. arvense (asteraceae) from the mainland had a significantly longer pappus than the achenes from the island populations. the conclusion of harper et al. (1970) that the achenes from islands have shorter pappi and lower dispersal ability than achenes from mainland was in agreement with our results (table 2). prickly lettuce is not yet completely settled throughout the territory of southern sweden, local populations are partly isolated, so from this regard we can consider them as »islands«, conversely to the situation in slovenia where this species is distributed throughout whole country. this phenomenon was described by dole@alová et al. (2001) and confirmed during recent field observations in 2009 and 2010. strykstra et al. (1998) showed that pappus size of arnica montana was significantly, but weakly positively correlated with achene mass. heavier achenes with high germination and seedling quality, therefore, stay closer to the point of release than lighter ones. long distance dispersal as a 14 acta bot. croat. 73 (1), 2014 k�ístková e., lebeda a., novotná a., dole@alová i., berka t. 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:44 color profile: generic cmyk printer profile composite default screen prerequisite of the establishment of new populations of a. montana depends on the transport by human activity. a lower proportion of pappused achenes were found in older populations of lactuca muralis (synonym. mycelis muralis) than in newer colonies (cody and overton 1996). they stated that it is unclear whether the observations were due to genetic differences or phenotypic plasticity; however, the existence of variation between populations had been established. according to sheldon and burrows (1973), the efficiency of dispersal is determined more by the fine details of pappus geometry, which directly affects its aerodynamic properties, than by the size ratio of pappus to achene. the pappus is generally thought of primarily as a long-distance seed dispersal mechanism. sheldon and burrows (1973) cited the suggestion of goebel (1905) that the primary function of the pappus could be to be a transpiration apparatus for the fruit. stevens at al. (1983) suggested that the pappus of chrysothamnus nauseosus plays an important role in orientation of achene on the soil surface and adhesion, both of which increase survival when achenes surface-germinate. this theory is at least partly supported by meyer (1997) who did not observe a significant correlation between absolute achene mass and relative pappus mass of chrysothamnus nauseosus. associations of evaluated morphological characters of achenes with geographical factors among geographical factors, latitude had the greatest influence on length and width of achene body. despite this, at higher altitudes, longer and wider achenes were found. gravuer et al. (2003) investigated the dispersal capability of a rare grassland species liatris scariosa var. novae-angliae in new england. they found that dispersal ability of that taxon was reduced for longer and heavier achenes and it increased for wider achenes. according to their study, the diaspore mass was one of the strongest predictors of dispersal ability. at higher altitudes, wider achenes are preferred because their dispersal ability increases. variation within local populations and within samples in our current study significant differences in the length and width of achene body, index of both parameters, length of pappus bristles, pappus area, diameter of discus, and number of ribs were found between populations within countries and between samples within populations. andersen (1992) hypothesizes that the high intraspecific variability in seed settling velocities of eight wind–dispersed asteraceae is connected with risk-spreading strategy of species. the highly significant between-plant differences in achene mass found in populations of chrysothamnus nauseosus (asteraceae) are genetically fixed, as reported by meyer (1997). significant variability within the populations observed in the current study probably has a genetic basis indicating sufficient variation within populations to permit continuous selection. future research experiments with only a few species may lead to tentative conclusions, but can hardly be generalized for a larger group of species. in our study we did not examine physical acta bot. croat. 73 (1), 2014 15 morphology of lactuca serriola achenes from slovenia and sweden 742 kristkova et al.ps u:\acta botanica\acta-botan 1-14\742 kristkova et al.vp 19. o ujak 2014 16:39:45 color profile: generic cmyk printer profile composite default screen parameters of achenes of l. serriola regarding their setting velocity, but we did document the significant differences in crucial traits of l. serriola achenes from two eco-geographically distinct areas in europe. we discussed each parameter separately, however, their integration and interpretation need a more comprehensive treatment. data from the current study will be combined with those obtained by novotná et al. (2011), helping to contribute to the elucidation of evolutionary and taxonomical concepts for l. serriola. acknowledgements critical reading and valuable remarks by dr. gerald seiler (fargo, north dakota, usa) are gratefully acknowledged. the research was supported by grant msm 6198959215 (ministry of education, czech republic) and by the internal grant of palacký university in olomouc (iga_prf_2013_001). references andersen, m. c., 1992: an analysis of variability in seed settling velocities of several wind-dispersed asteraceae. american journal of botany 79, 1087–1091. andersen, m. c., 1993a: diaspore morphology and seed dispersal in several wind-dispersed asteraceae. american journal of botany 80, 487–492. andersen, u. v., 1993b: dispersal strategies of danish seashore plants. ecography 16, 289–298. anonymous, 2011: climate and temperature. retrieved june 22, 2011 from http://www. climatetemp.info/sweden/ augspurger, c. k., 1986: morphology and dispersal potential of wind-dispersed diaspores of neotropical trees. american journal of botany 73, 353–363. baker, g. a., dowd, d. j., 1982: effect of parent plant density on the production of achene types in the annual hypochoeris glabra. journal of ecology 70, 201–215. blumenthal, d., jordan, n., 2001: weeds in field margins: a spatially explicit simulation analysis of canada thistle population dynamics. weed science 49, 509–519. boukema, i. w., hazenkamp, th., van hintum, th. j. l., 1990: the cgn collection reviews: the cgn lettuce collection. centre for genetic resources, wageningen. brant, v., holec, j., 2004: locika kompasová (lactuca serriola l.) 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(2n = 2x = 16) and a. × cornutum clementi ex vis. (2n = 3x = 24). this paper reviews the results of studies of their morpho-anatomical characteristics and genetic structure. although all three taxa were determined as varieties of the common onion, only the shallot a. cepa aggregatum group (syn. a. ascalonicum l.) belongs to that species. the shallot a. × proliferum represents a hybrid between the two closely related species, a. cepa and a. fistulosum l. the third form of shallot, a. × cornutum is a still incompletely understood triploid hybrid between a. cepa and one or two closely related allium species, whose identity has not been fully elucidated. in contrast to shallot a. cepa aggregatum group, which has normal meiosis and produces fertile seed, hybrid shallots a. × proliferum and a. × cornutum are sterile, and reproduce exclusively vegetatively by underground bulbs or bulbils from the inflorescence. key words: allium cepa, shallot, hybrid, polyploidy, triploid viviparous onion, top onion, karyotype, genome, meiosis common onion, allium cepa l. the genus allium comprises about 750 species distributed all over the northern hemisphere except for its tropical regions (stearn 1992, fritch and friesen 2002). the common onion is the most widespread and economically important species of the genus allium (hanelt et al. 1992, fritch and friesen 2002). taxonomically, it belongs to allium section cepa (mill.) prokh, which consists of twelve species, most of which are used as condiment, vegetable or medicinal plants (gurushidze et al. 2007). the common onion is not known in the wild form. its initial domestication started more than 4000 years ago, probably in the middle east (hanelt et al. 1992). several wild relatives of the common onion originate from the mountainous regions of asia in the region beacta bot. croat. 72 (2), 2013 387 * corresponding, e-mail: puizina@pmfst.hr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. tween south siberia, the persian gulf, and the caspian sea (jones and mann 1963, hanelt 1992, fritch and friesen 2002). however, the wild progenitor(s) and origin of common onion are still not fully understood. due to their morphological similarities, the wild species allium oschaninii o. fedtsch (a. cepa var. silvestre regel) from central asia has been for a long time believed to represent the wild ancestor of the common onion (stearn 1980, hanelt et al. 1992). this hypothesis was rejected because of different heterochromatic (giemsa c-banding) patterns of their karyotypes (vosa 1976). additional experiments also rejected that hypothesis: severe crossing barriers appear between a. cepa and a. oschaninii (van raamsdonk et al. 1992); the chloroplast phylogeny of the section cepa placed a. oschaninii outside of the clade containing a. cepa (havey 1992). several recent molecular analysies indicated that the wild species a. vavilovii m. popov et vved. could be a possible progenitor of a. cepa. rflp analysis of nuclear dna revealed identical pattern in a. cepa and a. vavilovii (bradeen and havey 1995) and the sequences of three chloroplast loci also indicated a close genetic relationship between a. cepa and a. vavilovii (van raamsdonk et al. 2000). one of the main items of evidence suggesting that a. vavilovii is a progenitor of the common onion was the phylogenetic study of internal transcribed (its) region of the nuclear ribosomal dna (gurushidze et al. 2007). by application of selection and crossings a number of different a. cepa forms and varieties have been produced. they can be grown in different climates, on every continent, except in the extreme northern regions and humid tropical and subtropical regions. red onion cultivars are classified into three groups (brickell et al. 2009, jones and mann 1963, fritch and friesen 2002): a) common onion group: underground bulbs are large, flattened at the poles, usually single, inflorescence without bulbils, and reproduction by seeds. this group includes the largest number of commercially produced cultivars, b) aggregatum group: underground bulbs elongated with numerous lateral bulbs, no bulbils in the inflorescence, either producing seed or else sterile, and reproduction almost exclusively vegetatively, c) proliferum group: underground bulbs often poorly developed, inflorescence bears bulbils, do not produce seeds and reproduce vegetatively by bulbils in the inflorescence. the term shallot is most commonly used for a specific variety of a. cepa, and was formerly even classified as a distinct species, a. ascalonicum l. recent studies have shown that the term shallot in southern croatia is additionally used for diploid and triploid viviparous onions a. × proliferum (moench.) schrad and a. × cornutum clementi ex vis. (puizina 1992, 1997). this article provides an overview of the results of studies on the morpho-anatomical and genetic structure. a common morphological feature of all three taxa is the vegetative propagation using underground bulbs and a close relationship with the common onion. shallot, allium cepa aggregatum group shallots had previously been considered a separate species, a. ascalonicum, but today they are classified within the common onion under the currently accepted name a. cepa aggregatum group (rabinowitch and kamenetsky 2002). besides the best known name a. ascalonicum, there are several other synonyms in use: a. cepa var. aggregatum, a. cepa var. ascalonicum, a. cepa cv. 'shallot' (jones and mann 1963, hanelt et al. 1992, rabinowitch and kamenetsky 2002). 388 acta bot. croat. 72 (2), 2013 puizina j. although these plants look morphologically very similar to a. cepa, there are some differences. plants are perennials and generally have smaller flowers, inflorescences, bulbs and leaves than a. cepa. compared with theleaves of a. cepa, leaves of shallot are thin, tender, often bent and particularly flat, almost concave on the inner side of the leaves. underground bulbs are well developed; their shape is oblong, semi-cylindrical. a large number of bulbs are gathered in the clusters. after planting each bulb develops an entire group of side bulbs that are interconnected. each new bulb develops its own leaves, so that the whole plant has a bushy appearance. in contrast, a. cepa usually has a well-developed stem. the shallot cultivars examined by puizina (1992) had no stamen morphology typical of a. cepa (outer anthers simple, interior anthers with an expanded base and one small tooth on each side), but all six stamens were simple (tab. 1). all taxa of a. cepa aggregatum group are diploids (2n = 2x = 16) (hannelt et al. 1992). the structure of the shallot's karyotype could not be distinguished from the karyotype of a. cepa. the constitutive heterochromatin as identified by giemsa c-banding is located mainly at the end of the chromosomes. only a few interstitial c-bands were identified and their distribution was similar to the distribution of giemsa c-banding in a. cepa (figs. 4a, b) (vosa 1976, puizina 1992). in meiosis eight bivalents are regularly seen in diakinesis and metaphase i (fig. 5d). bivalents have terminal type of chiasmata and are generally of the same shape as a. cepa bivalents (rings and rods) (fig. 5a). the plant rarely produces flowers although the pollen is fertile and a seed is produced. it is mainly propagated vegetatively by dividing and planting underground bulbs. on a global scale, the shallot, a. cepa aggregatum group, is a minor allium crop. however in south east asia and some african countries where onion seed is hard to produce, where onion culture is difficult and where the growing season is too short for the production of bulb onion, the vegetatively propagated shallot is cultivated as an important substitute for bulb onion (rabinowitch and kamenetsky 2002). the relationship of shallots to common onion was investigated by application of random amplified polymorphic dna (rapd) markers and morphological traits (le thierry d'ennequin et al. 1997). sexually propagated shallots were more closely related to common onion than the vegetetively propagated shallots, which grouped separately. the grey shallot is a specific form of shallots long cultivated in france and italy. maass (1996) compared the isozyme profile of 30 accessions of the french grey shallot, with those of 466 bulb onions and other shallots, 15 accessions of a. oschaninii and 22 accession of a. vavilovii. the results suggested that the french grey shallots differ from other shallots and were more closely related to either to a. oschaninii or a. vavilovii, that to a. cepa and other shallots. according to the rapd analysis and gish results, friesen and klaas (1998) also concluded that grey shallots belong to a. oschaninii. shallot, allium × proliferum (moench) schrad. in the local dialect of south croatia, there are several different names for this shallot: kozjak, orija{, {kalonja, ljutika, ljutika-talijanka (puizina and pape[ 1999). the diploid viviparous onion is also known as top onion, tree onion or egyptian onion. previously it has been classified as a variety of common onion a. cepa var. proliferum (targioni, tozzetti) or a. cepa var. viviparum (metzg.) alef. (helm 1956, jones and mann 1963, mccollum 1974, puizina and pape[ 1996). since plants represent a spontaneous hybrid between a. cepa (common onion) and a. fistulosum (japanese bunching onion), schubert et al. (1983) acta bot. croat. 72 (2), 2013 389 shallots in croatia – genetics, morphology and nomenclature 390 a c t a b o t .c r o a t .72 (2),2013 p u iz in a j. tab. 1. morphological characteristics of shallots in croatia. morphological character a. cepa shallots a. fistulosum a. cepa aggregatum group a. × cornutum a. × proliferum bulbs round, flattened at the poles, 5–10 cm in diameter semicylindrical, well developed and gathered in clusters, 2–5 cm diameter elongated, pear-shaped, developed and gathered in clusters, 2–5 cm diameter elongated, less developed, 2–4 cm in diameter elongated, less developed, 1–2 cm in diameter stem up to 100 cm tall, hollow, conical, inflated at the base, 3 cm diameter up to 70 cm tall, hollow, conical, inflated at the base, 1–1.5 cm diameter up to 100 cm tall, hollow, conical, slightly inflated at the base, diameter 3 80–150 cm tall, hollow, conical, inflated at the base, 3–5 cm diameter 12–70 cm tall, inflated in the middle, 1–2 cm diameter leaves up to 40 cm long, diameter up to 2 cm, semicircular and flattened on the upper side up to 40 cm long, diameter up to 1 cm, semicircular and flattened on the upper side up to 40 cm long, diameter up to 2 cm, tubular, circular in cross-section up to 50 cm long, diameter up to 3 cm, semicircular and flattened on the upper side from 7–30 cm long, diameter 0.5–1.5 cm, cylindrical, circular in cross-section spathe shorter than inflorescence, divided into 2–4 parts, mostly triform shorter than inflorescence, divided into 2–4 parts shorter than inflorescence, usualy triform significantly longer than the inflorescence, 1–2 parts almost the same length as the inflorescence, 1–2 parts inflorescences hemispherical, diameter, 4–9 cm hemispherical, diameter, 3–5 cm, dense spherical to semispherical, with bulbils, diameter 4–5 cm hemispherical, numerous bulbils and rare flowers, diameter 3–5 cm hemispherical, diameter 1.5–5 cm, dense perianth star like shape of a flower, segments white with a green stripe, size of segments 3–4.5 × 2– 2.5 mm star like shape of a flower, segments white with a green stripe, size of segments 3 × 2 mm star like shape of a flower, segments white with a green stripe, size of segments 3.5–4 × 3 mm shape of a flower is more campanulate than starlike, segments white with a green stripe, size of segments 5–7 × 3 mm closed shape of a flower, campanulate, segments yellowish-white, size of segments 5–7 × 2 mm flower pedicels equal, up to 4 cm long equal up to 2 cm long equal 1–2 cm long unequal 0.3–7 cm long unequal, 0.3–2 cm long stamens filaments 4.5 mm, outer simple, interior with extended toothed base. filaments 3–4 mm, outer and inner simple filaments 3–4 mm, outer simple filaments 4.5 mm, inner simple, with a toothed inner base filaments 4–7 mm, outer and inner simple filaments 8–12 mm long, the outer and inner simple pistils lower than the stamens lower than the stamens taller than the stamens taller than the stamens lower than the stamens pointed out that for nomenclatural reasons this onion should be named a. × proliferum (moench) schrad. plants have a relatively poorly developed bulb, similar to the parental species a. fistulosum. a sort of gigantism that characterizes this cultivar is its most striking feature, in which it also differs from other varieties of onions. due to the rapid vegetative development, these perennial plants can grow up in height up to 130 cm, and their flower pedicels can be 3–5 cm long. their inflorescences regularly contain flower bulbils with some sterile flowers. bulbils often sprout within the transformed umbel (figs. 1c, f), sometimes in two or more tiers. with respect to the shape and structure of the stamen's filaments, the plant more closely resembles a. fistulosum. specifically, the shape of the flower is more campanulate and closed as in a. fistulosum, rather than stellar and open as in a. cepa. stamen filaments are long, thin, without or with barely noticeable expansion on the basis of internal stamens. anthers are often rudimentary or sterile (fig. 2d). given the form of leaf, this type of shallot resembles a. cepa (flattened on the inner side). a special feature of this shallot is the long spathe that envelops the inflorescence, which it surmounts considerably.. this novel feature is not characteristic of a. cepa or a. fistulosum. as the underground bulbs are poorly developed, and the plant does not produce seeds, plants mostly reproduce vegetatively by bulbils from the inflorescences (usually 4–8 bulbils per inflorescence). acta bot. croat. 72 (2), 2013 391 shallots in croatia – genetics, morphology and nomenclature fig. 1. allium × cornutum: a – appearance; b – plants in the soil; d – young inflorescence, e – mature inflorescence carrying two bulbils which are already sprouting. allium × proliferum: c – plants in the soil; f – bulbils often sprout within the transformed inflorescence before its opening. top onions, a. × proliferum, are a vegetatively propagated garden crop in europe, north east asia and north america. several karyological studies indicated its hybrid (allodiploid) nature and showed that the karyotype consists of one haploid a. cepa and one haploid a. fistulosum set of chromosomes (bozzini 1964, mccollum 1974, fiskesjo 1975, schubert et al. 1983, puizina and pape[ 1999). chromosomes preserved their original c-banding pattern as in the parental species with the exception of a. cepa satellite chromosome that lost its satellite (fig. 4d). meiosis of a. × proliferum is characterized by chromosome pairing of nonhomologous chromosomes into heterobivalents (fig. 5d). however, univalents were frequently seen and very rarely some multivalents. bivalents with terminally located chiasmata dominate, but occasionally bivalents with localized chiasmata (near the centromere) can be seen (puizina 1997). the hybrid origin has been proved by isozyme analysis (maass 1997a), rapd markers (friesen and klaas 1998) as well as by genomic in situ hybridization (gish) experiments using labeled total genomic dnas from a. cepa and a. fistulosum as probes. each probe labeled one genome (eight chromosomes of top onion; friesen and klaas 1998, puizina and pape[ 1999). allium wakegi araki is a sexually sterile ancient garden crop in japan and china. its hybrid nature (a. cepa × a. fistulosum) has been confirmed by gish and by localization of 5s rrna loci at chromosomal positions corresponding to a. cepa and a. fistulosum (hizume 1994). shallot, allium × cornutum clementi ex vis. the triploid shallot is traditionally cultivated in south and coastal croatia under the name 'ljutika' and it is very popular as a spice and condiment due to its tasty bulbs and leaves. it was first named a. cepa var. viviparum (langer and koul 1983; puizina and pape[ 1996, 1997), but friesen and klaas (1998) suggested that this name is connected with the other viviparous onion a. × proliferum and proposed using the name a. cornutum 392 acta bot. croat. 72 (2), 2013 puizina j. fig. 2. flower and stamen morphology: a, b – a. fistulosum; c, d – a. × proliferum; e, f – a. × cornutum; g, h – a. cepa. in contrast with a. cepa, which is characterized by the broadened base of the inner filament (h), the triploid a. × cornutum possesses rather pronounced triangular teeth at the bases of the inner filaments (f). clementi ex vis. (visiani 1842), the only name which is unambiguously connected with the triploid onion (stearn 1980, maass 1997b). however, taking into account its hybridogenic origin, it was modified to: a. × cornutum. interestingly, this name was first used by visiani (1842) for a dalmatian bulbiferous taxon, which was observed for the first time on the rocks of dubrovnik (stearn 1980). in contrast to most flowering species of allium in which the leaves are already dying back at flowering time, triploid shallots are perennials, their leaves remain green and suitable for use during entire year. it blooms, like the majority of other allium species, from may and june. plants are sterile and do not produce seeds. they propagate vegetatively by underground bulbs and bulbils from inflorescence. phenotypically, triploid shallots (viviparous onions) closely resemble a. cepa, and sometimes it is difficult to distinguish them if they are planted both in the soil, before developing their inflorescences (figs. 1 a, b). the triploid shallot's flower is star-shaped; the segments are white with a green stripe. three inner stamens have a broadened base with two prominent teeth (one from each side) (figs. 2e, f). outer stamens are simple. inflorescences of the triploid shallot initially contain only the flowers, which are by their dimensions slightly larger than the flowers of a. cepa and generally les numerous in the inflorescence than in a. cepa (figs. 1a, 3a). during the maturation of inflorescences, small reproductive bulbils begin to appear (figs. 1d, e) and flowers gradually disappear. at the end, the mature inflorescence is composed only of the bulbils (up to 20–30 bulbils per inflorescence). it has never been noticed that bulbils from the inflorescence develop their own stems and inflorescences, which is a regular case in the diploid hybrid shallot, while occasionally triploid bulbils from the inflorescences develop only a few leaves (fig. 1e). these small reproductive bulbils, which regularly develop among sterile flowers in the inflorescence of the triploid shallot are a reliable sign for its recognition, as they never develop in the diploid a. cepa. another reliable sign of the triploid shallot as against a. cepa are the elongated underground bulbs (fig. 1a). bulbs are gathered in large numbers, sometimes 10–20 or more individual bulbs are held together by the same basal plate. from each bulb leaves emerge and the plant has a bushy form. the triploid shallot can also be distinguished from a. cepa with respect to the shape of leaves. leaves are not semicircular and flattened on one side as they are in a. cepa, nor are they round in shape. they are intermediate between the semicircular and the round shape. the stalk that bears the inflorescence is only slightly flattened at the bottom, whereas the stalk in a. cepa is inflated at the base of the stem. the triploid shallot does not exhibit the same effect of gigantism as top onions. it is even slightly smaller than the diploid a. cepa (they grow up to 100 cm). although its bulbs and leaves are very tasty and are used in the diet, there is a problem of their prolonged storage and keeping after harvesting, because they tend to rather quickly produce new leaves and roots, thus losing weight. that is probably the reason why the bulbs are usually kept by being preserved in vinegar. the karyotype of a. × cornutum (2n = 3x = 24) consists of 24 chromosomes (singh et al. 1967, puizina and pape[ 1996). the homology between the chromosomes is weak and occasional, and it is very difficult, and even impossible, to identify homologous pairs of chromosomes. therefore the chromosomes are usually lined up with respect to size. the exceptions are the three subtelocentric chromosomes, which are put at the end of the karyotype (figs. 3a, b). the most common chromosome associations in diakinesis and metaphase i of acta bot. croat. 72 (2), 2013 393 shallots in croatia – genetics, morphology and nomenclature a. × cornutum are heterotrivalents, which suggests a partial homology of the three genomes (puizina and pape[ 1997). additionally, the frequent occurrence of complex multivalents was observed (4–11 paired chromosomes) suggesting that intergenomic pairing, translocations and chromosomal rearrangements were involved in the evolution of the triploid karyo394 acta bot. croat. 72 (2), 2013 puizina j. fig. 3. mitotic chromosomes of root tip cells in a. × cornutum (a), 2n = 3x = 24, stained by feulgen. arrows indicate the three satellite schromosomes. chromosomes aligned in the kariogram (b). scale bar=10 mm. fig. 4. schematic representation of giemsa c-banding karyotypes in shallots. type (fig. 5e). identification of the constitutive heterochromatin by giemsa and fluorochrome banding in the karyotype of a. x cornutum (lepen and puizina 2011) indicated the hybrid structure of the karyotype, which contained only one genome originating from a. cepa (fig. 4e). previously, triploid viviparous onions have been suspected to be either an allotriploid (aab) (singh et al. 1967), or a segmental allotriploid (aa'a'') (koul and gohil 1971). the chloroplasts dna (cpdna), the nuclear fragment of rrna genes and the rapd molecular markers indicated a. cepa as one parent (havey 1991, 1992, 1993; klaas and friesen 2002). in order to determine the parental allium species of a. × cornutum, genomic fluorescent in situ hybridization (gish) was applied (puizina et al. 1999). biotinylated genomic dnas from six diploid allium species (a. cepa, a. fistulosum, a. roylei stearn, a. vavilovii, a. galanthum kar. et kir. and a. oschaninii) were used as probes. while probes obtained from genomic dna of a. cepa, a. vavilovii and a. roylei hybridized to somatic chromosomes of a. × cornutum, probes from a. fistulosum, a. galanthum, and a. oschaninii did not. the dna probes of a. cepa and a. roylei each completely or predominantly labeled one genome (eight chromosomes). a few chromosomes, the markers of the triploid karyotype, were not completely labeled by any of the probes applied. these gish results indicated that triploid viviparous onions might possess a complex triparental genome organization (puizina et al. 1999). using a similar approach friesen and klaas (1998) reached a different conclusion. they could verify only a. cepa as a parental species and according to these authors, the majority of dna and chromosomes of a. × cornutum originate from a. cepa. further progress in the identification of the parental species of a. × cornutum, an 'enigmatic plant' as noted by klaas and friesen (2002), was hampered by the lack of more molecular data about wild relatives of common onion as well as suitable molecular markers. recently, the its sequences (internal transcribed spacer of the 18s-5.8s-26s rdna) of large number of common onion relatives originating from central asia, were deposited in genbank (gurushidze et al. 2007) as well as the sequences of the 5s rrna (son et al. acta bot. croat. 72 (2), 2013 395 shallots in croatia – genetics, morphology and nomenclature fig. 5. meiosis, metaphase i: a – allium cepa; b – a. fistulosum; c – a. cepa aggregatum group; d – a. × proliferum; e – a. × cornutum. 2012). our preliminary results detected several types of its and 5s rrna sequences in a. × cornutum. detailed analysis of these sequences is in progress and the results achieved will help us to understand the origin and the genome structure of that interesting triploid hybrid better. the triploid viviparous shallot a. × cornutum is a rather widespread garden crop in south-east asia, europe and other parts of the world. based on several morphological, cytogenetic and molecular studies, it is now recognized as a separate allium crop and a new taxonomic unit. since almost all its clones are practically identical (maass 1997b, friesen and klaas 1998, puizina et. al 1999), the monophyletic origin of this widely distributed crop is likely, and northern india (kashmir) might be the place of origin. although humans must have contributed to the maintenance and spread of this vegetatively reproduced crop, findings of a. × cornutum in abandoned vineyards and other less-favored agricultural and even landfill areas suggest that this plant is very tolerant to drought and poor soil and is able to persist even in wild habitats. acknowledgements this work received funding from the croatian ministry of science, education and sport through a grant to jasna puizina (no. 177-11911196-0829). author thanks to dr. juraj kamenjarin and dr. mirko ru{~i} for providing pictures of shallots. the author also thanks the two anonymous reviewersfor their helpful suggestions and comments, which improved the final version of the manuscript. references bozzini, a., 1964: on the karyotype of a viviparous onion known as allium cepa var. viviparum (metzg.) alef. caryologia 17, 459–464. bradeen, j. m., havey, m., 1995: restriction fragment length polymorphisms reveal considerable nuclear divergence within a well-supported maternal clade in allium section cepa (alliaceae). american journal of botany 82, 1455–1462. brickell, c. d., alexander, c., david, j. c., hetterschleid, w. l. a., leslie, a. c., malecot, v., xiaobai, j., 2009: international code of nomenclature for cultivated plants. scripta horticulturae 10, 1–184. fiskesjo, g., 1975: chromosomal relationships between three species of allium as revealed by c-banding. hereditas 81, 23–32. friesen, n., klass, m., 1998: origin of some minor vegetatively propagated allium crops studied with rapd and gish. genetic resources and crop evolution 45, 511–523. fritch, r. m., friesen, n., 2002: evolution, domestification and taxonomy. in: rabinowitch, h. d., urrah, l. 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(eds.), flora europaea 5. university press, cambridge. stern, w. t., 1992: how many species of allium are known? kew magzine 9, 180–182. van raamsdonk, l. w. d., de vries, t., 1992: biosystematic studies in allium l. section cepa. botanical journal of the linnean society 109, 131–43. van raamsdonk, l. w. d., vrielink-van ginkel, m., kik, c., 2000: phylogeny reconstruction and hybrid analysis in allium subgenus rhizirideum. theoretical and applied genetics 100, 1000–1009. schlosser, j. c., farka[-vukotinovi], l., 1869: flora croatica, zagreb visiani, r., 1842: flora dalmatica 1. hofmeister, lipsiae. vosa, c. g., 1976: heterochromatic patterns in allium. the relationship between the species of the cepa group and its allies. heredity 36, 383–392. 398 acta bot. croat. 72 (2), 2013 puizina j. 625 pise and gaikwad.vp acta bot. croat. 72 (2), 287–294, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/botcro-2013-0007 grapevine yellows affecting the croatian indigenous grapevine cultivar grk marin je@i]1, jasminka karoglan konti]2, darko preiner2, edi maleti]2, mirna ]urkovi]-perica1* 1 university of zagreb, faculty of science, division of biology, department of microbiology, marulicev trg 9a, zagreb, croatia 2 university of zagreb, faculty of agriculture, department of viticulture and enology, svetosimunska 25, zagreb, croatia abstract – the grapevine cultivar grk, a close relative of crljenak ka{telanski/zinfandel, is grown exclusively in southern croatia. grapevine yellows-like symptoms were observed on vines in the vineyards in lumbarda (southern croatia) and in propagated grapevines near zadar and zagreb. the majority of the detected phytoplasma isolates belonged to the 16sri group. however, rflp pattern and r16f2n/r2 fragment sequence assigned one isolate to the 16sriii group. thus far, on cv. grk, phytoplasmas belonging to three different groups have been detected: 16sri, 16sriii, and 16srxii, which was confirmed previously. aside from the 16sri, 16srv and 16srxii phytoplasma groups previously found on grapevines in croatia, the finding of 16sriii group, which is not common on grapevines in europe, adds to the diversity of phytoplasmas in a very small geographic region. key words: molecular detection, phytoplasma, rflp, vitis vinifera cv. grk introduction grk is an old croatian grapevine cultivar grown almost exclusively on the island of kor~ula. its name (grk in croatian language means greek) and long cultivation suggests that the cultivar was brought from the east at the time of the greek colonization of the island (4th century bc). however, because of its close genetic relationship with crljenak ka{telanski/zinfandel (as parent and progeny, respectively), and thus its relationship with a group of dalmatian cultivars (peji] et al. 2000, maleti] et al. 2004), it is most probable that cv. grk arose in this area. the population of cv. grk is rather small and the most productive vineyards are located on sandy soils near the town of lumbarda, where its grape achieves the highest quality and gives the famous wine quality. acta bot. croat. 72 (2), 2013 287 * corresponding author, e-mail: mirna.curkovic-perica@biol.pmf.hr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. in recent years a decrease in the productive potential of cv. grk vines in lumbarda region has been observed. symptoms like leaf folding, yellowing and drying long before the fall, poor fruit set (cv. grk has female flowers) and yield lowering indicates either a physiological or a phytosanitary disorder of the vines. thus, the cv. grk population has been screened for the economically most important viruses: grapevine fanleaf virus (gflv), arabis mosaic virus (armv), grapevine leafroll-associated virus 1 (glrav-1) and grapevine leafroll-associated virus 3 (glrav-3). using elisa, testing of viruses was carried on 70 selected vines from five vineyards more than 15 years old, out of which 37 vines were free of the tested viruses (preiner et al. 2010). these selected mother stocks of cv. grk, were propagated and planted in a mother vineyard in ba{tica near zadar in year 2008 for further propagation and clonal selection. the obtained results have shown that infection of the cv. grk population with those grapevine-associated viruses tested for is much lower than the average for grapevine cultivars in dalmatia,which reaches about 90% of virus-infected vines (karoglan konti] et al. 2009). this indicated that there had to be some other cause of the symptoms observed in lumbarda vineyards. one of the possible causes for the decline of the lumbarda vineyards might be phloem-restricted prokaryotes called phytoplasmas. phytoplasmas are classified by their 16s ribosomal gene into less then 20 clusters (montano et al. 2001, irpcm 2004, bertaccini and duduk 2009). infected grapevines develop very characteristic symptoms – leafyellowing and curling, aborted clusters or shriveling berries, incomplete lignification of shoots, as well as vein necrosis (constable 2010). diseases known as »grapevine yellows« are caused by phytoplasmas from the groups 16sri-b (aster yellows), 16sri-c (clover phyllody), 16sr-ii (peanut witches' broom), 16sriii (x-disease), 16srv-c and 16srv-d (elm yellows) and 16srxii-a and 16srxii-b (stolbur) (firrao et al. 2005). studies of grapevine yellows started in croatia in the 1950s (panjan 1957), but the first molecular confirmation of the causal agent of the disease – phytoplasma – came later with [ari] et al. (1997). since then, a few studies on phytoplasma occurrence on grapevine in croatian vineyards have been conducted ([eruga et al. 2000; [eruga musi] et al. 2009, 2011a, b; [kori] et al. 1998, 2011), but only one of them included the testing of one indigenous cv. grk vine from lumbarda in kor~ula (mikec et al. 2006). the aim of this research was (i) to confirm phytoplasmas as the causal agent of the grapevine yellows-like symptoms on cv. grk vines, (ii) to determine the disease prevalence at three locations where this cultivar is planted in croatia and (iii) to determine the diversity of the suspected pathogen (phytoplasmas). materials and methods samples of leaf main veins were collected in late summer of 2010 at three locations in croatia where grk grapevines can be found: production vineyards in lumbarda on island kor~ula with 10,023 plants, the national grapevine collection in jazbina near zagreb (six plants) and a mother vineyard in ba{tica near zadar (400 plants) (fig. 1). the disease symptoms were assessed on site and samples for further analysis chosen accordingly: from 11 plants with pronounced symptoms in lumbarda and five plants with mild symptoms in ba{tica. only two plants showed mild symptoms in jazbina, while the rest were asymptom288 acta bot. croat. 72 (2), 2013 je@i] m., karoglan konti] j., preiner d., maleti] e., ]urkovi]-perica m. atic. therefore, in that location, the three asymptomatic plants were also included in the analysis. the procedure for total nucleic acid extraction was performed as previously described by [eruga et al. (2003). dna concentration was determined using nanodrop (themo fisher scientific 2000) and adjusted to 20 ng ml–1 for the polymerase chain reaction (pcr). direct amplification of phytoplasma 16s rdna, was performed with a r16f1/r0 primer pair (lee et al. 1995), and was followed by nested pcr assay, utilizing a r16f2n/r2 primer pair (gundersen and lee 1996) with 0.5 ml from the first pcr reaction used as a template (making the template 50 x diluted in the nested pcr reaction mixture). in all experiments positive (dna isolated from various phytoplasma strains maintained in catharanthus roseus grown in vitro) as well as negative (water and healthy c. roseus dna isolate) controls were used. approximately 5 ml of pcr mixture were analyzed on 1% agarose gel, stained with ethidium bromide and visualized with uv. all experiments were done in triplicates. restriction fragment length polymorphism (rflp) analysis was performed on r16 f2n/r2 primer pair obtained amplicons. besides isolates from lumbarda, ba{tica and jazbina, reference strains of phytoplasmas (maintained in c. roseus plants) were included in rflp analysis: hydb ('candidatus phytoplasma asteris', 16sri-b), kvi (peach x disease, 16sriii-b), fd (flavescence dorée, 16srv-c), gsfy/2 ('ca. p. prunorum', 16srx-b), sa-1 (stolbur, 16srxii-a). using restriction endonucleases msei, alui and kpni, digestion was performed on 200 ng of dna according to manufacturer instructions (fermentas, vilnius, lithuania) and 10 ml of reaction mixture was loaded on 5% polyacrilamide gel electrophoacta bot. croat. 72 (2), 2013 289 phytoplasmas in grapevine fig. 1. geographical locations in croatia from which the grapevine samples were collected. resis (page) vertical gels. ethidium bromide-stained gels were photographed and restriction fragment patterns of phytoplasma isolates from cv. grk were compared with patterns of standard phytoplasma strains. amplicon (obtained with r16f2n/r2 primer pair) from sample 02l was sequenced using macrogen dna sequencing service (macrogen, amsterdam, netherlands). results in lumbarda on kor~ula, 79% of 10,023 vines showed pronounced disease symptoms while 11% had mild symptoms. the rest of the vines were asymptomatic. the vineyard in ba{tica near zadar is much smaller, and only mild grapevine yellows symptoms were observed on eight out of 400 plants. in a vineyard in jazbina near zagreb, which serves as the national collection of grapevine cultivars, only six cv. grk vines are grown and mild symptoms were observed on two vines. 290 acta bot. croat. 72 (2), 2013 je@i] m., karoglan konti] j., preiner d., maleti] e., ]urkovi]-perica m. tab. 1. detection of phytoplasmas belonging to two different ribosomal groups in cv. 'grk' samples collected from three locations in croatia and expressing different symptom intensity. pronounced symptoms are indicated with (++), mild symptoms with (+) and symptomless grapevines with (–) in the third column. fourth and fifth column indicate presence or absence of specific amplicon revealed by agarose gel electrophoresis after the direct and nested pcr. location sample symptoms direct pcr r16f1/r0 nested pcr r16f2n/r2 phytoplasma ribosomal group lumbarda (kor~ula) 01l ++ – + 16sri 02l ++ – + 16sriii 23l ++ – + 16sri 04l ++ – + 16sri 05l ++ – + 16sri 06l ++ – + 16sri 07l ++ – + 16sri 08l ++ – + 16sri 09l ++ – + 16sri 10l ++ – + 16sri 11l ++ – + 16sri jazbina (zagreb) 1j + – + 16sri 2j + – + 16sri 3j – – + 16sri 4j – – + 16sri 5j – – + 16sri ba{tica (zadar) 170 + – + 16sri 289 + – + 16sri 290 + – + 16sri 369 + – + 16sri 370 + – – n/a in the direct pcr, only positive controls produced visible amplicons and therefore nested pcr was performed yielding 20 positive samples out of 21 tested. dna fragments of expected length (1.2 kb) were obtained. in all our experiments, only one sample from ba{tica was consistently negative, despite the mild grapevine yellows-like symptoms observed on that tested vine. on the other hand, phytoplasma was detected in 3 asymptomatic vines from jazbina (tab. 1). in rflp analysis we digested all dna products obtained from the first nested pcr. all rflp patterns were consistent with 16sri phytoplasma group, except for sample 02l (lumbarda) which had a 16sriii-like pattern (fig. 2). the partial sequence obtained for that sample (jq046414) was very similar to the italian 16sriii-b group phytoplasma 16srdna sequence available in the database (hq589194) with only one nucleotide mismatch. there were four nucleotide mismatches between our 16srdna sequence and the sequence of an isolate belonging to16sriii from the usa (af060875) (davis et al. 1998). acta bot. croat. 72 (2), 2013 291 phytoplasmas in grapevine fig. 2. restriction fragment length polymorphism patterns of phytoplasmas detected in croatian vineyards with additional five standard strains as references. amplicons obtained with r16f2n/r2 were digested with alui, kpni and msei restriction enzymes over night and analyzed on 5% polyacrylamide gels. marker jx174 haeiii digest. samples: 02l and 11l (lumbarda), 2j (jazbina), 170 (ba{tica); reference strains: hydb ('candidatus phytoplasma asteris', 16sri-b), kvi (peach x disease, 16sriii-b), fd (flavescence dorée, 16srv-c), gsfy/2 ('ca. p. prunorum', 16srx-b), sa-1 (stolbur, 16srxii-a). discussion phytoplasmas were detected in all locations included in our study, in plants with pronounced and also in plants without any visible disease symptoms. in samples from lumbarda, pronounced symptoms, observed on 79% of the cv. grk vines, were unambiguously connected with phytoplasma infection. such prevalence of the disease on a high number of vines might lead to wine production from this particular cultivar in southern croatia becoming inefficient. even more, in the tested vines sampled from two other locations, the same phytoplasma was detected even in asymptomatic or vines with only mild grapevine yellows-like symptoms. this is the first study of the prevalence of phytoplasmoses on the grapevine cv. grk after the initial report of one vine of this cultivar sampled in lumbarda, which was infected by phytoplasma from 16srxii group (mikec et al. 2006). infections of grapevines with phytoplasmas belonging to three groups 16srxii, 16sri and 16srv were reported in croatia previously (mikec et al. 2006; [eruga et al. 2000; [eruga musi] et al. 2009, 2011a, b; [kori] et al. 1998, 2011). phytoplasma belonging to group 16srv was detected recently in two vineyards in north-western continental croatia, near the slovenian-croatian border ([eruga musi] et al. 2011b); 16srxii was found to be widely distributed, in both continental and littoral croatia while 16sri was detected only sporadically, mostly in southern croatia (mikec et al. 2006), the geographical origin of cv. grk vines. phytoplasmas from this group were reported previously on some other grapevine cultivars in southern littoral croatia, namely in ^ara on island of kor~ula and on the nearby pelje{ac peninsula (mikec et al. 2006), but neither in northern dalmatia (where our samples from ba{tica were collected) nor in jazbina near zagreb. therefore it is an interesting finding that all tested cv. grk vines from three locations were infected by the phytoplasma belonging to the 16sri group. since grapevines from lumbarda were transplanted to ba{tica near zadar and jazbina near zagreb for enological studies, there is a high possibility that grapevines were infected before they were transplanted. another possibility is a novel infection of grk cultivar grapevines, initiated by insect vectors in our research areas. however, in that case infections by the much more widespread 16srxii group phytoplasma would be more probable. yet in jazbina in continental croatia where infections by phytoplasmas belonging to 16srxii were previously repeatedly confirmed on other cultivars ([eruga 2003, [eruga et al. 2000, [eruga musi] et al. 2009), on cv. grk only phytoplasma belonging to 16sri group was found. rflp pattern and sequence of r16f2n/r2 obtained amplicon of one sample affiliated that phytoplasma to a distinctive 16sriii phytoplasma group, rather rare and uncommon on grapevine in europe. phytoplasmas from this group were found to infect grapevines in usa, israel and northern italy (musetti 2008). phytoplasma belonging to 16sriii group was found previously in croatia on a weed plant – chenopodium album near drni{ ([eruga 2003), but this is the first report on vitis vinifera infected with phytoplasma belonging to this particular group. this phytoplasma was found in a single grapevine only in southern croatia, on a geographically small and isolated area. phytoplasmas that belong to ribosomal groups 16sri and 16sriii infect a broad range of plant hosts. therefore, there is a high probability that other plant species in croatia might be infected with these phytoplasmas as well. 292 acta bot. croat. 72 (2), 2013 je@i] m., karoglan konti] j., preiner d., maleti] e., ]urkovi]-perica m. conclusion this study has shown an unusually high prevalence of the 16sri group phytoplasma on cv. grk in all three geographically distant locations where it is planted. this oddity is especially prominent in plants transplanted to the continental croatia where phytoplasmas belonging to the 16srxii group are well established and widespread in vineyards. we also report on the first occurrence of the 16sriii group phytoplasma, rather uncommon in european grapevines. thus far phytoplasmas belonging to three ribosomal groups (16sri, 16sriii and 16srxii) were detected on cv. grk, increasing the total number of phytoplasma groups detected in croatian vineyards to four (16sri, 16sriii, 16srv and 16srxii). acknowledgement this research was supported by croatian ministry of science education and sport, projects no. 119-1191192-1215 and 178-1781844-2758. references bertaccini, a., duduk, b. 2009: phytoplasma and phytoplasma diseases: a review of recent research. phytopatologia mediterranea 48, 355–378. constable, f. e., 2010: phytoplasma epidemiology: grapevines as a model. in: wein traub, p. g., jones p. 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[ari], a., [kori], d., bertaccini, a., vibio, m., murari, e., 1997: molecular detection of phytoplasmas infecting grapevines in slovenia and croatia. proceedings 12 meeting icvg, lisabon, 77–78. [eruga, m., 2003: molecular detection and identification of phytoplasmas in croatian grapevines (vitis vinifera l.) (in croatian with english summary). msc thesis, university of zagreb, zagreb. [eruga, m., ]urkovi]-perica, m., [kori], d., kozina, b., miro[evi], n., [ari], a., bertaccini, a., kraja^i], m., 2000: geographic distribution of bois noir phytoplasma infecting grapevines in croatia. journal of phytopathology 148, 239–242. [eruga, m., [kori], d., kozina, b., mitrev, s., kraja^i], m., ]urkovi] perica, m., 2003: molecular identification of a phytoplasma infecting grapevine in the republic of macedonia. vitis 42, 181–184. [eruga musi], m., [kori], d., budin[^ak, @., kri@anac, i., mikec, i., 2009: survey of phytoplasma diversity in heavily grapevine yellows affected areas of croatia. le progres agricole et viticole hors serie, 206–207. [eruga musi], m., pu[i], p., fabre, a., [kori], d., foissac, x., 2011a: variability of stolbur phytoplasma strains infecting croatian grapevine by multilocus sequence typing. bulletin of insectology 64, 39–40. [eruga musi], m., [kori], d., halu[ka, i., kri@anac, i., plavec, j., mikec, i., 2011b: first report of flavescence dorée -related phytoplasma affecting grapevines in croatia. plant disease 95, 353–353. [kori], d., [ari], a., vibio, m., murari, e., kraja^i], m., bertaccini, a., 1998: molecular identification and seasonal monitoring of phytoplasmas infecting croatian grapevines. vitis 37, 171–175. [kori], d., [eruga musi], m., plavec. j., kri@anac, i., 2011: geographical distribution of 'flavescence dorée' phytoplasmas in croatian grapevines. bulettin of insectology 64, 243–244. 294 acta bot. croat. 72 (2), 2013 je@i] m., karoglan konti] j., preiner d., maleti] e., ]urkovi]-perica m. acta bot. croat. 82 (1), 2023 27 acta bot. croat. 82 (1), 27–33, 2023 coden: abcra 25 doi: 10.37427/botcro-2022-028 issn 0365-0588 eissn 1847-8476 first record of alien naturalized populations of the crop cucurbita moschata (cucurbitaceae) in spain, with remarks on typification status ana juan1, joaquín moreno2*, alejandro terrones1 1 university of alicante, environmental sciences and natural resources, carretera de san vicente s/n, 03690 san vicente del raspeig (alicante), spain 2 miguel hernández university of elche, department of applied biology, avda. universidad s/n. edf. torreblanca, 03202 elche (alicante), spain abstract – as a result of a floristic survey carried out in riparian ecosystems of the south-eastern part of the iberian peninsula (spain), the first report of well-established populations of the alien cultivated plant species cucurbita moschata duchesne for the iberian peninsula is provided here. data about the morphological description (compared to other cucurbita species), certain clarification aspects about the typification status of this name and related synonyms, and the ecological and climatic conditions of the riparian area are given together with an identification key of the cucurbita species to facilitate further identification. the alien status and distribution of c. moschata together with its relatives c. ficifolia, c. pepo and c. maxima are reviewed for the spanish references. this study outlines the first record of a naturalized population of c. moschata in spain, well supported by the stability of the population over the years and in the ecological conditions. finally, detailed ecological data indicate that agricultural activities together with riparian habitatsare starting points and corridors, respectively, for seed dispersal for the process of the invasion of alien plants in the south-eastern iberian peninsula. keywords: alien plants, cucurbita, mediterranean, riparian habitats, spain, typification, xenophyte introduction cucurbita l. (cucurbitaceae) comprises about 12-13 species widely distributed on the american continents, of which five species are cultivated (paris 2016). in europe, the most important cultivated species are c. pepo l., c. maxima duchesne, c. moschata duchesne and c. ficifolia bouché (teppner 2004, henning et al. 2017). tardío et al. (2018) recently included these four species in the spanish list of the traditional crops for agricultural biodiversity, with c. pepo as the main crop species of the genus due to its great commercial importance. in the framework of the flora iberica project, fernandes (2005) reported c. pepo, c. maxima and c. ficifolia as crop species, without any mention of the possible findings of spontaneous specimens of them, in any spanish or portuguese geographical area. in addition, fernandes (2005) did not include the existence, not even as a crop, of c. moschata for the iberian peninsula and balearic islands. according to the euro+med plant base (henning et al. 2017), crops of c. moschata are mostly reported from eastern european countries, though the cultivation of this species is also mentioned for mediterranean countries including spain (tardío et al. 2018) and italy (lust and paris 2016). however, as previously stated by quintero (1981), c. pepo and c. maxima were the most widely cultivated species along the south-eastern iberian territories. probably due to the extensive agricultural use of c. pepo and c. maxima, there are some reports of the existence of non-cultivated subspontaneous individuals of these two species, but always close to their original field cultures (e.g., serra 2007, herrera and campos 2010). there are a couple of mentions of noncultivated individuals of c. moschata, reported to be ephemerals, for portugal and spain (verloove and alves 2016, gómez-bellver et al. 2019), otherwise no specific geographic reference to the presence of this species outside of cultivation (mateo et al. 2015). although species of the genus cucurbita have been considered as invasive alien flora (sanz elorza et al. 2004), any specific geographical indications of * corresponding author e-mail: joaquin.morenoc@umh.es juan a., moreno j., terrones a. 28 acta bot. croat. 82 (1), 2023 their presence, degree of naturalization, abundance and habitat as alien species are remarkably lacking. on the basis of the ongoing study of the alien flora of the river vinalopó (alicante province, south-eastern spain), the fieldwork conducted on this river has recently revealed the presence of a high number of casual and even naturalized alien plants (juan et al. 2019, terrones et al. 2021). the aims of this study are (i) to report the presence of well-established populations of c. moschata outside cultivation, which would correspond to the first record of the species as a naturalized alien population, (ii) to update the presence and alien status of the species of cucurbita in spain, and (iii) to identify ecological conditions that would favour the fruit dispersal and seed germination of these species, especially focused on c. moschata. in addition, the typification of the name c. moschata and related synonyms is reviewed. materials and methods the present study was based on fieldwork carried out during the period 2016-2020 on the river vinalopó (southeastern iberian peninsula, spain). the ecological features of this river dramatically change from its source, in a mountainous area characterized by a high-water quality, to its mouth, located in saline wetlands close to the mediterranean sea. most of the river flows throughout semiarid territories, which causes the salinization of the waters (josé ramón coves, pers. comm.) and even a seasonal total lack of flow in several non-continuous areas. the quality and physical-chemical properties of the waters of the river were described by josé ramón coves (pers. comm.). climate features of the closest meteorological station to the studied area (elda) were obtained from the climate-data online database (https://www.es.climate-data.org), which compiles data from the last 30 years. based on these data, the bioclimatic characteristics of the area followed the classification of rivas-martínez et al. (2001). information on species distribution was based on herbarium specimens from gbif (global biodiversity information facility – www.gbif.org) and literature. morphological features were based on the specialized literature (merrick and bates 1989, nee 1990, lira and rodríguez arévalo 1999, teppner 2004, paris 2016, oecd 2016), together with the observations based on the population found during this research. the collected plant material of c. moschata is preserved in the abh herbarium (thiers 2020), which was used to draw up detailed morphological pictures of this species. in this contribution, an identification key is reported to facilitate further identification. the typification status of c. moschata and related names was updated based on original descriptions and the inspection of available material from various herbaria (f, l, mo, nl, u, wag; thiers 2020). ecological data of the alien cucurbita populations are described, including remarks about the origin, degree of naturalization, habitat and the importance of the plant dispersal processes. results description and typification notes cucurbita moschata duchesne, essai hist. nat. courges: 7 (1786) gymnopetalum calyculatum miq. in fl. ned. ind., eerste bijv. (2): 332 (1861) lectotypus (designated here): label 1: “gymnopetalum ? calyculatum mq, banka, (j. amand)”; label 2: a. cogniaux monogr. cucurb. h “cucurbita moschata duch.” (probably cogniaux’s handwriting); u (u0001457!). an annual species characterized by softly hairy, nonto shallowly 3-5 lobed leaves, 15-30 × 20-40 cm, without or with whitish blotches; tendrils bifids or trifids; sepals free, mostly linear, but sometimes lanceolate together with flowers with broadened apices (fig. 1); corolla yellow-orange, 8-12 cm long; fruiting peduncle 5-ribbed thickened and dilated or cylindrical, 5-10 cm long, widely flat at the fruit attachment; fruits covered by a wipeable waxy layer, dumbbell and solid green or pyriform and cream-coloured with light green longitudinal reticulate mottled stripes (usually in bands), up to 35 cm length; seeds elliptical, 8-15 × 4-7 mm, with a rounded marginal bulge, margin mostly with a colour shade slightly different from the surface, marginal wings developed very strongly. most of these morphological features correspond to the main features used to distinguish it from the other domesticated species of this genus, c. ficifolia, c. maxima and c. pepo (tab. 1). regarding the protologue of c. moschata, paris (2000) already stated that this name was validly published by duchesne (1786) in the essai sur l’historie naturelle des courges, and hence, duchesne was solely responsibility for the authority. on the gbif database (www.gbif.org), three different entries were found related to the type specimens of the name c. moschata. firstly, the mention of the type specimen of this name held at the missouri botanical garden (mo1722018) should be considered a mistake, since no voucher is apparently held at mo only at f, lpb and ny (teisher, pers. comm.). the voucher information corresponds to the collection of t.j. killien (nº 1267) made in bolivia during 1989, but no publication has been found about this likely typification and, at least, the voucher at f (barcode f2013050!; bolivia, santa cruz, cordillera, tatarenda, 30 km s of rio grande on road to camiri, 19º08’s 63º15’w, 700 m, first range of andes with semi-deciduous forest and slash and burn agriculture, soils generally sandy, tim killeen 1267, 16 oct 1985) does not bear any identification of type material. therefore, the typification of the name c. moschata is still an uncompleted task, as with many other cultivated species, which were initially described without any direct reference to a herbarium voucher or collection. the second mention concerns the accepted synonym gymnopetalum calyculatum miq., the description of which (miquel 1860) was based on material collected from bangka by j. amann (pseudonym of w.s. kurz, see van steeniscucurbita moschata, naturalized populations in spain acta bot. croat. 82 (1), 2023 29 kruseman and van steenis 1950). de wilde and duyfjes (2010) designated a voucher kept at the herbarium u (barcode u0001457!) as the holotype. however, the use of the term holotype by these authors cannot be adequate, as miquel (1860) only referred to amann’s gathering activity but not to a specific sheet. hence, there is no certainty that fig. 1. cucurbita moschata duchesne. a – habit, b – flower (side view), c – flower (front view), d – mature fruit (drawing by joaquín moreno). tab. 1. main morphological features differentiating the four species of cucurbita reported in spain (based on lira 1995, lira and rodríguez arévalo 1999, teppner 2004). c. ficifolia c. maxima c. moschata c. pepo habit perennial annual annual annual leaves lobed not lobed/ shallowly lobed not lobed/ shallowly lobed shallowly to deeply lobed segment leaves rounded rounded acute acute indument with short glandular hairs stiff-haired/ hispid soft-haired spiculate sepals linear, apex non broadened linear, apex non broadened linear or with apex often broadened like leaves linear, apex non broadened seed color dark brown to black orange or white tan to brown pale tan fruit peduncle ribbed, moderately broadened at attachment cylindrical, non broadened at attachment ribbed, at attachment widely broadened (flat) ribbed, slightly, broadened at attachment juan a., moreno j., terrones a. 30 acta bot. croat. 82 (1), 2023 other duplicate material was not used to prepare the description of this name by the original author (icn, art. 9.1), though no additional specimens were found at nl, l, u and wag (m. scherrenberg, pers. comm.). therefore, the voucher u0001457 should be considered as the lectotype of the name g. calyculatum. finally, the third reference corresponds to a specimen of the name c. sulcata blanco kept at l (barcode l0585284!; cucurbita sulcata, merrill, species blancoanae nº 152, labelled as neotype by j.k. veldkamp 2/03, labelled as c. moschata by w.j.j.o de wilde & duyfjes, 2011), which was selected as neotype by veldkamp on the specimen; however, veldkamp never published his designation, which is thus ineffective. the name c. sulcata is currently considered a synonym of the species c. maxima, but the identification of this particular voucher was corrected to c. moschata on the specimen by de wilde and duyfjes. to avoid the effect of destabilizing the nomenclature of the name c. sulcata, an effective typification should be based on a specimen whose identification corresponds to the original description (blanco 1837). habitat and populations the populations of c. moschata were found growing in the central part of the course of the river vinalopó (named the middle vinalopó district), which runs across the inner central part of alicante province (spain), crossing semiarid and arid areas. at this geographical zone of the river, the watercourse is not continuous, and it can remain totally dry for long periods of time, even in excess of one year. this ecological peculiarity, added to the climatic characteristics of the area, contributes to soil salinity. four scattered populations, with up to 10 specimens each, of c. moschata have been discovered along five kilometres of this river basin, most of them growing clearly away from the direct influence of the main course of the water. the largest population, about 10 individuals, appeared on sandy soils on the upper part of the terrace of the river. at a distance of approximately two kilometres downstream, another well-established population with 5 individuals grew close to secondary dry channels of this river, characterized by gravel and sandy soils. the other two populations, formed by 2–3 individuals, and some isolated individuals of c. moschata were placed on the shallow area of meander scars and along the edges of the riverbed, both upstream and downstream in reference to the largest populations. the first observations of them were noted in 2016, when only the two above-mentioned largest populations were initially identified with five and two reproductive individuals, respectively. over the years these populations of c. moschata have developed autonomously, with increasing numbers of individuals, while new subpopulations or isolated individuals have been found far from them, being fully reproductive. although the size of the population may be different from year to year, the annual appearance of new specimens seems to be entirely independent, based on the development of functional seeds, which seem to germinate under the ecological conditions of this area. in addition, no crops were found close to the observed populations. the populations of c. moschata, in general, appear intermixed with wild shrubby and grassland vegetation (e.g., atriplex halimus l., cynodon dactylon (l.) pers., tamarix gallica l., together with other naturalized alien species along the river, such as physalis peruviana l., stenotaphrum secundatum (walt.) kuntze, solanum lycopersicum l. and s. sisymbriifolium lam. independently of the size and location of the population, the observed individuals were fully reproductive, and well-developed mature fruits were easily found. climate and watercourse features on the basis of the recent studies of josé ramón coves (pers. comm.), the main features of the water of the river vinalopó along the studied area are an average water temperature of about 20 °c, with an electric conductivity ranging from 2.8 to 5.9 ms cm–1, ph 7.66 (7.3–7.8) and an average nitrate concentration of 75 mg l–1 (25–180 mg l–1). the weather conditions of the studied area are characterized by typically mediterranean climate, with remarkably dry summers. the annual average rainfall is 345 mm, with maximum values in september (43 mm) and minimum in july (6 mm). the average monthly values of temperatures are always above 0 °c, january being the coldest month with minimum average temperatures of 7.7 °c whereas the maximum values were registered in both july and august (mean values of 25.1 °c and 24.9 °c, respectively). according to these rainfall and temperature data, the studied area corresponds truly to a semiarid area belonging to an inferior mesomediterranean belt. naturalized specimens observed of cucurbita moschata hs, alicante: elda, river vinalopó, 38°27’09’’n 0°48’12’’w, 337 m, 12 nov 2017, a. juan aj117 (abh 82475). elda, river vinalopó, left side of the river, upper part of the terrace of the river, 38°27’06’’n 0°48’13’’w, 335 m, 20 nov 2017, a. juan & i. juan aj118 (abh 82476). elda, river vinalopó, upper part of the terrace of the river, 38°27’04’’n 0°48’12’’w, 335 m, 14 sept 2018, a. juan, j. moreno & a. terrones (abh 82477). elda, river vinalopó, secondary dry channel, 38°26’51’’n 0°48’13’’w, 330 m, 27 october 2018, a. juan, j. moreno & a. terrones (v.v.). elda, river vinalopó, terrace of the river, 38°28’16’’n 0°48’15’’w, 370 m, 24 nov 2019, a. juan, j. moreno & a. terrones (v.v.). distribution of the cucurbita moschata in spain specific geographic references to cucurbita moschata as crop have been reported only from the northeast of the iberian peninsula (barcelona province), where it also behaves as an occasional alien growing on the margin of the river (gómez-bellver et al. 2019). the newly found c. moschata populations in alicante are very distant. the populations cucurbita moschata, naturalized populations in spain acta bot. croat. 82 (1), 2023 31 of c. moschata found in alicante province have been documented since 2016 and most recently have been observed to be composed of at least 25 reproductive individuals. discussion morphological aspects the collected specimens of cucurbita moschata show morphological features that fully fit the typical diagnostic characteristics of the species (teppner 2004, de wilde and duyfjes 2010), though certain variabilities related to the leaves, sepals and fruits were detected among the studied specimens. the leaves were shallowly lobed, and either without or with whitish blotches. most of the samples were characterized by the unique presence of linear sepals, but some specimens showed flowers with lanceolate sepals together with flowers with broadened sepals. two types of fruits were observed: (i) dumbbell and solid green with a fruiting pedicel widely flat at the fruit attachment, and (ii) more rarely, pyriform and cream-coloured with light green longitudinally reticulated mottled stripes (usually in bands) with 5-ribbed thickened and dilated peduncles at the fruit attachment. the observed minor morphological variations are likely derived from the existence of numerous cultivars of this species, which vary greatly in fruit shape and colour (lira 1995, teppner 2004, de wilde and duyfjes 2006). the main morphological characters distinguishing c. moschata plants or fruits from the closely related species, c. pepo and c. maxima, are basically related to the indument, leaves and fruit stalk (lira and rodríguez arévalo 1999, teppner 2004, tab. 1). while c. moschata is a soft-haired plant with nonto shallowly lobed leaves, smoothly grooved stems and a hard, smoothly angled fruit stalk widened at the apex, c. maxima is characterized by hispid, unlobed (or slightly lobed) leaves and rounded stems, with the fruit stalk soft and rounded, not enlarged at the apex. finally, c. pepo is typically spiculate with grooved stems and palmately lobed, often deeply cut and prickly leaves, with a hard and markedly angular fruit stalk sometimes slightly widened at the apex. the following dichotomous key is based on fernandes (2005) to which c. moschata was added to facilitate the plant identification: 1. plants with short glandular hairs, generally climbing; leaves 3-5-lobed, with rounded or obtuse segments; fruit with white flesh, seeds black . . . . . . . . . . . . . c. ficifolia – plants hispid with short, stiff hairs, prostrate or climbing; leaves from entire to clearly lobed, with triangular segments; fruit with pale or yellow or yellow-orange flesh, seeds white or orange . . . . . . . . . . . . . . . . . . . . . . 2 2. leaves entire or slightly lobed; rounded stems; fruit stalk subcylindrical, rounded, soft . . . . . . . . . . . c. maxima – leaves shallowly to deeply lobed; grooved stems; fruit stalk different . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. plant prickly, spiculate; leaves palmate, shallowly to deeply lobed; fruit stalk notably angled, slightly broadened at attachment on the fruit apex; fruit with lightcoloured yellow to orange flesh; seed margin with similar colour and texture as the surface . . . . . . . . . c. pepo – plant softly hairy; leaves shallowly lobed; fruit stalk slightly angled, mostly abruptly broadened at attachment on the fruit apex; fruit with orange flesh; seed margin with a different colour and texture than the surface . . c. moschata distribution and alien status of c. moschata and related species the presence of c. moschata out of cultivation is quite scarce not only around the mediterranean basin but also around the european continent. ardenghi and mossini (2015) have reported the presence of two non-cultivated populations in italy, located along an irrigation canal among rice fields, together with scattered vegetable refuse. according to these authors, the populations almost certainly grow from seeds rejected as food waste, since the observed seedlings likely derived from fruits produced in previous years. in addition, occasional reports of c. moschata from northern european territories have been also indicated (jonsell and karlsson 2010, verloove 2018). jonsell and karlsson (2010) stated the doubtful mention of this taxon in sweden during the 1950s and no new mention is available, and, verloove (2022) reported it on a dump in belgium. in spain, gómez-bellver et al. (2019) recently reported alien plants of c. moschata growing on the margin of the river llobregat. based on richardson et al. (2000), these mentions might be considered as ephemeral casual alien plants. contrary to these previous reports, our direct field observations yielded the lack of any vegetable crops close to this stretch of the river, including cultivated plants of c. moschata. therefore, the existence of these non-cultivated populations of c. moschata might have originated from seeds of old discarded agricultural waste in some remote area upstream of the river, as neither farming litter nor current cultivation has been observed close to the studied area. the existence of a large number of individuals cut off from the riverbed together with their repetitive presence during several years would denote a certain population stability. consequently, the presence of c. moschata populations along the basin of the river vinalopó, and their subsequent propagation over the years, would be nowadays considered as autonomous and well established, and not directly bound to any current agricultural activity. although most free-living plants of the cultivated species of cucurbita are reported as casual offspring from nearby fields, on the basis on our observations we consider the described alicante populations of c. moschata as the first ones to be naturalized in spain. regarding closely related species (c. ficifolia, c. maxima and c. pepo), only two mentions (barcelona and valencia, based on herbarium specimens) were reported for c. ficifolia, growing in fields of carob trees. conversely, the species c. pepo and c. maxima both have been largely reported for juan a., moreno j., terrones a. 32 acta bot. croat. 82 (1), 2023 different spanish provinces (e.g., serra 2007, sanz elorza et al. 2009, and more than 250 occurrences on gbif), including the balearic and the canary islands. most of the observations of these three species based on the labels of vouchers and existing literature reported the presence of scarce specimens out of the crops (e.g., serra 2007, gómez-mercado 2009, herrera and campos 2010, among others). they were considered as casual alien plants as they typically grew in disturbed habitats near crop fields. ecological aspects the existence of numerous small parcels of farmland on which are grown tomatoes, broad beans, peas, onions, and melons, among others things, basically for subsistence, is fairly extensive along the vinalopó valley, though they were not the main agricultural use for this territory (juárez 2010). nowadays, the discovered naturalized populations of c. moschata did not grow close to any pumpkin crops, and therefore, the river would be the main method of seed dispersal along the vinalopó valley. in fact, riparian habitats can act as ‘conveyor belts’ for propagules (richardson et al. 2007), and they might be important corridors for seed dispersal, both for native and alien species (pyšek and prach 1995, stohlgren et al. 1998). many alien species spread along watercourses (richardson et al. 2000), and their invasion success largely depends on their dispersal ability (pyšek and prach 1995). ecological conditions also play an important role on the entrance into and stabilization of alien plant populations along watercourses (iamonico 2021), especially for annual cucurbita species (lira 1995, oecd 2016). cucurbita fruits can be buoyant in the watercourse (oecd 2016), and hence, the river would represent a potential means of long seed dispersal. under the appropriate environmental conditions, including no severe frost, seasonably warm temperatures and well-drained soils (oecd 2016), the germination of seeds and development of offspring of c.  moschata would be favoured, even in the existence of relative drought. the observed climatic conditions of the studied area coincided with the ecological requirements, although the water quality of this river is slightly alkaline and saline (josé ramón coves, pers. comm.). in fact, a high number of noncultivated individuals of solanum lycopersicum (solanaceae) belonging to different cultivars, including cherry and plum tomatoes, appear clearly naturalized along the vinalopó river channel and its terrace, comprising well-established populations without direct intervention by humans (pers. obs.). the number of individuals of s. lycopersicum is quite high and their presence is so frequent that this species already coexists with the riparian natural vegetation of the river, and the long-time permanence of these individuals would be autonomous and even might be considered as invasive. similarly, other alien non-agricultural species, as drosanthemum hispidum (l.) schwanthes, ulmus pumila l., rumex cristatus dc., cotula coronopifolia l. or datura inoxia mill., have also been observed and their populations are easily found along the river vinalopó ( serra 2016, juan et al. 2019). among them, the species d. hispidum clearly shows an invasive behaviour, being the dominant species along the studied upper fluvial terraces of the river vinalopó and close slopes (pers. obs.). conclusions agricultural activities are confirmed again as a starting point for the process of naturalization of alien plants, which together with the presence of a river greatly favours the dispersion of these alien plants. conversely to the casual alien populations of cucurbita pepo, c. ficifolia and c. maxima, the herein identified populations of c. moschata are able to reproduce regularly without any human activities and they are well-established along the central area of the river vinalopó (southern iberian peninsula, spain), where no regular crops of this species have been located. therefore, the non-cultivated spanish populations of c. moschata are catalogued as naturalized alien populations, which would be the first reference for western mediterranean countries. nevertheless, further investigations are needed to identify new possible localities of this taxon, both upstream and downstream of the sites discussed. acknowledgments thanks to dr. marnel scherrenberg (collector manager of the naturalis biodiversity center) for his help about the amann’s specimens kept at the herbaria l, nl, u and wag, and to dr. jordan k. teisher (curator and director of the herbarium missouri botanical garden) for his comments about the material kept at mo, and their suggestions given. finally, we want to thank the two anonymous reviewers for their interesting comments and suggestions to improve the manuscript. no specific funding was obtained for this study. references ardenghi, n.m.g., mossini, s., 2015: cucurbitaceae. in: raabstraube, e. von, raus, th. 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and new contributions. phyton 44, 245– 308. terrones, a., moreno, j., juan, a., 2021: dna barcoding supports an easier identification of alien plants: the case of the genus physalis (solanaceae) in the iberian peninsula (spain). annali di botanica 11, 105–120. thiers, b., 2020: index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden’s virtual herbarium. retrieved 2022 from http://sweetgum.nybg.org/science/ih/. van steenis-kruseman, m.j., van steenis, c.g.g.j., 1950: malaysian plant collectors and collections: being a cyclopaedia of botanical exploration in malaysia and a guide to the concerned literature up to the year 1950. flora malesiana series 1. spermatophyta, 1, 2–639. verloove, f., 2018: cucurbita moschata. in: manual of the alien plants of belgium. botanic garden of meise, belgium. retrieved january 2, 2022 from https://alienplantsbelgium.be. verloove, f., alves, p., 2016: new vascular plant records for the western part of the iberian peninsula (portugal and spain). folia botanica extramadurensis 10, 5–23. opce-str.vp acta bot. croat. 70 (2), 209–214, 2011 coden: abcra 25 issn 0365-0588 diversity and distribution of the dinoflagellates brachidinium, asterodinium and microceratium (brachidiniales, dinophyceae) in the open mediterranean sea fernando gómez* instituto cavanilles de biodiversidad y biología evolutiva, universidad de valencia, po box 22085, 46071 valencia, spain abstract – brachidiniacean dinoflagellates have been investigated in the open waters of the mediterranean sea, along a transect from the south of france to the south of cyprus (20 june–18 july 2008). brachidinium and karenia papilionacea often co-occurred, b. capitatum predominating in the surface waters. the highest abundance of brachidinium were found in the upper 25 m in the western mediterranean with a maximum (24 cells l–1) at a depth of 5 m in the balearic sea. asterodinium (up to 4 cells l–1) was recorded below of deep chlorophyll maxima. the genus microceratium, only known from the tropical indo-pacific region, is reported for the first time in the mediterranean sea. microceratium was found below 100 m in the eastern mediterranean sea, with the highest abundance of 8 cells l–1 at 125 m depth, in the levantine basin. this study also illustrates for the first time specimens under the division of brachidinium and microceratium. this first occurrence of microceratium in the mediterranean sea should be considered an indicator of climate warming. however, it should not be considered a non-indigenous taxon. microceratium is the 'tropical morphotype', the adaptation of a local species (a life stage of karenia – brachidinium – asterodinium) to the tropical environmental conditions that prevail in summer in the open mediterranean sea. key words: biodiversity, brachydinium, brachydiniales, dinophyta, introduced species, invasive phytoplankton, karenia papilionacea, non-indigenous taxa. introduction the order bachidiniales encompasses a group of highly flattened unarmoured dinoflagellates with elongate extensions radiating from the episome and hyposome. brachidinium f.j.r. taylor has four elongate extensions radiating from the hyposome and an apical process on the episome (taylor 1963). asterodinium sournia differs from brachidinium acta bot. croat. 70 (2), 2011 209 * corresponding author, email: fernando.gomez@fitoplancton.com copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\gomez.vp 9. rujan 2011 10:26:03 color profile: disabled composite 150 lpi at 45 degrees in having two elongate extensions radiating from the hyposome, and one central and other two lateral extensions from the episome. microceratium sournia has only one central extension in the episome and two extensions from the hyposome (sournia 1972a, b). gómez et al. (2005) based on light and scanning electron microscopy revealed the morphological similarities between brachidinium and asterodinium and species of the genus karenia g. hansen et moestrup, especially with k. papilionacea a. j. haywood et k. a. steidinger (haywood et al. 2004). these taxa coincided in distinctive morphological characters such as the straight apical groove, cingulum-sulcus juncture, prominent nucleus in the left hyposome, numerous yellow-green chloroplasts, among other characters. the morphological similarity and the occurrence of intermediate stages between these genera suggested that asterodinium, brachidinium, microceratium and karenia papilionacea constitute a single or several closely related species (gómez 2006). species with high morphological versatility are able to project body extensions as an adaptation to environmental conditions. the hypothesis of a phylogenetic relation between these taxa remains to be tested with molecular methods. all the species of brachidiniales were described from the tropical indian ocean (taylor 1963, sournia 1972a, b). within the context of global warming, the expansion of tropical species is of especial interest as biological indicators of the changes in the marine ecosystem. about 700 species dinoflagellates have been reported in the mediterranean sea including ca 90% of the dinoflagellate genera of the world's oceans (gómez 2003, 2005). despite the historical tradition of taxonomic studies, some genera remain unreported in the mediterranean basin. this study describes the diversity and distribution of bachidiniacean dinoflagellates in the open waters of mediterranean sea. the genus microceratium is reported for the first time, and the first illustration of dividing cells of brachidinium and microceratium is given. materials and methods samples were collected during the boum (biogeochemistry from the oligotrophic to the ultra-oligotrophic mediterranean) cruise on board r/v l'atalante from the south of france to the south of cyprus (20 june–18 july 2008) (fig. 1) (for details see: http://www. biogeosciences-discuss.net/special_issue63.html). seawater samples were collected by niskin bottles from 30 stations. at each station 6 depths were sampled between 5 and 125 m and an additional sample at 250 m depth. these were preserved with acid lugol's solu210 acta bot. croat. 70 (2), 2011 gómez f. fig. 1. map of the station locations in the mediterranean sea during the boum cruise in june–july 2008. u:\acta botanica\acta-botan 2-11\gomez.vp 9. rujan 2011 10:26:03 color profile: disabled composite 150 lpi at 45 degrees tion and stored at 5 °c. samples of 500 ml were concentrated via sedimentation in glass cylinders. the top 450 ml of sample was slowly siphoned off with small-bore tubing during 6 days. the remaining 50 ml of concentrate, representing 500 ml whole water, was then settled in a composite settling chamber during one day. the sample was examined at 100 × magnification with a nikon inverted microscope (nikon eclipse te200) and the specimens were photographed with a digital camera (nikon coolpix e995). results a total of 50 specimens of brachidinium were observed from the 212 samples analysed. the highest abundance was recorded in the surface waters (0–25 m depth) of the balearic (up 24 cells l–1) and tyrrhenian seas. the hydrological and biochemical conditions during the cruise are detailed at: http://www.biogeosciences-discuss.net/special_issue63.html. brachidinium and karenia coincided in the distribution, with a wider vertical distribution of karenia (0–50 m depth) (figs. 2, 3). the figure 3o illustrates for the first time dividing cells of brachidinium that were collected from the tyrrhenian sea at a depth of 5 m. in all the known brachidiniaceans, including k. papilionacea, the nucleus (dark oval area in the lugol-fixed specimens) was invariably located in the left hyposome. in the recently divided specimens, the right extension of the hyposome of one of the daughter cells contacted with the hyposome of the other daughter cell. the ventral side of the daughter cell was opposed to the dorsal side of another cell (fig. 3o). ten specimens of asterodinium were recorded (figs. 4a–g). the highest abundance (4 cells l–1) was recorded at 100 m depth in the tyrrhenian sea. the records were higher in the western mediterranean sea. all the records were below 100 m depth, with the exception of one specimen collected at 50 m depth (fig. 2). acta bot. croat. 70 (2), 2011 211 brachidiniales in the mediterranean sea fig. 2. distribution of temperature, salinity, density, fluorescence and abundance of brachidiniaceans in the open mediterranean sea. a – temperature (°c), b – salinity, c – fluorescence (relative units) and density, st, d – karenia spp., e – brachidinium, f – asterodinium and microceratium, the latter indicated by filled circles. u:\acta botanica\acta-botan 2-11\gomez.vp 9. rujan 2011 10:26:04 color profile: disabled composite 150 lpi at 45 degrees a total of 13 specimens of microceratium were found (figs. 4h–r). these records can be grouped in two forms, one with shorter extensions (fig. 4h–l) and other forms with longer extensions (figs. 4m–r). all the specimens were encountered in the eastern mediterranean (fig. 2). the highest abundance (8 cells l–1) was recorded at a depth 125 m in the levantine basin. a couple of recently divided specimens were observed (fig. 4k). one of the daughter cells lacked the apical extension. this shows that the central extension is formed after the cell division. the position of the nucleus was similar that in the divided cells of brachidinium. the dorsal side of one daughter cell was opposed to the ventral side of the other daughter cell (fig. 4k). discussion previous studies based on ultrastructural similarities (gómez et al. 2005) and the occurrence of intermediate stages (gómez 2006) suggested a close relation, if not the conspecificity, of karenia papilionacea and the genera brachidinium and asterodinium. in this study, we found specimens of asterodinium lacking one of the antapical extensions (fig. 4a–b) or specimens of microceratium with a short left antapical extension (fig. 4o). these observations supported the view of the retraction or regeneration of the body extensions. in contrast to the antapical or lateral extensions, the central extension of the episome has a different morphology because it harbours the straight apical groove (gómez et al. 2005). figure 4k shows two daughter cells after a recent division, with one the daughter cells lacking the apical extension. this evidences the change of the cell contour from karenia into the morphologies of asterodinium/microceratium. the number of body ex212 acta bot. croat. 70 (2), 2011 gómez f. fig. 3. photomicrographs of karenia spp. (a–h), and brachidinium (i–u), bright field optics. a–e – specimens of karenia, d – karenia bidigitata, e – an unknown species. f–g – karenia papilionacea, o – brachidinium after cell division. scale bar: 20 mm. u:\acta botanica\acta-botan 2-11\gomez.vp 9. rujan 2011 10:26:06 color profile: disabled composite 150 lpi at 45 degrees tensions, which has been used for the generic separation of the brachidiniales, has a rare diagnostic value. the western mediterranean sea has been historically one of the most investigated regions for phytoplankton studies. the first record of asterodinium in the mediterranean sea was considered to be a biological indicator of the progressive warming of the mediterranean sea (gómez and claustre 2003, lejeusne et al. 2009). asterodinium was described from the tropical indian ocean (sournia 1972a, b). for this reason, asterodinium was further listed as an alien or invasive species in the mediterranean sea (zenetos et al. 2006). as it happened with asterodinium, this first occurrence of microceratium in the mediterranean sea could be considered an indicator of global warming. however, it should not be considered as a non-indigenous or exotic taxon. microceratium is the 'tropical morphotype' of a local species that appears in the mediterranean when environmental conditions resemble those in tropical waters. microceratium is another of the life stages of the morphotypes karenia – brachidinium – asterodinium that appears under warm and highly stratified conditions. microceratium is acta bot. croat. 70 (2), 2011 213 brachidiniales in the mediterranean sea fig. 4. photomicrographs of asterodinium (a–g) and microceratium (h–s), bright field optics. note the lack of the left antapical extensions in asterodinium (a–b). h–l – specimens of microceratium with short extensions. k – two cells after the division. note the lack of the apical extension in one of the daughter cells. m–s – specimens of microceratium with long extensions. o – note the scarcely developed left antapical extension. scale bar: 20 mm. u:\acta botanica\acta-botan 2-11\gomez.vp 9. rujan 2011 10:26:09 color profile: disabled composite 150 lpi at 45 degrees a biological indicator of 'tropicalization', an adaptation of a local species to the near tropical environmental conditions that prevail in summer in the open mediterranean sea. acknowledgements this research has been supported by the contract jci-2010-08492 of the ministerio español de ciencia y tecnologia. i thank k. leblanc and v. barthaux for the boum samples. this is a contribution of the boum (biogeochemistry from the oligotrophic to the ultraoligotrophic mediterranean) project of the french national lefe-cyber program and of the european ip sesame. references gómez, f. 2003: checklist of mediterranean free-living dinoflagellates. botanica marina 46, 215–242. gómez, f., claustre, h., 2003: the genus asterodinium (dinophyceae) as a possible biological indicator of warming in the western mediterranean sea. journal of the marine biological association of united kingdom 83, 173–174. gómez, f., 2005: a list of dinoflagellates in the world's oceans. acta botanica croatica 64, 1–82. gómez, f., nagahama, y., takayama, h., furuya, k., 2005: is karenia a synonym of asterodinium-brachidinium? (gymnodiniales, dinophyceae). acta botanica croatica 64, 263–274. gómez, f., 2006: the dinoflagellate genera brachidinium, asterodinium, microceratium and karenia in the open se pacific ocean. algae 21, 445–542. haywood, a. j., steidinger, k. a., truby, e. w., bergquist, p. r., bergquist, p. l., adamson, j., mackenzie, l., 2004: comparative morphology and molecular phylogenetic analysis of three new species of the genus karenia (dinophyceae) from new zealand. journal of phycology 40, 165–179. lejeusne, c., chevaldonné, p., pergent-martini, c., boudouresque, c. f., pérez, t. 2009: climate change effects on a miniature ocean: the highly diverse, highly impacted mediterranean sea. trends in ecology and evolution 25, 250–260. sournia, a., 1972a: quatre nouveaux dinoflagellés du plancton marin. phycologia 11, 71–74. sournia, a., 1972b: une période de poussées phytoplanctoniques prés de nosy-bé (madagascar) en 1971. espèces rares ou nouvelles du phytoplancton. cahiers o.r.s.t.o.m., série océanographique 10, 151–159. taylor, f. j. r., 1963: brachydinium, a new genus of the dinococcales from the indian ocean. journal of south africa botany 29, 75–78. zenetos, a., zibrowius, h., cinar, m. e., harmelin, j. g., furnari, g., andaloro, f., pancucci, m. a., streftaris, n., bellou, n., 2006: annotated list of alien marine species in the mediterranean with emphasis on worst invasive species. mediterranean marine science 6, 63–118. 214 acta bot. croat. 70 (2), 2011 gómez f. u:\acta botanica\acta-botan 2-11\gomez.vp 9. rujan 2011 10:26:09 color profile: disabled composite 150 lpi at 45 degrees 626 kaya et al.vp acta bot. croat. 72 (1), 157–168, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 regulation of growth and some key physiological processes in salt-stressed maize (zea mays l.) plants by exogenous application of asparagine and glycerol cengiz kaya1*, salih aydemir1, osman sonmez1, muhammed ashraf2, murat dikilitas3 1 harran university, agriculture faculty, soil science and plant nutrition department, sanliurfa, turkey 2 university of agriculture, department of botany, faisalabad, pakistan 3 harran university, agriculture faculty, plant protection department, sanliurfa, turkey abstract – maize seedlings were subjected to concentrations of 0 and 100 mm of nacl in hoagland’s nutrient solution medium in plastic pots containing perlite. two levels of asparagine (5 and 10 mm) and glycerol (20 and 40 mm) were sprayed onto the leaves of maize seedlings 10 days after germination. saline stress caused considerable decline in total dry mass, chlorophyll content and relative water content in the maize plants. it increased the activities of superoxide dismutase, catalase and polyphenol oxidase as well as electrolyte leakage, but did not alter the activity of non-specific peroxidise. foliar application of asparagine or glycerol was found to be effective in checking shoot growth inhibition under nacl stress. exogenously applied asparagine or glycerol reduced superoxide dismutase, non-specific peroxidase and polyphenol oxidase activities in salt-stressed maize plants compared to those not treated with these organic compounds. salinity increased na+ contents but reduced those of k+, ca2+ and p in the roots of the used genotype of maize. foliar application of asparagine or glycerol increased the contents of k+, ca2+ and p, but it reduced that of na+ in salt-stressed maize plants as compared to those of the salt-stressed plants not supplied with glycerol or asparagine. glycerol was more effective than asparagine in improving salinity tolerance of maize plants in terms of growth and physiological attributes measured in the present study. keywords: asparagine, corn, glycerol, maize, salinity tolerance abbreviations: asn – asparagine, cat – catalase, g – glycerol, ppo – polyphenol oxidase, rwc – relative water content, sod – superoxide dismutase acta bot. croat. 72 (1), 2013 157 * corresponding author, e-mail: c_kaya70@yahoo.com copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:41 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees introduction soil salinity limits agricultural productivity, particularly in arid and semi-arid regions of the world (yamaguchi and blumdwald 2005, ashraf et al. 2008; ashraf 2009). excessive amounts of soluble salts in soil decrease the osmotic potential of the soil solution which impairs uptake of water and nutrients thereby causing ion imbalance and toxicity in plants, while disturbing a variety of other key physiological and biochemical phenomena (läuchli and grattan 2007). the tolerance mechanisms plants employ to offset the adverse effects of saline stress have been extensively studied in the past (greenway and munns 1980, cushman et al. 1990). one of the key plant responses to salinity stress is the synthesis and accumulation of compatible organic osmolytes including quaternary ammonium compounds (storey and wyn jones 1977, ashraf and foolad 2007), amino acids the most promising of which are proline (jager and meyer 1977, fougère et al. 1991, thomas et al. 1992), polyols such as sorbitol and mannitol (cheeseman 1988, stoop et al. 1996), and organic acids (albert and popp 1977), etc.. all these organic osmolytes are known to play a vital role in osmotic adjustment, a phenomenon which enables a plant cell to maintain turgor. in plants, asparagine, one of the most common amides, has been reported to be the primary source of n for protein synthesis, particularly in actively growing tissues (brouquisse et al. 1992). it is synthesized from aspartate and glutamine by the action of asparagine synthase in germinating seeds, where n released as a result of protein disintegration is transferred to aspartate, or in roots where different forms of n such as nitrates or dinitrogen are utilized (sieciechowicz et al. 1988). some reports show that the synthesis of most of the amides including asparagine, is up-regulated by some environmental cues (rabe 1990, chaffei et al. 2004). asparagine accumulation in plants in response to environmental adversaries could be an ammonium detoxification mechanism and a means to stock up n when protein synthesis is impaired in plants due to stressful environments (herrera-rodriguez et al. 2007). one of the most striking biochemical events taking place in plants exposed to adverse environmental factors is the generation of reactive oxygen species (dionisio-sese and tobita 1998, ashraf 2009). these reactive oxygen species are known to perturb normal metabolism by causing considerable damage to nucleic acids, lipids, proteins (davies 1987). however, plants possess a variety of antioxidant enzymes which can easily counteract ros. of these antioxidative enzymes, superoxide dismutase (sod) is known to scavenge o2 – thereby producing h2o2 and o2. the hydrogen peroxide (h2o2) so produced can be scavenged by various peroxidases (pod) (dionisio-sese and tobita 1998). polyphenol oxidase (ppo) is usually used as a molecular marker for ensuring the adequacy of high temperature treatment of fruit purees (williams et al. 1986). however, it also acts as a potential selection criterion for salt tolerance for example, while appraising the effect of salinity on senna (agarwal and pandey 2004) and bean seedlings (demir and kocaliskan 2001) ppo was found to be a good indicator of salinity tolerance in these plants. very few reports can be found in the literature on the effect of exogenous application of glycerol or asparagine on growth of plants under salt stress. for example, in ricinus communis presoaking seed treatment with glycerol or aspartic acid, one of the precursors of asparagine synthesis, improved growth and secondary products (ali et al. 2008), and in rice seedlings, exogenous application of d-asparagine offset the inhibitory effects of salinity on shoot growth, whereas l-asparagine improved root growth (lin and kao 1995). these 158 acta bot. croat. 72 (1), 2013 kaya c., aydemir s., sonmez o., ashraf m., dikilitas m. 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees studies suggested a reason to examine the effects of these two organic substances on other crops so as to reinforce the database on the regulatory effects of these compounds on different potential crops. so, the present investigation was undertaken to assess whether exogenous application of asparagine or glycerol to maize plants could improve growth and mineral nutrition. also assessed was whether exogenously applied asparagine or glycerol could alter the activities of some key enzymes of oxidative defence system involved in counteracting the reactive oxygen species produced in maize plants under salt stress. materials and methods plant material and treatments before the initiation of the experiment, the caryopses of maize cv. dk 847 f1 were surface-sterilized in 0.1% (w/v) sodium dodecyl sulfate solution for 20 min and then thoroughly rinsed with sterile deionized water. the caryopses were then placed on sterile filter paper (two sheets) moistened with deionised water after they had been pre-soaked in deionised water for 6 h at 28 °c. five sterilized caryopses were planted in 8 kg peat contained in each plastic pot and all pots placed in a growth room at 27±2 °c with light intensity 350 mmol photons m–2 s–1 and rh ranging from 60 to 70%. soon after germination, the seedlings were thinned to three per pot. after 7-day growth, treatments of salt and growth regulators were initiated. the salt treatments consisted of control (without nacl) and 100 mm of nacl in full strength hoagland’s nutrient solution. two levels (5 and 10 mm) of asparagine (asn) and two (20 and 40 mm) of glycerol (g) were sprayed onto the leaves of maize seedlings 10 days after germination. the asn or g solutions mixed with 0.01% of a surfactant solution (tween-20) were sprayed once a week from day 10 after germination up to day 35, on the leaves of maize plants at a rate of 50 ml per pot. the control plants received an equal amount of water containing only 0.01% tween-20. the samples were harvested at 35 dap (days after planting). the composition of hoagland’s nutrient solution used was (mg l–1): 270 n, 31 p, 234 k, 200 ca, 64 s, 48 mg, 2.8 fe, 0.5 mn, 0.5 b, 0.02 cu, 0.05 zn and 0.01 mo. the ph of the treatment solutions was maintained at 6.5 by the addition of a few drops of 0.1 mm koh. each treatment had four replicates (each containing 3 plants) arranged in a completely randomised design. leaf relative water content (rwc) a fully expanded youngest leaf (3rd leaf) from each plant was excised and used to measure rwc following the method described by barrs and weatherley (1962). entire fully expanded leaves were used so as to minimize solute leakage due to excision effect. chlorophyll determination one plant per replicate was used for measuring chlorophyll content. fresh leaf samples (1.0 g each) were triturated in 90% acetone. the optical density of each filtered sample was read at 645 and 663 with a pye unicam uv-visible spectrophotometer (model sp6-550, pye unican, uk) and chlorophyll concentrations worked out following strain and svec (1966). acta bot. croat. 72 (1), 2013 159 asparagine and glycerol mitigate salinity stress in maize 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees free proline determination the protocol devised by bates et al. (1973) was used to determine free proline in fresh leaves (3rd leaf from top). proportions of fresh leaf samples (0.5 g each) were triturated each in 10 cm3 of aqueous sulfosalicylic acid solution (3%). an aliquot (2 cm3) of the filtrate was treated with 2 cm3 acid-ninhydrin and 2 cm3of glacial acetic acid. afterthe mixture was heated for 1 h at 100 °c in a water bath, it was extracted with 4 cm3 toluene. the chromophore containing toluene was aspirated, cooled down to room temperature, and the optical density was read at 520 nm. electrolyte leakage the electrolyte leakage (el) was estimated following dionisio-sese and tobita (1998). fresh leaf samples (3rd leaf) were cut into small pieces (5 mm length) and placed in 10 cm3 distilled deionized water contained in a test tube. the tubes were heated to 32 °c in a water bath. after 2 h of incubation, the initial electrical conductivity of the medium (ec1) was measured using a digital conductivity meter (s47-k, mettler toledo). thereafter, all samples were autoclaved at 121 °c for 20 min to kill the tissues so as to release all the electrolytes. the samples were then cooled to 25 °c to record final electrical conductivity (ec2). el was calculated using the following formula: el = (ec1/ec2) x 100 soluble protein content soluble protein content in the enzyme extracts was determined following bradford (1976) using bovine serum albumin fraction v as a standard. enzyme determination fresh leaves (0.5 g each sample) were triturated in 50 mm sodium phosphate buffer (ph 7.0) containing 1% soluble polyvinylpyrolidine (pvp). the extract was centrifuged at 20,000 g for 15 min at 4 °c and the supernatant used for the assays of the activities of superoxide dismutase (sod; ec 1.15.1.1), polyphenol oxidase (ppo; ec 1.10.3.1), catalase (cat; ec 1.11.1.6) and non-specific peroxidase (pod; ec 1.11.1.7). the activity of sod was assayed following the assay described by beauchamp and fridovich (1971) for appraising the activity of sod, which entails monitoring the enzyme’s ability to hinder the photochemical reduction of nitro-blue tetrazolium (nbt). one unit of sod was defined as the amount of enzyme used to inhibit the reduction of cytochrome c by 50%. catalase activity was appraised following kraus and austin-fletcher (1994) by assessing the utilization of h2o2 at 405 nm. the pod activity was determined following chance and maehly (1955) by adding the tissue extract (100 ml) to 3 ml of assay solution. the enzyme assay solution consisted of 3 ml of the reaction mixture containing 13 mm guaiacol, 5 mm h2o2 and 50 mm sodium-phosphate buffer (ph 6.5). an increase in absorbance at 470 nm for 1 min at 25 °c 160 acta bot. croat. 72 (1), 2013 kaya c., aydemir s., sonmez o., ashraf m., dikilitas m. 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees was read on a uv-visible spectrophotometer. the increase in a470 was measured for 3 min and activity expressed as da470 per mg of protein per minute polyphenol oxidase (ppo) activity was appraised following zaubermann et al. (1991). fresh leaf samples (0.5 g each) were triturated in 10 ml of 0.1 mol l–1 sodium phosphate buffer (ph 6.8) and 0.2 g of polyvinylpyrrolidone (pvp). the samples were centrifuged at 19,000 g for 20 min, and the supernatant was used for enzyme assay. the increase in absorbance at 410 nm at 25 °c was read automatically for 5 min. one unit of enzyme activity was defined as an increase of 0.01 in absorbance per min per mg protein. analysis of nutrients and measurement of dry weight all shoot and root samples were dried at 65 °c in an oven to constant dry weights. for chemical analyses, the dried and ground samples were ashed in a muffle furnace at 550 °c for 6 h, and the white ash so obtained was dissolved in 5 ml of 2 m hot hcl. after all samples had been properly filtered, the volume of each sample was raised to 50 ml by adding distilled deionized water. phosphorus in these sample digests was determined by the vanado-molybdate method. the concentrations of na+, ca2+ and k+ in these digested samples were analyzed using inductively coupled plasma (icp) spectometry (chapman and pratt 1982). statistical analysis of data the experiment was conducted twice under the same environmental conditions. statistical analysis of the data for two different experiments showed that there were no significant differences between the two experiments. hence, the data presented here are the means of the two individual experiments. each experiment was arranged in a completely randomized design and each treatment had four replicates (each containing 3 plants). data for each attribute were subjected to a one-way analysis of variance and the lsd worked out at p £ 0.05 using the statview software programme to compare the treatment means. results salinity stress caused a marked suppression in shoot and root dry mass of the maize plants. however, asn or g foliar-applied to salinized maize plants proved to be effective in significantly increasing plant dry biomass (tab. 1). glycerol was more effective than asparagine in increasing dry mass in salt-stressed maize plants. root:shoot dry weight ratio increased markedly under salt stress reflecting the fact that shoot growth was more adversely influenced by salt than root growth. however, application of asn or g resulted in a decrease of the root:shoot ratio. there was a marked reduction in leaf rwc in the maize plants under saline stress compared to that under the control treatment (tab. 1). however, foliar application of asn or g caused a significant improvement in rwc in the salt-stressed maize plants and there was more improvementwith glycerol. chlorophyll (chl a and b) contents decreased significantly in plants grown under nacl stress. however, exogenously applied asn or g increased chlorophyll contents in the salt-stressed plants compared to those in the salt-stressed plants not supplied with asn or g (tab. 2). glycerol was again more effective than asparagine in increasing chlorophyll conacta bot. croat. 72 (1), 2013 161 asparagine and glycerol mitigate salinity stress in maize 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees tents of the salt-stressed maize plants. electrolyte leakage (el) is considered to be a promising indicator of stress-induced membrane damage. electrolyte leakage was markedly greater in the salt-stressed plants than that in the control plants (tab. 2). however, asn or g foliar-applied to the salinized plants considerably reduced el in the leaves compared with the salinity-stressed plants that received neither asn nor g (tab. 2). glycerol was more effective than asparagine in reducing el in the salt-stressed maize plants. free proline levels were higher in the maize plants grown under saline stress than in those in the control plants. however, asn or g foliar-applied to the salinized plants decreased proline levels compared to the salt-stressed-maize plants receiving no treatment of asn or g (tab. 2). the effect of foliar application of g in reducing the proline content was more striking than that of asn. salinity stress up-regulated the activities of sod, cat and ppo, but it did not alter that of pod. exogenously applied asn or g reduced the activities of sod, pod and ppo in the 162 acta bot. croat. 72 (1), 2013 kaya c., aydemir s., sonmez o., ashraf m., dikilitas m. tab. 1. shoot, root and total plant dry weights, root: shoot ratio and relative water content (rwc) of salt-stressed and non-stressed maize plants, to which asparagine or glycerol were foliar applied (values followed by different letters, in the same column, are significantly different at p £ 0.05). control treatment (nutrient solution alone); salt, 100 mm nacl; a1 and a2: 5 and 10 mm asparagine, respectively; g1 and g2: 20 and 40 mm glycerol, respectively. treatments shoot dry wt. (g per plant) root dry wt. (g per plant) root:shoot ratio total plant dry wt. (g per plant) rwc (%) control 9.72 a 1.23 a 0.127 d 10.95 a 88 a salt 5.36 d 1.09 c 0.203 a 6.45 d 65 d salt +a1 7.24 b 1.15 bc 0.159 b 8.39 b 72 c salt +a2 6.89 c 1.12 c 0.163 b 8.01 c 74 c salt + g1 7.32 b 1.15 bc 0.157 b 8.47 b 79 b salt + g2 7.26 b 1.16 b 0.160 b 8.42 b 82 b tab. 2. chlorophyll a and chlorophyll b (mg per kg of fresh leaf tissue), electrolyte leakage (%) and proline content (mmol per g fresh weight) of salt-stressed and non-stressed maize plants, to which asparagine or glycerol were foliar applied (values followed by different letters, in the same column, are significantly different at p £ 0.05). control treatment: see table 1. treatments chl a chl b electrolyte leakage proline control 1289 a 912 a 12.3 d 1.12 d salt 905 d 596 d 38.2 a 2.32 a salt +a1 1056 c 705 b 26.5 b 1.32 c salt +a2 1078 c 712 b 25.8 b 1.56 b salt + g1 1109 b 778 c 23.6 c 1.08 d salt + g2 1117 b 784 c 22.3 c 1.01 d 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees stressed plants compared to those of the salt treated plants not supplied with asn or g (tab. 3). although pod activity was not altered due to salt stress, it was reduced considerably due to exogenous application of asparagine or glycerol and there was greater reduction with glycerol application. glycerol application was again more effective than that of asparagine in reducing the activities of all enzymes. saline growth medium caused a marked increase in leaf na+ but a decrease in those of leaf k+, ca2+ and p in maize plants. the maize plants grown under saline regime accumulated considerably lower na+ and higher k+, ca2+ and p upon foliar applications of asn or g than the salt-stressed plants that did not receive asn or g (tab. 4). sodium:potassium (na:k) ratio was found to be elevated significantly in the leaves of maize plants exposed to nacl stress, but exogenously applied asn or g to salinized plants significantly decreased the na:k ratio. asparagine was found to be more effective than glycerol in improving the contents of ca2+ and k+ in the leaves of salt-stressed maize plants, but it was less effective in reducing leaf na+. acta bot. croat. 72 (1), 2013 163 asparagine and glycerol mitigate salinity stress in maize tab. 3. activities of superoxide dismutase (sod: unit per mg of protein), polyphenol oxidase (ppo: 100 units per mg of protein), catalase (cat: 100 units per mg of protein) and peroxidase (pod: da470 per minute, per mg of protein) in salt-stressed and non-stressed maize plants to which asparagine or glycerol were foliar applied (values followed by different letters, in the same column, are significantly different at p £ 0.05). control treatment: see table 1. treatments sod ppo pod cat control 29.0 c 0.35 d 0.41 a 1.02 d salt 41.0 a 0.53 a 0.39 a 1.33 b salt +a1 33.0 b 0.44 b 0.21 c 1.20 c salt +a2 32.0 b 0.41 bc 0.38 a 1.40 a salt + g1 28.0 cd 0.38 cd 0.31 b 1.17 c salt + g2 26.0 d 0.38 cd 0.20 c 0.97 d tab. 4. na+, ca2+, k+ and p content (mmol per kg of dry matter) in leaves and roots of salt-stressed and non-stressed maize plants, to which asparagine or glycerol were foliar applied. values followed by different letters, in the same column, are significantly different at p £ 0.05. control treatment: see table 1. treatments leaf root na+ ca2+ k+ p na+:k+ na+ ca2+ k+ p 29 c 168 b 956 c 122 a 0.030 e 99 c 165 a 122 a 21 ab salt 328 a 112 e 673 d 86 d 0.487 a 278 a 78 d 72 d 14 c salt +a1 320 a 178 a 887 b 105 b 0.360 b 179 b 103 c 106 b 21 ab salt +a2 300 b 182 a 898 b 99 c 0.334 c 187 b 105 c 107 b 23 a salt + g1 265 c 148 d 765 d 109 b 0.346 bc 189 b 117 b 98 c 18 b salt + g2 243 d 159 c 812 e 111 b 0.299 d 176 b 114 b 103 b 23a 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees discussion from the data for different growth attributes such as shoot and root dry weights and total plant dry biomass, it is amply clear that foliar-applied asparagine or glycerol improved the growth of salt-stressed maize plants as compared to those not supplied with asparagine or glycerol. however, growth improvement was more marked with glycerol than with asparagine. the relatively greater effectiveness of glycerol in promoting the growth of salt-stressed maize plants can be related to the data for rwc, plant pigments (chl. a and b) and electrolyte leakage. it is pertinent to note, however, that both asparagine and glycerol increased rwc and chlorophyll a and b contents, and reduced electrolyte leakage in the salt-treated maize plants compared with those not treated with asparagine or glycerol. thus, the greater improvement in the growth of maize plants with glycerol application could have been due to a substantial glycerol-induced increase in rwc and chlorophyll a and b contents, and a decrease in electrolyte leakage. glycerol is a common cellular metabolite that acts effectively as both an osmoregulator and an osmoprotectant (shen et al. 1999, ashraf and harris 2004, courchesne et al. 2011). it is also one of the vital intermediates involved in energy metabolism in both prokaryotes and eukaryotes (ferreira et al. 2005). in a recent study, exogenous application of glycerol as a pre-sowing seed treatment to salt-stressed ricinus communis plants resulted in a marked improvement in growth (ali et al. 2008). electrolyte leakage from cellular membranes is one of the prominent salinity-induced effects on most plants. this membrane characteristic is contemplated as one of the viable indicators of salt tolerance (sairam et al. 2002, ashraf and ali 2008, mansour 2012). the considerable effect of glycerol on checking electrolyte leakage in the salt-stressed maize plants could be because exogenously applied glycerol might increase intra-cellular glycerol concentration, which in turn, alters membrane permeability, as earlier reported by brown (1990) and albertyn et al. (1994). it is also possible that glycerol stabilized cell membrane proteins including transport proteins to check electrolyte leakage (schein 1990). the growth of salt-stressed maize plants was also improved with asparagine application, though less than with glycerol. such improvement in growth due to exogenous application of asparagine was earlier observed in rice (lin and kao 1995). like other important amino acids and amides, asparagine serves as a vital nitrogen and carbon reservoir during osmotic stress (martinelli et al. 2007, sulieman et al. 2010). despite the involvement of asparagine in the storage of reduced n in salt-stressed plants (stewart and larher 1980, rabe 1990), it plays a central role in osmoregulation of plants exposed to osmotic stress (colmer et al. 1995, maaroufi-dguimi et al. 2011). accumulation of compatible osmolytes is one of the distinctive responses of plants subjected to salt stress. among different compatible osmolytes known in plants, proline has been widely studied because it plays a multitude of functions in the metabolism of plants subjected to salt stress, in addition to its significant role in osmoregulation as well as osmoprotection (ashraf and foolad 2007). the data for proline content shows that proline content decreased markedly in salt-stressed maize plants supplied with asparagine or glycerol. however, more reduction took place in proline due to glycerol application. a strong body of evidence shows that both asparagine (ashraf and harris 2004, lea et al. 2007) and glycerol (ferreira et al., 2005, martinelli et al. 2007) are also potential osmolytes and analogous to proline in regulating many physio-biochemical processes. it is highly likely that the reduction in proline content in salt-stressed plants may have been due to an antagonistic effect of asparagine and glycerol with proline. 164 acta bot. croat. 72 (1), 2013 kaya c., aydemir s., sonmez o., ashraf m., dikilitas m. 626 kaya et al.ps u:\acta botanica\acta-botan 1-13\626 kaya et al.vp 14. o ujak 2013 11:39:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees although salt stress enhanced the activities of some key antioxidant enzymes, sod, ppo and cat, the exogenously applied asparagine and glycerol considerably reduced the activities of these enzymes while glycerol was more effective in reducing the antioxidant enzyme activities. it is now evident that glycerol could act as a scavenger of reactive oxygen species (ros) possibly generated by different stresses including salt stress (smirnoff and cumbes 1989, sheveleva et al. 1997, shen et al. 1999). thus, in cases in which other alternative ros scavengers are available to plants, a reduction in the levels of antioxidant enzymes determined in the present study can be expected. although asparagine also caused reduction in the activities of antioxidant enzymes in salt-stressed maize plants, its role as an antioxidant has not been established (liu et al. 2004). exogenous application of asparagine and glycerol was also found to be effective in regulating the levels of different elements such as na+, k+, ca2+ and p in the leaves and roots of salt-stressed maize plants. exogenously applied glycerol reduced leaf na+ more effectively than asparagine, although both osmolytes remained almost equally effective in reducing root na+. this could also be one of the reasons for the greater effectiveness of glycerol than asparagine in improving growth of salt-stressed maize plants. however, in contrast, asparagine was more effective in improving leaf ca2+ and k+ as well as root k+ in salt-stressed maize plants. the differential effectiveness of glycerol and asparagine in regulating the levels of important inorganic nutrients may have been one of the factors in the differential improvement of growth in salt-stressed maize plants. overall, foliar application of asparagine or glycerol caused a substantial improvement in growth of maize plants of genotype dk 647 f1 under salt stress. this asparagineor glycerol-induced growth improvement was found to be associated with improved relative water content and photosynthetic pigments and decreased electrolyte leakage from cellular membranes in this maize genotype. both glycerol and asparagine significantly reduced leaf proline content and the activities of sod, ppo and cat and differentially regulated the levels of different inorganic nutrients in salt-stressed plants of the genotype. glycerol was found to be more effective than asparagine in regulating growth and physiological processes in salt-stressed plants of the genotype because it has a dual function i.e. as an osmolyte and as a scavenger of reactive oxygen species, whereas the function of asparagine as an antioxidant has not yet been established. we have used only one genotype to examine the effects of glycerol and asparagine, and the results presented here cannot be generalized unless such effects are observed in a number of maize genotypes. 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(2000, 2001); kasom (2003, 2004); had@iablahovi] and kasom (2005). the data provided in the present paper are based on all published and unpublished records of species of the family russulaceae in montenegro. materials and methods the survey of taxa is given in alphabetical order. along with each taxon presented, the published sources of records (in chronological order), collections in which the collected material is deposited, and our unpublished data (material examined) for every species found on the territory of montenegro are specified. all synonyms that the authors of records have used in montenegro are also specified as well as those used in modern taxonomic literature. our identification or revision of the records has been carried out using the following literature: neuhoff (1955), phillips (1981), singer (1986), hansen and knudsen (1992), arnolds et al. (1995), heilmann-clausen et. al (1998), basso (1999), horak (2005), and kränzlin (2005). for the interpretation of species we have also used the paper of tkal^ec and me[i] (2003). taxonomic nomenclature followed that of robert et al. (2005). a number of available records by other authors have been revised during this research. the material known to us has been deposited in the following herbaria and fungaria: f.m.m.c. – fungarium of montenegrin mycological centre, podgorica, montenegro; had`i} – private fungarium of ibrahim had`i}, ro`aje, montenegro; m.n.f. – montenegrin national fungarium, institute for nature protection, podgorica, montenegro. the material collected during our investigation is deposited in m.n.f. the chapter excluded records comprises records of species that we have not been able to interpret in the sense of the modern classification. marks and abbreviations * – taxon reported for the first time for montenegro; • – at least part of records of the taxon revised; = – synonym; () – empty brackets indicate that the author of the record has also quoted the taxon under the name used herewith; (x) – number in brackets indicates a synonym under which the author of the record has quoted the taxon; =? – taxon not found in modern taxonomic literature; exs. – collections in which the dried materials (exsiccata) are deposited; leg. – collector; mt – mountain; nom. nud. – nomen nudum; np – national park; ref. – references (sources of records of the taxon in montenegro); rev. – revised (when record have been revised by the authors of this paper). 286 acta bot. croat. 71 (2), 2012 kasom g., karadelev m. results in this paper 83 species belong to two genera of the family russulaceae: 38 species of the genus lactarius and 45 species of the genus russula. the taxa lactarius intermedius, l. subdulcis, and russula subfoetens are reported for the first time for the territory of montenegro. survey of taxa lactarius pers. • lactarius acris (bolton: fr.) gray ref.: peri] and peri] (1995b: 63, 1996a: 154, 1997a: 60, 2004: 20), kasom (2003: 251). exs.: f.m.m.c., m.n.f. material examined: 29 october 2010, mt dragi{nica, forest of fagus sylvatica, m.n.f. 3/10. • lactarius aurantiacus (pers.: fr.) gray = lactarius mitissimus (fr.: fr.) fr. (1) ref.: had@i] (1995: 23), karad@i] (1995: 82–83), peri] and peri] (1997a: 61 (1)). exs.: had`i}, m.n.f. material examined: 27 october 2008, mt ljubi{nja, forest of picea abies, m.n.f. 142/08; 01 october 2009, mt milogora, forest of abies alba and fagus sylvatica, m.n.f. 53/09. lactarius azonites (bull.) fr. ref.: peri] and peri] (1995b: 63, 1997a: 60). exs.: f.m.m.c. • lactarius blennius (fr.: fr.) fr. ref.: peri] and peri] (1996c: 73, 1997a: 60, 2003: 81), kasom (2003: 252, 2004: 25). exs.: f.m.m.c., m.n.f. material examined: 29 october 2010, mt dragi{nica, forest of fagus sylvatica; 29 october 2010, np lov}en: mt tre{tenik, forest of fagus sylvatica. lactarius camphoratus (bull.) fr. ref.: had@i] (1995: 23), peri] and peri] (1996c: 73). exs.: f.m.m.c., had`i}. • lactarius chrysorrheus fr. ref.: peri] and peri] (1997a: 60, 1997b: 283), peri] et al. (2000: 159), kasom (2003: 252). exs.: f.m.m.c., m.n.f. • lactarius circellatus fr. ref.: torti] (1988: 131), had@i] (1995: 23), peri] and peri] (1997a: 60), kasom (2003: 252). exs.: had`i}, m.n.f. acta bot. croat. 71 (2), 2012 287 russulaceae in montenegro • lactarius controversus pers.: fr. ref.: had@i] (1995: 23), karad@i] (1995: 78–79 (rev.)), peri] and peri] (1997a: 60), peri] et al. (2001: 15). exs.: f.m.m.c., had`i}. • lactarius deliciosus (l.: fr.) gray ref.: had@i] (1995: 23), karad@i] (1995: 80–81 (rev.)), peri] and peri] (1996b: 36–37 (rev.), 1997a: 60, 1997b: 283, 1998b: 75, 1999a: 92, 1999b: 52, 2000: 108), peri] et al. (2000: 159), kasom (2003: 252), had@iablahovi] and kasom (2005: 133–134). exs.: f.m.m.c., had`i}, m.n.f. material examined: 01 october 2006, lov}en np, ivanova korita, forest of pinus nigra. • lactarius deterrimus gröger ref.: had@i] (1995: 23), karad@i] (1995: 80–81 (rev.)), peri] and peri] (1996b: 42–43 (rev.), 1997a: 60, 1998b: 75), had@iablahovi] and kasom (2005: 135–136). exs.: f.m.m.c., had`i}, m.n.f. material examined: 20 and 21. july 2009, mt hajla, forest of picea abies, leg. ibrahim had`i}, m.n.f. 33/09. • lactarius fluens boud. 1899 ref.: peri] et al. (2000:159). exs.: f.m.m.c. lactarius fuliginosus (krapf: fr.) fr. ref.: peri] and peri] (1995b: 63, 1997a: 60, 2004: 20). exs.: f.m.m.c *• lactarius intermedius (krombh.) berk. et broome exs.: m.n.f. material examined: 01 october 2009, mt milogora, forest of abies alba and fagus sylvatica, m.n.f. 52/09. lactarius lignyotus fr. ref.: had@i] (1995: 23). exs.: had`i}. • lactarius lilacinus (lasch: fr.) fr. ref.: torti] (1988: 131), had@i] (1995: 23), peri] and peri] (1997a: 60). exs.: beo, m.n.f. material examined: 09 september 2005 and 16 october 2006, biogradska gora np, near biogradska river and biogradsko lake, forest of alnus sp., m.n.f. 18/05. annotation: torti] (1988: 131) quoted this taxon from torti] (1974), but in this article these data are not mentioned. lactarius mairei malençon ref.: peri] et al. (2000: 159), peri] et al. (2001: 15), peri] and peri] (2004: 21). exs.: f.m.m.c. 288 acta bot. croat. 71 (2), 2012 kasom g., karadelev m. lactarius musteus fr. ref.: had@i] (1995: 23). exs.: had`i}. • lactarius pallidus pers.: fr. ref.: peri] and peri] (1995b: 63, 1997a: 61), kasom (2003: 252). exs.: f.m.m.c., m.n.f. • lactarius picinus fr. ref.: had@i] (1995: 23), karad@i] (1995: 84–85), peri] and peri] (1996a: 154, 1997a: 61). exs.: f.m.m.c., had`i}. lactarius piperatus (l.) pers. sensu lato ref.: torti] (1974: 210, 1988: 131). • lactarius piperatus (l.) pers. ref.: had@i] (1995: 23), karad@i] (1995: 84–85 (rev.)), peri] and peri] (1995b: 63, 1996c: 73, 1997a: 61, 1999a: 92, 2000: 108–109, 2005: 94), peri] et al. (2000: 159). exs.: f.m.m.c., had`i}. • lactarius pubescens fr. ref.: had@i] (1995: 23), karad@i] (1995: 86–87 (rev.)), peri] and peri] (1997a: 61). exs.: had`i}, m.n.f. material examined: 17 october 2008, prokletije np, mt visitor; under betula sp., m.n.f. 64/08; 30 september 2009, mt golija, forest of betula sp., m.n.f. 55/09. lactarius pyrogalus (bull.: fr.) fr. ref.: had@i] (1995: 23). exs.: had`i}. • lactarius rufus (scop.: fr.) fr. ref.: had@i] (1995: 23), karad@i] (1995: 86–87), peri] and peri] (1997a: 61), kasom (2003: 252). exs.: had`i}, m.n.f. • lactarius salmonicolor r. heim et leclair ref.: had@i] (1995: 23), karad@i] (1995:88–89 (rev.)), peri] and peri] (1996b: 38–39, 1997a: 61). exs.: f.m.m.c., had`i}, m.n.f. material examined: 30 september 2009, mt golija, under abies alba; 01 october 2009, mt milogora, forest of abies alba and fagus sylvatica, under abies albe, m.n.f. 60/09; 29 october 2010, mt dragi{nica, forest of abies alba and fagus sylvatica, under abies albe, leg. d. roganovi} and s. had`iablahovi}, m.n.f. 2/10. • lactarius sanguifluus (paulet) fr. ref.: karad@i] (1995: 90–91 (rev.), peri] and peri] (1996b: 40–41, 1996c: 73, 1997a: 61, 1997b: 283, 1999a: 92, 1999b: 52), peri] et al. (2000: 159). exs.: f.m.m.c. acta bot. croat. 71 (2), 2012 289 russulaceae in montenegro • lactarius scrobiculatus (scop.: fr.) fr. ref.: had@i] (1995: 23), karad@i] (1995: 88-89 (rev.)), peri] and peri] (1997a: 61, 1998b: 75). exs.: f.m.m.c., had`i}, m.n.f. material examined: 30 july 2008, mt bogi}evica, forest of picea abies; 20 july 2009, mt hajla, forest of picea abies, leg. ibrahim had`i}, m.n.f. 32/09. • lactarius semisanguifluus r. heim et leclair ref.: had@i] (1995: 23), karad@i] (1995: 90–91 (rev.)), peri] and peri] (1996b: 44–45, 1996c: 73, 1997a: 61, 2005: 94). exs.: f.m.m.c., had`i}. lactarius serifluus (dc.: fr.) fr. ref.: peri] and peri] (1996c: 73). exs.: f.m.m.c. lactarius sphagneti (fr.) neuhoff ref.: had@i] (1995: 23). exs.: had`i}. *• lactarius subdulcis (pers.: fr.) gray exs.: m.n.f. material examined: 22 september 2010, canyon of su{ice river, forest of fagus sylvatica, leg. s. had`iablahovi}, m.n.f. 25/10. • lactarius torminosus (schaeff.: fr.) pers. ref.: karad@i] (1995: 92–93 (rev.)), peri] and peri] (1997a: 62). exs.: m.n.f. material examined: 30 september 2009, mt golija, forest of betula sp., m.n.f. 54/09. • lactarius turpis (weinm.) fr. misapplied name: lactarius necator (bull.: fr.) p. karst. sensu auct. (1) ref.: had@i] (1995: 23), karad@i] (1995: 82–83 (1) (rev.)), peri] and peri] (1997a: 61 (1), 1999a: 92 (1)). exs.: f.m.m.c., had`i}. • lactarius uvidus (fr.: fr.) fr. ref.: peri] and peri] (1996a: 154, 1997a: 62), kasom (2003: 252). exs.: f.m.m.c., m.n.f. material examined: 20 july 2009, mt hajla, forest of picea abies, m.n.f.34/09. • lactarius vellereus (fr.: fr.) fr. (1) ref.: karad@i] (1995: 92–93), peri] and peri] (1996c: 73, 1997a: 62, 1999a: 92), kasom (2003: 252). exs.: f.m.m.c., m.n.f. lactarius vinosus (quél.) bat. ref.: peri] and peri] (1997a: 62, 1997b: 283). exs.: f.m.m.c. 290 acta bot. croat. 71 (2), 2012 kasom g., karadelev m. • lactarius volemus (fr.: fr.) fr. ref.: had@i] (1995: 23), karad@i] (1995: 94–95), peri] and peri] (1995a: 40–41, 1995b: 63, 1997a: 62, 1999a: 92, 2004: 21,?: 40–41), peri] et al. (2000: 159). exs.: f.m.m.c., had`i}, m.n.f. material examined: 09 september 2005, biogradska gora np, forest of fagus sylvatica; m.n.f. 19/05; 09 july 2009, andrijevica town, forest of qurcus cerris, m.n.f. 17/09. • lactarius zonarioides kühner et romagn. = lactarius bresadolanus singer nom. nud. (1) ref.: karad@i] (1995: 78–79 (1) (rev.)), peri] and peri] (1997a:60 (1)). exs.: m.n.f. material examined: 20 july 2009, mt hajla, forest of picea abies, m.n.f. 31/09. • lactarius zonarius (bull.) fr. = lactarius zonarius var. scrobipes kühner et romagn. (1) ref.: had@i] (1995: 23), peri] and peri] (1997a: 62, 1997b: 283 (1)), kasom (2003: 252 (rev.)). exs.: f.m.m.c., m.n.f. russula pers. russula aeruginea lindblad ref.: had@i] (1995: 22). exs.: had`i}. russula albonigra (krombh.) fr. ref.: had@i] (1995: 22). exs.: had`i}. russula alutacea (pers.: fr.) fr. ref.: torti] (1988: 131), peri] and peri] (1997a: 62). exs.: m.n.f. material examined: 20 july 2009, mt hajla, forest of picea abies, m.n.f. 35/09. russula amoena quél. ref.: had@i] (1995: 22), peri] and peri] (1995b: 63, 1996a: 154, 1997a: 62, 1998b: 75). exs.: f.m.m.c., had`i}. russula anthracina romagn. ref.: peri] and peri] (1995b: 63, 1997a: 62, 1997b: 283, 1999a: 92), peri] et al. (2000: 159). exs.: f.m.m.c. • russula aurea pers. = russula aurata (with.) fr. (1) ref.: had@i] (1995: 22 (1)), peri] and peri] (1998a: 54–55 (1), 1998b: 75 (1), 1999a: 92, 1999b: 52 (1), 2004: 25 (1), 2005: 94 (1)), kasom (2003: 252 (1)). acta bot. croat. 71 (2), 2012 291 russulaceae in montenegro exs.: f.m.m.c., had`i}, m.n.f. material examined: 19 june 2008, mt orjen, grahovac village, forest of quercus cerris. • russula azurea bres. ref.: karad@i] (1995: 120–121 (rev.)), peri] and peri] (1997a: 62). exs.: /. russula brunneoviolacea crawshay ref.: had@i] (1995: 22). exs.: had`i}. russula carminipes j. blum ref.: peri] and peri] (1999a: 92). exs.: f.m.m.c. • russula cyanoxantha (schaeff.) fr. = russula cutefracta cooke (1) ref.: had@i] (1995: 22 (1) ()), karad@i] (1995: 122–123), peri] and peri] (1995a: 38–39, 1995b: 63, 1996a: 154, 1996c: 73, 1997a: 62, 1998b: 75, 1999a: 92, 1999b: 52, 2000: 110–111, 2002: 138,?: 38–39), peri] et al. (2000: 159), kasom (2004: 25). exs.: f.m.m.c., had`i}, m.n.f. material examined: 20 july 2009, mt hajla, forest of picea abies, m.n.f. 36/09. • russula delica fr. ref.: torti] (1988: 131), had@i] (1995: 22), karad@i] (1995: 122–123), peri] and peri] (1995b: 63, 1997a: 62, 1999a: 92), kasom (2003: 252, 2004: 26). exs.: f.m.m.c., had`i}, m.n.f. russula emetica (schaeff.) pers. ref.: had@i] (1995: 22), peri] and peri] (1995b: 63, 1996c: 73, 1997a: 63, 1999a: 92, 2005: 94). exs.: f.m.m.c., had`i}. russula foetens pers. sensu lato ref.: torti] (1974: 211, 1988: 131). • russula foetens pers.: fr. ref.: had@i] (1995: 22), karad@i] (1995: 124–125), peri] and peri] (1997a: 63), kasom (2003: 253). exs.: had`i}, m.n.f. russula fragilis (pers.: fr.) fr. ref.: had@i] (1995: 22). exs.: had`i}. russula gracillima jul. schäff. misapplied name: russula gracilis burl. sensu auct. (1) ref.: had@i] (1995: 22 (1)). exs.: had`i}. 292 acta bot. croat. 71 (2), 2012 kasom g., karadelev m. russula grata britzelm. = russula laurocerasi melzer (1), = russula laurocerasi var. fragrans romagn. (2) ref.: peri] and peri] (1996a: 154, 1997a: 63 (1)(2)). exs.: f.m.m.c. russula grisea fr. ref.: karad@i] (1995: 124–125), peri] and peri] (1997a: 63). exs.: /. russula heterophylla (fr.) fr. ref.: had@i] (1995: 22). exs.: had`i}. russula illota romagn. ref.: had@i] (1995: 22). exs.: had`i}. russula integra (l.) fr. ref.: had@i] (1995: 22), karad@i] (1995: 126–127), peri] and peri] (1995b: 63, 1997a: 63, 1999a: 92). exs.: f.m.m.c., had`i}. russula ionochlora romagn. ref.: had@i] (1995: 22). exs.: had`i}. russula maculata quél. et roze ref.: peri] and peri] (1999a: 92). exs.: f.m.m.c. russula mairei singer ref.: had@i] (1995: 22). exs.: had`i}. russula mustelina fr. ref.: had@i] (1995: 22). exs.: had`i}. • russula nigricans fr. ref.: had@i] (1995: 22), karad@i] (1995: 128–129 (rev.)), peri] and peri] (1997a: 63). exs.: had`i}. russula ochroleuca pers. ref.: had@i] (1995: 22). exs.: had`i}. russula olivacea (schaeff.) pers. ref.: had@i] (1995: 23), karad@i] (1995: 128–129), peri] and peri] (1995b: 63, 1997a: 63, 2004: 25), kasom (2003: 253). exs.: f.m.m.c., had`i}. acta bot. croat. 71 (2), 2012 293 russulaceae in montenegro russula paludosa britzelm. ref.: torti] (1988: 131), peri] and peri] (1997a: 63). exs.: /. russula puellaris fr. ref.: had@i] (1995: 23), kasom (2003: 253). exs.: had`i}. • russula queletii fr. ref.: had@i] (1995: 23), peri] and peri] (1997a: 63, 1997b: 283, 1999a: 92). exs.: f.m.m.c., had`i}, m.n.f. russula romellii maire ref.: peri] and peri] (1995b: 63, 1996b: 46–47, 1997a: 63). exs.: f.m.m.c. • russula rosea pers. = russula lepida fr. (1), = russula rosacea (pers.) gray (2) ref.: torti] (1988: 131 (2)), karad@i] (1995: 126–127 (1)), peri] and peri] (1997a: 63 (1), 2005: 94 (1)), kasom (2003: 253 (1)). exs.: f.m.m.c., m.n.f. • russula sanguinea (bull.) fr. = russula sanguinaria (schumach.) rauschert (1) ref.: had@i] (1995: 23 (1)), karad@i] (1995: 130–131 (1)), peri] and peri] (1996c: 73 (1), 1997a: 64 (1), 1997b: 283 (1), 1999b: 53 (1), 2005: 94 (1)), peri] et al. (2000: 159 (1)), kasom (2003: 253 (1), 2004:26 (1) (rev.)). exs.: had`i}, f.m.m.c., m.n.f. russula sardonia fr. = russula drimeia cooke (1) ref.: had@i] (1995: 23), peri] et al. (2000: 159 (1)). exs.: had`i}, f.m.m.c. russula solaris ferd. et winge ref.: peri] and peri] (1999a: 92). exs.: f.m.m.c. russula sororia (fr.) romell ref.: had@i] (1995: 23). exs.: had`i}. *• russula subfoetens wm.g. sm. exs.: m.n.f. material examined: 20 june 2011, andrijevica town, forest of quercus cerris, m.n.f. 04/11. russula turci bres. ref.: had@i] (1995: 23). exs.: had`i}. 294 acta bot. croat. 71 (2), 2012 kasom g., karadelev m. russula undulata velen. = russula atropurpurea (krombh.) britzelm. (1) ref.: had@i] (1995: 22 (1)). exs.: had`i}. • russula vesca fr. ref.: had@i] (1995: 23), peri] and peri] (1995a: 36–37, 1995b: 63, 1996a: 154, 1997a: 64, 1998b: 75,?: 36–37). exs.: had`i}, f.m.m.c. russula vinosa lindblad = russula obscura (romell) peck (1) ref.: had@i] (1995: 22 (1) and 23 ()). exs.: had`i}. russula violeipes quél. ref.: had@i] (1995: 23). exs.: had`i}. • russula virescens (schaeff.) fr. ref.: jaap (1916) in torti] (1988:131), had@i] (1995: 23), karad@i] (1995: 130–131 (rev.)), peri] and peri] (1995a: 34–35, 1995b: 63, 1996a: 154, 1997a: 64, 1999a: 93, 2005: 95,?: 34–35), peri] et al. (2000: 159). exs.: had`i}, f.m.m.c. russula vitellina gray. ref.: peri] and peri] (1996a: 154, 1997a: 64). exs.: f.m.m.c., had`i}. russula xerampelina (schaeff.) fr. = russula xerampelina var. erythropus (pelt.) konrad et j. favre (1) ref.: had@i] (1995: 23 (1)), karad@i] (1995: 132–133), peri] and peri] (1997a: 64). exs.: had`i}, f.m.m.c. excluded records lactarius pargamenus (sw.: fr.) fr. ref.: peri] and peri] (1999a: 92). exs.: f.m.m.c. annotation: for determination of this taxon peri] and peri] used the book of focht (1986: 223–224). according to focht’s description, the montenegrin record could represent l. glaucescens crossl. however, without revision of this record it is not possible to know which species the authors have found. lactarius semisanguifluus deformans r. heim et leclair =? ref.: had@i] (1995: 23). russula cloroflava (schaeff.) fr. =? ref.: had@i] (1995: 22). acta bot. croat. 71 (2), 2012 295 russulaceae in montenegro russula lutea (huds.: fr.) gray ref.: had@i] (1995: 22). exs.: had`i}. annotation: this name cannot be interpreted in the sense of modern taxonomy (nomen dubium). it has been used for two species: russula risigallina and r. vitellina (arnolds et al., 1995: 411, hansen and knudsen, 1992: 391, tkal^ec and me[i], 2003: 295). without revision of the cited record it is not possible to know which species the authors have found. discussion in this paper we present 84 species of russulaceae that have been found in montenegro. the genus lactarius is represented by 39 species, and the genus russula by 45 species. for each presented species, the published and unpublished sources of data are provided, as well as those from the collections in which the material is deposited. the three taxa lactarius intermedius, l. subdulcis, and russula subfoetens are reported for the first time for the territory of montenegro. acknowledgements a great debt of gratitude is owed to doctors zdenko tkal~ec, armin me{i}, vladimír antonín, katerina rusevska, branislav peri}, olgica peri}, ibrahim had`i}, zlatko buli}, danijel vincek and sead had`iablahovi} for their assistance during the preparation of this paper. references arnolds, e., kuyper, t. w., noordeloos, m. e. 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maruli}ev trg 9a, 10000 zagreb, croatia quercetin is known to possess antimicrobial activity against bacteria, fungi, and viruses. the activity of this flavonoid against phytoplasmas, non-cultivable plant pathogenic bacteria that cause numerous plant diseases, has never been examined before. the aim of this research was to examine the effect of different concentrations of quercetin (10 mm, 100 mm and 1 mm) on 'candidatus phytoplasma asteris' and on phytoplasma-infected periwinkle shoots grown in vitro. the addition of quercetin neither supported the growth of the shoots nor induced the remission of symptoms in the infected plants. on the contrary, addition of quercetin induced browning of leaves and the appearance of black spots on the leaves of treated infected and non-infected shoots. it also had no curative effect against the pathogen. phytoplasma presence was confirmed by nested pcr in infected shoots treated with quercetin through three subcultures. key words: phytoplasma, bacteria, antimicrobial activity, flavonoid, quercetin, periwinkle, plant tissue culture introduction flavonoids, natural substances present in all vascular plants, possess an antimicrobial activity against different bacteria, fungi, and viruses (harborne and williams 2000, cushnie and lamb 2005). when the antibacterial properties of flavonoids are discussed, most of the data come from in vitro studies of human pathogenic bacteria. those studies revealed that many different flavonoids, including quercetin, inhibit bacterial growth. however, different studies on the same bacterial species produced ambiguous results, possibly due to the different inoculum sizes and different assays used (disk diffusion assay, broth macrodilution assay, broth microdilution assay, agar well diffusion assay, agar dilution assay) as well as to different diffusion rates of various flavonoids, formation of precipitates or salts and structural alternations of flavonoids inside the applied inoculums. whether flavonoids act as bacteriostatic or bactericidal agents is under debate because some research groups have shown that flavonoids reduce the number of colony-forming units, while others speculate that this is the consequence of the formation of bacterial aggregates under flavonoid-exposure rather than bactericidal effect (cushnie and lamb 2005). several acta bot. croat. 69 (2), 2010 155 * corresponding author, e-mail: mirna@botanic.hr u:\acta botanica\acta-botan 2-10\334 curkovic perica.vp 11. listopad 2010 11:21:00 color profile: disabled composite 150 lpi at 45 degrees hypotheses have been proposed for the mechanisms of the antibacterial action of flavonoids: intereference with dna synthesis (plaper et al. 2003), inhibition of membrane function and inhibition of energy metabolism (cushnie and lamb 2005). data on flavonoid activity against plant pathogens encompass mostly antifungal and antiviral activity. flavonoids are able to suppress fungal infection or inhibit spore germination, thus protecting plants from fungal attacks (harborne and williams 2000). their antiphytoviral activity encompasses reduction of virus infectivity by interfering with initiation of virus infection (rusak et al. 1997), weakening of interactions among coat-protein subunits (verma 1973, malhotra et al. 1996) or altering interactions between viral nucleic acid and capside proteins (french et al. 1991). activity of quercetin against phytoplasmas, plant pathogenic bacteria, has not hitherto been tested. phytoplasmas are wall-less prokaryotes that have a two-host cycle involving plants and insect vectors (christensen et al. 2005). these uncultivable bacteria are the main cause of many economically important diseases infecting several hundred plant species worldwide. different techniques and treatments have been applied in attempts to cure diseases caused by different phytoplasma species: treatments with tetracyclines, b-amino-butyric acid, polyamines, terpenes, auxines, tissue culture and/or heat or hot water treatment (]urkovi] perica and [eruga musi] 2005, ]urkovi] perica 2008a). the aim of this research was to test whether quercetin treatment could eliminate phytoplasmas from the host or at least induce recovery of phytoplasma-infected plants. material and methods material and plant tissue culture methods catharanthus roseus (l.) g. don shoots infected with 'candidatus phytoplasma asteris' (strain hydb), belonging to the 16sri-b subgroup, were grown in vitro on ms (murashige and skoog 1962) basal nutrient medium supplemented with 100 mgl–1 myo-inositol, 1 g l–1 casein hydrolysate, 30 g l–1 sucrose, 9 g l–1 agar and 0.5 mg l–1 benzylaminopurine (ba). in such a system, phytoplasmas are present in a high titer and all shoots express symptoms of infection (proliferations, internode shortening, stunting). phytoplasma-infected shoots were obtained from phytoplasmology laboratory of the university of bologna (irpcm 2004). healthy in vitro-grown c. roseus shoots were also included in experiments as controls. each shoot was grown in a test tube (20 ´ 150 mm) filled with 15 ml of ms nutrient medium. the ph of the medium was adjusted to 5.7 before autoclaving at 118 kpa and 120 °c for 20 min. in order to test the effect of quercetin (sigma) on 'ca. p. asteris' – infected and healthy shoots, they were transferred to the medium mentioned above, which did not contain agar. this medium was, after autoclaving, supplemented with 10 mm, 100 mm or 1 mm of quercetin. quercetin stock solutions were prepared in ethanol. therefore, in the control experiment, ethanol without quercetin was also added to the medium in order to rule out the possibility of the antiphytoplasmal activity of the alcohol. untreated infected and non-infected controls were also transferred to liquid medium. the cultures were incubated at 22±2 °c under a 16-h photoperiod and subcultured in a 3 week-culture period. twenty-four infected c. roseus shoots per treatment, positive control (untreated infected shoots on the medium with ba), non-infected treated shoots, and negative control (non-infected shoots 156 acta bot. croat. 69 (2), 2010 ]urkovi]-perica m., je@i] m. u:\acta botanica\acta-botan 2-10\334 curkovic perica.vp 11. listopad 2010 11:21:00 color profile: disabled composite 150 lpi at 45 degrees on the medium with ba) were included in the experiment (tab. 1). shoots were placed on paper bridges with their stems 2–3 mm immerged in the liquid medium weight (g) and length (cm) were measured on 16 shoots per treatment to insure availability of plant material for phytoplasma detection. measurements were performed at the beginning and at the end of the first three subcultures. fresh weight increase and shoot length increase were calculated as a ratio of the final and the initial fresh weight or shoot length, respectively. mean values, analysis of variance and duncan's test were used for analysis and interpretation of the data. phytoplasma detection eight samples (0.5 g each) of randomly chosen infected shoots treated with different concentrations of quercetin, positive control and non-infected shoots were taken after the 3rd subculture in order to be tested for the presence of 'ca. p. asteris'. exceptions were infected shoots grown on 1 mm quercetin, which started to decay in the first subculture and were therefore tested after the first subculture. the procedure for phytoplasma detection, including total nucleic acid isolation, pcr amplification and product analysis was previously described (]urkovi] perica et al. 2007; ]urkovi] perica 2008a, b). also, serial dilutions of total dna (i.e. 20, 10 and 5 ng µl–1) were used as templates in pcr to determine a possible lower titer of phytoplasma in the treated shoots. direct pcr assays were performed using universal phytoplasma primer pair r16f1/r0 (lee et al. 1995), for the amplification of highly conserved 16s rdna. the amplification products were diluted and reamplified in the first nested pcr with primers r16f2n/r2 (gundersen and lee 1996). the additional second nested pcrs were performed using r16(i)f1/r1 (lee et al. 1994) only for the shoots treated with 1 mm of quercetin. results shoot length and fresh weight increase were used as parameters for measuring the effect of quercetin on 'ca. p. asteris'-infected and non-infected c. roseus shoots (tab. 1). none of the used quercetin concentrations showed any beneficialeffect on phytoplasma-infected plants. on the contrary, quercetin (100 µm and 1000 µm) added to the medium resulted in increased severity of symptoms, accompanied by leaf yellowing and browning, and emergence of black spots on leaf edges. healthy periwinkle shoots treated with quercetin (100 µm) also expressed stunting accompanied by leaf yellowing and slight browning. at the highest concentration of quercetin (1 mm), plants infected with 'ca. p. asteris' started to decay shortly after the transfer to the quercetin-supplemented medium, during the first subculture. shoot elongation and fresh weight increase of both, infected and non-infected plants, treated with quercetin (100 mm or more), were lower than those of untreated controls (tab. 1), and there was no remission of symptoms in infected plants. after 'ca. p. asteris'-infected c. roseus shoots were grown through three subcultures on media with different quercetin concentrations, molecular analyses using consecutive pcr reactions with r16f1/r0 and r16f2n/r2 primer pairs showed the presence of 'ca. p. asteris' in all of the tested samples on media supplemented with 10 mm or 100 mm quercetin and in positive control (fig. 1). when total dna in the concentration of 20 ng µl–1 or 10 ng µl–1 was used as template, all samples were positive in the direct pcr, but pcr using template acta bot. croat. 69 (2), 2010 157 effect of quercetin on phytoplasma u:\acta botanica\acta-botan 2-10\334 curkovic perica.vp 11. listopad 2010 11:21:00 color profile: disabled composite 150 lpi at 45 degrees 158 acta bot. croat. 69 (2), 2010 ]urkovi]-perica m., je@i] m. tab. 1. effect of different quercetin concentrations on shoot elongation and fresh weight increase on phytoplasma-infected catharanthus roseus shoots in vitro. substance and concentration shoot elongation1 fresh weight increase1 phytoplasma infected quercetin2 (10 mm) quercetin2 (100 mm) quercetin2 (1000 mm) etoh (1‰) control 1.16±0.13 c 1.01±0.07 d decay 1.19±0.11 c 1.21±0.12 c 1.22±0.1 c 1.04±0.06 d decay 1.33±0.23 c 1.39±0.27 c non-infected quercetin2 (100 mm) etoh (1‰) control 1.48±0.2 b 1.82±0.31 a 1.86±0.45 a 2.48±0.26 b 3.16±0.59 a 3.2±0.48 a 1 mean ± standard deviation. shoot elongation and fresh weight increase were calculated as the ratio of the final and the initial shoot length or fresh weight, respectively. means labeled with the identical letters are not significantly different at the 95% level of confidence. 2 quercetin stock solution was prepared in ethanol (1‰ final concentration in the medium fig. 1. agarose gel (1%) electrophoresis of nested pcr amplification products of phytoplasma 16s rdna obtained using primer pair r16f1/r0. m – molecular weight marker fx174 bsuri digested; ba – 'ca. p. asteris' –infected catharanthus roseus shoot from the medium supplemented with: 2.2 mm 6-benzylaminopurine. 1 – pcr positive control, 2 – 10 mm quercetin, 3 – 100 mm quercetin, 4 – ethanol; 5 – healthy c. roseus shoot from the medium supplemented with: 2,2 mm ba, 6 – 100 mm quercetin, 7 – ethanol, 8 – water control. u:\acta botanica\acta-botan 2-10\334 curkovic perica.vp 11. listopad 2010 11:21:03 color profile: disabled composite 150 lpi at 45 degrees in the concentration of 5 ng µl–1 dna did not amplify visible pcr product in samples treated with 10 mm or 100 mm quercetin (fig. 2). however, phytoplasma presence was again confirmed in all samples in nested pcr (fig. 3). for the shoots treated with 1 mm quercetin an additional second nested pcr, using the primer pair r16(i)f1/r1, was performed after the first subculture. phytoplasma was detected in seven out of eight tested samples, although shoots were already decaying. therefore, the fact that phytoplasma was detected in the second nested pcr or was not detected at all in one shoot is irrelevant since this high quercetin concentration (1 mm) also caused the decay of the host. discussion reports on quercetin toxicity in plants are quite ambiguous, and while some researchers found no deleterious effects of quercetin in the concentration range of 10–1000 µm on arabidopsis plants (reigosa and pazos-malvido 2007), others found that quercetin in the concentration of 100 µm and 333 µm was toxic to the same species (parvez et al. 2004). basile et al. (2000) found out that quercetin inhibits seed germination of raphanus sativus. in our experiments, quercetin supplemented to the medium in the concentration of 100 mm, and higher, caused leaf yellowing and browning in both non-infected and infected periwinkle shoots. the experience from other experimental systems shows that the mode of application can greatly influence the outcome of quercetin treatment (cushnie and lamb 2005). acta bot. croat. 69 (2), 2010 159 effect of quercetin on phytoplasma fig. 2. agarose gel (1%) electrophoresis of direct pcr amplification products of phytoplasma 16s rdna obtained using primer pair r16f0/r1. m – molecular weight marker 1kb ladder (frementas); ba – 'ca. p. asteris' –infected catharanthus roseus shoot from the medium supplemented with: 2.2 mm 6-benzylaminopurine. 1 – pcr positive control, 2 – 10 mm quercetin, 3 – 100 mm quercetin, 4 – ethanol, 5 – water control. dna concentration is indicated by the letter a=20 ng ml–1, b=10 ng ml–1, c=5 ng ml–1 u:\acta botanica\acta-botan 2-10\334 curkovic perica.vp 11. listopad 2010 11:21:04 color profile: disabled composite 150 lpi at 45 degrees since in our experiment quercetin was supplied to plants via liquid medium, which would allow better uptake of this flavonoid through the plant's vascular system, it is quite plausible that concentrations used in this experiment could have been toxic to healthy and infected plants. although quercetin treatment proved to have bacteriostatic or bactericidal effect against many bacteria (cushnie and lamb 2005), the titer of phytoplasma was only slightly affected in the shoots treated with 10 mm or 100 mm quercetin. moreover, the severity of the symptoms increased in treated phytoplasma-infected plants. the undesirable effect of quercetin treatment, resulting in the stunting of non-infected and infected plants and in increased severity of symptoms in infected plants, might be explained by several hypothesized mechanisms. first of all, quercetin may interfere with auxin transport through plants by binding to auxin transport membrane protein complexes (murphy at al. 2000) thus disturbing the balance of plant growth regulators in healthy and infected shoots. phytoplasmas also interfere with auxin transport in plants (aldaghi et al. 2009). the addition of quercetin might have increased severity of symptoms in phytoplasma-infected shoots by additionally interfering with auxin transport. periwinkle shoots infected by 'ca. p. asteris' exhibit symptoms like proliferations, leaf curling and internode shortening. however, stunting was even more pronounced in quercetin-treated than in non-treated infected plants, moreover leaf browning and appearance of black spots on the leaf edges appeared on quercetin treated plants. 160 acta bot. croat. 69 (2), 2010 ]urkovi]-perica m., je@i] m. fig. 3. agarose gel (1%) electrophoresis of nested pcr amplification products of phytoplasma 16s rdna obtained using primer pair r16f2/r2. m – molecular weight marker 1kb ladder (frementas); ba – 'ca. p. asteris' –infected catharanthus roseus shoot from the medium supplemented with: 2.2 mm 6-benzylaminopurine. 1 – pcr positive control, 2 – 10 mm quercetin, 3 – 100 mm quercetin, 4 – ethanol, 5 – water control. dna concentration is indicated by the letter a=20 ng ml–1, b=10 ng ml–1, c=5 ng ml–1 u:\acta botanica\acta-botan 2-10\334 curkovic perica.vp 11. listopad 2010 11:21:06 color profile: disabled composite 150 lpi at 45 degrees spencer et al. (2003) have showed that quercetin, when present in tissue culture media in concentrations higher than 10 µm, actually promoted oxidative damage of cells. in our experiment, healthy and infected plants performed worse on quercetinthan on control medium, with phytoplasma-infected shoots showing greater susceptibility to potentially toxic effects of quercetin. the results presented in this paper do not reveal the mechanism by which quercetin increased severity of the symptoms in phytoplasma–infected periwinkles, but it is possible that its negative effect is a consequence of several mechanisms. the supplement of quercetin had no curative effect on 'ca. p. asteris'. although quercetin slightly reduced phytoplasma titer in c. roseus tissues, its' detrimental effect on plantlets outweighs the possible benefits. the addition of quercetin made symptoms of phytoplasma infection even worse, probably due to synergistic effect of phytoplasma and quercetin as an auxin transport inhibitor. acknowledgements we gratefully acknowledge professor assunta bertaccini for phytoplasma reference strains. references aldaghi, m., massart, s., bertaccini, a., jijakli, m. h., lepoivre, p., 2009: identification of host genes potentially implicated in the malus pulmila and 'candidatus phytoplasma mali' interactions. proceedings 21 icvf conference, neustadt, 43–44. basile, a., sorbo, s., giordano, s., ricciardi, l., ferrara, s., montesano, d., castaldo cobianchi, r., vuotto, m. l., ferrara, l., 2000: antibacterial and allelopathic activity of extract from castanea sativa leaves. fitoterapia 71, 110–116. christensen, n. m., axelsen, k. b., nicolaisem, m., schultz, a., 2005: phytoplasmas and their interactions with hosts. trends in plant science 10, 526–535. cushnie, t. p. t., lamb, a. j. 2005: antimicrobial activity of flavonoids. international journal of antimicrobial agents 26, 343–335. ]urkovi] perica, m., 2008a: auxin-treatment induces recovery of phytoplasma-infected periwinkle. 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metabolites on arabidopsis thaliana germination and root growth. journal of chemical ecology 33, 1456–1466. rusak, g., kraja^i], m., ple[e, n., 1997: inhibition of tomato bushy stunt virus infection using a quercetagetin flavonoid isolated from centaruea rupestris l. antiviral research 36, 125–129. spencer, j. p. e., kuhnle, g. g. c., williams, r. j., rice-evans, c., 2003: intracellular metabolism and bioactivity of quercetin and its in vivo metabolites. biochemical journal 372, 173–181. verma, v. s., 1973: study on the effect of flavonoids on the infectivity of potato virus x. zentralblatt für bakteriologie, parasitenkunde, infektionskrankheiten und hygiene. zweite naturwissenschaftliche abt.: allgemeine, landwirtschaftliche und technische mikrobiologie 128, 467–472. 162 acta bot. croat. 69 (2), 2010 ]urkovi]-perica m., je@i] m. u:\acta botanica\acta-botan 2-10\334 curkovic perica.vp 11. listopad 2010 11:21:06 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 71 (1), 187–193, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 short communication natural occurrence of cucumber mosaic virus infecting water mint (mentha aquatica) in antalya and konya, turkey mehmet a. sevik department of plant protection, faculty of agriculture, university of ondokuz mayis, samsun, turkey abstract – a virus causing a disease in mint (the aromatic and culinary plant) has recently become a problem in the taurus mountains, a mountain range in the mediterranean region of turkey. to detect the virus and investigate its distribution in the region, mint leaf samples were collected from the vicinity of spring areas in the plateaus of antalya and konya in 2009. it was found that cucumber mosaic virus (cmv) was detected in 27.08% of symptomatic samples tested by das-elisa. to the best of our knowledge, this is the first report of cmv on mint plants in this region of turkey. key words: disease, cucumber mosaic virus, detection, mentha aquatica introduction mentha is a genus of flowering plants in the family lamiaceae including about 30 species found in the temperate regions of the world (dorman et al. 2003). species such as m. aquatica l. and m. longifolia (l.) are used as wild vegetables and culinary herbs (naghibi et al. 2005). m. aquatica (water mint) is a perennial plant. as the name suggests, it occurs in the shallow margins and channels of streams, rivers, pools, dikes, ditches, canals, wet meadows, marshes and fens (ling 2011). several viruses have been associated with disease symptoms in mint including cucumber mosaic virus, alfalfa mosaic virus, tomato spotted wilt virus, impatiens necrotic spot virus, arabis mosaic virus, strawberry latent ringspot virus, tobacco mosaic virus, tobacco ringspot virus, tomato leaf curl virus, tomato aspermy virus, cherry rasp leaf virus, lychnis ringspot virus, mint virus 1, mint virus-2, mint virus x, mint vein banding associated virus, peppermint latent virus, and peppermint stunt virus (vicchi and bellardi 1988, sether et al. 1991, de angelis et al. 1993, fletcher 2001, postman et al. 2004, tzanetakis et al. 2004, samad et al. 2008, tzanetakis et al. 2010). acta bot. croat. 71 (1), 2012 187 * corresponding author, e-mail: malis@omu.edu.tr copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\436 sevik.vp 26. o ujak 2012 14:49:09 color profile: disabled composite 150 lpi at 45 degrees cucumber mosaic virus (cmv), the type member of the plant virus genus cucumovirus (family bromoviridae), has a wide host range and infects a great variety of important crop plants, making it one of the most economically significant plant viruses (palukaitis et al. 1992). cmv infects more than 1200 plant species (roossinck et al. 2001). its ubiquitous nature may be attributed to its broad host range, non-persistent transmission by more than 86 aphid species in the field (edwardson and christie 1991) and transmission through seed in some hosts (o’keefe et al. 2007). seed transmission plays a pivotal role in the survival of the virus from season to season (johansen et al. 1994). therefore, the management of a cmv outbreak is difficult in the field (grube et al. 2000). ornamental plants, especially those propagated vegetatively as bulbs or rhizomes may be virus reservoirs (flasinski et al. 1995). interestingly, water mint is a herbaceous rhizomatous perennial plamt. the rhizomes are wide-spreading, fleshy, and bear fibrous roots. moreover, an important number of plant species, including many weeds, have been reported to serve as reservoirs for cmv during the intercropping season (chatzivassiliou et al. 2004). in previous studies, cmv infections have been frequently reported from turkey on different plant families showing mild to severe symptoms (erdiller and ertunç 1988, yilmaz et al. 1995, arli-sokmen et al. 2005). in recent years, in mint-growing areas around water springs of antalya and konya plateaus, a virus-like disease has become a major problem causing severe symptoms. the virus-like symptoms are exhibited by many mint plants, but little is known about the etiology of the disease. the objectives of the study were to identify the virus and characterize the disease it caused. materials and methods surveys and sample collection in 2009, surveys were performed in 14 natural spring water areas in some plateaus of antalya and konya provinces containing mint plants. leaf samples showing virus-like symptoms were taken from mint plants exhibiting symptoms, placed in polythene bags and stored at –20 °c until use. serological testing das-elisa method was used to detect the virus in mint leaf samples and performed according to clark and adams (1977) and the instructions of the cmv antiserum manufacturer (bioreba, switzerland). leaf samples with typical symptoms of virus infections were ground (1g leaf/5 ml buffer) in extraction buffer (pbs: 0.13 m nacl, 0.014 m kh2po4, 0.08 m na2hpo4, 0.002 m kcl, ph: 7.4, containing 0.05% tween-20), added to microplate wells (nunc microwell, denmark) after coating with cmv-specific polyclonal antiserum diluted in carbonate buffer (ph: 9.6) and incubated at 4 °c overnight. plates were washed three times with pbs/tween-20 buffer and coated with alkaline phosphatase-conjugated antibody diluted in extraction buffer and incubated for 4h at 37 °c. after washing, p-nitrophenyl phosphate (sigma) in diethanolamine substrate buffer (0.5 mg ml–1, ph: 9.8) was added to the wells and incubated at room temperature for 30–180 min. absorbance values were read at 405 nm using a microplate reader (tecan spectra ii). extraction buffer and healthy plants were used as negative controls. samples were considered to be 188 acta bot. croat. 71 (1), 2012 sevik m. a. u:\acta botanica\acta-botan 1-12\436 sevik.vp 26. o ujak 2012 14:49:09 color profile: disabled composite 150 lpi at 45 degrees positive when the absorbance values at 405 nm (a405) values exceeded the mean of the negative controls by least a factor of three (kutluk-yilmaz 2010). biological testing nicotiana tabacum l. 'samsun' was grown in a growth chamber at 23 °c with a 16 h light/8 h dark photoperiod cycle. the samples of water mints collected (previously used for elisa) were used for preparation of inoculum in the buffer and all fully expanded leaves of plants at 2–4 leaves growth stage were inoculated. results and discussion most of the farmers and people of antalya and konya regions complained of an increased incidence of mosaic disease in mint plants over the past few years. besides their culinary use, mint plants are also used during livestock grazing. it is well known that mint has been used for centuries for its medicinal properties and in the food and fragrance industries (mimica-dukic et al. 2003). visual surveys of the mint plants revealed a high incidence of leaf mosaic (fig. 1), deformation and severely affected apical plant growth. the newly growing apical leaves were small with very conspicuous symptoms. during the surveys, most spring areas contained plants with virus-like symptoms. virus-like symptoms were observed in all 14 mints grown areas surveyed. cmv was detected by das-elisa in the mint plants from all surveyed areas. the incidence of cmv in the symptomatic plants varied in the different springs. serological assay results revealed a variation in virus incidence among the different springs surveyed. the incidence of cmv was estimated as 27.08% in antalya and konya provinces, ranging from 14.2% to 42.8% in the springs visited. acta bot. croat. 71 (1), 2012 189 detection of cucumber mosaic virus in mint plants fig. 1. symptoms associated with natural infections by cmv on water mint plants. u:\acta botanica\acta-botan 1-12\436 sevik.vp 26. o ujak 2012 14:49:10 color profile: disabled composite 150 lpi at 45 degrees in the study, tests were conducted using several negative controls for cmv. therefore, the range of absorbance values of negative controls varied from 0.066–0.118 at 405 nm. positive samples gave absorbance values of 0.452– to 1.842 after 2 h substrate incubation (table 1). the presence of cmv in water mint plant was verified in samples by transmission to indicator test plants, tobacco (n. tabacum). tobacco plants mechanically inoculated with extracts of elisa-positive plants showed local necrotic spot on leaves 1 week after inoculation (fig. 2). these symptoms were similar to those that were described previously for the virus (choi et al. 2004). the cucumovirus cmv has been reported as the virus infecting different plants worldwide (varveri, and boutsika 1999) and different regions of turkey (erdiller and ertunç 1988, unlu and guldur 2004), but infection of mint with cmv is reported for the first time in these regions. the report of cmv affecting mentha was in 1966 in germany, where it was found to be the cause of a mosaic disease of peppermint. cmv was found in cultivated medicinal plants, including mint, in hungary (tzanetakis et al. 2010). hani (1971) studied the epidemiology of cmv in switzerland and reported mentha sp. as a weed host for the virus in the vicinity of tobacco fields where several cmv isolates were causing problems. cmv was also isolated from mentha sp. (vicchi and bellardi 1988), m. piperita and m. palustris in italy by crescenzi et al. (1993). m. pulegium for cmv was found infected for the first time by elisa in greece (chatzivassiliou et al. 2004). weeds, native plants and seed transmission may have a significant effect on virus epidemiology (duffus 1971) and knowledge of weed reservoirs and vectors of viruses is essential for understanding the epidemiology. cmv-infected weeds may also play a significant role in its spread to crops. in 1998, 28 fields in samsun, turkey were surveyed and 222 190 acta bot. croat. 71 (1), 2012 sevik m. a. tab. 1. range of absorbance values of positive and negative samples and incidence of cmv. no. of springs surveyed no. of samples tested the means das-elisa absorbance values incidence (%) negative samples positive samples 1 12 0.084–0.215 0.767–1.231 16.6 2 4 0.090–0.193 0.829 25.0 3 8 0.059–0.330 0.452–0.669 37.5 4 10 0.104–0.364 0.860–1.076 20.0 5 6 0.093–0.316 1.290–1.842 33.3 6 5 0.110–0.299 1.082 20.0 7 7 0.075–0.317 0.773–0.803 42.8 8 7 0.060–0.262 1.318 14.2 9 9 0.124–0.355 0.793–0.916 22.2 10 8 0.078–0.326 0.510–0.599 37.5 11 7 0.066–0.303 0.685–0.697 28.5 12 6 0.088–0.249 0.860 16.6 13 3 0.115–0.354 0.965 33.3 14 4 0.128–0.338 0.495–0.852 28.5 u:\acta botanica\acta-botan 1-12\436 sevik.vp 26. o ujak 2012 14:49:10 color profile: disabled composite 150 lpi at 45 degrees weed samples were collected from pepper fields. of the samples tested, 7.7% were found to be infected with cmv. cmv was found to be the virus most frequently detected, in 13 weed species out of 24 species (arli-sokmen et al. 2005). in experimental work, the virus is most frequently transmitted mechanically, and sap, purified virions, and viral rna are all infectious via mechanical transmission (roossinck 2001). in addition, dheepa and paranjothi (2010) reported that cmv from banana could be transmitted by rhizome inoculation and mechanical methods. to help prevent cmv from spreading, farmers are recommended to remove any mint plants infected with cmv near springs. the results obtained in this investigation clearly demonstrate that cmv is widely distributed in different mint growing areas of antalya and konya, turkey. this is the first report of cmv from water mint plants (m. aquatica) in turkey. acknowledgments i thank to d. sevik for helping and assistance during surveys of these regions and sample collections. references arli-sokmen, m., mennan h, sevik m. a., ecevit o., 2005: occurrence of viruses in field-grown pepper crops and some of their reservoir weed hosts in samsun, turkey. phytoparasitica 33, 347–358. chatzivassiliou, e. k., efthimiou, k., drossos, e., papadopoulou, a., poimenidis, g., katis, n. i., 2004: a survey of tobacco viruses in tobacco crops and native flora in greece. european journal of plant pathology 110, 1011–1023. choi, g. s., kim, j. h., kim, j. s., choi, j. k., 2004: characterization of cucumber mosaic virus isolated from water chickweed (stellaria aquatica). plant pathology journal 20, 131–134. acta bot. croat. 71 (1), 2012 191 detection of cucumber mosaic virus in mint plants fig. 2. virusinoculated tobacco (nicotiana tabacum l. 'samsun') showing local necrotic spots on leaves, characteristic of cmv infection. u:\acta botanica\acta-botan 1-12\436 sevik.vp 26. o ujak 2012 14:49:12 color profile: disabled composite 150 lpi at 45 degrees clark, m. r., adams, a. m., 1977: characteristics of the microplate method of enzyme linked immunosorbent assay for the detection of plant viruses. journal of general virology 34, 475–483. crescenzi, a., barbarossa, l., gallitelli, d., martelli, g. p., 1993: cucumber mosaic cucumovirus populations in italy under natural epidemic conditions and after a satellite-mediated protection test. plant disease 77, 28–33. de angelis, j. d., sether, d. m., rossignol, p. a., 1993: survival, development, and reproduction in western flower thrips (thysanoptera, thripidae) exposed to impatiens necrotic spot virus. environmental entomology 22, 1308–1312. dheepa, r., paranjothi, s., 2010: transmission of cucumber mosaic virus (cmv) infecting banana by aphid and mechanical methods. emirates journal of food and agriculture 22, 117–129. dorman, h. j, kosar, m., kahlos, k., holm, y., hiltunen, r., 2003: antioxidant properties and composition of aqueous extracts from mentha species, hybrids, varieties, and cultivars. journal of agricultural and food chemistry 51, 4563–4569. duffus, j. e., 1971: role of weeds in the incidence of virus diseases. annual review of phytopathology 9, 319–340. edwardson, j. r., christie, r. g., 1991: cucumoviruses. in: crc handbook of viruses infecting legumes, 293–319. crc press, boca raton. erdiller, g., ertunç, f., 1988: identification of muskmelon viruses in ankara province. journal of turkish phytopathology 17, 47–56. flasinski, s., scott, s. w., barnett, o. w., sun, c., 1995: diseases of peperomia, impatiens and hibbertia caused by cucumber mosaic virus. plant disease 79, 843–848. fletcher, j. d. 2001: new hosts of alfalfa mosaic virus, cucumber mosaic virus, potato virus y, soybean dwarf virus, and tomato spotted wilt virus in new zealand. new zealand journal of crop and horticultural science 29, 213–217. grube, r. c., zhang, y., murphy, j. f., loaiza-figueroa, f., lackney, v. k., provvidenti, r., jahn, m. k., 2000: new source of resistance to cucumber mosaic virus in capsicum frutescens. plant disease 84, 885–891. hani, a. 1971: on the epidemiology of cucumber mosaic virus in tessin. phytopathologische zeitschrift 72, 115–144. johansen, e., edwards, m. c., hampton, r. o., 1994: seed transmission of viruses: current perspectives. annual review of phytopathology 32, 363–386. kutluk-yilmaz, n.d. 2010: interactions between beet necrotic yellow vein virus and beet soilborne virus in different sugar beet cultivars. anadolu journal of agricultural sciences 25, 68–74. ling, c., 2011: mentha aquatica. usgs nonindigenous aquatic species database, gainesville, fl. mimica-dukic, n., bozin, b., sokovic, m., mihajlovic, b., matavulj, m., 2003: antimicrobial and antioxidant activities of three mentha species essential oils. planta medica 69, 413–419. 192 acta bot. croat. 71 (1), 2012 sevik m. a. u:\acta botanica\acta-botan 1-12\436 sevik.vp 26. o ujak 2012 14:49:12 color profile: disabled composite 150 lpi at 45 degrees naghibi, f., mosaddegh, m., motamed, s. m., ghorbani, a., 2005: labiatae family in folk medicine in iran: from ethnobotany to pharmacology. iranian journal of pharmaceutical research 2, 63–79. o’keefe, d. c., berryman, d. i., coutts, b. a., jones, r. a. c., 2007: lack of seed coat contamination with cucumber mosaic virus in lupin permits reliable, large-scale detection of seed transmission in seed samples. plant disease 91, 504–508. palukaitis, p., roossinck, m. j., dietzgen, r. g., francki, r. i. b., 1992: cucumber mosaic virus. in: maramorosch, k., murphy, f. a. and shatkin, a. j. (eds.), advances in virus research, 281–348. academic press, san diego. postman, j. d., tzanetakis, i. e., martin, r. r. 2004: first report of strawberry latent ringspot virus in mentha sp. from north america. plant disease 88, 907. roossinck, m. j., 2001: cucumber mosaic virus, a model for rna virus evolution. molecular plant pathology 2, 59–63. samad, a., gupta, m. k., shasany, a. k., ajayakumar, p. v., alam, m., 2008: begomovirus related to tomato leaf curl pakistan virus newly reported in mentha crops in india. new disease reports 18, 11. sether, d. m., deangelis, j. d., rossignol, p. a. 1991: first report of tomato spotted wilt virus in peppermint (mentha piperita). plant disease 75, 644. tzanetakis, i. e., postman, j. d., martin, r. r., 2004: identification, detection and phylogenetic analysis of new viruses infecting mint. phytopathology 94, 104. tzanetakis, i. e., postman, j. d., samad, a., martin, r. r., 2010: mint viruses: beauty, stealth, and disease. plant disease 94, 4–12. unlu, s., guldur, m. e., 2004: determination of infection ratio of cucumber mosaic virus (cmv) on pepper in sanliurfa. journal of faculty of agriculture, harran university 8, 83–89. varveri, c., boutsika, k., 1999: characterization of cucumber mosaic cucumovirus isolates in greece. plant pathology 48, 95–100. vicchi, v., bellardi, m. g., 1988: the role of weeds in the epidemiology of gladiolus viruses and mlo. acta horticulture 234, 371–378. yilmaz, m. a., baloglu, s., özaslan, m., güldür, m. e., 1995: plant viruses detected in horticultural crops in the gap region (in turish). proc symposium of plant protection problems and prevention, suggestions of gap region, sanliurfa, turkey, 241–250. acta bot. croat. 71 (1), 2012 193 detection of cucumber mosaic virus in mint plants u:\acta botanica\acta-botan 1-12\436 sevik.vp 26. o ujak 2012 14:49:12 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 71 (1), 159–175, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 arbuscular mycorrhizal and dark septate fungal associations in shallot (allium cepa l. var. aggregatum) under conventional agriculture perumalsamy priyadharsini1, radha raman pandey2, thangavelu muthukumar1* 1 root and soil biology laboratory, department of botany, bharathiar university, coimbatore 641 046, tamilnadu, india. 2 department of life sciences, manipur university, canchipur, imphal 795 003, india abstract – we examined roots of the shallot (allium cepa l. var. aggregatum), one of the most popular cultivated crops of the family aliaceae, cultivated under conventional agriculture for arbuscular mycorrhizal fungal (amf) and dark septate fungal endophyte (dse) associations. all the plants had dual colonization of both amf and dse associations. the intermediate-type amf morphology in the shallot is the first report of this amf type for the family aliaceae. the extents of total amf and dse colonization ranged from 20.7 to 67.3% and 3.6 to 35.3% respectively and varied significantly among fields. though no significant relationship existed between total amf and dse variables, they were correlated to the soil variables. significant correlations existed between soil p and microscelerotia and also between soils n and k and amf spore numbers. a total of six amf spore morphotype belonging to glomus and scutellospora were identified. scutellospora calospora was the most dominant morphotype in the studied fields. keywords: allium, arbuscular, endophyte, mycorrhiza, glomus, scutellospora abbreviations: amf – arbuscular mycorrhizal fungus, dse – dark-septate endophyte introduction the herbaceous biennial allium cepa l. (onion) is the most widely cultivated taxon in the family alliaceae. the onion a. cepa var. aggregatum is native to south west asia, but cultivated worldwide. crops benefit from arbuscular mycorrhizal fungus (amf) through enhanced uptake of nutrients with low mobility, especially phosphorus. the non-nutritional benefits of amf include alleviation of plant stresses caused by biotic and abiotic facacta bot. croat. 71 (1), 2012 159 * corresponding author, e-mail: tmkum@yahoo.com copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:38 color profile: disabled composite 150 lpi at 45 degrees tors, and the stabilizing of soil aggregates (smith and read 2008, bolandnazar et al. 2007, jaime et al. 2008, gianinazzi et al. 2010). the onion has an inefficient, mostly unbranched, shallow root system with sparse root hairs, and cannot maintain adequate uptake of nutrients such as phosphorus (p) that diffuse slowly through the soil solution, which has a negative effect on yields (mengel and kirkby 2001). onions are highly mycorrhizal-dependent (deressa and schenk 2008). previous studies do indicate that the onion is highly responsive to mycorrhization, resulting in improved plant growth and yield under normal as well as stressed conditions (jaime et al. 2008, bolandnazar et al. 2007, goussous and mohammad 2009, galván et al. 2011). a significant correlation between natural amf colonization and onion yields in conventionally managed onion farmlands has recently been reported (galván et al. 2009). in conventional agriculture, large amounts of fertilizers are used to increase the yield of onion (bosch-serra and currah 2002). additionally large amounts of biocides are used in shallot cultivation as this plant is very susceptible to pests and diseases (anonymous 1986, suhardi 1996). large scale use of fertilizers, biocides and tillage affect both arbuscular mycorrhizal formation and function (see plenchette et al. 2005). diversity of amf species seems to be essential for sustainable functioning of the ecosystem in the event of sudden changes in environmental conditions (wang et al. 1985, abbott and gazey 1994). studies indicate the existence of variation in life histories, reproduction abilities and morphology among amf species (dickson 2004, hart et al. 2001, hart and reader 2002). agricultural practices may induce selection pressure in such a way that a certain amf group could adapt to changes, establish and proliferate better than others (singh et al. 2008). for example, singh et al. (2008) indicated that camellia sinensis growing under natural conditions harboured a greater diversity of amf species than those under cultivation. further the composition of amf community could be strongly influenced by the individual plant species through differential effects on mycorrhizal establishment and sporulation (sanders and fitter 1992, bever et al. 1996). it is becoming increasingly important to gain a better understanding of amf diversities under field conditions, as suggested by husband et al. (2002). limited studies have examined amf diversities associated with specific plant species like vitis vinifera (balestrini et al. 2010), solanum tuberosum (das and kayang 2010b), michelia champaca (das and kayang 2010a), phaseolus aureus (valsalakumar et al. 2007) and camellia sinensis (singh et al. 2008). recently, galván et al. (2009) reported amf polytypes in onion roots under organic and conventional farming systems using rdna sequencing. crop roots are also colonized by a diverse group of melanaceous, septate fungi known as dark septate fungal endophytes (dse). the function of dse in plant roots is unclear and may range from pathogenic or saprophytic to mutualistic (jumpponen and trappe 1998, jumpponen 2001). the occurrence of dse on several temperate and tropical crop species has been reported (jumpponen and trappe 1998, muthukumar and tamilselvi 2010). addy et al. (2005) speculated that the nature of dse association with roots may be broader than nutrient transfer as there is no evidence of any specific fungal requirements from the host. an analysis of the role of dse in ecosystems (mandyam and jumpponen 2005) indicated facilitation of nutrient uptake of the host plant, alterations in host water uptake, stress tolerance and utilization of wider nutrient pools by the host through dse. mandyam and 160 acta bot. croat. 71 (1), 2012 priyadharsini p., pandey r. r., muthukumar t. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:39 color profile: disabled composite 150 lpi at 45 degrees jumpponen (2008) also suggested that a knowledge of dse abundance in relation to root-associated fungi is essential to understand this significance. in this study, we examined root colonization by amf and dse in shallots under conventional agriculture. the three main questions addressed in this study were (i) does amf and dse colonization vary within and between cultivated fields? (ii) is there any relationship between amf and dse within roots? (iii) do soil factors influence amf and dse colonization or structures within roots? in addition we also assessed the amf spore numbers to see if they were related to colonization levels and assessed their diversity with the shallot. materials and methods sampling onion root and soil samples were collected during december 2009 from 20 different onion fields (hereafter referred to as f1 to f20) from sathyamangalam (11°28'n and 77°59'e, 540 m a.s.l), tamil nadu, southern india. the average annual rainfall of this area is 360–600 mm. all the fields selected had uniform cultivation practices and cultivation periods (november to january). the fields were canal-irrigated. fertilization began with the application of diammonium phosphate (dap, 16–18% n, 40–48% p) during tillage. fertilizers were further sequentially applied at 25 days (urea-46%n and dap), 50 days (ammonium sulphate-21% n, 24% s) and 60 days (muriate of potash) after planting. the crop duration was 70–75 days. five randomly selected plants were sampled from each field resulting in a total of 100 plants. root samples were collected by uprooting the plants. the roots were gently washed free of soil with water, fixed in formalin-acetic acid-70% alcohol (5:5:90; v:v:v) and brought to the laboratory for further processing. soil shaken from roots and next to plants was collected and shade dried. the dried soil was divided into two halves. one half of the air-dried soil collected from individual plants was packed separately in polythene bags and stored at 4°c to enumerate and extract amf spores. the second half of the soil of all the individuals from a field was mixed together to form a composite soil sample. these composite soil samples were used to assess the soil chemistry. determination of soil characters soil ph was determined in 1:1 soil: water (v:v) using a digital ph meter soon after the soil samples arrived in the laboratory. soil samples were analyzed for sand, silt and clay fractions by the hydrometer method (bowels 1992). the total nitrogen (n) and available p were determined according to jackson (1971) and exchangeable potassium (k) was determined after extraction with ammonium acetate (jackson 1971). the iron (fe), manganese (mn), zinc (zn) and copper (cu) were determined according to dtpa method (lindsay and norvell 1978). preparation of roots for amf and dse assessment fixed roots were washed thoroughly in tap water. the roots were cut into 1-cm bits, cleared in 2.5% koh at 90 °c (koske and gemma 1989) for 60 min, acidified with 5n hcl acta bot. croat. 71 (1), 2012 161 root fungal associations in shallot onion u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:39 color profile: disabled composite 150 lpi at 45 degrees and stained with tryphan blue (0.05% in lactoglycerol) overnight. the stained roots were examined with an olympus bx 51 compound microscope (400×) for amf and dse structures. aseptate inter or intracellular, linear or coiled fungal hyphae accompanied with arbuscules or arbusculate coils were used to designate amf colonization. melanized, regularly septate fungal hyphae with microsclerotia or moniliform cells characterized dse colonization. the percentage of root length colonized by amf and dse was estimated according to the magnified intersection method (mcgonigle et al. 1990). classification of amf morphology of the shallot was as per dickson (2004). enumeration and isolation of amf spores spores were extracted from 100 g of soil by modifications of the wet-sieving and decanting techniques. after wet-sieving and decanting, the residues on the sieves (710 and 37 mm) were washed into beakers. the beaker contents were collected over a filter paper, spread on petri dishes and scanned under a dissection microscope at 40× magnification. all intact spores (non-collapsed with cytoplasmic content) free from parasitic attack were counted. sporocarps and spore clusters were considered as one unit. for identification, spores with similar morphology were mounted on slides with polyvinyl alcohol-lactic acid-glycerol (pvlg) or pvlg melzer’s reagent for identification (koske and tessier 1983). spores were identified based on spore morphology and sub-cellular characters (schenck and perez 1990) using descriptions available on schüsslers web site (http://www.lrz.de/~schuessler/ amphylo/amphylogeny.html), and invam (http://www.invam.caf.wvu.edu). frequency of occurrence (%) for each amf spore morphotype was calculated as the percentage of the number of the field soil possessing spores of that morphotype. amf species richness represents the number of taxa occurring in a field. statistical analysis data on amf and dse colonization and amf spore numbers were subjected to analysis of variance (anova) to assess the significance of variance among the plants and fields. pearson’s correlation was used to assess the relationship between the root colonization, spore numbers and the soil parameters (spss, windows version 9). spore numbers were log transformed and the percentage data on root colonization was arcsine transformed prior to analysis. results soil characteristics seventy percent of the fields studied had sandy loam soil with 30% of the remaining fields having sandy clay loam (tab. 1). soil chemistry varied between fields. the soil was moderately basic with the ph ranging from 7.9 (f4-6, 9) to 8.5 (f19). the ec ranged between 0.18 (f16) and 0.52 (f9) ds m–1. the total n, available p and exchangeable k range observed were 67 (f7, 18) to 118 (f19) mg kg–1, 5 (f7) to 8 (f17) mg kg–1 and 200 (f16) to 300 (f19) mg kg–1 respectively. similarly, soils had micronutrient concentration ranges of 3.82 (f1, 11) to 7.94 (f19) mg kg–1 fe, 2.08 (f1, 11) to 2.99 (f18) mg kg–1 mn, 1.33 (f1, 17) to 1.98 (f13) mg kg–1 zn and 0.69 (f16) to 1.66 (f3) mg kg–1 cu. 162 acta bot. croat. 71 (1), 2012 priyadharsini p., pandey r. r., muthukumar t. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:39 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .71 (1),2012 163 r o o t f u n g a l a s s o c ia t io n s in s h a l l o t o n io n tab 1. soil characteristics of shallot onion cultivated fields in tamil nadu. field sand silt clay soil type ph ec total n available p exchangeable k fe mn zn cu (d sm–1) (mg kg–1) f1 58 13 29 sandy clay loam 8.3 0.26 73 5.4 225 3.82 2.08 1.33 1.41 f2 40 16 44 sandy loam 8.0 0.30 76 6.4 215 4.22 2.16 1.43 1.32 f3 35 30 35 sandy loam 8.1 0.23 78 5.2 220 4.30 2.18 1.63 1.66 f4 62 19 19 sandy loam 7.9 0.29 70 5.6 235 4.72 2.19 1.52 1.23 f5 55 23 23 sandy loam 7.9 0.31 76 6.8 215 3.96 2.57 1.72 1.42 f6 60 16 24 sandy clay loam 7.9 0.50 80 7.6 205 3.90 2.52 1.59 1.37 f7 45 32 23 sandy loam 8.2 0.26 67 5.0 205 5.12 2.47 1.63 1.40 f8 45 23 32 sandy loam 8.1 0.27 72 5.8 220 5.69 2.52 1.78 1.52 f9 57 10 33 sandy loam 7.9 0.52 80 6.2 225 4.42 2.62 1.72 1.52 f10 57 24 19 sandy loam 8.0 0.34 77 5.8 220 3.85 2.67 1.66 1.12 f11 25 33 42 sandy loam 8.1 0.43 71 7.2 230 3.82 2.08 1.53 1.63 f12 42 29 29 sandy loam 8.2 0.26 76 6.4 215 4.22 2.36 1.46 1.52 f13 43 29 29 sandy loam 8.3 0.22 74 5.8 220 4.17 2.64 1.98 1.43 f14 55 20 25 sandy loam 8.1 0.23 71 7.2 235 4.20 2.64 1.55 1.27 f15 52 9 39 sandy clay loam 8.2 0.33 78 6.2 210 3.85 2.60 1.92 1.36 f16 36 44 20 sandy loam 8.1 0.18 81 7.5 200 4.72 2.84 1.54 0.69 f17 46 19 35 sandy clay loam 8.4 0.27 84 8.0 220 5.92 2.55 1.33 0.93 f18 76 12 12 sandy loam 8.2 0.38 67 7.0 230 6.88 2.99 1.42 0.98 f19 53 20 27 sandy clay loam 8.5 0.35 118 6.0 300 7.94 2.50 1.53 0.84 f20 52 19 30 sandy clay loam 8.2 0.32 84 7.0 235 6.16 2.74 1.40 0.79 u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 8 7 p r i y a d h a r s i n i n o v o . v p 2 6 . o u j a k 2 0 1 2 1 3 : 5 9 : 3 9 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s determination of amf and dse associations roots of all the onion plants examined from different fields had dual colonization of both amf and dse. amf colonization was characterized by the formation of an appressorium on the root surface (fig. 1a). a colonization peg arising from the appressorium spread within the roots to form intraradical colonization. colonization is characterized by intracellular hyphae with arbuscules (fig. 1b), intracellular hyphal coils or arbusculate coils (fig. 1c), intercellular hyphae and vesicles (fig. 1d). arbuscules were present in all the root samples examined. septate hyphae (fig. 1e) along with moniliform cells and/or microsclerotia (fig. 1f) characterized dse colonization. microsclerotia was absent in onion roots collected from three fields (f11, f12, f14), whereas moniliform cells were not observed in shallot roots collected from five fields (f1-3, f15, f17) (tab. 2). 164 acta bot. croat. 71 (1), 2012 priyadharsini p., pandey r. r., muthukumar t. fig. 1. arbuscular mycorrhizal (am) and dark septate endophyte (dse) fungal colonization in the shallot. a – appressorium (ap) of amf on the root surface; b – arbuscule (a) and intracellular hyphae (arrows) in root cell; c – arbusculate coil (ac) within a root cell; d – vesicles (v) and intercellular hyphae; e – septation (arrow heads) in dse hyphae; f – microsclerotia (ms) of dse in cortical cell. scale bars = 50µm. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:44 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .71 (1),2012 165 r o o t f u n g a l a s s o c ia t io n s in s h a l l o t o n io n tab. 2. extent of arbuscular mycorrhizal (am) and dark septate endophyte (dse) fungal association in rhizosphere soils of shallot onion. fields am dse colonization (%) spore number (100g–1) am fungal species colonization (%) rlh/rlhc rla/rlac rlv rltc rldh rlmo rlmi rldtc f1 31.8 ± 6.2 5.7 ± 2.2 11.0 ± 4.3 48.5 ± 11.7 11.2 ± 2.5 ge, gs1, sc 13.5 ± 3.0 – 0.7 ± 0.5 14.2 ± 3.3 f2 20.5 ± 4.8 2.4 ± 0.2 2.7 ± 1.6 25.6 ± 6.1 14.3 ± 0.9 gs, ga, sc 33.8 ± 4.7 – 1.5 ± 0.8 35.3 ± 5.0 f3 37.0 ± 5.2 10.1 ± 2.2 0.2 ± 0.1 47.3 ± 6.6 8.9 ± 2.6 ga, gs1 14.1 ± 4.9 – 0.2 ± 0.1 14.3 ± 5.0 f4 37.1 ± 2.5 7.4 ± 2.7 1.0 ± 0.8 45.5 ± 4.6 10.3 ± 1.6 ge, ga 12.4 ± 3.4 0.4 ± 0.5 0.7 ± 0.4 13.5 ± 4.0 f5 15.7 ± 3.2 3.9 ± 0.7 1.1 ± 0.7 20.7 ± 3.7 8.6 ± 1.4 ge, ga, sc 3.8 ± 1.4 1.4 ± 1.0 0.2 ± 0.1 5.4 ± 2.4 f6 31.2 ± 4.2 15.2 ± 3.6 0.4 ± 0.3 46.8 ± 6.3 16.7 ± 1.6 gs, gv, sc 4.3 ± 0.9 0.1 ± 0.1 0.1 ± 0.1 4.5 ± 0.9 f7 34.8 ± 3.0 9.5 ± 3.3 1.7 ± 1.2 46.0 ± 4.9 15.3 ± 2.2 gv, ge, sc 14.4 ± 3.2 0.8 ± 0.6 1.3 ± 0.5 16.5 ± 3.1 f8 34.5 ± 4.5 18.4 ± 3.7 0.2 ± 0.1 53.1 ± 4.9 17.1 ± 2.8 ga, sc 4.5 ± 0.7 0.3 ± 0.2 0.9 ± 0.3 5.7 ± 1.3 f9 35.1 ± 6.5 16.7 ± 5.2 0.3 ± 0.1 52.1 ± 10.8 15.1 ± 0.8 gs1, sc 3.1 ± 1.0 1.7 ± 1.2 0.3 ± 0.1 5.1 ± 1.9 f10 38.1 ± 4.3 12.9 ± 4.0 0.1 ± 0.1 51.1 ± 6.6 13.9 ± 2.2 ge, sc 6.2 ± 2.9 0.9 ± 0.5 0.2 ± 0.1 7.3 ± 3.1 f11 35.9 ± 4.6 16.3 ± 5.0 – 52.2 ± 8.2 8.9 ± 2.3 ge, gs1 4.7 ± 0.6 1.0 ± 0.9 – 5.7 ± 1.2 f12 33.5 ± 3.4 18.1 ± 4.5 0.5 ± 0.4 52.1 ± 5.5 11.9 ± 2.8 ga, gs, sc 4.4 ± 1.4 0.4 ± 0.5 – 4.8 ± 1.6 f13 38.7 ± 2.8 19.1 ± 3.0 0.1 ± 0.1 57.9 ± 5.6 8.8 ± 2.1 gs1 3.5 ± 1.3 1.9 ± 1.0 0.4 ± 0.6 5.8 ± 1.6 f14 30.6 ± 5.7 14.9 ± 3.2 0.6 ± 0.3 46.1 ± 8.5 7.4 ± 1.5 ge, gs1 3.8 ± 1.2 0.4 ± 0.4 – 4.2 ± 1.6 f15 30.3 ± 1.8 15.1 ± 2.3 0.1 ± 0.1 45.5 ± 3.5 8.1 ± 1.3 gs1 3.5 ± 1.2 – 0.1 ± 0.1 3.6 ± 1.2 f16 28.0 ± 3.5 21.6 ± 4.2 0.2 ± 0.1 49.8 ± 7.8 12.6 ± 2.7 ga, ge, gs, gs1, sc 21.7 ± 1.8 0.6 ± 0.4 0.2 ± 0.1 22.5 ± 1.8 f17 41.1 ± 2.0 22.8 ± 3.7 2.9 ± 1.4 66.8 ± 3.4 12.8 ± 3.2 ge, gs, gv,sc 18.1 ± 1.7 – 0.4 ± 0.3 18.5 ± 1.8 f18 38.8 ± 1.9 24.5 ± 1.9 1.4 ± 1.0 64.7 ± 2.1 13.3 ± 3.3 ga, gs, sc 19.7 ± 1.1 0.2 ± 0.2 0.5 ± 0.4 20.4 ± 0.7 f19 42.1 ± 1.1 23.9 ± 3.8 1.3 ± 0.7 67.3 ± 4.2 3.5 ± 0.7 ge, sc 19.5 ± 3.1 0.8 ± 0.6 0.2 ± 0.1 20.5 ± 3.8 f20 35.1 ± 3.6 8.5 ± 1.7 – 43.6 ± 5.2 3.7 ± 1.0 ge, gs, sc 13.0 ± 2.1 1.1 ± 0.7 0.3 ± 0.2 14.4 ± 2.3 plants(p4,200) 1.6 ** 2.4** 5.9** 1.8** <1 ns <1 ns 3.7** 2.6* <1 ns fields(f19,200) 12.0** 17.5** 20.9** 17.0** 3.71** 47.8** 3.0** 1.7* 34.4** p×f (f99,200) 2.81** 2.7** 3.0** 13.7** <1 ns 2.5** 1.6** <1 ns 2.2** rlh/rlhc – root length with hyphae or hyphal coils, rla/rlac – root length with arbuscles or arbusculate coils, rlv – root length with vesicles, rltc – total colonization, rldh – root length with dark septate hyphae, rlmo – root length with moniliform hyphae, rlmi – root length with microsclerotia, rldtc – dark septate endophyte total colonization, gs1 – glomus species 1, ga – glomus aggregatum, ge – glomus etunicatum, gs – glomus sinuosum, gv – glomus viscosum, sc – scutellospora calospora, numbers represent mean ± se **significant at 1% level, *significant at 5% level, ns-non significant. u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 2 \ 4 8 7 p r i y a d h a r s i n i n o v o . v p 2 6 . o u j a k 2 0 1 2 1 3 : 5 9 : 4 4 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s extent of amf colonization the extent of amf colonization and the root length with different amf structures varied significantly among roots collected from various fields (tab. 2). the percentage of total root length with amf colonization (%rltc) ranged from 20.7 (f5) to 67.3 (f19). total amf colonization and the percentage of root length with amf structures significantly varied among plants collected from a field and from different fields. the ranges of percentage root length with amf hyphae/ hyphal coils (%rlh/ rlhc), arbuscules/ arbusculate coils (%rla/ rlac) and vesicles (%rlv) were 15.7 (f5) to 42.1 (f19), 2.4 (f2) to 24.5 (f18) and 0.1 (f10, f13, f15) to 11.0 (f1) respectively. compared with average %rlh/ rlhc (33.5) and %rla/ rlac (14.3), %rlv was lower (1.3) and vesicles were not observed in onion roots collected from two fields (f11 and f20). correlation analysis indicated %rlh/ rlhc, %rla/ rlac and %rltc to be significantly and positively correlated to soil ph and fe. similarly, a significant positive correlation also existed between %rla/ rlac and soil mn. in contrast %rlv was significantly and negatively correlated to soil zn content. extent of dse colonization significant variation in percentage of root length with dse colonization (%rldtc) was evident among onion roots collected from different fields and ranged between 3.6 (f15) and 35.3 (f2). the percentage of root length with dark septate hyphae (%rldh) ranged from 3.1 (f9) to 33.8 (f2) and varied significantly among fields. likewise, the percentage of root length with moniliform hyphae (%rlmo) and microsclerotia (%rlmi) varied significantly both within and among fields ranged from 0.1 (f6) to 1.9 (f13) and 0.1 (f6, f15)to 1.5 (f2) respectively. the %rlmi was significantly and negatively correlated with soil p. similarly %rldh and %rldtc was also significantly and negatively correlated to soil zn and cu. in contrast, %rlmo was significantly and positively correlated to zn. neither %rldtc nor root length with dse structures were correlated to %rltc or amf structures (tab. 3). distribution of amf spores total spore counts significantly varied among the fields and ranged from 4 (f19, 20) to 17 (f18) spores per 100g soil. correlation analysis indicated the lack of correlation between %rltc and spore numbers (r = –0.022; p > 0.05). however, spore numbers were significantly and positively correlated to soil k and negatively to soil n (tab. 3). a total of six amf morphotypes of two genera were distinguished on the basis of spore morphology; they included glomus species 1, glomus aggregatum n. c. schenck et g. s. sm., glomus etunicatum w. n. becker et gerd., glomus sinuosum (gerd. et b.k. bakshi) almeida et schenck, glomus viscosum t. h. nicolson and scutellospora calospora (t. h. nicolson et gerd.) c. walker et f.e. sanders, one of which could not be identified at species level (fig. 2). scutellospora calospora (70%) was the most frequent amf morphotype associated with onion (figs. 3, 4). 166 acta bot. croat. 71 (1), 2012 priyadharsini p., pandey r. r., muthukumar t. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:44 color profile: disabled composite 150 lpi at 45 degrees discussion taxa in allium are known to form typical arum-type morphologies (smith and smith 1997, dickson et al. 2007) and this is the first report of the intermediate-type amf in the genus allium and family aliaceae. the intermediate-type amf morphology of the shallot is intermediate to the i3 and i4 subtypes described by dickson (2004). although the exact factors influencing amf morphology are not known, it has been suggested that both host root structure and amf fungal genotypes influence amf morphology (dickson et al. 2007). shallot roots in conventional agricultural fields had amf colonization levels within the ranges of those reported in other studies (muthukumar and tamilselvi 2010, aliasgharzad et al. 2009, goussous and mohammad 2009, jaime et al. 2008). however, the average amf colonization (49.12%) is lower than those reported for onions under conventional cultivation in fevoland (91%) and zeeland (72%) of the netherlands (galván et al. 2009). similarly, the %rla/rlac in the present study was threeto five-fold lower than reported for conventional onion cultivation in the netherlands (galván et al. 2009). generally, low levels of amf colonization in conventionally managed soils were explained by high soil p concentrations. this was not clearly the case of the onion fields in the present study as we found those amf colonization parameters were not correlated to the concentrations of p in the soil. our observations on the lack of correlation between soil p and amf variables suggests that the soil p concentrations in the field soils were well below the threshold levels that could influence amf. the average soil p in the present study (6.4 mg kg–1) was almost seven fold lower than p concentrations reported for dutch soils used for conacta bot. croat. 71 (1), 2012 167 root fungal associations in shallot onion tab. 3. pearson’s correlation coefficients between various arbuscular mycorrhizal fungi (amf), dark septate fungal endophyte (dse) and soil variables (sv). sv amf dse rlh/ rlhc rla/ rlac rlv rltc sp rldh rlmo rlmi rldtc ph 0.519* 0.477* 0.306 0.638** –0.414 0.252 –0.118 –0.041 0.241 ec 0.085 0.095 –0.166 0.068 0.204 –0.256 0.185 –0.212 –0.252 n 0.246 0.328 –0.057 0.318 –0.466* 0.230 0.087 –0.283 0.224 p –0.161 0.374 –0.203 0.083 –0.023 0.050 –0.082 –0.446* 0.026 k 0.442 0.258 0.041 0.410 –0.608** 0.146 0.129 –0.173 0.148 fe 0.488* 0.498* –0.064 0.553* –0.242 0.426 –0.048 0.091 0.425 mn 0.099 0.525* –0.403 0.275 0.014 –0.085 0.264 –0.277 –0.080 zn –0.074 0.072 –0.487* –0.105 0.029 –0.577** 0.459* –0.118 –0.551* cu –0.184 –0.341 0.016 –0.299 0.267 –0.507* 0.055 0.079 –0.500* significance at 1% level (**) and 5% level (*) rlh/ rlhc root length with hyphae or hyphal coils rla/rlac root length with arbuscles or arbusculate coils rlv root length with vesicles rltc total colonization sp spore number rldh root length with dark septate hyphae rlmo root length with moniliform hyphae rlmi root length with microsclerotia rldtc dark septate endophyte total colonization u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:44 color profile: disabled composite 150 lpi at 45 degrees 168 acta bot. croat. 71 (1), 2012 priyadharsini p., pandey r. r., muthukumar t. fig. 2. arbuscular mycorrhizal spores isolated from the soils of shallot onion. a – glomus aggregatum; b – fractured sporocarp of glomus sinuosum; p, peridium; sp, spores; c – glomus etunicatum; d – glomus viscosum with attached soil particles (arrowheads); e – glomus sp.1; f – scutellospora calospora; g – membranous walls (mw1, mw2) of s. calospora; h – germination shield of s. calospora. scale bars: a, b, e, f = 100 µm; c, d, g, h = 50µm. 0 10 20 30 40 50 60 70 80 ga gs gv ge g1 sc amf species f re q u e n c y (% ) fig. 3. arbuscular mycorrhizal fungal (amf) frequency in the soils used for shallot cultivation. for amf species abbreviations see tab. 2. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:50 color profile: disabled composite 150 lpi at 45 degrees ventional cultivation of onions (galván et al. 2009). the lack of correlation between soil p and mycorrhizal parameters agreed with the observation of galván et al. (2009), but contrasted with studies where an inverse correlation had been reported between these variables (ananthakrishnan et al. 2004, lingfei et al. 2005, khanam et al. 2006, valsalakumar et al. 2007, das and kayang 2010b). soil ph has a great relevance for plant growth as it influences nutrient mobilization as well as availability (marschner 1995). many studies reporting the influence of soil factors on amf colonization have failed to find any relationship between soil ph and colonization (zahka et al. 1995, khade and rodrigues 2008, valsalakumar et al. 2007, oliveira and oliveira 2010, khanam et al. 2006). in the present study ph of the soil was positively correlated to all amf colonization parameters except %rlv. lingfei et al. (2005) found results similar to ours, where soil ph was found to be correlated with the extent of hyphal and arbuscular colonization in grasses. colonization and extraradial mycelium formation by amf are known to be altered by soil ph (van aarle et al. 2002). coughlan et al. (2000) inferred that the tendency for colonization levels to increase with ph was due to the stimulation of the additional amf taxa and/or a greater ability of the taxa present to colonize host roots. however, the positive influence of ph on amf colonization contradicts the observations of wang et al. (2008) and fortin et al. (2002) where increasing soil ph had detrimental effects on amf spore germination and mycorrhization. though less studied than those of other soil nutrients, fe deficiencies at high soil ph are known to suppress colonization by amf (wang et al. 2008). the positive correlation between soil fe and amf variables indicate that fe could stimulate colonization by amf. a similar correlation has been reported in paullinia cupana during the rainy season by oliveira and oliveira (2010). michelini et al. (1993) indicated that fe interacts with other soil nutrients like ca, mn or p to influence amf colonization and can greatly be influenced by soil ph. in the present study, soil fe was also significantly and positively correlated to soil ph (r = 0.565; p<0.01; n = 20). contrarily, to the observations of audet and charest (2006), zn and %rlv had an inverse correlation in the present study. the negaacta bot. croat. 71 (1), 2012 169 root fungal associations in shallot onion 0 1 2 3 4 5 6 f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 f13 f14 f15 f16 f17 f18 f19 f20 fields a m f s p e c ie s fig. 4. arbuscular mycorrhizal fungal (amf) diversity in shallot soils. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:50 color profile: disabled composite 150 lpi at 45 degrees tive effect of soil zn can be due to the directly suppressive effect of zn on amf propagules or the indirect effect on host roots. the average amf spore number (11 spores per 100g soil) recorded in the present study is lower than the range of 54–3920 spores per 100g soil reported for tropical soils (valsalakumar et al. 2007, khanam et al. 2006, khade and rodrigues 2008, oliveira and oliveira 2010, ananthakrishnan et al. 2004, das and kayang 2010b). major portions of amf spores occurring in field soils are either dead or spore cases (muthukumar and udaiyan 1999) and the spore numbers presented in this study are only for intact spores. the lack of correlation between %rltc and spore numbers agrees with the observations of valsalakumar et al. (2007) in phaseolus aureus, zahka et al. (1995) in acer saccharum, khalil and loynachan (1994) in glycine max and sasai (1992) in cultivated plants of miyagi prefecture, japan. such a lack of correlation between amf colonization and spore numbers indicate that the factors influencing these variables are totally different. in this study amf spore numbers were negatively correlated to soil n and k. this contradicts the suggestions that soil k could stimulate the production of amf spores (oliveira and oliveira 2010). this influence of soil n and k on amf spore numbers can be attributed to their influence on soil ph as soil n (r = 0.463) and soil k (r = 0.464) were correlated (p<0.05) to soil ph. a total of one to five amf taxa were detected in conventionally cultivated onion field soil. this is lower than the 8 to 20 species that are usually reported for arable lands (land and schönbeck 1991, douds and millner 1999, fitter, 2001, jansa et al. 2002). however, the amf diversity in the present study is within ranges observed for a site by ananthakrishnan et al. (2004) and sjöberg et al. (2004). valsalakumar et al. (2007) also reported very low amf diversities of one to three taxa for the 21 sampling locations under phaseolus aureus cultivation in tamil nadu and karnataka of south india. the presence of low amf diversity could be due to the selection pressure imposed by cultivation practices resulting in the dominance of fast growing species (oehl et al. 2004) and species that are able to tolerate stresses like tillage, fertilizer and biocide applications (gosling et al. 2006, galván et al. 2009). dse colonization had been reported in roots of several crop species and in roots also colonized by amf (jumpponen and trappe 1998, muthukumar and tamilselvi 2010). in this study, the root systems of all shallot onions examined were commonly colonized by both dse and amf. dse colonization has been reported in onions from both temperate and tropical agroecosystems (jumpponen and trappe 1998, muthukumar and tamilselvi 2010). it is interesting to note that the extent of %rldtc was always lower (except f2) than %rltc. further, the lack of correlation between root lengths colonized by amf and dse clearly suggest that these two fungal types do not compete for resources within roots. however, the results of a recent study by scervino et al. (2009) show that dse could modify mycorrhizal status of plants. the correlation analysis showed that soil zn and cu influenced dse colonization and structures, which is in line with the observation of christie and kilpatrick (1992). in contrast, lingfei et al. (2005) found no significant correlations between dse colonization and soil factors. the response of dse to soil nutrients appears to vary with soil conditions. for example, christie and kilpatrick (1992) found an inverse correlation between cu and zn to dse colonization in soil amended cow slurry but not in soil amended with pig slurry. at present, the ecology of dse association in crop plants has not been documented. as grow170 acta bot. croat. 71 (1), 2012 priyadharsini p., pandey r. r., muthukumar t. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:50 color profile: disabled composite 150 lpi at 45 degrees ing evidence suggests that dse may influence plant growth in a way similar to amf (wu et al. 2010, wu and guo 2008, fumiaki and kazuhiko 2007), an understanding of the factors influencing their associations and function would enable their possible exploitation in agriculture. references abbott, l. k., gazey, c., 1994: an ecological view of the formation of va mycorrhizas. plant and soil 159, 69–78. addy, h. d., piercey, m. m., currah, r. s., 2005: macrofungal endophytes in roots. canadian journal of botany 83, 1–13. aliasgharzad, n., bolandnazar, s. a., neyshabouri, m. r., chaparzadeh, n., 2009: impact of soil sterilization and irrigation intervals on p and k acquisition by mycorrhizal onion (allium cepa). biologia 64, 512–515. ananthakrishnan, g., ravikumar, r., girija, s., ganapathi, a., 2004: selection of efficient arbuscular mycorrhizal fungi in the rhizosphere of cashew and their application in cashew nursery. scientia horticulturae 100, 369–375. anonymous, 1986: pest control in tropical onions. tropical development and research institute, london. audet, p., charest, c., 2006: effects of am colonization on wild tobacco plants grown in zinc contaminated soil. mycorrhiza 16, 277–283. balestrini, r., magurno, f., walker, c., lumini, e., bianciotto, v., 2010: cohorts of arbuscular mycorrhizal fungi (amf) in vitis vinifera, a typical mediterranean fruit crop. environmental microbiology reports 12, 593–604. bever, j. d., morton, j. b., antonovics, j., schultz, p. a., 1996: host-dependent sporulation and species diversity of arbuscular mycorrhizal fungi in a mown grassland. journal of ecology 84, 71–82. bolandnazar, s., aliasgharzad, n., neishabury, m. r., chaparzadeh, n., 2007: mycorrhizal colonization improves onion (allium cepa l.) yield and water use efficiency under water deficit condition. scientia horticulturae 114, 11–15. bosch-serra, a. d., currah, l., 2002: agronomy of onions. in: rabinowitch, h. d., currah, l. 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(eds.), ecological interactions in soil, 219–224. blackwell scientific publications, oxford. 174 acta bot. croat. 71 (1), 2012 priyadharsini p., pandey r. r., muthukumar t. u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:50 color profile: disabled composite 150 lpi at 45 degrees wang, y. y., vestberg, m., walker, c., hurme, t., zhang, x., lindström, k., 2008: diversity and infectivity of arbuscular mycorrhizal fungi in agricultural soils of the sichuan province of mainland china. mycorrhiza 18, 59–68. wu, l., guo, s., 2008: interaction between an isolate of dark septate fungi and its host plant saussurea involucrata. mycorrhiza 18, 79–85. wu, l. q., lv, y. l., meng, z. x., chen, j., guo, s. x., 2010: the promoting role of an isolate of dark-septate fungus on its host plant saussurea involucrata kar. et kir. mycorrhiza 20, 127–135. zahka, g. a., baggett, k. l., wong, b. l., 1995: inoculum potential and other vam fungal parameters in four sugar maple forests with different levels of stand dieback. forest ecology and management 75, 123–134. acta bot. croat. 71 (1), 2012 175 root fungal associations in shallot onion u:\acta botanica\acta-botan 1-12\487 priyadharsini novo.vp 26. o ujak 2012 13:59:50 color profile: disabled composite 150 lpi at 45 degrees 544 vizintin et al.vp acta bot. croat. 71 (2), 279–284, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 tuber donnagotto, a new winter truffle species from istria, croatia romano bo@ac, ivan [iri]*, ivica kos university of zagreb faculty of agriculture, sveto{imunska 25, 10000 zagreb, croatia abstract – tuber donnagotto is a new winter black truffle belonging to the order pezizales and the family tuberaceae. it grows in winter in the calcareous gravel soil (ph 7.6–7.8) near the adriatic sea (rovinj, istria, croatia) in predominantly pine forests (pinus halepensis). although similar to other black truffles, it has very irregular and hard fruit bodies, lobate and knotted in form, with deep irregular cavities reaching the middle of the fruit bodies. these cavities are clearly evident in the cross-section of the fruit bodies. a distinctive characteristic of this truffle is the fact that when it is hermetically closed it can be kept in a refrigerator (2–4 °c) for more than 60 days. tuber donnagotto has a slight but pleasant odor, reminiscent of boletus (boletus reticulates, b. edulis). furthermore, t. donnagotto has yellow-brownish and reticulate-alveolate spores, measuring 20–30 × 20–25 mm. key words: mushroom, pinus, spores, tuber donnagotto, istria, croatia introduction a major scientific contribution to underground mushrooms was made many years ago by the italian (vittadini 1831, sacardo 1895, mattirolo 1887) and french researchers (tulasne and tulasne 1851). ceruti et al. (2003) made a historical revision of the european tuber species from the 18th to the 20th century. according to this study, 35 valid species, varieties and forms that belong to the genus tuber are known today in europe. based on the revision of the genus tuber and the analysis of its sequences of rdna (roux et al. 1999), ceruti et al. (2003) do not recognize tuber aestivum var. uncinatum chatin (chevalier et al. 1979) as a valid species. they think it is just a synonym for tuber aestivum vittad. montecchi and sarasini (2000) described 11 species, varieties and forms that belong to a group of black colored truffles (tuber brumale vittad., t. brumale var. moscatum (ferry) montecchi and lazzari, t. melanosporum vittad., t. indicum cooke and masse, t. macrosporum vittad., t. malenconii donadini, riousset and chevalier, t. bellonae quel., t. mesentericum vittad., t. regianum montecchi and lazzari, t. aestivum and t. aestivum acta bot. croat. 71 (2), 2012 279 * corresponding author, e-mail: isiric@agr.hr copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. var. uncinatum (chatin) montecchi and borelli. all these species grow in europe except tuber indicum (which grows in china) and are used for commercial purposes. scientific research into underground fungi began in italy, where it was undertaken by vittadini (1831), saccardo (1892), mattirolo (1910), ceruti (1961). researchers in france include tulasne (1844), quelet (1873,1883) and later many other mycologists worldwide. in croatia underground mushrooms have not been scientifically studied and no research papers on truffles and other mushrooms found below the surface of the soil have been published to this day. in other countries of the former yugoslavia (serbia, montenegro and macedonia) the ecological specificities and molecular diversity of truffles have been investigated by marjanovi] et al. (2010). the authors described twelve tuber species, including five varieties of tuber rufum pico and concluded that the ecosystem conditions of balkan peninsula support truffle production. a new species of truffle, tuber donnagotto, belonging to a group of black colored truffles, has been described for the first time in this paper. it was found on the northern adriatic coast (rovinj, istria, croatia) in predominantly pine forest (pinus halepensis mill.) on the calcareous-gravel soil (ph 7.6–7.8). materials and methods the truffles were found on november 15 and december 18, 2010, january 15, february 20, march 13, and april 3, 2011, on the adriatic coast (rovinj, istria, croatia). the concentration of hydrogen ions (ph) of soil was measured in an aqueous solution using the iq 150 ph meter (iq scientific instruments, usa). the microscopy and measurement of morphological details of the truffles were carried out using a carl zeiss jena microscope. the recording of the morphological structure was performed using a carl zeiss jenamed 2 microscope, and the camedo c-5050 zoom, olympus camera (japan). results description of the new species tuber donnagotto bo`ac, [iri} et kos, sp. nov. mycobank mb 563709 etymology. in honor of the dogs (donna et gotto) of the breed lagotto romagnolo that found the truffles. diagnosis latina tuber donnagotto est species nova tuberis nigri, autumno crescentis, mycorrhizas cum pinibus (pinus halepensis mill.) formantis. peridium eius verrucis atris irregularibus et polygonalibus, 150–450 mm crassum, structura – pseudoparenchymatica – , cellulis polygonalibus 9–12 mm latis. ascocarpia atra, admodum irregularia, tuberiformia, lobos aut eminentias habentia. gleba cerea, venis albis densis et sinuosis marmorata, cuius in sectione transversa cavae irregulares manifeste visibiles. odor lenis et iucundus, boletus edulis bull.ex fries. asci globosi aut subglobosi, 55–90 × 65–75 mm, 1–7 ascosporas 280 acta bot. croat. 71 (2), 2012 bo@ac r., [iri] i., kos i. continentes. ascosporae ipsae fulvo-fuscae, late ellipsoideae aut subglobosae, 20–30 × 20–25 mm, ornamentum reticulato-faveolatum habentes. morphology the fruit bodies are hypogeous or subepigeous, black, warted, very irregular, rounded, tuberiform, lobed or knotty, irregular and deeply furrowed (fig. 1a), with a more or less evident apical opening; irregular cavities of various shapes and sizes are clearly visible on the cross-section (fig. 1b), the average diameter is 2–7 cm, they are as hard as stone and are similar to tuber excavatum vittad. the peridium is 150–450 mm thick, the structure is pseudoparenchymatous with polygonal cells 9–20 mm wide, on the peripheral part they are black-brown, the yellowish-brown inner layers are composed of irregular polygonal warts of different sizes, in some places they are higher and in some places smaller, flattened or slightly depressed at the center, sometimes reddish at the base, only rare warts have one or two radial ridges or fissures, dense parallel transversal striate which form a polygonal surface, clearly visible with a lens (fig. 1d). the gleba is whitish when very young, at full maturity it is light brown, marbled by dense white veins. the veins are short and very meandriform at the cross-section (fig. 1c). the odor of the young fruit bodies is very slight or completely absent, at full maturity it is pleasant and reminiscent of boletus (boletus reticulatus shaeff., boletus edulis bull. ex fries). most asci are spherical, a few of them (about 5%) are subglobose hyaline, measuring 55–80–90 × 65–75 mm, containing 1–7 spores, usually 4–6, the length of the pedicle is 15–20 mm. the spores are yellowish-brown, ellipsoid to spherical, size 20–30 × 20–25 mm measured excluding ornamentation. they have a reticulate-alveolate ornament with irregular polygonal meshes (fig. 1e). the reticulum has slightly prominent aculei in correspondence with knots, the meshes are 3–4 mm high and 5–8 mm wide, numbering 3–4 along the bigger spore dimension. q (length / width) = 1.1–1.2–1.5. habitat their habitat is predominantly pine forests (pinus halepensis) near the sea, from november to april in gravelly red soil, ph 7.6–7.8. at the first harvest (november 15, 2010) almost all fruit bodies were on the surface of the soil (semi-epigeous), at the second harvest (december 18, 2010) the truffles were bigger (4–7 cm) and were found deeper in the soil (5–10 cm), while at the third harvest (february 20, 2011) almost all the fruit bodies were on the surface (semi-epigeous) on which grass does not grow. during its growth the tuber donnagotto probably produces substances (allelochemicals) that inhibit growth and reproduction of grasses. additional specimens examined. croatia. istrian peninsula: rovinj, 35 km ne of pula (45° 04' 17.19" n, 13° 38' 15.5" e), 9 meters above the mean sea level, in symbiosis with pinus halepensis, leg. r. bo`ac, november 15, 2010, december 18, 2010, february 20, 2011, march 13, and april 3, 2011. acta bot. croat. 71 (2), 2012 281 tuber donnagotto, a new tuber species discussion the new underground species belongs to a group of black truffles 10 species, varieties and forms of which were previously known in europe (montecchi and sarasini 2000, ceruti et al. 2003). due to the great external similarity of black truffles, commercial collectors often mistake one for another. the well known black truffle tuber melanosporum grows at the same time and can be found in the same habitat as the tuber donnagotto. the most significant morphological differences of tuber donnagotto in relation to similar black truffles (tuber brumale and t. melanosporum) that grow at the same time (autumn and winter) are color (black, black-brown), form (warted, very irregular, rounded, tuberiform, lobed or knotty, and deeply furrowed), size and ornamentation of spores (20–30 × 20–25 mm and reticulate-alveolate ornament with irregular polygonal meshes), size and 282 acta bot. croat. 71 (2), 2012 bo@ac r., [iri] i., kos i. fig. 1. tuber donnagotto (holotype). a – fruit bodies; b – cross section of fruit bodies; c – cross section of matured fruit body; d – warts of peridium; e – asci and ascospores. bars denote 1 cm (a, c), 2 cm (b), 2 mm (d), 30 mm (e). hardness of fruit bodies (the average diameter 2–7 cm and hard fruit bodies), cross section (irregular cavities of various shapes and sizes are clearly visible), gleba (whitish when very young, at full maturity light brown, marbled by dense white veins), odor and taste (the odor of young fruit bodies is very slight or completely absent, at full maturity it is pleasant and reminiscent of boletus) and per time of viability in a refrigerator (more than 60 days). all the other european species of black truffles grow in the spring, summer and autumn and have a more regular form, significantly different odor and taste and significantly lower viability in a refrigerator. during growth tuber melanosporum produces allelochemicals that inhibit the growth and reproduction of grass (riousset et al. 2001). tuber melanosporum is significantly different from tuber donnagotto because it has regular and bigger fruit bodies, its peridium has much bigger warts, the gleba is blackish-brown with purple tinges, its odor is characteristic and pleasant, the spores are dark colored, blackish-brown and densely ornamented with single spines (ceruti et al. 2003). other similar black truffles, which grow at the same time and in similar habitats, are tuber brumale and tuber brumale var. moscatum. however, the quality of the new species in comparison with these black truffles is unquestionable. the mentioned truffles have more regular fruit bodies, tuber brumale has a gray-black gleba and form moscatum has a beige-hazel-brown gleba. the odor is strong and persistent: according to vittadini (1831) it is similar to that of the cortex of cornus sanguinea l., the spores are hazel-brown and densely ornamented with single spines. the tuber bellone is also similar in color and can grow at the same time (it was found on january 15, 2011, rovinj, istria, croatia), but in symbiosis with quercus ilex l. what is more, the peridium has pyramidal warts with smooth side surfaces, never transversally striate (ceruti et al. 2003), the asci are ovoid, 77–115 × 77–95 mm, the spores are yellow-ochre, spherical and reticulate-alveolate, the odor is distinctly phenolic when is fresh, and then more pleasant (montechi and sarasini 2000). the tuber aestivum is significantly different because it grows in spring and summer, the fruit bodies are rounded and more or less regular, the peridium has much larger pyramidal warts whose base is never reddish. the asci have saccate form, shortly pedununculate, 80–100 × 50–80 mm, the odor is pleasant, reminiscent of roast barley malt or fermenting matter. the somewhat similar tuber malenconii grows in symbiosis with quercus ilex, q. ilex subsp. ballota (desf.) samp, and q. coccifera l. its fruit bodies are much smaller, the peridium is composed of small and not very prominent pyramidal warts which turn red or yellow when rubbed. the asci are 60–80 mm wide and contain up to 6–8 spores. with time the odor of this species becomes more and more unpleasant, fecal or cabbage-like or garlicky. the spores are reticulate-alveolate with very small and dense meshes, the average numbering 6–8 along the bigger dimension (donadini et al. 1978). the tuber mesentericum has distinctly coarser (larger) pyramidal verrucae, the gleba is light hazel-brown at full maturity, the odor is generally very strong in just collected fruit bodies, reminiscent of tar and iodine (montecchi and sarasini 2000). tuber regianum has tuberiform or subglobose fruitbodies, 1.5–3 cm in diameter, dark brown or blackish red, the surface is glabrous or finely areolate-verrucose or with very small papillae (montecchi and lazzari 1987). tuber donnagotto is substantially different from all known species of black truffles. no other known species of black truffle has such irregular fruit bodies with deep and irregular grooves (fig. 1a, b). moreover, unlike all other black truffles, the fruit bodies of the new species can be kept in a refrigerator (2–4 °c) for more than 60 days. the fruitbodies are very hard, similar to tuber excavatum and are never attacked by insects or nematodes. acta bot. croat. 71 (2), 2012 283 tuber donnagotto, a new tuber species references ceruti, a., 1961: revisione di alcune specie di elafomicetali e di tuberali dell america del nord. allionia 7, 1–25. ceruti, a., fontana, a., nosenzo, c., 2003: le specie europee del genere tuber una revisione storico, museo regionale di scienze naturali, torino. chevalier, g., delmas, c., frochot, h., riousset l., 1979: l'espèce tuber aestivum vitt.: i. definition. mushroom science 109, 57–975. donadini, j. c., riousset, l., riousset, g., chevalier, g., 1978: tuber malenconii nov. sp. bulletin de la société mycologique de france 94, 351–358. marjanovi], @., grebenec, t., markovi], m., gli[i], a., milenkovi], m., 2010: ecological specificities and molecular diversity of truffles (genus tuber) originating from mid-west balkan peninsula. sydowia 62, 67–87. mattirolo, o., 1887: sul parassitismo dei tartufi e sulla questione mycorrhizae. malpighia 1, 359–369. mattirolo, o., 1910: i tartufi, loro coltivazione e rapporti con il rimboschimento e colla crisi viticola. l' alpe 8, 1–31. montecchi, a., lazzari, g., 1987: un nuovo tartufo di montogna: tuber regianum s.sp. rivista di micologia 30, 3–11. montecchi, a., sarasini, m., 2000: funghi ipogei d'europa, centro studi micologici, associazione micologica bresadola, trento. quelet, l., 1873: les champignons du jura et des vosges. memoires de la societe d'emulation 5, 333–427. quelet, l., 1883: quelques espécés critiques ou nouvelles de la flore mycologique de france. comptes rendus de l'association francaise pour l'avancemet des sciences 11, 387–412. riousset, l., riousset, g., chevalier, g., bardet, m., 2001: truffes d'europe et de chine. inra editions, paris. roux, c., sejalon-delmas, n., martins, m., parguey-leduc, a., dargent, r., becard, g., 1999: phylogenetic relationships between european and chinese truffles based on parsimony and distance analysis of its sequences. fems microbiology letters 164, 147–155. saccardo, p. a., 1892: tuberoidae vitt., sylloge fungarum ommium hucusque cognitorum. supplementum universale 2, 10, 80–83. saccardo, p. a., 1895: tuberoidae vitt., sylloge fungarum ommium hucusque cognitorum. supplementum universale 3, 11, 411–445. tulasne, l. r., tulasne, c., 1844: fungi nonnulli hypogaei, novi vel minus cogniti. giornale botanico italiano 2, 55–63. tulasne, l. r., tulasne, c., 1851: fungi hypogaei: historie et monographie champignons hypoges, friedrich kfincksieck, paris. vittadini, c., 1831: monographia tuberacearum ex typographia. felicis rusconi, milano. 284 acta bot. croat. 71 (2), 2012 bo@ac r., [iri] i., kos i. opce-str.vp acta bot. croat. 71 (1), 125–138, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 epiphytic metazoans on emergent macrophytes in oxbow lakes of the krapina river, croatia: differences related to plant species and limnological conditions maria [poljar*1, jelena fressl2, tvrtko dra@ina1, matija meseljevi]3, zlatko gr^i]4 1 department of zoology, division of biology, faculty of science, university of zagreb rooseveltov trg 6, hr-10000 zagreb, croatia 2 dvokut ecro ltd., environmental protection and sustainable development, trnjanska 37, hr-10000 zagreb, croatia 3 ant ltd., lab for analyses and toxicology, medarska 69, hr-10 090 susedgrad-zagreb, croatia 4 sanatio ltd, bolni~ka cesta 34c, hr-10 090 susedgrad-zagreb, croatia abstract – this study investigated the structure of the epiphytic metazoans on emerged macrophytes in the littoral zone of two oxbow lakes with different trophic levels. differences in the diversity and density of the epiphytic metazoans were analyzed in relation to plant architecture (simple or complex stems), food resources (algae and detritus) and water characteristics (transparency and derived trophic state index). a significant negative correlation was found between detritus on plants as food resource, and diversity and density of epiphytic metazoans, indicating grazing of microphagous species. rotifers dominated in diversity and density in the epiphyton on all habitats. total density of metazoans, rotifers and copepods in epiphyton were significantly higher on mentha in mesotrophic lake than on iris in a eutrophic lake. we presume that macrophyte belt width and trophic state governed biotic interactions and consequently epiphytic assemblages more strongly than macrophyte architecture. however, a mentha habitat showed a slightly higher density and diversity of epiphytic metazoans in relation to iris at the same site, but these differences were not significant. key words: macrophyte, mentha, iris, epiphyte, metazoans, trophic state, trasnparency, biotic interactions introduction shallow lakes are unique fresh water ecosystems, long overlooked in limnological research, despite their great biodiversity of algae, macrophytes, plankton, nekton and benthos acta bot. croat. 71 (1), 2012 125 * corresponding author, e-mail: mspoljar@zg.biol.pmf.hr copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:29 color profile: disabled composite 150 lpi at 45 degrees (castro et al. 2005). many of these shallow lakes are endangered by eutrophication, acidification and introduction of invasive species (kalff 2002). anthropogenic eutrophication is one of the main triggers in shifting shallow lakes from a clear-water macrophyte-dominated state to a turbid-water phytoplankton-dominated state (scheffer et al. 1993, köhler et al. 2005, hilt et al. 2010). macrophytes often characterize littoral zones of shallow lakes and have an important role in water biocenoses, structuring and modifying the physical-chemical features by photosynthesis, decomposition and mineralization (duggan et al. 2001, jeppesen et al. 2002, joniak et al. 2007). they reduce water movement and sediment resuspension, provide refuge and protection to zooplankton, macroinvertebrates and small fish against predators (chambers et al. 2008) and serve as oviposition habitats for fish, water birds and invertebrates (walsh 1989, beccera-munoz and schramm 2007, klassen and nolet 2007). so far, studies on the role of macrophytes in littoral habitats have mostly been focused on submerged (horppila and nurminen 2001, kuczy�ska-kippen and klimaszyk 2007, søndergaard et al. 2007, bogut et al. 2010, hilt et al. 2010) and less on emerged macrophytes (cazzanelli et al. 2008, [poljar et al. 2011). this can be explained by reference to several circumstances. the greater surface area of well dissected submerged macrophytes may benefit invertebrates by offering better food resources and protection against predators than macrophytes with simple stems (meerhoff et al. 2007, tessier et al. 2008). moreover, complex macrophytes respond promptly to eutrophication effects and are obligate in lake restoration (moss et al. 1997, hilt et al. 2006). the epiphyton community has an important role in the primary production of shallow lakes (cattaneo et al. 1998, laguste and reunanen 2005). epiphyton is composed of detritus, bacteria, algae, fungi, protozoan and metazoan invertebrates attached to aquatic macrophytes (wetzel 2001). it is a result of several factors and has a keystone position in the food web and nutrient circulation since it is sensitive to both bottom-up and top-down control mechanisms (jeppesen et al. 1999). macrophyte architecture (simple or complex stem with dissected leaves) and surface structure are important factors in epiphyton development (vieira et al. 2007, tessier et al. 2008). macrophytes with dissected leaves and more structural complexity provide a suitable area for epiphyton growth characterised by higher biodiversity than those with undissected leaves (laguste and reunanen 2005). light together with nutrients (phosphate, nitrate) are the main abiotic limiting factors, and they affect growth, development, density and diversity of aquatic macrophytes and epiphyton (cattaneo et al. 1998, hilt et al. 2010). some studies suggested that biotic factors, competition and predation, have a significant influence on epiphyton distribution (walsh 1995, lauguste and reunanen 2005). thus far epiphyton community ecology has received less attention than plankton communities, despite the diversity and density, probably due to methodological problems regarding quantitative sampling, which is not standardized (duggan 2001). our study was carried out in two shallow, eutrophic lakes with narrow macrophyte belts. previous study in these lakes suggested that differences in transparencies between two lakes caused significant differences in horizontal distribution of the zooplankton assemblage. even narrow helophyte belts offered a refuge to zooplankton, although lower transparencies reduced the effectiveness of macrophytes as a refuge from predators ([poljar et al. 2011). the present study aims to explore the effects of the water transparency and its derived trophic state index as well as effects of macrophyte species on 126 acta bot. croat. 71 (1), 2012 [poljar m., fressl j., dra@ina t., meseljevi] m., gr^i] z. 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:29 color profile: disabled composite 150 lpi at 45 degrees epiphytic metazoans diversity and density. concordantly, our goals were to analyse: (i) impact of environmental parameters and food resources on epiphyton; (ii) influence of different macrophyte architecture on the epiphytic metazoan assemblage. we presume that results of this study will reveal epiphytic metazoans to be an adequate indicator of eutrophication and change in environmental conditions. study area the main features of the two oxbow lakes on the krapina river (nw croatia) were well documented in a previous paper ([poljar et al. 2011). both lakes were formed by river-straightening operations and are situated approximately 500 m apart (fig. 1). no submerged macrophytes were present on the bottom, which consists of alluvial silt substrate. the water level depends mainly on precipitation and groundwater, with the lowest levels being recorded in the summer. the location, morphometric features and macrophyte composition of each of the lakes, krapina oxbow lake 1 (ko1) and krapina oxbow lake acta bot. croat. 71 (1), 2012 127 epiphytic metazoans on emergent macrophytes fig. 1. map of the investigated krapina river oxbow lakes (ko1 and ko2), showing study sites in littoral zones (l1 and l2). in l1 iris stems were sampled (habitat i1) and in l2 iris (habitat i2) and mentha (m2) stems were sampled. 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:30 color profile: disabled composite 150 lpi at 45 degrees 2 (ko2), are summarized in table 1a. lake ko1 had significantly higher conductivity and algal biomass but lower transparency, ph and percentage of macrophyte cover than lake ko2 ([poljar et al. 2011). on each sampling occasion, epiphyton samples were collected from the littoral zone of each lake (l1 in ko1 and l2 in ko2). environmental conditions in l1 and l2 are shown in table 1b. in the two oxbow lakes, the fish communities were 128 acta bot. croat. 71 (1), 2012 [poljar m., fressl j., dra@ina t., meseljevi] m., gr^i] z. tab. 1. a) main morphometric features, macrophyte composition and significantly different environmental parameters of the studied krapina oxbow lakes ko1 and ko2 (source [poljar et al. 2011); b) mean values of environmental parameters on the investigated study sites l1 and l2 (mean±sd). a) parameters ko1 ko2 coordinates 45°57'96'' n; 15°50'78'' e 45°57'37'' n; 15°50'63'' e lengthmax (m) 150 81 widthmean (m) 37 48 surface area (ha) 1.7 1.0 max. depth (m) 4.0 3.0 shore slope steep gradual transparencysd (m) 0.3–1.1 0.6–1.2 ph 7.12–7.41 7.13–7.75 conductivity (ms cm–1) 345–404 285–325 chl a (mg m–3) 1.18–23.38 2.37–10.65 macrophyte coverage % 3.2–5.5 5.0–7.8 surronding area ploughed-fields meadows macrophyte type emergent emergent macrophyte composition (%) typha latifolia (40%) iris pseudacorus (30%) carex sp. (15%) sparganium ramosum (15%) typha latifolia (40%) iris pseudacorus (20%) carex sp. (15%) mentha aquatica (25%) b) l1 l2 parameters mean±sd mean±sd temperature (°c) 20.7±4.4 22.2±4.5 dissolved oxygen (mg l–1) 6.2±2.4 7.6±2.9 conductivity (ms cm–1)* 370.4±18.3 302.9±11.7 ph * 7.26±0.09 7.48±0.13 alkality (mg l–1) 111.1±4.9 113.3±5.6 nitrate (mg n-no3 – l–1) 0.344±0.52 0.338±0.51 orthophospate (mg p-po4 3– mg l–1) 0.026±0.01 0.025±0.01 chl a (mg m–3) 11.2±3.81 6.5±3.0 afdw (mg m–3) 1809±1388 8324±10103 nl, l1=9; * significant difference p<0.05 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:30 color profile: disabled composite 150 lpi at 45 degrees similar (mrakov^i] and mar^i] 2006). among fish present species carp prevailed (cyprinus carpio) followed by black bullhead (ameiurus melas), pike (esox lucius), pikeperch (sander lucioperca), roach (rutilus rutilus), bleak (alburnus alburnus), bream (abramis brama), sunfish (lepomis gibbosus) and chub (squalius cephalus). materials and methods all measurements and sampling related to environmental variables, food resources and epiphyton were collected between april and october 2008. two seasons were considered in the analyses: spring (april–june) and summer (july–september). in september the water level was low and emergent macrophyte belts were above the water level and thus epiphyton samples could not be taken. samples were collected at monthly intervals, whereas in may, june and july, samples were collected twice per month. we sampled macrophytes on the east bank of each oxbow lake, as that side was flooded for a longer period than the others. on these sites only iris pseudacorus and mentha aquatica were present. iris contains sword-shaped leaves tightly packed at the base, while mentha has a square stem with alternating opposite pairs of leaves. epiphyton was sampled from these two macrophyte species, differing in their habitus architecture: simple, i.e., iris pseudacorus in the littoral zone of both lakes (i1 in l1 and i2 in l2) and complex, i.e., mentha aquatica (m2), only in l2. triplicate samples of each species and site (each sample included a single plant) were taken with a plastic hand cylinder sampler (30 cm high, diameter 8 cm, mesh net 26 mm) according to kornijów and kairesalo (1994). epiphyton sampling was provided by cutting submerged part of macrophytes into 10 to 15 cm long parts, which were scraped using a small brush, rinsed with distilled water, collected and transported in plastic bottles to the laboratory. pertaining macrophyte stems were deposited in other bottles and brought to the laboratory, where dry mass (dm) was measured after drying in a thermostat at 60 °c for 24 h (cattaneo et al. 1998). parallel with epiphyton sampling another set of triplicate macrophyte parts was taken for determination of algal biomass in epiphyton.. specimens of epiphytic metazoans were determined and counted on live material under an opton-axiovert 35 inverted microscope (100 to 450´). before counting the whole sample was thoroughly mixed in order to achieve homogenous distribution of specimens. the entire volume (c. 5 ml) of collected epiphyton was counted in a petri dish under an inverted microscope. in the case of high density, half of the sample was checked, and counting was adjusted for the entire sample. for species determination, we consulted the following monographs: koste (1978) rotifera, einsle (1993) copepoda and margaritora (1983) cladocera. bdelloidea were counted, but not identified, and densities of polyarthra dolichoptera and polyarthra vulgaris were aggregated into a single category (polyarthra spp.). cladocera were divided into two groups according to the body size: small-bodied (length 500 mm to 1 mm) and large-bodied (length > 1 mm to 6 mm). epiphyton assemblages for each station and each month were quantified as density and were expressed as the number of individuals per 1 g of macrophyte dry mass. after metazoan specimens counting, samples were used for determination of epiphyton ash free dry mass (afdme). these data were obtained after drying of each sample at 104 °c for 4 h in ceramic dishes and ashing at 600 °c for 6 h. acta bot. croat. 71 (1), 2012 129 epiphytic metazoans on emergent macrophytes 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:30 color profile: disabled composite 150 lpi at 45 degrees all physicochemical measurements (alkalinity, concentrations of nitrate and orthophosphate) as well as records related to investigated area and the period of investigation were presented in the study by [poljar et al. (2011). algal biomass (measured as chlorophyll a, chl a) and detritus or particulate organic matter, pom (measured as ash free dry mass, afdm) were considered to be possible food resources in plankton and epiphyton. chl a in epiphyton (chl a) was determined using an ethanol extraction method by nusch (1980). macrophyte coverage (%) was estimated from the ratio of transect length occupied by macrophytes to total transect length at five locations in each lake (lau and lane 2002). similarity among epiphyton samples was calculated using the sørensen index (si) according to equation si=2c/a+b, where a and b are the number of species in samples a and b, respectively, and c is the number of species shared by the two samples (sørensen 1948). according to the secchi disc transparency, we calculated trophic state index and distinguished trophic states by carlson (1977). rotifera and cladocera density in littoral water was computed from the study [poljar et al. (2011). in further analyses the mean of triplicate samples was used as a single data point for a given date and site. prior to statistical analysis, all biotic and abiotic parameters were logarithmically transformed [log (x+1)] and their normality was checked using shapiro-wilk’s test. as this test suggested that the data did not follow a normal distribution, even after transformation (p>0.05), a nonparametric kruskal-wallis test (comparison between multiple habitats) and mann-whitney u test (comparison between two seasons) were used. results according to trophic state index mean value (67±5.5) in ko1 highly eutrophic conditions prevailed while in ko2 values were much lower (38±5.3) which suggested mesotrophic conditions (fig. 2). these values significantly varied between the two oxbow lakes 130 acta bot. croat. 71 (1), 2012 [poljar m., fressl j., dra@ina t., meseljevi] m., gr^i] z. fig. 2. seasonal oscillations of transparency and trophic state index (tsisd) in two krapina river oxbow lakes (ko1 and ko2). 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:30 color profile: disabled composite 150 lpi at 45 degrees (z=3.59, n= 18, p=0.0003). further results of analyses suggested that epiphytic metazoan diversity and cladoceran density increased at higher transparency (tab. 2). these interactions were also significant particularly on m2 (r=0.84 to 0.89, n=8, p<0.05). ash free dry mass was deposed significantly more (p<0.05) on i1 than on i2 and m2 (fig. 3a), and significantly negatively correlated with biodiversity, higher rotifer and total metazoans density in epiphyton (tab. 2). algal biomass (chl a) did not oscillate significantly in macrophyte epiphyton during the investigated period (fig. 3b). it had a significant and positive relation with rotifer and total metazoan density in epiphyton (tab. 2). also, two oxbow lakes were significantly different in ph values (z=–3.04, n=18, p=0.002) and conductivity (z=3.58, n=18, p= 0.0003) (tab. 1b). among dm and nutrients, as well as between epiphytic community and environmental parameters in the surrounding water, no significant correlations were established (p>0.05). a total of 48 epiphytic metazoan taxa were recorded in this study, where rotifers prevailed in diversity (38 taxa) and density (70 %). habitats i2 and m2 (each 41 taxa) had significantly higher diversity than i1, where only 16 taxa were recorded (fig. 2c, tab. 3). sørensen similarity index between epiphytic metazoans at different sampling sites (i1 and i2 42%; i1 and m2 38%) was lower than between different macrophytes at the same sampling site (i2 and m2 75%). density of epiphytic metazoans as well as densities of rotifers and copepods in epiphyton reached significantly higher values on m2 compared to i1 (fig. 3d, f, g). total density acta bot. croat. 71 (1), 2012 131 epiphytic metazoans on emergent macrophytes tab. 2. significant spearman correlations (p<0.05) between food resources and biotic parameters (n=24). afdme – ash free dry mass. g afdm g–1 dm mg chl a g–1 dm epiphytic metazoans species richness (number of taxa) total epiphytic metazoans abundance (ind. g–1 dm) cladocerans abundance in epiphyton (ind. g–1 dm) transparency (sd m) 0.47 0.66 epiphytic metazoans species richness (number of taxa) –0.71 0.49 total epiphytic metazoans abundance (ind. g–1 dm) –0.51 0.43 0.47 rotifera abundance in epiphyton (ind. g–1 dm) –0.42 0.46 0.94 copepoda abundance in epiphyton (ind. g–1 dm) 0.77 0.52 cladocera abundance in plankton (ind. l–1) 0.41 0.62 rotifera abundance in plankton (ind. l–1) –0.45 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:30 color profile: disabled composite 150 lpi at 45 degrees 132 acta bot. croat. 71 (1), 2012 [poljar m., fressl j., dra@ina t., meseljevi] m., gr^i] z. fig. 3. mean, minimum and maximum values of analysed parameters among different habitats. results of significant differences among habitats according to kruskal-wallist test (df=2, n=24) and post-hoc multiple comparison are incorporated in graph titles. 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:34 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 133 epiphytic metazoans on emergent macrophytes tab. 3. mean densities (mean ± sd, ni1,i2,m2=24) of epiphytic metazoans at different habitats i1, i2 and m2. i1 (ind g–1 dm) i2 (ind g–1 dm) m2 (ind g–1 dm) group taxa mean sd mean sd mean sd cladocera acroperus elongatus (sars, 1862) 0.2 ± 1 1 ± 4 alona costata sars, 1862 38 ± 102 2 ± 4 1 ± 3 alona rectangula sars, 1862 10 ± 31 alona weltneri keilhack, 1905 1 ± 2 1 ± 4 bosmina longirostris (o. f. müller, 1776) 2 ± 3 7 ± 9 16 ± 26 ceriodaphnia laticaudata o. f. müller, 1867 1 ± 2 2 ± 5 ceriodaphnia quadrangula o. f. müller, 1785 0.4 ± 1 chydorus ovalis kurz, 1875 2 3 18 ± 26 chydorus sphaericus (o. f. müller, 1776) 2 ± 7 7 ± 20 daphnia cuculata sars, 1862 2 ± 3 pleuroxus denticulatus birge, 1879 0.2 ± 1 scapholeberis kingi sars, 1888 2 ± 6 cladocera total 40 ± 102 25 ± 43 51 ± 57 copepoda copepodites 5 ± 10 2 ± 7 6 ± 13 nauplii 17 ± 20 17 ± 10 71 ± 68 copepoda total 22 ± 29 20 ± 14 77 ± 73 insecta diptera larvae 3 ± 7 5 ± 8 nematoda nematoda 6 ± 14 61 ± 100 ostracoda ostracoda 0.5 ± 1 2 ± 5 rotifera ascomorpha saltans bartsch, 1870 35 ± 54 1 ± 4 asplanchna priodonta (goose, 1850) 0.1 ± 0.4 5 ± 12 1 ± 2 bdelloidea 36 ± 91 9 ± 17 brachionus angularis goose, 1851 0.2 ± 0.5 brachionus patulus (o. f. müller, 1786) 6 ± 12 2 ± 4 brachionus quadridentatus hermann, 1783 6 ± 12 brachionus urceolaris o. f. müller, 1773 33 ± 93 cephalodella forficata (ehrenberg, 1832) 1 ± 2 cephalodella gibba (ehrenberg, 1832) 0.1 ± 0.4 1 ± 4 colurella obtusa (goose, 1886) 10 ± 27 126 ± 208 colurella uncinata (o. f. müller, 1773) 1 ± 4 filinia longiseta (ehrenberg, 1834) 0.1 ± 0.4 2 ± 3 24 ± 41 gastropus stylifer imhof, 1891 5 ± 12 4 ± 9 keratella cochlearis (goose, 1851) 17 ± 19 17 ± 24 107 ± 132 keratella cochlearis tecta goose, 1851 0.2 ± 0.5 keratella quadrata (o. f. müller, 1786) 1 ± 1 10 ± 19 12 ± 19 lecane cornuta (müller, 1786) 3 ± 9 15 ± 42 lecane luna (o. f. müller, 1776) 3 ± 4 12 ± 17 27 ± 43 lecane lunaris (ehrenberg, , 1832) 5 ± 10 24 ± 43 55 ± 74 lepadella patella (o. f. müller, 1786) 8 ± 20 4 ± 6 ploesoma hudsoni (ehrenberg, , 1891) 1 ± 4 1 ± 3 polyarthra spp. 4 ± 7 2 ± 4 4 ± 9 pompholyx sulcata hudson, 1885 7 ± 19 3 ± 9 scaridium longicaudum (müller, 1786) 0.1 ± 0.2 1 ± 2 squatinella rostrum (schmarda, 1846) 0.2 ± 1 15 ± 44 squatinella lamellaris (o. f. müller, 1786) 0.2 ± 1 testudinella mucronata (goose, 1886) 1 ± 3 59 ± 140 trichocerca bicristata (goose, 1887) 1 ± 2 1 ± 2 6 ± 14 trichocerca capucina (wierzejski et zacharias, 1893) ± 1 ± 2 trichocerca longiseta (schrank, 1802) 2 ± 3 6 ± 8 18 ± 16 rotifera total 67 ± 56 160 ± 160 537 ± 570 grand total 128 ± 124 216 ± 205 733 ± 654 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:34 color profile: disabled composite 150 lpi at 45 degrees of epiphytic metazoans was significantly positively affected by rotifers and copepods density (tab. 2). separately, on habitat m2 rotifer abundance significantly positively affected epiphytic metazoan abundance (r=0.95, n=8, p<0.05). in general, prevailing among epiphytic rotifers were the microphagous species, keratella cochlearis, lecane lunaris and colurela obtusa. cladoceran density in epiphyton did not show significant differences (p>0.05) among investigated habitats (fig. 3e). their epiphytic abundance was positively affected by cladocerans in surrounding water and negatively by rotifer density in surrounding water (tab. 2). small-bodied microphagous cladocerans i.e., alona, bosmina and chydorus species contributed mostly in total epiphytic metazoan density (tab. 3). among cladocerans in m2, the presence of large-bodied (i.e. daphnia, ceriodaphnia) and of small-bodied species (tab. 3) was recorded. copepods, represented by nauplii and copepodites, also reached their highest density on m2 (fig. 3g). among studied epiphytic metazoans only copepod density in epiphyton showed a significant difference (z=2.21, nspring,summer=8, p=0.03) in seasonality with higher density in spring (46±50 ind. g –1dm) and lower in summer (28±56 ind. g–1 dm). discussion transparency is a well known indicator of the trophic state in aquatic systems (karabin 1985) and a main driver in the outcome of predator-prey relations (horpila and nurminen 2005, estlander et al. 2009). we assume that more intensive agriculture on ploughed fields and fishing around/in ko1 than ko2 contribute to decreased transparency via increasing particulate and dissolved organic matter indicating eutrophic conditions in lake ko1. significantly lower ph and higher conductivity in the higher trophic lake, ko1, indicate phosphorous release from sediment at lower ph, which leads simultaneously to increasing ionic concentration measured as electroconductivity (review kalff 2002). biela�ska-grajner and g�adysz (2010) also concluded that higher electroconductivity often cooccurs with anthropogenic eutrophication reflected as higher trophic levels. results of analyses in this study suggested that transparency positively influenced cladoceran diversity and density, as well as total metazoan diversity in epiphyton. this could be explained by intensive fish predation at higher transparency in pelagial, with cladocerans migrating to the littoral and becoming attached to macrophytes (nurminen et al. 2007, estlander et al. 2009). similar results were established in previous study in these oxbow lakes as increasing density of small and large-bodied cladocerans in littoral zone of ko2 at higher transparency in the pelagial ([poljar et al. 2011). we recorded the highest value of organic matter in epiphyton on a simple iris stem in ko1. this could be explained by the decreasing role of macrophytes as shelter for zooplankton at a higher trophic level, as water turbidity increased and transparency decreased (castro et al. 2005, estlander et al. 2009). thus iris belt in ko1 was not a favourable habitat for epiphytic metazoans and consequently low grazing on algae and detritus was expressed as higher afdme. results of correlations suggested decreasing afdme amount at higher epiphytic metazoan density and diversity, especially caused by higher rotifers density. it indicated metazoans grazing upon detritus in epiphyton and the development of few abundant microphagous species among rotifers (bdelloids, colurella, lecane) (review, macinnis 1997) and cladocerans (alona, bosmina, chydorus) (hart and lovvorn 2000, cazzanelli et al. 2008). 134 acta bot. croat. 71 (1), 2012 [poljar m., fressl j., dra@ina t., meseljevi] m., gr^i] z. 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:34 color profile: disabled composite 150 lpi at 45 degrees algal biomass in epiphyton did not vary significantly among the investigated macrophytes. thus our results do not confirm the results of other authors that macrophytes with complex architecture harbour a higher amount of epiphyton (duggan 2001, tessier et al. 2008). we explain our results on assessment that mentha surface area does not exceed that of iris. this is expressed in similar epiphytic algal biomass between habitats and sites. positive relation between algal biomass and epiphytic metazoans and rotifer densities could be explain by the feeding guilds of these organisms. as microphagous species dominated in epiphyton, we suppose that they influenced the grazing of organic matter. rotifer (ascomorpha, gastropus, trichocerca) and crustacean (nauplii, copepodites, daphnia) algivorous species just temporarily fed on epiphyton and did not significantly influence on algal grazing (armengol and miracle 2000, horppila and nurminen 2008). we think that in our study different densities among epiphytic metazoans were not derived from plant architecture but from interaction between width of macrophyte belt and turbidity in each lake. for instance, iris stems in the less transparent and higher trophic lake, ko1, hosted significantly fewer species than iris stems in the lake of higher transparency and lower trophic state, ko2. in ko1 the macrophyte belt is significantly narrower than in ko2 ([poljar et al. 2011). these results indicate that higher turbidity and trophic level together with narrow macrophyte belt probably reduce the influence of macrophyte belt as a zooplankton shelter against predators (estlander et al. 2009, [poljar et al. 2011). in lake ko2, iris and mentha recorded equal total diversity of epiphytic metazoans. however, species composition indicates that mentha epiphyton hosted some large-bodied cladocerans, i.e. ceriodaphia, daphnia, while on iris small-bodied cladocerans were attached. this is in agreement with records that macrophytes of complex architecture offer better shelter than those with simple architecture (vieira et al. 2007). namely, large-bodied cladocerans are first under attack from fish predators and need safe shelter against fish (balayla and moss 2003, cazzanelli et al. 2008). thereby, significant correlations among diversity and cladoceran density in mentha epiphyton derived presumably from a wider macrophyte belt in ko2 than in ko1. moreover, total epiphyton diversity positively correlated with cladoceran density. we presumed that at higher transparency, i.e. ko2, there was an increased risk of fish predation, which caused copepods to shift to the littoral zone, where they found more complex mentha a more suitable habitat provided by simple iris. this resulted in significant differences in their spatial distribution which is in accord with results of miracle et al. (2007). as in other studies (duggan et al. 2001, arora and mehra 2003), rotifers contributed most to the total density in epiphyton at each habitat. taxa restricted to vegetation, bdelloids and lecane, but also keratella cochlearis mainly contributed to this higher density. the latter species is characterised as common in vegetation and open water. in the more complex mentha habitat k. cochlearis developed a higher density, in contrary to the more simple iris habitat. according to romare et al. (2003) the littoral zone and its connection with the pelagial is important in structuring fish and plankton assemblage. the epiphyton community could also indicate the consequences of environmental and biocoenotic changes. a better insight would be revealed by further comprehensive studies of aquatic communities. acta bot. croat. 71 (1), 2012 135 epiphytic metazoans on emergent macrophytes 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:34 color profile: disabled composite 150 lpi at 45 degrees references armengol, x., miracle, m. r., 2000: diel vertical movements of zooplankton in lake la cruz (cuenca, spain). journal of plankton research 22, 1683–1703. arora, j., mehra, n. k., 2003: species siversity of planctonic and epiphytic rotifers in the backwaters of the delhi segment of the yamuna river, with remarks on new rwcords for india. zoological studies 42, 239–247. balayla, d. j., moss, b., 2003: spatial patterns and 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[poljar, m., dra@ina, t., habdija, i., meseljevi], m., gr^i], z., 2011: contrasting zooplankton assemblages in two oxbow lakes with low transparencies and narrow emergent macrophyte belts (krapina river, croatia). international review of hydrobiology 96, 175–190. tessier, c., cattaneo, a., pinel-alloul, b., hudon, c., bocard, d., 2008: invertebrate communities and epiphytic biomass associated with metaphyton and emergent and submerged macrophytes in a large river. aquatic sciences 70, 10–20. vieira, l. c. g., bini, l. m., velho, l. f. m., mazao, g. r., 2007: influence of spatial complexity on the density and diversity of periphytic rotifers, microcrustaceans and testate amoebae. fundamental and applied limnology – archiv für hydrobiologie 170, 77–85. walsh, e. j., 1989: ovoposition behaviour of the littoral rotifer euchlanis dilatata. hydrobiologia 186/187, 157–161. walsh, e. j., 1995: habitat specific predation susceptibilities of a littoral rotifer to two invertebrate predators. hydrobiologia 313/314, 205–211. wetzel, r. g. 2001: limnology. lake and river ecosystems. academic press, san diego. 138 acta bot. croat. 71 (1), 2012 [poljar m., fressl j., dra@ina t., meseljevi] m., gr^i] z. 536 spoljar_verzija-10.prn u:\acta botanica\acta-botan 1-12\536 spoljar_verzija-10.vp 26. o ujak 2012 11:38:35 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (1), 1–8, 2011 coden: abcra 25 issn 0365–0588 morphogenesis, volume and number of hop (humulus lupulus l.) glandular trichomes, and their influence on alpha-acid accumulation in fresh bracts of hop cones sini[a sre^ec1, vesna zechner-krpan2*, sanja marag2, igor [poljari]3, ivka kvaternjak1, gordan mr[i]3 1 kri`evci college of agriculture, m. demerca 1, hr-48260 kri`evci, croatia 2 faculty of food technology and biotechnology, university of zagreb, department of biochemical engineering, pierrotijeva 6, hr-10000 zagreb, croatia 3 forensic science centre »ivan vu~eti}«, ilica 335, hr-10000 zagreb, croatia abstract – the esem investigations revealed the morphogenesis of peltate glandular trichomes, which was divided into five phases. in phase one, new peltate glandular trichomes were initiated; in phase two, they were differentiated; in phase three trichomes grew vigorously; in phase four they were determined; in the fifth and final phase they came to maturity. volume of glandular trichome during the different phases of morphogenesis varied from 0.25 ´ 10–2 mm3 in phase 1, to 1.95 ´ 10–2 mm3 in phase 5. more glandular trichomes are placed on the base of the adaxial side of bracts (average 7 mm–2) than on the base of abaxial side (average 5.8 mm–2). in this research, positive spearman's rank order correlations were found between the average number of glandular trichomes and content of a-acids as well as between the average volume of glandular trichomes and content of a-acids. key words: humulus lupulus, trichome, morphogenesis, esem, alpha-acid introduction peltate glandular trichomes are micro bodies of spheroid and spherical shape on the epidermis of hop cone bracts in which the hop metabolites are accumulating (oliveira and pais 1988, 1990; ^eh et al. 2007; wang et al. 2008). seven morphological development stages of peltate glandular trichomes are described and the relation between their morphogenesis and accumulation of secondary metabolites was clarified (saito et al. 1995, hirosawa et al. (1995). kim and mahlberg (2000) described the early development of the secretory cavity of chemically fixed peltate glands in hop. the same authors found light and gray secretions on the irregular inner surface of the cuticle. moreover, they described acta bot. croat. 70 (1), 2011 1 * corresponding author, e-mail: vzkrpan@pbf.hr copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-11\srecec.vp 28. o ujak 2011 15:48:39 color profile: disabled composite 150 lpi at 45 degrees how secretions contributed to the thickening of the cuticle and created a network of striae distributed throughout the cuticle. the development of lupulin glands is strictly divided into a growth phase and biosynthetic-secretory phase (sugiyama et al. 2006). activity of vps gene (valerophenone synthase), which is involved in the first steps of bitter resin (a-acid) biosynthesis, was strong when the cuticle was slightly detached from the glandular head cells. wang et al. (2008) described three biosynthetic pathways for terpene-derived natural products found in hop trichomes and activity of vps gene is obvious in c pathway of terpene-derived natural products. the final step of carbon pathway is the biosynthesis of humulone or a-acids. the primarily goal of this research was to define the stages of peltate glandular trichome morphogenesis and also to consider the changes in their volume and morphology during the most important phases. the secondary goal was to define the influence of peltate glandular trichome number and volume on level of a-acids, the most important chemical compound of hops. materials and methods hop (humulus lupulus l. cv. aurora) grown in the hop garden of the gregurovec hop co-operative near kri`evci college of agriculture was used as experimental material. the hop cones or female inflorescence were successively collected every week during the hop harvest in the phenological phase of technological maturity. from the beginning of technological maturity till the end of hop harvest, five samples of hop cones were selected and treated further as dependent samples. the esem observations of peltate glandular trichome morphogenesis and shape were provided on bracts separated from the central string (rachis) of the hop cones. the bracts were of approximately equal length and diameter. the remaining bracts were used for analysis of a-acid content. in esem studies, a philips xl 30 esem (detector: edax, type pv 9760/68 me, resolution 134.30 ev, bse detector: philips pw 6848/00) and edax genesis v.5.21 software were used. photographs were taken at an accelerating voltage of 25 kv under recording time of 5 seconds. diameter of the observed area was 10 mm. at the same time the analyses of a-acids were also provided using the method of lead conductance value (lcv) of hops, powders and pellets, according to analytica – european brewery convention 7.4 (anonymous 1998). this method was chosen due to following reasons. first, the time for preparation of samples and standard solution of lead acetate is very short. second, the analyses of a-acids were provided from the bracts separated from the same hop cones from which the bracts for esem observations were taken (required weight of the hop bract sample for lcv analyses is 5 g only). third, the results of the lcv method are comparable with the results of other analytical methods prescribed by the analytical committee of european brewery convention (anonymous 2006) including hplc analysis (forster 1987, 2001; briggs et al. 1981; ko[ir 1996). fourth, according to wang et al. (2008), biosynthesis of humulone or a-acids is the final step in the carbon pathway of terpene-derived natural products. for these reasons, use of the lcv method is acceptable for the determination ofthe relationships or possible correlations between the number or volume of glandular trichome (independent variables) and the content of a-acids in the same hop cones from which the bracts for esem studies were taken. 2 acta bot. croat. 70 (1), 2011 sre^ec s., zechner-krpan v., marag s., [poljari] i., kvaternjak i., mr[i] g. u:\acta botanica\acta-botan 1-11\srecec.vp 28. o ujak 2011 15:05:36 color profile: disabled composite 150 lpi at 45 degrees volume of peltate glandular trichomes was calculated by following equation for volume calculation of spheroid bodies: v = 4 3 p · a 2 b a= width (distance between two points on x-axis in mm) b= height (distance between two points on y-axis in mm) the data analyses were provided using the t-test for dependent samples and calculating the least significant differences in order to find significant differences between the number of glandular trichomes on the abaxial and adaxial sides of the bracts and also between the volumes of glandular trichomes in different phases of morphogenesis. spearman’s rank order correlation was also used as nonparametric statistical method (hill and lewicki 2006) for the determination of correlations between dimensions and number of peltate glandular trichomes (independent variables) and level of a-acids. transformation of percents of a-acids was done by angular transformation (chanter 1975). during this research a total of 150 esem observations of morphology and dimensions of peltate glandular trichome were provided and also 18 chemical analyses of a-acid content from six samples of hop cones. sampling of hop cones was provided every sixth day during the last five weeks of hop vegetation, which corresponded with the phenological phases of hop cone formation and technological maturity. results after esem observations the morphogenesis of peltate glandular trichomes was divided into five phases. the first phase is the initiation of new peltate glandular trichome (plate 1, fig. 1, position 1); second phase is the differentiation of the young trichome (fig. 1, position 2); third phase is intensive growth (fig. 2); fourth phase is the phase of determination (fig. 3); the fifth phase is peltate glandular trichome maturity (fig. 4). in this research, significant differences were found between the number of glandular trichomes per mm² on the base and flat areas of the abaxial side of bracts and also on the base area of the adaxial sides of bracts (tab. 1). more glandular trichomes are to be found on the bases of the adaxial sides of the bracts (average 7 mm–2 of glandular trichomes) than on the base of the abaxial sides (average 5.8 mm–2 of glandular trichomes) (tab. 1). the smallest number of glandular trichomes is found on the flat area of the abaxial side of a bract (average 3.12 mm–2 of glandular trichomes). according to our studies, we assume that the bulbous glandular trichome is just the final stage of peltate glandular trichome development during the phenological phase of hop maturity (plate 2, fig. 5). average volume of glandular trichome during the different phases of morphogenesis varied from 0.25 ´ 10–2 mm3 in phase 1, to 1.95 ´ 10–2 mm3 in phase 5 (tab. 2). in the same observed area peltate glandular trichomes in different phases of morphogenesis (initiation, differentiation, determination, intensive growth and maturity) were found lying next to each other (plate 2, fig. 6). acta bot. croat. 70 (1), 2011 3 alpha acid accumulation in glandular trichomes u:\acta botanica\acta-botan 1-11\srecec.vp 28. o ujak 2011 15:05:36 color profile: disabled composite 150 lpi at 45 degrees 4 acta bot. croat. 70 (1), 2011 sre^ec s., zechner-krpan v., marag s., [poljari] i., kvaternjak i., mr[i] g. plate 1. morphogenesis and differentiation of glandular trichomes. fig. 1 – first phase of morphogenesis – phase of initiation of young glandular trichome (position 1) and second phase of morphogenesis – phase of differentiation of young glandular trichome (position 2); fig. 2 – third phase of morphogenesis – phase of intensive growth of peltate glandular trichomes; fig. 3 – fourth phase of morphogenesis – phase of peltate glandular trichome determination; fig. 4 – fifth phase of morphogenesis – phase of peltate glandular trichome maturity tab. 1. differences between the number of glandular trichomes (mm–2) on abaxial and adaxial side of bracts (n = 121) descriptive statistics flat area of abaxial side of bracts base of abaxial side of bracts base of adaxial side of bracts mean 3.12 5.83 6.95 st. dev. 2.02 4.32 2.62 st. error 0.18 0.39 0.24 comparisons base of abaxial side vs. flat area of abaxial side of bracts base of adaxial side vs. flat area of abaxial side of bracts base of adaxial vs. base area of abaxial side of bracts diff. of means 2.71** 3.82** 1.16* st. dev. diff. 0.43 0.30 0.45 lsd(p=0.05) lsd(p=0.01) 0.84 1.11 0.58 0.77 0.88 1.16 *: 95 % of significance and **: 99 % of significance u:\acta botanica\acta-botan 1-11\srecec.vp 28. o ujak 2011 15:05:37 color profile: disabled composite 150 lpi at 45 degrees in this study strong positive spearman rank order correlations were found between weeks of hop cone maturity vs. number and volume of glandular trichomes found (fig. 7). that phenomenon is also visible on microphotographs of hirosawa et al. (1995). positive spearman rank order correlations were found between the average number of glandular trichomes and the content of a-acids (fig. 8) and also between the average volume of glandular trichomes and the a-acid content (fig. 9). discussion these results are in line with the results of hirosawa et al. (1995) who found increasing values of total a-acids from the early a-phase to the late g-phase of peltate glandular trichome development. according to this study we assume that peltate glandular trichomes after the fifth phase of morphogenesis are transformed into bulb glandular trichomes (fig. 5). acta bot. croat. 70 (1), 2011 5 alpha acid accumulation in glandular trichomes plate 2. glandular trichomes, morphogenesis and bulbs. fig. 5 – peltate glandular trichomes in maturity phase (position 1) and formation of bulbs (position 2); fig. 6 – glandular trichomes in different phases of morphogenesis on same observed area. tab. 2. volume of peltate glandular trichomes (10–2 mm3) in different phases of morphogenesis (n = 36) descriptive statistics phase 1 phase 2 phase 3 phase 4 phase 5 mean 0.25 0.37 1.09 1.54 1.88 st. dev. 0.0075 0.049 0.085 0.154 0.063 st. error 0.0043 0.021 0.038 0.068 0.023 comparisons phase 2 vs. phase 1 phase 3 vs. phase 2 phase 4 vs. phase 3 phase 5 vs. phase 4 phase 5 vs. phase 3 diff. of means 0.12** 0.72** 0.45** 0.34** 0.79** st. dev. diff. 0.0015 0.016 0.025 0.027 0.013 lsd (p=0.01) 0.004 0.04 0.068 0.001 0.036 **: 99 % of significance u:\acta botanica\acta-botan 1-11\srecec.vp 28. o ujak 2011 15:05:38 color profile: disabled composite 150 lpi at 45 degrees the cause of such a phenomenon could be an increase of the partial pressure of liquids and gasses on the cell walls inside peltate glandular trichomes during the biosynthesis of terpene-derived natural products, particularly a-acids which are the final product in the carbon pathway of terpene-derived natural products (wang et al. 2008). this could be an 6 acta bot. croat. 70 (1), 2011 sre^ec s., zechner-krpan v., marag s., [poljari] i., kvaternjak i., mr[i] g. fig. 7. spearman rank order correlations between weeks of hop cones maturity during vegetation and hop harvest average number and volume of glandular trichomes fig. 8. spearman’s rank order correlations between average number of glandular trichomes and content of a-acids u:\acta botanica\acta-botan 1-11\srecec.vp 28. o ujak 2011 15:05:42 color profile: disabled composite 150 lpi at 45 degrees explanation for the results of oliveira and pais (1988), who found that number of peltate trichomes decreases with the expansion of the leaves (bracts). however, accumulation of hop polyphenols, xanthohmol (^eh et al. 2007) and finally humulone or a-acids (sre^ec et al. 2008, mozny et al. 2009, ku^era and krofta 2009) depends on water supply and air temperatures during the hop vegetation and particularly during the technological maturity of hop cones. the phases of morphogenesis in general correspond with the results of oliveira and pais (1988, 1990) and the sem observations of saito et al. (1995) and hirosawa et al. (1995). oliveira and pais (1988) described the morphological differences between peltate and bulbous trichomes and also found that the number of peltate trichomes decreases with the expansion of the leaves (bracts). it can thus be concluded with some certainty that the number and volume of peltate glandular trichomes as well as their morphogenesis and consequently accumulation of a-acids are greatly influenced by climatic factors. references anonymous, 1998: analytica-ebc, methods of analysis, ebc analysis committee, nurnberg, germany. anonymous, 2006: analytica – european brewery convention 7.4 http://www.european breweryconvention.org/pdf/analytica-ebc-contents 2006 + last amendment.pdf briggs, d. e., hough, j. s., stevens, r., young, t. w. 1981: malt and brewing science, 1. malt and sweet worth, chapman and hall, london. acta bot. croat. 70 (1), 2011 7 alpha acid accumulation in glandular trichomes fig. 9. spearman’s rank order correlations between average volume of glandular trichomes and content of a-acids u:\acta botanica\acta-botan 1-11\srecec.vp 31. o ujak 2011 13:16:34 color profile: disabled composite 150 lpi at 45 degrees chanter, d. o., 1975: modifications of the angular transformation. journal of the royal statistical society. series c (applied statistics) 24, 354–359. ^eh, b., ka^, m., ko[ir, i. j., abram, v., 2007: relationship between xanthohumol and polyphenol content in hop leaves and hop cones with regard to water supply and cultivar. international journal of molecular sciences 8, 989–1000. forster, a., 1987: conductometric methods for hops and hop products. european brewery convention symposium on hops, freising-weihenstephan, 17–19. forster, a., 2001: the quality chain from hops to hop products. proceedings 48 international hop growers congress, canterbury, 6–10. hill, t., lewicki, p. 2006: statistics: methods and applications. statsoft, inc., tulsa. hirosawa, t., saito, t., tanaka, t., matasushima, h., 1995: sem observation and hplc analysis of the accumulation of alphaand betaacids in the fresh developing hop (humulus lupulus l.) peltate glandular trichomes. journal of electron microscopy 44, 145–147. kim, e. s., mahlberger, p. g., 2000: early development of the secretory cavity of peltate glands in humulus lupulus l. (cannabaceae). molecules and cells 10, 487–492. ko[ir, i., 1996: the chemistry and analytics of hops. proceedings 33 hop seminar, @alec. hop bulletin supplement 1, 73–83. ku^era, j., krofta, k., 2009: mathematical model for prediction of alpha acid contents from meteorological data for 'saaz' aroma variety. acta horticulturae 848, 131–139. mozny, m., tolasz, r., nekovar, j., sparks, t., trnka, m., zalud, z., 2009: the impact of climate change on the yield and quality of saaz hops in czech republic. agricultural and forest meteorology 149, 913–919. oliveira, m. m., pais, m. s., 1988: glandular trichomes of humulus lupulus var. brewers gold: ontogeny and histochemical characterization of the secretion. nordic journal of botany 8, 349–359. oliveira, m. m., pais, m. s., 1990: glandular trichomes of humulus lupulus var. brewers gold (hops): ultrastructural aspects of peltate trichomes. journal of submicroscopic cytology and pathology 22, 241–248. saito, t., hirosawa, t., horiuchi, s., murakami, a., matsushima, h., 1995: a study of sem examination on fresh hop (humulus lupulus l.) peltate glandular trichomes. journal of electron microscopy 44, 39–44. sre^ec, s., kvaternjak, i., kau^i], d., [poljar, a., erhati], r., 2008: influence of climatic conditions on accumulation of a-acids in hop cones. agriculturae conspectus scientificus 73, 161–166. sugiyama, r., oda, h., kurosaki, f., 2006: two distinct phases of glandular trichome development in hop (humulus lupulus l.). plant biotechnology 23, 493–496. wang, g., tian, l., aziz, n., broun, p., dai, x., he, j., king, a., zhao, p. x., dixon, r. a., 2008: terpene biosynthesis in glandular trichomes of hop. plant physiology 148, 1254–1266. 8 acta bot. croat. 70 (1), 2011 sre^ec s., zechner-krpan v., marag s., [poljari] i., kvaternjak i., mr[i] g. u:\acta botanica\acta-botan 1-11\srecec.vp 28. o ujak 2011 15:05:44 color profile: disabled composite 150 lpi at 45 degrees 625 pise and gaikwad.vp acta bot. croat. 72 (2), 399–406, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/botcro–2013–0001 short communication herbaceous periwinkle, vinca herbacea waldst. et kit. 1799 (apocynaceae), a new species of the croatian flora jános csiky1, dragica purger2* 1 department of plant taxonomy and geobotany, institute of biology, faculty of sciences, university of pécs, hungary 2 national institute for the environment h–7623 pécs, köztársaság tér 7, hungary abstract – populations of herbaceous periwinkle, vinca herbacea waldst. et kit., were found on april 2007 on bansko hill (baranja, croatia), which lies on the south-western edge of the range of this pontic-pannonian species. since v. herbacea was included neither in the handbooks for plant identification nor in the current croatian flora database, a new key for the determination of vinca l. species of croatia is presented herein. the herbaceous periwinkle should be treated as a critically endangered (cr) species in croatia, considering the low number of individuals and the small extent of its occurrence in extremely rare habitats at the margin of its distribution. new recordings of some very rare or »data deficient« (dd) taxa of croatia are also presented here: scorzonera hispanica l. and inula germanica l. key words: bansko hill, continental, loess, pannonic, steppe. introduction the herbaceous periwinkle vinca herbacea waldst. et kit. 1799 (apocynaceae) (syn. vinca mixta velen., vinca pumila e.d.clarke) is a plant native to south-western asia from the caucasus mountains around the black sea through anatolia and the balkan peninsula to the eastern part of the danube valley and the carpathian basin, but its area is disjunctive (meusel et al. 1978). it is distributed in southern and central europe: the southern-central part of former sssr: russian federation and ukraine (pobedimova 1952), bulgaria (delipavlov 1983), serbia (obradovi] et al. 1986), hungary (soó 1966, király 2009), austria (fischer et al. 2008), slovakia (randu[ka and kri@o 1986), romania, greece and turkey (stearn 1972). vinca herbacea is a lowland-colline, pontic-pannonian flora element (jávorka 1925, soó 1966, meusel et al. 1978). the occurrence of this species was indicated for the eastern part of croatia in jávorka (1925), but this record was never given acta bot. croat. 72 (2), 2013 399 * corresponding author, e-mail: dragica.purger@neki.gov.hu copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. any precise location. it cannot be found in the handbooks for determination of plants in croatia (domac 1984, 2002), it is absent from the red book of vascular flora (nikoli] and topi] 2005) as well as from the current national database of the croatian flora (nikoli] 1997, 2012). there are no data about this plant in the papers dealing with the flora of bansko hill (godicl 1980, sturc 1988, purger and csiky 2008) or baranja (panjkovi] 1990, zahirovi] 2000). therefore vinca herbacea should be treated as a new taxon and included in the current croatian flora database. vinca herbacea is a herbaceous perennial vascular plant with procumbent or ascending stems, and a length up to 60(–80 cm), dying back at the end of autumn. leaves are narrowly lanceolate or narrowly elliptical, the lowermost sometimes obovate, cuneate at base, with margins minutely ciliate. pedicels are shorter than the subtending leaves (stearn 1972). flowers are well spaced along the stems. calyx-lobes are 4–8 mm, very narrowly triangular, with the margins smooth, ciliate or scabrid. corolla is deep blue in colour. corolla-tube is 10–15 mm, limb is 20–35 mm in diameter, blue, lobes are acute or very obliquely truncate. the flowers are produced in spring (april – may) (waldstein and kitaibel 1802, király 2009). there are two other native periwinkle species occurring in croatia: the bigleaf periwinkle vinca major and the common periwinkle v. minor (domac 1984). the latter species is similar in the vegetative stage to the herbaceous periwinkle and therefore our aim was to make a new key for determination of the vinca l. species of croatia based on the descriptions of waldstein and kitaibel (1802), stearn (1972), bényei-himmer et al. (1999) and király (2009). in this paper we also present new records from the same steppe habitats of some very rare or »data deficient« (dd) taxa of croatia. material and methods study area bansko hill (bansko brdo) is an elevation consisting of loess sediments (243 m a.s.l.), 21 km long, stretching in a ne–sw direction in ne croatia (bognar 1990). its south-eastern part is a sheer, 25–58 m high loess cliff, facing onto the floodplain of the danube. in consequence of the steep slope the precipitation cannot wet the soil sufficiently. the loess cliffs, exposed to sunshine (irradiation) and drying by winds, are extremely hot and dry in the summer time, cold and dry in the winter, so they function as extrazonal semi-deserts and orographic deserts in the zone of the closed oak forests and forest steppes (pócs 1999). the climate of ne croatia is moderately continental: the mean annual temperature is 10–11 °c, precipitation is 600–700 mm with two maxima (late spring and beginning of autumn) that show the sub-mediterranean feature of this climate (seletkovi] and katu[in 1992). plant identification and mapping plant identification was done by using recent hungarian (király 2009) and croatian (domac 1984, 2002) handbooks. in line with the central european flora mapping system (nikoli] et al. 1998) we compiled the distribution map of vinca herbacea in croatia. examples of plants were collected on bansko hill and deposited in the zagreb herbarium (za). geocoding of the site was performed with the use of a gps device. 400 acta bot. croat. 72 (2), 2013 csiky j., purger d. for classification of v. herbacea into iucn red list categories we used criteria from nikoli] (2005). results and discussion as a result of a survey of the loess flora and vegetation of bansko hill (ne croatia) from zmajevac to batina four populations of herbaceous periwinkle were recorded in the 0178/4 central european flora mapping grid (figs. 1–3). wgs 84 coordinates, altitude, population size and brief habitat descriptions: 1. lat. 45.854603, lon. 18.843011, 127 m a.s.l., small patches, in a secondary xero-mesophilous wood, 2. lat. 45.837268, lon. 18.840533, 113 m a.s.l., small patches, in a semi-natural steppe-forest complex, 3. lat. 45.833397, lon. 18.839234, 103 m a.s.l., small patches, in a semi-natural forest-steppe wood, 4. lat. 45.827463, lon. 18.833783, 105 m a.s.l., large patches, on a steep loess wall with secondary scrub and grassland vegetation. key for determination of vinca species 1.a stems rooting on peaks, dying back in the end of autumn. upper leaves narrowly lanceolate or narrowly elliptical, cuneate at base, (2–)3(–5) cm long, 0.5–1.5 cm wide, almost sessile. margins of calyx-lobes smooth, ciliate or scabrid. fruit apex acute, almost rostrate…………………………………………………………v. herbacea waldst. et kit. 1.b stems are over-wintering, rooting on nodes. petiole 1–9 mm long………………… 2 2.a upper leaves are lanceolate or elliptical, (2–)4(–5) cm long, 1–2.5 cm in width, petiole 1–4 mm long. margins of calyx lobes are smooth. fruit apex blunt……… v. minor l. 2.b upper leaves are ovate, near the base cordial-ovate, 4–8 cm long, 2–6 cm in width, petiole 5–9 mm long. margins of calyx lobes are ciliate. fruit apex acute……. . v. major l. ecology of vinca herbacea vinca herbacea is a xero-mezophytic species, populations of which were found on steep south-facing slopes as well as on plateau of bansko hill. this species mostly grows in steppe habitats, in dry rocky places, on shallow soils, humic or sandy soils in dryand in xero-mesophilous steppe grasslands, forest steppes as well as in shrubberies and oak forests (soó 1966, randu[ka and kri@o 1986). this is a plant with narrow ecological stress tolerance (borhidi 1995). the herbaceous periwinkle is an entomophilous species, while its propagules are spread by anemochory, and it flowers from april to the beginning of june (soó 1966). most of the individuals were flowering in april in 2007 in bansko hill, much earlier than the average in hungary. some of the individuals were infected by yellow fungus on the back of the leaves, but this fungus does not kill its host. acta bot. croat. 72 (2), 2013 401 vinca herbacea – a new species in the croatian flora distribution in the neighbouring area and conservation status closest to the croatian locality, vinca herbacea was found in southern hungary – baranya county (soó 1966) on the villány hills and mecsek mountains (farkas 1999). in serbia it occurs in fru{ka mountains, in the deliblato sand area, in the vicinity of belgrade (vi{nji~ka kosa, ko{utnjak) and near kragujevac (resnik) (stjepanovi]-veseli^i] 1973). it was also found in a sandy area near subotica, vr{a~ke mountains and titel hill (obradovi] et al. 1986). the herbaceous periwinkle is a relict from the boreal, having persisted in steppe habitats (obradovi] et al. 1986). 402 acta bot. croat. 72 (2), 2013 csiky j., purger d. fig. 1. distribution of vinca herbacea (*) in bansko hill (croatia). abbreviations: ba – batina; bm – beli manastir; br – branjina; bv – branjinski vrh; dr – dra`; ga – gaji}; ka – kamenac; kr – karanac; ko – kotlina; kv – kne`evi vinogradi; pd – podolje; pp – popovac; su – suza; zm – zmajevac (drawn by jános csiky and toni nikoli}) fig. 2. and 3. flowering vinca herbacea and its habitat on bansko hill, near zmajevac (photo by jános csiky) in the carpathian basin vinca herbacea is a scarce and in hungary a protected species (farkas 1999) but not endangered (németh 1989, király 2007), rare in austria (fischer et al. 2008), endangered in slovakia (feráková et al. 2001) and common in romania (oprea 2005). in serbia it is protected, but not threatened (stevanovi] 1999). threatening factors in the whole area of distribution are the small populations, the low number of flowering individuals, intensive pasturing, burning of grasslands in spring, clearings of shrubs and trees (pobedimova 1952). the habitat type of vinca herbacea is extremely restricted in croatia; the extent of its occurrence is less than 10 km2. there are fewer than 1000 adult individuals in the population. based on these two criteria (nikoli] 2005) vinca herbacea should be treated as a critically endangered (cr) species of the croatian flora. a survey of the populations performed in 2010 shows that the populations had not been significantly changed in the previous three years; nevertheless, a continued monitoring is needed (csiky and purger 2008). occurrence of some rare plants in the same area in the same steppe habitats on bansko hill (lat. 45.837268, lon. 18.840533, 113 m a.s.l., in a semi-natural steppe-forest complex) we found some heliophilous and xerotolerant species which are »data deficient« vascular plant taxa of croatia (nikoli] 2012): scorzonera hispanica l. is extremely rare, found in only a few localities in mediterranean regions of croatia. it is distributed in the eastern part of the illyrian province (jávorka 1925). in serbia it was found in the vicinity of novi sad (zorkóczy 1896) but this information was not confirmed later (obradovi] and butorac 1975). in hungary this species occurs in the southern part of the country, in the mecsek mountains (soó 1970). inula germanica l. is a very rare plant in croatia. this species was found closest to the croatian locality in hungary, in the mecsek mountains and very rarely occurs in baranya-, in the geresdiand szekszárdi-hills (farkas 1999, purger 2002). these are rare sub/mediterranean, and sub-continental species that occur on the southern loess slopes of bansko hill as well as other extremely rare continental species in croatia (csiky et al. 2008, purger et al. 2008). acknowledgement we thank julia török for proofreading the english version of the article. this survey was carried out with permission given by ministry of culture of republic croatia. it was supported by interreg iii a, slo-hu-cro 2006/01/167/hu project. references bényei-himmer, m., facsar, g., udvardy, l., 1999: data on the reproductive 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(ed.), flora of serbia (in serbian), 394–400. serbian academy of sciences, belgrade. sturc, b., 1988: floristic and geobotanical data from loess hills in baranya (in hungarian). pécsi muszaki szemle 33, 19–24. waldstein, f. a., kitaibel, p., 1802: descriptiones et icones plantarum rariorum hungariae 1. schmidt, viennae. acta bot. croat. 72 (2), 2013 405 vinca herbacea – a new species in the croatian flora zahirovi], @. 2000: rare and endangered plants of north-eastern part of croatia (in croatian). msc thesis. university of zagreb, zagreb. zorkóczy, l., 1896. flora of novi sad and surroundings (in hungarian). popovits. m. testvérek könyvnyomdája, ujvidék. 406 acta bot. croat. 72 (2), 2013 csiky j., purger d. 625 pise and gaikwad.vp acta bot. croat. 72 (2), 221–235, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 chlorophyll content, photosynthetic efficiency and genetic markers in two sour cherry (prunus cerasus l.) genotypes under drought stress marija viljevac1*, krunoslav dugali]1, ines mihaljevi]1, domagoj [imi]1, rezica sudar1, zorica jurkovi]1,2, hrvoje lepedu[3 1 agricultural institute osijek, ju`no predgra|e 17, hr-31000 osijek, croatia 2 croatian food agency, i. gunduli}a 36b, hr-31000 osijek, croatia 3 faculty of humanities and social sciences, j.j. strossmayer university of osijek, l. jägera 9, hr-31000 osijek, croatia abstract – drought is a limiting factor in fruit production today. identification of sour cherry genotypes tolerant to drought will enable the sustainability of fruit production. the aim of our study was to select sour cherry genotypes according to their genetic background as well as drought tolerance and investigate possible mechanisms of drought tolerance through the changes in photosynthetic apparatus (i.e. photosynthetic pigment content) and photosynthesis process assessed through the chlorophyll fluorescence transient. all of them together with molecular markers (ssrs and aflps), relative water content (rwc) as indicator of plant water status distinguish two genotypes (kelleris 16 and os), which are the opposite in regards to drought tolerance. down-regulation of photosynthesis in drought-treated kelleris 16 plants was seen as changes in antenna complexes of psii (decreased total chlorophylls content (a+b) and chlorophylls ratio (a/b)). despite unchanged maximum quantum yield of psii in drought-treated leaves of genotype os, overall photosynthetic performance expressed as piabs was down-regulated in both investigated genotypes. however, decrement of piabs was much pronounced in genotype kelleris 16, mainly because of changes in a certain fraction of rcs, which become dissipative centres, seen as increase in abs/rc and di0/rc, in order to avoid photooxidative damage of photosynthetic apparatus. also, electron transport, seen as decrease in et0/(tr0–et0) and et0/rc, was impaired which lead to impaired co2 fixation and photosynthesis. the described changes in the functioning of photosynthetic apparatus in drought-treated plants of kelleris 16 constitute the main distinction between the two investigated genotypes regarding drought adaptation mechanisms. key words: chlorophyll fluorescence, drought tolerance, genetic variability, leaf water content, photosynthetic pigments, prunus cerasus, sour cherry acta bot. croat. 72 (2), 2013 221 * corresponding author, e-mail: marija.viljevac@poljinos.hr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. abbreviations: abs/rc – absorption per active reaction centre; aflp – amplified fragment length polymorphism; chl a – chlorophyll a; chl b – chlorophyll b; chl a+b – total chlorophylls; dw – dry weight; ctab – cetyl trimethylammonium bromide; di 0 /rc – dissipation per active reaction centre; et 0 /rc – electron transport per active reaction centre; et 0 /(tr 0 /et 0 ) – electron transport beyond qa -; f v /f m – maximum quantum yield of photosystem ii; fw – fresh weight; lhc – light harvesting complex; ojip test – chlorophyll a fluorescence transient measurement; pcr – polymerase chain reaction; pi abs – photosynthetic performance index; psii – photosystem ii; pvp – polyvinylpyrrolidone; q a – primary plastoquinon acceptor; q b – secondary plastoquinon acceptor; rapd – random amplified polymorphic dna; rc/abs – density of reaction centers on chlorophyll basis; rflp – restriction fragment length polymorphism; rwc relative water content; ssr – simple sequence repeats; tr 0 /di 0 – flux ratio trapping per dissipation; tr 0 /rc – trapping per active reaction centre; tw – turgid weight; upgma – unweighted pair group method with arithmetic mean introduction the sour cherry (prunus cerasus l.) is a significantly represented fruit in croatian orchards, growing on 2400 ha in 2010. (fao statistical databases 2011). phenological, pomological and chemical variability was found within the population (pu[kar 2005). therefore, it is necessary to perform clonal selection in order to isolate and characterize genotypes with good agronomic traits in order to create seedlings that will ensure uniformity and quality of sour cherry plantations. while phenological, pomological and chemical properties of individual sour cherry genotypes are under the influence of environmental factors, it is necessary to conduct the analysis at the genome level. among the many molecular markers, ssr and aflp markers have proved to be reliable methods for determination of genetic variability in most species of the genus prunus (dirlewanger et al. 2002, hormaza 2002, pedersen 2006). since drought is one of the limiting factors in fruit production today, identification of sour cherry genotypes tolerant to drought will enable the sustainability of fruit production with respect to climate change in the future. reduced water content and leaf water potential, loss of turgor and stomata closure ultimately lead to arrest of photosynthesis, metabolic disorders and plant death (jaleel et al. 2009). leaf relative water content (rwc) is a good criterion for the primary selection of wheat and tomato genotypes tolerant and sensitive to drought (rampino et al. 2006, sánchez-rodríguez et al. 2010). also, rwc is better indicator of plant water status than water potential as it is associated with cell volume and precisely shows the balance between the absorbed and transpirated water (rosales-serna et al. 2004, hassanzadeh et al. 2009). efficiency of the photosynthetic apparatus in response to drought was studied in many plant species (clavel et al. 2006, christen et al. 2007). the measurement of chlorophyll fluorescence is a method commonly used to study photosynthetic efficiency (maxwell and johnson 2000). measurement of the chlorophyll fluorescence transient, followed by ojip-test, gives information on the functioning of the photosynthetic apparatus through parameters describing the absorption of photons, trapping of excitons, electron transport and energy dissipation in both stressful and in optimal conditions for plants (strasser et al. 2004). force et al. (2003) have shown the advantage of the usage of the ojip test in the evaluation 222 acta bot. croat. 72 (2), 2013 viljevac m., dugali] k., mihaljevi] i., [imi] d., sudar r., jurkovi] z., lepedu[ h. of psii efficiency, especially of the index of photosynthetic performance (piabs), since it provides information on overall energy flow through the psii. photosynthetic pigments, especially chlorophyll a and b, have a significant role in the process of photosynthesis because they are responsible for the absorption of light and transfer excitation energy to the chlorophyll molecules of reaction centres. in drought, photosynthetic pigment content decreases from optimal conditions in many plant species. plants reduce photosynthetic pigment content in dry periods because this is a mechanism for the prevention of photosynthetic apparatus damage by allowing less light to be absorbed (kauser et al. 2006, abdalla and el-koshiban 2007). the aim of our study was to select different sour cherry genotypes according to their genetic background as well as drought tolerance. we hypothesized that genotypes with opposite reactions to drought, based on leaf rwc, have different mechanisms of adaptation. in order to investigate changes in psii photochemistry of sour cherry genotypes in response to drought, two sour cherry genotypes with opposite reactions to drought were chosen according to their rwc for further studies of photosynthetic features through the photosynthetic pigments contents and chlorophyll fluorescence. material and methods plant material seven different sour cherry genotypes (genotypes kelleris 16 – kel 16, maraska – ma, cigany – cg and obla~inska represented with four genotypes os, bor, 18 and d6) were analyzed in this study. plant material was obtained from tovlja~ orchard of agricultural institute osijek. young leaves for dna analysis were picked in the spring of 2007 and stored at –80 °c and lyophilized. physiological experiments were performed on micropropagated seedlings produced in vitro by meristem isolation from axillary buds of same genotypes as dna analysis. photosynthetic efficiency under drought stress – greenhouse experiment an experiment was set up in a greenhouse of the agricultural institute osijek during june 2011. uniform two-year old in vitro propagated sour cherry plants in pots (~1.2 l of floradure supstrate, floragard, france) were selected randomly and placed on three movable tables with automatic irrigation systems in greenhouse. ten plants of each genotype were placed on one table and served as controls. control plants were treated with the optimal amount of water every other day according to optimized propagation process. another ten plants of each genotype were placed on the other two tables (two identical experiments). these plants were exposed to drought stress due to lack of water. within 10 days of the experiment, greenhouse daily temperatures were reaching up to 35 °c while humidity varied from around 75% in the morning to 35–40% during a day. these conditions led to the complete desiccation and decay of plants exposed to water deficiency i.e. drought stress. molecular markers analysis dna extraction: immediately before dna extraction, lyophilized tissue was ground in an oscillatory mill and 40 mg of sample was used for dna extraction conducted by ctab acta bot. croat. 72 (2), 2013 223 sour cherry (prunus cerasus l.) genotypes under drought stress method (doyle and doyle 1987) slightly modified by the addition of 4 % pvp to the extraction buffer. the dna quality and quantity was double checked by band confrontation with ldna on 1 % agarose gels and by spectrophotometer (biophotometer, eppendorf, germany). original extracts of genomic dna were diluted to a final working solution of 50 ng ml–1 which was used as template for pcrs. microsatellite analysis the twelve ssr primer pairs used for pcr amplification, previously described by cantini et al. (2001) and clarke and tobutt (2003), were provided by metabion (germany). pcr amplification for ssr analysis was carried out in 15 ml of the reaction mix containing 50 ng of template dna, pcr buffer (10 mm tris-hcl, 50 mm kcl, ph 8.3), 0.2 mm of each primer fluorescently labelled with cy5, 0.2 mm of each dntp, 1.5 mm mgcl2, and 0.25 u of taq dna polymerase (applied biosystems, usa). the dna was amplified in geneamp pcr system 2700 (applied biosystems, usa). »touchdown« pcr conditions were used: 94 °c for 5 min followed by 10 cycles of 94 °c for 30 s, 60 °c for 45 s (–0.5 °c per cycle), 72 °c for 1 min and then 25 cycles of 94 °c for 30 s, 55 °c for 45 s, 72 °c for 1 min with an elongation step of 72 °c for 5 min. aflp assay aflp reactions were performed as described by vos et al. (1995) using ecor i (5’-gactgcgtaccaattc-3’) and msei (5’-gatgagtcctgagtaa-3’) primers with one selective base in pre-amplification (e-a and m-c) and with three selective bases fluorescently labelled with cy5 in amplification (e-aaa/m-ctt; e-aaa/m-cct; e-act/ mcat; e-act/m-cag; e-aca/m-caa; e-aca/m-cac; e-aca/m-cat; e-aca/m-cta; e-aca/m-ctc; e-aca/m-ctg). separation and visualisation of ssr and aflp products amplification products were separated on a 0.5 mm thick and 6 or 7.5% polyacrylamide denaturing gel (for ssrs and aflps, respectively), containing 7 m urea. electrophoresis was performed using an automated alfexpress dna sequencing system (amersham biosciences). separation was done at 1200 v, 40 ma, and 50 w for 360 min at 50 °c. fluorescence signals were collected every 1 s and stored in a computer. a fluorescence labelled molecular marker size (cy5 sizer 50–500; pharmacia biotech, sweden) comprising 10 fragments in the size range of 50 to 500 bp was used as an external size marker. for ssrs an additional two internal standards (50, 115, 250 or 334 bp) were used depending on the size of the expected ssrs. amplified ssr and aflp markers were then analysed using the software package allelelinks (pharmacia biotech, sweden). data analysis of molecular markers only strong markers were scored as binary data, either with presence (1) or absence (0). all calculations were performed by ntsyspc 2.1 software (exeter software co., new york). the genetic similarity among genotypes was calculated using dice’s coefficient (dice 1945) for both types of markers. comparison between ssr and aflp matrices was 224 acta bot. croat. 72 (2), 2013 viljevac m., dugali] k., mihaljevi] i., [imi] d., sudar r., jurkovi] z., lepedu[ h. estimated by mantel’s test (mantel 1967), with which matrix comparison analysis was performed. because of the high correlation between the matrices (r = 0.878), cluster analysis was generated from the similarity matrix of both ssrs and aflps data, by the unweighted pair group method, using an arithmetic averages (upgma) algorithm. relative water content every two days, leaf rwc was measured in control and stressed plants. fully expanded leaves were excised and fresh weight (fw) was immediately recorded. leaves were then soaked for 24 h in distilled water at 8 °c in the dark, and the turgid weight (tw) was recorded. after drying for 24 h at 80 °c total dry weight (dw) was recorded. rwc was calculated according to the formula: rwc (%) = (fw–dw) / (tw–dw) × 100. according to rwc analysis, genotypes with opposite reactions to drought would be identified and taken for further investigations. chlorophyll a fluorescence transient chlorophyll fluorescence transient was measured on the eighth day of the experiment. it was induced by applying a pulse of saturating red light (peak at 650 nm, 3000 mmol m–2 s–1) to leaves dark-adapted for 30 min before measurements. changes in fluorescence were measured using plant efficiency analyser (pea, hansatech, uk) for 1s, starting from 50 ms after onset of illumination. during the first 2 ms, changes were recorded every 10 ms and every 1 ms afterward. the obtained data were used in the ojip test in order to calculate several biophysical parameters of psii functioning previously described by strasser et al. (2004). determination of photosynthetic pigments concentration concentrations of photosynthetic leaf pigments were measured on the eighth day of the experiment. photosynthetic pigments were extracted from about 0.1 g of liquid-nitrogen-powdered leaves with ice-cold absolute acetone in the presence of magnesium hydroxide carbonate. chlorophyll a (chl a) and chlorophyll b (chl b) per dry weight of tissue (dw) were determined spectrophotometrically (specord 200 analytic jena) at 470, 644.8 and 661.6 nm and total chlorophyll (chl a+b) and chlorophyll a/b ratio were calculated (lichtenthaler 1987). statistical analysis the results of the rwc were presented as adendrogram made by a single linkage method on the basis of euclidean distances (cesar et al. 2008, pedisi] et al. 2010) calculated from parameter difference between control and drought treatment during ten days of experiment. data on ojip parameters and photosynthetic pigment concentrations measured in control and drought treated plants were subjected to t-test. differences were considered significant at p £ 0.05. all statistical analyses were performed by statistica 7.1. (statsoft, inc. 2005, usa). acta bot. croat. 72 (2), 2013 225 sour cherry (prunus cerasus l.) genotypes under drought stress results the cluster analysis conducted based on the ssr and aflp data revealed two main groups on dendrogram (fig. 1). one of the groups (group 1) included only one genotype, kelleris 16 from a danish breeding program. the second group which is further divided into two major subgroups (2a and 2b) contained the remaining analyzed genotypes with a very high coefficient of genetic similarity (> 0.96). subgroup 2a included genotypes maraska and cigany, while subgroup 2b included all investigated genotypes of genotype obla~inska (os, 18, d6, bor). the dendrogram, based on euclidean distances calculated from rwc difference between control and drought treatment during ten days of experiment (fig. 2) is constituted by four main groups. the results, according to the grouping of genotypes, suggest that genotypes kelleris 16 and os have the opposite responses to drought, i.e. drought tolerance with genotype kelleris 16 as less and genotype os as more tolerant. 226 acta bot. croat. 72 (2), 2013 viljevac m., dugali] k., mihaljevi] i., [imi] d., sudar r., jurkovi] z., lepedu[ h. fig. 1. dendrogram based on genetic similarity from ssr and aflp markers. fig. 2. dendrogram based on euclidean distances calculated from rwc difference between control and drought treatment during ten days of experiment. drought stress down-regulated the photochemical efficiency of psii (figs. 3, 4; tab. 1). maximum quantum yield of psii (fv/fm) in leaves of genotype kelleris 16 (fig. 3a; tab. 2) in drought treatment was low compared to control with values 0.38 in drought and 0.84 in control treatment. values of fv/fm in leaves of genotype os were 0.82 and 0.81 in control and drought treatment, respectively. performance index (piabs) was also decreased in drought-treated leaves (fig. 3b; tab. 2) of both investigated genotypes. the values of piabs in genotypes kelleris 16 and os were 0.07 and 1.62, respectively, in drought-treated leaves. in control plants the values of piabs were 3.09 and 2.24, in genotypes kelleris 16 and os, respectively. acta bot. croat. 72 (2), 2013 227 sour cherry (prunus cerasus l.) genotypes under drought stress tab. 1. mean values (± standard deviation) of performance index components on the 8th day of the experiment in control and drought-treated leaves of genotypes kelleris 16 and os. genotype treatment kelleris 16 os control drought control drought rc/abs 0.47±0.02 0.20±0.08 0.43±0.02 0.40±0.01 tr0/di0 5.19±0.25 0.82±0.76 4.60±0.28 4.23±0.65 et0/(tr0–et0) 1.27±0.20 0.44±0.09 1.11±0.18 0.95±0.12 tab. 2. t-value and the probability levels in the t-test for maximum quantum yield of psii (fv/fm), performance index (piabs) and components of piabs measured on the 8 th day of the experiment in control and drought-treated leaves of genotypes kelleris 16 and os. significance level is marked at p £ 0.05 (*), 0.01 (**) and 0.001 (***). ns – no significance. genotype / parameter kelleris 16 os t p(t) t p(t) fv/fm 6.47 *** 1.57 ns piabs 12.23 *** 2.57 * rc/abs 8.94 *** 4.17 *** tr0/di0 15.54 *** 1.49 ns et0/(tr0–et0) 10.72 *** 2.04 ns 0.0 0.2 0.4 0.6 0.8 1.0 kel 16 os f v /f m (r e l. u n it s ) a 0.0 1.0 2.0 3.0 4.0 kel 16 os p ia b s (r e l. u n it s ) b fig. 3. maximum quantum yield of psii – fv/fm (a) and performance index – piabs (b) in leaves of sour cherry genotypes kelleris 16 and os (control treatment – white columns, drought treatment – grey columns) on the 8th day of the experiment. vertical bars indicate ± standard deviation. values of performance index components (tab. 1), such as density of reaction centres on chlorophyll basis (rc/abs), ratio of trapping and dissipation fluxes (tr0/di0) and efficiency of the conversion of excitation energy to electron transport (et0/(tr0–et0), in leaves of drought and control treatment revealed that drought-treated leaves of genotype kelleris 16 had significantly lower values (p < 0.001) of all piabs components than control leaves (tab. 2), while drought stress had a significant effect only on decrease of parameter rc/abs in drought-treated leaves of genotype os compared to control leaves. mean values and significant differences between drought and control treatment leaves according to t-test for parameters that describe specific fluxes or specific activities per active reaction centre areshown in fig. 4 and tab. 3, respectively. parameters describing ab228 acta bot. croat. 72 (2), 2013 viljevac m., dugali] k., mihaljevi] i., [imi] d., sudar r., jurkovi] z., lepedu[ h. 0 2 4 6 8 10 kel 16 os a b s /r c (r e l. u n it s ) a 0.0 0.5 1.0 1.5 2.0 2.5 kel 16 os t r 0 /r c (r e l. u n it s ) b 0.0 0.2 0.4 0.6 0.8 1.0 1.2 kel 16 os e t 0 /r c (r e l. u n it s ) c 0 1 2 3 4 5 6 7 8 kel 16 os d i0 /r c (r e l. u n it s ) d fig. 4. specific fluxes or specific activities per active reaction centre in leaves of sour cherry genotypes kelleris 16 and os on the 8th day of the experiment (control treatment – white columns, drought treatment – grey columns): a) abs/rc, b) tr0/rc, c) et0/rc, d) di0/rc. vertical bars indicate ± standard deviation. tab. 3. t-value and the probability levels in the t-test for specific fluxes or specific activities per active reaction centre measured onf the 8th day of the experiment in control and drought-treated leaves of genotypes kelleris 16 and os. significance level is marked at p £ 0.05 (*), 0.01 (**) and 0.001 (***). ns – no significance. genotype / parameter kelleris 16 os t p(t) t p(t) abs/rc –4.39 *** –4.15 *** tr0/rc –0.72 ns –2.84 ** et0/rc 15.70 *** –0.17 ns di0/rc –4.02 ** –2.97 ** sorption (abs/rc) and dissipation (di0/rc) of light energy per active reaction centre were significantly higher in drought treated leaves than in control leaves of both genotypes (fig. 4a and d). trapping per active reaction centre (tr0/rc) shown in fig. 4b was significantly increased under drought treatment compared to control in leaves of genotype os. by contrast, electron transport per active reaction center (et0/rc) was significantly lowered in drought treatment leaves of genotype kelleris 16 while the same parameter in drought treatment leaves of genotype os remain unchanged (fig. 4c). mean values and standard deviations of photosynthetic pigment concentrations in drought and control treatment leaves of genotypes kelleris 16 and os are shown in tab. 4. the mean values of chlorophyll a and total chlorophylls in drought-treated leaves were significantly lower than in control leaves of both investigated genotypes (tab. 5). drought stress did not decreased chlorophyll b concentration in leaves of genotype kelleris 16, unlike in genotype os, while the opposite situation appeared for chlorophyll a/b ratio. discussion in this study we used two types of molecular markers, microsatellite markers in order to identify sour cherry genotypes (kaçar et al. 2006, antonius et al. 2012) and aflp markers in order to identify genotypes within the cultivar obla~inska (zhou et al. 2002, tavaud et al. 2004). a significant correlation (r =0.878), almost identical to the correlation (r = 0.873) obtained by krichen et al. (2010), was revealed between the results of ssr and aflp acta bot. croat. 72 (2), 2013 229 sour cherry (prunus cerasus l.) genotypes under drought stress tab. 4. mean values (± standard deviation) of photosynthetic pigments concentrations on the 8th day of the experiment in control and drought-treated leaves of genotypes kelleris 16 and os. genotype treatment kelleris 16 os control drought control drought chl a (mg g–1 dw) 3.72±0.23 2.48±0.12 2.97±0.07 2.26±0.05 chl b (mg g–1 dw) 1.11±0.06 1.11±0.11 0.97±0.04 0.77±0.05 chl a+b (mg g–1 dw) 4.83±0.25 3.58±0.03 3.94±0.11 3.03±0.10 chl a/chl b 3.35±0.20 2.26±0.33 3.06±0.07 2.97±0.17 tab. 5. t-value and the probability levels in the t-test for photosynthetic pigments concentrations measured on the 8th day of the experiment in control and drought-treated leaves of genotypes kelleris 16 and os. significance level is marked at p £ 0.05 (*), 0.01 (**) and 0.001 (***). ns – no significance. genotype / parameter kelleris 16 os t p(t) t p(t) chl a 9.72 *** 16.27 *** chl b 0.09 ns 5.97 *** chl a+b 9.80 *** 12.18 *** chl a/chl b 5.61 ** 0.97 ns markers in this investigation. cluster analysis generated from the similarity matrix of both ssrs and aflps data differentiated only genotypes, but not genotypes of the cultivar obla~inska (fig. 1). these findings are in accordance with venturi et al. (2006) who showed that ssrs and aflps were not able to distinguish genotypes (or clones) of apple genotypes gala and braeburn. abiotic stressors, especially drought, negatively affect the growth and development of plants and reduce the productivity or fertility of plants. plant response to the stress caused by drought is a complex biological and physico-chemical process that involves a number of micro and macro molecules (khakwani et al. 2011). the study of drought influence on plant water status in association with photosynthetic parameters is important for understanding the physiological mechanisms of drought tolerance and identification of tolerant genotypes of some species (omae et al. 2007). ability to retain the water during dehydration is an important strategy for plant tolerance to stress caused by drought (sánchez-rodríguez et al. 2010). leaf rwc was measured in order to quantify water status in leaves of investigated genotypes. according to rwc results (fig. 2), genotypes kelleris 16 and os were proven to have the opposite reaction to drought. genotype os revealed a higher tolerance to drought than genotype kelleris 16. their photosynthetic efficiency and photosynthetic pigment contents were further investigated in order to understand the physiological mechanisms of drought tolerance. down-regulation of psii photochemical efficiency caused by drought stress was manifested as a significant decrease of maximum quantum yield of psii in drought-stressed leaves of genotype kelleris 16, 45.23% of the value in control leaves (fig. 3a). the fv/fm in drought-treated leaves of kelleris 16 was 0.38, which is much below the considered boundary value (0.75) for fully functional psii (bolhár-nordenkampf et al. 1989). a reduction of fv/fm in drought stress was reported by many authors (bauerle et al. 2003, bertamini et al. 2007). performance index (piabs) was decreased in drought-treated leaves of both investigated genotypes (fig. 3b). unlike fv/fm, piabs is much more sensitive photosynthetic parameter and it can detect stress in plants even before visible symptoms appear on the leaves (clavel et al. 2006, christen et al. 2007, @iv^ák et al. 2008). piabs describes overall photosynthetic performance through the parameters that describe three main functional characteristics of psii reaction centre (strasser et al. 2000), namely rc/abs (density of reaction centres on chlorophyll basis), tr0/di0 (ratio of trapping and dissipation fluxes) and et0/(tr0–et0) (efficiency of the conversion of excitation energy to electron transport). drought-treated leaves of genotype kelleris 16 had all components of piabs significantly lowered (42.55, 15.79 and 34.64% of the value in control leaves for rc/abs, tr0/di0 and et0/(tr0–et0), respectively) in comparison to control leaves (tab. 1 and 2). it is very likely that drastic reduction of tr0/di0 (to 15.79% of the value in control leaves) contributed the most to the decrease of photosynthetic efficiency, namely piabs. a similar conclusion about the impact tr0/di0 on piabs in different stress conditions was deduced by lepedu[ et al. (2009) and nussbaum et al. (2001). they reported that the reduction of tr0/di0 was significantly influenced by a large increase in energy dissipation (di0/rc) apart from trapping of electrons per active reaction centre (tr0/rc). the same situation occurred in our investigation. in drought-treated leaves of kelleris 16 energy dissipation per active reaction centre (di0/rc) was significantly increased, while trapping of electrons per active reaction centre (tr0/rc) was unaffected (fig. 4b, d). 230 acta bot. croat. 72 (2), 2013 viljevac m., dugali] k., mihaljevi] i., [imi] d., sudar r., jurkovi] z., lepedu[ h. drastic increase of energy dissipation is a consequence of enlarged absorption (abs/rc) in drought treatment leaves of both genotypes (fig. 4a). parameter abs/rc represents the functional size of the antenna complexes respectively, provides information on the average amount of absorbing chlorophyll molecules, hence increasing this parameter indicates a reduction in the number of active reaction centres. specifically, due to the high proton gradient through the thylakoid membranes, a portion of the active reaction centre gets a dissipation role, transforming violaksantin to zeaxanthin and thereby emitting energy in the form of heat in order to avoid oxidative damage of psii (critchley 2000). equal energy flow through the photosystems in terms of increasing the abs/rc and di0/rc occurs also in the stress caused by chromium (appenroth et al. 2001), and in phosphorus-depleted leaves (lin et al. 2009). the conversion of excitation energy in electron transport, i.e. parameter et0/(tr0–et0), was decreased in leaves of genotype kelleris 16 exposed to drought stress (tab. 2). further, electron transport beyond qa – expressed per active reaction centre (et0/rc) was also significantly impaired in drought-treated leaves of genotype kelleris 16 (fig. 4c) due to reduced transport from qa to qb. these results suggest that the conversion of excitation energy in electron transport caused the decrease of the piabs and overall photosynthetic efficiency since the connection between electron transport and co2 fixation has been proved (krall and edwards 1992), and thus the connection of piabs with co2 fixation (van heerden et al. 2007). decreased photosynthetic efficiency was not only due to changes in energy flow through the psii but also due to decreased pigment content. impaired content of photosynthetic pigments in leaves of drought treatment, especially chlorophyll a and total chlorophylls (a+b) may be due to reduced protein synthesis at the lhc complexes or destruction within complexes which, among other functions, protect the photosynthetic apparatus (kauser et al. 2006). also, degradation of photosynthetic pigments can be result in oxidative damage of chloroplast lipids, pigments and proteins, particular proteins in thylakoid membranes, which leads to the degradation of ps ii and consequently, reduced trapping and electron transport, as well as reduced production of atp and nadph, and and ultimately reduced co2 fixation (abdalla and el-koshiban 2007). reduced chlorophylls ratio (a/b) indicates that the antenna complexes of psii were increased allowing increased absorption of photons, leading to excess of the electrons in psii and to the modification of the active reaction centres in dissipative (lepedu[ et al. 2009). from the presented results, it can be concluded that photosynthesis was down-regulated in leaves of the genotype kelleris 16 exposed to drought, while genotype os appears to be more tolerant. an indication of impaired photosynthetic efficiency in leaves of genotype kelleris 16 was found in reduced photosynthetic pigment contents. changes of antenna complexes in psii, seen as decreased chlorophylls ratio (a/b), altogether with decreased total chlorophylls content (a+b), indicate the adaptation of psii to drought conditions. overall photosynthetic performance expressed as piabs was down-regulated in both investigated genotypes, although maximum quantum yield of psii remained unchanged in drought-treated leaves of genotype os. however, decrement of piabs was much more pronounced in genotype kelleris 16 mainly because of changes in a certain fraction of rcs which become dissipative centres, seen as an increase in abs/rc and di0/rc, in order to avoid photooxidative damage of photosynthetic apparatus. also, energy flow through the psii i.e. acta bot. croat. 72 (2), 2013 231 sour cherry (prunus cerasus l.) genotypes under drought stress electron transport was impaired, seen as a decrease in et0/(tr0–et0) and et0/rc, which leads to depleted co2 fixation and photosynthesis according to krall and edwards (1992). the described changes in the functioning of photosynthetic apparatus in drought-treated plants of kelleris 16 are the main distinction between the two investigated genotypes regarding the drought adaptation mechanisms of photosynthesis. acknowledgments this work was supported by the scientific research grants to z. j. 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fine scales (between communities) and on coarse scales (between biomes), while boundaries of intermediate scales (e.g. between community complexes) are quite neglected. in this study, we analysed boundaries between mesic and xeric community complexes in a sub-mediterranean karst area of south hungary. we applied the moving split window (msw) technique for boundary analysis. first, since the behaviour of msw concerning complex vegetation patterns is not fully understood, we prepared artificial datasets (simulated communities) to test its capacities. second, we established north-south oriented belt transects across mountain ridges of the villány mts, and investigated the transition between the community complexes of differently exposed slopes. using msw, we were able clearly to distinguish between transitional zones and zones that do not represent real transitions: peaks in the z-score profile of msw merge only in the case of transitional zones. moreover, we found that peaks merge depending on the independence (distinctness) of the transitional zone: when it is distinct, peaks merge only at the largest window widths. in the villány mts, transitions seem to occur mostly in the grasslands north of the ridges. we demonstrated that these grasslands can be regarded as boundaries between mesic and xeric complexes or as zones in their own right, with their own two boundaries. interpretation depends upon the scale of observation. keywords: msw, edge, simulation, gradient, transition abbreviations: msw – moving split window, sed – squared euclidean distance introduction the study of ecological gradients and boundaries has been in the focus of research efforts for decades (cf. whittaker 1967, austin 2005). boundaries are of particular importance, since they separate and connect neighbouring ecological units along a gradient acta bot. croat. 73 (1), 2014 63 * corresponding author, e-mail: erdos.laszlo@bio.u-szeged.hu copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:48 color profile: generic cmyk printer profile composite default screen (erd�s et al. 2011b) and they control the flow of organisms, materials, energy, and information (wiens et al. 1985, wiens 1992, cadenasso et al. 2003). ecological boundaries exist over a wide variety of spatial scales and organization levels (e.g. jagomägi et al. 1988, gosz 1993, peters et al. 2006). unfortunately, while boundaries on some scales have been intensively scrutinized, other scales are almost entirely neglected: most studies focus on boundaries between communities (fine-scale) or those between biomes (coarse scale) (kent et al. 1997, peters et al. 2006). however, boundaries and transitional phenomena between vegetation complexes (intermediate scale) may also be important from an ecological perspective (cf. westhoff 1974, van der maarel 1976). community complexes (also mentioned as phytocoenose complexes) are either mosaics of two or more communities (e.g. forest patches embedded in a grassland matrix) or series of different communities in one spatial direction (e.g. along decreasing moisture). in the case of ecological boundaries, the study of intermediate scales is especially important: knowledge on transitions between community complexes may contribute to the understanding of boundaries of lower and higher scales (kolasa and zalewski 1995, kent et al. 1997, laurance et al. 2001). it is widely known that vegetation features differ considerably in north-facing and south-facing slopes (e.g. armesto and martínez 1978, szabó 1990, kutiel and lavee 1999, sternberg and shoshany 2001, bátori et al. 2011, erd�s et al. 2012). however, differences are pronounced under some climatic conditions, but they are negligible in others. for example, humid atlantic conditions in western europe favour the existence of closed forests on both north-facing and south-facing slopes (jakucs 1972), while all slopes tend to be mostly treeless in arid and extreme-arid environments (kutiel and lavee 1999). in contrast, under mediterranean and continental (as well as sub-mediterranean and sub-continental) conditions, trees are much more abundant on north-facing than on south-facing slopes, resulting in great structural and compositional differences between the community complexes of the different slopes (cf. jakucs 1972, kutiel and lavee 1999, sternberg and shoshany 2001). it can be hypothesized that transition occurs somewhere near the ridge. however, knowledge on spatial processes and boundary properties across mountain ridges is limited. the few studies that focus on transitions occurring on mountain ridges use classical coenological methods with a limited capacity to characterize boundaries and transitional phenomena (e.g. kevey and borhidi 1998, 2002, 2005). several methods have been developed to study ecological boundaries (hufkens et al. 2009). moving split window (msw) analysis is considered one of the best methods (kent et al. 1997, also see ludwig and cornelius 1987, choesin and boerner 2002), and has been widely used in ecology (e.g. mu�os-reinoso 2001, zalatnai and körmöczi 2004, hennenberg et al. 2005, kröger et al. 2009, torma and körmöczi 2009, erd�s et al. 2011a). msw compares neighbouring areas of a transect using a dissimilarity function. comparison is performed in a window consisting in the simplest case of two adjacent plots. window width can be increased, so comparison can be made at several spatial resolutions. although there have been some studies analysing the behaviour of the msw method under different boundary conditions, using different window widths (brunt and conley 1990, körmöczi 2005), msw behaviour with respect to more complex vegetation patterns is little understood. moreover, promising as the method may seem at intermediate scales, it has not been used to study boundaries between community complexes. 64 acta bot. croat. 73 (1), 2014 erd�s l., zalatnai m., bátori z., körmöczi l. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:48 color profile: generic cmyk printer profile composite default screen in this study, we used the msw technique to examine boundary zones between community complexes of differently exposed slopes in a sub-mediterranean karst area of hungary. the strongly deviating vegetation types in the north-facing and the south facing slopes of the villány mts provided an excellent opportunity for the analysis of transitions between community complexes. first, we applied simulated communities to test the properties of the msw method. second, we analysed our field data gathered along north-south facing belt transects running through the mountain ridges. our main goal was to provide a case study on boundary characteristics between community complexes, but we also aimed to contribute to knowledge on the behaviour of the msw method under different boundary situations. materials and methods study site our study was conducted in the villány mountains (hungary) (fig. 1). the bedrock consists mainly of limestone, to a lesser degree of dolomite (lovász 1977). mean annual temperature is 10.5 °c (fodor 1977), mean annual precipitation is 680 mm (ambrózy and kozma 1990). the area is situated in the temperate forest belt, but it is very close to the forest steppe zone (pócs 2000, magyari et al. 2010). therefore, a complex of small shrubforest patches embedded in a dry grassland matrix occupies the warm and dry southern slopes (simon 1964, dénes 1995), whereas the cooler and moister northern slopes are covered mostly by mesic forests (horvát 1968) (fig. 2). near the ridges, on the northern slopes, a transitional grassland community has formed, with both mesic and xeric plant species (erd�s et al. 2010). acta bot. croat. 73 (1), 2014 65 transitions between community complexes along a gradient fig. 1. location of the study site in southern hungary. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:49 color profile: generic cmyk printer profile composite default screen for the present study, the three most intact areas of the villány mountains were chosen, where vegetation can be considered natural: mt szársomlyó, mt fekete and mt tenkes. mt szársomlyó and mt fekete are strictly protected nature reserves, whereas mt tenkes is being slated for legal protection. all of them have very high species richness (with a lot of relict and endemic species) and great habitat diversity (dénes 2000). field studies five north-south facing belt transects were established perpendicular to the mountain ridges (3 transects on mt szársomlyó: s1–s3, one transect on mt fekete: f, and one transect on mt tenkes: t). all transects were 200 m long, consisting of 1 m2 contiguous permanent quadrats. during field works in the period 2006–2010, the presence of all vascular plant species of the field layer was recorded twice (in april and in july). spring and summer records were combined for each quadrat before the analyses. artificial datasets since performance of the msw method on multiscale complex patterns is not sufficiently known, first we wanted to test how this technique performs on artificial data with known properties. artificial datasets were prepared so as to resemble the real field situations in the villány mts. five datasets were used, representing five possible species distribution patterns. each set consists of 200 plots and either 150, or 120 or 90 species (fig. 3). species number depends on the structure of the artificial gradient. species distributions near the boundaries were generated randomly so that probability of species occurrence decreased gradually away from the patch centre. central patch or transition zone (in the middle of the gradient) is 20 m wide in each case. 66 acta bot. croat. 73 (1), 2014 erd�s l., zalatnai m., bátori z., körmöczi l. fig. 2. typical vegetation of the villány mts. on the left, a mesic forest of the northern slope can be seen. on the right, there is part of the xeric shrubforest-grassland complex. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:52 color profile: generic cmyk printer profile composite default screen the first dataset consists of three distinct communities along a gradient, with no species overlaps (fig. 3a). it is a rather hypothetical case with totally sharp boundaries. in this case, the central patch cannot be considered a transitional one, since its species composition does not show a transitional character (i.e. there is no species overlap with the neighbouring communities). total species number was set to 150, which is close to the real field situations (the average species number along a 200 m long transect was 169 during field works). along the artificial gradient, communities have 60, 30 and 60 species, respectively (the central community is shorter and so has fewer species). the second dataset is more realistic, since the central community is transitional (fig. 3b). although it has its own species (occurring exclusively in the central patch), boundaries towards the neighbouring communities are blurred, with considerable overlaps (that is, the central zone has its own species as well as species from both neighbouring communities). total species number was again 150 (60 + 30 + 60). in the third dataset, boundaries are blurred, and the central patch does not have its own species any more (fig. 3c). here, total species number is 120 (60 + 60), since there are only two communities. species number is not even along the gradient, but it is higher in the central patch than in the other sections. in the fourth dataset, the boundaries between the communities are blurred, and the species number is roughly the same along the whole gradient (fig 3d). total species number is 120 (60 + 60). it should be noted that independence (i.e. distinctness) of the central patch decreases from the first to the fourth dataset. the fifth dataset contains blurred boundaries (fig 3e). in this case, the central patch is not transitional, it is, rather, a habitat patch situated in a homogeneous matrix. here, total species number is only 90 (60 for the matrix and 30 for the small patch). we hypothesized that real transect data from the villány mts would resemble either the second, or the third or the fourth simulated gradient, because a transition is expected near the ridge in the case of field data. moving split window analysis we used moving split window (msw) technique (webster 1978) to study the transitional phenomena on both the artificial and the field data. squared euclidean distance (sed) was applied as comparative function, since it is the most commonly used distance metric in msw studies (johnston et al. 1992). significance of the boundaries was tested using the z-score transformation. overall mean and standard deviation were computed from 99 randomizations and for the whole transect. random reference was made with random shift of species. the distribution of each species was shifted a random number of units along the transect. occurrences that are shifted beyond the end of the transect were wrapped back on to the opposite end (palmer and van der maarel 1995, horváth 1998). in order to gain a better understanding of the behaviour of the msw as well as of the spatial processes occurring near the ridge, we applied three spatial scales by using different window widths: for comparison, z-scores were averaged over 4–10, 10–20, 30–40 and 50–60 window widths, respectively (since we used 1 m2 quadrats, window width always means meters). average z-scores were plotted against window midpoint position, resulting in a z-score profile. in the profile, vegetation boundaries appear as peaks. msw profiles for window widths 4–10 acta bot. croat. 73 (1), 2014 67 transitions between community complexes along a gradient 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:52 color profile: generic cmyk printer profile composite default screen were basically very similar to the profiles for window widths 10–20 along each simulated gradient and field transect. thus, the results of the smallest window widths were not further evaluated in this study. the significance test of this statistic is not well developed. most of the studies do not deal with significance, and mark only the largest peak. in some instances, peaks above expected mean + 2sd are suggested as significant without further consideration (e.g. cornelius and reynolds 1991). we intended to reveal a verifiable test, therefore we determined the distribution of z-transformed distance values from randomized data. this is the distribution of expected values for random case. on the basis of the analysis of several hundred simulations (10,000 randomizations in each case), the distribution of z-scores for randomized data deviated from normal distribution: it was slightly right-skewed. therefore, confidence limits used for normal distributions were not applicable. instead, we determined the upper 5% confidence limit for each simulation, the value of which was less than 1.85 in the most conservative case. we suggested 1.85 be used as the 5% confidence limit in the case of sed and random shift (other randomizations may result in slightly different confidence limits, especially in the case of the smallest window sizes). accordingly, peaks above 1.85 were regarded as indicating significant (p < 0.05) boundaries in this study. the program for the msw-computations with random reference was written in the statistical language r 2.10.1 (r development core team 2009). r-source code is given as supplementary material (on-line supplement tab. 1). results behaviour of the msw on different artificial datasets analysis of the artificial datasets showed that the msw method was able to distinguish between transitional zones (second, third and fourth datasets) and zones that did not have a transitional character (first and fifth datasets). in the case of transitional zones, msw-peaks began to merge with the increase of window width (figs. 3b, c, d). however, if the central patch was not a transitional one, peaks did not merge even if the largest window widths were applied (figs. 3a, e). for example, boundaries in the second dataset were as blurred as in the fifth. nevertheless, while peaks merged in the case of the second dataset (fig. 3b), peaks did not coalesce in the case of the fifth (fig. 3e). in addition, msw indicated the distinctness of the central zone: the more independent this zone was, the later the peaks merged. in the case of the first artificial dataset (where communities were distinct, without species overlaps), the two peaks remained highly prominent and separate even at the largest window widths (fig. 3a). in the case of the second artificial dataset, msw showed two comparatively high peaks at smaller window widths, which began to merge at window widths 30–40. when window widths between 50 and 60 were applied, the two peaks disappeared and a plateau developed (fig. 3b). this plateau kept its shape even when window width reached 100 (data not shown). in the third case, peaks in the z-score profile merged at smaller window widths, with no plateau (fig. 3c). in the case of the fourth dataset, when independence of the central patch was the lowest, double peaks did not appear even at the smallest window widths (fig. 3d). 68 acta bot. croat. 73 (1), 2014 erd�s l., zalatnai m., bátori z., körmöczi l. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:52 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 69 transitions between community complexes along a gradient fig. 3. species distribution patterns in the artificial datasets along a simulated gradient and corresponding z-score profiles from the msw. a three entirely distinct communities with totally sharp boundaries. b three communities with moderately blurred boundaries. c communities with blurred boundaries and high species richness in the central zone. d communities with blurred boundaries and constant species number along the gradient. e habitat patch in a homogeneous matrix. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:56 color profile: generic cmyk printer profile composite default screen transitional phenomena between the north-facing and the south-facing slopes we found a total of 284 species along the five transects. species number and most frequent species of the transects are shown in table 1. when msw was applied to field data, three of the five transects resembled our second artificial dataset: transects f, s2 and s3 showed clear double peaks near the ridge, which merged at the largest window widths (figs. 4a, c, d). the area between the two peaks in the z-score profile coincided with the area occupied by the grassland community festuco rupicolae-arrhenatheretum in the real space (near the mountain ridge). in the case of transect s2, a wider and internally inhomogeneous transitional zone can be presumed when the largest window widths are considered (fig. 4c). this zone contains parts of a top-forest and of a shrubforest community. 70 acta bot. croat. 73 (1), 2014 erd�s l., zalatnai m., bátori z., körmöczi l. table 1. species number and most frequent species of the transects. transect species number most frequent species norhthern slope southern slope f 183 corydalis cava euphorbia cyparissias ranunculus ficaria orlaya grandiflora ruscus aculeatus festuca dalmatica anemone ranunculoides helianthemum ovatum gagea lutea teucrium chamaedrys s1 164 galanthus nivalis orlaya grandiflora corydalis cava calepina irregularis helleborus odorus melica ciliata anemone ranunculoides viola arvensis ranunculus ficaria allium sphaerocephalon s2 170 veronica hederifolia orlaya grandiflora corydalis cava geranium rotundifolium lamium purpureum lamium amplexicaule anthriscus cerefolium melica ciliata acer platanoides viola arvensis s3 164 veronica hederifolia orlaya grandiflora anthriscus cerefolium myosotis stricta ranunculus ficaria cerastium brachypetalum lamium purpureum lamium amplexicaule corydalis cava viola arvensis t1 166 corydalis cava euphorbia cyparissias anemone ranunculoides orlaya grandiflora ranunculus ficaria potentilla arenaria galanthus nivalis fraxinus ornus gagea lutea teucrium chamaedrys 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:53:56 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 71 transitions between community complexes along a gradient fig. 4. species distribution patterns along the transects from field studies and corresponding z-score profiles from the msw. in the distribution patterns, only species occurring in at least five plots are displayed. a transect f. b transect s1. c transect s2. d transect s3. e transect t. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:54:00 color profile: generic cmyk printer profile composite default screen in the case of transect s1, double peaks appeared near the ridge: one of them was very high, whereas the other one was comparatively low (fig. 4b). peaks did not merge at greater window widths. instead, the most prominent peak indicates a rather abrupt transition on the mountain ridge. this transition occurs between a small north-exposed shrubforest patch and a south-exposed rock sward. as for transect t, a conspicuous peak emerged north of the ridge (fig. 4e). this indicates the boundary between a top-forest community and a more open, xeric shrubforest patch. discussion simulated communities can be used to gain a better understanding of the properties of msw, which in turn helps to interpret results originating from field data (brunt and conley 1990). our simulations showed that using msw, one can distinguish between transitional zones and zones that do not represent real transitions. if a zone does not have a transitional character, peaks in the z-score profile will not merge even at the greatest window-widths (this was illustrated with the first and the fifth datasets) (figs. 3a, e). in contrast, if a patch is transitional between two communities along a gradient, peaks will merge at greater window widths. it was observed by körmöczi (2005) that msw produces different results when a transitional community is being studied and when the community in the middle is rather a patch in a homogeneous matrix. however, körmöczi (2005) used only two simulated gradients. our results reinforce his findings and suggest that the observed phenomenon is general. influence of the distinctness of a community on the coalescence of the msw peaks has not been studied formerly (cf. brunt and conley 1990, körmöczi 2005). we found that distinctness (independence) of the transitional zone determines the coalescence of the peaks of the profile. if the transitional unit is relatively independent (e.g. it has its own species occurring only in the transitional community), the peaks will merge only at larger window widths, and a plateau forms (fig. 3b). this plateau does not disappear, but it keeps its shape even at much greater window widths. if independence of the transitional unit is lower, the peaks merge at smaller window widths forming a single peak, and no plateau appears (fig. 3c). if distinctness is even lower, double peaks will not appear even at the smallest window widths (fig. 3d). this finding contradicts körmöczi (2005), who stated that coalescence of the peaks depends on the width of the transitional zone. for example, in the case of the third and fourth artificial datasets, width of the transition was exactly the same (20 plots). nevertheless, peaks did not merge at the same window widths in these two cases (figs. 3c, d). analyses of the field data showed that in three of the five transects (f, s2, s3), the transition between the mesic north-exposed slope and the xeric south-exposed slope occurs in the grassland community festuco rupicolae-arrhenatheretum. our study also demonstrated that this vegetation unit can be conceived as a boundary or as a zone in its own right, with its own two boundaries. interpretation may solely depend on the resolution of the study. in the case of transects f, s2 and s3, at finer resolutions (at smaller window widths) the transitional grassland unit north of the ridge has its own two boundaries, indicated by the peaks in the z-score profile (figs. 4a, c, d). however, at the greatest window widths (i.e. at a coarse resolution), peaks merge. this shows that this vegetation unit may be regarded as a boundary between the mesic northern slope and the xeric southern slope. 72 acta bot. croat. 73 (1), 2014 erd�s l., zalatnai m., bátori z., körmöczi l. 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:54:00 color profile: generic cmyk printer profile composite default screen similar grasslands have been studied in south hungary (near the villány mts) by lengyel et al. (2012). they examined mesic meadows of the arrhenatheretalia order and some related syntaxonomic units. interestingly, they found some grasslands that represented transitional states between mesic (arrhenatheretalia) and semi-dry (brometalia erecti) meadows, with the occurrence of both mesic and xeric species. species composition of those grasslands seems to resemble the community festuco rupicolae-arrhenatheretum (e.g. the dominance of arrhenatherum elatius, festuca rupicola and teucrium chamaedrys, high constancy of dianthus giganteiformis and sanguisorba minor, and a number of other common species). however, there is a fundamental difference: the position of festuco rupicolae-arrhenatheretum is near the ridges, while the similar relevés of lengyel et al. (2012) are not situated near mountain ridges (csiky personal communication). in the case of the villány mts, the transitional character of festuco rupicolae-arrhenatheretum communities can be explained by their being located near mountain ridges. consequently, they form a transition between the community complexes of differently oriented slopes. in the case of the relevés of lengyel et al. (2012), ecological indicator values suggest that factors such as temperature and light regimes are higher than in mesic but lower than in semi-dry grasslands, while the reverse is true for moisture and nutrient availability. since these stands are situated either in lowlands or on south-facing slopes, but never near ridges, overshadowing by neighbouring forest stands could be responsible for their transitional environmental conditions (the grasslands themselves have formed in the place of cleared forests, which probably also influences their special state). the transitional grassland community festuco rupicolae-arrhenatheretum has already aroused our interest, and we examined it using classical coenological methods (erd�s et al. 2010). the present study reinforces the idea that it is a distinct vegetation unit, as it is bordered by relatively sharp boundaries (high peaks in the z-score profile at small window widths). on the other hand, it has a strong transitional character, and represents a boundary zone between the northern and the southern slopes (merging peaks at greater window widths). however, we have to note that, concerning other areas in the region, stands of this community do not necessarily form boundary zones between community complexes of differently exposed slopes. similar transitional zones were identified using the msw method by zalatnai and körmöczi (2004) in alkaline grasslands of hungary. transitional zones were visually detected during field studies. these were examined in detail using the msw method. smaller window widths showed double peaks indicating the boundaries of the transitional zones, whereas double peaks merged at larger window widths, emphasizing that the zone represents an intermediate type between the two neighbouring units. the results were quite different in transects s1 and t. in the case of transect s1, the transitional character of the community festuco rupicolae-arrhenatheretum is less prominent, as peaks do not merge (fig. 4b). the grassland resembles the north-exposed communities, probably because this is the steepest north-facing slope of the five transects (30°), and the grassland is partly overshadowed by the neighbouring trees. these factors probably entail cooler and more humid conditions, enabling the establishment of a larger number of species typical of mesic forests. in the case of transect t, a conspicuous boundary is situated north of the ridge (fig. 4e). in this case, the grassland community north of the ridge does not represent a transitional state, but it resembles the xeric communities of the southern slope. this is probably due to acta bot. croat. 73 (1), 2014 73 transitions between community complexes along a gradient 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:54:01 color profile: generic cmyk printer profile composite default screen the fact that slope inclination at this section is small (8°). moreover, soil is shallow and stony, resembling conditions of the south-facing slope. transitions between steep southern and northern slopes have been studied in the transdanubian mountain ranges (kevey and borhidi 2002) and in the mecsek mts (kevey and borhidi 1998, 2005). it was found that transitions occur within ecotonal-type forests, where mesic and xeric species co-occur. in contrast, in the villány mts, transitions seem to occur mostly in the grasslands north of the ridges. this pattern was found in three of the five transects under scrutiny (f, s2, s3). in the remaining two transects (s1, t), transition was rather abrupt, occurring either on the mountain ridge, or at the edge of the mesic forests. it was recognized in vegetation ecology that a vegetation unit of transitional character can be considered a boundary or a zone in its own right, depending on the spatial resolution (cf. armand 1992, kolasa and zalewski 1995). thus, multi-scale approaches are necessary to scrutinize such transitional units. our analyses suggest that msw provides an appropriate tool for these studies. it can identify spatial resolutions at which the transitional vegetation unit should be regarded as a boundary, and resolutions at which it is rather a unit in its own right. our study serves as an example to show that msw can effectively support classical coenological methods: if msw shows that the vegetation unit under study has significant boundaries with the neighbouring communities, it may be treated as a separate unit at the given resolution. acknowledgements the authors would like to thank the inspectorate for environment, nature and water for allowing us to carry out these studies in the strictly protected nature reserves of the villány mountains. we are also thankful to jános csiky, tamás morschhauser and the department of plant taxonomy and geobotany of the university of pécs for the technical support. this work was supported by the project támop-4.2.1/b-09/1/konv-2010-0005 (»kutatóegyetemi kiválósági központ 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(eds.), landscape boundaries: consequences for biotic diversity and ecological flows, 217–235. springer-verlag, new york. zalatnai, m., körmöczi, l., 2004: fine-scale pattern of the boundary zones in alkaline grassland communities. community ecology 5, 235–246. acta bot. croat. 73 (1), 2014 77 transitions between community complexes along a gradient 603 erdos et al.ps u:\acta botanica\acta-botan 1-14\603 erdos et al.vp 19. o ujak 2014 16:54:01 color profile: generic cmyk printer profile composite default screen opce-str.vp acta bot. croat. 68 (2), 443–454, 2009 coden: abcra 25 issn 0365–0588 what’s in a name? – diatom classification should reflect systematic relationships. eileen j. cox department of botany, the natural history museum, cromwell road, london, sw7 5bd, uk. large numbers of diatom taxa are currently being described each year and molecular data sets are providing phylogenetic evidence that challenges the traditional systematic arrangement of diatoms, but is such information being integrated into the classification? the traditional diatom classification originated as an aid to identification rather than as an arrangement expressing perceived relationships, and characters for identification continue to bias taxonomic descriptions. reference to types for nomenclatural purposes has resulted in overly narrow taxon descriptions; i.e. types have been considered representative specimens (typical) of taxa, whereas they may not lie at the centre of the range of variation of a taxon. this paper discusses how taxonomic concepts are subject to change in the light of new data and that such changes should be reflected in the systematic arrangement. it presents some thoughts on character choice and the need to make appropriate comparisons before new taxa are erected. the importance of the suprageneric classification is also discussed. keywords: identification, classification, diatom, type specimens, terminology introduction in the introduction to their catalogue of diatom genera, fourtanier and kociolek (1999), showed how the rate of publication of new diatom genera had varied over the preceding two centuries, but was, at the time of writing, in an exponential growth phase. a high rate has been maintained (95 new genera since 1999), while more than 2,000 new species have been described over the same period (fourtanier and kociolek, 2007). however the integration of such new taxa into a meaningful systematic framework is much rarer. even if an author refers a new genus to a family, e.g. naviculaceae kützing, it is unclear whether such an allocation refers to the family as originally defined (kützing 1844), or in the sense of a particular publication, e.g. round et al. (1990). whereas the description of species and genera has proceeded apace, consideration of generic and suprageneric relationships has been largely neglected. some attempts have been made to revise higher groups in the light of molecular phylogenetic analyses, cytological and reproductive characters (medlin and kaczmarska 2004, mann in adl et al. 2005), but these efforts appear acta bot. croat. 68 (2), 2009 443 * corresponding author: e.j.cox@nhm.ac.uk u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:46 color profile: disabled composite 150 lpi at 45 degrees to have had little impact on those investigating previously unexplored or understudied areas and habitats. most interest seems to have been directed to recording new taxa (using traditional criteria) rather than understanding how they relate to each other (using a variety of approaches). while recording and describing new taxa is important, and correct identification underpins all other research (e.g. ecological, life history, phylogenetic, stratigraphical) into those taxa, the description of new taxa cannot occur in a vacuum, but must take account of what is already recognised and described. with the apparent dichotomy between traditional approaches to describing new species (e.g. lange-bertalot and colleagues, see metzeltin and kusber 2001) and phylogenetic analyses based on molecular biology (medlin and kaczmarska 2004, bruder and medlin 2007, kooistra et al. 2007, medlin et al. 2008, rampen et al. 2009), it is appropriate to evaluate whether the current paradigms of diatom classification are helping or hindering diatom taxonomy and systematics. paradigms williams (2007) provided an overview of the development of diatom classification, showing how, with its emphasis on valve morphology and particular wall features, it has been dominated by a desire to aid identification (explicitly or implicitly) rather than reflect relationships. h.l. smith’s explicitly artificial classification (smith 1872) modified by schütt (1896), formed the basis of the modern diatom classifications (hustedt 1930, 1927–1966; patrick and reimer 1966; hendey 1937, 1964; simonsen 1979; round et al. 1990), and although medlin and kaczmarska (2004) and mann in adl et al. (2005) presented new classifications, their groups are still described largely in terms of their shape and symmetry. it may be argued that the latter simply reflects the evolution of the groups, but on the other hand it may indicate that even when molecular studies underlie an analysis, diatomists interpret the data in terms of superficial (shape, symmetry) rather than structural (type of pores, presence of raphe, fultoportulae, rimoportulae) features. alternatively, perhaps we still know too little about some of our specimens to compare and discuss the structural features adequately. because h.l smith’s classification was developed to help identify specimens (and most other morphologically-based classifications are essentially variants on this), diagnoses and artificial keys are inextricably linked. species and generic descriptions focus on the characters that allow discrimination of taxa (at whatever level), rather than shared characters that group specimens into species, species into genera, and so on. thus they tend to rely on frustule morphology at the lm level, particularly shape, symmetry and the presence or absence of distinctive features. for example, the traditional separation of the freshwater araphid genera, asterionella, diatoma, fragilaria, hannaea, martyana, meridion, synedra, tabellaria, and tetracyclus rests on the particular combinations of valve symmetry, the presence or absence of septa, presence or absence of costae, mode of colony formation or its absence in each genus (tab. 1). however, more detailed studies of valve structure and the use of cladistic analysis revealed the heterogeneity of some of these genera, and the likelihood that generic boundaries were incorrectly drawn for others (williams 1985; williams and round 1986, 1987). a recent molecular analysis (medlin et al. 2008) indicates that there may also be problems with the genera as defined by williams and round (1986, 1987), highlighting the need for more studies on these taxa. 444 acta bot. croat. 68 (2), 2009 cox e. j. u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:46 color profile: disabled composite 150 lpi at 45 degrees identification v. classification thirty years ago, i pointed out that there has been confusion between the use of characters for diatom identification and their use for systematics (cox 1979). whereas shared characters are fundamental to recognising relationships expressed by systematics (cladistic theory demands that these are shared derived characters), distinctive differences (that may or may not have systematic significance) are required for identification. thus the presence of a raphe system defines the monophyletic raphid diatoms, but it may be subtle differences in details of the raphe system, e.g. its external path including details of the polar fissures, presence or absence and type of internal ribs beside the raphe slits, that are needed to discriminate between raphid diatoms. thus, oppositely deflected central raphe fissures combined with forked external polar raphe fissures aid identification of neidium spp., while the presence of strong internal ribs beside the raphe slits are characteristic of frustulia spp. valve outline and symmetry were critical characters in h.l. smith’s artificial system. both are relatively easy to define and to observe, but particular shape and symmetry characters can be shared by structurally diverse taxa. thus, the possession of lunate valves allows hannaea to be discriminated from other non-raphid, fragilarioid diatoms, but lunate valves are also found in a diverse range of raphid diatoms, e.g. amphora, climaconeis, cymbella, encyonema, epithemia, rhopalodia, seminavis. valve outline may aid the identification of a number of genera, but is not a systematically reliable, defining character. nor are all members of a genus necessarily the same shape, e.g. climaconeis includes both straight and lunate-valved species (cox 1982) and slight dorsiventrality is seen in some members of otherwise 'bilaterally symmetrical' genera, e.g. lyrella (mann and stickle 1997), placoneis (cox 2003). acta bot. croat. 68 (2), 2009 445 diatom classification and systematic relationships tab. 1. distribution, as presence (+) or absence (–), of characters traditionally used to distinguish freshwater araphid diatom genera. valve symmetry septa costae habit is op ol ar he te ro po la r b il at er al ly sy m m et ri ca l do rs iv en tr al pr es en t / ab se nt pr es en t / ab se nt so li ta ry ce ll s at ta ch ed su bs tr at um at on e en d at ta ch ed by pa ds of m uc il ag e, zi gza g or st el la te co lo ni es at ta ch ed va lv e fa ce to va lv e fa ce asterionella + + + – – – – – + – diatoma + – + – – + – – + + fragilaria + – + – – – – – – + hannaea + – – + – – + – – – martyana – + + – – – + + – – meridion – + + – – + – – – + synedra + – + – – – + + – – tabellaria + – + – + – – – + – tetracyclus + – + – + + – – – + u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:46 color profile: disabled composite 150 lpi at 45 degrees over the last thirty years there have been major developments in systematic methods, with a shift from phenetics to cladistics and the development of molecular biology. however, discovering shared (derived) homologous characters remains critical to the delimitation of 'natural' groups. in this search for informative characters it is necessary to understand diatom structure and development rather than simply relying on lm morphology. the subdivision of navicula sensu lato reflects the discovery that similarly shaped, bilaterally symmetrical raphid diatoms have different underlying frustule structures (raphe, areolae, cingulum), as well as types and numbers of chloroplast, modes of sexual reproduction, etc. as more functional characteristics linked to the survival of the organism, these are potentially more accurate guides to relationships than shape, symmetry and stria arrangement. typological method accompanying the process of systematics as it seeks to reflect the relationships between organisms, the codes of nomenclature provide rules that govern how taxa (of any rank) are named. the rules are concerned with the validity and legitimacy of names, their application and the choice of the correct name (ross 1993). names are fixed to specimens, the oldest name has priority, and the rules govern how to decide what should be the correct name of a taxon when several names have been applied to it. the rules do not address the definition of the taxon concerned, but only apply to the name. as ross (1993) stated, »it is names, not the taxa to which they apply that have types and, although such expressions as »the type of the species« are often used, these are shorthand for »the type of the specific name«. it is therefore possible that the »type specimen« of a species is not the most typical (representative) example of that species; the author may not have been aware of the full morphological range of that species when describing it. this is particularly likely if a species is described from only one or a few specimens in a single sample. if on the other hand, large populations from several sites are available for study before describing a new species, it is possible (and advisable) to choose a representative specimen as the nomenclatural type. a generic name is similarly linked to a specimen via the generitype, the species on which it was based. with respect to early generic names, this often creates a problem because the author may have included a number of unrelated species in the genus, which would now be separated into two or more different genera. it then becomes necessary to distinguish and name the newly recognised taxa. the concept of the genus has changed, and this must be expressed via its systematic treatment, but the original names remain linked to the same specimens (fig. 1). with closer examination it may become apparent that the generitype too is not the most representative species within the genus, but because of the rules its status cannot be changed. changing taxonomic concepts because taxonomic descriptions are based on the information available at the time of writing and may be heavily influenced by prevailing paradigms (such as the previously held 'live characters are uninformative or misleading'), they should be considered open to amendment in the light of new information and new analyses. however, it seems that some prefer to describe new genera rather than to amend existing ones. for example, if the interpretation of a genus is too tightly limited to the characters seen in the generitype, closely re446 acta bot. croat. 68 (2), 2009 cox e. j. u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:46 color profile: disabled composite 150 lpi at 45 degrees lated species may be unnecessarily separated into new genera. was it assumed that the generitype is typical of the genus and is not just one member of a group defined by particular characters? the extreme case is of course the creation of tightly defined monotypic genera. in the case of navicula, navicula tripunctata shows a suite of characters that helped to define navicula section lineolatae (hustedt 1930, 1927–1966) and navicula subgenus navicula (patrick 1959). however, if we compare it with other species that have generally been accepted as members of the same group, it is possible to recognise a range of variation in a number of characters, including raphe fissures, accessory ribs, stria arrangement and pores around the valve apices (cox 1999b). does this mean that we should restrict navicula to those species that share all aspects of their characters with n. tripunctata, or do we look for the group that contains the latter and describe the genus for the group? if we circumscribe the group before seeking to describe its salient characters, some of the features of the generitype may not appear in the description. but the generic description should be wider than the generitype description. when a new species with a unique combination of characters is discovered, establishing its closest relatives requires careful comparison with potentially a large number of different taxa, a process that should be undertaken with as open a mind as possible. it is also important to avoid a priori judgements on the relative importance of particular characters until all the evidence has been assembled, as well as ensuring that morphological characters are carefully evaluated from specimens to ensure that previous ways of describing characters do not mislead. thus, as in the case of climaconeis, the inclusion of both acta bot. croat. 68 (2), 2009 447 diatom classification and systematic relationships fig. 1. relevance of type specimens to changing taxon concepts. a – distribution of specimens of a variable taxon a in morphological space, with the historical type (t) at the edge of the morphological spectrum. b – taxon a has been subdivided, but is still defined by its historical type. in defining taxon b, a type is chosen at the centre of its morphological space. u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:47 color profile: disabled composite 150 lpi at 45 degrees dorsiventral and bilaterally symmetrical taxa in the genus (cox 1982) was based on the shared structural and chloroplast characters, rather than allowing valve symmetry to be an over-riding character that would split the genus. but if the original generic description of okedenia had been tightly adhered to, okedenia inflexa would not have been transferred to climaconeis, but have remained in its own monotypic genus. in an elegant morphological study of attheya, crawford et al. (1994) investigated four new species, and two taxa previously placed in chaetoceros, one of which had been transferred to gonioceros. the study revealed the characteristic structure of the horns at the corners of the cells, one of the diagnostic characters of attheya, providing a link among these taxa that is supported by reproductive and ecological evidence. however attheya species differ variously from each other on characters that are often considered genus specific. nevertheless, relying on such character differences would generate several monotypic or very small generic groupings. crawford et al. (1994) argued against this, treating them all as members of one genus. subsequent investigations of the phylogenetic position of attheya using molecular techniques (kooistra et al. 2007, rampen et al. 2009) confirm that its species form a distinct group, although its phylogenetic position varies between different analyses. characters and character states whatever the group of organisms the choice of characters on which they are compared is critical to the outcome of that comparison. comparative biology relies on the evaluation of homologous characters, those shared as a result of common ancestry, rather than analogous characters, which may be functionally similar, but derived independently. and homologous features can be expected to display detailed similarities, for example in position, structure and development. molecular biology similarly relies on the comparison of presumed homologous sites along sequences. whereas there are only four possible character states for any one site along a sequence, and these can be unequivocally identified as either a, t, c or g (although secondary structure and codon motifs are also considered when aligning), defining morphological characters and character states is ostensibly a more subjective exercise. but, even for diatoms, it is possible to utilise positional, structural and developmental information to interpret cytological phenomena and wall features. however, the way in which such features are described can have a significant impact on their interpretation. the terms used to describe particular features across a number of taxa will affect the comparisons between those taxa and hypotheses of relationships. for example, mann (1981) used the term vola (originally for hand-like occluding structures in centric diatoms [ross and sims 1972]) to describe any flap-like pore occlusion, whereas cox (2004) found that there are two types of flap-like occlusion in the cymbellales that differ from volae in centric diatoms and are better discriminated by the use of two terms, foricula and tectulum. following mann (1981), volate areolae would be recorded for a wide range of diverse diatoms, whereas cox’s (2004) terminology facilitates differentiation between the different types of occlusion. similarly, discrimination between a stauros and a fascia reflects underlying differences in the way the solid transverse areas are formed, a stauros being a solid structure ab initio, while a fascia is formed by silica infilling between virgae later in valve development (cox 2001). whether solid areas of silica interrupting transverse striae are similarly the product of silica infilling has not been investigated, but could be relevant to determining the affiliations of genera such as fogedia and stenoneis. 448 acta bot. croat. 68 (2), 2009 cox e. j. u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:47 color profile: disabled composite 150 lpi at 45 degrees as mentioned above (paradigms) superficial morphological appearance (lm) rather than structural information (as revealed by sem) has often been used to define taxa, but even when structural features have been incorporated, their evaluation has usually ignored underlying developmental processes and the possibility of phenotypic plasticity. the occurrence of janus cells, in which the valves of a single cell (genotype) differ significantly, reveals the potential phenotypic response (stoermer 1967, mcbride and edgar 1998), but the findings have not been incorporated into species descriptions. similarly, size reduction may have such a marked effect on valve outline that the extremes would be identified as different taxa (cox 1985). supra-generic classification while many new species and genera are being described, there have been very few recent revisions to the higher level classification since round et al. (1990). the most notable was that by medlin and kaczmarska (2004), who delineated two new subdivisions (coscinodiscophytina and bacillariophytina) and recognised the mediophyceae as a new class, based primarily on molecular data, although they integratedcytological and reproductive information where this was available. these major groups were adopted by mann (adl et al. 2005), albeit noting that the coscinodiscophytina and the mediophyceae are both paraphyletic. mann also divided the former subdivision into six groups, again based on molecular phylogenetics, the characteristics of the groups being very briefly circumscribed with reference to wall features (adl et al. 2005). on the other hand, for one of the relatively few family descriptions of the late 20th century, the cymatosiraceae, the authors considered reproductive behaviour important for its systematic position (hasle et al. 1983).this family was erected to include two previously known genera, cymatosira and campylosira, and seven new genera, plagiogrammopsis, brockmanniella, minutocellus, leyanella, arcocellulus, papiliocellulus and extubocellulus, four of them based on previously known species, and three on new species. despite their elongate valve shape, the previously known species had not been considered pennate diatoms (simonsen 1979), but had been placed in the 'centric' biddulphiaceae. interestingly, the primary reasons for placing the new family among the centric diatoms were the possession of flagellate male gametes and auxospore formation, not the more traditional wall structure or valve outline (hasle et al. 1983). with respect to the raphid diatoms, the inclusion of chloroplast characters in morphological analyses (cox and williams 2000, 2006) can produce interesting differences in suprageneric groupings compared to their treatment by round et al. (1990), often correlated with structural differences, e.g. areola type. thus, craspedostauros, which would traditionally have been placed within stauroneis, grouped with mastogloia and aneumastus (cox and williams 2000), while genera of the berkeleyaceae were separated in different parts of the tree (cox and williams 2006) based on different numbers and arrangements of chloroplasts. the latter study also indicated that achnanthes sensu stricto should be moved to the mastogloiales. based on molecular phylogenies (e.g. sims et al. 2006, rampen et al. 2009), achnanthes sensu stricto is close to the bacillariales rather than most naviculoid diatoms, but two »stauroneis« species, s. constricta and s. simulans are close to achnanthes in one study (rampen et al. 2009). the former has been transferred to craspedostauros (cox 1999a), whereas the generic affinity of the latter is uncertain, but probably not to stauroneis. acta bot. croat. 68 (2), 2009 449 diatom classification and systematic relationships u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:47 color profile: disabled composite 150 lpi at 45 degrees in discussing the systematic position of achnanthes sensu stricto it was suggested that the other genera of the achnanthales are probably more closely related to different biraphid genera than to each other (cox 2006). developmental studies have revealed that the monoraphid condition is derived; monoraphid diatoms initiate raphe slits on both new valves after mitosis, but one is subsequently infilled with silica (boyle et al. 1984). the association of the monoraphid condition with a variety of pore types suggests that raphe loss has occurred on more than one occasion, and hence the order achnanthales should be rejected. this is also supported by the relatively limited molecular data available on monoraphid diatoms (sims et al. 2006, bruder and medlin 2007). higher level classifications are assumed to reflect and interpret perceived relationships between genera and are essential to furthering our understanding of the group because they present hypotheses of relationships that can be tested. the description of new taxa without any reference to their relationship to other taxa is little more than stamp-collecting. we currently have more names and types than ever but unless they are integrated into a higher level classification they do not contribute to our understanding of the interrelationships and evolution of the group. nor in many cases do we have detailed ultrastructural, let alone cytological, reproductive or molecular, information on them. making appropriate comparisons the history of diatom studies reveals how the acquisition of new data (sem, tem, cytological, ontogenetic, reproductive, molecular) can challenge established assumptions, but there have been relatively few, recent, significant changes to the systematic arrangement of diatoms to reflect that knowledge. round et al. (1990) introduced many new orders and families and included monoraphid diatoms within the sub-class bacillariophycidae, rather than according them the same rank alongside the eunotiophycidae, but the old, centric, araphid pennate, raphid pennate groupings remained. medlin and kaczmarska (2004) made more radical changes by splitting the traditional centric group into two classes and combining all the pennate diatoms in another class. but while a number of research groups and individuals consider how to reflect perceived phylogenetic relationships in formal classifications, there is a steady stream of publications that seem to be directed simply to recording and describing taxa, (particularly non-centric diatoms) without integrating them into a systematic framework (e.g many issues within the iconographia diatomologica series). furthermore there often seems to be a reliance on the generitype as the guide to the characters (as representative) of a genus, rather than referring to the spectrum of variation within the genus. comparisons between species and/or genera are also often restricted to examples occurring in the same type of habitat, e.g. all freshwater or all marine, rather than considering that the closest living relatives might be found in another habitat, or that the environmental stresses experienced by inland subaerial or terrestrial diatoms might be closer to those experienced in the marine littoral than in freshwater. the effects of environment should also be considered, including whether particular types of variation are linked to particular environmental variables. for example, changing conductivity may be responsible for the phenotypic plasticity exhibited by janus cells. the valves within mastogloia janus cells were identified as m. grevillei (biseriate striae) and m. elliptica var dansei (uniseriate striae) (stoermer 1967). the former is considered a freshwater and the latter a brackish water diatom. other types of heterovalvy, particularly in monoraphid and filamentous diatoms have less frequently been allocated to different taxa, probably because heterovalvy is not a function of environment, or valves and frustules of filamentous diatoms remain linked together. 450 acta bot. croat. 68 (2), 2009 cox e. j. u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:47 color profile: disabled composite 150 lpi at 45 degrees comparisons of new material against published descriptions and illustrations can provide clues to identity and relationships, but any published description is that author’s interpretation of their material, and illustrations may not provide all the relevant information on morphology and structure. it must also be remembered that the author wrote at a particular time and will have interpreted the material in the light of prevailing paradigms, may have used terminology in a different way, and may not have investigated characters that were considered uninformative at that time. in other words, there is no substitute for looking at specimens, and evaluating them character by character. for example, two skeletonema taxa were present in the material from which skeletonema costatum was originally described but, although he illustrated both morphs, greville (1866) did not describe them as different species. skeletonema costatum thus became a catch-all for many slightly different taxa, all with the characters described by greville. molecular studies highlighted the existence of more than one taxon (sarno et al. 2005), and because it was possible to link one of them to his drawings and hence to an individual specimen, that name could be fixed to one morph (designated as the lectotype of s. costatum) while the other could be described as a new species (zingone et al. 2005). conclusions while recording and describing new taxa constitute an important contribution to evaluating diatom diversity, they should not be regarded as an end in itself, but part of the process of discovering how organisms have evolved and are adapted to the habitats they occupy. to that end, we should consider not only the entities we discover, but how they relate to each other, their ancestors and the rest of the biosphere. i therefore suggest that, in describing new taxa (at whatever taxonomic level) as many as possible of the following points should be addressed: � obtain as much morphological information as possible, such as live structure, lm and sem of frustules (valves + cingulum), including exterior and interior details, tem of pore substructure. � assess the morphological variability within the taxon, comparing within and between sites if multiple samples are available. � compare all the above features with as wide a range of taxa as possible (from specimens, not just illustrations or published descriptions), across a range of habitats, without assuming (a priori) that any one character is more significant than any other. � ensure that the patterns of variation in different characters have been documented (lm and em). � determine what groups of specimens can be delimited (use appropriate morphometric and molecular phylogenetic analyses) and how those groupings are supported. � give a full description of all features of the new taxon (taxa) and their range of variation (not just those typically given in differential diagnoses). � place new taxa in relation to existing ones, and give reasons for the placement. � if new taxa group with members of an existing higher taxon, but exhibit characters not previously recorded for the higher taxon, be prepared to modify the diagnosis of the latter. acta bot. croat. 68 (2), 2009 451 diatom classification and systematic relationships u:\acta botanica\acta-botan 2-09\cox.vp 6. listopad 2009 13:21:47 color profile: disabled composite 150 lpi at 45 degrees references adl, s. m., simpson, a. g. b., farmer, m. a., andersen, r. a., anderson, o. r., barta, j. r., bowser, s. s., brugerolle, g., fensome, r. a., fredericq, s., james, t. y., karpov, 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jasmina kamberović1*, anđelka plenković-moraj2, koraljka kralj borojević3, marija gligora udovič2, petar žutinić2, dubravka hafner4, marco cantonati5 1 university of tuzla, faculty of natural sciences and mathematics, univerzitetska 4, 75000, tuzla, bosnia and herzegovina, jasmina.kamberovic@untz.ba 2 university of zagreb, faculty of science, department of biology, rooseveltov trg 6, 10000 zagreb, croatia 3 minnesota drive, great sankey, warrington, uk, wa5 3sy 4 bartulovići 4, 20357 blace, croatia 5 muse – museo delle scienze, limnology and phycology section, corso del lavoro e della scienza 3, i-38123 trento, italy abstract – the biodiversity of algal communities and environmental conditions were investigated in the springs of mt. konjuh. the assemblages of 20 springs emerging from different lithologies (limestones and ophiolites, respectively) comprised 234 algal taxa. diatoms and cyanobacteria were the most species-rich groups. the most common alkaliphilic, circumneutral, and eutraphentic diatoms were represented by the genera gomphonema, nitzschia, navicula, cymbella, and achnanthidium, and by the cyanobacterial genus phormidium. hierarchical clustering and simprof analysis based on relative algal abundance clustered springs into six groups, separating them mainly according to spring type and lithology. indicator species for groups and springs on different lithological substrata were singled out, revealing 33 taxa with preferences for ophiolites, and 20 taxa with preferences for carbonates. the values of the shannon-wiener diversity index were moderately high per spring location, and similar for the two groups of springs on different lithologies. a higher similarity in species composition was noted between springs on ophiolites and limestones than between springs on ophiolites and other types of siliceous substrata. the present study suggests that algal assemblages in springs emerging from ophiolites, even those made up by a preponderance of silicates, should be analyzed separately from those related to springs on other siliceous substrata. the results obtained showed that most of the springs studied are affected by anthropogenic impacts and morphological alterations leading to the dominance of highly competitive mesoand eutraphentic algal species, thus emphasizing the importance of further investigation and conservation of these habitats. keywords: biodiversity, diatoms, cyanobacteria, springs, geological substratum, ecological preferences, trophic status * corresponding author, e-mail: jasmina.kamberovic@untz.ba introduction the importance of spring habitats as biodiversity hotspots and refugia for biodiversity conservation (cantonati et al. 2012a, taxböck et al. 2017) has been emphasized by the description of a large number of rare and endangered algal species (sabater and roca 1990, 1992, cantonati 1998, werum 2001, poulíčková et al. 2005, nascimbene et al. 2011). half of the pennate freshwater diatoms described in europe were recorded from spring ecosystems (werum and lange bertalot 2004). studies dealing with the algal biodiversity of european springs have increased since the mid 1990s (sabater and roca 1990, cantonati 1998, falasco and bona 2011, werum and lange-bertalot 2004, fránková et al. 2009). some of them resulted in the discovery of new diatom genera and species (cantonati and lange-bertalot 2006, 2009, 2010, 2011, cantonati et al. 2009, 2010). studies on algae in bosnia and herzegovina date back to the late 19th century and the early 20th century (protić 1897, 1899, 1901, 1908, gutwinski 1899, beck 1928), whereas the first detailed studies on diatoms were carried out in the framework of a wider study of diatom assemblages in several lakes and springs of algae in springs of different lithology acta bot. croat. 78 (1), 2019 67 the balkan peninsula (hustedt 1945). however, crenic algal assemblages in bosnia and herzegovina have been investigated only at a few karst spring locations (blagojević 1974, 1976, 1979, hafner 2009, dedić et al. 2015). results deriving from the study of algal ecology in springs (cantonati et al. 2012b, c) have application in the monitoring of freshwater ecosystems. as a commonly studied algal group, diatoms are a useful proxy for water quality, reflecting both physical and chemical characteristics of a water body (kelly and whitton 1995, rott et al. 1999, werum and lange-bertalot 2004, wojtal 2013). diatom assemblages can be grouped according to different lithology (werum and lange-bertalot 2004). for instance, cymbella tridentina lange-bertalot, m. cantonati & a. scalfi is a typical crenophilous species living on carbonate substrate (cantonati et al. 2010), while achnanthidium dolomiticum m. cantonati et lange-bertalot is more common in springs fed by drainage basins dominated by dolomite lithology (cantonati and lange-bertalot 2006). diatom genera such as achnanthes s.l. and cymbella are generally more represented on carbonate substrates, whilst eunotia and pinnularia are more frequent on silicate rocks (cantonati 1998). algal assemblages in springs on ophiolitic or ultramafic rocks have been rarely studied (werum 2001). although widespread on several continents (dilek and furnes 2011), these substrata cover less than 1% of the earth's surface (coleman and jove 1992). dinaric ophiolites constitute one of the largest exposures of mantle rocks on earth (lugović et al. 1991). ophiolites from bosnia belong to the western belt of ophiolites in the balkans as fragments of a long chain which passes through western serbia (mt. zlatibor), kosovo and the western belt of the mirdita ophiolite in albania to the pindos and othris ophiolites in central greece. the ophiolites and associated sediments (mostly carbonates and ophiolitic melange) cover a surface of tens of thousands square kilometers in bosnia and herzegovina, with the krivajakonjuh mountain complex, which is the largest ophiolite zone in this area, positioned in the north-eastern part of the country (trubelja et al. 1995, babajić 2009). still, despite its wide-ranging coverage, freshwater algal assemblages on dinaric ophiolite substrata are largely unexplored. unlike other siliceous rocks, ophiolites have less than 45% concentrations of silica, generally contain high amounts of fe, and trace metals such as ni and cr (alexander et al. 2007, trubelja et al. 1995). results of chemical studies on ultramaphic rocks of the krivaja-konjuh ophiolitic complex demonstrated modal composition with high content of mgo, low content of cao, and a high mgo: feo ratio of about 5 and more (operta 2017). the main objectives of this study were to: (i) explore algal biodiversity in crenic habitats of mt. konjuh, north-eastern bosnia and herzegovina, including rarely investigated springs on ophiolites, (ii) describe ecomorphological, physical and chemical properties of these spring habitats, (iii) analyze the influence of geological substrata on algal assemblages, and (iv) describe the ecological preferences of dominant algal groups. materials and methods study area konjuh mountain is a part of the central dinarides on the balkan peninsula, with an altitude of 1326 m a.s.l. in 2009 it was designated a protected landscape (iucn category v) with ecosystems characterized as tertiary relicts. it belongs to a zone with a moderately continental climate, with a mean annual temperature varying between 9.2–10 °c, and annual precipitation ranging from 900 to 1250 mm (kudumović dostović 2012). as part of the ophiolitic complex, mt. konjuh consists mainly of peridotite and serpentinite rocks. in addition, ophiolitic melange, gabbro, shale and limestones are also found (ristić et al. 1967). physical and chemical analyses ecomorphological characteristics of springs (shading, current velocity, spring type) were assessed using methods described in spitale (2007). discharge was measured by a graduated bucket. the flow discharge for large rheocrenic springs was estimated by multiplying the cross section area, obtained from the values of average depth and width of the stream channel, and average velocity, which was measured by the float method. temperature, dissolved oxygen, ph, and conductivity were measured directly on site with a hanna hi 9828 multimeter (hanna instruments inc., u.s.a.). water for analyses of chemical parameters was taken at the same time. total alkalinity, calcium, and magnesium were analyzed via titration, and sulphates were measured using the turbidimetric method (apha, awwa and wef, 1999). ammonia, nitrates and nitrites were analyzed via a spectrometric method according to the standard procedures (bas iso 7150-1: 2002, bas iso 7890-3:2002, bas en 26777:2000) in bosnia and herzegovina (institute for standardization of bosnia and herzegovina, 2002a, b, 2000, respectively). algal sampling and identification a total of 196 algal samples were collected at the springhead and 15 meters downstream during the spring, summer and autumn of 2013. altogether 20 springs were seasonally studied: nine springs on ophiolites (2 rheocrenes, 6 rheohelocrenes, and one hygropetric spring), one (rheohelocrenic) spring on ophiolitic melange, and ten springs on carbonate (limestone) substrate (7 rheocrenes and 3 rheohelocrenes). the springs are situated at altitudes ranging between 402 and 1003 m a.s.l (fig. 1). epilithic algae were taken by brushing the surfaces of 5–6 stones (kelly et al. 1998), samples of epibryon were collected from aquatic bryophytes, while epipelon was collected from the sandy bottom with a cylinder. abundance of diatom and non-diatom algae was estimated as number of cells per square centimeter (epilithon and epipelon) or number of cells per gram of dried bryophyte mass. non-diatom species were identified in fresh samples, whilst diatoms were acid cleaned (hustedt 1930) and mounted in naphrax (brunel microscopes ltd., u.k.). at least 400 diatom valves were identified in each slide using random kamberović j., plenković-moraj a., kralj borojević k., gligora udovič m., žutinić p., hafner d., cantonati m. 68 acta bot. croat. 78 (1), 2019 transects under an olympus bx41 light microscope (olympus corporation, japan) at a magnification of 1000x. quantitative data on diatoms and non-diatoms were integrated according to stevenson and bahls (1999). because of their estimation per different units, and the need for uniformity for statistical analysis, the absolute cell numbers were transformed into relative (percentage) abundances. in addition to the abundance, which shows the average percentage of the taxa within samples, data on the taxa frequency are also given to provide information on the occurrence of taxa in the analyzed group of samples. identification was done following starmach (1985), cvijan and blaženčić (1996), krammer and lange-bertalot (1986, 1988, 2000, 2004 a,b), komárek and anagnostidis (1998, 2005), krammer (2000, 2002), john et al. (2002), hofmann et al. (2011) and lange-bertalot et al. (2017). data processing and statistical analyses diversity was calculated by using species richness (s) as the number of identified taxa, the shannon-wiener index of species diversity, h'(ln), (shannon and weaver 1949) and pielou’s index of equitability, j' (krebs 1999). ecological requirements of diatoms for moisture, ph and trophic status were obtained from van dam et al. (1994). the diatom trophic index (ti) by rott et al. (1999), the croatian trophic diatom index (tidhr) and the ecological quality ratio – eqr (anonymous 2013, 2016) were used in the evaluation of trophic and ecological status of spring ecosystems. all data were transformed by using the square root function prior to statistical analyses. non-metric multidimensional scaling (nmds) and hierarchical group average clustering based on mean values of the relative algal abundance for each spring were used for spring ordering. similarity profile analysis (simprof) with iterative permutation procedure (999 permutations) was used for the detection of boundaries among clusters. the ordination was conducted on the bray-curtis similarity matrix of species data (legendre and legendre 1998). normal distribution of the main physical and chemical parameters and values of diversity indices were tested by the kolmogorov-smirnov test in order to select parametric or nonparametric test. the significance of differences in the main physical and chemical factors between springs emerging from two different lithologies (ophiolites vs. limestone) was analyzed using the nonparametric mann-whitney u test (p < 0.05). parametric t-test was used to test for differences between diversity indices in springs from contrasting lithologies. the indval analysis (dufrene and legendre 1997) was used to identify characteristic species of: i) springs on different lithological substrata, and ii) different clusters. the significance of indicator value of each species was estimated by monte carlo permutation tests. statistical analyses were performed in ibm spss statistics for windows version 22.0 (ibm corp., u.s.a.), primer 6.0 (clarke and warwick 2001), and pc-ord 5 software packages (grandin 2006). results environmental variables ecomorphological characteristics of springs, including spring names with short spring codes, location, shading, velocity and discharge are described in on-line suppl. tab. 1. springs on ophiolites were characterized by low but relatively steady discharge (≤ 2 l s–1), water temperature below 8 °c, low turbidity, low calcium content, high magnesium, and high dissolved oxygen concentrations (on-line suppl. tab. 2). on the other hand, springs on carbonates (limestone) had an unstable discharge varying from complete drying up to 150 l s–1 (on-line suppl. tab. 1). carbonate rheocrenic springs had higher values of oxygen concentration than the rheohelocrenes, neutral to slightly alkaline water, and high calcium content. high nitrate concentrations (up to 18 mg l–1) were noted in several springs independently from the lithology. the values of alkalinity and conductivity ranged from 16 to 70 mg caco3 l–1, and 136–584 µs cm–1, respectively (on-line suppl. tab. 2). springs emerging from ophiolites showed higher values of temperature, ph, magnesium and lower values of conductivity, discharge, sulphates and calcium than springs on carbonates (on-line suppl. tab. 3). during field studies many negative anthropogenic influences were noted. the most pronounced effects are alterations of morphology of the spring area for the purpose of water abstraction, deforestation with accompanying effects fig. 1. location of the study sites presented by spring codes on the mt. konjuh in bosnia and herzegovina. algae in springs of different lithology acta bot. croat. 78 (1), 2019 69 of tree transport, road infrastructure building and trampling by cattle. species composition, diversity, ecological preferences of diatoms and trophic status a total of 234 algal taxa were identified in 196 samples collected from 20 springs. all identified taxa are listed in tab. 1. the most abundant algal groups were diatoms and cyanobacteria, with 187 and 34 taxa, respectively. the other algal classes were xanthophyceae (3), florideophyceae, ulvophyceae, chlorophyceae and conjugatophyceae (2 in each), chrysophyceae and klebsormidiophyceae (1 in each). total number of recorded taxa ranged between 46 (rheocrenic spring on ophiolite – 2ke) and 76 (rheohelocrenic spring on ophiolite – sk). the most common algal genera were gomphonema (17), nitzschia (15), navicula (11), phormidium (9), cymbella (9), and achnanthidium (8). the species achnanthidium minutissimum, planothidium lanceolatum, meridion circulare, cocconeis pseudolineata and amphora pediculus were recorded in more than a half of all investigated springs. besides the above mentioned, also very abundant were cocconeis lineata, gomphonema micropus, cocconeis euglypta, odontidium mesodon and tapinothrix varians. hierarchical group average clustering and the simprof test identified 6 assemblages (fig. 2) mainly related to spring type (rheocrenes and rheohelocrenes) and geological substratum (ophiolites, carbonates, and ophiolitic melange). species contribution to the clusters was described by the simper analysis (on-line suppl. tab. 4), and statistically significant indicator species (p < 0.05) for each cluster provided by the indval analysis are listed in fig. 2. species indicator values and contribution of the most abundant species could not be technically estimated for springs mv (cluster 1) and zl (cluster 6), because the first and the sixth cluster included only one spring. however, the most frequent and abundant species in spring mv (cluster 1) were achnanthidium minutissimum, achnanthidium pyrenaicum, odontidium mesodon, nitzschia fonticola and amphora pediculus, whilst the most abundant and frequent species in spring zl (cluster 6) were meridion circulare, gomphonema micropus, eunotia soleirolii, nitzschia dubia and ammatoidea sp. cluster 2 (rheocrenes on ophiolites) was characterized by the xerotolerant diatom humidophilla perpusilla, distinguished by both indval and simper analysis. taxa distinguished by indval were the eutraphentic diatom gomphonema parvulum and the cyanoprokaryote pseudanabaena sp. simper analysis singled out several species with high contributions: planothidium lanceolatum, chlorogloea microcystoides, meridion circulare, phormidium formosum, and other taxa listed in on-line suppl. tab. 4. rheocrenes on carbonates (cluster 3) were characterized by rhoicosphenia abbreviata and nitzschia fonticola (given by indval), and a high contribution of the rheophilic cyanobacterium tapinothrix varians, the crenophilous diatoms m. circulare and odontidium mesodon, and the epiphytic diatom species p. lanceolatum and cocconeis lineata. cluster 4 comprised rheohelocrenes on ophiolites and carbonates, and was characterised by the diatoms cocconeis pseudolineata, encyonopsis cesatii, diploneis fontanella, and tryblionella angustata. indval also singled out achnanthidium exile, amphora lange-bertalotii var. tenuis, cymbella hantzschiana, gomphonema subclavatum, and nitzschia capitellata. representatives of shaded rheocrenes (cluster 5) were eutraphentic diatoms, such as amphora pediculus, encyonema minutum, fallacia subhamulata, gomphonema angustatum, achnanthidium affine, and cymbella diminuta. according to nmds, springs were separated by lithology, with carbonate springs distributed on one side and ophiofig. 2. hierarchical group average clustering based on algal assemblages in the studied springs (presented by spring codes) on the mt. konjuh and characteristic species for each cluster identified by the indval analysis. number of clusters was calculated with the simprof analysis. kamberović j., plenković-moraj a., kralj borojević k., gligora udovič m., žutinić p., hafner d., cantonati m. 70 acta bot. croat. 78 (1), 2019 tab. 1. algae of springs in the mt. konjuh, gs – geological substrata (o – ophiolites, c – carbonates, om – ophiolitic melange); a – mean abundance per sample, %; f – frequency per sample, %; max. – maximum relative abundance per sample, %; n – number of springs where the taxon was found. taxa gs a f max. n cyanophyceae ammatoidea sp. o-c-om 8.5 7.1 50.0 4 aphanocapsa minuta (kylin) whitton o-c 0.4 1.0 0.7 2 chamaesiphon confervicola a.braun c 32.5 1.0 58.5 1 chamaesiphon fuscus (rostafinski) hansgirg o-c 1.9 2.0 5.9 3 chamaesiphon incrustans grunow c 1.1 1.5 3.1 1 chamaesiphon polonicus (rostafinski) hansgirg o 9.1 3.6 28.6 5 chamaesiphon sp. c 3.5 2.0 5.9 3 chamaesiphon subglobosus (rostafinski) lemmermann o 0.2 0.5 0.2 1 chlorogloea microcystoides geitler o-c 23.0 9.2 78.6 6 chroococcaceae o-c-om 21.2 4.1 72.7 5 chroococcus sp. o 0.1 0.5 0.1 1 chroococcus turgidus (kützing) nägeli o-c 2.2 4.6 5.0 5 clastidium setigerum o.kirchner o 0.3 0.5 0.3 1 cyanothece aeruginosa (nägeli) komárek o 0.03 0.5 0.0 1 hassallia pulvinata frémy o-c 8.0 2.0 20.1 2 hyella sp. c 0.3 0.5 0.3 1 leptolyngbya perforans (geitler) anagnostidis et komárek c 28.8 1.0 56.2 1 leptolyngbya sp. o-c-om 7.3 10.2 39.6 9 lyngbya martensiana meneghini ex gomont o-c 1.9 3.1 6.1 3 lyngbya natans hansgirg o 0.9 0.5 0.9 1 oscillatoria sp. c 18.0 0.5 18.0 1 phormidium corium gomont ex gomont o 4.8 1.0 8.6 1 phormidium formosum (bory de saint-vincent ex gomont) anagnostidis et komárek o-c 22.2 16.8 99.0 9 phormidium griseoviolaceum (skuja) anagnostidis c 9.9 0.5 9.9 1 phormidium incrustatum gomont ex gomont c 5.0 1.0 8.7 1 phormidium kuetzingianum (kirchner ex hansgirg) anagnostidis et komárek c 1.5 1.0 1.6 1 phormidium retzii kützing ex gomont o-c 22.6 9.7 89.3 7 phormidium rotheanum itzigsohn o 2.4 1.0 2.8 1 phormidium subfuscum kützing ex gomont o-c 32.2 2.0 81.3 3 phormidium tinctorium kützing ex gomont c 31.0 3.1 66.7 3 pleurocapsa aurantiaca geitler o 0.3 0.5 0.3 1 pleurocapsa minor hansgirg o-c 8.7 3.6 35.6 4 pseudanabaena sp. o-c 6.1 6.1 19.6 3 tapinothrix varians (geitler) bohunická et j.r.johansen o-c 36.9 28.1 98.7 14 florideophyceae audouinella sp. c 21.4 5.1 70.0 3 batrachospermum sp. c 6.8 0.5 6.8 1 chrysophyceae phaeodermatium rivulare hansgirg c 10.8 3.6 58.4 3 xanthophyceae ophiocytium majus nägeli o 0.1 0.5 0.1 1 tribonema sp. o-c 4.2 7.1 17.8 5 vaucheria sp. c 4.9 8.7 24.7 3 bacillariophyceae and fragillariophyceae achnanthidium affine (grunow) czarnecki o-c-om 2.8 14.8 28.4 9 achnanthidium caledonicum (lange-bertalot) lange-bertalot o-c 8.8 4.6 36.5 5 achnanthidium dolomiticum m. cantonati et h. lange-bertalot o 2.4 1.5 3.3 1 achnanthidium exile (kützing) heiberg o-c 4.0 9.2 10.8 8 achnanthidium minutissimum (kützing) czarnecki o-c-om 20.6 87.8 82.1 20 achnanthidium pyrenaicum (hustedt) h.kobayasi o-c-om 7.4 29.1 53.4 13 achnanthidium rosenstockii (lange-bertalot) lange-bertalot c 0.8 0.5 0.8 1 achnanthidium straubianum (lange-bertalot) lange-bertalot o-c 1.5 4.6 4.2 4 adlafia bryophila (j.b.petersen) gerd moser, lange-bertalot et d.metzeltin o-c 0.6 3.1 1.4 5 adlafia minuscula (grunow) lange-bertalot o-c-om 0.9 12.2 2.8 11 amphipleura pellucida (kützing) kützing o-c 1.3 11.7 4.4 7 amphora copulata (kützing) schoeman et r.e.m.archibald c 1.5 0.5 1.5 1 amphora indistincta levkov o-c-om 2.2 21.9 16.6 12 amphora lange-bertalotii var. tenuis levkov et metzeltin o-c 1.4 10.2 7.3 7 amphora ovalis (kützing) kützing o-c-m 3.0 14.8 15.9 8 amphora pediculus (kützing) grunow ex a.schmidt o-c 3.6 65.8 28.9 19 algae in springs of different lithology acta bot. croat. 78 (1), 2019 71 taxa gs a f max. n brachysira vitrea (grunow) r.ross c 0.7 0.5 0.7 1 brebissonia lanceolata (c.agardh) mahoney et reimer o-c 3.0 8.2 27.6 7 caloneis alpestris (grunow) cleve c 0.1 0.5 0.1 1 caloneis fontinalis (grunow) lange-bertalot et reichardt o-c-m 0.8 28.6 4.1 18 caloneis silicula (ehrenberg) cleve o-c 0.4 3.1 0.5 4 caloneis sp. c 0.5 0.5 0.5 1 caloneis tenuis (w.gregory) krammer o-c-m 0.5 4.6 0.9 7 chamaepinnularia parsura (hustedt) c.e.wetzel et ector c 0.2 0.5 0.2 1 cocconeis disculus (schumann) cleve o 3.0 3.1 8.2 1 cocconeis pediculus ehrenberg c 0.2 1.0 0.2 1 cocconeis euglypta ehrenberg o-c-om 5.2 39.8 38.0 15 cocconeis placentula var. klinoraphis geitler o 0.5 0.5 0.5 1 cocconeis lineata ehrenberg o-c-om 6.9 58.7 85.5 18 cocconeis pseudolineata (geitler) lange-bertalot o-c-om 6.7 57.7 74.7 20 cymatopleura solea var. apiculata (w.smith) ralfs c 0.4 1.5 0.8 3 cymbella affinis kützing o-c 1.7 8.7 12.5 5 cymbella cymbiformis c.agardh o-c 7.4 4.6 29.2 2 cymbella excisiformis krammer o 0.8 2.0 1.4 1 cymbella diminuta (grunow) reichardt c 0.9 3.1 2.1 2 cymbella hantzschiana krammer o-c 1.7 13.8 4.7 10 cymbella laevis nägeli c 1.2 1.0 2.1 1 cymbella parva (w.smith) kirchner o-c 1.3 12.2 5.5 9 cymbella tridentina lange-bertalot, m.cantonati et a.scalfi o 5.1 3.1 20.1 2 cymbella vulgata krammer c 0.2 1.0 0.3 2 cymbopleura amphicephala (nägeli) krammer o 0.8 2.6 1.6 1 cymbopleura florentina (grunow) k.krammer o 0.4 1.5 0.7 1 cymbopleura subaequalis (grunow) krammer c 0.7 0.5 0.7 1 cymbopleura subaustriaca krammer o-c-om 1.9 5.1 7.0 3 decussata hexagona (torka) lange-bertalot c 0.4 0.5 0.4 1 delicata minuta k.krammer o-c 0.4 1.5 0.6 2 denticula kuetzingii grunow o-c 0.9 4.1 2.2 5 denticula tenuis kützing o-c 3.2 16.8 21.8 9 humidophila contenta (grunow) lowe, kociolek, johansen, van de vijver, lange-bertalot et kopalová o-c 0.5 8.7 1.9 9 humidophila paracontenta var. magisconcava (lange-bertalot) lowe, kociolek, johansen, van de vijver, lange-bertalot et kopalová o-c 1.3 9.2 6.7 11 humidophila perpusilla (grunow) lowe, kociolek, johansen,van devijver, lange-bertalot et kopalová o-c 5.7 27.0 76.7 14 humidophila sp. om 0.5 0.5 0.5 1 odontidium hyemale (roth) kützing o-c 1.1 3.6 2.2 5 odontidium mesodon (ehrenberg) kützing o-c-om 7.4 35.2 65.2 17 diploneis elliptica (kützing) cleve o-c 1.0 4.6 4.1 4 diploneis fontanella lange-bertalot o-c-om 1.0 19.9 3.4 14 diploneis krammeri lange-bertalot et e.reichardt o-c-om 1.3 26.0 13.2 16 diploneis minuta j.b.petersen o-c 0.5 3.1 0.7 3 encyonema hebridicum (gregory) grunow o-c 0.6 2.6 1.2 3 encyonema minutum (hilse) d.g.mann o-c 0.7 4.6 2.3 5 encyonema neogracile krammer o 0.4 0.5 0.4 1 encyonema silesiacum (bleisch) d.g.mann o-c 0.7 5.6 1.6 5 encyonema triangulum (ehrenberg) kützing o 0.6 1.0 0.7 1 encyonema ventricosum (c.agardh) grunow o-c-om 2.5 16.3 30.5 9 encyonopsis cesatii (rabenhorst) krammer o-c 3.9 35.2 24.3 15 encyonopsis fonticola (hustedt) krammer o-c 2.9 10.2 10.2 9 encyonopsis krammeri reichardt o-c-om 1.0 16.8 5.5 12 encyonopsis microcephala (grunow) krammer o 1.1 3.1 2.5 1 encyonopsis minuta krammer et e.reichardt m 0.7 3.1 1.8 1 epithemia argus (ehrenberg) kützing o-c-om 1.4 2.0 4.3 1 epithemia frickei krammer o-c 1.0 1.5 2.2 3 epithemia turgida (ehrenberg) kützing c 1.1 2.6 3.3 1 eucocconeis flexella (kützing) meister o 0.6 1.5 0.9 2 eucocconeis laevis (østrup) lange-bertalot o-c-om 0.7 6.1 2.7 8 eunotia ambivalens lange-bertalot et tagliaventi o 1.4 1.0 1.6 1 eunotia arcubus nörpel et lange-bertalot o-c 7.8 11.2 60.7 7 eunotia soleirolii (kützing) rabenhorst o-om 1.4 9.2 4.1 4 fallacia lange-bertalotii (e.reichardt) e.reichardt c 0.7 2.0 1.2 2 tab. 1. continued kamberović j., plenković-moraj a., kralj borojević k., gligora udovič m., žutinić p., hafner d., cantonati m. 72 acta bot. croat. 78 (1), 2019 taxa gs a f max. n fallacia subhamulata (grunow) d.g.mann c 1.4 4.1 7.8 4 fragilaria amphicephaloides lange-bertalot o 1.4 2.0 1.9 1 fragilaria recapitellata h. lange-bertalot et d. metzeltin c 4.1 5.6 26.9 3 frustulia vulgaris (thwaites) de toni c-om 0.3 3.1 0.5 4 gomphonema angustatum (kützing) rabenhorst c-om 1.5 12.8 11.6 8 gomphonema angustivalva e.reichardt o-c 3.3 1.0 6.2 2 gomphonema angustum c.agardh o-c-om 1.7 27.6 10.5 14 gomphonema brebissonii kützing o 0.4 1.0 0.4 1 gomphonema elegantissimum reichardt et lange-bertalot o-c 4.7 18.4 42.6 8 gomphonema exilissimum (grunow) lange-bertalot et e.reichardt o-c 1.4 15.8 7.1 11 gomphonema micropus kützing o-c-om 2.0 44.9 23.3 17 gomphonema occultum e.reichardt et lange-bertalot c 0.6 2.0 1.2 2 gomphonema olivaceum (hornemann) brébisson c 0.6 4.6 1.8 2 gomphonema olivaceoides hustedt c 0.8 0.5 0.8 1 gomphonema parvulum (kützing) kützing o-c-om 1.8 18.4 11.7 15 gomphonema procerum reichardt et lange-bertalot o-c 0.2 1.5 0.5 2 gomphonema pseudoaugur lange-bertalot o 0.3 0.5 0.3 1 gomphonema pseudobohemicum lange-bertalot et e.reichardt c 1.6 2.6 3.7 2 gomphonema pumilum (geitler) bohunická et j.r.johansen om 3.1 24.5 46.0 12 gomphonema subclavatum (grunow) grunow o-c-om 0.6 12.2 1.3 10 gomphonema tergestinum (grunow) fricke o-c 2.0 12.8 7.0 10 grunowia sinuata (thwaites) rabenhorst o-c 1.6 3.1 5.9 2 gyrosigma acuminatum (kützing) rabenhorst c 6.1 4.1 14.5 1 gyrosigma obtusatum (sullivant & wormley) c.s.boyer c 1.5 2.0 2.4 1 halamphora normanii (rabenhorst) levkov o-c 1.2 3.1 3.6 4 halamphora veneta (kützing) levkov o 0.7 0.5 0.7 1 hannaea arcus (ehrenberg) r.m.patrick c 0.1 0.5 0.1 1 hantzschia amphioxys (ehrenberg) grunow o-c 0.5 4.1 0.9 3 luticola mutica (kützing) d.g.mann c-om 0.8 3.1 2.0 4 meridion circulare (greville) c.agardh o-c-om 5.9 64.3 38.7 20 navicula antonii lange-bertalot c 1.7 21.9 7.5 10 navicula cariocincta lange-bertalot c 1.0 3.6 3.1 3 navicula cataracta-rheni lange-bertalot o 2.0 6.1 8.9 3 navicula cryptotenella lange-bertalot o-c-om 1.7 12.8 22.4 8 navicula cryptotenelloides lange-bertalot o 0.8 0.5 0.8 1 navicula gregaria donkin c 1.1 0.5 1.1 1 navicula leistikowii lange-bertalot o-c 0.9 13.8 2.7 9 navicula oblonga (kützing) kützing o 0.6 0.5 0.6 1 navicula radiosa kützing o-c 1.2 17.9 4.0 11 navicula sp. c 1.9 4.1 10.3 3 navicula tripunctata (o.f.müller) bory de saint-vincent o-c 1.2 17.3 6.2 9 naviculadicta geisslerae (jahn) jahn o 0.7 0.5 0.7 1 naviculadicta tridentula (krasske) lange-bertalot o 0.6 2.6 1.6 3 naviculadicta sp. c 4.4 0.5 4.4 1 neidiomorpha binodiformis (k.krammer) m.cantonati, h.lange-bertalot et n.angeli o-c 0.3 1.5 0.4 2 neidium longiceps (gregory) ross o-c 0.3 2.0 0.5 3 nitzschia amphibia grunow o-c 1.6 7.1 7.2 8 nitzschia capitellata hustedt o-c 0.8 5.6 1.8 7 nitzschia dissipata (kützing) grunow o-c 1.0 15.3 4.1 11 nitzschia dubia w.smith o-c-om 2.1 14.3 11.2 7 nitzschia fonticola (grunow) grunow c 4.5 10.2 33.5 6 nitzschia frustulum (kützing) grunow o-c-om 1.2 10.2 3.4 9 nitzschia heufleriana grunow c 0.6 1.5 0.8 1 nitzschia inconspicua grunow o-c 0.7 3.6 1.3 6 nitzschia linearis (c.agardh) w.smith o-c 2.7 31.6 58.5 16 nitzschia palea (kützing) w.smith o-c 2.4 14.8 12.0 12 nitzschia palea var. tenuirostris grunow c-om 4.8 7.1 31.6 5 nitzschia oligotraphenta (lange-bertalot) lange-bertalot c 0.5 0.5 0.5 1 nitzschia sp.1 c 2.6 0.5 2.6 1 nitzschia sp.2 o 0.9 0.5 0.9 1 nitzschia umbonata (ehrenberg) lange-bertalot c 1.1 0.5 1.1 1 tab. 1. continued algae in springs of different lithology acta bot. croat. 78 (1), 2019 73 taxa gs a f max. n nupela lapidosa (krasske) lange-bertalot o 0.7 0.5 0.7 1 orthoseira roeseana (rabenhorst) o'meara o-c 0.7 2.0 1.4 3 pinnularia acutobrebissonii kulikovskiy, lange-bertalot et metzeltin o 0.9 2.0 1.1 1 pinnularia borealis ehrenberg o 0.5 2.6 0.7 2 pinnularia gibba ehrenberg c 0.4 0.5 0.4 1 pinnularia kuetzingii k.krammer o 0.7 2.0 0.8 1 pinnularia sp. om 0.5 0.5 0.5 1 pinnularia subcapitata var. elongata krammer o 1.0 2.0 2.0 1 pinnularia subrupestris k.krammer o-c-om 1.1 6.1 3.3 7 placoneis paraelginensis lange-bertalot o-m 0.3 3.1 0.5 5 platessa conspicua (a. mayer) lange-bertalot m 2.8 1.5 8.1 1 platessa lutheri (hustedt) potapova c 0.9 1.5 1.6 1 planothidium dubium (grunow) round et bukhtiyarova m 1.4 22.4 10.3 8 planothidium frequentissimum (lange-bertalot) round et l.bukhtiyarova o-c-om 1.8 21.9 6.6 18 planothidium joursacense (héribaud-joseph) lange-bertalot o 3.6 0.5 3.6 1 planothidium lanceolatum (brébisson ex kützing) lange-bertalot  o-c-om 10.7 86.7 64.9 20 planothidium minutissimum (krasske) e.a.morales c-om 10.1 1.0 15.8 2 planothidium reichardtii lange-bertalot et werum o-c 1.0 5.1 3.9 8 platessa holsatica (hust.) lange-bertalot c-om 4.4 4.1 28.9 5 psammothidium grischunum (wuthrich) l.bukhtiyarova et round c 3.7 13.3 15.7 6 psammothidium subatomoides (hustedt) bukhtiyarova et round o-c-om 1.5 8.7 10.7 7 pseudostaurosira brevistriata (grunow) williams et round c 0.1 0.5 0.1 1 reimeria sinuata (gregory) kociolek et stoermer c 0.3 1.0 0.4 1 rhoicosphenia abbreviata (c.agardh) lange-bertalot c 0.7 9.2 2.9 5 rhopalodia parallela (grunow) o. müller o-c 0.5 5.1 0.8 3 rossithidium anastasiae (kaczmarska) potapova o-c-om 2.8 11.2 15.3 7 rossithidium petersenii (hustedt) round et bukhtiyarova o-c 1.4 6.6 3.0 6 rossithidium pusillum (grunow) round et l.bukhtiyarova c 0.2 1.0 0.3 1 sellaphora seminulum (grunow) d.g. mann o-c 0.6 4.6 1.7 6 sellaphora pseudopupula (krasske) lange-bertalot c 0.4 1.5 0.7 3 sellaphora pupula (kützing) mereschkovsky c 0.7 9.2 2.4 6 sellaphora stroemii (hustedt) h.kobayasi o 0.6 0.5 0.6 1 sellaphora nigri (de notaris) c.e. wetzel et l. ector o-c-om 2.9 25.0 22.5 17 simonsenia delognei (grunow) lange-bertalot c 1.1 1.5 3.1 1 staurosirella martyi (héribaud) morales et manoylov c 0.1 0.5 0.1 1 stauroneis anceps ehrenberg o-om 0.3 2.6 0.4 3 stauroneis jarensis lange-bertalot o 0.3 0.5 0.3 1 stauroneis phoenicenteron (nitzsch) ehrenberg c 0.3 0.5 0.3 1 stauroneis smithii grunow o-c 0.8 6.1 3.6 5 surirella angusta kützing o-c-om 1.1 10.2 5.0 9 surirella brebissonii var. kuetzingii krammer et lange-bertalot o 0.2 0.5 0.2 1 surirella linearis w.smith o-c 2.1 18.4 12.8 12 surirella minuta brébisson o-c 1.0 4.6 2.9 5 surirella spiralis kützing o 0.7 0.5 0.7 1 surirella terricola lange-bertalot et e.alles o-c-om 0.9 8.7 3.3 9 tryblionella angustata w.smith o-c 3.0 10.7 13.9 11 tryblionella angustatula (lange-bertalot) cantonati et lange-bertalot o 1.2 3.6 3.1 3 ulnaria ulna (nitzsch) p.compère o-c-om 0.8 24.5 4.1 16 chlorophyceae chaetophora sp. o 12.9 1.0 20.0 1 microspora sp. o 0.6 0.5 0.6 1 klebsormidiophyceae klebsormidium flaccidum (kützing) p.c. silva, k.r. mattox et w.h. blackwell o 3.2 1.5 5.1 1 ulvophyceae ulothrix sp. o 0.3 0.5 0.3 1 ulotrichales c 0.8 1.0 1.7 1 conjugatophyceae cosmarium obtusatum (schmidle) schmidle o 0.1 1.5 0.1 1 spirogyra sp. c 0.5 1.5 1.0 1 tab. 1. continued kamberović j., plenković-moraj a., kralj borojević k., gligora udovič m., žutinić p., hafner d., cantonati m. 74 acta bot. croat. 78 (1), 2019 lite springs on the other side of the plot (fig. 3). the highest shannon-wiener diversity and the highest number of taxa were recorded from the epibryon sample of the spring sk on ophiolitic substratum (2.95 and 47, respectively). the lowest diversity (0.08) and the lowest number of taxa (5) were found in the epilithon summer sample in the spring tu on carbonate substratum. the rheocrenic spring st, characterised by the species tapinothrix varians and cocconeis lineata, had the lowest values of pielou's index, indicating several dominant and subdominant species in the sample and huge unevenness in species abundances. parametric t-test performed on diversity indices of springs on different geological substrata did not show statistical differences (p > 0.05). however, statistical differences for used trophic indices between two groups of springs were found (rott's trophic index – ti, t = – 2.4, p = 0.02, croatian diatom trophic index – tidhr, t = 2.75, p = 0.007, and the ecological quality ratio – eqr which is based on tidhr). higher mean values of trophic indices were noted in samples from ophiolites (tab. 2). spring zl on ophiolitic melange was not considered for statistical analysis as it was the sole site on this geological substratum. values of ti varied between 1.22 (spring mv) and 2.59 (spring zl). meso-eutrophic and eutrophic conditions were detected for eleven and eight springs, respectively. according to the ti, an oligotrophic status was assessed only for the spring mv. assessment of ecological status of springs based on values of eqr and tidhr indicated good status for all localities, except for the spring mv (excellent status) and spring po (relatively good status) (tab. 2). values of ph preferences were found for 58.3% of identified diatoms: 51.4% alkaliphilous (most frequent taxa: amphora pediculus, cocconeis lineata, c. euglypta, gomphonema angustum, g. micropus, meridion circulare, navicula tripunctata, planothidium lanceolatum), 33.9% circumneutral (e.g., a. minutissimum, odontidium mesodon, encyonema minutum, encyonopsis cesatii, navicula radiosa, etc.), 9.2% acidophilous (e.g. neidium affine var. longiceps, pinnularia subcapitata var. elongata, tryblionella angustata), and 5.5% alkalibiontic (rarely occurring species such as achnanthidium exile and halamphora veneta). moisture-condition preference indicator values were found for 50.2% of the diatom taxa: 17% of taxa were classified as ‘never, or only very rarely occurring outside water bodies’ (meridion circulare, nitzschia fonticola, ulnaria ulna), 21.3% occurred ‘mainly in water bodies, sometimes on wet places’. the highest number of species (46.8%) ‘occurred mainly on wet and moist places’, while a low number of species (11.7%) ‘occurred mainly on wet and moist or temporarily dry places’ (caloneis tenuis, diploneis fontanella, luticola mutica). the lowest number of taxa (3.2%) belongs to the category ‘occurring nearly outside of water bodies’ (adlafia bryophila, diploneis minuta, halamphora veneta). geological preferences geological preferences of algae for the two main geological substrata, carbonates and ophiolites were analyzed. as only one site on this geological substratum was available, the spring on ophiolitic melange could not be included in statistical analyses. a total of 49 species (31%) were found exclusively in samples taken from springs on ophiolites, and were mainly identified from one site only, and the most common (relative frequency > 3%) were: navicula cataracta-rheni, chamaesiphon polonicus, tryblionella angustatula, cymbella tridentina, cocconeis disculus and encyonopsis microcephala. a total of 73 taxa (40%) were found exclusively in samples from carbonate substratum, and the most common (frequency > 5%) were: planothidium dubium, navicula antonii, n. cariocincta, psammothidium grischunum, gomphonema angustatum, rhoicosphenia abbreviata, sellaphora pupula, vaucheria sp., nitzschia palea var. tenuirostris, n. fonticola, fragilaria recapitellata, audouinella sp., gomphonema olivaceum, gyrosigma acuminatum, phaeodermatium rivulare, platessa holsatica, cymbopleura diminuta, encyonopsis minuta and phormidium tinctorium. results of the indval analysis of algal assemblages for the factor ‘lithology’ (tab. 3) comprised statistically significant (p ≤ 0.01) indicator species with algal frequency in the samples > 10%. although many species were identified in the springs on both investigated substrata, they showed higher abundance or frequency on one substratum. a preference for ophiolitic substrata, with indval values > 30, was established for: achnanthidium minutissimum, humidophila perpusilla, diploneis krammeri, encyonopsis cesatii, gomphonema elegantissimum, navicula radiosa, planothidium lanceolatum and nitzschia linearis. by contrast, a preference fig. 3. non-metric multidimensional scaling (nmds) of sites (presented by spring codes) based on bray-curtis matrix of similarities of algal assemblages in relation to geological substratum and clusters. trophic preferences were found for 101 species (54%), and were as follows: oligotraphentic (13.95%, encyonopsis cesatii, gomphonema angustum, surirella spiralis), oligo-mesotraphentic (13.9%), mesotraphentic (9.9%), meso-eutraphentic (12.9%), eutraphentic (32.75%, amphora copulata, a. pediculus, cocconeis spp., navicula tripunctata), hypereutraphentic (3%, nitzschia palea, nitzschia umbonata) and oligo – to eutraphentic (13.9%). algae in springs of different lithology acta bot. croat. 78 (1), 2019 75 tab. 2. the values of diversity and trophic indices of studied springs on the mt. konjuh with ranges of minimum (min), maximum (max), mean and standard deviation (sd), t – test values and p – statistical significance for groups of springs on carbonates (101 samples) and ophiolites (95 samples), n – number of samples per spring, s – number of taxa, j' – pielou's index, h'(ln) – shannon – wiener diversity index, ti rott – rott's trophic index, tidhr – croatian trophic index, eqr – ecological quality ratio with water quality ranges. substrata spring code n s h’ j' ti rott tidhr eqr ophiolites 1ke 12 48 1.65 0.63 2.61 2.23 0.71 (ii) 2ke 12 46 1.50 0.6 2.14 2.31 0.67 (ii) 1bo 9 55 1.90 0.67 2.40 2.41 0.64 (ii) 2bo 6 63 1.93 0.57 1.77 2.48 0.61 (ii) 1vu 9 63 2.07 0.65 2.32 2.49 0.6 (ii) 2vu 6 49 2.01 0.66 2.29 2.44 0.62 (ii) mi 5 56 2.09 0.7 2.14 2.28 0.69 (ii) sk 10 76 2.22 0.69 2.32 2.41 0.64 (ii) ka 9 54 1.76 0.62 2.03 2.30 0.68 (ii) min. 6 0.32 0.14 1.39 1.49 0.49 (iii) max. 47 2.95 0.91 3.27 2.77 1 (i) mean 19.21 1.79 0.62 2.23 2.36 0.65 (ii) sd   9.09 0.59 0.17 0.49 0.19 0.77 carbonates gb 12 66 2.04 0.7 2.32 2.19 0.72 (ii) kr 8 62 1.34 0.47 2.32 2.21 0.72 (ii) ks 12 64 1.77 0.64 2.31 2.16 0.74 (ii) mv 9 54 1.69 0.58 1.22 1.76 0.9 (i) po 12 70 1.58 0.56 1.89 2.54 0.59 (iii) st 12 46 1.30 0.51 1.90 2.10 0.76 (ii) ta 12 55 1.30 0.5 1.98 2.28 0.69 (ii) tu 12 65 1.50 0.52 2.11 2.38 0.65 (ii) ub 8 67 2.23 0.74 2.56 2.41 0.63 (ii) zp 12 58 2.23 0.75 2.19 2.34 0.66 (ii) min. 5 0.08 0.03 0.70 1.05 0.47 (iii) max. 43 2.83 0.91 3.18 2.82 0.96 (i) mean 19.19 1.74 0.61 2.06 2.24 0.7 (ii) sd   9.23 0.67 0.2 0.51 0.34 0.13 ophiolitic melange (mean) zl 9 48 1.61 0.61 2.57 2.25 0.7 (ii) t (ophiolites vs. carbonates) –0.02 –0.56 –0.56 –2.4 2.75 –2.75 p (ophiolites vs. carbonates)   0.99 0.57 0.57 0.02 0.007 0.007 for carbonate substrata (indval > 30) was noted for the following species: amphora pediculus, odontidium mesodon, gomphonema micropus, g. pumilum, navicula antonii, and planothidium dubium. discussion diatoms were the dominant algal group in the investigated springs. our results were mostly compared with those obtained from the alps, due to similarities in the sampling approach based on different spring types. a comparison of the presented results with previous investigations conducted in the south-eastern alps (cantonati 1998) revealed many species in common, such as achnanthidium minutissimum, odontidium (diatoma) mesodon, achnanthidium pyrenaicum, cocconeis euglypta, gomphonema pumilum s.l. and meridion circulare. however, several species frequent in alpine springs (brachysira brebissonii, encyonema minutum, and odontidium hyemalis) were either rarely present or not detected in our study. a higher similarity was found with the results of angeli et al. (2010), who investigated diatoms in anthropogenically influenced, low altitude springs, where many eutraphentic taxa were recorded (cocconeis, planothidium, and nitzschia). the highest number of taxa identified, excluding diatoms, belongs to the genera phormidium and chamaesiphon, which is in accordance with the results of cantonati et al. (1996). several species frequently recorded in this study (chlorogloea microcystoides, chamaesiphon polonicus, tapinothrix varians, audouinella sp., vaucheria sp. and tribonema sp.) were also found in previous investigations (e.g., cantonati et al. 2012c). on the other hand, species frequently noted in alpine springs, such as xenotholos kerneri, hydrurus foetidus, chamaesiphon starmachii, tapinothrix janthina and plectonema tomasinianum were not recorded in our study, presumably due to their preference for higher elevations with lower water temperature and/or siliceous substratum. for instance, a typical rheobiontic species of crenic habitats, hydrurus foetidus, was not found in this study, possibly due to the lower water velocity of the springs investigated. our results can be well compared with those kamberović j., plenković-moraj a., kralj borojević k., gligora udovič m., žutinić p., hafner d., cantonati m. 76 acta bot. croat. 78 (1), 2019 tab. 3. indicator values (indval) and frequency of indicative species of ophiolitic and carbonate springs on the mt. konjuh at p <0.01 (4999 permutations using monte carlo test). taxa indval frequency in group 1 frequency in group 2 gr ou p 1 op hi ol ite s achnanthidium minutissimum (kützing) czarnecki 58.8 96 81 planothidium lanceolatum (brébisson ex kützing) lange-bertalot  58.5 92 82 encyonopsis cesatii (rabenhorst) krammer 50.5 58 22 humidophila perpusilla (grunow) lowe, kociolek, 46.8 49 14 nitzschia linearis (c. agardh) w. smith 40 47 23 diploneis krammeri lange-bertalot et e. reichardt 38.9 45 13 navicula radiosa kützing 34.6 37 6 gomphonema elegantissimum e. reichardt et lange-bertalot 30.6 33 9 cymbella hantzschiana krammer 28.1 29 4 gomphonema parvulum (kützing) kützing 23.8 27 12 gomphonema tergestinum (grunow) fricke 23.8 27 4 diploneis fontanella lange-bertalot 22.5 32 12 encyonopsis krammeri reichardt 21.1 26 11 gomphonema exilissimum (grunow) lange-bertalot et e. reichardt 20.9 26 10 amphora lange-bertalotii var. tenuis levkov et metzeltin 20.4 22 3 navicula leistikowii lange-bertalot 20.2 24 7 encyonopsis fonticola (hustedt) krammer 19.3 21 4 cymbella parva (w.smith) kirchner 17.5 21 7 achnanthidium exile (kützing) heiberg 16.8 19 3 navicula cataracta-rheni lange-bertalot 15.4 15 0 tryblionella angustata w. smith 15.1 18 6 chlorogloea microcystoides geitler 14.6 17 5 pseudanabaena sp. lauterborn 13.6 14 1 nitzschia amphibia grunow 13.3 14 3 surirella terricola lange-bertalot et e. alles 12.6 14 5 eunotia soleirolii (kützing) rabenhorst 11.5 12 0 rhopalodia parallela (grunow) o. müller 10.7 12 1 encyonema silesiacum (bleisch) d.g.mann 10.1 12 2 diploneis elliptica (kützing) cleve 9.9 10 1 eucocconeis laevis (østrup) lange-bertalot 9.6 10 3 nitzschia capitellata hustedt 9.2 12 2 tryblionella angustatula (lange-bertalot) cantonati et lange-bertalot 9.0 9 0 hantzschia amphioxys (ehrenberg) grunow 8.3 9 1 gr ou p 2 ca rb on at es amphora pediculus (kützing) grunow ex a. schmidt 56.1 50 83 odontidium mesodon (ehrenberg) kützing 44.3 18 50 navicula antonii lange-bertalot 39.4 0 39 planothidium dubium (grunow) round et bukhtiyarova 39.4 0 39 gomphonema micropus kützing 34.7 29 52 gomphonema pumilum (geitler) bohunická et j. r. johansen 31.2 12 35 achnanthidium pyrenaicum (hustedt) h. kobayasi 29.1 21 38 navicula tripunctata (o. f. müller) bory de saint-vincent 26.1 3 29 psammothidium grischunum (wuthrich) l. bukhtiyarova et round 23.9 0 24 nitzschia oligotraphenta (lange-bertalot) lange-bertalot 22.5 4 24 achnanthidium affine (grunow) czarnecki 20.0 5 22 gomphonema angustatum (kützing) rabenhorst 18.3 0 18 nitzschia fonticola (grunow) grunow 18.3 0 18 navicula cryptotenelloides lange-bertalot 18.0 4 19 rhoicosphenia abbreviata (c.agardh) lange-bertalot 16.5 0 17 sellaphora pupula (kützing) mereschkovsky 16.5 0 17 vaucheria sp. a. p. de candolle 15.6 0 16 phormidium retzii kützing ex gomont 14.9 3 16 nitzschia palea var. tenuirostris grunow 11.0 0 11 fragilaria recapitellata h. lange-bertalot et d. metzeltin 10.1 0 10 algae in springs of different lithology acta bot. croat. 78 (1), 2019 77 from 16 springs and streams of the dolomiti bellunesi national park in northern italy (cantonati 2008), where a total of 65 algal taxa apart from diatoms, and 1–12 taxa per spring location were recorded. the exceptional heterogeneity of springs, resulting in richness of rare and endangered species already highlighted in several studies (aboal et al. 1998, werum and lange-bertalot 2004, cantonati et al. 2006) was confirmed for the area investigated. overall, one third of all taxa identified in the present study (36.3%) was noted in one spring only, which is slightly lower than 40% recorded by cantonati and spitale (2009) and clearly less than 58% noted by bertrand et al. (2004). all of the aforementioned studies refer to springs as unique habitats, each requiring a specific diversity estimation. hierarchical clustering based on relative algal abundance clustered springs into six groups. these results were in partial agreement with cantonati et al. (2012b, c), whose spring classification included 6 diatom-based and 7 groups based on algal taxa except diatoms. species typical for rheocrenes on carbonates, such as achnanthidium pyrenaicum and nitzschia fonticola (cantonati et al. 2012b), have been detected with high abundance in the spring mv. groups of rheocrenes on carbonates (3rd and 5th cluster) showed greatest similarity with rheocrenes on carbonate substrata with no3– enrichment or shading described by cantonati et al. (2012b). diatom species shared by alpine and dinaric carbonate springs were odontidium mesodon, achnanthidium pyrenaicum, amphora pediculus, meridion circulare, cocconeis lineata, c. pseudolineata, and sellaphora nigri. this congruence was expected, since the measured increased nitrate concentrations in these springs determined a higher number of eutraphentic species (e.g., amphora pediculus). the cyanoprokaryote tapinothrix varians and the red alga audouinella spp. showed affinity for shaded springs, as detected in some earlier studies (cantonati et al. 1996, cantonati et al. 2012c). however, certain differences were observed when the springs on ophiolites were compared with springs emerging from siliceous aquifers. in a comparison of algal assemblages, the highest resemblance was observed between rheocrenes on ophiolites and group 4 (rheocrenes on carbonate substrata with lower conductivity or seasonal desiccation) by cantonati et al. (2012b). species in common were planothidium frequentissimum, achnanthidium dolomiticum, meridion circulare and several indicators of desiccation, e.g. humidophila perpusilla and h. contenta. moreover, two new records for the diatom flora of bosnia and herzegovina were found in the investigated ophiolite springs: achnanthidium dolomiticum, a species previously recorded in springs on dolomite substrata (cantonati and lange-bertalot 2006), and cymbella tridentina, a rheophilic species of carbonate headwaters (cantonati et al. 2010). a common feature of dolomite and ophiolite springs is a relatively high magnesium concentration, presumably a key determinant for the distribution of a. dolomiticum. the largest group, rheohelocrenic springs on ophiolites, was compatible with hygropetric rheocrenes on carbonate substrata in cantonati et al. (2012b), with several species in common: denticula tenuis, encyonopsis cesatii, and e. microcephala. despite different lithology, springs on ophiolites and springs on carbonate substrata had a very similar algal composition. the greatest concurrence in algal communities recorded in this study with springs on siliceous substrata from previous studies was noted for the sole spring on ophiolitic melange (zl), with several species of gomphonema and eunotia in common. the influence of lithology on algal assemblages has been emphasized by several authors (cantonati 1998, werum 2001, cantonati et al. 2006, wojtal 2013), typically considering springs on different, carbonate and siliceous, lithologies. moreover, phycological studies focusing on springs on ophiolites (silicate rocks) were very rare. when algal assemblages of springs on peridotite and serpentinite (ophiolites) from this study were compared with springs on amphibolite in germany (werum 2001), a high similarity in species composition was noted. however, many species commonly occurring on ophiolites in this study, such as a. minutissimum, humidophila perpusilla, diploneis fontanella, nitzschia linearis and planothidium lanceolatum, were not good indicators of aquifer lithology because of their widespread distribution (werum 2001). on the contrary, species that had lower abundance and higher frequency, as detected by indval analysis, proved to be more reliable indicators of an aquifer’s lithology. the indval analysis singled out the following species as having a preference for ophiolites: amphora langebertalotii var. tenuis, cymbella hantzschiana, c. parva, encyonopsis cesatii, e. krammeri, gomphonema parvulum, g. tergestinum and navicula leistikowii. some of the species listed are typical for calcareous fens, such as e. cesatii (fránková et al. 2009), others are widespread such as g. parvulum (abarca et al. 2014). results of the indicator-species analysis for carbonate substrata were in accordance with previous studies (cantonati et al. 2012b, c), in which several rheophilic species (achnanthidium pyrenaicum, odontidium mesodon, gomphonema micropus, g. pumilum, planothidium dubium) or species with a preference for nitrogen enrichment (navicula tripunctata and amphora pediculus) were noted. a low resemblance has been found when our results on ophiolite-spring algal assemblages were compared with results from previous investigations in springs emerging from other types of siliceous substrata such as granite and rhyolite (werum 2001, kapetanović and hafner 2007). species typical for rheocrenes and helocrenes on siliceous substrata of the south-eastern alps (cantonati et al. 2012c) or detected in the pyrenees (sabater and roca 1992), were not found in the ophiolite springs studied. in the latter, genera typical for siliceous substrata, such as tabellaria, eunotia and pinnularia, were either missing or occurring only with a few species. nevertheless, a large number of shared species (110) suggests fewer differences in the structure of algal communities between carbonates and ophiolites (this study) than between ultramaphites (this study) and other types of siliceous substrata (e.g. granite, rhyolite; algal community composition data obtained from the literature). in addition, a species typical for carbonate substrata, tapinothrix varians (cantonati kamberović j., plenković-moraj a., kralj borojević k., gligora udovič m., žutinić p., hafner d., cantonati m. 78 acta bot. croat. 78 (1), 2019 et al. 1996), was found in all springs regardless of lithological substratum, and especially in springs with higher current velocity. on the other hand, tapinothrix janthina, the vicariant species typical for springs on silicates, was not found in the present study. our results suggest that algal assemblages in springs emerging from ophiolites and springs emerging from other types of siliceous substrata should be separately analyzed in future studies. the total number of species in springs can greatly depend on the lithology or the heterogeneity of microhabitats. nascimbene et al. (2011) recorded higher numbers of diatoms, plants and lichens from springs on siliceous substrata, whilst other algal groups (including cyanoprokaryotes) were more numerous on carbonate substrata. on the other side, frankovà et al. (2009) pointed out that the number of diatom species decreased significantly from mineral-rich to mineral-poor spring fen sites. although a higher number of taxa was noted in springs on carbonates than in those on ophiolites, differences between averages of diversity indices were not significant. the total number of diatom taxa found in the 20 springs of mt. konjuh (187) was higher than in 17 swiss springs (118 taxa; taxböck and preisig 2007), very similar to pyrenean springs (194 taxa; sabater and roca 1992), and lower than in 30 springs in south-eastern alps (250 taxa; cantonati 1998). although the average value of the shannon-wiener diversity index indicated a moderately high diversity of algal species (h` = 1.75), it was lower than the value obtained for alpine springs (h` = 2, cantonati 1998). several authors emphasized a relationship between lower altitudes and higher nitrate concentrations in springs (aboal et al. 1998, cantonati and spitale 2009), which can result in a decrease of diatom species (bertrand et al. 2004). a wide range in the total number of species per individual sample (from 5 to 47) and a high number of taxa per spring location (from 46 to 76) indicate the need to sample different microhabitats to achieve a more accurate biodiversity assessments of spring habitats. the algal assemblages in wetland habitats around springs and streams of the vranica mountain, bosnia and herzegovina (kapetanović and hafner 2007), especially those on siliceous substrata, differ from those of the springs studied on mt. konjuh by the preponderance of acidophilous taxa (brachysira brebissonii, eunotia incisa, tabellaria flocculosa, and many species from the genera neidium and eunotia). acidophilous species were rarely found on mt. konjuh, due to ultramafic and calcareous lithologies resulting in higher ph values. on the other hand, many eutraphentic species were noted (32.7%). this proportion was higher than that recorded by cantonati and spitale (2009) in the dolomiti bellunesi national park in italy (22%). altough natural diatom distribution could be altered by antrhopogenic influences, wojtal (2013) pointed out that springs with high nutrient concentrations also show heterogeneity of diatom assemblage composition indicating the great potential of springs for biodiversity assessment. angeli et al. (2010) pointed out that any morphological alteration of springs leads to great disturbance of their diatom communities, which was also the case in many springs studied herein. phormidium retzii, an indicator of nitrogen enrichment (cantonati et al. 2012c), was found in seven springs on mt. konjuh, indicating anthropogenic impact and a deteriorated ecological status. values of rott’s ti were high due to the dominance of highly competitive mesoand eutraphentic algal species, which is compatible with the results of some previous investigations of low-altitude springs in germany (werum 2001), and anthropogenically impacted springs in austria (gesierich and kofler 2010), thus emphasizing that an increase in nitrate leads to decreasing numbers of sensitive and rare species. the mean values of the rott ti (2.06 and 2.23 in carbonate and ophiolitic springs, respectively) belong in the same trophic class as a half of the investigated springs from the river adige basin, and are higher than rott’s ti from springs of trentino (angeli et al. 2010). adversely, the eqr and tidhr values indicated for one level lower trophic class than rott’s ti in the present investigation, presumably due to geographical adaptation of the species indicator values. however, the use of indices developed for rivers in the trophic-status assessments of springs could undergo certain limitations, in particular in the case of spring types markedly differing from streams. conclusion the study provides the first information on the biodiversity of algal communities in the mountain springs of north-eastern bosnia and herzegovina. although springs had long been neglected in hydrobiological investigations, recent studies emphasize the importance of these habitats in biodiversity conservation. despite the visible anthropogenic impacts in springs of mt. konjuh, a moderately high diversity of algal species per spring location was detected. results of this study revealed that springs on ophiolites should be analyzed separately from springs on other siliceous substrata in future studies dealing with community structure in different spring types. due to the high heterogeneity of spring habitats, in order to preserve spring species diversity of mt. konjuh the conservation of springs as a group of habitats is required, and not only the protection of individual spring sites. special focus should be directed towards preserving small rheohelocrenic springs, which are usually ignored in management and protection plans, even though they have a great potential in species conservation. acknowledgements we are grateful to elvir babajić for help in the identification of the lithology of substrata, to selvir kamberović for help in data processing, and the federal ministry of education and science in bosnia and herzegovina for providing funding for a research visit to the muse – museo delle scienze, trento, italy. algae in springs of different lithology acta bot. croat. 78 (1), 2019 79 references abarca, n., jahn, r., zimmermann, j., enke, n., 2014: does the cosmopolitan diatom gomphonema parvulum (kutzing) kutzing have a biogeography? 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(ed.), iconographia diatomologica 13. a. r. g. gantner verlag k. g., ruggell, 1–479. wojtal, z. a., 2013: species composition and distribution of diatom assemblages in spring waters from various geological formations in southern poland. bibliotheca diatomologica 59, 1–436. opce-str.vp acta bot. croat. 71 (1), 51–86, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 southeastern-alpine endemic leontodon hispidus subsp. brumatii (cichoriaceae) in the sava valley (central slovenia) igor dakskobler1*, andrej seli[kar2, branko vre[2 1 institute of biology, scientific research centre of the slovenian academy of sciences and arts, regional unit tolmin, brunov drevored 13, si-5220 tolmin, slovenia 2 institute of biology, scientific research centre of the slovenian academy of sciences and arts, novi trg 2, si-1000 ljubljana, slovenia abstract – in the spring and summer of 2010 a number of new localities of the southeastern-alpine endemic leontodon hispidus subsp. brumatii were found on temporarily flooded riparian rocks in the gorge of the sava river between the village of sava and zidani most (central slovenia). the species has so far been known only in northeastern italy and western slovenia (the so~a valley). in order to obtain more specific information its sites were studied phytosociologically and the communities in which it grows in the sava and the so~a valleys compared. two new associations were described on the basis of these comparisons: triseto argentei-leontodontetum brumatii ass. nov. and leontodonti brumatii-seslerietum calcariae ass. nov. as this endemic taxon and its endemic communities are a characteristic of riparian flora and vegetation of some slovenian mountain rivers and as its localities in the sava valley are explicitly disjunct and the southeasternmost in the entire known distribution area, they deserve to be studied and protected. key words: leontodon brumatii, phytogeography, synsystematics, sava, so~a, slovenia introduction leontodon hispidus l. subsp. brumatii (schiede ex reichenb) t. wraber [leontodon hispidus var. brumatii (schiede ex rchb.) fiori = l. brumatii schiede ex rchb.] is a southeastern-alpine endemic, known so far only in the foothills of the julian and carnic alps and along some rivers in the friuli lowland, western slovenia and northeastern italy (mayer 1952, 1958, 1960; wraber 1998, 2007; poldini 1991, 2002, 2009; jogan et al. 2001; ^u[in 2001, 2006; ^u[in and dakskobler 2001; dakskobler 2005). its localities in slovenia are on riparian rocks and river boulders in the nadi`a riverbed in the breginjski kot, on riparian rocks along the so~a river between bovec and solkan (relatively often acta bot. croat. 71 (1), 2012 51 * corresponding author, e-mail: igor.dakskobler@zrc-sazu.si copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:00 color profile: disabled composite 150 lpi at 45 degrees only along the middle so~a between podselo and plave), on similar sites along the u~ja river. reliable sites are only on the italian side of the former border crossing u~ja (wraber 1998), very likely also downstream in the slovenian territory, along the rivers idrija (rarely, golo brdo, under kostanjevica) and the idrijca (very rarely, a rocky eyot on slap ob idrijci, under the dam at the new, small hydroelectric power plant, 9848/4, leg. et det. i. dakskobler, 30. 5. 2010, herbarium ljs, relevé 1 in table 2). during our inventory of the flora in the sava valley, between the village of zidani most and sava, this taxon was found at several spots on riparian rocks on both sides of the river; first on 12 may 2010 on the right bank between trbovlje and hrastnik, downstream from the hamlet ribnik (dole`ak) – 9856/3 (leg. i. dakskobler et b. vre{); on the same day on the same bank of the sava downstream from hrastnik, in the village of podkraj, between the farmsteads rus and tohar (9856/4); two days later, on 14 may 2010, on the left bank of the sava near hrastnik – 9856/4 (leg. a. seli{kar). the article presents the localities and sites of this endemic taxon in the sava valley and the communities in which it grows and compares them with its sites and communities in the so~a valley. the issue of its conservation is discussed in view of the fact that its sites along the sava are in an area, designated for the construction of new hydroelectric power plants. materials and methods flora and vegetation along the sava and the so~a rivers (figs. 1, 2) were studied according to the established central-european methods (braun-blanquet 1964, ehrendorfer and hamann 1965). floristic records and phytosociological relevés were entered into the flovegsi database (seli[kar et al. 2003). the same application was used to make the distribution map (fig. 2). when processing the relevés we transformed the combined cover-abundance values with numerical values (1–9) according to van der maarel (1979). numerical comparisons were performed with the syn-tax 2000 program package (podani 2001). the relevés were compared by means of »(unweighted) average linkage method« – upgma and principal coordinates analysis (pcoa). wishart’s similarity ratio was used in all the methods. the nomenclature source for the names of vascular plants is the mala flora slovenije (martin^i^ et al. 2007), martin^i^ (2003) for the names of mosses and theurillat (2004) for the names of the syntaxa. ecological description of the study area the study area with leontodon hispidus subsp. brumatii belongs to the pre-alpine phytogeographical region (m. wraber 1969). in terms of landscape regions it forms a part of the sava valley or, more widely speaking, of the posavsko hribovje region (perko and oro@en adami^ 1998). from litija towards zidani most the sava initially still runs through a relatively wide valley which narrows at the village of sava and remains narrow with steep banks throughout its course. on the right bank of the river, up to hrastnik, runs a regional road, and on the left bank the double track ljubljana–zagreb railway. the prevailing geological bedrock consists of triassic dolomite and limestone, in some sections (in the vicinity of litija, podkraj pri hrastniku) also permian-carbon slate claystones and sandstones (buser 1990). the climate is moderate continental. mean annual air temperature in 52 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:00 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 53 southeastern-alpine endemic leontodon hispidus... fig. 1. distribution of the taxon leontodon hispidus subsp. brumatii in slovenia with new localities in the sava valley. fig. 2. localities of the taxon leontodon hispidus subsp. brumatii in the sava valley (central slovenia). 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:01 color profile: disabled composite 150 lpi at 45 degrees the period 1961–1990 was between 8 °c and 10 °c, mean january temperature –2 °c to 0 °c and mean july temperature 18 °c to 20 °c (cegnar 1998). mean annual precipitation in the same period was between 1200 mm and 1400 mm (zupan^i^ 1998). the sava river is torrential in this section and its level rapidly increases after heavy rain; rocky ledges along the river are partly or entirely flooded at least ten times a year (fig. 3). normally, there is a short period in may when the water level is low, and a longer period favourable for the development of the vegetation in the summer months from july to september (hydrological data archive 2008). dolomite slopes above the sava are overgrown with basophilic beech forests (ostryo-fagetum, arunco-fagetum, hacquetio-fagetum), and in areas where clay slates are the dominant bedrock also with acidophilous beech forests (blechno-fagetum). on the colluvium (hillside scree) in the gorges and at the foot of slopes there are some sites of valuable broad-leaved species (hacquetio-fraxinetum, veratro nigri-fraxinetum, tilio cordatae-aceretum platanoidis ostryetosum). the steepest, rockiest sites at the right bank of the sava (reber between the village of sava and mo{enik; reber between zagorje and trbovlje) are overgrown with basophilic forests of scots pine and black pine (genisto januensis-pinetum sylvestris). on similarly steep, rocky sites on both banks of the river there are also sites of the community of pubescent oak and hop hornbeam (querco pubescenti-ostryetum carpinifoliae). on smaller areas on rare gravel sites grows a community of grey and red willow (salicetum eleagno-purpureae). the banks of the sava between litija and zidani most have been changed considerably, often reinforced with dry stone walls. towards zidani most the river course becomes noticeably more placid and the banks flooded, due to the dam of the bo{tanj hydroelectric power plant downstream. the best preserved natural slopes with rocky ledges are in the gorge between hrastnik and trbovlje and it is there that most of localities of leontodon hispidus subsp. brumatii were found. results overview of the new localities of leontodon hispidus subsp. brumatii in the sava valley 9856/3 (utm 33twm00) slovenia, dolenjska, the sava valley, the right bank of the sava downstream from the hamlet of ribnik (dole`ak), riparian rocks and pioneer grey willow stands (salicetum eleagno-purpureae), 200 m a.s.l., leg. et det. i. dakskobler et b. vre{, 12. may 2010, herbarium ljs; the right bank of the sava, upstream from the hamlet of ribnik (dole`ak) and the outfall of the ribnik stream into the sava, 205 m a.s.l. det. b. vre{, 8. june 2010. 9856/3 (utm 33t wm00) slovenia: [tajerska, trbovlje, the left bank of the sava, a rocky ledge at the trbovlje thermal power plant; also to the east and southeast of this thermal power plant, under ringa railway tunnel and further on to the foot of the hill vi{tov vrh (opposite ribnik or dole`ak), 200 m a.s.l., det. a. seli{kar, 19. may 2010. 9856/3 (utm 33t wm00) slovenia: [tajerska, the sava valley, trbovlje, hamlet za savo, on several spots on the banks of the sava under the farmsteads vrstov{ek, frajle, frankovi~ and kos, 200 m a.s.l. det. b. vre{, 27. july 2010. 9856/3 (utm 33t wm00) slovenia: dolenjska, the sava valley, mitov{ek, ribogojnica (fish hatchery), riparian rocks on the right bank of the sava, 205 m a.s.l. det. b. 54 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:01 color profile: disabled composite 150 lpi at 45 degrees vre{, 26. july 2010; spodnji [klendrovec, under the road prusnik – mitov{ek, 210 m a.s.l.; det. b. vre{, 15. september 2010. 9856/4 (utm 33t wm00) slovenia: dolenjska, the right bank of the sava downstream from hrastnik, podkraj, at the homesteads rus and tohar and several spots between them (under the ravenski hrib hill), riparian rocks, 200 m a.s.l., leg. et det. i. dakskobler et b. vre{, 12. may 2010, i. dakskobler et b. ^u{in, 17. june 2010, b. vre{, 26. july 2010 and 7. october 2010, herbarium ljs. 9856/4 (utm 33t wm00) slovenia: [tajerska, hrastnik, the left bank of the sava near the hamlet of za savo, to the east of the bridge across the river, riparian rocks, 200 m a.s.l., leg. et det. a. seli{kar, 14. may 2010 and 20. july 2010, herbarium ljs. 9856/4 (utm 33t wm00) slovenia: [tajerska, the sava valley, hrastnik, the foot of the vi{tov vrh hill, riparian rocks and ledges on the left bank of the sava (under the railway track) between trbovlje and hrastnik, at several spots, 200 m a.s.l., leg. et det. a. seli{kar et i. dakskobler, 18. may 2010, herbarium ljs. 9856/4 (utm 33t wm00) slovenia: dolenjska, the right bank of the sava to the west of hrastnik, under the filling station along the main road zagorje–hrastnik, 200 m a.s.l. det. i. dakskobler et a. seli{kar, 18. may 2010. 9856/4 (utm 33twm10) slovenia: [tajerska, the left bank of the sava, below the village of suhadol, flood bank along the sava, 195 m a.s.l. det. a. seli{kar, 20. july 2010. 9955/2 (utm 33tvm90) slovenia: dolenjska, the sava valley, renke, the right bank of the sava downstream from [u{tarski most, a bridge across the sava, 220 m a.s.l., riparian rocks., leg. et det. i. dakskobler et b. ^u{in, 17. may 2010, herbarium ljs. 9955/2 (utm 33tvm90) slovenia: [tajerska, the sava valley, mo{enik, the left bank of the sava, to the west of the [u{tarski most (a footbridge), rocks along the sava, at the outfall of a tufa-forming stream, 220 m a.s.l. det. a. seli{kar and b. vre{, 12. october 2010; mo{enik, the left bank of the sava, to the west of the bridge[u{tarski most, riparian rocks below the abandoned mars homestead, 220 m a.s.l. det. a. seli{kar et b. vre{, 12. october 2010. leontodon brumatii was recorded on the banks of the sava between litija and zidani most at the altitude of 195 to 220 m, at 38 microlocalities in four sections: between the villages of sava and zagorje (the westernmost localities are at renke on the right and at mo{enik at the left bank of the river), between zagorje and trbovlje (at mitov{ek on the right and under the hamlet of za savo on the left bank of the river), between trbovlje and hrastnik (at ribnik and the hrastnik filling station on the right bank and at the foot of vi{tov vrh on the left bank of the river, where it grows abundantly on riparian rocks) and between hrastnik and zidani most or rade~e (the easternmost locality is under the village suhadol on the left bank and at the homestead tohar in podkraj on the right bank) and in three quadrants – 9955/2, 9856/3 and 9856/4 (figs. 1, 2). phytosociological description of the sites of l. brumatii in the sava valley and in the so~a valley conspectus of the established syntaxa: asplenietea trichomanis br.-bl. in meier et br.-bl. 1934 corr. oberdorfer 1977 acta bot. croat. 71 (1), 2012 55 southeastern-alpine endemic leontodon hispidus... 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:01 color profile: disabled composite 150 lpi at 45 degrees potentilletalia caulescentis br.-bl. in br.-bl. et jenny 1926 cystopteridion fragilis richard 1972 triseto argentei-leontodontetum brumatii ass. nov. hoc loco var. typica var. nov. var. chamaecytisus purpureus var. nov. var. deschampsisa cespitosa var. nov. festuco-brometea br.-bl. et tüxen 1943 scorzonero-chrysopogonetalia horvati} et horvat in horvati} 1958 = scorzoneretalia villosae horvati} 1975 saturejion subspicatae horvati} 1975 centaurenion dichroanthae (pignatti 1953) poldini et feoli chiapella in feoli chiapella et poldini 1993 leontodonti brumatii-seslerietum calcariae ass. nov. hoc loco chamaecytisetum purpureae subass. nov. hoc loco saxifragetum crustatae subass. nov. hoc loco salicetea purpureae moor 1958 salicetalia purpureae moor 1958 salicion eleagno-daphnoidis (moor 1958) grass 1993 salicetum eleagno-purpureae sillinger 1933 petasitetosum hybridi ([ilc et ^u{in 2000) oriolo et poldini 2002 var. populus nigra dakskobler 2010 leontodon hispidus subsp. brumatii is an endemic with a small distribution area. there are very few data on the communities in which it grows. it is known to grow on rocks (poldini 1991). the subspecies is not discussed on its own but together with leontodon hispidus in the flora alpina (aeschimann et al. 2004). wraber (1998) claims that it usually grows in a very characteristic environment, on riparian rocks that are periodically washed by river water. he stresses that the plant can also grow outside the direct influence of the water splashing, which was confirmed in our research in the so~a valley, where the studied taxon was recorded also on rocks and steep gravelly slopes that the river cannot reach even when the water level is at its highest. the specimens growing the highest above the sava river level were found in a crevice of the retaining wall under the railway track, some 3.5 m above the low spring water level, and on a stony road slope approximately 5 m above the low spring water level. however, even these specimens are flooded when the water level is at its highest. the rivers so~a and idrijca are torrential and have a similar water regime to that characteristic for the sava river (fig. 3). interesting is the frequent occurrence of the subspecies l. brumatii under the doblar hydroelectric power plant where the fluctuation of the water level and in turn inundation of the subspecies’ sites occurs on a daily basis. so far, no comprehensive phytosociological inventory of all its known localities in the so~a valley has been made, but we have made 15 relevés of this species at different locations over a longer time period. these relevés were compared to those from the sava valley and thus the following results were obtained (figs. 4, 5). in all of the comparisons the relevés from the sava valley (zs) were grouped separately from the relevés from the so~a valley (po). two phytosociological tables were made for both regions (tabs. 1, 2). 15 relevés made in the sava valley on the both banks of sava be56 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:01 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 57 southeastern-alpine endemic leontodon hispidus... fig. 3. water level (in cm) of the rivers sava, so~a and idrijca in 2008 (according to hydrological data archive 2008). the red line marks the threshold of flooding of the sites of leontodon hispidus subsp. brumatii. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:01 color profile: disabled composite 150 lpi at 45 degrees 58 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. fig. 4. dendrogram of communities with leontodon hispidus subsp. brumatii from the so~a valley (po) and from the sava valley (za) – upgma, similarity ratio. fig. 5. two-dimensional scatter-diagram of communities with leontodon hispidus subsp. brumatii from the so~a valley (po) and from the sava valley (za) – pcoa, similarity ratio. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:02 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 59 southeastern-alpine endemic leontodon hispidus... tab. 1. communities with leontodon hispidus subsp. brumatii in the sava valley (central slovenia). fl – fluvisols, li – lithosols. number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pr. fr. database number of relevé 23 68 27 23 74 03 23 57 95 23 53 37 23 58 27 23 58 28 23 73 87 23 54 01 23 54 48 23 54 49 23 58 30 23 54 06 23 71 60 23 52 13 23 58 29 altitude in m 200 220 180 200 200 200 220 200 200 200 200 200 200 200 200 aspect n s n s s s 0 0 sw sw 0 n 0 n 0 slope in degrees 2 70 35 2 2 5 0 0 3 3 0 10 0 2 0 parent material da da da da da da da da da da da da da alu alu soil fl li li li li li li li li li li li li fl fl stoniness in % 30 100 80 90 100 100 100 100 80 80 100 100 70 30 30 cover in %: tree layer e3 . . . . . . . . . . . . . . 20 shrub layer e2 70 5 . 20 20 20 10 30 20 5 10 10 20 70 80 herb layer e1 50 40 30 60 60 30 40 30 60 40 15 40 40 50 70 moss layer e0 10 30 20 10 10 10 20 10 30 20 50 30 10 10 10 relevé area (m2) 100 10 20 15 15 9 50 50 50 50 50 20 50 100 200 number of species 30 13 13 11 16 20 16 32 25 20 18 24 12 57 44 date of taking relevé 6. 9. 20 10 12 .1 0. 20 10 17 .6 .2 01 0 18 .5 .2 01 0 18 .5 .2 01 0 18 .5 .2 01 0 12 .1 0. 20 10 18 .5 .2 01 0 19 .5 .2 01 0 19 .5 .2 01 0 18 .5 .2 01 0 18 .5 .2 01 0 20 .7 .2 01 0 12 .5 .2 01 0 18 .5 .2 01 0 locality r ib ni k r en ke h ra st ni k h ra st ni k h ra st ni k h ra st ni k m oš en ik h ra st ni k t rb ov lj e t rb ov lj e h ra st ni k h ra st ni k h ra st ni k r ib ni k (d ol e` ak ) h ra st ni k quadrant 98 56 /3 99 55 /2 98 56 /4 98 56 /4 98 56 /4 98 56 /3 99 55 /2 98 56 /3 98 56 /3 98 56 /3 98 56 /3 98 56 /4 98 56 /4 98 56 /3 98 56 /3 character and differential species of the syntaxa at leontodon hispidus subsp. brumatii e1 + 2 2 2 2 2 2 2 3 2 2 2 2 1 + 15 100 m brachythecium rutabulum e0 . 3 1 1 1 + 2 + 2 1 1 3 1 + 1 14 93 m cinclidotus fontinaloides e0 . . . . + 1 . 1 3 2 3 1 . + . 8 53 tr trisetum argenteum e1 + . 1 . . + . . + + . 1 . . . 6 40 ep chamaecytisus purpureus e1 . . . 2 2 1 . . . . . . . . . 3 20 fb peucedanum oreoselinum e1 . . + 1 1 . . . . . . . . . + 4 27 es sesleria caerulea subsp. calcaria e1 . + . . 2 + + . . . . . . . + 5 33 fb inula ensifolia e1 . . . . . + . . . . . . . . . 1 7 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:02 color profile: disabled composite 150 lpi at 45 degrees 60 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pr. fr. ma deschampsia cespitosa e1 . . + . . 1 1 1 3 2 2 2 3 1 1 11 73 pm phalaris arundinacea e1 . . . . . + + 1 + + + + + 1 + 10 67 sp populus nigra e3 . . . . . . . . . . . . . . 2 1 7 sp populus nigra e2b . . . 1 1 1 + 2 2 + + + 2 3 2 12 80 sp populus nigra e2a . . . . . . . + . . 1 . . 1 1 4 27 sp salix alba e2 . . . . + . . . + + + . . r + 6 40 sp salix eleagnos e2b . . . . . + . . . . . 1 . + 1 4 27 sp salix eleagnos e2a . . . . . . . + . . . . . + + 3 20 sp salix purpurea e2b . . . . . + . 1 . + . . . 3 . 4 27 sp salix purpurea e2a . + . . . . . . + . + . . 1 . 4 27 sp salix purpurea e1 . . . . . . . . . . . . 2 . . 1 7 gu petasites hybridus e1 . . . . . . . . . . . . . + . 1 7 fb festuco-brometea genista tinctoria e1 . 2 . . . . . . . . . . . . + 2 13 pa potentillion anserineae rorippa sylvestris e1 . . 1 . . . . + + . + . 1 + + 7 47 barbarea vulgaris e1 . . . . . . . + . + + . . + + 5 33 pp potentillo-polygonetalia agrostis stolonifera e1 . . . . . . 1 + + + . . + + . 6 40 plantago major e1 . . . . . . . + + . + . . r + 5 33 rumex crispus e1 . . + . . . . . . . . . . + . 2 13 ma molinio-arrhenatheretea taraxacum officinale e1 . + . . . + . . + . . + . + + 6 40 trifolium pratense e1 . . . . . + . + + + . . . + + 6 40 plantago lanceolata e1 . . . + + . . + . . . + . + . 5 33 achillea millefolium e1 . . . . . . . + . + . . . r . 3 20 centaurea carniolica e1 + . . . . . . . . . . . . + + 3 20 dactylis glomerata e1 . . . . . . . + . . . + . . + 3 20 galium mollugo e1 . + . . . . . . . . . . . + + 3 20 ranunculus acris e1 . . . . . . . . . . . + . + + 3 20 veronica chamaedrys e1 . . . . . . . . + . . . . + + 3 20 pastinaca sativa e1 + . . . . . . . . . . . . r . 2 13 lotus corniculatus e1 . . . . . + . . + . . . . . . 2 13 av artemisietea vulgaris artemisia vulgaris e1 + . . + + . . + + . . . . + + 7 47 erigeron annuus e1 + . . . . . . + . . + + + + + 7 47 rumex obtusifolius e1 . . . . . . + + + + . + . + + 7 47 euphorbia esula e1 . . . . . . . . + . . + . . + 3 20 picris hieracioides e1 + . . . . . . . . . . . . . + 2 13 tab. 1. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:02 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 61 southeastern-alpine endemic leontodon hispidus... number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pr. fr. pm phragmiti-magnocaricetea mentha aquatica e1 . . + . . . . . . + . . . . . 2 13 sp salicetea purpureae salix triandra e2b . . . . . + . . . . . . . . 1 2 13 ai alnion incanae rubus caesius e2a . + + 1 1 1 1 1 + . . + . 1 1 11 73 ulmus laevis e2b . . . + + + . + . + . . . + 1 7 47 ulmus laevis e2a . . . . . . . + . . + + . + . 4 27 chaerophyllum hirsutum e1 . . . . . . . . . . . . . + + 2 13 ag alnus glutinosa e2 . + . . . . 1 . . . . . . . . 2 13 fs fagetalia sylvaticae fraxinus excelsior e2a . . . . . . . + . . . . . + . 2 13 scrophularia nodosa e1 . . . . . . . . . . . + . + . 2 13 brachypodium sylvaticum e1 . . . . . . . . . . . . . + + 2 13 qf querco-fagetea clematis vitalba e1 . . . . . . . + . . . . . . 1 2 13 rp rhamno-prunetea crataegus monogyna e2b . . . . . . . . . + . . . . . 1 7 crataegus monogyna e2a . . . . . . + + . . + . . . . 3 20 cornus sanguinea e2a . . . . . . . . . . . . . + + 2 13 ar agropyretea intermedii-repentis poa compressa e1 . . . . . . . + . . . + . . . 2 13 bt bidentetea tripartitae rorippa palustris e1 + . . . . . . . . . 1 . . . . 2 13 fc filipendulo-convolvuletea helianthus tuberosus e1 + . . . . . . + . . + . . + 1 5 33 saponaria officinalis e1 . . + . . . . + . . . . . + + 4 27 echinocystis lobata e1 + . . . . . . . . . . . . + . 2 13 mentha longifolia e1 . . . . . . . . . . . . . + + 2 13 gu galio-urticetea solidago gigantea e1 + + . + + + + + + + + + . 1 1 13 87 aegopodium podagraria e1 . . . . . . . . . . . . . + 1 2 13 urtica dioica e1 . + . . . . . . . . . . . . + 2 13 o other species fallopia japonica e1 + . . . . . + . . . . . . 1 1 4 27 poa sp. e1 . . + . . . . . . . . . . . + 2 13 euphorbia nutans e1 . . . . . . . . . . . + . . + 2 13 malus domestica e2a . . . . . . . . . . . . . + + 2 13 m mosses tortella sp. e0 . . . 1 1 1 . 1 + + 1 + . . . 8 53 tab. 1. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:02 color profile: disabled composite 150 lpi at 45 degrees tween mo{enik and hrastnik were grouped together (tab. 1). in this area, l. brumatii mainly overgrows riparian rocks, rarely also coarse, riparian gravel, both being at least periodically flooded (figs. 6, 7). phanerograms and some moss species grow only rarely on these rocks. in addition to l. brumatii the most abundant grass among the phanerograms is deschampsia cespitosa. other species, which are mostly the character species of cultivated meadows and nitrophilic ruderal communities, have only scattered and individual occurrences. only three of the relevés in table 1 (relevés 4 to 6) slightly resemble those in the so~a valley (tab. 2), which is mainly due to the occurrence of chamaecytisus purpureus, peucedanum oreoselinum and inula ensifolia. even the subspecies sesleria caerulea subsp. calcaria, which usually dominates in the relevés in the so~a valley (in addition to l. brumatii), is relatively rare on the riparian rocks in the sava valley and is more abundantly represented in only one relevé. most stands with l. brumatii in the sava valley are described as a new association triseto argentei-leontodontetum brumatii ass. nov. in the floristic composition as a whole there are several species which indicate a partial similarity of the studied community with the communities with dominant phalaris arundinacea that overgrow periodically flooded sites immediately along the mid-course of rivers subject to considerable fluctuation of water level and that are classified into the alliance phalaridion arundinaceae kopecký 1961 and the association rorippo-phalaridetum kopecký 1961 (balátová-tulá^ková et al. 1993). so far, this association in slovenia has been documented with only three relevés (petrinec 1999). in addition to its relevés the synoptic table (tab. 3) includes also the relevés of the same association from the border region between slovakia and hungary (hrivinák and ujházy 2003). the synoptic table and numerical comparison of the three syntaxa (figs. 8, 9) clearly demonstrate that our stands cannot be classified into the association rorippo-phalaridetum. they comprise only a few of the species diagnostic for the class phragmiti-magnocaricetea klika in klika et novák 1941 (mentha aquatica, lycopus europaeus and phalaris arundinacea) and only p. arundinacea 62 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. appendix to tab. 1. sporadic species: festuco-brometea: petrorhagia saxifraga + (9), medicago lupulina + (14), potentillo-polygonetalia: ranunculus repens + (12), potentilla reptans + (14), molinio-arrhenatheretea: leontodon hispidus + (1), leucanthemum ircutianum + (9), cerastium holosteoides + (12), crepis biennis + (14), leucanthemum vulgare + (14), rumex acetosa + (14), vicia cracca + (14), vicia sepium + (15), artemisietea vulgaris: cichorium intybus + (1), tanacetum vulgare + (8), phragmiti-magnocaricetea: lycopus europaeus + (8), salicetea purpureae: salix fragilis /e2b/ + (1), salix fragilis /e2a/ + (2), humulus lupulus + (1), alnion incanae: ulmus laevis /e1/ + (9), peucedanum verticillare + (1), cardamine impatiens + (14), knautia drymeia subsp. intermedia + (14), quercetalia pubescentis: campanula rapunculoides + (3), fraxinus ornus + (5), ostrya carpinifolia + (14), querco-fagetea: hieracium sabaudum + (1), carex digitata + (2), ficaria verna subsp. bulbifera + (14), rhamno-prunetea: crataegus monogyna /e2b/+ (10), rhamnus catharticus + (5), rosa canina agg. + (14), agropyretea intermedii-repentis: equisetum arvense (14), bidentetea tripartitae: polygonum lapathifolium + (1), polygonum mite + (1), filipendulo-convolvuletea: calystegia sepium + (8), lythrum salicaria + (13), stellarietea mediae: amaranthus retroflexus + (1), chenopodium album + (1), digitaria sanguinalis + (1), echinochloa crus-galli + (1), polygonum persicaria + (1), setaria pumila + (1), solanum nigrum + (1), poa annua + (10), setaria viridis + (13), cerastium glomeratum r (14), other species: acer negundo + (1), ambrosia artemisiifolia + (1), brassica sp. + (1), agrostis sp. + (3), ribes rubrum + (14), physocarpus opulifolius + (7), mosses: musci div. + (2). 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:02 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 63 southeastern-alpine endemic leontodon hispidus... tab. 2. communities with leontodon hispidus subsp. brumatii in the so~a valley (western slovenia). li – lithosols number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pr. fr. database number of relevé 23 54 65 23 76 27 23 76 28 21 85 15 21 85 17 21 85 16 23 76 29 23 76 30 23 76 31 23 76 32 23 76 33 23 76 37 23 76 38 23 76 39 23 76 35 altitude in m 176 200 200 83 84 83 120 120 120 120 120 230 220 220 160 aspect 0 s e s e s w s w w n e n n w s w s w n e n n e n n e n n slope in degrees 0 90 90 35 40 40 10 20 10 10 70 45 40 40 30 parent material a ar ar al al al a a a a a ar ar ar ar soil li li li li li li li li li li li li li li li stoniness in % 100 100 100 100 100 100 90 100 90 60 100 30 30 30 90 cover in %: tree layer e3 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 shrub layer e2 10 10 5 0 0 10 30 5 10 5 10 20 30 40 5 herb layer e1 20 25 30 25 30 30 30 20 40 40 40 70 70 80 30 moss layer e0 0 5 5 5 10 30 5 5 5 5 10 20 10 10 15 relevé area (m2) 50 50 50 10 10 10 10 10 50 10 15 100 100 100 15 number of species 14 29 18 20 13 18 25 22 25 22 36 37 37 33 17 date of taking relevé 30 .5 .2 01 0 8. 5. 19 92 8. 5. 19 92 28 .4 .2 00 8 28 .4 .2 00 8 28 .4 .2 00 8 4. 10 .2 00 2 24 .7 .2 00 2 24 .7 .2 00 2 24 .7 .2 00 2 24 .7 .2 00 2 19 .6 .2 00 1 17 .5 .1 99 9 17 .5 .1 99 9 19 .6 .2 00 1 locality s la p ob id ri jc i l o{ ki po ld an l o{ ki po ld an a nh ov o a nh ov o a nh ov o v og r{ ~e ks po pd nj i l og v og r{ ~e ks po pd nj i l og v og r{ ~e ks po pd nj i l og v og r{ ~e ks po pd nj i l og v og r{ ~e ks po pd nj i l og b u~ en ic a b u~ en ic a b u~ en ic a b u~ en ic a quadrant 98 48 /4 98 48 /3 98 48 /3 99 47 /2 99 47 /2 99 47 /2 98 48 /3 98 48 /3 98 48 /3 98 48 /3 98 48 /3 98 48 /2 98 48 /2 98 48 /2 98 48 /2 character and differential species of the syntaxa es sesleria caerulea subsp. calcaria e1 1 1 + 2 2 2 2 2 2 2 2 3 3 3 + 15 100 at leontodon hispidus subsp. brumatii e1 1 + + 2 2 1 1 1 + r + + + r + 15 100 ep calamagrostis varia e1 . . . . . + + + + + 1 + + + 1 10 67 at hieracium porrifolium e1 . . . + . . 1 + + + + + + + . 9 60 es aster bellidiastrum e1 . . . . . . + + + . 1 + + + 1 8 53 at athamanta turbith e1 . 1 2 . . + + . + . + . + + . 8 53 at campanula carnica e1 . 1 + . . . . . . . . . . . . 2 13 at campanula pyramidalis e1 . + 1 . . . . . . . . . . . . 2 13 at sedum album e1 . + + . . . . . . . . . . . . 2 13 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:02 color profile: disabled composite 150 lpi at 45 degrees 64 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pr. fr. ep chamaecytisus purpureus e1 . . . + 1 + 1 2 2 2 2 . . . . 8 53 fb carex humilis e1 . . . + + + . + 1 2 1 . . . . 7 47 fb centaurea scabiosa subsp. fritschii e1 . . . + . 1 1 + + + + . . . . 7 47 ep allium ericetorum e1 . . . + . . 1 1 2 2 1 . . . . 6 40 fb inula ensifolia e1 . . . . . . + 1 1 1 . . . . . 4 27 ep erica carnea e1 . . . . . . + . . . . 2 2 1 + 5 33 at saxifraga crustata e1 . . . . . . . . . . . 2 2 2 r 4 27 mo laserpitium prutenicum e1 . . . . . . . . . . . 1 1 1 + 4 27 tr petasites paradoxus e1 . . . . . . . . . . . 1 1 1 1 4 27 tr campanula cespitosa e1 . . . . . . . . . . . 1 1 1 . 3 20 es rhinanthus aristatus e1 . . . . . . . . . . . + 1 + . 3 20 fb stachys recta e1 . . . . . . . . . . . + + + . 3 20 at asplenietea trichomanis asplenium rutamuraria e1 . + + . + . . . . . . . . . . 3 20 phyteuma scheuchzeri subsp. columnae e1 . . . . . . + + . . + . . . . 3 20 es elyno-seslerietea globularia cordifolia e1 . . . + . . . . + . . . + . . 3 20 fb festuco-brometea genista tinctoria e1 . . + + + + + . . . . 1 + + . 8 53 euphorbia cyparissias e1 + . . . 1 . + . + . + . . . . 5 33 satureja montana subsp. variegata e1 . 1 1 . . . . . + + . . . + . 5 33 peucedanum oreoselinum e1 . . . . . . 1 + + + . . . . + 5 33 buphthalmum salicifolium e1 . . + . . . . . . . . 1 + 1 . 4 27 bromopsis erecta e1 . + . + . . . . . + . . . . . 3 20 koeleria pyramidata e1 + . . . . . . . . + . . . . . 2 13 inula hirta e1 . . . . . . + . . . r . . . . 2 13 ononis spinosa e1 . . . . . . . + . + . . . . . 2 13 medicago falcata e1 . . . . . . . . + + . . . . . 2 13 gymnadenia conopsea e1 . . . . . . . . . . r . + . . 2 13 mo molinion caeruleae scf parnassia palustris e1 . . . . . . . . . . . + + . . 2 13 ma molinio-arrhenatheretea taraxacum officinale e1 . . . r . + . r . . . . . . . 3 20 lotus corniculatus e1 1 . . . . . . + . . . . . . . 2 13 tab. 2. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:02 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 65 southeastern-alpine endemic leontodon hispidus... number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pr. fr. tg trifolio-geranietea peucedanum cervaria e1 . + . . . + 1 1 . 1 + . . . . 6 40 tr thlaspietea rotundifolii achnatherum calamagrostis e1 . 1 1 . . . . . . . . + . . . 3 20 peucedanum verticillare e1 . . . . . . . . . . r + . . . 2 13 biscutella laevigata e1 . . . . . . . . . . . + 1 . . 2 13 ep erico-pinetea aster amellus e1 . + 1 . . . + . + + 1 . + + . 8 53 leontodon incanus e1 . + + . . . + 1 1 + 1 . . . . 7 47 carex ornithopoda e1 . . . . . + . . . . . + + + . 4 27 epipactis atrorubens e1 . . . . . . . . . . . 1 + . + 3 20 pinus nigra e2b . . . . . . . . . . . 1 r r . 3 20 pinus nigra e2a . . . . . . . . . . . r + . . 2 13 polygala chamaebuxus e1 . . . . . . . . . . . 1 1 1 . 3 20 chamaecytisus hirsutus e2a . . . + . + . . . . . . . . . 2 13 mua mulgedio-aconitetea salix appendiculata e2a . . . . . . + . . . + + + + + 6 40 qp quercetalia pubescentis fraxinus ornus e2b . . . . . . + . . . . . + 1 . 3 20 fraxinus ornus e2a . + + . . + . + 1 + 1 1 1 1 . 10 67 fraxinus ornus e1 . . . . . . . . . . . 1 1 1 + 4 27 ostrya carpinifolia e2b . . . . . . . . . . . 1 1 2 . 3 20 ostrya carpinifolia e2a . + . . . . . . + . . + 1 1 . 5 33 lembotropis nigricans e1 . . . . . . . . . . + + + . . 3 20 carex flacca e1 . . . . . . . . . . . 1 1 1 . 3 20 campanula rapunculoides e1 . . . . r . . . . . + . . . . 2 13 clematis recta e1 . . . . . . . . . . + . + . . 2 13 fs fagetalia sylvaticae mycelis muralis e1 . + + . . . . . . . . . . . . 2 13 anemone trifolia e1 . . . . . . . . . . + . . . . 1 7 lathyrus vernus e1 . . . . . . . . . . + . . . . 1 7 ranunculus lanuginosus e1 . . . . . . . . . . r . . . . 1 7 tilia cordata e2a + . . . . . . . . . r . . . . 2 13 qf querco-fagetea clematis vitalba e2a . + + + + + . . + + . . . . . 7 47 carex digitata e1 . . . . . . . . . . . . + 1 . 2 13 tab. 2. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:03 color profile: disabled composite 150 lpi at 45 degrees occurs at a higher frequency, but in modest abundance (mainly +). the decisive factor for the synsystematic classification of the studied stands is therefore the dominant l. brumatii, which is characteristic of wet rocks. based on the available material the most sensible classification of the new association is therefore into the alliance cystopteridion fragilis, order potentilletalia caulescentis and class asplenietea trichomanis, even though no other diagnostic species of the alliance, order and class have been recorded in these stands. 66 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. number of relevé 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pr. fr. sp salicetea purpureae populus nigra e2a + . . . + + . . . . . . . . . 3 20 salix eleagnos e2 1 . . . . . . . . . . . + + . 3 20 salix purpurea e2a + . . . . + . . . . . . . . + 3 20 av artemisietea vulgaris erigeron annuus e1 + . . r . . . . . . . . . . . 2 13 o other species festuca sp. e1 . . + . . . . . + . . . . . . 2 13 m mosses schistidium apocarpum e0 . 1 . . . . 1 + + 1 + . . . . 6 40 tortella sp. e0 . + . + 1 . . . . . . 1 1 1 . 6 40 tortella tortuosa e0 . . . . . . 1 1 1 1 + . . . 1 6 40 cinclidotus fontinaloides e0 . . . + 1 2 . . . . . . . . . 3 20 ctenidium molluscum e0 . . . . . . . . . . . 1 1 + . 3 20 fissidens dubius e0 . . . . . . . . . . . + + + . 3 20 collema sp. e0 . + + . . . . . . . . . . . . 2 13 dicranum sp. e0 . . . . . . . . . . . + + . . 2 13 musci div. e0 . . . . . . . . . . . + . . 2 2 13 appendix to tab. 2. sporadic species: asplenietea trichomanis: hieracium austriacum + (1), calamintha einseleana + (2), sedum maximum + (2), asplenium trichomanes + (2), ceterach javorkeanum + (2), paederota lutea + (15), elyno-seslerietea: erigeron glabratus + (8), festuco-brometea: allium carinatum subsp. pulchellum + (2), thymus praecox + (4), salvia pratensis + (7), trifolium montanum + (11), trifolio-geranietea: digitalis grandiflora + (2), verbascum lychnitis r (2), vincetoxicum hirundinaria + (7), lilium bulbiferum r (11), thlaspietea rotundifolii: hieracium bifidum + (1), hieracium piloselloides + (1), trisetum argenteum + (4), silene vulgaris subsp. glareosa + (13), erico-pinetea: molinia caerulea subsp. arundinacea + (14), pinus sylvestris r (14), vaccinio-piceetea: solidago virgaurea + (11), picea abies + (14), quercetalia pubescentis: ostrya carpinifolia /e1/ 1 (12), arabis turrita + (2), coronilla emerus subsp. emeroides r (6), sorbus aria + (11), coronilla emerus subsp. emerus + (14), fagetalia sylvaticae: anemone trifolia + (11), lathyrus vernus + (11), ranunculus lanuginosus r (11), tilia cordata /e2a/ r (11), tilia cordata /e1/ + (12), fagus sylvatica /e2/ r (12), fagus sylvatica /e1/ + (12), querco-fagetea: ulmus minor + (4), pyrus pyraster + (9), listera ovata + (11), corylus avellana r (12), quercus robur r (12), salicetea purpureae: salix eleagnos /e1/ + (12), salix purpurea /e1/ + (4), artemisietea vulgaris: artemisia vulgaris + (1), melilotus albus r (8), other species: mentha sp. + (15), robinia pseudacacia r (5), mosses: homalothecium sericeum + (2), encalypta streptocarpa + (11) tab. 2. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:03 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 67 southeastern-alpine endemic leontodon hispidus... fig. 7. typical site (habitat) of the triseto argentei-leontodontetum brumatii community in the sava valley (photo a. seli{kar). fig. 6. leontodon hispidus subsp. brumatii on temporarily flooded riparian rocks in the sava valley (central slovenia) (photo i. dakskobler). 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:09 color profile: disabled composite 150 lpi at 45 degrees 68 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. tab. 3. synoptic table of the three riparian communities. succesive number 1 2 3 number of relevé 12 3 10 author dsv vp hu sign tl rp rp1 asplenietea trichomanis leontodon hispidus subsp. brumatii e1 100 . . thlaspietea rotundifolii trisetum argenteum e1 42 . . phragmiti-magnocaricetea phalaris arundinacea e1 67 100 100 mentha aquatica e1 17 . . lycopus europaeus e1 8 100 10 veronica anagallis-aquatica e1 . 66 . glyceria notata e1 . 66 . leersia oryzoides e1 . 66 . rumex hydrolapathum e1 . 66 . veronica becabunga e1 . 33 . scrophularia umbrosa e1 . 33 . berula erecta e1 . 33 . carex pseudocyperus e1 . 33 . iris pseudacorus e1 . . 50 galium palustre e1 . . 30 carex acuta e1 . . 20 glyceria maxima e1 . . 20 poa palustris e1 . . 10 filipendulo-convolvuletea helianthus tuberosus e1 17 . . saponaria officinalis e1 17 . . calystegia sepium e1 8 . 50 lythrum salicaria e1 8 33 40 epilobium hirsutum e1 . 66 . mentha longifolia e1 . 33 . myosoton aquaticum e1 . 33 10 galega officinalis e1 . 33 . stachys palustris e1 . . 20 echinocystis lobata e1 . . 10 bidentetea tripartitae rorippa palustris e1 8 . . polygonum hydropiper e1 . 100 30 polygonum lapathifolium e1 33 20 ranunculus sceleratus e1 . 33 . succesive number 1 2 3 number of relevé 12 3 10 author dsv vp hu sign tl rp rp1 bidens frondosa e1 . . 20 atriplex prostrata e1 . . 10 galio-urticetea solidago gigantea e1 83 . . urtica dioica e1 8 33 90 glechoma hederacea e1 . . 10 lamium maculatum e1 . . 10 artemisietea vulgaris rumex obtusifolius e1 42 . 10 artemisia vulgaris e1 33 . . erigeron annuus e1 33 . . euphorbia esula e1 20 . . tanacetum vulgare e1 8 . . stellarietea mediae poa annua e1 8 . . setaria viridis e1 8 . . polgonum persicaria e1 . 33 . galeopsis bifida e1 . 33 . galium aparine e1 . 33 30 cirsium arvense e1 . . 40 lemnetea minoris lemna minor e1 . 33 . potametea pectinati elodea canadensis e1 . 33 . agropyretea intermedii-repentis poa compressa e1 17 . . elytrigia repens e1 . . 10 potentillion anserineae rorippa sylvestris e1 42 . 10 barbarea vulgaris e1 25 66 . alopecuros geniculatus e1 . 66 . potentillo-polygonetalia agrostis stolonifera e1 42 66 . plantago major e1 25 . . rumex crispus e1 8 . 10 ranunculus repens e1 8 . . 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:09 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 69 southeastern-alpine endemic leontodon hispidus... succesive number 1 2 3 number of relevé 12 3 10 author dsv vp hu sign tl rp rp1 molinietalia caeruleae myosotis scorpioides e1 . 100 . equisetum palustre e1 . . 10 scirpus sylvaticus e1 . . 10 molinio-arrhenatheretea deschampsia cespitosa e1 75 . . taraxacum officinale e1 33 . . trifolium pratense e1 33 . . plantago lanceolata e1 33 . . achillea millefolium e1 17 . . dactylis glomerata s.str. e1 17 . . lotus corniculatus e1 17 . . galium mollugo e1 8 . . ranunculus acris e1 8 . 10 veronica chamaedrys e1 8 . . leucanthemum ircutianum e1 8 . . cerastium holosteoides e1 8 . . poa trivialis e1 . 66 50 alopecurus pratensis e1 . . 20 symphytum officinale e1 . . 20 lysimachia nummularia e1 . . 20 carex hirta e1 . . 20 potentilla anserina e1 . . 10 vicia cracca e1 . 10 festuco-brometea s. lat. peucedanum oreoselinum e1 25 . . genista tinctoria e1 8 . . petrorhagia saxifraga e1 8 . . inula ensifolia e1 8 . . salicetea purpureae populus nigra e2 83 . . salix alba e2 33 . . salix eleagnos e2 25 . . salix purpurea e2 58 . . salix triandra e2 8 . . salix fragilis e2 8 . . succesive number 1 2 3 number of relevé 12 3 10 author dsv vp hu sign tl rp rp1 alnion incanae rubus caesius e2 75 . 10 ulmus laevis e2 67 . . alnus glutinosa e2 17 . . solanum dulcamara e1 . . 20 equisetum arvense e1 . . 10 humulus lupulus e1 . . 10 fagetalia sylvaticae carpinus betulus e2 17 . . fraxinus excelsior e2 8 . . scrophularia nodosa e1 8 . . quercetalia pubescentis campanula rapunculoides e1 8 . . fraxinus ornus e1 8 . . querco-fagetea clematis vitalba e1 8 . . carex digitata e1 8 . . rhamno-prunetea crataegus monogyna e2 33 . . rhamnus catharticus e2 8 . . erico-pinetea sesleria caerulea subsp. calcaria e1 33 . . chamaecytisus purpureus e1 25 . . calamagrostis varia e1 8 . . other species fallopia japonica e1 8 . . poa sp. e1 8 . . agrostis sp. e1 8 . . physocarpus opulifolius e2 8 . . galium rivale e1 . . 10 mosses brachythecium rutabulum e0 100 . . cinclidotus fontinaloides e0 58 . . tortella sp. e0 67 . . 1: triseto-leontodontetum – this paper; 2: rorippo-phalaridetum – petrinac 1999; 3: rorippo-phalaridetum – hrivinák and ujházy 2003 tab. 3. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:09 color profile: disabled composite 150 lpi at 45 degrees 70 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. fig. 8. dendrogram of the two riparian communities (tl – triseto-leontodontetum, rp and rp1 – rorippo-phalaridetum) – upgma, similarity ratio. fig. 9. two-dimensional scatter-diagram of two riparian communities (tl – triseto-leontodontetum, rp and rp1 – rorippo-phalaridetum) – pcoa, similarity ratio. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees diagnostic species of the new association are leontodon brumatii (dominant species), trisetum argenteum and some riparian mosses of which we established two – brachythecium rutabulum and cinclidotus fontinaloides. trisetum argenteum is a character species of scree communities, especially in the subalpine and alpine belts (aeschimann et al. 2004). it is relatively frequently deposited and preserved on riparian rocks along the central or lower course of alpine rivers, e.g. in slovenia along the so~a and the sava, so it is also a good indicator of the site or community of l. brumatii on riparian rocks. the nomenclature type of the new association, holotypus, is relevé no. 12 in table 1. the new association is subdivided into three variants: the typical (relevés 2 and 3 in table 1); the variant with chamaecytisus purpureus (the differential species are also peucedanum oreoselinum, sesleria caerulea subsp. calcaria and inula ensifolia) – relevés 4 to 6 in table 1– these stands indicate a possible transition into the community leontodonti brumatii-seslerietum calcariae described hereinafter, i.e. into the initial riparian grassland; and the variant with deschampsia cespitosa (the differential species is also phalaris arundinacea) – relevés 7 to 13 in table 1. everywhere sand accumulates in the runnels carved into the rocks or in coarse gravel, conditions are created that facilitate successional development towards riparian willow stands with black poplar, which respond particularly well to sufficient amount of fine particles (bo@i^ et al. 2008). the stands of this variant therefore indicate potential successional development into the community salicetum eleagno-purpureae var. populus nigra, into which relevés no. 14 and 15 were classified (see dakskobler 2010). individual shoots of tree species populus nigra, ulmus laevis and salix alba occur also in other relevés. most of the relevés in table 2 (the so~a valley) are classified into the new association leontodonti brumatii-seslerietum calcariae ass. nov. its stands characterize the initial stony riparian grassland on undeveloped soil (lithosol). the species composition belongs to very different phytosociological groups (tab. 4); the largest, some 21 %, is the acta bot. croat. 71 (1), 2012 71 southeastern-alpine endemic leontodon hispidus... tab. 4. structure of the association leontodonti brumatii-selserietum calcariae according phytosociological groups (in %). asplenietea trichomanis 13 elyno-seslerietea 10 festuco-brometea 21 molinion caeruleae 1,4 molinio-arrhenatheretea 1,4 scheuchzerio-caricetea fuscae 0,7 trifolio-geranietea 2,5 thlaspietea rotundifolii 5,1 erico-pinetea 21 vaccinio-piceetea 0,7 mulgedio-aconitetea 2,2 quercetalia pubescentis 9,5 fagetalia sylvaticae 2,2 querco-fagetea 4,4 salicetea purpureae 2,9 artemisietea vulgaris 0,7 other species 1,1 total 100 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees proportion of species of dry grasslands from the class festuco-brometea and species of basophilic pine forests from the class erico-pinetea, followed by chasmophytic species (asplenietea trichomanis), species of subalpine grasslands (elyno-seslerietea) and thermophilic oak forests (quercetalia pubescentis), which have a proportion of 10 %. varied species composition also indicates a certain similarity with the communities of thermophilic forest edges from the class trifolio-geranietea, although it features a small proportion of diagnostic species of this class (2.5 %). in support of the synsystematic classification a synoptic table was made (tab. 5), into which two riparian meadow communities – centaureo dichroanthae-globularietum cordifoliae pignatti 1953 (feoli 72 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. tab. 5. synoptic table of pioneer grassland and fringe vegetation. succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp asplenietea trichomanis leontodon hispidus subsp. brumatii e1 100 . . . . hieracium porrifolium e1 75 36 . . . athamanta turbith e1 50 . . . . saxifraga crustata e1 33 . . . . phyteuma scheuchzeri subsp. columnae e1 25 . . . . asplenium ruta-muraria e1 8 . . . . paederota lutea e1 8 . . . . seseli gouanii e1 . 43 . . . dianthus sylvestris e1 . 7 . . 10 silene hayekiana e1 . . 33 . . elyno-seslerietea . . . . . sesleria caerulea subsp. calcaria e1 100 86 aster bellidiastrum e1 67 7 . . . globularia cordifolia e1 25 100 33 . . rhinanthus aristatus e1 25 14 11 . . erigeron glabratus e1 8 . . . . phyteuma orbiculare e1 8 . . . . carex mucronata e1 . 93 . . . helianthemum alpestre e1 . 36 . . . dryas octopetala e1 . 14 . . . gentiana clusii e1 . 7 . . . carlina acaulis s. lat. e1 . 7 89 . 20 helianthemum nummularium subsp. grandiflorum e1 . . 89 . . ranunculus carinthiacus e1 . . 56 . . betonica alopecuros e1 . . 44 . . galium anisophyllon e1 . . 33 . . alchemilla glaucescens e1 . . 22 . . gentiana verna e1 . . 11 . . 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 73 southeastern-alpine endemic leontodon hispidus... succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp hieracium valdepilosum e1 . . 11 . . hieracium villosum e1 . . 11 . . festuca calva e1 . . 11 . . phyteuma orbiculare e1 . . 11 . . festuco-brometea carex humilis e1 58 86 11 70 10 centaurea scabiosa (inc. subsp. fritschii) e1 58 . 44 10 70 genista tinctoria e1 58 . 11 10 10 peucedanum oreoselinum e1 42 93 56 70 60 inula ensifolia e1 33 86 . . 10 euphorbia cyparissias e1 33 21 44 50 90 satureja montana subsp. variegata e1 25 14 . 10 . stachys recta e1 25 . . 30 50 buphthalmum salicifolium e1 25 7 . 50 80 bromopsis erecta e1 17 . 100 50 30 inula hirta e1 17 . . 50 10 ononis spinosa e1 17 . 22 . . medicago falcata e1 17 . . 40 10 gymnadenia conopsea e1 17 29 78 10 . koeleria pyramidata e1 8 . 100 30 40 thymus praecox s. lat. e1 8 7 78 10 50 salvia pratensis e1 8 . 78 30 70 trifolium montanum e1 8 . 56 50 20 allium carinatum s. lat. e1 7 . 44 . 30 centaurea dichroantha e1 . 100 . . . teucrium montanum e1 . 93 11 10 10 fumana procumbens e1 . 93 . . . stipa eriocaulis e1 . 93 . 10 . galium lucidum e1 . 93 . 30 . plantago holosteum e1 . 86 67 . . thesium divaricatum e1 . 71 . . . bromopsis condensata e1 . 71 . . . genista sericea e1 . 64 . . . thymus longicaulis e1 . 64 . . . chrysopogon gryllus e1 . 64 . 10 10 helianthemum nummularium subsp. obsurum e1 . 64 . 10 40 scorzonera austriaca e1 . 57 . 50 . linum tenuifolium e1 . 50 . . . cytisus pseudoprocumbens e1 . 50 . . . potentilla australis e1 . 43 . . . leontodon crispus e1 . 43 . 10 . tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees 74 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp asperula cynanchica e1 . 43 22 10 70 orchis morio e1 . 43 . . . scabiosa graminifolia e1 . 36 . . . dianthus monspessulanus e1 . 36 . 10 10 hippocrepis comosa e1 . 36 78 10 . teucrium chamaedrys e1 . 29 . 30 90 plantago argeneta e1 . 21 33 30 . sanguisorba muricata (inc. s. minor) e1 . 21 100 10 40 galium verum e1 . 21 89 . 80 carex liparocarpos e1 . 14 . . . asperula purpurea e1 . 14 . . . scabiosa triandra e1 . 14 67 . 40 festuca rupiciola (inc. f. pseudovina, f. ovina agg.) e1 . 14 100 30 50 anacamptys pyramidalis e1 . 14 . . . carex caryophyllea e1 . 14 78 . . hypochoeris maculata e1 . 14 . . 10 globularia elongata e1 . 14 . . . allium senescens e1 . 14 11 . . botriochloa ishaemum e1 . 7 . . . ophrys apifera e1 . 7 . . . centaurea bracteata (c. gaudinii) e1 . 7 . . . betonica serotina e1 . 7 . . . gentianella pilosa e1 . 7 56 . . hieracium pilosella e1 . 7 11 10 . brachypodium rupestre e1 . . 100 70 80 pimpinella saxifraga e1 . . 100 30 60 plantago media e1 . . 100 10 30 prunella grandiflora e1 . . 89 10 20 briza media e1 . . 89 30 30 medicago lupulina e1 . . 89 10 20 polygala comosa e1 . . 89 . . silene vulgaris subsp. vulgaris e1 . . 89 . 20 orchis ustulata e1 . . 89 . . campanula rotundifolia e1 . . 79 . . campanula glomerata e1 . . 78 . . orchis militaris e1 . . 67 . . linum catharticum e1 . . 56 . 20 ranunculus bulbosus e1 . . 44 . . euphrasia stricta e1 . . 22 . . potentilla pusilla e1 . . 11 . . arabis hirsuta e1 . . 11 . . tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 75 southeastern-alpine endemic leontodon hispidus... succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp astragalus onobrychis e1 . . 11 . . cuscuta epithimum e1 . . 11 . 10 carlina vulgaris e1 . . 11 . . filipendula vulgaris e1 . . 11 70 60 orobanche gracilis e1 . . 11 . . gentianella ciliata e1 . . 11 . . rhinanthus freynii e1 . . 11 . . hypochoeris maculata e1 . . 11 30 10 cirsium pannonicum e1 . . . 70 30 euphorbia verrucosa e1 . . . 70 30 knautia illyrica e1 . . . 50 . dorycnium germanicum e1 . . . 50 60 vicia tenuifolia e1 . . . 30 . genista sylvestris e1 . . . 30 . centaurea rupestris e1 . . . 30 . scorzonera villosa e1 . . . 30 . veronica barrelieri e1 . . . 10 . euphorbia nicaeensis e1 . . . 10 . thesium linophyllum e1 . . . 10 . pulsatilla montana e1 . . . 10 . lychnis viscaria e1 . . . . 60 scabiosa hladnikiana e1 . . . . 30 prunella laciniata e1 . . . . 20 cuscuta epithymum e1 . . . . 20 ornithogalum sphaerocarpum e1 . . . . 20 anthyllis vulneraria e1 . . . . 10 thero-brachypodietea koeleria lobata e1 . 100 . . . artemisia alba e1 . 29 . . . eryngium amethystinum e1 . 21 . . 10 campanula sibirica e1 . 21 . . . medicago prostrata e1 . 7 . . . melica ciliata e1 . 7 . . . koelerio-corynephoretea cardaminopsis arenosa e1 . . 33 . . sedum sexangulare e1 . . 22 . . cerastium brachypetalum e1 . . 22 . . petrorhagia saxifraga e1 . . 11 . . molinion caeruleae laserpitium prutenicum e1 33 . . . . molinia caerulea subsp. caerulea e1 . . 11 . . tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees 76 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp carex distans e1 . . 11 . . carex tomentosa e1 . . 11 . . sanguisorba officinalis e1 . . 11 . . herminium monorchis e1 . . 11 . . inula salicina e1 . . . 10 20 selinum carvifolia e1 . . . . 10 molinio-arrhenatheretea taraxacum officinale e1 25 . . . . lotus corniculatus agg. e1 8 29 89 50 30 dactylis glomerata e1 . 29 89 10 80 senecio jacobea e1 . 14 . 10 . leontodon hispidus subsp. hispidus e1 . . 100 . 10 trifolium pratense e1 . . 89 . . centaurea jacea e1 . . 78 10 70 achillea millefolium e1 . . 78 . 50 galium mollugo (inc. g. album) e1 . . 78 . 30 helictotrichon pubescens e1 . . 78 . . prunella vulgaris e1 . . 78 . . rhinanthus minor e1 . . 67 . 10 trifolium repens e1 . . 67 . . vicia cracca e1 . . 67 . . leucanthemum ircutianum (inc. l. vulgare) e1 . . 67 10 30 plantago lanceolata e1 . . 56 . . orchis coriophora subsp. coriophora e1 . . 56 . . ranunculus acris e1 . . 44 . . euphrasia rostkoviana e1 . . 44 . . lathyrus pratensis e1 . . 44 10 40 leontodon autumnalis e1 . . 44 . . tragopogon pratensis e1 . . 44 . 10 ranunculus nemorosus e1 . . 33 . . rumex acetosa e1 . . 33 . . luzula campestris e1 . . 33 . . festuca pratensis e1 . . 22 . . achillea roseoalba e1 . . 22 . . carex hirta e1 . . 11 . . trifolium campestre e1 . . 11 . . knautia arvensis e1 . . 11 . 30 festuca arundinacea e1 . . 11 . . heracleum sphondylium e1 . . 11 . . pimpinella major e1 . . 11 . . allium scorodoprasum e1 . . 11 . . tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 77 southeastern-alpine endemic leontodon hispidus... succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp stellaria graminea e1 . . 11 . . bromus hordeaceus e1 . . 11 . . veronica chamaedrys e1 . . . 10 20 arrhenatherum elatius e1 . . . 10 . knautia arvensis e1 . . . . 30 festuca rubra e1 . . . . 30 poa angustifolia e1 . . . . 20 rumex acetosa e1 . . . . 10 trisetum flavescens e1 . . . . 10 calluno-ulicetea potentilla erecta e1 . . 78 . 10 chamaecytisus supinus e1 . . . 30 . chamaespartium sagittale e1 . . . 10 . genista germanica e1 . . . . 10 polygala vulgaris e1 . . . . 10 scheuchzerio-caricetea fuscae parnassia palustris e1 17 . 33 . . schoenus nigricans e1 . 79 . . . carex panicea e1 . . 11 . . trifolio-geranietea peucedanum cervaria e1 42 . . 100 100 vincetoxicum hirundinaria e1 8 7 22 30 20 lilium bulbiferum e1 8 . . . . anthericum ramosum e1 . 43 . 50 80 silene nutans e1 . . 89 30 30 thalictrum minus e1 . . 33 70 40 valeriana collina e1 . . 11 . 10 ferulago galbanifera e1 . . . 90 . dictamnus albus e1 . . . 70 . iris illyrica e1 . . . 50 . geranium sanguineum e1 . . . 50 70 polygonatum odoratum e1 . . . 50 10 paeonia officinalis e1 . . . 30 . trifolium alpestre e1 . . . 30 . viola hirta e1 . . . 30 70 trifolium rubens e1 . . . 30 50 coronilla coronata e1 . . . 20 . trifolium medium e1 . . . 10 20 ruta divaricata e1 . . . 10 . verbascum austriacum e1 . . . 10 . lathyrus latifolius e1 . . . 10 . tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees 78 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp lilium bulbiferum e1 . . . 10 . hypericum perforatum e1 . . . 10 . potentilla recta e1 . . . 10 . origanum vulgare e1 . . . . 70 clinopodium vulgare e1 . . . . 50 coronilla varia e1 . . . . 20 rosa gallica e1 . . . . 20 veronica jacquiniii e1 . . . . 10 melampyrum nemorosum e1 . . . . 10 dianthus barbatus e1 . . . . 10 inula conyza e1 . . . . 10 veronica teucrium e1 . . . . 10 lathyrus sylvestris e1 . . . . 10 vicia tenuifolia e1 . . . . 10 thlaspietea rotundifolii petasites paradoxus e1 33 7 22 . . campanula cespitosa e1 25 14 . . . peucedanum verticillare e1 17 . . . . biscutella laevigata e1 17 50 78 . . achnatherum calamagrostis e1 8 14 . . . trisetum argenteum e1 8 . . . . silene vulgaris subsp. glareosa e1 8 7 . . . euphorbia triflora subsp. kerneri e1 . 93 . . . gypsophila repens e1 . 79 . . . trinia glauca e1 . 71 . . . matthiola carnica e1 . 57 . . . hieracium piloselloides e1 . 50 22 . . brassica glabrescens e1 . 36 . . . crambe tataria e1 . 36 . . . reseda lutea e1 . 21 . . . diplotaxis tenuifolia e1 . 21 . . . euphrasia cuspidata e1 . 14 . . . rumex scutatus e1 . 7 . . . rumex scutatus e1 . . 33 . . hieracium glaucum e1 . . 11 . . dianthus sternbergii e1 . . 11 . . erico-pinetea calamagrostis varia e1 83 . 11 10 10 chamaecytisus purpureus e1 67 57 11 . . allium ericetorum e1 50 7 . . . erica carnea e1 42 93 . . 20 tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 79 southeastern-alpine endemic leontodon hispidus... succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp aster amellus e1 50 . 11 . 10 leontodon incanus e1 42 7 . . . carex ornithopoda e1 33 . . . . epipactis atrorubens e1 25 . 11 . . pinus nigra e2 25 . . . . polygala chamaebuxus e1 25 7 . . . chamaecytisus hirsutus e2 17 . . . 20 molinia caerulea subsp. arundinacea e1 8 7 11 30 . pinus sylvestris e2 8 . . . . polygala nicaeensis subsp. forojulensis e1 . 43 11 10 . daphne cneorum e1 . 14 . . . genista januensis e1 . . . . 10 vaccinio-piceetea solidago virgaurea e1 8 . . 10 60 picea abies e2a 8 . 11 . . mulgedio-aconitetea salix appendiculata e2a 50 . . . . rhamno-prunetea cornus sanguinea e1 . . . 30 . prunus mahaleb e1 . . . 20 . crataegus monogyna e1 . . . 10 . rhamnus rupestris e1 . . . 10 . prunus spinosa e1 . . . 10 . rosa canina e1 . . . 10 . rubus fruticosus agg. e1 . . . . 10 quercetalia pubescentis fraxinus ornus e2a 75 7 . 30 . ostrya carpinifolia e2a 33 7 . 10 30 lembotropis nigricans e1 25 . . 10 20 carex flacca e1 25 . 22 . 70 campanula rapunculoides e1 17 . . 10 20 clematis recta e1 17 7 . . 10 coronilla emerus subsp. emeroides e1 8 . . . . sorbus aria e2a 8 . . 10 . coronilla emerus subsp. emerus e2a 8 . . . . primula veris subsp. columnae e1 . . 89 . . helleborus multifidus subsp. istriacus e1 . . . 50 . sesleria autumnalis e1 . . . 30 . mercurialis ovata e1 . . . 30 . melittis melissophyllum e1 . . . 30 40 lathyrus niger e1 . . . 20 10 tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:10 color profile: disabled composite 150 lpi at 45 degrees 80 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp cnidium silaifolium e1 . . . 10 . quercus pubescens e1 . . . 10 . hypericum montanum e1 . . . 10 20 tanacetum corymbosum e1 . . . . 20 buglossoides purpurocaerulea e1 . . . . 20 campanula persicifolia e1 . . . . 10 quercus cerris e1 . . . . 10 calamintha menthifolia e1 . . . . 10 fagetalia sylvaticae tilia cordata e2 17 . . . . anemone trifolia e1 8 . . . . lathyrus vernus e1 8 . . . . ranunculus lanuginosus e1 8 . . . . fagus sylvatica e2 8 . . . . knautia drymeia e1 . . 11 . 30 cyclamen purpurascens e1 . . . 10 . salvia glutinosa e1 . . . . 20 querco-fagetea clematis vitalba e2 42 . . 10 10 carex digitata e1 17 . . . . ulmus minor e1 8 . . . . pyrus pyraster e2 8 . . . . listera ovata e1 8 . 11 . . corylus avellana e1 8 . . 20 . quercus robur e1 8 . . . . cruciata glabra e1 . . 67 . 20 potentilla alba e1 . . . 70 . betonica officinalis e1 . . . 70 70 serratula tinctoria e1 . . . 30 30 hepatica nobilis e1 . . . 10 . carex montana e1 . . . 10 . helleborus odorus e1 . . . . 30 festuca heterophylla e1 . . . . 20 hieracium racemosum e1 . . . . 10 pteridium aquilinum e1 . . . . 10 hedera helix e1 . . . . 10 salicetea purpureae salix eleagnos e2 25 7 . . . salix purpurea e2 25 . . . . populus nigra e2 17 . . . . tab. 5. – continued 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:11 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 81 southeastern-alpine endemic leontodon hispidus... succesive number 1 2 3 4 5 number of relevé 12 14 9 21 10 author dsv cfp dz gkl ac sign ls cg gb cp gp artemisietea vulgaris erigeron annuus e1 8 . . . . melilotus albus e1 8 . . . . other species festuca sp. e1 8 . . . . mentha sp. e1 8 . . . . robinia pseudacacia e1 8 . . . . phalaris arundinacea e1 . . 11 . . salix cinerea e1 . . 11 . . fragaria vesca e1 . . . . 80 verbascum nigrum e1 . . . . 30 geranium phaeum e1 . . . . 20 juniperus communis e1 . . . . 30 1: leontodonti-seslerietum calcariae, this paper 2: centaureo dichroanthae-globularietum cordifoliae, feoli chiapella and poldini 1993 3: gentianello pilosae-brometum erecti, dakskobler and zavr{nik 2009 4: cirsio-peucedanetum cervariae, van gils, keysers and launspach 1975 5: geranio-peucedanetum cervariae, ^arni 1998 tab. 5. – continued fig. 10. dendrogram of pioneer grassland and fringe vegetation (ls – leontodonti-seslerietum calcariae, cg – centaureo dichroanthae-globularietum cordifoliae, gb – gentianello pilosae-brometum erecti, cp – cirsio pannonicae-peucedanetum cervariae, gp – geranio-peucedanetum cervariae) – upgma, similarity ratio. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:11 color profile: disabled composite 150 lpi at 45 degrees chiapella and poldini 1993), gentianello pilosae-brometum erecti dakskobler et zavr{nik 2009 (dakskobler and zavr[nik 2009), and two thermophilic forest edge communities – cirsio pannonicae-peucedanetum cervariae van gils et al. 1975 (van gils et al. 1975) and geranio-peucedanetum cervariae (kuhn 1937) t. müller 1961 var. geogr. knautia drymeia ^arni 1998 (^arni 1998) were ranged and compared using numerical methods. the results (figs. 10, 11) clearly demonstrate that our stands are the most similar to the initial grassland on gravel sites of mountain rivers and streams in the foothills of the southeastern alps (association centaureo dichroanthae-globularietum cordifoliae) and are considerably different from thermophilic forest edge communities. their floristic composition is also clearly different from riparian stands of the association gentianello pilosae-brometum erecti. this community is much more species-rich and grows on more developed soils, rendzinas. the stands of the association leontodonti brumatii-seslerietum calcariae are a successional stage in the afforestation of riparian rocks towards scrub and forest communities (seslerio albicantis-ostryetum, ostryo-fagetum). in addition to some meadow species they are mainly characterized by diagnostic species or rock crevices, screes and basophilic pine forests. diagnostic species of the new association are leontodon hispidus subsp. brumatii, sesleria caerulea subsp. calcaria, calamagrostis varia, hieracium porrifolium, aster bellidiastrum and athamanta turbith. the holotypus of the new association is relevé no. 7 in table 2. the new association is classified as an explicitly fringe association (non-typical; with its species composition it is close also to the classes elyno-seslerietea, thlapietea rotundifolii and asplenietea trichomanis), into the illyrian-pre-alpine 82 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. fig. 11. two-dimensional scatter-diagram of pioneer grassland and fringe vegetation (ls – leontodonti-seslerietum calcariae, cg – centaureo dichroanthae-globularietum cordifoliae, gb – gentianello pilosae-brometum erecti, cp – cirsio pannonicae-peucedanetum cervariae, gp – geranio-peucedanetum cervariae) – pcoa, similarity ratio. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:11 color profile: disabled composite 150 lpi at 45 degrees suballiance centaurenion dichroanthae, into the alliance saturejion subspicatae, order scorzonero-chrysopogonetalia and class festuco-brometea. it is subdivided into two subassociations. the differential species of the subassociation leontodonti-seslerietum chamaecytisetum purpureae subass nov. are chamaecytisus purpureus, carex humilis, allium ericetorum, centaurea scabiosa subsp. fritschii and inula ensifolia. its nomenclatural type is the holotypus of the association (relevé no. 7 in table 2); its sites are limestone riparian rocks. it is still flooded when the water level is high, but soil has already started accumulating in the runnels, which allows for a progressive development of the vegetation. the differential species of the subassociation leontodonti-seslerietum saxifragetum crustatae subass. nov. are saxifraga crustata, erica carnea, laserpitium prutenicum, campanula cespitosa, petasites paradoxus and stachys recta. the nomenclatural type, holotypus, is relevé no.13 in table 2. the sites of this subassociation are steep, gravelly, erosion slopes on platy limestone with addition of chert on the northeastern slopes of bu~enica, some ten metres above the so~a between tolmin and most na so~i. the species composition indicates the development from a scree community through stony grassland into a thermophilic forest (ostryo-fagetum). the first three relevés in table 2 could not be classified into the new association. the floristic composition of relevé no. 1 in table 2 (rocky eyot in the idrijca at slap ob idrijci) shows some similarities with the stands of the association triseto-leontodontetum, while relevés 2 and 3 in the same table represent a chasmophytic community, for the time being synsystematically still undefined, at the road cut between podsela and doblar, dominated by athamanta turbith, campanula carnica and c. pyramidalis. discussion the new localities of the southeastern-alpine endemic leontodon hispidus subsp. brumatii in the sava valley (central slovenia, the black sea river basin), in the gorge of the sava between the village of sava and zidani most, are rather surprising, because so far this taxon was known in slovenia only in the so~a basin (on riparian rocks along the so~a, nadi`a, u~ja, idrija and idrijca), which belongs the adriatic sea river basin, but not along other alpine rivers. nevertheless, the new localities are situated in the region where quite a few species with predominantly alpine distribution grow (e.g. rhododendron hirsutum, paederota lutea, pinguicula alpina, rosa pendulina, cerastium subtriflorum, tephroseris pseudocrispa, aster bellidiastrum, campanula carnica, c. cespitosa, trisetum argenteum, arabis alpina subsp. alpina, astrantia carniolica, acinos alpinus, aconitum degenii subsp. paniculatum, tofieldia calyculata, myrrhis odorata – the latter is mentioned by petkov[ek 1939). the flora of the sava valley also comprisessome species that most frequently occur in the so~a valley, but are usually much rarer in other landscape regions of slovenia. such species are for example saxifraga petraea, veratrum nigrum, hemerocallis lilioasphodelus, spiraea chamaedryfolia. there are also some similarities between the forest communities of valuable broad-leaved species (e.g. the probable occurrence of the stands of two associations described in the so~a valley, veratro nigri-fraxinetum and saxifrago petraeae-tilietum, also in the sava valley). it is also likely that the taxon l. brumatii occurs on suitable sites elsewhere in the sava basin (and other slovenian alpine rivers), but has been overlooked. riparian sites are usually difficult to access and are therefore not the subject of a more detailed floristic inventory. the established differences in the communities acta bot. croat. 71 (1), 2012 83 southeastern-alpine endemic leontodon hispidus... 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:11 color profile: disabled composite 150 lpi at 45 degrees of l. brumatii between the so~a and the sava valleys do not rule out the possibility that the stands of the association triseto-leontodontetum that was described in the sava valley occur also in the so~a valley (which is very likely, because the sites in the so~a valley comprise a higher number of this endemic than those in the sava valley). similarly, the occurrence of the stands of the association leontodonti-seslerietum described in the so~a valley, is likely also in the sava valley (already demonstrated in one of the relevés). both newly described associations include phytocoenoses that are usually distributed in smaller areas and are more or less long-term successional stages. these are by nature subject to the river dynamics and can disappear from one spot only to reappear in another. they are extremely exposed to human interventions of any kind. the endemic leontodon brumatii and its communities are a characteristic of riparian flora and vegetation of some of slovenian mountain rivers. according to current knowledge, the localities in the sava valley are explicitly disjunct and the southeasternmost in the entire known distribution area, so they deserve our attention and protection. the envisaged interventions along the central sava, i.e. construction of new hydroelectric power plants, would most probably lead to destruction of suitable habitats for leontodon brumatii. with a higher water level between zagorje and hrastnik, this endemic is likely to disappear from the river’s riparian zone, as has probably already happened downstream, before zidani most or rade~e and further south. acknowledgements the research in the sava valley was conducted in the framework of the research programme gradients and biodiversity: flora, fauna and vegetation (grant no. p1-0236), funded by slovenian research agency, and of the assignment inventory of plant species in the middle sava area (commissioned by the hse d.o.o.). bo{ko ^u{in, msc, helped us in the field. associate professor andra` ^arni, phd and urban [ilc, phd, helped us with literature sources. iztok sajko prepared figures 1 and 2 for print. sincere thanks to two anonymous reviewers for their helpful remarks, instructions, corrections and improvements. english translation by andreja [alamon verbi~. references aeschimann, d., lauber, k. d. m., moser, d. m.,theurillat, j.-p., 2004: flora alpina, 2: gentianaceae–orchidaceae. haupt verlag, bern, stuttgart, wien. bo@i^, g., vilhar, u., urban^i^, m., kobal, m., ferreira, a., kraigher, h.,grebenc, t., sinjur, i., [tupar, b. hrenko, m., verli^, a., jarni, k., brus, r., ^arni, a., [ilc, u., ko[ir, p., marin[ek a., dakskobler, i., 2008: population genetic studies and sites investigation of autochthonous european black poplar (populus nigra l.) along river basins and floodplain forest areas in slovenia and 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(eds.), flora alpina 3: register, 301–313. haupt verlag, bern. van gils, h., kaysers, e., launspach, w., 1975: saumgesellschaften im klimazonalen bereich des ostryo-carpinion orientalis. vegetatio 31,1, 47–64. wraber, m., 1969: pflanzengeographische stellung und gliederung sloweniens. vegetatio 17, 176–199. wraber, t., 1998: notulae ad nomenclaturum editionis mala flora slovenije anni 1999 spectantes. hladnikia (ljubljana) 10, 41–43. wraber, t., 2007: cichoriaceae [compositae subfam. cichorioideae]. in: martin^i^, a. (ed.), mala flora slovenije, 687–716. tehni{ka zalo`ba slovenije, ljubljana. zupan^i^, b., 1998: average annual precipitations between 1961 and 1990 (in slovenian). in: fridl, j., kladnik, d., oro@en adami^, m., perko, d. et al. (eds.), geografski atlas slovenije: dr`ava v prostoru in ~asu, 99, dzs, ljubljana. 86 acta bot. croat. 71 (1), 2012 dakskobler i., seli[kar a., vre[ b. 457 dakskobler et al.ps u:\acta botanica\acta-botan 1-12\457 dakskobler_verzija-10.vp 26. o ujak 2012 10:49:11 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (2), 339–349, 2009 coden: abcra 25 issn 0365–0588 new diatom taxa from the world’s first marine bioblitz held in new zealand: skeletomastus a new genus, skeletomastus coelatus nov. comb. and pleurosigma inscriptura a new species margaret a. harper*, john e. patterson, john f. harper school of geography, environment and earth sciences, victoria university of wellington, p.o. 600 wellington 6140, new zealand diatoms were investigated as part of the world’s first marine bioblitz held on the south coast of wellington, new zealand, in october 2007. two unusual diatoms were associated with the red alga herposiphonia ceratoclada. they were examined by light and scanning electron microscopy. the first, previously described as »cocconeis coelata« arnott ex greville, is a thick-ribbed biraphid diatom that appears to have an undular raphe and marginal septum like some mastogloia species, but it does not have partecta in the valvocopula. instead it has short marginal ribs that support a pseudoseptum. its solid lyre and multiseriate striae with cibrate pores could be modifications of similar structures in aneumastus aksaraiensis spaulding et al. it typifies the new genus skeletomastus which appears to be close to both mastogloia and aneumastus, hence the name. the second is a proposed new pleurosigma species, p. inscriptura with naviculoid symmetry and a markedly sigmoid raphe system with deflected ends. it appears bright blue in darkfield illumination which helped us locate it in other samples from the wellington region. we compare it with some other pleurosigma species and consider their diffraction properties. keywords: diatom, mastogloiaceae, pseudoseptum, costae, lyre, sigmoid, multiseriate, ultrastructure, cocconeis, aneumastus, skeletomastus, pleurosigma introduction bioblitzes aim to document biodiversity in a limited space and time. experts collect samples and identify species while an invited public watches. in october 2007 a bioblitz was held in the taputeranga marine reserve situated near wellington city on the coast of cook strait, new zealand. the reserve covers 840 hectares from the foreshore south to 41°22.00’ s and from 174°44.67’ e to 174° 47.37’ e. high energy tidal flows of alternating warmer and cooler water passing through cook strait have formed a rocky shore with little shingle or sand. the diatom flora of the reserve is rich, with at least 164 taxa, the commonest genus being cocconeis (19 taxa). acta bot. croat. 68 (2), 2009 339 * corresponding author: margaret.harper@vuw.ac.nz u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:20 color profile: disabled composite 150 lpi at 45 degrees the diatoms described in this paper were found on subtidal seaweed. one diatom resembled an unusual mastogloia and we propose a new genus for it. the other is a new species of pleurosigma unknown to two experts on pleurosigma: stidolph from new zealand (stidolph 1992, 2002) and sterrenberg from the netherlands (sterrenburg 1990; 1991a, b; 2001; 2003). material and methods seaweeds and other organisms growing in the subtidal zone were collected by divers. macroalgae were lightly sonicated in a tank (50 khz, 10 min) to free any epiphytic diatoms. the loose material was partially oxidised with warm hydrogen peroxide and disaggregated with hydrochloric acid before washing. some of the resulting suspension was mounted in naphrax (ri 1.73) and examined on a light microscope (lm) (leitz diaplan) under direct light and differential interference contrast. diatoms were photographed with a leica camera and photographs improved in picasa (cropped, converted to greyscale and sharpened). standard darkfield (sterrenburg 1991a) was obtained using an olympus chb student grade microscope with an abbé condenser and tungsten bulb with the addition of a black disc to obscure the cone of direct lighting at 40x and 100x magnification. this revealed pleurosigma valves on the slides and allowed their darkfield colour to be recorded. their bright blue colour was used to locate further valves. part of the suspension was subjected to stronger sonication with an immersed probe (20 khz, 5 min) to break apart the valves of »cocconeis coelata«. the suspensions were dried down onto aluminium stubs. they were then plasma-ashed for about 1 hour at 5 watts in air at a pressure of about 0.5 millibars. the sample was positioned about 50 mm downstream of the discharge zone to avoid rf heating of the aluminium stub. the procedure effectively removes any organic material without altering the silicate structure. the stub was gold-coated and first examined with a jeol 733 microprobe in scanning electron mode to see whether the diatom originally called »cocconeis coelata« had obvious partecta. it and a specimen of the pleurosigma were then photographed using a jeol 6500f scanning electron microscope (sem). theoretical ratios for angles between oblique striae versus oblique pore-spacing are as follows: if p pores per 10 mm were counted in an oblique line then a = 10/p mm, and if q (transverse) lines of pores per 10 mm were counted crossing a transapical (longitudinal) line then c = 10/q mm. in this case cos(q /2) = c/a = p/q so q/p = sec(q /2) for q <132° changing q by a given percentage changes q/p or p/q by a smaller percentage: p/q = cos(q /2); therefore ( )d p / q dq = ½ sin(q /2); so ( )q p d p / q dq = –1/2 tan(q /2); 1/2 tanq /2<1 if tanq /2<2; therefore ½ q <66° and q <134°. so q/p is obtained more accurately from a measurement of q than q is by a measurement of p/q. 340 acta bot. croat. 68 (2), 2009 harper m. a., patterson j. e., harper j. f. u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:20 color profile: disabled composite 150 lpi at 45 degrees results skeletomastus cocconeis coelata arnott ex greville is a species of neither cocconeis nor mastogloia so we erect a new genus for it. skeletomastus (m. a. harper gen.nov.) monotypic genus latin diagnosis: valvae costis radialibus munitae inter se longitudinaliter connexis ita in sterna laterales duo (lyrae similia) coalescentes. costae breves marginales (costulae) pseudoseptum fulcrant, quod est (in frustulo) a valvocopula tectum. striae multiseriatae ex poris circularibus cribratis constantes. rami raphis extra circa centrum opposite undulati, intra recti. valves fortified by radial ribs connected to each other longitudinally thus coalescing into two lateral sterna (similar to a lyre). short marginal ribs (riblets) support a pseudoseptum, with this being covered (in the frustule) by the valvocopula. striae multiseriate consisting of circular cribrate pores. branches of the raphe externally, with respect to the centre oppositely undulate, internally straight. etymology: greek, skeletos – skeleton and mastus – nipple, the first part based on the comment on its skeleton-like structure in greville (1862), the second on the similarities in its structure to aneumastus (particularly a. aksaraiensis). skeletomastus coelatus (arnott ex greville) m. a. harper, comb. nov. basionym: cocconeis coelata g. a. walker arnott ex greville, r. k. (greville 1862: page 234, plate 10, figures 5 and 6). lectotype: on british museum slide, g. a. w. arnott 56, (s 631) bm 1492 labelled eupleuria ocellata on ballia. this slide contains syntypes and i concur with david william’s choice of a lectotype (williams 1988: 103, fig. 4). eupleuria ocellata is a synonym of entopyla ocellata (arnott) greville also present on the slides. synonyms: skeletomastus coelatus (arnott ex greville) m. a. harper (this paper), cocconeis coelata arnott ex greville 1862, campyloneis coelata (arnott ex greville) chase (habirshaw 1885: 79), navicula coelata (greville) cleve 1894 (schmidt 1894, pl.193, figs. 5, 3). cocconeis coelata erroneously attributed to gregory (grunow 1867: 9). some records give »caelata« an orthographical variant of the epithet and others »coelatus« (van landingham 1968). skeletomastus coelatus has raphes on both valves so it does not fit the genera cocconeis and campyloneis. it was synonymised with a query by cleve (1894) with diploneis campylodiscus (grunow in schmidt) cleve (van landingham 1968). we disagree with this placement because this diatom does not have solid lyres or undular raphes, (see navicula campylodiscus grunow (schmidt 1875, pl. 8, figs. 9, 12). the figures of the original navicula campylodiscus show that the areas each side of the axial rib differ from the multiseriate areas near the margin, giving the appearance of longitudinal canals that have large pores where they meet the outer multiseriate areas. these features indicate navicula campylodiscus really does belongs to the genus diploneis. acta bot. croat. 68 (2), 2009 341 new taxa skeletomastus coelatus and pleurosigma inscriptura u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:20 color profile: disabled composite 150 lpi at 45 degrees illustrations: originals in greville (1862: pl. 10, figs. 5 and 6) include an aberrant rapheless valve. multiseriate striae are shown in schmidt (1894: pl. 193 fig. 53) and on in the lectotype photograph (williams 1988: fig. 4, p. 103). exsiccata: van heurck (h.v.h.) slides issued as #244 »cocconeis coelata gregory« (erroneous authority). we have examined one in the healey collection (slide # 2682) held at niwa, wellington, new zealand. 342 acta bot. croat. 68 (2), 2009 harper m. a., patterson j. e., harper j. f. plate 1. skeletomastus coelatus [cocconeis coelata] in lm (figs. 1–4) and sem (figs. 6–12). fig.1 – high focus, undulate raphe and multiseriate striae; fig. 2 – same view low focus, marginal pseudoseptum with short ribs; fig. 3 – new valve without pseudoseptum showing that this forms later; fig. 4 – larger valve; fig. 5 – external view of pores and raphe; fig. 6 – oblique apical view of split valvocopula and internal costae, fig. 7 – oblique side view of hypovalve sunk inside epicingulum; fig. 8 – external pores on edge of valvocopula; fig. 9 – internal view cribrate pores; fig. 10 – internal apical view of valvocopula overlapping pseudoseptum; fig. 11 – internal cibrate valvocopula pores; fig. 12 – internal view, valvocopula over pseudoseptum. scale bars: 10 mm (figs. 1–7); 1 mm (figs. 10–12), 0.25 mm (fig 9). u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:25 color profile: disabled composite 150 lpi at 45 degrees the elliptical valves of skeletomastus coelatus have multiseriate striae (figs. 1, 3, 4), lyrate areas (figs. 1–4) and a marginal »septum« (fig. 2) visible under lm. all our valves (>108, including 16 frustules) were fully raphid so this diatom is not a cocconeis. their length in the original description is 13 to 46 mm (greville 1862), some of the island bay valves were longer (range 22 to 53 mm). our valves were 11.5 to 34 mm wide. unlike most diatoms larger valves are wider in proportion, though their lyres are not (figs. 1, 4). the undular external raphe slits (fig. 1), become straight inside (fig. 2). under the marginal »septum« are projections between the costae (fig. 2), these appear too short to be partecta walls. valves without »septa« (fig. 3) indicate that these form later. in sem the flat valves have round pores, wide virgae and undulate raphe slits with expanded ends but no terminal fissures (fig. 5). internal views of the valve (figs. 6, 16) show that thick costae underlie the hyaline areas and pores have cibra (figs. 9, 14). the raphe slit is in a thick axial ridge (figs. 6, 13) and a fascia connects this ridge to the solid (fig. 13) lateral costae of the lyrate area (figs. 6, 13). the valve mantles have scattered pores (figs. 5, 6), and some have plaques at their edge (fig 13). part of the split valvocopula (fig 6) overlaps the pseudoseptum (figs. 10–12), and it has simple pores outside (fig. 8) and cibrate pores inside (fig. 11). there are at least two more plain copulae (fig. 7). removal of the valvocopula shows the pseudoseptum forms a shelf round the valve edge (figs. 13 –15). this shelf is supported by short inward-branching ribs (figs. 14, 15) that appear to be outgrowths of the pseudoseptum. when the pseudoseptum is broken off, the ribs come off with it tearing the valve (fig. 16). costae have »shoulders« where they meet the pseudoseptum and only a narrow strap-like neck continues to the edge (fig. 17); this could well form a connected space under the pseudoseptum. evidence for this is in figure 14, where the floor of the right-hand intercostal space (plus arrow) has more highlights than its neighbour to the left. lighting in the latter is obstructed by a particle under its left rib. habitats skeletomastus coelatus has been collected from cook strait and stewart island. the original material on ballia sent to g. a. w. arnott by w. h. harvey (arnott 1858) came from the hms acheron collections from one of these areas or from fiordland. it was recorded on coralline algae from lyall’s bay (now called lyall bay), cook strait by petit (1877). our material was on herposiphonia ceratoclada from a neighbouring bay (island bay). it has been found at traills bay, stewart island (stidolph pers. comm..) in a mixed sample rm 135. this is preserved on the s.r.s. slides 996–1000 at niwa (wellington) and also on british museum slide 100875. it is epiphytic on finely-branched red algae. pleurosigma this pleurosigma species is unlike other pleurosigma species with naviculoid symmetry so we erect a new species for it. pleurosigma inscriptura m. a. harper, sp. nov. latin diagnosis: valvae lanceolatae polis rotundatis, ambito respectu axem apicalem transapicalemve symmetrico, 70–110 µm longae, 16–21 µm latae. striae decussatae, striis obliquis ad angulum c. 70° intersecantis et areolis 16–20 in 10 µm praeditis, striis transapicalibus 20–23 in 10 µm. raphe sigmoidea, ad centrum respectu lineam inter nodulos polares angulo c. 3° inclinata. acta bot. croat. 68 (2), 2009 343 new taxa skeletomastus coelatus and pleurosigma inscriptura u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:25 color profile: disabled composite 150 lpi at 45 degrees valves lanceolate with rounded poles, symmetrical in outline with respect to the apical and transapical axes, 70–110 µm long, 16–21 µm wide. striae decussate, with the oblique striae intersecting at an angle of c. 70° and provided with 16–20 areolae in 10 µm, 20–23 transapical striae in 10 µm. raphe sigmoid, inclined at the centre by an angle of c. 3° with respect to a line drawn between the polar nodules. locus typicus: island bay, wellington, new zealand. 344 acta bot. croat. 68 (2), 2009 harper m. a., patterson j. e., harper j. f. plate 2. skeletomastus coelatus [cocconeis coelata] in sem (figs. 13–17), pleurosigma inscriptura in lm (figs. 19–20), in sem (figs. 18, 20–21). fig. 13 – oblique view of broken valve (arrow indicates solid lyrae); fig.14 – view across centre (arrow indicates short rib); fig. 15 – detail of figure 13 arrow points to branched marginal rib; fig. 16 – broken pseudoseptum (arrow shows tear in valve made by lost rib); fig.17 – detail of broken valve (arrow indicates end of costa); fig. 18 – sem internal view of p. inscriptura valve; fig. 19 – large specimen; fig. 20 – smaller specimen; fig. 21 – internal view, central nodule; fig. 22 – internal view of apex. scale bars: 10 mm (figs. 13, 18–20), 1 mm (figs. 14–17, 20, 21). u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:31 color profile: disabled composite 150 lpi at 45 degrees holotype: pleurosigma inscriptura slide chr 593684 allan herbarium, lincoln, new zealand. isotypes: slide 939 – pleurosigma inscriptura niwa herbarium, national institute of water and atmospheric research, riccarton, new zealand. etymology: named for the appearance of its sigmoid raphe, as if handwritten. the lanceolate valves (figs. 18–20) are so symmetrical that the outline of flat-lying valves can be flipped about either the apical or transapical axis and will still coincide with the original, unlike sigmoid valves (which have one pervalvular axis of symmetry). most naviculoid pleurosigma species are slightly sigmoid in outline (round et al. 1990); some examples are listed in table 1. pleurosigma inscriptura valves range from 70–110 mm (based on 22 valves) with a length to width ratio of c. 5 (range 4.2 to 6.3). the oblique striae have c. 18.5 areolae in 10 mm (range 16 to 20). in pleurosigma the areolar centres alternate between lying on the apical axis and to one side of it; both sets form part of transverse lines of striae and all lines are included in estimates of striae frequency. transverse striae have a mean frequency of 22 striae in 10 mm (range 19.5 to 23). the ratios of counts on transverse striae to oblique striae have a mean of 1.18 (range 1.16 to 1.22). the mean angle of intersection for oblique striae is 69° (range 66° to 73°). the raphe system is clearly sigmoid near its centre as well as at its ends, unlike many other naviculoid pleurosigma species. this results in a larger raphe angle (ca 3° to the line joining its polar ends) and its ends are more deflected. in lm the central raphe endings appear joined and deflected in the same direction. the polar raphe ends appear like small triangles (sterrenburg 1991a). details of the valve interior in sem (figs. 21, 22) show that most areolae open internally by double poroids and are »obex« (stidolph 1992) without rims, but a few are single near the centre (fig. 21). the centre has no hyaline areas outside a small oval central nodule acta bot. croat. 68 (2), 2009 345 new taxa skeletomastus coelatus and pleurosigma inscriptura tab. 1. characteristics of p. inscriptura compared with some other symmetrical pleurosigma species with clearly sigmoid raphes. characteristic p. inscriptura nov sp. p. strigosum w. smith p. rigidum w. smith p. williamsii reid 2002 p. obtusum mann darkfield bright teal bright blue indigo unknown none oblique angle 65–73° 64–70° 52–57° c.50° 68° "striae / 10 mm 17–20 16–18 17–21 18–25 14, 20 transverse " / " 19.5–23 17–20 16–20 18–25 22 raphe angle 2.5–4° 6–10° 4–8° c.5° c. 12° " central ends close small close close deflected terminal areas triangle small triangle funnel (obscured) shape lanceolate lanceolate lanceolate lanceolate rhombic symmetry 3 axes 3 axes < 3 axes 3 axes < 3 axes length mm 70–132 120–200 200–360 90–320 126, 131 width mm 16–21 28–37 38–45 15–38 29, 30 reference this paper sterrenburg 2003 sterrenburg 2001 reid 2002 stidolph 2002 u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:31 color profile: disabled composite 150 lpi at 45 degrees and its ridges (fig. 21). raphe slits lie between two equally thickened ribs, their internal central ends are rounded with slight deflections to the same side. the apical raphe ends have helictoglossae that are turned to opposite sides (fig. 22). habitats most valves of the new p. inscriptura were collected with red algae from wellington’s south coast. some came from herposiphonia ceratoclada growing subtidally on the brown alga halopteris, but they also occurred occasionally on gigartina circumcincta from island bay, and on a coralline alga (cf. arthocardia sp.) collected at low spring tide from owhiro bay (next to island bay). pleurosigma inscriptura also occurred among epiphytic diatoms collected from titahi bay (cochran 2002: slide mtb-1ep). pleurosigma stidolphii was also present in some samples. discussion skeletomastus early workers (schmidt 1894) realised »cocconeis coelata« was biraphid, so not a cocconeis. initially we interpreted our lm views of skeletomastus coelatus as being those of a species of mastogloia. these have raphe slits that undulate but in the same direction (paddock and kemp 1990), can be multiseriate and may have a solid lyre; but always have a loculate valvocopula (cox 2006). sem views show skeletomastus coelatus has no valvocopular chambers, rather a pseudoseptum supported by short marginal ribs. comparison with other genera and species show its cibra are like those of craspedostauros britannicus (cox 1999), but positioned as in m. danseii; its valvocopula split overlaps the pseudoseptum as in mastogloia danseii (cox 2006) and its pseudoseptum is continuous as in achnanthes coarctata. these are all members of the natural group mastogloiales (cox 2006, cox and williams 2006), which is largely defined by the presence of the two fore and aft h-shaped plastids such taxa possess. we have not seen the plastids of s. coelatus. lyrate genera outside this group differ from s. coelatus: fallacia has a hollow lyre, diploneis has channels next to the raphe and lyrella does not have costae (thickened interstriae) and has internal siliceous flaps on its pores. although s. coelatus appears to fit in the mastogloiales, it does not fit easily in any genus: unlike aneumastus it has no flaps on external pores, nor does it have a stauros-like craspedostauros, nor is it monoraphid 346 acta bot. croat. 68 (2), 2009 harper m. a., patterson j. e., harper j. f. tab. 2. comparison of spectral colour wavelengths with mean spacing of oblique striae and mean angles in three main colour groups and with p. inscriptura. colour wavelength nm oblique striae per 10 mm. mean (range) spacing nm (difference) oblique angle mean (range means) indigo (5 spp.) 425–450 19.6 (18–21.3) 510 (70) 57.5° (55–63) blues (11 spp.) 450–500 19.3 (16–25) 518 (40) 67.6° (60–81) p. inscriptura 18.3 (17–20) 546 (36) 68.6° (65–73) greens (0 spp.) 500–570 gold (5 spp.) 570–590 12.6 (10–18) 790 (200) 90.8° (80–100) u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:31 color profile: disabled composite 150 lpi at 45 degrees like achnanthes (cox 2006). therefore we have erected a new genus skeletomastus for it. it has most in common with a. aksaraiensis spaulding, akbulut and kociolek which has biseriate striae with round pores except next to the raphe, connections between costae forming a zig-zag lyre and terminal pseudosepta (spaulding et al. 2003). spaulding et al. (2003) suggest a. akaraiensis is either basal to mastogloia or derived from it – this could well be true of skeletomastus. pleurosigma inscriptura this species differs from most pleurosigma species in combining a naviculoid valve with a clearly sigmoid raphe; otherwise its features are typical of pleurosigma in general (round et al. 1990, reid 2002). its raphe and oblique angle, terminal areas and darkfield colour are the characteristics (sterrenburg 1991a), rather than dimensions and stria frequency, that distinguish it from the other species (tab. 1). the bright greenish-blue colour seen in standard darkfield is typical of pleurosigma species with striae of similar frequency and oblique striae at an angle of 60–70° (tab. 2). this angle is a more sensitive parameter (sterrenburg 1991a) than the ratios. theoretical ratios for angles between oblique striae versus oblique pore-spacing (see methods) give a more sensitive measure of differences between species than the ratio of frequency of transverse and oblique striae. the external pores of pleurosigma species are slits and their darkfield colours are mainly due to diffraction through these. mean oblique stria spacing is approximately related to observed colour (tab. 2). other factors that could affect this are whether slits overlap, type of internal pores, variable spacing in different parts of the valve and other properties of pores and walls. acknowledgements the authors thank david mann of the royal botanic garden, edinburgh, for useful comments and the latin diagnoses, ursula cochran of gns science, wellington, for editing the manuscript, david flynn of the electron microscopy unit, science faculty, victoria university of wellington, for scanning electron micrographs, stuart stidolph for information and frithjof sterrenburg, sijbekarspel, netherlands for help with the taxonomy of pleurosigma and phil garnock-jones of the school of biological sciences, victoria university of wellington for help with the synonymy of skeletomastus coelatus. references arnott, g. a. w., 1858: on rhabdonema, and a new allied genus. quarterly journal of microscopical science 6, 87–93. cleve, p. t., 1894: synopsis of the naviculoid diatoms, 1. konliga svenska vetenskaps-akademiens handlingar 26, 1–194. cochran, u. a., 2002: detection of large holocene earthquakes in the sedimentary record of wellington, new zealand using diatom analysis. phd thesis, victoria university, wellington. acta bot. croat. 68 (2), 2009 347 new taxa skeletomastus coelatus and pleurosigma inscriptura u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:31 color profile: disabled composite 150 lpi at 45 degrees cox, e. j., 1999: craspedostauros gen. nov., a new diatom genus for some unusual marine raphid species previously placed in stauroneis ehrenberg and stauronella mereschkowsky. european journal of phycology 34, 131–147. cox, e. j., 2006: achnanthes sensu stricto belongs with genera of the mastogloiales rather than with other monoraphid diatoms (bacillariophyta). european journal of phycology 41, 67–81. cox, e. j., williams, d. m., 2006: systematics of naviculoid diatoms (bacillariophyta): a preliminary analysis of protoplast and frustule characters for family and order level classification. systematics and biodiversity 4, 385–399. greville, r. k.,1862: descriptions of new and rare diatoms. series, 7. quarterly journal of microscopical science, new series 2, 234–236. grunow, a. 1867: reise seiner majestät fregatte novara um die erde. botanischer theil, band i, algen, wien, aus der kaiselich-königlichen hof-und-staasdruckerei. habirshaw, f. 1885. catalogue of the diatomaceae with reference to the various published descriptions and figures. h. h. chase, geneva. paddock, t. b. b., kemp, k.-d., 1990: an illustrated survey of the morphological features of the diatom genus mastogloia. diatom research 5, 73–103. petit, p., 1877: catalogue des diatomées d’île campbell et de la nouvelle zelande. fonds de la mer 3, 164–198 reid, g. 2002: four new species of pleurosigma (bacillariophyta) from alexandria, egypt. botanical journal of the linnean society 140, 77–92. round, f. e.; crawford, r. m.; mann, d. g., 1990: the diatoms. biology and morphology of the genera. cambridge university press, cambridge. schmidt, a., 1875: atlas der diatomaceen-kunde series 1, 2. aschersleben, leipzig. schmidt, a., 1894: atlas der diatomaceen-kunde series 5, 49. aschersleben, leipzig. spaulding, s. a., akbulut, a., kociolek, j. p., 2003: a new diatom species of aneumastus d. g. mann and stickle from central turkey. diatom research 18, 149–160. sterrenburg, f. a. s., 1990: studies on the genera gyrosigma and pleurosigma bacillariophyceae: a new phenomenon: co-existence of dissimilar raphe structures in populations of several species. in: ricard, m. (ed.), ouvrage dédié à la mémoire du professeur henry germain 1903–1989: koeltz, koenigstein, germany. sterrenburg, f. a. s., 1991a: studies on the genera gyrosigma and pleurosigma bacillariophyceae: light microscope criteria for taxonomy. diatom research 6, 367–389. sterrenburg, f. a. s., 1991b: studies on the genera gyrosigma and pleurosigma bacillariophyceae: the typus generis of pleurosigma, some presumed varieties and imitative species. botanica marina 34, 561–573. sterrenburg, f. a. s., 2001: studies on the genera gyrosigma and pleurosigma: the types of shadbolt and related taxa. proceedings of the academy of natural sciences of philadelphia 151, 121–127. sterrenburg, f. a. s., 2003: studies on the diatom genera gyrosigma and pleurosigma (bacillariophyceae): pleurosigma strigosum w. smith and some presumptive relatives. micropaleontology 49, 159–169. 348 acta bot. croat. 68 (2), 2009 harper m. a., patterson j. e., harper j. f. u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:31 color profile: disabled composite 150 lpi at 45 degrees stidolph, s. r., 1992: observations and remarks on the morphology and taxonomy of the diatom genera gyrosigma hassall and pleurosigma w. smith. iii. gyrosigma sterrenburgii sp. nov. and pleurosigma amara sp. nov. diatom research 7, 345–366. stidolph, s. r., 2002: observations and remarks on the morphology and taxonomy of the diatom genera gyrosigma hassall and pleurosigma w. smith. 5. pleurosigma types of a. mann (1925): a critical re-investigation. micropaleontology 48, 273–284. van landingham, s. l., 1968. catalogue of the fossil and recent genera and species of diatoms and their synonyms. 2. bacteriastrum through coscinodiscus. j. cramer, lehre. williams, d. m., 1988: an illustrated catalogue of the type specimens in the greville diatom herbarium. bulletin of the british museum (natural history), botany 18, 1–148. acta bot. croat. 68 (2), 2009 349 new taxa skeletomastus coelatus and pleurosigma inscriptura u:\acta botanica\acta-botan 2-09\harper.vp 6. listopad 2009 11:30:31 color profile: disabled composite 150 lpi at 45 degrees 582 roy et al.vp acta bot. croat. 72 (1), 23–33, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 amino acids through developmental stages of sunflower leaves nayan roy1, subrata laskar2, anandamay barik1* 1 ecology research laboratory, department of zoology, the university of burdwan, burdwan 713 104, west bengal, india 2 department of chemistry, the university of burdwan, burdwan 713 104, west bengal, india abstract – the pico-tag analysis of proteins revealed that 17 protein-bound and 18 free amino acids were present throughout the developmental stages of sunflower leaves. the total protein-bound amino acid content was much higher than total free amino acid content throughout the development of sunflower leaves. the contents of protein-bound and free amino acids as well as essential and non-essential ones displayed different patterns with leaf maturation, suggesting that total protein levels are poor predictors of the nutritive status of leaves. key words: amino acids, helianthus annuus, sunflower introduction studies in plant-insect interactions have mainly concentrated on the effects of insect performance of plant secondary metabolites or nutritive compounds (schoonhoven et al. 2005). the nutritional quality of plants is generally characterized in terms of total nitrogen or protein content (ruuhola et al. 2003), and the growth efficiency of a variety of insects is closely related to plant nitrogen content. further, the nutritive quality of plant tissues for insects may be affected by the amino acid composition of protein (schoonhoven et al. 2005). low protein level in the insect diet, i.e., poor food resources from a nutritional point of view possibly may lead to ingestion of some amount of toxic secondary compounds (broadway and duffey 1988, haukioja et al. 1991, slansky and wheeler 1992, ruuhola et al. 2003), which may affect optimal insect growth, survival and fecundity. the balance of amino acids that constitute plant proteins differs from that of insects and deficiency in even one essential amino acid in a herbivore diet may cause an unbalanced nitrogen metabolism in insects (berenbaum 1995). acta bot. croat. 72 (1), 2013 23 * corresponding author, e-mail: anandamaybarik@yahoo.co.in copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees diacrisia casignetum kollar (lepidoptera: arctiidae) is polyphytophagous and damages numerous field crops (i.e., sunflower, jute, sesame, castor, etc.) in india and many other asian countries (roy and barik 2012 a, b). it has been a serious pest of the sunflower (helianthus annuus l.) in india for many years (banerjee and haque 1984). it feeds gregariously on sunflower leaves leaving the mid ribs only. variation in performance and abundance of phytophagous insects is mainly due to variation in qualitative and quantitative amounts of amino acids among host plants, including changes in the nutritional quality of leaves within a particular host plant during its different developmental stages (schoonhoven et al. 2005). herbivores often show a preference for young leaves within a plant because of the increasing toughness along with decreased water and protein content of mature leaves (mattson and scriber 1987; schoonhoven et al. 2005). further, the amino acid composition of the proteins indicates the nutritive quality of plant tissues, which may affect insect’s growth and development because total protein levels are poor predictors of the nutritive status of leaves (berenbaum 1995). therefore, the present investigation was undertaken to determine the qualitative and quantitative variations in free and protein-bound amino acids in sunflower (helianthus annuus l.) leaves throughout the developmental stages of sunflower leaves, which will be used as experimental diets in further studies of the herbivorous insect, d. casignetum to understand the nutritional ecology of this insect pest for the purpose of developing better control strategies. there have been a number of studies on the amino acid content of sunflower leaves (cabello et al. 2006, dulermo et al. 2009, aguera et al. 2010); but the available data mainly focuses on metabolic changes during natural ageing in sunflower leaves (cabello et al. 2006), amino acid changes in sunflower cotyledon during a necrotrophic fungus (i.e., botrytis cinera) interaction (dulermo et al. 2009) and leaf development in sunflower plants grown with varying nitrate concentrations (aguera et al. 2010). but, there have been no reports on the differences in free and protein-bound amino acids throughout the developmental stages of sunflower leaves. materials and methods plant material fresh young (1–2 weeks old), mature (2–4 weeks old) and senescent (5–7 weeks old) sunflower cv. pac-36 leaves were harvested randomly during january, 2011 from sunflower plants (cv. pac-36) growing in the field near chinsurah rice research center (22° 53' n, 88° 23' e), west bengal, india (roy and barik 2012a). total amino acid content measurement the variability of total amino acid content of sunflower leaves throughout the development state of sunflower leaves was estimated taking 1g each of fresh young, mature and senescent leaves by the method of moore and stein (1948). each determination was repeated three times. one gram of each kind of fresh leaf was placed separately in a hot-air oven at 50 ± 1 °c temperature for 72 h, materials were removed from the oven, and weighed in a digital monopan balance. one portion (25 mg) of the oven-dried sample was taken in a covered heat-resistant porcelain crucible (50 ml) and placed in a muffle furnace (sunvic, uk) for burning. material was initially allowed to smoke slowly and to lose organic matter gradually by increasing the furnace temperature at the rate of 5 °c min –1 to 450 °c and burnt 24 acta bot. croat. 72 (1), 2013 roy n., laskar s., barik a. 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees to ash at 450 ± 5 °c for 30 min. the resultant ash was reconstituted with distilled water, dried to a constant dry weight in the hot air oven at 100 ± 0.5 °c for 2 h, and weighed in a digital monopan balance. this procedure was repeated three times for each kind of leaf tissue. the total amino acid content was presented as mean µg mg –1 ash-free leaf tissue ± standard error. protein-bound amino acid measurement a sufficient amount of freshly collected leaves (young, mature and senescent) was rinsed with double distilled water and dried on a paper towel. one hundred g fresh leaf samples of each kind were dipped in 3 l n-hexane in a 5 l cotton-plugged conical flask and kept in the laboratory at room temperature for 21 days. the flask was then vigorously shaken daily for 30 min. the leaves were removed from n-hexane and dried in air at room temperature (27 ± 1 °c). the dried leaf material was extracted with phosphate buffer (ph 7) for 30 min, kept for 30 min in a –20 °c freezer, and was filtered through whatman no. 41 filter paper (maidstone, uk). each kind of water extract was dialyzed in deionized water and then placed in a lyophilizer. the powdered protein obtained from each kind of leaf was weighed in a digital balance (± 0.01 mg). this process was repeated three times for each kind of leaf and values were expressed as mean ± standard error. these nine powdered protein samples were used for amino acid analysis separately. the powdered protein sample (20 µg) was hydrolyzed by 6n hydrochloric acid containing 5% thioglycolic acid (matsubara and sasaki 1969). the solution was sealed in a tube under nitrogen and incubated in a hot-air oven at 110 °c for 24 h in the pico.tag work station. the hydrolyzed sample and the authentic amino acids internal standard – 'standard h' (0.005 ml), were taken in respective tubes, introduced into the reaction vial and dried completely. these were then separately derivatised in a solution mixture of ethanol: triethyl amine: water: phenyl isothiocyanate (7: 1: 1: 1 v/v) in a nitrogen atmosphere at 25 °c for 20 min (ghosh et al. 1997). the samples were dried and reconstituted in a diluent solution (na2hpo4, 0.071% w/v in distilled water with ph 7.4; ph was adjusted by 10% h3po4 containing 5% v/v acetonitrile). amino acids were analyzed at 38 °c as per the pico.tag manual using a pico-tag c18 hydrophobic column (5µm, 3.9 × 150 mm; waters) and detected at 254 nm (chart speed – 2 cm/min). amino acids present in the unknown sample was characterized by comparing the peaks of the amino acids in the 'standard h' (pierce, rockford, il, usa), and the actual amount of each amino acid present was determined from the area under the individual curve. all solvents used were of analytical grade and purchased from e. merck (india). free amino acid measurement freshly collected leaves of each kind (young, mature and senescent) were rinsed with double distilled water and dried by paper towel. one hundred g of fresh leaf samples of each kind were dipped in 2 l millipore water in a 5 l cotton-plugged conical flask and kept at room temperature (27 ± 1 °c) for 20 min using a magnetic stirrer. the extract of each kind was filtered through whatman no. 41 filter paper. the filtrate was kept for 30 min in a –20 °c freezer and was again filtered through whatman no. 41 filter paper (maidstone, uk). the water extract from each kind of leaf was placed in lyophilizer. the powdered proteins obtained were weighed in a digital monopan balance. this procedure was repeated acta bot. croat. 72 (1), 2013 25 amino acids in sunflower leaves 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees three times for each of the samples of young, mature and senescent sunflower leaf. the total free amino acid content was presented as mean ± standard error. these powdered protein samples were used for amino acid analysis separately by the above mentioned procedure. results total amino acid content total amino acid content varied throughout the developmental stages of sunflower leaves. total amino acid content was the highest in mature leaves (3.61 ± 0.442 µg per mg ash-free leaf tissue) followed by young leaves (2.88 ± 0.378 µg per mg ash-free leaf tissue) and senescent leaves (2.287 ± 0.268 µg per mg ash-free leaf tissue). amino acids bound in proteins amino acid bound in proteins was greatest in mature leaves (0.521 ± 0.014 mg g–1 leaf tissue) followed by young leaves (0.441 ± 0.012 mg per g leaf tissue) and senescent leaves (0.383 ± 0.015 mg per g leaf tissue) (tab. 1). the pico.tag analysis of protein-bound amino acids demonstrated that 17 different types of amino acids were present throughout the developmental stages of sunflower leaves (tab. 2). the quantitative analysis revealed that bound monocarboxylic amino acids, aromatic amino acids, heterocyclic amino acids and sulphur-containing amino acids were present in highest level in young leaves, whereas dicarboxylic amino acids and hydroxy amino acids were present in the largest amount in mature and senescent sunflower leaves. the total monocarboxylic amino acid content gradually decreased throughout the developmental stages of sunflower leaves (from young leaf to senescent leaf). in this group, glycine was found to be absent during the development of sunflower leaves (tab. 2). further, alanine and leucine were present in the highest amount in young and mature leaves, respectively, whereas isoleucine was detected in trace amounts in senescent leaves. the two amino acids of the dicarboxylic group, aspartic acid and glutamic acid and their amides, were present in moderate amounts since they represent almost 20% of the total amino acids. further, these two amino acids increased slightly throughout the developmental age of sunflower leaves. the amount of hydroxy amino acids increased from young leaf (22.03 ± 0.814%) to mature leaf (34.02 ± 0.756%) and then slightly decreased in senescent 26 acta bot. croat. 72 (1), 2013 roy n., laskar s., barik a. tab. 1. total protein-bound and free amino acid content (mg per g fresh leaf tissue) throughout the developmental stages of sunflower leaves leaf stages protein-bound amino acids free amino acids young 0.441 ± 0.012 0.280 ± 0.007 mature 0.521 ± 0.014 0.304 ± 0.010 senescent 0.383 ± 0.015 0.226 ± 0.006 mean ± se, n = 3. 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 27 amino acids in sunflower leaves tab. 2. percentage of amino acids (g per 16 g n**) bound in proteins throughout the developmental stages of sunflower leaves group young mature senescent monocarboxylic alanine 11.14 ± 0.398 5.21 ± 0.133 3.44 ± 0.144 amino acids glycine – – – valine* 7.03 ± 0.300 6.59 ± 0.225 8.55 ± 0.292 leucine* 9.60 ± 0.518 11.11 ± 0.543 8.18 ± 0.265 isoleucine* 3.07 ± 0.132 4.14 ± 0.133 0.16 ± 0.012 total 30.84 ± 0.277 27.05 ± 0.318 20.33 ± 0.159 dicarboxylic glutamic acid 10.40 ± 0.219 10.69 ± 0.318 11.78 ± 0.514 amino acids + glutamine aspartic acid 9.40 ± 0.398 9.65 ± 0.179 11.14 ± 0.416 + asparagine total 19.80 ± 0.618 20.34 ± 0.139 22.92 ± 0.098 hydroxy amino threonine* 7.13 ± 0.217 17.55 ± 0.497 17.48 ± 0.364 acids serine 14.90 ± 0.537 16.47 ± 0.259 15.60 ± 0.248 total 22.03 ± 0.814 34.02 ± 0.756 33.08 ± 0.612 diamino acids arginine* 3.37 ± 0.139 5.67 ± 0.352 3.66 ± 0.214 lysine* 2.77 ± 0.058 3.56 ± 0.225 3.82 ± 0.156 total 6.14 ± 0.081 9.23 ± 0.577 7.48 ± 0.369 aromatic amino tyrosine 5.25 ± 0.237 5.02 ± 0.398 5.65 ± 0.179 acids phenylalanine* 8.66 ± 0.144 0.35 ± 0.017 5.92 ± 0.352 total 13.91 ± 0.093 5.37 ± 0.381 11.57 ± 0.173 heterocyclic histidine* – – – amino acids proline 1.29 ± 0.092 – 0.7 ± 0.011 total 1.29 ± 0.092 – 0.7 ± 0.011 sulphur containing cysteine 0.79 ± 0.015 0.35 ± 0.023 0.43 ± 0.012 amino acids methionine* 5.20 ± 0.012 3.64 ± 0.358 3.49 ± 0.163 total 5.99 ± 0.005 3.99 ± 0.381 3.92 ± 0.176 essential 46.83 ± 0.162 52.61 ± 0.109 51.26 ± 0.207 non-essential 53.17 ± 0.162 47.39 ± 0.109 48.74 ± 0.207 * essential amino acid; mean ± se, n= 3. ** g per 16 g n = amino acid composition data were reported as grams amino acid per 100 g of sample for each amino acid. the nitrogen content of the sample was used to convert amino acid per 16 g nitrogen and the values are expressed as g per 16 g n. for calculation of protein content, the nitrogen content is multiplied by 6.25, the practice originated from early research of proteins that were found to contain 16% nitrogen (100/16= 6.25). 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees leaf (33.08 ± 0.612 %). threonine was found to be present in the highest amount in mature (17.55 ± 0.497%) and senescent (17.48 ± 0.364%) leaves, whereas serine was present in the highest amount in young leaves (14.90 ± 0.537%) among all the amino acids. the total content of diamino acids increased from young leaf to mature leaf and then decreased in senescent leaf, but this pattern is not followed in lysine content which increased slightly throughout the developmental stages of leaves. aromatic amino acids were lower in mature leaves than young and senescent leaves. tyrosine was almost same throughout the developmental age of sunflower leaves, whereas phenylalanine drastically reduced to a trace amount from young leaf to mature leaf and then it increased almost seventeen fold in senescent leaf. in the heterocyclic amino acids group, histidine was found to be absent throughout the developmental state of leaves, whereas proline was absent in mature leaf. among the sulphur-containing amino acids, cysteine was present in a small amount at all stages of leaf development, whereas methionine decreased from young leaf to senescent leaf. free amino acids total free amino acid content was highest in mature leaves (0.304 ± 0.010 mg g–1) followed by young leaves (0.280 ± 0.009 mg g–1) and senescent leaves (0.226 ± 0.006 mg g–1) (tab. 1). the amount of the total monocarboxylic amino acids group gradually decreased throughout the development of sunflower leaves, like bound amino acids (tab. 3). though, the amount of this group was higher throughout the development of sunflower leaves in comparison with bound amino acids. unlike bound amino acids, alanine was found to be absent, and glycine was present throughout the development stages of sunflower leaves. further, glycine also formed a large portion of amino acids in mature leaves. the amount of aspartic acid and glutamic acid (and their amides) increased from young leaf to mature leaf stage and then a decrease was observed in senescent leaf (tab. 3). the amount of hydroxy amino acid was lower throughout the developmental stages of sunflower leaves in comparison with bound amino acids, but serine content was high in mature and senescent leaves in comparison with bound forms. the diamino acid content was higher than the bound amino acid content in young and senescent leaves. in mature leaves a decreased free amino acid content was observed due to a drastic reduction of arginine. tyrosine gradually decreased throughout the developmental stages of sunflower leaves. the percentage of phenylalanine was almost doubled from young leaf to senescent leaf except in the mature leaf where a large decrease (i.e., 2.26 fold from young leaf) was noticed. phenylalanine was very high in mature and senescent leaves in comparison with the bound amino acid form. histidine, which formed a major portion of heterocyclic amino acids in young and senescent leaves, was absent in bound amino acids. the percentage of proline content was almost equal in young and senescent leaf, but increased or decreased almost 3.5 fold in mature leaf from that in young leaf or senescent leaf, respectively. among the sulphur-containing amino acids, methionine followed almost the same pattern as the bound form whereas cysteine content was higher in the free amino acid form. the drastic decrease in the contents of essential free amino acids in mature leaves was due to an increase in the contents of non-essential amino acids. 28 acta bot. croat. 72 (1), 2013 roy n., laskar s., barik a. 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 29 amino acids in sunflower leaves tab. 3. percentage of free amino acids (g per 16 g n**) throughout the developmental stages of sunflower leaves group young mature senescent monocarboxylic alanine – – – amino acids glycine 7.74 ± 0.243 20.69 ± 0.508 6.25 ± 0.248 valine* 12.14 ± 0.490 6.51 ± 0.323 11.90 ± 0.473 leucine* 9.48 ± 0.363 2.92 ± 0.207 4.97 ± 0.185 isoleucine* 3.55 ± 0.133 2.26 ± 0.121 3.47 ± 0.121 total 32.91 ± 0.249 32.38 ± 0.502 26.59 ± 0.658 dicarboxylic glutamic acid 8.82 ± 0.341 9.22 ± 0.266 4.22 ± 0.162 amino acids + glutamine aspartic acid 2.43 ± 0.109 8.34 ± 0.213 3.84 ± 0.109 + asparagine total 11.25 ± 0.450 17.56 ± 0.479 8.06 ± 0.271 hydroxy amino threonine* 1.19 ± 0.075 4.33 ± 0.167 1.27 ± 0.121 acids serine 6.29 ± 0.265 23.28 ± 0.554 19.69 ± 0.487 total 7.48 ± 0.341 27.61 ± 0.722 20.96 ± 0.609 diamino acids arginine* 12.17 ± 0.421 0.94 ± 0.012 10.77 ± 0.179 lysine* 1.82 ± 0.185 1.06 ± 0.139 1.00 ± 0.040 total 13.99 ± 0.236 2.00 ± 0.150 11.77 ± 0.219 aromatic amino tyrosine 9.48 ± 0.440 6.56 ± 0.150 3.19 ± 0.092 acids phenylalanine* 6.66 ± 0.162 2.94 ± 0.080 13.83 ± 0.306 total 16.14 ± 0.375 9.5 ± 0.231 17.02 ± 0.398 heterocyclic histidine* 10.85 ± 0.312 0.84 ± 0.017 9.61 ± 0.145 amino acids proline 1.14 ± 0.069 4.17 ± 0.103 1.23 ± 0.069 total 11.99 ± 0.381 5.01 ± 0.121 10.84 ± 0.214 sulphur containing cysteine 1.66 ± 0.133 2.03 ± 0.138 1.41 ± 0.133 amino acids methionine* 4.58 ± 0.248 3.91 ± 0.133 3.35 ± 0.132 total 6.24 ± 0.381 5.94 ± 0.272 4.76 ± 0.266 essential 62.44 ± 0.092 25.71 ± 0.519 60.17 ± 0.216 non-essential 37.56 ± 0.092 74.29 ± 0.519 39.83 ± 0.216 * essential amino acid; mean ± se, n= 3. ** g per 16 g n = amino acid composition data were reported as grams amino acid per 100 g of sample for each amino acid. the nitrogen content of the sample was used to convert amino acid per 16 g nitrogen and the values are expressed as g per 16 g n. for calculation of protein content, the nitrogen content is multiplied by 6.25, the practice originated from early research of proteins that were found to contain 16% nitrogen (100/16= 6.25). 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees discussion the overall total amino acid concentrations in plants vary extremely, depending, however, on environmental conditions (shobana et al. 2010). amino acid is an important factor affecting the feeding behaviour of herbivorous insects. the total content of protein-bound amino acids was much higher than that of total free amino acids throughout the developmental stages of sunflower leaves, indicating that the role of protein-bound amino acids is probably more important than that of free amino acids (ruuhola et al. 2003). the critical importance of amino acid composition of diet to the growth and reproduction of insects is well documented in the literature (horie and watanable 1983, nation 2001). while considering variation in amino acid composition, it is important to distinguish among the developmental stages of plant leaves (karley et al. 2002). this study demonstrated that changes in the contents of both free and protein-bound amino acids varied considerably throughout the development of sunflower leaves. the protein-bound amino acid content, i.e., dicarboxylic amino acids and hydroxy amino acids, is higher than free amino acid content throughout the developmental stages of sunflower leaves, whereas monocarboxylic amino acids, aromatic amino acids, heterocyclic amino acids and sulphur containing amino acids are higher in free amino acids, indicating that the roles of free and protein-bound amino acids are both important in the nutrition of herbivores. a decrease in the contents of monocarboxylic protein-bound or free amino acids occurred in senescing leaves which indicate the breakdown of cellular proteins and withdrawal of amino acids (ruuhola et al. 2003). glycine was detected in large quantities in free amino acid forms in mature leaves, being the most abundant amino acid in this group. this amino acid, which is involved in many metabolic processes in the cell, apart from being a component in many proteins, was absent in bound forms. the relative levels of the two nitrogen-rich essential protein-bound amino acids, diamino acids, lysine and arginine, increased from young leaf to mature leaf and then decreased in senescent leaf. this decrease indicates the reduction of nutritive quality in senescent leaves (weibull et al. 1990), since these two amino acids are target sites for proteolysis by trypsin which is a common protease of insect gut (broadway and duffey 1988). the high level of free amino acids in young and mature leaves, especially of free glutamic acid (and its amides), reflect the active metabolism of growing tissues (weibull 1987). interestingly, the relative content of free essential amino acids decreased at the expense of non-essential amino acids from young leaf to mature leaf in sunflower plants, suggesting that the quality of the amino acid pool actually decreased. though the relative content of free essential amino acids increased again in senescent leaves, which is due to the higher content of histidine, arginine, valine and phenylalanine. serine in free amino acid forms was found to be most active amino acid to promote senescence of leaves, while cysteine and phenylalanine had similar but less effect (martin and thimann 1972). senescent sunflower leaves also demonstrated a higher percentage of serine and phenylalanine than young leaves in free amino acid forms. free aromatic amino acids are used for the synthesis of phenolic compounds and lignin (strack 1997). further, phenylalanine is suggested to be a limiting factor for both the biosynthesis of phenolics and plant growth (jones and hartley 1999). the absolute content of free phenylalanine decreased 2.26 fold from young leaf to mature leaf and then increased almost five fold in senescent leaf. this suggests that phenylalanine is most active in senescent leaves and herbivorous insects do not prefer this kind of leaf. 30 acta bot. croat. 72 (1), 2013 roy n., laskar s., barik a. 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees the process leading to developmental changes in amino acid composition might be due to developmental regulation of transporter expression from phloem loading of amino acids in leaf vascular tissue (fischer et al. 1995, karley et al. 2002). however, there are other processes, i.e., metabolism, unloading and xylem-phloem transfer pathway which might be responsible for developmental changes of amino acid composition during leaf ageing (rentsch and frommer 1996, hirner et al. 1998, ruuhola et al. 2003). in the literature, clear information is available that the amino acid composition changes throughout the development of plant leaves (karley et al. 2002, amiard et al. 2004). the reasons for this variation are probably related to fundamental aspects of plant physiology, i.e., the changes reflect the role of amino acid in both the form of nitrogen transported and the portioning of nitrogen during development (karley et al. 2002). the value of leaves for its insect pests is known to decline rapidly with leaf maturation due to decrease in water and protein content as well as increased toughness of leaves (haukioja et al. 2002). but changes in the profiles of the protein-bound and free amino acids may further change the nutritive value of leaves because the dietary value of proteins may be inferior due to the absence of appropriate levels of needed amino acids (brodbeck and strong 1987, schoonhoven et al. 2005). in conclusion, the amino acid composition of sunflower leaves in free and bound forms displayed different patterns throughout the developmental stages of sunflower leaves, which may provide useful information to clarify the quality of sunflower leaves as total protein levels are poor predictors for the nutrition for d. casignetum (ruuhola et al. 2003, schoonhoven et al. 2005). it will be interesting to follow the behavior of the herbivorous insect d. casignetum when feeding on the three stages of leaves with different quantities of amino acids in free and bound forms. acknowledgement this study was supported financially by the university grants commission, new delhi, india through a minor research project (no. f. no. 37/615/2009). references aguera, e., cabello, p., de la haba, p., 2010: induction of leaf senescence by low nitrogen nutrition in sunflower (helianthus annuus) plants. physiologia plantarum 138, 256–267. amiard, v., morvan-bertrand, a., cliquet, j. b., billard, j. p., huault, c., sandstrom, j. p., prud’homme, m. p., 2004: carbohydrates and amino acid composition in phloem sap of lolium perenne l. before and after defoliation. canadian journal of botany 82, 1594–1601. banerjee, t. c., haque, n., 1984: defoliation and yield loss in sunflower caused by caterpillars of diacrisia casignetum kollar (lepidoptera: arctiidae). indian journal of agricultural science 54, 137–141. berenbaum, m. r., 1995: turnabout is fair play: secondary roles for primary compounds. journal of chemical ecology 21, 925–940. acta bot. croat. 72 (1), 2013 31 amino acids in sunflower leaves 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees broadway, r. m., duffey, s. s., 1988: the effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. journal of insect physiology 34, 1111–1117. brodbeck, b., strong, d., 1987: amino acid nutrition in herbivorous insects and stress to host plants. in: barbosa, p., schultz, j. c., (eds.), insect outbreaks, 347–364. academic press, london. cabello, p., aguera, e., haba, p. d. l., 2006: metabolic changes during natural ageing in sunflower (helianthus annuus) leaves: expression and activity of glutamine synthetase isoforms are regulated differently during senescence. physiologia plantarum 128, 175– 185. dulermo, t., bligny, r., gout, e., cotton, p., 2009: amino acid changes during sunflower infection by necrotrophic fungus b. cinerea. plant signaling and behavior 4, 859–861. fischer, w. n., kwart, m., hummel, s., frommer, w. b., 1995: substrate specificity and expression profile of amino acid transporters (aaps) in arabidopsis. journal of biological chemistry 270, 16315–16320. ghosh, a. k., naskar, a. k., sengupta, s., 1997: characterization of a xylanolytic amyloglucosidase of termitomyces clypeatus. biochimica et biophysica acta 1339, 289–296. horie, y., watanable, k., 1983: effect of various kinds of dietary protein and supplementation with limiting amino acids on growth, haemolymph components and uric acid extraction in silk worm, bombyx mori. journal of insect physiology 19, 187–199. haukioja, e., ruohomaki, k., suomela, j., vuorisalo, t., 1991: nutritional quality as a defense against herbivores. forest ecology and management 39, 237–245. haukioja, e., ossipov, v., lempa, k. 2002: leaf maturation vs. phenolics as modifiers of insect consumption and growth. entomologia experimentalis et applicata 104, 125– 136. hirner, b., fischer, w. n., rensch, d., kwart, m., frommer, w. b., 1998: developmental control of h1/amino acid permease gene expression during seed development of arabidopsis. the plant journal 14, 535–544. jones, c. g., hartley, s. e., 1999: a protein competition model of phenolic competition. oikos 86, 27–44. karley, a. j., douglas, a. e., parker, w. e., 2002: amino acid composition and nutritional quality of potato leaf phloem sap for aphids. journal of experimental botany 205, 3009–3018. martin, c., thimann, k. v., 1972: role of protein synthesis in the senescent leaves ii. the influence of amino acids in senescence. plant physiology 50, 432–437. matsubara, h., sasaki, r. m., 1969: high recovery of tryptophan from acid hydrolysis of proteins. biochemical and biophysical research communication 35, 175–181. mattson, w. j., scriber, j. m., 1987: nutritional ecology of insect folivores of woody plants: nitrogen, water, fiber, and mineral conditions. in: slansky, f., rodriquez, j. g. (eds.), nutritional ecology of insects, mites, spiders and related invertebrates, 105–146. john wiley and sons, new york. 32 acta bot. croat. 72 (1), 2013 roy n., laskar s., barik a. 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees moore, r. a., stein, w. h., 1948: photometric ninhydrin method for use in the chromatography of amino acids. journal of biological chemistry 176, 367–388. nation, j. p., 2001: insect physiology and biochemistry. crc press, boca raton. rentsch, d., frommer, w. b., 1996: molecular approaches towards an understanding of loading and unloading of assimilates in higher plants. journal of experimental botany 47, 1199–1204. roy, n., barik, a., 2012 a. the impact of variation in foliar constituents of sunflower on development and reproduction of diacrisia casignetum kollar (lepidoptera: arctiidae). psyche 2012, article id 812091, 9 pages. roy, n., barik, a., 2012 b. influence of four host plants on feeding, growth and reproduction of diacrisia casignetum (lepidoptera: arctiidae). entomological science doi. 1111/j.1479-8298.2012.000546.x ruuhola, t., ossipov, v., lempa, k., haukioja, e., 2003: amino acids during development of mountain birch leaves. chemoecology 13, 95–101. schoonhoven, l. m., van loon, j. j. a., dicke, m., 2005: insect-plant biology. oxford university press, oxford. shobana, k., murugan, k., naresh kumar, a., 2010: influence of host plants on feeding, growth and reproduction of papilio polytes (the common mormon). journal of insect physiology 56, 1065–1070. slansky, f., wheeler, g. s., 1992: caterpillars’ compensatory feeding response to diluted nutrients leads to toxic allelochemical dose. entomologia experimentalis et applicata 65, 171–186. strack, d., 1997: phenolic metabolism. in: dey, p. m., harborne, j. b. (eds.), plant biochemistry 387–416. academic press, gb-london. weibull, j., 1987: seasonal changes in the free amino acids of oat and barley phloem sap in relation to plant growth stage and growth of rhopalosiphum padi. annals of applied biology 111, 729–736. weibull, j., renquist, f., brishammar, s., 1990: free amino acid composition of leaf exudates and phloem sap. plant physiology 92, 222–226. acta bot. croat. 72 (1), 2013 33 amino acids in sunflower leaves 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 582 roy et al.ps u:\acta botanica\acta-botan 1-13\582 roy et al.vp 14. o ujak 2013 10:34:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 465 acta bot. croat. 73 (2), 465–470, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication muhlenbergia schreberi j. f. gmel (poaceae), a new naturalized species in croatia nejc jogan department of biology, biotechnical faculty, university of ljubljana, večna pot 111, si-1000 ljubljana, slovenia abstract – muhlenbergia schreberi, nimblewill, is a widespread north american perennial grass species, slowly spreading in european countries, where it has been recorded in spain, switzerland, italy, and slovenia. in addition, a well naturalized population was discovered in opatija (northwestern croatia, croatian littoral) in 2011 as described herein. it has been recognized as a persistent weed in some north american states, and in the last few decades its secondary european distribution range has been slowly increasing. thus most probably it will also spread in croatia and become classifi ed as invasive. keywords: croatia, muhlenbergia schreberi, neophyte, nimblewill, potentially invasive species introduction in the last decades, neophytes have been at the forefront of not only fl oristic research but also of nature conservation, which is becoming more and more aware of their potential importance as threats to indigenous biodiversity. so countries mostly have drawn up their lists of aliens or invaders (boršić et al. 2008, celesti-grapow et al. 2010, jogan et al. 2012). unfortunately, further from the mere act of listing, »the path becomes rough« and so alien species are spreading steadily and more and more new taxa are entering our nature. not all neophytes are easily recognizable in the fi eld and sometimes their secondary spread remain unnoticed for decades. this is certainly the case with some umbellifers, such as ammi visnaga (ruščić and nikolić 2011) and even more with some of the grasses. such is the case of sporobolus neglectus, recorded for the fi rst time in croatia soon after world war ii (marković 1973), but without any further records until recently, when it has been observed to be already widespread along several main roads in lowland croatia. most probably the case of muhlenbergia schreberi, recently recorded for the fi rst time in croatia in opatija, will be similar. * corresponding author, e-mail: nejc.jogan@bf.uni-lj.si copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. jogan n. 466 acta bot. croat. 73 (2), 2014 muhlenbergia schreberi j. f. gmel [m. diffusa willd.] the genus muhlenbergia schreb. belongs to the subfamily chloridoideae with majority of taxa in dry and warmer parts of the world. of the approximately 160 species, the majority are confi ned to america and only about a dozen are also native in the warmer parts of asia (clayton and renvoize 1986, mabberley 1990, watson and dallwitz 1992). the genus was named after the lutheran pastor heinrich ludwig mühlenberg (1756–1817), a famous researcher into north american fl ora, especially grasses (ascherson and graebner 1898–1902). in europe, the genus muhlenbergia is represented by only some ephemerophytes (ascherson and graebner 1898–1902, ryves et al. 1996, conert 1998) and by m. schreberi, which is locally naturalized. in addition, some authors include sporobolus vaginifl orus (a. gray) a. w. wood, a naturalized north american annual, in the genus muhlenbergia as m. vaginifl ora (a. gray) jogan (jogan 1999, poldini et al. 2002). muhlenbergia schreberi, nimblewill, is a perennial grass 0.1–0.3 m tall with a muchbranched geniculate culm rooting at the lower nodes, somehow cespitose. leaf sheaths are shorter than internodes, with ligule < 0.5 mm long, and leaf blades narrowly lanceolate, fl at, 2–3 mm wide and 3–7 cm long, more or less perpendicular to the culm. infl orescence is a distinctly contracted interrupted panicle, up to 20 cm long, with short branches, nodding, spikelets are numerous, on short pedicels, one-fl owered, < 2.5 mm long (awn not included). glumes are very reduced, < 0.5 mm long, lemma appressedly hairy, 2–2.5 mm long, tapering into a straight weak 2–5 mm long awn, distinctly 3-nerved, and a palea slightly shorter than lemma, visible. stamens are only slightly protruding at the top of the spikelet during anthesis, with yellow anthers 0.2–0.4 mm long. after anthesis, ripe spikelets disarticulate above the persistent glumes, caryopsis 1.3 × 0.3 mm. 2n = 40. flowering time is late, september and october. the primary distribution of m. schreberi is in the eastern part of north america, from central mexico to south-east canada, and south america southwards to north argentina. the centre of its distribution range seems to be in the eastern half of the usa. in south america it is reported for the warm temperate belt, i.e. for north argentina (nicora and rugolo de agrasar 1987), and is much rarer in colombia, south uruguay and south brazil. in its secondary distribution range, in switzerland nimblewill is restricted to the ticino/ tessin canton in the south fi rst recorded by becherer in 1963 (conert 1998) where it is still locally naturalized (aeschimann and burdet 1994, lauber and wagner 2001). in neighboring north italy it is reported for piedmont, lombardy, trentino-alto adige (aeschimann et al. 2005), and bolzano/südtirol (fischer et al. 2008). in slovenia it has been naturalized at least since the 1980s in the sub-mediterranean part on the junction between kras plateau and vipava valley (jogan 1990) and has been recently recorded naturalized in litija (central slovenia, unpublished). there was a neglected occurrence in north-eastern spain (catalunya, bordils 10 km north-east of girona) where the species was collected in 1932 in alluvial forest plantations of platanus and populus but it was discovered again in 2007 (pyke 2008). on the one hand this shows the persistence of the population and on the other hand the general fl oristic neglect of grasses. because of its occurrence in spain and italy, it could be expected to be found in france. it has also been recorded as a casual in united kingdom (ryves et al. 1996). muhlenbergia schreberi in croatia acta bot. croat. 73 (2), 2014 467 in the former ussr (cvelev 1976) m. schreberi is reported from the caucasian region (north ossetia, west transcaucasia, today georgia) and armenia, but its occurrence there is defi nitely not native as it is wrongly reported to be in euro+med plantbase (http://ww2. bgbm.org/europlusmed). in japan it is listed among invasive alien species as established (http://www.nies.go.jp/biodiversity/invasive/). its occurrence seems secondary also in the western usa, where nimblewill is treated as a noxious weed in california (peterson 2003). in south america the ecological conditions of m. schreneri are reported as hygrophilous subtropical forests and gallery forests (nicora and rugolo de agrasar 1987). in north america its ecology seems very diverse (peterson 2003, 2007). judging from the 89 herbarium labels from smithsonian (http://collections. si.edu/) of which about half mention the habitat type, it has mostly been collected in ruderal places (17/43), various types of wetlands (12/43), grasslands (8/43) and woods or shaded places (6/43), which is certainly only a rough estimation of the author, but clearly shows the habitat type preferences. hitchcock and chase 1971 simplify its ecology to »damp shady places«. in the caucasus region the grass was reported as a weed in plantations of subtropical plants (cvelev 1976). in switzerland m. schreberi is reported as a new-comer to an abandoned landfi ll site together with several other neophytes (bellosi et al. 2011) and in general it is scattered on road banks, lawns and among shrubs (lauber and wagner 2001) and several other ruderal places and forests (conert 1998). it prefers weakly acid substrates with enough humus, slightly sandy. in northeast spain, where m. schreberi populations have persisted for more than 70 years, it is reported to grow in river banks, wet and ruderal places (pyke 2008). in slovenia, the habitat types of m. schreberi are ruderal, forest fringes on shady slopes and meadows (jogan 1990). in the whole wider area of the alps m. schreberi is recognized as a species of geo-alliarion type of vegetation (aeschimann et al. 2005). methods the newly discovered locality of m. schreberi is the outcome of the author’s systematic fl oristic fi eld work focused on grasses. mapping is conducted in the frame of the informal international project »flora of istria« (starmühler 1998) comprising the wider territory of istria south of the line trieste-rijeka including the northern part of the kvarner archipelago, coordinated by w. starmühler (graz, austria). the fl oristic activities of several dozen collaborators have been carried out for roughly 15 years and preliminary results such as checklists and herbarium revisions have been published in a series of articles starting with starmühler 1998. herbarium vouchers are deposited in public herbaria and duplicates are offered to collaborating institutions. the fi rst collection of m. schreberi is deposited in herbarium lju (university of ljubljana, biotechnical faculty). results and discussion the fi rst known population of m. schreberi in croatia (fig. 1) was recorded in the kvarner littoral, in opatija, on lawns, forest fringes and ruderal places near the hotel ambasador, 45°20'26"n, 14°18'41"e, 0651/4 (leg. n. jogan 11. 12. 2011, herbarium lju). the population is hard to analyze because it is scattered over a frequently mown lawn on the south oriented slope in front of the hotel. the lawn is about 100 × 30 m in size and a few jogan n. 468 acta bot. croat. 73 (2), 2014 dozen stands of m. schreberi of different sizes up to a few square meters are scattered all over the lawn. due to very frequent mowing, only some culms can be found fl owering, but with its stolons this perennial plant is slowly spreading and it can be recognized quite easily during the winter by its dry whitish leaf blades or during the vegetation period by the quite dense leaves distinctly in two rows on vegetative shoots. propagation and further spread at the locality might be enhanced by frequent mowing that results in small parts of the stolons being dispersed and rooting at the nodes; propagation by seed is not to be excluded, however. in the autumn, the fl owering period of m. schreberi, only a couple of accompanying plant species are fl owering, e.g. cynodon dactylon, bothriochloa ischaemum, on more ruderal margins digitaria sanguinalis, setaria viridis and also an interesting american ephemerophyte cyclospermum leptophyllum (pers.) sprague ex britton and p. wilson which had not been recorded in croatia previously. in its native range m. schreberi is treated as a troublesome weed (brown 1979) especially in lawns, as it slowly spreads by stolons and patches on lawns gradually increase, but in autumn does not remain green, like other lawn grasses. it will at fi rst turn brown, and then, until the late spring the withered pale brown leaves are easily noticed as »dry« patches in the lawn. green leaves develop later in spring. because of the vegetative spread of the manybranched stolons, m. schreberi stands are almost without other plant species. also in several other weed lists m. schreberi has a status ranging from »casual alien« to »noxious weed« (compilation in global compendium of weeds, http://www.hear.org/gcw/species/ muhlenbergia_schreberi/), e.g. in california, kentucky, nebraska and great plains it is listed as invasive weed. fig. 1. muhlenbergia schreberi. a) typical summer aspect with densely growing shoots bearing almost perpendicular linear leaf blades; b) autumn aspect, when long narrow nodding panicles are developed; c) detail of panicle from herbarium specimen; d) spikelet, awned lemma and palea visible. pictures a) and b) were taken at a locality in litija, slovenia, as it was not possible to get such well developed plants in the opatija locality, while pictures c) and d) were taken from a herbarium specimen collected at the opatija locality. all photos were taken by n. jogan. muhlenbergia schreberi in croatia acta bot. croat. 73 (2), 2014 469 some authors recognize m. schreberi in europe as invasive or at least naturalized, but judging from the situation in its homeland, america, where the species is described as »noxious weed« or »environmental weed« (see above), in its secondary distribution range too it can slowly become at least a local weed of the ruderal places, if not an invasive grassland species. in europe it is listed as neophytic threat in italy (as »invasive« in lombardy and veneto, »naturalized« in piedmont, »casual« in alto adige; celesti-grapow et al. 2010), switzerland (status »unknown« by wittenberg ed. 2006 and later corrected to »naturalized« on web version of the same document, see http://www.sib.admin.ch/uploads/media/ uw-0629-e.pdf) and slovenia (»potentially invasive«, jogan et al. 2012). we can conclude that for several decades nimblewill has obviously been a well naturalized species in several european countries with some mediterranean climatic impact. it is slowly spreading by seeds and vegetatively, and as a late-fl owering grass its occurrence is often overlooked. most probably also in the newly recorded locality in opatija the species has been present for several years already and thorough fi eld work in autumn will certainly reveal more populations scattered in the kvarner littoral. at the moment, its occurrence seems confi ned to ruderal places, but diversity of microhabitats and conditions similar to those in natural habitat types (e.g. forest fringes, wet grasslands, even rocks) suggests its possible spreading in the vicinity. in croatia m. schreberi can be listed as a naturalized neophyte, most probably not (yet) as an invasive species, but the wider geographical area adjacent to the discovered locality needs to be explored. references aeschimann, d. lauber, k, moser, d. m., theurillat, j.-p., 2005: flora alpina 2. haupt verlag, bern, stuttgart, wien. aeschimann, d., burdet, h. m., 1994: flore de la suisse et des territories limitrophes. 2. aufl . le nouveau binz, neuchatel. ascherson, p., graebner, p. 1898–1902: synopsis der mitteleuropäischen flora 2/1. wilhelm engelmann, leipzig. bellosi, b, selldorf, p., schoenenberger, n., 2011: exploring the fl ora on inert landfi ll sites in southern ticino (switzerland). bauhinia 23, 1–16. boršić, i., milović, m., dujmović, i., bogdanović, s., cigić, p., rešetnik, i., nikolić, t., mitić, b. 2008: preliminary check-list of invasive alien plant species (ias) in croatia. natura croatica 17, 55–71. brown, l., 1979: grasses, an identifi cation guide. houghton miffl in co., boston. celesti-grapow, l., pretto, f., carli, e., blasi, c.:2010: flora vascolare alloctona e invasiva delle regioni d’italia. casa editrice universita la sapienza, roma. clayton, w. d., renvoize, s. a., 1986: genera graminum. hmso, london. conert, h. j., 1998: gramineae. in: hegi, g. (ed.), illustrierte fl ora von mitteleuropa. paul parey, hamburg. cvelev, n. n., 1976: zlaki sssr. nauka, leningrad. fischer, m. a., adler, w., oswald k., 2008: exkursionsfl ora. österreich, liechtenstein, südtirol. biologiezentrum der oberösterreichischen landesmuseen, linz. jogan n. 470 acta bot. croat. 73 (2), 2014 hitchcock, a. s., chase a., 1971: manual of the grasses of the united states. dover, new york. jogan, n., bačič, t., strgulc krajšek, s., 2012: neobiota from slovenia, final report of the project. retrived october, 2012 from http://www.bioportal.si/neobiota.php. jogan, n., 1990: contribution to the knowledge of grasses (poaceae) in slovenia (in slovenian). biološki vestnik 38, 27–38. jogan, n. 1999: grass – poaceae. in: martinčič, a., wraber, t., ravnik, v., jogan, n., podobnik, a., turk, b., vreš, b. (eds.), little fl ora of slovenia (in slovenian), 711–813. tehniška založba slovenije, ljubljana. lauber, k., wagner, g., 2001: flora helvetica, 3. aufl . haupt verlag, bern, stuttgart, wien. mabberley, d. j., 1990: the plant-book. cambridge university press, cambridge. marković, l., 1973: sporobolus neglectus nash, new adventitious species in yugoslavia (in croatian). acta botanica croatica 32, 237–242. nicora, e. g., rugolo de agrasar, z., 1987: los generos de gramineas de america austral. editorial hemisferio sur, buenos aires. peterson, p. m., 2003: muhlenbergia schreb. in: barkworth, m. e., capels, k. m., long s., piep, m. b. (eds.), flora of north america 25. magnoliophyta: commelinidae (in part): poaceae, part 2., 145–200. oxford university press, new york. peterson, p. m., 2007: muhlenbergia schreb. in: barkworth, m. e., anderton l. k., capels k. m., long s. (eds.), manual of grasses for north america, 219–230. usu press, logan. poldini, l. vidali, m., oriolo, g., 2002: nuovo atlante corologico delle piante vascolari nel friuli venezia giulia. regione autonoma friuli venezia giulia, azienda parchi e foreste regionali & universita degli studi di trieste, dipartimento di biologia. udine. pyke, s., 2008: contribución al conocimiento de la fl ora alóctona catalana, collectanea botanica 27, 95–104. ruščić, m., nikolić, t., 2011: ammi visnaga (l.) lam. (apiaceae), a new taxon in croatian fl ora. acta botanica croatica 70, 301–306. ryves, t. b., clement, e. j., foster, m. c., 1996: alien grasses of the british isles. bsbi, london. starmühler, w., 1998: vorarbeiten zu einer »flora von istrien« i. carinthia ii 188/108, 535–576. watson, l., dallwitz, m. j., 1994: grass genera of the world: descriptions, illustrations, identifi cation, and information retrieval; including synonyms, morphology, anatomy, physiology, phytochemistry, cytology, classifi cation, pathogens, world and local distribution, and references. retrived from http://delta-intkey.com/grass. wittenberg, r., 2006: an inventory of alien species and their threat to biodiversity and economy in switzerland. cabi bioscience switzerland centre report to the swiss agency for environment, forests and landscape. the environment in practice no. 0629. federal offi ce for the environment, bern. opce-str.vp acta bot. croat. 71 (1), 1–12, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 protein glycosylation in sugar beet cell line can be influenced by dna hyperand hypomethylating agents dubravko pavokovi]*, marijana krsnik-rasol department of molecular biology, division of biology, faculty of science, university of zagreb, horvatovac 102a, hr-10000 zagreb, croatia abstract – protein glycosylation is a coand post-translational modification that influences protein function, stability and localization. changes in glycoprotein pattern during differentiation/dedifferentiation events exist in animal cells and dna methylation status is closely related to the changes. however, in plant cells this relationship is not yet established. in order to verify whether such a relation exists, hypermethylating drugs 2,4-dichlorophenoxyacetic acid and hydroxyurea, or hypomethylating drug 5-azacytozine were applied to sugar beet (beta vulgaris l.) cells during 14 days of in vitro subculture, and the glycoprotein patterns of the cells were compared. the applied drugs were not toxic, as observed from cell phenotype and by measuring growth of the control and treated cells. hyper and hypomethylating treatments influenced the activity of enzymes related to differentiation state of the cells: peroxidases and esterases, and their isoform patterns. electrophoretic patterns of soluble and membrane proteins were similar between control and treatments, but the treatments modified nand o-linked glycoprotein patterns as visible from gna and pna lectin blots. this suggested that hypermethylation and hypomethylation of genomic dna in sugar beet cells affect protein glycosylation patterns and cellular metabolism, possibly in a mechanism similar to that existing in animal cells. key words: cell differentiation, dna methylation, glycoproteins, sds-page, sugar beet abbreviations: 2,4-d – 2,4-dichlorophenoxyacetic acid, 5-a – 5-azacytozine, gna – galanthus nivalis agglutinin, hno – habituated nonorganogenic cell line, ho – habituated organogenic cell line, hu – hydroxyurea, n – normal cell line, pna – peanut agglutinin sds-page – sodium dodecyl sulphate polyacrylamide gel electrophoresis introduction protein glycosylation is the gradual attachment of different carbohydrates to a nascent protein carried out in endoplasmic reticulum and golgi apparatus. carbohydrates may vary but the common ones attached are mannose, glucose, galactose, n-acetylgalactosamine, xylose, fucose, and n-neuraminic acid or, sialic acid (spiro 2002). the attachment site is acta bot. croat. 71 (1), 2012 1 * corresponding author, e-mail: dubravko@zg.biol.pmf.hr copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:17 color profile: disabled composite 150 lpi at 45 degrees on the specific asparagine (n-linked glycosylation) or on serine/threonine residues (o-linked glycosylation). in animal tissues, nand o-linked glycans are involved in a number of normal and aberrant physiological functions such as cell adhesion, signal transduction, protein conformational stabilization and targeting, protease resistance, charge umbrella, water-binding capacity, cellular recognition and developmental regulation (for a review see varki 2008). changes in protein glycosylation are observed during normal cell and tissue differentiation but also during malignant transformation of animal cells (peracaula 2007, hedlund et al. 2008). epigenetics, like dna methylation level, has important roles in this process by affecting activities of protein glycosyltransferases (kannagi 2004, kawamura et al. 2008, kannagi et al. 2010). in plants, nand o-linked glycans strongly influence protein conformation, stability, biological activity (rayon et al. 1998). the glycan part of the protein serves as a subcellular localization signal (fitchette et al. 2007). disruption of normal protein glycosylation severely affects plant development causing embryo lethality, severe defects in cell wall biogenesis or plant growth retardation (vitale 2001, klein et al. 2006). protein glycosylation is influenced by metabolic changes occurring in the tissues, such as acclimatization to in vitro conditions (balen et al. 2008) or by the developmental state and the age of the plants (elbers et al. 2001). however, it is not known whether using agents that change the dna methylation level in plant cells will change protein glycosylation, as it does in animal cells. sugar beet cell lines have been thoroughly investigated in culture for more than 20 years and are established as a suitable model for studies of plant metabolism and cell differentiation (de greef and jacobs 1979, causevic et al. 2005, causevic et al. 2006). the n (normal) line is photosynthetic, differentiated but not organogenic. the ho (habituated organogenic) line is photosynthetic, differentiated and organogenic, shoot-producing. the hno (habituated non-organogenic) is etiolated, and dedifferentiated. the cell lines, coming from the same mother plant, have distinct genomic dna methylcytosine content, cell differentiation level and morphology. using hypomethylating drugs like 5-azacytozine (5-a) or hypermethylating drugs like 2,4-dichlorophenoxyacetic acid (2,4-d) and hydroxyurea (hu) it is possible to affect dna methylation levels, modify metabolism, phenotype and ultimately differentiation state of the cells (causevic et al. 2005). this model system seemed suitable to test whether externally applied agents that modify dna methylation levels will affect nand o-glycosylation patterns of proteins in sugar beet n cell line. as a result of previous observations, the n line was chosen because its dna cytosine methylation level is normally at 22% and it is between ho, at 18%, and hno, at 29% (causevic et al. 2005), which should give more flexibility in changes of methylation level and differentiation status during the experiment. both, hypermethylating and hypomethylating treatments induced cell dedifferentiation in the n line, causing a part of the cells to change their morphology and look like hno cells, white and very friable (causevic et al. 2005). a suitable way to discern changes in metabolism and differentiation state indirectly is to monitor activity and isozyme patterns of certain enzymes. peroxidases but also esterases are markers of cellular or organ differentiation (krsnik-rasol et al. 1999). in sugar beet, peroxidase activities were related to the dna methylcytosine percentage (causevic et al. 2005). in cactus mammillaria gracilis, as well as in cotton embryos, the number of esterase isoforms depended on the morphological state of the tissue (balen et al. 2003, hilaire et al. 2007). 2 acta bot. croat. 71 (1), 2012 pavokovi] d., krsnik-rasol m. u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:17 color profile: disabled composite 150 lpi at 45 degrees in this work, the n cells were treated with 5-a, 2,4-d and hu during the period of one subculture. toxicity of the treatments was evaluated by measurement of the growth of the cells. changes in metabolism and protein glycosylation pattern were observed, which could be related to the action of the drugs used. material and methods plant material, growth conditions and treatments the cell lines of sugar beet originate from the same mother plant and were cultivated in vitro on a solid pg0 nutrient media (negrutiu et al. 1975, de greef and jacobs 1979). the n cell line was grown in a growth chamber at 22 °c under a 16 h photoperiod (80 µmol photons m–2 s–1). for treatments, the toxicity of the chemicals was evaluated first using concentrations in the range of 25–200 µmol l–1 of 2,4-d, 100–1000 µmol l–1 of 5-a, and 200–400 µmol l–1 of hu (all purchased from sigma, germany) (data not shown). the chemicals were added as sterile solutions immediately before casting the nutrient medium in petri dishes. based on the obtained results we analyzed the enzyme activity and glycoprotein pattern in plant material upon treatment with optimal concentrations of chemical agents: 2,4-d (100 µmol l–1), and hu (200 µmol l–1) that resulted in maximal increase in fresh weight of cells in comparison to control, as well as 5-a (200 µmol l–1) that minimally inhibited the plant growth in comparison to control. tissue samples were collected at days 7, and 14 of subculture, weighted for growth analysis, frozen in liquid nitrogen and stored at –80 °c. protein extracts and enzyme analysis sugar beet n tissue was grinded in a bead mill retsch 200 (retsch gmbh, germany) for 2 min at 30 hz. tissue was supplemented with ice-cold potassium phosphate buffer ph 7.0 or tris-hcl buffer ph 7.4. crude protein extracts were clarified by centrifugation at 20 000 x g for 20 min and again at 20 000 x g for 10 min, all at 4 °c. protein content of supernatant was determined according to bradford (1976). guaiacol peroxidase activity of protein extracts was determined spectrophotometrically (ati unicam uv4, cambridge, uk) at room temperature (25 °c) by measuring the increase in absorbance at 470 nm. the test solution was prepared after siegel and galston (1967), with 5 mmol l–1 guaiacol and 5 mmol l–1 h2o2, in a 100 mmol l–1 potassium phosphate buffer ph 7.0. a broad spectrum esterase substrate 1-naphthylacetate was used for esterase activity measurement according to burlina and galzigna (1972). esterase activity was determined spectrophotometrically at room temperature (25 °c) by measuring the increase in absorbance at 322 nm. the reaction medium contained 1.5 ml of 100 mmol l–1 tris-hcl ph 7.4 and 30 ìl of 100 mmol l–1 1-naphthylacetate dissolved in methanol. for each measurement 150 ìl of crude extract was used. measurements were performed every 15 seconds over a three min period. the esterase activities were corrected for spontaneous hydrolysis of 1-naphthylacetate. the blank contained only the buffer and 1-naphthylacetate. the activities of peroxidases and esterases were expressed as a percentage of the control. experiments were performed at least in triplicates. acta bot. croat. 71 (1), 2012 3 dna methylation and glycoproteins of sugar beet cells u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:17 color profile: disabled composite 150 lpi at 45 degrees statistical analysis of enzyme activities by means of one-way anova was performed using embedded functions in sigmaplot 12 software (systat software inc. ca, usa) and results were considered significant when p < 0.05. protein gel electrophoresis soluble proteins were extracted according to pavokovi] et al. (2007). pellet left after extraction of soluble proteins was washed three times in extraction buffer and then 0.5 ml of extraction buffer containing 130 mmol l–1 tris-hcl ph 6.8, 4.6% (w/v) sodium dodecyl sulphate (sds), 16% glycerol and 0.6% (w/v) dithiothreitol, preheated to 80 °c was added to the pellet to disrupt the membranes and to extract membrane proteins. homogenates were incubated at 80 °c for 10 min on a shaker, and clarified by centrifugation at 20000 x g for 10 min. supernatants were decanted and another 0.5 ml of the same pre-heated sds-extraction buffer was added to the pellet. samples were incubated again under the same conditions as above. after centrifugation, supernatants were pooled and used in subsequent electrophoretic analyses as membrane protein fraction. sds polyacrylamide gel-electrophoresis of protein samples was carried out in a vertical gel system in 12% polyacryamide gels with the buffer system of laemmli (1970). proteins were visualized using coomassie brilliant blue stain. soluble protein samples were also separated in native conditions by vertical electrophoresis in 8–18% polyacrylamide gradient gels with the same buffer system, but without sds. in-gel activity of peroxidase and esterase isoforms were performed as described earlier (balen et al. 2003), based on the methods developed by siegel and galston (1967). electroblot and lectin hybridization following sds-pag electrophoresis, proteins were electroblotted onto nitrocellulose membrane for 1h, at 60v. to detect glycoconjugates, lectins from galanthus nivalis (gna) and peanut (pna) were used. lectins were conjugated with digoxigenin (dig), and anti-dig alkaline phosphatase was used as a secondary anti-body (roche diagnostics gmbh, germany). site of the hybridization was visualized using bcip/nbt as a substrate for the alkaline phosphatase. gels and membranes were scanned on a flatbed scanner (hp, usa) at 300 dpi, and protein and glycoprotein expression profiles were analyzed by uthscsa image tool v3.0 (tx, usa). results here we report that the use of externally applied drugs hu, 2,4-d and 5-a, which change dna methylation level, affects glycoprotein patterns in sugar beet n cell line. eventual toxicity of the dna modifying treatments was verified by comparing cell growth (fig. 1). cells treated with 200 µmol l–1 hu proliferated similarly to the control, while cells treated with 100 µmol l–1 2,4-d and 200 µmol l–1 5-a proliferated slowly but without any apparent necrosis or growth arrest. however, a concentration of 200 µmol l–1 of 2,4-d in the medium was toxic: growth stopped and cells quickly became necrotic (data not shown). 4 acta bot. croat. 71 (1), 2012 pavokovi] d., krsnik-rasol m. u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:17 color profile: disabled composite 150 lpi at 45 degrees the treatments had a profound effect on cellular metabolism, which was visible from measurements of esterase and peroxidase activities, and by in-gel visualization of their isoforms at day 7 and day14 of the subculture (fig. 2). esterase activity was estimated using 1-naphtylacetate as a substrate (fig. 2b). when compared with the control, all treatments significantly increased esterase activity at days 7 and 14 of the subculture. after native electrophoresis, in-gel staining of esterases showed five isoforms: e1–e5 (fig. 2a). treatments differentially induced esterase isoforms, but the changes of the signal of isoforms e3–e5 were the most remarkable. isoform e5 was particularly induced by the 2,4-d and 5-a treatments. the guaiacol peroxidase activities were also modified by treatments in both data points (fig. 2c). at day 7, significant decrease in activity was observed in 5-a and hu treated cells. at day 14, 2,4-d and 5-a significantly decreased peroxidase activity, but the hu treatment significantly increased its activity. however, in-gel activity of peroxidases could not be obtained under the same conditions, probably due to the isoelectric nature of the peroxidase isoforms. application of agents that modify dna methylation level changed glycosylation patterns, which was observed by differential binding of gna and pna lectins to glycoproteins. expression of soluble and membrane proteins remained unchanged, as shown by coomassie brilliant blue staining (fig. 3a, b). we observed that fewer soluble proteins were glycosylated than membrane proteins (fig. 3c–f), which is in accord with the fact that most glycosylated proteins are destined to plasma membranes and to the extracellular space. in soluble protein fraction, no differences were observed in the glycosylation pattern of high-mannose glycans (fig. 3c). in the membrane fraction (fig. 3d), a glycoprotein of 20 kda was induced at days 7 and 14 with 2,4-d and at day 7 with hu. at day 14 hu the signal of this glycoprotein was lost, together with that of 22 kda. in soluble protein fraction pna lectin revealed two o-linked glycoproteins with sizes around 40 and 42 kda (fig. 3e). acta bot. croat. 71 (1), 2012 5 dna methylation and glycoproteins of sugar beet cells fig. 1. growth of the sugar beet n cell line treated with hyperor hypomethylating drugs. data are mean ± se for three replicates. u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:19 color profile: disabled composite 150 lpi at 45 degrees at 7 days all treatments decreased the signal from the 42 kda glycoprotein. at day 14, in the control and in the 2,4-d and 5-a the 40 and 42 kda glycoproteins were not detected; however, with hu, both of those glycoproteins reappeared. in the membrane fraction pna lectin also revealed more o-linked glycoproteins than in the soluble fraction. induction of a 18 kda glycoprotein appeared after 7 and 14 days in cells treated with 2,4-d. a 40 and 20 kda glycoproteins were downregulated by 5-a and hu at 14 days of the treatment (fig. 3f). discussion here we have shown that the application of chemicals that change genomic dna methylation level affected cellular metabolism and glycosylation of cellular and membrane proteins in sugar beet cells. significant genomic dna hypermethylation was successfully induced in several plant species, including sugar beet, with 2,4-d and hu, and dna hypomethylation with 5-a, with concentrations similar to or higher than those applied here, and without any apparent toxicity (causevic et al. 2005, yamamoto et al. 2005). 6 acta bot. croat. 71 (1), 2012 pavokovi] d., krsnik-rasol m. fig. 2. the effect of hyperor hypomethylating drugs on esterase and peroxidase activities and its isoenzyme patterns in sugar beet n line. a – esterase izoenzyme patterns visualized with 1-naphtylacetate after native electrophoresis of 100 mg of protein samples in a 12% polyacrylamide gel; b – esterase activities expressed as a percentage of control; c – peroxidase activities expressed as a percentage of control; ctrl – control; 2,4d – 2,4-dichlorophenoxyacetic acid; 5-a – 5-azacytozine; hu – hydroxyurea. data are mean ± se for at least three replicates. asterisks denote statistically different enzyme activity at p<0.05. u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:20 color profile: disabled composite 150 lpi at 45 degrees since the morphological changes in n cells were minimal after treatments, as an indirect proof of changes in differentiation status we monitored the changes of activities of peroxidases and esterases. here we measured statistically significant differences in activities of esterases and peroxidases after the treatments. these enzymes have been established acta bot. croat. 71 (1), 2012 7 dna methylation and glycoproteins of sugar beet cells fig. 3. metabolic changes induced by hyperor hypomethylating drugs affect glycosylation pattern of cellular and membrane proteins in sugar beet n line. proteins were electroblotted onto nitrocellulose membrane and different carbohydrates as glycoconjugates were detected using plant lectins. a – cbb stained gel of soluble proteins; b – cbb stained gel of membrane proteins; c – high-mannose glycoproteins detected with gna lectin in soluble protein fraction; d – high-mannose glycoproteins detected with gna lectin in membrane protein fraction; e – o-linked glycoproteins detected with pna lectin in soluble protein fraction; f – o-linked glycoproteins detected with pna lectin in membrane protein fraction. numbers on the left show protein size in kda. c – control; 2,4d – 2,4-dichlorophenoxyacetic acid; 5-a – 5-azacytozine; hu – hydroxyurea. u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:22 color profile: disabled composite 150 lpi at 45 degrees as biochemical markers of cell differentiation and morphogenesis in plants (krsnik-rasol et al. 1999, balen et al. 2003, passardi et al. 2005). peroxidase activity can be affected by dna methylation status, which was already reported in bryonia dioica internodes and in sugar beet cells, both treated with dna hypoor hypermethylating agents (galaud et al. 1993, causevic et al. 2005). while causevic et al. (2005) reported an increase in activity of guaiacol peroxidases after treatment with 5-a and a decrease after hu treatment, we reported here the opposite situation. the cause for such discrepancy could lie in different extraction methods, which could yield different peroxidase isoforms. it is known that there are a huge number of peroxidases in plant cells, each with a specific function assigned. for example, in arabidopsis, there are 73 predicted peroxidase genes (oliva et al. 2009). we also observed in native gels that esterase isoforms, and especially isoform e5, was more intensively colored with the treatments 5-a and hu. it is tempting to propose that the e5 isoform might be an indirect proof of changes of dna methylation. changes in the number and intensity of esterase isoforms occur when tissues of identical origin change their morphological status, as in mammillaria plant, callus or tumor tissues (balen et al. 2003). similarly, the number of esterase isoforms increased when cotton cells differentiated during embryogenesis (hilaire et al. 2007). it seems that the changes in activity of peroxidases and esterases reflected metabolic changes caused by the drugs used. to detect changes in glycoprotein patterns, gna and pna lectins were used. gna lectin binds to the terminal mannose of the n-glycans (shibuya et al. 1988), and pna specifically recognizes the galactosy1 â-1,3-n-acetyl galactosamine sequence present in the core unit of o-glycans (goldstein and heyes 1978). using these lectins it was possible to detect a large number of nand o-linked glycans and make a proper screening of changes in the glycosylation pattern. in mammalian cells a possible explanation behind altered protein glycosylation was proposed in which the dna methylation and/or histone deacetylation alter cellular carbohydrate metabolism, sugar transportation and expression of genes for sugar transporters (kannagi 2004). subsequent experiments on gastrointestinal cancer cells revealed that inhibition of dna methylation with 5-aza-2’-deoxycytidine silences some genes belonging to the glycosyltransferase and glycosidase families (kawamura et al. 2008). this repressed aberrant glycosylation and cancer-associated carbohydrate antigens. in plants, 2,4-d can strongly modulate activity of some glycosyltransferases and induce glycosylation of biological molecules such as scopoletin and quercetin in tobacco and vitis sp. cell cultures, respectively (kokubo et al. 2001, taguchi et al. 2001). it remains to be seen whether application of 2,4-d affects the activity of protein glycosyltransferases. in carrot 2,4-d transiently induced dna methyltransferase gene met1–5 before the induction of somatic embryogenesis in epidermal cells (yamamoto et al. 2005). hydroxyurea, like the 2,4-d increases dna methylation level, but by a mechanism that involves inhibition of the enzyme ribonucleotide reductase (koç et al. 2004). this is most probably different mechanism, than the one by which 2,4-d acts on dna methylation, which could explain the differences in glycoprotein pattern after treatments with these two different dna hypermethylators. in plants, changes in glycosylation pattern are observed during developmental processes in which cell differentiation is affected, like embryogenesis in pumpkin or establishment of in vitro tissues of cactus mamillaria sp. (leljak-levani] et al. 2011, balen et al. 8 acta bot. croat. 71 (1), 2012 pavokovi] d., krsnik-rasol m. u:\acta botanica\acta-botan 1-12\469 pavokovic.vp 26. o ujak 2012 10:09:22 color profile: disabled composite 150 lpi at 45 degrees 2005, balen et al. 2008). in mamillaria sp. the changes lead to the appearance of novel protein n-glycan structures (balen et al. 2005). unfortunately, the function of these glycan structures on protein stability and location is not known. a specific protein expression pattern after establishment and/or maintenance of the differentiation state in plant cells is enabled by the changes in dna methylation status, most probably by the methylation of promoters regions (berdasco et al. 2008). in conclusion, we have shown that chemicals that change the methylation status of genomic dna affect cellular metabolism and glycosylation protein pattern in sugar beet n cell line, strengthening the relationship between dna methylation level and protein glycosylation. this process could be similar to the process already reported in mammalian cells. by following an approach similar to that used for mammalian cells, e.g. bisulfite pcr, it could be possible to pinpoint specific promoters that are differentially methylated during changes in genomic dna methylation, and to gain more information how dna methylation status controls protein glycosylation in plants. additionally, research into protein glycosylation in plants presents an opportunity to identify protein biomarkers of the specific developmental state of a cell or a tissue. acknowledgements financial support for this work was provided by the ministry of science, education and sports of the republic of croatia (project no. 119-1191196-1200). references balen, b., krsnik-rasol, m., simeon-rudolf, v., 2003: isoenzymes of peroxidase and esterase related to morphogenesis in mammillaria gracillis pfeiff. tissue culture. journal of plant physiology 160, 1401–1406. balen, b., zamfir, a., vakhrushev, s. y., krsnik-rasol, m., peter-katalini], j., 2005: determination of mammillaria gracillis n-glycan patterns by esi q-tof mass spectrometry. croatica chemica acta 78, 463–477. balen, b., peharec, p., krsnik-rasol, m., 2008: developmentally specific soluble and membrane proteins and glycoproteins in mammillaria gracillis pfeiff. 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lpi at 45 degrees 80 acta bot. croat. 82 (1), 2023 acta bot. croat. 82 (1), 80–82, 2023 coden: abcra 25 doi: 10.37427/botcro-2022-026 issn 0365-0588 eissn 1847-8476 short communication new bryophyte taxa for bosnia and herzegovina jovana p. pantović1*, svetlana n. grdović2, marko s. sabovljević1,3 1 university of belgrade, faculty of biology, institute of botany and botanical garden, takovska 43, 11000 belgrade, serbia 2 university of belgrade, faculty of veterinary medicine, bulevar oslobođenja 18, 11000 belgrade, serbia 3 pavol jozef šafárik university, institute of biology and ecology, faculty of science department of botany, mánesova 23, 040 01 košice, slovakia abstract – bosnia and herzegovina has a long history of bryophyte flora research. however, it is still considered insufficiently investigated, and until recently bryophyte investigations were completely neglected. hence new records for the country are expected with novel explorations. here, we report one liverwort (porella obtusata) and four moss species (bryum klinggraeffii, cinclidotus danubicus, habrodon perpusillus and imbribryum subapiculatum) new for the country’s bryophyte flora. with these new records, the bryoflora of bosnia and herzegovina numbers 673 taxa (no hornworts, 134 liverworts and 539 mosses). keywords: balkans, bryoflora, liverwort, mosses, new records introduction the balkan peninsula is characterized by a diversity of geological history, climate and habitat types, which all resulted in a diverse and rich bryophyte flora in a relatively small area (sabovljević et al. 2011). lately, numerous new records have been published within the balkan region, i.e. albania, croatia, north macedonia, montenegro and serbia (e.g., sabovljević et al. 2010). in spite of that, distributional data of many species are still incomplete, especially for ephemeral species and those that are taxonomically difficult and unresolved. furthermore, certain parts of many regions and countries are to date completely unexplored in terms of bryology. bryological research in bosnia and herzegovina started with sendtner in the middle of the 19th century (kummer and sendtner 1849). however, explorations were sporadic, with long interruptions (grgić 1985), hence there is a lack of recent and updated floristic data, in particular of certain areas. for example, only a few studies were published recently for bosnia and herzegovina (e.g., pantović et al. 2016, 2017). at present, the bryophyte flora of the country numbers 133 liverworts and 535 moss taxa (hodgetts and lockhart 2020, ellis et al. 2021a,b). materials and methods the subject of the bryological research was the tributaries of the lower course of the neretva river in the region of southern herzegovina (bosnia and herzegovina, se europe), namely the rivers buna, bunica, bregava, tihaljina and trebižat (fig. 1). the area of southern herzegovina is influenced by a mediterranean and sub-mediterranean climate. this area has approximately 2,291 hours of sunshine per year, while the vegetation period lasts around or more than 240 days. although the precipitation is high, with an average rainfall of 1,515 mm y-1, owing to the porous nature of its karstic soil, there is a general lack of surface water (galić 2011). the main features of the investigated area result in the richness of natural geomorphological, hydrological and biological values (redžić et al. 2008, lasić and jasprica 2016). the bryophyte samples were collected in august 2020. all main habitat types alongside the river courses were investigated, and specimens were collected from various substrata, e.g. soil, rocks, and tree bark. the list of species localities with details is given below. voucher specimens were deposited in the bryophyte collection of the herbarium of university of belgrade (beou). nomenclature for liverworts and mosses follows hodgetts and lockhart (2020). the investigated sites include: 1 – peć mlini, 43.33747 n, 17.32592 e, 143 m a.s.l., date 11.08.20., leg: jovana pantović (jp) & gordana čokanović (gč), det: jp, 2 – peć mlini, 43.33698 n, 17.32373 e, 137 m, 11.08.20., leg: jp & gč, det: marko sabovljević (ms), 3 – bagin most, humac, 43.18675 n, 17.51575 e, 69 m, 11.08.20., leg: jp & gč, det: ms, 4 – struge, 43.09229 n, 17.69733 e, 7 m, 12.08.20., leg: jp & gč, * corresponding author e-mail: jpantovic@bio.bg.ac.rs new bryophyte taxa for bosnia and herzegovina acta bot. croat. 82 (1), 2023 81 det: ms, 5 – trebižat river, near hacijenda bar, 43.12312 n, 17.67339 e, 17 m, 12.08.20., leg: jp & gč, det: ms, 6 – bagin most, 43.18342 n, 17.52270 e, 64 m, 13.08.2020., leg: jp & gč, det: jp, 7 – ljubuški, baščine, 43.18040 n, 17.52745 e, 65 m, 13.08.20., leg: jp & gč, det: jp, 8 – bregava river, near the confluence, 43.10134 n, 17.73029 e, 8 m, 13.08.20., leg: jp & gč, det: jp, 9 – bregava river, near mini hydroelectric power plant “do”, 43.08491 n, 18.00514 e, 94 m, 14.08.20., leg: jp & gč, det: jp, 10 – buna and neretva confluence, 43.23527 n, 17.83394 e, 25 m, 15.08.20., leg: jp & gč, det: jp, 11 – buna and neretva confluence, 43.23570 n, 17.83407 e, 30 m, 15.08.20., leg: jp & gč, det: jp, 12 – buna river, dokića pond, 43.24556 n, 17.84653 e, 367 m, 15.08.20., leg: jp & gč, det: ms, 13 – bunica river, malo polje, 43.23239 n, 17.88093 e, 42 m, 15.08.20., leg: jp & gč, det: jp, 14 – bunica river, 43.23667 n, 17.86907 e, 38 m, 15.08.20., leg: jp & gč, det: jp, 15 – tekija, buna river source, 43.25667 n, 17.90298 e, 38 m, 15.08.20., leg: jp & gč, det: jp. results and discussion here we report five new species for the bryophyte flora of bosnia and herzegovina: one liverwort and four mosses (number referring to sites given in text of materials and methods). bryum klinggraeffii schimp.: site 13: wet rocks by the river. the ruderal moss b. klinggraeffii is widespread through europe, but it is red-listed in some countries like portugal (critically endangered – cr), romania (endangered – en), and slovenia (data deficient – dd) (hodgetts and lockhart 2020). cinclidotus danubicus schiffn. & baumgartner: site: 3: rocks in the water; site 4: the platanus sp. roots by the water; site 6: rock by the river; site 7: roots by the water; site 8: limestone in dry riverbed; site 10: rocks in the water; site 11: salix sp. bark; site 13: rocks in the water; site 15: rocks in the water. this species is endemic for europe, and in the balkan region is known only from croatia and slovenia as well as from hungary. (hodgetts and lockhart 2020). habrodon perpusillus (de not.) lindb.: site: 5: fraxinus sp. bark; site 9: populus nigra bark. this species is common in the mediterranean region; however, it is rare and red-listed in some non-mediterranean countries, e.g. romania (cr), norway, great britain, slovenia (en), canary islands and switzerland (vulnerable – vu) (hodgetts and lockhart 2020). imbribryum subapiculatum (hampe) d.bell & holyoak: site 1: rock crevice by the water; site 2: tufa; site 13: wet rocks by the river; site 14: wet soil by the river. imbribryum subapiculatum is a temperate species somewhat less frequent in the balkan peninsula, probably due to misidentification with other species of small tuber-bearing bryum species. porella obtusata (taylor) trevis.: site 12: populus nigra bark. porella obtusata is a liverwort with a southwestern distribution in europe. it is considered a threatened species in some european countries, for example it is endangered (en) in norway, vulnerable (vu) in serbia and near threatened (nt) in italy and the canary islands (hodgetts and lockhart 2020). the bryoflora of bosnia and herzegovina, together with the new records reported here numbers 673 taxa (134 liverworts and 539 mosses). further new findings of bryophytes are expected with the intensification of field investigation. fig. 1. position of the investigated area of the lower course of the neretva river within bosnia and herzegovina. all recorded localities of five new species records for the country (liverwoth porella obtusata and mosses bryum klinggraeffii, cinclidotus danubicus, habrodon perpusillus and imbribryum subapiculatum) are marked on the map with a unique symbol. pantović j. p., grdović s. n., sabovljević m. s. 82 acta bot. croat. 82 (1), 2023 acknowledgments this research was conducted as a part of a project “steps towards the protection of neretva tributaries of the neretva: buna, bunica, bregava and trebižat, bosnia and herzegovina” financially supported by the critical ecosystem partnership fund (cepf). the list of bryophyte species from this area will be published in a popular educational booklet on biodiversity from this area as a part of project outcomes. references ellis, l.t., ah-peng, c., aslan, g., bakalin, v.a., bergamini, a., callaghan, d.a., campisi, p., raimondo, f.m., choi, s.s., csiky, j., csikyné radnai, e., cykowska-marzencka, b., czernyadjeva, i.v., kalinina, y.m., afonina, o.m., domina, g., drapela, p., fedosov, v.e., fuertes, e., gabriel, r., kubová, m., soares albergaria, i., gospodinov, g., natcheva, r., graulich, a., hedderson, t., hernández-rodríguez, e., hugonnot, v., hyun, c.w., kırmacı, m., çatak, u., kubešová, s., kučera, j., la farge, c., larraín, j., martin, p., mufeed, b., manju, c.n., rajesh, k.p., németh, c., nagy, j., norhazrina, n., syazwana, n., o’leary, s.v., park, s.j., peña-retes, a.p., rimac, a., alegro, a., šegota, v., koletić, n., vuković, n., rosadziński, s., rosselló, j.a., sabovljević, m.s., sabovljević, a. d., schäfer-verwimp, a., sérgio, c., shkurko, a.v., shyriaieva, d., virchenko, v.m., smoczyk, m., spitale, d., srivastava, p., omar, i., asthana, a.k., staniaszek-kik, m., cienkowska, a., ștefănuţ, m.m., ștefănuţ, s., tamas, g., bîrsan, c.c., nicoară, g.r., ion, m.c., pócs, t., kunev, g., troeva, e.i., van rooy, j., wietrzyk-pełka, p., węgrzyn, m.h., wolski, g.j., bożyk, d., cienkowska, a., 2021a: new national and regional bryophyte records, 65. journal of bryology 43, 67–91. ellis, l.t, alatas, m., alvaro alba, w.r, charry giraldo, a.m., amatov, v., batan, n., becerra infante, d.a, burghardt, m., czernyadjeva, i.v., kuzmina, e.y., doroshina, g.y., erata, h., garilleti, r., gradstein, s.r., jukoinene, i., karaman erkul, s., keksin, a., ezer, t., lara, f., draper, i., maksimov, a.i., mammandova, a.v., natcheva, r., nemeth, c., pantović, j., sabovljević, m. s., papp, b., poponessi, s., cogoni, a., porley, r.d., reiner-drehvald, m.e., schafer verwimp, a., schmotzer, a., segota, v., alegro, a., rimac, a., stefanut, s., szurdoki, e., vilk, e.f., virchenko, v.m., bijlsma, r.j., callaghan, d.a., 2021b: new national and regional bryophyte records, 67. journal of bryology 43, 301–311. galić, a., 2011: hydrogeological conditions of the area of water reservoirs in western herzegovina. phd thesis. faculty of mining, geology and civil engineering, university of tuzla, bosnia and herzegovina. grgić, p., 1985: istraženost briofita u bosni i hercegovini i njene karakteristike. godišnjak biološkog instituta univerziteta sarajevo 38, 33–41. hodgetts, n., lockhart, n., 2020: checklist and country status of european bryophytes –update 2020. irish wildlife manuals, no. 123. national parks and wildlife service, department of culture, heritage and the gaeltacht, ireland. kummer, a., sendtner, o., 1849: enumeratio plantarum in itinere sendtneriano in bosnia lectarum, cum definitionibus novarum specierum et adumbrationibus obscurarum varietatumque. flora oder allgemeine botanische zeitung, 32, 1-10. lasić, a., jasprica, n., 2016: vegetation diversity of the two dinaric karstic rivers in bosnia and herzegovina. biologia 71, 777–792. pantović, j., milanović, đ., sabovljević, m., 2016: three novelties for the bryophyte flora of bosnia and herzegovina. herzogia 29, 801–804. pantović, j., milanović, đ., janković, i., sabovljević, m., 2017: towards the bryophyte flora of the sutjeska national park (the republic of srpska, bosnia and herzegovina). glasnik šumarskog fakulteta univerziteta u banjoj luci 26, 51–74. redžić, s., barudanović, s., radević, m. (eds.), 2008: bosnia and herzegovina a country of diversity. overview and status of biological and landscape diversity of bosnia and herzegovina: the first report of bosnia and herzegovina for the convention on biological diversity. federalno ministarstvo okoliša i turizma, sarajevo (in bosnian). sabovljević, m., alegro, a., sabovljević, a., marka, j., vujičić, m., 2011: an insight into diversity of the balkan peninsula bryophy te f lora in the european background. revue d’écologie 66, 399–413. sabovljević, m., papp, b., szurdoki, e., 2010: new bryophyte records to some countries of the south-eastern europe. cryptogamie, bryologie 31, 289–292. opce-str.vp acta bot. croat. 68 (2), 221–230, 2009 coden: abcra 25 issn 0365–0588 two new mesodictyopsis species, m. akitaensis sp. nov. and m. miyatanus sp. nov., from a late miocene to pliocene freshwater sediment, japan hiroyuki tanaka1*, tamotsu nagumo2 1 maebashi diatom institute, 57-3 kawamagari, maebashi, gunma 371-0823, japan 2 department of biology, the nippon dental university, fujimi 1-9-20, chiyoda-ku, tokyo 102-8159, japan two new mesodictyopsis species, m. akitaensis sp. nov. and m. miyatanus sp. nov., are described from freshwater sediment of the late miocene to pliocene miyata formation, northern honshu, japan. the species are characterized by small size, veluma on inner side of areolae, one fultoportula (sometimes two in m. akitaensis) on central valve face with two (in rare cases three in m. miyatanus) satellite pores, mantle fultoportulae with three satellite pores located on upper mantle close to valve face/mantle junction. single rimoportula located in a row of areolae on marginal valve face near valve face/mantle junction in m. akitaensis or valve face/mantle junction on a strip in m. miyatanus. the veluma of m. akitaensis are cribra in inner view and sometimes volae on marginal valve face in outer view while those of m. miyatanus are cribra in both inner and outer view. the two species are compared with two similar reported mesodictyopsis taxa. key words: fossil, diatom, mesodictyopsis, ultrastructure, lake tazawa, miyata formation, japan introduction the miyata formation is located in the eastern part of akita prefecture, northern honshu, japan and is known to contain plant fossils (huzioka and uemura 1973). the formation also contains freshwater diatoms and two reports have been published (tanaka and nagumo 2002, tanaka 2007). recently, the authors found two small centric diatoms characterized by velum on the inner side of areolae, one fultoportula (sometimes two in m. akitaensis) with two (in rare cases three in m. miyatanus) satellite pores on the central valve face and mantle fultoportulae with three satellite pores. the authors concluded that the taxa belong to the genus mesodictyopsis proposed by khursevich et al. (2004) with seven species, six species found from upper miocene–lower pliocene deposits of lake baikal and one species from miocene sediments of the transbaikal area. it was not possible, however, to match the two acta bot. croat. 68 (2), 2009 221 * corresponding author: guntana@green.ocn.ne.jp u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:32 color profile: disabled composite 150 lpi at 45 degrees new taxa with any of the seven mesodictyopsis species reported thus far. the authors, therefore, propose the two new species as mesodictyopsis akitaensis h.tanaka et nagumo and mesodictyopsis miyatanus h.tanaka. the two species described here are the first finding in the genus mesodictyopsis outside the lake baikal area. materials and methods samples were collected from an outcrop on the northeast shore of lake tazawa in senboku city (former tazawako town), akita prefecture (fig. 1). all samples are laminated siltstone of the miyata formation estimated to be late miocene, according to the plant fossils (uemura 1988), or pliocene by k-ar and fission track methods (tsuchiya 1999). samples were boiled in a 30% h2o2 solution to separate the sediment particles from the diatom valves. after cooling, the sediment was washed several times with distilled water, hydrochloric acid and nitric acid were added, then the solution was boiled to dissolve the calcium salts in the sample and washed again several times with distilled water. the cleaned material was mounted in pleurax or styrax. lm observations were made using a nikon apophot microscope with a plan-apochromat 100´ oil immersion objective lens (na = 1.4). sem observations were made using a hitachi s-4000 field emission microscope. terminology used is that of ross et al. (1979) and khursevich et al. (2004). descriptions of new species mesodictyopsis akitaensis h. tanaka et nagumo sp. nov. (figs. 2–8, 18–29) valves circular, flat valve face, ranging from 6.5–8.0 mm in diameter. punctae rows ca. 24 in 10mm at valve margin. vela located on inner side of areolae sometimes forming volae in outer view. one fultoportula (sometimes two) with two satellite pores located near the valve center. mantle fultoportulae located on thickened strips of upper mantle, 6–8 in each valve, each with a short tube externally and three satellite pores internally. single rimoportula lacking outer tube, internal sessile labia, within a row of areolae on marginal valve face near valve face/mantle junction. valve mantle shallow, consisting of two or three areolae in a vertical row. 222 acta bot. croat. 68 (2), 2009 tanaka h., nagumo t. fig. 1. location of sampling site, lake tazawa, senboku city, akita prefecture, japan. u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:32 color profile: disabled composite 150 lpi at 45 degrees holotype: mpc-003017, micropaleontology collection, national museum of nature and science, japan. type locality: northeastern shore of lake tazawa, senboku city, akita prefecture, japan (39° 44’ n, 140° 41’ e). type material: taz-f03, siltstone of miyata formation, late miocene to pliocene, collected by one of authors (h. tanaka) on 26 september 1992. etymology: the species name refers to the type locality, akita prefecture. lm observation of mesodictyiopsis akitaensis shows a small circular valve face with radiating rows of single punctae from valve center to margin (figs. 2–8). short, dark (or bright) lines appear on valve margin (mantle fultoportulae in sem observation). external sem observation shows flat circular valve face with radiating areolae rows (figs. 18, 19). areolae of valve face are usually from round to oval in shape. sometimes vela are volae in the outer view of marginal valve face (figs. 23, 24), but always cribra in the inner view (figs. 25–29). one opening of fultoportula (sometimes two) located near valve center (figs. 18, 19). a small depression near valve center corresponds with a valve face fultoportula of sibling valve (figs. 19, 20), as in cyclostephanos invisitatus (m. h. hohn et hellerman) theriot et al. (kobayasi and inoue 1985). mantle fultoportula openings with short tubes are located high on the mantle at the end of a costa (fig. 19). a rimoportula opening, shaped round to slit, is located in a row of areolae on marginal valve face near valve face/mantle junction (figs. 19–21, 23). internal sem observation shows cribra located inside of areolae (figs. 26–29), a valve face fultoportula (sometimes two) with two satellite pores (figs 26, 28, 29), the mantle fultoportulae with three satellite pores each (figs. 27, 28) located every four to eight costa, and a single sessile rimoportula located on the marginal valve face near valve face/mantle junction in a row of areolae (figs. 28, 29). areolae of marginal valve face are generally quadrilateral or polygon in shape. mesodictyopsis miyatanus h. tanaka sp. nov. (figs. 9–13, 30–39) valves circular, flat valve face, ranging from 4.0–8.0 mm in diameter. punctae rows ca. 20 in 10 mm at valve margin. cribra located on inner side of areolae. one fultoportula with two (in rare cases three) satellite pores occurs near the valve center. mantle fultoportulae acta bot. croat. 68 (2), 2009 223 mesodictyopsis akitaensis and m. miyatanus sp. nov pl. 1. mesodictyopsis akitaensis and mesodictyopsis miyatanus (figs 2–17). figs. 2–8 – mesodictyopsis akitaensis lm. fig. 2 – holotype, mpc-003017, national museum of nature and science, japan. figs. 3–8 – the same valves shown at different focal planes. figs. 9–17 – mesodictyopsis miyatanus. lm. fig. 9 – holotype, mpc-003018, national museum of nature and science, japan. figs. 10–17 – the same valves shown at different focal planes. u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:32 color profile: disabled composite 150 lpi at 45 degrees located at the end of the costa of the upper mantle, 5–7 in each valve, each with a short tube externally and three satellite pores internally. single rimoportula lacking outer tube, internal sessile labia, on valve face/mantle junction. valve mantle shallow, consisting of two or three areolae in a vertical row. 224 acta bot. croat. 68 (2), 2009 tanaka h., nagumo t. pl. 2. mesodictyopsis akitaensis. sem, external views (figs. 18–24). fig. 18 – whole valve. fig. 19 – oblique view of figure 18 showing opening of valve face fultoportula (arrow), opening of rimoportula (arrowhead). fig. 20 – enlarged view of figure 19 showing opening of valve face fultoportula (arrow), opening of rimoportula (arrowhead) and central depression. fig. 21 – detailed view of figure 18 showing cribra and opening of rimoportula (arrowhead). fig. 22 – detailed view of opening tube of mantle fultoportula. fig. 23 – valve margin showing volae and opening of rimoportula (arrowhead). fig. 24 – enlarged view of valve margin showing volae. scale bars = 1 mm (figs. 18–20, 23), and 0.5 mm (figs. 21, 22, 24). u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:36 color profile: disabled composite 150 lpi at 45 degrees holotype: mpc-003018, micropaleontology collection, national museum of nature and science, japan. type locality: northeastern shore of lake tazawa, senboku city, akita prefecture, japan (39° 44’ n, 140° 41’ e). type material: taz-f03, siltstone of miyata formation, late miocene to pliocene, collected by one of the authors (h. tanaka) on 26 september 1992. etymology: the species name refers to the miyata formation where the new species was found. lm observation of mesodicyiopsis miyatanus shows a small circular valve face with approximately radiating rows of single punctae from valve center to margin but having a slightly irregular arrangement at the valve center (figs. 9–17). short dark (or bright) lines are located on the valve margin (mantle fultoportulae in sem observation). external sem observation shows flat circular valve face with radial areolae rows (figs. 30, 32). areolae acta bot. croat. 68 (2), 2009 225 mesodictyopsis akitaensis and m. miyatanus sp. nov pl. 3. mesodictyopsis akitaensis. sem, internal views (figs. 25–29). fig. 25 – whole valve. fig. 26 – detailed view of a valve face fultoportula with two satellite pores and areolae with cribra inside. fig. 27 – detailed view of mantle fultoportula with three satellite pores and areolae with cribra inside. fig. 28 – enlarged oblique view of figure 25 showing rimoportula (arrowhead) in an areolae row and cribra. fig. 29 – enlarged view showing two valve face fultoportulae with two satellite pores each and rimoportula (arrowhead) located in an areolae row. scale bars = 1 mm (figs. 25, 28, 29) and 0.5 mm (figs. 26, 27). u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:38 color profile: disabled composite 150 lpi at 45 degrees of valve face and mantle are from round to oval in shape. diameters of central valve face areolae are usually slightly larger than those of margin. vela are always cribra. a valve face fultoportula opening located near valve center (fig. 30). mantle fultoportulae openings with short tubes are located high on the mantle atop every (four) five to eight (nine) costa (fig. 32). a rimoportula opening is located on valve face/mantle junction on a costa (figs. 32, 33). internal sem observation shows the cribra located inside of areolae (fig. 39), one valve face fultoportula with two (rarely three) satellite pores (fig. 36), mantle fultoportulae always with three satellite pores each (fig. 37), and a single sessile rimoportula located on valve face/mantle junction on a costa (figs. 37, 38). areolae of internal valve face are usually from round to oval in shape but generally quadrilateral in marginal valve face. discussion the presence of cribra within the areolae is a characteristic shared by both the freshwater genera mesodictyon and mesodictyopsis of stephanodiscaceae makarova (khursevich et al. 2004). however, mantle fultoportulae with three satellite pores each and fulto226 acta bot. croat. 68 (2), 2009 tanaka h., nagumo t. pl. 4. mesodictyopsis miyatanus. sem, external views (figs. 30–33). fig. 30 – whole valve (arrow: opening of valve face fultoportula). fig. 31 – enlarged view of figure 30 showing opening of valve face fultoportula (arrow). fig. 32 – oblique view of figure 30 showing opening of valve face fultoportula (arrow) and opening of rimoportula (arrowhead). fig. 33 – detailed view of valve face showing opening of valve face fultoportula (large arrow), openings of two mantle fultoportulae (small arrows) and opening of rimoportula (arrowhead). scale bars = 1 mm. u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:40 color profile: disabled composite 150 lpi at 45 degrees portula(e) on the valve face are characteristic of mesodictyopsis (khursevich et al. 2004). mesodictyon has no valve face fultoportulae and its mantle fultoportulae have two satellite pores (theriot and bradbury 1987). the two new species described here belong to mesodictyopsis because they both have cribra inner sides of the areolae, one valve face fultoportula (m. akitaensis: sometimes two) with two (m. miyatanus: in rare cases three) satellite pores and mantle fultoportulae always with three satellite pores. khursevich et al. (2004) reported three morphological groups in mesodictyopsis. the first group is characterized by the location of a single rimoportula at the valve face/mantle acta bot. croat. 68 (2), 2009 227 mesodictyopsis akitaensis and m. miyatanus sp. nov pl. 5. mesodictyopsis miyatanus. sem, internal views (figs. 34–39). fig. 34 – whole valve (arrow: valve face fultoportula, arrowhead: rimoportula). fig. 35 – oblique view of figure 34 showing valve face fultoportula (arrow) and rimoportula (arrowhead). fig. 36 – detailed view of figure 34 showing valve face fultoportula with two satellite pores and areolae with cribra inside. fig. 37 – enlarged view of figure 35 showing rimoportula (arrowhead) and two mantle fultoportulae with three satellite pores (arrows). fig. 38 – oblique view of valve (arrow: valve face fultoportula, arrowhead: rimoportula). fig. 39 – cross section of valve face showing cribra inside the areolae. scale bars = 1 mm (figs. 34, 35, 38) and 0.5 mm (figs. 36, 37, 39). u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:43 color profile: disabled composite 150 lpi at 45 degrees junction or in the submarginal zone of the valve face. this puts both m. akitaensis, single rimoportula on marginal valve face close to valve face/mantle junction, and m. miyatanus, single rimoportula on valve face/mantle junction, in this first morphological group. mesodictyopsis akitaensis mesodictyopsis akitaensis differs from the other two members of the first morphological group in khursevich et al. (2004), mesodictyopsis singularis khursevich, iwashita et fedenya and mesodictyopsis peculiaris khursevich, kociolek et fedenya, in significant ways. first, m. akitaensis has only one (rare cases, two) valve face fultoportulae with two satellite pores while m. singularis may have one to several with three satellite pores. also, the mantle fultoportulae of m. akitaensis are fewer with one every five to eight strips and those of m. singularis every two to rarely three strips (tab. 1). m. peculiaris has a rimoportula located between two closely placed mantle futoportulae while the others are widely spaced. m. akitaensis, on the other hand, has mantle fultoportulae always located every five to eight strips. mesodictyopsis has cribra inside of the areolae (khursevich et al. 2004) and while this is true of m. akitaensis, it sometimes has volae, especially on marginal valve face in external views. mesodictyopsis miyatanus m. miyatanus differs from m. singularis in that it has a higher density of punctae rows and more punctae in each row, only one valve face fultoportula usually with two satellite pores and mantle fultoportulae located every five to eight strips. m. miyatanus is distinguished from m. peculiaris by mantle fultoportulae located on every five to eight costae and its rimoportula is located between two of these spaced mantle fultoportulae, not close together as in m. peculiaris. with the exception of the vela inside the areolae, other similar species such as thalassiosira dispar (perag. et hérib.) serieyssol (serieyssol et al. 1998) exist having a valve face fultoportula with two or three satellite pores, mantle fultoportulae with three satellite pores and a rimoportula at the valve face mantle border. the presence of vela inside the areolae then is the defining characteristic of m. miyatanus. in japan, tanaka and nagumo (2006) reported mesodictyon sp. from the late miocene mitoku formation, tottori prefecture. the species, however, has vela inside the areolae, one valve face fultoportula located near center and mantle fultoportulae with three satellite pores, actually features of mesodictyopsis. mesodictyopsis taxa therefore have been found in two formations in japan, the mitoku formation and the miyata formation. acknowledgments we are grateful to dr. kazuhiko uemura of the national museum of nature and science, tokyo for information concerning the geology and fossils of the miyata formation and dr. galina k. khursevich of academy of sciences of belarus, minsk for information on the genus mesodictyopsis. 228 acta bot. croat. 68 (2), 2009 tanaka h., nagumo t. u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:43 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .68 (2),2009 229 m e s o d ic t y o p s is a k it a e n s is a n d m . m iy a t a n u s s p . n o v tab. 1. comparison of mesodictyopsis akitaensis, m. miyatanus, m. peculiaris, m. singularis. characteristics m. akitaensis m. miyatanus m. peculiaris m. singularis valve face diameter (mm) 6.5–8.0 4.0–8.0 3.0–9.5 4.0–17.2 punctae rows (in 10 mm) ca. 24 ca. 20 15–20 14–18 punctae (in 10 mm) ca. 24 ca. 20 ca. 20 12–16 fultoportulae valve face location near valve center near valve center near valve center near valve center number 1(2) 1 1 1-several satellite pores 2 2(3) 2 3 mantle location every 4–8 strip every (4)5–8(9) strip two are placed close to one another, the rest widely spaced every 2(3) strip number (/valve) 6–8 5–7 4–6 ca. 18 satellite pores 3 3 3 3 rimoportula position marginal valve face (near, valve face/mantle junction), on punctae row valve face/mantle junction, on strip marginal valve face (near valve face/mantle junction), between two closely located mantle fultoportulae marginal valve face (near valve face/mantle junction) number 1 1 1 1 references this paper this paper khursevich et al. (2004) khursevich et al. (2004) u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ t a n a k a . v p 5 . l i s t o p a d 2 0 0 9 1 4 : 5 7 : 4 3 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s references huzioka, k., uemura. k., 1973: the late miocene miyata flora of akita prefecture, northeast honshu, japan. bulletin of the national science museum, tokyo 16, 661–738. kobayasi, h., inoue, h., 1985: fine structure and taxonomy of the small and tiny stephanodiscus (bacillariophyceae) species in japan 1. stephanodiscus invisitatus hohn & hell. the japanese journal of phycology 33, 149–154. khursevich, j. p., kociolek, j. p., iwashita, t., fedenya, s. a., kuzumin, m. i., kawai, t., williams, d. f., karabanov, f. b., prokopenko, a. a., minoura, k., 2004: mesodictyopsis khursevich, iwashita, kociolek and fedenya a new genus of diatoms in the class coscinodiscophyceae (bacillariophyta) from upper miocene sediments of lake baikal, siberia. proceedings of the california academy of sciences 55, 338–357. ross, r., cox, e. j., karayeva, n. i., mann, d. g., paddock, t. b. b., simonsen, r., sims, p. a., 1979: an amended terminology for the siliceous components of the diatom cell. nova hedwigia 64, 513–533. serieyssol, k. k., garduno, i. i., gasse, f., 1998: thallassiosira dispar comb. nov. and t. cuitzeonensis spec. nov. (bacillariophyceae) found in miocene sediments from france and mexico. nova hedwigia 66, 177–186. theriot, e., bradbury, j. p., 1987: mesodictyon, a new fossil genus of the centric diatom family thalassiosiraceae from the miocene chalk hills formation, western snake river plain, idaho. micropaleontology 33, 356–367. tanaka, h., 2007: pliocaenicus nipponicus h.tanaka & nagumo from miyata formation in plant fossil siltstone, akita prefecture. diatom 23, 119–120. tanaka, h., nagumo, t., 2002: cyclotella pliostelligera sp. nov., a new fossil freshwater diatom from japan. proceedings 15 international diatom symposium, perth, 351–358. tanaka, h., nagumo, t., 2006: late miocene freshwater diatoms from mitoku area in misasa town, tottori prefecture, japan. diatom 22, 17–25. tsuchiya, n., 1999: late miocene to pliocene volcanism and reservoir formation in the akita–yamagata oil field, northeast japan. bulletin of the geological survey japan 50, 17–25. uemura, k., 1988: late miocene floras in northeast honshu, japan. national science museum, tokyo. 230 acta bot. croat. 68 (2), 2009 tanaka h., nagumo t. u:\acta botanica\acta-botan 2-09\tanaka.vp 5. listopad 2009 14:57:43 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 73 (1), 21–35, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 succession rates and patterns twelve years after land use abandonment in the estuary of the river aliakmon, n. greece fotios xystrakis1*, kostantinos theodoropoulos2, eleni eleftheriadou2, dimitrios a. samaras3, christos damianidis2, theodoros papadopoulos2 1 department of environmental and natural resources management, university of patras, g. seferi 2, gr-30100 agrinio, greece 2 faculty of forestry and natural environment, aristotle university of thessaloniki, gr-54124 thessaloniki, greece. 3 gr. xenopoulou 44, gr-16346 athens, greece. abstract – vegetation succession is a key element for research studying biodiversity losses, effects of climatic change on ecosystems, invasive species and restoration of ecosystems in which human activities have shifted their natural or semi-natural vegetation. surrogate variables like pignatti's bioindicator values or dissimilarity indices can provide further insights regarding succession trajectories aggregating the combined effects of changes in the cover/abundance of taxa. the land-use abandonment in an area in the estuary of the river aliakmon, n. greece, provides an opportunity to study medium-term rates and patterns during the first twelve years of vegetation succession. cluster and time-series analyses of turnover rates, sørensen's dissimilarity index and pignatti's bioindicator values revealed clear differences in succession patterns and rates among permanent plots. succession rates and patterns in the study area were found to be dominated by two ecological factors. on the one hand, availability of fresh water, assumed by the proximity to the river, allows the fast growing populus alba to develop a forest canopy that radically alters the shading environment which, in its turn, controls succession trajectories. increased soil salinity on the other hand, allows salt-tolerant taxa to be quickly established, and defines the species inventory on these sites as early as the first years of succession. key words: forest canopy, halophytic vegetation, pignatti's bioindicator, soil salinity, succession, riverine forest, time-series introduction vegetation succession is a key element for research studying biodiversity losses, effects of climatic change on ecosystems, invasive species and restoration of ecosystems in which acta bot. croat. 73 (1), 2014 21 * corresponding author, e-mail: fotios.xystrakis@gmail.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:42:55 color profile: generic cmyk printer profile composite default screen human activities have shifted the natural or semi-natural vegetation (prach et al. 2001, prach and walker 2011). ecological restoration can, in addition to its main components of planning, implementation and evaluation, be regarded as the implementation of succession concepts (zedler 2005, hobbs et al. 2007) or as a form of manipulated succession (matthews and endress 2010). to exemplify, information concerning long-term succession assists rational planning that reduces the possibility of undesirable results; or else allows for the definition of a framework of possible trajectories within which restoration can occur and defines the time frames within which restoration should be evaluated and further intervention is needed (hobbs et al. 2007, �ehounková and prach 2008). this linkage between restoration ecology and succession implies that as much input as possible concerning ecological processes and functioning should be available (prach et al. 2001). management practises can be based on the knowledge of vegetation dynamics including succession rates and patterns and estimated final succession stages (bonet 2004). knowledge of succession stages can also support conservation practices for endangered taxa of fauna and flora (laurence et al. 2010, tropek et al. 2010). restoration practices could be evaluated on the basis of the similarity of the restored ecosystem to a reference biocoenosis, species composition and abundance, among other criteria. succession can be studied by means of chronosequences or permanent research plots, combined, or not, with experimental manipulations and meta-analyses (austin 1977, prach and walker 2011). direct observations over time should be preferred over chronosequence methods because the latter assume similar environmental conditions for different ages and this is not always valid (fastie 1995). nevertheless, chronosequences and permanent plots can be complementary and simultaneously used to test different hypotheses (bakker et al. 1996). widely adopted approaches for succession analysis include the identification of cluster transitions; direction in terms of species composition; individual species analysis at the level of presence/absence or abundance; and changes in plant traits or syntaxonomic elements (bakker et al. 1996). various applications of permanent plots that are used to study the internal causes and mechanisms of succession can be found in bakker et al. (1996). to measure the pace of succession two approaches are usually adopted: (1) to measure the rate at which species composition changes from earlyto late-successional stages, and (2) to measure the deceleration of species gain and turnover rates over time (matthews and endress 2010). successional trends and trajectories are often more apparent when plot data are aggregated to the level of functional groups, plant strategy types, or indicator values (bakker et al. 1996, kahmen and poschlod 2004, matthews and endress 2010, prévosto et al. 2011), as was also shown for the study area (xystrakis et al. 2011). ellenberg's indicator values, or similar approaches, can be used as surrogates for changes in environmental factors in plant communities (dzwonko 2001). despite the criticisms and the problems that often occur with their use (smart and scott 2004), under certain circumstances, they can adequately replace measurements of environmental variables (diekmann 2003, ewald 2003). estuaries and wetlands in general reflect a history of long term human influence. fertile soils and availability of irrigation are among the main factors supporting intensified agriculture which in its turn, stresses wetland functions (gerakis and kalburtji 1998). a considerable area of wetland ecosystems has been lost over the last decades in the northern mediterranean region with the major threat consisting of the alteration in their hydrological 22 acta bot. croat. 73 (1), 2014 xystrakis f., theodoropoulos k., eleftheriadou e., samaras d. a., damianidis c., papadopoulos t. 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:42:55 color profile: generic cmyk printer profile composite default screen status to provide for the needs of farming (brinson and malvárez 2002) as well as of direct loss of wetland area (zalidis et al. 1997). the latter specifically affected more than 50% of greek marshes and estuaries (zalidis et al. 1997). given the ecological importance of wetlands, alongside their over-exploitation, especially in greece (zalidis et al. 1997, gerakis and kalburtji 1998, smardon 2009), it is not surprising that the development of protection and restoration strategies accompanied by appropriate legislation gained importance at both international and national levels (smardon 2009). the tight relation between restoration and vegetation succession explains why the latter is widely discussed in the relevant literature (roozen and westhoff 1985, rossell et al. 2009, matthews and endress 2010). nevertheless, in greece there is still a considerable lack of experience regarding the successional pattern of wetland vegetation and within this concept, our objective is to analyze the vegetation succession after 12 years of land use abandonment (agriculture and grazing) using a case study approach in the estuaries of the river aliakmon, n. greece. we try to provide insights regarding changes in succession rates and the different succession trajectories that are observed in the study area. study area the study area, 'stergiou ecopark' (fig. 1), an area of ca 3.54 km2, is part of a larger complex of wetlands that are listed under the ramsar convention and are included in the natura 2000 network of directive 92/43/eec (site name: delta axiou-loudia-aliakmona-evryteri periochi-axioupoli. gr1220002), indicating the high conservation significance of the area. the land use in the ecopark, until the year 1999, was mainly rice cultivation and partly cattle grazing in some uncultivated, halophytic communities. in 1999 'stergiou ecopark' was included in the greek ministry of agriculture project 'long-term pause of cultivation of agricultural land' based on council regulation (eec) 2078/1992 and land use (agriculture and grazing) was abandoned. soils are mainly alluvial deposits with high salinity levels over most of the ecopark's area. the climate of the area, based on köppen's classification, is characterized as 'mediterranean with mild winters and warm, dry summers (csa)' (köppen 1931). according to the natural potential vegetation map of europe (bohn et al. 2000), the natural vegetation of the wider study area is characterized by a mosaic of aegean halophytic vegetation (salicornia europaea, sarcocornia perennis, halocnemum strobilaceum, juncus maritimus, j. subulatus, schoenoplectus litoralis, aeluropus littoralis) (code p28) and patches of east mediterranean brackish water tamarisk scrub (tamarix hampeana) with juncus maritimus and j. heldreichianus (code u39). the actual vegetation of the wider study area consists of patches of populus alba and alnus glutinosa canopy forests in the vicinity of the aliakmon, reed beds (phragmites australis) along the irrigation canals, tamarisk-dominated communities (t. hampeana) and where the soil salinity is high, made apparent from the existence of salt crystals on the surface of the soil, communities of halophytic vegetation dominate. the latter include, for example, the taxa sarcocornia perennis, halimione portulacoides, salicornia europaea, hordeum marinum and puccinellia distans subsp. distans. acta bot. croat. 73 (1), 2014 23 succession after land use abandonment in an estuary 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:42:55 color profile: generic cmyk printer profile composite default screen materials and methods six permanent plots of 100 m2 were established during 2000 in the dominant vegetation types occurring in the area (fig. 1). more specifically, two were placed at rather short distance from the aliakmon, where seedlings of populus alba and salix spp. were already present from the first year (plots 1, 2), three in halophilous vegetation types that among others included the taxa salicornia europaea, salsola soda, suaeda maritima, puccinellia distans, hordeum marinum at different cover values (plots 3, 4, 5), and finally one in tamarix hampeana-dominated scrubland (plot 6). plots 1 to 4 were established in ex rice cultivation areas and plot 5 in rangeland. vegetation was sampled based on braun-blanquet's method (braun-blanquet 1964). all vascular plant species occurring were identified and their cover/abundance was estimated by means of the 7-class braun-blanquet ordinal scale, after having been classified in three different layers: tree layer (height > 5 m), shrub layer (0.5 m < height < 5 m) and ground layer (height < 0.5 m). additional environmental variables included the visual estimation of tree height and the total cover of each 24 acta bot. croat. 73 (1), 2014 xystrakis f., theodoropoulos k., eleftheriadou e., samaras d. a., damianidis c., papadopoulos t. fig. 1. the 'stergiou ecopark'. distribution of plots is annotated. tamarisk scrublands are located at the north-east part of the ecopark, while riverine forest (mainly poplars) is distributed in the vicinity of river aliakmon. halophytic communities dominate the study area, yet a large part near the centre of the ecopark is periodically flooded, restricting further sampling. background map: s.a. coordinate reference system: ggrs'87 epsg code: 2100) (copyright © 2012, ktimatologio s.a.). 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:42:59 color profile: generic cmyk printer profile composite default screen vegetation layer. vegetation data can be found in the annex in xystrakis et al. (2011). specimen identification was made by means of tutin et al (1968, 1972, 1976, 1980), tutin et al. (1993) and strid and tan (1997, 2002). nomenclature follows (in order) euro+med (2006–2010), strid and tan (1997, 2002), jahn and schönfelder (1995), greuter et al. (1984, 1986, 1989), tutin et al. (1968, 1972, 1976, 1980) and tutin et al. (1993). to study succession, various turnover rates and the changes on taxa indicator values were analysed, using the following methods: cluster analysis was performed in order to assess the similarity in vegetation composition through the succession years as well as among plots. relative euclidean distance and ward's linkage method were used in the matrix in such a way that species abundances were log transformed and species appearing in two plots or less were excluded. we decided though to preserve taxa occurring at different layers (ground, shrub, tree) as distinct taxa, since vegetation layering provides insights in succession processes. the vegetation matrix consisted of 72 plots and 128 species. the analysis was performed in pc-ord (mccune and mefford 2006). turnover rate short-term turnover rates were estimated adopting the methods from matthews and endress (2010). therefore for each plot, taxa abundances were transformed into presence/ absence and the measures for proportional loss (lp), proportional gain (gp) and proportional turnover (tp) were estimated using the following equations: gp = g s s t t 1 2 1+ +( ) (1) lp = l s s t t 1 2 1+ +( ) (2) tp = g l s s t t + + +( )1 (3) with: st = number of taxa in year t g = number of taxa in year t+1, not present in year t l = number of taxa in year t, not present in year t+1 since these turnover rates, based on presence/absence data for taxa, do not include changes on species cover values, the succession rates were also displayed as the year to year change in the vegetation composition by means of the calculation of the dissimilarity (sørensen distance measure) among all the plots of each year and the earliest year plots (du et al. 2007). dissimilarity values were also aggregated to the level of the defined groups that were derived from the cluster analysis. to estimate the sørensen distance measure, taxa abundance/cover values were transformed according to the transformations proposed by maarel, van der (2007). it has to be pointed that the form of sørensen's distance measure, as implemented in pcord, uses taxa cover/abundance values. here we adopt the nomenclature followed in pcord (mccune and mefford 2006). acta bot. croat. 73 (1), 2014 25 succession after land use abandonment in an estuary 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:42:59 color profile: generic cmyk printer profile composite default screen alongside the study of the changes on the basis of cover/abundance of plant species in each plot, succession was also studied on the basis of the changes in weighted-average of pignatti's bioindicator values for each plot, each succession year (pignatti 2005). this approach has been questioned with respect to its ability to provide information independent of the floristic census results, since cover/abundance values of plant species and bioindicator values have the problem of circular argumentation. nevertheless, it is the only method able partially to fill the gaps occasioned by the lack of measured ecological data. salinity values were excluded from the analysis because they were available for only a limited number of taxa. inter-annual differences in dissimilarity index (sørensen's distance) as well as inter-annual change in the pignatti bioindicator values can be considered and treated as univariate time series. therefore, the nonparametric mann-kendall trend test (hipel and mcleod 1994) was performed in order to investigate possible trends along the succession years. the analysis was performed by means of the 'kendall' package in r (r development core team 2011). moreover, since it is argued that early years of succession reflect higher turnover rates than later phases (odland and del moral 2001), for the time series reflecting trends as shown from the mann-kendall trend test, we tested whether the time-series were trend-stationary, allowing for a structure break in both their level and slope or were unit root processes. the analysis was performed by means of the zivot and andrews unit root test implemented in the 'urca' package in r (r development core team 2011). the analyses were performed for every plot as well as for the averaged values of the defined groups from the cluster analysis (plots 1, 2, 3, 4, 5) that showed similar responses in terms of succession but only the latter presented in this paper. results cluster analysis showed that plots can be grouped in three distinct groups (fig. 2): group a, which includes plots 1 and 2, group b which includes plots 3, 4 and 5 and finally, group c which only includes plot 6. it is interesting to observe that group b could be further divided in two distinct clusters, the first containing plot 3 (all years of succession) and plot 4 (three first succession years) and the second containing plot 5 (all years of succession) and plot 4 (latest nine succession years). this indicates that plot 4 resembled plot 3 during the first years of succession but then, its succession trajectory resembled that of plot 5. moreover, it has to be pointed out that these results allow us to consider plots 3, 4 and 5 on a common basis, regardless of the differences in land use before land abandonment. finally, it has to be mentioned that since plot 6 shows distinct patterns of succession and floristic composition from the rest of the plots (the vegetation consists of dense tamarisk scrubland, and it was actually never managed as rice field) it was decided it would be excluded from further analyses. based on the species turnover rates, and the analysis of species gain and loss for each plot (fig. 3) it can be observed that changes on species occurrence may not result in changes in overall turnover rates. during the first years of succession, there is a generally great loss of species and only a few new species are introduced in the plots, while in the third year of succession, the turnover rates are mostly based on the introduction of new species in the floristic inventory of the plots. nevertheless, there is fluctuation between annual gain and loss of species, leading to respective fluctuations in annual turnover rates. 26 acta bot. croat. 73 (1), 2014 xystrakis f., theodoropoulos k., eleftheriadou e., samaras d. a., damianidis c., papadopoulos t. 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:42:59 color profile: generic cmyk printer profile composite default screen the trend of the sørensen dissimilarity index was significant at the 0.05 level (tau = 0.848 and tau = 0.788 for groups a and b respectively) (fig. 4). structural change tests identified possible breaks in both level and slope in the years 2006 and 2008 for plots of group a and b respectively. it is worth noting that the intervals where the possible break may occur (t statistic in zivot and andrews test significant at the 0.05 level) for group b show a great amplitude, expanding from year 2004 to 2011. regarding plots of group a (plots 1 and 2), there is a clear trend in some of the weighted averaged pignatti bioindicator values during the succession process (fig. 5). we present only the outputs for pignatti's values of light, humidity and nutrients as they are the more striking examples. only changes in temperature, continentality and soil reaction values are discussed in the text. the mann-kendall trend test identified a significant linear trend at 95% confidence for light (tau = –0.879) nutrients (tau = –0.646) and humidity (tau = –0.473). structural change tests revealed structural changes for humidity and nutrient (fig. 5), pignatti's bioindicator values for light reflect a clear decreasing trend without any structural breaks, indicating that during the first 12 years of succession they were constantly acta bot. croat. 73 (1), 2014 27 succession after land use abandonment in an estuary fig. 2. cluster dendrogram of plots. three main groups can be identified, distinguishing plots during succession process: group a, including plots 1 and 2 (poplar stands), group b, including plots 3, 4 and 5 (halophytic communities) and group 6, only including plot 6 (dense tamarisk shrubland). 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:43:00 color profile: generic cmyk printer profile composite default screen decreasing and that the flattening of the curve after the year 2009 is not yet sufficient for it to be identified as possible break in level and slope. the lack of a clear trend in pignatti's temperature, reaction and continentality values indicates that succession and changes in 28 acta bot. croat. 73 (1), 2014 xystrakis f., theodoropoulos k., eleftheriadou e., samaras d. a., damianidis c., papadopoulos t. fig. 3. gain and loss of taxa and turnover rates during succession fig. 4. sørensen's dissimilarity index for plots of group a (left) and of group b (right). all values refer to differences from the first year (2000). vertical lines indicate the year during which a possible shift in both level and slope occurs. horizontal lines around the year of possible shift indicate the period when t statistic of zivot and andrews test falls below the 0.05 level 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:43:03 color profile: generic cmyk printer profile composite default screen species cover/abundance values only slightly influenced these parameters, or that there are intense interannual fluctuations, not allowing for the distinction of a clear trend. similar outputs were obtained for the aggregated values of plot 3, 4 and 5 (fig. 6), where only the outputs for pignatti's values of light, humidity and nutrient are presented. changes in temperature, continentality and soil reaction values are considered. some weighed averaged pignatti's bioindicator values show clear indications of linear trends, while other rather reflect inter-annual fluctuations. the mann-kendall trend test identified significant trends at 95% confidence level for light (tau = 0.545); humidity (tau = –0.626); soil reaction (tau = –554) and continentality (tau = –626), while temperature and nutrient values were not found to have significant linear trends. structural change tests revealed significant changes in level and slope only for pignatti's light values. values of humidity and continentality, although showing clear decreasing trends, do not show patterns of structural breaks in slope and level, while values of soil reaction probably have a break only in level. acta bot. croat. 73 (1), 2014 29 succession after land use abandonment in an estuary fig. 5. pignatti's bioindicator values (y axis) of aggregated plots of group a during succession (x axis). annual fluctuations can be observed, yet trends are also apparent. vertical lines indicate the year during which a possible shift in both level and slope occurs. horizontal lines around the year of possible shift indicate the period when t statistic of zivot and andrews test falls below the 0.05 level fig. 6. pignatti's bioindicator values (y axis) of aggregated plots of group b during succession (x axis). annual fluctuations can be observed, yet trends are also apparent. vertical lines indicate the year during which a possible shift in both level and slope occurs. horizontal lines around the year of possible shift indicate the period when t statistic of zivot and andrews test falls below the 0.05 level 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:43:05 color profile: generic cmyk printer profile composite default screen discussion our results firstly demonstrate that the use of turnover rates estimated from equations 1, 2, and 3 cannot describe the succession patterns in 'stergiou ecopark'. the consideration of only the presence/absence of taxa resulted in large inter-annual fluctuation of gains and losses of species which dominated the outputs. conclusions cannot be drawn if cover/abundance values are not used and succession patterns remain unrevealed. the changes in sørensen's dissimilarity index showed that during the first years of succession, dissimilarity rapidly increased, reaching a milestone at the succession age of 7 and 9 years for groups a and b respectively (fig. 4). patterns of succession indicating a decline in rates with time have been widely discussed in the literature (foster and tilman 2000) and are explained by the general succession mechanism as described in myster and pickett (1994). it is argued that on abandoned agricultural fields, species gain increases with time, while species loss decreases, resulting in a decelerating decrease in overall species turnover (anderson 2007), yet in the present study, dissimilarity is not due to gain or loss of species, as these patterns rather reflect random fluctuations (fig. 3), and it should mostly be attributed to changes in species cover/abundance values resulting in changes in dissimilarity index. cluster analysis, right from the first years of succession, identified rather pure clusters, consisting of the same plots each and there were only a few 'exchanges' observed, mainly between plots 3 and 4 at early and late stages of succession. it has to be pointed out, though, that succession rates are different in the plots of group a and b. in the former group, differences in both dissimilarity and pignatti's values are pronounced, reflecting a clear gradient of vegetation evolution. dissimilarity between the first and the last examined year increases up to 0.8, while weight-averaged bioindicator values show a difference of up to one unit, indicating a shift in class as defined in pignatti (2005). plots of group b do not reflect such pronounced differences either in dissimilarity or in changes in pignatti's values, although the identified trends in group b are also significant. this indicates that plots of group b, from the very first year of succession, are 'stabilised' and their floristic composition does not radically change along the time trajectory. changes in taxa cover/abundance values in the study area are controlled, rather, by soil salinity and canopy cover. the presence of salicornia europaea, sarcocornia perennis, hordeum marinum and tamarix hampeana in the plots of the group b, indicate higher salt concentrations in these plots. since irrigation has stopped, soil salinity is expected to increase and these salt-tolerant taxa quickly replaced other ruderal, salt-intolerant taxa and finally became dominant. high soil salinity in plots of group b restricts the potential rates of change through restricting the potential pool of species capable of establishing themselves after land abandonment. salt tolerant taxa dominate the plotsright from the first years of succession, forming plant communities that remain quite stable over the years. similar results were reported by mesléard et al. (1991). in such special environments, the distinct abiotic conditions control the patterns of vegetation succession (anderson 2007). in contrast, plots of group a, due to their proximity to the bank of the aliakmon, allowed populus alba and salix spp. to become established, most likely due to the high fresh-water table which is easily available to plant roots (mesléard et al. 1991). these differences in the floristic composition of the defined groups can also indirectly demonstrate the effect of the distance to the river in succession trajectories. certainly, reliable conclusions cannot be 30 acta bot. croat. 73 (1), 2014 xystrakis f., theodoropoulos k., eleftheriadou e., samaras d. a., damianidis c., papadopoulos t. 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:43:05 color profile: generic cmyk printer profile composite default screen drawn from only two plots, but similar results, which support our argumentation, can be found in mesléard et al. (1991), where forest canopy was only found to develop in the vicinity of rivers and irrigation canals. differences in succession trajectories along salinity gradients have been reported previously (crain et al. 2008), succession speed being negatively associated with an increasing salinity gradient. species turnover was also found to be higher during the first years of succession in odland and del moral (2001) with stabilising indications after the 8th–9th year. species diversity resembled the respective diversity of adjacent vegetation types five years after the beginning of succession but the colonisation from perennial species like puccinellia maritima and halimione portulacoides increased after the third year in an estuary under restoration in southern england (wolters et al. 2008). a longer time series, a prerequisite for robust time series analysis, would allow us better to examine and reliably to conclude whether the indications of abrupt shifts are part of a constant trend in the succession or whether they are true shifts that indicate radical changes in succession rates. fluctuations in environmental conditions may further result in fluctuations in species cover/abundance values that co-occur with directional changes (odland and del moral 2001), and that could impede their discrimination. pignatti's bioindicator values revealed distinct succession patterns between groups a and b. group a not only started from lower values for light (7.5 against 9.8) but unlike in group b, these values rapidly decreased with a magnitude of 1.65 units in pignatti's scale for light, indicating a shift toward classes of intermediate shade tolerance. this decrease is directly connected to the increasing density of shrub, during the early years, and tree canopy during the later years. populus alba and salix spp. (only the first years) rapidly colonised plots 1 and 2 and formed a dense canopy, forcing light demanding early-successional species to reduce their cover and abundance values until their reduction or extinction from the plots. striking examples are the taxa polypogon monspeliensis, centaurium erythraea, epilobium tetragonum, conyza canadensis. in contrast, for group b (plots 3, 4, 5), where the canopy has not closed up to date, light-demanding species were favoured. the effect of stand closure in succession had been previously discussed and it is apparent in various ecosystems of the world, as for example pine plantations (jones et al. 2012). the abrupt shift of moisture bioindicator values of plots of group a (fig. 5), which is observed during the year 2005 (6 years after land abandonment) indicates a shift of floristic composition from moisture-demanding towards more water-stress tolerant taxa. this can be attributed to the high rates of moisture consumption from the poplars that exhaust the available soil moisture, reflecting rapid growth rates and biomass build-up from may up to june, depending on soil water availability (liang et al. 2006). moreover, the lack of irrigation following land abandonment could have also affected hydrological conditions. plots of group b maintained higher bioindicator values of moisture availability, although these plots are exposed to direct solar radiation due to canopy absence. nevertheless, a decrease was similarly observed during 2005. pignatti's bioindicator values for nutrient availability also decreased after the first years of succession for the plots of group a, by a magnitude of ca one degree, revealing the possible lagged effect of fertilisation. agricultural practices of rice cultivation included soil fertilisation and therefore, during the first years of succession more nutrient-demanding taxa were able to become established. lack of fertilisation alongside the rapid growth of acta bot. croat. 73 (1), 2014 31 succession after land use abandonment in an estuary 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:43:05 color profile: generic cmyk printer profile composite default screen poplars rapidly exhausted soil nutrients resulting in the decrease of bioindicator values. in contrast, in plots of group b, increased soil salinity did not allow nutrient-demanding, fast-growing species to be established and bioindicator values only slightly fluctuated. based on our results, we can conclude that succession rates and patterns in the estuary of the aliakmon are dominated by two ecological factors, which, although not measured, can be discussed on the basis of species composition. fresh water availability, assumed by the proximity to the river, allows fast-growth populus alba to develop a forest canopy that radically alters the shading environment which, in its turn, controls succession trajectories. increased soil salinity, on the other hand, allows salt tolerant taxa to be quickly established, and defines species composition on these sites as early as the first years of succession. changes in species cover/abundance values are rather restricted and succession follows slower rates. there are serious limitations regarding the present study. the number of plots is small, yet, all vegetation types occurring within the study area were recorded and the plot size allowed for the complete inventory of the most dominant species in the study area. ecological data could provide valuable information enabling reliable conclusions to be drawn concerning environmental control of succession trajectories. bioindicator values cannot be used as independent variables for the development of models, yet they stand as good proxies for a generalised description of the dominant ecological conditions of a site. we can infer, therefore, that in spite of the drawbacks our results provide a valid generalisation of the succession processes for the 'stergiou ecopark' and, combined with further data, could provide valuable insights to the processes of succession in estuarine ecosystems. acknowledgements the authors would like to 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(1), 2014 35 succession after land use abandonment in an estuary 812 xystrakis et al.ps u:\acta botanica\acta-botan 1-14\812 xystrakis et al.vp 19. o ujak 2014 16:43:05 color profile: generic cmyk printer profile composite default screen opce-str.vp acta bot. croat. 70 (2), 245–258, 2011 coden: abcra 25 issn 0365–0588 impact of riparian trees shade on aquatic plant abundance in conservation islands magdi m. ali1*, samar a. hassan2, abdel-samei m. shaheen1� 1 department of botany, faculty of science at aswan, south valley university, aswan 81528, egypt 2 first cataract conservation islands, natural protectorates sector, egyptian environmental affairs agency, egypt temperature, acidity, light conditions, total dissolved salts, conductivity, dissolved oxygen, submerged macrophytes and shade and sun path directions were measured at 23 sites along the river nile banks with acacia nilotica growing at water’s edge around the first cataract conservation islands. ceratophyllum demersum and potamogeton crispus were common in the shaded and unshaded zones, myriophyllum spicatum and vallisneria spiralis were found only in the unshaded zone and azolla filiculoides only in the shaded zone. banks of the sites surveyed were oriented to five directions (nw, se, ne, sw, n). there is a significant difference in both the type and density of submerged plants growing under the shade of riparian trees (acacia nilotica) as compared to unshaded areas. water column irradiance is the most influential variable dictating the distribution of submerged plants. the area of the shade provided by riparian trees was affected by environmental and/or plant variables. environmental variables comprised the daily course of the exposition to sun; and plant variables included the area of the tree crown, the height of the tree and geographical position of the tree in relation to sun exposition. trees on the west bank of the islands at the sw-ne direction have the highest shading effect. the management of tree vegetation might control incoming solar radiation affecting submerged macrophytes. key words: acacia nilotica, ceratophyllum demersum, potamogeton crispus, myriophyllum spicatum, vallisneria spiralis, riparian vegetation, submerged macrophytes, nile, egypt abbreviations: dwsc – dry weight standing crop, zeu – eutrophic depth introduction aquatic macrophytes provide food, shelter, and substrate for a variety of organisms in aquatic systems (ali et al. 2007). however, when aquatic plants grow in undesired abundance they tend to become troublesome aquatic weeds and cause severe aquatic environacta bot. croat. 70 (2), 2011 245 * corresponding author, e-mail: magdi_ali_23361@yahoo.com � dr abdel-samei shaheen died before the completion of this study in september 2008. copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:21 color profile: disabled composite 150 lpi at 45 degrees mental problems. profuse infestations of aquatic weeds can restrict boat traffic, interfere with fisheries, affect water flow, increasing sedimentation by trapping silt particles and displace more desirable species (pieterse and murphy 1990, sand-jensen 1998). in egypt, the nile extends for about 1200 km from aswan at the south to the mediterranean coast at the north. the main stream of the nile and its rosetta and damietta branches includes some 500 islands, which constitute a complex ecosystem. some of these islands exhibit great diversity in origin, size and structure (el-hadidi and hosni 2000); therefore, 144 islands were declared as a protected area (prime minister’s decree under law no. 1969/1998). twelve of these islands are in the province of aswan. the nile at aswan (24° 05’ n, 32° 55’ e) is interrupted by about 30 islands varying in size and structure, where the river is deflected westwards and the current becomes stronger, indicating the real beginning of the first cataract. before the construction of the old aswan dam in 1902 and the aswan high dam in the 1960s, most of the first cataract islands were intermittently under water. however, now most of them are permanently exposed with loamy-sand deposits between them. these granite islands, among the oldest known islands, support unique assemblages of native species characteristic of the aquatic life in the river nile (el-hadidi and springuel 1978). in addition, the islands offer an excellent habitat for many birds, including migratory water birds (barsy 1993). the area of the first cataract islands (saluga, ghazal and the small surrounding islands) were declared a conservation area by the prime minister’s decree under law no. 928 in 1986 and has had protected status since then within the egyptian environmental affairs agency. the distribution and abundance of aquatic plants are influenced by many factors. nutrients are the most important factor for submerged plant growth and distribution, although, nutrient enrichment in water could inhibit the growth of some aquatic plants. johnson and ostrofsky (2004) demonstrate the importance of sediment characteristics in determining macrophyte community structure. van donk and otte (1996) report that fish grazing on macrophytes affected the internal balance among autotrophic components by changing composition and lowering the macrophyte standing crop. middelboe and markager (1997) and armengol et al. (2003) reported that water depth is the most important factor influencing water transparency and hence distribution of submerged plants varies with depth. water velocity not only affects the abundance of submerged plants (ali et al. 1999), but also controls gas exchange processes (sorrell and downes 2004). springuel (1981) indicates that definite changes in plant distribution in the area of the first cataract islands are associated with the increasing depth of soil deposits due to the sedimentation of fine material suspended in water. this is fundamentally caused by changes in water regimes (e.g. steady water level and slowing water current velocity) due to the damming of the nile south of aswan (ali et al. 1995). increasing sedimentation by the trappingof silt particles by submerged plants is one of the most significant effects of submerged vegetation (sand-jensen 1998) on the aquatic habitat of the first cataract conservation islands. sediment deposition in the narrow passages between these islands could merge small islands together, which in turn may lead to the disappearance of this unique aquatic microhabitat, which has been protected for the past 25 years. in addition, it will threaten the distinctive natural aquatic communities characterising them, which are already important from a wildlife or landscape perspective (el-hadidi and springuel 1978, el-hadidi and hosni 2000). environmental managers of the first cataract conservation islands are therefore faced 246 acta bot. croat. 70 (2), 2011 ali m. m., hassan s. a., shaheen a.-s. m. u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:21 color profile: disabled composite 150 lpi at 45 degrees with the problem of losing habitat due to deposition, the disappearance of aquatic plant communities and the merging of islands. they may need to manage excessive plant growth to maintain and conserve habitat quality and therefore the unique flora around the islands. according to the bioclimatic map of the mediterranean zone (1963), the study area is in a true desert climate. the rainfall of this area is not only scanty, but also extremely irregular and variable (kassas 1955). the first cataract islands are characterised by shallow water of low current velocity and support rich floristic diversity (ali et al. 1999). the study area supports abundant growth of submerged macrophyte communities, dominated by ceratophyllum demersum l. and potamogeton crispus l. (springuel 1981, ali et al. 1999). conservation area of the first cataract islands exhibits the lowest eutrophication level and alkalinity of all localities in the nile (ali et al. 1999). ali et al. (1995) provided evidence of the significant relation between plant community composition and structure, and hydrosoil texture (mainly sandy loam) that is strongly associated with water level regime. their results revealed a sediment-related effect of water level regime on submerged plant community. the important environmental factors are concentrations of hydrosoil calcium, magnesium, organic matter and nitrate (ali et al. 1995). aquatic macrophyte control methods (murphy 1988a, b; pieterse and murphy 1990; jadhav et al. 2008; palmer et al. 2010) provide studies of aquatic macrophyte abundance and growth (spence 1981, smart and barko 1986, sand-jensen and madsen 1991, schwarz et al. 1996, middelboe and markager 1997, binzer and sand-jensen 2002, raeder et al. 2010). these authors identified light as a primary factor limiting growth. moreover, the reduction of light has been considered as a technique for the limitation of excess growth of aquatic macrophytes (dawson and kern-hansen 1979, dawson 1978, dawson and haslam 1983, dawson 1981, bunn et al. 1998). a vinyl-coated fiberglass screen with a mesh size of 64 apertures cm–2 (aquascreen menadri-southern corp.commercial product) provided effective control of weeds when applied on the bottom (mayer 1978, perkins et al. 1980, nichols and shaw 1993). when applied on the surface, there are negative effects of artificial shading application; water could become stagnant and depleted of oxygen. alternatively, riparian vegetation, if planned correctly, can provide valuable habitat for wildlife and erosion protection for the bank. in addition, it affects water temperatures by decreasing the warming of water (scottish environment protection agency 2009). this study was aimed to examine the effect of shade produced by riparian trees on submerged aquatic macrophytes growing in the river nile at the first cataract conservation islands, in upper egypt. the analysis of the effects of riparian woody vegetation consisting of acacia nilotica trees on submerged plant communities, might contribute to the optimisation of their potential for wildlife in the river corridor. materials and methods sampling regime in total, 23 sites were randomly selected along the river nile banks around the first cataract islands, namely: ghazal island (3 sites), saluga island (5 sites), awad island (2 sites), basma island (3 sites), seheil island (9 sites) and one site barbar at the west bank of acta bot. croat. 70 (2), 2011 247 aquatic plants management by riparian trees shade u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:21 color profile: disabled composite 150 lpi at 45 degrees the nile (fig. 1). all sites selected are colonized by acacia nilotica growing on the edge of the islands. the survey was made in spring (april to june 2008). the canopies of a. nilotica trees have various shapes and heights above the water, as well as of different orientations to the sun. data on environmental variables, aquatic macrophytes and a. nilotica trees were collected from each site. aquatic plants sampling at each site, samples were collected in the morning from 9:00 to 11:00 am, along fixed transects, positioned rectangular to the shore. a transect extended from shore water edge to cover both zones, shaded and unshaded, at each a. nilotica tree. each sampling site was divided into two sampling zones, the tree-shaded zone and the unshaded zone. at each zone, submerged plant communities were assessed using the grapnel method to collect relative standing crop samples (five grapnel-hauls per sampling zone). dry weight standing crop (dwsc) of each species per grapnel haul was determined after air-drying (ambient temperature 45 c) for a week. this method gives good comparative values of abundance, but does not estimate absolute standing crop per unit area of substrate (murphy and eaton 1983). environmental variables water samples were collected to get indications of conditions at each site. seven physico-chemical parameters, namely water depth; temperature and ph (using a jenway 3070 portable ph meter); total dissolved salts and electric conductivity (using engineered 248 acta bot. croat. 70 (2), 2011 ali m. m., hassan s. a., shaheen a.-s. m. fig. 1. location of sites at aswan; ghazal island (1–3); basma island (4–6); awad island (7–8); barber (9); saluga island (10–14); seheil island (15–23). u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:22 color profile: disabled composite 150 lpi at 45 degrees system device model 76) and dissolved oxygen (winkler’s method; apha 1985), were measured in situ between 09:00 and 11:00 am. underwater light attenuation was measured using a twin-sensor skye par meter, model skp 200 with skp 215 sensors, and light extinction coefficient, a measure of turbidity (moss 1988), was calculated using the beer-lambert equation: k m–1 = loge (io/i) / d, where io = subsurface par, i = par at depth (d). this equation concerns the exponential decline of light intensity with depth (armengol et al. 2003). these readings were also used to calculate the depth of the euphotic zone (zeu) (zeu = 3.51 / k). the water column irradiance encountered by the submerged plants is the ratio between the euphotic zone depth and the maximum depth (zmax) (abernethy 1994). in addition, diurnal sun path direction was estimated in situ by determining the opposite direction of tree shade using a brunton’s compass. the direction of each site surveyed and shaded by a. nilotica trees was estimated, using a google map, by drawing a tangent on the bank at each site and at the waterside. data analysis similarity percentages (simper) were calculated to determine which species contribute most to the observed similarity between the sampling sites surveyed using community analysis package version 4.1.3 (seaby and henderson 2007). this method uses the bray-curtis measure (equation 1) of similarity, related to the complement of the steinhaus’ similarity measure (beals 1984): s s s s i i i i n i i i i n 1 2 1 1 2 1 − + = = = = ∑ ∑ ( ) where, si1 and si2 are the abundances of species i in samples 1 and 2 respectively. kruskal-wallis h-test was carried out to measure the difference in mean of the total dwsc among the various direction groups in shaded and unshaded sampling sites, using winstat for excel software (fitch 2009). this a non-parametric 'analysis of variance' test, which is similar to a one-factor analysis of variance, in that the values for a given dependent variable are placed into, groups according to a given independent variable. kruskal-wallis test is applicable to fully randomised designs and can accept groups of unequal size (wardlaw 1997). canonical correspondence analysis (cca) was carried out using canoco for windows version 4.5 (ter braak and smilauer 1998) to explain the relationship between aquatic macrophytes and sampling sites in the light of the measured water variables. the critical value for water column irradiance (zeu/zmax) indicates whether light condition is suitable for net macrophyte production was used as useful variable of assessing the role of light in stimulating growth of macrophytes. total dissolved salts reflect the range of total acta bot. croat. 70 (2), 2011 249 aquatic plants management by riparian trees shade u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:22 color profile: disabled composite 150 lpi at 45 degrees ions; therefore, it was retained in preference to conductivity. direction of the site bank was dealt with as multiple regression (montgomery and peck 1982) and represented by five dummy (nominal) variables »nw«, »se«, »ne«, »sw«, »w«, and »n«. results aquatic macrophytes in the water zone shaded by acacia nilotica trees, only three submerged macrophytes of low dry weight standing crop (dwsc) varied between 0.02 and 3.24 g sample–1, mean 0.36 g sample–1, se 0.17) were recorded in seven sites only; the rest of the sites had no submerged macrophytes (fig. 2). ceratophyllum demersum dominated four sites (ghazal island (gh1), basma island (ba5, ba6) and barbar island (br9)) with dwsc ranged between 0.02 and 2.22 g sample–1 (mean 0.18 g sample–1, se 0.1); potamogeton crispus was only found in two sites in seheil island (se20; dwsc 1.11 g sample–1 and se21; dwsc 0.68 g sample–1) and azolla filiculoides dominated gh3 in ghazal island with dwsc 2.4 g sample–1 (fig. 2). on the other hand, the unshaded water zone contained more biomass (dwsc varied from 4.4 to 72.1 g sample–1,mean 29.8 g sample–1, se 3.9) and more species of (four species) submerged macrophytes (fig. 3). ceratophyllum demersum was noticed in all sites and was the dominant in 20 sites (dwsc varied from 1.9 to 72.1 g sample–1,mean 22.1 g sample–1, se 3.9); myriophyllum spicatum l. was the dominant in barbar island (br9; dwsc 54.6 g sample–1) and was recorded in another six sites (awad island (aw7), saluga island (sa10 and sa14) and seheil island (se18 and se19)) with dwsc that ranged between 0.9 and 18.6 g sample–1 (mean 3.4 g sample–1, se 2.4); and p. crispus was found in six sites (basma island ba5, seheil island se15-se19) with dwsc that varied between 1.6 and 13.2 g sample–1 and dominated two sites in seheil island (se20 and se21) with dwsc 13.6 and 16.6 g sample–1 (mean 4.1 g sample–1, se 1.3), respectively. also, vallisneria spiralis was recorded in basma island (ba5) and seheil island (se22 and se 23) having dwsc 1.5, 1.9 and 1.7 g sample–1, respectively (fig. 3). comparing aquatic macrophytes in the two zones, c. demersum and p. crispus were in common, m. spicatum and v. spiralis were found only in the unshaded zone and a. filiculoides was only noticed in the shade zone. 250 acta bot. croat. 70 (2), 2011 ali m. m., hassan s. a., shaheen a.-s. m. 0 1 2 3 4 g h 1 g h 2 g h 3 b a4 b a5 b a6 a w 7 a w 8 b r9 s a1 0 s a1 1 s a1 2 s a1 3 s a1 4 s e1 5 s e1 6 s e1 7 s e1 8 s e1 9 s e2 0 s e2 1 s e2 2 s e2 3 sites ceratophyllum demersum potamogeton crispus azolla sp. d w s c (g s a m p le ) – 1 fig. 2. dry weight standing crop (dwsc) of submerged macrophytes recorded in the water zone shaded by acacia nilotica trees. u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:23 color profile: disabled composite 150 lpi at 45 degrees environmental variables at the sampling dates water variables varied slightly between sites (ph ranged between 7.58 and 7.73 (mean 7.7, se 0.007); temperature fluctuated between 19.3 and 21.0 c (mean 20.35, se 0.097); conductivity varied from 228 to 286 ms cm–1 (mean 258.78, se 3.96); and total dissolved salts ranged between 263 and 280 mg l–1 (mean 273.7, se 0.65). however, the rest of the variables varied considerably. dissolved oxygen ranged from 0.96 to 4.08 mg l–1 (mean 2.1, se 0.204). water column irradiance varied between 0.26 and 2.03 (mean 0.94, se 0.096) in shaded zones and fluctuated from 0.35 and 2.34 (mean 1.17, se 0.113) in unshaded zones. if zeu equals zmax, then the macrophytes are constantly illuminated and photosynthesis is continuous during the daylight period. if the maximum depth is greater than the depth of the eutrophic zone then the macrophytes spend a proportion of their daylight period in the dark. water depth ranged between 1.2 to 6.5 m (mean 2.92 m, se 0.3) in shaded zones and from 1.5 to 6.5 m (mean 3.26 m, se 0.3) in unshaded zones, water depth was the same in both zones in sites ba5, ba6, aw8, sa10, sa14, se17, se19, se18 and se22. during the period of the study (april – june) the shadow-sun path direction was running southeast – northwest with an angle that was 100° in april, 120° in may and 160° in june. banks of the sites surveyed were of five directions; namely nw (gh3, ba4, ba5, ba6, sa13, se15, se17, and se18), se (gh1, gh2, aw7, br9, and se16), ne (se20 and se22), sw (aw8 and se23), w (se19 and se21), and n (sa10, sa11, sa12 and sa14). the simper analysis identifies those species, which contribute the most to the similarity between samples (tab. 1). the simper analysis indicates that c. demersum and p. crispus are the commonest species in the sites surveyed. their abundance explains 99% of the similarity between the sampling sites. c. demersum contributes 90% to the similarity between the sampling sites, while p. crispus only contributes 9.2 % in both shade and unshaded water zones. kruskal-wallis test indicated no significant differences between the five direction groups using total dwsc of macrophytes collected from the shaded zones (h-value = 3.617, and p = 0.606). however, differences were very significant between direction groups and total dwsc of macrophytes collected from the unshaded zones (h-value = 13.465, p = 0.019). acta bot. croat. 70 (2), 2011 251 aquatic plants management by riparian trees shade 0 20 40 60 80 g h 1 g h 2 g h 3 b a4 b a5 b a6 a w 7 a w 8 b r9 s a1 0 s a1 1 s a1 2 s a1 3 s a1 4 s e1 5 s e1 6 s e1 7 s e1 8 s e1 9 s e2 0 s e2 1 s e2 2 s e2 3 sites ceratophyllum demersum potamogeton crispus myriophyllum spicatum vallisneria spiralis d w s c (g s a m p le ) – 1 fig. 3. dry weight standing crop (dwsc) of submerged macrophytes recorded in the unshaded water zone. u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:23 color profile: disabled composite 150 lpi at 45 degrees canoco programme encountered only vegetated sampling sites (30 sites) into cca, while sites without macrophytes (16 sites from the shaded zone) were not included in the analysis. the environmental variables considered in the cca explained only 38.5 % of the total plant distribution and the species-environmental biplot explained 86.2 % of the total variation contained in the first two axes. the cca ordination diagram (fig. 4) indicates that water column irradiance is the most influential variable dictating the distribution of submerged macrophytes in the first cataract islands, while dissolved oxygen (do) is of least importance. the cca diagram indicates that sites from either shaded or unshaded zones that have high water column irradiance, i.e. of low water depth and high eutrophic 252 acta bot. croat. 70 (2), 2011 ali m. m., hassan s. a., shaheen a.-s. m. tab. 1. summary of simper analysis of 46 sampling sites from both shaded and unshaded zones. species average abundance average similarity contribution % cumulative % ceratophyllum demersum 11 11 90 90 potamogeton crispus 2.1 1.1 9.2 99 unassigned average similarity = 12 –0.6 0.8 –1.0 1.0 c. demersum p. crispus m. spicatum v. spirals a. filiculoides ph ec tdo sl nw se ne sw w n gh1u gh2u gh3u ba4u ba5u ba6u aw7u aw8u br9u sa10u sa11u sa12u sa13u sa14u se15u se16u se17u se18u se19u se20u se21u se22u se23u gh1s gh3s ba5s ba6s br9s se20s se21s fig. 4. canonical correspondence analysis (cca) ordination triplot of submerged macrphytes (ê) recorded in shaded (s) and unshaded (u) zones in the sampling sites (¡) surveyed in relation to the water variables measured (arrows): sl – water column irradiance, t – temperature, ec – electrical conductivity, do – dissolved oxygen; site directions (<): nw, se, ne, sw, w, and n. u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:23 color profile: disabled composite 150 lpi at 45 degrees light values (zeu/zmax ratio > 1) are characterised by p. crispus (e.g. se19u, se20s, se21s, se20u, seu21u) and those of lower water column irradiance (i.e. of high water depth and low eutrophic light values) are characterised by c. demersum and/or m. spicatum (e.g. ba5s, ba5u, b6s, ba6u, ch1s, gh1u, br9s, br9u). sites with p. crispus are of ne and w and have the highest ph and low dissolved oxygen values. c. demersum, situated in the middle of the diagram, is the most common species in both shaded and unshaded sites. these sites are mainly located on banks facing the nw direction. this is indicated by the position of the nominal variable nw, which is also, situated close to the centre of the programme close to c. demersum point. these sites were characterised by moderate values of all the environmental variables measured. the cca diagram showed that sites of se direction (right bottom) were distinguished by having m. spicatum. these sites were differentiated by having low water column irradiance and high temperature (t) and dissolved oxygen (do). v. spiralis was found in sites from the unshaded zones of nw and sw directions and of low water dissolved oxygen and temperature, but of high water electrical conductivity (ec). a. filiculoides was only recorded in one sampling site (gh3), which was of nw direction and characterised by the highest light extinction coefficient (i.e. lowest transparency), temperature and dissolved oxygen. discussion several studies have reported the beneficial functions of trees on the edges of water bodies such as thermal buffering, provision of shade, nutrient interception, storage and release enhancement of bank stability, provision of habitat and food for terrestrial and aquatic species and improving sediment regimes (bunn et al. 1999, pusey and arthington 2003, hudson and harding 2004, søvik and syversen 2008). riparian zones represent areas of strong biological, physical and chemical interaction between terrestrial and aquatic ecosystems. habitat structure is more diverse in sites where natural riparian vegetation is present (beltrão et al. 2009). water shaded by riparian trees is cooler and contains more dissolved oxygen; roots of riparian vegetation provide spawning sites for fish and the associated invertebrates are a significant food resource for them (harper et al. 1995); however, too dense a tree cover completely eliminates submerged macrophytes and hence other aquatic biota associated with them. similarly, in the present study cca indicates that site banks facing the nw direction (e.g. gh3, ba5, ba6, sa13), the opposite direction to sun path (se) that characterised by low water column irradiance, have the lowest water temperature and high dissolved oxygen. most of the shaded zones (five out of eight) in sites of nw direction, opposite direction to the sun path (se), have no vegetation (e.g. ba4s, sa13s, se15s, se17s, and se18s). surveys of natural shading by riparian trees have shown significant effects on aquatic plants (young et al. 2000). too much shading results in a suppression of submerged vegetation that is of vital importance to many aquatic biota (e.g. juvenile fish, invertebrates, algae). the scottish environment protection agency (2009) indicated that the use of shaded and unshaded sections of banks depresses or eliminates aquatic plant growth in the shaded sections and encourages plant growth in the unshaded sections. similarly, in the present study, aquatic plants growth was depressed or plants were absent in shaded zones, but they were abundant in unshaded zones. there was a significant difference in both the type and density between submerged plants growing shaded and unshaded by riparian trees (acacia nilotica). m. spicatum and v. spiralis were only found in the unacta bot. croat. 70 (2), 2011 253 aquatic plants management by riparian trees shade u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:23 color profile: disabled composite 150 lpi at 45 degrees shaded zones. we found ceratophyllum demersum dominating in both shaded and unshaded zones. it has been previously published (gupta and chandra 1996), that the growth rate of this species is attributed to an adequate co2 level and the tolerance to low light intensity. in the present study, cca triplot suggested that site banks of se directions that facing the sun path are of low water column irradiance, which was due to deep water characterising them. these deep-water sites are dominated by c. demersum or m. spicatum. similarly, johnson and ostrofsky (2004) found that c. demersum was the most frequently encountered species at all depths with highest relative importance in the deeper sites. the findings of the present study also agree with those of kautsky (1988) who pointed to the high ability of c. demersum to grow in adverse condition and stated that c. demersum is abundant in all sites where other aquatic species are rarely present. ceratophyllum demersum has a suitable morphology for light interception and longevity (grime 1979). these results suggest that c. demersum and m. spicatum are stress-tolerant, which are in line with other researches (frank 1975, segretain 1996). water variables varied slightly between sites, which could be attributed to the physical conditions in the river nile at the first cataract conservation islands, as well as the fixed sampling time (between 9:00 – 11:00 am). rate of oxygen released by submerged macrophytes are enhanced by light (sand-jensen et al. 1982, carpenter et al. 1983). this may explain high dissolved oxygen values recorded in sites associated with macrophyte dwsc (e.g. at ba6 dwsc is 72.1 g sample–1). in these sites, euphotic depth (zeu) almost equals the maximum depth (zmax), which allows constantly illumination and continuous photosynthesis of submerged macrophytes during the daylight period. high light intensity promotes growth of submerged macrophytes and allows higher photosynthetic rate, converting co2 to o2 (kitaya et al. 2003). sites having high macrophyte dwsc are dominated by c. demersum, which is well known as oxygen generator (han and rundquist 2003). we can conclude that sites of various light intensities supported vegetation of different composition and abundance. in the present study, riparian acacia nilotica trees provided natural shading limiting the growth of submerged aquatic plants, otherwise difficult to manage. the type and height of tree required for shade is dependent on the width and the cross-section of the water body (dawson 1978, williamson et al. 1990). in the present study, the area of shade provided by a riparian tree is affected by environmental and/or plant variables. in addition to the width and the cross-section of the water body, environmental variables comprise daily earth rotation around its axis and seasonal variation in the angle between the earth’s axis and the sun-earth path; and plant variables that include the projected area of the tree-crown, height and geographical position of the tree in relation to the sun-earth path. the present study indicated that highest shade effect of riparian trees could be achieved by planting trees on the west bank of the islands at the southwest-northeast direction, which perpendicularly obstructs the sun path that runs southeast-northwest. acknowledgements this work was supported by the faculty of science at aswan, south valley university and the first cataract conservation islands, eeaa. the authors thank mr. mahmoud hasseib, director of the southern protectorates sector for facilities provided during the fieldwork. 254 acta bot. croat. 70 (2), 2011 ali m. m., hassan s. a., shaheen a.-s. m. u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:23 color profile: disabled composite 150 lpi at 45 degrees we also thank the referees for their critical reviews and helpful suggestions which greatly improved the manuscript. this paper is dedicated to the memory of our friend and colleague references abernethy, v., 1994: the functional ecology of euhydrophyte communities of european riverine wetland ecosystems. phd thesis, university of glasgow, scotland. ali, m. m., hamad, a., springuel, i. v., murphy, k. j., 1995: environmental factors affecting submerged macrophyte communities in regulated waterbodies in egypt. archiv für hydrobiologie 133, 107–128. ali, m. m., springuel, i. v., yacoub, h. a., 1999: submerged plants as bioindicators for aquatic habitat qulity in the river nile. journal of union arab biologist 9 (b), 403–418. ali, m. m., mageed, a. a., heikal, m., 2007: importance of aquatic macrophyte for invertebrate diversity in large subtropical reservoir. limnologica 37, 155–169. apha, 1985: standard methods for the examination of water and wastewater. american public health association, new york. armengol, j., caputo, l., comerma, m., feijoó, c., garcía, j.c., marcé, r., navarro, e., ordoñez, j., 2003: sau reservoir’s light climate: relationships between secchi depth and light extinction coefficient. limnetica 22, 195–210. barsy, s. a., 1993: ecological study on bird life in aswan area. msc thesis, faculty of science at aswan, assiut university, assuit, egypt. beals, e. w., 1984: bray-curtis ordination: an effective strategy for analysis of multivariate ecological data. advances in ecological research 14, 1–55. beltrão, g. b. m., medeiros, e. s. f., ramos, r. t. c., 2009: effects of riparian vegetation removal on the structure of the marginal aquatic habitat and the associated fish fauna in a tropical brazilian reservoir. biota neotropica 9/4, 37–44. binzer, t., sand-jensen, k., 2002: production in aquatic macrophyte communities: a theoretical and empirical study of the influence of spatial light distribution. limnology and oceanography 47, 1742–1750. bunn, s. e., davies, p. m., mosisch, t. d., 1999: ecosystem measures of river health and their response to riparian and catchment degradation. freshwater biology 41, 333–345. bunn, s. e., davies, p. m., kellaway, d. m., prosser, i. p., 1998: influence of invasive macrophytes on channel morphology and hydrology in an open tropical lowland stream, and potential control by riparian shading. freshwater biology 39, 171–178. carpenter, s. r., elser, j. j., olson, k. m., 1983: effects of roots of myriophyllum verticillatum on sediment redox conditions. aquatic botany, 17, 243–249. dawson, f. h., 1978: aquatic plant management in seminatural streams: the role of marginal vegetation. journal of environmental management 6, 213–221. dawson, f. h., 1981: the reduction of light as a technique for the control of aquatic plants an assessment. proceedings of the association of applied biologists symposium aquatic weeds and their control, oxford, l57–164. acta bot. croat. 70 (2), 2011 255 aquatic plants management by riparian trees shade u:\acta botanica\acta-botan 2-11\ali.vp 26. rujan 2011 14:00:49 color profile: disabled composite 150 lpi at 45 degrees dawson, f. h., haslam, s. m., 1983: the management of river vegetation with particular reference to shading effects on marginal vegetation. landscape planning 10, 147–169. dawson, f. h., kern-hansen, u., 1979: the effect of natural and artificial shade on the macrophytes of lowland streams and the use of shade as management technique. internationale revue der gesamten hydrobiologie 64, 437–455. el-hadidi, m. n., hosni, h. a., 2000: flora aegyptiaca. in: el hadidi, m. n. 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ecology of spring creek cawthron. report no. 611. cawthron institute, nelson, new zealand. 258 acta bot. croat. 70 (2), 2011 ali m. m., hassan s. a., shaheen a.-s. m. u:\acta botanica\acta-botan 2-11\ali.vp 9. rujan 2011 12:56:23 color profile: disabled composite 150 lpi at 45 degrees 625 pise and gaikwad.vp acta bot. croat. 72 (2), 323–335, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/v10184-012-0019-3 proline enhances antioxidative enzyme activity, photosynthesis and yield of cicer arietinum l. exposed to cadmium stress shamsul hayat1,2*, qaiser hayat2, mohammed nasser alyemeni1, aqil ahmad2 1 department of botany and microbiology, king saud university, riyadh, saudi arabia 2 plant physiology section, department of botany, aligarh muslim university, aligarh 202 002, india abstract – seeds of chickpea inoculated with rhizobium were sown in pots supplemented with different doses of cadmium (0, 25, 50 or 100 mg per kg of soil). at the stage of 30 days after sowing (das), the plants were sprayed with 20 mm solution of proline and were sampled at 90 das to assess the various parameters. the foliar treatment of proline resulted in the alleviation of the adverse effects generated by metal exposure, which was expressed in terms of the increase in plant growth. the activity of carbonic anhydrase in the cadmium-fed plants sprayed with proline was higher than that of control. the proline applied as foliar spray increased the photosynthetic attributes and yield characteristics in the cadmium-stressed plants. the activity of antioxidative enzymes increased with increasing concentration of cadmium. maximum values were recorded in the plants exposed to 100 mg cadmium per kg of soil. key words: antioxidative enzymes, cadmium stress, cicer arietinum, growth, photosynthesis, proline, yield introduction plant growth and development is under the fine control of the genetic information contained in a cell, where some genes are up-regulated and some genes are down-regulated. however, only a limited portion of this information is expressed at any given time of plant growth and development. there are certain factors that play a significant role in regulating the repression and de-repression of these genes. out of these factors, light (thompson and white 1991), pollutants such as heavy metals (royals et al. 1992), phytohormones (cleland 1999) and metabolites such as proline (ashraf and foolad 2007) are noteworthy. plants grow well in soil, not only because it provides them with anchorage but also acta bot. croat. 72 (2), 2013 323 * corresponding author, e-mail: hayat_68@yahoo.co.in copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. with essential nutrients for their growth and development. out of the various heavy metals, cd is the most toxic. although present in traces in soil, its level has enormously increased in recent times especially in areas of heavy automobile traffic, near smelters and by the use of sewage sludge, city refuse and cd-containing phosphatic fertilizers. this increased concentration of cd in soil causes growth inhibition associated with various metabolic dysfunctions and even plant death (hasan et al. 2009). in order to cope with these conditions plants accumulate a wide array of metabolites, mainly proline (yang et al. 2009). proline, a multifunctional amino acid that besides acting as an excellent osmolyte is also known for stabilizing sub-cellular structures such as proteins and cell membranes, scavenging free radicals, balancing cellular homeostasis and signaling events and buffering redox potential under stress conditions (szabados and savoure 2009). proline may also function as a protein-compatible hydrotrope (srinivas and balasubramanian 1995), alleviating cytoplasmic acidosis and maintaining appropriate nadp+/nadph ratios compatible with metabolism during stress (hare and cress 1997). keeping in view the diverse physiological roles played by proline (hayat et al. 2012), the present study was aimed at elucidating whether exogenous proline could confer resistance against the damaging effects of cd. the hypothesis tested is that exogenous application of proline will alleviate the damaging effects of cd in plants and thereby enhance the growth, photosynthesis and yield attributes. materials and methods certified seeds of chickpea (cicer arietinum l.) cv. avarodhi were purchased from the national seed corporation ltd., new delhi, india. the seeds were surface sterilized with 0.01% mercuric chloride solution followed by inoculation with rhizobium and were sown in five sets of earthen pots (10 inch diameter) filled with sandy loam soil and farmyard manure (6:1) arranged under a simple randomized block design. at the start of the experiment, out of these five sets of prepared pots, four sets were supplemented with different doses (0, 25, 50 or 100 mg per kg of soil) of cd, and one set of pots was left untreated, serving as control. at 30 days after sowing (das), the foliage of the resulting plants was sprayed with 20 mm proline, except control which received double distilled water instead of proline. the concentration of proline was selected on the basis of our earlier experiments (data not shown). the plant samples were collected at 60 and 90 das to assess various growth and physiological parameters. all the parameters studied followed a similar trend at both the sampling stages; however, the magnitude of the data for these parameters was higher at 90 das, and therefore, the data obtained at this stage of plant growth are presented in the present study. plant growth analysis and enzyme activities the plants were uprooted and washed under running tap water. the root and shoot length of these plants was measured with the help of a graduated scale. these plants were blotted in blotting sheets to remove the adhering water and weighed on electronic balance to record their fresh weight. these plants were kept in an oven run at 80 °c for 72 hrs and then weighed to obtain their dry weight. the ca activity in the leaves was measured as described by dwivedi and randhawa (1974). the leaf samples were cut in small pieces in cysteine 324 acta bot. croat. 72 (2), 2013 hayat s., hayat q., alyemeni m. n., ahmad a. hydrochloride solution. these leaf samples were blotted and poured in a test tube, followed by addition of phosphate buffer (ph 6.8), 0.2 m nahco3, bromothymol blue and methyl red indicator, at the last. this reaction was titrated against hcl. the activity of the enzyme was expressed on fresh mass basis. for the assays of peroxidase (pox), catalase (cat) and superoxide dismutase (sod) the leaf tissue was homogenized in 50 mm sodium phosphate buffer (ph 7.0) containing 1% soluble polyvinylpyrrolidone. the homogenate was centrifuged at 15.000 rpm for 10 min at 5 °c and the supernatant obtained was used as an extract for pox, cat and sod. the reaction mixture for cat consisted of phosphate buffer (ph 6.8), 0.1 m h2o2 and 1.0 ml enzyme extract. h2so4 was added to the reaction mixture, and after being incubated for 1 min at 25 °c, it was titrated against potassium permanganate solution (chance and maehly 1956). for the estimation of pox activity the enzyme extract (0.1 ml) was added to the reaction mixture consisting of pyrogallol phosphate buffer (ph 6.8) and 1% h2o2. the change in the absorbance was read for 2 min at the interval of 20 sec, at 420 nm on a spectrophotometer. the activity of sod was measured according to beauchamp and fridovich (1971). a 3 ml of reaction mixture containing 50 mm phosphate buffer (ph 7.8), 13 mm methionine, 75 mm nitroblue tetrazolium, 2 mm riboflavin, 0.1 mm edta and 0–50 mm of enzyme extract was prepared. riboflavin was added last. this reaction mixture was exposed to low fluorescent light and the decrease in absorbance of the reaction mixture was read at 560 nm on a spectrophotometer. 50% inhibition was considered as one enzyme unit. the proline content in the fresh leaf sample was measured following the method described by bates et al. (1973). fresh leaf sample (0.5 g) was homogenized in a mortar with 5 ml of 3% sulphosalicylic acid. the homogenate was filtered through whatman no. 2 filter paper and collected in a test tube with two washings each with 5 ml of sulphosalicylic acid. two ml each of glacial acetic acid and acid ninhydrin were added to 2 ml of the above extract. this mixture was heated in a boiling water bath for 1 h. the reaction was terminated by transferring the test tube to an ice-bath. four ml of toluene was added to the reaction mixture with vigorous shaking for 20–30 seconds. the chromophore (toluene) layer was aspirated and warmed to room temperature. the absorbance of red colour was read at 520 nm against a reagent blank. photosynthetic measurements, leaf water potential, yield characteristics and seed protein content the stomatal conductance (gs), intercellular co2 concentration (ci), transpiration rate (e), water use efficiency (wue) and net photosynthetic rate (pn) in intact leaves were measured by li-6400 portable photosynthesis system (li-cor lincoln, ne, usa), between 11:00 and 12:00 hrs under bright sunlight. the atmospheric conditions during measurement were photosynthetically active radiation (par), 1016±6 µmolphotons m –2 s–1, relative humidity 60±3%, atmospheric temperature 22±1 °c and atmospheric co2, 360 mg l –1. the ratio of atmospheric co2 to intercellular co2 concentration was constant. leaf water potential, at each selected stage, was measured in fresh, detached leaves of the sample plants by using psypro water potential system (wescor inc. usa). at harvest (160 das), 3 plants (replicates) from each treatment were randomly sampled and counted for the number of pods per plant. 25 pods from each treatment were randomly selected for computing the number of seeds per pod. the pods from three plants, representing each treatment, were acta bot. croat. 72 (2), 2013 325 proline and cadmium stress crushed, cleaned to assess the seed weight per plant. the total protein content in the dry seeds, at harvest, was estimated by the method of lowry et al. (1951). statistical analysis the experiment was conducted according to randomized block design technique in the years 2009 and 2010. the data of both the experiments were pooled. each treatment was represented by ten pots where each pot was considered as a replicate. three observations (from three different pots) were recorded per treatment. the treatment means were compared by analysis of variance using spss software version 10 (spss, chicago, il, usa). least significant difference (lsd) was calculated at 5% level of probability. results growth characteristics the spraying of plants with proline resulted in a significant increase in all the growth parameters, by 30.2% (root length); 29.8% (shoot length); 33.6% (plant fresh mass); 41.2% (plant dry mass), over that of the control (fig. 1). the plants grown in 25, 50 or 100 mg of cd per kg of soil possessed lower values for root length, shoot length, plant fresh and dry mass as compared to control and response of all the growth characteristics was dependent on the concentration of cadmium in the soil. all the growth characteristics (root length, shoot length, plant fresh and dry mass) decreased as the concentration of cadmium was increased from 25 to 100 mg of cd per kg of soil. further, the exogenous application of proline completely alleviated these ill effects generated by 25 mg of cd per kg of soil on all the growth characteristics. however, the plants grown in the soil supplemented with 50 mg of cd per kg soil and also sprayed with proline revealed values for all the growth characteristics that were not statistically different from those of control. however, the plants fed with cd at the rate of 100 mg per kg of soil and also sprayed with proline, showed significantly lower values for these parameters than the control. carbonic anhydrase (ca) activity the ca activity in unstressed plants was significantly increased (40.9%) by the foliar application of proline as compared to that of control (fig. 2). the activity of ca in plants fed with cd supplemented at the rate of 25 mg per kg of soil and also sprayed with proline was found to be 18.9% higher. however, the application of proline to the foliage of plants fed with cd (50 mg per kg of soil) generated the values of ca that were statistically equal to that of the control. the activity of ca in plants exposed to cd supplied at the rate of 100 mg per kg of soil and also sprayed exogenously with proline possessed significantly lower values (16.1%) than the control. photosynthetic parameters exogenous application of proline to the unstressed plants resulted in significant increases of 24.0% (gs), 20.3% (ci), 64.4% (wue), 29.0% (e), 22.9% (pn) as compared to the untreated control (fig. 2). further, the foliar application of proline alleviated the stress generated by cd (25 mg per kg of soil) where a significant increase of 10.9% (gs), 11.8% (ci), 26.7% (wue), 8.1% (e) and 11.2% (pn) was recorded, compared to the control. the 326 acta bot. croat. 72 (2), 2013 hayat s., hayat q., alyemeni m. n., ahmad a. values of almost all these photosynthetic parameters, in the plants fed with cd supplied at the rate of 50 mg per kg of soil, were found to be statistically equal to those of the control. however, like other parameters, the foliar application of proline to the plants receiving cd (100 mg per kg of soil) showed significantly lower values for photosynthetic attributes, than the control (fig. 2). antioxidative enzyme activities the foliar application of proline to unstressed plants significantly enhanced the antioxidative enzyme activity by 11.9% (cat), 25.0% (pox), and 26.4% (sod), over that of the control (fig. 3). the exogenous application of proline to the cd fed plants further acta bot. croat. 72 (2), 2013 327 proline and cadmium stress fig. 1. effect of proline on the cadmium-induced (0, 25, 50 or 100 mg cd per kg of soil) changes in root length (a), shoot length (b), fresh mass (c), dry mass (d) in cicer arietinum at 90 das. data are the mean of three independent replicates. vertical bars represent the standard error (±). 328 acta bot. croat. 72 (2), 2013 hayat s., hayat q., alyemeni m. n., ahmad a. fig. 2. effect of proline on the cadmium-induced (0, 25, 50 or 100 mg cd per kg of soil) changes in ci (a), e (b), pn (c), wue (d), ca activity (e) and gs (f) in cicer arietinum at 90 das. data are the mean of three independent replicates. vertical bars represent the standard error (±). elevated the activity of these enzymes over unstressed plants sprayed with proline as well as of the control plants. the treatment resulted in a significant increase of 17.1% (cat), 42.8% (pox), 31.4% (sod) in plants fed with cd supplied at the rate of 25 mg per kg of soil; 24.1% (cat), 57.9% (pox), 42.1% (sod) in those fed with cd (50 mg) and 31.5% (cat), 82.9% (pox) and 61.4% (sod) in the plants receiving 100 mg cd per kg of soil, respectively, over that of the control (fig. 3). proline content the foliar application of proline significantly increased the endogenous proline content in the unstressed plants by 38.7%, compared to control (fig. 4a). further the endogenous acta bot. croat. 72 (2), 2013 329 proline and cadmium stress fig. 3. effect of proline on the cadmium-induced (0, 25, 50 or 100 mg cd per kg of soil) changes in cat (a), pox (b) and sod activities (c) in cicer arietinum at 90 das. data are the mean of three independent replicates. vertical bars represent the standard error (±). proline level experienced a sharp increase in the cd-fed plants when proline was sprayed on their foliage. the endogenous proline level in plants fed with 25, 50 or 100 mg cd per kg of soil and sprayed with proline was increased significantly by 52.0%, 59.2% and 81.1% over their respective controls. leaf water potential as is evident from figure 4(b), the leaf water potential increased significantly by 58.8%, compared to the control, in the unstressed plants sprayed with proline. the application of proline to the foliage of plants exposed to cd (25, 50 or 100 mg per kg of soil) also resulted in a significant increase of leaf water potential over control. (fig. 4b). 330 acta bot. croat. 72 (2), 2013 hayat s., hayat q., alyemeni m. n., ahmad a. fig. 4. effect of proline on the cadmium induced (0, 25, 50 or 100 mg cd per kg of soil) changes in proline content (a) and leaf water potential (b) in cicer arietinum at 90 das. data are the mean of three independent replicates. vertical bars represent the standard error (±). number of pods per plant and number of seeds per pod the exogenous application of proline to the unstressed plants and to those fed with 25 mg of cd per kg of soil significantly enhanced the number of pods per plant, by 50.0% and 25.8% respectively, compared to the control (tab. 1). further the number of pods was found to be statistically equal to control in the plants exposed to cd (50 mg per kg of soil) and also sprayed with proline, whereas it was significantly lower in the plants exposed to cd (100 mg per kg of soil). on the other hand, the number of seeds per pod was found to be statistically non-significant, irrespective of the treatments. seed yield and mass of 100 seeds the seed yield and mass of 100 seeds also followed the same pattern observed in number of pods per plant. a significant increase of 88.6% (seed yield) and 31.9% (mass of 100 seeds) was recorded in unstressed plants sprayed with proline (tab. 1). foliar spray of proline on the plants exposed to cd (25 mg per kg of soil) also resulted in a significant increase of 34.7% (seed yield) and 7.0% (mass of 100 seeds) over control. seed protein content the application of proline to the foliage of unstressed plants and to those exposed to 25 mg cd per kg of soil, increased the seed protein content significantly, by 11.2% and 6.8%, respectively, over that of control (tab. 1). further, the proline spray onto the plants exposed to 50 mg cd per kg of soil revealed values for seed protein content that were statistically equal to that of the control. however, the value of seed protein content in the 100 mg cd-fed plants and also treated with proline was found to be statistically lower than that of the control. discussion cadmium causes multiple direct and indirect effects on plant growth and metabolism by forming complexes with o, n and s ligands and also inhibits net photosynthesis in various crops (hasan et al. 2009). cadmium becomes associated with the cell wall and middle acta bot. croat. 72 (2), 2013 331 proline and cadmium stress tab. 1. effect of proline on the cadmium-induced (0, 25, 50 or 100 mg cd per kg of soil) changes on yield characteristics and seed protein content in cicer arietinum. treatments pod number number of seeds aper pod seed yield (g per plant) mass of 100 seeds (g) seed protein content (%) control 19.40 2 5.80 15.55 18.55 cd (0) + proline 29.10 2 10.94 20.51 20.63 cd (25) + proline 24.40 1.33 7.81 16.63 19.81 cd (50) + proline 19.61 2 5.10 14.00 18.44 cd (100) + proline 16.49 2 3.51 10.00 16.75 lsd = 0.05 1.4 ns 0.5 1.0 0.7 n.s. – non significant lamellae, which increases the cross linking of pectins and results in the inhibition of cell growth (barcelo and poschenreider 1990) and therefore, the plant as a whole. however, the follow-up treatment with proline improved the growth of plants expressed in terms of increased length of root and shoot and fresh and dry mass of the plant (fig. 1). the concentration of proline was based on our preliminary experiment in which 10, 20, 30, 40, 50 mm of proline was tested (hayat and hayat 2011). in view of some earlier reports (hayat et al. 2012), it is suggested that the application of proline increases endogenous proline content (fig. 4a) under heavy metal stress conditions. this will help to protect enzymes (islam et al. 2009), 3-d structure of proteins (paleg et al. 1981) and organelle and cell membranes by reducing the lipid peroxidation (okuma et al. 2004). besides this it also supplies energy for growth and survival, thereby helping the plant to tolerate stress (hoque et al. 2007) thereby enhancing growth characteristics (fig. 1). cd brings about the closure of stomata by decreasing the partial pressure of co2 in the stroma (barcelo and poschenreider 1990), which becomes a direct cause of the reduction of the gs, ci, wue and e. further, cd is known to enhance the activity of the enzyme chlorophyllase, which brings about the degradation of chlorophyll (reddy and vora 1986) and also reduces the synthesis of d-amino-levulinic acid and protochlorophyllide reductase complex (gadallah 1995), leading to decreased pigment concentration. the reduced photosynthesis in cd-treated plants might also be due to the reduced activity of ca (hasan et al. 2008). the activity of this enzyme is largely determined by photon flux density, concentration of co2, the availability of zn (tiwari et al. 2005) and gene expression (kim et al. 1994). the stress generated by cd decreases the partial pressure of co2 in the stroma by inducing the stomatal closure (barcelo and poschenreider 1990) resulting in the loss of ca activity. however the follow-up treatment of cd-stressed plants with proline resulted in enhanced ca activity and thereby increased pn (fig. 2). it is well documented that the exogenous application of proline protects plants against stress by stabilizing the complex ii of electron transport chain in mitochondria (hamilton and heckanthorn 2001), membranes and 3-d structure of proteins (holmstrom et al. 2000) and enzymes such as rubisco and ca (makela et al. 2000) thereby increasing photosynthetic attributes (fig. 2). therefore, the follow-up treatment with proline resulted in enhanced activity of these enzymes, which may be due to the ros scavenging potential of proline (zhou et al. 2010) through enhancement of the activity of antioxidative enzymes (islam et al. 2009) i.e. cat, pox and sod (fig. 3) and the endogenous level of proline (fig. 4a). these results are supported by previous findings that exogenous proline elevated the free proline content eliciting its biosynthetic pathways (hayat et al. 2012) and the activity of the antioxidative enzymes (cat, pox and sod) by acting at the level of transcription and/or translation. since cd causes membrane degradation by increasing the lipid peroxidation, thereby leading to significant electrolyte leakage (hasan et al. 2009), it will obviously result in a lowering of leaf water potential (fig. 4b). however, the follow-up treatment with proline enhanced the leaf water potential by protecting the membranes from metal induced oxidative damage (okuma et al. 2004) thereby decreasing the electrolyte leakage and increasing the leaf water potential (fig. 4b). the increased growth, photosynthetic efficiency and elevated activity of antioxidative enzymes under the influence of exogenous proline are likely to increase the yield (tab. 1). a plausible reason for the increase in yield is the delay of senescence of plant organs (particularly leaves and flowers) in response to exogenous proline (balestrasse et al. 2004), which will help the plant to improve the efficiency of photosynthetically active 332 acta bot. croat. 72 (2), 2013 hayat s., hayat q., alyemeni m. n., ahmad a. sites and also prevent the premature loss of flowers and fruits. this consequently resulted in the observed increase in the number of pods per plant (tab. 1). proline is known to increase the total phenolic content in plants (kwok and shetty 1998) and phenolics are known to prevent auxin degradation (schneider and whitman 1974), which might be responsible for the increase in the seed yield. this gets additional support from the observations of hayat et al (2001) that phytohormones increase the degree of sink at the level of seeds, directing the flow of metabolites to the developing seeds thereby improving the mass of 100 seeds and consequently the seed yield per plant at harvest (tab. 1). conclusion it can be concluded from the present investigation that the foliar application of proline to cadmium-treated plants decreased the negative effects generated by the cadmium. all the growth and photosynthetic parameters increased significantly when proline was applied as foliar spray. the activity of antioxidative enzymes and the proline content in the cd fed plants increased further with foliar spray of proline. acknowledgements the authors extend their appreciation to the deanship of scientific research at king saud university for funding the work through the research group project no. rgp-vpp-199. references ashraf, m., foolad, m. r., 2007: roles of glycine betaine and proline in improving plant abiotic stress resistance. environmental and experimental botany 59, 206–216. balestrasse, k. b., gallego, s. m., tomaro, m. l., 2004: cadmium-induced senescence in nodules of soybean (glycine max l.) plants. plant and soil 262, 373–381. barcelo, j., poschenrieder, c., 1990: plant water relations as affected by heavy metal stress: a review. journal of plant nutrition 13, 1–37. bates, l. s., walden, r. t., tearse, i. d., 1973: rapid determination of 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fu, j. j., 2010: the physiological response and sub-cellular localization of lead and cadmium in iris pseudacorus l. ecotoxicology 19, 69–76. acta bot. croat. 72 (2), 2013 335 proline and cadmium stress 529 zia-ul-haq et al.vp acta bot. croat. 72 (1), 133–144, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 chemical composition and antioxidant potential of acacia leucophloea roxb. muhammad zia-ul-haq1, sanja ]avar2,*, mughal qayum3, inamullah khan4, shakeel ahmad5 1 research institute of pharmaceutical sciences, department of pharmacognosy, university of karachi, karachi 75270, pakistan 2 university of sarajevo, faculty of science, department of chemistry, 71000 sarajevo, bosnia and herzegovina 3 department of pharmacy, kohat university of science and technology, kohat 2600, pakistan 4 department of pharmacy, university of peshawar, peshawar 25120, pakistan 5 department of agronomy, bahauddin zakariya university, multan 60800, pakistan abstract – the aim of this study was to evaluate the compositional and nutritional potential of methanolic extracts of various parts of acacia leucophloea roxb. concerning the chemical composition and antioxidant potential of which limited information is available. compositional studies indicated carbohydrates as major components in both seed and pods. despite differences in mineral content among the leaves, pods and seeds, calcium was found in the highest amount and zinc in the lowest. the amino acid profile indicated aspartic acid as the major amino acid and proline as the minor. among protein fractions, globulin was present in higher amounts than other fractions. linoleic acid was the major fatty acid detected in the oil from both pods and seeds, while g-tocopherol was the major component of the tocopherol observed from same oil. moreover, significant antioxidant potential was observed from the extracts of all three parts investigated. the results obtained in this study clearly indicate that a. leucophloea has a sufficient potential for use as a natural antioxidant agent. further phytochemical studies will be performed for specification of the biologically active principles. key words: acacia leucophloea, mineral content, amino acids, fatty acids, tocopherols, sterols, antioxidant activity abbreviations: aoac – association of official analytical chemists; bha – butylated hydroxy anisole; bht – butylated hydroxy toluene; frap – ferric reducing antioxidant power; ros – reactive oxygen species; tac – total antioxidant capacity; teac – trolox equivalent antioxidant capacity; trap – radical-trapping antioxidant parameter. acta bot. croat. 72 (1), 2013 133 * corresponding author, e-mail: sanja_cavar@yahoo.com copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:55 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees introduction the leguminosae form the third largest plant family in the world, comprising about 650 genera and 18000 species. the subfamily mimosoideae, with 3000 species in 50–60 genera, is distributed worldwide and is abundant in tropical, subtropical and warm-temperate areas (polhill and raven 1981). acacia is the most significant genus of the subfamily mimosoideae. it is estimated that there are roughly 1380 species of acacia worldwide, about two-third of them native to australia and rest spread around tropical and subtropical regions of the world (saini et al. 2008). acacia species although distributed throughout pakistan, are mostly found in sindh and punjab province. in pakistan 34 species of acacia are reported, most of them growing wild (nasir and ali 1974, akhtar 1992). acacia contains organic compounds, many of which are psychoactive in humans. the alkaloids found include dimethyltryptamine (dmt), 5-methoxydimethyltryptamine (5-meo-dmt) and n-methyltryptamine (nmt). similarly, many cyanogenic glycosides have also been isolated from this species (david et al. 1987). acacia is effective when used as a coating on pills to make the bitterness easier to swallow or digest. it is important to the food industry as a flavor fixative and emulsifier. this plant has been used in traditional medicine to treat high cholesterol, diabetes, cancer, gingivitis, mouth sores, pharyngitis, and indigestion in children. acacia leucophloea roxb. is a moderate-sized dry-season fodder and pasture tree, found in various parts of pakistan and is known for its nutritional and medicinal potential. its germinated seeds are eaten after cooking. its leaves, tender shoots, and pods are readily consumed by goats, sheep and cattle. accacia leucophloea bark has a foul smell and its fibers are used to make fish nets and rough rope. the bark yields water soluble gum of fair quality (troup 1983) which is demulcent and used as emulsifying agent. bark is used to purify liquor and yields a reddish-brown stain which is used for the preparation of dyes. moreover, the bark is used against snake bites (selvanayagam et al. 1995, walter et al. 2000). the seed sprouts are consumed as a vegetable in some parts of indonesia. the species is treasured as firewood and is appropriate for charcoal production. acacia leucophloea is a good reforestation species for poor soils in low rainfall areas (orwa et al. 2009). traditionally, parts of this plant are used against diarrhoea, cancer, inflammation, ophthalmia, hemorrhoid, leprosy, bleeding piles, and leucoderma problems. its young leaves and pods are used as an astringent. the leaves are believed to possess hypotensive, cns-depressent, antisyphilitic and antimicrobial principles, while the gum possesses demulscent properties (khare 2007). the human body has numerous mechanisms to shield biomolecules against reactive oxygen species (ros) induced damage. however, the instinctive protection may not be adequate for rigorous or continuous oxidative stress. hence, certain amounts of exogenous antioxidants are frequently required to maintain sufficient antioxidants level to balance the ros-pressure in the human body. synthetic antioxidants such as butylated hydroxanisole (bha) and butylated hydroxytoluene (bht) although very effective, are not without side-effects and should be replaced with natural antioxidants. various natural antioxidants like flavonoids, phenols, tannins, and terpenoids, reported from crude herbal extracts, may lessen ros-pressure due to their inherent ability to scavenge reactive oxygen/nitrogen radical species (ros/rns), chelating metal catalysts, activating antioxidant enzymes, and inhibiting oxidases (grace 2005). in this context, as part of our continuous studies on 134 acta bot. croat. 72 (1), 2013 zia-ul-haq m., ]avar s., qayum m., khan i., ahmad s. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:55 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees exploring the hidden potential of indigenous flora of pakistan (zia-ul-haq et al. 2007; 2008a, b; 2009; 2010; 2011), this investigation focuses mainly on exploring a. leucophloea as a source of food, feed, and antioxidant source to provide baseline data for future commercial exploitation and for overcoming malnutrition-associated problems. materials and methods plant material acacia leucophloea (seeds, leaves, flower and pods) plant material was collected from khanewal, a district of multan, punjab, pakistan in june 2008, and authenticated by saima shehzadi, a taxonomist in charge of the herbarium at the institute of pure and applied biology, bahauddin zakariya university, multan and a voucher specimen was deposited for future identification. extraction the plant material (acacia leucophloea seeds, leaves, flower and pods) was air-dried in shade and crushed to coarse powder separately with help of pestle and mortar. plant material (0.5 kg each) was macerated with an aqueous methanolic mixture (80:20; v/v, 1l), at room temperature for fifteen days with occasional shaking. the process was repeated three times with the same quantity of solvent mixture. the extracts so obtained were combined, filtered through filter paper under vacuum and concentrated under reduced pressure in a rotary evaporator (model q-344b – quimis, brazil) using a warm water bath (model q-214m2 – quimis, brazil) to obtain a thick gummy mass, which was further dried in a desiccator and stored in an air-tight vial until tests for antioxidant activities were performed. proximate analysis moisture, lipids, ash, protein, and carbohydrates of seeds were determined according to aoac (1990). mineral content the samples (100 mg) after acid digestion (a nitric/perchloric acid (2:1) mixture) were used for elemental analysis. sodium and potassium were estimated by flame photometer (flame photometer model-eel). an atomic absorption spectrometer (perkin–elmer model 5000) was used to measure calcium, manganese, magnesium, zinc, copper, and iron, while phosphorus was determined by the phosphovanado-molybdate method (zia-ul-haq et al. 2007). the samples were quantified against standard solutions of known concentration that were analyzed concurrently. estimation of seed protein fractions cold trichloroacetic acid 20% was used for extracted protein (rajaram and janardhanan 1990) purification by precipitation. the seed protein fractions, albumins and acta bot. croat. 72 (1), 2013 135 phytochemical studies of acacia leucophloea roxb. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:55 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees globulins were extracted (murray 1979). the remaining pellet was treated with 80% ethanol (1:5 w/v) overnight and centrifuged at 20,000 x g for 20 min, and the supernatant containing prolamine was air-dried and dissolved in 0.1 n naoh. the final remaining pellet was extracted with 0.4 n naoh (1:10 w/v) overnight and centrifuged at 20,000 x g for 20 min and the supernatant thus obtained was the glutelin proteins. the fractions obtained were determined for protein content (lowry et al. 1951). amino acid content hydrochloric acid (6m) was used to hydrolyze samples in an evacuated test tube for 24 h at 105 °c. the dried residue was dissolved in citrate buffer (ph 2.2) after flash evaporation. aliquots were analyzed in an automatic amino acid analyzer (hitachi perkin–elmer model kla 3b), using the buffer system described earlier. methionine and cysteine were analyzed separately after performic acid treatment and subsequent hydrolysis with hcl (khalil et al. 1990). tryptophan was determined after alkali (naoh) hydrolysis by the colorimetric method (freidman and finely 1971). fatty acid composition fatty acid composition of extracted oils was determined following the iso draft standard iso/fids 5509 (1997). one drop of the oil was dissolved in1 ml of n-heptane, 50 ml 2m sodium methanolate in methanol were added, and the closed tube was agitated vigorously for 1 min. after addition of 100 ml of water, the tube was centrifuged at 4500 x g for 10 min and the lower aqueous phase was removed. after that, 50 ml of 1 m hcl were added to the n-heptane phase, the two phases were briefly mixed and the lower aqueous phase was rejected. about 20 mg of sodium hydrogen sulphate were added, and after centrifugation at 4500 g for 10 min the top n-heptane phase was transferred into a vial and injected in a varian 5890 gas chromatograph with a capillary column, cp-sil88 (100 m long, 0.25 mm id, film thickness 0.2 mm). the temperature programme was: from 155 °c heated to 220 °c (1.5 °c min–1), 10 min isotherm; injector 250 °c, detector 250 °c; carrier gas 1.07 ml min– h2; split ratio 1:50; detector gas 30 ml min–1 h2; 300 ml per min air and 30 ml per min n2; manual injection volume less than 1 ml. the integration software computed the peak areas and percentages of fatty acid methyl esters (fame) were obtained as weight percent by direct internal normalization. tocopherol content for determination of tocopherols a solution of 250 mg of oil from the samples studied in 25 ml n-heptane was directly used for hplc. the hplc analysis was conducted using a merck-hitachi low-pressure gradient system, fitted with an l-6000 pump, a merck-hitachi f-1000 fluorescence spectrophotometer (detector wavelengths for excitation 295 nm, for emission 330 nm) and a d-2500 integration system; 20 ml of the samples were injected by a merck 655-a40 autosampler onto a diol phase hplc column 25 cm x 4.6 mm id (merck, darmstadt, germany) using a flow rate of 1.3 ml min–1. the mobile phase used was n-heptane / tert-butyl methyl ether (99+1, v/v). calibration was done by external standards with a-, b-, gand d-tocopherol (calbiochem, bad soden, germany) (balz et al. 1992). 136 acta bot. croat. 72 (1), 2013 zia-ul-haq m., ]avar s., qayum m., khan i., ahmad s. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:55 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees sterol composition the determination of sterols was made following the official method of the association of official analytical chemists (aoac 1990). seed oil was saponified, while unsaponifiable matter was extracted with diethyl ether, and solvent was removed under vacuum. then sterols were separated from other unsaponifiables by thin layer chromatography (tlc silica gel 60 f254; merck, darmstadt, germany) using a solvent system of hexane:diethyl ether (70:30 v/v). the spots were visualized with 2, 7-dichlorofluorescein (0.2% in alcohol). the sterols band was identified under uv light by comparing the rf value with that of authentic cholesterol (sigma aldrich co., st. louis, mo, usa). the recovered sterol from silica gel was converted to trimethylsilyl ether derivatives using a mixture of hexamethyldisilane-trimethylchlorosilane (99:1, v/v). analysis of transformed sterols was carried out on a perkin elmer gas chromatograph model 8700, equipped with methyl phenyl polysiloxane coated capillary column ov-17 (30 m × 0.25 mm, 0.20 mm film thickness) and a flame ionization detector (fid). the column was isothermally operated at a temperature of 255 °c. injector and fid temperatures were set at 275 and 290 °c, respectively. extra pure n2 at a flow rate of 3 ml min–1 was used as a carrier gas. the internal standard used was 5-a-cholestane. identification and quantification of the unknown sterol components were performed using a pure sterol standard mixture. total antioxidant capacity determination plant extracts were analyzed for their antioxidant capacity by three different tac assays: 1) trolox equivalent antioxidant capacity (teac) assay, 2) ferric reducing antioxidant power (frap) assay and 3) total radical-trapping antioxidant parameter (trap) assay. trolox equivalent antioxidant capacity (teac) assay a stable stock solution of abts·+ was produced by reacting a 7 mmol l–1 aqueous solution of abts with 2.45 mmol l–1 potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. at the beginning of the analysis day, an abts·+ working solution was obtained by the dilution in ethanol of the stock solution to an absorbance of 0.70 ± 0.02 au at 734 nm, verified by a hewlett-packard 8453 diode array spectrophotometer (hp, waldbronn, germany), and used as mobile phase in a flow-injection system. results were expressed as micromoles of trolox per g of plant extract (pellegrini et al. 2003). ferric reducing antioxidant power (frap) assay the principle of this method is based on the reduction of a ferric-tripyridyltriazine complex to its ferrous coloured form in the presence of antioxidants. briefly, the frap reagent contained 5 ml of a (10 mmol l–1) tptz (2,4,6-tripyridyl-s-triazine) solution in 40 mmol l–1 hcl plus 5 ml of fecl3 (20 mmol l –1) and 50 ml of acetate buffer (0.3 mol l–1, ph=3.6) and was prepared freshly and warmed at 37 °c. aliquots of 100 ml sample were mixed with 3 ml frap reagent and the absorbance of reaction mixture at 593 nm was measured spectrophotometrically after incubation at 37 °c for 10 min. for construction of calibration curve five concentrations of feso x 7h2o (1000, 750, 500, 250, 125 mmol l –1) were used and the absorbencies were measured as sample solution. the values were expressed as the acta bot. croat. 72 (1), 2013 137 phytochemical studies of acacia leucophloea roxb. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees concentration of antioxidants having a ferric reducing ability equivalent to that of 1 mmol l–1feso4. results were expressed as micromoles of fe 2+ equivalents per g of plant extract (benzie and strain 1999). total radical-trapping antioxidant parameter (trap) assay this method is based on the protection provided by antioxidants on the fluorescence decay of r-phycoerythrin (lag-phase) during a controlled peroxidation reaction. briefly, 120 ml of diluted sample were added to 2.4 ml of phosphate buffer (ph 7.4), 375 ml of bidistilled water, 30 ml of diluted r-pe and 75 ml of abap; the reaction kinetics at 38 °c were recorded for 45 min (or more, if necessary) by a ls-55 luminescence spectrometer (perkin elmer, wellesley, ma). trap values were calculated from the length of the lag-phase due to the sample compared with that of trolox and expressed ass micromoles of trolox per g of plant extract (ghiselli et al. 1995). statistical analysis analyses were performed in triplicate. data analysis was carried out using the analysis of variance and lsd test using the »mstatc« statistical computer package. results and discussion the wide variation in geological and climatic conditions and agronomic practices of pakistan offers the broadest array of flora harboured by the forests, deserts, mountains and rivers. this rich floral biodiversity of pakistan constitutes an impressive pool for ‘natural pharmacy’ from which the indigenous communities select ingredients to prepare herbal recipes (phytomedicines) for the treatment, management and control of their various ailments. much of the potential of these botanicals is however still untapped. in current research, a less explored medicinal tree found in pakistan has been investigated for various nutritional parameters. proximate composition is an important indicator and the first step during nutritional evaluation of fruits and seeds of plants and crops; it also dictates further studies of any component that seems more interesting. proximate composition indicated the presence of higher amounts of proteins (25.81 ± 0.93% and 13.81 ± 0.93%) and carbohydrate (52.30 ± 1.16% and 46.75 ± 0.89%) in seed than pod, respectively (tab. 1). similarly, a higher content of fibres indicates its potential use in bakery and pastry products. our results are in agreement 138 acta bot. croat. 72 (1), 2013 zia-ul-haq m., ]avar s., qayum m., khan i., ahmad s. tab. 1. proximate chemical composition of acacia leucophloea roxb. component seeds (%) pods (%) crude protein 25.81 ± 0.93 13.81 ± 0.93 total lipids 5.03 ± 0.07 4.75 ± 0.09 total carbohydrates 52.30 ± 1.16 46.75 ± 0.89 crude fiber 6.70 ± 0.38 19.11 ± 0.44 moisture 5.61± 0.23 9.57 ± 0.79 ash 4.55 ± 0.56 6.01 ± 0.19 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees with those reported earlier for the seeds (vijayakumari et al. 1994). to the best of our knowledge, no previous study exists on pod proximate composition. ash content was higher in pod (6.01 ± 0.19% and 4.55 ± 0.56%) indicating a higher mineral content in pods, while lipid content was lower in pod (5.03 ± 0.07 % and 4.75 ± 0.09%) than in seeds (tab. 1). pods usually have lower levels of protein and lipids, and greater fiber content than found in seeds. usually the proximate composition of plants and crops seeds varies depending on the cultivars, climatological conditions, maturity and collection time of seed, water and fertilizer application as well as acceptability, selectivity and intake of nutrients by plants and crops (zia-ul-haq et al. 2011). mineral contents indicated potassium was present in the highest concentration, 11.47 and 13.09 g kg–1, in seed and pod, respectively (fig. 1), while leaves had a higher concentration of calcium (17.32 g kg–1). sufficient amounts of calcium, magnesium and phosphorus were present in all three parts analyzed. potassium was observed to be the main element in pods. since leaves and pods of the acacia species investigated are is a rich source of minerals, they may be added as mineral supplement in ruminant diets as high mineral contents are considered essential for various physiological functions of browsing animals. high levels of calcium are required during growth, gravidity and lactation of animals. results are comparable with another closely related species, a. nilotica seed (siddhuraju et al. 1996), and leaves (rubanza et al. 2007, kumar et al. 2010). the differences, however, may be due to the different unit for the expression of mineral content. mineral content found in all parts are promising, as all these parts are eaten by animals, providing their mineral requirements. to the best of our knowledge, no previous studies of the micronutrients of pods of a. leucophloea exist. presented results for seed protein fractions indicated the highest amounts of globulins 13.01 ± 1.27% and 6.12 ± 0.25%, while prolamines, 1.21 ± 0.54% and 1.09 ± 0.46%, were present in the lowest amounts in both seed and pods, respectively (tab. 2). mostly seed proteins of legumes are higher in globulin and lower in prolamines. lesser content of albumins acta bot. croat. 72 (1), 2013 139 phytochemical studies of acacia leucophloea roxb. fig. 1. mineral contents of acacia leucophloea roxb. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees (sulphur containing) in pods is responsible for the lesser content of sulphur-containing amino acids. the presented results are comparable to those reported earlier for the seeds (vijayakumari et al. 1994). however, no previous studies of the pods exist. amino acid content (table 3) indicated that glutamic acid (17.29 ± 0.75% and 15.46 ± 0.89%) and aspartic acid (13.07 ± 0.69% and 10.21 ± 0.42%) are present in the highest concentrations, while methionine (0.63 ± 0.84% and 0.17 ± 0.23%), and cysteine (1.21 ± 0.72% and 1.03 ± 0.19%) are present in the lowest concentrations. our results are comparable to those reported earlier for seeds (vijayakumari et al. 1994). however, like other parameters, no previous studies pods exist of. fatty acid composition (fig. 2) indicated the presence of linoleic acid in the highest proportion (49.60 ± 1.01% and 35.92 ± 1.01%), while sufficient amounts of oleic acid (22.67 ± 0.89% and 24.12 ± 0.44%), and palmitic acid (17.80 ± 0.17% and 26.09 ± 0.75%) are also present in seeds and pods. the presented results are close to those reported earlier (vijaya140 acta bot. croat. 72 (1), 2013 zia-ul-haq m., ]avar s., qayum m., khan i., ahmad s. tab. 2. protein fractions of seed and pods of acacia leucophloea roxb. protein fractions seeds (%) pods (%) albumins 9.67 ± 0.39 5.18 ± 0.89 globulins 13.01 ± 1.27 6.12 ± 0.25 prolamines 1.21 ± 0.54 1.09 ± 0.46 glutelins 2.32 ± 1.08 2.03 ± 0.77 tab. 3. amino acid composition of acacia leucophloea roxb. amino acid seeds (%) pods (%) alanine 6.86 ± 0.33 8.77 ± 0.21 arginine 6.35 ± 0.19 5.16 ± 0.62 aspartic acid 13.07 ± 0.69 10.21 ± 0.42 cysteine 1.21 ± 0.72 1.03 ± 0.19 glutamic acid 17.29 ± 0.75 15.46 ± 0.89 glycine 8.02 ± 0.05 6.91 ± 0.34 histidine 4.36 ± 0.52 3.41 ± 0.62 isoleucine 3.89 ± 0.41 4.13 ± 0.49 leucine 8.01 ± 0.29 9.17 ± 0.32 lysine 6.08 ± 0.11 8.03 ± 0.92 methionine 0.63 ± 0.84 0.17 ± 0.23 phenylaniline 4.62 ± 0.71 4.62 ± 0.71 proline 5.24 ± 0.64 2.01 ± 0.44 serine 4.37 ± 0.73 6.39 ± 0.23 threonine 3.86 ± 0.48 5.96 ± 0.48 tryptophan 0.32 ± 0.08 0.91 ± 0.22 tyrosine 2.03 ± 0.55 2.03 ± 0.14 valine 4.73 ± 0.07 5.63 ± 0.11 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees kumari et al. 1994; zia-ul-haq et al. 2007, 2008). linoleic acid, have been reported to prevent cardiovascular disorders such as coronary heart disease, atherosclerosis, as well as high blood pressure. oils containing more unsaturated fatty acids are believed to reduce cholesterol-related heart diseases. g-tocopherol was found in the highest amount (169.81 ± 1.44 mg per 100 g) in the oils of this plant species while b-tocopherol was found in the lowest amount (1.04 ± 0.76 mg per 100 g) (fig. 3). since it is known from the literature that tocopherols significantly increase the stability of vegetable oils, we suppose that acacia oil will also have a long shelf life time because of its high tocopherol contents. no previous study reports tocopherol contents of acacia species. high amounts of tocopherols can be interesting for the production of naturally occurring tocopherols for the stabilization of fats and oils against oxidative deterioration and for applications in dietary, pharmaceutical, or biomedical products. sterol profile of seed oil indicated that b-sitosterol was the major sterol (73.07 ± 0.90 mg per 100 g), while d7acta bot. croat. 72 (1), 2013 141 phytochemical studies of acacia leucophloea roxb. fig. 2. fatty acid profile of acacia leucophloea roxb. fig. 3. tocopherol and sterol profile of oils from seeds of acacia leucophloea roxb. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:58 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees avenasterol was present in the lowest amount (1.04 ± 0.76 mg per 100 g). like other parameters, this is the first report on the sterol content of a. leucophloea. however, the values are close to those reported earlier for other acacia species (falade et al. 2008). the results from the antioxidant assays (tab. 4) showed that all examined extracts might to a certain extent act as radical scavengers. the distinct scavenging activities of different extracts can be due to the diverse chemical nature of various phytochemicals. our results vary to a large extent from those reported previously (shang-tzen et al. 2001). this may be due to the differences in assay performed and cultivar used. all parts of a. leucophloea may be good candidatse for further development as an antioxidant remedy. in-depth investigations are necessary, covering at least two or three years’ data to permit insight into yearly variations of seeds grown under defined conditions (agronomic practices, pesticide use, fertilization, irrigation and climatic data). conclusion in conclusion, the results obtained in this study clearly indicate that a. leucophloea has a sufficient potential for use as a source for a herbal recipe for the treatment, management and control of various ailments, as well as a natural antioxidant agent. further phytochemical studies will be performed for specification of the biologically active principles. references akhtar, s., 1992: chemical and nutritional evaluation of genus acacia of the family leguminosae of pakistan. phd thesis, university of punjab lahore, pakistan. aoac, 1990: official methods of analysis of the association of official analytical chemists. association of official analytical chemists, washington, dc. balz, m., shulte, e., their, h. p., 1992: trennung von tocopherolen und tocotrienolen durch hplc. lipid/ fett 94, 209–213. benzie, i. f. f., strain, j. j., 1999: ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. methods in enzymology 299, 15–27. david, s., seigler, j., ebinger, e., 1987: cyanogenic glycosides in ant-acacias of mexico and central america. the southwestern naturalist 32, 499–503. falade, o. s., adekunle, a. s., aderogba, m. a., atanda, s. o., harwood, c., adewusi, s. r., 2008: physicochemical properties, total phenol and tocopherol of some acacia seed oils. journal of science of food and agriculture 88, 263–268. 142 acta bot. croat. 72 (1), 2013 zia-ul-haq m., ]avar s., qayum m., khan i., ahmad s. tab. 4. in vitro antioxidant activity by various assays acacia leucophloea roxb. sample teac (mmol g–1) frap (mmol g–1) trap (mmol g–1) leaves 543.03 ± 1.17 233.17 ± 1.24 68.39 ± 1.12 pods 683.23 ± 1.42 254.42 ± 1.86 76.02 ± 1.37 seeds 529.66 ± 1.55 178.14 ± 1.14 49.14 ± 1.34 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:58 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees freidman, m., finely, j. w., 1971: methods of tryptophan analysis. journal of agriculture food chemistry 19, 626–631. ghiselli, a., serafini, m., maiani, g., azzini, e., ferro-luzzi, a., 1995: a fluorescence-based method for measuring total plasma antioxidant capability. free radical biology and medicine 18, 29–36. grace, s. c., 2005: phenolics as antioxidants. in smirnoff, n. 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(cicer arietinum l.) seed oil. tarim bilimeri dergisi 15, 25–30. zia-ul-haq, m., ahmad, s., chiavaro, e., mehjabeen m., ahmed, s., 2010: studies of oil from cowpea (vigna unguiculata (l.) walp.) cultivars commonly grown in pakistan. pakistan journal of botany 42, 214–220. zia-ul-haq, m., iqbal, s., ahmad, m., 2008a: characteristics of oil from seeds of 4 mungbean (vigna radiate l. wilczek) cultivars grown in pakistan. journal of american oil society 85, 851–856. zia-ul-haq, m., iqbal, s., ahmad, s., bhanger, m. i., wiczkowski, w., amarowicz, r., 2008b: antioxidant potential of desi chickpea varieties commonly consumed in pakistan. journal of food lipids 15, 326–342. zia-ul-haq, m., iqbal, s., ahmad, s., imran, m., niaz, a., bhanger, m. i., 2007: nutritional and compositional study of desi chickpea (cicer arietinum l.) cultivars grown in punjab, pakistan. food chemistry 105, 1357–1363. 144 acta bot. croat. 72 (1), 2013 zia-ul-haq m., ]avar s., qayum m., khan i., ahmad s. 529 zia-ul-haq et al.ps u:\acta botanica\acta-botan 1-13\529 zia-ul-haq et al.vp 14. o ujak 2013 11:36:58 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 70 (2), 215–244, 2011 coden: abcra 25 issn 0365-0588 ecological assessment of wetland ecosystems of northern kazakhstan on the basis of hydrochemistry and algal biodiversity sophia s. barinova1*, eibi nevo1, tatiana m. bragina2 1 institute of evolution, university of haifa, mount carmel, haifa 31905, israel 2 russian university of kazakhstan, kustanaiskaya oblast, naurzum rayon, karamendy, altynsarin str., 45-1, kazakhstan abstract – we studied diversity of algae and cyanobacteria in the wetlands of protected natural lakes with salinity ranging from 0.19 up to 32.7 in the arid/semiarid regions of northern kazakhstan. in plankton and periphyton of 34 lakes, we found 254 species belonging to 113 genera of 8 algal divisions. the diversity in arid regions is represented by widespread species of diatoms, green algae, and cyanobacteria in similar proportions. alkaliphiles, among the indicators of acidification, and betamesosaprobionts, among the indicators of saprobity, predominated. the indices of saprobity in lakes varied from 1.47 to 2.7, reflecting low-trophic and low anthropogenically disturbed wetlands. oligohalobes-indifferents are most common. highly diverse algal communities were found irrespective of various levels of mineralization. as a consequence of aridization, salinity increase suppressed algal diversity. the mineralization was the most important variable defining the diversity levels, irrespective of the type and location of wetland lakes in the arid regions. keywords: algae, cyanobacteria, aridization, diversity, salinity, wetland, water quality, kazakhstan introduction in arid regions, aquatic environments experience a stressful impact of high concentrations of mineral and organic substances due to high evaporation rates (subyani 2005). algal habitats are characterized by a high amplitude salinity variation that in large lakes suppresses algal diversity (hammer 1986). for example lower bacterial diversity (rusznyák et al. 2008) and algal species richness (ács et al. 2003) were found in the open water area of lake velencei (where the conductivity is about 2.5–3.5 ms cm–1 averagely) than in the bog-like area of the lake (where the conductivity is about 1.5–2.3 ms cm–1 on average). many algal species are indicators of environmental conditions reflecting the influence of acta bot. croat. 70 (2), 2011 215 * corresponding author, e-mail: barinova@research.haifa.ac.il copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:26 color profile: disabled composite 150 lpi at 45 degrees salinity on aquatic communities and the regional flora as a whole. a decrease of algal diversity is in turn related to reduced productivity of aquatic ecosystem and thereby of the trophic level of wetlands. it is well known that an increase of salinity to 20, suppress the diversity of lake biota (hammer 1986). the arid regions of central asia and middle east occupy a considerable part of eurasia (köppen and geiger 1953). in kazakhstan, the arid and semiarid dry grasslands to deserts are widespread in the upper reaches of the ob’ river basin and the turkestan desert (bragina and bragin, 2002). the hydrographic network of this territory is developed slightly, closed and has no constant drainage (skliarenko, 2006). during spring high water, channels are filled with water that reaches lakes and spreads wide over the steppe. the freshwater and the salt waterlakes have depths not exceeding 2.5–3 m and have the typical features of all reservoirs of arid territories, a cyclic hydrological mode where the periods of filling and drying repeat each 12–15 years. large highly mineralized lakes balkhash, tengiz, issyk kul’ and karakul’, as well as the aralian and caspian seas, are confined to this climatic area (hammer 1986). phytogeographically, this region is situated near the boundary of the irano-turanian province and the province north of it (takhtajan 1978). a large number of lakes in this area are protected on account of their importance for biodiversity conservation (bragina and bragin 2002). we previously studied algal biodiversity in respect to ecological assessment of the wetlands (barinova et al. 2002) and compared it with other arid regions (barinova et al. 2009). here we continue studying the diversity of algal communities and its relation to salinity. material and methods this research was based upon 98 samples of phytoplankton and periphyton collected in the north kazakhstan region in october 1999 and may–june 2000. altogether 34 lakes were sampled, as well as the mouth of karasu river near lake tuntugur, jailmo well near lake kulykol, and jarsor brook near lake jarsor located in the northern kazakhstan arid area (fig. 1). the water level in the studied shallow lakes depends on the climate and on snow melt, and is related to the complete or partial drying of some lakes in dry years. the studied shallow lakes have a permanent depth of 0.5–2.0 m. they become overgrown by underwater and surface vegetation, and exhibit high hydrogen sulphide content in their water, which varies in seasons and in lake gulfs (bragina and bragin 2002). water salinity in the lakes varies from fresh to salty, with sulfates and chlorides prevailing. the samples were taken by scooping up for phytoplankton and by scratching for periphyton and were fixed in 3% formaldehyde (whitton et al. 1991). algae were studied with swift and amplival dissecting microscopes under magnifications of 740–1850 and were photographed with the digital camera inspector 1. the diatoms were prepared with the peroxide technique (swift 1967) modified for glass slides (barinova 1988, 1997). in parallel with sampling for algae we measured electrical conductivity, total dissolved solids, and ph with hanna hi 9813, and dpc2, concentration of n-no3 with hanna hi 93728 and phosphates with hach spectrophotometer, as well as salinity by the argentometric method (apha 1998); ph measurements were made down to 0.15. 216 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:26 color profile: disabled composite 150 lpi at 45 degrees the assessment of b+g radioactive pollution of water samples was performed with detector-indicator of radioactivity quartex rd 8901. the algal abundances were assessed on the basis of a 6-score scale (korde 1956, barinova et al. 2006). the taxonomy follows the systems adopted in the »süswasserflora von mitteleuropa« (ettl 1978; starmach 1985; ettl and gartner 1988; krammer and lange-bertalot 1991a, b, c, d; komárek and anagnostidis 1998) and mattox and stewart (1984) system for green algae, with additions for individual taxa in gollerbach et al. (1953), kisselev (1954), popova (1966), vinogradova et al. (1980), palamar-mordvintseva (1982), krammer (1985, 2000), moshkova and gollerbach (1986), lange-bertalot and krammer (1987), meffert (1987), komárek and anagnostidis (1989), popovsky and pfiester (1990), barber and carter (1996), hegewald (2000), rumrich et al. (2000). the ecological and geographic characteristics of algae are obtained from the database compiled by the author for freshwater algae as a basis for statistical analysis of algal biodiversity distribution over ecological gradients (barinova 2000, barinova et al. 2000, 2006). our ecological analysis has revealed a grouping of freshwater algae in respect to ph, salinity and saprobity as well as the other habitat conditions. each group was separately assessed with respect to its significance for bioindications. species that respond predictably to environmental variables can be used as bioindicators reflecting the response of aquatic ecosystems to eutrophication, ph levels (acidifications), salinity and organic pollutants. acta bot. croat. 70 (2), 2011 217 algal biodiversity in kazakhstan wetlands fig. 1. lakes and wetland region in the northern kazakhstan. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:28 color profile: disabled composite 150 lpi at 45 degrees distribution of species sensitive to ph and suitable as bioindicators for acidity is analyzed in accordance with the classification of hustedt (1938–1939). this classification system is divided into ph-related groups, from alkalibiontes to acidobiontes. the bioindication of salinity is based on the classification system by hustedt (1957) with groups ranging from polyhalobes to oligohalobes-halophobes according to kolbe’s (1927) system of halobity. there are several alternative approaches to assessment of saprobity (adaptation to excessive levels of nutrients), with that of pantle and buck (1955) modified by sláde^ek (1973, 1986) being found most suitable for the present analysis. the indicators of saprobity are assigned to four groups according to their saprobity index values (s) ranging from polysaprobes (s=3.5–4.0) to xenosaprobes (s=0–0.5). the indices of saprobity are obtained as a function of saprobic species numbers and their relative abundances: s = ssh / sh (eq. 1) where s indicates index of saprobity for algal community; s – species-specific saprobity level; h – abundance on the 6-scores scale (after korde 1956). for phytogeographic analysis, species ranges were plotted and assigned to phytogeographic divisions of takhtajan (1978). statistical methods were used in comparative floristic approaches (novakovsky 2004) for clarifying of algal flora similarity in the natural protected wetlands in the semi-arid climate of the northern kazakhstan. the percent disagreement was calculated by ward’s method in statistica 6.0 program. ecological classification of water quality was based on a combination of hydrochemical variables and the indices of saprobity (romanenko et al. 1990, barinova et al. 2006). the status of water objects was assessed as sum of all data integrated in the functional model of an aquatic ecosystem (barinova et al. 2006). gamma and beta radiation were studied as background variables. results influence of ecological conditions on biodiversity of algae in the arid region wetlands of kazakhstan because of the remoteness of kazakhstan’s protected areas, algal diversity there has remained virtually unstudied. the lakes that we studied can be considered as typical for this region. their salinity (as the saltiness or dissolved salt content of a body of water) varies from 0.19 to 32.7 nacl g l–1 (tab. 1) increasing during the summer dry period and contains not only chlorides but also sulphates as regional norm. the acidity varies from slightly acid to alkaline, whereas the concentration of nitrates and phosphates attests to a sufficient trophic base for algal development (tab. 1). at the same time, the saprobity indices of pantle and buck (1955) modified by sláde^ek (1973) varied from 1.65 in the gr. karakamys lake and 1.47 in the karasu river mouth to 2.7 in the lakes tahtakul and aksuat, which indicates a lack of appreciable anthropogenic impact. 218 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:28 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .70 (2),2011 219 a l g a l b io d iv e r s it y in k a z a k h s t a n w e t l a n d s tab. 1. environmental conditions, species number and index of saprobity in the lakes of the kazakhstan arid regions in 1999–2000; classes of salinity and groups of salinity indicators according to hustedt (1957): mh, mesohalobes; hl, oligohalobes – halophilous; i, oligohalobes – indifferent; saprobity index s according to sláde^ek (1973), and equation (1). no of lake lake conductivity, msm/cm nacl g l–1 class of salinity ph p–po4 3–, mg l–1 n-no3 –, mg l–1 no. of algal species saprobity index s group of salinity indicators group of similarity on the tree 1 aike 11.73 6.79 iii 7.01 0.16 3.4 19 1.98 hl 1 2 aksuat 1.16–6.95 0.69–3.94 iv 6.16–6.56 0.01–0.02 1.0–1.4 1 2.70 – 2 3 alpash 8.72 4.92 iv 7.05 0.01 1.0 13 1.74 i 2 4 annovskoe 0.7–0.79 0.43–0.45 iv 6.87–7.11 0.01 1.2–2.0 22 1.71–1.81 hl 2 5 balykty 4.33 2.43 iv 7.84 0.02 1.1 26 2.21 i 1 6 batpakkol 0.37 0.21 iv 7.29 0.26 2.3 2 2.0 hl 2 7 biesoygan 0.851 0.52 iv 6.76 0.39 2.1 7 2.55 i 2 8 bozshakol 0.62–0.91 0.38–0.55 iv 6.43–7.37 0.20–2.25 1.7–8.5 63 1.95–2.13 hl 3 9 chushkaly 16.4 9.35 iii 6.94 0.28 1.7 18 1.97 hl+mh 1 10 gr. kak 57.3 32.7 ii 6.36 0.05 2.2 6 2.11 hl 2 11 gr. karakamys 0.81–2.88 0.46–1.7 iv 6.59–6.66 0.01–0.68 0.8–1.2 28 1.65–1.8 i+hl 2 12 gr. sankebay 14.29 8.17 iii 7.40 0.09 1.6 3 2.49 hl 2 13 jaltyr 11.47 6.52 iii 6.77 1.0 1.0 6 1.86 i 2 14 jaman 0.37–0.41 0.22–0.23 iv 6.54–7.53 0.03–0.08 1.2–1.6 57 1.78–2.10 i+hl 3 15 jarken 0.86 0.49 iv 6.74 0.03 0.9 22 2.00 i+hl 2 16 jarkol 5.45–5.67 3.1–3.42 iv 7.26–7.38 0.17–1.08 2.3–4.1 8 1.7–1.96 i 2 17 jilandy 1.48 0.85 iv 7.15 0.68 1.2 4 2.05 i+hl 2 18 kamyshovoe 0.37–0.43 0.21–0.26 iv 6.29–7.14 0.02–0.11 1.2–1.9 79 1.86–1.9 i+hl 3 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 1 \ b a r i n o v a . v p 9 . r u j a n 2 0 1 1 1 1 : 2 1 : 2 8 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s 220 a c t a b o t .c r o a t .70 (2),2011 b a r in o v a s .s .,n e v o e .,b r a g in a t .m . tab. 1. – continued no of lake lake conductivity, msm/cm nacl g l–1 class of salinity ph p–po4 3–, mg l–1 n-no3 –, mg l–1 no. of algal species saprobity index s group of salinity indicators group of similarity on the tree 19 karasu river, near lake tuntugur 0.398 0.24 iv 6.86 0.23 1.4 2 1.47 hl 2 20 koybagor 1.1–1.16 0.66–0.69 iv 6.83–7.35 0.07–0.27 1.3–2.0 112 1.95–2.04 i+hl 3 21 kulagul 0.87–1.16 0.50–0.69 iv 7.48–7.57 0.17–0.98 0.0–0.6 18 2.38–2.44 i 1 22 kulykol 10.4–10.9 5.94–6.43 iii 6.73–7.18 0.03–0.3 1.1–2.1 20 1.85–2.3 i 1 23 kulykol, well jailma 1.01 0.58 iv 7.28 0.06 1.0 3 2.35 i+hl 2 24 kushmurun 30.1 18.0 iii 6.77 0.1 3.5 8 2.08 i+hl 2 25 majbalyk 1.95–3.23 1.11–1.78 iv 6.92–7.4 0.0–0.02 1.4–2.2 4 2.04 i 2 26 sankebay 14.68 8.8 iii 8.15 0.54 5.4 5 1.93 i 2 27 sarykol 1.14–1.17 0.67–0.69 iv 6.97–6.98 0.0–0.06 0.9–1.5 37 1.71–2.15 i 1 28 shoshkaly 6.14–6.56 3.49–3.92 iv 6.33–7.15 0.03–0.32 1.1–1.3 31 1.87–2.53 i+hl 1 29 stream jarsor 0.63–0.79 0.38–0.44 iv 6.78–6.86 0.01 1.0 19 1.76–1.99 i+mh 2 30 sultan 3.04 1.73 iv 6.73 0.0 0.9 5 2.15 i 2 31 suly 0.328 0.19 iv 7.13 1.77 1.4 7 1.84 i 2 32 tahtakul 0.99–2.3 0.56–1.38 iv 6.35–6.79 0.03–0.48 1.1–1.8 7 1.8–2.7 i 2 33 teniz 2.52–6.99 1.43–3.98 iv 6.66–6.69 0.0–0.04 1.0–1.5 61 1.87–1.96 i+hl 3 34 tyuntugur 1.03–1.16 0.63–0.7 iv 6.66–7.53 0.01–0.26 1.5–2.0 58 1.88–2.12 i+hl 3 u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 1 1 \ b a r i n o v a . v p 9 . r u j a n 2 0 1 1 1 1 : 2 1 : 2 9 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s our taxonomic analysis revealed 254 species from eight taxonomical divisions, among which diatoms slightly prevail over green and blue-green species (tab. 2, fig. 2a). for 13 species we indicate only generic assignments because certain critical features, such as sexual forms in spirogyra, are lacking in our material. acta bot. croat. 70 (2), 2011 221 algal biodiversity in kazakhstan wetlands tab. 2. ecology and geographical distribution of algae and cyanobacteria in kazakhstan lakes. ecological types: habitat: b – benthic; p – planktonic; p-b – planktonic-benthic; t – temperature; temp – temperate; eterm – eurytermic; warm – warm-water; cool – cool-water; reo – reophility and oxygenation; st – standing water; str – stream; d – saprobity categories of watanabe (1986); es – eurysaprob; sx – saproxen; sp – saprophil; s – saprobity categories of sláde^ek (1986); o – oligosaprob; o-a – oligo-alfa-mesosaprob; o-b – oligo-beta-mesosaprob; b – beta-mesosaprob; b-a – beta-alfa-mesosaprob; a – alfa-mesosaprob; a-b – alfa-beta-mesosaprob; x – xenosaprob; a-r – alfa-meso-polysaprob; r – polysaprob; hal – halobity; mh – mesohalobe; i – oligohalobious-indifferent; hl – oligohalobious-halophilous; hb – oligohalobious-halophobous; ph – polyhalobe; ph – acidophility; ind – indifferent; alf – alkaliphil; acf – acidophil; alb – alkalibiont; geo – chorological types; mt – mediterranean; k – cosmopolite; b – boreal; pt – paleotropical; nt – neotropical; ha – holarctic. »–« no data. numbers of lakes as in table 1. no taxon no of lake habitat t reo d s hal ph geo cyanoprokaryota 1 anabaena constricta (szaf.) geitl. 21 p–b – – – p – – ha 2 anabaena contorta bachm. 1 p – st-str – – – – k 3 anabaena flos-aquae (lyngb.) bréb. f. flos-aquae 18 p – st – b i – k 4 anabaena flos-aquae f. jacutica (nyg.) elenk. 8 p – – – – i – b 5 anabaena minima tschernov 33 – – – – – – – b 6 anabaena sp. 18, 28, 33 – – – – – – – – 7 anabaena spiroides kleb. 34 p – st-str – o i – k 8 anabaena variabilis kütz. 9 p-b – st – – mh – k 9 aphanizomenon flos-aquae (l.) ralfs 6, 8, 20 p – – – b hl – k 10 aphanothece clathrata w. et g. s. west 1, 4, 8, 14, 20, 24, 27 p – – – b hl – k 11 aphanothece stagnina (spreng.) a. br. 8, 20, 21 p-b – – – o hl ind k 12 chroococcus limneticus lemm. 20 p – – – o – – k 13 chroococcus minor (kütz.) näg. 33 b – – – o-b – – k 14 chroococcus minutus (kütz.) näg. 20 p – – – – – – k 15 chroococcus turgidus (kütz.) näg. 1, 3, 5, 8, 21, 33, 35 p-b – – – o hl alf k 16 chroococcus vacuolatus skuja 33 p-b – – – – – – b, mt u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees 222 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. no taxon no of lake habitat t reo d s hal ph geo 17 chroococcus varius a. br. 4, 8 b – – – o-b – – k 18 coelomoron pusillum (van goor) komárek 1, 4, 8, 14, 21, 33 p temp st – a – – k 19 coelosphaerium kuetzingianum näg. 15 p – – – – i – k 20 coelosphaerium minutissimum lemm. 21 p – – – – hl – k 21 gloeotrichia pisum (ag.) thur. 4, 18 b – – – – hl ind k 22 gomphosphaeria aponina kütz. 28, 33 p – – – – hl alf k 23 lyngbya aestuarii (mert.) leibm. 28 p-b – – – – – – k 24 lyngbya contorta lemm. 1 – – – – – hl – ha 25 lyngbya sp. 15, 18 – – – – – – – – 26 merismopedia minima beck 18, 20, 28, 33, 34 b – – – – – – ha 27 merismopedia smithii de toni 14, 18, 20 p cool – – – – – ha 28 merismopedia tenuissima lemm. 1, 8, 9, 14, 20, 33 p-b – – – b hl – k 29 microcystis aeruginosa (kütz.) kütz. 7, 8 p – – – b hl – k 30 microcystis pulverea f. delicatissima (w. et g. s. west) elenk. 1 p – – – – i – k 31 nostoc kihlmanii lemm. 30 p cool st – – i ind ha 32 oscillatoria brevis kütz. ex gom. 10 p-b – st – a – – k 33 oscillatoria limosa (roth) ag. 11 p-b – st-str – a hl – k 34 oscillatoria princeps vauch. ex gom. 10, 22 p-b – st-str – a – – k 35 phormidium ambiguum gom. 11 b – st-str – – i ind k 36 phormidium autumnale (ag.) gom. 20, 30, 33 b – st-str – b – – k 37 phormidium paulsenianum b.-peters. 28 b – – – – ph – ha 38 phormidium retzii ag. ex gom. 10 b – st-str – o – – k 39 rhabdogloea scenedesmoides (nyg.) komárek et anagn. 8 p cool st – – – – ha 40 spirulina major kütz. ex gom. 10 p – st – – – – k 41 synechocystis sallensis skuja 20 p-b cool st – o – acf ha, nt 42 tolypothrix sp. 21 – – – – – – – – 43 woronichinia compacta (lemm.) komárek et hindák 1, 18, 21, 27 p cool st – – – – ha, pt tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 223 algal biodiversity in kazakhstan wetlands no taxon no of lake habitat t reo d s hal ph geo bacillariophyta 44 achnanthes brevipes ag. 11, 13, 28 b – – – – hl alf k 45 achnanthes gibberula var. interrupta poretzky et anisimova 20 b – – – – hl – k 46 achnanthes lanceolata (bréb. in kütz.) grun. in cl. et grun. 5, 8, 22, 29, 34 p-b warm st-str sx o i alf k 47 achnanthes minutissima kütz. 3, 11, 18, 20, 27–29, 33 b eterm st-str es o i alf k 48 amphipleura pellucida (kütz.) kütz. 5, 33 b – st – o-a i alf k 49 amphora coffeaeformis (ag.) kütz. 3, 22, 33 b – st-str – – mh – k 50 amphora commutata grun. in v. h. 3 b – – – – hl – k 51 amphora holsatica hust. in pasch. 11 p – st-str – – hl – k 52 amphora ovalis (kütz.) kütz. 3, 8, 11, 14, 18, 20, 33, 34 b temp st-str sx a-b i alf k 53 amphora pediculus (kütz.) grun. ex a. schmidt 5, 8, 16, 20, 21, 33, 34 b temp – es a-b i alf k 154 amphora veneta kütz. 4, 8, 10, 14, 28 b – – es a-p i alf k 55 asterionella formosa hass. 4, 8, 18 p – – sx i alf k 56 aulacoseira granulata (ehrb.) sim. 8, 33, 34 p-b cool st-str es b i alf k 57 aulacoseira italica (ehrb.) sim. 34 p-b cool st-str es b i alf k 58 caloneis amphisbaena (bory) cl. 5, 8, 12, 20, 28–30, 34 b – – – – hl alf k 59 caloneis silicula (ehrb.) cl. 5, 8, 11, 18, 20, 32 b – st sp b i alf k 60 caloneis westii (w. sm.) hendey 5, 33 b – – – – mh – k 61 campylodiscus noricus ehrb. 26, 33 b – – – o i alf k 62 chaetoceros sp. 9, 33 p – – – – – – – 63 cocconeis placentula ehrb. 9, 11, 15, 18–20, 33 p-b temp st-str es o i alf k 64 craticula cuspidata (kütz.) d. g. mann 5, 20, 33 b temp st – – i alf k 65 cyclostephanos dubius (fricke in a. schmidt) round 20 – – – – b-a hl alf k 66 cymatopleura librile (ehrb.) pant. 5, 7, 89, 14, 18, 20, 25, 27, 28, 34 p-b – – – – – – k tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees 224 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. no taxon no of lake habitat t reo d s hal ph geo 67 cymbella cornuta (ehrb.) r. ross 8, 14, 18, 27, 31, 34 b – – – – i alf k 68 cymbella neocistula krammer 14 b – st-str sx b i alf k 69 cymbella tumida (bréb.) v. h. 8, 18, 20 b temp – sx o i alf k 70 cymbella turgidula grun. 5, 14, 18, 20, 33 b – st-str es – – ind k 71 diatoma vulgare bory var. vulgare 18, 20, 22, 27, 33, 34 p-b – st-str – x i ind k 72 diatoma vulgare var. ehrenbergii (kütz.) grun. 34 b – – – – i alf k 73 diploneis elliptica (kütz.) cl. 23 b eterm – sx o i alf k 74 diploneis ovalis (hilse) cl. 24 b – – sp i alb b 75 encyonema silesiacum (bleisch. in rabenh.) d. g. mann in round et al. 8, 18, 27, 34 b – st-str sx o-b i ind k 76 epithemia adnata (kütz.) bréb. 5, 8, 14, 18, 20, 31, 34 b temp – sx b i alf k 77 epithemia sorex kütz. 14, 18, 20, 33 b temp st sx b i alf k 78 epithemia turgida (ehrb.) kütz. 4, 8, 11, 14, 15, 18, 20, 29, 31, 34 b temp – – – i alf k 79 eunotia exigua (bréb. ex kütz.) rabenh. 11 b – – es o hb acf k 80 eunotia minor (kütz.) grun. in v. h. 18 – – – es b-a – ind k 81 eunotia monodon ehrb. 15 b – – o hb acf k 82 eunotia pectinalis (o. müll.) rabenh. 18, 27 b – – sx – hb acf k 83 eunotia praerupta ehrb. var. praerupta 18 b cool st-str sx – hb acf k 84 eunotia praerupta var. bidens (ehrb.) grun. in cl. et grun. 18 b cool – – – hb acf k 85 eunotia sibirica cl. in cl. et grun. 11, 31 b – – – – i – b 86 fallacia pygmaea (kütz.) stikle et mann 5, 24 b – – es a mh alf k 87 fragilaria crotonensis kitton 3, 8 p – st es – hl alf k 88 fragilaria sp. 15, 24 – – – – – – – – 89 fragilaria ulna (nitzsch) l.-b. 4, 5, 8, 11, 13, 14, 18–21, 26, 27, 29, 33, 34 p-b temp st-str es b i ind k 90 fragilaria vaucheriae (kütz.) b. peters. 8, 14, 16, 17, 18, 20, 21, 27, 28, 33, 34 p – – – – i alf ha tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 225 algal biodiversity in kazakhstan wetlands no taxon no of lake habitat t reo d s hal ph geo 91 fragilariforma constricta (ehrb.) williams et round 14 b – – – – i acf ha 92 fragilariforma virescens (ralfs) williams et round 14, 18 p-b – st es x i ind k 93 fragilariopsis separanda (hust.) hasle 24 – – – – – hl – ha 94 frustulia sp. 24 – – – – – – – – 95 geissleria schoenfeldii (hust.) l.-b. et metzetlin in l.-b. 20 b – – – – i alf b 96 gomphonema acuminatum ehrb. var. acuminatum 11, 20 p-b – st es b i alf k 97 gomphonema acuminatum var. coronatum (ehrb.) w. sm. 14, 18, 20 p-b – st – b i ind k 98 gomphonema augur ehrb. 14, 18 b – – es – i ind k 99 gomphonema clavatum ehrb. 8, 18, 33 b – – es – i – k 100 gomphonema parvulum (kütz.) kütz. 8, 11, 33 b temp str es b i ind k 101 gomphonema truncatum ehrb. 8, 14, 18, 20 p-b – – es b-a – – k 102 gyrosigma acuminatum (kütz.) rabenh. 5, 8, 18, 22, 26, 34 b cool – – b i alf k 103 gyrosigma spenceri (w. sm.) cl. var. spenceri 9, 16, 22, 33 b – – es o mh alf k 104 gyrosigma spenceri var. nodiferum grun. 3, 9, 13, 14, 28 b – – – – i ind b 105 hippodonta hungarica (grun.) l.-b., metzeltin et witkowski 14, 16, 18, 20, 21, 34 b – st-str es b i alf k 106 luticola mutica (kütz.) mann 33, 34 b – – sp o-b – – k 107 mastogloia sp. 11, 20, 33 – – – – – – – 108 melosira varians ag. 34 p-b temp st-str es b hl alf k 109 navicula exigua grun. 5, 7–9, 14, 16, 20, 22, 25–28, 33, 34 b – – es i alf k 110 navicula gregaria donk. 8 b – – es b mh alf k 111 navicula peregrina (ehrb.) kütz. 34 b – – es o mh alf k 112 navicula rhynchocephala kütz. 8, 14, 18, 28 b – – – a hl alf k 113 navicula sp. 3, 20 – – – – – – – – 114 navicula viridula (kütz.) ehrb. 5, 8, 14, 18, 20, 28, 33, 34 b – – es a hl alf k 115 neidium dubium (ehrb.) cl. 10, 20 b – – b i alf k 116 neidium iridis (ehrb.) cl. 8, 18, 20 b – st es o hb ind k 117 nitzschia acicularis (kütz.) w. sm. 2, 5, 9, 14, 15, 18, 20–22, 24, 27, 28, 33, 34 p-b temp – es a i alf k tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees 226 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. no taxon no of lake habitat t reo d s hal ph geo 118 nitzschia clausii hantzsch 11, 29 b – es o-a mh acf k 119 nitzschia dissipata (kütz.) grun. 11, 18, 20, 22, 25, 34 b – st-str sx b i alf k 120 nitzschia filiformis (w. sm.) v. h. 8, 9, 14, 28, 34 b – – es a-b hl – k 121 nitzschia linearis (c. ag.) w. sm. 3, 8, 20, 28, 31, 34 b temp – es b i alf k 122 nitzschia macilenta greg. 14, 18, 20, 34 – – – – – hl – – 123 nitzschia palea (kütz.) w. sm. 5, 7, 8, 12–15, 18, 20, 21, 27, 28, 33, 34 p-b temp – sp b-a i ind k 124 nitzschia reversa w. sm. 5 – – – – – hl – k 125 nitzschia sigmoidea (nitzsch) w. sm. 20 p-b – – – b i alf k 126 nitzschia sp. 22, 24 – – – – – – – – 127 nitzschia vermicularis (kütz.) hantzsch in rabenh. 3, 5, 11, 12 b – – – b i alf k 128 pinnularia gibba ehrb. var. gibba 18, 20, 33 b – – es – i ind b 129 pinnularia gibba var. subundulata a. mayer 20, 34 b – – – – i – b 130 pinnularia microstauron (ehrb.) cl. var. microstauron 20, 29 b temp – sp o i ind k 131 pinnularia microstauron var. brebissonii (kütz.) mayer 8 b – st-str es b i ind k 132 pinnularia viridis (nitzsch) ehrb. 3, 14, 20, 22, 29, 31, 32, 34 p-b temp – es b i ind k 133 pinnularia sp. 18 b – – – – – – – 134 rhoicosphenia abbreviata (c. ag.) l.-b. 4, 8, 9, 14, 15, 17, 27, 28, 33, 34 p-b – – es b i alf k 135 rhopalodia gibba (ehrb.) müll. 14, 18, 20, 29 b temp – es o i alb k 136 rhopalodia musculus (kütz.) o. müll. 29 p-b – – x mh alb k 137 sellaphora pupula (kütz.) mereschkowsky 18, 34 b eterm st sp a hl ind k 138 stauroneis anceps ehrb. var. anceps 6, 8, 9, 11, 16, 18, 20, 22, 28, 33 p-b – – sx b i ind k 139 stauroneis anceps var. gracilis rabenh. 8 b – – sx – i ind k 140 stauroneis phoenicenteron (nitzsch) ehrb. f. phoenicenteron 18, 20 b temp – s b i ind k tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 227 algal biodiversity in kazakhstan wetlands no taxon no of lake habitat t reo d s hal ph geo 141 stauroneis phoenicenteron f. gracilis (ehrb.) hust. 20 – – – – – – ind – 142 stauroneis sp. 20 b – – – – – – – 143 stephanodiscus hantzschii grun. 1, 7–9, 18, 20, 33, 34, p-b temp st es a i alf k 144 stephanodiscus sp. 5 – – – – – – – 145 surirella brebissonii kram. et l.-b. 20, 27, 33 b – st-str b-a i alf k 146 surirella linearis w. sm. 14 p-b – – es b i ind ha 147 surirella ovalis bréb. 16, 26, 29, 33 p-b – – es o mh alf k 148 surirella ovata var. pinnata (w. sm.) rabenh. 20, 27, 33, 34 b – – es b i alf k 149 surirella tenera greg. 34 p-b – st es b i alf k 150 tryblionella gracilis w. sm. 5, 17, 20, 23, 29 b – – – – – – k 151 tryblionella hungarica (grun.) d. g. mann – p-b – – sp a mh alf k chlorophyta 152 actinastrum hantzschii lagerh. 8, 14, 21, 34 p-b – st-str – b i – k 153 ankistrodesmus falcatus (corda) ralfs 20 p-b – st-str b hb – k 154 ankistrodesmus fusiformis corda sensu korsch. 14, 20 p-b – st-str – – i – k 155 binuclearia lauterbornii (schmidle) pr.-lavr. 1, 23 p – – – – – – ha 156 chaetophora pisiformis (roth) ag. 18, 32 b – st-str – – – – k 157 chlamydomonas sp. 20 – – – – – – 158 cladophora fracta (müll. ex vahl.) kütz. 4, 5, 9, 13, 14, 22, 27, 28, 33, 34 p-b – st-str – b – – k 159 cladophora glomerata (l.) kütz. 11 p-b – st-str – b i alf k 160 closterium acerosum (schrank) ehrb. 28 p-b – st-str – a i ind k 161 closterium dianae ehrb. 5, 18, 20, 33, 34 p-b – st-str – o – – k 162 closterium ehrenbergii menegh. 15 p-b – st-str – b hb ind k 163 closterium gracile bréb. in chevalier 8, 22 p-b – st-str – – hb acf k 164 closterium sp. 8 – – – – – – – 165 coelastrum microporum näg. in a. br. 8, 33 p-b – st-str – b i ind k tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees 228 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. no taxon no of lake habitat t reo d s hal ph geo 166 coelastrum sphaericum näg. 8, 14, 20 p-b – st-str – – i – k 167 coenochloris pyrenoidosa korsch. 11, 14, 18 p – – – – hl – ha 168 coenococcus polycoccus (korsch.) hind. 4, 11, 15, 18, 20, 33, 34 p – st – – – – k 169 coenocystis planktonica korsch. 12 p – st – b i – k 170 coenocystis subcylindrica korsch. 15, 27 p – – – – i – b 171 cosmarium punctulatum bréb. 4, 18, 27 p-b – – – – hb acf k 172 cosmarium sp. 4, 14, 18, 27, 34 – – – – – – – 173 crucigenia tetrapedia (kirchn.) w. et g. s. west 4, 8, 20, 27, 33, 34 p-b – st-str – b i ind k 174 crucigeniella apiculata (lemm.) komárek 34 p-b – st-str – – – – k 175 desmodesmus brasiliensis (bohlin) hegew. 8, 20, 33 p-b – st-str – b – – k 176 desmodesmus denticulatus (lagerh.) an, friedl et hegew. 20 p-b – st-str – b i – k 177 desmodesmus opoliensis (p. richt.) hegew. 20 p-b – st-str – – – – k 178 desmodesmus spinosus (k. biswas) hegew. 18 p-b – st-str – o-b – – ha, nt 179 dictyosphaerium pulchellum wood 4, 8, 14, 20, 21 p-b – st-str – – i ind k 180 elakatothrix acuta pasch. 20 p – – – – i – k 181 elakatothrix gelatinosa wille 20 p – st-str – o i – k 182 eremosphera gigas (w. archer) fott et kalina 8, 15, 34 p – – – – i acf k 183 franceia tenuispina korsch. 18 p – – – – – ha 184 lagerheimia ciliata (lagerh.) chod. 20 p-b – st-str – – – – k 185 lagerheimia genevensis chod. 1, 20 p – – – i – k 186 micrasterias sp. 18 – – – – – – 187 monoraphidium arcuatum (korsch.) hind. 1, 20, 28, 34 p-b – st-str – – – – k 188 monoraphidium contortum (thur.) kom.-legn. 1, 8, 9, 14, 15, 18, 20, 21, 27, 28, 33 p-b – st-str – – – – k 189 monoraphidium griffithii (berk.) kom.-legn. in fott 7, 8, 9, 14, 18, 20, 21, 28, 33, 34 p-b – st-str – – – – k tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:29 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 70 (2), 2011 229 algal biodiversity in kazakhstan wetlands no taxon no of lake habitat t reo d s hal ph geo 190 monoraphidium komarkovae nyg. 22 p – st-str – – – – k 191 monoraphidium minutum (näg.) kom.-legn. 1 p-b – st-str – – – – k 192 mougeotia sp. 11, 18, 20, 28, 33 – – – – – – 193 nephrochlamys rotunda korsch. 20 p-b – st-str – – – – ha 194 oedogonium sp. 3, 11, 14, 15, 20, 29–32 – – – – – – – 195 oocystis lacustris chod. 4 p-b – st-str – b hl – k 196 oocystis submarina lagerh. 1, 8, 27, 33 p-b – st – – i – k 197 palmodictyon lobatum korsch. 15 b – st-str – – – – k 198 pediastrum boryanum (turp.) menegh. 1, 14, 18, 20, 27, 33, 34 p-b – st-str – b i ind k 199 pediastrum duplex meyen 4, 14, 27 p-b – st-str – b i ind k 200 pediastrum kawraiskyi schmidle 4, 8 p-b – st-str – – – – ha 201 pediastrum tetras (ehrb.) ralfs 14, 20, 34 p-b – st-str – b i ind k 202 penium sp. 18 – – – – – – – 203 raphidocelis sigmoidea hind. 20 p – st-str – – – – b, mt 204 raphidocelis subcapitata (korsch.) nyg. 8, 33 p-b – st-str – – – – ha, nt 205 scenedesmus acuminatus (lagerh.) chod. 18, 20, 27 p-b – st-str – b i ind k 206 scenedesmus acutiformis var. costatus (hub.-pest.) pankow 18 p-b – – – – – – k 207 scenedesmus acutus meyen 14, 18, 20, 28 p-b – st-str – o-b i – k 208 scenedesmus apiculatus (w. et g. s. west) chod. var. apiculatus 18, 28 p – st-str – – – – ha, pt 209 scenedesmus apiculatus var. indicus (hortob.) tzarenko 20 p-b – st-str – – – – ha, nt 210 scenedesmus arcuatus (lemm.) lemm. 14, 20 p-b – st-str – b i – k 211 scenedesmus bijugatus (turp.) kütz. 27 p – – – i ind k 212 scenedesmus disciformis (chod.) fott. et kom. 20 p-b – st-str – – – – k 213 scenedesmus ellipticus corda 18, 27, 34 p-b – st-str – o-b – – k 214 scenedesmus gutwinskii chod. 20 p – – – – – ha 215 scenedesmus incrassatulus bohl. 20 p-b – st-str – – – – k tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:30 color profile: disabled composite 150 lpi at 45 degrees 230 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. no taxon no of lake habitat t reo d s hal ph geo 216 scenedesmus obliquus (turp.) kütz. 1, 8, 11, 20, 33 p-b – st – – i – k 217 scenedesmus obtusus meyen 30 p-b – st-str – – – – ha 218 scenedesmus quadricauda (turp.) bréb. 5, 14, 18, 20, 27, 33, 34 p – – – – i ind k 219 schroederia setigera (schrod.) lemm. 8 p – st-str – – i – ha, nt 220 selenastrum gracile reinsch 14, 20 p-b – st-str – b – – k 221 spirogyra sp. 4, 15, 18, 27–30, 33 – – – – – – – – 222 spirogyra weberi kütz. 29 – – – – – – – k 223 staurastrum gracile ralfs 18, 27 p – st – – i – k 224 staurastrum sebaldii reinsch. 4 p-b – – – – – acf k 225 staurastrum sp. 18 – – – – – – – – 226 staurodesmus sp. 14 – – – – – – – – 227 stigeoclonium tenue (ag.) kütz. emend. cox et bold 20 b – st-str – a – – k 228 tetrachlorella alternans (g. m. smith) korsch. 14 p-b – – – – – ha 229 tetraedron caudatum (corda) hansg. 34 p-b – st-str – b i ind k 230 tetraedron incus (teil.) g. m. smith 18, 20 p-b – st-str – – i – k 231 tetraedron minimum (a. br.) hansg. 1, 18, 20, 27, 34 p-b – st-str – b i – k 232 tetrastrum elegans playf. 8, 20, 33 p – st-str – – i – k 233 tetrastrum triacanthum korsch. 20 p – st-str – – – – ha 234 ulothrix tenerrima kütz. 29 b – – – – i – k 235 ulothrix zonata (weber et mohr) kütz. 13, 22, 27, 34 p-b – st-str – o i ind k 236 ulothrix sp. 27 b – – – – – – – 237 volvox aureus ehrb. 20 p – st – b i – k 238 zygnema sp. 18 b – – – – – – – chrysophyta 239 dinobryon sertularia ehrb. 11 p – – – – i – k cryptophyta 240 cryptomonas sp. 1, 9, 16, 18, 20–22, 27, 32 p – – – – – – – dinophyta 241 glenodinium quadridens (stenis) schiller. 20 p – – – – – – k tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:30 color profile: disabled composite 150 lpi at 45 degrees the lacustrine algoflora mainly consists of geographically widespread species (85% cosmopolitan, 9% of pan-holarctic distribution, and 4.5% of boreal distribution (tab. 2). in the lakes of arid regions, algae occur over the water column and on hard substrates, with some preference for the latter (fig. 2b). the most abundant periphyton species are cyanoprokaryotic anabaena flos-aquae f. flos-aquae (kamyshovoe lake), coelosphaerium minutissimum (kulagul lake), phormidium retzii (lake great kak), diatoms amphora pediculus (great kak lake), and green algae binuclearia lauterbornii (aike lake) and spirogyra weberi (jarsor stream) with respect to ph, the indicator species (hustedt 1938–1939) are segregated into four groups among which the alkaliphiles prevail (fig. 2c). such distribution is characteristic of slightly alkalic conditions (tab. 1). the most common alkaliphiles are chroococcus turgidus, (cyanoprokaryota) and achnanthes minutissima, amphora ovalis, a. pediculus, caloneis amphisbaena, epithemia turgida, fragilaria vaucheriae, navicula exigua, nitzschia acicularis, n. palea, rhoicosphenia abbreviata (bacillariophyta). the diversity of ph indicators reflects the great amplitude of this variable. salinity indicators (hustedt 1957) are assigned to five ecological groups (fig. 2d), with oligohalobes-indifferents as a dominant group, although the oligohalobes-halophiles and mesohalobes are also common, as well as a single species of polyhalobes (tab. 2). among the oligohalobes-indifferents the most common are amphora ovalis, epithemia turgida, fragilaria ulna, f. vaucheriae, nitzschia acicularis, n. palea, rhoicosphenia abbreviata (bacillariophyta), crucigenia tetrapedia, pediastrum boryanum (chlorophyta), trachelomonas hispida, t. volvocina (euglenophyta). remarkably, the blue-greens are acta bot. croat. 70 (2), 2011 231 algal biodiversity in kazakhstan wetlands no taxon no of lake habitat t reo d s hal ph geo euglenophyta 242 astasia inflata klebs 19 p – st – – – – ha, nt 243 euglena acus ehrb. var. acus 20, 25, 34 p eterm st – b i ind k 244 euglena oxyuris schmarda 14, 17 p st-str – a mh ind k 245 euglena viridis ehrb. 7 p-b eterm st-str – p mh ind k 246 euglena sp. 3, 4, 15 – – – – – – – 247 phacus caudatus hübn. 14, 20, 33 p-b eterm st-str – b i alf k 248 phacus longicauda (ehrb.) duj. 20 p-b – st – a i ind k 249 phacus orbicularis hübn. 20 p-b – st-str – b i ind k 250 trachelomonas hispida (perty) stein emend. delf. 4, 8, 18, 20, 27–29 p-b eterm st-str – b i – k 251 trachelomonas volvocina ehrb. 8, 14, 18, 20, 27, 29, 34 p-b eterm st-str – b i ind k xanthophyta 252 ophiocytium majus missing 15, 33 p – st – – – acf k 253 tribonema viride pascher 11, 15, 29, 33 p-b – – – – i – k 254 tribonema sp. 22 b – – – – – – – tab. 2. – continued u:\acta botanica\acta-botan 2-11\barinova.vp 26. rujan 2011 13:58:16 color profile: disabled composite 150 lpi at 45 degrees 232 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. 0 50 100 150 cyanoprokaryota bacillariophyta chlorophyta euglenophyta xanthophyta cryptophyta dinophyta chrysophyta a y = -24x 2 + 111x 35 r 2 = 1 0 10 20 30 40 50 60 70 80 90 100 p p-b b ecological group n , s p e c ie s connection to substrate b y = -21x 2 + 103x 71 r 2 = 0,9571 0 10 20 30 40 50 60 acf ind alf alb ecological group n , s p e c ie s ph c y = -11,214x 2 + 55,786x 11,6 r 2 = 0,39 0 20 40 60 80 100 120 hb i hl mh ph ecological group n , s p e c ie s salinity d y = -0,7992x 2 + 7,7432x + 0,0833 r 2 = 0,1527 0 10 20 30 40 50 60 70 x o o-â o-á â â-á á-â á á-p p ecological group n , s p e c ie s sladecek's saprobity e y = -29x 2 + 113x 70 r 2 = 1 0 5 10 15 20 25 30 35 40 sx es sp ecological group n , s p e c ie s watanabe's saprobity f y = -4,25x 2 + 17,35x 1,75 r 2 = 0,8027 0 5 10 15 20 25 cool temp eterm warm ecological group n , s p e c ie s temperature g y = -69x 2 + 261x 161 r 2 = 1 0 10 20 30 40 50 60 70 80 90 100 st st-str str ecological group n , s p e c ie s streaming and oxygenation h fig. 2. algal species diversity and ecology in the wetland lakes of kazakhstan: a – distribution of species richness per taxonomic divisions; b – distribution of species per habitat ecological groups; c – distribution of species per groups of ph indicators; d – distribution of species per groups of salinity indicators; e – distribution of species per groups of saprobity indicators (after sláde^ek 1973); f – distribution of species per groups of saprobity indicators (after watanabe 1986); g – distribution of species per groups of temperature indicators; h – distribution of species per groups of streaming and oxygenation indicators. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:30 color profile: disabled composite 150 lpi at 45 degrees mostly halophilic, but include also the only palyhalobic species phormidium paulsenianum. all the halophobes are diatoms, among them several species of eunotia. algal indicators of organic pollution are assigned to nine ecological groups (figs. 2e, f; tab. 2), representing the entire spectrum of indication systems (sláde^ek 1973, 1986; watanabe et al. 1986). beta-mesosaprobionts prevail with phormidium autumnale, microcystis aeruginosa (cyanoprokaryota), fragilaria vaucheriae, surirella ovata, pinnularia acta bot. croat. 70 (2), 2011 233 algal biodiversity in kazakhstan wetlands tab. 3. chemical variables (with standard deviation) and water quality classification based on concentrations of nitratenitrogen and phosphorus in the wetland lakes during 1–20 october 1999. lake n-no3 (mg l –1) p-po4 3– (mg l–1) rank of water quality n-no3 p-po4 3– aike 3.4±0.14 0.16±0.01 5a 4a kulykol 1.5±0.1 0.06±0.00 3b 3b kulykol (n2) 1.1±0.0 0.05±0.00 3b 3a kulykol, kordon jailma 2.1±0.1 0.30±0.01 3b 4b kulykol, well jailma 1.0±0.0 0.06±0.00 3a 3b stream jarsor 1.0±0.0 0.01±0.00 3a 2a jarsor 8.6±0.3 4.06±0.12 5b 5b batpakkol 2.3±0.1 0.26±0.01 4b 4b kulagul 0.0±0.0 0.98±0.03 1 5b sankebay 5.4±0.2 0.54±0.20 5b 5a jarkol, naurzum natural reserves* 4.1±0.2 1.08±0.03 5b 5b kushmurun, south part 3.5±0.1 0.10±0.00 5a 3b kojbagor, coast 2.0±0.1 0.13±0.00 4a 4a kojbagor 1.9±0.1 0.27±0.01 4a 4b tuntugur 1.9±0.1 0.12±0.00 4a 4b tuntugur, coast 2.0±0.1 0.26±0.01 4a 4b river karasu, near l. tuntugur 1.4±0.1 0.23±0.01 3b 4b bozshakol 2.7±0.1 0.20±0.01 5a 4a bozshakol, north-east part 8.5±0.3 2.25±0.07 5b 5b biesoygan 2.1±0.1 0.39±0.01 4b 5a sarykol 1.5±0.1 0.06±0.00 3b 3b taly 1.7±0.1 0.15±0.00 4a 4a kamyshovoe 1.9±0.1 0.11±0.00 4a 4a jaman 1.6±0.1 0.08±0.00 4a 3b shoshkaly 1.3±0.1 0.32±0.01 3b 5a annovskoe 2.0±0.1 0.01±0.00 4a 2a maybalyk 1.4±0.1 0.02±0.00 3b 2b tahtakul 1.8±0.1 0.48±0.01 4a 5a sarybalyk 1.8±0.1 0.03±0.00 4a 2b aksuat 1.0±0.0 0.01±0.00 3b 2a gr. karakamys 1.2±0.0 0.01±0.00 3b 2a note: the assessment of b+g radioactive pollution of water samples with detector-indicator of radioactivity quartex rd 8901 shows that its level does not exceed 20 mkr/h. * – naurzum biosphere reserve. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:30 color profile: disabled composite 150 lpi at 45 degrees viridis (bacillariophyta), cladophora, crucigenia tetrapedia (chlorophyta), trachelomonas volvocina (euglenophyta). indicators of temperature conditions reflect a wide range of temperature fluctuations. a group of temperate species prevails, but species of cold and warm waters are also present (fig. 2g). among indicators of streaming and oxygenation, species of slightly turbulent waters – moderate oxygenation prevail, yet in figure 2h, the summit of the trend is displaced toward the indicators of streaming highly oxygenized waters. 234 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. tab. 4. saprobity indices, species richness and classification of water quality in the wetland lakes of kazakhstan during october 1999. lake no. of algal species index of saprobity (s) rank of water quality based on biological variables rank of water quality based on chemical variables water ecosystem state index (wesi) aike 20 1.98 3a 5a 0.5 kulykol 2 2.3 3b 3b 1.0 kulykol (n2) 2 1.85 3a 3b 0.8 kulykol, kordon jailma 3 2.3 3b 4b 0.7 kulykol, well jailma 3 2.35 3b 3b 1.0 stream jarsor 7 1.99 3a 3a 1.0 jarsor – – – 5b – batpakkol 2 2.0 3a 4b 0.6 kulagul 4 2.38 3b 5b 0.5 sankebay 5 1.93 3a 5b 0.4 jarkol. naurzum natural reserves 2 1.7 3a 5b 0.4 kushmurun, south part 9 2.08 3b 4a 0.8 kojbagor, coast 29 2.03 3b 4b 0.7 kojbagor 26 1.95 3a 4a 0.6 tuntugur 6 1.93 3a 4b 0.6 tuntugur, coast 26 1.88 3a 4a 0.6 karasu river near l. tuntugur 2 1.47 2b 4b 0.7 bozshakol 23 1.99 3a 5a 0.5 bozshakol, north-east part 31 1.95 3a 5b 0.4 biesoygan 7 2.55 4a 5a 0.7 sarykol 10 2.15 3b 3b 1.0 taly 6 2.05 3b 4a 0.8 kamyshovoe 22 1.9 3a 4a 0.6 jaman 8 1.78 3a 4a 0.6 shoshkaly 2 2.53 4a 5a 0.7 annovskoe 5 1.71 3a 4a 0.6 maybalyk – – – 3b – tahtakul 2 2.7 4a 5a 0.7 sarybalyk – – – 4a – aksuat – – – 3b – karakamys – – – 3b – u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:30 color profile: disabled composite 150 lpi at 45 degrees assessment of wetland lacustrine ecosystems according to the hydrochemical and hydrobiological variables hydrochemical data for autumn of 1999 (dry period) are represented in tables 1 and 3. the analysis of water conductivity and mineralization reveals a group of highly mineralized lakes – jarsor, sankebay and kushmurun – with salinity level above 7. the other lakes are brackish or freshwater. practically all the investigated lakes show the neutral or slightly alkalic reaction typical of natural water bodies with active self-purification processes. sulacta bot. croat. 70 (2), 2011 235 algal biodiversity in kazakhstan wetlands tab. 5. chemical variables (with standard deviation) and water quality classification based on concentrations of nitrate nitrogen and phosphorus in the wetland lakes during may–june 2000. lake n-no3 (mg l -1) p-po4 3 (mg l-1) rank of water quality n-no3 p-po4 3alpash 1.0±0.0 0.01 3a 2a kulykol 1.2±0.0 0.03 3b 2b gr. kak 2.2±0.1 0.05 4b 3a suly 1.4±0.1 1.77 3b 5b balykty 1.1±0.0 0.02 3b 2b stream jarsor 1.0±0.0 0.01 3a 2a jarsor 2.9±0.1 0.32 5a 5a maybalyk 2.2±0.1 0.00 4b 1 kulagul 0.6±0.0 0.17 3a 4a gr. sankebay 1.6±0.1 0.09 4a 3b jarkol, naurzum natural reserves* 2.3±0.1 0.17 4b 4a chushkaly, naurzum natural reserves* 1.7±0.1 0.28 4a 4b tounsor (teniz) 1.5±0.1 0.00 3b 1 kojbagor 1.3±0.1 0.07 3b 3b tuntugur 1.5±0.1 0.01 3b 2a teniz 1.0±0.0 0.04 3a 3a sultan 0.9±0.0 0.00 3a 1 bozshakol 1.7±0.1 0.00 4a 1 jarken 0.9±0.0 0.03 3a 2b jaltyr 1.0±0.0 1.00 3a 5b sarykol 0.9±0.0 0.00 3a 1 jilandy 1.2±0.0 0.68 3b 5b kamyshovoe 1.2±0.0 0.02 3b 2b jaman 1.2±0.0 0.03 3b 2b shoshkaly, western part 1.1±0.0 0.03 3b 2b annovskoe 1.2±0.0 0.00 3b 1 gr. karakamys 0.8±0.0 0.04 3a 3a tahtakul 1.1±0.0 0.03 3b 2b sarybalyk 1.5±0.1 0.00 3b 1 aksuat 1.4±0.1 0.02 3b 2b karakamys 1.0±0.0 0.68 3a 5b u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:30 color profile: disabled composite 150 lpi at 45 degrees phide (h2s plus the acid-soluble sulfides of metals) concentration 2.005 mg l –1 was found in the northeastern part of bozshakol lake only, which is evidence of periodic anoxia. the saprobity index s varies from 1.47 to 2.70, which corresponds to 2b – 4a ranges of water quality (tab. 4). the biotic component of lake ecosystems provides for a high level of self-purification. 236 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. tab. 6. saprobity index s, species richness and water quality classification in wetland lakes of northern kazakhstan in may–june 2000. lake maximum no. of algal species, (per sample) index of saprobity (s) rank of water quality based on the biological variables rank of water quality based on the chemical variables water ecosystem state index (wesi) alpash 14 1.74 3a 3a 1.0 kulykol 8 2.04 3b 3b 1.0 gr. kak 9 2.11 3b 4b 0.7 suly 7 1.84 3a 4b 0.6 balykty 26 2.21 3b 3b 1.0 stream jarsor 10 1.76 3a 3a 1.0 jarsor – – – 5a – maybalyk 4 2.04 3b 4b 0.7 kulagul 13 2.44 4a 4a 1.0 gr. sankebay 2 2.49 4a 4a 1.0 jarkol, naurzum natural reserves 6 1.96 3a 4b 0.6 chushkaly, naurzum natural reserves 12 1.97 3b 4b 0.7 tounsor (teniz) 30 1.87 3a 3b 0.8 kojbagor 64 2.04 3b 3b 1.0 tuntugur 23 2.12 3b 3b 1.0 teniz 25 1.96 3a 3a 1.0 sultan 5 2.15 3b 3a 1.25 bozshakol 30 2.13 3b 4a 0.8 jarken 23 2.00 3b 3a 1.25 jaltyr 6 1.86 3a 5b 0.4 sarykol 23 1.71 3a 3a 1.0 jilandy 6 2.05 3b 5b 0.5 kamyshovoe 53 1.86 3b 3b 1.0 jaman 40 2.10 3b 3b 1.0 shoshkaly, western part 23 1.87 3b 3b 1.0 annovskoe 10 1.81 3a 3b 0.8 gr. karakamys 6 1.65 3a 3a 1.0 tahtakul 4 1.80 3a 3b 0.8 sarybalyk 5 1.48 2b 3b 0.6 aksuat 1 2.70 4a 3b 1.2 karakamys 18 1.80 3a 5b 0.4 u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:30 color profile: disabled composite 150 lpi at 45 degrees in spring, the lakes kak, jarsor, sankebay and sarybalyk were strongly mineralized, with salinity above 7 (tabs. 5, 6). the other lakes remained freshwater or brackish. the ph reaction was neutral or slightly acidic in all the lakes, characteristic of natural waters with active self-purification processes. the defined background radioactivity in the studied lakes during 1999–2000 was stable, with a level not exceeding 20 mkr h–1, presenting the regional norm and unable to impact lake communities. discussion the overall diversity is the highest in the lakes bozshakol, kamyshovoe, kojbagor (63 – 112 species), and some other freshwater lakes (tab. 1) of iv water salinity class. bio-indicational analysis of algal diversity shows that the dominant indicator species are alkaliphiles, oligohalobes-indifferents and beta-mesosaprobes conveying the integrity of major ecological variables. similarity analysis (fig. 3) is based on the distribution matrix of 254 revealed species over 34 water bodies and calculated as the percent disagreement by ward’s method. the dendrogram shows that the algal taxonomic diversity is divided into three different clusters, with most of the kazakhstan lakes separated at the 74% similarity level. the group designated at the 82% similarity level (cluster 2) comprises algal assemblages of lakes with great amplitude of salinity fluctuations (ii to iv classes), with species numbers 1 – 28. the dominant indicators are oligohalobes-indifferent, halophiles and mesohalobes. the second group discriminated at 82% similarity level comprises assemblages of moderately mineralized lakes of iii–iv salinity classes, with the species numbers 18 – 37. the dominant indicators are oligohalobes-indifferent, halophiles and occasionally mesohalobes. cluster 3 of low similarity level comprises assemblages of slightly mineralized lakes of iv salinity class: bozshakol, tuntugur, jaman, teniz, kamyshovoe, and kojbagor, with species numbers 57 (jaman) to 112 (kojbagor), dominated by oligohalobes-indifferent and halophiles; mesohalobes are lacking in these lakes. therefore, the dendrogram clustered all the revealed diversity around three major variables: species richness of algal communities, salinity class, and the dominant salinity indicators. the most similar are the species-rich communities of slightly mineralized lakes, as well as the species-poor communities of highly mineralized lakes (ács et al. 2003). these regularities indicate that, other conditions remaining the same, salinity is the main depressing factor of algal diversity irrespective of the type and distribution of the water body. in other words, the compositions of algal communities reflect in the first place the salinity level related to climatic aridity. because the species diversity in protected wetlands is mostly influenced by natural factors, floristic cores can reflect historical natural impact on algal biodiversity. comparative floristics help summarize regional algal diversity in major floristic cores (fig. 4). we used comparative floristic approaches also for revealing the major factors influencing the lacustrine flora enriching process. in the statistical program graphs (novakovsky 2004) which presented not only tables of calculation but also constructed visual graphs, we anaacta bot. croat. 70 (2), 2011 237 algal biodiversity in kazakhstan wetlands u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:31 color profile: disabled composite 150 lpi at 45 degrees lyzed presence-absence of 254 species in 34 lakes with serensen-chekanovsky indices calculation. as a result, a dendrite of similarity (fig. 4) shows five floristic cores, which are marked by dashed lines. most lakes with species rich communities and fresh water combined into central core (a). the lakes bozshakol with 63 species and kojbagor with 112 species placed in the center of core a. all lakes from core (a) are 3–4 salinity class with high species diversity, low to medium dissolved solids and seasonally fluctuating electrical conductivity, neutral to low acidic range of ph, low to middle nutrient concentration, and iii–iv class of water pollution. this means that ecosystems in core (a) lakes are well developed. core (b) formed 9 freshwater lakes with middle species diversity, medium dissolved solids and nutrients concentration, clearer than in core (a), but with neutral to low alkalic water. 238 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. ward`s method percent disagreement kojbagor kamyshovoe teniz jaman tuntugur bozshakol gr.karakamys jarken annovskoe str.jarsor alpash suly tahtakul sultan kushmurun jarkol majbalyk biesoygan gr.kak jaltyr sankebay kulykol-jailma jilandy gr.sankebay r.karasu batpakkol aksuat sarykol shoshkaly balykty kulykol chushkaly kulagul aike 1 2 0.2 0.4 0.6 0.8 3 linkage distance fig. 3. tree diagram for algal species diversity in the wetland lakes of kazakhstan, ward’s method, percent disagreement. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:31 color profile: disabled composite 150 lpi at 45 degrees core (c) included two lakes only that connected with core (a) and characterized as freshwater, low alkaline, low organic polluted with low to medium dissolved solids and nutrients concentration. core (d) also formed two lakes, which have conditions similar to those lakes of core (c) and also closely related with the diversity of core (a). the last core (e) included three freshwater lakes, which have similar conditions with lakes from the major core (a). a few lakes that are not included in the mentioned above cores have intermediate (as in tahtakol or kushmurun) or extreme environmental conditions such as in the great kak lake: high salinity and electrical conductivity, low acidic water with low phosphates and medium nitrates concentration and as a result low species diversity. therefore, comparative floristic analysis pointed to salinity as the most important factor that has had a historical influence on algal diversity in the studied wetland lakes. the assessment of the aquatic ecosystems state is based on a correlation of hydrochemical data and their ranges for nitrogen and phosphorus (the trophic elements) with those for saprobity indices (the biota’s self-purification capacity). we calculated the index of the water ecosystem state, wesi (barinova 2000, barinova et al. 2006) relating the ranges of self-purification to those of trophic elements. wesi reflects the potentials of the aquatic ecosystem to regenerate after anthropogenic impacts. it also conveys the intensity of anthropogenic impacts if such occurred. in the case of wesi above or equal to 1.0, the ecosystem is assessed as balanced and buffered from anthropogenic impacts. at wesi below 1.0, the biotic components are under toxic influences. a smaller wesi reflects a greater toxicity. sometimes the latter can be natural rather than anthropogenic. acta bot. croat. 70 (2), 2011 239 algal biodiversity in kazakhstan wetlands fig. 4. dendrite of similarity constructed on the base of serensen-checkanovsky indices. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:32 color profile: disabled composite 150 lpi at 45 degrees our analysis showed a normal (balanced) state for the ecosystems of the lakes kulykol, the spring jailma, the spring jarsor and sarykol lake (tab. 4) in dry autumn of 1999. most of the samples reveal a slight natural toxic influence which may be come from sulphides. more significant toxic influence is recognized for the lakes aike, sankebay, jarkol, and bozshakol. however, the composition of their algal communities gives no evidence of heterotrophy. normal state (wesi above or equal 1.0) was found in the spring season of 2000 in the lakes alpash, kulykol, balykty, kulagul, great sankebay, kojbagor, tuntugur, teniz, sultan, jarken, sarykol, kamyshovoe, jaman, shoshkaly, karakamys, aksuat, and the stream jarsor. this group includes most of the lakes. at the same time, a slight toxic suppression of photosynthetic activity was revealed for the lakes suly, majbalyk, jarkol, chushkaly, taunsor (teniz), bozshakol, jaltyr, jilandy, annovskoe, tahtakul, sarybalyk, karakamys. therefore, the toxicity might have been temporary, during the dry period, unrelated to any constant anthropogenic impact. according to the functional model of aquatic ecosystems (barinova 2000, barinova et al. 2006), ecosystems of majority of lakes are at the regenerating stage and most influenced by evaporation in summer season. algal species and index saprobity s dynamic in communities during the 1999–2000 study period show that the maximal taxonomic diversity (number of species) for algal communities in 1999 was observed in the lakes kojbagor (64), bozshakol (30), kamyshovoe (53) and jaman (40). the saprobity index s varied from 1.48 to 2.70, which corresponds to 2b–4a ranks of water quality. although sulfides were periodically revealed in bozshakol lake, algal species richness is rather high because this lake is fresh and provides the best environment not only for higher aquatic plant development, but also for algal diversity in plankton and submerged plants. the means of s for these lakes in the spring revealed a high self-purification capacity. the most species-rich in the year 2000 were the lakes balykty (26), tounsor (teniz) (30), kojbagor (64), tuntugur, bozshakol (30), kamyshovoe (53), jaman (40), and other swith more than 20 species per each sample. on the basis of nitrate concentration, most of the lakes in 1999 were assigned to 4b and 5a–b water quality ranges (tab. 3), which indicates a reduced consumption of this element by the lacustrine biota. for phosphate concentrations, 19 samples fall in the same high ranges. only in the kulagul (kulykol) lake is there a high concentration of phosphates against the minimal nitrate concentration. in this lake the algal productivity is limited by nitrogen which explains the low consumption of phosphates. during the study period only in the northeastern part of bozshakol lake was a high concentration of sulfides (h2s) found, which can be explained by anaerobic decomposition of dead matter produced by the lake ecosystem during the periods of water bloom or a decay of aquatic macrophytes. this biota toxic variable periodically formed a reduction zone in the bottom, but in the thin layer of water under the surface life is flourishes. on account of their nitrate and phosphate concentrations, several lakes were assigned to 4b and 5a water quality classes in 2000 (tab. 5), indicating under-consumption of these components by the biota. in lakes maybalyk, tounsor (teniz), sultan, bozshakol, sarykol, annovskoe, and sarybalyk, a relatively high concentration of nitrates was associated with a low concentration of phosphates; the nitrates were under-consumed, because the development of algal community was limited by phosphorus. nitrogen was not a limiting factor 240 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:32 color profile: disabled composite 150 lpi at 45 degrees in these lakes. as a whole, the biotic communities were actively developing, although occasionally restricted by the deficit of phosphorus in freshwater lakes and in a single brackish lake, sarybalyk. conclusion the calculated indices of the environmental quality of kazakhstan’s arid region wetlands ranged within the expected natural variations. toxic influence is recognized in jaltyr, jilandy, and karakamys. a slight natural influence was revealed for most of the lakes. the organic matter enriching the water after aquatic plant death regulates the production of sulfides as toxic substances for algae. yet the algal communities provide no evidence of heterotrophy. the toxicity might have been temporary, unrelated to a constant anthropogenic impact. according to the functional model of aquatic ecosystems (barinova et al. 2006), lakes jaltyr, jilandy, and karakamys are assigned to the regeneration stage. in comparison to 1999 and 2000, salinity (mineralization) in the studied lakes slightly decreased (by 0.01–0.02), with the exception of sarybalyk and aksuat, where it increased from 1.47 and 0.69 respectively in autumn 1999 to 8.88 and 3.94 respectively in spring 2000. salinity as a consequence of aridization suppressed algal diversity, and thereby decreased the productivity of the first trophic level, undermining the trophic base of wetlands as water fowl habitat. lake ecosystems are insignificantly disturbed, only few of them revealing an appreciable toxic effect. the saprobity indices calculated for each of the lakes attest to a high self-purification capacity. by comparing of the ecosystem state indices, wesi, for 1999 and 2000 we are led to the conclusion that self-purification activity increased from dry to wet seasons in kulagul, sankebay, jarkol, kojbagor, tuntugur, bozshakol, annovskoe, and tahtakul. a seasonally increase of salinity in lakes aksuat, sarybalyk, and jarsor did not affect their general state. therefore, we concluded that salinity in the lakes is the most important factor that has also had a historical influence on the algal diversity of the arid region wetland lakes. acknowledgements we thank e. a. bragin, n. n. berezovikov, s. n. erokhov, v. s. vilkov and v. i. drobovtsev for help in the sampling trips as well as a. g. karlsen and a. solovieva for help in laboratory analysis. this work was partly supported by the israeli ministry of absorption. references ács, é., borsodi, a. k., makk, j., molnár, p., mózes, a., rusznyák, a., reskóné, m. n., kiss k. t., 2003: algological and bacteriological investigations on reed periphyton in lake velencei, hungary. hydrobiologia 506–509, 549–557. apha, awwa, wef, 1998. standard methods for the examination of water and 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(in russian). nauka press, leningrad. vinogradova, k. l., gollerbach, m. m., zauer, l. m., sdobnikova, n. v., 1980: chlorophyta, rhodophyta, phaeophyta. flora plantarum cryptogamarum urss, 13 (in russian). nauka press, leningrad. watanabe, t., asai, k., houki, a., 1986: numerical estimation to organic pollution of flowing water by using the epilithic diatom assemblage – diatom assemblage index (daipo). the science of the total environment 55, 209–218. whitton, b. a., rott, e., friedrich, g., 1991: use of algae for monitoring rivers. institute of botany. innsbruck university press, innsbruck. 244 acta bot. croat. 70 (2), 2011 barinova s. s., nevo e., bragina t. m. u:\acta botanica\acta-botan 2-11\barinova.vp 9. rujan 2011 11:21:32 color profile: disabled composite 150 lpi at 45 degrees 544 vizintin et al.vp coden: abcra 25 issn 0365–0588 eissn 1847-8476 short communication distribution of buxbaumia viridis (moug. ex lam. et dc.) brid. ex moug. et nestl. (bryophyta) in montenegro sne@ana dragi]evi]1*, beáta papp2, peter erzberger3 1 natural history museum of montenegro, trg vojvode be}ir bega osmanagi}a 16, 81000 podgorica, montenegro 2 hungarian natural history museum, botanical department, budapest, pf 222. h-1476, hungary 3 belziger str. 37, d-10823 berlin, germany abstract – the present paper is a contribution to the knowledge of the distribution of the moss species buxbaumia viridis in montenegro. the records are from 14 known sites at elevations over 1300 m a.s.l. in the northern and north-eastern parts of the country. population size is remarkable in durmitor national park at crno jezero lake, where sporophytes can be found on ca 50 tree trunks. keywords: moss, buxbaumia viridis, distribution, montenegro introduction the genus buxbaumia hedw. is represented by two species in europe: buxbaumia aphylla hedw. and buxbaumia viridis (moug. ex lam. et dc.) brid. ex moug. et nestl. (hill et al. 2006), both of which occur in montenegro. according to sabovljevi] and stevanovi] (2000), buxbaumia aphylla hedw. was recorded in 1998 at two localities within the durmitor national park, while buxbaumia viridis was found much earlier in the same area by martin^i^ (1964). buxbaumia viridis is a boreal-montane species (düll 1984). it is sparsely distributed throughout the northern hemisphere, from europe to southwest asia and china to western north america (smith 2004). it has been recorded from all southeast european countries except the european part of turkey (sabovljevi] et al. 2008). buxbaumia viridis is found growing on well-decayed wood in constantly humid forests, sheltered or shaded places, rarely on humus-rich acidic soil. during most of the year it lives as protonema in the substrate and can usually be recognized only, when sporophytes are acta bot. croat. 71 (2), 2012 365 * corresponding author, e-mail: sneza.dragicevic@t-com.me copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. acta bot. croat. 71 (2), 365–370, 2012 produced. these can be found singly or in small groups on the surface of decaying trunks. the sporophyte is of remarkable size; the seta (5–10 mm long) supports a large asymmetrical capsule, which when mature bears an »indusium«, (a characteristic feature of this species, expressed in its synonym buxbaumia indusiata). the indusium is formed by the cuticle of the capsule when it splits longitudinally and peels back, persisting in large, ragged pieces (smith 2004). the species is endangered by forestry practices, especially by clear-cutting huge areas of forest, and other forms of commercial exploitation that have reduced the extent of old-growth forests, the number of well-decayed logs, and changes in the microclimate. buxbaumia viridis appears on the red list of most countries where it occurs. the european committee for the conservation of bryophytes rates buxbaumia viridis as vulnerable in europe (stewart 1995). the species is listed in annex ii of the ec habitats and species directive and in appendix i of the council of europe bern convention. it is treated as critically endangered in the bryophyte red list of serbia and montenegro (sabovljevi] et al. 2004). the moss is protected by law in montenegro, but seems to be at extremely high risk of extinction owing to its fragmented distribution, and small total number of plants. study area the study area is located in the northern and north-eastern parts of montenegro. it includes 8 mountainous regions: the bjelasica mts, durmitor mts, hajla mts, komovi mts, ljubi{nja mts, prokletije mts, sinjajevina mts and visitor mts (fig. 1). some characteristics about the regions investigated are given in table 1. materials and methods our investigations were carried out mainly in the high mountain regions of montenegro between 2004–2009. in each locality, in order to detect the presence of the species, we searched for large, well-decayed trunks in old-growth conifer, beech or deciduous (mixed) forests. the specimens collected are deposited in the herbarium of the natural history museum of montenegro, the hungarian natural history museum, budapest (bp) and in the herbarium of botanisches museum berlin-dahlem (b). in order to estimate the threat status according to the new iucn categories (iucn 1994, 2001) the following criteria should be taken into consideration (hallingbäck et al. 1998): a. large decline (major population decline observed, estimated, inferred or suspected in the last 10 years or 3 generations, whichever is the longer) b. restricted area of occupancy, few localities, decline c. small population and decline, and d. very small or restricted populations for the estimation of population size of buxbaumia viridis the trees colonized can be counted as individuals, because the destruction of the substrate will affect all plants growing on it (hallingbäck et al. 1998). in the field we counted the number of trees on which sporophytes of the species were detected. 366 acta bot. croat. 71 (2), 2012 dragi]evi] s., papp b., erzberger p. results and discussion during our fieldwork in montenegro, more than 40 years after the first record of buxbaumia viridis for the country, we found populations on mt durmitor as well as in several other mountain areas. the species was collected at 14 localities in on eight mountains in the northern part of the country. the sites were situated at high elevations, over 1300 m a.s.l., usually in boreal types of forests. acta bot. croat. 71 (2), 2012 367 distribution of buxbaumia viridis brid. ex moug. et nestl. in montenegro fig. 1. the distribution of known localities of buxbaumia viridis in montenegro (1-bjelasica mts; 2-durmitor mts; 3-hajla mts; 4-komovi mts; 5-ljubi{nja mts; 6-prokletije mts; 7-visitor mts; 8-sinjajevina mts). 368 a c t a b o t .c r o a t .71 (2),2012 d r a g i] e v i] s .,p a p p b .,e r z b e r g e r p . tab. 1. some characteristics about investigated regions. no in fig. 1 geographical name maximum hight (m asl.) bedrock climate conservational status flora, vegetation 1 bjelasica mts 2137 m (crna glava) silicate, volcanic cliffs alpine, moderatecontinental-meditter-ranean – rich endemic vascular flora 2 durmitor mts 2523 m (bobotov kuk) carbonate, clastic and siliceous rocks (mostly triassic) alpine, moderate-mediter-ranean national park, ipa rich vascular flora 3 hajla mts 2280 m (dermandoski greben) limestone and volcanic cliffs alpine, moderate-mediter-ranean ipa oak and beech forests, spruce or spruce and macedonian pine forests 4 komovi mts 2484 m (ku~ki kom) limestone alpine, moderate-mediter-ranean ipa extensive scree 5 ljubi{nja mts 2238 m (dernja~i{ta) limestone and silicate alpine ipa spruce forest, pastures 6 prokletije mts 2528 m (maja kolata) limestone alpine, moderate-mediter-ranean national park rich endemic and relict vascular flora 7 sinjajevina mts 2277 m (babji zub or torna) carbonate mediter-ranean and continental ipa rich flora 8 visitor mts 2211 m (plana) limestone alpine, moderate-mediter-ranean ipa proposed mixed thermophi-lous forests, beach, beach-silver fir and macedonian pine forests; mountain meadows, cliffs and screes the localities were as follows: bjelasica mountain, at svinja~a stream near jezerine at kola{in, 42°49'49,5'', 19°37'06,7'' e, ca 1360 m, conifer and deciduous (mixed) forest, on decaying wood, leg. s. dragi}evi}, b. papp and p. erzberger, 18.07.2007. durmitor mountain, durmitor national park, around crno jezero lake at @abljak, 43°08'52,0'' n, 19°05'48,0'' e, ca 1400 m, conifer forest, on decaying trunk, leg. b. papp and p. erzberger, 05.10.2004. (papp and erzberger 2010) and leg. s. dragi}evi} 10.07.2007.; by military resort, ca 1400 m, on decaying trunk, leg. s. dragi}evi}, 10.07.2007.; along mlinski potok, 43°08'58,2'' n, 19°05'23,8'' e, ca 1400 m, leg. a. martin~i~ (martin^i^ 1964) and leg. b. papp and p. erzberger, 07.10.2004. (papp and erzberger 2010); around barno jezero lake, 43°09'19,6'' n, 19°05'25,8'' e, ca 1500 m, conifer forest, on decaying trunk, leg. b. papp and p. erzberger, 10.10.2004. (papp and erzberger 2010) and leg. s. dragi}evi}, 12.07.2007. hajla mountain, forest track towards band`ov, 42°47,430' n, 20°07,602' e, ca 1336 m, conifer forest (picea abies), on decaying trunk, leg. s. dragi}evi}, 28.07.2009. komovi mountain, valley of the ljuba{tica, 42°41'44,3'' n, 19°33'43,5'' e, ca 1770 m, beech forest, on decaying trunk, leg. b. papp and p. erzberger, 28.07.2008. ljubi{nja mountain, near pljevlja, ca 1650 m, conifer forest (picea abies), on decaying trunk, leg. s. dragi}evi}, 26.08.2007.; kosanica, near pljevlja, 43°10'39,6'' n, 19°18'13,5'' e, ca 1301 m, conifer and deciduous (mixed) forest, on decaying trunk, leg. s. dragi}evi}, 07.07.2011. prokletije mountains, along the road from zastan koliba to maja jezerce, 42°29'21,1'' n, 19°48'56,7'' e, ca 1500 m, on decaying trunk, leg. b. papp and p. erzberger, 11.10.2006.; tre}a livada in grebaje valley at gusinje, 42°30'41,6'' n, 19°46'40,9'' e, ca 1220 m, beech forest, on decaying trunk, leg. b. papp, 12.10.2006. visitor mountain, along the road to visitorsko jezero lake, 42°38'07,7'' n, 19°53'28,3'' e, ca 1600 m, leg. b. papp and p. erzberger, 09.10.2006. sinjajevina mountain, region [aranci, along the road to kr{ village, 43°7'16,9'' n, 19°15'39,4'' e, ca 1410 m, conifer forest, on decaying trunk, leg. s. dragi}evi}, 28.06.2007. and 07.07.2011.; zmini~ko jezero lake, 43°06' n, 19°05' e, ca 1285 m, conifer and deciduous (mixed) forest, on decaying trunk, leg. s. dragi}evi}, 29.06.2007. owing to the threat status of the species, criterion a cannot be applied to the species in montenegro, as there is insufficient information about earlier occurrences; most of the available data are less than five years old. the species falls into criterion b, the vulnerable category, as it was recently recorded in five to ten 10 km x 10 km squares and found in five to ten localities with most of the populations severely fragmented and in decline. however, applying criteria c and d places the species in the critically endangered category (cr), since the population sizes are small, even the largest population in durmitor national park at crno jezero lake consists of not more than 50 colonized trunks. in the other sites, only a few sporophytes could be found on a small number of trunks. as the highest iucn category has to be taken into consideration, we can conclude that, in spite of the new records, buxbaumia viridis must be treated as a critically endangered (cr) species in montenegro. acta bot. croat. 71 (2), 2012 369 distribution of buxbaumia viridis brid. ex moug. et nestl. in montenegro acknowledgements we wish to thank dr. vicente mazimpaka and dr. marko karaman for helpful comments on an earlier version of the manuscript. references düll, r., 1984: distribution of the european and macaronesian mosses (bryophytina) i. bryologische beitraege 4, 1–109. hallingbäck, t., hodgetts, n., raeymaekers, g., schumacker, r., sérgio, c., söderström, l., stewart, n., vána, j., 1998: guidelines for application of the revised iucn threat categories to bryophytes. lindbergia 23, 6–12. hill, m. o., bell, n., bruggeman-nannenga, m. a., brugués, m., cano, m. j., enroth, j., flatberg, k. i., frahm, j. p., gallego, m. t., garilleti, r., guerra, j., hedenäs, l., holyoak, d. t., hyvönen, j., ignatov, m. s., lara, f., mazimpaka, v., muñoz, j., söderström, l., 2006: bryological monograph. an annotated checklist of the mosses of europe and macaronesia. journal of bryology 28, 198–267. iucn, 1994: iucn red list categories. international union for the conservation of nature, gland, switzerland. iucn, 2001: iucn red list categories and criteria, 3.1. international union for the conservation of nature species survival commission, gland, switzerland and cambridge, uk. martin^i^, a., 1964: contribution to the knowledge of moss flora of yugoslavia i. durmitor (montenegro) (in slovenian). biolo{ki vestnik 12, 43–49. papp, b., erzberger, p., 2010: contributions to the bryophyte flora of durmitor national park, montenegro. nova hedwigia 138, 147–163. sabovljevi], m., stevanovi], v., 2000: buxbaumia aphylla hedw., new to montenegro (fr yugoslavia), and some notes on its ecology. cryptogamie bryologie 21, 87–89. sabovljevi], m., cveti], t., stevanovi], v., 2004: bryophyte red list of serbia and montenegro. biodiversity and conservation 13, 1781–1790. sabovljevi], m., natcheva, r., dihoru, g., tsakiri, e., dragi]evi], s., erda , a., papp, b., 2008: check-list of the mosses of se europe. phytologia balcanica 14, 207–244. smith, a. j. e., 2004: the moss flora of britain and ireland. university press, cambridge. stewart, n. (ed.), 1995: red data book of european bryophytes. european committee for the conservation of bryophytes, trondheim. 370 acta bot. croat. 71 (2), 2012 dragi]evi] s., papp b., erzberger p. 60 acta bot. croat. 82 (1), 2023 acta bot. croat. 82 (1), 60–70, 2023 coden: abcra 25 doi: 10.37427/botcro-2022-029 issn 0365-0588 eissn 1847-8476 acer velutinum bioss. (velvet maple) seedlings are more tolerant to water deficit than alnus subcordata c.a. mey. (caucasian alder) seedlings mokarram ravanbakhsh1, babak babakhani1*, mahmood ghasemnezhad1,2, fariba serpooshan1, mohamad hassan biglouie1,3 1 department of biology, tonekabon branch, islamic azad university, tonekabon, iran. 2 department of horticultural sciences, faculty of agricultural sciences, university of guilan, rasht, iran. 3 department of water engineering, faculty of agricultural sciences, university of guilan, rasht, iran. abstract – drought stress is a major environmental factor limiting plant growth. selection of drought-tolerant plants is of critical importance in vegetation restoration and forestation programs. alnus subcordata and acer velutinum are two valuable, dominant, and endemic species in the hyrcanian forests. there are fast-growing species and significant diffuse-porous hardwood in afforestation and reforestation. one-year old seedlings of both species were exposed to four water shortage treatments (100, 75, 50 and 25% of field capacity (fc) chosen as control, mild, moderate, and severe) for 12 weeks. thereafter, their morphological characteristics such as height and basal area, total and organ biomass (root, stem, and leaf ), leaf area (la), specific leaf area (sla), leaf area ratio (lar), as well as physiological and biochemical characteristics such as relative water content (rwc), content of chlorophyll, free proline and malondialdehyde (mda), and superoxide dismutase (sod) and peroxidase (pod) activity were measured. the results showed that when exposed to reduced water availability, plant height, basal diameter, total and organ biomass, la, lar, rwc and chlorophyll content decreased, but their proline concentration, mda content, sod, and pod activity increased in both species. the root to shoot ratio (r/s) and root mass ratio (rmr) increased at 50 and 25% fc treatments in a. subcordata, whereas no significant difference was found in a. velutinum under drought treatments. sla increased significantly at 50% fc in a. velutinum and decreased in a. subcordata under drought treatments compared to control treatment. a. velutinum showed more proline content, rwc, pod, and lower increase in mda content than a. subcordata under moderate treatment. therefore, a. velutinum appears to possess a better mechanism to cope with drought stress. the drought tolerance of a. velutinum may enhance its potential for climatic adaptations under drier conditions with the ongoing climatic change. keywords: alnus subcordata, acer velutinum, antioxidant enzymes, biomass, growth, water deficit introduction the impacts of climate change on vegetation will appear as a combination of stress factors, including high temperatures, reduction of rainfall, and alterations in wildfire regimes. the principal aspect of global climate change, the frequency, and intensity of drought stress will increase in the future (wu et al. 2017). drought can damage afforestation and reforestation programs because seedlings are more prone to drought than mature trees. drought-tolerant species should be considered so as to contribute to sustainable forest ecosystems (bhusal et al. 2020). selection of droughttolerance plants has a critical role in vegetation restoration and silvicultural strategies (khaleghi et al. 2019). drought affects various aspects of the plant; the roots are the first part to be affected in the face of drought. the chemical signals (abscisic acid) produced in the roots along with decreased leaf turgor and atmospheric vapor pressure can reduce stomatal conductance. the limitation associated with increased stomatal resistance (under mild to moderate water deficit), is known as a stomatal limitation. limitation due to non-stomatal disturbance under severe drought stress (non-stomatal limitation) can be induced by the limited diffusion of co2 from the intercellular spaces to the chloroplasts or by metabolic factors such as a decrease in rubisco activity, disturbances in the regeneration of ribulose diphosphate and reactive oxygen species (ros) production from the excess excitation energy. low growth can be * corresponding author e-mail: babakhani_babak@yahoo.com mailto:babakhani_babak@yahoo.com responses to water deficit in acer velutinum and alnus subcordata acta bot. croat. 82 (1), 2023 61 due to suppression of the photosynthetic process that eventually reduces biomass (du et al. 2010, dulai et al. 2014). chlorophyll content can directly influence photosynthetic potential and primary production. reduction in chlorophyll content under water deficit has been regarded as a typical feature of oxidative stress (liu et al. 2019). photosynthetic pigment stabilization under stress conditions increases resistance to drought stress (ge et al. 2014). decreased chlorophyll content under water deficit was reported in such tree species as fagus sylvatica (gallé and feller, 2007), quercus variabilis (wu et al. 2013), alnus cremastogyne (tariq et al. 2018), and acer davidii (guo et al. 2019), while no change in chlorophyll content was found in melia azedarach (dias et al. 2014). relative water content (rwc) is a key indicator of degree of hydration and vital for optimal physiological functions and growth processes. rwc in woody and shrubby species reached 50 to 40% and seldom was it as low as 30 to 20% under severe water stress, which eventually causes leaf senescence (wu et al. 2013). relatively high rwc maintenance in water shortage is an indicator of drought tolerance (ying et al. 2015, toscano et al. 2016). quercus variabilis seedlings could maintain sufficient rwc and slight growth at 40% field capacity (fc) (wu et al. 2013). rwc of alnus cremastogyne signifcantly decreased by 32.6% under drought (tariq et al. 2018). decrease of rwc in response to moderate (50% fc) and severe (30% fc) drought treatment in maclura pomifera has been reported (khaleghi et al. 2019). resistance to biotic and abiotic stress in plants increases by the accumulation of significant amounts of free proline, soluble sugars (sucrose, glucose and fructose), and soluble proteins (maturation proteins). these compatible solutes are able to maintain the concentration of cell sap and prevent the loss of water in plasma (mohammadkhani and heidari 2008, farooq et al. 2009, guo et al. 2018). proline functions not only as an osmolyte, but also as an antioxidant, thus helping ros detoxification by membrane integrity protection and enzyme/protein stabilization (ghaffari et al. 2019, khaleghi et al. 2019). the intercellular concentration of malondialdehyde (mda), a breakdown product of lipid peroxidation, has been measured as an indicator of oxidative damage (ge et al. 2014, abid et al. 2018). to scavenge ros, plants maintain an efficient antioxidant defense system including non-enzymatic antioxidants and antioxidant enzymes (khaleghi et al. 2019). peroxidase (pod) and superoxide dismutase (sod) disintegrate ros, and therefore, protect plants from drought stress (geng et al. 2019). sod catalyzes the conversion of superoxide radical (o2•−) to molecular oxgen (o2) and hydrogen peroxide (h2o2). this h2o2 is detoxified to o2 and h2o through the activities of catalase (cat) and pod as well as the ascorbate-glutathione (asa-gsh) cycle (wang et al. 2012, abid et al. 2018). based on climate modeling, the air temperature in iran will rise by 2.7 °c up to 2050, which will increase the water needs of plants (attarod et al. 2017). the caspian forest climate has become warmer and the vegetation growth trend has been upwards of about one hundred meters in the last half-century (taleshi et al. 2018). reforestation by alnus subcordata c.a. mey. (caucasian alder), and acer velutinum bioss. (persian or velvet maple) to increase production capacity reduced the pressure of wood exploitation on hyrcanian forests (abdolahi et al. 2017). a. subcordata and a. velutinum are the most valuable endemic species and are indigenous to the hyrcanian province in the euro-siberian region. due to their importance, numerous studies have done on the quantitative and qualitative characteristics of the species, mechanical properties of wood and nutrient elements (naghdi et al. 2016, naji et al. 2016, tavankar et al. 2017, ghorbani et al. 2018, jourgholami et al. 2020). according to a few recent studies, nano priming technique increased drought tolerance of a. subcordata seeds (rahimi et al. 2016). a. subcordata as an urban tree showed limited tolerance to water deficit by determination of midday leaf water potential (ψl) and stomatal conductance (gs) (sjöman et al. 2021). however, their response to drought and the mechanism of these two species in artificial cultivation are still unclear and poorly understood. therefore, the objectives of the present study were (i) to evaluate the effects of drought stress on a. subcordata and a. velutinum seedlings which are dominant species in hyrcanian forests and have a high commercial value in wood industries, to discover their capacity to handle water deficit in the initial vegetative growth period by morphological, physiological and biochemical responses; and (ii) to determine these two species’ different adaptive responses to drought stress. materials and methods plant material and drought treatments the experiment was carried out in a greenhouse at university of guilan, iran (37°15́ n, 49°36́ e). the average annual temperature was 15.9 °c and cumulative precipitation 1329.1 mm (allahyari et al. 2016). one-year-old a. subcordata c.a. mey. and a. velutinum boiss. seedlings were obtained from a local nursery called pilambara (37°35′ n, 49°05′ e) in resvanshahr, guilan province, iran. the seedlings were transplanted to 9 l plastic pots filled with homogenized topsoil. the plants were grown in a naturally lit greenhouse (tem perature range: 18–28 °c; relative humidity range 73–94%) in a semi-controlled environment (only sheltered from rainfall) from july 10 to october 10, 2019. the greenhouse was well ventilated by plastic side films being rolled around it (guo et al. 2013). drought treatments were performed three months after the planting of the seedlings (an acclimatization period, and when plants had produced fully expanded leaves) (guo et al. 2013, medeiros et al. 2013, meng et al. 2013). a randomized complete design with two factors (two species and four watering regimes) was employed with three replications for four water shortage treatments (100, 75, 50 and 25% of field ravanbakhsh m., babakhani b., ghasemnezhad m., serpooshan f., biglouie m. h. 62 acta bot. croat. 82 (1), 2023 capacity performed as a control, mild, moderate, and severe, respectively). using a scale with a capacity of 40 kg, transpiration water loss was measured gravimetrically by weighing all pots and re-watering with tap water every two days. the water added to each pot during the experimental period was 27, 18, 10.8 and 6.75 l for control, mild, moderate, and severe treatments respectively for seedlings of a. subcordata and 22.5, 15, 9, and 6 l for seedlings of a. velutinum. the evaluation was performed after three months at the end of the experiment. growth parameters seedling height (cm) was measured from the soil surface to the terminal bud of the main stem using a measuring tape; also, the basal diameter (mm) was measured at the ground line by electronic calipers. plant height, basal diameter and biomass (total dry mass) were recorded at the end of the experiments. three seedlings were harvested randomly from each treatment. the leaves, stems, and roots were cut and dried in an oven at 65 °c for 48 hours to calculate root, stem, and leaf biomass (the average weight of three samples per treatment). biomass contribution including leaf mass ratio (lmr), stem mass ratio (smr) and root mass ratio (rmr) was calculated by dividing the stem, leaf, and root biomass by the total biomass (root, stem, and leaf), respectively. root: shoot ratio (r/s) was calculated using root biomass by total leaf and stem biomass in percentage. leaf area (la) was determined with a leaf scanner (model a3 light box gcl bubble etch tanks), and windias 3.2. software. specific leaf area (sla) was estimated by dividing the leaf area by leaf biomass, while leaf area ratio (lar) was determined by dividing the total leaf area by every seedling total biomass (wu et al. 2017, zhang et al. 2019). relative water content ten leaf discs with a diameter of 5 mm were cut from the interveinal parts of each plant and fresh weight (fw) was determined. after that, turgor weight (tw) was calculated by weighing discs dipped in water for 24 hours in the dark. finally, leaf discs were oven dried for 24 hours at 65 °c to determine dry weight (dw). relative water content (rwc) was measured as follows: rwc (%) = (fw-dw) / (tw-dw) × 100 (toscano et al. 2016). photosynthetic pigment content for the extraction of photosynthetic pigments, 200 mg liquid nitrogen frozen tissue was ground by pestle and mortar and pigments were extracted by adding 10 ml of 80% cold acetone. the content of chlorophyll a (chl a) and b (chl b), total chlorophyll (chl a+b) and carotenoids was measured spectrophotometrically at 663, 645 and 470 nm respectively by spectrophotometer (ltd t80 + uv/vis; pg instruments, leicestershire, uk) according to lichtenthaler (1987). the chlorophyll and carotenoid concentrations expressed as mg g–1 fw were calculated as:  = × − × × × 663 645chl (12.7 ) (2.69 ) /1000a a a v w  = × − × × × 645 663chl (22.9 ) (4.68 ) /1000b a a v w  + = × + × × × 645 663chl (20.2 8.02 ) / (1000 )a b a a v w carotenoids = × − × − × × × 4701000 2.27 chl 81.4 chl 227 1000 a a b v w where: a ‒ absorbance at specific wavelengh v ‒ final volume of chlolophyll extract in 80% acetone w ‒ fresh weight of tissue extracted free proline concentration free proline concentration was estimated according to bates et al. (1973). in this method, 0.5 g of frozen leaf samples was extracted with 10 ml of 3% (w/v) sulfosalicylic acid; 2 ml of an aliquot of the supernatant was mixed with 2 ml of acetic acid and 2 ml of ninhydrin acid incubated for 40 minutes at 100 °c. the reaction was stopped in an ice bath and the reaction mixture was obtained with 4 ml of toluene and absorbance of the top layer was measured at 520 nm. proline concentration was calculated by a standard curve, ranging from 0 to 400 µg ml-1 that was plotted with l-proline. free proline concentration in tissue was calculated as: proline (mmol g–1) = (mmol proline/ml) ´ (ml toluene/115) ´ 5/w malondialdehyde (mda) content the extent of lipid peroxidation was evaluated as malondialdehyde (mda) content. 100 mg leaf tissue was extracted in 2 ml 0.1% (w/v) trichloroacetic acid (tca) and centrifuged at 12000 g for 15 min and then 0.5 ml of the upper phase was mixed with 1.5 ml tca 20% (w/v) containing 0.5% (w/v) thiobarbituric acid (tba). the mixture was heated for 90 min at 90 °c and then rapidly cooled in an ice bath. afterwards, the mixture was centrifuged at 10000 g for 5 min and the absorbance (a) of the supernatant was recorded at 532 and 600 nm. the mda content in tissue was calculated by an extinction coefficient of 155 mm-1 cm-1 as nmol g-1 (chakhchar et al. 2015): mda (nmol g–1 fw) = − × × ×532 600 1000 155 a a v d w where: v ‒ final volume of extract w ‒ fresh weight of tissue extracted d ‒ dilution factor enzyme activities 100 mg fresh leaves was ground in liquid nitrogen using a mortar and pestle, and the ground samples were homogenized with 1 ml 50 mm sodium phosphate buffer at neutral ph containing 2 mm α-dithiothreitol, 2 mm edta, 0.2% triton x-100, 50 mm tris-hydrochloric acid and 2% polyvinylpyrrolidone. the homogenate was centrifuged at responses to water deficit in acer velutinum and alnus subcordata acta bot. croat. 82 (1), 2023 63 14000 g for 30 min at 4 °c and the supernatant was collected and stored at −80 °c for sod and pod activity analysis (yang and miao 2010, ghaffari et al. 2019). sod activity (ec 1.15.1.1) was evaluated by inhibition ability of the photochemical reduction of nitroblue tetrazolium (nbt) reduction to formazan by o2•−. one unit of sod was considered as the amount of enzyme required to cause 50% inhibition of nbt photochemical reduction which can be measured at 560 nm (giannopolitis et al. 1977). guaiacol peroxidase activity (pod) (ec 1.11.1.7) was assayed according to the guaiacol method (plewa et al. 1991). pod catalyzes guaiacol to tetraguaiacol by h2o2. absorbance was read at 465 nm for 2 min. the calculation were done through the following formulas:   =   mol pod activity g fw min m − × − × × 465 465 t s a (t2) a (t1) v t2 t1 e v w where: a – absorbance at specific wavelength vt – total volume vs – enzyme volume e – extinction coefficient   =   u sod activity g fw −  − ×   × (od control od sample) 100 100 od control 50 w where: od controlabsorbance in the absence of sod od sampleabsorbance in the presence of sod. statistical analysis a randomized complete design was employed with three replications (n = 3). first, the variables were analyzed using one-way anova (analysis of variances) with water supply regimes as factors for each species, then the main effects of drought stress and species and their interactions were determined by two-way anova. when significant differences occurred among treatments, means were separated by duncan’s multiple range tests at p ≤ 0.05. pearson’s correlation coefficients were used to calculate the bivariate relationships between some morphophysiological and biochemical traits. results growth parameters the highest plant growth parameters (height, basal diameter, total and organ biomass and leaf area) were observed in the well-watered 100% fc treatment, while drought treatments significantly decreased plant height, basal diameter, total and organ biomass in both species (p ≤ 0.05). plant height decreased by 30.9, 26.6 and 16.9% when exposed to 25, 50 and 75% fc in a. subcordata respectively, and 23.3 and 17.8% in a. velutinum at 25 and 50% fc treatments, respectively in comparison with control treatment. basal diameter decreased by 29.2, 32.7 and 13.8% at 25, 50 and 75% fc treatments in a. subcordata respectively, and 19.8% at 25% fc in a. velutinum, compared to control condition. biomass traits showed a decreasing trend in both species under water treatment; namely, leaf biomass reduction was 79.1 and 80.8%, that of stem biomass was 40.5 and 75.8%, root biomass 60.9 and 64.2%, and finally total biomass 61.6 and 64.2% at 25% fc in a. velutinum and a. subcordata respectively compared to control condition (tab. 1 and tab. 2). drought stress significantly decreased leaf area in both species. leaf area decreased 71.9 and 83.6% in a. velutinum and a. subcordata, respectively, when exposed to 25% fc. specific leaf area tended to increase with decreasing soil water contents and significantly increased by 70.9% when exposed to 50% fc in a. velutinum. in contrast, it decreased 19.3, 26.3 and 49.6% in a. subcordata at 75, 50 and 25% fc, tab. 1. effect of drought stress on height, basal diameter, leaf area (la), special leaf area (sla), and leaf area ratio (lar) of a. velutinum and a. subcordata seedlings. values are means of three replicates ± standard deviation (sd). different capital letters indicate significant (p ≤ 0.05) differences between a. velutinum and a. subcordata subjected to the same treatment. different lowercase letters indicate significant (p ≤ 0.05) differences among different treatments applied to the same species. fs: species effect, fd: drought effect, fs×fd: species × drought interaction effect. *, **, and ***: significant at p ≤ 0.05, 0.01, and 0.001, respectively. field capacity (fc, %) plant height (cm) basal diameter (mm) leaf area (cm2) special leaf area (cm2 g–1) leaf area ratio (cm2 g–1) acer velutinum 100 52.75±1.96da 14.51±0.37bca 123.40±11.21ba 117.23±5.22eb 30.51±1.53ba 75 50.75±1.24da 14.06±0.52ca 78.55±3.41cb 131.84±10.34deb 24.49±0.85bcb 50 43.33±1.44db 13.35±0.59cda 60.80±4.30cb 200.35±14.53ca 29.00±0.21ba 25 40.44±2.23db 11.63±0.38db 34.62±0.81dc 126.79±11.11deb 17.74±1.49cc alnus subcordata 100 132.67±7.97aa 18.69±0.80aa 153.66±12.50aa 356.28±22.99aa 57.95±3.74aa 75 110.12±5.54bb 16.11±0.87bb 78.94±7.08cb 287.35±5.33bab 57.46±2.35aa 50 97.33±6.32cab 12.58±0.62cdc 37.36±3.90dc 262.53±30.17bb 31.78±7.17bb 25 90.89±5.37cc 13.23±0.70cdc 25.13±2.39dc 179.33±0.05cdc 18.70±0.90cb fs 328.49*** 15.95*** 0.01ns 98.39*** 53.38*** fd 13.68*** 65.12*** 93.90*** 8.89** 28.64*** fs × fd 3.98* 18.00** 5.40** 11.69*** 14.17*** ravanbakhsh m., babakhani b., ghasemnezhad m., serpooshan f., biglouie m. h. 64 acta bot. croat. 82 (1), 2023 respectively. leaf area ratio significantly decreased by 41.85 and 67.7% at 25% fc in a. velutinum and a. subcordata, respectively (tab. 1). the biomass contribution was significantly affected by changes in water availability. r/s increased by 45 and 53.3% in a. subcordata under moderate and severe treatments, while no significant difference among drought treatments was found in a. velutinum. rmr increased with reduced water availability in a. subcordata. the enhancement was 24.4% at 50% fc and 28.2% at 25% fc in comparison with control treatment, whereas no significant diffrence was observed in a. velutinum. drought stress markedly decreased lmr by 45.9 and 44.1% when exposed to 25 and 50% fc in a. velutinum respectively, and 32.1 and 27.3% in a. subcordata in the 25 and 50% fc treatments, respectively in comparison with control treatment. smr in a. velutinum significantly increased in all treatments in comparison with control treatment, while it showed a reduction tendency in a. subcordata (tab. 3). relative water content and photosynthetic pigment content rwc showed significant decreases of 24.9 and 33.5% respectively at 50 and 25% fc in a. subcordata, whereas in a. velutinum the only significant decrease was of 27.3% at 25% fc compared with the well-watered seedlings (tab. 4). chl a content was reduced by 24 and 28% at 50 and 25% fc in a. velutinum, respectively, and 21.9, 60.9 and 53.3% in a. subcordata in the 75, 50 and 25% fc treatments, respectively, compared to control condition. chl b content detab. 2. effect of drought stress on biomass in a. velutinum and a. subcordata seedlings. values are means of three replicates ± standard deviation (sd). different capital letters indicate significant (p ≤ 0.05) differences between a. velutinum and a. subcordata applied to the same treatment. different lowercase letters indicate significant (p ≤ 0.05) differences among different treatments applied to the same species. fs: species effect, fd: drought effect, fs×fd: species × drought interaction effect. *, **, and ***: significant at p ≤ 0.05, 0.01, and 0.001, respectively. field capacity (fc, %) root biomass (g) leaf biomass (g) stem biomass (g) total biomass (g) acer velutinum 100 33.33±3.38ba 16.00±0.58ba 12.33±1.20dea 61.67±3.76ca 75 22.67±1.45cdb 8.00±0.58cb 12.00±0.58dea 42.67±1.45deb 50 13.00±0.58ec 3.67±0.33dc 8.33±0.33deb 25.00±0.58fgc 25 13.00±0.58ec 3.33±0.88dc 7.33±0.33eb 23.67±1.45gc alnus subcordata 100 46.66±2.90aa 20.33±0.66aa 58.00±4.16aa 125.00±4.00aa 75 27.33±1.66bb 14.33±1.20bb 30.00±3.05bb 71.66±5.48bb 50 21.33±2.02cdbc 5.33±0.33dc 19.00±1.15cc 45.66±2.40dc 25 16.66±2.18dec 3.90±0.92dc 14.00±0.57cdc 34.56±3.47efc fs 26.21*** 44.43*** 211.64*** 185.72 *** fd 60.19*** 195.72*** 61.62*** 162.89*** fs × fd 2.23ns 7.25 ** 39.88 *** 25.16*** tab. 3. effect of drought stress on biomass partitioning rate of a. velutinum and a. subcordata seedlings. values are means of three replicates ± standard deviation (sd). different capital letters indicate significant (p ≤ 0.05) differences between a. velutinum and a. subcordata subjected to the same treatment. different lowercase letters indicate significant (p ≤ 0.05) differences among different treatments applied to the same species. fs: species effect, fd: drought effect, fs×fd: species × drought interaction effect. *, **, and ***: significant at p ≤ 0.05, 0.01, and 0.001, respectively. field capacity (%) root to shoot ratio (r/s) leaf mass ratio (lmr) stem mass ratio (smr) root mass ratio (rmr) acer velutinum 100 1.18±0.12aa 26.19±2.12aa 19.96±1.33ca 53.85±2.60aba 75 1.13±0.07aba 18.73±1.09bcb 28.25±2.14bb 53.01±1.62aba 50 1.09±0.08abca 14.62±1.07cdeb 33.36±1.50bb 52.01±2.00abca 25 1.22±0.05aa 14.16±1.48deb 30.78±1.92bb 55.05±1.04aa alnus subcordata 100 0.60±0.06db 16.26±0.01bcda 46.33±2.42aa 37.39±2.43db 75 0.62±0.03db 19.97±0.44ba 41.75±1.60aa 38.26±1.31db 50 0.87±0.08ca 11.82±1.40eb 41.64±2.25aa 46.53±2.25ca 25 0.92±0.05bca 11.03±1.80eb 41.03±2.91aa 47.94±1.37bca fs 56.18 *** 14.74 *** 105.59 *** 65.76 *** fd 2.90 ns 20.74 *** 1.63 ns 4.58* fs × fd 2.58 ns 5.90** 8.20 ** 4.09 * responses to water deficit in acer velutinum and alnus subcordata acta bot. croat. 82 (1), 2023 65 creased 20.4 and 53% in a. velutinum and 56.8 and 52% in a. subcordata at 50 and 25% fc respectively. chl a+b decreased by 20, 60 and 53.3% when exposed to 75, 50 and 25% fc in a. subcordata, respectively, and 25.5 and 36.2% in a. velutinum in the 25 and 50% fc treatments respectively, in comparison with control treatment. the content of carotenoids significantly decreased under drought in a. subcordata, where the reduction was 50 and 38.5% at 50 and 25% fc, whereas a. velutinum showed a tendency to increase in carotenoids under drought stress (tab. 4). biochemical responses in the leaves of both species, increase in proline content was recorded upon stress treatments. proline content in a. velutinum leaves increased 22.1 and 132.6% at 75 and 50% fc, respectively and 136.8% at 25% fc. in a. subcordata the increase was 34.9 and 62.2% at 75 and 50% fc, respectiely and 169.8% at 25% fc in comparison with control treatment (fig. 1a). the mda content increased substantially as drought stress progressed in both species. in a. subcordata the increase was 93.7 and 133.8% at 75 and 50% fc, respectab. 4. effect of drought stress on photosynthetic pigments content, and rwc of a. velutinum and a. subcordata seedlings. values are means of three replicates ± standard deviation (sd). different capital letters indicate significant (p ≤ 0.05) differences between a. velutinum and a. subcordata subjected to the same treatment. different lowercase letters indicate significant (p ≤ 0.05) differences among different treatments applied to the same species. fw: fresh weight, rwc: relative water content; fs: species effect, fd: drought effect, fs×fd: species × drought interaction effect. *, **, and ***: significant at p ≤ 0.05, 0.01, and 0.001, respectively. field capacity (%) chlorophyll a (mg g–1 fw) chlorophyll b (mg g–1 fw) total chlorophyll (mg g–1 fw) total carotenoids (mg g–1 fw) rwc (%) acer velutinum 100 1.00±0.05abab 0.49±0.13aab 1.49±0.07abab 0.18±0.02bcda 72.58±3.82aa 75 1.30±0.20aa 0.53±0.03aa 1.84±0.23aa 0.22±0.03abca 75.08±2.66aa 50 0.76±0.07bcb 0.39±0.09abcab 1.11±0.01cbc 0.25±0.02aba 69.20±3.82aa 25 0.72±0.10bcdb 0.23±0.03bcb 0.95±0.12cdc 0.25±0.02aba 52.77±1.36bb alnus subcordata 100 1.05±0.08aba 0.44±0.03aa 1.50±0.06aa 0.26±0.02aa 70.38±3.00aa 75 0.82±0.12bcb 0.40±0.06aba 1.23±0.11bcb 0.21±0.00abcb 67.61±2.73aa 50 0.41±0.00dc 0.19±0.01cb 0.60±0.02dc 0.13±0.0 dc 52.83±3.08bb 25 0.49±0.04cdc 0.21±0.02bcb 0.70±0.07dc 0.16±0.00cdc 46.77±3.08bb fs 11.55** 5.21 * 18.36 ** 5.54 * 13.99** fd 12.69 *** 8.13 ** 23.72 *** 0.82 ns 23.10 *** fs × fd 2.42 ns 0.81 ns 3.03ns 7.69 ** 1.94 ns fig. 1. changes in proline (a), malondialdehyde (mda) (b), superoxide dismutase (sod) (c) and guaiacol peroxidase (pod) measured in leaves from a. velutinum and a. subcordata seedlings subjected to four drought treatments (100, 75, 50 and 25% of field capacity – fc). values are means of three replicates ± standard deviation (sd). different capital letters indicate significant (p ≤ 0.05) differences between a. velutinum and a. subcordata subjected to the same treatment. different lowercase letters indicate significant (p ≤ 0.05) differences among the different treatments to which the same species were subjected. ravanbakhsh m., babakhani b., ghasemnezhad m., serpooshan f., biglouie m. h. 66 acta bot. croat. 82 (1), 2023 tively and 142.7% at 25%, whereas in a. velutinum the increase was 60.5 and 65% at 50 and 25% fc (fig. 1b). in a. velutinum, sod activity increased 12 and 8.9% at 50 and 25% fc, respectively. in a. subcordata, sod activity was significantly increased by 36, 25 and 20.9% at 75, 50 and 25% fc, respectively (fig. 1c). pod activity in a. velutinum increased by 113 and 327% at 75 and 50% fc , respectively and 40% at 25% fc, whereas the values in a. subcordata were increased by 148 and 140% at 75 and 50% fc, respectively (fig. 1d). correlation analysis correlation analysis indicated that there was a significant and positive correlation between sla and chl a, chl b and chl a+b in a. subcordata, but there was no significant correlation between sla and chl concentration in a. velutinum. correlation analysis revealed that there was a significant and positive correlation between sod and pod activities also, between proline and chl a, chl a+b in both species. according to correlation analysis there was no significant correlation between rwc and proline in a. velutinum but also, there was a negative correlation between rwc and proline in a. subcordata. correlation analysis also revealed that there was a significant and positive correlation between carotenoid content and sod activity in a. velutinum (tab. 5 and tab. 6). discussion drought stress is the most adverse abiotic stress to plant growth. permanent or temporary water shortage causes detrimental effects on plant growth and development (tariq et al. 2018; du et al. 2019). height, total and organ biomass of both species signifcantly declined under moderate and severe treatments (50 and 25% fc) in comparison with control treatment. basal diameter signifcantly decreased under moderate and severe treatments (50 and 25% fc) in a. subcordata and just reduced under severe treatments (25% fc) in a. velutinum. these results are in accordance with tab. 5. correlation analysis among some morphophysiological and biochemical traits in acer velutinum under drought stress conditions. each square indicates the pearson correlation coefficient of a pair of parameters. leaf area: la, specific leaf area: sla, relative water content: rwc, chlorophyll a: chl a, chlorophyll b: chl b, total chlorophyll: chl a+b, and carotenoids: car, free proline: pro, malondialdehyde: mda, peroxidase: pod, superoxide dismutase: sod. ** and * indicate a significant correlation between control and drought treatments at p ≤ 0.01 and p ≤ 0.05, respectively. la sla car sod pod mda pro rwc chl a chl b chl a+b la 1.000 –0.293 –0.476 –0.695* –0.306 –0.719** –0.803** 0.607* 0.466 0.426 0.524 sla 1.000 0.300 0.632* 0.779** 0.323 0.427 0.047 –0.256 –0.196 –0.294 car 1.000 0.605* 0.365 0.343 0.415 –0.213 –0.096 –0.229 –0.181 sod 1.000 0.624* 0.638* 0.771** –0.150 –0.224 –0.298 –0.314 pod 1.000 0.366 0.487 0.151 –0.332 –0.042 –0.293 mda 1.000 0.910** –0.524 –0.585* –0.523 –0.651* pro 1.000 –0.540 –0.651* –0.420 –0.667* rwc 1.000 0.596* 0.768** 0.735** chl a 1.000 0.473 0.939** chl b 1.000 0.745** chl a+b 1.000 tab. 6. correlation analysis among some morphophysiological and biochemical traits in alnus subcordata under drought. each square indicates the pearson correlation coefficient of a pair of parameters. leaf area: la, specific leaf area: sla, relative water content: rwc, chlorophyll a: chl a, chlorophyll b: chl b, total chlorophyll: chl a+b, and carotenoids: car, free proline: pro, malondialdehyde: mda, peroxidase: pod, superoxide dismutase: sod. ** and * indicate a significant correlation between control and drought treatments at p ≤ 0.01 and p ≤ 0.05, respectively. la sla car sod pod mda pro rwc chl a chl b chl a+b la 1.000 0.836** 0.873** –0.566 –0.314 –0.826** –0.757** 0.775** 0.869** 0.758** 0.889** sla 1.000 0.648* –0.300 0.094 –0.797** –0.746** 0.746** 0.632* 0.607* 0.665* car –0.559 –0.330 –0.716** –0.626* 0.799** 0.944** 0.698* 0.925** sod 1.000 0.705* 0.444 0.295 –0.250 –0.538 –0.210 –0.466 pod 1.000 0.281 –0.191 0.113 –0.309 –0.182 –0.288 mda 1.000 0.543 –0.702* –0.630* –0.697* –0.692* pro 1.000 –0.812** –0.688* –0.655* –0.721** rwc 1.000 0.749** 0.684* 0.776** chl a 1.000 0.725** 0.974** chl b 1.000 0.861** chl a+b 1.000 responses to water deficit in acer velutinum and alnus subcordata acta bot. croat. 82 (1), 2023 67 previous studies on salix paraqplesia and hippophae rhamnoides (fang et al. 2012) as well as prunus sargentii and larix kaempferi seedlings (bhusal et al. 2020) which demonstrated that drought significantly reduced seedling growth and biomass. we found that drought treatment significantly increased the r/s and rmr in a. subcordata. it was statistically ineffective in a. velutinum. the increase in r/s is the result of declining growth rate and biomass production and increased water uptake (wu et al. 2008, du et al. 2010). many studies have shown that there is an increase in r/s ratio under water stress (fang et al. 2012; guo et al. 2019, zhang et al. 2019). more biomass allocation to belowground organs and maintainance of higher r/s can be indicated as an important adaptive trait (fang et al. 2012). in the present study, drought decreased la in both species under drought stress. sla showed an increasing trend in a. velutinum under drought stress treatments. however, it decreased in all drought treatments in a. subcordata. also, lar significantly decreased under drought conditions in both species. decreased la usually occurs due to inhibition of leaf development, loss of access to photosynthetic products to make new cells (tariq et al. 2018). some plant species adjust la to prevent transpiration or a relative increase in root water uptake capacity (guo et al. 2019). sla and lar increased under severe stress compared to the control in jatropha curcas seedlings, which is considered a drought-tolerant plant (díaz-lópez et al. 2012). in our study, a. velutinum significantly increased the sla under moderate treatment (50% fc), which indicates that it probably has been able to cope with drought stress by increasing photosynthetic capacity and carbon assimilation (wu et al. 2017, barros et al. 2020). correlation analysis indicated that there was a significant and positive correlation between sla and chl a, chl b and chl a+b in a. subcordata, but there was no significant correlation between sla and chl concentration in a.velutinum. we found that chl a, chl b, and chl a+b content significantly decreased under drought stress in both species. a. velutinum had a higher chlorophyll content (chl a, chl b, and chl a+b) than a. subcordata under moderate and severe treatment (50 nd 25% fc). according lei et al. (2006), the dry climate population of populus przewalskii had higher chlorophyll content than the wet climate population under the drought treatment. drought stress also significantly decreased chlorophyll content of juglans mandshurica, juglans nigra and juglans regia seedlings (liu et al. 2019). our results also showed that the carotenoid content was not significantly increased by drought in a. velutinum, while it was significantly decreased under moderate and severe treatment (50 and 25% fc) in a. subcordata. reduction of carotenoids suggested that drought stress caused noticeable oxidative stress by ros accumulation (lei et al. 2006). the slight increase in carotenoid content in a. velutinum could suppress photosynthetic apparatus damage by oxygen consumption in xanthophyll cycle or detoxification of ros (ashraf and harris, 2013, medeiros et al. 2013). correlation analysis also revealed that there was a significant and positive correlation between carotenoids content and sod activity in a. velutinum. in our study, a. velutinum seedlings showed a decline in rwc only under the severe treatment (25% fc), whereas a. subcordata showed a significant decrease in the moderate and severe treatments (50 and 25% fc, respectively). díazlópez et al. (2012) indicated that jatropha curcas can be considered a drought-resistant species as it has been able to sustain its rwc level under mild to severe stress drought treatments. moreover, ying et al. (2015) suggested that provenance kunming (km) had higher rwc than provenance nanchang (nc) of camptotheca acuminate under moderate and severe treatments (50 and 30% fc) and exhibited greater drought stress tolerance as expected given the natural habitat of this provenance. proline content of both the species, investigated in this study, was significantly increased under drought treatments with respect to the well-watered plants although the higher increase was recorded in a. velutinum comparied to a. subcordata under moderate treatment (50% fc), whereas the increment was significantly greater in a. subcordata than in a. velutinum under the severe treatment (25% fc). according to correlation analysis, there was no significant correlation between rwc and proline content in a. velutinum, while negative correlation between rwc and proline was recorded in a. subcordata. ashrafi et al. (2018) reported a negative correlation between rwc and osmoprotectants in thymus vulgaris and t. kotschyanus, and found that osmoprotectants accumulate by reduction of rwc to maintain plant water. similarly, bangar et al. (2019) found that proline content was negatively associated with rwc in vigna radiate. mda is a product of poly-unsaturated fatty acid degeneration in phospholipids of cellular membrane, and is used as an index of oxidative stress magnitude under drought (wang et al. 2012, guo et al. 2018). mda content increased along with the drought stress in both species in this study. the significant increase of mda content with progressive drought stress, suggests that drought stress caused oxidative damage. our results, according to wu et al. (2013) in quercus variabilis and tariq et al. (2018) in alnus cremastogyne subjected to drought stress, showed an increase of mda content. in a. velutinum, the values increased under moderate and severe treatment (50 and 25% fc), while in a. subcordata mda content was elevated upon all drought treatments. the increases in mda content in a. velutinum were lower than those in a. subcordata. this indicated that drought led to more damage in the cellular membranes under stress treatments in a. subcordata. similarly, ying et al. (2015) found that drought stress significantly increased mda content in camptotheca acuminata provenance km and nc and the increases in mda content in provenance km were lower than those in provenance nc. they suggested that the less production of ros in provenance km under water deficit led to better membrane integrity. the ability of antioxidant enzymes to eliminate ros and reduce its harmful effects may be related to plant ravanbakhsh m., babakhani b., ghasemnezhad m., serpooshan f., biglouie m. h. 68 acta bot. croat. 82 (1), 2023 drought resistance (anjum et al. 2011). high accumulation of ros initiated and accelerated lipid peroxidation. pod plays an essential role in reducing the accumulation of h2o2, reducing mda content and maintaining cell membrane integrity. increased sod and pod activity in stress treatments reflects an increase in ros removal capacity and thus a reduction in membrane lipid damage (ge et al. 2014, guo et al. 2018). toscano et al. (2016) suggested that eugenia uniflora and photinia × fraseri subjected to mild and moderate water stress showed increasing activities of antioxidant enzymes. we found that drought stress induced pod and sod activity in both species under drought treatments in our study, although the highest activities were measured under mild and moderate treatments (75 and 50% fc) compared to the control. our results are in good accordance with those published by ge et al. (2014), who reported an increase of pod and sod activities in phoebe bournei subjected to mild and moderate water stress and a decrease under severe drought. in addition, ge et al. (2014) demonstrated that the increase in mda content acts as a feedback mechanism to control the activities of antioxidant enzymes. in our study, a. veltinum showed higher pod activity and lower increment of mda than a. subcordata under the moderate and severe treatments. similarly, wang et al. (2012) found that a stronger protective mechanism by a drought-tolerant apple rootstock (malus prunifolia) than in a sensitive-tolerant apple rootstock (malus hupehensis) can be ascribed to lower mda content, higher values for leaf rwc, and greater antioxidative defense system. wu et al. (2013) has also shown that the mda content at 60% fc treatment kept a lower increase compared with 40 and 20% fc treatments, indicating better protection against membranes lipid peroxidation, more efficient repairing mechanisms, including the antioxidative system, osmotic adjustment, and photosynthetic pigments in quercus variabilis seedlings. conclusion the present study concluded that although there were common responses in investigated parameters between two hyrcanian endemic species i.e., a. velutinum and a. subcordata, certain different responses were also recorded under drought stress. our results demonstrated that drought stress significantly reduced growth, biomass and photosynthetic pigment content, but increased free proline content, pod and sod activities in both species. a. velutinum showed a slight reduction in seedlings height, basal diameter, biomass and had higher rwc and photosynthetic pigment than a. subcordata. a. velutinum also showed more efficient antioxidant systems with higher activities of pod, and a lower increase in mda content under drought stress. our results highlight that a. velutinum maintained stronger drought tolerance based on the measured parameters. according to these findings, it is recommended that a. velutinum plantation should have priority over a. subcordata in water deficit regions. references abdolahi, a., ali arab, a. r., parhizkar, p., pourmalekshah, a. a. m. a., 2017: effect of gap size and position within gaps on growth characters and survival of chestnut-leaved oak (quercus castaneifolia c. a. mey.), cappadocian maple (acer cappadocicum gled.) and caucasian alder (alnus subcordata c. a. mey.). iranian journal of forest and poplar research 25, 275–285. 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(campanulaceae) ivana b. janković1, milica m. drobac2, dmitar v. lakušić1* 1 institute of botany and botanical garden, faculty of biology, university of belgrade, takovska 43, 11000 belgrade, serbia 2 department of pharmacognosy, faculty of pharmacy, university of belgrade, vojvode stepe 450, 11000 belgrade, serbia abstract – during the past few years, the isophylloid campanula pyramidalis complex has been the subject of studies aimed at an improved understanding of the relationships within it. the center of distribution of the c. pyramidalis complex is in the balkan peninsula with some smaller parts of the area located in the south apennines. although 21 taxa of the c. pyramidalis complex were described, only four species are accepted: c. pyramidalis, c. versicolor, c. secundifl ora and c. austroadriatica. in the present study, we propose compounds of the methanolic leaf extract as possible chemotaxonomic markers for the c. pyramidalis complex. eleven fl avonoids and two phenolic acids were detected in leaf extract using high-performance liquid chromatography with diode-array detection analysis. the investigated taxa of the c. pyramidalis complex differ in terms of the composition of the methanolic leaf extract. clustering of investigated taxa is not completely consistent with the previously reported molecular and morphometric data. keywords: campanula pyramidalis, chemotaxonomy, fl avonoids, methanolic leaf extract, phenolic acids introduction many studies have been performed to elucidate some of the complicated relationships that exist within the campanulaceae family. however, due to the large number of taxa it is hard to make a comprehensive study and a single classifi cation system. endemic or rare campanula, as well as smaller groups, aggregates and complexes are at the focus of interests, since usually they are molecularly, morphologically, karyologically, and biogeographi* corresponding author, e-mail: dlakusic@bio.bg.ac.rs copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. janković i. b., drobac m. m., lakušić d. v. 482 acta bot. croat. 73 (2), 2014 cally distinctive (kovanda and ančev 1989, eddie and ingrouille 1999, eddie et al. 2003, shulkina et al. 2003, cosner et al. 2004, nikolov 2005, kovačić and nikolić 2006, cupido et al. 2011, mansion et al. 2012, stamenković et al. 2012). such a group is constituted by the balkan »isophylloid« bellfl owers (kovačić 2004, tkalec et al. 2004) that belong to the campanula pyramidalis complex also informally referred to as the »pyramidalis« aggregate (geslot 1984) or subsection pyramidalis (kolakovsky 1992). the balkan peninsula is the center of the distribution of the c. pyramidalis complex with some small disjunct parts of the range that lay in the south apennines (fig 1.) (lakušić et al. 2013). the campanula pyramidalis complex is traditionally represented with three generally accepted species campanula pyramidalis linnaeus (1753: 164), campanula versicolor andrews (1804: 396) and campanula secundifl ora visiani et pančić (1862: 20) (fedorov and kovanda 1976, greuter et al. 1984). according to the results of molecular phylogenetic study, the south adriatic populations represent separate species, recently described as new – campanula austroadriatica d. lakušić et kovačić (2013: 519) (lakušić et al. 2013). apart from the above mentioned species, there are 13 taxa at specifi c and at intraspecfi c level and four hybrids described within the c. pyramidalis complex (lakušić et al. 2013). however, they are not geographically and taxonomically well-defi ned (lakušić et al. 2013). in this paper we rely on groups of populations that are presented with fi ve clades (p. 1, p. 2, p. 3, s, v), well-supported on the phylogenetic networks and trees (lakušić et al. 2013): p. 1 clade − c. pyramidalis s. str. − northern and central adriatic coast (fig. 1), p. 2 clade − c. austroadriatica sp. nova – southern adriatic coast from the neretva river canyon to northern albania (fig. 1), p. 3 clade − c. »montenegrina« prov. − populations from the continental part of montenegro traditionally considered as part of c. pyramidalis s. lato (fig. 1). molecularly this clade is much closer to c. secundifl ora. therefore, we apply the concept of r. lakušić, who fig. 1. distribution of the campanula pyramidalis complex in the investigated area. symbols p.1–3, s and v correspond to molecular clades (lakušić et al. 2013). the methanolic leaf extract of the c. pyramidalis complex acta bot. croat. 73 (2), 2014 483 originally recognized these groups of populations as c. secundifl ora subsp. montenegrina r. lakušić (lakušić et al. 2013), s clade − c. secundifl ora s. lato − which includes c. secundifl ora subsp. secundifl ora from the gorge of the panjica river in south-west serbia, and c. secundifl ora subsp. limensis r. lakušić (lakušić et al. 2013) from the canyons of the lim and the mileševka in south-west serbia and northern montenegro (fig. 1), and v clade − c. versicolor − southern parts of the balkan peninsula (macedonia, albania, greece, bulgaria) (fig. 1). previous phytochemical studies on the plants of the genus campanula revealed the presence of different secondary metabolites such as fl avonoids, phenolic acids, anthocyanins and triterpenoids (cuendet et al. 2001, yayli et al. 2003, yayli et al. 2005, touafek et al. 2011). the presence of volatile oils in some campanula species was described as well (tosun et al. 2011, kadriye et al. 2012, politeo et al. 2013). isoenzyme variability among nine campanula taxa from croatia and bosnia and herzegovina, including campanula pyramidalis, has been studied (tkalec et al. 2004). the main goal of our present phytochemical analysis is to investigate the possibility of the use compounds of the methanolic leaf extract as possible chemotaxonomic markers for the c. pyramidalis complex. furthermore, our goal is to determine if phytochemical data provide any correspondence with and support for the establishment of currently recognized or previously described taxa within this complex, especially for the unrecognized taxa c. »montenegrina« and c. »limensis«. materials and methods plant material the aerial parts of campanula species were collected from different locations in croatia, serbia, montenegro and macedonia (tab. 1, fig. 1). voucher specimens of each sample have been deposited in the herbarium of the institute of botany and botanical garden (beou), faculty of biology, university of belgrade (tab. 1). in addition to the formal names c. pyramidalis, c. versicolor, c. secundifl ora and c. austroadriatica that are registered in the international plant names index (ipni), in this paper we have used informal names c. »montenegrina« and c. »limensis«. these informal names are used in order to emphasize morphological (janković et al. 2013) and molecular specifi cities of the population from the canyon of the morača river and the mountains in its surroundings (c. »montenegrina«), as well as the populations from the canyons of the rivers lim and mileševka (c. »limensis«) (lakušić et al. 2013). phytochemical analysis the air-dried and fi nely ground leaves were extracted twice with methanol (1:15 w/v) using an ultrasonic bath for 30 min, left to stand for 24 h and fi ltered. the extract was combined, concentrated in a rotary evaporator under reduced pressure for total solvent removal and dissolved in methanol prior to analysis in the concentration of 10 mg ml–1. high-performance liquid chromatography (hplc) analysis was performed on an agilent 1100 series system consisting of a g 1312a binary pump, a g1328b injector (20 μl janković i. b., drobac m. m., lakušić d. v. 484 acta bot. croat. 73 (2), 2014 ta b. 1 . si te s, v ou ch er n um be rs , d at es o f co lle ct in g an d cl ad es ( ac co rd in g to l ak uš ić e t a l. 20 13 ) of e xa m in ed p op ul at io ns o f th e c am pa nu la p yr am id al is co m pl ex . c om po un ds p re se nt in th e m et ha no lic le af e xt ra ct s: 1 ) l ut eo lin -7 -o -h et er os id e, 2 ) c hl or og en ic a ci d, 3 ) l ut eo lin 7o -r ut in os id e, 4 ) l ut eo lin 7o -g lu co si de , 5 ) fl a vo no id 1 ( lu te ol in d er iv at iv e) , 6 ) fl a vo no id 2 ( 7o he te ro si de o f lu te ol in o r its d er iv at iv e) , 7 ) ap ig en in -7 -o -g lu co si de , 8 ) fl a vo no id 3 ( ap ig en in d er iv at iv e) , 9 ) ap ig en in -7 -o -d ih et er os id e, 1 0) fl av on oi d 4 (h et er os id e of a pi ge ni n or it s de ri va tiv e) , 1 1) fl av on oi d 5 (a pi ge ni n de ri va tiv e) , 1 2) lu te ol in , 1 3) c af fe ic a ci d. c om po un ds a re m ar ke d as p re se nt (+ ) o r a bs en t ( –) . c la de s ta xo n si te vo uc he r d at e c om po un ds 1 2 3 4 5 6 7 8 9 10 11 12 13 σ p. 1 c . p yr am id al is v el eb it 30 29 7 18 .1 1. 20 09 . + – – – – – – – – – – – + 2 p. 2 c . a us tr oa dr ia tic a o ri je n 30 29 8 18 .1 1. 20 09 . + – – – – – – – – – – – – 1 p. 2 c . a us tr oa dr ia tic a r is an 30 29 1 28 .1 1. 20 09 . + + – – – – – – – – – – – 2 p. 2 c . a us tr oa dr ia tic a o ra ho va c 30 28 9 28 .1 1. 20 09 . + + – – – – – – – – – – – 2 p. 2 c . a us tr oa dr ia tic a b ud va 30 28 8 28 .1 1. 20 09 . + + – – – – – – – – – – – 2 p. 2 c . a us tr oa dr ia tic a st ar i b ar 30 28 7 28 .1 1. 20 09 . + – + – – – – – – – – – – 2 p. 2 c . a us tr oa dr ia tic a r um ija 30 29 9 30 .1 2. 20 09 . + – + – – – – – – – – – – 2 p. 2 c . a us tr oa dr ia tic a v ir pa za r 30 28 6 28 .1 1. 20 09 . + + + – – – – – – – – – – 3 p. 3 c . » m on te ne gr in a« m or ač a 30 28 5 28 .1 1. 20 09 . + + + + – – – – – – – – – 4 s c . » lim en si s« d ob ru n 30 28 4 28 .1 1. 20 09 . + + – + + + + + + + + + – 11 s c . s ec un di fl o ra pa nj ic a 30 30 0 06 .1 2. 20 09 . – + – + + + + + – – – + – 7 v c . v er si co lo r v el es 27 62 3 06 .0 8. 20 08 . + – + + + + + – – – – – – 6 σ 11 7 5 4 3 3 3 2 1 1 1 2 1 the methanolic leaf extract of the c. pyramidalis complex acta bot. croat. 73 (2), 2014 485 sample loop) and g1315b dad detector, equipped with zorbax eclipse xdb-c18 column (4.6 × 250 mm, 5 μm). a gradient elution was performed with solvent a (0.03% (v/v) h3po4, ph = 2.8) and solvent b (solvent a:acetonitrile = 10:90) as follows: in 0 min, 15% b; in 25 min 25% b; in 30 min 35% b; in 35 min 50% b; in 40 min 70% b and in 45 min 15% b. the column temperature was 25 ºc, fl ow rate 1.0 ml min–1 and the injection volume was 30 μl. the spectra were acquired from 190 to 400 nm. detection was performed at 250, 320, 340, 350 and 370 nm. all analyses were carried out in triplicate. identifi cation of compounds was carried out by comparing their spectra and their retention times with those of standards (luteolin, luteolin 7-o-glucoside, apigenin 7-o-glucoside, chlorogenic acid and caffeic acid) as well as with literature spectral data (mabry et al. 1970, markham 1982). statistical analysis flora software (karadžić et al. 1998) was used for cluster analysis (fig. 2) in order to determine the structure and separation of the populations based on presence / absence of compounds of methanolic leaf extract. populations were classifi ed using the optimal cluster method based on jaccard’s distance as a heterogeneity measure. the relation between populations and components of methanolic leaf extract originated by principal component analysis (pca) was also obtained by using flora software and it is presented in form of bi-plot (fig. 3). fig. 2. cluster analysis for whole data set. symbols p.1–3, s and v correspond to molecular clades (lakušić et al. 2013). janković i. b., drobac m. m., lakušić d. v. 486 acta bot. croat. 73 (2), 2014 results and discussion this study represents one of the fi rst insights into the chemical compounds of the campanula pyramidalis complex. a total of 11 fl avonoids and 2 phenolic acids were detected in the methanolic leaf extract of 12 populations of 6 taxa (tab. 1). the fl avonoids present are fl avones, either free aglycon (luteolin) or heterosides of apigenin, luteolin and its derivatives. luteolin and its glycosides were also identifi ed previously in the genus campanula (teslov 1990, yayli et al. 2003, touafek et al. 2011). concerning the phenolic acids, chlorogenic acid was identifi ed in the methanolic leaf extract of c. austroadriatica, c. »montenegrina«, c. »limensis« and c. secundifl ora whereas caffeic acid was present only in c. pyramidalis. the mentioned acids were previously reported in some species of the genus campanula (c. cephalotes, c. maleevii, c. rotundifolia, c. persicifolia) (teslov and blinova 1973, teslov et al. 1983, teslov and podushkin 1988). the result of the cluster analysis has shown that two main clusters stand out (fig. 2): (i) cluster 1 – c. pyramidalis, c. austroadriatica and c. »montenegrina«; (ii) cluster 2 c. secundifl ora, c. »limensis« and c. versicolor. luteolin 7-o-heteroside is present in almost all populations but absent only in c. secundifl ora. generally, we can conclude that populafig. 3. bi-plot principal component analysis (pca) for populations and compounds detected in the methanolic leaf extract of the campanula pyramidalis complex. numbers from 1 to 13, next to the white rectangles, correspond to number of compounds from table 1. other symbols correspond to clades as explained in fi gure 1. the methanolic leaf extract of the c. pyramidalis complex acta bot. croat. 73 (2), 2014 487 tions are characterized by the regular presence of luteolin 7-o-heteroside and the occasional presence of chlorogenic acid and/or luteolin 7-o-rutinoside (tab. 1). therefore, we can conclude that luteolin 7-o-heteroside, chlorogenic acid and luteolin 7-o-rutinoside are less responsible for separation of taxa than other compounds (fig. 3). a luteolin derivative (labeled as fl avonoid 1 in tab. 1), 7-o-heteroside of luteolin or its derivative (fl avonoid 2 in tab. 1) and apigenin 7-o-glucoside are the compounds that separate cluster 1 from cluster 2. campanula pyramidalis s.s. is unique in producing caffeic acid besides luteolin 7-oheteroside. caffeic acid is a common phenolic constituent of campanulaceae (lammers 2007). luteolin 7-o-glucoside is present in taxa that belong to cluster 2. however, luteolin 7-o-glucoside has also been detected in c. »montenegrina« from cluster 1. the presence of luteolin 7-o-glucoside is another evidence that c. »montenegrina« is associated with c. secundifl ora and c. versicolor as molecular data has showed. campanula secundifl ora and c. »limensis« differ from other taxa by the presence of two fl avonoids, luteolin and an apigenin derivative (fl avonoid 3 in tab. 1). campanula »limensis« is distinguished by the presence of three compounds: apigenin 7-o-diheteroside, heteroside of apigenin or its derivative (fl avonoid 4 in tab. 1) and an apigenin derivative (fl avonoid 5 in tab. 1), which are not found in other taxa examined. the chemical data indicate that within the c. pyramidalis complex the mediterranean and sub-mediterranean populations c. pyramidalis s. s. (p.1), c. austroadriatica (p.2) and c. »montenegrina« (p.3) form one group, while other group is represented by the continental populations of c. secundifl ora (s), c. »limensis« (s) and c. versicolor (v). however, the results of methanolic leaf extract analysis are inconsistent with molecular phylogenetic data. in fact, molecular data has showed that c. pyramidalis s. s. (p.1) is the most distant taxon at genetic level, forming one main cluster, while c. austroadriatica (p.2) forms a second main cluster. the third main cluster is represented by c. »montenegrina« (p.3) and c. secundifl ora (s) (lakušić et al. 2013). especially interesting is the position of c. »montenegrina« (p.3), due to its chemical characteristics nested within cluster 1 with c. pyramidalis s. str. (p.1) and c. austroadriatica (p.2). this relation of c. »montenegrina« (p.3) with other investigated taxa is inconsistent with respect not only to molecular but also to morphometric data, which suggest that c. »montenegrina« (p.3) is part of c. secundifl ora s. l. (janković and lakušić 2011, janković et al. 2013, lakušić et al. 2013). campanula »montenegrina« occupies a transition zone between the adriatic and the continental bellfl owers belonging to the c. pyramidalis complex (janković et al. 2013). also, it inhabits a wider range of altitudes from canyons to high mountains. the chemotaxonomic, molecular phylogenetic and morphometric results emphasize the necessity to investigate the populations that lie in the continental part of montenegro more fully. those populations are morphologically and geographically very specifi c and therefore they require a new taxonomic treatment (lakušić et al. 2013). the second cluster, to which belong c. secundifl ora s. s. (s), c. »limensis« (s) and c. versicolor (v), is differentiated in the same way chemically and molecularly (lakušić et al. 2013). from all taxa investigated in this study, c. »limensis« is highly differentiated in the composition of the methanolic leaf extract by the number of different fl avonoids. eleven different compounds were detected in the methanolic leaf extract of c. »limensis«. it differs even from its closest taxon c. secundifl ora s. s. morphometric data (janković and lakušić 2011, janković et al. 2013) also suggest that c. »limensis« is separate entity and its taxonomic status should be seriously reconsidered in future studies. janković i. b., drobac m. m., lakušić d. v. 488 acta bot. croat. 73 (2), 2014 chemotaxonomic results based on the analysis of methanolic leaf extract demonstrated that investigated taxa of the c. pyramidalis complex are different. therefore, our results suggest that the presence or absence of certain compounds in the methanolic leaf extract can be used as chemotaxonomic markers for further studies of the c. pyramidalis complex. acknowledgements the authors gratefully acknowledge the fi nancial support provided by the serbian ministry of science and technological development (project no. 173021 – examination of potential medicinal plants: morphological, chemical and pharmacological characterization and 173030 – biodiversity of the plant life of serbia and balkan peninsula – assessment, sustainable use and conservation). also, we would like to offer our special thanks to dijana dobrota (magdeburg, germany) for improving the language of the manuscript. references andrews, h. c., 1804–1805: the botanist’s repository 6, 396. cosner, m. e., raubeson, l. a., jansen, r. k., 2004: chloroplast dna rearrangements in campanulaceae: phylogenetic utility of highly rearranged genomes. bmc evolutionary biology 4, 27. cuendet, m., potterat, o., hostettmann, k., 2001: flavonoids and phenylpropanoids derivatives from campanula barbata. phytochemistry 56, 631–636. cupido, c. n., eddie, w. m. m., tiedt, l. r., 2011: systematic and ecological signifi cance of seed coat morphology in south african campanulaceae sensu stricto. edinburgh journal of botany 68, 351–371. eddie, w. m. m., ingrouille, m. j., 1999: polymorphism in the aegean »fi ve-loculed« species of the genus campanula, section quinqueloculares (campanulaceae). nordic journal of botany 19, 153–169. eddie, w. m. m., shulkina, t., gaskin, j., haberle, r. c., jansen, r. k., 2003: phylogeny of campanulaceae s. str. inferred from its sequences of nuclear ribosomal dnk. annals of the missouri botanical garden 90, 334–375. fedorov, a. a., kovanda, m., 1976: campanula l. in: tutin, t. g., burges, n. a., chater, a. o., edmondson, j. r., heywood, v. h., moore, d. m., valentine, d. h., walters, s. m., webb, d. a. 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rariores aut novae. a prof. roberto de visiani et prof. josepho pančić descriptae et iconibus illustratae. decas, i. memorie del reale istituto veneto di scienze 10, 20. yayli, n., yildirim, n., doğan, n., usta, a., altun, l., 2005: triterpenes from campanula lactifl ora. journal of asian natural product research 7, 771–775. yayli, n., yildirim, n., usta, a., zkurt, s., akgün, v., 2003: chemical constituents of campanula lactifl ora. turkish journal of chemistry 27, 749–755. opce-str.vp acta bot. croat. 68 (2), 465–472, 2009 coden: abcra 25 short communication issn 0365–0588 oligotrophy: the forgotten end of an ecological spectrum john p. kociolek1*, eugene f. stoermer2 1 museum of natural history and department of ecology and evolutionary biology, university of colorado, boulder, co, 80309 u.s.a. 2 school of natural resources and environment, university of michigan, ann arbor, mi 48109, u.s.a. most studies that consider the relationship of diatoms and water quality have focused their attention on the questions and practicalities of water pollution or perturbations in some form or another. many models and studies have demonstrated the environmental tolerances or changes in structure of diatom communities related to environmental challenges. this focus on the impacted end of the water quality spectrum has led, of necessity, to issues ultimately related to habitat restoration. we contend that a concentrated effort to develop more fully the theory and there is a need for practice related to oligotrophy, the other, ignored end of the water quality spectrum. we explore the historical usages of the term oligotrophy, as well as the challenges and promise of autecological and community approaches to understanding oligotrophy, and the possibility of focusing more on conservation rather than restoration in water quality issues. keywords: diatom, water quality, oligotrophy, conservation some background oligotrophy refers to »poor food«, a situation where nutrients are in low quantity. the term has been used to describe a wide range of environments, including soils and other terrestrial environments (raven et al. 2005), as well as almost all aquatic ecosystems; marine, estuarine and freshwater (hutchinson 1967). the meaning of low quantities of nutrients (including in some cases salts and other minerals) has become synonymous with early, primitive and natural conditions of freshwater. an exception is when oligotrophic conditions are culturally derived (»cultural oligotrophication« stockner et al. 2000). the concept of oligotrophy has been compared to eutrophy (»good food«), where systems have large quantities of nutrients (usually nitrogen and phosphorus). in the development of these concepts in limnology in particular, they were seen as a continuum from acta bot. croat. 68 (2), 2009 465 * corresponding author: patrick.kociolek@colorado.edu u:\acta botanica\acta-botan 2-09\kociolek.vp 6. listopad 2009 13:47:59 color profile: disabled composite 150 lpi at 45 degrees oligotrophy through an intermediate phase (mesotrophy) to eutrophy. extremes of the ends of this spectrum (»ultra-oligotrophic« and »hyptereutrophic«) have also been recognized and discussed. in addition to these concepts representing a continuum of conditions, they were also thought to represent an ontogenetic trajectory, with natural systems being oligotrophic and through development over time, naturally becoming eutrophic. this concept of lake trophy was applied widely to lakes (naumann 1931; järnfeldt 1952, 1953; rhode 1969; hutchinson 1969, 1973), and the ontogenetic sequence initially to bogs (e.g. naumann 1931), but then more generally (e.g. rhode 1969; hutchinson 1969). the concept was conceived initially by naumann (1919, 1929) and popularized by thienemann (e.g. 1926) (moss et al. 1994). hasler (1947) adapted the term eutrophication to include »cultural eutrophication«, the notion being that addition of nutrients by human agencies accelerated a natural process. this implied the process could be reversed by controlling nutrient loadings (hasler 1969). in many cases the original terms have been expanded to the point where the word oligotrophic is used to describe any desirable condition and eutrophic to describe any undesirable condition. determining the trophic status of freshwater ecosystems within the conceptual framework of trophic status, it soon became of interest to be able to develop vital signs or indicators of trophic condition. soon, a wide range of parameters was being developed to help classify aquatic systems relative to trophic status. while the chemical parameters focused on the »food« (i.e. concentrations of nitrogen, phosphorus and other constituents) in the ecosystems, the physical and biological measures focused on the implications of nutrient levels (water clarity, numbers of kinds of primary producers). at the time of this growing interest in the trophic status of aquatic ecosystems, the 1960s and 1970s, ecological disasters where being uncovered and described. hence, much of the attention was focused on the »eutrophic« end of the spectrum – those indicators of pollution. in biological terms, some investigators confined use of the terms to total abundance and biomass. these measures include chlorophyll a, cells/ml and other quantifiable approaches without consideration of the taxa present (carlson 1977, 1979, 1981). these »black box« approaches claim to be closer to the original trophy concepts. but beyond these approaches, early investigators of the concepts of oligotrophy and eutrophy did examine both autecological and synecological points of view. naumann (1919) discussed phytoplankton associations, relating different communities of phytoplankton with trophic condition. pearsall (1921, 1932), ruttner (1952) and järnfeldt (1956) all discussed the types of plankton found across a wide range of lake types and geography. for individual species, the focus was to identify those species that were indicators of eutrophy (e.g. palmer 1969) – species that had preferences for or tolerances of a wide range of ecological perturbations (nutrients, but also temperature, salts, metals, ph to name a few). from a community approach, a wide range of indices and metrics were developed to help identify degraded systems and the types and levels of degradation (i.e. thunmark 1945, nygaard 1949, rawson 1956, stockner and benson 1967). 466 acta bot. croat. 68 (2), 2009 kociolek j. p., stoermer e. f. u:\acta botanica\acta-botan 2-09\kociolek.vp 6. listopad 2009 13:47:59 color profile: disabled composite 150 lpi at 45 degrees oligotrophy and diatoms from a biological point of view, those working on diatom communities were in the forefront of the development of many of the models and of conduct of the »on the ground« research to identify and distinguish impacted and polluted ecosystems. due to the large number of separate taxa, and large population sizes, diatom communities lent themselves to descriptive and experimental approaches to determining relative impacts on aquatic ecosystems. ruth patrick (patrick et al. 1954) helped to develop conceptual foundations for the application of diatom communities, including her famous saying, »diversity is the hallmark of a clean environment.«large, nation-wide projects to assess the status and trends of u.s. freshwaters were undertaken at this time in rivers (williams 1964, 1972), lakes (taylor et al. 1978), but the coarseness of the taxonomy of the day (most workers used hustedt 1930 as their taxonomic guide) render these studies difficult to interpret according to present taxonomic understanding. the large number of floristic and ecologic works that ensued allows compilations and syntheses of the ecological distributions and tolerances of diatoms (lowe 1974; a tradition that continues today, e.g. van dam et al. 1994). more recently, national scale programs to assess the »health« of u.s. freshwaters using diatoms, (environmental monitoring and assessment program »emap« for lentic systems, national water quality assessment program »nawqa« for lotic systems) have continued to focus more on recognizing impacted or polluted sites than recognizing oligotrophic conditions. the two programs are sponsored and managed by two different federal agencies, which leads to some inconsistencies in philosophy, emphasis, and implementation. there is important past focus on the degradation of freshwater, protocols, procedures and development of indices and metrics that allow time-efficient and cost-effective identification of impacted systems. thus, instead of counts of several thousand to ten(s) of thousands of valves, as performed during the 1950s and 1960s, most protocols call for only 500 to 600 valves, in which dominant species swamp out the identification of rare species in counts (potapova and charles 2004). it may be that it is the rare species that are indicators of oligotrophic conditions. from the diatom floras produced during this period (e.g. patrick and reimer 1966, 1975) it is remarkable that almost no species that were characterized as being associated with oligotrophic conditions were identified or described. the refined taxonomy that has been developed over the last 30 years begins to make past characterizations of taxa dated (e.g. the oligotrophic »cyclotella« group of hutchinson 1967). more recently, high diversity in the phytoplankton has been attributed to intermediate disturbances versus system stability (e.g. padisák et al. 1993). a remarkable floristic work on what was thought to be a well-known (the best known?) freshwater diatom flora, that of central europe, has sparked a renewed interest in oligotrophic diatoms. in this work by lange-bertalot and metzeltin (1996), based on modest samples from three oligotrophic, or nearly oligotrophic lakes, one each in finland, austria and germany, over 800 taxa were identified (the entire flora of central europe was thought to be comprised of 1600 taxa; krammer and lange-bertalot 1986, 1988, 1991a, b). over 100 taxa were either described as new or could not be attributed to any previously described species. the remarkable species richness and the uniqueness of the taxa were thought to be the hallmark of these oligotrophic lakes. acta bot. croat. 68 (2), 2009 467 oligotrophy: the forgotten end of an ecological spectrum u:\acta botanica\acta-botan 2-09\kociolek.vp 6. listopad 2009 13:47:59 color profile: disabled composite 150 lpi at 45 degrees a further innovation of lange-bertalot (1996) as applied to diatoms was the creation of a »red list« of diatom species that are rare and indicative of clean (oligotrophic) conditions. like the higher plant and animal lists on which it is based, species on this red list are indicators of habitats worth protecting. oligotrophy, diatoms and a new research paradigm conservation of habitats while the past decades have focused on identifying and stopping the environmental insults inflicted upon freshwater ecosystems around the world, there is a great opportunity to attempt to identify and protect those that are left. an old adage goes that »an ounce of prevention is worth a pound of cure« and thus, as freshwater issues around the world become more important, being able to identify places that are pristine and in need of protection will be more important. how will we identify these systems – by physical and chemical parameters alone? stevenson (1998) has suggested that diatoms are better indicators of water quality than water chemistry. how will information about diatoms to help us preserve fragile freshwater ecosystems? the ecology of the 21 st century will be one of restoration ecology once abatement of pollution is accomplished, a next critical step is to restore habitats, to bring them back from the brink of irreversible change. but what will we restore them to? how will we know what that condition is? and why shouldn’t we strive to achieve an oligotrophic endpoint? again, how is that determined? by the suite of species present and/or the structure of their communities? paleontology a role that paleontology might play in this regard is to help us understand the natural course of a lake’s ontogeny, from oligotrophic to eutrophic, without the intervention of the human species. excellent exposures of freshwater diatomites, and core from holocene systems may hold clues to this evolution of communities. it is too bad that the discipline of fossil freshwater diatoms is perhaps the least populated in terms of taxonomic expertise in the world of diatom science. documenting biodiversity as the work of lange-bertalot and metzeltin (1996) illustrates well, we still have a lot to do in terms of describing the diversity of diatoms from freshwater ecosystems. in many large-scale programs, from the count data of 600 valves, many species appear to be new to science. in the nawqa datasets, nearly 25% of the taxa are undescribed. and rare species have not been sought in these samples. further development of the concept of oligotrophy we believe there is a strong case to be made for a focused investigation into the concept of oligotrophy. it may be necessary to develop new terminology adequately to describe the range of conditions and causalities present in the modern world. multivariate statistical ap468 acta bot. croat. 68 (2), 2009 kociolek j. p., stoermer e. f. u:\acta botanica\acta-botan 2-09\kociolek.vp 6. listopad 2009 13:47:59 color profile: disabled composite 150 lpi at 45 degrees proaches allow estimation of departures from ecosystem baselines and assignment of causality for known and well-measured parameters. whether this approach is applicable across systems types, across different time scales and across geographic space is uncertain. additional work is needed to understand the theoretical and practical aspects of identifying pristine places, and restoring degraded systems to their original physical, chemical, biological and functional conditions. there may also be historical perspectives that need to be taken into account. for example, what might be considered »clean« or »oligotrophic« in continental europe might be deemed mesotrophic in areas of north america. as new measures to assess the condition of waters are developed (e.g. index of biological integrity), understanding of the baseline condition(s) is imperative. as stevenson (1998) points out, the currently popular concept of »reference« site does not necessarily imply »oligotrophy«, but understanding those differences is essential as we weigh the costs/benefits (and losses) to achieving one or the other. it is time for ecologists, taxonomists, systematists, restoration biologists and aquatic scientists of many interests and specializations to take up this challenge. it may also prove to be advantageous to consider other aspects of diatom biology to best determine trophic status. for example, the robust appearance of diatoms that grew in some habitats least affected by human interventions has been noted historically (e. g. lewis 1865) and in paleolimnological studies (stoermer et al. 1985). in some cases it is possible to trace morphological changes in a species beginning before appreciable human impact until its eventual local extinction (stoermer et al. 1989). to fully assess status of an aquatic environment it may also be advantageous to pay greater attention to the cytology and morphology of diatom species present. there are numerous examples in the literature of apparent morphological effects of various environmental perturbations and, in our opinion, this approach deserves further attention. references carlson, r. e., 1977: a trophic state index for lakes. limnology and oceanography 22, 361–369. carlson, r. e., 1979: a review of the philosophy and construction of trophic state indices. in maloney, t. e. 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u:\acta botanica\acta-botan 2-09\kociolek.vp 6. listopad 2009 13:47:59 color profile: disabled composite 150 lpi at 45 degrees thunmark, s., 1945: zur soziologie des süsswasser-planktons. eine methodologisch-ökologische studie. folia limnologica scandanavica 3, 1–66. van dam, h., mertenes, a., sinkeldam, j., 1994: a coded checklist and ecological indicator values of freshwater diatoms from the netherlands. netherlands journal of aquatic ecology 28, 117–133. williams, l. g., 1964: possible relationships between plankton-diatom species numbers and water-quality estimates. ecology 45, 809–823. williams, l. g., 1972: plankton diatom species biomasses and the quality of american rivers and the great lakes. ecology 53, 1038–1050. 472 acta bot. croat. 68 (2), 2009 kociolek j. p., stoermer e. f. u:\acta botanica\acta-botan 2-09\kociolek.vp 6. listopad 2009 13:47:59 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (1), 11–27, 2009 coden: abcra 25 issn 0365–0588 phytoplankton composition of the ebro river estuary, spain maría del carmen pérez1*, nora i. maidana2, augusto comas3 1 universidad politécnica de valencia, av. naranjos s/n°, 46022 valencia, españa 2 dpto. de biodiversidad y biología experimental, universidad de buenos aires, ciudad universitaria, pab. ii, 4° piso, c1428eha buenos aires, argentina 3 centro de estudios ambientales, apdo. 202, 55100 cienfuegos, cuba the composition of phytoplankton in the ebro river estuary (spain) was analyzed at six sampling stations in april, july and october 1999 and february 2000, based on plankton net and bottle samples. a total of 304 taxa belonging to 13 classes were identified. bacillariophyceae and chlorophyceae were the most important groups. the diatom genera nitzschia and navicula provided most species (25 and 17, respectively). thalassiosira duostra is recorded for the first time for this ecosystem. we propose the following new combinations: monactinus simplex (meyen) corda var. echinulatum (wittrock) comb. nov. and monactinus simplex (meyen) corda var. sturmii (reinsch) comb. nov. keywords: phytoplankton, estuary, taxonomy, ebro, spain. introduction estuaries are highly productive systems in which nutrients are supplied from the land (dyer 1973, mc lusky 1989). phytoplankton study in the ebro river was earlier concentrated on seasonality (sabater and muñoz (1990), small cyclotella species (sabater and klee 1990), and chlorophyceae (pérez et al. 2002, comas et al. 2006). research into phytoplankton has been performed in the ebro delta (comin 1984) and near bays (lópez and arté 1973; delgado 1987; delgado et al. 1990, 1995, 1996). in the framework of the pioneer european project, seasonal collecting data campaigns were undertaken during 1999–2000 to understand the impact of nutrients in the lower ebro river and in its plume. in this study we show the results of phytoplankton composition at six collecting sites from april 1999 to february 2000. acta bot. croat. 68 (1), 2009 11 * corresponding author, e-mail: perez.baliero@gmail.com u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 10:59:58 color profile: disabled composite 150 lpi at 45 degrees materials and methods study area the ebro river is 960 km long and is one of the biggest spanish rivers discharging in the mediterranean sea (fig. 1). the ebro estuary is classified as a »salt wedge estuary« or »type 4« after hansen-rattray’s classification (ibañez et al. 1997). it has a strong and clearly marked halocline due to the low tidal range. circulation is primarily affected by the river discharge. the annual discharge (424 m 3 s –1 mean) is smoothed by the presence of about 170 dams (ibañez et al. 1999). in particular, the mequinenza and riba-roja dams, located 100 km upstream from the mouth, have an important regulatory effect over the discharge in the delta. furthermore, these dams condition the suspended inert solids concentration (usually low) and the phytoplankton populations of the water. in the ebro delta, rice crops and cultivated fields alternate with coastal lagoons. the region has a dry mediterranean climate with a mean annual temperature around 16–17 °c, with around 550 mm of precipitation per year (martinez et al. 1999). samplings four sampling campaigns on the ebro estuary, april, july and october 1999 and february 2000, were performed within the framework of the european pioneer project. electric conductivity, temperature and ph were measured in situ with a multiparametric sounding hydrolab surveyor 3. phytoplankton samples for qualitative analyses were collected 12 acta bot. croat. 68 (1), 2009 pérez m. c., maidana n. i., comas a. 295000 300000 305000 310000 315000 320000 4495000 4500000 4505000 4510000 4515000 4520000 r1 r2r3 r4 r5 r6 mediterranean sea spain africa fig. 1. location of sampling stations in the ebro estuary. u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 10:59:59 color profile: disabled composite 150 lpi at 45 degrees with bottles at different depths trying to cover the whole water column and with 20μm mesh plankton net by horizontal tows in the centre of the stream at every sampling station (fig. 1). all samples were fixed in situ with glutaraldehyde (2% final concentration) according to sournia (1978), and they were deposited at the laboratorio de tecnologías del medio ambiente of the upv, valencia, spain. detailed examinations and drawings of the material were made with leitz laborlux and nikon optiphot light microscopes with phase contrast optics. part of each sample was prepared according to conventional methodology for the qualitative study of diatoms (hasle and fryxell 1970). observations were made with a jeol jsm 6300 scanning electron microscope (sem). for cryo-sem analysis a drop of concentrated sample was placed on an isopore membrane-filter gttp millipore. the sample was frozen with liquid n2, coated with gold and examined using a jeol jsm-5410 scanning microscope equipped with cryo-station. according to new results based on cytological studies and gene sequence analysis, the traditional chlorophyceae is divided into different classes (melkonian 1983, mattox and stewart 1984, graham and wilcox 2000); but, not all nomenclatural changes have been published, and therefore we are still using chlorophyceae in the traditional sense. we accept new concept, the separation of scenedesmus meyen in two genera: desmodesmus and scenedesmus s.str. (an et al. 1999, hegewald 2000). according to hegewald in buchheim et al. (2005) based on inferences from rdna data, pediastrum meyen could be divided into 5 independent genera. we follow this concept in this paper, with the exception of p. willei (fig. 6-a), because molecular genetic data about this taxon are not yet available. in the same way, the taxonomy of bacillariophyceae at species, genus and higher levels, has been undergoing deep changes in the last decades (round et al. 1990, medlin and kaczmarska 2004, among others), introducing many changes and also leaving a high amount of incertitude that has to be settled, and therefore in this paper we use the criteria of round et al. (1990). results the water ph ranged between 7.86 and 8.55 (mean value 8.2), surface temperature between 9.81 and 27.26 °c (mean value 18.5) and the conductivity between 928 and 9071 μs cm –1 (mean value 3777). the surface salinity was measured on july 12 th and october 5 th 1999 and varied between 2.3 and 5.1. three hundred and four taxa included in 13 algal classes were identified (tab. 1, fig. 2): bacillariophyceae 175, chlorophyceae 75, chrysophyceae 2, cryptophyceae 2, cyanobacteria 12, dinophyceae 17, dictyochophyceae 2, ebriidae 1, euglenophyceae 9, prasinophyceae 1, prymnesiophyceae 2, xanthophyceae 2 and zygnemaphyceae 4. considering the species numbers, bacillariophyceae and chlorophyceae were the most important classes with 57 % and 25 % of the recorded species, respectively (fig. 2). the genera navicula sensu stricto and nitzschia presented the highest number of species, 25 and 17 species, respectively (tab. 1). from the total of 149 observed genera, pediastrum, pseudopediastrum, monactinus, stauridium and monoraphidium (chlorophyceae), cryptomonas acta bot. croat. 68 (1), 2009 13 phytoplankton of the ebro estuary u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 11:00:00 color profile: disabled composite 150 lpi at 45 degrees 14 acta bot. croat. 68 (1), 2009 pérez m. c., maidana n. i., comas a. tab. 1. list of algae species observed in the ebro estuary (1999–2000). * diatoms observed in only one sample. bacillariophyceae achnanthes cf. amoena hustedt achnanthidium minutissimum (kützing) czarnecki achnanthidium sp. actinocyclus normanii (gregory) hustedt amphora copulata (kützing) schoeman et archibald a. cf. normanii rabenhorst a. ovalis (kützing) kützing a. pediculus (kützing) grunow in schmidt et al. a. veneta kützing asterionella formosa hassall asterionellopsis glacialis (castracane) f.e. round aulacoseira ambigua (grunow) simonsen a. granulata var. granulata (ehrenberg) simonsen a. granulata var. angustissima (otto müller) simonsen a. cf. nyassensis (otto müller) simonsen bacillaria paradoxa gmelin in linneaeus bacteriastrum cf. elongatum cleve b. delicatulum cleve b. hyalinum lauder caloneis amphisbaena (bory) cleve *c. bacillum (grunow) cleve cerataulina cf. pelagica (cleve) hendey chaetoceros cf. laciniosus schütt ch. cf. pseudocurvisetus mangin ch. didymus ehrenberg ch. muellerii lemmermann chaetoceros sp. cocconeis pediculus ehrenberg c. placentula var. euglypta (ehrenberg) grunow c. placentula var. lineata (ehrenberg) van heurck *c. scutellum ehrenberg cocconeis sp. corethron criophyllum castracane craticula cuspidata (kützing) mann in round, crawford et mann *c. molestiformis (f. hustedt) s. mayama ctenophora pulchella (ralfs ex kuetzing) williams et round cyclostephanos dubius (fricke) round c. invisitatus (hohn et hellerman) theriot, stoermer et hakansson cyclotella atomus hustedt c. choctawhatcheeana prasad c. meduanae germain c. meneghiniana kützing c. ocellata pantocsek *c. aff. polymorpha meyer et håkansson c. rossii håkansson cylindrotheca closterium (ehr.) reimann et lewin *cymatopleura elliptica (brebisson ex kutzing) w. smith c. solea (brebisson in brebisson et godey) w. smith cymbella helvetica kützing c. tumida (brébisson in kützing) van heurck diadesmis confervacea kützing diatoma moniliformis kützing d. tenue agardh d. vulgaris bory diatoma sp. discostella glomerata (bachmann) houk et klee d. stelligeroides (hustedt) houk et klee d. wolterecki (hustedt) houk et klee ditylum brightwellii (t. west) grunow encyonema minutum (hilse in rabenhorst) mann in round, crawford et mann e. prostratum (berk.) kütz. *encyonopsis microcephalum (grunow) krammer *entomoneis paludosa (w. smith) reimer in patrick et reimer eolimna minima (grunow in van heurck) h. lange-bertalot in g. moser, h. lange-bertalot et d. metzeltin e. subminuscula (manguin) g. moser, h. lange-bertalot et d. metzeltin eucampia zodiacus ehrenberg u:\acta botanica\acta-botan 1-09\perez.vp 16. travanj 2009 8:41:19 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 15 phytoplankton of the ebro estuary fallacia cf. insociabilis (krasske) mann in round, crawford et mann *f. subhamulata (grunow in van heurck) mann in round, crawford et mann fistulifera pelliculosa (brebisson) lange-bertalot fragilaria capucina desmaziéres f. cf. constricta ehrenberg f. crotonensis kitton gomphonema olivaceum (hornemann) ehrenberg g. parvulum (kützing) kützing gomphonema sp. gomphosphenia lingulatiformis (h. lange-bertalot et e. reichardt) h. lange-bertalot (=gomphonema lingulatiforme) guinardia striata (stolterfoth) hasle gyrosigma acuminatum (kützing) rabenhorst g. attenuatum (kützing) cleve g. sciotense (sulliv. et wormley) cl. ks: curtis hemiaulus sinensis greville karayevia clevei (grunow in cleve et grunow) round et bukhtiyarova leptocylindrus danicus cleve l. minimus gran lithodesmium cf. undulatum ehrenberg luticola goeppertiana (bleisch in rabenhorst) mann in round, crawford et mann l. mutica var. ventricosa (kützing) hamilton in hamilton et al. *l. nivalis (ehrenberg) mann in round, crawford et mann melosira varians c. a. agardh navicula antonii lange-bertalot in rumrich et al. (=n. menisculus var. grunowii) n. capitatoradiata germain n. cryptotenella lange-bertalot n. erifuga lange-bertalot n. gregaria donkin n. cf. hambergii hustedt n. lanceolata (agardh) kützing n. phyllepta kützing n. recens (lange-bertalot) lange-bertalot n. rhynchocephala kützing n. rostellata kützing *n. salinarum grunow *n. schroeteri meister n. tripunctata (o. f. müller) bory n. veneta kützing n. viridula (kützing) ehrenberg navicula sp. nitzschia acicularis (kützing) w. smith n. amphibia grunow n. angustatula lange-bertalot *n. brevissima grunow n. cf. capitellata hustedt n. cf. rautenbachiae cholnocky n. constricta (gregory) grunow n. dissipata (kützing) grunow *n. dubia w. smith n. filiformis (w. smith) van heurck *n. fonticola grunow n. frustulum (kützing) grunow n. gracilis hantzsch n. inconspicua grunow *n. intermedia hantzsch n. linearis (agardh) w. smith n. longisima var reversa grunow n. microcephala grunow n. palea (kützing) w. smith n. recta hantzsch *n. sigma (kützing) w. smith n. sigmoidea (nitzsch) w. smith *n. sinuata var. tabellaria (grunow) grunow n. sublinearis hustedt nitzschia sp. odontella mobiliensis bailey *planothidium ellipticum (cleve) round et bukhtiyarova p. rostratum (østrup) round et bukhtiyarova pleurosigma sp. pleurosira laevis (ehrenberg) compère proboscia alata (brightwell) sundström psammothidium subatomoides (hustedt) bukhtiyarova et round pseudo-nitzschia cf. pseudodelicatissima (hasle) hasle tab. 1. – continued u:\acta botanica\acta-botan 1-09\perez.vp 16. travanj 2009 8:41:19 color profile: disabled composite 150 lpi at 45 degrees 16 acta bot. croat. 68 (1), 2009 pérez m. c., maidana n. i., comas a. pseudo-nitzschia sp. pseudosolenia calcar-avis (schultze) sundstrom pseudostaurosira brevistriata (grunow in van heurck) williams et round puncticulata bodanica (grunow in schneider) håkansson *p. radiosa (lemmermann) håkansson reimeria uniseriata sala, guerrero et ferrario rhizosolenia sp. rhoicosphaenia abbreviata (c. agardh) lange-bertalot sellaphora pupula (kützing) mereschkowsky seminavis cf. gracilenta (a. grunow ex a. schmidt) dg mann skeletonema cf..costatum (grev.) cleve s. potamos (weber) hasle staurosira construens var. subsalina (hustedt) bukhtiyarova s. construens var. venter (ehrenb.) p. b. hamilton in hamilton, poulin, charles et angell stephanodiscus hantzschii grunow s. hantzschii morphotype »tenuis« (hustedt) håkansson et stoermer s. parvus stoermer et håkansson s. rotula (kützing) hendey surirella brebissonii var. kuetzingii krammer et lange-bertalot *s. linearis var. helvetica (brun) f. meister *s. minuta (brun) f. meister tabularia fasciculata (c. agardh) d. m. williams et round t. cf. tabulata (c. a. agardh) snoeijs thalassionema nitzschioides (grunow) mereschkowsky thalassiosira baltica (grunow in cleve et grunow) ostenfeld t. duostra pienaar in pienaar et pieterse t. cf. faurii (gasse) hasle t. pseudonana hasle et heimdal t. cf. visurgis hustedt t. weissflogii (grunow) g. fryxell et hasle thalassiosira sp. 1 thalassiosira sp. 2 tryblionella hungarica (grunow) d. g. mann *t. suevica grunow (=n. levidensis var. salinarum grunow) ulnaria acus (kützing) aboal u. ulna (nitzsch) p. compère chlorophyceae s. l. chlorococcales s. l. actinastrum hantzschii var. hantzschii lagerh. a. hantzschii var. subtile wolosz. ankyra ancora (g.m. smith) fott botryococcus cf. braunii kütz. chlorotetraedron incus (teil.) kom. et kovac. closteriopsis longissima (lemm.) lemm. coelastrum indicum turn. c. microporum näg. c. pseudomicroporum kors. c. reticulatum var. polychordum kors. coenococcus fottii hind. crucigenia smithii (bourr. et manguin) kom. c. tetrapedia (kirchn.) w. et g.s. west crucigeniella cf. pulchra (w. et g.s. west) kom. desmodesmus abundans (kirchn.) hegew. d. armatus s.l. (chod.) hegew. d. brasiliensis (bohl.) hegew. d. communis (hegew.) hegew. d. denticulatus var. fenestratus (teil) hegew. d. intermedius (chod.) hegew. d. maximus (meyen) hegew. d. opoliensis (richter) an et al. d. pannonicus (hortob.) hegew. d. spinosus (chod.) hegew. dictyosphaerium ehrenbergianum näg. d. tetrachotomum printz dictyosphaerium sp. golenkinia radiata chod. kirchneriella obesa (w. west) schmidle lagerheimia ciliata (lagerh.) chod. l. subsalsa lemm. micractinium pusillum fres. monactinus simplex var. simplex (meyen) corda m. simplex var. sturmii (reinsch) comb. nov. tab. 1. – continued u:\acta botanica\acta-botan 1-09\perez.vp 16. travanj 2009 8:41:19 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 17 phytoplankton of the ebro estuary m. simplex var. echinulatun (wittr.) comb. nov. monoraphidium arcuatum (kors.) hind. m. contortum (thur.) kom-legn. m. griffithii (berk.) kom-legn. nephrocytium schilleri (kamm.) com. oocystidium ovale kors. oocystis lacustris chod. o. marssonii lemm. pediastrum duplex var. duplex meyen pediastrum willei comas et al. planktosphaeria gelatinosa g.m. smith pseudopediastrum boryanum var. boryanum (turp.) hegew. pseudoschroederia antillarum (kom.) hegew. et schnepf p. robusta (kors.) hegew. et schnepf raphidocelis contorta (schmidle) marvan et al. scenedesmus acuminatus (lagerh.) chod. s. arcuatus (lemm.) lemm. s. ellipticus corda s. obliquus var. dimorphus (turp.) hansg. s. obtusus meyen schroederia setigera (schröd.) lemm. selenastrum bibraianum reinsch. s. gracile reinsch. siderocelis ornata (fott) fott stauridium tetras (ehrenb.) hegew. tetrachlorella alternans (g.m. smith) kors. tetraedron caudatum (corda) hansg. tetrastrum komarekii hind. t. staurogeniaeforme (schröd.) lemm. treubaria triappendiculata bern. westella botryoides de wild o. ulotrichales s. l. elakatothrix genevensis (reverdin) hind. e. subacuta kor{. klebsormidium subtile (kütz.) silva et al. koliella cf. spiculiformis (vischer) hind. planctonema lauterbornii schmidle o. volvocales chlamydomonas sp. eudorina elegans ehrenb. gonium pectorale o. f. müller pandorina morum (müller) bory pseudoscourfieldia marina (throndsen) manton chrysophyceae dinobryon sp. mallomonas sp. cryptophyceae cryptomonas sp. rhodomonas sp. cyanophyceae aphanizomenon flos-aquae (l.) ralfs ex bornet et flahault coelomoron pusillum (van goor) kom. chroococcus limneticus lemm. c. cf. microscopicus kom-legn. et cronberg cyanobium sp. geitlerinema splendidum (grez. ex gom.) anagn. et kom. merismopedia sp. microcystis aeruginosa kütz. planktolyngbya cf. brevicellularis cronberg et kom. planktothrix isothrix (skuja) kom. et komark. pseudanabaena sp. spirulina major kütz. ex gom. dinophyceae ceratium candelabrum (ehrenb.) stein c. furca (ehrenb.) claparède et lachmann c. fusus (ehrenb.) duj. c. hirundinella (o.f. müller) schrank c. pentagonum gourr. c. tripos (o.f. müller) nitzsch dinophysis sacculus stein dinophysis sp. gymnodinium sp. peridinium sp. podolampas sp. prorocentrum micans ehrenb. prorocentrum sp. 1 protoperidinium cf. conicum (gran) balech tab. 1. – continued u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 11:00:00 color profile: disabled composite 150 lpi at 45 degrees (cryptophyceae), and cyclotella (bacillariophyceae) (figs. 3: a–d) were very frequent in our samples. we identified 175 diatoms belonging to 69 genera (25 centrics and 44 pennates). centric diatoms (figs. 3, 4, 5) were less numerous than pennate. a high percentage of the identified species (82.3%) are known from non-marine habitats and only 34.8% of diatoms were planktonic. twelve species: amphora pediculus, bacillaria paradoxa, cocconeis placentula var. euglypta, cyclostephanos dubius (fig. 4-a), cyclotella atomus, c. meduanae (figs. 3: a–b), c. meneghiniana, nitzschia palea, skeletonema potamos (fig. 4-b), stephanodiscus rotula (fig. 4-e), thalassiosira weissflogii (fig. 5-b) and ulnaria ulna were present in all the stud18 acta bot. croat. 68 (1), 2009 pérez m. c., maidana n. i., comas a. p. cf. depressum (bailey) balech protoperidinium sp. 1 scrippsiella trochoidea (stein) balech dictyochophyceae dictyocha crux ehrenb. d. fibula ehrenb. ebriidea hermesinum adriaticum zacharias euglenophyceae euglena spirogyra ehrenb. e. tripteris (duj.) klebs euglena sp. eutreptiella sp. lepocinclis texta (duj.) lemm. emend. conrad lepocinclis sp. phacus acuminatus stokes p. tortus lemm. trachelomonas sp. prasinophyceae pyramimonas sp. prymnesiophyceae periphyllophora mirabilis (schiller) kamptner syracosphaera sp. xanthophyceae goniochloris cf. muticum (a. braun) fott pseudostaurastrum hastatum (reinsch) chod. zygnemaphyceae closterium sp. cosmarium sp. staurastrum gracile var. gracile ralfs s. smithii (g.m. smith) teiling tab. 1. – continued bacillariophyceae 57.6% cyanobacteria 3.9% dinophyceae 5.6% chlorophyceae 24.7% euglenophyceae 3% zygnemaphyceae 1.3% other classes 3.9% fig. 2. relative contribution of species among classes to total number of phytoplankton species in the ebro estuary (1999–2000). u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 11:00:00 color profile: disabled composite 150 lpi at 45 degrees ied samples, while 22 diatoms were found in only one sample during the studied period (tab. 1). thalassiosira duostra (figs. 5 c–f) is recorded for the first time in this ecosystem. the present paper reports four new chlorococcales s.l. and three ulotrichales s.l. the new findings are: botryococcus cf. braunii, lagerheimia ciliata, lagerheimia subsalsa, westella botryoides and the ulotrichales s.l: planctonema lauterbornii, klebsormidium subtile and koliella cf. spiculiformis. we found two different varieties of pediastrum simacta bot. croat. 68 (1), 2009 19 phytoplankton of the ebro estuary fig. 3. sem micrographs of cyclotella and discostella. a–b – cyclotella meduanae (a – external view, b – internal view), c – c. rossii, d – c. aff. polymorpha, e – discostella pseudostelligera, f – d. woltereckii u:\acta botanica\acta-botan 1-09\perez.vp 16. travanj 2009 8:41:21 color profile: disabled composite 150 lpi at 45 degrees plex (var. sturmii and var. echinulatum), which should be transferred to monactinus simplex (meyen) corda (chlorococcales s.l.) due to cyanobacteria, a population probably belonging to cyanobium was observed. this species was a representative of the picophytoplankton fraction for the whole of the study period. among dinophyceae, several ceratium and protoperidinium marine species were observed near the river mouth (stations 1 and 2, fig. 1). with respect to potentially harmful species, during this period were observed the dinoflagellates dinophysis sacculus and prorocentrum cf. micans (fig. 6 b–c) in the samples closest to the sea. 20 acta bot. croat. 68 (1), 2009 pérez m. c., maidana n. i., comas a. fig. 4. sem micrographs of cyclostephanos, skeletonema and stephanodiscus. a – cyclostephanos dubius, b – skeletonema potamos, c – stephanodiscus hantzschii morphotype »hantzschii«, d – st. parvus, e – st. rotula, f – st. hantzschii morphotype »tenuis« u:\acta botanica\acta-botan 1-09\perez.vp 16. travanj 2009 8:41:26 color profile: disabled composite 150 lpi at 45 degrees discussion the taxonomic inventory and quantitative description of organisms are important prerequisites for understanding the status and functioning of ecosystems (john 1994). the high species richness of chlorophyceae and bacillariophyceae in the lower ebro river (75 and 175 taxa respectively) may be interpreted as typical of freshwater ecosystems. when compared to other regional ecosystems, the species richness of chlorophyceae in the ebro estuary is evident (sabater 1990, montesanto et al. 2000). acta bot. croat. 68 (1), 2009 21 phytoplankton of the ebro estuary fig. 5. sem micrographs of thalassiosira. a – thalassiosira pseudonana, b – t. weissflogii, c – f – t. duostra (c–e – external views with different arrangements of the central groups of fultoportulae, f – internal view) u:\acta botanica\acta-botan 1-09\perez.vp 16. travanj 2009 8:41:31 color profile: disabled composite 150 lpi at 45 degrees with respect to chlorococcales, this in its present concept comprises only the family chlorococcaceae and is transferred to chlamydophyceae (ettl and gärtner 1988), however several other genera have been replaced in sphaeropleales (hegewald and hanagata 2000) or in the quite different class of trebouxiophyceae (friedl 1995, hepperle et al. 2000, wolf et al. 2002). monactinus simplex (meyen) corda (= pediastrum simplex meyen) is more widely distributed in warm regions, but in recent years the area is a little more enlarged. according to the last monograph on the genus pediastrum (komárek and jankovská 2001) few taxonomical varieties were accepted, p.i. the vars. echinulatum wittr and sturmii (reinsch) wolle. in this study both taxa are very well represented, and, since they have not been yet transferred to monactinus we propose here the following new combinations: monactinus simplex (meyen) corda var. echinulatum (wittrock) comb. nov. (basionym: pediastrum simplex var. echinulatum wittrock in wittrock, v. et nordsted, o., alg. aq. dulc. exsicc. praec. scand. fasc. 5, no. 235, 1833). a variety characterized by cell walls ornamented by prominent granules and coenobia usually with small and irregular holes (fig. 7a). monactinus simplex (meyen) corda var. sturmii (reinsch) comb. nov. (basionym: pediastrum sturmii reinsch, die alg. fl. mittl. theil. frank., p. 90, tab. 7: 1, 1867.) is characterized by the lateral sides of lobes in old coenobia distinctly convex and cell wall with very fine and regular granulations (fig. 7-b). 22 acta bot. croat. 68 (1), 2009 pérez m. c., maidana n. i., comas a. fig. 6. sem micrographs pediastrum, dinophysis and prorocentrum. a – pediastrum willei, b – dinophysis sacculus, c – prorocentrum cf. micans u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 11:00:12 color profile: disabled composite 150 lpi at 45 degrees the genera scenedesmus and desmodesmus are represented by 5 and 11 species, respectively, being two of the richest genera (pérez et al. 2002), resembling the situation in the danube river (schmidt et al. 1994, kiss and schmidt 1998). on the other hand, the number of chlorophyceae found in our study is higher (71 taxa) in comparison with the number found by sabater and muñoz (1990) between 1986 and 1987 in four sites located along the last 60 km of the river ebro (41 taxa). it could indicate a higher trophic level. in our results, as compared with those of sabater and muñoz (1990), 18 chlorophyceae species were not observed in our samples. these authors observed that diatoms and green algae were the most abundant algae. they concluded that the phytoplankton dynamics of the ebro river is based upon two major factors: water flow and water salinity. montesanto et al. (2000) recorded a total of 122 taxa of suspended algae in the aliakmon river in greece between february 1995 and january 1996. these authors observed that the two most important groups were chlorophyta with 43.4% of total species number (53 representatives) and bacillariophyceae with 19.7% containing a great number of tychoplanktonic species either from the phytobenthos or the littoral zone. on the other hand, vili^i] et al. (2000) observed that the marine diatoms and dinoflagellates provided the dominant phytoplankton groups in the eastern adriatic zrmanja river estuary (croatia). as was expected, diatoms were represented by more non-marine (80%) than marine (20%) taxa. of the non marine diatoms, pennates were well represented with a high number of species (124) while centrics had the lower species numbers (16). on the other hand, marine diatoms were represented by similar numbers of centrics and pennates (20 and 15, respectively). among centrics, cyclotella species are not easy to distinguish from each other due to their high degree of polymorphism (håkansson 2002, rivera et al. 2003) and the possible convergence of some ultrastructural features and they were usually included in species complex such is the case of some taxa recorded for the ebro river. cyclotella rossii (fig. 3-c) was present in all sampling sites along the study period. it clearly differs from c. ocellata and other related species due to the regularly aligned series of pores at the central part of the valves and by the marginal striae with similar length. acta bot. croat. 68 (1), 2009 23 phytoplankton of the ebro estuary fig. 7. light microphotographs of monactinus simplex. a – monactinus simplex var. echinulatum, b – monactinus simplex var. sturmii. u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 11:00:12 color profile: disabled composite 150 lpi at 45 degrees cyclotella aff. polymorpha (fig. 3-d) was found only in station r5 and was represented by only few valves that could be distinguished from c. rossii by the irregular length of the marginal striae and from c. ocellata mainly by the radially oriented rows of puncta or orbiculi in the central part of the valve. although c. ocellata provides a variable cyclotella ocellata-krammeri-rossii complex (knie and hübener 2007), the differentiating features of c. rossii is that the valves are the regular length of the marginal striae. we agree with knie and hübener (2007) and genkal and popovskaya (2008) about the need to perform further investigations into the autoecology as well as dna sequence analyses to clarify the taxonomic status of this species, also including c. polymorpha in this group of taxa. stephanodiscus rotula (fig. 4-e) and s. parvus (fig. 4-d) were present in all the sampling stations. s. rotula should not be confused with other related species, such as s. alpinus because of the valves have the central fultoportulae in heterotopic position (according to håkansson 2002). on the other hand, s. parvus (fig. 4-d) differs from the very closely related s. minutulus because it always has the single central fultoportula in an isotopic position. remarkable in the ebro river samples was the presence of thalassiosira duostra (fig. 5: c–f), a diatom with a scarcely known geographical distribution. it was mentioned for south africa (the type locality), and found in some european rivers and ponds and in a reservoir in brazil (pienaar and pieterse 1990, torgan et al. 2004). the ebro material agrees in every respect with the morphological characteristics of the species. the higher relative abundance of benthic diatoms in february 2000 was probably related to rainfall occurring in the catchment area during the previous days to sampling. sabater (1990) observed a great influence of diatom benthic species in the phytoplankton of the medium-sized mediterranean river the ter (spain). previous studies dealing with the phytoplankton composition in the ebro river, reported 59 freshwater diatoms (sabater and klee 1990, sabater and muñoz 1990), which is lower number in comparison to 137 freshwater diatoms identified in this study. the absence of marine taxa in those previous studies could be explained by the differences in sampling methodologies, i.e. the sub-superficial water samples in sabater and klee (1990) and in sabater and muñoz (1990) as against the. covering of all the water column in this study including the salt wedge. the picocyanobacteria were represented by an important population of cyanobium sp. and its distribution and abundance were studied in perez and carrillo (2005). with respect to potentially harmful dinoflagellates, we observed two interesting species: dinophysis sacculuss (fig. 6-b), recorded as toxic in the mediterranean sea by zingone et al. (1998, 2006), and prorocentrum cf. micans (fig. 6-c), a very common species in coastal and estuarine waters (steidinger and tangen 1996). ebridian flagellates have only two extant species, ebria tripartita and hermesinum adriaticum, and this latter was observed in our study (tab. 1) in one sample from the river mouth. this flagellate species is heterotrophic and possibly mixotrophic and is restricted to warmer waters (hargraves 2002). acknowledgemens the authors are greatly indebted to prof. dr. f. round of the university of bristol for his valuable comments about some diatom species, to m. rodilla, s. falco, i. romero and r. martínez for their technical assistance with sampling and to the technicians of the elec24 acta bot. croat. 68 (1), 2009 pérez m. c., maidana n. i., comas a. u:\acta botanica\acta-botan 1-09\perez.vp 22. travanj 2009 11:00:13 color profile: disabled composite 150 lpi at 45 degrees tronic microscopy service of the upv (valencia) for their assistance with sem images. we thank the reviewers, who improved the quality of the manuscript. this project has been financed by european community mas3-ct98-0170 (dg12-voma): pioneer »preparation and integration of analysis tools towards operational forecast of nutrients in estuaries of european rivers«. references an, s. s., friedl, t., hegewald, e., 1999: phylogenetic relationship of scenedesmus and scenedesmus-like coccoid green algae as inferred from its-2 rdna sequence comparisons. plant biology 1, 1–11. buchheim, m., buchheim, j., carlson, t., braband, a., hepperle, d., wolf, m., hegewald, e., 2005: phylogeny of the hydrodictyaceae (chlorophyceae) inferences from rdna data. journal of phycology 41, 1039–1054. comas gonzález a., pérez baliero, m. c., gonzález del río rams, j., 2006: pediastrum willei nom. et sp. nov. 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(zingiberaceae) siju senan1, dhanya kizhakayil1, thotten e. sheeja1*, bhaskaran sasikumar1, alangar i. bhat2, villupanoor a. parthasarathy1 1 division of crop improvement and biotechnology, indian institute of spices research, calicut, kerala 673 012, india 2 division of crop protection, indian institute of spices research, calicut, kerala 673 012, india abstract – twenty one polymorphic microsatellite loci were isolated and characterized from turmeric (curcuma longa l.). these markers were screened across thirty accessions. the number of alleles observed for each locus ranged from two to eight with an average of 4.7 alleles per locus. the discrimination power of these markers ranged from 0.25 to 0.67 (average 0.6). the simple sequence repeat (ssr) markers can complement the currently available ssr markers and would be useful for the genetic analysis of turmeric accessions. keywords: curcuma longa, microsatellites, triploid, turmeric. abbreviations: ssr – simple sequence repeat, pcr – polymerase chain reaction, est – expressed sequence tags. introduction turmeric (curcuma longa l.) is an important spice and medicinal plant cultivated widely in south-east asian countries, with india as its prime producer. curcumin, the main bioactive component of turmeric has been shown to exhibit a wide range of biological actions (chattopadhyay et al. 2004) and its medicinal properties are well documented (aggarwal et al. 2007). turmeric is mainly propagated through rhizomes and is reported to be a triploid [2n=3x=63; x=21] (ramachandran 1961, islam 2004) and a nonaploid (2n=9x=63; x=7) (skornickova et al. 2007). conservation and utilization strategies require fundamental knowledge of the extent of genetic diversity of the crop. conventionally, turmeric accessions are characterized using morphological and agronomical traits. microsatellite or simple sequence repeat (ssr) constitutes a robust set of molecular markers acta bot. croat. 72 (2), 2013 407 * corresponding author, e-mail: teshee@rediffmail.com copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. widely used for population genetic analyses, germplasm characterization, parentage analysis and marker-assisted selection in plants. so far only 17 est-ssr (siju et al. 2010a) and 35 genomic ssr markers (sigrist et al. 2010, siju et al. 2010b) have been reported in turmeric. this limited availability warrants the need to expand the existing repertoire of microsatellite markers for future studies aiming at better estimation of genetic variability for the effective conservation of the genetic resources of turmeric. the present study was conducted to develop novel microsatellite markers from genomic dna libraries of turmeric. materials and methods total genomic dna was extracted from a wild turmeric accession maintained at the germplasm repository of the indian institute of spices research, calicut, india using the modified ctab protocol (syamkumar et al. 2003). genomic libraries enriched for microsatellite repeats were constructed following the protocol of glenn and schable (2005) with four sets of 3' biotinylated probes(tg)12, (aac)6, (aag)8 and (acag)6. plasmids were isolated from 268 recombinant clones and sequenced at bioserve biotechnologies, hyderabad, india. the sequences were assembled into contigs using egassembler (masoudi-nejad et al. 2006) and the identification of ssrs within the sequenced clones was performed using websat (martins et al. 2009). primers targeting the amplification of unique microsatellite repeats were designed using the web-based computer programprimer3 (rozen and skaletsky 2000). polymorphism was assessed by genotyping thirty turmeric accessions maintained in the repository. each 25 µl of pcr mixture contained 1 x taq buffer (sigma, missouri, usa), 1.5 mm mgcl2, 0.2 mm of dntps, 5.0 pmol each of primers, 50 ng genomic dna and 1 u taq dna polymerase (sigma, missouri, usa). pcr amplification was performed on a master cycler ep gradient s thermocycler (eppendorf, germany) with the following profile: 1 cycle of 94 °c for 5 min; followed by 35 cycles of 94 °c for 30 s, annealing at optimal temperature (tab. 1) for 45 s, 72 °c for 1 min; and 1 cycle of 72 °c for 20 min. pcr products were sized on 8.0% denaturing page along with a 10-bp dna ladder (invitrogen, carlsbad, ca) and silver stained (benbouza et al. 2006). the efficiency of microsatellite loci in genotype identification was evaluated with a discriminating power parameter (d), which represents the probability that two randomly chosen individuals have different patterns, and thus are distinguishable (tessier et al. 1999). results and discussion sequencing of these 268 clones revealed a total of 123 sequences containing microsatellites. after redundancy elimination, only 92 sequences contained unique ssrs having repeat units ranging from 4 to 21. thus the efficiency of microsatellite isolation using the enrichment protocol accounted for 34% (92 unique sequences out of 268 sequenced clones). high proportions of dna fragments lacking microsatellite repeats is a significant issue with the improved protocols of microsatellite isolation protocols, due to the high level of non-specific binding of streptavidin-coated magnetic beads to the target dna (st. john and quinn 2008). however the redundancy (25 %) observed in the present study might be due to the application of pcr steps during enrichment, which might have amplified the same genomic dna fragments prior to cloning. though efficient enrichment protocols 408 acta bot. croat. 72 (2), 2013 senan s., kizhakayil d., sheeja t. e., sasikumar b., bhat a. i., parthasarathy v. a. a c t a b o t .c r o a t .72 (2),2013 409 p o l y m o r p h ic m ic r o s a t e l l it e m a r k e r s f r o m c u r c u m a l o n g a tab.1. characteristics of 21 microsatellite markers for curcuma longa l. marker forward primer (5'–3') repeat motif ta (°c) allele size range (bp) na d genbank accession numberreverse primer (5'–3') cumisat -19 catgcaaatggaaattgacac (ac)16 (at)6 65 204–154 8 0.67 hq154119 tgataaattgacacatggcagtc cumisat -20 cgatacgagtccatctcttcg (ac)6 65 158–148 8 0.64 hq154120 ccttgctttggtggctagag cumisat -21 tcattcaaagtccgatggaa (aag)9 62 164–146 6 0.67 hm438970 ttcgagtgcagaaggagaatta cumisat -22 aatttattagcccggaccac (ctt)10 64 158–122 7 0.67 hm438971 aagaaagtgagtagaaaccaaagc cumisat -23 cgtggaaggtgagtttgac (aag)4 65 165–132 3 0.66 hm438972 cagaagggaactgagatgg cumisat -24 aggtattctactcgaccaag (aac)10 58 141–123 4 0.60 hm438973 aaattcatatagccccatc cumisat -25 tacatgagaaacaacaaagccc (aac)7 65 146–140 3 0.60 hm438974 agttagccaagtcccaatttagc cumisat -26 cattccgatgaattgtatg (aac)9 58 208–188 5 0.61 hm438975 gcagttgttttgcttcag cumisat -27 tatagatagccatgctgaag (ac)8 63 121–111 4 0.25 hm438976 ccattttagttcattacgtg cumisat -28 ttcaacttctcctcgctcag (aag)7(gat)5 65 160–139 7 0.67 hm438977 gcaaggtctgcatctatttctc cumisat -29 gtggtatccccatgaagagc (aag)10 65 177–150 8 0.67 hm438978 atgaccaagccctttcacc 410 a c t a b o t .c r o a t .72 (2),2013 s e n a n s .,k iz h a k a y il d .,s h e e ja t .e .,s a s ik u m a r b .,b h a t a .i.,p a r t h a s a r a t h y v .a . marker forward primer (5'–3') repeat motif ta (°c) allele size range (bp) na d genbank accession numberreverse primer (5'–3') cumisat -30 ctctaatgtcgcctctcacg (aag)5 65 157–142 5 0.64 hm438979 gcatctcccgttcttctcc cumisat -31 ggaggaggagaagcagaag (agc)6…(aag)4 65 169–148 2 0.51 hm438980 gacaggcgaaggaagaaac cumisat -32 tgttgtaggtagaagcaaatgac (aag)9 64 135–120 4 0.54 hm438981 ttggtgtcctaattctttcaac cumisat -33 atggatggatacaacaacaac (aac)8 65 170–140 3 0.61 hm438982 tataaacacactccctcttgg cumisat -34 aagttggtgaaggattagagctac (aac)6 62 144–117 2 0.58 hm438983 cacctagtgggataaatcttgg cumisat -35 ggttcgtcgctggaaagtaat (ctt)10 60 214–187 7 0.67 hm438984 gcatctcaacaggggctg cumisat -36 tgggctcaatggttgatacg (aag)6 65 220–208 4 0.58 hq154121 ctcctcatcgctatccgagg cumisat -37 ccattggcgaggatgaagc (aaacac)4 65 218–192 4 0.60 hq154122 cctgccaagcaaagccaag cumisat -38 tcatcataaacactcctg (actg)4 58 126–118 3 0.58 hq154123 gaagaagaggctaagttc cumisat -39 tatcccctgaaaactaatcc (tg)6 64 180–176 2 0.55 hq154124 aaaatgtcacgaactattgc ta – annealing temperature of primer pair; na – total number of alleles; d – discrimination power tab.1. – continued might give higher number of positive clones this could also lead to higher levels of redundancy (squirrell et al. 2003). out of the eighty primers designed and targeted for the amplification, twenty one polymorphic markers were identified (tab 1). these markers generated a maximum of three alleles per genotype, tallying with the triploid status of turmeric (ramachandran 1961, islam 2004) and characteristics of ssrs reported earlier (sigrist et al. 2010; siju et al. 2010a, b). a total of 99 alleles were detected across 30 turmeric accessions with an average of 4.7 alleles/ locus. the number of alleles per locus varied from 2 (cumisat 31, 34, 39) to 8 (cumisat 19, 20, 29). to evaluate the efficiency of ssr markers for discriminating turmeric accessions/varieties, the discrimination power (d) of markers was calculated according to tessier et al. (1999). the d value for these markers ranged from 0.25 (cumisat 27) to 0.67 (cumisat 19, 21, 22, 28, 29, 35) with an average value of 0.6. this is almost on a par with the previous report of turmeric microsatellites (siju et al. 2010b), suggesting their potential utility in future studies aimed at genetic diversity analysis and marker-assisted selection in the species. acknowledgements this research was supported by a research grant from the department of biotechnology (dbt), new delhi, india. references aggarwal, b. b., bhatt, i. d., ichikawa, h., ahn, k. s., sethi, g., sandur, s. k., sundaram, c., seeram, n., shishodia, s., 2007: curcumin – biological and medicinal properties. in: ravindran, p. n., babu, k. n., sivaraman, k. (eds.), turmeric: the genus curcuma, 297–368. crc press, new york. benbouza, h., jacquemin, j. m., baudoin, j. p., mergeai, g., 2006: optimization of a reliable, fast, cheap and sensitive silver staining method to detect ssr markers in polyacrylamide gels. biotechnology agronomy society and environment 10, 77–81. chattopadhyay, i., biswas, k., bandyopadhyay, u., banerjee, r. k., 2004: turmeric and curcumin: biological actions and medicinal applications. current science 87, 44–53. glenn, t. c., schable, n. a., 2005: isolating microsatellite dna loci. methods in enzymology 395, 202–222. islam, m. a., 2004: genetic diversity of the genus curcuma in bangladesh and further biotechnological approaches for in vitro regeneration and long-term conservation of c. longa germplasm. phd thesis, university of hannover. martins, w. s., lucas, d. c. s., neves, k. f. s., bertioli, d. j., 2009: websat – web software for microsatellite marker development. bioinformation 3, 282–283. masoudi-nejad, a., tonomura, k., kawashima, s., moriya, y., suzuki, m., itoh. m., kanehisa, m., endo, t., goto, s., 2006: egassembler: online bioinformatics service for large-scale processing, clustering and assembling ests and genomic dna fragments. nucleic acids research 34, w459–462. ramachandran, k., 1961: chromosome numbers in the genus curcuma linn. current science 30, 194–196. acta bot. croat. 72 (2), 2013 411 polymorphic microsatellite markers from curcuma longa rozen, s., skaletsky, h. j., 2000: primer 3 on the www for general users and for biologist programmers. in: krawetz, s., misener, s. (eds.), bioinformatics methods and protocols: methods in molecular biology, 365–386. humana press, totowa, nj. sigrist, m. s., pinheiro, j. b., azevedo-filho, j. a., colombo, c. a., bajay, m. m., lima, p. f., camilo, f. r., sandhu, s., souza, a. p., zucchi, m. i., 2010: development and characterization of microsatellite markers for turmeric (curcuma longa). plant breeding 129, 570–573. siju, s., dhanya, k., syamkumar, s., sasikumar, b., sheeja, t. e., bhat, a. i., parthasarathy, v. a., 2010a: development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (curcuma longa l.). molecular biotechnology 44, 140–147. siju, s., dhanya, k., syamkumar, s., sheeja, t. e., sasikumar, b., bhat, a. i., parthasarathy, v. a., 2010b: development, characterization and utilization of genomic microsatellite markers in turmeric (curcuma longa l.). biochemical systematics and ecology 38, 641–646. skornickova, j. l., sida, o., jarolimova, v., sabu, m., fer, t., travnicek, p., suda, j., 2007: chromosome numbers and genome size variation in indian species of curcuma (zingiberaceae). annals of botany 100, 505–526. squirrell, j., hollingsworth, p. m., woodhead, m., russell, j., lowe, a. j., gibby, m., powell, w., 2003: how much effort is required to isolate nuclear microsatellites from plants? molecular ecology 12, 1339–1348. st. john, j., quinn, t. w., 2008: rapid capture of dna targets. biotechniques 44, 259–264. syamkumar, s., lowarence, b., sasikumar, b., 2003: isolation and amplification of dna from rhizomes of turmeric and ginger. plant molecular biology reporter 21, 171a– 171e. tessier, c., david, j., this, p., boursiquot, j. m., charrier, a., 1999: optimization of the choice of molecular markers for varietal identification in vitis vinifera l. theoretical and applied genetics 98, 171–177. 412 acta bot. croat. 72 (2), 2013 senan s., kizhakayil d., sheeja t. e., sasikumar b., bhat a. i., parthasarathy v. a. 544 vizintin et al.vp acta bot. croat. 71 (2), 249–260, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 soil seed bank of the invasive robinia pseudoacacia in planted pinus nigra stands imre cseresnyés*, péter csontos institute for soil sciences and agricultural chemistry, centre for agricultural research, hungarian academy of sciences, herman o. út 15, budapest, h-1022, hungary abstract – pinus nigra and robinia pseudoacacia are exotic trees used for afforestation in hungary. pinus nigra was non-invasive, however r. pseudoacacia escaped from cultivation and invaded several vegetation types including pine plantations. it has recently been planned to cut p. nigra plantations and replace them by native tree stands, especially in nature reserves. the scattered presence of r. pseudoacacia specimens in pine stands might place constraints on planned tree replacement because of their vegetative resprouting and recolonization from an established seed bank. the aim of this study was to investigate the soil seed bank under the canopy of solitary r. pseudoacacia specimens found in p. nigra plantations. altogether 250 soil samples were collected from the 0–6 and 6–12 cm soil layers under solitary robinia trees of varying ages (with basal areas between 62.4 and 1089.3 cm2). seeds were separated by sieving then scarified and germinated. seed bank density ranged between 640 and 2285 seeds m–2 with an average distribution of 82.7% and 17.3% in the upper and lower soil layer, respectively. total density of the seed bank and also the seed bank ratio of the lower soil layer increased with tree age. the accumulated seed bank of r. pseudoacacia should be considered in the careful planning of tree replacement operations in pinus nigra stands. keywords: afforestation, dormancy, pinus nigra, plantation, robinia pseudoacacia, seed germination, soil seed bank abbreviations: ba – basal area of tree, dsb – density of seed bank, sbr – seed bank ratio of the lower soil layer, usb – seed bank density in the upper soil layer introduction black locust (robinia pseudoacacia l.) is native to the eastern part of north america. its introduction to hungary dates back to 1710 and it has been intensively used in afforestation practices since then, due to its diversified utilization (walkovszki 1998). at present black locust stands cover about 400,000 hectares, or 23% of the total forested lands of hungary, a higher coverage than in other european countries (rédei et al. 2008). the black loacta bot. croat. 71 (2), 2012 249 * corresponding author, e-mail: cseresnyes.imre@rissac.hu copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. cust has a remarkable spreading capacity due to its rapid vegetative propagation, high adaptability and nitrogen-fixing character (swamy et al. 2002, rice et al. 2004). the invasion success of robinia and of other woody fabaceae species is further enhanced by their persistent soil seed bank and the physical dormancy of seeds (richardson and kluge 2008). the life expectancy of robinia seeds in soil is considerably prolonged by antimicrobial proteins accumulated in the seed tissues, making them resistant to most pathogens (talas-ogras et al. 2005). this species has accordingly become one of the most important woody plant invaders (cronk and fuller 1995) and naturalized in asia and australia as well as in the western and central part of north america (holle et al. 2006). in hungary, black locust spreads spontaneously mainly in semi-arid sandy areas with average annual rainfall around 550–600 mm (rédei et al. 2001). its ever increasing use in afforestation gives a further impulse to its spread by creating new sources of invasion. as a consequence, the black locust is now recorded as one of the most dangerous invasive neophyte species in hungary (balogh et al. 2004). the austrian pine (pinus nigra arn.) is an indigenous tree in the balkan-mediterranean region. in hungary, it was first introduced in the second half of the 19th century (tamás 2003), and nowadays its stands cover 63,000 hectares (source: hungarian forest management inventory). alien tree plantations are usually characterized by a poorly developed herb layer due to the absence of a well organized community of accompanying grasses, forbs and other species. this phenomenon is especially characteristic of hungarian p. nigra plantations, where the strong canopy shading and litter accumulation eliminate the species-rich vegetation existing prior to afforestation with the pine (csontos et al. 1996, 2007). the lack of competitive herband shrub-layers makes austrian pine plantations more susceptible to invasion by aggressively spreading aliens than diverse native forest communities (alpert et al. 2000, mandryk and wein 2006). in hungary, the widespread invasion of r. pseudoacacia in austrian pine stands is a typical example of this phenomenon, but further species like ailanthus altissima (mill.) swingle, asclepias syriaca l. and phytolacca americana l. could also be mentioned in this respect. the low quality timber of the austrian pine has a limited applicability and it has lost any economic importance during recent decades. therefore in hungary – as in other european countries (augusto et al. 2001) – the replacement of alien pine plantations by native forests or grass vegetations has been begun and intensively executed, especially in nature reserves and national parks. obviously, the soil seed banks of alien invasive plants form a real threat to successful conversion of austrian pine plantations to native vegetation types. among the invasive species listed above, r. pseudoacacia deserves especial attention because of its pronounced ability to form a long-term persistent seed bank in the soil (thompson 1993), due to the physical dormancy of seeds caused by hardseededness (czimber 1980). the soil seed bank of the non-native black locust may cause potential nature conservation and forest management problems on the clear-felled pine stands, even if stubs are pulled out to prevent re-sprouting from roots and trunks. therefore, the first aim of our studies was to quantify the seed bank of the black locust in the soil austrian pine plantations that it has invaded. black locust seeds are known to remain viable for some decades (up to 40 years) in the soil (toole and brown 1946), and thus their accumulation under mature specimens is ex250 acta bot. croat. 71 (2), 2012 cseresnyés i., csontos p. pected. the second aim of our studies was to highlight the relationship between the age of individual trees (represented by basal area) and the soil seed bank density beneath their canopy. seeds buried in deeper soil layers generally remain viable for longer period of time than those positioned close to the soil surface (fenner and thompson 2005). if this applies for black locust, then the ratio of viable seeds in the lower soil layer compared to the total amount of soil seed bank should be increased under older tree individuals. investigation of this presumption formed the third aim of the present studies. materials and methods for soil seed bank studies, five austrian pine stands invaded by black locust in the north hungarian region covered by sandy soil were selected: on the boundary of the settlements of ács, isaszeg, csévharaszt, tárkány and komárom (fig. 1). the information about localities and main characteristics of the pine plantations were obtained from local forest inventories (tab. 1). acta bot. croat. 71 (2), 2012 251 robinia pseudoacacia seed bank in austrian pine stands fig. 1. geographical position of pinus nigra stands involved in soil seed bank sampling. 1 – ács; 2 – isaszeg; 3 – csévharaszt; 4 – tárkány; 5 – komárom tab. 1. characteristics of the studied pinus nigra stands invaded by robinia pseudoacacia in hungary. no. of sites location gps positions stand age (years) stand area (hectares) 1 ács n 47°44'51.6"; e 18°01'00.2"; 120 m asl. 28 5.12 2 isaszeg n 47°31'27.1"; e 19°21'25.4"; 205 m asl. 48 4.23 3 csévharaszt n 47°16'53.3"; e 19°24'56.2"; 129 m asl. 51 2.47 4 tárkány n 47°35'06.6"; e 17°56'05.1"; 143 m asl. 57 7.76 5 komárom n 47°44'52.4"; e 18°02'14.8"; 127 m asl. 68 12.35 soil sampling was carried out between 17 july and 12 august 2009. in each sampling site five black locust trees were chosen in the interior of the plantation, i.e. at least 20 m distance from the edge of the pine plantation (to avoid the edge effect). also, attention was paid to selecting solitary black locust individuals, thus ensuring that the soil seed bank beneath the targeted tree was not influenced by neighbouring black locust specimens. afterwards the basal area at breast height (ba) was determined by trunk perimeter measurement. five sampling points were marked out around each tree at a distance of 1.5–2.0 m from the trunk base. in the sampling points leaf litter and freshly fallen legumes were removed from the soil surface, then soil cores of 80 cm2 surface area and 480 cm3 volume were cut from the upper (0–6 cm) and the lower (6–12 cm) soil layers. the five-five subsamples originating from the same vertical layers were bulked, thus forming a 2400 cm3 total soil volume per layer (4800 cm3 per tree). penetration of robinia pseudoacacia seeds into soil layers deeper than 12 cm is negligible (marjai 1995), thus the applied sampling depth was considered to be sufficient. samples were transported to the laboratory and washed through a metal sieve with mesh size of 1.5 mm. after room-temperature (22 °c) drying, black locust seeds were hand-sorted from the debris and counted. the number of viable seeds (which actually forms the seed bank) was calculated after performing germination tests. prior to the germination procedure, seeds were surface sterilized by soaking them for two minutes in a 20% ethanol solution to deal with mould (chuanren et al. 2004). hardseededness was reduced by mechanical scarification carried out with emery paper (baskin and baskin 1998). seeds were placed on filter paper moistened with tap water in petri dishes and kept at 24 °c temperature for 21 days. evaporated water was supplied as necessary, and the germinated seeds were removed daily. on the fifth day the non-swollen seeds were re-scarified and re-germinated. numbers of germinated and non-germinated seeds were determined on the 21st day. the mean seed bank density of each studied austrian pine stand was calculated (as seeds m–2) by averaging the results of the five black locust trees sampled. consequently, these values can be regarded as the outgrowths of a 12,000 cm3 sample volume, which amount exceeds 2–3-fold the required minimal volume, which is generally 4000–6000 cm3 in climax forest vegetation (csontos 2007). morimoto et al. (2010) collected a 12,500 cm3 sample volume from the upper 5 cm soil layer for investigation of the r. pseudoacacia seed bank. the relationship between basal area of tree (ba; cm2) and density of seed bank (dsb; seeds m–2) was evaluated by standard regression methods: linear, power and logarithmic regression. dsb was calculated by adding the numbers of viable seeds found in the upper and lower soil layer. further regression analyses were also carried out in order to relate the ba (i) to density of seed bank found in the upper layer only (usb), and (ii) to seed bank ratio of lower soil layer (sbr; %). in the latter case, sbr was calculated as seed density of the lower soil layer divided by the dsb. statistical significance was assessed at p = 0.05, and among the regression types the one serving the best fit (greatest r2) was accepted. results altogether 250 soil core subsamples were collected (125 from each soil layer), thus 50 samples (25 from each soil layer) were analysed after bulking 5–5 subsamples of each tree sampled. the black locust seed bank was present in both soil layers of each sampled aus252 acta bot. croat. 71 (2), 2012 cseresnyés i., csontos p. trian pine stands. on average 48.6 (ranging from 20 to 96) and 10.1 (ranging from 0 to 38) seeds per tree were found in the upper (0–6 cm) and the lower (6–12 cm) soil layer, respectively. among the 25 black locust trees sampled there was only one in the pine stand of ács, and below it no seeds were found in the lower soil layer. this black locust tree has the smallest ba among the studied specimens. altogether 1466 seeds were washed out of soil samples and subjected to germination testing. the seeds expressed a high germination rate: 1371 seeds, or 93.5% of the total amount, proved to be viable. seeds showed a greater germination rate (95.6%) in the lower than in the upper soil layer (93.1%). on the basis of germination results, 1398 seeds m–2, the mean soil seed bank density was calculated: 1156 and 242 seeds m–2 in the upper and the lower soil layer, respectively (fig. 2). seed bank densities varied among different austrian pine stands. the greatest seed bank, 2285 seeds m–2 was developed in the pine stand of komárom (in 0–6 cm soil layer: 1660 seeds m–2; in 6–12 cm soil layer: 625 seeds m–2), while the smallest, 640 seeds m–2, was found in the pine stand of ács (only 600 and 40 seeds m–2 average densities were calculated in the upper and lower soil layer, respectively). seed distribution between the two soil depths also had a great variability. the mean percentage ratio of seeds found in the upper soil layer was 82.7% of the total amount, consequently 17.3% of seeds appeared from the lower soil layer (fig. 3). the highest (27.4%) and smallest (6.3%) seed bank ratio in 6–12 cm soil depths were detected in the pinewoods of komárom and ács, respectively (the same plantations that were responsible for the highest and the smallest total seed bank densities). the basal area of the sampled black locust trees ranged from 62 to 1089 cm2 (average value was 488 cm2). the simple regression analysis showed a positive correlation (p < 0.001) between the the basal area of tree (ba, cm2) and the total density of seed bank (dsb; seeds m–2, fig. 4). the obtained regression equation [1] indicates a curvilinear relationship: acta bot. croat. 71 (2), 2012 253 robinia pseudoacacia seed bank in austrian pine stands fig. 2. average densities of soil seed bank in the upper (0–6 cm) and lower (6–12 cm) soil layers under robinia pseudoacacia trees growing in pinus nigra stands, in hungary. sampling sites are ordered according to age of the plantations (solid bars show the average values of the five sites). dsb = 61.87 ba0.504 [1] adjusted r2 = 0.7003. relationship between ba and seed bank density in the upper soil layer (usb) proved to be similar (usb = 78.33 ba0.433) but at a lower fit (r2 = 0.5596). 254 acta bot. croat. 71 (2), 2012 cseresnyés i., csontos p. fig. 3. percentage share of soil seed bank distribution between the upper (0–6 cm) and lower (6–12 cm) soil layers under robinia pseudoacacia trees growing in pinus nigra stands. sampling sites are ordered according to age of the plantations (right end bar shows the average of the five sites). fig. 4. correlation between the total density of seed bank (dsb; seeds m–2) and the basal area (ba; cm2) of robinia pseudoacacia trees growing in pinus nigra stands, in hungary. the equation of curve: dsb = 61.87 ba0.504 (r2 = 0.7003) regression analysis resulted in a linear and positive relationship (p < 0.001) between the ba and the seed bank ratio of lower soil layer (sbr) (fig. 5). increase in ba contributes to the increase of sbr (in %), according to equation [2]: sbr = 0.0204 ba + 3.0576 [2] adjusted r2 = 0.5579. two sbr data had to be excluded from statistical evaluation, because of the performed outlier analysis: one black locust tree in the sampled pinewood of komárom and tárkány as well. discussion our investigation verified the ability of the black locust to form a persistent soil seed bank in austrian pine stands, and also showed a correlation between seed bank density and age of tree. the density of robinia pseudoacacia seed banks ranged from 640 to 2285 seeds m–2 in the pine plantations studied; its mean value was about 1400 seeds m–2. under black locust trees in several of the city parks of budapest, an 871 seeds m–2 soil seed bank density and – after mechanical scarification – a 94% germination rate were reported by simkó and csontos (2009). our mean germination result (93.5%) also agrees with the 92–98% value stated by masaka and yamada (2009) on the basis of their ecophysiological research executed in japan. an extensive field study showed a 96% germination rate and a 2000–12 000 seeds m–2 seed bank density in monodominant black locust stands acta bot. croat. 71 (2), 2012 255 robinia pseudoacacia seed bank in austrian pine stands fig. 5. correlation between the seed bank ratio of lower (6–12 cm) soil layer (sbr; %) and the basal area (ba; cm2) of robinia pseudoacacia trees growing in pinus nigra stands, in hungary. (empty squares show outliers excluded from the regression analysis.) the equation of line: sbr = 0.0204 ba + 3.0576 (r2 = 0.5579). in hungary (marjai 1995). in this case, the monodominant character of the studied robinia plantations can explain the relatively high seed bank density. as opposed to marjai’s (1995) research, our investigation was carried out under the canopies of solitary black locust trees in austrian pine stands, where the amount of seeds incidentally scattered to greater distances, was not counterbalanced by seed rain of neighbouring trees. black locust seed bank was detected in both soil layers. since the penetration of seeds to deeper soil layers is time-consuming, a high seed density ratio in the 6–12 cm soil depth was mainly observable under old black locust trees, i.e. having large basal area (fig. 5). the extremely high seed density ratios (shown by the two outlier data) was probably due to local disturbances caused by forest animals or human activities, that by turning up lower soil layers resulted in a local »seed bank profile inversion« (csontos 2007). higher germination rate was detected in the lower (95.6%) than in the upper one soil layer (93.1%). most probably this resulted (i) partly from the favourable environmental conditions for seed survival associated with deep burial (witkowski and garner 2000, fenner and thompson 2005), and (ii) partly from the higher decomposition rate of non-viable seeds in the deeper soil layer. both the growing seed production of elderly trees and the long-term accumulation of dormant seeds may interpret the age-dependent increase in soil seed bank density (marjai 1995). though the fecundity of r. pseudoacacia generally declines after about 40–50 years, corresponding to the decrease in tree vigour, the soil seed bank density may continue to increase owing to the considerable accumulation of dormant seeds (masaka et al. 2010). robinia seed longevity does not exceed 40 years in general (toole and brown 1946), therefore the net seed accumulation rate becomes regressive in the course of time, leading to a curvilinear relationship between seed bank density and basal area of tree (fig. 4). different natural and human disturbances also facilitate the spreading of the black locust. the lifetime of austrian pine is up to hundreds of years in its natural area, but in the hungarian stands the decline of trees begins much earlier, mainly as a consequence of the suboptimal environmental conditions. the resistance of 40–50 year old pines declines quickly in shallow-soiled habitats and thus rapid destruction can be initiated by a permanent drought or infested pathogen fungi (koltay 1990, 1997). these factors promote the penetration and spread of invaders in austrian pine stands. the black locust was naturalized in east asia in the second half of the 19th century; nowadays the invader regularly appears in indigenous pinus thunbergii stands and broad-leaved forests, chiefly in the vicinity of severely disturbed urban regions (maekawa and nakagoshi 1997, lee et al. 2004, song et al. 2005, taniguchi et al. 2007, morimoto et al. 2010). frequent fire events prove that pine plantations in hungary are highly susceptible to forest fires in view of their accumulated resinous needle litter (cseresnyés and csontos 2004; cseresnyés et al. 2006, 2011). the heat effect is capable of breaking the physical dormancy of black locust seeds (marjai 1995, masaka and yamada 2009), thus initiating a quick colonization by the invader in the burnt area (auld and denham 2006, jung et al. 2009). within the scope of sustainable forest management, the replacement of austrian pine stands by natural vegetation types began in nature reserves of hungary in the past few decades. after coniferous stands are clear cut, the recolonisation of indigenous species from nearby native forests and grasslands is generally slow, even if the appropriate propagulum sources are available in its surroundings (matlack 1994, rydgren et al. 1998, tamás 256 acta bot. croat. 71 (2), 2012 cseresnyés i., csontos p. 2001). under these circumstances regeneration of the native vegetation primarily happens from the locally available soil seed bank. in soil of pinus nigra stands the seed bank of species of the native flora (that existed prior to afforestation by pine) became impoverished; its density and richness steadily declined because of their short-term persistent seeds (csontos et al. 1996, augusto et al. 2001). consequently the diverse natural seed bank could be replaced gradually by the high density persistent seed bank of non-indigenous species including the black locust. the germination of r. pseudoacacia seeds can be continued for decades, strongly inhibiting or preventing completely the restoration of the native flora after the removal of an austrian pine stand. the effective root sprouting also promotes the rapid spread of locust trees, especially after local disturbances, and thus finally it can happen that an area just cleared from the non-native pine is occupied by another non-native tree, the locust, which would be a rather unacceptable result. since both the density of the seed bank and the ratio of seeds detected in the lower soil layer increases with the diameter at breast height of the tree, the threat of a spontaneous black locust stand establishment is particularly to be expected in areas already sporadically occupied by old black locust trees. the black locust is responsible for irreversible changes in the physicochemical and biological soil properties by n-fixation, leading to the formation of species-poor nitrophilous weed associations in the herb layer (tobisch et al. 2003). therefore, establishment of the black locust should be prevented by careful planning of the replacement of austrian pine stands by native tree species. acknowledgements many thanks are due to erika cseresnyés-bózsing for her help in seed germination tests and for useful suggestions on the manuscript. we thank foresters józsef mák and ferenc vadas for their help in data collection concerning austrian pine stands. we are grateful to the anonymous referees for their comments. references alpert, p., bone, e., holzapfel, c., 2000: invasiveness, invasibility and the role of environmental stress in the spread of non-native plants. perspectives in plant ecology, evolution and systematics 3, 52–66. augusto, l., dupouey, j-l., picard, j-f., ranger, j., 2001: potential contribution of the seed bank in coniferous plantations to the restoration of native deciduous forest vegetation. acta oecologica 22, 87–98. auld, t. d., denham, a. j., 2006: how much seed remains in the soil after a fire? plant ecology 187, 15–24. balogh, l., dancza, i., király, g., 2004: actual list of neophytes in hungary and their classification according to their success. in: mihály, b., botta-dukát, z. 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(eds.), methods in comparative plant ecology, 199–202. chapman and hall, london. tobisch, t., csontos, p., rédei, k., führer, e., 2003: comparisons of black locust (robinia pseudoacacia l.) stands based on the herb-layer vegetation (in hungarian). tájökológiai lapok 1, 193–202. toole, e. h., brown, e., 1946: final results of the duvel buried seed experiment. journal of agricultural research 72: 201–210. walkovszki, a., 1998: changes in phenology of the locust tree (robinia pseudoacacia l.) in hungary. international journal of biometeorology 41, 155–160. witkowski, e. t. f., garner, r. d., 2000: spatial distribution of soil seed banks of three african savanna woody species at two contrasting sites. plant ecology 149, 91–106. 260 acta bot. croat. 71 (2), 2012 cseresnyés i., csontos p. 625 pise and gaikwad.vp acta bot. croat. 72 (2), 237–256, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/v10184-012-0020-x flora of spoil heaps after hard coal mining in trzebinia (southern poland): effect of substratum properties marcin w. woch1*, magdalena radwañska1, anna m. stefanowicz2 1 department of botany, institute of biology, pedagogical university of kraków, podchor¹¿ych 2, kraków 31-084, poland 2 department of ecology, institute of botany, polish academy of sciences, lubicz 46, 31-512 kraków, poland abstract – the aim of the present study was to investigate the composition of spontaneous plant cover and the physicochemical properties of the substratum of spoil heaps of the siersza hard coal mine in trzebinia (southern poland) abandoned in 2001. floristic and soil analyses were performed in 2011. the substratum was very diverse in terms of texture (sand: 55–92 %, clay: 6–38 %), nutrient content (total c: 1.3–41.0 %, total n: 0.05–0.49 %, total ca: 0.5–7.3 %) and ph (3.7–8.7). moreover, total thallium concentration in the substratum was high, ranging from 6.0 to 14.6 mg kg–1. plant cover varied from 50 to 95 %. the number of plant species per 4 m2 varied from 6 to 29 and correlated negatively with total carbon content (r = –0.85, p < 0.01), and positively with sand content in the substratum (r = 66, p < 0.05). the highest number of species per area unit was observed on a humus substratum, where initial soil has developed on the part of carboniferous waste rock spoil under 20–30 year old trees, and the lowest on carbon shale with coal and culm. among 197 plant species, most belong to asteraceae, fabaceae, poaceae and rosaceae families. hemicryptophytes (49%) and terophytes (18%) predominated. the investigated area was primarily colonized by native species spread by the wind. however, invasive alien species also had a significant share (8%) in the plant cover. key words: carbon, coal spoil heap, seed dispersion, species richness, succession, thallium, trzebinia, poland introduction the increasing number of post-industrial wastelands are resulting in considerable changes in the landscape and vegetation cover. the large hard coal deposits and the fact that coal is the main energy raw material in poland have encouraged mining with its related spoil storage. heaps of waste rock have become a frequent part of the landscape in southern poland, especially in the upper silesia coalfield as well as in the smaller kraków coalfield acta bot. croat. 72 (2), 2013 237 * corresponding author, e-mail: jurania@o2.pl copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. adjoining it from the east. the pioneer flora and vegetation of the hard coal mining waste dumps have been the subject of numerous papers (e.g. caba£a and sypieñ 1987; tokarska-guzik et al.1991; caba£a and jarz¥bek 1999; rostañski 2000, 2006; woryna and rostañski 2003; wo�niak 1998, 2010). however, there is still a need for more floristic research analyzing the properties of specific substrata of these anthropogenic habitats. previously, little botanic research has dealt with the issue of substratum characteristics (baig 1992, rostañski 2006, wo�niak 2010, makineci et al. 2011). the relatively wide variety of species of post-mining dumping grounds includes both native species as well as those alien to central european flora, which have ecological amplitudes enabling them to colonize such places. it is especially vital to discover the edaphic reasons for the differentiation of the flora growing on the post-mining dumping grounds, and whether the changes in the substratum in time or its spatial heterogeneity are more decisive (gibson et al. 1985, picket et al.1987, luken 1990, holl and cairns 2002, wo�niak 2010). the aim of the research presented in this paper was to study the species composition of the plants growing on the post-mining spoil heaps and wastelands of the disused siersza mine (kwk siersza) in trzebinia, where spontaneous plant succession (colonization and change in the community structure over time) have been taking place (krebs 2009). the most important types of substratum occurring in the studied area were classified, for which analyses of physicochemical properties as basic factors determining the flora makeup were performed. material and methods study site the wastelands of the siersza hard coal mine (kwk siersza) studied lie in trzebinia in north-western malopolska (50° 19' 072"n, 20° 44' 6938"e). the beginnings of this mine date back to the year 1808 and it operated until 1999 (kiryk 1994). in 2001, the post-mining waste left behind on the surface was condensed with a vibrating roller, the upper layer being left loose in order to facilitate plant colonization. the area was leveled off, the slope was moderated in the direction of the rail tracks and soil was distributed in some parts. the research area was constituted by post-mining waste dumps as well as wastelands remaining after the buildings had been demolished; altogether the site covers an area of about 20 ha. the remaining habitats studied were also illegal rubbish dumps, areas storing debris from demolished administrative buildings as well singular ash mounds from the siersza power plant. the studied area is located in the œl¹sk-kraków highlands (pagóry myœlachowickie), located at 340 m above sea level (kondracki 2000). post-mining wastelands are mostly surrounded by forest areas – directly from the north, while in the south the forests are situated behind a local railway line delivering coal to the nearby siersza power plant and a street. in the west and east, the area studied borders with the low-density residential areas of the siersza and kopalnia estates (fig.1). climate and vegetation the kraków coalfield is in a transitional climate zone, between a moderate oceanic climate in the west and a moderate continental climate in the east. various air masses collide in this area, which is a result of the location in the centre of europe as well as a latitudinal system of geographical lands. the average annual air temperature is 7.8 °c, while the annual rainfall 770–811 mm. the vegetation season spans between 205 and 215 days. in 238 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. this area, westerly winds are the most frequent, followed by easterly winds (witkowska-kita et al. 2010). trzebinia has been traditionally strongly connected with industry, therefore its plant cover has been largely transformed. in the flora of trzebinia there are 650 plant species, 40 of which are protected in poland (suder and caba£a 2004; woch 2005, 2007, 2012). a high share of alien species (anthropophytes) – 19.8% (suder and caba£a 2004) indicates significant synanthropization. hemicryptophytes are the dominating life forms (39.2%), followed by terophytes (12%) (suder and caba£a 2004). artificially planted pine monocultures dominate the forests; there are also impoverished beech forests as well as riparian deciduous forests. generally, forests cover 43.6% of the municipality (witkowska-kita et al. 2010). field survey the anthropogenic substratum occurring in the studied area can be divided into six types – two spatially dominating: (1) carboniferous shale with coal (about 12 ha), (2) mixed substratum (about 7 ha) and four spatially marginal (no more than 1 ha): (3) culm substratum; (4) humus substratum under 20–30 year old trees of betula pendula, pinus sylvestris, populus tremula and salix caprea; (5) illegal dumps of construction debris with rubbish and single mounds of (6) coal ashes from the power plant. floristic data were compiled during the 2011 vegetation season in patches of each substratum type. to assess the number of plant species per area unit, 10 plots of 4m2 were established in the centers of the most typical patches of each substratum type. a floristic list was drawn up at each plot. additionally, 3 samples of substratum/soil to a depth of 15 cm were collected and bulked to obtain one composite sample from each plot. acta bot. croat. 72 (2), 2013 239 flora of spoil heaps after coal mining fig. 1. study site with two dominating types of substratum: cc – carboniferous shale with coal, mx – mixed substratum. retrieved in 2012 from googleearth and modified. floristic analyses to make a complete list of plant species occurring on 20 ha area of the wastelands of the siersza hard coal mine, the occurrence of each plant species was estimated, taking 6 substratum types into consideration. while analyzing the flora, species frequency, share of geographical and historical groups (rutkowski 2004, sudnik-wójcikowska 2011), share of life forms (rutkowski 2004) as well as the seed proliferation (podbielkowski 1995, sudnik-wójcikowska 2011) were taken into account. the latin nomenclature follows rutkowski (2004). soil analyses before analysis, soil samples were sieved (2 mm mesh). soil texture was determined by a combination of sieving and sedimentation (international organization for standardization 1998). soil ph was measured with a ph-meter after extraction with h2o at a 1:5 (w:v) ratio (international organization for standardization 1994). total carbon was assessed by dry combustion technique with a leco rc-612 (international organization for standardization 1995a), and sulfur with a leco sc-144 dr. nitrogen was measured by a method based on kjeldahl digestion (kjeltec 2300, foss tecator; international organization for standardization 1995b). total cd, pb, zn, tl, mn and mg in soil were measured with the use of atomic absorption spectrometry (varian 220 fs) after digestion in hot concentrated hclo4, exchangeable cd, pb, zn, tl, mn, fe, na, k, ca, mg after extraction with 0.1 m bacl2 (ph 7.0) (international organization for standardization 1995c, modified), and water-soluble cd, pb and zn after extraction with deionized water. phosphorus was measured with the molybdenum blue method after digestion in hot hclo4 (total) or after extraction with 0.5 m nahco3 (olsen et al. 1954) with a colorimeter (hach-lange dr 3800). results the study area was differentiated in terms of the physicochemical properties of the substratum (tab. 1). for example, sand and clay content ranged from 55 to 92% and from 6 to 38%, respectively. soil ph varied from 3.7 to 8.7, total c content from 1.3 to 41.0%, total n from 0.05 to 0.49%, total ca from 0.5 to 7.3%, and total p from 221 to 914 mg kg–1. large differences between sites were also found in concentrations of available element forms. for example, exchangeable ca ranged from 52 to 3039 mg kg–1, exchangeable mg from 1.8 to 1132 mg kg–1, and available p from 0.1 to 14.8 mg kg–1. total content of heavy metals such as cd, pb and zn in the substratum of many sites was slightly elevated, exceeding 5 mg cd kg–1, 260 mg pb kg–1 and 500 mg zn kg–1. in turn, total tl concentration was high at all studied sites and ranged from 6.0 to 14.6 mg kg–1. characteristics of main types of substratum carboniferous shale with coal the prevailing substratum type in the studied area consisted of carboniferous shale and coal. some physicochemical properties of soil developed on this substratum were diverse. this was especially observed in the case of s, exchangeable fe, mn and zn as well as water240 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. acta bot. croat. 72 (2), 2013 241 flora of spoil heaps after coal mining tab. 1. physicochemical properties of substratum and vegetation parameters at studied sites. values for pbex, tlex and cdws were below detection limits in all soils, so they are not shown in the table; d – illegal dumps of construction debris with rubbish, cc – carbon shale with coal, co – coal ashes, hu– humus subsoil, cm – culm subsoil, mx – mixed substratum; s'plant – number of plant species, cov – plant cover; tot – hclo4-extractable, ex – bacl2-extractable, ws – water soluble, av – available (olsen method); bdl – below detection limit. site 1 2 3 4 5 6 7 8 9 10 subsoil type d cc d cc cc co hu cm mx hu sand (%) 82 59 79 71 76 79 90 55 92 81 silt (%) 3 9 5 4 1 10 4 7 2 5 clay (%) 15 32 16 25 23 11 6 38 6 14 ctot (%) 1.5 23.9 2.2 14.5 11.6 5.9 5.8 41.0 1.3 3.3 ntot (%) 0.49 0.05 0.07 0.20 0.28 0.05 0.17 0.48 0.12 0.06 stot (%) 0.42 0.02 0.94 0.04 1.00 0.98 0.19 0.12 0.98 0.04 catot (%) 6.02 0.51 2.58 1.35 1.32 2.68 7.31 0.68 1.81 0.79 cdtot (mg kg –1) 3.45 1.45 5.31 3.15 5.56 1.59 2.76 2.97 1.28 1.83 fetot (%) 0.95 1.50 0.60 2.22 2.01 2.30 2.15 1.48 1.00 2.11 ktot (%) 0.33 0.57 0.17 0.39 0.58 0.26 0.14 0.49 0.09 0.25 mgtot (%) 0.49 0.27 0.38 0.41 0.53 1.04 0.82 0.49 0.09 0.20 mntot (mg kg –1) 238 125 259 180 346 255 1509 321 134 448 natot (mg kg –1) 132 282 123 233 296 914 107 258 163 132 ptot (mg kg –1) 914 351 493 342 365 838 651 336 221 367 pbtot (mg kg –1) 84 69 163 263 240 49 101 107 32 85 tltot (mg kg –1) 9.91 12.37 7.79 11.06 14.01 11.73 14.59 12.51 5.98 9.65 zntot (mg kg –1) 415 137 510 417 456 207 272 343 84 169 caex (mg kg –1) 1716 1140 1460 2792 2288 646 3039 52 885 2428 cdex (mg kg –1) bdl 0.22 0.22 0.07 0.77 0.62 0.63 bdl 0.64 0.63 feex (mg kg –1) 2.04 29.36 2.11 2.52 3.19 2.14 2.06 2.43 2.24 1.97 kex (mg kg –1) 58.3 36.5 93.8 48.0 25.9 38.5 61.9 70.9 40.0 59.9 mgex (mg kg –1) 77.8 330 62.2 432 480 137 364 1132 1.8 110 mnex (mg kg –1) bdl 21.19 0.31 2.48 14.68 bdl 1.27 18.64 bdl 0.52 naex (mg kg –1) 11.1 16.0 11.9 22.5 14.4 11.0 10.3 27.7 105.9 10.4 znex (mg kg –1) 0.36 40.39 0.73 2.41 28.53 bdl bdl 8.90 bdl 0.18 pav (mg kg –1) 6.01 13.32 10.52 2.89 14.77 6.83 2.64 0.07 0.08 0.18 znws (mg kg –1) 0.18 22.71 2.27 0.64 4.11 1.42 0.16 0.54 0.21 0.19 pbws (µg kg –1) 484 290 883 353 257 822 365 388 412 239 ph (h2o) 8.4 3.7 8.2 6.3 5.8 8.2 7.7 6.0 8.7 7.4 s'plant 23 7 24 8 20 20 18 6 23 29 cov (%) 50 90 60 80 95 75 65 80 85 60 -soluble zn and ph. the soil was an acidic sandy clay loam, characterized by high carbon content. total k, pb and zn as well as exchangeable fe, mn, zn, available p and water-soluble zn contents in soil were high in comparison to other soils. these sites were dominated by agrostis stolonifera, calamagrostis epigejos and tanacetum vulgare (tab. 1, sites 2, 4 and 5). mixed substratum a mixed substratum was the second dominant substratum type. it was a mixture of carboniferous shale and coal, debris originating from demolished mine buildings, brought-in soil, sand and dolomite stones. soil was sandy and alkaline, with a relatively low content of total and/or exchangeable forms of many studied elements, both nutrients and xenobiotics (c, k, mg, p, pb, tl and zn). this substratum was characteristic for the most xerothermic habitat of the studied area, covered mainly by agrostis stolonifera, calamagrostis epigejos, melilotus officinalis and solidago canadensis (tab. 1, site 9). culm substratum a substratum consisting of culm left after coal production. because it is not very permeable, numerous shallow water pools have come into being. sandy clay soil was slightly acidic and contained the highest percent of carbon among all the tested soils. moreover, concentrations of exchangeable forms of k, mg, mn and na were elevated. however, the soil was poor with respect to ca and available p content. juncus tenuis, phragmites australis, typha latifolia and t. laxmannii were dominant plant species growing at this site (tab. 1, site 8). humus substratum initial soil developed on the oldest and poorly disturbed (even during mine operation) part of carboniferous waste rock spoil, under 20–30 year old trees of betula pendula, pinus sylvestris, populus tremula and salix caprea. sand or loamy sand soils were alkaline, although total ca and mg contents were diverse. additionally, total mn content was the highest among all tested soils. calluna vulgaris, deschampsia flexuosa and vaccinium myrtillus dominated the undergrowth (tab. 1, sites 7 and 10). illegal dumps of construction debris with rubbish unstable substratum of concrete-brick debris originating from building demolition mixed with household rubbish, resulting from the illegal activities of the local people. alkaline sandy loam or loamy sand contained relatively low amounts of total c and fe. total ca and p contents were relatively high, while n content was diverse. it was covered mainly with annual plant species such as chenopodium album, species from polygonum genus (p. aviculare, p. persicaria) and setaria viridis (tab. 1, sites 1 and 3). coal ashes deposited separately, unstable mounds of ashes after coal burning in the nearby siersza power plant. sandy loam soil developed on the site was alkaline and had the highest total fe, mg, na content among studied soils, and relatively high s, total p and water-soluble pb content. on the other hand, n and exchangeable ca levels were relatively low. the site was cov242 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. ered mainly with grasses such as agrostis stolonifera, calamagrostis epigejos and poa compressa (tab. 1, site 6). floristic properties the number of plant species per 4 m2 varied from 6 to 29, and plant cover from 50 to 95%. the highest number of species per area unit was observed on humus substratum, on illegal dumps of construction debris with rubbish and on a mixed substratum. the number of plant species decreased with increasing total carbon content in the substratum (r = –0.85, p < 0.01; fig. 2) and increased with sand content (r = 0.66, p < 0.05; fig. 3). high carbon and clay contents (relatively low sand content) are related with coal and culm substrata (tab. 1). culm material (site 8) and coal substratum (sites 2 and 4) were found to be unfavorable for vegetation. the only exception was coal site 5, characterized by a relatively high nutrient content (n, s, mg, p) and high number of species. acta bot. croat. 72 (2), 2013 243 flora of spoil heaps after coal mining fig. 2. correlation between total carbon content in substratum and a number of plant species (spearman correlation: r = –0.85, p < 0.01). fig. 3. correlation between sand content in substratum and a number of plant species (spearman correlation: r = 0.66, p < 0.05). in the approximately 20 ha of the studied area of the former siersza hard coal mine, 197 species of plants belonging to 53 families were reported (tab. 2). the highest number of species belonged to asteraceae (36), followed by fabaceae (19), poaceae (17), rosaceae (16), polygonaceae (8), scrophulariaceae (7), salicaceae (7), brassicaceae (6), lamiaceae (6), apiaceae (5), caryophyllaceae (5). some families were represented by 2 or 3 species: aceraceae (3), betulaceae (3), dipsacaceae (3), euphorbiaceae (3), balsaminaceae (2), boraginaceae (2), caprifoliaceae (2), ericaceae (2), fagaceae (2), juncaceae (2), onagraceae (2), orchidaceae (2), papaveraceae (2), pinaceae (2), plantaginaceae (2), ranunculaceae (2), typhaceae (2), violaceae (2). a lot of families were represented by 1 species: amaranthaceae, anacardiaceae, chenopodiaceae, clusiaceae, convolvulaceae, cornaceae, corylaceae, crassulaceae, cyperaceae, equisetaceae, oleaceae, geraniaceae, hippocastanaceae, hypolepidaceae, liliaceae, linaceae, malvaceae, oxalidaceae, resedaceae, rhamnaceae, rubiaceae, solanaceae, urticaceae and vitaceae. as far as life forms are concerned, it was observed that hemicryptophytes predominated (49%). second group were therophytes (18%). quite a few were also mega-phanerophytes (10%), geophytes (9%) and nano-phanerophytes (7%). the remaining species (3%) belonged to non-ligneous chamaephytes, ligneous chamaephytes and hydrophytes (2% each), 1 species was a liana (fig. 4). despite the prevalence of native species (69%), one third of the flora was represented by alien plants (anthropophytes) (31%). among anthropophytes the most numerous were archaeophytes (plants that were introduced up to the end of the 15th century) (13%). the next groups of alien species were: kenophytes (newcomers, after 15th century) (8%), epecophytes (established only in ruderal and/or segetal communities) (4%), hemiagriophytes (established in semi-natural communities) (1%), agriophytes (established in natural 244 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. tab. 2. list of plant species occurring in the study area. substratum: cc – carboniferous shale with coal, co – coal ashes, cm – culm substratum, d – illegal dumps of construction debris with rubbish, hu – humus substratum, mx – mixed substratum (see tab. 1), a order of substratum short-cuts means a descending frequency of species records on a given substratum; form – life forms from raunkiaer: g – geophyte, h – hemicryptophyte, m – megafanerophyte, t – therophyte, c – non-ligneous chamaephyte, ch – ligneous chamaephyte, hy – hydrophyte, n – nano-phanerophyte, l – liana. geographic-historical classification in polish flora: r – native species, k – kenophyte, a – archaeophyte, hemi – hemiagriophyte, agr – agriophyte, erg – ergaziophyte, ep – epecophyte, ef – ephemerophyte, dispersion – types of seed dispersion: an – anemochory, zooch – zoochory, hy – hydrochory, antr – anthropochory, au – autochory, b – barachory; juv. – juvenile specimen. species no of records substratum form status dispersion acer campestre (juv.) 31 mx, d m r an acer negundo (juv.) 35 mx, d m k an acer platanoides (juv.) 34 mx, hu m r an achillea millefolium 144 mx, d h r zooch acinos arvensis 45 mx, d t/h r an aegopodium podagraria 31 d g/h r zooch aesculus hippocastanum (juv.) 1 mx m k zooch/b agrostis gigantea 15 mx, d, cc h r an acta bot. croat. 72 (2), 2013 245 flora of spoil heaps after coal mining species no of records substratum form status dispersion agrostis stolonifera 159 mx, d, hu, cm h r an alnus glutinosa (juv.) 1 hu m r an alnus incana (juv.) 8 cc m r an amaranthus retroflexus 2 d t ep an anthemis arvensis 67 d t a zooch anthemis tinctoria 1 mx h r zooch antirrhinum majus 1 d, cc t ef an/antr arctium lappa 1 mx h r zooch artemisia absinthium 3 mx ch a an artemisia vulgaris 99 mx, d c r an astragalus glycyphyllos 12 mx, hu h r zooch bellis perennis 2 d, cc h r zooch berteroa incana 1 mx, d t/h a an betula pendula 130 mx, cc, hu m r an bidens frondosa 63 cm, d t agr an/antr/ hy/zooch brachypodium pinnatum 25 cc, mx h/c r an bromus hordeaceus 11 mx t r an bromus tectorum 4 mx, d, cc t a an calamagrostis epigejos 179 mx, d, hu, cc, cm g/h r an calendula officinalis 4 d t erg zooch calluna vulgaris 9 hu, mx, cc ch r an capsella bursa-pastoris 18 mx, d t/h a an cardaminopsis arenosa 44 mx, d h/t r an carex hirta 14 d, mx g r an carlina vulgaris 1 hu h r an/zooch carpinus betulus 2 hu m r an cenaturea jacea 63 mx, d, hu h r an cenaturea stoebe 67 mx, d h r an chamaecytisus ratisbonensis 3 mx ch/n r zooch chamaenerion angustifolium 6 d, hu, mx h r an chamomilla recutita 10 mx, d, hu, cc t a an chamomilla suaveolens 13 mx, cc t k an chelidonium majus 27 d h r zooch chenopodium album 47 d, cc t r an cichorium intybus 73 mx, d, hu, cc, co h a an cirsium vulgare 97 mx, d, hu h r an/zooch clinopodium vulgare 52 mx h r an tab. 2. – continued 246 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. species no of records substratum form status dispersion convallaria majalis 6 mx, d g r zooch convolvulus arvensis 2 mx, cc h r an/antr conyza canadensis 111 mx, d, co, co t/h ep an cornus alba 4 mx n r zooch coronilla varia 73 mx, cc h r au corynephorus canescens 24 mx, d h r an crataegus monogyna 1 hu n/m r zooch dactylis glomerata 12 mx h r an daucus carota 127 mx h r zooch deschampsia flexuosa 2 hu h r an dianthus deltoides 2 mx h/c r an dipsacus fullonum 1 mx h a an techium vulgare 71 mx, d, cc h a an/zooch elymus repens 42 mx, d, cc g r an epipactis atrorubens 1 hu g r an epipactis helleborine 3 hu g r an equisetum arvense 32 mx, hu, d g r an erigeron annuus 105 mx, d, hu, cc, cm h k an erysimum cheiranthoides 13 d, cc t a an eupatorium cannabinum 91 d, co h r an euphorbia cyparissias 33 mx, hu h r zooch euphorbia esula 1 d h r zooch euphorbia helioscopia 2 d t a zooch euphrasia rostkoviana 5 hu, mx t r an fallopia convolvulus 16 d t a an fragaria vesca 60 mx, cc h r zooch fragaria x ananassa 1 d h ef zooch frangula alnus 1 hu n r zooch galeopsis tetrahit 1 d t r an galinsoga parviflora 6 d t k an galium mollugo 18 mx h r an genista germanica 4 mx n r zooch geranium pratense 1 hu h r au geum urbanum 4 mx, d h r zooch helianthus annuus 1 mx h ef zooch heracleum sphondylium 32 hu, d h r zooch hieracium vulgatum 2 mx h r an hieracium pilosella 12 mx, hu h r zooch tab. 2. – continued acta bot. croat. 72 (2), 2013 247 flora of spoil heaps after coal mining species no of records substratum form status dispersion holcus mollis 1 mx h/g r an hypericum perforatum 96 mx, hu, d h r an/zooch impatiens glandulifera 2 d t k au impatiens parviflora 7 mx, cc, d t k au juncus conglomeratus 7 cm, mx h r an/zooch juncus tenuis 4 cm, mx, d h k an/zooch knautia arvensis 7 mx h r an lactuca serriola 6 mx, hu h a an lamium album 2 cc, d h a zooch larix decidua 1 d m r an lathyrus pratensis 1 hu h r au leontodon hispidus 33 mx, cc h r an ligustrum vulgare 2 mx n k zooch linum catharticum 2 mx t h r lolium perenne 4 mx, d, cc h r an lotus corniculatus 143 mx, hu, d, cc h r au lupinus polyphyllus 1 mx h ep au malva alcea 1 mx h a au medicago lupulina 141 mx, d t h r medicago sativa 4 d, mx h k au melampyrum pratense 2 mx t r an melandrium album 66 mx, d, co t r an melilotus alba 114 mx, d, cc h/t a an/zooch melilotus officinalis 103 mx, d, cc h a an/zooch molinia caerulea 67 mx h r an myosoton aquaticum 1 hu g r an modontites serotina 4 mx t r an oenothera biennis 112 mx, d h k an ononis spinosa 1 mx h r au origanum vulgare 27 mx h c zooch oxalis europaea 2 d g ep au papaver rhoeas 26 d, mx t a an parthenocissus inserta 1 mx l ef antr pastinaca sativa 96 mx, d, cc h r zooch phragmites australis 129 cm, d, cc g/hy r an picris hieracioides 91 mx, cc h r an pinus sylvestris 145 mx, hu, d, cc m r an plantago lanceolata 82 d, mx h r zooch tab. 2. – continued 248 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. species no of records substratum form status dispersion plantago major 98 d, mx h r zooch poa compressa 105 mx, cc, d, hu h r an poa nemoralis 1 hu h r an polygonum aviculare 31 d t r an/antr polygonum lapathifolium 12 cc, d t r an/antr polygonum persicaria 68 cc, d t r an/antr populus nigra 'italica' 4 cc, d m r an populus tremula 129 mx, cc m r an populus wilsonii 1 mx m erg an populus x canadensis 1 mx m erg an potentilla anserina 17 mx, d, co h r an potentilla argentea 2 mx h r an potentilla reptans 2 mx h r an potentilla supina 28 mx t/h r an prunella vulgaris 63 mx h r hy/zooch prunus avium 3 hu m/n r zooch prunus serotina 2 hu m agr zooch prunus spinosa 1 hu n r zooch pteridium aquilinum 14 hu g r an quercus petraea 6 hu m r zooch quercus rubra 29 hu m hemi zooch ranunculus acris 3 d, hu h r au ranunculus bulbosus 1 mx g h au reseda lutea 16 d, mx h k an rhus typhina 1 cc m/n erg zooch robinia pseudacacia 136 mx, cc m hemi an/zooch rosa canina 3 mx n r zooch rubus ceasius 64 mx, d, hu n/ch r zooch rumex acetosa 14 mx, d, hu h r an rumex acetosella 8 mx, d, co g/h r an rumex obtusifolius 3 mx, co h k an rumex thyrsiflorus 4 mx, co, cc h r an salix alba 66 mx, cc, d m r an salix caprea 75 mx, cc, d n/m r an rin0salix cinerea 1 hu n r an sambucus nigra 2 hu n/m r zooch sanguisorba officinalis 1 cc h r zooch scabiosa ochroleuca 8 mx h r an tab. 2. – continued acta bot. croat. 72 (2), 2013 249 flora of spoil heaps after coal mining species no of records substratum form status dispersion sedum acre 12 mx c r hy/zooch senecio jacobaea 14 hu, cc, d h r an senecio vulgaris 1 d t/h a an setaria viridis 29 d, mx, cc t a an silene vulgaris 74 mx, cc, d, co h/c r an sinapis arvensis 5 d, mx t a an sisymbrium loeselii 3 d t/h ep an solanum tuberosum 1 d g ef zooch solidago canadensis 183 mx, hu, d h/g agr an solidago gigantea 1 mx h/g agr am solidago virgaurea 3 cc h r an sonchus asper 28 mx, d t a an sorbus aucuparia 3 hu m r zooch spiraea media 1 hu n r an stellaria graminea 12 mx, hu h r an symphoricarpos albus 3 hu n ef zooch symphytum officinale 2 cc, d h r an/antr tanacetum vulgare 148 mx, cc, d, hu h r an taraxacum officinale 138 mx, cc, d h ep an torilis japonica 31 hu t/h r zooch tragopogon pratensis 1 cc h ep an trifolium arvense 31 mx, cc t r an/zooch trifolium medium 4 mx h r an/zooch trifolium pratense 137 mx, d h r an trifolium repens 51 mx h r zooch tussilago farfara 144 mx, cc, co, cm g/h r an typha latifolia 16 cm, cc hy/h r an typha laxmannii 3 cm hy/h k an urtica dioica 44 d, mx, hu, cc h r an vaccinium myrtillus 2 hu ch r zooch verbascum lychnitis 1 mx h r an/zooch verbascum thapsus 14 mx, d h r an/zooch veronica chamaedrys 1 cc, hu c r hy vicia angustifolia 1 mx t a au vicia cracca 71 mx, hu h r au viola arvensis 5 mx, d, hu t a zooch viola riviniana 1 hu h r zooch tab. 2. – continued communities) (2%), ephemerophytes (alien species that have been casually introduced into the territory) (3%) and ergaziophytes (1%) (cultivated plants, running wild) (fig. 5). in the studied area the wind was the main vector of seed spreading (anemochory) (58%), the second was animals (zoochory) (31%). other ways of dissemination were: autochory (6%), anthropochory (4%), hydrochory (3%) and barochory (1 species) (fig. 6). among the noted 250 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. geophytes 9% hemicryptophytes 49% megafanerophytes 10% nanofanerophytes 7% terophytes 18% not-ligneous chamaeophytes 3% ligneous chamaeophytes 2% hydrophytes 2% liana 1 % fig. 4. raunkiaer life forms in the flora (%). native species 69% archaeophytes 13% kenophytes 8% epecophytes 4% hemiagriophytes 1% agriophytes 2% ephemerophytes 3% ergaziophygophytes 1% fig. 5. geographic-historical classification of the flora (%). anemochore 58% zoochore 31% antropochore 4% autochore 6% hydrochore 3% barochore 1% fig. 6. types of seed dispersion of the plant species (%). species two – epipactis atrorubens and e. helleborine – are strictly protected in poland, and three are partly protected: convallaria majalis, frangula alnus and ononis spinosa. interesting was the presence of a few 2–3 m patches of a new expansive kenophyte of south-eastern origin – typha laxmannii. discussion the large variety of habitat conditions in these post-mining areas was influenced by the variation in substratum physicochemical parameters, such as humidity, exposure, texture, acidity, concentration of elements indispensable to plants and xenobiotics. the six discerned basic substratum types were characterized by distinct flora. the variability and instability of the substratum influences the diversity of the habitats, and various degrees of disintegration of the soil substrate may occur at different depths (rostañski 2006). carbon shale with coal as well as culm substrata of sparsely overgrown areas in early succession stages contain more exchangeable magnesium in comparison to mixed or humus substrata. carbon content, coarseness and compactness of the substratum were important factors determining plant diversity in the studied area. plant species richness was highest on light calcareous and sandy soils with low content of clay fractions. in turn, it was lowest on heaps dominated by dust fraction with low content of calcium but a high content of coal, as in the case of culm and coal substrata. this relationship is corroborated by the results of cohn et al. (2000) and wo�niak (2010). the high number of plant species on humus substratum may be connected with the advanced age (20–30 years) of succession in these places. generally, total fe, pb, zn and tl as well as exchangeable fe, mn, zn and available p and water-soluble zn content in soil increased with increasing share of coal wastes in a substratum. it is caused by high content of iron sulfides, mainly pyrite and traces of chalcopyrite, galena, marcasite, and sphalerite in coals and their associated sediments (lottermoser 2010). thallium is often found accompanying these minerals. total tl concentrations in the studied soils varied from 5.98 to 14.59 mg kg–1. this indicates that tl concentrations in soils significantly exceeded tl concentrations found in unpolluted soils. kabata-pendias and pendias (1993) stated that unpolluted soils worldwide contain from 0.02 to 2.8 mg tl kg–1, while in poland they contain from 0.01 to 0.4 mg tl kg–1. this means that the most polluted substratum in our study contained ca 37 times more thallium than unpolluted soils in poland. thallium is an element rarely studied in europe, to a much lesser degree than other toxic elements. high tl concentrations in such sites may threaten the environment, because thallium is a highly toxic metal and it may be accumulated in plants and animals living at polluted sites (dmowski and badurek 2002). in the approximately 20 ha of the studied area of the former siersza hard coal mine, 197 species of plants belonging to 53 families have been reported, which indicates a high species richness in this small area. the species composition of the spontaneously emerging plant cover on the dumps and wastelands is conditioned by the occurrence of a certain pool of species in its closest vicinity as well as some of them having an ecological amplitude allowing effective colonization of such places (brändle et al. 2003). this is why the majority of the flora of the studied area was mostly constituted by species occurring within the surrounding 5 km (holl 2002, suder and caba£a 2004, woch 2007). this is a typical regularity for various types of post-industrial places, where primary succession on the bare acta bot. croat. 72 (2), 2013 251 flora of spoil heaps after coal mining substratum will occur, such as quarries, sandpits, sedimentation tanks, dumps of various industrial materials (e.g. jochimsen 1996; cohn et al. 2000; grodziñska et al. 2001; rostañski 2006; woch 2007, 2012; wo�niak 2010). the predominance of anemochoric plants indicates that wind dispersal is one of the most important features of plants that conquer the area first. apart from the production of a large number of seeds, it is a basic feature of pioneer species expanding into a distant area, which direct their main effort towards migration (krebs 2009). the role of zoochory increases considerably together with the time of the succession, playing an ever greater role in the later stages of plant cover formation. it is most probably connected with a parallel colonization of such areas by an ever greater number of animal species as well as an increase in their population. native species dominated the flora of the studied areas (69%), a share comparable to similar areas in central europe, where it is 70% on average (caba£a and jarz¥bek 1999, rostañski 2000, lacina and koutecký 2005). species alien to the flora of a certain area are more frequently encountered on newly anthropogenically affected areas, where they encounter weaker competition from native species. hence a third of the studied flora was constituted by species alien to the polish flora – anthropophytes (31%). this group included new arrivals after the 15th century (neophytes) (19%), as well as arrivals from early historical times (archaeophytes) (12%). such a relatively high participation of neophytes is characteristic for young manmade structures with unstabilized plant cover. species alien to europe, such as robinia pseudacacia and solidago canadensis, were frequent and common in the research area, as well as the less frequent acer negundo, impatiens glandulifera, i. parviflora, padus serotina and quercus rubra. the abundance of these plants is characteristic for similar anthropogenic sites (caba£a and sypieñ 1987, rostañski 2006). the rapid diffusion of these species is result of their increased ecological flexibility in a new environment; especially anthropogenic soils. northern american trees and shrubs are especially invasive, including robinia pseudacacia, quercus rubra and padus serotina, which were introduced into polish industrial areas in great numbers in the second half of the 20th century. robinia pseudacacia coexists with root nodule bacteria significantly increasing the content of nitrates in the substratum (sudnik-wójcikowska 2011). this has a positive effect on the soil formation process, but it can also hinder the appearance of native species and favor the succession of other alien species, which are mostly nitrophilous; similarly, diasporas of quercus rubra and padus serotina easily infiltrated the researched area zoochorically from the nearby forests. when it comes to plants, the formation of a stable mosaic of patches dominated by expansive native grasses (apophytes) such as agrostis stolonifera, calamagrostis epigejos, and invasive alien species as well as solidago canadensis and s. gigantea within the first years of succession is a characteristic phenomenon. such a superficially dominating vegetation type may in turn persist for many years. the presence of the new, expansive neophyte typha laxmannii was interesting. in poland, 29 localities are known, the first being discovered in 1988 near kielce. in europe t. laxmannii occurs naturally in bulgaria, romania, and the ukraine, as well as the south-western part of russia. in the south-western part of the range of occurrence it is a synanthropic species, noted in the czech republic, france, germany, slovakia, slovenia as well as italy (baryla et al. 2005). the next three found localities show that the species is spreading rapidly in anthropogenically modified habitats. t. laxmannii colonization may be connected with anemochoric penetration of the species from the abundant 252 acta bot. croat. 72 (2), 2013 woch m. w., radwańska m., stefanowicz a. m. nearby populations occurring in a locality 3 km west of the researched area (woch 2005). on the other hand, the discoveries of garden-grown ephemerophytes such as antirrhinum majus, calendula officinalis, convallaria majalis, fragaria x ananassa, or solanum tuberosum are connected with the illegal storage of various types of waste. perennial species were predominant in the flora of the researched area (82%, 49% of hemicryptophytes); the second group was constituted by annual plants (therophytes) (18%). these values are comparable with the results of earlier studies performed in similar places, where in general the participation of hemicryptophytes is between 33 and 60%, while the participation of therophytes is between 19 and 30% (caba£a and sypeñ 1987; tokarska-guzik et al. 1991; caba£a and jarz¥bek 1999; woryna and rostañski 2003; rostañski 1997, 2000, 2006). the large participation of therophytes in initial habitats and xeric areas is connected with the fact that completing a life cycle within an often short vegetation season is an important stress tolerance adaptation (madon and médail 1997). habitats with coal ashes and carboniferous shale with coal substratum were only slightly overgrown with plants, the large participation of therophytes in it signifies that it is in early succession stages. on a mixed substratum, the largest number of xerothermic species was reported; this is connected with easy water permeability, quick heating, as well as an increased content of calcium carbonate. ononis spinosa grew on it – a xerothermic grassland species protected in poland. in some of the waste dumps with the mostly advanced stages of succession, communities similar to mixed forests have been formed, in which the layer of trees included betula pendula, pinus sylvestris, populus tremula and salix caprea, and undergrowth connected with calluna vulgaris, deschampsia flexuosa, poa nemoralis and vaccinium myrtillus forest species. compact turf was formed and a 5–10 cm layer of topsoil was accumulated (humus substratum). greater shading in this part of the area, the humidity of the northern slopes, as well as the deposition of organic matter from the nearby forest was responsible for rapid forestation. the location on the outskirts of the former mining plant as well the age of the oldest trees estimated on the basis of the number of branch whorls to be between 20 and 30 years implies that the succession in these places already started when the mine was operating. in the humus substratum only, in the favorable climate under the trees there were forest species strictly protected by the polish law: epipactis atrorubens, e. helleborine as well as the partially protected frangula alnus. plant patches near the forest with rare species of orchids occurred also on the most advanced succession stages in post-mining dumps in the upper silesia coalfield (caba£a and sypieñ 1987, caba£a and jarz¥bek 1999, rostañski 2006), lower silesia coalfield (kuczyñska et al. 1984), as well as in post-mining dumps in england and germany (ash et al. 1994, esfeld et al. 2008). the studies carried out in various post-mining plants reveal their high species abundance as well the occurrence there of rare protected plants (wo�niak and rostañski 2001, rostañski and michalska 2003, rostañski 2006). they can be very important in the protection of biodiversity on the condition that their habitat variety is preserved, leaving them without carrying out recultivation (lundholm and richardson 2010). post-mining areas such as waste dumps remaining after the siersza hard coal mine should be protected in various ways and used as research and education 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275–280, 2014 2014coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication responses of trifoliate orange (poncirus trifoliata (l.) raf.) to continuously and gradually increasing nacl concentration christos chatzissavvidis1*, chrysovalantou antonopoulou2, ioannis therios2, kortessa dimassi2 1 laboratory of pomology, department of agricultural development, democritus university of thrace, pantazidou 193, 68200 orestiada, greece 2 laboratory of pomology, school of agriculture, aristotle university, 54124 thessaloniki, greece abstract – the effect of continuous or gradual stress due to nacl on in vitro growth, proline and sugar accumulation and nutrient acquisition of trifoliate orange (poncirus trifoliata (l.) raf.) explants was studied. apical shoot tips obtained from previous subculture were transferred to a murashige and skoog nutrient medium for proliferation and were exposed to continuous or gradual salinity stress for 42 days. the salt used to induce salinity was nacl added in six concentrations: 0, 50, 100, 150, 200 and 300 mm. gradual salinization was achieved by transferring the explants sequentially every week to the above mentioned nacl concentrations. most salt treatments had a negative effect on the growth parameters of explants. sodium concentration of explants increased in all nacl treatments compared to control and it was higher in the treatments with gradual exposure to salinity. potassium concentration was reduced, mostly in the treatments with continuous exposure. calcium and mg concentrations increased in all saline treatments. in general, the high salinity level in the substrate enhanced the proline and sugar concentrations of the studied explants. in conclusion, salinity had significant impacts on the growth and chemical status of p. trifoliata. keywords: carbohydrates, micropropagation, poncirus trifoliata, proline, salinity stress introduction citrus is one of the most economically important crops worldwide. however, in arid and semi-arid regions drought and saline irrigation water are a common problem and a major consideration in citrus production (ferguson and grattan 2005). the damage to plants exposed to salinity has been attributed to ion toxicity, nutrient imbalance, osmotic and acta bot. croat. 73 (1), 2014 275 * corresponding author, e-mail: cchatz@agro.duth.gr copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 921 chatzissavvidis et al.ps u:\acta botanica\acta-botan 1-14\921 chatzissavvidis et al.vp 24. o ujak 2014 11:44:14 color profile: generic cmyk printer profile composite default screen oxidative stress (grattan and grieve 1998, zhu 2001). compared with other crops, citrus and poncirus are among the most sensitive to soil salinity, although the ability of citrus trees to tolerate salinity varies among species and depends on the rootstock. for orange, the values of electrical conductivity of the soil solution that correspond to a 100%, 50%, or zero yield potential are 1.7, 4.8 and 8.0 ds m–1, respectively (maas 1984). the genera citrus and poncirus are commonly used rootstocks and ornamental species worldwide. field screening in saline sites to select salt tolerant genotypes of citrus rootstocks is complicated because of differential ontogenic reactions of the plant to salinity and a large genotype in combination with environment interaction. in vitro tissue culture is a simple technique that offers a suitable alternative for the study of physiological mechanisms of tolerance to salinity, since it provides relatively fast responses, needs a short testing time and a controlled environment, especially in tree species that have long reproductive cycles (perez-tornero et al. 2009). the aim of this experiment was to examine nutritional, physiological and growth responses of in vitro cultivated trifoliate orange (poncirus trifoliata (l.) raf.) to continuously and gradually increasing external nacl concentration. materials and methods apical shoot tips (length approximately 20 mm) of trifoliate orange (poncirus trifoliata (l.) raf.) obtained from previous subculture, were transferred to a murashige and skoog nutrient medium (murashige and skoog 1962) for proliferation and were exposed to continuous or gradual salinity stress in vitro. the salt used to induce salinity was nacl added in six concentrations: 0 (control), 50, 100, 150, 200 and 300 mm (or 0, 3, 6, 9, 12 and 16 g l–1, respectively). gradual salinization was achieved by transferring the explants sequentially every week to the above mentioned nacl concentrations. all media contained 2 mg l–1 ba (6-benzyladenine), 0.1 mg l–1 iba (3-indolebutyric acid), 30 g l–1 sucrose and 6 g l–1 agar (oxoid no3). the ph of the medium was adjusted to 5.8 prior to autoclaving at 121 °c for 20 minutes. cultures were incubated at 25±2 °c under cool white fluorescent tube lamps (90 mmol m–2 s–1), with a 16-hours photoperiod, for six weeks. each treatment consisted of 15 replicates (tubes). for the explants grown under continuous salt stress, growth characteristics (shoot number and length, fresh and dry matter weight) were recorded at the end of the experiment, while for the explants exposed to gradual salinity stress, growth parameters were recorded at each nacl treatment after each transfer. at the same time, samples of explants were analyzed for proline and carbohydrates according to khan et al. (2000). furthermore, after growth assessment, explants were dried at 75 °c for 48 hours, ground to a fine powder and ashed at 500–550 °c. subsequently, the shoot samples were analyzed for concentration of k, ca, mg, mn, zn and na ions by atomic absorption spectroscopy (perkin-elmer 2380) and for concentration of cl ions by titration with agno3. the experimental layout was completely randomized and the experiment was repeated twice. the reported data are the means of the two experiments. means were compared using the duncan multiple range test for p £ 0.05. results and discussion some explants treated continuously with 300 mm nacl showed signs of chlorosis and/or wilting (data not shown). these salt injuries could be due to accumulation of na or cl 276 acta bot. croat. 73 (1), 2014 chatzissavvidis c., antonopoulou c., therios i., dimassi k. 921 chatzissavvidis et al.ps u:\acta botanica\acta-botan 1-14\921 chatzissavvidis et al.vp 24. o ujak 2014 11:44:14 color profile: generic cmyk printer profile composite default screen (or both) resulting in excessive levels of transpiring leaves, building up rapidly in the cytoplasm and inhibiting enzyme activity, or in the cell walls, altering their functioning (flowers and yeo 1986). it is well established that salinity stress is followed by chlorophyll degradation that leads to a chlorotic appearance in explants. high concentrations of nacl applied under in vitro conditions reduced the chlorophyll content in citrus (perez-tornero et al. 2009) and other woody species (chatzissavvidis et al. 2008). similarly, ghaleb et al. (2010) experimenting with sour orange cultivated under in vitro conditions recorded complete leaf damage at 300 mm nacl. in all nacl treatments, a decreased number of shoots per explants was observed (tab. 1). similarly, in explants of citrus macrophylla a significant decrease in the shoot number was observed at 30 mm nacl (perez-tornero et al. 2009). also, it was reported that continuous or gradual exposure to high nacl in nutrient medium led to a reduced number of shoots in sour orange (shiyab et al. 2003). the length of shoots produced at and above 150 mm nacl was found to be significantly decreased in continuous maintenance treatments and increased in the gradual transfer treatments, except for the treatment with 300 mm nacl (tab. 1). likewise, shiyab et al. (2003) observed that shoot length of sour orange explants was negatively affected by continuous and gradual exposure at a salinity level of 150 mm nacl or higher. similarly, bitter almond (prunus dulcis (miller) d. webb.) explants showed decreased shoot length when cultured on a substrate with high nacl content (shibli et al. 2003). the weight of fresh and dry matter of shoots was decreased by nacl treatments (tab. 1). the fact that the explants gradually exposed to 150–300 mm nacl had higher dry matter weight than those exposed to 50–100 mm seems to be an unexpected result. a possible explanation is that the explants grown under high salinity benefit from gradual exposure (as compared to continuous one) and develop a mechanism that directs salt ions to build up in the apoplast of cells, or be isolated within the vacuole. in any case, further research on gradual and continuous exposure of plants to salinity would elucidate this point. in agreement to these results, shiyab et al. (2003), experimenting with sour orange cultured under nacl stress, observed a negative effect on explant weight, mainly in continuous exposure treatments. the inclusion of nacl in the nutrient medium also led to a decrease in dry acta bot. croat. 73 (1), 2014 277 in vitro response of trifoliate orange to salinity tab. 1. effect of continuously (c) and gradually (g) increased nacl levels on certain growth parameters, total sugar and proline concentrations of trifoliate orange explants. mean values followed by different letters in each column are statistically different (duncan’s multiple range test, p £ 0.05). t re at m en ts n ac l (m m ) number of shoots length of shoots shoot weight (mg) total sugars proline (cm) fresh matter dry matter (mmol g–1 f.w.) (mmol g–1 f.w.) c g c g c g c g c g c g 0 8.1a 8.1a 0.86ab 0.86d 580a 580a 93a 93a 27.03b 27.03c 37.13b 37.13b 50 4.3b 3.9cd 0.99a 0.94c 432b 262e 75b 59d 35.58ab 36.47c 46.80ab 38.45b 100 2.1cd 2.7cd 0.59bc 0.34f 237c 187f 62bc 51e 51.86a 41.87bc 54.51a 38.79ab 150 2.9bc 5.1bc 0.26cd 1.09a 206c 409b 55c 81b 40.60ab 41.24bc 49.95ab 38.60ab 200 1.7cd 5.7b 0.28cd 1.03b 154c 400c 48c 75bc 46.38a 65.48a 55.85a 49.30a 300 0.7d 3.9cd 0.11d 0.68e 141c 313d 52c 69c 40.63ab 50.73ab 56.40a 42.74a 921 chatzissavvidis et al.ps u:\acta botanica\acta-botan 1-14\921 chatzissavvidis et al.vp 24. o ujak 2014 11:44:14 color profile: generic cmyk printer profile composite default screen weight of bitter almond explants (shibli et al. 2003). the reduced vegetative growth of the nacl treated explants may partly be attributed to impairment of their photosynthetic efficiency. photosynthesis, together with cell growth, is among the primary processes to be affected by salinity (munns et al. 2006). moreover, since under in vitro conditions relative humidity is very high, the toxic effects of salinity are more likely to be due to ion toxicity rather than to water stress (mills et al. 2001). sodium concentration in the trifoliate orange explants increased significantly in all nacl treatments compared to control and, specifically, it was higher in the treatments with gradual exposure to salinity. in the treatments above 150 mm nacl (continuous exposure) and those above 50 mm nacl (gradual exposure), na concentration reached excessive or toxic levels (> 0.25%) for citrus leaves. this pattern, the result of increasing nacl in the medium, is a common and expected response and has been reported for many citrus species (dionisio and antonii 1997, shiyab et al. 2003, perez-tornero et al. 2009, ghaleb et al. 2010). moreover, it is well documented that tissue concentrations of cl in citrus increase in response to nacl treatments (dionisio and antonii 1997, perez-tornero et al. 2009, ghaleb et al. 2010). the concentration of chloride in explants was several times higher than that of na, and a similar response was also observed for jojoba (simmondsia chinensis (link) schneid) explants under high salinity conditions (roussos et al. 2007). interestingly, cl increased more than na; cl was 578% higher at 300 mm nacl with respect to 0 mm nacl, whereas na was only 24.5% higher (tab. 2). according to perez-tornero et al. (2009), these results suggest that the important deleterious effects in the poncirus trifoliata explants grown in vitro at increasing nacl concentration could be due to toxic intracellular levels of saline ions, mainly cl. since, in general, cl concentration in orange plants above 1% may be toxic causing leaf burn and defoliation (koo et al. 1984), cl concentrations found in the explants treated with nacl in the present experiment are considered to be high. as regards k concentration, it was reduced in all saline treatments compared to control (tab. 2). specifically, in high nacl treatments, the decline of k concentration was higher in the treatments with continuous exposure. a decline of k+ concentration in citrus explants caused by the inclusion of nacl into the nutrient medium has also been reported by other researchers (dionisio and antonii 1997, shiyab et al. 2003, perez-tornero et al. 2009, ghaleb et al. 2010). what is more, this reduction was less in gradually shocked cultures, as was also found by shiyab et al. (2003) for sour orange. the decrease of k concentrations of 278 acta bot. croat. 73 (1), 2014 chatzissavvidis c., antonopoulou c., therios i., dimassi k. tab. 2. effect of continuously (c) and gradually (g) increased nacl levels on na, cl, k, ca and mg concentrations (% d.w.) of trifoliate orange explants. mean values followed by different letters in each column are statistically different (duncan’s multiple range test, p £ 0.05). treatments nacl (mm) na cl k ca mg c g c g c g c g c g 0 0.220f 0.220e 0.84f 0.84e 1.69a 1.69a 0.91e 0.91d 0.15e 0.15e 50 0.235c 0.247d 2.56e 2.50d 1.53b 1.27c 1.04d 1.76a 0.18d 0.27a 100 0.228e 0.252c 2.64d 2.55d 1.44c 1.43b 1.13c 1.49b 0.19cd 0.25b 150 0.232d 0.253c 3.27c 3.16c 0.88d 1.29bc 1.36b 1.21c 0.20c 0.21c 200 0.261b 0.265b 4.55b 4.74b 0.84d 1.31bc 1.61a 1.14c 0.23a 0.18d 300 0.270a 0.278a 5.66a 5.73a 0.77e 1.31bc 1.64a 1.21c 0.21b 0.18d 921 chatzissavvidis et al.ps u:\acta botanica\acta-botan 1-14\921 chatzissavvidis et al.vp 24. o ujak 2014 11:44:14 color profile: generic cmyk printer profile composite default screen plantlets in the presence of na may be attributed to na-k competition. growth and k concentration in gradual exposure treatments declined less than in cases of continuous exposure, or even increased slightly, which may be an indication of the high adaptability of trifoliate orange to high salinity. in addition, it shows that, for salinity tolerance studies, gradual transfer is preferable to continuous exposure. calcium and mg concentration increased in all saline treatments (tab. 2). these results are in agreement with those of other researchers experimenting with carrizo (poncirus trifoliata × citrus sinensis) hybrids and citrus macrophylla explants (garcia-sanchez et al. 2003, perez-tornero et al. 2009). calcium concentration in explants presented a lower increase in the treatments with gradual transfer than in those with a continuous exposure to high nacl concentration in the nutrient medium. it has been reported that mg concentration in plants is diminished, enhanced or not affected by salinity (dionisio and antonii 1997, roussos et al. 2007). this discrepancy found in proliferated explants, lacking a root system, could be related to differences in the diffusion rates of mineral nutrients in the culture medium with high nacl concentrations (perez-tornero et al. 2009). in all nacl treatments, mn and zn concentrations of explants were significantly reduced by salinity (data not shown). it is possible that the diffusion rate of the above ions decreases with increasing osmotic potential of the medium due to salinity, resulting in lower ion availability. finally, to counter increasing external salinity, plant cells must adjust osmotically, either by accumulating ions or by increasing their synthesis of organic solutes such as proline and sugars (ferguson and grattan 2005). in the present experiment, proline concentration presented a significant increase in the explants cultured with 200 or 300 mm nacl (and with 100 mm nacl under continuous exposure) (tab. 1). similar results were observed in citrus macrophylla explants where even at 60 mm nacl the proline levels increased significantly, rising in line with the external salt concentration (perez-tornero et al. 2009). accordingly, there was a positive effect of salinity on sugar concentration (except the treatment 300 mm nacl in continuous exposure) (tab. 1). increased proline and sugar levels were also reported by rochdi et al. (2003) working with sour orange explants treated with 137 mm nacl. in conclusion, the inclusion of nacl in the nutrient medium leads to significant alterations in the growth, chemical status and proline and sugar concentrations of trifoliate orange explants. acknowledgements we would like to thank s. kouti and v. tsakiridou for their assistance in chemical analyses. references chatzissavvidis, c., veneti, g., papadakis, i., therios, i. n., 2008: effect of nacl and cacl2 on the antioxidant mechanism of leaves and stems of the rootstock cab-6p (prunus cerasus l.) under in vitro conditions. plant cell tissue and organ culture 95, 37–45. dionisio, r. m., antonii, c., 1997: citrus response to salinity: growth and nutrient uptake. tree physiology 17, 141–150. acta bot. croat. 73 (1), 2014 279 in vitro response of trifoliate orange to salinity 921 chatzissavvidis et al.ps u:\acta botanica\acta-botan 1-14\921 chatzissavvidis et al.vp 24. o ujak 2014 11:44:14 color profile: generic cmyk printer profile composite default screen ferguson, l., grattan, s. r., 2005: how salinity damages citrus: osmotic effects and specific ion toxicities. horttechnology 15, 95–99. flowers, t. j., yeo, a. r., 1986: ion relations of plant under drought and salinity. australian journal of plant physiology 13, 75–91. garcia-sanchez, f., carvajal, m., cerda, a., martinez, v., 2003: response of 'star ruby' grapefruit on two rootstocks to nacl salinity. journal of horticultural science and biotechnology 78, 859–865. ghaleb, w. s., sawwan, j. s., akash, m. w., al-abdallat, a. m., 2010: in vitro response of two citrus rootstocks to salt stress. international journal of fruit science 10, 40–53. grattan, s. r., grieve, c. m., 1998: salinity-mineral nutrient relations in horticultural crops. scientia horticulturae 78, 127–157. khan, a. a., mcneilly, t., collins, j. c., 2000: accumulation of amino acids, proline and carbohydrates in response to aluminum and manganese stress in maize. journal of plant nutrition 23, 1303–1314. koo, r. c. j., anderson, c. a., steward, i., tucker, d. p. h., calvert, d. v., wutscher, h. k., 1984: recommended fertilizers and nutritional sprays for citrus. florida agricultural experiment station bulletin 536d. maas, e. v., 1984: salt tolerance of plants. in: christie, b. r. (ed.), the handbook of plant science in agriculture. crc press, boca raton, florida. mills, d., zhang, g., benzioni, a., 2001: effect of different salts and of aba on growth and mineral uptake in jojoba shoots grown in vitro. journal of plant physiology 158, 1031–1039. munns, r., james, r. a., lauchli, a., 2006: approaches to increasing the salt tolerance of wheat and other cereals. journal of experimental botany 57, 1025–1043. murashige t., skoog f., 1962: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum 15, 473–497. perez-tornero, o., tallon, c. i., porras, i., navarro, j. m., 2009: physiological and growth changes in micropropagated citrus macrophylla explants due to salinity. journal of plant physiology 166, 1923–1933. rochdi, a., el yacoubi, h., rochdi, a. r., 2003: responses to nacl stress of citrus aurantium, citrange troyer and poncirus trifoliata in callus cultures: assessment of characters for evaluating salt stress responses in citrus rootstocks. agronomie 23, 643–649. roussos, p. a., gasparatos, d., tsantili, e., pontikis, c. a., 2007: mineral nutrition of jojoba explants in vitro under sodium chloride salinity. scientia horticulturae 114, 59–66. shibli, r. a., shatnawi, m. a., swaidat, i. q., 2003: growth, osmotic adjustment, and nutrient acquisition of bitter almond under induced sodium chloride salinity in vitro. communications in soil science and plant analysis 34, 1969–1979. shiyab, s. m., shibli, r. a., mohammad, m. m., 2003: influence of sodium chloride salt stress on growth and nutrient acquisition of sour orange in vitro. journal of plant nutrition 26, 985–996. zhu, j. k., 2001: plant salt tolerance. trends in plant science 6, 66–71. 280 acta bot. croat. 73 (1), 2014 chatzissavvidis c., antonopoulou c., therios i., dimassi k. 921 chatzissavvidis et al.ps u:\acta botanica\acta-botan 1-14\921 chatzissavvidis et al.vp 24. o ujak 2014 11:44:14 color profile: generic cmyk printer profile composite default screen 635 kabas et al.vp acta bot. croat. 72 (1), 169–184, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 stipetum novakii ass. nova – a new association of serpentine rocky grassland vegetation (halacsyetalia sendtneri) in serbia eva n. kaba[1*, antun a. alegro2, nevena v. kuzmanovi]1, ksenija m. jakovljevi]1, sne@ana s. vukoji^i]1, dmitar v. laku[i]1 1 institute of botany and botanical garden, faculty of biology, university of belgrade, takovska 43, 11000 belgrade, serbia 2 department of botany, faculty of science, university of zagreb, maruli}ev trg 20/ii, 10000 zagreb, croatia. abstract – phytosociological characteristics of grassland communities above serpentines (order halacsyetalia sendtneri h. ritter-studni~ka 1970) in serbia, are analyzed according to braun-blanquet methodology. in order to detect the basic floristic differentiation of analyzed communities ordinary correspondence analysis was applied. cluster analysis was also performed to see the structure and separation of the communities based on the floristic composition. in order to determine diagnostic species, fidelity indices with presence/ absence data and the size of all groups standardized to equal size were calculated. the new association stipetum novakii is described in open rocky serpentine grasslands in brdjani gorge. key words: stipetum, serpentine, ordination, fidelity, serbia introduction serpentine (ophiolithic, ultramafic) rocks represent a group of siliceous rocks which are characterized by calcium deficiency, high concentrations of aluminium, iron, magnesium, nickel, cobalt and chromium, and a few plant nutrients. in contrast to other acid siliceous rocks, the ph values of the serpentine substrate vary from basic to ultrabasic (ph 5.5–8). serpentine flora and vegetation differ from those occurring on other types of siliceous substrates or limestone. open serpentine habitats are characterized by pronounced thermophilous character and xeric conditions. the xerothermic character of serpentine plants is also enhanced by the specific chemical composition of the serpentine substrate (stevanovi] et al. 2003). all of this results in a great number of specific adaptations (nanism, purpuracta bot. croat. 72 (1), 2013 169 * corresponding author, e-mail: ekabas@bio.bg.ac.rs copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees escence, glaucescence, stenophyllism, plagiotropism etc.) as well as the occurrence of numerous relic and endemic species. the largest serpentine areas in europe are in the balkan peninsula (epirus, thessaly and sterea ellas in greece, n, nc and se albania, c and e bosnia, w, sw and c serbia, e and c rhodopes mountains in bulgaria and gorge of river p~inja and se slopes of mt [ar planina in the ne part of republic of macedonia). in the territory of serbia, the most frequent serpentine zones are in its c, w, nw and sw parts (jakovljevi] et al 2011). general overviews of the rich and interesting endemic serpentine flora in the balkans are given by tati] and veljovi] (1990) and stevanovi] et al. (2003). in serbia 19 associations were described in serpentine rocky grasslands (jovanovi] et al. 1986, laku[i] and sabovljevi] 2005) within the alliance centaureo-bromion fibrosi ble~i} et al. 1960 (order halacsyetalia sendtneri h. ritter-studni~ka 1970. since the ultrabasic serpentine substrate appears between 500 to 1900 m a.s.l., their syntaxonomic diversity might be considerable, and this kind of vegetation requires a more objective revision of all syntaxa within the alliance centaureo-bromion fibrosi and order halacsyetalia sendtneri. the aim of this paper is to present the phytosociological analyses of the community dominated by stipa novakii martinovsky and compare it with the other communities of this type in serbia, as well as to give an insight into the ecological characteristics in order to clarify its syntaxonomic position. materials and methods study area the community dominated by stipa novakii has been studied on serpentine rocky soils and small screes in different phases of genesis in br|ani gorge in serbia. br|ani gorge is located high between mt vujan and ilijak hill. it is 5 km long, 5 km away from the town of gornji milanovac, extending in a north to south direction, passing through the ibarska highway. the gorge was cut by the western morava 3rd rank tributary, the despotovica. the sides of the gorge are slanted at an angle of 10–70°, at an altitude around 300 m. geologically, the gorge is mostly composed of the serpentine habsburgite–serpentinite. rock erosion led to the formation of a pedological layer. these are rather shallow soils of low fertility and rock particles are abundantly and inherently present. the soil is eroded and has a mildly alkaline to neutral reaction (vi]entijevi]-markovi] 2004). the xerophilous oak forests of the alliance quercion frainetto represent the potential of vegetation in this place (horvat et al. 1974). however, since the major parts of the forests are completely degraded, the dominant vegetation type in this place is steppe–like grassland vegetation. climate type in this area is temperate-continental, but in its slighter subhumid variant (fig. 1.). the annual mean temperature is 10.8 °c and the annual precipitation is 796 mm. january is the coldest month with a temperature of –3.8 °c, while august is the warmest with atemperature of 26.5 °c. the wettest month is may with a precipitation of 89 mm and february is the driest, with 50 mm (hijmans et al. 2005). the vegetation period lasts eight months. it is important to say that the greatest amount of precipitation comes precisely during the vegetation season (from april to june), winter is the driest season, and winds are not frequent. 170 acta bot. croat. 72 (1), 2013 kaba[ e. n., alegro a. a., kuzmanovi] n. v., jakovljevi] k. m., vukoji^i] s. s., laku[i] d. v. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees field sampling sampling was done according to the methodology and protocols traditionally used in phytosociology. relevés were made according to braun-blanquet (1964). plot size was usually 25 square meters, with the exception of three stands which were made on smaller relevé area (6, 20 and 14 m2) because of the terrain features. the stands were recorded on serpentine soil on altitudes between 320 and 360 m on south, east and south-east exposed slopes with an inclination of 10 to 40 °. cover of rocks within the stand area varied from 10–70%. the sampling period was spring, 2011. nomenclature of plant taxa, with a few exceptions, follows the flora europaea database. all taxa with authors’ names quoted in the paper are given in table 1. data analysis in order to detect specificity and resolve the syntaxonomy of the community dominated by stipa novakii, we processed 185 relevés belonging to 18 associations traditionally included in the alliance centaureo-bromion fibrosi (halacsyetalia sendtneri), distributed throughout the territory of serbia (table 1, fig 2). the communities eryngio-brometum fibrosi z. pavlovi}, 1962 ex v. randjelovi} 2004 and halascya sendtneri-potentilla mollis z. pavlovi} 1962, are described just on the basis of two relevés and hence not included in numerical analyses. after transformation of braun-blanquet abundance-cover scale according to westhoff and van der maarel (1973), the relevés were subjected to correspondence analysis (ca) in order to detect the basic structure of the floristic composition. relevés that showed a significant deviation from the main core of the relevés typical of the analyzed vegetation were excluded from further numerical analysis. finally, the main core of relevés was classified using the upgma (unweighted pair-group average linkage) cluster method based on chord distance as a heterogeneity measure. these analyses were performed using flora software (karad@i] et al. 1998). due to the fact that traditionally recognized characteristic and differential species, in most cases, have mainly regional validity, the same species can occur in different species acta bot. croat. 72 (1), 2013 171 a new serpentine rocky grassland association 0 5 10 15 20 25 30 35 40 45 50 i ii iii iv v vi vii viii ix x xi xii 0 10 20 30 40 50 60 70 80 90 100 temperature rainfall ra in fa ll (m m ) te m p e ra tu re (° c ) 10.76 °c 796 mm fig. 1. the climate diagram for br|jani gorge in serbia (from walter and leith 1964). 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 172 acta bot. croat. 72 (1), 2013 kaba[ e. n., alegro a. a., kuzmanovi] n. v., jakovljevi] k. m., vukoji^i] s. s., laku[i] d. v. tab. 1. communities traditionally included in alliance centaureo-bromion fibrosi of order halacsyetalia sendtneri, that are used to compare. no. associations utm (10×10 km) localities reference 1. festuco duriusculaeeuphorbietum glabriflorae s. jov. et r. jov. cp75 tara, zlatibor, 980–1060 m a.s.l. jovanovi] et al. (1992) 2. festuco sulcataepotentilletum zlatiborensis z. pavlovi} 1951 ct94 zlatibor, 890–1020 m a.s.l. pavlovi] (1951) 3. poo molinieriplantaginetum holostei z. pavlovi} 1951 dp72 studena mt., 910–1250 m a.s.l. pavlovi] (1951) 4. artemisio-teucrietum montani bla`en~i}, vu~kovi}, r. 1983 dp91 go~, mitrovo polje, 710 –810 m a.s.l. bla@en^i] and vu^kovi] (1986) 5. carici humilis-festucetum pancicianae r. jovanovi} et s. jovanovi} 1987 dm79 kopaonik, 1650–1900 m a.s.l. jovanovi]-dunji] and jovanovi] (1987) 6. potentillo tommasinianaefestucetum pancicianae d. laku{i} 1989 dm79 kopaonik, krmeljica, 1050–1070 m a.s.l. laku[i] and ran\elovi] (1996) 7. artemisio-silenetum armeriae d. laku{i} 1989 dm99 kopaonik, vlajkovci, 700–750 m a.s.l. laku[i] and ran\elovi] (1996) 8. sedo-dianthetum serbici z. pavlovi} 1967 dn77 rogozna, 500–850 m a.s.l. pavlovi] (1967) 9. potentillo-fumanetum bonaparti rexhepi 1979 dn85 ibar valley – kosovo, 600–900 m a.s.l. rexhepi (1979) 10. hyperico-euphorbietum glabriflorae rexhepi 1978 en05 kopaonik, barelj, 1500m a.s.l. rexhepi (pers. comm.) 11. onosmato-scabiosetum fumarioides rexhepi 1978 dn60 kozni~ka boka, 800–960 m a.s.l. rexhepi (1985) 12. polygalo-genistetum hassertianae ble~i} et al. 1969 dn60 kozni~ka boka, 620–810 m a.s.l. ble^i] et al. (1969) 13. hyperico-euphorbietum glabriflorae rexhepi 1978 dn80 drenica, nekoc, 730–895 m a.s.l. krasniqi and millaku (2007). 14. sedo-bornmüellerietum dieckii ble~i} et al. 1969 dn97 [arplanina, ostrovica, 1280–1380 m a.s.l. ble^i] et al. (1969) 15. bornmüellero dieckiiseslerietum latifoliae s. jov. et v. stev. dn97 [arplanina, ostrovica, 1700 m a.s.l. jovanovi] et al. (1992) 16. cynancho-saponarietum intermediae ble~i}, tati} et krasni}i 1969 em07 [ara mt., brezovica, 820–1270 m a.s.l. ble^i] et al. (1969) 17. stipeto-convolvuletum compacti millaku et al. 2011 em16 kosovo, gurane, 650–735 m a.s.l. millaku et al. (2011) 18. festuco-plantaginetum serpentini randjelovi} et ru`i} 1982 em57 pre{evo, miratovac, 600–960 m a.s.l. ran\elovi] and ru@i] (1983) 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:31 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees combinations in different regions (e.g. in other mountain ranges as a consequence of a different florogenesis). this is the case with all the mentioned lists of balkan syntaxa in which characteristic and differential species are characterized with a high degree of acta bot. croat. 72 (1), 2013 173 a new serpentine rocky grassland association fig. 2. the map of the localities of analyzed communities. the distribution is mapped using an underlying 10 x 10 utm coordinate grid 100 x 100 km (utm grid zone 34, the black dots with the corresponding ordinal numbers represents the localities of the communities listed in table 1. the black square represents the locality of the stipa novakii community. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:34 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees subjectivity, rather intuitive than objective. therefore, in this article, we used the concept of diagnostic and dominant species proposed by chytrý et al. (2002) and chytrý and tichý (2003). using the statistical measures of fidelity, we quantified concentrations of species occurrences in groups of classified sites in order to determine diagnostic species (chytrý et al. 2002). the size of the site groups in the data set is virtually equalized, while the relative frequencies of species occurrence within and outside of these groups are kept constant (tichý and chytrý 2006). then, fidelity is calculated using the f-values of the association. with the use of this approach, species fidelity to a community types does not depend on the number of available field records. since these values of fidelity measures are not the functions of statistical significance of fidelity, we performed the monte carlo test of significance of observed maximum indicator value for species with 4999 permutations. for the calculation of f-values, pcord 6.0 software (mccune and mefford 2011) was used. in order to determine dominant species we calculated the coverage index (ic) according to lausi et al. (1982). in the purpose of defining the geoelements we used the groupings made by meusel et al. (1965, 1978), meusel and jäger (1992), modified for the serbian territory by stevanovi] (1992). results ordination correspondence analysis has shown that the relevés of ass. festuco sulcatae-potentilletum zlatiborensis are completely separated from the rest of the relevés along the negative part of the first principal axes (fig.3), which correlates with the highest extent of variability (about 85%). at the same time, this analysis completely separated the relevés with the domination of stipa novakii from the rest of the communities along the positive part of the second axis. it can also be seen that the kosovan site groups festuco-plantaginetum serpentini, hyperico-euphorbietum glabriflorae and stipeto-convolvuletum compacti are well separated along the negative part of the second axis. the rest of the relevés representing the centaureo-bromion fibrosi communities are concentrated in the centre of correspondence space. classification the results of the cluster analysis, performed on the data set from which relevés of ass. festuco sulcatae-potentilletum zlatiborensis were excluded has shown that three main »stand groups« (clusters) could be differentiated. the cluster that separates at the highest heterogeneity level consists of two relevés from the ass. poo molinieri-plantaginetum holostei. these two stands are separated from the community they belong to, probably because they were recorded in open rocky grasslands, covering up to 25% and at higher altitudes of 1250 m a.s.l. from 91 species in total in these stands, only 10 and 12 species of the association are present. at the next level two main clusters can be seen, one of them representing the community dominated by stipa novakii, and the other one representing the rest of the centaureo-bromion fibrosi communities. within this last cluster, at different heterogeneity levels, 18 small subclusters can be distinguished. each of them represents a more or less discrete community (fig 4). 174 acta bot. croat. 72 (1), 2013 kaba[ e. n., alegro a. a., kuzmanovi] n. v., jakovljevi] k. m., vukoji^i] s. s., laku[i] d. v. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:34 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees dominant and diagnostic species five taxa out of 476 were exclusively present in the stipa novakii community from br|ani gorge, whereas 445 taxa were exclusive for the rest of the analyzed communities (centaureo-bromion fibrosi) and absent from the relevés from br|ani gorge. only 26 taxa were present in both groups. the most frequent and the most abundant species with large coverage indices in stipa novakii community are: stipa novakii chamaecytisus hirsutus (l.) link, erysimum linariifolium tausch, silene bupleuroides l., artemisia lobelii all., galium purpureum l., chrysopogon gryllus (l.) trin., polygala supina schreber and cheilanthes marantae (l.) domin (tab. 2). species with the highest f-values, and a high degree of fidelity to this community are: stipa novakii, chamaecytisus hirsutus, erysimum linariifolium, polygala supina, chrysopogon gryllus which again indicates that these species could be diagnostic for this community. a set of species characterized by relatively high ic-values, but very low f-values was the following: leontodon crispus subsp. asper (waldst. et kit.) rohlena, halacsya sendtneri (boiss.) dörfler and teucrium montanum l. (tab. 2). high ic-values and low f-values indicate that they are common (with relatively large frequency, abundance and cover) in the investigated community, but without a diagnostic value for the stipa novakii community. their fidelity values are much greater in respect to some other communities of centaureo-bromion fibrosi, where they can even be a diagnostic species. acta bot. croat. 72 (1), 2013 175 a new serpentine rocky grassland association fig. 3. correspondence analysis (ca) of 19 analyzed communities of serpentine rocky ground vegetation traditionally included in alliance centaureo-bromion fibrosi of order halacsyetalia sendtneri in serbia. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:36 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 176 acta bot. croat. 72 (1), 2013 kaba[ e. n., alegro a. a., kuzmanovi] n. v., jakovljevi] k. m., vukoji^i] s. s., laku[i] d. v. t a b . 2 . d ia gn os ti c ta bl e of ne w as so ci at io n s ti p e tu m n o v a k ii as s. no va .f ro m b r| an sk a g or ge in s er bi a. (* h o lo ty p u s h o c lo c o ) u t m gr id 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 34 t , d p 57 a lt it ud e (m ) 32 0 32 1. 9 32 1. 9 32 7. 1 33 1. 9 33 1. 9 35 5. 1 33 5. 9 33 8. 9 34 2. 9 36 0. 9 e xp os it io n s e s e s e s e s e e s e s s s s e s lo pe (° ) 35 -4 0 40 30 10 -1 5 30 30 15 -2 0 30 30 35 30 c ov er of ro ck s (% ) 45 10 15 70 30 65 30 50 45 40 50 r el ev é ar ea (m ²) 25 6 20 14 25 25 25 25 25 25 25 n um be r of sp ec ie s pe r re le vé 20 8 12 18 11 11 12 13 12 13 17 r el ev é n o. 1 1 2 3 4* 5 6 7 8 9 10 11 p o n t s u b m s ti p a n o v a k ii m ar ti no vs ký 3 1 1 3 1 3 1 3 2 2 2 10 0 v (1 -3 ) 55 .5 6 0. 97 0. 00 02 s u b m c h a m a e c y ti su s h ir su tu s (l .) l in k 1 + 1 1 1 2 1 1 2 1 91 v (+ -2 ) 33 .3 3 0. 91 0. 00 02 s e p e ry si m u m li n a ri if o li u m t au sc h 1 1 1 + + 1 1 1 1 82 v (+ -1 ) 25 .2 5 0. 83 0. 00 02 p o n t s u b m s il e n e b u p le u ro id e s l . 1 1 1 + + 1 1 1 1 82 v (+ -1 ) 25 .2 5 0. 52 0. 00 14 e a z c h e il a n th e s m a ra n ta e (l .) d om in 1 1 1 1 1 1 1 64 iv (1 ) 21 .2 1 0. 55 0. 00 06 s u b m a rt e m is ia lo b e li i a ll . + 1 1 1 1 2 2 1 73 iv (+ -1 ) 27 .2 7 0. 5 0. 00 20 p o n t s u b m c h ry so p o g o n g ry ll u s (l .) t ri n. + 1 1 1 1 1 1 1 73 iv (+ -1 ) 23 .2 3 0. 71 0. 00 02 e a p g a li u m p u rp u re u m l . 1 1 1 1 1 + 1 1 73 iv (+ -1 ) 23 .2 3 0. 65 0. 00 02 p o n t s u b m p o ly g a la su p in a s ch re be r 1 + 1 + 1 1 1 1 73 iv (+ -1 ) 22 .2 2 0. 72 0. 00 02 m e d s u b m f ra x in u s o rn u s l . r + 1 + + 45 ii i (r -1 ) 14 .1 4 0. 52 0. 00 02 m e d p o n t l e o n to d o n c ri sp u s v il l. su b sp . a sp e r (w al ds t. et k it .) r oh le na r + r 1 1 + 55 ii i (r -1 ) 12 .1 2 0. 18 0. 32 49 m e d s u b m s c ro p h u la ri a c a n in a l . su b sp . tr is ti s (k .m al ý) n ik ol i} 1 1 1 1 1 1 55 ii i (1 ) 18 .1 8 0. 58 0. 00 02 p o n t s u b m b ro m u s p a n n o n ic u s k um m er et s en dt ne r 2 1 1 1 + 45 ii i (+ -2 ) 16 .1 6 0. 41 0. 00 30 e a z t h y m u s g la b re sc e n s w il ld .s u b sp . gl ab re sc en s 1 1 + 1 1 1 55 ii i (+ -1 ) 17 .1 7 0. 58 0. 00 02 frequency(%) constancyclass coverindexaccording tolausi(ic) fidelityindex(f) p(montecarlotest) areatype 2 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:36 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 177 a new serpentine rocky grassland association je p t e u c ri u m m o n ta n u m l . 1 + 1 1 1 1 55 ii i (+ -1 ) 17 .1 7 0. 07 0. 76 60 s e g a li u m a lb u m m il le r 1 + 1 1 1 36 ii i (+ -1 ) 10 .1 0 0. 53 0. 00 02 h o l a sp le n iu m ru ta -m u ra ri a l . 1 r + 27 ii (r -1 ) 6. 06 0. 34 0. 00 68 p o n t s u b m m e d ic a g o p ro st ra ta ja cq . r 1 + 27 ii (r -1 ) 6. 06 0. 07 0. 70 37 m e d s u b m h a la c sy a se n d tn e ri (b oi ss .) d ör fl er 3 1 2 27 ii (1 -3 ) 15 .1 5 0. 17 0. 37 49 m e d s u b m a e th io n e m a sa x a ti le (l .) r .b r. su b sp . sa xa ti le 1 + + + 36 ii (+ -1 ) 9. 09 0. 36 0. 00 88 p o n t s u b m s ta c h y s re c ta l . 1 + + 27 ii (+ -1 ) 7. 07 0. 25 0. 02 88 p o n t s u b m c e n ta u re a st o e b e l . su bs p. rh e n a n a (b or ea u) s ch in z et t he ll . r + 18 i (r -+ ) 3. 03 0. 24 0. 06 86 s je p a sp le n iu m c u n e if o li u m v iv . 1 + 18 i (+ -1 ) 5. 05 0. 20 0. 14 64 p o n t s u b m p ru n u s m a h a le b l . + + 18 i (+ ) 4. 04 0. 32 0. 00 50 p o n t s u b m c o ti n u s c o g g y g ri a s co p. + + 18 i (+ ) 4. 04 0. 30 0. 01 42 s e a ju g a g e n e v e n si s l . + 9 i (+ ) 2. 02 0. 22 0. 06 02 p o n t s u b m c a rd u u s c a n d ic a n s w al ds t. et k it . su bs p. c a n d ic a n s + 9 i (+ ) 2. 02 0. 22 0. 06 02 m e d s u b m q u e rc u s c e rr is l . + 9 i (+ ) 2. 02 0. 22 0. 06 78 m e d s u b m q u e rc u s p u b e sc e n s w il ld .s ub sp . p u b e sc e n s + 9 i (+ ) 2. 02 0. 22 0. 06 78 m e d s u b m s c a b io sa fu m a ri o id e s v is .e t p an ~i } + 9 i (+ ) 2. 02 0. 04 10 .0 00 m e d s u b m s il e n e p a ra d o x a l . + 9 i (+ ) 2. 02 0. 00 10 .0 00 1 r el ev és 111 : s er bi a, b r| an sk a kl is ur a, 43 .9 93 n ,2 0. 42 0 e 2 p o n t -s u b m p on ti cs ub m ed it er ra ne an ,s u b m s ub m ed it er ra ne an ,s e p c en tr al e ur op ea n m ou nt ai n, e a z e ur oa si an ,e a p e ur oa si an m ou nt ai n, m e d -s u b m m ed it er ra ne an -s ub m ed it er ra ne an ,j e p s ou th e ur op ea n m ou nt ai n, s e c en tr al e ur op ea n, h o l h ol ar ct ic ,s je p c en tr al -s ou th er n e ur op ea n m ou nt ai n 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:36 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 178 acta bot. croat. 72 (1), 2013 kaba[ e. n., alegro a. a., kuzmanovi] n. v., jakovljevi] k. m., vukoji^i] s. s., laku[i] d. v. fig. 4. cluster analysis of the second data set of 18 analyzed communities of serpentine rocky ground vegetation traditionally included in the alliance centaureo-bromion fibrosi of the order halacsyetalia sendtneri in serbia. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:39 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees phytogeographic analysis floristic composition of stipa novakii community showed the dominance of species of mediterranean-sub-mediterranean and pontic-sub-mediterranean geoelements (11 taxa i.e. 35% in each chorological type). taxa with centres of distribution primarily in the central european or mountain regions are present in much lower numbers (tab. 2). since the community is developed in serpentine open rocky grasslands, such a floristic composition was expected. these habitats are known to have extremely thermophilous character, conditioned by the serpentine geological substratum, and the deriving shallow and poor soils, developing on more or less steep slopes with southern expositions. all the results presented suggest that the community dominated by stipa novakii developed on open serpentine rocky soils in br|ani gorge is well differentiated from other associations within the alliance centaureo-bromion fibrosi, and we propose a new association under the name stipetum novakii ass. nova hoc loco. syntaxonomical treatement ass. stipetum novakii ass. nova hoc loco (holotypus tab. 2, rel. 4, fig. 5) dominant species: stipa novakii (1–3) (ic = 56), chamaecytisus hirsutus (+–2) (ic = 33), erysimum linariifolium (+–1) (ic = 25), silene bupleuroides (+–1) (ic = 25), artemisia lobeli (+–1) (ic = 27), galium purpureum (+–1) (ic = 23), chrysopogon gryllus (+–1) (ic = 23), polygala supina (+–1) (ic = 22), cheilanthes marantae (1) (ic = 21). diagnosis: stipetum novakii is secondary rocky grassland developed on mostly se and s (rarely e) exposed cliffs, with an inclination between 10° and 40°. the dominant and characteristic species stipa novakii with its dense tufts, forms poorly to richly developed stands up to 60 cm high, covering 10–70 % (average 40.9 %) of the relevé area (tab. 2). the very poorly developed, thin pioneer soil layer with a large concentration of heavy metals acta bot. croat. 72 (1), 2013 179 a new serpentine rocky grassland association fig. 5. grassland of the association stipetum novakii in br|ani gorge serpentines (photo: s. vukoji~i}) 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees results in there being relatively low plant diversity. in eleven relevés representing this community only 31 species were found. the average number of species per relevé was 13. diagnostic species of the association are: stipa novakii, chamaecytisus hirsutus, erysimum linariifolium, polygala supina, chrysopogon gryllus, galium purpureum. however, such a situation enables the development of a certain number of endemic serpentine species such as stipa novakii, halacsya sendtneri (boiss.) dörfler, scrophularia canina l. subsp. tristis (k. malý) nikoli} etc. since the other characteristic species, as well as the differential ones, appear to be common accompanying species, we have chosen only stipa novakii as a name giving species. the fact that it is endemic for very small serpentine area in western serbia is also the criteria for stipa novakii to be elected for the nominal species and also points up the endemic status of the community itself. the occurrence of some differential shrub and forest species, such as chamaecytisus hirsutus, prunus mahaleb l., cotinus coggygria scop., quercus cerris l., quercus pubescens willd., distinctly characterizes this grassland community. the community basically represents a relatively short-term transitional syngenetic stage in the succession of vegetation, performing its role in binding and fixing the mobile substrate of mature screes on serpentine slope bedrock. the progressive succession will probably result in secondary grasslands of the chrysopogonetum grylli type, after which shrubs and short trees will prevail in the physiognomy of the vegetation cover. nomenclatural remark holotypus: tab. 2, relevé 4 – holotypus hoc loco syntaxonomical scheme festuco-brometea br.-bl. et r. tx. 1943 halacsyetalia sendtneri h. ritter-studni~ka 1970 all. non defined stipetum novakii kaba{ et d. laku{i} ass. nova hoc loco discussion in the analyzed serpentine rocky grasslands vegetation, only 5 out of 476 taxa were exclusively presented in stands dominated by stipa novakii. all of the investigated communities had 26 taxa in common, with a certain number of balkan serpentine endemics among them, such as: scrophularia canina subsp. tristis, silene bupleuroides, scabiosa fumarioides vis. et pancic, halacsya sendtneri and others, suggesting that they really do belong to the same higher syntaxa. of the taxa, 454 were not recorded in the community from br|ani gorge, and were present only in the rest of the centaureo-bromion fibrosi communities. the most frequent and the most abundant species present in the stands from br|ani gorge is stipa novakii. this is an endemic species for west serbian serpentines. its range descends southwards kosovo, where it is gradually replaced with another endemic species, stipa mayeri martinovský. the endemic status of its dominant species implies the endemic status of the community stipetum novakii itself. correspondence analysis has shown, firstly, that ass. festuco sulcatae-potentilletum zlatiborensis is completely separated from all other analyzed communities, representing well-supported syntaxa. in addition to its obvious detachment from other communities, we have noticed that the community was listed within halacsyetalia sendtneri and centau180 acta bot. croat. 72 (1), 2013 kaba[ e. n., alegro a. a., kuzmanovi] n. v., jakovljevi] k. m., vukoji^i] s. s., laku[i] d. v. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees reo-bromion fibrosi by mistake. due to ecological, physiognomic and structural characteristics such as closed plant cover with ground cover of 90–100%, a typical grassland look, the fact that it is developed on plateaus (slope = 0–5°, extremely 15°) with the thicker soil layer with more humus, a higher floristic richness (86 registered species), in the original paper in which it was published, it was considered to be an intermediate syngenetic stage connecting the open serpentine vegetation of poo molinieri-plantaginetum holostei and the dry mountainous grasslands of koelerietum montanae type (pavlovi] 1951). the authors consider that this association does not belong to the order halacsyetalia sendtneri and should be listed within festucetalia valesiacae br.-bl. et r. tx. 1943 and chrysopogoni-danthonion alpinae koji} 1957, but this exceeds the aim of this paper. furthermore, both numerical analyses have also shown that stipetum novakii ass. nova significantly differs from the rest of the communities representing centaureo-bromion fibrosi. the fact that species bromus erectus hudson subsp. fibrosus (hackel) stojan. et stefanov. is absent from the community from br|ani gorge, as are most of the diagnostic species of alliance centaureo-bromion fibrosi, such as centaurea kosaninii hayek, sedum serpentini janchen, alyssum montanum l. subsp. serbicum novák, thymus lykae degen et jáv., stachys recta l. subsp. baldaccii (k. malý) hayek, stachys scardica (griseb.) hayek, euphorbia glabriflora vis., poa badensis haenke ex willd., alyssum markgrafii o. e. schulz, plantago holosteum scop. etc., indicates the high specificity and distinctiveness of this community and may lead us to think that its place is not at all within centaureo-bromion fibrosi. regarding this, there is a high probability that stipetum novakii ass.nova should belong to a new alliance, yet to be defined by further research. on the other hand, high f-values in the species scrophularia canina subsp. tristis, cheilanthes marantae, silene bupleuroides, artemisia lobelii, bromus pannonicus kummer et sendtner, halacsya sendtneri, and negative f-values within the species festuca duriuscula l., potentilla arenaria borkh. var. tommasiniana (f. w. schultz) hegi, koeleria splendens c. presl, danthonia provincialis dc., festuca valesiaca schleicher ex gaudin subsp. sulcata (hackel) hegi, festuca valesiaca subsp. pseudovina (hackel ex wiesb.) hegi, indicate that the stipetum novakii community should not be listed within xerophilous rocky grasslands of the order festucetalia valesiacae, and its syntaxonomical position is, without any doubt, within the endemic order of the central balkans, halacsyetalia sendtneri. since our numerical analysis showed that the heterogeneity level that separates stipetum novakii ass. nova from the rest of the analyzed communities provides a sufficient basis for conferring on it the status of the association and also indicates that a new alliance should be introduced, further analyses are needed in order to get a more consistent syntaxonomic classification within the order halacsyetalia sendtneri. acknowledgements the authors are grateful to the serbian ministry of science and technological development (project no. 173030 biodiversity of the plant life of serbia and balkan peninsula – assessment, sustainable use and conservation (2011–2014)) for financial support. the authors also wish to thank the editor and reviewers for their valuable comments and suggestions, which significantly improved this manuscript. acta bot. croat. 72 (1), 2013 181 a new serpentine rocky grassland association 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:42 color profile: generic cmyk printer profile composite 150 lpi at 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(ed.), ordination and classification of communities, handbook of vegetation science 5, 619–726. w. junk, hague. 184 acta bot. croat. 72 (1), 2013 kaba[ e. n., alegro a. a., kuzmanovi] n. v., jakovljevi] k. m., vukoji^i] s. s., laku[i] d. v. 635 kabas et al.ps u:\acta botanica\acta-botan 1-13\635 kabas et al.vp 14. o ujak 2013 11:42:42 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 187 acta bot. croat. 74 (1), 187–193, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication first record of inocutis tamaricis in romania with comments on its cultural characteristics vasilică c. chinan*, lucian fusu, ciprian c. mânzu alexandru ioan cuza university, faculty of biology, bd. carol i, no. 20a, 700505, iaşi, romania abstract – inocutis tamaricis is a lignicolous basidiomycete associated exclusively with tamarix species. the fi rst romanian record of this species is reported from constanţa city near the black sea coast where it was detected on tamarix tetrandra. we noticed that in pure culture it forms swollen hyphae in the aerial mycelium, which have not been reported so far for i. tamaricis. keywords: inocutis tamaricis, romania, swollen hyphae, tamarix tetrandra introduction inocutis tamaricis (pat.) fiasson & niemelä is a thermophilous basidiomycete typical for the mediterranean region that grows exclusively on tamarix species (ryvarden and gilbertson 1993) and causes a white rot of the heartwood (bondartseva and parmasto 1986). this taxon was renamed by fiasson and niemelä (1984) from the widely known inonotus tamaricis (pat.) maire to inocutis tamaricis. later, on the basis of molecular data, wagner and fischer (2001) confi rmed this new combination. the global distribution of i. tamaricis includes the southern part of the palearctic. it is particularly widely distributed in the countries bordering the mediterranean sea and the black sea (piątek 2001). reviewing the distribution of this species, klán (1978) mentions its presence in portugal, spain, france, italy, greece, croatia, montenegro, bulgaria, ukraine, georgia, algeria, morocco, syria, and kazakhstan. furthermore, i. tamaricis was reported to be present in egypt (hejný and kotlaba 1984), uzbekistan (bondartseva and parmasto 1986), macedonia (karadelev 1993), south of european russia, central asia, israel, senegal (ryvarden and gilbertson 1993), china (dai et al. 1997), canary islands (beltrán-tejera and rodríguez-armas 1999), turkey (doğan et al. 2005) and iran (ghobad-nejhad and kotiranta 2008). * corresponding author, e-mail: vasilechinan@yahoo.com copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. chinan v. c., fusu l., mânzu c. c. 188 acta bot. croat. 74 (1), 2015 inocutis tamaricis is sporadically present in some countries and although it is a pathogen of tamarix spp., in macedonia (karadelev 2000) and bulgaria (gyosheva et al. 2006) it is included in the red list of fungi. in this study we present the fi rst record of i. tamaricis in romania and provide new data on its cultural characteristics. materials and methods in the fi eld, three basidiomata were collected from two trunks of living tamarix tetrandra pall. ex m. bieb. observations of microscopic characters were made in 5% koh and in 1% aqueous solution of congo red using a compound microscope. spores and swollen hyphae measurements were based on thirty observations. statistical data (length and width) are given as arithmetic mean ± standard deviation (sd). species identifi cation was performed based on bondartsev 1953, bondartseva and parmasto 1986, jülich 1989, ryvarden and gilbertson 1993, and bernicchia 2005. the analyzed material was dried and deposited in the herbarium of the faculty of biology, alexandru ioan cuza university, romania (index herbariorum acronym: i). in order to investigate the cultural and morphological characteristics of i. tamaricis, dikaryon culture isolated from one basidioma (voucher i 137309, leg. chinan and mânzu) was grown on 9 cm petri dishes containing the following nutrient media: – sabouraud: 10 g peptone, 40 g dextrose, 15 g agar, 1000 ml distilled water; – potato dextrose agar (pda): 200 g unpeeled potatoes, 20 g dextrose, 20 g agar, 1000 ml distilled water); – malt extract agar (mea): 15 g malt extract, 15 g agar, 1000 ml distilled water; – czapek: 30 g sucrose, 20 g agar, 2 g nano3, 1 g kh2po4, 0.5 g mgso4 × 7 h2o, 0.5 g kcl, 0.01 g feso4 × 7 h2o, 1000 ml distilled water. the culture media were inoculated with small blocks of actively growing mycelium at a distance of 1.5 cm from the edge of the petri dish and incubated at 25 °c in the dark. three replicates were used for each medium. species identifi cation and the identity of the cultured mycelium were confi rmed using molecular markers (its, internal transcribed spacer region). dna sequences were generated from a dried basidioma (voucher i 137309) and cultured mycelium derived from it using primers its1 (5’-tcc gta ggt gaa cct gcg g-3’) and its4 (5’-tcc tcc gct tat tga tat gc-3’) (white et al. 1990) and sequenced at a commercial facility (alvalab, santander, spain). the obtained its sequence (genbank accession number kj755854) was compared with those of inocutis rheades (pers.) fiasson & niemelä, i. dryophila (berk.) fiasson & niemelä and i. tamaricis available in genbank. the fi rst two species were used in the analysis because of a possible confusion in identifi cation due to the morphological resemblance between these species and i. tamaricis (ghobad-nejhad and kotiranta 2008). a sequence of inonotus tricolor (bres.) y. c. dai was used as out-group. sequences were aligned using clusatlw (larkin et al. 2007) followed by minor alignment corrections by eye. phylogenies and p-distances were estimated in mega 6.06 (tamura et al. 2013). the phylogenetic analysis was run using maximum likelihood and a k2p + g model of nucleotide substitution as this was determined to be the most appropriate model with mega 6.06 using the bayesian information criterion (bic). support for monophyly of clades was evaluated by bootstrap values (1000 replicates). inocutis tamaricis in romania acta bot. croat. 74 (1), 2015 189 results and discussion during a fi eld trip to the black sea coast in south east romania, i. tamaricis was detected in june 2012 on two cultivated t. tetrandra bushes, in the park of the natural sciences museum complex from constanţa city (44°12′19.19″n, 28°38′20.05″e). this is the fi rst record of this fungus in romania. macroscopic and microscopic description the basidiomata are fan-shaped, semicircular, 7–22 cm across, 4–9 cm wide, 3–4 cm thick, surface rusty-brown, zonate, hispid to villose (fig. 1a). hymenial surface is pale ochraceous at fi rst, later becoming brown, pores angular, 1–3 per mm. tube layer is rustybrown 5–30 mm long. context is rusty-brown, fi brous, zonate, with granular, marbled core at base. hyphae of the fi brous context with simple septa, yellow to rusty brown, thin to thick-walled, 4–10 μm wide. hyphae of mycelial core are brown, encrusted, branched, thick-walled, dark brown mixed with lobed and branched sclerids. basidia are clavate, 4-sterigmate, 14–18 × 7–9 μm. basidiospores are rusty brown, thick-walled, ellipsoid, 6.5– 8.5 μm long (mean 7.6 ± 0.6), and 5–6 μm wide (mean 5.3 ± 0.4). inocutis tamaricis is easy to identify in the fi eld due to its habitat on the wood of various tamarix species and the presence of a marbled core at the base of the basidioma. the macroscopic and microscopic characteristics of the specimens from romania are in accordance with the published descriptions of this species (bondartseva and parmasto 1986, ryvarden and gilbertson 1993, jülich 1989, bernicchia 2005). cultural characteristics on sabouraud agar, the colony is initially white, then yellowish to ochraceous (fig. 1b). the aerial mycelium is cottony with concentric zones. the reverse in the old part of the colony is brownish-ochraceous, but in the recently covered zone it is unchanged. on other nutrition media, such as czapek, mea, and pda the colony is whitish with yellowish tints, and the reverse is unchanged to cream-yellowish. this strain of i. tamaricis preferred pda, on which we recorded the highest growth rate (with a mean of 23 mm per week), followed by mea (18 mm per week), sabouraud (17 mm per week), and czapek (15 mm per week). regarding the microstructural characteristics we observed that on all tested media the aerial mycelium presents numerous swollen hyphae (fig. 1c). these are spherical to fusiform, 7–25 μm long (mean 13.1 ± 6) and 7–20 μm wide (mean 10.4 ± 4.3), hyaline to yellowish, single or in chains (monilioid). the swellings on czapek, mea, and sabouraud media appeared after about three weeks and on pda after two weeks. the cultural and structural characteristics of this species were previously investigated by stalpers (1978) and ţura et al. (2009) on different nutrient media (cherry decoction agar, mea, pda, beer wort, corn-meal agar, and czapek medium). although the morphological characteristics of the colonies examined by us are similar to the previous descriptions, our observations showed that this strain forms numerous infl ated hyphae in the aerial mycelium. these were not mentioned so far for i. tamaricis. chinan v. c., fusu l., mânzu c. c. 190 acta bot. croat. 74 (1), 2015 molecular analysis the identical its sequences obtained from the basidioma and cultured mycelium confi rmed that observations were made on i. tamaricis mycelium and not on a contaminant species. the its sequence of i. tamaricis from romania clustered with very high bootstrap support with all other i. tamaricis sequences available in genbank (fig. 2), confi rming thus the identifi cation based on microscopic characters and host preference. the p-distances between i. tamaricis from romania and other conspecifi c sequences, varying from 0.004 to 0.036, are well below or comparable with the mean intraspecifi c sequence divergence of 0.025 reported for fungi (schoch et al. 2012). ecology in romania, i. tamaricis was found only on living plants. although this species has been reported from both living and dead tamarix trunks (ryvarden and gilbertson 1993, beltrán-tejera and rodríguez-armas 1999, ghobad-nejhad and kotiranta 2008), the vast majority of authors report that this species was only detected on living tamarix species (pegler 1964, intini 1977, klán 1978, hejný and kotlaba 1984, bondartseva and parmasto 1986, jülich 1989, llimona et al. 1995, tentori 1997, bernicchia 2005, dai et al. 2007, dai et al. 2009, assyov et al. 2010, ţura et al. 2010, zhou et al. 2011, alexov et al. 2012, dai 2012). our results show that i. tamaricis occurs in romania as a parasite. fig. 1. inocutis tamaricis: a – basidioma on tamarix tetrandra; b – colony on sabouraud agar; c – aerial mycelium with swollen hyphae (in congo red solution). scale bar = 20 μm (photo by v. c. chinan). inocutis tamaricis in romania acta bot. croat. 74 (1), 2015 191 the host plant, t. tetrandra, is not a native species in romania, but is present in urban areas and parks as an ornamental shrub. to the best of our knowledge, i. tamaricis has not yet been recorded living on this host plan t. interestingly, this inocutis species has not been found yet in romania on t. ramosissima ledeb., which is a widespread native species, including in the danube delta (ciocârlan 2011) where the ecological conditions could be favorable for this fungus. in the published data, not all of the authors mention the species of tamarix on which it grows. the most frequent host mentioned in the literature is t. gallica l. (intini 1977, lópez-prada and castro cerceda 1996, tentori 1997, zervakis et al. 1998, bernicchia 2005, and ţura et al. 2009, 2010). other species of tamarix reported as hosts for this fungus are: t. arborea (sieber ex ehrenb.) bunge (piątek 2001), t. canariensis willd. (llimona et al. 1995, beltrán tejera and rodríguez-armas 1999), t. chinensis lour. (dai 2012), t. nilotica (ehrenb.) bunge (hejný and kotlaba 1984), t. parvifl ora dc. (karadelev 1993), and t. ramosissima (sinadskii and bondartseva 1960). as the species was already signaled in bulgaria and ukraine, our record of i. tamaricis from romania contributes to the knowledge on its distribution in the western black sea region. acknowledgements we sincerely thank dr. daniel ţura for important discussions on the subject and the anonymous reviewers, whose suggestions helped improve this manuscript. references alexov, r., vassilev, d., nedelev, p., traikov, i., 2012: new records of seven rare and noteworthy basidiomycetes from bulgaria. trakia journal of sciences 10, 10–16. fig. 2. maximum likelihood tree based on its sequences. bootstrap values (support > 65%) based on 1,000 replications are shown near nodes. the sequence of inocutis tamaricis from romania is indicated by a black triangle. chinan v. c., fusu l., mânzu c. c. 192 acta bot. croat. 74 (1), 2015 assyov, b., stoykov, d., nikolova, s., 2010: new records of some rare and noteworthy larger fungi from bulgaria. trakia journal of sciences 8, 1–6. beltrán tejera, e., rodríguez-armas, j. l., 1999: aphyllophorales (basidiomycotina) of arid habitats of the canary islands. preliminary data. mycotaxon 70, 111–125. bernicchia, a., 2005: polyporaceae s.l., fungi europaei. edizioni candusso, italy. bondartsev, a. s., 1953: the polyporaceae of the european part of ussr and of caucasia [in russian]. academy of sciences of ussr, leningrad. bondartseva, m. a., parmasto, e. h., 1986: clavis diagnostica fungorum ussr. ordo aphyllophorales 1. familiae hymenochaetaceae, lachnocladiaceae, coniophoraceae, schizophyllaceae. nauka, leningrad. ciocârlan, v., 2011: vascular fl ora of the danube delta. analele ştiinţifi ce ale universităţii „alexandru ioan cuza” iaşi, s. ii a. biologie vegetală 57, 41–64. dai, y. c., 2012: pathogenic wood-decaying fungi on woody plants in china. mycosystema 31, 493–509. dai, y. c., cui, b. k., yuan, h. s., li, b. d., 2007: pathogenic wood-decaying fungi in china. forest pathology 37, 105–120. dai, y. c., niemelä, t., zang, m., 1997: synopsis of the genus inonotus (basidiomycetes) sensu lato in china. mycotaxon 65, 273–283. dai, y. c., yuan, h. s., wang, h. c., yang, f., wei, y. l., 2009: polypores (basidiomycota) from qin mts. in shaanxi province, central china. annales botanici fennici 46, 54–61. doğan, h. h., öztürk, c., kasik, g., aktas, s., 2005: a checklist of aphyllophorales of turkey. pakistan journal of botany 37, 459–485. fiasson, j. l., niemelä t., 1984: the hymenochaetales: a revision of the european poroid taxa. karstenia 24, 14–28. ghobad-nejhad, m., kotiranta, h., 2008: the genus inonotus sensu lato in iran with keys to inocutis and mensularia worlwide. annales botanici fennici 45, 465–476. gyosheva, m. m., denchev, c. m., dimitrova, e. g., assyov, b., petrova, r. d., stoichev, g. t., 2006: red list of fungi in bulgaria. mycologia balcanica 3, 81–87. hejný, s., kotlaba, f., 1984: the discoveries of polypores inonotus tamaricis and ganoderma resinaceum in egipt. mykologické listy 14, 9–11. intini, m., 1977: alcuni agenti di carie del genere inonotus karst. micologia italiana 6, 13–19. jülich, w., 1989: guida alla deteminazione dei funghi vol. 2. aphyllophorales, heterobasidiomycetes, gastromycetes. saturnia, italy. karadelev, m., 1993: contribution to the knowledge of wood-destroying fungi in the republic of macedonia, fungi macedonici i. young explorers of macedonia, skopje. karadelev, m., 2000: a preliminary red list of macromycetes of the republic of macedonia. european council of conservation of fungi, newsletter 10, 7–11. klán, j., 1978: inonotus tamaricis (pat.) maire in greece, its general distribution and taxonomic notes on the section phymatopilus donk. česká mykology 32, 47–54. larkin, m. a., blackshields, g., brown, n. p., chenna, r., mcgettigan, p. a., mcwilliam, h., valentin, f., wallace, i. m., wilm, a., lopez, r., thompson, j. d., gibson, t. inocutis tamaricis in romania acta bot. croat. 74 (1), 2015 193 j., higgins, d. g., 2007: clustal w and clustal x version 2.0. bioinformatics 23, 2947– 2948. llimona, x., vila, j., hoy0, p., aguasca, m., àngel, f., gràcia, e., llistosella, j., martín, m. p., mayoral, a., rocabruna, a., sierra, d., tabarés, m., 1995: el programa biodiversitat micològica de les terres de ponent. notícia i primers resultats. revista catalana de micologia 18, 103–136. lópez-prada, m. i., castro cerceda, m. l., 1996: aportación al conocimiento de las especies del orden aphyllophorales s.l. 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(eds.): pcr protocols: a guide to methods and applications, 315– 322. academic press inc., new york. zervakis, g., dimou, d., balis, c., 1998: a check-list of the greek macrofungi including hosts and biogeographic distribution: i. basidiomycotina. mycotaxon 66, 273–336. zhou, l. w., hao, z. q., wang, z., wang, b., dai, y. c., 2011: comparison of ecological patterns of polypores in three forest zones in china. mycology 2, 260–275. 625 pise and gaikwad.vp acta bot. croat. 72 (2), 211–219, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 growth and photosynthesis of lemna minor l. exposed to different light conditions and sucrose supplies @eljka vidakovi]-cifrek1, sonja sori]2, marija babi]1* 1 department of botany and botanical garden, faculty of science, university of zagreb, rooseveltov trg 6, hr-10000 zagreb, croatia 2 krijesnice 40, hr-10000 zagreb, croatia abstract – duckweed (lemna minor l.) is a model plant suitable for investigation into plant physiology, biochemistry and ecotoxicology. depending on the type of the experiment, duckweed is cultivated on different nutrient media under various chamber conditions. in our work, duckweed was cultivated on pirson-seidel’s nutrient solution supplemented with 5, 7.5 or 10 g l–1 sucrose under cool white (cw) or gro-lux (gl) light sources. when different light sources and sucrose supplies are compared, a significant stimulative effect of gl light on duckweed grown on 7.5 and 10 g l–1 sucrose was seen to start on day 9. considering photosynthetic performance the results showed that there were no significant differences in maximum quantum yield of psii (fv/fm) after 7 and 16 days of exposure, regardless of light source and sucrose supply. effective quantum yield of psii (fpsii) decreased only after 16 days of exposure to 5 g l –1 sucrose under cw light. the higher growth rate and photosynthetic performance in plants exposed to gl light is a consequence of its spectral distribution resembling the action spectrum of photosynthesis. furthermore, enhanced growth noticed in plants cultivated on higher sucrose contents (7.5 and 10 g l–1) indicated the promotive effect of sucrose in plants grown under low light conditions. key words: chlorophyll fluorescence, growth rate, light, lemna minor, photosynthesis, photosystem ii, sucrose abbreviations: chl – chlorophyll, cw – cool white light source, gl – grolux light source, gr – growth rate, ppfd – photosynthetic photon flux density, f v /f m – maximum quantum yield of psii, f psii – effective quantum yield of psii, psii – photosystem ii, ps – pirson and seidel’s nutrient solution introduction duckweed (lemna minor l.) is a small, free-floating freshwater macrophyte. due to its small size, high multiplication rate, vegetative propagation, high sensitivity and easy handling, duckweed has been commonly used as model plant in evaluating the effects of a wide acta bot. croat. 72 (2), 2013 211 * corresponding author, e-mail: mbabic@zg.biol.pmf.hr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. range of substances (wang 1990, lewis 1995, naumann et al. 2007, zhang et al. 2010). moreover, lemna bioassay has been recommended by the epa, astm, oecd, sis and iso (eberius 2001) in order to make it possible to use duckweed for reproducible toxicological studies in different laboratories (naumann et al. 2007). as well as for ecotoxicological studies, duckweed is an excellent plant model for molecular (mardanov et al. 2008), biochemical (sherameti et al. 2002) and physiological studies (krajn^i^ and devidé 1980, kondo 1988, chi et al. 2012), as well as for phytoremediation of polluted waters (mkandvire and dudel 2007), environmental biomonitoring (wang and williams 1988) and biotechnology (gyunter et al. 2008). furthermore, it has also been used to evaluate the effects of nutrient deficiency (rapparini et al. 2002), extreme temperatures, radiofrequency radiation (germ and gaber[^ik 1999, tkalec et al. 2007) and different light conditions (artetxe et al. 2002, somsri et al. 2010). according to the concept of an experiment, duckweed is cultivated on different nutrient media, e.g. bristol’s, hunter’s, hoagland’s and schenk and hildebrandt’s (wang 1990, lewis 1995, zhang et al. 2010). besides mineral elements, some nutrient media contain organic compounds, usually amino acids and carbohydrates (wang 1990, frick 1994, tkalec et al. 2007). depending on the concentration, organic substances can stimulate plant growth but also can cause osmotic stress and down-regulation of photosynthesis (hdider and desjardins 1994, balen et al. 2012, and references therein). plant growth is dependent not only on the composition of nutrient media but also on chamber conditions – temperature, photoperiod and light quality. artificial light sources differ from sunlight in both spectral characteristics and intensity. in growth chambers, usually cool white (cw) or day-light fluorescent tubes with a wide range of light intensities (40–180 mmol photons m –2 s–1) are used. some of the commercially available light sources are not the appropriate for photosynthesis due to light intensities and spectral characteristics that are incompatible with the absorption spectrum of photosynthetic pigments. the aim of this work was to evaluate the impact of sucrose supply and light conditions on the growth rate and photosynthetic efficiency of lemna minor l. duckweed was grown on pirson-seidel’s (ps) nutrient solution (pirson and seidel 1950) under cool white (cw) or gro-lux (gl) fluorescent light sources that have different spectral energy distributions. the light energy of cw sources is mainly distributed in the blue, green and yellow parts of the spectrum while gl sources emit most of energy at the blue and red which are the spectral regions most effective in photosynthesis. ps medium originally contains 10 g l–1 of sucrose (pirson and seidel 1950). in our experiment, growth and photosynthetic efficiency in duckweed cultivated on media supplemented with lower sucrose concentrations (5 and 7.5 g l–1) were also tested. according to the recommendation of huebert and shay (1993), we previously tested the duration of exponential growth on ps medium. since the exponential growth during 16 days was confirmed, this period was taken for the maximum duration of the experiment. material and methods plant material and growth conditions duckweed (lemna minor l.) was collected from the botanical garden, faculty of science, university of zagreb in 1996, and sterilised according to krajn^i^ and devidé (1980). 212 acta bot. croat. 72 (2), 2013 vidakovi]-cifrek @., sori] s., babi] m. axenic stock cultures were maintained on pirson-seidel (ps) nutrient solution (pirson and seidel 1950) containing 3.95 mmol l–1 kno3, 5.46 mmol l –1 cacl2 ´ 2 h2o, 1.47 mmol l–1 kh2po4, 1.21 mmol l –1 mgso4 ´ 7 h2o, 49 mmol l –1 na2edta ´ 2 h2o, 20 mmol l –1 fe-citrate, 1.5 mmol l–1 mncl2 ´ 4 h2o, 8.1 mmol l –1 h3bo3, 29.2 mmol l –1 sucrose and 0.66 mmol l–1 asparagine. the ph value of nutrient solution was adjusted to 4.55 using koh. plants were grown at 24 ± 2 °c under an illumination provided by cw fluorescent light (f36w/33-640, sylvania), ppfd = 45 mmol photons m –2 s–1 at plant level and a light:dark cycle of 16:8 hours. cultures were grown under static conditions and subcultured biweekly. experimental conditions experimental cultures were started by inoculating a healthy duckweed colony consisting of three or four fronds into 100 ml erlenmeyer flasks containing 60 ml of ps nutrient solution supplemented with sucrose in concentrations of 10 g l–1 (29.2 mmol l–1), 7.5 g l–1 (21.9 mmol l–1) or 5 g l–1 (14.6 mmol l–1). the experimental plants were grown under cw fluorescent lights (f36w/33-640, sylvania), ppfd = 50±5 mmol photons m –2 s–1 or grolux (gl) (f36w/gro, sylvania), ppfd = 50±5 mmol photons m –2 s–1. experimental plants were exposed to the same photoperiod and temperature as stock cultures. growth assessment growth rate (gr) on the basis of frond number was calculated on days 2, 5, 7, 9, 12, 14 and 16, according to the equation: gri = ln lnn n t t t ti 0 i 0 − − gri is the growth rate per day; nt 0 is the frond number at day t0 (the beginning of the experiment); nt i is the frond number at day ti (i = 2, 5, 7, 9, 12, 14, 16); ti – t0 is the time period between ti and t0, expressed in days (iso 20079, 2004). the frond number doubling time (t) was calculated for days 2, 5, 7, 9, 12, 14 and 16, using the equation (iso 20079, 2004): ti = ln 2 gr i ti is the frond number doubling time for day i (i = 2, 5, 7, 9, 12, 14, 16). each result was expressed as an average of 20 replicates (10 replicates from two independent experiments) ± standard error (se). in vivo measurement of chlorophyll fluorescence duckweed photosynthetic efficiency was assessed by measuring in vivo chlorophyll fluorescence on day 7 and day 16 of the experiment. chlorophyll fluorescence was measured from the upper face of dark-adapted fronds by pulse-modulated chlorophyll fluorometer (qubit systems inc., canada). minimal fluorescence (f0) was measured using a weak red modulated light, resulting in 0.2 – 0.25 mv f0 signal for dark-adapted plants. then, a satuacta bot. croat. 72 (2), 2013 213 effect of sucrose and light on lemna minor rating light pulse (ppfd = 3760 mmol photons m –2 s–1, t = 0.7 s) was applied to induce maximal fluorescence (fm). the difference between fm and f0 was used for calculation of variable fluorescence (fv). after quenching of maximal chl fluorescence in the dark, plants were illuminated by continuous actinic light (ppfd = 90 mmol photons m –2 s–1). steady-state fluorescence (f) and maximum fluorescence (f’m) of the illuminated samples were measured. saturating pulses were applied at 40 s intervals. after 5 min of irradiation by actinic light, fluorescence signal reached the steady-state. finally, f0’ value was determined after turning off the actinic light. all the values determined during the measurement (f0, fm, f’m, f and f0’) allowed the calculation of psii maximum quantum yield (fv/fm) and psii effective quantum yield (fpsii) according to maxwell and johnson (2000). the results were given as the average of four replicates ± standard error (se). data analysis data were analysed by one-way analysis of variance (anova) and subsequent newman-keul’s test. differences between treatments were considered statistically significant at p < 0.05. all statistical analyses were performed using the statistica 7.1 (statsoft, inc., usa) software package. results at the beginning of the experiment (after 2 days of exposure), the light quality and sucrose content did not affect duckweed frond number, which resulted in similar growth rates in all treatment groups. after 5 and 7 days of exposure to cw light, the duckweed growth rates did not change with variation of sucrose content. the same was noticed in plants exposed to gl light. however, when different light sources were compared, a stimulative effect of gl light was noticed in plants grown on 7.5 and 10 g l–1 sucrose. the growth rates of 214 acta bot. croat. 72 (2), 2013 vidakovi]-cifrek @., sori] s., babi] m. ab ab bc ab bc b c b b b bbb a b a c a c a c a b a b a c a c a c a b a b a 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 2 5 7 9 12 14 16 exposure (d) g ro w th ra te (d ) – 1 a aaa a a 5 g l–1 sucrose 7.5 g l–1 sucrose 10 g l –1 sucrose gl gl gl gl gl gl glcw cwcwcwcwcwcw cw fig. 1. growth rates (gr) of l. minor during 16 days of cultivation on ps nutrient solution containing different sucrose supply under different light sources are shown as the mean of 20 replicates ± se. mean values were compared within each exposure time and were considered statistically significant at p < 0.05. significantly different values for each treatment period (days) are marked with different letters. plants grown on 5 g l–1 sucrose under gl light did not differ from those obtained under cw light. starting with day 9, the growth rates increased at higher sucrose supply (7.5 and 10 g l–1) but only in plants exposed to gl light. however, in plants grown on 5 g l–1 sucrose under gl light the growth rates were low and similar to those obtained under cw light at all sucrose supplies tested. the exception was growth rate observed after 12-days exposure to gl light, when even the lowest sucrose supply (5 g l–1) allowed significant increase of growth rate in comparison to that obtained in plants exposed to cw light (fig. 1). at the end of the experiment (on day 16), cultivation on 5 g l–1 sucrose resulted in similar frond-number doubling times (2.77 and 2.65) under both tested light sources (cw and gl, respectively; tab. 1). in plants exposed to cw light, addition of higher sucrose supplies did not change the doubling time. however, in plants grown under gl light, increase of sucrose supply (7.5 and 10 g l–1) shortened the frond-number doubling time (6.0 and 6.3%, respectively). the stimulative effect of gl light on duckweed grown on higher sucrose supplies started as early as day 7 (data not shown), and continued until the end of the experiment. on day 16 the doubling time of plants grown on 7.5 and 10 g l–1 sucrose under gl light shortened by 9.7 and 10.1%, respectively, in comparison to plants exposed to cw light. chlorophyll fluorescence was measured on days 7 and 16. on both days, the values of maximum quantum yield of psii (fv/fm) ranged from 0.67 to 0.72 and showed no significant influence of sucrose supply and light source (tab. 2). like fv/fm, effective quantum yield of psii (fpsii) also showed no significant influence of sucrose supply and light source on day 7. however, on day 16 it decreased in plants grown on 5 g l–1 sucrose under cw light, the decrease being significant in comparison to plants grown on 7.5 and 10 g l–1 sucrose under gl lights. discussion in natural conditions, duckweed prefers nutrient-reach waters, where the conditons of its exposure to light ranges from full sunlight to dense shade. like many other species, it possesses a highly plastic photosynthetic apparatus allowing quick adaptation to changes in the light environment (demmig-adams and adams 1992). in order to interpret the results properly, one has to take into consideration the effects of chamber conditions as well as the composition of the nutrient medium on the physiological processes of experimental plant. the influence of light spectral distribution and intensity on plant growth rate and photosynthesis was proved a long time ago. the plants achieve optimal growth and productivity when exposed to light sources ensuring adequate spectral distribution. the acta bot. croat. 72 (2), 2013 215 effect of sucrose and light on lemna minor tab. 1. doubling time (t) after 16 days of cultivation of lemna minor l. on ps nutrient solution containing different sucrose supply under different light sources. each value represents the mean of 20 replicates ± se. comparisons between mean values were considered statistically significant at p < 0.05. significantly different values are marked with different letters. 5 g l–1 sucrose 7.5 g l–1sucrose 10 g l–1sucrose cw light 2.770a ± 0.041 2.763a ± 0.038 2.765a ± 0.047 gl light 2.653a ± 0.047 2.493b ± 0.025 2.485b ± 0.033 photosynthetic pigments (chlorophylls and carotenoids) absorb light most strongly in blue and red regions of the spectrum, which coincides with the action spectrum of photosynthesis. so, it was not surprising that in our experiment plant growth was more pronounced in plants exposed to gl light, the spectral distribution of which corresponds more to the action spectrum of photosynthesis than cw light. since plants as autotrophic organisms have the ability to convert light into chemical energy and produce organic compounds, nutrient media containing essential macroand micronutrients should suffice for their growth. however, the light intensity used in this experiment (50 ± 5 mmol photons m –2 s–1) was insufficient to ensure optimal photosynthetic rate. iso and oecd protocols suggest performing 7-day lemna assay on media containing mineral elements only, using light intensities of 85– 125 mmol photons m –2 s–1 (iso 20079, 2004; oecd 221, 2006). during the first week of the experiment, the growth rates of plants exposed to the same light source (cw or gl) did not depend on the sucrose content of the nutrient medium. however, in a comparison of the effect of different light sources on plants supplied with the same amount of sucrose, the stimulative effect of gl light became evident. hence, for example, after 5 day cultivation on 7.5 and 10 g l–1 sucrose, gl light revealed higher growth rates than cw light. this trend started on day 5 and continued until the end of the experiment. the results confirmed the stimulative effect of gl light on the growth of l. minor cultivated on higher sucrose supplies under low light conditions. at low-light conditions, 5 g l–1 sucrose did not satisfy the growth requirements under both, cw and gl light. during the second week of the experiment, plants grown on 5 g l–1 sucrose under gl light showed significantly lower growth rates than those obtained on higher sucrose supplies. however, under cw light the growth rate remained low even at 7.5 and 10 g l–1 sucrose. iso and oecd protocols (iso 20079, 2004; oecd 221, 2006) recommend a doubling time of up to 2.5 days. in our experiment the doubling time of duckweed grown on 7.5 and 10.0 g l–1 under gl light, which showed the most pronounced growth, amounted 2.49 and 2.48 days, respectively. however, the doubling time of all other treatments exceeded 2.5 (tab. 1) indicating suboptimal growth conditions. 216 acta bot. croat. 72 (2), 2013 vidakovi]-cifrek @., sori] s., babi] m. tab. 2. maximum quantum yield of psii (fv/fm) and effective quantum yield of psii (fpsii) in lemna minor l. after 7 and 16 days of cultivation on ps nutrient solution containing different sucrose supply under different light sources. values represent means of four replicates ± se which were compared within each exposure time and considered statistically significant at p < 0.05. significantly different values are marked with different letters. light source sucrose content day 7 day 16 fv/fm fpsii fv/fm fpsii cw 5 g l–1 0.671a ± 0.007 0.572a ± 0.008 0.698a ± 0.002 0.586b ± 0.005 7.5 g l–1 0.688a ± 0.004 0.600a ± 0.014 0.693a ± 0.002 0.607ab ± 0.017 10 g l–1s 0.693a ± 0.033 0.567a ± 0.031 0.707a ± 0.009 0.616ab ± 0.005 gl 5 g l–1 0.689a ± 0.004 0.581a ± 0.010 0.705a ± 0.002 0.620ab ± 0.006 7.5 g l–1 0.687a ± 0.007 0.600a ± 0.014 0.709a ± 0.006 0.633a ± 0.014 10 g l–1 0.695a ± 0.003 0.622a ± 0.006 0.720a ± 0.011 0.637a ± 0.003 photosynthetic performance of duckweed grown under cw and gl light was evaluated using chlorophyll fluorescence. the fv/fm ratio is often used as a parameter reflecting the maximum (i. e. potential) quantum efficiency of psii in dark-adapted plants (wu et al. 2003). the fv/fm values obtained in this experiment varied between 0.67–0.72, and as such they reached the lower limit of optimal fv/fm, for most plant species ranging between 0.7–0.83 (ritchie 2006). therefore, fv/fm values obtained were still within the optimal fv/fm range. as opposed to fv/fm, effective quantum yield of psii (fpsii) is considered an indicator of actual photochemical efficiency of psii in plants exposed to light (wu et al. 2003). in this experiment, fpsii attained values of 0.57–0.64 which is in accordance with the normal fpsii range, varying between 0.4–0.6, as proposed by ritchie (2006). our results showed that there were no significant differences in duckweed fv/fm and fpsii after 7 and 16 days of exposure to cw and gl light, regardless of the sucrose content. the only exception was fpsii obtained in plants grown on 7.5 and 10 g l–1 sucrose after 16 days of exposure to gl light, which showed the highest values. fpsii of the remaining treatment groups showed a downward trend, with the lowest value obtained in plants grown on 5 g l–1 sucrose under cw light (tab. 2). as plants acclimated to low light conditions maximize photon energy capture due to more efficient light-harvesting complex (anderson et al. 1988), they highly depend on the light source. in our experiment the effect of inappropriate spectral characteristics of cw light source was most obvious in plants grown on medium supplied with the lowest energy source in the form of sucrose, where the minimum value of fpsii was noticed. the most efficient photosynthetic performance of the plants grown on 7.5 and 10 g l–1 sucrose under gl light was probably due to the spectral distribution of gl light which favours photosynthesis more than cw light. plants grown on nutrient media containing sucrose depend on light intensity less than plants grown on media containing mineral nutrients only. it has been shown previously that sucrose supply decreased photosynthetic performance, probably because it provides enough energy for other metabolic activities thus supporting heterotrophic and photomixotrophic growth (frick 1994, jo et al. 2009 and references therein). this is in agreement with the slight increase of photosynthetic performance in duckweed grown under low light intensity (50 mmol photons m–2 s–1) on media containing mineral nutrients only (artetxe et al. 2002) when compared with values we obtained on medium containing sucrose. according to the results obtained, it may be concluded that the higher growth rate and photosynthetic performance in plants exposed to gl light were related to its spectral energy distribution favouring photosynthesis. since plants cultivated on higher sucrose content (7.5 and 10 g l–1) showed better growth than those on 5 g l–1 sucrose, it indicated the promotive effect of sucrose in plants grown under low light conditions. acknowledgments this work was supported by the ministry of science, education and sports of the republic of 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of industrial effluents using phytotoxicity tests. environmental toxicology and chemistry 7, 645–652. wu, f. b., zhang, g. p., yu, j. s. 2003: genotypic differences in effect of cd on photosynthesis and chlorophyll fluorescence of barley (hordeum vulgare l.). bulletin of environmental contamination and toxicology 71, 1272–1281. zhang, y., hu, y., yang, b., ma, f., lu, p., li, l., wan, c., rayner, s., chen, s., 2010: duckweed (lemna minor) as a model plant system for the study of human microbial pathogenesis. plos one 5, e13527. acta bot. croat. 72 (2), 2013 219 effect of sucrose and light on lemna minor 554 surina.vp acta bot. croat. 72 (1), 113–132, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 heaths with dwarf ericaceous shrubs and alpine juniper (juniperus alpina) in the dinaric alps: a nomenclatorial and synsystematic re-appraisal bo[tjan surina1, 2,* 1 university of primorska, faculty of mathematics, natural sciences and information technologies, glagolja{ka 8, si-6000 koper, slovenia 2 natural history museum rijeka, lorenzov prolaz 1, 51000 rijeka, croatia abstract – the ecology and phytosociology of north-western dinaric heaths of the association rhododendro hirsuti-juniperetum alpinae horvat ex horvat et al. 1974 nom. corr. prop. as well as the syndynamics and synsystematics of heaths in the dinaric alps are discussed. while the structure (physiognomy) of these stands is very homogenous and dominated by few species, the flora is heterogeneous, since ecotonal areas, where heaths are most frequently developed, represent a contact zone of elements of different syntaxa. due to an abrupt reduction in pasture activities strong encroachments of shrubs and trees have become common, which additionally contribute to the floristic heterogeneity of the heaths. although the identification and circumscription together with synecology and synchorology of heaths in general are more or less easily understood and straightforward, their floristic affinities, in relation to structure homogeneity and syndynamics, are complicated, which led to the proposal of several synsystematic schemes depending on interpretation of the relationship between flora and structure of stands. dinaric heaths are classified into three classes, erico-pinetea, vaccinio-piceetea and festuco-brometea and a classification scheme is proposed together with nomenclatorial revision of the analyzed heaths with dwarf ericaceous shrubs and alpine juniper (juniperus alpina) in the dinaric alps. key words: dinaric alps, ericaceae, juniperus alpina, phytosociology, rhododendron hirsutum, synsystematics introduction an accurate and updated vegetation map is an essential tool for successful conservation planning, monitoring and managing biological diversity and other natural resources both within and outside the existing protected areas. in order to delimit areas with various vegetation types accurately, while assigning them to objective categories that can be easily acta bot. croat. 72 (1), 2013 113 * corresponding author, e-mail address: bostjan.surina@prirodoslovni.com copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:43 color profile: generic cmyk printer profile composite default screen recognized in the field and that reliably reflect fundamental biological differences (primarily the floristic composition and physiognomy), we started with detailed vegetation mapping in the obru~ area (liburnian karst, dinaric alps) in north-western adriatic (fig. 1), one of the most important plant areas in croatia (see randi] 2009). vegetation studies in the obru~ mountain range were initiated by croatian botanist ivo horvat and resulted in a worked-out vegetation typology (e.g. horvat 1930, 1931, 1938) and a vegetation map (in collaboration mostly with z. pelcer, z. matan and s. bertovi}) of the broader area (horvat 1962). based on a vegetation map of horvat and co-workers (horvat 1962), the study area represents a mosaic of forest and non-forest vegetation types where (subalpine) beech forests of the association polysticho lonchitis-fagetum (=fagetum croaticum australe subalpinum) generally prevail. southern slopes are covered by thermophilic stands of the association seslerio autumnalis-fagetum (=fagetum croaticum australe seslerietosum) and fir-beech stands of the association omphalodo-fagetum var. geogr. calamintha grandiflora (=fagetum croaticum australe abietetosum) seslerietosum autumnalis. stony and steep slopes, intercepted with boulders, gravels and cracks, are covered with more or less pure fir stands of the association calamagrostido variae-abietetum (piceetosum). recently, though not included in the vegetation map, beech stands from the summit were segregated into a new beech association calamagrostido arundinaceae-fagetum (cerove^ki 2009). among non-forest vegetation types, botanists mapped three syntaxa: (a) mountain pine scrubs with pinus mugo of the association hyperico grisebachii-pinetum mugo var. geogr. arabis scopoliana (=pinetum mughi croaticum [p.p.]), (b) dry and stony grasslands of the subassociation carici humilis-centaureetum rupestris seslerietosum tenuifoliae (=cariceto-centaureetum rupestris [p.p.]), and (c) heaths with rhododendron hirsutum and juniperus alpina (=juniperus communis l. subsp. sibirica (suter) ^elak., j. nana willd.; rhodereto-juniperetum). syntaxonomy of »heaths« besides the many typological and nomenclatorial issues among most of the above-mentioned syntaxa (compare weber et al. 2000), we find the synsystematics and syndynamics of stands dominated by hairy alpenrose (rhododendron hirsutum), alpine juniper (juniperus alpina) and large-leaved willow (salix appendiculata), although of homogenous structure, the most difficult problem to solve. these heaths are usually established in a phytoclimatic belt which is characterized by the timberline and/or isolated tree lines, within more or less large ecotone areas, once used for pastures but, due to overall changes in the socioeconomic situation, now almost completely abandoned (poldini et al. 2004). as a result, these areas are nowadays exposed to rapid dynamic phenomena and abrupt changes in vegetation cover. the climatogenic timberline in the liburnian karst exceeds the highest peaks due to the overall relatively low altitude (the highest peak, mt sne`nik, reaches 1796 m a.s.l.). however, under anthropogenic influence, and rarely due to extreme environmental site conditions (e.g. steep slopes, sites exposed to strong bora etc.), the man-made limit of the timberline and/or isolated tree, due to pasture and logging economy, might lie considerably lower than the climatogenic one (e.g. wraber 1997). horvat (1962) found structurally homogenous and ecologically well defined heaths with rhododendron hirsutum and juniperus alpina developed on exposed ridges of lower altitude (between 1200–1400 m a.s.l.). he placed them into the association rhododendro114 acta bot. croat. 72 (1), 2013 surina b. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:43 color profile: generic cmyk printer profile composite default screen -juniperetum nanae and afterwards supported this with an incomplete synoptic table (horvat et al. 1974, tab. 135, col. 1, 16 relevés). these stands, according to vegetation map, are more or less frequent in the obru~ (the main ridge, pakleno, mts suhi vrh, fratar and gornik), snje`nik (bjela [kalja and ceclje area, medvejci, planina and guslice) and risnjak area above 1200 m a.s.l. similar stands were observed on sites with similar ecological conditions in the slovenian part of the liburnian karst as well, on the sne`nik plateau, between 1200–1600 m a.s.l., in the area of gornji du`ovec, zatrep, grdobe, planinc, ilovca, stani{~e, @drocle etc. and mapped as rhododendro hirsuti-salicetum appendiculatae. structurally similar, but strikingly different in floristic composition are thermophilic stands with juniperus alpina and sesleria robusta from the biokovo mountain range (croatia, central dalmatia) described by domac as seslerio robustae-juniperetum sibiricae (domac 1962, vrdoljak 1983). besides hosting a considerable number of dinaric tussock (seslerion tenuifoliae) and sub-mediterranean grassland taxa (satureion subspicatae), these stands are characterized by the absence of rhododendron hirsutum and other de-alpine taxa. within these stands, a subassociation –pinetosum dalmaticae – was recognized on the basis of the presence and/or dominance of pinus nigra ssp. dalmatica, the only real differential taxon for the floristically and structurally based subassociation. on jahorina mountain range (central part of bosnia and herzegovina) bjel^i] (1966) placed stands dominated by juniperus alpina into the association junipero sibiricae-semperviretum schlechanii, while similar stands on mt bjelasica in montenegro, developed on somewhat more acidic soils, were studied by laku[i] (1966) who at that time recognized two new associations: roso pendulinae-juniperetum nanae and hyperici-vaccinietum montenegrini. on vranica mountain range (central bosnia and herzegovina), a geologically diverse area characterized by a mixture of calcareous and siliceous bedrocks, laku[i] et al. (1979) studied more acidophytic (hyperici-vaccinetum bosniacum, vaccinio-callunetum subalpinum) and relatively basiphytic stands (aquilegio-rhododendretum hirsuti, arctostaphylletum uvae-ursi) dominated by dwarf ericaceous species. while the structure (physiognomy) of these stands is very homogenous and dominated by few species such as rhododendron hirsutum, juniperus alpina, salix appendiculata, erica carnea, calamagrostis varia and rosa pendulina, the flora is heterogeneous since this ecotonal area represents a contact zone of elements of different syntaxa. additionally, due to a conspicuous reduction in pasture activities, strong encroachments of phanerophytes have become common contributing to a mixture of floristic composition of stands. although the identification and circumscription together with synecology and synchorology of the heaths in general are more or less simple and straightforward, their floristic affinities, in relation to structure homogeneity and syndynamics, are complicated, which led to the proposal of several syntaxonomic schemes depending on the interpretation of the relationship of the two aspects, focusing either on the flora (e.g. wallnöfer 1993a, 1993b), the structure (e.g. horvat 1962, horvat et al. 1974, theurillat et al. 1995, stanisci 1997), or both (poldini et al. 2004). theurillat et al. (1995) placed all mountain pine scrubs, regardless of the substrate, into a new class roso pendulinae-pinetea mugi, while they classified all the orotemperate heaths into the class loiseleurio-vaccinietea. on the other hand, wallnöfer (1993a, 1993b) differentiated acidophilic (vaccinio-piceetea) and basiphilic mountain pine scrubs (erico-pinetea), while acidophilic heaths were included into the class loiseleurio-vaccinietea and basiphilic into the class seslerietea albicantis. in the apennines (stanisci acta bot. croat. 72 (1), 2013 115 ericaceous shrubs and alpine juniper in the dinaric alps 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:43 color profile: generic cmyk printer profile composite default screen 1997), mountain pine scrubs and formations with juniperus alpina lack many boreo-alpine elements and were therefore referred to the class pino-juniperetea, a class consisting of orophilous communities dominated by conifers in the circum-mediterranean area. in the dinaric alps (e.g. ble^i] 1957, 1958, laku[i] 1966, bjel^i] 1966, laku[i] et al. 1979, zupan^i^ et al. 2004, 2006, vukeli] et al. 2008), mountain pine scrubs and dwarf ericaceous scrubs, strongly influenced by the researches of braun-blanquet (1931) and horvat (e.g. 1938, 1962, 1974), were classified in different subordinate syntaxa into the class vaccinio-piceetea without an exception. in our study we aim to: (1) elucidate the phytosociological characteristics and site conditions of structurally similar but floristically different stands of north-west dinaric heaths with hairy alpenrose rhododendron hirsutum and alpine juniper juniperus alpina, (2) identify the potential vegetation cover and syndynamic relationships between the north-west dinaric heaths and surrounding forest and non-forest vegetation types and (3) to propose a sensible syntaxonomic scheme for the heaths of the dinaric alps. methods in years 2005 and 2011, we recorded 22 relevés dominated by rhododendron hirsutum and juniperus alpina in the liburnian karst (fig. 1) applying the standard central-european method (braun-blanquet 1928, westhoff and van der maarel 1973, dierschke 1994). the plot size used for sampling averaged 30 m2 and further details on the phytosociological parameters of sites are given in appendix i. a complete floristic inventory is given in table 2, while taxa occurring in a single relevé are listed in appendix ii. coverage index (d%, e.g. 116 acta bot. croat. 72 (1), 2013 surina b. fig. 1. map showing the study area (hatched line) and approximate localities of the syntaxa from table 1 with corresponding acronyms. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:44 color profile: generic cmyk printer profile composite default screen surina 2005) was calculated for each taxon in table 2. the nomenclature and taxonomic source for the names of vascular plants was mala flora slovenije (martin^i^ et al. 2007). syntaxonomic groups in tables 2 and 3 were assigned according to flora alpina (aeschimann et al. 2004), dakskobler (2006) and poldini et al. (2004) and the list of syntaxa with full names is given in appendix iii. prior to numerical analysis, the original cover-abundance values for individual taxa were transformed into an ordinal scale as proposed by van der maarel (1979). groups of vegetation types were ascertained using cluster and ordination analysis with the help of the programme package past (hammer et al. 2001). the arrangement of relevés in table 2 was done according to the results of cluster analysis (fig. 2a) and diagnostic groups of species were subsequently tested by means of the simper analysis, an algorithm implemented in programme package past. in order to explain the acta bot. croat. 72 (1), 2013 117 ericaceous shrubs and alpine juniper in the dinaric alps 20 18 16 14 12 10 8 6 4 2 distance ty p ic u m se sl e ri e to su m te n u if o li a e 21 20 19 18 17 16 15 14 13 12 11 10 01 02 03 04 05 06 07 08 09 rajn jsss rf rfpm vvcv hvb hvm pmi rj rhpm ecpm scpm rcja aopm ec fagr anrh rh pmm2 hgpm pmc pmm1 -240 -180 -120 -60 60 120 180 240 component 1-300 -250 -200 -150 -100 -50 50 100 150 c o m p o n e n t2 c d a b -1.0 1.0 -1 .0 1 .0 alt incl. s( )% c( )% no taxa. l t c u r n typicum seslerietosum tenuifoliae 1 .0 no taxa. l t c environmental var. suppl. var. u n -1.0 1.0 -1 .0 alt r a b c 22 fig. 2. the results of (a) cluster (upgma), (b) pca (eigenv.: 1 – 0.883, 2 – 0.687, 3 – 0.679 4 – 0.695; cumulative percentage variance of species-environment relation [in %]: 1 – 45.1, 2 – 58.5, 3 – 68.7, 4 – 77.6; (c) ordination (euclid distances, eigenv.: 1 – 19,7%, 2 – 12%, 3 – 10,5%; acronyms correspond to those in fig. 1 and tab. 1) and (d) dca (eigenv.: 1 – 0.885, 2 – 0.703, 3 – 0.759, 4 – 0.585; cumulative percentage variance of species-environment relation [in %]: 1 – 42.8, 2 – 50.0, 3 – 76.9, 4 – 82.2) analysis of stands with dominating rhododendron hirsutum and juniperus sibirica in the liburnian karst (north-west dinaric alps) and structurally similar stands from the south-eastern calcareous and dinaric alps. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen 118 acta bot. croat. 72 (1), 2013 surina b. t a b .1 . l is to ff lo ri st ic al ly an d/ or st ru ct ur al ly si m il ar sy nt ax a w it h e ri c a c e a e ,a lp in e ju ni pe ra nd m ou nt ai n pi ne in th e s ou th -e as te rn c al ca re ou s an d d in ar ic a lp s. sy nt ax a ac ro ny m co un tr y re gi on m ou nt .r an ge re fe re nc e t ab . no .r el . r h o d o d e n d ro h ir su ti -j u n ip e re tu m si b ir ic a e r j s l o ,h r v n w d in ar ic a lp s s ne `n ik -r is nj ak th is w or k 2 22 r o so -j u n ip e re tu m n a n a e r aj n m n e s e d in ar ic a lp s b je la si ca l a k u [ i] 19 66 23 10 h y p e ri c iv a c c in ie tu m m o n te n e g ri n i h v m m n e s e d in ar ic a lp s b je la si ca l a k u [ i] 19 66 22 12 j u n ip e ro -s e m p e rv iv e tu m sc h le c h a n ii js s s b h c d in ar ic a lp s ja ho ri na b je l ^ i] 19 66 2 10 r h o d o d e n d re tu m h ir su ti r h s l o s e c al ca re ou s a lp s ju li an a lp s s u r in a 20 05 25 7 h y p e ri c o g ri se b a c h ii -p in e tu m m u g o h gp m s l o n w d in ar ic a lp s s ne `n ik z u pa n ^ i^ et al .2 00 4 1 20 s e sl e ri o ro b u st a e -j u n ip e re tu m si b ir ic a e s rj s h r v c d in ar ic a lp s b io ko vo d o m a c 19 62 v r d o l ja k 19 83 1, 2 25 + 1 a q u il e g io -r h o d o d e n d re tu m h ir su ti a nr h b ih c d in ar ic a lp s v ra ni ca l a k u [ i] et al .1 97 9 24 6 h y p e ri c iv a c c in ie tu m b o sn ia c u m h v b b h c d in ar ic a lp s v ra ni ca l a k u [ i] et al .1 97 9 23 9 p in e tu m m u g i m o n te n e g ri n u m p m m 1 m n e s e d in ar ic a lp s l ju bi {n ja b l e ^ i] 19 57 2 7 p in e tu m m u g i m o n te n e g ri n u m p m m 2 m n e s e d in ar ic a lp s m ag li }. g ol ij a. d ur m it or b l e ^ i] 19 58 9 8 p in e tu m m u g o il ly ri c u m p m i b h c d in ar ic a lp s v ra ni ca l a k u [ i] et al .1 97 9 29 11 p in e tu m m u g i c ro a ti c u m p m c h r v n w d in ar ic a lp s g ol a p lj e{ iv ic a. v el eb it h o r v a t 19 38 9 8 r h o d o d e n d ro h ir su ti p in e tu m p ro st ra ta e r hp m i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 1 13 e ri c o c a rn e a e -p in e tu m p ro st ra ta e e cp m i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 2 14 s o rb o c h a m a e m e sp il ip in e tu m m u g o s cp m i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 3 7 r h o d o th a m n o -j u n ip e re tu m a lp in i r cj a i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 4 14 a m e la n c h ie ro -p in e tu m m u g o a op m i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 5 6 e ri c e tu m c a rn e a e e c i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 6 12 f e st u c o a lp e st ri sg e n is te tu m ra d ia ta e f ag r i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 7 21 r h o d o d e n d re tu m fe rr u g in e i r f i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 8 20 r h o d o d e n d ro fe rr u g in e ip in e tu m p ro st ra ta e r fp m i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 9 13 v a c c in io v it is -i d e a e -c a ll u n e tu m v u lg a ri s v vc v i s e c al ca re ou s a lp s . p o l d in i et al .2 00 4 10 8 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen variation by specific environmental and structural (phytosociological) variables, unconstrained (pca, dca) and constrained (rda) ordination analyses were performed, using the canoco computer programme (braak ter and [milauer 2002). in order to determine the lengths of gradients, dca analyses, detrended by segments, were initially performed and the models (linear, unimodal) used accordingly. the statistical significance of the site parameters (p<0.05) was tested using the monte carlo test, with 499 permutations. only the significant parameters were then analyzed together, in order to produce a general view of the environmental impact on floristic composition and structure of stands. for estimating the environmental affinities of the relevés, we used pignatti’s indicator values for vascular plants (pignatti 2005). the environmental value in a relevé (evw) was estimated as the weighted average of the indicator values of all present species, their abundances being used as weights (lep[ and [milauer 2003). heaths from the liburnian karst were compared with mountain pine scrubs and structurally similar stands with dwarf ericaceous scrubs and/or alpine juniper from other parts of the dinaric alps and south-eastern calcareous alps between italy and montenegro (tab. 1, fig. 1). results floristic composition and structure of the liburnian (the north-western dinaric) heaths we recorded 98 taxa of phanerogams in 22 relevés with a median number of 27 per relevé (min=17, max=37; tab. 2). coefficient of variation of the number of taxa per relevé is 19.63%. rhododendron hirsutum+–4 (d%=7.7), juniperus alpina+–4 (6.7), salix appendiculata1–3 (6.4) and calamagrostis varia1–2 (5.4) occurred in all relevés with high or the highest coverage of all recorded taxa. almost one quarter of the all registered taxa occurred in more than 50% of relevés, with the highest presence and coverage shown by cyclamen purpurascens+–2 (3.5), rosa pendulina+–2 (3.9), allium ericetorum+–2 (3.7), erica carnea1–4 (4.5), campanula cochleariifolia+–2 (2.6), thesium linophyllum+–2 (2.2), picea abies+–1 (1.7), clematis alpina+–2 (2.2) and aster bellidiastrum+–2 (2.5). the structure of stands is defined by their edificators, rhododendron hirsutum, juniperus alpina and salix appendiculata, forming more or less dense scrubby vegetation type not exceeding 1 m in height. as a rule, these stands are developed on very shallow organogenic soil – lithosol or even over a bare, compact to fragmented limestone bedrock on gravelly slopes of mountain ridges above 1200 m a.s.l., with long-lasting snow cover where northerly exposed sites prevail. here, the studied stands are in close contact with subalpine beech (polysticho-fagetum), fir-spruce (calamagrostido-abietetum) and dinaric fir-beech forest stands (omphalodo-fagetum) as well as with scrubby stands of mountain pine (hyperico-pinetum mugo), forming transitions toward forest vegetation types. in frost hollows, an ecologically extreme habitat with specific microsite conditions (e.g. martin^i^ 1977, surina and vre[ 2004, 2009, modri] surina and surina 2010), the studied stands prefer southerly exposed gravelly slopes and close contact with azonal spruce (lonicero caeruleae-piceetum, hacquetio-piceetum) and extrazonal subalpine beech forest stands with hairy alpenrose – polysticho-fagetum rhododendretosum (surina and rakaj 2007), but more frequently with non-forest stands with carex ferruginea, salix appendiculata stands, doronico austriaci-adenostyletum alliariae, and drepanoclado uncinati-heliospermetum pusilli, the last representing the most cryophilic stands developed specifically in frost hollows of north-western dinaric alps. acta bot. croat. 72 (1), 2013 119 ericaceous shrubs and alpine juniper in the dinaric alps 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen 120 acta bot. croat. 72 (1), 2013 surina b. t a b . 2 . a na ly ti ca l ta bl e of th e as so ci at io n r h o d o d e n d ro h ir su ti -j u n ip e re tu m a lp in a e h or va t ex h or va t et al . 19 74 n o m . c o rr . p ro p . in th e l ib ur ni an ka rs t (n or th -w es t d in ar ic a lp s) . 1 1 1 1 1 1 1 1 1 1 2 2 2 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 c h a r a c te r is ti c g r o u p o f sp e c ie s fo r th e a ss o c ia ti o n d % ty p d % se s d to t % e p r h o d o d e n d ro n h ir su tu m 2 3 3 4 4 3 3 3 4 + 3 3 3 3 3 3 2 3 3 3 3 3 7. 4 7. 6 7. 7 e p j u n ip e ru s a lp in a 4 3 4 3 4 2 4 3 3 3 3 3 3 3 3 2 2 1 1 1 1 + 7. 6 5. 8 6. 7 m a s a li x a p p e n d ic u la ta 2 2 3 2 2 2 2 1 2 3 3 3 3 3 3 2 1 3 2 2 3 2 5. 3 7. 1 6. 4 e p c a la m a g ro st is v a ri a 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 1 2 2 1 2 2 2 5. 0 5. 5 5. 4 v p r o sa p e n d u li n a 2 1 2 1 . 2 2 2 2 1 2 2 2 1 1 . . + 2 2 1 1 4. 3 3. 8 3. 9 e p e ri c a c a rn e a 4 1 3 3 3 . . . . 1 1 3 3 1 2 1 1 2 2 2 1 2 3. 8 5. 2 4. 5 d if fe r e n ti a l sp e c ie s fo r th e su b a ss o c ia ti o n se sl e r ie to su m te n u if o li a e e s s e sl e ri a ju n c if o li a /t e n u if o li a . . . . . . . . . + . 3 1 + 2 4 3 3 3 4 4 3 6. 7 4. 0 t r a th a m a n ta c re te n si s s. l. . . . . . . . . . . . . + + + 2 2 2 2 1 1 1 3. 3 2. 1 a t d a p h n e a lp in a . . . . . . . . . 2 2 2 . + + . + + . . . . 2. 0 1. 2 e p e ri c o -p in e te a a ll iu m e ri c e to ru m . . 2 2 + 2 2 + 1 + . 2 1 + 1 + 2 1 2 2 2 2 3. 1 4. 2 3. 7 r u b u s sa x a ti li s . 2 1 1 1 1 + 2 3 . 1 . 1 1 + . . . . 1 . . 3. 6 1. 1 2. 2 c ir st iu m e ri si th a le s 1 1 2 + 2 1 1 1 . . . 1 2 + 2 . . . . . 1 . 3. 2 1. 5 2. 1 b u p h ta lm u m sa li c if o li u m 1 . 2 1 2 + + 1 . + . + + . . . . . . . . . 2. 7 0. 6 1. 4 c o to n e a st e r in te g e rr im u s . 1 1 1 + . . + . . + + . . . . . . . + + . 1. 5 0. 8 1. 0 a m e la n c h ie r o v a li s . + . . . . . . . + . 1 . . + . . . + . . . 0. 3 0. 8 0. 6 a q u il e g ia n ig ri c a n s + . . + + . 1 . . . . . . . . . . + . . . . 1. 0 0. 3 0. 5 c o to n e a st e r to m e n to su s . . . . . . . . . . . + . . . . . . 2 . . . 0. 7 0. 4 p in u s m u g o . . . . + . . . . . . + . . + . . . . . . . 0. 2 0. 3 0. 3 c a re x a lb a . . . . . . . . . + . . + . . . . . . . . . 0. 3 0. 2 v p v a c c in io -p ic e e te a c le m a ti s a lp in a + . . 1 . + . 2 1 + 2 . . + . + + 1 . 2 1 1 1. 7 2. 5 2. 2 p ic e a a b ie s + 1 + + 1 + + . + . + . + . . + + . + + + . 2. 1 1. 3 1. 7 h o m o g y n e sy lv e st ri s + . . 1 1 + 1 . + . . . 1 + + . . . + + 1 1 1. 6 1. 5 1. 6 s o li d a g o v ir g a u re a s. l. 1 1 . 1 1 . . 1 + + + . + . + . . . . . . . 1. 9 0. 7 1. 2 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen acta bot. croat. 72 (1), 2013 121 ericaceous shrubs and alpine juniper in the dinaric alps 1 1 1 1 1 1 1 1 1 1 2 2 2 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 l o n ic e ra c a e ru le a + + . . . . 1 + . 1 1 + + . . . . . + + + 1. 1 1. 4 1. 2 a b ie s a lb a . . . . . . . . + + + + + + . + + + + + . 2. 0 1. 1 h ie ra c iu m m u ro ru m a g g . . . . + + 1 . 1 . . + . . . + . . + + + + 1. 1 1. 2 1. 1 v a le ri a n a tr ip te ri s 1 . + 1 . . + . + . + . . . . . . . . . . . 1. 4 0. 2 0. 7 p o ly st ic h u m lo n c h it is . + + . . . . 1 . . . . . . . . . . . . . 0. 9 0. 3 v a c c in iu m v it is -i d a e a . . 1 . . . . + . . . . . . . . . . . . . 0. 6 0. 2 f s f a g e ta li a sy lv a ti c a e (i n c l. a re m o n io -f a g io n ) a f c y c la m e n p u rp u ra sc e n s 2 1 2 1 2 2 2 1 + + . 1 2 + 1 . 1 1 1 1 2 + 4. 2 3. 2 3. 5 l o n ic e ra a lp ig e n a . . . . . . . + . . + . . . . . . . . + . . 0. 2 0. 3 0. 3 m e rc u ri a li s p e re n n is . . . + + . . . . . . . . . . . . . . . . . 0. 4 0. 2 r a n u n c u lu s p la ta n if o li u s . . + + . . . . . . . . . . . . . . . . . . 0. 5 0. 2 f a g u s sy lv a ti c a . . . + . . . . . . . + . . . . . . . . . . 0. 2 0. 2 0. 2 q p q u e rc e ta li a p u b e sc e n ti s c o n v a ll a ri a m a ja li s . . + . . . . + . . . . . . . . . . . . . . 0. 5 0. 2 q f q u e rc o -f a g e te a c a re x d ig it a ta . 1 + . 1 . 1 . + . . + . . . + . + . + . . 1. 5 0. 9 1. 1 a n e m o n e n e m o ro sa . . . . . . . 1 . . . + . . . . . . . . . . 0. 3 0. 2 0. 2 t g t ri fo li o -g e ra n ie te a l a se rp it iu m si le r . . . . . + . . . + . 1 1 + 1 . + . . . . . 0. 2 1. 2 0. 8 l a se rp it iu m la ti fo li u m . . + . . . . + . . + . + . . . . . . . . . 0. 5 0. 3 0. 4 t r t h la sp ie te a ro tu n d if o li i c a m p a n u la c o c h le a ri if o li a 1 . 1 . 1 2 . + 1 + . 1 + . 1 1 2 1 1 1 + + 2. 2 2. 9 2. 6 g y m n o c a rp iu m ro b e rt ia n u m . . . . . + 1 + . + 1 + . 2 1 + . . . + 1 + 0. 8 2. 0 1. 6 a d e n o st y le s g la b ra + . . . . 1 + + + + . . + . . . + . . . . . 1. 3 0. 5 0. 9 b is c u te ll a la e v ig a ta . . . . . . . . . . . . 1 . 1 . + + + . . . 1. 1 0. 6 c a m p a n u la c a e sp it o sa 1 1 . . . . . . . . . . . . . . . . . . . . 0. 8 0. 3 p e lt a ri a a ll ia c e a . . . . . . . . . . + + . . . . . + . . . . 0. 6 0. 3 v a le ri a n a m o n ta n a . . . . . . . . . . + + . . + . . . . . . . 0. 5 0. 3 t a b . 2 . – co nt in ue d 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen 122 acta bot. croat. 72 (1), 2013 surina b. 1 1 1 1 1 1 1 1 1 1 2 2 2 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 a t a sp le n e te a tr ic h o m a n is a st e r b e ll id ia st ru m 2 1 . 2 2 2 2 + 1 + 1 2 2 + . . . . . . + . 3. 7 1. 7 2. 5 a sp le n iu m ru ta -m u ra ri a . . 1 . . + 1 . . . . . + . + + . . . . . . 1. 0 0. 5 0. 7 c a re x b ra c h y st a c h y s . 1 . + . + 1 . . . . . . . . . . . . + . . 1. 1 0. 2 0. 6 a sp le n iu m v ir id e . . 1 + . . + . 1 . . . . . . . . . . . . . 1. 2 0. 4 h ie ra c iu m b u p le u ro id e s . . . . . . . . . . + . . . . . 1 . . . . . 0. 4 0. 3 c a m p a n u la ju st in ia n a . . . + . . . + . . . . . . . . . . . + . . 0. 4 0. 2 0. 3 a q u il e g ia k it a ib e li a n a . . . . . 1 + . . . . . . . . . . . . . . . 0. 5 0. 2 m a m u lg e d io -a c o n it e te a p le u ro sp e ru m a u st ri a c u m . . . 1 1 . . . . . . . . . . . . . . . . . 0. 6 0. 3 e s e ly n o -s e sl e ri e te a p h y te u m a o rb ic u la re . . 1 + 1 . . . . . . . 2 1 1 1 2 2 2 2 2 2 0. 9 4. 0 2. 8 g a li u m a n is o p h y ll u m . . . . . . . . . + 2 . . 1 + . . . . . . . 0. 9 0. 6 e ri g e ro n g la b ra tu s . . . . . 2 . . . . + . . . . . . . . . . . 0. 5 0. 2 0. 3 r a n u n c u lu s c a ri n th ia c u s . . . . . . . . . . . . + . + . . . . . . . 0. 3 0. 2 p t p o o a lp in a e -t ri se ta li a c a m p a n u la sc h e u c h ze ri . . . . . . . . . . . + . + . + . . . . . + 0. 7 0. 5 c a re x fe rr u g in e a . . . 2 2 . . . . . . . . . . . . . . . . . 1. 0 0. 0 0. 4 f b f e st u c o -b ro m e te a t h e si u m li n o p h y ll u m 1 + 1 1 1 2 2 . . . . + 1 + 1 . . . 1 + 1 1 2. 7 1. 9 2. 2 g e n ti a n a lu te a /s y m p h y a n d ra 2 1 . . . 2 2 . . 1 1 + 1 + + . . . . + + . 2. 1 1. 6 1. 9 c a rl in a a c a u li s s. l. + + + . 1 . + . . . . . + . . . . . . . . . 1. 3 0. 2 0. 6 g a li u m lu c id u m . . . . . . . . . 1 2 1 + + + . . . . 2 1 + 2. 3 1. 3 o th e r ta x a g a li u m m o ll u g o . . 1 . . . . . 2 . . . . . . . . . . . . . 1. 1 0. 4 f ra g a ri a v e sc a . . + + . . . . 1 . . . . . . . . . . . . . 0. 9 0. 3 h ie ra c iu m sp . . . . . + . . . . . . . . . + . . . . . . . 0. 2 0. 1 0. 2 t a b . 2 . – co nt in ue d 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen generally, species of the class erico-pinetea completely dominate in stands, being the most frequent and achieving the highest coverage indices (d%=44.4, tabs. 2, 3), followed by spruce forest species (vaccinio-piceetea, 15.5) and petrophytic species of screes and rock crevices (thlaspietea rotundifolii, 9.6 and asplenietea trichomanis, 6.7, respectively). dry grassland species of the class festuco-brometea (6.2) are represented by six species, the most frequent, occurring in more than 50% of relevés, being thesium linophyllum and gentiana lutea subsp. symphyandra+–2 (1.9). there are 11 beech forest species (fagetalia sylvaticae, 5.2) recorded in the studied stands, but although cyclamen purpurascens+–2 (3.5), occurs in more than 90% of relevés, the others, a negligible coverage, are much less frequent. although structurally very similar, the studied stands form floristically and ecologically two well circumscribed vegetation types (tabs. 2, 3, fig. 2 a, b, d). stands from the first group (»typical« ones) are developed at more elevated, cooler, moister, shadier, sheltered and more nutrient-rich sites. these stands are characterized by higher number and coverage of spruce forest species which is in line with overall lower values for the substrate ph reaction and cooler sites. on the other hand, stands from the second group (-seslerietosum tenuifoliae) are developed on warmer and lighter sites at lower altitudes but, also thrive on northerly exposed slopes of the ridge, more exposed to strong winds. these stands host a considerably higher number and greater coverage of species preferring open habitats (screes – thlaspietea rotundifolii, subalpine dinaric tussock grasslands – elyno-seslerietea). among the phytosociological parameters, the altitude (p=0.002) and the number of species per relevé (p=0.006) turned out to be statistically significant factors in floristic differentiation of stands. the first group of stands is clearly separated from the second one along the altitudinal gradient. results of the simper analysis showed the overall average dissimilarity between the two groups to be 51.84 with the taxa that contributed most to the dissimilaracta bot. croat. 72 (1), 2013 123 ericaceous shrubs and alpine juniper in the dinaric alps tab. 3. coverage indices (d%) according to syntaxonomic groups within the association rhododendro hirsuti-juniperetum alpinae horvat ex horvat et al. 1974 nom. nov. prop. in the liburnian karst (north-west dinaric alps). syntaxa typical stands seslerietosum association erico-pinetea 38.2 47.8 44.4 vaccinio-piceetea 17.2 14.4 15.5 thlaspietea rotundifolii 6.0 11.7 9.6 elyno-seslerietea 1.5 12.2 1.6 mulgedio-aconitetea 7.1 7.2 7.1 asplenietea trichomanis 7.2 6.6 6.7 festuco-brometea 6.1 6.1 6.2 fagetalia sylvaticae 6.7 4.2 5.2 trifolio-geranietea 0.7 1.9 1.4 querco-fagetea 2.1 1.1 1.4 poo alpinae-trisetalia 1.0 0.7 0.9 other taxa 2.1 0.2 0.8 quercetalia pubescentis 0.7 . 0.3 molinio-arrhenatheretea 0.3 . 0.1 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen ity being sesleria juncifolia subsp. tenuifolia (3.1), erica carnea (1.8), phyteuma orbiculare (1.7), aster bellidiastrum (1.5), athamanta cretensis (1.5), rubus saxatilis (1.4) etc. even the coefficient of variation of the number of taxa per relevé showed differences, being in the first group (»typical« stands) significantly lower (16.5%) than in the second one (21.5%) which might indicate more homogenous floristic composition of stands within the first group. based on the results of the simper analysis and ecological preferences of taxa in general, as a differential group of species for the second group of stands we chose sesleria juncifolia subsp. tenuifolia+–4 (6.7), athamanta cretensis+–2 (3.3) and daphne alpina+–2 (2), exclusively differentiating the two groups. a taxonomic note: the taxonomic status of specimens of athamanta cretensis (var. mutellinoides?) from the liburnian karst is uncertain; specimens at lower altitudes (1000–1300 m) differ morphologically and physiognomically from typical ones found on higher altitude summits (e.g. mt. sne`nik, 1796 m, or mt. snje`nik, 1524 m), and are similar to specimens of a. haynaldii borb. et uechtr. syntaxonomic position and notes on nomenclature of dinaric heaths comparison of the floristic inventory in table 2 and that in the reduced synoptic table 135 in horvat et al. (1974) clearly showed that the studied stands belong to the association rhododendro hirsuti-juniperetum horvat 1962. however, in his original diagnosis (horvat 1962), the author failed to designate the type relevé (art. 2b, 7 and 17 of the phytosociological code), nor did he provide an analytical and/or synoptic table, although subsequently (horvat et al. 1974) he did publish a synoptic table (tab. 135, col. 1), which, according to art. 7 (weber et al. 2000), is the first valid publication of a name (definition iii) – rhododendro hirsuti-juniperetum alpinae horvat ex horvat et al. 1974 nom. corr. prop. (=rhododendro hirsuti-juniperetum horvat 1962 nom. nud.). as a neotype (definition viii) we chose a relevé no. 4 in table 2, neotypus hoc loco. within the studied stands, we recognized a new subassociation rhododendro hirsuti-juniperetum alpinae seslerietosum tenuifoliae subass. nova hoc loco, and as a differential group of species for the subassociation we chose sesleria juncifolia subsp. tenuifolia, athamanta cretensis and daphne alpina. nomenclature type for the new subassociation is relevé no. 17 in table 2, holotypus hoc loco. results of the ordination analysis (fig. 2c) and cluster analyses using various similarity measures (not shown) suggest great floristic similarity between stands of the association rhododendro hirsuti-juniperetum from the liburnian karst (rj) and stands from the south-eastern calcareous alps, belonging to the associations rhododendro hirsuti-pinetum prostratae (rhpm), erico carneae-pinetum prostratae (ecpm) and rhododendretum hirsuti (rh; group a). in all three associations, species from the class erico-pinetea prevail both in number and coverage over the species of the other syntaxa and we classified stands from the liburnian karst into class erico-pinetea, order erico-pinetalia and alliance ericion carneae (see below). stands of the association aquilegio-rhododendretum hirsuti (anrh) were nested in the same group of relevés (fig. 2c, group a) indicating close floristic and ecological similarity. hence, we classified them into the alliance ericion carneae. although the syntaxon aquilegio-rhododendretum hirsuti is supported with the analytical table (tab. 24 in laku[i] et al. 1979), the type relevé has not been designated and is, according to definition iv of the code (compare also articles 5, 15–18), treated as an unpublished name. as a lectotype of the association aquilegio nigricantis-rhododendretum hirsuti laku{i} et al. ex 124 acta bot. croat. 72 (1), 2013 surina b. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen surina ass. nova (=aquilegio -rhododendretum hirsuti laku{i} et al. 1979 nom. inv.), we chose relevé no. 2 in tab. 24 in laku[i] et al. 1979, lectotypus hoc loco. dinaric scrubs of mountain pine (hgpm, pmc, pmm 1, 2) formed a group c (fig. 2c), while more acidophilic syntaxa, either heaths (vvcv, rf, hvb, hvm, rajn, jsss) or mountain pine scrubs (rfpm, pmi), formed another homogenous group (group b, fig. 2c). we classified the later, being distinctly acidophilic, into the class vaccinio-piceetea, order vaccinio-piceetalia and alliance rhododendro-vaccinion. the associations hyperico-vaccinietum bosniacum, hyperici-vaccinietum montenegrinum and vaccinio-callunetum subalpinum are invalidly published, violating the principles of phytosociological nomenclature in several articles (e.g. art. 5, 10, 15–18, 46). hence, validly published names with selected lectotypes are proposed: hyperico maculati-vaccinietum myrtilli laku{i} et al. ex surina ass. nova (=hyperico-vaccinietum bosniacum laku{i} et al. 1979 nom. inv.), lectotypus hoc loco: relevé no. 2 in table 23 in laku[i] et al. (1979); hyperico grisebachii-vaccinietum myrtilli laku{i} ex surina ass. nova (=hyperici-vaccinietum montenegrinum laku{i} 1966 nom. inv.), lectotypus hoc loco: relevé no. 6 in table 22 in laku[i] (1966); vaccinio myrtilli-callunetum vulgaris laku{i} et al ex surina ass. nova (=vaccinio-callunetum subalpinum laku{i} et al. 1979 nom. inv.), lectotypus hoc loco: relevé no. 10 in table 23 in laku[i] et al. (1979). according to the results of the analyses, stands of the associations roso pendulinae-juniperetum alpinae laku{i} 1966 nom. corr. prop. (art. 41; =roso pendulinae-juniperetum nanae laku{i} 1966) and sempervivo schlechanii-juniperetum alpinae bjel~i} 1966 nom. invers. et corr. prop. (art. 42; =junipero-semperviretum schlechanii bjel~i} 1966), gathered in group c together with stands of the associations hyperico maculati-vaccinietum myrtilli, hyperico grisebachii-vaccinietum myrtilli and vaccinio myrtilli-callunetum vulgaris, showed considerable floristic similarities and are thus classified accordingly. for the two mentioned syntaxa, lectotypes were here designated: roso pendulinae-juniperetum alpinae laku{i} 1966 nom. corr. prop., lectotypus hoc loco: relevé no. 4 in table 22 in laku[i] (1966); sempervivo schlechanii-juniperetum alpine bjel~i} 1966 nom. invers. et corr. prop., lectotypus hoc loco: relevé no. 6 in table 2 in bjel^i] (1966). in all the numerical analyses we performed, relevés of the association seslerio robustae-juniperetum alpinae domac 1962 nom. corr. prop. formed a completely separated and floristically clearly distinct group of stands, being the most thermophilic and composed of a significant number of species of the class festuco-brometea. therefore, we omitted this syntaxon from the subsequent analyses and classified it within the class festuco-brometea, order scorzonero-chrysopogonetalia and alliance satureion subspicatae. erico-pinetea horvat 1959 erico-pinetalia horvat 1959 ericion carneae rübel ex grabherr et al. 1993 aquilegio nigricantis-rhododendretum hirsuti laku{i} et al. ex surina ass. nova hoc loco (=aquilegio-rhododendretum hirsuti laku{i} et al. 1979 nom. inv.) rhododendro hirsuti-juniperetum alpinae horvat ex horvat et al. 1974 nom. corr. prop. (=rhododendro-juniperetum horvat 1962 nom. nud.) -seslerietosum tenuifoliae surina subass. nova hoc loco vaccinio-piceetea br.-bl. in br.-bl. et al. 1939 emend. zupan~i~ (1976) 2000 piceetalia excelsae pawlowski in pawlowski et al. 1928 rhododendro-vaccinion (br.-bl. in br.-bl. et jenny 1926) br.-bl. 1948 roso pendulinae-juniperetum alpinae laku{i} 1966 nom. corr. prop. acta bot. croat. 72 (1), 2013 125 ericaceous shrubs and alpine juniper in the dinaric alps 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen (=roso pendulinae-juniperetum nanae laku{i} 1966) sempervivo schlechanii-juniperetum alpinae bjel~i} 1966 nom. invers. et corr. prop. (=junipero-sempervivetum schlechanii bjel~i} 1966) hyperico maculati-vaccinietum myrtilli laku{i} et al. ex surina ass. nova hoc loco (=hyperici-vaccinietum bosniacum laku{i} et al. 1979 nom. inv.) hyperici grisebachii-vaccinietum myrtilli laku{i} ex surina ass. nova hoc loco (=hyperici-vaccinietum montenegrinum laku{i} 1966 nom. inv.) vaccinio myrtilli-callunetum vulgaris laku{i} et al. ex surina ass. nova hoc loco (vaccinio-callunetum subalpinum laku{i} et al. 1979 nom. inv.) festuco-brometea br.-bl. et tx. 1943 scorzonero-chrysopogonetalia horvati} et horvat (1956) 1958 satureion subspicatae horvat 1961 seslerio robustae-juniperetum alpinae domac 1962 nom. corr. prop. discussion according to our observations, heaths of the association rhododendro hirsuti-juniperetum alpinae persist in sites with extremely low winter temperatures, but with long duration of snow cover, where stands covered with snow are sheltered from low temperature extremes, winter desiccation, ice blast and solar radiation. the same pattern with structurally similar stands on mt. jahorina was thoroughly discussed by bjel^i] (1966). it seems that in our case extreme winter temperatures on sites with ericaceous heaths are buffered with deep and persistent snowpacks originating either from blasts of snow along the mountain ridges or specific microclimate of frost dolines (temperature inversion!). the specific origin of snowpacks, as well as specifics in relief, are well reflected in floristic composition of stands and distinction of the two subassociations: (a) –typicum, with cryophilic stands developed in moister and cooler sites at the margins or on slopes of frost dolines, and (b) –seslerietosum tenuifoliae, with relatively thermophilic stands developed on warmer, lighter and wind exposed sites on mountain ridges. although within a rather restricted area of the liburnian karst, structurally identical but, in terms of their origin, floristic composition and specifics in site ecology, nevertheless different groups of stands were identified. due to site ecology and pronounced human impact (deforestation due to intensive logging and subsequent soil erosion and pasture activities), these stands, which are in contact with various forest and non-forest stands, but in general developed within the stands of zonal association polysticho lonchitis-fagetum, host a plethora of species of different vegetation types (classes, see poldini et al. 2004), rendering synsystematic and syndynamic analyses particularly troublesome. although there were earlier attempts to explain the origin and syndynamics of studied heaths (e.g. horvat 1962, bjel^i] 1966), none of the proposed schemes actually suffice and the question of heath syndynamics, at least in the dinaric alps, remains an open question. this is followed with uncertainties in heath synsystematics where several authors proposed various synsystematic schemes based either on floristic principle, structure of stands of both. in our proposal we followed the compromise made by poldini et al. (2004), placing heaths on the basis of floristic principle into two classes: erico-pinetea and vaccinio-piceetea, and only latter, while classifying the associations into the syntaxa ranked bellow the class, took into the consideration their structure. hence, the dinaric associations rhododendro hirsuti-juniperetum alpinae and aquilegio nigricantis-rhododendretum hirsuti (their stands being de126 acta bot. croat. 72 (1), 2013 surina b. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:45 color profile: generic cmyk printer profile composite default screen veloped on calcareous soils) were placed within the class erico-pinetea, while associations roso pendulinae-juniperetum alpinae, sempervivo schlechanii-juniperetum alpinae (their stands being developed on non-calcareous soils or on sites with close contact to spruce forests, respectively), hyperico maculati-vaccinietum myrtilli and vaccinio myrtilli-callunetum vulgaris (their stands being developed on non-calcareous soils) within the class vaccinio-piceetea. this scheme is well supported by the results of the numerical analyses (fig. 2c). the association arctostaphylletum uvae-ursi silicicolum laku{i} et al. 1979 nom. inv., since documented with only a single relevé, was not analyzed, but most probably belongs to the same group of syntaxa. the stands of the association seslerio robustae-juniperetum alpinae from the biokovo mountain range in central dalmatia, structurally similar, but floristically quite distinct, synsystematically better fit within the class festuco-brometea than erico-pinetea or even vaccinio-piceetea (for further discussion see trinajsti] 1987) and to ascertain their syntaxonomic position more accurately would require much more comprehensive analyses comparing syntaxa not only of two, but three or four classes. being aware that their syntaxonomic position is not entirely clear we followed the proposal of trinajsti] (1987, 2008), although sedlar and co-workers placed similar stands with pinus nigra subsp. dalmatica dominating in a tree layer within the class pino-juniperetea (sedlar et al. 2011). although resolving the syntaxonomy of mountain pine scrubs communities was far beyond the scope of the present paper, the results of our preliminary analyses showed a certain (phyto)geographical structure (fig. 3c), where dinaric stands represented a distinct group of syntaxa in all of the performed analyses. however, only a thorough synoptic approach taking into account all the south-eastern-european stands of the mountain pine would properly challenge their current syntaxonomy. acknowledgements we thank friends and colleagues andra` @nidar{i~ (slovenia forest service, slovenia), andrej radalj (the jezero society, croatia) for the help during the field work and @eljka modri} surina (natural history museum rijeka, croatia), marko randi} (public institution »priroda«, croatia), igor dakskobler (science and research centre of the slovenian academy of sciences and arts, slovenia), academ. mitja zupan~i~ (slovenian academy of sciences and arts, slovenia), emerit. livio poldini (university of trieste, italy), romeo di pietro (sapienza university of rome, italy), vladimir hr{ak and joco vukeli} (university of zagreb, croatia) for fruitful discussions on syntaxonomy of heaths. special thanks go to igor dakskobler for valuable comments and discussions on nomenclatorial issues, while @eljka modri} surina commented on a previous version of the manuscript. the research was financially supported by the public institution »priroda« (project no. 112-07/11-02/01 – 2170-52-01/1-11-19). references aeschimann, d., lauber, k., mosser, d. m., theurillat, j.-p., 2004: flora alpina, 1–3. haupt verlag, bern, stuttgart, wien. acta bot. croat. 72 (1), 2013 127 ericaceous shrubs and alpine juniper in the dinaric alps 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:46 color profile: generic cmyk printer profile composite 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(eds.), die pflanzengesellschaften österreichs, 3, wälder und gebüsche, 244–282. fischer verlag, jena-stuttgart-new york. wallnöfer, s., 1993a: vaccinio-piceetea. in: grabherr, g., greimler, j., mucina, l. (eds.), die pflanzengesellschaften österreichs, 3, wälder und gebüsche, 283–337. fischer verlag, jena-stuttgart-new york. weber, h. e., moravec, j., theurillat, j.-p., 2000: international code of phytosociological nomenclature, 3. journal of vegetation science 11, 739–768. westhoff, v., van der maarel, e., 1973: the braun-blanquet approach. in: whittaker, r. h. (ed.), ordination and classification of communities. handbook of vegetation science 5, 619–726. dr. w. junk b.v. publishers, the hague. wraber, t., 1997: bupleurum exaltatum, mt. sne`nik and – (?) sheeps (in slovenian). proteus 59, 374–377. zupan^i^, m., wraber, t., @agar, v., 2004: dinaric mountain pine association hyperico grisebachii-pinetum mugo on mt. sne`nik (in slovenian). razprave 4 razreda sazu 45, 185–261. zupan^i^, m., @agar, v., culiberg, m., 2006: slovene alpine pinus mugo scrub in comparison with european pinus mugo scrab. dela 4 razreda sazu 40, 1–111. appendix i – phytosociological and site parameters of relevés in table 1 (rel. no. and field no., altitude, exposition, inclination, coverage: s – stoniness, c – herb layer 1–8 – mt. sne`nik, 9–22 – mt. obru~ 1 (20111004/07), 1413 m, nne, 36 m2, 200, s 40%, c 60%, leg. a. radalj et b. surina; 2 (20111004/08), 1412 m, se, 36 m2, 100, s 60%, c 40%, leg. a. radalj et b. surina; 3 (20111004/04), 1390 m, s, 36 m2, 250, s 60%, c 40%, leg. a. radalj et b. surina; 4 (20111004/03), 1375 m, nne, 30 m2, 350, s 20%, c 80%, leg. a. radalj et b. surina; 5 (20111004/05), 1400 m, e, 25 m2, 400, s 30%, c 70%, leg. a. radalj et b. surina; 6 (20111004/01), 1391 m, e, 20 m2, 400, s 50%, c 50%, leg. a. radalj et b. surina; 7 (20111004/02), 1400 m, nne, 25 m2, 600, s 40%, c 60%, leg. a. radalj et b. surina; 8 (20050724/05), 1340 m, s, 25 m2, 200, s 30%, c 70%, leg. a. @nidar{i~ et b. surina; 9 (20111004/06), 1350 m, nne, 50 m2, 300, s 40%, c 60%, leg. a. radalj et b. surina; 10 (20110907/08), 1242 m, ese, 36 m2, 300, s 40%, c 60%, leg. b. surina; 11 (20110907/12), 1220 m, e, 30 m2, 350, s 60%, c 40%, leg. b. surina; 12 (20110907/11), 1260 m, nne, 30 m2, 300, s 40%, c 60%, leg. b. surina; 13 (20110907/07), 1239 m, n, 25 m2, 300, s 30%, c 70%, leg. b. surina; 14 (20110907/06), 1243 m, nne, 20 m2, 300, s 40%, c 60%, leg. b. 130 acta bot. croat. 72 (1), 2013 surina b. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:46 color profile: generic cmyk printer profile composite default screen surina; 15 (20110907/10), 1248 m, n, 25 m2, 400, s 50%, c 50%, leg. b. surina; 16 (20110830/10), 1260 m, nne, 30 m2, 250, s 60%, c 40%, leg. @. modri} surina et b. surina; 17 (20110907/05), 1252 m, ene, 30 m2, 300, s 60%, c 40%, leg. b. surina; 18 (20110907/09), 1244 m, nne, 25 m2, 350, s 60%, c 40%, leg. b. surina; 19 (20110907/04), 1260 m, ne, 50 m2, 250, s 50%, c 50%, leg. b. surina; 20 (20110907/01), 1255 m, ene, 20 m2, 300, s 40%, c 60%, leg. b. surina; 21 (20110907/02), 1246 m, ene, 30 m2, 350, s 30%, c 70%, leg. b. surina; 22 (20110907/03), 1250 m, ne, 36 m2, 400, s 40%, c 60%, leg. b. surina. appendix ii – taxa occurring once in table 1 erico-pinetea: arctostaphyllos uva-ursi 1 (12), gymnadenia conopsea + (18), peucedanum austriacum s.l. + (8); vaccinio-piceetea: huperzia selago + (4), luzula sylvatica + (4); fagetalia sylvaticae (incl. aremonio-fagion*): hacquetia epipactis* + (8), euphorbia carniolica + (8), melica nutans + (8), dryopteris filix-mas + (9), scrophularia nodosa + (9), acer pseudoplatanus + (13); querco-fagetea (incl. quercetalia pubescentis*): melittis melissophyllum* + (9), hepatica nobilis + (8); trifolio-geranietea: ligusticum seguerii 2 (11); thlaspietea rotundifolii: petasites paradoxus 2 (18), dryopteris submontana 1 (9), festuca nitida 1 (9), scrophularia laciniata + (10), trisetum argenteum + (4); asplenietea trichomanis: asplenium fissum + (6), kernera saxatilis + (6), cystopteris fragilis + (9); mulgedio-aconitetea: viola biflora + (6), aconitum ranunculifolium + (9), hypericum maculatum ssp. maculatum + (9), veratrum album s.l. + (3), tephroseris ovirensis + (4); elyno-seslerietea: polygala alpestris ssp. croatica + (20); festuco-brometea: teucrium montanum + (10), ruta divaricata 1 (10); molinio-arrhenatheretea: leontodon hispidus 1 (4). appendix iii – list of syntaxa mentioned in text and table 1 amelanchiero-pinetum mugo minghetti in pedroti 1994; aquilegio nigricantis-rhododendretum hirsuti laku{i} et al. ex surina ass. nova hoc loco (=aquilegio-rhododendretum hirsuti laku{i} et al. 1979 nom. inv.); arctostaphylletum uvae-ursi laku{i} et al. 1979 nom. inv.; aremonio-fagion (horvat 1938) borhidi in török, podani et borhidi 1989; asplenietea trichomanis br.-bl. in meier et br.-bl. 1934; berberido creticae-juniperion foetidissimae brullo et al. 2001; calamagrostido arundinaceae-fagetum cerove~ki 2009; calamagrostido variae-abietetum horvat 1950 piceetosum; carici humilis-centaureetum rupestris horvat 1931 seslerietosum tenuifoliae horvat 1962 nom. nud.; doronico austriaci-adenostyletum alliariae horvat ex horvat 1974; drepanoclado uncinati-heliospermetum pusilli surina et vre{ 2004; elyno-seslerietea br.-bl. 1948; ericetum carneae rübel 1911; ericion carneae rübel ex grabherr et al. 1993; erico carneae-pinetum prostratae zötl 1951 nom. inv.; erico-pinetea horvat 1959; erico-pinetalia horvat 1959; fagetalia sylvaticae pawlowski in pawlowski et al. 1928; festuco-brometea br.-bl. et tx. 1943; festuco alpestris-genistetum radiatae peer ex poldini et al. 2004; hacquetio-piceetum zupan~i~ (1980) 1999; hyperico maculati-vaccinietum myrtilli laku{i} et al. ex surina ass. nova hoc loco (=hyperici-vaccinietum bosniacum laku{i} et al. 1979 nom. inv.); hyperico grisebachii-vaccinietum myrtilli laku{i} ex surina ass. nova hoc loco (=hyperici-vaccinietum montenegrinum laku{i} 1966 nom. inv.); acta bot. croat. 72 (1), 2013 131 ericaceous shrubs and alpine juniper in the dinaric alps 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:46 color profile: generic cmyk printer profile composite default screen hyperico grisebachii-pinetum mugo wraber et al. in zupan~i~ et al. 2004 var. geogr. arabis scopoliana (=pinetum mughi croaticum horvat 1938 p.p.; pinetum mughi montenegrinum ble~i} 1957 nom. inv.); juniperetalia haemisphaericae rivas-martinez et molina 1999; junipero sibiricae-semperviretum schlechanii bjel~i} 1966 (=sempervivo schlechanii-juniperetum sibiricae bjel~i} 1966 nom. invers. prop.); lonicero caeruleae-piceetum zupan~i~ (1980) 1999; loiseleurio-vaccinietea eggler ex schubert 1960; mulgedio-aconitetea hada~ et klika in klika et hada~ 1944; molinio-arrhenatheretea r. tx. 1937 em. r. tx. 1970; omphalodo-fagetum (tregubov 1957 corr. puncer 1980) marin~ek et al. 1993 var. geogr. calamintha grandiflora surina 2001 seslerietosum autumnalis nom. nud. (=fagetum croaticum australe abietetosum horvat 1938, abieti-fagetum dinaricum tregubov 1957); piceetalia excelsae pawlowski in pawlowski et al. 1928; pinetum mughi illyricum laku{i} et al. 1979 nom. inv.; pino-juniperetea rivas-martinez 1964; polysticho lonchitis-fagetum (horvat 1938) marin~ek in poldini et nardini 1993 (=fagetum croaticum australe subalpinum horvat 1938) rhododendretosum hirsuti surina et rakaj 2007; poo alpinae-trisetalia ellmauer et mucina 1993; quercetalia pubscentis klika 1933; querco-fagetea br.-bl. et vlieg. 1937; rhododendretum ferruginei rübel 1911; rhododendro ferruginei-pinetum prostratae zötl 1951 nom. inv.; rhododendro hirsuti-juniperetum sibiricae horvat ex horvat et al. 1974 (=rhododendro hirsuti-juniperetum sibiricae horvat 1962 nom. nud.); rhododendro hirsuti-pinetum prostratae zötl 1951 nom. inv.; rhododendro hirsuti-salicetum appendiculatae toma`i~ nom. nud. (=salicetum appendiculatae horvat ex horvat et al. 1974); rhododendro-vaccinion (br.-bl. in br.-bl. et jenny 1926) br.-bl. 1948; rhodothamno chamaecisti-juniperetum alpini poldini et al. 2004; roso-juniperetum nanae laku{i} 1966 (=roso pendulinae-juniperetum sibiricae laku{i} 1966 nom. corr.); roso pendulinae-pinetea mugi theurillat in theurillat et al. 1995; satureion subspicatae horvat 1962; scorzonero-chrysopogonetalia horvati} et horvat (1956) 1958; seslerio autumnalis-fagetum (horvat 1938) m. wraber ex borhidi 1963 (=fagetum croaticum australe seslerietosum autumnalis horvat 1938); seslerion tenuifoliae horvat 1962; seslerietea albicantis oberdorfer 1978 corr. oberdorfer 1990; seslerio robustae-juniperetum sibiricae domac 1962; sorbo chamaemespili-pinetum mugo minghetti 1996; thlaspietea rotundifolii br.-bl. in br.-bl. et jenny 1926; trifolio-geranietea th. mueller 1961; vaccinio myrtilli-callunetum vulgaris laku{i} et al. ex surina ass. nova hoc loco (vaccinio-callunetum subalpinum laku{i} et al. 1979 nom. inv.); vaccinio-piceetea br.-bl. 1939 emend. zupan~i~ (1976) 2000; vaccinio vitis-ideae-callunetum vulgaris poldini et al. 2004. 132 acta bot. croat. 72 (1), 2013 surina b. 554 surina.ps u:\acta botanica\acta-botan 1-13\554 surina.vp 14. o ujak 2013 11:35:46 color profile: generic cmyk printer profile composite default screen opce-str.vp acta bot. croat. 70 (2), 289–300, 2011 coden: abcra 25 issn 0365-0588 new localities of the subendemic species berberis croatica, teucrium arduini and micromeria croatica in the dinaric alps dario kremer1, marko randi]2, ivan kosalec1, ana brklja^i]3, gordan luka^4, irena kru[i]5, dalibor ballian6, faruk boguni]6, ksenija karlovi]7* 1 faculty of pharmacy and biochemistry, university of zagreb, schrottova 39, hr-10000 zagreb, croatia 2 public institution »priroda«, grivica 4, hr-51000 rijeka, croatia 3 public institution »velebit nature park«, kani`a gospi}ka 4b, hr-53000 gospi}, croatia 4 public institution »paklenica national park«, f. tu|mana 14a, hr-23244 starigrad paklenica, croatia 5 public institution »northern velebit national park«, krasno 96, hr-53274 krasno, croatia 6 faculty of forestry, university of sarajevo, zagreba~ka 20, bih 71000 sarajevo, bosnia and herzegovina 7 faculty of agriculture, university of zagreb, sveto{imunska 25, hr-10000 zagreb, croatia. abstract – new localities of three subendemic species (berberis croatica, teucrium arduini and micromeria croatica) have been found in the dinaric alps. berberis croatica was found at ten new locations, nine of them in croatia and one in bosnia and herzegovina. teucrium arduini was found on mt u~ka, mt velebit, mt biokovo and mt snije`nica, at nine new locations while micromeria croatica was found at four new locations, only on mt velebit. key words: berberis croatica, teucrium arduini, micromeria croatica,, velebit, u~ka, biokovo, snije`nica, dinaric alps abbreviations: nhc – national habitat classification acta bot. croat. 70 (2), 2011 289 * corresponding author, e-mail: karlovic@agr.hr copyright® 2011 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:54 color profile: disabled composite 150 lpi at 45 degrees introduction berberis croatica horvat is a mountainous deciduous shrub distinguished by its small, thick, rigid leaves, 1–3 cm long, and short and erect inflorescences with fewer flowers (5–12 in inflorescence) than the common barberry. the occurrence of this species in croatia was first reported by borbás (1886) under the name berberis aetnensis presl var. brachyacantha strobl. its taxonomic status still remains uncertain (karlovi] et al. 2010, zovko kon^i] et al. 2010), and akeroyd and webb (1993) in flora europaea recognize only b. vulgaris l. and b. cretica l. as european barberries. according to [ili] (2005) the distribution range of b. croatica includes the western part of the balkan peninsula (croatia, bosnia and herzegovina, montenegro and macedonia). in croatia it could be found on mt u~ka, mt obru~, gorski kotar (mt risnjak, mt vi{evica, mt bjelolasica, ^abar, prezid, vrbovsko), mt velebit, mt li~ka plje{ivica, mt dinara, mt kame{nica and mt biokovo (degen 1938:151, ku[an 1969b, martinis 1994, nikoli] 2008) at altitudes ranging from 1000 (even below) to 1700 m a.s.l. kremer et al. (2008) described eleven new localities of b. croatica in croatia with altitudes ranging from 840 to 1600 m a.s.l. the distribution of b. croatica on mt kame{nica was not clear until now, because there was no specific information concerning whether croatian barberry grows in croatia, bosnia or both. teucrium arduini l. is a semi-woody plant from the lamiaceae family with erect or ascending stems, ovate leaves and dense inflorescences with small whitish flowers. it is an endemic illyrian balkan species with a distribution range extending from the istria peninsula in the north of croatia to albania, within the restricted area of the western balkans ([ili] 1990, laku[i] et al. 2007). populations of t. arduini mainly grow in localities with mediterranean (adriatic mediterranean) and sub-mediterranean influence, even though some isolated populations inhabit limestone canyons and gorges in the transitional sub-mediterranean-central-european climatic zone with strong mediterranean influence (laku[i] et al. 2007). altitudinal distribution stretches from the sea level up to 1600 m a.s.l. from about 70 species belonging to the genus micromeria benth (meimberg et al. 2006), 21 are recognized for the european region (chater and guinea 1972) and nine for croatia (lova[en-eberhardt 2000). one of the species documented in croatia is micromeria croatica (pers.) schott for which two varieties and four forms, published so far, are listed by bräuchler et al. (2008). micromeria croatica is a perennial plant with numerous, 5–30 cm long stems; hairy, stalkless leaves and flowers with pink-purple corollas appearing from (june) july till august (september). micromeria croatica grows in the crevices of carbonate rocks predominantly in the alpine and the sub-alpine range, extending from the altitude of 150 m to more than 2000 m a.s.l ([ili] 1979, [ili] 1990). it is an endemic species of the dinaride mountains. in croatia it could be found in gorski kotar, lika, krbava, klek, along the massif of velebit and on mt po{tak (degen 1938:635, [ili] 1979, forenbacher 1990). also, it was mentioned in literature for mt risnjak ([egulja et al. 1994, pelivan 1997), mt dinara (alegro and ru[^i] 2010) and the lower part of the cetina basin (ru[^i] 2010). data about its presence on mt kalnik (kranj~ev, personal communication) need confirmation. the aim of this paper is to present new localities of berberis croatica, teucrium arduini and micromeria croatica which are so far unpublished but also to report the lack of these species at some of the localities that are mentioned in the older literature but were not found during our field work. 290 acta bot. croat. 70 (2), 2011 kremer d., randi] m., kosalec i., brklja^i] a., luka^ g., kru[i] i., et. al. u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:54 color profile: disabled composite 150 lpi at 45 degrees materials and methods the new localities were discovered during several field trips undertaken from may 2008 till september 2010. all the locations, except one (kame{nica) are in croatia. several specimens of each species were taken from each locality for identification and for further research (unconnected with this paper). voucher specimens of berberis croatica were deposited in the herbarium collection of the department of ornamental plants, landscape architecture and history of garden art, faculty of agriculture university of zagreb, zagreb while voucher specimens of teucrium arduini and micromeria croatica were deposited in the herbarium of the department of pharmaceutical botany with the »fran ku{an«, pharmaceutical botanical garden faculty of pharmacy and biochemistry, university of zagreb, zagreb. standard keys for identification were used (chater and guinea 1972, trinajsti] 1973, [ili] 1979, domac 1994). lova[en-eberhardt (1994) and erhardt (2002) were employed as a standard for the nomenclature of the species. each locality was described by providing data about the altitude and position: latitude and longitude were obtained using 1:25000 maps, gauss-krüger coordinates system and the software package mapsource – garmin. habitat types are defined by national habitat classification codes (narodne novine 2009, flora croatica database – habitats 2009). since all three analyzed species are listed as strictly protected native taxa (narodne novine 2006) the general condition of the plants is given as well as the problems observed and possible causes for any reduction in numbers of plants. absence of the species at the localities that have already been mentioned in the literature was also recorded. results and discussion all newly found localities of the three investigated species, with their specific identification numbers are listed in table 1. 1. localities of berberis croatica new localities of berberis croatica were found at a few distinct geographical sites: the hinterland mountains of rijeka, mt velebit, mt biokovo and mt snije`nica. in the hinterland mountains of rijeka, b. croatica was found at three locations (fig. 1). mli~ni vrh between platak and gornje jelenje several small groups of shrubs of berberis croatica grow on grassy, rocky ridge of eastern branch of mli~ni vrh (45°24' n, 14°35' e) at 1190 m a.s.l. this locality is exposed to strong winds and is weakly vegetated. plants of croatian barberry are up to 30 cm high, in poor health and with reduced fructification. they are surrounded by grassland formed by narrow-leaved moor grass (sesleria tenuifolia schrad.). among the plants of b. croatica grow: satureja subspicata bartl ex vis. ssp. liburnica [ili}, daphne alpina l., carex humilis leyss., cotoneaster nebrodensis (guss.) k. koch, teucrium montanum l., knautia l. sp. and allium ericetorum thore. habitat type, according to the national habitat classification (nhc), belongs to east-mediterranean rocky grassland of the epi-mediterranean zone (saturejon subspicatae h-i} 1975 community) (c.3.5.2.) on its upper elevation limit and in a distinctive succession phase of overrun by the small shrubs. acta bot. croat. 70 (2), 2011 291 berberis, teucrium and micromeria in the dinaric alps u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:54 color profile: disabled composite 150 lpi at 45 degrees one group of more vital shrubs grows at the edge of a common beech forest (fagus sylvatica l.). these plants of b. croatica are up to 60 cm high and more vital than the previously mentioned group of plants. plant species that grow nearby are: lonicera alpigena l., satureja subspicata ssp. liburnica, clematis alpina (l.) mill., carex humilis, sesleria tenuifolia, knautia illyrica g. beck, dianthus monspessulanus l., convallaria majalis l., pimpinella saxifraga l., vicia cracca l., phyteuma orbiculare l., ligusticum lucidum mill., seseli libanotis (l.) k. koch, rubus saxatilis l. and aconitum l. sp. according to the nhc, this habitat type belongs to the forest edge of southeastern alpine-illyrian thermophile beech forest (e.4.6.). unnamed rocky peak between crni vrh and gornik this unnamed rocky peak (45°27' n, 14°31' e; 1333 m a.s.l.) is situated between crni vrh (1335 m) and gornik (1320 m) in the mountains above grobni~ko polje. a few shrubs of berberis croatica grow about ten meters below the peak, on a somewhat sheltered site exposed to the sun, covering an area of several square meters. the biggest shrub of b. croatica is about 40 cm high while the others are stunted. the plants grow surrounded by 292 acta bot. croat. 70 (2), 2011 kremer d., randi] m., kosalec i., brklja^i] a., luka^ g., kru[i] i., et. al. tab. 1. newly found localities of berberis croatica, teucrium arduini and micromeria croatica. loc. no. location description taxon x coordinate y coordinate 1 mli~ni vrh, between platak and gornje jelenje b. croatica 5467377 5029483 2 unnamed rocky peak, between crni vrh and gornik b. croatica 5462190 5035069 3 mala kosa, near railway station drivenik (gorski kotar) b. croatica 5477764 5014619 4 unnamed peak, between kita and ze~jak b. croatica 5499795 4950940 5 budakovo brdo b. croatica 5506784 4937308 6 cliff kuk od [pilji} plane b. croatica 5513901 4933556 7 \u|inovac near prezid b. croatica 5565395 4901709 8 kame{nica b. croatica 6412673 4841060 9 vo{ac (mt biokovo) b. croatica 6421497 4790947 10 mt snije`nica b. croatica 6528739 4714640 11 argun t. arduini 5438524 5014879 12 [u{anj cesari~ki t. arduini 5509300 4931298 13 panos t. arduini 5522823 4920284 14 veliki vaganac t. arduini 5535451 4910288 15 veliko rujno t. arduini 5534823 4912368 16 bojinac t. arduini 5533647 4911924 17 \u|inovac near prezid t. arduini 5565395 4901709 18 [trbina (mt biokovo) t. arduini 6421561 4796502 19 mt snije`nica t. arduini 6528739 4714640 20 cliff rossijev kuk m. croatica 5499399 4958006 21 cliff ruji~in kuk m. croatica 5509937 4935696 22 cliff kuk od [pilji} plane m. croatica 5513901 4933556 23 \u|inovac near prezid m. croatica 5565395 4901709 u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:54 color profile: disabled composite 150 lpi at 45 degrees grassland of sesleria tenuifolia. in the nearby vegetation fagus sylvatica, rhododendron hirsutum l. and salix appendiculata vill. dominate. this narrow micro-locality with shrubs of b. croatica, according to nhc could be described as overrun grassland of saturejon subspicatae (c.3.5.2) community, at the upper limit of its altitudinal distribution, with many characteristic thermophile species lacking. mala kosa near railway station drivenik (gorski kotar) based on altitude (840 m a.s.l.) and habitat conditions, this population of berberis l. connects the b. croatica population on vi{evica with b. vulgaris population in li~ko polje. shrubs of b. croatica grow near the railway station drivenik, on the eastern slope of mala kosa (45°16' n, 14°43' e), on big limestone rocks, surrounded by shrubby vegetation and european silver fir (abies alba mill.) forest. habitat type is dinaric fir forest on calcareous block (e.7.1.1.), i.e. the narrow micro-locality belongs to the rocky glade within this forest type. croatian barberry was found on four new localities on mt velebit. acta bot. croat. 70 (2), 2011 293 berberis, teucrium and micromeria in the dinaric alps fig. 1. newly found localities of berberis croatica (triangles; a), teucrium arduini (crosses; b) and micromeria croatica (circles; c) in croatia and bosnia and herzegovina. for number explanation see table 1. u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:55 color profile: disabled composite 150 lpi at 45 degrees unnamed peak between kita and ze~jak the first prominent rocky peak (44°42' n, 14°60' e) on the left side of the hiking trail from kita to ze~jak is a newly found locality of b. croatica. plants of b. croatica grow from the fissures in limestone on the peak top covering a discontinuous area of about 20 m2. plants are vigorous, up to 80 cm high and are growing on the site exposed to the southeast together with juniperus communis ssp. alpina (sm.) ^elak, clematis alpina, rosa pendulina l., lonicera alpigena, cotoneaster nebrodensis, mercurialis perennis l. and cirsium mill. sp. common beech (fagus sylvatica) dominates in the nearby vegetation. according to the nhc this habitat type belongs to subalpine scrub (d.2.). budakovo brdo budakovo brdo (44°35' n, 15°05' e; 1318 m a.s.l.) is one of the peaks situated in the middle section of mt velebit. two bushes of b. croatica were found near the hiking trial (in the valley) in the direction of soline peak (1267 m a.s.l.). plants grow on the southeastern exposure, at 1215 m a.s.l. this locality is sheltered from the wind and plants are vigorous, up to 1.20 m high. plant species that grow nearby are: corylus avellana l., arctostaphylos uva-ursi (l.) spreng, cotoneaster integerrimus medik., lonicera glutinosa vis., viburnum lantana l., fagus sylvatica, clematis recta l., vicia cracca, vincetoxicum hirundinaria medik., iris variegata l., lilium bulbiferum l., anemone ranunculoides l. and linum narbonense l. habitat type is difficult to define since there is a visible transition between forest edge, scrub and overrun grassland in advanced phase of secondary succession. kuk od [pilji} plane kuk od [pilji} plane (44°33' n, 15°11' e; 1255 m a.s.l.) is situated in the middle section of mt velebit, not far away from the village ba{ke o{tarije. plants of berberis croatica are spread among the limestone rocks exposed to the southeast. plants are vigorous, up to 50 cm high and grow together with juniperus communis ssp. alpina, juniperus sabina l., corylus avellana, amelanchier ovalis medik, micromeria thymifolia (scop.) fritsch, gentiana lutea l. ssp. symphyandra (murb.) hayek, satureja subspicata, actaea spicata l. and campanula l. sp. habitat type, according to the nhc, is submontane rocks overgrown with elements of micromerion croaticae ht. 1931 community and, in certain extent, thermophile shrubs which can be included in the habitat of lower altitudinal belt of subalpine scrub (d.2.). \u|inovac near prezid \u|inovac is an area near prezid pass in the south section of mt velebit. a few plants of b. croatica were found on the left side of the trail, at about 850 m a.s.l. (44°16' n; 15°50' e). plants are about 1 m high and surrounded by ribes alpinum l., juniperus sabina, ostrya carpinifolia scop. and acer pseudoplatanus l. among other species fagus sylvatica, satureja montana l. and origanum vulgare l. were noticed. the habitat represents a transitional zone between scrub vegetation (d.2.) and forest edge. beyond the northwest dinaric alps new localities of b. croatica were found on mt kame{nica, mt biokovo and mt snije`nica. 294 acta bot. croat. 70 (2), 2011 kremer d., randi] m., kosalec i., brklja^i] a., luka^ g., kru[i] i., et. al. u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:55 color profile: disabled composite 150 lpi at 45 degrees kame{nica a few plants of berberis croatica were found in the part of kame{nica mountain that belongs to bosnia and herzegovina. berberis croatica grows about 400 m away from the locality »klanac«, at the altitude of 1421 m a.s.l. (43°42' n, 16°55' e). plant species that grow nearby are fagus sylvatica, abies alba, ostrya carpinifolia, acer pseudoplatanus, juniperus communis ssp. alpina, teucrium arduini, rhamnus intermedia steud. et hochst., daphne alpina and gentiana lutea ssp. symphyandra. habitat type is subalpine scrub (d.2.). vo{ac (mt biokovo) vo{ac (43°18' n, 17°02' e; 1422 m a.s.l.) is one of the famous peaks of mt biokovo. b. croatica grows on the peak top, near the climbers' lodge, and on the southeast-exposed slope. the plants cover an area of a few square meters; they are averagely 40 cm high, vigorous and with regular fructification. several plants were also found about one hundred meters below the top, near the hiking trail, in the direction of a car-park. another group of plants grows below the rock situated about 200 m meters northeast of vo{ac peak. plant species that grow nearby are: satureja montana, juniperus communis ssp. alpina, arctostaphylos uva-ursi, globularia cordifolia l., carex humilis, sesleria robusta schott, nyman et kotschy, edraianthus graminifolius (l.) a. dc., helianthemum mill. sp. and hieracium l. sp. habitat belongs among transitional types i.e. the transition between east-adriatic rocky grassland of the epi-mediterranean zone (c.3.5.2.) and subalpine scrub (d.2.) whose elements are mosaically spread over the habitat. mt snije`nica mt snije`nica is the southernmost mountain in croatia, situated near the border with bosnia and herzegovina and montenegro. berberis croatica was found on the right (several plants) and left (one plant) side of a trail (heading from the peak towards kuna konavoska), approximately at 1125 m a.s.l. (42°34' n, 18°21' e). plants grow from fissures in limestone, they are up to 1 m high, vigorous and fructifying. plant species that grow nearby are: teucrium arduini, rhamnus intermedia, frangula rupestris (scop.) schur, ostrya carpinifolia, moltkia petraea (tratt.) griseb. and viburnum lantana. the habitat of poorly vegetated rocks with elements of dalmatian calcareous rocks (b.1.4.2.) and vegetation of thermophile scrub could be recognized. 2. localities of teucrium arduini new localities of teucrium arduini were also found at a few distinct geographical sites of the dinaric alps: mt u~ka, mt velebit, mt biokovo and mt snije`nica. during the collection of plant material of t. arduini on mt kozjak we failed to find plants between the climbers' lodge called »putalj« and st. ivan biranj church (and a few hundred meters more in the direction on mala~ka pass) although this locality is mentioned in the references (kamenjarin 1996). acta bot. croat. 70 (2), 2011 295 berberis, teucrium and micromeria in the dinaric alps u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:55 color profile: disabled composite 150 lpi at 45 degrees argun argun is one of the peaks on suhi vrh ridge (1321 m a.s.l.) on mt u~ka, situated south of vela u~ka. several plants of teucrium arduini grow on the rocky southeast slope of argun (45°16' n, 14°13' e) at the altitude of about 1200 m. they are up to 35 cm high and vigorous. nearby vegetation belongs to thermophilous fagus sylvatica forest with sesleria autumnalis (scop.) f. w. schultz. habitat type represents poorly vegetated and weakly differentiated illyrian-adriatic littoral calcareous scree (b.2.2.) surrounded by littoral beech forest (e.4.6.3). teucrium arduini was found at six new localities on mt velebit. [u{anj cesari~ki locality [u{anj is known from the literature (degen 1938:583, forenbacher 1990), but previously there was no precise information where t. arduini really grows. teucrium arduini was found in an old quarry (44°32' n, 15°07' e; 600 m a.s.l.) situated near the position where the road for ravni dabar diverges from the karlobag – ba{ke o{tarije – gospi} road. several plants of t. arduini grow separately on rocks, on the south-eastern facing slope. the locality has been almost completely destroyed by the building of a car-park on a small lookout point and the carting of sawdust from a nearby saw-mill. plants of t. arduini are, generally, in a bad state. they are up to 30 cm high and grow almost completely alone on rocks. among other plant species that grow nearby, satureja montana is the most frequent. other plant species that grow on the nearby rocks and the lookout spot are: pinus nigra j. f. arnold, satureja subspicata, juniperus communis l., marrubium incanum desr., onosma l. sp., campanula fenestrellata feer, silene saxifraga l., scrophularia heterophylla willd. ssp. laciniata (waldst. et kit.) maire et petitm, cephalaria leucantha (l.) schrad. ex roem. et schult., centaurea deusta ten. ssp. concolor (dc.) hayek and achillea l. sp. habitat type, according to the nhc, is poorly vegetated calcareous rock (b.1.), more precisely bare karren and abandoned quarry (j.4.3.2.1.). panos panos (1258 m a.s.l.) is one of the notable peaks in the south section of mt velebit. teucrium arduini was found on both sides of a gravel road in the direction of panos (44°26' n; 15°17' e), approximately 500 m after the gravel road crossing jelova ruja – [ugarska duliba or fifteen minutes before the trail crossing panos – [ugarska duliba. plants of t. arduini grow on, and near the road, on a rocky slope, with south – southeastern exposure. they are very vigorous, up to 50 cm high and with regular fructification. plant species that grow nearby are: micromeria thymifolia, satureja subspicata, rhamnus fallax boiss., fagus sylvatica and campanula sp. habitat type belongs to infrastructure areas (j.4.4.) i.e. forest road edge. veliki vaganac the locality veliki vaganac (44°19' n, 15°28' e; 700 m a.s.l.) is situated in the south section of mt velebit, above the small town starigrad paklenica. teucrium arduini grows individually on an area of a several dozen square meters, between rocks and gravel, on a site exposed predominantly to the south and southeast, along a small car-park and nearby gravel road. plants of t. arduini are in a good state in spite of the fact that the habitat is ruined by the building of a gravel road. among other species we noticed rhamnus intermedia, daphne alpina, frangula rupestris, drypis spinosa l., teucrium chamaedrys l., satureja 296 acta bot. croat. 70 (2), 2011 kremer d., randi] m., kosalec i., brklja^i] a., luka^ g., kru[i] i., et. al. u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:55 color profile: disabled composite 150 lpi at 45 degrees montana, cotinus coggygria scop., ostrya carpinifolia and campanula pyramidalis l. habitat type belongs to infrastructure areas (j.4.4.) veliko rujno veliko rujno is a famous shrine (gospa od rujna) situated in the south section of mt velebit, several kilometers from the locality veliki vaganac. teucrium arduini was found most abundantly at the end of a gravel road leading from veliki vaganac to veliko rujno (44°22' n; 15°26' e; 880 m a.s.l.). plants grow on the rocks near the gravel road, on sites with predominantly south and south eastern exposure. plant species that grow nearby are satureja montana, fraxinus ornus l., prunus spinosa l., cornus mas l., acer monspessulanum l. and eryngium amethystinum l. habitat type, according to the nhc, belongs to infrastructure areas (j.4.4.), and poorly vegetated calcareous rocks (b.1.). bojinac bojinac is a small area in the western part of paklenica national park. teucrium arduini was found on a trail, 25 minutes from veliko rujno at approximately 1000 m a.s.l. (44°21' n; 15°26' e). plants grow on the rocky slope, predominantly on the eastern exposure. they are up to 50 cm high, vigorous and with regular fructification. among other species we noticed satureja subspicata, rhamnus intermedia, fraxinus ornus, ostrya carpinifolia, stachys recta l., campanula waldsteiniana schult., campanula fenestrellata and scrophularia l. sp. habitat type is poorly vegetated rock (b. 1.), i.e. round rocky calcareous mass with scarce elements of vegetation of rocky crevices. \u|inovac near prezid only two plants of teucrium arduini were found on a huge rock on the left side of a trail, about 45 minutes from the beginning of the hiking trail to crnopac (44°16' n, 15°50' e; 880 a.s.l.). the plants are up to 35 cm high, in a good state and with regular fructification. among other species we noticed fagus sylvatica, ostrya carpinifolia, satureja montana, juniperus sabina and sorbus aria (l.) crantz. habitat type is poorly vegetated rock (b.1.). [trbina (mt biokovo) [trbina (43°18' n, 17°02' e) is a small belvedere situated a few hundred meters southeast of vo{ac peak. the locality vo{ac is known from the literature (ku[an, 1969a), but during our field work teucrium arduini was not found there. it is also possible that ku[an (1969a) found t. arduini bellow [trbina but described the new locality using the name of a more famous peak vo{ac. the population of t. arduini is located five to ten minutes (on foot) from the lookout point called [trbina in the direction of makarska town. t. arduini grows on scree and rocks beside the trail, predominantly on sites with southern exposure. plants which grow on the scree are mostly up to 25 cm high and stunted, while a few plants which grow on rocks are up to 40 cm high and more vigorous. among other plants, we noticed fagus sylvatica, ostrya carpinifolia, sorbus aria, satureja montana, moltkia petraea, valeriana montana l., rumex l. sp. and onosma l. sp. teucrium arduini was also noticed on a few sites near the road which connects entrance of nature park »biokovo« and vo{ac (43°18' n, 17°03' e). on these sites t. arduini grows on a scree, but plants are very vigorous and fructify. also, t. arduini grows from the fissures in limestone and in the gravel near the road, as well as on the rocks. habitat type predominantly is classified as illyrian-adriatic littoral scree (b.2.2.1.). acta bot. croat. 70 (2), 2011 297 berberis, teucrium and micromeria in the dinaric alps u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:55 color profile: disabled composite 150 lpi at 45 degrees mt snije`nica on mt snije`nica teucrium arduini was found on the left side of the trail (heading from the peak towards kuna konavoska), approximately at 1125 m a.s.l. (42°34' n; 18°21' e). several plants grow on rocks with a southeastern exposure. they are up to 40 cm high and relatively vigorous. another group of plants grows on the right side of the trail (heading from the peak towards kuna konavoska). plants from this group grow on rocks with southern exposures. they are more robust, up to 65 cm high, very vigorous and with regular fructification. plant species that grow nearby are: berberis croatica, rhamnus intermedia, frangula rupestris, ostrya carpinifolia, moltkia petraea and viburnum lantana. habitat type belongs to poorly vegetated rocks with elements of dalmatian calcareous rocks (b.1.4.2.). 3. localities of micromeria croatica during the field work we failed to find micromeria croatica on mt risnjak, a location mentioned in the literature ([egulja et al. 1994, pelivan 1997). nor was m. croatica found on samarske stijene in gorski kotar, nor did other field researches confirm the presence of m. croatica on samarske stijene (topi}, personal communication). new localities of this species were recorded only on mt velebit. rossijev kuk rossijev kuk (44°46' n, 14°60' e; 1615 m a.s.l.) is one of the peaks situated beside the footway premu`i}eva staza in the northern section of mt velebit (northern velebit national park), 5–10 minutes on foot from the climbers' shelter rossijeva koliba (1580 m a.s.l.). plants of micromeria croatica grow on a rocky slope with a southwestern exposure. among other species we noticed pinus mugo turra, rosa pendulina, achillea clavennae l., scutellaria alpina l., gentiana lutea ssp. symphyandra, ranunculus platanifolius l., lamium galeobdolon (l.) l., melittis melissophyllum l., lilium carniolicum bernh. ex w. d. j. koch and silene l. sp. habitat type is illyrian-dinaric calcareous rocks (micromerion croaticae ht. 1931 community) (b.1.3.3.). ruji~in kuk ruji~in kuk (44°34' n, 15°08' e; 946 m a.s.l.) is one of the peaks situated near the gravel road which connects ku}i{ta cesari~ka and kugina ku}a. this peak is close to ravni dabar (723 m a.s.l.). plants of m. croatica were found on the rocks at the base of ruji~in kuk (eastern exposure), beside the gravel road heading in the direction towards ravni dabar. plant species that grow nearby are: fagus sylvatica, micromeria thymifolia, rubus fruticosus l. agg., campanula waldsteiniana, vincetoxicum hirundinaria and campanula fenestrellata. habitat type, according to nhc is rock, partly shaded by beech forest and poorly vegetated by elements of illyrian-dinaric calcareous rock vegetation (micromerion croaticae community); (b.1.3.3.). kuk od [pilji} plane several plants of micromeria croatica can be found on a slope with a southeastern exposure, on limestone rocks open to the influence of strong winds (44°33' n, 15°11' e; 1255 m a.s.l.). plant species that grow nearby are: juniperus communis ssp. alpina, juniperus sabina, corylus avellana, amelanchier ovalis, micromeria thymifolia, gentiana lutea ssp. 298 acta bot. croat. 70 (2), 2011 kremer d., randi] m., kosalec i., brklja^i] a., luka^ g., kru[i] i., et. al. u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:55 color profile: disabled composite 150 lpi at 45 degrees symphyandra, satureja subspicata, actaea spicata and campanula sp. as well as on kuk od [pilji} plane, m. croatica grows individually at several micro locations in this area, especially abundantly on rocks on the left side of the gravel road from stupa~inovo to jadi~evac and polo`ine. the habitat could be characterized as poorly vegetated calcareous rock with elements of micromerion croaticae (b.1.3.3.) community. \u|inovac near prezid a few plants of m. croatica were found on the huge rocks on the both sides of a trail to crnopac, about one hour on foot from the beginning of the hiking trail (44°16' n, 15°50' e; 900 m a.s.l.). plant species that grow nearby are: fagus sylvatica, ostrya carpinifolia, juniperus communis ssp. alpina, juniperus sabina, sorbus aria, satureja subspicata and sedum l. sp. habitat type belongs to poorly vegetated calcareous rock (b.1.). acknowledgements this work was supported by the ministry of science, education and sports of the republic of croatia (project no 006-0000000-3178). references akeroyd, j. r., webb, a., 1993: berberis l. in: tutin, t. g., heywood, v. h., burges, n. a., valentine, d. h., moore, d. m. 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[kolska knjiga, zagreb. flora croatica database of habitats, 2009: http://hirc.botanic.hr/fcd/ kamenjarin, j., 1996: vascular flora of mount kozjak above split. natura croatica 5, 119–144. karlovi], k., kremer, d., liber, z., [atovi], z., vr[ek, i., 2009: intraand interpopulation variability and taxonomic status of berberis croatica horvat. plant biosystems 143, 40–46. acta bot. croat. 70 (2), 2011 299 berberis, teucrium and micromeria in the dinaric alps u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:55 color profile: disabled composite 150 lpi at 45 degrees kremer, d., randi], m., kosalec, karlovi], k., 2008: new localities of berberis croatica horvat in croatia. acta botanica croatica 67, 237–244. ku[an, f., 1969a: vegetation cover of mt. biokovo (in croatian). jazu, zagreb. ku[an, f., 1969b: new barberry (berberis) species in croatian flora (in croatian). acta botanica croatica 28, 423–434. laku[i], b., laku[i], d., slavkovska, v., stevanovi], v., stevanovi], b., 2007: morpho-anatomical differentiation of the balkan endemic species teucrium arduini l. 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(lamiaceae) on the canary islands as inferred from chloroplast and nuclear dna sequences and issr fingerprint data. molecular phylogenetics and evolution 41, 566–578. narodne novine, 2006: regulation on designating wild taxa protected and strictly protected (in croatian). narodne novine 7, 157. narodne novine, 2009: habitat types in croatia (national habitat classification– nks) (in croatian). narodne novine 119, 21–52. nikoli], t., ur., 2008: flora croatica database (http://hirc.botanic.hr/fcd). prirodoslovno-matemati~ki fakultet, sveu~ili{te u zagrebu. pelivan, a., 1997: national park risnjak (in croatian). ekolo{ki glasnik 10, 6–20. ru[^i], m., 2010: lower flow of cetina river (in croatian). in: nikoli], t., topi], j., vukovi], n. (eds.), botani~ki va`na podru~ja hrvatske, 103–108. prirodoslovno-matemati~ki fakultet i [kolska knjiga, zagreb. [egulja, n., lova[en-eberhardt, @., hr[ak, v., luka^, g., 1994: review of the state of research of flora in national park 'risnjak'. proceedings of the symposium 40 years of the national park 'risnjak' (1953–1993), crni lug, 71–77. [ili], ^., 1979: monography of genera: satureja l., calamintha miller, micromeria bentham, acinos miller and clinopodium l. in flora of yugoslavia (in croatian). zemaljski muzej bih, sarajevo. [ili], ^., 1990: endemic plants (in croatian). ip svjetlost, sarajevo. [ili], ^., 2005: atlas of woody plants (trees and shrubs) of bosnia and herzegovina (in croatian). matica hrvatska ^itluk and franjeva~ka ku}a masna luka, ^itluk. trinajsti], i., 1973: berberis l. (in croatian). analiti~ka flora jugoslavije 1, 377–381. zovko kon^i], m., kremer, d., schühly, w., brantner, a., karlovi], k., kalo\era, z., 2010: chemical differentiation of berberis croatica and b. vulgaris using hplc fingerprinting. croatica chemica acta 83, 451–456. 300 acta bot. croat. 70 (2), 2011 kremer d., randi] m., kosalec i., brklja^i] a., luka^ g., kru[i] i., et. al. u:\acta botanica\acta-botan 2-11\kremer et al.vp 9. rujan 2011 13:07:56 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 73 (1), 131–147, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 assessment of macro-micro element accumulation capabilities of elodea nuttallii under gradient redox statuses with elevated nh4-n concentrations tanjeena zaman, takashi asaeda* department of environmental science and technology, saitama university, 255 shimo-okubo, sakura-ku, saitama 338-8570, japan abstract – aquatic plants often encounter various redox conditions in their natural environment. elodea nuttallii (planch.), a submerged aquatic macrophyte, has a flexile ability to use different nutrient sources from various environments. in the present study, elodea nuttallii was subjected to various redox conditions (+400 mv to –180 mv) at both normal (2.5 ppm) and high (10 ppm) ammonium concentrations and evaluated for macro and micro element accumulation. a reduced environment was prepared by adding glucose to growth medium and nitrogen gas bubbling, while an oxic environment was executed by atmospheric air bubbling. plants in oxygen-deprived conditions manifested heavy metal (hm) toxicity, such as reduction of biomass and photosynthetic pigments, excess generation of reactive oxygen species (ros), lipid peroxidation and reduction of major macro elements. in reduced treatments, the bioaccumulation sequence for micro elements was cu>mn>zn>al>cd>fe>pb at both normal and high nh4-n concentrations. the combined effect of low redox state and high ammonium concentration had a strong physiological impact on the submerged macrophyte. however, macroand micronutrient accumulation was more significantly affected by reduced environment than by a high nh4-n concentration. keywords: anoxia, ammonium, elodea nuttallii, macro-micro elements, accumulation, translocation introduction metal mobility and availability in sediments and in wetlands is governed by a number of sediment factors and processes; e.g. adsorption/desorption reactions, precipitation/dissolution and complexation/decomplexation, salinity, organic matter content, sulphur (s) and carbonate content, plant growth, ph and redox potential (eh) as well as microorganism activity (du laing et al. 2009, maría-cervantes et al. 2010). oxidation and reduction processes subsequently affect ph (yu et al. 2007), which is directly related to stability and solacta bot. croat. 73 (1), 2014 131 * corresponding author, e-mail: takasaeda@yahoo.co.jp copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen ubility of various metals and nutrient elements in soil and sediment, and to their availability in plants (reddy and patrick 1977). according to devai and delaune (1995), eh of soil or sediment can range from –300 to + 700 mv and anaerobic soil or sediment exhibit redox potentials from + 350 mv to as low as –300 mv. sediments/soil tend to undergo a series of sequential redox reactions in a homogenous environment when sediment redox status changes from aerobic (high eh) to anaerobic (low eh) conditions and vice versa. major reactions, in order of decreasing eh, are nitrification, denitrification, manganic manganese [mn (iv)] reduction, ferric iron [fe (iii)] reduction, sulfate (so4 2-) reduction, and methanogenesis (patrick et al. 1996). in anoxic conditions, by reduction reactions, oxide elements such as phosphorus (p), molybdenum (mo), cobalt (co), copper (cu), zinc (zn) are often transformed to a more mobile and plant-available form (francis and dodge 1990). lower sediment ph under mildly oxic conditions increase the bioavailability of al, cu, fe, mn and zn to rooted aquatic plants (jackson et al. 1993). submerged aquatic plants adapt to detoxify reduced elements by releasing root oxygen to the rhizosphere, which is also governed by eh condition and microbial oxygen demand (laskov et al. 2006). the concentration of metals in plants can be more than 100,000 times greater than in the associated water (albers and camardese 1993). recent studies on heavy metal (hm) toxicity revealed that these metals may cause molecular damage to plant cells either directly or indirectly through the excessive generation of reactive oxygen species (ros), such as hydrogen peroxide (h2o2), hydroxyl radicals (oh·) and superoxide radicals (o2· –). these ros can damage membranes and inactivate several enzymes by reacting very rapidly with dna, lipids, pigments and proteins (weckx and clijsters 1996). thus, variation in redox conditions exerts a substantial influence on the physiological processes of plants. elodea nuttallii, a submerged aquatic rooted macrophyte, can absorb nutrients either by roots or shoots or by both together in varying proportions (barko et al. 1991). this species is well known as a hyper-accumulator of various metals and elements as well as being stress resistant to various environmental factors (mishra and tripathi 2008). the capability of the shoots and roots of submerged macrophytes to accumulate trace metals allows their use in trace-element biomonitoring in lake ecosystems (baldantoni et al. 2004). physical factors that fluctuate temporarily include ph, redox potential, temperature, salinity or light and in addition to the presence of other metal ions in the surrounding aquatic environment strongly affect metal uptake by submerged plants (fritioff et al. 2005). sediment redox status and its effect in wetland plants and crops have been vigorously studied in the last three decades. the effect of a reduced environment on aquatic macrophytes is very slight (delaune et al. 1999). increased ammonium concentration and low redox status (reduced condition) in the natural habitat (due to pollution or eutropication) are two characteristics prominently associated with eutropic lakes, such as plesne lake in central europe (kopáèek et al. 2004). furthermore, in a reduced environment different oxidized elements become available in the surrounding environment. trace elements like cu, fe, mn and zn are essential minerals for normal growth of aquatic macrophytes but excessive concentration might have a deleterious effect by disordering physiological and biochemical processes in the cells. these elements give especially grounds for concern as they are not biodegradable (lu et al. 2007) and contribute to the food chain. the aim of the present work was to assess the significance of reducing conditions on (i) the release of inorganic contaminants, (ii) their concentration and translocation, and (iii) the oxidative damage caused by these elements under normal and high nh4-n concentrations in elodea nuttallii. 132 acta bot. croat. 73 (1), 2014 zaman t., asaeda t. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen materials and methods sediment and plant collection the sediment was collected from a pond in oaso park near tokyo, in december, 2010. the organic-rich sediment (organic matter content >5%) was derived from the top surface (<15 cm depth) of pond sediment. elodea nuttallii (planch.) was collected from moto-arakawa river, saitama, japan, in april, 2011. collected plants were allowed to adapt to laboratory conditions for 2 weeks in the experimental tanks, where the temperature was maintained at 25 °c, with a relative air humidity of 90% and a photon flux density of approximately 100 mmol m–2 s–1 was provided by fluorescent lamp in a 12h light/12h dark cycle. experimental set-up elodea nuttallii was subjected to gradient redox potentials under normal and high nh4-n concentrations. since it was difficult to keep a constant redox potential throughout the experiment period, a range of potentials was maintained. three levels of redox potential were used, as (i) +400 mv ~ +440 mv, (oxic; o1), (ii) –5 mv ~ +5 mv (hypoxic/moderately reduced; o2) and (iii) –180 mv ~ –120 mv (anoxic/highly reduced; o3) (fig. 1). in the case of a nitrogen source, the suitable nh4-n concentration for the plant is 2.5 ppm (ozimek et al. 1993). here, two different nh4-n concentrations [2.5 (n1) and 10 (n2) ppm] were used (fig. 1). the experiment was conducted in microcosms (mcs), each consisting of a 6 l (15.7 × 15.7 × 24.5 cm3) glass vessel which was hermetically sealed with an air-tight lid. each mc was filled with 600 g of air-dried sediment and deionized water in a 1:5 ratio. then, growth medium contained 5% hoagland nutrient solution (hoagland and arnon 1950) was mixed, and ammonium sulfate was added to adjust the required nh4-n concentration. highly reduced and moderately reduced microcosms were prepared following the method developed by yu et al. (2007). glucose, a simple carbon source, was used in this experiment during the 22-day incubation period. at the beginning of incubation, 8.16 g glucose was added to the reduced (mc 3) and highly reduced microcosms (mc 4) on days 1 and 3, and twice that amount was added on day 5. on day 14, again, 8.16 g glucose was added to mc 4. continuous flushing of n2 gas was carried out for the last 3 days for a acta bot. croat. 73 (1), 2014 133 macro-micro element accumulation capabilities of elodea nuttallii r e d o x le v e l d e c re a se nh4-n concentration increase n1o1 n2o1 n1o2 n2o2 n1o3 n2o3 c o n tr o l fig. 1. layout of the experimental set-up (seven microcosms per treatment). nh4-n concentration and redox level are presented as n and o, respectively. microcosms were randomly distributed with equal spacing in growth chamber. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen hypoxic/moderately reduced (mc3) condition and for the last 7 days for anoxic/highly reduced (mc4) condition to reduce the redox potential (eh) values to approximately 5 mv and 180 mv, respectively. for oxic treatments, continuous bubbling with atmospheric air was used. eh and ph were measured four times a day using four portable ph/orp meters (pot-101 m, sibata, japan). for control, 5% hoagland nutrient medium was used without any further treatment. the temperature was maintained at 23 ± 2 °c in a room with fluorescent lighting. no attempt was made to control the ph of the sediment suspensions. after the incubation period (22 days), eight plants (approximately 12–14 cm in height) were planted in each experimental tank. then, the experiment was continued for 14 days, with continued n2 gas flushing to maintain the required eh potential. in total, three treatments, each with 7 microcosms, were applied. sediment, plant and water analysis sediment samples were air-dried, homogenized and sieved to < 2 mm. the particle sizes of the sediment samples (in terms of d50) were determined using sieves according to the american society for testing and materials protocol (astm d422-63, 2002). plants were carefully washed using tap water and finally with distilled water, and were separated into leaves, shoots and roots. plant materials were dried using an oven drier at 60 °c until constant weight. plant materials were reweighed (for dry weight) and homogenized by grinding into fine powder using a mortar and pestle. powdered samples were stored in airtight vials for subsequent analysis. total nitrogen (tn) and total carbon (tc) of powdered plant samples were measured by chn coder (yanaco mt-3). about 10 mg of dried plant sample and 200 mg of dried sediment sample were digested at 200 °c with di-acid mixture (nitric acid : perchloric acid; 1:2) until evolution of nitrous gas was stopped and the digest became clear. the digests were diluted with distilled water to a total of 100 ml and passed through whatman 42 filter paper. organic matters in the sediment were measured by the walkley and black (1934) method. the concentrations of the following elements were measured in the sediment and in the plant samples: fe, mn, zn, pb, ca, mg, cu and k with atomic absorption spectrophotometer (aas; shimadzu aa-660 g) using the direct air-acetylene flame method, and the concentration of al and cd were determined with a graphite furnace atomizer (gfa-4b), according to the instructions and procedure. total phosphorus (tp) and total sulphur (ts) were measured using the ascorbic acid method and the barium chloride method respectively. replicate samples were analyzed separately, analyses were done in duplicate, and results for plant materials and sediments were calculated on a dry weight basis. water samples were collected at 7 day intervals and were passed through whatman glass microfibre filters gf/c and stored at 4 oc until analysis. the concentrations of fe, mn, zn, pb, ca, mg, cu, k, al, cd and ts of water sample were measured following the methods used for sediment and plant sample analysis. ammonium nitrogen was determined by autoanalyzer (technicon ii traac 800). biomass increment on the14th day after treatment (dat), two plants from each tank were harvested, and cleaned with tap water, and fresh biomass was measured after blotting with laboratory towel. the fresh biomass increment was calculated as the percent increment of plant mass relative to initial fresh mass at the time of transplanting, using the following equation: 134 acta bot. croat. 73 (1), 2014 zaman t., asaeda t. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen where bt is the increased biomass (% relative to initial fresh biomass) at the 14th dat, ft is the fresh biomass at 14th dat and fo is the initial fresh biomass of the plant. chlorophyll content, carotenoid content and chlorophyll flurorescence photosynthetic pigments were extracted in 95% ethanol in the dark for 24 h. afterwards, the sample was centrifuged for 10 min at 8000 × g. finally, supernatants were read at 665 and 649 nm for chlorophyll a and chlorophyll b, respectively, and at 470 nm for carotenoid content using spectrophotometer (shimadzu uv-1700, japan). the contents of chlorophylls and carotenoid were calculated according to lichtenthaler (1987). chlorophyll a fluorescence measurements were performed with a handy flurocam (fc 1000-h, photon systems instruments, czech republic) using auto image segmentation. maximum photochemical efficiency of psii (fv/fm), the activity of psii (fv/fo) and electron transport rate (etr) through psii (fm/fo) were determined and used as a stress indicator for plants. h2o2 concentration and peroxidase activity endogenous h2o2 concentrations were analyzed following the method of cervilla et al. (2007), where samples were extracted with cold acetone. phosphate buffer (0.1 mol l–1) at ph 6 was used to make extracts suitable for peroxidase (pod) activity measurements. pod was determined according to the method described by goel et al. (2003). lipid peroxidation and proline concentration the level of lipid peroxidation was measured in terms of malondialdehyde (mda), a product of lipid peroxidation in the plant samples estimated by thiobarbituric acid (tba) reaction (heath and packer 1968). the concentration of proline was measured with the bates et al. (1973) method. plant material was homogenized with 10 ml of 3% (v/v) sulfosalicylic acid. free proline present in the supernatant was treated with acid-ninhydrin at 80 °c for 1 h and measured spectrophotometrically at 520 nm. bioconcentration factor and translocation factor the bioconcentration/bioaccumulation factor (bcf) is an index to express the ability of a plant to accumulate metal with respect to metal concentration in substrate. bcf (for whole plant) was calculated by the following formula: the translocation factor (tf) is an indication of the ability of the plant to translocate metals from the roots to the aerial parts of the plant. tf was calculated by the following formula: translocation factors (tf) for trace elements between sediment and roots and within a plant were expressed by the ratios of [trace element] sediment/ [trace element] root and [trace eleament] root/ [trace element] (shoot + leaves) to show trace elements translocation properties from sediment to roots and roots to shoots, respectively. acta bot. croat. 73 (1), 2014 135 macro-micro element accumulation capabilities of elodea nuttallii 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen statistical analysis the experimental set up was a completely randomized design, and average values of three treatments were considered. data were analyzed statistically using the spss 13.0 software package, by anova and by tukey’s multiple range tests to determine differences between means. before performing a statistical analysis, data were checked for normal distribution. pearson correlation analysis was carried out to explore the correlations. results biomass increment plants subjected to a high concentration of nh4-n (10 ppm) along with hypoxic/anoxic treatments showed brown-black discoloration of the leaves and biomass increment values were negative. increment of ammonium even in oxic treatment considerably reduced biomass (fig. 2). when oxygen level decreased, biomass was more affected at both ammonium levels. at 2.5 ppm, nh4-n nutrition condition by hypoxic and anoxic treatment, the fresh biomass declined by 73.02 and 80%, respectively. photosynthetic pigments and chlorophyll fluorescence photosynthetic pigments including chl a, chl b and carotenoid content showed a slight falling trend with the increment of nh4-n concentration in oxic treatments (tab. 1). at hypoxic and anoxic treatments both chlorophyll and carotenoid levels significantly declined even when nh4-n concentration was at a normal level (2.5 ppm), suggesting that hypoxia itself was sufficient to affect both chlorophyll and carotenoid content. moreover, carotenoid seemed to be affected more severely and found absent at high reduced treatment at 10 nh4-n concentration. maximum photochemical efficiency of psii (fv/fm), the activity of psii (fv/f0) and electron transport rate (etr) through psii (fm/f0) are presented in table 1. their values were not significantly affected by high nh4-n concentration in oxic treatment but were 136 acta bot. croat. 73 (1), 2014 zaman t., asaeda t. -5 15 35 55 75 95 115 135 155 control oxic reduced highly reduced b io m a ss in c re a m e n t (% in c re a se d re la ti v e to in it ia l w e ig h t) 2.5 ppm 10 ppm fig. 2. effect of nh4-n concentrations under various redox conditions on biomass of elodea nuttallii. the data are presented as the mean ± sd. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen significantly decreased in plants grown in hypoxic and anoxic treatments. increment of nh4-n concentration in hypoxic and anoxic treatments more significantly affected their values (tab. 1). lipid peroxidation rate and proline content lipid peroxidation rate was determined by measuring mda content. this parameter was significantly increased (fig. 3a) in plants that were exposed to reduced treatments (p < 0.01). moreover, increment of nh4-n concentration in reduced treatments accelerated the increment of mda level, and hence, maximum mda content was observed in plant under highly reduced treatment at 10 ppm nh4-n concentration. proline level declined slightly in plants under oxic treatments with high nh4-n concentrations (fig. 3b), suggesting high ammonium concentration has a weak effect on the proline content of the plant. in reduced and highly reduced treatments, the proline content was considerably reduced (fig. 3b). plants in oxic treatment with 2.5 ppm nh4-n concentration showed the highest proline content (1.21 mg g–1 fw), whereas plants in anoxic treatment with 10 ppm nh4-n concentration showed the lowest proline content (0.22 mg g–1 fw). endogenous h2o2 generation and pod activities a significantly higher h2o2 concentration (p < 0.05) was found throughout the experimental period in reduced and highly reduced treatments (figs. 4a). similar up-regulation was also observed for pod activity (fig. 4b). in oxic treatments, increment of nh4-n concentration exhibited a slight increasing trend in both h2o2 level and pod activity (figs. 4a, 4b). h2o2 concentration and pod activity were positively correlated in all treatments and conditions (oxic, r = 0.847, n = 16, p < 0.01; reduced, r = 0.948, n = 16, p < 0.001 and highly reduced r = 0.929, n = 16, p < 0.001). element bioaccumulation and translocation in plant bcf and tf were calculated to study the accumulation characteristics of different essential and non-essential elements in different body parts (leaf, shoot and root) of the plant. sigacta bot. croat. 73 (1), 2014 137 macro-micro element accumulation capabilities of elodea nuttallii tab. 1. photosynthetic pigments and chlorophyll fluorescence parameters (mean ± sd) of elodea nuttallii under different conditions (nh4-n concentrations) and treatments (oxic to highly reduce). parameters control 2.5 ppm nh4-n 10 ppm nh4-n oxic reduced highly reduced oxic reduced highly reduced chla 2.9 ± 0.1 3.2 ± 0.2 1.7 ± 0.2* 1.4 ± 0.2** 2.7 ± 0.1 1.2 ± 0.1** 1.0 ± 0.0*** chlb 1.4 ± 0.1 1.7 ± 0.2 1.1 ± 0.2* 1.0 ± 0.0** 1.3 ± 0.2 1.0 ± 0.1** 0.9 ± 0.1** carotenoid 1.1 ± 0.0 1.3 ± 0.2 0.9 ± 0.0* 0.5 ± 0.1** 0.9 ± 0.1* 0.3 ± 0.0** 0.0 ± 0.0*** fv/fm 0.7 ± 0.0 0.8 ± 0.0 0.6 ± 0.0 0.5 ± 0.0* 0.7 ± 0.0 0.5 ± 0.0* 0.4 ± 0.0* fv/f0 4.3 ± 0.1 4.7 ± 0.1 2.5 ± 0.2 ** 1.8 ± 0.2** 3.9 ± 0.3* 2.0 ± 0.0** 1.3 ± 0.2*** fm/ f0 5.6 ± 0.2 5.9 ± 0.1 3.7 ± 0.1 ** 3.1 ± 0.3*** 5.2 ± 0.2 3.4 ± 0.2** 2.9 ± 0.2*** significance at: p < 0.05*; p < 0.01**; p < 0.001***. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen 138 acta bot. croat. 73 (1), 2014 zaman t., asaeda t. 0 10 20 30 40 50 60 70 control oxic reduced highly reduced m d a c o n te n t (µ m o l g -1 f w ) 2.5 ppm 10 ppm(a) b b b b a a ab a 0 0.2 0.4 0.6 0.8 1 1.2 1.4 control oxic reduced p ro li n e c o n te n t (m g g -1 f w ) 2.5 ppm 10 ppm(b) b b b ab bc c cd d highly reduced fig. 3. effects of nh4-n concentrations under various redox conditions on mda content and proline concentration of elodea nuttallii; (a) mda content and (b) proline concentration. the data are presented as the mean ± sd. one-way anova followed by tukey’s test was used to determine the significance of difference between treatments (p < 0.05). tab. 2. bioaccumulation factor of elements in elodea nuttallii (mean ± sd) under different conditions (nh4-n concentrations) and treatments (oxic to highly reduce). elements control 2.5 ppm 10.0 ppm oxic reduced highly reduced oxic reduced highly reduced ca 3.6 ± 0.6cd 3.3 ± 0.3cd 0.4 ± 0.1e 0.2 ± 0.1ef 2.9 ± 0.2cd 0.3 ± 0.1e 0.3 ± 0.1e mg 9.6 ± 3.3b 10.9 ± 3.8b 4.9 ± 1.1c 3.8 ± 0.8c 10.1 ± 3.1b 4.2 ± 0.5c 3.1 ± 0.2cd k 14.3 ± 4.6a 13.9 ± 4.1a 4.9 ± 1.6c 4.2 ± 1.1 c 12.6 ± 4.8a 4.2 ± 1.0 c 3.6 ± 0.8 cd s 2.3 ± 1.1c 2.6 ± 0.8c 3.9 ± 1.2bc 4.0 ± 1.2bc 2.7 ± 1.0c 3.3 ± 0.8c 3.5 ± 0.6c cu 0.6 ± 0.1cd 0.7 ± 0.2cd 4.6 ± 0.5ac 5.9 ± 0.6 ac 0.7 ± 0.2cd 5.5 ± 0.4 ac 6.3 ± 0.8ad mn 0.5 ± 0.2c 0.5 ± 0.1c 2.8 ± 0.2b 2.9 ± 0.4b 0.5 ± 0.1c 3.4 ± 0.1a 3.1 ± 0.0ab zn 0.8 ± 0.1de 0.9 ± 0.1de 1.1 ± 0.1de 1.2 ± 0.0cd 0.9 ± 0.1de 1.3 ± 0.1cd 1.5 ± 0.2c fe 0.3 ± 0.0c 0.3 ± 0.0c 0.7 ± 0.1b 0.8 ± 0.1b 0.3 ± 0.0c 0.9 ± 0.1b 1.0 ± 0.0a al 0.7 ± 0.2de 0.7 ± 0.1de 1.1 ± 0.2cd 1.1 ± 0.2cd 0.7 ± 0.1de 1.1 ± 0.1cd 1.1 ± 0.1cd pb 0.1 ± 0.0e 0.1 ± 0.0e 0.5 ± 0.1c 0.6 ± 0.0c 0.1 ± 0.0e 0.6 ± 0.1c 0.6 ± 0.0c cd 0.1 ± 0.0e 0.0 ± 0.0e 0.9 ± 0.1c 1.1 ± 0.1b 0.0 ± 0.0e 0.8 ± 0.1c 0.9 ± 0.2c different letter superscripts indicate significant differences between treatments, and same superscript letter as control indicate no significant difference. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:27 color profile: generic cmyk printer profile composite default screen nificant bcf differences (p < 0.001) were found between different redox treatments at both nh4-n conditions for both macro and micro elements (tab. 2). in both nh4-n concentrations with hypoxia/anoxia, bcf was downregulated for ca, mg and k, but upregulated for s, fe, cu, mn, zn, cd, pb and al. in an oxic treatment, the highest bioaccumulation was found for k, but in a highly reduced treatment cu showed the highest value. translocation of elements from sediment to roots seems more significantly affected by redox treatments than by nh4-n conditions (tab. 3). in reduced (hypoxic and anoxic) treatments, the translocation factor from sediment to root was increased for ca, mg and k, whereas, downregulated tf was observed for fe, cu, mn, cd, pb and al (tab. 3). moreover, in a highly reduced treatment, the tf of ca was mostly increased, while the tf of cd mostly declined (tab. 3). however, translocations of s and zn were not significantly affected by redox treatments and nh4-n conditions (tab. 3). on the other hand, translocation of elements from roots to shoot and leaf was not affected by nh4-n conditions but was affected by redox treatments (tab. 4). translocation of ca, fe, cu and mn was decreased by reduced treatment; however, translocation of cd was decreased in oxic treatment under both nh4-n conditions (tab. 4). acta bot. croat. 73 (1), 2014 139 macro-micro element accumulation capabilities of elodea nuttallii 0 50 100 150 200 250 control oxic h 2 o 2 c o n c e n ta rt io n (m g g -1 f w ) 2.5 ppm(a) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 control oxic p o d a c ti v it y (u n it s m in -1 g -1 f w ) 2.5 ppm(b) reduced highly reduced 10 ppm reduced 10 ppm highly reduced fig. 4. variations in h2o2 concentration and pod activity of elodea nuttallii, grown at different nh4-n concentrations under various redox statuses; (a) h2o2 concentration and (b) pod activity. the data are presented as the mean ± sd. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:28 color profile: generic cmyk printer profile composite default screen discussion reducing sediment conditions comprehend sediment oxygen deprivation, at the same time producing various compounds in sediment, many of which are considered highly phytotoxic (pezeshki and delaune 2012). however, concentration of elements as well as 140 acta bot. croat. 73 (1), 2014 zaman t., asaeda t. tab. 3. translocation factor (sediment/root) of elements in elodea nuttallii under different conditions (nh4-n concentrations) and treatments (oxic to highly reduce). elements control 2.5 ppm 10.0 ppm oxic reduced highly reduced oxic reduced highly reduced ca 2.9 ± 0.9d 3.0 ± 0.8d 13.6 ± 9.0b 30.2 ± 3.0a 3.0 ± 0.6d 13.6 ± 9.0b 39.2 ± 9.8a mg 0.5 ± 0.2d 0.6 ± 0.1d 1.4 ± 0.2c 1.4 ± 0.2c 0.6 ± 0.1d 1.6 ± 0.3b 1.6 ± 0.1b k 0.3 ± 0.0d 0.3 ± 0.0d 0.9 ± 0.2c 0.9 ± 0.1c 0.3 ± 0.0d 0.9 ± 0.0c 1.0 ± 0.2c s 1.4 ± 0.5c 1.1 ± 0.1c 1.0 ± 0.2d 1.0 ± 0.2d 1.2 ± 0.3c 1.2 ± 0.3c 1.3 ± 0.2c fe 9.3 ± 1.6c 7.9 ± 0.9c 6.9 ± 0.2d 6.6 ± 0.1d 7.0 ± 0.0d 5.8 ± 1.3de 5.9 ± 2.1de cu 4.8 ± 0.9b 3.2 ± 0.4c 1.4 ± 0.3cd 1.2 ± 0.1cd 3.9 ± 1.4bc 1.3 ± 0.3cd 1.4 ± 0.4cd mn 4.9 ± 1.7b 4.9 ± 0.3b 1.2 ± 0.1c 1.1 ± 0.1c 4.6 ± 0.4b 1.2 ± 0.1c 1.0 ± 0.1cd zn 0.7 ± 0.0a 0.7 ± 0.0a 0.6 ± 0.0a 0.8 ± 0.1a 0.7 ± 0.0a 0.7 ± 0.1a 0.8 ± 0.1a cd 18.6 ± 7.6b 23.8 ± 4.2ab 1.9 ± 0.7d 1.5 ± 0.3d 28.7 ± 5.2a 1.9 ± 0.4d 1.7 ± 0.1d pb 34.2 ± 4.0b 32.2 ± 5.7b 24.2 ± 2.4ab 22.4 ± 2.3ab 31.9 ± 6.1b 19.8 ± 1.1a 23.5 ± 2.0ab al 4.0 ± 0.0c 4.1 ± 0.1c 2.8 ± 0.0cd 2.8 ± 0.0cd 4.1 ± 0.0c 3.0 ± 0.0cd 2.9 ± 0.0cd different letter superscripts indicate significant differences between treatments, and same superscript letter as control indicate no significant difference. tab. 4. translocation factor (root/(shoot+leaf) of elements in elodea nuttallii under different conditions (nh4-n concentrations) and treatments (oxic to highly reduce). elements control 2.5 ppm 10 ppm oxic reduced highly reduced oxic reduced highly reduced ca 0.5 ± 0.1c 0.5 ± 0.1c 0.3 ± 0.1cd 0.2 ± 0.0d 0.5 ± 0.0c 0.3 ± 0.1cd 0.1 ± 0.0d mg 0.3 ± 0.1c 0.2 ± 0.0cd 0.2 ± 0.0cd 0.2 ± 0.0cd 0.2 ± 0.0cd 0.2 ± 0.0cd 0.3 ± 0.0c k 0.4 ± 0.1c 0.4 ± 0.1c 0.3 ± 0.0cd 0.3 ± 0.1cd 0.3 ± 0.0cd 0.3 ± 0.0cd 0.3 ± 0.1cd s 0.5 ± 0.3c 0.5 ± 0.2c 0.4 ± 0.1c 0.3 ± 0.0cd 0.5 ± 0.2c 0.3 ± 0.1cd 0.3 ± 0.0cd fe 0.7 ± 0.2c 0.6 ± 0.0c 0.3 ± 0.1d 0.3 ± 0.0d 0.7 ± 0.1c 0.3 ± 0.0d 0.2 ± 0.0d cu 0.5 ± 0.1c 0.7 ± 0.2bc 0.2 ± 0.1d 0.2 ± 0.0d 0.6 ± 0.1c 0.2 ± 0.0d 0.1 ± 0.0de mn 0.8 ± 0.2b 0.6 ± 0.0bc 0.4 ± 0.0c 0.4 ± 0.0c 0.6 ± 0.0bc 0.4 ± 0.0c 0.4 ± 0.0c zn 0.3 ± 0.0d 0.5 ± 0.0cd 0.4 ± 0.0d 0.3 ± 0.1de 0.5 ± 0.0cd 0.3 ± 0.0d 0.3 ± 0.0d cd 1.8 ± 1.0c 0.9 ± 0.5d 2.3 ± 0.8bc 1.9 ± 0.2c 0.8 ± 0.3d 1.9 ± 0.3c 1.7 ± 0.0c pb 0.3 ± 0.0b 0.4 ± 0.1ab 0.1 ± 0.0d 0.1 ± 0.0d 0.4 ± 0.1ab 0.1 ± 0.0d 0.1 ± 0.0d al 0.5 ± 0.0c 0.5 ± 0.0c 0.4 ± 0.0cd 0.4 ± 0.0cd 0.5 ± 0.0c 0.4 ± 0.0cd 0.4 ± 0.0cd different letter superscripts indicate significant differences between treatments, and same superscript letter as control indicate no significant difference. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:28 color profile: generic cmyk printer profile composite default screen their speciation (physiochemical form and associations with sediment constituents) also affects their mobility and toxicity (kabata-pendias and pendias 1992). the results of the present study revealed the combined effects of low redox condition and high ammonium concentration on macro-micro nutrient accumulation in the plant. fe, mn, cu, zn, cd, pb and al were soluble at low ph, and alteration in redox conditions affects their speciation as well as solubility (takeno 2005, du laing et al. 2009, miller et al. 2010). in oxic treatments, low concentrations of fe and mn were found, which might be the result of the formation of fe and mn (hydrate) oxides at high eh (yu et al. 2007), and these oxides are very slightly soluble (gambrell 1994). the phyotoxicity due to different elements depends on metal type, metal concentration and duration of exposure (odjegba and fasidi 2007). the metal uptake and distribution in submerged plant species vary according to the relative concentration of the elements in the environment, the growth of the plant, type of absorption mechanism, metal speciation, metal stability and constants with ligands, redox potential and ph at water-sediment interface, light, and microbial activity (nagajyoti et al. 2010). according to markert and wtorova (1992), the presence of a high concentration of heavy metals (micro elements or trace metals) seems to be directly associated with the exclusion of nutritional elements. in our study, plants in reduced treatment were observed for exclusion phenomenon for macro elements (k, ca and mg), and consequently, their concentration declined below critical level in the plant (tab. 2), and increased in water sample (tab. 5). among k, ca and mg, the most significant decrease was observed in ca (0.3 ppm) (tab. 2). the bcf sequence for bioaacumulated micro elements was cu>mn>zn>al>cd>fe>pb in both nh4-n conditions under reduced treatments (tab. 2). trace metal concentrations in aquatic plants vary considerably according to the part of the plant as well as chemical characteristics of the elements. baldantoni et al. (2004) concluded that a submerged macrophyte takes up the elements in acta bot. croat. 73 (1), 2014 141 macro-micro element accumulation capabilities of elodea nuttallii tab. 5. concentration (mg l–1, mean ± sd) of elements in water of experimental tank under different conditions (nh4-n concentrations) and treatments (oxic to highly reduce). elements control 2.5 ppm nh4-n 10 ppm nh4-n oxic reduced highly reduced oxic reduced highly reduced ca 11.3 ± 1.1c 10.6 ± 2.8c 16.6 ± 3.2b 18.7 ± 1.9a 11.9 ± 2.8c 17.6 ± 3.8b 20.4 ± 2.6a mg 9.1 ± 1.3c 10.7 ± 1.1c 12.9 ± 1.7ab 15.2 ± 1.5a 11.1 ± 1.0c 13.3 ± 1.4b 17.0 ± 0.8a k 12.7 ± 2.1d 12.6 ± 1.6c 19.7 ± 3.4b 23.3 ± 1.7a 12.5 ± 2.3c 21.0 ± 3.0b 25.5 ± 4.1a s 15.7 ± 3.2d 15.4 ± 1.8d 19.2 ± 2.0cd 22.1 ± 3.9b 16.3 ± 3.2d 23.8 ± 2.2b 28.7 ± 3.6a cu 0.0 ± 0.0b 0.0 ± 0.0b 0.9 ± 0.2a 1.3 ± 0.3a 0.0 ± 0.0b 1.1 ± 0.2a 1.4 ± 0.3a mn 1.3 ± 0.1c 1.2 ± 0.2c 7.2 ± 0.7b 10.0 ± 1.4a 1.4 ± 0.1c 9.1 ± 2.4b 14.6 ± 0.9a zn 2.5 ± 0.4c 2.6 ± 0.3c 5.2 ± 1.5b 8.4 ± 1.9a 2.7 ± 0.3b 10.7 ± 1.1a 11.8 ± 1.2a fe 4.1 ± 1.7d 4.4 ± 1.2d 41.4 ± 3.7a 57.8 ± 7.2a 4.6 ± 2.1d 50.9 ± 5.4a 72.7 ± 8.2a al 0.0 ± 0.0c 0.0 ± 0.0c 3.2 ± 0.1b 4.3 ± 0.7a 0.0 ± 0.0c 4.0 ± 0.2b 5.1 ± 0.5a pb 0.0 ± 0.0b 0.0 ± 0.0b 0.2 ± 0.0a 0.3 ± 0.0a 0.0 ± 0.0b 0.3 ± 0.0a 0.4 ± 0.0a cd(mg/l) 0.0 ± 0.0b 0.0 ± 0.0b 0.1 ± 0.0a 0.2 ± 0.0a 0.0 ± 0.0b 0.2 ± 0.0a 0.3 ± 0.0a different letter superscripts indicate significant differences between treatments, and same superscript letter as control indicate no significant difference. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:28 color profile: generic cmyk printer profile composite default screen the shoots from water by the roots. in reduced treatments, trace metal contents of water sample have also been increased, which is probably due to eh and ph effects, because at low eh and ph such elements were solubilized in water from sediment (tabs. 5, 6). plant uptake of metal is mainly dependent on metal mobility and availability in sediment. uptake of different metals also depends on protein transporters (foulkes 2000). pb, zn, cd, fe, cu are taken up at the cell surface through the cation channel (welch and norvell 1999, cosio et al. 2004), so they compete with each other and exclude ca ion. in our experiment, inreduced treatments, cu+2 was more considerably bioaccumulated than fe+2, which might be due to fe+2 and cu+2 competing with each other for binding sites on the cell wall and being taken into the cell walls of plants (fox and guerinot 1998). the accumulation of zn by the plant was also low though this element was bioavailable in the surrounding environment. the uptake of metals was also found to be ph dependent (wang et al. 2006) although in certain cases no ph effect was seen. the elements al and pb were found to be less accumulated in plants, which might be due to the above reason. in reduced treatments, metal accumulation in shoot and leaf was found to be higher than that in root, which might be due to the direct uptake by the shoot and leaves or from root to shoot by acropetal transport. since roots degenerate and are greatly reduced in size due to metal toxicity (basiouny et al. 1977), their potential for metal uptake might be limited. heavy metals could lead to oxidative damage to aquatic plants through ros generation (mittler 2002). this was particularly crucial for photosynthetic organisms which generate ros constantly during normal photosynthesis. chlorophyll concentration was higher in plants in oxic treatments with a normal nh4-n concentration (2.5 ppm), whereas at higher nh4-n concentrations, the chlorophyll level declined significantly. conversely, low redox potential affects chlorophyll synthesis at normal to high nh4-n concentrations. the loss of chlorophyll contents consequently disrupts the photosynthetic machinery, thus the electron transport rates of psi and psii are disturbed, which leads to the generation of ros. in the present study, decrease of chlorophyll content was probably achieved both by reaction with biosynthetic enzymes as well as peroxidase mediated degradation (asada 1994). in addition, carotenoid represents the other group of photosynthetic pigments that are highly effective in quenching chlorophyll triplet states and singlet oxygen (lichtenthaler 1987). the degree of anoxia damage to the photosynthetic apparatus in different oxygen-deprived conditions was determined by chlorophyll fluorescence of psii in dark-adapted leaf, where the fv/fm values were decreased with increased oxygen deprivation (<0.4). pronounced fluorescence decay in plants was observed under reduced environments, which might be due to 142 acta bot. croat. 73 (1), 2014 zaman t., asaeda t. tab. 6. redox potential and ph (mean ± sd) values of growth medium under different conditions (nh4-n concentrations) and treatments (oxic to highly reduce). parameters control 2.5 ppm nh4-n 10 ppm nh4-n oxic reduced highly reduced oxic reduced highly reduced eh 288 ± 16.4 b 440 ± 11.3a –4 ± 1.1d –150 ± 17.4f 432 ± 14.1a –2 ± 3.8d –157 ± 18.3f ph 6.9 ± 0.5b 7.4 ± 1.0ab 4.5 ± 0.3c 4.1 ± 0.1c 7.1 ± 0.8ab 4.2 ± 0.4c 4.1 ± 0.6c different letter superscripts indicate significant differences between treatments, and same superscript letter as control indicate no significant difference. 806 zaman and asaeda.ps u:\acta botanica\acta-botan 1-14\806 zaman and asaeda.vp 19. o ujak 2014 17:19:28 color profile: generic cmyk printer profile composite default screen the substitution of mg by other metals (such as cu, pb, cd). the fv/fm ratio, the maximum quantum yield of psii photochemistry, is frequently used as an indicator of photoinhibition or of other kinds of stress to photosystem ii (calatayud and barreno 2004). membrane lipids and proteins are especially prone to attack by free radicals. proline accumulation is considered to be involved in stress resistance mechanisms (lutts et al. 1999). decrease in proline content in the plant under reduced treatments might be due to the dysfunction of sulphydryl groups during heavy metal transportation into the plants (nagoor 1999), which affects protein synthesis. the increased activity of protease or other catabolic enzymes that are activated by heavy metals might be another reason (gupta et al. 1996). the results of the present study indicate that plants under reduced treatments seemed to be more vulnerable to metal toxicity as more than one metal was present at a toxic level in reduced treatments. lipid peroxidation profoundly alters the structure of membranes and modifies their enzymatic and transport activities (rai 1995). increased mda levels in plant tissue indicate an increased lipid peroxidation in cell membrane. the high concentration of cellular h2o2 and elevated pod activity in our experimental plants suggested that the ros scavenging system was activated under such stressed conditions. reduced biomass increment observed in our experiment suggested the plant growth was inhibited. noticeable declines of e. nuttallii populations in japan (nagasaka 2004) and elodea canadensis in europe (sculthorpe 1967) were reported, and scientists have suggested different stress (biotic and abiotic) factors regarding this decline (hamabata 1991, kadono et al. 1997). our results also supported by brix and sorrell (1996), who reported that wetland plants grown in reducing treatments stopped growing, some of them losing mass. conclusions by subjecting elodea nuttallii to high ammonium concentration in hypoxic/anoxic environments we experienced a number of symptoms, such as suppression of growth, chlorosis of leaves, increased shoot : root ratio, increased lipid peroxidation, decreased proline level, decreased concentrations of mineral cations (such as k, ca and mg in the tissues), increased micro elements and decreased photosynthetic pigments etc. most of these symptoms were reported for ammonium toxicity (britto and kronzucker 2002, cao et al. 2004) as well as for metal toxicity in a reduced environment (mittler 2002, nagajyoti et al. 2010, monferrán et al. 2012). however, it is difficult to distinguish between high ammonium concentration effect and metal toxicity in a reduced environment. overall, the combined effect of a low redox state and high ammonium concentration has stronger physiological impact on submerged macrophytes than high ammonium concentration (10 ppm nh4-n in oxic treatments) acting alone. at the same time the balance of macro-micro nutrients was found more significantly affected by low redox status than by the applied high ammonium concentration in oxic treatment. acknowledgements the authors would like to thank prof. takeshi fujino for his assistance. this research was financially supported by a research grant-in-aid from the ministry of education, culture, sports, science and technology, japan, and the river basin environment foundation. acta bot. croat. 73 (1), 2014 143 macro-micro element accumulation 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sampling of bioaerosols has been carried out using a rotorod sampler as well as by exposing culture plates. the screening of some common vegetables was also done for the isolation of fungi as market pathogens to study post-harvest diseases. altogether, fifty nine fungal spore types and 78 species of 33 genera belonging to different groups were recorded respectively on the rotorod strips and on exposed petri dishes. many saprophytic and pathogenic fungi were found to be associated with sampled vegetables from the market. in all forty-six fungal species belonging to 26 genera were recovered from five varieties of vegetables collected from the same market. the most dominant forms of fungi were of aspergillus followed by cladosporium, penicillium, alternaria, fusarium, curvularia, trichoderma, and rhizopus. aspergillus niger, a. flavus, a. fumigatus, penicillium spp. and cladosporium herbarum, found to be dominant during the period of investigation. important mycotoxin-producing fungi such as a. flavus, a. fumigatus and fusarium moniliforme were isolated from the vegetables collected from the market. keywords: bioaerosols, vegetable, saprophyte, pathogen, mycotoxin, post-harvest disease introduction in many market places, spoiled material, dumped plant material and debris are often present and are likely to act as reservoirs of plant pathogens due to the constant build up of the spore population from fungi growing on them. the activity of nearly all fungi is to carry out biodegradation and deterioration because of their requirements for prime sources of carbon, nitrogen and other nutrients (pitt and hocking 1985). the deterioration of raw vegetables may also be caused by physical damage, the action of their own enzymes, microbial action or a combination of these factors. acta bot. croat. 71 (1), 2012 147 * corresponding address: b-12, gagan mahal, sir pochkhanwala road, worli, mumbai, 400025, india, e-mail: drumeshkakde@gmail.com copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:00 color profile: disabled composite 150 lpi at 45 degrees the spoilage occurring during the processing, distribution, and sale of fresh vegetables is often referred to as post-harvest spoilage or marketing disease. as a general rule, vegetables become more susceptible to infection by post-harvest pathogens as they ripen. vegetable and fruits are perishable products with active metabolisms during the postharvest period (eckert 1975). therefore, proper handling and environmental conditions after harvesting are essential in maintaining product quality. mechanical damage has dual importance because it not only reduces the value of vegetables but an also increases the chance for microbial spoilage. post-harvest diseases are responsible for significant loss of vegetable and fruit production (frazier 1967). a study of bio-aerosols in a market environment showed that most of the fungi found on vegetables and fruits originated in the field or developed during transportation (panduranjan and suryanarayanan 1985). airborne spores can cause infection to salable articles. although many types of microbial spoilage result from invasion by parasitic microorganism, much spoilage also results when normally saprophytic microorganisms behave as opportunistic parasites. for example tomatoes can be spoiled by members of the fusarium and geotrichum families (brackett 1987). the spraying of water to give a fresh appearance to vegetables and fruits in the market also provides a moist surface that encourages the growth of molds in storages. bioaerosols in market environments may have some implications for the health of the people working in there. most of the fungi present in high concentrations in market environments have been suspected of being causative agents of respiratory diseases in humans and infections of produce. the adverse effects of inhaled fungal propagules on the immune system have been well documented by various workers (day 1986; burge 1985, 1989; lacey 1991). fungal infection can also increase the chances of contamination by mycotoxins, which can cause neurological disorders, liver cancers, lung cancers and other diseases. many molds capable of producing mycotoxins are also frequent contaminants of food and agricultural commodities. molds, which are of importance in food because of potential mycotoxin production, include members of the genera aspergillus, trichothecium, fusarium. the mycotoxins that are currently receiving the most attention as potential hazards to human and animal health include aflatoxin, ochratoxin a, sterigmatocystin, patulin, penicillic acid, citrinin, zearalenone and the toxic trichothecenes. all these compounds cause some degree of acute toxicity when received in high amounts (kramer et al. 1960; hudson 1969; willie and morehouse 1978; bullerman 1979; wicklow and shottwell 1983; miller 1990, 1992). no systematic studies on the incidence of post-harvest disease and airborne fungal spores in a vegetable market have been published. however, such information is needed to evaluate the relationship between the prevalence of fungal spores and post-harvest diseases of vegetables due to airborne fungi in markets, and to understand the type of mold exposure that can cause health effects. hence, the present study was undertaken in one of the major vegetable markets of nagpur city to investigate post-harvest disease due to the prevalence of airborne fungi in the market. in this paper we describe the measurement of airborne fungal spores and post-harvest diseases made in a vegetable market in nagpur. 148 acta bot. croat. 71 (1), 2012 kakde u. b., kakde h. u. u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:01 color profile: disabled composite 150 lpi at 45 degrees material and methods the geographical location of nagpur is 79 degrees 8 minutes east and 21 degrees 8 minute north. thus it can be said that nagpur falls in the deccan plateau region. the city is located in maharashtra state and lies in the center of india; the zero milestone of india is situated within this city. an aero-mycological survey was carried out in a vegetable market at fule market (21° 8’ 45” n/79° 5’ 26”e) in nagpur city. it is one of the main outdoor vegetable markets of nagpur city (m.s.), from which a number of vegetables are marketed and transported to different local and outstation markets. this market is situated in the heart of the city and surrounded on all sides by schools, quarters, gardens, railway station etc. the study was undertaken at different seasonal climatic conditions i.e., monsoon (june– september), winter (october–february) and summer (march–may) for the period of two years. bioaerosols were sampled using a rotorod air sampler and settle plates (9 cm diameter perti dishes) to provide quantitative and qualitative measurements of fungal spores respectively. the rotorod air sampler with a sampling rate of 100 l min–1 (tilak air sampler, 1982) was used to analyze the total spore counts. the sampler was operated for 15 min. at the height of one meter above the ground level at fifteen day intervals. the sampling was done during morning (07 am to 10 am) when the market was most active. exposed cellophane tapes were mounted with glycerin jelly and examined under a microscope. the trapped spores were identified with the help of reference slides and available standard literature (ainsworth et al. 1972; tilak 1982, 1989; nair et al. 1986). for the qualitative analysis of fungi three different media were used: czapek’s dox agar (cz), potato dextrose agar (pda) and martin’s rose bengal agar (mrba). two petri dishes of each medium were exposed for 3 minutes at intervals of 15 days during morning when market activities like loading unloading, agitation, weighing of vegetables were at their peak. all the petri dishes were kept at different heights (0.5–1 m) above the ground. after exposure to the air the petri dishes were brought to the laboratory in pre-sterilized polythene bags and incubated at 25 °c for 5–7 days. colonies were counted and identified. the identification of colonies was based on their color, size, shape and other morphological features (gilman 1957, barnett 1960, raper and fenell 1965, raper and thom 1968, smith 1969, ainsworth et al. 1972, ellis 1971). the temperature and relative humidity of the air during the experiments were also recorded. samples of fresh as well as previously infected or rotten vegetables (cabbage, beans, onion, potato, tomato) were collected in pre-sterilized polythene bags from the same market to examine post-harvest fungi. for the growth of fungi, these vegetables were incubated using a sterile moist blotter method for 5 to 7 days. fungi were isolated from these vegetable samples and observed directly by preparing lacto-phenol cotton blue mounts. fungi isolated by this technique were sub-cultured to sabouraud’s agar and cz medium and colonies grown for further identification up to species level. results and discussion quantitative and qualitative analysis of air samples showed that the market environment was contaminated by some major fungal spore types such as aspergillus, penicillium, cladosporium, alternaria. the spore population of fungi followed a definite seasonal acta bot. croat. 71 (1), 2012 149 incidence of post-harvest disease and airborne fungal spores u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:01 color profile: disabled composite 150 lpi at 45 degrees variation in the weather prevailing over the market environment. the highest incidence of spores was observed during august to january (monsoon and winter) while march-may (summer) was the lean period. quantitative and qualitative analysis of bioaerosols: altogether 59 spore types and 78 fungal species were identified from the air of vegetable market by the rotorod sampler and settle plate method respectively. these belonged to 33 genera. three spore types from the zygomycotina, 13 from the ascomycotina, 3 from the basiodiomycotina and 30 from the deuteromycotina were recovered by rotorod sampling. unidentified, less frequent spore types, pollen grains and other bioerosols were classified as other types. sixty-six species belonging to the class deuteromycotina were identified from the settle plates, 10 from the zygomycotina and 2 from the ascomycotina. unidentified colonies including yeasts and less frequent taxa were grouped as unidentified colonies (tab. 1, 2). more spore types and fungal species were found in the months of august, november and january than in april and may (summer). during the monsoon and winter seasons more varieties of vegetables and fruits are available in the market than at other time. the greater concentration of fungal spores during the winter and rainy seasons may be due to the greater availability of dead and decaying material in the market yard acting as sources 150 acta bot. croat. 71 (1), 2012 kakde u. b., kakde h. u. tab. 1. incidence of the dominant spore types collected in air samples in a vegetable market. spore types average spores/m3 per cent contribution range (spores/m3) ascomycotina chaetomium 24 0.7 0–85 basidiomycotina smuts 46 1.3 0–120 uredospores 32 0.9 0–95 deuteromycotina alternaria 464 13.2 120–800 aspergillus/penicillium* 1260 35.9 985–5200 cercospora 41 1.2 0–100 cladosporium 623 17.8 425–2000 curvularia 202 5.8 85–520 drechslera 40 1.1 0–95 fusarium 53 1.5 0–120 helminthosporium 126 3.6 10–300 nigrospora 65 1.9 0–150 pithomyces 24 0.7 0–95 hyphal fragments 54 1.5 20–150 other types# 456 13.0 125–900 * small, spherical and similar type of spores, aspergillus, penicillium, trichoderma, mucor, rhizopus. # – less frequent, unidentified spores, pollen grains and other bioaerosols. u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:01 color profile: disabled composite 150 lpi at 45 degrees of inoculum. this coupled with moisture caused by dew in winter and rain in the monsoon, may promote profuse growth of fungi and subsequent sporulation and discharge of spores into the atmosphere. in the two-year study aspergillus-penicillium type spores (small rounded) were the major spore type found, contributing 35.9% for the first year and 34.8% for the second year of the investigation to the total bioaerosols. rest of the bioaerosols were made up of cladosporium, alternaria, curvularia and helminthosporium (tab. 1). among the total counts of viable fungi identified, we recognized aspergillus, penicillium, cladosporium, alternaria and curvularia as the most abundant genera. aspergillus acta bot. croat. 71 (1), 2012 151 incidence of post-harvest disease and airborne fungal spores tab. 2. incidence of the most common (contribution > 5%) fungal species identified from settle plate samples in a vegetable market fungal species total colonies per cent contribution zygomycotina cunninghamella echinulata 25 0.5 c. elegans 33 0.7 mucor globosus 27 0.5 rhizomucor pusillus 28 0.6 rhizopus oryzae 25 0.5 r. stolonifer 76 1.5 ascomycotina chaetomium globosum 26 0.5 deuteromycotina alternaria brassicae 81 1.6 a. solani 112 2.2 a. tenuis 140 2.8 aspergillus candidus 129 2.6 a. flavipes 38 0.8 a. flavus 258 5.1 a. fumigates 126 2.5 a. niger 321 6.4 a. ochraceous 44 0.9 a. sydowi 42 0.8 a. terreus 46 0.9 a. versicolor 25 0.5 botrytis cinerea 25 0.5 candida albicans 33 0.7 cladosporium epiphyllum 215 4.3 c. herbarum 242 4.8 fungal species total colonies per cent contribution c. lignicola 160 3.2 colletotrichum sp. 61 1.2 curvularia geniculata 39 0.8 c. lunata 117 2.3 c. oryzae 55 1.1 c. tetramera 45 0.9 drechslera australiensis 42 0.8 fusarium chlamydosporum 29 0.6 f. moniliforme 140 2.8 f. oxysporum 47 0.9 f. solani 136 2.7 helminthosporium nodulosum 45 0.9 h. oryzae 80 1.6 nigrospora sphaerica 40 0.8 penicillium citrinum 131 2.6 p. chrysogenum 169 3.4 p. italicum 104 2.1 p. nigricans 124 2.5 p. oxalic 40 0.8 phoma lingam 93 1.9 sporotrichum roseum 36 0.7 trichoderma harzianum 76 1.5 t. viride 86 1.7 trichothecium roseum 25 0.5 verticillium glaucum 25 0.5 sterile colonies 25 0.5 unidentified colonies* 943 18.7 (* less frequent (<.5%), unidentified colony forming units) u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:01 color profile: disabled composite 150 lpi at 45 degrees species were the most prevalent throughout the seasons. the most dominant species isolated were aspergillus niger, a. flavus, a. fumigatus, alternaria solani, cladosporium herbarum, curvularia lunata, rhizopus stolonifer, penicillium chrysogenum, p. italicum. the majority of these fungal species were present throughout the year. aspergillus species followed a seasonal trend. meteorological conditions such as high temperature and low humidity during the summer contribute fewer fungi while in the rainy season the concentration of fungi is significantly increased in the atmosphere of market. high concentrations of these fungal spores have been reported in different working environments (sumbali and badyal 1991, sulia and khan 1980, rosas et al. 1992, kakde et al. 2001). spores of some fungi (aspergillus, penicillium, cladosporium etc.) become airborne as a consequence of active liberation mechanism or more often in working environments by agitation of their substrates (ingold 1971). handling of unwashed vegetables was responsible for the increase of the spore load concentration in the air especially of cladosporium and penicillium (lehtonen et al. 1993). these genera were also isolated from the surface samples of vegetables. the seasonal pattern of aspergillus, cladosporium, penicillium, alternaria and other dominant fungi are governed by the availability of dead and decaying organic matter and their prevalence throughout the year was because of abundant substrata available for their growth in all the seasons in the market. in the present investigation variation in the spore concentrations was recorded in the different months (fig. 1). the difference in the spore concentration and types could be due to the prevailing environmental conditions and presence of agricultural waste, plant debris and infected vegetables and fruits in the market environments. the release of fungal spores and consequently their concentrations in the atmosphere are the result of the action of many biological and environmental factors. the occurrence of fungal spores in the air is markedly seasonal because of these organisms’ sensitivity to weather changes (hjelmroos 1993, craig and levetin 2000, stepalska and wolek 2005). 152 acta bot. croat. 71 (1), 2012 kakde u. b., kakde h. u. 0 2 4 6 8 10 12 july aug sept oct nov dec jan feb mar april may june months p e r c e n t c o n tr ib u ti o n spore types fungal species fig. 1. seasonal distribution of fungal spores and species during the sampling period in a vegetable market yard. u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:01 color profile: disabled composite 150 lpi at 45 degrees altogether forty-six fungi from 26 genera were isolated from selected common vegetables collected. there were 5 zygomycotina species; one ascomycotina and the rest of the species were deuteromycotina. the largest number of species (46) was found on cabbage and cauliflower, 27 on tomato, 26 on bean, 24 on potato, and 15 on onion (tab. 3). some of the species found are presented in figure 2. in the present investigation a few more fungal species were isolated from the air during august, november, december and january (fig. 1). the incidence of post-harvest diseases in the vegetables was also higher than in other months. as the concentration of fungal spores in the air decreases, the frequency of occurrence of fungi on the vegetables also decreases, and vice-versa. aspergillus species were found to be the chief contaminant from the total species isolated. the most frequently isolated fungal species from vegetables were aspergillus niger, a. flavus, penicillium crysogenum, curvularia spp., cladosporiun herbarum, rhizopus stolonifer, alternaria spp., fusarium spp. aspergillus has been reported as the dominant spore type in markets and other environments by many researchers (sullia and khan 1980, pandurajan and surryanarayanan 1985, santra and chanda 1989, abdel-hafez et al. 1989, sumbali and badyal 1991, duchaine et al. 2001, kakde et al. 2001). in the last few years it has been reported that the spores and sclerotia of toxic fungi contain very large concentrations of mycotoxins. the spores of aspergillus flavus and a. parasiticus have been reported to contain 1100 mg g–1 of aflatoxin, which is carcinogenic to human beings (wicklow and shotwell 1983). acta bot. croat. 71 (1), 2012 153 incidence of post-harvest disease and airborne fungal spores tab. 3. fungi isolated from different vegetables collected from a vegetable market vegetables major fungal species associated cabbage (brassica oleracia) aspergillus niger, a. fumigatus, a. versicolor, a. flavus, alternaria brassicae, a. alternata, a. solani, botrytis cinerea, cercospora brassicae, cladosporium herbarum, curvularia lunata, fusarium brassicae, f. moniliforme, penicillium chrysogenum, phoma brassicae, phoma lingam, rhizopus sp., trichoderma herzianum, t. viride bean (dolichos lablab) aspergillus niger, a. flavus, a. fumigatus, alternaria solani, a. brassicae, botrytis cinerea, cladosporium species, fusarium spp., rhizopus sp., penicillium crysogenum, penicillium species, onion (allium cepa) alternaria tenuis, a. solani, aspergillus niger, a. flavus, botrytis cinerea, cladosporium herbarum, colletotrichum sp., fusarium moniliforme, rhizopus sp., penicillium digitatum, trichoderma spp. potato (solanum tuberosum) aspergillus niger, a. flavus, alternaria brassicae, a. solani, a alternata, botrytis cinera, cercospora brassicae, cladosporium herbarum, curvularia lunata, fusarium spp., f. moniliforme, rhizopus stolonifer, penicillium chrysogenum, phoma lingam, rhizoctonia solani, trichoderma herzianum tomato (lycopersicon esculentum) aspergillus niger, a. flavus, alternaria alternata, a. solani, botrytis cinera, cladosporium herbarum, cladosporium sp., colletotrichum sp., fusarium moniliforme, fusarium solani, rhizopus stolonifer, penicillium chrysogenum, saccharomyces sp. u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:01 color profile: disabled composite 150 lpi at 45 degrees a series of experiments on fungal species isolated both from vegetables and air were carried out. one such experiment reported the correlation coefficient (r) between percent occurrence of 15 fungal species on selected vegetables (x) and percent occurrence in air (y) as 0.7595. this indicates a strong positive linear dependence between x and y. 154 acta bot. croat. 71 (1), 2012 kakde u. b., kakde h. u. fig. 2. some common fungi isolated from the vegetables collected from the market: a – alternaria solani, b – aspergillus niger, c – aspergillus flavus, d – aspergillus fumigatus, e – cladosporium herbarum, f – curvularia lunata, g – chaetomium gobosum, h – fusarium moniliforme, i – helminthosporium orza, j – rhizopus sp, k – penicillium citrinum, l – penicillium chrysogenum u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:06 color profile: disabled composite 150 lpi at 45 degrees can it be concluded that, in the population of these fungal species, the population correlation coefficient, r = 0.50 is significant? the problem in this situation is to test the null hypothesis h0: r = 0.50 against the alternative hypothesis h0: r ¹ 0.50. we are given n = 15, r = 0.7595 and r0 = 0.50. z r r e e � � � � �� � � � � � � �� � � � 1 2 1 1 1 2 1 0 795 1 0 795 0log log . . .9950 w e e � � � � � � � � � � � � �� � � � 1 2 1 1 1 2 1 0 50 1 0 50 00 0 log log . . . 5493 t z w n� � � � � � � �( ) ( . . ) .3 0 9950 0 5493 12 15440 the table value at 5% level of significance is 1.96. since, t �196. , we accept h0 at the 5% level of significance and conclude that the data support the hypothesis. hence, using information on the percentage isolation of fungal species on vegetables, the percentage isolation in air can be predicted. conclusion the most frequent associated fungi isolated from the vegetable and fruits were aspergillus niger, a. flavus, penicillium crysogenum, curvularia spp., alternaria spp., fusarium spp. aspergillus, penicillium, cladosporium, fusarium, alternaria etc. these fungi were most prevalent in the air of market environment and also found to be responsible for most of the decay of the vegetables during storage. hence, there is probably a cyclic relationship existing between the prevalence of fungal bioaerosols and spoilage diseases in market environments. that may be due to the growth of fungi on dumped plant materials, packing leaves, infected vegetables and fruits, where fungi grow saprophytically and produce spores profusely, consequently contaminating the environment. there was a good correlation between the number of visible mold growths on collected vegetables from the market and the concentration of airborne molds in the market environment. although there were fewer fungal species in the air, all the dominant fungal spores recovered from the air were also isolated from the samples of vegetables collected. hence, both air and surface sampling are necessary to evaluate mold problems in such environments. the number of mold growths on salable articles is a good predictor of airborne mold concentration. references abdel-hafez, s. i. i., el-said, a. h. m., 1989: seasonal variation of airborne fungi in wadi quena, eastern desert, egypt. grana 28, 193–203. ainsworth, g. c., sparrow, f. k., sussman, a. s., 1972: the fungi, 4 a. academic press, new york. acta bot. croat. 71 (1), 2012 155 incidence of post-harvest disease and airborne fungal spores u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:06 color profile: disabled composite 150 lpi at 45 degrees barnett, h. l., 1960: illustrated genera of imperfect fungi. burgess publishing co. minneapolis. brackett, r. e., 1987: vegetables and related products. in: beuchat, l. r. (ed.), food and beverage mycology, 129–154. van nostrand reinhold, new york, bullerman, l. b., 1979: significance of mycotoxins to food safety and human health. journal of food protection 42, 65–86. burge, h. a., 1985: fungus allergens. clinical reviews in allergy 3, 319–229. burge, h. a., 1989: airborne allergenic fungi: classification, nomenclature, and distribution. immunology and allergy clinics of north america 9, 307–319. craig, r. l., levetin, e., 2000: multi-year study of ganoderma aerobiology. aerobiologia 16, 75–81. day, j. h., 1986: ufei-fungal interaction-working paper provided to the health and welfare canada. ottawa, ontario, k1aol2. duchaine, c., meriaux, a., 2001: the importance of combining air sampling and surface analysis when studying problematic houses for mold biodiversity determination. aerobiologia 17, 121–125. eckert, j. w., 1975: general principles, 1. in: pantastica, e. b. (ed.), post-harvest physiology, handling and utilization of tropical and subtropical fruits and vegetables, 393– 414. avi publishing co., westport. ellis, m. b., 1971: dematiaceous hyphomycetes. publisher commonwealth agricultural bureaux. commonwealth mycological institute. kew, surrey (uk). frazier, w. c., 1967: food microbiology. mcgraw-hill book co., inc, new york. gilman, j. c., 1957: a manual of soil fungi. iowa state uni. press. ames. hjelmroos, m., 1993: relationship between airborne fungal spore presence and weather variables. grana 32, 40–47. hudson, h. j., 1969: aspergilli in the airspora at cambridge. transactions british mycological society 52, 153 – 159. ingold, c. t., 1971: fungus spores: their liberation and dispersal. oxford university press, london and new york. kakde umesh, b., kakde hemalata, u., saoji aarti, a., 2001: seasonal variation of fungal propagules in a fruit market environment, nagpur (india). aerobiologia 17, 177– 182. kramer, c. l., pady, s. m., rogerson, c. t., 1960: kansas aeromycology, 5. penicillium and aspergillus. mycologia 52, 545–551. lacey, j., 1991: aerobiology and health the role of airborne fungal spores in respiratory disease in: hawks worth, p. l. (ed.), frontiers in mycology. c.a.b. international, kew. lehtonen, m., reponen, t., 1993: everyday activities and variation of fungal spore concentration in indoor air. international journal of biodeterioration and biodegradation 31, 25–39. miller, j. d., 1990: contamination of food by fusarium toxin studies from austria-asia proceeding. japanese association of mycotoxicology 32, 17–24. 156 acta bot. croat. 71 (1), 2012 kakde u. b., kakde h. u. u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:06 color profile: disabled composite 150 lpi at 45 degrees miller, j. d., 1992: fungi contaminants in indoor air. atmospheric environment 26 a, 2163–2172. nair, p. k. k., joshi, a. p., gangal, s. v., 1986: airborne pollen spores and other plant materials of india. csiro centre biochemicals, delhi and lucknow. pandurajan, a. g., surryanarayanan, j., 1985: a survey of mycoflora associated with some fresh vegetable and fruits in a market at saidpeth market. journal of economic and taxonomic botany 7, 309–315. pitt, j. i., hocking, a. d., 1985: fungi and food spoilage. academic press, sidney, australia. raper, k. b., fennell, d. i., 1965: the genus aspergillus. the williams and wilkins co., baltimore. raper, k. b., thom, c., 1968: a manual of penicillia. hafner publishing co., new york. rosas, i., calderon, c., escamilla, b., ulloa, m., 1992: seasonal distribution of aspergillus in the air of an urban area: mexico city. grana 31, 315–319. santra, s. c., sunirmal chanda, 1989: airborne fungal flora in indoor environment of the calcutta metropolis, india. grana 28, 141–145. smith, g., 1969: an introduction to industrial mycology. edward arnold ltd, london. stepalska, d., wolek, j., 2005: variation in fungal spore concentrations of selected taxa associated to weather conditions in cracow, poland, in 1997. aerobiologia 21, 43–52. sulia, s. b., khan, k. r., 1980: aerospora of bangalore city market and its relation to occurrence of market diseases. journal advances in pollen and spore research 5–7, 157–159. sumbali, g., badyal, k., 1991: relationship between fungal aerospora of fruit shop and incidence of fruit rots. journal indian phytopath 44, 214–218. tilak, s. t., 1982: aerobiology. vaijayanti prakashan, 'ushakal' saraswati colony, (west) aurangabad. tilak, s. t., 1989: airborne pollen and fungal spores. vaijayanti prakashan, aurangabad. wicklow, d. t., shottwell, o. l., 1983: intra-fungal distribution of aflatoxin among conidia and sclerotia of aspergillus flavus and a. parasiticus. canadian journal of microbiology 29, 1–5. willie, t. d., morehouse, l. g., 1978: mycotoxic fungi, mycotoxins, and mycotoxicosis. mareal dekkar inc., new york. acta bot. croat. 71 (1), 2012 157 incidence of post-harvest disease and airborne fungal spores u:\acta botanica\acta-botan 1-12\474 kakde and kakde.vp 26. o ujak 2012 13:42:06 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 68 (1), 127–146, 2009 coden: abcra 25 issn 0365–0588 the nutritive value and role of panicum turgidum forssk. in the arid ecosystems of the egyptian desert selim z. heneidy1*, marwa w. halmy2 1 botany department, faculty of science, alexandria university, egypt 2 environmental sciences department, faculty of science, alexandria university, egypt panicum turgidum is one of the most drought resistant plants in the egyptian desert. five habitat types were selected for the study: sand dunes (north sinai and in wady el-natrun), gravel desert, coastal plain, and water runnels of a wadi bed (south sinai and eastern desert). about 47.4% of the studied sites were subjected to high grazing pressure, 26.3% to moderate grazing pressure; the remaining 26.3% were subjected to low grazing pressure. concentration of the nutrients (k, na, ca, p and ca/p ratio) in the grazeable parts of p. turgidum populations exhibited insignificant variability between habitats and phytogeographical regions. meanwhile, concentration of potassium showed significant variability between the phytogeographical regions. ca/p ratio of the grazeable parts of p. turgidum was significantly related to the soil silt and organic matter content. the percentage of crude protein was higher than the minimum required for the maintenance of the grazing animals, i.e. p. turgidum has a good or excellent forage quality. due to its high nutritive value and ease of cultivation, p. turgidum grains could be considered as a potential crop, which may serve as a supplementary food to the common cereals. it worth noting that the variation in the nutritive values among the cultivated populations was insignificant as all the cultivated populations had a high nutritive value, indicating the effect of edaphic factors on the wild population nutritive value which was proved also from the correlation between the edaphic factors and nutritive variable. keywords: arid, grazing, protein, nutrients, desert, egypt. introduction grasses play an important role in land stabilization and animal nutrition due to their protein, carbohydrates, fats, fibers and mineral contents. therefore, planting grasses constitutes an important part of any rangeland rehabilitation program. panicum turgidum forssk. (poaceae) is one of the widely distributed and most drought resistant grasses of the egyptian desert (migahid and el shourbagy 1961). according to täckholm (1974), this species is widely distributed in all phytogeographical regions of egypt except the western mediterranean coastal desert. the success and wide distribution of this plant species indiacta bot. croat. 68 (1), 2009 127 * corresponding author, e-mail: drsheneidy@yahoo.com u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:35 color profile: disabled composite 150 lpi at 45 degrees cates its ability to tolerate certain unfavorable conditions (ismail and seed 1983). panicum turgidum has the merit of being very resistant to drought and also a very effective sand-binding xerophyte (migahid and el shourbagy 1961). it is also considered to be tolerant to high soil salinity levels as well as to sand accumulation. therefore, it is a good species for stabilizing loose soil (el shourbagy 1958, heneidy and waseem 2007). due to the high palatability of this grass it is considered an important fodder and grazing plant for many animals, especially in summer when annuals disappear and shortage in natural forage occurs. also, in dry conditions, p. turgidum provides grazing as standing hay (mandaville 1990). since many native grasses of the coastal strip of egypt are spring growers, the summer growth of p. turgidum may make this species suitable as complementary forage for the deteriorated lands of the western coastal desert of egypt. the present study aims at assessing the variation in the nutritive value among the studied populations of p. turgidum and its relation to soil characters. consequently, it has successfully been used in rehabilitation trials in the western coastal deserts of egypt (heneidy and waseem 2007). materials and methods fresh specimens of p. turgidum were collected from 19 sites, selected to represent a wide variety of habitats within the range of distribution of p. turgidum in the eastern desert, wady el-natrun and sinai (tab. 2). according to täckholm (1974) these sampling sites are located in four different phytogeographical regions: the eastern desert region; mediterranean region (north sinai); oases of the western desert (wady el-natrun); and sinai proper (fig. 1). the sampling site represents five types of habitats: sandy plains; sand dunes (sampling populations located in north sinai and in wady el-natrun), gravel desert, coastal plain, and water runnels of the wadi bed (sampling populations located in south sinai and eastern desert). pasture condition, grazing pressure and the relative abundance of p. turgidum were assessed for the nineteen sampling sites, by direct observations during the field visits. relative accessibility of the aboveground parts of the studied species for grazing was assessed using the ocular estimate method (etienne 1989). the aboveground parts of p. turgidum in each site were assigned to one of the relative accessible parts classes; 40–59, 60–89 and 90–100% (jensen and scotter 1977). for the chemical analysis and nutritive value estimation, samples of the fresh grazeable aboveground parts of the studied species populations were collected from at least five bushes from each of the sampling sites. the collected plant materials were cleaned, oven dried at 65 °c to constant weight, powdered before chemical analysis. mature grains collected from all the studied populations were pooled together to form a composite sample. the grains were powdered ready for chemical analysis. the mixed-acid digestion was used for preparing the sample solution for determination of element content. na, k, ca and mg were analyzed using the flame photometer. phosphorous was determined according to eaton et al. (1995). ash and ether extract (i.e. crude fats) was determined according to allen et al. (1974). total protein was determined according to the modified lowery method (hartreé 1972). the crude fiber was determined according to aoac (1960). nitrogen free extract was calculated according to the following equation (le houérou 1980): nfe (in % dm) = 100 – (cp + cf +ee + ash) where cp = crude protein, cf = crude fibers, ee = ether extract (i.e. total fats). 128 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:35 color profile: disabled composite 150 lpi at 45 degrees digestible crude protein (dcp) was determined using the equation of demarquilly and weiss (1970): dcp (in % dm) = 0.93 cp – 3.52 this equation is only valid in the case of nitrogen concentration from 0.61 to 3% (or cp > 3.81%). total digestible nutrients (tdn) were estimated according to abu el-naga and el-shazly (1970). the gross plant energy was calculated from its chemical analysis components multiplied by the average of heat of combustion (lofgreen 1951). digestible and metabolized energy (de and me) were estimated according to crampton and harris (1969). net energy was estimated according to garrett (1980). three soil samples were taken down to the depth of 0–50 cm for each site. soil samples were air dried, thoroughly mixed, passed through a 2 mm sieve before physical and chemical analysis (black 1965, allen et al. 1974). soil texture analysis was conducted using the bouyoucus hydrometer method. determination of calcium carbonate content was carried out using a calcimeter. total organic matter was determined by loss-on-ignition. for determination of soil reaction (ph), electric conductivity (e.c.), bicarbonates and some of the available nutrients 1: 2.5 (w/v) soil: water extracts were prepared. sulfates were carried out using the »gravimetric with ignition of residue method«. calcium and magnesium were determined using direct titration against 0.1n edta. a flame photometer was used for deteracta bot. croat. 68 (1), 2009 129 panicum turgidum in the egyptian desert fig. 1. map of northern egypt showing the locations of the sampling sites from which p. turgidum populations were collected. 1bir lahfen; 2el-koseima road; 3abu aekela; 4geble libni; 5spika; 6ras el-deeb (el zaranik protected area); 7el zaranik protected area; 8el-qaa plain (15 km from el-tur city); 9el-qaa plain (35 km from el-tur city); 10wadi kid, rahila road (nabq protected area); 11wadi kid (nabq protected area); 12wadi kid, khashm el-fakh (nabq protected area); 13wadi el sokhen (abu galum protected area); 14wadi mamlah (abu galum protected area); 15wadi el rasassa (abu galum protected area); 16km 101, cairo-suez road; 17km 75, cairo-suez road; 18west of al-gaar lake (wady el natrun); 19east of um-risha lake (wady el natrun). u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:35 color profile: disabled composite 150 lpi at 45 degrees mination of k and na. phosphorous was determined using the ascorbic acid method according to eaton et al. (1995). the significance of variation in the soil parameters, chemical constituents and nutritive variables of the plant materials in relation to habitat and to the phytogeographical regions was assessed using one-way anova test. simple linear correlation analysis was applied to detect the relationship between the chemical composition and nutritive variables of the studied populations and the soil parameters of the sampling sites to investigate the influence of soil characteristics on the plant chemical composition and hence on its nutritive values. two multivariate analysis techniques (classification and ordination) were conducted on the data of the chemical composition and the nutritive variables of the plant materials of the studied populations. one way-anova test was applied for the soil parameters of the resulting groups from cluster analysis. the significance of variation in chemical composition and nutritive variables among the cultivated populations were assessed using one-way anova test. all the statistical analyses were conducted with the minitab 13.1 release-pc computer program (minitab 2000). results habitat characteristics the soil characteristics of the studied habitats and the phytogeographical regions from which p. turgidum populations were collected showed variation among the different habitats and the phytogeographical regions (fig. 2, 3). the soil of sampling sites was generally alkaline with ph ranging from 7.71 to 8.22. the phvalue exhibited significant variation between habitats while it showed insignificant variation between phytogeographical regions (tab. 1). the soil texture varied from loamy sand to sandy. calcium carbonate content, sand, silt and clay percentages showed significant variation between different habitats and also between phytogeographical regions. potassium content showed significant variation between habitats and insignificant variation between phytogeographical regions. bicarbonate and phosphorus content of the soil showed significant variation between habitats and also between phytogeographical regions. generally, all the studied soil parameters showed significant difference between the studied habitats and also between the phytogeographical regions from which p. turgidum was collected, with the exception of the ph and the potassium content, which did not show any significant variation between the phytogeographical regions. 130 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. tab. 1. one way-anova testing the significance of variation in soil characteristics among the phytogeographical regions and among the investigated habitats from which p. turgidum was sampled. · significant p < 0.05; ·· very significant p < 0.01; and ··· highly significant p < 0.001 ph (%) mg/ 100g caco3 sand silt clay k hco3 p phytogeographical regions 3.21 4.69 · 8.49 ··· 6.75 ·· 6.45 ·· 3.01 5.16 · 4.99 · habitats 3.92 · 29.02 ··· 6.01 ·· 4.90 · 9.96 ··· 3.86 · 11.71 ··· 16.24 ··· u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:35 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 131 panicum turgidum in the egyptian desert fig. 2. the variation in soil parameters between the studied habitats (cp: coastal plain; gd: gravel desert; sd: sandy dunes; sp: sandy plain; and wb: wadi bed) of the studied p. turgidum populations. u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:35 color profile: disabled composite 150 lpi at 45 degrees 132 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. fig. 3. the variation in soil parameters between the studied phytogeographical regions (ns: north sinai; ss: south sinai; ed: eastern desert and wn: wady el-natrun) from which p. turgidum populations was sampled. u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 133 panicum turgidum in the egyptian desert tab. 2. description of the sampling sites from which p. turgidum populations were collected, the relative accessibility of the above-ground parts and the relative abundance of p. turgidum in each site (*cc= very common, c; common). s it e r e g io n h a b it a t s o il te x tu r e p a st u r e c o n d it io n g r a z in g p r e ss u r e r e la ti v e a c c e ss ib il it y (% ) * r e la ti v e a b u n d a n c e 1 n o r th s in a i sandy plain, with deep very fine sand deposits sandy good high 60–89 c 2 sandy plain, with deep very fine sand deposits sandy good high 90–100 c 3 sandy plain, with very deep sand deposits sandy good high 60–89 c 4 sandy plain, with very deep sand deposits sandy poor high 60–89 cc 5 sand dune sandy fair moderate 60–89 cc 6 sand dune sandy fair low 40–59 cc 7 sand dune sandy fair low 40–59 cc 8 s o u th s in a i coastal plain, drainage lines of the plain covered with alluvial deposits loamy sand good moderate 90–100 c 9 coastal plain, drainage lines of the plain covered with alluvial deposits sandy good high 60–89 cc 10 wadi bed, drainage lines of the wadi covered with alluvial deposits loamy sand fair moderate 90–100 cc 11 wadi bed, mouth of the wadi covered with alluvial deposits sandy good high 90–100 cc 12 wadi bed, drainage lines of the wadi covered with alluvial deposits sandy good high 60–89 cc 13 wadi bed, mouth of the wadi covered with alluvial coarse deposits mainly boulders and gravels loamy sand fair high 60–89 cc 14 wadi bed, mouth of the wadi covered with alluvial coarse deposits mainly boulders and fine gravels sandy fair high 60–89 cc 15 wadi bed, mouth of the wadi covered with alluvial coarse deposits mainly boulders and gravels loamy sand fair moderate 90–100 cc 16 e a st e r n d e se r t gravel and sand formations, drainage lines of the gravel desert covered with fine deposits sandy fair moderate 60–89 cc 17 gravel and sand formations, drainage lines of the gravel desert covered with fine deposits loamy sand fair low 60–89 c 18 w a d y e l n a tr u n sandy plain, with fine deep sand deposits sandy fair low 40–59 cc 19 sandy plain, with fine deep sand deposits sandy good low 60–89 cc u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees grazing status and pasture condition generally, pasture conditions can be classified as poor, fair and good. about 42.1% of the studied sites (1, 2, 3, 8, 9, 11, 12, and 19) are considered good pastures. at the same time 52.6% of the studied sites (5, 6, 7, 10, 13, 14, 15, 16, 17 and 18) were fair in pasture condition, while site 4 had a poor pasture condition. grazing pressure in the studied populations ranged from low to high (tab. 2). about 47.4% of the studied sites (1, 2, 3, 4, 9, 11, 12, 13 and 14) were subjected to high grazing pressure (tab. 2), while 26.3% of the studied sites (5, 8, 10, 15 and 16) were subjected to moderate grazing pressure. the remaining 26.3% of the studied sites (6, 7, 17, 18 and 19) were subjected to low grazing pressure. the relative accessibility of the aboveground parts of p. turgidum for grazing (by which we mean the ratio of the green to the dry old shoots in each plant bush) ranged from 60 to 89% in most of the sampling sites (1, 3, 4, 5, 9, 12, 13, 14, 16, 17 and 19). the highest relative accessibility (>90%) was recorded in sites number 2, 8, 10, 11 and 15, while sites number 6, 7 and 18 acquired the lowest values (40–59%). the coastal plain, the water runnels of the wadi bed and the gravel desert showed a higher percentage of accessible parts than the sandy plain and sand dunes habitats. the relative abundance of the studied species in all the examined sampling sites was very common in 73.7% of the studied sites and common in the rest of the populations. the relative accessibility of the aboveground parts of the plant was high in moderately and highly grazed sites, while it was low in areas with low grazing pressure. chemical composition of the studied populations the variation of the different nutrients in the grazeable parts of p. turgidum populations between the investigated habitats and phytogeographical regions in nutrients content of the studied populations were insignificant except for potassium content, which exhibited significant variation in the different phytogeographical regions (tab. 3). it is obvious that populations of p. turgidum collected from eastern desert region attained a maximum mean sodium and potassium contents (1.04, 1.32% respectively), while those collected from north sinai region attained maximum mean calcium, magnesium and phosphorus contents (1.37, 0.91 and 0.29% respectively). populations collected from south sinai region attained the maximum mean ca/mg and ca/p ratios (1.70 and 5.71 respectively; fig. 4). generally, the studied populations collected from the eastern desert region attained the highest mean values of ether extract and crude protein content (2.7 and 7.6% respectively; fig. 4). north sinai populations attained the highest mean values of ash and crude fiber content (9.6 and 37.5% respectively). on the other hand, the studied p. turgidum populations collected from wady el-natrun region attained the highest mean values of nitrogen free extract (46.4%). a highly significant variation in the total carbohydrates content (nfe) was established among the phytogeographical regions, while the variation in crude fiber and in crude protein content in the different habitats was significant (tab. 3). the variation in the ash and fat content of the grazeable parts of the studied p. turgidum populations is not significant among either the habitats or the phytogeographical regions. nutritive value assessment of the studied population in general, eastern desert populations showed the maximum mean of digestible crude protein content (3.53%; fig. 4). the studied p. turgidum populations of wady el-natrun re134 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 135 panicum turgidum in the egyptian desert fig. 4. the variation in the nutrients in mg/g, ash (%), ca/mg, ca/p ratio, metabolic constituents (ee: ether extract (%), cf: crude fiber (%), cp: crude protein (%), nfe: nitrogen free extract (%) and om: total organic matter (%) and nutritive variables (dcp: digestible crude protein (%), tdn: total digestible nutrients (%), ge: gross energy (mcal kg–1 dm), de: digestible energy (mcal kg–1 dm), me: metabolized energy (mcal kg–1 dm), and ne: net energy (mcal kg–1 dm) among (a) the studied habitats (sp: sandy plain; sd: sandy dunes; cp: coastal plain; wb: wadi bed; and gd: gravel desert) and (b) the studied phytogeographical regions (ns: north sinai; ss: south sinai; ed: eastern desert and wn: wady el-natrun). u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees gion had the maximum of mean total digestible nutrients (64.91%), gross energy (4.27 mcal kg –1 dm), digestible energy (2.85 mcal kg –1 dm), metabolized energy (2.44 mcal kg –1 dm), and net energy (1.22 mcal kg –1 dm). the digestible crude protein showed significant variation among the studied habitats (tab. 3). the correlation analysis between the chemical compositions, nutritive variables of the studied wild populations of p. turgidum and the soil parameters of the sampling sites from which these populations were collected (tab. 8) reveals significant negative relationship between the bicarbonate content of the soil, the digestible crude protein content and the crude protein content of the plant. a highly significant negative relationship existed between the crude fiber content of the studied population and the calcium carbonate content of the soil. the ca/mg ratio of the studied population showed a significant negative relationship with the ph of the soil, while exhibiting a significant positive relationship with magnesium and potassium content of the soil. the ca/p ratio of the studied populations exhibited a significant positive relationship with the organic matter and the silt content of soil. the digestible energy and the metabolized energy of the studied populations exhibited a significant positive relationship with the phosphorous content of the soil. 136 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. tab. 3. one way-anova testing the significance of variation in the content of nutrients, ca/mg, ca/p ratios, ash, ether extract (ee), crude fiber (cf), crude protein (cp), nitrogen free extract (nfe), total organic matter (om), digestible crude protein (dcp), total digestible nutrients (tdn), gross energy (ge), digestible energy (de), metabolized energy (me) and net energy (ne) in the aboveground parts of p. turgidum among the phytogeographical regions and among the investigated habitats from which p. turgidum was sampled. · significant, p < 0.05; ·· very significant, p < 0.01; and ··· highly significant, p < 0.001. parameter phytogeographical regions habitats m g g – 1 na 0.65 0,09 k 3.81 · 0.42 ca 0.89 0.21 mg 0.57 0.47 p 2.34 1.61 ca/mg 0.22 0.48 ca/p 0.61 0.67 % ash 1.94 0.64 ee 0.81 0.26 cf 2.58 6.82 ·· cp 2.11 3.60 · nfe 7.38 ·· 1.92 om 1.95 0.58 dcp 2.11 3.60 · tdn 1.93 1.41 m c a l k g – 1 ge 1.35 0.38 de 1.99 1.63 me 1.99 1.63 ne 1.8 1.26 u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees discussion the variation in the accessible aboveground parts of p. turgidum among the different sites with different grazing levels may be attributed to the variation in the physical configuration of the plant (the ratio between the green to dry old shoots) in the highly grazed sites compared to low-grazed sites. the plant bushes in the low-grazed sites were characterized by being very tangled, with large accumulations of dry old branches. this is in agreement with valentine (1990). he indicated that some of the positive effects of grazing are the increase in nutritive value of grasses by increasing the ratio of new growth: old growth, removal or prevention of thatch buildup and reduction of the accumulation of dry old shoots that may stunt new growth. in the present study, the forage value of p. turgidum was evaluated according to its chemical composition. according to le houérou (1980) the forage value of the consumed plant is the result of its nutritive value, i.e. chemical composition and digestibility. it is highly affected by the stage of maturity, edaphic influence; climate and range condition. in addition, the nutritive value of any forage is dependent upon its content of energy-producing nutrients as well as its contents of physiologically essential nutrients (shaltout and el-beheiry 1997). the concentration of nutrients varied from population to another, which may be attributed to the variation in the edaphic and climatic factors. the results of the present study showed that the ca/p ratio of the grazeable parts of p. turgidum was significantly related to the soil silt and organic matter content (tab. 8). on other hand, ca/mg ratio was significantly related to soil magnesium, potassium content and soil reaction (ph). this agrees with the reported accumulation of nutrients in the grazeable phytomass of a species that may differ considerably from one habitat to other (abdel-razik et al. 1988b). it is worth noting that the ca/mg ratio in the grazeable parts of p. turgidum in the present study was comparable to the mean of the natural forage in the western mediterranean coastal rangelands (tab. 4). however, according to boudet (1975) the na, k, ca, mg and p contents of p. turgidum meet the level required for the maintenance of animals. in addition, acta bot. croat. 68 (1), 2009 137 panicum turgidum in the egyptian desert tab. 4. nutrients contents (%), ca/mg and ca/p ratios of the studied wild p. turgidum populations compared to other related studies, to the maximum tolerable level and to the maintenance level of these nutrients required for animal feed. panicum turgidum determined by (%) ca/p ca/mg na k ca mg p mean of the studied wild populations present study 0.92 1.14 1.15 0.76 0.24 5.07 1.57 wadi eithely (eastern desert) kabiel (2001) 0.31 0.69 0.78 0.26 0.25 – – western mediterranean coastal rangelands (el omayed) abdel-razik et al. (1988b) 0.87 1.68 2.95 2.23 0.09 32.6 1.34 maintenance levels boudet (1975) 0.08 0.4 0.2 0.2 0.12 – – maximum tolerable level nrc (1984) 10 3 2 4 1 – – u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees according to nrc (1984) the p. turgidum na, k, ca and p content does not exceed the maximum tolerable level (tab. 4). comparing the mean of the composition of nutrients of p. turgidum in all the studied wild populations with the mean of natural forage in the western mediterranean coastal rangeland – the most important rangelands in egypt according to el-kady (1987) – showed that the concentration of k, ca, and mg is lower than that of the mean of natural forage in the western mediterranean coastal rangeland (tab. 4). however, the phosphorus content is much higher than that of the mean of natural forage in the western mediterranean coastal rangeland. p. turgidum is characterized by the association of mycorrhizae (vesicular-arbuscular type) with its roots as stated by khaliel (1989) and srivastava et al. (1997), which may explain the high content of phosphorous in p. turgidum. it was asserted by penning de vries et al. (1980) that if too little p is available, the n absorption, and hence production are reduced. this demonstrates the importance of introducing p. turgidum in the western mediterranean coastal rangelands to compensate for the deficiency in phosphorous, the only element in which the forage of this region is deficient according to abdel-razik et al. (1988a). the importance of the adequate ca/p ratio of 2–3 is considered by ayyad and le floc’h (1983) to be a major factor affecting the utilization of the whole animal diet. in addition, the high ca/p ratio leads to lower utilization of both ca and p. according to abdel-razik et al. (1988b) the mean ca/p ratio of the natural forage in the western mediterranean coastal rangelands is notably high (32.6). in the present study, ca/p ratio ranged from 3.36 in p. turgidum populations collected from wady el-natrun to 5.71 in those of south sinai; this low ca/p ratio gives a further reason for the introduction of p. turgidum as a natural forage plant in the western mediterranean region. crude protein (cp) and crude fibers (cf) are viewed classically as an indicator of the nutritional value of food for grazing animals (bryant and kuropat 1983, heneidy 2002). the percentage of crude protein in the grazeable parts of the studied wild p. turgidum populations is higher than the minimum (6% of forage dry matter) required for the maintenance of animals, if compared with many tropical grasses (tab. 5). the crude fiber (cf) content of the grazeable parts of the studied p. turgidum populations was quite high. it ranged from 34.8% in eastern desert to 37.5% in north sinai (fig. 4). the mean value of the studied wild populations (36.7%) was higher than that obtained by kabiel (2001), but lower than that obtained by farooq et al. (1989) and heneidy (1996). the importance of lipids (ether extract or ee) to plants in terms of structure and use in metabolism is well known. 138 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. tab. 5. ash, ether extract (ee), crude protein (cp), crude fiber (cf) and nitrogen free extract (nfe) contents (%) of the studied wild p. turgidum populations compared to other related studies and to cultivated p. turgidum. panicum turgidum determined by (%) ash ee cp cf nfe mean of the studied wild populations present study 8.8 2.5 6.7 36.7 45.2 mean of cultivated populations 7.7 3.7 9.2 31.4 48.1 peshawar (pakistan) farooq et al. (1989) 12.2 2.2 6.5 37.4 37.4 gulf of aqaba (sinai) heneidy (1996) 9.1 5.3 6.6 39.9 39.4 wadi eithely kabiel (2001) 12.1 2.3 4.8 35.1 45.7 u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees the development of mankind has reached the point at which a variety of new resources need to be tapped in order to fill the basic needs for food and feed, especially in the developing countries (lieth 2002). information on the seeds of wild species and wild relatives of cultivated crops is scarce but with the increasing interest in new food sources and the use of genetic diversity in breeding programs, the seeds of wild plants are now receiving more attention (iriondo and pérez 1997). wild plants have provided a safety net for human beings from time immemorial, whenever crop harvests were poor. they are consumed daily and traded at markets through special rural-urban marketing channels. in addition, some of the wild plants play an important food security role in the time of famine (e.g. cenchrus biflorus and p. turgidum). the results of this study indicated that p. turgidum grains are characterized by high nutritive value comparable to that of the common cereals (corn, wheat and barley, tab. 6) and other cultivated grain-producing grasses. many scientists (irvine 1952, williams and farias 1972, harlan et al. 1989) reported that the local inhabitants of the western sahara use and eat the grains of p. turgidum, grinding the grains into flour, using it in making porridges and baking it into bread. grain shattering is one of the main problems, resulting in low yield. generally, in many grasses the cleaning of the grains is necessary to obtain adequate grain quality. unfortunately grains of most species of tropical pasture grasses are characterized by being very difficult to thresh and to clean because of the strong attachment of palea and lemma to the caryopsis (e.g. stipagrostis plumose, lasuirus sindicus and cenchrus ciliaris). this makes these species difficult to domesticate (le houérou 1985). however, it was noticed that p. turgidum grains are very easy to thresh and to clean. accordingly, p. turgidum grains could be considered as a potential crop, and it may serve as a supplementary food, alongside the common cereals. this might help to alleviate the current food gap in the arid regions. the quality of forage can be expressed in several parameters, such as total digestible nutrients, digestible crude protein and caloric value (duivenbooden 1985). the total digestible nutrients (tdn) is an appropriate measure of the food energy available to animals only after the digestion losses have been deducted (lofgreen 1951). therefore, tdn is a measure of energy requirement of animals and energy value of feeds (heneidy 2002). the mean value of tdn (64.03%; tab. 7) of the grazeable parts of the studied wild p. turgidum acta bot. croat. 68 (1), 2009 139 panicum turgidum in the egyptian desert tab. 6. ash, crude protein (cp), fat (ee) contents (%) and gross energy (ge) in mcal kg –1 dm of p. turgidum grains compared to some common cereals and to grain-producing species. species determined by (%) (mcal/kgdm) ash cp ee ge panicum turgidum present study 18,1 10,92 7,31 4,01 panicum antidotale kearl et al. (1979) 12,3 10,9 1,9 – panicum miliaceum 7,2 10,4 3,2 – echinochloa crusgalli 19,7 7,6 2,2 – zea mays 3,21 8,06 13,84 4,94 triticum vulgare 4,73 9,5 8,69 4,63 hordeum vulgare 4,38 9,92 2,02 4,13 u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees populations was lower than that obtained by heneidy (1996). however, it meets the requirements for sheep diet (61.7%; nrc 1975) and breeding cattle diet (50.0%; nrc 1984). and it is higher than that of the common fodder crops (barseem (clover) and barley, tab. 7) recorded by soliman and el-shazly (1978). the tdn value of p. turgidum in the four studied phytogeographical regions can be considered fair for grazing animals compared with common fodder crops. the feeding value of forage depends primarily on the magnitude of its contribution to the energy need of an animal. difference in this respect between forages is almost completely a consequence of the relative amounts in which they are voluntarily consumed (petrusewicz 1976). energy contained in food is called gross energy (ge), energy intake or consumption. after subtracting the caloric value of the feces (f) 140 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. tab. 7. the nutritive variables (dcp: digestible crude protein, tdn: total digestible nutrients (%), ge: gross energy, de: digestible energy and ne: net energy in mj kg –1 dm) of the studied wild p. turgidum populations compared to other related studies, rangelands, common fodder crops and to cultivated p. turgidum. panicum turgidum determined by (%) (mj kg –1 dm) dcp tdn ge de ne mean of the studied wild populations present study 2.73 64.03 17.6 11.79 5.02 mean of cultivated populations 5.05 63.48 18.06 11.7 4.97 gulf of aqaba (sinai) heneidy (1996) 2.65 65.67 17.51 11.83 5.04 rangelands (western mediterranean coastal region) el omayed abdel-razik et al. (1988a) 5.4 75 – – – 4.9 72 15.09 10.91 4.77 common fodder crops soliman and el-shazly (1978) barseem – 56 – – – barley – 64 – – – corn – 68 – – – tab. 8. simple correlation coefficient between the soil parameters, the chemical composition (ca/p, ca/mg, cf: % crude fiber and cp: % crude protein) and the nutritive variables (dcp: digestible crude protein, de: digestible energy and me: metabolized energy) of the aboveground parts of the studied wild p. turgidum populations; · significant, p < 0.05; ·· very significant, p < 0.01; and ··· highly significant, p < 0.001. plant materials soil parameters ph om caco3 silt mg k hco3 p chemical composition ca/p –0.31 0.50 · –0.08 0.47 · 0.24 0.4 0.34 0.34 ca/mg –0.5 · 0.15 –0.06 0.02 0.45 · 0.45 · 0.17 0.27 cf 0.09 –0.03 –0.74 ·· –0.27 0.19 0.1 0.2 0.23 cp 0.3 –0.24 0.4 0.04 –0.41 –0.4 –0.51 · –0.4 nutritive variables dcp 0.3 –0.24 0.4 0.04 –0.41 –0.4 –0.51 · –0.4 de –0.09 0.15 –0.26 0.07 0.3 0.33 0.29 0.46 · me –0.09 0.15 –0.26 0.07 0.3 0.33 0.29 0.46 · u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:36 color profile: disabled composite 150 lpi at 45 degrees from the food energy and the losses through urine and methane, the reminder is called metabolized energy (me) or assimilation energy (drodz 1975). the mean of gross energy (ge), digestible energy (de) and the metabolized energy (me) of the studied wild populations of p. turgidum (tab. 7) was higher than mean of the natural forage in the western mediterranean coastal rangelands (heneidy not published). the principal components analysis (pca) applied to the chemical composition and nutritive variables data of the studied p. turgidum populations showed that the first three vectors accounted for 92.7% of the total variance (14.98). the contribution of the investigated variables in the variation accounted for by the first three vectors was limited to a number of variables including ash, crude protein, carbohydrate contents (nfe), total digestible nutrients (tdn) and ca/p ratio content of the plant materials (tab. 9). pc1 expressed the greatest proportion of variation (42.8% of the total variance) with an eigenvalue of 6.41. it was related to ash, carbohydrate contents, organic matter, total digestible nutrients and ca/p ratio content of the plant materials (tab. 9). pc2 accounted for 32.8% of the variation with an eigenvalue of 4.91. it was related to crude protein, carbohydrate contents and ca/p ratio content of the plant materials. pc3 accounted for 17.1% of the variation with an eigenvalue of 2.56. this pc corresponded to the crude protein, total carbohydrates, total digestible nutrients and ca/p ratio content of the plant materials. when these three vectors were used as axes for the scatter diagram (fig. 5) of the examined populations the same grouping was obtained as that of the cluster analysis of the p. turgidum studied populations using the chemical composition and nutritive variables data exemplified by the dendrogram resulting from the complete linkage method of sorting (fig. 6). the studied populations were acta bot. croat. 68 (1), 2009 141 panicum turgidum in the egyptian desert tab. 9. the loadings of the chemical composition and nutritive variables of the studied wild p. turgidum populations on the first three eigenvectors of the pca analysis (*values of the character with major contribution in the three vectors). character eigen vectors pc1 pc2 pc3 ash *–0.50 0.2 –0.21 cf 0.02 –0.02 –0.02 cp 0.05 *0.29 *0.74 ee 0.04 –0.036 –0.12 nfe *0.39 *–0.43 *–0.39 om *0.50 –0.2 *0.21 tdn *0.34 –0.1 *0.25 dcp 0.02 0 0.01 ge 0.02 –0.01 0.01 de 0.04 –0.03 –0.11 me 0.02 0 0.01 ne 0.01 0 0.01 nr 0 0 0 ca/mg 0.02 0 0.02 ca/p *0.48 *0.80 *–0.36 u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:37 color profile: disabled composite 150 lpi at 45 degrees separated into two main groups »i« and »ii« at 0.97 dissimilarity distance. group »i« included populations 2, 3, 4, 5, 6, 7, 8, 9, 11, 16, 17, 18 and 19, while group »ii« included populations 1, 10, 12, 13, 14 and 15. group »i« was split into two subgroups »a« and »b« at 0.81 dissimilarity distance. subgroup »a« included populations 2, 3, 4, 5 and 6, while subgroup »b« included populations 7, 8, 9, 11, 16, 17, 18 and 19. the results obtained from applying both classification and ordination numerical analyses separated the studied wild p. turgidum populations into two major groups (i and ii) on the basis of eight characters (the ash, crude fiber, crude protein, total carbohydrates, organic matter and digestible crude protein content and ca/p ratio). while group »ii« included six populations (five populations 142 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. fig. 5. three-dimensional scatter plot of the first three eigenvectors of the pca analysis based on data of chemical composition and nutritive variables of the studied wild p. turgidum populations. the solid line enclosed the main groups (i and ii). the dashed line enclosed the two subgroups (a and b) differentiated within group i. fig. 6. dendrogram resulted from application of complete linkage method of sorting, using chemical composition and nutritive variables of the studied wild p. turgidum populations. u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:37 color profile: disabled composite 150 lpi at 45 degrees collected from south sinai-gulf of aqaba coastal plain and one population from north sinai) characterized by the lowest ash content, highest crude fiber, organic matter, total digestible nutrients content. group »i« comprised the rest of the studied populations with relatively high nutritive value (higher protein, fat, carbohydrates and mineral content together with lower fiber content). group »i« was further subdivided into two subgroups (»a« and »b«). subgroup »b« was distinguished by having the highest values of carbohydrates, crude protein, digestible crude protein and energy content. moreover it is characterized by low crude fiber contents and ca/p ratio. this group included eight populations collected from wady el-natrun, eastern desert, north sinai and south sinai. it is evident that subgroup »b« comprised populations posses the best nutritive characters concerning low fiber content, high crude protein, carbohydrates and ash content. the variation in the nutritive value among the studied populations may be attributed to the effect of some edaphic parameters. the variations in ec, organic matter content, calcium, potassium, bicarbonate, chloride, and phosphorous content of the soil were significant among the three groups (tab. 10). it worth noting that, the variation in the nutritive value among the cultivated populations was insignificant. all the cultivated populations acquired high nutritive value, which indicate the effect of edaphic factors exerted on the wild population nutritive value as was illustrated also from the correlation between some edaphic factors and some nutritive variable. despite the variation in the edaphic, climatic and other factors such as grazing pressure factors among the studied phytogeographical regions, there was slight variation in the chemical composition and the nutritive variables of the studied populations, especially in the content of the organic constituents. however this did not significantly affect the nutritive value of the plant across the different studied populations. these relatively low variaacta bot. croat. 68 (1), 2009 143 panicum turgidum in the egyptian desert tab. 10. one way-anova testing the significance of variation in the soil characteristics among the main groups resulting from the cluster analysis of the studied wild p. turgidum populations; ·significant, p < 0.05; ·· very significant, p < 0.01; and ··· highly significant, p < 0.001. soil characters f-value ec (mmohs cm –1 ) 4.5 · ph 2.1 (%) om 4.4 · sand 3.2 silt 2.9 clay 2.2 caco3 3 (mg/100g) ca 3.9 · mg 0.3 na 3.5 k 8.4 ·· hco3 7.3 ·· cl 4.0 · so4 1.0 p 11.2 ·· u:\acta botanica\acta-botan 1-09\heneidy.vp 16. travanj 2009 12:42:37 color profile: disabled composite 150 lpi at 45 degrees tions in the nutritive value among the investigated populations indicate the considerable plasticity of the studied species to the environmental conditions. however, a notable difference was recorded between the wild and cultivated p. turgidum populations, in the chemical composition and the nutritive variables. the cultivated populations attained higher levels in crude protein, fats, carbohydrates, digestible crude protein and lower crude fiber content than the wild populations (tab. 5 and 7). based on the value of dcp and net energy, considering the scale suggested by boudet and riviere (1968), p. turgidum has good or excellent forage quality. references abdel-razik, m., ayyad, m. a., heneidy, s. z., 1988a: preference of grazing mammals for forage species and their nutritive value in a mediterranean desert ecosystem (egypt). journal of arid environments 15, 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(eds.), microbial biotechnology. prof. k. s. bilgrami commemoration volume, 87–96. täckholm, v., 1974: student’s flora of egypt. cairo university press. valentine, j. f., 1990: grazing management. academic press inc., san diego. williams, j. t., farias, r. m., 1972: utilization and taxonomy of the desert grass panicum turgidum. economic botany 26, 13–20. 146 acta bot. croat. 68 (1), 2009 heneidy s. z., halmy m. w. u:\acta botanica\acta-botan 1-09\heneidy.vp 22. travanj 2009 12:29:52 color profile: disabled composite 150 lpi at 45 degrees london. 544 vizintin et al.vp issn 0365–0588 eissn 1847-8476 host range and host choice of cuscuta species in hungary kornél baráth*, jános csiky department of plant systematics and geobotany, institute of biology, faculty of sciences, university of pécs, h-7624 pécs, ifjuság str. 6, hungary abstract – extensive field studies were carried out in hungary to get a picture of the host range and host choice of the cuscuta species under natural conditions. we examined both parasitised and unparasitised plant species and found some aspects in which they are different. compiling the host spectra of the various cuscuta species based on herbaria, literature and our own observations, we can say that dodders infest at least 26% of the vascular flora of the country. in our study, the hungarian cuscuta species parasitised all plants that had a coverage of more than 25% in the sampling sites. we prepared a list of the most frequent host species for the parasites and revealed the importance of exclusive hosts. the results suggest that the habitat differences of the cuscuta species can be responsible for the different host ranges. furthermore, it was found that the reason why dodders parasitise plants from various life-forms in different proportions is not (only) the active host choice, but the characteristic features of the habitats. key words: cuscuta, parasitic plant, host, habitat introduction approximately 3900 parasitic plant species are known in the world, which is more than 1% of flowering plants (nickrent 2002). the genus cuscuta (dodders) comprises approximately 170 parasitic plant species widely distributed in tropical, subtropical and temperate regions (dawson et al. 1994). hitherto eight cuscuta species have been reported from hungary: cuscuta campestris yuncker, c. epithymum (l.) nath. and c. europaea l. are considered quite frequent, while c. lupuliformis krock. and c. australis r. br. are rare parasites in the country. cuscuta approximata bab. is an extremely rare species, known only from one locality in south hungary (csiky 2003). baráth (2004) reported that c. epilinum weihe probably became extinct in the country in the 1920s. c. suaveolens ser. was only a temporary element of the hungarian flora about 100 years ago (balogh et al. 2004). acta bot. croat. 71 (2), 2012 215 * corresponding author, e-mail: barikori@yahoo.com copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. acta bot. croat. 71(2), 215–227, 2012 coden: abcra 25 although several cuscuta species are dangerous agricultural pests (dawson et al. 1994), their host range and host choice have not been sufficiently investigated. the majority of the references regarding the host spectra of dodders concern mainly cultivated plants, while most observations in natural or seminatural habitats are accidental supplements of other (taxonomical, physiological) studies on cuscuta (gaertner 1950). in the past it was believed that each cuscuta species is host-specific and able to infest only one species (cf. dean 1934, gaertner 1950). the names of some dodders (c. epilinum weihe., c. epithymum (l.) nath., c. polygonorum engelm., c. cephalanti engelm. etc.) refer to the species or genera of the host on which they were found (cf. gaertner 1950, erd�s 1971). later, when it was realised that dodders can parasitise several species, and that even most of them have considerable host ranges, this conception changed (engelmann 1843, soó 1968). engelmann (1859) and yuncker (1932) listed the prominent host species or genera for many dodders in their monograph and reported some cuscuta species that are able to grow on any host(s) within reach. mirande (1900) divided the cuscuta species into two classes based on the size of their host range. the distinction of the categories can be found in recent studies as well, under the names of host-generalist and host-specialist or host-specific. although engelmann (1859), hildebrand (1908), thompson (1911) and degen (1911) examined the host species of dodders, the first extensive host index was prepared by gertz (1928, 1933) for c. europaea. dean (1934, 1935) systematically investigated the host range of dodders in the united states and compiled a substantial host list for c. gronovii willd. ex schult. and c. glomerata choisy. gaertner (1950) realised that knowledge of the host species of dodders can be important for agronomy, plant pathology and plant taxonomy, and reviewed the literature on the host range of cuscuta species. she compiled an extensive host list for 9 cuscuta species based on literature and her own observations. later erd�s (1971), kuoh and chiang (1989), koji] and vrbni^anin (2000), jayasinghe et al. (2004), liao et al. (2005), sarma et al. (2008) and baráth (2004, 2009, 2010) also systematically investigated the host range of dodders, but all of them excluded unparasitised plants from consideration and did not discuss the question of host choice. krohn (1934) realised the importance of unparasitised plants and reported that dodders are able to grow in close proximity to several plants without parasitising them. musselman (1986) investigated the genus cuscuta in virginia and stated that each species may be characterised by what it does not attack. although some excellent laboratory experiments focused on the active host choice of some dodders (kelly 1992, sanders et al. 1993, alers-garcía 2005, runyon et al. 2006), our knowledge about the underlying mechanisms of the host selection under natural conditions is quite insufficient. csiky et al. (2004) supposed that the hungarian cuscuta species are not host specific. in the cases of c. epithymum and c. campestris they found that the number of host species was positively correlated with the size of the parasites. in this study, we wanted to test the unspecific hypothesis with two other methods. firstly, if a dodder species indiscriminately infests the surrounding plants, then the greater the species diversity is, the more species are parasitised by cuscuta. secondly, if a dodder is not host specific then the frequency of the host species and the frequency of their infestations must be correlated. the purpose of the present study was also to explore the qualitative and quantitative differences of the host ranges of cuscuta species in hungary and reveal some aspects in which the parasitised and unparasitised plant species are different. 216 acta bot. croat. 71 (2), 2012 baráth k., csiky, j. materials and methods the study was carried out on the recent cuscuta species (c. europaea, c. campestris, c. epithymum, c. lupuliformis, c. australis, c. approximata) between 2003–2009 at 186 localities in hungary (fig. 1). both parasitised and unparasitised plants were examined in the habitats using altogether 407 representative quadrats. at most two relevés were taken at the same locality, but even in these cases at least 50% of the plant species were different from each other. in the cases of the rare species (cuscuta approximata, c. australis, c. lupuliformis) relevés were taken in the location of each known population. the percentage cover and the parasitism status were recorded for each plant species in the quadrats. only those plants were considered hosts (parasitised plants) on which the dodder haustorium was found. in doubtful cases (often on grasses) the haustorium was cut and the penetration was checked with a magnifier or, rarely, using a stereo microscope. since different dodder species occur in dissimilar vegetation types in hungary (csiky et al. 2004) and the size of the species also varies (baráth 2004) the size of the sampling quadrats has been defined separately for each cuscuta species in compliance with its habitat (lájer et al. 2007). the parasites were smaller than the sampling quadrat in every case. altogether 116 relevés of 5m x 5m size were taken for c. europaea, 50 relevés of 5m x 5m size for cuscuta lupuliformis, 122 relevés of 2m x 2m size for c. campestris, 87 relevés of 2m x 2m size for c. epithymum, 29 relevés of 1m x 1m size for c australis, and 3 relevés of 1m x 1m size in the only habitat of c. approximata. despite the different sizes, the sampling quadrats faithfully represent the different habitats of the various cuscuta species; consequently, they are comparable with each other in many aspects (hortobágyi and simon 2000, otypková and chytry 2006, lájer et al. 2007). acta bot. croat. 71 (2), 2012 217 host range and choice of cuscuta fig. 1. location of the sampling quadrats in hungary according to the grid of the central european mapping system. species were considered exclusive hosts of a given dodder if they were never found to be parasitised by other cuscuta species. the overlapping of the host ranges of dodders was calculated as jaccard coefficient: n n n ab a b� �100 where nab is the number of the species that were parasitised by both cuscuta a and b. na is the number of the host species of cuscuta a, while nb is the same for cuscuta b. the habitat similarities were computed in the same way based on the overlapping of all plant species of the habitats. the homogeneity between the proportions of the overlapping of the host ranges and of the overlapping of the total plants species was tested by pearson’s chi-square test. the qualitative differences between the host ranges of the various cuscuta species were investigated according to the raunkiaer life-form system (raunkiaer 1934, horváth et al. 1995). the chi-square test was used to compare the distribution of the life-forms of the host species and of the total plant species. the differences between the constancy and mean covering values of the parasitised and unparasitised plant species were investigated separately in the case of each cuscuta species using the brunner-munzel test. the degree of correspondence between two variables was measured in every case by kendall’s t rank correlation coefficient. the brunner-munzel test was performed with ropstat (vargha 2008), while other statistical analyses (kendall’s t and chi-square test) were performed with the past statistics software package (hammer et al. 2001). only 3 relevés of c. approximata were not evaluated by statistical methods. the host spectra of the hungarian cuscuta species was compiled based on our field studies, on data of several herbaria [herbarium carpato-pannonicum of the natural history museum (bp), university of pécs (jpu), university of debrecen (de), janus pannonius museum (pecs), universitatis napocensis, cluj-napoca (cl)] and on the few literature sources concerning hungary (erd�s 1971, csiky 2003). during the herbarium studies we found host plants for c. epilinum, but in the case of c. suaveolens there were not any reliable data. results during the field study 459 plant species were found to be parasitised by the examined cuscuta species. since an additional 113 hosts were found in the course of the herbarium study and in the literature, it can be said that dodders parasitise at least 572 plant species in hungary, which is approximately 26% of the vascular flora of the country (simon 2000). cuscuta epithymum displayed the widest spectrum with 341 hosts, while c. approximata parasitised only 15 species (tab. 1). the host range of dodders can be characterised not only by size, but also by the proportion of the exclusive host species (tab. 1). for example, cuscuta europaea was found to parasitise at least 183 different plants in hungary, but 129 of them were attacked by other dodders as well and only 54 host species were infested solely by c. europaea. the proportion of the exclusive and the total host plants reflects the degree of the peculiarity of the host ranges. this value is 29.51% for cuscuta europaea, which is average among the hungarian dodders. regarding c. epithymum and c. approximata, the high proportions 218 acta bot. croat. 71 (2), 2012 baráth k., csiky, j. (58.36%, 46.67%) of exclusive hosts are remarkable, while the host range of c. lupuliformis and c. epilinum can be characterised by only a few exclusive host species (13.13%, 12.50%). the degree of the overlapping of the host ranges revealed both differences and similarities among the dodders (tab. 2). the species c. europaea and c. lupuliformis had the greatest proportion of the common hosts, since 66 species were parasitised by both dodders, which is 23.40% of the total host species of the parasites. the divergence among the species composition of the habitats (host availability) of dodders can be evaluated in the same way based on the overlapping of all plant species of the habitats. comparing the proportions, we found that the overlapping of the host species and that of the total plant species were almost the same (c2 = 0.711, p = 0.999, df = 11). the classification of the host species based on the raunkiaer life-form system also revealed dissimilarities among the host ranges of dodders (tab. 3). we observed also that the distribution of the life-forms was almost the same in the cases of the host species and of acta bot. croat. 71 (2), 2012 219 host range and choice of cuscuta tab. 1. the number of the host species and the proportions of the exclusive host species of the hungarian dodders. cuscuta europaea c. campestris c. epithymum c. lupuliformis c. australis c. approximata c. epilinum number of the host species (a) 183 224 341 99 72 15 16 number of the exclusive host species (b) 54 68 199 13 12 7 2 b/a x 100 (%) 29.51 30.36 58.36 13.13 16.67 46.67 12.50 tab. 2. overlapping of the host ranges of the hungarian cuscuta species. 572 c. europaea (183) c. campestris (224) c. epithymum (341) c. lupuliformis (99) c. australis (72) c. approximata (15) c. epilinum (16) c. europaea (183) 91 73 66 35 0 7 c. campestris (224) 22.36% 106 55 43 1 10 c. epithymum (341) 13.93% 18.76% 38 25 8 12 c. lupuliformis (99) 23.40% 17.03% 8.64% 37 0 3 c. australis (72) 13.73% 14.53% 6.05% 21.64% 0 2 c. approximata (15) 0 0.418% 2.25% 0 0 0 c. epilinum (16) 3.52% 4.17% 3.27% 1.74% 2.27% 0 the total plant species. (c2 = 0.959 with p = 0.999, df = 9 for cuscuta europaea, c2 = 0.228 with p = 0.999, df = 9 for c. campestris, c2 = 0.629 with p = 0.999, df = 9 for c. epithymum, c 2 = 2.149 with p = 0.988, df = 9 for c. lupuliformis and c2 = 0.559 with p = 0.999, df = 8 for c. australis). according to the results, hungarian cuscuta species parasitised all plants that had a coverage of more than 25% in the sampling sites. moreover we found strong positive correlation between the numbers of the host species and the number of the total plant species of the sampling quadrats (kendal tb value was t = 0.741 with p < 0.001 for c. europaea, t = 0.791 with p < 0.001 for c. campestris, t = 0.746 with p < 0.001 for c. epithymum, t = 0.623 with p < 0.001 for c. lupuliformis and t = 0.731 with < 0.001 for c. australis). the frequency of the species and the frequency of their infestations were also significantly correlated (kendal tb value was t = 0.709 with p < 0.001 for cuscuta europaea, t = 0.783 with p < 0.001 for c. campestris, t = 0.755 with p < 0.001 for c. epithymum, t = 0.577 with p < 0.001 for c. lupuliformis and t = 0.814 with p < 0.001 for c. australis). a substantial difference was found between the frequency of the parasitised and unparasitised plant species in the habitat (the values of the brunner-munzel test were calculated bm = 6.49 with p < 0.001 for c. europaea, bm = 7.07 with p < 0.001 for c. campestris, bm = 8.11 with p < 0.001 for c. epithymum, bm = 2.98 with p = 0.003 for c. lupuliformis and bm = 3.27 with p = 0.002 for c. australis). moreover, the differences between the mean cover of parasitised and unparasitised plants were also significant (bm = 5.73 with p < 0.001 for c. europaea, bm = 4.51 with p < 0.001 for c. campestris, bm = 8.98 with p < 0.001 for c. epithymum, bm = 9.76 with p < 0.001 for c. lupuliformis and bm = 3.17 with p = 0.003 for c. australis). we drew up the list of the ten most frequent host plants of each hungarian cuscuta species based on the frequency of infestation of the host species (tab. 4). however, it should not be forgotten that the more frequently parasitised host species are not necessarily the most preferred ones. 220 acta bot. croat. 71 (2), 2012 baráth k., csiky, j. tab. 3. distribution of the host plants of the hungarian dodders based on the raunkiaer life-form system (%). cuscuta europaea c. campestris c. epithymum c. lupuliformis c. australis c. approximata c. epilinum mesophanerophytes 5.78 2.14 1.47 11.63 3.33 0 0 microphanerophytes 5.78 2.86 2.44 10.08 3.33 0 0 nanophanerophytes 1.78 0.36 2.44 3.10 2.22 10.00 0 chamaephytes 3.56 3.93 8.56 3.88 3.33 15.00 4.76 hemikryptophytes 34.22 31.07 52.08 26.36 33.33 45.00 42.86 hemitherophytes 13.33 13.21 7.82 5.43 6.67 5.00 19.05 therophytes 23.56 37.50 18.83 19.38 28.89 10.00 28.57 geophytes 7.56 6.07 5.13 7.75 4.44 15.00 4.76 epiphytes 0.44 0.36 0.24 2.33 0 0 0 helo-. hidrophytes 4.00 2.50 0.98 10.08 14.44 0 0 discussion in agreement with some previous studies (csiky et al. 2004, baráth 2004), we observed a positive correlation between the number of the host species and the distribution frequency of the cuscuta species. drawing on six years of extensive field studies, we can say that c. epithymum and c. campestris are the most frequent cuscuta species in hungary and their host ranges are the widest (341 and 224 host species). cuscuta europaea is less common and its host spectrum is also smaller (183). cuscuta lupuliformis and c. australis are rare parasites, their host ranges comprising 99 and 72 different plants, respectively. acta bot. croat. 71 (2), 2012 221 host range and choice of cuscuta tab. 4. the most frequent host plants of the cuscuta species in hungary and the percentage frequency of their infestation. c. europaea c. campestris 85.3 % urtica dioica l. 68.0 % polygonum aviculare l. 40.6 % elymus repens (l.) gould. 37.7 % lolium perenne l. 37.9 % humulus lupulus l. 36.0 % convolvulus arvensis l. 31.0 % rubus caesius l. 36.0 % ambrosia artemisifolia l. 24.1 % arrhenatherum elatius (l.) presl 32.8 % chenopodium album l. 22.4 % galium aparine l. 23.8 % cichorium intybus l. 22.4 % lactuca serriola l. 21.3 % elymus repens (l.) gould. 21.5 % sambucus ebulus l. 20.4 % artemisia vulgaris l. 21.5 % melandrium album (mill.) garcke 19.6 % atriplex tatarica l. 18.9 % calystegia sepium (l.) r. br. 18.8 % tripleurospermum perforatum (mérat) laínz c. lupuliformis c. australis 58.0 % urtica dioica l. 72.4 % 2polygonum lapathifolium l. 52.0 % rubus caesius l. 72.4 % plantago major l. 46.0 % salix triandra l. 65.5 % bidens tripartita l. 40.0 % calystegia sepium (l.) r. br. 58.6 % chenopodium ficifolium sm. 40.0 % echinocystis lobata (michx.) torr. et gray 58.6 % myosoton aquaticum (l.) mönch 28.0 % elymus repens (l.) gould. 55.1 % chenopodium polyspermum l. 24.0 % lactuca serriola l. 41.3 % potentilla supina l. 20.0 % phragmites australis (cav.) trin. ex. steud. 34.5 % alopecurus aequalis sobol. 20.0 % galium aparine l. 34.5 % tanacetum vulgare l. 16.0 % aristolochia clematitis l. 27.6 % polygonum aviculare l. c. epithymum c. approximata 42.2 % achillea millefolium l. s.l. 100 % genista pilosa l. 40.2 % galium verum l. 66.7 % rumex acetosella l. 40.2 % arrhenatherum elatius (l.) presl 33.3 % luzula luzuloides (lam.) dandy et wilm. 35.6 % plantago lanceolata l. 33.3 % lotus corniculatus l. 24.1 % sanguisorba minor scop. 22.9 % festuca rupicola heuff. 21.8 % teucrium chamaedrys l. 21.8 % daucus carota l. 20.6 % convolvulus arvensis l. only 3 host species were found in the unique habitat of c. approximata, but another 12 parasitised plant species were recognised during the herbarium studies, so the host range of this dodder includes 15 species in hungary. despite the fact that c. epilinum had been considered a strict host specialist (phippen 1867, tóth and cagá� 2001, hamed 2005) we found 16 host species for this dodder during the herbarium studies. besides the frequency, the taxonomic complexity could be the reasons for the large host range of cuscuta epithymum. several authors (engelmann 1859, yuncker 1932, miguel and martín 2007 etc.) reported that this dodder is not a homogeneous species, while babington (1843), degen (1911), buia (1960) and soó (1968) separate some c. epithymum taxa as distinct species. according to the recent taxonomical books, two subspecies of c. epithymum occur in hungary (simon 2000, baráth and csiky 2009). csiky et al. (2004) and baráth (2007) reported that the two taxa prefer different habitats. while c. epithymum subsp. epithymum is widespread in mesophilous meadows and pastures, c. epithymum subsp. kotschy (desmoulins) arcangeli can be found mainly in dry grasslands. although these habitats are different from each other in many aspects, the species richness of both habitat types is high (baráth 2004). several taxonomic books display some species or genus as frequent hosts of the cuscuta species (simon 2000, koji] 1973). although this can be useful information, the knowledge of the exclusive hosts of dodders likewise facilitates the identification. in our study, 355 (62% of total) plant species were found to be parasitised only by one cuscuta species. the significance of this phenomenon is that the knowledge of the majority of the parasitised plants (frequent and exclusive hosts) in a habitat is mostly enough for the identification of the species of cuscuta itself. in the case of c. epithymum 97% of the relevés included at least one, and as many 17 exclusive host species. the ten most common hosts of this dodder are also rather particular, not frequently parasitised by others (tab. 4). although these proportions are somewhat lower for the other dodders, each cuscuta species can be identified by the host species in hungary. the investigation of the overlapping of the host ranges revealed that the host species of hungarian dodders are quite different. the degree of the overlapping was less than 25% in every case. the fact that c. europaea and c. lupuliformis had the greatest proportion of common hosts can be explained by the similarity of their habitats. cuscuta europaea is widespread along streams and wet ditches, but sometimes it occurs in floodplains, which is the typical habitat of c. lupuliformis in hungary (baráth 2004, csiky et al. 2004). in these cases, it is observable also that they parasitise each other. comparing the proportions of the overlapping of the host species and of the total plant species, we did not find significant differences. this indicates that the more similar the habitats of two dodders are, the higher the number of common hosts that can be found; consequently, the habitat differences of the parasites can be responsible for the different host ranges. classification of the host species based on the raunkiaer life-form system revealed substantial dissimilarities among the host spectra. we found the highest proportion of phanerophytes in the case of cuscuta lupuliformis (meso-micro-nanophanerophytes altogether is 24.81%). this result confirms the observation that c. lupuliformis often parasitises trees and shrubs (rajput and tahir 1988, rhui-cheng et al. 1995, simon 2000, baráth 2004, 2009, baráth and csiky 2006). the considerable proportion of heloand hydrophytes (10.08%) is also a characteristic feature of the host spectrum of this dodder. the 222 acta bot. croat. 71 (2), 2012 baráth k., csiky, j. relatively large proportion (52.08%) of perennials is the most important feature of the host spectrum of c. epithymum. in our study, this dodder parasitises dwarf shrubs more often (8.56%) than other host species. this statement is in agreement with several field studies (soó 1968, strid and tan 1991, baráth 2004, meulebrouck et al. 2007). erd�s (1971) reported that therophyte species dominate among the hosts of both cuscuta epithymum and c. campestris. in contradiction of this observation, we found only 18.83% proportion of annuals for c. epithymum. the reason why his results are different from ours could be that his field studies were carried out mostly in agricultural fields and in some ruderal habitats. although glück (1911) reported that c. epithymum var. alba infests several waterand uliginous -plants in nature, in our study, the frequency of heloand hydrophytes infested by c. epithymum is very low. we found the highest helo-, hydrophyte proportion in the case of c. australis (14.44%). regarding c. campestris, our result confirms erd�s’ observation (1971), as the majority of the host plants of this dodder were found to be annuals (37.50%). we also observed that both c. epithymum and c. campestris are able to parasitise trees only in their seedling and juvenile stages. this finding corresponds with the results of jayasinghe et al. (2004) and meulebrouck et al. (2007). by contrast, c. europaea was found to infest fully developed low trees, although it is not a common phenomenon either. the proportion of the hemitherophytes is remarkable in the host spectra of both c. europaea and c. campestris (13.33% and 13.21%). the correspondence between the life-form proportions of the hosts and of the total plant species confirms the explanation that dodders parasitise plants from various life-forms in different proportions not (only) because of active host choice, but also because of the characteristic features of the habitats. cuscuta australis often infests waterand uliginousplants, because in the swamps and fens where it occurs (csiky et al. 2004) those plants are common. c. epithymum can be found mainly on perennial plants, because hemicryptophyte plants are the most frequent in its habitat. cuscuta campestris infests plants mostly on the edge of roads and agricultural fields (erd�s 1971, csiky et al. 2004) and these habitats are dominated mainly by therophyte species which are the most important hosts of this parasite. we could see that c. lupuliformis parasitises trees and shrubs more often than other dodders, but the proportion of phanerophytes is also the highest in its habitat. although krohn (1934) stated that dodders are able to grow in close proximity to several plants without parasitizing them, dean (1934) and yuncker (1921) reported that cuscuta gronovii willd. ex schult infests each plant that comes into contact with it in nature. in the cases of c. europaea and c. campestris the same was observed by bentham (1878), yuncker (1932) and baráth (2009, 2010). in our study, hungarian cuscuta species parasitised all plants that had coverage of more than 25% in the sampling sites. based on the correlation between the numbers of the hosts and of the total plants, we can say that the greater the species diversity is, the more species are parasitised by cuscuta. we observed that some plants were parasitised more often than others in the same type of habitat. the significant positive correlation between the frequency of the species and the frequency of their infestations proved that the more often parasitised plants are the more frequent in the habitat. the results also suggest that parasitised plants are more frequent and they occur in greater abundance in the habitat than unparasitised plants. in conclusion, the hungarian cuscuta species are not host specific and are able to parasitise almost each plant that comes into contact with them. since the habitats of hungarian acta bot. croat. 71 (2), 2012 223 host range and choice of cuscuta dodders are strongly different from each other in species diversity and composition and the host range is mostly determined by host availability, we can say that habitat differences can be responsible for the different host ranges. acknowledgements we wish to express our thanks to zoltán bátori, dávid schmidt and dániel pifkó for assistance in field studies. special appreciation goes to konrád lájer, sándor csete and zoltán csabai for help in the statistical analyses. sincere thanks to szilvia kocsis and dragica purger for useful comments and suggestions for the manuscript. references alers-garcia, j., 2005: active host choice and parasitism by cuscuta gronovii: its effects on host individuals, population and mutualistic interaction. phd thesis, indiana university, bloomington. babington, c. c., 1843: note on a supposed new british cuscuta. the phytologist 1, 467. balogh, l., dancza, i., király, g., 2004: actual list of neophytes in hungary, and their classification according their invasiveness (in hungarian). in: mihály, b., bottadukát, z. 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(in hungarian). pszichológia 28, 81–103. yuncker, t. g., 1921: notes on our indiana dodders. proceedings of indiana academy of science 1919, 157–163 yuncker, t. g., 1932: the genus cuscuta. memoirs of the torrey botanical club 18, 113–331. acta bot. croat. 71 (2), 2012 227 host range and choice of cuscuta acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 419 acta bot. croat. 73 (2), 419–435, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 plant diversity and chemical soil composition of rocky pastures in relation to the sheep grazing intensity on the northern adriatic islands (croatia) ivica ljubičić1*, mihaela britvec1, sven d. jelaska2, stjepan husnjak3 1 university of zagreb, faculty of agriculture, department of agricultural botany, svetošimunska 25, 10000 zagreb, croatia 2 university of zagreb, faculty of science, department of botany, marulićev trg 20, zagreb, croatia 3 university of zagreb, faculty of agriculture, department of soil science, svetošimunska 25, zagreb, croatia abstract – optimal grazing pressure on rocky pastures is benefi cial to the development of plant species and maintenance of plant diversity. both abandonment of grazing and overgrazing gradually reduce plant diversity. this paper correlated abundance patterns of the fl ora on rocky pastures with the values of the chemical composition of the soil resulting from the degree of sheep grazing intensity. the study was carried out in the period from 2008 to 2010 on the islands of pag, krk and cres. at 30 sites, 310 taxa of vascular plants were found. the highest plant diversity and 220 plant taxa were found on moderately grazed pastures. abandoned pastures with a total of 93 plant taxa observed show the dominance of phanerophytes (35.5%) and the highest proportion of the mediterranean fl oral element when compared to pastures of moderate and heavy grazing intensity. the highest concentration of total nitrogen in the soil (0.71%) was recorded on plots of heavy grazing intensity. the results of the study indicate that moderate grazing intensity, from 1 to 1.5 sheep ha–1, can be recommended on the northern adriatic islands. this should contribute not only to the preservation of plant diversity, but also to the improvement of ecological sheep farming. keywords: chemical composition of the soil, mediterranean fl ora, rocky pasture, sheep grazing intensity introduction pastures are extremely important habitats that signifi cantly increase plant diversity because they are inhabited by many rare plant species. nevertheless, they are also endangered * corresponding author, e-mail: iljubicic@agr.hr copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. ljubičić i., britvec m., jelaska s. d., husnjak s. 420 acta bot. croat. 73 (2), 2014 due to overgrazing resulting from high livestock density or due to the abandonment of traditional sheep breeding resulting in progressive succession. the method of pasture management is particularly important for sustainability of plant diversity. in the northeastern part of spain three sheep grazing intensities were studied on pastures along climatic gradients, and it was shown that the increase of grazing intensity may reduce or increase plant diversity, depending on the amount of water and nutrients in the soil available to plants (de bello et al. 2007). high grazing intensity treatment and pasture abandonment cause a reduction in fl oristic diversity and biomass (škornik et al. 2010). depending on climatic conditions and soil fertility, various models have been developed to show that the intensity of sheep grazing affects plant diversity (milchunas et al. 1988, proulx and mazumder 1998, cingolani et al. 2005). škornik et al. (2010), found that for the preservation of plant diversity in dry grassland in the northern adriatic karst pastures moderate grazing intensity with 4–7 sheep ha–1 was the most appropriate. furthermore, high intensity of sheep grazing increased the dominance of ruderal plants and reduced the proportion of chamaephytes and geophytes. overgrazing may in extreme cases lead to total destruction of plant communities (horvat 1949, puerto et al. 1990, montalvo et al. 1993), and is often perceived as a major factor in the degradation of mediterranean ecosystems and landscapes (noy-meir and oron 2001). however, many such ecosystems, when they are undergrazed or entirely abandoned, tend to become impenetrable woody thickets with low species diversity. overgrazing in hot and dry areas with shallow soils on a rocky substrate will result in the development of barren, rocky ground as is the case today in large areas of the mediterranean coastline (peco et al. 1998, eler et al. 2005). such habitats with rocky soil have a limited amount of water and nutrients available to plants, thus the increase of grazing pressure adds to the reduction of plant species diversity (proulx and mazumder 1998). in recent decades, the cessation of extensive method of pasture management has been related to the modernization of livestock breeding (rook et al. 2004). abandonment of grazing in some parts of europe has led to the loss of grassland and the disappearance of certain plant species, so the remaining, preserved grasslands tend to be granted protected status (cousins and eriksson 2001). furthermore, he et al. (2011), carried out a study on the importance of grasslands as a potential source of nitrogen and carbon and suggested that the accumulation of these nutrients in the soil at two depths (0–30 and 30–60 cm) decreased linearly with the increase of grazing pressure. this indicates that the accumulation of nutrients at a depth down to 30 cm is signifi cantly higher in moderately grazed than in more heavily grazed grasslands, thus showing that moderate grazing intensity brings about the mobilisation of nutrients in the soil, while high grazing intensity results in loss of carbon and nitrogen, the threshold for the transformation being 4.5 sheep ha–1. overgrazing may hamper productivity and cause serious loss of nutrients in the soil, thereby decreasing soil fertility (rietkirk and van de koppel 1997, su et al. 2005, han et al. 2008). grazing may also affect the mechanical properties of the soil because the increase of grazing pressure increases soil compaction (knapp et al. 1999). overgrazing, trampling and pollution favor the creation of anthropogenic communities which with constant accumulation of manure are transformed into extremely nitrophilous vegetation cover (derner and schuman 2007, ingram et al. 2008). basically, high grazing pressure tends to decrease plant diversity and biomass and facilitates spatial homogeneity (augustine and mchaughton 1998, frank and groffman 1998). sheep grazing and plant diversity of rocky pastures acta bot. croat. 73 (2), 2014 421 changes in the intensity of grazing may lead to an increasing number of uncultivated and overgrown soils (biondi et al. 2005). over the past years, the process of abandonment of rocky pastures on the island of pag has proceeded rapidly and large pasture areas have been turned into macchia, the most common taxa coming from the genus juniperus (ljubičić 2008). the aim of the present study was to compare the composition of the fl oras on the rocky pastures of the northern adriatic islands of pag, krk and cres, to determine the dependence of the fl oristic composition on the chemical composition of the soil resulting from the degree of sheep grazing intensity, and to determine the optimal grazing pressure, an essential factor in the maintenance of plant diversity. materials and methods study area and data sampling the studied areas were rocky pastures of the islands of pag, krk and cres, belonging to the group of northern adriatic islands (fig. 1). this is a transitional area between the eumediterranean zone and the submediterranean zone. with some exceptions, it still contains the basic characteristics of the mediterranean climate. due to the climatic conditions, evergreen and deciduous vegetation types are present together. average annual air temperature is 15.5 °c with an average annual precipitation of 1,113 mm. geological bedrock is composed mainly of carbonate sediments with inclusion of limestone dolomite rocks, and the fig. 1. overview of studied area of rocky pastures on the northern adriatic islands of pag, krk and cres. ljubičić i., britvec m., jelaska s. d., husnjak s. 422 acta bot. croat. 73 (2), 2014 islands have predominantly the following soil types: rocky soil, limestone dolomite black soil, rendzina, brown soil on limestone and dolomite, terra rossa soil and anthropogenic karst soils. these types of soils are often composed of more or less heterogeneous soil combinations (husnjak et al. 2008). the studied area belongs to the alliance chrysopogoni-saturejon ht. et h-ić 1934, namely eastern adriatic rocky pastures vegetation of the submediterranean zone in the mediterranean littoral vegetation belt. within this alliance most prominent are the following communities: koelerio splendenti-festucetum illyricae trinajstić 1992, stipo eriocauli-salvietum offi cinalis h-ić (1956) 1958 and asphodelo aestivo-chrysopogonetum grylli h-ić (1956) 1958 (trinajstić 2008). the study was carried out during the growing seasons of 2008, 2009 and 2010. during the sampling we tried to cover the largest range of variability in nature possible. the data are organized within two matrices of data. the fi rst matrix represents the abundance (the cover of certain species on the plot), while in the second matrix each plot is assigned to the values measured of the following variables: total nitrogen in the soil (n), physiologically active phosphorus (p2o5) and potassium (k2o), soil ph, humus content, organic matter content, method of land use, grazing pressure, altitude, terrain slope and exposure. vascular plants were studied at 30 sites. the choice of plots was made according to the intensity of sheep grazing, and therefore 10 sites on rocky pastures abandoned for more than 20 years were selected, as well as 10 with moderate intensity (1–1.5 sheep ha–1) and 10 localities with heavy grazing intensity (≥ 8 sheep ha–1). each site was represented by two plots, which makes a total of 60 plots. the plots of abandoned pastures are numbered 1 to 8 (island of pag), 21 to 26 (island of krk) and 45 to 50 (island of cres), moderate-intensity grazing plots are numbered: 9 to 14 (island of pag), 27 to 34 (island of krk) and 51 to 56 (island of cres) while plots of heavy intensity grazing are numbered 15 to 20 (island of pag), 35 to 44 (island of krk) and 57 to 60 (island of cres). the plot area was chosen with respect to the intensity of sheep grazing: for abandoned pastures 15 ´ 15 m, moderate 10 ´ 10 m and heavy intensity 10 ´ 10 m plots. the fl oristic composition was sampled at full fl owering (may, june) and before release of sheep to pasture. the life forms of plants were aligned according to pignatti (1982), and the endemic, endangered and allochthonous taxa according to nikolić (2013). a fl oristic list with the respective abundances was made according to braun-blanquet method (horvat 1963). nomenclature of plant taxa follows flora croatica database (nikolić 2013). soil sampling was carried out during and after sheep grazing within the growing season. this methodology has been used in similar studies of grazing pressure (eler et al. 2005; de bello et al. 2007; škornik et al. 2010). the studied rocky pastures are located mainly on skeletoidal soils and the mentioned nutrient content applies only to fi ne-grained soil. therefore, when assessing the content of total nitrogen and other nutrients, analytical data must be considered within this context. soil sampling was conducted to measure the long-term role of sheep as vectors of nutrients on rocky pastures. samples were taken up to a depth of 25 cm in the upper layer of the pedosphere according to the method of salminen et al. (1998). the studied pasture plots were grazed by landrace sheep breeds (from pag, krk and cres). a modifi ed nonlinear method was used to calculate the livestock units (lu), based on metabolic weight defi ned by the ratio of livestock body mass and metabolically calibrated livestock exponentiated to 0.75. an animal of 1,000 lbs (libra) = 453.6 kg daily eats dry matter equivalent to 2% of its own body weight (holechek et al. 1989), with the proviso that this was recalculated according to the croatian standard in which the body weight of a livestock sheep grazing and plant diversity of rocky pastures acta bot. croat. 73 (2), 2014 423 unit is 500 kg. the number of livestock units used for the evaluation of the pasture capability was calculated using the following formula: number of lu = [livestock quantity ´ average body mass of sheep ´ (days of livestock grazing / 365)] / 500 the average body mass of a sheep (from pag, cres and krk) is 38.59 kg (mioč et al. 2004, 2007, pavić et al. 2006). according to these fi gures, sheep grazing intensities are converted into the occupation of pasture by a livestock unit: heavy grazing intensity (≥ 0.62 lu), moderate grazing intensity (0.10 lu), and rocky pastures abandoned for more than 20 years (0 lu). data analysis the differences between the three grazing intensities for measured soil properties were tested using the analysis of variance (anova), and the data collected in the fi rst and second matrix (fl ora vs. soil) were analyzed by using correlation, cluster and ordination analysis. degree of similarity among the studied sites of rocky pastures with respect to the fl oristic composition was compared with the use of cluster analysis according to the data on the presence of taxa and its abundance. the binary similarity index according to bray-curtis (bray and curtis 1957) was used in the analysis of the similarities between the fl ora of the studied sites. in the analysis of similarity/diversity of fl ora, agglomerative upgma algorithm (unweighted paired group method using arithmetic averages) was used. floristic composition of sampled plots was analyzed by applying metric multidimensional scaling – pcoa (principle coordinate analysis) (gower 1966), using the square root of the complement of jaccard index as a measure of diversity (mr): where, a – the number of species common to both plots compared, b and c – the number of species occurring only in the fi rst or in the second plot; for this purpose the program canoco 4.5 (ter braak and šmilauer 2002) was used. the fi rst step consisted of dca analysis (detrended correspondence analysis), which was performed in order to determine the length of ordinal axis gradients, based on which direct gradient analysis (redundancy analysis) was selected in the second step; this assumes a linear response of species to habitat factors (within the observed range of their values). environmental variables were included in the redundancy analysis iteratively based on the explained variance of species and statistical signifi cance (p < 0.05) of the contribution of the variable to the total explained variance using the monte carlo permutation test. results in fl oristic studies (ljubičić 2012) carried out on rocky pastures of the northern adriatic islands of pag, krk and cres, 310 taxa of vascular fl ora were found on 60 plots (281 species, 27 subspecies and two varieties). the greatest number of taxa was recorded on pastures of moderate sheep grazing intensity, varying from 68 to 90 taxa per plot, on heavy intensity pastures the number of taxa varied between 49 and 74, while on abandoned pastures from 32 to 45 taxa were recorded per plot (fig. 2). ljubičić i., britvec m., jelaska s. d., husnjak s. 424 acta bot. croat. 73 (2), 2014 in our study the mediterranean fl oral element (53.9%) was dominant on the rocky pastures of the northern adriatic islands. analysis of life forms showed dominance of hemicryptophytes with 38.4% of the studied plants. abandoned pastures are dominated by phanerophytes, at 35.5%, unlike those characterized by moderate and heavy grazing intensity (fig. 3). fig. 2. the number of taxa observed on plots, according to the different grazing intensity of rocky pas tures on the northern adriatic islands; symbols from 1 to 60 mean numbers of experimental plot. fig. 3. abundance of plant life forms on rocky pastures of northern adriatic islands according to the different grazing intensities; ch – chamaephytes, he – hemicryptophytes, ge – geophytes, ph – phanerophytes, th – therophytes. signfi icantly more endemic taxa were found on plots of heavy and moderate grazing intensity, compared to plots on abandoned pastures studied. from a total of 23 established endangered taxa (ljubičić 2012), 18 were found on plots of moderate grazing intensity, 11 on plots of heavy grazing intensity, and only six taxa were found on plots of abandoned pastures. an equal number of allochthonous species was found on plots of moderate and heavy grazing intensity, while no allochthonous species were found on plots of abandoned pastures. the results of the measurement of soil composition on plots of rocky pastures of varying sheep grazing intensity are shown in table 1. the highest concentration of total nitrogen and humus in the soil was found on pastures of heavy sheep grazing intensity. statistically signifi cant differences in the concentration of physiologically active phosphorus were determined in pastures of heavy sheep grazing intensity, pastures of moderate grazing intensity sheep grazing and plant diversity of rocky pastures acta bot. croat. 73 (2), 2014 425 and abandoned pastures. the average value of physiologically active phosphorus (p2o5) at the level of all plots amounts to 2.57 mg/100 g of soil. mean value of the physiologically active potassium content (k2o) at the level of all plots amounts to 85.03 mg/100 g. table 2 shows the correlations of the measured properties of the soil parameters, where a signifi cantly strong positive correlation (r = 0.897) in the relationship of total nitrogen and humus content was found. tab. 1. mean values of the basic chemical soil properties on the studied rocky pastures of the northern adriatic islands, according to grazing intensity. results are expressed as the arithmetic mean of 20 plots and grazing intensity. average values within the same row marked with different letters are statistically signifi cantly different at the level of p ≤ 0.05. soil composition no grazing moderate intensity heavy intensity n (%) p2o5 (mg/100 g) k2o (mg/100 g) ph humus (%) 0.58 a, c 1.37 a, c 84.32 a, b 7.191 12.45 b, c 0.50 a 0.94 a 65.0 a11 7.351 9.82 a 0.71 b 5.41 b 105.76 b11 7.481 13.54 b, c tab. 2. correlation between the properties of the soil composition on the studied rocky pastures of the northern adriatic islands in all 60 plots and their p values. the cells below the diagonal contain the correlation coeffi cients and the cells above the diagonal the corresponding p values. statistically signifi cant correlations (p ≤ 0.05) are in bold. n p2o5 k2o ph humus n 0.0139 0.0697 0.0468 <0.0001 p2o5 0.316 0.0002 0.0627 0.0018 k2o 0.235 0.4601 0.6184 0.2428 ph 0.257 –0.24111 0.0651 0.5994 humus 0.897 0.3941 0.1531 0.0691 the results of cluster analysis based on the values of the bray-curtis similarity index are graphically represented by a upgma dendrogram (fig. 4). the diagram clearly shows the separation and formation of three groups according to the intensity of pasture use: 1 – abandoned pastures, 2 – pastures of heavy grazing intensity and 3 – pastures of moderate sheep grazing intensity. pastures of moderate and heavy intensity are relatively similar, while abandoned pastures form a separate cluster unlike either of them (fig. 4). iterative inclusion of environmental variables on the basis of their statistical signifi cance and proportion of explained species variance was carried out by redundancy analysis with nine environmental variables: total soil nitrogen (n), physiologically active phosphorus (p2o5), potassium (k2o), reaction soil-ph, humus content, altitude, terrain slope, northness and eastness. aggregate examination of redundancy analysis shows that four canonical axes explained 23.3% of species variability, and 76.7% of species variability was explained by environmental variables used in the analysis (tab. 3). ljubičić i., britvec m., jelaska s. d., husnjak s. 426 acta bot. croat. 73 (2), 2014 t-values of canonical coeffi cients on ordinate axis which show the signifi cance of a certain variable on axes are listed in table 4. the fi rst and the second canonical axis are mostly infl uenced by soil parameters (humus content, nitrogen – n and physiologically active phosphorous – p2o5), the third by northness, while the fourth is mostly infl uenced by altitude and potassium – k2o. the length of the vector on the diagram of the fi rst and second canonical axis depicting plots and environmental factors (fig. 5a) shows that humus, p2o5, k2o and nitrogen have the greatest infl uence on the changes in the fl oristic composition structure. the second quadrant of the biplot is dominated by plots of abandoned pastures and the drop in the value of soil reaction (ph) is clearly noticeable. the increase in the value of p2o5 and humus is marked in the fi rst quadrant, dominated by pastures of high sheep grazing intensity and the declivity of the terrain. fig. 4. bray-curtis similarity upgma dendrogram of the fl oristic composition of 60 rocky pasture study plots (s1–s60) on the northern adriatic islands shows the separation of clusters into three groups, which typically belong to: 1 – abandoned pastures, 2 – pastures of heavy grazing intensity and 3 – pastures of moderate sheep grazing intensity. tab. 3. results of the redundancy analysis of the fi rst four canonical axes carried out on 60 plots, 310 species and nine environmental variables: total soil nitrogen (n), physiologically active phosphorus (p2o5), potassium (k2o), reaction soil – ph, humus content, altitude, terrain slope, northness and eastness. axes 1 2 3 4 total inertia eigenvalues 0.104 0.061 0.040 0.028 1.000 species-environment correlations 0.704 0.701 0.786 0.797 cumulative percentage variance of species 10.4 16.5 20.5 23.3 of species-environment relation 34.4 54.3 67.5 76.7 sum of all eigenvalues 1.000 sum of all canonical eigenvalues 0.304 sheep grazing and plant diversity of rocky pastures acta bot. croat. 73 (2), 2014 427 fig. 5. ordinate diagram of the fi rst two canonical axes of redundancy analysis depiciting (a) experimental plots and environmental variables (indicated with dark arrows) and (b) species (the fi rst quadrant: sp112 – euphorbia cyparissias, sp56 – carduus nutans, sp53 – c. micropterus, sp60 – carlina corymbosa, etc.; the second quadrant: sp217 – pistacia lentiscus, sp1 – acer monspessulanum, sp233 – quercus ilex, sp123 – fraxinus ornus, sp85 – coronilla emerus ssp. emeroides, sp265 – smilax aspera, sp240 – rubia peregrina, sp75 – clematis fl ammula, sp136 – geranium molle, sp244 – ruscus aculeatus, etc.; the third and fourth quadrants: sp45 – bromus erectus, sp119 – festuca valesiaca, sp181 – melica ciliata, sp17 – anthoxanthum odoratum, etc.,) indicated by grey arrows and environmental variables. simbols (circles, triangles and squares) represent experimental plots from 1 to 60. arrow direction marks the increase of the value, and length of arrow marks the degree of infl uence on the explanation of total variability on the fi rst two axes. tab. 4. t-values of the canonical coeffi cients of environmental variables on the fi rst four canonical axis; the highest values are marked in bold; % variance – proportion of explained variance of certain axis. axes 1 2 3 4 variance (%) 34.39 19.93 13.15 9.21 n 2.99 –1.99 –3.24 1.91 p2o5 3.96 –0.40 0.70 –3.47 k2o –1.07 3.30 0.95 4.51 ph 1.94 –1.40 0.75 –0.90 humus –3.60 3.63 2.74 –1.44 organic matter 0.00 0.00 0.00 0.00 height –0.73 –0.64 2.38 5.83 inclination –1.20 –0.58 2.80 –2.69 northness 1.96 –0.57 5.22 –1.23 eastness –0.63 0.10 0.50 –2.49 ljubičić i., britvec m., jelaska s. d., husnjak s. 428 acta bot. croat. 73 (2), 2014 the diagram of the fi rst and second canonical axis depicting species and environmental factors (fig. 5b) shows vectors affected by statistically most signifi cant species for the formation of the ordinate diagram. it is also visible that certain taxa group into clusters corresponding to specifi c agro-ecological factors. the fi rst quadrant is characterized by grouping of unpalatable species: euphorbia cyparissias l., carduus nutans l., c. micropterus (borbás) teyber, carlina corymbosa l., etc. the second quadrant of the biplot is dominated by species comprising garrigue, macchia and mediterranean region forests: pistacia lentiscus l., acer monspessulanum l., quercus ilex l., fraxinus ornus l. and coronilla emerus l. ssp. emeroides boiss. et spruner, comprised of trees and shrubs; smilax aspera l., rubia peregrina l. and clematis fl ammula l., showing the presence of climbing plants; geranium molle l., ruscus aculeatus l., etc. in the ground layer. the third and fourth quadrants show a grouping of typical grassland species: bromus erectus huds, festuca valesiaca schleich. ex gaudin, melica ciliata l., anthoxanthum odoratum l., etc. (fig. 5b). discussion sheep grazing intensity and plant diversity sustainability of plant diversity on rocky pastures mainly depends on the sustainability of habitat, which is affected by a number of agro-ecological factors. according to a study of vegetation dynamics (vrbek 2005), habitat sustainability is not possible without anthropogenic action. thus, saïd (2001) and saïd and gegout (2000), found that large areas of rocky pastures are, due to the weakening of anthropogenic infl uence, in the process of progressive succession threatened with being overgrown. each succession is directly related to and infl uences the change in the fl oristic composition of plant communities (oosting 1942, keever 1950). using the method of remote observation ljubičić (2008) examined the overgrowing of rocky pastures on the island of pag and found that over the past 40 years there has been signifi cant loss of rocky pasture area, and the successive stage of macchia was signifi cantly increased at the expense of rocky pastures. changes in the socio-economic conditions of life on the islands of pag, krk and cres have been refl ected in changes in the vegetation and fl oristic composition. kotnik and vidrih (1994) also found that the abandonment of agricultural production gradually led to the loss of the traditional patterns of rocky pastures. successive stage of macchia has been in constant expansion in the last decade. the distribution area of macchia is mostly the rocky pasture area that is today used increasingly less for grazing. only 30% of the overall plant diversity is related to forest habitat, 70% to open areas, and most of the latter to grassland. this shows the importance of the management of rocky pastures on the northern adriatic islands in order to preserve the habitat and therefore the plant diversity. the greatest number of taxa was recorded on plots of moderate grazing intensity; there was moderate diversity on plots of heavy sheep grazing intensity and on abandoned pastures the lowest number of taxa was found. these results show that the abandonment of grazing on rocky pastures encourages progressive succession and development of macchia characterized by reduced plant diversity. fuhlendorf et al. (2001) and floyd et al. (2003) also confi rm that very intensive grazing reduces the number of species and lays bare the soil surface. by examining the response of semi-natural mediterranean pastures to the grazing intensity noy-meir et al. (1989) found changes in most of the studied taxa observed within 15 fenced plots. thus, for example, perennial species performed better in fenced plots, and sheep grazing and plant diversity of rocky pastures acta bot. croat. 73 (2), 2014 429 highly grazed pastures were dominated by thorny species. studies of rocky pastures on the northern adriatic islands also demonstrated that thorny taxa of the family asteraceae are signifi cantly more abundant on plots of higher grazing intensity (ljubičić 2012). puerto et al. (1990) studied the mediterranean grasslands of spain and found the largest number of species on moderately grazed pastures. in addition, on these pastures they revealed the highest biomass yield. in the northern adriatic karst grassland škornik et al. (2010), found the greatest plant diversity on moderately grazed pastures with 4–7 sheep ha–1. for the area of rocky pastures on the northern adriatic islands on the basis of our research there is greatest plant diversity on moderately grazed pastures with 1–1.5 sheep ha–1. the difference in the optimal grazing intensity values between the study carried out by škornik et al. (2010) and the present study can be explained by the greater fertility of the soil and better condition of pasture, which allows a greater number of sheep/ha as compared to our study which was conducted mainly on poor and skeletoidal soil. basically, moderate sheep grazing intensity favors the development of plant diversity, and therefore may be considered optimal for the proper management of pastures and the preservation of plant diversity. the presence of taxa of the family poaceae is higher on plots of moderate sheep grazing intensity than on plots of heavy intensity while the fewest taxa of the family were found in abandoned pastures. this study showed a higher presence of taxa of the family fabaceae on grazed plots than on ungrazed plots (ljubičić 2012). while studying anthropogenic impacts on natural pastures, pantisa and tzanoudakis (2001), also found a higher presence of taxa of the family fabaceae. thus, in semi-natural systems, i.e. where grazing as an anthropogenic factor is involved, a higher presence of legume species was found in relation to allnatural habitats. on areas of abandoned pastures no taxa of the family asteraceae were found (ljubičić 2012). likewise, in the study of succession processes and the invasion of allochthonous species on the islands of galapagos, itow (2003) found during the process of progressive succession a gradual decrease in numbers of species of the family asteraceae. the above phenomenon can be explained by the heliophilia of most members of the family asteraceae and their disabled development due to the large proportion of phanerophytes in this habitat type. floral elements and plant life forms the mediterranean fl oral element is the most frequent in the present study of the northern adriatic islands. in the vegetation study of the southern adriatic islands such as the island of svetac (pavletić 1979) and brusnik (bogdanović and mitić 2003) it was found that the mediterranean fl oral element was there also the most dominant. from the above it can be concluded that the high percentage of plants belonging to mediterranean fl oral elements groups confi rms the mediterranean character of rocky pastures in the study area, which is typical of other adriatic islands as well. the endemic species (onosma echioides (l.) l. ssp. dalmatica (scheele) peruzzi et n. g. passal., aurinia sinuata (l.) griseb., rhamnus intermedius steud. et hohst., etc.) were the least numerous on abandoned pastures where the process of progressive succession has been visible in the last 20 years. in contrast, on open habitats such as plots of optimal grazing pressure (in the present case 1.5 sheep ha–1) there is a favourable infl uence on the development and preservation of endemic species. according to pignatti (1982) and ellenberg (1992), the percentage of certain plant life forms in an area indicates the local climatic and soil conditions. on abandoned pastures the proportion of phanerophytes is higher than on pastures of moderate and heavy grazing inljubičić i., britvec m., jelaska s. d., husnjak s. 430 acta bot. croat. 73 (2), 2014 tensity. the present study also confi rmed the expectedly lowest number of therophytes on abandoned pastures. the disabled development of therophytes may be explained by the progressive succession stage and the dominance of phanerophytes. in line with this are studies carried out by škornik et al. (2010), who also found that there are no therophytes on abandoned pastures. in addition, the latter authors found a signifi cantly higher proportion of therophytes on pastures of heavy grazing intensity. the present study determined an equal presence of geophytes, regardless of the grazing intensity, which is in line with the average proportion of geophytes on the mediterranean area and indicates the species-rich spring aspect. such presence of life forms is a clear indication of the habitat environmental conditions and clearly separates the open pasture habitats from their progressive succession stages. according to the list of invasive species in croatia (boršić et al. 2008), a low presence of allochthonous species was found in the study area, which indicates a very low rate of rocky pasture utilization and the isolation of the study area by its sea barrier. invasive species are often pioneer species in habitats exposed to disturbances/stress (ruščić 2010). therefore, it is to be expected that increasing urbanization, increasing tourism activities and other anthropogenic factors will also cause a future increase of the number of allochthonous and invasive taxa on the rocky pastures of the northern adriatic islands. endangered taxa from our above results, it can be concluded that moderate grazing (1.5 sheep ha–1) is benefi cial to the development and preservation of endangered taxa. on the other hand, overgrazing leads to changes in habitat conditions, over-fertilization by grazing animals causes further nitrifi cation of the soil, both leading to increased abundances of nitrophilic and thorny species, which due to their unpalatability permanently remain dominant and suppress the development of endangered species. it was also found that the endangered taxa of the family orchidaceae have the higest presence on moderately occupied pastures (ljubičić 2012). according to the list of croatian fl ora (nikolić 2013), 7.85% of plants are considered endangered, which is consistent with this study where the proportion of endangered plants on rocky pastures of the northern adriatic islands is 7.42%. soil composition all soil parameters except the ph value showed the lowest values in the moderately and the highest in the intensively grazed pastures. the concentration of nitrogen in the soil is statistically signifi cantly higher in heavily grazed compared to the moderately grazed pastures. in contrast, the study by he et al. (2011) showed that the concentration of nitrogen (in the soil layer 0–10 and 10–30 cm) decreases linearly with an increase in the number of livestock per unit area, which can be explained by a greater amount of grazed grass and therefore less biomass for the process of humifi cation. these authors also found that the amount of carbon in the surface soil layer (0–10 cm) is signifi cantly higher in the case of moderately grazed pastures in relation to pastures of higher grazing intensity after fi ve years of sheep grazing. these results refer to the transformation and loss of carbon from the soil under the infl uence of high grazing intensity, given that the threshold for this transformation is 4.5 sheep ha–1. from the above, it can be concluded that the increase in concentration of these nutrients is expected only to a certain degree of sheep grazing intensity. based on the results obtained in present study, it was found that these soils are poor in physiologically sheep grazing and plant diversity of rocky pastures acta bot. croat. 73 (2), 2014 431 active phosphorus p2o5. the highest concentration of p2o5 was measured on heavily grazed pastures, which results from the high number of sheep as nutrient vector. the results of the physiologically active potassium content (k2o) (> 40.1 mg/100 g of soil) indicate the richness of the study area in this element. the high presence of physiologically active potassium may be explained as a result of fi res that occurred at certain study sites as well as by the release from organic matter and sheep manure (španjol et al. 2003, rosavec et al. 2013). the measured humus content corresponds to the category of high humus content soil. a strong positive correlation in the relation of humus content in the soil and total nitrogen is explained by the number of sheep as nutrient vectors. the studied plots of the rocky pastures are part of skeletal and skeletoidal soils and the very high humus content relates only to fi negrained soil, therefore the obtained data are to be considered within this context. interconnection of studied plots similarity analyses based on the presence of species and values of the bray-curtis index show groupings of rocky pastures with respect to grazing intensity, so there is greater similarity between pastures of moderate and heavy sheep grazing intensity than between abandoned pastures and either of these two. our results confi rm what has been documented in some other studies of mediterranean and sub-mediterranean grasslands (škornik et al. 2010). this is in concordance with fi ndings of de bello et al. (2007), although they have studied a gradient of environmental factors, particularly soil moisture, which has proven to be very important as well. our plots were situated in an environmentally more homogenous area with respect to soil moisture, and therefore the infl uence of grazing pressure was dominant. similarity analyses based on abundance and values of bray-curtis similarity index show grouping with respect to grazing intensity, clearly separating three groups: 1 – pastures of moderate sheep grazing intensity, 2 – pastures of heavy sheep grazing intensity and 3 – abandoned pastures. these results fully refl ect the conditions on the rocky pasture plots, where it is clearly visible that the abandoned pasture plots are in the process of succession and overgrowth, as shown in a previous study of the island of pag (ljubičić et al. 2008). redundancy analysis studied the interconnection of plant species with respect to environmental factors and these have accordingly been allocated into three directions from the source followed by environmental variables which could indicate different habitat types. there was a separation and grouping into three clusters according to the occupation of pastures, given that there is an overlap in certain parts between pastures of moderate and heavy sheep grazing intensity. in their canonical correspondence analysis dobrović et al. (2006) found that the changes in altitude, ph and inclination had the strongest infl uence on changes of fl oristic structure, but they did not study the soil composition. in our study among other parameters, humus, p2o5, k2o and n have the greatest infl uence on structural changes in fl oristic composition. the nutrient content in the soil is the most affected by an increase of the grazing pressure and a lower terrain slope, which was expected in the present study. conclusion from a study of the rocky pastures of the northern adriatic islands of pag, krk and cres it is concluded that the intensity of sheep grazing has a signifi cant impact on plant diversity and chemical composition of the soil. the greatest plant diversity was determined on pasljubičić i., britvec m., jelaska s. d., husnjak s. 432 acta bot. croat. 73 (2), 2014 tures of moderate sheep grazing intensity. with the increase of grazing intensity the concentration of total nitrogen (n) and humus in the soil also increases and facilitates the development of nitrophilic and unpalatable species. by contrast, the abandonment of sheep grazing leads to a progressive succession process and a growing proportion of woody plants (phanerophytes) which, due to the canopy produced, results in a signifi cant reduction in plant diversity. determination of the largest proportion of plants of the mediterranean fl oral element confi rms the mediterranean character of the study area. moderate grazing intensity is benefi cial to the development and preservation of endemic and endangered taxa. the analysis of the similarities between the fl ora of the studied plots clearly distinguished three clusters with respect to the intensity of grazing. humus, total nitrogen (n), physiologically active phosphorus (p2o5) and potassium (k2o) best explain the differences in fl oristic composition obtained by ordinate analyses in relation to other environmental variables. grazing pressure and lower slope of the terrain contribute to the 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berberis croatica horvat and berberis vulgaris l. dario kremer1, renata juri[i] grube[i]1, zvjezdana popovi]2, ksenija karlovi]3* 1 faculty of pharmacy and biochemistry, university of zagreb, schrottova 39, hr-10000 zagreb, croatia. 2 public institution »nature park biokovo«, trg tina ujevi}a 1, hr-21300 makarska, croatia. 3 faculty of agriculture, university of zagreb, sveto{imunska 25, hr-10000 zagreb, croatia. abstract – the three year variability of fruits and seeds was investigated in berberis croatica (vo{ac, mt biokovo, rakov potok near zagreb) and in b. vulgaris (fran ku{an pharmaceutical botanical garden in zagreb). berberis croatica had the following dimensions of fruits (seeds): length 7.28–7.88 (4.57–5.03) mm; width 3.85–3.99 (width 1: 2.06–2.20; width 2: 1.44–1.63) mm; weight 0.065–0.078 (0.0116–0.0134) g. dimensions of b. vulgaris fruits (seeds) were: length 10.20–11.29 (5.71–6.24) mm; width 5.29–5.83 (width 1: 2.40–2.71; width 2: 1.60–1.98) mm; weight 0.1602–0.2199 (0.0146–0.0235) g. the fruit shape of both species was similar and the length/width ratio was 1.91–2.04 in b. croatica and 1.77–2.07 in b. vulgaris. the number of seeds per fruit was 1.23–1.58 in b. croatica and 1.36–1.54 in b. vulgaris. generally, fruits and seeds of b. vulgaris were significantly longer, wider and heavier than fruits and seeds of b. croatica. anova showed significant statistical differences between populations for all analyzed fruit and seed traits while the species significantly differed in all traits, except in the fruit shape and number of seeds in fruit. keywords: berberis croatica, berberis vulgaris, fruit, seed introduction interand intraspecific variability in fruit and/or seed traits has been in the focus of interest of many authors (jordano 1984, hendrix and sun 1989, willson et al. 1990, herrera 1992, obeso and herrera 1994, eriksson 1999) but only few of them address the genus berberis. at the same time, when dealing with the fruit and seed properties of acta bot. croat. 71 (1), 2012 115 * corresponding author, e-mail: karlovic@agr.hr copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:00 color profile: disabled composite 150 lpi at 45 degrees barberry species in relation with interannual variation (herrera 1998, allen and wilson 1992), papers are mainly concentrated on the seed and/or fruit production. even though data regarding basic fruit and seed related traits for some of the european species of the genus berberis, namely b. vulgaris (rudolf 1974, akbulut et al. 2009) and b. hispanica (obeso and herrera 1994) could be found, those concerning b. croatica are lacking. b. croatica is a species with a still unresolved taxonomic status and is still not recognized as a distinctive species by the leading authorities on european flora. several attempts have been made to crystallize the morphological (karlovi] et al. 2009) and chemical difference (zovko kon^i] et al. 2010) between b. croatica and b. vulgaris. however, fruit and seed characters have not been included in the analysis to date yet they could help in the characterization of the distinctive morphological traits of these species. material and methods fruits of berberis croatica horvat and b. vulgaris l. were randomly collected during three years (from 2008 to 2010). b. croatica fruits were collected in one natural habitat (vo{ac, mt biokovo, in text as abbreviation vo; 43°18’ n; 17°02’ e; 1422 m a.s.l.), while b. vulgaris fruits were collected in one natural (rakov potok near zagreb, in text as abbreviation rp; 45°47’ n; 15°42’ e; 160 m a.s.l.) and one cultivated habitat (fran ku{an pharmaceutical botanical garden, zagreb, in text as abbreviation pbg; 45°50’ n; 15°59’ e; 195 m a.s.l.). twenty plants were harvested in the locality vo{ac, ten in the locality rakov potok and six in the cultivated habitat. according to our permit from the ministry of culture of the republic of croatia, we were able to analyse 100 fruits for the locality vo{ac and so the same number of b. vulgaris fruits (100) was collected for each population. length, width and weight of fruits, as well as seeds, were measured. since the seeds of the researched berberis species have irregular shapes, the width of the seed was measured on two axes (width 1 and width 2). the ratio between length and width of fruits and length and width (width 1, width 2) of seeds was obtained through mathematical calculation, as well as their mutual (fruit-seed) ratio, which included length, width and weight. a greater ratio indicates narrower fruits (or seeds), while a smaller ratio indicates more orbicular fruits (or seeds). the differences in fruit and seed traits between species and populations were analyzed by the general linear models (glm) procedure using repeated measures anova and tuckey’s post-hoc test at the p £0.05 level. the statistical analysis was performed using statistica 7 software (statsoft inc., tulsa, ok, usa). results table 1 presents descriptive statistics for the analyzed traits of fruits and seeds of berberis croatica and b. vulgaris. the average length of b. croatica fruits ranged from 7.28 mm (vo 2010) to 7.88 mm (vo 2009) while the width ranged from 3.85 mm (vo 2010) to 3.99 mm (vo 2008). weight of b. croatica fruits ranged from 0.065 g (vo 2008) to 0.078 g (vo 2010). fruits of b. vulgaris were the largest and their length ranged from 10.20 mm (pbg 2009) to 11.29 mm (rp 2010), while the width ranged from 5.29 mm (rp 2008) to 5.83 mm (pbg 2009). weight of b. vulgaris fruits ranged from 0.1602 g (rp 2009) to 0.2199 g (pbg 2009). the shapes of the two species were similar and the ratio between 116 acta bot. croat. 71 (1), 2012 kremer d., juri[i] grube[i] r., popovi] z., karlovi] k. u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:00 color profile: disabled composite 150 lpi at 45 degrees length and width of fruits was from 1.91 (vo 2010) to 2.04 (vo 2009) in b. croatica and from 1.77 (pbg 2009) to 2.07 (rp 2010) in b. vulgaris. the most variable trait was fruit weight, ranging from 25.61 % (vo 2010) to 28.55 % (vo 2008) and from 22.84 % (rp 2010) to 36.54 % (pbg 2008) in b. croatica and b. vulgaris, respectively. the least variable trait was fruit length, which ranged from 5.84 % (vo 2009) to 9.62 % (vo 2010) and from 6.37 % (pbg 2009) to 9.83 % (rp 2008) in b. croatica and b. vulgaris, respectively (tab. 1). the number of seeds per fruit ranged from 1.23 (vo 2010) to 1.58 (vo 2009) in b. croatica and from 1.36 (rp 2008, rp 2010) to 1.54 (pbg 2010) in b. vulgaris. acta bot. croat. 71 (1), 2012 117 berberis croatica and b. vulgaris fruit and seed traits tab. 1. statistics for analyzed traits of fruits and seeds of berberis croatica – vo{ac (vo) and b. vulgaris – rakov potok (rp) and pharmaceutical botanical garden »fran ku{an« (pbg). min, max and std. dev. are in mm (length, width) and g (weight); cv (coefficient of variability) is in %. minimum and maximum are bolded. statistics b. croatica b. vulgaris vo 2008 vo 2009 vo 2010 rp 2008 rp 2009 rp 2010 pbg 2008 pbg 2009 pbg 2010 fruit traits length n mean min max std. dev. cv 100 7.83 7.00 9.04 0.50 6.39 100 7.88 6.89 9.07 0.46 5.84 100 7.28 5.26 9.43 0.70 9.62 100 10.89 5.34 12.74 1.07 9.83 100 10.39 8.64 12.10 0.79 7.60 100 11.29 5.84 13.26 0.91 8.06 100 10.42 8.94 12.54 0.74 7.10 100 10.20 9.01 12.47 0.65 6.37 100 10.41 7.33 12.12 0.67 6.44 width n mean min max std. dev. cv 100 3.99 3.03 5.03 0.42 10.53 100 3.91 2.86 5.06 0.44 11.25 100 3.85 2.23 5.01 0.51 13.25 100 5.29 3.91 6.90 0.69 13.04 100 5.30 4.22 6.90 0.62 11.70 100 5.55 4.16 7.04 0.54 9.73 100 5.42 3.76 7.76 0.94 17.34 100 5.83 4.59 7.58 0.68 11.66 100 5.71 3.50 7.43 0.73 12.78 length/ width n mean min max std. dev. cv 100 1.98 1.46 2.88 0.25 12.63 100 2.04 1.52 2.88 0.25 12.25 100 1.91 1.40 3.05 0.22 11.52 100 2.05 1.22 2.77 0.19 9.27 100 1.98 1.55 2.78 0.22 11.11 100 2.07 1.30 2.40 0.19 9.18 100 1.97 1.49 2.60 0.30 15.23 100 1.77 1.47 2.07 0.16 9.04 100 1.85 1.12 2.66 0.25 13.51 weight n mean min max std. dev. cv 100 0.065 0.034 0.119 0.018 27.06 100 0.071 0.032 0.113 0.020 28.55 100 0.078 0.036 0.132 0.020 25.61 100 0.182 0.066 0.307 0.050 27.48 100 0.160 0.082 0.250 0.037 22.85 100 0.209 0.107 0.328 0.048 22.84 100 0.180 0.062 0.362 0.066 36.54 100 0.220 0.102 0.376 0.0531 24.15 100 0.206 0.051 0.348 0.062 30.05 u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:01 color profile: disabled composite 150 lpi at 45 degrees 118 acta bot. croat. 71 (1), 2012 kremer d., juri[i] grube[i] r., popovi] z., karlovi] k. statistics b. croatica b. vulgaris vo 2008 vo 2009 vo 2010 rp 2008 rp 2009 rp 2010 pbg 2008 pbg 2009 pbg 2010 seed traits length n mean min max std. dev. cv 145 5.03 4.13 5.72 0.31 6.16 158 4.92 3.97 5.84 0.30 6.10 123 4.57 3.58 5.74 0.38 8.32 136 5.88 4.30 6.94 0.47 7.99 138 5.71 4.23 6.88 0.52 9.11 136 5.99 4.29 6.99 0.47 7.85 142 6.14 4.81 7.29 0.45 7.33 149 5.94 5.14 6.64 0.36 6.06 154 6.24 4.51 7.75 0.49 7.85 width 1 n mean min max std. dev. cv 145 2.10 1.28 2.90 0.26 12.38 158 2.06 1.22 2.51 0.23 11.17 123 2.20 1.30 2.60 0.18 8.18 136 2.57 1.43 3.09 0.27 10.51 138 2.55 1.54 3.16 0.35 13.73 136 2.71 2.03 3.14 0.23 8.49 142 2.46 1.78 3.28 0.33 13.41 149 2.40 1.87 3.14 0.32 13.33 154 2.50 1.68 3.32 0.31 12.40 width 2 n mean min max std. dev. cv 145 1.49 0.82 2.27 0.26 17.45 158 1.44 0.92 1.97 0.21 14.58 123 1.63 0.85 1.96 0.22 13.50 136 1.94 0.87 2.48 0.30 15.46 138 1.82 1.09 2.75 0.25 13.74 136 1.98 0.96 2.57 0.30 15.15 142 1.61 0.50 2.36 0.34 21.12 149 1.60 0.79 2.24 0.34 21.25 154 1.66 0.83 2.66 0.36 21.69 length/ width 1 n mean min max std. dev. cv 145 2.43 1.87 3.62 0.30 12.35 158 2.41 1.93 3.53 0.26 10.79 123 2.09 1.66 2.83 0.20 9.57 136 2.30 1.88 3.30 0.23 10.00 138 2.26 1.87 2.96 0.22 9.73 136 2.22 1.71 2.67 0.21 9.46 142 2.53 1.88 3.40 0.31 12.25 149 2.50 2.01 3.07 0.24 9.60 154 2.52 1.90 3.96 0.30 11.90 length/ width 2 n mean min max std. dev. cv 145 3.48 2.04 5.09 0.57 16.38 158 3.48 2.45 4.95 0.51 14.66 123 2.86 2.17 5.01 0.45 15.73 136 3.12 2.22 5.65 0.59 18.91 138 3.18 2.15 4.71 0.42 13.21 136 3.10 2.15 5.46 0.59 19.03 142 3.95 2.62 6.11 0.80 20.25 149 3.88 2.58 6.67 0.84 21.65 154 3.89 2.44 6.72 0.83 21.34 width 1/ width 2 n mean min max std. dev. cv 145 1.45 0.56 2.12 0.29 20.00 158 1.46 0.92 1.91 0.22 15.07 123 1.37 1.01 1.89 0.16 11.68 136 1.35 1.07 2.40 0.19 14.07 138 1.42 0.76 2.20 0.20 14.08 136 1.40 0.98 2.29 0.21 15.00 142 1.58 1.02 2.51 0.32 20.25 149 1.56 1.03 2.96 0.34 21.79 154 1.55 0.93 2.81 0.30 19.35 tab. 1. – continued u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:01 color profile: disabled composite 150 lpi at 45 degrees seeds of b. vulgaris were also larger than seeds of b. croatica. the length of b. croatica seeds ranged from 4.57 mm (vo 2010) to 5.03 mm (vo 2008), width 1 from 2.06 mm (vo 2009) to 2.20 mm (vo 2010), width 2 from 1.44 mm (vo 2009) to 1.63 mm (vo 2010) and the weight from 0.0116 g (vo 2008) to 0.0134 (vo 2010) g. in b. vulgaris seeds the length ranged from 5.71 mm (rp 2009) to 6.24 mm (pbg 2010), width 1 from 2.40 mm (pbg 2009) to 2.71 mm (rp 2010), width 2 from 1.60 mm (pbg 20) to 1.98 mm (rp 2010) and the weight from 0.0146 g (pbg 2010) to 0.0235 g (rp 2010). the most variable trait was seed weight, ranging from 20.15 % (vo 2010) to 24.14 % (vo 2008) and from 22.13 % (rp 2010) to 57.72 % (rp 2009) in b. croatica and b. vulgaris, respectively. the least variable trait was seed length and it ranged from 6.10 % (vo 2009) to 8.32 % (vo 2010) and from 6.06 % (pbg 2009) to 9.11 % (rp 2009) in b. croatica and b. vulgaris, respectively (tab. 1). anova showed significant statistical differences between species for most analyzed fruit and seed traits (tab. 2). the only exception was the ratio between fruit length and width as well as the number of seeds in the fruit. the analysis of fruit traits between species showed that fruits of b. vulgaris were significantly longer, wider and heavier than fruits of b. croatica (tab. 3). nevertheless, fruit shape (based on ratio between length and wide) was similar in the two species. acta bot. croat. 71 (1), 2012 119 berberis croatica and b. vulgaris fruit and seed traits statistics b. croatica b. vulgaris vo 2008 vo 2009 vo 2010 rp 2008 rp 2009 rp 2010 pbg 2008 pbg 2009 pbg 2010 weight n mean min max std. dev. cv 145 0.012 0.001 0.018 0.003 24.14 158 0.012 0.001 0.021 0.003 23.97 123 0.013 0.005 0.019 0.003 20.15 136 0.022 0.004 0.035 0.007 31.78 138 0.022 0.007 0.034 0.013 57.72 136 0.024 0.010 0.035 0.005 22.13 142 0.018 0.003 0.038 0.008 44.32 149 0.017 0.003 0.046 0.007 43.79 154 0.015 0.002 0.033 0.006 42.47 seed and fruit traits seeds in fruit n mean min max std. dev. cv 100 1.45 1.00 2.00 0.50 34.49 100 1.58 1.00 3.00 0.52 32.91 100 1.23 1.00 2.00 0.42 34.15 100 1.36 1.00 2.00 0.48 35.29 100 1.38 1.00 2.00 0.49 35.51 100 1.36 1.00 2.00 0.48 35.29 100 1.42 1.00 2.00 0.50 35.21 100 1.49 1.00 2.00 0.50 33.56 100 1.54 1.00 2.00 0.50 32.47 seed/fruit length 0.64 0.62 0.63 0.54 0.55 0.53 0.59 0.60 0.60 seed width 1/fruit width 0.61 0.62 0.54 0.43 0.43 0.40 0.47 0.43 0.44 seed width 2/fruit width 0.87 0.89 0.74 0.59 0.60 0.56 0.73 0.67 0.68 seed/fruit weight 0.177 0.170 0.172 0.119 0.137 0.113 0.098 0.077 0.071 tab. 1. – continued u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:01 color profile: disabled composite 150 lpi at 45 degrees 120 acta bot. croat. 71 (1), 2012 kremer d., juri[i] grube[i] r., popovi] z., karlovi] k. tab. 2. results of anova for analysed traits of fruit and seed. asterisks indicate significant p values at p£0.05. effect sum of squares degr. of freedom mean square f p-level fruit length population 1765.34 2 882.67 1608.8 <0.0001* species 1725.57 1 1725.57 2536.49 <0.0001* fruit width population 523.84 2 261.92 643.30 <0.0001* species 513.08 1 513.08 1161.04 <0.0001* fruit length/width population 4.504 2 2.252 40.52 <0.0001* species 0.202 1 0.202 2.89 0.0904 fruit weight population 2.99660 2 1.49830 832.78 <0.0001* species 2.94489 1 2.94489 1497.422 <0.0001* seed length population 347.93 2 173.97 977.2 <0.0001* species 335.54 1 335.54 1608.2 <0.0001* seed width 1 population 49.319 2 24.659 300.74 <0.0001* species 43.625 1 43.625 454.14 <0.0001* seed width 2 population 31.795 2 15.898 189.02 <0.0001* species 15.041 1 15.041 119.48 <0.0001* seed length/width 1 population 16.161 2 8.081 126.2 <0.0001* species 1.598 1 1.598 15.92 0.0001* seed length/width 2 population 142.38 2 71.19 165.92 <0.0001* species 18.93 1 18.93 25.67 <0.0001* seed width 1/width 2 population 6.560 2 3.280 48.44 <0.0001* species 0.826 1 0.826 10.08 0.0016* seed weight population 0.023150 2 0.011575 44.116 <0.0001* species 0.016446 1 0.016446 59.0474 <0.0001* seeds in fruit population 2.047 2 1.023 4.241 0.0153* species 0.005 1 0.005 0.020 0.8870 u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:01 color profile: disabled composite 150 lpi at 45 degrees anova of seed traits showed similar results (tab. 4). in general, seeds of b. vulgaris were significantly longer, wider and heavier than fruits of b. croatica. interspecies and acta bot. croat. 71 (1), 2012 121 berberis croatica and b. vulgaris fruit and seed traits tab. 3. p values from tukey post-hoc test for variability of fruits of berberis croatica in vo{ac and b. vulgaris in rakov potok and pharmaceutical botanical garden »fran ku{an« (pbg) populations. asterisks indicate significant p values at p£0.05. population vo{ac rakov potok population vo{ac rakov potok length length/width vo{ac vo{ac rakov potok <0.0001* rakov potok 0.1867 pbg <0.0001* 0.0001* pbg 0.9058 0.0757 width weight vo{ac vo{ac rakov potok <0.0001* rakov potok <0.0001* pbg <0.0001* 0.4388 pbg <0.0001* 0.9738 tab. 4. p values from tukey post-hoc test for variability of seeds of berberis croatica in vo{ac and b. vulgaris in rakov potok and pharmaceutical botanical garden »fran ku{an« (pbg) populations. asterisks indicate significant p values at p£0.05. population vo{ac rakov potok length vo{ac rakov potok <0.0001* pbg <0.0001* <0.0001* width 1 vo{ac rakov potok <0.0001* pbg <0.0001* 0.0058* width 2 vo{ac rakov potok <0.0001* pbg 0.0031* <0.0001* length/width 1 vo{ac rakov potok 0.0008* pbg 0.0163* <0.0001* population vo{ac rakov potok length/width 2 vo{ac rakov potok 0.0002* pbg <0.0001* <0.0001* width 1/width 2 vo{ac rakov potok 0.0198* pbg 0.0004* <0.0001* weight vo{ac rakov potok <0.0001* pbg <0.0001* <0.0001* seeds in fruit vo{ac rakov potok 0.4001 pbg 0.9030 0.6651 u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:01 color profile: disabled composite 150 lpi at 45 degrees intraspecies variability for seed traits was something higher than for fruit traits. the least interand intraspecies variability was recorded for the number of seeds in fruit. discussion the least variable traits in this analysis were fruit and seed length while the most variable traits were fruit and seed weight. that was expected since dimensionality considerations predict that, all else being equal, the coefficients of variability (cvs) of mass-related traits will be larger than those of linear or surface measurements (lande 1977), such as length and width of fruits and seeds. the largest variability for fruit and seed weight was also reported by obeso and herrera (1994) which state cvs for these traits ranging generally from 20–30 % for eight investigated species, including berberis hispanica. the same authors also reported fruit length and width variability ranging generally between 5 and 12 % (7.6 % and 7.4 % in b. hispanica for fruit length and fruit width, respectively). these results are in keeping with the results reported in this paper. regarding fruit traits of b. vulgaris, akbulut et al. (2009) reported much lower values for the wild population of b. vulgaris growing in turkey. for example, average fruit length, width and mass are reported to be 7.69 mm, 3.32 mm, and 0.07 g, (compared with the average value range for the same traits in this investigation, that is, 10.20–11.29 mm, 5.29–5.83 mm and 0.1602–0.2199 g). this could be explained by environmental factors but also by great intraspecific variation in berberis already reported by landrum (1999). generally, fruits and seeds of b. vulgaris were significantly longer, wider and heavier than fruits and seeds of b. croatica. this is in congruence with the results of karlovi] et al. (2009) who, when analyzing distinguishing morphological traits between these two species, stated that b. croatica is characterized by reduced growth/size traits compared with b. vulgaris. our results support this thesis. results regarding fruit and seed traits show significant differences between species for most analyzed traits (10 out of 12). nevertheless, whether the observed differences between b. croatica and b. vulgaris are merely non-hereditary modifications caused by environmental factors or species-related differences, should be further tested by means of genetic analysis. acknowledgements this work was supported by the ministry of science, education and sports of the republic of croatia (project »micromorphological and chemotaxonomic researches on some species of the family lamiaceae«). references akbulut, m., calisir, s., marakoglu, t., coklar, h., 2009: some physicomechanical and nutritional properties of barberry (berberis vulgaris l.) fruits. journal of food process engineering 32, 497–511. allen, r. b., wilson, j. b., 1992: fruit and seed production in berberis darwinii hook., a shrub recently naturalised in new zealand. new zealand journal of botany 30, 45–55. 122 acta bot. croat. 71 (1), 2012 kremer d., juri[i] grube[i] r., popovi] z., karlovi] k. u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:01 color profile: disabled composite 150 lpi at 45 degrees hendrix, s. d., sun, i. f., 1989: interand intraspecific variation in seed mass in seven species of umbellifer. new phytologist 112, 445–451. herrera, c. m., 1992: interspecific variation in fruit shape: allometry, phylogeny and adaptation to dispersal agents. ecology 73, 832–1841. herrera, c. m., 1998: population-level estimates of interannula variability in seed production: what do they actually tell us? oikos 82, 612–616. eriksson, o., 1999: seed size variation and its effect on germination and seedling performance in the clonal herb convallaria majalis. acta oecologica 20, 61–66. jordano, p., 1984: seed weight variation and differential avian dispersal in blackberries rubus ulmifolius. oikos 43, 149–l53. karlovi], k., kremer, d., liber, z., [atovi], z., vr[ek, i., 2009: intraand interpopulation variability and taxonomic status of berberis croatica horvat. plant biosystems 143, 40–46. lande, r., 1977: on comparing coefficients of variation. systematic zoo1ogy 26, 214–217. landrum, l. r., 1999: revision of berberis (berberidaceae) in chile and adjacent southern argentina. annals of the missouri botanical garden 86, 793–834. obeso, j. r., herrera, c. m., 1994: inter and intraspecific variation in fruit traits in co-occuring vertebrate dispersed plants. international journal of plant science 155, 382–387. rudolf, p. o., 1974: berberis, barberry, mahonia. in: schopmeyer, c. s. (tech. coord.), seeds of woody plants of the united states, 247–251. usda forest service, washington, dc. zovko kon^i], m., kremer, d., karlovi], k., kosalec, i., 2010: evaluation of antioxidant activities and phenolic content of berberis vulgaris l. and b. croatica horvat. food and chemical toxicology 48, 2176–2180. willson, m. f., michaels, h. j., bertin, r. i, benner, b., rice, s., lee, t. d., hartgerink, a. p., 1990: intraspecific variation in seed packaging. american midland naturalist 123, 179–185. acta bot. croat. 71 (1), 2012 123 berberis croatica and b. vulgaris fruit and seed traits u:\acta botanica\acta-botan 1-12\432 kremer.vp 26. o ujak 2012 11:25:01 color profile: disabled composite 150 lpi at 45 degrees 544 vizintin et al.vp acta bot. croat. 71 (2), 299–310, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 influence of environmental and spatial factors on the distribution of surface sediment diatoms in chaohu lake, southeast china xu chen1, 2, xiangdong yang2*, xuhui dong2, enfeng liu2 1 department of geography, faculty of earth sciences, china university of geosciences, wuhan 430074, people’s republic of china 2 state key laboratory of lake science and environment, nanjing institute of geography and limnology, chinese academy of sciences, nanjing 210008, people’s republic of china abstract – the spatial distribution of surface sediment diatoms in chaohu lake (southeast china), and their relationships with environmental and spatial variables were analyzed in this study. the diatom assemblages were dominated by planktonic species. three dominant species cyclostephanos dubius, aulacoseira granulata and aulacoseira alpigena are unevenly distributed across the lake. the distribution of surface sediment diatoms must be subject to trophic status, hydrodynamics and other spatial variables in the lake. keywords: aulacoseira, cyclostephanos, diatom, sediment, distribution, chaohu lake abbreviations: c fd – the percentage frequency dependent susceptibility, loi – loss on ignition, fpf – fine particle fraction, md – median grain size, pcnm – principal coordinates of neighbour matrices introduction the spatial structure of biological populations and communities plays a central role in many ecological theories, such as the theories of succession, adaptation, competition and so on (legendre and fortin 1989). in order to untangle the spatial patterns and include space as an explicit variable in ecological modeling, many methods have been proposed during the recent years, such as the geographic coordinate method, trend-surface analysis and principal coordinates of neighbour matrices (pcnm) analysis (cf. borcard et al. 2004). among these methods, pcnm analysis can achieve a spectral decomposition of the spatial relationships among the sampling sites (borcard and legendre 2002). and acta bot. croat. 71 (2), 2012 299 * corresponding author, e-mail: xdyang@niglas.ac.cn copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. pcnm analysis can be easily incorporated into canonical analysis models, providing a useful tool to assess the influence of spatial structure (dray et al. 2006). for instance, recent studies argued that the spatial factor had an important effect on the distribution of fish in lakes based on pcnm analysis (brind’amour et al. 2005, sharma et al. 2011). the spatial patterns of not only large organisms but also of various microorganisms (e.g., phytoplankton, cladoceran and chironomid) have attracted much attention recently (beisner et al. 2006, sweetman et al. 2010, cao et al. 2012). diatoms are unicellular and eukaryotic organisms, occurring throughout the world, growing in almost all aquatic environments (battarbee et al. 2001). the relationships between diatoms and environmental variables act a vital role in limnological and paleolimnological researches (smol and cumming 2000). most studies ignore the effect of spatial variables on diatom distribution. however, yang et al. (2009) indicated that spatial variables were a further influence on diatom distribution in the round loch of glenhead, a small lake in south-west scotland. unlike small lakes, large lakes always exhibit complex spatial gradients (nöges et al. 2008, cózar et al. 2012). it is assumed that spatial factors should impose an important role in diatom distribution in large lakes. in this study, we consider the importance of environmental and spatial factors on diatom distribution in chaohu lake, a large (770 km2) shallow lake in southeast china. diatoms and sedimentary proxies from surface sediments were analyzed in the laboratory. meanwhile, spatial factors were calculated using pcnm analysis. thereafter, we estimated the effects of spatial and environmental variables on surface sediment diatoms quantitatively, using the canonical analysis model. materials and methods study site chaohu lake (n 31°25’–31°43’, e 117°17’–117°51’) is the fifth largest freshwater lake located in the yangtze floodplain of southeast china (fig. 1). the chaohu watershed is located in a subtropical monsoon climate zone with an annual average temperature of ca. 16.1 °c, an annual average rainfall of ca. 996 mm and an annual mean wind speed of ca. 4.1 m s–1 (wang and dou 1998). the lake has an open water area of 770 km2 with a mean water depth of 3.0 m. between 1984 and 2006, the average concentrations of total phosphorus (tp), total nitrogen (tn) and chlorophyll a (chl a) were 256 mg l–1, 2850 mg l–1 and 20–40 mg l–1 respectively (xie 2009). the main sources of nutrients come from the inputs of the rivers located in the western region, including nanfei river and paihe river (fig.1) (shang and shang 2005). due to the increasing nitrogen and phosphorus inputs from industrial wastewater, domestic sewage and agricultural fertilizers since the late 1970s, the lake has suffered serious eutrophication in the past several decades. sampling and laboratory analyses thirty nine surface sediment samples were collected in june 2009 using a kajak gravity corer. the top 1 cm sediment was removed for surface sediment samples. all samples were transported back to the laboratory and stored at 4 °c. sedimentary proxies were analyzed in the state key laboratory of lake science and environment, including particle size, magnetic susceptibility, loss on ignition (loi) and diatoms. particle size spectra of the samples 300 acta bot. croat. 71 (2), 2012 chen x., yang x., dong x., liu e. were determined using a malvern automated laseroptical particle-size analyzer (mastersixer-2000). magnetic susceptibility was measured using a bartington instruments ms2 sensor. loi of samples was measured after 4 h of exposure at 550 °c (heiri et al. 2001). diatom samples were treated using hydrogen peroxide and hydrochloric acid in order to remove all organic and carbonate components (battarbee et al. 2001). microspheres were added to calculate the diatom concentration (battarbee and kneen 1982). all samples were mounted on microscope slides using the high refraction mountant naphrax®. diatoms were identified and counted using an olympus bx51 microscope with an oil immersion objective at a magnification of 10×100. diatom taxonomy mainly followed krammer and lange-bertalot (1986; 1988; 1991a, b). a minimum of 500 valves were identified and counted for each sample. spatial data gps coordinates of 39 sampling sites were recorded in the field. the original coordinate values were z-score transformed and these standard coordinates were used to create the dataset of spatial variables derived from pcnm using the program spacemaker2 (borcard and legendre 2004). a matrix of euclidean distances between samples was computed and subsequently truncated based on truncation distance, which was equal to or larger than the largest distance between neighbours. and then, a principal coordinate analysis (pcoa) was performed on the truncated distance matrix. thereafter, eigenvectors associated with positive eigenvalues were kept and used in the subsequent ordination analysis. acta bot. croat. 71 (2), 2012 301 distribution of surface sediment diatoms fig.1. location and map of chaohu lake (southeast china) and the sampling sites. data analysis all statistical analyses of diatom assemblages were based on percent abundances and included diatom taxa with ³ 1% abundance in at least one sample. diatom distribution data were represented in contour plots, performed using interpolation by a kriging approach with surfer software (golden software inc.). and the percentage data of diatom were square root transformed prior to ordination analyses. detrended correspondence analysis (dca) was applied to the diatom percentage data to explore the patterns of species changes and biological species turnover (the gradient length) (jongman et al. 1995). redundancy analysis or canonical correspondence analysis was selected based on gradient length to explore the relationships between diatom assemblages and explanatory variables (jongman et al. 1995). to reduce the possible effects of the difference in the number of variables included in each set of explanatory variables, we used only those variables that were significant based on a forward selection (p<0.05; n=499 unrestricted permutations). the significant variables were then classified into spatial category [s] and environmental category [e]. three steps were required to partition variance in diatoms between spatial and environmental categories. first, an rda with no covariables was used to measure the total amount of variance (as sum of canonical eigenvalues) in the diatom assemblages attributable to all explanatory variables [s+e] and the total unexplained variance (100– [s+e]). second, a partial rda was used to calculate variance explained by the unique effects of each category ([s] or [e]). in this step, ordination of one explanatory category was run with the other category as covariable. third, the interaction term [se] was calculated by subtracting appropriate terms generated during steps 1 and 2 (i.e., [se]=[s+e]–[s]– [e]). variance partitioning analysis is based on standard ordination methods (borcard et al. 1992). the ordination was performed using the program canoco version 4.5 (ter braak and [milauer 2002). results distribution of surface sediment diatoms a total of 24 genera and 68 species were identified in the 39 surface sediment samples. diatom assemblages were characterized by planktonic diatoms such as cyclostephanos dubius (hustedt) round, aulacoseira granulata (ehrenberg) simonsen and aulacoseira alpigena (grunow) krammer with few epiphytic or benthic species. and smaller abundances of cyclotella meneghiniana kützing, nitzschia palea (kützing) w. smith, tryblionella levidensis w.smith, gyrosigma acuminatum (kützing) rabenhorst and stephanodiscus sp. were observed in the samples. the total percentage of all three dominant species together (i.e., c. dubius, a. granulata and a. alpigena) was above 81% in each sample. the western part of the lake was dominated by c. dubius (fig. 2). in the central region of the lake, a. granulata was more abundant than the other species. a. alpigena, n. palea and stephanodiscus sp. were mainly found in the western and eastern basins. g. acuminatum was concentrated in the eastern region. t. levidensis and c. meneghiniana were widely distributed in the lake but with small higher abundance patches in different regions. environmental and spatial factors the environmental and spatial characteristics in the sampling sites are presented in figure 3. the percentage frequency dependent susceptibility (cfd%) showed minor differences 302 acta bot. croat. 71 (2), 2012 chen x., yang x., dong x., liu e. across the lake. unlike cfd, both loi and particle size showed visible spatial variations. the values of loi in the western basin were much higher than those in other basins. sediments in the central basin were characterized by coarse particles. spatial variables pcnm1 and pcnm2 were calculated and both of them were included in the later analysis. pcnm1 decreased from the eastern basin to the western basin. high values of pcnm2 occurred in the central basin. acta bot. croat. 71 (2), 2012 303 distribution of surface sediment diatoms fig. 2. spatial distribution of surface sediment diatoms concentrations (104 valves dry weight g–1) in chaohu lake. ordination analyses the results showed that the gradient length of dca axis 1 was 1.04 standard deviations, indicating that redundancy analysis (rda) was suitable for exploring the relationships between diatom assemblages and spatial and environmental factors (jongman et al. 1995). the environmental variables included cfd, loi, fine particle fraction (fpf) and median grain size (md). forward selection identified loi, fpf and spatial variables (i.e. pcnm1 and pcnm2) as significant environmental variables for the later ordination analysis (fig. 4). rda captured the variance in the species-environment and space relationship quite well; 77.2% by the first two axes (tab.1). the first ordination axis was positively related to loi (r=0.703), while it was negatively related to pcnm1 (r=–0.766) and pcnm2 (r= –0.197). the second ordination axis was negatively related to fpf (r=–0.581) and pcnm1 (r=–0.260) (tab.1). in the rda biplot (fig. 4), most of the samples in the eastern and central region were present in the ordination space characterized by high values of pcnm1 and pcnm2. samples in the western region of the lake were present in the space characterized by high values of loi. some of the samples in the central part of the lake were present in the space characterized by low values of fpf. variation partitioning spatial and environmental variables together were able to account for 30% of the variance in diatom data. pure environmental variance [e] (non-spatially structured environmental factors) accounted for 12.4%, pure spatial variance [s] for 11.2%, and 6.4% was atributed to the spatially structured components of the environmental variables [se] included in the analysis. seventy per cent of the variance in diatom data was unexplained by either the spatial or environmental variables considered in the analysis. 304 acta bot. croat. 71 (2), 2012 chen x., yang x., dong x., liu e. fig. 3. variations of environmental and spatial variables in the sampling sites. cfd – the percentage frequency dependent susceptibility, loi – loss on ignition, fpf – fine particle fraction, md – median grain size, pcnm – principal coordinates of neighbour matrices. discussion environmental factors and diatom distribution the diatom assemblages were dominated by three planktonic species (i.e., c. dubius, a. granulata and a. alpigena) in chaohu lake. the dominant species varied according to different parts in the lake. for example, the percentage of c. dubius was steadily above 60% in the samples of the western region. however, the diatom assemblages in the central region acta bot. croat. 71 (2), 2012 305 distribution of surface sediment diatoms tab.1. summary statistics for the redundancy analysis of diatom-environment and space. only environmental variables selected in the forward selection procedure are presented in the ordination. axes 1 2 3 4 eigenvalue 0.137 0.095 0.049 0.019 species-environment and space correlations 0.866 0.757 0.653 0.647 cumulative percentage variance of species data 13.7 23.1 28.1 30.0 of species-environment and space relation 45.6 77.2 93.6 100.0 correlation with species axes loi 0.703 –0.051 –0.211 –0.313 fpf 0.071 –0.581 –0.374 –0.180 pcnm1 –0.766 –0.260 –0.163 –0.128 pcnm2 –0.197 –0.077 0.632 –0.019 fig. 4. redundancy analysis ordination plot (axes 1 and 2). the arrows represent the significant explanatory variables explaining variation in the diatom assemblage. were characterized by aulacoseira granulata whose abundance was above 50% in each sample. compared with the two dominant species, a. alpigena was less abundant in each sample. and a. alpigena was mainly found in the western and eastern basins. the total of percentages of other species was less than 20% in each sample. loi has been widely used as a method to estimate the amount of organic matter in sediments, and it can be used as a proxy for paleoproductivity (santisteban et al. 2004). and in the yangtze floodplain lakes, loi can be indirectly indicative of lake trophic level (yao and xue 2009). rda indicated that there was a significantly positive correlation between loi and rda axis 1 (r=0.703), suggesting the influence of trophic level on the diatom assemblages. cyclostephanos dubius is a typical eutrophic diatom in many shallow nutrient-rich lakes all over the world (bradshaw and anderson 2003). higher nutrient concentrations (e.g, total nitrogen and total phosphorus) were observed in the western part of the lake due to the great amount of industrial and domestic sewage from hefei city (xu et al. 2003, xie 2009). therefore, the aggregation of c. dubius in the western region was in response to the high nutrient level in this area. in addition, the western region was also one aggregated area of other species (e.g., a. alpigena, t. levidensis, n. palea and cyclotella meneghiniana). these species were adapted to the mesotrophic or eutrophic status in the yangtze floodplain lakes (yang et al. 2008). fine particle fraction was another significant factor influencing diatom distribution. particle size fraction can provide information on hydrodynamic intensity in the yangtze floodplain lakes (chen et al. 2011, liu et al. 2012). coarser grains may indicate strong currents that are able to transport fine particles away; in contrast, finer grains may be indicative of weak hydrodynamic intensity. fpf strongly correlated to axis 2 (r=–0.581), suggesting the effect of hydrodynamic intensity on diatom distribution. for instance, the growth of a. granulata was sensitive to flow condition due to its fast sinking rate (hotzel and croome 1996). the strong hydrodynamic condition in the central basin, suggested by the coarser grains, would favor the development of a. granulata. the influence of spatial variables spatial structures observed in ecological communities can arise from two independent processes: (1) species’ response to environmental factors, which are usually spatially structured; and (2) spatial autocorrelation can also be created directly as a result of contagious biotic processes such as growth, seed dispersal or competition dynamics (fortin and dale 2005, dray et al. 2006). in most situations, the spatial structure of communities is due to the synchronous effect of these two processes. variance partitioning analysis (borcard et al. 1992) can be used to distinguish the effect of these two sources of spatial structure. in this study, we use partial ordination analysis to untangle the effects of »pure« spatial variance and spatially structured components of the environmental variables. the 11.2% of total variance in diatom data explained by the sole effect of spatial variables suggests that spatial factors must have a significant effect on diatom distribution in chaohu lake. generally, the first pcnm variables represent coarse patterns, and the last ones represent finer-scale patterns (borcard et al. 2004). pcnm1 showed the large-scale spatial gradient from the eastern to the western basin, while pcnm2 exhibited the finer-scale pattern. in addition, some environmental factors showed a visible spatial gradient, such as loi declining from the western to the eastern basin. it is assumed that the combined effect of 306 acta bot. croat. 71 (2), 2012 chen x., yang x., dong x., liu e. spatial and environmental variables must exert an influence on diatom distribution. the result indicated that 36.4% (=6.4/(6.4+11.2)) of the variance in spatial sub-models was due to the interactive effect of spatial and environmental factors. further research in this study, the combined contribution of environmental and spatial variables to the variance in diatom data was only 30%. the high unexplained contribution may result from inadequate measurement of limnological variables. in shallow lakes, wind-driven resuspension may be responsible for the spatial distribution of diatoms (yang et al. 2009). in addition, other chemical variables (e.g. tn, tp) of sediment can provide more information on microecological conditions influencing diatom distribution. therefore, more data about the hydrodynamic condition and chemical factors would improve the conclusions about the spatial distribution of diatoms in chaohu lake. conclusions surface sediment diatom assemblages were dominated by three planktonic species (i.e. cyclostephanos dubius, aulacoseira granulata and a. alpigena) in chaohu lake. these three dominant species were unevenly distributed in the lake. the results of ordination analyses indicated that loss on ignition, fine particle fraction, pcnm1 and pcnm2 were four significant factors influencing diatom distribution. it is assumed that the distribution of surface sediment diatoms must be subject to trophic status, hydrodynamic intensity and other spatial variables in the lake. the unique effect of spatial variables (i.e., pcnm1 and pcnm2) captured 11.2% of the variance in diatom data. although this value is not very high, the results recommend that spatial variables should be considered in the studies of diatom distribution in large shallow lakes. acknowledgements we would like to thank zhang enlou, pan hongxi, yao min, meng xianghua and du chenchang from nanjing institute of geography and limnology, chinese academy of sciences for their help in the field. this study was supported by the national natural science fund of china (40972217) and by national basic research program of china (2012 cb956100). references battarbee, r. w., jones, v. j., flower, r. j., cameron, n. g., bennion, h., carvalho, l., juggins, s., 2001: diatoms. in: smol, j. p., birks, h. j. b., last, w. m. 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(eds), 1998: memoirs of lakes in china. science press, beijing. xie, p., 2009: reading about the histories of cyanobacteria, eutrophication and geological evolution in lake chaohu (in chinese). science press, beijing. xu, f. l., tao, s., dawson, r. w., xu, z. r., 2003. the distributions and effects of nutrients in the sediments of a shallow eutrophic chinese lake. hydrobiologia 429, 85–93. yao, s., xue, b., 2009: recent environmental evolution of shijiuhu lake inferred from lake sediments (in chinese). quaternary research 29, 248–255. yang, x. d., anderson, n. j., dong, x. h., shen, j., 2008: surface sediment diatom assemblages and epilimnetic total phosphorus in large, shallow lakes of the yangtze acta bot. croat. 71 (2), 2012 309 distribution of surface sediment diatoms floodplain: their relationships and implications for assessing long-term eutrophication. freshwater biology 53, 1273–1290. yang, h., flower, r. j., battarbee, r. w., 2009. influence of environmental and spatial variables on the distribution of surface sediment diatoms in an upland loch, scotland. acta botanica croatica 68, 367–380. 310 acta bot. croat. 71 (2), 2012 chen x., yang x., dong x., liu e. opce-str.vp acta bot. croat. 73 (1), 51–62, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 salt stress tolerance in cowpea is poorly related to the ability to cope with oxidative stress sidney c. praxedes1, fábio m. damatta2, claudivan f. de lacerda3, josé t. prisco4, enéas gomes-filho4* 1 unidade acadêmica especializada em ciências agrárias, universidade federal do rio grande do norte, rn 160, km 03, distrito de jundiaí, 59280-000 macaíba-rn, brazil 2 departamento de biologia vegetal, universidade federal de viçosa, 36571-000 viçosa, minas gerais, brazil 3 departamento de engenharia agrícola, universidade federal do ceará, 60455-760 fortaleza, ceará, brazil 4 departamento de bioquímica e biologia molecular and instituto nacional de ciência e tecnologia em salinidade (inctsal/cnpq), universidade federal do ceará, caixa postal 6039, 60440-970 fortaleza, ceará, brazil abstract – we have previously demonstrated that salt tolerance in cowpea could be associated with lesser impairments of the photosynthetic capacity. taking into account that photosynthesis is the main sink for reducing power consumption, our central working hypothesis is that a salt-sensitive cultivar is more prone to suffer from oxidative stress. we analyzed the long-term effects of salt stress on oxidative damage and protection against reactive oxygen species in both leaves and roots of a salt-tolerant (pitiúba) and a salt-sensitive (tvu) cowpea cultivar. two salt treatments (0 and 75 mm nacl) were applied to 10-day-old plants grown in nutrient solution for 24 days. significant salt-induced oxidative damage as demonstrated via increases in malondialdehyde concentration were noted, particularly in leaves at the end of the experiment, although such damage was found earlier in pitiúba. in salt-stressed plants, superoxide dismutase (sod) activity increased only in pitiúba at 24 days from the start of salt additions (dssa). in pitiúba, catalase (cat) was not significantly affected by the treatments, whereas in tvu its activity was dramatically lower in salt-stressed plants at 10 dssa onwards. in general salt stress led to significant increases, much more pronounced in ascorbate peroxidase (apx), glutathione reductase (gr) and guaiacol peroxidase (gpx), at the end of the experiment in both cultivars. in roots, salt-induced increases in enzyme activities were particularly noted at 24 dssa, as found for sod and apx in pitiúba, cat in tvu and gr and gpx in both cultivars. therefore, in contrast to our expectations, the present results argue, to a great extent, against a functional link between salt stress tolerance and the expression of the antioxidant system. acta bot. croat. 73 (1), 2014 51 * corresponding author, e-mail: egomesf@ufc.br copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:41 color profile: generic cmyk printer profile composite default screen we also demonstrated that leaves and roots should be evaluated for a full assessment of whole plant acclimation to salt stress. key words: antioxidative defense, oxidative stress, salt tolerance, vigna unguiculata non-standard abbreviations: apx – ascorbate peroxidase; cat – catalase; dssa – days from the start of salt additions; gpx – guaiacol-type peroxidase; gr – glutathione reductase; mda – malondialdehyde; ros – reactive oxygen species; sod – superoxide dismutases introduction soil salinity is a major threat to global food security. up to 20% of the world’s irrigated land, which produces one third of the world’s food, is salt-affected. tolerance to salt stress is a complex trait involving the coordinated action of many gene families that perform a variety of functions such as control of water loss through stomata, ion sequestration, metabolic adjustments, osmotic adjustments and antioxidative defense (abogadallah 2010). in fact, salt-induced alterations in the physiology of the plants may provoke metabolic disturbances that trigger an excessive production of reactive oxygen species (ros) (hasegawa et al. 2000, ahmad et al. 2008). to cope with ros, plants are endowed with a complex nonenzymatic and enzymatic antioxidant system including superoxide dismutases (sod), which catalyze the reaction from superoxide to h2o2, and catalase (cat) and enzymes of the ascorbate/glutathione cycle, including ascorbate peroxidase (apx) and glutathione reductase (gr), which function to detoxify the h2o2 produced (asada 1999, mittler 2002). eventually, a guaiacol-type peroxidase (gpx) also participates in h2o2 scavenging (zhang and kirkham 1996). failure of the antioxidant defense system may result in oxidative damage to several cell constituents such as proteins, enzymes, dna and membrane lipids (asada 1999, mittler 2002). in spite of the large number of reports on the role of antioxidative defense under salt stress, the relative importance of the antioxidant system to the overall plant salt tolerance is still a contentious matter (parida and das 2005, munns and tester 2008, abogadallah 2010). on the one hand, the enzymatic ability to scavenge ros is assumed to be an essential component of salt tolerance, based on studies on mutant and transgenic plants with greater capacities to detoxify ros that showed improved salt tolerance (hasegawa et al. 2000, parida and das 2005). on the other hand, it has been suggested that genetic differences in salt tolerance are not necessarily due to differences in the enzymatic ability to scavenge ros (munns and tester 2008) probably because the defense mechanisms pathways are redundant (palatnik et al. 2002, munns and tester 2008). cowpea is one of world’s most important crops (ehlers and hall 1997) that are grown mostly in regions prone to salt stress. however, this species is as yet genetically underexploited (timko et al. 2008). little information on the basic processes involved in salt stress tolerance in cowpea is available. to the best of our knowledge, only two studies have explored the cowpea’s antioxidative responses to salt stress, either by analyzing only leaves (maia et al. 2010) or both leaves and roots (cavalcanti et al. 2007), though limited to short-term evaluations without consideration of the duration of exposure of the plants to salt stress. we believe that studies involving both leaves and roots are important for a better understanding of the long-term physiological performance of the whole plant under salt stress conditions. 52 acta bot. croat. 73 (1), 2014 praxedes s. c., damatta f. m., de lacerda c. f., prisco j. t., gomes-filho e. 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:41 color profile: generic cmyk printer profile composite default screen recently, we demonstrated that salt tolerance in cowpea was associated with restricted na+ accumulation in leaves, improved leaf area and lesser impairments of the photosynthetic capacity (praxedes et al. 2010). because photosynthesis is the main sink for reducing power consumption, our central working hypothesis is that a salt-sensitive cultivar is more prone to suffer from oxidative stress that, in turn, will probably aggravate its susceptibility to salt stress. we also hypothesized that leaves and roots display different antioxidative abilities to cope with salt stress. therefore, the present study was conducted to analyze the long-term effects of salt stress in both leaves and roots of two cowpea cultivars with contrasting tolerance to salt stress. material and methods plant material and growth conditions two cowpea [vigna unguiculata (l.) walp] cultivars displaying varying tolerance to salt stress were used: pitiúba (salt-tolerant) and tvu 2331 (salt-sensitive). these cultivars have been selected according to their growth responses to salinity, i.e., the tolerant cultivar displays less impaired growth and photosynthesis under salt conditions than does its sensitive counterpart (costa et al. 2003, praxedes et al. 2010). seeds were surface-sterilized in 1% sodium hypochlorite for 5 min, followed by rinsing three times in distilled water, after which they were sown in vermiculite moistened with distilled water. five days after sowing, uniform seedlings were transplanted into trays containing half-strength hoagland’s nutrient solution, and acclimated for 5 days. then, seedlings of each cultivar were transferred to 3-l pots containing the same nutrient solution and randomly subjected to two salt treatments: 0 (control) and 75 mm nacl. salt additions (at the rate of 25 mm d–1, starting 24 h after transferring the plants to the pots) were split over time in an attempt to avoid osmotic shock. all nutrient solutions were replaced every 6 days and kept aerated. their ph was checked daily and adjusted to 5.5, with 0.1 n naoh or 0.1 n hcl, when necessary. the amount of transpired water was checked daily by weighing the pots and was replaced with distilled water. the experiment was conducted in fortaleza (3.74°s, 38.58°w), northeastern brazil, under greenhouse conditions with mean midday photosynthetically active radiation of about 1,500 mmol m–2 s–1, temperature of 31.2 ± 5.8 °c, and relative humidity of 72 ± 10%. sample procedures on the day the plants were transferred to 3-l pots (day 0) and at 3, 10, 17, and 24 days from the start of salt additions (dssa), leaf (third fully expanded from the apex) and root (one-third of the apical portion) segments were collected between 10:00 and 11:00 a.m., flash frozen in liquid nitrogen and then freeze-dried, as described in azevedo-neto et al. (2006). lyophilized leaf (20 mg) and root (10 mg) powder were homogenized in a mortar and pestle with 1 ml of ice-cold extraction buffer (100 mm potassium phosphate, ph 7.0, 0.1 mm edta and 1 mm ascorbate). the homogenate was filtered through muslin cloth and centrifuged (20,000 g for 15 min). the supernatant fraction was used as crude extract for lipid peroxidation and enzyme activity assays. all the steps described above were carried out at about 4 °c. each replicate represented the mean of two determinations. acta bot. croat. 73 (1), 2014 53 salt tolerance in cowpea 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:41 color profile: generic cmyk printer profile composite default screen biochemical assays lipid peroxidation was determined by measuring the amount of malondialdehyde (mda) produced by the thiobarbituric acid reaction as described by heath and packer (1968). total superoxide dismutase (sod; ec 1.15.1.1) activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (nbt), as described by giannopolitis and ries (1977). the reaction mixture (1.5 ml) contained a suitable amount of extract and 50 mm potassium phosphate, ph 7.8, 0.1 mm edta, 13 mm methionine, 75 mm nbt and 2 mm riboflavin, which was the last to be added. the tubes were shaken and illuminated with two 20 w cool-white fluorescent lights. the reaction was allowed to proceed for 10 min and its absorbance was read at 560 nm. one unit of sod activity (u) was defined as the amount of enzyme required to cause 50% inhibition of the nbt photoreduction rate. other enzymes were assayed with suitable amount of extract and minor changes in the reaction media (described below): catalase (cat; ec 1.11.1.6), 100 mm potassium phosphate, ph 7.0, 0.1 mm edta, and 20 mm h2o2 (beers jr. and sizer 1952); ascorbate peroxidase (apx; ec 1.11.1.1), 50 mm potassium phosphate, ph 6.0, 0.1 mm edta, 0.5 mm ascorbate, and 1.0 mm h2o2 (nakano and asada 1981); glutathione reductase (gr; ec 1.6.4.2), 100 mm potassium phosphate, ph 7.8, 0.1 mm edta, 0.05 mm nadph, and 3 mm oxidized glutathione (foyer and halliwell 1976); and guaiacol peroxidase (gpx; ec 1.11.1.7), 100 mm potassium phosphate, ph 7.0, 0.1 mm edta, 5 mm guaiacol, and 15 mm h2o2 (urbanek et al. 1991). for cat and gpx the reactions were started by adding the crude extract. apx and gr reactions were started by the addition of h2o2 and oxidized glutathione, respectively. all enzymatic assays were carried out at 30 °c and were previously checked for linearity in relation to time of reaction and amount of extract used. experimental design and statistical analysis the plants were distributed over a completely randomized single-plant plot design, with four treatment combinations (two cultivars and two salt treatments) and five replicates. differences among cultivars, salt treatments and times of collection (0, 3, 10, 17 and 24 dssa) were tested using a three-way analysis of variance. student’s t test was used to compare the differences in salt treatments at the same dssa. to examine in further detail the coupling between oxidative stress and protection, pearson’s product-moment correlation was performed using mda pool and antioxidative enzyme activity data. data analysis and graph generation were made using the software r (r development core team 2012), version 2.13.1, according to sokal and rohlf (1995). results lipid peroxidation and antioxidant enzymes in leaves the salt stress treatments had similar effects on the leaf mda concentration in both cultivars (non-significant cultivar x stress interaction), but this effect was dependent on the time of exposure to salts (significant time x stress and time x stress x cultivars interactions) (tab. 1). regardless of cultivar, significant salt-induced increases in mda were noted par54 acta bot. croat. 73 (1), 2014 praxedes s. c., damatta f. m., de lacerda c. f., prisco j. t., gomes-filho e. 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:41 color profile: generic cmyk printer profile composite default screen ticularly at the end of the experiment, although such increases were found earlier in pitiúba (at 17 dssa) than in tvu 2331 (hereafter referred to as tvu) (figs. 1a, b). the salt stress also affected the antioxidant enzyme activities which was dependent on the time of exposure and cultivar (tab. 1). on average, all the enzymes that we analyzed displayed higher activities in tvu than in pitiúba (tab. 1; figs. 1, 2). in salt-stressed plants, sod activity increased (p < 0.01) at 24 dssa in pitiúba (fig. 1c), but not in tvu (fig. 1d), in which sod did not consistently change along the experiment. in pitiúba, cat was not significantly affected by the treatments, whereas in tvu cat activity was dramatically lower in salt-stressed plants as compared to their control counterparts, particularly at 10 dssa onwards (figs. 1e, f). with the exception of apx in pitiúba (at 3 dssa) and gpx in tvu (at 10 dssa onwards), salt stress brought about significant increases in apx, gr and gpx only at the end of the experiment, though such increases were much more pronounced in tvu than in pitiúba (figs. 1g–l). regardless of cultivars, both apx and gpx correlated positively (p < 0.01) with mda, whereas sod was also positively correlated with mda, but only in pitiúba. in contrast, cat correlated negatively with mda (p < 0.01) in both cultivars (tab. 2). lipid peroxidation and antioxidant enzymes in roots salt stress per se did not affect the mda concentration in either cultivar, even though the mda pools were significantly lower at 24 dssa in salt-treated plants than in control individuals for both cultivars (significant stress x time and cultivar x time interactions) (tab. 1; acta bot. croat. 73 (1), 2014 55 salt tolerance in cowpea tab. 1. three-way analysis of variance (f values) of the effect of cultivar (c), treatment (t) and time (tm) on malondialdehyde (mda) concentration and oxidative stress enzyme activities in leaves and roots. sod – superoxide dismutase; cat – catalase; apx – ascorbate peroxidase; gr – gluthatione reductase; gpx – guaiacol peroxidase. level of significance: p = 0.05 (*), p = 0.01 (**), n.s. – non-significant effect; cv – coefficient of variation. traits c t tm cxt cxtm txtm cxtxtm cv (%) leaves mda 0.01n.s. 47.52** 52.36** 0.22n.s. 0.78n.s. 6.58** 3.53 * 11.35 sod 65.83** 7.75** 4.97** 0.26n.s. 0.94n.s. 6.62** 4.09** 18.84 cat 8.67** 22.37** 9.28** 5.18 * 4.32** 1.58n.s. 1.74n.s. 35.13 apx 50.63** 43.49** 23.56** 12.54** 12.78** 15.83** 9.12** 30.16 gr 83.06** 1.06n.s. 4.73** 1.62n.s. 2.40n.s. 12.03** 4.47** 29.99 gpx 86.48** 69.10** 56.76** 36.36** 19.80** 12.47** 6.81** 36.60 roots mda 16.52** 0.05n.s. 11.21** 0.90n.s. 3.39** 6.77** 1.01n.s. 19.23 sod 6.54** 10.33** 7.48** 0.00n.s. 4.95** 8.92** 2.38n.s. 28.15 cat 0.01n.s. 1.70n.s. 2.04n.s. 0.00n.s. 2.03n.s. 3.69* 1.17n.s. 34.46 apx 6.07** 11.21** 10.12** 0.00n.s. 6.06** 9.29** 1.73n.s. 25.84 gr 2.29n.s. 0.01n.s. 13.75** 0.37n.s. 4.11** 10.45** 3.44* 25.54 gpx 103.41** 34.99** 265.86** 0.35n.s. 94.11** 38.53** 0.29n.s. 37.93 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:41 color profile: generic cmyk printer profile composite default screen 56 acta bot. croat. 73 (1), 2014 praxedes s. c., damatta f. m., de lacerda c. f., prisco j. t., gomes-filho e. fig. 1. leaf concentrations of malondialdehyde (mda; a, b), and activities of superoxide dismutase (sod; c, d), catalase (cat; e, f), ascorbate peroxidase (apx; g, h), gluthatione reductase (gr; i, j), guaiacol peroxidase (gpx; k, l) at different times from the start of salt additions, of two cowpea cultivars grown in nutrient solutions containing 0 (control, �) or 75 (salt stress, �) mm nacl. each point is the mean ± se of five replicates. level of significance: p £ 0.05 (*), p £ 0.01 (**) (student’s t test). 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:44 color profile: generic cmyk printer profile composite default screen figs. 2a, b). on average, the mda concentration was significantly lower in tvu than in pitiúba. regardless of the salt treatments, the average activities of sod, apx and gpx were slightly larger in tvu than in pitiúba, with no cultivar difference found for cat and gr activities (tab. 1, fig. 2). in general, salt-induced increases in enzyme activities were particularly noted at 24 dssa, as found for sod and apx in pitiúba, cat in tvu and gr and gpx in both cultivars (significant cultivar x time (except for cat) and stress x time interactions) (tab. 1, figs. 2c-l). notably, the activities of some antioxidative enzymes, especially sod and apx, were remarkably larger in roots than in leaves regardless of cultivar and salt treatment (cf. figs. 1, 2). irrespective of cultivar, no significant correlation was found between antioxidant enzymes and mda pools (tab. 2). discussion the effects of oxidative stress on membranes are frequently evaluated through the increases in mda concentration, a byproduct of lipid peroxidation (bor et al. 2003, pinheiro et al. 2004, sánchez-rodríguez et al. 2010). therefore, increases in mda levels have commonly been reported along with the exposition time to salts (bor et al. 2003, banu et al. 2009). here, we noted that the mda pools increased earlier in the leaves of the salt-tolerant pitiúba than in the salt-sensitive tvu, suggesting increased oxidative stress in that cultivar which could correlatively be associated with a less robust enzymatic antioxidant system in pitiúba than in tvu. on the other hand, the elevated antioxidant enzyme activities in tvu could be interpreted as an intrinsic higher tolerance to oxidative stress (overall, higher enzyme activities in both leaves and roots regardless of salt treatments). an upregulation of some antioxidant enzymes (particularly apx, gr and gpx) under salt stress conditions could also play an important role in efficiently scavenging ros that are expected to be produced at higher rates in tvu than in pitiúba due to larger decreases in both growth and photosynthetic rates and higher na+ accumulation in the former (praxedes et al. 2010) so that mda accumulation could be held in check. the ratio between the activity of sod and the activities of apx and cat has a pivotal role in controlling the cellular levels of the superoxide radical and hydrogen peroxide (mittler 2002). apx has higher affinity by hydrogen peroxide than cat, thus apx should acta bot. croat. 73 (1), 2014 57 salt tolerance in cowpea tab. 2. pearson’s product-moment correlations between malondialdehyde (mda) concentration and oxidative stress enzyme activities in leaves and roots. sod – superoxide dismutase; cat – catalase; apx – ascorbate peroxidase; gr – gluthatione reductase; gpx – guaiacol peroxidase. level of significance: p < 0.05 (*), p < 0.01 (**), n.s. – non-significant effect. pitiúba tvu leaves roots leaves roots sod 0.52** –0.18n.s. 0.13n.s. 0.09n.s. cat –0.37* 0.09n.s. –0.53** –0.15n.s. apx 0.42** 0.16n.s. 0.40** 0.02n.s. gr –0.29n.s. 0.07n.s. 0,10n.s. –0.39n.s. gpx 0.72** 0.08n.s. 0.67** 0.02n.s. 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:44 color profile: generic cmyk printer profile composite default screen 58 acta bot. croat. 73 (1), 2014 praxedes s. c., damatta f. m., de lacerda c. f., prisco j. t., gomes-filho e. fig. 2. root concentrations of malondialdehyde (mda; a, b), and activities of superoxide dismutase (sod; c, d), catalase (cat; e, f), ascorbate peroxidase (apx; g, h), gluthatione reductase (gr; i, j), guaiacol peroxidase (gpx; k, l) at different times from the start of salt additions, of two cowpea cultivars grown in nutrient solutions containing 0 (control, �) or 75 (�) mm nacl. see further details in legend to figure 1. 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:48 color profile: generic cmyk printer profile composite default screen be involved primarily in fine signal modulation against ros, while cat would be involved in the removal of ros excess during oxidative stress (mittler 2002). here, we found that whereas sod and apx activities were either stimulated or unchanged in response to salt stress in the leaves of both cultivars, cat activity dramatically decreased under salt-stress conditions, as noted in tvu. some studies have previously reported inhibition in cat activity in leaves of cowpea seedlings challenged with salt stress growing under soil conditions (cavalcanti et al. 2007, maia et al. 2010). cavalcanti et al. (2007) suggested that such inhibition was associated with irreversible damages suffered by the enzyme, which is markedly sensitive to salt excess (hertwig et al. 1992). therefore, we contend that damage in cat might have occurred here in tvu, which accumulated salts in leaves to a greater extent than pitiúba (praxedes et al. 2010). this might lend some support to explain the stronger negative correlation between cat and mda in tvu than in pitiúba. in any case, the significance of particular activities of ros scavenging enzymes should be interpreted based on the overall plant response to salt stress, the cellular levels of ros and the activities of other enzymes involved in ros scavenging in order to make a precise correlation between enzyme activity and plant acclimation to salt stress (abogadallah 2010). in this regard, higher activities of apx, gr and gpx could compensate for the decreased cat activity in salt-treated tvu plants in order to restrain the development of oxidative damage. this is circumstantially supported by the correlation analysis between the antioxidant enzymes and mda concentrations in both cultivars, that is, the greater the oxidative pressure (higher mda), the greater the activities of sod, apx and gpx and sod. in contrast with leaves, the levels of mda in roots ultimately decreased at the end of the experiment in salt-treated plants from both cultivars as compared with untreated individuals, which was accompanied by increased activities of some antioxidant enzymes. notably, the mda concentration tended to be lower in roots than in leaves in both cultivars, which could be related to the higher activities of some enzymes such as sod and apx in roots than in leaves. overall, our results contrast with those reported by maia et al. (2010). these authors found positive correlations between leaf sod, apx or gpx activities with salt stress tolerance, but these results were obtained using another cowpea genotype as a salt-sensitive cultivar with different age and grown under conditions different from those used in this current study. although we have highlighted the importance of antioxidant enzymes in cowpea acclimation to salt stress, no positive correlation between increases in the activities of enzymes and salt stress tolerance was found in this current long-term study. therefore, the enzymatic protection mechanism against salt stress might be a good selection marker for some species or genotypes but not for others, as had already been shown by abogadallah (2010). conclusions despite some changes found in the expression of the enzymatic antioxidant system, particularly in leaves, the present results argue against our working hypothesis that the salt-sensitive cultivar is more prone to suffer from oxidative stress. however, the nonenzymatic system was not evaluated and accordingly merits attention in future studies. we also demonstrated that leaves and roots displayed varying antioxidant abilities to cope with salt stress and so acta bot. croat. 73 (1), 2014 59 salt tolerance in cowpea 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:48 color profile: generic cmyk printer profile composite default screen both types of organs should be evaluated for a full assessment of whole-plant acclimation to salt stress. acknowledgement the authors would like to thank the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for their financial support and the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for a phd scholarship to s. c. praxedes. references abogadallah, g. m., 2010: antioxidative defense under salt stress. plant signaling and behavior 5, 369–374. ahmad, p., sarwat, m., sharma, s., 2008: reactive 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environmental safety 60, 324–349. pinheiro, h. a., damatta, f. m., chaves, a. r. m., fontes, e. p. b., loureiro, m. e., 2004: drought tolerance in relation to protection against oxidative stress in clones of coffea canephora subjected to long-term drought. plant science 167, 1307–1314. praxedes, s. c., lacerda, c. f., damatta, f. m., prisco, j. t., gomes-filho, e., 2010: salt tolerance is associated with differences in ion accumulation, biomass allocation and photosynthesis in cowpea cultivars. journal of agronomy and crop science 196, 193–204. r development core team, 2012: r: a language and environment for statistical computing. r foundation for statistical computing, vienna. retrieved april 18, 2012 from http://www.r-project.org sánchez-rodríguez, e., rubio-wilhelmi, m. m., cervilla, l. m., blasco, b., rios, j. j., rosales, m. a., romero, l., ruiz, j. m., 2010: genotypic differences in some physiological parameters symptomatic for oxidative stress under moderate drought in tomato plants. plant science 178, 30–40. sokal, r. r., rohlf, f. j., 1995: biometry: the principles and practice of statistics in biological research. w. freeman and company, new york, usa. timko, m. p., rushton, p. j., laudeman, t. w., bokowiec, m. t., chipumuro, e., cheung, f., town, c. d., chen, x., 2008: sequencing and analysis of the gene-rich space of cowpea. bmc genomics 9, 103–123. acta bot. croat. 73 (1), 2014 61 salt tolerance in cowpea 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:48 color profile: generic cmyk printer profile composite default screen urbanek, h., kuzniak-gebarowska, e., herka, k., 1991: elicitation of defense responses in bean leaves by botrytis cinerea polygalacturonase. acta physiologiae plantarum 13, 43–50. zhang, j., kirkham, m. b., 1996: antioxidant responses to drought in sunflower and sorghum seedlings. new phytologist 132, 361–373. 62 acta bot. croat. 73 (1), 2014 praxedes s. c., damatta f. m., de lacerda c. f., prisco j. t., gomes-filho e. 708 praxedes et al.ps u:\acta botanica\acta-botan 1-14\708 praxedes et al.vp 19. o ujak 2014 16:48:48 color profile: generic cmyk printer profile composite default screen 625 pise and gaikwad.vp acta bot. croat. 72 (2), 295–310, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/v10184-012-0021-9 interactions between leaf macronutrients, micronutrients and soil properties in pistachio (pistacia vera l.) orchards prodromos koukoulakis1, christos chatzissavvidis2*, aristotelis papadopoulos1, dimitrios pontikis3 1 national agricultural research foundation, soil science institute, 57001 thermi, greece 2 democritus university of thrace, department of agricultural development, pantazidou 193, orestiada 68200, greece 3 agromichaniki ltd, anthilis 13, 35100 lamia, greece abstract – the interactions between: (i) leaf dry matter macronutrietns, micronutrients and soil chemical properties, (ii) leaf macroand micronutrients, (iii) soil macroand micronutrients and (iv) soil chemical properties, and soil microand macronutrients in 50 pistachio orchards were investigated in leaves and soils by means of regression analysis. most of the soils were deficient in plant-available p, zn, mn, fe, and b, while they were excessively supplied with cu. leaf analysis showed that most of the trees were sufficient in k, mg, mn and b, but deficient in n, p and fe, and excessive in zn and cu. it was found that almost all the significant elemental interactions occurring in pistachio leaves or soils were synergistic, contributing considerable quantities of available nutrients and, therefore, improving the nutrient status of pistachio trees, and the level of soil fertility. on the other hand, the interactions between k and mg in leaves, and between soil ph and leaf n or soil fe, mn and b, were antagonistic. it is suggested that these results must be taken into account during fertilization of pistachio trees, in order to avoid nutritional disorders and to promote plant growth, productivity and nut quality. keywords: pistacia vera, plant nutrition, leaf, macronutrients, micronutrients, soil introduction in greece, pistachio (pistacia vera l.) trees are cultivated on about 5,000 ha and produce 9,000 tons of fruit (fao 2008). it is an economically important species that produces nuts of high nutritional value. the proper nutrition of pistachio trees is a basic prerequisite for the production of nuts of excellent quality and high commercial value. the high producacta bot. croat. 72 (2), 2013 295 * corresponding author, e-mail: cchatz@agro.duth.gr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. tivity of pistachio trees makes necessary, among other factors, knowledge of: a) the interactions between leaf dry matter (d.m.) nutrient content, and chemical soil characteristics and b) the interrelations among nutrient elements in leaves. the interactions between leaf macroand micronutrients, also between leaf nutrients and the soil properties, as well as between soil macroand micronutrients, and between nutrients in soil and soil properties have been studied sporadically and in a very general way (brady and weil 2002). according to relatively recent work, there is a direct relationship between the plant nutrient content and soil properties (kizilgoz et al. 2001). usually, the interactions between soil characteristics and nutrient elements contribute negatively to the available level of nutrients in pistachio, with serious consequences to nut yield and quality. the low ph is correlated positively with the availability of most micronutrients (e.g. zn, mn, fe) and p (olsen 1972). a high caco3 concentration in combination with a highly alkaline soil accentuates plant growth and yields, notwithstanding any reduction in nutrient availability (mengel and kirkby 1987). organic matter in soil is related positively to an increase of nutrient availability due either to the favorable effect on decomposition of the rocks and minerals or to the increase of cation exchange capacity (cec) (tisdale et al. 1993, koukoulakis et al. 2000). moreover, clay positively affects the increase of available cations (ca++, mg++, k+) (brady and weil 2002). the interactions between nutrients have been studied to a certain extent in several crops, but not in pistachios. there is a particular shortage of relevant data concerning the elemental contribution of these interactions in pistachio cultivation, at least under the conditions of greece.. according to dibb and thompson (1985), the nutrient concentrations in plants depend on the rate of their absorption, translocation and accumulation, and during these processes the elemental interactions within the plants may play an important role (kalavrouziotis and koukoulakis 2009a). the aim of this work was to investigate the elemental contribution of the interactions between nutrients in leaves, and in soils, as well as between soil properties and nutrients, and their effect on soil fertility and on the nutrient status of pistachio trees. materials and methods planting material and sampling procedure fifty productive pistachio (pistacia vera cv. aegenes) orchards were selected in various locations of the fthiotida district (south greece). the trees were grafted on the rootstock 'tsikoudia' (pistacia terebinthus cv. tsikoudia), trained as a vase and they were mostly planted at a spacing of 6.5–7.0 m between and within rows. standard commercial cultural practices were followed during the survey. representative soil samples from a depth of 0 to 0.30 m were collected from 20 cores in each orchard. the sampling points (four cores per tree) were located 2 m from the trunk, along and between tree rows. at the end of july, ten mature leaves per tree were collected from the middle node of non-bearing 1-year-old shoots and from all around the periphery of the canopy. soil analyses each sample was air-dried, crushed, sieved through a 2-mm mesh sieve and tested for ph, free caco3 and organic matter (om). the basic soil characteristics were determined 296 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. according to the accepted methods (jackson 1958). the clay content of the soil samples was determined using the bouyoucos method (boujoucos 1962). most soils of the orchards studied (92%) had a high clay content, which ranged between 44 and 74%. the remaining 8% of the soils contained clay 30–38%, and they are characterized as sand-clay-loamy (scl) and clay-loamy (cl) soils. as regards ph, 82% of the studied soils may be characterized as moderately alkaline, with 74% of them presenting ph values between 7.5 and 8.0. interestingly, 79% of the studied orchards presented the optimum range of ph values (7.5–8.5) for pistachio tree growth. seventy four percent of the soils had a high caco3 content (above 4%), while the organic matter content was low (below 1%) for 54% of the studied soils and medium (1–2%) for 38% of the studied soils. moreover, soil electrical conductivity was low and ranged between 0.06 and 0.70 ms cm–1 with a mean value of 0.40 ms cm–1. the available soil nutrients were extracted by the following methods: p by olsen’s procedure, exchangeable k and mg by ammonium acetate, b by hot water, and the micronutrients (mn, zn, fe and cu) by dtpa (diethylenetriamine pentaacetic acid). analytical determination of the elements k, mg, fe, mn, zn and cu in the soil samples was performed by atomic absorption spectroscopy (perkin-elmer 2340) using standard methods (chapman and pratt 1961). phosphorus was analyzed by the vanado-molybdo-phosphate yellow complex (chapman and pratt 1961). finally, b was determined by the azomethine-h method (wolf 1971). leaf tissue analyses the leaf samples were washed twice with distilled water, air-dried at 85 oc for 48h (till constant weight was obtained) and ground in a mill to pass through a thirty-mesh screen. tissue b extraction was made by dry ashing of a 0.5 g sample in a muffle furnace at 500 oc for 6 h. the ash was dissolved in 10 ml of 0.1 n hydrochloric acid (hcl) and b was determined colorimetrically (420 nm) by the azomethine-h method (wolf 1971). the analytical determination of n was performed by the kjeldahl method (chapman and pratt 1961). phosphorus, k, mg, fe, mn, zn and cu analyses were conducted by dry ashing of 0.5 g of dried tissue for 6h at 550 oc. subsequently, the ash was dissolved in 3 ml of 6n hcl and the solution was diluted with deionised water to 50 ml final volume. phosphorus concentration was determined by the vanado-molybdo-phosphate yellow complex method, while k, mg, fe, mn, zn and cu were determined by atomic absorption spectroscopy (perkin-elmer 2340) using standard methods (chapman and pratt 1961). statistical analysis in each orchard, thirty uniform size pistachio trees were selected and they were separated in five blocks. each replicate block consisted of six trees. regression models were developed for each interaction found among soil properties, i.e. ph, caco3, clay, organic matter and leaf n, p, k, mg, zn, fe, mn, b and cu concentrations, as well as among leaf and soil nutrients. the estimators in the models were tested by t-test and the overall regression model by f-test at a level of significance of 0.05 and 0.001. moreover, regression coefficients of the models were determined. all the regression models were obtained by using the statistical package of spss version 17. acta bot. croat. 72 (2), 2013 297 nutrient elements in pistachio trees in order to quantify the percent elemental contribution (pec) of an interaction, the procedure developed by kalavrouziotis et al. (2010), was modified with respect to the calculation of pec. more specifically, the quantification procedure and the modification mentioned are explained below in four steps. 1) regression analysis is run between the concentrations of elements of soil or of plant dry matter, as given by the available analytical experimental data, and the statistically significant regression equations are chosen. 2) the interactions corresponding to the statistically significant regression equations, are classified on the basis of the same dependent variable, i.e. x0*y, x1*y, x2*y etc., where x and y represent a different element, for example if x0 is p, x1 is zn, x2 is mn, and y fe, then the interactions will be p-fe, zn-fe, and mn-fe. as can be seen, the dependent variable is written on the right to avoid confusion. obviously, each one of the elements studied can be a dependent or independent variable according to which one is to be contributed. the number of interactions with the same dependent variable is not constant, but it varies according to the interactive capacity of the elements that participate in the interactions, and their respective concentration levels. in calculating the pec, the mean value of the calculated dependent variables is found by dividing their total sum by the number of interactions involved. 3) the statistically significant equations are solved for the maximum (xmax) and the minimum (xmin) values of the independent variable, as given by the analytical data. thus, the calculated maximum and minimum values are the dependent variables ymaxcl and ymincl, respectively, are found, and the difference between them is determined. 4) also, the maximum analytical value of the dependent variable (ymaxan) and minimum values (yminan) are taken respectively from the analytical experimental data, and the pec is calculated by means of the relation 1: pec = (ymaxcl – ymincl) ´ 100/(ymaxan – yminan) (1) where ymaxcl represents the calculated maximum value of the dependent variable in mg kg–1, ymincl represents the calculated minimum value of the dependent variable in mg kg–1, ymaxan represents the maximum value of the dependent variable obtained from the set of the existing analytical data of soil or plant tissue analysis (in mg kg–1), and yminan represents the minimum value of the dependent variable, obtained from the set of the existing analytical data of soil or plant tissue analysis (in mg kg–1). it is noted that the relation 1 can be used for the calculation of pec for interactions occurring either in the soil or in the plant tissues. it should be mentioned at this point that the mathematical relation for the calculation of pec, given in the publication of kalavrouziotis et al. (2010), was the following: pec= (ymaxcl– ymincl) ´100/emc (2) where ymaxcl, ymincl represent the maximum and minimum calculated values of the dependent variable in mg kg–1, and emc represents the mean total plant dry matter or soil content of the element representing the dependent variable. as it can be seen, relation 2 did not take into account the observed actual difference (ymaxan – yminan), but only the difference between the calculated values of the dependent variables, and the mean dry matter concentration (eec), of the dependent variable, giving not very satisfactory results. therefore, after a de298 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. tailed consideration, we modified the calculation of pec and replaced the above relation 2 with the relation 1, as mentioned above. results interactions between soil properties and leaf nutrient concentration regression equations of the statistically significant interrelations between leaf nutrient concentration and soil properties (ph, organic matter (om), caco3 (cc), and soil clay content (c)), are given in table 1. the interactions ph ´ n (fig. 1c) and caco3 ´ cu (fig. 1d) and c ´ mn (fig. 1e) are antagonistic, suggesting that the increase of ph or of caco3 or of c decreases the leaf n, cu and mn concentrations, respectively. on the other hand, the interactions om ´ zn (fig. 1a), and om ´ b (fig. 1b) are synergistic contributing to pistachio leaf zn and b, respectively. the results of the interaction quantification procedure, as expressed by the percent elemental contribution (pec), are shown in table 2. the negative figures, being the result of antagonistic interactions, reflect a decrease of the respective element in the soil or in the plant respectively. on the other hand, the positive figures show an increase of the nutrient level. it is noted that most of the figures are positive, a fact that emphasizes the synergistic nature of most of the interactions that occurred in the soil and in plants, underlining the importance of the elemental interactions. interactions between leaf nutrients seventeen interactions (regression equations) among pistachio leaf macroand micronutrients were statistically significant out of a total 81 interactions (9 ´ 9) (tab. 3). from these 17 interactions 12 were synergistic (s), meaning that they contributed to leaves the respective quantities of the interacting elements, one was antagonistic (a), and four synergistic-antagonistic (s-a). according to interactions between macroand micronutrients occurring in pistachio leaves (figs. 2 a–f), it is obvious that the elemental interactions among leaf nutrients are mainly described by quadratic, and secondarily by logarithmic regression equations. in addition, concerning the above interactions, the respective pec values are presented in table 2. acta bot. croat. 72 (2), 2013 299 nutrient elements in pistachio trees tab. 1. interactions between pistachio leaf nutrients and soil properties. no interactions regression equations r sig. type 1 ph ´ n n = 0.275 ´ (ph)2 – 3.969 ´ (ph) + 16.258 0.439 0.007 aa 2 cc ´ cu cu = –0.048 ´ (cc)2 – 0.101 ´ (cc) + 5.73 0.356 0.041 a 3 om ´ zn zn = –7.593 ´ (om)2 + 21.67 ´ (om) + 8.037 0.363 0.036 sb 4 om ´ b b = –8.287 ´ (om)2 + 18.054 ´ (om) + 78.905 0.341 0.055 s 5 c ´ mn mn = 0.037 ´ (c)2 – 4.501 ´ (c) + 162.202 0.350 0.046 a cc=caco3, om=organic matter, c= clay a antagonistic b synergistic 300 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. 10 20 30 40 50 60 0 0.5 1 1.5 2 2.5 l e a f z n ( µ g g 1 ) organic matter (%) (a) 60 80 100 120 140 0 0.5 1 1.5 2 2.5 l e a f b ( µ g g 1 ) organic matter (%) (b) 1.4 1.9 2.4 2.9 6.5 7 7.5 8 8.5 l e a f n ( % ) soil ph (c) 0 20 40 60 80 0 5 10 15 20 25 l e a f c u ( µ g g 1 ) soil caco 3 (%) (d) 0 20 40 60 80 25 35 45 55 65 75 85 l e a f m n ( µ g g 1 ) soil clay (%) (e) fig. 1. interactions of pistachio leaf zn, b, n, cu and mn concentrations with soil organic matter, organic matter, ph, caco3 and clay, respectively. elemental contribution to soil by the interactions between soil nutrients, and soil properties according to regression equations of the interactions between soil nutrients and soil properties (tab. 4) the interactions om ´ k, c ´ k, c ´ mg, caco3 ´ zn and om ´ zn were synergistic while those of caco3 ´ fe, ph ´ fe, caco3 ´ mn, ph ´ mn, ph ´ b, caco3 ´ b acta bot. croat. 72 (2), 2013 301 nutrient elements in pistachio trees tab. 2. percent elemental contribution values by the interactions among i) soil properties and leaf nutrients, ii) leaf nutrients, iii) soil properties and soil nutrients, and iv) soil nutrients. elements i ii iii iv n –7.45 41.80 0.0 0.0 p 0.0 0.0 0.0 15.21 k 0.0 –14.33 29.20 47.09 mg b fe mn zn cu 0.0 5.18 0.0 –26.80 13.53 –8.16 12.90 22.73 4.64 3.14 20.07 23.07 –3.67 31.15 –31.97 –18.61 4.56 0.0 17.76 0.0 13.83 2.10 13.59 13.15 tab. 3. interactions among nutrients in pistachio leaves. no interaction regression equations r sig. type 1 k ´ n n = –0.37 ´ (k)2 + 1.046 ´ (k) + 6.169 0.350 0.046 sa 2 mg ´ n n = 0.173 ´ (mg) + 1.717 0.286 0.044 s 3 mn ´ n n = 8.11 ´ 10–5 ´ (mn)2 + 0.009 ´ (mn) + 1.645 0.371 0.031 (s-ab) 4 mg ´ k k = ln(mg) ´ (–0.184) + 0.905 0.278 0.050 a 5 n ´ mg mg = ln(n) ´ (0.863) + 0.125 0.280 0.049 s 6 zn ´ fe fe = 0.018 ´ (zn)2 + 0.261 ´ (zn) + 50.438 0.440 0.006 s 7 mn ´ fe fe = 0.002 ´ (mn)2 + 0.403 ´ (mn) + 46.988 0.535 0.000 s 8 mg ´ zn zn = –13.297 ´ (mg)2 + 35.126 ´ (mg) + 3.827 0.375 0.028 (s-a) 9 fe ´ zn zn = 0.147 ´ (fe) + 10.373 0.429 0.002 s 10 mn ´ zn zn = 0.008 ´ (mn)2 – 0.105 ´ (mn) + 18.822 0.496 0.001 s 11 cu ´ zn zn = –0.003 ´ (cu)2 + 0.371 ´ (cu) + 10.781 0.377 0.027 s 12 fe ´ mn mn = –0.002 ´ (fe)2 + 0.835 ´ (fe) – 8.556 0.540 0.000 s 13 zn ´ mn mn = 0.033 ´ (zn)2 – 0.621 ´ (zn) + 31.975 0.470 0.003 s 14 b ´ mn mn = –0.021 ´ (b)2 + 4.415 ´ (b) – 186.661 0.368 0.033 s 15 mg ´ cu cu = –29.57 ´ (mg)2 + 72.103 ´ (mg) + 3.038 0.416 0.011 (s-a) 16 zn ´ cu cu = –0.021 ´ (zn)2 + 1.889 ´ (zn) + 6.997 0.400 0.017 s 17 mn ´ b b = –0.003 ´ (mn)2 + 0.471 ´ (mn) + 75.14 0.196 0.006 (s-a) a synergistic b antagonistic and c ´ b were antagonistic. most of the interactions between soil properties and soil nutrients are described by quadratic regression equations. in order to have a more concrete idea about the elemental contribution to soil, by the interactions mentioned in table 4, a quantification procedure was applied and the results are shown in table 2. 302 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. 0 20 40 60 80 100 120 140 60 70 80 90 100 110 l e a f m n ( m g g 1 ) leaf b ( mg g -1 ) (a) 0 20 40 60 80 100 120 140 0 50 100 150 l e a f m n ( m g g 1 ) leaf fe ( mg g -1 ) (b) 0 40 80 120 160 0 40 80 120 l e a f f e ( m g g 1 ) leaf mn ( mg g -1 ) (c) 0.5 1 1.5 2 0 0.5 1 1.5 2 2.5 l e a f k ( % ) leaf mg (%) (d) 1.4 1.6 1.8 2 2.2 2.4 0 0.5 1 1.5 2 2.5 l e a f n ( % ) leaf mg (%) (e) 1.4 1.6 1.8 2 2.2 2.4 0.5 1 1.5 2 l e a f n ( % ) leaf k (%) (f) fig. 2. interactions among nutrients in pistachio leaves. elemental contribution to soil by the interactions between soil nutrients most of the statistically significant interactions between soil macroand micronutrients (tab. 5) were described by quadratic (fig. 3 a, d, e, g, h) and some of them by logarithmic equations (fig. 3 b, c, f). it can be seen that almost all of these interactions were synergistic with the exception of p ´ zn (fig. 3 d), which was 'synergistic-antagonistic' (s-a). therefore, the elemental contribution of these interactions was positive, i.e. significant quantities acta bot. croat. 72 (2), 2013 303 nutrient elements in pistachio trees tab. 4. interactions among soil properties and nutrients occurring in pistachio orchard soils. no interactions regression equations r sig. type 1 om ´ k k = –23.505 ´ (om)2 + 195.03 ´ (om) + 86.12 0.549 0.000 sa 2 c ´ k k = in(c) ´ 410.1 – 1325.823 0.500 0.000 s 3 c ´ mg mg = 0.714(c)2 – 61.268(c) + 1759.68 0.432 0.007 s 4 cc ´ fe fe = 0.01 ´ (cc)2 – 0.365 ´ (cc) + 4.977 0.373 0.029 ab 5 ph ´ fe fe = 6.616 ´ (ph)2 – 95.376 ´ (ph) + 371.1 0.653 0.000 a 6 cc ´ mn mn = –0.079 ´ (cc) + 2.219 0.286 0.044 a 7 ph ´ mn mn = ln(ph) ´ (–9.86) + 21.802 0.339 0.016 a 8 cc ´ zn zn = 0.023 ´ (cc)2 – 0.34 ´ (cc) + 2.932 0.359 0.039 s 9 om ´ zn zn = 0.394 ´ (om)2 + 2.04 ´ (om) – 0.219 0.548 0.000 s 10 ph ´ b b = 0.066 ´ (ph)2 – 1.03 ´ (ph) + 4.47 0.447 0.005 a 11 cc ´ b b = –0.003 ´ (cc) + 0.480 0.398 0.004 a 12 c ´ b b = ln(c) ´ (–0.079) + 0.76 0.342 0.015 a c=clay, cc=caco3, om =organic matter asynergistic bantagonistic tab. 5. interactions among soil nutrients occurring in pistachio orchards. no interactions regression equations r sig. type 1 fe ´ p p = –0.204 ´ (fe)2 + 3.579 ´ (fe) + 10.076 0.363 0.036 sa 2 p ´ k k = –0.315 ´ (p)2 + 20.165 ´ (p) + 33.757 0.525 0.001 s 3 cu ´ k k = ln(cu) ´ 67.828 + 214.352 0.657 0.000 s 4 p ´ mg mg = ln(p) ´ 114.341 + 212.76 0.274 0.054 s 5 fe ´ mn mn = 0.03 ´ (fe)2 – 0.108 ´ (fe) + 1.511 0.529 0.000 s 6 p ´ fe fe = ln(p) ´ 1.105 + 0.038 0.303 0.033 s 7 p ´ zn zn = –0.009 ´ (p)2 + 0.434 ´ (p) – 1.505 0.386 0.023 (s-ab) 8 k ´ zn zn = –1.81 ´ 10–5 ´ (k)2 + 0.021 ´ (k) – 1.255 0.461 0.004 s 9 cu ´ zn zn = –0.01 ´ (cu)2 + 0.509(cu) + 0.811 0.481 0.002 s 10 k ´ cu cu = –0.0000472 ´ (k)2 + 0.043 ´ (k) – 2.944 0.465 0.003 s a synergistic b antagonistic of macroand micronutrients have been contributed to soil. more specifically, in terms of various nutrients, the pec values are presented in table 2. 304 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. 0 100 200 300 400 500 600 0 20 40 60 s o il k ( m g g 1 ) soil p ( mg g -1 ) 0 200 400 600 800 1000 1200 0 10 20 30 40 50 s o il m g ( m g g 1 ) soil p ( mg g -1 ) 0 5 10 15 0 10 20 30 40 50 s o il f e ( m g g 1 ) soil p ( mg g -1 ) 0 5 10 15 20 0 10 20 30 40 50 s o il z n ( m g g 1 ) soil p ( mg g -1 ) 0 10 20 30 40 50 0 100 200 300 400 500 600 s o il p ( m g g 1 ) soil k (mg g -1 ) 0 100 200 300 400 500 600 700 0 10 20 30 40 s o il k ( m g g 1 ) soil cu ( mg g -1 ) 0 5 10 15 20 0 500 s o il z n ( m g g 1 ) soil k (mg g -1 ) 0 5 10 15 20 0 10 20 30 40 s o il z n ( m g g 1 ) soil cu ( mg g -1 ) (a) (b) (c) (d) (e) (f) (g) (h) fig. 3. interactions among soil available nutrients discussion the critical ranges of soil nutrients are the following (in mg g–1): k 140–280, p 15–25, ca 300–750, mg 50–100, b 0.50–1.0, fe 4–25, mn 15–25, zn 1.0–2.5 and cu 0.9–1.5 (koukoulakis 1995). regarding the previous values, most of the studied soils were deficient in p, zn, mn, fe and b content (46%, 44%, 100%, 82% and 100%, respectively). on the other hand, the soils of 62% of the orchards were excessively supplied with available cu. interestingly, according to baslar et al. (1999) pistacia terebinthus subsp. palaestina, a species related to the rootstock used in the present work, generally prefers neutral soils and thrives in the presence of variable caco3 and p concentrations. therefore, lime and p content in soil are not expected to be limiting factors for pistachio trees growth in the fthiotida district. on the other hand, the recommended critical values for pistachio nutritional status (crane and maranto 1988, mills and benton jones 1996) are the following: n 2.5–2.9%, p 0.14–0.17%, k 1.0–2.0%, mg 0.6–1.2%, b 50–230 mg g–1, fe 30–125 mg g–1, mn 30–80 mg g–1, zn 7–14 mg g–1and cu 3–4 mg g–1. based on the previous values, leaf analysis showed that k, mg, mn and b were sufficient in 60%, 52%, 54% and 100% of the studied orchards, respectively, while, n, p and fe were deficient in 100%, 96% and 44% of the orchards, respectively. on the other hand, zn and cu were excessive in 68% and 100%, respectively, of the orchards under consideration. some soil properties seem to correlate significantly with the concentration of nutrients in the leaves. such antagonistic interaction caco3 ´ cu (fig. 1 d) has also been reported by kalavrouziotis et al. (2010) for this soil which was planted with brassica oleracea var gemmifera (brussels sprouts). in another study with pistacia lentiscus trees, caco3 concentration in soil was found to be negatively correlated with leaf n and k concentrations (dogan et al. 2003). also, the interaction om ´ b (fig. 1b) is in line with findings of stevenson and cole (1999), who found that b is the only non metal which can combine with om, primarily as organic complexes, with compounds that contain cis-hydroxyl groups such as saccharides, the plant-available b being released after mineralization of the organic matter. similarly, in relation to zn (fig. 1a), it has been stated by hodgson et al. (1966) that 28–90% of this element may be organically bound in the soil. it has generally been found in the present work that the elemental interactions between leaf nutrients are mainly described by quadratic, and secondarily by logarithmic regression equations. similar results have been reported by kalavrouziotis and koukoulakis (2009b) and kalavrouziotis et al. (2010) experimenting with brussels sprout plants. similar interactions between leaf nutrients (tab. 3) have also been found by other researchers. more specifically, experiments with broccoli (brassica oleracea var italica) plants resulted in the following synergistic interactions in leaves: n ´ k, mg ´ n, fe ´ mn, mn ´ b and b ´ mn (kalavrouziotis et al. 2008, 2009). in accordance with the above findings, kalavrouziotis and koukoulakis (2009b) reported the synergistic interactions of n ´ k and zn ´ fe occurring in the leaves of brussels sprout plants. on the other hand, haldar and mandal (1981) showed that the application of zn decreased fe concentration in shoots, but they mention that this decrease was not due to dilution effect or to reduced rate of translocation from roots to tops. acta bot. croat. 72 (2), 2013 305 nutrient elements in pistachio trees in relation to the interaction of mn ´ fe, contrary to our results, srivastava and gupta (1996) state that the chlorosis of fe, which is induced by increased levels of mn, is related to the enzymatic disturbance due to the fact that mn may compete with fe for binding sites of the enzymes. similarly, mn may interfere with the translocation of fe from roots to shoots. the interaction zn ´ cu found in the present study to be synergistic was said by hewitt (1983) to be antagonistic, each of the interacting elements affecting their respective concentration in the plant, competing for common carrier sites. also, during metabolism they replace each other in some metalloenzymes. haldar and mandal (1981) found that high zn concentration in soil accentuates cu deficiency, each element competitively inhibiting the uptake of the other (giordano et al. 1974). these workers also stated that the interaction zn ´ fe is mutually antagonistic, because excess zn or fe may cause reduction in the absorption of fe or zn, respectively. this mutual effect of zn and fe increases the possibility of their deficiency in the growing plants (kausar et al. 1976). a careful study of the results about pec points out that the elemental synergistic interactions occurring within the pistachio leaves may contribute significant quantities of nutrients, while the antagonistic interactions can also deprive the plants of these nutrients, as in the case of k, which was found to be negative, suggesting a 14.33% decrease of k contribution to leaves (tab. 2). moreover, the generally high pec to soil found, suggests that the synergistic interactions mobilize from non-available sources significant quantities of nutrients, while the antagonistic interactions immobilize available nutrients to plants, unfavorably affecting soil fertility. studying these results, the following may be concluded: the interactions between soil nutrients and soil properties play a significant role in supplying or decreasing the soil available plant nutrients, and therefore, they determine to a great extent the level of soil fertility. obviously, the interactions among soil nutrients favored significantly soil fertility, and hence, plant growth and nut yield. in detail, the synergistic and statistically significant interaction p ´ mg (fig. 3b) found in the soil of pistachio orchards has also been found to occur between soil p and leaf mg (merhaut 2007). this interaction seems to be associated with the ionic balance, related to cation and anion uptake by plants as well as to the increased root growth, sometimes observed with increased p fertilization. the effect of p fertilization increasing mg uptake has also been documented in rice (oryza sativa l.), wheat (triticum aestivum l.), bean (phaseolus vulgaris l.), and corn (zea mays l.) (fageria et al. 1995). in relation to k, it has been suggested that the interaction of this element with other elements presents a great variability among plant species (dibb and thompson 1985). this interaction should be studied more scrupulously, considering that many other factors, such as the level of available mg (very high in the present study), fertilization and age of plants, moisture and temperature in soil, could affect the above interaction (arnon 1975). the macronutrient k is a very strong competitor, when it is present in high concentration. it particularly affects the uptake of mg. however, in the present work, the interaction mg ´ k was found to be antagonistic due to the high level of mg in the leaves (2.4%); the mean k concentration in leaves was only 1.2%, close to the low limit of the critical range (1.0–2.0%) (crane and maranto 1988). it must be noted that, generally, the statistically significant interactions are usually biphasic, that is, the elements involved affect each other negatively or positively but at high concentrations. for example koukoulakis et al. (1988) observed that increasing the supply of k reduced leaf mg concentration in tomato and cucumber, with an 306 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. increase of k availability in the soil, due to k ´ mg antagonism. however, increasing k concentrations of nutrient solutions in hydroponically grown tomatoes resulted in increased mg concentration in fruits and seeds (mengel and kirkby 1987). in the present work, it has been shown that in general, the increase of soil cu concentration significantly increased k level in the soil, suggesting that the interaction cuxk is synergistic (fig. 3f). similar results have been reported by kalavrouziotis and koukoulakis (2009b) according to which the interaction cu ´ k was synergistic in the roots, leaves and sprouts of brussels sprouts. in contrast to the above findings, sonmez et al. (2007) reported that increasing cu applications resulted in a decline of leaf and root k concentration in tomato seedlings. on the other hand, alva et al. (1999) found that the application of increased levels of cu decreased the zn content of leaves and roots of citrus seedlings, grown in a sand substrate. however, in the present work, the interaction cu x zn was synergistic and statistically significant in both, soil and pistachio leaves (tabs. 3, 5). the availability of nutrients, especially of micronutrients, in soil was strongly influenced by ph, caco3 and organic matter (tab. 4). it is known that zn solubility increases at low ph values (lindsay 1991, brady and weil 2002). in particular, the optimum availability of zn in soil is observed at a ph range 5–7. indeed, we found that the interactions ph ´ n, caco3 ´ cu (tab. 1), and caco3 ´ fe, ph ´ fe, caco3 ´ mn, ph ´ mn, ph ´ b, and caco3 ´ b (tab. 4) were antagonistic, as they were negatively interrelated. on the other hand, the interactions om ´ b and om ´ zn (tab. 1) as well as om ´ k and om ´ zn (tab. 4) were synergistic, suggesting the favorable effect of organic matter on the availability of soil plant nutrients. the fact that the interaction caco3 ´ zn was synergistic was unexpected, as most of the soils studied in this work were calcareous with high ph. a possible explanation could be the formation of znco3 due to the high zn level in 13 cases of orchards out of a total of 50, varying from 3–17.5 mg kg–1 soil. the znco3 is a relatively soluble compound, which could even be used as fertilizer (tisdale et al. 1993). in conclusion, almost all the significant elemental interactions occurring in pistachio leaves or soils were synergistic. they contribute considerable quantities of available macroand micronutrients and therefore improve the nutrient status of pistachio leaves, and the level of soil fertility. on the other hand, any antagonistic or synergistic interactions must be taken into account during fertilization of pistachio, because among other consequences, they may possibly lead to plant deficiencies. soil analytical data showing the nutrient concentration levels, and their relationships, may be helpful in predicting possible antagonistic or synergistic interactions. furthermore, the significant interactions between soil physical or chemical properties and nutrient content of the leaves or soils found in the present study, suggest that altering these properties (e.g. improving the organic matter or ph), could favorably modify the level of soil fertility, and therefore enhance plant growth, productivity and nut quality. references alva, a. k., huang, b., prakash, o., paramasivam, s., 1999: effects of copper rates and soil ph on growth and nutrient uptake by citrus seedlings. journal of plant nutrition 22, 1687–1699. arnon, i., 1975: mineral nutrition of maize. international potash institute, bern, switzerland. acta bot. croat. 72 (2), 2013 307 nutrient elements in pistachio trees baslar, s., dogan, y., mert, h. h., 1999: studies on the ecology of pistacia terebinthus l. subsp. palaestina (boiss.) engler in west anatolia. journal of the faculty of science, ege university 22, 1–12. boujoucos, g. j., 1962: hydrometer method improved for making particle size analysis of soils. journal of agronomy 54, 464–465. brady, n. c, weil, r. r., 2002: the nature and properties of soils. prentice hall, n.j., usa. chapman, h. d., pratt, p. f., 1961: methods of analysis for soils, plants and waters. division of agricultural sciences, university of california, riverside, usa. crane, j. c., maranto, j., 1988: pistachio production. cooperative extension university of california, division of agricultural and natural resources. publication 2279, oakland, ca. dibb, d. w., thompson, jr. w. r., 1985: interaction of potassium with other nutrients. in: munson, r. d. (ed.), potassium in agriculture, 515–533. sssa, madison, wisconsin. dogan, y., baslar, s., aydin, h., mert, h., 2003: a study of the soil-plant interactions of pistacia lentiscus l. distributed in the western anatolian part of turkey. acta botanica croatica 62, 73–88. fao (food and agriculture organization), 2008. retrieved september 5, 2012, from http://faostat.fao.org. fageria, n. k., zimmermann, f. j. p., baligar, v. c., 1995: lime and phosphorus interactions on growth and nutrient uptake by upland rice, wheat, common bean, and corn in an oxisol. journal of plant nutrition 18, 2519–2532. giordano, p. m., noggle, j. c., mortvendt, j. j., 1974: zinc uptake by rice as affected by metabolic inhibitors and competing cations. plant soil 41, 637–646. haldar, m., mandal, l. n., 1981: effect of phosphorus and zinc on the growth and phosphorus, zinc, copper, iron and manganese nutrition of rice. plant soil 59, 415–425. hewitt, e. j., 1983: the essential and functional mineral elements. in: robinson, j. b. d., bould, c., hewitt, e. j., needham, p. (eds.), diagnosis of mineral disorders in plants, vol 1, 7–53. her majesty’s stationary office, london, uk. hodgson, j. f., lindsay, w. l., trierweiler, j. f., 1966: micronutrient cation complexing in soil solution. ii. complexing of zinc and copper in displacing solution from calcareous soils. soil science society of america proceedings 30, 723–726. jackson, m. l., 1958: soil chemical analysis. prentice hall inc., englewood cliffs, n.j., usa. kalavrouziotis, i. k., koukoulakis, p. h., 2009a: environmental implications of soil properties and essential nutrient interactions, under the effect of treated municipal wastewater. water air soil pollution 197, 267–276. kalavrouziotis, i. k., koukoulakis, p. h., 2009b: distribution of elemental interactions in brussels sprout plants, under the treated municipal wastewater. journal of plant interactions 4(3), 219–231. kalavrouziotis, i. k., koukoulakis, p. h., robolas, p., papadopoulos, a. h., pantazis, v., 2008: macroand micronutrient interactions in soil under the effect of brassica oleracea var italica, irrigated with treated municipal wastewater. fresenius environmental bulletin 17, 1–15. 308 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. kalavrouziotis, i. k., koukoulakis, p. h., sakellariou-makrantonaki, m., papanikolaou, c., 2009: effects of treated municipal wastewater on the essential nutrients interactions in the plant of brassica oleracea var italica. desalination 242, 297–312. kalavrouziotis, i. k., koukoulakis, p. h., mehra, a., 2010: quantification of elemental interactions effects on brussels sprouts under treated municipal wastewater. desalination 254, 6–11. kausar, m. a., chaudhary, f. m., rashid, a., latif, a., alam, s. m., 1976: micronutrient availability to cereals from calcareous soils. i. comparative zn and cu deficiency and their mutual interaction in rice and wheat. plant soil 45, 397–410. kizilgoz, i., kizilkaya, r., acar, i., kaptan, h., 2001: nutrient contents of pistachio trees (pistacia vera l.) growing in district of sanliurfa and the relationship between their microelement deficiency and some soil properties. cahiers options méditerranéennes 56, 47–52. koukoulakis, p., 1995: principles of rational fertilization of crops (in greek). agriculture and husbandry 9, 43–61. koukoulakis, p. h., simonis, a. d., bladenopoulou, s., 1988: potassium-magnesium antagonism in tomato and cucumber, grown in plastic greenhouse. proceedings of athens academy 63, 130–139. koukoulakis, p. c., simonis, a. d., gertsis, a., 2000: the organic matter in soils. the problem of greek soils (in greek). stamoulis publications, athens. lindsay, w. l., 1991: inorganic equilibria affecting micronutrients in soil. in: mortdvedt, j. j., giordano, m., lindsay, w. c. (eds.), micronutrients in agriculture, 90–111. sssa, madison, wisconsin, usa. mengel, k., kirkby, e. a., 1987: principles of plant nutrition. international potash institute, berne, switzerland. merhaut, d. j., 2007: magnesium. in: barker, a. v., pilbeam, d. j. (eds.), handbook of plant nutrition, 146–181. crc press, boca raton, fl, usa. mills, h. a., benton jones jr, j., 1996: plant analysis handbook. micromacro publishing inc., athens, usa. olsen, s. r., 1972: micronutrient interactions. in: mortdvedt, j. j., giordano, m., lindsay, w. c. (eds.), micronutrients in agriculture, 243–264. sssa. madison, wisconsin, usa. sonmez, s., kaplan, m., somnez, n. k., kaya, h., uz, i., 2007: effect of both soil copper applications and foliar copper application frequencies on macronutrients contents of tomato plants. asian journal of chemistry 19, 5372–5384. srivastava, p. c., gupta, u. c., 1996: trace elements in crop production. science publishers inc., lebanon, usa. stevenson, f. j., cole, m. a., 1999: cycles of soil. carbon, nitrogen, phosphorus, sulfur, micronutrients. john wiley and sons inc., new york, usa. tisdale, s. l., nelson, w. l., beaton, j. d., havlin, j. l., 1993: soil fertility and fertilizers. macmillan publishing co, new york, usa. acta bot. croat. 72 (2), 2013 309 nutrient elements in pistachio trees wolf, b., 1971: the determination of boron in soil extracts, plant materials, composts, manures, water and nutrient solutions. communications in soil science and plant analysis 2, 363–374. 310 acta bot. croat. 72 (2), 2013 koukoulakis p., chatzissavvidis c., papadopoulos a., pontikis d. opce-str.vp acta bot. croat. 73 (1), 171–207, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 a new sesleria juncifolia association from south-eastern italy and its position in the amphi-adriatic biogeographical context romeo di pietro1*, robert p. wagensommer2 1 department of planning design technology architecture, section of landscape and the environment, sapienza university of rome, via flaminia 72, i-00198 rome, italy 2 viale aldo moro 39, i-71013 san giovanni rotondo (fg), italy abstract – the sesleria juncifolia calcareous grasslands in the apulia region (southern italy) were studied on the basis of 24 phytosociological relevés. according to upgma cluster analysis division and nmds ordination the relevés were classified into four major groups which gave rise to three sub-associations (gargano) and a geographically impoverished variant (alta murgia). the new association stipo austroitalicae-seslerietum juncifoliae ass. nova was proposed. due to the relict and scattered distribution of sesleria juncifolia in apulia region, the variances in species composition amongst the different subassociations are mainly influenced by local factors. the community stipo-seslerietum should be included in the south-eastern italian alliance hippocrepido-stipion austroitalicae while at the rank of order it exhibits intermediate coenological features between the central-south apennine endemic suborder festuco-seslerienalia nitidae and the north-west balkan order scorzonero-chrysopogonetalia. key words: apulia, balkans, biogeography, grassland, italy, phytosociology, sesleria juncifolia, syntaxonomy, vegetation introduction the genus sesleria is one of the most important south-eastern european grass groups, especially in the mountain areas where sesleria species often play a dominant role. of all the various taxa belonging to this genus, the sesleria juncifolia complex is restricted to southern europe, where it exhibits a typically amphi-adriatic disjunct range (fig. 1a) composed of two different sub-units: the western balkans (from the italian karst to the southern albania) and the apennines (from the northern tuscany to the northern calabria). the taxonomical debate about the overall number of taxa belonging to the sesleria juncifolia complex has always been a critical issue (see deyl 1946, 1980; ujhelyi 1959; strgar 1981; ubaldi 2006; acta bot. croat. 73 (1), 2014 171 * corresponding author, e-mail: romeo.dipietro@uniroma1.it copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:28 color profile: generic cmyk printer profile composite default screen alegro 2007) that has still not been completely resolved. as far as the italian peninsula is concerned, the occurrence of four distinct taxa (s. juncifolia suffren, s. kalnikensis jáv., s. apennina ujhelyi, s. calabrica (deyl) di pietro) has been recently hypothesised on the basis of karyological, morphological and biogeographical features (di pietro et al. 2005, di pietro 2007). likewise all the other species belonging to the same complex, sesleria juncifolia is a montane species having its synecological optimum in the upper montane and subalpine belts of the major central-southern apennines limestone massifs where it is the guide species in several types of dry grasslands (bruno and furnari 1966, petriccione and persia 1995, biondi et al. 1999, blasi et al. 2005, costanzo et al. 2009, di pietro 2010). furthermore the wide ecological amplitude of this species allows it to give rise to grassland communities not only in the lower montane belt, as is the case of carici macrolepidis-seslerietum of mount cucco (biondi et al. 2004) and seslerio-stipetum appenninicolae of sibillini mountains (catorci et al. 2007), but in the hilly belt too, as is the case with genisto-seslerietum juncifoliae in the rosandra valley in the friuli venezia-giulia region and adjacent slovenia (poldini 1980, kaligari^ 1994) and in the rossa gorges and furlo gorges in the marches region (brilli-cattarini 1972). scattered residual populations of sesleria juncifolia are even found on the coastal and sub-coastal cliffs of capri island, circeo promontory and mount leano (tyrrhenian side of the italian peninsula) at about 300 m a.s.l. under typical thermo-mediterranean bioclimatic conditions. this wide coenological and altitudinal pattern of the sesleria juncifolia s.l. grasslands is comparable to that occurring in the western balkans for both the high and for the low altitude zones (krk and pag islands) (horvat et al. 1974, redzic 1999). according to pignatti (1982) the presence of sesleria juncifolia in apulia region was restricted to the gargano peninsula, where it was known for monte degli angeli, monte 172 acta bot. croat. 73 (1), 2014 di pietro r., wagensommer r. p. fig. 1. amphi-adriatic distribution of the sesleria juncifolia complex (a). distributional map of the populations of s. juncifolia occurring within the italian peninsula showing the isolation of the apulian populations from the sourrounding s. juncifolia sites (b). geographical location of the sesleria juncifolia s.l. associations included in the synoptic table (c). 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:29 color profile: generic cmyk printer profile composite default screen sant’angelo, monte calvo and pulsano (fenaroli 1974). subsequently this species was found in other sites of the gargano area (wagensommer and di pietro 2006, wagensommer 2010) and in few isolated sites in alta murgia national park (fig. 6). these new records have resulted in s. juncifolia being removed from the list of endangered species at a regional level. nevertheless, according to the subcriteria b1 (100 km2 < eoo < 5,000 km2) and b2 (10 km2 < aoo < 500 km2) and to the option »a« (7 locations in the whole region) of the iucn rules (iucn, 2006), in the apulia region sesleria juncifolia is to be considered as a species at risk of becoming »endangered« (according to the iucn »b« criterion s. juncifolia is: nt b1+2ab iii). during field research into the ecology and conservation of the rare species of gargano national park (di pietro and wagensommer 2008), several dry grasslands communities dominated by s. juncifolia were found (green squares in fig. 2). in the present research some new s. juncifolia communities stands were also identified (the red squares in fig. 2). because the occurrence of these sesleria juncifolia communities was unknown to italian botanists, no phytosociological data were available on this topic in italian botanical literature. the presence of s. juncifolia communities in apulia is a very peculiar fact, especially considering the prominent role of the mediterranean climate in the region, the low altitudes of the main apulian ranges (mount cornacchia, 1,151 m, is the highest regional peak) and their isolation from the other mountainous massifs of the apennines. in fact the gargano and alta murgia s. juncifolia communities are those that exhibit the longest distance as the crow flies from the nearest other s. juncifolia population occurring in peninsular italy (fig. 1b). the geographical isolation of gargano and alta murgia areas sums up to the lithological and ecological isolation of these areas which is due to the occurrence of large silty-clayey plains which surround the gargano and alta murgia limestone systems. the aim of the present paper is to provide a phytosociological description of the apulian sesleria juncifolia communities and to make a floristic, coenological and syntaxonomical comparison with the similar sesleria juncifolia communities described so far for the italian and the balkan peninsulas. study area the study area is divided into two parts: gargano promontory and alta murgia plateau (fig. 2b). the promontory of gargano, known as the »spur« of the italian peninsula, is a succession of broad plains and low-lying hills jutting into the adriatic sea from the east coast of italy, in foggia province. it is 65 km long and 40 km at its widest, with an area of about 2,100 square km and it acts as an island emerging from the tavoliere plain. mount gargano is composed entirely of limestone, surrounded by terraces of various geologic periods. the highest peak is mount calvo (1,065 m), while the only other peaks exceeding one thousand meters are montenero (1,014 m) and mount spigno (1,008 m). the intense karst activity affecting the limestone bedrock leads to the superficial water drainage being restricted to torrential rivers, while the coastal lakes (lesina, and varano) in the north-western side of the promontory are very important. according to blasi (2003) the apulian sesleria juncifolia sites are included in the following phytoclimatic units: dry meso-mediterranean; subhumid meso-mediterranean; humid/subhumid mesotemperate (fig. 2a). the potential vegetation of gargano is almost completely composed of oak woodlands acta bot. croat. 73 (1), 2014 173 sesleria juncifolia grasslands in apulia (southern italy) 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:29 color profile: generic cmyk printer profile composite default screen 174 acta bot. croat. 73 (1), 2014 di pietro r., wagensommer r. p. fig. 2. upper part: a – bioclimatic map of apulia region (modified after blasi 2003) b – apulia region physical map with the areas including sesleria juncifolia population circled in red. c – apulia region in the italian political map. lower part: 1 km × 1 km grid of distribution of sesleria juncifolia stands in the gargano and alta murgia territories. 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:31 color profile: generic cmyk printer profile composite default screen (quercus ilex, q. pubescens, q. cerris) except for the important site of »umbra foresta« which is covered by a wide beech forest and of pine woods along the coasts. the sesleria juncifolia communities are restricted to the southern slope of gargano, which is characterized by the presence of deep vertical valleys (vallone di pulsano, valle dell’inferno, valle del surdo, etc). the alta murgian relief is a wide limestone plateau included in the territory of bari and barletta-andria-trani provinces and it is the core of the alta murgia national park, which covers an area of about 680 square kilometres. the bedrock is formed by cretaceous limestone usually covered with pleistocene calcarenite and this geological character has determined a lot of karstic phenomena represented by swallow holes, dolinas, poljes. the highest peaks are torre disperata (686 m) and mount caccia (680 m). the deep changes caused by the human action have shaped the physiognomy of the original vegetation to the extent that it is impossible or very difficult to carry out a precise and certain analysis of the potentialities of this territory. at present the alta murgia park is marked by very extended steppe-like grasslands which in the 92/43/eec directive are for the most included in the prior habitat 62a0 (eastern sub-mediterranean dry grasslands), and for a minor part in the other prior habitats 6220 and 6210. materials and methods the study was carried out via a phytosociological and statistical analysis of relevés of dry grasslands with the dominance of sesleria juncifolia performed in apulia region (south-eastern italy). subsequently a phytogeographical and phytosociological comparison with the literature data regarding the sesleria juncifolia communities of the rest of italy and of the western balkan peninsula was provided. twenty-four relevés, according to the braun-blanquet (1964) approach, were performed in may 2007–2009. all the cover data were recorded according to the braun-blanquet scale and transformed into the scale proposed by van der maarel (1979) and noest et al. (1989). a matrix of 24 relevés × 148 species was subjected to a divisive hierarchical classification (using the chord distance algorithm to produce the dissimilarity matrix and the minimum variance linkage as agglomeration criterion on quantitative data) and to nmds ordination (syn-tax 2000 package, podani 2001). four main types of vegetation were distinguished from a coenological point of view and a syntaxonomical assignment was subsequently proposed. moreover a synoptic table composed of the frequency columns of all sesleria juncifolia s.l. associations described in italy and in the western balkans (on-line supplement appendix 4; fig. 1c) was prepared and subsequently subjected to cluster analysis using the same statistical procedures mentioned above. in order to attenuate the influence of the high cover/abundance values of the grasslands’ guide species, in the cluster analysis procedures the various taxa belonging to the sesleria juncifolia complex were reported under the single collective name of s. juncifolia s.l. owing to the thermophilous character of the apulian communities, comparison with the balkan sesleria juncifolia communities was restricted to those associations which showed similar ecological features. on the contrary the s. juncifolia communities belonging to the elyno-seslerietea class and occurring in the subalpine and alpine belts of the balkans were not included in this comparative cluster analysis. for the identification of plant taxa the diagnostic key published in licht (2008) was used. the nomenclature of the taxa follows conti et al. (2005) and euro+med plantbase, whilst life form and chorotype systems follow pignatti (1982). life form and chorological analyses consider whether a particular species acta bot. croat. 73 (1), 2014 175 sesleria juncifolia grasslands in apulia (southern italy) 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:31 color profile: generic cmyk printer profile composite default screen occurs or not in a cluster (normal values), its frequency (frequency values), and its cover values (cover). the physiognomical role of each species was investigated by calculating its specific cover index (braun-blanquet 1964). in naming the phytosociological syntaxa, we adhered to the rules contained in the third edition of the international code of phytosociological nomenclature (icpn) (weber et al. 2000). results phytosociological relevés the dendrogram resulting from the hierarchical classification (fig. 3) highlighted two main clusters: cluster »a« included the s. juncifolia communities of gargano promontory, while cluster »b« included the residual impoverished sesleria juncifolia communities of the alta murgia area. cluster »a« is subdivided into two further subclusters. subcl. a2 includes the sesleria grasslands occurring within the steep slopes of the pulsano gorge. subcl. a1 includes the sesleria grasslands placed at higher altitude. in particular, cluster a1–1 includes the grasslands of the top of mount calvo while cluster a1–2 is related to the summit area of mount s. angelo. the result of the cluster analysis was confirmed by the nmds ordination (fig. 4), although no any correlation between axis and environmental parameters was identified. the geographical isolation from the rest of the sesleria juncifolia communities occurring in the apennines and in the balkans have resulted in the apulian sesleria juncifolia dry grasslands developing very peculiar floristic and coenological features for which none of the syntaxa already published is suitable at present. for this reason we propose the new association: stipo austroitalicae-seslerietum juncifoliae di pietro et wagensommer ass. nov hoc loco holotypus: table 1, rel. 2. for locations and dates of relevés in tab. 1, see on-line supplement appendix 1; sporadic species are listed in the online supplement appendix 2. 176 acta bot. croat. 73 (1), 2014 di pietro r., wagensommer r. p. fig. 3. cluster analysis dendrogram of the apulian sesleria juncifolia communities relevés. 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:33 color profile: generic cmyk printer profile composite default screen diagnosis: stipo austroitalicae-seslerietum juncifoliae dry grasslands are developed on limestone outcrops, ridges and carbonate-rich talus slopes, and are strongly dominated by sesleria juncifolia which is joined by a complex of common calcicolous stress-tolerant species such as plantago holosteum, pimpinella tragium, galium corrudifolium, koeleria splendens, anthyllis vulneraria subsp. rubriflora and by a group of species having a very restricted distribution such as stipa austroitalica, leontodon apulus, centaurea subtilis, satureja cuneifolia, genista michelii (etc.). the sites are characterised by nutrient-poor calcareous xeric soils (especially lithosols) and are typically exposed to extreme microclimatic conditions such as drought and high temperature amplitude. stipo-seslerietum occurs, in form of isolated and highly fragmented stands, at altitudes which range between 400 and 1,050 m. bioclimate: meso-mediterranean/meso-temperate thermotypes; lower dry/upper sub-humid umbrotype. characteristic species: stipa austroitalica, leontodon apulus, dianthus tarentinus, anthyllis vulneraria subsp. rubriflora. dynamics: stipo-seslerietum forms primary grasslands on the small-size talus slopes developed within gargano southern slope gorges. on deep soils stipo-seslerietum behaves like a secondary stage of ostryo-quercetum ilicis or cyclamino-quercetum virgilianae at lower altitudes or ostrya carpinifolia woods at higher altitudes. distribution: stipo-seslerietum is endemic to the apulia region. the majority of the sites occur in the gargano promontory while only few relic populations occur in the alta murgia plateau. stipo-seslerietum is divided into three sub-associations: typicum (gargano promontory southern slopes gorges); helianthemetosum apennini (top of mount calvo); seslerietosum autumnalis (mount s. angelo ridges): stipo austroitalicae-seslerietum juncifoliae typicum di pietro et wagensommer subass. nov hoc loco. holotypus: table 1, rel. 2 the most typical aspect of stipo-seslerietum is found within the steep slopes of the gorges which cut across the southern side of the gargano promontory (fig. 5a) where small acta bot. croat. 73 (1), 2014 177 sesleria juncifolia grasslands in apulia (southern italy) fig. 4. nmds ordination of the apulian sesleria juncifolia communities relevés with the partition of the clusters identified by the cluster analysis. 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:33 color profile: generic cmyk printer profile composite default screen 178 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 1. stipo austroitalicae-seslerietum juncifoliae di pietro et wagensommer ass. nov. relevés number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 typ. typ. typ. altitude m a.s.l. 510 500 500 500 490 500 500 1000 1040 1020 1040 1025 1030 1025 1015 1015 860 860 870 865 860 580 582 578 aspect w w wsw s w sw wsw se se e e s s s w w n n n n n n e ne slope (°) 80 15 5 30 10 35 15 75 70 65 0 60 40 35 70 10 50 55 5 70 50 50 55 30 rockyness: a=high; m=med.; s=scarce aa m a a m m m 65 60 55 a aa a a aa s aa aa a aa aa aa aa aa detritus: a=high; m=med.; s=scarce . a m m a a a . . . s . . . . s s s . . . . . . cover (%) 70 70 80 70 75 70 75 55 65 60 85 40 60 80 70 90 75 70 70 70 65 65 60 60 area m2 20 10 7 10 8 45 30 45 50 60 15 9 9 7 10 9 40 35 15 40 50 25 20 25 sesleria juncifolia 2 4 5 4 4 4 4 2 3 3 2 3 3 3 4 5 4 4 4 4 3 3 3 3 stipa austroitalica 1 1 1 1 2 1 2 1 1 1 . + + 1 + 1 2 1 + 1 . 1 . 1 leontodon apulus + + + . + + . 1 1 2 1 . + + . + 2 1 1 2 + 1 2 2 anthyllis vulneraria subsp. rubriflora + + . 1 . . + 1 1 1 2 . + + + 1 2 1 2 1 . . . . dianthus tarentinus + + . . . . + . + + . . + + . . + + + . . . + . stipo austroitalicae-seslerietum juncifoliae subass. typicum centaurea subtilis 2 2 2 1 2 2 2 . . . . . . . . . . . . . . . . . genista michelii 1 2 2 + 2 2 2 . . . . . . . . . . . . . . . . . satureja cuneifolia 1 + 2 + 1 2 2 . . . . . . . . . . . . . . . . . fumana ericifolia 1 + 1 . + + 1 . . . . . . . . . . . . . . . . . thesium humifusum + + + 1 + + . . . . . . . . . . . . . . . 1 . . stipo austroitalicae-seslerietum juncifoliae helianthemetosum apennini helianthemum apenninum . . . . 1 . . 2 2 2 + + + + 1 1 . . . . . . . . plantago holosteum . . . . . . . 3 3 2 + . 1 1 + + . . . . . . . . medicago prostrata . . . . . . . 2 2 + + . + . . 1 . . . . . . . . stipo austroitalicae-seslerietum juncifoliae seslerietosum autumnalis sesleria autumnalis . . . . . . . . . . . . . . . . + 1 + 1 + . . . doronicum columnae . . . . . . . . . . . . . . . . + + + 2 2 . . . aubrieta columnae subsp. italica . . . . . . . . . . . . . . . . + + . + 2 . . . alyssum diffusum subsp. garganicum . . . . . . . . . . . . . . . . 1 1 1 1 + . . . viola merxmuelleri . . . . . . . . . . . . . . . . + + + . . . . . stipo austroitalicae-seslerietum juncifoliae residual alta murgia form helianthemum oelandicum subsp. incanum . . . . . + . . . . . . . . . . 1 . . . . 2 2 3 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 3 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 179 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) relevés number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 elaeoselinum asclepium . . . . . . . . . . . . . . . . . . . . . 2 + + euphorbia nicaeensis subsp. japygica . . . . . . . . . . . . . . . . . . . . . + + + hippocrepido glaucae-stipion austroitalicae hippocrepis glauca + + . + . . . 1 + . . 1 2 2 + 2 1 . . . . . . . cytisus spinescens + + + + 1 1 + . . . . . . . . . . . . . . . . . thymus spinulosus . . . . . . . + . . 1 . . . . . . . . . . 1 + + scorzonera villosa subsp. columnae . . . . . . . . + + . . . . . + . . . . . . + + transgr. cytiso-bromion sideritis italica . . . . . . . 1 + 1 . . + 2 2 . . 1 + 1 + . . . onobrychis alba subsp. alba . 1 . 1 + + 1 + . + + . . . . . . . . . . . . . centaurea deusta . . . + . . . + . + . . . . . . . + . + + . . . crepis lacera . . . . . . . . . + . . . . . . . 1 + 1 . . . . scorzonero-chrysopogonetalia/artemisio-brometalia galium corrudifolium . + + 1 1 + + + 2 2 + 1 + + 2 2 + 1 + 1 2 2 + . koeleria splendens + 1 . . . . 1 1 1 + + . + + . 1 2 . . + . . + 1 pimpinella tragium . + . . + + + . + 2 . 2 . + 1 . 2 2 1 2 1 . . . euphorbia spinosa . . . + . . . 1 1 + . + + . + . . . . . . . . . cytisus decumbens . . . . . . . + . . . . . + . 1 1 2 2 1 . . . . onosma angustifolia . 2 . 1 + . . . . . . . . . . . . . . . . . . . onosma echioides . . . . . . . . . . . . . . . . . . . . . 2 + 1 convolvulus elegantissimus . . . + . . . . . . . . . . + . . . . . . 1 + + muscari neglectum . . . . . . . . . . + . . + . . + 1 . + . . . . aethionema saxatile . . . . . . . + + + . . . . . . . . + . . . . . jurinea mollis . . . . . . . + . 1 . . . . + + . . . . . . . . phagnalon rupestre subsp. illyricum + + . 1 . . . . . . . . . . . . . . . . . . . . stachys recta subsp. subcrenata + . . + . . . . . . . . . . . . . . . . . . . . linum austriacum subsp. tommasinii . . . . . . . + + . . . . . . . . . . . . . . . cephalaria leucantha . . . . . . . + . . . . . . + . . . . . . . . . teucrium chamaedrys . . . . . . . 2 . . . . . . . . . + . . . . . . carex liparocarpos . . . . . . . . . . . . . . . . 1 + . . . . . . allium sphaerocephalon . . . . . . . . . . . . . . . . . . + + . . . . tab. 1. – continued 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 3 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 180 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 1. – continued relevés number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 arabis collina subsp. collina . . . . . . . . . . . . . . . . . . + . + . . . scabiosa columbaria . . . . . . . . . . . . . . . . . . . + + . . . euphorbia myrsinites . . . . . . . . . . . . . . . . . . . . . . + + eryngium amethystinum . . . . . . . + . . . . . . . . . . . . . . . . artemisia alba . . . . . . . . + . . . . . . . . . . . . . . . ranunculus illyricus . . . . . . . . . . + . . . . . . . . . . . . . thlaspi praecox . . . . . . . . . . + . . . . . . . . . . . . . orchis pauciflora . . . . . . . . . . . . . . . . . + . . . . . . alyssum diffusum s.l. . . . . . . . . . . . . . . . . . . . . . + . . silene paradoxa . . . . . . . . . . . . . . . . . . . . . . + . leontodon cichoraceus . . . . . . . . . . + . . . . . . . . . . . . . festuco-brometea festuca circummediterranea . . + + + . . 1 1 1 . . . . 1 1 . . . . . 2 + . minuartia verna subsp. attica . . . . . . . 1 1 . + + . . . + . + . . . . . . bromus erectus . . . . . . . 2 1 + 3 + + + . + 1 . . . . . . . valeriana tuberosa . . . . . . . . . . + . . . . . + + + . . . . . polygala nicaeensis . . . . . . . . . . . . . . . . + + + + . . . . bromus hordeaceus . . . . . . . + + 1 . . . . . . . . . . . . . . sanguisorba minor . . . . . . . + 1 1 . . . . . . . . . . . . . . brachypodium rupestre . . . . . . . . . . . . . . . . . 2 + 2 . . . . inula montana . . . . . . . . . . . . . . . . . + 1 . . . . . centaurium erythraea . . + . . . . . . . . . . . . . . . . . . . . . carduus nutans . . . . . . . . . + . . . . . . . . . . . . . . carex caryophyllea . . . . . . . . . . 1 . . . . . . . . . . . . . hieracium pilosella . . . . . . . . . . + . . . . . . . . . . . . . orchis antropophora . . . . . . . . . . . . . . . . . + . . . . . . anthoxantum odoratum . . . . . . . . . . . . . . . . . . . . . 2 . . cisto-micromerietea rhamnus saxatilis subsp. infectoria . 1 + 1 1 1 + . . . . . . + . . . . + . . + + + sedum ochroleucum + + . 1 . . + . . . . . . . . + . . . . . . + + satureja montana subsp. montana . . . . . . . . 1 . . . 1 . . . 1 2 + 1 . . . + 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 4 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 181 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 1. – continued relevés number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 teucrium capitatum . . . . . . . + + . . . . + . + . . + . . . . + ceterach officinarum . . . . . . . . + + . 1 . . . . . . . + + . + . teucrium montanum 1 . + . + + + . . . . . . . . . . . . . . . . . asperula aristata subsp. longiflora . . . . . + + + . . . . . . . + . . . . 1 . . . emerus major subsp. emeroides . . . . . + . . . . . . . . . . + . 2 1 + . . . athamanta sicula . . . . . . . . . + . + . . + . . . . 1 + . . . cistus creticus subsp. eriocephalus . + . . . + + . . . . . . . . + . . . . . . . . ruta graveolens . + . . . . + . . . . . . . . . . . . . . + + . cistus salvifolius . . . . 1 + + . . . . . . . . . . . . . . . . . fumana thymifolia . 1 . . . . + . . . . . . . . . . . . . . . . . micromeria graeca . . . . . . . . . . . . . . . . . . . . . 1 . + helichrysum italicum subsp. italicum + . . . . . . . . . . . . . . . . . . . . . . . genista tinctoria . . . . . . . . + . . . . . . . . . . . . . . . asparagus acutifolius . . . . . . . . . . . . . . . . . . . . . . + . asplenietea trichomanes inula verbascifolia + . + . . + + . . . . . . . . . . . . . . . . . lomelosia crenata subsp. dallaportae 1 . . . . 1 + . . . . . . . . . . . . . . . . . sedum caespitosum . . . . . . . . . . . . . . . . . . . . . . + + hieracium acanthodontoides . . . . . . . . . . . . . . . . . . + . . . . . scabiosa taygetea subsp. garganica . . . . . . . . . . . . . . . . . . + . . . . . asplenium trichomanes . . . . . . . . . . . . . . . . . . . . 1 . . . polypodium interjectum . . . . . . . . . . . . . . . . . . . . 1 . . . aurinia saxatilis subsp. megalocarpa . . . . . . . . . . . . . . . . . . . . . . + . hellenocarum multiflorum . . . . . . . . . . . . . . . . . . . . . . 1 . sedum hispanicum . . . . . . . . . . . . . . . . . . . . . . + . sedum dasyphyllum . . . . . . . . . . . . . . . . . . . . . . . + lygeo-stipetea & tuberarietea reichardia picroides + + + . . . + . 1 + . . . . . . . . . . . 1 . + brachypodium retusum . + . + 2 . 1 . . . . . . . . . . . . . . . . . trachynia distachya . . . . . . . + + + . . . . . . . . . . . . . + briza maxima . . . . . . . + + . . . . . . . . . . . . 1 + . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 4 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 182 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 1. – continued relevés number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 urospermum dalechampii . . . . . . . + + + . . . . . . . . . . . . . . crupina crupinastrum . . . . . . . + + . . . . . . + . . . . . . . . cynosurus echinatus . . . . . . . + + . . . . . . . . . . . . . . + catapodium rigidum . . . . . . . . . + . . . . . . . . . . . . + + bromus madritensis . . . . . . . . . . . . . . . . . . . . . 1 + + bartsia trixago . + . + . . . . . . . . . . . . . . . . . . . . crepis sancta . . . . . . . 1 + . . . . . . . . . . . . . . . xeranthemum inapertum . . . . . . . + . + . . . . . . . . . . . . . . poa annua . . . . . . . . + + . . . . . . . . . . . . . . linum strictum subsp. strictum . . . . . . + . . . . . . . . . . . . . . . . . hypochoeris achyrophorus . . . . . . . + . . . . . . . . . . . . . . . . bombycilaena erecta . . . . . . . . + . . . . . . . . . . . . . . . asterolinon linum-stellatum . . . . . . . . . + . . . . . . . . . . . . . . bupleurum baldense . . . . . . . . . + . . . . . . . . . . . . . . ononis ornithopodioides . . . . . . . . . . . . . . . . . . . . . + . . avena barbata . . . . . . . . . . . . . . . . . . . . . . . + euphorbia exigua subsp. exigua . . . . . . . . . . . . . . . . . . . . . . . + other species ornithogalum comosum + + . . . . . . . . + . . . . . . . . . + . . + acinos alpinus . . . . . . . . + + . . . + . . . . . 1 + . . . hirschfeldia incana subsp. incana . . . . . . . + + 1 . . . . . . . . . . . . . . triticum ovatum . . . . . . . . . . . . . . . . . . . . . 1 + + diplotaxis tenuifolia . . . . + . . . . . . . . . . . . . . . . . . + silene vulgaris . . . . + . . . . . . . . . . . . . . . . . 1 . carlina corymbosa . . . . . . . + + . . . . . . . . . . . . . . . silene conica . . . . . . . 1 + . . . . . . . . . . . . . . . polygala monspeliaca . . . . . . . . . . + . . . . + . . . . . . . . cerastium pumilum . . . . . . . . . . + . . . . . . . . . . . + . geranium columbinum . . . . . . . . . . . . . . . . . . . + + . . . vicia pubescens . . . . . . . . . . . . . . . . . . . . . + + . sporadic species . 1 . 1 1 . . 2 . . 1 . . . . 1 . . . . . 4 2 1 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 4 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n rocky outcrops emerge from a matrix composed of limestone debris. this subassociation is developed at the lowest altitude (400–520 m). the characteristic species of this subassociation are genista michelii, centaurea subtilis and satureja cuneifolia, whose distribution in the gargano area is almost only restricted to this habitat type. stipo austroitalicae-seslerietum juncifoliae helianthemetosum apennini di pietro et wagensommer subass. nov hoc loco. holotypus: tab. 1, rel. 13 as this sub-association is restricted to the top of mount calvo, the highest summit of gargano, it is the aspect of stipo-seslerietum which is developed at the highest altitude (exceeding 1,000 m) and which best exhibits the physiognomical features of a typical dry grassland (fig. 5b). although it is mainly developed on rocky habitats, it can also be found on flat surfaces where bromus erectus can play a co-dominant role together with anthyllis vulneraria subsp. rubriflora and plantago holosteum. the characteristic species of this sub-association are helianthemum apenninum, plantago holosteum and medicago prostrata. stipo austroitalicae-seslerietum juncifoliae seslerietosum autumnalis di pietro et wagensommer subass. nov hoc loco. holotypus: tab. 1, rel. 18 this subassociation is restricted to the north facing slopes of monte degli angeli (fig. 5c) where sesleria juncifolia become dominant in the steepest part of the slopes, at altitudes ranging between 820 and 870 m, where a higher degree of rockiness is found. aspects that are exclusively north-facing together with the vicinity of ostrya carpinifolia woodlands lead to the peculiar co-existence of sesleria juncifolia and sesleria autumnalis which has never been recorded elsewhere in the italian peninsula. the characteristic species of this subassociation are sesleria autumnalis, doronicum columnae and aubrieta columnae subsp. italica. acta bot. croat. 73 (1), 2014 183 sesleria juncifolia grasslands in apulia (southern italy) fig. 5. stipo-seslerietum typicum in pulsano valley (a), stipo-seslerietum helianthemetosum on the top of mount calvo (b), stipo-seslerietum seslerietosum autumnalis on the ridges of monte degli angeli (c). acta botanica 1-2014 str187.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 24. o ujak 2014 11:11:53 color profile: generic cmyk printer profile composite default screen stipo austroitalicae-seslerietum juncifoliae impoverished form the alta murgia sesleria juncifolia communities exhibit a floristic composition slightly different from that of the gargano communities due to the presence of some endemics or amphi-adriatic species the distribution of which is restricted to southern apulia and the southern balkans (e.g. hellenocarum multiflorum, euphorbia nicaeensis subsp. japygica). this fact, however, does not change the coenological and biogeographical features of the community as well as this does not allow the use of any other syntaxonomical references than stipo-seslerietum. the sporadic occurrence of sesleria juncifolia populations in alta murgia restricted to a few isolated rocky outcrops has led us to classify these communities as an impoverished variant of stipo-seslerietum rather than as a distinct sub-association. synoptic columns sporadic species in synoptic table 4 are listed in the online supplement appendix 3; the associations included are listed in the online supplement appendix 4. as far as the synoptic table is concerned, the dendrogram resulting from the hierarchical classification of the selected s. juncifolia associations (fig. 8) highlighted two main clusters: cluster »a« which includes the s. juncifolia communities of the subalpine and the alpine belt of the apennines and cluster »b« which includes the associations of the hilly and the montane belt of both the apennines and the western balkans. cluster b exhibits a further division in sub-cluster b1, which includes most of the apennine and balkan associations, and sub-cluster b2, which is restricted to two associations (the apulian stipo-seslerietum and the dalmatian salvio-seslerietum). a further subdivision of the subcluster b1 separates the apennine (b1a) from the balkan communities (b1b). discussion most of the phytosociological literature concerning the gargano grasslands has discussed the steppe-like vegetation (especially dominated by stipa austroitalica) of the limestone plateaus (forte et al. 2005, terzi et al. 2010) or the chasmophytic communities rich 184 acta bot. croat. 73 (1), 2014 di pietro r., wagensommer r. p. 0 5 10 15 20 25 30 35 40 % chorotypes flora frq. cover fig. 6. chorological spectrum of stipo austroitalicae-seslerietum juncifoliae calculated on the presence (flora), frequency (frq.), cover of a given chorotype in the phytosociological table. 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:36 color profile: generic cmyk printer profile composite default screen a c t a b o t .c r o a t .73 (1),2014 185 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 2. chorological spectrum of the sesleria juncifolia s.l. associations included in the synoptic table. amphi-adriat. 16.8 15.7 16.9 28.9 20.3 10.8 7.7 5.6 13.6 9.8 9.6 4.3 3.0 2.8 15.7 4.2 8.8 10.3 balkan endem. . . . . . . . . . . 5.3 . . 1.1 8.2 5.0 . 9.0 boreal 19.4 10.1 18.3 7.8 5.3 4.2 . . 0.6 1.5 . 6.7 7.3 3.4 0.9 6.1 . 1.4 centr.-europ. 10.3 5.6 5.6 3.3 1.5 8.4 7.7 5.1 1.9 3.0 8.5 3.7 14.0 11.3 2.7 10.3 0.2 6.9 subcosmop. . . . . . . . . 0.6 . . . . . . 1.1 0.8 . eurasiat 7.1 5.1 5.6 4.4 6.0 15.0 11.6 14.4 9.3 4.5 13.8 21.3 13.4 20.3 9.1 17.2 6.5 11.0 eurimedit. . 3.9 2.8 2.2 9.8 7.2 11.6 15.3 13.6 6.0 7.4 6.1 3.7 5.6 26.4 6.1 25.6 13.8 europ-caucas. 2.6 5.6 4.9 8.9 5.3 7.8 9.4 6.5 5.6 3.8 2.1 14.6 15.2 10.7 2.7 8.0 1.0 2.8 ital endemics 13.5 15.2 12.0 8.9 20.3 13.8 12.2 13.5 15.4 24.1 . 0.6 . . . . 19.5 . medit-mont. 4.5 5.6 2.8 2.2 2.3 3.6 8.8 4.2 5.6 9.8 20.2 12.2 1.2 4.0 4.5 8.4 7.3 6.2 oroph s-europ. 25.8 29.2 30.3 31.1 26.3 13.2 12.2 12.6 7.4 31.6 7.4 16.5 18.3 15.8 8.2 17.6 3.2 13.1 se-europ . 3.9 0.7 2.2 2.3 13.8 16.6 17.2 14.8 5.3 16.0 10.4 20.1 20.9 10.9 12.6 10.8 20.7 stenomedit. . . . . 0.8 . . 2.3 9.9 . 5.3 . . . 10.9 1.5 13.5 3.4 sub-atlantic . . . . . 2.4 2.2 3.3 2.5 0.8 4.3 3.7 3.7 4.0 . 1.9 2.8 1.4 l eo nt -s es l s ca bs es l. s es l. ap en ni na e s es ld ry ad et um a nt hs es l. ca l. c ar ic i hu m .s es l. c ar ic i m ac r. -s es l. s es l. -s ti pe tu m ju ri ne os es l. ca l. s es l. -d ra be tu m g en is to -s es l. ju n s es lc ar ic et um hu m s es l. ju nc if . s es l. ka ln ik . s al vi os es l. g en is to -s es l. ka ln s ti po au ss es l. b ro -s es l. in te rr . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n in endemic and rare species (bianco et al. 1988, di pietro and wagensommer 2008, terzi and d’amico 2008). by contrast, the grassland types developed in other habitats such as rocky slopes, stable talus slopes or mountain ridges (which are those in which s. juncifolia dominates more frequently) have been overlooked. this is why sesleria juncifolia is known only for its role of »companion« species, as in some centaureo-campanuletalia chasmophitic communities, while it has never been associated with the role of guide species in a grassland community. it is not easy, however, to provide an unequivocal syntaxonomical framework for these apulian sesleria juncifolia communities. although their ecological features are similar to those of the other s. juncifolia communities of the rest of the apennines, their floristic composition is quite different. the low average altitude of the apulian relief and the high incidence of the mediterranean climate result in stipo-seslerietum being characterized by higher percentages of therophytes and a lower percentages of boreals and south-european orophytes when compared to the sesleria communities occurring within the true apennines (tabs. 2 and 3). furthermore stipo-seslerietum hosts several species (e.g. lomelosia crenata subsp. dallaportae, centaurea subtilis, genista michelii, satureja cuneifolia, inula verbascifolia, aubrieta columnae subsp. italica etc.) which exhibit an italian distribution more or less restricted to the apulian region. these chorological features are due to the past and present geographical isolation of the gargano and murge ranges. the phytosociological literature reports that the majority of the italian primary and secondary sesleria juncifolia s.l. associations have been described for the montane and subalpine belts of the apennines and are to be included in the seslerion apenninae alliance and in the elyno-seslerietea class (bruno and furnari 1966; biondi et al. 1988; petriccione and persia 1995; biondi et al. 1999, 2004; blasi et al. 2003, 2005). the jurineo mollis-seslerietum calabricae of the pollino-orsomarso massif (di pietro 2010) and seslerio-stipetum appenninicolae of the sibillini mountains (catorci et al. 2007) have alone been included in the cytiso-bromion erecti alliance (ex phleo ambigui-bromion erecti) and in festuco-brometea class. the complete lack of the subalpine endemic and southeastern european orophitic components does not allow seslerion apenninae to be used as a good reference for the apulian stipo-seslerietum. the strong occurrence of the steno-mediterranean therophytic component mixed with a significant percentage of amphi-adriatic hemycryptophytic and chamaephytic components suggests that other syntaxonomical references could be hypothesised (e.g.cytiso-bromion erecti and hippocrepido-stipion austroitalicae, 186 acta bot. croat. 73 (1), 2014 di pietro r., wagensommer r. p. tab. 3. life form spectrum of the sesleria juncifolia s.l. associations included in the synoptic table. l eo nt -s es l s ca bs es l. s es l. ap en ni na e s es ld ry ad et um a nt hs es l. ca l. c ar ic i hu m .s es l. c ar ic i m ac r. -s es l. s es l. -s ti pe tu m ju ri ne os es l. ca l. s es l. -d ra be tu m g en is to -s es l. ju n s es lc ar ic et um hu m s es l. ju nc if . s es l. ka ln ik . s al vi os es l. g en is to -s es l. ka ln s ti po au ss es l. b ro -s es l. in te rr . ch 28.4 27.0 28.9 37.8 37.6 27.5 22.1 26.0 30.7 34.6 30.9 28.7 17.1 18.1 30.9 20.6 25.5 31.2 g 1.9 3.4 6.3 3.3 0.8 6.6 7.2 6.0 5.5 . 13.8 5.5 5.5 10.2 10.0 6.1 4.2 4.2 h 67.7 61.8 60.6 54.4 55.6 62.9 63.5 63.7 52.1 65.4 53.2 61.6 76.8 70.6 57.3 64.1 41.5 61.8 ph 0.6 1.7 1.4 . 2.3 . 0.6 . 9.2 . 2.1 3.7 . . . 8.1 3.2 1.4 t 1.3 6.2 2.8 4.4 3.8 3.0 6.6 4.2 2.5 . . 0.6 0.6 1.1 1.8 1.0 25.6 1.4 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:36 color profile: generic cmyk printer profile composite default screen which are typical alliances of the lower altitudinal belts). the possible choice of cytiso-bromion (endemic alliance of the central and southern italy) would be supported by the presence of the two apennine endemics sideritis italica and crepis lacera and by a group of southeastern-european species (centaurea deusta, onobrychis alba subsp. alba and cytisus spinescens), which various authors considered in the role of differentials for this alliance in their syntaxonomical synthesis. these same species, however, are frequently found also in the communities of the alliance hippocrepido-stipion austroitalicae, this latter including the steppe-like vegetation of the easternmost side of the italian peninsula (apulia, eastern basilicata and eastern molise regions). hippocrepido-stipion is represented in stipo-seslerietum by the majority of its characteristic component (stipa austroitalica, thymus spinulosus, hippocrepis glauca, and scorzonera villosa subsp. columnae). for this reason we have preferred to include stipo-seslerietum in the alliance hippocrepido-stipion austroitalicae rather than in cytiso-bromion. at a higher syntaxonomical rank, the choice of hippocrepido-stipion would result in stipo-seslerietum being included in the order scorzonero-chrysopogonetalia the presence of which in the italian peninsula was known for the karst territories of ne italy (feoli-chiapella and poldini 1993) and has been subsequently recorded for the apulia and molise regions in southern italy (fanelli et al. 2001, forte et al. 2005, di pietro and wagensommer 2008, terzi et al. 2010). precisely in the sub-mediterranean zones of friuli venezia-giulia karst, poldini (1980) described the association genisto sericeae-seslerietum juncifoliae which exhibits ecological and structural features very similar to those of the apulian stipo-seslerietum. both communities are developed at relatively low altitudes, where there is a greater influence of the mediterranean climate, and are characterized by a group of species which behave as geographical vicariants (genista michelii / g. sylvestris, satureja montana / s. variegata; satureja cuneifolia / s. subspicata; athamanta sicula / a. turbith; centaurea subtilis / c. rupestris; stipa austroitalica / s. eriocaulis). dry grasslands dominated by the stipa-sesleria taxa association are not a novelty for the italian peninsula since they are known also for the central apennines (montane belt of the sibillini mountains in marches region) with the community seslerio juncifoliae-stipetum appenninicolae (catorci et al. 2007). notwithstanding their physiognomical similarities, the apulian stipo-seslerietum and the marches seslerio-stipetum are easily distinguishable from a floristic point of view due to the presence of a significantly higher mediterranean component in the apulian association (tab. 2). the cluster analysis of the synoptic table (fig. 8) including most of the sesleria juncifolia s.l. associations described for both sides of the adriatic sea (fig. 1c) reports the geographical location of these associations) – shows that stipo austroitalicae-seslerietum is clearly separated from the sesleria juncifolia s.l. associations described for the subalpine and upper montane belts of the italian peninsula (seslerion apenninae; cluster a) whereas it is included in the cluster which encompasses the central and southern apennine sesleria juncifolia associations belonging to the festuco-brometea class (cluster b). the thermophilous sesleria juncifolia associations described for the western balkans form a clearly-distinguishable eastern-adriatic sub-cluster (b1b) in the festuco-brometea cluster, except for salvio-seslerietum juncifoliae of coastal croatia which segregates together with stipo-seslerietum in the dendrogram. this amphi-adriatic link between stipo-seslerietum and salvio-seslerietum, which could suggest a possible inclusion of stipo-seslerietum in scorzonero-chrysopogonetalia, is only due to the sharing of a group of thermophilous species (cephalaria leucantha, teucrium capitatum, convolvulus elegantissimum,, brachypodium retusum, helichrysum italicum etc.) that do not occur in the sesleria juncifolia communities of the high altitude zones. by acta bot. croat. 73 (1), 2014 187 sesleria juncifolia grasslands in apulia (southern italy) 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:36 color profile: generic cmyk printer profile composite default screen contrast, there are some other typical amphi-adriatic species which can be expected on both the sides of the adriatic sea (e.g. helianthemum oelandicum subsp. incanum, festuca circummediterranea, onosma echioides, jurinea mollis, etc.) which curiously occur only in the italian sesleria juncifolia grasslands tables. the only taxon which testifies to an amphi-adriatic connection between stipo-seslerietum and salvio-seslerietum is the chasmophitic inula verbascifolia, which occurs in both these associations with the role of »companion«. as mentioned before, one of the most distinctive characters of stipo-seslerietum is that of exhibiting a percentage of therophytes which is significantly higher than those found in the other sesleria juncifolia associations (tab. 3). the presence of a strong mediterranean therophytic component, however, is also a diagnostic character of the alliance hippocrepido-stipion austroitalicae in comparison with other similar alliances (cytiso-bromion, satureion subspicatae, scorzonerion villosae), and this supports our decision to choose it as syntaxonomical reference. it is more complicated to identify a proper reference at the rank of order. koeleretalia splendentis which was proposed by horvati] (1973) for separating mediterranean from the temperate steppe-like grasslands (scorzoneretalia villosae) would be suitable both in physiognomical and bioclimatic terms but it was subsequently reported to scorzonero-chrysopogonetalia, and, for a minor part, to cymbopogono-brachypodietalia. owing to the impossibility of including stipo-seslerietum in cymbopogono-brachypodietalia, or in any other thero-brachypodietea syntaxon (as reported in figure 7) the presence of a high number of therophytes in stipo-seslerietum is not accompanied by an equally important role in terms of cover percentages), the choice necessarily falls upon scorzonero-chrysopogonetalia, although there still are many unsolved questions concerning the coenological and syntaxonomical features of this order and many doubts on the possibility that its distribution area could be extended to the southern italy. several of the species identified as characteristic of scorzonero-chrysopogonetalia by the various authors (e.g. horvati] 1973, 1975; horvat et al. 1974; feoli-chiapella and poldini 1993; red@i] 1999) such as potentilla zimmeteri, leucanthemum atratum subsp. platylepis, knautia illyrica, knautia resmannii, salvia pratensis subsp. saccardiana, pseudolisymachion barrelieri subsp. nitens, centaurea scabiosa subsp. fritschii, scorzonera villosa subsp. villosa exhibit a prevailing northern adriatic distribution and do not occur in southern italy. at the same time other species often associated to scorzonero-chrysopogonetalia dry grasslands (plantago holosteum, polygala nicaeensis subsp. mediterranea, cyanus triumfetti, koeleria 188 acta bot. croat. 73 (1), 2014 di pietro r., wagensommer r. p. 0 10 20 30 40 50 60 70 80 ch g h ph t % life forms flora frq. cover fig. 7. life form spectrum of stipo austroitalicae-seslerietum juncifoliae calculated on the presence (flora), frequency (frq.), cover of a given life form in the phytosociological table. 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:37 color profile: generic cmyk printer profile composite default screen splendens s.l., eryngium amethystinum, leontodon crispus, thlaspi praecox, thymus longicaulis, sanguisorba minor etc.) are considered characteristic of artemisio-brometalia or closely related syntaxa by other authors (royer 1991, biondi et al. 1995, mucina et al. 2009, di pietro 2011). on the one hand the high presence of italian endemic species (fig. 6), most of which are endemic to the central-southern apennine range, would clearly suggest the inclusion of stipo-seslerietum and hippocrepido-stipion in the suborder festuco-seslerienalia nitidae which has been recently proposed in the coenological range of artemisio-brometalia for central and southern italy (di pietro 2011). on the other hand the presence of several species which are endemic to the apulian limestone platform (centaurea subtilis, lomelosia crenata subsp. dallaportae, aubrieta columnae subsp. italica, genista michelii, viola merxmullerii, euphorbia nicaeensis subsp. japygica etc.) and of several thermophilous southeastern european species advises against the use of a western and central-european order such as artemisio-brometalia. conclusion the description of stipo austroitalicae-seslerietum juncifoliae is a step towards filling a gap in the phytosociological knowledge of the apulian vegetational pattern and in defining the syntaxonomical and distributional framework of sesleria juncifolia communities in peninsular italy. this association is unusual in peninsular italy in being located at significantly lower altitudes than the other sesleria juncifolia communities and in its mediterranean bioclimatic context. although several amphi-adriatic species occur in this association the floristic similarities with the thermophilous s. juncifolia communities occurring in the western coastal side of the balkan peninsula are rather low. in syntaxonomical terms stipo-seslerietum has been included here in the southern italy endemic alliance hippoacta bot. croat. 73 (1), 2014 189 sesleria juncifolia grasslands in apulia (southern italy) fig. 8. cluster analysis of the synoptic columns of the main sesleria juncifolia associations occurring in the italian and balkan peninsula (list of the associations in on-line supplement appendix 4). a – peninsular italy sesleria juncifolia microthermic associations belonging to elyno-seslerietea class. b1a – »thermophilous« sesleria juncifolia association of the italian peninsula; b1b – »thermophilous« sesleria juncifolia associations of the dinaric and karst territories; b2 – stipo-seslerietum + salvio-seslerietum. 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:37 color profile: generic cmyk printer profile composite default screen 190 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 4. synoptic table of the main sesleria juncifolia s.l. communities of the italian and balkan peninsulas. relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 association name number of species per column 54 68 65 36 52 57 84 78 81 70 45 66 62 80 49 111 124 77 number of relevés per column 13 10 9 13 21 9 13 11 19 29 9 10 15 11 6 7 24 13 seslerion apenninae avenula praetutiana 4 3 2 3 3 2 1 . . 3 . . . . . . . . dianthus brachycalyx . 4 2 . 5 3 1 5 3 2 . . . . . . . . trinia dalechampii 5 3 2 1 . . 5 . . 2 . . . . . . . . pedicularis elegans (s.l.) 5 2 5 1 . . . . . . . . . . . . . . festuca violacea subsp. italica 4 3 3 . 2 . . . . . . . . . . . . . poa molineri 4 . . . 4 . 1 2 . . . . . . . . . . cerastium arvense subsp. suffruticosum 4 . . . . 5 4 4 . . . . . . . . . . carum heldreichii . 2 1 2 . 4 . . . . . . . . . . . . leontopodium nivale 5 . 1 2 . . . . . . . . . . . . . . ranunculus breyninus 5 . 5 . . 3 . . . . . . . . . . . . arenaria bertoloni . 2 . . 3 . . . . 4 . . . . . . . . carduus carlinifolius . . . . 1 . 1 . . 2 . . . . . . . . cerastium tomentosum . 3 . . 2 . . . . . . . . . . . . . lomelosia graminifolia . . . 4 . . . . . 3 . . . . . . . . campanula scheuchzeri subsp. pollinensis . . . . 4 . . . 1 . . . . . . . . . androsace vitaliana subsp. praetutiana . . 1 . . . . . . . . . . . . . . . cynoglossum magellense . . 1 . . . . . . . . . . . . . . . seslerietalia tenuifoliae . . . . . . . . . . . . . . . . . . sesleria juncifolia s.l. (junc., apenn., calab., kaln.) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 edraianthus graminifolius subsp. graminifolius 5 5 4 4 5 5 2 . 1 . . 2 . . . . . . carex kitaibeliana 5 5 4 5 5 2 . . . . . . . . . . . . anthyllis montana subsp. jaquinii 1 . 3 4 5 4 . 4 . . . . . . . . . 4 l eo -s .j un c s ca -s .a pe nn s es l. ap en n s es ld ry a a nt hs .c al c .h um .s .a pe c .m ac r. -s .a pe s es ls ti p ap p. ju rs .c al s es ld ra b g en -s .j un c s es lc .h um s es l. ju nc if . s es l. ka ln ik . s al vs .j un c. g en -s .k al n s ti po -s .j un c b ro -s .i nt er r. 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 7 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 191 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 globularia meridionalis . 3 2 4 . 3 . 1 . 5 . . . . . . . . androsace villosa subsp. villosa 3 3 5 5 . . . . . 2 . . . . . . . . anthyllis vulneraria subsp. pulchella 5 . 1 . . . 2 . . . . . . . . . . . gentiana dinarica . 4 4 4 . . . . . . . . . . . . . . paronychia kapela subsp.kapela . 3 2 . 2 . . . . . . . . . . . . 2 elyno-seslerietea . . . . . . . . . . . . . . . . . . minuartia verna subsp. verna 4 5 4 2 4 4 . . . . . 1 . . . . . 1 draba aizoides subsp. azoides 5 5 3 1 2 2 . . . . . . . . . . . . pulsatilla alpina subsp. alpina 2 3 3 1 . . . . . 1 . 1 . . . . . . phyteuma orbiculare . 3 3 1 . . . . . . . 2 2 1 . . . . saxifraga paniculata subsp. paniculata 5 2 1 . 4 2 . . . . . . . . . . . . helianthemum oelandicum subsp. alpestre 5 3 5 2 . . . . . . . . . . . . . 2 polygonum persicaria 1 2 2 1 . . . . . . . . . . . . . . gentiana verna subsp. verna 2 1 2 . . . . . . . . 4 . . . . . . dryas octopetala 1 3 . 5 . . . . . 1 . . . . . . . . thymus praecox subsp. polytrichus 3 . 5 . 5 . . . . . . 4 . . . . . . pedicularis comosa . 2 . . 4 3 2 . . . . . . . . . . . linum alpinum . 1 . . . . . 1 . 2 . 1 . . . . . . silene acaulis s. l. 4 2 4 . . . . . . . . . . . . . . . juncus monanthos 2 2 2 . . . . . . . . . . . . . . . aster alpinus 5 . 1 . . . . . . . . 1 . . . . . . anthyllis montana subsp. montana . 3 . . . . . . . 3 . 4 . . . . . . festuca laevigata subsp. laevigata . . . . . 1 . 3 . 3 . . . . . . . . astragalus depressus . . . . . . 1 . 1 1 . . . . . . . . erigeron epiroticus 3 2 . . . . . . . . . . . . . . . . oxytropis campestris subsp. campestris 5 . 1 . . . . . . . . . . . . . . . potentilla crantzii subsp. crantzii 2 . 1 . . . . . . . . . . . . . . . elyna myosuroides 1 . 1 . . . . . . . . . . . . . . . galium anisophyllum . . 2 . 2 . . . . . . . . . . . . 1 gentianella crispata . . . . 1 . . . . . . . . . . . . 1 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 7 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 192 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 festuca laevigata subsp. crassifolia 1 . . . . . . . . . . . . . . . . . saxifraga oppositifolia subsp. speciosa 2 . . . . . . . . . . . . . . . . . anemone narcissiflora ssp. narcissiflora . . 1 . . . . . . . . . . . . . . . oxytropis neglecta . . 2 . . . . . . . . . . . . . . . pedicularis verticillata . . 1 . . . . . . . . . . . . . . . sedum atratum . . 2 . . . . . . . . . . . . . . . carex firma . . . 1 . . . . . . . . . . . . . . carex rupestris . . . 1 . . . . . . . . . . . . . . cerastium alpinum . . . . 2 . . . . . . . . . . . . . draba aspera . . . . . . . . . 4 . . . . . . . . antennaria dioica . . . . . . . . . . . . 1 . . . . . allium lusitanicum . . . . . . . . . . . . . . . . . 2 cytiso-bromion erecti, festuco-seslerienalia nitidae carex macrolepis . . . 2 . 5 4 2 5 1 . . . . . . . . brachypodium genuense . 2 . 2 1 . 2 . . 1 . . . . . . . . festuca circummediterranea . . . . . . 1 2 1 1 . . . . . . 3 . centaurea ambigua . . . . . 1 2 4 . 1 . . . . . . . . erysimum pseudorhaeticum . . . . . 2 3 5 . 2 . . . . . . . . crepis lacera . . . . . . 1 4 1 . . . . . . . 1 . linum austriacum subsp. tommasinii . . 1 . 1 . . . . . . . . . . . 1 . laserpitium siler subsp. siculum . . . . . 5 2 . 3 . . . . . . . . . scabiosa holosericea . . . . 2 . . . . 3 . . . . . . . . stipa dasyvaginata subsp. appenninicola . . . . . . . 4 3 . . . . . . . . . leontodon cichoraceus . . . . . . . 1 . . . . . . . . 1 . onobrychis alba subsp. alba . . . . . . . 2 . . . . . . . . 2 . orchis pauciflora . . . . . . . 1 . . . . . . . . 1 . centaurea deusta . . . . . . . . 2 . . . . . . . 2 . cytisus spinescens . . . . . . . . 5 . . . . . . . 2 . ornithogalum gussonei . . . . . . . . . . . . . . 3 . . 2 asperula calabra . . . . 4 . . . . . . . . . . . . . onobrychis alba subsp. pentelica . . . . 4 . . . . . . . . . . . . . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 7 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 193 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 sesleria nitida . . . . 2 . . . . . . . . . . . . . rhinantus wettsteinii . . . . . 2 . . . . . . . . . . . . festuca inops . . . . . . 4 . . . . . . . . . . . potentilla rigoana . . . . . . 1 . . . . . . . . . . . cachrys ferulacea . . . . . . . 2 . . . . . . . . . . leontodon rosani . . . . . . . 2 . . . . . . . . . . phleum hirsutum subsp. ambiguum . . . . . . . 5 . . . . . . . . . . euphorbia barrelieri . . . . . . . . 4 . . . . . . . . . cerastium scaranii . . . . . . . . . 3 . . . . . . . . crepis rubra . . . . . . . . . . . . . . . . 1 . elaeoselinum asclepium . . . . . . . . . . . . . . . . 1 . sideritis italica . . . . . . . . . . . . . . . . 3 . satureion subspicatae / scorzonerion villosae . oreoselinum nigrum . . . . . . . . 1 . . . 5 5 . 2 . . satureja montana subsp. variegata . . . . . . . . . . 3 5 . . 1 3 . 1 veronica jacquinii . . . . . . . . . . . 1 2 4 2 . . . festuca stricta subsp. sulcata . . . . . . . 1 . . . . 1 1 . . . . potentilla zimmeteri . . . . . . . . . . 1 1 . . 2 . . . genista sericea . . . . . . . . . . 5 2 . . . 4 . 1 globularia cordifolia . . . . . . . . . . 1 5 . . . 3 . 5 veronica barrelierii . . . . . . . . . . 1 3 . . . 4 . 1 inula ensifolia . . . . . . . . . . 3 5 . . . . . 1 genista sylvestris . . . . . . . . . . 3 . . . 3 . . . stipa eriocaulis s.l. . . . . . . . . . . 2 . . . 3 . . 1 iris pallida subsp. illyrica . . . . . . . . . . 2 . . . . 2 . . satureja subspicata subsp. liburnica . . . . . . . . . . 1 . . . . 3 . 4 seseli elatum subsp. gouanii . . . . . . . . . . 4 . . . . 3 . . lilium carniolicum . . . . . . . . . . . 1 . 1 . . . . bupthalmum salicifolium . . . . . . . . . . . . 5 5 . . . . cirsium pannonicum . . . . . . . . . . . . 2 3 . . . . euphorbia brittingeri . . . . . . . . . . . . 4 4 . . . . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 7 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 194 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 leontodon incanus . . . . . . . . . . . . 5 5 . . . . polygala comosa . . . . . . . . . . . . 4 2 . . . . dianthus sylvestris subsp. tergestinus . . . . . . . . . . . . . . . 4 . 1 potentilla australis . . . . . . . . . . . . . . . 1 . 2 potentilla tommasiniana . . . . . . . . . . . . . . . 4 . 2 medicago prostrata . . . . . . . . . . . . . . . . 2 2 lomelosia crenata subsp. crenata . . . . . . . . 5 . . . . . . . . . hypochaeris maculata . . . . . . . . . . . . 3 . . . . . dianthus giganteum . . . . . . . . . . . . . 2 . . . . centaurea cristata . . . . . . . . . . . . . . 1 . . . sideritis syriaca . . . . . . . . . . . . . . 2 . . . tanacetum cinerarifolium . . . . . . . . . . . . . . 4 . . . centaurea pannonica . . . . . . . . . . . . . . . 2 . . centaurea tommasinii . . . . . . . . . . . . . . . . . 1 edrajanthus tenuifolius . . . . . . . . . . . . . . . . . 2 euphorbia fragifera . . . . . . . . . . . . . . . . . 2 euphrasia illyrica . . . . . . . . . . . . . . . . . 2 genista dalmatica . . . . . . . . . . . . . . . . . 2 leucanthemum liburnicum . . . . . . . . . . . . . . . . . 1 onosma javorkae . . . . . . . . . . . . . . . . . 1 seseli montanum subsp. tommasinii . . . . . . . . . . . . . . . . . 2 hippocrepido-stipion austroitalicae . convolvulus elegantissimus . . . . . . . . . . . . . . 2 . 2 . carduus micropterus subsp. perspinosus . . . . . . . . 1 . . . . . . . . . alyssum diffusum subsp. garganicum . . . . . . . . . . . . . . . . 2 . centaurea subtilis . . . . . . . . . . . . . . . . 2 . crepis apula . . . . . . . . . . . . . . . . 1 . cytisus decumbens . . . . . . . . . . . . . . . . 2 . dianthus tarentinus . . . . . . . . . . . . . . . . 3 . euphorbia nicaeensis subsp. japigica . . . . . . . . . . . . . . . . 1 . genista michelii . . . . . . . . . . . . . . . . 2 . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 7 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 195 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 hippocrepis glauca . . . . . . . . . . . . . . . . 3 . leontodon apulus . . . . . . . . . . . . . . . . 5 . satureja cuneifolia . . . . . . . . . . . . . . . . 2 . scabiosa taygetea subsp. garganica . . . . . . . . . . . . . . . . 1 . scorzonera villosa subsp. columnae . . . . . . . . . . . . . . . . 2 . stipa austroitalica . . . . . . . . . . . . . . . . 5 . thymus spinulosus . . . . . . . . . . . . . . . . 2 . viola merxmuelleri . . . . . . . . . . . . . . . . 1 . artemisio-brometalia + scorzonero-chrysopogonetalia teucrium montanum . 2 . 2 . 4 2 3 5 1 2 4 . 2 1 4 2 5 teucrium chamaedrys . . . . . 3 3 4 2 . . 1 3 4 1 2 1 . carex humilis 4 . . 2 . 5 . 5 . . 4 5 5 4 . 4 . 4 helianthemum oelandicum subsp. incanum . 3 2 4 5 5 5 5 5 . . . . . . . 2 . stachys recta s.l. . . . . . . 1 . 2 1 1 1 2 4 . 4 1 . hippocrepis comosa . 2 . . . 2 1 . 1 1 . 1 2 . . 1 . 1 galium lucidum . . 1 . . 4 5 . . 2 2 4 . 3 . 5 . . asperula cynanchica . 4 3 . . 5 4 3 . . . . 4 4 . . . . koeleria splendens aggr. . 3 . . 1 4 5 5 2 . . . . . . . 3 4 cyanus triumfetti . . 1 . . 2 5 2 . . . 5 4 . . 5 . . anthyllis vulneraria ssp. weldeniana . . . 2 . 4 4 . . . . . 4 2 1 3 . 3 biscutella laevigata . 3 2 . 1 . . . 1 . . 1 1 . . . . . dianthus sylvestris subsp. sylvestris . . . . . . 5 . . 2 3 1 . 1 1 . . . carex caryophyllea . . 2 . . . . . . . . 1 1 . 1 . 1 . eryngium amethystinum . . . . . . 3 5 1 . 1 . . . . . 1 4 muscari neglectum . . . . . . 2 3 1 . . . . . 5 . 2 . thalictrum minus . . . . . . . . . . 1 4 3 2 . 2 . . anthyllis vulneraria ssp. rubriflora . . . . 5 . . 5 3 . . . . . . . 4 . allium sphaerocephalon . . . . . 2 4 4 . . . . . . . . 1 . trinia glauca s.l. . . . . . 4 . 5 . . 1 5 . . . . . . asperula purpurea . . . . . . 3 5 . . 1 . . . . 1 . 2 plantago holosteum . . . . . . . 4 1 . 1 . . . . . 2 5 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 196 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 galium corrudifolium . . . . . . . 4 3 . . . . . 4 . 5 3 artemisia alba . . . . . . . 2 . . 2 . . . . 5 1 . anthericum ramosum . . . . . . . . . . 4 3 5 5 . . . . silene saxifraga . 2 . . 1 . . . . . . . . . . 2 . . coronilla vaginalis . 2 . . . . . . . 2 . 4 . . . . . . leontodon crispus subsp. crispus . . . . 1 . 3 . 3 . . . . . . . . 2 knautia purpurea . . . . . 2 4 5 . . . . . . . . . . polygala major . . . . . 3 1 . 1 . . . . . . . . . thesium linophyllon . . . . . 2 . . 4 . . . 4 . . . . . petrorhagia saxifraga . . . . . . 1 2 4 . . . . . . . . . potentilla incana . . . . . . 2 3 . . 2 . . . . . . . helianthemum apenninum . . . . . . 2 4 . . . . . . . . 3 . globularia bisnagarica . . . . . . 1 . . . . . 4 4 . . . . thesium humifusum . . . . . . . 1 . . 1 . . . . . 2 2 fumana procumbens . . . . . . . . 1 . 2 . . . 1 . . 3 asperula aristata . . . . . . . . 5 . . 5 . . . . 1 2 aethionema saxatile . . . . . . . . 1 . . . . . 1 . 1 . laserpitium siler subsp. siler . . . . . . . . . . . . 5 5 . 4 . . thlaspi praecox . . . . . . . . . . . . . . 2 1 1 1 carlina acaulis subsp. caulescens . 3 . . . . . . 1 . . . . . . . . . allium flavum . . . . 1 . . . 3 . . . . . . . . . thymus striatus . . . . . 1 . 2 . . . . . . . . . 1 euphrasia stricta . . . . . 2 . . . . . . . 1 . . . . aira caryophyllea . . . . . . 1 1 . . . . . . . . . . bunium bulbocastanum . . . . . . 1 1 . . . . . . . . . 1 silene otites . . . . . . 1 5 . . . . . . . . . . arabis collina . . . . . . 2 . . . . . . . . . 1 . centaurea rupestris . . . . . . . 5 . . 3 . . . . . . 2 inula hirta . . . . . . . 1 . . . . . 1 . . . . inula montana . . . . . . . 2 . . . . . . . . 1 . echinops ritro s.l. . . . . . . . . 5 . 1 . . . . . . 2 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 197 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 jurinea mollis . . . . . . . . 5 . . . . . . . 1 . scabiosa columbaria . . . . . . . . 2 . . . . . . . 1 . leucanthemum heterophyllum . . . . . . . . . 1 . 1 . . . . . . pimpinella tragium . . . . . . . . . 2 . . . . . . 4 . centaurea jacea . . . . . . . . . . . 3 . 1 . . . . cephalaria leucantha . . . . . . . . . . . . . . 3 . 1 . euphorbia spinosa . . . . . . . . . . . . . . 4 . 2 1 polygala nicaeensis . . . . . . . . . . . . . . 4 . 1 2 astragalus monspessulanus . . . . . . . . . 3 . . . . . . . 1 anthericum liliago . . . . . . . . 2 . . . . . . . . 1 knautia illyrica . . . . . . . . . . . 5 . . . . . 1 plantago argentea . . . . . . . . . . . 1 . . . . . 1 koeleria cristata . . . . . . . . . . . . . . 1 . . 1 seseli montanum subsp. montanum . 2 . . . . . . . . . . . . . . . . alyssum diffusum subsp. calabricum . . . . 1 . . . . . . . . . . . . . alyssum diffusum subsp. diffusum . . . . . . 4 . . . . . . . . . . . petrorhagia prolifera . . . . . . 1 . . . . . . . . . . . alyssum campestre . . . . . . . 3 . . . . . . . . . . convolvulus cantabrica . . . . . . . 1 . . . . . . . . . . ranunculus millefoliatus . . . . . . . 1 . . . . . . . . . . linum tenuifolium . . . . . . . . 1 . . . . . . . . . ononis pusilla . . . . . . . . 1 . . . . . . . . . astragalus sempervirens . . . . . . . . . 1 . . . . . . . . euphorbia nicaeensis . . . . . . . . . . 1 . . . . . . . potentilla hirta . . . . . . . . . . . . . . 1 . . . carduus nutans . . . . . . . . . . . . . . . . 1 . carex liparocarpos . . . . . . . . . . . . . . . . 1 . euphorbia myrsinites . . . . . . . . . . . . . . . . 1 . orchis antropophora . . . . . . . . . . . . . . . . 1 . ranunculus illyricus . . . . . . . . . . . . . . . . 1 . ruta graveolens . . . . . . . . . . . . . . . . 2 . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 198 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 festuco-brometea . bromus erectus . 3 . . 3 5 4 5 3 1 2 5 3 4 5 5 3 5 lotus corniculatus . . . . . 2 1 . . 1 1 4 4 2 . 2 . 2 gymnadenia conopsea . . . . . 5 2 . 1 . . 2 1 1 . 1 . . thymus longicaulis . . . . . 3 4 . 2 . 1 . 4 . 2 . . 1 brachypodium rupestre . . . . . 4 . 1 2 . . . 1 1 . . 1 . helianthemum nummularium subsp. obscurum . . . . . . . 2 1 . . 4 4 5 . 2 . . euphrasia salisburgensis 2 4 2 2 . . 2 . . . . . . . . . . . hieracium pilosella 1 . . . . 2 2 . . . . 1 . . . . 1 . linum catharticum . 3 . 2 . 1 . . . . . . 1 1 . . . . leontodon hispidus ssp. hispidus . 2 . 2 . . . 2 1 . . 1 . . . . . 1 sanguisorba minor s.l. . . . . . . 3 . . . 2 . 1 . . 4 1 2 thymus pulegioides s.l. . . . . . . . 3 . 3 . . . 5 . 3 . . dorycnium penthaphyllum subsp. germanicum . . . . . . . . . . 2 1 2 . . 2 . 1 minuartia verna subsp.collina . . 1 . . . 4 3 . . . . . . . . . . campanula glomerata . . . . . . . 1 . . . 2 . 2 . . . . pimpinella saxifraga . . . . . . . . . 1 . 3 . . . 1 . . koeleria pyramidata . . . . . . . . . . . . 2 2 . 2 . . plantago media . . . . . . . . . . . . 2 1 . 2 . . festuca rubra . . . . . 2 . . . . . . . . . 2 . . dactylorhiza sambucina . . . . . . 1 2 . . . . . . . . . . anthoxantum odoratum . . . . . . 1 . . . . . . . . . 1 . scorzonera austriaca . . . . . . . 3 . . 3 . . . . . . . carlina acaulis subsp. acaulis . . . . . . . . . 1 . 1 . . . . . . filipendula vulgaris . . . . . . . . . . . 1 . 1 . . . 1 festuca valesiaca . . . . . . . . . . . . . . 1 1 . 1 centaurea scabiosa . . . . . . . . . . . . 3 3 . . . . galium verum . . . . . . . . . . . . 3 1 . . . . leucanthemum vulgare . . . . . . . . . . . . 2 1 . . . . potentilla recta . . . . . . . . . . . . 1 1 . . . . prunella vulgaris . . . . . . . . . . . . 2 1 . . . . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 199 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 ranunculus bulbosus . . . . . . . . . . . . 1 1 . . . . salvia pratensis . . . . . . . . . . . . 4 3 . . . . briza media . . . . . . . . . . . . 1 . . 1 . . festuca rupicola . . . . . . . . . . . . . . . 3 . 3 orchis mascula . . . . . 4 . . . . . . . . . . . . dianthus carthusianorum . . . . . . 1 . . . . . . . . . . . narcissus poeticus . . . . . . 1 . . . . . . . . . . . orchis morio . . . . . . 2 . . . . . . . . . . . carex flacca . . . . . . . . . . . . 1 . . . . . dactylis glomerata . . . . . . . . . . . . . 1 . . . . knautia arvensis . . . . . . . . . . . . . 1 . . . . prunella laciniata . . . . . . . . . . . . . 1 . . . . orchis provincialis . . . . . . . . . . . . . . 2 . . . dianthus monspessulanum . . . . . . . . . . . . . . . 4 . . lathyrus pratensis . . . . . . . . . . . . . . . 1 . . phleum hirsutum subsp. hirsutum . . . . . . . . . . . . . . . 3 . . trifolium montanum . . . . . . . . . . . . . . . 2 . . trifolium pratense . . . . . . . . . . . . . . . 1 . . bromus hordeaceous . . . . . . . . . . . . . . . . 1 . ononis spinosa . . . . . . . . . . . . . . . . . 2 onobrichis montana . . . . . . . . . . . . . . . . . 1 cisto-micromerietea + rosmarinetea . cytisus hirsutus subsp. polytrichus . . . . . . 1 3 2 . . . 4 . . 2 . . genista januensis . . . . . . . 1 . . . . 5 2 . . . 2 satureja montana subsp. montana . . . . . . . . 1 1 . . . . . . 2 . genista radiata . . . . . 1 2 . . . . . . . . . . . onosma echioides ssp. echioides . . . . . . . . 4 . . . . . . . 1 . helichrysum italicum . . . . . . . . . . . . . . 2 . 1 . teucrium capitatum . . . . . . . . . . . . . . 1 . 2 . emerus major subsp. emeroides . . . . . . . . . . . . . . . 3 2 . cotoneaster tomentosum . . . . . . . . 1 . . . . . . . . . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 200 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 matthiola fruticulosa subsp. fruticulosa . . . . . . . . 3 . . . . . . . . . salvia officinalis . . . . . . . . . . . . . . 5 . . . cistus creticus subsp. eriocephalus . . . . . . . . . . . . . . . . 1 . cistus salvifolius . . . . . . . . . . . . . . . . 1 . fumana ericifolia . . . . . . . . . . . . . . . . 2 . fumana thymifolia . . . . . . . . . . . . . . . . 1 . micromeria graeca . . . . . . . . . . . . . . . . 1 . onosma echioides subsp. angustifolia . . . . . . . . . . . . . . . . 1 . rhamnus saxatilis subsp. infectoria . . . . . . . . . . . . . . . . 3 . tuberarietea guttatae . bupleurum baldense . . . . . . 1 . . . . . . . . . 1 . crepis sancta . . . . . . 1 . . . . . . . . . 1 . bombycilaena erecta . . . . . . . 1 . . . . . . . . 1 . crupina vulgaris . . . . . . . 1 . . . . . . . . . . asterolinon linum-stellatum . . . . . . . . . . . . . . . . 1 . briza maxima . . . . . . . . . . . . . . . . 2 . bromus madritensis . . . . . . . . . . . . . . . . 1 . catapodium rigidum . . . . . . . . . . . . . . . . 1 . crupina crupinastrum . . . . . . . . . . . . . . . . 1 . cynosurus echinatus . . . . . . . . . . . . . . . . 1 . euphorbia exigua . . . . . . . . . . . . . . . . 1 . hypochoeris achyrophorus . . . . . . . . . . . . . . . . 1 . linum strictum subsp. strictum . . . . . . . . . . . . . . . . 1 . ononis ornitopodioides . . . . . . . . . . . . . . . . 1 . trachynia distachya . . . . . . . . . . . . . . . . 1 . urospermum dalechampii . . . . . . . . . . . . . . . . 1 . vulpia ciliata . . . . . . . . . . . . . . . . 1 . xeranthemum inapertum . . . . . . . . . . . . . . . . 1 . chasmophytic species . sempervivum tectorum . . . . 4 4 1 5 . . 3 . . . . 4 . . sedum album . . . . 2 2 2 1 . . . . . 1 . 3 . . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 201 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 saxifraga caesia . 4 1 2 . . . . . 2 . . . . . . . . sedum rupestre . . . . . 3 2 5 1 . . . . . . . . . sedum ochroleucum . . . . 3 . . . . . . . . . 5 . 2 . potentilla apennina 1 2 . . . . . . . . . . . . . . . . sedum dasyphyllum . . . . 2 . . . . . . . . . . . 1 1 athamanta turbith . . . . . . . . . . 2 . . . . 3 . . inula verbascifolia . . . . . . . . . . . . . . 4 . 1 . asplenium trichomanes . . . . . . . . . . . . . . . 3 1 . ceterach officinarum . . . . . . . . . . . . . . . 2 2 . scabiosa silenifolia . 3 . . . . . . . . . . . . . . . . galium paleoitalicum . . . . . . . . . 2 . . . . . . . . saxifraga callosa . . . . . . . . . 1 . . . . . . . . asplenium ruta-muraria . . . . . . . . . . . . . . . 2 . . saxifraga crustata . . . . . . . . . . . . . . . 3 . . athamanta sicula . . . . . . . . . . . . . . . . 2 . aubrieta columnae subsp. italica . . . . . . . . . . . . . . . . 1 . aurinia saxatilis subsp. megalocarpa . . . . . . . . . . . . . . . . 1 . hellenocarum multiflorum . . . . . . . . . . . . . . . . 1 . hieracium acanthodontoides . . . . . . . . . . . . . . . . 1 . lomelosia crenata subsp. dallaportae . . . . . . . . . . . . . . . . 1 . other species . acinos alpinus . . . . 1 2 1 . . 2 . . . . . 2 2 . poa alpina subsp. alpina 3 3 4 . . 3 . . . 1 . . . . . . . . tanacetum corymbosum . . . . . 1 1 3 1 . . . . 2 . . . . silene vulgaris . . . . . . . 1 . . . . 1 2 3 . 1 . thesium parnassi 1 4 2 2 . . . . . . . . . . . . . . viola eugeniae subsp. eugeniae 1 2 2 . . 2 . . . . . . . . . . . . helianthemum nummularium subsp. grandiflorum 1 1 . . 2 . . . . . . . . . 2 . . . juniperus communis subsp. nana . 3 2 . 1 . . . 1 . . . . . . . . 1 bupleurum falcatum subsp. cernuum . . . . 1 . . . 2 . . 1 . . . 5 . . valeriana tuberosa . . . . . . 1 2 . . . . . . 2 . 1 . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 202 a c t a b o t .c r o a t .73 (1),2014 d ip ie t r o r .,w a g e n s o m m e r r .p . tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 aster bellidiastrum . 2 2 1 . . . . . . . . . . . . . . ranunculus thora . 2 1 2 . . . . . . . . . . . . . . arctostaphylos uva-ursi . 1 1 . . . . . . . . 5 . . . . . . cuscuta epithymum . . . . 1 . . . 1 . . . . . . . 1 . thymus glabrescens subsp. decipiens . . . . . 2 2 3 . . . . . . . . . . cerastium pumilum . . . . . . 1 1 . . . . . . . . 1 . senecio doronicum . . . . . . 2 . . 1 . 2 . . . . . . galium album . . . . . . 1 . . . . . 1 . 1 . . . sorbus aria . . . . . . . . 3 . . 1 . . . 1 . . inula salicina . . . . . . . . . . . 5 2 3 . . . . euphorbia cyparissias . . . . . . . . . . . . 3 4 . 4 . 1 geranium sanguineum . . . . . . . . . . . . 4 3 . 2 . . laserpitium latifolius . . . . . . . . . . . . 3 2 . 1 . . hypericum perforatum . . . . . . . . . . . . . 1 . 1 1 . gentianella columnae 1 2 . . . . . . . . . . . . . . . . polygala alpestris 1 . 1 . . . . . . . . . . . . . . 1 armeria canescens subsp. canescens 3 . . . . . 1 . . . . . . . . . . 1 astrantia pauciflora subsp. tenorei . 2 1 . . . . . . . . . . . . . . . linum capitatum ssp. serrulatum . 1 1 . . . . . . . . . . . . . . . primula auricola subsp. ciliata . 3 . . . . . . . . . . . . . 2 . . carex mucronata . . . 4 . . . . . 3 . . . . . . . . euphrasia italica . . . . 3 . . . 2 . . . . . . . . . pinus leucodermis . . . . 2 . . . 1 . . . . . . . . . sedum hispanicum . . . . 1 . . . . . . . . . . . 1 . arenaria serpyllifolia . . . . . . 2 . . . . 1 . . . . . . seseli libanotis . . . . . . 1 . . . . . . . . 5 . . ornithogalum comosum . . . . . . . 1 . . . . . . . . 1 . plantago lanceolata subsp. sphaerocephala . . . . . . . . 1 . . 1 . . . . . . amelanchier ovalis subsp. cretica . . . . . . . . 1 . . . . . . 1 . 1 fagus sylvatica . . . . . . . . 1 . . . . . . 1 . . carlina corymbosa . . . . . . . . 1 . . . . . . . 1 2 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 203 s e s l e r ia j u n c if o l ia g r a s s l a n d s in a p u l ia (s o u t h e r n it a l y ) tab. 4. – continued relevée nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 centaurium erythraea . . . . . . . . 1 . . . . . . . 2 . genista tinctoria . . . . . . . . 1 . . . . . . . 1 . quercus ilex . . . . . . . . 1 . . . . . . . 1 . anthyllis vulneraria ssp. polyphylla . . . . . . . . . 3 1 . . . . . . . erica carnea . . . . . . . . . 1 . . . 5 . . . . helianthemum oelandicum subsp. italicum . . . . . . . . . 4 . . . . 1 . . . cyclamen purpurascens . . . . . . . . . . 2 . . 1 . . . . frangula rupestris . . . . . . . . . . 2 . . . . 2 . . ruta divaricata . . . . . . . . . . 3 . . . . 4 . . stachys officinalis . . . . . . . . . . . 3 . 1 . . . . vincetoxicum hirundinaria . . . . . . . . . . . 1 . 2 . . . . juniperus communis . . . . . . . . . . . 5 . . . 2 . . verbascum chaixii . . . . . . . . . . . 1 . . . 2 . . cirsium acaule . . . . . . . . . . . . 1 . . . . 2 clematis recta . . . . . . . . . . . . 1 1 . . . . daphne cneorum . . . . . . . . . . . . 3 1 . . . . hieracium bauhini . . . . . . . . . . . . 2 2 . . . . primula vulgaris . . . . . . . . . . . . 1 1 . . . . polygonatum odoratum . . . . . . . . . . . . 2 . . 2 . . allium carinatum . . . . . . . . . . . . . 1 . 5 . . fragaria vesca . . . . . . . . . . . . . 1 . 2 . . mercurialis ovata . . . . . . . . . . . . . 4 . 1 . . trifolium rubens . . . . . . . . . . . . . 1 . 1 . . brachypodium retusum . . . . . . . . . . . . . . 5 . 1 . carex halleriana . . . . . . . . . . . . . . . . . 2 crepis chondrilloides . . . . . . . . . . . . . . . . . 1 poa bulbosa . . . . . . . . . . . . . . . . . 2 podospermum roseum . . . . . . . . . . . . . . . . . 1 klasea radiata . . . . . . . . . . . . . . . . . 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 6 7 7 d i p i e t r o a n d w a g e n s o m m e r . v p 2 0 . o u j a k 2 0 1 4 9 : 5 5 : 3 8 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n crepido-stipion austroitalicae but its placing at higher syntaxonomical ranks remains uncertain. the lack of any pan-european synthesis on this topic makes the inclusion of stipo-seslerietum in scorzonero-chrysopogonetalia nothing but provisional. the general revision of south-european dry grasslands is currently in progress will certainly provide new elements for the solution of this syntaxonomical and biogeographical issue. syntaxonomical scheme festuco-brometea br.-bl. et tüxen ex br.-bl. 1949 scorzonero villosae-chrysopogonetalia grylli horvati} et horvat in horvati} 1963 (prov.) festuco circummediterraneae-seslerienalia nitidae di pietro 2011 hippocrepido glaucae-stipion austroitalicae forte et terzi in forte, perrino et terzi 2005 stipo austroitaliacae-seslerietum juncifoliae di pietro et wagensommer ass. nov. stipo austroitaliacae-seslerietum juncifoliae typicum subass. nov. stipo austroitaliacae-seslerietum juncifoliae helianthemetosum apennini subass. nov. stipo austroitaliacae-seslerietum juncifoliae seslerietosum autumnalis subass. nov. syntaxa quoted in the text are listed in the online supplement appendix 5. acknowledgements the authors thank la sapienza university for financial support (grant prot. c26f07x4my – 2007). we are also grateful to all colleagues who contributed some references that were 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university of florence. wagensommer, r. p., di pietro, r., 2006: notulae alla checklist della flora italiana 1244–1245. informatore botanico italiano 38, 205–206. weber, h. e., moravec, j., theurillat, j. p., 2000: international code of phytosociological nomenclature. journal of vegetation science 11, 739–768. acta bot. croat. 73 (1), 2014 207 sesleria juncifolia grasslands in apulia (southern italy) 677 di pietro and wagensommer.ps u:\acta botanica\acta-botan 1-14\677 di pietro and wagensommer.vp 20. o ujak 2014 9:55:39 color profile: generic cmyk printer profile composite default screen opce-str.vp acta bot. croat. 69 (1), 107–121, 2010 coden: abcra 25 issn 0365–0588 role of antioxidant enzyme responses and phytochelatins in tolerance strategies of alhagi camelorum fisch growing on copper mine masoud m. a. boojar*, zahra tavakoli department of biology, university of tarbiat moalem, 49 mofateh avenue, tehran, p.o. box: 15614, iran. this study was performed to clarify some aspects of tolerance mechanisms against excess copper (cu) in alhagi camelorum fisch, a dominant wild type plant growing in a cu-contaminated zone and its vicinity. total and available copper was at toxic levels for plants growing on the contaminated soil. there were no visual and conspicuous symptoms of cu toxicity in this plant species. most of the excess cu in soil was transferred to and accumulated in plant leaves in which the storage rate in vacuoles and chloroplasts was 48% and 7% respectively. there was an insignificant decrease in chlorophyll content and a significant increase in tissues phytochelatins and antioxidant enzyme activities in plants collected from the contaminated zone as compared to plants of the same species growing on uncontaminated soil. we also observed significant elevation in oxidative damage biomarkers, malondialdehyde and dityrosine, when the aerial parts of alhagi camelorum were compared with the same parts of the plant collected from an uncontaminated zone. alhagi camelorum elevated its antioxidative enzyme activities, phytochelatins and accumulated the excess of cu in leaf vacuoles in response to cu-toxicity as tolerance strategy. key words: copper, metal, toxicity, antioxidant, phytochelatin, tolerance abbreviations: cat – catalase, dtpa – diethylene triaminepenta acetic acid, ec – electric conductivity, gpx – glutathione peroxidase, gsh – glutathione, hepps – (3-[4(2-hydroxyethyl)-1-piperazinyl] propanesulfonic acid), mda – malondialdehyde, msa – methanesulfonic acid, nbt – nitroblue tetrazolium, pc – phytochelatin, ros – reactive oxygen species, sod – superoxide dismutase, tba – thiobarbituric acid, tfa – trifluoro acetic acid introduction copper is an essential micronutrient for plants and a component of several enzymes mainly participating in electron flow, catalyzing the redox reactions in mitochondria and chloroplast. in addition, this element is also a cofactor or a part of a prosthetic group of many key enzymes that play important roles in photosynthesis, respiration, co2 assimilation, atp synthesis and nitrogen metabolism (demirevska-kepova et al. 2004). the abacta bot. croat. 69 (1), 2010 107 * corresponding author, e-mail: aboojar@yahoo.com u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees sorption of copper from soil by plants and its transport to the above-ground parts depend on the ability of plants to transfer this metal across the soil-root interface and to the total amount of cu, present in the soil (agata and ernest 1998, baker and proctor 1990). on the other hand, copper is a widespread contaminant originating from different human activities including mining and smelting of copper ores. mining activities generate a large amount of waste rocks and tailings, which get deposited on the surface. in this condition, a network of sequestration activities and immobilization functions regulate the uptake, distribution and detoxification of excess metal ions in plants (clemens 2001). an active detoxification mechanism acts by means of glutathione (gsh) and of peptides synthesized at the expense of gsh, the phytochelatins (pcs), (de vos et al. 1992). glutathione (gsh) is a low-molecular weight thiol tripeptide, involved in cellular defense against the toxic action of xenobiotics, oxyradicals as well as of metal cations (meister and anderson 1983). it is able to modify metal toxicity by chelating metal ions in cells; it plays a key role in protecting macromolecules from damage by free radicals by trapping them in an aqueous phase (freedman et al. 1989). on the other hand, pcs are polymers of g-glu-cys units with the terminal glycine and capable of binding heavy metals via thiolate coordination. they are induced in response to many heavy metals including copper (morelli and scarano 2004) when the non-pc based mechanism of detoxification gets exhausted and free metal ions become available to induce pc synthesis (schat et al. 2002). exposure to excess cu is capable of stress induction in which the role of oxidative stress and reactive oxygen species (ros) production may be involved (stadman and oliver 1991, waldermar et al. 1994). on the other hand, under cu toxicity, excess copper is an efficient generator of ros in fenton-type reactions, leading to disturbance in metabolic pathways and macromolecule damages (hegedus et al. 2001). ros are partially reduced forms of atmospheric oxygen and under normal conditions their production in plant cells is tightly controlled by the scavenging system. ros can oxidize biomolecules such as dna, proteins and lipids, creating oxidative injury that results in a reduction of plant growth and development (hernandez-jimenez et al. 2002, ogawa and iwabuchi 2001). since the half-lives of ros are extremely short, their stable end products of oxidative damage to cellular macromolecules can be used for oxidative stress monitoring (orhanl et al. 2004). dityrosine, a hallmark of oxidized proteins and malondialdehyde (mda) a biomarker of lipid peroxidation are closely correlated with oxidative stress level (halliwell and gutteridge 1998). to control the level and effects of ros, cells have developed various antioxidant defenses including antioxidant enzymes and low molecular mass radical scavengers. they regenerate the active form of antioxidants and eliminate or reduce the damage caused by ros (alsher et al. 1997). among plants growing on cu-contaminated soils, cu-tolerant genotypes evoke antioxidant enzyme induction as a general response to the toxic effects of copper to protect them against metabolic disturbance and cellular damage (van assche and clijsters 1990). superoxide dismutase (sod), the first major enzyme found in all aerobes, catalyses dismutation of superoxide anion to h2o2 and molecular oxygen. glutathione peroxidase protects the membrane lipids from oxidative damage and detoxifies the organic peroxides; 108 acta bot. croat. 69 (1), 2010 boojar m. m. a., tavakoli z. u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees it can also act on organic hydroperoxides (kantol et al. 1998). the intracellular level of h2o2 is regulated by a wide range of enzymes, the most important being catalase and peroxidase. catalase inactivates h2o2 to oxygen and water (rusina et al. 2004). in the present study, field surveys have been carried out on alhagi camelorum plant growing in the area of a copper mine in kerman state. the aim of this study was to investigate the cu-accumulating ability of this wild type plant growing around the mine. we also evaluated the corresponding status of the antioxidant enzyme activities, the levels of phytochelatins, ghs and oxidative damage products of lipids and proteins to clarify some aspects of this plant toxicity tolerance. materials and methods this study was carried out at sarcheshmeh, located in sirjan at kerman province in iran (longitude: 55o, 52’, 20’’ e, latitude: 29o, 56’, 40’’ n). a maximum temperature of +32 oc and an average annual air temperature of 15 oc were recorded. the annual rainfall was about 516 mm and there was no industry nearby. after a geobotanical survey, two zones were considered for plant and soil sampling. zone 1 was located in center of the cu-mine area and zone 2 was approximately 9.7 km south of the waterlogged area of the cu-mine and the ecological conditions were similar in both zones. the soil of zone 2 was taken as a control because it had never received sources of cu. zone 1 was one of the well-known copper mines where the main activity was copper extraction at %1.16 grade of copper. tailings had been abandoned for 11 years at the time of sampling. the levels of cd, co, zn and pb in zone 1 were generally below the usa maximum tolerant concentrations (kabata-pendias 1995). the ratio of total cu in zone 1 to that in zone 2 was about 52-fold. the ratio for available cu was about 83 fold, showing that the available level was higher than toxicity threshold levels (icrcl 1987). soil and plant sampling alhagi camelorum fisch was endemic and widespread in the study area. at each zone, plant samples were collected at a determined time of a single growing season and according to the actual landform of the copper-mine and the distribution of vegetation before flowering period. care was taken to collect plant species samples from both zones while they were in growth period. we established three random regions in each study zone. we collected at least 5–8 plants of our species from each area. fresh tissues including roots, stems and mature leaves of collected plants were considered for three replicate analysis. plants were cleaned in abundant deionised fresh water, rinsed with distilled water and identified by an expert botanist. care was taken to avoid sample contamination with other sources of cu during sampling, washing, transporting and analysis. corresponding soil samples were also collected at the location of plant sampling from the rooting zone (maximum sampling depth about 30 cm) and transferred to a polythene bags. excess air was squeezed out, the bags were sealed, transferred to the laboratory and stored at 4 oc for a maximum of 48 h prior to analysis. these samples were then air-dried and sieved through a 2 mm plastic screen. there were 6 replicates for each soil sample. acta bot. croat. 69 (1), 2010 109 antioxydant enzyme response and phytochelatins u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees soil analysis dried soil samples were digested with hcl + hno3 + hclo4 (3:1:1, w.v.) (yuan 1988). total cu and other metals were determined by atomic absorption spectrophotometer (analyst 100, perkin elmer, usa), using an acetyleneair flame. diethylenetriaminepentaacetic acid (dtpa)-extractable cu, cd, co, zn and pb contents of 10 g soil samples (sample: dtpa, 1:2, w.v.) were determined by atomic absorption spectrophotometer (page et al. 1982). the reagents and standards for aas were ultra pure. the detection limits in mg kg–1 for total and extractable metals in soils were: 0.06 for cd, 0.15 for co, 0.17 for pb, 0.08 for cu and 0.11 for zn. this step represents the fraction that is water soluble and most easily available to plants and easily leachable into the ground water (sieb 1995). soil nitrate (no3–) was analysed colorimetrically (keeney and nelson 1982). the total nitrogen was determined by the kjeldhal digestion method (duchaufour 1970). the ph and electrolytic conductivity (ec) were determined in a water:soil extract 1:1 using a beckman ph-meter and a conductivity meter model hi8633, hanna instruments co., respectively. plant biomass and cu content analysis the washed plant samples were separated into roots and shoots, and dried in an oven at 60 oc for 48 h; then the biomass presented as dry weight was measured. for elemental analysis, the dried plant tissues were ashed in a muffle furnace at 550 oc for 24 h. the ash was digested with a mixture of hno3 and hclo4 (5:3 wet weight), heated in an oven. after cooling, the extracts were diluted and made up to 25 ml with 1 m hno3. copper concentration of the extract was determined by atomic absorption spectrophotometer. chlorophyll determination fresh and mature leaves (0.5 g) were extracted with 10 ml 80% acetone as described by alan (1994). the absorbance of extract was measured at 663 and 645 nm in the uv-vis light spectrophotometer (model uv-9100). the chlorophyll content was calculated using the equation as follows: ct = 20.2 a645 + 8.02 a663 chloroplast isolation fresh and mature leaves (5 g) were homogenized for 15 s with a homogenizer in 50 ml ice-cold grinding medium containing: 0.33 m sorbitol, 1mm edta, 0.1% bsa, 2 mm sodium ascorbate and 50 mm k2hpo4, ph 7.5. the homogenate was filtrated through miracloth and centrifuged for 1 min at 1000 g at 4 oc to remove whole cells and cell debris. the intact chloroplasts were pelleted through centrifugation at 4500 g for 30 s and were gently resuspended in the same buffer without bsa and centrifuged again at the same conditions. this washing procedure was repeated twice and pelleted chloroplasts were isolated (rusina et al. 2004). vacuole isolation leaves were floated on an enzyme solution containing 1 mm cacl2, 500 mm sorbitol, 0.05% (w.v.) polyvinyl pyrrolidone, 15 mm mes/tris ph 5.5, 0.2% (w.v.) bovine serum 110 acta bot. croat. 69 (1), 2010 boojar m. m. a., tavakoli z. u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees albumin, 1% (w.v.) cellulose, 0.5% (w.v.) macerozym, 0.01% (w.v.) pectolyase, and agitated for 30 min. vacuoles were released into the recording chamber by hyposmotic shock treatment of protoplasts in 100 mm kcl, 5 mm mgcl2, 2mm egta, 1 mm dithiotheritol (dtt) and 5 mm tris/mes, ph 7.5, adjusted to p = 300 mosm with d-sorbitol. after setting of the vacuoles, the hypotonic solution was carefully replaced by standard bath solution (scholz-starke et al. 2004). measurement of dityrosine: 1.2 grams of fresh tissue material were homogenized with 5 ml of ice-cold 50 mm hepes-koh, ph 7.2, containing 10 mm edta, 2 mm pmsf, 0.1 mm p-chloromercuribenzoic acid, 0.1 mm dl-norleucine and 100 mg polyclar at. the plant tissue homogenate was centrifuged at 5000 g for 60 min to remove debris. purification of o,o’-dityrosine in the clear tissue homogenized supernatant fluid was accomplished by preparative hplc. o,o’-dityrosine was recovered by gradient elution from the c-18 column (econosil c18, 250 mm ´ 10 mm) (orhanl et al. 2004). the composition of eluent varied linearly from acetonitrile–water–tfa (1:99:0.02) to acetonitrile–water– tfa (20:80:0.02) over 25 min. the gradient was started 5 min after the injection. a flow rate of 4 ml/min was used. o,o’-dityrosine was analyzed by reversed-phase hplc with simultaneous uv-detection (280 nm) and fluorescence-detection (ex. 280 nm, em. 410 nm). a phenomenex inertsil ods 2 (150 mm ´ 4.6 mm, 5 mm) hplc column (bester, amsterdam, the netherlands) equipped with a guard column was used for these analyses. a gradient was formed from 10 mm ammonium acetate, adjusted to ph 4.5 with acetic acid, and methanol, starting with 1% methanol and increasing to 10% over 30 min. the flow rate was 0.8 ml min–1. a standard dityrosine sample was prepared according to amado et al. (1984). dityrosine was quantified by assuming that its generation from the reaction of tyrosine with horseradish peroxidase in the presence of h2o2 was quantitative (using the extinction coefficient e315 = 4.5 mm–1 cm–1 at ph 7.5). malondialdehyde analysis proteins of tissue homogenate were precipitated with 40% trichloracetic acid (tca), w.v. the mda assay was based on the condensation of one molecule malondialdehyde with two molecules of thiobarbituric acid (tba) in the presence of reduced reagent volumes to increase sensitivity, generating a chromogen with uv absorbance. the tba + mda complex was analyzed by hplc (bird et al. 1983). hplc system consisted of a hewlett + packard 1050 gradient pump (avondale, pa) equipped with an automatic injector, a 1050 diode-array absorption detector and a personal computer using chem station software from hewlett + packard. aliquots of the tba + mda samples were injected on a 5 mm supelcosil lc-18 reversed phase column (30 ´ 4.6 mm). the mobile phase consisted of 15% methanol in double-distilled water degassed by filtering through a 0.5 mm filter (millipore, bedford, ma). the flow rate was 2 ml min–1. mda + tba standards were prepared using tetraethoxypropane. the absorption spectra of standards and samples were identical, with a characteristic peak at 540 nm. measurements were expressed in terms of malondialdehyde (mda) normalized to the sample protein content. protein content was determined by the method of bradford, with standard curves prepared using bsa (bradford 1976). extraction and determination of pcs and gsh were performed according to sneller et al. (2000). frozen plant tissues were homogenized with a pestle and mortar with quartz sand in 2 ml of 6.3 mm diethylene triaminepenta acetic (dtpa) with 0.1% trifluoro acetic acta bot. croat. 69 (1), 2010 111 antioxydant enzyme response and phytochelatins u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees acid (tfa) at 4 oc. the homogenate was centrifuged at 14,000 g at 4 oc for 12 min. the clear supernatants were collected for the assay by hplc using pre-column derivatization with a fluorescent probe, monobromobiane (mbrb). 250 ml of supernatant was mixed with 450 ml of 200 mm hepps (3-[4-(2-hydroxyethyl)-1-piperazinyl] propane sulfonic acid) at ph 8.2, with 6.3 mm dtpa, and 10 ml of 25 mm (mbrb). derivatization was carried out in the dark at 45 oc for 30 min. the reaction was terminated by the addition of 300 ml of 1 m methane sulfonic acid (msa). the samples were stored in the dark at 4 oc until hplc analysis. blank samples were used to identify the reagent peaks. the bimane derivatives were separated using a binary gradient of mobile phase a (0.1% tfa) and b (100% acetonitrile) at room temperature (22 ± 2 oc). fluorescence was detected at 380 nm excitation and 470 nm emission wavelengths. the flow rate was 0.5 ml /min. fifty ml of the derivatives sample was run in a linear gradient from 12% to 25% b for 15 min, then 25% to 35% b for 14 min and next 35% to 50% b for 21 min. before a new sample was injected, the column was cleaned (5 min, 100% b) and equilibrated (10 min, 12% b) and post-time was 5 min. total analysis time was 70 min. analytical data were integrated by using the hp chemstation. retention times of pcs and gsh in biological samples were checked with pcs and gsh standards, respectively. individual pc subtypes were quantified by using the relationship peak vs. concentrations of gsh standard solutions. corrections for differential derivatization efficiencies were made according to the method stated by sneller et al. (2000). preparation of enzyme extracts whole tissue (leaves, stems and/or roots) were homogenized (1:5 w.v.) separately in an ice cold mortar using 50 mm sodium phosphate buffer, ph 7.0, containing 1 m nacl, 1% polyvinylpyrrolidone and 1 mm edta. after centrifugation (20,000 g, 15 min), the supernatant (crude extract of leaves) was used to determine enzyme activities, which were measured at 25 oc. enzyme assays catalase (ec 1.11.1.6) activity was determined by following the consumption of h2o2 (extinction co-efficient 0.0394 mm ml–1) at 240 nm for 30s (aeby 1984). the assay mixture contained 100 mm potassium phosphate buffer (ph 7.0), 15 mm h2o2 and 50 ml leaf extract in a 3 ml volume. unit was defined as mmol h2o2 decomposed per 1 min. to detect glutathione peroxidase [ec 1.11.1.9 (gsh-px)] activity, the method of hopkins and tudhope, with t -butyl hydroperoxide as a substrate was used (hopkins and tudhope 1973). the reaction mixture comprised 50 mm potassium phosphate buffer, ph 7.0, 2 mm edta, 0.28 mm nadph, 0.13 mm gsh, 0.16 u gr, 0.073 mm t-butyl hydroperoxide and enzyme extract (50 mg protein). one unit of gsh-px activity was defined as the amount of enzyme that catalyzed the oxidation of nadph [mmol min–1 mg–1 protein]. sod activity was determined according to minami and yoshikawa (1979), with 50 mm tris-ca-codylic sodium salt buffer, ph 8.2, containing 0.1 mm edta. the reaction mixture was composed of 1.42% triton x-100, 0.055 mm nitroblue tetrazolium (nbt), 16 mm pyrogallol and enzyme extract (50 mg protein). the principle of this reaction is based on the measurement of the concentration of the reduced form of nbt determined at 540 nm. the 50% inhibition was established according to mccord and fridovich (1969). this 112 acta bot. croat. 69 (1), 2010 boojar m. m. a., tavakoli z. u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees inhibition was defined as the quantity of enzyme required to inhibit the reduction of nbt by 50% per 1 min. statistical analysis all statistical analysis was carried out by using the procedure available in the spss v.10 (spss inc., chicago, il) statistical package. each experiment was run on each sample at least in three replicates from all samples collected from all parts for each zone. student‘s t-test was applied to determine the significance of results between different samples. results evaluations of ph and ec level revealed that the water extracts of the soils in both zones were mild acidic and there was no problem with salinity; however the soil samples associated with plants in zone 1 had a slightly lower ph than zone 2. to better characterize nitrogen species levels, we evaluated total nitrogen and nitrate levels, and the results indicated that both parameters were slightly lower in zone 1 than zone 2, although they did not differ significantly. total contents of each metal; cd, co, zn and pb in the soil samples of zone 1 were significantly higher than those from zone 2 soils. the available cu concentration for plants in zone 1 consisted of 39% of total cu level. there were no significant differences in the available levels of the studied metals between soils of the zones except for cu. the soils of zone 2 did not display higher concentrations of other evaluated metals, among which co and cd were very low (tab. 1). accordingly, their levels could not be toxic for plants. measurement of cu contents in roots, leaves and stems of alhagi camelorum collected from the different sites showed that all tissues of our studied plant species in zone 1 contained significantly higher cu levels as compared with samples collected from zone 2 soils. in addition, analysis showed significantly high cu contents in leaf, stem and roots, in which, the level of cu in leaves of a. camelorum rose up to around three fold of its total content in roots. the ratio of cu in roots of this species growing on zone 1 to that in the roots of the same plants in zone 2 was approximately 7. distribution of cu in leaf acta bot. croat. 69 (1), 2010 113 antioxydant enzyme response and phytochelatins tab. 1. chemical characteristics of soils of the studied zones. data were presented as mean ± sd. * – significant difference with respect to zone 2 (p < 0.05). t – total content; e – dtpa – extractable content; ec – electric conductivity, # – electrolytic conductivity in water: soil extract (1:1). z on e heavy metal content [mg (kg dw)–1] ph e c # (ms cm–1) nitrogen (g (kg dw–1)cu zn co cd pb t e t e t e t e t e no3 – total 1 842.7 ±* 38.5 332.4 ±* 15.9 38.64 ±* 7.4 4.08 ± 0.36 9.6 ± 2.34 <0.11 3.05 ± 0.81 <0.17 12.8 ±* 3.61 <0.28 5.82 ± 0.44 2.37 ± 0.24 0.27 ± 0.03 1.41 ± 0.10 2 16.36 ± 3.52 4.21 ± 0.77 26.41 ± 4.9 6.24 ± 0.61 3.140 ± 1.83 <0.15 1.68 ± 0.56 <0.12 6.9 ± 1.82 <0.28 6.65 ± 0.25 1.92 ± 0.16 0.31 ± 0.02 1.68 ± 0.14 u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees organelles revealed that the plant species in zone 1 had significantly higher cu content in their vacuoles and/or chloroplasts than the same plant species in zone 2. the concentration of copper in vacuoles of alhagi camelthorn was 29.67; this comprised 48% total leaf cu levels and was over six times higher than the chloroplast cu content (tab. 2). the assay of biomass and chlorophyll contents showed that these parameters in plant species from zone 1 were insignificantly lower than in the same plant species associated with zone 2 (tab. 3). 114 acta bot. croat. 69 (1), 2010 boojar m. m. a., tavakoli z. tab. 2. copper bioconcentration in tissues [g (kg dw)–1] and in organelle [(mg (g dw–1)] of leaves. data are presented as mean ± sd; b – statistically different with respect to roots; c – significant difference as compared with vacuoles; statistically different with respect to stems; # – the ratio of leaf organelle cu to total leaf cu zone tissue organelle leaf stem root chloroplasts cu/cu% vacuoles cu/cu% 1 61.54 ± 5.68 b 29.86 ± 3.24 b 17.05 ± 0.22 4.27 ± 0.71 c 7 29.67 ± 2.88 48 2 5.61 ± 1.05 b 3.28 ± 0.65 2.54 ± 0.49 0.28 ± 0.02 c 5 1.25 ± 0.18 22 tab. 3. chlorophyll content, and biomass of different plant tissues. data are presented as mean ± sd. zone biomass mg (g fw)–1 chlorophyll mg (g fw)–1shoot root 1 98.17 ± 5.64 76.41 ± 4.32 2.18 ± 0.24 2 112 ± 6.82 83.52 ± 5.16 2.71 ± 0.32 tab. 4. antioxidant enzyme activities and biomarkers of lipid peroxidation and protein oxidation in tissues of studied plants. data are presented as mean ± sd. * – significant difference as compared with zone 2; #-significant difference as compared with roots. tissue z on e sod (u/mg protein) gpx (u/mg protein) cat (um/min/mg) mda (nmol/mg protein) dityrosine (nmol/mg protein) gsh (nmol/gfw) leaf 1 28.10 ± 2.91 *# 5.41 ± 1.30 # 57.43 ± 5.32 *# 52.10 ± 4.12 4.36 ± 0.53 416 ± 26 * 2 17.28 ± 2.62 # 6.26 ± 1.12 # 33.51 ± 2.81 23.62 ± 2.30 2.71 ± 0.22 670 ± 41 stem 1 31.58 ± 3.04 *# 8.10 ± 1.63 * 66.04 ± 5.84 * 45.70 ± 3.66 3.92 ± 0.26 285 ± 22 * 2 12.77 ± 2.55 4.31 ± 0.82 40.55 ± 3.61 40.15 ± 3.85 3.21 ± 0.28 512 ± 38 root 1 38.26 ± 3.16 * 11.07 ± 1.74 * 87.12 ± 6.47 * 42.80 ± 3.30 3.10 ± 0.21 110 ± 14 * 2 9.62 ± 1.64 3.72 ± 0.63 23.38 ± 2.31 47.32 ± 3.72 3.81 ± 0.35 367 ± 27 u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:57 color profile: disabled composite 150 lpi at 45 degrees as a comparison, the antioxidant enzyme activities in tissues of alhagi camelorum from zone 1, were significantly higher than those of zone 2. on the other hand, the increases in the enzyme activities of this species growing on zone 1 were considerable for roots, stems and then leaves, in that order, but in zone 2, the pattern of increase was observed in leaves, stems and then roots. moreover, a. camelorum showed significant increase in sod and cat activities of roots as compared stems and/ or leaves in zone 1. gpx activity of the plant increased significantly only in the roots. with regard to the levels of oxidative damage biomarkers of lipids as mda and proteins as dityrosine, these parameters were considerable in leaves, stems and roots in order in zone 1, but in zone 2 the pattern of increase was for roots, stems and leaves in that order. the studied parameters in the leaves of this plant species growing in zone 1 were significantly higher (about two fold) in comparison with the same plants collected from zone 2 (tab. 4). in addition, the evaluated plant tissues from zone 1 revealed significantly lower concentrations of gsh than those from zone 2. by contrast, the levels of both pc2 and pc3 in each evaluated tissues of the studied plant from zone 1 were significantly higher than the same plant tissues collected from zone 2 (fig 1). discussion this investigation definitely showed that the study area was significantly contaminated with cu at toxic levels and the rate of available concentration of this metal was high (about 39%) for plant growth. the copper levels in leaves of a. camelorum were above the critical level for copper toxicity (robson and reuter 1981). the soil analysis revealed normal levels of other heavy metals (pb, co and cd). the high cu availability may be attributed to the soil ph characteristic. studies have confirmed that a low level of ph causes an increase in cu solubility and its release from the soil phase leading to an elevation in copper uptake by roots (watmough and dickinson 1995). our results were in accordance with the findings reported in the cyprus skouriotissa cu mine, where ph was mildly acidic and contained copper up to 787 mg [kg (dry weight)]–1 (johansson et al. 2005). in our zones of investigation, the normal growth of our studied plant samples in metalliferous soils without any visual and conspicuous symptoms of cu toxicity (lewis et al. 2001) implied that a. camelorum was tolerant to toxic levels of cu. acta bot. croat. 69 (1), 2010 115 antioxydant enzyme response and phytochelatins * * * * * * 0 10 20 30 40 50 60 70 80 90 100 leaf stem root leaf stem root phytochelatin 2 n m o l/ g r. f w zone 1 zone 2 phytochelatin 1 fig. 1. the levels of phytochelatins in tissues of studied plants. * – significant difference as compared with zone 2. u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:58 color profile: disabled composite 150 lpi at 45 degrees this plant species was endemic around the cu mine and had adapted to contaminated soils by developing tolerance mechanisms to this metal stress. most of these mechanisms have been recognized previously as exclusion, accumulation of metals and internal protective responses that vary among plant species and among different tissues affected by their intrinsic characteristics (freitas et al. 2004, nicolau et al. 2005, shu et al. 2002, brun et al. 2001). alhagi camelorum had the ability to accumulate copper in its tissues at levels that were lower than 1000 mg g–1 tissue, a threshold limit that is prescribed for hyperaccumulators (reeves and baker 2000). accordingly, our studied plant could not be considered as hyperaccumulator. with regard to the importance of cu distribution, the patterns of copper bioaccumulation and partitioning among different parts of tolerant plants including paspalum distichum and cynodon dactylon, have been reported in many studies (marschner 1995, mulligan et al. 2001, pyatt 2001, stoltz and greger 2002). in these two species that were collected from a cu mine in china, the authors reported higher copper contents in roots of cynodon dactylon and in the shoots of paspalum distichum than in their other tissues. as another indication, pistacia terebinthus and cistus creticus collected from the skouriotissa cu mine accumulated a considerable amount of the absorbed cu in their roots, although bosea cypria accumulated most cu in the leaves (johansson et al. 2005). in agreement with these documents, the studied plant, a. camelorum, showed cu accumulation and partitioning in which cu was transported and stored into the above ground parts, particularly into the leaves. the immobilization of excess heavy metals via their storage in cell walls (hughes and williams 1988) or accumulation in vacuoles (mccain and markley 1989), were suggested as another strategy to increase plant internal tolerance to cu toxicity. we found that a. camelorum from zone 1 accumulated about 48% of total cu in the leaves vacuoles that was considerable as compared with the same plant collected from zone 2. many studies have confirmed that the main portion of metals including copper enter the vacuoles in the form of a phytochelatin complex, where they gain acid-labile sulphur and form high molecular weight complexes (sanita di toppi et al. 2002). potentially toxic heavy metal ions are firstly chelated by gsh and then transferred to pcs for eventual sequestration. however, gsh act as a first line of defense against metal toxicity by complexing metals before the induced synthesis of pcs arrives at effective levels (freedman et al. 1989, singhal et al. 1987). pc synthesis induced by metals is accompanied by a rapid depletion of total gsh in plants, because gsh is the direct precursor/substrate for the synthesis of pcs (gupta et al. 1998). in conformity with these documents, we observed an elevation in pc levels and decrease in gsh concentrations in plants of zone 1 as compared to plants of zone 2. on the other hand and in agreement with our study, sudhakar et al. (2006), showed that exposure of hydrilla verticillata (l.f.) royle to high doses of copper led to decrease in gsh and an elevation in pc levels. in addition, pcs usually play only a part in integrated mechanisms of copper homeostasis and detoxification. tolerance strategies of plants to copper exposure may include other responses such as variation in antioxidative enzyme activities. it has also been confirmed in many investigations that when copper is in excess, it can promote and stimulate generation of fenton-type reactive oxygen species leading to increase in mda and dityrosine as biomarkers of oxidative damages (weckx and clijesters 1996, devi and prasda 1998, lombardi and sebastiani 2005). in response to ros gener116 acta bot. croat. 69 (1), 2010 boojar m. m. a., tavakoli z. u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:58 color profile: disabled composite 150 lpi at 45 degrees ation, antioxidant enzyme activities are elevated as defense system. these responses to excess copper can vary among plant species and among different tissues (lombardi and sebastini 2005). in this study, cu-accumulation by vacuoles caused a decrease in the level of copper outside this organelle, leading to decrease in ros production and low induction on antioxidative enzymes in the leaves as compared with roots of this plant. accordingly, the limited but significant increase in mda and dityrosine in leaves as compared to the roots of a. camelorum could be the consequence of considerably low activities of antioxidative enzymes in leaves. however, the slight loss in chlorophyll content may be due to lipid peroxidation of chloroplast and thylakoid membranes and chlorophyll degradation mediated by cu (baszynski et al. 1988, vinit-dunand et al. 2002). in agreement with our findings, chettri et al. (1998), reported a considerable decrease in total chlorophyll levels in cladonia convoluta collected from a copper mine. they found a copper content in this plant species exceeding 175 mg. g–1 dry weight. moreover, although a. camelorum leaves in zone 1 showed a significant increase in oxidative damage parameters with respect to zone 2, the parameter of biomass decreased insignificantly in this tissue of the plant in zone 1. this could be attributed to insufficient activities of antioxidant enzymes within tissues of a. camelorum to protect them completely against ros damage. however, the non-enzymatic antioxidant defense system comprises phytochelatins and gsh, functioning in free radical entrapment and metal detoxification, and restricted both the severe increase in mda and dityrosine and a considerable decrease in the biomass parameter. in conclusion, the present study revealed the involvement of the detoxification potential of phytochelatins, gsh, antioxidant enzyme activities and the ability to accumulate cu in the leaf vacuoles in a. camelorum and protection against cu-toxicity. acknowledgments this work was supported in part by a conjoint grant from research deputy of tarbiat moallem university. references aeby, h., 1984: catalase in vitro. methods in enzymology 105, 121–126. agata, f., ernest, b., 1998: metal–metal interactions in accumulation of v5+, ni2+, mo6+, mn2+ and cu2+ in under and above ground parts of sinapis alba. chemosphere 36, 1305–1317. alan, r. w., 1994: the spectral determination of chlorophyll a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. plant physiology 144, 307–313. alscher, r. g., donahue, j. l., cramer, c. l., 1997: reactive oxygen species and antioxidants: relationships in green cells. physiologia plantarum 100, 224–233. amado, r., aeschbach, r., neukom, h., 1984: dityrosine: in vitro production and characterization. methods in enzymology 107, 377–388. acta bot. croat. 69 (1), 2010 117 antioxydant enzyme response and phytochelatins u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:58 color profile: disabled composite 150 lpi at 45 degrees baker, a. j. m., proctor, j., 1990: the influence of cadmium, copper, lead and zinc on the distribution and evolution of metallophytes in british island. plant systematics and evolution 173, 91–108. baszynski, t., tukendrof, m., ruszkowska, m., 1988: characteristics of the photosynthetic apparatus of copper non-tolerant spinach exposed to excess copper. journal of plant physiology 132, 708–713. duchaufour, p., 1970: précis de pedologie. masson y cie, paris. bird, b. r., hung, s. s. o., hadley, m., draper, h. h., 1983: determination of malonaldehyde in biological materials by high-pressure liquid chromatography. analytical biochemistry 128, 240–244. bradford, m. m., 1976: a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. analytical biochemistry 72, 248–254. brun, l. a., maillet, j., hinsinger, p., pepin, m., 2001: evaluation of copper-contaminated vineyard soils. environmental pollution 111, 293–302. chettri, m., cook, c. m., varadaka, e., sawidis, t., 1998: the effect of cu, zn and pb on the chlorophyll content of the lichens cladonia convoluta and cladonua rangformis. environmental and experimental botany 39, 1–10. clemens, s., 2001: molecular mechanisms of plant metal tolerance and homeostasis. planta 212, 475–486. demirevska-kepova, k., simova-stoilova, l., stoyanova, z., holzer, r., feller, u., 2004: biochemical changes in barely plants after excessive supply of copper and manganese. environmental and experimental botany 52, 253–266. devi, s. r., prasd, m. n. v., 1998: copper toxicity in ceratophyllum demersum l. a free floating macrophyte. plant science 138, 157–165. de vos, c. h. r., vonk, m. j., vooijs, r., schat, h., 1992: glutathione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in silene cucubalus. plant physiology 98, 853–858. duchaufour, ph., 1970: precis de pedologie. masson y cie, paris. freedman, j. h., ciriolo, m. r., peisach, j., 1989. the role of glutathione in copper metabolism and toxicity. journal of biological chemistry 264, 5598–5605. freitas, h., prasad, m. n. v., pratas, j., 2004: plant community tolerance to trace elements growing on the degraded soils of sao domingos mine in the south east of portugal. environment international 30, 65–72. gupta, m., tripathi, r. d., rai, un., chandra, p., 1998: role of glutathione and phytochelatin in hydrilla verticillta (l. f.) royle and vallisneria spiralis under mercury stress. chemosphere 37, 785–800. halliwell, b., gutteridge, j. m. c., 1998: mechanisms of damage to cellular targets by oxidative stress: lipid peroxidation. in: halliwell, b., gutteridge, j. m. c. 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copper. physiology plantarium 96, 506–512. yuan, d. w., 1988: compared study on the pre-treatment methods for measuring soil total copper, zinc, lead, cadmium, nickel and manganese. agro-environmental protection science 7, 34–36. acta bot. croat. 69 (1), 2010 121 antioxydant enzyme response and phytochelatins u:\acta botanica\acta-botan 1-10\boojar.vp 9. travanj 2010 13:21:58 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 73 (1), 149–158, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 phenotyping of rice in salt stress environment using high-throughput infrared imaging zamin s. siddiqui2*, jung-il cho1, sung-han park1, taek-ryoun kwon1, byung-ok ahn1, gang-seob lee1, mi-jeong jeong1, kyung-whan kim1, seong-kon lee1, soo-chul park1 1 national academy of agricultural sciences, rural development administration, suwon 441-707, republic of korea 2 stress and crop physiology laboratory, department of botany, university of karachi, karachi 75270, pakistan abstract – phenotyping of rice (oryza sativa l. cv. donggin) in salt stress environment using infrared imaging was conducted. results were correlated with the most frequently used physiological parameters such as stomatal conductance, relative water content and photosynthetic parameters. it was observed that stomatal conductance (r2 = –0.618) and relative water content (r2 = –0.852) were significantly negatively correlated with average plant temperature (thermal images), while dark-adapted quantum yield (fv/fm, r 2 = –0.325) and performance index (r2 = –0.315) were not consistent with plant temperature. advantages of infrared thermography and utilization of this technology for the selection of stress tolerance physiotypes are discussed in detail. keywords: infrared imaging, phenotype, rice, salt stress introduction plants of the same genotype may have different phenotypes, depending on the growing environment. phenotyping is a technique dealing with plant visible characteristics (phenotypes), or trait analysis. conventional phenotyping has been hard, time consuming and destructive. recent development of high-tech imaging systems and their computation enables modern, fast and non-destructive phenotyping research. depending on the plant traits, high-throughput phenotyping techniques can be useful because they can reduce phenotyping time from weeks to minutes, or even seconds. high technology in phenomics accelerates the procedure for selecting plant varieties that perform better in the field when affected by drought or salt. in the past, physiological attributes like stomatal conductance, osmotic potential, dark-adapted quantum yield and biomass allocation were frequently used in phenotyping techniques under salt and drought stress environments (davis et al. 2005, burke acta bot. croat. 73 (1), 2014 149 * corresponding author, e-mail: usdapark@korea.kr copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:48 color profile: generic cmyk printer profile composite default screen et al. 2006, siddiqui et al. 2008, munns et al. 2010, richards et al. 2010, siddiqui 2013). however, these physiological attributes were time consuming, laborious, and destructive. more common physiological approaches and modern high-tech infrared phenotyping techniques are being used to identify stress tolerant plants. therefore, it is important to correlate the two techniques and to standardize the protocol for testing favourable phenotypes under salt stress environment (munns et al. 2010). although literature showing phenotyping using an infrared (ir) camera is available, these techniques need to be closely monitored and should be correlated with physiological data that might have some role in the regulation of plant temperature in abiotic stress environment (merlot et al. 2002, jones et al. 2009, collins et al. 2010). mainly, high-tech phenotyping through an ir camera is based on the temperature/heat produced in stress-plant (munns et al. 2010). therefore, the present study has been designed to examine the phenotyping of rice cv. donggin by highly sensitive ir thermal camera and find out its correlation with some physiological parameters in salt stress environment. stomatal regulation and plant water status are important aspects in stress environment and these physiological attributes stabilize the temperature inside the plant leaf. the hypothesis that physiological parameters related to plant temperature are affected by the salt stress was tested using infrared imaging. material and methods germination and growth seeds of rice (oryza sativa l. cv. donggin) were collected from national center for genetically modified crops, national academy of agricultural science, rural development administration, south korea. seeds were washed with distilled water several times before sowing, then allowed to germinate in 90 mm diameter petri dishes. six-day old equal size seedlings were transferred to a hydroponic system. after transplanting, the seedlings were left for a further four days in half-strength yoshida nutrient solution prior to the imposition of nacl (tab. 1). plants were treated with 75, 150 and 225 mm nacl in full-strength 150 acta bot. croat. 73 (1), 2014 siddiqui z. s., cho j.-i., park s.-h., kwon t.-r., ahn b.-o., lee k.-s. et al. tab. 1. yoshida solution composition. chemical amount (g/5 l) nh4no3 475 nah2po4 ´ h2o 201 k2so4 357 cacl2 443 mgso4 ´ 7 h2o 1620 mncl2 ´ 4 h2o 7.50 (nh4)6mo7o24 ´ 4 h2o 0.37 h3bo3 4.67 znso4 ´ 7 h2o 0.18 cuso4 ´ 5 h2o 0.16 fecl3 ´ 6 h2o 38.5 c6h8o7 ´ h2o 59.5 1 m h2so4 250 ml 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:48 color profile: generic cmyk printer profile composite default screen yoshida nutrient solution, while control plants were treated with only yoshida solution. treated and control plants were grown in a growth chamber (eyela) at a temperature of 25–28 ± 2 °c, 60–80% humidity and a photoperiod of 14/10 hours (day/night). light intensity varied from 200–350 mmol photon m–2 s–1. experiments were replicated four times. relative water content six randomly selected leaves from each treatment and control were sampled and 4 × 2 cm2 mid-vein and the edge sections were cut with scissors. after fresh weight measurement, each sample was placed in a 90 mm air-tight plastic petri plate containing distilled water. after 12-hours hydration in the dark, the leaf samples were taken out of the water and their surfaces were well dried quickly and lightly with filter/tissue paper and immediately weighed to obtain fully turgid weight (tw). leaf samples were then oven dried at 80 °c for 24 h and weighed to determine dry weight (dw). relative water content (rwc) was calculated using the following formula: rwc (%) = [(fw – dw) / (tw – dw)] × 100 where fw is sample fresh weight, tw is sample turgid weight, and dw is sample dry weight. stomatal conductance and psii quantum yield stomatal conductance of twenty randomly selected leaves of each treatment and control were examined using a leaf porometer (model sc-1, decagon, usa). measurements of chlorophyll a fluorescence emissions from the twenty randomly selected leaves were monitored with a fluorescence monitoring system (company handy pea) in the pulse amplitude modulation mode. a leaf adapted to dark conditions for 30 minutes using leaf-clips was initially exposed to a modulated measuring beam of far-red light (led source with a typical peak at wavelength 735 nm). original (f0) and maximum (fm) fluorescence yields were measured under weak modulated red light (< 0.5 mmol m–2 s–1) with 1.6 s pulses of saturating light (> 6.8 mmol m–2 s–1, photosynthetically active radiation). the variable fluorescence yield (fv) was calculated by the equation of fm – f0. the ratio of variable to maximum fluorescence (fv/fm), calculated as maximum quantum yield of psii photochemistry as well as photosynthesis performance index were determined as described by maxwell and johnson (2000). ir thermal images we used flir-sc-620 (flir systems, usa) for thermal imaging experiments. the system was optimized 30 minutes before measurements. to test the temperature difference between treated and untreated plants in salt stress environment, plants of each treatment and control were examined. plant images were taken using a rectangular box of an area about 46 × 30 cm2. temperature of 24 ± 2 °c inside the box and relative humidity of 60–70% were recorded. the images were taken at 10 a.m. using a flir sc-620 series camera with 640 × 480 pixel ir resolution. images from each treatment and control were directly extracted from the camera into computer and a report was generated using thermacam researcher pro 2.10 software. acta bot. croat. 73 (1), 2014 151 phenotyping of rice in salt stress environment 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:48 color profile: generic cmyk printer profile composite default screen statistical analysis all data from treated and control plants were subjected to analysis of variance using spss 17.0 software. the values were expressed as the mean of four replicates ± standard error (se). student t-test (p < 0.05) was used to check statistical significance. correlation analysis was computed between average plant temperatures (ir image) and physiological attributes. results infrared thermography phenotyping technique was used to identify plant response in salt stress environment showing significant difference between salt stress and unstressed plants (fig. 1). for this study, plants were subjected to various salt concentrations and therefore plant temperatures and color patterns were recorded. image colors represent the temperature pattern and were in the following order: blue (less temperature) < green < yellow < red (high temperature). plants in a saline environment showed substantially less blue color expression than those in a non-saline environment. it was observed that blue color intensity changed from blue to green, then yellow and red color as salinity increased as compared to the control. leaves of each treated and control plant showed substantial variations in color and temperature. however, maximum leaf temperature was recorded in a 225 mm nacl treated 152 acta bot. croat. 73 (1), 2014 siddiqui z. s., cho j.-i., park s.-h., kwon t.-r., ahn b.-o., lee k.-s. et al. fig. 1. infrared images of plants treated with nacl in the concentration range 0–225 mm, observed by a flir-sc-620 camera. images were analyzed by thermacam researcher pro 2.10 software. first row shows the whole plants set treated with salt in comparison to the control. second row represents a single plant treated with same salt and control solutions. third row shows the roots of treated and control plants. 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:50 color profile: generic cmyk printer profile composite default screen plant compared to control (tab. 2). likewise, the roots of salt stress plants also showed significant variations in color. roots of highly salt stressed plants (225 mm nacl) showed higher temperatures than the control plants. root size was also greatly reduced in salt treated plants. performance index of salt treated and untreated plants were examined and were expressed on a graph (fig. 2). performance indices gradually declined due to salt stress, as compared to the control. the lowest performance index values as compared to the control acta bot. croat. 73 (1), 2014 153 phenotyping of rice in salt stress environment tab. 2. temperatures of salt-treated and control plants and leaves calculated based on the ir thermal images. student t-test was done to compare control and salt treated samples. different letters present significantly different values at p < 0.05. nacl plants temperature (°c) leaf temperature (°c) min max avg min max avg control 23.8a 32.9a 28.4a 27.6a 28.2a 27.9a 75 mm 22.9b 32.8a 27.9b 27.7a 29.3b 28.5b 150 mm 25.1c 33.7b 29.4c 29.4b 30.4c 29.9c 225 mm 26.1d 32.8a 29.5c 29.4b 31.4d 30.4d student t-test was done to compare control and salt treated samples. similar alphabets are non-significantly differed at p < 0.05. avg – average. p e rf o rm a n c e in d e x 0 1 2 3 4 5 6 7 r e la tv ie w a te r c o n te n t (% ) 0 20 40 60 80 100 0 75 150 225 s to m a ta l c o n d u c ta n c e (m m o l m – 2 s – 1 ) 0 20 40 60 80 100 120 140 160 0 75 150 225 d a rk a d a p te d q u a n tu m y ie ld (f v /f m )0.0 0.2 0.4 0.6 0.8 1.0 1.2 salinty (nacl mm) a b b c a a b c a b c d a b b c fig. 2. relative water content, stomatal conductance, performance index and dark-adapted quantum yield (fv/fm) in saline-treated plants in comparison to the control. values are mean ± se, n = 4. different letters represent statistically significant values (p < 0.05). 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:50 color profile: generic cmyk printer profile composite default screen were found in a 225 mm nacl treated plant. the relative water content as compared to the control plant decreased upon salt stress in a dose-dependent manner (fig. 2). maximum decrease in relative water content as compared to the control was found in 225 mm nacl treated sample. likewise, stomatal conductances in plants as compared to the control were significantly decreased in a salt-treated sample (fig. 2). moreover, the study showed that the decrease in stomatal conductance was related to salt concentrations resulting in a maximum decrease at 225 mm nacl treatment. similarly, dark-adapted quantum yield (fv/fm) was reduced in a salt-treated plant. maximum decrease as compared to the control was observed in a 225 mm-treated plant while decrease in quantum yield was somewhat similar and was non-significant in 75 and 150 mm treated plants. correlations between the average ir image temperature pattern and physiological attributes like relative water content, stomatal conductance, performance index and dark-adapted quantum yield were significant (fig. 3). significant negative correlation was observed between average image temperature and relative water content (r2 = –0.852) as well as stomatal conductance (r2 = –0.612) while correlation between plant temperature and performance index (r2 = –0.315) as well as dark-adapted quantum yield (r2 = –0.325) was not significant. discussion comparison and correlation between conventional and modern phenotyping of rice plants in salt stress environment were conducted. conventional phenotyping provided physiological attributes like relative water content, stomatal conductance, dark-adapted quan154 acta bot. croat. 73 (1), 2014 siddiqui z. s., cho j.-i., park s.-h., kwon t.-r., ahn b.-o., lee k.-s. et al. r e la ti v e w a te r c o n te n t (% ) 30 40 50 60 70 80 90 s to m a ta l c o n d u c ta n c e (m m o l m – 2 s – 1) 20 40 60 80 100 120 140 160 27 28 29 30 31 q u a n tu m y ie ld (f v /f m ra ti o ) 0.3 0.4 0.5 0.6 0.7 0.8 0.9 average plant temperature (°c) 28 29 30 31 p e rfo rm a n c e in d e x 1 2 3 4 5 6 7r 2 = -0.33 r 2 = -0.32 r 2 = -0.62r 2 = -0.85 fig. 3. correlations between average plant temperature and relative water contents, stomatal conductance, performance index and dark-adapted quantum yield (fv/fm) tested in saline and non-saline environment. r2 stands for linear regression values. 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:51 color profile: generic cmyk printer profile composite default screen tum yield and performance index. a modern approach using an ir thermal camera provided thermal variations in plants upon salt stress. it was found that physiological attributes like relative water content and stomatal conductance were significantly correlated with ir thermal-image temperature. on the other hand, performance index and quantum yield were not significantly correlated with the result obtained by ir thermography. among several parameters obtained from the chlorophyll fluorescence measurements, the dark-adapted quantum yield (fv/fm) and the performance index were selected for the comparison between salt stress and unstressed plant. the reason for this choice was that the fv/fm ratio is the most extensively used photosystem ii (psii) efficiency indicator. this parameter has been shown to correlate with a number of functional psii complexes. many studies have used this ratio as an indicator for stress tolerance or sensitivity (penuelas and boada 2003). performance index was introduced to quantify the effects of environmental factors like chilling, heat, drought, chromate, ozone, or urban injuries to photosynthesis (hermans et al. 2003, de ronde et al. 2004, strauss et al. 2006). in this study, neither parameter was significantly correlated with the result obtained by ir thermography. the reason behind this may be that ir sensing is based on heat generation of plant which is linked with water status rather than photosynthetic performance. stomatal conductance, relative water content and dark-adapted quantum yield, performance index are affected by salt stress and these physiological attributes are linked with the leaf temperature (munns et al. 2010). generally, water loss from the leaf needs a substantial amount of energy to convert each molecule of water from liquid to vapor. this energy is then taken away from the leaf in the evaporating water for cooling purpose (jones et al. 2009). thus, for a given environmental or stress condition, leaf transpiration is an important determinant of leaf temperature. in the case of stress caused by either salt or drought, an immediate plant response is a reduction in transpiration to reduce water loss, and an increase in the leaf temperature (woo et al. 2008, munns et al. 2010). moreover, garrity and o’toole (1994) have shown that ir thermography could be used to determine leaf and canopy temperature, although as an indirect estimation of plant water status. it is presumed that a direct and an indirect relationship between physiological parameters and ir images are based on the type and nature of stress, plants and research area. for instance, genotypes with a higher tolerance to stress uptake soil water efficiently by maintaining higher stomatal conductance and therefore can be identified as plants with cooler leaves (jones et al. 2009, berger et al. 2010, lu et al. 2011). further, sirault et al. (2009) has suggested that leaf temperature is an indicator of stomatal conductance and it was increased with high salt concentration (munns et al. 2010). the ranking of the genotypes based on the growth study and thermal ir measurements was consistent (james et al. 2008) and both have been successfully deployed in wheat breeding for both drought and heat screening (fischer et al. 1998, reynolds et al. 1998, brennan et al. 2007). in this study, high salt concentration (225 mm nacl) caused a substantial reduction in stomatal conduction and relative leaf water content and subsequently increased average plant leaf temperature. it was shown that dark-adapted quantum yield and ir images were non-significantly correlated. hence, it could be stated that decrease in stomatal conductance and relative water content in leaf could generate more heat, causing leaf temperature to increase in a given leaf area. leaf temperature is a proximate indicator of stomatal conductance and water status which are often analyzed with an ir sensor (sirault et al. 2009, munns et al. 2010). physiological attributes like stomatal conductance and ir images were found consistent in previous studies (jones et al. 2009, munns et al. 2010). acta bot. croat. 73 (1), 2014 155 phenotyping of rice in salt stress environment 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:51 color profile: generic cmyk printer profile composite default screen dark-adapted quantum yield, fv/fm (a measure of the intrinsic photochemical efficiency of light harvesting in photosystem ii) is the most easily measured and commonly used fluorescence parameter in the stress studies (baker et al. 2008). photosynthesis ability of a plant under stress condition is attributed to stomata factors, which not only regulate leaf water content but also maintain carbon dioxide concentration inside the leaf (brougnoly and lauteri 1991, tourneux and peltier 1995, khan and panda 2008, siddiqui et al. 2008). water status in a plant is highly sensitive to salinity and therefore it is dominant in determining plant responses to stress (stepien and klobus 2006). dark-adapted quantum yield responses to salt or drought stress environment are rather slow and can be detectable in a large tray experiment using very small seedlings (woo et al. 2008, jansen et al. 2009). since this experiment was carried out using small trays, it was presumed that dark-adapted quantum yield may not produce significant change in leaf temperature and thus it could not be detected by ir thermal images sensing. therefore, based on the working principal of the ir camera, it could be suggested that ir may not be related to the photosynthesis performance of a plant under saline environment. meanwhile, plant temperature allows the indication of the degree of stress in a crop on the basis of relative water content and stomatal conductance. in stress, plant temperature and water stress are perhaps linked to soil water availability, leaf water potential, and stomatal conductance. ir thermography was proved to be related to soiland plant-based measures of water stress. it was also observed that ir thermography can be potentially used for identifying the differences between genotypes and single crop in variable plant irrigation and stress environment (romano et al. 2011, zia et al. 2011). however, high technology utilization in field experiment needs to be developed in order to identify the best protocol to optimize the data accuracy. conclusion correlation analysis between conventional and modern phenotyping showed that plant or leaf temperature variation could be a useful tool to identify stress tolerant physiotype/genotype in the stress environment. a modern analysis performed by highly sensitive ir camera (ir thermography techniques) may be less time consuming, non-destructive and cover a larger scale. acknowledgments this work was supported by a grant from the next-generation bio-green 21 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measured using 18o2 and mass spectrometry in solanum tuberosum leaf disks. planta 195, 570–577. woo, n., badger, m. r., pogson, b., 2008: a rapid non-invasive procedure for quantitative assessment of drought survival using chlorophyll fluorescence. plant methods 4, 27. zia, s., spohrer, k., wenyong, d., spreer, w., romano, g., xiongkui, h., mller, j., 2011: monitoring physiological responses to water stress in two maize varieties by infrared thermography. international journal of agricultural and biological engineering 4, 7–15. 158 acta bot. croat. 73 (1), 2014 siddiqui z. s., cho j.-i., park s.-h., kwon t.-r., ahn b.-o., lee k.-s. et al. 856 siddiqui et al.ps u:\acta botanica\acta-botan 1-14\856 siddiqui et al.vp 19. o ujak 2014 17:21:51 color profile: generic cmyk printer profile composite default screen acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 299 acta bot. croat. 73 (2), 299–314, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 effect of water defi ciency and potassium application on plant growth, osmolytes and grain yield of brassica napus cultivars murad ali1, jehan bakht2*, gul daraz khan1 1 department of water management, the university of agriculture, peshawar, pakistan 2 institute of biotechnology and genetic engineering, the university of agriculture, peshawar, pakistan abstract – one of the major issues with brassica oil seed production is the water requirement of the brassica crop. to address the problem, fi eld experiments were conducted to evaluate the effect of potassium (k) and water defi ciency levels on canola (brassica napus l.). analysis of the data revealed that application of k, irrigation and interactions between irrigation and cultivar (i × c), irrigation and potassium (i × k), potassium and cultivar (k × c), and irrigation and cultivar and potassium (i × c × k) had a signifi cant (p < 0.05) effect on shoot proline content, relative water content, plant fresh weight and grain yield. potassium application, irrigation and interaction between i × c, k × c, and i × c × k had a signifi cant (p < 0.05) effect on shoot sugar content. water defi ciency increased shoot proline and sugar contents and decreased relative water content. potassium application increased shoot proline level in a dose dependent manner. minimum proline and sugar contents and maximum relative water content, plant fresh and dry weight and yield were obtained when 100% irrigation was applied. maximum grain yield was obtained upon application of 100% irrigation in combination with 120 kg ha–1 k. keywords: brassica napus, grain yield, potassium, proline, relative water content, water defi ciency introduction plants can cope with drought stress through genetic and adaptive mechanisms. plants possess the mechanisms to escape, avoid and/or resist to drought. they can also escape drought by adjusting their physiological and biochemical development according to the availability of water in their habitat (arraudeau 1989). water stress induces a signifi cant decrease in metabolic factors such as decrease in chlorophyll content and enhanced accumu* corresponding author, e-mail: jehanbakht@yahoo.co.uk copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. ali m., bakht j., khan g. d. 300 acta bot. croat. 73 (2), 2014 lation of proline (gibon et al. 2000, din et al. 2011). free proline accumulation and sugar in high concentration under abiotic stress has been reported to have an adaptive mechanism (sarker et al. 1999, ronde et al. 2004, sofo et al. 2004, monreal et al. 2007, javadi et al. 2008, keyvan, 2010, mafakheri et al. 2010, moustakas et al. 2011, bakht et al. 2013). potassium plays a vital role in photosynthesis, translocation of photosynthates, protein synthesis, control of ionic balance, regulation of plant stomata and water use, activation of plant enzymes and many other processes (reddy et al. 2004). potassium is not only an essential macronutrient for plant growth and development, but also a primary osmoticum in the maintenance of the low water potential of plant tissues. therefore, accumulation of k in plant tissues under drought stress may play an important role in water uptake along a soil-plant gradient (glenn and brown 1998). the accumulation and release of potassium by stomatal guard cells lead to changes in their turgor, resulting in stomatal opening and closing (outlaw 1983). in water-stressed plants, increased abscisic acid (aba) levels are known to stimulate the release of potassium from guard cells, giving rise to stomatal closure (assmann and shimazaki 1999). numerous studies have shown that application of k fertilizer mitigates the adverse effects of drought on plant growth (anderson et al. 1992, sangakkara et al. 2001). fusheing (2006) has revealed that the lower water loss from plants supplied with suffi cient k is due to the reduction in transpiration, which not only depends on the osmotic potential of mesophyll cells, but is also controlled to a large extent by the opening and closing of stomata. the objective of this study was to test the effectiveness of potassium application in mitigation of water defi ciency, with reference to the physiological changes like proline, sugar and relative water content in four canola cultivars. these fi ndings could be used as screening basics for further study in breeding programs. materials and methods site description the experimental site of the university of agriculture peshawar kpk pakistan is situated at 34° n, 72° e and an altitude of 290 meters above sea level. field experiments were conducted at malakandher research farm (the university agricultural peshawar, kpk pakistan) using a randomized complete block design with split plot arrangement. four cultivars of brassica napus were investigated: ‘wester’, ‘rainbow’, ‘oscar’, and ‘legend’. the following treatments were studied during the course of study: (1) irrigation levels: 100% replacement of evapotranspiration (eta), 80% replacement of eta, and 60% replacement of eta; (2) potassium (k) levels: 60 kg ha –1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). biochemical analysis moisture content was determined through the gravimetric method by taking samples at each treatment at 3 depths i.e. 0–30, 30–60 and 60–90 cm. the moisture content on mass and volume basis, bulk density, depth of water needed and duration of irrigation were determined as described by james (1993). moisture content was fi rst determined on mass basis (θm) and was converted to moisture content on volume basis (θv) by multiplying with bulk density and dividing by water density. from this the depth of water needed was calculated for each treatment. fresh and dry weights of all samples were determined two months after response of b. napus to water deficiency and potassium application acta bot. croat. 73 (2), 2014 301 sowing. after the taking of fresh weight, plant samples were dried in an oven at 80 °c for three days to record plant dry weight. plant height was measured two months after sowing. in each treatment, plant heights of three plants were measured in cm and then average values were calculated. grain yield was calculated by threshing all pods of the plants in each treatment and then converted into grain yield kg ha–1. shoot proline content was determined 60 days after sowing using the method of bates et al. (1973) with minor modifi cations. briefl y, frozen plant material of 500 mg was homogenized in 1.0 ml of sterilized iron free water and centrifuged at 5000 rpm for 5 minutes. extract (100 µl) was mixed with acid ninhydrin for 1 h at 100 °c and the reaction was terminated in an ice bath. the reaction mixture was thoroughly shaken and the optical density was determined at 520 nm. level of shoot proline content was determined from a standard curve in the range of 0–20 µg ml–1 of l-proline. sugar concentration was measured 60 days after sowing as described by dubois et al. (1956). briefl y, 20 mg plant tissue was crushed in 4 ml of extraction buffer, centrifuged for 5 minutes at 9000 rpm and the pellet was re-extracted. two ml each of chloroform and distilled water were added to the pooled supernatant. two ml of the sample was mixed with 1 ml of distilled water, 1 ml phenol (c6h5oh) (5%) and 5 ml sulfuric acid. after the appearance of a dark pink color, the mixture was kept at 80 °c for 30 minutes inside the oven. the samples were then shaken and placed in a water bath at 30 °c for 20 minutes and absorbance was measured at 490 nm. standard of d-glucose (0, 0.2, 0.4, 0.6, 0.8 µmol) was also prepared using the same procedures. relative water contents were determined 60 days after sowing adopting the standard procedure of bajji et al. (2001). the leaf samples (about 5 cm2 each) from all the treatments were weighed to obtain fresh weight (fw). turgid weight (tw) was measured by completely immersing the samples in distilled water and placing them in the dark at 4 °c for 24 h. the samples were blotted on fi lter paper and weighed. the samples were dried at 70 °c for 48 h and fi nally dry weight (dw) was obtained. relative water content was calculated by the following formula: rwc = [(fw – dw) / (tw – dw)] × 100 statistical analysis data were analyzed according to the randomized complete block (rcb) design with split plot arrangements using anova (gomez and gomez 1984). mstat computer software was used for the analysis of the data (bricker 1991). interaction between the variables was measured by analysis of variance. differences in means were separated by the least signifi cant difference (lsd) test (steel and torrie 1997). results plant growth and development analysis of the data indicated that k application, irrigation and interaction between i × c, i × k, k × c, and i × c × k had a highly signifi cant (p < 0.01) effect on shoot fresh weight of canola (fig. 1). maximum shoot fresh weight was observed in 100% irrigated plants. similarly, maximum shoot fresh weight was produced by the ‘legend’ when 100% irrigaali m., bakht j., khan g. d. 302 acta bot. croat. 73 (2), 2014 tion of actual evapotranspiration was applied. in the case of potassium treatments, maximum shoot fresh weight was gained by plants treated with 120 kg k ha–1. further, ‘wester’ produced maximum shoot fresh weight when treated with 120 kg k ha–1. interaction between i × k showed that maximum shoot fresh weight was produced by the ‘legend’ at 100% irrigation level and 60 kg k ha–1. irrigation, cultivars and interaction between i × c had a signifi cant (p < 0.01) effect on shoot dry weight of canola whereas the effect of k application and all other interactions were non-signifi cant (p > 0.05) (fig. 2). maximum shoot dry weight was observed in those treatments where a 100% irrigation level was applied. similarly, maximum shoot dry weight was produced by ‘legend’ when 100% irrigation of actual evapotranspiration was applied. analysis of the data revealed that irrigation application and interaction of i × c × k had a signifi cant (p < 0.01) effect on plant height 60 days after sowing and at physiological maturity (fig. 3). the effect of k application and all other interactions were non-signifi cant (p > 0.05). taller plants were observed in those treatments where 100% irrigation was applied followed by 80% irrigation level. cultivars ‘rainbow’ and ‘legend’ attained maximum plant height when treated with 100% irrigation level. potassium application produced non-signifi cantly taller plants in those treatments where 120 kg k ha–1 was applied. maximum plant height was noted in ‘wester’ and ‘rainbow’ when they were treated with 120 kg k ha–1. i × k interaction revealed maximum plant height in 100% irrigated plants treated with 60 kg k ha–1. fig. 1. effect of water defi ciency and potassium application and their interactions on shoot fresh weight (g) 60 days after sowing of brassica napus cultivars (‘wester’, ‘rainbow’, ‘oscar’ and ‘legend’). potassium application: 60 kg ha–1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). bar represents ± lsd at p < 0.05. response of b. napus to water deficiency and potassium application acta bot. croat. 73 (2), 2014 303 fig. 2. effect of water defi ciency and potassium application and their interaction on shoot dry weight (g) 60 days after sowing of brassica napus cultivars (‘wester’, ‘rainbow’, ‘oscar’ and ‘legend’). potassium application: 60 kg ha–1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). bar represents ± lsd at p < 0.05. fig. 3. effect of water defi ciency and potassium application and their interactions on plant height (cm) after 60 days of sowing of brassica napus cultivars (‘wester’, ‘rainbow’, ‘oscar’ and ‘legend’). potassium application: 60 kg ha–1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). bar represents ± lsd at p < 0.05. ali m., bakht j., khan g. d. 304 acta bot. croat. 73 (2), 2014 shoot proline statistical analysis of the data revealed that the application of k, irrigation and interaction between i × c, i × k, k × c and i × c × k had a signifi cant (p < 0.01) effect on shoot proline content of b. napus (fig. 4). maximum shoot proline content was observed in those treatments where 60% irrigation of actual evapotranspiration (eta) was applied. maximum shoot proline content was produced by ‘rainbow’ at 60% irrigation of actual evapotranspiration. similarly, k application also increased proline concentration in all cultivars in a dose dependent manner, although, the magnitude of increase varied among the different cultivars. maximum shoot proline content was noted in plants treated with 120 kg k ha–1. our results also indicated that maximum shoot proline content was produced in plants treated with 60% irrigation of actual evapotranspiration (eta) and 120 kg k ha –1. fig. 4. effect of water defi ciency and potassium application and their interaction on shoot proline contents (μmol g–1 fw) of brassica napus cultivars (‘wester’, ‘rainbow’, ‘oscar’ and ‘legend’). potassium application: 60 kg ha–1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). bar represents ± lsd at p < 0.05. shoot sugar content analysis of the data revealed that k application, irrigation and interaction between i × c, k × c and i × c × k had a signifi cant (p < 0.05) effect on shoot sugar content of canola whereas the effect of interaction of i × k was non-signifi cant (p > 0.05) (fig. 5). maximum shoot sugar content was produced by ‘rainbow’ at 60% irrigation of actual evapotranspiration (eta). in case of k application, maximum shoot sugar content was produced in plants treated with 120 kg k ha–1. similarly, maximum shoot sugar content was produced by ‘oscar’ at 120 kg k ha–1. response of b. napus to water deficiency and potassium application acta bot. croat. 73 (2), 2014 305 relative water content of shoot statistical analysis of the data showed that k application, irrigation and interaction between i × c, i × k, k × c, and i × c × k had a signifi cant (p < 0.01) effect on relative water content (fig. 6). the data revealed that maximum relative water content was observed in those treatments in which a 100% irrigation level was applied. further, maximum relative water content was maintained by ‘wester’ when applied with a 100% irrigation level. in the case of interaction between i × k, maximum relative water content was observed in plants treated with a 100% irrigation level and treated with 60 kg k ha–1. similarly, maximum relative water content was retained by ‘wester’ when it was treated with 60 kg k ha–1 and applied with 100% irrigation of actual evapotranspiration (eta). furthermore, it can be also inferred from the data that k application resulted in a decrease in rwc under full irrigation, but had a protective role under water defi cit conditions, resulting in improvement of rwc. grain yield grain yield was signifi cantly (p < 0.01) affected by k, irrigation and interaction between i × c, i × k, k × c, and i × c × k (fig. 7). maximum grain yield (2093.9 kg ha–1) was noted in 100% irrigated treatments. similarly, grain yield was maximum (2287.62 kg ha–1) in ‘oscar’ when applied with 100% irrigation. potassium application at 120 kg k ha–1 produced maximum grain yield (1816.71 kg ha–1). the data also suggested that maximum grain yield (1995.62 kg ha–1) was produced by ‘wester’ when treated with 120 kg k ha–1. in the fig. 5. effect of water defi ciency and potassium application and their interaction on shoot sugar contents (μmol g–1 fw) of brassica napus cultivars (‘wester’, ‘rainbow’, ‘oscar’ and ‘legend’). potassium application: 60 kg ha–1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). bar represents ± lsd at p < 0.05. ali m., bakht j., khan g. d. 306 acta bot. croat. 73 (2), 2014 fig. 6. effect of water defi ciency and potassium application and their interaction on shoot relative water content (rwc) of brassica napus cultivars (‘wester’, ‘rainbow’, ‘oscar’ and ‘legend’). potassium application: 60 kg ha–1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). bar represents ± lsd at p < 0.05. fig. 7. effect of water defi ciency and potassium application and their interactions on grain yield (kg ha–1) of brassica napus cultivars (‘wester’, ‘rainbow’, ‘oscar’ and ‘legend’). potassium application: 60 kg ha–1 (k1), 90 kg ha–1 (k2), and 120 kg ha–1 (k3). bar represents ± lsd at p < 0.05. response of b. napus to water deficiency and potassium application acta bot. croat. 73 (2), 2014 307 case of i × k interaction, maximum grain yield (2335.1 kg ha–1) was noted in those plants which received 100% irrigation level and 120 kg k ha–1. it is clear from the data that maximum grain yield (2549.47 kg ha–1) was produced by ‘oscar’ at 100% irrigation level and 120 kg k ha–1. discussion water defi ciency increased proline concentration in all the cultivars of canola; however, this increase varied between the different cultivars. accumulation of proline under stress conditions is used as an adaptive mechanism by many plant species (chu et al. 1974, francisco et al. 2007, ali et al. 2008, hasan et al. 2008, bakht et al. 2011, shafi et al. 2011, bakht et al. 2012, bakht et al. 2013, hayat et al. 2013). enhancement of proline concentration was noted in canola plants under drought stress at different growth stages (din et al. 2011). difference in proline content of different cultivars may be due to up-regulation of degrading enzymes such as proline dehydrogenase. proline is also considered as a storage compound of reduced n and carbon skeletons for post-stress growth recovery (vartanian et al. 1992). similarly, k application also increased proline concentration in all the cultivars in a dose dependent manner. however, the magnitude of increase was variable among the cultivars. similar results are also reported by mukherjee (1974), wiemberg et al. (1988), and aziz et al. (1999) who concluded that proline did not start to accumulate in leaves until the concentration of total monovalent cations, particularly of k, in leaves reached a certain threshold levels. this might have happened in our study when the k level was increased from 60 to 120 kg per ha. the vital role of potassium in photosynthesis, translocation of photosynthates, protein synthesis, ionic balance, regulation of plant stomata and water use, activation of plant enzymes and many other processes is well recognized (marschner 1995, reddy et al. 2004). adequate k fertilization of crop plants facilitates osmotic adjustment, which maintains turgor pressure at lower leaf water potentials and improves the ability of plants to tolerate drought stress (egilla et al. 2001, mengel and arneke 1982). the promotion of root growth and protection of photosynthetic machinery and osmotic adjustment after enhanced k application under drought stress resulted in continued carbon fi xation. consequently, k application increased proline concentration at all water levels, the maximum being at 60% eta. though k is the major osmoticum, accumulation of compatible solutes is an important component of the adaptive mechanism under drought stress conditions (mccue and hanson) 1990). our results indicated that water defi cit and interaction of k and water applied resulted in an increase in the sugar concentration in different cultivars. fixation of assimilated carbon into a particular plant metabolite is determined by the capacity of the plant for photo assimilates production, which decreases under drought stress conditions. akatsuka and nelson (1966) indicated that k increased starch synthetase activity and protected the enzyme from thermal inactivation. biswas et al. (1992) reported increased sugar and starch content of sugarcane, tomatoes and potatoes under water stress. this increase in sugar concentration under drought stress may be due to increased production of starch. however, a negative correlation between sugar accumulation and starch content was noted in many plant species (silva and arrabaca 2004). furthermore, k application enhances the ability of plants to produce assimilates resultantly increasing its ability of sugar production. proali m., bakht j., khan g. d. 308 acta bot. croat. 73 (2), 2014 tection of photosynthetic machinery and production of assimilates may be the major reasons for the increased accumulation of sugars under drought stress conditions. allan et al. (2008) and rogiers et al. (2011) reported that accumulation of sucrose was inversely correlated to leaf and root water status. relative water content of different canola cultivars decreased with a lowering of the irrigation levels under fi eld conditions. potassium application positively affected the rwc in all genotypes. similarly, k application negatively affected rwc at full irrigation, but had a positive effect on rwc during decreasing water availability. decreasing water availability and the continued need for gas exchange and cooling results in greater loss of water under drought stress conditions. resultantly, a decrease in rwc was noted in this experiment, and even the enhanced production of proline and sugars under drought stress did not manage to maintain rwc. kage et al. (2004) attributed the difference in the leaf relative water content of rapeseed varieties to their root system variations. decrease in the leaf relative water content in plants under water defi cit has been reported (allan et al. 2008). furthermore, it can be also inferred from the data that k application resulted in a decrease in rwc under full irrigation, however, had a protective role under water defi cit conditions resulting in improvement of rwc. the maintenance of plant water economy by k application in terms of a high rwc level under water defi ciency condition could be ascribed to the supposed role of k in stomatal resistance, water use effi ciency and lowered transpiration rate. these results are supported by umar and din (2002), who reported that application of k improves rwc of plants under water stress conditions. high drought stress intensity increases the potassium requirement for improving the water status and maintaining photosynthesis (umar 2006). apart from the disorder in photosynthesis electron transport chain, the production of active oxygen formed by potassium defi ciency is increased by nadph oxidation (an important source for active oxygen production in plants subjected to potassium defi ciency stress). it has been reported that plants become more susceptible to environmental stress in potassium defi cient conditions (cakmak 2005). it can be inferred from the data that shoot fresh and dry weights were negatively affected by water defi cit, but positively affected by k application. furthermore, there was a negative effect of k application on shoot fresh and dry weight under full irrigation, and yet a positive effect was observed under water defi cit conditions. irrigation and irrigation and k interaction had signifi cant effects whereas k application had a non signifi cant effect on plant height of b. napus cultivars under study. it was further observed that there was a decrease in plant height with decreasing availability of irrigation water and an increase in plant height with increasing k application. when the different rates of k were applied with different irrigations, there was a decrease in plant height with increasing k application at full irrigation, but an increase in plant height with increasing k application at 80% or 60% eta supplementation through irrigation. potassium has a positive role in turgidity maintenance and continual cell growth (egilla et al. 2005, fusheing 2006). stomata closer in response to leaf turgor decline to high vapor pressure defi cit in the atmosphere or to root-generated chemical signals, the latter being common in drought conditions (chaves et al. 2009). thus photosynthesis is one of the key processes to be affected by water defi cit via decreased co2 diffusion to the chloroplast and metabolic constraints. the relative impact of those limitations varies with the intensity of the stress, the occurrence (or not) of superimposed stresses, and the response of b. napus to water deficiency and potassium application acta bot. croat. 73 (2), 2014 309 species. furthermore, at the cellular level, though moderate water defi cits had opposite effects on cell number and cell size, a more severe stress reduced both variables (aguirrezabal et al. 2006). the decreased photosynthate production and assimilation thus reduced the growth of the seedlings, as evident in the lower fresh and dry weights. fresh and dry weight data were collected at 60 days after sowing. at 60 days after sowing, most of the photosynthates are directed towards grains. similarly, k application also affected photosynthate translocation positively, which might have reduced fresh and dry weight of the subject plant species (marschner et al. 1996). the osmotic adjustment through production of compatible solutes (proline and sugar) and increased k availability with application facilitates the maintenance of cell turgor during periods of water stress (turner and jones 1980, morgan 1984) and contributes to leaf survival by maintaining higher rwc at low water potentials (flower and ludlow 1986, basnayake et al. 1993) and during recovery. the leaf survival also results in an increase in the absorption of sunlight, increasing the photo assimilates produced, which results in increased biomass production. therefore, for plants growing in drought conditions, accumulation of abundant k in their tissues may play an important role in water uptake along a soil-plant gradient (fanaei et al. 2009). numerous studies have shown that the application of k fertilizer mitigates the adverse effects of drought on plant growth (anderson et al. 1992, tiwari et al. 1998, sangakkara et al. 2001, egilla et al. 2005, singh and kuhad 2005, fanaei et al. 2009). similarly, early maturation is one among the different mechanisms known to increase plant survival under water stress conditions (levitt 1980). consequently, a decrease in number of days to 50% fl owering and physiological maturity was noted in this experiment as well (data not shown). the acceleration of the fl owering and/or maturity processes probably contributed to a reduction of the impact of drought stress in canola genotypes (moghadam et al. 2009). grain yield was negatively affected when the quantity of irrigation water was reduced. potassium application positively increased grain yield at all irrigation levels; however, it is evident from the slope of the regression line that the unit increase with increasing quantity of k was more pronounced under drought stress conditions. the different physiological traits like photosynthesis, velocity and rate of assimilate transfer from source to sink determine seed yield (chhabra et al. 2007, albarrak 2006). furthermore, at any stage of crop growth such as germination, moisture stress can cause an irreversible loss in yield potential (reginato 1983, hosseini and hassibi 2011, khalil et al. 2012). genotypic differences in effi ciency of k uptake and utilization have been reported for all major economically important plants (rengel and damon 2008). niknam et al. (2003) also showed that compared with b. napus, b. juncea genotypes maintained good yield under water defi cit when osmotically adjusted. potassium application under drought stress resulted in the protection of membranes resulting in increase in photosynthesis and enhanced partitioning of photo-assimilates to the roots (cakmak 2005). this would result in enhanced fi xation of carbon and acquisition of nutrients from the soil. resultantly, an increase in grain yield was noted with enhanced k application under drought stress. furthermore, in addition to photosynthate production, the partitioning between different organs is also an important component of drought adaptation. under stress conditions, the metabolic process are altered to produce substances involved in conferring protection and consequently, fewer resources are allocated to grain production. ali m., bakht j., khan g. d. 310 acta bot. croat. 73 (2), 2014 references aguirrezabal, l., combaud, s. b., radziejwoski, a., dauzat, m., cookson, s. j., granier c., 2006: plasticity to soil water defi cit in arabidopsis thaliana: dissection of leaf development into underlying growth dynamic and cellular variables reveals invisible phenotypes. plant, cell and environment 29, 2216–2227. akatsuka, t., nelson, o. e., 1966: starch granule-bound adenosine diphosphate glucosestarch glucosyltransferases of maize seeds. journal of biological chemistry 241, 2280– 2286. albarrak, k. m., 2006: irrigation interval and nitrogen level effects on growth and yield of canola (brassica napus l.). science journal of king faisal university 7, 87–99. aziz, a., martin-tanguy, j., larher, f., 1999: salt stress-induced proline accumulation and changes in tyramine and polyamine levels are linked to ionic adjustment in tomato leaf discs. plant science 145, 83–91. ali, b., hayat, s., fariduddin, q., ahmad a., 2008: 24-epibrassinolide protects against the stress generated by salinity and nickel in brassica juncea. chemospere 72, 1387–1392. allan, k. s. l., candido, f. l. n., benedito, g. s. f., roberto, c. l. c., flavio, j. r. c., hadrielle, k. b. n., monick, j. s. l., 2008: physiological and biochemical behavior in soybean (glycine max cv. sambaiba) plant under water defi cit. australian journal of crop science 2, 25–32. anderson, m. n., jensen, c. r., losch, r., 1992: the interaction effects of potassium and drought in fi eld-grown barley. i. yield, water-use effi ciency and growth. acta agriculturae scandinavica, section bsoil and plant science 42, 34–44. arraudeau, m. a., 1989: breeding strategies for drought resistance in cereals. in: baker, f. w. g., (ed.), drought resistance in cereals, 107–116. cab international, wallingford, uk. assmann, s. m., shimazaki, k., 1999: the multisensory guard cell. stomatal responses to blue light and abscisic acid. plant physiology 119, 809–816. bajji, m., lutts, s., kinet, j. m. 2001: water defi cit effects on solute contribution to osmotic adjustment as a function of leaf ageing in three durum wheat (triticum durum desf) cultivars performing differently in arid conditions. plant science 160, 669–681. bakht, j., shafi, m., jamal, y., sher, h., 2011: response of maize (zea mays l.) to seed priming with nacl and salinity stress. spanish journal of agricultural research 9, 252– 261. bakht, j., javed, m., shafi, m., mohammad, a. k., mohammad, s., 2012: effect of salinity and aba application on proline production and yield in wheat genotypes. pakistan journal of botany 44, 873–878. bakht, j., bano, a., shafi, m., dominy, p., 2013: effect of abscisic acid application on cold tolerance of chick pea (cicer arietinum l.). european journal of agronomy 44, 10–21. basnayake, j., ludlow, m. m., cooper, m., henzell, r. g., 1993: genotypic variation of osmotic adjustment and desiccation tolerance in contrasting sorghum inbred lines. field crops research 35, 51–62. bates, l. s., waldren, r. p., teare, i. d., 1973: rapid determination of free proline for water stress studies. plant and soil 39, 205–208. response of b. napus to water deficiency and potassium application acta bot. croat. 73 (2), 2014 311 biswas, b. c., prasad, n., tewatia, r. k., 1992: fertilizer use and crop quality. fertilizer news 39, 47–49. bricker, b., 1991: a micro-computer program for the design, management and analysis of agronomic experimentation. michigan state university east lansing mi 48824 usa. cakmak, i., 2005: the role of potassium in alleviating detrimental effects of abiotic stresses in plants. journal of plant nutrition and soil science 168, 521–530. chaves, m. m., flexas, j., pinheiro, c., 2009: photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. annals of botany 103, 551–561. chhabra, m. l., sinha, b. k., singh, d., dhawan, k., sharma, r., 2007: physiological constraints to productivity in indian mustard (b. juncea l.). in: tingdong f., chunyun g. 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(lamiaceae: sub-family nepetoideae: tribe mentheae: sub-tribe salviinae) taxa from turkey, including five endemic taxa, was examined using scanning electron microscopy: salvia bracteata banks et sol., s. cadmica boiss., s. blepharoclaena hedge et hub.-mor., s. cryptantha montbret et aucher ex bentham, s. aethiopis l, s. ceratophylla l., s. candidissima vahl subsp. candidissima., s. cyanescens boiss et bal., s. virgata jacq, s. halophila hedge, s. verticillata l. subsp. verticillata, s. verticillata l. subsp amasiaca (freyn et bornm.) bornm. the mericarps examined exhibited variation in size, shape, colour, and surface sculpturing. mericarp size ranged between 1.6–3.5 mm length and 1.0–2.9 mm width. observed shapes included spherical, trigonous-prolate spheroidal and prolate spheroidal. mericarp surface sculpturing revealed three distinct types: reticulate, foveate and verrucate. our study supports further work across the genus. key words: nutlet, mericarp, morphology, salvia, lamiaceae, sem introduction salvia l. belongs to the family lamiaceae, representing a diverse cosmopolitan assemblage of nearly 1000 species (walker et al. 2004, walker and sytsma 2007). the genus comprises at least 500 species in central and south america, 250 species in central asia and the mediterranean and 90 species in eastern asia (walker et al. 2004). turkey is a major centre of diversity for salvia in asia (vural and adigüzel 1996). since the most recent reviews of the genus in turkey, four new species have been described and the total has now reached 90. forty seven of these salvia species in turkey are endemic (hedge 1982, davis et al. 1988, duman 2000, dönmez 2001, hamzaogh lu et al. 2005). five of the 12 taxa investigated here are endemic to turkey. acta bot. croat. 68 (1), 2009 105 * corresponding author, e-mail: kamuran.aktas@bayar.edu.tr u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:43:45 color profile: disabled composite 150 lpi at 45 degrees salvia and its fruits have economic and cultural significance. in this paper we present results on investigation of the mericarps and answers to the question if there are variations among taxa and whether taxa can be distinguished by fruits. furthermore, there is strong evidence about polyphyletic origin of salvia from several different lineages that possess two fertile stamens and the staminal lever mechanism (walker et al. 2004, walker and sytsma 2007). mericarp characters have been shown to be phylogenetically informative in other lamiaceae genera (e.g. teucrium l. (marin et al. 1994); hemigenia r.br. and microcorys r.br. (guerin 2005) and preliminary investigations sampling across groups in salvia are needed to assess evolutionary patterns and phylogenetic utility. the mericarp anatomy and surface ultrastructure of some salvia species from the jordan region were reported by oran (1996, 1997). the variation observed was found to be useful at the species level but higher level utility was not assessed. some researchers correlated their major clades of salvia with stamen types identified from examination of fresh material or obtained from the literature (walker and sytsma 2007). three stamen types for turkish salvia species were identified (hedge 1982). types a (with a reduced theca borne on the lower connective arm) and b (lower connective arm with variously shaped connective tissue blocking access to nectar) of both accounts are equivalent. type c (lower connective arm reduced, connective and filament not articulated) of hedge (1982) has no equivalent in walker and sytsma (2007) although it is at least superficially similar to type c (rosmarinus l.) and h (part of salvia sect. audibertia) in which the lower connective arm is entirely aborted. given that the classification of salvia has been shown to be inadequate and that stamen types appear to correlate to major clades, it will be important to determine whether the fruit types examined here correlate with stamen type, particularly since the phylogenetic placement of most of the species is unknown. there are a number of accounts of the fruits of lamiaceae including a small but growing number of micromorphological studies (see isley 1947, hedge 1967, wojciechowska 1958, 1961, 1966, 1972, husain et al. 1990, rejdali 1990, ryding 1992a, b, marin et al. 1994, oran 1996, turner and delprete 1996, zhou et al. 1997, duleti]-lau[evi] and marin 1999, guerin 2005, moon and hong 2006, kaya and dirmenci 2008). the observed morphological characters have consistently been found to be useful at various taxonomic levels. this is valuable in view of the need for identification of mericarps in economic and non-scientific contexts and for additional characters to assess the morphological radiation of a polyphyletic salvia and potentially to aid in delimiting segregate genera in a revised classification of salvia s.l. although mericarp characters, particularly exomorphic features, are used in many taxonomic treatments, they are far from being fully exploited. seldom does one find keys or synopses for mericarp identification, and understanding of microcharacters requires detailed laboratory work. it has been pointed out that this is partly due to the rather scanty literature relating to mericarps and lack of a suitable descriptive terminology which can be applied universally (rejdali 1990). thus, while considerable literature is available on the seeds of some plant families, such as the umbelliferae and cyperaceae, little, other than incidental notes in manuals or species descriptions, has been published for many other families. most of the groups dealt with are of economic importance, particularly the weedy and cultivated species. wild plants have been relatively ignored. more recent account of species may be found in bartholtt (1984). 106 acta bot. croat. 68 (1), 2009 özkan m., aktasz k., özdemir c., guerin g. u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:43:45 color profile: disabled composite 150 lpi at 45 degrees the tribes ajugaea, scutellarieae and stachyeae as represented in the usa have been distinguished, with the use of mericarp characters (isley 1947). this author also noted that it was possible to recognize most species by mericarp characters. it may be possible to produce a key to species or species-groups based solely on the texture, size and areole (abscission scar) shape of the mericarps (rejdali 1990). this author reported that the mericarps of sideritis l. were ovate to longitudinally elongate, oblong, with the surface varying tremendously between ridged, tuberculate or reticulate (rejdali 1990). he examined nutlets of hemigenia and microcorys using sem and reported significant infrageneric variation in mericarp shape and the nature of the attachment (abscission) scar, surface sculpturing, exocarp cells and indumentum (guerin 2005). typical nutlets were ovoidal, strongly reticulate or rugose, with the exocarp cells isodiametric and convex to papillate. also common were cylindrical nutlets, often with longitudinal ridging and papillate exocarp cells. surface pitting and concave exocarp cells were rare. the homologies of these characters were assessed and provide important data within phylogenetic analyses within the westringieae and important generic and infrageneric diagnostic characters. detailed studies on the mericarps of most of the salvia taxa investigated here have not been reported in the literature. there have been reports of the anatomical and surface ultrastructure studies on the mericarps of salvia from the jordan region, including three of the species examined here: s. bracteata banks et sol., s. ceratophylla l. and s. verticillata l., although few data were reported for the latter (oran 1996, 1997). to the extent possible, these results are compared with ours. detailed comparisons with the surface ultrastructure study are problematic due to the lack of tabulation of characters and because some observed surface types were not categorised and some species were not assigned to listed types (e.g. s. bracteata). in oran (1996, 1997) there are some apparent inconsistencies between categories of surfaces and more detailed descriptions and images given in the plates. observed shapes were reported as spherical, ovoid or ovoid-spherical. by contrast, harley et al. (2004) described the mericarps of salvia as trigonous, ovoid or sub-orbicular. the pattern of mericarp morphology across the infrageneric grouping of the genus is presently unknown. the fruit of lamiaceae is typically a schizocarp consisting of indehiscent locules which separate to form fruitlets (harley et al. 2004). the entire fruiting body may remain entire or split into only two fruitlets (e.g. many taxa within tribe chloantheae (sub-family prostantheroideae), but typically, four separating, indehiscent fruitlets result that should properly be referred to as mericarps. although the term ’nutlet’ has commonly been applied in reports of fruitlet morphology in lamiaceae (marin et al. 1994, oran 1996, turner and delprete 1996, guerin 2005, moon and hong 2006, kaya and dirmenci 2008), this is a somewhat informal term. mericarps were the unit of study and this is strictly the correct term. we use the term ’mericarp’ throughout. the point of attachment of the mericarps to the schizocarpic receptacle is here referred to as an ’abscission scar’ rather than an ’attachment scar’ (e.g. as in guerin 2005). the term ’areole’ is perhaps best applied to the attachment point of seeds rather than indehiscent fruits. acta bot. croat. 68 (1), 2009 107 mericarp morphology of salvia u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:43:45 color profile: disabled composite 150 lpi at 45 degrees materials and methods material used for this study was collected from wild populations in turkey (tab. 1). the plant samples were dried as herbarium samples and stored in celal bayar university herbarium (cbuh). in order to assess infraspecific variation, 9–10 mericarps from each taxon were measured. for sem studies, mericarps were directly mounted on stubs and coated with gold. micrographs were taken with a jeol 5600 sem. all the specimens were examined, but only the clearest photographs representing each mericarp sculpturing type were selected and illustrated. terminology for descriptions of morphological characteristics of the mericarps follows stearn (1978) and bojnanfský and farga[ová (2007). results the characteristics of mericarp morphology (i.e., size, shape, surface sculpturing and colour) for the investigated taxa are given in table 2. infraspecific variation was restricted 108 acta bot. croat. 68 (1), 2009 özkan m., aktasz k., özdemir c., guerin g. tab. 1. localities where research of salvia l. was performed taxon voucher locality collection number s. bracteata banks et sol. amasya : kirklar mountain, 900 m, 16.vi. 1998. ozkan 1025 s. cadmica boiss. (e) ankara: bala, beynam national park, under forest, 1200 m, 14.vi.2004. ozkan 4034 s. blepharoclaena hedge et hub.mor. (e) kirsehir : çiçekdagi, ahmet veli mausoleum, in the forest, 1500 m, 28.v.2004. ozkan 4024 s. cryptantha montbret et aucher ex bentham (e) kirsehir : çiçekdagi road 50 km, slopes of mountain, 1150 m, 6.vi.2005. ozkan 5020 s. aethiopis l. amasya: merzifon, tavsan mountain, 950 m, 28.vi.1998. ozkan 1032 s. ceratophylla l. kastamonu: tosya, roadside, 700 m, 25.v. 1998. ozkan 1014 s. candidissima vahl. subsp. candidissima kayseri: develi, roadside, 1150 m, 15.vii. 2004. ozkan 4054 s. cyanescens boiss et bal. (e) kastamonu: tosya, gavurdagi, 1050 m, 17. vii. 1998. ozkan 1054 s. virgata jacq. amasya: merzifon, tavsan mountain, west slopes, 500 m, 25.vii.2001. ozkan 2018 s. halophila hedge (e) ankara: eskisehir road, 70 km, riversi-des, 1000 m. 27.vii.2004. ozkan 4074 s. verticillata l. subsp. verticillata amasya: merzifon, between büyük radar and derealan village, 1000 m, 7.vii.1998. ozkan 1038 s. verticillata subsp. amasiaca (freyn et bornm.) bornm. amasya: merzifon, derealan village, roadside, 1100 m, 7.vii.1998. ozkan 1044 e: endemic for turkey. u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:43:45 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 68 (1), 2009 109 mericarp morphology of salvia t a b . 2 . c o m p a ri so n o f m e ri c a rp sh a p e , si z e , sc u lp tu ri n g a n d c o lo u r in s a lv ia ta x a s : s p h e ri c a l, t -p s : t ri g o n o u s a n d p ro la te s p h e ro id a l, p s : p ro la te s p h e ro id a l; f : f o v e a te , r : r e ti c u la te , v : v e rr u c a te t a x o n s ta m e n ty p e 1 s h a p e l e n g th (m m ) w id th (m m ) l /w ra ti o s c u lp tu ri n g m id ri d g e c o lo u r m s d r a n g e m s d r a n g e m r a n g e s . b ra c te a ta a s 3 .2 5 ± 0 .1 7 3 .0 – 3 .5 2 .5 5 ± 0 .1 3 2 .2 – 2 .7 1 .3 1 .1 – 1 .5 f – d a rk -b ro w n s . c a d m ic a a s 3 .0 6 ± 0 .0 7 3 .0 – 3 .2 2 .3 8 ± 0 ,1 2 2 .2 – 2 .6 1 .3 1 .2 – 1 .4 f – d a rk b ro w n s . b le p h a ro c la e n a a s 2 .5 0 ± 0 .1 2 2 .4 – 2 .7 1 ,9 5 ± 0 .1 1 1 .8 – 2 .1 1 .3 1 .2 – 1 .4 f – b la c k s . c ry p ta n th a a s 3 .1 2 ± 0 .0 7 3 .0 – 3 .2 2 .7 5 ± 0 ,1 4 2 .5 – 2 .9 1 .1 1 .0 – 1 .3 f – p a le -b ro w n s . a e th io p is b t -p s 2 .6 0 ± 0 .1 2 2 .1 – 2 .9 1 .6 7 ± 0 .1 1 1 .5 – 1 .8 1 .6 1 .4 – 1 .8 f – p a le -b ro w n s . c e ra to p h y ll a b s 2 .6 4 ± 0 .1 2 2 .5 – 2 .9 2 .2 2 ± 0 .1 4 1 .9 – 2 .3 1 .2 1 .2 – 1 .4 r – b la c k s . c a n d id is si m a su b sp . c a n d id is si m a b t -p s 2 .4 0 ± 0 .1 1 2 .2 – 2 .6 1 .6 1 ± 0 .1 4 1 .4 – 1 .8 1 .5 1 .3 – 1 .7 f – p a le -b ro w n s . c y a n e sc e n s b t -p s 2 .6 1 ± 0 .0 9 2 .5 – 2 .8 1 .8 5 ± 0 .1 4 1 .6 – 2 .0 1 .4 1 .3 – 1 .6 v – d a rk b ro w n s . v ir g a ta b t -p s 2 .3 1 ± 0 .1 5 2 .0 – 2 .4 1 .5 7 ± 0 .1 2 1 .4 – 1 .7 1 .5 1 .2 – 1 .7 r + b la c k s . h a lo p h il a b s 1 .7 7 ± 0 .1 1 1 .6 – 2 .0 1 .3 7 ± 0 ,1 4 1 .2 – 1 .6 1 .3 1 .0 – 1 .6 r – d a rk b ro w n s . v e rt ic il la ta su b sp . v e rt ic il la ta c p s 1 .8 8 ± 0 .1 0 1 .8 – 2 .0 1 .1 4 ± 0 .1 1 1 .0 – 1 .3 1 .6 1 .4 – 2 .0 r – d a rk b ro w n s . v e rt ic il la ta su b sp . a m a si a c a c p s 2 .2 5 ± 0 .2 0 2 .0 – 2 .5 1 .2 0 ± 0 .1 3 1 .0 – 1 .4 1 .9 1 .5 – 2 .4 r – d a rk b ro w n m : m e a n ; s d : s ta n d a rd d e v ia ti o n 1 a a n d b e q u iv a le n t to h e d g e (1 9 8 2 ) a n d w a l k e r a n d s y t s m a (2 0 0 7 ); c e q u iv a le n t to h e d g e (1 9 8 2 ). u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:43:45 color profile: disabled composite 150 lpi at 45 degrees to minor differences in mericarp size. the studied salvia mericarps ranged in length from 1.6 mm to 3.5 mm with the minimum value in s. halophila hedge and the maximum value in s. bracteata, and in width from 1.0 mm to 2.9 mm with the minimum value in s. verticillata subsp. verticillata and the maximum value in s. cryptantha montbret et aucher ex benth. mericarps of salvia bracteata, s. cadmica boiss., s. blepharoclaena hedge et hub.mor., s. cryptantha, s. ceratophylla and s. halophila were spherical (fig 1a-d, f, j), s. aethiopis l., s. candidissima vahl. subsp. candidissima, s. cyanescens boiss. et bal.and s. virgata jacq. were trigonous and prolate spheroidal (fig 1e, g, h, i) and s. verticillata subsp. verticillata and s. verticillata subsp. amasiaca (freyn et bornm.) bornm. were prolate spheroidal in shape (fig 1k, l). 110 acta bot. croat. 68 (1), 2009 özkan m., aktasz k., özdemir c., guerin g. fig. 1. scanning electron micrographs of mericarp in salvia. a – salvia bracteata; b – s. cadmica; c – s. blepharoclaena; d – s. cryptantha; e – s. aethiopis; f – s. ceratophylla; g – s. candidissima subsp. candidissima; h – s. cyanescens; i – s. virgata; j – s. halophila; k – s. verticillata subsp. verticillata; l – s. verticillata subsp. amasiaca. u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:44:20 color profile: disabled composite 150 lpi at 45 degrees mericarps of investigated taxa exhibited three types of surface sculpturing, and are present in the following species: type i. foveate: surface with small pits: salvia bracteata, s. cadmica, s. blepharoclaena, s. cryptantha, s. aethiopis and s. candidissima subsp. candidissima (figs. 2a-e, g). type ii. reticulate: surface finely reticulate: s. ceratophylla, s. virgata, s. halophila, s. verticillata subsp. verticillata and s. verticillata subsp. amasiaca (figs. 2f, i, j, k, l). type iii. verrucate: surface with irregular projections or knobs: s. cyanescens (fig 2h). the mericarps of salvia bracteata, s. cadmica, s. cyanescens, s. halophila, s. verticillata subsp. verticillata and s. verticillata subsp. amasiaca were dark brown while those acta bot. croat. 68 (1), 2009 111 mericarp morphology of salvia fig. 2. scanning electron micrographs of mericarp surfaces in salvia. a – salvia bracteata; b – s. cadmica; c – s. blepharoclaena; d – s. cryptantha; e – s. aethiopis; f – s. ceratophylla; g – s. candidissima subsp. candidissima; h – s. cyanescens; i – s. virgata; j – s. halophila; k – s. verticillata subsp. verticillata; l – s. verticillata subsp. amasiaca u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:44:26 color profile: disabled composite 150 lpi at 45 degrees of s. cryptantha, s. aethiopis and s. candidissima subsp. candidissima were pale brown and those of s. blepharoclaena, s. ceratophylla and s. virgata were black. discussion the shape, size, sculpturing and colour of mericarps examined varied, and character combinations identified or defined taxa or groups of taxa. the length to width ratio of mericarps varied with the perhaps obvious pattern of being larger in prolate mericarps than in spherical mericarps. significantly, character combinations correlated with stamen type (i.e. type a, b or c) for each taxon. taxa with stamen type a had spherical-foveate mericarps, taxa with stamen type b had spherical-reticulate mericarps or were trigonous and prolate spheroid with variable sculpturing diagnostic at lower levels; taxa with stamen type c had prolate-spheroidal-reticulate mericarps (although only the subspecies of a single species were examined for type c). this correlation is supported by molecular phylogenetic evidence, at least for salvia aethiopis and salvia candidissima, which have more or less identical mericarps, stamen type b and were shown to be closely related in all consensus trees of walker and sytsma (2007). the mericarps of both subspecies of salvia verticillata were prolate spheroidal, reticulate with small pits and were dark brown in colour. mericarp size was given as 2.2 mm ´ 1.3 mm for both subspecies (hedge 1982). we measured the mericarp sizes as 1.8–2.0 ´ 1.0–1.3 mm for s. verticillata subsp. verticillata and 2.0–2.5 ´ 1.0–1.4 mm for s. verticillata subsp. amasiaca. although the mericarps of both subspecies were reticulate with small pits, the size, particularly the length, differed and this character could be used to discriminate the subspecies. the mericarps of salvia virgata had a prominent mid-ridge extending distally from the abscission scar. the same characteristic were observed on the mericarps of sideritis (rejdali 1990). this apomorphic characteristic of s. virgata distinguishes it from all other investigated taxa and may remain an important character for identification when more taxa are sampled. although salvia is the largest genus of lamiaceae, its mericarp morphology and anatomy have been poorly reported. there has been a report of the only other known external micromorphological study on the mericarps of salvia (oran 1996). detailed comparisons with the conclusions of this previous study are hampered by the excessive number of surface types given, including two separate but very similar types listed for s. viridis l., the lack of types being assigned to all species in the study, the lack of tabulation of characters, and the lack of a clear conclusion as to the pattern of characters within and between groups and sections. the observed shapes, reported as spherical, ovoid or ovoid-spherical, are equivalent to our spherical and prolate-spheroidal respectively, with the third shape intermediate. exocarp cellular features such as cell shape and concavity/convexity are not reported here separately from surface sculpturing as the individual cells are somewhat obscured in the taxa examined. the mericarp surfaces were relatively smooth with sculpturing at lower levels created by the outer walls of the exocarp cells. the nature of the individual exocarp cells has shown to be an informative character in other groups and certainly varies significantly across the lamiaceae with smooth or pitted cells common and convex to conical or papillose cells present in sub-family prostantheroideae (guerin 2005). 112 acta bot. croat. 68 (1), 2009 özkan m., aktasz k., özdemir c., guerin g. u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:44:26 color profile: disabled composite 150 lpi at 45 degrees the only available data on some of the investigated taxa are a few notes given in flora of turkey (hedge 1982). our results showed some similarities and differences with these notes (tab. 3). some of the differences in the description of shape are apparently due to use of different terminology rather than any difference in observations or interpretation. for example, the »rounded-trigonous ovoid« of hedge (1982) is clearly equivalent to »trigonous and prolate spheroidal«. the differences in mericarp size are mostly insignificant and probably attributable to sampling error and differences in the accuracy of the measurement methods. however, a number of measurements are very different, for example for salvia blepharoclaena, which had dimensions reported by hedge (1982) over 1.5´ larger than in this study. the shape was given as rounded-trigonous ovate whereas the mericarps examined here were clearly spherical (see figure 1c). the only available notes concerning the mericarps of s. cryptantha is a record of them being pale brown in hedge (1982), which agrees with our observations. we observed the same colour for s. cryptantha. some researchers found that the density of glands on mericarp was the most useful character in teucrium (sub-family ajugoideae) and husain et al. (1990) found that mericarp shape and the nature of the abscission scar were invariable in representative members of tribe saturejeae (marin et al. 1994). we found that in salvia the mericarps were glabrous but that the shape was a significant character. the abscission scar of the taxa examined was very small, simple and positioned in the typical basal position, offering no utility at lower levels. conversely, the abscission scar in some members of tribe westringieae (sub-family prostantheroideae) and ajuga l. is baso-lateral and occupies a significant fraction of the length of the mericarp (often over half). the nature of the abscission scar is variable and phylogenetically informative at an infrageneric level in the westringieae (guerin 2005). clearly, even characters that are invariable within particular groups may be of systematic value at higher levels and hence worth reporting in the literature. some terms that appear to be analogous at best have been applied to characters across several sub-families and tribes of the lamiaceae. for example, the reticulate surfaces of mericarps examined here are net-like at a very fine scale possibly relating to the walls of individual exocarp cells and the surface overall is not distinctly sculptured, whereas guerin (2005) applied the term to mericarps with relatively large, prominent ridges when the ridges joined into a net-like arrangement around depressed lacunae. conversely, in some cases different terms have been applied to the same character. for example, harley et al. (2004) described the mericarps of salvia as trigonous, ovoid or sub-orbicular, terms which clearly describe shapes comparable to those reported here for the genus. the previous comparisons with the shape terminology of hedge (1982) and oran (1996) are further examples. the above suggests a need for an acceptable and unifying descriptive terminology for mericarps to be applied across lamiaceae. author number four is currently collating terminology which has been used in the family or which should properly be used, but wider sampling of mericarp morphology across sub-families will be required to propose a comprehensive terminology. in summary, we observed variation in the external morphology of mericarps in salvia taxa representing the morphological diversity (particularly with regard to stamen types) of the genus in turkey, including endemic taxa, and found that characters of shape, size, surface sculpturing and colour varied and were useful in distinguishing groups, species and acta bot. croat. 68 (1), 2009 113 mericarp morphology of salvia u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:44:26 color profile: disabled composite 150 lpi at 45 degrees subspecies with regard to apomorphies but also with character combinations defining groups. character combinations correlated with stamen types, suggesting mericarps may be useful phylogenetically and in classification. we conclude that a wider sampling of salvia species from turkey, indeed across the entire genus and even the tribe mentheae, would be advantageous and informative. the mericarp micromorphology of taxa nested within salvia s.l. such as rosmarinus also needs to be examined and compared. this would provide data towards an understanding of the evolutionary and morphological radiation of the group but also towards building practical identification tools. references barthlott, w., 1984: microstructural features of seed surfaces. in: heywood, v. h., moore, d. m. (eds.), current concepts in plant taxonomy. academic press, london. bojnanfský, v., farga[ová, a., 2007: atlas of seeds and fruits of central and east european flora (the carpathian mountains region). springer, stuttgart. davis, p. h., mill, r. r., tan, k., (eds) 1988: flora of turkey and the east aegean islands. edinburgh university press, edinburgh. dönmez, a. a., 2001: a new turkish species of salvia l. (lamiaceae). botanical journal of the linnean society 137, 413–416. duleti]-lau[evi], s., marin, p. d., 1999: pericarp structure and myxocarpy in selected genera of nepetoideae (lamiaceae). nordic journal of botany 19, 435–446. duman, h., 2000: salvia l. in: güner, a., özhatay, n., ekim, t., baszer, k. h. c. (eds.), flora of turkey and the east aegean islands 11, 197 – 209. edinburg university press, edinburg. guerin, g. r., 2005: nutlet morphology in hemigenia r.br. and microcorys r.br. 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(ed.), flora of turkey and the east aegean islands 7, 400 – 461. edinburgh university press, edinburgh. husain, s. z., marin, p. d., [ili], c., qaiser, m., petkovi], b., 1990: a micromorphological study of some representative genera in the tribe satureae (lamiaceae). botanical journal of the linnean society 103, 59–80. iseley, d., 1947: investigations in seed classification by family characteristics. iowa agricultural experimental university journal of research 12, 247–260. kaya, a., dirmenci, t., 2008: nutlet surface micromorphology of the genus nepeta l. (lamiaceae) in turkey. turkish journal of botany 32, 103–112. 114 acta bot. croat. 68 (1), 2009 özkan m., aktasz k., özdemir c., guerin g. u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:44:26 color profile: disabled composite 150 lpi at 45 degrees marin, p. d., petkovi], b., duleti], s., 1994: nutlet sculpturing of selected teucrium species (lamiaceae): a character of taxonomic significance. plant systematics and evolution 192, 199–214. moon, h. k., hong, s. p., 2006: nutlet morphology and anatomy of the genus lycopus (lamiaceae: mentheae). journal of plant research 119, 633–644. oran, s. a., 1996: ultrastructure of nutlet surface of the genus salvia l. in jordan and the neighbouring countries. dirasat, natural and engineering sciences 23, 393–408. oran, s. a., 1997: nutlet anatomy of the genus salvia l. in jordan. flora mediterranea 7, 27–40. rejdali, m., 1990: seed morphology and taxonomy of the north african species of sideritis l. 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(eds), advances in labiate science, 85 – 96. the royal botanical garden, london. ryding, o., 1992b: pericarp structure and phylogeny within lamiaceae subfamily nepetoideae tribe ocimeae. nordic journal of botany 12, 273–298. stearn, w. t., 1978: botanical latin. redwood burn limited, london. turner, b. l., delprete, p. g., 1996: nutlet sculpturing in scutellaria sect. resinosa (lamiaceae) and its taxonomic utility. plant systematics and evolution 199, 109–120. vural, a., adiguzel, n., 1996: a new species from central anatolia: salvia aytachii m. vural et n. adigüzel (labiatae). turkish journal of botany 20, 531–534. walker, j. b., sytsma, k. j., treutlein, j., wink, m., 2004: salvia (lamiaceae) is not monophyletic: implications for the systematics, radiation, and ecological specializations of salvia and tribe mentheae. american journal of botany 91, 1115–1125. walker, j. b., sytsma, k. j., 2007: staminal evolution in the genus salvia (lamiaceae): molecular phylogenetic evidence for multiple origins of the staminal lever. annals of botany 100, 375–391. wojciechowska, b., 1958: taxonomy, morphology and anatomy of seeds in the genus salvia l. (polish with english summary). monographie botanicae 6, 1–56. wojciechowska, b., 1961: fruits in the middle european species of some genera of stachyoideae (fam. labiatae). monographie botanicae 12, 89–120. wojciechowska, b., 1966: morphology and anatomy of fruits and seeds in the family labiatae with particular respect to medicinal species. monographie botanicae 21, 119–243. wojciechowska, b., 1972: morphology and anatomy of fruits of scutellaria chaiturus, galeobdolon and sideritis of the family labiatae. monographiae botanicae 37, 137– 168. zhou, s. l., pan, k. y., hong, d. y., 1997: pollen and nutlet morphology in mosla (labiatae) and their systematic value. israel journal of plant sciences 45, 343–350. acta bot. croat. 68 (1), 2009 115 mericarp morphology of salvia u:\acta botanica\acta-botan 1-09\ozkan.vp 16. travanj 2009 11:44:26 color profile: disabled composite 150 lpi at 45 degrees acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 447 acta bot. croat. 73 (2), 447–463, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 the infl uence of selected factors on the distribution of epilithic diatoms in a torrential river the kamniška bistrica (slovenia) mihael j. toman, ana m. grošelj, igor zelnik* university of ljubljana, biotechnical faculty, department of biology, večna pot 111, si-1000 ljubljana abstract – physical and chemical characteristics of habitats and species diversity in streams and rivers are strongly infl uenced by the catchment area. we analysed the infl uence of selected environmental and spatial variables on the diversity and species composition of epilithic diatom communities in periphyton. samples were collected along the river course in a torrential river the kamniška bistrica. sampling sites were selected in reaches distributed from the source to the outlet of the river and were under different infl uences from the catchment area and with different physical and chemical characteristics. the most common and dominant diatom species in the periphyton community were achnanthes biasolettiana and a. minutissima. achnanthes species often inhabit rivers and springs with moderate organic pollution. another common diatom taxon was gomphonema pumilum – a key species indicating oligosaprobic conditions. the results of the canonical correspondence analyses revealed that variance of the periphytic diatom community was explained by water temperature and conductivity as well as altitude. diatom species richness was positively correlated with saprobic index values and abundance of fi lamentous algae in the river bed indicating a relatively low organic matter and nutrient input into the river system. keywords: diatoms, periphyton, spatial distribution, torrential river introduction benthic algae and cyanobacteria are the main primary producers in torrential rivers. benthic diatoms are frequently used for evaluating the ecological status of running waters (virtanen et al. 2011), since they are various, dominant in phytobenthos, and since the ecological preferences of several species are well known (beltrami et al. 2012). habitat and species diversity of biotic communities in running waters are strongly infl uenced by the properties of the catchment area, land use and pollution sources. * corresponding author, e-mail: igor.zelnik@bf.uni-lj.si copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. toman m. j., grošelj a. m., zelnik i. 448 acta bot. croat. 73 (2), 2014 diatoms colonize different aquatic habitats and substrates. they are a taxonomically diverse group with high sensitivity to chemical stressors (martínez de fabricius et al. 2003, frankovich et al. 2006, kiss et al. 2012), and vary spatially and temporally (passy 2007, soininen 2007). the relationships between diatoms and environmental variables were shown by many authors (jasprica and hafner 2005, passy 2007, soininen 2007, lange et al. 2011, jasprica et al. 2012, virtanen and soininen 2012, xu et al. 2012). species diversity and abundance of diatoms are controlled by environmental factors like nutrients, temperature, light intensity, grazing pressure, substrate stability and discharge (izagirre and elosegi 2005). major environmental determinants for diatom distribution in streams, as reported by soininen (2007) are: ph, conductivity, total phosphorus, temperature, alkalinity, altitude, nitrates, calcium, biological oxygen demand (bod), chlorophyll a and substrate type. lange et al. (2011) found that light availability, nutrient concentrations and grazing pressure determined the stream diatom community composition. beltrami et al. (2012) stressed that altitude and geology were the main factors determining diatom communities in alpine streams. biggs and close (1989) suggested that disturbances such as spates reduce the effect of grazing pressure, because recolonization of invertebrates is usually slow compared to periphyton growth. for the effective implementation of the european water framework directive (wfd) the member states are supposed to provide the unimpacted reference state of the system (kelly et al. 2012, várbíró et al. 2012). the potential of diatoms as bioindicators for the assessment of water quality and reference conditions is well known (almeida and feio 2012) samples were collected along the river’s course in different seasons. hydrological conditions in the kamniška bistrica are well known; several times per year the fl ow increases 10–20 fold compared to the basal fl ow. studies dealing with the relationship between environmental variables and algal communities in slovenia have been performed only in extreme environments (krivograd klemenčič et al. 2010, krivograd klemenčič and toman 2010), while the distribution of diatoms along the gradients in running waters is poorly known. also, no seasonal studies of environmental and spatial effects on diatom communities in this river or other similar rivers in the region have been completed, and therefore no comparisons with previous studies were possible. the main goal of this research was to investigate the infl uence of selected environmental, spatial and temporal factors on the species composition and diversity of epilithic diatom assemblages to investigate their use as indicators in torrential rivers. materials and methods study area the kamniška bistrica river is a left tributary of the sava river and an important part of the danube catchment area, collecting water from the southern belt of the limestone alps. the length of the river is 32.8 km, drainage area 546.1 km2 and the average (annual) discharge is about 7 m3 s–1. the karstic source (46°19'35.83''n, 14°35'15.45''e) is at an altitude of 600 m a.s.l. the riverbed mainly consists of limestone and magnesium limestone (dolomite) of middle to early jurassic age (buser 2009). the catchment area is characterized by dispersed settlements, agricultural land, farms and different kinds of industry, all of which are potential sources of inorganic and organic compounds (tab. 1). distribution of epilithic diatoms in a torrential river acta bot. croat. 73 (2), 2014 449 sampling and laboratory analyses samples were taken at fi ve sampling sites (tab. 1) in march, july and august. the sample from the beginning of march is considered a winter sample due to the very low temperatures. the sample from the beginning of july represents a transition between spring and summer, since there was a longer period of rainy and relatively cold weather and elevated discharge as well. the sample from the end of august is a typical summer sample. epilithic diatoms in the periphyton communities were sampled from stones (φ = 6–20 cm) at each site, by scraping and brushing off the stone surface (5 cm2 per stone). samples for diatom identifi cation were preserved in 4% formaldehyde, samples for chlorophyll a analyses were stored at 4 °c. for taxonomic identifi cation all samples were diluted to 50 ml, homogenized with a magnetic stirrer and subsamples were treated with concentrated nitric acid (hno3). permanent slides of diatom frustules were prepared using the high refraction mountant naphrax®. diatom taxa were identifi ed and counted using an olympus cx41 microscope with an oil-immersion objective at a magnifi cation of 1000×, taxonomy followed kramer and lange-bertalot (1991). the proportion of diatoms, cyanobacteria and other algae were analysed at a magnifi cation of 400×. cyanobacteria and other non-diatom algae were identifi ed using hindák et al. (1978), biggs and kilroy (2000), komárek and anagnostidis (2002). proportions of diatom taxa among all algae were obtained by further division of their total share according to the proportions of counted frustules at a magnifi cation of 1000×. tab. 1. sampling sites on the kamniška bistrica river and some of their characteristics; l – left, r – right. site coordinates altitude (m) distance from source (km) channel width (m) description and land-use k1 46°19'35.8''n 14°35'15.5''e 582 0.8 5.3 l-bank: forest; r-bank: forest, dry channel, grassland and pastures, parking area with cottage and restaurant. k2 46°16'57.0''n 14°36'54.5''e 477 4.7 11.0 l-bank: meadow, vegetable-garden, road and parking place, restaurant, forest; r-bank: forest. k3 46°14'47.4''n 14°36'08.2''e 402 9.1 14.7 l-bank: fl oodplain wood; r-bank: fl oodplain wood, meadows, road, downstream settlements, factory, quarry. k4 46°07'37.6''n 14°36'25.3''e 289 24.8 22.1 regulated channel; remnants of lowland oak-hornbeam forest, riparian zone with clearings overgrown with invasive alien plant species, settlements. k5 46°05'18.9''n 14°37'34.3''e 269 30.9 22.7 regulated channel; degraded banks, overgrown with invasive alien plants and pioneer shrubs and trees; improved grassland, settlement, remnants of lowland oak-hornbeam forest. toman m. j., grošelj a. m., zelnik i. 450 acta bot. croat. 73 (2), 2014 at each sampling site selected physical and chemical parameters were measured (water depth, current velocity, ph, o2 concentration, o2 saturation, temperature and conductivity), using the portable multi-meter pcd 650 (eutech instruments, singapore). light intensity was measured with the point sensor li-1000 (li-cor). the cover of inorganic and organic substrate was estimated according to the aqem (2002) protocol (tab. 2). shading was calculated as the ratio between the height and density of riparian vegetation and the width of the channel. tab. 2. the structure of organic and inorganic substrates at the sampling sites (k1–k5). mean coverage (%) of each fraction of the substrate; cpom – coarse particulate organic matter, fpom – fi ne particulate organic matter. type of substrate k1 k2 k3 k4 k5 filamentous algae 12 3 3 20 22 mosses 17 5 4 4 5 terrestrial plants 3 3 3 3 3 xylal 4 3 5 7 4 cpom 3 3 3 3 3 fpom 0 0 3 3 3 megalithal 50 35 3 3 3 macrolithal 20 45 25 30 25 mesolithal 10 15 45 50 45 microlithal 5 5 10 10 15 akal 15 0 5 3 5 psammal 0 0 3 0 5 argyllal 0 0 0 0 3 water samples were analyzed in the laboratory. water transparency and the chlorophyll a content were measured spectrophotometrically. concentration of nitrates were measured with the na-salicylate method (monteiro et al. 2003), while the soluble reactive phosphorus (srp) was measured using the sncl2 method (apha 1998). periphyton biomass was also determined as dry weight (at 105 °c) of the biofi lm covering the sampled stones scratched from the 5 cm2 rectangle. results of these measurements are given in table 3. data analyses relative abundance (percentage values) of the diatom taxa were calculated for each sample (tab. 4) and used in further analyses. the shannon-wiener (s-w) diversity index was used to estimate diatom diversity and the saprobic index (si) was calculated using saprobic (si) and indicator values (gi) according to kosi et al. (2006) to determine water quality using the following formula: (hi – abundance of the taxon i; n – number of taxa) distribution of epilithic diatoms in a torrential river acta bot. croat. 73 (2), 2014 451 ta b. 3 . m ea su re d en vi ro nm en ta l v ar ia bl es fr om fi ve s am pl in g si te s (k 1– k 5) in cl ud in g m ea n va lu es , m in im um a nd m ax im um . k 1 k 2 k 3 k 4 k 5 v ar ia bl e m ea n m in . m ax . m ea n m in . m ax . m ea n m in . m ax . m ea n m in . m ax . m ea n m in . m ax . sh ad in g 0. 69 0. 57 0. 75 0. 30 0. 18 0. 36 0. 29 0. 20 0. 34 0. 20 0. 14 0. 23 0. 07 0. 04 0. 09 l ig ht in te ns ity a t s ur fa ce (µ m ol m –2 s– 1 ) 11 1 84 14 5 15 47 12 80 17 80 12 02 14 5 17 31 10 18 11 3 15 11 86 8 41 15 23 r el at iv e lig ht in te ns ity 0. 4 0. 1 0. 6 1. 0 0. 9 1. 0 0. 9 0. 7 1. 0 0. 9 0. 6 1. 0 0. 9 0. 7 1. 0 b io m as s of p er ip hy to n (g m –2 ) 81 22 15 1 30 17 37 45 27 58 88 22 17 8 91 22 22 6 c ur re nt v el oc ity (m s –1 ) 0. 43 0. 39 0. 46 0. 53 0. 35 0. 67 0. 58 0. 44 0. 84 0. 42 0. 29 0. 57 0. 71 0. 43 0. 93 w at er d ep th (m ) 0. 33 0. 23 0. 45 0. 43 0. 26 0. 53 0. 40 0. 19 0. 50 0. 23 0. 09 0. 45 0. 29 0. 17 0. 50 d is ch ar ge (m 3 s –1 ) 0. 54 0. 46 0. 67 1. 94 1. 66 2. 39 2. 80 2. 39 3. 44 1. 34 1. 15 1. 65 2. 23 1. 90 2. 74 te m pe ra tu re (° c ) 5. 4 5. 3 5. 6 7. 5 6. 0 8. 4 10 .5 6. 6 12 .9 14 .8 6. 0 19 .6 13 .2 6. 7 17 .5 ph 7. 7 7. 4 8. 3 8. 0 7. 7 8. 4 8. 0 7. 7 8. 5 8. 3 8. 0 8. 7 7. 5 7. 3 7. 8 c on du ct iv ity (μ s cm –1 ) 16 4 15 5 17 3 20 8 19 0 23 0 23 3 21 5 26 0 36 0 31 9 40 0 48 5 43 5 51 7 o 2 ( m g l –1 ) 10 .7 8. 9 11 .6 11 .4 11 .0 12 .1 11 .4 10 .6 12 .7 11 .8 9. 5 14 .6 10 .7 9. 4 12 .6 sr p (m g l –1 ) * * 0. 01 * * 0. 02 * * * * * 0. 03 0. 13 0. 06 0. 22 n itr at e (m g l –1 ) 1. 4 1. 1 2. 0 3. 5 1. 9 5. 9 2. 2 1. 7 2. 6 3. 0 2. 6 3. 4 8. 9 8. 6 9. 4 * c on ce nt ra tio n of s ol ub le re ac tiv e ph os ph or ou s (s r p) w as b el ow d et ec tio n. toman m. j., grošelj a. m., zelnik i. 452 acta bot. croat. 73 (2), 2014 ta b. 4 . n um be r of d ia to m t ax a, s ha nn on -w ie ne r (s -w ) di ve rs ity i nd ex , sa pr ob ic i nd ic es , th e pe rc en ta ge ( % ) of d ia to m s in p hy to be nt ho s an d re la tiv e ab un da nc es (% ) o f d ia to m s pe ci es o n sa m pl in g si te s (k 1– k 5) in d iff er en t s ea so ns /m on th s (m – m ar ch , j – j ul y, a – a ug us t) . a bu nd an ce s re ac hi ng at le as t 5 % a re in b ol d; + , s pe ci es p re se nt w ith re la tiv e ab un da nc e < 1% ; – , n ot d et ec te d. si te k 1 k 2 k 3 k 4 k 5 n um be r o f d ia to m ta xa p er s ite 25 18 25 44 35 m on th m j a m j a m j a m j a m j a n um be r o f d ia to m ta xa p er s am pl e 17 18 17 13 13 10 19 17 21 31 31 34 27 28 24 sw d iv er si ty in de x 2. 45 1. 45 1. 73 1. 35 1. 50 1. 58 1. 55 1. 45 1. 90 3. 09 2. 12 3. 74 2. 59 3. 36 1. 20 sa pr ob ic in de x (a ll al ga e) 1. 88 1. 83 1. 84 1. 24 1. 72 1. 82 1. 79 1. 88 1. 71 1. 99 1. 88 2. 01 1. 92 1. 41 2. 14 sa pr ob ic in de x (d ia to m s) 1. 69 1. 79 1. 90 1. 70 1. 22 1. 80 1. 75 1. 77 1. 77 1. 90 1. 77 2. 07 1. 94 1. 88 2. 21 % o f d ia to m s in p hy to be nt ho s 59 53 28 43 36 43 50 56 39 92 63 77 85 29 70 % o f d ia to m s pe ci es : a ch na nt he s m in ut is si m a k üt zi ng 32 38 50 20 32 56 23 21 34 9 11 20 3 22 5 a ch na nt he s bi as ol et tia na g ru no w 28 56 37 8 55 30 9 70 53 5 64 13 1 26 1 g om ph on em a pu m ilu m g ru no w 23 3 7 69 12 11 63 2 2 + + – + + – d ia to m a vu lg ar is b or y + + – – + – + – + 12 1 3 3 1 + n av ic ul a m en is cu lu s sc hu m an n – – – + – – + – + + 1 7 2 4 1 n av ic ul a la nc eo la ta (a ga rd h) e hr en be rg – + + + – 1 + 1 1 38 – 1 58 4 – n itz sc hi a fo nt ic ol a g ru no w 5 – 1 + – – + + + 15 2 5 5 4 6 c oc co ne is p la ce nt ul a e hr en be rg + + + + + + 1 1 4 + 10 16 + 17 1 g om ph on em a ol iv ac eu m (h or ne m an n) b ré bi ss on 2 + 1 2 + 1 1 1 1 4 1 9 7 2 1 n itz sc hi a di ss ip at a (k üt zi ng ) w . s m ith + + + + + – + + + 3 + + 7 2 + n av ic ul a at om us (k üt zi ng ) g ru no w – – 2 – – 2 – – 1 – – + – 2 82 a m ph or a pe di cu lu s e hr en be rg + + + + + – + 1 + 3 3 2 2 5 + distribution of epilithic diatoms in a torrential river acta bot. croat. 73 (2), 2014 453 the cluster analyses were performed with the program syn-tax (podani 2001) to establish the similarity between diatom communities from different sampling sites/seasons. as a method of linkage, unweighted pair group method with arithmetic mean (upgma) was used and the bray-curtis index served as a similarity measure for the creation of a dendrogram. detrended correspondence analysis (dca) was applied to the diatom percentage data to explore the patterns of species changes and biological species turnover (the gradient length). the eigenvalue for the fi rst dca axis was > 0.4 (0.76), while gradient length was 3.54 sd (standard deviation units of species turnover) and indicated strong unimodality (ter braak and verdonschot 1995) and therefore canonical correspondence analysis (cca) was chosen to explore the relationships between diatom assemblages and explanatory variables. separate ccas for each environmental variable were performed to test the signifi cance of its infl uence on the variation of species composition. variables were gathered in three groups representing spatial, temporal and environmental gradients. forward selection of explanatory variables was used to provide a ranking of the relative importance of the specifi c variables and to avoid co-linearity. unrestricted monte carlo test with 999 permutations was used to test the statistical signifi cance of the variables and canonical axes. another cca was done with a subset of only signifi cant variables (p < 0.05) and the proportions of variance explained by these variables were calculated. variance partitioning by the mentioned groups of variables and specifi c signifi cant variables was performed with a series of pccas. some of the variables were not normally distributed so the data were log-transformed. ordination of the samples according to the most important environmental parameters was made using cca. the whole set of analyses was performed using canoco 4.5 (ter braak and šmilauer 2002). relationships between the diatom diversity and environmental factors were explored with spearman correlation coeffi cients in spss version 17. results distribution and diversity of diatoms a total of 52 diatom taxa were identifi ed in the samples from the kamniška bistrica river. the majority of these taxa (44) were found at the sampling site k4, in the regulated channel. the highest number of diatom species (40) was found in summer samples (a), and the lowest (34) in winter (m) samples. two species occurred with a high share in all seasons, namely achnanthes biasolettiana (5–64%) and a. minutissima (9–21%). the similarity of diatom assemblages is presented in fig. 1, which indicates that about one third of taxa were common to all samples (tab. 4). the dendrogram shows that two larger clusters are formed according to locations/spatial factors. the left group uniting the upper three locations (k1, k2, k3) shows a higher sample similarity than downstream locations (k4, k5). the total number of diatom taxa at the source site (k1) was 25 (tab. 4). the most abundant species (≥ 5%) were achnanthes biasolettiana, a. minutissima and gomphonema pumilum. the lowest number of diatom taxa (18 species) was detected downstream at the site k2 (tab. 4). the most abundant were the co-dominant taxa gomphonema pumilum, achnanthes minutissima and a. biasolettiana with shares between 96% and 99%. toman m. j., grošelj a. m., zelnik i. 454 acta bot. croat. 73 (2), 2014 the total number of diatom taxa at site k3 was 25 (tab. 4), with the same co-dominant species as in site k2 (gomphonema pumilum, achnanthes biasolettiana and a. minutissima). the site in the regulated channel (k4) exhibited the most species-rich diatom assemblage with 44 taxa (tab. 4). eight taxa reached ≥ 5% of the relative abundance, two species (achnanthes biasolettiana and a. minutissima) occurred with a high share in all seasons. other common species included: cocconeis placentula (a and j), diatoma vulgaris, navicula lanceolata (m), gomphonema olivaceum and nitzschia fonticola. in the august sample the highest number of taxa were observed (34) and the highest s-w diversity index value (3.74) was calculated (tab. 4) – the sample also contained the highest number of taxa (6) with a relative abundance ≥ 5% (achnanthes minutissima, cocconeis placentula, a. biasolettiana, etc). the total number of diatom taxa downstream of the regulated channel at site k5 was 35 (tab. 4). the s-w diversity index value was low (1.2) due to the dominance of the species navicula atomus (82%). eight other taxa occurred with a relative abundance ≥ 5%: navicula lanceolata, achnanthes biasolettiana, a. minutissima, cocconeis placentula, nitzschia dissipata, n. fonticola, gomphonema olivaceum and amphora pediculus. fig. 1. dendrogram showing the similarity of diatom communities in different sampling sites (k1– k5) in different seasons/months (m – march, j – july, a – august). left cluster unites the following samples: 1_m and 1_j (k1 from march and july), 2_j (k2 from july), 1_a and 2_a (k1 and k2, both from august), 3_j and 3_a (k3 from july and august), 2_m and 3_m (k2 and k3, both from march). cluster on the right side includes the samples: 4_m and 5_m (k4 and k5, both from march), 4_j and 4_a (k4 from july and august), 5_j and 5_a (k5 from july and august). distribution of epilithic diatoms in a torrential river acta bot. croat. 73 (2), 2014 455 saprobic index and water quality the average si value for all sampling sites was 1.81 which indicates a 2nd quality class or β-mesosaprobic level with moderate organic loading. that was also the most common quality class in our survey. samples from the sites close to the source were classifi ed into 1st or 2nd class which is the oligoto β-mesosaprobic class (tab. 4). infl uence of environmental and spatial factors on diatom species composition the greatest share of variance of the diatom community was explained by water conductivity (30.3%), water temperature (14.2%) and altitude (12.3%). variance partitioning revealed (tab. 5) that the highest share of diatom species composition variation is infl uenced by environmental factors (25%), namely water temperature and conductivity, followed by altitude (8.5%), which is the only spatial factor with signifi cant infl uence. water conductivity and altitude are gradients that correlate with the 1st axis of the cca (fig. 2a, tab. 6), while water temperature correlates with the 2nd axis of the cca. tab. 5. results of forward selection calculated with canonical correspondence analysis (cca), 999 permutations. signifi cant environmental variables (p < 0.05) and the percentage of the total variance of species data explained (%tve) – gross effect of the variable and percentage as a result of variance partitioning when pccas were performed – net effect of the variable. variable gross effect net effect p % tve p % tve conductivity environmental 0.001 30.3 0.013 11.4 temperature environmental 0.004 14.2 0.006 14.2 altitude spatial 0.006 12.3 0.040 8.5 tab. 6. the results of canonical correspondence analysis, showing the eigenvalues and percentage of the explained variance of diatom assemblages for the fi rst two axes and their correlations with signifi cant environmental variables. total inertia was 1.055 and sum of all canonical eigenvalues was 0.596. axis 1 axis 2 eigenvalue 0.325 0.161 explained variance of species-environment relation (%) 54.5 27 correlation with variables: conductivity –0.9156 –0.1322 temperature –0.3359 –0.7993 altitude 0.8918 0.2143 diatoms presented on the left side of fig. 2b are found in the downstream sites k4 and k5 (fig. 2a) and prefer water with high content of electrolytes (conductivity), while diatoms in the sites closer to the source, which are found on the right side, prefer water with lower conductivity. toman m. j., grošelj a. m., zelnik i. 456 acta bot. croat. 73 (2), 2014 correlation between environmental factors and diatom community diversity correlations between environmental, spatial (altitude, distance from source, channel width) and temporal (seasons) factors were calculated. a correlation of very high signifi cance was calculated between altitude and distance from source. conductivity was signifi cantly correlated with concentrations of nitrate, srp and distance from source. temperature was negatively correlated with altitude and content of o2, but positively with distance from source and with relative light intensity. diatom species richness was positively correlated (p < 0.05) with the saprobic index calculated on the basis of diatoms and other algae, with the cover value of organic substrate (fi lamentous algae and xylal) and quality class (tab. 7). on the other hand, diversity of diatoms was negatively correlated (p < 0.05) with the share of macrolithal, average water depth and light intensity. the most signifi cant correlations are presented in figs. 3a–e. fig. 2. canonical correspondence analysis ordination diagrams of diatom assemblages from various seasons and sites of the kamniška bistrica river. only signifi cant variables are included. (a) distribution of the samples along the environmental variables, where sites are represented with numbers (1–5) and seasons/months with letters (m – march, j – july, a – august); (b) distribution of diatom species present in at least three samples are shown: ach bia – achnanthes biasolettiana, ach min – achnanthes minutissima, amp ped – amphora pediculus, coc ped – cocconeis pediculus, coc pla – cocconeis placentula, cym aff – cymbella affi nis, cym min – cymbella minuta, cym sil – cymbella silesiaca, cym sin – cymbella sinuata, dia mon – diatoma moniliformis, dia vul – diatoma vulgaris, fra arc – fragilaria arcus, fra cap – fragilaria capucina, fra con – fragilaria construens, fra uln – fragilaria ulna, gom ang – gomphonema angustatum, gom oli – gomphonema olivaceum, gom par – gomphonema parvulum, gom pum – gomphonema pumilum, mel var – melosira varians, mer cir – meridion circulare, nav ato – navicula atomus, nav crt – navicula cryptotenella, nav gre – navicula gregaria, nav lan – navicula lanceolata, nav men – navicula menisculus, nav tri – navicula tripunctata, nit dis – nitzschia dissipata, nit fon – nitzschia fonticola, nit pal – nitzschia palea. distribution of epilithic diatoms in a torrential river acta bot. croat. 73 (2), 2014 457 fig. 3. statistically signifi cant correlations between the number (n) of diatom taxa in the sample and water depth (a), % of macrolithal in the substrate (b), % of cover with fi lamentous algae (c), saprobic index (si) calculated on the base of diatoms (d) and si calculated on the base of all algae (e). tab. 7. summary of regression analyses between diatom species richness (nr. of taxa) and shannonwiener diversity index (h') and some of the environmental parametres; ** p < 0.01, * p < 0.05. parameter h’ nr. of taxa water quality class 0.508 0.596* saprobic index (all algae) 0.217 0.788** saprobic index (diatoms) 0.307 0.627* algae 0.437 0.800** xylal 0.127 0.565* macrolithal –0.2780 –0.662*** water depth –0.2130 –0.763*** light intensity –0.2430 –0.588*0 toman m. j., grošelj a. m., zelnik i. 458 acta bot. croat. 73 (2), 2014 discussion in the temperate zone, diatoms are the most abundant algae in the periphyton community, especially in streams with a stony substrate, with the highest share in spring and autumn periods. in the samples from the investigated torrential river, diatoms represented from 28 to 92% of the primary producers (tab. 4). the highest number of the species was found in august – when low abundances of chlorophyta and cyanobacteria were detected. the similarity of diatom assemblages (fig. 1) refl ected the locations / spatial factors. the left group uniting the upper three locations shows a higher sample similarity than downstream locations. a lower rate of similarity is a consequence of greater variation in natural factors such as water temperature, as well as the variety of human impacts on the river ecosystem. about a third of taxa were common to all samples (see tab. 4). for instance, species achnanthes minutissima, a. biasolettiana, gomphonema pumilum, g. olivaceum and cocconeis placentula were most common and present in all samples; moreover, they often reached high shares in the epilithic community (tab. 4). these taxa have the ability to attach fi rmly to substrata, which increases the temporal occurrence of these diatoms (virtanen et al. 2011). authors from different parts of the world report that a. minutissima is one of the most frequent taxa in epilithic diatom communities in running waters (hoffman et al. 2011, soltanpour-gargari et al. 2011, virtanen et al. 2011), mainly because of the wide ecological amplitude, making it a pioneer taxon. according to hoffman et al. (2011) achnanthes biasolettiana and gomphonema pumilum prefer ca-rich, oligotrophic to mesotrophic running waters on limestone bedrock of the alpine and pre-alpine regions where they often reach high abundance. this is also the case in our research area and in accordance with our results (tab. 4). besides, hoffman et al. (2011) and virtanen et al. (2011) emphasize that a. minutissima and g. pumilum inhabit periphyton communities as a complex of different species (hoffman et al. 2011) so the same species is not necessarily present in all samples. these facts explain their common occurrence and relatively wide ecological niches. the most abundant winter diatoms with relative abundance ≥ 5% in the winter period, beside the common taxa (achnanthes biasolettiana, a. minutissima, gomphonema pumilum) were: navicula lanceolata, diatoma vulgaris, gomphonema olivaceum, nitzschia fonticola, and nitzschia dissipata. the taxa achnanthes biasolettiana and a. minutissima were also the most abundant in spring and summer. their ability to fi rmly attach to the substratum in changeable water fl ows (virtanen et al. 2011) as well as their quick recolonization could be the reasons for their constant presence in the periphyton community. some other species that attach to substrata, such as cocconeis placentula and gomphonema pumilum also exceeded the share of 5% in that period. the mentioned situation in august was similar in the fi rst three sites, while in the downstream section the most common species were a. minutissima, cocconeis placentula, navicula atomus and nitzschia fonticola. the most diverse genus was navicula with 14 species found, which was in accordance with fi ndings in soltanpour-gargari et al. (2011) who reported navicula and nitzschia to be the most diverse genera. samples from the upper three sites (source region) showed low values (1.35–2.5) of the s-w index as a result of lower species richness and species dominance (tab. 4). low concentrations of nitrates, srp and total dissolved solids in the water, as well as low temperadistribution of epilithic diatoms in a torrential river acta bot. croat. 73 (2), 2014 459 tures and unstable substrate could be the main ecological factors for very low productivity, which causes low species richness and/or diversity. a relatively low value of the diversity index (1.20) was calculated from summer samples at the downstream site (k5) due to the dominance of the species navicula atomus (82%). this species occurrs frequently in warmer periods and is very tolerant of organic loading (hürlimann and schanz 1993, goma 2005). the facts that in august temperature reached the highest values and site k5 lies downstream of the water treatment plant, the farming and urban area might explain the dominance of the mentioned species, which reduced the diversity of diatom community. the greatest diversity index value (3.74) was calculated for the summer downstream sample k4, where nutrient concentrations were high. this sample was the most species-rich and had the highest number of taxa (6) exceeding a relative abundance of 5%, i.e., achnanthes minutissima, cocconeis placentula and a. biasolettiana, together comprising about 50% of the diatom assemblage. however, no signifi cant correlations between shannonwiener diversity index and any of the included variables was calculated. water quality was evaluated using the saprobic index. most of the sites belong to oligosaprobic and oligo-β-mesosaprobic states, characteristic of moderate organic loading. the most abundant taxa at sites k2, k1 and k3 (such as achnanthes minutissima and a. biasolettiana) were characteristic of a β-mesosaprobic state, while gomphonema pumilum is characteristic of an oligosaprobic state (hoffman et al. 2011). samples from the sites close to the source were classifi ed into 1st or 2nd class which is the oligoto β-mesosaprobic class (tab. 4) and indicates low organic loading. infl uences of environmental and spatial factors on the diatom community were tested using cca. the signifi cant variables explained almost 57% of diatom species composition (tab. 5), which is similar to results published by passy (2007), where the share reached 60%. on the other hand, there is a great difference in results of variance partitioning, since the share of variance explained with pure environmental factors in our case (25.6%) was almost three times higher than calculated by passy (2007) (9%), while in case of the spatial factors our result was about twice as low. a possible reason for mentioned differences could be the size of research area, since the river we studied was four times as short as the river studied by passy (2007); soininen (2007) reports that the relative importance of environmental and spatial factors varies with study scale. moreover, soininen (2007) claims that distance effects are negligible over small scales, while at broader scales they may overwhelm the importance of local environmental factors. in our study, the highest share of variance of the epilithic diatom community was explained by water conductivity and temperature (environmental factors) and altitude (topographic factor). their pure (net) effects on diatom species composition were slightly different (tab. 5), as water temperature explained the highest share of the variance. temperature, conductivity and altitude were also found to be major determinants of diatom communities by soininen (2007), temperature was emphasised by izagirre and elosegi (2005), altitude by beltrami et al. (2012) and conductivity by virtanen and soininen (2012). the share of total variance explained with pure selected factors dropped to 34%, most probably due to the high correlation of conductivity with altitude. the spatial variation in communities may also refl ect the effects of unmeasured environmental variables (virtanen and soininen 2012). considering the net effects of variables the highest share of variance is explained by temperature, which highly correlates with relative light intensity, while no signifi cance was calculated with seasons. toman m. j., grošelj a. m., zelnik i. 460 acta bot. croat. 73 (2), 2014 diatoms distributed on the left side of the diagram (fig. 2b) are found in the downstream sites k4 and k5 (fig. 2a) and prefer water with high content of electrolytes (conductivity). species like navicula cryptotenella, n. gregaria, n. veneta, nitzschia palea, surirella angusta have been defi ned as indicators for eutrophic and polluted streams and found in streams with high conductivity and phosphorous and nitrogen content (virtanen and soininen 2012). exogenous factors that infl uence the periphytic community often have synergistic effects making the infl uence of a single variable on the species composition, diversity and other community characteristics hard to defi ne. there is also the question of size of the studied area (soininen 2007), which determines the order of specifi c factors. furthermore, there is also the infl uence of biotic interactions (grazing, competition) which are exceedingly diffi cult to quantify. the mentioned facts could be possible reasons for the differing conclusions on the importance of various factors in structuring epilithic diatom communities (soininen 2007, lange et al. 2011, beltrami et al. 2012). a highly signifi cant correlation was calculated between altitude and distance from source indicating that these spatial variables are very closely related. conductivity is signifi cantly correlated with concentrations of nitrate, srp and distance from source, which also represent increased human infl uence (tab. 1). on the other hand conductivity also correlates with cover of fi lamentous algae and periphyton biomass as dependant elements, indicating that conductivity could suitably refl ect trophic conditions, which is very simple to measure. as expected, temperature was negatively correlated with altitude and content of o2, but positively with distance from source. the highest correlation was calculated with relative light intensity, which is also an important factor determining the structure and function of the periphyton community (lange et al. 2011). species diversity is determined by the diversity of microhabitats and effects like amount of nutrients, water temperature, fl ow regime and stability of the ecosystem, which depends mainly on hydrological disturbances and pollution (moss 2010). velghe et al. (2012) calculated negative correlation between diatom species richness and amount of phosphorous. in our case diatom species richness was unexpectedly positively correlated (p < 0.05) with the saprobic index calculated on the basis of diatoms / all algae, with water quality class and with the cover value of fi lamentous algae (tab. 7, fig. 3). these fi ndings indicate low content of organic matter and nutrients, too low even to enable the thriving of species-rich epilithic diatom community, which needs higher amounts of nutrients and/or organic matter to support high number of species. conclusion our results revealed considerable changes in diatom species composition along the river, which much outweighed the changes during the year. other researchers observed minor changes in diatom community composition between seasons. the highest share of variance of the periphytic diatom community was explained by environmental factors – by water temperature and conductivity. considering that temperature mostly depends on climatic conditions, the conductivity is greatly infl uenced by human impact, for instance it strongly correlates with nitrates and srp. besides, it also strongly correlated with periphyton biomass and cover of fi lamentous algae, so it seems to be a good indicator of nutrient loading. distribution of epilithic diatoms in a torrential river acta bot. croat. 73 (2), 2014 461 water framework directive as the offi cial monitoring system in the european union is more focused on the evaluation of ecosystem state, rather than water quality state. diatom communities are good indicators of an ecological state and should be used further in monitoring. acknowledgements we would like to thank gorazd kosi, phd. for his help with diatom determination. references almeida, s. f. p., feio, m. j., 2012: diatmod: diatom predictive model for quality assessment of portuguese running waters. hydrobiologia 695, 185–197. apha (american public health association), 1998: standard 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virtanen, l., soininen, j., 2012: the roles of environment and space in shaping stream diatom communities. european journal of phycology 47, 160–168. xu, c., xiangdong, y., xuhui, d., enfeng, l., 2012: infl uence of environmental and spatial factors on the distribution of surface sediment diatoms in chaohu lake, southeast china. acta botanica croatica 71, 1–12. opce-str.vp acta bot. croat. 73 (1), 107–129, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 early spring ephemeral therophytic non-nitrophilous grasslands as a habitat of various species of romulea in the southern balkans andra@ ^arni1*, vlado matevski2, urban [ilc1, renata ]u[terevska2 1 institute of biology, scientific research center of the slovenian academy of sciences and arts, novi trg 2, si 1000 ljubljana, slovenia 2 institute of biology, faculty of natural sciences of st. cyril and methodius university in skopje, gazi baba b/b, mk 1000 skopje, republic of macedonia abstract – the work deals with habitats of romulea bulbocodium and romulea linaresii ssp. graeca in the southern balkans. both species appear in early spring ephemeral therophytic non-nitrophilous grasslands in regions under the influence of the mediterranean climate. these communities are classified within the romulion alliance, which encompasses such communities from the eastern mediterranean area. it was established that the main climatic factor causing the diversity of these communities is seasonality in precipitation and temperature. two associations are presented, as lagopo-poetum bulbosae and romuleo graecae-poetum bulbosae. key words: balkans, climate, grassland, nomenclature, romulea, vegetation abbreviations: esetg – early spring ephemeral therophytic non-nitrophilous grasslands, icpn – international code of phytosociological nomenclature introduction in the early spring, from the end of february to the end of march, carpets of flowering plants belonging to the genus romulea appear in areas under the influence of the mediterranean climate (fig. 1). later, at the end of april and beginning of may, when these species-rich communities are optimally developed for sampling, romulea species are already in fruit and can no longer be identified. in summer, early spring ephemeral therophytic non-nitrophilous grasslands (esetg) dry out due to the hot and dry mediterranean climate. only a few drought-resistant species can then be found, e.g., achnatherum bromoides, due to c4 assimilation syndrome (pyankov et al. 2010), and only green-grey remains of the colorful spring carpet. acta bot. croat. 73 (1), 2014 107 * corresponding author, e-mail: carni@zrc-sazu.si copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:14 color profile: generic cmyk printer profile composite default screen the genus romulea comprises about 95 taxa, of which 80 occur in south africa and the arabian peninsula (manning and goldblatt 2008), while the others can be found in the mediterranean basin (peruzzi et al. 2011). our study deals with the habitats of two species: romulea bulbocodium, which is widespread in the mediterranean basin, and romulea linaresii ssp. graeca, an endemic species of the balkan peninsula (frignani and iiriti 2011) (fig. 2). two other subspecies of r. linaresii, subsp. linaresii and abyssinica, are endemic to sicily and ethiopia, respectively (pignatti 1982). romulea bulbocodium is a quite tall plant up to 15 cm and has 1–6 flowers that are lilac or violet, often greenish outside with a yellow throat and tube (fig. 2a). it has a wider distribution in the balkans than r. linaresii, being found along the adriatic coast, in macedonia, bulgaria, greece and turkey (nikoli] 2000, ste[evi] 2002, gussev 2011, natcheva and ivanova 2011). 108 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. fig. 1. view of esetg in the end of march. romulea in full flower. at the time of sampling in the beginning of may up to 100 plant species can be found in a single plot. fig. 2. romulea bulbocodium (a) is found in the interior part of the region, whereas romulea linaresii ssp. graeca (b) appears in the coastal part. a b 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:19 color profile: generic cmyk printer profile composite default screen romulea linaresii ssp. graeca is smaller than r. bulbocodium. it can be up to 5 cm tall with 1–2 dark violet-purple flowers (fig. 2b) and is distributed only in the southern part of the balkans and turkey (e.g. ste[evi] 2002, özdemir et al. 2007, petrova and vladimirov 2009, bergmeier et al. 2011, gussev 2011, rakaj 2011). there is lively interest in the taxonomical peculiarities and distribution of romulea species in the balkans but there have been only a few investigations dealing with the habitats of these species: esetg (oberdorfer 1954, bolòs et al. 1996). sampling of these communities is rather difficult due to high biological and phenological diversity. grasslands are species-rich communities established over centuries of permanent grazing (kaligari^ et al. 2006, catorci et al. 2012). as well as by grazing, grasslands in the mediterranean area are also maintained by periodic fires (kavgaci et al. 2010, türkmen and dünzenli 2011). in recent years, due to land use change (bracchetti et al. 2012), grasslands have become among the most threatened habitats (jani[ová et al. 2011, vassilev et al. 2011), since the communities often become overgrown (barbero and quezel 1976, ^arni et al. 2010). oberdorfer (1954) sampled such habitats in 1944 in the areas around thessaloniki, southern macedonia, thrace, thessaly, attica and close to corinth. he designated these communities that are mainly dominated by poa bulbosa ssp. div. and which can be found on fresh, fine argillous soils around intensively grazed places. he described the alliance romulion within this framework. he also stated that such vegetation is rarely found on limestone since cisto-micromerietea vegetation mainly appears there. bolòs et al. (1996) later also elaborated such habitats on cephalonia (ionian islands, greece). in addition to esetg found in areas under the influence of the mediterranean climate, other types of therophytic grasslands exist in more continental parts of the balkans (e.g., in bulgaria and the republic of macedonia). these communities are classified within trifolion cherleri. the classification of the latter alliance into higher rank syntaxa is still under consideration, since they mediate between the group of (sub-) mediterranean therophytic grasslands (helianthemetea) and more continentally influenced perennial grasslands (festuco-brometea) (micevski 1970, tzonev et al. 2009). there are even discrepancies at the association level, for instance within erysimo-trifolietum (micevski 1977, sopotlieva and apostolova 2007). there is a well recognizable climatic gradient in the southern balkans: mediterranean – sub-mediterranean – continental, which provides the opportunity to study in detail the turn-over of plant species and communities under changing climatic conditions (]u[terevska et al. 2012). rodwell et al. (2002) defined the alliance romulion as eastern mediterranean ephemeral vegetation on humid salty soils. they place this alliance within saginetea maritimae, a class of ephemeral vegetation with winter annuals on bare or disturbed salt-marsh mud and sand. the aim of the study was to sample esetg in the southern balkans and define the habitats of romulea species in the region. we have tried to present in the paper the floristic composition of communities and to analyze their structural and choraological spectrum. we have determined the most important climatic factor for diversity within communities and correlated it with their geographical distribution. we have attempted to discover the ecological and syntaxonomic position of esetg and have performed a nomenclatural revision of these syntaxa. acta bot. croat. 73 (1), 2014 109 early spring therophytic grasslands 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:20 color profile: generic cmyk printer profile composite default screen material and methods the research area was the southern part of the balkans (fig. 3). according to the map of natural vegetation of europe (bohn et al. 2003), the northern part of the area in which romulea species appear lies in the zone of thermophilous deciduous broadleaved forests (carpinus orientalis-quercus pubescens zone), whereas the southern part along the aegean coast lies in the zone of mediterranean evergreen forests dominated by quercus ilex. the northern limit of growth of romulea species corresponds to the northern boundary of thermophilous deciduous broadleaved forests as defined by horvat et al. (1974) and the distribution of quercus coccifera scrub or forest (oberdorfer 1947). the climatological station in valandovo (southern part of the republic of macedonia) reports that there is 611 mm mean annual precipitation and the mean annual temperature is 14.6 °c; in thessaloniki, on the coast, there is 458 mm of mean annual precipitation and a mean annual temperature of 15.8 °c. in both stations, the highest peak of precipitation is in november with a less pronounced peak in may in valandovo and in march and may in thessaloniki (matevski and ^arni 2003). 110 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. fig. 3. distribution of communities: communities with romulea bulbocodium are distributed in the inland part and represented by full circles; communities with romulea linaresii ssp. graeca are distributed in coastal areas and represented by empty circles. in the map two layers generated through interpolation of climate data stored in worldclim database are presented. the numbers in bio 4 present the coefficient of variation as standard deviation of the weekly mean temperatures expressed as a percentage of the annual mean, in this case multiplied by 100, whereas bio 15 also presents the coefficient of variation as previously, although in this case precipitation is multiplied by 10 (hijmans et al. 2005). 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:21 color profile: generic cmyk printer profile composite default screen sampling was performed according to the standard braun-blanquet method (braunblanquet 1964). the layer is not indicated, because all species appear in the herb layer. since it is practically impossible to determine romulea species when they are fruiting, we visited the sites three times. in early spring, we surveyed the whole area, identified potential plots for sampling communities and noted geophytes, such as romulea, gagea, ornithogalum etc. at the end of april and beginning of may, we performed field sampling of vegetation. at this time, we made an inventory of plants and estimated coverage. we visited plots once more in summer, in order to find plants, above all grasses, which had not been in flower at sampling time. these species mainly possess a c4 photosynthesis pathway and do well under high light intensity and high temperatures. they include achnatherum, bothriochloa, chysopogon, and also the non-grass portulaca. after elaboration of the plant material, we constructed a table (tab. 1), which was subjected to numerical analysis. classification was then carried out by pc-ord (mccune and mefford 2011), run in the juice 7.0 programme (tichý 2002). a dendrogram was drawn using ward’s method and euclidean distance as a measure of resemblance (fig. 4). the characteristic species of individual groups were determined by calculating the species’ fidelity and they are presented in the analytic table (tab. 1). the phi coefficient was used as a fidelity measure and calculated in the juice program. the threshold phi value (multiplied by 100 in the juice program) for species to be considered characteristic was set at 40 (chytrý et al. 2002). all the climatic data available in the worldclim database (hijmans et al. 2005, available at http://worldclim.org/) were extracted for each sample plot. before elaboration, we firstly performed detrended correspondence analysis (dca) of data and discovered that the gradient is less than 3 sd, indicating linearity, and we therefore used prinicipal component analysis (pca) and redundancy analysis (rda) in further analysis. since climatic data were provided from an external source and not from a species inventory of esetg (such as life forms and chorotypes), rda was used to test the possible effect of climatic variables on vegetation composition. each variable was entered separately, each in turn, for analysis and acta bot. croat. 73 (1), 2014 111 early spring therophytic grasslands fig. 4. dendrogram of samples (relevés). two main clusters can be seen, inland communities with romulea bulbocodium are indicated by number 1–12, whereas the coastal communities with romulea linaresii ssp. graeca are indicated by number 13–23. 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:22 color profile: generic cmyk printer profile composite default screen 112 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. tab. 1. analytical table of the early spring ephemeral therophytic non-nitrophilous grasslands in the southern balkan. 1 1 1 1 1 1 1 1 1 1 2 2 2 2 relevé number 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 lagopo-poetum bulbosae romulea bulbocodium 3 3 3 3 3 3 2 2 + 3 4 3 . . . . 2 . 1 1 . . . myosotis ramosissima + + + + + + . . + . . . . . + . . . . . . . . teucrium capitatum . . . . . + + + + + + + . . . . . . . + . . . alyssum desertorum . . . + + . + + + . + . . . . . . . . . . . . achillea coarctata + . . + + + . . + . . . . . . . . . . . . . . carex caryophyllea . + . + + + . . . . + . . . . . . . . . . . . romuleo graecae-poetum bulbosae romulea linaresii ssp. graeca . + + . . . . . . . . . 1 2 1 1 . 1 1 + 1 1 1 galium murale . . . . . . . . + . . + + + + + + + . . . + + hordeum murinum ssp. leporinum . . . . . . . . . + . . + + + + . + . + + + . lagurus ovatus . . . . . . . . . . . . + . 1 . . . . . 1 1 + crepis zacintha . . . . . . . . . . . . . . 1 . . + . + . + + urospermum picroides . . . . . . . . . . . . . . + . . + . . + + + romulion hedypnois rhagadioloides . . + . . . . . + + + + + + + + + 1 + + + + + hypochoeris cretensis . . 1 + + + + + + . . . + + + . + . + + . . + allium guttatum . . . . + + . . . . + + . . . + . + + + + + . linaria simplex . . . . . . . + + . . . + + . . . . + . . . + ornithogalum collinum . . . . . + . . . . . . . 1 . . . . 2 2 . + + lotus angustissimus . . + + + . . . . . . . . . . + . . . . . . . helathemetalia,heliathemetea poa bulbosa 3 2 3 2 3 2 4 4 4 3 3 2 4 3 3 3 3 4 3 2 2 3 2 psilurus incurvus . + + + + 1 + + . + 2 + + 2 + + + + + + + + + vulpia ciliata . + 1 + + + + + + . . + + . 1 1 + + 1 + 3 1 + tuberaria guttata . 1 2 + 3 2 + . . . . + 1 + 2 2 2 1 1 + 2 1 2 trifolium campestre + . 1 2 2 + . . . + . + + + 1 . + 2 1 + + + 1 ornithopus compressus + . 1 1 + . + + . . . + 1 + + 2 + + + + . + + trifolium angustifolium + . + + + + . . + 1 . 2 . + + + . 1 + + + 1 + hypochaeris glabra . 1 + + + + + + . . . + + . . + + . + + + + + trifolium arvense . . + 1 1 + + + . + . 1 . . . + + + + + + 1 + plantago bellardii . . . 1 + 2 + . . . + 2 . 1 + 2 1 + + 1 1 2 1 linaria pelisseriana . . + + + + + . . . . . . + + + 1 + + + + + + trifolium subterraneum 3 2 2 + + . + . . . . . 2 + + 2 + . . + . 1 + trifolium cherleri . . 1 . 1 2 + . . . . 1 . . + + + 3 2 4 + + 2 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:22 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 113 early spring therophytic grasslands 1 1 1 1 1 1 1 1 1 1 2 2 2 2 relevé number 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 aira elegantissima . . 2 1 + + . . . . . . + . 1 + + + + . + 1 + asterolinon linum-stellatum . . 1 . . . . + + + + + + . + . . 1 . + + + + galium divaricatum . + + + + + . . . . + . . . . + . + + + + . . medicago minima . . . . 1 + 2 2 1 + . + 1 + . . . + . 1 . . . arenaria leptoclados + . . . . + + + + + 1 + . + + . . . . . . . . filago gallica . + + . . . . . . . + + . . . . . + + + + + + trifolium stellatum + . . . . . . 1 + . . . + + 2 . . . . . + 1 + briza maxima . . . . . . . + . . . + . . + + . + + . 1 1 + tolpis umbellatum . . . . + + . . . . . . . . + . + + . + + + . helianthemum salicifolium . . . . . + + 2 2 + + . . . . . . . . 1 . . . velezia rigida . . . . . . . + + . + + + + + . . . . . . . . taeniatherum caput-medusae . . . . . . . . . + + . . . + . . + + + . . + sedum caespitosum + . . . . . . + + . . . + + . . + . . . . . . trachynia distachya . . . . . 2 2 1 . + 1 . . . . . . + . . . . . hymenocarpos circinnatus . . . . . . . . 2 . . 2 1 + + . + . . . . . . viola kitaibeliana + + . . . . . + . . . . + + . . . . . . . . . arenaria serpyllifolia . + . + + . . . . . . . . . . . . . + + . . . vulpia myuros . + . . . . . . . . . . . . . . . + . . + + + rostraria cristata . . . . + . . + . + . . + . + . . . . . . . . crucianella latifolia . . . . . . + + + . . . . . . . + . . . . . + alkanna tinctoria . . . . . . + + . . . . + + + . . . . . . . . medicago disciformis var. disciformis . . . . . . . . 1 + . + + + . . . . . . . . . helianthemum aegyptiacum + . . . . . 1 . . . . . . . . . . . + + . . . petrorhagia prolifera . + . . + + + . . . . . . . . . . . . . . . . medicago disciformis var. strumensis . . . . . . + 2 . . . . . . + . . + . . . . . clypeola jonthlaspi . . . . . . + + + . . . + . . . . . . . . . . dasypyrum villosum . . . . . . . . + + . + . . . . . . + . . . . festuco-brometea trifolium scabrum + + + + + + + + + 1 1 + + + 1 + . 1 + 1 + + + eryngium campestre + + . + . + . + + . + . + . . . + . 1 + . . . aegilops triuncialis . . + . . . + . + + + 1 . . . . + 1 . + + . + chrysopogon gryllus . . . + . 1 + + + + + . . . . . + . + + . . + medicago rigidula . . . . + + + + 1 + + + + + . . . . . + . . . thymus substristus + + + + + . + + + . . . . + . . . . + . . . . scleranthus verticillatus + + . + + + + + . . + . . . . . + . . + . . . tab. 1. – continued 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:23 color profile: generic cmyk printer profile composite default screen 114 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. 1 1 1 1 1 1 1 1 1 1 2 2 2 2 relevé number 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 chondrilla juncea . + + . + + . . . . . . . . . . . + + + + . . potentilla recta ssp. laciniosa . . + + + + . . . + . + . . . . . + + + . . . bromus squarrosus . . . . + + + + . + . + . . . . . + . . + . . arabidopsis thaliana . + + . . + . + . + . . . . . . . + + . . . . centaurea grisebachii . . + + + + + . . . . . . . . . . + . . . . . hypericum perforatum . . + . . . + + + . . . . . . . . + + . . . . koeleria splendens . . . . + . + + + + . . . . . . . . + . . . . achillea setacea + + + . + . . . . . . . . . . . . . . + . . . sanguisorba minor . + + . + + . . . . . . . . . . . . . . . . . convolvulus cantabrica . . . + . + + + . . . . . . . . . . . . . . . astragalus onobrychis . . . + . + . . . . . . . . . . . . + 1 . . . thymus striatus . . . . . + . . . . + . . . . . . . 1 + . . . allium sphaerocephalon . . . . . . + + . + . . . . . . . . . + . . . other species cynodon dactylon + + + + 1 1 . . + . + 1 + + + + 1 . + + + + + parentucellia latifolia + 1 1 1 + 1 + + + . + . + + . 2 + . + + . + 1 filago germanica + + + + + + + . . + + + + + + + + + + + . . . anthemis auriculata + + + . . . 1 1 + 1 . + 1 + 1 1 + . 1 + 1 + 1 petrorhagia velutina . + + . + + + + . . . + 1 + + + + 1 + + 1 + 1 sherardia arvensis + + 1 . + + . . 1 + + + + . + + . 1 . + + + + plantago lagopus + + + . + . . . 2 3 + 3 1 3 + . 1 . + 1 2 + 3 erodium cicutarium + + . . + + + . + + + + 1 + + + 2 . + + . . + achnatherum bromoides . . + . + . + + 1 + . | . . + . + + + + . ++ avena barbata . . + . + . . + + + + + . . . . + + + + + + + veronica arvensis . + + + + . . . + . . . + . + + 1 + . . . + + cistus incanus ssp. creticus . + + . . . . . + + . + + + . + + + . . + . + trifolium retusum . . + + + . . . . . . + . . + 1 . 1 + + + + + valerianella turgida . + + . + + . . + . + + . . . . + + . + . . + senecio vulgaris . . + . . . + + + . . . + + . + + . + + . . + trifolium nigrescens + . + + + + . . . . . . + . . + . . + + + . . trifolium tenuifolium + . + + + . + . . . . + . . + + . . + . . 1 . carthamus lanatus + + . . + + + + . + . . . . . . . . + + . + . dactylis glomerata + . + . + . . . + . . + . . . . . + + . + + + vicia lathyroides + . . + + . + + . . . + + + . . . . . . + . + anagallis arvensis . . + . + . + + + + + + . . + . . + . . . . . bothriochloa ischaemum . . . . + . + + . + + + . . . + + . + + . . . polycarpon tetraphyllum 1 . + . . . . . + . . + 1 + . + + . . . . . 1 tab. 1. – continued 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:23 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 115 early spring therophytic grasslands 1 1 1 1 1 1 1 1 1 1 2 2 2 2 relevé number 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 plantago lanceolata + 1 + . + + . . . + . . . . . . . . . + + + . plantago coronopus + + . . . . . . . . . . + . . + + + . . + + + cerastium semidecandrum + . . . . . + 1 + . + . 1 + . + . . . . . + . moenchia erecta . + + + + . . . . . . . . . + . + . + + . . + bromus hordeaceus . + . . 1 + . . . + . + . . . . . + 1 + + . . aegilops geniculata . . . . + + . + . 1 . . . . + . . + + 1 . . + onobrychis caput-galli . . . . . + 2 + . . + 1 + + + . . . 1 . . . . asparagus acutifolius . . . . . . + + . + + . . . . + . + . + . + + verbascum sinuatum + . + . . . + + + . . . + . . + . . . . + . . veronica verna + . . + . + . + + . . + + + . . . . . . . . . cerastium glomeratum . + + . . . . . . . . 1 . . + + . + . + . . + portulaca oleracea . + . . . . . . . + . . . . . + . + + . + + + eragrostis minor . . + . + . . . . . . + . . . . . + + . + + + cynosurus echinatus . . + . + . . . . . . + . . . . . + + . + + + crupina crupinastrum . . + . . . + + . + + + . . + . . . + . . . . catapodium rigidum . . + . . . . . + + 1 + + . + . . + . . . . . silene gallica . . + . . . . . . + . + . . + + . + . . + . + crepis neglecta . . . . + . + . . . . . + . . . + . . + + + + linum corymbulosum . . . . . + + . + + + . . . + . . + . + . . . aphanes microcarpa + 1 . + + . . . . . . . . . . . . + . + . . + rumex acetosella + + + + + + . . . . . . . . . + . . . . . . . capsella bursa-pastoris . + . . + . . . . . . . + + . . + . + + . . . crepis foetida ssp. rhoedifolia . . + . . + + . . . . + . . + . . . . . + + . valerianella coronata . . + . . . . . . + . . + + + . . + . + . . . echium plantagineum . . + . . . . . . . . . . . . . + + + . + + + daucus guttatus . . . . + + . . . . . . . . + + + . . . + + . astragalus pelecinus ssp. pelecinus . . . . + . + 2 . . . . + + . . . . + . . . + centaurea benedicta . . . . . . . . . + . . + + . + + . . + . + . holosteum umbellatum + . . . . . + + + . + . . . . . + . . . . . . herniaria incana + . . . . + . . . + . . . . . + . + + . . . . erysimum crassistylum + . . . . . . . + + + . . + . . . . + . . . . lolium perenne . . + . . . . . . + . + . . . . . + . . + + . anisantha tectorum . . . . . . + . . . . . . + . . + . . . + + + crepis sancta . . . . . . . + + + . . . + + + . . . . . . . medicago praecox . . . . . . . . . . + + . + . + . + . + . . . anisantha madritensis . . . . . . . . . . . + . . + + . + . . + + . ranunculus ficaria + . . + + + . . . . . . + . . . . . . . . . . tab. 1. – continued 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:23 color profile: generic cmyk printer profile composite default screen 116 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. 1 1 1 1 1 1 1 1 1 1 2 2 2 2 relevé number 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 euphorbia helioscopia . + . . + . . . + . . . + . . + . . . . . . . minuartia hirsuta ssp. falcata . + . . . + . . . + . . . . . + . . . + . . . crepis setosa . + . . . + . . . . . . . . . . + . 1 + . . . teesdalia coronopifolia . . + + . . . . . + . . . . . . + . . . . . + vicia sativa ssp. nigra . . + . . . . + . . . . . . . + . . . . + + . thymus heterotrichus . . . + . . . . + + . + . . . . . . . . . . + anthoxanthum odoratum . . . . + . . . . . . . . . . . . . + . 2 2 + neatostema apulum . . . . . 1 . . + + + . . + . . . . . . . . . allium flavum . . . . . . . . + + + . . . . . . . . + . . + melica ciliata . . . . . . . . + . . + . . . . . + . + . + . rorippa thracica 1 + . . . . . . . . . . . . + + . . . . . . . geranium molle ssp. brutium + + . . . . . . . . . . . . . + . . . + . . . filago arvensis + . . + + + . . . . . . . . . . . . . . . . . teucrium chamaedrys + . . . + . . . . + . + . . . . . . . . . . . nigella arvensis . . + . + + . . . . + . . . . . . . . . . . . convolvulus elegantissimus . . + . . . . . + . . . 2 + . . . . . . . . . sedum hispanicum . . + . . . . . . . . . . + + . . . . . . . + anthemis ruthenica . . . + + . . . . . + . . . . . . + . . . . . sedum rubens . . . . + + . . . . . . . . . . . . . + . . + colchicum autumnale . . . . . + . . . + . . . . . + . . . + . . . galium setaceum . . . . . . . . + . + . + + . . . . . . . . . leopoldia comosa . . . . . . . . . + . . . . . + . + . + . . . calicotome villosa . . . . . . . . . . . . . . . + . . . . + + + centaurea orphanidea . . . . . . . . . . . . . . . . . + + + . . + other species: agrostis stolonifera 14: +, 13: +; alyssum corymbosoides 18: +; aurinia corymbosa 16: +; alyssum foliosum 5: +; alyssum murale 18: 1, 10: +; alyssum strigosum 19: +, 6: +; alyssum umbellatum 8: +, 7: +; amaranthus albus 14: +, 13: +, 16: +; anemone pavonina 12: +; apera spica-venti 16: +; aphanes arvensis 3: +, 16: +; asperula aristata ssp. scabra 3: +; asphodeline lutea 2: +, 1: 1; asphodelus ramosus ssp. ramosus 2: +, 1: +, 4: +; astragalus spruneri 20: +; bellis perennis 11: +, 14: +; berteroa orbiculata 9: +; anisatha sterilis 19: +, 10: +; buglossoides arvensis 3: +; bunias erucago 6: 1, 7: +; bupleurum commutatum 23: +, 9: +; calendula arvensis 5: +, 6: +, 13: +; campanula phrygia 14: +, 15: +, 16: +; cardamine hirsuta 14: +, 22: +, 19: +; carex divisa 8: 3; carex divulsa ssp. divulsa 14: +, 22: +; carex flacca ssp. serrulata 19: +; centaurea salonitana 2: +, 3: +, 5: +; centaurium erythraea ssp. rumelicum 13: +; centaurium maritimum 16: +; cerastium brachypetalum ssp. tenoreanum 21: +, 22: 1, 23: +; cerastium brachypetalum ssp. roeseri 2: +, 19: +; cerastium comatum 14: +, 12: +; cerastium pumilum 4: 1, 9: +, 7: +; glebionis segetum 16: +; cichorium intybus 8: +, 14: +, 3: +; cistus incanus ssp. incanus 2: +, 1: +; cistus salvifolius 17: +; clinopodium arvensis 2: +, 1: +; clinopodium vulgare 21: +; convolvulus arvensis 14: +; crassula tillaea 6: +, 4: +, 7: +; crupina vulgaris 3: +, 7: +; cuscuta epithymum 20: +; daucus broteri 2: +, 1: +, 7: +; dianthus monadelphus ssp. pallens 3: +; dianthus viscidus 19: +, 9: +; dittrichia viscosa ssp. viscosa 13: +, 17: +; draba muralis 14: +, 22: +, 18: +; echinaria capitata 3: +; echinops sphaerocephalus 2: +, 1: +; ephedra major 14: +; epilobium hirsutum 13: +; erodium hoefftianum 13: +; erophila verna ssp. macrocarpa 5: +; erophila verna ssp. praecox 6: +; erophila verna ssp. verna 8: tab. 1. – continued 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:24 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 117 early spring therophytic grasslands +, 21: +; erysimum diffusum 23: +, 1: +; euphorbia barrelieri ssp. thessala 21: +, 23: +, 9: +; euphorbia chamaesyce 17: +; euphorbia exigua 3: +, 18: +; euphorbia falcata 20: +; euphorbia myrsinites 2: +, 1: +; euphorbia peplus 12: +, 16: +, 7: +; euphorbia seguierana ssp. niciciana 8: +, 23: +; euphorbia taurinensis 2: +, 1: +; festuca callieri 19: +; festuca valesiaca 21: +, 19: +; fumana thymifolia 18: +, 20: +; gagea pusilla 11: +; galium tenuissimum 8: +, 3: +; galium tricornutum 11: +, 21: +; gastridium ventricosum 13: +, 17: +; genista carinalis 21: +; geocaryum cynapioides 16: +; geranium lucidum 14: +, 22: +; geranium pusillum 2: +; geranium robertianum 14: +, 19: +; globularia alypum 18: +, 20: +; gymnadenia conopsea 16: +; hainardia cylindrica 3: +, 20: +; herniaria glabra 21: +; herniaria hirsuta 11: +, 22: +, 4: +; herniaria hirsuta ssp. cinerea 5: +, 6: +; hippocrepis ciliata 3: +, 20: +; holcus annuus ssp. setiglumis 13: +; hyparrhenia hirta 22: +, 9: +; hypericum olympicum 21: +; hypericum rumeliacum 21: +, 23: +, 3: +; inula oculus-christi 23: +; jasione heldreichii 15: +; juncus capitatus 4: +; kickxia elatine 16: +; knautia species 3: +; lagoecia cuminoides 14: +, 12: +; lamium purpureum 8: +, 11: +; lathyrus cicera 2: +; lathyrus sphaericus 19: +; legousia falcata 14: +, 3: +, 15: +; lens nigricans 19: +; leontodon saxatilis 4: 1; limodorum abortivum 16: +; linum nodiflorum 18: +; filago minima 2: +, 4: +; luzula forsteri 14: +, 21: +, 22: +; lychnis coronaria 14: +; matricaria chamomilla 10: +; medicago arabica 8: 1, 11: +; medicago coronata 14: +; medicago falcata 14: +, 19: +; medicago lupulina 1: +, 3: 1, 18: +; medicago orbicularis 18: +, 19: +, 13: +; melilotus neapolitanus 1: +; mibora minima 4: +; micromeria juliana 14: +; microthlaspi perfoliatum 3: +; micropyrum tenellum 8: +, 5: +; minuartia glomerata ssp. macedonica 2: +, 1: +; minuartia hybrida 3: +, 19: +, 4: +; minuartia viscosa 8: +, 21: +; moenchia mantica 14: 2, 19: +, 16: +; myosotis incrassata 4: +, 7: +; myosotis stricta 4: +; neslia panicultata ssp. thracica 2: +, 1: +; oenanthe silaifolia 14: +; onobrychis aequidentata 3: 1; ononis reclinata 20: +; orchis species 13: +; orlaya daucoides 19: +; ornithogalum armeniacum 5: +; paliurus spina-christi 14: +, 22: +; pallenis spinosa 22: +, 20: +; papaver rhoeas 10: +; parvotrisetum myrianthum 22: +, 4: +; petrorhagia illyrica ssp. illyrica 23: +, 2: +; petrorhagia saxifraga 4: +, 9: +; phleum phleoides 10: +; piptatherum coerulescens 18: +; plantago afra 20: +; pleurochaete squarrosa (only species in moose layer) 7: +; poa angustifolia 11: +, 10: +; poa pratensis 8: 1; polygonum aviculare 4: +; potentilla argentea 8: +, 11: +, 14: +; potentilla pedata 8: +, 3: +, 10: +; prunella laciniata 14: +; ranunculus isthmicus 6: +; ranunculus millefoliatus 8: +, 4: +, 7: +; ranunculus muricatus 22: +; ranunculus psilostachys 14: +; ranunculus rumelicus 8: +, 11: +, 5: +; raphanus raphanistrum 7: +; rhagadiolus stellatus 3: +; rorippa lippizensis 22: +; rubia tinctorum 14: +; salvia verbenaca 10: +; scabiosa argentea ssp. ucranica 8: +, 23: +, 10: +; scabiosa sicula 18: +, 19: +; scabiosa triniifolia 22: +; prospero autumnale 2: +, 1: +, 4: +; scolymus hispanicus 4: +; podospermum canum 11: +, 3: +, 10: +; securigera parviflora 12: +; sedum amplexicaule 19: +; sedum annuum 16: +; senecio leucanthemifolius ssp. vernalis 8: +; sideritis montana 18: +; sideritis romana 5: +; silene subconica 1: +; silene cretica 19: +; sisymbrium officinale 11: +, 5: +, 6: +; sonchus arvensis 13: +; spergula arvensis 11: +, 4: +; spergula pentandra 5: +, 6: +, 4: +; stachys angustifolia 19: +; stachys cretica ssp. cassia 23: +; stellaria pallida 14: +, 3: +; stipa capensis 14: +, 20: +; stipa capillata 20: +; taraxacum sect. ruderalia 8: +, 5: +; thymus odoratissimus 23: +, 10: +; thymus sibthorpii 15: +, 4: +; tolpis virgata 7: +; tordylium apulum 12: +; tordylium maximum 3: +; torilis africana 19: +, 13: +; tragopogon balcanicus 18: +; tremastelma palaestinum 3: +, 18: +, 20: 1; tribulus terrestris 4: +, 16: +, 7: +; trifolium globosum 7: 1; trifolium hirtum 19: +, 16: +; trifolium pallidum 8: +, 3: 1; trifolium repens 11: 1; trifolium grandiflorum 19: +, 16: +; trifolium vesiculosum 14: +; medicago monspeliaca 18: +, 20: +, 5: +; valantia muralis 14: +, 12: +; valerianella dentata 2: +, 1: +, 3: +; valerianella discoidea 3: +; verbascum blattaria 21: +, 22: +; verbascum densiflorum 21: +, 22: +; vicia articulata 17: +; vicia barbazitae 8: +; vicia hirsuta 21: +; vicia villosa 10: +, 17: +, 16: +; xeranthemum annuum 23: +, 1: +, 20: +; localites of relevés, details of relevé are indicated in the following order: relevé number, country code, date (year/month/day), relevé area (m2), altitude (m), aspect (degrees), slope (degrees), cover herb layer (%), description of locality, latitude, longitude. 1. gr, 20060428, 50, 340, 270, 1, 100, between vrasna and sohos, 40.71429, 23.62406; 2. gr, 20070513, 30, 426, 45, 1, 100, near to askos, 40.75845, 23.41808; 3. gr, 20070513, 25, 162, 135, 2, 100, above vrasna, 40.70946, 23.64963; 4. mk, 20070516, 30, 514, 225, 3, 95, strumica, novo selo, 41.43231, 22.90296; 5. mk, 20070516, 30, 361, 180, 4, 100, strumica, novo selo, 41.42425, 22.89463; 6. mk, 20070516, 30, 571, 180, 5, 90, radovi{, pla~kovica, 41.65541, 22.47287; 7. mk, 20060424, 30, 247, 180, 8, 80, nikoli~, pasture in q. coccifera zone, 41.26343, 22.73831; 8. mk, 20060424, 35, 265, 180, 15, 90, nikoli~, pasture in q. coccifera zone, 41.26646, 22.73807; 9. gr, 20060426, 30, 225, 135, 5, 100, kokinochori, q. tab. 1. – continued 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:24 color profile: generic cmyk printer profile composite default screen its significance was assessed using the monte carlo permutation test with 999 permutations. the analysis was run with scaling for inter-sample distances to relate the gradient in the floristic composition to explanatory variables (ter braak and [milauer 2002). forward selection of explanatory variables was used to provide a ranking of the importance of specific variables and to avoid co-linearity (ter braak and [milauer 2002). variables with p=0.001 were excluded from further analysis. we also calculated the total variance explained by individual variables and all variables together (lep[ and [milauer 2003) (tab. 2). we estimated only the total explained variance, since it was impossible to make a model because, due to the high correlated climatic variable, the process would cause multi-co-linearity (sullivan et al. 2011, alahuta et al. 2011). we then presented the most explanatory statistical variables in a rda diagram (fig. 5). 118 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. coccifera zone, 40.82193, 24.02571; 10. gr, 20070515, 50, 320, 180, 2, 95, chalkidiki, between vavdos and simantra,, 40.37739, 23.33592; 11. gr, 20070515, 30, 233, 225, 5, 95, thesaloniki, panorama, 40.57318, 23.03191; 12. gr, 20070515, 30, 316, 180, 1, 100, chalkidiki, vasilika between agios antonios and monopigado, 40.43548, 23.11874; 13. gr, 20060407, 30, 390, 180, 2, 90, thasos, above prinos, 40.73500, 24.60870; 14.gr, 20060427, 40, 338, 180, 8, 90, thasos, megalos prinos, 40.73328, 24.60799; 15. gr, 20070512, 30, 11, 180, 2, 90, thasos, aliki, 40.17827, 24.60262; 16. gr, 20070513, 30, 255, 180, 3, 100, chalkidiki, between olimpiada and stratoni, 40.52979, 23.83467; 17. gr, 20060426, 40, 67, 135, 7, 90, kokkinochoma (kavala), 40.92415, 24.29526; 18. gr, 20070513, 30, 200, 225, 2, 90, mirtofito, 40.78824, 24.21881; 19. gr, 20070513, 30, 210, 180, 2, 100, askos, along the road to highway, 40.72011, 23.38634; 20, gr, 20070513, 30, 486, 180, 6, 100, askos, 40.75688, 23.40912; 21. gr, 20070514, 30, 213, 180, 2, 100, chalkidiki, between porto koufo and kalamitsi, 39.97878, 23.96423; 22. gr, 20070514, 30, 52, 90, 2, 90, chalkidiki, skala sikias, 40.02384, 23.99353; 23. gr, 20060428, 30, 41, 180, 4, 100, kavala, nea peramos, 40.85714, 24.31423. tab. 1. – continued tab. 2. results of forward selection: significant environmental variables and percentage of the total variance of species data explained (% tv), calculated with rda. variable p f % tv temperature seasonality 0.001 2.75 11.6 precipitation seasonality 0.001 2.75 11.6 temperature annual range 0.001 2.72 11.5 precipitation of coldest quarter 0.001 2.65 11.2 precipitation of driest month 0.001 2.65 11.2 precipitation of driest quarter 0.001 2.59 11 precipitation of wettest month 0.001 2.55 10.8 min temperature of coldest month 0.001 2.65 10.6 mean diurnal range 0.001 2.44 10.4 precipitation of wettest quarter 0.001 2.54 10.4 precipitation of warmest quarter 0.001 2.32 9.9 mean temperature of coldest quarter 0.001 2.25 9.7 total 60.1 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:24 color profile: generic cmyk printer profile composite default screen we plotted the layers of seasonality of temperature and seasonality of precipitation on a geographical map of distibution of samples (relevés) in the southern balkans (eliá[ et al. 2013) to test the significance of results provided by previous analyses. life forms and the chorological spectra of groups were also determined following raunkiaer 1934, pignatti et al. 2005, micevski (1993–2001), micevski and matevski (2005) and were passively projected onto the pca diagram (fig. 6). acta bot. croat. 73 (1), 2014 119 early spring therophytic grasslands fig. 6. principal component analysis (pca) of samples (relevés) with passive projection of life forms (a) and chorotypes (b). communities with romulea bulbocodium are represented by full circles and communities with romulea linaresii ssp. graeca with empty circles. eigenvalues of axes 1 and 2 are 0.279 and 0.189. a b fig. 5. ordination diagram of sampling plots based on redundancy analysis (rda). only the two most important variables are included: seasonality in precipitation and temperature. communities with romulea bulbocodium are distributed in the interior part and represented by full circles; communities with romulea linaresii ssp. graeca are distributed in coastal areas and represented by empty circles. first and second canonical axes have eigenvalues 0.117 and 0.050. 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:25 color profile: generic cmyk printer profile composite default screen the nomenclature of species follows the euro+med plantbase (euro+med 2006), except amaranthaceae, cistaceae, dipsacaceae, linaceae, polygonacaea, ranunculaceae, rhamnaceae, valerianaceae and berteroa orbiculata following flora europaea and medicago disciformis var. strumensis vel~. et bond., centaurium erythraea ssp. rumelicum (velen.) melderis and pleurochaete squarrosa (brid.) lindb. syntaxonomic nomeclature follows international code of phytosociological nomenclature (icpn) (weber et al. 2000). results esetg appear in areas under the influence of the mediterranean climate (fig. 3). their northern limit corresponds to the distribution area of quercus coccifera. esetg appear in the coastal region of the aegean sea and in the interior along the rivers struma and strumica, where the influence of the mediterranean climate can penetrate into the continent. esetg do not appear in either agricultural areas or forests, only in areas with extensive grazing. they cannot therefore be found in the fertile plains along the strymonas or axios rivers, but appear generally on the edges of these areas, where grazing is still maintained. the dendrogram (fig. 4) shows two well defined groups of communities. the first represents esetg in which romulea bulbocodium appear. these communities thrive in the interior part of the area and are co-dominated by carex divisa, plantago lagopus, poa bulbosa, trifolium subterraneum and tuberaria guttata. romulea linaresii ssp. graeca appears in the other group of esetg. these communities are found along the coast of the aegean sea and are co-dominated by plantago lagopus, poa bulbosa, trifolium cherleri and vulpia ciliata. 120 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. fig. 7. pca ordination of species. fit for inclusion of species into ordination diagram was set to 45. the species are presented by 4 letters for genus and 3 for species, for explanation compare table1. 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:26 color profile: generic cmyk printer profile composite default screen since the topological variables and management are fairly unique throughout the region, we tried to identify the main factor within all the climatic factors that causes the diversity of communities. we tested the significance of climatic data from worldclim database. since the significance of the following variables was low (p=0.001), they were excluded from analysis: annual precipitation, isothermality, annual mean temperature, mean temperature of warmest quarter, mean temperature of wettest quarter, max temperature of warmest month and mean temperature of driest quarter. these variables mainly sum up annual averages. the results show that the most important climatic feature that enables the diversity of esetg in the region is pronounced changes in precipitation and temperature i.e. seasonality (tab. 2). higher precipitation seasonality can be found in the coastal regions, where there is pronounced summer hydric stress, which is less pronounced in the interior. precipitation of the warmest quarter is higher in the interior, more precipitation can be found on the coast in the wettest period (i.e., autumn) (graph not presented in the text). precipitation is more evenly distributed in the interior (fig. 5). the situation is the opposite with temperature, with which seasonality is more pronounced in the interior, where lower temperatures are found during the winter. among significant variables are the minimal and mean temperature of the coldest month and quarter, respectively. this shows that the diversity of esetg is also caused by cooler winter temperatures (tab. 2, fig. 5). structural analysis showed that communities in the coastal area contain more therophytes (e.g., briza maxima, urospermum pycroides, hedypnois rhagadioloides, galium murale)(fig. 6a). on the other hand, hemicryptophytes and chamaephytes are more abundant in the interior (scleranthus verticillatus, carex caryophyllea, rumex acetosella). in terms of chorological spectrum, there are more species with a mediterranean distribution pattern (e.g., galium murale, hedypnois rhagadioloides, velezia rigida) in the coastal communities, whereas euroasiatic species are more common (carex caryophyllea, rumex acetosella, filago arvensis) inland (fig. 6b). it was decided to assign the plant communities elaborated to two associations. communities from the interior part of the region in which romulea bulbocodium appears were classified as lagopo-poetum bulbosae and the other one, found in the coastal region, as romuleo graecae-poetum bulbosae. diagnostic species as well as typification are given in the section nomenclature. discussion mediterranean vegetation is limited to the north, with a continental climate dominating the continental parts of the balkans. the main climatic factor preventing penetration of mediterranean vegetation into the interior of the balkan peninsula is winter frosts (^arni and matevski 2005, medail and diadema 2009). at the same time, the mediterranean climate, with summer droughts, is a strong ecological filter that does not allow continental flora to settle in the region (filibeck et al. 2012). esetg, often dominated by poa bulbosa, can be found mainly on fresh fine clay soils on intensively grazed sites around settlements. such vegetation is rarely found on carbonate bedrock, since erosion and degradation is faster (oberdorfer 1954). it can be found on carbonate bedrock only in places (e.g., in the bottom of valleys) where colluvium offers a refacta bot. croat. 73 (1), 2014 121 early spring therophytic grasslands 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:26 color profile: generic cmyk printer profile composite default screen uge for these communities. garrigue dominated by cistus creticus is often found around these stands (^arni et al. 2010). the vegetation growth of these communities correlates with the annual precipitation regime. germination of spring annuals and growth of most perennials begins soon after the first autumn rains. growth is fairly slow during the winter months but vegetation is usually well established by february. growth is fast in spring, with the peak of growth and seed set in may–june. by the end of june, most of the herbaceous vegetation is dry and the seed dispersed. summer species, in this case c4 grasses, such as achnatherum, bothriochloa, chrysopogon, grow during the summer and seed in september–october (pérez-camacho et al. 2012). analysis of climatic variables derived from worldclim database shows that they are the most important variables for explaining the variance among esetg (60%) and are highly correlated (tab. 3). it must be borne in mind that the mediterranean climate provides a climatic envelope for esetg on the large scale (gilingham et al. 2012). on a smaller scale, the analysis showed that the most important factor for the variability of esetg within the research area is seasonality. variables that give information about average climatic conditions (mean temperature and precipitation) are of fairly low importance. inter-annual changes in precipitation and temperature also appear to be responsible for variability in other grazed herbaceous communities (zhang et al. 2011, lohmann et al. 2012). the rda diagram shows that the highest seasonality of precipitation is within esetg in which romulea linaresii ssp. graeca appears and the lowest within that with romulea bulbocodium. the situation is the reverse with temperature (fig. 5). temperature seasonality (fig. 3) is lowest on the coast of the aegean sea and at higher altitudes (esetg do not appear there). on the other hand, precipitation seasonality is highest in the coastal area and diminishes gradually towards the continent (fig. 3). in the analysis of climatic variables, two outliers differing from the result of the classification (fig. 4), can be found, relevés 19 and 20 in table 1. both species of romulea appear in these relevés. they were sampled above lake volvi (chalkidiki, greece) and the proximity of the lake probably influences the local climate. the life forms spectrum shows the mediterranean character of esetg. in these communities, therophytic species form 65 % of the floristic inventory of inland (romulea bulbocodium) communities and 73 % of coastal (romulea linaresii) communities. comparison with similar therophytic grasslands from bulgaria (sopotlieva 2009) and the republic of macedonia (]u[terevska et al. 2012) showed that there are 47 % of therophytes in such grasslands in bulgaria and 53–64% in those of the republic of macedonia. more therophytes can be found in the coastal communities, where summer drought is more pronounced. summer drought is a limiting factor for perennials but they can extend their biological activity if water is available (peréz-camacho et al. 2012). more evenly distributed precipitation gives an advantage to perennials in inland communities. more elements of perennial dry grasslands from the class festuco-brometea can be found in the inland communities (tab. 1). analysis of chorotypes (geo-elements) showed that 66% of species with a mediterranean (in the widest sense) distribution pattern appear in coastal communities and 59 % in inland communities. comparing these data with regions with more pronounced continentality 122 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:26 color profile: generic cmyk printer profile composite default screen (sopotlieva 2009, ]u[terevska et al. 2012), it can be seen that there are 41% of mediterranean species in bulgarian communities and 46–53% in those of the republic of macedonia. this indicates that mediterranean are quite different from continental communities. relevés of such communities further to the south have not been published but similar therophytic communities of olive grove grassland show 65 % of mediterranean species (^arni and matevski 2005). in a comparison with the synthetic table made by oberdorfer (1954) in the same region, many differences in the floristic composition of esteg can be found. however, these differences are very difficult to evaluate since changes appear in the management regime (abandonment), climatic changes, sometimes even differences in taxonomical concepts. in the period 1950–1999, precipitation decreased and summer temperatures rose in the eastern mediterranean (nastos et al. 2013) while land use change and abandonment of traditional agriculture is one of the crucial problems in the mediterranean landscape (bajocco et al. 2012). nomenclature oberdorfer (1954) classified esetg within the order helianthemetalia and class therobrachypodietea. the class thero-brachypodietea is nowadays limited to mediterranean perennial pseudosteppe communities (rodwell et al. 2002). the vegetation under consideration is classified within the class of mediterranean terrestrial plant communities dominated by annual low-growing herbs and grasses helianthemetea guttati (br.-bl. in br.-bl. et al. 1952) rivas goday et rivas-mart. 1963 and order of mediterranean and sub-mediterranean ephemeral communities on acid soils and fire-prone habitats helianthemetalia guttatii br.-bl. in br.-bl. et al. 1940 (rodwell et al. 2002, allegrezza et al. 2006, fanelli et al. 2010, ribeiro et al. 2012). it would be also possible to classify the alliance romulion within the class poetea bulbosae and order poetalia bulbosae, which is found in mediterranean and sub-mediterranean heavily grazed pastures, trampled and manured by sheep (rodwell et al. 2002, farris et al. 2010) known till now only from the western mediterranean region (cano et al. 2007). as there appear climatic and floristic differences, the possible occurrence of these syntaxa in the eastern mediterranean region should be studied in the future. esetg cannot be classified within the class of ephemeral vegetation with winter annuals on bare or disturbed salt-marsh mud and sand saginetea maritimae, since no halophilous plant species appear (e.g., tomaselli et al. 2011) and classification of the alliance romulion in the list of alliances (rodwell et al. 2002) should be reconsidered. esetg should be classified within the alliance romulion, as proposed by oberdorfer (1954). however, at first glance, oberdorfer (1954) mentions only poa bulbosa (that means a typical subspecies and not subspecies timoleontis) in list 1 (»liste i«) as the dominant species of »lagopeto-poetum timolentis« and »tortileto-poetum timoleontis«, which would cause an invalid publication of the associations and, consequently, the alliance (icpn, art. 3f, 8). however, according to theurillat (pers. com.), oberdorfer (1954: 88) says about synthetic lists 1–3 (»synthetische listen i–iii«) »... brachypodium ramosum oder b. phoenicoides treten vollkommen zurück und werden durch poa bulbosa (div. ssp.) oder stipa tortilis ersetzt.« it is true that in »list i« poa bulbosa is indicated as a species but acta bot. croat. 73 (1), 2014 123 early spring therophytic grasslands 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:26 color profile: generic cmyk printer profile composite default screen in the other two lists (»liste ii« and »liste iii«), it is »poa bulbosa coll.«. we would therefore be inclined to consider that a printing error occurred in the first list and that here, too, it should be interpreted as »poa bulbosa coll.« (including also the subspecies timoleontis) on the basis of what is said on page 88. in this sense, oberdorfer would use the name of the aggregate species in the list (poa bulbosa coll.) but, on the other hand, would use a more narrowly defined taxon in the association name (poa bulbosa ssp. timoleontis). from the point of view of articles 3f and 43, both association names can thus be considered formally to be validly published, and so also the alliance name romulion oberdorfer 1954. the correction of the name »romulion« to »romulion graecae« by bolòs et al. (1996) cannot be accepted. names such as »romulion« should stay without the addition of a specific name, since there is no rule in icpn about the correction of these names, and recommendation 10c cannot be applied. romulion oberdofer 1954 lectotype: lagopo-poetum bulbosae oberdorfer 1954 corr. ^arni et al. 2014 – lectotypus hoc loco. diagnostic species (oberdorfer 1954): allium guttatum, alyssum minutum, a. repens, campanula ramosissima, gagea reticulata, g. chrysantha, hedypnois rhagadioloides, hypochoeris cretensis, lagoecia cuminioides, linaria simplex, lotus angustissimus, ornithogallum collinum, ornithogalum armeniacum, picris pauciflora, romulea bulbocodium, r. linaresii ssp. graeca, r. columnae, sedum aetnense, silene graeca and ziziphora capitata. ecological conditions: early spring ephemeral therophytic grasslands on deeper soils in the eastern mediterranean area. the critical point of consideration of the nomenclature is the doubtful appearance of poa bulbosa ssp. timoleontis (i.e., poa timoleontis) in communities in the research area. during the field survey carried out in the southern part of the republic of macedonia and around thessaloniki in greece, we could not confirm poa bulbosa ssp. timoleontis as appearing in the communities under consideration; only poa bulbosa s. str. was identified. since the area of research and also the species composition match oberdorfer’s »lagopeto-poetum timoleontis«, it was decided to correct the name of »lagopeto-poetum timoleontis« to lagopo-poetum bulbosae, on the basis of art. 43 of icpn. we have not corrected the name of »toriletum-poetum timoleontis« appearing further to the south, since poa timoleontis may appear there. lagopo-poetum bulbosae oberdorfer 1954 corr. ^arni et al. 2014 nom. corr. hoc loco neotype: tab. 1, rel. 5 – neotypus hoc loco. diagnostic species: achillea coarctata, alyssum desertorum, carex caryophyllea, poa bulbosa, romulea bulbocodium and teucrium capitatum. ecological conditions: early spring ephemeral grasslands in the regions with less pronounced summer hydric stress. oberdorfer described this community under the name »lagopeto-poetum timolentis« [recte: »lagopeto-poetum timoleontis«]. since the genus lagopus has been validly published, the name »lagopeto-poetum timoleontis« must be maintained according to art. 14, and corrected to lagopo-poetum timoleontis oberdorfer 1954 according to art. 41. at the same time, we would like to add a comment to the name »tortileto-poetum timoleontis«. since no genus »tortilis« exists, this must mean stipa tortilis (art. 14), and the name should be corrected with the genus stipa, i.e., stipo tortilis-poetum timoleontis oberdorfer 1954. 124 acta bot. croat. 73 (1), 2014 ^arni a., matevski v., [ilc u., ]u[terevska r. 821 carni et al.ps u:\acta botanica\acta-botan 1-14\821 carni et al.vp 19. o ujak 2014 17:06:26 color profile: generic cmyk printer profile composite default screen we also describe a new association, as: romuleo graecae-poetum bulbosae ass. nova hoc loco holotype: tab. 1, rel. 15 – holotypus hoc loco. diagnostic species: crepis zacyntha, galium murale, hedypnois rhagadioloides, hordeum murinum ssp. leporinum, lagurus ovatus, poa bulbosa, romulea linaresii ssp. graeca, urospermum pycroides. ecological conditions: early spring ephemeral grasslands in the regions with more pronounced summer hydric stress. conclusions esetg are typical elements of mediterranean landscapes that are maintained by traditional land use. since these are among the most diverse habitats in the region, the class heliathemetea and alliance romulion for the eastern mediterranean, respectively, therefore also deserve to be listed in syntaxonomic interpretation manuals of the habitat directive among habitats 6220 pseudosteppe with grasses and annuals of the thero-brachypodietea (farris et al. 2007, biondi et al. 2012). acknowledgement we are grateful to jean-paul theurillat for his nomenclature clarification. we thank iztok 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(piperaceae) has been used by brazilian traditional communities against several illnesses including rheumatism, bronchitis, sexually transmitted diseases and complaints of the urinary tract. medicinal plants are a source of several remedies used in clinical practice to combat microbial infections. in this study, ethanol extract and fractions of piper arboreum leaves were used to assay antimicrobial and modulatory activity. the minimum inhibitory concentration (mic) was determined using microdilution method of ethanol extract and fractions from the leaves of p. arboreum ranging between 8 and 1024 mg ml–1. the capacity of these natural products to enhance the activity of antibiotic and antifungal drugs was also assayed. in these tests, natural products were combined with drugs. the natural products assayed did not demonstrate any clinically relevant antimicrobial activity (mic ³ 1024 mg ml–1). however, the modulation of antibiotic activity assay observed a synergistic activity of natural products combined with antifungal (such as nystatin and amphotericin b) and antibiotic drugs (such as amikacin, gentamicin and kanamycin). according to these results, these natural products can be an interesting alternative not only to combat infectious diseases caused by bacteria or fungi, but also to combat enhanced resistance of microorganisms to antibiotic and antifungal drugs. acta bot. croat. 73 (1), 2014 281 * corresponding author, e-mail: hdmcoutinho@gmail.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 922 tintino et al.ps u:\acta botanica\acta-botan 1-14\922 tintino et al.vp 24. o ujak 2014 11:46:45 color profile: generic cmyk printer profile composite default screen keywords: antimicrobial activity, synergism, piper arboretum, staphylococcus aureus, escherichia coli, candida abbreviations: atcc – american type culture collection; mic – minimum inhibitory concentration introduction the genus piper (piperaceae) presents almost 1,000 species, distributed mainly in the tropical and subtropical regions of asia and america. plants from this genus are used for commercial and medicinal purposes (nunes et al. 2007) and demonstrate several biological activities (ruiz and roque 2009). antibacterial and trypanocidal activities have been reported for piper arboreum aub. and this plant is used by traditional communities in brazil in the form of a decoct against rheumatism, bronchitis, and sexually transmitted diseases (agra et al. 2007, ramos and kato 2009, regasini et al. 2009). the world health organization (who 1998) defines a medicinal plant as any plant that contains any active compounds that can be used for therapeutic or chemical purposes. substances produced by plants are their defence mechanism against insects, herbivores, and phytopathogenic microorganisms (gotlieb 1981). some researchers have named these phytocompounds »antibiotic like-substances« (geismann 1963). the use of plant extracts as antimicrobial agents represents a low risk for the development of resistance by the microorganisms because these products are composed of several phytocompounds of different groups (daferera et al. 2003). concerning resistance to antibiotics, many researchers have expressed their opinion that natural products from plants could be interesting alternatives (dancer 2001, nostro et al. 2004, coutinho et al. 2005, georgopapadakou 2005). several plant extracts and phytochemicals are known for their antimicrobial properties. many studies have been conducted in different countries to demonstrate such activities (salvagnini et al. 2008, simões et al. 2008, silva et al. 2008). the staphylococcus genus is found in skin and mucosal microbiota of animals and birds. some staphylococcus species are etiological agents of many animal and human infections (nostro et al. 2004, coutinho et al. 2009a, b). staphylococcus aureus, s. epidermidis, s. saprophyticus and s. haemolyticus are the most important etiological agents of community and nosocomial human infections. besides, this bacterium is responsible for different types of intoxications, being the most common etiological agent of infections on different tissues and/or organs (e.g. furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, meningitis, bacterial arthritis) (verhoeff et al. 1999). escherichia coli is one of the most important human infectious agents. this bacterium is an enterotoxin producer and the causal agents of diarrhoea outbreaks (konowalchuk et al. 1977, scotland et al. 1980) and urinary tract infections (hughes et al. 1982). candidiasis is the most frequent mycosis caused by opportunistic fungi. the main species associated with this disease are candida albicans, c. tropicalis, c. parapsilosis, c. glabrata and c. krusei (coutinho 2009). these yeasts are found in skin and in mucosal microbiota, becoming pathogenic in patients with immunodeficiency or immunosuppression (dignani et al. 2003). 282 acta bot. croat. 73 (1), 2014 tintino s. r., souza c. e. s., guedes g. m. m., costa j. i. v., duarte f. m. et al. 922 tintino et al.ps u:\acta botanica\acta-botan 1-14\922 tintino et al.vp 24. o ujak 2014 11:46:45 color profile: generic cmyk printer profile composite default screen many plants have been evaluated not only for direct antimicrobial activity but also as resistance-modifying agents (gibbons 2004). several chemical compounds, synthetic or from natural sources, have demonstrated a direct activity against many species of bacteria, enhancing the activity of a specific antibiotic, reversing the natural resistance of bacteria to a specific antibiotic, causing the elimination of plasmids and inhibiting the active efflux of antibiotics through the plasma membrane (coutinho et al. 2009a, b). the potentiation of antibiotic activity or the reversal of antibiotic resistance allows the classification of these compounds as modifiers of antibiotic activity (coutinho et al. 2009a). the aim of this work was to evaluate the antimicrobial and modulatory activity of ethanol extract and fractions of piper arboreum associated with antibiotic and antifungal drugs against multiresistant bacteria and fungi. materials and methods microbial strains the bacteria used in the minimum inhibitory concentration assay were the strains of e. coli atcc25923 and s. aureus atcc10536. in the antifungal assays, the strains of candida albicans atcc40006, c. krusei atcc2538 and c. tropicalis atcc40042 were used. to evaluate the modulatory activity of the ethanol extract and fractions the multiresistant bacterial strains isolated from clinical environments: e. coli 27 and s. aureus 358 with the resistance profile were used (tab. 1). all strains were obtained from the laboratory of clinical mycology – universida de federal da paraíba. plant material stems of piper arboreum were collected in march/2010 in the gaiambira reserve, bananeiras county, paraíba state (brazil). the plant material was identified by prof. carlos alberto garcia and a voucher (c. a. garcia 205) was deposited in the education and health center herbarium, universida de federal de campina grande. the leaves of piper arboacta bot. croat. 73 (1), 2014 283 antimicrobial activity of piper extracts tab. 1. bacterial source and antibiotic resistance profile. bacteria source antibiotic resistance escherichia coli 27 surgical wound ast, ax, ami, amox, ca, cfc, cf, caz, cip, clo, im, can, szt, tet, tob escherichia coli atcc10536 – staphylococcus aureus atcc25923 – staphylococcus aureus 358 surgical wound oxa, gen, tob, ami, can, neo, para, but, sis, net ami – amicilina; amox – amoxillin, amp – ampicillin; ast – aztreonam; ax – amoxacillin; but – butirosina; ca – cefadroxil; can – canamycin; caz – ceftazinidima; cfc – cefaclor; cf – cephalothin; cip – ciprofloxacin; clo – chloramphenicol; com – cefepime; ctz – ceftazidime; gen – gentamicin; imi – imipenem; lev – levofloxacin; mer – merpenem; neo – neomycin; net – netilmicin; oxa – oxacillin; sis – sisomicin; szt – sulfametrim; tet – tetracycline; tob – tobramycin; para – paramomicina; ptz – piperacilina-tazobactam 922 tintino et al.ps u:\acta botanica\acta-botan 1-14\922 tintino et al.vp 24. o ujak 2014 11:46:45 color profile: generic cmyk printer profile composite default screen reum, after collection, were dried at 40 °c by 72 hours and powdered, obtaining 1.5 kg. the powdered material was extracted by maceration using 1 l of 95% (v/v) methanol as solvent at room temperature by three days, this process being repeated five times. the solution was then filtered and concentrated under vacuum in a rotary evaporator under 60 °c and 760 mm/hg of temperature and pressure, respectively. this amount of powdered material yielded 350 g of ethanol extract. the ethanol extract from the leaves (50 g) of piper arboreum was dissolved in a mixture of etoh:h2o (7:3), generating a hydroalcohol solution which was submitted to a liquid/ liquid partition using the following solvents: hexane, dichloromethane and ethyl acetate. the obtained solutions were mixed with sodium sulphate anhydrous (na2so4) and filtered. after this process, the solutions were concentrated using a rotary vacuum evaporator at temperatures below 50 °c, yielding the following fractions: hexane fraction (3 g), dichloromethane fraction (3.5 g) and ethyl acetate fraction (9 g). drugs the drugs used in the tests were the aminoglycosides kanamycin, amikacin, neomycin and gentamicin, and the antifungals mebendazole, amphotericin b, nystatin and benzoylmetronidazole (sigma co., st. louis, usa). all drugs were diluted in sterile water. minimum inhibitory concentration the broth microdilution method was used in order to determine minimum inhibitory concentration (mic). the ethanol extract and fractions were dissolved using dimethylsulfoxide and diluted to 1024 mg ml–1 using sterile distilled water. the bacterial inoculum was diluted using brain heart infusion broth to a final concentration of 105 colony forming units per ml. each inoculum (100 ml) was distributed in each well of a microtiter 96 wells plate and then submitted to a twofold serial dilution using 100 ml of the ethanol extract, with concentrations ranging between 8 and 1024 mg ml–1. the plates were incubated for 24 hours at 35 °c (javadpour et al. 1996). bacterial mic was determined using resazurin, with blue changing to red indicating bacterial growth. the fungal mic was determined by turbidity, observing the growth of fungal strains. the mic was defined as the lowest concentration in which no growth can be observed, according to clsi (2008). positive controls of growth were performed using the culture medium with the microbial inoculum. drug modulation test to observe how the ethanol extract and fractions affect the action of antimicrobial drugs against the assayed strains, the method proposed by coutinho et al. (2008) was used. the ethanol extracts and fractions were tested using a sub-inhibitory concentration (mic/8 = 128 mg ml–1). a sample with 100 ml of the solution containing brain heart infusion broth, the microbial inoculums and the extract or fractions was placed in each well. after this, 100 ml of the antimicrobial drug was mixed in the first well, following the twofold dilution. concentrations of aminoglycosides and antifungals ranged between 5000 and 2.44 mg ml–1 and 1024 and 2 mg ml–1, respectively. the assays were performed in triplicate. 284 acta bot. croat. 73 (1), 2014 tintino s. r., souza c. e. s., guedes g. m. m., costa j. i. v., duarte f. m. et al. 922 tintino et al.ps u:\acta botanica\acta-botan 1-14\922 tintino et al.vp 24. o ujak 2014 11:46:45 color profile: generic cmyk printer profile composite default screen results and discussion the mic results of the extract and fractions alone and combined with antifungals are demonstrated in the table 2. against all fungal strains assayed, the mic observed was ³ 1024 mg ml–1, these results not being clinically relevant. the same results were observed by regasini et al. (2009). however, when the antifungal drugs were associated with the extract and fractions, a synergistic activity was observed against c. krusei, enhancing the antifungal drug activity. the mic results of the natural products alone and combined with aminoacta bot. croat. 73 (1), 2014 285 antimicrobial activity of piper extracts tab. 2. minimum inhibitory concentration (mic) of antifungal drugs alone and associated with extract and fractions of piper arboreum against c. albicans, c. krusei and c. tropicalis (mg ml–1). dfpa c. albicans c. krusei c. tropicalis natural product/antifungal alone + dfpa alone + dfpa alone + dfpa dfpa ³ 1024 – ³ 1024 – ³ 1024 – amphotericin b ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 mebendazole ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 nystatin ³ 1024 ³ 1024 64 ³ 1024 ³ 1024 ³ 1024 benzoilmetronidazole ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 eepa natural product/antifungal alone + eepa alone + eepa alone + eepa eepa ³ 1024 – ³ 1024 – ³ 1024 – amphotericin b ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 mebendazole ³ 1024 ³ 1024 ³ 1024 64 ³ 1024 ³ 1024 nystatin ³ 1024 ³ 1024 64 64 ³ 1024 ³ 1024 benzoilmetronidazole ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 hfpa natural product/antifungal alone + hfpa alone + hfpa alone + hfpa hfpa ³ 1024 – ³ 1024 – ³ 1024 – amphotericin b ³ 1024 ³ 1024 ³ 1024 2 ³ 1024 ³ 1024 mebendazole ³ 1024 ³ 1024 ³ 1024 2 ³ 1024 ³ 1024 nystatin ³ 1024 ³ 1024 64 1 ³ 1024 ³ 1024 benzoilmetronidazole ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 efpa natural product/antifungal alone + efpa alone + efpa alone + efpa efpa ³ 1024 – ³ 1024 – ³ 1024 – amphotericin b ³ 1024 ³ 1024 ³ 1024 2 ³ 1024 ³ 1024 mebendazole ³ 1024 ³ 1024 ³ 1024 0.5 ³ 1024 ³ 1024 nystatin ³ 1024 ³ 1024 64 64 ³ 1024 ³ 1024 benzoilmetronidazole ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 ³ 1024 dfpa – dichloromethane fraction; eepa – ethanol extract from the leaves; hfpa – hexane fraction; efpa – ethyl acetate fraction 922 tintino et al.ps u:\acta botanica\acta-botan 1-14\922 tintino et al.vp 24. o ujak 2014 11:46:45 color profile: generic cmyk printer profile composite default screen glycosides are shown in the table 3. as observed with the antifungals, the natural products also demonstrated a mic ³ 1024 mg ml–1, but when the natural product was associated with the aminoglycosides, a synergistic or antagonistic antibiotic activity was detected against both bacterial strains assayed. when compared with the results of suffredini et al. (2006), the results observed on this work indicate a better antibacterial activity. 286 acta bot. croat. 73 (1), 2014 tintino s. r., souza c. e. s., guedes g. m. m., costa j. i. v., duarte f. m. et al. tab. 3. minimum inhibitory concentration (mic) of antibiotics alone and associated with extract and fractions of piper arboreum against escherichia coli strain 27 and staphylococcus aureus strain 358 (mg ml–1). dfpa ec27 sa358 natural product/antibiotic alone + dfpa alone + dfpa dfpa ³ 1024 – ³ 1024 – kanamycin 1250 1250 156 39 amikacin 2500 2500 78 78 78 neomycin 1250 1250 78 gentamicin 156 625 39 39 eepa natural product/antibiotic alone + eepa alone + eepa eepa ³ 1024 – ³ 1024 – kanamycin 1250 1250 156 39 amikacin 2500 625 78 78 neomycin 1250 1250 78 78 gentamicin 156 156 39 39 hfpa natural product/antibiotic alone + hfpa alone + hfpa hfpa ³ 1024 – ³ 1024 – kanamycin 1250 ³ 5000 156 39 amikacin 2500 78 78 78 neomycin 1250 1250 78 78 gentamicin 156 39 39 39 efpa natural product/antibiotic alone + efpa alone + efpa efpa ³ 1024 – ³ 1024 – kanamycin 1250 1250 156 39 amikacin 2500 312 78 78 neomycin 1250 1250 78 78 gentamicin 156 39 39 39 dfpa – dichloromethane fraction; eepa – ethanol extract from the leaves; hfpa – hexane fraction; efpa – ethyl acetate fraction 922 tintino et al.ps u:\acta botanica\acta-botan 1-14\922 tintino et al.vp 24. o ujak 2014 11:46:45 color profile: generic cmyk printer profile composite default screen all these different mic results can be attributed to the environmental conditions related to the differences in the plants studied (cysne et al. 2005). however, the natural products of p. arboreum enhanced the antimicrobial activity of the assayed drugs, acting in a synergistic form (tabs. 2, 3). this is the first report of synergism between natural products of p. arboreum and antimicrobial agents. there are several mechanisms involved in the inhibition of microorganism growth, especially due to the hydrophobic nature of some phytocompounds. these compounds may interact with the lipid bilayer of the cell membrane, affecting the respiratory chain and the production of energy (wendakoon and sakaguchi 1995) or enhancing the membrane permeability, including permeability to antimicrobials (burt 2004). this permeability enhancement can be obtained from a combination of antimicrobial drugs with natural products in a sub-inhibitory concentration (coutinho et al. 2008, 2009b). this strategy refers the usage of natural products and drugs in an approach with monoor multi-extract combinations that will affect not only a single but several targets (hemaiswarya et al. 2008, wagner et al. 2009). conclusions as demonstrated in our work, natural products of piper arboreum showed an expressive capacity to modulate the activity of antimicrobial drugs in a synergistic or antagonistic manner, representing an alternative to the efforts to control infectious diseases caused by multidrug-resistant strains of fungi and bacteria. references agra, m. f., freitas, p. f., barbosa-filho, j. m., 2007: synopsis of the plants known as medicinal and poisonous in northeast of brazil. brazilian journal of pharmacognosy 17, 114–140. burt, s., 2004: essential oils: their antibacterial properties and potential application sin foods: a review. international journal of food microbiology 94, 223–253. clsi – clinical and laboratory standards institute (2008). performance standards of antimicrobial disk susceptibility test: ninth informational supplement. nccls document m100-s9. national committee for clinical laboratory standards, wayne, pa, 120– 126. coutinho, h. d. m., cordeiro, l. d., bringel, k. p., 2005: antibiotic resitance of pathogenic bacteria isolated from the population of juazeiro do norte-ceará. revista brasileira de ciências da saúde 9, 127–138. coutinho, h. d. m., costa, j. g. m., lima, e. o., falcão-silva, v. s., siqueira-júnior, j. p., 2009a: herbal therapy associated with antibiotic therapy: potentiation of the antibiotic activity against methicillin-resistant staphylococcus aureus by turnera ulmifolia l. bmc complementary and alternative medicine 9, 13–17. coutinho, h. d. m., costa, j. g. m., lima, e. o., falcão-silva, v. s., siqueira-júnior, j. p., 2009b: in vitro interference of hyptis martiusii benth. and chlorpromazine against an acta bot. croat. 73 (1), 2014 287 antimicrobial activity of piper extracts 922 tintino et al.ps u:\acta botanica\acta-botan 1-14\922 tintino et al.vp 24. o ujak 2014 11:46:45 color profile: generic cmyk printer profile composite default screen aminoglycoside-resistant escherichia coli. indian journal of medical research 129, 566–568. coutinho, h. d. m., 2009: factors influencing the virulence of candida spp. west indian medical journal 58, 160–163. coutinho, h. d. m., costa, j. g. m., siqueira júnior, j. p., lima, e. o., 2008: in vitro anti-staphylococcal activity of hyptis martiusii benth against methicillin-resistant staphylococcus aureus-mrsa strains. revista brasileira de farmacognosia 18, 670–675. cysne, j. b., canuto, k. m., pessoa, o. d. l., nunes, e. p., silveira, e. r., 2005: leaf essential oils of four piper species from the state of ceará – northeast of brazil. journal of the brazilian chemical society 16, 1378–1381. daferera, d. j., ziogas, b. n., polissou, m. g., 2003: the effectiveness of plant essential oils on the growth of botrytis cinerae, fusarium sp. and clavibacter michiganensis subs. michignensis. crop protection 22, 39–44. dancer, s. j., 2001: the problem with cephalosporins. journal of antimicrobial chemotherapy 48, 463–478. dignani, m. c., solomkin, j. s., anaissie, e., 2003: candida. in: anaissie, e., mcginnis, m. r., pfaller, m. a. 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(2009) have used castanea sativa among other trees for different approaches in biomonitoring. taking into consideration only acta bot. croat. 72 (2), 2013 337 * corresponding author, e-mail: helmut.mayrhofer@uni-graz.at copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. castanea sativa and quercus ssp. giordani (2012) assessed the effects of forest management on epiphytic lichens in coppiced forests in humid mediterranean liguria. aprile et al. (2011) reported on monitoring of air pollution in relation to land use patterns in the campania region of italy using castanea sativa and quercus pubescens as phorophytes. the knowledge of the lichen biota of montenegro has improved because of extensive field work in biogradska gora national park (bilovitz et al. 2009) and at other sites (bilovitz et al. 2008, 2010). kne@evi] and mayrhofer (2009) have provided a catalogue based on a comprehensive evaluation of published sources. this paper contributes a new lichen species (xylographa soralifera) for the balkan peninsula, a new lichenicolous fungus (monodictys epilepraria) for montenegro and new distribution data for the interesting species caloplaca monacensis, collema subflaccidum and fuscopannaria mediterranea. the species composition is compared with a site in switzerland (bergell), sites in piedmont, tuscany and the island of elba (tab. 1). 338 acta bot. croat. 72 (2), 2013 mayrhofer h., drescher a., ste[evi] d., bilovitz p. o. tab. 1. list of taxa from livari in comparison with the sites in tuscany (t), elba (e) and piedmont (p) in italy, bergell (b) in switzerland and from various sites in the western alps and northern apennines (l) of italy. * – species occurring only on morus alba; ° – as lecidella elaeochroma. taxa t e p b l amandinea punctata (hoffm.) coppins et scheid. x x x x arthonia radiata (pers.) ach. x x x x caloplaca herbidella (hue) h. magn. x x *caloplaca monacensis (leder.) lettau *candelariella reflexa (nyl.) lettau x x x cladonia coniocraea (flörke) spreng. x x x x cladonia pyxidata (l.) hoffm. x collema flaccidum (ach.) ach. x collema subflaccidum degel. x degelia plumbea (lightf.) p. m. jørg. et p. james x x x flavoparmelia caperata (l.) hale x x x x x fuscopannaria mediterranea (c. tav.) p. m. jørg. (see fig. 4) x hypocenomyce scalaris (ach.) m. choisy x x x hypogymnia tubulosa (schaer.) hav. x lecanora chlarotera nyl. x x x x lecidella achristotera (nyl.) hertel et leuckert x x x x x lepraria leuckertiana (zedda) l. saag lepraria rigidula (de lesd.) tønsberg x lepraria vouauxii (hue) r. c. harris x leptogium lichenoides (l.) zahlbr. x x leptogium saturninum (dicks.) nyl. x leptogium teretiusculum (wallr.) arnold acta bot. croat. 72 (2), 2013 339 lichenized fungi of a chestnut grove in livari (rumija, montenegro) taxa t e p b l lobaria amplissima (scop.) forssell x x lobaria pulmonaria (l.) hoffm. x x x x lobarina scrobiculata (scop.) nyl. ex cromb. x megalospora tuberculosa (fée) sipman melanelixia fuliginosa (duby) o. blanco et al. x melanelixia glabra (schaer.) o. blanco et al. x x x melanohalea exasperata (de not.) o. blanco et al. micarea prasina fr. (on lignum of root) nephroma laevigatum ach. x x ochrolechia androgyna (hoffm.) arnold pachyphiale carneola (ach.) arnold x parmelia saxatilis (l.) ach. x x x x parmelia sulcata taylor x x x x x parmeliella triptophylla (ach.) müll. arg. parmelina quercina (willd.) hale x x x parmelina tiliacea (hoffm.) hale x x x x parmeliopsis ambigua (wulfen) nyl. x x peltigera collina (ach.) schrad. peltigera praetextata (flörke ex sommerf.) zopf x x pertusaria albescens (huds.) m. choisy var. albescens x x x x x pertusaria albescens var. corallina auct. pertusaria coccodes (ach.) nyl. x x pertusaria flavida (dc.) j. r. laundon x x x pertusaria hymenea (ach.) schaer. x x x pertusaria leioplaca dc. x x x x pertusaria pertusa (weigel) tuck. x x x phaeophyscia chloantha (ach.) moberg *phaeophyscia orbicularis (neck.) moberg x x x phlyctis argena (spreng.) flot. x x x x physconia perisidiosa (erichsen) moberg x x physconia venusta (ach.) poelt x placynthiella icmalea (ach.) coppins et james x *punctelia subrudecta (nyl.) krog x x x ramalina farinacea (l.) ach. x x x rinodina sophodes (ach.) a. massal. x x x strigula affinis (a. massal.) r. c. harris (on lignum) trapeliopsis flexuosa (fr.) coppins et p. james (on lignum) xylographa soralifera holien et tønsberg (on lignum) tab. 1. – continued study area – physical settings the investigated locality on the eastern slopes of the rumija mountain range is situated in the small rural settlement called gornja briska (coordinates 42°06’58–59’’n / 19°13’ 16–18’’e) c. 1.5 km southeast of the village of livari at an elevation of 475–490 m above sea level (fig. 1). the rumija range stretches in the nw-se direction, forming a natural barrier between the adriatic sea and lake skutari. in contrast to the steep slopes facing the adriatic sea, the eastern slopes facing skutari lake are discontinued by plateaus, mainly in the southern part. the regional climatic situation is characterized by the climatic diagrams from the two nearby stations virpazar at the northwestern lakeside and shkodra (albania) at the southeastern shore of lake skutari (fig. 3). the sub-mediterranean (supra-mediterranean) character is expressed by the precipitation curve with a maximum in october and a very indistinct second peak in may (station shkodra, furlan 1977). the mean dry period in summer (dotted polygon in the diagram with the precipitation curve below the temperature line) lasts less than two months. this character is not only altered by the geomorphological situation on a plateau, but also by the water storage capacity of the deep soil, which mitigates the dry period during the summer months (furlan 1977). these conditions enable the development of deciduous broadleaved forests in this altitudinal belt: in general a coppiced forest of white hornbeam (carpinion orientalis alliance horvat 1958), mixed forests with oak and chestnut (castaneo-quercetum submediterraneum wraber 1954) and chestnut plantations. the open structure of the chestnut stands (fig. 2) originates from plantation for crop production and results in a rather irregular canopy. material and methods field studies were carried out in an old castanea sativa grove on a slope flattening. about 40 trunks of castanea sativa rich in species abundance and diversity were randomly selected and investigated. some remarkable species were taken from one single trunk of a 340 acta bot. croat. 72 (2), 2013 mayrhofer h., drescher a., ste[evi] d., bilovitz p. o. fig. 1. the investigated locality is situated in the small rural settlement called gornja briska, 1.5 km southeast of the village livari. background: lake skutari and albanian alps. fig. 2. the open structure of the chestnut stands originates from plantation for crop production and results in a rather irregular canopy. roadside morus alba. the list presented is based on collections made by the first author in 2004 and supplemented with studies by the first and second author in 2009. the specimens have been identified with the aid of poelt (1969), poelt and vìzda (1977, 1981), clauzade and roux (1985) and wirth (1995). some of the identifications required verification using standardized thin-layer chromatography (tlc) following the protocols of acta bot. croat. 72 (2), 2013 341 lichenized fungi of a chestnut grove in livari (rumija, montenegro) fig. 3. climatic diagrams from the two nearby stations virpazar at the northwestern lakeside (data from the hydrometeorological institute of montenegro) and shkodra (albania) at the southeastern shore (data from furlan 1977). fig. 4. fuscopannaria mediterranea (mayrhofer 18438; photo p. o. bilovitz), a species rare in montenegro. white and james (1985) and orange et al. (2010). the specimens are preserved in the herbarium of the institute of plant sciences, karl-franzens-university graz (gzu). the nomenclature follows nimis and martellos (2008) and other modern treatments. results fifty nine species and one variety of lichenized fungi and the lichenicolous fungus monodictys epilepraria are recorded (tab. 1). below we give remarks on the noteworthy species caloplaca monacensis, collema subflaccidum, fuscopannaria mediterranea, xylographa soralifera and monodictys epilepraria: caloplaca monacensis is a neglected name for caloplaca cerina var. cyanolepra (vondrák et al. 2009). it is also known from two localities around pljevlja in northern montenegro occurring on pyrus communis and fraxinus excelsior (bilovitz et al. 2010). further records from the balkan peninsula are summarized by abbott (2009) for greece, bilovitz and mayrhofer (2011) for bosnia and herzegovina, ku[an (1953) for croatia and suppan et al. (2000) for the dinaric region of slovenia. the species is also reported by vondrák et al. (2009) and [oun et al. (2011) from bulgaria. collema subflaccidum was only known from the adriatic coast from the surroundings of herceg novi (degelius 1954, as collema subfurvum) and petrovac (vìzda 1968, as collema furfuraceum; vìzda 1971, as collema subfurvum). vìzda (1971) corrected his record of collema furfuraceum published in vìzda (1968) as c. subfurvum. kne@evi] and mayrhofer (2009) did not consider this correction and filled the first citation under collema furfuraceum. fuscopannaria mediterranea (fig. 4) was also only known from the environs of herceg novi (jørgensen 1978, as pannaria mediterranea) and budva (bilovitz et al. 2008) at the adriatic coast. xylographa soralifera was described from north america by holien and tønsberg (2008). they cited one record from italy. heininger and spribille (2009) recorded it from several localities in austria and also a second specimen from italy. the sample from livari is unusually richly fertile with only few soralia. monodictys epilepraria diederich et m. s. christ. is a lichenicolous fungus on lepraria rigidula. this is the first record from montenegro of the species recently described by kukwa and diederich (2005). they report it from the czech republic, great britain, lithuania, poland, spain and sweden. subsequently it was also mentioned from estonia (suija et al. 2006), austria (hafellner and obermayer 2007), slovenia (kukwa 2008, bilovitz et al. 2011), germany (brackel 2009, eichler et al. 2010, schiefelbein 2013) and bosnia and herzegovina (bilovitz and mayrhofer 2010). discussion roth and scheidegger (1997) reported 111 species from 95 investigated trunks distributed within an area of c. 2 km2 of the region of bergell in switzerland. dietrich and bürgi-meyer (2011) mentioned 77 epiphytic lichens including 38 species occurring on the trunks and twigs of castanea sativa from one site in the canton of lucerne in central switzerland. castanea sativa was the best substrate followed by quercus sp. with 34 species in 342 acta bot. croat. 72 (2), 2013 mayrhofer h., drescher a., ste[evi] d., bilovitz p. o. their study area. matteucci et al. (2012) investigated 67 plots in orchards and coppices of castanea sativa in three bioclimatic regions in the western alps and northern apennines of italy, where they recorded 152 taxa. temperate oceanic plots with high precipitation host species of the lobarion communities in this area. loppi et al. (1997) recorded 76 taxa from montieri in tuscany, while matteucci et al. (2010) reported 63 species from 77 trees including sixty-three castanea sativa ones at four sites in piedmont. the diversity in livari (60 taxa) is similar. the species composition is most similar to the tuscany site with species of the lobarion, but parmeliella triptophylla and peltigera collina occur only in livari. lignicolous species like micarea prasina, strigula affinis or trapeliopsis flexuosa are not considered in the cited papers from italy and switzerland. medium aged trees with a diameter at breast height of 40 to 60 cm host the highest number of species in livari, whereas the oldest and thickest trees with extremely deeply incised bark host hardly any lichen species. moderate disturbance does not damage the lichens and most of them respond positively to increased light availability. eight species from livari are mentioned in the red list of epiphytic lichens of italy (nascimbene et al. 2013): megalospora tuberculosa as »critically endangered«, caloplaca herbidella, degelia plumbea, lobaria amplissima, lobarina scrobiculata, pachyphiale carneola and parmeliella triptophylla as »near threatened« as well as lobaria pulmonaria as »least concern«. wirth (2010) introduced a procedure to assess the eco-climatic oceanity (ko) with the help of the two indicator values of continentality and moisture. according to this procedure the chestnut grove in livari harbors a lot of species colonizing highly oceanic (ko 7) and extremely oceanic (ko 8–9) localities. caloplaca herbidella, leptogium saturninum, lobarina scrobiculata and rinodina sophodes are included in the category ko 7, degelia plumbea, lobaria amplissima and parmeliella triptophylla in the category ko 8–9. nephroma laevigatum and peltigera collina are between these two categories. on the other hand we did not find any lichen species of the categories ko 1–2 (colonizing extremely continental localities) and ko 3 (colonizing highly continental localities). the arithmetic mean of all species with regard to eco-climatic oceanity comes to 5 (ko 5 = colonizing moderately oceanic localities). acknowledgements we would like to thank peter kosnik for his technical support with tlc, pramodchandra harvey for valuable linguistic suggestions, toby spribille for the determination of xylographa soralifera, branka kne`evi}, bojana nedovi}, danka petrovi}, miladin jaki} for their assistance during the field work and the hydrometeorological institute of montenegro for supplying climate data. financial support from the world university service (wus) austria and from the austrian science foundation (fwf project p20842-b16) is gratefully acknowledged by h.m. and p.o.b. references abbott, b. f. m., 2009: checklist of the lichens and lichenicolous fungi of greece. bibliotheca lichenologica 103, 1–368. aprile, g. g., catalano, i., migliozzi, a., mingo, a., 2011: monitoring epiphytic lichen biodiversity to detect environmental quality and air pollution: the case study of acta bot. croat. 72 (2), 2013 343 lichenized fungi of a chestnut grove in livari (rumija, montenegro) roccamonfina park (campania region – italy). in: moldoveanu, a. m. 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[oun, j., vondrák, j., søchting, u., hrouzek, p., khodosovtsev, a., arup, u., 2011: taxonomy and phylogeny of the caloplaca cerina group in europe. lichenologist 43, 113– 135. suija, a., jüriado, i., leppik, e., randlane, t., 2006: new estonian records. new lichens and lichenicolous fungi. folia cryptogamica estonica 42, 103–105. suppan, u., prügger, j., mayrhofer, h., 2000: catalogue of the lichenized and lichenicolous fungi of slovenia. bibliotheca lichenologica 76, 1–215. tretiach, m., ganis, p., 1999: hydrogen sulphide and epiphytic lichen vegetation: a case study on mt. amiata (central italy). lichenologist 31, 163–181. vefzda, a., 1968: lichenes selecti exsiccate. fasc. xxix (no. 701–725). instituto botanico academiae scientiarum ^echoslovacae, pru° honice prope pragam. vefzda, a., 1971: lichenes selecti exsiccate. fasc. xxxix (no. 951–975). instituto botanico academiae scientiarum ^echoslovacae, pru° honice prope pragam. vondrák, j., [oun, j., redchenko, o., lökös, l., khodosovtsev, a., 2009: populations of two caloplaca species with peculiar ecology observed in the bükk mts, hungary. bryonora 44, 8–12. white, f. j., james, p. w., 1985: a new guide to microchemical techniques for the identification of lichen substances. bulletin of the british lichen society 57 (suppl.), 1–41. wirth, v., 1995: flechtenflora. bestimmung und ökologische kennzeichnung der flechten südwestdeutschlands und angrenzender gebiete. ulmer, stuttgart. wirth, v., 2010: ökologische zeigerwerte von flechten – erweiterte und aktualisierte fassung. herzogia 23, 229–248. 346 acta bot. croat. 72 (2), 2013 mayrhofer h., drescher a., ste[evi] d., bilovitz p. o. 625 pise and gaikwad.vp acta bot. croat. 72 (2), 347–358, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 doi: 10.2478/botcro-2013-0011 germination characteristics of salicornia patula duval-jouve, s. emerici duval-jouve, and s. veneta pign. et lausi and their occurrence in croatia nina šajna1, marjana regvar2, simona kaligari^3, @eljko škvorc4, mitja kaligari^1* 1 department of biology, faculty of natural sciences and mathematics, university of maribor, koroška 160, 2000 maribor, slovenia 2 department of biology, biotechnical faculty, university of ljubljana, ve~na pot 111, 1000 ljubljana, slovenia 3 the institute of the republic of slovenia for nature conservation, regional unit of maribor, pobreška 20a, 2000 maribor, slovenia 4 faculty of forestry, university of zagreb, svetošimunska 25, 10000 zagreb, croatia abstract – according to recent molecular analyses of salicornia, we revised the annual glassworts from the croatian coast, classified until now only as salicornia europaea. two species, a diploid salicornia patula and a tetraploid s. emerici were recognized. they can be easily distinguished by floral characters, but not only by their habitus, which varies extremely according to environmental factors. both species differ also in seed morphology. salicornia patula has dimorphic seeds, with larger central seeds reaching high germination rates. germination patterns helped to explain the habitat preferences. the species rarely co-occur, however. the rare s. patula occupies drier habitats, on coastal mudflats or sands that are irregularly inundated. it occurs within the assoc. suaedo maritimae-salicornietum patulae. salicornia emerici occupies the lowest coastal mudflats, regularly inundated, where nutrient-rich conditions prevail, and forms an almost monotypical assoc. salicornietum emerici. due to the synonymy of s. veneta with s. emerici, we exclude the occurrence of s. veneta in croatia as an independent taxon. keywords: salicornia patula, salicornia emerici, salicornia veneta, species description, habitat preference, thero-salicorniatea introduction the genus salicornia l. comprises annual glassworts of the tribe salicornieae dumort. (chenopodiaceae). they are halophytic herbs with articulated succulent stems (davy et al. acta bot. croat. 72 (2), 2013 347 * corresponding author, e-mail: mitja.kaligaric@uni-mb.si copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 2001) exhibiting extreme phenotypic plasticity (ingruille and pearson 1987). the reduced leaf and flower morphology provides few taxonomical characters (kadereit et al. 2007), which cause difficulties in determination with standard determination keys. another obstacle is inbreeding on one side and hybridization on the other side. although numerous taxa have been described in the last 250 years, ball (1993) and stace (1997) recognized only three species: salicornia pusilla j. woods (with one-flowered cymes), s. europaea l. aggr. (incl. s. ramosissima j. woods, s. europaea l. and s. obscura p. w. ball et tutin) and s. procumbens smith aggr. (incl. s. nitens p. w. ball et tutin, s. fragilis p. w. ball et tutin and s. dolichostachya moss). the aggregate s. europaea represents a diploid group (2n = 18), a very variable group due to autogamy. the aggregate s. procumbens represents the tetraploid group (2n = 36). many morphometric studies have been aimed at defining european taxa (huiskes et al. 1985, ingruille and pearson 1987, ingruille et al. 1990) while a comprehensive taxonomic classification with determination key of species found along the french coast is described by lahondère (2004). recently, the taxonomy and phylogeny of glassworts, based on its sequences and atpb-rbcl spacer (kadereit et al. 2006), showed that the annual and perennial glassworts (salicornia / sarcocornia) are one lineage. a narrower phylogenetic analysis showed that within four species of salicornia, only two groups emerged, which corresponded with the ploidy level – diploids and tetraploids respectively (papini et al. 2004). the latest most comprehensive molecular study, where ets sequences of 164 accessions belonging to 31 taxa were elaborated, showed that all adriatic diploids clustered in one clade (s. perennans clade) and that all italian diploids within this clade are named s. patula (kadereit et al. 2007). all eurasian tetraploids belong to the same clade (s. dolichostachya clade), where s. emerici and s. veneta (distributed in italy) are clustered together. the newest study by kadereit et al. (2012) simplified the classification of annual eurasian glassworts to only four species and ten subspecies. salicornia patula became a synonym for s. perennans willd. and both tetraploids, s. emerici and s. veneta (also previously identified as synonyms by kaligari^ et al. 2008), became a synonym for s. procumbens subsp. procumbens. our previous molecular and morphological study from the gulf of trieste, comprising the slovenian and italian coastlines, including the »locus classicus« of s. veneta in the grado lagoon, has resulted in a clear distinction between diploids and tetraploids by flow cytometry (kaligari^ et al. 2008). additionally, its analysis of nrdna showed only two species present (s. patula and s. emerici), though we recognized four morphs, based on vegetative traits, in the beginning (»patula«, »emerici«, »veneta«, and »saline type«). the levels of ploidy matched to a high extent only with traits concerning floral morphology (length of the middle fertile segment, length of the lateral flower and, width of the scarious margin of the fertile segment). additionally, beer et al. (2010) rejected the long-believed correlation between the presence of one or two stamens and ploidy level. the morph named »veneta«, collected in the locus classicus and on the slovenian coast near ankaran, was not supported as a separate entity either on a molecular or on a morphological basis and it was classified in the frame of the tetraploid s. emerici (kaligari^ et al. 2008). results mentioned above give a new perspective. tetraploids (s. emerici) appear to be more common than diploids (s. patula) in the northern adriatic. they occupy lower mudflats, with higher moisture and inundation by regular tides (with additional flow of 348 acta bot. croat. 72 (2), 2013 šajna n., regvar m., kaligari^ s., škvorc @., kaligari^ m. nutrients) than diploids, which occupy higher elevations in the salt marsh flooded only occasionally or even exposed to periods of drought and nutrient limitations. the commonness of tetraploids can be explained by the fact, that the sedimentary coast of the friuli plain offers suitable conditions for tetraploid assemblages. optimal habitats are also developed in the form of the »barene«, mostly tabular mudflats of the lagoons (e.g. of grado and venice), periodically flooded by the tide with nutrient-rich seawater, which are occupied by a special morph described by lausi (1969) as salicornia veneta. on the eastern adriatic coast, comprising also the croatian coast, the annual glassworts were classified only roughly as s. europaea or s. herbacea with no information on ploidy level (domac 1984, 2002). in the red book of the vascular flora of croatia a tetraploid s. veneta is also mentioned, however, it is noted as »data deficient« taxon (nikoli] and topi] 2004). in the actual national database of the croatian flora (nikoli] 2007) s. veneta is cited from two localities, one from the island of rab and another from the close proximity of stari grad. in the same database many localities all along the adriatic coast are recorded for s. europaea. in this study, several questions were asked concerning the determination, habitat preference, and the distribution of glassworts in croatia: (1) which characteristics of the seed, besides seed size, are significant for the determination of diploids and tetraploids?; (2) do seeds from the s. emerici, »s. veneta« morphotype, and s. patula vary in their germination rate?; (3) which species of salicornia appear further along the southeast in croatia, and how are they distributed?; (4) in which halophile associations do they grow? to address these questions, we combined known and new species characteristics in an extended species description with photographs. we evaluated whether differences in germination rates support the genetic distinction between diploids and tetraploids. in order to find and to determine annual glassworts further to the east, we checked some of the known croatian coastal wetlands, where we collected data about the distribution and compared the phytosociological observations of species composition. materials and methods seed characteristics seeds of salicornia emerici, s. patula, and »s. veneta« morphotype were collected late september to october 2005 from ankaran (45°34’n, 13°44’e), strunjan (45°32’n, 13°36’e), and laguna di grado – primero (45°43’n, 13°26’e) (the locations are presented in fig. 1). we measured seed length and width for 20 central and lateral seeds of, s. patula, s. emerici and the »s. veneta« morphotype. additionally, we weighed 50 seeds of each species four times to obtain the seed mass. we were also prompted by the work of iberite (1996, 2004), who mentioned the curving of the trichomes on the seed testa as a diagnostic character, to perform observations of 10 trichomes covering the seed testa on three seeds. observations were performed under the microscope and their length and width were noted. we did not distinguish between main and lateral seeds except in the case of s. patula. multiple comparisons of seed characteristics were done with non-parametric kruskal – wallis anova median test. acta bot. croat. 72 (2), 2013 349 germination of salicornia species germination tests seeds from the same samples mentioned above were used in germination studies. seeds did not undergo cold treatment and were dry stored at 20 °c until germination tests in march 2006. we investigated germination responses of seeds originating from the central flower and those from lateral flowers separately, since germination patterns can differ between dimorphic seeds of a single species (imbert 2002). germination rate was tested in a petri dish on a filter paper watered with 10 ml of deionized water to achieve the best germination response. we tested 20 seeds per dish and 3 dishes in every test, conducted in a growing chamber at 23 °c with a 16/8 h photoperiod. the effect of salinity was tested by adding sodium chloride to deionized water. berger (1985) stressed, in his study about germination of s. patula, that in the 340 mm nacl solution central seeds were still able to germinate, while lateral seeds were not. we used the above mentioned 340 mm nacl solution, though. germinated seeds were counted daily and the germination rate was followed for 18 days. investigations in croatia the study areas were the following coastal wetlands in the kvarner and northern dalmatia (fig. 1): ancient salt pans near osor (44°41’39’’n, 14°23’49’’e), three locations on the island of pag: zr~e (44°32’29’’n, 14°54’51’’e), pag (44°25’59’’n, 15°03’51’’e) and dinjiska (44°21’47’’n, 15°10’09’’e), wetland near rtina (44°15’51’’n, 15°18’22’’e), wetland and estuary near ljuba~ki stanovi (near ljubac 44°14’45’’n, 15°17’15’’e), nin – dunes (44°14’53’’n, 15°11’05’’e) and nin – salt pans (44°14’21’’n, 15°11’16’’e). plants were determined according to the determination key kaligari^ et al. (2008). nomenclature follows iberite (2004) for taxa and frondoni and iberite (2002) for syntaxonomy. on each site a short vegetation survey was made. 350 acta bot. croat. 72 (2), 2013 šajna n., regvar m., kaligari^ s., škvorc @., kaligari^ m. croatia slovenia italy 3 1 adriatic sea 20 km 2 4 5 6 7 8 910 fig. 1. plants originating from italian (3), slovenian (1, 2), and croatian (5–10) salicornia populations. results seed characteristics seed length and width for central seeds did not significantly differ among salicornia emerici, »s. veneta« morphotype or s. patula (kruskal – wallis anova median tests; length: c2 = 2.828, df = 2, p = 0.243; width: c2 = 2.54, df = 2, p = 0.280). significant differences were shown for the length and width of lateral seeds (kruskal – wallis anova median tests; length: c2 = 15.758, df = 2, p < 0.001; width: c2 = 7.60, df = 2, p = 0.03), herewith confirming the dimorphism of s. patula seeds. the highest seed mass was found in the central seeds (0.51 ± 0.011 mg) from s. veneta, followed by seeds from s. patula (0.45 ± 0.003 mg) and s. emerici (0.42 ± 0.077 mg). lateral seeds had a lower seed mass on general »s. veneta« morphotype: 0.32 ± 0.010 mg; s. patula: 0.39 ± 0.035 mg; s. emerici: (0.34 ± 0.005 mg). seed dimorphism was not observed in seed mass for seeds of s. patula. trichome length, like width, did not significantly differ among seeds from s. emerici, »s. veneta« morphotype and dimorphic s. patula seeds (central, lateral seeds) according to kruskal – wallis anova median tests (length: c2 = 3.445, df = 3, p = 0.328; width: c2 = 7.250, df = 3, p = 0,064). extended species description the presented species description is based on the determination key from kaligari^ et al. (2008) with added seed characteristics and photographs. the new nomenclature is inserted in parentheses following kadereit et al. (2012). salicornia patula (= s. perennans willd.) is an erect plant, a few to 40 cm high. the branching is simple to profuse, always from the base (fig. 3a). generative parts are small. short fertile shoots (16–22 mm) bear 7 to 9 short (c. 3 mm) and thin (c. 2.5 mm) fertile segments, which have distinctly convex sides (fig. 4a). end of a segment has a narrow scarious edge (<0.3 mm). the inflorescence has unequal flowers with a larger central flower and distinctly smaller lateral flowers (<2 mm). seed dimorphism is present with smaller lateral seeds (<1 mm) and bigger central seed (1.3–1.5 mm). smaller seeds are dark brown, while bigger seeds are light brown. salicornia emerici (= s. procumbens subsp. procumbens) is usually erect, 35 to 45 cm high, densely branched with lower branches curving upwards (fig. 3b). long fertile shoots (27–33 mm) consist of 8 to 9 fertile segments, which are cylindrical, >4 mm long and >3 mm wide (fig. 4b). scarious edge is wider (about 0.4 mm) as in s. patula. flowers in the fertile segments are nearly equal in size (>2 mm) and seeds are not dimorphic. plants of »salicornia veneta« morphotype (= s. procumbens subsp. procumbens) have floral characteristics similar to those of s. emerici, however, the plants of this morphotype of s. emerici are less branched, narrowed and 30 to 40 cm tall. they exhibit a pyramidal form of the shoot, probably shaped by the tidal regime of open seacoasts. germination response the germination response of seeds from central and lateral seeds in s. patula was different from seeds of s. emerici and s. veneta (fig.2). the overall highest germination rates and the greatest germination velocity (100% in 6 to 7 days) were shown for central acta bot. croat. 72 (2), 2013 351 germination of salicornia species seeds of s. patula in non-saline conditions (tab. 1). both tetraploid species, with no obvious seed dimorphism, showed a very similar germination pattern: slightly better germination of lateral seeds in deionized water (fig. 2b) and slightly better germination of the central seed 352 acta bot. croat. 72 (2), 2013 šajna n., regvar m., kaligari^ s., škvorc @., kaligari^ m. 0 2 4 6 8 10 12 14 16 18 0 20 40 60 80 100 a 0 2 4 6 8 10 12 14 16 18 0 20 40 60 80 100 b 0 2 4 6 8 10 12 14 16 18 0 20 40 cg e rm in a ti o n [% ] days 0 2 4 6 8 10 12 14 16 18 0 20 40 d s. emerici s. veneta s. patula days fig. 2. germination percentage of main (a, c) and lateral (b, d) seeds in non-saline (a, b) and saline (c, d) condition in salicornia. tab. 1. results of germination test in dh2o and saline conditions (340 mm nacl) for main seeds and lateral seeds of diploid salicornia patula (= s. perennans) and tetraploids salicornia emerici and the »salicornia veneta« morphotype (both synonyms for s. procumbens subsp. procumbens). time (day) germination rate of main seed/lateral seed [%] conditions: dh2o salinity (340 mm nacl) s. emerici s. veneta s. patula s. emerici s. veneta s. patula 1 7.5/2.5 2.5/0 5/0 5/0 0/0 0/0 2 17.5/7.5 20/7.5 5/0 7.5/0 7.5/0 0/0 3 20/10 30/10 30/0 7.5/0 10/0 5/0 4 22.5/10 35/12.5 90/0 7.5/0 10/0 10/0 5 22.5/10 35/12.5 90/0 7.5/0 10/0 10/0 6 25/20 40/15 95/0 7.5/0 10/0 10/0 7 25/30 47.5/30 100/5 7.5/0 10/0 25/5 8 27.5/32.5 50/32.5 100/5 7.5 20/0 25/5 9 37.5/50 52.5/57.5 100/5 12.5 20/0 25/5 10 37.5/50 52.5/57.5 100/5 12.5 20/0 25/5 11 40/50 52.5/57.5 100/5 12.5 22.5/2.5 25/5 12 40/52.5 52.5/60 100/5 12.5 22.5/2.5 30/5 13 40/52.5 52.5/60 100/5 12.5 22.5/2.5 30/5 14 40/55 52.5/60 100/5 15 22.5/2.5 30/5 15 40/57.5 52.5/65 100/5 15 22.5/2.5 30/5 16 40/57.5 52.5/67.5 100/5 15 22.5/2.5 30/5 17 40/57.5 52.5/67.5 100/5 15 22.5/2.5 30/5 18 40/57.5 55.5/70 100/5 15 22.5/2.5 30/5 in saline conditions (fig. 2c). in each species the germination rate was inhibited and the germination of lateral seeds was especially slowed down with salinity. acta bot. croat. 72 (2), 2013 353 germination of salicornia species fig. 3. plant architecture of salicornia patula (a) and salicornia emerici (b). fig. 4. the differences of the shape (red arrow), length (blue arrow), and the scarious margin width (green arrow) of fertile segments between salicornia patula (a) and salicornia emerici (b). phytosociological classification salicornia patula grows on the border or even together with the perennial halophyte stands of sarcoccornetea fruticosae and juncetea maritimae. salicornia patula often forms small stands where it is the dominant species but shows low cover percentage. it grows together with suaeda maritima, puccinellia festuciformis, salsola soda, sarcocornea fruticosa, halimione portulacoides, atriplex prostrata etc. it penetrates stands of limonio-artemisietum coerulescentis horvati] (1933) 1934 and into puccinellio festuciformis-arthrocnemetum fruticosi (br.-bl. 1928) géhu 1976 (= salicornietum fruticosae br.-bl. 1928) (pand a et al. 2007). small patches of s. patula-dominated stands with suaeda maritima were also found in the locality nin – sand dunes and near the estuary of ljuba~. these stands could be attributed to the association suaedo maritimae-salicornietum patulae brullo et furnari 1976 ex géhu et géhu-franck 1984. this association is cited to be one of the most widespread glasswort associations in the mediterranean (frondoni and iberite 2002), but not in the eastern adriatic. stands of s. emerici are almost monocultures, with some suaeda maritima, phragmites australis, limonium angustifolium aggr. etc. these stands are easily attributed to association salicornietum emerici o. bolós ex brullo et furnari 1976. salicornia emerici is also widespread in the gulf of trieste (e.g. grado lagoon, slovenian coast; own unpublished data). on the croatian coast this association has been observed in the salt pans of nin, in the estuaries near rtina, in salt pans near dinjiska (pag) and in the coastal lagoon of zr~e (pag). among the observed glassworts in croatia we did not find any resembling the »s. veneta« morphotype. discussion taxonomic determination even though salicornia species show considerable phenotypic plasticity often related to environmental gradients, at least some morphological differences have a genetic basis (teege et al. 2011, vanderpoorten et al. 2011). our previous study showed that morphometrical data of generative parts of the plants corresponded better with ploidy level as vegetative parts (kaligari^ et al. 2008). additionally, tetraploids and diploids remained easy to recognize after seed shedding, since dried generative shoots of the latter were smaller. therefore we were expecting some correspondence of seed characteristics with ploidy. however, besides seed dimorphism in s. patula no other seed characteristic was useful for species determination. the curving of trichomes used by iberite (1996, 2004) turned out to be a very doubtful as a diagnostic character, for trichomes vary according to the position on the testa. on each seed we observed straight and spirally curved trichomes and we did not observe any trichome characteristics exclusive to any species. germination and habitat preference salicornia patula and s. emerici have a characteristic habitat preference. they occupy different microhabitats and rarely co-occur. salicornia patula inhabits the upper levels of alluvial mudflats or sandy soils or artificial structures (like ditches in salt pans). this means the driest, hypersaline and nutrient poor stands during summer (jefferies et al. 1979). davy and smith (1985) described a similar distinction in habitats between diploid s. pusilla (now 354 acta bot. croat. 72 (2), 2013 šajna n., regvar m., kaligari^ s., škvorc @., kaligari^ m. s. europaea subsp. europaea; kadereit et al. 2012) and the tetraploid pioneer s. dolichostachya further seawards (now s. procumbens susp. procumbens; kadereit et al. 2012), along the salt-marsh gradient of the norfolk coast of britain. it seems that there is a general rule of habitat distinction between diploids, occupying the upper part, and tetraploids, forming pioneer communities at the lower part of the salt-marsh gradient. our results show high germination rate of the central seed and the highest rate among all investigated species, while the lateral, most abundant seeds on the plant, hardly germinated. central seeds are also more salt tolerant than the same seed type of s. emerici or s. veneta. lateral seeds are smaller, they are more likely to form a persistent seed bank (carter and ungar 2003) and represent a source for plant population recovery after drastic disturbance events such as flooding (imbert 2002). this could explain the rarity of s. patula. since persistent seeds poorly germinate, the species finds it hard to cope with disturbances and is only able to occupy more stable habitats, even though drought and high saline conditions might prevail. additionally, more stable habitats are also scarcely distributed and are more frequently anthropogenically affected. salicornia emerici inhabits lower parts of the sea-exposed mudflats (avoiding thicker granulates, e.g. sand), often exposed to regular tide regime and rich nutrient flow. the estuaries or muddy shallow coasts are typical natural habitats, while the wet parts of the salt pans are man-made (where technology allows its development). our germination studies show no differences between the germination rate of the central and lateral seeds of s.emerici, even though the lateral germinate slightly better. however, s.emerici plants released more seeds that germinated per fertile shoot than s. patula (around 2 times as many germinating seeds). salicornia veneta the status of s. veneta as a separate species has been clearly put in doubt already by kaligari^ et al. (2008), however, kadereit et al. (2012) finally classified this taxon as a synonym for s. procumbens subsp. procumbens (which includes also s. emerici). an addi4tional characteristic contradicting the endemic status of s.veneta, besides molecular analysis and floral morphology, is the incongruence with the assumed restricted northern adriatic endemism distribution. namely, the »s. veneta« morphotype was found in several other parts of italy (iberite 1996) and on sardinia as well (filigheddu et al. 2000). according to teege et al. (2011), a new species of salicornia should only be accepted when besides their morphological distinctness, their monophyly has been shown by molecular data. however, synonymizing s. veneta could raise several conservation questions, because s. veneta has been selected as a flagship species currently listed in annex i of the bern convention (anonymous 1979) and in the annex ii of the habitat directive (anonymous 1992). even though we recognized s. veneta in the field by its pyramidal plant architecture the main certainty of the determination was underlined by the site of plant collection, which was the type locality (locus classicus). our results (kaligari^ et al. 2008) show that generative parts of s. veneta are not distinct from tetraploid s. emerici (now s. procumbens subsp. procumbens; kadereit et al. 2012). furthermore, here we present the lack of functional distinctness related to germination. neither germination pattern nor germination rate of the »s. veneta« morphotype differed from the values observed for s. emerici, either for the main acta bot. croat. 72 (2), 2013 355 germination of salicornia species seed, or for the lateral seed, irrespective of conditions beng saline or non-saline. these findings suggest that tetraploids »s. veneta« morphotype and s. emerici could be described as ecotypes, which was suggested for other tetraploid european salicornia species by teege et al. (2011). on the other hand, continuous patterns of habitual variation might be explained by the phenotypic plasticity. according to our results plants recognized as »s. veneta« would be better described as morphotypes since we did not study ecological factors and we did not prove delineation between s. emerici and »s. veneta« based on ecological characteristics. conclusion considering the new insights given from molecular studies we can so far recognize and confirm only salicornia emerici (now s. procumbens subsp. procumbens; kadereit et al. 2012) and s. patula (now s. perennans willd.; kadereit et al. 2012) as taxa of glassworts present on the croatian coast. we propose salicornia veneta should not be retained as a separate taxon and its conservation value should be expanded to the s. procumbens group (sensu kadereit et al. 2012). we would like accodingly to encourage further phytosociological research on the basis of a new taxonomic delineation, enriched with environmental and functional data. acknowledgements the authors would like to thank b. livio for the fieldwork and k. cafuta for laboratory assistance. the authors acknowledge valuable comments and suggestions of the anonymous referee. the research for this paper was funded by the program group »biodiversity« (p1-0078) and the research projects j1-6577, l1-7001-006 and p4-0077 all founded by the slovenian research agency. references anonymous, 1979: bern convention on the conservation of european wildlife and natural habitats (bern convention): annex i. council of the european communities. anonymous, 1992: council directive 92/43/eec of 21 may 1992 on the conservation of natural habitats and of wild fauna and flora: annex ii. council of the european communities. ball, p. w., 1993: salicornia l. in: webb, d. a. 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n., regvar m., kaligari^ s., škvorc @., kaligari^ m. acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 291 acta bot. croat. 73 (2), 291–298, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 pcr detection assay for sex determination in papaya using scar marker kanupriya chaturvedi1, padmakar bommisetty1, arpita pattanaik1, vasugi chinnaiyan2, dinesh m. ramachandra2, aswath chennareddy1* 1 division of biotechnology, indian institute of horticultural research, hesaraghatta lake post, hesaraghatta, bangalore 560 089, india. 2 division of fruit crops, indian institute of horticultural research, hesaraghatta, bangalore, india abstract – papaya (carica papaya l., 2n = 18), a polygamous angiosperm, is a major fruit crop in tropical and subtropical regions. it is trioecious with three sex forms: male, female, and hermaphrodite, where sex determination is controlled by the xy chromosome pair with two slightly different y chromosomes i.e. y for male and yh for hermaphrodite. sex type determination in papaya, which cannot be determined either by embryo shape or morphology at the juvenile developmental stage, is an essential pre-requisite for crop improvement processes as it helps in identifi cation of fruitful plants. hence, molecular profi ling could be used as an alternative that provides a quick and reliable identifi cation of sex types in plantlets at initial stages only. in the present study we have validated the sexlinked sequence characterized amplifi ed region (scar) marker w11 using pcr detection assay among different cultivars of papaya i.e. dioecious with either female or male and gynodioecious with either female or hermaphrodites and also performed a double-blind test for validating the seedlings of 84 f1 plants, which resulted in their sex determination. the assay clearly gives 800 bp band in male plants in dioecious types and hermaphrodite in gynodioecious plants. keywords: carica papaya, dioecious, double-blind test, gynodieocious, papaya, scar marker, sex determination introduction carica papaya l., a native of latin america, is a diploid (2n = 18) belonging to the family caricaceae. papaya is one of the most important fruit crops of india with a production of 4.71 million tons grown over an area of 0.1 million ha (national horticulture board 2012). it is widely grown in many other countries also (tab.1). papaya is widely cultivated * corresponding author, e-mail: aswathiihr@gmail.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. chaturvedi k., bommisetty p., pattanaik a., chinnaiyan v., ramachandra d. m., chennareddy a. 292 acta bot. croat. 73 (2), 2014 for its edible fruits and milky latex that yields papain, a proteolytic enzyme, and carpain alkaloids that are of great economic importance. as the yields and proteolytic activity of the crude papain obtained from the female fruits are greater than that obtained from hermaphrodites (madrigal et al. 1980), dioecious papaya cultivars are preferred for the extraction of papain. the papaya is polygamous plant species with three main sex types, namely pistillate or female, staminate or male, and hermaphrodite (storey 1941). the results of earlier reports (hofmeyr 1938, storey 1938, 1941, 1953) on the genetic analysis of sex determination in papaya suggest that sex determination is controlled by three alleles. the genetics of sex is very intriguing in the papaya. among several hypotheses, the genetic balance hypothesis explaining sex determination in papaya (where factors determining femaleness are present in the sex chromosome and that of maleness in the sum total of autosomes) has been widely accepted (hofmeyr 1938). it is further assumed that m1 and m2 respectively represent the inert segments of sex chromosomes. the m1 is slightly larger than the m2 segment. it is presumed that genes responsible for life are eliminated in these inactive regions and cause the lethality of the genotypes m1m1, m1m2, and m2m2. however, the genotypes m1m and m2m will be viable due to the presence of an active sex chromosome. recent reports have proven that sex determination in papaya is controlled by a recently evolved xy chromosome pair, with two slightly different y chromosomes controlling the development of males (y) and hermaphrodites (yh) (gschwend et al. 2011, na et al. 2012, wang et al. 2012). traditionally, the propagation of papaya plants is through seeds, which would give rise to a population of generally 1:3 for male to female. however, the sex of these plants cannot be deduced from the external morphology of embryonic or juvenile forms. the sex of the seedlings can be detected only after the plants attain reproductive maturity, i.e. after 5–8 months. however, if the sex of the dioecious plants is identifi ed at the seedling stage, prior to their transplantation to the fi eld, then a desired ratio of male to female plants (5% males/95% females) can be achieved, thereby saving on the input cost. in tab. 1. global papaya production in million tons (source: national horticulture board 2012). countries 2002 2003 2004 2005 2006 2007 2008 2009 2010 india 2.15 1.69 2.54 2.14 2.48 2.91 3.63 3.91 4.71 brazil 1.60 1.71 1.61 1.57 1.90 1.81 1.89 1.79 1.87 indonesia 0.61 0.63 0.73 0.55 0.64 0.62 0.72 0.77 0.70 nigeria 0.76 0.80 0.86 0.76 0.76 0.77 0.69 0.76 0.70 mexico 0.88 0.96 0.79 0.71 0.80 0.92 0.64 0.71 0.62 ethiopia 0.23 0.23 0.26 0.30 0.26 0.23 0.25 0.26 0.23 democratic republic of the congo 0.21 0.21 0.21 0.22 0.22 0.22 0.22 0.22 0.23 colombia 0.09 0.09 0.10 0.14 0.16 0.22 0.21 0.19 0.26 thailand 0.35 0.31 0.28 0.03 0.13 0.20 0.20 0.21 0.21 guatemala 0.05 0.07 0.08 0.10 0.11 0.18 0.19 0.20 0.20 other 1.41 1.46 1.39 1.49 1.43 1.37 1.41 1.44 1.49 total 8.32 8.17 8.85 8.01 8.90 9.45 10.050 10.460 11.220 sex determination assay in papaya acta bot. croat. 73 (2), 2014 293 the case of gynodieocious types, the hermaphrodite plants give pyriform fruits and the female round fruits. however, from the marketing point of view, pyriform fruits are desirable. as the segregation in this case is also 1:1 for female to hermaphrodite, identifi cation at the early stage is desirable. molecular markers have been studied earlier for sex determination in papaya (sondur et al. 1996, somsri et al. 1998, parasnis et al. 1999, 2000, niroshini et al. 2000, 2008, urasaki et al. 2002a, b, deputy et al. 2002, lemos et al. 2002). recently a transcriptomics study has been conducted for sex determination in papaya (urasaki et al. 2012). however, none of these studies validated the use of sequence characterized amplifi ed region (scar) markers across a large population of dioecious and gynodieocious cultivars and a population with an intergeneric cross. in this paper the scar marker w11 (deputy et al. 2002) was initially tested among dioecious and gynodieoecious cultivars and was later used to determine the sex of 84 seedlings of an intergeneric cross of carica papaya × vasconcella caulifl ora containing a mixture of male, female and hermaphrodite plants. materials and methods a total of 98 papaya plants of which a known set of 14 cultivars comprising 6 dioecious and 8 gynodieocious plants (tab. 2) and unknown set of 84 f1 seedlings of carica papaya × vasconcella caulifl ora maintained at the indian institute of horticultural research in bangalore were taken for the study. in the unknown set all the seedlings were marked to confi rm scar marker results when the seedlings produced their fi rst fl ower. healthy and mature leaf tissue (2 g) was ground in liquid nitrogen until it formed a very fi ne powder, and genomic dna was extracted by using modifi ed ctab (hexadecyltrimethylammonium bromide) method (doyle and doyle, 1990). purifi ed dna was quantifi ed using genequant uv-spectrophotometer (ge health care bio-sciences ltd, u.k.) and diluted accordingly for further analysis. tab. 2. list of dioecious and gynodioecious papaya (carica papaya l.) varieties used for validation. type sample number name of the variety and sex type dioecious 1 2 3 4 5 6 line 21 male line 21 female shilong male shilong female nigeria male nigeria female gynodioecious 7 8 9 10 11 12 13 14 arka prabhat female arka prabhat hermaphrodite surya hermaphrodite surya female thailand hermaphrodite thailand female dwarf lilly female dwarf lilly hermaphrodite chaturvedi k., bommisetty p., pattanaik a., chinnaiyan v., ramachandra d. m., chennareddy a. 294 acta bot. croat. 73 (2), 2014 pcr amplifi cation profi le was carried out in 25 ml volume containing 10 mm tris-hcl, ph 8.3, 1.5 mm mgcl2, 50 mm kcl, 0.2 mm each dntp, 0.5 mm each primer, 50 ng genomic dna and 0.6 units of taqdna polymerase (bangalore genei, bangalore). reactions were carried out in an eppendorf mastercycler gradient thermocycler (eppendorf, hamburg, germany) using the following temperature profi le: an initial step of 5 min at 95 °c, 30 cycles of 1 min at 95 °c, 1 min at 58.5 °c and 1 min at 72 °c, and a fi nal step of 7 min at 72 °c. amplifi cation products were screened by electrophoresis using 1.5% agarose gel. results diverse dioecious and gynodioecious papaya cultivars were initially screened for linkage of w11 to sex type. the reaction profi le of this scar marker assay was optimized by modifying the earlier mentioned protocol (deputy et al. 2002) through a change of the concentrations of pcr ingredients, such as a decrease in mgcl2 concentration, an increase in the concentration of dntps, primers, dna and taq polymerase. amplifi ed fragment of 800 bp were obtained in hermaphrodite and male papaya plants only, but not in female plants. this amplifi cation pattern was confi rmed by initial screening and validated in true male, female and hermaphrodite plants of both dioecious and gynodioecious cultivars of carica papaya and visconcella caulifl ora (fig. 1 a, b), which were used as controls for later validation assay. fig. 1. amplifi cation pattern of w11 marker among male, female and hermaphrodite specimens of dioecious and gynodioe cious papaya plants.w11 scar marker giving an amplicon of 800 bp size only in male and hermaphrodite, but not in female plants; m – 1 kb ladder; a) from 1 to14: male, female and hermaphrodite papaya cultivars (refer to table 2 for details); b) 1 – ‘surya’ (female), 2 – ‘shillong’ (male), 3 – ‘surya’ (hermaphrodite), 4 – v. caulifl ora (female), 5 – v. caulifl ora (male). we used 84 f1 two month old seedling plants of an intergeneric cross of carica papaya × visconcella caulifl ora, information about the sex of which was unknown, for validation purpose. genomic dna was extracted from tender and healthy leaves of 10 day old leaves. these seedlings were maintained until adult stage and used for cross-verifi cation in the fi eld. of 84 papaya seedlings, a screened amplifi ed 800 bp fragment was obtained in only three sex determination assay in papaya acta bot. croat. 73 (2), 2014 295 plants (results not shown) indicating that they might be either hermaphrodite or male, while those lacking 800 bp fragment were considered to be females. the analysis was repeated three times, confi rming the reproducibility of results. internal controls were used during each step of the analysis. after fl owering we observed that the plants that showed the band and were tagged were all hermaphrodites and rest were females. discussion prior information about the sex type in papaya is necessary before an elite breeding program is planned. from studies on controlled pollinations over years (singh 1990), it was established that in the progenies of a cross whose parents are known (pure line), the proportion of males and bisexual or female follows a defi nite ratio. in the dioecious types, if fl owers of female plants are pollinated with the fl ower of male plants the progenies obtained will have approximately 50% males and 50% females. in the gynodioecious types where either bisexual plants are selfed or the female is cross pollinated by bisexual fl owers, the progenies obtained will be 50% females and 50% hermaphrodites. the gynodioecious types are better in fruit production as both female and hermaphrodite plants produce fruits, while in dioecious types only female plants produces fruits. previous studies have provided preliminary data indicating that random amplifi ed polymorphic dna (rapd) markers might be useful for detecting sex expression in papaya (sondur et al. 1996, somsri et al. 1998, niroshini et al. 2000, 2008, parasnis et al. 2000, lemos et al. 2002, urasaki et al. 2002a, b), but these reports utilized a relatively small number of plants. however, deputy et al. (2002) reported scar markers that are specifi c for male and hermaphrodite plants in a large number of plants. deputy et al. (2002) cloned three rapd-pcr products showing linkage to the gene that determines fl ower sex, sex1, in papaya. two of these rapd products, t12 and t1, had been previously mapped to 7 cm fl anking the sex1 gene. the third product w11, which we validated, was chosen because w11 is closer to sex1 than either t12 or t1. scar w11 and scar t12 were mapped in a papaya ‘sun-up’ and ‘kapoho’ cross. both of these cultivars are of the hawaiian type. all hawaiian types have previously been shown to be quite similar at the dna level (stiles et al.1993). scar w11 and scar t12 showed linkage in all 182 plants, indicating these markers are within 0.3 cm of sex1. there were no crossovers between scar t12 and sex1 in the 182 plants, indicating a linkage signifi cantly closer than the 7.0 cm previously reported by sondur et al. (1996). scar w11 produced products almost exclusively in males and hermaphrodites but not in females; however, it is not clear at this time whether the difference is the result of a limited number of base changes in the scar binding sites or more substantial alterations such as deletions of the binding sites or even the entire regions. in the present study the w11 scar marker validated in the cultivars of carica papaya and visconcella caulifl ora, amplifi ed a discriminating band in hermaphrodites and male papaya plants, but not in females, (figs. 1 a, b). the reason may be that the specifi c chromosomal region for sex type determination in papaya shares an identical segment of the y chromosome of males and hermaphrodite plants and is absent in females. this result is consistent with the expected result and as reported by deputy et al. (2002). liu et al. (2004) identifi ed the male specifi c (msy) region in hermaphrodite and male papaya plants and found that some sequences of this region chaturvedi k., bommisetty p., pattanaik a., chinnaiyan v., ramachandra d. m., chennareddy a. 296 acta bot. croat. 73 (2), 2014 are common in both male and hermaphrodite plants. in our studies the fragments of 800 bp shared identical sequences between male and hermaphrodite type papaya, and this may support the above study (data not shown). detection of sex linked rapd markers as well as the scar markers have been attempted in several dioecious species. thirty two male-specifi c rapd bands were identifi ed in hop (humulus lupulus l.) by the screening of 900 random primers (polley et al. 1997). others found one rapd fragment of 400 bp size, closely linked with the male sex type of hemp (cannabis sativa l.) (mandolino et al. 1999). the pointed gourd (trichosanthes dioica roxb.) has also been studied and found to have a rapd marker associated with females that is absent in all male plants (singh et al. 2002). similarly, the presence of a female-specifi c band in nutmeg (myristica fragrans houtt.) has also been reported by the screening of 60 operon primers (shibu et al. 2000). others have detected two rapd markers linked to m locus (maleness) in asparagus (asparagus offi cinalis l.) and successfully converted one of these bands to a scar marker (jiang and sink 1997). the results illustrate the possibility of developing a molecular marker based method to identify sex at seedling stage in other agriculturally important dioecious plants including nutmeg (m. fragrans houtt.), hemp (c. sativa l.), pistachio (pistacia vera l.), kiwifruit (actinidia chinensis p.), asparagus (a. offi cinalis l.). farming of these crops could greatly benefi t from the development of methods for sex detection at an early stage as a suffi ciently larger number of productive hermaphrodite or female (depending on the market preference) plants could be cultivated by minimizing the number of unproductive male trees. in our study the assay kit has been successfully used to identify the hermaphrodite plants in the segregating population (f1) of carica papaya × vasconcellea caulifl ora, which was developed for papaya ring spot virus (prsv) resistance. the results of this molecular based detection of sex type in papaya were in congruent with the morphological observations among 84 f1 papaya plants. the three plants that showed an amplifi cation of the 800 bp fragment turned out to be hermaphrodites while the remaining 81 plants were females. thus the present assay clearly identifi ed the true sex type in the seedling stage of the papaya plants. this assay is more like a double blind test where the plants were marked before fl owering as per our test and validated once the fl owering occurred. it saved time and also increased the confi dence level of the marker. this can go a long way in reducing the period taken for developing resistant lines for prsv. in the intergeneric hybridization, the female parent used was gynodioecious carica papaya and the male was the dioecious type vasconcellea caulifl ora. the sex of the segregants if identifi ed in the f1 or in f2 generation after screening would help in reducing the population size for further sib mating, which would be easily manageable. references deputy, j. c., ming, r., ma, h., liu, z., fitch, m. m. m., wang, m., manshardt, r., stiles, j. i., 2002: molecular markers for sex determination in papaya (carica papaya l.). theoretical and applied genetics 106, 107–111. doyle, j. j., doyle, j. l., 1990: isolation of plant dna from fresh tissue. focus 12, 13–15. sex determination assay in papaya acta bot. croat. 73 (2), 2014 297 gschwend, a. r., qingyi, y., eric, j. t., fan-chang, z., han, j., vanburen, r., aryal, r., charlesworth, d., 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gschwend, a. r., han, j., zeng, f., aryal, r., vanburen, r., murray, j. e., zhang, w., navajas-péreze, r., feltus, f. a., lemke, c., tong, e. j., chen, c., wai, c. m., singh, r., wang, m-l., jia min, x., alam, m., charlesworth, d., moore, p. h., jiang, j., paterson, a. h., ming, r., 2012: sequencing papaya x and yh chromosomes reveals molecular basis of incipient sex chromosome evolution. proceedings of the national academy of sciences of the united states of america 109, 13710–13715. 625 pise and gaikwad.vp acta bot. croat. 72 (2), 311–322, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 augmentation of major isoflavones in glycine max l. through the elicitor-mediated approach ramesh k. saini, muthu k. akitha devi, parvatam giridhar*, gokare a. ravishankar plant cell biotechnology department, csir-central food technological research institute, mysore 570 020, india. abstract – isoflavone content in soybean seeds was enhanced by the elicitor-mediated approach under field conditions through the floral application of abiotic elicitors-salicylic acid, methyl jasmonate and biotic elicitors-aspergillus niger and rhizopus oligosporus. among isoflavones, daidzein and glycitein were found to be highly responsive to elicitors, with an increase of 53.7% and 78.7%, respectively as compared to control. highest total isoflavone content (1276.4 mg g–1 of seeds) was observed upon the administration of 0.1 mm salicylic acid, which is 92.7% higher than in control. this study would be valuable for augmentation of the isoflavone content in soybean seeds in field grown plants for better nutraceutical potential. keywords: glycine max l, isoflavone, elicitation abbreviations: dpph – 2,2-diphenyl-1-pycrilhydrazil hydrate introduction isoflavones belong to a broad class of phytoestrogens and are the naturally occurring and abundant bioactive components in soybean. they have antiestrogenic, antioxidant, anti-inflammatory activities and are also associated with lower incidence of chronic diseases such as cancers, heart and kidney diseases and in addition prevent bone loss (messina 1999, parr and bolwell 2000, atkinson et al. 2004). estrogenic and anti-estrogenic effects of isoflavone depend on the natural levels of estrogen. at low level of natural estrogen, isoflavones mimic estrogen by activating the estrogen receptors (barnes et al. 2000), whereas, at high estrogen levels, they bind with estrogen receptors, decrease the availability of estrogen receptors and act as anti-estrogens (kuiper et al. 1998). in view of the various beneficial effects attributed to isoflavones, consumer interest in soybean products has been considerably enhanced, leading to the incorporation of soybean in a wide range of foods acta bot. croat. 72 (2), 2013 311 * corresponding author, e-mail: parvatamg@yahoo.com copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. (setchell and cole 2003). soybean and soy products contain twelve different isoflavone isomers including three principal aglycones (daidzein, genistein, and glycitein), their glycosides, and their corresponding acetyl and malonyl forms. several factors such as the genetic, the environmental, geographical location, storage and plant variety might influence the isoflavone content and composition in soybean seeds (wang and murphy 1994, hoeck et al. 2000, lee et al. 2003). recently, variations in the isoflavone levels and antioxidant activity in the soybean cultivars of india and bulgaria along with different soy products have been reported (sakthivelu et al. 2008, akitha devi et al. 2009). isoflavone content in soybean seeds (and other soybean tissues) is influenced by biotic and abiotic factors including physical and chemical damages, uv light, low temperature, wounding, pathogens and plant microbe interactions as well as elicitor treatment (wegulo et al. 2005, caldwell et al. 2005, zhang et al. 2006, phommalth et al. 2008). it is also found that these factors are involved in the up-regulation or induction of the phenyl propanoid pathway genes for the biosynthesis of isoflavones. elicitation is an efficient strategy that by means of compounds or treatments induces plants to synthesize phytoalexins at enhanced levels (rao and ravishankar 2002). flavonoid production is stimulated when the plant recognizes certain elicitor molecules or structures that characterize a pathogen or a symbiont (tahvonen 1988, al-tawaha et al. 2005). elicitors thus, have been used to increase plant resistance to various pests (beausejour et al. 2003), to enhance the content of beneficial phytochemicals in various plant species (kim et al. 2006, perez-balibrea et al. 2011) as well in nutraceutical industries (zhao et al. 2001). the dynamic mechanisms of elicitors are considered to be complex and the effect of elicitors depends on numerous factors, such as the concentration and type of the elicitor and the growth stage of the culture at the time of elicitation. both abiotic and biotic elicitors were previously studied for their efficacy in enriching isoflavones in mature seeds (al-tawaha et al. 2005, boue et al. 2008). however, all these studies were confined to either matured stage beans or cotyledonary seedlings. recently, isoflavones have attracted great interest due to their potent antioxidant activities through the stabilization of free radicals (gordon, 1990). significant losses of isoflavones (50% or more) occur during processing of the soybean in traditional soy foods (wang and murphy 1996), which is a bottleneck in the maintenance of effective levels of isoflavones in soy products. this problem could be addressed by increasing the isoflavone content and reducing the variability in soybean seeds. a novel elicitor mediated approach thus seems possible for increasing isoflavone synthesis and content in mature soybean seeds. in the present study, the efficacy of floral administration of selective elicitors to enhance the isoflavone content in glycine max and antioxidant potential is explored. materials and methods plant materials the soybean (glycine max l.) breeding line js-335 used in this study was selected because of its early maturity (90 days), maximum sown area in india and resistance to major diseases and pest. authentic seeds were obtained from the national seeds corporation, mysore. js-335 soybean is derived from the cross of js78-77 (kohar ´ p.s. 73-22) ´ 71 – 05. 312 acta bot. croat. 72 (2), 2013 saini r. k., akitha devi m. k., giridhar p., ravishankar g. a. preparation and application of elicitors biotic elicitors were prepared according to giridhar and parimalan (2010), using two fungal cultures aspergillus niger and rhizopus oligosporus that were obtained from the microbial culture facility of food microbiology department of csir-cftri. fresh cultures of a. niger and r. oligosporus were grown on potato dextrose agar (himedia, mumbai) slants and incubated for 7 days at 37 °c. then the spores of the respective fungi were used to prepare a spore suspension in 0.1% sodium lauryl sulfate (w/v) and diluted with sterile distilled water under sterile conditions to obtain a spore density of ~2.5 ´106 spores ml–1. later the same was inoculated into 150 ml erlenmeyer conical flasks containing 40 ml of potato dextrose agar and the cultures were incubated in the dark for 10 days. after the incubation, the cultures were autoclaved and the mycelium was separated from the culture broth by filtration and its fresh weight was recorded. an aqueous extract was made by homogenizing with a mortar and pestle using neutralized sand. the extract was filtered through whatman no. 1 filter paper, and kept as the stock solution from which the individual fungal mycelial extracts at a working concentration of 0.1, 0.25 and 0.5 % w/v (wet weight of fungal mycelium in 1000 ml of distilled water) were prepared in sterile water and used for the elicitation experiment. abiotic elicitors, salicylic acid and methyl jasmonate dissolved in distilled water and diluted to three concentrations (0.1 mm, 0.25 mm, and 0.5 mm) were used for the study. the field experiment was conducted at the plant cell biotechnology department of this institute. the soil type was red silt loam with fine-silty characteristics which is the best for soybean cultivation. js-335 soybean was planted in complete random block design (crbd) layout at the spacing of 1.5 m in length and 40 cm wide rows, with four replicates of control and each treatment. in each row, 5 plants were grown, with a plant to plant spacing of 20 cm. all the plants were maintained according to recommended management practice (icar (2006) throughout the growing season. all the elicitors were sprayed on the fully opened flower of js-335 soybean plants between 10 and 11 am. an equal quantity of distilled h2o was sprayed on control plants. a total 1000 ml of elicitor solution was sprayed in each treatment (four rows of total 20 plants) isoflavone extraction after complete maturity, approximately 60 days after treatments, seeds were harvested and stored at ambient temperature for 30 days. before extraction, seeds were dried at 37 °c for 48 hours. seed samples with the seed coat (400 mg) were finely ground and extracted with 2 ml of concentrated hcl and 10 ml of absolute ethanol (99.9% pure) for 2 hours in a boiling water bath using a standard method (vyn et al. 2002), which relies on acid hydrolysis of 12 endogenous isoflavone isomers to their respective aglycone forms, mostly daidzein, glycitein and genistein. the resulting suspension were cooled and centrifuged at 10,000 ´ g for 10 min. the supernatant obtained after centrifugation was filtered through a syringe filter (whatman 0.5 µm, 13 mm diameter). isoflavone standards standards of isoflavones, daidzein, genistein and daidzein were purchased from sigma-aldrich, bangalore. isoflavone stock solutions were prepared by dissolving the standards acta bot. croat. 72 (2), 2013 313 isoflavone enrichment in soybean in absolute ethanol at concentration of 1 mg ml–1. calibration curves were made for each standard in concentration range 0.01–0.1 mg ml–1. the content of individual isoflavones in the sample was calculated manually on the basis of each peak area. individual and total isoflavone content was expressed as micrograms per gram of dry weight. chromatographic conditions the hplc analysis of the respective extracts for isoflavones was performed using a shimadzu chromatograph (lc 20-ad, hplc), equipped with dual pump, uv detector (spd 20a) and a c-18 column (sunfire; 5 µm with dimension of 250 × 4.6 mm). the separation and elution of isoflavones was modified by employing a binary gradient mode using solvent a (10% acetonitrile) and solvent b (38% acetonitrile) with injection volume of 20 µl of the sample and at a flow rate of 0.8 ml min–1 for 40 min (kumar et al. 2009). the solvent system was run as follows (% solvent a/solvent b): 0 min (0/100), 5 min (10/90), 35 min (0/100), and 40 min (0/100). isoflavones were monitored at 260 nm. free radical scavenging activity dpph (2,2-diphenyl-1-pycrilhydrazil hydrate) radical-scavenging activity of elicitor treated and untreated seed extracts were evaluated by using the method of khan et al. (2011). an aliquot of 0.2 ml of the methanolic seed extract (2 mg ml–1) was added to a 3.8 ml absolute ethanol (99.9%) solution of dpph (a stable free radical) to a final concentration of 0.1 mm. the mixture was shaken vigorously for 1 min by vortexing and left to stand at room temperature in the dark for 30 min. subsequently, the absorbance of the sample (asample) was measured at 517 nm using the uv-visible spectrophotometer against ethanol as blank. a negative control (acontrol) was taken after adding dpph solution to 0.2 ml of the respective extraction solvent. the percent of dpph inhibition and scavenging of the sample was calculated according to the following equation: percent of inhibition and scavenging =[1 – a a sample control � � � � � � � �× 100] the free radical-scavenging activity of the extracts was expressed as percent of inhibition at 100 µg ml–1 seed extract. total phenolic content total phenolic content of treated and untreated seed extracts was determined by folin–ciocalteu assay using gallic acid as standard (kim et al. 2003). in brief, 50 µl of the sample was mixed with distilled water (3 ml), followed by 7% sodium carbonate (750 µl) solution and folin– ciocalteu reagent (250 µl). subsequently the solution was vortexed and incubated for 8 min at room temperature. to this, 950 µl distilled water was added and the reaction mixture was allowed to stand for 2 hours at room temperature, and finally the absorbance was measured at 725 nm using a uv–visible spectrophotometer (shimadzu uv 160) against distilled water as blank. the total phenolic content was expressed as mg of gallic acid equivalents g–1 of seeds. linearity range of the calibration curve was 50–1000 µg ml–1 (r = 0.98). 314 acta bot. croat. 72 (2), 2013 saini r. k., akitha devi m. k., giridhar p., ravishankar g. a. statistical analysis the isoflavone content, dpph scavenging activity and total phenolic content from all four replicates were measured separately, and represented as mean ± standard deviation (s.d.). data were analyzed statistically by spss 17.0 software by analysis of variance (one way anova), least significant difference (lsd) was calculated between the mean values of different treatments. different alphabetical letters were assigned to demonstrate significant difference between the treatments. results effect of elicitors on isoflavones in soybean seeds the effect of floral administration of several abiotic and biotic elicitors on the isoflavone levels of soybean seeds was determined in the present study. aglycone forms as well as total isoflavones were evaluated in elicitor-treated and untreated soybean seeds (tab. 1). the isoflavone content increased with an increase in the concentration of methyl jasmonate whereas it decreased with an increase in the concentration of salicylic acid, aspergillus niger and rhizopus oligosporus. among the major isoflavones analysed, daidzein and glycitein showed higher responses to elicitors with a mean increase of 53.7% and 78.7%, while, genistein increased negligibly (2.9%) compared to control. a maximum of 54.7% increase in total isoflavone content was observed in the experiment and a minimum of 27.3% in case of a. niger, compared to control. elicitors at different concentrations had a varying influence on isoflavone levels in soybean seeds. the total isoflavone response to salicylic acid was 14.7% greater then average. acta bot. croat. 72 (2), 2013 315 isoflavone enrichment in soybean tab. 1. isoflavone content (µg g–1) in mature seeds of soybean plants (var. js-335) treated with biotic and abiotic elicitors. values are mean ± sd of four independent replicates. different letters indicate statistically significant differences between the means (p < 0.05) for total and individual isoflavone content. treatment concentration daidzein glycitein genistein total isoflavone salicylic acid 0.1 mm 503.5 ± 25.1a 389.0 ± 35.7a 384.0 ± 35.0c 1276.4a 0.25 mm 283.6 ± 15.2g 191.3 ± 9.0e 438.1 ± 25.5b 913.1c 0.5 mm 393.3 ± 16.0de 270.2 ± 18.1c 221.1 ± 18.7g 884.7c methyl jasmonate 0.1 mm 331.9 ± 9.8f 173.5 ± 12.5ef 178.0 ± 14.7h 683.4e 0.25 mm 369.1 ± 16.9e 138.0 ± 15.9gh 256.5 ± 24.0f 763.5d 0.5 mm 442.9 ± 20.9bc 296.2 ± 25.1b 313.0 ± 39.8de 1052.0b aspergillus niger 0.10% 422.2 ± 20.7cd 161.3 ± 13.5f 524.0 ± 26.5a 1107.4b 0.25% 376.9 ± 44.8e 229.0 ± 12.5d 239.5 ± 8.9fg 845.5c 0.50% 201.7 ± 10.6i 123.3 ± 17.6h 342.6 ± 20.0d 667.7de rhizopus oligosporus 0.10% 465.3 ± 16.6b 271.7 ± 16.6c 335.5 ± 17.5d 1072.6b 0.25% 308.4 ± 17.2fg 225.0 ± 22.5d 225.2 ± 22.5g 758.7bd 0.50% 228.0 ± 13.0hi 155.7 ± 14.8fg 314.0 ± 21.0de 697.6de control – 234.6 ± 12.5h 122.4 ± 10.6h 305.5 ± 10.3e 662.5e highest total isoflavone content (1276.4 mg g–1 of seeds) was observed following the floral application of 0.1 mm salicylic acid, which represented a 92.7% increase in comparison to the untreated control. maximum daidzein and glycitein content (i.e. 503.5 and 389.0 mg g–1 of seeds, respectively) was observed in the same treatment, whereas maximum genistein content (i.e. mean of 524.0 mg g–1 of seeds) was observed in 0.1% a. niger treated seeds. in this study, salicylic acid as abiotic elicitor, and a. niger as biotic elicitor were found to be most effective for enhancement of isoflavone content in soybean seeds. antioxidant activity antioxidant activities of the elicitor-treated soybean seed extracts were evaluated by free radical scavenging assay and also by total phenolic content (tab. 2). in the present study, there was no significant difference in free radical scavenging activities between the control and the treated soybean seed extracts (p < 0.05). on the whole, the average free radical scavenging activity of elicitor treated and control seeds were approximately 80.0 %. the dpph scavenging activity did not correlate significantly with total isoflavones or with total phenolic content (r2 = 0.153). the total phenolic contents of elicitor-treated and control seed extracts are shown in table 2. the total phenolic content correlated well with total isoflavone (r2 = 0.767), as expected. thus, the trend in total phenolic content upon treatments is in agreement with trend in total isoflavone content. salicylic acid treatment (0.1 mm) produced the highest total phenolic content reaching 9.73 mg of gallic acid equivalent g–1 of seeds. 316 acta bot. croat. 72 (2), 2013 saini r. k., akitha devi m. k., giridhar p., ravishankar g. a. tab. 2. free radical scavenging activity and total phenolic contents of seed extracts from different treatment. free radical scavenging activity was represented as percent of dpph inhibition and scavenging at 100 µg ml–1 seed extract. different letters indicate statistically significant differences between the means (p < 0.05) for total phenolic content. treatment concentration dpph (% free radical scavenging activity) tpc (mg of gae/g) salicylic acid 0.1 mm 82.22 ± 1.7 9.73 ± 0.23a 0.25 mm 82.22 ± 2.0 8.42 ± 0.17c 0.5 mm 82.44 ± 2.4 8.22 ± 0.23cd methyl jasmonate 0.1 mm 80.18 ± 1.8 7.79 ± 0.2ef 0.25 mm 78.93 ± 1.4 7.89 ± 0.26de 0.5 mm 80.18 ± 1.6 8.42 ± 0.3c aspergillus niger 0.10% 80.41 ± 1.4 8.42 ± 0.29c 0.25% 80.52 ± 1.4 8.18 ± 0.21cd 0.50% 80.52 ± 1.2 8.0 ± 0.35de rhizopus oligosporus 0.10% 77.78 ± 1.4 9.32 ± 0.20b 0.25% 76.16 ± 1.3 8.17 ± 0.29cd 0.50% 80.41 ± 0.3 7.8 ± 0.23ef control – 80.52 ± 1.6 7.5 ± 0.17f dpph – 2,2-diphenyl-1-picrylhydrazyl, tpc – total phenolic contents, gae – gallic acid equivalent. discussion in this study, a floral spray of various elicitors was found effective in increasing the isoflavone content in mature seeds of soybean plants. both abiotic (i.e. salicylic acid and methyl jasmonate) and biotic elicitors (a. niger and r. oligosporus) caused an increased level of aglycones and total isoflavone in soybean seeds and this result is analogous to the studies of zhang et al. (2006), that aglycone could be enhanced by selecting the appropriate and optimal concentration of elicitor. upon elicitation, daidzein were more responsive than genistein and this observation was similar to the results of al-tawaha et al. (2005). several researchers observed that, genistein is the least responsive to elicitors (seguin et al. 2004, shinde et al. 2009). however, zhang et al. (2006) demonstrated that salicylic acid treatment significantly affected malonyl-daidzein and glycitein levels at different developmental stages in soybean plant. moreover, our findings were in contrast to the result of kneer et al. (1999), who demonstrated that salicylic acid, soluble chitosan and potassium cyanide stimulated production of genistein in yellow lupin roots (lupinus luteus l.). in the present study, we found that the a. niger elicitor proved to be better for the induction of isoflavones than r. oligosporus. however, lee et al. (2010) suggested that rhizopus oligosporus was the best elicitor for the induction of glyceollins in korean soybean varieties. similarly, the studies of prasad et al. (2006) suggested that r. oligosporus mycelial-extract-treated cultures of capsicum frutescens showed a 6-fold elicitation of capsaicinoids. interestingly, soybean isoflavones act as inducers for infection and nodulation of rhizobium spp. (phillips and tsai 1992) and also induce disease resistance to pathogens, especially in phytophthora sojae (rivera-vargas et al. 1993). in general, concentration is a more important factor than treatment period in elicitation, where a lower concentration is more effective on most isoflavones (zhang et al. 2006). applied concentration of elicitor and content of elicited molecule obtained generally showed a distinct bell-shaped dose–response curve (kneer et al. 1999), which means that elicitor molecules are effective at optimum concentration while higher doses are toxic to the plants by inducing cell death and apoplastic oxidative stress (mur et al. 2006). some studies also have demonstrated that the timing of floral administration of elicitors havs a significant effect on isoflavone content in soybean (zhang et al. 2006). in general, methyl jasmonate and salicylic acid are key signaling molecules, modulating several physiological events such as defense response to environmental stresses in plants (creelman and mullet 1997, draper 1997). lu et al. (2006) reported that the activation of a set of defense genes resulted after exogenous application of salicylic acid and methyl jasmonate, which has been shown to move systematically through plants. significant evidence showed that exogenous supply of methyl jasmonate led to an increase in various classes of secondary metabolites of interest in several plants (modolo et al. 2002, wei 2010, perez-balibrea et al. 2011). as suggested by earlier reports, the use of methyl jasmonate might activate the respective genes in the phenylpropanoid (pp) pathway (dixon and paiva 1995). the varied expression of ifs genes (ifs1 and ifs2) is responsible for the changes in level of isoflavones in seeds under elicitor treatment (dhaubhadel et al. 2007, cheng et al. 2008). also, yeast extract elicited the accumulation of isoflavones via elevated level of l-phenylalanine ammonia lyase and chalcone synthase transcripts in cell suspension of medicago truncatula (suzuki et al. 2005) and transcripts of flavanone 2-hydroacta bot. croat. 72 (2), 2013 317 isoflavone enrichment in soybean xylase, isoflavone 2‘-hydroxylase, and isoflavone synthase in glycyrrhiza echinata cell suspension cultures (nakamura et al. 1999). in contrast, long-term drought stress in soybean plants has been established to result in the down-regulation of ifs2 gene coinciding with a decrease in isoflavone content (gutierrez-gonzalez et al. 2010). herein may be explanation for the increased production of isoflavones. koleva et al. (2002) described the dpph method as a simple, rapid and convenient method for screening the free radical scavenging activity of various samples. in the present study, free radical scavenging activity was not correlated with isoflavone content, which suggests that isoflavones are less potent in the free radical scavenging activity compared to some phenolic acids present in soybean seeds (cos et al. 2003). however, soybean isoflavones are potent antioxidants and free radical scavengers, as shown by several reports (lee et al. 2002, lin and lai 2006, boue et al. 2008, sakthivelu et al. 2008, akitha devi et al. 2009). the soybean contains many phenolic acids such as syringic, ferulic, sinapic, coumaric, gentisic, vanillic, hydroxybenzoic, caffeic, and chlorogenic acids (kozlowska et al. 1983). out of these, caffeic and chlorogenic acids are strong free radical scavengers (sroka et al. 2003). in the present study, we are able to show the improvement in the isoflavone content in soybean seeds through an elicitor-mediated approach. significant losses of isoflavones (50% or more) occur during processing of the soybean in traditional soy foods, which is a bottleneck in the maintenance of effective levels of isoflavones in soy products. accordingly, a higher level of isoflavones in the harvested seeds is advantageous in that it will retain the seeds’ nutraceutical benefits in processed foods. acknowledgements rks and mka are thankful to ugc and csir new delhi, india for their research fellowships. this study was undertaken undera department of science and technology, government of india, funded grant. references akitha devi, m. k., mahendranath, g., sakthivelu, g., giridhar, p., rajasekaran, t., ravishankar, g. a., 2009: functional attributes of soybean seeds and products, with reference to isoflavone content and antioxidant activity. food chemistry 114, 771–776. al-tawaha, a. m., seguin, p., smith, d. l., beaulieu, c., 2005: biotic elicitors as a means of increasing isoflavone concentration of soybean seeds. annals of applied biology 146, 303–310. atkinson, c., compston, j. e., day, n. e., dowsett, m., bingham, s. a., 2004: the effects of phytoestrogen isoflavones on bone density in women: a double-blind, randomized, placebo-controlled trial1–3. american journal of clinical nutrition 79, 326–333. barnes, s., kim, h., darley-usmar, v., patel, r., xu, j., boersma, b., luo, m., 2000: beyond era and erb: estrogen receptor binding is only part of the isoflavone story. the journal of nutrition 130, 656s–657s. beausejour, j., clermont, n., beaulieu, c., 2003: effect of streptomyces melanosporofaciens strain ef-76 and of chitosan on common scab of potato. plant and soil 256, 463–468. 318 acta bot. croat. 72 (2), 2013 saini r. k., akitha devi m. k., giridhar p., ravishankar g. a. boue, s. m., shih, f. f., shih, b. y., daigle, k. w., carter-wientjes, c. h., cleveland, t. e., 2008: effect of biotic elicitors on enrichment of antioxidant properties and induced isoflavones in soybean. journal of food science 73, h43– h49. caldwell, c. r., britz, s. j., mirecki, r. m., 2005: effect of temperature, elevated carbon dioxide and drought during seed development on the isoflavone content of dwarf soybean [glycine max (l) merrill] grown in controlled environment. journal of agricultural and food chemistry 53, 1125–1129. cheng, h., yu, o., yu, d., 2008: polymorphisms of ifs1 and ifs2 gene are associated with isoflavone concentrations in soybean seeds. plant science 175, 505 – 512. cos, p., hermans, n., calomme, m., maes, l., de bruyne, t., pieters, l., et al., 2003: comparative study of eight well-known polyphenolic antioxidants. journal of pharmacy and pharmacology 55, 1291–1297. creelman, r. a., mullet, j. e., 1997: biosynthesis and action of jasmonates in plants. annual review of plant physiology and plant molecular biology 48, 355–381. dhaubhadel, s., gijzen, m., moy, p., farhangkhoee, m., 2007: transcriptome analysis reveals a critical role of chs7 and chs8 genes for isoflavonoid synthesis in soybean seeds. plant physiology 143, 326–338. dixon r. a., paiva, n. l., 1995: stress-induced phenylpropanoid metabolism. the plant cell 7, 1085–1097. draper, j., 1997: salicylate, superoxide synthesis and cell suicide in plant defence. trends in plant science 2, 162–165. giridhar, p., parimalan, r., 2010: a biotechnological perspective towards improvement of annatto color production for value addition – the influence of biotic elicitors. asian pacific journal of molecular biology and biotechnology 18, 77–79. gordon, m. h., 1990: the mechanism of antioxidant action in vitro. in: hudson, b. j. f. 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croat. 71 (2), 311–363, 2012 coden: abcra 25 issn 0365–0588 eissn 1847-8476 centric diatoms of large rivers and tributaries in hungary: morphology and biogeographic distribution keve t. kiss1*, rolf klee2, luc ector3, éva ács1 1 danube research institute, centre for ecological research of hungarian academy of science, h-2131 göd, jávorka s. u. 14, hungary 2 pähler str. 8, d-82346 andechs, germany 3 public research centre gabriel lippmann, department of environment and agro-biotechnologies (eva), 41 rue du brill, l-4422 belvaux, luxembourg centric diatoms of 107 different hungarian running waters were investigated. among them the largest was the river danube, from which more than one hundred plankton samples were analysed by scanning electron microscopy (sem). only one sample was analysed from creeks, which were the smallest running waters analysed in this study. there were also channels with slow currents flowing out of rivers or connecting different rivers. in total, 41 centric taxa belonging to 11 genera were found during this study. the average number of taxa found in a single watercourse was 7, the maximum 40 and the minimum 1. cyclotella meneghiniana was the most frequently encountered species (present in 60% of sites). twelve taxa were found in more than 20% of sites, 7 taxa between 5–10% and 6 taxa only in one site. key words: diatoms, diversity, distribution, actinocyclus, aulacoseira, conticribra, cyclostephanos, cyclotella, discostella, melosira, skeletonema, stephanocostis, stephanodiscus, thalassiosira, hungary introduction the presence of centric diatoms is characteristic of european lakes especially in spring, but they are also dominant and frequently abundant in large, slowly flowing rivers from early spring to autumn. therefore, this is an important group of primary producers and water quality indicators. the european union water framework directive (wfd) requires that the ecological status of water bodies in member states should be examined, and that a good ecological acta bot. croat. 71 (2), 2012 311 * corresponding author, e-mail: kiss.keve@okologia.mta.hu copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. status should be attained by 2015. according to wfd, among other mandatory actions, it is obligatory to investigate phytoplankton and periphyton communities. typically, several centric diatom taxa can be found in such samples, often reaching huge abundances and they should therefore be identified at species level. diatoms are effective tools for the assessment of the ecological status of wetland habitats as well. the occurrence of species with declining populations in a given habitat is characteristic of vulnerable areas. the reason for such decline is generally the decreasing diversity of habitats (e.g., as a consequence of eutrophication of water) or the decreasing number of optimal habitats. the occurrence of diminishing and presumably endangered species in the water is important from floristic and conservation respects since they can serve as a reason for a given area to be protected. the establishment of a red list is also important for microscopic organisms (lange-bertalot and steindorf 1996). among the centric diatoms we can also find some rare (e.g., orthoseira roseana) and some alien or potentially invasive species (e.g., actinocyclus normanii, cyclostephanos delicatus, skeletonema potamos, thalassiosira gessneri) (kiss et al. 2002, ka[tovský et al. 2010), so their study is also important from the point of view of conservation. the diatom flora of the river danube is well studied (kiss 1984, 1986, 1987, 1988, 2005, kiss and nausch 1988, kiss et al. 1990, kiss and genkal 1993, 1996, ács et al. 2006), while there have been only a few electron microscopic examinations of the centric diatoms of other hungarian rivers. the revision of previous light microscopic investigations (szemes 1967, uherkovich 1971) was necessary in order to compare these with the results obtained with the electron microscopic analyses by kiss (1986). these two factors motivated us to summarize the most recent data about the centric diatoms of the hungarian stretches of the rivers danube and tisza and their tributaries. the aim of this work was to study the species composition and biodiversity of centric diatoms in hungarian water flows. a 4-year grant was received to study the centric diatoms in the eastern part of hungary; therefore, we examined more samples from this region than from the western part of the country. a few hundred samples were collected from more than 100 water bodies of the hungarian watershed area of the river danube and the river tisza (including tributaries, channels, backwaters and side arms). the morphology and biogeographic distribution of 41 taxa are presented here. in the case of rare or less widespread species (like conticribra guillardii, thalassiosira duostra, cyclostephanos delicatus, cyclotella balatonis, c. choctawhatcheeana, etc.) the recent literature is cited, in other cases (like melosira varians, aulacoseira granulata, conticribra weissflogii, cyclostephanos dubius, etc), only the most important references. materials and methods more than twenty large hungarian rivers (berettyó, bodrog, bódva, danube, dráva, hármas-körös, hernád, hortobágy, ipoly, kettös-körös, kraszna, marcal, maros, rába, sajó, sebes-körös, szamos, tarna, tisza, túr, zagyva) and their tributaries, related channels, backwaters and side arms (altogether 107 different hungarian running waters) were examined (fig. 1a). the mean water discharge of the large rivers has a wide range (the largest, the river danube, has a discharge of 2300 m3 s–1 and the smallest streams of ~ 5 m3 s–1). channels flowing out of rivers or connecting different rivers were also investigated; usually 312 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. they are slowly flowing channels of different sizes (discharges between 0.1–1 m3 s–1 and 10–40 m3 s–1). samples were taken 1–3 times for the great majority of these running water ecosystems. if a sample was free from centric diatoms the sampling was repeated later. among the largest rivers like danube and tisza rivers several tens of samples were taken and the diatoms analysed by em. the smallest sampling sites investigated were several creeks from which only one sample was analysed. one-litre samples were taken from the main current 20–30 cm below the water surface and preserved with formaldehyde. samples were treated with hydrochloric acid and hydrogen peroxide, washed several times with distilled water and coated with gold-palladium for sem investigations. the micrographs were taken with a hitachi s-2600-n scanning electron microscope in digital form. data of valve size (diameter, number of striae/10 mm, etc) for each taxa are integrated from our own measurements and from literature data. the structural elements of the valves were measured and analysed on the basis of the terminology provided by genkal (1977), which is in accordance with anonymous (1975), ross et al. (1979) and theriot and serieyssol (1994). the taxonomic part of the paper is mostly based on the traditional classification system of round et al. (1990), but some newly described genera were also added (e.g., conticribra, discostella). the species distribution is shown on a sketch map of hungary prepared using the esri arcinfo 9.3 gis program (fig. 1a); the small, 1–4 order, streams are not drawn on the map. sampling site coordinates and detailed occurrence data of species are available upon request to the first author. results the morphological and ecological characteristics, as well as distributional data of the 41 centric diatom taxa found in hungarian watercourses are presented below. the morphological characterization of each taxon is complemented with em micrographs and distribution maps. our long term experience and the hungarian database (http://okir.kvvm.hu/fevi/) were used to characterise several ecological preferences of a given taxon. coscinodiscophyceae melosirales melosiraceae melosira c. agardh 1827 melosira varians c. agardh 1827 (figs. 1 b-e) the frustules of melosira varians are cylindrical and form chains (8–35 mm in diameter). chains can be long (even 300–500 mm or more in the periphyton, but can be short or »unicellular chains« in the plankton of rivers). valve mantles are finely punctuated with irregularly arranged openings of rimoportulae. the girdle band comprises a few copulae (fig. 1 b). valve face is flat and regularly finely punctuated (fig. 1. c, d) with small acta bot. croat. 71 (2), 2012 313 centric diatoms from large rivers 314 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 1. a – sampling sites (positions available upon request). b-d – melosira varians in girdle-, inside-, outside view. e – distribution of m. varians. scale bar 10 µm. granules in the central part (fig. 1 d). near the margin, short, thin connecting spinulae can be seen by sem (figs. 1 b, c). melosira varians is a cosmopolitan species that occurs both in benthos and plankton. it can be found in dystrophic and oligotrophic to eutrophic waters. it is frequent in hungarian running waters with a 36% occurrence rate (fig. 1 e). aulacoseirales aulacosiraceae aulacoseira thwaites 1848 aulacoseira ambigua (grunow) simonsen 1979 (figs. 2 a-c) aulacoseira ambigua was first illustrated in van heurck (1882) as a variety of melosira crenulata. in 1903, müller proposed the separation of this taxon from the base species, using the name m. ambigua. later on, simonsen (1979, p. 56) transferred m. ambigua to the genus aulacoseira. important new results are summarised about melosira and aulacoseira species in the papers of houk (2003, tab. 16) and houk and klee (2007). the cylindrical frustules form long chains. cell sizes: diameter 4–17 mm and mantle length 3.5–15.0 mm. the areolae are more or less round or elongated, arranged in spiral rows, running dextrorse on the mantle, 24–26 rows in 10 mm. the row of areolae always begins between each spine (fig. 2. a). the spines of the separation cells are simple, conical and those of the linking cells are small, spatulate (fig. 2 b). the valve face is flat, with randomly arranged pores at the margin. the collar is medium high. the hollow ringleist (pseudoseptum) is semicircular; its external opening is well visible on the mantle. aulacoseira ambigua is cosmopolitan, widely distributed in the plankton of mesotrophic/eutrophic lakes and slow running rivers. it has a medium frequency in hungarian running waters with a 14% occurrence rate (fig. 2 c). aulacoseira granulata (ehrenberg) simonsen 1979 (figs. 2 d-f) ehrenberg described this species in 1843 as gallionella granulata. ralfs in pritchard et al. (1861: 815–820) transferred the taxon to the genus melosira and simonsen (1979) to aulacoseira. frustules are cylindrical, in long straight chains, diameter 3–30 mm, mantle length 4–24 mm. the rows of coarse areolae on the valve mantle of the separation valves are arranged parallel to the pervalvar axis, 7–15 rows in 10 mm (fig. 2 d). in contrast, the linking valves have dextrorse rows of areolae (fig. 2 e). areolae can vary in shape (round, oval, nearly square) even on different valves of one chain. there are short, cone-shaped spines at the margin of the valve face. characteristic long spines (2–4) are situated in the furrows of the next separation valve (fig. 2 d). there are linking valves with short, spatulate or flared spines. the valve face is flat with irregular pores near the margin, absent in the centre. the collar is short, the ringleist is almost undeveloped. aulacoseira granulata is a cosmopolitan species occurring in eutrophic rivers, ponds and lakes. it has a medium frequency in hungarian running waters with a 20% occurrence rate (fig. 2 f). acta bot. croat. 71 (2), 2012 315 centric diatoms from large rivers aulacoseira italica (ehrenberg) simonsen 1979 emend. r.m. crawford, likoshway et r. jahn 2003 (figs. 3 a-c) ehrenberg (1838) described this species as gallionella italica from fossil diatomite from santa fiora, tuscany, italy, collected by klaproth (crawford et al. 2003). later, it was transferred to the genus melosira as m. italica (ehrenberg) kützing (kützing 1844) and to aulacoseira by simonsen (1979). finally, crawford et al. (2003) described and illustrated in detail the type material of santa fiora. frustules are cylindrical, forming long straight chains with a diameter of 3–30 mm and a mantle length of 8–20 mm. valve face flat to slightly convex with small, irregularly arranged 316 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 2. a-b – aulacoseira ambigua outside view and girdle view (external opening or tube of rimoportula or internally the rimoportula with arrowhead on all micrographs where it is seen). d – chain; e – girdle view of a. granulata: c – distribution of a. ambigua: f – a. granulata. scale bar 20 µm (d), 10 µm (b, e) and 5 µm (a). acta bot. croat. 71 (2), 2012 317 centric diatoms from large rivers fig. 3. a-b – aulacoseira italica outside and girdle view. d-f – a. aff. muzzanensis outside view, chain and inside view; c – distribution of a. italica; g – a. aff. muzzanensis. scale bar 10 µm (a, b, e) and 5 µm (d, f). pores (fig. 3 a). rows of mostly fine, pervalvar elongated areolae on the valve mantle, arranged parallel or slightly curved to the pervalvar axis (sinistrorse), 3–18 rows in 10 mm. linking spines are robust and relatively long, spatulate and often jagged at the tips; spines of the sibling valves are interconnected (fig. 3 b). the long conical, pointed separating spines are situated in a thin ring. the collar is medium high; the ringleist is almost absent. girdles composed by several slight girdle bands. aulacoseira italica is a cosmopolitan species in eutrophic rivers, ponds and lakes. it is a rare species in hungarian running waters with a 1% occurrence rate (fig. 3 c). aulacoseira aff. muzzanensis (f. meister) krammer 1991 (figs. 3 d-g) meister (1912) described the species as melosira muzzanensis but it was transferred in 1991 to the genus aulacoseira by krammer, who illustrated the isotype from a lake in switzerland, lago di muzzano (krammer 1991). the specimens found in hungary correspond well to the illustrations given by houk (2003, pl. 27, figs. 1–7), aulacoseira aff. muzzanensis (f. meister) krammer, for populations of south moravia and bohemia. frustules cylindrical, forming short chains with connection of valves and long acute connection spines, but frequently occurring in a double cell form, too (fig. 3 e). diameter is 8–25 mm, mantle length 4–8 mm. the more or less straight rows of coarse, mainly rectangular, areolae on the valve mantle are arranged parallel to the pervalvar axis, 7–10 rows in 10 mm (fig. 3 d-f). there is one row of areolae below each spine and one row of areolae between each spine. separating valves have cone-shaped spines at the margin of the valve face, different in length. characteristic long spines (2–4) are situated in the furrows of the next separation valve (fig. 3 e). linking valves are with short, spatulate or bifurcated spines, originating from each interstria. the valve face is flat with marginally dispersed fine areolae on the discus. the collar is short; the ringleist is almost absent. the rimoportulae are close to the valve face (fig. 3 f). aulacoseira muzzanensis is a cosmopolitan species found in plankton and benthos, also in mesotrophic/eutrophic rivers, ponds and lakes. it is rare in hungarian running waters with a 5% occurrence rate (fig. 3 g). aulacoseira pusilla (f. meister) tuji et houki 2004 (figs. 4 a, b, e) melosira pusilla was described by meister (1913) from lake suwa (nagano prefecture, japan). tuji and houki (2004) proposed a new combination, aulacoseira pusilla, and tuji and williams (2006) claimed that aulacoseira subborealis (nygaard) denys, muylaert et krammer (denys et al. 2003) is a synonym of this taxon. the frustules are cylindrical, in short chains, frequently in a double cell form (fig. 4 b). cell sizes: 5.5–9.0 mm in diameter (mostly 6–7 mm) and 2–4 mm in mantle length. the more or less round areolae on the mantle are generally somewhat inclined (sinistrorse and up to ca. 20°) and curved but may sometimes be almost straight and parallel to the pervalvar axis (fig. 4 b). the spines are rather small and simple, pointed, without anchors or projections. the number of pervalvar ribs at the spine base is 2. mantle areolae 28–38/10 mm; pervalvar striae 23–28/10 mm. areolae on the valve face are spread over the entire surface or a broad marginal zone (fig. 4 a). the rimoportula is situated at the base of a row of areolae on the inner side of the pseudoseptum. its external opening is inconspicuous and probably represented by or associated with an areola. the collar is short and the ringleist is almost absent. 318 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. aulacoseira pusilla is cosmopolitan, can be found in the plankton of rivers and also in lakes. it has a medium frequency in hungarian running waters, with a 14% occurrence rate (fig. 4 e). aulacoseira subarctica (o. müller) e.y. haworth 1990 (figs. 4 c, d, f) the species was described by müller (1906) as melosira italica subsp. subarctica and the type material has been investigated and illustrated by tuji and houki (2004). simonsen (1979) transferred the taxon to the aulacoseira genus. haworth (1988) named it a. subarctica as a separate species and validated this new name later (haworth 1990). acta bot. croat. 71 (2), 2012 319 centric diatoms from large rivers fig. 4. a-b – aulacoseira pusilla outside and girdle view. c-d – a. subarctica outside and girdle view; e – distribution of a. pusilla; f – a. subarctica. scale bar 10 µm (b) and 5 µm (a, c, d). frustules are cylindrical, forming long chains. cell sizes: 3–15 mm in diameter and 3.5– 18 mm in mantle length. areolae 12–20/10 mm on the mantle in curved, dextrorse rows 17–21/10 mm (figs. 4 c, d). one of the characteristic features of a. subarctica is the relatively long, strong, conical, pointed spines (fig. 4 d). every second row of round or slightly elongated areolae clearly begins in the spine base. the valve face is flat with randomly arranged fine pores at the valve face margin (tuji and houki 2004). the collar is low, and the ringleist is strongly developed. aulacoseira subarctica is a planktonic species in oligotrophic to eutrophic lakes, ponds and slowly running rivers. it is widely distributed across northern europe, north america, japan, china, australia and new zealand but rare in the tropics (gibson et al. 2003). it is a scarce species in hungarian running waters with a 7% occurrence rate (fig. 4 f). thalassiosirales thalassiosiraceae conticribra stachura-suchoples et d.m. williams 2009 conticribra guillardii (hasle) stachura-suchoples et d.m. williams 2009 (figs. 5 a-c) the species was described by hasle (1978) from a brackish water locality (helsinki, gulf of finland) and was transferred to the conticribra genus by stachura-suchoples and williams (2009). additional data were published about its morphology by makarova et al. (1979), kiss et al. (1984), genkal (1992), trigueros et al. (2000). frustule rectangular in girdle view, diameter is 5–14 mm. the length of the pervalvar axis is 1/3 of the diameter. solitary cells, colonies not observed. valve face with radial, somewhat irregular rows of pores (fig. 5 a). the pores can form tiny areolae on the valve margin. a fine siliceous ring irregular in shape can be located in the centre of the valve face from which slightly elevated, repeatedly dichotomously branched interfascicles extend towards the valve margin. their number on the valve face is 3–5 in 1 mm, near the valve mantle 7–8 in 1 mm. valve mantle with pores and very thin striae. in some specimens, a single valve face fultoportula with three satellite pores is located at 1/3 radius from valve centre on the side opposite to the rimoportula. on the valve margin, a regular ring of fultoportulae is visible 6–10 in 10 mm. the marginal fultoportulae internally are surrounded by 4 satellite pores. a single rimoportula is situated on valve margin, the orientation of labium varies (fig. 5 b). its external tube is longer than those of the marginal fultoportulae. conticribra guillardii occurs in both fresh and brackish waters. it is scarce in hungarian running waters with a 7% occurrence rate (fig. 5 c). conticribra weissflogii (grunow) stachura-suchoples et d.m. williams (figs. 5 d-f) the first valid description of this species was given in van heurck (1885) as micropodiscus weissflogii grunow in van heurck 1885 and this taxon was illustrated later by van heurck (1896). hustedt (1926) described the same species as thalassiosira fluviatilis. based on lm examination of grunow’s material from slide 416 of the van heurck collection, fryxell and hasle (1977) compared micropodiscus weissflogii from schleswig (germany) with the material of thalassiosira fluviatilis from the wümme river (hustedt collection) and they stated that the two species are identical and proposed to synonymise both taxa making the new combination thalassiosira weissflogii (grunow) fryxell et hasle. micropodiscus weissflogii was transferred to the new genus conticribra by stachura-suchoples and williams (2009). 320 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. frustules are drum-shaped or cylindrical, diameter 15–23 mm. the pervalvar axis is about half of the diameter. valve face with radial areolation, areolae are polygonal 3–4/1 mm and 6–7/1 mm on valve mantle. external openings of the regularly arranged marginal fultoportulae (9–12/10 mm) with elongated tubes are situated between the valve-face and the shallow mantle; they have four satellite pores. one single, relatively large rimoportula is visible near the valve edge above the ring of the marginal fultoportulae (fig. 5 d). its acta bot. croat. 71 (2), 2012 321 centric diatoms from large rivers fig. 5. a-b – conticribra guillardii outside and inside view. d-e – c. weissflogii outside and inside view. c – distribution of c. guillardii; f – c. weissflogii. scale bar 10 µm (d, e) and 5 µm (a, b). external tube is longer than that of the marginal fultoportulae. the lip is usually perpendicular to the mantle (fig. 5 e). four to 9 or more valve-face fultoportulae with 4 (3) satellite pores are arranged in a more or less ring-like structure in the centre of the valve. the number of valve face fultoportulae increases with increasing salinity. large valves in sea-water have more fultoportulae (10–28) than valves in brackish or freshwater (2–10). conticribra weissflogii is considered a cosmopolitan species. it has been recorded in polluted fresh and brackish waters, first of all rivers and streams. we consider it an euryhaline species. it is a frequent species in hungarian running waters with a 27% occurrence rate (fig. 5 f). thalassiosira cleve 1873 thalassiosira duostra c. pienaar in pienaar and pieterse 1990 (figs. 6 a-c) thalassiosira duostra was described by pienaar and pieterse (1990) from the vaal river in south africa. later several new data concerning the species in europe and south america were published (szabó et al. 2004, torgan et al. 2006, wojtal and kwandrans 2006, pérez et al. 2009). the frustule is drum-shaped, with a flat valve face. diameter varies 7–26 µm (average 14 µm); pervalvar axis 6–9 µm. the valve face areolae are round (or polygonal), they can be very small or relatively large; 10–30/10 µm (fig. 6 a). the rows of areolae usually form 6–9 triangular sectors. there are areolae with external foramina and internal cribra. valve mantle areolae are small, sometimes elongated, 15–40/10 µm. a ring of the external tubes (0.1–1.1 µm long) of marginal fultoportulae is situated on the edge of the valve face, 8–11/10 µm (fig. 6 a). the arrangement (position and orientation) of the tubes is not strictly regular. the internal tubes of marginal fultoportulae are very short, surrounded by four satellite pores (fig. 6 b). among the fultoportulae, 1–2 rimoportula are situated with well developed, radially oriented labium. the valve face fultoportulae (2–8) are situated in a different position, frequently in groups, and form a loose semicircle or are disposed on different sides of the valve face. internally, they have 4 satellite pores. their number can differ on the epiand hypovalve. thalassiosira duostra can be characterized as a freshwater, probably mesohalobous species and has been found in several eutrophic or polluted lakes and rivers. our data seem to confirm its wide ecological range. it has a medium frequency in hungarian running waters with a 13% occurrence rate (fig. 6 c). thalassiosira gessneri hustedt 1956 (figs. 6 d-f) thalassiosira gessneri was described by hustedt (1956) from the lake maracaibo (venezuela). hasle and lange (1989), and later kiss et al. (2002) published papers about its lm and sem morphology. the frustules are drum shaped. the remarkable tangential undulation of the valve face is well visible in semilateral view. the diameter is 20–37 µm, the pervalvar axis 10–15 µm. the valve face areolae are small (10–15/10 µm, fig. 6 d), they are larger on the elevated part of the tangential undulation than on the depressed part and are covered by cribra internally (fig. 6 e). sometimes the valve face is smooth; the structures of the small areolae are not seen. the external pores of valve face fultoportulae are situated mainly on the elevated part of the undulation (fig. 6 d). internally, the valve face has a very fine striation (fig. 6. e). the interstriae are very thin; they are cribrumless lines on the internal valve 322 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. surface, radially arranged, not straight and branched. a ring of well developed fultoportulae is situated on the valve margin. their external tube is less than 1 µm. the number of marginal fultoportulae is 5–7 in 10 µm. they are operculate and usually have five satellite pores. a single rimoportula is situated between the marginal fultoportulae. its external tube is longer and larger than those of the fultoportulae (fig. 6 e). the rimoportula is short-necked, differently twisted with an elongated, compressed narrow lip. the position of lip can be situated parallel, perpendicularly or in different angle to the valve mantle. the rimoportula is situated in front of the open part of the valve face fultoportulae’ semicircle. acta bot. croat. 71 (2), 2012 323 centric diatoms from large rivers fig. 6. a-b – thalassiosira duostra outside and inside view. d-e – t. gessneri outside and inside view; c – distribution of t. duostra; f – t. gessneri. scale bar: 20 µm (e) and 10 µm (a, b, d). the valve face fultoportulae have four satellite pores. the shape of the semicircular valve face fultoportulae is variable (half-ring, or more or less complete-ring). sometimes there are additional fultoportulae inside or outside of the ring (fig. 6. e). the number of valve face fultoportulae varies between 5–15 and increases with the diameter. thalassiosira gessneri can be characterized as a freshwater species, found in several eutrophic or polluted rivers in phytoplankton and periphyton samples. it is rare in hungarian running waters with a 1% occurrence rate (fig. 6 f). thalassiosira incerta makarova 1961 (figs. 7 a-c) thalassiosira incerta was described by makarova (1961) from the caspian sea and found later also in freshwater and brackish localities (hasle 1978, belcher and swale 1986, clarke 1992). the frustule is cylindrical, diameter 13–25 µm. the valve face is flat, areolae are round and relatively large (fig. 7 a), 10–19/10 µm (small on valve mantle: 15–28/10 µm) and covered by cribra internally (fig. 7 b). the rows of areolae usually form 5–8 triangular sectors, interstriae are not seen. three to 6 valve face fultoportulae are situated in the centre with three satellite pores. a ring of well developed fultoportulae is situated on the valve margin; 3–6/10 µm. they are operculate and usually have five satellite pores (fig. 7 b). a single rimoportula is situated between the marginal fultoportulae. its external tube is longer and larger than those of the fultoportulae. the rimoportula is short-necked with an elongated, compressed narrow lip usually perpendicular to the margin. thalassiosira incerta was found in eutrophic fresh and brackish water lakes and rivers. it is rare in hungarian running waters with a 2% occurrence rate (fig. 7 c). thalassiosira lacustris (grunow) hasle in hasle et fryxell 1977 (figs. 7 d-f) this species was described by smith (1856) as cyclotella punctata from freshwater near wisbech. grunow in cleve and grunow (1880) transferred c. punctata to coscinodiscus with the epithet lacustris. later c. lacustris was transferred to thalassiosira by hasle and fryxell (1977). this species is considered an exotic and invasive diatom in north america (smucker et al. 2008) and has been found in european waters of various salinities, from freshwater to brackish (hustedt 1928, hasle and lange 1989, kiss et al. 2002). disc-shaped frustules distinctly tangentially undulated; diameter, 17–62 (70) mm (fig. 7 d). at the base of the mantle, the valve has a marginal thickened flat rim to which the valvocopula is attached with 3–6 copulae. the coarse texture of the external valve surface formed by small or large, round or polygonal areolae is very characteristic of this taxon, 9–14 areolae/10 mm. sometimes areolae are arranged radially, sometimes their radial arrangement is not clear. the valve face fultoportulae are arranged like a ring or half-ring between the central area and the margin on the elevated part of the tangential undulation (fig. 7 d). sometimes there are additional fultoportulae inside or outside the ring. the number of valve face fultoportulae varies between 3 and 22. near the margin, distinct tubular occluded processes are arranged irregularly above the ring of the marginal fultoportulae. their external tube is about 0.5 µm long. the number of marginal fultoportulae is 4–7/10 µm, and increases with the diameter of the frustule. the number of occluded processes is about half that of the marginal fultoportulae. a single rimoportula is situated above the marginal fultoportulae. its external tube is longer and larger than that of the fultoportulae and it is found between the occluded processes. the rimoportula is short324 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. -necked, differently twisted, with an arched, relatively large lip (fig. 7 e). the rimoportula is situated in front of the open part of the semicircle of the valve face fultoportulae. all fultoportulae are surrounded by four satellite pores (fig. 7 e). internally on the valve face, very fine striation is visible. there are four-seven rows of small cribra in the striae near the margin and one-two in the centre. the interstriae are very thin, radially arranged, not straight and branched (fig. 7 e). the edge of the valve mantle is large (thick) internally. thalassiosira lacustris has been recorded in fresh to brackish waters, widely distributed in lowland rivers, ponds, estuaries and coasts. it is a scarce species in hungarian running waters with a 6% occurrence rate (fig. 7 f). acta bot. croat. 71 (2), 2012 325 centric diatoms from large rivers fig. 7. a-b – thalassiosira incerta outside and inside view. d-e – thalassiosira lacustris outside and inside view; c – distribution of t. incerta; f – t. lacustris. scale bar 10 µm (a, b, d, e). thalassiosira pseudonana hasle et heimdal 1970 (figs. 8 a-c) although described from a freshwater locality (river wümme), this species is apparently distributed in coastal waters (hasle and heimdal 1970). papers about its em morphology were published by several authors: hargraves and levandowsky (1971), lowe and busch (1975), hasle (1976), belcher and swale (1977), makarova et al. (1979), kiss (1984). alverson et al. (2011) explained well the nomenclatural history, sem morphology and dna structure of thalassiosira pseudonana. 326 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 8. a-b – thalassiosira pseudonana outside and inside view. d-e – cyclostephanos delicatus outside and inside view; c – distribution of t. pseudonana; f – c. delicatus. scale bar 5 µm (b, d, e) and 2 µm (a). the frustule is cylindrical; the valve face is flat (fig. 8 a). the diameter is 2.5–9.0 mm, fine, and tiny pores or a polygonal areola-like structure are seen on the valve face. the exact pattern depends on the degree of silicification. heavily silicified frustules have only fine irregular rows of pores; weakly silicified valves have a polygonal areola-like structure with interfascicles which are branched two-three times. the number of interfascicles is 2–7 in 1 mm. a ring of numerous fultoportulae is seen on the valve margin with three satellite pores. slightly below the fultoportulae a small rimoportula is found (fig. 8 b). rarely, a single valve face fultoportula can be observed, especially on specimens from brackish water. thalassiosira pseudonana has been recorded from many fresh, brackish and marine water habitats; it seems to be a euryhaline species. sometimes it is abundant in the phytoplankton of the danube. it has a medium frequency in hungarian running waters with a 16% occurrence rate (fig. 8 c). skeletonemataceae skeletonema greville 1865 skeletonema potamos (c. i. weber) hasle in hasle and evensen 1976 (figs. 9 a-c) the species was described as stephanodiscus subsalsus (cleve-euler) hustedt from lake jensen in germany (hustedt 1928). a second description was published from freshwater habitats by weber (1970) under the new genus and species name microsiphona potamos weber. hasle and evensen (1976) established the correct taxonomical position of the species with the new combination skeletonema potamos. cell diameter is 3.0–6.5 mm, length of pervalvar axis varies between 5–18 mm. the valve face is flat in the centre and slightly rounded near the valve mantle (fig. 9 a). there are fine radial striae on the valve face, consisting of irregular polygonal »areolae« and two-three times branched interfascicles (very thin rib-like structure). near the margin, a ring of 3–8 fultoportulae is situated. the fultoportulae are tubular, cleft at the distal tip. they are »thick-walled« like the valve face with the rib-like elevations extending from the valve surface onto the fultoportulae. a single rimoportula is visible between the marginal fultoportulae. it is situated in the middle of the ring of fultoportulae, but sometimes it is near the valve centre. the structure of the valve mantle is the same as that of the valve face. numerous bands compose the very thin girdle. the valve is more silicified than the girdle (fig. 9 b). skeletonema potamos has been recorded in the last 40 years from more and more localities in rivers and reservoirs in europe (belcher and swale 1978, steinberg et al. 1987, chang and steinberg 1988, kiss and nausch 1988) and south america (torgan et al. 2009), both from freshwater to slightly brackish habitats, such as river estuaries. in many cases, it is one of the most abundant species of river phytoplankton in summer; it is a warm stenothermic species with high light demand (kiss et al. 1994). it is scarce in hungarian running waters with a 9% occurrence rate (fig. 9. c). skeletonema subsalsum (cleve-euler) bethge 1928 (figs. 9 d-f) the species was described by cleve-euler (1912) as melosira subsalsa and bethge (1928) transferred it later to skeletonema. hasle and evensen (1975), klee and steinberg (1987) and aké castillo et al. (1995) characterised its lm and em morphology. skeletonema subsalsum is a typical chain-forming planktonic diatom. adjacent cells are usually very close. cells occurring tightly together have flattened valve faces. valve is acta bot. croat. 71 (2), 2012 327 centric diatoms from large rivers circular, diameter 3–6 mm. the valve face is flat with typical skeletonema structure with radially arranged striae of polygonal »areolae« and sometimes branched interfascicles (fine costa-like structure; fig. 9 d). valve mantle is different in length with parallel rows of rectangular areolae and interfascicles (fig. 9 e). frustules are connected with 7–14 marginal fultoportulae, which are flat (squeezed), flat-bifurcated or flat-spoon like, and provide a very close connection between valve mantles. even when the fultoportulae are not really distinct from the mantle in their distal parts, the circular holes and the undulated distal outline of the fultoportulae are distinct (fig. 9 e). the fultoportulae have short perpendi328 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 9. a-b – skeletonema potamos outside and semi-girdle view. d-e – s. subsalsum outside and inside view. c – distribution of s. potamos; f – s. subsalsum. scale bar 5 µm (e), 2.5 µm (d) and 2 µm (a, b). cular, tubular parts internally with no longitudinal slits. the girdle consists of many narrow, weakly silicified bands. among the fultoportulae a single rimoportula is situated. skeletonema subsalsum has been mostly recorded from saline and brackish water environments and is occasionally reported from freshwater localities (gibson et al. 1993). krammer and lange-bertalot (1991) think its distribution is cosmopolitan. it is a rare species in hungarian running waters with a 4% occurrence rate (fig. 9 f). stephanodiscaceae cyclostephanos round in theriot et al. 1987 cyclostephanos delicatus (genkal) casper et scheffler 1990 (figs. 8 d-f) this species was described as stephanodiscus delicatus by genkal (1985) from the river volga in the rybinsk reservoir, russia. kobayasi and kobayashi (1987) and casper et al. (1988) published interesting complementary data about its lm and em characteristics. stephanodiscus delicatus was transferred to the genus cyclostephanos by casper and scheffler (1990). valves circular; diameter 5–14 mm, number of striae is 14–25/10 mm (fig. 8 d). the valve face has a clearly visible small central elevation or depression (frequently with a small annulus). the interfascicles can be branched (fig. 8 e) and are generally slightly elevated on the external part of the valve face. on the internal valve face, interstriae are slightly elevated near the margin. they do not have a structure resembling real costae or alveolar chamber-like structure. interstriae usually end in spines. this feature is less conspicuous on more heavily silicified valves. in this case the external valve face is almost smooth and the external openings of the areolae are smaller than on weakly silicified valves. a single valve face fultoportula can be seen in slightly eccentric position, internally with two satellite pores. its round external opening is slightly elevated like a tiny volcano. opposite to this elevated opening a small depression can be seen (the space of connecting valve face fultoportula). there are marginal fultoportulae on each interstria from 4th to 7th with a short tube externally and with two satellite pores internally. among the marginal fultoportulae, a single rimoportula with small sessile labium can be found; its external opening is slightly elevated from the ring of marginal fultoportulae. cyclostephanos delicatus has been recorded from several eutrophic rivers and lakes; it has a medium frequency in hungarian running waters with an 11% occurrence rate (fig. 8 f). cyclostephanos dubius (fricke) round in theriot et al. 1987 (figs. 10 a-c) this species was shown by fricke in schmidt et al. (1874–1956) as cyclotella dubia, and then was transferred by hustedt (1928) to stephanodiscus dubius. round (1982) established the new genus cyclostephanos and transferred s. dubius (fricke) hustedt to this genus. theriot et al. (1987) made a final validation of the genus name cyclostephanos. frustule is short, cylindrical, 4.5–35 mm in diameter. the valve face is concentrically undulate. there are radial rows of areolae (striae), which are multiseriate (2–4) near the margin and uniseriate in the centre (fig. 10 a). interfascicles can be relatively fine, as in stephanodiscus or strong costa-like as in cyclotella, 12–18 in 10 mm. internally, they form alveolar chambers (this is the most important characteristic of the genus). areolae on valve mantle in groups, separated by thin end of interstriae or not separated. there are solid spines on the interfascicles near the margin, on some specimens on each interfascicle, on others only on a acta bot. croat. 71 (2), 2012 329 centric diatoms from large rivers few. marginal fultoportulae are seen on every second to fifth costa with external openings below spines (fig. 10 b), the short internal tube with two satellite pores is on the costae. a few valve face fultoportulae with two satellite pores are situated close to the central or marginal area of the valve face (fig. 10 b). their external openings are smaller than areolae. a single rimoportula can be seen between the marginal fultoportulae with a small labium on a costa. cyclostephanos dubius is cosmopolitan; it has been recorded frequently as an abundant species of river and shallow lake phytoplankton. it is frequent in hungarian running waters with a 44% occurrence rate (fig. 10 c). 330 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 10. a-b – cyclostephanos dubius outside and inside view. d-e – c. invisitatus outside and inside view; c – distribution of c. dubius; f – c. invisitatus. scale bar 5 µm (a, b, d, e). cyclostephanos invisitatus (m. h. hohn et hellerman) stoermer, theriot et håkansson in theriot, stoermer and håkansson 1987 (figs. 10 d-f) this species was described by hohn and hellerman (1963) from the river auglaize (putman co., ohio) and also found in river potomac (maryland) and ridley creek (pennsylvania). lowe and crang (1972) published important data about the ultrastructure of this species with tem and sem, investigating material from susquehanna river near berwick, pennsylvania, usa. theriot et al. (1987) transferred this species to the genus cyclostephanos. the frustule is cylindrical, diameter 5–14 mm, the length of the pervalvar axis is half-three quarters of the diameter (fig. 10 d). the valve face is flat; it consists of radial rows (striae) of poroid areolae (one areola in the centre and 2–3 near the margin). number of striae is 14–22/10 mm, that of areolae is 20–40/10 mm. in the centre a small, more or less regular rosette is situated, formed by an annulus around some areolae. near this annulus one, rarely two, valve face fultoportula(e) internally with two satellite pores. interstriae are more or less straight, some of them with bifurcation on the valve face. they are bifurcated or straight under spines on the valve mantle. there are small spines at valve mantle, valve face junction on each interstria; marginal fultoportulae with two satellite pores are situated on each stria from 3 to 6 and a rimoportula among them with a small sessile labium. the external opening of the rimoportula is a slightly elevated pore in the ring of marginal fultoportulae. on the internal valve face, interstriae are slightly elevated near the margin (fig. 10 e). they do not have a structure resembling real costae or an alveolar chamber-like structure. cyclostephanos invisitatus was described as a unicellular form. in the river danube, however, especially in summer, short chains of a few cells are frequently recorded, even up to 10–15 cells. chains are formed by connection of spines. frustules of the chains have normal or flat spines, sometimes with spoon-like broadenings or bifurcations resembling s. binderanus (kiss 1988). cyclostephanos invisitatus is a cosmopolitan species and has been recorded from many eutrophic rivers and lakes. it is the third most frequent species in hungarian running waters with a 45% occurrence rate (fig. 10 f). cyclotella (kützing) brébisson 1838 nom. cons. cyclotella atomus hustedt 1937 var. atomus (figs. 11 a-c) the species was described by hustedt (1937) from the plankton of the sindanglaja reservoir situated in west java, indonesia. detailed data were published on the em structure of species, description of a new variety by genkal and kiss (1993). frustule cylindrical, the valve face is flat (smooth) or slightly undulated (fig. 11 a). the diameter is 3.5–8.0 mm. length of pervalvar axis is similar to or smaller, and occasionally bigger, than the diameter. striated marginal part of the valve face is larger than the half radius. the central hyaline area is small. striae have 1–3 areolae close to the centre and 5–7 close to the margin (fig. 11 a). number of striae is 15–20/10 mm. there is no »real« alveolar structure (alveoli on the marginal part of the valve face are not clearly distinguished from the central hyaline area), on the internal valve face, only »open« alveoli (the alveolar chamber is not closed in direction of centre (fig. 11 b). on the valve margin, there is a ring of 5–9 fultoportulae, which have small pores externally near the valve face and valve mantle junction and they acta bot. croat. 71 (2), 2012 331 centric diatoms from large rivers are situated on each costa from 2nd to 4th and have two satellite pores. on the valve margin, a single rimoportula is visible internally and on the central hyaline area one (rarely two) valve face fultoportula with three satellite pores in eccentric position. cyclotella atomus var. gracilis genkal et k. t. kiss 1993 (figs. 11 d-f) the striated marginal part of the valve face is about half the length of the radius. the central hyaline area is relatively large. striae have 2–3 areolae close to the centre and 5–6 close to the margin (fig. 11 d). number of striae is 15–20/10 mm. this variety has a »real« 332 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 11. a-b – cyclotella atomus var. atomus outside and inside view. d-e – c. atomus var. gracilis outside and inside view. c: – distribution of c. atomus var. atomus; f – c. atomus var. gracilis. scale bar 5 µm (d, e) and 2 µm (a, b). alveolar structure (alveoli on the marginal part of the valve face are clearly distinguished from the central hyaline area). internally alveolar chambers are circularly closed in direction of centre (fig. 11 e). there is a ring of 5–9 fultoportulae on the valve margin, which have small pores externally near the valve face and valve mantle junction and they are situated on each costa from 2nd to 4th and have two satellite pores. a single rimoportula can be seen internally positioned on the valve margin and on the central hyaline area one valve face fultoportula with three satellite pores in an eccentric position. both varieties of c. atomus were recorded from different eutrophic rivers and lakes in europe. it is a euryhaline, cosmopolitan species. it has a medium frequency in hungarian running waters, c. atomus var. atomus with a 24%, c. atomus var. gracilis with a 20% occurrence rate (figs. 11 c, f). cyclotella choctawhatcheeana a. k. s. prasad in prasad, nienow and livingston 1990 (figs. 12 a-c) the species was described by prasad et al. (1990) from the choctawhatchee estuary from the gulf of mexico. later it was newly described as c. hakanssoniae wendker (wendker 1991) from the schlei estuary (germany); this taxon is a synonym of c. choctawhatcheeana. a recent paper presents many details about the valve morphology (buri] et al. 2007). unicellular, but can form short chains occasionally. the diameter of the cells varies between 6–15 µm with 20–27 striae in 10 µm. the valve face has a tangential undulation of the central area with a colliculate structure (fig. 12 a). these features are lightly marked on small valves and pronounced on large ones. the striated marginal area consists of striae and interstriae continuing into the mantle. the striae have 1–3 very small areolae (pores) near the undulated central area and 2–5 close to the margin. the interstriae are thin and terminate close to the edge of the valve face. the openings of the alveolar chambers are slightly elongated. there are 1–8 valve face fultoportulae, arranged in a semicircle, internally with three satellite pores (fig. 12 b). the external pores of valve face fultoportulae are situated on the elevated part of the tangential undulation. there are openings of the mantle fultoportulae on every second to fourth interstria near the edge of the valve with a small round pore externally and two satellite pores internally. the rimoportula has an elongated external hole and an elongated slit internally. the lip of the rimoportula is in radial position or at different angles to the costae. cyclotella choctawhatcheeana has been found in different localities around the world in brackish waters and rivers connected with saline lakes. it seems an invasive species in eutrophic brackish waters. it is a rare species in hungarian running waters with a 2% occurrence rate (fig. 12 c). cyclotella delicatula hustedt 1952 (figs. 12 d-f) the species was described by hustedt (1952) from the plankton of a small groundwater lake (seebachlacke, lower austria). detailed morphology based on lm and sem studies from the austrian type material was published by scheffler et al. (2003) and houk et al. (2010). it was also found in a spanish karstic lake and illustrated by kiss et al. (2007). frustules are cylindrical, diameter 3.2–11.0 mm, length of the pervalvar axis is 1.5–3.0 mm. the central part of the valve face is flat and slightly domed towards the mantle. the central area is smooth, round or polygonal (fig. 12 d) decorated with only the opening of the valve face fultoportula or with round to irregular small hollows, which never penetrate the silica acta bot. croat. 71 (2), 2012 333 centric diatoms from large rivers layer of the valve face. outside of central hyaline area the valve face is structured by striae formed by areolae and interstriae 15–26/10 mm. at the edge of the interstriae two or three small areolae are normally situated between two large areolae but striae with three or six to seven areolae have also been found. the striae are usually unequal in length; therefore the central area is frequently polygonal (fig. 12 d). interstriae are relatively narrow and never straight, sometimes decorated with one or two rings of small granules near the periphery of the valve. several stria and interstria are branched. the valve has one valve face fultoportula 334 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 12. a-b – cyclotella choctawhatcheeana outside and inside view. d-e – c. delicatula outside and inside view; c – distribution of c. choctawhatcheeana; f – c. delicatula. scale bar 5 µm (a, d) and 2 µm (b, e). close to the centre, its external opening is relatively large and sometimes difficult to distinguish from small hollows by sem. internally it has a short tube and usually two satellite pores (fig. 12 e). a ring of fultoportulae is situated on the valve margin with two satellite pores. the external opening of the marginal fultoportulae is a relatively large pore on four to six costae. the inner aperture of the alveolar chamber is round to elongate. the costae bearing fultoportulae can be slightly depressed. the single rimoportula is situated on the valve face. its external opening is a relatively large pore at about one third of the radius, usually on a branched interstria. the position of the labium varies. cyclotella delicatula has been recorded first of all from mesotrophic lakes and from rivers with connection to lakes. it is a scarce species in hungarian running waters with a 6% occurrence rate (fig. 12 f). cyclotella distinguenda hustedt 1927 (figs. 13 a-c) the species was described by hustedt (1927) from the lower lunz lake (lunzer untersee) in austria. håkansson (1989) published high quality micrographs from the original hustedt material and she compared cyclotella distiguenda with c. operculata (c. agardh) kützing and c. plitvicensis hustedt. new illustrations were published on hustedt’s original type material and kützing’s material from tennstädt in germany by håkansson (2002) and houk et al. (2010), and from lake la cruz in spain by kiss et al. (2007). valve is circular, diameter 9–24 mm, length of pervalvar axis 4–6 mm. the central part of the valve face has a tangential undulation (fig. 13 a), which can be pronounced or slight. it is smooth or decorated with verrucae, with smaller or larger pores and frequently with special shaped wavy lines at the base of the undulation. the marginal part of the valve is structured by striae and interstriae, 10–14/10 mm. interstriae are relatively narrow and on a few valves they are quite inconspicuous. striae are formed by areolae, usually two larger areolae are situated at both sides of the interstriae and one or two small ones are located between them. the number of areolae increases towards the margin and on the valve margin there are 4–5/striae. striae are equal in length and never diverge on the flat part of the valve face, however they do diverge at the valve mantle border and on the mantle in ~ 25% of the valves and lose their distinctness. there is no valve face fultoportula but valves display a ring of marginal fultoportulae situated near the junction between the valve face and the mantle, their external openings are round pores, larger than the areolae and are visible on interstriae. internally fultoportulae have three satellite pores and a relatively long cowling (fig. 13 b). the position of the marginal fultoportulae varies, usually on every second to fourth costa. costae are more or less equal in length. the alveolar chambers are usually round on small valves or elongated on large ones. the single rimoportula is situated on the same level as the fultoportulae. it has a short tube with a large labium, usually parallel, but can be at a slight oblique to the valve face (fig. 13 b). the external opening of the rimoportula varies from round to elongate. cyclotella distinguenda has been recorded from mesotrophic lakes and from rivers with connection to lakes. it is rare in hungarian running waters with a 1% occurrence rate (fig. 13 c). cyclotella meduanae h. germain 1981 (figs. 13 d-f) the species was described and illustrated in lm and tem by germain (1981) from the lower part of the river mayenne at angers (france); germain also found this cyclotella in acta bot. croat. 71 (2), 2012 335 centric diatoms from large rivers the river maine, another tributary of the river loire. no sem pictures from the type material have ever been published (houk et al. 2010). unicellular but can form short chains occasionally. frustule cylindrical, valve face flat (smooth), diameter 5–8 mm. the length of the pervalvar axis is about half the length of the diameter. the striated part of the valve face is clearly divided from the central hyaline area (fig. 13 d). diameter of central hyaline area is less than half the length of the diameter. number of striae is 13–16 in 10 mm. alveoli consist of 7–10 radial rows of fine laminate areolae. interfascicles form costae or costae-like structures on the internal valve face and 336 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 13. a-b – cyclotella distinguenda outside and inside view. d-e – c. meduanae outside and inside view; c – distribution of c. distinguenda; f – c. meduanae. scale bar 10 µm (b) and 5 µm (a, d, e). smooth alveolar chambers. marginal fultoportulae with three satellite pores are situated on every second-third interstria (costa). external openings of marginal fultoportulae and rimoportula are well-developed pores. rimoportula is situated between marginal fultoportulae; it has a small labium (fig. 13 e). many granules and spinules can be seen on the valve margin. central area is without fultoportula. this species resembles a small c. meneghiniana concerning the structure of the valve face but it has no central fultoportula and there are no marginal fultoportulae in each interstria. it can be distinguished from c. atomus by the distinct central and marginal area and by the lack of a central fultoportula. cyclotella meduanae has been recorded from different rivers and lakes with different halobity and trophic status. it has a medium frequency in hungarian running waters with a 14% occurrence rate (fig. 13 f). cyclotella meneghiniana kützing 1844 (figs. 14 a-c) the species was described and illustrated by kützing (1844) in material from ferrara (emilia-romagna region, northern italy) and the illustrations were made after meneghini’s specimens. håkansson (1990) presented detailed morphological criteria reinvestigating the italian type material of cyclotella meneghiniana (figs. 1–3, 5–17) and also the type material of the synonym cyclotella kuetzingiana (figs. 19–30), collected in shirehampton near bristol. frustules drum-shaped, the valve face in girdle view more or less tangentially undulate. valve face is circular, 5–43 mm in diameter, with well-defined marginal, striate zone and structureless or more or less radially streaked central hyaline area (fig. 14 a). marginal zone is about one half of the length of the radius with prominent radial striae 6–10 in 10 mm. striae consist of an alveolus, closed externally by an arched areolate layer forming the external ridges of the marginal zone. alveoli are distinctly separated by solid costae (fig. 14 b), corresponding externally to the furrows between the arched outer layer of striae. there are 1–7 valve face fultoportulae on one side of the slight tangential fold, internally surrounded by three satellite pores. marginal fultoportulae with three satellite pores project slightly onto the inner side of the valve, while the external opening is more or less flush with the valve mantle. interfascicles distinct with well-developed spines near the margin (in some cases there are spinules and/or small granules near the spines). below the spines marginal fultoportulae penetrate each interstria. a single rimoportula with a well-developed labium is present on one of the costae at the same level as marginal fultoportulae. cyclotella meneghiniana is cosmopolitan, frequent and abundant in rivers and lakes, under different trophic conditions. houk et al. (2010) defined it as a common species in the littoral and pelagic zone of eutrophic stagnant waters or slowly running rivers. it is the most frequently encountered species in hungarian running waters with a 61% occurrence rate (fig. 14 c). cyclotella scaldensis muylaert et sabbe 1996 (figs. 14 d-f) the species was described by muylaert and sabbe (1996) from plankton of the estuary of the river schelde in belgium. genkal et al. (2008) described and illustrated with sem the species cyclotella ambigua grunow from the phytoplankton of the kuibyshev reservoir (river volga) and considered this taxon conspecific with cyclotella scaldensis, but they never investigated material from the type locality (river yenisei, siberia, russia). the acta bot. croat. 71 (2), 2012 337 centric diatoms from large rivers type population of cyclotella ambigua has been illustrated only with lm by houk et al. (2010), but there is no type material for sem investigation in the grunow collection in vienna. for these reasons we prefer to keep the denomination of cyclotella scaldensis for the specimens found in hungarian watercourses. frustules are cylindrical with a diameter of 7–34 mm. valve face in girdle view with a strong tangentially undulate central part which is colliculate, with more or less radially arranged colliculae (fig. 14 d). the marginal striated zone comprises less than 1/2 of the 338 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 14. a-b – cyclotella meneghiniana outside and inside view. d-e – c. scaldensis outside and inside view; c – distribution of c. meneghiniana; f – c. scaldensis. scale bar 10 µm (a) and 5 µm (b, d, e). valve radius. marginal zone with prominent radial striae, 7–13 in 10 mm. striae have 3-7 radial rows of pores; external are larger than internal pores. each stria consists of a single alveolus closed externally by an arched areolate layer forming the external ridges of the marginal zone. internally, the alveoli are distinctly separated by solid costae (fig. 14 e), corresponding externally to the furrows between the arched outer layer of the alveoli (striae). interstriae terminate with spines. not every interstria has spines. below the spines, marginal fultoportulae are situated on every second to fourth interstria. the marginal fultoportulae with three satellite pores project slightly onto the inner side of the valve. the external opening of marginal fultoportulae are more or less flush with the valve mantle. the single rimoportula is located at the same level as, or slightly lower than the marginal fultoportulae. the rimoportula is elongated and flattened, appears bell-like from the edge of the valve and varies from a simple to spirally flattened tube with its aperture positioned parallel with, or twisted to the edge of the valve. externally the rimoportula has a thickened round aperture. the valve face fultoportulae (1–9) with three satellite pores are located on the convex part of the central area and externally look like round pores. cyclotella scaldensis is a planktonic species, found in brackish to freshwaters, rather eutrophic and slowly running rivers and estuaries (houk et al. 2010). it is a rare species in hungarian running waters with a 2% occurrence rate (fig. 14 f). cyclotella ocellata pantocsek 1901 (figs. 15 a-c) pantocsek (1901) described cyclotella ocellata (and his synonym c. crucigera) from sediment of lake balaton collected in siófok. investigations of type slides and type material by håkansson (1993), kiss et al. (1999) and houk et al. (2010), and of populations of the gallbergweiher, germany (hegewald and hindákova 1997) and lake khubsugul, mongolia (genkal and popovskaya 2008) helped us to understand better the large morphological variability of this species. frustules are disc-shaped, solitary or rarely in short chains, 4–44 mm in diameter, length of pervalvar axis 2–6 µm. the valve face has orbiculi depressi zero to seven; their numbers can vary on the epiand hypovalve. their inverse place is the papilla postament (the round spot on the place where the papilla was fixed; fig. 15 a). besides orbiculi depressi, there are a few relatively small punctae. the central part of the valve is more or less flat, it can be relatively small or large, not depending on the diameter, and contains different-sized and spaced colliculate protuberances. the marginal part of the valve is structured by 14 to 20 striae and interstriae in 10 µm. usually a row of small areolae is situated between two rows of large areolae on the valve face. interstriae different in length, a few of them forked. small granules are observed on the interstriae near the margin on most valves and found sporadically on the striae. valve face fultoportulae (1–13, rarely any fultoportula) are usually surrounded by two satellite pores. in many cases, the external openings of these fultoportulae are very difficult to observe, because many valves have some small holes, which are arranged irregularly in the central part. generally, every third to fifth interstria bears a marginal fultoportula but it may occur on each to every 6th interstria. the internal openings are surrounded by two satellite pores. one rimoportula is situated in the marginal area (fig. 15 b). costae are usually equal in length but those bearing a fultoportula are often shorter. the inner aperture of the alveoli can be round or elongated. the valve usually has a single rimoportula (sometimes two or more) and the orientation of the lip varies. houk et al. (2010) define this centric species as a littoral and pelagic species of mostly standing, oligoto eutrophic waters, particularly with sandy or gravelly bottom, also found in acta bot. croat. 71 (2), 2012 339 centric diatoms from large rivers slowly flowing rivers. it seems to be in close correlation with relatively high ca2+ ion concentration. it has a medium frequency in hungarian running waters with a 23% occurrence rate (fig. 15 c). cyclotella balatonis pantocsek 1901 (figs. 15 d-f) cyclotella balatonis was described by pantocsek (1901) from lake balaton (hungary). houk et al. (2010) published a detailed morphological description of the species based on lm and sem investigation of the type material; it has been found in hungary 340 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 15. a-b – cyclotella ocellata outside and inside view. d-e – c. balatonis outside and inside view; c – distribution of c. ocellata; f – c. balatonis. scale bar 10 µm (d, e), 5 µm (a) and 2 µm (b). (lake balaton), germany (lakes abtsdorfer see, grosser uleisee, tachinger see, waginger see), and the czech republic (@elivka reservoir, river vltava). according to budzyñska and wojtal (2011), the limited number of citations of this centric diatom may be related to its misidentification or confusion with other cyclotella species; they observed it in the plankton of the eutrophic-hypertrophic rusa�ka lake (western poland) and proposed the new combination puncticulata balatonis (pantocsek) wojtal et budzyñska. cells solitary or in short, loosely united chains of 2–8-cells. valve face is flat or slightly concave or convex, diameter 16–22 µm (fig. 15 d). central area is frequently reticulate-verrucose with areolae (internally with domed cribra) and small openings of valve face fultoportulae with three satellite pores. areolae are not strictly radially arranged. marginal area with striae 14–17/10 µm having two radial rows of areolae with tiny punctae between them and increase in number towards the valve margin. striated part takes about half of the radius (or a little bit less). interstriae slightly unequal in length, continue onto the mantle, become narrow below the marginal fultoportula opening. several interstriae divide (this is not very visible with lm). every third-fourth interstria is thicker (shadow-line) and bears externally the round opening of the marginal fultoportulae. the fultoportula’ tubules have two satellite pores which are situated internally on the thickened ribs that are separated by 2–4 thinner costae (fig. 15 e). usually 2–5 (sometimes only one) round rimoportula opening is situated on the valve face at the end of a shortened stria close to the margin, internally with a sessile labium positioned radially or slightly diagonally. cyclotella balatonis can be characterized as a freshwater species of mesotrophic or slightly eutrophic lakes, reservoirs and slowly flowing rivers. it is rare in hungarian running waters with a 5% occurrence rate (fig. 15 f). cyclotella praetermissa j. w. g. lund 1951 (figs. 16 a-c) the species was described by lund (1951) from the plankton of blelham tarn (lake district, cumbria, north west england). it was transferred to the new genus puncticulata by håkansson (2002). houk et al. (2010) proposed to retain the previous generic name cyclotella for the puncticulata taxa and we agree with their proposal. cells solitary or in short, loosely united chains of 2–7-cells. valves concentrically undulate, diameter 8–25 µm (fig. 16 a). central area is frequently reticulate-verrucose with areolae (internally with domed cribra) and small openings of valve face fultoportulae in circular pattern (internally with three satellite pores). the areolae are not strictly radially arranged. marginal area with striae 11–19(22)/10 µm having two radial rows of areolae with tiny punctae between them and increase in number towards the valve margin. interstriae slightly unequal in length continue onto the mantle, become narrow below the marginal fultoportula opening. every fourth (3–6) interstria is thicker (shadow-line) and bears externally the round opening of the marginal fultoportulae. the fultoportula tubules have two satellite pores internally on the thickened ribs, which are separated by 3–7 thinner costae (fig. 16 b). usually one (sometimes two) round rimoportula opening is situated on the valve face at the end of a shortened stria, internally with a sessile labium positioned radially or slightly diagonally. cyclotella praetermissa can be characterized as a freshwater, probably mesohalobous species and can be found in several eutrophic or polluted lakes and rivers. it is rare in hungarian running waters with a 1% occurrence rate (fig. 16 c). acta bot. croat. 71 (2), 2012 341 centric diatoms from large rivers cyclotella radiosa (grunow) lemmermann 1900 (figs. 16 d-f) the species was found in lake mondsee (vöcklabruck district, upper austria) and illustrated as cyclotella comta var. radiosa grunow (van heurck 1882). lemmermann (1900) proposed the specific name c. radiosa (grunow) lemmermann. håkansson (2002) transferred this taxon to the genus puncticulata but houk et al. (2010) preferred to retain the previous generic name cyclotella. central area of valves slightly concave or convex, characterised by radial rows of punctae (loculate areolae, fig. 16 d) internally with domed cribra and irregularly arranged external 342 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 16. a-b – cyclotella praetermissa outside and inside view. d-e – c. radiosa outside and inside view; c – distribution of c. praetermissa; f – c. radiosa. scale bar 10 µm (a) and 5 µm (b, d, e). openings of valve face fultoportulae (internally surrounded by 3 satellite pores, fig. 16 e). diameter is 7–35 µm; marginal area with striae with three (four) punctate rows of areolae (13–16 striae/10 mm) and interstriae continuing onto the mantle. several interstriae bifurcate. every third to fifth interstria is thicker (shadow-line) and has externally the opening of the marginal fultoportulae (internally the short tubules have two satellite pores in a circumferential direction). usually one (sometimes two) rimoportula opening is situated on the valve face at the end of a shortened stria, internally with a sessile to slightly stalked labium. cyclotella radiosa is a pelagic species of lakes and lowland rivers; it prefers eutrophic conditions with higher conductivity and alkalinity but can also be found in mesotrophic waters. it has a medium frequency in hungarian running waters with a 13% occurrence rate (fig. 16 f). discostella houk et klee 2004 discostella pseudostelligera (hustedt) houk et klee 2004 (figs. 17 a-c) the species was first described by hustedt (1939) from rivers ems as cyclotella pseudostelligera. houk and klee (2004) transferred it to discostella genus as d. pseudostelligera. frustules are cylindrical 4–10 mm in diameter. the valve face is nearly flat (the central part sometimes slightly domed or depressed), weakly silicified (fig. 17 a). the marginal area has radial interfascicles (18–20/10 mm) which can be branched. fascicles have usually two rows of areolae. central area can be with or without a star-shaped (stellate) structure. there is a zone with no ornamentation between the short striae and the star-like pattern. the valve mantle has 2–3 lines of areolae. the marginal openings of fultoportulae are located on every (2nd) 3rd to 6th stria and characterized by a unique elongated tube (the end of these tubes can be flat or bifurcated). the internal openings of the marginal fultoportulae are situated between costae and surrounded by 2 satellite pores. there is no valve face fultoportula. one rimoportula is situated in the mantle area close to the valve end between costae, at slightly lower position then the marginal fultoportulae (fig. 17 b), with a sessile labium. among the costae alveolar chambers can be seen. discostella pseudostelligera is a cosmopolitan species, found in lakes or lowland rivers. it prefers nutrient-rich waters but it has been found in oligotrophic to eutrophic waters. it is the second most frequently found species in hungarian running waters with a 53% occurrence rate (fig. 17 c). discostella stelligera (cleve et grunow) houk et klee 2004 (figs. 17 d-f) the species was described from lake rotoaira (north island, new zealand) by grunow in cleve (1881) as cyclotella meneghiniana var. stellulifera, and then raised to cyclotella stelligera by cleve and grunow in van heurck (1882). houk and klee (2004) transferred it to discostella genus as d. stelligera. frustules are circular, disk-shaped, 5–40 mm in diameter. the centre of the valve face is concave or convex (fig. 17 d). the convex central area always displays a star-shaped (stellate) structure usually composed of elongated alveoli, open to the valve inside and with an areolate outer layer (1–2 lines of areolae). the concave central area always lacks alveoli (so the central area is a large hyaline part in inside view); has a not well defined stellate pattern externally, consisting of radially arranged costae. marginal area is with regular radial interstriae (internally thick costae), 9–16/10 mm (fig. 17 e). among the costae there acta bot. croat. 71 (2), 2012 343 centric diatoms from large rivers are well-developed alveolar chambers. the mantle has 1 line of areolae. marginal fultoportulae are located on every second, third or fourth stria. the internal openings of the marginal fultoportulae are surrounded by 2 satellite pores. the external openings are structured as very short tubes or pores, surrounded by silicified rings (tubular structure). there is no valve face fultoportula. one rimoportula occurs at the valve edge between two costae (a little bit higher than the fultoportulae), internally with a sessile labium in the ring of marginal fultoportulae, externally with a simple pore between two costae. 344 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 17. a-b – discostella pseudostelligera outside and inside view. d-e – d. stelligera outside and inside view; c: – distribution of d. pseudostelligera; f – d. stelligera. scale bar 10 µm (e) and 5 µm (a, b, d). discostella stelligera is a cosmopolitan species, it can be found in lakes or lowland rivers. it prefers oligotrophic, mesotrophic waters. it has a medium frequency in hungarian running waters with a 17% occurrence rate (fig. 17 f). stephanocostis genkal et a. e. kuzmina 1985. stephanocostis chantaicus genkal et a. e. kuzmina (figs. 18 a, b) genkal and kuzmina (1985) described this species from lake khantaiskoye in siberia. casper and scheffler (1986) described this species as pleurocyclos stechlinensis from the oligotrophic lake stechlin, brandenburg, germany; the description of genkal and kuzmina acta bot. croat. 71 (2), 2012 345 centric diatoms from large rivers fig. 18. a – stephanocostis chantaicus outside view. c-d – stephanodiscus binderanus outside view and chain; b – distribution of s. chantaicus; e – s. binderanus. scale bar 10 µm (d), 3 µm (c) and 2 µm (a). has nomenclatural priority. scheffler and morabito (2003) provided very good sem illustrations of this stephanocostis from the plankton of lago di como (italy) and stechlinsee (germany). frustules are cylindrical, solitary or in short chains, 3.5–8.0 mm in diameter. the valves are flat or, rarely, concentrically undulated. the characteristic feature of this species is the strongly elevated interstriae on the valve face (figs. 18 a), they can be branched and become a little bit wider near the margin. some of them are shorter and do not reach the centre of the valve face. the areolae between the interstriae are irregularly arranged. there is one areola in striae near the centre and 3–4 close to the margin. position of central fultoportula is eccentric (with three satellite pores), difficult to differentiate among the areolae in the central part. the small external openings of the marginal fultoportulae are slightly elevated. one rimoportula is situated on an interfascicle near the margin. stephanocostis chantaicus has been found in different mesotrophic lakes and rivers, sometimes as an abundant species. it is rare in hungarian running waters with a 1% occurrence rate (fig. 18 b). stephanodiscus ehrenberg 1846 stephanodiscus binderanus (kützing) willi krieger 1927 (figs. 18 c-e) this species was described by kützing (1844) as melosira binderana, and was transferred to the genus stephanodiscus by krieger (1927) on the basis of its frustular morphology. a detailed description about the sem and tem morphology of stephanodiscus binderanus and s. binderanus var. oestrupii was published by round (1972) and genkal (1997). the barrel-like frustules form filaments (strong chains; fig. 18 c). valve is flat, 4–24 mm in diameter. radial rows of areolae form striae, between them distinct interfascicles are situated. number of areolae 2 (3) near the valve margin, 12–13/10 mm. there are characteristic spines at the end of each interstria (fig. 18 d). spines are bifurcated and they connect the frustules together. marginal fultoportulae with three satellite pores have an external tube. a single fultoportula is situated with two satellite pores on the valve face near the centre (it is not seen well in many cases). the internal openings of areolae have domed cribra, characteristic for many stephanodiscus species. the girdle region consists of two short bands adjacent to the two valve mantles and intermediate wider band(s). the girdle region is delicate and often collapses. the connection between valves by spines is so strong that frustules can more easily break at valve mantle – girdle junction than between two valves. stephanodiscus binderanus is a cosmopolitan species and has been recorded from many rivers and lakes with different trophic status. it is a rare species in hungarian running waters with a 1% occurrence rate (fig. 18 e). stephanodiscus hantzschii grunow in cleve and grunow 1880 (figs. 19 a-f) this species has been variously interpreted and illustrated by several authors since its description by grunow in cleve and grunow (1880). frustule is cylindrical; valve circular, almost flat, with moderately sloped mantle margins, 5–30 mm in diameter. valve face consisting of areolae arranged in striae separated by distinct interstriae. interstriae subtended by a well-developed spine. there is no valve face fultoportula. marginal fultoportulae with three satellite pores occur below every third to fifth spine. between the marginal fultoportulae a rimoportula is located centripetally from 346 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. the level of the spine insertion. the internal opening of the rimoportula has a well developed labium and the external opening is an elongated tube (frequently not seen well), somewhat similar to the spines in appearance. valve ornamentation occurs in two distinct forms (f. hantzschii and f. tenuis), with occasional intergrades between them. the areolae of the valve face are arranged in biseriate striae near the margin and becoming uniseriate near the centre. there are 8–10 striae in 10 mm near the valve margin, separated by a wide and distinct interstria (fig. 19 a). number of areolae 18–25 in 10 mm. valves frequently appear strongly silicified under em. areolae of valve face with domed cribra are acta bot. croat. 71 (2), 2012 347 centric diatoms from large rivers fig. 19. a-b – stephanodiscus hantzschii f. hantzschii outside and inside view. d-e – s. hantzschii f. tenuis outside and inside view; c – distribution of s. hantzschii f. hantzschii, f: s. hantzschii f. tenuis. scale bar 5 µm (a, b, d, e). variously formed (fig. 19 b), depending on the degree of silicification, they can be circular, slit-like or partially occluded. areolae of valve mantle are much finer and are often arranged in line. stephanodiscus hantzschii f. tenuis (hustedt) håkansson et stoermer 1984 (figs. 19 d-f) hustedt (1957) described stephanodiscus tenuis with a fine silicified valve resembling to s. hantzschii. based on a detailed observation of the type material of s. hantzschii, håkansson and stoermer (1984) transferred s. tenuis to s. hantzschii as its forma. areolae of the valve face are arranged in multiseriate (3–5) striae near the valve margin, becoming uniseriate near the centre (figs. 19 d, e). striae 8–10/10 mm near the valve margin and separated by a thin but distinct interstria. number of areolae 25–35/10 mm. the striae and interstriae can become sinuous towards the valve centre. in the centre an annulus, a rosette-like structure is differentiated. there are long spines on each interstria at the valve margin (fig. 19 d). stephanodiscus hantzschii is a cosmopolitan species, recorded in rivers and lakes with different trophic levels and halobity. stephanodiscus hantzschii f. hantzschii has a medium frequency in hungarian running waters with a 24% occurrence rate (fig. 19 c), s. hantzschii f. tenuis is frequent with a 37% occurrence rate (fig. 19 f). stephanodiscus minutulus (kützing) cleve et j. d. möller 1882 (figs. 20 a-c) this species was described as cyclotella minutula by kützing (1844) and was transferred by cleve and möller (1882) to the genus stephanodiscus as s. minutulus. important data were published in the papers of håkansson (1986, 2002), genkal and håkansson (1990). frustule is short, cylindrical, 4–12 mm in diameter (fig. 20 a). the valve face is flat or slightly concave or convex. there are radial rows of areolae (striae), which are disorganized in the centre, then uniseriate and multiseriate (2–3) near the margin. the central area with relatively large, usually round areolae, is characteristic but depending on degree of valve silicification slit-like or elongated openings of areolae occur, too. the valve mantle is very shallow, about 2 to 3 areolae at most. short, solid spines are situated on each interstria at the margin. just below the spines, the round openings of marginal fultoportulae with two satellite pores can be seen on each second to fifth interstria and a single rimoportula with small sessile labium among them (fig. 20 b). a single valve face fultoportula with two satellite pores is visible towards the centre; its external opening is elevated a little. the internal valve face is very characteristic with domed cribra of areolae. stephanodiscus minutulus is a cosmopolitan species; it has been recorded as frequent and abundant in rivers and shallow lakes with different trophic status. it is a characteristic species in the river danube first of all during the cold period (from november to april). it is a frequent species in hungarian running waters with a 44% occurrence rate (fig. 20 c). stephanodiscus neoastraea håkansson et b. hickel 1986 emend. casper, scheffler et augsten 1992 (figs. 20 d-f) stephanodiscus neoastraea was described by håkansson and hickel (1986) from the bornhöveder see (germany). casper et al. (1992) made an amended description of the species, and casper and klee (1992) made a clear differentiation of s. rotula and s. neoastraea based on the valve face fultoportula arrangement håkansson (2002) confirmed the absence of valve face fultoportula in the latter species. genkal (2009) considered s. agassizensis, s. heterostylus and s. maximus as synonyms of s. neoastraea, and defined its autecology as a 348 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. freshwater-brackish water, widespread planktonic species, present in spring-autumn at water temperatures of 10–24 °c, salinity up to 7.5‰, transparency 50–90 cm, and ph 7.6–8.2. frustules are circular, isoor heterovalvate, solitary or in short chains. the centre of the valve face is concave or convex (fig. 20 d), 11–54 mm in diameter. areolae with domed cribra arranged in fascicles. these fascicles are biseriate (rarely triseriate) at the edge of the valve face and uniseriate towards the centre, interfascicles 8–11/10 mm. the areolation of the valve centre can be irregular or the fascicles end in a rosula (annulus). relatively long acta bot. croat. 71 (2), 2012 349 centric diatoms from large rivers fig. 20. a-b – stephanodiscus minutulus outside and inside view. d-e – s. neoastraea outside and inside view; c – distribution of s. minutulus; f – s. neoastraea. scale bar 10 µm (d, e) and 5 µm (a, b). spines are arranged irregularly on every 2nd–4th interfascicle at the valve edge. there are marginal fultoportulae on the mantle below the spines on 1–3 interstriae. their external opening has a short tube, internally with three satellite pores (fig. 20 e). valve face fultoportulae 0–5, on convex valves they are on the central part of the valve face (fig. 20 d), while on concave valves they are near the marginal part where the fascicles become biseriate (fig. 20 e). their external openings are slightly elevated and have two satellite pores. a single rimoportula is visible among the marginal fultoportulae with a long external tube and a well-developed stalked labium. stephanodiscus neoastraea can be found in eutrophic lakes and lowland rivers, but it has also been found in the oligotrophic lake stechlin, germany. it has a medium frequency in hungarian running waters with a 13% occurrence rate (fig. 20 f). stephanodiscus vestibulis håkansson, e. c. theriot et stoermer 1986 (figs. 21 a-c) håkansson et al. (1986) described stephanodiscus vestibulis from a man-made canal »lazy lagoon«, connected with the eutrophic west okoboji lake (dickinson country, iowa, usa). frustules are cylindrical, 4–11 mm in diameter. the centre of the valve face is concave or convex. from the central area uniseriate rows of areolae (14–30/10 mm) run towards the valve face/mantle junction, becoming biseriate after the central elevation/depression (fig. 21 a). internally, the areolae are covered by domed cribra. there is a single valve face fultoportula on the central area with an external thickened opening and internally with three satellite pores (fig. 21 b). every interfascicle bears a solid spine on the valve face/mantle junction. the mantle has 3–4 lines of areolae. the marginal fultoportulae are located on every third-fourth interfascicle. the external opening of fultoportulae is hidden by a vestibule (a porch-like structure). internally, the short tubules of fultoportulae are surrounded by three satellite pores. one rimoportula with a tubular-like extension is in the ring of spines, internally with a sessile, slit-like labium, externally with a longer tube than the spines. stephanodiscus vestibulis was recorded from eutrophic freshwaters of western north america, japan and korea; it is a probably a widespread cosmopolitan species, invasive in europe. it is actually a scarce species in hungarian running waters with a 6% occurrence rate (fig. 21 c). coscinodiscales hemidiscaceae actinocyclus ehrenberg 1837 actinocyclus normanii (w. gregory ex greville) hustedt 1957 (figs. 21 d-f) the species was described by gregory ex greville (1859) as coscinodiscus normanii and was transferred to actinocyclus by hustedt (1957) as a. normanii var. subsalsus (juhlin-dannfelt) hustedt. kiss et al. (1990) suggested that the two forms of this species (f. normanii and f. subsalsa) have no taxonomical value and should not be treated as separate taxa. the frustule is drum shaped; the valve face is flat or slightly concave or convex. the diameter is 13–66 mm, the length of the pervalvar axis 11–33 mm. the valve face has a characteristic areola pattern. areolae are positioned into sectors (fig. 21 d). the sectors are delimited on both sides by a single row of areolae, which are radial and reach the centre of the valve face. the rows of areolae within a sector are parallel to the delimiting row of areo350 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. lae and gradually decrease in length. on the valve face the areolae are occluded externally by a cribrum with very tiny pores and they are opened internally with a well-developed foramen (fig. 21 e); 6–12 areolae in 10 mm on the valve face and 11–22 on the valve mantle. the number of areolae depends on the diameter; it is higher on small valves (15–25 mm) and lower on large valves (50–66 mm). areolae run down straight on the valve mantle, parallel to the pervalvar axis. the external openings (pores) of the rimoportulae are clearly visible on the curved outer surface of the mantle; they have an elongated neck and a large labium. the acta bot. croat. 71 (2), 2012 351 centric diatoms from large rivers fig. 21. a-b – stephanodiscus vestibulis outside and inside view. d-e – actinocyclus normanii outside and inside view (pseudonodulus white arrow). c – distribution of s. vestibulis; f: – a. normanii. scale bar 10 µm (d, e), 5 µm (a) and 2 µm (b). pseudonodulus is located above the openings of the rimoportulae (fig. 21 d). it has a slight depression and a larger opening than those of the rimoportulae. the most typical morphological feature of the inner part of the valve is the ring of rimoportulae, 3–11 in number, depending on the diameter. actinocyclus normanii has been recorded from several eutrophic lakes and rivers with different halobity. its increase during the last decades could be the result of eutrophication (kiss et al. 1990). actinocyclus normanii is considered a warm stenothermic species because its density is high in the summer. it might be an invasive species. it is a rare species in hungarian running waters with a 3% occurrence rate (fig. 21 f). discussion during this study we examined altogether 107 different watercourses. among them the largest was the river danube from which more than 100 samples were analysed by em from 10 hungarian sampling sites. only one sample was analysed from creeks, which were the smallest streams analysed in this study. there were also channels out flowing from rivers or connecting different rivers. usually they are slowly flowing channels of different size. in total, 41 centric taxa, belonging to 11 genera, were found during this study. the average taxon number found in a single watercourse was 7, the maximum 40 and the minimum 1. cyclotella meneghiniana was the most frequently encountered species (more than 60% of all sites, fig. 22). the following 12 taxa were found in more than 20% of sites (between 20–53%): aulacoseira granulata, conticribra weissflogii, cyclostephanos dubius, c. invisitatus, cyclotella atomus var. atomus, c. atomus var. gracilis, c. ocellata, discostella pseudostelligera, melosira varians, stephanodiscus hantzschii f. hantzschii, s. hantzschii f. tenuis, s. minutulus. eight taxa were found in 5–10% of the investigated sites (aulacoseira 352 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. fig. 22. the occurrence frequency of centric diatom species in the investigated hungarian rivers, expressed as the percentage of all sampling sites in this study where the given species occurred (abbreviations of species names correspond to the omnidia program). aff. muzzanensis, a. subarctica, conticribra guillardii, cyclotella balatonis, c. delicatula, skeletonema potamos, stephanodiscus vestibulis, thalassiosira lacustris) and 6 taxa were found in only one site (aulacoseira italica, cyclotella distinguenda, c. praetermissa, stephanocostis chantaicus, stephanodiscus binderanus, thalassiosira gessneri). there is a correlation between river size (river order) and the species richness of centric diatoms (fig. 23). the number of species in rivers increases with the size of the catchment area. there are several reasons behind this phenomenon. a large river has many affluents and they enrich the host river with their algal flora. large rivers have more connections with lakes and many lacustrine species originate from there. they have many side arms and oxbows which are also a source of centric diatoms. reservoirs can frequently be found on large rivers where centric diatoms can proliferate easily in the still water. there are man-made channels between rivers which can transport algae between them. we believe that riverine species can be found among the frequently and moderately frequently occurring centric species (fig. 22) but there are also riverine species among the rarer species, too. we consider the following species true riverine species (real potamoplanktic species, defined as living and proliferating in slowly flowing rivers with a current of about 1 m s–1 or less): aulacoseira granulata, conticribra weissflogii, cyclostephanos dubius, c. invisitatus, cyclotella atomus, c. meduanae, c. meneghiniana, skeletonema potamos, stephanodiscus hantzschii, s. minutulus, thalassiosira duostra, t. pseudonana. the most abundant taxa in large rivers are among these potamoplanktonic species. here are a few examples from the river danube: s. minutulus is one of the most abundant species in the cold season (early spring and late autumn). cyclostephanos invisitatus is abundant from april to late september; s. hantzschii f. tenuis in the summer and autumn. we consider skeletonema potamos a warm stenothermic species, it may be the most abundant centrales acta bot. croat. 71 (2), 2012 353 centric diatoms from large rivers fig. 23. taxon numbers in different sized rivers (s: watershed <100 km2, m: 100.1-1000 km2, l: 1000.1–10000 km2, xl: > 10000.1 km2). species during the summer. cyclotella atomus, c. meduanae, c. meneghiniana, t. pseudonana have medium abundance from early may to late september (kiss 1984, 1987, 1988; kiss and genkal 1993, 1996; kiss et al. 1994). there are several periphytic species living in rivers which are washed out from the periphyton, like most of the aulacoseira species and melosira varians. several lacustrine species were found during this study in rivers and channels, like actinocyclus normanii, cyclotella balatonis, c. distinguenda, c. ocellata, c. praetermissa, c. radiosa, discostella pseudostelligera, d. stelligera. among these species, d. pseudostelligera can be abundant in summer time; a. normanii was abundant in summer at the end of 1980th and it became rare during the past three decades (kiss and nausch 1988, kiss et al. 1990). cyclotella choctawhatcheeana is a brackish water species and probably has a salt lake origin in hungarian rivers. there are several salt lakes in transylvania (romania) and these lakes could be the origin of this species (further studies can prove this hypothesis). it is important to evaluate centric diatoms of hungarian rivers from a nature conservation point of view. hungary does not have an accepted, comprehensive red list that includes centric diatoms. the demand for such a red list has already been acknowledged, however, targeted research has only just started (ács et al. 2002, 2004; szabó et al. 2004, 2005; kiss 2005, németh 2005, kiss and ács 2009). the first, internationally recognised red list of diatoms was published by lange-bertalot and steindorf (1996) about the freshwater species of germany. this list includes several hundred species from the ones that are at the brink of extinction to the ones that are not endangered momentarily. recognising the nature conservation value of algae, another comprehensive list has been published in slovakia, which includes algae and cyanobacteria as well (hindák and hindáková 2001). in this publication, the authors mainly list rare species from slovakia, moreover species that have been described from slovakia. for this reason, there are only a few centric species in this list, which also occur in hungary. it has to be emphasised in connection with algological red lists, that rare, conservationally valuable algae – like any other microscopical and macroscopical aquatic organisms – can only be protected by protecting the whole lake and its nearest surroundings, its catchment area. also in the case of rivers, a selected part of the catchment area has to be treated as nature conservation area. several taxa were found in hungarian rivers that are valuable or potentially valuable from a nature conservation perspective, based on lange-bertalot and steindorf (1996), szabó et al. (2004), kiss (2005), németh (2005), kiss and ács (2009). several of the rare species could be listed in some future hungarian red list of algae. these are the following: endangered taxa: cyclotella choctawhatcheeana, c. distinguenda, stephanocostis chantaicus. vulnerable taxa: aulacoseira subarctica, conticribra guillardii, cyclotella delicatula, thalassiosira gessneri, t. incerta, t. lacustris. taxa with decreasing stocks: actinocyclus normanii, aulacoseira pusilla, cyclotella scaldensis, c. praetermissa, skeletonema subsalsum, stephanodiscus binderanus. taxa not yet classified: aulacoseira pusilla, cyclostephanos invisitatus, cyclotella atomus var. gracilis, cyclotella meduanae, cyclotella scaldensis. 354 acta bot. croat. 71 (2), 2012 kiss k. t., klee r., ector l., ács é. acknowledgements this study was supported by hungarian national science foundation (otka k 68327, and partly by ktia-otka 80140). we gratefully acknowledge dr. eduardo a. morales (universidad católica boliviana san 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(bacillariophyceae) – eine kleine cyclotella-art aus dem schlei-ästuar (brd). nova hedwigia 52, 359–363. wojtal, a. z., kwandrans j., 2006: diatoms of the wyzyna krakowsko-czestochowska upland (s poland) – coscinodiscophyceae (thalassiosirophycidae). polish botanical journal 51, 177–207. acta bot. croat. 71 (2), 2012 363 centric diatoms from large rivers acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 385 acta bot. croat. 73 (2), 385–400, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 distribution patterns and fl oristic analysis of the colymbada tauromenitana (guss.) holub populations in sicily (italy) saverio sciandrello*, sonia d’agostino department of biological, geological and environmental sciences, university of catania, via a. longo 19, i – 95125 catania, italy abstracts – colymbada tauromenitana (guss.) holub (asteraceae) is a rare paleoendemic, chasmophyte species, occurring on calcareous cliffs in the eastern part of sicily (italy). the aim of this work is to analyze the structure and fl oristic composition of the c. tauromenitana community, in order to characterize the diversity of populations in relation to different ecological data. in all, 61 plots were examined. for each plot, the fl oristic composition and the cover of the species were determined using the standard relevé method. three vegetation types emerged from canonical components analysis (cca), correlated to a gradient of environmental conditions ranging from the coast to inland areas. the fi rst group with lomelosia cretica and dianthus rupicola subsp. rupicola was correlated to thermo-xerophilous conditions (lower thermo-mediterranean belt), the second group with silene fruticosa and colymbada tauromenitana was linked to thermophilous conditions (upper thermo-mediterranean belt) and the third with dianthus siculus and odontites bocconei was correlated to mesophilous conditions (meso-mediterranean belt). altitude is the main factor infl uencing both species richness and fl oristic composition. the density of c. tauromenitana is infl uenced mainly by rainfall. finally, we propose a new risk status for this rare species. keywords: colymbada tauromenitana, conservation status, distribution, ecology, italy, paleoendemic, plant diversity, sicily introduction the high level of plant endemism in the mediterranean area is related to the environmental heterogeneity, particular climatic conditions, geological and paleogeographic factors as well as millennial human land use (cowling et al. 1996, médail and verlaque 1997, carmel and flather 2004, minissale and sciandrello 2013). * corresponding author, e-mail: s.sciandrello@unict.it copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. sciandrello s., d’agostino s. 386 acta bot. croat. 73 (2), 2014 plant conservation strategies are a crucial issue in the mediterranean area, due to excessive human impact on the natural landscape which in several cases has led to the loss of habitats and endemic species (schröter et al. 2005, pisanu et al. 2011, tomaselli et al. 2012). sicily is one of the most important centers of plant diversity in the mediterranean region (médail and quézel 1999). with about 3,300 vascular taxa (species and subspecies) according to giardina et al. (2007), it accounts for nearly 43% of the entire vascular fl ora of italy (7,634 species, according to conti et al. 2005). the importance of the vascular fl ora of sicily lies not only in the total number of taxa but also in the considerable number of endemic species (about 13% according to brullo et al. 2011). chasmophytes represent one of the most important groups of plants within the fl ora of sicily (davis 1951, thompson 2005). this ecological group includes one very interesting taxon, colymbada tauromenitana (guss.) holub (= centaurea tauromenitana guss.), a huge plant which grows on the limestone cliffs of the peloritani district (pirola 1961, gramuglio et al. 1985, brullo et al. 1995). this species is a paleoendemic species exclusive to eastern sicily (arena et al. 1975). currently, populations of c. tauromenitana are threatened by human activities, mainly represented by excavations for building, and by invasive plants, especially the expansion of opuntia fi cus-indica. the aim of this study is the analysis of the structure and fl oristic composition of the c. tauromenitana community, in order to characterize the diversity of populations in relation to different environmental conditions. these results will be useful for a proper evaluation of its conservation status and for the improvement of actions aimed at the conservation of this rare taxon. materials and methods study area the study area includes the peloritani district, in the eastern part of sicily (fig. 1). this area is characterized by mountains of moderate altitude, comprising, from south to north, monte tauro (245 m), monte petraro (475 m), castelmola (496 m), roccella (602 m), monte ziretto (381 m), costa ogliastro (466 m), monte veneretta (884 m), monte lapa (771m), monte castelluccio (501 m), monte pernice (712 m) and monte galfa (1,000 m). from a geological viewpoint the study area belongs to the longi-taormina and capo s. andrea unit. the lower subunit is formed by an epimetamorphic basement capped by mesozoic-cenozoic sedimentary cover made up, from bottom to top, of continental redbeds evolving upwards to platform carbonates (lentini et al. 2000). all colymbada tauromenitana populations are protected by natura 2000 areas (european community 1992) with the exception of the m. petraro site. annual precipitation at the sites ranges from 600 to 1,000 mm and mean annual temperature varies between 19–20 °c (along the coast) and 13–14 °c (inner area) (brullo et al. 1996). according to the bioclimatic classifi cation proposed by rivas-martínez (1993, 2004), the area under study is referred to the mediterranean pluviseasonal oceanic bioclimate, with thermotypes ranging from the thermo-mediterranean to the meso-mediterranean and ombrotypes from the semiarid to lower humid. ecology of colymbada tauromenitana in the eastern sicily (italy) acta bot. croat. 73 (2), 2014 387 data analysis the published data on the distribution of this species (gussone 1827, lojacono 1908, pirola 1961, gramuglio 1967, gramuglio et al. 1985) were reviewed and specimens of herbaria from catania and palermo were also examined. on the basis of these starting data, all the sites of sicily in which c. tauromenitana has been reported were visited; in addition, fig. 1. study area; red dots show the distribution area of erucastretum virgati centauretosum tauromenitanae; blue stars indicate the distribution area of erucastretum virgati dianthetosum siculi; blue line indicat es natura 2000 areas; yellow grid of 2 × 2 km to assess area of occupancy. sciandrello s., d’agostino s. 388 acta bot. croat. 73 (2), 2014 other potentially suitable sites for the species were investigated. our assessment of the sicilian population size was based on a census in those sites where we found the species. the assignation of c. tauromenitana to an iucn (international union for conservation of nature) risk category (iucn, 2001), was largely made on a quantitative assessment. the threats to c. tauromenitana in each locality were determined from the fi eld observations. a grid of 2 × 2 km was used to assess area of occupancy (aoo, defi ned as the area within the extent of occurrence, following iucn (2008) and rossi and gentili (2008). conservation status was assessed following iucn (2008) and the conservation measures adopted or proposed for the species were based on our fi eld observations. the fi eld work, carried out in order to analyze the structure and composition of the c. tauromenitana plant community, was done in 2009–2012 during which 61 plots (10 × 10 m) were made using the standard method of relevés (westhoff and van der maarel 1978). numerical analysis (cluster analysis – wpgma method, chord coeffi cient) of relevé was performed using the software package syn-tax 2000 (podani 2001). environmental gradients and plant communities were examined with canonical component analysis (cca), using the pc-ord, v6, software. in particular, the cca takes into account different quantitative data such as the distance from the sea (ds), altitude (altit.), slope of terrain, number of species (n. sp.) and rock type. the original braun-blanquet sampling scale (braunblanquet 1964) has been transformed into an ordinal scale, according to van der maarel (1979). for the nomenclature of the species, giardina et al. (2007), raimondo and spadaro (2009) were followed, while phytosociological nomenclature follows brullo et al. (2002). for each taxon, life form and chorological element are reported. the life form follows the raunkiaer system as proposed by pignatti (1982), while for chorological elements brullo et al. (1998a) are followed. the exsiccata (preserved in botanical museum of catania) were studied with the help of flora europaea (tutin et al. 1964–80), the italian fl oras (fiori 1923–29, pignatti 1982). two indexes were chosen to estimate diversity: (1) species richness (sr), i.e., the total number of plant species recorded in a sample plot, and (2) shannon-wiener’s diversity index (h’). this index takes into account the degree of equitability (j) of the species distribution. sr and h’ represent the α-diversity level of the vegetation in relation to the size of the relevé. spearman rank correlation coeffi cients were used to evaluate the importance of environmental factors in the distribution of the plant diversity. a p-value of < 0.05 was taken as indicating a statistically signifi cant difference or correlation. the rock type classifi cation follows lentini et al. (2000), which for this area recognizes four types: type 1 – limestone and dolomite: detritic limestones with rudite clasts of quartz at the base (ut); type 2 – limestone and dolomite: detritic limestones, biocalcarenites with brachiopods gray and reddish veined (ug); type 3 – limestone and marl alternation in »medolo facies« (utm) and type 4 – »flysch of capo d’orlando« (omc). the study of the species and its habitat c. tauromenitana is a perennial herb or dwarf shrub, with erect stems, 50–100 (120) cm high, branched above. the species is diploid (2n = 2× = 20) (de santis et al. 1976) and fl owers from the second half of may through the fi rst half of july. dissemination occurs between late july and early august. c. tauromenitana does not reproduce vegetatively (pers. observation). the current conservation status of c. tauromenitana in sicily (conti el al. 1997) is ecology of colymbada tauromenitana in the eastern sicily (italy) acta bot. croat. 73 (2), 2014 389 »low risk« (lr). according to our data, single individuals of c. tauromenitana can survive for several decades. c. tauromenitana grows on limestone cliffs of the peloritani district (sicily) with other endemic species (arena et al. 1975, sciandrello et al. 2013a), as erucastrum virgatum (j. et c. presl) c. presl, dianthus rupicola biv. subsp. rupicola, brassica incana ten., brassica raimondoi sciandr., c. brullo, brullo, giusso, miniss. et salmeri, anthyllis vulneraria l. subsp. busambarensis (lojac.) pignatti, antirrhinum siculum mill., dianthus siculus c. presl in j. et c. presl, galium aetnicum biv., micromeria graeca (l.) benth. ex rchb. subsp. consentina (ten.) guinea, etc.; it is a member of the highly specialized rupicolous communities of the association erucastretum virgati brullo et marcenò 1979 centauretosum tauromenitanae pirola ex brullo et marcenò 1979 (asplenietea trichomanis (br.-bl. in meier et br.-bl. 1934) oberd. 1977, dianthion rupicolae brullo et marcenò 1979). the vegetation pattern of the area is characterized by perennial dry grasslands dominated mostly by caespitose hemicryptophytes, by rupicolous vegetation, rarely shrubby elements that are adapted to different habitats and restricted woods (sciandrello et al. 2013b). results distribution and population size our investigations confi rmed 8 sites with colymbada tauromenitana (fig. 1): monte petraro (castelmola); villagonia, isola bella and monte tauro (taormina); castelmola and roccella (castelmola); monte veneretta (castelmola); monte ziretto and costa ogliastro (castelmola); monte lapa (mongiuffi -melia); monte pernice (mongiuffi -melia); monte castellaccio (letojanni, gallodoro, mongiuffi melia). the locality of capo s. alessio (giardina et al. 2007) is not confi rmed, while the m. galfa site, characterized by potentially suitable cliffs for the growth of c. tauromenitana, has been carefully explored and the species has not been found. the areas in which the species occur vary between altitudes of 20–800 m, on slopes of 40–90° exposed mainly east, and a distance from the coastline that varies between 30 and 6,000 m. the entire population of c. tauromenitana consisted of fewer than 7,200 plants, distributed with variable density (tab. 1). the current populations of c. tauromenitana occupy (aoo) about 28 km2 (fig. 1), distributed along the cliffs with the exception of the populations of m. petraro and m. castellaccio. the highest density is distributed on the cliffs facing east between m. veneretta and m. lapa with about 2,500 individuals (fig. 2). other interesting populations with high values of individuals were found in the area of castelmola, on the cliffs facing east of m. ziretto-costa ogliastro and on the south facing rocks of the castello di taormina, while low values were found along the coast, to m. pernice and m. petraro. overall, the narrow distribution area of c. tauromenitana is linked to the substrate type (mainly limestone), with the exception of a small population of a few individuals, growing on conglomerate substrates to m. petraro. we may therefore consider c. tauromenitana a narrow endemic of the carbonatic district of taormina. finally, considering the number of individuals, the narrow area and several threats (invasive species, fi re, natural collapses and stone extraction), we may conclude that the distribution area of c. tauromenitana is highly localized and at risk of further reduction. therefore, based on the fi eld investigation carried out in the present survey, we propose a new iucn category, vulnerable (vu a2ce, b1ab (ii,iii,iv)). s c ia n d r e l l o s., d ’a g o st in o s. 390 a c ta b o t. c r o a t. 73 (2), 2014 tab. 1. sites where colymbada tauromenitana has been located. no. plots – number of plots; no. ind. – number of individuals of c. tauromenitana; no. endemic – number of endemic plants; rock type: ut – limestone and dolomite with rudite clasts of quartz at the base, ug – limestone and dolomite with brachiopods gray and reddish veined, utm – limestone and marl alternation, omc – flysch; no. species, number of species; t med – mean temperature; rainfall (mm) – annual precipitation. locality n o. p lo ts n o. in d. c . t au ro m en ita na co ve r ( % ) n o. e nd em ic r oc k ty pe a lti tu de (m ) se a di st an ce (m ) n o. s pe ci es (a ve ra ge ) t m ed (° c ) r ai nf al l ( m m ) m ai n th re at s monte petraro (castelmola) 3 80 26 5 omc 460 2210 21 16–17 700–800 natural collapses, fi re villagonia (taormina) 3 280 16 7 ut 25–35 95–130 19 18–19 600–700 invasive plants rupe antistante isola bella (taormina) 3 350 16 4 ut 20–70 35–75 16 18–19 600–700 invasive plants rupi di capotaormina 1 30 5 5 ut 60 90 19 19–20 600–700 invasive plants monte tauro e castello di taormina 3 750 30 6 ut 350 600 19 17–18 700–800 building, invasive plants rupi di castelmola 5 10000 30 7 ut 480 16000 24 16–17 800–900 invasive plants rupi antistanti castelmola 3 600 26 6 ut 530 18500 24 16–17 800–900 fi re and pasture roccella (castelmola) 2 100 25 5 ut 420 16800 22 16–17 800–900 invasive plants monte veneretta (castelmola) 120 12000 31 9 ut 760–850 27000 26 14–15 900–1000 fi re, pasture monte ziretto (castelmola) 5 800 26 4 ut 530 1200–1400 25 15–16 800–900 fi re, pasture costa ogliastro (castelmola) 5 450 30 5 ut 400–450 1550–1850 20 15–16 800–900 fi re, pasture monte lapa (mongiuìffi melia) 4 12000 32 6 ut 620–650 3000–3100 28 14–15 900–1000 fi re, pasture monte pernice (mongiuìffi melia) 4 250 20 8 utm 500–530 420000 15 15–16 800–900 stone extraction monte castellaccio (letojanni, gallodoro, mongiuffi melia) 3 350 26 5 ut 250 250000 21 17–18 700–800 natural collapses monte galfa (roccafi orita, mongiuffi melia) 5 0 0 7 ut-ug 720–920 6300–6700 21 13–14 1000–1200 fi re, pasture ecology of colymbada tauromenitana in the eastern sicily (italy) acta bot. croat. 73 (2), 2014 391 plant communities and species diversity in total, 72 species of vascular plants have been recorded on the cliffs of the study area. most species belong to mediterranean elements, with the dominant life forms being chamaephytes (35%) and hemicryptophytes (33%), while the therophytes show low values (3%). the ecological features of the rupicolous plant community may be highlighted by the fl oristic composition and its variations along a gradient of conditions from the coast to the inland. the results of the cluster analysis (fig. 3) show two main vegetation groups, each with specifi c indicator species. group a (56 relevés) represents the coastal plant communities with colymbada tauromenitana, while group b (5 relevés) represents inland plant communities without c. tauromenitana. within group a, two sub-groups were distinguished: a1, comprising the thermo-mesophilous plant communities, and a2, which includes the thermo-xerophilous plant communities. within sub-group a1 we can distinguish the rupicolous vegetation of the conglomeratic outcrops (a11) and plant communities of the limestone outcrops (a12), while the sub-group a2 includes the strictly coastal plant communities with sub-halophilous species (a21) and the thermophilous plant communities linked to marine ecological conditions (a22). the species richness and shannon-wiener diversity index indicate that the plant communities near the coast show a moderate diversity with an average sr of 21.7 and an average shannon-wiener diversity index of 2.3 (j = 0.73). both values increase by altitude in fig. 2. rupicolous vegetation with colymbada tauromenitana on limestones outcrops of m. veneretta (castelmola) (a); colymbada tauromenitana with the presence of opuntia fi cus-indica on cliffs of taormina (b); plant in its peak fl owering time (c, d). sciandrello s., d’agostino s. 392 acta bot. croat. 73 (2), 2014 middle zones with an average of 24 species and a shannon-wiener diversity index of 2.5 (j = 0.79). the highest areas (m. galfa) have a moderate sr of 21 and shannon-wiener diversity index of 2.6 (j = 0.84) (tab. 2). tab. 2. relationship between species richness, standard deviation, equitability, shannon-wiener’s diversity index and bioclimate zones. 1 – monte petraro; 2 – villagonia, capotaormina, isola bella, m. tauro and castello (taormina); 3 – roccella and castelmola; 4 – m. veneretta; 5 – m. ziretto and costa ogliastro; 6 – m. lapa; 7 – m. pernice; 8 – m. castellaccio; 9 – m. galfa. 36 plots (2, 3, 5, 6, 7, 8) 20 plots (1, 4, 5) 5 plots (9) thermomediterranean lower zone thermomediterranean upper zone mesomediterranean zone 0–500 m 400–800 m 800–1000 m species richness 21.75 24.1 20.8 standard deviation 5.5 6.8 3.4 equitability 0.73 0.79 0.84 shannon-wiener’s index 2.3 2.5 2.6 fig. 3. dendrogram resulting from cluster analysis of the plots (wpgma method, chord coeffi cient). plant communities: a1 – erucastretum virgati centauretosum tauromenitanae facies with silene fruticosa; a2 – erucastretum virgati centauretosum tauromenitanae facies with lomelosia cretica; b – erucastretum virgati dianthetosum siculi. 1–61 indicates the plots (relevés). ecology of colymbada tauromenitana in the eastern sicily (italy) acta bot. croat. 73 (2), 2014 393 our assumption of plant community differentiation along a gradient of environmental conditions ranging from the coast to inland areas, highlighted by the fl oristic diversity, is better supported when some concrete environmental variables are introduced and a canonical component analysis (cca) is conducted. the result of the cca shows a main gradient of distance of the sea and altitude on axis 1 and a secondary gradient of slope on axis 2 (fig. 4). the cca clearly separates on axis 1 (on the left) the strictly coastal plant communities belonging to the thermo-mediterranean lower bioclimatic range, which prefer thermo-xefig. 4. canonical correspondence analysis (cca) plot. total variance (»inertia«) in the species data: 2.0486; eigenvalues: axis 1 = 0.272; axis 2 = 0.087. plant communities (comm.): 1 – erucastretum virgati centauretosum tauromenitanae facies with silene fruticosa (polygon marked by triangles and centroid 1); 2 – erucastretum virgati centauretosum tauromenitanae facies with lomelosia cretica (polygon marked by squares and centroid 2); 3 – erucastretum virgati dianthetosum siculi (polygon marked by dots and centroid 3). main gradient: distance from the sea (ds), altitude (altit.) and slope of terrain. p1–61 indicates the plots (relevés). sciandrello s., d’agostino s. 394 acta bot. croat. 73 (2), 2014 rophilous conditions, as demonstrated by the presence and dominance of lomelosia cretica and dianthus rupicola subsp. rupicola; in the central part are plant communities belonging to the thermo-mediterranean upper bioclimatic range that prefer thermophilous conditions, as demonstrated by the presence and dominance of silene fruticosa, athamanta sicula and colymbada tauromenitana, while on the right there are the mesophilous plant communities belonging to the meso-mediterranean bioclimatic range, as demonstrated by the dominance of dianthus siculus, ballota hispanica and odontites bocconei. on axis 2, which correlate with slope, from the bottom up we can observe a reduction of the inclination which refl ects a lower number of chasmophilous species. the spearman correlation shows a signifi cant correlation between species richness and elevation (r = 0.62; p < 0.05), but a low correlation with distance from the sea (r = 0.35). moreover, the species richness was signifi cantly correlated with temperature (r = –0.52; p < 0.05) and rainfall (r = 0.67). an important correlation was also observed between c. tauromenitana density and rainfall (r = 0.67; p < 0.05), as well as between c. tauromenitana density and species richness (r = 0.66; p < 0.05), whereas a low correlation with sea distance (r = 0.23; p < 0.05). discussion our results indicate that altitude is the main factor infl uencing both species richness and vegetation composition. this important ecological factor signifi cantly affects also the life forms of the species in response to the hydrothermic gradient linked with altitude (mahdavi et al. 2013). the results shows that the lower zones (0–500 m a.s.l.) are characterized by the predominance of chamaephytes (37%), while in the thermo-mediterranean upper zone (500– 800 m a.s.l.) we fi nd a high rate of hemicryptophytes (41%). probably the predominance of chamaephytic life form in lower zones suggests a particular adaptation to marine conditions (mainly represented by coastal moisture and marine aerosols). in the meso-mediterranean belt (800–1000 m a.s.l.), due to cold winds, once more the chamaephytic life form predominates (36%) mixed with hemicryptophytes (32%). the results show no signifi cant differences in fl oristic elements in relation to the three areas identifi ed. in fact from a chorological viewpoint, in these three zones, the fl oristic element is represented mainly by species with a wide mediterranean distribution (about 70%), while the endemic element rate is quite constant. therefore, in accordance with odland (2009), we can consider the altitudinal gradient a complex environmental gradient associated with variation in several environmental parameters (slope, rocky type, and distance from sea). the moderate diversity and density of the vegetation in the lower zone (lower thermo-mediterranean belt) may be related to the severe environmental conditions, that on the one hand limit the growth and productivity of some species, and yet on the other can favor the development of a few highly competitive and specialized species, such as dianthus rupicola subsp. rupicola, lomelosia cretica, limbarda crithmoides, etc. another serious problem regarding the lower zones is linked to the presence of invasive species such as opuntia fi cus-indica (fig. 2), that, facilitated by man, has colonized the cliffs there at the expense of the natural populations of c. tauromenitana. however, high temperature, low precipitation, high evaporation and high salinity are the main environmental constraints that plants face in the lower zone. these ecophysiological stress factors (especially high temperature and low rainfall) explain the moderate species richness at low altitudes. the high diversity and density in the upper zones may largely be ecology of colymbada tauromenitana in the eastern sicily (italy) acta bot. croat. 73 (2), 2014 395 related to the high precipitation and moderate temperature that favor the development of many moderately competitive species, such as dianthus siculus, odontites bocconei, colymbada tauromenitana, silene fruticosa, athamanta sicula etc. the extent of the cliffs is another factor that may affect the plant diversity, in particular through habitat heterogeneity and resource availability (romdal and grytnes 2007). it is generally expected that in a larger area there is higher habitat heterogeneity and a better chance that larger populations of the species may survive. most likely, this very signifi cant number of individuals is linked to the large surface area of the rock walls, optimum climatic conditions, the highest degree of naturalness and especially the reduced possibility of anthropogenic habitat destruction. indeed, the cliffs of m. veneretta, the largest in the study area, have a high species richness (26) and shannon-wiener diversity index (2.57) and probably represent the speciation area of colymbada tauromenitana and its upper altitudinal limit in the peloritani district. therefore, we found that the presence of c. tauromenitana was strongly correlated to total species richness and especially hot-humid climatic conditions (arena et al. 1975, kallimanis et al. 2010). c. tauromenitana grows mainly on limestone outcrops at altitudes between 20 and 800 m, with several other endemic species. it is a characteristic species of the association erucastretum virgati centauretosum tauromenitanae. the primary habitat of this species is mainly the vertical cliff listed as »calcareous rocky slopes with chasmophytic vegetation« (cod. 8210, european community 1992). moreover, in the study area, this species also grows on rocky slopes (secondary habitat) together with semi-rupicolous low shrub of two different communities: euphorbietum dendroidis at low altitudes and micromerio consentinae-phlomidetum fruticosae at higher altitudes (sciandrello et al. 2013b), listed as »thermo-mediterranean and pre-desert scrub (cod. 5330, european community 1992). considering that dissemination of the some species is favored by the wind and the proximity of a suitable habitat (such as m. galfa), we might deduce that the altitudinal limit of c. tauromenitana (about 800 m) is probably related to the diffi culty of the seeds germinating at low temperatures (13 °c) and high rainfall (1,200 m). as concerns the seed dispersal strategy of the rupicolous species of the study area, the strategy of short distance is the most represented on cliffs. the seeds simply fall from the mother plant or are dispersed by swaying movements of the infructescence or by rain drops hitting the capsules. other possible ways of seed dispersal on cliffs are exozoochory or anemochory over short distances. from a phytosociological point of view, the classifi cation of relevés (on-line supplement tabs. 1–3), using cluster analysis and cca (figs. 3, 4), displayed three clear vegetation groups, each with typical species that are useful in characterizing the communities. choosing just one indicator or guiding species for each community, the fi rst is characterized by the presence of dianthus rupicola biv. subsp. rupicola and lomelosia cretica (l.) greuter et burdet (indicating a specifi c altitude range between 0–500 m), the second by the presence of silene fruticosa and colymbada tauromenitana (indicating a specifi c altitude range between 400– 800 m), and in the third the presence of dianthus siculus c. presl in j. et c. presl and odontites bocconei (guss.) walp. subsp. bocconei (indicating a specifi c altitude range between 800–1,000 m) is signifi cant. therefore, in relation to the fl oristic composition differences related to altitude, the populations referred to erucastretum virgati (brullo and marcenò 1979) can be divided into three facies, here proposed as: 1. erucastretum virgati brullo et marcenò 1979 centauretosum tauromenitanae pirola ex brullo et marcenò 1979, facies with lomelosia cretica: cluster a2 (0–400 m altitude) sciandrello s., d’agostino s. 396 acta bot. croat. 73 (2), 2014 within the thermo-mediterranean lower belt. this facies is characterized by the dominance of dianthus rupicola subsp. rupicola and lomelosia cretica, together with other thermorupicolous species of the dianthion rupicolae alliance, such as antirrhinum siculum, brassica incana, colymbada tauromenitana ect. and other several thermo-xerophilous species of the oleo-ceratonion alliance, such as euphorbia dendroides, phlomis fruticosa, olea europaea var. sylvestris, prasium majus, teucrium fruticans, ruta chalepensis, ect. (on-line supplement tab. 2). 2. erucastretum virgati brullo et marcenò 1979 centauretosum tauromenitanae pirola ex brullo et marcenò 1979, facies with silene fruticosa: cluster a1 (400–800 m altitude) within the thermo-mediterranean upper belt. this facies is characterized by the dominance of silene fruticosa and colymbada tauromenitana, together with hypochoeris laevigata, athamanta sicula, ballota hispanica, teucrium fl avum, ect., and some thermo-mesophilous species, such as bupleurum fruticosum, emerus major subsp. emeroides, fraxinus ornus, pistacia terebinthus, quercus ilex, ect. (on-line supplement tab. 1). 3. erucastretum virgati brullo et marcenò 1979 dianthetosum siculi subass. nova, characteristics species: dianthus siculus and odontites bocconei subsp. bocconei, holotypus: relevé 61, cluster b (800–1,000 m altitude) within the meso-mediterranean belt. this facies is characterized by the dominance of dianthus siculus and odontites bocconei subsp. bocconei, together with other rupicolous species of the asplenietalia glandulosi order, such as silene fruticosa, brassica incana, anthyllis vulneraria subsp. busambarensis, teucrium fl avum, athamanta sicula, hypochoeris laevigata, ballota hispanica, sedum dasyphyllum, ect. (on-line supplement tab. 3). the limestone cliffs are populated by chasmophilous vegetation ascribed to erucastretum virgati, widespread in the southern sector of the calabria and eastern part of sicily with several subassociations geographically well delimited, highlighting the geology and paleogeography connections of the calabrian peloritani arc (brullo et al. 1998b, brullo et al. 2001). overall, endemic species play a central role in biodiversity conservation, because of the habitat loss and climate change, due to their often small population sizes, low genetic diversity and specifi c habitat requirements (gargano et al. 2007, brullo et al. 2011, trigas et al. 2012, brullo et al. 2013, vandepitte et al. 2013). in line with the objectives of the habitats directive, their preservation therefore becomes a priority (médail and quézel 1999, myers et al. 2000). in southern italy the cliffs preserve endemic species of very high phytogeographical and ecological value, used also to characterize well-defi ned territorial units (brullo et al. 1998b, wagensommer and di pietro 2007, di pietro and wagensommer 2008, terzi and d’amico 2008, minissale et al. 2013). conclusion in conclusion, the priority conservation measures proposed, based on fi eld observations, for the rupicolous species are: (1) protection of the rupicolous habitat with a high number of endemic species; (2) creation of plant micro-reserves (pmr) or sites of community importance (sci); (3) planning and land management (regulation of human activities, limitation of fi re, eradication of alien species such as opuntia fi cus-indica on the cliffs); (4) restoration of damaged habitats; (5) collection of local germplasm for ex situ conservation; (6) promoting the integration of some threatened endemic species (in situ/ex situ); (7) a regional law on ecology of colymbada tauromenitana in the eastern sicily (italy) acta bot. croat. 73 (2), 2014 397 the conservation of endemic species and (8) genetic analysis 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(ed.), classifi cation of plant communities, second ed. dr junk, the hague, the netherlands, pp. 287–399. 544 vizintin et al.vp acta bot. croat. 71 (2), 261–277, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 spatial and temporal plant phenological niche differentiation in the wadi degla desert ecosystem (egypt) ahmad k. hegazy1*, abdelrahman a. alatar1, jon lovett-doust2, hosam a. el-adawy3 1 department of botany and microbiology, college of science, king saud university, riyadh 11451, p.o. box 2455, saudi arabia 2 department of biological sciences, university of windsor, windsor, ontario, canada 3 natural resources surveying department, environmental studies and research institute, minofiya university, sadat city, egypt abstract – twenty dominant plant species representing different life forms were investigated phenologically over a period of 36 months (january 2004 to december 2006). plant populations were sampled at down-, mid-, and upstream sites in a desert wadi ecosystem. the results were analyzed using twinspan, dca and cca techniques. five phenological niches were apparent: (1) species flowering all year round, with peaks in spring and autumn such as ochradenus baccatus; (2) species flowering during winter including lycium shawii and tamarix nilotica; (3) species flowering during spring, e.g., zilla spinosa, zygophyllum coccineum and capparis spinosa; (4) species flowering during summer including iphiona mucronata and deverra triradiata; and (5) species flowering during autumn that include atriplex halimus and two anabasis species. the climatic variables, including temperature, rainfall and relative humidity, affect the phenological niches and between-species differences. within-species variations occurred between years and there were no between-site variations for most study species. the different plant species exhibited phenological diversity along the course of the wadi ecosystem. the phenological niches are species-specific and environmentally dependent rather than local selective pressures. keywords: desert, environment, flowering, egypt, life forms, phenology, niche introduction whereas an understanding of plant phenology may be best interpreted in the context of the larger flowering community (augspurger 1983), there have been few studies that exacta bot. croat. 71 (2), 2012 261 * corresponding author permanent address: botany department, faculty of science, cairo university, giza, egypt. e-mail: akhegazy@yahoo.com copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. amined patterns among or within species (goulart et al. 2005). few studies have compared species located at sites having marked environmental differences (borchert 1980, lobo et al. 2003, vitasse et al. 2009). a comparative approach is useful for understanding the phenological rhythm as a species survival strategy, and showing how abiotic factors may generate differentiation among species in their phenology. the extent of synchrony between phenological and ecological events is often shown by the correlation of population sizes at different localities, where synchrony tends to decrease with distance (hudson and keatley 2010). wadi vegetation dynamics as a whole and plant phenologies in particular have been little studied, especially following the episodic flash floods that characterize these ephemeral watercourses. in the desert, plant growth is almost always triggered by rainfall, and hence occurs infrequently or irregularly during rain. vegetation tends to develop in wadis and other habitats receiving runoff water. given that rainstorms do not follow precise annual patterns, and there are frequently decades between storm events, the perennial plant seasonal phenologies are products of the whole environmental setting, and thus tend to have great variability and variation in response (kassas 1966). desert plants often exhibit phenotypic plasticity in their ability to adjust the timing of phenological events to accommodate differences in habitat, microsite and other variations in local environmental factors (ayyad et al. 1985, gunster 1994, ghazanfar 1997). phenological records on desert plants can be used as environmental indicators to determine the length of the actual growing season, where plants may compete for an advantage. the phenological phases of plants within different environmental conditions may explain how desert species are adapted to, and often restricted by, particular local conditions, and how plant life cycles are adjusted in accordance with environmental stressors. this approach can be used to explain features in a plant population or community that must change for survival and growth under a given set of environmental conditions (seghieri and simier 2002, goulart et al. 2005). the phenology of 54 desert plants in a gravel desert wadi in northern oman was studied by ghazanfar (1997) in relation to climatic variables. the onset and duration of growth and flowering were significantly associated with the timing and abundance of rainfall, as was plant growth form. the variation in total rainfall did not affect the onset of flowering in phanerophytes and chamaephytes due to their adaptive perennial habit and water availability in the extended rhizosphere. alternatively, due to their ephemeral habit, the phenology of therophytes and geophytes was significantly affected by variations of rainfall. despite the differences in timing and amount of rainfall, the sequence of flowering for the majority of species was more or less constant among years. marco et al. (2000) studied three populations of larrea divaricata in dry chaco systems, in argentina, and showed that patterns of flowering and fruiting in especially heterogeneous habitats differed significantly among populations of a single species. ayyad et al. (1985) studied phenology in eight desert perennials having different life forms, in the non-saline depressions of the western mediterranean coastal region. they found major effects of climatic fluctuation and of controlled grazing on the phenology in these plants. recently, lesica and kittelson (2010) suggested that studies have only just begun to document phenological changes and their relationship to climate change. phenological studies at vegetation-level are scarce in arid lands, which are likely early indicators of climate 262 acta bot. croat. 71 (2), 2012 hegazy a. k., alatar a. a., lovett-doust j., el-adawy h. a. change. previous studies on plant-environment relationships have demonstrated that population dynamics and phenological events are considered good indicators of the bioclimatic character in desert regions (shaltout 1987, hegazy 1990, ågren et al. 2008). in this study we argue that hyperarid desert wadi conditions may lead to phenological specialization and diversity, including temporal partitioning of the flowering and reproductive niche around the year in both space and time. phenological specialization may be a product of competition among plants for pollinators and pollination activities, and we expect, in principle, some evolutionary divergence along a resource axis (i.e., resource partitioning), reducing the negative interaction between coexisting species. stone et al. (1998) studied partitioning of pollinators during flowering in an african acacia community. they showed major functional partitioning was achieved among some acacia species through separation of flowering in space and seasonal time, and through inter-specific differences in pollinator guilds. the aim of this paper is to examine whether there are different phenological patterns among the different plant populations in a desert wadi ecosystem and, whether the phenology of the different plant species is constrained by local selective pressure and whether it is to any extent influenced by the climatic variables. material and methods wadi degla is located in the north of the eastern desert of egypt. it extends some 30 kilometres in a southeast to northwest direction, between longitude 31° 19' to 37' and latitude 29° 53' to 57'. the wadi has a significant gradient, dropping c. 10 m km–1 along the course of the stream, and draining from gabal (mountain) abu shama, at 578 m a.s.l. and debouching into the nile valley, at 21 m a.s.l. (fig. 1). the study sites of the wadi differ in elevations, with the downstream section ranging from 85 to 200 m a.s.l., midstream from 220 to 330 m a.s.l., upstream from 340 to 420 m a.s.l., and the (downstream) tributaries from 140 to 250 m a.s.l. the main wadi is generally wide, though differs in its width among the four locations between 50 to 600 m. the highest values of rainfall in the study site are 25.3±14.23 mm, relative humidity 64.37±5.54%, temperature 27.66±1.2 °c and wind speed 15.71±5.1 km h–1. the lowest values of temperature are 16.63±4.44 °c, dew point 8.74±2.7, relative humidity 47.27±0.01% and wind speed 11.14±0.44 km h–1 with absence of rainfall. wadi degla is an ancient and steep limestone valley in egypt's eastern desert. in 1999, the downand midstream part of wadi was declared a protected area (fig. 1). the soil inwadi degla is mostly composed of rock waste varying in texture from silt to gravels and boulders. the bed of the wadi is covered with layers of differing textures, influencing water availability (hassan 2002). aridity prevails throughout the area: low and irregular rainfall, sharp diurnal changes of temperature and atmospheric humidity. the array of major climatic variables is collected at the local helwan climate station for the study period of 2004–2006 (tab. 1). the wettest year was 2004 and 2006 the driest. analysis of the rainfall data for period 1998–2007 (trmm, tropical rainfall measurement data 2007, el-gamal et al. 2008) demonstrate the hyperaridity and severe scarcity of rainfall over the wadi degla ecosystem. the total accumulated depth of rainfall varies from 100 mm (c. 10 mm year–1) in the eastern (upstream) region of the catchment area to more than 400 mm (c. 40 mm year–1) acta bot. croat. 71 (2), 2012 263 plant phenological niches in a desert wadi ecosystem 264 acta bot. croat. 71 (2), 2012 hegazy a. k., alatar a. a., lovett-doust j., el-adawy h. a. fig. 1. map showing the location of wadi degla (wadi degla downand midstream (protected area) and wadi el-thamah that represents wadi degla upstream) and its tributaries. plots sampled for study are represented by black squares. acta bot. croat. 71 (2), 2012 265 plant phenological niches in a desert wadi ecosystem tab. 1. monthly average temperature, relative humidity (%), wind speed (km h–1), wind direction (degrees from north) and dew point (°c) at helwan station during the field study years 2004–2006 (data source from egyptian meteorological organization). accumulated rainfall (mm month–1) calculated according to tropical rainfall measurement mission technique (el-gamal et al. 2008). month/ year rainfall (mm) temperature (°c) relative humidity (%) wind speed (km h1) wind direction (°) dew point (°c) jan-2004 27 11.9 70.6 21.5 202 6.36 feb-04 6 15.9 58.9 9.4 66 7.44 mar-04 8 19.2 54.1 13.9 109 8.78 apr-04 <1 20.9 50.6 14.1 70 8.50 may-04 <1 25.3 51.6 14.1 93 12.18 jun-04 0 26.8 47.2 11.4 135 12.41 jul-04 0 28.5 47.2 10.8 146 14.35 aug-04 <1 28.2 51.9 10.5 149 14.94 sep-04 2 26.0 54.6 12.6 126 13.89 oct-04 3 24.6 57.4 11.5 89 13.52 nov-04 39 20.6 62.1 12.2 122 11.67 dec-04 10 17.3 60.3 13.2 92 8.19 annual total 96 annual mean 8 jan-2005 8 13.5 66.3 18.0 177 6.18 feb-05 15 14.1 58.1 13.5 157 4.90 mar-05 5 18.2 56.4 12.9 116 7.00 apr-05 20 21.7 45.3 16.5 142 8.02 may-05 <1 25.2 47.6 14.0 102 12.22 jun-05 <1 27.7 46.2 11.5 124 14.44 jul-05 0 30.9 49.4 10.4 152 17.66 aug-05 0 30.8 54.6 10.7 121 18.76 sep-05 0 27.2 52.4 11.1 139 16.10 oct-05 0 24.1 59.0 11.5 139 15.24 nov-05 1 18.7 57.1 10.6 126 10.03 dec-05 2 16.1 56.2 12.1 126 7.84 annual total 52 annual mean 4 jan-2006 3 14.8 55.3 8.8 142 6.10 feb-06 5 15.8 52.1 11.9 142 5.97 mar-06 6 19.4 47.7 11.6 143 7.60 apr-06 2 22.3 51.6 11.8 100 8.10 may-06 <1 25.1 98.5 12.0 57 8.59 jun-06 0 28.0 39.5 13.4 106 12.59 jul-06 0 29.6 43.4 10.3 196 15.14 aug-06 0 31.8 48.8 10.3 127 17.93 sep-06 <1 28.6 51.9 10.9 96 16.56 oct-06 0 25.8 54.4 8.9 162 15.28 nov-06 1 18.0 57.7 9.0 183 9.58 dec-06 1 13.4 58.0 9.7 161 5.64 annual total 20 annual mean 2 in the downstream, western part of the catchment area, near the outlet, while it ranged between 200 to 250 mm (c. 20–25 mm year–1) in the midstream locations. the total accumulated ten-year depth of rainfall of the three downstream tributaries varied from 250 mm to 350 mm (in the west), according to position (fig.1). for phenological observations, a total of 114 (10 × 10 m) permanent plots were placed at the three sampling sites (down-, midand upstream) along wadi degla stream. population phenology of the dominant twenty perennial species present at one or more site was investigated. these constituted the major components of the wadi vegetation (el-adawy 2011), and represented an array of different life forms (tab. 2). species identification followed boulos (1999–2005). phenological observations of individuals of each population were recorded on 50 marked and randomly selected individual plants. when there were fewer than 50 individuals in a population, observations were made on all individuals available in that population. periodic observations of phenological phases were carried out every 10 days in the peak growing season (spring), or monthly in other seasons, during three consecutive years (2004 to 2006). phenological phases were defined on a scale from 1 to 6 called the phenology in266 acta bot. croat. 71 (2), 2012 hegazy a. k., alatar a. a., lovett-doust j., el-adawy h. a. tab. 2. major species represented in populations at wadi degla habitats. ch – chamaephyte (subshrub); nph – nano-phanerophyte (shrub); ph – phanerophyte (tree); + – present; – – absent species habitat type life formdown-stream mid-stream up-stream agathophora alopecuroides (chenopodiaceae) + + – ch anabasis articulata (chenopodiaceae) – + + ch anabasis setifera (chenopodiaceae) + + – ch atriplex halimus (chenopodiaceae) + + + nph capparis spinosa (capparaceae) + + – ch cocculus pendulus (menispermaceae) – + – ph deverra tortuosa (apiaceae) + – + ch deverra triradiata (apiaceae) + + – ch echinops spinosus (asteraceae) + – – ch ephedra aphylla (ephedraceae) + – – nph fagonia mollis (zygophyllaceae) – + – ch farsetia aegyptia (brassicaceae) + + – ch gymnocarpos decandrus (caryophyllaceae) + – – ch iphiona mucronata (asteraceae) + + – ch lycium shawii (solanaceae) + + + ph ochradenus baccatus (resedaceae) + + – nph retama raetam (fabaceae) – + + nph tamarix nilotica (tamaricaceae) – + – nph zilla spinosa (brassicaceae) + + + ch zygophyllum coccineum (zygophyllaceae) + + – ch dex. these phases were: 1 = vegetative, 2 = flower buds, 3 = flowering, 4 = fruiting, 5 = seed dispersal and 6 = senescence or dormancy (hegazy and eesa 1991). the mean value of phenological index for individuals of different plant sizes was determined for each location per month. within each population, an individual was recorded when visual estimation indicated that at least 25% of the canopy had reached a particular phase. the classification technique known as two-way indicator species analysis (twinspan) (hill 1979) was used to analyze the plant phenological traits, using the community analysis package (cap 2002). twinspan was used to separate the phenological traits of the different species into groups. ordination techniques were performed by using detrended correspondence analysis (dca), community analysis package (cap) and canonical correspondence analysis (cca) (ter braak and [milauer 2002). dca was performed to allow overall assessment of variance between the different phenological groups obtained from twinspan. cca was used for showing the relation between the phenological groups and environmental variables. twinspan, dca and cca were based on the phenological index (pi) of the twenty species at different sites across the twelve months of 2004, which was representative in regard to temperature, and also the rainiest of the three years of observation. the ordination using cca at the population level (site) gave similar results and is not reported here (see el-adawy 2011). data used in the classification and ordination of population phenology are spatially based and considered as different values of phenological index in different species at different sites. yet these data were also temporal in nature. here, sites were represented by month, environmental variables by six climatic factors (collected at the local helwan climate station near the study area) and the phenological index was considered according to the number of sites where the species occurred. thus phenological index was considered as a temporal index that changed with time (monthly) and space (site) along the wadi. shannon-wiener diversity index (h') was calculated to measure the flowering phenology of the twenty plant species throughout 2004–2006, following ludwig and reynolds (1988). analysis of variance (anova) (spss 15 for windows) was used to test the variation in phenological traits of the study species according to population locality (downvs. midvs. upstream) and year (2004 vs. 2006) as sources of variation. results phenological diversity according to the time of flowering and fruiting, four plant phenological groups are derived from twinspan classification (fig. 2). group a covered the three months november to january, here called the winter season. this group includes populations of lycium shawii and tamarix nilotica. group b included the three months august to october, which approximates the autumn season. populations of the four species agathophora alopecuroides, anabasis articulata, anabasis setifera and atriplex halimus are included in this group. group c comprised the four months february to may, representing the spring season. this group contains populations of capparis spinosa, cocculus pendulus, deverra tortuosa, echinops spinosus, ephedra aphylla, fagonia mollis, farsetia aegyptia, gymnocarpos decandrus, ochradenus baccatus, retama raetam, zilla spinosa and zygophyllum acta bot. croat. 71 (2), 2012 267 plant phenological niches in a desert wadi ecosystem coccineum. the last group, d, represents the hot summer season covering the two months june and july. this group was characterized by populations of deverra triradiata and iphiona mucronata. the dca ordination diagram of the different populations of the twenty species around the year (fig. 3) demonstrated the segregation of four phenological groups, which repre268 acta bot. croat. 71 (2), 2012 hegazy a. k., alatar a. a., lovett-doust j., el-adawy h. a. 12 anar u casp d faae d nov dec jan aug sep oct feb mar apr may jun jul a b c d fig. 2. dendrogram of 12 months based on phenological index of twenty species in different populations in wadi degla. four phenological groups are defined from the twinspan technique (a, b, c, d). indicator species are abbreviated to the first two letters of both genus and species followed by capital letter representing habitat (population) (d – downstream; m – midstream; u – upstream). distribution of species demonstrated through dca diagram. -160 -110 -60 -10 40 90 140 190 240 -125 -75 -25 25 75 125 175 dca-axis 1 d c a -a x is 2 agal d agal m atha d atha m atha uocbah m ocbaf m ocbaf d ocbah d anar u anar m anse d anse m ipmu m ipmu d detr d detr m epapf d epapm d famo m ecsp d zyco m zyco d deto d deto u gyde d cope m casp m casp d rera m faae m faae d rera u zisp d zisp u zisp tani m lysh u lysh m lysh d b d c a fig. 3. dca ordination of species in different populations in 12 months with phenological groups defined from twinspan classification. species which flower and fruit in autumn, summer, spring and winter are further separated by the appearance of a fifth pattern of o. baccatus. indicator species are abbreviated to the first two letters of both genus and species followed by capital letter representing habitat (population) (d – downstream; m – midstream; u – upstream). the fifth letter for e. aphylla and o. baccatus, m = male, f = female, h = hermaphrodite. sent four phenological events. populations of species in group a represent a phenological event characterized by flowering in winter months, which are characterized by the lowest temperature and highest rainfall and relative humidity values (tab. 1). species in group d flower in the summer months, characterized by the highest temperature, almost no rainfall and the lowest humidity values. different populations of species of group b represent a phenological group characterized by flowering in autumn. most of the populations of the species included in group c represent the most typical phenological group, characterized by flowering during the spring. months of groups b and c were characterized by intermediate values of temperature, rainfall and relative humidity. populations of female and hermaphrodite ochradenus baccatus took a particular location at the middle and lower side of the ordination diagram (fig. 3), that lying between spring and autumn groups, flowering across most of the year, with two peaks, in spring and autumn, representing the fifth phenological group. e. aphylla, e. spinosus and f. mollis flower in spring, with some individuals delaying, and starting flowering at the beginning of summer. alternatively, some individuals of d. triradiata and i. mucronata may not flower until the end of spring. populations of l. shawii and t. nilotica typically flowered in late winter and early spring. a. alopecuroides populations (group b) are located at the upper part of the array and near group d, and flower in late summer. the twenty species represent 18 genera and 13 families and showed inter-specific variation in the relative timing of the phenological phases during 2004 and 2006. for example, the five species, a. halimus, d. tortuosa, d. triradiata, r. raetam and i. mucronata showed no difference between years, while the six species a. alopecuroides, d. tortuosa, d. triradiata, f. aegyptia, r. raetam and z. coccineum differed significantly in phenophases among the three study sites. female and male individuals of e. aphylla showed significant phenological variations. some species exhibited vegetative activity throughout the entire rainy year 2004, including o. baccatus and c. pendulus, while others persisted in vegetative activity for periods between 6 months (e.g., e. spinosus) and 11 months (e.g., c. spinosa). reproductive activity extended across the entire year in some species, with bimodal peaks in flowering and fruiting, e.g., o. baccatus, or it may extend across most months of the year, e.g., c. pendulus and d. tortuosa). most species exhibited reproductive activity during a period of two to four months with a unimodal peak of flowering and fruiting. phenology – environment relationship the environmental variables representing different phenological groups all over the year are shown in table 3. there is significant variation among the major four phenological groups in response to rainfall, temperature, relative humidity and dew point. species in group a are phenologically active through the winter season, while group d species are active in the summer months. the remaining groups b and c are phenologically active in autumn and spring. the correlation between phenological dormancy or senescence and climatic variables is shown in the ordination diagram produced by cca with biplot of the environmental variables (fig. 4). the length and direction of an arrow representing a given environmental variable provide an indication of the importance and direction of the gradient of environmental variable change. the angle between, an arrow and each axis is a reflection of the deacta bot. croat. 71 (2), 2012 269 plant phenological niches in a desert wadi ecosystem gree of correlation with the axis. by dropping a perpendicular to the arrow from each species-point an indication is provided of the relative position of species along the variable gradient. temperature, dew point, relative humidity and rainfall were the major climatic variables exhibiting significant correlations with the first two axes of the analysis (tab. 4, fig. 4). species of groups a and c flower in winter, spring and come to dormancy in the hot season. in contrast, species of group d, i. mucronata and d. triradiata which flower in the hot season and become senescent in the wet season, are present on the high gradient of rainfall and relative humidity and low gradient of temperature and dew point (see the arrow length in fig. 4). species of group b, characterized by flowering in autumn and senescence in winter and spring seasons, are present on an intermediate gradient of rainfall and relative humidity and 270 acta bot. croat. 71 (2), 2012 hegazy a. k., alatar a. a., lovett-doust j., el-adawy h. a. tab. 3. environmental variables of the different phenological groups obtained by twinspan classification in wadi degla. values are means followed by standard deviation. significance is according to anova (p£0.05), ns = non significant environmental variable phenological group significance a b c d rainfall (mm) 25.3±14.2 1.4±1.3 3.8±4.0 0±0 < 0.05 temperature (°c) 16.6±4.4 26.3±1.8 20.3±3.8 27.6±1.2 < 0.05 relative humidity (%) 64.3±5.5 54.6±2.7 53.8±3.6 47.2±0.01 < 0.01 wind speed (km h–1) 15.7±5.0 11.5±1.0 12.9±2.3 11.1±0.4 ns wind direction (°) 138.8±56.8 122.0±30.6 85.0±20.1 140.9±8.3 ns dew point (°c) 8.7±2.7 14.1±0.7 9.2±2.0 13.3±1.3 < 0.05 tab. 4. results of cca and correlation coefficient of the six parameters with the first four cca axes. axes 1 2 3 4 total inertia eigenvalues 0.031 0.024 0.003 0.002 0.070 species-environment correlations 0.947 0.973 0.943 0.822 cumulative percentage variance of species data 43.8 78.5 83.0 86.5 of species-environment relation 50.0 89.5 94.7 98.6 sum of all unconstrained eigenvalues 0.070 sum of all canonical eigenvalues 0.061 temperature –0.7670*** –0.5370* 0.1789 –0.0472 relative humidity 0.3175 0.8500*** –0.2928 0.0490 wind speed 0.4180 0.2695 –0.2882 0.4496 dew point –0.8930*** –0.2571 0.1424 –0.0971 rainfall 0.2035 0.6079* –0.3618 –0.0144 wind direction –0.2366 0.3254 0.2996 0.5390* significance levels: * p£0.05, ** p£0.01, *** p£0.005 low gradient of temperature and dew point. two species were significantly associated with wind direction, both of them characterized by having separate sex forms, i.e., male and female individuals, e. aphylla, and female and hermaphrodite individuals, o. baccatus. in the year 2004, the highest shannon diversity index (h') of flowering phenology for the study species (2.63) was achieved in april, followed by march, may and then february, with h' values 2.58, 2.48 and 2.13, respectively (figs. 5a, b). alternatively, the lowest values of h' were achieved in the dry months: july (0.6) and august (0.97) in the year 2006. during the remaining months, values of h' ranged between 1.09 (september 2006) and acta bot. croat. 71 (2), 2012 271 plant phenological niches in a desert wadi ecosystem -0,8 -0,6 -0,4 -0,2 0 0,2 0,4 0,6 0,8 1 -1,2 -1 -0,8 -0,6 -0,4 -0,2 0 0,2 0,4 0,6 cca-axis 1 c c a -a x is 2 ipmu detr famo cope deto agal atha anar anse ecspepapm epapf zyc o casp gyde faae rera zisp lyshtani ocbah ocbaf relative humidity temperature rainfall dew point wind speed wind direction fig. 4. cca biplots of the twenty species represented by points and environmental variables indicated by arrows. the species are abbreviated to the first two letters of both genus and species. the fifth letter in e. aphylla and o. baccatus: m – male, f – female and h – hermaphrodite. 0 0,5 1 1,5 2 2,5 3 1 2 3 4 5 6 7 8 9 10 11 12 month (a) 0 0,5 1 1,5 2 2,5 3 3,5 a b c d phenological group s h a n n o n in d e x (h ') (b) s h a n n o n in d e x (h ') fig. 5. shannon diversity index (h') of flowering phenology of (a) twelve months and (b) phenological groups in each of two years, 2004 (white column) and 2006 (grey column). calculations based on the number of flowering individuals. vertical bars are the standard deviations. phenological groups: a – winter, b – autumn, c – spring, d – summer flowering. 1.79 (june 2006). the diversity of flowering phenologies was higher in all months through 2004 than 2006, except in june and november. in a comparison of flowering phenology at the level of phenological groups, the c species group, representing spring reproduction, attained the highest h' values through 2004 and 2006 (2.8±0.63 and 2.74±0.72, respectively). the lowest values were attained in group d (dry season) reproduction through 2004 (1.86±0.17) and group b (autumn season) through 2006 (1.58±0.15). phenological group a (winter flowering species) attained intermediate values through 2004 (1.99±0.22) and 2006 (1.88 ±0.16). discussion for plant phenology in desert wadi ecosystems, plants' responses may involve phenological niche differentiation in space and time, which explains the phenological diversity in desert plants. the twenty study species have demonstrated a complex temporal phenological behaviour. the different plant phenological groups, plus the particular (bimodal) trend of o. baccatus, represent an array of phenological niche differentiation according to the time of flowering of most individuals in the different locations of wadi degla. the diversity of flowering phenology was higher in spring than in summer, suggesting greater synchrony among the flowering periods of most of the study species in spring. this phenological niche differentiation around the year is subject to seasonally variable biotic and abiotic factors and agrees with the observations of opler et al. (1980) in tropical forests, and marques et al. (2004) in subtropical forests, who reported on the different phenological patterns according to plant life forms and rainfall events. adaptive phenology is considered an important reproductive strategy in arid deserts, where many plant species effectively 'escape' from drought in one way or another (ward 2009). biotic factors potentially driving plant phenology include physiological and morphological adaptations for the utilization of resources (rathcke and lacey 1985, pavon and briones 2001), competition for pollinators or pollinator attraction, and competition for seed dispersers (lobo et al. 2003), and the avoidance of herbivory (coley and barone 1996). on the other hand, significant environmental factors such as temporal variations in rainfall (opler et al. 1976), changes in water level sequestered by plants (borchert 1994), changes in temperature (pfeifer et al. 2006), photoperiod (rivera et al. 2002), irradiance (hamann 2004) and sporadic or 'accidental' climatic events (kassas 1966, sakai et al. 1999) have all been reported as key factors affecting the phenological niche differentiation of populations and communities. plant species belonging to different life forms in particular phenological groups exhibited similar phenological niches. this may be attributed to several different factors, including their occurrence under the same climatic drivers. moreover, they exhibited the same niche independent of particular physiological and morphological adaptations, or phylogenetics, e.g., group b which flower in summer-autumn included four species in the chenopodiaceae (ayyad et al. 1985, wright and calderon 1995, marques et al. 2004). some other species having the same life forms exhibited different phenological niches. the populations of female and hermaphrodite ochradenus baccatus included in group c took different trends in their phenological niche. this may be due to the complex responses to the climatic factors, or other factors such as pollinators, or phylogeny. the par272 acta bot. croat. 71 (2), 2012 hegazy a. k., alatar a. a., lovett-doust j., el-adawy h. a. ticular phenological niche of o. baccatus includes some individuals flowering over most of the year, with major peaks in spring and autumn (hegazy et al. 2011). this pattern in o. baccatus was also reported by wolfe and burns (2001) at a wadi located at about 5 km south of ein gedi in israel. the six species a. alopecuroides, d. tortuosa, d. triradiata, f. aegyptia, r. raetam and z. coccineum demonstrated important between-site differences in their phenology. moreover, most species included in our study showed significant differences in the phenological phases between the two years of observation. such phenotypic differences in response to environmental cues are an obvious benefit to success in accommodating plant species and differentiation of their flowering times in the different sites of the wadi ecosystem. as produced from cca results, more than one factor affected the initiation and duration of the different phenological events, particularly the flowering phase. abbad and benchaabane (2004) studied phenology in populations of atriplex halimus at three locations in western north-africa, characterized by different climatic conditions. they concluded that phenology was significantly influenced by climatic conditions, where high temperature and rainfall together tended to accelerate formation of flower buds. phenological events involving reproductive activity in plants are highly affected by temperature and rainfall patterns (jackson 1966, inouye et al. 2003, lesica and kittelson 2010). moreover, plants in arid ecosystems respond to the availability of water at particular soil depths and temperatures, accumulated by the location over time. other factors may also be important. for example, in the present study, the two dioecious and gynodioecious species, e. aphylla and o. baccatus were both located on the wind direction gradient, which is considered an important factor influencing their phenological niche. topographic features in wadi degla may also influence the water availability, with downstream sites receiving more water and developing better soil. the down-stream sites received actual rainfall amounts up to four times as much as the upstream sites, as observed through the tropical rainfall measuring mission, during the recent ten year period (trmm 2007). penuelas et al. (2004) examined the effects of experimental, historical and geographical changes in rainfall and showed highly significant effects on flowering phenology. their results showed that changes in rainfall and water availability, as an important driver of climate change, can cause complex phenological changes. the width of the wadi bed and its elevation can be considered limiting factors for light incidence, where photoperiod extends for longer periods in the upstream site (width 220–600 m). soil type plays a role in water availability for plants, where the soil was a mixture of sandy and rocky types in downand midstream parts but was sandier in the upstream part of a wadi (hassan 2002). gilbert et al. (1996) studied spatial variation in patterns of selection in a plant-pollinator system in four desert wadis in sinai, egypt, and demonstrated significant local (upstream-downstream) specialization in flowering phenology in alkanna orientalis. they concluded that pollinator behavior was probably responsible for the selection gradient in maternal fitness. patterns of flowering formed a spectrum, from continuous to very infrequent flowering (newstrom et al. 1994). the diversity of flowering phenology of the different plant species fluctuated around the year where these species use resources in different periods, which relaxes inter-specific competition among co-existing species. there were various phenological niches under the same type of climate. other factors rather than acta bot. croat. 71 (2), 2012 273 plant phenological niches in a desert wadi ecosystem climate may be also responsible for the high flowering phenological disparity. phytogeographical origin, phylogenetic constraints, structural and architectural traits, and high environmental heterogeneity typical of arid ecosystems seem to be the main drivers of the flowering phenological diversity in wadi degla. in conclusion, plant populations in wadi ecosystems, e.g., wadi degla, can be classified with respect to their phenological niche differentiation into an array of patterns: (1) species flowering/fruiting all the year round with two peaks during spring and autumn, e.g., o. baccatus; 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a gynodioecious plant. american journal of botany 88, 1419–1423. wright, s. j., calderon, o., 1995: phylogenetic patterns among tropical flowering phenologies. journal of ecology 83, 937–948. acta bot. croat. 71 (2), 2012 277 plant phenological niches in a desert wadi ecosystem opce-str.vp acta bot. croat. 73 (1), 159–170, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 some peltigericolous microlichens from southern poland pawe� czarnota*, emil hernik department of agroecology, university of rzeszów, ]wikli skiej 2, 35–601 rzeszów, poland abstract – ten microlichens growing on more or less moribund thalli of several terricolous peltigera spp. are reported from wy yna �l�sko-krakowska upland in southern poland. the list includes: absconditella delutula, agonimia vouauxii, bacidia pycnidiata (for the first time as a lichenicolous lichen), bacidina chloroticula, placynthiella dasea, scutula sp., steinia geophana, thelocarpon epibolum, vezdaea aestivalis and v. retigera. notes on the taxonomy, ecology and distribution of each species are briefly provided and polish samples discussed. keywords: ascomycota, ecology, lichenized fungi, lichenicolous lichens, post-industrial region introduction lichenicolus fungi growing on representatives of the genus peltigera were investigated many times (e.g. alstrup and hawksworth 1990, kümmerling and alstrup 1992, mi dlikowska and alstrup 1995, hawksworth and mi dlikowska 1997, martínez and hafellner 1998, martínez 1999, kocourková 2000, czy�ewska and kukwa 2009, candan et al. 2010, puolasmaa et al. 2012). the genus peltigera, with members usually forming large lichen thalli with cyanobacteria (e.g. nostoc spp.), is widely considered one of these lichen hosts that are inhabited by the highest number of fungal specialists (hawksworth and mi dlikowska 1997). data on lichenicolous lichens and 'four-biont symbioses' are rare (hawksworth 1988, czarnota 2009). in poland lichenicolous lichens are still insufficiently known, but recently some of them have been included within the national list of lichenicolous fungi (czy�ewska and kukwa 2009). study area materials were collected in 2004–2005 by the second author in the three geographical mesoregions of the wy yna �l�sko-krakowska upland (2,615 km²) in southern poland, the acta bot. croat. 73 (1), 2014 159 * corresponding author, e-mail: pawczarnota@poczta.onet.pl copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. wy yna cz�stochowska upland, the obni enie górnej warty depression and the garb tarnogórski hummock (fig. 1; kondracki 2000). the three mesoregions are different from geomorphological, hydrological and floral points of view, and they also differ in the level of anthropogenic impact. various types of limestones dominate there, sometimes appearing above the ground as big outcrops often covered by xerothermic plants (urbisz 2004), and several endemic vascular plants including galium cracoviense ehrend. (mirek 2001), potentilla silesiaca uechtr. (ko�odziejek 2001a), cochlearia polonica fröhlich (kwiatkowska 2001) can be found there. the lichen-forming fungus verrucaria polonica nowak (nowak 1959) was described from its locus classicus the wy yna �l�sko-krakowska upland. in the obni enie górnej warty depression there are different natural metal-enriched habitats and post-mining iron wastes to be found. because of the former mining activity there are a lot of local small elevations and depressions covered by post-ruderal grasslands and plant communities of wet habitats (ko�odziejek 2001b). 160 acta bot. croat. 73 (1), 2014 czarnota p., hernik e. fig. 1. localities of examined peltigericolous lichens based on atpol grid square system and geographical mesoregions of poland. abbreviations of mesoregions: 341.12 – garb tarnogórski plateau, 341.31 – wy yna cz�stochowska upland, 341.25 – obni enie górnej warty depression. materials and methods collected material was investigated by standard microscopy. hand-made sections of ascocarps and pycnidia mounted in water were prepared for detailed examination of their internal pigmentation and dimensions of anatomical characters. sometimes internal tissues were mounted in hno3 or koh for better recognition of the spore septation and the separation of particular structures. habits, internal characters of fruit-bodies and some hamathecial features of noteworthy species have been photographed (figs. 2–3). nomenclature of lichenized fungi follows index fungorum (date of searching march 12, 2013). examined specimens are deposited in the herbarium of rzeszów university (res) and duplicates in the private herbarium of e. hernik. their localities are mapped (fig. 1) according to the atpol grid square system (zaj c 1978) modified by cie�li�ski and fa�tynowicz (1993). results absconditella delutula (nyl.) coppins et h. kilias (figs. 2 c–e). this microlichen usually grows on siliceous rocks; it is very rarely found on other substrates, e.g. decaying wood (czarnota and kukwa 2008). its thallus is almost invisible and looks like a thin gelatinous film, like other representatives of the genus. 1-septate, ellipsoid to fusiform spores are characteristic of this species, distinguishing it from the similar in general appearance a. lignicola, with 3-septate ascospores. considering the shape and the spore septation a. delutula resembles a. sphagnorum, but in the later case ascospores are smaller. moreover a. sphagnorum occurs usually in completely different habitats, inhabiting sphagnum mosses and is found on or close to peat bogs. similar, 1-septate ascospores and pale, translucent when wet apothecia are also known in the common and widespread dimerella pineti. however, this species usually grows on the bark of trees or occasionally on humus. a. delutula has only recently been reported quite frequently in europe (zimmermann et al. 2011), and is also known from asia, australia and greenland (czarnota and kukwa 2008). list of localities: wyz· yna czeu stochowska upland, atpol fd-17, przy�ubsko village, 50°32’ n, 19°36’ e, roadside in pinus sylvestris forest, on thallus of peltigera sp., november 7, 2005, leg. e. hernik 18 (res; hb. e. hernik). agonimia vouauxii (b. de lesd.) m. brand et diederich (figs. 2 a–b). this species was collected in poland in the metal-enriched post-mining habitats in upper silesia (bielczyk 2012, guzow-krzemi�ska et al. 2012). other reports of a. vouauxii show that this ephemeral species is ecologically tolerant (schifelbein et al. 2010, vondrák et al. 2010). a. vouauxii inhabiting peltigera is reported only by diederich et al. (2013) from luxembourg. among other representatives of the genus, a. vouauxii is distinguished by 2-spored asci containing ascospores of 47–52 × 15–20 mm and its verrucose to warted, non squamulosae, green thallus. considering the 2-spored asci, only a. tristicula could be confused with this species, but its ascospores, perithecia and the subsquamules of the thallus are much larger. another species similar to a. vouauxii, polyblastia agraria, found directly on clayey soil, has a thin, gelatinous thallus (ceynowa-gie�don 2001). acta bot. croat. 73 (1), 2014 161 peltigericolous microlichens from poland list of localities: wyz· yna czeu stochowska upland, atpol fd-06, between rz�dkowice and hucisko villages, 50°34’ n, 19°28’ e, on the site of a small fire at the edge of a pine forest, on peltigera sp., august 1, 2005, leg. e. hernik 13 (res); atpol fd-07, kostkowice village, 50°34’ n, 19°35’ e, roadside ditch, on moribund peltigera didactyla, november 3, 2005, leg. e. hernik 14 (res); atpol fd-17, przy�ubsko village, 50°32’ n, 19°36’ e, on peltigera cf. didactyla over calcareous soil, november 7, 2005, leg. e. hernik 3a (res); ibid., roadside ditch, on peltigera didactyla, august 29, 2005, leg. e. hernik 10 (res); ibid., siedliszowice village near pilica town, 50°32’ n, 19°40’ e, on peltigera rufescens, september 5, 2005, leg. e. hernik 9 (res); atpol fd-26, �rubarnia village, lachowizna settlement, 50°27’ n, 19°30’ e, roadside pine forest, on moribund thallus of peltigera sp. january 10, 2005, leg. e. hernik 12 (res). 162 acta bot. croat. 73 (1), 2014 czarnota p., hernik e. fig. 2. peltigericolous lichens: a – habit of agonimia vouauxii (e. hernik 14); b – ascospores of a. vouauxii (e. hernik 9); c – habit of absconditella delutula (e. hernik 18); d – apothecial section of a. delutula (e. hernik 18); e – asci and ascospores of a. delutula (e. hernik 18); f – habit of anamorphic stage of bacidia pycnidiata (partially on plant debris) (e. hernik 8a). scale bars: a, c, f – 1 mm; b, e – 10 mm; d – 100 mm. bacidia pycnidiata czarnota et coppins (fig. 2f). this species recently described by czarnota and coppins (2006) from the czech republic and poland is currently also known from belgium (ertz et al. 2008), lithuania (motiejunaite et al. 2011), finland (pykälä 2008) and estonia (suija et al. 2007). in the acta bot. croat. 73 (1), 2014 163 peltigericolous microlichens from poland fig. 3. peltigericolous lichens: a – habit of bacidina chloroticula (e. hernik 17); b – habit of placynthiella dasaea (e. hernik 11); c – habit of anamorphic stage of scutula sp. (e. hernik 2); d – pycnidial section of scutula sp. and its peltigera host (e. hernik 2); e – mesoconidia of scutula sp. (e. hernik 2); f – habit of steinia geophana (e. hernik 6); g – apothecial section of s. geophana and its peltigera host (the apothecium surrounded by goniocysts of vezdaea retigera) (e. hernik 6); h – habit of thelocarpon epibolum (e. hernik 4); i – habit of vezdaea aestivalis (e. hernik 5). scale bars: a, b, c, f, h, i – 1 mm; d, g – 100 mm; e – 10 mm. meantime several new localities of b. pycnidiata in central poland (�ubek 2009, 2012) and the czech republic (vondrák et al. 2010) have been discovered, but samples inhabiting the thalli of peltigera species have never been shown to date anywhere. bacidia pycnidiata was recorded in different circumstances, in more or less managed woodlands as well as in old-growth forests, mostly as an epiphyte or inhabiting terricolous and corticolous bryophytes. it is reasonably tolerant to human impact (motiejunaite et al. 2011). indeed, data presented here, from a strongly industrialized region of poland and other unpublished author’s records on metal-rich wastes, show that b. pycnidiata has a large ecological plasticity. in the studied area, particularly in the wy yna cz�stochowska upland, this species seems to prefer dry sandy habitats at the edge of pine forests close to natural limestone rocks. perhaps it is more common elsewhere, but it grows mostly without apothecia, forming long-necked diagnostic pycnidia, usually immersed within green, minutely granular thallus, and more or less straight conidia. list of localities: wyz· yna czeu stochowska upland, atpol fd-06, between rz�dkowice and hucisko villages, 50°34’ n, 19°28’ e, close to an electric line at the edge of pine forest, on moribund thallus of peltigera didactyla, july 12, 2005, leg. e. hernik 8a-d (res). bacidina chloroticula (nyl.) v�zda et poelt (fig. 3 a). the identification of several members of bacidina forming small, pale, orange-pink or beige apothecia (e.g. b. phacodes (körber) v�zda, b. delicata (larbal. ex leight.) v. wirth et v�zda, b. chloroticula, b. contexta s. ekman et t. sprib., b. californica s. ekman and b. crystalifera s. ekman) is sometimes very difficult. this is due their non specific diagnostic characters referring to the size of excipular hyphae, the septation and dimensions of their thin, acicular ascospores and conidia and the structure of their thalli. the varying morphology of some of these characters can be considered as some kind of infraspecies variability within most of the above mentioned species depending on their individual ontogeny. for b. chloroticula the wide lumina of excipular hyphae resulting in the entirely paraplectenchymatic excipulum are the main diagnostic characters, and, based on this feature, it has been determined here despite the distinct orange apothecia resembling b. delicata instead (see fig. 3 a). peltigericolous b. chloroticula has already been recorded once in poland by mi dlikowska (1999), but on other substrates (plant debris, wood, natural and artificial rocks, sometimes bark of trees and roots) it is commonly reported in europe, and known also in north america and asia (coppins and aptroot 2009). list of localities: obniz· enie górnej warty depression, atpol ed-94, d�bowiec village, 50°41’ n, 19°13’ e, on peltigera canina, november 6, 2005, leg. e. hernik 19 (res); wyz· yna czeu stochowska upland, atpol fd-06, between rz�dkowice and hucisko villages, 50°34’ n, 19°28’ e, roadside pine forest, on moribund thallus of peltigera cf. didactyla, july 12, 2005, leg. e. hernik 17 (res); garb tarnogórski hummock, atpol fd-16, kotlina mitr�gi basin, józefów village near zawiercie town, by the tributary of ogrodzieniecki stream to czarna przemsza river, 50°27’ n, 19°14’ e, by road in spruce forest, on peltigera cf. didactyla, december 29, 2004, leg. e. hernik 16a–b (res; hb. hernik). placynthiella dasaea (stirt.) tønsberg (fig. 3 b). this is a commonly widespread species in the northern hemisphere growing lignicolous, corticolous or rarely terricolous (hitch and purvis 2009), but only sometimes inhabiting 164 acta bot. croat. 73 (1), 2014 czarnota p., hernik e. thalli of peltigera. p. dasaea usually grows as a sterile crust forming a granular, green to brown thallus fleetingly reacting c+ red due to the gyrophoric acid. for more taxonomic details and affinities to other taxa see tønsberg (1992). list of localities: garb tarnogórski hummock, atpol fd-16, kotlina mitr�gi basin, józefów village (fugasówka settlement) near zawiercie town, by the tributary of ogrodzieniecki stream to czarna przemsza river, under an electric line in pine forest, on thallus of peltigera sp., december 27, 2004, leg. e. hernik 1 (res); wyz· yna czeu stochowska upland, atpol fd-26, �rubarnia village, lachowizna settlement, 50°27’ n, 19°30’ e, roadside pine forest, on moribund thallus of peltigera sp. january 10, 2005, leg. e. hernik 11 (res). scutula sp. (figs. 3 c–e). the sample examined here probably represents an undescribed anamorphic mesoconidial state of some peltigericolous species of scutula, better known as libertiella. a similar specimen has already been found in n poland on peltigera didactyla by m. kukwa (czy�ewska and kukwa 2009), who gave a brief description without any nomenclatural innovation. our lichenized specimens have well developed, slightly warted, pale green thalli and more or less immersed, obpyriform pycnidia of 120–170 mm width, producing ovoid to almost globose conidia, (3–)3.5–4 × 4–5 mm (fig. 3 c–e). upper external parts of pycnidia are slightly brownish with distinctly pale collar around the ostiolum, but usually pycnidia are wide open and concave showing the pale conidial mass and excipulum (fig. 3 d). then they look like pycnidia strongly damaged by snails. scutula sp. was found in the company of other peltigericolous lichen-forming fungi: steinia geophana and vezdaea retigera. list of localities: wyz· yna czeu stochowska upland, atpol fd-07, by the road between huta szklana settlement and prad�a village, 50°34’ n, 19°39’ e, roadside ditch, on moribund thallus of peltigera cf. didactyla, august 19, 2005, leg. e. hernik 2a–d (res; hb. hernik). steinia geophana (nyl.) stein (figs. 3 f–g). this usually terricolous worldwide microlichen (fletcher et al. 2009) has already been found also to be peltigericolous on peltigera didactyla by alstrup and laessoe (1986) in denmark, alstrup and hawksworth (1990) in greenland and on peltigera rufescens in poland by mi dlikowska (1999). in the cases included here, besides the more or less moribund thalli of some peltigera, s. geophana was found also on other substrates such as a humus, plant debris or soil. however, inhabiting peltigera this lichenized fungus probably plays the role of an additional component (together with its photobiont) in occasional four-biont symbioses (fig. 3 g). this relationship may have a parasitic character and finally, s. geophana can grow on a dead thallus of peltigera. s. geophana mostly resembles some black-coloured species of micarea, for example m. deminuta and m. contexta, by its immarginate strongly convex apothecia and a membrane-like, often inconspicuous thallus. this species develops, however, 16-spored asci with almost globose ascospores (fig. 3 g), while members of micarea are permanently 8-spored. for detailed description of s. geophana see fletcher et al. (2009). acta bot. croat. 73 (1), 2014 165 peltigericolous microlichens from poland list of localities: wyz· yna czeu stochowska upland, atpol fd-06, between rz�dkowice and hucisko villages, 50°34’ n, 19°28’ e, close to an electric line at the edge of pine forest, on moribund thallus of peltigera sp., july 12, 2005, leg. e. hernik 6, 7 (res); atpol fd-07, by the road between huta szklana settlement and prad�a village, 50°34’ n, 19°39’ e, roadside ditch, on moribund thallus of peltigera cf. didactyla, august 19, 2005, leg. e. hernik 20 (res). thelocarpon epibolum nyl. (fig. 3 h). this perithecioid microlichen was recorded many times in poland as lichenicolous (czy�ewska and kukwa 2009), as well as lignicolous or occasionally overgrowing other substrates (fa�tynowicz 2003). it usually develops an external, thin, gelatinous thallus with coccomyxa-algae and is often found on different representatives of peltigera, but its strong trophic relationship to this lichen host has not been observed. however, more than any other species of the genus, t. epibolum tends to be peltigericolous (kocourková 2000). there are some differences in ascospore dimensions between specimens determined under the name t. epibolum elsewhere, but due to the lack of molecular support to confirm delimitation of the new taxon, this name is used here. examined specimens produce ascospores 4–6 × 2–2.5 mm, thus they correspond to the type of t. epibolum nyl. var. epibolum (kocourková 2000). list of localities: obniz· enie górnej warty depression, atpol ed-94, mas�onskie natalin village, 50°39’ n, 19°13’ e, at the edge of pine forest, on peltigera canina, november 5, 2005, leg. e. hernik 23 (res); wyz· yna czeu stochowska upland, atpol fd-06, between rz�dkowice and hucisko villages, 50°34’ n, 19°28’ e, roadside at the edge of pine forest, on moribund thallus of peltigera didactyla, july 12, 2005, leg. e. hernik 7 (res, together with steinia geophana) and e. hernik 22a–b (res); ibid., july 8, 2005, leg. e. hernik 24 (res); atpol fd-07, kostkowice village, 50°34’ n, 19°35’ e, roadside ditch, on moribund peltigera didactyla, november 3, 2005, leg. e. hernik 15 (res); by the road between huta szklana settlement and prad�a village, 50°34’ n, 19°39’ e, roadside ditch, on moribund thallus of peltigera cf. didactyla, august 19, 2005, leg. e. hernik 25 (res); atpol fd-17, przy�ubsko village, 50°32’ n, 19°36’ e, roadside ditch, on moribund thallus of peltigera sp., november 7, 2005, leg. e. hernik 4 (res). vezdaea aestivalis (ohlert) tscherm.-woess et poelt (fig. 3 i). vezdaea aestivalis was reported from poland on the thalli of several species of peltigera by mi dlikowska and alstrup (1995), czarnota and kiszka (2004) and czy�ewska et al. (2008), however, it is reported only rarely to be peltigericolous. most often it is found overgrowing bryophytes on the bark of trees, various rocky substrates and on soil. this ephemeral species has a huge ecological plasticity, as it can occur in strongly managed or as well in 'ancient' environments (czarnota and kiszka 2004). among other species of the genus vezdaea, v. aestivalis can be recognized by large (visible without microscope) apothecia, 2-septate ascospores, strongly branched and anastomosed paraphyses entwining individual asci and well developed, non-granular, external thallus. list of localities: wyz· yna czeu stochowska upland, atpol fd-07, kostkowice village, 50°34’ n, 19°35’ e, at the edge of pine forest, on peltigera sp., november 4, 2005, leg. e. hernik 5, 5a (res; hb. hernik). 166 acta bot. croat. 73 (1), 2014 czarnota p., hernik e. vezdaea retigera poelt et döbbeler this toxitolerant ephemeral species is frequently found in western europe and north america in metal-enriched habitats on base-rich grounds, as well as on terricolous bryophytes often in post-mining areas or on waste heaps (chambers and purvis 2009). it was already recorded on some peltigera (palice 1999, alstrup et al. 2004, chambers and purvis 2009), also in poland (czy�ewska and kukwa 2009); however, in this country it is regarded as rather rare (czarnota and kukwa 2009). surely, v. retigera is more frequent especially in post-industrial sites, but overlooked or undercollected as it usually forms only sterile thalli composed of minutely granular pale green and translucent goniocysts. in the case of specimens mentioned in this paper, v. retigera is a peltigericolous lichen-forming species mostly accompanied with steinia geophana. list of localities: wyz· yna czeu stochowska upland, atpol fd-07, by the road between huta szklana settlement and prad�a village, 50°34’ n, 19°39’ e, roadside ditch, on moribund thallus of peltigera cf. didactyla, august 19, 2005, leg. e. hernik 2a–b,d (res, together with scutula sp.); atpol fd-06, between rz�dkowice and hucisko villages, 50°34’ n, 19°28’ e, on small post-fire place at the edge of pine forest, on peltigera sp., august 1, 2005, leg. e. hernik 26a, b (res). discussion the investigation carried out in contaminated industrialized areas of southern poland revealed ten lichenized fungi inhabiting thalli of several terricolous representatives of the genus peltigera. most of them have already been reported as peltigericolous lichens, but the recently described bacidia pycnidiata has been found on this kind of substrate for the first time. this shows its larger ecological plasticity, contradicting the previous state of knowledge. almost all species belong to the group of ephemeral microlichens or early colonizers. therefore, they are often overlooked. they all are possibly much more common in the study area, but except for the probably new member of the genus scutula, rather confined to other substrates, e.g. plant debris, decaying wood, humus, siliceous pebbles and soil. thus they should be regarded as occasional lichenicolous lichens, mostly commensal but in the course of time some of them, e.g. steinia geophana may become even parasitizing. the undescribed scutula, found here in its second locality in poland, however seems to be obligatorily peltigericolous. acknowledgements a part of this study was financially supported by the university of rzeszów task grant no. wbr/ka/ds/5/2013. references alstrup, v., laessoe, t., 1987: a late-season parasitic flora on peltigera didactyla at allerod, denmark. graphis scripta 1, 56–57. alstrup, v., hawksworth, d., 1990: the lichenicolous fungi of greenland. meddelelser om grønland, biosience 31, 1–90. acta bot. croat. 73 (1), 2014 167 peltigericolous microlichens from poland alstrup, v., svane, s., søchting, u., 2004: additions to the lichen flora of denmark vi. graphis scripta 15, 45–50. bielczyk, u., 2012: lichens of zinc-lead post-mining areas in the olkusz region – state of preservation, threats and needs for protection. in: lipnicki, l. 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green 1884; weill 1903; siersch 1927; metcalfe et al. 1950; curtis and lersten 1990; nahrsted and butterweck 1997; baroni fornasiero et al. 1998; baroni fornasiero et al. 2000; ciccarelli et al. 2001a, b; onelli et al. 2002) in both vegetative and reproductive organs. this phytochemical diversity has been investigated in taxonomic and morphological studies (crockett and robson 2011, nurk and crockett 2011). only metcalfe et al.(1950) have made specifically anatomical studies, and all the most recent work has investigated the secretory structures (lotocka and osinska 2010, gitea et al. 2011). this study contributes to knowledge of vegetative organ anatomy in hypericum species from heterogeacta bot. croat. 72 (2), 2013 269 * corresponding author, e-mail: paolo.colombo@unipa.it copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. neous environments. our aim is thus to provide a detailed description of the micromorphological and anatomical characteristics of the leaf and stem in hypericum species. materials and methods plant sampling the samples were collected directly from the sites in the literature (giardina et al. 2007) during the flowering period. the following were examined (tutin et al. 1972, pignatti 1982): hemicryptophyte scapose (h scap) mesophytes hypericum perforatum l. – present throughout europe except in the far north, present throughout italy up to an altitude of 1400 m; h. perfoliatum l. – grows in mediterranean regions, present throughout italy up to an altitude of 1400 m; h. pubescens boiss. – colonizes humid environments, salty habitats at times, and is present in portugal and southern spain, sicily and malta; meso-hygrophyte hypericum tetrapterum fr. – colonizes marshes and cane-brakes, present in south-central europe, extends into sweden and grows throughout italy; xerophyte hypericum triquetrifolium turra – grows in the eastern mediterranean, in italy present only in the southernmost regions. nanophanerophytes (np) mesophytes hypericum androsaemum l. – present in western europe, locally in southern europe, and is found throughout italy up to an altitude of 1400 m; h. hircinum l. – in moist, shady localities, present in the mediterranean region and found throughout central and southern italy. chamaephyte fruticose (ch frut) xero-halophyte hypericum aegypticum l. – colonizes maritime cliffs and is present on central-eastern mediterranean islands from sardinia to crete. tissue analysis some samples were sectioned when fresh and stained with phloroglucinol in alcoholic solution and 2% hydrochloric acid for lignified components of the cell wall, with chlor-zinc iodide for the presence of cellulose in plant tissue, sudan iii to ethyl alcohol at 80% saturated solution for cutin and suberin (catalano 1925), iodine iodide solution (lugol) for starch (johansen, 1940), ruthenium red for pectic-like substances (jensen 1962) and potassium bichromate for tannins (faure 1914). other samples were fixed with faa (90% 270 acta bot. croat. 72 (2), 2013 perrone r., de rosa p., de castro o., colombo p. ethanol, 5% formalin, 5% acetic acid) (sass 1958) and after dehydration in graded ethanol were embedded in paraffin, following the protocol of beccari and mazzi (1966). as a counterstain, 1% safranin in alcoholic solution was used in addition to the more specific reagents in plant microtechnique. transverse sections of fresh stem were made with bare-handed microtome (a.m.g. diagnostici). the sections, about 10 mm each, were made with jung-r 2050 – supercut microtome (reichert-jung/leica). the basal and cauline leaves and the stem were cut to about half the maximum vegetative development for each species. the foliar morphological parameters (fmp) were measured for each species i.e. average thickness of the foliar lamina in correspondence with the median vein, average thickness of the lamina in the section between the margin and the median vein, of the adaxial and abaxial surfaces (cuticular and epidermal), of the mesophyll, of the palisade and spongy tissue, as well as the foliar epidermal parameters (fep) i.e. length, width, and thickness of the epidermal cells, the number of cells and stomatal density × mm2, stomatal polar and equatorial axis, stomatal complex. epicuticular wax morphology was investigated and compared using three different methods: 1.epidermal epoxy replicas according to the laroche (1982) protocol; 2.extraction of the cuticle according to the christophel et al. (1996) protocol; 3.sem (scanning electron microscope) observations. fresh and permanent preparations were photographed using lm leica dmls, while digital images were obtained using a nikon ds camera head ds-fi1. the s.e.m. images were obtained using a leo 420 cambridge. data analysis the dimensions of the epidermal cells, the stomatal polar and equatorial axes, and cell and stomatal density × mm2 were measured using an image analyzer program integrated with a ds camera head ds-fi1. plants were collected on reaching their maximum vegetative development. on each sampling date, ten individuals were collected randomly. all morphometric data were statistically analysed to obtain mean, median and mode as well as standard deviation and variance. epicuticular wax morphology was classified according to wilkinson (1979). the anatomical terminology used is that according to esau (1965). results foliar micromorphology hypericum perforatum and h. perfoliatum. the epidermis is glabrous on both leaf surfaces. the adaxial surface (figs. 1a, c) shows very convex cells range, the longitudinal and radial walls are slightly wavy. these surfaces have no stomata. there are very evident veins consisting of long, narrow cells in rows, the lower the order of the vein the more rows. the abaxial surface (figs. 1b, d) is composed of cells from irregularly isodiametric to more or less elongate, but all with slightly wavy longitudinal and radial walls.the stomata are at the same level as the other epidermal cells and are numerous and arranged irregularly. the abaxial surface is covered with numerous depressions corresponding to the location of translucent secretory reservoirs and the stomata found in the vicinity of these structures are arranged in a circular pattern around them, while those between the depressions have an acta bot. croat. 72 (2), 2013 271 leaf and stem anatomy in hypericum species 272 acta bot. croat. 72 (2), 2013 perrone r., de rosa p., de castro o., colombo p. fig. 1. general characteristics of the adaxial and abaxial epidermis in frontal views (lm). h. perforatum; a – adaxial surface showing slightly wavy wall cells; b – abaxial surface with circular depressions consisting of 2 or more slightly larger epidermal cells and stomata (scale bars 50 mm). h. perfoliatum; c – adaxial surface with slightly wavy wall small cells; d – abaxial surface with depressions corresponding to the translucent glands (scale bars 50 mm). h. pubescens; e – adaxial surface showing irregular polyhedral convex cells mixed with small anisocytic stomata and vestiges of trichomes; f – abaxial surface with stomata and vestiges of trichomes (scale bars 100 mm). h. tetrapterum; g – adaxial surface with isodiametric cells and wide elongated depressions of various shapes; h – abaxial surface with depressions and several anisocytic stomatal complexes (scale bars 100 mm). h. triquetrifolium, i – adaxial surface showing veins formed by numerous rows of rectangular elongated cells and stomata; j – abaxial surface with stomata and depressions (scale bars 50 mm). h. androsaemum; k – adaxial surface (scale bars 100 mm); l – rectangular and elliptical cells with highly corrugated walls, abaxial surface (scale bars 25 mm), a polarized light, showing enlarged detail of anisocytic stomatal types and cuticular thickening of stomatal rims. h. hircinum; m – adaxial surface (scale bars 100 mm) showing a large number of small isodiametric cells contained by ribs; n – abaxial surface (scale bars 50 mm) showing areolas with internal stomata end depressions. h. aegypticum; o – adaxial surface with isodiametric cells with linear walls; p – cells with rounded corners, stomata and abaxial surface (scale bars 50 mm) open pattern. in h. perfoliatum, the stomata are very numerous and are confined inside the areolae demarcated by the veins found in great numbers on the abaxial surface; the stomata are anisocytic and slightly sunken, strongly elliptic with the polar axis oriented parallel to the major leaf axis. in h. perforatum, these depressions are circular, far from perforated, consisting of two or more slightly larger epidermal cells, with less undulating, less convex walls, even concave in some cases. for this reason the whole structure appears depressed in relation to the average level of the epidermis. also in h. perfoliatum there are depressions corresponding to the translucent glands, which penetrate to various depths throughout the mesophyll. sem observations confirm those from the light microscope. in h. perforatum (fig. 2a), cuticular ornamentation on the adaxial surface presents type »b« granules, while the abaxial surface is characterized by cells with cuticular ornamentation of a cross-linked mixed type with waxy laminar flake deposits (fig. 2b). in h. perfoliatum (fig. 2c) the adaxial surface presents rod and filament type »e« ornamentation, dense, very small and in tiny isolated outgrowths or in groups. the abaxial surface (fig. 2d) is morphologically very similar to the adaxial but with much less obvious cuticular ornamentation mostly confined to the grooves between cells. the epicuticular wax is more abundant. once again, there are depressions, commonly found on the adaxial surface, corresponding to the translucent glands located in the mesophyll. hypericum pubescens. trichomes are present on all leaf surfaces, the veins are mostly long, the pubescence »marked and felted« (pignatti 1982). the trichomes are simple with a widened and stubby base inserted on the epidermis and surrounded by 8–9 epidermal cells arranged in a rosette. the elongated body is made up of 5–6 sloping uniseriate items, of which the distal one appears narrow and pointed. both adaxial (fig. 1e) and abaxial (fig. 1f) surfaces are composed of irregular polyhedral convex cells, mixed with small anisocytic stomata deeply embedded in the epidermis. all adaxial and abaxial surface cells are covered with conspicuous cuticular ornamentation. sem confirms observations obtained with epoxy replicas but focuses on the very dense cuticular ornamentation on both epidermises (figs. 2e, f), including the basal items which constitute the foot of all the hairs; even the body of the hairs is scattered with the same type »j« plate and scale ornamentation, miniscule scales arranged more or less vertically. on the adaxial epidermis we also find the characteristic depressions which indicate the presence of translucent glands in the mesophyll, the base of which is enclosed by two adjacent cells, similarly ornamented. hypericum tetrapterum and h. triquetrifolium. trichomes are absent on both leaf surfaces. the adaxial surface (figs. 1g, i) is composed of vaguely polygonal isodiametric cells with slightly corrugated longitudinal and radial walls in h. tetrapterum, a rectangular-sinuous pattern in h. triquetrifolium, but strongly convex in both species. on both leaf surfaces the veins are characterized by numerous rows of rectangular cells, more or less elongated and paired, but offset by about half a cell. the anisocytic stomata are randomly arranged on both epidermal surfaces, except around the characteristic depressions, which are fairly numerous and complex; they indicate the presence of translucent glands in the mesophyll and are arranged in a circle. these depressions have one to five cells at the base, from which others radiate out, larger, elongated and arranged in a star shape. the abaxial surface (figs. 1h, j) is characteristic for its epidermal cells, which are always very convex, wider and more regular in h. tetrapterum and with strongly undulating longitudinal and radial walls in h. triquetrifolium. sem shows the epidermal morphology of the adaxial and abaxial surface to be rich in cuticular ornamentation. in h. tetrapterum this ornamentation is mainly confined acta bot. croat. 72 (2), 2013 273 leaf and stem anatomy in hypericum species towards the margins between adjacent cell walls and is of plate and scale type »j« according to the classification of wilkinson (1979). thus, the epidermal cells present »flake« ornamentation arranged vertically, very densely on the body of the epidermal cells and on the guard cells in h. tetrapterum (figs. 2g, h), while in h. triquetrifolium these are strongly waxy, with type »b« granules on both surfaces (figs. 2i, j). 274 acta bot. croat. 72 (2), 2013 perrone r., de rosa p., de castro o., colombo p. fig. 2. leaf blade epidermis scanning electron microscopy (sem) images. h. perforatum; a – adaxial surface showing convex isodiametric cells with a waxy coating (scale bars 60 mm); b – abaxial surface with depressed stomata characterized by ornamented guard cells (scale bars 20 mm). h. perfoliatum; c – adaxial surface (scale bars 6 mm) detail of cells at the base of depression; d – abaxial surface (scale bars 20 mm). h. pubescens; e – adaxial surface (scale bars 4 mm) detail of the epidermis with »plates and scales« ornamentation and depressed stomata; f – abaxial surface (scale bars 20 mm) with trichomes. h. tetrapterum; g – adaxial surface (scale bars 6 mm) showing isodiametric cells with marked waxy coatings; h – abaxial surface (scale bars 2 mm) detail of ornamentation and slightly sunken stomata. h. triquetrifolium; i – adaxial surface showing cells with waxy body and anisocytic stomata, (j) abaxial surface (scale bars 20 mm). h. androsaemum; k – adaxial surface; l – abaxial surface (scale bars 20 mm) with several stomata elevated compared to level of epidermis. h. hircinum; m – adaxial surface (scale bars 60 mm) with marked epicuticular ornamentation; n – abaxial surface (scale bars 20 mm) showing stomata with highly cutinized rims. h. aegypticum; o – adaxial surface (scale bars 20 mm) showing cuticles with thick ornamentation; p – abaxial surface (scale bars 60 mm) with depressed stomata hypericum androsaemum and h. hircinum. trichomes are absent on both leaf surfaces. the adaxial surface is composed of rectangular and elliptical isodiametric cells which are strongly convex but with highly undulating longitudinal and radial walls; there is no sign of areolae that delimit these cells, nor stomata in h. androsaemum. veins are present rarely, with a pattern from undulating to linear; the cells are small and numerous but convex (figs. 1k, l). in h. hircinum, the adaxial leaf surface (fig. 1m) shows a mosaic of small areolae of various dimensions. these areolae are constituted by veins that run through the mesophyll. within each areola there are elliptical or polygonal cells with slightly wavy walls, the stomata are absent. the abaxial surface of h. hircinum also presents a strong mosaic pattern, with the presence of more or less irregular areolae (fig. 1n). anisocytic stomata are present on the abaxial surface of both species and are slightly sunken, especially compared to the convex character of the surrounding epidermal cells. sem shows that the cuticular ornamentations in h. androsaemum are scarce and barely detected and are type »k« crusts and layers (figs. 2k, l), while in h. hircinum these ornamentations are fairly minute granules of type »c« and »b« on the adaxial and abaxial surface respectively, and are also present on the stomatal guard cells (figs. 2m, n). the surfaces of these two species present depressions of various shapes which correspond to the translucent glands present in the mesophyll at various depths. hypericum aegypticum. trichomes are absent on both leaf surfaces. the adaxial surface (fig. 1o) of both basal and cauline leaves presents an epidermis of isodiametric cells (generally circular, elliptical, rarely rectangular) with linear walls and rounded corners, strongly convex and smooth. the leaves are amphistomatic, with few anisocytic stomata. the abaxial surface (fig. 1p) is made up of cells with the same morphological structure as those on the adaxial surface; the stomata are more numerous and also anisocytic. under sem (fig. 2o), the micromorphology of the tangential walls of adaxial epidermal cells is richer in detail. these walls are composed of cuticle with thick ornamentation which, according to wilkinson (1979), is of type »d« rods and filaments, and extends over the entire epidermal surface, even where very convex. even the guard cells have this ornamentation, and are thus barely distinguishable. the abaxial surface (fig. 2p) is characterized by the presence of densely ornamented epicuticular wax. thus, the surface appears encrusted by these deposits which increase the impermeability. on neither surface were found the characteristic depressions indicating the presence of translucent glands. even when present, these are found only deep into the spongy tissue and thus far from leaf surfaces. the foliar epidermal parameters (fep) are shown in table 1, the characteristics of the stomatal complex in table 2. leaf anatomy dorsiventral laminae are present in h. perforatum (fig. 3a), h. perfoliatum (fig. 3b), h. tetrapterum (fig. 3d), h. androsaemum (fig. 3f) and h. hircinum (fig. 3g), isobilateral in h. pubescens (fig. 3c), h. triquetrifolium (fig. 3e) and h. aegypticum (fig. 3h). when the leaves are dorsoventral, they present a protrusion corresponding with the median veins, while the two halves of the lamina are generally more or less convex on the adaxial side. both the adaxial and abaxial epidermis are mono-stratified. the adaxial chlorenchyma is also mono-stratified, with the exception of h. hircinum, which is bi-stratified, and the spongy tissue is generally thin (tab. 3). acta bot. croat. 72 (2), 2013 275 leaf and stem anatomy in hypericum species 276 a c t a b o t .c r o a t .72 (2),2013 p e r r o n e r .,d e r o s a p .,d e c a s t r o o .,c o l o m b o p . tab. 1. foliar epidermal parameters (f.e.p.) of hypericum l. species cell lenght mm cell width mm cell thickness mm cells freq./mm2 morphology of epicuticular waxes measures of central tendency measures of dispersion measures of central tendency measures of dispersion measures of central tendency measures of dispersion measures of central tendency measures of dispersion s standard deviation s 2 variance s standard deviation s 2 variance s standard deviation s 2 variance s standard deviation s 2 variance hypericum perforatum ad 72.0 72.0 72.0 3.59 12.89 28.0 30.0 28.0 3.16 10.00 17.0 16.0 17.0 1.83 3.33 546 546 20.78 432 granules type »b«ab 60.0 60.0 60.0 4.69 22.00 25.0 24.0 25.0 1.33 1.78 13.0 12.0 13.0 1.49 2.22 1010 1008 32.89 1082 h. perfoliatum ad 61.0 62.0 61.5 6.68 44.67 24.0 24.0 24.0 2.26 5.11 12.0 12.0 12.0 1.49 2.22 1263 1266 72.19 5211 rods and filaments type »e« ab 48.0 43.0 47.5 5.60 31.33 26.0 26.0 26.0 2.49 6.22 8.0 8.0 8.0 1.83 3.33 1317 1324 51.72 2675 h. pubescens ad 72.1 70.0 71.5 4.31 18.54 42.0 40.0 42.5 4.29 18.44 21.0 20.0 20.5 2.05 4.22 480 483 43.97 1933 plates and scales type »j« ab 46.0 46.0 46.0 3.02 9.11 26.0 26.0 26.5 2.91 8.44 17.0 16.0 17.0 1.25 1.56 571 568 50.33 2533 h. tetrapterum ad 57.0 56.0 56.0 5.62 31.56 40.0 41.0 40.5 6.31 39.78 13.0 12.0 13.0 1.56 2.44 457 471 42.10 1772 plates and scales type »j ab 80.0 78.0 79.0 8.03 64.44 44.0 47.0 46.0 7.79 60.67 17.0 16.0 17.0 1.56 2.44 684 700 61.13 3737 h. triquetrifolium ad 64.0 60.0 63.5 7.48 56.00 28.0 29.0 28.0 3.16 10.00 21.0 24.0 21.0 2.26 5.11 691 710 61.25 3752 granules type »b«ab 54.1 54.0 54.0 5.82 33.88 24.0 27.0 24.5 3.13 9.78 13.0 13.0 13.0 1.41 2.00 1124 1122 68.38 4676 h. androsaemum ad 50.1 49.0 49.5 3.03 9.21 27.0 26.0 26.5 2.45 6.00 14.0 15.0 14.5 1.70 2.89 1404 1411 76.05 5784 layers and crusts type »k« ab 41.0 40.0 40.5 3.71 13.78 50.1 49.0 49.5 4.70 22.10 16.1 19.0 16.0 1.97 3.88 821 836 65.09 4237 h. hircinum ad 34.1 37.0 34.0 3.18 10.10 20.0 19.0 19.5 1.41 2.00 8.1 8.0 8.0 0.99 0.99 2568 2587 93.35 8713 granules type »c« ad /»b«ab ab 29.0 30.0 29.0 1.83 3.33 17.1 18.0 17.5 1.85 3.43 4.0 4.0 4.0 0.67 0.44 3538 3561 80.70 6513 h. aegypticum ad 34.1 35.0 34.0 2.51 6.32 22.1 21.0 21.5 1.60 2.54 15.1 15.0 15.0 1.45 2.10 2283 2305 90.97 8275 rods and filaments type »d« ab 35.0 34.0 34.5 1.76 3.11 22.1 21.0 21.5 1.85 3.43 13.0 13.0 13.0 1.15 1.33 2321 2343 75.95 5769 symbols: ad =adaxial surface; ab = abaxial surface. m ea n m od e m ed ia n s ur fa ce m ea n m od e m ed ia n m ea n m od e m ed ia n m ea n m ed ia n on the isobilateral lamina, the epidermises are mono-stratified, the mesophyll appears homogeneous and not distinguishable in the palisade and spongy tissue. veins of each order pass through the mesophyll and in particular the spongy tissue. the median veins are particularly large and protrude from the abaxial surface. they have a parenchymatous sheath and acta bot. croat. 72 (2), 2013 277 leaf and stem anatomy in hypericum species tab. 2. characteristics of the stomatal complex in hypericum species stomatal complex types middle dimensions arrangement of stomata on the leaf blade p ar ac yt ic a ni so cy ti c polar diameter mm equatorial diameter mm density (× mm2) hypericum perforatum – * 22ab 12ab 358ab hypostomatic h. perfoliatum – * 23ab 6ab 275ab hypostomatic h. pubescens – * 19ad/15ab 8ad/7ab 115ad/ 420ab amphistomatic h. tetrapterum – * 8ad/9ab 6ad/5ab 23ad/ 457ab amphistomatic h. triquetrifolium + * 27ad/23ab 13ad/11ab 168ad/345ab amphistomatic h. androsaemum – * 21ab 16ab 228ab hypostomatic h. hircinum – * 21ab 16ab 211ab hypostomatic h. aegypticum – * 25ad/20ab 20ad/20ab 380ad/263ab amphistomatic + – present; * – predominant; – – absent; ad – adaxial surface; ab – abaxial surface. tab. 3. leaf typologies and foliar morphological parameters (fmp) in hypericum species mesophyll structure foliar morphological parameters (fmp) d or si ve nt ra l is ob il at er al middle thickness of leaf blade mm middle thickness in proximity to midrib mm middle thickness epid. ad mm middle thickness epid. ab mm m es op hy ll m m palisade mm s po ng y ti ss ue m m hypericum perforatum + – 170 243 19 15 112 48 64 h. perfoliatum + – 78 230 15 10 40 23 17 h. pubescens – + 142 250 17 21 103 60ad/32ab 39 h. tetrapterum + – 105 156 13 17 74 30 44 h. triquetrifolium – + 165 325 22 14 132 50ad/48ab 33 h. androsaemum + – 116 158 14 15 116 55 61 h. hircinum + – 126 315 9 5 90 40 50 h. aegypticum – + 450 690 25 30 400 120ad/91ab 112 + – present; – – absent; ad – adaxial surface; ab – abaxial surface 278 acta bot. croat. 72 (2), 2013 perrone r., de rosa p., de castro o., colombo p. fig. 3. transversal sections of the leaf middle portion. h. perforatum; a – dorsiventral leaf, thick adaxial surface, large palisade chlorenchyma, thin abaxial surface (scale bars 25 mm). h. perfoliatum; b – dorsiventral leaf, highly thickened adaxial surface with tanniferous cells, undifferentiated mesophyll (scale bars 50 mm). h. pubescens; c – isobilateral leaf, thin adaxial surface with many large multicellular trichomes, thin abaxial surface with very pronounced vein and numerous trichomes (scale bars 100 mm). h. tetrapterum; d – dorsiventral leaf, thin adaxial and abaxial surface, mesophyll with large chlorenchyma (scale bars 100 mm). h. triquetrifolium; e – isobilateral leaf, thin epidermis with tanniferous cells, mesophyll with translucent glands and type a secretory canals (scale bars 50 mm). h. androsaemum; f – dorsiventral leaf, extremely thin epidermis, spongy tissue with translucent glands (scale bars 25 mm). h. hircinum; g – dorsiventral leaf, large mesophyll with veins in the spongy tissue and type b secretory canals associated with the phloem (scale bars 25 mm). h. aegypticum; h – plano-convex isobilateral leaf, coriaceous and thin epidermis, wide and mucilaginous chlorenchyma with several translucent glands (scale bars 50 mm) a collenchyma cap, which make the vein even more prominent and conspicuous. the chlorenchyma is interrupted in this particular section of the lamina. translucent glands are found in the mesophyll of all the taxa, always very numerous and of various sizes, floating within the thickness; when present below the adaxial epidermis these glands are larger than those found above abaxial epidermis, while those at the center of the mesophyll are the largest of acta bot. croat. 72 (2), 2013 279 leaf and stem anatomy in hypericum species fig. 4. transverse section of fresh stem of mature plant made with bare-handed microtome. h. perforatum; a – lignified stem with elliptical cross-section and two small slightly prominent wings (scale bars 100 mm). h. perfoliatum; b – lignified stem with slightly wavy circular profile and two small evident wings (scale bars 1000 mm). h. pubescens; c – very lignified stem with almost circular profile (scale bars 1000 mm). h. tetrapterum; d – lignified stem with rectangular profile and four evident longitudinal triangular wings (scale bars 100 mm). h. triquetrifolium; e – lignified stem with very wavy elliptical profile in correspondence with two small wings (scale bars 100 mm). h. androsaemum; f – slightly lignified stem with circular profile and two small evident wings (scale bars 1000 mm). h. hircinum; g – lignified stem with wavy elliptical profile and two long, thin wings (scale bars 1000 mm). h. aegypticum; h – very lignified stem with circular suberous profile (scale bars 100 mm) all and even partially invade the palisade. in some cases (h. perforatum, h. perfoliatum) these are at a tangent to the two epidermises. stem anatomy from a morphological point of view the stem presents: in hemicryptophyte scapose species, lignified and prostrate at the base, briefly creeping, in h. perforatum and h. perfoliatum respectively; lignified creeping with ascending branches in h. pubescens; prostrate then erect, ligneous in h. tetrapterum and h. triquetrifolium, in the latter strongly branching (pignatti 1982). an evergreen shrub in the two nano-phanerophytes, h. hircinum and h. androsaemum; a ligneous shrub with contorted branches in h. aegypticum, a chamaephyte fruticose. in cross-section, the hemicryptophyte scapose stem has a profile from circular to elliptical with two small wings, along which are found black nodules, respectively, in h. perforatum (fig. 4a), h. perfoliatum (fig. 4b), and h. triquetrifolium (fig. 4e). in h. perforatum and h. triquetrifolium the outline also appears slightly wavy, particularly in correspondence with the two small wings; h. tetrapterum presents four triangular wings (fig. 4d). wings are absent in h. pubescens. in h. hircinum and h. androsaemum two wings are evident (figs. 4g, f), in h. aegypticum the stem profile is circular without wings (fig. 4h). anatomically, independently of the presence of more or less prominent wings, including the longitudinal triangular wings of h. tetrapterum, the young stem presents a mono-stratified epidermis with thin cuticle followed, in a centripetal direction, by a multilayered chlorophyll parenchyma (3–4 layers), a reserve parenchyma with rounded cells and, finally, by a mono-stratified endodermis with lenticular thickenings on the radial walls. the stele begins with a pericycle which is also mono-stratified, followed centripetally by concentric rings of phloem and xylem; in the phloem at this juvenile stage, type a and b secretory canals present in the cortex at various depths cannot yet be distinguished; the xylem is composed predominantly of protoxylem and a few metaxylematic elements in radial position, separated by numerous medullary rays. in the mature stem of h. perforatum, h. perfoliatum, h. tetrapterum and h. triquetrifolium we always find an outer mono-stratified epidermis where, however, the constituent cells become smaller and the thickness of the outer tangential walls increases through enrichment with cutin. in h. pubescens, the stem trichomes dry out and are partly lost because the outermost layers tend to become enriched with suberin. the sub-epidermal chlorenchyma thins, but persists, while the cortex is enriched with a reserve parenchyma mixed with tanniferous cells. centripetally, the cortex is delimited by a closed endodermal sheath, followed by a pericycle with some sclerenchymatous elements. the stele is composed of phloem and numerous, scattered type a secretory canals in a ring. in h. pubescens in particular, black nodules are found in the outer cortex, in the phloem, at the border between xylem and phloem and also at the end of the xylematic ring tangential to the medulla. h. tetrapterum has a rectangular profile, with rounded edges on each side, there is a triangular wing expansion; a mono-stratified epidermis with large cells and black nodules underlies the entire stem, including the wings. centripetally there follows a chlorenchyma and, finally, more internally, a reserve parenchyma, which thins and surrounds the stem, giving rise to a thin cortex containing a few type b secretory canals. we then find an endodermoide, a pericycle with numerous sclerenchymatous elements and finally, the phloem, where many irregularly distributed type b secretory canals are highlighted externally, and 280 acta bot. croat. 72 (2), 2013 perrone r., de rosa p., de castro o., colombo p. more regularly arranged type a canals in the centripetal direction. h. perforatum has a slightly elliptical profile, a wider medulla, and is rich in reserve parenchyma. externally there is a mono-stratified epidermis consisting of more or less large rounded cells, a thin cortical parenchyma and a wide cortex with numerous lacunae, which are more numerous below the two small wings. the phloem underneath contains type a and b secretory canals, interspersed with the lacunae. the xylem is generally robust and often ring-porous, except in h. pubescens (fig. 5c) and h. aegypticum (fig. 5h), where it is diffuse-porous. the difacta bot. croat. 72 (2), 2013 281 leaf and stem anatomy in hypericum species fig. 5. details of secondary xylem: diffuse-porous xylem (a, b, d, e, f, g) and ring-porous xylem (c, h). hypericum perforatum (a), h. perfoliatum (b), h. pubescens (c), h. tetrapterum (d), h. triquetrifolium (e), h. androsaemum (f), h. hircinum (g), h. aegypticum (h). scale bars 100 mm. 282 a c t a b o t .c r o a t .72 (2),2013 p e r r o n e r .,d e r o s a p .,d e c a s t r o o .,c o l o m b o p . tab. 4. stem morpho-anatomy in hypericum taxa n. middle vessels × mm2 æ middle vessels mm secondary xylem habitus environment secretory structures wings/stem cross-section hypericum perforatum 4 20 ring-porous creeping ascending lignified dry meadows scrubland. uncultivated land type a and b secretory canals in chlorenchyma and phloem. type b secretory canals in cortex two small / slightly elliptical h. perfoliatum 6 21 ring-porous creeping ascending lignified scrubland. woods. hedges type a secretory canals in phloem. type b secretory canals in cortex two small / circular h. pubescens 8 27 diffuse-porous creeping ascending branches lignified wet and sometimes salty habitats type a secretory canals in phloem (small and rare). type b secretory canals in cortex (few). black nodules in outer cortex. between xylem and phloem and between xylem and medulla absent / circular h. tetrapterum 3 19 ring-porous prostrate lignified marshes and cane-brakes type a and b secretory canals in phloem. type b secretory canals in cortex (few). black nodules on wings four triangular / rectangular h. triquetrifolium 6 20 ring-porous prostrate very branching lignified uncultivated ground type a secretory canals in phloem (small). type b secretory canals in outer cortex two small / slightly wavy elliptical h. androsaemum 3 19 ring-porous erected shrub woods. wet and shady localities type a secretory canals in phloem (few). type b secretory canals in outer cortex two small evident / circular h. hircinum 3 19 ring-porous erected shrub ravines. wet and shady localities type a secretory canals with rhomboidal and circular lumen (2:1) and large type b secretory canals in phloem two long. thin / wavy elliptical h. aegypticum 2 15 diffuse-porous depressed shrub with contorted branches maritime cliffs type a secretory canals in phloem (small). type b secretory canals in cortex (large and numerous) absent / circular fuse-porous xylem is characterized by two extreme types: that of h. pubescens, where the number of vessels per mm2 is generally high, with a peak of 8 per mm2, and large diameter of 27 mm; and that of h. aegypticum, which presents the lowest number of vessels (2 per mm2) and at the same time a significantly lower average vessel diameter of 15 mm. in ring-porous xylem in h. perforatum, h. perfoliatum, h. tetrapterum and h. triquetrifolium (tab. 4) the medullary rays are uniseriate, while the vessels that make up most of the ring xylem are well lignified with a small lumen in h. tetrapterum (fig. 5d). in h. perforatum (fig. 5a), h. perfoliatum (fig. 5b) and h. triquetrifolium (fig. 5e) these vessels are less lignified, with wider lumens. the medullary rays of h. pubescens are always uniseriate, while the vessels are numerous and well-lignified. there are also black nodules on the outer cortex, in the phloem, in the border between the xylem and phloem as well as at the end of the xylematic ring tangential to the medulla. further details are reported in table 4. discussion the species investigated colonize diverse environments, from mesic to wet conditions, from marsh and reed to sea cliffs, and are therefore characterized by a remarkable eco-morphological plasticity. however, all are adapted to withstand prolonged drought for the duration of the dry season, which in the mediterranean climate extends from april to october, with a variable thermopluviometric performance that has been more marked in recent years. the species are mostly mesophytes (h. perforatum, h. perfoliatum, h. pubescens, h. androsaemum, h. hircinum), but there are also meso-hygrophytes such as h. tetrapterum, and xerophytes and xero-halophytes such as h. triquetrifolium and h. aegypticum respectively. with the arrival of spring and summer, the sudden increase in temperature and drastic reduction in rainfall means all the species suffer thermal water stress, even those in wetlands where the regime is temporary or fluctuating. all this is reflected in the micromorphological features of the epidermal cells, which are constant and genetically controlled (carr et al. 1971, faggetter 1987), although influenced by environmental conditions (bartholott 1981), together with the overall structure of the stomatal complex. the mesophyte can face moderate water deficiency due to its cuticular thickening, waxy coating and variable cell and stomatal frequency. the adaxial and abaxial epidermal cells mainly show convex surfaces, with various types of ornamentation (tab. 1), even at the level of the stomatal guard cells. there are thin pruinose coatings with a water-repellent function in taxa from humid or marshy environments and thick coatings in those growing in sunny, arid or salty habitats. all stomatal complexes are anisocytic, with high density per mm2 in species with hypostomatic leaves (h. perforatum, h. perfoliatum, h. androsaemum, h. hircinum) as some grow at altitude, where they are subject to temperature fluctuations, others in very sunny and arid habitats. the leaves, with their limited surface area, present thick, waxy cuticular coatings that distinguish one individual from another (tab. 1). the presence of epicuticular waxes, regardless of type, is of great importance for the prevention of water loss. these waxes increase the capacity of the leaves to bind water and exchange gases through their water-repellency (polací et al. 2004) and reduce the interception of solar rays in exposed environments. a further property of the wax is to minimize mechanical damage and inhibit fungal and insect attack (eglinton and hamilton 1967). the arrival of summer drastically reduces the grassy mantle, and leaves that are still green at this point are a good resource for insects. consequently, the wax layer tends to vary with season and age of species (eglinton acta bot. croat. 72 (2), 2013 283 leaf and stem anatomy in hypericum species and hamilton 1967). species with hypostomatic leaves, as well as having a high stomatal frequency, present a similarly elevated frequency of epidermal cells on both leaf surfaces; these cells are also long, narrow and fairly thin (tab. 1). species with amphistomatic leaves show lower stomatal frequency on the adaxial surface and a high frequency on the abaxial surface with reduced leaf surfaces (h. pubescens, h. tetrapterum). they also possess, like the stomata, a low frequency of epidermal cells which are short, wide and thick, in agreement with their growth habitat. h. triquetrifolium, a xerophyte with amphistomatic leaves and anisocytic stomatal complexes, is characterised by a high frequency of cells and stomata, especially on the abaxial surface of the leaf. moreover, the particular morphology of the epicuticular waxes of type »b« granules (tab. 1) ensures the efficient reflection of light and heat. as a xero-halophyte exposed to sea spray, h. aegypticum is moderately crassulent and has a high stomatal frequency on both leaf surfaces; the stomata also have polar and equatorial axes similar to those of h. triquetrifolium, a species that grows in arid and uncultivated environments. the presence of trichomes on all foliar surfaces in h. pubescens slows down transpiration, reduces thermal load and photo-induced lesions as well as repelling small insects and pathogenic agents. considering that these taxa grow in open, bright environments, their trichomes may fulfil most of the potential roles mentioned above. comparison of stem anatomy is useful for characterization, both for the distribution of type a secretory structures in the entire thickness of the cortex, and the type b secretory structures present in the phloem at different depths, and even more so for the xylem type, which lends itself to phylogenetic interpretation and can be either ring-porous or diffuse-porous. ring-porous xylem, not very common and typical of temperate zone species, can be interpreted as evidence of greater evolutionary specialization, influenced also by environmental characteristics. diffuse-porous xylem, present in h. aegypticum and h. pubescens, possessing a parenchyma mixed with mechanical and conduction elements, is apotracheal, a typology considered to be more primitive than the paratracheal type. the »apotracheal diffuse« type is even more primitive than other typologies. being shorter, the vessels of diffuse-porous xylem show a less efficient flow velocity. in the case of h. pubescens, this eco-physiological feature is subordinate to the average diameter and the number of vessels × mm2, which are the highest of all the taxa examined. in conclusion, interspecific differences can be seen in density, type and position of the stomatal complex, the amount and morphology of epicuticular wax, secondary wood features such as ring-porous or diffuse-porous xylem as well as the number and average diameter of vessels. hence, foliar and stem anatomy can be considered an additional tool for taxonomic and evolutionary studies in hypericum. references baroni fornasiero, r., bianchi, a., pinetti, a., 1998: anatomical and ultrastructural observations in hypericum perforatum l. leaves. journal of herbs, spices and medicinal plants 5, 21–33. baroni fornasiero, r., maffi, l., benvenuti, s., bianchi, a., 2000: morphological and phytochemical features of secretory structures in hypericum richeri (clusiaceae). nordic journal of botany 20, 427–434. 284 acta bot. croat. 72 (2), 2013 perrone r., de rosa p., de castro o., colombo p. bartholott, w., 1981: epidermal and seed surface character of plants: systematic applicability and some evolutionary aspects. nordic jurnal of botany 1, 345–354. beccari, n., mazzi, v., 1966: manuele di tecnica microscopica. guida pratica alla ricerca istologica e istochimica.vi edizione. società editrice libraria. blenk, p., 1884: über die durchsichtigen punkte in den blättern. flora 67, 97–144. carr, s. m. g., milkovits, l., carr, d. j., 1971: eucalypt phytoglyphs: the microanatomical features of epidermis in relation to taxonomy. australian journal of botany 19, 173–190. catalano, g., 1925: guida pratica di anatomia e fisiologia vegetale. dott. francesco vallardi, milano. christophel, d.c., kerrigan, r., rowett, a.i., 1996: the use of cuticular features in the taxonomy of the lauraceae. annals of the missouri botanical garden 83(3), 419–432. ciccarelli, d., andreucci, a. c., pagni, a. m., 2001a: the black nodules of hypericum perforatum l. subsp. perforatum: morphological, anatomical and histochemical studies during the course of ontogenesis. israel journal of plant sciences 49, 33–40. ciccarelli, d., andreucci, a. c., pagni, a. m., 2001b: translucent glands and secretory canals in hypericum perforatum l. 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ames iowa. siersch, e., 1927: anatomie und mikrochemie der hypericumdruesen (chemie des hypericins). planta 3, 481–489. tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. a., 1972: flora europaea. cambridge press, cambridge. weill, g., 1903: recherches histologiques sur la famille des hypéricacées 1. a. joanin et cie, paris. wilkinson, h. p., 1979: the plant surface (mainly leaf). in: metcalfe, c. r., chalk, l. anatomy of the dicotyledons 1, 97–165. clarendon press, oxford. 286 acta bot. croat. 72 (2), 2013 perrone r., de rosa p., de castro o., colombo p. opce-str.vp acta bot. croat. 73 (1), 37–50, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 influence of crop species and edaphic factors on the distribution and abundance of trichoderma in alfisol soils of southern india vellaisamy muniappan, thangavelu muthukumar* root and soil biology laboratory, department of botany, bharathiar university, coimbatore 641 046, tamilnadu, india abstract – the effect of crop species and edaphic factors on the distribution of trichoderma species in alfisol soil under different agrosystems was evaluated. each soil sample was assayed for nine abiotic factors and culturable microfungal populations. fungal abundance was determined by dilution plate technique, and the identification of fungi was based on morphological characteristics. pearson’s correlation coefficient was used to determine the relationship of association between these factors and the presence and abundance of trichoderma species in each soil type. the abundance of soil fungi ranged between 7.0 × 103 and 13.6 × 103 colony forming units (cfus) per gram of dry soil. the population densities of the two trichoderma species (t. koningii and t. viride) isolated in the present study varied significantly with crop species and their abundance (varied from 0.6 to 3.6 × 103 cfus g–1 dry soil). twenty-two other colony-forming fungal types with an abundance ranging between 7.0 × 103 and 13.6 × 103 cfus g–1 dry soil were also isolated in the present study. as soil ph negatively influenced relative abundance of t. koningii, soil p and relative abundance of t. viride were significantly and positively correlated to each other. further, relative abundance of t. koningii was significantly and positively correlated to relative abundance of aspergillus fumigatus but negatively correlated to relative abundance of stachybotrys atra. likewise, a significant negative correlation existed between relative abundance of t. viride and absidia glauca. key words: alfisol soil, crops, edaphic factors, fungal abundance, soil ph, trichoderma introduction trichoderma are abundant free living soil fungi that are highly interactive in root, soil and foliar environments. their significance resides in their potential for control of soil borne plant pathogens. trichoderma act as biocontrol agents through competition for nutrients or space, mycoparasitism and antibiosis (wells 2000). some strains also act as plant growth promoters through solubilization and sequestration of organic nutrients (harman et al. acta bot. croat. 73 (1), 2014 37 * corresponding author, e-mail: tmkum@yahoo.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:56 color profile: generic cmyk printer profile composite default screen 2004a). there is a reasonable amount of information about the behavior of many soil borne plant pathogens in the soil environment. however, little attention has been given to factors affecting growth of antagonistic organisms such as trichoderma in the soil (wakelin et al. 1999). factors affecting the growth of trichoderma under laboratory conditions have been extensively studied, but the correlation between various growth responses in in situ and in vivo conditions are dubious (cook and baker 1983, papavizas 1985). this limited knowledge on the ecology of these biocontrol agents coupled with the environment variability limits the success of trichoderma as a biological control agent under field situations (lewis and papavizas 1991). there is an urgent need for more information on the factors affecting the distribution of trichoderma, in agro ecosystems. the alfisol soils that constitute around 10% of the ice-free land area are the dominant soil types in many tropical and subtropical areas, including peninsular india (rust 1983). alfisols are characterized by high fertility and water-holding capacity, moderate leaching and have at least 35% base saturation (soil survey staff 1994). therefore, extensive areas of alfisols have been used for cultivation or forestry (rao et al. 1991). as high yields and sustainable agriculture are targeted by farmers, researchers, politicians and society, knowledge of the fungal population present in the soil and their functions in the corresponding ecosystems is vital. although some soil fungi (e.g., mycorrhizal fungi) are intensively studied, little is known about fungal community structures and community dynamics in agricultural soils. it is generally agreed that only a small percentage of the 1.5 million fungi world-wide are culturable (hawksworth and rossman 1997). nevertheless, up to now, many studies have investigated fungal diversity by using culture–dependent approaches (attitalla et al. 2012, okoth et al. 2009, sariah et al. 2005). in spite of studies examining the bioinoculant potential of trichoderma against various plant pathogens in alfisol soils (manjula et al. 2004, kumar et al. 2012), information on the survivability and proliferation of fungi in relation to soil type and factors is limited. the ability of trichoderma to dispose and to colonize the rhizosphere will determine its effectiveness as a biocontrol agent. thus, an understanding of the quantitative distribution of the fungus in different agro-ecosystems is essential before it can be developed into biological formulations for field application. the following study was undertaken with the objectives of (i) quantifying the populations of trichoderma spp., from cultivated ecosystems, (ii) to determine if crop species had any significant influence on the distribution or abundance trichoderma spp. and (iii) to characterize the influence of edaphic factors and co-occurring fungal species on populations of trichoderma. materials and methods sampling alfisol soil samples were collected from agrosystems within a 2 km radius of coimbatore (11°04’ n and 76°92’ e, altitude 426.7–550 m a.s.l.), tamil nadu, india. these included soil samples under ten different crop species: tomato (lycopersicon esculentum, soil 1), coconut (cocus nucifera, soil 2), sorghum (sorghum bicolor, soil 3), ground nut (arachis hypogea, soil 4), maize (zea mays, soil 5), sugarcane (saccharum officinarum, soil 6), rice (oryza sativa, soil 7), turmeric (curcuma longa, soil 8), areca nut (areca catechu, soil 9) and papaya (carca papaya, soil 10). 38 acta bot. croat. 73 (1), 2014 muniappan v., muthukumar t. 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:56 color profile: generic cmyk printer profile composite default screen ten undistributed soil cores measuring 3 cm diameter were randomly taken from the depth of 0–15 cm of the top horizon adjacent to the plants, and combined. the composite samples were placed in airtight sterile ziploc polythene bags and transported to the laboratory. half of the soil samples used to assess the soil microfungal populations was stored at 4 °c, while the other half was air dried, passed through a 2-mm screen to remove coarse debris and plant materials, and used to analyze the physico-chemical properties of the soil. determination of soil characteristics the composite soil sample from each agrosystem was divided into five equal parts and assessed for various properties. one hundred ml of sterile distilled water was added to 100 g of soil sample, thus yielding a 1:1 soil suspension. the samples were stirred briefly and allowed to equilibrate for 1h. the ph of each sample was then measured using a digital ph meter (elico li 610, hyderabad, india) by immersing the electrode in the sediment at the bottom of the soil slurry. one hundred ml of distilled water was added to 10 g of dry soil to make a suspension of 1: 10 (w/v) dilution. the electrical conductivity ec was measured using a digital ec tds analyzer (elico cm 183, hyderabad, india). the total nitrogen (n) and phosphorus (p) in the soils were determined by micro-kjeldahl and molybdenum blue methods respectively (jackson 1971). soil k was estimated by the flame photometric method after extraction in ammonium acetate solution (jackson 1971). other mineral (fe, zn, cu and mn) element composition was also analyzed according to jackson (1971). evaluation of soil micro fungal population soil dilution technique was used to evaluate the total number of fungal colony forming units (cfus) in the soil samples and to isolate pure cultures (danielson and davey 1973). each composite soil intended for the assessment of micro fungal populations was divided into five equal parts. five grams (on dry weight basis) of the composite soil subsample were added to 250 ml sterile conical flasks containing 100 ml of sterilized distilled water. flasks were shaken vigorously for five minutes. one ml aliquots of the 1:103 dilutions were spread evenly on each of the five petri dishes (100 ´ 15 mm) containing 20 ml of agar rose bengal medium (martin 1950) supplemented with 0.06 g l–1 of streptomycin sulphate for each composite soil subsample. petri plates were incubated at room temperature (26–30 °c) till the development of fungal colonies. after four days, all fungal colonies were counted and the plates were incubated again for another three days. many colonizers sporulated after seven days, so at that time individual colony types were counted, including those of trichoderma. for each of the colony types counted, representative colonies were isolated and brought under pure culture for identification. results were recorded as the number of colonies per plate. averages of composite soil subsamples were used to determine the abundance and frequency of soil micro fungal populations. semi-permanent slides were prepared by mounting fungi in lactophenol containing 0.5% cotton blue. the characterization of trichoderma isolates into species aggregates were based on cultural and morphological characters (bissett 1984, 1991, 1992; bissett et al. 2003; chaverri et al. 2001, 2004; chaverri and samuels 2002, 2003; rifai 1969; webster 1964; webster and rifai 1968). the other co-existing soil fungi were also identiacta bot. croat. 73 (1), 2014 39 influence of crop species and edaphic factors on trichoderma 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:56 color profile: generic cmyk printer profile composite default screen fied according to the descriptions by barron (1972), domsch et al. (1980), ellis (1971), nelson et al. (1983), pitt (1979), and subramanian (1970). abundance (isolates of a species as percent of total isolates) and frequency of occurrence (percent from total plate samples) for each soil was calculated using the formulae: abundance = number of colonies of a particular fungus in the sample ´ 100 total number of fungal colonies of the sample frequency = number of agrosystems in which a particular fungus was present ´ 100 total number of agrosystems examined statistical analysis data on soil characteristics and microfungal populations were subjected to analysis of variance and the means were separated using duncan’s multiple range test (dmrt) (spss for windows, version 9). the relationships between soil factors and microfungal populations and among fungal populations were analyzed by pearson’s correlation. data were log transformed prior to statistical analysis. results soil factors measurement of ph, ec, soil n, p, k, fe, zn, mn, cu and fe are presented in table 1. the soils in the present study were near neutral to alkaline with a ph range of 7.23 to 8.23. the ec ranged from 0.245 to 1.05 dsm–1. the soils were nutrient deficient with various nu40 acta bot. croat. 73 (1), 2014 muniappan v., muthukumar t. tab. 1. characteristics of the alfisol soils used in the study. soil ph ec (ds m–1) soil nutrients (mg kg–1) nitrogen phosphorus potassium copper iron zinc manganese s1 7.23 a 0.245 a 9.8 b 0.25 a 42.00 c 1.41 a 26.44 f 1.24 d 8.24 c s2 7.64 b 0.636 f 10.1 b 0.75 b 50.00 c 1.31 a 10.42 d 1.52 f 2.84 a s3 7.93 c 0.299 b 14.0 e 1.15 c 40.00 c 1.21 a 5.44 a 1.28 de 4.24 b s4 7.89 c 0.536 e 11.5 c 0.26 a 51.00 d 1.80 b 8.32 c 0.86 b 8.22 c s5 8.23 e 0.315 b 6.4 a 0.28 a 42.00 c 1.24 a 6.78 b 0.76 ab 4.24 b s6 8.10 d 0.302 b 14.0 e 0.45 a 18.00 a 3.64 e 10.60 d 1.48 f 18.60 e s7 7.74 b 1.050 g 12.6 d 1.20 c 51.00 d 2.82 c 8.50 c 1.08 c 7.06 c s8 8.12 d 0.408 c 10.4 b 1.55 d 31.00 b 4.06 f 14.80 e 0.64 f 15.56 d s9 7.99 c 0.448 d 10.6 b 1.30 c 19.00 a 3.08 d 9.41 cd 1.40 ef 16.20 d s10 7.93 c 0.548 e 14.0 e 1.35 c 51.00 d 4.24 f 10.36 d 1.60 f 20.24 f means in a column followed by same letter(s) are not significantly (p > 0.05) different according to duncan’s multiple range test. soil types s1–s10 are explained in methods. 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:56 color profile: generic cmyk printer profile composite default screen a c t a b o t .c r o a t .73 (1),2014 41 in f l u e n c e o f c r o p s p e c ie s a n d e d a p h ic f a c t o r s o n t r ic h o d e r m a tab. 2. distribution and frequency (percent from total plate samples) of soil microfungi associated with different crop species. soil types s1 – s10 are explained in methods. fungal species soils frequency (%) s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 absidia glauca hagem + + + + + + + 70 aspergillus candidus link ex. fr. + + + + + + + 70 aspergillus flavipes (bain. et sart.) thom et chruch + + + + + + + 70 aspergillus fumigatus fresen. + + + + + + 60 aspergillus sp. + + + + + 50 aspergillus pulverulentus (mcalpine) thom. + + + + + + 60 aspergillus terreus thom. + + + + + + + + + 90 cladosporium cladosporioides (fres.) de vries. + + + + + + 60 curvularia lunata (wakker) boedijn. + + + + 40 drechslera halodes (drechsler) subram. et b.l. jain + + + + + + 60 mucor sp. + + + + + + 60 fusarium coeruleum (lib.) sacc. + + + + + 50 fusarium solani (mart.) sacc. + + + + + + + 70 humicola grisea traeen + + + + + 50 nigrospora sphaerica (sacc.) e.w. mason + + + + 40 penicillium adametzi zaleski + + + + + + 60 penicillium citrinum sopp + + + + + 50 penicillium rubrum stoll + + + + + + + + 80 rhizopus stolonifer (ehrenb.) vuill. + + + + + + + + 80 scopulariopsis brevicaulis (sacc.) bainier + + + + + + 60 stachybotrys atra corda. + + + + + + + + 80 trichoderma koningii oudem + + + + + + + + 80 trichoderma viride pers. ex. fries + + + + + + + + + 90 verticillium albo-atrum reinke et berth + + + + 40 7 4 1 m u n i a p p a n a n d m u t h u k u m a r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 7 4 1 m u n i a p p a n a n d m u t h u k u m a r . v p 1 9 . o u j a k 2 0 1 4 1 6 : 4 6 : 5 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n trient levels ranging as follows: 6.4 to 14.0 mg kg–1 of total n, 0.25 to 1.55 mg kg–1 of total p, 18 to 51 mg kg–1 of exchangeable k, 5.44 to 26.44 mg kg–1 of fe, 0.60 to 1.60 mg kg–1 of zn, 2.84 to 20.24 mg kg–1 of mn and 1.21 to 4.24 mg kg–1 of cu. trichoderma populations trichoderma was isolated from all the alfisol soils (tab. 2). these include trichoderma koningii oudem and trichoderma viride pers. ex. fries. the average population density of t. koningii ranged between 0.6 × 103 cfus and 3.6 × 103 cfus g–1 of soil, t. koningii was not found in s. bicolor and c. papaya-cultivated soils. trichoderma viride average populations ranged between 0.6 × 103 cfus and 1.6 × 103 cfus g–1 of soil. trichoderma viride was absent from a. hypogea soil. populations of both t. koningii (f7,36 = 2.45, p < 0.05) and t. viride (f8,36 = 2.31, p < 0.05) significantly varied with plant species. based on the overall occurrence in different soils, t. viride was the most frequent trichoderma species occurring in 90% of the soil samples examined. co-occurring microfungal population the 22 colony-forming soil microfungi, apart from t. koningii and t. viride were classified into 20 species belonging to 14 genera and two could not be identified to species level (tab. 2). total fungal colonies significantly varied among crop species (f9,40 = 4.485, p < 0.001) and were maximum in s. bicolor soil (14 × 103 cfu g–1 soil) and minimum in s. officinarum soil (7 × 103 cfu g–1 of soil) (fig.1). aspergillus terreus and t. viride occurred in the soils of most crop species examined, whereas nigrospora sphaerica and verticillium albo-atrum were present in a minimum number of soil samples (tab. 2). among the 10 soil samples examined trichoderma species (t. koningii) was abundant only in three soils (a. hypogea, c. nucifera, and l. esculentum soils) (tab. 3, fig. 2). relative abundance of other soils was shared by aspergillus terreus (s. officinarum, a. catechu and c. papaya soils), stachybotrys atra (s. bicolor soil), drechslera halodes (c. longa soil), mucor sp. (z. mays soil) and rhizopus stolonifer (o. sativa soil). aspergillus was the most diverse genus in the present study with six species followed by penicillium (three species). 42 acta bot. croat. 73 (1), 2014 muniappan v., muthukumar t. c f f e b a d e c b 7 9 11 13 15 s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 soils 0 t o ta l fu n g a l a b u n d a n c e (1 0 c fu g s o il ) 3 – 1 // fig. 1. total soil micro fungal populations in different agrosystems. vertical bars indicate ± 1 s.e. bars bearing same letter(s) are not significantly different according to duncan’s multiple range test (p > 0.05). soil types s1–s10 are explained in methods. 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:57 color profile: generic cmyk printer profile composite default screen a c t a b o t .c r o a t .73 (1),2014 43 in f l u e n c e o f c r o p s p e c ie s a n d e d a p h ic f a c t o r s o n t r ic h o d e r m a tab. 3. abundance of soil microfungal species (isolates as a percent of total isolates isolated from agrosystems of different crop species). abundant species in each agrosystem is presented in bold. soil types s1 – s10 are explained in methods. fungus name soils s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 absidia glauca 4.00 8.82 1.67 2.86 1.82 1.67 4.88 aspergillus candidus 6.00 2.94 2.86 2.86 5.00 2.00 4.88 aspergillus flavipes 2.00 3.33 2.38 5.71 7.27 5.00 10.00 aspergillus fumigatus 12.00 13.24 4.29 5.00 7.14 12.73 aspergillus pulverulentus 4.41 7.14 1.82 6.67 2.00 2.44 aspergillus sp. 6.67 5.71 1.82 6.67 6.00 aspergillus terreus 14.00 17.65 8.57 16.67 17.14 9.09 5.00 20.00 17.07 cladosporium cladosporioides 2.86 3.33 7.14 11.43 1.82 7.32 curvularia lunata 1.47 2.86 2.38 2.44 drechslera halodes 2.00 8.57 1.67 1.82 15.00 2.00 fusarium coeruleum 1.47 4.76 2.86 1.67 4.00 fusarium solani 2.00 11.43 10.00 7.14 8.33 14.00 14.63 humicola grisea 2.00 6.67 5.71 1.67 6.00 mucor sp. 1.47 7.14 16.67 5.45 3.33 4.88 nigrospora sphaerica 2.94 2.86 5.00 4.76 penicillium adametzi 18.00 1.47 8.33 2.38 3.64 5.00 penicillium citrinum 1.47 2.38 8.57 2.00 4.88 penicillium rubrum 2.00 5.88 14.29 3.33 2.86 5.45 5.00 4.88 rhizopus stolonifer 4.00 2.94 1.67 2.86 14.55 6.67 8.00 7.32 scopulariopsis brevicaulis 2.00 5.71 5.00 9.52 11.43 2.44 stachybotrys atra 14.29 5.00 9.52 2.86 7.27 3.33 6.00 7.32 trichoderma koningii 22.00 26.47 16.67 7.14 8.57 12.73 8.33 4.00 trichoderma viride 8.00 4.41 11.43 9.52 8.57 12.73 11.67 12.00 12.20 verticillium albo-atrum 2.94 2.86 2.00 2.44 7 4 1 m u n i a p p a n a n d m u t h u k u m a r . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 7 4 1 m u n i a p p a n a n d m u t h u k u m a r . v p 1 9 . o u j a k 2 0 1 4 1 6 : 4 6 : 5 7 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n relationship between soil factors and microfungal populations correlation analysis revealed an absence of significant influence of soil factors on total soil microfungal populations. but abundance of t. koningii was significantly and negatively correlated to soil ph (r = –0.670; p < 0.05; n = 10) and relative abundance of t. viride was significantly and positively correlated to soil p (r = 0.697; p < 0.05; n = 10). further, relative abundance of t. koningii was significantly and positively correlated to relative abundance of a. fumigatus (r = 0.749; p < 0.05; n = 10), but was significantly and negatively correlated to stachybotrys atra (r = –0.766; p < 0.05; n = 10). relative abundance of t. viride was significantly and negatively correlated to relative abundance of absidia glauca (r = –0.727; p < 0.05; n = 10). relative abundance of t. koningii and t. viride was significantly and negatively correlated to each other (r = –0.666; p < 0.05; n = 10). among the co-occurring fungi, relative abundance of penicillium adametzi was significantly and negatively correlated to soil ph (r = –0.712; p < 0.05; n = 10). in contrast, a significant positive correlation existed between soil ec and relative abundance of rhizopus stolonifer (r = 0.756; p < 0.01; n = 10). as soil cu was significantly and negatively correlated to relative abundance of aspergillus fumigatus (r = –0.678; p < 0.05; n = 10), soil fe was significantly and positively correlated to relative abundances of aspergillus candidus (r = 0.713; p < 0.05; n = 10) and penicillium adametzi (r = 0.826; p < 0.001; n = 10). the relative abundance of the unidentified aspergillus sp. was significantly and positively correlated to relative abundances of aspergillus flavipes (r = 0.683; p < 0.05; n = 10) and humicola grisea, (r = 0.817; p < 0.01; n = 10) and negatively to relative abundance of curvularia lunata (r = –0.708; p < 0.01; n = 10). likewise, relative abundance of cladosporium cladosporioides was significantly and positively correlated to relative abundances of penicillium citrinum (r = 0.841; p < 0.01; n = 10) and scopulariopsis brevicaulis (r = 44 acta bot. croat. 73 (1), 2014 muniappan v., muthukumar t. 0% 20% 40% 60% 80% 100% s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 trichoderma koningii trichoderma viride other fungi abundance s o il s fig. 2. percent abundance (%) of trichoderma koningii and trichoderma viride in relation to other soil fungi in different agrosystems. soil types s1 – s10 are explained in methods. 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:57 color profile: generic cmyk printer profile composite default screen 0.844; p < 0.001; n = 10). as the relative abundance of curvularia lunata was significantly and positively correlated to the relative abundance of mucor sp., (r = 0.669; p < 0.05; n = 10), it was significantly and negatively correlated to the relative abundance of humicola grisea (r = –0.648; p < 0.05; n = 10). a significant positive correlation existed between relative abundances of mucor sp. and stachybotrys atra (r = 0.637; p < 0.05; n = 10). in contrast, a significant negative correlation existed between the relative abundances of rhizopus stolonifer and scopulariopsis brevicaulis (r = –0.632; p < 0.05; n = 10). discussion the frequent occurrence of trichoderma species in alfisol soils under different crop species is in accordance with the fact that species of trichoderma are an abundant and biologically important component of soil micro-flora, though differences in their abundance can occur (samuels 2006). in contrast, attitalla et al. (2012) isolated trichoderma harzianum only from the five of the 23 soil samples they examined from al-jabal al-akhdar region of libya. the population of trichoderma in the present study varied from 0.6 to 3.6 × 103 cfus g–1 of soil, which is in agreement with published ranges. nearly all temperate and tropical soils contain trichoderma populations of 101 – 103 cfus g–1 of soil (see harman et al. 2004a, b and references therein). studies have shown that short-term changes in trichoderma populations tend to occur in response to temporary changes in soil moisture, nutrient availability or soil temperature (kredics et al. 2003; wakelin et al. 1999). in the present study, populations of trichoderma significantly varied with crop species. however, to our knowledge, there is only one previous study (okoth et al. 2009) that has examined the role of crop species on trichoderma populations in addition to the substrate specificity of a few trichoderma species. nevertheless, the influence of plant species on trichoderma has been mostly restricted to endophytic species. for example, trichoderma stromaticum is found only in association with theobroma species in tropical america (samuels et al. 2000). it has been shown that plant species play a major role in determining the occurrence and distribution of fungal species (grayston et al. 1998; ibekwe et al. 2002). in spite of their coexistence in 70% of the soil samples, populations of both the trichoderma spp. were negatively related to each other. this suggests that both these soil fungi compete in the soil environment or the fungi may be reacting variedly to the given environmental conditions. the first view is supported by the fact that strains of t. viride are known to be highly competitive and are known to proliferate best in the presence of healthy plant roots, whereas, t. koningii tends to occur in diverse soil conditions (hjeljord and tronsmo 1998; samuels 2006). the second view is supported by the fact that soil ph has been thought to be of great importance to the activity and occurrence of trichoderma species (kredics et al. 2003, küçük and kivanç 2003, harman et al. 2004a). kredics et al. (2003) showed that species of trichoderma grow optimally around ph 4.0 to 5.0, and exhibit little or no growth below ph 2.0 or above ph 6.0. nevertheless, the ph of the soils in the present study varied between 7.23 and 8.23, and t. koningii was abundant in soils with a ph of 7.23 and 7.89. this corroborates the findings of gherbawy et al. (2004) reporting that trichoderma was isolated from the nile valley soil in egypt with a ph range of 7.4 to 8.4. in the present study, soil ph was found to have significant influence on the relative abundance of t. koningii but not t. viride. this clearly shows that even the taxa within a genus can respond differently to the same environmental factors. eastburn and butter (1988) also reported that soil ph acta bot. croat. 73 (1), 2014 45 influence of crop species and edaphic factors on trichoderma 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:57 color profile: generic cmyk printer profile composite default screen had no significant influence on the distribution of trichoderma harzianum in a wide ph range of 6.2 to 7.9. application of ammonia to the soil was found to increase the populations of trichoderma spp. (simon et al. 1988). however, no significant relationship existed between soil nutrient contents and populations of trichoderma spp. indicating that the occurrence of trichoderma spp. is not strongly influenced by these soil nutrients, within the range encountered. this is in line with okoth et al. (2009) who also failed to find any relationship between trichoderma spp., and soil nutrients in different land use types of kenya. gherbawy et al. (2004) also reported a lack of correlation between the abundance of trichoderma to the physical and chemical properties of the soils or ph. it is therefore, possible to infer that the indigenous trichoderma spp. in the present study might have adapted to the changes in the physical and chemical properties of these cultivated soils. although there is reasonable information about the behavior of many soil fungi in their soil environment, little attention has been given to factors affecting the growth of trichoderma in field soils. factors affecting growth of trichoderma spp. on artificial cultured media have been extensively studied, but the correlation between various growth responses on agar and in soil is dubious (cook and baker 1983). though most of the abiotic factors studied did not significantly affect the abundance of trichoderma spp. except soil ph and p, some biotic factors were found to be important in determining the distribution of both t. koningii and t. viride. although the occurrence of t. koningii and t. viride was not significantly influenced by changes in the total fungal populations, the populations of the two trichoderma spp. were associated with changes in certain fungal species like absidia glauca, aspergillus fumigatus and stachybotrys atra. the existence of an inverse relation between trichoderma spp. abundance and populations of absidia glauca and stachybotrys atra is interesting as these fungi may be competing with trichoderma spp. for nutrients or space (calvet et al. 1992). there have been relatively few studies examining the competition of fungal species for nutrients, space or infection sites in the rhizosphere. competition for carbon, n and fe has been shown to be a mechanism associated with suppression of fusarium wilt by trichoderma species (whipps 2001). further, antibiotic production by isolates of trichoderma / gliocladium has been reported (howell 1998). eastburn and butler (1988) indicated species of aspergillius (a. ustus, a. tamarii) and penicillium (p. citrinum, p. chrysogenum, p. grieoroseum) to be positively related with a population of t. harzianum suggesting the tendency for the co-existence between these species. the relative abundance of t. koningii had a positive influence on the relative abundance of aspergillius fumigatus in the present study. this positive relation between populations of t. koningii and a. fumigatus could be due to the lack of antagonism or synergism among these fungi or it could be an indirect response resulting from the similar response of the two organisms to certain environmental conditions. an understanding of the organisms occupying the same microsites as t. koningii and t. viride might give us insight into what types of habitats these trichoderma spp. prefer. however, the varied responses of t. koningii and t. viride to the same environmental factors clearly explain how a species within the same genus can respond diversely to similar environmental conditions. unfortunately, there is little information available on the habitat requirements of the co-occurring species of trichoderma, especially aspergillus and penicillium especially at microsite level. the frequent occurrence of the co-occurring a. fumigatus is in agreement with attitalla et al. (2012) who also found aspergillus spp. to be more fre46 acta bot. croat. 73 (1), 2014 muniappan v., muthukumar t. 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:57 color profile: generic cmyk printer profile composite default screen quent in cultivated and non-cultivated soils of libya. interestingly, fast-growing zygomycetes were not frequent in the present study. this corroborates the general observation that fast-growing zygomycetes are not frequently isolated from agricultural soil in general, and from arable soils, in particular (hagn et al. 2003). trichoderma species are known to increase the concentration of a variety of nutrients under axenic conditions. however, the population of neither t. koningii nor t. viride was related to any soil nutrients except soil p. evidence does exist that trichoderma species can solubilize various plant nutrients such as rock phosphate, fe3+, cu2+, mn4+ and zn, that can be unavailable to plants in certain soils (harman et al. 2004a). though there may be important abiotic or biotic factors, which were not accounted for in this study, the establishment and wide-spread occurrence of trichoderma sp., appears to be more directly determined by the environment. a similar conclusion was reached by widden (1984, 1987) and eastburn and butler (1988). they found that the environment had the greatest effect on the densities of several species of trichoderma. thus, it appears that in soils where the abiotic environment is not greatly limiting, the factors of primary importance are those involving competition, antagonism and synergism. in conclusion, our study clearly showed that crop species could influence the structure of the soil fungal communities. the results of the present study also indicated that the fungal populations in the studied soils are very stable and are not easily influenced by edaphic factors. however, further studies involving a dual approach of culture-dependent and -independent techniques would throw more light on our understanding on the actual fungal diversity of the studied soils. furthermore, such a study would also help us to deduce the influence of agricultural management practices on shifts in the indigenous fungal community structure. references attitalla, i. h., abdelrawaf, s. s., omar, k. s., el-komy, h. m. a., sarwar, m., 2012: occurrence and microbiological characteristics of trichoderma in al-jabal al-akhdar region, libya. journal of biological sciences 12, 209–217. barron, g. l., 1972: the genera of hypomycetes from soil. robert e. krieger publishing company, new york. bissett, j., 1984: a revision of the genus trichoderma.i. section longibrachiatnum sect. nov. canadian journal of botany 62, 924–931. bissett, j., 1991: a revision of the genus trichoderma. ii. intrageneric classification. canadian journal of botany 60, 2357–2372. bissett, j., 1992; 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(eds.), trichoderma and gliocladium, 2. enzymes, biological control and commercial applications, 131–151. taylor and francis ltd., london. howell, c. r., 1998: the role of antibiosis in biocontrol. in: harman, g. e., kubicek, c. p. 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(eds.), pedogenesis and soil taxonomy: ii. the soil orders, 253–282. elsevier, new york. samuels, g. j., 2006: trichoderma: systematics, the sexual state and ecology. phytopathology 96, 195–206. samuels, g. j., pardo-schultheiss, r., hebbar, p., lumsden, r. d., bastos, c. n., costa, j. c., bezerra, j. l., 2000: trichoderma stromaticum sp. nov., a parasite of the cocoa witches broom pathogen. mycological research 104, 760–764. sariah, m., choo, c. w., zakaria, h., norihan, m. s., 2005: quantification and characterization of trichoderma spp. from different ecosystems. mycopathology 159, 113–117. acta bot. croat. 73 (1), 2014 49 influence of crop species and edaphic factors on trichoderma 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:57 color profile: generic cmyk printer profile composite default screen simon, a., sivasithambaram, k., macnish, g. c., 1988: effect of application of soil nitrogenous fertilizers and lime on biological suppression of gaeumannomyces graminis var. tritici. transactions of the british mycological society 91, 287–294. soil survey staff, 1994: key to soil taxonomy. sixth edition, pocahontas press., inc., blacksburg, virginia. subramainan, c. v., 1970: hypomycetes. icari, new delhi. wakelin, s. a., sivasithamparam, k., cole, a. l. j., skipp, r. a., 1999: saprophytic growth in soil of a strain of trichoderma koningii. new zealand journal of agricultural research 42, 337–345. webster, j., rifai, m. a., 1968: culture studies on hypocrea and trichoderma iv. hypocrea piluifera sp.nov. transactions of the british mycological society 51, 511–514. webster, j., 1964: culture studies on hypocrea and trichoderma i. comparison of perfect and imperfect states of h. gelatinosa, h. rufa and hypocrea sp. transactions of the british mycological society 47, 75–96. wells, h. d., 2000: trichoderma as a biochemical agent. in: mukerji, k. g., garg, k. l. (eds.), biocontrol of plant diseases, 1, 71–82. crc press inc., boca raton, florida. whipps, j. m., 2001: microbial interactions and biocontrol in the rhizosphere. journal of experimental botany 52, 487–511. widden, p., 1984: the effects of temperature on competition for spruce needles among sympatric species of trichoderma. mycologia 76, 873–883. widden, p., 1987: fungal communities in soils along an elevation gradient in northern england. mycologia 79, 298–309. 50 acta bot. croat. 73 (1), 2014 muniappan v., muthukumar t. 741 muniappan and muthukumar.ps u:\acta botanica\acta-botan 1-14\741 muniappan and muthukumar.vp 19. o ujak 2014 16:46:57 color profile: generic cmyk printer profile composite default screen opce-str.vp acta bot. croat. 68 (2), 251–262, 2009 coden: abcra 25 issn 0365–0588 diatom biodiversity in mongolia: a new amphoroid diatom from saline lakes in western mongolia, amphora soninkhishigae sp. nov. mark b. edlund1*, avery l. c. shinneman2, zlatko levkov3 1 st. croix watershed research station, science museum of minnesota, marine on st. croix, minnesota 55047, usa 2 department of geology, university of minnesota, 310 pillsbury dr. se, minneapolis, minnesota 55455, usa 3 institute of biology, faculty of natural sciences, gazi baba bb, 1000 skopje, republic of macedonia a new amphora species, amphora soninkhishigae sp. nov. is described from the saline lakes, oigon nuur and uvs nuur, in western mongolia. amphora soninkhishigae is characterized by its small size (valves 12–28 mm long, 2.9–3.8 mm wide), fine ornamentation, and a broad, internally thickened central area on the dorsal side of the valve (dorsal stauros) that branches along the dorsal margin. among the amphoroid diatoms, amphora soninkhishigae belongs in the subgenus oxyamphora cleve, where it is allied with other small amphora taxa bearing a dorsal stauros including amphora staurophora juhlin-dannfelt, amphora abludans simonsen, amphora laevissima var. perminuta grunow, and amphora laevis var. minuta cleve. key words: diatom, taxonomy, ultrastructure, biogeography, systematics, amphora soninkhishigae, mongolia introduction the amphoroid diatoms have long been recognized to be an unnatural group. early workers recognized morphological groups within "amphora" (e.g., 23 groups by smith 1873) and cleve (1895–1896) formally recognized nine groups at the subgenus level (e.g., subgenera amphora, halamphora cleve, psammamphora cleve). round et al. (1990) recognized that amphora needed revision and described the segregate marine genus seminavis d.g. mann in round et al. to accommodate the amphora angusta-group (subgenus cymbamphora cleve) and resurrected catenula mereschowsky (mereschowsky 1902– 1903) to accommodate the tiny-celled c. adhaerens and c. pelagica-group. vyverman et al. (1998) erected the new eunotioid genus eunophora, a southern hemisphere endemic that only superficially resembled amphora. williams and reid (2006) described collicuacta bot. croat. 68 (2), 2009 251 * corresponding author, e-mail: mbedlund@smm.org u:\acta botanica\acta-botan 2-09\edlund.vp 9. listopad 2009 13:11:49 color profile: disabled composite 150 lpi at 45 degrees loamphora, a new eunotioid genus, to accommodate two amphoroid taxa, including the previously known a. reichardtiana grunow. eunophora and colliculoamphora are only superficially related to amphora sensu stricto, and are more closely allied with the eunotioid diatoms. most recently, levkov (2009), in the first step toward a revision of amphora, elevated the subgenus halamphora cleve to the genus level. the amphoroid diatoms are species-rich in mongolia with more than 25 taxa reported to date (edlund et al. 2001, shinneman 2008, levkov 2009) including five taxa formally described from mongolia’s ancient lake hövsgöl (amphora mongolica østrup, a. dentata edlund et levkov, a. hovsgoliana levkov et edlund, a, neglectiformis levkov et edlund, and a. paracopulata levkov et edlund). in a recent hydrobiological survey of the valley of the great lakes in western mongolia, we undertook a first detailed study of the diatom flora from lakes in this ecologically and culturally significant region (dulmaa 1979, olson and dinerstein 1998, fernandez-gimenez 2000, shinneman et al. 2009). among the 15 amphoroid taxa reported by shinneman (2008), an unidentified amphora was noted in two saline lakes and is herein formally described. study area the valley of the great lakes lies in the far west of mongolia, bounded by the altai mountains to the west, the khangai mountains to the east, and the gobi desert to the south. the great lakes region is part of the endorheic central asian basin and includes several smaller closed drainage basins with lakes ranging from fresh to hypersaline (dulmaa 1979, shinneman et al. 2009). many of the large terminal basins in the valley are believed to be remnants of large tertiary or quaternary paleo-lakes (grunert et al. 2000). there are three large terminal basins in the area, khyargus, uvs, and uureg nuur, and numerous large and small ephemeral ponds, playa lakes, floodplain lakes, and dune-blocked lakes. lakes sampled in summer 2004–2005 ranged from dilute to hyper-saline (40–200,000 ms cm–1). saline lakes (sc>3000 ms cm–1) were common in the region (shinneman et al. 2009). terminal large lakes in the central valley were saline and many small lakes and pools were also highly saline. at the highest concentrations, ionic composition was dominated by sodium chloride, whereas dilute lakes were more commonly composed of sulfate and carbonate salts. trophic status index (tsi) calculations (carlson 1977) made using tp and secchi depth measurements indicated that most lakes, fresh and saline, were eutrophic to hyper-eutrophic at the time of sampling in late summer. nitrogen to phosphorus ratios showed that most lakes were strongly p-limited (shinneman et al. 2009). material and methods during two field seasons (august 2004 and august 2005) we sampled over 60 lakes in western mongolia. at each lake a surface sediment sample (0–1 cm sediment depth) was collected from the deepest site accessible by canoe using a line-operated "wiegner" gravity corer. the sediment was preserved in 10% formaldehyde solution and prepared for microscopy following renberg (1990). cleaned material was mounted on microslides with zrax and random transects examined with an olympus bx50 microscope (magnification 1250 times and n.a. 1.40) until 300–400 valves were counted. digital light micrographs were captured using a spot insight qe camera (diagnostic instruments). cleaned material was 252 acta bot. croat. 68 (2), 2009 edlund m. b., shinneman a. l. c., levkov z. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:34 color profile: disabled composite 150 lpi at 45 degrees also mounted on carbon coated aluminum stubs, coated with 20 nm au, and examined with a jeol jsm-6060lv scanning electron microscope (sem) at 15–20 kv operating voltage. descriptive terminology follows levkov (2009). results amphora soninkhishigae edlund, shinneman, levkov sp. nov. (figs. 1–12) holotype: marked specimen (fig. 6) on ansp slide m1468 (g.c. 36334), ansp material m1468 (g.c. 24046). isotypes: slides and material of collection m1468 deposited in california academy of sciences (m1468c), national university of mongolia diatom herbarium (m1468b), and in m.b. edlund collection (m1468a; science museum of minnesota). paratypes: slides and material of collection m1461 deposited in cas (m1461c), m1461b in national university of mongolia diatom herbarium, m1461a in m.b. edlund collection (science museum of minnesota). uvs nuur, uvs aimag, mongolia, site 63, surficial sediment from 6.3 m depth, 759 m elevation, (50.25172°n, 93.26403°e), coll: m1461 by m. edlund, a.l.c. shinneman, n. soninkhishig, 08 august 2005 (figs. 13–24). type locality: oigon nuur (lake), zavkhan aimag, mongolia, site 82, surficial sediment from 5 m water depth, 1680 m elevation, (49.20750°n, 96.61932°e), coll: m1468 by m. edlund, a.l.c. shinneman, n. soninkhishig, 12 august 2005. etymology: this taxon is named in honor of dr. n. soninkhishig, biology faculty, national university of mongolia, for her long friendship and pivotal role in developing a diatom research and training program in mongolia. descriptio: cellulae solitariae, valde dorsiventrales, aspectu cincurae lanceolatae ad anguste elipticae. valvae semilanceolatae quoad specimina maxima, ad semi-lanceolatae quoad specimina minima, margine dorsali leniter convexa, margine ventrali leviter recta vel leviter inflata. apices valvarum acuti quoad specimina maxima anguste rotundati quoad specimina minima. longitudo valvae 12–28 mm, latidudo 2.9–3.8 mm. area axialis angusta, linearis. area centralis ad latus dorsale angustam fasciam formans ad marginem valvae extensa, ad latus ventrale indistincta. ramis raphis rectis, excentrica ad marginem ventralem positis, fissuris proximalibus rectis. striae ventrales vix aspectabiles microscopio photonico, ca. 50 in 10 mm. chromatophora ignotae. description: cells solitary, frustules lanceolate to narrowly elliptical with amphoroid symmetry (fig. 24). valves semi-lanceolate in larger specimens to semi-elliptical in smaller specimens (figs. 1–12). dorsal margin convex, ventral margin straight to slightly gibbous (figs. 1–12). valve ends acute in larger specimens to narrowly rounded in smaller specimens (figs. 1–12). valve length 12–28 mm, valve breadth 2.9–3.8 mm (figs. 1–12). axial area narrow, linear (figs. 1–12). central area on dorsal side narrow fascia expanding and branching near dorsal margin, on ventral side indistinct (figs. 1–12). raphe branches straight, running near the ventral margin, proximal raphe endings straight (figs. 1–12). striae indistinct, hard to resolve with lm, ca. 50 in 10 mm. plastids unknown. ultrastructure: in the sem, frustules are lanceolate to narrowly elliptical, sometimes gibbous in the middle (fig. 25). a very narrow marginal ridge is present along the junction of the valve face and dorsal margin (fig. 27). the transition from valve face to valve mantle acta bot. croat. 68 (2), 2009 253 amphora soninkhishigae sp. nov. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:34 color profile: disabled composite 150 lpi at 45 degrees 254 a c t a b o t .c r o a t .68 (2),2009 e d l u n d m .b .,s h in n e m a n a .l .c .,l e v k o v z . figs. 1–24. light micrographs (dic) of amphora soninkhishigae. 1–12 – size diminution series from oigon nuur, mongolia (fig. 6 is the holotype). 13–23 – size diminution series from uvs nuur, mongolia. 24 – complete frustule, uvs nuur, mongolia. scale bar = 10 mm u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ e d l u n d . v p 5 . l i s t o p a d 2 0 0 9 1 5 : 5 6 : 3 6 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s is abrupt (figs. 25, 27). the raphe is located near the ventral valve margin. a distinct raphe ledge is absent; however, a slightly elevated sternum is apparent in the mid-valve on the dorsal valve side. the raphe branches are straight with simple, straight or weakly dorsally bent proximal endings and strongly dorsally deflected distal fissures (figs. 25–27). the central area on dorsal side is narrow, rectangular, and bordered with strongly shortened striae near the dorsal margin (fig. 27). these striae are composed of elongated areolae which are occluded internally ("ghost" areolae). the central area on ventral side is semi-lanceolate and extends to the ventral margin (fig. 27). striae are uniseriate, composed of small round areolae, except for a few on the central dorsal side that comprise strongly transapically elongated areolae (fig. 27). ventral striae are interrupted in the mid-valve acta bot. croat. 68 (2), 2009 255 amphora soninkhishigae sp. nov. figs. 25–30. scanning electron micrographs of a. soninkhishigae from oigon nuur, mongolia. 25 – ventral side of complete frustule. 26 – end of frustule showing dorsal deflection of terminal raphe endings (arrow). 27 – ventral side of frustule, mid-valve, showing slight dorsal deflection of proximal raphe endings, uniseriate dorsal and ventral striae, central area as a dorsal stauros with transapically elongated areolae along dorsal margin of valve (arrow). 28 – internal view of valve with linear internal raphe slits and branching dorsal stauros (arrow). 29 – internal view of valve center showing dorsal stauros that branches at the dorsal margin of valve, absence of central helictoglossae on proximal raphe ends, and internal character of striae. 30 – internal view of valve end with distal raphe ending in a small helictoglossa (arrow). scale bar: 5 mm (fig. 25), 2 mm (fig. 28), 1 mm (figs. 26, 27, 29, 30). u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:39 color profile: disabled composite 150 lpi at 45 degrees (fig. 29). internally, the most remarkable feature is the strongly thickened central area that extends costa-like as a dorsal stauros onto the dorsal mantle, as where it is branches and is even more thickened (figs. 28, 29). this feature can be observed also externally as a slight inflation in the valve middle (fig. 25). internally, the raphe branches are linear and terminate distally in poorly developed helictoglossae (fig. 30). the internal proximal raphe endings are simple (fig. 29) without tongue-like extensions (central helictoglossae). striae are uniseriate and the areolae are not occluded with hymens on the valve interior (fig. 29). diagnosis: it is the combination of characters associated with frustule shape, the linear raphe branches, and the central area or dorsal stauros that branches at the dorsal margin that differentiates amphora soninkhishigae from other amphora species. only few small-celled amphora taxa have a lanceolate-elliptical frustules with a prominent dorsal stauros similar to amphora soninkhishigae. two taxa that bear a strong resemblence to a. soninkhishigae are a. staurophora juhlin-dannfelt 1882 [=a. dannfeltii berg (berg 1952), non a. staurophora (castracane) cleve (cleve 1895–1896), the latter has rostrate valve ends] and a. laevissima var. perminuta grunow. amphora staurophora is illustrated by bérard-therriault et al. (1986: fig. 85) with elliptical frustules of length 9–14 mm, breadth 5–8 mm, and valve width of 2–3 mm, which corresponds closely to the juhlindannfelt (1882) description, but the raphe branches are slightly dorsally arched and the dorsal stauros broadens approximately mid-valve on the dorsal side without branching. in contrast, the dorsal stauros of a. soninkhishigae widens and branches only very near the dorsal margin and the raphe branches are linear. schoeman and archibald (1986: fig. 19) illustrate a specimen identified as amphora laevissima var. perminuta that is possibly conspecific with a. soninkhishigae. however, they compare it to the type of amphora laevissima var. perminuta grunow (in van heurck 1884–1887, see archibald and schoeman 1986: figs. 22, 23) which has raphe branches that are slightly dorsally arched and a dorsal staursos more similar to a. staurophora. amphora abludens simonsen (simonsen 1960) in the type description and as illustrated by bérard-therriault et al. (1986: figs. 50–52) is larger and more coarsely ornamented than a. soninkhishigae and has weakly dorsally arched raphe branches. the virgae of a. abludens also become fibulae-like along the entire dorsal margin, a character not present in a. soninkhishigae. amphora sublaevis hustedt (hustedt 1955) is more coarsely ornamented and has dorsally arched raphe branches. lastly, an illustration of amphora laevis var. minuta cleve by bérardtherriault et al. (1986: 417, fig. 76) is larger and does not have the obvious branching of the dorsal stauros seen in a. soninkhishigae. however, there is taxonomic confusion with amphora laevis var. minuta. cleve (1895–1896: 130) notes that this taxon is based on h. l. smith (1876–1888) exsiccatum no. 615 (as a. laevissima gregory); however, examination of smith’s slide (edlund unpublished) shows this taxon to have rectangular frustules, recurved raphe branches, and a convex ventral margin, which is quite different than the specimen illustrated by bérard-therriault et al. (1986: fig. 76). apart from the thickened, branching dorsal stauros, amphora soninkhishigae shares a combination of characters with amphora spec. (j) sensu lee et al. (1989: fig. 2: 9) including: straight raphe branches with weakly dorsally bent proximal raphe fissures; a weakly developed raphe ledge; dorsal striae opposite the central area composed of strongly transapically elongated areolae; and ventral striae composed of a series (2–4) of small round poroids. the internal structure of amphora spec. (j) view is not known. differences between these two taxa include valve size, with amphora spec. (j) having smaller valves 256 acta bot. croat. 68 (2), 2009 edlund m. b., shinneman a. l. c., levkov z. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:39 color profile: disabled composite 150 lpi at 45 degrees a c t a b o t .c r o a t .68 (2),2009 257 a m p h o r a s o n in k h is h ig a e s p .n o v . tab. 1. morphological and habitat characteristics of amphora soninkhishigae and allied amphora species. taxon author habitat frustule shape length frustule breadth valve breadth striae density stauros raphe branches (mm) (mm) (mm) (no/10 mm) amphora soninkhishigae edlund, shinneman et levkov 1 inland saline lakes, mongolia lanceolate to narrow-elliptical 12–28 – 2.9–3.8 50 branching near dorsal margin straight a. staurophora juhlin-dannfelt 2 baltic sea elliptical-oval 14 9.2 3.3 not resolved widens toward dorsal side slightly dorsally arched a. staurophora juhlin-dannfelt 3 gulf of st. lawrence smalllanceolate 9–14 5–8 2–3 indistinct widens toward dorsal side slightly dorsally arched a. laevissima v. perminuta grunow in van heurck 4 swansea dock, wales lanceolatetruncate ends 16–21.3 7.6–8 3–3.3 not resolved widens toward dorsal side slightly dorsally arched amphora abludens simonsen 5 baltic sea elliptical 13–30 10–14 2.5–5 38 present weakly dorsally arched amphora sublaevis hustedt 6 e. coast usa ellipticaltruncate 25–60 10–20 5–6 40 widens toward dorsal side dorsally arched amphora laevis v. minuta cleve 7 gulf of st. lawrence lanceolate 13–42 7–20 3–8 not resolved widens toward dorsal side straight amphora sp. 8 endosymbiont lanceolate 8.8 5.4 2.1 >60 not present straight authors: 1 edlund et al. (this study) 2 juhlin-dannfelt (1882, type) 3 bérard-therriault et al. (1985) 4 schoeman and archibald (1986) 5 simonsen (1960) 6 hustedt (1955) 7 bérard-therriault et al. (1895–1896) 8 lee at al. (1989) u : \ a c t a b o t a n i c a \ a c t a b o t a n 2 0 9 \ e d l u n d . v p 5 . l i s t o p a d 2 0 0 9 1 5 : 5 6 : 3 9 c o l o r p r o f i l e : d i s a b l e d c o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s (~10 mm), higher striae density (~60 per 10 mm), and different habitat preference. amphora spec. (j) is endosymbiotic in large foraminifera (lee et al. 1989). distribution and ecology: amphora soninkhishigae was abundant (>5% relative abundance) in the epipelon of only two of the survey lakes in western mongolia. in oigon nuur and uvs nuur, a. soninkhishigae was found at 13.5% and 6.25% relative abundance, respectively. it was also found in lower abundance in a third saline lake, khyargus nuur. oigon nuur is a large, shallow terminal basin with total phosphorus 0.058 mg l–1, specific conductivity 32276 ms cm–1, and ph 9.26. uvs nuur is the largest (by surface area) lake in mongolia; in 2005, it had total phosphorus of 0.023 mg l–1, specific conductivity 20363 ms cm–1, and ph 9.43. khyargus nuur had total phosphorus of 0.031 mg l–1, specific conductivity 9400 ms cm–1, and ph 9.41. discussion recent studies confirm the long-held view that amphora sensu lato is a heterogeneous group and have resulted in recognition or resurrection of several segregate genera (mereschkowsky 1902–1903, round et al. 1990, vyverman et al. 1998, williams and reid 2006). initial steps have also been taken to formally reconsider or recognize cleve’s amphora subgenera at the genus level (levkov 2009). furthermore, targeted survey efforts on several geographic regions have highlighted the underappreciated diversity of this group and resulted in the recent discovery and description of many new amphoroid taxa (nagumo 2003, levkov 2009). in mongolia, recent surveys have resulted in the description of four new amphora species from ancient lake hövsgöl (levkov 2009), numerous new distributions (edlund et al. 2001, shinneman 2008, levkov 2009), and the discovery of a. soninkhishigae in saline lakes in western mongolia. the systematic position of amphora soninkhishigae among the amphoroid diatoms must be considered. taxa that are closely allied with a. soninkhishigae, including a. staurophora, a. abludans, a. sublaevis, and a. laevis var. minuta, have generally been placed in the marine subgenus oxyamphora cleve. these taxa share lanceolate to elliptical frustules, +/– a dorsal stauros, linear to weakly dorsally arched raphe branches near the ventral margin, no longitudinal lines or keels, and fine ornamentation. these characters, in part, define the subgenus oxyamphora (see cleve 1895–1896), the subgenus where we propose a. soninkhishigae is best accommodated. however, a solid, strongly thickened central structure or dorsal stauros, which characterizes amphora soninkhishigae, is present in several species belonging to different amphoroid subgenera (sensu cleve 1895–1896). for example, in the subgenus psammamphora cleve, the central structure of amphora delphinea l.w. bailey (see metzeltin and lange-bertalot 1998: figs 145: 3 and 164: 5) continues onto the dorsal valve mantle where it is perforated by few striae. a similar structure was observed in amphora delphineiformis levkov (levkov 2009: fig. 253: 1). in members of psammamphora, the dorsal structure differs from a. soninkhishigae by continuing onto the dorsal mantle; in broken valves it appears as a structure extending and widening towards the valve mantle (levkov unpublished observation). several representatives of halamphora cleve also have a semi-stauros on the dorsal side. in amphora montana krasske the semi-stauros is spatulate and flattened internally, but it is perforated with a few strongly shortened striae 258 acta bot. croat. 68 (2), 2009 edlund m. b., shinneman a. l. c., levkov z. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:40 color profile: disabled composite 150 lpi at 45 degrees (carter and round 1993: figs. 25, 26). similar features can also be observed in a. normanii rabenhorst (carter and round 1993: fig. 35). additional features aligning amphora soninkhishigae with oxyamphora include lack of tongue-like extensions (central helictoglossae sensu levkov 2009) on the internal proximal raphe endings. in most halamphora species, the proximal raphe endings terminate internally with fused central helictoglossae, except in a. chilensis (sala et al. 2007), whereas in amphora sensu stricto (i.e. subgenus amphora cleve) each raphe branch terminates with separate central helictoglossae (see krammer 1980). in some, but not all representatives of other subgenera (e.g. oxyamphora, diplamphora and psammamphora) the proximal raphe endings are also simple without extensions. on the other hand, in representatives of psammamphora and oxyamphora the raphe distally terminates with fully developed helictoglossae, a feature that is poorly developed in a. soninkhishigae. lastly, in amphora soninkhishigae the raphe lies in a weakly elevated external raphe ledge that is present only dorsally, a feature shared by the subgenera halamphora and oxyamphora (archibald and barlow 1983). in most subgenus amphora taxa the raphe lies in a raphe ledge elevated along both sides (guslyakov 1985: figs. a, b). a strongly developed raphe ledge on both valve sides is also present in representatives of subgenus diplamphora (guslyakov 1985: fig. 1), while in subgenus psammamphora this structure is absent. what is perhaps more intriguing is that a member of the subgenus oxyamphora can be found in mongolia over 2000 km from any marine habitat. oxyamphora taxa are generally considered marine, although some representatives inhabit estuarine habitats (bérard-therriault et al. 1986). inland or athallassic saline lakes, although not always sodium chloride-dominated systems, can provide inland habitats for more typical marine species of microalgae. the larger mongolian saline lakes where amphora soninkhishigae was found are sodium chloride-dominated but also have ample magnesium and sulfate ion content. typical marine diatom taxa such as achnanthes brevipes c.agardh, melosira moniliformis var. octogona (grunow) hust., and haslea spicula (hickie) bukht. can be found in mongolia’s valley of the great lakes (edlund et al. 2001). other saline amphora species in mongolia include amphora commutata grunow in van heurck, a. coffeaeformis (c.agardh) kütz., and a. coffeaeformis var. angularis (van heurck) cleve; however, a. soninkhishigae is the first member of subgenus oxyamphora reported from mongolia. the valley of the great lakes is a culturally and ecologically significant region (dulmaa 1979, olson and dinerstein 1998, fernandez-gimenez 2000) that is currently under threat from climate warming and political and economic drivers of change (shinneman 2008). people of the region follow the millennia-old traditions of pastoral nomadism; however, increases in herd size, changes in herd makeup, and more sedentary households have resulted in increased eutrophication of surface waters in the region (shinneman 2008). the valley is also home to several critically endangered wildlife species and an important stopover for migratory birds (olson and dinerstein 1998). threats to the ecology and culture of the region make biodiversity surveys, especially those targeting important bioindicator groups, critical and time-sensitive. initial results from our hydrobiological surveys indicate that the aquatic biodiversity of this region of mongolia can be used for assessment of modern and historical ecological assessment (shinnemann et al. 2009), and that the biodiversity is underappreciated, as evidenced by the discovery of new diatom taxa (shinneman 2008) including amphora soninkhishigae. acta bot. croat. 68 (2), 2009 259 amphora soninkhishigae sp. nov. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:40 color profile: disabled composite 150 lpi at 45 degrees acknowledgements we thank our mongolian colleagues, drs n. soninkhishig, y. khand, and g. tserenkhand, and especially our drivers, cooks, and field crew for their help and generosity during two field expeditions. we thank dr. matthew julius, st. cloud state university, for providing scanning electron microscope facilities. this material is based upon work supported by the national science foundation (nsf) under grants deb-0316503 and deb-0431529 to mbe. any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the nsf. dr. levkov acknowledges support from the alexander von humboldt foundation. references archibald, r. e. m., barlow, d. j., 1983: on the raphe ledge in the genus amphora (bacillariophyta). bacillaria 6, 257–266. bérard-therriault, l., cardinal, a., poulin m., 1986: les diatomées (bacillariophyceae) benthiques de substrats durs des eaux marines et saumâtres du québec. 6. naviculales: cymbellaceae et gomphonemaceae. le naturaliste canadien 113, 405–429. berg, å., 1952: eine diatomeengemeinschaft an der schwedischen ostküste. arkiv för botanik, series 2, 2, 1–39. carlson, r. e., 1977: a trophic state index for lakes. limnology and oceanography 22, 361–369. carter, j. r., round, f. e., 1993: studies on freshwater amphora species. v. a. montana and a. normanii. diatom research 8, 1–11. cleve, p. t., 1895–1896: synopsis of the naviculoid diatoms. kungliga svenska vetenskaps-akademiens handlingar, 27, 1–220. dulmaa, a., 1979: hydrobiological outline of the mongolian lakes. international revue der gesamten hydrobiologie 64, 709–736. edlund, m. b., soninkhishig, n., williams, r. m., stoermer, e. f., 2001: biodiversity of mongolia: checklist of diatoms, including new distributional reports of 31 taxa. nova hedwigia 72, 59–90. fernandez-gimenez, m. e., 2000: the role of mongolian nomadic pastoralists’ ecological knowledge in rangeland management. ecological applications 10, 1318–1326. grunert, j., lehmkuhl, f., walther, m., 2000: paleoclimatic evolution of the uvs nuur basin and adjacent areas (western mongolia). quaternary international 65, 171–192. guslyakov, n. e., 1985: o morfologii pantsirei nekotorykh predstabitelei roda amphora (bacillariophyta) from the black sea. botanicheskii zhurnal 70, 1478–1481. hustedt, f., 1955: marine littoral diatoms of beaufort, north carolina. duke university press, durham, north carolina. juhlin-dannfelt, h., 1882: on the diatoms of the baltic sea. academical dissertation. kongl. boktryckeriet, p.a. norstedt and söner, stockholm. krammer, k., 1980: morphologic and taxonomic investigations of some freshwater species of the diatom genus amphora ehr. bacillaria 3, 197–226. 260 acta bot. croat. 68 (2), 2009 edlund m. b., shinneman a. l. c., levkov z. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:40 color profile: disabled composite 150 lpi at 45 degrees lee, j. j., mcenergy, m. e., ter kuile, b., erez, j., roetger, r., rockwell, r. f., faber, w., lagziel, a., 1989: identification and distribution of endosymbiotic diatoms in larger foraminifera. micropaleontology 35, 353–366. levkov, z., 2009: amphora sensu lato. in: lange-bertalot, h. (ed.), diatoms of europe: diatoms of european inland waters and comparable habitats elsewhere. koeltz, koenigstein. mereschkowsky, c., 1902–1903: le types de l’endochrome chez les diatomées. scripta botanica, horti universitalis imperialis petropolitanae 21, 1–106 (in russian); 107–193 (in french). metzeltin, d., lange-bertalot, h., 1998: tropical diatoms of south america, 1. iconographica diatomologica 5, 1–695. metzeltin, d., lange-bertalot, h., garcia-rodriguez, f., 2005: diatoms of uruguay. iconographica diatomologica 15, 1–737. nagumo, t., 2003: taxonomic studies of the subgenus amphora cleve of the genus amphora (bacillariophyceae) in japan. bibliotheca diatomologica 49, 1–265. olson, d. m., dinerstein, e., 1998: the global 200: a representation approach to conserving the earth’s most biologically valuable ecoregions. conservation biology 12, 502–515. renberg, i., 1990: a procedure for preparing large sets of diatom slides from sediment cores. journal of paleolimnology 4, 87–90. round, f. e., crawford, r. m., mann, d. g., 1990: the diatoms. biology and morphology of the genera. cambridge university press, united kingdom. sala, s., guerrero, j. m., coste, m., 2007: valve morphology of amphora chilensis hustedt (bacillariophyceae). nova hedwigia 85, 353–364. schoeman, f. r., archibald, r. e. m., 1986: observations on amphora species (bacillariophyceae) in the british natural history museum i. some species from the subgenus oxyamphora cleve. nova hedwigia 43, 113–127. shinneman, a. l. c., 2008: climatic and anthropogenic influences on aquatic ecosystems in the valley of the great lakes, mongolia. ph.d. thesis university of minnesota, minneapolis, minnesota. shinneman, a. l. c., edlund, m. b., almendinger, j. e., soninkhishig, n., 2009: diatoms as indicators of water quality in western mongolian lakes: a 54-site calibration set. journal of paleolimnology. 42, 373–389. simonsen, r., 1960: neue diatomeen aus der ostsee ii. kieler meeresforschungen 16, 126–130. smith, h. l. 1873: conspectus of the diatomaceae. analysis of the species of the genus amphora. the lens 2, 65–91. smith, h. l., 1876–1888: diatomacearum species typicae. centuries 1–6, numbers 1–600; supplement numbers 601–750. stodder, boston. van heurck, h., 1884–1887: types de synopsis de belgique. serie 1–22. 550 slides, text of a. grunow. acta bot. croat. 68 (2), 2009 261 amphora soninkhishigae sp. nov. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:40 color profile: disabled composite 150 lpi at 45 degrees vyverman, w., sabbe, k., mann, d., vyverman, r., hodgson, d., vanhoutte, k., kilroy, c., 1998: eunophora gen. nov. (bacillariophyta), a new genus of amphoroid diatoms from tasmanian highland lakes with close affinities to the genus eunotia. european journal of phycology 33, 95–111. williams, d. m., reid, g., 2006: fossils and the tropics, the eunotiaceae (bacillariophyta) expanded: a new genus for the upper eocene fossil diatom eunotia reedi and the recent tropical marine diatom amphora reichardtiana. european journal of phycology 41, 147–154. 262 acta bot. croat. 68 (2), 2009 edlund m. b., shinneman a. l. c., levkov z. u:\acta botanica\acta-botan 2-09\edlund.vp 5. listopad 2009 15:56:40 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 71 (1), 31–50, 2012 coden: abcra 25 issn 0365-0588 eissn 1847-8476 classification of mesic grasslands and their transitions of south transdanubia (hungary) attila lengyel1,*, dragica purger2, jános csiky3 1 department of plant systematics, ecology and theoretical biology, eötvös loránd university, pázmány p. s. 1/c, h-1117 budapest, hungary 2 south transdanubian environment protection institute, köztársaság tér 7, h-7623 pécs, hungary 3 department of plant taxonomy and geobotany, university of pécs, ifjúság u. 6, h-7624 pécs, hungary abstract – relevés from meadows and pastures of south transdanubia (hungary) are evaluated by clustering and ordination methods. the relevé selection focused on the arrhenatheretalia order but its transitions towards other types were also included. the groups of relevés are delimited and described according to differential, dominant and constant species. ecological conditions of the groups were compared using indicator values. nine groups were distinguished, four of them belonging strictly to the order arrhenatheretalia. each alliance of arrhenatheretalia presented in the study area (cynosurion, arrhenatherion) was represented by two groups. groups from these two alliances are separated along a light gradient, while groups of the same alliance differ in nutrient values. within cynosurion, the nutrient-poor group cannot be identified unambiguously as any syntaxa previously known from hungary. the nutrient-rich cynosurion meadows are similar to lolio–cynosuretum, however, they show a stronger relationship with wet meadows. within arrhenatherion, pastinaco–arrhenatheretum is recognised as a hay meadow of nutrient-rich soils. the other meadow type is similar to filipendulo–arrhenatheretum, thus raising syntaxonomical problems. there are transitional groups towards semi-dry and wet meadows, one dynamic phase and one outlier group among the other five clusters. keywords: vegetation, meadows, pastures, indicators, phytosociology, syntaxonomy introduction mesophilous grasslands are among the most species-rich herbaceous vegetation types in hungary. their presence is linked with the traditional agricultural system but with the intensification of farming, the area of these valuable habitats is decreasing. it is an urgent acta bot. croat. 71 (1), 2012 31 * corresponding author, e-mail: lengyelat@caesar.elte.hu copyright® 2012 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:05 color profile: disabled composite 150 lpi at 45 degrees challenge to reveal and document the diversity of mesic grasslands in order to help their conservation. despite their conservation importance, mesophilous meadows are a neglected category in the hungarian vegetation classification. according to the current syntaxonomy (borhidi 2003), mesophilous meadows are included into the arrhenatheretalia order. this order is represented by three alliances in hungary: arrhenatherion comprising lowland and colline hay meadows, cynosurion covering lowland to montane pastures and phyteumo–trisetion comprising montane hay meadows; the last one is certainly not present in south transdanubia. previously, works aimed at synthesizing the variability of the arrhenatheretalia order (soó 1971, 1973; borhidi 2003) were based on data collected unevenly from local or subregional studies (e.g. juhász-nagy 1959; máthé and kovács 1960; jeanplong 1960; kovács 1994, 2002; lájer 2002) without the application of more objective methods. there have been a few papers dealing with the meadows of the southern part of transdanubia. however, they either only focus on the description of the stands of a restricted area or meadow type (dénes 1997, lájer 2002) or their sampling and analytical methodology is unclear (horvát 1962, 1972). there are more comparative publications about other meadow types which have transitions towards mesic meadows. a classification of semi-dry grasslands has recently been prepared by illyés et al. (2009) either exclusively for hungary, or together with other central european countries as well (illyés et al. 2007). the classification of wet meadows of hungary has been clarified by botta-dukát et al. (2005). in a few neighbouring countries, the syntaxonomy of mesophilous meadows has been investigated more thoroughly. the mesic meadows of austria have been classified by mucina et al. (1993) and ellmauer (1994). in slovakia, a classification has been proposed by uhliarová et al. (2007) and jani[ová et al. (2007) and recently refined by focusing on the western carpathians by rozbrojová et al. (2010). in romania meadow communities are reviewed by coldea et al. (1991) and sanda et al. (1999). short descriptions of croatian meadow associations are presented in trinajsti] (2008), while recent broad-scale classification studies have been published by stan^i] (2000, 2008). there are papers containing reviews of previously surveyed meadows and pastures in serbia (e.g. koji] et al. 2004, bla@en^i] et al. 2005), yet they have not been evaluated nor have their results been synthesised. some brief analyses are available for slovenia (e.g. ^arni 2001, zelnik 2007, zelnik and ^arni 2008). our aim is to prepare the classification of the meadows belonging to the arrhenatheretalia order in south transdanubia and to relate our results to the existing syntaxonomical system. we intend both to differentiate among associations of this order and to delimit them from types of other orders. materials and methods study area the study area is situated in the southern part of hungary (fig. 1). the major, northern part of the area belongs to the south transdanubian geographic district, while the southern lowlands (e.g. drava plain) are parts of the great hungarian plain (dövényi 2010). for simplicity, hereafter we use the term 'south transdanubia' for the entire study area, including its plain subregion. the southern border of the examined area is the drava river and the 32 acta bot. croat. 71 (1), 2012 lengyel a., purger d., csiky j. u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:06 color profile: disabled composite 150 lpi at 45 degrees national border. the other limits are defined according to the distribution of mesophilous meadows (molnár et al. 2008), which approximately match the 700 mm isohyet in the east and in the north, and are delimited by the dominance of sand substratum in the west (belsõ-somogy). at the northern part of the study range there are the mecsek mts, which consist of several kinds of bedrock (mainly jurassic and triassic limestone). this insular mountain is surrounded by hills covered by young (pannonian, pleistocene) sediments. south from this colline-montane landscape lies the alluvial plain of drava river. the highest peak of the area is 682 m in the mecsek mts, while the lowest point is 87 m on the drava plain. there is a climatic continentality gradient increasing from the northwest towards the southeast (lovász 1977). the study range is influenced by the sub-mediterranean climate the most strongly in hungary. the potential vegetation of the sites where field sampling was carried out is deciduous forest, dominated by quercus spp., carpinus betulus and fagus sylvatica. some sites located along the drava are on drained felled alluvial forests with quercus robur, fraxinus angustifolia and carpinus betulus. vegetation data field sampling was carried out from mid-may to late-june between 2004 and 2009. in total, 211 phytosociological relevés were collected according to the zürich-montpellier method (dengler et al. 2008). plots were located in vegetation stands corresponding with the mesic meadow categories (e1, e2, e34) of the hungarian habitat guide (bölöni et al. 2003) which is also used for the recently finished country-wide habitat mapping project (molnár et al. 2007). according to this system, the category of mesic meadows is somewhat wider than that of the syntaxonomic system. in terms of syntaxonomy, this allows the inclusion of violion caninae grasslands and transitional stands towards brometalia erecti and molinietalia meadows, beyond arrhenatheretalia which forms the core of this vegetaacta bot. croat. 71 (1), 2012 33 mesic grasslands of south transdanubia fig. 1. the study area u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:07 color profile: disabled composite 150 lpi at 45 degrees tion type. during sampling, the authors preferred stands under regular and traditional management (grazing, mowing) and avoided obvious within-plot heterogeneity in factors affecting vegetation (e.g. small-scale disturbances). however, finding 'typical' stands according to the previously described associations was not aimed at since we wanted to represent the total variation as much as possible. relevés were taken in 4×4 or 5×5 m2 plots which are the recommended sizes for this vegetation type (lájer et al. 2008). species cover was estimated on a percentage scale but they were log-transformed before analysis. poorly distinguishable taxa were merged (a list is available from the authors upon request). the data set of 211 relevés was stratified geographically (knollová et al. 2005) into ca. 3×3 km2 grid squares and 5 relevés were selected from each cell using the heterogeneity-constrained random resampling method (hcr, lengyel et al. 2011). the number of trials in hcr resampling was 10 000 and the complement of the ruzicka index (which is the jaccard index generalized to abundance data, podani 2000) was used for calculating pair-wise dissimilarities between plots. the resampled data set consisted of 155 relevés. data analysis before classification, a noise elimination procedure was applied according to botta-dukát et al. (2005). the data set was analysed by principal coordinates analysis (applying the ruzicka index again) and the scores of the relevés on the significant ordination axes were used as input variables for classification. the first 19 axes were considered significant because their eigenvalues exceeded the expectations based on the broken stick model (legendre and legendre 1998). relevé coordinates (155 relevés as objects, each with 19 scores as variables) were classified based on their pair-wise euclidean distances using ward's agglomerative method (podani 2000). the optimal level of the clustering was determined by the optimclass 1 method (tichý et al. 2010) using p<10–3 threshold for characteristic species. correspondingly, the dendrogram was cut at the nine-cluster level and relevé groups were described by their differential, constant and dominant species (dengler et al. 2008). species with high affinity to a cluster were treated as differential species rather than truly characteristic species because in a wider comparison fidelity values may change significantly. species fidelities to clusters were measured by the phi coefficient (chytrý et al. 2002) adjusted to equal group sizes (tichý and chytrý 2006) and those reaching phi = 0.2 and p<10–3 significance by fisher's exact test were considered differential. species occurring in at least 80% of the plots of a cluster were listed among the constant species. a species was considered dominant if it reached 30% cover in at least 10% of the relevés of a given cluster. unweighted mean ecological indicator values (borhidi 1995, horváth et al. 1995) were calculated for each plot and clusters were compared on box-and-whisker plots. relationships of clusters were also examined by ordination. canonical analysis of principal coordinates (anderson and willis 2003) with cluster memberships as nominal explanatory variables was applied in order to concentrate more on cluster separation. ordination results are shown on spider plots which represent ordination and classification together. each relevé is linked to the centroid of its group on the scatter plot, thus the diagram resembles spiders with 'legs' starting from the centroid (the 'body') and ending in the relevés' positions. mean indicator values of relevés were passively projected onto the diagram. the goodness of fit of the projection was estimated by a permutation test. 34 acta bot. croat. 71 (1), 2012 lengyel a., purger d., csiky j. u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:07 color profile: disabled composite 150 lpi at 45 degrees all analyses were performed in r environment (version 2.10.0, r development core team 2009), using vegan (oksanen et al. 2010) and cluster (maechler et al. 2005) packages. the nomenclature of plant species follows király (2009). results description of groups the dendrogram of relevés is shown in figure 2. differential, constant and dominant species are summarized in tables 1, 2 and 3 respectively. group 1 number of relevés: 31; mean species number per relevé: 41.0. most relevés of group 1 originate from moderately-grazed extensive pastures from hilltops and hillsides. in the syntaxonomic system, this type would be placed into the cynosurion alliance within the arrhenatheretalia order. beyond typical elements of mesic meadows, these stands are also characterised by species tolerating nutrient-poor and dry soil (e.g. agrostis capillaris, anthoxanthum odoratum, festuca pseudovina & valesiaca, f. rubra, hypochoeris radicata). group 2 this group consists of two outlier relevés, and has therefore not been interpreted. group 3 number of relevés: 24; mean species number per relevé: 37.0. relevés classified to this group were taken in stands similar to but more disturbed than those of group 1. the most common forms of disturbance are overgrazing, drying and soil erosion with additional leaching. some of the stands are probably old fields which are irregularly or just recently mown or grazed. accordingly, there are many weeds among the differential species of this group (e.g. convolvulus arvensis, trifolium arvense). we consider this type a dynamic state of other, more regularly managed mesic meadows, mostly of cynosurion types. acta bot. croat. 71 (1), 2012 35 mesic grasslands of south transdanubia fig. 2. dendrogram of relevés u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:08 color profile: disabled composite 150 lpi at 45 degrees 36 acta bot. croat. 71 (1), 2012 lengyel a., purger d., csiky j. tab. 1. differential species of the groups with the values of phi coefficients. group: 1 2 3 4 5 6 7 8 9 centaurium erythraea 0.49 ––––––––leontodon autumnalis 0.37 ––––––––holcus lanatus 0.36 ––––––––festuca pseudov. & valesiaca 0.36 ––––––––anthoxanthum odoratum 0.32 ––––––––trifolium dubium 0.31 ––––––––agrostis capillaris 0.31 ––––––––luzula campestris 0.29 –––––0.26 ––festuca rubra 0.29 ––––––––daucus carota 0.28 ––––––––cynosurus cristatus 0.28 ––––0.42 –––trisetum flavescens 0.28 –––––0.27 ––trifolium pratense 0.27 ––––––––picris hieracioides 0.26 –0.29 ––––––lotus corniculatus 0.26 ––––––––ranunculus bulbosus 0.26 ––––––––trifolium repens 0.25 ––––––––hypochoeris radicata 0.21 ––––––––leontodon hispidus 0.20 ––––––––centaurea stoebe –1.00 –––––––danthonia decumbens –0.98 –––––––peucedanum oreoselinum –0.95 –––––––sherardia arvensis ––0.55 ––––––moenchia mantica ––0.49 ––––––trifolium arvense ––0.48 ––––––odontites vernus ––0.43 ––––––trifolium campestre ––0.42 ––––––agrimonia eupatoria ––0.41 ––––––convolvulus arvensis ––0.39 ––––––geranium columbinum ––0.38 ––––––ranunculus polyanthemos –––0.62 –––––festuca rupicola –––0.48 –––––pimpinella saxifraga –––0.44 –––––pastinaca sativa –––0.44 –––––medicago falcata –––0.41 –––––thymus glabrescens ––––0.93 ––––sanguisorba minor ––––0.88 ––––brachypodium pinnatum ––––0.88 ––––teucrium chamaedrys ––––0.78 ––––trifolium montanum ––––0.76 ––––thesium linophyllon ––––0.76 ––––u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:08 color profile: disabled composite 150 lpi at 45 degrees acta bot. croat. 71 (1), 2012 37 mesic grasslands of south transdanubia group: 1 2 3 4 5 6 7 8 9 vicia tenuifolia ––––0.76 ––––koeleria cristata ––––0.76 ––––centaurea scabiosa ––––0.73 ––––dianthus pontederae ––––0.69 ––––asperula cynanchica ––––0.68 ––––lathyrus latifolius ––––0.61 ––––peucedanum cervaria ––––0.61 ––––anthyllis vulneraria ––––0.61 ––––onobrychis viciifolia ––––0.61 ––––salvia pratensis ––––0.61 ––––plantago media ––––0.59 ––––lolium perenne –––––0.63 –––agrostis stolonifera –––––0.62 –––cynodon dactylon –––––0.56 –––bromus racemosus agg. –––––0.50 –––carex distans –––––0.44 –––bellis perennis –––––0.43 –––verbena officinalis –––––0.42 –––filipendula vulgaris ––––––0.74 ––betonica officinalis ––––––0.57 ––carex pallescens ––––––0.49 ––campanula patula ––––––0.48 ––sanguisorba officinalis ––––––0.47 ––vicia cracca ––––––0.40 ––lathyrus pratensis ––––––0.40 ––cruciata glabra ––––––0.40 ––knautia drymeia ––––––0.40 ––rumex acetosa ––––––0.38 ––saxifraga bulbifera ––––––0.36 ––colchicum autumnale ––––––0.31 ––carex hirta ––––––0.29 ––crepis biennis –––––––0.28 –ranunculus acris –––––––0.26 0.40 ranunculus repens ––––––––0.71 symphytum officinale ––––––––0.68 cirsium canum ––––––––0.64 sonchus arvensis ––––––––0.60 lychnis flos-cuculi ––––––––0.56 lysimachia vulgaris ––––––––0.47 potentilla reptans ––––––––0.46 poa trivialis ––––––––0.41 tab. 1. – continued u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:08 color profile: disabled composite 150 lpi at 45 degrees group 4 number of relevés: 9; mean species number per relevé: 42.6. this cluster contains transitional relevés towards semi-dry meadows, mainly from lowlands. syntaxonomically this group should be interpreted as a cluster of transitional stands between arrhenatheretalia and brometalia erecti orders. its stands are mown, grazed or overgrazed, but species-rich. differential and dominant species are generalists and disturbance tolerants which occur both on mesic and xeromesic meadows (e.g. festuca rupicola, medicago falcata, pimpinella saxifraga, ranunculus polyanthemos). 38 acta bot. croat. 71 (1), 2012 lengyel a., purger d., csiky j. tab. 2. constant species of the groups with percentage frequency values. group: 1 2 3 4 5 6 7 8 9 anthoxanthum odoratum 97 100 ––––83 ––trifolium pratense 97 –––––––85 holcus lanatus 94 100 –––––––lotus corniculatus 94 ––––––––plantago lanceolata 94 100 83 89 –––––trisetum flavescens 94 –––––93 ––achillea millefolium agg. 90 100 88 100 80 92 –93 85 galium verum 90 –92 100 –83 100 83 85 poa pratensis agg. 87 –100 –80 83 83 93 100 daucus carota 84 ––––––––veronica chamaedrys 81 –––––90 83 –agrostis capillaris –100 –––––––ambrosia artemisiifolia –100 –––––––centaurea stoebe –100 –––––––danthonia decumbens –100 –––––––helictotrichon pubescens –100 –––––––peucedanum oreoselinum –100 –––––––festuca rupicola –––100 80 ––––pimpinella saxifraga –––89 –––––brachypodium pinnatum ––––100 ––––plantago media ––––100 ––––salvia pratensis ––––100 ––––thymus glabrescens ––––100 ––––trifolium montanum ––––100 ––––dianthus pontederae ––––80 ––––sanguisorba minor ––––80 ––––teucrium chamaedrys ––––80 ––––alopecurus pratensis ––––––86 83 92 betonica officinalis ––––––86 ––lathyrus pratensis ––––––83 ––ranunculus acris –––––––80 100 potentilla reptans ––––––––100 u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:08 color profile: disabled composite 150 lpi at 45 degrees group 5 number of relevés: 5; mean species number per relevé: 32.4. group 5 is a semi-dry meadow group of the brometalia order present in the mecsek mts. stands are grazed or mown, and harbour far less species than the previous, transitional group. this cluster is characterised by dry grassland species (e.g. asperula cynanchica, dianthus pontederae, teucrium chamaedrys, vicia tenuifolia). the syntaxonomic position of groups 4 and 5 should be discussed in more detail in an analysis focusing on both semi-dry and mesic meadows. group 6 number of relevés: 12; mean species number per relevé: 30.5. relevés of group 6 originate from intensively grazed lowland pastures with nutrient-rich and well-watered soils. these plots have the lowest species numbers. the differential species of this group (e.g. agrostis stolonifera agg., cynosurus cristatus, lolium acta bot. croat. 71 (1), 2012 39 mesic grasslands of south transdanubia tab. 3. dominant species of the groups with percentage frequency values. group: 1 2 3 4 5 6 7 8 9 lotus corniculatus 23 ——11 —————trifolium pratense 19 ———————23 agrostis capillaris 16 50 ———————trisetum flavescens 16 ————————festuca rubra 10 ——————10 —holcus lanatus 10 —————17 —15 leontodon hispidus 10 ————————danthonia decumbens —50 ———————helictotrichon pubescens —50 ————10 ——peucedanum oreoselinum —50 ———————festuca rupicola ——25 22 40 ————poa pratensis agg. ——17 ————23 15 arrhenatherum elatius ———11 ———23 —brachypodium pinnatum ———11 40 ————cynodon dactylon ———11 —————festuca pratensis ———11 ———17 69 medicago falcata ———11 —————securigera varia ———11 —————teucrium chamaedrys ————40 ————bromus erectus ————20 ————vicia tenuifolia ————20 ————trifolium repens —————42 ———bromus racemosus agg. —————17 ———carex distans —————17 ———alopecurus pratensis ——————10 17 —centaurea jacea ——————10 ——u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:08 color profile: disabled composite 150 lpi at 45 degrees perenne) tolerate grazing and trampling well but demand nutrient-rich and semi-humid or humid soil. most of the sites are located on the drava plain where they probably originated from the overgrazing and drying of wet meadows. in the syntaxonomical system this group belongs to the cynosurion alliance. group 7 number of relevés: 29; mean number of species per relevé: 40.7. group 7 contains special, transitional meadows that belong to the arrhenatherion alliance of the arrhenatheretalia order. these stands are characterized by the co-existence of species that tolerate temporary drought and flood as well (e.g. betonica officinalis, filipendula vulgaris, helictotrichon pubescens) and moisture-demanding species (e.g. alopecurus pratensis, carex pallescens, sanguisorba officinalis). besides grassland species generally characterising the mesophilous meadows (e.g. campanula patula, rumex acetosa, saxifraga bulbifera, trisetum flavescens), plants of forest edges are also present (e.g. cruciata glabra, knautia drymeia). this variety of species characters is explained by these meadows usually being situated on packed soils with a fluctuating water supply, because of which the moist and dry periods alternate over the year. these meadows resemble drying molinion coeruleae stands, however, the latter is a moister grassland type. moreover, many of these meadows are both grazed and mown, which also contributes to their diversity. these are ones of the most species-rich meadows of the region. this type is distributed in lowlands and hills. group 8 number of relevés: 30; mean number of species per relevé: 34.6. this cluster consists of relevés from regularly mown, productive, tall grass stands. these meadows grow on nutrient-rich and steadily humid soils from the lowlands to the mecsek mts. they are 'typical' oat grass meadows, which harbour many generalist grassland species but have very few differential species (e.g. crepis biennis, ranunculus acris). in the syntaxonomical system, this group corresponds to the pastinaco–arrhenatheretum association, which is distributed widely in other central european countries as well. this group has transitions to almost all the others. from our preliminary analysis, we found that this group could have been delimited differently, which would slightly alter its syntaxonomic interpretation, depending on how transitional stands are classified. group 9 number of relevés: 13; mean number of species per relevé: 33. relevés classified into group 9 originate from the most humid sites, and should be considered as members of molinietalia meadows. these stands are located in lowlands and valleys and they can be flooded in spring, which is not usual for mesic (arrhenatheretalia) but typical for wet (molinietalia) grasslands. differential species are wet meadow plants (e.g. cirsium canum, poa trivialis, ranunculus repens, symphytum officinale). since this group is at the periphery of the scope of our analysis, it is not discussed in more detail here. indicator values and ordination box-and-whisker plots reveal the strong outlier character of group 5 because it considerably differs from all other groups regarding all indicator values (figs. 3, 4, 5). other 40 acta bot. croat. 71 (1), 2012 lengyel a., purger d., csiky j. u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:09 color profile: disabled composite 150 lpi at 45 degrees groups show gradual differences in temperature (fig. 3a), soil reaction (fig. 4a) and continentality (fig. 5b) values because the positions of the medians and the proportions of overlaps resemble random patterns. in contrast, groups are aggregated into 'levels' along the gradients of moisture, nutrient and light values. groups on the same level have similar medians and highly overlapping interquartile ranges, while the levels differ significantly from each other. in moisture values (fig. 3b), between the two extremes (group 5 and group 9, dry and wet, respectively) a drier level consisted of groups 1, 3 and 4, while a moister level is formed of groups 6, 7 and 8. there are two levels along the nutrient gradient as well (fig. 4b). more nutrient-rich clusters are groups 6, 8 and 9, while groups 1, 3, 4 and 7 have on average lower nutrient status. two levels are differentiated according to light availability values (fig. 5a). groups 1, 3, 4 and 6 are characterised by more light, and acta bot. croat. 71 (1), 2012 41 mesic grasslands of south transdanubia fig. 3. box-and-whisker plots of mean temperature (a) and moisture (b) indicator values of clusters. tb – borhidi's temperature values, wb – borhidi's moisture values. fig. 4. box-and-whisker plots of mean soil reaction (a) and nutrient status (b) indicator values of clusters. rb – borhidi's soil reaction values, nb – borhidi's nutrient demand values. u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:12 color profile: disabled composite 150 lpi at 45 degrees groups 7, 8 and 9 with less. this pattern corresponds with their differences in management because relevés of groups 1, 3, 4, 5 and 6 originate mostly from grazed sites but the others from mown meadows. the relative positions of the groups can be seen on the ordination diagram (fig. 6). the first canonical axis explains 23.6% of the within-group variation, which is 5.0% of the total variation. the second axis of canonical analysis of principal coordinates accounts for 20.9% of the canonical variation, which is 4.5% of the total variation of species data. indicator val42 acta bot. croat. 71 (1), 2012 lengyel a., purger d., csiky j. fig. 5. box-and-whisker plots of mean light availability (a) and continentality (b) indicator values of clusters. lb – borhidi's light demand values, cb – borhidi's climatic continentality fig. 6. canonical analysis of principal coordinates ordination diagram with spider plot of the clusters and with passively projected mean indicator values u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:14 color profile: disabled composite 150 lpi at 45 degrees ues such as: temperature indicator value, moisture, nutrient status and light availability are projected onto the scatterplot with high (p<0.001) continentality indicator value, with weaker but still significant (p<0.05) fit, while soil reaction could not be fitted to the first two axes (tab. 4). moisture and nutrient status correlate with both ordination axes, temperature indicator value and light availability with only the first axis, continentality indicator value with only the second axis. the ordination diagram supports the importance of moisture, nutrient status and light availability gradients in the species composition. discussion ours is the first attempt on the classification of hungarian mesophilous meadows using multivariate methods, even if our work focuses on a minor part of the country. therefore, our syntaxonomic results generate rather new questions and hypotheses about the current syntaxonomic system. from our classification four clusters emerged as important types certainly belonging to the arrhenatheretalia order. two of these four clusters belong to the cynosurion alliance (group 1, group 6) and the other two to arrhenatherion (group 7, group 8). these alliances are separated along the first axis of the canonical principal coordinate ordination that corresponds also with light availability (fig. 6), with groups of cynosurion getting higher mean light availability values. this is in line with the differences in their management (i.e. grazing for cynosurion and mowing for arrhenatherion), and accordingly borhidi's light values seem to differentiate well between grazed and mown communities. within both alliances, clusters differ in nutrient status (figs. 4b, 6). these patterns were more or less incorporated previously in the syntaxonomic concept but species compositions of the groups were not in accordance with the association descriptions in every case. in the hungarian syntaxonomic system (borhidi 2003), mesophilous pastures on nutrient-poor soils are included in the festuco commutatae–cynosuretum distributed in colline and montane areas, which contains species associated with the montane climate in hungary (carlina acaulis, rhinanthus alectorolophus, rh. wagneri, danthonia decumbens, alchemilla spp.). however, even the highest altitudes of our study area do not reach the montane belt and most of the montane characteristic species of the above association are not present in the regional species pool. thus, pastures with low nutrient availability in acta bot. croat. 71 (1), 2012 43 mesic grasslands of south transdanubia tab. 4. pearson’s correlation of indicator values with the ordination axes and the goodness of fit of their passive projections. tb – temperature, wb – moisture, rb – soil reaction, nb – nutrient status, lb – light availability, cb – continentality indicator value correlation with the axes fit cap 1 cap 2 r-squared p tb –0.996 0.095 0.209 <0.001 wb 0.744 0.669 0.544 <0.001 rb –0.167 0.986 0.006 n.s. nb 0.450 0.893 0.548 <0.001 lb –0.997 –0.072 0.533 <0.001 cb 0.093 –0.996 0.059 <0.05 u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:14 color profile: disabled composite 150 lpi at 45 degrees south transdanubia did not correspond with festuco commutatae–cynosuretum; however, this can be explained by the lack of data from this region in the past, which prevented the hungarian description (borhidi 2003) of this association being extended to the southern stands. as borne out by rozbrojová et al. (2010), extensive pastures have species compositions similar to low production hayfields. there is a low productive hay meadow association in hungary in the arrhenatherion alliance, anthyllido–festucetum rubrae, which is distributed through the whole country in colline and montane ranges according to borhidi (2003). however, we could not recognise this association based on our data. it seems time to revise these two association names and descriptions (festuco commutatae–cynosuretum and anthyllido–festucetum rubrae) based on statistical analyses, limit their application to narrower geographic areas and find appropriate names for the others. moreover, if uniqueness and distinctness of narrow-range associations are not supported enough, it is advisable to adopt names from the syntaxonomic systems of neighbouring countries in order to standardise the nomenclature of syntaxa over the countries (knollová et al. 2006). based on archive data and available literature of central european countries (hájková et al. 2007, janisová et al. 2007), festuco–cynosuretum, anthoxantho–agrostietum and festuco–agrostietum seem equally applicable names for southern hungarian extensive pastures. the group of nutrient-rich cynosurion pastures (group 6) resembles the lolio–cynosuretum association that is already reported from hungary. according to borhidi (2003) this association occurs in colline and montane ranges. however, hájková et al. (2007) extended its altitude range to the lowlands in the czech republic. in our study area, similar stands are the most common in the drava plain, but rarer in the higher altitudes, contrary to borhidi (2003). lowland stands are not only situated at lower altitudes but also in southern geographic position affected more by sub-mediterranean climate and they harbour a few of the sub-mediterranean species that are rare or sparse in the study region (e.g. hordeum secalinum). therefore, we think an urgent task to explore the relationship between the nutrient-rich cynosurion pastures of south transdanubia and the bromo–cynosuretum stands of croatia. it should also be further investigated how they are delimited from trampled and grazed wet meadows. this problem is also recognised by zuidhoff et al. (1995) who suggested applying the name junco–cynosuretum sougnez 1957 to relevés that they classified to a transitional cluster between calthion and cynosurion (mainly lolio–cynosuretum). noteworthily, their analysis is based mostly on western european relevés with disproportionately few central european data. we identified group 8 as a pastinaco–arrhenatheretum elatioris association that is a common meadow community in central europe and also included in the hungarian system. nevertheless, there are some other associations characterised by arrhenatherum elatius which are difficult to distinguish from pastinaco–arrhenatheretum. in hungary, such an association is alopecuro–arrhenatheretum which is a mountainous type described from the northern part of the country (máthé and kovács 1960). in croatia, ononido–arrhenatheretum is distributed along the drava and mura rivers and is considered a moister and less productive meadow than pastinaco–arrhenatheretum (ilijani] and [egulja 1983, trinajsti] 2008). though, these associations are not clearly delimited from pastinaco–arrhenatheretum (stan^i] 2008) and the differences are probably so slight that the applied hierarchical classification methods are not able to distinguish them as separate clusters of our current data set. here we suggest defining these associations by supervised 44 acta bot. croat. 71 (1), 2012 lengyel a., purger d., csiky j. u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:14 color profile: disabled composite 150 lpi at 45 degrees classification tools (bruelheide 2000) and/or calculating similarity measures (tichý 2005, van tongeren et al. 2008) between reliable reference plot data and the unidentified relevés, and using the resulting assignments for mapping their distribution. we expect that a few relevés would be identified as any of these rarer associations but the majority of group 8 should be assigned to pastinaco–arrhenatheretum. it is also worth mentioning that pastinaco–arrhenatheretum is characterised by generalist meadow species, which are common on drying wet meadows and other dynamic types, thus the species composition of pastinaco–arrhenatheretum can easily appear on various sites. it is another question whether differences among the above narrow associations (alopecuro–arrhenatheretum and ononido–arrhenatheretum) and the core type (pastinaco–arrhenatheretum) are bigger than the differences among dynamic phases. the low productive part of arrhenatherion alliance had previously been covered by two associations: anthoxantho–festucetum rupicolae from lowlands and anthyllido–festucetum rubrae from colline and montane ranges (borhidi 2003). anthoxantho–festucetum rupicolae is originally described from this area (drava plain) by dénes (1997) and some of our relevés are clearly identifiable with the published data. anthyllido–festucetum rubrae is considered common in deciduous forest ranges (borhidi 2003), but we could not recognise it in our data. instead, our group 7 seemed the most similar to filipendulo– –arrhenatheretum described originally from austria (hundt and hübl 1983) and detected also in croatia (trinajsti] 2002). similar stands are reported from the bakonyalja region in transdanubia (bauer et al. 2001), but no phytosociological relevés are published. the presence of this association in hungary should be validated by analysing our data together with relevés of the above countries. if this community is accepted as present in hungary, the position of anthoxantho–festucetum rupicolae should also be revised because filipendulo–arrhenatheretum would be an association with a wide distribution and many variants, and anthoxantho–festucetum rupicolae may be only its dry variant with subregional distribution. in austria three subassociations of filipendulo–arrhenatheretum are described in hundt and hübl (1983): typical, wet (with cirsium oleraceum) and dry (with centaurea scabiosa), although the dry subassociation differs from anthoxantho–festucetum rupicolae floristically. recently, an ecologically similar association was described (zelnik 2007) with the name triseto–centaureetum macroptili. this community represents transitions between wet and mesic grasslands and it occurs on elevated part of alluviae where the water table fluctuates, as in the locations of group 7. however, we are not aware of any occurrences of its dominant species, centaurea jacea subsp. macroptilon, in the studied region. moreover, triseto–centaureetum harbour some acidophilous species that are also very rare or absent in south transdanubia (e.g. nardus stricta, festuca filiformis). we also identified a dynamic phase of several types but mainly of nutrient-poor pastures. such clusters are usually not interpreted syntaxonomically. however, these degraded stands show continuous transition to other, more 'stable' types and their representation in an unsupervised classification is also a question of sampling decisions. three of our clusters are identified as belonging to or representing transitions to orders other than arrhenatheretalia. aware that the transitions between arrhenatheretalia and brometalia or arrhenatheretalia and molinietalia meadows are continuous and they can only be delimited quite arbitrarily, we are careful with the syntaxonomic interpretation of acta bot. croat. 71 (1), 2012 45 mesic grasslands of south transdanubia u:\acta botanica\acta-botan 1-12\464 lengyel et al.vp 26. o ujak 2012 10:25:14 color profile: disabled composite 150 lpi at 45 degrees transitional groups. these clusters are hard to characterise because they often include stands from various transitional pathways between broader syntaxa. for example, group 4 comprises a broad variety of transitions between mesic and semi-dry meadows, its diagnostic species thus being the generalists, which have in common only their moisture preference. heterogeneity of this group is also supported by festuca rupicola being the only species that dominates more than one stand. there are three associations considered transitional between semi-dry and mesic meadows by borhidi (2003): anthoxantho–festucetum rupicolae dénes 1997 in the arrhenatherion alliance, onobrychido viciaefoliae–brometum erecti müller 1966 and carlino acaulis–brometum oberdorfer 1957 in the bromion erecti, however, the presence of the latter two is not validated by statistical analysis in a recent review (illyés et al. 2009). literatures of neighbouring countries also consider ranunculo bulbosi–arrhenatheretum ellmauer in mucina et al. 1993 and filipendulo vulgaris–arrhenatheretum hundt et hübl 1983 of arrhenatherion as such intermediate types. there are several associations interpretable as transitions between wet and mesic meadows as well (e.g. alopecuro–arrhenatheretum (máthé et kovács 1960) soó 1971, cirsio cani–festucetum pratensis májovsky et ru`i~ková 1975, arrhenathero–molinietum arundinaceae lájer 2002, holcetum lanati issler 1934). in 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(b) seslerio autumnalis-campanuletum tommasinianae ass. nov., representing stands predominantly developed within thermophytic beech stands, semito fully-shaded by the tree canopy; (c) cystopteri fragilis-campanuletum tommasinianae, sciophytic, stands adapted to moisture and cold with high frequency and coverage of bryophytes. results of dca analyses using a unimodal model suggest that campanula tommasiniana is primarily a plant of open and exposed sites of higher elevation despite being most frequently found in rock crevices within thermophytic and altimontane beech forests. key words: campanula tommasiniana, campanulaceae, dinaric alps, ecology, endemic species, liburnian karst, mt. u~ka, phytosociology introduction the kaleidoscopic complexity of topographic, climatic and geological conditions in the mediterranean results in a high degree of overall biodiversity and a rapid species turnover. the flora of the mediterranean is, for example, the species-richest area in the old world. although the mediterranean region represents less than 1.5% of the land area of the world, its flora comprises around 30,000 species and subspecies of flowering plants (quezél 1985, greuter 1991), more than 160 species of ferns, and hosts approximately 10% of all known species of ferns and flowering plants on earth (blondel and aronson 1999). around 80% acta bot. croat. 73 (1), 2014 221 * corresponding author, e-mail: bostjan.surina@prirodoslovni.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen of all endemic taxa are to be found in the area (gomez-campo 1985). however, the main reason for such a high degree of biodiversity of flowering plants in mediterranean is not the overall species richness of the area, but the high number of endemics, many of them restricted to one or only a few known localities with specifics in ecology (e.g., geology, soil type, relief, etc.) or geography (e.g., islands, mountains). here, almost every island, regardless of its size, hosts at least some (indigenous) endemic taxa. ecologically similar to islands, mountains promote speciation events too, which may result in there being up to 42% of endemics among the higher plants (medail and verlaque 1997), although this rate may vary a lot (blondel and aronson 1999). one of the many local or regional endemics of the genus campanula along the adriatic coast (for the review see kova^i] 2004, park et al. 2006) is the chasmophytic c. tommasiniana koch (in f. schultz, arch. fl. fr. et allemagne 6. cent., 229, 1852), a narrow endemic of mt. u~ka, located above kvarner bay in the liburnian karst (north-western adriatic). along with edraianthus wettsteinii subsp. lovcenicus and e. dinaricus (campanulaceae), two narrow endemics of mt lov}en, above kotor bay, and mt mosor in central dalmatia (wettstein 1887, janchen 1910, mayer and ble^i] 1969, laku[i] et al. 2009), respectively, and a few predominantly island populations of ambiguous taxonomic rank, e.g., centaurea friderici, c. kartschiana, c. cuspidata (asteraceae; e.g., teyber 1913, ginzberger and teyber 1921, ginzberger 1921, lovri] 1971, radi] 1981, lovri] 1992), brassica incana (brassicaceae; e.g., ginzberger 1921) and asperula staliana (rubiaceae; e.g., korica 1975, lovri] and korica 1981, korica 1986) aggregates in north-western adriatic and dalmatia, c. tommasiniana is one of the most range-restricted taxa among the all adriatide (sensu trotter 1912), occupying an area of only 6.5 km2 (surina 2013). the data on ecology and niche assembly of campanula tommasiniana, despite its being a prominent narrow endemic, are surprisingly scarce. according to horvati] (1963b), c. tommasiniana is a chasmophyte growing in calcareous rock crevices within thermophytic beech forests of the association seslerio autumnalis-fagetum, building an association campanuletum tommasinianae-justinianae. however, results of a detailed survey of its distribution range and habitat preferences (surina 2013) suggest its great ecological plasticity, for it thrives in rock crevices of rocky outcrops and cliffs as well as within various types of forest stands (e.g., associations fraxino orni-quercetum ilicis, querco-carpinetum orientalis, aristolochio luteae-quercetum pubescentis, seslerio autumnalis-ostryetum, seslerio autumnalis-fagetum and ranunculo platanifolii-fagetum) along the whole elevational (vegetational) profile of the mountain’s eastern slopes (for detailed comments on vegetation profiles see [ugar 1970, 1984). on western slopes, in contrast to the eastern, it rarely descends below 1000 m a.s.l. while commenting on the site ecology of leontopodium alpinum on mt. u~ka, c. tommasiniana (as c. waldsteiniana!) has been recorded within the alliance micromerion croaticae ([ugar 1971). with our research we aimed to get insights into site ecology, habitat preferences and niche assembly of campanula tommasiniana. research area the u~ka mountain range rises above kvarner bay; between the poklon pass (920 m a.s.l.) and plomin bay it forms a distinct ridge in a north-south direction along the north-eastern istrian coast (ne adriatic). the mountain’s highest peaks, vojak (1396 m 222 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen a.s.l.), suhi vrh (1332 m a.s.l.) and plas (1285 m a.s.l.), are located in its northern part, which is generally much higher than its southern part. while the macrorelief of the mountain is rather simple, characterized by a prominent ridge, forming distinct eastern and western slopes of the mountain, the microrelief is diversified, being the most picturesque with the solitary limestone towers and cliffs in the vela draga canyon and beyond the peak plas on the mountain’s western slopes. some precipitous rock faces are to be found on the eastern slopes as well; the most distinct are those located north of the grdi breg slope, east of vojak peak, the eastern rock face of the suhi vrh peak and the south-western rock face of argun peak. according to geological maps ([iki] et al. 1963, [iki] et al. 1967) and explanatory texts ([iki] and pol[ak 1973, [iki] and pleni^ar 1975), lower and upper cretaceous limestones and dolomites prevail in the area. along the lovranska draga valley, limestones and dolomites are intercepted by quaternary sediments, marlstones and sandstones, conglomerates, breccias and foraminiferan limestones. as indicated by various but pronounced climatogenic vegetation types along an elevational gradient of the mountain, the climate is diverse. the foothills are rather warm with mean annual temperatures between 14–15 °c, while the values gradually decrease with increasing elevation, being only 4–6 °c at the summits (zaninovi] 2008). the higher elevations of the mountain receive from 2000 to 2500 mm of precipitation yearly, which is the highest amount of precipitation in istria (gaji^-^apka et al. 2008). however, despite being positioned on the adriatic coast, the eastern foothills of the mountain receive still more than 1500 mm of precipitation yearly (ibid.). the precipitation regime is a mediterranean one. mt. u~ka is one of the most renowned botanical sites in the adriatic area (topi] et al. 2009) with a remarkable tradition of botanical exploration (e.g., brana 2012, trinajsti] and pavleti] 2012). as a consequence, the mountain’s flora is rather well known, although the data on vegetation are somewhat scarce. from a biogeographical aspect and according to [ugar (1970, 1984, but with phytosociological nomenclature according to vukeli] 2012), vegetation types along an elevational profile of the mountain belong to two phytogeographical regions: (a) mediterranean, further divided into eumediterranean (ass. fraxino orni-quercetum ilicis), submediterranean (ass. querco-carpinetum orientalis), both restricted to a narrow elevational belt on the mountain’s foothills, and epimediterranean (ass. aristolochio luteae-quercetum pubescentis) zones, and (b) eurosibiric-northamerican region, with paramediterranean (ass. seslerio autumnalis-fagetum) and illyric (altimontane beech forests, ass. ranunculo platanifolii-fagetum) zones, both covering majority of the mountain range. within eumediterranean and submediterranean subzones, stands with dominating laurus nobilis in a tree layer (fraxino orni-quercetum ilicis fac. laurus nobilis and querco-carpinetum orientalis lauretosum nobilis) are well developed locally on deeper and moister soils (horvati] 1963a, pelcer 1983). in comparison to other eastern adriatic mountain ranges of the dinaric alps, the outstanding features of mt. u~ka are the homogenous and extensive forests both from the inland (western) and seaward (eastern) sides. since 1999, the natural and cultural heritage of the area has been protected within u~ka nature park (narodne novine 1999). materials and methods in summer 2011, we recorded 112 relevés with campanula tommasiniana through the whole distribution range of the species. the cover-abundance estimates were made accordacta bot. croat. 73 (1), 2014 223 ecology of campanula tommasiniana 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen ing to the domin scale (sensu dahl and hada^ 1941) and the plot size used for sampling averaged 9 m2 (the standard plot size for chasmophytic and scree stands, but see also chytry and otýpková 2003). details of the phytosociological parameters of sites are given in appendix 1 to on-line supplement tabs. 2–4. with each relevé, light conditions were roughly estimated as: open sites (no shading), semi-open sites (medium shading) and fully covered sites (full shading) by the canopy of the nearby forest vegetation. a complete floristic inventory is given in table 4, while taxa occurring only once in the analyses are listed in appendix 2 to on-line supplement tabs. 2–4. the nomenclature and taxonomic source for the names of vascular plants was flora europaea (tutin et al. 2001), while the names of bryophytes were in agreement with the catalogue of mosses of slovenia (martin^i^ 2003) and the annotated checklist of slovenian liverworts and hornworts (martin^i^ 2011). all collected and identified bryophytes are stored in the herbarium of the natural history museum rijeka (nhmr). raunkiær’s life forms (raunkiaer 1907), subsequently revised and modified by müller-dombois and ellenberg (1974), were adopted from pignatti (2005) for vascular plants and hill and preston (1998), düll et al. (1999) and martin^i^ (1966, 2003) for the bryophytes. syntaxonomic groups in tab. 4 were assigned primarily according to flora alpina (aeschimann et al. 2004), while in some cases we followed our own criteria. coverage index (d%, e.g. surina 2005) was calculated for life forms (tab. 1) and each taxon (tab. 4), respectively. prior to numerical analysis, the original cover-abundance values for individual taxa were transformed accordingly (curral 1987). groups of vegetation types were ascertained using cluster and ordination analyses with the help of the programme package past (hammer et al. 2001). the arrangement of relevés was done according to the results of cluster analysis and diagnostic groups of species were tested by means of the simper analysis (on-line supplement tabs. 1–4), an algorithm implemented in the programme package past, and constrained ordination analyses using species fit (of 25%) as an inclusion rule, characterized as the quality of the description of the »behaviour« of species values, derived from the particular combination of ordination axes (lep[ and [milauer 2003). in order to explain the variation in niche assembly by specific environmental and structural (phytosociological) parameters, unconstrained (dca) and constrained (rda, cca) ordination analyses were performed, using the canoco computer programme (braak ter and [milauer 2002). in order to determine the lengths of gradients, dca analyses, detrended by segments, were initially performed and the models (linear, unimodal) used accordingly. the statistical significance (p < 0.02) of the site parameters was tested using the monte carlo test, with 499 permutations. only the significant parameters were then analyzed together, with the aim of producing a general view of the environmental impact on floristic composition and structure of stands. for estimating the general environmental affinities of the relevés, indicator values (co-variables) for vascular plants were assigned according to pignatti (2005) and passively projected into the ordination diagrams. the environmental value in a relevé (evw) was estimated as the weighted average of the indicator values of all present species, their abundances being used as weights (lep[ and [milauer 2003). the kruskal–wallis non-parametric test was used to test whether samples were taken from groups with equal median environmental values and a post-hoc test was carried out using mann-whitney’s pairwise comparisons. while defining the syntaxa we followed the sigmatistic-braun-blanquet approach (braun-blanquet 1928), subsequently improved by westhoff and van der maarel (1973), and based on a revised association concept proposed by willner (2006). 224 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen a c t a b o t .c r o a t .73 (1),2014 225 e c o l o g y o f c a m p a n u l a t o m m a s in ia n a tab. 1. stand parameters of chasmophytic niche assambleges with campanula tommasiniana on mt. u~ka (nw adriatic) groups of assemblages sjc sac cfc s gm gs c s m elevation (m)* 1116 1237 1260 1258 405 1033 1143 (760–1216) (1188–1296) (1140–1316) (965–1380) (396–420) (827–1355) (47–1375) coverage (%)* stoniness 70 80 70 70 45 60 50 (50–80) (70–90) (60–80) (30–90) (40–70) (40–80) (20–70) herb layer 30 20 25 30 35 25 30 (20–50) (10–30) (20–40) (10–40) (30–50) (10–40) (10–40) moss layer 1 1 1 8 35 20 40 (0–10) (1–10) (1–40) (1–40) (1–40) (30–70) no. of vascular plants per rel.* 12 9 10,5 9 15 10 10 (8–17) (6–17) (6–20) (6–15) (11–23) (5–21) (4–16) no. of bryophytes per rel.* 7 2 4 8 7,5 11 11 (0–13) (1–8) (0–8) (4–10) (6–14) (5–16) (6–17) total no. of vascular plants 46 45 45 52 45 81 54 total no. of bryophytes 25 18 20 29 26 47 50 life forms (no. of taxa/d%) hemicryptophytes 24 (52%)/89.2 26 (58%)/66.2 24 (53%)/71.3 62 (31%)/76.2 18 (40%)/46.4 54 (67%)/76.2 65 (35%)/79.9 phanerophytes 9 (20%)/11.3 6 (13%)/3.2 4 (9%)/3.3 5 (10%)/2.1 18 (40%)/38.5 12 (15%)/5.2 7 (13%)/1.4 chamaephytes 6 (13%)/27.8 9 (20%)/27.5 12 (27%)/20.9 8 (16%)/12.7 6 (13%)/12.5 4 (5%)/4.9 / geophytes 6 (13%)/12.3 3 (7%)/4 2 (4%)/3.1 4 (8%)/4.1 3 (7%)/2.6 7 (9%)/9.1 8 (15%)/5.2 therophytes 1 (2%)/3.3 1 (2%)/1.4 3 (7%)/1.4 2 (4%) 4.1 / 4 (5%)/4.6 4 (7%)/13.3 no. of relevés 9 17 18 14 25 23 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 4 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n results niche selection and floristic assembly of stands campanula tommasiniana, inhabiting calcareous rock crevices and cracks from low elevated sites (47 m) to mountain tops (1390 m), is an edificatory vascular plant for chasmophytic assemblages. the total floristic assembly of stands, covering 10–80% (me=35%) of the sampling plots, counts for 155 taxa of vascular plants and 77 taxa of bryophytes (two of them are lichenicolous fungi), with a median number of 12 (min=6, max=23) and 8 (min=0, max=17) taxa per plot, respectively (tab. 4, on-line supplement tabs. 2–4). the coverage and number of taxa of vascular plants and bryophytes varies a lot between the sites (tab. 1) and depends heavily on site ecology. among the vascular plants, typical chasmophytes occur in more than one third of the relevés: campanula tommasiniana (100%), asplenium ruta-muraria (71), a. trichomanes (65), sesleria juncifolia (57), athamanta turbith (46) and cymbalaria muralis (40), while among bryophytes the most frequent were tortella tortuosa (71), ctenidium molluscum (54), schistidium sp. (54), neckera crispa (51), homalothecium sericeum (50), h. philippeanum (36) and plasteurhynchium striatulum (35; tab. 4). in number and coverage of vascular plant taxa, hemicryptophytes completely prevail and represent more than a half of all registered vascular plant taxa (d%=46.4– 89.2), followed by phanerophytes (18%, d%=1.4–38.5), geophytes (15%, d%=2.6–12.3), therophytes (5%, d%=0–13.3) and chamaephytes (3%, d%=0–27.8). cluster and simper analyses application of various algorithms and (dis)similarity measures yields very similar clustering topology. a dendrogram of chasmophytic stands from mt. u~ka shows two 226 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. tab. 2. overall average dissimilarities (lower left hand corners) and taxa contribution (up to 50% of overall dissimilarities, upper right hand corners) between the three general types of chasmophytic niche assemblages with campanula tommasiniana on mt u~ka (nw adriatic) according to simper analysis. taxa abbreviations as in table 4 and text below. syntaxa are explained in the text following figure 3. syntaxa sjc cfc sac sjc nec cri (5.5), cte mol (4.6), ses jun (3.6), asp tri (2.5), cys fra (2.3), cym mur (2.1), pla str, hom phy, myc mur, cam tom (2), ath tur (1.7), glo cor, rad com (1.4), tor tor, sil sax, sch sp, asp rtm (1.3), hom ser, ara alp (1.2) ses jun (4), nec cri (2.8), ano vit (2.5), pla str (2.3), hom ser (2.2), asp tri, cam tom (2), nec bes, cte mol (1.9), ses aut (1.8), ath tur, hom phi (1.7), glo cor, sch sp, asp rtm (1.5), tor tor, sil sax (1.4), mic thy, por pla, nec com (1.2), sat mon (1.1), cyc pur (1) cfc 80.44 nec cri (3.1), cte mol (2.8), ano vit (2), cys fra (1.8), hom ser (1.7), pla str, cym mur, hom phi (1.6), nec bes (1.5), ses aut, cam tom, myc mur (1.3), asp rtm, tor tor (1.2), rad com, sch sp, pse alb, nec com (1.1), por pla, asp tri, ara alp, fis dub, cyc pur (1) sac 78.63 65.92 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen groups of relevés (fig. 1). in cluster »a«, 97 taxa of vascular plants, 34 taxa of bryophytes and 2 taxa of lichenicolous fungi are surveyed. beside campanula tommasiniana2–5(100%), sesleria juncifolia3–6(90), asplenium ruta-muraria+–3(71) and athamanta turbith+–6(69) completely prevail (tab. 4, on-line supplement tabs. 2–4). other relatively frequent vascular plants are silene saxifraga subsp. hayekiana+–3(55), micromeria thymifolia1–3(48), asplenium trichomanes1–4(40) and scrophularia laciniata+–3(36). the most frequent bryoacta bot. croat. 73 (1), 2014 227 ecology of campanula tommasiniana distance 90 80 70 60 50 40 30 20 10 a sjcs sjcc sjcg sjcgm sjcgs sacs sacm cfc b fig. 1. dendrogram of chasmophytic assemblages with campanula tommasiniana on mt. u~ka (nw adriatic; ward’s method, euclidean distances). syntaxonomy is explained in the text following figure 3. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen phytes are tortella tortuosa1–3(79), schistidium sp.1–3(45) and homalothecium sericeum1–3(38). in cluster »a« the following taxa occur exclusively: globularia cordifolia2–4(57), senecio abrotanifolius1–3(29), stachys subcrenata1–3(26), arabis scopoliana1–3(19), sempervivum tectorum+–3(16), rosa pimpinellifolia+–2(14%), euphrasia illyrica+–3(12), campanula marchesettii1–3(10), c. cochleariifolia+–2(10), rhamnus saxatilis1–3(9), gentiana lutea subsp. symphyandra+–2(9), teucrium arduinii1–4(9), satureja subspicata subsp. liburnica+–2(7), primula auricula1–4(5), leontopodium alpinum1–2(5) and others, while silene saxifraga subsp. hayekiana, sedum album1–3(16), micromeria thymifolia, hieracium bupleuroides1–3(31), scrophularia laciniata, allium saxatile subsp. tergestinum+–3(24), teucrium montanum+–3(24), saxifraga paniculata1–3(16) occur almost exclusively. bryophytes, found relatively frequently only in this cluster, are tortella densa2–3(10), ditrichium flexicaule2–3(17), while syntrichia ruralis var. ruralis1–3(16) and scapania aspera1–3(7) occur in cluster »a« almost exclusively. cluster »a« further divides into three sub-clusters: (a) sjcg (on-line supplement tab. 2, rel. 10–44) is characterized by high frequency and coverage of globularia cordifolia2–4(89) and hieracium bupleuroides1–3(43), and the two groups of stands: micromeria thymifolia2–3(94), scrophularia laciniata+–3(56; sjcgm) and stachys subcrenata1–3 (59), tortella densa2–3(35; sjcgs), respectively (rel. 10–27, 28–44 in on-line supplement tab. 2); (b) sjcc (on-line supplement tab. 2, rel. 45–58), characterized by high constancy, frequency and coverage of bryophytes, specially ctenidium molluscum1–4(93), neckera crispa1–4(36) and fissidens dubius1–3(43); (c) sjcs (on-line supplement tab. 2, rel. 1–9), where sedum album1–3(89), satureja montana subsp. variegata3–5(67) and syntrichia ruralis var. ruralis1–3(56) occur almost exclusively. cluster »b« represents less homogenous but more diverse floristic assembly; a total of 130 taxa of vascular plants and 56 taxa of bryophytes are registered. along the campanula tommasiniana2–6(100), the most frequent vascular plants are: asplenium trichomanes2–5 (100), a. ruta-muraria2–4(96), cymbalaria muralis2–4(91), mycelis muralis+–3 (87), cystopteris fragilis2–4(83), geranium robertianum1–3 (57), plagiochilla porelloides1–3(43), cyclamen purpurascens2–3(39), valeriana tripteris1–3(39), senecio fuchsii+–3(35), pseudofumaria alba1–7(30), saxifraga rotundifolia2–4(30), adenostyles glabra1–3(30), sesleria juncifolia+–3 (30). in comparison to cluster »a«, bryophytes are much more frequent and abundant in cluster »b«. the most common taxa are ctenidium molluscum3–8(100), neckera crispa3–8 (96), schistidium sp.1–3(65), homalothecium philippeanum3–5(57), plasteurhynchium striatulum1–5(57), radula complanata1–3(57), tortella tortuosa1–3(52), fissidens dubius1–4(48), porella platyphylla1–3(45), neckera complanata1–4(39), pseudoleskeela catenulata1–4(39), bryum sp.1–3(35), homalothecium sericeum1–4(35), anomodon viticulosus1–5(30), cololejeunea calcarea1–4(30), pedinophyllum interruptum3(30), mnium thomsonii2–3(30) etc. in cluster »b«, mycelis muralis, plagiochila porelloides1–3(43), mnium thomsonii, quercus ilex1–2(13), galeobdolon flavidum+–3(26) occur exclusively, while cystopteris fragilis, arabis alpina1–3(52), coronilla emerus subsp. emeroides2–3(19), asparagus acutifolius1–2 (19), hedera helix1–3(13) occur almost exclusively. here, three floristically well defined sub-clusters are recognized: (a) sacm (on-line supplement tab. 4, rel. 7–31), representing floristic assemblages characterized by the presence and high coverage of homalothecium sericeum1–5(84), sesleria autumnalis1–5(68) and neckera besseri1–6(52), cyclamen purpurascens+–3(72), galeobdolon flavidum+–3(32) and mycelis muralis+–3(48); (b) sacs (on-line supplement tab. 4, rel. 1–6), a group of floristically quite distinct stands with high frequency, coverage and (almost) exclusive occurrence of coronilla emerus subsp. emeroides2–3(100), asparagus acutifolius1–2(100), quercus ilex1–2(66), hedera helix1–3(66), lep228 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen todon smithii3–6(66) and salvia officinalis1–2(50); (c) cfc, stands characterized by exclusiveness, highest frequency and coverage of mycelis muralis+–3(87), cystopteris fragilis2–4(83), geranium robertianum1–3(57), arabis alpina1–3(52), plagiochila porelloides1–3(43) and mnium thomsonii2–3(30; on-line supplement tab. 3). floristically, the cluster cfc which is the most different from cluster sjc (80.44%); the taxa that contribute most to dissimilarity are sesleria juncifolia, asplenium trichomanes, cystopteris fragilis, cymbalaria muralis, mycelis muralis, athamanta turbith, globularia cordifolia, asplenium ruta-muraria, silene saxifraga subsp. hayekiana and arabis alpina among the vascular plants, while among the bryophytes there are necera crispa, ctenidium molluscum, homalothecium philippeanum, h. sericeum, radula complanata, tortella tortuosa and schistidium sp. (tab. 2). cluster cfc differs significantly less from cluster sac (65.92%) and the taxa that contribute most to dissimilarity are neckera crispa, ctenidium molluscum, anomodon viticulosus and many other bryophytes, while among the vascular plants the most important differential taxa are cystopteris fragilis, cymbalaria muralis, sesleria autumnalis, mycelis muralis, asplenium trichomanes, a. ruta-muraria, arabis alpina and cyclamen purpurascens. the average dissimilarity between clusters sjc and sac is 78.63%, and the most important differential taxa are sesleria juncifolia, s. autumnalis, asplenium trichomanes, a. ruta-muraria, globularia cordifolia etc. among vascular plants, and neckera crispa, n. besseri, anomodon viticulosus, plasteurhynchium striatulum, ctenidium molluscum etc. among bryophytes. specifically, groups of assemblages within the cluster sjc (sjcs, sjcgm, sjcgs and sjcc (fig. 1) appear to be floristically most similar with overall average dissimilarities ranging between 55.61 (between sjcgm and sjcgs) and 72.82% (between sjcs and sjcgs; on-line supplement tab. 1). average dissimilarities between the cluster cfc and all the other groups of assemblages are in general high (72.26–84.52%), while the most distinct group of assemblages is represented by the group sacs with the highest recorded dissimilarities (86.5% to sjcgm, 86.38% to sjcgs and 80.37% to sjcc). sacs shows the lowest dissimilarity to sacm (70.56%). ecology and vegetation typology of stands cluster topology (fig. 1) reflects the ecology of chasmophytic assemblages well (fig. 2). cluster »a« (sjc) includes stands of sunny and exposed rocky outcrops and cliffs, while cluster »b« represents stands fully(cfc) to semi-shaded (sac) by the tree canopy (fig. 2f). unconstrained ordination analysis (fig. 2a) explains 25.3% of variance along the first axis, where assemblages preferring sunny and exposed sites (sjc) are located on the left, while those occupying shaded and moist rock crevices (sac, cfc) are on the lower right side of the diagram. the ecology of chasmophytic assemblages is even better reflected in results of constrained ordination analyses (figs. 2b–f). cca analysis of all assemblages (fig. 2b) yields coverage (f = 5.56) and number of bryophyte taxa (2.42), elevation (5.47) and number of vascular plants (1.85) as statistically significant explanatory variables (p < 0.002). in coverage and number of respective bryophyte taxa assemblages of the cluster cfc, and partly of sac dominate. these stands prefer sciophytic, moist, nutrient-rich and higher-elevated sites. in contrast, assemblages of cluster sjc, depauperate in number and coverage of bryophyte taxa, prefer open sites along the broad elevational range. assemblages of the cluster sac seem to be ecologically somewhat intermediate, preferring semiacta bot. croat. 73 (1), 2014 229 ecology of campanula tommasiniana 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:44 color profile: generic cmyk printer profile composite default screen 230 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. -0.5 4.5 -2 3.5 -2 3 -0 .5 3 .5 expl. var. covariables sat mon sed alb syn rur ath tur mic thy sil sax sta sub glo cor tor den ses jun scr lac cam tom hie bup gal fla cte mol nec cri cys fra ara alp mni tho ger rob pla por fis dub cyc pur nec bes ses aut lep smi hed hel asp acu cor eme que ilesal off hom ser myc murstc sac cfc -2 2 -1.5 2-0 .5 -1 .5 3 .5 1 .5ssjc sac m gm s gs c a b c e f d 50 50 50 sac sjc cfc open sites semi-shaded sites fully-shaded sites -3 4 -4 6 all pul asp acu bro ere cet off cor eme cys fra fra orn ger rob hed hel myc mur que ile sal off ses aut bra tom col cal cte mol lep smi nec cri pla por pla str cam tom ath tur glo cor ses jun sil sax cfc sacm sacs sjc elevation no. of bryoph. no. of vasc. plants t c l r n u elevation elevation no. of bryophytes no. of bryoph. no. of vasc. plants t c c cover. of stoniness l r n n u u t r l cover. of bryoph. cover. of bryoph. n c t u l r 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:45 color profile: generic cmyk printer profile composite default screen or fully shaded sites most commonly within thermophytic beech forests of the association seslerio autumnalis-fagetum. elevation is negatively correlated with the number of vascular plant taxa, where a group of sac assemblages from lower elevated sites is well differentiated from all other stands in floristic richness, distinct composition as well as most thermophytic site conditions. using an inclusion rule in order to define a subset of species that fit the quality criterion of representing the percentage of variability in species values explained by the explanatory variables, the following groups of species are identified (fig. 2e): sesleria juncifolia, athamanta cretensis, silene saxifraga subsp. hayekiana, globularia cordifolia and campanula tommasiniana for the group sjc; cystopteris fragilis, mycelis muralis, neckera crispa, ctenidium molluscum, geranium robertianum, plagiochila porelloides, brachythecum tommasinii and plasteurhynchium striatulum for the group cfc; sesleria autumnalis, fraxinus ornus, ceterach officinarum, leptodon smithii, bromus erectus and cololejeunea calcarea for the group sacm, and quercus ilex, salvia officinalis, coronilla emerus subsp. emeroides, allium pulchellum subsp. carinatum, asparagus acutifolius and hedera helix for the group sacs. the results of the non-parametric test for equal medians of environmental values of chasmophytic assemblages (tab. 3) show significant differences (p < 0.05) in both site parameters and pignatti’s indicator values (tab. 3). generally, moisture (u, h=87.94) and light conditions (l, 84.91) show the highest probability of non-equal medians of environmental values in the studied assemblages, followed by soil reaction (r, 80.78), coverage of bryophytes (76.61), nutrients (n, 67.85) and elevation (50.82). post-hoc pairwise comparisons suggest that stands from the cluster »a« (sjc) significantly differ from all the other groups in light conditions, moisture, soil reaction, amount of nutrients and coverage of bryophytes. on the other hand, group cfc differs significantly from all the other groups in moisture and soil reaction, while the group sac represents relatively mesophytic assemblages. according to initial cluster, and subsequent unconstrained and constrained ordination analyses, further easily recognized groups of assemblages are recognized based on specifics in site ecology and floristic assembly. within the group sjc, a subset of stands sjcs, characterized by the presence and high coverage of sedum album, satureja montana acta bot. croat. 73 (1), 2014 231 ecology of campanula tommasiniana fig. 2. unconstrained and constrained analyses of chasmophytic assemblages with campanula tommasiniana on mt. u~ka (nw adriatic) according to site parameters and pignatti’s indicator values. a – dca diagram, length of gradient 4.071, eigenvalues 0.533, 0.340, 0.212, 0.159; cumulative percentage variance of species data: 6.4, 10.6, 13.1, 15.0; b – cca diagram according to site parameters and pignatti indicator values; eigenvalues: 0.429, 0.362, 0.151, 0.122; cumulative percentage variance of species data: 5.2, 9.6, 11.4, 12.8; c – cca diagram of stands of the assemblage group sjc; eigenvalues: 0.284, 0.248, 0.164, 0.140; cumulative percentage variance of species data: 5.4, 10.1, 13.3, 15.9; d – cca diagram of stands of the assemblage group sac; eigenvalues: 0.386, 0.231, 0.297, 0.257; cumulative percentage variance of species data: 8.8, 14.1, 20.9, 26.8; e – cca diagram of species-environmental data using inclusion rule (25%) option; f – ternary plot for the three types of assemblages according to light condition of sites. full lines – explanatory variables: site parameters (elevation, number of vascular plants, number of bryophytes, coverage of bryophytes, coverage of stoniness); dashed lines – co-variables: passively projected pignatti indicator values (l – light conditions, t – temperature, r – soil reaction, c – continentality, u – moisture, n – nutrients). syntaxonomy is explained in the text following figure 3. � 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:45 color profile: generic cmyk printer profile composite default screen 232 a c t a b o t .c r o a t .73 (1),2014 s u r in a b .,m a r t in ^ i^ a . tab. 3. results of kruskal-wallis tests and mann-whitney’s pairwise comparisons (post-hoc tests) of environmental affinities of the studied groups of chasmophytic assemblages with campanula tommasiniana on mt. u~ka (nw adriatic), based on environmental data, phytosociological parameters and pignatti’s indicator values parameter syntaxa mann-whitney’s pairwise comparisons bonferroni corrected/uncorrected kruskal-wallis test h/psjcs sjcgm sjcgs sjcc cfc sacs sacm elevation sjcs 0.0001 0.0001 0.0001 0.1159 0.002 0.6103 50.82/p < 0.05 sjcgm 0.0001 0.904 0.4871 0.0025 0.0004 0.0001 sjcgs 0.01432 1 0.6768 0.0074 0.0001 0.0002 sjcc 0.0198 1 1 0.0251 0.0001 0.0001 cfc 1 0.5219 0.1539 0.5279 0.0001 0.0057 sacs 0.0376 0.0085 0.0085 0.0130 0.0161 0.0001 sacm 1 0.0016 0.0001 0.0078 0.1195 0.0038 cover. (%) of bryoph. sjcs 0.0499 0.4075 0.0363 0.0001 0.075 0.0001 76.61/p < 0.05 sjcgm 1 0.0801 0.0001 0.0001 0.0001 0.0001 sjcgs 1 1 0.0001 0.0001 0.0001 0.0001 sjcc 0.7628 0.0003 0.0133 0.0001 0.2373 0.0019 cfc 0.0002 0.0001 0.0001 0.0001 0.136 0.0001 sacs 1 0.0079 0.1817 1 1 0.4016 sacm 0.0026 0.0001 0.0001 0.0403 0.0001 1 l sjcs 0.0015 0.0025 0.2439 0.0001 0.0018 0.0001 84.91/p < 0.05 sjcgm 0.0309 0.0983 0.0027 0.0001 0.0001 0.0001 sjcgs 0.0534 1 0.0276 0.0001 0.0001 0.0001 sjcc 1 0.0573 0.5788 0.0001 0.0001 0.0001 cfc 0.0001 0.0001 0.0001 0.0001 0.1386 0.3069 sacs 0.0376 0,0086 0.0085 0.0130 1 0.3442 sacm 0,0012 0.0001 0.0001 0.0001 1 1 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 5 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 233 e c o l o g y o f c a m p a n u l a t o m m a s in ia n a tab. 3. – continued parameter syntaxa mann-whitney’s pairwise comparisons bonferroni corrected/uncorrected kruskal-wallis test h/psjcs sjcgm sjcgs sjcc cfc sacs sacm r sjcs 0.0001 0.0001 0.0025 0.0003 0.5169 0.1682 80.78/p < 0.05 sjcgm 0.0042 0.1734 0.0289 0.0001 0.0011 0.0001 sjcgs 0.0001 1 0.0001 0.0001 0.0001 0.0001 sjcc 0.052 0,6078 0,0037 0.0001 0.0057 0.0004 cfc 0,0066 0,0001 0.0001 0.0002 0.0098 0.0001 sacs 1 0.0235 0.0097 0.1203 0.2048 0.7172 sacm 1 0.0003 0.0001 0.0009 0.0273 1 n sjcs 0.4502 0.5714 0.9246 0.0004 0.0445 0.0001 67.85/p < 0.05 sjcgm 1 0.4485 0.5656 0.0001 0.0018 0.0001 sjcgs 1 1 0.9209 0.0001 0.0063 0.0001 sjcc 1 1 1 0.0001 0.0012 0.0002 cfc 0.0001 0.0001 0.0001 0.0002 0.067 0.9122 sacs 0.9436 0.0384 0.1325 0.2475 1 0.1110 sacm 0.0039 0.0004 0.0009 0.0004 1 1 u sjcs 0.0019 0.0983 0.1472 0.0001 0.008 0.0001 87.94/p < 0.005 sjcgm 0.0407 0.0313 0.0001 0.0001 0.0004 0.0001 sjcgs 1 0.6571 0.0023 0.0001 0.0001 0.0001 sjcc 1 0.0012 0.0608 0.0001 0.2479 0.0001 cfc 0.0003 0,0001 0,0001 0,0001 0,0004 0,001 sacs 0.1682 0.0085 0.0085 1 0.0088 0,012 sacm 0.0011 0.0001 0.0006 0.0014 0.0213 0.0252 sjcs – seslerio juncifoliae-campanuletum sedetosum; sjcgm – seslerio juncifoliae-campanuletum globularietosum var. micromeria thymifolia; sjcgs – seslerio juncifoliae-campanuletum globularietosum var. stachys subcrenata; cfc – cystopteri fragilis-campanuletum; sacs – seslerio autumnalis-campanuletum var. salvia officinalis; sacm – seslerio autumnalis-campanuletum var. mycelis muralis. l – light conditions, r – soil reaction, n – nutrients, u – moisture. values in bold are statistically significant. 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 5 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n subsp. variegata and syntrichia ruralis var. ruralis, prefers warmer, open to semi shaded and significantly lower-elevated sites and relatively lower soil ph reaction in an otherwise broad elevational range (tab. 3, figs. 2c, 3). sjcc subset is characterized by a significantly higher number and coverage of bryophytes, particularly ctenidium molluscum, neckera crispa and fissidens dubius and prefers moister rock crevices in higher elevated sites. the two subsets, sjcgm and sjcgs, respectively, although differing well floristically, show the only significant differentiation in site moisture where the subset sjcgm, characterized by the presence and high coverage of globularia cordifolia, hieracium bupleuroides, micromeria thymifolia and scrophularia laciniata, prefers slightly dryer and more exposed sites. among the explanatory variables, four statistically significant parameters are identified by 234 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. 1,2 1,6 2 2,4 2,8 3,2 3,6 4 4,4 0 200 400 600 800 1000 1200 1400 1600 1800 0 8 16 24 32 40 48 56 64 72 d % 4,2 4,8 5,4 6 6,6 7,2 7,8 8,4 9 9,6 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5 2,1 2,4 2,7 3 3,3 3,6 3,9 4,2 4,5 4,8 elevation (m) bryophyte coverage (%) l r un s jc s s jc s s jc g m s jc g m s jc g s s jc g s s jc c s jc c c fc c fc s a c m s s a c s s a c m s a c m s jc s s jc s s jc g m s jc g m s jc g s s jc g s s jc c s jc c c fc c fc s a c m s s a c s s a c m s a c m s jc s s jc s s jc g m s jc g m s jc g s s jc g s s jc c s jc c c fc c fc s a c m s s a c s s a c m s a c m fig. 3. box plots of environmental parameters (elevation, coverage of bryophytes) and pignatti’s indicator values (l – light conditions, r–soil reaction, n – nutrients, u – moisture) of floristically distinct chasmophytic assemblages with campanula tommasiniana on mt. u~ka (nw adriatic).syntaxonomy (x-axis) is explained in the text below. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:45 color profile: generic cmyk printer profile composite default screen cca analysis (fig. 2b) : coverage (f=2.69) and number (1.80) of bryophytes, elevation (2.70) and number of vascular plants (2.00). two subsets of assemblages is are recognized within the group sac (fig. 2d, tabs.1, 3): sacs, characterized by the exclusive occurrence, high frequency and coverage of coronilla emerus subsp. emeroides, asparagus acutifolius, quercus ilex, hedera helix and salvia officinalis, developed on significantly drier and lower elevated sites; and sacm, where the differential group of taxa is represented by cyclamen purpurascens, galeobdolon flavidum and mycelis muralis, species otherwise frequent in the beech forest understorey. here, cca analysis (fig. 2d) gives only elevation (f = 2.64) and number of bryophytes (1.74) as statistically significant explanatory variables. hemicryptophytes achieve the highest coverage in group sts and the lowest in groups sjcc and cfc (tab. 1). chamaephytes are most abundant in groups sjc, while they are absent in group cfc. in number and coverage of phanerophytes, the group sacs departs significantly from all the other groups. accodingly, no therophytes were surveyed in this group. syntaxonomy, nomenclature and typification of the syntaxa we propose three new associations, seven new lower ranked syntaxa and the following classification scheme: asplenietea trichomanis br.-bl. et maire 1934 corr. oberd. 1977 potentilletalia caulescentis br.-bl. in br.-bl. et jenny 1926 potentillion caulescentis br.-bl. in br.-bl. et jenny 1926 physoplexido-potentillenion caulescentis theurillat in theurillat et al. 1995 seslerio juncifoliae-campanuletum tommasinianae ass. nov. (sjc) sedetosum albae subass. nov. (sjcs) globularietosum cordifoliae subass. nova (sjcg) var. micromeria thymifolia var. nova (sjcgm) var. stachys subcrenata var. nova (sjcgs) ctenidietosum mollusci subass. nova (sjcc) moehringion muscosae horvat et horvati} 1962 seslerio autumnalis-campanuletum tommasinianae ass. nova (sac) (=campanuletum tommasinanae-justinianae horvati} 1960 nom. nud.) var. salvia officinalis (sacs) var. mycelis muralis (sacm) cystopteri fragilis-campanuletum tommasinianae ass. nova (cfc) characteristic group of taxa for the association seslerio juncifoliae-campanuletum are campanula tommasiniana, sesleria juncifolia, athamanta turbith and silene saxifraga subsp. hayekiana (tab. 4, on-line supplement tab. 2); differential group of taxa for the subassociation sedetosum are sedum album, satureja montana subsp. variegata and syntrichia ruralis var. ruralis; differential group of taxa for the subassociation globularietosum are globularia cordifolia and hieracium bupleuroides (micromeria thymifolia, scrophularia laciniata and stachys subcrenata, tortella densa for the variants micromeria thymifolia and stachys subcrenata, respectively); differential group of taxa for the subassociation ctenidietosum mollusci are ctenidium molluscum, neckera crispa and fissidens dubius. a characteristic group of taxa for the association seslerio autumnalis-campanuletum are acta bot. croat. 73 (1), 2014 235 ecology of campanula tommasiniana 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:45 color profile: generic cmyk printer profile composite default screen 236 a c t a b o t .c r o a t .73 (1),2014 s u r in a b .,m a r t in ^ i^ a . tab. 4. synoptic table of chasmophytic syntaxa with campanula tommasiniana on mt. u~ka (nw adriatic) association sjc cfc sac subassociation s g c variant m s s m characteristic and differential groups of taxa ppc cam tom campanula tommasiniana 100 19 100 12 100 13 100 15 100 14 100 13 100 18 es ses jun sesleria juncifolia 44 9 100 15 100 16 93 15 30 2 . 20 2 ppc ath tur athamanta turbith 78 13 53 4 72 6 79 8 9 . 36 2 ppc sil sax silene saxifraga subsp. hayekiana 67 7 76 8 72 6 . . . 4 1 kc sed alb sedum album 89 10 . . 7 2 . . 20 1 ss sat mon satureja montana 67 8 18 1 6 1 14 2 . 83 7 24 2 b syn rur syntrichia ruralis var. ruralis 56 10 12 3 . 14 2 . . 24 2 es glo cor globularia cordifolia . 88 9 89 9 14 1 . . . pc2 hie bup hieracium bupleuroides 11 2 35 2 50 4 14 2 . . 4 1 ppc mic thy micromeria thymifolia 44 6 94 10 17 1 36 3 . . 4 1 at scr lac scrophularia laciniata 33 2 53 4 17 1 43 3 . 12 1 ss sta sub stachys subcrenata . 12 2 56 5 21 2 . . . b tor den tortella densa . . 33 10 . . . . b cte mol ctenidium molluscum 11 2 18 5 11 3 93 13 100 14 50 7 64 6 b nec cri neckera crispa 22 4 6 2 17 4 64 9 96 15 17 2 76 10 b fis dub fissidens dubius . . 11 2 43 6 48 3 50 4 24 2 fs myc mur mycelis muralis . . . . 87 7 . 48 4 cf cys fra cystopteris fragilis . . . 7 1 83 8 . 8 2 tr ara alp arabis alpina . . . 14 2 52 4 . . b pla por plagiochila porelloides . . . . 43 3 . . b mni tho mnium thomsonii . . . . 30 2 . . b hom ser homalothecium sericeum 22 5 35 11 39 12 50 7 35 3 83 8 84 8 qp ses aut sesleria autumnalis 11 1 6 1 . 14 2 22 1 100 6 60 6 nec bes neckera besseri 22 3 6 1 . . 13 2 50 9 52 5 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 5 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 237 e c o l o g y o f c a m p a n u l a t o m m a s in ia n a tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m co cor eme coronilla emerus subsp. emeroides . . . . 4 1 100 8 . pra asp acu asparagus acutifolius . . . . 4 1 100 6 . qi que ile quercus ilex . . . . . 67 3 . qf hed hel hedera helix . . . . 4 1 67 3 . b lep smi leptodon smithii 11 2 12 3 . . . 67 8 8 2 ss sal off salvia officinalis . . . . . 50 2 . af cyc pur cyclamen purpurascens 22 2 6 1 . 7 2 39 3 33 1 72 4 fs gal fla galeobdolon flavidum . . . . 9 1 . 32 2 other vascular plants at asp tri asplenium trichomanes 67 10 35 3 22 2 50 4 100 11 83 6 92 11 at asp rtm asplenium ruta-muraria 100 8 76 7 44 3 79 7 96 8 17 2 64 6 gs tha min thalictrum minus 33 2 12 2 44 4 29 2 4 1 33 2 12 1 crp cym mur cymbalaria muralis 33 2 24 1 11 1 50 3 91 8 . 32 3 bv rha rup rhamnus rupestris 33 5 18 1 6 1 7 2 . 33 2 16 2 ep cal var calamagrostis varia 22 2 24 2 11 2 14 2 9 2 . 8 2 ss gal luc galium lucidum 44 2 12 2 6 1 29 3 . 33 1 gs ant ram anthericum ramosum 11 2 . 17 2 29 3 9 1 17 2 24 2 ss all ter allium saxatile subsp. tergestinum 22 2 18 1 39 2 14 2 . . 24 2 es ran car ranunculus carinthiacus . 6 1 11 1 36 2 22 2 . 8 1 qp sor ari sorbus aria 11 1 . 11 1 7 1 . 17 2 4 1 fb teu mon teucrium montanum 22 2 18 2 50 4 . . 33 2 . qp fra orn fraxinus ornus 11 1 . . . 4 1 50 5 20 2 qp ost car ostrya carpinifolia 44 3 . . 7 1 . 50 2 16 2 at sax pan saxifraga paniculata . 6 1 22 1 29 2 4 1 . . car ten carduus tenuiflorus 11 1 35 2 17 2 . . . 8 1 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 238 a c t a b o t .c r o a t .73 (1),2014 s u r in a b .,m a r t in ^ i^ a . tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m vp hie mur hieracium murorum . . 6 1 14 2 . 17 2 16 1 lps ros dum rosa dumalis 44 2 . . 7 1 4 1 . 8 1 pc1 ker sax kernera saxatilis . 12 2 6 1 . 9 2 . 8 1 tr sen rup senecio rupestris 11 1 . . 7 2 4 1 . 8 1 at cet off ceterach officinarum 56 6 . . . . 67 6 28 2 eup ill euphrasia illyrica 11 2 . 11 2 29 2 . . . es sen abr senecio abrotanifolius . 29 2 22 2 57 4 . . . cf pse alb pseudofumaria alba 22 1 . . . 30 4 . 12 2 vp val tri valeriana tripteris . . . 14 2 39 3 . 20 2 thy sp thymus sp. . 35 3 28 2 7 1 . . . at sem tec sempervivum tectorum . 12 2 17 2 29 2 . . . qp ara tur arabis turrita . . . . 4 1 33 2 36 2 fb bro ere bromus erectus agg. . . . 7 1 . 67 3 8 2 pp ade gla adenostyles glabra . . . 7 1 30 2 . 16 2 ss cam mar campanula marchesettii 11 2 12 2 17 1 . . . . cf moe mus moehringia muscosa 22 2 . . . 9 2 . 20 1 tr pel all peltaria alliacea 33 2 . . 7 1 . . 12 1 ep bup sal buphthalmum salicifolium . 6 2 . 14 2 . . 12 2 qf poa nem poa nemoralis . . . 7 2 13 2 . 8 1 bv rha sax rhamnus saxatilis . 6 1 17 1 7 2 . . . es gen sym gentiana lutea subsp. symphyandra . 12 2 6 1 14 2 . . . vp cle alp clematis alpina . 6 1 . 7 2 . . 4 1 at asp vir asplenium viride . . . 7 1 13 2 . 4 1 qf car dig carex digitata 11 1 . . . 4 1 . 8 1 m gal mol galium mollugo 11 1 . . 7 1 . . 4 1 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 239 e c o l o g y o f c a m p a n u l a t o m m a s in ia n a tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m cp cle vit clematis vitalba . . . 7 1 . 17 2 . ma ver alb veratrum album . 6 1 . . 4 1 . 4 1 tr ger mac geranium macrorhyzum 44 6 . . . . . 4 1 ppc ara sco arabis scopoliana . . 39 3 29 3 . . . ta ger rob geranium robertianum . . . . 57 4 . 12 1 ma sax rot saxifraga rotundifolia . . . . 30 3 . 4 2 tr teu aur teucrium arduinii 33 3 12 1 . . . . . ma sen fuc senecio fuchsii . . . . 35 2 . 20 2 fb car hum carex humilis . . . . . 50 2 8 1 gs ros pim rosa pimpinellifolia . 12 2 33 2 . . . . ppc cam jus campanula justiniana . . . . 26 2 . 4 1 ep all eri allium ericetorum 33 2 . . . . . 4 1 pra jun oxy juniperus oxycedrus . . . . . 33 2 4 1 tr cam coc campanula cochleariifolia . . 17 1 21 2 . . . pc1 pri aur primula auricula . . 6 1 14 2 . . . at pol vul polypodium vulgare agg. . . . . 13 2 . 8 2 ss sat lib satureja subspicata subsp. liburnica . 12 1 11 2 . . . . qp euo ver euonymus verrucosus 11 1 . . . . . 20 2 qf gal syl galium sylvaticum . . . . 9 1 . 16 2 vp las kra laserpitium krapfii . . . . 9 1 . 16 2 fb all pul allium carinatum subsp. pulchellum . . . . 4 1 33 1 . sc lig seg ligusticum seguerii 22 2 6 1 . . . . . es leo alp leontopodium alpinum . . 6 1 14 2 . . . qf pyr pir pyrus piraster 11 1 . . . . 17 2 . ppc cam pyr campanula pyramidalis . . . . . 17 2 4 1 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 240 a c t a b o t .c r o a t .73 (1),2014 s u r in a b .,m a r t in ^ i^ a . tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m qp ace obt acer obtusatum 11 1 . . . . . 4 1 es phy orb phyteuma orbiculare . 6 1 6 1 . . . . af cal gra calamintha grandiflora . . . . 4 1 . 4 1 fs fag syl fagus sylvatica . . . . 4 1 . 4 1 ma tha aqu thalictrum aquilegiifolium . . . . 4 1 . 4 1 ap lam mac lamium maculatum 22 4 . . . . . . kc sed sex sedum sexangulare 11 2 . . . . . . eup sp euphorbia sp. 11 1 . . . . . . o lil bul lilium bulbiferum 11 1 . . . . . . m lot cor lotus corniculatus 11 1 . . . . . . af rha fal rhamnus fallax 11 1 . . . . . . fb glo wil globularia willkomii . 12 2 . . . . . fb dor ger dorycnium germanicum . 6 1 . . . . . fes sp festuca sp. . 6 1 . . . . . ps jun com juniperus communis subsp. communis . 6 1 . . . . . fs lab alp laburnum alpinum . 6 1 . . . . . fb mel cil melica ciliata . 6 1 . . . . . at dia syl dianthus sylvestris agg. . . 11 1 . . . . ss gen syl genista sylvestris . . 6 1 . . . . ppc dap alp daphne alpina . . 6 1 . . . . pc2 pot cau potentilla caulescens . . 6 1 . . . . ep epi atr epipactis atrorubens . . . 7 1 . . . es eri pol erigeron polymorphus . . . 7 1 . . . fs mer per mercurialis perennis . . . . 13 2 . . fs epi mon epilobium montanum . . . . 9 1 . . 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 241 e c o l o g y o f c a m p a n u l a t o m m a s in ia n a tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m at sed his sedum hispanicum . . . . 4 1 . . ta ace pse acer pseudoplatanus . . . . 9 1 . . ma aco var aconitum variegatum . . . . 4 1 . . tr ant fum anthriscus fumarioides . . . . 4 1 . . ta aru dio aruncus dioicus . . . . 4 1 . . hier sp hieracium sp. . . . . 4 1 . . ta pol acu polystichum aculeatum . . . . 4 1 . . qp tam com tamus communis . . . . 4 1 . . qp cot cog cotinus coggygria . . . . . 17 2 . qp mel mel melittis melissophyllum . . . . . 33 2 . rub sp2 rubus sp. . . . . . 33 2 . pra lon etr lonicera etrusca . . . . . 17 1 . co vio sco viola alba subsp. scotophylla . . . . . 33 1 . sc aet sax aethionema saxatile . . . . . 17 2 . ppc cam istr campanula fenestrellata subsp. istriaca . . . . . 17 2 . ss cen rup centaurea rupestris . . . . . 17 2 . ss cre cho crepis chondrylloides . . . . . 17 2 . r hel ita helichrysum italicum . . . . . 17 2 . crp par jud parietaria judaica . . . . . 17 2 . gs peu cer peucedanum cervaria . . . . . 17 2 . qp que pub quercus pubescens . . . . . 17 2 . qf ros arv rosa arvensis . . . . . 17 2 . rub sp1 rubus sp. . . . . . 17 2 . rub ulm rubus ulmifolius . . . . . 17 2 . qp dig lae digitalis laevigata . . . . . . 16 2 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 242 a c t a b o t .c r o a t .73 (1),2014 s u r in a b .,m a r t in ^ i^ a . tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m fb dia mon dianthus monspessulanus . . . . . . 8 2 qp mer ova mercurialis ovata . . . . . . 12 2 fb lin cat linum catharticum . . . . . . 4 1 qp cam per campanula persicifolia . . . . . . 4 1 vp ros pen rosa pendulina . . . . . . 4 1 tg vin hir vincetoxicum hirundinaria . . . . . . 8 1 fb ara hir arabis hirsuta . . . . . . 4 1 qp cni sil cnidium silaifolium . . . . . . 4 1 tr cys mon cystopteris montana . . . . . . 4 1 af eup car euphorbia carniolica . . . . . . 4 1 qp hyp mon hypericum (montanum?) . . . . . . 4 1 fb koe pyr koeleria pyramidata . . . . . . 4 1 m leo his leontodon hispidus . . . . . . 4 1 ros sp rosa sp. . . . . . . 4 1 fs sal glu salvia glutinosa . . . . . . 4 1 at sed max sedum maximum . . . . . . 4 1 qp tan cor tanacetum corymbosum . . . . . . 4 1 b bryophyptes and lichens tor tor tortella tortuosa 67 9 82 31 78 21 86 13 52 4 67 7 72 6 sch sp schistidium sp. 44 6 47 17 50 15 36 4 65 5 33 3 68 6 hom phi homalothecium philippeanum 22 5 6 2 17 4 43 7 57 6 33 4 52 6 tor nit tortella nitida 33 6 18 5 17 5 7 2 4 1 50 5 20 2 bry sp bryum sp. 44 6 6 2 6 2 29 3 35 2 17 2 24 2 hyp cup hypnum cupressiforme var. cupressiforme 11 2 6 2 6 2 21 2 4 1 33 3 24 2 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 243 e c o l o g y o f c a m p a n u l a t o m m a s in ia n a tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m pla str plasteurhynchium striatulum 11 2 6 1 . 7 1 57 6 50 6 80 8 enc str encalypta streptocarpa 11 2 12 4 . 29 4 17 1 33 3 24 2 por pla porella platyphylla 11 2 . . 14 2 13 1 33 3 48 4 wei sp weissia sp. . . 6 2 14 2 4 1 33 3 20 1 pse cat pseudoleskeela catenulata . . 6 2 14 2 39 3 17 2 4 2 ano vit anomodon viticulosus 33 7 . . . 30 3 33 4 68 8 ort cup orthotrichum cupulatum 44 10 18 6 . . 4 1 . 36 3 rad com radula complanata . . . 29 4 57 5 17 2 36 2 col cal cololejeunea calcarea . . . 14 2 30 2 50 5 8 1 nec com neckera complanata . . . 7 1 39 2 17 2 56 5 dit fle ditrichum flexicaule 11 2 . 28 8 29 4 . . . sca sp scapania sp. . . 17 5 7 2 4 1 . . wei con weissia controversa var. controversa 22 3 6 2 . . . . 4 1 hyp lac hypnum cupressiforme var. lacunosum . 6 2 11 3 . . . 4 1 enc vul encalypta vulgaris . . 11 2 7 1 9 1 . . nec pen neckera pennata . . . . 13 2 17 1 8 1 ort ano ortotrichum anomalum . . . . 4 1 33 2 4 1 myu jul myurella julacea . 6 1 . 7 1 13 2 . . sco cir scorpiurum circinatum . . . . 4 1 33 5 . sca asp scapania aspera . . 6 2 21 4 . . . ped int pedinophyllum interruptum . . . . 30 3 . 4 1 ano att anomodon attenuatus . . . . 9 2 . 4 1 tor fra tortella tortuosa var. fragillifolia 11 2 . . . . . 8 2 bra tom brachythecium tommasinii . . . . 22 2 . 8 1 cir cra cirriphyllum crassinervium . . . . 22 2 . 4 1 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n 244 a c t a b o t .c r o a t .73 (1),2014 s u r in a b .,m a r t in ^ i^ a . tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m sci flo eurhynchium flotowianum . . . 7 2 . . 4 1 ort int orthothecium intricatum . . . . 4 1 . 4 1 fru dil frullania dilatata . . . . 4 1 . 4 1 lep sp leptogium sp. 22 3 . . . . . . tor bre tortella tortuosa var. brevifolia 11 2 . . . . . . fis tax fissidens taxifolius ssp. taxifolius 11 2 . . . . . . tor mur tortulla muralis var. muralis 11 2 . . . . . . gri pul grimmia pulvinata . . 6 2 . . . . cam elo campylium elodes . . 6 2 . . . . bar cro barbula crocea . . . 14 2 . . . ort sp ortotrichum sp. . . . 7 2 . . . dis cap distichium capillaceum . . . 7 1 . . . jun sub jungermannia subulata . . . 7 2 . . . mni mar mnium marginatum . . . . 13 2 . . apo pub apometzgeria pubescens . . . . 4 2 . . pla cus plagiomnium cuspidatum . . . . 4 2 . . pla und plagiomnium undulatum . . . . 4 2 . . tha alo thamnobryum alopecurum . . . . 4 2 . . thu tam thuidium tamariscinum . . . . 4 2 . . euc ver eucladium verticillatum . . . . 4 1 . . eur str eurhynchium striatum . . . . 4 1 . . mni ste mnium stellare . . . . 4 1 . . hom lut homalothecium lutescens . . . . 4 1 . . bra sta brachythecium starkei . . . . 4 1 . . eur sch eurhynchium schleicheri . . . . 4 1 . . 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n a c t a b o t .c r o a t .73 (1),2014 245 e c o l o g y o f c a m p a n u l a t o m m a s in ia n a tab. 4. – continued association sjc cfc sac subassociation s g c variant m s s m met con metzgeria conjugata . . . . 4 1 . . sol sac solorina saccata . . . . 4 1 . . syn mon syntrichia montana . . . . . 17 2 . leu sci leucodon sciuroides . . . . . . 20 2 bar sp barbula sp. . . . . . . 4 1 bra sal brachythecium salebrosum . . . . . . 4 1 pre qua preissia quadrata . . . . . . 4 1 hyp and hypnum andoi . . . . . . 4 1 hyp jut hypnum jutlandicum . . . . . . 4 1 pte fil pterigynandrum filiforme . . . . . . 4 1 sca aeq scapania aequiloba . . . . . . 4 1 wei bra weissia brachycarpa . . . . . . 4 1 af-aremoniio-fagion; ap-aegopodion podagrariae; b-bryophytes and lichens; bv-berberidion; cf-cystopteridion fragilis; co-carpinion orientalis; cp-crataego-prunetea; crp-centrantho rubri-parietarion; ep-erico-pinetea; fb-festuco-brometea; gs-geranion sanguinei; lps-ligustro-prunenion spinosae; pc1-potentilletalia caulescentis; pp-petasition paradoxi; ppc-physoplexido-potentillion caulescentis; qf-querco-fagetea; qi-quercetea ilicis; qp-quercetalia pubescentis; es-elyno-seslerietea; kc-koelerio-corynephoretea; m-mollinio-arrhenatheretea; ma-mulgedio-aconitetea; ss-satureijon subspicatae; pc2-potentillion caulescentis; o-origanetalia vulgaris; pra-pistacio-rhamnetea alaterni; ps-prunetalia spinosae; r-rosmarinetea; sc-stipetalia calamagrostis; at-asplenietea trichomanis; fs-fagetalia sylvaticae; ta-tilio-acerion; tg-trifolio-geranietea; tr-thlaspietea rotundifolii; tr-thlaspietalia rotundifolii; vp-vaccinio-piceetea. sjcs – seslerio juncifoliae-campanuletum sedetosum; sjcgm – seslerio juncifoliae-campanuletum globularietosum var. micromeria thymifolia; sjcgs – seslerio juncifoliae-campanuletum globularietosum var. stachys subcrenata; cfc – cystopteridi fragilis-campanuletum; sacs – seslerio autumnalis-campanuletum var. salvia officinalis; sacm – seslerio autumnalis-campanuletum var. mycelis muralis. 8 1 5 s u r i n a a n d m a r t i n c i c . p s u : \ a c t a b o t a n i c a \ a c t a b o t a n 1 1 4 \ 8 1 5 s u r i n a a n d m a r t i n c i c . v p 2 4 . o u j a k 2 0 1 4 1 1 : 2 8 : 4 6 c o l o r p r o f i l e : g e n e r i c c m y k p r i n t e r p r o f i l e c o m p o s i t e d e f a u l t s c r e e n campanula tommasiniana, sesleria autumnalis, homalothecium sericetum and neckera besseri (tab. 4, on-line supplement tab. 4); a differential group of taxa for the variant salvia officinalis are coronilla emerus subsp. emeroides, quercus ilex, asparagus acutifolius, hedera helix, leptodon smithii and salvia officinalis, and for the variant mycelis muralis: cyclamen puprurascens, mycelis muralis and galeobdolon flavidum. a characteristic group of taxa for the association cystopteri fragilis-campanuletum are campanula tommasiniana, mycelis muralis, cystopteris fragilis, geranium robertianum, arabis alpina, plagiochila porelloides and mnium thomsonii (tab. 4, on-line supplement tab. 3). characteristic and differential taxa for the newly described syntaxa are indicated in fig. 2b. seslerio juncifoliae-campanuletum tommasinianae ass. nov. globularietosum cordifoliae subass. nova var. micromeria thymifolia var. nova. nomenclatorial type for the association, subassociation and variant (holotypus): croatia, nw adriatic, liburnian karst, mt. u~ka, eastern slope; elev. 1287 m, exp. ese, incl. 90°; relevé area: 8 m2, coverage of the relevé area: vascular plants-10%, bryophytes-1%; calcareous rocky outcrop not shaded by the tree canopy (rel. no 22 in on-line supplement tab. 2): sesleria juncifolia 3, campanula tommasiniana 2, globularia cordifolia 2, homalothecium sericeum 2, micromeria thymifolia 2, saxifraga paniculata 2, scrophularia laciniata 2, silene saxifraga subsp. hayekiana 2, thymus sp. 2, tortella nitida 2, athamanta turbith 1, carduus tenuiflorus 1, hieracium bupleuroides 1, syntrichia ruralis var. ruralis 1. seslerio juncifoliae-campanuletum tommasinianae globularietosum cordifoliae var. stachys subcrenata var. nova. nomenclatorial type for the variant (holotypus): croatia, nw adriatic, liburnian karst, mt. u~ka, western slope; elev. 1278 m, exp. ssw, incl. 90°; relevé area: 10 m2, coverage of the relevé area: vascular plants-20%, bryophytes-1%; calcareous rocky outcrop not shaded by the tree canopy (rel. no 38 in on-line supplement tab. 2): campanula tommasiniana 4, sesleria juncifolia 4, asplenium ruta-muraria 3, athamanta turbith 3, ditrichium flexicaule 3, globularia cordifolia 3, tortella densa 3, t. tortuosa 3, schistidium sp. 3, senecio abrotanifolius 2, silene saxifraga subsp. hayekiana 2, stachys subcrenata 2, teucrium montanum 2, carduus tenuiflorus 1, sempervivum tectorum 1, primula auricula 1. seslerio juncifoliae-campanuletum tommasinianae sedetosum albae subass. nova. nomenclatorial type for the subassociation (holotypus): croatia, nw adriatic, liburnian karst, mt. u~ka, eastern slope; elev. 760 m, exp. ne, incl. 85°; relevé area: 20 m2, coverage of the relevé area: vascular plants-20%, bryophytes-10%; calcareous rock not shaded by the tree canopy (rel. no. 3 in on-line supplement tab. 2): campanula tommasiniana 4, sesleria juncifolia 4, allium saxatile subsp. tergestinum 3, anomodon viticulosus 3, asplenium ruta-muraria 3, athamanta turbith 3, ceterach officinarum 3, ctenidium molluscum 3, ditrichium flexicaule 3, homalothecium sericeum 3, hypnum cupressiforme var. cupressiforme 3, leptodon smithii 3, neckera besseri 3, n. crispa 3, orthotrichum cupulatum 3, schistidium sp. 3, silene saxifraga subsp. hayekiana 3, syntrichia ruralis var. ruralis 3, tortella tortuosa 3, rhamnus rupestris 2, sedum album 2, allium ericetorum 1, asplenium trichomanes 1, satureja montana subsp. variegata 1. seslerio juncifoliae-campanuletum tommasinianae ctenidietosum mollusci subass. nova. nomenclatorial type for the subassociation (holotypus): croatia, nw adriatic, liburnian karst, mt. u~ka, eastern slope; elev. 1235 m, exp. se, incl. 85°; relevé area: 10 m2, coverage of the relevé area: vascular plants-40%, bryophytes-10%; calcareous rock not shaded by the 246 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:46 color profile: generic cmyk printer profile composite default screen tree canopy (rel. no. 45 in on-line supplement tab. 2): calamagrostis varia 4, sesleria juncifolia 4, asplenium ruta-muraria 3, a. trichomanes 3, athamanta turbith 3, campanula tommasiniana 3, galium corrudifolium 3, anthericum ramosum 2, bryum sp. 2, ctenidium molluscum 2, cymbalaria muralis 2, euphrasia illyrica 2, fissidens dubius 2, homalothecium sericeum 2, micromeria thymifolia 2, neckera crispa 2, saxifraga paniculata 2, schistidium sp. 2, scrophularia laciniata 2, sedum album 2, senecio abrotanifolius 2, syntrichia ruralis var. ruralis 2, tortella tortuosa 2, homalothecium philippeanum 1, hypnum cupressiforme var. cupressiforme 1, ranunculus carinthiacus 1, silene saxifraga subsp. hayekiana 1, thalictrum minus 1, gentiana lutea subsp. symphyandra +. seslerio autumnalis-campanuletum tommasinianae var. mycelis muralis ass. and var. nova. nomenclatorial type for the association and the variant (holotypus): croatia, nw adriatic, liburnian karst, mt. u~ka, western slope; elev. 1055 m, exp. e, incl. 85°; relevé area: 16 m2, coverage of the relevé area: vascular plants-40%, bryophytes-30%; calcareous rock fully shaded by the tree canopy (rel. no. 28 in on-line supplement tab. 4): anomodon viticulosus 5, neckera crispa 5, campaula tommasiniana 4, pseudofumaria alba 4, asplenium trichomanes 3, ctenidium molluscum 3, cyclamen purpurascens 3, galeobdolon flavidum 3, geranium robertianum 3, homalothecium sericeum 3, mercurialis ovata 3, moehringia muscosa 3, mycelis muralis 3, neckera besseri 3, n. complanata 3, plasteurhynchium striatulum 3, porella platyphylla 3, valeriana tripteris 3, arabis turrita 2, athamanta turbith 2, brachythecium tommasinii 1, galium sylvaticum 1, hypnum cupressiforme var. cupressiforme 1, laserpitium krapfii 1, radula complanata 1, senecio fuchsii 1, sesleria autumnalis 1, s. juncifolia 1, ceterach officinarum +, peltaria alliacea +. seslerio autumnalis-campanuletum tommasinianae var. salvia officinalis var. nova. nomenclatorial type for the variant (holotypus): croatia, nw adriatic, liburnian karst, mt. u~ka, eastern slope; elev. 396 m, exp. se, incl. 90°; relevé area: 18 m2, coverage of the relevé area: vascular plants-30%, bryophytes-1%; calcareous rock semi shaded by the tree canopy (rel. no. 1 in on-line supplement tab. 4): campanula tommasiniana 4, coronilla emerus subsp. emeroides 3, homalothecium sericeum 3, leptodon smithii 3, satureja montana subsp. variegata 3, schistidium sp. 3, teucrium montanum 3, tortella nitida 3, weissia sp. 3, arabis turrita 2, asparagus acutifolius 2, asplenium trichomanes 2, bromus erectus agg. 2, ceterach officinarum 2, juniperus oxycedrus 2, ostrya carpinifolia 2, rhamnus rupestris 2, sesleria autumnalis 2, galium corrudifolium 1, hedera helix 1, neckera besseri 1, ortotrichum anomalum 1, quercus ilex 1, salvia officinalis 1, thalictrum minus 1. cystopteri fragilis-campanuletum tommasinianae ass. nova. nomenclatorial type for the association (holotypus): croatia, nw adriatic, liburnian karst, mt. u~ka, eastern slope; elev. 1297 m, exp. ne, incl. 90°; relevé area: 12 m2, coverage of the relevé area: vascular plants-40%, bryophytes-60%; calcareous rock fully shaded by the tree canopy (rel. no. 3 in on-line supplement tab. 3): neckera crispa 7, pseudofumaria alba 7, campanula tommasiniana 6, ctenidium molluscum 6, anomodon viticulosus 4, brachythecium tommasinii 4, homalothecium sericeum 4, mnium marginatum 4, plasteurhynchium striatulum 4, saxifraga rotundifolia 4, arabis alpina 3, asplenium trichomanes 3, bryum sp. 3, cymbalaria muralis 3, cystopteris fragilis 3, geranium robertianum 3, mnium thomsonii 3, mycelis muralis 3, neckera pennata 3, pedinophyllum interruptum 3, plagiochila porelloides 3, pseudoleskeela catenulata 3, schistidium sp. 3, asplenium ruta-muraria 2, adenostyles glabra 1, anomodon attenuatus 1, epilobium montanum 1, neckera complanata 1, acta bot. croat. 73 (1), 2014 247 ecology of campanula tommasiniana 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:47 color profile: generic cmyk printer profile composite default screen discussion niche assembly and ecology campanula tommasiniana inhabits rock crevices that are remarkably diverse in the number of vascular plants and bryophytes. the number of coverage vascular plants vs. bryophytes varies substantially and depends much on microsite conditions; bryophytes prevail over vascular plants in fully shaded and moist microsites, e.g. in group of assemblages cfc (cystopteridi-campanuletum), while in open and exposed sites (sjc – seslerio juncifoliae-campanuletum), vascular plants completely prevail over bryophytes. in our survey, one new bryophyte, tortella densa, was recorded for croatia for the first time and the finding was reported in detail elsewhere (surina and martin^i^ in ellis et al. 2012). in comparable studies (but inferred from a different number of samples/relevés; bryophytes and lichens excluded) botanists found a considerably lower number of vascular plant taxa in niche assemblies of chasmophytic narrow endemics. for example: pignatti and pignatti (1978), for campanula morettiana, restricted to the italian dolomites, in an elevational range between 1730–2450 m, recorded 38 taxa of vascular plants in 15 relevés (5–15 per rel., median=8), while the accompanying flora of geographically even more restricted moehringia tommasinii (caryophyllaceae) from northern istria, in an elevational range between 150–360 m, numbers 32 taxa of vascular plants recorded in 9 relevés (4–13 per rel., median=11) (martini 1990). however, in a detailed study on the phytosociological characteristics of the sites of moehringia villosa (dakskobler 2000), restricted to the southern julian alps and the adjacent prealps, 156 taxa of vascular plants in 88 relevés (5–30 per rel., median=15) sampled in an elevational range between 430–1830 m were recorded, which is comparable to the results of our study. both campanula tommasiniana and moehringia villosa occupy rock crevices of relatively broad elevational range, forming floristically and ecologically well characterized but different syntaxa. according to the results of an extensive study (lavergne et al. 2004), endemic taxa compared to widespread congeners differ in a number of ecological characteristics (their habitat in terms of abiotic and community characteristics) and biological traits (floral traits and the size and maternal fertility of individual plants), but not in levels of herbivory and levels of ecophysiological traits (specific leaf area, leaf dry matter content, leaf nitrogen concentration, and rates of photosynthesis). the principal component of the difference concerns the occurrence of endemic taxa in rocky and steep slopes in low, open habitats rather than forest vegetation (compare grove and rackham 2003, quezél and medail 2003), with low species richness, regardless of geological bedrock. highly specialized conditions in the physical environment select for these differences in small, ecologically and taxonomically isolated (endemic) populations, since rock crevices and screes experience a range of microclimatic conditions that are very different from both level rocky terrain and horizontal landscapes with soil. however, campanula tommasiniana, despite being almost an obligate chasmophyte (see surina 2013), indicates a remarkable ability to adapt to various abiotic factors, but a relatively low number of taxa per microsite suggests low biotic competitiveness. in contrast to the similarly rich floristic assembly of moehringia villosa, the niche assembly of campanula tommasiniana constitutes only three taxa of vascular plants, all rockdwellers: campanula tommasiniana, asplenitum trichomanes, a. ruta-muraria), occurring in more than 50% of all samples, which additionally speaks for its high abiotic plasticity and niche differentiation. the most important ecological gradients in shaping the floristic assemblies are light 248 acta bot. croat. 73 (1), 2014 surina b., martin^i^ a. 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:47 color profile: generic cmyk printer profile composite default screen conditions and moisture. according to field diagnostics, results of numerical and statistical analyses based on groups of characteristic and differential taxa among vascular plants, bryophytes as well as site ecology, three floristically and ecologically well defined groups of assemblages are identified. the assemblage sj-c – seslerio juncifoliae-campanuletum is the most homogenous in terms of floristical composition, while the assemblage cfc – cystopteri-campanuletum has the most uniform site ecology. this reflects also the life form spectrum, where chamaephytes, adapted to open sites, do not thrive at all in the assemblage cysopteri-campanuletum. our research proved the high diagnostic values of bryophytes for the delimitation of groups of assemblages based on floristic composition. several taxa occur exclusively or almost exclusively in floristically and ecologically well differentiated groups of assemblages. however, their diagnostic values for site conditions are less significant, a fact already stressed and discussed by surina and martin^i^ (2012). results of our analyses suggest that campanula tommasiniana is a chasmophyte of open and exposed habitats in the altimontane to subalpine vegetation belt, a similar niche preference (see horvat 1931) also for the closely related (see park et al. 2006, liber et al. 2008) but allopatric c. waldsteiniana, another endemic of the north-western dinaric alps. campanula tommasiniana experiences a range of microclimatic conditions along ecological gradients building three floristically and ecologically well defined and differentiated groups of chasmophytic assemblages, suggesting its high ecological plasticity and abiotic stress tolerance. hence, the reason for its limited range, restricted to an area of 6–7 km2, remains a puzzling question. another stenoendemic, hladnikia pastinacifolia (apiaceae), a monotypic species with even narrower distribution area, restricted to a few localities of the trnovski gozd plateau in north-western dinaric alps (mayer 1960, ^u[in 2004), colonizes an even broader range of habitats – stony grasslands, rock crevices and screes ([ajna et al. 2012), reflecting an ability to adapt to a variety of both abiotic and biotic factors. nevertheless, h. pastinacifolia, like campanula tommasiniana, remains confined to an extremely narrow distribution area, despite the many available habitats in the vicinity, also without any reasonable explanation. nomenclatorial and synsystematic issues stands with campanula tommasiniana were first mentioned by horvati] (1944), who recognized its diagnostic value and distinct floristic assemblages, naming the association campanuletum tommasinianae horvati} 1944. in the 1960s horvati} mentioned and cited the association’s name as campanuletum tommasinianae-justinianae horvati} 1960 (horvati] 1960, 1963b). he found campanula tommasiniana, c. justiniana, hieracium bifidum and h. amplexicaule subsp. petraeum (recte h. bupleuroides) to be regionally characteristic taxa for the association occurring on calcareous cliffs and blocks within the thermophytic beech forests of the association seslerio autumnalis-fagetum. he classified it within the alliance moehringion muscosae. to that end, trinajsti] (2008) in his plant communities of croatia, without giving a specific reason, found the valid publication for the association horvati}’s treatise from 1963. however, neither a supplementary analytical and/or synoptic table nor designation of a type relevé were provided, and both botanists failed to describe the association according to the rules of phytosociological nomenclature (compare weber et al. 2000). beside purely nomenclatorial issues, our results show significantly more comacta bot. croat. 73 (1), 2014 249 ecology of campanula tommasiniana 815 surina and martincic.ps u:\acta botanica\acta-botan 1-14\815 surina and martincic.vp 24. o ujak 2014 11:28:47 color profile: generic cmyk printer profile composite default screen plex problems in syntaxonomics of communities with campanula tommasiniana. since three groups of assemblages are easily recognized, both floristically and ecologically, and even classified in two different alliances, we find the validation of the name and the description of additional syntaxa based on a sound association concept (willner 2006), and following the rules of phytosociological nomenclature (weber et al. 2000), to be fully justified. while we find the differentiation and status of the newly described syntaxa not questionable, their syntaxonomic position within higher ranked syntaxa remains problematic. hence our proposal is still only preliminary since it is evident that earlier attempts (e.g., horvat 1962, horvati] 1963b, trinajsti] 1980, trinajsti] 2008) at stabilization of a chasmophytic syntaxonomic system in the northern adriatic do not suffice, and therefore a thorough numerical revision is required. acknowledgements florijan tomc and filip star~evi} (both u~ka nature park, liganj), marko randi} (public institution »priroda«, rijeka) and @eljka modri} surina (natural history museum rijeka, rijeka) helped substantially during the field work. borut kru`i} (natural history museum rijeka, rijeka) offered technical help in data digitalization. igor dakskobler (institute of biology, science and research centre of the slovenian academy of sciences and arts, ljubljana) helped with some literature data and gave plenty of advice in floristic data treatment, while @eljka modri} surina, antun alegro and toni nikoli} (both university of zagreb, faculty of science, department of botany and the botanical garden, zagreb) commented on previous version of the manuscript. many thanks to all of them. the research was financially supported by u~ka nature park and natural 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(umbelliferae juss.) family (šajna et al. 2012). furthermore, h. pastinacifolia is found only on the trnovski gozd karst plateau in western slovenia where it has a very narrow distribution area of 4 km2. because of its rarity, it has been placed on the red list and is among the species mentioned in annexe ii of the habitat directive (council directive 1992). the entire distribution area is included in the natura 2000 network as a site of community interest (čušin 2004). molecular analyses showed that h. pastinacifolia is a pleistocene survivor in situ and has widened its distribution little since the end of glaciation (šajna et al. 2012). however, the species is not a habitat specialist and can be found * corresponding author, e-mail: nina.sajna@uni-mb.si copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. šajna n., šuštar-vozlič j., kaligarič m. 376 acta bot. croat. 73 (2), 2014 growing in various habitats such as stony grasslands, rock crevices and screes. among habi tats, screes are priority habitats listed in annexe i of the habitat directive (bern convention 1979). since h. pastinacifolia is not a habitat specialist (wraber 1990) it could be expected elsewhere in the dinaric mountains further to the southeast (nikolić and topić 2004). the concise description of h. pastinacifolia is based on sušnik (1964). hladnikia pastinacifolia is monocarpic annual or biennial plant (hegi 1975), also characterized as monocarpic perennial species (reichenbach 1830–1833). plants are herbaceous, 15–30 cm high with long lignifi ed root. plants form only basal rosettes of numerous glossy and leathery leaves for several years before fl owering. the fl owering stem is erect. at fi rst only one compound umbel is formed and later the stem branches and forms several lateral compound umbels. a single umbel consists of twenty rays. the involucre and involucel are multifoliate. petals are white, heart-shaped, 1 mm long. pistil has a prolonged style, which ends with a claviform stigma. stylopodium is shorter than the style and rounded. the fruit is a typical schizocarp, which separates into two mericarps 4 mm long and 2 mm wide. seed testa and fruit pericarp are fused. most of the seed is occupied by endosperm, embryo is small and underdeveloped. cross-sections through mericarps show protruding ribs and numerous smaller oil ducts. knowledge about the biology and ecology of rare and protected species is essential for their protection. furthermore, anatomical characteristics, especially fruit anatomy, are very important for the taxonomy of apiaceae. the doctoral dissertation of sušnik (1964) was the leading work dealing with h. pastinacifolia morphology, anatomy, and taxonomy, relying on the means available at the time. according to his interest in taxonomic questions, the most important contributions of his thesis were the determination of the chromosome number (2n = 22) and the comparison of h. pastinacifolia fruit extracts with falcaria vulgaris bernh. and representatives of the genera oenanthe l. and athamanta l. with paper chromatography. additionally, he described the morphology and placement of fl oral elements and the anatomy of the mericarp (fertile mericarp sensu sušnik (1964), since he hypothesized that in each fruit only one mericarp contains an embryo as a consequence of a mutation). further on, he studied the morphology of leaf lamina and its changes during the plant’s development. plant parts that had no taxonomic value were addressed very briefl y. sušnik (1964) reported results about morphology and anatomy in a descriptive manner, and hardly any measurements were made. later research concerning h. pastinacifolia was mainly focused on syntaxonomy (martinčič 1958, martinčič 1961, poldini 1978, kaligarič 1997, kaligarič and poldini 1997, dakskobler 1998, dakskobler 2006). recently, research into the species’ ecology was undertaken (šajna et al. 2009, šajna et al. 2011, šajna et al. 2012), as well as population genetics and phylogeny (šajna et al. 2012). sušnik (1964) did not perform anatomical studies on h. pastinacifolia, and accordingly we have endeavoured to complete the anatomical investigation with the use of contemporary techniques. we focus on characteristics, which are ecologically important from the point of view of species persistence in time. we have described new characteristics concerning leaf stomata and gathered new information about pollen characteristics and root anatomy. material and methods sampling quantities and sampling locations were determined by a permit issued by the republic of slovenia’s institute for nature conservation, which allowed us to sample only anatomy of hladnikia pastinacifolia acta bot. croat. 73 (2), 2014 377 a few plants and their parts e.g. 30 leaves, 50 fruits. the proposed sampling location was a low-intensity pasture at predmeja (trnovski gozd, slovenia). mature fruits were sampled on 17th september 2004, leaves on 19th may 2005, and roots, as well as fl owers on 28th june 2005. all voucher materials are preserved in faa (formalin–acetic acid–alcohol), except dry fruits, and are deposited in the herbarium of the faculty of natural sciences and mathematics, university in maribor (slovenia). for the preparation of microscopic slides, we used fresh material or material fi xed with faa. sections were performed by hand and observed in water. for the indication of root lignifi ed tissues phloroglucinol dissolved in ethanol and concentrated hcl were used. during the histochemical reaction the cell walls, containing lignin, show a red coloration (dietz and ullmann 1998). we collected 3 roots from a fl owering specimen. part of the root 1 cm below the transition zone with the stem was sectioned. we studied the sections under a nikon eclipse 50i microscope. additionally, we observed primary fl uorescence of unfi xed, unstained material with epi-illumination with blue light excitation. the source was a c-lhg1 mercury lamp hg 100 w. autofl uorescence in fruit sections (n = 10) was observed after 10 minutes of irradiation. measurements under the microscope were performed with eclipse net software (nikon, waver, belgium). to check whether in each fruit one mericarp fails to develop an embryo, we choose 30 fruits randomly from 6 main umbels. we used hand-made epidermal peels to obtain micrographs for calculations of stomata density (sd; number of stomata per mm2), density of epidermal cells (ed; number of epidermal cells per mm2) and stomata index (si% = sd/(sd + ed) × 100). we conducted one count for the abaxial and adaxial sides for 30 fully developed leaves, which had lobate laminae with 5 segments. we chose the lowest leaf segments to study. leaf petiole was sectioned 5 mm before the fi rst leaf segments. we described pollen shape with the ratio of the length of the polar axis to the equatorial diameter (p/e ratio). by counting pollen grains of a single anther (n = 5), we quantifi ed the number of pollen grains and calculated the pollen–ovule ratio (p/o ratio). results flowers of h. pastinacifolia are protandric which means that the stamen ripens before the gynoecium at the time of anthesis. pollen grains (fig. 1) are trizonocolporate, oval, 44.0±1.1 mm long and 18.9±0.9 mm wide (n = 15). pollen shape according to p/e ratio was fig. 1. pollen grains of h. pastinacifolia. a – pollen grains inside the anther (autofl uorescence of fi xed material); b – trizoncolporate pollen grains. šajna n., šuštar-vozlič j., kaligarič m. 378 acta bot. croat. 73 (2), 2014 2.33±0.11. inside the anthers, there were 805±45 pollen grains. average p/o ratio for the whole fl ower with 5 stamens and 2 ovules was 4025/2. at the time of anther maturity, both styles are positioned in parallel before the stigma becomes receptive (fig. 2a). when the stigma does become receptive, it widens and takes on a claviform shape (fig. 2b), while both styles edge away from each other. fig. 2. micrographs of stigma shape during development. a – non-receptive stigma; b – receptive stigma with various pollen grains and fungal sclerotium (arrow). on the mature fruit the calyx teeth, style, and stylopodium remain present. at the time of maturity the schizocarp splits into two mericarps which remain attached to the carpophore (fig. 3). the groups of sclerenchyma cells of the dual carpophore are positioned opposite each other in the ventral part of the commissure. the pericarp, measured between the ribs, is 130 mm wide. the exocarp is formed of a single layer of isodiametric cells with a cutinized cell wall. the mesocarp is parenchymatous with 5 protruding ribs. intrajugal oil ducts are round. below them a sclerenchyma stereome with vascular tissue is found. two vascular bundles are in each commissural half. under the valeculae (depressions between ribs) six fl attened oil ducts (vittae) are present. after the drying out of the mericarp at maturity, inside a rib the intrajugal oil ducts become tightly compressed and the secretory ducts shrink (fig. 3d). in a dry mericarp, the vittae measure 40–60 mm in diameter. from longitudinal sections of mericarps we recognized an axial-linear embryo type (nomenclature after martin 1946), which is narrow, fl at and much longer than it is wide (picture not shown). fruits are homomericarpic or heteromericarpic if one mericarp does not develop properly (fig. 4). cross-sections of dry mature schizocarps from the main umbel showed that in approximately 60% of fruits both mericarps are fully developed and contain embryos. in 35% of fruits only one mericarp contains an embryo, while in 6% neither mericarp has an embryo. in a single umbellet all three combinations of fruits can be observed (fig. 4). leaves of h. pastinacifolia are amphistomatal. stomata are evenly distributed in upper and lower epidermis. stomata are mostly of the anisocytic type, since one of the accompanying cells is smaller than the rest. stomata of abaxial epidermis are 34.6±5.5 mm long, while in the adaxial side they measure 37.7±4.9 mm in length. stomata density for the abaxial side is between 25 and 31 stomata per mm2, ed was about 113 mm–2, si was on average 20%. calculated values for the adaxial epidermis were: sd = 15 mm–2, ed = 91 mm–2, si = 14%. five to nine vascular bundles are found in leaf petiole (fig. 5). numerous secretory ducts are present in the petiole above and under each vessel, while a single secretory duct is presanatomy of hladnikia pastinacifolia acta bot. croat. 73 (2), 2014 379 ent next to and a single one inside the phloem (fig. 5c). collenchyma is positioned subepidermally. the petiole parenchyma does not have any very big intercellular spaces. root cross-sections of h. pastinacifolia showed that secondary xylem and secondary phloem are well developed (fig. 6). secondary xylem exhibited characteristics that could be used for the age estimation of individuals, since we could distinguish delineated annual growth rings (fig. 6b). we were able to recognize growth rings according to groups of largefig. 4. variety of schizocarps in an umbellet of h. pastinacifolia. a – one mericarp does not contain an embryo (*), both mericarps fail to develop an embryo (arrow), and both mericarps are fully developed containing embryos (two arrows); b – cross-section of a shizocarp with one fully developed mericarp and one without embryo (*). fig. 3. cross-section of h. pastinacifolia schizocarp. a – cross-section of an immature schizocarp; b – autofl uorescence of immature schizocarp; c – detail of rib section; d – mature dry mericarp in detail (note the high accumulation of anthocyanins). letters indicate marginal rib (mr), median rib (mer), lateral rib (lr), carpophore (ca), exocarp (e), mesocarp (m), endocarp (en), secretory duct inside the rib (sd), valecular oil duct (vod), sclerenchyma tissue (st), vascular bundle (v). šajna n., šuštar-vozlič j., kaligarič m. 380 acta bot. croat. 73 (2), 2014 sized vessels formed in the beginning of each growing season. therefore we could distinguish early wood and late wood. the rays of vascular elements were wide and often branched at the beginning of the growing season. primary xylem in the center is followed by three annual growth rings (fig. 6b). therefore, we can determine that this specimen was 4 years fig. 5. leaf petiole of h. pastinacifolia. a – cross-section showing 7 vascular bundles; b – autofl uorescence of petiole showing yellow fl uorescence of secretory ducts (sd); c – numerous secretory ducts are present in parenchyma (sdp); some accompany vascular bundles (sdv) and some are found within the phloem (sdph). letters indicate vascular bundles (v), collenchyma (c), xylem (xy), phloem (ph). fig. 6. cross-section of h. pastinacifolia root. a – autofl uorescence showing numerous bright yellow spots in the root cortex indicating essential oils inside secretory ducts; b – in the secondary xylem, according to phloroglucinol/hcl reaction with lignin, annual growth rings are visible (indicated by lines). letters indicate secondary phloem (p), secondary xylem (x). anatomy of hladnikia pastinacifolia acta bot. croat. 73 (2), 2014 381 old at the time of harvesting. older secondary xylem is lignifi ed, while younger growth rings include much specialized xylem parenchyma. secondary phloem does not form layers and numerous smaller secretory ducts are visible. discussion the new insight into h. pastinacifolia anatomy gave interesting new results which could help us to interpret the survival ability and fi tness constraints of this rare taxon. the pollenovule ratio of h. pastinacifolia fl ower is 2013 on average. it has been stressed before that p/o ratio is inversely proportional to self-pollination within species (cruden 1976). the umbels of h. pastinacifolia were frequently visited by insects because of nectar (excreted by stylopodium) and pollen. high p/o ratio leads to the conclusion that such fl owers are visited by less effective pollinators and at the same time, they are less effective at self-pollination. even though pollinators from the main umbel differed from those from the lateral umbels, fl owering later in the season, the prevailing groups of pollinating insects were dipterans (anthomyiidae and syrphidae) and coleoptera (own observation). attraction of these groups of pollinators is explained by the low amount of sucrose in apiaceae nectar (petanidou 2005). since many of the observed beetles were palinophagous, the high p/o ratio is benefi cial for successful pollination of h. pastinacifolia. sušnik (1964) observed that some mericarps of h. pastinacifolia do not contain an embryo and concluded that this could be a fi xed trait, which would add to species’ rarity. however, our results show that mericarps without embryos are present in 35% of schizocarps. we did not fi nd a reason for some mericarps not to develop fully, whether this is a consequence of ovule abortion or lack of fertilization. immature fruits of h. pastinacifolia accumulate anthocyanins in the endocarp (figs. 3d, 4a). the mesocarp of one mericarp consists of 6 vallecular oil ducts and numerous secretory ducts associated with the synthesis and accumulation of biologically active substances and essential oils (atia et al. 2009). autofl uorescence microscopy reveals secretory tissues in apiaceae fruits. zobel and march (1993) reported intense yellow autofl uorescence of parts containing coumarins, furanocoumarins, and fl avonoids. oil ducts in h. pastinacifolia fruits show high yellow fl uorescence, which probably determines the location of essential oils. new fi ndings include the presence of amphistomatal leaves in h. pastinacifolia. the stomata density in the abaxial epidermis is greater than in the adaxial epidermis. amphistomatal leaves are a morpho-physiological and not a taxonomic plant trait. such leaves are typical for plants from habitats with high irradiance since leaves do not grow horizontally according to light source, but at an angle (carlquist and miller 1999). in all habitats of h. pastinacifolia high irradiance is characteristic for at least part of the day. amphistomatal leaves are occasionally associated with dry habitats as well (parkhurst 1978). the dark green and glossy leaves of h. pastinacifolia already visually indicate drought tolerance. even though the trnovski gozd plateau represents an orographic barrier resulting in very high precipitation, the measurements of soil humidity in the habitats of h. pastinacifolia in summer (data not shown) were very low, only 3–4%. in the beginning of july 2006, we observed smaller parts with dried out vegetation in stony pastures with h. pastinacifolia. according to our observation, microtopography was of key importance for the survival of plants from different species. this is also the case with h. pastinacifolia, since slightly elešajna n., šuštar-vozlič j., kaligarič m. 382 acta bot. croat. 73 (2), 2014 vated parts had a lower density of vegetative rosettes and seedlings were absent. therefore drought tolerance is an important trait that enables survival and persistence despite the generally prevailing humid conditions. according to our results, h. pastinacifolia is a monocarpic perennial species. time of fl owering and subsequent dying of the plant occurs in plants older than 4 years. the distribution of vascular elements within h. pastinacifolia roots shows the annual formation of distinct growth rings, which could be used for the method described by dietz and ullmann (1997) to estimate a specimen’s age. however, because root chronology is an invasive method and because in general, demographic characteristics like growth, probability of survival and fl owering, as well as reproductive output of perennial plants are strongly size-dependent (werner 1975, metcalf et al. 2003), for the crude estimation of age the diameter of the root, measured with calipers, could be used in the fi eld. the root of h. pastinacifolia is a storage organ which allocates resources to aboveground organs. long and thick single roots, forming a rootstock, which grows deep, are characteristic for species which are better adapted to substrate coverage as well as to substrate erosion (miyanishi and johnson 2007). roots represent an important organ for persistence in the scree habitats of h. pastinacifolia, and also enable greater water uptake during dry conditions. the autofl uorescence microscope observations showed the presence of numerous secretory ducts in the secondary phloem of h. pastinacifolia root, emitting bright yellow fl uorescence. the considerable presence of autofl uorescent substances, which could be attributed to essential oils and phenolic compounds, are most likely the reason we did not observe signs of herbivory on leaves and why fruits were rarely attacked by animals except heteroptera. whenever we study extremely rare and protected species, we can face ethical and legal dilemmas because we have to interfere with populations and perform sampling, sometimes even with invasive methods (ceriani et al. 2008). however, especially for such species, studies and knowledge are pivotal for their protection. in this paper we have shown that anatomical features can provide additional information for a better understanding of the species’ biology and ecology. our results about h. pastinacifolia contribute to a better understanding of species’ pollination characteristics, drought tolerance, life cycle, and unattractiveness to herbivores. acknowledgements we thank the republic of slovenia institute of nature conservation for issuing the sampling permit. the study was partly supported by the slovene ministry of high education, science and technology within the biodiversity research program (p1-0078). nina šajna acknowledges a grant from the society for the advancement of plant sciences in vienna (austria). mitja kaligarič was supported by program group p1-0164 and infrastructural 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(in slovenian). phd thesis, university of ljubljana, ljubljana. werner, p. a., 1975: predictions from rosette size in teasel (dipsacus fullonum l.). oecologia 20, 197–210. wraber, t., 1990: čaven, ein botanisch beruhmter berg in slowenien. carinthia 180, 195– 210. zobel, a. m., march, r. e., 1993: autofl uorescence reveals different histological localizations of furanocoumarins in fruits of some umbelliferae and leguminosae. annals of botany 71, 251–255. acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 315 acta bot. croat. 73 (2), 315–332, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 leaf rolling reduces photosynthetic loss in maize under severe drought aykut saglam1*, asim kadioglu2, mehmet demiralay2,3, rabiye terzi2 1 karadeniz technical university, faculty of science, molecular biology and genetics, 61080 trabzon, turkey 2 karadeniz technical university, faculty of science, biology, 61080 trabzon, turkey 3 artvin çoruh university, faculty of sciences and letters, biology, 08000, artvin, turkey abstract – effects of leaf rolling (lr) on maize photosynthesis under severe drought stress were studied in two cultivars with opposite drought responses, batem 56-55 (drought tolerant) and batem 51-52 (drought sensitive). drought stress and artifi cial prevention of leaf rolling (plr) were applied at grain fi lling stage for 30 days. lr in batem 56-55 occurred later than in batem 51-52. leaf water potential (ψleaf) did not change in batem 56-55 but decreased in batem 51-52 at lr. maximum quantum yield of photosystem ii (fv/fm), effective quantum yield of photosystem ii (φpsii) and electron transport rate (etr) of the cultivars decreased during lr more signifi cantly in batem 56-55 in comparison to batem 51-52. the same was observed for the decrease in net photosynthetic rate (pn), stomatal conductance (gs), transpiration (e) and intracellular level of co2 (ci). rubisco activity and content were reduced at lr, but were less affected in batem 56-55 than in batem 51-52. ear and kernel weights also decreased at lr. all parameters at plr were more reduced than those of lr. these results implied that lr was an important and necessary mechanism protecting photosynthesis and reducing yield loss under drought stress by maintaining the leaf hydration, preventing loss of the photosynthetic pigments, sustaining the activity of psii, keeping the stomata open, and conserving the activity of rubisco. keywords: drought, leaf rolling, maize, photosynthesis, yield abbreviations: car – carotenoid, chl – chlorophyll, ci – intracellular co2, dw – dry weight, e – transpiration, etr –electron transport rate, fv/fm – maximum quantum yield of psii, φpsii – effective quantum yield of psii photochemistry, gs – stomatal conductance, lr – leaf rolling, npq – nonphotochemical quenching, pn – net photosynthetic rate, psii – photosystem ii, plr – prevention of leaf rolling, ψleaf – leaf water potential introduction maize is one of the key sources of human nutrition and is becoming an increasingly valuable crop due to the decrease in rice production and an enhanced demand for meat products * corresponding author, e-mail: aykut_saglam@yahoo.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. saglam a., kadioglu a., demiralay m., terzi r. 316 acta bot. croat. 73 (2), 2014 and dairy (salvi et al. 2007). maize is grown mostly under rain-fed conditions and is more susceptible to drought than other cereals. in addition drought is unpredictable. therefore drought tolerance is an important factor in maize breeding (banziger and araus 2007). grain yield is reduced by drought but depends on plant species, developmental stages, variety of the plants, and intensity of the stress (grzesiak et al. 2013). maize is especially vulnerable to soil drought during grain-fi lling periods (bai et al. 2006). the decrease of grain yield in maize under drought stress has been the subject of recent studies. for instance, a fi ve-day drought stress during pollination has caused formation of aborted embryos and decrease in kernel numbers in maize (zinselmeier et al. 1999). drought stress and shading during pre and post pollination stages have decreased kernel set in the apical regions of maize ears (setter et al. 2001). the photosynthetic process is impaired mostly by drought stress (chaves et al. 2009) due to both stomatal and non-stomatal limitations, which lead to a decrease in photosynthetic activity (zlatev and yordanov 2004). stomatal closure is one of the earliest responses of the plants to drought, reducing transpiration, co2 uptake and photosynthetic activity (de souza et al. 2013). the limitation of co2 uptake causes an imbalance between psii activity and the calvin cycle, increasing the excitation energy on psii, and subsequent photodamage (baker and rosenqvist 2004), mainly to the psii oxygen-evolving complex, and the disruption of d1 protein, resulting in inactivation of psii reaction centers (ashraf and harris 2013). a number of mechanisms are involved in plant defense strategies against drought stress such as reduction of leaf size, leaf movement or leaf rolling (lr) (kadioglu et al. 2012). although, there are many studies about the effects of drought on photosynthesis in plants, there are still few studies showing changes in photosynthesis during lr under drought stress. for example, a relation between lr and susceptibility to photoinhibition of sorghum photosynthesis under drought was reported (corlett et al. 1994). in ctenanthe setosa, lr was reported to protect psii against damage under severe drought (nar et al. 2009). furthermore, moderate lr increased effi ciency of photosynthesis in rice (lang et al. 2004). however, the effects of lr on photosynthetic yield regarding agronomic parameters and cultivars have not been studied. thus, in the present study, the effects of lr on photosynthesis yield and drought-response mechanisms are elucidated. we hypothesized that lr could protect photosynthesis from drought stress and preserve maize yield. in addition, delayed or late lr has been associated with drought tolerance in rice cultivars (singh and mackill 1990). on the other hand, how delayed lr affects photosynthesis and yield has not yet been fully elucidated. therefore, the effects of artifi cial prevention of leaf rolling (plr) on photosynthesis under drought stress was investigated as well as the way in which lr affects photosynthesis in cultivars with contrasting drought tolerance. material and methods growth of the plants and stress treatments the experiment was carried out between may and august 2008, 2009 and 2010 at karadeniz technical university, greenhouse field (40°59´n, 39°46´e, 179 m). a rainout shelter (8 m width × 15 m length × 3 m height) was constructed as a tunnel open at both ends and kept 150 cm above ground to allow free airfl ow and avoid rises in temperature due to leaf rolling and photosynthesis acta bot. croat. 73 (2), 2014 317 the greenhouse effect on an open fi eld and covered with polyethylene transmitting more than 90% of sunlight. the plants were grown in the rainout shelter under sunlight. average temperature of the fi eld was 21.8 ± 4.1 °c between may and august in 2008–2010. seeds of the maize cultivars (batem 51-52 and batem 56-55) were obtained from western mediterranean agricultural research institute, antalya, turkey; batem 51-52 was reported as drought sensitive and batem 56-55 as drought tolerant (saruhan et al. 2012). cultivars were determined as drought tolerant/sensitive based on their leaf water potentials, stomatal conductance, proline and malondialdehyde (mda) contents after 30 days of drought stress, to which the plants were exposed at grain fi lling stage (r2). eight seeds were sown in pots (30 cm diameter and 50 cm deep) fi lled to 47.5 cm height with 14 kg of fully water-saturated sandy soil. there were twelve pots for each cultivar. after full germination, seedlings were reduced to two in each pot. plants were kept well irrigated and grown to stage r2 by adding one and half liters of water to each pot every other day. at stage r2, half of the plants were kept well watered (control), while the others were subjected to drought stress by withholding water for 30 days until maturity was reached. to investigate the effect of lr on photosynthesis during drought, lr was artifi cially inhibited (plr) by clamping leaves with plastic wires in the beginning of drought application. leaves (6th leaf from top) were harvested on the 30th day of drought. leaf rolling degree, water status and temperature were measured, in addition to photosynthetic pigment content, chlorophyll a fl uorescence, photosynthetic gas exchange parameters, rubisco activity, rubisco content and some agronomic characteristics. leaf rolling degree and water status leaf rolling degree was measured according to premachandra et al. (1993) as a percentage reduction in the width of the leaf mid-portion. leaf water potential (ψleaf) was measured with a thermocouple psychrometer (c-52, wescor) after it had been calibrated by using nacl solutions of known water potentials. discs about 6 mm in diameter were cut from the leaves of three plants and sealed in the c-52 sample chamber. samples were equilibrated for 60 min before the readings were recorded by a water-potential data logger (psypro, wescor inc., logan, ut usa) in the psychrometric mode. lipid peroxidation lipid peroxidation was measured in terms of malondialdehyde (mda) content (ε = 155 mm–1 cm–1), a product of lipid peroxidation following the method of heath and packer (1968). leaf samples (0.5 g) were homogenized in 10 ml of 0.1% (w/v) trichloroacetic acid (tca). as much as 4 ml of 0.5% (w/v) thiobarbituric acid containing 20% (w/v) of tca was added to 1 ml aliquot of supernatant. the absorbance of the supernatant was recorded at 532 and 600 nm and mda content was expressed as nmol mda per g fresh weight (nmol mda g–1 fw). photosynthetic pigments total chlorophyll (chl) and carotenoid (car) contents were determined following the method of arnon (1949). the 6th leaf from the tops of different plants of the same age were selected randomly and homogenized in a mortar in 80% acetone. the extract was centrisaglam a., kadioglu a., demiralay m., terzi r. 318 acta bot. croat. 73 (2), 2014 fuged at 5,000 g for 5 min. absorbance of the supernatant was recorded at 663, 645, and 450 nm by spectrophotometer (nicolet evolution 100, thermo scientifi c, usa). content was expressed as mg of pigments per g dry weight (mg g–1 dw). thermal imaging thermal images were obtained using a thermovision a20 infrared camera (flir systems, usa) according to vollsnes et al. (2009). the infrared camera was mounted vertically ~10 cm above the leaf. each leaf was positioned in the camera view fi eld to focus the image and to maximize the image area covered by the leaf. the images of the 6th leaf from the tops of six different plants were captured. images were analyzed for temperature determination using the thermacam researcher software (liu et al. 2011). chlorophyll a fl uorescence measurements chlorophyll a fl uorescence measurements were performed by pulse modulated fl uorometer (os1-fl, optiscience corporation, tyngsboro, ma, usa) according to nar et al. (2009) between 10:00 and 14:00 h at the grain-fi lling stage in the rainout shelter under sunlight. for each treatment, the 6th leaf from six different plants was selected and clamped at the center point with the os1-fl leaf clip holder. leaves were dark-adapted for 20 min and the chl fl uorescence was then measured. minimum chl fl uorescence yield (f0) was determined under weak modulated λ660 irradiation (< 0.1 μmol m –2 s−1). maximum chl fl uorescence yield (fm) was reached by exposing psii to saturating λ690 pulse (0.8 s) of white light (8,000 μmol m–2 s–1). after dark measurements, the leaves were exposed to the red actinic light of the fl uorometer together with the sunlight functioning as actinic light source. steady state chl fl uorescence (fs) was achieved after exposure to the actinic light. a simultaneous saturating pulse (0.8 s) of white light (8,000 μmol m–2 s–1) was applied to determine maximum chl fl uorescence in the light (fm’). maximum quantum yield of psii photochemistry (fv/fm) and effective quantum yield of psii photochemistry (φpsii) were calculated according to the following equations (fv/fm = (fm – f0)/fm and φpsii = (fm’ – fs)/fm’) of genty et al. (1989). nonphotochemical quenching (npq) was calculated according to the equation, npq = (fm – fm’)/fm’. electron transport rate (etr) was calculated according to following equation, etr = (фpsii × par × 0.5 × 0.84). par was measured with a par clip. value 0.5 indicates partitioning of energy between psii and psi, while value 0.84 presents proportional light absorbance by photosynthetic tissue. photosynthetic gas exchange measurements stomatal conductance (gs) was monitored on the 6 th leaf from six different plants by using a dynamic diffusion porometer (ap4, delta-t devices, burwell, cambridge, uk) after it had been calibrated with a standard calibration plate following the manufacturer’s instructions. other gas exchange parameters were measured using a portable photosynthesis system (tps-2 photosynthesis system, pp systems, amesbury, ma, usa) equipped with a 25 × 18 mm plc6 automatic universal leaf cuvette on the 6th leaf from six different plants under natural sunlight. middle parts of the selected leaves were measured from 10:00 to 14:00 h. the parameters measured were transpiration (e), net photosynthetic rate (pn) and intracellular co2 (ci). leaf rolling and photosynthesis acta bot. croat. 73 (2), 2014 319 agronomic characteristics grain yield and yield components were evaluated through ear weight and 100-kernel weight in six plants. determination of rubisco activity about 0.25 g of frozen maize leaves were ground to a powder using a chilled mortar and pestle with liquid n2, a small amount of quartz sand, and insoluble polyvinylpolypyrrolidone (pvp). tissues were homogenized with cooled extraction buffer containing 50 mm of tris–hcl (ph 7.5), 1 mm of ethylenediaminetetraacetic acid (edta), 10 mm of mgcl2, 12% (v/v) of glycerol, 0.1% (v/v) of β-mercaptoethanol and 1% (w/v) of pvp-40 (soluble pvp) at 0–4 °c. the homogenate was centrifuged at 15,000 g for 15 min at 4 °c. protein contents of the homogenates were determined as described by bradford (1976) using bovine serum albumin as a standard. the rubisco activity in the supernatant was assayed according to sawada et al. (2003) with minor modifi cations. the rubisco activity was measured at 30 °c for 10 minutes by adding 100 µl of supernatant into 900 µl of assay buffer containing 50 mm of hepes–koh (ph 8.0), 1 mm of edta, 20 mm of mgcl2, 25 mm of dithioerythritol, 10 mm of nahco3, 5 mm of atp, 0.15 mm of nicotinamide adenine dinucleotide (nadh), 5 mm of creatine phosphate, 0.6 mm of ribulose-1,5-bisphosphate (rubp), 10 units of phosphocreatine kinase, 10 units of glyceraldehyde-3-phosphate dehydrogenase and 10 units of phosphoglycerate kinase. total rubisco activity was expressed in units per milligram protein. determination of rubisco content to determine rubisco content, leaves were homogenized as described in the rubisco activity assay. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds–page) of leaf protein was carried out in a vertical slab gel discontinuous buffer system following the method of laemmli (1970) using a 7% acrylamide gel concentration. a total volume of 20 μl protein extract solution including 30 μg of total protein was loaded into each well. in addition, to quantify the amount of rubisco in the leaf homogenates, rubisco recombinant protein standards ranging from 0.0625 to 0.25 pmol were also loaded. protein molecular mass standard (thermo fisher scientifi c, waltham, ma) was used. the gels were run at approximately 80 ma for 30–40 min in electrophoresis buffer by using a mini-protean tetra cell electrophoretic unit (biorad, germany). proteins were then transferred to polyvinylidene fl uoride (pvdf) transfer membrane (whatman, dassel, germany) in mcfarland’s electroeluting buffer (12 mm tris base, 96 mm glycine, and 20%, v/v, methanol) at < 25 v for 45 min. the blotted membrane was subjected to immunodetection of rubisco according to ma et al. (2009). the pvdf blot was placed in 10 ml of blocking solution (tbst; 5%, w/v, nonfat dry milk in 24.8 mm tris, ph 7.4, 150 mm nacl, 2.7 mm kcl, and 0.5%, v/v, tween 20) and rocked gently on a rotary shaker or platform rocker for 30 min then washed four times for 5 min each with tbst and incubated for 1 h on a shaker-rocker with polyclonal rabbit anti-rubisco antibody (anti-rubisco large subunit global antibody, diluted 1:6,000; agrisera, vännäs, sweden). after four 2.5-min washes with tbst, the blot was incubated with goat anti-rabbit immunoglobulin g (igg) polyclonal antibody conjugated to horseradish peroxidase (diluted 1:2,000; agrisera, vännäs, sweden) for 30 min on the shaker. after four 2.5-min washes, chromogenic substrate solution for horseradish peroxidase saglam a., kadioglu a., demiralay m., terzi r. 320 acta bot. croat. 73 (2), 2014 (0.015%, w/v, of 4-chloro-1-napthol and 0.05%, v/v, hydrogen peroxide in tbs containing 16.7%, v/v methanol) was added, and the blot was allowed to develop 15 min. the blot was then rinsed three times in water for 2 min each. the developed blot was allowed to dry on a clean piece of fi lter paper and stored in a ziplock bag at −20 °c. the western blot image of protein bands was captured with a scanner (hp scanjet 4850; hewlett packard, palo alto, ca). a comparison of the average intensity of each band from western blot analysis was quantifi ed with the use of the imagej analysis program (imagej 1.34s; http://rsbweb.nih. gov/ij/). statistical analysis each measurement was conducted three times with six biological replicates. variance analysis of mean values was performed with duncan multiple comparison test (two-way anova) using spss software for microsoft windows (ver. 13.0, spss inc., usa) and signifi cance was determined at p < 0.05 level. results leaf rolling degree and water status leaves of batem 56-55, drought tolerant cultivar, rolled two days later than batem 5152, drought sensitive cultivar, under drought. in addition, lr degree (%) in batem 51-52 (77.0 ± 0.5) was higher than batem 56-55 (65.0 ± 2.7). the leaf water potential (ψleaf) control values were –0.30 ± 0.01 and –0.50 ± 0.03 mpa in control groups of batem 51-52 and batem 56-55, respectively. the ψleaf signifi cantly decreased (6-fold) in batem 51-52 at lr (–1.81 ± 0.03 mpa) compared to the control (fig. 1). however, the ψleaf did not change in batem 56-55 (–0.51 ± 0.02 mpa). at plr, decreases in ψleaf were measured in both batem 51-52 (–2.30 ± 0.02 mpa) and batem 56-55 (–1.20 ± 0.03 mpa) compared to the controls (fig. 1). ψleaf in batem 56-55 at plr was 7.8 fold lower than its control, while ψleaf in batem 51-52 was 2.4 fold lower than its control. fig. 1. changes in leaf water potential of maize cultivars. c – control, lr – leaf rolling, plr – prevention of leaf rolling. different small letters show statistical differences at p < 0.05 among c, lr and plr of same cultivar. asterisk denotes signifi cant differences between cultivars. vertical bars are standard deviation of means. leaf rolling and photosynthesis acta bot. croat. 73 (2), 2014 321 photosynthetic pigments the total chl content in batem 51-52 decreased from 5.20 ± 0.01 mg g–1 dw (control) to 1.50 ± 0.01 mg g–1 dw at lr (fig. 2a) while the total chl content increased from 5.9 ± 0.03 mg g–1 dw (control) to 6.50 ± 0.01 mg g–1 dw in batem 56-55 at lr. total chl content in batem 51-52 was 3.5 fold lower at lr than in the control. on the other hand, the chl content in batem 56-55 was 1.1 fold higher at lr than in the control. in addition, the total chl content at plr was also lower than that of the control. the chl content in batem 51-52 was 1.00 ± 0.01 mg g–1 dw at plr, a 5.2-fold decrease as compared to control. it was 3.10 ± 0.06 mg g–1 dw in batem 56-55 at plr, a 1.9 fold decrease as compared to control. total carotenoid (car) content increased in batem 51-52 and batem 56-55 at lr compared to the controls. total car contents in rolled leaves of batem 51-52 (1.30 ± 0.01 mg g–1 dw) and batem 56-55 (1.9 ± 0.01 mg g–1 dw) were 2.2 and 2.1-fold higher than the controls (0.6 ± 0.01 mg g–1 dw and 0.9 ± 0.02 mg g–1 dw) respectively. in addition, a signifi cant decrease in the car contents was determined in batem 51-52 at plr. the car content in batem 51-52 (0.2 ± 0.04 mg g–1 dw) at plr was 7.5-fold lower than the control (0.60 ± 0.01 mg g–1 dw) (fig. 2b). fig. 2. changes in photosynthetic pigment contents of maize cultivars. a) total chl content and b) total car content; c – control, lr – leaf rolling, plr – prevention of leaf rolling. different small letters show statistical differences at p < 0.05 among c, lr and plr of same cultivar. asterisk denotes signifi cant differences between cultivars. vertical bars are standard deviation of means. thermal imaging leaf temperature increased in both tolerant and sensitive cultivars at lr compared to the controls. leaf temperature was 21.3 ± 0.2 °c in the control group of batem 51-52 and 20.7 ± 0.1 °c in the control group of batem 56-55. it increased to 21.7 ± 0.05 °c in batem 51-52, and to 21.4 ± 0.2 °c in batem 56-55, at lr. in addition, the leaf temperature at plr was determined to be higher than that of lr in batem 56-55. there was no signifi cant difference between lr and plr in batem 51-52. the leaf temperature was 21.7 ± 0.06 °c in batem 51-52 and 21.9 ± 0.1 °c in batem 56-55 at plr (fig. 3). saglam a., kadioglu a., demiralay m., terzi r. 322 acta bot. croat. 73 (2), 2014 chlorophyll fl uorescence parameters maximum quantum yield of psii photochemistry (fv/fm) decreased in both cultivars at lr (fig. 4a). the decrease in the fv/fm was higher in batem 51-52 than in batem 56-55. fv/fm in the rolled leaves of the batem 51-52 (0.26 ± 0.04) was 2.9-fold lower than in the control (0.76 ± 0.03) while fv/fm in batem 56-55 (0.7 ± 0.01) was 1.1-fold lower at lr than in the control (0.76 ± 0.03). moreover, signifi cant decreases in fv/fm were determined in both cultivars at plr compared to the controls. fv/fm values decreased from 0.76 (control) to 0.16 and 0.6 in batem 51-52 and batem 56-55 at plr, respectively. fv/fm values in batem 51-52 and batem 56-55 were 4.8 and 1.3 fold lower than in their controls (fig. 4a). effective quantum yield of psii photochemistry (φpsii) decreased in batem 51-52 at lr compared to the control while it did not change in batem 56-55 (fig. 4b). as compared to control (0.6 ± 0.004), a 3-fold decrease in φpii2 (0.2 ± 0.01) was determined in batem 51-52 at lr. a signifi cant decrease in φpsii was also determined at plr. the φpsii in batem 51-52 and batem 56-55 diminished to 0.02 ± 0.003 and 0.36 ± 0.01 at plr, respectively. the values of φpsii in batem 51-52 and batem 56-55 were 30.0 and 1.5 fold lower than in the control groups, respectively (fig. 4b). non-photochemical quenching (npq) increased in rolled leaves of both cultivars compared to the controls (fig. 4c). furthermore, the increase in npq was higher in batem 5655 than that of batem 51-52 at lr. npq in batem 51-52 increased from 0.200 ± 0.001 to 0.400 ± 0.004 (a 2-fold increase). npq in batem 56-55 also increased (from 0.400 ± 0.001 to 0.60 ± 0.01) compared to control, a 1.3 fold increase. in the case of plr, npq decreased in both cultivars signifi cantly compared to the controls. npq in batem 51-52 was 0.100 ± 0.004 at plr. it was 0.2 ± 0.01 in batem 56-55. the rates of the decreases in npq were 2.0 fold in both batem 51-52 and batem 56-55 compared to the controls (fig. 4c). electron transport rate (etr) decreased in batem 51-52 at lr (fig. 4d). etr decreased from 58.0 ± 0.8 µmol m–2 s–1 (control) to 20.5 ± 1.2 µmol m–2 s–1 at lr, which was 2.8 fold lower than in the control. on the other hand, etr did not change in batem 56-55 during lr. fig. 3. thermal images of maize leaves. a) batem 51-52, sensitive control, b) batem 56-55, tolerant control, c) batem 51-52, upon drought stress and d) batem 56-55, upon drought stress. c – control, lr – leaf rolling, plr – prevention of leaf rolling. leaf rolling and photosynthesis acta bot. croat. 73 (2), 2014 323 in the case of plr, etr decreased in both cultivars compared to the controls. etrs in batem 51-52 and batem 56-55 were 5 ± 0.6 µmol m–2 s–1 and 35.3 ± 1.1 µmol m–2 s–1. there were 12.0 and 1.6-fold decreases in batem 51-52 and batem 56-55 as compared controls, respectively (fig. 4d). photosynthetic gas exchange measurements stomatal conductance (gs) diminished in both cultivars at lr. gs in batem 51-52 decreased from 180 ± 2.2 mmol m–2 s–1 (control) to 23 ± 0.5 mmol m–2 s–1 (a 7.8-fold decrease). the decrease in gs was greater in batem 51-52 than in batem 56-55 at lr (fig. 5a). gs in batem 56-55 diminished from 170 ± 1.3 mmol m–2 s–1 to 92 ± 2.7 mmol m–2 s–1, which was a 1.9-fold decrease. remarkable decreases in gs were measured in both cultivars at plr compared to the controls. gs values in batem 51-52 and batem 56-55 were 5 ± 0.4 mmol m –2 s–1 and 50 ± 2.5 mmol m–2 s–1, respectively at plr. gs in batem 51-52 and batem 56-55 were 36 and 3.4-fold lower than the controls, respectively. transpiration rate (e) decreased in both cultivars at lr compared to the controls (fig. 5b). e in batem 51-52 decreased from 5.3 ± 0.1 mmol m–2 s–1 (control) to 1.3 ± 0.1 mmol fig 4. changes in chl fl uorescence parameters of maize cultivars. a) fv/fm – maximum quantum yield of psii photochemistry b) φpsii – effective quantum yield of psii photochemistry, c) npq – non-photochemical quenching and d) etr – electron transport rate; c – control, lr – leaf rolling, plr – prevention of leaf rolling. different small letters show statistical differences at p < 0.05 among c, lr and plr of same cultivar. asterisk denotes signifi cant differences between cultivars. vertical bars are standard deviation of means. saglam a., kadioglu a., demiralay m., terzi r. 324 acta bot. croat. 73 (2), 2014 m–2 s–1. e in batem 56-55 was reduced from 3.7 ± 0.2 mmol m–2 s–1 (control) to 2.6 ± 0.3 mmol m–2 s–1. decrease in e was higher in batem 51-52 than in batem 56-55 at lr. the rates of decreases were 1.4 and 4.1 in batem 56-55 and batem 51-52 compared to control, respectively. the decreases in e were more at plr than those of lr as compared to controls. e values were 0.110 ± 0.007 mmol m–2 s–1 and 0.37 ± 0.02 mmol m–2 s–1 in batem 51-52 and batem 56-55 at plr, respectively (fig. 5b). e values in batem 51-52 and batem 56-55 were 48 and 10-fold lower than the control groups, respectively. photosynthetic rate (pn) decreased in both cultivars at lr compared to the controls (fig. 5c). pn values were reduced from 11.5 ± 0.4 µmol m –2 s–1 (control) to 1.2 ± 0.04 µmol m–2 s–1 in batem 51-52. it was reduced from 15.9 ± 0.6 µmol m–2 s–1 (control) to 7.1 ± 0.4 µmol m–2 s–1 in batem 56-55 at lr. pn values were reduced at the rates of 9.6 and 2.3 in batem 51-52 and batem 56-55 at lr compared to their controls, respectively. pn decreased more in both cultivars at plr than lr compared to control. pn values were 1.03 ± 0.06 µmol m –2 s–1 and 6.3 ± 0.14 µmol m–2 s–1 in batem 51-52 and batem 56-55 at plr, respectively (fig. 5c). the rates of decrease in pn were 11.1 and 2.5 in batem 51-52 and batem 56-55 at plr compared to the controls, respectively. intracellular co2 concentration (ci) was reduced by drought at lr (fig. 5d). ci in batem 51-52 decreased from 428.0 ± 8.6 µmol mol–1 (control) to 378.0 ± 11.6 µmol mol–1. it was reduced from 347.0 ± 4.9 µmol mol–1 to 305.0 ± 16.3 µmol mol–1 in batem 56-55. the rates of the decrease in ci values were 1.1 and 1.1 in batem 51-52 and batem 56-55 at lr compared to their controls. the reductions in ci at plr were higher than those of lr compared to control. ci values were 358.0 ± 9.0 µmol mol –1 and 275.0 ± 2.1 µmol mol–1 in batem 51-52 and batem 56-55 at plr, respectively. the rate of the decrease in ci was 1.2 in batem 51-52 compared to the control while it was 1.3 in batem 56-55 at plr. fig. 5. changes in photosynthetic gas exchange parameters of maize cultivars. a) gs – stomatal conductance, b) pn – net photosynthetic rate, c) e – transpiration and d) ci – intracellular co2; c – control, lr – leaf rolling, plr – prevention of leaf rolling. different small letters show statistical differences at p < 0.05 among c, lr and plr of same cultivar. asterisk denotes signifi cant differences between cultivars. vertical bars are standard deviation of means. leaf rolling and photosynthesis acta bot. croat. 73 (2), 2014 325 rubisco activity rubisco activity in batem 51-52 was higher than batem 56-55. rubisco activity decreased in both cultivars at lr compared to the controls. rubisco activity in batem 51-52 decreased from 1286.0 ± 15.0 u mg–1 protein (control) to 869.0 ± 24.0 u mg–1 protein. it was reduced from 464.0 ± 13.0 u mg–1 protein (control) to 303 ± 11.0 u mg–1 protein for batem 56-55 (fig. 6a). there was a 1.5-fold reduction in the rubisco activity in both cultivars at lr compared to their controls. rubisco activities determined at lr were higher than plr for both cultivars. rubisco activities were 557 ± 19.0 u mg–1 protein and 128.0 ± 9.0 u mg–1 protein in batem 51-52 and batem 56-55 at plr, respectively. the reductions in the activities were higher at plr than lr compared to the controls (fig. 6). the reductions in the activities were 2.3-fold and 3.6-fold in batem 51-52 and batem 56-55 at plr compared to the controls, respectively. rubisco content rubisco content in batem 51-52 was higher than batem 56-55 in control plants (fig. 6b). it was 0.9 ± 0.05 pmol µg–1 protein in batem 51-52 and 0.4 ± 0.01 pmol µg–1 protein in batem 56-55. rubisco contents of both cultivars decreased at lr as compared to control. they were 0.2 ± 0.008 pmol µg–1 protein in batem 51-52 (4.5-fold lower than control) and 0.2 ± 0.002 pmol µg–1 protein in batem 56-55 (2-fold lower than control). rubisco contents of the cultivars at plr were lower than those of lr plants. there was 0.002 ± 0.0005 pmol µg–1 protein in batem, 450-fold lower than control and 0.002 ± 0.0009 pmol µg–1 protein in batem 56-55, 200-fold lower than control. fig. 6. changes in rubisco activity (a) and content (b) of maize cultivars; c – control, lr – leaf rolling, plr – prevention of leaf rolling. different small letters show statistical differences at p < 0.05 among c, lr and plr of same cultivar. asterisk denotes signifi cant differences between cultivars. vertical bars are standard deviation of means. agronomic characteristics as compared to control, ear weights were reduced in both cultivars at lr. ear weights decreased from and 64.0 ± 0.5 g to 40.0 ± 0.5 g in batem 51-52 and 46.0 ± 0.4 g to 25.0 ± 0.7 g in batem 56-55 at lr, respectively. the reduction in the ear weight was higher in saglam a., kadioglu a., demiralay m., terzi r. 326 acta bot. croat. 73 (2), 2014 batem 51-52 (2.6 fold) than batem 56-55 (1.2 fold) at lr (fig. 7a). ear weights decreased more at plr than lr compared to the controls. ear weights in batem 51-52 and batem 5655 were 21 ± 1.1 g and 40 ± 0.5 g at plr, respectively. the decreases in ear weight were 3.0 and 1.5 fold in batem 51-52 and batem 56-55 as compared to their controls, respectively. one hundred-kernel weight also declined at lr. the kernel weight in batem 51-52 diminished from 45.0 ± 2.3 (control) to 15 ± 0.4 g at lr. it was not changed in batem 56-55. the reduction in 100-kernel weight in batem 51-52 was 3.0 fold compared to the control (fig. 7b). the decreases were more pronounced in both cultivars at plr compared to the controls. decreases in 100-kernel weights of batem 51-52 and batem 56-55 were 3.5 and 1.2 fold at plr compared to the controls, respectively (fig. 7b). fig. 7. changes in agronomic characteristics of maize cultivars. a) ear weight and b) 100-kernel weight; c – control, lr – leaf rolling, plr – prevention of leaf rolling. different small letters show statistical differences at p < 0.05 among c, lr and plr of same cultivar. asterisk denotes signifi cant differences between cultivars. vertical bars are standard deviation of means. discussion in the present study, leaf water potential in rolled leaves of drought sensitive maize cultivar was reduced by drought. confi rming our study, decreases in leaf water potential during lr were also reported in maize and c. setosa plants subjected to drought stress (terzi et al. 2009, saruhan et al. 2012). on the other hand, plants having lr mechanism were reported to exhibit a resistance to drought and high temperature and had higher water use effi ciency (kadioglu et al. 2012). that the decrease in leaf water content in our study was enhanced by plr compared to lr suggested that lr might be a water-saving regulatory mechanism and may protect maize photosynthesis from drought stress. to evaluate effects of environmental stress on growth and yield, chl content and chl fl uorescence parameters can be measured because these traits are related with carbon exchange rate (fracheboud et al. 2004). the effect of lr on photosynthetic pigment content was studied in maize under drought stress. chl content decreased under drought in the sensitive cultivar at lr in our study while it increased in the tolerant one. similarly, a high decrease in pigment content was reported in a sensitive maize cultivar under drought stress (chugh et al. 2013). the increase in pigment concentration could be related to decreased leaf surface and assembling chlorophylls on less area of leaf (tourian et al. 2013). relation leaf rolling and photosynthesis acta bot. croat. 73 (2), 2014 327 between lr and photosynthetic pigment content was shown under drought stress in c. setosa (nar et al. 2009). during early lr, a decrease in photosynthetic pigment content was observed. however, it approached control values in the late lr situation under severe drought stress. in our study, signifi cant decreases in pigment content were observed at plr compared to lr. these signifi cant reductions in the pigment content could be related to changes in microclimate parameters such as irradiance and temperature differences, which might be created by the lr. however, the plr may disrupt humid environment formed in rolled leaves, which have low temperature inside. thermal images of maize leaves indicated that rolled leaves were cooler than artifi cially opened leaves. leaf temperature of rolled maize leaves was 21.4 ± 0.2 °c while the temperature of artifi cially opened leaves was 21.9 ± 0.1 °c. in addition, reduced irradiance upon the leaf surfaces (par) due to lr was reversed by plr in our study. par in control (1081 ± 0.2) decreased to 295 ± 42 during lr then increased to 1092 ± 20 during plr. therefore we concluded that lr might prevent light damage on psii by reducing leaf exposure to light. chlorophyll a fl uorescence provides information about the effects of lr on photosynthetic apparatus under drought stress (nar et al. 2009). thus, changes in fl uorescence parameters were investigated in this study. one of those parameters, fv/fm has been widely used to detect changes in the photosynthetic apparatus due to stress (baker and rosenqvist 2004). in other words, changes in the fl uorescence yield refl ect changes in photochemical effi ciency. low fv/fm values in plants under stress indicate damage to the psii reaction center (nar et al. 2009). in this study, the decrease of fv/fm in the sensitive cultivar was more pronounced than in the tolerant cultivar during lr, indicating a higher drought tolerance in the latter cultivar. in addition, higher decreases in fv/fm at plr indicated that lr might have a protective role on photosynthetic apparatus. however, no change in fv/fm was found in c. setosa, a drought tolerant plant (terzi and kadioglu 2006), during lr (nar et al. 2009). increase in npq resulting from drought stress is a well-known phenomenon (efeoğlu et al. 2009, huang et al. 2013). in the present study, npq increase during lr indicated the thermal dissipation of energy load on the leaves, perhaps preventing photo damage. maize genotypes were found to conserve their photosystems from adverse effects of drought by increasing npq (de souza et al. 2013). carotenoids are known as accessory photosynthetic pigments protecting the chlorophylls from oxidative stress damage during photosynthesis, dissipating energy through non-photochemical quenching and having an important role in the xanthophyll cycle (de souza et al. 2013). the increases in npq and car content on rolled leaves of maize indicated thermal dissipation of excess energy in our study, which supported this hypothesis. decreases in car content and npq at plr also supported the protective role of lr on the photosynthetic system. reduction in effective quantum yield of psii photochemistry was observed in sensitive cultivar at lr in this study. down-regulation of electron transport can be estimated by decreased in φpsii. this decrease can also be related with increases in excitation energy quenching in the psii antennae (horton et al. 1996). consequently, the increase of npq in the sensitive cultivar was lower than in the tolerant, resulting in more energy on psii and also more damage under drought. the decreases in φpsii showed increased excitation energy quenching process in the psii antennae and the regulation of electron transport. therefore, the increase in npq during lr may cause a decrease in φpsii. on the other hand, high npq in the tolerant cultivar did not affect φpsii at lr. increased chl content in the tolerant cultivar saglam a., kadioglu a., demiralay m., terzi r. 328 acta bot. croat. 73 (2), 2014 at lr might compensate for energy loss through npq in the tolerant cultivar. in addition, φpsii at plr was reduced more than lr in our study. the decrease in φpsii at plr could also be a proof for a protective role of lr on psii. a decline in electron transport rate was determined in the present study. this decrease in etr may be associated with the increased trans-thylakoid ∆ph. this change in ∆ph causes the xanthophyll de-epoxidation that follows increasing thermal dissipation of energy when photosynthesis is reduced (lu and zhang 1999). in addition, photochemical down-regulation under stress conditions could cause reductions in etr. drought stress was recorded to limit photosynthetic machinery of six maize genotypes (cruz de carvalho et al. 2011). on the other hand, higher decreases in etr during plr supported the hypothesis that lr could be a protective mechanism for photosystems under drought. stomatal conductance is one of the parameters that affect photosynthesis under drought stress (siddique et al. 1999, jiang et al. 2006). because gs restricts co2 uptake, it is considered to be one of the main reasons for reduced photosynthesis (ashraf and harris 2013). in this respect, reduction in photosynthesis probably depends more on co2 in the chloroplast than on leaf water potential (medrano et al. 2002). in our study, gs was reduced by drought in rolled leaves of maize plants probably due to reduction in leaf water potential. a decrease in gs and ψleaf and a positive correlation between them were reported in maize plants under water stress (cruz de carvalho et al. 2011; zia et al. 2011). on the other hand, sharp decreases in gs and increases in leaf temperature following plr in our study suggest that lr may maintain co2 uptake by creating a cool and humid environment inside the rolled leaf thus allowing some of stomata to remain open. similarly, gs values were found to be higher at adaxial (inner) surfaces of the rolled leaves than those of plr during drought stress (nar et al. 2009) in the present study, the net photosynthetic rate decreased in rolled leaves of both maize cultivars under drought. however, the reduction in pn of the tolerant cultivar was less than in the sensitive cultivar. similarly, photosynthesis of drought-tolerant and sensitive maize cultivars responded to drought in different ways (cruz de carvalho et al. 2011). in addition to pn and transpiration rate, intracellular co2 concentration also decreased in rolled leaves of maize cultivars in the present study. in addition, similar results were reported for drought tolerant and sensitive maize cultivars subjected to drought stress (zhang and liu 2009). stomatal and non-stomatal factors cause reduction in photosynthesis. in the present study, decreases in pn, gs, ci and e indicated that reduction in photosynthesis was due to stomatal factors. however, as compared to lr, notable reductions in all gas exchange parameters occurred following plr. these differences may indicate that lr may minimize photosynthetic losses due to drought stress, functioning as a protective mechanism. the effect of lr on the activity of the co2-fi xing enzyme rubisco was investigated in the current study. reductions in the rubisco activity were observed in rolled leaves of both cultivars. furthermore, rubisco activity was higher in the sensitive than in the tolerant cultivar. this might be related to the high amount of rubisco in the drought sensitive cultivar. on the other hand, high content of inactive rubisco in tolerant cultivar at lr might be a reason for low activity even if tolerant and sensitive cultivar has same amount of rubisco. in addition the decrease in rubisco activity in our study may be a result of stomatal closure due to long-term drought stress. stomata closure reduces photoassimilates and concentration of ci (baczek-kwinta et al. 2010) leading to rubisco deactivation (chaves and oliveira 2004). decrease in ci in our study supports the hypothesis that the reduction in rubisco leaf rolling and photosynthesis acta bot. croat. 73 (2), 2014 329 activity may be a result of stomatal closure. as compared to lr, higher decreases in rubisco activity after plr indicate that the lr mechanism may preserve activity of co2 fi xation enzymes. drought stress causes reductions in maize development and growth (shirazi et al. 2011). xiao et al. (2005) and ti-da et al. (2006) reported that cob characters especially number of kernels of corncob, ear weight and 100-kernel weight were decisive factors for maize yield loss under drought stress. decreases in kernel weight of corn cultivars subjected to drought were also observed previously (khalily et al. 2010). decreases in ear weight and 100-kernel weights at plr indicated that lr could be a mechanism to reduce yield loss in our study. in conclusion, leaves were rolled in drought tolerant maize cultivars later than in sensitive cultivars under drought stress. the results showed that monitoring of lr would be useful for the early detection of drought stress. signifi cant decreases in chl fl uorescence parameters of plr plants compared to lr implied that lr might protect the photosynthesis under severe drought. also, the study showed that lr allowed photosynthesis to occur by protecting psii effi ciency and rubisco activity under severe drought by maintaining leaf water content. 100-kernel and ear weights at lr were less affected by drought as compared to plr. lr might also be important for maize yield under drought stress. therefore, we suggest that maize cultivars with late lr may be planted in order to avoid yield losses resulting from drought. acknowledgements this work was supported by the scientifi c and technological research council of turkey (tubitak, tbag 110t472). the authors are grateful to dr şemsettin kulaç (faculty of forestry, düzce university) for his help on the rain-out-shelter construction. references arnon, d. i., 1949: copper enzymes in isolated chloroplasts. polyphenoloxidase in beta vulgaris. plant physiology 24, 1–15. ashraf, m., harris p. j. c., 2013: photosynthesis under stressful environments: an overview. photosynthetica 51, 163–190. baczek-kwinta, r., adamska, a., seidler-lozykowska, k., tokarz, k., 2010: does the rate of german chamomile growth and development infl uence the response of plants to soil drought? biologia 65, 837–842. bai, l. p., sui, f. g., ge, t. d., sun, z. h., lu, y. y., zhou, g. s., 2006: effect of soil drought stress on leaf water status, membrane permeability and enzymatic antioxidant system of maize. pedosphere 16, 326–332. baker, n. r., rosenqvist, e., 2004: application of chlorophyll fl uorescence can improve crop production strategies: an examination of future possibilities. journal of experimental botany 55, 1607–1621. banziger, m., araus, j. l., 2007: recent advances in breeding maize for drought and salinity stress tolerance. in: jenks, m. a., hasegawa, p. m., jain, s. m. 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(euphorbiaceae), a new alien species for the eurasian area with nomenclatural, taxonomical, morphological and ecological notes mauro iberite1*, duilio iamonico2 1 department of environmental biology, university of rome sapienza, piazzale aldo moro 5, 00185 rome, italy 2 laboratory of phytogeography and applied geobotany, department pdta, section environment and landscape, university of rome sapienza, via flaminia 72, 00196 rome, italy abstract – manihot is a native genus of the northern and southern america with diversity centres in brazil, mexico and guatemala. some taxa have colonized other continents (except europe) where they are considered aliens. during recent fl oristic surveys we found the genus in the agro pontino area (lazio region, central italy, southern europe). this is the second record in europe for the genus and the fi rst of m. grahamii for the eurasian area. at present this taxon is to be considered as naturalized alien species in agro pontino (and thus in italy and europe). to better clarify the taxonomic and nomenclatural data, the names janipha loefl ingii var. (ß) multifi da (≡ m. grahamii) and jatropha carthaginensis (≡ m. carthaginensis) were lectotypifi ed respectively on a specimen from k and an iconography by jacquin. key words: asia, europe, italy, lectotypifi cation, manihot mill., manihot carthaginensis (jacq.) müll. arg., status of naturalization, synonymy introduction manihot mill. (euphorbiaceae, crotonoideae) is a genus of about 75–100 species distributed in america, from arizona to argentina and west indies (rogers and appan 1973, chacón et al. 2008, apgiii 2009). its distribution area is disjunct: none of the north and central american species occur in south america. centres of diversity are two: brazil, with about 80 species (66 endemic, according to cordeiro and secco 2010) and from mexico to guatemala with 18 species. molecular studies confi rm this disjunction, showing a * corresponding author, e-mail: mauro.iberite@uniroma1.it copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. iberite m., iamonico d. 144 acta bot. croat. 74 (1), 2015 dichotomy between the mesoamerican and south american manihot species (chacón et al. 2008). out of its native range, manihot was recorded as alien genus, in north america (11 species; usda 2012), asia (4 species; radcliffe-smith 1986; li and gilbert 2008; ibin 2012), africa (7 species; sanbi 2005–2010, biota africa 2012) and australia (6 species; apni 2012). recent fl oristic surveys in the agro pontino area (lazio region, central italy, southern europe) allowed to fi nd a population referring to the genus manihot and in particular to m. grahamii hook. this gathering represents the second record of the genus manihot in europe and the fi rst of m. grahamii in the eurasian area. due to the diffi culties in the identifi cation, it was necessary to investigate the nomenclatural aspects of this species and m. carthaginensis (jacq.) müll. arg., being m. grahamii related to this name in the past. taxonomical notes, morphological data in comparison with the related species m. infl ata mueller and m. janiphoides mueller and ecological observations were provided. material and methods the present study was the result of personal fi eld investigations, examination of the specimens kept in the herbaria ro, linn, k, p, w (abbreviations according to thiers 2011) and the analysis of literature (protologues included). the nomenclature follows rogers and appan (1973). the status of naturalization was evaluated according to pyšek et al. (2004), richardson and pyšek (2006), ricciardi and cohen (2007). the descriptions are based on personal observations of diagnostic characters (leaves shape and infl orescence structure). results and discussion according to rogers and appan (1973) m. grahamii is included in the sect. heterophyllae pax emend. rogers & appan (18 sections are recognized in the genus manihot) a south american group characterized by low to medium tall plants (shrubs or small tree), with not tomentose abaxial surface of the leaves blade and leaves lobes entire to pandurate, infl orescence in panicle, bracts and bracteoles foliaceous and entire. plants from italy (agro pontino area, central italy) clearly refer to this section. among the taxa included into the sect. heterophyllae, m. grahamii seems to be related to m. infl ata and m. janiphoides. all these species are included in the group of taxa with bracts and bracteoles setaceous less than 25 mm wide and infl orescence in a panicle with one main axis and spaced fl owers. the analyses of the morphological features and qualitative characters shows that m. grahamii is characterized by leaves blade glabrous abaxially, 7-lobed or more with lobes at the base more than 25 mm wide, while m. janiphoides has leaves lobe densely pubescent and m. infl ata has leaves 3–5 lobed each lobe at the base less that 25 mm wide. typifi cation of manihot grahamii manihot grahamii was described from paraná, south america (hooker 1842). the protologue consists of a diagnosis with habitat, provenance and collector (»hab. woods of the parana. tweedie«) and one synonym (»janipha loefl ingii...(excl. syn.)―β multifi da«) cited from graham (1840: 172–173). hooker (l. c.) dedicated the taxon to r. graham, observing that m. grahamii partially refers to janipha loefl ingii humb., bompl. et kunth var. multifi manihot grahamii hook. in eurasian area acta bot. croat. 74 (1), 2015 145 da graham. anyhow a new combination of this name cannot be proposed since a previous homonym was published by crantz (1766: 67), so hooker (l. c.) proposed a nomen novum avoiding an illegitimate combination. rogers and appan (1973: 88) stated the existence of three specimens at k (»type syntypes, k-3«). we found seven sheets at k (the herbarium catalogue, royal botanical garden, kew 2015), two of which (codes k000600382 and k000600383) cannot be considered for the typifi cation since they were collected by e. hassler, not by j. tweedie. the other fi ve exsiccata include tweedie’s handwriting and signature. one of these (code k000600377) came from »portalegre« (now the city of porto alegre) a locality placed along the south-eastern coast of south america, in rio grande do sul state (brazil). since this locality was not cited in the protologue by hooker (l. c.) the specimen cannot be eligible as the lectotype. the remains exsiccata (codes k000600378381) were collected by j. tweedie from »parana« (it probably refers to the current paraná state, brazil). however, only one (code k000600379) includes the date of collection (»1837«) and it can be considered certainly original material, while the sheets with codes k000600378, -380, -381 lack of this information. so, they might refer to a later collection (post 1842, the year of hooker’s publication) and could not be original material. since the exsiccatum k000600379 is the only extant and certain original material, matching with the diagnosis, it is here designated as the lectotype of m. grahamii. manihot grahamii hook., icon. pl., 6: t. 530. 1842. lectotype (here designated): argentina, »a beautiful decidious tree of the parana«, 1837, j. tweedie s. n. (k000600379!). image of the lectotype available at http://apps.kew. org/herbcat/getimage.do?imagebarcode=k000600379 ≡ janipha loefl ingii kunth var. (ß) multifi da graham, edinburgh new philos. j. 29: 172. 1840 non crantz (1766: 67). ≡ manihot tweediana müll. arg., fl. bras. (martius) 11(2): 450. 1873–1874, nom. illeg., nom. superfl . (art. 52.1 of the icn). description: arborescent shrubs to low trees to 3.5 m tall, forming an umbrella-like dense canopy of foliage at the top. not tuberous roots, light brown epidermis. trunk up to 10 cm in diameter; bark smooth, reddish brown, peeling readily from trunk, latex in small quantity, light yellowish white. young stems, glabrous, moderate olive green, internal stem colour of younger stems brilliant yellow green. leaves alternate, stipules up to 1.0 cm long, fi liform, glabrous, caducous; petioles up to 15 cm in length, glabrous, petiole attachment to lamina basal, nonpeltate; lamina greenish without any purplish pigmentation, glabrous, abaxial surface wax pattern smooth, veins on the adaxial lamina surface conspicuous, bright yellowish, glabrous; palmately 7–9 lobed; median lobes oblong pandurate, rarely entire, gradually widening from a narrow base to a prominently dilated apical region which abruptly narrows down and terminates in an acuminate apex, usually as long as 10 cm, base of lobes ca 0.3 cm wide, width between base of sinus and petiole-lamina junction ca 0.8 cm, lowest lobes more or less similar in outline as median lobes but smaller. infl orescence is a monoecious raceme, as long as 15.0 cm, accompanied by one or two individual pedunculated fl owers and an umbel with three fl owers; all parts glabrous. bracteoles and bractlets setaceous. bell-shaped fl owers with fi ve petals fused to one third of the length; pistillate fl owers restricted to base of the infl orescence, pedicels ca 1.2 cm long, tepal 1.25 cm long. the fuit is a subspherical three-carpellate capsule, not winged, 1.5 cm long, dehiscence septicidal. seeds are gray, brown or mottled and oblong in shape, 1.2 cm long, with rib-like projections along the lateral edges, caruncle moderately prominent. iberite m., iamonico d. 146 acta bot. croat. 74 (1), 2015 iconography: fig. 1 in hooker (1842, available from http://www.biodiversitylibrary. org/item/54440#page/257/mode/1up). chromosome number: 2n = 36 (cruz 1968, sub m. tweedieana muell.). status of naturalization: m. grahamii was intentionally introduced in italy (agro pontino area) from seeds collected in natural environment around buenos aires (argentina) by an italian emigrant in 1971 (direct evidence token by m. iberite). m. grahamii was cultivated in private gardens, in agro pontino, during the 70’s and 80’s. the plants have always produced fl owers, fruits and fertile seeds (pers. obs. by m. iberite). the cultivated plants were eradicated during the fi rst years of the 80’s. despite of this, from then to nowadays, the occurring of the species has been casual always deriving from seeds. the population found (fig. 1) consists of 34 individuals of different ages (stem diameter ranging from 1.5 to 10 cm) and it has occurred on the site for about ten years. the young individuals prove that the population replaces itself. so, according to pyšek et al. (2004), richardson and pyšek (2006), ricciardi and cohen (2007) m. grahamii is to be considered a naturalized alien species. fig. 1. manihot grahamii hook. in agro pontino (lazio region, italy), on clay substrate along ditches near latina. a) detail of leaves and fruits. manihot grahamii hook. in eurasian area acta bot. croat. 74 (1), 2015 147 habitat and climate of the site: m. grahamii grows on clay substrate along ditches. the altitude is about 20 m a.s.l. flowering time is june to august. the climate of the agro pontino is characterized by average annual temperatures of 15.5 °c and rainfall of 931 mm; the average temperature in january is 8.4 °c. the absolute minimum temperature was –9.2 °c (january 1985; aeronautica militare italiana 2013). it is interesting to note that m. grahamii is one of the two manihot taxa that is able to thrive in regions out of tropical and subtropical areas with occasional but predictable frost (roger and appan 1973: 7). the climate conditions of the agro pontino area match with the ecological range of the species. occurring in europe and asia: the genus manihot was not reported in any of the italian and european fl oras (e. g. tutin 1968, fiori 1923–1929, pignatti 1982, benedí, 1997, conti et al. 2005, 2007, daisie 2008, lambdon et al. 2008, celesti-grapow et al. 2009, 2010, anzalone et al. 2010), while it was recently recorded in southern italy (stinca et al. 2014 – manihot esculenta crantz). thus, the record in agro pontino area (lazio region, central italy) represents the second record of the genus for europe. as regard the species m. grahamii, since no records appears in the asian comprehensive fl oras (e. g. li and gilbert 2008, editorial committee of the flora of taiwan 2008, ibin 2012, radcliffe-smith 1986), our gathering can be considered the fi rst in the eurasian area. as graham (l. c.) described its variety under j. loefl ingii kunth and hooker (l. c.) reported it as partial synonym, it appears necessary to investigate this name. j. loefl ingii was published by kunth (1817: 107) as nomen novum pro jatropha janipha l., avoiding the publication of the tautonym janipha janipha (see art. 23.4 of the icn, mcneill et al. 2012). kunth (l. c.) cited four synonyms from linnaeus (1767: 126), jacquin (1763: 256–257) and loefling (1758: 309, 1766: 309). the loefl ing’s polynomial was cited by linnaeus (l. c.) and jacquin (l. c.) as synonym of jatropha janipha l. and j. carthaginensis jacq. respectively. the linnaean name is a nomen novum pro j. carthaginensis. since the jacquin’s name was validly published, the linnaean name is superfl uous and it is illegitimate under the art. 52.1 of the icn (mcneill et al. 2012). the jacquin’s name appears to be as not typifi ed. jacquin (l. c.) provided a short diagnosis, a long and detailed description, and the provenance (»habitat passim carthagenae«) and included a plate (»tab. clxii. fig. i.«, image available at http://www.botanicus.org/item/31753002894886) that can be considered original material. no specimens of j. carthaginensis were found in the jussieu’s collection at p. there is one specimen at w (barcode 0045823,) that bears a plant identifi able as j. carthaginensis. however, no date of collection is reported on the original label (at the centre of the sheet), so it could be referred to a later collection (post 1763, the year of publication of jacquin’s americanarum historia) and could not be original material. so, the image by jacquin is the only extant original material and it is here designated as the lectotype of the name j. carthaginensis. the comparison between the types of j. janipha l. and j. carthaginensis jacq. clearly shows that the name refers to the same plant and they can be considered synonyms. manihot carthaginensis (jacq.) müll. arg., prodr. [a. p. de candolle] 15(2): 1073. 1866. ≡ jatropha carthaginensis jacq., stirp. amer., 2: 256–257. 1763. lectotype (here designated): [icon] tab. clxii fig. i (jacquin, 1763). = jatropha janipha l., mant. pl. : 126. 1767, nom. superfl . nom. illeg. (art. 52.1 of the icn) ≡ janipha loefl ingii kunth, nov. gen. sp. [h.b.k.] 2: 107. 1817, nom. nov. pro jatropha janipha l. iberite m., iamonico d. 148 acta bot. croat. 74 (1), 2015 manihot carthaginensis is morphologically different from m. grahamii on the basis of the leaves blade shape: m. carthaginensis has leaves lobes deeply pandurate, while m. grahamii has entire lobes. this character is also diagnostic at section level, distinguishing the sect. heterophyllae pax emend. roger & appan (14 species) from the sect. carthaginensis (2 species) (rogers and appan 1973). conclusions floristic surveys in lazio region (central italy), extensive analysis of literature and herbarium investigations allowed us to fi nd a population identifi able as m. grahamii. the genus manihot is here recorded in europe for the second time, while m. grahamii can be considered the fi rst record for the eurasian fl ora. in order to fi x the hooker’s name a lectotype and an epitype are designated. for the nomenclatural purpose, the name jatropha carthaginensis is also lectotypifi ed. the morphological observations show that m. grahamii is clearly distinct from the related m. esculenta, m. infl ata and m. janiphoides. as regards the status of naturalization, threats and distribution, further investigations are needed to verify the possible change of the status of naturalization (invasion?) and the presence of m. grahamii in other italian and european regions. according to the observed and native ecological features, we can expect to fi nd this species mainly in disturbed areas (e.g. ditches or roadsides) of central and southern european countries. acknowledgements thanks are due to directors and curators of all quoted herbaria for their support during our visits or loan of specimens/photographs. references aeronautica militare italiana, 2013: servizio meteorologico: dati 1971–2000. retrieved march 30, 2013, from www.meteoam.it a. p. g. iii, 2009: an update of the angiosperm phylogeny group classifi cation for the orders and families of fl owering plants: apg iii. botanical journal of the linnean society 161, 105–121. apni, 2012: australian plant name index. retrieved march 30, 2013 from http://www. anbg.gov.au/cgi-bin/apni biota africa, 2012: westcentral and east african plants. retrieved march 30, 2013 from http://www.biota-africa.org/index.php?page_id=l900 benedi, t. g., 1997: euphrobiaceae. in: castroviejo, s., laínz, m., lópez gonzáles, g., montserrat, p., muñoz garmendia, f., paiva, j., villar, l. 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(eds.), flora of pakistan, 172, 1–170. university of karachi, karachi. ricciardi, a., cohen, j., 2007: the invasiveness of an introduced species does not predict its impact. biological invasions 9, 309–315. richardson, d. m., pyšek, p., 2006: plant invasion: merging the concepts of species invasiveness and community invasibility. progress in physical geography 30, 409–431. rogers, d. j., appan, s. j. 1973: manihot manihotoides. flora neotropica 13, 1–271. sanbi, 2005–2010: plants of southern africa: an online checklist. retrieved march 30, 2013 from http://posa.sanbi.org/searchspp.php stinca, a., d’auria, g., motti, r., 2014: manihot esculenta (euphorbiaceae), a new alien species in italy. hacquetia 13, 355–357. the herbarium catalogue, royal botanical garden, kew 2015: retrived march 30, 2013 from http://apps.kew.org/herbcat/gethomepageresults.do;jsessionid=e3fcabb 679a844ef52ff6bebdd83ee0a?homepagesearchtext=manihot+grahamii&x=0& y=0&homepagesearchoption=scientifi c_name&nameofsearchpage=home_page thiers, b. 2011: index herbariorum: a global directory of public herbaria and associated staff. new york botanical garden’s virtual herbarium. retrievd march 30, 2013 from http://sweetgum.nybg.org/ih/ tutin, t. g., 1968: euphrobiaceae. in: tutin, t. g., heywood, v. h., burges, n. a., moore, d. m., valentine, d. h., walters, s. m., webb, d. e. (eds.), flora europaea, 2, 211– 226. cambridge university press, cambridge. usda, 2012: united states department of agriculture. natural resources conservation service. retrieved march 30, 2013 from http://plants.usda.gov/java/namesearch?keyw ordquery=manihot&mode =sciname&submit.x=0&submit.y=0 opce-str.vp acta bot. croat. 73 (1), 79–92, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 antioxydant response to biotic and abiotic inducers for the resistance against fusarium wilt disease in eggplant (solanum melongena l.) hacer h. altinok1*, murat dikilitas2 1 erciyes university, faculty of agriculture, department of plant protection, kayseri, turkey 2 harran university, faculty of agriculture, department of plant protection, sanliurfa, turkey abstract – acibenzolar-s-methyl as an abiotic plant activator and a non-host isolate of fusarium oxysporum on eggplant (f. oxysporum f. sp. melonis) as a biotic inducer were applied to eggplant seedlings in order to confer increased resistance to f. oxysporum f. sp. melongenae, the causal agent of fusarium wilt of eggplant. acibenzolar-s-methyl and f. oxysporum f. sp. melonis were applied 72 h before pathogen inoculation and the development of disease symptoms was assessed with a fusarium yellow rating at 7th, 11th, 14th, 17th and 21th day after inoculation. pretreatment of eggplants with acibenzolar-s-methyl and f. oxysporum f. sp. melonis significantly reduced the severity of fusarium wilt disease. the severity of the disease in positive control plants reached to 92.50% whereas that of acibenzolar-s-methyl and f. oxysporum f. sp. melonis-pretreated seedlings of eggplants was only 32.21% and 21.13%, respectively, 21 days after inoculation. acibenzolar-s-methyl and f. oxysporum f. sp. melonis pretreatments resulted in a hypersensitive reaction and triggered the elaboration of histological barriers such as callose and h2o2 synthesis. in situ studies demonstrated that the hydrogen peroxide (h2o2) accumulation and the callose deposition as responses to the pathogen attack started 24 h after inoculation. acibenzolar-s-methyl and f. oxysporum f. sp. melonis-pretreated plants also showed significant increases in the activity of catalase and polyphenol oxidase enzymes along with the increase of proline and h2o2 content when compared to f. oxysporum f. sp. melongenae-infected plants. keywords: acibenzolar-s-methyl, catalase, fusarium wilt disease, fusarium oxysporum, hydrogen peroxide, induced resistance, polyphenol oxidase, solanum melongena abbreviations: asm – acibenzolar-s-methyl, dai – days after inoculation, cat – catalase, ppo – polyphenol oxidase, fom – fusarium oxysporum f. sp. melonis, fomg – fusarium oxysporum f. sp. melongenae, hr – hypersensitive reaction acta bot. croat. 73 (1), 2014 79 * corresponding author, e-mail: ahandan@gmail.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:00 color profile: generic cmyk printer profile composite default screen introduction fusarium wilt caused by fusarium oxysporum schlecht. f. sp. melongenae (fomg) is an economically important soil-borne disease limiting eggplant production worldwide. this pathogen is one of the most important fungal pathogens of eggplants (solanum melongena) in the mediterranean region of turkey, which causes serious yield losses. infected plants exhibit leaf chlorosis and slight vein clearing on outer leaflets initially, then yellowing and leaf-dropping, followed finally by vascular discoloration of stem and death of aboveground portions (altinok 2005). current strategies for the control of this disease are based mostly on soil disinfection, use of resistant cultivars and efficient fungicides. the most effective method for control of the disease worldwide has been suggested to be the use of resistant cultivars; however, no variety so far has been reported as being resistant to fomg. although physiological races of this pathogen have not been reported, however, only one vegetative compatibility group (vcg) has been identified (katan 1999, altinok and can 2010). a pathogenic strain applied to a non-host plant is able to protect it against further infection by its specific formae speciales, which is a well-established phenomenon and has been described as cross-protection or premonition (matta 1989). this phenomenon is known as an expression of induced systemic resistance (isr), a defense mechanism of plant response to microbial infections. plants exhibit a wide variety of defense strategies against pathogen attack. they form physical barriers and biochemical defenses before and after pathogen attacks. however, barriers formed depend on the ability and structure of the plants rather than the severity of the pathogen (dikilitas 2003). for example, a type of plant defense system such as hypersensitive response (hr), also called plant cell death, due to host-pathogen incompatibility prevents crop plants from pathogen infection (mehdy 1994). systemic acquired resistance (sar) is another defense mechanism which plays an important role in protecting plants from pathogen attack and this mechanism is often accompanied by hr (hammerschmidt and kuc 1995). the responses of induced resistance in plants can be activated by using biotic inducers or by some chemicals such as acibenzolar-s-methyl (bth or asm), salicylic acid (sa), and 2,6-dichloroisonicotinic acid (ina) (graham and graham 1999). asm as a potential plant activator has no antimicrobial activity but it activates the sar signal transduction pathway against a broad spectrum of fungal, bacterial and viral pathogens in many plants (ishii et al. 1999). induced resistance needs an inducer prior to the pathogen attack; however, the time between the inducer and challenger inoculation is an important factor for the development of induced resistance; it ranges from one to three days for fusarium wilt diseases (matta 1989). the induction of sar is related to various cellular defense responses which are a synthesis of pathogenesis-related proteins (pr), phytoalexins, localized cell death, the accumulation of wall-bound phenolic compounds and synthesis of hydrolytic enzymes such as chitinase, b-1,3-glucanase and peroxidase (dikilitas et al. 2009). the characteristics of hr include rapid generation of active oxygen species (aos), described as the oxidative burst, which occurs in the early phase of plant-pathogen interactions. in this stage, h2o2 is formed as a signaling molecule, and is one of the main components of aos. apart from h2o2, superoxide (o2 –) and hydroxyl (oh) radicals are also produced to protect the plants from attacking organisms (borden and higgins 2002). accumulation of aos in the organism contributes to the defense system as an antimicrobial compound, which helps to form cell wall barriers, and as a secondary signaling molecule to activate defense responses (de gara 80 acta bot. croat. 73 (1), 2014 altinok h. h., dikilitas m. 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:00 color profile: generic cmyk printer profile composite default screen et al. 2003). for example, the generation of h2o2 results in the formation of lignin and callose; their deposition with proteins and phenolics at sites of penetration is recognized as an early defense response of a host to microbial pathogens (nicholson and hammerschmidt 1992, hammerschmidt and kuc 1995, jacobs et al. 2003). however, it should be remembered that the excess of aos production can significantly damage plant tissues at structural and functional levels (djebali et al. 2007). as a response to pathogen attack, plants are protected against the damage of reactive oxygen species by an antioxidant system which includes enzymes such as superoxide dismutase (sod) and peroxidase (pod). for example, sod activity has been shown to change in response to salt stress lee et al. (2001) and to pathogen attack of plants de gara et al. (2003). peroxidase activity in diseased plants was also observed to have resistance involved in many host-pathogen interactions (alcazar et al. 1995). the mechanisms involved in the induced resistance against fomg have not been adequately studied. the purpose of the present study was to evaluate the potential plant activator asm and a non-host fusarium oxysporum f. sp. melonis strain for the inhibition of fusarium wilt disease of eggplant caused by fomg and to investigate their possible modes of action through changes in proline accumulation and in situ localization of h2o2 and callose, and changes in specific activity of the antioxidant enzymes such as catalase (cat) and polyphenol oxidase (ppo). materials and methods plant material eggplant (solanum melongena l. cv. »kemer«, susceptible to fomg) seeds were sterilized by being dipped in 1% sodium hypochlorite (v/v) for 30 min and sown in a soil mix containing sand, perlite, and peat compost (1:1:2), and kept in a growth chamber (25 °c, 60–70% rh, 12 h photoperiod, 50 to 60 klux m–2). the eggplant seedlings were transplanted when they were 6 week old to pots 8.5 cm in diameter. the seedlings were then watered on demand and fertilized with npk (15:15:15). the pretreatment of asm and a non-host fusarium oxysporum isolate on eggplants an abiotic plant activator, acibenzolar-s-methyl (benzo [1, 2, 3] thiadiazole-7-carbothioic acid-s-methyl ester, asm; actigard 50 wg, syngenta crop protection, inc., basel switzerland) and the non-host fungus of the eggplant fusarium oxysporum f. sp. melonis (fom) as a biotic inducer were used in this study. fusarium oxysporum f. sp. melongenae (fomg), the most virulent isolate, according to the severity of the disease reported in a former study, was selected for pathogen inoculation (altinok and can 2010). asm was dissolved in distilled water to obtain a concentration of 0.2 mg ml–1 and then sprayed onto eggplant seedlings at 7–8 leaf stage. in parallel, a set of seedlings were pretreated with fom by root-dipping into a conidial suspension of fom (106 conidia ml–1) for 10 min. seventy two hours after the pretreatments (asm, fom, or water), the seedlings were then dipped into the conidial suspension of the pathogen fomg (106 conidia ml–1) for 10 min. two fusarium strains (the pathogen and the inducer) were cultured on fusarium-minimal medium; for 7 days in the dark at 25 °c (nelson et al. 1983). in the control set, the wateracta bot. croat. 73 (1), 2014 81 antioxydants and resistance against fusarium wilt disease in solanum 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:00 color profile: generic cmyk printer profile composite default screen -treated plants (positive control) were dipped into the conidial inoculum of fomg for the same time period. the healthy controls (negative control) were also treated with sterile distilled water instead of treatments. in our previous study, the optimum time interval for sar induction was determined to be 72 h for these inducers (altinok 2009). after the pathogen inoculation, the seedlings were transplanted into plastic pots and kept in the growth chamber as described above. microscopy studies in order to detect early accumulation of h2o2 and callose deposition, the samples were examined 24, 48 and 72 h after inoculation. samplings were then continued 7, 1, 14 and 21 days after inoculation. the fresh leaf tissues were immediately used for microscopic examination. the microscopic examination of the infected plants was carried out on a microscope (nikon optiphot) equipped with differential interference contrast (dic). the detection of callose accumulation during the infection is discussed by nicholson and hammerschmidt (1992), and hammerschmidt and kuc (1995), referring to fluorescence microscopy and histochemical studies. autofluorescence of the callose in infected tissue was detected with uv light irradiation (350–450 nm; excitation-emission). the cell-wall bound callose components fluoresced bright pale blue under uv light. the tissues were observed with 40x optic zoom and recorded with a digital camera (dp71; olympus). the accumulation of h2o2 was visualized using 3,3 n-diaminobenzidine tetrahydrochloride liquid substrate system (dab; sigma, st. louis, usa). the infected leaves were incubated in a solution of dab (1 ml of dab liquid chromogen to 9 ml of dab liquid buffer) for 20 min in the dark and the reaction was stopped by gentle washing of the leaf sample in water. the plant tissues were then transferred to 100% methanol and incubated overnight at room temperature to remove chlorophyll, followed by soaking in an aqueous solution saturated with chloral hydrate (2.5 g ml–1) for 12–24 h to soften and clear the tissue. the samples were subsequently mounted in 50% glycerol and a cover-slip placed over the samples to produce semi-permanent preparations. the accumulation of h2o2 appeared as dark brown pigmentation (soylu 2006). the deposition of the callose substances in the leaves of infected plants was determined with the aniline blue fluorescence method. the infected leaves were incubated in a solution of 1% phloroglucinol in 100% methanol overnight at room temperature. following further incubation for clearing tissues in chloral hydrate (2.5 g ml–1), they were then transferred to aniline blue (0.05% w/v) reagent prepared in sodium phosphate buffer (1 m, ph 8.0). after this stage, the samples were mounted on slides with a few drops of hoyer’s solution [30 g gum arabic (sigma g-9752), 200 g chloral hydrate (sigma c-8383), 20 g glycerol, 50 ml water], and the slides were covered with a coverslip for the microscopic examination (bougourd et al. 2000). sample preparation for enzymatic studies for enzyme analyses, leaf tissues were sampled 7, 14 and 21 days after inoculation (dai). the leaf samples for enzyme analysis were stored at –80 °c until use. 82 acta bot. croat. 73 (1), 2014 altinok h. h., dikilitas m. 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:00 color profile: generic cmyk printer profile composite default screen a one-gram leaf sample was ground with an ice-cold pestle and mortar with 10 ml 50 mm phosphate buffer (ph 7.0). the homogenates were centrifuged at 12000 g for 20 min at 4 °c. the supernatant filtered through two layers of the cheese-cloth was used for the assays of the enzymatic activities as well as for protein and h2o2 determination. determination of h2o2 content the h2o2 content was measured colorimetrically after reaction with ticl4 as described by tsai et al. (2004) with slight modifications. the reaction mixture consisted of 1.8 ml of 50 mm phosphate buffer (ph 7.0), 0.2 ml of leaf extract supernatant and 1 ml reagent [0.1% (v/v) ticl4 in 20% (v/v) h2so4]. the samples without leaf extract were used as blank. the absorbance was measured at 410 nm. the amount of h2o2 was calculated by use of a standard curve prepared with known concentration of h2o2. the h2o2 content was expressed as mmol g–1 fresh weight (fw). proline measurement the proline content was measured according to the method of bates et al. (1973). proline was extracted from 0.5 g of leaf sample by grinding in 10 ml of 3% sulphosalicylic acid and the mixture was centrifuged at 10000 g for 10 min. two ml of the supernatant was mixed with 2 ml freshly prepared acid-ninhydrin and 2 ml glacial acetic acid and heated at 90 °c in a water bath for 1 h. the reaction was terminated in an ice-bath, and then 5 ml toluene was added to the mixture and vortexed for 15 sec. after standing at least 20 min in darkness at room temperature to separate the toluene and the aqueous phase, the toluene phase was carefully collected into test tubes and the absorbance of the fraction was read at 520 nm with a spectrophotometer (shimadzu uv-1700). the proline concentration in the sample was determined from a standard curve using analytical grade l-proline and calculated on fw basis. the measurement of cat activity the catalase (cat, ec. 1.11.1.6) activity was assayed according to kato and shimizu (1987) by monitoring the consumption of h2o2 in a uv spectrophotometer (shimadzu uv-1700) at 240 nm. the reaction mixture consisted of 0.2 ml enzyme extract, 1.5 ml phosphate buffer (ph 7.4, 0.1 mol l–1), and 1 ml distilled water. the reaction was started by adding 0.3 ml 0.1 mol l–1 h2o2. samples without h2o2 were used as blank. the decrease in absorbance was recorded every 30 seconds for 2 min. the enzyme activity was expressed as mmol h2o2 min –1 mg–1 protein by using the extinction coefficient of 40 mm–1 cm–1 for h2o2. one cat unit is defined as the amount of enzyme necessary to decompose 1 mmol min–1 h2o2 under the above mentioned conditions. the measurement of ppo activity polyphenol oxidase (ppo, ec 1.14.18.1) activity was assayed with 4-methylcatechol as a substrate according to the method described by zaubermann et al. (1991). the reaction mixture consisted of 0.5 ml diluted enzyme extract, 2 ml phosphate buffer (ph 7.0, 0.1 mol acta bot. croat. 73 (1), 2014 83 antioxydants and resistance against fusarium wilt disease in solanum 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:00 color profile: generic cmyk printer profile composite default screen l–1) and 0.5 ml of 100 mmol l–1 4-methylcatechol. the increase in absorbance at 410 nm at 25 °c was recorded every 30 seconds for 2 min. the enzyme activity was expressed as changes in absorbance in min–1 mg–1 protein. protein determination protein determination of the samples was made according to the coomassie brilliant blue g250 method at 595 nm colorimetric wavelength (bradford 1976). disease assessment disease symptom development was assessed 7, 11, 14, 17 and 21 dai with a fusarium yellow rating from 0 to 4, in which 0 = no lesions, 1 = slight leaf chlorosis and necrosis, 2 = vein clearing on outer leaflets, 3 = yellowing and dropping of leaves, 4 = dead plant. the plants were evaluated individually and the percentage of mean disease severity index (% dsi) was calculated (townsend and heuberger 1943). statistical analyses the disease severity data were subjected to analysis with levene’s homogeneity of variance test then grouped by duncan’s multiple range test (p < 0.05) contained in the spss software (spss inc., chicago, il, usa). the experiment was conducted for each treatment from the scores of 60 plants (three replicates of 20 plants for each treatment) and repeated twice; representative results of two experiments for each treatment are presented in the results section. results the effect of asm and fom pretreatments on the reduction of fusarium wilt symptoms in a previous study, the lowest disease ratings were detected at a time interval of 72 h between treatment and pathogen inoculation (altinok 2009). from this work, this interval was taken into consideration in order to determine the disease development, histochemical study and enzyme assay experiments. resistance induced in eggplant seedlings by asm and fom is presented in figure 1. initial symptoms appeared seven days after inoculation as yellowing of the older leaves on asm-, fom-pretreated and positive control plants; the mean disease severity was 6.32%, 5.07% and 11.25%, respectively. the systemic progress of the disease in control plants rapidly increased with time and by 21 dai, most of the positive control plants showed severe wilting and eventually collapsed at the end of the experiment. areas of browning were observed in the xylem of the infected stems. the pretreatment of eggplants with asm and fom before inoculation with fomg did not inhibit pathogen penetration into the root tissues, but the progress of disease was much slower than those in only fomg-inoculated plants. non-inoculated seedlings (negative control plants) showed no symptoms and appeared healthy throughout the course of the experiment. at the end of the experiment, the mean disease severity in positive control plants reached 92.50% whereas in asmand fom-pretreated plants it was 32.21% and 21.13%, respectively (fig. 1). 84 acta bot. croat. 73 (1), 2014 altinok h. h., dikilitas m. acta botanica 1-2014 str088.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 24. o ujak 2014 11:05:22 color profile: generic cmyk printer profile composite default screen accumulation of h2o2 at the reaction sites light microscopy studies demonstrated that the applications of asm and fom enhanced a systemically induced resistance to fomg infection in the susceptible eggplant cultivar. the localization and the timing of the accumulation of h2o2 to determine aos accumulation was investigated using the technique of dab staining. the tissue staining method used is based on the reaction of h2o2 with dab to produce a dark brown precipitate at the reaction sites. in the present study, we demonstrated rapidly inducible responses including cell death after the attack of fomg. in leaf epidermal cells, brown staining, an indicative sign of localized h2o2 production, was evident as early as 24 h after pretreatments with asm and fom (figs. 2a, b). while no browning was detectable in non-infected epidermal cells, h2o2 accumulation in tissues stained with dab was clearly localized to the sites of reaction in leaf epidermal cell walls (figs. 2a, b). the accumulation of h2o2 was still evident after 11 days following pretreatments of asm and fom inducers (figs. 2c, d). callose deposition at reaction sites histochemical localization of callose was previously described by bougourd et al. (2000). in our study, the deposition of callose after pretreatments with asm and non-host fusarium oxysporum was closely associated with the accumulation of h2o2 (figs. 3a, b). callose was determined by fluorescence under uv light after aniline-blue staining as a marker of defense response after the pathogen attack. in mesophyll cells, callose accumulation was observed as blue autofluorescence after 24 h of the pathogen inoculation in asmand fom-pretreated plants (figs. 3a, b). the mesophyll cells of the eggplant that had not been inoculated with the pathogen did not show any autofluorescence. the deposition of callose was still detectable after 11 days of pretreatments with asm and fom (figs. 3c, d). enzyme activities in asm and fom pretreated plants for the time convenience, the results obtained from the microscopy studies led us to perform biochemical analysis on 7, 14 and 21 dai on test plants. when the accumulation of biochemical metabolites were considered, proline and h2o2 contents were found signifiacta bot. croat. 73 (1), 2014 85 antioxydants and resistance against fusarium wilt disease in solanum fig. 1. effect of asm and fom pretreatments on the severity of fusarium wilt by fomg. 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:01 color profile: generic cmyk printer profile composite default screen 86 acta bot. croat. 73 (1), 2014 altinok h. h., dikilitas m. fig. 2. accumulation of h2o2 in the epidermal cells of asmand fom-pretreated eggplant seedlings inoculated with fomg; a – early accumulation (24 h) of h2o2 in the epidermal cells of eggplant following pretreatments of asm; b – early accumulation (24 h) of h2o2 in the epidermal cells of eggplant following pretreatments of fom; c – late accumulation (11 dai) of h2o2 in the epidermal cells of eggplant following pretreatments of asm; d – late accumulation (11 dai) of h2o2 in the epidermal cells of eggplant following pretreatments of fom. fig. 3. localization of callose in mesophyll cells of asmand fom-pretreated eggplant seedlings inoculated with fomg; a – early accumulation (24 h) of callose in the mesophyll cells of eggplant following pretreatments of asm; b – early accumulation (24 h) of callose in the epidermal cells of eggplant following pretreatments of fom; c – late accumulation (11 dai) of callose in the epidermal cells of eggplant following pretreatments of asm; d – late accumulation (11 dai) of callose in the epidermal cells of eggplant following pretreatments of fom. 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:04 color profile: generic cmyk printer profile composite default screen cantly higher in asmand fom-pretreated plants than those of fomg-inoculated plants (tab. 1, p £ 0.05). the accumulation of those metabolites was significantly higher when compared to control plants throughout the course of experiment indicating that asm and fom pretreated plants not only accumulated these metabolites earlier but also kept their level higher than those of control plants in which the accumulation of proline and h2o2 were also evident to a lower extent. when antioxidant enzymes were measured, cat and ppo activities were also found significantly higher in asmor fom-pretreated plants than those found in fomg-inoculated plants (tab. 1, p £ 0.05). the activities of the enzymes were significantly higher than those of positive control plants inoculated with fomg throughout the course of the experiment although a general trend to decline was evident in the enzymes both in pretreated and treated plants. for example, the enzymatic activities in the second week declined and this trend carried on towards the end of experiment. we demonstrated that the early production of h2o2 as well as the deposition of callose and the synthesis of the antioxidant enzymes and the stress metabolites such as proline in asmand fom-pretreated plants played important roles in the early stages of defense against fomg. the histochemical changes related with the synthesis of antioxidant enzymes acta bot. croat. 73 (1), 2014 87 antioxydants and resistance against fusarium wilt disease in solanum tab. 1. effect of asm and fom pretreatments on the biochemical activities of eggplants inoculated with fomg. days fom + fomg asm + fomg (+) control* proline (mmol g–1 fw) 7 34.1 ± 1.3b** 49.6 ± 4.4a 30.1 ± 1.9c 14 45.2 ± 2.2a 40.4 ± 3.8ab 35.5 ± 2.6c 21 26.6 ± 3.1ab 30.5 ± 2.9a 16.9 ± 1.8c h2o2 (mmol g –1 fw) 7 30.1 ± 7.3b 40.2 ± 5.2a 22.1 ± 3.1c 14 35.2 ± 5.2ab 39.1 ± 3.2a 17.7 ± 3.2c 21 25.1 ± 4.1ab 30.3 ± 4.1a 10.2 ± 2.4c cat (µmol min–1 mg–1 protein h2o2) 7 0.21 ± 0.3b 0.27 ± 0.4a 0.15 ± 0.1c 14 0.15 ± 0.1a 0.17 ± 0.1a 0.06 ± 0.1b 21 0.10 ± 0.2a 0.08 ± 0.1a 0.04 ± 0.1b ppo (d od410 min –1 mg–1 protein) 7 0.26 ± 0.2a 0.25 ± 0.3a 0.20 ± 0.2b 14 0.11 ± 0.2b 0.14 ± 0.3a 0.08 ± 0.2c 21 0.12 ± 0.3a 0.11 ± 0.3ab 0.09 ± 0.1b * positive control plants were inoculated with fomg. **data are means (± se) of at least three replicates. means followed by different letters in lines are significantly different at p £ 0.05 as determined by duncan’s multiple range test. acta botanica 1-2014 str091.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 24. o ujak 2014 11:07:33 color profile: generic cmyk printer profile composite default screen and biochemical metabolites were well established and correlated. the results of the study showed that susceptible eggplants were enhanced with a systemically induced resistance to fomg infection after pretreatments of asm and fom applications. asm and fom pretreatments resulted in hr and triggered the elaboration of histological barriers like callose and h2o2. discussion in this study, the potential roles of asm and fom on the induction of resistance to fomg were studied through biochemical and histological analysis. the microscopic observations indicated that the pretreatment of eggplants with asm and fom resulted in an increased rapid response and temporary production of aos by the plant known as »oxidative burst« (mehdy 1994, low and merida 1996). h2o2 from a potential oxidative cross-linking of the cell wall at the plant cell surface has been previously reported to be a characteristic response of plant cells to microbial elicitors or challenge with an avirulent pathogen (grant and loake 2000). h2o2 has also been observed in cell walls of leaves undergoing hr. similar observations have been highlighted by benhamou and belanger (1998) in tomato; they suggested that the commercial use of benzo-(1,2,3)-thiadiazole-7-carbothioic acid s-methyl ester (bth) would reduce the extent of fungal colonization in the plants, which was associated with a massive accumulation of structural barriers such as phenolic compounds. in our study, h2o2 and proline accumulation were quite effective in preventing the spread and further infection of the disease although the contents of proline and h2o2 declined towards the end of the experiment. the case in fomg-inoculated plants followed the similar pattern with a low trend. the earlier accumulation of h2o2 is well correlated with the defense system. the increased production of radicals such as h2o2 is a common feature of defense responses to avirulent pathogens. for example, levine et al. (1994) stated that the application of h2o2 not only increased the triggering of hypersensitive cell death, but also limited the spread of cell death by inducing cell protectant genes in surrounding cells. there are similar reports about an increased level of h2o2 contributing to the stimulation of disease resistance in plant; however, this high level was reduced by scavenging by antioxixant enzymes such as cat and pod, which prevented the most harmful effects of excess h2o2 on cells (baker and orlandi 1995, cao and jiang 2006). histological events associated with cell death response involves the appearance of brown staining which is an indicative sign of h2o2 production in mesophyll cells undergoing pathogen attack (vanacker et al. 2000). histological events also involve the deposition of callose at reaction sites. for example, hauck et al. (2003) used histochemical staining with aniline blue followed by uv fluorescence microscopy to determine the callose at reaction sites after challenge with a hrcc mutant of pseudomonas syringae pv. phaseolicola. brammall and higgins (1988) reported that the response of tomato plants against f. oxysporum f. sp. radicis-lycopersici correlated with with genetic resistance. the thickening of host cell walls and the deposition of polymers such as b-1,3 glucan generally form appositions (papillae) in the cell wall (bolwell and daudi 2009). although the activities of antioxidant enzymes did not show any significant differences in asm and fom pretreated plants, they were significantly higher than those of fomg-inoculated group throughout the experiment. 88 acta bot. croat. 73 (1), 2014 altinok h. h., dikilitas m. 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:04 color profile: generic cmyk printer profile composite default screen the increased concentrations in metabolites such as proline and h2o2 were in agreement with the increase of antioxidant enzymes. a similar case was also observed in the work of cao and jiang (2006), which reported a very strong correlation between the increment of peroxidase and h2o2 accumulation, which eventually increased phenolic compounds. these findings were also supported by the earlier work of soylu et al. (2003). they reported that the increases in pox activity and h2o2 were closely associated with progressive incorporation of phenolic compounds within the cell wall. in general, the reinforcement of the cell wall reduces susceptibility to wall-degrading enzymes secreted by pathogens. it is also possible that the diffusion of pathogen-derived toxins is restricted and forms a mechanical barrier to the penetration of fungal hyphae (djebali et al. 2007). in this study, evidence is provided that asm-pretreatments could improve the condition of eggplants inoculated with fomg by stimulating the many defense reactions, such as physiological and biochemical responses. therefore, asm-pretreatment could be used in practice against fusarium wilt disease of eggplant caused by fomg. the current study with fom on fomg-inoculated plants also showed that the activation of plant resistance might possibly activate the genes that might in turn provide valuable information concerning fungal resistance mechanisms by using nonpathogenic fusarium oxysporum formae speciales on eggplant. it is known that pathogenic strains display protective behavior on non-host plants against their specific fusarium pathogens (alabouvette et al. 2009). in their capacity to protect plants against their pathogens, there is a great deal of diversity among soil-borne nonpathogenic strains of f. oxysporum, and some resistance-inducing strains have been isolated not only from soil, but also from the stems of healthy plants. as demonstrated by minerdi et al. (2008), one reason for the lack of pathogenicity in fusarium strains is due to the associated ectosymbiotic bacteria complex that modulates the expression of pathogenicity genes. although histochemical and enzymatic studies conducted here revealed the inductive effect of fom against fomg, fom cannot conveniently be considered a biocontrol agent due to its pathogenic effect on other plant species. therefore, further studies to elicit underlying mechanisms involved in the protective behaviors of pathogenic fusarium species on non-host plants are necessary. in conclusion, with the use of asm and fom, we showed that the increase in enzymatic activities corresponded to the accumulation of proline and h2o2 contents as well as h2o2 and callose accumulation microscopically in which all played significant roles and contributed to the increased levels of eggplant resistance to the attacking pathogen fomg. it would be very important to determine the status and levels of pathogen-related (pr) proteins and enzymes such as glucanase and chitinase produced by the host plant as well as by the pathogen for their amounts and the specific activity (increased or decreased) would increase our insight into asmor fom-mediated resistance. although asm appear not to have any antimicrobial characteristics, determining the production of pr proteins by the pathogen would enable us to gain a better insight under induced resistance conditions into whether this chemical could be used along with the other plant activators. acknowledgements this study was supported by research fund of the erciyes university. project number: fba-0736. the authors are thankful to the late dr. yeter canihos for her kind support; dr. acta bot. croat. 73 (1), 2014 89 antioxydants and resistance against fusarium wilt disease in solanum 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:04 color profile: generic cmyk printer profile composite default screen m. kamberoglu and dr. s. soylu for kind assistance and dr. saim ozdamar for laboratory facilities. the part of this study was carried out in the central science laboratory of harran university-turkey. references alabouvette, c., olivain, c., mighelo, q., christian, s., 2009: microbiological control of soil-borne phytopathogenic fungi with special emphasis on wilt-inducing fusarium oxysporum. new phytologist 184, 529–544. alcazar, d. m., egea, c., espin, a., candela, m. e., 1995: peroxidase isoenzymes in defense response of capsicum annuum to phytophtora capsici. physiological plant pathology 94, 736–742. altinok, h. h., 2005: first report of fusarium wilt of eggplant caused by fusarium oxysporum f. sp. melongenae in turkey. plant pathology, 54, 577. altinok, h. h., 2009: activation of systemic disease resistance by acibenzolar-s-methyl and a non-pathogen fusarium oxysporum melonis (fom) strain against fusarium wilt disease in eggplant seedlings. journal of turkish phytopathology 38, 21–32. altinok, h. h., can, c., 2010: characterization of fusarium oxysporum f. sp. melongenae isolates from eggplant in turkey by pathogenicity, vcg and rapd analysis. phytoparasitica 38, 149–157. bates, l. s., waldeen, r. p., teare, i. d., 1973: rapid determination of free proline for water stress studies. plant and soil 39, 205–207. baker, c. j., orlandi, e. w., 1995: active oxygen in plant pathogenesis. annual review of phytopathology 33, 299–321. benhamou, n., bélanger, r. r., 1998: benzothiadiazole-mediated induced resistance to fusarium oxysporum f. sp. radicis-lycopersici in tomato. plant physiology 118, 1203–1212. bolwell, g. p., daudi, a., 2009: reactive oxygen species in plant-pathogen interaction. in: del rio, l. a., puppo, a. 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dikilitas m. 754 altinok and dikilitas.ps u:\acta botanica\acta-botan 1-14\754 altinok and dikilitas.vp 19. o ujak 2014 16:56:04 color profile: generic cmyk printer profile composite default screen acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 333 acta bot. croat. 73 (2), 333-345, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 seasonal differences in growth, photosynthetic pigments and gas exchange properties in two greenhouse grown maize (zea mays l.) cultivars iqbal hussain1*, abdul wahid2, rizwan rasheed1, hafiz muhammad akram3 1 department of botany, govt. college university, faisalabad-38000, pakistan 2 department of botany, university of agriculture, faisalabad-38040, pakistan 3 plant physiology section, agronomic research institute, aari, faisalabad-38850, pakistan abstract – the greenhouse (gh) effect has emerged as a major factor in changing cropping patterns and limiting crop yields. this study was conducted to determine the comparative growth and photosynthetic responses of selected heat-resistant (cv. sadaf) and heat-susceptible (cv. agatti-2002) cultivars of maize to simulated gh conditions during spring and autumn seasons at seedling, silking and grain fi lling stages in 2007. fifteen day old plants were shifted to plexiglass-fi tted canopies to create gh conditions and data were recorded at each growth stage. the results revealed that the seasons, gh conditions and cultivars had large effects on plant growth and photosynthetic attributes. simulated gh conditions increased the canopy temperature 4–7 °c in spring and 3–5 °c in autumn, but increased relative humidity by 2–3% in spring and 5–9% in autumn season. although gh reduced the growth of both cultivars, shoot dry mass was reduced more in spring grown heat-susceptible maize at all growth stages. although the cultivars showed a decrease in growth and photosynthesis, gh conditions resulted in less damage to cv. sadaf than cv. agatti-2002 in both seasons. major indicators of sensitivity to gh effect were loss of chlorophyll b and carotenoids, reductions in net photosynthesis and stomatal conductance, and possibly reduced ability of rubisco to fi x co2 in sensitive maize. keywords: canopy temperature, gas exchange, greenhouse effect, growth, maize, photosynthesis abbreviations: car – carotenoid, ci – leaf substomatal co2 concentration, chl-a – chlorophyll a, chl-b – chlorophyll b, chls – total chlorophylls, e – leaf transpiration rate, gh – greenhouse, gs – stomatal conductance, par – photosynthetically active radiations, pn – net photosynthesis * corresponding author, e-mail: iqbalbotanist1@yahoo.com copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. hussain i., wahid a., rasheed r., akram h. m. 334 acta bot. croat. 73 (2), 2014 introduction the greenhouse (gh) effect on plants arising from the increased concentration of greenhouse gases and resulting in increased ambient temperature has emerged as a major growthlimiting factor for most crop plants. the gh effect results in increased canopy temperature that causes reduction of plant biomass (blum 1988, hussain et al. 2010). heat stress directly alters photosynthetic acclimation and physiological processes and indirectly changes the pattern of development (mahmood et al. 2012, arbona et al. 2013). in different studies, high temperature caused a decrease in growth, transpiration, respiration and photosynthesis and fi nal economic yield (karim et al. 2000, stone 2001). optimal photosynthesis has a fundamental importance for the carbon accumulation, growth and biomass production of different plant species. sub-optimal growth conditions affect all the aspects of photosynthesis. high temperature signifi cantly inhibits net photosynthesis (pn) and stomatal conductance (gs) in many plant species (crafts-brandner and salvucci 2002, morales et al. 2003). the pn in developed and nearly developed leaves was more sensitive than in developing leaves (karim et al. 1999). photosynthetic apparatus is highly sensitive to high temperature and inhibited when the leaf temperature exceed 38 °c (crafts-brandner and salvucci 2002). high temperature reduces the activation state of rubisco, which is the most susceptible component of the photosynthetic apparatus in both c3 and c4 plants (crafts-brandner and salvucci 2002, salvucci and crafts-brandner 2004, luo et al. 2011). extensive studies have shown that both photosystems are damaged by increasing temperature during photosynthesis, thus leading to reduced photosynthetic effi ciency (szilvia et al. 2005, du et al. 2011). maize (zea mays l.) is a c4 plant and has a distinctive leaf anatomy and photosynthetic metabolism (taiz and zeiger 2010). both photosynthesis and growth of c4 plants respond positively towards elevated ambient co2 (ghannoum et al. 2000). the rate of leaf development and pn in maize is at maximum near 31 to 34 °c (tollenaar 1989, yan and hunt 1999, kim et al. 2007) at ambient co2, but decreased at temperatures above 37 °c while complete inhibition occurred near 45 °c (crafts-brandner and salvucci 2002). however, there are great intraspecifi c differences in maize for tolerance to ambient environmental changes. in the era of climate change, the gh effect has attained major importance and thus led scientists to study precisely its infl uence on the photosynthetic properties of important crop species. to adapt to changes in the environment, plants have evolved a number of physiological and biochemical strategies. leaf photosynthesis plays an important role in the adaptation of plants to changing environmental conditions, but this also depends upon the type of species and the growth stage. limited reports have highlighted the specifi c growth and photosynthetic responses of plants to current and upcoming changes in the climatic conditions. the present study was undertaken to determine the changes in growth, pigment composition and gas exchange attributes of selected differentially heat-responsive maize cultivars grown in gh conditions. materials and methods source of maize seed, treatment and plant growth conditions seeds of selected maize (zea mays l.) cultivars sadaf (heat-tolerant) and agatti-2002 (heat-sensitive) were obtained from the maize and millets research institute (mmri), effect of greenhouse conditions on two maize cultivars acta bot. croat. 73 (2), 2014 335 yousafwala, sahiwal, pakistan. the experiments were conducted in the wire-house of the department of botany, university of agriculture, faisalabad, pakistan during the spring and autumn seasons of 2007. seeds of both cultivars were grown in plastic pots, 30 cm high, 82 cm in circumference at the top and 70 cm at the bottom. a hole was made in the bottom for leaching during replacement of the soil solution. each pot contained 13 kg of dry sand, which was washed thoroughly with tap water followed by distilled water before fi lling in the pots. ten seeds of both cultivars were sown. after germination, the pots were given half strength nutrient solution (hoagland and arnon 1950) after four days in an amount to drain the previous solution. five healthy and equal sized, three-day old seedlings were retained in each pot for making determinations at seedling, silking and grain fi lling stages. greenhouse conditions were created by shifting the pots containing growing plants to the canopies that were placed in a wire-house at the above growth stages, whilst the control set was kept outside the canopies in a wire-house. the top of the wirehouse was covered with polythene sheeting to produce the light transmission index of 75 to 80% in and outside the canopy. moreover, by measuring par using an open system portable infrared gas analyzer (irga; lca-4, analytical development company, hoddesdon, england), it was noticed that the plants inside and outside the canopy had par (400–700 nm) at the leaf surface in the range 1185–1204 µmol m–2 s–1 between 10 and 11 am. the temperatures and relative humidity inside and outside the canopies were recorded in both the seasons just above the plant height (fig. 1). the plants were kept inside the canopies for 20 days at each of the growth stages, and harvesting was done after 15 days of treatment application. fig. 1. variation in the temperature and relative humidity inside and outside the plexiglass fi tted canopy during an experiment in spring and autumn seasons in 2007. hussain i., wahid a., rasheed r., akram h. m. 336 acta bot. croat. 73 (2), 2014 for the determination of chl-a, chl-b, their total and total carotenoid (car) contents, the third leaf (0.5 g) from the top was excised and homogenized with pestle and mortar in 80% acetone; volume was made up to 5 ml and fi ltered. the absorbance was measured at 480 nm for car, and at 645 and 663 nm for chl-a and chl-b, respectively, using a spectrophotometer (hitachi-u-2001, japan). chl-a, chl-b, their total content and their chl-a:chl-b ratio were calculated as described by yoshida et al. (1976), while car were computed with the formula of davies (1976). gas exchange characteristics were measured by irga of the third leaf (from the top) of each plant. measurements were performed from 10:00 am to 11:00 am with the following leaf chamber adjustments: leaf surface area 11.35 cm2, ambient co2 concentration 357 µmol mol–1, temperature of leaf chamber varied from 32.5 to 37 °c, leaf chamber gas fl ow rate 392.8 ml min–1, molar fl ow of air per unit leaf area 440 µmol m–2 s–1, ambient pressure 99.6 kpa, water vapor pressure in to chamber ranged from 20.5 to 23.1 mbar, par (q leaf) on leaf surface ranged from 975 to 1250 µmol m–2 s–1. statistical analysis the experimental design was completely randomized with four replications per treatment. the presence or absence of signifi cant differences between different factors at p = 0.05 was ascertained with analysis of variance (anova). the means were compared to fi nd signifi cant differences among them using least signifi cant difference. computer software costat (cohort software, 2003, monterey, california, usa) was used for all statistical analysis and ms-excel was used to graphically present the data. results plant dry mass data for changes in shoot dry weight indicated signifi cant (p < 0.01) difference in cultivars and growth stages during spring but not during autumn. however, for root dry weight, such an interaction was notable at silking and grain fi lling stages in spring only (tab. 1). shoot dry weight during spring was reduced at seedling stage in both cultivars, although cv. sadaf indicated a lower reduction (10%) than cv. agatti-2002 (39%) in gh conditions. at silking stage, cv. sadaf showed an increase (5%) and cv. agatti-2002 a decrease (36%) in shoot dry weight. at grain fi lling stage, shoot dry weight was reduced in both cultivars, but this reduction was lower (3%) in cv. sadaf than in cv. agatti-2002 (30%). in autumn, although the trend of changes in shoot dry weight was similar to that observed during spring, except at the grain fi lling stage when cv. sadaf indicated an increase while cv. agatti-2002 showed a decrease in shoot dry weight under gh conditions. root dry weight declined in both cultivars although the reduction was less (~18%) in cv. sadaf than in cv. agatti-2002 (~26%) during spring at the seedling stage. at silking and grain fi lling stages, cv. sadaf showed an increase (~8 and 5%, respectively) and cv. agatti-2002 a decrease (34 and 32%, respectively) in root dry weight. in autumn, there was a reduction of root dry weight in both cultivars but cv. agatti-2002 suffered more than cv. sadaf (~22 and 6%, respectively). at silking and grain fi lling stages, cv. sadaf showed an increase (~41 and 4%, respectively) but cv. agatti-2002 a decrease (~20 and 34%, respectively) in root dry weight. effect of greenhouse conditions on two maize cultivars acta bot. croat. 73 (2), 2014 337 photosynthetic pigments the gh conditions differentially modulated the contents of photosynthetic pigments in both maize cultivars. in the case of chl-a, no signifi cant (p > 0.05) interaction of cultivars and growth conditions under the gh effect was noted at seedling stage in the spring season, at silking stage in both seasons and grain fi lling stage during autumn. the seedling stage data showed that cv. sadaf showed no signifi cant differences in both seasons, whilst cv. agatti-2002 indicated a decline of ~7 and 21%, respectively in both of the seasons in gh conditions. at silking stage, chl-a was not changed in cv. sadaf but decreased (~18%) in cv. agatti-2002 during spring while it increased by 7 and 11% in both cv. sadaf and cv. agatti-2002 in autumn, respectively. at grain fi lling stage in spring, cv. sadaf exhibited an increase (4%) but cv. agatti-2002 a decrease (~14%) of chl-a. however, the responses of the two cultivars were similar in autumn in gh conditions (fig. 2). data for changes in chl-b indicated no signifi cant interaction (p > 0.05) of cultivars and growth conditions at any growth stages under the gh effect. data recorded at seedling stage indicated an increased chl-b by cv. sadaf (~4%) during spring and autumn seasons, but cv. agatti-2002 showed a decreased chl-b during spring (10%) but no change during autumn. at silking stage, chl-b increased in cv. sadaf during spring (~7%) but declined in autumn (~13%), whilst in cv. agatti-2002 it declined in both seasons (~16 and 47% in spring and autumn, respectively). at grain fi lling stage, chl-b was reduced less in cv. sadaf (~11%) than in cv. agatti-2002 (~21%) during spring. in autumn, chl-b increased in cv. sadaf (~14%) but did not change in cv. agatti-2002 in gh conditions (fig. 2). data for leaf total chls content showed no signifi cant (p > 0.05) interaction of seasons and cultivars under prevailing gh conditions at any growth stage except during the spring at grain fi lling stage, when there was a signifi cant (p < 0.01) interaction for cv. agatti-2002 tab. 1. effect of greenhouse conditions on dry weight of maize plants grown during spring and autumn in 2007. the comparisons have been made of the cultivars separately for seasons and growth stages. the values sharing same letter differ non-signifi cantly (p > 0.05). seasons growth stages cultivars shoot dry weight (g) root dry weight (g) control greenhouse control greenhouse spring seedling sadaf 3.82a 3.43 b 1.65a 1.36a agatti-2002 3.02 c 1.84 d 1.89a 1.40a silking sadaf 17.52 a 18.44 a 3.82 a 4.12 a agatti-2002 15.85ab 10.12c 3.27 b 2.16c grain fi lling sadaf 60.93a 58.89ab 11.67abc 12.24ab agatti-2002 49.52bc 34.64d 11.86abc 8.06c autumn seedling sadaf 4.90a 4.09a 1.80a 1.69a agatti-2002 3.18a 3.03a 1.84a 1.44a silking sadaf 13.84b 15.52ab 3.24a 4.57a agatti-2002 13.05bc 12.83bc 2.88a 2.63a grain fi lling sadaf 50.02b 51.40ab 12.90ab 13.44a agatti-2002 55.90a 39.50cd 13.78a 9.16bc hussain i., wahid a., rasheed r., akram h. m. 338 acta bot. croat. 73 (2), 2014 fig. 2. changes in photosynthetic pigments in the control and greenhouse grown plants of maize cultivars during spring and autumn seasons at three growth stages. the comparisons have been made of the cultivars separately for spring and autumn seasons, the data bars carrying same letter in a season differ non-signifi cantly (p > 0.05); fw – fresh weight. effect of greenhouse conditions on two maize cultivars acta bot. croat. 73 (2), 2014 339 cultivar. at seedling, silking and grain fi lling stages, cv. sadaf showed similar amounts of total chls in spring and autumn seasons, while cv. agatti-2002 indicated a marked reduction in total chls in autumn at seedling stage (~13%) and in spring at silking (~17%) and grain fi lling (~16%) stages under the gh effect (fig. 2). for chl-a/chl-b ratio, the interaction of cultivars and gh conditions were non-signifi cant (p > 0.05) at both seasons and all the growth stages. at seedling stage, cv. sadaf revealed a signifi cant reduction in the chl-a/chl-b ratio during both seasons, while cv. agatti-2002 exhibited an increased chl-a/chl-b ratio (~3%) in spring and declined (~21%) in autumn under gh effect. at silking stage, there was an insignifi cant reduction (~3%) in chla/chl-b in cv. sadaf during spring, but an increase (~23%) during the autumn, while agatti-2002 showed an insignifi cant reduction (~3%) in chl-a/chl-b ratio in spring and increased (~18%) during autumn under gh effect. at grain fi lling stage, cv. sadaf showed an increased (~17%) chl-a/chl-b ratio during spring, but a reduction (~16%) during autumn, while in cv. agatti-2002, this ratio was insignifi cantly increased (~8%) in spring and declined (~3%) during autumn. leaf car content was affected signifi cantly in cv. agatti-2002 at silking stage during spring due to the gh effect. the interaction of cultivars and gh conditions was not signifi cant (p > 0.05) at seedling stage during either season, it was signifi cant (p < 0.01) during both seasons at silking stage, while at the grain fi lling stage the cultivars and gh condition interactions were signifi cant during spring and non-signifi cant (p > 0.05) during autumn. at seedling stage, cv. sadaf indicated an increase (~6%) of the car during spring, but a reduction (~14%) during the autumn season, while cv. agatti-2002 indicated a reduction of ~12% during spring and of ~21% in autumn under gh effect. at silking stage there was an insignifi cant reduction (~5%) in car in cv. sadaf during spring, but an increase (~23%) during autumn, while cv. agatti-2002 showed a marked reduction (~48%) in car in spring and an insignifi cant reduction (~3%) during autumn under gh effect. at grain fi lling stage, cv. sadaf again exhibited an increase (~14%) of car during spring but an insignifi cant reduction (~4%) during the autumn season, while in agatti-2002 a marked reduction was noted during spring (~22%) and autumn (~39%) (fig. 2). gas exchange parameters leaf pn was signifi cantly affected due to the gh effect and the response of cultivars was different at different growth stages attained in both the seasons. for this attribute, the interaction of cultivars and gh conditions were signifi cant (p < 0.05) at seedling stage in both seasons. at the seedling stage in the spring season, leaf pn decreased by ~28% in cv. sadaf and showed a marked decline (~37%) in cv. agatti-2002, while in autumn season there was a nominal reduction (~ 4%) in cv. sadaf and a marked reduction (~21%) in cv. agatti-2002. at silking and grain fi lling stages, although both the cultivars showed a reduction in pn, a particularly signifi cant reduction (~60% and ~57%) was noted in cv. agatti-2002 under the gh effect in both seasons (fig. 3). for leaf transpiration rate (e), the interactions of cultivars and gh conditions were nonsignifi cant (p > 0.05) at both of seasons and growth stages (seedling and silking) except for a signifi cant (p < 0.05) interaction of cultivars and gh conditions at grain fi lling stage. gh conditions did not have effect in spring-grown plants in any of the maize cultivars at seedling stage (fig. 3). at silking stage in the spring, no specifi c reduction in e in any of the hussain i., wahid a., rasheed r., akram h. m. 340 acta bot. croat. 73 (2), 2014 fig. 3. changes in gas exchange parameters of the control and greenhouse grown plants of maize cultivars during spring and autumn at three growth stages. the comparisons have been made of the cultivars separately for spring and autumn, the data bars carrying same letter in a season differ non-signifi cantly (p > 0.05). effect of greenhouse conditions on two maize cultivars acta bot. croat. 73 (2), 2014 341 cultivars was recorded, while in autumn, a greater reduction (~20%) was evident in cv. sadaf and a lesser one (~9%) in cv. agatti-2002 in gh conditions. at the grain fi lling stage e was the same as for silking stage in the spring for both cultivars and just slightly less than in the seedling stage. only in autumn was e reduced (~25%) in cv. agatti-2002 from the previously mentioned stages (fig. 3). in leaf water use effi ciency, measured as pn/e, a signifi cant interaction of cultivars and gh conditions was evident during the autumn at seedling stage (p < 0.05) and at silking (p < 0.01) and grain fi lling (p < 0.01) stages during both seasons. a greater reduction at seedling (~34%) and silking stage (~50%) was recorded in pn/e due to gh effect in cv. agatti-2002 than in cv. sadaf in the spring. however, in the autumn, pn/e was not affected in cv. sadaf, but markedly reduced at seedling (~28%) and silking (~17%) stage in cv. agatti-2002. at grain fi lling stage, this reduction in pn/e was recorded in both maize cultivars in both the seasons, being much greater (~57%) during spring and (~43%) in autumn in cv. agatti-2002 under the gh effect (fig. 3). the interaction of cultivars and gh conditions for leaf stomatal conductance (gs) was non-signifi cant (p > 0.05) for both seasons and all growth stages except during the autumn for seedlings and both seasons for the grain fi lling stage, which exhibited a signifi cant (p < 0.05) interaction of these factors. at the seedling stage gs was reduced slightly (~7%) in cv. sadaf and greatly (23%) in cv. agatti-2002 during spring, while in autumn there was no great reduction in this attribute in cv. sadaf although there was a marked reduction (41%) in cv. agatti-2002 in the gh. at the silking stage in the spring season, the gs increased (7%) in cv. sadaf but declined (~10%) in cv. agatti-2002, while in the autumn season, both cultivars indicated declines in this attribute. at grain fi lling stage, gh conditions produced a little decline in the gs of cv. sadaf (~14%) while cv. agatti-2002 was more affected (~41%) in the spring, while in autumn the gs was not affected in cv. sadaf but markedly declined (30%) in cv. agatti-2002 in the gh conditions (fig. 3). the interaction of cultivars and gh conditions for leaf substomatal co2 concentration (ci) was non-signifi cant (p > 0.05) for both seasons and all growth stages except during the autumn at seedling stage, when a signifi cant (p < 0.05) interaction of these factors was observed. leaf ci was increased in both cultivars at all growth stages due to gh effect but cv. sadaf indicated a lower value of this parameter than cv. agatti-2002. the ci increased greatly in cv. agatti-2002 in autumn at seedling stage (23%), during the spring at silking stage (29%) and during the autumn at grain fi lling stage (23%), while in cv. sadaf it increased slightly at all these stages (fig. 3). discussion signifi cant interactions of the cultivars and treatments for different attributes appeared in one season and disappeared in another season at different phenological stages (figs. 2 and 3; tab. 1). this showed that the prevailing gh conditions produced a lot of changes in maize growth pattern as well as in the photosynthetic system, even though the effects were less in the heat tolerant (cv. sadaf) than the sensitive cultivar (cv. agatti-2002). in such conditions, plants undergo a depression in visual growth and development, but the extent of reduction depends greatly upon the type of stress, its severity and duration (ahmed et al. 2012, arbona et al. 2013, galani et al. 2013). greater dry weight results from the extent of the available hussain i., wahid a., rasheed r., akram h. m. 342 acta bot. croat. 73 (2), 2014 photosynthetic area together with an enhanced capacity of leaves to photosynthesize (karim et al. 2000, huve et al. 2006, suarez and medina 2006). the results of this experiment revealed that both of maize cultivars indicated quite a lot of changes in chl-a, chl-b, total chls, chl-a/chl-b ratio and car contents in both growing seasons and all growth stages (fig. 2). of two chlorophyll species, chl-b was more damaged than chl-a by prevailing gh high temperature condition (fig. 2), leading to an overall loss of chlorophylls (fig. 2), thereby causing more yellowing of leaves in cv. agatti-2002 than in cv. sadaf. these changes resulted in an increased chl-a/chl-b ratio (fig. 2), which was slightly higher in spring at the seedling stage. it has been shown that high temperature enhances chlorophyllase activity, which degrades the chlorophylls and reduces their contents (todorov et al. 2003, wahid et al. 2007). of the two chlorophyll species, chl-b is more prone to degradation by heat stress, especially during the spring when the temperature is suffi ciently high and leaf nitrogen contents (not reported here) might have been reduced (matile and hörtensteiner 1999). from these changes in the chlorophyll concentrations, it can be deduced that the sensitivity of chl-b to gh condition is mainly responsible for the yellowing of leaves, particularly in spring grown plant. from the prevailing temperature conditions in the canopy grown plants in spring compared to autumn, it can be seen that plants sown in spring months had to face more adverse temperatures at later growth stages (silking and grain fi lling), than the autumn grown plants, which do not experience such a high temperature and greater relative humidity during these growth stages (hussain et al. 2010). thus, it can be inferred that gh conditions are more detrimental to the photosynthetic machinery of the spring sown plants in the warmer months. carotenoids have dual roles in plants. by acting as accessory light harvesting pigments, they harvest the light and funnel it onto the photosystem. the other important role of carotenoids remains the alleviation of oxidative damage to the biological membranes via the xanthophyll cycle (havaux 1998). environmental stress to the tolerant plants is reported to increase car contents as compared to control counterparts, which suggests they have a role in the stress tolerance (wahid 2007, arbona et al. 2013). in the present research, it was noted that tolerant maize (cv. sadaf) in the gh conditions either showed increased, steady state or minimal decrease in car contents during both the seasons as compared to sensitive maize (cv. agatti-2002), which displayed decreased car content in both the seasons. however, these decreases were more remarkable in the springthan in the autumn-grown maize plants (fig. 2). thus, in concurrence with previous reports (wahid et al. 2007), these data substantiated a crucial and profound role of carotenoids in the relatively adverse condition like gh, where increased temperature and changes in gh are the main determinants of growth. plant productivity is assessed on the basis of effi ciency of a plant to fi x co2 and production of photo assimilates by the leaves (source tissue) for export to various sinks for utilization and storage (rajcan and tollenaar 1999, luo et al. 2011). maize, like a number of other crop plants, also shows great changes in co2 fi xation under suboptimal growth conditions (tollenaar 1989, sinsawat et al. 2004). in this study, the leaf gas exchange data revealed that the growing season and gh conditions had a great infl uence on these attributes of both the cultivars. leaf pn, e and gs were little affected at seedling, reduced more at silking and reduced the most at grain fi lling stage under gh conditions, while agatti-2002 showed greater sensitivity to gh conditions during all the stages (fig. 3). the pn/e increased more in spring-grown maize than in autumn-grown maize (fig. 3), whilst ci indicated a effect of greenhouse conditions on two maize cultivars acta bot. croat. 73 (2), 2014 343 greater decrease in spring than in autumn (fig. 3). the data suggested that such declines in co2 fi xation by the sensitive maize was mainly due to reduced gs and reduced activity of rubisco in absorbing co2 and reduction of assimilate production via the calvin cycle in the mesophyll cells (douthe et al. 2011). as mentioned above, both photosynthetic pigments and gas exchange parameters are the fundamental processes involved in dry matter yield. therefore, optimal operation of reactions in both these processes is important. studies highlighting the proportionate changes in these processes are scanty. in the present study, we noted that the pattern of changes in chlb was similar to the patterns of gs and ci, which indicated that prevailing seasonal conditions were equally deterrent to all these attributes. despite the fact that the photosynthetic pigment and gas exchange parameters are entirely different in nature and composition, these results show that parallel changes in both are important determinants of maize growth. in conclusion, changes in the canopy temperature and relative humidity due to gh conditions were responsible for alteration in plant dry masses and photosynthetic attributes in maize, across spring and autumn season, although the latter was less adverse. the results have great implications in the production of such crop cultivars, which may be well adapted to upcoming changes due to gh effect. acknowledgement this work 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stone, p., 2001: the effects of heat stress on cereal yield and quality. in: basra, m. a., (ed.), crop responses and adaptation to temperature stress, 243–291. food products press, binghamton, new york. suarez, n., medina, e., 2006: infl uence of salinity on na+ and k+ accumulation, and gas ex change in avicennia germinans. photosynthetica 44, 268–274. szilvia, z. t., schansker, g., kissimon, j., kovács, l., garab, g., strasser, r., 2005: biophysical studies of photosystem ii-related recovery processes after a heat pulse in barley seedlings (hordeum vulgare l.). journal of plant physiology 162, 181–194. taiz, l., zeiger, e., 2010: plant physiology, 4th ed. sinauer associates inc. publ. massachusetts, usa. todorov, d. t., karanov, e. n., smith, a. r., hall, m. a., 2003: chlorophyllase activity and chlorophyll content in wild type and eti5 mutant of arabidopsis thaliana subjected to low and high temperatures. biologia plantarum 46, 633–636. tollenaar, m., 1989: response of dry matter accumulation in maize to temperature. dry matter portioning. crop science 29, 1239–1246. wahid, a., 2007: physiological implications of metabolites biosynthesis in net assimilation and heat stress tolerance of sugar cane (saccharum offi cinarum) sprouts. journal of plant research 120, 219–228. wahid, a., gelani, s., ashraf, m., foolad, m. r., 2007: heat tolerance in plants: an overview. environmental and experimental botany 61, 199–223. yan, w., hunt, l. a., 1999: an equation for modelling the temperature response of plants using only the cardinal temperatures. annals of botany 84, 607–614. yoshida, s., forno, d. a., cock, j. h., gomez, v., 1976: laboratory manual for physiological studies of rice. irri, los banos. 655 nikolova et al.vp acta bot. croat. 72 (1), 13–22, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 comparative study of in vitro, ex vitro and in vivo grown plants of arnica montana – polyphenols and free radical scavenging activity milena nikolova1*, mariya petrova2, ely zayova2, antonina vitkova1, ljuba evstatieva1 1 institute of biodiversity and ecosystem research, acad. g. bonchev 23, 1113 sofia, bulgaria 2 institute of plant physiology and genetics, 21 g. bonchev street, 1113 sofia, bulgaria abstract – arnica montana l. is an endangered species rich in sesquiterpene lactones, phenolic acids and flavonoids with high pharmaceutical value. the polyphenolic content and free radical scavenging activity of plants that had passed all stages of cultivation: micropropagation and rooting (in vitro), adaptation in greenhouse (ex vitro) and mountain conditions (in vivo) were evaluated. four surface flavonoid aglycones [scutellarein 6-methyl ether (hispidulin), scutellarein 6,4’-dimethyl ether (pectolinarigenin), 6-oh luteolin 6-methyl ether and kempferol-6-methyl ether] were detected in the acetone exudates of the studied samples by means of thin layer chromatography. no differences in the accumulation of surface flavonoids were found among the tested leaf extracts of in vitro, ex vitro and in vivo samples. however, the extracts from the flowers were richer in surface flavonoids than extracts from the leaves. the methanol extracts of the samples from ex vitro and in vivo grown a. montana plants had significantly higher radical scavenging activity and polyphenolic content than the extracts of in vitro samples. the observed differences in the contents of these biologically active compounds were related to different growth conditions and stages of plant development. the biotechnological method of a. montana established holds promise for the future production of antioxidants. keywords: antioxidant, flavonoid, phenol, arnica montana introduction arnica montana l. (asteraceae) is a valuable perennial herb. the species contains sesquiterpene lactones (e.g. helenalin), phenolic acids (caffeic acid derivatives) and flavonoids (quercetin 3-o-glucuronic acid) with significant antiseptic, anti-inflammatory, antibacterial and antioxidant effects (woerdenbag et al. 1994, lyss et al. 1997, iauk et al. acta bot. croat. 72 (1), 2013 13 * corresponding author, email: milena_n@bio.bas.bg copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 2003, santos et al. 2006). the plant has been used mainly as anti-inflammation drug and is applied topically for skin, bruises, rheumatic and muscle pains. the species is used as ingredient in many homeopathic remedies. arnica montana is a rarely found in bulgaria and has been reported to grow only on rila mountain (assyov and petrova 2006), however so far its distribution has not been confirmed. therefore the development of a biotechnological method for in vitro propagation of the species and subsequently ex vitro and in vivo acclimatization is very important for its cultivation and sustainable use. phenolic compounds and flavonoids possess multiple biological effects including antioxidant, free radical scavenging abilities, inhibition of hydrolytic and oxidative enzymes, anti-inflammatory, anticarcinogenic, antibacterial, hypolipidemic, antimutagenic and other activities (burda and oleszek 2001, cai et al. 2004, rocha-guzman et al. 2007). they are also associated with the prevention and treatment of cardiovascular and cerebrovascular diseases (gan et al. 2010). arnica montana flavonoids are supposed to act synergistically with sesquiterpene lactones (willuhn 1991). they also serve as chemosystematic markers and are used to assess identity and purity of a. montana according to the european pharmacopoeia (hänsel and sticher et al. 2007). the total polyphenol content and antioxidant activity of a. montana extracts have been reported in several publications (haãrmãnescu et al. 2008, fraisse et al. 2011, gawlik-dziki et al. 2011, moldovan et al. 2011). the literature survey showed that there is no information about the antioxidant activity of in vitro cultured a. montana plant material. the purpose of this study was to evaluate the polyphenol content and antioxidant capacity of plants derived from in vitro culture and then adapted in greenhouse (ex vitro) and mountain conditions (in vivo). material and methods plant material the origin of arnica montana plants was botanical garden, germany (ag). six samples were analyzed: two in vitro samples: agm (multiple plants), agr (rooted plants); two ex vitro samples ag1: ag1_l (leaves), ag1_f (flower heads) of greenhouse plants from sofia region 553 m a.s.l. and two in vivo sample ag2: ag2_l (leaves), ag2_f (flower heads) of cultivated plants from the rhodope mountains, beglika. preparation of extracts acetone exudates. air-dried (but not ground) 1g plant material was briefly (2–3 min) rinsed with acetone at room temperature to dissolve the lipophilic components accumulated on the surface. the obtained acetone filtrate was then dried using a rotary-evaporator to give a crude extract which was suspended in meoh and then subjected on tlc. methanol extracts. dry, ground plant material (1 g) was extracted with 80% (3 x 30 ml) methanol by classic maceration for 24 h. after evaporation of the solvent the crude extract was subjected to subsequent analysis. biotechnological tools: the in vitro propagation protocol of a. montana plants consisted of four stages: 1) an initial stage – obtaining seedlings on basal ms medium (murashige and skoog 1962) with 40 mg l–1 gibberellic acid; 2) a propagation stage – 14 acta bot. croat. 72 (1), 2013 nikolova m., petrova m., zayova e., vitkova a., evstatieva lj. 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees shoot formation on ms medium supplemented with 1 mg l–1 6-benzylaminopurine and 0.1 mg l–1 indole-3-acetic acid; 3) rooting stage – shoot rooting on half strength ms medium containing 0.5 mg l–1 indole-3-butyric acid; 4) ex vitro acclimatization of plants was carried out on a mix of peat, perlite and coconut fiber (2:1:1 v/v/v) for 4 weeks. then the plants were adapted in the experimental field in the rhodopes mountains at an altitude of 1500 m a.s.l (in vivo plants). cultural conditions in vitro cultures were maintained in a growth room at temperature of 22 ± 2 °c under a 16 h photoperiod with light intensity of 40 mm m–2 s–1 provided by cool white fluorescent tubes. the micropropagated plants were cultivated in ex vitro conditions ata temperature of 24 ± 2 °c under a 16 h photoperiod with a light intensity of 50 mm m–2 s–1 with subsequent transfer to greenhouse. then the plants were adapted in the experimental field in the rhodopes mountains at an altitude of 1500 m a.s.l (in vivo plants). thin layer chromatographic analysis of flavonoid aglycones the acetone exudates were screened for surface flavonoids by tlc analysis. three tlc sorbents and three mobile phases were used for the analysis of the flavonoid exudates. toluene-dioxan-acetic acid (95:25:4, v/v/v) was applied for the development of the aglycones mixture on silica gel plates kiselgel 60 f254 (10 x 20 cm, 0.2 mm layer). toluene-methylethylketone-methanol (60:25:15, v/v/v) was used for dc-alufolien polyamid 11 f254 plates (10 x 20 cm, 0.15 mm layer). acetic acid–water (30:70, v/v) was used for cellulose plates dc-alufolien cellulose 5552 (10 x 20 cm, 0.1 mm layer). chromatograms were viewed under uv light before and after spraying with »natural product reagent a«, 1% solution of diphenylboric acid 2-aminoethyl ester complex in methanol. the identification of the compounds was achieved by co-chromatography with authentic markers obtained from prof. eckhard wollenweber. determination of total phenolic content. total phenolic content of the methanol extracts was determined by employing the methods given in the literature involving folin-ciocalteu reagent and gallic acid as standard (giorgi et al. 2009, ni]iforovi] et al. 2010). plant extracts were diluted to a concentration of 1 mg ml–1, and aliquots of 0.5 ml were mixed with 2.5 ml of folin–ciocalteu reagent (previously diluted 10-fold with distilled water) and 2 ml of na2co3 (6%). after 1 h at room temperature, the absorbances of the samples were measured at 765 nm on spectrophotometer versus blank sample. total phenols were determined as gallic acid equivalents (mg ga per g of extract) by the following formula: c = c × v / m where c is total content of phenolic compounds, mg g–1 plant extract, in gae; c is the concentration of gallic acid established from the calibration curve in mg ml–1; v is the volume of extract in ml; m is the weight of pure plant methanolic extract in grams. determination of total flavonoid content. total flavonoid content was determined according to miliauskasa et al. (2004), using rutin as a reference compound. one ml of plant acta bot. croat. 72 (1), 2013 15 arnica montana polyphenols and free radical scavenging activity 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees extract in methanol (10 g l–1) was mixed with 1 ml aluminium trichloride in ethanol (20 g l–1) and diluted with ethanol to 25 ml. the absorption at 415 nm was read after 40 min at room temperature. blank samples were prepared from1 ml plant extract and 1 drop acetic acid and diluted to 25 ml. the absorption of rutin solutions was measured under the same conditions. standard rutin solutions were prepared from 0.05 g rutin. all determinations were carried out in duplicate. the amount of flavonoids in plant extracts in rutin equivalents (re) was calculated by the following formula: c = absample × mcontrol × 10 / abcontrol × msample where c is flavonoid content (mg g–1 plant extract) in re; absample is the absorption of plant extract solution; abcontrol is the absorption of standard rutin solution; msample is the weight of plant extract in grams; mcontrol is the weight of rutin in the solution in grams. dpph radical scavenging activity. the effect of methanolic extracts on dpph radical was estimated according to choi et al. (2002) and stanojevi] et al. (2009). different concentrations of plant extract (10, 20, 50, 100 and 200 mg/ml), in methanol were added at an equal volume (2.5 ml) to methanol solution of dpph (0.3 mm, 1 ml). after 30 min at room temperature, the ab values were measured at 517 nm on a spectrophotometer (jenway 6320d) and converted into the percentage antioxidant activity using the following equation: dpph antiradical scavenging capacity (%) = [1–(absample–abblank)/abcontrol] × 100 methanol (1.0 ml) plus plant extract solution (2.5 ml) was used as a blank, while dpph solution plus methanol was used as a control. the ic50 values were calculated by sigmoid non-linear regression model using plots, where the abscissa represented the concentration of tested plant extracts and the ordinate the average percent of scavenging capacity (software prizm 3.00). ic50 values denote the concentration of sample required to scavenge 50% of dpph radical. statistical analysis statistical analysis was carried out using excel. all experiments were performed in triplicate. results were presented as a value ± standard deviation (sd). significant levels were defined at p < 0.05 as analyzed by t-test. results surface flavonoid aglycones four surface flavonoid aglycones were detected in the acetone exudates of the studied samples (fig. 1): scutellarein 6-methyl ether (hispidulin) (1), scutellarein 6,4’-dimethyl ether (pectolinarigenin) (2), 6-oh luteolin 6-methyl ether (3) and kempferol-6-methyl ether (4). three of detected flavonoid structures belong to the flavone class of flavonoids and one belongs to the flavonol class. thin layer chromatographic data – rf (rate of flow) and color of detected flavonoid aglycones are presented in table 1. the flavonoid aglycones (1) and (2) were the main flavonoids in all studied exudates. the flavonoid aglycones (3) and (4) 16 acta bot. croat. 72 (1), 2013 nikolova m., petrova m., zayova e., vitkova a., evstatieva lj. 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees were detected in trace amounts in the leaf exudates while in the flower exudates they were present in high quantities. differences among the leaf exudates of studied samples were not observed. total phenolic content the results of total phenolic content determination in the methanol extracts of studied samples, evaluated using folin – ciocalteu method, are presented in table 2. the content of phenols in extracts of in vitro samples agm and agr expressed as gallic acid equivalents (gae) was 23.65 to 24.78 mg g–1 of dry extract, respectively. there were no significant differences in content of total phenols (p > 0.05) among the methanol extracts of in vitro acta bot. croat. 72 (1), 2013 17 arnica montana polyphenols and free radical scavenging activity oh o o r1 ho r2 ch 3 o 2 3 45 6 7 8 9 10 1' 2' 3' 4' 5' 6' oh o ho oh o o h ch3o 2 3 4 10 5 6 7 8 9 1' 2' 3' 4' 5' 6' (1, 2, 3) ( )4 (1) r1=h; r2=oh scutellarein 6-methyl ether (hispidulin) (2) r1=h; r2=och3 scutellarein 6,4'-dimethyl ether (pectolinarigenin) (3) r1=oh; r2=oh6-oh luteolin 6-methyl ether (3) (4) kempferol-6-methyl ether fig. 1. structures of the identified flavonoid aglycones 1–4. tab. 1. thin layer chromatographic data on detected flavonoid aglycones in the studied samples of arnica montana flavonoid aglycones thin layer chromatographic data – rf (rate of flow) and color s1 s2 s3 scutellarein 6,4’-dimethyl ether (pectolinarigenin) 0.52 (brown/ brown) 0.57 (brown/ brown) 0.67 (brown/ brown) scutellarein 6-methyl ether (hispidulin) 0.33 (brown/ brown) 0.21 (brown / brownish-yellow) 0.52 (brown/ brown) kempferol-3-methyl ether 0.39 (brown / brownish-yellow) 0.15 (brownish-yellow / yellow) 0.28 (brownish-yellow / yellow) 6-oh luteolin 6-methyl ether 0.16 (brown / orange) 0.06 (brown / yellow) 0.21 (brown / light orange) s1 – sorbent = silicagel, eluent = toluene:dioxan:acetic acid (90:25:4); s2 – sorbent = polyamid, eluent = toluene:methylethylketone:methanol (60:25:15); s3 – sorbent – cellulose, eluent = 30% acetic acid 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees samples. the leaf extracts of ex vitro and in vivo (ag1 l and ag2 l) developing plants contain higher levels of phenols compared to extracts of in vitro samples, respectively 38.13 and 57.80 mg g–1 of dry extract. the extracts from flower heads of the same samples (ag1 f and ag2 f) contained phenols of respectively 36.41 and 51.25 mg g–1 of dry extract. the extracts of in vivo (ag2 l and ag2 f) samples contain higher phenols than extracts of ex vitro samples (ag1 l and ag1 f). the marked differences were statistically significant (p < 0.05). there were no significant differences in the phenolic content (p > 0.05), between the extracts of flower heads and leaves of a separate samples. total flavonoid content flavonoid contents is expressed as rutin equivalents: mg re per g of dry extract. the amount of flavonoids was the lowest in the leaf extracts from in vitro grown plants agm and agr – 1.48 and 1.49 mg g–1 (tab. 2). no differences in the content of total flavonoids among leaf extracts from ex vitro and in vivo grown plants (ag1 l and ag2 l) were observed – 4.20 and 4.54 mg g–1 of dry extract, respectively. however, a significant difference (p < 0.05) was found in the content of flavonoids between extracts of leaves and flowers of a sample. the extracts of flower heads of ex vitro and in vivo grown plants (ag1 f and ag2 f) contain flavonoids of respectively 9.21 and 9.50 mg g–1 of dry extract. dpph free-radical scavenging activity the degree of discoloration of violet colour of dpph·, as it gets reduced, indicated the radical scavenging potential of the antioxidant. results of the dpph scavenging activity of studied samples, expressed as ic50 value that represent the concentration of the sample required to scavenge 50% of dpph radicals, are given in table 2. it was found that the leaf extracts of ex vitro and in vivo samples had the strongest radical scavenging activity, respectively ic50 = 64.01 and 33.79 mg ml –1. the extracts of in 18 acta bot. croat. 72 (1), 2013 nikolova m., petrova m., zayova e., vitkova a., evstatieva lj. tab. 2. polyphenol content and free radical scavenging activity of samples of in vitro, ex vitro and in vivo grown arnica montana plants sample plant part total phenols* mg gae/g extract total flavonoids* (mg re/g extract) dpph scavenging activity ic50 (mg/ml) agm in vitro leaves 23.65±1.6a 1.48±0.8d >200k agr in vitro leaves 24.78±1.1a 1.49±0.7d >200k ag1_l ex vitro leaves 38.13±3.5b 4.20±1.4f 64.01±5,51h ag1_f ex vitro flower heads 36.41±5.9b 9.21±1.1e 85.73±8,11j ag2_l in vivo leaves 57.80±4.2c 4.54±1.6f 33.79±6,32g ag2_f in vivo flower heads 51.25±2.6c 9.5±2.7e 60.22±7,51h agm – in vitro sample multiple stage; agr – in vitro sample, plant rooting stage; ag1_l – ex vitro grown plants, leaves, ag1_f – ex vitro grown plants, flower heads; ag2_l – in vivo grown plants, leaves; ag2_f – in vivo grown plants, flower heads; gaegallic acid equivalents; re – rutin equivalents. * values represent mean ±sd, n=3. values with the same letter are not significantly different, p ³ 0.05 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees vitro samples showed low activity and their ic50 values were above 200 mg ml –1. the extracts of flower heads of ex vitro and in vivo samples showed slightly lower activity than the corresponding leaf extracts ic50 = 85.73 and 60.22 mg ml –1 respectively. discussion the commercial importance of polyphenols has led to attempts to develop alternative systems for their production. the advantage of using the tissue and organ cultures is more stable production of secondary metabolites than cultures of undifferentiated cells, such as cells in callus or suspension culture (ramachandra rao and ravishankar 2002). in the present study surface flavonoids, total phenols and flavonoids as well as the antioxidant potential of extracts from in vitro, ex vitro and in vivo grown plants of arnica montana were analyzed. it was found that there was no difference in the synthesis of surface flavonoids among in vitro, ex vitro and in vivo grown plants. differences were observed in the composition of surface flavonoids between the exudates of leaves and flower heads. the exudates of flower heads contained a high quantity of the flavonoids 6-oh luteolin 6-methyl ether and kempferol-6-methyl ether. this is in accordance with reports of other authors for differences in surface flavonoid composition in different plant organs (williams et al. 1999). the results of quantitative analysis showed that the total content of phenols and flavonoids in the extracts of in vitro cultures were lower than in the extracts of ex vitro and in vivo grown plants. the differences in the content of these secondary metabolites might be due to the different growth conditions and stages of plant development. the lower level of phenols in extracts of in vitro samples compared to extracts of field grown plants has been already reported (stanly et al. 2001, rames et al. 2009, parsaeimehr et al. 2010, sagwan et al. 2011, singh et al. 2011). ramesh et al. (2009) explained the increased synthesis of phenols in natural conditions as a defensive reaction to environmental stress. the higher content of total phenols in in vivo grown plants than in those from ex vitro adapted plants could be explained by the different altitudes at which they were grown, respectively central rhodopes – beglica 1500 m a.s.l. and greenhouse, sofia region 553 m a.s.l. the positive correlation between altitude and the content of caffeic acid derivatives in a. montana cv arbo flower heads was reported (spitaler et al. 2006, 2008). higher levels of flavonoids in the extracts of flower heads of ex vitro and in vivo grown plants were observed compared to corresponding leaf extracts. this result is in agreement with other studies reporting flavonoid concentration in different plant parts (savickiene et al. 2002, tomczyk and gudej 2003, khatiwora et al. 2010) the extracts of ex vitro and in vivo samples had a radical scavenging activity greater than that of the extracts from in vitro samples. higher activity of the extracts of these samples corresponds to a high content of phenols in them. a positive correlation between total phenolic contents and antioxidant activity has been reported by many authors (cai et al. 2004, shan et al. 2005, wong et al. 2006, wu et al. 2006, moldovan et al. 2011, maizura et al. 2011). moreover, the results presented confirm earlier observations that the antioxidant activity of extracts is correlated more strongly with the content of phenolics than that of flavonoids (miliauskasa et al. 2004, ni]iforovi] et al. 2010, nikolova 2011). to the best of our knowledge the present study is the first report of polyphenol content and free radical scavenging activity of in vitro grown plants of arnica montana. acta bot. croat. 72 (1), 2013 19 arnica montana polyphenols and free radical scavenging activity 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees in conclusion, four surface flavonoid aglycones were identified in the acetone exudates of the studied samples of a. montana. no differences were observed in the synthesis of surface flavonoids among the extracts of in vitro, ex vitro and in vivo samples. but there was a difference between different organs – the extracts of the flower heads were richer in surface flavonoids than leaf extracts. the antioxidant capacity and total flavonoid and phenolic content of the studied samples increased in the order in vitro, ex vitro and in vivo grown plants of a. montana. the extracts of flower heads were the richest in total flavonoids, but leaf extracts had the highest content of phenols and exhibited the highest free radical scavenging activity. a. montana plants obtained by in vitro technique and grown under natural conditions could be an appropriate source of antioxidants. the selection of highly productive lines as well as the optimization of cultivation environments is a prerequisite for the application of this system. acknowledgements the authors are grateful for the financial support provided by the bulgarian national science fund, ministry of education, youth and science (project dtk-02/38). also the authors thank prof. e. wollenweber for the kind gift of the flavonoid standards. references assyov, b., petrova, a., 2006: conspectus of the bulgarian vascular flora (in bulgarian). bulgarian biodiversity foundation, sofia. burda, s., oleszek, w., 2001: antioxidant and antiradical activities of flavonoids. journal of agricultural and food chemistry 49, 2774–2779. cai, y., luo, q., sun, m., corke, h., 2004: antioxidant activity and phenolic compounds of 112 chinese medicinal plants associated with anticancer. life sciences 74, 2157–2184. choi, c. w., kim, s. c., hwang, s. s., choi, b. k., ahn, h. j., lee, m. y., park, s. h., kim, s. k., 2002: antioxidant activity and free radical scavenging capacity between korean medicinal 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h., konings, a., 1994: cytotoxicity of flavonoids and sesquiterpene lactones from arnica species against the glc-4 and the colo 320 cell lines. planta medica 60, 434–437. wong, c., li, h., cheng, k., chen, f., 2006: a systematic survey of antioxidant activity of 30 chinese medicinal plants using the ferric reducing antioxidant power assay. food chemistry 97, 705–711. wu, c. q., chen, f., wang, x., kim, h. j., he, g. q., haley-zitlin, v., huang, g., 2006: antioxidant constituents in feverfew (tanacetum parthenium) extract and their chromatographic quantification. food chemistry 96, 220–227. 22 acta bot. croat. 72 (1), 2013 nikolova m., petrova m., zayova e., vitkova a., evstatieva lj. 655 nikolova et al.ps u:\acta botanica\acta-botan 1-13\655 nikolova et al.vp 14. o ujak 2013 10:31:56 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta botanica 1-2015 za web.indd acta bot. croat. 74 (1), 2015 1 acta bot. croat. 74 (1), 1–17, 2015 coden: abcra 25 issn 0365-0588 eissn 1847-8476 morphometric variation and taxonomic identifi cation of thirteen wild rose populations from tunisia zohra ben cheikh-affene1*, faouzi haouala2, fethia harzallah-skhiri3 1 department of horticultural science and landscape, higher agronomic institute, university of sousse, 4042 chott-mariem, sousse, tunisia 2 department of agronomy and plant biotechnology, national institute of agronomy of tunisia, university of carthage, 43 avenue charles nicolle, 1082 tunis mahrajène, tunisia 3 laboratory of genetic, biodiversity and valorisation of bioresources (lr11se14), high institute of biotechnology of monastir, university of monastir, rue tahar haddad, monastir 5000, tunisia abstract – thirteen populations of wild roses (rosa l.) growing in northern and central tunisia have been used for studies on the discrimination between accessions and populations. thirty-eight morphological characters related to the branches, prickles, leaves and corymbs were measured on the collected accessions to study the phenotypic diversity among and within species. principal component and hierarchical cluster analyses (pca and hca) separated rose accessions into two distinctive groups and eight subgroups. a taxonomic interpretation of the morphological variability has shown that tunisian rose populations belong to two sections (synstylae and caninae) of the genus rosa. moreover, they have been identifi ed as seven separated taxa: r. sempervirens l., r. sempervirens var. submoshata rouy., r. sempervirens var. prostrata lindl. belonging to synstylae section and r. canina l., r. agrestis savi., r. micrantha smith. and r. dumetorum thuill. belonging to caninae section. pca and hca proved that morphological characters used in taxonomic identifi cation such as styles form, leaf and leafl ets length, number of fl owers by corymb, leafl et serration, presence of glands in leafl et, peduncle, receptacle and sepal have a high value of discrimination, and have been very successful in morphological identifi cation. keywords: caninae section, morphometry, synstylae section, taxonomic differentiation, wild roses. * corresponding author, e-mail: bencheikhz@hotmail.fr copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. ben cheikh-affene z., haouala f., harzallah-skhiri f. 2 acta bot. croat. 74 (1), 2015 introduction roses are native to diverse habitats within the northern temperate hemispheres (europe, asia, the middle east, north africa and north america) and have been introduced and naturalized through the world. they have been planted around human habitats for at least two millennia for the ornamental and medicinal values of their fl owers (fl owering landscape, cut fl owers, perfume, oil and rose water) (hummer and janick 2009). the genus rosa l. is characterized by an enormous phenotypic plasticity that has challenged botanists. indeed, the taxonomic treatment of this highly diverse subgenus is complicated and it is caused by an enormous phenotypic, genotypic and ecological variability and plasticity due to some evolutionary process, such as hybridization, introgression, etc. (de cock 2008). rose species exhibit a highly diverse ploidy level from diploid to octoploid, while decaploidy was observed in r. prealucens (jian et al. 2010) and pentaploidy (2n = 5x = 35) in species from section caninae. those species evolved a mode of reproduction unique in the plant kingdom a distinctive unbalanced meiosis termed ‘canina meiosis’(klasterska and natarajan 1974, kovarik et al. 2008). the evidence for apomixis in wild rosa l. is extremely limited and confi ned to the section caninae (dickinson et al. 2003). czapik (1994) mentioned that apomictic taxa are highly polymorphic with numerous microspecies leading to a diffi cult and controversial taxonomic treatment. the classifi cation system adopted by rehder (1940), divided this genus in four subgenera, three of which are monotypic; hulthemia (dumort) focke, platyrhodon (hurst) rehder and hesperhodos cockerell. rosa, the fourth subgenus, contains about 95% of all species (about 200 species) and was subdivided into 10 sections: pimpinellifoliae (dc.) ser, gallicanae (dc.) ser, caninae (dc.) ser, carolinae crép, cinnamomeae (dc.) ser, synstylae (dc.), chinensis (dc.) ser, banksiae lindl, laevigatae michx. and bracteatae thory., each of the four latter sections have only one to three species (ritz et al. 2005). rehder’s system was widely accepted and is still used as a basis for modern treatises (bailey 1963, wisseman 2003, nybom 2009). during recent years, wild roses have acquired great importance in research, not only in genetic but in other domains. in fact, rose hips, the fruits of the rose plant are in many european countries used in food products (gao et al. 2005) and for medicinal purposes (warholm et al. 2003, rein et al. 2004, boskabady et al. 2006). indeed, rose hips are rich in phyto-nutrients such as phenols, carotenoids (lycopene), vitamins (c, a, e and p) (erenturk et al. 2005, olsson et al. 2005, barros et al. 2010) and carbohydrates (demir and ozcan 2001), as well as other bioactive compounds that have benefi cial medical effects. recently, anti-infl ammatory properties (winther et al. 1999), anti-ulcerogenic (gurbuz et al. 2003), anti-oxidant (gao et al. 2005, ozmen et al. 2005) and anti-mutagenic activities have been demonstrated (karakaya and kavas 1999). in tunisia, native rose bushes used to grow in the ravines, in brush fi elds and hedges and in the forest from the north to the tunisian dorsal (jebel chaâmbi) (pottier-alapetite 1979). but in the last ten years, climate changes and the programs of rural development and the expansion of the cities have threatened the biodiversity in tunisia and caused the risk of the extinction of some species particularly rosa accessions from northern and central humid zones. those populations were subjected to genetic erosion and the information about the species is limited. in this way it is urgent to prospect the tunisian rose populations, to identify and essentially to conserve them for subsequently valorisation. morphological diversity of tunisian wild roses acta bot. croat. 74 (1), 2015 3 in this work we aim to study the variability within the genus rosa accessions growing wild in the northern and the central part of tunisia. to accomplish this we have analysed a plant material collection from 13 populations. the main objectives of this work were: 1) evaluation of morphometric variability and 2) to place the identifi ed groups in a hierarchical classifi cation. materials and methods plant material field collections of rose populations were carried out in april and may 2007–2010. the collection sites were selected according to the flora of tunisia (pottier-alapetite 1979). from fi ve provinces, nine localities were chosen to cover the geographical range of wild roses. in the northwest regions of tunisia, nine populations were found to be naturally distributed in seven localities. in each of the localities tabarka, aïn drahem, tebaba, wechtata, djeba and kessra we found one type of population but in the teboursouk locality we found three types. in the northeast we found two populations in one locality (sidi median from zaghouan). in the center we found also two populations in the locality of haffouz (kairouan). the latitude longitude and altitude of each location were determined using the geographical positioning system (gps) using a magellan explorist (tab. 1). morphological characters studies were based on vegetative and fl oral characters. in each population fi ve accessions were sampled. thirty samples of one year old branches of 100 cm of length (we respect all different positions and directions) were collected randomly in the spring (april– may). thirty samples of leaves, infl orescences and fl owers in full bloom were taken to measure morphological characters (qualitative and quantitative characters). all voucher specimens are deposited in the high institute of agronomy of chott-meriem botanic laboratory (herbarium isach) and each one was assigned a corresponding number and code rr110-rr123. twenty one vegetative characters that are associated with the branches, prickles, leaves and leafl ets and gland density on those organs were defi ned. seventeen fl oral characters that are associated with the corymb and with all other fl ower parts, and gland density on those parts were also defi ned (fig. 1). taxonomic differentiation hierarchical classifi cation was based on baily’s, crépin’s and maire’s classifi cations (boulenger 1924, 1932, 1937, crépin 1869, bailey 1963, maire 1980, silvestre and montserrat 1998). in addition to the characters already established and to defi ne sections, we are also interested in styles (styles exerted beyond the mouth of receptacle, long or short, exerted styles free or connate into a column), in stipules (free, adnate only at the base or adnate more than one half of their length) and in the kind of infl orescence (solitary or corymbose). ben cheikh-affene z., haouala f., harzallah-skhiri f. 4 acta bot. croat. 74 (1), 2015 ta b. 1 . pr ov in ce , l oc al ity , s pe ci es , c or re sp on di ng c od e an d ec oge og ra ph ic al p ar am et er s of s ite s ha rv es t. pr ov in ce l oc al ity sp ec ie s c od e n um be r of s am pl es b io cl im at ic st ag e l at itu de l on gi tu de a lti tu de (m ) ja nd ou ba (n or th w es t) ta ba rk a r . s em pe rv ir en s ta b 10 h um id 36 °5 2’ 8 °4 3’ 9 1 a in d ra he m r . s em pe rv ir en s a in d 10 h um id 36 °4 0’ 8 °4 0’ 29 0 b ej a (n or th w es t) te ba ba r . s em pe rv ir en s te b 10 su bh um id 36 °5 7’ 8 °5 7’ 11 4 w ec ht at a r . s em pe rv ir en s w ec h 10 su bh um id 36 °5 4’ 9 °0 8’ 7 3 te bo ur so uk r . c an in a te bs 1 5 su bh um id 36 °2 9’ 9 °1 8’ 22 8 r . a gr es tis te bs 2 5 su bh um id 36 °2 9 9 °1 8’ 22 8 r . s em pe rv ir en s te bs 3 10 su bh um id 36 °2 8’ 9 °1 6’ 29 0 d je ba r . s em pe rv ir en s d jb . 10 su bh um id 36 °2 8’ 9 °0 5’ 29 8 se lia na (t ra ns iti on z on e be tw ee n th e n or th w es t a nd th e n or th ea st ) k es sr a r . c an in a k es s 5 se m iar id 35 °5 0’ 9 °2 2’ 94 2 z ag ho ua n (n or th ea st ) si di m ed ia n r . s em pe rv ir en s z ag h1 10 se m iar id 36 °2 0’ 10 °2 0’ 33 5 r . c an in a z ag h2 5 se m iar id 36 °2 0’ 10 °0 4’ 22 8 k ai ro ua n (c en te r) h af fo uz r . m ic ra nt ha h af z1 10 se m iar id 35 °3 8’ 9 °3 9’ 26 4 r . a gr es tis h af z2 5 se m iar id 35 °3 8’ 9 °3 9’ 26 4 morphological diversity of tunisian wild roses acta bot. croat. 74 (1), 2015 5 statistical analysis the data were analysed using analysis of variance (anova) and the signifi cance of the differences between means were determined at p < 0.05 using duncan’s multiple range test. to evaluate the approximation or the removal of species all the data were subjected to principal components analysis (pca) and hierarchical clusters analysis (hca) (crisci and lopez armengol 1983, cottin 1988) using the spss 16 software (statistical package for the social sciences) and excel 2007 software, allowing classifi cation of individuals into groups (molina cano 1977). results morphological diversity mean values of morphological characters studied are reported in tabs. 2, 3, 4. data show large variability among accessions for almost characters. among the examined characters diameter and length of prickles (dia. pr, l. pr) were highly correlated with the length of prickle’s diagonal (l. pr. dig) (75 and 88%, respectively) (data not reported). the length of leaf (l. lf) was highly correlated with the width (w. lf) and the area of the leaf (lf. a) and with the length of rachis (l. ra) (84, 81 and 73%, respectively). the number of fl owers per corymb (nb. fl. co) was correlated with the length of corymb (l. co) (78%), and this last fig. 1. morphological and fl oral characters of wild roses (rosa l.) evaluated in this study. ben cheikh-affene z., haouala f., harzallah-skhiri f. 6 acta bot. croat. 74 (1), 2015 character was correlated with the length of pedicel (l. ped) and of stylar column (l. sty) (86 and 75%, respectively). this last character (l. sty) was negatively correlated with the length of sepal (l. s), the number of lobes per sepal (nb. lob. s) and number of lobulated sepals (nb. s. lob) (87, 71 and 70%, respectively). the presence of glands in pedicel, receptacle and sepal (gl. ped, gl. rep, gl. s) was positively correlated with the length of corymb (l. co) (74%) and the length of stylar column (l. sty) (95%) and negatively correlated with the diameter of prickles (dia. pr) (71%), number of lobulated sepals (nb. s. lob), number of lobes per sepal (nb. lob. s) and length of sepal (l. s.) (78, 91 and 72%, respectively). principal component analysis and hierarchical cluster analysis the 38 morphological characters were used for the pca and hca. the hca based on the euclidean distances between groups indicated two groups of accessions: a and b, with six and seven accessions, respectively, identifi ed by their fl oral and vegetative characters with a dissimilarity > 20 (figs. 2, 3). when the dissimilarity was higher than 16, the a group was divided into two sub groups, a1 (hafz1, hafz2 and tebs2) and a2 (zagh2, tebs1 and kess). with a dissimilarity > 5, subgroup a1 was further divided into two subgroups a11 (tebs2, hafz2) and a12 (hafz1) and the second subgroup a2 was divided in two subgroups a21 (tebs1, kess) and a22 formed by one accession (zagh2). the b group with dissimilarity > 8 was divided into 2 subgroups: b1, b2. when the dissimilarity was higher than two, the subgroup b1, was divided in three subgroups b11 (tebs3, tab. 2. mean values of vegetative characters related to branches and prickles of each rose accessions growing wild in tunisia. for the abbreviations of rose accessions (codes) see tab. 1; dia. br – diameter of branches, dt. pr – density of prickles on a branch length of 10 cm, dia. pr – diameter of prickle’s base, l. pr – length of prickle, l. pr. dig – length of prickle’s diagonal. values with the same letters within the same column are not different at p > 0.05. accessions dia. br (cm) dt. pr dia. pr (mm) l. pr (mm) l. pr. dig (mm) l. pr/ dia. pr hafz2 0.6±0.1cd 6.2±6.8e 6.8±1.5d 4.8±0.6a 6.1±0.9ab 0.7±0.1a tebs2 0.5±0.2b 15.6±4.4cd 7.5±1.4e 6.0±0.7bcd 7.9±0.9e 0.7±0ai0 hafz1 0.7±0.1cd 25.1±6.5e 8.6±1.5g 6.8±0.9d 8.7±1.1f 0.8±0.1ab kess 0.6±0.1c 11.9±5.3b 6.7±1.0d 6.2±0.9cd 7.0±1.0cd 0.9±0.1bc tebs1 0.6±0.2c 13.1±3.7c 8.2±1.3ef 11.0±3.2f 10.8±2.2g 1.5±0.3g zagh2 0.7±0.2d 36.5±13.9f 7.9±1.7ef 9.2±2.7e 10.3±2.2g 1.2±0.3ef tebs3 0.4±0ai0 23.3±4.0e 4.9±0.7ab 6.3±1.8cd 6.5±1.1bcd 1.3±0.3f djb 0.5±0.1a 17.3±3.4d 4.7±0.7ab 4.9±1.3a 5.9±0.8ab 1.0±0.3cde teb 0.4±0.1a 16.5±5.3d 5.2±0.8bc 5.0±1.0a 6.2±1.0ab 0.9±0.1cd tab 0.4±0.1a 18.3±3.7d 5.7±0.7c 6.2±1.4cd 7.2±0.6d 1.0±0.2de aind 0.4±0.1a 24.2±5.6e 5.2±0.8bc 5.4±1.6abc 6.5±1.0bc 1.0±0.3cde wech 0.6±0.1bc 16.8±5.2d 5.6±0.7c 6.5±1.9d 7.0±0.9cd 1.1±0.3ef zagh1 0.4±0.1a 21.7±6.9e 4.5±0.6a 5.1±1.0ab 5.7±0.8a 1.1±0.2ef morphological diversity of tunisian wild roses acta bot. croat. 74 (1), 2015 7 ta b. 3 . m ea n va lu es o f v eg et at iv e ch ar ac te rs re la te d to le af , l ea fl e t, gl an d an d br is tle o f e ac h ro se a cc es si on s gr ow in g w ild in t un is ia . f or th e ab br ev ia tio ns of r os e ac ce ss io ns ( co de s) s ee t ab . 1 ; n b. l ft – n um be r of le afl e ts , l . l f – le ng th o f le af , w . l f – w id th o f le af , l . r a – le ng th o f ra ch is , l . l ft – le ng th o f l ea fl e t, w . l ft – w id th o f l ea fl e t, l . p et – le ng th o f p et io le , l . s t – le ng th o f s tip ul e, l f. a – le af a re a, n b. p r. ra – n um be r o f p ri ck le s on th e ra ch is , l ft . s er – le afl e t s er ra tio n (n um be r of te et h in 1 c m o f le afl e t m ar gi n) , g l. ul ft – g la nd p re se nt ( gl an du la r; 5 ) or a bs en t ( eg la nd ul ar ; 0 ) on up pe r s id e le afl e t, b s. u l ft – w ith (p ub es ce nt ; 5 ) o r w ith ou t ( gl ab ro us ; 0 ) b ri st le o n up pe r s id e le afl e t. v al ue s w ith th e sa m e le tte rs w ith in th e sa m e co lu m n ar e no t d iff er en t a t p > 0 .0 5. a cc es si on s n b. l ft l . l f (c m ) w . l f (c m ) l . r a (c m ) l . l ft (c m ) w . l ft (c m ) l . l ft / w . l ft l . p et (c m ) l . s t (c m ) l f. a (c m 2 ) n b. pr . r a l ft . se r g l. ul ft b s. ul ft h af z2 6. 8± 0. 6e 8. 4± 0. 9c de 6. 7± 1. 0d 4. 6± 0. 5c de 3. 8± 0. 6d e 2. 2± 0. 4c d 1. 7± 0. 0d 1. 1± 0. 3a 1. 1± 0. 1b c 25 .0 ± 7. 5c d 4. 9± 2. 0d e 10 .2 ± 2. 2d 0a 0a te bs 2 6. 0± 1. 0c d 8. 5± 1. 1c de 6. 0± 0. 8c 4. 9± 0. 6e 3. 6± 0. 5d 2. 5± 0. 4e f 1. 4± 0. 1c 1. 7± 0. 4c d 0. 9± 0. 1b 24 .9 ± 7. 1c d 6. 5± 2. 3f 11 .5 ± 2. 1e 0a 0a h af z1 7. 0± 0e 8. 0± 1. 1b cd 5. 9± 0. 8c 5. 0± 0. 8e 2. 1± 0. 4a 1. 9± 0. 2b 1. 0± 0. 2a 1. 3± 0. 2b 0. 8± 0. 1a 24 .7 ± 6. 0c d 5. 3± 1. 5e 9. 2± 1. 2c 5b 5b k es s 5. 2± 0. 6a 7. 4± 0. 7a b 5. 4± 0. 6c 4. 3± 0. 5a bc 3. 0± 0. 4c 2. 3± 0. 2d e 1. 3± 0. 1b 2. 2± 0. 3f 1. 7± 0. 32 f 18 .9 ± 4. 7b 1. 7± 1. 5a 6. 9± 1. 7b 0a 5b te bs 1 5. 8± 1. 0c d 7. 8± 0. 8b c 3. 1± 0. 6a 4. 7± 0. 5d e 5. 6± 0. 5j 2. 0± 0. 6b c 2. 8± 0. 4g 1. 9± 0. 5e 1. 0± 0. 1b c 18 .0 ± 2. 9b 3. 6± 1. 3b c 7. 4± 1. 6b 0a 5b z ag h2 6. 7± 0. 6e 6. 9± 1. 3a 4. 3± 1. 1b 4. 4± 0. 8b cd 2. 5± 0. 5b 1. 4± 0. 3a 1. 7± 0. 2d 1. 6± 0. 3c 1. 0± 0. 2b c 12 .9 ± 5. 5a 6. 5± 2. 7f 9. 3± 3. 2c 0a 0a te bs 3 5. 6± 0. 9b c 8. 5± 1. 6c de 7. 1± 1. 5d ef 4. 4± 0. 9b cd 4. 1± 0. 7e f 2. 2± 0. 2b cd 1. 9± 0. 3e 1. 7± 0. 3c 1. 1± 0. 2c 24 .7 ± 8. 8c d 3. 8± 1. 5b c 7. 4± 1. 2b 0a 0a d jb 5. 3± 0. 7a b 8. 6± 1. 2d e 7. 5± 1. 2e f 4. 4± 0. 6b cd 4. 2± 0. 6f 2. 6± 0. 3f 1. 6± 0. 1d 1. 9± 0. 4d e 1. 1± 0. 1c 30 .7 ± 8. 0e 3. 7± 1. 2b c 7. 7± 0. 8b 0a 0a te b 5. 2± 0. 7a b 8. 5± 0. 9c de 6. 9± 0. 8d e 4. 0± 0. 5a 4. 6± 1. 3g h 2. 1± 0. 3b cd 2. 1± 0. 3f 1. 5± 0. 3c 1. 0± 0. 1b 21 .6 ± 8. 6b c 3. 8± 1. 4b c 5. 8± 1. 0a 0a 0a ta b 5. 2± 0. 6a b 9. 4± 0. 4f 8. 9± 1. 5g 4. 0± 0. 6a b 5. 2± 0. 8i 2. 5± 0. 4e f 2. 1± 0. 2f 1. 6± 0. 2c 1. 1± 0. 2c 28 .8 ± 10 .5 de 2. 9± 1. 2b 5. 8± 1. 1a 0a 0a a in d 5. 3± 0. 7a b 8. 8± 0. 9e f 7. 8± 0. 7f 4. 3± 0. 5a bc 4. 3± 0. 5f g 2. 5± 0. 3e f 1. 7± 0. 2d 1. 9± 0. 2d e 1. 4± 0. 2d 25 .4 ± 5. 4c d 3. 7± 1. 2b c 5. 8± 0. 8a 0a 0a w ec h 5. 2± 0. 6a b 10 .5 ± 1. 5g 9. 7± 1. 5h 4. 9± 0. 7e 5. 3± 0. 8i j 2. 5± 0. 4e f 2. 1± 0. 2f 1. 9± 0. 2e 1. 5± 0. 2d e 30 .3 ± 9. 8e 4. 4± 1. 5c d 5. 5± 1. 1a 0a 0a z ag h1 6. 1± 1. 0d 10 .4 ± 1. 8g 9. 3± 1. 6g h 5. 4± 0. 8f 5. 0± 1. 0h i 2. 5± 0. 6e f 2. 0± 0. 3e f 1. 9± 0. 3d e 1. 6± 0. 2e f 42 .5 ± 17 .1 f 4. 8± 1. 4d e 7. 3± 1. 2b 0a 0a ben cheikh-affene z., haouala f., harzallah-skhiri f. 8 acta bot. croat. 74 (1), 2015 ta b. 4 . m ea n va lu es o f fl o ra l c ha ra ct er s of e ac h ro se a cc es si on s gr ow in g w ild in t un is ia . f or th e ab br ev ia tio ns o f ro se a cc es si on s (c od es ) se e ta b. 1 ; n b. fl co – n um be r o f fl o w er s pe r c or ym b, l . c o – le ng th o f c or ym b, l . p ed – le ng th o f p ed ic el , n b. l ft . b lt – nu m be r o f l ea fl e t i n fl o w er in g br an ch le ts , l . r ep – le ng th o f re ce pt ac le , w . r ep – w id th o f re ce pt ac le ( m ed iu m ), l . s ty – le ng th o f st yl ar c ol um n, w . s ty – w id th o f st yl ar c ol um n, d ia . r ep . o – di am et er o f r ec ep ta cl e or ifi ce , n b. s . l ob – n um be r o f s ep al lo bu la te d, n b. lo b. s – n um be r o f l ob es p er s ep al , l . s – le ng th o f s ep al , w . s – w id th of s ep al , l . p – le ng th o f p et al , w . p – w id th o f p et al , g l. pe d – gl an du la r ( 5) o r e gl an du la r ( 0) p ed ic el , g l. re p – gl an du la r ( 5) o r e gl an du la r ( 0) re ce pt ac le , g l. s – gl an du la r ( 5) o r e gl an du la r ( 0) s ep al s. v al ue s w ith th e sa m e le tte rs w ith in th e sa m e co lu m n ar e no t d iff er en t a t p > 0 .0 5. a cc es si on n b. fl co l . c o (c m ) l . p ed (c m ) n b. l ft . b lt l . r ep (c m ) w . r ep (c m ) l . s ty (m m ) w . s ty (m m ) d ia . r ep . o (m m ) n b. s. lo b n b. lo b. s l . s (c m ) w . s (c m ) l . p (c m ) w . p (c m ) g l. pe d g l. re p g l. s h af z2 2. 8± 1. 1a 3. 7± 0. 8a 1. 5± 0. 7a 5. 1± 0. 5a 0. 9± 0. 1e 0. 5± 0. 0d e 2. 6± 0. 2a 1. 9± 0. 0a 4. 6± 0. 7c 2. 5± 0d 7. 1± 0. 9f 2. 6± 0. 6g 0. 5± 0. 4c 2. 1± 0. 2e f 2. 4± 0. 2e 0a 0a 0a te bs 2 2. 8± 1. 1a 3. 7± 0. 7a 1. 5± 0. 7a 5. 1± 0. 4a 0. 9± 0. 1e 0. 5± 0d e 2. 3± 0. 2a 2. 7± 0a 5. 1± 0. 7e 2. 5± 0d 7. 0± 0. 9f 2. 6± 0. 6g 0. 6± 0. 4c 2. 1± 0. 2e f 2. 4± 0. 1e 0a 0a 0a h af z1 2. 9± 1. 1a 3. 6± 0. 9a 1. 2± 0. 5a 7. 0± 0c 1. 1± 0. 1g 0. 4± 0c 1. 9± 0. 2a 2. 6± 0a 4. 2± 1. 2b d 2. 5± 0d 5. 0± 0. 6d 1. 9± 0. 3e 0. 6± 0. 3d 2. 0± 0. 2d e 2, 0± 0. 2d 0a 0a 0a k es 2. 9± 1. 5a 4. 0± 1. 2a 1. 3± 0. 8a 6. 6± 0. 7b 1. 0± 0f 0. 5± 0d e 2. 3± 0. 3b 2. 6± 0. 3e 5. 1± 0. 4e 2. 4± 0d 6. 1± 1. 2e 1. 7± 0. 1d 0. 5± 0a b 1. 7± 0. 2b 2. 0± 0. 2c d 0a 0a 0a te bs 1 2. 9± 1. 4a 4. 0± 1. 2a 1. 3± 0. 8a 6. 6± 0. 7b 1. 0± 0f 0. 5± 0d e 2. 3± 0. 2b 2. 6± 0. 2e 5. 1± 0. 4e 2. 4± 0. 1d 6. 1± 1. 1e 1. 7± 0. 1d 0. 5± 0a b 1. 7± 0. 1b 2. 0± 0. 2c d 0a 0a 0a z ag h2 3. 0± 1. 2a 4. 8± 1. 0a 1. 4± 0. 6a 5. 0± 0a 1. 1± 0h 0. 5± 0e 2. 6± 0. 4c 4. 0± 0. 3f 5. 2± 0. 3e 2. 5± 0d 5. 2± 2. 3d 2. 1± 0. 2f 0. 4± 0a 1. 9± 0. 2c d 1. 9± 0. 2b c 0a 0a 0a te bs 3 4. 7± 1. 7b c 8. 3± 2. 3c 2. 7± 1. 4b 5. 0± 0a 0. 8± 0d 0. 4± 0a 5. 6± 0. 5d 1. 8± 0. 2c 3. 9± 0. 3a 0± 0a 0± 0a 1. 3± 0. 2c 0. 5± 0a b 2. 0± 0. 1c d 2. 3± 0. 1d e 5b 5b 5b d jb 4. 1± 1. 7a b 8. 0± 1. 7c 3. 2± 1. 2c 5. 0± 0a 0. 7± 0b c 0. 4± 0a 5. 6± 0. 7d e 1. 6± 0. 2b 3. 7± 0. 4a 0. 2± 0. 4b 0. 2± 0. 4a 1. 1± 0. 1b 0. 5± 0a b 1. 7± 0. 2b 1. 8± 0. 2b 5b 5b 5b te b 8. 4± 5. 5d 12 .2 ± 3. 1e 4. 6± 1. 3d 5. 0± 0a 0. 7± 0. 1a b 0. 4± 0b c 6. 1± 0. 7f g 1. 6± 0. 2b 4. 0± 0. 5a b 1. 1± 1. 0c 0. 8± 0. 8b 0. 9± 0. 1a 0. 4± 0. 1a 1. 9± 0. 2c d 2. 0± 0. 2b cd 5b 5b 5b ta b 6. 0± 3. 2c 9. 5± 2. 8d 3. 7± 1. 2c 5. 0± 0a 0. 7± 0c 0. 4± 0b c 6. 0± 0. 8f 1. 6± 0. 1b 3. 8± 0. 3a 0± 0a 0± 0a 1. 3± 0. 1c 0. 6± 0b 2. 1± 0. 3f 2. 3± 0. 2e 5b 5b 5b a in d 8. 1± 4. 3d 12 .3 ± 2. 9e 3. 4± 0. 7c 5. 0± 0a 1. 0± 0f 0. 5± 0d 6. 3± 0. 6g 2. 1± 0. 2d 4. 3± 0. 3c 2. 5± 0. 0d 2. 2± 0. 5c 1. 5± 0. 1c 0. 5± 0a b 2. 4± 0. 2g 2. 7± 0. 2f 5b 5b 5b w ec h 7. 9± 3. 5d 10 .5 ± 2. 5d 3. 5± 0. 8c 5. 0± 0a 0. 7± 0a 0. 5± 0a c 5. 9± 0. 7e f 1. 7± 0. 3b c 4. 2± 0. 3b 0. 4± 0. 8b 0. 3± 0. 6a b 0. 9± 0. 1a 0. 5± 0a b 1. 6± 0. 21 a 1. 6± 0. 1a 5b 5b 5b z ag h1 3. 9± 2. 3a b 6. 4± 2. 3b 2. 2± 1. 0b 5. 0± 0a 0. 7± 0a 0. 4± 0b 5. 6± 0. 6d e 1. 6± 0. 2b 4. 3± 0. 4b d 0± 0a 0± 0a 1. 1± 0. 1b 0. 5± 0a 1. 8± 0. 1b c 1. 9± 0. 1b c 5b 5b 5b morphological diversity of tunisian wild roses acta bot. croat. 74 (1), 2015 9 fig. 2. repartition of the vegetative and fl oral characters on the two principal axes of the principal components analysis. identifi cation of the groups and subgroups corresponding to the 13 tunisian rose accessions studied. for the abbreviations of rose accessions (■) and the characters (�) see tabs. 1, 2, 3, 4. fig. 3. dendogram of the 13 tunisian rose accessions studied, obtained by cluster analysis based on the euclidean distances relating to the fl oral and vegetative characters. for the abbreviations of rose accessions see tab. 1. ben cheikh-affene z., haouala f., harzallah-skhiri f. 10 acta bot. croat. 74 (1), 2015 djb), b12 (tab, teb), and b13 (aind). the subgroup b2 was divided in 2 subgroups b21 (wech) and b22 (zagh1). the pca horizontal axis explained 34.42% of the total variance and the vertical axis 11.58%. the groups a and b clearly stand out, forming separate groups in pca (fig. 2) and a deep dichotomy in hca (fig. 3). the horizontal axis (pc1) was positively correlated with groups a1 and a2 and negatively correlated with group b. the variables most highly correlated were diameter of prickles (dia. pr), the presence of glands and of bristles on the upper side of leafl et (gl. ulft, bs. ulft), length of receptacle (l. rep), number of lobulated sepals and of lobes per sepal (nb. s. lob, nb. lob. s), the length and the width of sepals (l. s, w. s) (on-line supplement tab. 1). this axis 1 was moderately correlated with dia. br, l. pr. dig, lft. ser, nb. lft. blt, w. rep, w. sty. the width of leaf (w. lf), the length of leafl et (l. lft), the lengths of corymb, peduncle and stylar column (l. co, l. ped, l. sty) and the presence of glands in pedicel, receptacle and sepal (gl. ped, gl. rep, gl. s) were highly negatively correlated with pc1. the vertical axis (pc2) was positively correlated with subgroup a2 and negatively with a1. it was positively correlated with the length of prickle and the ratio of length and diameter of prickle (l. pr, l. pr/dia. pr) (on-line supplement tab. 1, fig. 2) and negatively with the width of sepal (w. s) and the presence of glands in the upper side of leafl et (gl. ulft). all accessions from group a were correlated with those characters: dia. br, dia. pr, l. pr. dig, w. rep, l. rep, w. s, l. s, w. sty, gl. ulft, bs. ulft, lft. ser, nb. lob. s, nb. s. lob, nb. lft. blt, (figs. 2, 3) and were characterized by large branches (dia. br = 0.5–0.7 cm), dotted with a large prickle having a long diagonal (dia. pr = 6.7–8.6 mm; l. pr. dig = 6.1– 10.8 mm), leaves with a high number (nb. lft = 5.2–7.0) of short leafl ets (l. lft = 2.1–5.6 cm) with high serration (6.9–11.5). leaves on fl owering branchlets were formed by a high number of leafl ets (nb. lft. blt = 5 to 7), short corymbs (l. co = 3.6–4.8 cm) with few fl owers (nb. fl. co = 2.8–3.0). a short pedicel (l. ped = 1.2–1.5 cm) bearing a fl ower also having a short column stylar (l. sty = 1.8–2.6 mm), but a long receptacle (l. rep = 0.9–1.1 cm), long sepal (1.7–2.6 cm) with many appendix (nb. slob = 2.4–2.5; nb. lobs = 5.0– 7.1). receptacle, pedicle and sepals are all eglandular. the subgroupa1, was represented by hafz1, hafz2 and tebs2, all characterized by the highest values for the characters related to the serration of leafl et and sepals and the high values for length and wide of sepals (lft. ser, nb. lob. s, nb. s. lob, l. s, w. s). this subgroup was divided into two subgroups: subgroup a11: hafz2 and tebs2. those accessions have eglandular and glabrous leafl ets with short petiole, rachis with numerous prickles, short corymb (l. co = 3.7 cm) with the lowest number of fl owers (nb. fl. co = 2.8), the fl ower has a long petal, the longest sepal longer than petal (l. s = 2.6 cm, l. p = 2.1 cm). hafz2is characterized by highly serrated sepal (nb. lob. s = 7.1), the narrowest and the shortest scattered and shorter prickles (dt. pr = 6.2, l. pr = 4.8mm), the lowest number of fl owers by corymbs (nb. fl. co = 2.8), while tebs2 is characterized by rachis with the highest number of prickles (nb. pr. ra = 6.5), and highly serrated leafl et (lft. ser = 11.5). subgroup a12: hafz1 include accessions with a robust bush exhaling an apple scent. it differs in having its highest number of leafl ets in bloomy branchlets (nb. lft. blt = 7.0), in the largest thorny branches (dia. br = 0.7 cm, dt. pr = 25.1), with the shortest leafl et (l. lft morphological diversity of tunisian wild roses acta bot. croat. 74 (1), 2015 11 = 2.1 cm), glandular and pubescent (gl. ulft = 5, bs. ulft = 5), fl owers in the shortest corymb (l. co = 3.6 cm), dotted with the shortest pedicel (l. ped = 1.2 cm), the longest receptacle (l. rep = 1.1 cm), a high number of lobulated sepals (nb. s. lob = 2.5) and of lobes per sepal (nb. lob. s = 5.0). the subgroup a2 reported in the positive section for pc1 and pc2 (fig. 2), was correlated with diameter of branches and of prickle’s base (dia. br, dia. pr), lengths of prickle and of their diagonals (l. pr, l. pr. dig), number of leafl ets in fl owering branchlets (nb. lft. blt), length and width of receptacle (l. rep, w. rep), width of styles (w. sty), and number of lobulated sepals (nb. slob). this subgroup is divided into two subgroups: subgroup a21: kess and tebs1. those accessions were mainly characterized by an eglandular (gl. ulft = 0) and pubescent leafl et (bs. ulft = 5) and a long receptacle (l. rep = 1.0 cm). those accessions were characterized by bristly leafl ets with low serration (lft. ser = 6.9 and 7.4, respectively) with a rachis with few prickles. kess accession was characterized by the longest petiole (l. pet = 2.2 cm) and rachis with the lowest number of prickles (nb. pr. ra = 1.7). tebs1 accession is characterized by the longest prickle and the longest prickle diagonal (l. pr = 11.0 mm; l. pr. dig = 10.8 mm), the narrowest leaf (w. lf = 3.1 cm), the longest leafl et (l. lft = 5.6 cm), and the highest ratios for leafl et and prickle (l. lft/w. lft = 2.8; l. pr/dia. pr = 1.5). subgroup a22: zagh2 is an upright shrub, characterized by the largest thorny branches (dia. br = 0.7 cm, dt. pr = 36.5), rachis with numerous prickles (nb. pr. ra = 6.5), leafl ets eglandular (gl. ulft = 0), glabrous (bs. ulft = 0), and doubly serrated (lft. ser = 9.3), fl ower with the longest (l. rep = 1.2 cm) and the broadest (w. rep = 0.5 cm) receptacle, the largest receptacle orifi ce (dia. rep. o = 5.2 mm) and the widest style (w. sty = 4.0 mm). the group b was represented by zagh1, wech, aind, tab, teb, djb and tebs3, which were correlated with the length and diameter of prickle ratio (l. pr/dia. pr), the length of stipule (l. st), the number of fl owers per corymb (nb. flco), the length and the width of leaf (w. lf, l. lf), the length of leafl et, pedicel and stylar column (l. lft, l. ped, l. sty) (figs. 2, 3). all accessions from group b were characterized mainly by a long stylar column (l. sty. = 5.6 to 6.3 mm. they were also characterized by slender branches (dia. br = 0.4– 0.6 cm), long (8.5–10.5 cm) and width (9.7–6.9 cm) leaves, mainly with lanceolate (l. lft = 4.1–5.3 cm and w. lft = 2.1–2.6 cm), eglandular and glabrous leafl ets (gl. ulft = 0, br. ulft = 0), the lowest number of leafl ets by leaves in fl owering branchlets (nb. lft. blt = 5), long corymbs (l. co = 6.4–12.3 cm) with many small fl owers (nb. fl. co = 3.9–8.4), long and glandular pedicel (l. ped = 2.2–4.6 cm). receptacle (l. rep = 0.7–1.0 cm) and sepals (l. s = 0.9–1.5 cm) were glandular too but short. group b was divided into 2 subgroups: b1, b2. the subgroup b1was divided in three subgroups: b11, b12 and b13. subgroup b11 is represented by tebs3 and djb accessions which were characterized mainly by a medium number of fl owers (nb. fl. co = 4.1–4.7), and short leafl et (l. lft = 4.1–4.2 cm) with high leafl et serration (lft. ser = 7.4–7.7). djb accession presents a medium density for prickles, the broadest leafl et (w. lft = 2.6 cm), a low number of lobultated sepals and of lobes per sepal (nb. s. lob = nb. lob. s = 0.2), fl ower with the shortest receptacle (l. rep = 0.7 cm), having the lowest diameter for the receptacle orifi ce (dia. rep. o = 3.7 mm). tebs3 accession has a fl ower with a sepal with entire edges (nb. lob. s = 0, nb. s. lob = 0) and the narrowest branches (dia. br = 0.4 cm) but with high density of prickles (dt. pr = 23.3). ben cheikh-affene z., haouala f., harzallah-skhiri f. 12 acta bot. croat. 74 (1), 2015 subgroup b12 is represented by tab and teb accessions, characterized by prickles with a medium density (16.5–18.3) and a prickle base of medium diameter (dia. pr = 5.2–5.7 mm), the shortest rachis (l. ra = 4.0 cm) and short petiole (l.pet = 1.5–1.6 cm). its fl owers having the shortest and the narrowest receptacle (l. rep = 0.7 cm, w. rep = 0.4 cm). teb is characterized by the longest pedicel (l. ped = 4.6 cm), the highest number of fl owers by corymb (nb. flco = 8.4), the shortest (l. s = 0.9 cm) and narrowest (w. s = 0.4 cm), low number of lobulated sepals (nb. s. lob = 1.1) and low lobes per sepal (nb. lob. s = 0.8). in contrast, tab has a fl ower with long pedicel (l. ped = 3.7 cm) and sepals with entire edges (nb. s. lob = 0, nb. lob. s = 0). subgroup b13 is represented by aind accession, and it was characterized by thorny branches with a high number of prickles (dt. pr = 24.2), the longest corymbs (l. co = 12.3 cm), the broadest receptacle (w. rep = 0.5 cm), the longest stylar column (l. sty = 6.3 mm), the highest number of lobulated sepals (nb. s. lob = 2.5), the longest and widest petals (l. p = 2.4 cm, w. p = 2.7 cm). the subgroup b2 was divided in two subgroups each of them with one accession (b21; wech and b22; zagh1) characterized by long (l. lf = 10.5; 10.4 cm, respectively) and broad leaves (w. lf = 9.3; 9.7 cm, respectively), ratio of length and diagonal of prickles tends towards 1 (l. pr/dia. pr = 1.1, which means the length and the diagonal of prickles are equal) and the shortest receptacle (l. rep = 0.7 cm). wech accession has the longest (l. lf = 10.5 cm) and the broadest (w. lf = 9.7 cm) leaf, the broadest receptacle (w. rep = 0.5 cm), the lowest number of leafl ets (nb. lft = 5.2), a low leafl et serration (lft. ser = 5.5), the shortest sepal (l. s = 0.9 cm) with low number of lobulated sepals (nb. s. lob = 0.4), and lobes per sepal (nb. lobs = 0.3), the shortest and narrowest petal (l. p = 1.6 cm, w. p = 1.6 cm). zagh1 accession was characterized by the lowest diameter (dia. pr = 4.5 mm), and length of diagonal prickles (l. pr. dig = 5.7 mm). the highest length of rachis (l. ra = 5.4 cm) and the highest leaf area (lf. a = 42.5 cm2). a medium length for pedicel (l. ped = 2.2 cm) and corymb (l. co = 6.4 cm), with few fl owers (nb. fl. co = 3.9), sepal with entire edges (nb. s. lob = nb. lob. s = 0). discussion accessions from group a were characterized by a large branches dotted with long prickles, leaves, in general, with seven short leafl ets, the margins of which were highly serrated, leaves in fl owering branchlets with fi ve to seven leafl ets, short corymb with few fl owers, pedicel short too, bearing fl owers having short styles but long receptacles, sepals also long with many appendices. the receptacle, the pedicel and the sepals of those accessions were eglandular. all this characteristics described species of section caninae, according to the taxonomy of roses (baker 1871, boulenger 1937, bailey 1963, maire 1980). in this group, hafz2 and tebs2 accessions are characterized by big fl owers, leafl et presumed with few glands in the under surface but eglandular in the upper surface, a glabrous pedicel. according to baker (1871) and maire (1980) those accessions taxonomically should belong to rosa agrestis savi. hafz2 and tebs2, the slight variability within the two taxa can be related to the effect of the environment, indeed accession of tebs2 was collected from a river bank but hafz2 was collected from an arid region. the hafz1 accession is a robust bush that gives off a smell like apples. leaves with seven short leafl ets, glandular and pubescent in both sides, fl owers clustered in short cormorphological diversity of tunisian wild roses acta bot. croat. 74 (1), 2015 13 ymb with short pedicel. flowers with the broadest sepal are dotted with many appendices. this accession presents the same characteristics of r. villosa l. but differs by a glabrous pedicel and receptacle (bailey 1963). maire (1980) describes r. micrantha smith. with glandular leafl ets on both sides but did not give any information about the coloration and the scent of glands of leafl ets. nevertheless, this accession may be a hybrid, specifi c to the tunisian region, of r. canina l. and r. villosa. accessions tebs and kess were characterized by an eglandular leafl ets on both sides with glabrous pedicel, receptacle and sepal. those characteristics correspond to r. canina taxa (maire 1980), in addition those accessions present a pubescent leafl et on both sides and bristles are oppressed in the upper side of leafl ets which characterize r. canina ssp. dumetorum thuill. (syn r. dumetorum thuill.), (syn r. corymbifera borkh.) (le floc’h et al. 2010). the slight variability within the two species can be related to the effect of the environment, indeed accession of tebs1 was collected from a river bank. zagh2 accession is an upright shrub, characterized by the largest thorny branches, glabrous leafl ets, and doubly serrated edge. zagh2 accession should belong to rosa canina ssp. vulgaris gams (syn r. caninasensu strict) because of its glabrous leafl ets (maire 1980). accessions from group b are characterized by the longest stylar column, this character was a systematic determinant character only for synstylae section, indeed the styles connate into a slender exerted column (crépin 1869, boulenger 1932, bailey 1963, maire 1980, silvestre and montserrat 1998). they were also characterized by slender branches, leaves, generally, with fi ve lanceolate and glabrous leafl ets, leaves of fl owering branchlets with fi ve leafl ets also. they have a long corymb with many small fl owers. the pedicel, receptacle and sepals were glandular. those characters characterized rosa sempervirens l. (silvestre and montserrat 1998). in this group fi ve subgroups were defi ned with hca and we defi ned two varieties. tebs3 and djb accessions were characterized mainly by a medium number of fl owers and short leafl et with high leafl et serration. in addition, the stylar column was densely pubescent throughout the column length and reached the length of the internal stamens, the calyx and the fl oral pedicel were glandular; all those characters should indicate r. sempervirens var. genuina rouy. (syn r. sempervirens). teb and tab accessions are characterized by a medium density of prickles, prickle base with medium diameter, the shortest rachis and a short petiole, and by fl owers that have the shortest and the narrowest receptacle. according to maire (1980) those accessions present the characteristics of the variety genuina but they differ by the globular form of the receptacle. thus, we identifi ed them as the form f. scandens (maire 1980). so teb and tab accessions should be r. sempervirens var. genuina f. scandens (mill) batt. the accessions tebs3, djb, teb and tab were collected from the north east of tunisia (longitude: 36°28’–36°57’; latitude: 9°05’–8°57’). indeed, their descriptions suggest r. sempervirens and the slight morphological differences can be related to the effect of environment and we can consider those accessions as r. sempervirens ecotypes. aind accession was distinguished by its biggest fl owers with the largest style and lobulated sepals. this accession presents the characteristics of the r. sempervirens var. submoshata rouy. (silvestre and montserrat 1998). wech and zagh1 accessions presented a prostrate habit, which characterize r. sempervirens var. prostrate (dc) desv. (de candole 1815, gore 1832, boulenger 1932). ben cheikh-affene z., haouala f., harzallah-skhiri f. 14 acta bot. croat. 74 (1), 2015 as a result of this study, we can conclude that among the vegetative and fl oral characters studied, those showing the highest discriminating value were the sizes of prickle, leaf and leafl et, leaf area, lengths of style, pedicel and receptacle, sizes of sepal, petal, and receptacle, pedicel and sepal gland density. moreover, those discriminant characters are used in the taxonomic identifi cation of the genus rosa. the study revealed considerable phenotypic diversity in the tunisian wild rose population. accessions from the 13 populations studied belong to two different sections. the fi rst one is section caninae, into which the accessions from teboursouk1, 2, kessra, haffouz1, 2 and zaghouan2 were grouped, being related by some cases of synonymy but differing in some morphological traits such as the presence or absence of bristles and glands in leafl et. thus, this morphological criterion subdivided the six caninae accessions into four species such as r. canina (zagh2), r. agrestis (hafz2, tebs2), r. micrantha (hafz1) and r. dumetorum (tebs1, kess). the second section is syntylae, which is represented by accessions from tebaba, aïn drahem, wechtata, tabarka, djeba, teboursouk3 and zaghouan1, identifi ed as r. sempervirens. indeed, the morphologic analyses prove the presence of four ecotypes of r. sempervirens such as: ecotypetebs 3, ecotype djb, ecotype teb and ecotype tab and two varieties; r. sempervirens var. submoshata (aind) and r. sempervirens var. prostrata (zagh1, wech). phenotypic variation for quantitative characters is most likely non-genetic in origin. the most reliable estimates of genetic structure should therefore come from qualitative characters, especially when environmental effects can be minimized. we are now in the process of developing suitable molecular markers through dna fi ngerprinting with microsatellite dna hybridization ssr. these markers will eventually provide an additional data set on those accessions. determination key a. exerted styles, connate into a column usually long as stamens (l. sty = 5.6–6.3 mm)….......................................................................... section: synstylae leaves usually with 5 leafl e ts, lanceolate, long and slender branches..... rosa sempervirens 1. climbing shrub 1.1. stylar column densely pubescent throw the column length and reach the length of the internal stamens, receptacle, calyx and fl oral pedicel were glandular.......................................................................... r. sempervirens (tebs3, djb, teb and tab) 1.2. the same characters than as for var. genuina but sepals were lobulated with many appendix……..................................................... r. sempervirens var. submoshata (aind) 2. a prostrate habit................................................................................... r. sempervirens var. prostrate (zagh1 and wech) b. style reaching only the mouth of the receptacle and stigmas forming a sessile head over it (l. sty = 1.8–2.6 mm), leaves of fl owering branches usually with 7 leafl ets (5.0–7.0), short pedicel with long and smooth receptacle (l. rep = 0.9–1.1 cm), glabrous sepals with many appendices...................................................................................... section caninae 1. leafl et pubescent and glandular on both sides, copiously pinnate...... r. micrantha (hafz1) 2. leafl et glandular and pubescent in under side and longer than 2 cm..... r. agrestis (hafz2 and tebr2) 3. leafl et eglandular................................................................................. r. canina (zagh2) 4. leafl et pubescent in both sides with bristles oppressed in the upper side of leafl et........................................................................................ r. dumetorum (tebr1 and kess) morphological diversity of 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(eds.), encyclopaedia of rose science, 111–117. elsevier, oxford. opce-str.vp acta bot. croat. 73 (1), 209-220, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 interactions with mycorrhizal fungi in two closely related hybridizing orchid species alessia luca, francesca bellusci, giuseppe pellegrino* department of biology ecology and earth science, university of calabria, via bucci 6/b, i-87036 rende (cs), italy abstract – the nuclear ribosomal dna was used to identify the orchid mycorrhizal fungi found in roots of orchis xbivonae and its parental species orchis anthropophora and orchis italica. polymerase chain reaction products were sequenced and identified using the expanded database. we determined that closely related tulasnellaceae are mycorrhizal in the three orchid taxa, suggesting that the mycorrhizal partner does not impair hybrid survival. this study demonstrates that o. xbivonae displays few differences in comparison with its two parental species in identity of its associated mycorrhizal fungi, it is a short-term by-product of the hybridizing behavior of common pollinators, and thus it will not easily origin descendents with potential new genetic combinations and/or ecological preferences. keywords: mycorrhizal fungi, nrdna, orchis anthropophora, orchis italica, orchis xbivonae, tulasnellaceae introduction the orchidaceae is one of the largest plant families with close to 25,000 species and a circum-global distribution (dressler 1993, cribb et al. 2003). it is also one of the most complex plant families with many adaptations. one adaptive mechanism is related to reproductive biology, indeed orchid flowers evolved a close relationship with insect pollinators such that sympatric species can be pollinated by different insect species (van der pijl and dodson 1966), maintaining species isolation (scopece et al. 2007). interestingly, 30% of orchid species shows non-rewarding flowers (renner 2005), attracting pollinators by generalized food deception, mimicry or sexual deception (jersakova et al. 2006). another example of adaptive mechanism is related to mycorrhizal interactions that have allowed orchids to fit in several habitat types and has led to colonize worldwide. the main group of fungi inhabiting orchid roots is basidiomycetes (rasmussen 2002), though ascomycetes have been found (selosse et al. 2004). mycorrhizal fungi provide the basic acta bot. croat. 73 (1), 2014 209 * corresponding author, e-mail: giuseppe.pellegrino@unical.it copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:46 color profile: generic cmyk printer profile composite default screen organic carbon (smith 1966, cameron et al. 2006) and carbohydrates for seedling growth (leake 1994, rasmussen 1995). also adult individuals of achlorophyllous and photosynthetic orchids obtain part of carbon heterotrophically by mycorrhizal fungi (bidartondo et al. 2004, julou et al. 2005, selosse and roy 2009, jacquemyn et al. 2010). in addition, in an adult green-leaved orchid goodyera repens, there is a flow of carbon from the plant to the fungus (cameron et al. 2006). this suggests that also the green-leaved adult orchids, after the initially mycoheterotrophic phase of development, establish the general symbiotic relationship with fungi in which carbon from the plant is exchanged for mineral nutrients gathered by the fungus. in recent years, the application of modern molecular approaches has increased greatly the facility for identifying the mycobionts. from the beginning of 2000, a large number of orchid mycorrhizal has been identified directly from orchid roots, tubers and rhizomes through molecular biology techniques (shefferson et al. 2007, stockinger et al. 2010, jacquemyn et al. 2011a). the main widespread molecular markers used to identify orchid mycobionts are the internal transcribed spacers (itss) of the nuclear ribosomal dna using primer sets (white et al. 1990, taylor and mccormick 2008) for pcr amplification. the level of specificity between fungus and orchid is an important factor determining chances of successful seedling establishment (bidartondo and read 2008). early molecular studies have revealed that terrestrial orchids interact with a wide range of mycorrhizal fungi. some orchids are extreme specialists and are associated to a single fungal species (mccormick et al. 2004, taylor et al. 2004, dearlaney 2007, shefferson et al. 2008), while others are generalists, hosting many different fungi (mccormick et al. 2006, bonnardeaux et al. 2007, irwin et al. 2007, roy et al. 2009). the requirement of terrestrial orchids to form a symbiosis with mycorrhizal fungi may act on orchid distribution and diversification (waterman et al. 2008, phillips et al 2011). a large diversity and distribution of orchids is related to a broadly distributed fungi (otero et al. 2007), while rarity of orchids and narrow distribution is associated to a narrow mycorrhizal specificity (valadares et al. 2012). hybrid zones, in which hybrids live sympatrically with parental species, are highly suitable to verify mycorrhizal hybrid preferences, that means do hybrids form a symbiosis with the same fungi of one or both parents? or have totally different partners? there are only few studies that have investigated mycorrhizal associations in hybrid compare to parental species. previous studies showed that hybrids of genus caladenia had fungi genetically different from those associating with parental species (hollick et al. 2005), while adult individuals of orchis simia and o. anthropophora and their hybrid were associate to closely related tulasnellaceae fungi (schatz et al. 2010), such as three closely related hybridizing orchis species showed common mycorrhizal associations in protocorms and adult stage (jacquemyn et al. 2011b). in this study, we examined, with molecular analyses, a sympatric zone between orchis italica poir. and o. anthropophora l., which hybridize to form o. xbivonae tod. our main aim was to compare the identity of mycorrhizal associates in two parental species o. italica, o. anthropophora, and their hybrid o. xbivonae at the adult stage to determine if lack of appropriate fungal symbionts can be related to hybrid viability, and to study the role of mycorrhizal associations in allowing to the hybrid to exploit prospective new ecological niches different from parental habitat. 210 acta bot. croat. 73 (1), 2014 luca a., bellusci f., pellegrino g. 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:46 color profile: generic cmyk printer profile composite default screen materials and methods study area and orchid species the study on orchis italica poir., o. anthropophora (l.) all., and their hybrid orchis xbivonae tod. was conducted in a natural population located onto the »monte di cassano« (39°47’n, 16°18’e, 512 m a.s.l.), one kilometre north-west of the city of »cassano allo ionio« (northern calabria region, italy). the whole area covers roughly 1,500 m2 (25 m wide and 60 m long) of calcareous soil and is bounded on the west by a road and on the remainder by deep gorges. in the studied site, orchis italica and o. anthropophora overlap extensively in their spatial distribution and flowering time (flowering peak between the end of april and the middle of may) and grew together with o. xbivonae (pellegrino et al. 2009). orchis italica and o. anthropophora are closely related (bateman et al. 2003), have same chromosome number (2n = 42) (queiros 1985, cauwet-marc and balayer 1986, bianco et al. 1987, costantinidis et al. 1997) and have been both included in the o. militaris (delforge 2005) or »anthropomorphic« group (bateman et al. 2003). orchis italica show a pendent lip, deeply tri-lobed and with a cylindrical spur, while, o. anthropophora has a narrow lip and, differently from all the others orchis species, it lacks a spur (delforge 2005). the detected specimens of o. xbivonae are 20–40 cm high, with oblong leaves and cylindrical inflorescences. the pendent labellum is trilobate with median lobule reduced to a minuscule dent. spur is very short, saccate and pointing downwards, with a length of about the half of that of o. italica. molecular analysis molecular characterization of mycorrhizae involved: (i) extraction of dna from orchid roots, (ii) amplification of nuclear ribosomal dna regions useful in determining fungal identity, (iii) dna sequencing for identification of mycorrhizal fungi and assessment of specificity levels. small parts of roots were cut from 15 randomly selected individuals of orchis anthropophora and o. italica and for 8 individuals of o. xbivonae (all hybrids found) for molecular analysis. all roots were surface sterilized using 1% hypochlorite (30 s) followed by three rinses in distilled water (30 s). total dna was extracted from 1–2 cm length of root pieces per plant using the cetyltrimethyl ammonium bromide (ctab) method (doyle 1991). dna was resuspended in 50 ml of distilled water. to discriminate among fungal taxa colonizing orchid roots, the internal transcribed spacers (itss) of the nuclear ribosomal dna were amplified using fungal universal primer pairs its1-of and its4-of (taylor and mccormick 2008) chosen for their most constant and reliable amplification (jacquemyn et al. 2011b). pcrs were performed on a ptc-100 thermal cycler (mj research inc., watertown, ma, usa) according to the following thermal cycling profile: 94 °c for 3 min (1 cycle), 94 °c for 45 s, 58 °c for 45 s, 72 °c for 1 min (30 cycles); 72 °c for 5 min (1 cycle). amplification products were electrophoretically separated on a 1.8% agarose gel (methaphore, fms), photographed after ethidium bromide staining and purified with the qiaex ii gel extraction kit (qiagen) to remove unincorporated primers and dntps following the acta bot. croat. 73 (1), 2014 211 mycorrhizal fungi in orchid hybrids 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:46 color profile: generic cmyk printer profile composite default screen manufacturer’s instructions. the purified pcr fragments were sequenced directly in forward and reverse directions; fluorescent dye sequencing was performed on a 310 prism abi dna genetic analyzer (applied biosystems, carlsbad, california, usa). data analysis the clustalw algorithm (thompson et al. 1994) of the molecular evolutionary genetics analysis version 5 (mega5) program package was applied for the exact alignment of sequences (tamura et al. 2011). ambiguous sites were checked manually and corrected by comparing electropherograms from both strands. consensus sequences were obtained for each specimen (5’ and 3’ borders were identified using mycorrhizal sequences already available in dna data bank of japan (2013). sequence identity was determined using the blast algorithm available through the national center for biotechnology information (2013). in addition, since none of the its sequence types obtained had 100% identity with genbank sequences of identified rhizoctonia group, otus were identified comparing our its sequences and previously developed mycobionts otus. the identification of otus was performed comparing the sequences obtained in this study with those of jacquemyn et al. (2011b) using the clustalw algorithm of the mega 5 program package. our fungi sequences were classified as otus when its sequence similarity exceeded 97%. pairwise genetic distances between sequences were measured by the number of nucleotide substitutions occurring between them under the tamura and nei model (tamura and nei 1993). a neighbour-joining tree (saitou and nei 1987) was constructed using clustal and displayed by treeview v.1.6.6. differences in mycorrhizal assemblages between taxa were analysed using a one-way anova. when the f test was significant, means were compared using the tukey test at 5% error probability. results the identification of orchids associated fungi by molecular techniques was successful for all the collected samples. on the basis of less than 5% of genetic distance (tab. 1), out of 23 fungal otus identified previously (jacquemyn et al. 2011b) eight were observed (otu 2, 4, 6, 7, 10, 11, 12 and 17) in our investigated plants; six were related to tulasnellaceae, and two to ceratobasidiaceae (tab. 2). six different fungal otus were found in orchis italica (otu 2, 4, 7, 10, 12 and 17) and o. anthropophora (otu 2, 4, 6, 7, 10 and 11), and four (otu 2, 4, 7 and 10) in o. xbivonae (fig. 1). parental species showed different frequently dominant otus (otu 2 and 12 in o. italica and otu 7 and 10 in o. anthropophora). otu 2 and 4 were found in all three taxa, while o. italica and o. anthropophora showed two exclusive otus, otu 12 and 17, otu 6 and 11, respectively (fig. 1). phylogenetic analysis placed its sequences for orchis italica, o. anthropophora and o. xbivonae in eight distinct groups closely associated with otus (fig. 2). anova analysis showed no significant differences in mycorrhizal assemblage between parental species and hybrids, (o. anthropophora vs o. xbivonae: f1,21 = 1.004, p = 0.35, o. italica vs o. xbivonae: f1,21 = 0.785, p = 0.38), although the parental species have a slight different assemblages (o. anthropophora vs o. italica: f1,28 = 3.411, p < 0.05). 212 acta bot. croat. 73 (1), 2014 luca a., bellusci f., pellegrino g. 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:46 color profile: generic cmyk printer profile composite default screen acta bot. croat. 73 (1), 2014 213 mycorrhizal fungi in orchid hybrids tab. 1. genetic distance between fungal operational taxonomic units (otus) identified in orchis italica (i), o. anthropophora (a) and o. xbivonae (b). less than 5% of genetic distance is marked in bold. orchis species otu2 otu4 otu6 otu7 otu10 otu12 otu17 otu11 i 33 0.004 0.327 0.317 0.321 0.334 0.121 0.420 0.438 i 20 0.005 0.324 0.319 0.323 0.334 0.122 0.413 0.434 i 28 0.004 0.324 0.313 0.315 0.331 0.118 0.420 0.434 i 22 0.005 0.325 0.314 0.316 0.332 0.118 0.420 0.435 i 30 0.327 0.004 0.133 0.097 0.200 0.329 0.339 0.354 i 24 0.324 0.007 0.135 0.101 0.200 0.327 0.343 0.357 i 29 0.320 0.107 0.113 0.009 0.228 0.351 0.380 0.376 i 25 0.318 0.103 0.105 0.005 0.223 0.345 0.377 0.373 i 27 0.342 0.204 0.231 0.226 0.015 0.349 0.412 0.407 i 19 0.334 0.202 0.234 0.223 0.005 0.349 0.412 0.413 i 15 0.118 0.327 0.317 0.345 0.348 0.005 0.388 0.400 i 14 0.121 0.336 0.326 0.355 0.358 0.007 0.391 0.407 i 12 0.121 0.336 0.326 0.355 0.358 0.007 0.391 0.407 i 10 0.118 0.326 0.317 0.345 0.348 0.002 0.381 0.397 i 4 0.413 0.336 0.390 0.377 0.402 0.382 0.005 0.091 a 2 0.004 0.322 0.311 0.315 0.328 0.116 0.417 0.431 a 1 0.330 0.007 0.137 0.101 0.199 0.332 0.345 0.359 a 8 0.324 0.004 0.137 0.101 0.197 0.327 0.339 0.354 a 9 0.313 0.139 0.005 0.113 0.238 0.317 0.396 0.402 a 3 0.310 0.133 0.004 0.111 0.236 0.314 0.390 0.399 a 15 0.316 0.137 0.004 0.111 0.236 0.320 0.396 0.402 a 4 0.316 0.105 0.111 0.011 0.229 0.349 0.385 0.386 a 14 0.312 0.101 0.107 0.005 0.221 0.343 0.371 0.368 a 13 0.321 0.107 0.113 0.009 0.228 0.348 0.378 0.374 a 5 0.312 0.099 0.105 0.002 0.223 0.343 0.374 0.370 a 12 0.325 0.197 0.233 0.223 0.002 0.343 0.405 0.406 a 6 0.325 0.197 0.233 0.223 0.002 0.343 0.405 0.406 a 11 0.342 0.202 0.234 0.224 0.013 0.349 0.409 0.404 a 7 0.342 0.202 0.234 0.224 0.013 0.349 0.409 0.404 a 10 0.431 0.357 0.397 0.377 0.410 0.397 0.091 0.005 b 1 0.002 0.324 0.313 0.318 0.331 0.118 0.416 0.433 b 2 0.330 0.005 0.139 0.103 0.200 0.327 0.337 0.352 b 4 0.330 0.005 0.130 0.099 0.202 0.333 0.343 0.357 b 3 0.315 0.103 0.109 0.005 0.221 0.346 0.372 0.371 b 5 0.318 0.105 0.111 0.007 0.226 0.349 0.378 0.374 b 8 0.318 0.103 0.105 0.005 0.223 0.348 0.380 0.376 b 7 0.325 0.197 0.233 0.223 0.002 0.343 0.405 0.406 b 6 0.336 0.202 0.233 0.223 0.013 0.343 0.406 0.400 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:46 color profile: generic cmyk printer profile composite default screen 214 acta bot. croat. 73 (1), 2014 luca a., bellusci f., pellegrino g. tab. 2. fungal operational taxonomic units (otus) identified in orchis anthropophora (a), o. italica (i) and o. xbivonae (x)(marked by +). fungi were grouped into otus defined by 97% internal transcribed spacer (its) sequence similarity. target family closest match in genbank presence in examined orchids a i b otu-2 tulasnellaceae gq907254 + + + otu-4 tulasnellaceae gq907260 + + + otu-6 tulasnellaceae gq907266 + otu-7 tulasnellaceae gq907258 + + + otu-10 tulasnellaceae gu066935 + + + otu-11 ceratobasidiaceae gu066936 + otu-12 tulasnellaceae hq330992 + otu-17 ceratobasidiaceae hq331002 + orchis anthropophora (n = 15) orchis italica (n = 15) orchis xbivonae (n = 8) otu-7 otu-7 otu-7 otu-11 otu-10 otu-10 otu-4 otu-4 otu-4 otu-2 otu-2 otu-2 otu-11 otu-6 otu-17 otu-12 fig. 1. frequency distribution of identified operational taxonomic units (otus) in orchis anthropophora, o. italica and o. xbivonae. otu7 a 5 i 25 b 8 a 14 b 3 i 29 b 5 a 13 a 4 i 24 b 2 a 1 b 4 i 30 otu4 a 8 a 9 a 3 otu6 a 15 a 11 a 7 b 6 i 27 i 19 otu10 a 12 a 6 b 7 i 14 i 12 i 15 otu12 i 10 i 28 i 22 a 2 i 20 i 33 otu2 b 1 otu11 a 10 otu17 i 4 fig. 2. neighbour-joining phylogenetic tree showing the relationship between internal transcribed spacer (its) sequences for orchis anthropophora (a), o. italica (i) and o. xbivona (b) mycobionts and operational taxonomic units (otus). 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:46 color profile: generic cmyk printer profile composite default screen discussion we have shown that adult plants of the two parental species and their hybrid associated with several frequently different fungal otus. first, 75% of mycorrhizal fungi identified belong to tulasnellaceae, a large, common group of orchid mycorrhizal fungi that have already been recorded in o. anthropophora and o. italica (jacquemyn et al. 2011b). moreover, tulasnellaceae have been recognized as the most important association with other mediterranean orchids such as anacamptis (girlanda et al. 2011), cypripedium (shefferson et al. 2007), dactylorhiza (jacquemyn et al. 2012) and ophrys (girlanda et al. 2011). previous literature showed that the number of fungal otus of orchis sp. varied between one and nine; in detail o. anthropophora, o. italica, o. simia and o. militaris were associated with at least seven different otus, o.s purpurea with four, whereas o. anatolica, o. mascula, o. olbiensis and o. troodi with only one or two fungal otus (jacquemyn et al. 2010, jacquemyn et al. 2011a, b). in our case, in agreement with previous studies, o. italica and o. anthopophora were associated with six fungal otus, predominantly with otu 2 and 12 (o. italica) and with otu 7 and 10 (o. anthopophora) (fig. 1). orchis italica and o. anthopophora associated secondly with two different otus belonging to ceratobasidiaceae confirming previous results (jacquemyn et al. 2011b). orchis xbivonae associated with fewer mycorrhizal fungi in comparison with its two parental species. similar fungi occurred in the two parents, perhaps due to the close phylogenetic positions of the two parental species (bateman et al. 2003). similarly, micorrhizal fungi of orchis xbergonii mostly belonged to tulasnellales associated with the two parental species (o. simia and o. anthopophora) and that the fungi associated with hybrids had less-diverse sequences than those associated with the parental species (schatz et al. 2010). hybrid zones are notable for studying reproductive barriers among closely related species, role of selection in maintaining or eroding species differences, and role of hybridization in plant evolution (rieseberg and buerkle 2002, lexer et al. 2005). essential conditions for speciation by hybridization are that the hybrid exploits an ecological niche, either the parental one or a totally new one, shows an own reproductive success and produces a sufficient number of seeds (arnold 1997). compared to parental species, previous study demonstrated that o. xbivonae showed low fruiting values in open-pollinated flowers and the absence of any form of postmating isolation (pellegrino et al. 2009). the low levels of reproductive success, the lack of post-zygotic barriers and of f2 (or later) generations (pellegrino et al. 2009) suggest that the mycorrhizal symbiosis imposes no constraints on the survival of hybrids, and that the lack of pollinators appears to strongly limit hybrid fitness, as has previously been reported in parental species (pellegrino et al. 2010) and other deceptive orchids (mattila and kuitunen 2000, pellegrino et al. 2005, smithson 2006). the coexistence of orchis xbivonae with its parents suggests that this hybrid is a short-term by-product due to the behavior of shared pollinators (schatz 2006). mycorrhizal do not represent a limitation to o. xbivonae growth but at the same time do not offer an ecological opportunity to partially separate hybrid habitat from the parental ones. in our case the low specificity and divergent association pattern between hybrids and parental species do not represent an effective barrier to hybridization. indeed, hybrids and parental species share most of their mycorrhizal fungi, and thus no such incompatibilities are to be acta bot. croat. 73 (1), 2014 215 mycorrhizal fungi in orchid hybrids 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:46 color profile: generic cmyk printer profile composite default screen expected, and post-mating barriers at the seed germination stage should be weak, implying that mycorrhizal associations only play a minor role in affecting hybridization between orchids. overall, these results indicate that our hybrid zone represents a phenomenon of little evolutionary meaning and that the few hybrid plants will not easily origin descendents with potential new genetic combinations and/or ecological preferences. in conclusion, our results corroborate the idea that hybrid zone did not have a prominent role in speciation processes of mediterranean orchids. indeed, with the exclusion of one case (orchis xcolemanii) in which the hybrids showed high levels 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(eds.), pcr-protocols and applications. a laboratory manual, 315–322. academic press, new york. 220 acta bot. croat. 73 (1), 2014 luca a., bellusci f., pellegrino g. 692 luca et al.ps u:\acta botanica\acta-botan 1-14\692 luca et al.vp 24. o ujak 2014 11:22:47 color profile: generic cmyk printer profile composite default screen 571 rijal lebland et al.vp acta bot. croat. 72 (1), 35–47, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 pseudo-nitzschia peragallo (bacillariophyceae) diversity and domoic acid accumulation in tuberculate cockles and sweet clams in m’diq bay, morocco benlahcen rijal leblad*1, 2, nina lundholm4, didier goux5, benoît veron3, regia sagou6, hamid taleb6, hassan nhhala1, hassan er-raioui2 1 laboratoire de phytoplancton, institut national de recherche halieutique, 90000 tanger, morocco 2 faculté des sciences et techniques, université abdelmalek essadi, tanger, morocco 3 umr 100 pe2m, université de caen basse-normandie, 14032 caen, france 4 the natural history museum of denmark; sølvgade 83s; dk-1307 copenhagen, denmark 5 cmabio, université de caen basse-normandie, 14032 caen, france 6 laboratoire des biotoxines, institut national de recherche halieutique, casablanca, morocco abstract – the diversity of pseudo-nitzschia (bacillariophyceae) and accumulation of the neurotoxin domoic acid (da) in two types of shellfish; tuberculate cockles (acanthocardia tuberculata) and sweet clams (challista chione) was explored in m’diq bay, morocco during 2007. the highest abundances of pseudo-nitzschia were found during the period from march to october, with peaks occurring in may and september. toxin analysis showed an accumulation of domoic acid in shellfish sampled during spring and autumn. the maximum toxin concentration was 4.9 mg da g–1 of the whole tissue recorded in sweet clam during spring. using transmission electron microscopy, thirteen pseudo-nitzschia species were identified, eight of which are known as producers of domoic acid: p. multistriata, p. cuspidata, p. galaxiae, p. multiseries, p. pseudodelicatissima, p. pungens var. aveirensis, p. calliantha and p. fraudulenta. the five nontoxic species observed were p. subpacifica, p. arenysensis, p. dolorosa, p. subfraudulenta, and p. cf. caciantha. key words: diatoms, domoic acid, phytoplankton, pseudo-nitzschia, shellfish, morocco acta bot. croat. 72 (1), 2013 35 * corresponding author, e-mail: benlahcenr@yahoo.fr copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:02 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees introduction the planktonic diatom genus pseudo-nitzschia presently comprises 37 species (>20 mm long), several of which have a wide biogeographical distribution (hasle 2002). fourteen species are known to produce domoic acid (da), a toxin that may accumulate e.g. in shellfish, and cause amnesic shellfish poisoning (asp) in humans eating contaminated shellfish. human intoxication with da gives an array of gastro-intestinal and neurological symptoms such as vomiting, diarrhoea, mental confusion, memory loss (amnesia), disorientation and coma (wright et al. 1989). the first correlation between pseudo-nitzschia bloom and da contained in shellfish was confirmed in prince edward island, canada (bates et al. 1989), where p. multiseries abundance exceeded 15 x 106 cells l–1 and mussels were contaminated by 790 mg of da g–1 of shellfish meat. on the other hand, amzil et al. (2001) first reported da in relation to pseudo-nitzschia blooms on the french mediterranean coast. subsequently, other studies have reported detection of da in italy (sarno and dahlmann (2000)), greece (kaniougrigoriadou et al. 2005, moschandreou et al. 2010), tunisia (inès et al. 2006) and croatia (ujevi] et al. 2010). massuti and margalef (1950) and margalef (1969) have previously described nitzschia delicatissima, n. pungens, and n. seriata in the mediterranean coasts. in the last decade, several studies were conducted on the systematics of pseudo-nitzschia, and a number of species have been identified. species identified in spain are p. brasiliana, p. calliantha, p. delicatissima, p. fraudulenta, p. multistriata, p. pungens, p. galaxiae, p. cacciantha, p. mannii, p. arenysensis (quijano-scheggia et al. 2008, 2010) and p. australis (zapata et al. 2011). in italy, p. calliantha and p. delicatissima (caroppo et al. 2005, zingone et al. 2006), p. galaxiae, p. multistriata (sarno and dahlmann 2000), orsini et al. 2002, cerino et al. 2005, zingone et al. 2006), p. fraudulenta (zingone et al. 2006) were identified. pseudo-nitzschia calliantha (spatharis et al. 2007) was identified in greece. in the southern black sea (bargu et al. 2002) and in the eastern adriatic sea, p. calliantha, p. fraudulenta, p. pungens, p. pseudodelicatissima, p. manii (ljube[i] et al. 2011, mari] et al. 2011) were identified. the maghreb coasts were also investigated and some species of pseudo-nitzschia were identified such as p. calliantha in tunisia (inès et al. 2006) and p. calliantha in algeria (illoul et al. 2008). in morocco, only one study dealing with pseudo-nitzschia systematics was done, by akallal et al. (2002) in the atlantic coast; seven species was identified: p. fraudulenta, p. multiseries, p. multistriata, p. pungens var. cingulata, p. subpacifica, p. delicatissima and p. pseudodelicatissima. in morocco, psp (paralytic shellfish poisoning) intoxication by mussels was first reported in november 1971 and october 1975 (essaid 1977). subsequently, several intoxications by psp toxins were recorded: in october 1982 with two deaths (bourhili 1984), in november 1994 with high number of incidences of human intoxication and four deaths (taleb et al. 2001).the first human intoxication with domoic acid after ingestion of infected mussels probably dates back to 1978 in al-hoceima (mediterranean coast), where patients who had eaten mussels (mytilus galloprovincialis) suffered from loss of memory and disorientation. these symptoms are characteristic of da effects on human consumers; and of other shellfish poisonings, none results in amnesiac states. pseudo-nitzschia blooms have frequently been observed off the western mediterranean coast of morocco since 2002. . due to previous asp episodes in and outside morocco, it was judged important to explore pseudo-nitzschia diversity in local areas such as m’diq bay, which is subjected to commercial shellfish exploitation and to gather knowledge on 36 acta bot. croat. 72 (1), 2013 rijal leblad b., lundholm n., goux d., veron b., sagou r., et al. 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:03 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees species dynamics in order to highlight their effect on shellfish continuation, mainly tuberculate cockles and sweet clams. in this regard, the present study was undertaken in m’diq bay from january to december 2007, aiming at determining the biodiversity of pseudo-nitzschia. this study deals with their seasonal diversity follow-up and abundance and identification of putative da producers. material and methods location stations sampling was performed in m’diq bay, which is located in the west mediterranean coast of morocco, adjacent to the gibraltar strait (35°43’425 n – 05°19’841 w). this sampling point is 7–10 m deep (fig. 1). continental inputs reach m’diq bay through one temporal and torrential stream. this bay features important socio-economic activities, particularly tourism and fishing. among the latter, shellfish catching is important (559 tons year–1). seawater sampling seawater samples were taken on a weekly frequency basis; this was to allow a weekly estimation of pseudo-nitzschia abundance. seawater samples were taken using nansen bottles at depths of 0.5 m. monthly samplings of seawater were also carried out using a plankton net with a 20 mm mesh for pseudo-nitzschia species identification purpose. these samples were fixed with an appropriate solution (acetic lugol) and kept in conditions of darkness. phytoplankton analysis pseudo-nitzschia abundance was evaluated by counting using the inverteded microscope (utermöhl 1958). for electron microscope analysis, about 100 specimens were examined for each sample in order to identify its species and determine its composition. acta bot. croat. 72 (1), 2013 37 pseudo-nitzschia diversity in m’diq bay, morocco fig. 1. location of the sampling site in m’diq bay (morocco). 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:06 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees for ultra-structural examination of the frustules, the cells were rinsed by chemical oxidation (lundholm et al. 2002); ten ml of each sample was transferred into 50 ml conical tubes. caco3 was removed by adding 1 ml 10% hcl and overnight oxidation took place after addition of 2 ml 30% h2so4 and 10 ml saturated aqueous solution of kmno4, with periodic agitation. samples were cleared by the addition of 10 ml of saturated oxalic acid and rinsed with distilled water and centrifuged (3–4 times). after removal of the supernatant, 20 ml of the obtained material was placed on a millipore disc and left to dry. examination of the grids was done by transmission electron microscope (tem) using a jeol/ jem-1011. domoic acid analysis concentrations of da were determined by hplc (shimadzu 10vp type). this apparatus is composed of a scl-10vp controller, a lc-10advp quaternary pomp, a cto-10vp colonne four, a sil-10advp autosampler, a spd-m10avp photodiode array detector, a vydac c18 column (250 × 4.6 mm, with 5 mm) and the guard cartridge (vydac c18, 5 mm). da was assessed in periods of high pseudo-nitzschia abundance. it is measured in the whole meat of cockles and sweet clams according to the quilliam et al. (1995) protocol. threefold analysis was performed using about 100 g of shellfish meat (ten to fifteen individuals are required to have such an amount of meat). after being shredded and homogenated, four g of meat were added to 16 ml of solvent extraction (methanol-water, 1:1) and then homogenized (ultra-turrax for 3 minutes at about 10,000 rpm). the homogenate was centrifuged at least at 4,000 rpm for 10 min to obtain supernatant. the later was analyzed using the following chromatographic conditions: mobile phase flow rate of 1 ml min–1, detector wave length of 242 nm, injection volume of 20 ml and an oven temperature for the column of 40 °c. the determination of da content in samples was done with a detection limit of 0.3 mg g–1. results domoic acid and pseudo-nitzschia abundance during the sampling period (january to december 2007), pseudo-nitzschia in m’diq bay was present continuously in low abundance and higher abundance (ca. 10–20 cells ml–1) were observed from march to november (fig. 2). five proliferation periods (>30 cells ml–1) were recorded during this period, with two major peaks (88 cells ml–1 and 157 cells ml–1) occurring in may and in the end of september, respectively (fig. 2). hplc analysis showed the presence of da on five occasions (tab. 1, fig. 2). the highest da levels were recorded during spring. the highest da concentration found was 4.9 mg da g–1 recorded in sweet clam in may 2007. this study reports for the first time the presence of da in shellfish in the mediterranean coast of morocco. however, da concentration never exceeded the normative threshold of 20 mg ad g–1 of shellfish meat (tab. 1). pseudo-nitzschia diversity using scanning transmission electron microscopy, thirteen species were identified as toxic (tab. 2, fig. 3): pseudo-nitzschia cuspidata (hasle), p. fraudulenta (cleve), p. 38 acta bot. croat. 72 (1), 2013 rijal leblad b., lundholm n., goux d., veron b., sagou r., et al. 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:07 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees multistriata (takano), p. pseudodelicatissima (hasle), p. galaxiae (lundholm et moestrup), p. multiseries (hasle), p. calliantha (lundholm, moestrup et hasle) and p. pungens var. aveirensis (lundholm, churro, carreira et calado). the other five non-toxic species are p. dolorosa (lundholm et moestrup), p. arenysensis (quijano-scheggia, garcés, lundholm), p. subpacifica (hasle), p. subfraudulenta (hasle), and p. cf. caciantha (lundholm, moestrup et hasle). the composition of pseudo-nitzschia species varied greatly during the year (fig. 4). some species were found during a long period of the year while others appeared in specific short periods. p. cuspidata, p. fraudulenta, p. subpacifica and p. arenysensis / p. delicatissima were the most frequently recorded species. the spring and autumn periods are the acta bot. croat. 72 (1), 2013 39 pseudo-nitzschia diversity in m’diq bay, morocco 0 1 2 3 4 5 6 0 8 -0 1 -0 7 1 5 -0 1 -0 7 2 2 -0 1 -0 7 3 1 -0 1 -0 7 0 6 -0 2 -0 7 1 2 -0 2 -0 7 2 0 -0 2 -0 7 2 7 -0 2 -0 7 0 5 -0 3 -0 7 1 9 -0 3 -0 7 2 7 -0 3 -0 7 0 3 -0 4 -0 7 2 4 -0 4 -0 7 0 1 -0 5 -0 7 0 7 -0 5 -0 7 1 5 -0 5 -0 7 2 1 -0 5 -0 7 2 8 -0 5 -0 7 0 5 -0 6 -0 7 1 3 -0 6 -0 7 1 9 -0 6 -0 7 2 6 -0 6 -0 7 0 2 -0 7 -0 7 0 9 -0 7 -0 7 1 6 -0 7 -0 7 2 4 -0 7 -0 7 0 1 -0 8 -0 7 0 6 -0 8 -0 7 1 3 -0 8 -0 7 2 9 -0 8 -0 7 1 1 -0 9 -0 7 1 7 -0 9 -0 7 2 4 -0 9 -0 7 0 1 -1 0 -0 7 1 1 -1 0 -0 7 2 2 -1 0 -0 7 3 0 -1 0 -0 7 1 2 -1 1 -0 7 1 9 -1 1 -0 7 2 8 -1 1 -0 7 0 3 -1 2 -0 7 1 1 -1 2 -0 7 µ g d a g -1 o f m e a t 0 20 40 60 80 100 120 140 160 180 p s e u d o n it z s c h ia a b u n d a n c e (c e ll s m l -1 ) da cockle da sweet clam pseudo-nitzschia spp fig. 2. abundance of pseudo-nitzschia and domoic acid (da) concentrations in shellfish from m’diq bay during 2007. � sampling for domoic acid determination tab. 1. domoic acid concentration (mg da g–1 of meat) in shellfish acanthocardia tuberculata (tuberculate cockle) and callista chione (sweet clam). nd = not detected. date mg da g–1 of meat (acanthocardia tuberculata) mg da g–1 of meat (callista chione) 2007-01-22 0.85 (±0.10) 0.71 (±0.80) 2007-04-24 1.66 (±0.19) 0.59 (±0.7) 2007-05-21 2.11 (±0.25) 4.90 (±0.58) 2007-07-02 nd nd 2007-09-17 nd 1.16 (±0.13) 2007-09-24 nd nd 2007-10-01 0.75 (±0.09) 1.57 (±0.18) 2007-11-12 nd nd 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:07 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees two main seasons for proliferation of pseudo-nitzchia species, particularly those known to be producing da. eight and nine species were identified during spring and autumn, respectively (fig. 4). during pseudo-nitzschia bloom in may, p. arenysensis / p. delicatissima were significantly dominant at 65% while during the pseudo-nitzschia bloom in october, several species of pseudo-nitzschia proliferated but there were three dominating species (p. pseudodelicatissima, p. dolorosa and p. cuspidata) with lower values, reaching 20%, 18% and 17% respectively. discussion this study was conducted in the m’diq bay during 2007. the evolution of species composition of pseudo-nitzschia was studied in comparison with the local evolution of da detection in two shellfish species: tuberculate cockle (acanthocardia tuberculata) and sweet clam (challista chione). this study is the first for the mediterranean coastline of morocco. it has been shown that there is a continuous presence of pseudo-nitzschia spp. in m’diq bay, characterized by 5 proliferation periods (>20 cells ml–1) (march to november). the lower abundances were recorded during the rainy period (december – february). similar results were found by quijano-schegga et al. (2008) on the spanish mediterranean coast. according to loureiro et al. (2009), the pseudo-nitzschia abundance recorded in catalonia in 2007 was similar to that recorded in this study in autumn, while in april, their reported value of pseudo-nitzschia abundance is lower than in the present study. perhaps the pseudo-nitzschia bloom registered on the spanish coast during april could be conducted by ocean currents to the moroccan coast. 40 acta bot. croat. 72 (1), 2013 rijal leblad b., lundholm n., goux d., veron b., sagou r., et al. tab. 2. morphometric summary of pseudo-nitzschia species in m’diq bay. species name valve shape fibulae in 10 mm striae in 10 mm row of poroids poroids in 1 mm centrale nodule length (mm) width (mm) p. cuspidata lanceolate 20–24 34–41 1 5–6 + 58.5–65.3 1.6–2.3 p. subpacifuca linear 17–20 28–32 2(3) 9–10 + 45.2–60.1 4.9–6.1 p. arenysensis/ p. delicatissima lanceolate 20–24 36–38 2 9–12 + 39.5–45.1 1.8–2.1 p. fraudulenta linear 21–24 21–24 2(3) 6–7 + 65.4–70.1 4.3–5.2 p. multistriata lanceolate 26–30 38–40 2(3) 12 – 60.2–70.3 3.3–3.8 p. pseudodelicatissima linear 20–26 36–45 1 5–6 + 65.5–69.0 1.7–2.1 p. subfraudulenta linear 15–18 25–26 2 6–7 + 46.3–52.1 4.2–4.9 p. multiseries lanceolate 16–17 16–17 3(2–4) 6–7 – 80.0–85.2 2.8–3.2 p. calliantha linear 19–21 24–26 32–36 44–45 1 1 5–6 5–6 + + 58.2–68.1 1.7–1.9 p. dolorosa lanceolate 17–20 30–37 1(2) 6 + 73.0–90.3 2.2–2.8 p. galaxiae lanceolate 20–25 68–70 n.d. n.d. + 20.0–25.0 1.3–1.6 p. pungens var. aveirensis n.d. 16 16 2 4 – n.d. 2.9–3.1 p. cf. cacciantha lanceolate 18–21 33–34 1 5 + 72.0–75.0 2.6–2.7 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:07 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees in this study, the highest pseudo-nitzschia abundance and da concentration occurred in may and the end of september; both periods showing a transition between cold and warm seasons. environmental condition changes seem likely to have boosted pseudo-nitzschia development. it is possible that the m’diq bay area experienced an increase in nutrients during these periods. several studies suggested that pseudo-nitzschia blooms are associated with cool and high nutrient waters (trainer et al. 2000). pseudo-nitzschia abundance has a positive correlation with water temperature, phosphate and ammonium (ljube[i] et al. 2011). acta bot. croat. 72 (1), 2013 41 pseudo-nitzschia diversity in m’diq bay, morocco fig. 3. transmission electron micrographs of pseudo-nitzschia spp. from m’diq bay. a – p. galaxiae; b – p. multiseries; c – p. multistriata; d – p. arenysensis; e – p. subpacifica; f – p. cuspidata; g – p. pungens var. aveirensis; h, i – p. cf. cacciantha; j – p. fraudulenta; k – p. subfraudulenta; l, m – p. calliantha; n – p. dolorosa; o – p. pseudodelicatissima. 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:11 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees in spring bloom (88 cells ml–1), we recorded da presence in both the two studied shellfish species, showing a high value in sweet clam (4.9 mg da g–1) and low value in tuberculate cockle (2.11 mg da g–1). this variation of da contamination between these two shellfish species could be related to their different biological characteristics; it seems that their specific receptors for da could be different and have varying affinities for da. the same explanation was given by sagou et al. (2005) for contamination by psp toxins. during spring bloom, we observed four dominant pseudo-nitzschia species: p. arenysensis (65%) and p. cuspidata (19 %) as well as two species with lower abundance: p. fraudulenta (7%) and p. subpacifica (9%) (fig. 4). eight identified species (tab. 2, fig. 3) are considered producers of da (according to lundholm et al. 2011). domoic acid recorded in shellfish could be related to p. cuspidata and p. fraudulenta, as they are known to be da producers (rhodes et al. 1996, rhodes 1998, trainer et al. 2009, quijano-scheggia et al. 2010). for the other two species, p. subpacifica is not known to be a da producer while the case of p. arenysensis requires some clarification. pseudo-nitzschia arenysensis is quite similar to p. delicatissima; it is not possible to distinguish between them using microscopic observation, only by genetic analysis (quijano-scheggia et al. 2008). moreover, p. delicatissima is deemed to be toxic but p. arenysensis is not. thus, the 65% of spring bloom, apparently taken by p. arenysensis, could be also composed partially or totally of p. delicatissima, which could produce da and likely infect shellfish. 42 acta bot. croat. 72 (1), 2013 rijal leblad b., lundholm n., goux d., veron b., sagou r., et al. fig. 4. relative contributions of pseudo-nitzschia species in m’diq bay in the period january to december 2007. 571 rijal lebland et al.ps u:\acta botanica\acta-botan 1-13\571 rijal lebland et al.vp 14. o ujak 2013 10:36:14 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees in the autumn bloom, we recorded a pseudo-nitzschia abundance reaching 157 x 103 cells.l–1 and composed of 8 pseudo-nitzschia species, such as: p. pseudodelicatissima (20%), p. dolorosa (18%), p. cuspidata (17%), p. subpacifica (13%), p. arenysensis (10%), p. fraudulenta (9%), p. calliantha (6%) and p. multistriata (3%). among these species, p. dolorosa has never been recognized as a da producer while p. pseudodelicatissima (martin et al. 1990, pan et al. 2001, amzil et al. 2001), p. multistriata (rhodes et al. 2000, sarno and dahlmann 2000, amato et al. 2010, quijano-scheggia et al. 2010) and p. calliantha (besiktepe et al. 2008, alvarez et al. 2009, quijano-scheggia et al. 2010) are already known to be da producers. even if pseudo-nitzschia bloom in autumn was the highest, da concentration in both the two studied shellfish species was low and lower than that recorded in spring bloom: 1.57 mg da g–1 in sweet clam and 0.75 mg da g–1 in tuberculate cockle. this difference of contamination between spring and autumn could be explained by both the difference in environmental conditions during spring and autumn periods and the differences in pseudo-nitzschia species composing blooms. the production of da is dependent on the concentration of nutrients (pan et al. 1996, klein et al. 2010). the da detected in tuberculate cockle and sweet clam was due to the presence of some toxic species such as p. multistriata, p. cuspidata, p. galaxiae, p. multiseries, p. pseudodelicatissima, p. pungens var. aveirensis, p. calliantha and p. fraudulenta. in the present study, the da level measured in spring and autumn blooms did not exceed the normative threshold of 20 mg da g–1 of shellfish meat. in the literature, several blooms of pseudo-nitzschia resulting in shellfish contamination by da were studied. taking into account the available literature on the mediterranean sea, the results of the present study, particularly in terms of shellfish da concentrations, are not sufficient to suggest there is a danger to public health. during 2007, a large diversity of pseudo-nitzschia species was observed in m’diq bay. some of them have already been identified in mediterranean waters while others are described for the first time: p. cuspidata, p. multiseries, p. subpacifica and p. subfraudulenta. conclusion the present study has shown that there is a seasonal succession of thirteen species of pseudo-nitzschia all the year round. some of them have already been identified in mediterranean waters while others are described for the first time: p. cuspidata, p. multiseries, p. subpacifica and p. subfraudulenta. the highest abundance of pseudo-nitzschia species was recorded in spring and autumn. some of them are known to be producers of a large quantity of da. during spring and autumn seasons, da concentration in the two shellfish species studied, tuberculate cockle (acanthocardia tuberculata) and sweet clam (callista cheone), was higher in the latter than in the former. in m’diq bay, these two seasons have to be considered a potentially dangerous period for asp events. however, much more work needs undertaking for the mechanisms of pseudo-nitzschia species development in relationship with local environmental conditions to be understood. this study is in fact the first attempt at an assessment of pseudo-nitzschia species succession and domoic acid production on the moroccan 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jaime f. garcía1*, enrique jurado2 1 the autonomous university of nuevo león, school of agronomy, campus la ascension, km 3.5 sandia el grande, 67950 sandia el grande aramberri, nuevo león, méxico 2 the autonomous university of nuevo león, school of forest sciences, km 145, 67700 linares, nuevo león, méxico abstract – mortality in six plant species was examined in the vegetation of a mountain region in northeastern mexico and hypotheses of survival pathways within populations in the ecosystem were tested. signifi cant differences in the general mortality pattern were found among species indicating species-specifi c responses to stress gradients. average mortality differed among species: yucca carnerosana, 33.8%; pinus cembroides, 29.9%; larrea tridentata, 25.9%; hechtia podantha, 13.7%; agave lechuguilla, 13.0%; and thelocactus santaclarensis, 9.0%. within populations, mortality increased with water stress and survivorship increased with less stressful environments. results from this study may be useful for the development of a management plan to support the conservation and sustainable use of forest vegetation in this mountain community. keywords: agave lechuguilla, climate change, hechtia podantha, larrea tridentata, pinus cembroides, thelocactus santaclarensis, water stress, yucca carnerosana introduction climate change has been linked to drastic vegetation shifts (vitousek et al. 1997, hughes 2000, menzel et al. 2006), which could alter plant (mclachlan et al. 2005) and animal (peterson et al. 2002) distributions. projected changes include increased frequency and severity of droughts (ipcc 2001). this in turn could have numerous consequences for ecosystems (wright 1992, corlett and lafrankie 1998, curran et al. 1999, harrison 2000), because severe or frequent droughts may cause drastic changes in the vegetation, mainly in arid and semi-arid ecosystems (allen and breshears 1998, hanson and weltzin 2000). the research required to provide a better understanding of the effect of drought is necessarily complex (van nieuwstadt et al. 2005). plants in northeastern méxico are adapted to regularly occurring droughts and inter-annual variation in rainfall (garcía * corresponding author, e-mail: jaimefgarcia@hotmail.com copyright® 2015 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. garcía j. f., jurado e. 96 acta bot. croat. 74 (1), 2015 2011). for the mountain vegetation of northeastern méxico there are no studies examining the effects of drought on plant survival, and little is known about what factors affect the probability of mortality in woody plants during a severe drought. furthermore, it is unknown how the vegetation of the mountains in northeastern mexico will respond to climate change. mortality rates of species may be decisive for future predictions of climate change effects. the aim of this research was to examine the factors associated with plant mortality and pathways to survival (plant size, nursery, competition, soil type, shade, understory plants and nurse stones). nursery, shade and understory vegetation are mechanisms of plant -plant facilitation and promote plant establishment. two major hypotheses were tested: (1) mortality among species differs due to species-specifi c responses and to genetic variability in resistance to stressors, and (2) between populations, plant survival varies throughout the ecosystem due to environments that are more or less stressful. determining the effect of drought on mortality and survival of the species studied across the region will contribute to predicting future vegetation distributions on regional scales and incorporate climate change predictions into conservation efforts. materials and methods study site and species high mortality of trees of an entire ecosystem subjected to the adverse effects of drought can be exacerbated by climate change, but at the same time provides an opportunity to test hypotheses on present and future plant composition. to examine drought effects on plant species in this mountain community, a study was conducted in ascension aramberri nuevo leon mexico (24°20'14"n, 99°56'35.9"w, at 2190 m a.s.l.). the climate is cool, medium dry with an annual rainfall of 423 mm. rain falls mainly in late summer and early autumn. mean annual temperature is 14.9 °c, with a maximum of 22.8 °c in summer and a minimum of –3 °c in winter. examination of patterns of mortality and survival of plants was conducted between summer 2007 and the winter of 2012. the most frequent and abundant plant species distributed in the area were chosen: (1) pinus cembroides zucc. (pinaceae), which in the mountain region represents a dominant plant that provides habitat and ecosystem structure; (2) larrea tridentata (moç. & seseé ex dc.) coville (zygophyllaceae), dominant in the semiarid zone, and four species distributed in both ecosystems; (3) yucca carnerosana (trel.) mckelvey (agavaceae); (4) hechtia podantha mez (bromeliaceae); (5) agave lechuguilla torr. (agavaceae), and (6) thelocactus santaclarensis halda, kupcák & sladk. (cactaceae). the seeds of the fi rst species are collected for human consumption and species 3 and 5 are used for the extraction of natural fi ber. mortality among species to test for differences in mortality levels among species due to drought effects, in the fall of 2008 a minimum of 40 plots for each species were established. for all species eight different locations were selected in areas where the species are widely distributed; in each site, fi ve plots were established. plot size varied between species: (i) for yucca carnerosana 50 × 50 m; (ii) 40 × 40 m for pinus cembroides; (iii) 20 × 20 m for larrea tridentata, hechtia podantha and agave lechuguilla; and (iv) for thelocactus santaclarensis plots were of 10 × 10 m. the 240 plots were within a radius of 25 km. each plant was classifi ed as living or dead, with the assumption that a lack of living structures was evidence of mortality. drought and plant populations acta bot. croat. 74 (1), 2015 97 survival pathways within populations to determine survival pathways, mortality events and factors associated with increased survival such as: plant size, nursery, competition, soil type, shade, understory plants and nurse stones, were documented. according to franco et al. (1994), l. tridentata extracts ground water near the surface and small shrubs recover faster from drought than large shrubs. to test whether smaller plants were more resistant to drought, in spring 2010, in four randomly selected chaparral systems, mortality was estimated along six 50-m transects and plants were classifi ed each as living or dead and plant size for l. tridentata ≤ 1.0 and > 1.0 m was recorded. to test whether mortality of y. carnerosana is decreased if it grows in association with p. cembroides, mortality rates of y. carnerosana in an area visually dominated by p. cembroides were contrasted with those in an adjacent area nearly devoid of p. cembroides. in the spring of 2009 we chose twelve sites that included both p. cembroides sites and sites without p. cembroides. in each site we sampled along two 200 m by 50 m belt transects across the study area. plants were classifi ed as living or dead and whether they occurred: (i) with p. cembroides or (ii) not. to determine whether soil type had an effect on mortality and survival of h. podantha, three soil types were chosen: lithosols, soil with small trees; rendzina, soil with high organic matter associated with p. cembroides, and griese (gypsisol) without trees. in summer of 2011 ten random (20 × 20 m) plots were established, on each soil type in three communities. every h. podantha within a plot was classifi ed as living or dead, and mortality levels between the three soils were compared. to determine if a. lechuguilla grows more often under other plants, and whether shade is an important factor determining survival, in the fall of 2009 twenty plots of 20 × 20 m were established in areas where the studied species was abundant. each a. lechuguilla was recorded as growing: (i) in direct sunlight or (ii) under the canopy of another plant, and each individual in the plots was classifi ed as living or dead. to test the hypothesis that seedlings of p. cembroides growing in association with understory vegetation had a higher survival rate, in the winter of 2008, (a year with high seed production) four locations were selected in each site. seedlings of p. cembroides were located along twenty 100-m transects and classifi ed as growing in association with: (i) understory vegetation (forbs, grasses, shrubs and cacti, tab. 1), (ii) under the p. cembroides canopy, and (iii) on cleared soil, devoid of vegetation. seedling survival was monitored each season recording live and dead individuals in each environment until the winter of 2011. the percentage of seedling survival based on the number of live plants/total seedling × 100 was calculated. the nurse association may be biotic, as with nurse-plants, or abiotic, as with rocks, where there are some reports of cacti growing preferentially near rocks, and hence the rocks provide a microhabitat that favors seedling establishment and survival (parker 1987). rocks act as moisture-collectors and delay evaporation by shading (reyes-olivas et al. 2002). to test whether t. santaclarensis populations increased with distance to small semi-buried stones, or those growing at least 0.5 cm from a small stone fi xed to the ground, in the summer 2007 seven localities were chosen with similar elevations and slopes, and four 100 m-long transects 2 m wide were randomly placed. recruitment levels of t. santaclarensis with three levels of stoniness (low ≥ 25%; medium < 25 and ≤ 50% and high > 50%) were correlated. garcía j. f., jurado e. 98 acta bot. croat. 74 (1), 2015 tab. 1. taxonomical list of seedlings of understory vegetation found in the summers between 2009– 2011; seedlings were within a 20-cm radius of seedlings of pinus cembroides. family species 2009 2010 2011 acanthaceae justicia runyonii small 76 81 52 ruellia rudifl ora (gray) urban. 14 89 21 stenandrium dulce (cav.) nees 43 112 25 agavaceae agave lechuguilla torr. 37 61 55 agave striata zucc. 34 42 36 yucca carnerosana (trel.) mckelvey 17 22 19 yucca fi lifera chabaud 10 18 15 amaranthaceae amaranthus sp. 21 98 37 asteraceae aster subulatus michx. 55 143 66 helianthus annus l. 47 95 0 sanvitalia ocymoides dc. 109 222 56 verbesina encelioides (cav.) gray. 55 93 16 viguiera stenoloba blake. 45 211 55 boraginaceae heliotropium angiospermum murray 72 109 66 heliotropium currassavicum l. 23 69 17 cactaceae opuntia engelmannii salm-dyck ex engelm. 7 11 9 opuntia leptocaulis dc. 21 33 33 opuntia tunicata lehmann 10 13 12 thelocactus santaclarensis halda, kupcák & sladk. 56 57 56 chenopodiaceae chenopodium album l. 27 76 17 suaeda torreyana s. watson 74 145 62 convulvulaceae convolvulus arvensis l. 26 92 76 evolvulus alsinoides l. 18 54 21 euphorbiaceae croton torreyanus muelli. arg. 32 44 41 euphorbia dentata michx. 45 56 49 tragia ramosa torr. 29 45 31 fabaceae acacia greggii wrightii (benth.) isely 9 11 10 canavalia villosa benth. 17 20 18 desmanthus virgatus (l.) will 7 8 7 drought and plant populations acta bot. croat. 74 (1), 2015 99 to test whether microhabitat had an effect on t. santaclarensis survival, three experiments were conducted: (1) in early autumn of 2007 four sites were chosen; at each site, ten plots of 10 × 10 m were established. percentage of seedling survival was recorded, on three contrasting microhabitats where this species is distributed: (i) on ground with few stones, (ii) on bare soil, and (iii) under a dense canopy. in each environment (2) soil moisture and (3) dew were determined; the gravimetric method was used to determine moisture, and dew was determined with the method used by reyes-olivas et al. (2002), collecting water on pairs of 95 cm2 disks of whatman fi lter paper no. 1 placed on polyethylene plates; 10 plates were placed in bare soil and beneath the canopy and 10 at stony sites. disks were opened on one occasion in december 26, 2008, from 7.00 pm to 7.00 am. seedling survivors were monitored each month in each plot until the winter of 2010; each individual of t. santaclarensis in the plots was classifi ed as living or dead. to avoid double counting of new seedlings; seedlings were referenced on sketches in the winter of 2008 for p. cembroides and early autumn of 2007 for t. santaclarensis. family species 2009 2010 2011 lamiaceae salvia coccinea juss. 27 31 30 malvaceae abutilon sp. 47 61 41 hibiscus cardiophyllus gray. 12 34 29 sida neomexicana gray. 31 35 29 papaveraceae argemone echinata ownb. 66 79 74 poaceae aristida sp. 28 31 29 botriochloa sp. 42 45 33 botriochloa sp.2 28 38 36 bouteloua trifi da thurb 19 21 32 cenchrus incertus m.a. curtis 99 145 139 chloris submutica h.b.k. 30 29 32 pennisetum ciliare (l.) link. 45 138 131 tridens sp. 98 119 121 rhynchelitrum repens (willd) c.e. hubb. 79 99 84 solanaceae physalis pubescens l. 78 99 92 solanum rostratum dunn. 40 48 31 urticaceae urtica dioica l. 34 33 24 verbenaceae lantana velutina mart. & gal. 29 38 27 tab. 1. – continued garcía j. f., jurado e. 100 acta bot. croat. 74 (1), 2015 statistical analyses to contrast mortality levels among species, average percent mortality was compared using anova (α = 0.05). all data were arcsine transformed to conform to a normal distribution assumption, previous to anova (sokal and rohlf, 1995). tukey multiple means of comparisons were used when the anova indicated signifi cant differences. ninety-fi ve percent confi dence intervals were used in graphics to highlight differences for each species. to determine whether l. tridentata mortality occurred more often than expected by chance, mortality levels of l. tridentata ≥1.0 m and < 1.0 m were compared with goodness-of-fi t test for poisson χ2-test. wilcoxon test for paired samples was conducted to determine mortality and survival levels of y. carnerosana in and out of p. cembroides areas. the kruskalwallis test for comparison of k independent samples was used to determine mortality and survival between soil types for h. podantha. a t-test for paired means of two samples was used to determine whether individuals occurred under other plants more often than expected by chance, and to determine if a. lechuguilla grew more often under other plants. all data were log transformed to conform to the normal distribution assumption, previous to ttest. to test the hypothesis that seedlings of p. cembroides, in association with understory vegetation, had increased survival rates, friedman test was carried out. to test whether t. santaclarensis populations increased with distance to small stones, recruitment levels of t. santaclarensis with the percentage of t. santaclarensis associated to small stones were correlated. a regression analysis and an anova (α = 0.05) were carried out to test if the slope b1 differed from 0. anova (α = 0.05) was used to determine the effect of microhabitat on seedling survivors of t. santaclarensis; previous to anova (α = 0.05) data were transformed to conform to a normal distribution assumption. differences in soil moisture and dew were determined with friedman test. results plant mortality between species the effect of drought on plant mortality signifi cantly differed among species (f = 6.82, d. f. = 239, p < 0.001; fig. 1). y. carnerosana, p. cembroides and l. tridentata had greater mortality (33.8.0%, 29.9% and 25.9%), than h. podantha (13.7%), a. lechuguilla (13.0%), 0 25 50 75 100 m o rt a li ty (% ) species t. santaclarensis a. lechuguilla h. podantha l. tridentata * p. cembroides * y. carnerosana * fig. 1. mortality between species. * indicates signifi cant differences detected with tukey’s test. error bars represent the confi dence intervals (α = 0.05). drought and plant populations acta bot. croat. 74 (1), 2015 101 and t. santaclarensis (9.03%). percentage of mortality by species exhibited signifi cant variability, indicating that the effects of drought although widespread, differed among species. survival pathways within populations a total of 1324 l. tridentata plants were recorded, from which 105 plants > 1.0 m and 33 plants ≤ 1.0 m died, giving 7.93 and 2.49% mortality levels, respectively. plants > 1.0 m died more often than expected by chance (χ2 = 945, d. f. = 1, p < 0.0001, fig. 2a). the higher survival of plants ≤ 1.0 m, may be the result of a morphological modifi cation to provide protection from drought. a total of 1950 plants of y. carnerosana were recorded on the plots and 403 of them died (20.6 % mortality). plants had higher survival rates when growing with p. cembroides (approximately 70.2%); on bare soil areas only 29.7% survived (z = 6.48, p < 0.001; fig. 2b). in the association y. carnerosana-p. cembroides the mortality gradient of y. carnerosana may be sharply lower than that present on bare soil, so the increased facilitation by association with p. cembroides seems to be related to the provision of moisture. there were a total of 3158 h. podantha plants, of which 2498 survived, providing a 20.9% mortality. soils devoid of trees (lithosols and gypsisol) were signifi cantly associated with mortality (χ2 = 12.06, d. f. = 2, p < 0.0002; fig. 2c). in mountain vegetation when suitable microsites are common, low mortality of h. podantha on rendzina soil associated to p. cembroides indicates that favorable microsites increase survival. a positive interaction among p. cembroides and h. podantha may ameliorate stress caused by drought. 0 5 10 15 20 > 1.0 m ≤ 1.0 m o rt a li ty ( % ) χ2 = 945.06, p < 0.0001a 0 5 10 15 20 m o rt a li ty ( % ) non p. cembroides p. cembroides b z = 6.48, p < 0.001 0 5 10 15 20 lithosols gypsisol rendzina m o rt a ll it y ( % ) χ2 = 12.06, p < 0.0002 c 0 5 10 15 direct sunlight shade m o rt a li ty ( % ) t = 7.29, p < 0.0001 d 20 fig. 2. plant mortality is a function of: (a) plant size > 1.0 m for larrea tridentata; (b) non-association with pinus cembroides for yucca carnerosana; (c) soil type (lithosols and gypsisol) for hechtia podantha and (d) direct sunlight for agave lechuguilla. garcía j. f., jurado e. 102 acta bot. croat. 74 (1), 2015 a total of 1427 plants of a. lechuguilla were detected in shade and under direct sunlight from which 293 were recorded as dead (20.52% mortality). shaded plants showed low mortality (5.81%), whereas those exposed to direct sunlight showed high mortality (14.71%; t = 7.29, d. f. = 19, p < 0.0001; fig. 2d). survival of p. cembroides differed according to the environmental conditions (x2r = 12.31, d. f. = 2, p < 0.05; fig. 3a). it was higher for seedlings in understory vegetation (36.1%), lower under canopy (19.5%) and lowest on bare soil (8.8%). in this environment stress due mainly to drought might have caused the death of most of the seedlings. results indicate that survival was higher under nurse plants that might provide a shield against stressful environmental conditions. 0 20 40 60 80 100 3 9 7 8 3 3 9 8 7 3 3 9 9 6 3 4 0 0 5 3 4 0 1 4 3 4 0 2 3 3 4 0 3 2 3 4 0 4 1 3 4 0 5 0 3 4 0 5 9 3 4 0 6 8 3 4 0 7 7 3 4 0 8 6 3 s u rv iv a l (% ) months and years a understory vegetation p. cembroides bare soil 1 2 -0 8 0 3 -0 9 0 5 -0 9 0 8 -0 9 1 1 -0 9 0 2 -1 0 0 5 -1 0 0 8 -1 0 1 1 -1 0 0 2 -1 1 0 5 -1 1 0 8 -1 1 1 1 -1 1 r² = 0.8525 0 50 100 150 200 0 25 50 75 100 n u m b e r o f t . s an ta cl ar en si s small stones (%) b 0 20 40 60 80 100 s u rv iv a l (% ) months and years c stony soil dense canopy bare soil 0 6 -0 7 1 0 -0 7 0 2 0 8 0 6 0 8 1 0 0 8 0 2 -0 9 0 6 -0 9 1 0 -0 9 0 2 -1 0 0 6 -1 0 0 9 -1 0 fig. 3. plant recruitment and seedling survival as a function of understory vegetation for pinus cembroides (a) increased % of small stones (b) and association with stony soil for thelocactus santaclarensis (c). drought and plant populations acta bot. croat. 74 (1), 2015 103 recruitment of t. santaclarensis was positively correlated with small stones (f = 113.07, p < 0.001, r2 = 0.8525; fig. 3b). a total to 3167 individuals of t. santaclarensis were recorded on transects where there were a greater number of seedlings. there were a total of 1214 t. santaclarensis plants: 566 on stony soils, 390 under the canopy of p. cembroides and 258 on bare soil respectively. survival differed among habitats, with higher survival of t. santaclarensis (f = 49.63, d. f. = 15, p < 0.001, fig. 3c) on stony soil than under dense closed canopy and on bare soil. low moisture in the soil might have caused death of most of the seedlings. on stony soil, positive interactions between rocks and t. santaclarensis infl uenced seedling survival during consecutive years (fig. 3c). soil moisture differed according to the microenvironment (x2r = 7.45, d. f. = 2, p < 0.05) (stony soil 14.13 ± 1.11%; border 10.55 ± 0.97% and under a dense canopy 8.31 ± 1.45%). dew collected differed between microenvironments (x2r = 9.31, d. f. = 2, p < 0.05) with 1.41 ± 0.22 g collected on stony soil; 1.39 ± 0.30 g on bare soil and 0.71 ± 0.11 g under dense closed canopy. discussion mortality among species except for y. carnerosana mortality was lower for species growing in both ecosystems (h. podantha, a. lechuguilla and t. santaclarensis), with dominant species and y. carnerosana showing localized patterns of very high mortality (23 to 81%). water stress may cause changes in plant community composition and structure (shilo-volin et al. 2005, weigelt et al. 2005), and in some cases, they may also reverse the competitive hierarchies of plant species (fynn et al. 2005). drought effects caused widespread high mortality among species (9.03 to 33.8%, fig. 1). shrubs and tree species (y. carnerosana; p. cembroides and l. tridentata, fig. 1) were more susceptible to drought due to the inherent differences among species, to species-specifi c responses and to genetic variability in resistance to stressors. changes in patterns of mortality may also modify plant populations in this mountain community, and affect the entire ecosystem, which could have major impacts on local diversity (gitlin et al. 2006). survival pathways within populations l. tridentata plants ≤ 1.0 m were more resistant to drought effects (fig. 2a). low moisture and water limitation due to stressful conditions limited survival of plants > 1.0 m, because of size differences among plants (both above and belowground). plant response may vary according to microenvironmental differences (frazer and davis 1988). in the arid and semi-arid regions where the species is distributed, soil wetting does not penetrate to a depth of 0.6 m (franco et al. 1994). therefore, small plant roots may be exposed to greater soil water availability. previous studies have determined that adult trees are more sensitive than juveniles, with more positive responses to precipitation, and more negative responses to temperature (suarez et al. 2004). mortality was higher in y. carnerosana growing outside p. cembroides vegetation (fig. 2b), and this positive interaction between p. cembroides and y. carnerosana, suggests positive survival effects through increased water or nutrient availability (holzapfel and mahall 1999) inducing an increase in seedling survival. garcía j. f., jurado e. 104 acta bot. croat. 74 (1), 2015 mortality of h. podantha was higher on lithosol and gypsisol soils (fig. 2c), and survival was higher in rendzina, perhaps because this soil is rich in humus and soil fertility (joergensen 1991). death of a. lechuguilla plants was attributed to stress on bare soil (fig. 2d). shade provided by p. cembroides improved survival of a. lechuguilla. seedling survival of a. lechuguilla has been shown to be higher under reduced direct solar radiation (franco and nobel 1989), and lower soil temperatures (flores-martínez et al. 2004). shade from nurse plants reduces thermal amplitudes and decreases soil water evaporation (domingo et al. 1999), and also reduces thermal stress and transpiration of understory plants, thereby protecting them from photo-inhibition (nobel 1980, vetaas 1992, moro et al. 1997). this in turn may further increase plant survival. semchenko et al. (2012) determined a positive effect of shade on plant growth by amelioration of stress or active regulation of growth rate. moreover, high light intensity may promote seedling survival through fungal pathogen suppression (augspurger 1983).these fi ndings indicate stage-specifi c patterns of plant survival in environments that experience severe drought. however higher frequency of severe drought may increase plant mortality, which may occur in rapid pulses rather than gradual declines (gitlin et al. 2006). hence high plant mortality may inhibit the ability of local populations to recover and expand into more hospitable environments (hewitt and kellman 2004). understory plants increased the survival of p. cembroides (tab. 1; fig. 3a), where nurse plants ameliorated environmental conditions that affected seedling survival. previous studies have shown that the presence of vegetation may protect establishing seedlings against high radiation, high temperatures and losses of soil moisture, thereby increasing survival (callaway 1995, castro et al. 2002, gómez-aparicio et al. 2004). recruitment of t. santacl arensis was positively correlated with small stones (fig. 3b) due to the plants’ ability to develop root systems close to rocks (nobel et al. 1992; nobel and zutta 2005). a positive interaction between t. santaclarensis and small stones promoted survival (fig 3b), where small stones act as collectors of dew (larmuth and harvey 1978). higher soil moisture and dew under stones than under p. cembroides and on bare soil suggests that water availability (fig 3c) is key for survival. in dry habitats such as those found in this mountain vegetation, drought is the major constraint on seedling survival and competition for water is often more important than competition for light or nutrients (casper and jackson 1997). mortality patterns were a function of plant size > 1.0 m for l. tridentata; non-association with p. cembroides for y. carnerosana; soil type for h. podantha and direct sunlight for a. lechuguilla (figs. 2a–d). survival within populations increased with size ≤ 1.0 m for l. tridentata; association with p. cembroides for y. carnerosana; rendzina soil type for h. podantha and shade for a. lechuguilla (figs. 2a–d). the high variation in mortality within populations shows high-stress sites and might be utilized to investigate the role of sites of increased stressor conditions for seedling survival. the overall patterns of survivorship found are consistent with the hypothesis that greater survival is associated with less stressful environments. results from this study show that seedlings of p. cembroides died on bare soil and under p. cembroides, with understory plants having a higher survival (tab. 1; fig. 3a). when abiotic stress is high due to drought exacerbated by climate change, positive interaction by facilitation is important (bertness and callaway 1994, callaway et al. 2002) to mitigate those negative effects. drought and plant populations acta bot. croat. 74 (1), 2015 105 a positive interaction between small stones and t. santaclarensis increased survival (figs. 3b–c). stony soil favors plant survival due to its capacity to retain moisture (fig. 3b), while under dense closed canopy and in bare soil habitats (fig. 3c) mortality is attributed to lack of moisture. it has been suggested that rocks may act as good moisture-collectors (reyes-olivas et al. 2002). while facilitation of recruitment by ‘nurse’ plants has been widely documented (flores and jurado 2003), nurse rocks have been poorly documented. however research does report positive interactions between cacti and rocks (parker 1987, 1989, nobel et al. 1992), where rocks ameliorate stressor conditions; this provides a fresh and moist environment without reducing sunlight (peters et al. 2008). furthermore small rocks protect seedlings from freezing temperatures (nobel 1980). implications for management the effects of drought on plants can affect associated community members across trophic levels (gitlin et al. 2006, johnson et al. 2011) and can be expected to affect entire ecosystems (wimp et al. 2004, bangert et al. 2005). changes in above-ground–belowground linkages in response to drought may infl uence plant communities in the future (johnson et al. 2011) and have evolutionary consequences (grant and grant 1993, 2002, nevo 2001). in the context of climate change, ecosystem managers should consider the promotion of plant recruitment through intensifi cation of activates such as soil conservation. high stress sites could be used to determine implications on changes in plant diversity and abundance. the ecosystem of the mountains of this region could be especially sensitive to climate change (risser 1995, hanson and weltzin 2000), due to high tolerance to low water levels, and may act as barometers of change currently going on in other ecosystems (brown et al. 2001, gitlin et al. 2006). acknowledgments jfg would like to thank students of the 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^arni1,3, aleksander marin[ek1, urban [ilc1 1 jovan had`i institute of biology, research centre of the slovenian academy of sciences and arts, novi trg 2, si-1001 ljubljana, slovenia 2 university of primorska, up famnit, glagolja{ka 8, si-6000 koper, slovenia 3 university of nova gorica, nova gorica, slovenia abstract – ecological gradients along river banks of the mura river influence forest species composition. on the basis of 58 relevés of floodplain forests along the mura river, the classification of vegetation plots was performed with the pc-ord program. the diagnostic species combination for three clusters revealed after classification was calculated by fidelity measure (phi-coefficient) and presented in an analytic table. average ellenberg inidicator values, stream distances and relative elevations of the relevés were passively projected onto pca to show ecological relationships among them. correlations of plant functional type and stream distance gradients were calculated with regression analysis. for the main edifiers response curves to the stream distance gradients were made. the classification of the mura floodplain forests has revealed three ecologically interpretable vegetation types: salicetum albae (most humid and nutrient-rich sites), fraxino-ulmetum allietosum ursini (ecologically intermediate sites), f.-u. quercetosum robori (the driest and the least nutrient-rich sites). zonation of vegetation is connected to distance from the closest stream which influences species distribution through ecological gradients of moisture and nutrient. the proportion of therophytes is significantly negatively correlated with the distance from the closest stream and the proportion of neophytes is significantly negatively correlated with distance from the main stream. key words: floodplain, gradient, hardwood, neophytes, riparian, vegetation, softwood, zonation, mura river abbreviations: dcs – distance from the closest stream, dms – distance from the main stream. introduction floodplain forests are subject to periodic over/the-bank flooding and cycles of erosion and deposition of nutrient-rich sediment. well drained and nutrient-rich soils of floodplain acta bot. croat. 72 (1), 2013 71 * corresponding author, e-mail: petrako@zrc-sazu.si copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:14 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees forests distinguish these forests from the floodplain swamp forests on stagnant water sites with gleyic soils, which are periodically flooded predominately by ground water. floodplain forests are characterized by the highest species richness and diversity, density and productivity (deiller et al. 2001, mitsch and gosselink 2000, schnitzler 1994). they include several microhabitats harbouring numerous plant and invertebrate species unable to survive elsewhere (machar 2001). therefore floodplain forests, according to the habitat directive, belong among the habitats of the greatest importance for nature protection on the european scale. historically, natural floodplain forests have been fragmented and heavily impacted by watercourse regulations, timber harvesting and other human activities (klimo and hager 2001). like other large european rivers, the mura has undergone several hydraulic management phases since the 19th century. these regulation works and other human impacts, such as plantation and felling, have modified the floodplain forest ecosystem. therefore identification of vegetation types along the mura river is very difficult, undistinct and has not been done yet. despite many an intervention into the river and its current, the mura has still preserved so many natural treasures that it is treated as a nationally important value (gor[ak and bakan 2003). flooded forests have already been thoroughly studied in many parts of europe (müller 1992, schnitzler 1994, vicherek et al. 2000, paal et al. 2007, willner and grabherr 2007, kevey 2008, klimo et al. 2008, poldini et al. 2011) and also in the southeastern part of europe in the area of large rivers, such as the sava, drava and danube (horvat et al. 1974, vukeli] and bari^evi] 2004, 2005). two types of forests have usually been identified in the area of floodplain forests: softwood and hardwood forests. softwood forests are mainly composed of early successional and light-demanding species such as salix alba, salix fragilis, populus nigra and populus alba. hardwood forests are dominated by long-living species, with fraxinus excelsior/fraxinus angustifolia, quercus robur and ulmus minor/ulmus laevis. alluvial hardwood forests are found on flooded but well-drained and fertile soils of large european floodplain (schnitzler 1994) in slovenia there were some surveys of vegetation of river banks, about all white willow stands salicetum albae, along some other rivers, such as the drava (petrinec 1999), so~a (dakskobler et al. 2004), krka and mirna ([ilc 2003), mostly with a relatively narrow belt of riverine forest vegetation. the mura river has the best preserved floodplain in slovenia, but floodplain forests of river banks of the mura river remain so far unresearched. the main research questions of the study were: – which are the main forest vegetation types on the banks of the mura river and the main ecological gradients that influence species composition of these forests? – despite the big changes in flooding regime and human impact (plantation, cutting down) does zonation of vegetation along the mura river still exist? is zonation connected to distance from the main stream of the river (dms) or distance from the closest stream (dcs)? – what is the distribution of plant functional types (life forms, plant ecological strategies, origin and residence time status) along stream distance gradient? 72 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:14 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees material and methods study area the study took place along the mura river in the eastern part of slovenia, on the banks of the mura river and holocene terraces. this part of slovenia has a continental climate: the annual mean precipitation is 800 mm and annual mean temperature is 9.2 °c. the study area belongs to the sub-pannonian region, as we can find some pannonian features; flooded and swamp forests, some very rare pannonian elements. since the area is composed mainly of sandstone, the flora shows a mainly central european floristic character. the mura (mur) river is a central european river, springing at 1898 m a.s.l. in the eastern alps in austria. the mura river is the largest tributary of the drava river, that is the fourth largest tributary of the danube river. when reaching slovenia, the mura river forms a 34-km long state border line between austria and slovenia, flows through slovenian territory for 29 km and then becomes a state border again: first between slovenia and croatia (33 km), and then between croatia and hungary (globevnik and miko[ 2009). in slovenia it has riverbed in lowland with a small gradient. the level of the mura river increases in spring because of the melting snow in the high mountains and in autumn because of higher precipitation. regulation works on the mura river started in 19th century. before regulation, 40% of water flowed through main channel, but today almost all the water flows in the main channel. there are still some side channels in the forests along the mura river with connection to the main stream of the mura. flood protection dikes were constructed in the 1972–1990 period using a 100 year flood design to protect cultivated areas. the mura river corridor between the dikes is up to 1 km wide (globevnik and miko[ 2009). most of the forests range inside these dikes. sampling and measurements vegetation of floodplain forests was investigated according to the standard central european method (braun-blanquet 1964). we made 58 relevés of floodplain forests along the mura river. all the relevés were made inside levees in the area of periodic floods. we did not include floodplain swamp forests (class alnetea glutinosae) in our research as they have already been investigated (accetto 1994, nemesszeghy 1986, glava] 1975). for each plot, gps coordinates were collected and later on, distances from main stream of mura (dms) and distances from the closest stream (dcs), which may be the main or a side stream, were measured from the map in arc-gis program. in 14% of relevés (8 of 58) dcs value equalled dms value. this means that in 86% of the relevés the closest stream was a side stream. we also measured the relative elevation of each plot (elevation relative to average river level) with dmr of the area as it is known that elevation as well as distance from the river affects hydroperiod (flood frequency and duration) and thus influences species distribution and abundance (turner et al. 2004). data analysis 58 relevés made on flooded sites of the mura river were entered into the turboveg (hennekens and schaminée 2001) database. acta bot. croat. 72 (1), 2013 73 floodplain forests along the mura river 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees the numerical classification of vegetation plots, based on their species composition, was performed with pc-ord 5.0 program (mccune and mefford 2005). the data was square root transformed. euclidean (pythagorean) distance as the distance measure and flexible beta method for group linkage were used. we made several classifications with different numbers of clusters of relevés. we subjectively accepted classification with three clusters being most ecologically sound. 58 relevés are presented in the analytic table. the nomenclature of taxa is according to flora europaea (tutin et al. 1964–1980). diagnostic species for the clusters (corresponding associations and subassociations) were determined in the juice program (tichý 2002) by their fidelity values. fidelity was calculated using the phi coefficient, applied to the classified data set with cluster sizes equalized to 1/3 or 1/2 respectively (in the case of searching for species diagnostic for two clusters together) of the size of the entire data set, of the total data set size according to tichý and chytrý (2006). species with phi > 30 were considered as diagnostic for individual clusters, but species whose occurrence concentration in the plots of a particular cluster was not significant at p < 0.05 (fisher’s exact test) were excluded. unweighted average ellenberg indicator values of relevés made on flooded sites, distances of the relevés from the streams (dms, dcs) and relative elevation were passively projected onto a principal components analysis biplot (pca from canoco 4.5; ter braak and [milauer 2002) to show ecological relationship among these relevés and to explain environmental gradients underlying the main ordination axes. square root transformed cover data were used as the input data. for further interpretation of the three clusters of flooded forests of the mura river, unweighted average ellenberg indicator values of 3 clusters of flooded forests, moisture and nutrient, which indicate the biggest correlations with axis 1 of the pc-ord diagram, calculated in the juice program, and stream distances (dms, dcs) were presented with box-whiskers diagrams made in statistica program (statsoft 2007). boxes present mean and standard errors (se), whiskers indicate standard deviations (sd). correlations of plant ecological strategies, life forms and origin and residence time status (native, archaeophyte, neophyte) and stream distance gradient were calculated with regression analysis, also in statistica program. each of the patterns was fitted with a linear regression model. the plant ecological strategies, life forms and plant status have been defined using the biolflor database (klotz et al. 2002), that considers grime’s classification (grime 1979). for the five most important tree species of the forests in the area, responses to the environmental variables (dcs, dms) were fitted using the huisman-olff-fresco models (hof; huisman et al. 1993). hof is a hierarchical set of five species response models with increasing complexity: model i-flat with no response, ii-monotonously increasing or decreasing, iii-monotonously increasing or decreasing with a »plateau«, iv-symmetric unimodal and v-asymmetric unimodal response. four parameters of these models were estimated using a non-linear maximum likelihood estimation procedure (oksanen and minchin 2002), available in jari oksanen’s »gravy« library (http://cc.oulu.fi/%7ejraioksa/ softhelp/softalist.html) for the r program (http://www.r-project.org). this routine was run externally with r program (http://www.r-project.org) from the juice program (tichý 2002), using a procedure developed by zelený and tichý (2007) 74 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees results the classification of the flooded forests of the mura river is presented in the dendrogram (fig. 1), and in the analytic table (tab. 1). classification into three clusters was accepted as it represents three ecologically interpretable vegetation types; 1. salix alba and populus nigra stands, 2. fraxinus angustifolia subsp. oxycarpa stands and 3. quercus robur and carpinus betulus stands. the three clusters correspond to three communities; salicetum albae issler 1926, fraxino-ulmetum effusae slavni} 1952 allietosum ursini subass. nova hoc loco, fraxino-ulmetum effusae slavni} 1952 quercetosum robori subass. nova hoc loco. pca is presented of the 58 relevés of flooded sites of the mura river with ellenberg indicator values (fig. 2). eigenvalues of the first two axes are 0.161 and 0.114. the three communities (three clusters) thrive along a moisture and nutrient gradient. moisture and nutrient indicator values are negatively correlated with dcs (with moisture –0.308, with nutrients –0.341) and dms (with moisture –0.224, with nutrients –0.209). the association salicetum albae (cluster one) occurs on the most moist and nutrient-rich sites, the subassociation fraxino-ulmetum effusae quercetosum robori (cluster three), on the other hand, occurs on the least moist and the least nutrient-rich sites (fig 2, fig. 3). dcs is the lowest in cluster one (salicetum albae) and the highest in cluster three (fraxino-ulmetum effusae quercetosum robori). dms is also the highest in cluster three and lower in clusters one and two (fraxino-ulmetum effusae allietosum ursini). cluster three shows the highest range of dms indicating that the main stream of the mura has the lowest effect on the distribution of quercus robur stands, which can be also found relatively far (up to 3080 m far) from the main stream of mura. the proportion of neophytes is strongly significantly negatively correlated with dms (fig. 5), and not significantly correlated with dcs. the most abundant neophytes in our relevés are robinia pseudacacia, impatiens glandulifera, solidago gigantea and rudbeckia laciniata. acta bot. croat. 72 (1), 2013 75 floodplain forests along the mura river fig. 1. dendrogram of classification. cluster 1: salix alba and populus nigra stands, cluster 2: fraxinus angustifolia subsp. oxycarpa stands and cluster 3: quercus robur and carpinus betulus stands. for simplicity, the bottom part is not shown. 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 76 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. t a b . 1 . a na ly ti c ta bl e of th e th re e fo re st ty pe s on fl oo de d si te s of m ur a r iv er .d ia gn os ti c sp ec ie s of in di vi du al fo re st ty pe s ar e ra nk ed by de cr ea si ng fi de li ty co ef fi ci en t (³ 30 ). v eg et at io n la ye r ab br ev ia ti on s: t1 = up pe r tr ee la ye r, t2 = lo w er tr ee la ye r, s= sh ru b la ye r, h= he rb la ye r, m = m os s la ye r. n um be r of re le vé c lu st er nu m be r 1 2 3 s a li c e tu m a lb a e is sl er 19 26 s a p o p u lu s n ig ra t1 3 3 3 3 4 5 3 4 4 4 3 4 3 . . . 1 . . 2 . . . + 2 1 . . . + . . . . . . . . 2 . . . . . . . . . . . . . . . . . . . a g a ln u s g lu ti n o sa t1 . . . . . . . . . . . . . . . 4 . . . 3 4 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . a g a ln u s g lu ti n o sa t2 + . . + 1 . . . . + 2 . 4 + + . + + . + 2 1 1 1 2 2 . + . . + + . . . . + . . . . . . . . . . . . . . . + . . . . . a g a ln u s g lu ti n o sa s . . . . . . . . . . . . . + + . . . . . . . . + . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . m a a g ro st is st o lo n if e ra h + + . . . . + + + + . 2 . . + + + + . . . . + . . . 1 + + + . . . . . + . + . . . . . . . . . . . . . . . . . + . . g u im p a ti e n s g la n d u li fe ra h + 1 1 + + . + + 1 1 1 + 1 2 . 1 1 2 r + + 1 + + + 4 3 2 2 1 + + + . + + + + . . . + + + . + . + . + + . . . + + . . s a s a li x a lb a t1 . . . . . . . . . . . . . 3 4 . 3 . . . . 2 3 4 . 3 3 4 3 4 + . . . . . . . . . . . . . . . . . . . . . . . . . . . s a s a li x a lb a t2 . . . . . . . . . . . . . + 2 . . . . + . . + . . + . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . s a s a li x a lb a s . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s a s a li x a lb a h . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p m p h a la ri s a ru n d in a c e a h . . . . . . . + . + . + . 1 2 + + . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a e ly m u s c a n in u s h 2 + . . . . + . . 2 . + . . . . + + . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q f g a la n th u s n iv a li s h + 1 1 . 2 + 1 . + . + + 1 . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . g u g le c h o m a h e d e ra c e a h . + + . . . + . . 1 + 1 + . . . . + . . . 2 . + + . + 3 2 2 . . . . + 1 . . . . . . . . . . + . . . . . + . . . . . g u u rt ic a d io ic a h 3 2 3 1 2 1 3 2 2 2 3 4 2 1 + + 2 1 1 3 2 2 3 2 2 4 3 3 4 5 3 + 2 2 1 2 2 1 . + + + 3 + + + + + . . 3 . . 1 + + . + a i s c il la b if o li a h . . + . + + + . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f ra x in o -u lm e tu m e ff u sa e s la vn i} 19 52 q f v io la h ir ta h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 + 1 1 + + . . + 1 . . 1 1 1 2 + . 1 + . + . . . . + f a sa ru m e u ro p a e u m h . . . . . + . . . . . . . r . . . . . . . . . . . . . . . . . + 2 + . + . + + . 1 1 1 + 2 2 + 1 . . + 2 1 . . . + 1 a i c a rd a m in e im p a ti e n s h . . . . . + . . . . . . . 1 . + . . . . + . + . . . . . . . . . + + + . + + . + 1 2 + 1 1 + 1 + + 3 + 2 . + + 1 . + g u g e u m u rb a n u m h . . . . . . . . . + . + + + . + . . . . 1 . + . . . . . . + + + + + . + + + . 1 + + . + + + + + . + + 1 + + + + + + f v io la re ic h e n b a c h ia n a h . . . . + + 1 . . . . . . . . . . . . . + . . . . . . . . . . + . . . + + . + + + + . + + + + + + 1 . + + + + + 1 . m a a ju g a re p ta n s h . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . r + . . + . . + + . . + + . + . . 1 . + + + . + 1 + a i f ra x in u s a n g u st if o li a su bs p. o x y c a rp a t1 . . . . . . 1 . . . 1 + . 2 . . . . + . + + . 1 1 . . . . . 4 4 4 4 3 4 4 4 3 3 . + . 5 . + + + . . . 1 . 1 . . . 1 a i f ra x in u s a n g u st if o li a su bs p. o x y c a rp a t2 . . . . . . . + . . + . . + . . . . + . . . . . . . . . . . 2 1 1 + 1 2 + . 2 + . + 1 1 + . . + . . . + + . . . . . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 77 floodplain forests along the mura river n um be r of re le vé c lu st er nu m be r 1 2 3 a i f ra x in u s a n g u st if o li a su bs p. o x y c a rp a s . . . . + . + . . . . . . . . . 2 . . . . . . . . . . . . . . + . . . . . . . . . + . + + + . . . + . . + . . . + + a i f ra x in u s a n g u st if o li a su bs p. o x y c a rp a h . . . . . + . + . . . + . . . + + . . . . . . . . . . . . r . + . + + . . . . . r . . . 1 1 + . . . + + . + + + . . f l e u c o ju m v e rn u m h 1 . + + + . . 1 . + . + . 1 . . + . 2 . + . + + + + . . . + + + + + + + + + . 2 1 1 1 + + + + + + + 1 + + 1 1 1 + + o o rn it h o g a lu m u m b e ll a tu m h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + . . . + . . . + . + + + . . . + . . . . . . + + q f b ra c h y p o d iu m sy lv a ti c u m h . . . . . + + + + . . . . . . . . . . . . . . . . + . . . r + + + . . + . + . 1 + + . + 2 2 . + . + + + + . + . 2 1 f p u lm o n a ri a o ff ic in a li s h 2 + + 1 2 2 2 + 1 + . . 1 + . . . . + + 2 . . + + + . . + . + 1 1 + + + + 1 1 + 2 + 1 1 + + + + + 1 1 1 2 1 1 2 1 1 m a c o lc h ic u m a u tu m n a le h . . . . . . + . . . . . . 1 . . + . . . . . . . . . . . . . . + 2 1 . . . . . + 1 + + 2 2 + + . . . . . 1 . . . 1 + g u r u d b e c k ia la c in ia ta h . . . . . . . . . . . . . 2 . . 2 . r . . . + . . . . . . . + . 2 + . . 2 + . + 1 1 2 1 + r . . . + + . . . . . . + r p c ra ta e g u s m o n o g y n a t2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . r p c ra ta e g u s m o n o g y n a s . . . . . . + . . . . . . . . . . + . . . . . . . . . . . . . + . + . . . . . + . . . . 1 + 1 + . + . . . . . . 1 2 r p c ra ta e g u s m o n o g y n a h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . 1 + . . . . . . . . . . . . f p a ri s q u a d ri fo li a h + . . . . + . . + + . . . + . . . . . . + . . . . . . . . . . + + . + . + . 1 . + + . + . + + + . + . + + + + + . . o r o b in ia p se u d a c a c ia t1 . . . . . . . . . . . . . . . . . . 4 . . 1 . . . . . . . . + + 1 1 3 1 + . . 2 2 4 1 + . 1 1 2 . . + 1 + . . + . . o r o b in ia p se u d a c a c ia t2 2 2 . + 2 + 2 . 1 . . . + . . . . . 1 . . . . . . . + . . + . + 1 + 1 . . . . + 2 + 1 1 . + + + . + 1 . + . . . + 2 o r o b in ia p se u d a c a c ia s . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . + . . . . . . . . + . . . . . . . . . + . . . . . + . o r o b in ia p se u d a c a c ia h . . . . . . . . . . . . . . . . . + . . . + . . . . . . . + . . . . . . + + . . . . . . + . . . . . . . . . . . . . f c a re x sy lv a ti c a h . + . . . . + . . + . . . . r + . . . . . . + . + . . + . . . + . + + 1 + + . . . . . + 1 + 2 + 1 + . + . 1 + + 1 + a i u lm u s la e v is t1 . 1 1 . . . . . . . . . . . . . . . . . + . . . . . . . 1 . . . . . . . . . . . . . . . 1 . . . . . + . . . . . . . a i u lm u s la e v is t2 . 1 2 1 1 3 . 1 1 . . . . . . . + . . . 1 + . . + . . . 3 3 . + 1 3 + 2 . 2 . 1 2 + . + 1 + 1 2 3 3 2 1 2 . 1 . 2 + a i u lm u s la e v is s . . . + . 1 . . . . . . . . . . + . . . . . + . . . . . . . . . + + . + + . . + 1 + . 2 1 1 1 1 1 1 1 1 1 1 . . + + a i u lm u s la e v is h . . . 1 . + . . . . . . . . + . . . . . 1 . . . . . . . . . . . 1 + . . . . . . . . . . 1 1 + + . . + 1 + . + . + . q f a e g o p o d iu m p o d a g ra ri a h + 1 . + . + 3 2 2 . + . . 1 . . . . 3 . + 3 . . + . + 1 2 3 2 2 + 1 3 2 1 1 1 + 1 + + 2 1 3 . 3 2 1 2 + . 1 3 1 . 3 o p la g io m n iu m u n d u la tu m m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . + . . . . . . . . . + + . . . . . . + . . . . . . g u s o li d a g o g ig a n te a h . . . . . . + . + . . . . 2 + 2 2 . . . . . . . . . . . . r . + 1 1 + . . . . 2 + 4 3 3 + + . . . . 2 + + . . . + 1 m a d e sc h a m p si a c e sp it o sa h . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . + . . . + . . . . . . . . . r . . . + . 1 + 1 1 . . . f a ru m m a c u la tu m h 1 2 2 1 + 1 1 + 2 + + + 1 + . . + . 2 + 1 + 1 + + . . + + . + 1 1 + + + 1 1 1 1 2 1 1 1 + + + + + + 1 + + + + + + + f s a lv ia g lu ti n o sa h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . + . . . . . . . . + + . . + . . . . . . . . . f ra x in o -u lm e tu m e ff u sa e s la vn i} 19 52 a ll ie to su m u rs in i su ba ss .n ov a a i g e ra n iu m p h a e u m h . . . . . . . . . . + . . . . . . . 1 . + . . . . . . . . . + + + + . + + + . . . . . + + . . + . . + . . . . . . + t a b . 1 . – co nt in ue d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 78 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. n um be r of re le vé c lu st er nu m be r 1 2 3 f a ll iu m u rs in u m h . 1 + . . + . . + + + . . 2 . + + . 1 1 1 . 2 . 5 . . . . . 5 5 4 5 3 3 2 3 . . . + . + r . . . . . 1 . . . . 1 . + a i f e st u c a g ig a n te a h . . . . . 1 + 1 . . . . . . . + + . . . + . . . . + + . . . + + + . + + + . . . . . . . . . . + . . . . . . . . . . m a f il ip e n d u la u lm a ri a h . . . . + . . . . . . . . 1 . + + . . . . . . . . . . . . . . + + + + . + + . . . + . + . . . . + . . . 1 . . . . . m a l y si m a c h ia n u m m u la ri a h . . . . . . . . . . . . . . 1 + + . + . . + . . . . . . . . + . + + + + . 1 . + . . + + r + . . . + . . . . . . + . a i r u m e x sa n g u in e u s h . . . . . . . . . . . . . + . + + . + . . . . . . . . + . . . + . . . + + + . + . . . . . . . . . . . . . + . . . . m a p o a tr iv ia li s h . . . + . . + . . . . . . + r 1 1 + 2 + 2 2 + + + . + + . . + + + + + + . 2 . + . + + + . + + + . . . . . . . + . . g u a ll ia ri a p e ti o la ta h + . . + 1 . . 1 + . . . + r . . . . . . + . . . . . + . . . + + . + + + . + . . . . . . . + + + + 1 + . . + + . . + f ra x in o -u lm e tu m e ff u sa e s la vn i} 19 52 q u e rc e to su m ro b o ri su ba ss .n ov a q f q u e rc u s ro b u r t1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . + 1 1 . . 1 1 . 3 5 5 4 3 3 3 4 4 4 2 q f q u e rc u s ro b u r t2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . + . + . . . . 1 + q f q u e rc u s ro b u r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . q f q u e rc u s ro b u r h . . . . . . . . . . . . . . . . . . . . r . . . . . . . . . + . . . . . . . . . . + + + . . . . . + . . . . . . + . q f m o e h ri n g ia tr in e rv ia h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . . . + + . . 1 1 + 1 + . 2 . 1 . + 1 1 + 1 f p o ly g o n a tu m m u lt if lo ru m h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . + + + 1 1 . . + + . + . . . f c a rp in u s b e tu lu s t1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 3 . . . . . . + + . . . f c a rp in u s b e tu lu s t2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 3 . . . . 1 . 3 2 . + . f c a rp in u s b e tu lu s s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . + . . . f c a rp in u s b e tu lu s h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . + . + + . + . r p l ig u st ru m v u lg a re s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 1 + . . . . . . . . . 4 3 r p l ig u st ru m v u lg a re h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 + . . . + . . . . . . . . q f m e li c a n u ta n s h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . + + . . . . 2 q f a c e r c a m p e st re t1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . q f a c e r c a m p e st re t2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . + . . . . . r . . . . . q f a c e r c a m p e st re h . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . + . . . + . . . . + . + + . + . . . q f a c e r c a m p e st re s . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . + . . . . . . . + a i g a g e a lu te a h . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . + . 1 2 + . + . . + . . + . . . . . . + + s m e ri g e ro n a n n u u s h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . + . . . + + + + . . . . . . . . + . + g u g a le o p si s sp e c io sa h . . . . . . . . . . . . . . + . . . . . . . . . . . + + . . . . . . . . . . . + . + . + . . . . + 1 . . . . . . . + a i l is te ra o v a ta h . . . . . . . . . . . . . r . . . . . . . . . . . . . . . . . . . . . . + . . . . . . + + + . + . . . . + . . + + . f c o ry d a li s c a v a h . . . 1 . . . + + + . + . . . . . . . . . . . + 1 . . . . . . . . . . . . . 2 . + + . . . . 2 . . . + + . + + . . . t a b . 1 . – co nt in ue d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 79 floodplain forests along the mura river n um be r of re le vé c lu st er nu m be r 1 2 3 f a th y ri u m fi li x -f e m in a h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . r + . . s m a ri st o lo c h ia c le m a ti ti s h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . + + q f c a m p a n u la p e rs ic if o li a h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . . . . . . . . . . + . . . + . . f c o ry d a li s so li d a h + + + + . + . . . + + . . . . . . . . . . . . . + . . . . r + . . . . . . r + + + + + . + . + + . + . + . . + + . . s a s a li c e te a p u rp u ra e s a li x fr a g il is t1 . . . . . . . . . . . . . . . . + 4 . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s a li x fr a g il is t2 . . . . . . . . . . . . . . . . . + . . . . . . . + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s a li x fr a g il is s . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a i a ln io n in c a n a e a i r a n u n c u lu s fi c a ri a h 1 + 2 + 3 1 . + 2 1 + 2 2 + . 1 1 + 1 1 + + 2 2 1 + . + + + + + + + + + + . 1 1 1 2 . + + + 1 + . . + + + + . + . . a i a n e m o n e ra n u n c u lo id e s h 1 + + + . 2 2 + 1 2 1 2 + . . . . + . . + . . + + . . + . . + + + + + + . + + 1 . + + + + + + + . + 1 + + + + + . . a i s ta c h y s sy lv a ti c a h . + + + + + + . . . . . . . . . . . . + 2 + . + + + . + + + . + + 1 + . . + + . . . + . 2 + + 1 + 2 + + 1 . . + + . a i p ru n u s p a d u s t2 3 1 . . + . 1 1 1 . . . . . . 1 . . . . 2 + . 1 . . . + . . 1 1 . . 1 . 1 + . 3 2 . . + . . . 1 . . . 3 . . . 4 . 2 a i p ru n u s p a d u s s 2 + . . + 3 1 + 2 . . 1 . 1 . 1 1 + . . . . . 1 + . . . . . + 2 . . + + + . + 2 + + . + . . . 2 2 2 . 1 1 . . + + + a i p ru n u s p a d u s h + + . . . + + . . . . + . . . . + . . . + . . + . . . . . . . + . . + + . . . . + + . . . . . + . + . . + + . + . . a i c ir c a e a lu te ti a n a h + + + . + + . . . + . . + 1 . . . . . + 2 . . + . . . + . . 1 1 + . + + + . . . . . . 1 + + . + 1 1 1 . + + + . . . a i o m p h a lo d e s sc o rp io id e s h + + . . + + . . + . . . . + . . . . . . + . . . + + + . . . + . . . + . + + . . 2 . . + . + 1 + 1 . . . . + + + . 1 a i c a re x re m o ta h . . . . . . . . . . . . . + + + + + . + + . + . + . . . . . . . . . . . . + . . . . . . . . . . . . . . . + . . . . a i a c e r n e g u n d o t2 . . . . . . . 2 . . . . . . . . . . . . . . 1 . . . + . . . . . . . . . . + . . . . + . . . . . . . 1 . . . . . . . a i a c e r n e g u n d o s . . . . + . . . . . . . . . + . . . . . . . + . . . . . . . . . . . . . . 2 . . . . . 1 . . . . . . . . . . . . . . a i a c e r n e g u n d o h . . . . . . . + . . . . . . + . . . . . . . . . . + . . . . . . . + . . . + . . . + . . . . . . . . + . . . . . . . a i c h ry so sp le n iu m a lt e rn if o li u m h . . . . . . . . . . . . . . . . . . 3 + . + + . . . . . . . + . . . . . . 1 . . . . . . . . . . . . r . . . . . . . a i p o p u lu s a lb a t1 . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . . . . . . 3 . . . . . . . . . . . . . + 3 a i p o p u lu s a lb a s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . a i p o p u lu s a lb a h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + a i im p a ti e n s n o li -t a n g e re h . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . + + . . . . . . . . . r . . . . . . . . . . + . . . . . a i c a re x b ri zo id e s h . . . . . . . . + . + . . . . . . . . . . . + . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . a i d ry o p te ri s c a rt h u si a n a h . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . r . + . . . . . . . . . . + . . a i s te ll a ri a n e m o ru m h . . . . . . . . . . . . . . . . . . . . . . . + . . 1 r . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . t a b . 1 . – co nt in ue d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 80 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. n um be r of re le vé c lu st er nu m be r 1 2 3 a i u lm u s m in o r t2 + . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a i u lm u s m in o r s + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . a i u lm u s m in o r h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . f f a g e ta li a sy lv a ti c a e s a m b u c u s n ig ra t2 . . 1 2 . . . . 2 2 1 . + . . . . . . . . 1 . . 2 1 + 3 . . . . + + . + . + . . . . . . . . . . . . + . . . . . . . s a m b u c u s n ig ra s 2 3 2 3 2 1 + 2 2 2 1 . 1 + . . + . 3 1 1 3 . + 2 2 . 2 2 3 2 1 2 3 3 2 1 3 + + + . 1 + . 2 + + + + 3 . . + . + + . s a m b u c u s n ig ra h . . . . . . . . . . . . . . . . . . . . + + . . . . 3 + . + + + . + + . . . . + + + . . . + . + . . 1 . . + + . . . a d o x a m o sc h a te ll in a h 1 + + + . 1 + . + . + . . . . . . . 1 + + 2 . 1 + + . + + 1 + 1 1 1 2 1 1 2 2 3 2 1 1 . 1 1 + 2 2 1 1 1 . + 2 2 . . l a m ia st ru m g a le o b d o lo n su bs p. m o n ta n u m h + + 1 . . + . + + + + . . + . . . . 1 + 2 2 . 1 + + . . + + 2 1 1 2 1 + 1 . 2 + 2 + + + 1 + + 2 3 1 . 2 . 4 3 3 . 2 s y m p h y tu m tu b e ro su m h + + . . . . . + + . . . . . . . . . + . . . . . . . . . . . . + . + . + . + . . . + + . . . . . . . + . + + . . . . r a n u n c u lu s la n u g in o su s h . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . + . . . . . + . . . . . . . . . . . . . . m il iu m e ff u su m h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . 1 . . . . . . . + . . . o x a li s a c e to se ll a h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . + . . . . . . . . + . . . . . q f q u e rc o -f a g e te a a n e m o n e n e m o ro sa h . . . . . . + . . . . . . . . + . . . . . . . . . . . . . . . + . . . . . . + . . . . . . . 3 . . . . . + . . + . 1 c e ra st iu m sy lv a ti c u m h . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . + . . . . . . . . . . . + 1 . . + . . . + . . . . 1 . h e d e ra h e li x h . . . . + . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . c o ry lu s a v e ll a n a t2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . + . . . . . . . . . c o ry lu s a v e ll a n a s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . 1 . . . c o ry lu s a v e ll a n a h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . a g a ln e te a g lu ti n o sa e h u m u lu s lu p u lu s s . . . . . . . . + . . . . . . . + . . . . . . . . 1 . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . h u m u lu s lu p u lu s h . . . . . . . . . . . . . . . . + . . . . . . . . 1 . . + . . . r . . . . . . + + . 2 . + . . . . . + . . . . . . . c a lt h a p a lu st ri s h . . . . . . . . . . . . . + . . + . . + + . . . . . . . . . . . . . + . . + . . . . . . . . . . . . . . r . . . . . c a re x v e si c a ri a h . . . . . . . . . . . . . 2 1 . . . . . . . . . . . . . . . . . . . . . . . . . + + + . . . . . . . . . . . . . . . r p r h a m n o -p ru n e te a e u o n y m u s e u ro p a e u s t2 . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t a b . 1 . – co nt in ue d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 81 floodplain forests along the mura river n um be r of re le vé c lu st er nu m be r 1 2 3 e u o n y m u s e u ro p a e u s s + + + . + . + + + . + . + . . . + . . . . . . . . . . . . . . . . . . . . . . + + + 1 . . . . . . . . . . . . . . . e u o n y m u s e u ro p a e u s h . . . . . . + . . . . . . + . . + . . . + + . + . . . . . + . + 1 + + . . + . + + 1 1 + 1 + . . + + + + + + + + + + c o rn u s sa n g u in e a t2 . + . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . c o rn u s sa n g u in e a s . . 1 . . + 2 + 1 . . . . . + + 2 . . . + 1 . + 1 . . + . . . . . . . . . . + . . . + + 2 + . + + . . . . 1 + . 2 2 c o rn u s sa n g u in e a h . . . . . . . . + . . . . . + . . . . . . + . . . . . . . . . . . . . . . . . . + . + . + . + . . . . . . . . . . . v ib u rn u m o p u lu s s . . . . + + . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . v ib u rn u m o p u lu s h . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . c ra ta e g u s la e v ig a ta t2 . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c ra ta e g u s la e v ig a ta s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . + . . . . . . c ra ta e g u s la e v ig a ta h . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . + . . . g u g a li o -u rt ic e te a g a li u m a p a ri n e h 3 3 2 4 3 3 1 1 1 4 3 2 2 2 + 1 1 2 3 4 3 1 3 3 3 2 3 3 3 1 1 2 + 2 + 2 3 1 . 1 + + 1 + + + + 2 2 + 2 2 3 2 1 + + + s te ll a ri a n e g le c ta h 1 . . + + + . + . . . . . + . 1 + 3 1 3 1 2 4 1 + + 2 2 + + + + 1 1 2 4 4 4 . 2 . 3 2 1 . 3 + 1 + + 2 + . 1 + 1 + + r u b u s c a e si u s s . . . . 2 + 2 2 1 1 2 1 4 + . + 1 . . . . . . . . . . . . . . . . . . . . . + 1 1 . 1 . + . . . . . . . . . . . . . r u b u s c a e si u s h 1 . . . . . + . . . 1 . . 2 4 2 . + + + . . . 1 + 2 1 . . + + 1 1 1 1 . 1 . . . . + 1 2 2 + + + + + + 3 3 + + . . + l a m iu m m a c u la tu m h + + + . + + + 3 . . . . . . . . . . 2 + + + + 2 + . 2 . 1 + 2 . + + + + . 2 3 + . . 2 . . 1 + + 1 . + . . . . 1 . . g a le o p si s p u b e sc e n s h + . . + + + . . . . . . . . . + . + . . . . . . . . . . . . . . + . . . . . . . + . . . + . + . . . . . . + + . + . d u c h e sn e a in d ic a h . + . . . . . . . . . + . . . . . . + + + 1 . . . . . + + . . + . . . . . 1 . . . . . 1 . . . . . . . . . . . . . . m y o so ti s sp a rs if lo ra h . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . 2 . + . + + 2 . . . . . . . . . + . . . . a n th ri sc u s sy lv e st ri s h . . . . . . . . . . . . . . . . . . + . + . . . . . . . . . . . . + . . . + . . r . . . . . . . . + . . . + . . . . c a ly st e g ia se p iu m h . . . . . . . . . . . . . . . . + . . . . . . . . + . . . . . . . . . . . . . . . + . . . . + . . + . . . . . . . . c h e li d o n iu m m a ju s h . . . . . . . . . . . . . . . . . . . . . + . . . . + . . . . . . . 1 . . . . . . . . . . + . . . . . . . . . . . . e c h in o c y st is lo b a ta h . . . . . . . . + . . . . . . . . . . . . . . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c h a e ro p h y ll u m a u re u m h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . + + . . . . . . . . . . . . p m p h ra g m it im a g n o c a ri c e te a ir is p se u d a c o ru s h . . . . . . . . . . . + . + + + + + . . . . + r . . . . . . + . . . . . + + . . . . . . . . . . . . . . . . . . . . c a re x a c u ti fo rm is h . . . . . . . . . . . . . 2 2 2 . . . . . . . . . . . . . . . . + . . . + . . . . . . 1 . . . . . + . . 1 . . . . . p h ra g m it e s a u st ra li s h . . . . . . . . . . . . . . + . 3 + . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c a re x e la ta h . . . . . . . . . . . . . . + + 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t a b . 1 . – co nt in ue d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 82 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. n um be r of re le vé c lu st er nu m be r 1 2 3 m a m o li n io -a rr h e n a th e re te a d a c ty li s g lo m e ra ta h + . . . . . + + . + . + . . . . . . . . . . . . . + + . . . . . . . . + . . . + . . . . . . . . . + . . . . . . . . h e ra c le u m sp h o n d y li u m h . . . . . . . + . . . . . . . . . . . . . . . . + + . . . . . . . . . + . . . . . + . . + + . + . . . . . . . . . + s y m p h y tu m o ff ic in a le h . . . . . . . . . . . . . + . + 1 . . . . . . . . + . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . t a ra x a c u m o ff ic in a le h . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + r u m e x o b tu si fo li u s h . . . . + . + . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s e li n u m c a rv if o li a h . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . + . . . . . . . . . v e ro n ic a c h a m a e d ry s h . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . + a a rt e m is ie te a s il e n e a lb a h . . . . . . + . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . + . . . . . . + . + + . . + . . . . . . . s m s te ll a ri e te a m e d ia e v e ro n ic a h e d e ri fo li a h 2 4 4 2 3 3 1 + + 3 4 3 1 + . 1 + 1 2 2 2 2 2 3 2 . + + + + 1 + + + + 1 + + + 2 3 1 2 + . r 4 + + . + + . + + + . + e q u is e tu m a rv e n se h . . . . . . . . . . . . . + + . + . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . r + + . . . . o o th er sp ec ie s a n e m o n e sp e c ie s h . . . . . . . . . . . . . + . . . . + + . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . p o p u lu s x c a n a d e n si s t1 . . . + . . . . . . . . . . . . . . . + . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v io la sp e c ie s h . . . . . . . . . . . . . + . . . . . . + . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . a p p e n d ix to ta b le 1 r el ev é nu m be r, re le vé ar ea ,d at e, co or di na te s (u t m w g s ; la ti tu de ,l on gi tu de ), co ve ra ge : up pe r tr ee la ye r (t 1) ,l ow er tr ee la ye r (t 2) ,s hr ub la ye r (s ), he rb la ye r (h ). 1 . 30 0 m 2 ; 19 .6 .2 00 5; 58 80 42 ,5 16 20 13 ; t1 60 % ,t 2 60 % ,s 50 % ,h 10 0% ; 2 . 30 0 m 2 ; 19 .6 .2 00 5; 58 70 22 ,5 16 15 35 ; t1 70 % ,t 2 50 % ,s 40 % ,h 10 0% ; 3 . 40 0 m 2 ; 19 .6 .2 00 5; 58 70 95 ,5 16 15 23 ; t1 90 % ,t 2 30 % ,s 30 % ,h 90 % ; 4 . 30 0 m 2 ; 14 .5 .2 00 5; 59 00 73 ,5 16 01 65 ; t1 60 % ,t 2 60 % ,s 70 % ,h 90 % ; 5 . 20 0 m 2 ; 19 .6 .2 00 5; 59 93 06 ,5 15 41 47 ; t1 70 % , t2 50 % ,s 40 % ,h 10 0% ; 6 . 30 0 m 2 ; 19 .6 .2 00 5; 59 22 25 ,5 15 96 29 ; t1 90 % ,t 2 40 % ,s 50 % ,h 90 % ; 7 . 25 0 m 2 ; 19 .6 .2 00 5; 59 35 62 ,5 15 81 62 ; t1 70 % ,t 2 50 % ,s 60 % ,h 90 % ; 8 . 40 0 m 2 ; 19 .6 .2 00 5; 59 33 17 ,5 15 85 46 ; t1 80 % ,t 2 50 % ,s 40 % ,h 80 % ; 9 . 25 0 m 2 ; 19 .6 .2 00 5; 59 34 12 ,5 15 84 60 ; t1 80 % ,t 2 70 % ,s 50 % ,h 80 % ; 1 0 . 20 0 m 2 ; 19 .6 .2 00 5; 58 73 98 ,5 16 20 94 ; t1 80 % ,t 2 40 % ,s 20 % ,h 10 0% ; 1 1 . 40 0 m 2 ; 19 .6 .2 00 5; 59 37 80 ,5 15 82 40 ; t1 70 % ,t 2 50 % ,s 30 % ,h 90 % ; 1 2 . 30 0 m 2 ; 19 .6 .2 00 5; 58 74 46 ,5 16 20 65 ; t1 70 % ,t 2 20 % ,s 20 % ,h 10 0% ; 1 3 . 20 0 m 2 ; 19 .6 .2 00 5; 59 92 60 ,5 15 41 40 ; t1 40 % ,t 2 70 % ,s 60 % ,h 10 0% ; 1 4 . 20 0 m 2 ; 4. 5. 20 05 ; 58 60 54 ,5 16 27 68 ; t1 50 % ,t 2 10 % ,s 30 % , h 10 0% ; 1 5 . 40 0 m 2 ; 25 .5 .2 00 5; 58 72 31 ,5 16 10 82 ; t1 50 % ,t 2 30 % ,s 20 % ,h 90 % ; 1 6 . 40 0 m 2 ; 14 .5 .2 00 5; 59 19 85 ,5 15 98 89 ; t1 60 % ,t 2 20 % ,s 20 % ,h 10 0% ; 1 7 . 20 0 m 2 ; 6. 5. 20 05 ; 58 45 17 ,5 16 26 72 ; t1 60 % ,t 2 5% ,s 25 % ,h 10 0% ; 1 8 . 40 0 m 2 ; 25 .5 .2 00 5; 58 74 43 ,5 16 09 34 ; t1 70 % ,t 2 10 % ,s 10 % ,h 10 0% ; 1 9 . 40 0 m 2 ; 4. 5. 20 05 ; 58 60 17 , t a b . 1 . – co nt in ue d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:17 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 83 floodplain forests along the mura river 51 62 37 7; t1 80 % ,t 2 20 % ,s 50 % ,h 10 0% ; 2 0 . 40 0 m 2 ; 4. 5. 20 05 ; 58 60 46 ,5 16 24 37 ; t1 80 % ,t 2 10 % ,s 10 % ,h 10 0% ; 2 1 . 40 0 m 2 ; 25 .5 .2 00 5; 58 69 80 ,5 16 14 01 ; t1 80 % , t2 40 % ,s 20 % ,h 10 0% ; 2 2 . 40 0 m 2 ; 25 .5 .2 00 5; 58 72 59 ,5 16 14 66 ; t1 70 % ,t 2 50 % ,s 40 % ,h 10 0% ; 2 3 . 40 0 m 2 ; 5. 5. 20 05 ; 58 22 58 ,5 16 52 22 ; t1 40 % ,t 2 30 % ,s 5% ,h 10 0% ; 2 4 . 40 0 m 2 ; 13 .5 .2 00 5; 59 35 39 ,5 15 66 56 ; t1 75 % ,t 2 20 % ,s 20 % ,h 10 0% ; 2 5 . 40 0 m 2 ; 14 .5 .2 00 5; 58 99 37 ,5 16 03 69 ; t1 50 % ,t 2 40 % ,s 40 % ,h 10 0% ; 2 6 . 20 0 m 2 ; 13 .5 . 20 05 ; 59 37 11 ,5 15 81 07 ; t1 50 % ,t 2 40 % ,s 30 % ,h 10 0% ; 2 7 . 40 0 m 2 ; 17 .5 .2 00 5; 59 47 48 ,5 15 66 79 ; t1 60 % ,t 2 20 % ,s 40 % ,h 10 0% ; 2 8 . 40 0 m 2 ; 25 .5 .2 00 5; 58 74 83 , 51 61 30 3; t1 60 % ,t 2 50 % ,s 20 % ,h 10 0% ; 2 9 . 40 0 m 2 ; 25 .5 .2 00 5; 58 72 93 ,5 16 14 24 ; t1 70 % ,t 2 40 % ,s 20 % ,h 10 0% ; 3 0 . 40 0 m 2 ; 25 .5 .2 00 5; 58 74 42 ,5 16 14 42 ; t1 60 % , t2 40 % ,s 30 % ,h 10 0% ; 3 1 . 40 0 m 2 ; 5. 5. 20 05 ; 58 23 11 ,5 16 51 33 ; t1 70 % ,t 2 30 % ,s 20 % ,h 10 0% ,m 5% ; 3 2 . 40 0 m 2 ; 20 .5 .2 00 5; 59 35 71 ,5 15 79 63 ; t1 75 % ,t 2 20 % ,s 30 % , h 10 0% ,m 5% ; 3 3 . 40 0 m 2 ; 20 .5 .2 00 5; 58 10 12 ,5 16 62 18 ; t1 70 % ,t 2 30 % ,s 20 % ,h 10 0% ; 3 4 . 40 0 m 2 ; 20 .5 .2 00 5; 58 09 50 ,5 16 63 10 ; t1 80 % ,t 2 50 % ,s 30 % ,h 10 0% , m 5% ; 3 5 . 20 0 m 2 ; 13 .5 .2 00 5; 59 36 28 ,5 15 80 82 ; t1 60 % ,t 2 30 % ,s 40 % ,h 10 0% ; 3 6 . 20 0 m 2 ; 13 .5 .2 00 5; 59 34 51 ,5 15 78 97 ; t1 80 % ,t 2 40 % ,s 30 % ,h 10 0% ; 3 7 . 30 0 m 2 ; 6. 5. 20 05 ; 58 82 55 ,5 16 19 21 ; t1 80 % ,t 2 40 % ,s 20 % ,h 10 0% ; 3 8 . 20 0 m 2 ; 17 .5 .2 00 5; 59 47 52 ,5 15 69 56 ; t1 75 % ,t 2 35 % ,s 50 % ,h 10 0% ; 3 9 . 20 0 m 2 ; 19 .6 .2 00 5; 60 20 51 , 51 55 52 2; t1 80 % ,t 2 40 % ,s 40 % ,h 10 0% ; 4 0 . 40 0 m 2 ; 21 .4 .2 00 5; 58 82 12 ,5 16 22 49 ; t1 80 % ,t 2 50 % ,s 30 % ,h 10 0% ; 4 1 . 40 0 m 2 ; 4. 5. 20 05 ; 58 60 49 ,5 16 30 87 ; t1 60 % ,t 2 70 % ,s 30 % ,h 10 0% ; 4 2 . 40 0 m 2 ; 5. 5. 20 05 ; 58 22 50 ,5 16 53 96 ; t1 80 % ,t 2 20 % ,s 10 % ,h 10 0% ; 4 3 . 40 0 m 2 ; 5. 5. 20 05 ; 58 22 09 ,5 16 50 05 ; t1 60 % ,t 2 30 % ,s 30 % ,h 10 0% ; 4 4 . 40 0 m 2 ; 17 .5 .2 00 5; 59 47 01 ,5 15 71 31 ; t1 80 % ,t 2 20 % ,s 35 % ,h 10 0% ,m 5% ; 4 5 . 40 0 m 2 ; 20 .5 .2 00 5; 58 09 01 ,5 16 60 63 ; t1 70 % ,t 2 40 % ,s 30 % ,h 10 0% ,m 5% ; 4 6 . 40 0 m 2 ; 20 .5 .2 00 5; 58 10 73 ,5 16 62 02 ; t1 60 % ,t 2 60 % ,s 40 % ,h 90 % ; 4 7 . 40 0 m 2 ; 6. 5. 20 05 ; 58 51 35 ,5 16 35 46 ; t1 60 % ,t 2 60 % ,s 20 % ,h 10 0% ; 4 8 . 40 0 m 2 ; 13 .5 .2 00 5; 59 34 01 ,5 15 68 38 ; t1 70 % ,t 2 30 % ,s 40 % ,h 10 0% ; 4 9 . 40 0 m 2 ; 17 .5 .2 00 5; 59 69 65 ,5 15 56 29 ; t1 80 % ,t 2 50 % ,s 40 % ,h 10 0% ; 5 0 . 40 0 m 2 ; 17 .5 .2 00 5; 59 73 30 ,5 15 55 58 ; t1 90 % ,t 2 40 % ,s 40 % ,h 90 % ; 5 1 . 40 0 m 2 ; 20 .5 .2 00 5; 58 09 66 ,5 16 63 82 ; t1 60 % ,t 2 50 % ,s 40 % ,h 10 0% ; 5 2 . 30 0 m 2 ; 26 .5 .2 00 5; 58 86 41 ,5 16 16 73 ; t1 70 % ,t 2 50 % , s 30 % ,h 90 % ,m 5% ; 5 3 . 40 0 m 2 ; 26 .5 .2 00 5; 58 85 90 ,5 16 16 29 ; t1 60 % ,t 2 40 % ,s 20 % ,h 10 0% ; 5 4 . 40 0 m 2 ; 26 .5 .2 00 5; 60 25 33 ,5 15 40 03 ; t1 75 % ,t 2 50 % ,s 20 % ,h 10 0% ; 5 5 . 40 0 m 2 ; 26 .5 .2 00 5; 60 25 62 ,5 15 40 01 ; t1 80 % ,t 2 40 % ,s 30 % ,h 90 % ; 5 6 . 40 0 m 2 ; 6. 5. 20 05 ; 58 82 47 ,5 16 19 57 ; t1 90 % ,t 2 60 % ,s 10 % ,h 10 0% ; 5 7 . 40 0 m 2 ; 6. 5. 20 05 ; 58 45 21 ,5 16 33 41 ; t1 80 % ,t 2 40 % ,s 70 % ,h 90 % ; 5 8 . 40 0 m 2 ; 6. 5. 20 05 ; 58 44 94 ,5 16 27 88 ; t1 80 % ,t 2 40 % ,s 50 % ,h 90 % . t ax a oc cu ri ng on ly on ce in th e ta bl e 1 c ro c u s v e rn u s, h, 44 (+ ), 46 (+ ), g e ra n iu m ro b e rt ia n u m ,h ,4 5 (+ ), 47 (+ ), c a rd a m in e a m a ra ,h ,8 (+ ), c ir si u m e ri si th a le s, h, 31 (+ ), u lm u s g la b ra ,t 2, 50 (+ ), l o n ic e ra c a p ri fo li u m ,h ,4 5 (+ ), 50 (+ ), s c ro p h u la ri a n o d o sa ,h ,4 6 (r ), 50 (+ ), c le m a ti s v it a lb a ,h ,4 5 (+ ), 57 (+ ), p ru n u s a v iu m ,t 1, 45 (1 ), k n a u ti a d ry m e ia ,h ,4 5 (+ ), e u p h o rb ia d u lc is ,h ,5 3 (r ), a c e r p la ta n o id e s, t2 ,5 4 (+ ), a c e r p la ta n o id e s, h, 54 (+ ), h e ll e b o ru s o d o ru s, h, 45 (r ), m a ia n th e m u m b if o li u m ,h ,5 6 (+ ), v e ro n ic a o ff ic in a li s, h, 44 (+ ), c a rd a m in e p ra te n si s, h, 16 (+ ), 18 (+ ), p e u c e d a n u m p a lu st re ,h ,4 0 (+ ), r a n u n c u lu s re p e n s, h, 8 (+ ), g le c h o m a h ir su ta ,h ,4 (1 ), s ta c h y s p a lu st ri s, h, 17 (+ ), c ir si u m o le ra c e u m ,h ,1 7 (+ ), 44 (+ ), v a le ri a n a d io ic a ,h ,1 8 (+ ), a n g e li c a sy lv e st ri s, h, 17 (+ ), c a re x d iv u ls a ,h ,4 4 (+ ), 47 (+ ), c ru c ia ta g la b ra ,h ,4 0 (+ ), 45 (+ ), d ip sa c u s sp ec ie s, h, 32 (+ ), 36 (+ ), e ly m u s re p e n s, h, 8 (+ ), 26 (+ ), l y si m a c h ia p u n c ta ta ,h ,1 5 (1 ), 17 (+ ), p ip ta th e ru m v ir e sc e n s, h, 23 (+ ), 27 (+ ), p o ly g o n u m m it e ,h ,1 8 (4 ), 27 (+ ), r u m e x a c e to sa ,h ,4 0 (+ ), 56 (+ ), r u m e x c ri sp u s, h, 5 (+ ), 10 (+ ), s e n e c io n e m o re n si s, h, 27 (+ ), 44 (+ ), a e sc u lu s h ip p o c a st a n u m ,h ,3 5 (+ ), a e sc u lu s h ip p o c a st a n u m ,t 2, 7 (1 ), a li sm a p la n ta g o -a q u a ti c a ,h ,1 5 (+ ), a ll iu m sc o ro d o p ra su m ,h ,3 4 (+ ), a ln u s in c a n a ,t 2, 10 (+ ), a ln u s sp ec ie s, t2 ,1 2 (1 ), a rc ti u m la p p a ,h ,2 7 (+ ), a rt e m is ia v u lg a ri s, h, 27 (+ ), b ro m u s st e ri li s, h, 27 (+ ), c a rd a m in e fl e x u o sa ,h ,2 6 (+ ), c a re x m u ri c a ta ag g. ,h ,4 4 (+ ), c h e n o p o d iu m sp ec ie s, h, 21 (+ ), c ru c ia ta la e v ip e s, h, 58 (+ ), d ip sa c u s p il o su s, h, 29 (+ ), e u o n y m u s v e rr u c o su s, h, 1 (+ ), f ra g a ri a v e sc a ,h ,4 5 (+ ), f ra x in u s p e n n sy lv a n ic a ,h ,4 9 (+ ), f ra x in u s p e n n sy lv a n ic a ,t 1, 44 (+ ), g a g e a sp a th a c e a ,h , 55 (+ ), h o lc u s la n a tu s, h, 10 (+ ), im p a ti e n s p a rv if lo ra ,h ,1 (+ ), j u g la n s re g ia ,s ,5 (+ ), l a p sa n a c o m m u n is ,h ,5 4 (+ ), l e e rs ia o ry zo id e s, h, 46 (+ ), l it h o sp e rm u m o ff ic in a le , h, 17 (+ ), l o n ic e ra c a e ru le a ,h ,4 3 (r ), m e n th a a q u a ti c a ,h ,1 5 (+ ), m o ru s a lb a ,t 2, 12 (+ ), o rn it h o g a lu m n u ta n s, h, 52 (+ ), o x a li s st ri c ta ,h ,6 (+ ), 56 (+ ), p a rt h e n o c is su s in se rt a ,h ,3 0 (+ ), p o ly g o n u m la p a th if o li u m ,h ,1 8 (+ ), p o a p a lu st ri s, h, 8 (+ ), p o p u lu s sp ec ie s, t1 ,2 3 (+ ), p o te n ti ll a re p ta n s, h, 15 (+ ), p u lm o n a ri a m o ll is ss p. m o ll is ,h ,2 4 (+ ), r e y n o u tr ia ja p o n ic a ,h ,2 6 (+ ), s u c c is a sp ec ie s, h, 8 (+ ), t h a li c tr u m a q u il e g ii fo li u m ,h ,4 5 (r ), u lm u s sp ec ie s, t2 ,7 (+ ), v e rb a sc u m sp ec ie s, h, 18 (+ ), v ib u rn u m la n ta n a ,h , 57 (+ ), v ib u rn u m la n ta n a ,s ,1 7 (+ ). t a b . 1 . – co nt in ue d 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:17 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 84 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. fig. 2. passive projection of ellenberg indicator values and values of dcs and dms onto the pca diagram of relevés and species of flooded sites of the mura river. only indicator values with the highest correlations with the first two pca axes are shown. the highest correlations with the first axis are shown by the indicator values for nutrients (0.579), moisture (0.515) and dcs (–0.384), with the second axis the values for dms (–0.328). legend: � salix alba and populus nigra stands (salicetum albae issler 1926), � fraxinus angustifolia subsp. oxycarpa stands (fraxino-ulmetum effusae slavni} 1952 allietosum ursini subass. nova hoc loco), � quercus robur and carpinus betulus stands (fraxino-ulmetum effusae slavni} 1952 quercetosum robori subass. nova hoc loco) fig. 3. mean, standard error (box) and standard deviation (whiskers) of ellenberg indicator values for nutrients (a) and moisture (b) of the three communities of the flooded forests. communities (clusters) are numbered as in fig.1. 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:18 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees in the case of life forms, correlation with dcs was very highly significant only in the case of therophytes (fig. 6). correlation with dms was not significant in any case of life form. acta bot. croat. 72 (1), 2013 85 floodplain forests along the mura river fig. 4. mean, standard error (box) and standard deviation (whiskers) of distance from the closest stream (dcs) (a) and distance from the main stream (dms) (b) of the three clusters. clusters are numbered as in fig. 1. fig. 5. relation between the proportion of neophytes and increasing distance from the main stream (dms). 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:20 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees correlations of ecological strategists and dcs and dms was calculated with regression analysis. no analysis show statistical significant correlation. in all three clusters hemicryptophytes represent the biggest proportion of species, geophytes and phanerophytes follow. therophytes represent the smallest proportion of species in the relevés of three clusters (tab. 2). all three clusters consist of a mixture of different strategists (c-s-r, c, c-s, c-r and r). the proportions of csr and c species were the highest in all three clusters (tab. 3). species response curves fitted using hof models (fig. 7) describe the relationships among the main tree species of flooded forests of the mura river and environmental variables dcs (fig. 7a) and dms (fig. 7b). 86 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. fig. 6. relation between the proportion of therophytes and increasing distance from the closest stream (dcs). tab. 2. mean value of the share (%) of life forms with standard deviations (sd) of the three clusters. life form cluster 1 cluster 2 cluster 3 hemicryptophytes 37.6 ± 8.96 48.37 ± 6.88 41.67 ± 6.87 geophytes 22.16 ± 7.74 21.62 ± 2.94 22.79 ± 6.05 phanerophytes 20.95 ± 5.4 13.17 ± 3.61 20.47 ± 5.62 therophytes 11.8 ± 3.66 7.68 ± 1.57 7.55 ± 2.48 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:21 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees acta bot. croat. 72 (1), 2013 87 floodplain forests along the mura river tab. 3. mean value of the share (%) of c-s-r strategies with standard deviations (sd) of three clusters. strategy type cluster 1 cluster 2 cluster 3 c-s-r 36.25 ± 8.07 41.09 ± 3.79 41.14 ± 5.5 c 33.54 ± 6.87 27.07 ± 3.18 31.28 ± 5.35 c-s 10.8 ± 5.15 13.66 ± 3.98 11.81 ± 2.28 c-r 9.81 ± 4.06 6.81 ± 1.55 6.82 ± 2.45 r 4.25 ± 1.5 3.71 ± 1.59 3.67 ± 2.22 fig. 7. probability of occurrence of the main tree species of the area of the mura river on the gradients of dcs (a) and dms (b), modelled with the huisman-olff-fresco models. only species occurrences in the tree layer were considered for modelling. a b 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:22 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees carpinus betulus and quercus robur respond to both dcs and dms. they are found in areas distant from streams. fraxinus angustifolia subsp. oxycarpa does not respond to either dcs or dms. salix alba and populus nigra respond only to dcs and do not respond to dms. their response is the opposite of the response of carpinus betulus and quercus robur as they are found close to the stream. discussion zonation of plant communities and ecological gradients the three forest vegetation types that appear on river banks were detected with numerical analysis (fig. 1). the main ecological gradients that influence the species composition of these forests are nutrients and moisture, and they are negatively correlated with distance from the stream (dcs, dms) (fig.2). moistureand nutrient-rich plots are closer to the stream. distance from the first closest stream (dcs) shows bigger correlation to species composition than dms and it is the underlying gradient of vegetation zonation, which can be also seen from the dcs values of the three forest vegetation types (fig. 4a). three forest vegetation types are also ecologically delimited (fig. 3a,b) as there is a distinction between the ecological values of the three groups of relevés. the values of relative elevation of the plots were much equalized, (as forests in depressions – floodplain swamp forests – were not included), and therefore do not influence species composition. the three communities have been classified into syntaxa, as follows. syntaxonomical scheme: salicetea purpurae moor 1958 salicetalia purpurae moor 1958 salicion albae soó 1930 salicetum albae issler 1926 querco-fagetea br.-bl. et vlieger 1937 fagetalia sylvaticae pawlowski in pawlowski et al. 1928 alnion incanae lüdi 1921 fraxino-ulmetum effusae slavni} 1952 subass. allietosum ursini subass. nova hoc loco holotypus: relevé number 31 in tab. 1 holotypus hoc loco fraxino-ulmetum effusae slavni} 1952 subass. quercetosum robori subass. nova hoc loco holotypus: relevé number 48 in tab. 1 holotypus hoc loco in the central part of europe along big rivers the hardwood association querco-ulmetum minoris issler 1926 (syn. fraxino-ulmetum tx. ex oberd. 1953) is distinguished by the dominant species quercus robur, ulmus minor/laevis, fraxinus excelsior. in central eastern and south eastern europe a hardwood association with different fraxinus species fraxinus angustifolia subsp. oxycarpa (fraxino-ulmetum effusae slavni} 1952 (syn. fraxino pannonicae-ulmetum soó 1936 corr. 1963)) is the one that occupies the highest positions in floodplain areas along large rivers (willner and grabherr 2007, neuhäuslová et al. 2001, borhidi and kevey 1996, vicherek et al. 2000, vukeli] and bari^evi] 2004, 2005, rau[ 1976, 1992, slavni] 1952). that association was first correctly described by slavni} in 1952 in the floodplain area of vojvodina (serbia) along rivers drava and 88 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:23 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees danube. the reference of slavni] (1952) was overlooked by most european researchers who recognized the name fraxino pannonicae-ulmetum soó 1936 corr. 1963 given by soó (1963) as the correct one. description of the syntaxa (tab. 1, fig. 3a, b) salicetum albae issler 1926 this community occurs along streams, on floodplains and along gravel pits. along the mura the white willow (salix alba) and poplar tree (populus nigra) form a relatively wide belt of riparian vegetation. these tree species dominate the stands and are also diagnostic species of the association. in the tree layer there are also alnus glutinosa, salix fragilis, ulmus laevis, fraxinus angustifolia subsp. oxycarpa, prunus padus and robinia pseudacacia. the stands are often inundated and the ground water is high. the soil is structureless. sedimentation is the result of inundation and alluvium deposits and its layers are clearly defined. diagnostic species (tab. 1) such as impatiens glandulifera, agrostis stolonifera, elymus caninus, urtica dioica etc. indicate moist and nutrient-rich, regularly disturbed sites. the large surfaces of the sites of this community were reclaimed in the past and planted with different cultivars of hybrid poplar and alder. the association is widely accepted and described all over europe. fraxino-ulmetum effusae slavni} 1952 the community occurs in a wide belt along the mura above the belt formed by white willow forests. in the area of mura river we detected two subassociations of this association (subass. allietosum ursini and subass. quercetosum robori); fraxino-ulmetum effusae slavni} 1952 subass. allietosum ursini subass. nova. it occurs fragmentarily on finely structured alluvial soil. the sites are only occasionally flooded. the community is confined to the association salicetum albae. in the tree layer species fraxinus angustifolia subsp. oxycarpa dominates the stands, while ulmus laevis, robinia pseudacacia and prunus padus also frequently occur. the diagnostic species are characteristic for moist, fresh, nutrient rich sites: geranium phaeum, allium ursinum (dominates the herb layer), festuca gigantea, etc. in the vegetation map of forest communities of the area of murska sobota (^arni et al. 2008) forests of this subassociation were treated as a variant of fraxino-ulmetum effusae slavni} 1952 var. prunus padus vukeli} et bari~evi} 2004. fraxino-ulmetum effusae slavni} 1952 subass. quercetosum robori subass. nova. in terms of development, this forest type is the most mature and stable in the inundated region, it is the least nutrient rich and the least moist of the three vegetation types (fig. 3). the dominant tree species in the tree layer is quercus robur, sometimes also carpinus betulus, fraxinus angustifolia subsp. oxycarpa, robinia pseudacacia and ulmus laevis. the group of diagnostic species is large, and most species are of ordo fagetalia (alliance alnion incanae) and classes rhamno-prunetea and galio-urticetea. in the vegetation map of forest communities of the area of murska sobota (^arni et al. 2008) forests of this subassociation were treated as the association genisto elatae-quercetum roboris horvat 1938. later on, recent analysis showed that this vegetation type was more related to the association fraxino-ulmetum than to the association genisto elatae-quercetum. slavni] (1952) was among the first to point out that the community fraxino-ulmetum displays a significant floristic affiliation with the slavonian forest of pedunculate oak genisto elataeacta bot. croat. 72 (1), 2013 89 floodplain forests along the mura river 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:23 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees -quercetum roboris; however these are two separate systematic units. the community fraxino-ulmetum effusae inhabits the highest position of floodplain areas along large rivers, all these areas being exposed to periodical flooding. on the other hand, the community of genisto elatae-quercetum occurs in swamps along boggy backwaters, where mollic gleysol is formed (vukeli] and bari^evi] 2004). both the above described subassociations of association fraxino-ulmetum effusae correspond well to forests along the upper course of the river drava (around vara`din) in croatia near to slovenia described by vukeli] and bari^evi] (2004) as fraxino-ulmetum variant prunus padus (drier variant). as the difference between the variant prunus padus and variant typicum (more humid variant along the danube river and lower part of the sava river) described by the same authors, is significant, we propose a description of subassociations. zonation of forest vegetation can also be evidenced in a response curve of the main edifiers of plant communities (fig. 7a) on the gradient of dcs. salix alba and populus nigra, edifiers of association salicetum albae, are found close to streams and their occurrence decreases with the distance from the stream. the probability of occurrence for the tree species quercus robur and carpinus betulus, edifiers of the variant fraxino-ulmetum subass. quercetosum robori, increases with distance from the stream. the tree species fraxinus angustifolia does not respond to dcs, its probability of occurrence is equalized on the gradient of distances, and therefore gets a chance to become dominant in stands in the middle of the distance gradient (fraxino-ulmetum subass. allietosum ursini), where the occurrence of salix and populus falls and the increasing occurrence of quercus and carpinus is still low. response curve of main edifiers of plant communities in the gradient of dms on the other hand proved the already established fact that dms is not an underlying gradient for the zonation of forest vegetation. distribution of plant functional types along stream distance gradient the group of life forms is a good indicator of disturbance. short-living species (like therophytes) are favoured by continual disturbance, therefore cover value of therophytes is higher in flooded stand compared to unflooded stands. long-living species (geophytes, phanerophytes) on the other hand, benefit from the interruption of inundation or from long habitat continuity (glaeser and wulf 2009). in our case too, the proportion of therophytes in the relevés close to the stream is bigger and decreases with distance from it (fig. 6). that is because the stands close to the stream are more disturbed by floods, deposition and removal of nutrient material. other life forms show no statistical significant correlation with dcs as has been already found out also for some other plant communities along the moisture gradient (zelnik and ^arni 2008). riparian habitats are considered to be particularly prone to invasion by alien plant species (tickner et al. 2001). the expansion of neophytes (alien species introduced to the region after the year 1500 a.d.) in river-floodplain systems characterized the 20th century as human alterations to these systems, such as channel modifications, flow regulation, and floodplain drainage, intensified (schnitzler et al. 2007, tabacchi et al. 1996). many alien species are typically wetland species (zedler and kercher 2004, skaálova and py[ek 2009), and some of them have also high nutrient demands (schnitzler et al. 2007). close to the river the abundant supply of water and the high nutrient content of freshly deposited 90 acta bot. croat. 72 (1), 2013 ko[ir p., ^arni a., marin[ek a., [ilc u. 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:23 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees sediments combine with the typically plentiful light availability to promote the successful germination and establishment of alien species (sanchez-perez et al. 1993, schnitzler et al. 2003). as we tested the proportion of neophytes in the stands and their correlation to dcs and dms, surprisingly there were no significant correlations with dcs, but there was a significant correlation with dms (fig. 5). this means that bigger occurrence of neophytes is not connected to certain type of vegetation (willow stands because of their community architecture promote light in the understoreys) as has been reported (schnitzler and aumaitre 2008). also in the area of mura the best preserved willow stands are along side streams (and not along the main stream) as vegetation close to main stream is much more affected by different human impacts. a more important factor is the proximity of the main stream alone, which beside promotion of light (there are many open and disturbed areas near the main stream) assures active dispersal of rhizomes or stems (vegetative matter and seeds) deposited during floods and thereby encourages propagation (schnitzler et al. 2007, richardson et al. 2007). the flow of the main stream is quick and never drains up, in contrast to side streams. both groups of species (neophytes and therophytes) are characteristic of disturbed sites. the proportion of therophytes is significantly negatively correlated with the distance from the closest stream (dcs), which can be easily understood and has been observed and explained already by other authors. more surprising is that neophytes do not respond to environmental factors in the same way as therophytes. a proportion of neophytes does not correlate with the same distance – distance from the closest stream (dcs), but with distance from the main stream (dms). it seems that for establishment of neophytes, the propagation assured with the flow of the main stream is one of the most important factors beside disturbance itself. strong correlations of ecological strategy and moisture gradient have already been detected for some other vegetation types (zelnik and ^arni 2008, turner et al. 2004, glaeser and wulf 2009). relative elevation and distance from main channel, as indicators of flooding regime, were reported to be consistently important in predicting occurrence, community composition and abundance of species (turner et al. 2004, menges and waller 1983). in our case the distribution of plant ecological strategies indicates similar habitat conditions along transecst from the streams, as there is no statistical significant correlation of plant ecological strategies with dms and dcs. all relevés were made inside the area of frequent floods (inside dikes) on quite equalized elevations. in our study all three types of vegetation show similar proportions of plant ecological strategies in their compositions. conclusion the present study contributes basic data on the floristic composition and structure of floodplain forests of mura river. the zonation of floodplain forests is connected to distance from the closest stream (dcs) and not from the main stream (dms). distance from the closest stream influences species distribution through ecological gradients of moisture and nutrient. three types of forests have been detected along these gradients. the proportion of therophytes is significantly negatively correlated with the distance from the closest stream and the proportion of neophytes is significantly negatively correlated with distance from the acta bot. croat. 72 (1), 2013 91 floodplain forests along the mura river 608 kosir et al.ps u:\acta botanica\acta-botan 1-13\608 kosir et al.vp 14. o ujak 2013 10:41:23 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees main stream. the correlation of proportion of neophytes with dms can be explained by the importance of their effective propagation ensured by flow of the main stream. acknowledgements our study was supported by the ministry of agriculture, forestry and food and ministry of higher education, science and technology. it was carried out as part of a project v-40983: ecological circumstances as basis for forest economy and also as part of a research programme p1-0236: gradients and biodiversity. references accetto, m., 1994: moor and flooded forests. design of ecosystems division (in slovenian). oddelek za biologijo, biotehni{ka fakulteta, ljubljana. borhidi, a., kevey, b., 1996: an annotated checklist of the hungarian plant communities. in: borhidi, a. 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(1990) and set in the framework of a new suite of families and orders. a brief description was provided for each genus, but the format did not allow detailed discussion and more detailed accounts of each new or remodelled raphid genus were planned for separate publication. few of these have been completed, except for sellaphora (mann 1989), lyrella (mann and stickle 1993) and petroneis (jones et al. 2005). meanwhile, molecular biology has provided new tools to probe relationships at all taxonomic levels (e.g. mann and evans 2007), providing important new insights that quite frequently contradict the round et al. (1990) classification at the family level and above. among the new taxa described by round et al. (1990: 657) was the suborder sellaphorineae, which comprised two families: sellaphoraceae (sellaphora, fallacia, rossia and caponea) and pinnulariaceae (pinnularia, diatomella, oestrupia and the extinct genus dimidiata) (round et al. 1990: 128). small-subunit rdna (behnke et al. 2004, medlin and kaczmarska 2004, sorhannus 2007) and rbcl gene trees (evans et al. 2008) support the view that sellaphora, fallacia, rossia and pinnularia, together with some acta bot. croat. 68 (2), 2009 239 * corresponding author: d.mann@rbge.org.uk u:\acta botanica\acta-botan 2-09\mann.vp 9. listopad 2009 13:09:45 color profile: disabled composite 150 lpi at 45 degrees small-celled naviculoid species currently referred to mayamaea and eolimna (both described after 1990), comprise a monophyletic group. as noted by round et al. (1990: 657, 'sellaphorineae'), the raphe in sellaphoraceae and pinnulariaceae exhibits similar characteristics, with internal central endings deflected towards the primary side (except where hidden by extra silica deposition) and external central endings similarly deflected but also expanded. in this paper, we make a preliminary survey of protoplast structure in the sellaphorineae, as a first step to determine whether any characteristics are autapomorphic for the sellaphoraceae. material and methods sellaphorineae are generally epipelic. to obtain freshwater epipelon from shallow (< 1 m) sites, sediment and overlying water were collected using a glass tube as described by round (1953); deeper sediments were sampled using an ekman grab. for marine species, exposed damp sand was sampled at low tide. sample preparation was as described by mann and stickle (1993) and mann and chepurnov (2005). bright field (bf) and differential interference contrast (dic) light microscopy (lm: planapochromat lenses, nominal numerical aperture 1.32) were carried out using a reichert polyvar 1 photomicroscope and kodak technical pan film, or a polyvar 2 fitted with a polaroid dmc2 digital camera. film negatives were digitized using a nikon super coolscan 5000. contrast and brightness of digital and digitized images were manipulated in adobe photoshop cs2 (http://www. adobe.com/) by global application of the levels and curves tools; limited burn-in of the chloroplasts and valve detail in figures 14–28; and use of the unsharp mask filter at 60%, 4.5 pixels radius on final-size images at 300 dpi. the observations were made between october 1984 and august 2008, initially by a.j.s. (figs. 29–43) but after 1986 by d.g.m. (figs. 1–20, 44–52). results the interphase plastid structure was determined for selected species of sellaphora, fallacia and rossia within the sellaphoraceae. in order to check whether the characteristics found may occur outside the sellaphoraceae, we also studied pinnularia and diploneis species, to confirm and extend literature records. identifications follow mann et al. (2008) for sellaphora, hustedt (1964) for fallacia (as navicula spp.), and hendey (1964) for pinnularia. sellaphora bacillum (ehrenb.) d.g. mann (figs. 1–13): two optical sections of s. bacillum cells are provided, in girdle and valve views (figs. 1–7, 8–13, respectively), to facilitate understanding of chloroplast structure in sellaphoraceae. each cell contains a single saddle-like, h-shaped chloroplast (figs. 8, 9), which lies with its central isthmus against the epivalve. the single large pyrenoid is tetrahedral (here and in most other larger sellaphora species, it is usually close to being a regular tetrahedron) and lies eccentrically within the chloroplast with one vertex projecting through the central isthmus into the opposite chloroplast lobe. the outer edges of the pyrenoid are entire (figs. 1, 8), but the edge facing the nucleus is penetrated by a branching system of cavities and tubes (figs. 2–6, 9–11). the nucleus, which lies as a broad cytoplasmic bridge separating the two apical vac240 acta bot. croat. 68 (2), 2009 mann d. g., stickle a. j. u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:30 color profile: disabled composite 150 lpi at 45 degrees uoles is displaced towards the corner of the cell opposite the pyrenoid (figs. 7, 12). two prominent spherical volutin granules are present, one in each vacuole (figs. 5, 6, 13), usually associated with one of the raphe slits. acta bot. croat. 68 (2), 2009 241 cytology of sellaphoraceae figs. 1–13. sellaphora bacillum, living cells (ashford-in-the-water, derbyshire, england). 1–7 – successive foci of a cell in girdle view (epivalve at the top). the pyrenoid is entire and triangular in surface view (1) but penetrated by branching tubular channels (2–6) extending from the nucleus (6, 7); the cell also contains two prominent volutin granules (4–6, arrow). 8–13 – successive foci of a cell in valve view, from near the epivalve (8) to near the hypovalve (13). again, the pyrenoid is entire and triangular in surface view (8) and cytoplasmic invaginations become visible in deeper foci (10, arrow). the nucleus (with single spherical nucleolus, arrow 12) lies on the opposite side of the cell from the pyrenoid. scale bar = 10 mm. u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:34 color profile: disabled composite 150 lpi at 45 degrees sellaphora pupula (kütz.) mereschk. (fig. 49): cell structure in s. pupula and its allies (see mann et al. 2008 for a taxonomic review) is like that of s. bacillum (compare figures 8 and 49). the chloroplast is again saddle-like. the observation of this species led mereschkowsky (1902) to propose the genus. he unaccountably overlooked the prominent, tetrahedral and invaginated pyrenoid. the nucleus is eccentric. sellaphora pupula is the generitype. rossia (figs. 14–17): rossia valves bear lyre-shaped or linear, lateral areas, in which striae are present but more delicate than elsewhere. the lateral areas also bear small 'pegs', which appear to link to the edge of a very wide conopeum (sims and paddock 1979). however, in lm the raphe system does not possess the 'tear-drop' central endings that are characteristic of fallacia (compare figures 14, 21, 25 etc.). rossia was described by voigt (1960) and has rarely been reported. the species illustrated here is very similar to 'navicula hyalinula' sensu sims and paddock (1979) and needs to be transferred to (or described as a new species within) rossia. there is a single h-shaped chloroplast with an extremely narrow connection between the two sides (fig. 15). each side contains a slightly angular, invaginated pyrenoid (figs. 15–17). the nucleus is central, lying in a broad cytoplasmic bridge between the two principal vacuoles. near each pole, there is a ± spherical compartment of the vacuole surrounding a single large volutin granule (fig. 17). fallacia pygmaea (kütz.) stickle et d.g. mann (figs. 18–20): there is a single h-shaped chloroplast per cell, whose proximal (fig. 18) and distal (fig. 20) margins are plain or slightly undulate. the connection between the two sides of the chloroplast is narrow, but not as fine as in rossia. there is a small cushion-like pyrenoid embedded in the centre of each chloroplast lobe, penetrated by a small, little-branched cavity (fig. 19). the nucleus was slightly eccentric in the specimens seen (fig. 19). no volutin granules are present. fallacia pygmaea is the generitype. fallacia forcipata (grev.) stickle et d.g. mann type i (figs. 21–28): fallacia forcipata is a heterogeneous complex and we illustrate four demes here, each with its own chloroplast and frustule morphology (a valve is illustrated for each deme to facilitate future taxonomic revisions). in type 1, the chloroplast has a complex morphology. essentially, the chloroplast consists of two large, girdle-appressed plates. it is offset with respect to the apical plane, with the isthmus displaced to one side (fig. 24, arrow; contrast fig. 18) and the distal margins displaced in the opposite direction (fig. 22, arrow). the chloroplast isthmus bears two longitudinal extensions beneath the raphe system of the epivalve (figs. 24, 26), and two similar extensions are formed on the opposite side, by elaboration of the chloroplast margin (fig. 22). this arrangement persists during size reduction (figs. 21–24, 25–28). there are two pyrenoids per chloroplast, each flattened, angular and invaginated; they do not extend into the isthmus (fig. 24). the nucleus is eccentric (figs. 23, 28). no volutin granules are present. fallacia forcipata type ii (figs. 29–33): the chloroplast is simpler than in type i and has a simple or slightly undulate distal margin (compare figures 33 and 20); it is symmetrically placed with respect to the apical plane. unlike others studied here, it has a very wide isthmus (fig. 30), accommodating a transversely elongate invaginated pyrenoid (fig. 31) that is rectangular in valve view. there are two volutin granules, one in each vacuole. fallacia forcipata type iii (figs. 34–37): type iii cells (which have strikingly convergent striae at the centre: fig. 34) have a symmetrically placed chloroplast as in type ii, but possess two pyrenoids (fig. 36), as in type i. the pyrenoids are invaginated and have flat 242 acta bot. croat. 68 (2), 2009 mann d. g., stickle a. j. u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:34 color profile: disabled composite 150 lpi at 45 degrees tops, rather than the sloping profiles of type i (contrast figures 36 and 23). the chloroplast bears broad lobes alongside the isthmus (fig. 35) and on the opposite side (fig. 37); these have no longitudinal extension. the nucleus is eccentric (fig. 36). acta bot. croat. 68 (2), 2009 243 cytology of sellaphoraceae figs. 14–28. sellaphorineae, living interphase cells in valve view. 14–17 – rossia valve and successive foci (portobello beach, edinburgh); note the very narrow connection between the two sides of the chloroplast (15), the two invaginated pyrenoids (e.g. 16, arrow), and the two polar volutin granules (e.g. 17, arrow), each in a special vacuolar compartment. 18–20 – fallacia pygmaea (from a freshwater pond at ashford-in-the-water, derbyshire, england), with a simple h-shaped chloroplast and one small, cushion-like, invaginated pyrenoid (e.g. 19, arrow) on each side. 21–28 – fallacia cf. forcipata, type i: a large (21–24) and a small (25–28) cell from portobello beach, edinburgh. the longitudinal lobes of the complex chloroplast are connected beneath the epivalve (24, arrow) but not beneath the hypovalve (22, arrow). both sides of the chloroplast contain a flat, angular pyrenoid (e.g. 27, arrow), but the nucleus is nevertheless eccentric (28, arrow). scale bars = 10 mm (use the bar in 25 for all except 18–20). u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:36 color profile: disabled composite 150 lpi at 45 degrees 244 acta bot. croat. 68 (2), 2009 mann d. g., stickle a. j. figs. 29–43. fallacia, living cells in valve view. 29–33 – fallacia forcipata, type ii (cruden bay, near aberdeen, scotland), with a relatively simple chloroplast and ± central isthmus (30), a single central rectangular pyrenoid and prominent volutin granules (31), and eccentric nucleus (32). 34–37 – fallacia forcipata, type iii (cruden bay, near aberdeen, scotland), with convergent central striae (34), central plastid isthmus (35), transverse chloroplast lobes (35, 37) and one flat pyrenoid on each side (e.g. 36, arrow). 38–41 – fallacia forcipata, type iv (portobello beach, edinburgh), with narrow valves (38), longitudinal chloroplast lobes (39, 41), eccentric isthmus (39), and a single high tetrahedral pyrenoid (40, arrow). 42, 43 – postmitotic fallacia forcipata cells with valve-appressed chloroplasts (portobello beach, edinburgh). scale bars = 10 mm (use the bar in 43 for all except 42). u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:39 color profile: disabled composite 150 lpi at 45 degrees fallacia forcipata type iv (figs. 38–41): the principal cytological difference between this and type i is that type iv has only one pyrenoid, which is tetrahedral and lies to one side of the cell (fig. 40) as in sellaphora. longitudinal lobes are present beneath the raphe system (figs. 39, 41). fallacia cf. subhamulata (grun.) d.g. mann (figs. 44–48): the single h-shaped chloroplast is similar to that of sellaphora, except that the pyrenoid is more rounded (figs. acta bot. croat. 68 (2), 2009 245 cytology of sellaphoraceae figs. 44–48. fallacia cf. subhamulata, living cell in valve view (threipmuir reservoir, near edinburgh), showing the simple h-shaped chloroplast (45, 48), single eccentric, invaginated, cushion-like pyrenoid (45–47: arrow in 45), and eccentric nucleus (47, with nucleolus at arrow). 49 – sellaphora pupula sensu stricto (malham tarn, n england) with prominent tetrahedral pyrenoid and simple h-shaped chloroplast. 50 – pinnularia cf. cruciformis in girdle view (portobello beach, edinburgh): detail of pyrenoid, penetrated by numerous tubular channels. 51, 52 – marine diploneis species (portobello beach, edinburgh) in which the elongate (51) or cushion-like (52) pyrenoids are apparently invaginated (arrows). scale bars = 10 mm. u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:40 color profile: disabled composite 150 lpi at 45 degrees 45–47) with simpler invaginations. the chloroplast was asymmetrically placed with respect to the apical plane in the two specimens photographed (fig. 48). pinnularia cf. cruciformis (donkin) cleve (fig. 50): cells almost always lay in girdle view and possessed two girdle-appressed chloroplasts, each with a single panduriform pyrenoid at its centre (fig. 50), penetrated by > 100 simple tubular channels. diploneis spp. (figs. 51, 52): two marine diploneis species apparently had invaginated pyrenoids. the invaginations appeared as simple or branched fine striations. cell cycle changes: although the chloroplast morphologies described above were constant in tens to hundreds of interphase cells examined for each species, major changes in shape and position occur during the cell-cycle. these have been described in detail for sellaphora by mann (summarized 1989) and involve premitotic translational movement onto the girdle and postmitotic rotation during the development of the new chloroplast isthmuses. similarly, in fallacia, the chloroplast moves across the cell before cell division to lie beneath the valves with simultaneous simplification of shape (figs. 42, 43). discussion some of the observations reported here were used as the basis for the description of fallacia by round et al. (1990: 554), as follows: »plastid basically h-shaped, consisting of 2 girdle-appressed plates connected by a narrow isthmus lying against the epivalve; there may also be narrow lobes parallel to the raphe, extending out from the isthmus, and from one of the lateral plates. one or two invaginated pyrenoids present.« fallacia chloroplasts have also been described briefly by kuylenstierna (1989–1990), cox (1996) and sabbe et al. (1999), whose descriptions are consistent with our data, and by karsten (1899: 58, 59), who claimed that f. pygmaea and f. reichardtii (grun.) witkowski, lange-bertalot & metzeltin had two chloroplasts. we believe he was mistaken, because we collected several populations of the f. pygmaea species complex from fresh and brackish waters, all of which had a single h-shaped chloroplast, as does the morphologically similar f. hudsonis (cox 1996). cells kept in rough culture, as karsten's were, often accumulate large amounts of refractile reserve material in the vacuoles, which can obscure fine detail. fallacia, rossia and sellaphora therefore share the following cytological characteristics: (1) one chloroplast per cell, (2) chloroplast basically h-shaped, consisting of two large girdle-appressed plates connected by a central isthmus, and (3) pyrenoids invaginated. all sellaphora species studied so far have a simple chloroplast containing a single offset pyrenoid, which is clearly tetrahedral in the larger-celled species (> 10 mm long). fallacia is more variable than sellaphora in chloroplast shape and position (exactly centred with respect to the apical plane or slightly displaced); pyrenoid number (one or two) and shape (regular tetrahedron to flattened rectangular to rounded); and pyrenoid position (usually in the lateral plates but sometimes central). some fallacia species resemble sellaphora in having a simple chloroplast and a single eccentric pyrenoid (compare figures 44–48 with 49), but the agreement is not exact (e.g. f. cf. subhamulata has rounded, not strictly polyhedral pyrenoids). the single rossia species examined is unusual among sellaphoraceae for its extremely narrow chloroplast isthmus, the exactly central nucleus, and the special compartment in each vacuole, containing a volutin granule. whether this arrangement is typical of other rossia species is unknown (the type of rossia is r. elliptica m. voigt, which is apparently unreported since its discovery). 246 acta bot. croat. 68 (2), 2009 mann d. g., stickle a. j. u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:41 color profile: disabled composite 150 lpi at 45 degrees fallacia and lyrella were formerly classified together in navicula sect. lyratae because of the presence in both of a lyre-shaped area of plain or only faintly striated silica. the separation of these genera in round et al. (1990), which is supported by rbcl data (jones et al. 2005, evans et al. 2008), was based on the presence or absence of a conopeum, the kind of pore occlusion, nuclear behaviour during successive cell divisions, and a »completely different plastid structure and arrangement«. the single fallacia chloroplast occupies the whole of the girdle (except small areas close to the poles) and, as shown here, consists of two large girdle-appressed plates, connected by an isthmus lying against one valve (apparently always the epivalve, as in sellaphora). in lyrella, on the other hand, the chloroplast or chloroplasts lie almost wholly against the valves, leaving the girdle free (mann and stickle 1993, 1997), as in the related genus petroneis (jones et al. 2005), and the pyrenoids are never invaginated. the pinnulariaceae, closely related to the sellaphoraceae according to molecular data (see introduction), are not as uniform in cell structure. some species possess two large girdle-appressed chloroplasts per cell, including pinnularia cf. cruciformis (illustrated here) and other marine species (e.g. mereschkowsky 1901, pl. 1, figs. 14, 21, 22; mann 1996, fig. 8), and some freshwater species (e.g. heinzerling 1908, pl. 1, figs. 1, 12; tschermakwoess 1953, figs. 1, 7; cox 1996, fig. 22; schmid 2003; poulí^ková et al. 2007, poulí^ková and mann 2008). other pinnulariaceae have h-shaped chloroplasts with an isthmus against one valve (e.g. heinzerling 1908, pl. 1, fig. 16; tschermak-woess 1953, figs. 2g, 5, 6; cox 1996, fig. 26), as in sellaphoraceae, and the diplastidic caloneis amphisbaena exhibits a transient phase during the cell cycle in which there is a single valve-appressed, h-shaped chloroplast (thaler 1972, fig. 7e). many pinnulariaceae, both diplastidic and monoplastidic, possess invaginated pyrenoids (tschermak-woess 1953, thaler 1972, mann 1996, fig. 8, schmid 2001: 10, poulí^ková et al. 2007, poulí^ková and mann 2008). the invaginations of the c. amphisbaena pyrenoid were studied in thin sections by walker et al. (1979), edgar (1980) and schmid (2001). all shown by edgar (1980) contained extensions of the cytoplasm, but schmid (2001: 10) states that some are extensions only of the periplastidial compartment, i.e. they are lined by the two inner plastid membranes but not the two outer 'chloroplast endoplasmic reticulum' membranes (unfortunately, membranes are not visible in schmid’s figures 44 and 45). no other invaginated pyrenoids have been examined ultrastructurally. schmid (2001) suggested that this type of pyrenoid represents »significant surface enlargement for localized activity in bi-directional transport phenomena between host and chloroplast (endosymbiont)«, presumably largely in relation to the pyrenoid’s function as a key site for carboxylation (e.g. roberts et al. 2007). outside the sellaphorineae, invaginated pyrenoids are very rare. we have not detected them in any other marine or freshwater genera except diploneis, two species of which we illustrate here. s.j.m. droop also recorded invaginated pyrenoids in other diploneis species (archived photographs, royal botanic garden edinburgh) and tschermak-woess (1953) illustrated them in species identified as diploneis domblittensis var. subconstricta a. cleve (in which the invaginations were identical to those shown in our figure 51) and d. oculata (bréb.) cleve; however, the drawing of the d. oculata valve given by tschermakwoess (1953, fig. 4d) suggests misidentification of fallacia pygmaea or f. hudsonis. so far, no molecular data have been published for diploneis species, many of which are diffiacta bot. croat. 68 (2), 2009 247 cytology of sellaphoraceae u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:41 color profile: disabled composite 150 lpi at 45 degrees cult to establish in culture (our unpublished observations), and there are few other pointers to a close relationship between the sellaphorineae and diploneis, apart from the pyrenoid structure. it is not inconceivable, however, that the porous conopea of fallacia (round et al. 1990) and rossia (sims and paddock 1979) could be homologous to the outer wall in diploneis. hence it is unclear whether the invaginated pyrenoids of sellaphorineae are an autapomorphy for the suborder or symplesiomorphic for a broader grouping including diploneis. references behnke, a., friedl, t., chepurnov, v. a., mann, d. g., 2004: reproductive compatibility and rdna sequence analyses in the sellaphora pupula species complex (bacillariophyta). journal of phycology 40, 193–208. bruder, k., medlin, l. k., 2007: molecular assessment of phylogenetic relationships in selected species/genera in the naviculoid diatoms (bacillariophyta). i. the genus placoneis. nova hedwigia 85, 331–352. cox, e. j., 1996: identification of freshwater diatoms from live material. chapman & hall, london. edgar, l. a., 1980: fine structure of caloneis amphisbaena (bacillariophyceae). journal of phycology 16, 62–72. evans, k. m., wortley, a. h., simpson, g. e., chepurnov, v. a., mann, d. g., 2008: a molecular systematic approach to explore diversity within the sellaphora pupula species complex (bacillariophyta). journal of phycology 44, 215–231. heinzerling, o., 1908: der bau der diatomeenzelle mit besonderer berücksichtigung der ergastischen gebilde und der beziehung des baues zur systematik. bibliotheca botanica 69, 1–88. hendey, n. i., 1964: an introductory account of the smaller algae of british coastal waters, part v: bacillariophyceae (diatoms). h.m.s.o., london. hustedt, f., 1964: die kieselalgen deutschlands, österreichs und der schweiz. in: dr l. rabenhorsts kryptogamenflora von deutschland, österreich und der schweiz, 7 (3: 3), 349–556. akademische verlagsgesellschaft, leipzig. jones, h. m., simpson, g. e., stickle, a. j., mann, d. g., 2005: life history and systematics of petroneis (bacillariophyta), with special reference to british waters. european journal of phycology 40, 43–71. karsten, g., 1899: die diatomeen der kieler bucht. wissenschaftliche meeresuntersungen, new series (kiel), 4, 17–205. kuylenstierna, m., 1989–1990: benthic algal vegetation in the nordre älv estuary (swedish west coast). phd thesis, university of göteborg. mann, d. g., 1989: the diatom genus sellaphora: separation from navicula. british phycological journal 24, 1–20. mann, d. g., 1996: chloroplast morphology, movements and inheritance in diatoms. in: chaudhary, b. r., agrawal, s. b. (eds): cytology, genetics and molecular biology of algae, 249–274. spb academic publishing, amsterdam. 248 acta bot. croat. 68 (2), 2009 mann d. g., stickle a. j. u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:41 color profile: disabled composite 150 lpi at 45 degrees mann, d. g., chepurnov, v. a., 2005: auxosporulation, mating system, and reproductive isolation in neidium (bacillariophyta). phycologia 44, 335–350. mann, d. g., evans, k. m., 2007: molecular genetics and the neglected art of diatomics. in: brodie, j., lewis, j. r. (eds), unravelling the algae – the past, present and future of algal systematics, 231–265. crc press, boca raton, florida. mann, d. g., stickle, a. j., 1993: life history and systematics of lyrella. nova hedwigia 106, 43–70. mann, d. g., stickle, a. j. 1997: sporadic evolution of dorsiventrality in raphid diatoms, with special reference to lyrella amphoroides sp. nov. nova hedwigia 65, 59–77. mann, d. g., thomas, s. j., evans, k. m., 2008: revision of the diatom genus sellaphora: a first account of the larger species in the british isles. fottea 8, 15–78. medlin, l. k., kaczmarska, i., 2004: evolution of the diatoms: v. morphological and cytological support for the major clades and a taxonomic revision. phycologia 43, 245–270. méreschkowsky, c., 1901: études sur l'endochrome des diatomées. mémoires de l'académie impériale des sciences de st.-pétersbourg, ser. 8, 11, 1–40. mereschkowsky, c., 1902: on sellaphora, a new genus of diatoms. annals and magazine of natural history, ser. 7, 9, 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in the systematics of the diatoms: their mor1–31. schmid, a. -m. m., 2003: endobacteria in the diatom pinnularia (bacillariophyceae). i. »scattered ct-nucleoids« explained: dap–dna complexes stem from exoplastidial bacteria boring into the chloroplasts. journal of phycology 39, 122–138. sims, p. a., paddock, t. b. b., 1979: observations and comments on some prominent morphological features of naviculoid genera. nova hedwigia 64, 169–191. sorhannus, u., 2007: a nuclear-encoded small-subunit ribosomal rna timescale for diatom evolution. marine micropaleontology 65, 1–12. acta bot. croat. 68 (2), 2009 249 cytology of sellaphoraceae u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:41 color profile: disabled composite 150 lpi at 45 degrees phology and ultrastructure. proceedings 16 international diatom symposium, athens thaler, e., 1972: beitrag zur entwicklungsgeschichte und zum zellbau einiger diatomeen. österreichische botanische zeitschrift 120, 313–347. tschermak-woess, e., 1953: über auffallende strukturen in den pyrenoiden einiger naviculoideen. österreichische botanische zeitschrift 100, 160–178. voigt, m., 1960: some new diatoms from the far east. journal of the royal microscopical society, ser. 3, 78, 92–94. walker, g. k., sicko-goad, l., stoermer, e. f., 1979: an ultrastructural examination of the pennate diatom caloneis amphisbaena. microbios letters 12, 141–152. 250 acta bot. croat. 68 (2), 2009 mann d. g., stickle a. j. u:\acta botanica\acta-botan 2-09\mann.vp 5. listopad 2009 15:23:41 color profile: disabled composite 150 lpi at 45 degrees opce-str.vp acta bot. croat. 73 (1), 255–265, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 short communication colchicum cupanii guss. subsp. glossophyllum (heldr.) rouy, datura innoxia mill. and eclipta prostrata (l.) l., new floristic records in montenegro and western balkan danka cakovi]1*, danijela ste[evi]1, sne@ana vuksanovi]2, kit tan3 1 university of montenegro, faculty of science, department of biology, dzordza vashingtona bb, 81000 podgorica, montenegro 2 natural history museum of montenegro, trg vojvode be}ir bega osmanagi}a, 81000 podgorica, montenegro 3 institute of biology, university of copenhagen, øster farimagsgade 2d, dk-1353 copenhagen k, denmark abstract – during field investigations on long ulcinj beach, montenegrin coast, three taxa new to montenegrin flora were collected: colchicum cupanii guss. subsp. glossophyllum (heldr.) rouy, datura innoxia mill. and eclipta prostrata (l.) l. the first species is native to europe, while the others are alien. colchicum cupanii subsp. glossophyllum is an endemic of the balkan peninsula, with a distribution formerly restricted to greece and albania, so this new record extends its distribution to the north-west. the distribution of eclipta prostrata in the balkans is also enlarged by this new record for montenegro, in a westerly direction. key words: balkan, colchicum cupanii, datura innoxia, eclipta prostrata, flora, montenegro introduction if the richness of the flora of an area is expressed by the ratio of number of species per square meter, montenegro has the richest flora of all european countries (stevanovi] et al. 1995). the number of species and subspecies of vascular plants in montenegro exceeds 3600 (rohlena 1942, pulevi] 2005, ste[evi] et al. 2008). the flora of some parts of the country is not comprehensively known, or is not fully documented for all seasons, so new records are not uncommon in the country (guterman 2012, petrovi] and ste[evi] 2011). accordingly the number of 3600 vascular plant taxa may yet prove to be an underestimation for montenegro. acta bot. croat. 73 (1), 2014 255 * corresponding author, e-mail: danka.petrovic@t-com.me copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:01 color profile: generic cmyk printer profile composite default screen between april 2010 and november 2012 several field trips were undertaken in the coastal part of montenegro, with the goal of filling floristic gaps in former investigations and searching for additions to the flora of montenegro. the most interesting data were gathered during the survey of the long ulcinj beach and its hinterland and form the subject of this paper. this coastal region has attracted the attention of botanists since the beginning of the 19th century (rohlena 1942, pulevi] 1976, ble^i] and pulevi] 1979, trinajsti] 1989, mijovi] 1994, laku[i] et al. 2004, petrovi] and vuksanovi] 2005, vuksanovi] and petrovi] 2007), but due to absence of continual and systematic investigations its flora is still incompletely known. material and methods plant material was collected during field investigations of the montenegrin coast, in period from april 2010 to november 2012. each location where plants were recorded were geo-coded by using a gps device garmin e-trex vista c. field data about the habitats, population size and threat assessments for each species were also obtained. voucher specimens were deposited in the tgu (voucher numbers 319392-319396) and in the herbarium collection of the natural history museum of montenegro (voucher numbers 4082-4085). identification of materials was conducted according to rouy (1905), tutin et al. (1972, 1976), pignatti (1982) and persson (2007). the climate of the area is presented by the walter climate diagram (fig. 1) and additional climatic data have been obtained from the weather reporting station in ulcinj, for the period 1961–1990. natura 2000 habitats are adjusted according to the interpretation manual of european union habitats (european commission 256 acta bot. croat. 73 (1), 2014 cakovi] d., ste[evi] d., vuksanovi] s., tan k. fig. 1. walter climate diagram of ulcinj. 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:02 color profile: generic cmyk printer profile composite default screen directorates general 2007). red list was specified according to the iucn criteria (iucn 2011). the index of floristic richness is expressed by the ratio log n/log a (log of the number of species and subspecies/log of the surface area). study area at approximately 12 km, long ulcinj beach is the longest beach on the adriatic coast, at the south-eastern end of which it is located (fig. 2). this area is bordered by the channel of port milena to the west and the bojana river to the east. it belongs to the wide ulcinj plain, frequently exposed to very strong winds that carry sand from the shore inland, so sand covers a major part of the area (mijovi] 1994). the average width of the beach and the hinterland is 1.5 km, and the northern border is the regional road. within the area of long beach, quaternary formations are strongly developed, represented by alluvial formations and sands on the beaches. alluvial sediments develop in the estuary of the bojana river and in larger parts of long beach, where the alluvium is made of pebbles, sand, mud and sand-clay, i.e. materials which form the catchment area. sandy beach sediments form on places where the sea penetrates softer rocks and creates space for the accumulation of the result of the erosion of these rocks. these are present along the length of long beach. the climate is of the mediterranean type, characterized by cool, wet winters and hot, dry summers (fig. 1). in some years july and august are completely without rainfall. according to investigations ten natura 2000 habitats were recorded: annual vegetation of drift lines (1210), mediterranean salt meadows (juncetalia maritime 1410), embryonic shifting dunes (2110), shifting dunes along the shoreline with ammophila arenaria (white dunes 2120), humid dune slacks (2190), dunes with euphorbia terracina (2220), brachypodietalia dune grasslands with annuals (2240), wooded dunes with pinus pinea and/or pinus pinaster (2270), mediterranean temporary ponds (3170), and salix alba and populus alba galleries (92a0). in addition a number of anthropogenically modified habitats are present. this great habitat diversity results in huge floristic richness. acta bot. croat. 73 (1), 2014 257 new floristic records in montenegro fig. 2. geographical position of long ulcinj beach (basic map: afe_basemap, with permission of the atlas florae europaea). 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:03 color profile: generic cmyk printer profile composite default screen results and discussion during field investigations of the coastal part of montenegro, three new taxa for montenegrin flora were collected on long ulcinj beach: colchicum cupanii guss. subsp. glossophyllum (heldr.) rouy, datura innoxia mill. and eclipta prostrata (l.) l. colchicum cupanii guss. subsp. glossophyllum (heldr.) rouy in the field survey conducted at the beginning of november 2012, one unfamiliar colchicum species, producing leaves and flowers together, was found (fig. 3). prior to this investigation, the only colchicum species in montenegro that were known to produce postfloral leaves were c. autumnale l., c. bivonae guss, and c. haynaldii heuff. (rohlena 1942, pulevi] 2005). after detailed study of the collected material, the plant was confirmed as colchicum cupanii subsp. glossophyllum (rouy 1905; persson 2007, personal communication). in the european flora colchicum cupanii is represented by two subspecies: cupanii and glossophyllum. the distribution of the typical subspecies cupanii is in the apennine, s france, cyclades, s aegean islands, and nw africa, while the subspecies glossophyllum is considered an endemic of the balkan peninsula, with a distribution restricted to greece and albania (euro+med, 2006–). accordingly, this new record extends the known distribution area to the north-west. on long ulcinj beach c. cupanii subsp. glossophyllum was recorded in three localities, placed in the belt transect of 1.5 km. in each case the substrate was sandy, but the habitat type differed. in the first locality (n 41° 54’ 33”, e 19° 15’ 23.3”), four individuals of colchicum were present. the habitat was identified as natura 2000 – 2270 wooded dunes with pinus pinea and/or pinus pinaster. in the tree layer the dominant species were pinus halepensis miller. and pinus pinaster aiton, while in the shrub and herb layer the following species were common: quercus coccifera l., myrtus comminus l., smilax aspera 258 acta bot. croat. 73 (1), 2014 cakovi] d., ste[evi] d., vuksanovi] s., tan k. fig. 3. colchicum cupanii subsp. glossophyllum (photo: d. ste{evi}). 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:06 color profile: generic cmyk printer profile composite default screen l., crataegus monogyna jacq., rubus ulmifolius schott., ruscus aculeatus l., asparagus acutifolius l., pteridium aguilinum l. in the vicinity of the colchicum individuals, species such as stellaria media (l.) cirillo, teucrium capitatum l., teucrium chamaedrys l., geranium purpureum vill., geranium villosum ten., holoschoenus vulgaris link, verbascum sinuatum l. were found. in a second locality (distant ca 300 m from the first locality), up to 10 individuals of this plant were recorded as constituenst of roadside vegetation on the border with the natura 2000 habitat 2270 wooded dunes with pinus pinea and/or pinus pinaster. accompanying species: erodium cicutarium (l.) l’hér, tunica saxifraga (l.) scop., medicago arabica (l.) huds., helianthemum obscurum pers., alkanna tinctoria tausch, asphodelus microcarpus viv. the third locality (n 41° 53’ 18.6”, e 19° 18’ 52.7”) was also near the local road, but next to the fringe of a humid dune slack (natura 2000 habitat 2190). the number of individuals was up to 10. accompanying species: ephedra distachya l., artemisia campestris l., erodium cicutarium, tunica saxifraga, cynodon dactylon (l.) pers. voucher specimens: montenegro: long ulcinj beach n 41° 54’ 33”, e 19° 15’ 23.3”, 10 m a.s.l., 2270 wooded dunes with pinus pinea and/or pinus pinaster, (cakovi}, d., ste{evi}, d., 2.11. 2012., tgu319392); long ulcinj beach n 41° 54’ 12”, e 19° 16’ 11.2”, 10 m a.s.l., 2270 wooded dunes with pinus pinea and/or pinus pinaster, (cakovi}, d., ste{evi}, d., 2.11. 2012., tgu319393); long ulcinj beach n 41° 53’ 18.6”, e 19° 18’ 52.7”, 15 m a.s.l., 2190 humid dune slack, (vuksanovi}, s., ste{evi}, d., cakovi}, d. 9.11.2012., tgu319394). threats, endangered status and protection in the last 10 years long ulcinj beach and its hinterland has been exposed to intensive anthropogenic pressure, mainly tourism development and urbanization. in order to improve tourist facilities, the construction of buildings, roads, parking places and beach furniture has intensified. in some cases small buildings are constructed right on the beach sand. this has resulted in an increase of waste and the spread of alien species. taking into account all the named threats, the survival of this species is questionable, and the authors recommend its protection by national legislation. the population numbers less than 50 mature individuals, thus applying the iucn red list categories and criteria (iucn 2011) and red list categories and criteria at the regional level (iucn 2003) the current national status of colchicum cupanii subsp. glossophyllum in montenegro is critically endangered (cr d1). datura innoxia miller in may 2010, on waste ground in the hinterland of long ulcinj beach (n 41° 53’ 52.2”, e 19° 17’ 58.2”, fig. 4) a couple of individuals of datura, with significantly bigger flowers and more grayish appearance than neighboring individuals of datura stramonium l., were collected. this species was identified as datura innoxia. identification was confirmed during the fruiting period. the capsule was egg shaped, directed downwards and three cm in diameter, with numerous slender and spines of almost equal-length up to four mm. in the same localacta bot. croat. 73 (1), 2014 259 new floristic records in montenegro 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:06 color profile: generic cmyk printer profile composite default screen ity several alien species were present: phytolacca americana l., chenopodium multifidum l., datura stramonium, conyza albida spreng. and conyza canadensis (l.) cronquist. monitoring of this population since may 2010 to november 2012 did not show any significant invasion into the surrounding hinterland. this year another population of datura innoxia was found in dobre vode, 25 km away from the first locality. this annual plant originates from central america and is widespread in europe. although flora europaea (moore 1972) does not include species from the balkans, some literature sources indicate that this species has been known from the sofia district (bulgaria) since 1933 (greuter and raus 2005). in greece, the first documented voucher was by a. yannitsaros in october 1966. he collected plants from a rubbish-tip on waste ground on the south peloponnesian island of kithira (published in yannitsaros 1998: 88, as datura metel l.). in october 1972 the plant was recorded from the east coastal mainland of greece (nea makri in attikis, greuter 10614, ath). in crete, datura innoxia has been recorded from roadsides in the district of merabellou; it was collected by a. hansen in october 1973 (hansen 1063, c). later it was recorded from the ionian island of kerkira, n, s and e peloponnese, ne aegean islands of thasos and samothraki, e aegean islands and the cyklades. in greece the plant is mainly distributed in coastal places, along roads, on waste ground, in gardens, etc. in arkadias it has been found further inland and at a higher altitude (150 m) on waste ground by a fertilizer plant (kit tan and strid 18710, herb. strid). the species was reported in croatia approximately 30 years ago (he]imovi] 1981). populations of datura innoxia have spread not only to a number of locations in the littoral region of croatia but also within the continental parts (he]imovi] 1982, [tamol and markovi] 1985, franji] 1993, pand@a and stan^i] 1990), so the inland part of montenegro should not be excluded from the monitoring of the spread of this species in future. datura innoxia spreads along roads, in courtyards, gardens and in ruderal vegetation as an ergasiophygophyte (pand@a et al. 2001). in agricultural and forest lands where it appears as the principal weed, datura innoxia may cause extensive and expensive damage (trinajsti] et al. 1993, franji] 260 acta bot. croat. 73 (1), 2014 cakovi] d., ste[evi] d., vuksanovi] s., tan k. fig. 4. datura innoxia, habitus and fruits (photo: d. ste{evi}). 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:08 color profile: generic cmyk printer profile composite default screen and trinajsti] 1996, franji] et al. 1998). up to now, in montenegro this species has not been reported for cultivated land, but due to its capacity to invade such habitats it can be considered a potential threat. our records show that datura innoxia is spreading in the mediterranean part of the balkan peninsula and thus can be expected in albania as well. voucher specimens: montenegro: long ulcinj beach n 41° 53’ 52.2”, e 19° 17’ 58.2”, 15 m a.s.l., waste ground (cakovi}, d., ste{evi}, d., 9.11.2012., tgu319395). eclipta prostrata (l.) l. during the field survey in the hinterland of long ulcinj beach, eclipta prostrata (fig. 5) was found at only one location (n 42° 53’ 19.0” e 19° 18’ 42.6”), ca 300 m inland, in the small ruderal patch in the humid dune slack zone (natura 2000 habitat – 2190, ass. eriantho-schoenetum nigricantis (pignatti 1953) géhu in géhu et al. 1984). part of the population was in a small water-filled depression, with water up to 10 cm, while the remainder of the population was on humid sandy soil. approximately 30 mature individuals were found. in addition to eclipta l. numerous adventive species were present, indicating a strong anthropogenic influence at this site: conyza canadensis, paspalum paspalodes (michx) scribner, euphorbia maculata l., sporobolus poirettii (r. et s.) hitchc, oenothera biennis l., xanthium italicum moretti, aster squamatus (spreng) hieron. elements of native flora: medicago arabica, cynodon dactylon, holoshoenus vulgaris, potentilla reptans l., lythrum salicaria l. as long ulcinj beach hosts large numbers of migrating bird (it is one of the most important bird areas of montenegro, czip, 2008) we conclude that eclipta prostrata was probably introduced to this locality by birds. eclipta is a small genus in the family compositae (tribe heliantheae), of which several species are distributed primarily in the new world and some in the old world (umemoto acta bot. croat. 73 (1), 2014 261 new floristic records in montenegro fig. 5. eclipta prostrata (photo: d. ste{evi}). 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:10 color profile: generic cmyk printer profile composite default screen and koyama 2007). currently there is only one species in europe: eclipta prostrata (euro+med 2006). this species is widely distributed across all continents, excluding antarctica, and tends to remain in the tropics and sub-tropics, but can also be found in temperate regions (holm et al. 1977) and can therefore adapt to changing environmental conditions. it is most aggressively invasive in warm, low-lying areas with high rainfall but it can also tolerate saline conditions, higher altitudes and drier sites. prefers full or partial sun and tolerates both fine and coarse-textured soils. eclipta prostrata has low fertility requirements, high anaerobic tolerance, and low drought tolerance (usda). opinions about its origin differ significantly. tutin (1976) consider eclipta prostrata native for tropical and warm-temperate america, while stone (1970) suggested a probable asiatic origin. up to 1976 in europe this species was reported only for italy, spain and portugal (tutin 1976). it has since appeared in belgium, bulgaria, cyprus, greece, romania, turkey, and ukraine. (euro+med 2006). in the balkan peninsula e. prostrata was collected in greece for the first time in 2001, in a rice field south of kalamata airport in messinia, s. peloponnese (raus and raabe, 2002). a few years later it was recorded from the e. aegean island of rhodes (a collection by ristow and collaborators on 10 january 2004, personal communication). in september 2010, it was recorded from the island of poros, off the coast of the e. peloponnese in the se part of the main town, on a coastal embankment near an irrigation channel (seregrin and kit tan, personal communication). in cyprus it was reported by della and iatrou (1995). since then it has shown a trend of slow expansion along irrigation channels and cultivated land and its status in greece is now naturalized non-invasive species (hand 2009). in bulgaria e. prostrata was first reported in 2007, close to the riverbank in vegetation which belongs to the class isoeto-nanojuncetea br.-bl. et tuxen 1943 (tzonev 2007). up to now this species has not been found in any neighboring balkan countries, so this find represents the new western boundary of its balkan distribution and illustrates the tendency of this species to spread within the mediterranean region. somewhat surprisingly, this rather unsightly species has been used in various parts of tropical and sub-tropical regions like south america, asia, africa, as a medicinal and edible plant (bown 1995, cheryl 2007, khan and khan 2008, dalal et al. 2010). voucher specimens: montenegro: long ulcinj beach n 42° 53’ 19.0” e 19° 18’ 42.6”, 2190 humid dune slack, ruderal patch (cakovi}, d., ste{evi}, d., vuksanovi}, s., 9.11.2012., tgu319396). acknowledgements the authors would like to thank dr karin persson for valuable help in identification of colchicum cupanii subspecies, and discussions about its ecology and distribution, and dr anton dresher for help with the literature. the field investigation that took place in the autumn of 2012 was funded by undp office in montenegro (project number mne 12-057). references ble^i], v., pulevi], v., 1979: some new data in the flora of montenegro. glasnik republi~kog zavoda za za{titu prirode i prirodnja~kog muzeja 12, 189–193. 262 acta bot. croat. 73 (1), 2014 cakovi] d., ste[evi] d., vuksanovi] s., tan k. 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:10 color profile: generic cmyk printer profile composite default screen bown, d., 1995: encyclopedia of herbs and their uses. dorling kindersley, london. cheryl, l., 2007: comparison of plants used for skin and stomach problems in trinidad and tobago with asian ethnomedicine. journal of ethnobio and ethnomedicine 3, 1–12. czip, 2008: iba areas in montenegro. retrieved march 9, 2013 from http://www.birdwatchingmn.org/index.php?lng=cg&page=61#plaza dalal, s., kataria, s. k., sastry, k., rana, s. v. s., 2010: phytochemical screening of methanolic extract and antibacterial activity of active principles of hepatoprotective herb eclipta alba. ethnobotanical leaflets 14, 248–58. della, a., iatrou, g., 1995: new plant records from cyprus. kew bulletin 50, 387–396. euro+med, 2006: euro+med plantbase – the information resource for euro-mediterranean plant diversity. retrieved march 9, 2013 from http://ww2.bgbm.org/europlusmed european commission directorates general, 2007: interpretation manual of european union habitats, 2007. european commission directorate general for the environment nature and biodiversity, brussels. franji], j.,1993: new findings of datura innoxia miller (solanaceae) in croatia. acta botanica croatica 52, 97–100. franji], j.,trinajsti], i., 1996: current state of the distribution of the species datura inoxia miller (solanaceae) in croatia. fragmenta phytomedica et herbologica 24, 5–9. franji], j., trinajsti], i., [kvorc, @., 1998: a contribution to the knowledge of the spreding of some neophytes in croatia. fragmenta phytomedica et herbologica 26, 5–17. greuter, w., raus, t., 2005: med-checklist notulae, 23. willdenowia 35, 55–64. guterman, w., 2012: a note on pteris vittata l. 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[tamol, v., markovi], lj., 1985: a contribution to the flora of bra~ island. acta botanica croatica 44, 99–106. trinajsti] i., 1989: a contribution to the knowledge of the vegetation of coastal sandbars class ammophiletea b.bl. et tx 1943 in the montenegrian coast. canu – glasnik odjeljenja prirodnih nauka 7, 45–51. trinajsti], i., pavleti], z., frani], j., liber, z.,1993: contribution to the knowledge of the neophytic flora of makarska littoral (dalmacija, hrvatska). fragmenta phytomedica et herbologica 21, 57–62. 264 acta bot. croat. 73 (1), 2014 cakovi] d., ste[evi] d., vuksanovi] s., tan k. 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:10 color profile: generic cmyk printer profile composite default screen tutin, t. g., heywood, v., burges, n. a., moore, d. m., valentine, d. h., walters, s. m.,webb, d. a., (eds.) 1972: flora europaea 3. university press, cambridge. tutin, t. g., 1976: eclypta l. in tutin, t. g., heywood, v., burges, n. a., moore, d. m., valentine, d. h., walters, s. m.,webb, d. a., (eds.). flora europaea 4,141. university press, cambridge. tutin, t. g., heywood, v., burges, n. a., moore, d. m., valentine, d. h., walters, s. m.,webb, d. a., (eds.) 1976: flora europaea 4. university press, cambridge. tzonev, r., 2007: eclipta prostrata (asteraceae): a new alien species for the bulgarian flora. phytologia balcanica 13, 79–80. umemoto, s., koyama, h., 2007: a new species of eclipta (compositae: heliantheae) and its allies in eastern asia. thai forest bulletin (botany) 35, 108–118. usda. ars, national genetic resources program. germplasm resources information network – (grin). national germplasm resources laboratory, beltsville, maryland. retrieved march 9, 2013 from http://plants.usda.gov/java/namesearch?keywordquery= eclipta+prostrata&mode=sciname&submit.x=6&submit.y=7 vuksanovi], s., petrovi], d., 2007: the occurrence of kickxia cirrosa (l.) fritsch in montenegro supports the earliest of this species for the balkan peninsula. bocconea 21, 201–205. yannitsaros, a., 1998: additions to the flora of kithira (greece) i. willdenowia 28, 77– 94. acta bot. croat. 73 (1), 2014 265 new floristic records in montenegro 816 cakovic et al.ps u:\acta botanica\acta-botan 1-14\816 cakovic et al.vp 24. o ujak 2014 11:38:10 color profile: generic cmyk printer profile composite default screen acta botanica 2-2014.indd acta bot. croat. 73 (2), 2014 359 acta bot. croat. 73 (2), 359–373, 2014 coden: abcra 25 issn 0365-0588 eissn 1847-8476 responses of wheat (triticum aestivum l.) and maize (zea mays l.) plants to cadmium toxicity in relation to magnesium nutrition nataša nikolić*, milan borišev, slobodanka pajević, milan župunski, mirjana topić, danijela arsenov department of biology and ecology, faculty of sciences, trg dositeja obradovića 2, 21000 novi sad, serbia. abstract – the infl uence of cadmium (cd) on physiological processes in wheat (triticum aestivum l.) and maize (zea mays l.) plants exposed to either optimal mineral nutrition or the absence of magnesium (mg) as well as the accumulation of cadmium and magnesium in plant organs were studied using the method of water culture in a greenhouse. cd treatment reduced shoot fresh mass more strongly in mg-supplied than in mg-defi cient plants. negative effect of cd on photosynthetic activity was more pronounced in t. aestivum than in z. mays plants. cd treatment decreased leaf chlorophyll and carotenoid concentration in both z. mays and t. aestivum, irrespective of the mg supply. cd was preferentially accumulated in the roots of both species. catalase activity in t. aestivum leaves and roots was unaffected by cd and mg supply. cd treatment did not affect fe accumulation in the leaves of either species, while in the roots a considerable increase occurred, irrespective of the mg nutrition. higher tolerance of z. mays and t. aestivum plants to cd toxicity exposed to mg defi ciency could partly be ascribed to the preservation of fe nutrition. keywords: cadmium toxicity, growth, magnesium supply, photosynthetic activity, pigments, triticum aestivum, tolerance, zea mays introduction heavy metals are important and widespread pollutants in the environment. due to their toxicity to most living organisms, control of the emission and accumulation of these contaminants into the biosphere has become a main task worldwide. among other non-essential heavy metals, cadmium (cd) has attracted considerable scientifi c attention due to its potential to endanger human health (tran and popova 2013). soil contamination with cd may come either from different anthropogenic sources including application of sewage sludge, wastewater, fertilizers, pesticides, as well as traffi c and industrial activities, or from natural * corresponding author, e-mail: natasa.nikolic@dbe.uns.ac.rs copyright® 2014 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. nikolić n., borišev m., pajević s., župunski m., topić m., arsenov d. 360 acta bot. croat. 73 (2), 2014 sources. cd is readily taken up by roots and translocated to different plant organs (amirjani 2012). due to its high mobility in the phloem (benavides et al. 2005), cd may impede the acquisition of essential nutrients by various plant parts. considering the role of nutrients in the creation and maintenance of the structure and function of plant cells (wahid et al. 2007), optimal plant growth may be disrupted by the presence of cd. this metal is generally considered a highly toxic element with negative effects on plant growth and development, photosynthesis, transpiration, stomatal regulation, enzymatic activities, water relation, mineral elements uptake, protein metabolism, and membrane functioning (tran and popova 2013). possible mechanisms involved in the generation of the above disorders are induction of oxidative stress and replacement of essential elements such as zn, fe, and mn, as they are cofactors of many enzymes (seregin and ivanov 2001, lópez-millán et al. 2009). the antioxidant compounds and antioxidative enzymes play important roles in the improvement of stress tolerance in plants (noctor and foyer 1998). heavy metals are known to increase activity of antioxidant enzymes (siddiqui 2013). metalloenzymes (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase) protect cellular components such as proteins, membrane lipids and nucleic acids, against oxidative injury caused by reactive oxygen species (halliwell and gutteridge 1989). magnesium (mg) is an essential macroelement for plants as it is a constituent of the chlorophyll molecule (bose et al. 2011). in addition, it plays an important role in the metabolism and translocation of carbohydrates, the production of oils and fats, activation of enzymes involved in the synthesis of nucleic acids, regulation of the other essential elements uptake, and transport of phosphate compounds throughout the plant (tucker 1999). this implies that conservation of optimal mg levels in organs of plants exposed to cd stress postpones the toxicity effects of the heavy metal and improves plant performance. earlier research has pointed to perturbation of both uptake and transport of different cations, including mg, in various plant species exposed to cd (yang et al. 1996). extensive studies on the level of mg supply in alleviation of heavy metal toxicity have been undertaken recently. the results have shown that both mg supply (10 mmol l–1) and mg defi ciency may extenuate cd toxicity in brassica rapa l. var. pervirdis and arabidopsis thaliana, respectively (kashem and kawai 2007, hermans et al. 2011). negative effects of cd on growth, physiological traits and biochemical processes in triticum aestivum and zea mays plants have been described previously (florijn and van beusichem 1993, lagriffoul et al. 1998, ozturk et al. 2003, lin et al. 2007, ci et al. 2010). to our knowledge, these species have not been studied in relation to cd stress under mg defi ciency. therefore, the aim of the present study was to explore the role of this element in t. aestivum and z. mays tolerance to cd toxicity in relation to plant growth, rate of photosynthesis and transpiration, synthesis of chlorophylls and carotenoids, catalase activity, proline accumulation, and accumulation and translocation of cd, fe and mg. the study was conducted on plants exposed to optimal and defi cient mg supply. materials and methods plant growth and treatments wheat (triticum aestivum l.) and maize (zea mays l.) seeds were surface sterilized with 3% h2o2 for 15 minutes and washed extensively with distilled water. the seeds were germinated in plastic boxes fi lled with sterilized sand, particle size between 0.01 and 0.075 responses of wheat and maize to cadmium in relation to magnesium acta bot. croat. 73 (2), 2014 361 mm. the seedlings were grown in a growth chamber at constant temperature of 25 °c and 16 h light/8 h dark photoperiod for twelve days. after that period, morphologically uniform plants were selected and transferred to 0.4 l pots containing reid-york nutrient solution (reid and york 1958), and cultivated in a greenhouse for 21 days. the f ollowing treatments were set: a) suffi cient mg supply without cd (control), b) suffi cient mg supply with cd (cd), and c) mg starvation with cd (cd-mg). nutrient solution with suffi cient mg supply contained the following macroand micro-elements: 1.0 mmol l–1 kh2po4, 5.0 mmol l –1 kcl, 4.0 mmol l–1 cacl2 × 2 h2o, 2.0 mmol l –1 mgso4 × 7 h2o, and 7.0 mmol l –1 nh4no3 (reid and york 1958). defi ciency of mg was achieved by replacing mgso4 with 2.0 mmol l–1 k2so4 in order to maintain the supply of sulfur. cd stressed plants were supplied with 0.1 μmol l–1 of cadmium added as cdcl2. the nutrient solutions were continuously aerated and were replaced every 5 days. all parameters were measured after 21 days of the treatments. rates of photosynthesis and transpiration were measured in vivo. the fi rst three fully expanded leaves from the top of the plants were sampled and immediately used for analysis of photosynthetic pigments, catalase activity and proline accumulation. photosynthetic pigments, rates of photosynthesis and transpiration the chlorophyll a, chlorophyll b and carotenoid contents were determined according to wettstein (1957) after extraction in absolute acetone and expressed as mg g–1 dw. rates of photosynthesis and transpiration were measured in vivo on intact leaves, using a lc pro+ portable photosynthesis system (adc bioscientifi c ltd., uk). during the measurement, the saturated photosynthetically active radiation (par) with photon fl ux density of 1000 μmol m–2 s–1 was emitted, and the fl ow rate of ambient air to the leaf chamber was 100 μmol s–1. all parameters were measured between 10:00 and 12:00 a.m., during a clear sunny day. biomass production and tolerance index plants were harvested after 21 days of cultivation, and fresh mass of shoots and roots was measured. t. aestivum and z. mays susceptibility to cd stress in mg-supplied and mgstarved plants in relation to control was evaluated using the tolerance index (ti). this parameter was calculated as follows: ti = fresh weight of treated plants × 100 / fresh weight of control plants (wilkins 1978). determination of mg, cd and fe following the biomass measurements, leaves and roots were oven-dried at 80 °c till the constant weight. concentrations of mg, cd and fe were determined by employing fl ame atomic absorption spectrophotometry (varian, aas240fs). the plant material was digested using closed vessel high pressure microwave digestion (milestone d series microwave digestion system). for all treatments and both plant species, three independent replicates were used for analysis of metal accumulation in leaves and roots. the ability of plants to translocate mg, cd and fe from the roots to the shoots in relation to mg and cd supply is indicated by the translocation factor (tf). this factor was calculated according to zacchini et al. (2009): tf = concentration of the heavy metal in the shoot × 100 / concentration of the heavy metal in the root. nikolić n., borišev m., pajević s., župunski m., topić m., arsenov d. 362 acta bot. croat. 73 (2), 2014 determination of catalase activity and free proline accumulation in leaves the leaf samples were homogenized using mortar and pestle in the presence of liquid nitrogen, and extracted in 5 ml of 50 mm phosphate buffer, ph 7.0, containing 1 mm ethylenediaminetetraacetic acid (edta) and 1% (w/v) polyvinylpyrrolidone (pvp). the extract was centrifuged at 4 °c at 15,000 rpm for 15 minutes. the supernatants were used for determination of catalase (cat) activity according to the method of claiborne (1984). cat activity was determined by the rate of hydrogen peroxide disappearance measured spectrophotometrically at 240 nm. the total protein concentration in the supernatant was determined using the method of bradford (1976) with bovine serum albumin as a standard. free proline content in leaves was measured according to bates et al. (1973). leaf samples were homogenized in 3% (w/v) sulfosalicylic acid and centrifuged for 10 minutes at 10,000 rpm. the reaction mixture consisted of 2 ml of supernatant, 2 ml of acid ninhydrin reagent (1.25 g ninhydrin in 30 ml of glacial acetic acid and 60 ml of 6 m phosphoric acid) and 2 ml of glacial acetic acid. mixtures were boiled at 100 °c for 30 minutes and then they were transferred to an ice bath. toluene (4 ml) was added to each sample to extract the proline-ninhydrin compound. the absorbance of the chromophore-containing toluene was measured spectrophotometrically (spectrophotometer du-65 beckman) at 520 nm. the proline concentration was calculated from a standard curve and expressed on a fresh weight basis as μg proline g–1. statistical analysis the data were processed statistically by the analysis of variance. differences between treatments were analyzed following duncan’s multiple range test at p < 0.05. results biomass production and plant tolerance to cd cd treatment negatively affected shoot fresh mass in both species (tab. 1). in t. aestivum, a signifi cant reduction of 67.75% was recorded upon cd treatment in comparison to the control. z. mays shoots mass was lowered in treated plants, by 71.98 and 27.68% at cd and cd−mg treatments, respectively. shoot mass was less affected by cd toxicity in both z. mays and t. aestivum plants exposed to mg starvation. in t. aestivum, root fresh mass was not affected by cd, irrespective of mg supply. a considerable decrease of 35.24% of the z. mays root mass was observed at cd treatment. the tolerance indices based on the biomass production were higher in plants exposed to mg starvation (tab. 1). photosynthetic pigments, rates of photosynthesis and transpiration chlorophyll a concentration was considerably lower in cd stressed t. aestivum and z. mays plants, irrespective of mg supply (tab. 2). chlorophyll b was decreased in t. aestivum plants exposed to mg starvation; however, mg nutrition had no effect on the chlorophyll b level in z. mays plants experiencing cd stress. with respect to control, the chlorophyll b concentration was lower in both groups of z. mays plants exposed to cd stress. concentration of carotenoids was diminished in both species exposed to cd. responses of wheat and maize to cadmium in relation to magnesium acta bot. croat. 73 (2), 2014 363 tab. 1. effect of cd on plant growth and tolerance measured in mg-supplied (cd) and mg-starved (cd-mg) z. mays and t. aestivum plants. values are means of fi ve independent measurements ± sd. data in parentheses present the percentage of control values. statistically different values in each row are indicated by different letters according to duncan’s test (p < 0.05). control cd cd-mg t. aestivum shoot fresh mass (g) 1.12 ± 0.50 a 0.35 ± 0.06 b (31) 0.87 ± 0.06 a (78) root fresh mass (g) 0.30 ± 0.12 a 0.22 ± 0.08 a (73) 0.22 ± 0.06 a (73) tolerance index (%) 100.00 ± 0.00 aaa 41.99 ± 10.80 caa 80.57 ± 18.27 baa z. mays shoot fresh mass (g) 5.96 ± 0.53 a 1.67 ± 0.14 c (28) 4.31 ± 0.21 b (72) root fresh mass (g) 2.27 ± 0.31 a 1.47 ± 0.26 b (65) 2.08 ± 0.29 a (92) tolerance index (%) 100 ± 0.00 a 38.09 ± 3.50 caaa 77.50 ± 3.98 baaa tab. 2. effect of cd on photosynthetic pigments concentration, rate of photosynthesis (a) and transpiration (e), leaf proline accumulation and cat activity of leaves and roots in mg-supplied (cd) and mg-starved (cd-mg) z. mays and t. aestivum plants. values are means of at least three independent measurements ± sd. data in parentheses present the percentage of control values. statistically different values in each row are indicated by different letters according to duncan’s test (p < 0.05). control cd cd–mg t. aestivum chlorophyll a (mg g-1 dw) 13.83 ± 0.77 a 7.76 ± 0.80 b (56) 7.32 ± 0.03 b (53) chlorophyll b (mg g-1 dw) 3.80 ± 0.17 a 2.87 ± 0.34 ab (76) 2.26 ± 0.08 b (59) carotenoids (mg g-1 dw) 3.82 ± 0.18 a 2.43 ± 0.26 b (64) 2.30 ± 0.02 b (60) a (μmol of co2 m -2 s-1) 24.97 ± 0.29 a 8.92 ± 1.37 b (36) 10.05 ± 1.63 b (40) e (mmol of h2o m -2 s-1) 2.92 ± 0.26 a 2.54 ± 0.34 a (87) 2.51 ± 0.24 a (86) proline (μg g-1 fw) 4.51 ± 0.62 b 6.77 ± 0.05 b (150) 14.28 ± 1.83 a (317) cat activity (u mg-1 protein) of leaves 1.31 ± 0.29 a 0.96 ± 0.09 a (73) 1.04 ± 0.18 a (79) of roots 0.14 ± 0.07 a 0.30 ± 0.08 a (214) 0.21 ± 0.07 a (150) z. mays chlorophyll a (mg g-1 dw) 26.10 ± 1.66 a 13.22 ± 0.84 b (51) 14.49 ± 0.09 b (56) chlorophyll b (mg g-1 dw) 6.34 ± 0.34 a 3.89 ± 0.39 b (61) 3.76 ± 0.03 b (59) carotenoids (mg g-1 dw) 5.38 ± 1.63 a 2.85 ± 1.11 b (53) 4.77 ± 0.28 ab (89) a (μmol of co2 m -2 s-1) 33.03 ± 1.59 a 27.10 ± 1.80 b (82) 22.92 ± 1.63 c (69) e (mmol of h2o m -2 s-1) 3.47 ± 0.03 a 3.21 ± 0.16 b (92) 3.17 ± 0.15 b (91) proline (μg g-1 fw) 7.31 ± 1.05 a 6.13 ± 0.51 a (84) 6.36 ± 1.34 a (87) cat activity (u mg-1 protein) of leaves 0.21 ± 0.07 a 0.20 ± 0.08 a (95) 0.30 ± 0.07 a (143) of roots 0.35 ± 0.07 ab 0.41 ± 0.32 a (117) 0.28 ± 0.03 b (80) nikolić n., borišev m., pajević s., župunski m., topić m., arsenov d. 364 acta bot. croat. 73 (2), 2014 exposure of t. aestivum and z. mays plants to cd stress accompanied with different levels of mg elicited a decrease of photosynthetic rate (tab. 2). the negative effect of cd on photosynthetic activity was more pronounced in t. aestivum (decrease by 64.28 and 59.75% at cd and cd–mg treatments, respectively) than in z. mays (decrease by 47.95 and 30.61% at cd and cd–mg treatments, respectively). with respect to control, transpiration was not considerably changed in t. aestivum, while in z. mays plants a decrease of about 8% occurred in both treatments. accumulation and translocation of mg, cd and fe cd preferentially accumulated in the roots of t. aestivum and z. mays plants (tab. 3). mg defi ciency did not affect cd accumulation in leaves. cd concentration in roots was lower in t. aestivum plants exposed to mg starvation in relation to mg supplied plants, while the optab. 3. effect of cd on magnesium, iron and cadmium concentrations and translocation factors in mg-supplied (cd) and mg-starved (cd-mg) z. mays and t. aestivum plants. values are means of three independent measurements ± sd. statistically different values in each row are indicated by different letters according to duncan’s test (p < 0.05). control cd cd-mg t. aestivum magnesium leaves (μg g-1) 3571.67 ± 64.44 b 4660.33 ± 49.74 a 708.33 ± 49.79 c roots (μg g-1) 1819.00 ± 15.00 b 2484.00 ± 6.41 a 962.00 ± 32.06 c translocation factor 196.37 ± 4.60 a 187.63 ± 2.46 b 73.64 ± 5.17 c iron leaves (μg g-1) 396.67 ± 59.32 a 307.00 ± 8.39 a 320.33 ± 16.64 a roots (μg g-1) 1788.00 ± 17.41 c 3850.00 ± 45.51 b 5026.67 ± 168.39 a translocation factor 22.17 ± 3.26 a 7.97 ± 0.14 b 6.38 ± 0.48 b cadmium leaves (μg g-1) 0.00 ± 0.00 b 818.6 ± 17.02 a 894.00 ± 37.85 a roots (μg g-1) 0.00 ± 0.00 c 1468.67 ± 34.13 a 1338.67 ± 34.64 b translocation factor 0.00 ± 0.00 c 55.73 ± 0.22 b 66.79 ± 2.16 a z. mays magnesium leaves (μg g-1) 2005.80 ± 127.13 b 3736.13 ± 84.05 a 1156.42 ± 17.75 c roots (μg g-1) 2460.25 ± 29.00 a 2537.50 ± 351.81 a 751.42 ± 18.38 b translocation factor 81.57 ± 6.08 b 149.40 ± 23.49 a 154.00 ± 6.08 a iron leaves (μg g-1) 345.46 ± 98.08 a 288.58 ± 18.63 a 347.50 ± 57.72 a roots (μg g-1) 601.92 ± 7.62 c 2037.17 ± 10.85 a 1715.50 ± 181.01 b translocation factor 57.53 ± 16.92 a 14.16 ± 0.84 b 20.62 ± 5.52 b cadmium leaves (μg g-1) 0.00 ± 0.00 b 399.42 ± 6.39 a 416.58 ± 76.94 a roots (μg g-1) 0.00 ± 0.00 c 2618.17 ± 21.81 b 3611.33 ± 309.87 a translocation factor 0.00 ± 0.00 b 15.26 ± 0.37 a 11.72 ± 3.19 a responses of wheat and maize to cadmium in relation to magnesium acta bot. croat. 73 (2), 2014 365 posite trend occurred in z. mays roots. differences in root cd accumulation probably results from variability in translocation of the metal between these species. the translocation factor was higher in mg-starved t. aestivum plants. in t. aestivum, mg concentration in leaves was higher than in roots in control and cdtreated plants (tab. 3). a signifi cant increase of mg accumulation in t. aestivum in both shoots and roots was observed during cd treatment with respect to control as well in z. mays leaves (tab. 3). in t. aestivum plants experiencing mg starvation, higher concentration of mg was measured in roots than in leaves, suggesting lower translocation of the element from roots to shoots (tab. 3). accumulation of fe was stimulated in roots of t. aestivum and z. mays plants under cd stress, and translocation of this element was lower than in control plants. catalase activity in leaves and roots generally, higher cat activity has been observed in leaves than in roots of t. aestivum, while the opposite trend occurred in mg-supplied z. mays plants (tab. 2). cat activity in t. aestivum leaves and roots was unaffected by cd and mg supply. however, decreased cat activity was recorded in z. mays roots in plants exposed to mg starvation. discussion disorders in plant growth, photosynthetic activity, chlorophyll and carotenoid concentration, and mineral nutrition occurred in t. aestivum and z. mays plants exposed to cd. responses of the studied plants to the toxic effect of cd depended on the mg supply. growth inhibition is the most general non-specifi c symptom of heavy metal stress (pál et al. 2006). heavy metals affect plant growth either through direct interaction with cell wall polysaccharides or indirectly, provoking perturbation of metabolic processes (seregin and ivanov 2001). in the present study, cd affected shoot fresh mass of t. aestivum and z. mays plants (tab.1). shoot biomass production was lower in mg-supplied plants (cd) than in those exposed to mg starvation (cd–mg), suggesting the protective role of mg defi ciency. the results are in line with the fi ndings of chou et al. (2011), who examined the effect of mg starvation on cd toxicity in rice seedlings, using higher concentration of cd (5 µmol l–1 cdcl2). the authors concluded that mg starvation protected rice seedlings from the cd toxicity, estimating the decrease of biomass production and chlorophyll content, and induction of oxidative stress. root growth is more susceptible to heavy metals than shoot growth, which is correlated with the predominant accumulation of the metals in the roots of many plant species (reviewed in seregin and ivanov 2001). in the present work, cd did not affect root growth in t. aestivum plants, irrespective of mg supply. however, root growth in z. mays was reduced by 35.24% at cd treatment, with respect to control. furthermore, the tolerance index, based on biomass production of treated and control plants, suggested higher susceptibility to cd toxicity in both species cultivated under mg suffi cient conditions, than in plants exposed to mg starvation. according to the sensitivity scale proposed by lux et al. (2004), z. mays and t. aestivum plants exposed to cd under optimal mg nutrition and mg defi ciency showed medium and high tolerance, respectively. the protective effect of mg defi ciency against cd toxicity could be ascribed to the maintenance of fe status, the increase in antioxidative capacity, detoxifi cation and ⁄or protection of the photosynthetic apparatus (hermans et al. 2011). nikolić n., borišev m., pajević s., župunski m., topić m., arsenov d. 366 acta bot. croat. 73 (2), 2014 cd is a non-redox metal, unable to produce reactive oxygen species (ros) such as superoxide radicals (o2˙¯), hydrogen peroxide (h2o2) and hydroxyl radicals (˙oh) via fenton and/or haber weiss reactions (salin 1988). this metal was found to produce oxidative stress indirectly, by interfering with the antioxidant defense system (benavides et al. 2005). disorders in enzymatic and non enzymatic antioxidant mechanisms with concomitant increase in ros accumulation were observed in pisum sativum exposed to cd (rodríguezserrano et al. 2006). cd reduced the glutathione and ascorbate contents, and activities of catalase, glutathione reductase and guaiacol peroxidase. therefore, maintaining of the balance between ros generation and scavenging under heavy metal stress contributes to plant tolerance. cat activity was not considerably changed in t. aestivum and z. mays leaves exposed to cd (tab. 2). these results are not in line with the fi ndings of chou et al. (2011), who demonstrated activities of antioxidative enzymes, including cat, that were higher in mg-defi cient than in mg-suffi cient rice leaves. however, szalai and co-workers (2005) observed low and unchanged cat activity in z. mays inbred lines exposed to cd. in the present work, a negative effect of cd and mg starvation upon cat activity was evident in z. mays roots (tab. 2), and the result may be related to decrease of fe concentration. cat is a metalloenzyme, containing the porphyrin prosthetic group, and fe plays an important role in its activity. the strong correlation between the presence of fe and the activity of the metalloenzymes, cat and ascorbate peroxidase, has been previously observed in borage offi cinalis (mohamed and aly 2004) and in hydroponically cultivated rice (chalmardi and zadeh 2013). the cat activity was reduced in salvinia natans exposed to cd (mohan and hosetti 2006). proteins form complexes with cd, with concomitant changes of the protein conformation and solubility. the authors concluded that the decline in cat activity in salvinia natans was the consequence of the interaction between the protein and cd. considering the role of cat in the decomposition of toxic h2o2 to water and oxygen, decrease of its activity may result in the h2o2 increase in z. mays root cells, since cd stimulates h2o2 accumulation (semane et al. 2010). cd is known to affect photosynthetic pigments concentration in numerous plant species (amirjani 2012). in the present study, cadmium decreased the concentration of leaf chlorophylls and carotenoids in both z. mays and t. aestivum, and differences between cd and cd–mg treatments were not statistically signifi cant (tab. 2). these results suggest that effect of cd was more noticeable on the chlorophyll and carotenoid concentrations than that of the lack of mg, although it is the central atom of the chlorophyll molecule. in their work with water plants, kupper et al. (1998) reported that cd replaced the central mg ion in the chlorophyll structure. in the experiment with young z. mays plants, lagriffoul et al. (1998) concluded that the inhibition of electron transport at the level of the water-splitting complex and simultaneous decrease in chlorophyll pigment contents may have resulted from the interaction between cd and mn. concentration of carotenoids was higher in z. mays leaves during cd-mg treatment in comparison to cd treatment, but these differences were not statistically signifi cant (tab. 2). carotenoids are important light-harvesting pigments, able to quench triplet chlorophyll and remove reactive oxygen species, protecting chlorophyll and membranes from destruction (amirjani 2012). according to the present results, increased carotenoid concentration could have contributed to the higher tolerance of z. mays to cd toxicity in mg-starved plants. furthermore, it has been reported that cd may disturb fe metabolism in plants, resulting in induced fe defi ciency despite optimal availability of fe in the growth substrate (sidlecka and krupa 1999). fe is important in the formation of precurresponses of wheat and maize to cadmium in relation to magnesium acta bot. croat. 73 (2), 2014 367 sors of the chlorophyll molecule, i.e. δ-aminolevulinic acid and protochlorophyllide (mar schner 1986). in the present study, decreased chlorophylls content in z. mays and t. aestivum leaves exposed to cd was not attributable to fe defi ciency, since the concentration of fe in leaves of both species was unaffected by the treatments. previous studies showed that cd regulates chlorophyll synthesis due to interaction with chlorophyll-synthesizing enzymes. experiments with hordeum vulgare showed that interaction of cd with sh-groups may inhibit activity of protochlorophyllide reductase, with concomitant decrease of chlorophyll concentration (stobart et al. 1985). in the experiments with phaseolus vulgaris l. seedlings treated with different concentrations of cadmium acetate under both light and dark growth conditions, padmaja et al. (1990) showed that cd inhibited 5-aminolevulinic acid synthesis and ala-dehydratase activity. cd decreased photosynthetic rate more markedly in t. aestivum than in z. mays, irrespective of the mg supply (tab. 2). the decline of the photosynthetic rate under cd stress can result from various factors: the disturbance of chloroplast ultrastructure, synthesis of chlorophyll, plastoquinone, and carotenoids, electron transport, activity of the calvin cycle enzymes, co2 fi xation, and stomatal conductance (ernst 1980, seregin and ivanov 2001, xue et al. 2013). in the present work, a decrease in the photosynthetic activity may be partly due to the reduced chlorophyll content in both species. previous investigations depicted strong reduction of the stomatal conductance in cd treated plants (baryla et al. 2001). the depression of photosynthesis in z. mays plants may be attributed, at least partly, to stomatal closure, considering the decreased transpiration rate in plants exposed to cd. these results are in line with the report of bazzaz et al. (1974), who found a direct correlation between the cd concentration and the inhibition of net photosynthesis and transpiration in z. mays, indicating the impact of cd on stomatal resistance. in present work, the reduction of growth in z. mays and t. aestivum plants exposed to cd might be the consequence of inhibition of carbon fi xation due to a decrease of photosynthetic rate and chlorophyll content, as reported also by hassan et al. (2005). however, it seems that reduction of the photosynthetic rate in the cd-exposed t. aestivum plants results from non-stomatal limitations, which is in accordance with the observations of xue et al. (2013) in glycine max. cd affects the uptake, translocation, and subsequent distribution of nutrient elements in plants (tran and popova 2013). disorders of mineral nutrition evoked by high levels of cd lead to nutrient defi ciencies, oxidative burst, and consequent depression of plant growth and development (nazar et al. 2012). decreased photosynthesis and plant growth strongly correlated with nutrient disorders under cd exposure (sun and shen 2007). it is considered that disbalance in acquisition of nutrients results from the competition of cd with other cations in their transport across membranes (llamas et al. 2000). cd uses the same cation transport systems as the essential elements, because of the lack of specifi city of these proteins (lópezmillán et al. 2009), such as members of zip and nramp families (perfus-barbeoch et al. 2002). the uptake of cd seems to occur mainly via ca2+, fe2+, mn2+, and zn2+ transporters (clemens 2006). however, cd-induced inhibition of the uptake of macroand micronutrients relies on the size of metal ion radii and disorders in the cell metabolism with concomitant changes in membrane enzyme activities and membrane structure (seregin and ivanov 2001). in the present study, the cd concentration was higher in roots of both z. mays and t. aestivum than in their leaves (tab. 3). nontolerant plants and tolerant excluders mainly accumulate cd in roots (verbruggen et al. 2009). generally, the most of the cd accumulated nikolić n., borišev m., pajević s., župunski m., topić m., arsenov d. 368 acta bot. croat. 73 (2), 2014 in the root remains in this organ, and only 2% is transported to leaves in higher plants (xue et al. 2013). there were no considerable differences in cd concentration of leaves between mg-supplied and mg-starved t. aestivum and z. mays plants. these results suggest that mg supply did not considerably affect cd accumulation in the plant leaves and that the higher tolerance of mg-starved plants was not related to decreased accumulation of cd. the obtained results disagree with the fi ndings of chou et al. (2011), who reported a shoot cd concentration of mg–defi cient seedlings higher than that of mg-suffi cient rice seedlings. however, additional mg in the nutrient solution (10 mmol l–1) may enhance the growth of plants suffering from cd toxicity, due to reduction in cd concentration in the plant (kashem and kawai 2007). higher concentration of cd in roots of mg-supplied t. aestivum plants than in plants exposed to mg starvation probably results from lower translocation of the heavy metal. in z. mays, the translocation factors have not differed in relation to mg supply. higher cd concentration occurred in plants exposed to mg defi ciency. these results suggest a speciesspecifi c response of z. mays and t. aestivum plants experiencing cd toxicity with respect to mg nutrition. however, differences in cd distribution within plants occur also between genotypes. in the experiment with z. mays inbred lines florijn and van beusichem (1993) distinguished genotypes with higher cd concentration in the roots than in shoots from genotypes with similar cd concentration in the shoots and roots. furthermore, a considerable difference in cd concentration among plant parts and genotypes, with the highest concentration in roots, was described in t. aestivum genotypes in response to cd addition (zhang et al. 2002). in neither species did cd affect fe accumulation in the leaves, while a considerable increase occurred in the roots, irrespective of the mg supply (tab. 3). a contradiction exists between these results and the fi ndings of other authors. the increase in the fe concentration of leaves in cd-treated plants exposed to mg defi ciency was observed by hermans et al. (2011), while fodor et al. (2005) reported decreased fe concentration in shoots of cd treated populus alba. enhanced accumulation of fe in z. mays and t. aestivum roots observed in the present work could be the consequence of lower translocation of the element from roots to aerial plant parts. depression of fe translocation followed by increased root fe concentrations has been found in cucumis sativus exposed to cd (fodor et al. 1996). fe limits cd uptake and translocation, and prevents disturbances in plant growth, photosynthetic activity and photosynthetic pigments accumulation, and alleviates cd toxicity (nazar et al. 2012). however, increased fe concentration in roots of t. aestivum and z. mays plants suggests that cd treatments impeded assimilation of fe in the roots, considering the same translocation factors for both treatments. similar observations were reported for nicotiana tabacum plants exposed to cd (yoshihara et al. 2006). in the present work, signifi cant increase of mg concentrations in leaves occurred in cdtreated plants, with respect to the control (tab. 3). however, in t. aestivum and z. mays roots, the values were considerably and negligibly increased, respectively, during the same treatment. the literature data are inconsistent regarding the mg accumulation in certain plant organs. in brassica juncea, mg concentration of the roots was unaffected, while the concentration of the shoots increased slightly by cd exposure (jiang et al. 2004). in a pot experiment with different rice genotypes exposed to cd stress, liu et al. (2003) investigated the absorption and accumulation of cd, fe, zn, mn, cu and mg in roots and shoots, at both heading and ripening periods. no signifi cant correlation between cd and mg was found in responses of wheat and maize to cadmium in relation to magnesium acta bot. croat. 73 (2), 2014 369 roots at the two periods. however, while in the leaves cd showed no signifi cant correlation with mg at heading, a signifi cant positive correlation appeared at the ripening stage. soil contamination with cadmium increased the content of mg and ca in aboveground parts and roots of lupinus arboreus (wyszkowski 2002). no signifi cant change in mg content in z. mays leaves or roots in response to the cd accumulation was observed (lagriffoul et al. 1998). the incongruity of the literature data related to disorders in the mineral elements uptake and distribution is probably the result of several factors: specifi c response of certain species and genotypes exposed to cd stress, cd concentration applied, and experimental conditions. optimal plant nutrition is a good strategy in alleviation of the deleterious effects of cd on plants and can contribute to the increase of plant tolerance to cd (sarwar et al. 2010). maintenance of uptake and transport of mg to shoots may contribute to the chlorophyll biosynthesis and protect photosynthetic system under cd stress (gomes et al. 2013). heavy metals inhibit the formation of root hairs, causing disbalance in plant water relationships (pál et al 2006). the stimulation of proline accumulation in plants exposed to excess heavy metals has been reported in other species and might be related to cd induced changes of water regime. osmolytes, including proline, can improve leaf relative water content, scavenge reactive oxygen species and protect the integrity of membranes (changhai et al. 2010). in the present study, the accumulation of proline was stimulated by cd only in t. aestivum, irrespective of the mg supply (tab. 3). the osmotic adjustment under waterdefi cient conditions plays a crucial role in maintaining high transpiration rates (damatta et al. 2003), and this is in agreement with the results obtained in the present work. in conclusion, mg starvation contributed to higher tolerance of z. mays and t. aestivum plants to cd toxicity. the results obtained might partly be ascribed to the preservation of fe nutrition, as reported for other plant species. further investigations are needed to elucidate other mechanisms involved in alleviation of cd toxicity in z. mays and t. aestivum plants. acknowledgements this study was conducted within the project no. iii 043002 and iii 043007, supported by the ministry of education, science and technological development of the republic of serbia. references amirjani, м. r., 2012: effects of cadmium on wheat growth and some physiological factors. international journal of forest, soil and erosion 2, 50–58. baryla, a., carrier, p., franck, f., coulomb, c., sahut, c., havaux, m., 2001: leaf chlorosis in oilseed rape plants (brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth. planta 212, 696–709. bates, i. s., waldren, r. p., teare, i. d., 1973: rapid determination of free proline for water-stress studies. plant and soil 39, 205–207. bazzaz, f. a., rolfe, g. l., windle, p., 1974: differing sensitivity of corn and soybean photosynthesis and transpiration to lead contamination. journal of environmental quality 3,156–158. nikolić n., borišev m., pajević s., župunski m., topić m., arsenov d. 370 acta bot. croat. 73 (2), 2014 benavides, m. p., gallego, s. m., tomaro, m. l., 2005: cadmium toxicity in plants. brazilian journal of plant physiology 17, 21–34. bose, j., babourina, o., rengel, z., 2011: role of magnesium in alleviation of aluminium toxicity in plants. journal of experimental botany 62, 2251–2264. bradford, m. m., 1976: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analytical biochemistry 72, 248–254. chalmardi, z. k., zadeh, a. a., 2013: role of silicon in alleviation of iron defi ciency and toxicity in hydroponically-grown rice (oryza sativa l.) plants. journal of science and technology of greenhouse culture 3, 79–88. changhai, s., baodi, d., yunzhou, q., yuxin, l., lei, s., mengyu, l., haipei, l., 2010: physiological regulation of high transpiration effi ciency in winter wheat under drought conditions. plant, soil and environment 56, 340–347. chou, t.-s., chao, y.-y., huang, w.-d., hong, c.-y., kao, c. h., 2011: effect of magnesium defi ciency on antioxidant status and cadmium toxicity in rice seedlings. journal of plant physiology 168, 1021–1030. ci, d., jiang, d., wollenweber, b., dai, t., jing, q., cao, w., 2010: cadmium stress in wheat seedlings: growth, cadmium accumulation and photosynthesis. acta physiologiae plantarum 32, 365–373. claiborne, a., 1984: catalase activity. in: greenwald, r. a. 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f., 2006: cadmium inducible fe defi ciency responses observed from macro and molecular views in tobacco plants. plant cell reports 25, 365–373. zacchini, m., pietrini, f., mugnozza, g. s., iori, v., pietrosanti, l., massacci, a., 2009: metal tolerance, accumulation and translocation in poplar and willow clones treated with cadmium in hydroponics. water, air, & soil pollution 197, 23–34. zhang, g., fukami, m., sekimoto, h., 2002: infl uence of cadmium on mineral concentrations and yield components in wheat genotypes differing in cd tolerance at seedling stage. field crops research 77, 93–98. 611 siddiqui.vp acta bot. croat. 72 (1), 145–156, 2013 coden: abcra 25 issn 0365–0588 eissn 1847-8476 effects of double stress on antioxidant enzyme activity in vigna radiata (l.) wilczek zamin s. siddiqui* department of botany, university of karachi, karachi 75270, pakistan abstract – the effects of double stress environment i.e. lead (heavy metal) and nacl (saline) on the activity of antioxidant enzymes in vigna radiata seedling were studied. the antioxidant activities of enzymes, i.e of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and their activity proportions were examined. superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase and glutathione reductase activities were substantially increased in a combined stress environment as compared to catalase. further, in comparison with catalase and ascorbate peroxidase, glutathione reductase showed increased activities together with superoxide dismutase in a combined stress environment. superoxide dismutase and glutathione reductase showed higher activity proportion in combined treatment. physiological role of these enzymes in stress tolerance mechanism is discussed. keyword: antioxidant, catalase, enzyme activity, glutathione reductase, guaiacol peroxidase, scorbate peroxidase, seedling, stress, superoxide dismutase, vigna radiata abbreviations: apx – ascorbate peroxidase, cat – catalase, gpx – guaiacol peroxidase, gr – glutathione reductase, ros – reactive oxygen species, sod – superoxide dismutase introduction environmental stress factors like drought, temperature, high salinity and heavy metals are the major constraint that limit plant growth and productivity, by disturbing the intracellular water balance. usually, in fields or on agricultural land, unlike in a laboratory or even a greenhouse environment, plants are subjected to a manifold array of stress factor (siddiqui et al. 2008, nawaz et al. 2010). however, most of the studies have been devoted to assess the physiological response of plants in a single stress environment like salinity (sinha 1991, sharma and gills 1994, kumar and kumar 1996), drought (shinozaki and yamaguchi 2007) and heavy metals (hameed et al. 2000, jetley et al. 2004) and studies on the physiological responses of plants under a combination of such stresses are restricted to acta bot. croat. 72 (1), 2013 145 * corresponding address, e-mail: zaminss@uok.edu.pk copyright® 2013 by acta botanica croatica, the faculty of science, university of zagreb. all rights reserved. 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees just a few reports (wang et al. 2003, dudley and shani 2003, wang and huang 2004, jakab et al. 2005) and even they are not directly related to the combination of stress factors like heavy metal and salt stress. plants have utilized various mechanisms to combat with abiotic stresses. among them, stress tolerance gene expression, compatible solutes, phenols and antioxidant enzyme production are some examples (jakab et al. 2005, ahmed et al. 2010, siddiqui and khan 2011). antioxidant enzymes such as superoxide dismutase (sod) ascorbate peroxidase (apx), glutathione reductase, guaiacol peroxidase and catalase are well-known defense systems providing protection against the hazards of reactive oxygen species (ros) in different stressful conditions (allen et al. 1997, kwon et al. 2002, ahmed et al. 2010). activities of these antioxidant enzymes are frequently observed in a single stress environment but the response and the proportion of the relative activities of these enzymes in a combined heavy metal (lead) and salt stress environment have seldom been reported. to make up for this lack, the present study examines the response of antioxidant enzymes and the proportions of their relative activities in a combined heavy metal (lead chloride and lead nitrate) and salt (nacl) stress environment. lead is used in two forms to differentiate the effects of lead as chloride or nitrate together with sodium chloride environments. material and methods germplasm of vigna radiata was obtained from the pakistan agriculture research centre karachi university. seeds were sterilized with 50% clorox for 10 min, and then repeatedly washed with distilled water. the sterilized seeds were placed on moist whatman 1 filter paper in petri dishes and kept at 25 °c in the dark until germination. germinating seeds were transferred into small plastic pots in a plastic tray (diameter 10cm, height 12.5 cm) containing acid-washed sand. the whole set up waskept in a growth chamber with a 13-h photoperiod (28 °c, 600 ± 50 mmol m–2 s–1 par) and 11 h night (22 °c) at 60–70% humidity. plants were irrigated with 3 l of half-strength hoagland’s solution (hoagland et arnon 1950) until the plant developed five to six expanded leaves. treatments were prepared in this order (t1= 15 and 30 mg l–1 lead chloride and lead nitrate, t2 = 75, 150 and 225 mm nacl, t3 =15 mg l–1 and 3 mg l–1 lead chloride and nitrate in 75, 150 and 225 mm nacl, t4 = half strength hoagland solution (control). all the treatments were prepared in half strength hoagland solution and poured separately in large size (30.5 cm) plastic plates in which pots were kept on alternate days. plants were allowed to grow up to three weeks. leaf samples of three weeks old plants were randomly collected from the treatments and the control and subjected to antioxidant enzyme activity estimation. all the treatments and controls were replicated four times. extraction randomly collected 500 mg leaf samples were crushed in liquid nitrogen at 4 °c and homogenized in 10 ml protein extraction buffer containing tris-hcl ph 6.8, 50 mg pvp, 10 ml ddt, 0.1 mm edta. the contents were centrifuged at 10,000 rpm for 15 min. total protein was estimated by the method of bradford (1976) 146 acta bot. croat. 72 (1), 2013 siddiqui z. s. 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees catalase (enzyme number by nc iubmb: ec 1.11.1.6) catalase (cat) activity was estimated by the method of patterson et al. (1984). the decomposition of h2o2 was measured at 240 nm taking de at 240 nm as 43.6 mm cm –1 . reaction mixture (3.0 ml) consisted of 10.5 mm h2o2 in 0.05 m potassium phosphate buffer (ph 7.0) and the reaction was initiated after the addition of 0.1 ml enzyme extract at 25 °c. the decrease in absorbance at 240 nm was used to calculate the activity. one unit of cat activity is defined as the amount of enzyme that catalyzes the conversion of 1 mm of h2o2 min –1 at 25 °c. ascorbate peroxidase (enzyme number by nc iubmb: ec 1.11.1.11) ascorbate peroxidase (apx) activity was determined according to the method of nakano and asada (1981). the reaction mixture (2.0 ml) contained 0.05 m potassium phosphate buffer (ph 7.0), 0.2 mm edta, 0.5 mm ascorbic acid and 0.25 mm h2o2. the reaction was started after the addition of 0.1 ml enzyme extract at 25 °c. the decrease in absorbance at 290 nm for one minute was recorded and the amount of ascorbate oxidized was calculated from the extinction coefficient 2.8 mm cm –1 . the unit of activity is expressed as micromole of ascorbic acid oxidized min –1 at 25 °c. guaiacol peroxidase (enzyme number by nc iubmb: ec 1.11.17) guaiacol peroxidase (gpx) activity was measured spectrophotometrically at 25 °c by the method of tatiana et al. (1999). the reaction mixture (2.0 ml) consisted of 0.05 m potassium phosphate buffer (ph 7.0), 2 mm h2o2, and 2.7 mm guaiacol. the reaction was started by the addition of 0.1 ml enzyme extract. the initial rate of guaiacol oxidation was measured by the rate of formation of tetraguaiacol and was measured at 470 nm (de = 26.6 mm cm –1 ). one unit is defined as the amount of enzyme required to catalyze the conversion of one micromole of hydrogen peroxide, with guaiacol as hydrogen donor, per minute under specified conditions. glutathione reductase (enzyme number by nc iubmb: 1.6.4.2) glutathione reductase (gr) activity was determined at 25 °c by measuring the rate of nadph oxidation as a decrease in absorbance at 340 nm (e = 6.2 mm cm –1 ) according to the method of halliwell and foyer (1978). the reaction mixture (1.0 ml) consisted of 100 mm tris-hcl buffer (ph 7.8), 21 mm edta, 0.005 mm nadph, 0.5 mm oxidized glutathione, and the enzyme extract. nadph was added to start the reaction. unit activity is defined by the expression »one unit will reduce 1.0 mmol of oxidized glutathione per minute under standard assay conditions«. superoxide dismutase (enzyme number by nc iubmb: ec 1.15.1.1) the assay for superoxide dismutase (sod) activity was performed by following the method of beyer and fridovich (1987). the assay mixture consisted of 27.0 ml of 0.05 m potassium phosphate buffer (ph 7.8), 1.5 ml of l-methionine (300 mg per 2.7 ml), 1.0 ml of nitroblue tetrazolium salt (14.4 mg per 10 ml), and 0.75 ml of triton x-100. aliquots (1.0 ml) of this mixture were delivered into small glass tubes, followed by the addition of 20 ml enzyme extract and 10 ml of riboflavin (4.4 mg per 100 ml). the cocktail was mixed and then illuminated for 15 minutes in an aluminum foil-lined box, containing 25 w fluoresacta bot. croat. 72 (1), 2013 147 effect of salt and heavy metal stress on vigna seedling 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees cent tubes. in a control tube the sample was replaced by 20 ml of buffer and the absorbance was measured at 560 nm. the reaction was stopped by switching off the light and placing the tubes in the dark. increase in absorbance due to formation of formazan was measured at 560 nm. under the described conditions, the increase in absorbance in the control was taken as 100% and the enzyme activity in the samples were calculated by determining the percentage inhibition per minute. one unit of sod is the amount of enzyme that causes a 50% inhibition of the rate for reduction of nitroblue tetrazolium salt under the conditions of the assay. statistical analysis data were subjected to fanova using statistical package for social sciences, ver 17.0. the four factors control, heavy metals, salt and combination of heavy metal and salt were computed. results of the bonferoni test are expressed by alphabet letters on bar graphs. relative proportions of enzymes proportions of antioxidant enzymes were calculated by the following formula: proportion of antioxidant enzymes = specific activity of sod in combined environment specific activity of antioxidant enzyme results the effects of combined heavy metal (lead chloride and lead nitrate) and salt (nacl) stress on antioxidant enzyme activity in three week old vigna radiata plants were examined. applications of chloride and nitrate salt of lead significantly altered the superoxide dismutase (sod) and catalase (cat) activities in the saline (nacl) medium (fig. 1). sod activities were higher in a combined lead and salt stress environment than in an environment without nacl. optimum activity was recorded in a plant when it was treated with a combination of 15 mg l –1 lead chloride and 75 mm nacl. however, 15 mg l –1 lead chloride without nacl inhibited sod activities. likewise, sod activities in 15 mg l –1 lead chloride and nitrate together with 150 and 225 mm nacl solution were enhanced. it was observed that sod had a greater activity in a two stress than in a single stress environment. single stresses, such as lead without nacl, showed enhancement in cat activities, s optimum activity occurring in plants treated with 15 mg l –1 lead nitrate (fig 1). however, at 30 mg l –1 lead nitrate with or without nacl almost complete inhibition in the cat activity was seen. with the application of heavy metals (lead chloride and lead nitrate) in a high saline environment (150, 225 mm nacl) fewer cat than sod activities were recorded. it was observed that cat had less activity in a combined stress than in a single stress environment. when lead chloride and nitrate were combined with a nacl solution there was substantial improvement in ascorbate peroxidase (apx) activity compared to control (fig 2). maximum activities were measured when plants were treated with 15 mg l –1 lead nitrate together with all saline media (75, 150 and 225 mm nacl). however, lead chloride and lead nitrate showed significant inhibition when provided without nacl. 148 acta bot. croat. 72 (1), 2013 siddiqui z. s. 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees glutathione reductase (gr) activities improved in a combined stress environment, with some exceptions, showing maximum activities in a plant treated with 15 mg l –1 lead chloride and 30 mg l –1 lead nitrate together with all saline concentrations (fig 2). application of single-stress lead chloride in particular caused considerable reduction in gr activities. guaiacol peroxidase (gpx) activities were enhanced by the combination of lead and salt stress compared to control (fig. 3). however, optimum activities were recorded when plants were treated with 15 mg l –1 lead nitrate together with 75, 150 and 225 mm nacl. in general, a combination of heavy metal and salt stress improved gpx activities except when 30 mg l –1 lead nitrate was applied together with 150 and 225 mm nacl. acta bot. croat. 72 (1), 2013 149 effect of salt and heavy metal stress on vigna seedling nacl (75 mm) 0 50 100 150 200 250 300 without nacl with nacl nacl (150 mm) s u p e r o x id e d is m u ta s e s p e c if ic a c ti v it y (p e r m g o f p ro te in ) 0 50 100 150 200 250 300 nacl (225 mm) treatments c a1 a2 b1 b2 0 50 100 150 200 250 300 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 c a1 a2 b1 b2 0.0 0.1 0.2 0.3 0.4 0.5 0.6 a b a b a a a b a b a b a b a a a a a a a b a b a a a b a b a b a b b a a b a a a a a b a b a b a a a b a b a a a b a b c a ta la s e s p e c if ic a c ti v it y (p e r m g o f p ro te in ) fig. 1. antioxidant enzyme’s super oxide dismutase and catalase response in combined lead and saline environment. c – control, alead chloride, b – lead nitrate; b1 = 15, b2 = 30 mg l–1 lead concentrations. vertical lines on bar graphs represent mean ± se. similar alphabet letters show non significant difference at p<0.05 from corresponding ones measured on plant treated with lead salt with and without saline environment. 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees in order to compare the activity of antioxidant enzymes in combined stress environment, the relative enzyme activity performances in a combined stress environment were calculated. the study showed substantial activity of glutathione reductase (gr) after sod (fig 4). apx and gpx also had optimum activities compared to cat. the highest sod and gr enzyme units being found in a combined stress environment indicates a better tolerance against a combination of these stresses than found in other antioxidant enzymes. discussion antioxidant activities, in enzymes such as ascorbate peroxidase, catalase, superoxide dismutase activities are known to increase in a variety of environmental stresses like soil sa150 acta bot. croat. 72 (1), 2013 siddiqui z. s. nacl (75 mm) 0 1 2 3 4 without nacl with nacl nacl (150 mm) 0 1 2 3 4 nacl (225 mm) c a1 a2 b1 b2 0 1 2 3 4 0 5 10 15 0 5 10 15 treatments c a1 a2 b1 b2 0 5 10 15 a a a a a b a b a a a a a a a b a b a b a b a b a a a b a a a b a a a b a b a b a b a b a b a b a b a a a a a b a b a ba s c o rb a te p e ro x id a s e s s p e c if ic a c ti v it y (p e r m g o f p ro te in ) g lu ta th io n e re d u c ta s e s p e c if ic a c ti v it y (p e r m g o f p ro te in ) fig. 2. antioxidant enzyme’s ascorbate peroxidase and glutathione rductase response in combined lead and saline environment. for symbol explanation see figure 1. 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees linity, drought, extremes of temperature and heavy metals. physiologically these stresses cause oxidative damage to plants either directly or indirectly (comba et al. 1998, baisak et al. 1994, becana et al. 2000, shah et al. 2001, siddiqui et al. 2008). in fact, oxidative stress hazards are due to the production of reactive oxygen species which include superoxide radical (o2 +), hydroxyl radical (oh+) and hydrogen peroxide (h2o2). reactive oxygen species (ros) products in turn cause damage to the biomolecules by peroxidation, electrophilic substitution reaction, reduction of membrane lipids, proteins, chloroplast pigments, enzymes, nucleic acids, etc. (comba et al. 1998, becana et al. 2000, majeed et al. 2010). in a defense system, sod has a critical role in plants and functionally it dismutates superoxide radicals into water and o2. it has been reported that sod activities increased in rice plants growing under toxic levels of pb and salinity (verma and dubey 2003), water stress (zhang and kirkham 1994), cd, pb, al and cu toxicity (chongpraditnum et al. 1992, verma and dubey 2003, malecka et al. 2007, majeed et al. 2010). increase in sod activity in response to stress seems to be used in de-novo synthesis of the further antioxidant enzymatic protein (fadzilla et al. 1997, verma and dubey 2003). therefore, it is presumed that sod activities and synthesis of other antioxidant enzymatic protein like apx, cat, gr, gpx in a combined stress environment are linked. it may also be suggested that a combination of two or more antioxidant enzymes activities is required to provide complete tolerance (complete detoxification of ros) to plants under combined stress in particular. lower catalase activity in combined lead and salt stress environments is perhaps due to combined chloride toxicities, from both lead and sodium chloride. it is presumed that a combination of two chloride stresses may retard cat protein and thus reduce the activity. it has been observed that catalase activity under lead chloride toxicity was delayed in the removal of h2o2 and peroxides, which results in lipid peroxidation and thus in growth retardation. moreover, a similar decline in catalase activity was reported under salt stress (comba et al. 1998), chilling (mishra and singhal 1992), drought stress (lozano et al. acta bot. croat. 72 (1), 2013 151 effect of salt and heavy metal stress on vigna seedling nacl (75 mm) 0 1 2 3 without nacl with nacl nacl (150 mm) 0 1 2 3 nacl (225 mm) treatments c a1 a2 b1 b2 0 1 2 3 a a a a a a a b a b a a a b a a a b a a a a a b a b a b a b g u a ia c o l p e ro x id a s e s p e c if ic a c ti v it y (p e r m g o f p ro te in ) fig. 3. antioxidant enzyme’s guaiacol peroxidase response in combined lead and saline environment. for symbol explanation see figure 1. 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees 1996) and hypoxia stress (ushimaru 1992). physiologically, reduction in catalase activity under stressful conditions has been attributed to the inactivation of enzyme protein due to ros (ushimaru 1992) decrease in enzyme synthesis or change in the assembly of enzyme subunits (verma and dubey 2003). ascorbate peroxidase (apx) guaiacol peroxidase (gpx) and glutathione reductase (gr) are crucial components after superoxide dismutase (sod), which is required for complete neutralization of h2o2 (macrae and ferguson 1985, ushimaru 1992). it is presumed that a system of antioxidant enzymes in a combined stress environment acts as a chain reaction. for instance, sod first dismutated ros into hydrogen peroxide which was then further converted into water and molecular oxygen by the activities of apx, gpx or gr in different proportions. it was presumed that sod and cat might act together to combat combined stress environment as it was observed in single stress study. however, in this study, cat seems to be more sensitive under combined salt and lead stress. the better activity pattern of apx, gpx and gr in a combined stress environment showed a unique combination of activity patterns, not including cat. further, in a single stress study, apx exploits the reducing power of ascorbic acid more efficiently to get rid of potentially harmful h2o2 in response to abiotic stress (hertwig et al. 1992, jebara et al. 2005, siddiqui et al. 2008, majeed et al. 2010). likewise, glutathione reductase catalyzes the nadph-dependent reduction of glutathione by its redox active thiol group, and glutathione is involved in the re152 acta bot. croat. 72 (1), 2013 siddiqui z. s. cat a n ti o x id a n t e n z y m e s ra ti o o v e r s u p e r o x id e d is m u ta s e 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 75 mm nacl 150 mm nacl 225 mm nacl apx a1 a2 b1 b2 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 gr 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 gpx combined treatments a1 a2 b1 b2 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 a n ti o x id a n t e n z y m e s ra ti o o v e r s u p e r o x id e d is m u ta s e fig. 4. antioxidant enzymes proportion over super oxide dismutase (sod) activity in combined lead and saline environments. apx – ascorbate peroxidase, cat – catalase, gpx – guaiacol peroxidase, gr – glutathione reductase 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:15 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees dox regulation of the cell cycle (kubo et al. 1995, weckx and clijsters 1996, hideg et al. 1997, siddiqui et al. 2008) and has often been considered to play a significant role in plant defense mechanisms against oxidative stress. further the active role of gr activities has been reported by several workers in individual abiotic stress environments (kubo et al. 1995, weckx and clijsters 1996, hideg et al. 1997, siddiqui et al. 2008, majeed et al. 2010, ahmed et al. 2010). it is presumed that gr after the sod in a combined stress environment may be involved in regulation in a cell cycle, which may not only provide tolerance but also improve 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printer profile composite 150 lpi at 45 degrees weckx, j. e. j., clijsters, h. m. m., 1996: oxidative damage and defense mechanisms in primary leaves of phaseolus vulgaris as a result of root assimilation of toxic mounts of copper. physiologia plantrum 96, 506–512. zhang, j., kirkham, m. b.,1994: drought-stress induced changes in activities of superoxide dismutase, catalase and peroxidase in wheat species. plant cell physiology 35, 785–791. 156 acta bot. croat. 72 (1), 2013 siddiqui z. s. 611 siddiqui.prn u:\acta botanica\acta-botan 1-13\611 siddiqui.vp 14. o ujak 2013 11:38:16 color profile: generic cmyk printer profile composite 150 lpi at 45 degrees